SK6092001A3 - Composition consisting of influenza virus surface proteins 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

Technical field

The present invention relates to pharmaceutical active compound formulations, in particular antigens which are mixed with a mucosal adjuvant and a dispensing device formed as a spray applicator can be applied to the nasal mucosa so that efficacy so far unreachable can be achieved.

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

(a) proteins with an influential surface which are formulated liposomal (virosomes);

(b) mucosal adjuvant of bacterial origin;

c) a specific spray applicator, which is constructed such that nearly 100% of the sprayed dose can be applied in its entirety to the nasal mucosa important for efficacy.

BACKGROUND OF THE INVENTION

Antigens are preferably influenza-surface glycoproteins that are formulated liposomally (so-called virosomes). These virosomes are mixed with a mucosal adjuvant of bacterial origin. Ideally, it comes from a class of active or inactive toxins such as Heat Labile Toxin (HLT), choleratoxin (CT) or procholeragenoid (PCG). The nasal spray applicator is designed such that the active ingredient can be applied substantially all over the nasal mucosa. The formulation underlying the invention is

31704 / T can be used for various health indications such as vaccination against influenza or other infectious diseases as well as the therapeutic treatment of a nasal congestion, the reconstitution of a damaged nasal mucosa or a generally mucosal localized disease.

Various authors have described the immunostimulatory effect of liposomes (artificial membranes) (Gregoriadis, G .: Immunological adjuvants: A role for liposomes, Immunol. Today 11, (1990) 89-97). This immunostimulatory effect is obtained when the antigens attach to the surface of the liposome (GlOck, R., Mischler, R., Finkel, B., Que, JU, Scarpa, B., Cryz, SJ junior: Immunogenicity of a new virosome influenza vaccine in the elderly., Lancet 344 (1994), 160-163), encapsulated within the liposome (Gregoriadis, G., Davis, D., Davis, A., Liposomes as immunological adjuvants. Antigen incorporation Studies, Vaccine 5, (1987) 145 -151) or merely mixed with the liposome (De Haan, A., Geerligs., HJ, Huckshorn., JP., Van Schaarenburg., GJM, Palache., AM, Wilschut., J .: Mucosal immunoadjuvant activity of liposomes: induction of systemic IgG and secretory IgA responses in mice by intranasal immunization with influenza subunit vaccine and coadministered liposomes, Vaccine 13, 1995, 613-616). An additional immunostimulatory effect was observed when such liposomes were "spiked" by transmembrane and fusogenic glycoproteins (e.g., influenza glycoproteins (Glück, R., Mischler, R., Brantschen., S, Just, M., Althaus, B., Crzy, SJ). junior: Immunopotentiating reconstituted influenza virosome (IRIV) vaccine delivery system for immunization against hepatitis AJ Clin. Invest. 90, (1992) 2491-2495) or glycoprotein sendai (Gould-Fogerite, S., Mazurkiewicz, JE, Bhisitkul., D., Mannino, RJ: Reconstitution of biologically active glycoproteins into large liposomes Source: CH Kim, J. Diwan, H. Tedeschi and JJ Salerno (Publisher), Advances in Membrane Biochemistry and Bioenergetics, Plenum Publishing Corp., New York (1987), page 569.

So far, however, this effect has been reported to be highest after parenteral administration (i. M., S. C. Or i. P.). However, some authors have discovered that such formulations can be successfully administered by conventional methods (droppers or

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• conventional nasal sprays). However, the results relate almost exclusively to experiments with laboratory animals, mostly mice, which have a much larger nasal mucosa than comparable humans compared to body weight. In addition, the amounts of antigen administered were so high that, for economic reasons as well as for product safety reasons, human use would not be contemplated. It has also been shown that a satisfactory effect cannot be achieved with reasonable dosing of the antigen (influenza) via liposomal formulation and proper nasal administration.

Other authors have therefore attempted to develop an effective nasally administrable vaccine by mixing antigens with a mucosal adjuvant of bacterial origin instead of a liposomal formulation. In particular, HLT, CT or non-toxic derivatives of HLT or CT have been used (Elson, CO, Ealding, W .: Generalized systemic and mucosal immunity in mice after mucosal stimulation, with cholera toxin. J. Immunol. 132 (1984) 2736-2741) .

Results in the literature show promising effects after nasal application, which have been repeatedly achieved, especially in laboratory animals. However, few clinical trials in humans confirm some efficacy (Tamura, SJ., Ishihira, K., Miyata, K., Aizawa, C., Kurata, T .: Mechanism of enhancement of the immune responses to influenza vaccine with cholera toxin. Vaccine 13 (1995) 339-341, however, the amount of antigen and adjuvant used has been repeatedly very high, which again raises concerns about product safety and the possibility of commercial use.

Another disadvantage of the previously described attempts to develop a nasally applicable, prophylactic or therapeutically effective vaccine is the inadequate delivery systems available today.

Currently, there are carrier sprays as mono-, bi- and multidose applicators. Detailed investigations and trials of prior nasal sprays using colorants (methylene blue) and nasal endoscopy suggest that the injected substance (vaccine, pharmaceutical solution) reaches the mucosa of the mucoso-associated immune system by a maximum of 25

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The mucosa-associated immune system mucosa is located in the nasal cavity on the lateral nasal wall in the region of the turbines (nasal mussels), as shown in Figure 1, human turbines and doe-like turbines. Our findings show that with the nasal sprays so far due to significant losses in the vestibule (our vestibule) (see Figure 2) as well as on the wall of the nasal septum, only an insufficient amount of the pharmaceutical active substance reaches the main nasal cavity. These support structures are lined with immunologically inactive platelet epithelium and tallow 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 existing nasal applicators to spray an effective dose on the respiratory mucosa (instead of specific and non-specific defense) are of different kinds:

1) due to lack of knowledge of anatomy, the main spray beam vector is directed in the wrong direction when sprayed. In this case, the pharmaceutical active substance practically does not enter the main nasal cavity. The sprayed liquid partially flows down the upper tongue.

2) The spray nozzle in the carrier does not have a diameter adapted to the anatomical conditions. The inner support opening is naturally slit-shaped and has a nozzle function (see Figure 2). Carriers of normal width do not allow this anatomical narrowing to be overcome. When spraying in front of the Lumen, only the spray cone sector corresponding to the width of our Lumen reaches the main nasal cavity.

3) The optimum spray angle has not yet been scientifically proven. In conventional spray applicators, it varies between 30 and 80 ° to the horizontal at normal head position or perpendicular to the longitudinal axis of the human body. This also explains the significant losses, namely up to 90% of the applied volume. Our systematic research shows that the optimum spray angle lies

31704 / T •·································· € and 80 °.

4) Conventional nasal sprays have a carrier portion that is too short. The distance between the finger cuff and the spray port is too short. With the existing sprays, it is not possible at all to reach the inner support opening. The support part should be at least 0.7 cm longer, particularly preferably the support part has a length of 1 to 1.5 cm.

It is therefore an object of the present invention to provide an improved formulation of a pharmaceutical active ingredient as well as an improved delivery device for preventing and treating infectious diseases. This object is achieved by the features of the claims

The most effective preparation for preventive and therapeutic effect consists of the formulation of the surprisingly costimulating effect of liposomes mixed with a mucosal adjuvant and applied by a new dosing device or a new nasal spray.

Accordingly, the complex of the invention consists of the following components:

(a) active substance (antigen);

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

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

In particular, the invention relates to a vaccine for intranasal administration comprising:

(a) proteins with an influential surface which are formulated liposomal (virosomes);

(b) mucosal adjuvants of bacterial origin; and

(c) a specific spray applicator which is designed such that nearly 100% of the spray dose can be applied in its entirety to the carrier

31704 / T • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • Mucosal mucosa, important for efficacy.

The active substance therefore relates to antigens, in particular to influenza glycoproteins, which, because of their transdomain membrane, can easily be incorporated into the artificial membrane (liposomes). However, other antigens alone or in combination with influenza antigens can easily be coupled to the surface of the liposomes. Coupling according to the chemical properties of the antigens is accomplished spontaneously or using the crosslinker chemical molecules described previously (Martin, FJ, Papahadiopoulos, D .: Irreversible coupling of immunoglobulin fragments to preformed vesiclec, J. Bio. Chem. 257, (1982) 286) -288). It can also be a DNS plasmid or an RNS plasmid that is encoded for an antigen and can be encapsulated into a liposome.

The term "antigen" within the meaning of the present invention includes both complete molecules and fragments of these molecules having antigenic properties and / or can be used for immunization. Further, the term antigen includes molecules and / or fragments of molecules that are immunostimulatory.

The influenza-surface protein used in the vaccines of the invention preferably comprises hemagglutinin (HA). Further, the influenza-surface protein may comprise hemagglutinite (HA) in conjunction / combination with neuraminidase (NA).

In one preferred embodiment, the invention relates to a vaccine comprising further antigens. In particular, other antigens may be bound to the surface of virosomes.

In a further preferred embodiment, the invention relates to a vaccine according to the invention which, in addition to the antigens consisting of the influenza-surface protein (s), comprises further antigens, preferably pathogenic organisms. Here, the pathogenic organism may be a virus, a bacterium, a fungus or a parasite. These organisms contain germs of various diseases such as hepatitis A virus, hepatitis B virus, respiratory Syncitial virus (pneumovirus), parainfluenzavirus, mumps virus, HIV virus, diphtheria bacteria, (Corynebacterium diphtheriae), tetanus bacillus (Cl)

31704 / T pneumococci, Haemophilus influenzae, E. coli, Candida albicans, Candida tropicalis, Candida pseudotropicalis, Candida parapsilosis, Candida krusei, Aspergillus-arten, Trichomonas species, Trypanosoma species, Leishmania species, Toxoplasma gondii, Plasmodium falcip, Plasmodium falcip ovale, Plasmodium malaríae, species Trematoden and Nematoden. In particular, they comprise the species Trematoden Schistoma haematobium, S. mansoni, S. japoniaum and Nematoden species Tricharis trichiura, Ascaris lumbricoides and Trichinella spiralis.

The invention further includes a vaccine that is an influenza vaccine. In particular, the invention includes a vaccine which:. may be used to prevent and / or treat general infections / infectious diseases. Further, the vaccine is for the prevention and / or treatment of influenza diseases, for the prevention and / or treatment of nasal congestion, for the treatment of nasal mucosal surface injuries. Further preferably, the invention comprises a vaccine wherein the hemagglutinin content per dose (100 μΙ) is between 1 - 30 pg, preferably between 3 - 10 pg and most preferably 3.75 pg.

In one preferred embodiment, the invention refers to a vaccine wherein the ratio of liposome phospholipid to HA is between 1: 10 and 20: 1, preferably between 1: 1 to 10: 1 and particularly preferably 3: 1.

Accordingly, the invention preferably relates to a vaccine wherein the liposome phospholipid is selected from the group of neutral, cationic and / or anionic phospholipids.

In one preferred embodiment, the invention relates to a vaccine wherein the mucosal adjuvant is an active toxin, an inactive toxin and / or a non-toxic toxin. In particular, it contains a mucosal adjuvant, preferably a Heat Labile Toxin (HTL), choleratoxin (CT) and / or Procholeragenoid (PCG). In one further preferred embodiment, the mucosal adjuvant is a Heat Labile Toxin (HTL) from Escherichia Coli. In one further preferred embodiment, the toxins may be inactivated. This inactivation can be accomplished by recombinant technology.

In one further embodiment, the invention comprises a vaccine which:

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• • 4 • • 4444 • · · · 44 • · • 4 44 4 444 44 4 4 4 4 44 • 44 44 44 44

it comprises Heat Labile Toxin (THL) and / or choleratoxin (CT) in a ratio that is between 1: 2 to 20: 1, preferably between 1: 1 to 1: 10 and most preferably 7.5: 1.

The term "balanced mixture of liposomes and mucosal adjuvants" as used above refers, on the one hand, to the correct ratio of antigens to phospholipids, as well as to the mucosal adjuvant required for satisfactory efficacy. Clinical investigations have shown the following: the ideal ratio of phospholipid (e.g. phosphatidylcholine, phosphatidylethanolamine, neutral, anionic or cationic phospholipids) to the influenza antigen is between 1: 1 to 20: 1. Ideally, the ratio of influenza antigen to active mucosal adjuvant (Toxin) The THL or CT is between 1: 2 and 20: 1. The most preferred ratio is 7.5: 1.

As mentioned above, the ratio of the influenza antigen to the inactive mucosal adjuvant (inactive toxin) of PCG or a non-toxic derivative of HLT and CT is between 3: 1 and 1:20, ideally 1: 2.

Surprisingly, in vitro experiments to measure the immunostimulatory mucosal effect have shown that the mixture of liposome adjuvant and mucosal adjuvant does not cause an addition of effects, but the addition increases by a factor of at least

5th

The term "new medical device" refers to a spray applicator that is particularly suitable for intranasal immunization (vaccination). It is not enough to assemble a mucosal active substance if the application is poor or insufficient.

The new device is characterized by taking full account of the anatomical structure of the nose:

1) The distance from the finger cuff to the spray head is preferably at least 4.0 cm.

2) The front part of the carrier part consists of a substantially cylindrical extension which is, for example, at least 5 mm long and has a diameter of at most 5 mm.

3) The injection angle of the dispensing device described in the invention is preferably 50 ⁰ to 70 ⁰ to the horizontal.

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• · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·

4) The axis of the main direction of the dosing device or spray applicator is preferably determined by means of a special sleeve: the sleeve can preferably be fitted on the front part of the support part of the dosing device and is designed in such a way that is directed substantially towards the lateral wall of the nasal cavity.

Providing a cuff to support the upper lip is advantageous because the upper tongues are relatively close to the nose and yet the distance is large enough to provide an effective lever for convenient alignment. In addition, sensitive touch sensors on the user's lip allow the correct (centric or parallel) position of the cuff to be examined without the need to look into the mirror. In addition, however, the cuff may also be formed to support other parts of the body or head.

The narrow support portion preferably has a minimum length of 0.5 cm, more preferably 1 cm, and the next thicker support portion is preferably at least 1 cm long, preferably 2 cm. The distance between the dorsal finger area and the spray tip should be at least 2 cm, preferably 2.5 to 3 cm, or the distance from the finger area (cuff) to the spray tip should be at least 3 cm, preferably 4-5 cm. The spray spray angle should preferably be 50 ^° to 70 ° to the horizontal when the head is held in a normal perpendicular position.

In addition, the main vector of the injected dose should be directed between the middle and lower nose shells. This means that it should be directed to the middle part of the nose. This generally implies an almost horizontal direction of the applicator. Such a direction can be fixed using a special applicator cuff as described in the present invention. It is also possible to make the sleeve and the applicator one piece or integral. In this case, the fastening device / coupling device is rigidly connected to the applicator or is made in one piece.

In one preferred embodiment, the spray applicator is an apparatus whose spray head support part is preferably at most 5 mm thick and at least 5 mm, more preferably 7 mm long, whose distance

31704 / T medzi ······················ it is at least 3 cm long on the fingers up to the spray head and which, by means of a mountable cuff, allows introduction into the nasal opening such that the main sector of the nasal spray forms an angle of approximately 15 to 20 ° with the horizontal.

In another preferred embodiment, the antigen is a mixture of influenza surface antigens that is presented on the surface of liposomes. This composition may be combined with antigens of other pathogenic microorganisms. As mentioned above, therefore, the vaccine of the invention may contain other antigens, preferably antigens of other pathogenic microorganisms.

In another embodiment, the method described above is for the prophylaxis of infectious diseases, for the treatment of a nasal congestion and various injuries of the nasal mucosal surface.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 Comparative representation between human turbinalia and doe turbines

Figure 2 Schematic representation of the anatomical structure of the inner nasal opening

Figure 3 A comparative representation of the principle construction of a preferred embodiment of a dispensing device according to the invention

Figure 4 Representation of the correct spray angle in effective application

Figure 5 Representation of nasal cytology: quantification of different cell populations obtained by nasal smear technique in individuals (20 in each group) up to 29 days after three different types of intranasal vaccination. A marked increase in centroblasts should be noted as a sign of local immune response in Group A versus significantly less increase in Groups B and C (p <0.005),

Figure 6 A preferred embodiment of the dispensing device according to FIG

317047?

Invention without cuff a

7a and 7b show two views of a preferred embodiment of the sleeve for the dispensing device of the invention of FIG. 6th

DETAILED DESCRIPTION OF THE INVENTION

Hereinafter, preferred embodiments of the invention will be described with reference to the figures.

Example 1

Production of a mucosal virosomal influenza virus vaccine with HLT as an additional mucosal adjuvant.

The production of influenza virus vaccine is described in Gliick, R., Wegmann, A .: Liposomal presentation of influenza antigens, in Nicholson, KG, Webster, RG, Hay, AJ, published by Textbook of Influenza (1998) pages 400-409, London: Blackwell . Briefly, the production includes: strains H1N1 A / Singapore / 6/86, H3N2 A / Wuhan / 359/95 and B / Beijing / 184/93 influenza virus that are cultured in activated hen eggs and supplied from the National Institute of Biological Standards and Control, London, GB. Intact virions are isolated by chorioallantois fluid by zone centrifugation and inactivated by β-propiolactone. The purified virions are placed in a buffer containing 0.1 M octaethylene glycol mono (N-dodecyl) ether (OEG) (Nikko Chemicals, Japan) in PBSNaCl. The virions are incubated for 20 minutes at 21 ° C to allow complete decomposition of the viral component.

For extraction of hemagglutinin (HA) and neuramidinase (NA), the mixture is centrifuged for 60 minutes at 100,000 g. The residue that contains HA, NA and viral phospholipids (PL) is used to produce various intranasal vaccine formulations. Additional phospholipids (phosphatidylcholine (Lipoid, Germany)) were added and dissolved. Virosomes are formed spontaneously during OEG detergent removal by chromatography. To the mucosal dose

31704 / T • ······························ · A vaccine (100 µl) containing 3.75 µg HA of each of the three influenza virus strains, as recommended by the WHO and 35 µg lecithin, is added 0.5 µg Heat Labile Toxin (HLT) from E. coli from production strain E coli HE22VK (Vogel, FR, Powel, MF: Vaccine Design Compendium of Vaccine Adjuvants and Excipients: The Submit and Adjuvant Approach (MF Powel, MJ Newmann, Hrsg.), Plenum Press, New York (1995) page 141) as a mucosal adjuvant.

Example 2

E. coli cells are sedimented in a flow centrifuge (Westfalia AG) and suspended in "Phosphate buffered saline" (PBS, 6.06 g / L Na 2 HPO 4, 1.46 g / L KH 2 PO 4, 2.4 g / L NaCl pH 7 4).

The elevated temperature-labile intracellular toxin (HLT) release is accomplished by disintegrating cells in a ball mill (e.g., Dyna-Mill, W. Bachofen AG) or in French-Press. For example: 10 µl of cell suspension is pumped at a flow rate of 33 ml / minute by ball mill. The ball mill is filled with 500 ml glass balls, rotates 3000 rpm and the slot size is 0.05 mm.

Separation of the cell solids from the disintegrated solution is accomplished by tangential microfiltration. For example: 10 L of cell lysate is concentrated in the Prostak-System (Millipore AG) using a 0.2 µl filter to about 4 L, then washed with 24 L of PBS. The permeate is filtered with a sterile 0.2 µ filter (e.g. Gelman Supor DCF, Pall).

HLT is isolated from the semen by affinity chromatography. The stationary phase used is immobilized galactose (Galaktose-Gel, e.g. from Pierce) or immobilized lactose (Lactosyl-Gel, e.g. from Pharmacia). The mobile phase consists of PBS (buffer A) and 5% (g / v) lactose in PBS (buffer A) at half concentration. For example, the sterilized cell lysate is applied to a column pre-conditioned with buffer A and the column is then washed with buffer until the UV-absorption reaches the baseline of 280 nm. HLT is then eluted from the column with buffer B.

31704 / T • 4 · 4 · 4 · 4 · 4 · 4 · 4 · 4 ·· ··· ··· ·· ·· ·· ·

Example 3

Production of mucosal virosomal influenza vaccine with PCG as an additional mucosal adjuvant

The production of an influenza virosomal vaccine is described in Example 1. To a mucosal dose of vaccine (100 μί) containing 3.75 µg HA of each of the three influenza vaccine strains as recommended by the WHO and 35 µg lecithin, 6 µg of procholeragenoid is added as a mucosal adjuvant. produced by heating purified CT derived from V. cholerae (Inaba 569B) (Pierce, NF, Cray, WC, Sacci, JB, Craig, JP, Geramnier, R., Furer, E .: Procholeragenoid: A safe and effectiv antigen for oral immunization against experimental cholera, Inf., Immunity40, 3, (1983), pages 1112-1118).

Example 4

Production of mucosal vaccine hepatitis B- (HBs)

Phosphatidylethanolamine (R. Berchtold, Biochemiksches Labor, Bern, Schweiz) was dissolved in methanol and 0.1% (v / v) triethylamine was added. The solution is then mixed with gamma -aleinimidobutyric acid N-hydroxysuccinimide ester (GMBS) (Pierce Chemical Company, Rockford, IL) (phosphatidylethanolamine: GMBV = 2: 1), which is previously dissolved in dimethylsulfoxide (DMSO). After 15 minutes incubation at room temperature, the solvent is evaporated in a Speedvac centrifuge for 1 hour under vacuum. Recombinant HBs particles are produced in a known manner in CHO cell lines and purified.

In order to obtain a reduced HBs particle with free cysteine residues, the particles are treated with 40 mmol / l DL-dithiothreitol (DTT) for 5 minutes at room temperature. DTT was removed using a Sephadex-G10 column (Pharmacia LKB Biotechnology, Uppsala, Sweden) and octaethyl alcohol (Fluka Chemicals, Switzerland) was added at a final concentration of 100 mmol / L. evaporated

31704 / T • Phosphatidylethanolamine-GMBS is then stirred for 1 hour. with HBs solution (HBs: GMBS ratio = 1: 2), unbound GMBS is then captured by cysteine. The reactions were checked by thin layer chromatography.

To the pre-cross-linked phosphatidylethanolamine-GMBS-HBs, 32 mg of phosphatidylcholine (Lipoid GmbH, Ludwigshafen, Germany) was added and this mixture was dissolved in a total volume of 2.66 ml PBS containing 100 mM OEG (PBSOEG).

Influenza hemagglutinin A / Singapore is purified as described in Example 1 above. A solution containing 4 mg hemagglutinin is centrifuged for 30 minutes at 100,000 g and the sediment is dissolved in 1.33 ml PBS-OEG.

The phospholipids and hemagglutinin solution are mixed with the mixture and sonicated for 1 minute. The mixture is then centrifuged for 1 hour at 100,000 g and the residue is sterilized by filtration (0.22 µm).

The virosomes with adsorbed HBs are then removed by detergent removal using the BioRad-SM-Bio-Beads of Example 1. To a stock solution containing 50 µg of HBs antigen / ml, 10 µg / ml of HLT of Example 2 is added and filled into the spray applicator of Example 6th

Example 5

In vitro assay to determine mucosal adjuvant activity

The biological activities of the mucosal adjuvants are 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 are added to the Y-1 ATCC kidney cells: CCL 79 (Y-1 adrenal tumor, mouse steroid secreting) in the mini-culture according to Sack DA and Sack RB (Sack, DA and Sack, RB: Escherichia coli enterotoxigenic test using Y-1 adrenal cells in miniculture, Infect.Ommun. 11 (1974) pages 334-336:

(a) HLT (5 pg / ml), (b) PCG (60 pg / ml), (c) influenza virosomes and HLT: 37 pg / ml influenza hemagglutinin of three strains (H1N1, H3N2, B), 100

31704 / T ························ DπΙ phosphatidylcholine and 5 μς / ητιΙ as well as (d) influenza virosome and PCG: 37 μρ / ΓηΙ influenza hemagglutinin of three strains, 100 μg / ml phosphatidylcholine and 60 μg / ml PCG.

The following activities are determined (in units):

(a) 15 units, (b) 6 units, (c) 80 units, (d) 36 units. The highest biological activity of the adjuvant is achieved by a mixture of virosome and HLT or virosome and PCG. The effect of the adjuvant is determined by the cell toxicity parameter.

Example 6

Clinical evaluation of various spray applicators

The dosing device according to the invention is, by virtue of its basic construction, substantially comparable to conventional spray applicators, but is particularly well adapted to the anatomical structure for nasal application of any of the pharmaceutical active ingredients described in particular herein.

A preferred embodiment of the dispensing device 2 according to the invention is shown in Figures 3 and 6. In a side view shown in Fig. 3 of the dispensing device 2 according to the invention (spray applicator), the basic components are recognized. The metering device 2 essentially comprises a reservoir 4 with a pharmaceutical active substance (not shown) and a spray or pumping mechanism 6. This pumping mechanism 6 is non-fluidically connected to the reservoir 4 by a connecting element 8. Injection and pumping by the dosing device according to finger cuffs 10. The pharmaceutical active substance present in the container 4 extends through the opening 12 from the pumping mechanism into the outlet opening 14.

The metering device according to the invention 2 differs from the known spray applicators in particular in the construction of the carrier part 16, which plays an essential role for the effective application of the pharmaceutical active substance. carrier

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The portion 16 is preferably formed integrally with the pumping mechanism 6 and the finger cuff 10. The support portion is preferably divided into two sections 18 and 20, wherein the section 18 positioned in the direction of the finger cuff 10 has a larger diameter than the adjacent section 20 facing the outlet opening 14 of the carrier

16. The transition region between the two sections 16 and 20 forms a stop for the wear collar 24 on the wearer's upper lip, which can be fastened in the section 20 of the carrier portion 16 according to one preferred embodiment of the present invention.

The section 20 of the support 16 adjacent to the outlet port 14 is preferably formed substantially cylindrical and has a diameter d ₂ of the. at most 5 mm, preferably between 2 and 4 mm. The length of the front section 20 of the support part 16 is, for example, at least 5 mm, preferably at least 10 mm and particularly preferably 10 to 20 mm. The sleeve 24 is preferably slidable from the outlet opening 14 through a section 20 of the support portion 16 and is preferably secured with a rotation prevention device on the dispensing device 2. For example, the rotation prevention device can be realized in the form of a polygonal profile, for example elliptical cross section. 16 and the corresponding recess 26 in the cuff 24.

In a manner corresponding to another embodiment of the dispensing device 2 of the invention, the portion of the spray applicator entering the nose of the patient may also be formed on the cuff 24. Such an embodiment is shown, for example, in Figure 7b. As shown in Figure 7b, a substantially tubular extension 28 is formed on the sleeve 24, which can be mounted on a section 20 of the support portion 16 adjacent to the outlet opening 14. The dimensions of the tubular extension 28 preferably correspond essentially to the dimensions (d _2). and I20) of the above-described embodiment.

The section 18 of the support portion 16 preferably has a length l1 and at least 10 mm and preferably about 20 mm. The dimensions of the two sections 18 and 20 are preferably chosen such that the distance between the dorsal surface for the fingers of the user and the tip of the spray or outlet 14 is at least 2 cm, preferably 2.5 to 3 cm. This is particularly ensured if the distance between the sleeve 24 to the tip of the spray 14 is at least 3 cm, or preferably 4 to 5 cm.

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With the dosing device 2 according to the invention with these dimensions, the pharmaceutical active substance can be sprayed into the main nasal cavity or internal nasal opening substantially more precisely, so that a significantly higher efficiency can be achieved than with known systems.

The metering device 2 according to the invention is preferably provided with a collar 24, so that, in addition to the favorable dimensions, an optimum spray angle α can be set. However, independently of the spray applicator 2 described above, the sleeve 24 can be disposed on conventional dispensing devices so that even with such conventional spray applicators a substantially improved efficiency can be achieved by virtue of a precisely adjustable spray angle α.

The metering device 2 according to the invention can furthermore achieve improved efficiency either by selecting the dimensions of the support part 16 or by a suitable sleeve 24 for adjusting the optimum spray angle α. In particular, however, the combination of the new metering device 2 with the dimensions described above and the sleeve 24 for adjusting the optimum spray angle and achieve the best possible treatment results are achieved.

The sleeve 24, as previously briefly described, is displaced by the tubular extension 28 over the section 20 of the support portion 16. In the assembled state, the sleeve 24 is preferably connected to the metering device 2 without rotation. For example, the anti-rotation lock on the support part 16 can be flattened on at least one side, by means of which the sleeve 24 can be mounted without rotation in a correspondingly shaped opening. The sleeve 24 has a support 30 that can be supported on the upper tongue of the user to define a spray angle in a defined manner together with the support portion 16 provided with a section 20 that faces the patient's orifice. The optimum spray angle α is preferably between 50 ^° and 70 °.

The collar 30 of the collar 24 is preferably easily curved in the shape of an upper lip to provide an optimal area for placement and support. The sleeve 24 may be made of any suitable material, but all plastics capable of injection molding are preferred.

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Various dosing devices were tested for efficacy. To this end, 1% methylene blue solution was filled into five different types of carrier applicators:

a) a spray applicator according to the invention with a directional cuff that fixes the direction of the spray dose;

b) spray applicator according to the invention without directional cuff;

(c) a conventional commercial spray applicator without a narrow support 2 cm in length and 4 mm in diameter with a spray angle of 50 °;

d) spray applicator according to the invention, but with a spray angle of 50 °;

e) a spray applicator according to the invention, but with a finger cuff which is only 3 cm away from the spray head.

All spray applicators are so-called dual-dose applicators. 5 sprayers tested each spray applicator under medical supervision. Subjects injected one dose of methylene blue solution into their own right and left nostrils. Using nasal endoscopy, mucosal turbines are explained and photographically documented. The intensity of the blue color as well as the blue color on the turbines is evaluated

using the scale (geometric center): The evaluation gives the following results group a) 19 points group B) 16 points group c) 7 points group d) 9 points group e) 12 points.

Only in group a) the spray liquid was applied almost exclusively to the nasal mucosa. Also in group b) a large part of the spray content was sprayed onto the mucosa. In the other groups, a substantial portion of the spray content was sprayed into the nasal atrium. There the applied spray liquid cannot show

31704 / T •···························· · No efficiency.

Example 7

Comparison of the clinical efficacy of intranasal virosomal influenza vaccines with and without HLT used in the new spray device described in the present invention on human volunteers compared to a parenteral commercial influenza vaccine.

The open-label randomized clinical trial was conducted in full compliance with the principles of the Helsinki Statement and local laws and guidelines for clinical trials. After approval of the protocol by the Lucerne Canton Ethics Committee and notification to the Schweizer Bundesgesundheitsbehôrde, 80 healthy volunteers (aged 18-64) gave their informed consent to participate. Volunteers who showed symptoms of acute or chronic illness at the time of immunization and were concomitantly treated with immunosuppressive drugs or were known to weaken immunity were excluded.

Intranasal vaccine formulations are administered to each of three groups of 20 volunteers (Table 1). Groups A and B received 2 doses of formulation A and B, respectively, into each nostril on days 1 and 2 doses a week later. Group C received 2 doses of double-concentration formulation A on day 1. Group D was inoculated intramuscularly with a parenteral formulation into the deltoid region. Blood and saliva (Omnisal ^R , GB) samples were taken before the first vaccination and one month after the first immunization (day 29 ± 2). Due to technical problems, only the first 47 persons were able to assess saliva samples. Nasal cavity wiping cytology was performed prior to immunization and at days 4, 8, and 1 month after the first immunization.

For analysis, blood and saliva samples were coded.

The serum immune response to the HA vaccine component is determined by a standard assay using hemagglutinin units of the corresponding antigens. Sera are run at 56 ° C for 30 minutes prior to assay. Titres are

31704 / T, expressed as the reciprocal of the highest serum dilution that completely prevents hemagglutination. A titre of> 1:40 was found to be protective.

In general and influenza specific IgA antibodies were determined by known ELISA methods (Tamura, SI, Ito, Y., Asanuma, H. et al., Cross-protection against influenza virus infection afforded by trivalent inactivated vaccines inoculated intranasally with cholera toxin B subunit. J. Immunol., 149, (1992) pages 981-987). Virus specific IgA values were expressed as ELISA units of specific IgA per pg of total IgA concentration.

The nasal epithelial cells were taken exclusively from the Maxillo-Turbinate of both nasal cavities of each person by the same type of small nylon brush used in cytopathological examinations during bronchoscopy (Glúck, U., Gebbers, JO.). Nasal cytopathology in smokers: Am. J. Rhinol., 10, (1996), pages 55-57). Sampling was performed by the same investigator (U.G) under rhinoscopic control by rotational and translational movements along the bottom attachment of the turbinate. The cells were transferred to a glass preparation and immediately fixed in a solution of 200 ml of ethanol + 100 ml of acetone + 6 drops of trichloroacetic acid.

The slide with an object stained with papanicolaou was examined by a pathologist who had graduated from the Institute of Fur Pathologie und Cytopathologie, the hospital of the canton of Lucerne, and who was not informed of the vaccination status.

Mean numbers of cells, oscillating cells, goblet cells, lymphocytes, centroblasts, neutrophils, eosinophils and epithelial scale cells were determined in 25 representative regions of the object slide at a 10X magnification.

Signification between baseline titer and postimmunization titer was determined using the Paired-T assay. Differences in the ability of 4 vaccinations to elicit protective anti-HA antibodies in the study group were determined by χ ² .

All adverse events observed during the clinical study had to be recorded. An adverse event was defined as an adverse event change

31704 / T •··················· · Baseline (pre-vaccination) status of persons, whether or not the outcome is considered to be related to vaccination. Adverse events (local or systemic reaction) that occurred after immunization were recorded by the clinician on a special adverse event form. The baseline rate of adverse events was determined prior to immunization.

80 people with an average age of 40 years and comparable social status were enrolled. 27.5% of the participants were female. All 3 nasal vaccines as well as parenteral vaccine were well tolerated. Anamnetically, there were no significant differences between the three nasal vaccine groups and all three preparations were well tolerated. In individual cases, possible concomitant reactions were subsequently reported: fever, fatigue, nausea, rhinitis, nasal congestion and rhinopharyngitis. Also, the parenteral virosomal vaccine was extremely well tolerated.

Serological immune response is shown in Table 2. Significant titer increases were measured in Group A (2 nasal vaccinations at 7-day intervals), Group C (1 nasal vaccination, double dose) as well as Group D (parenteral vaccination against all three viral vaccines). strain). The highest geometric mean antibody titers (GMTs) were found in Groups A and D. Group D had significantly highest values (GMTs) against H1N1 strain (p <0.05). For H3N2 strain, there were no significant differences between groups A and D.

These groups reacted significantly better than groups B and C. There were no significant differences from groups A, C and D in strain B. However, these groups showed a significantly higher titer than group B. Serum conversion rates were highest in groups A and D. significantly higher than those in Groups B and C, and for all three strains they met the European Community serological requirements for parenteral influenza vaccine (Commission of the European Communities, Medical Device Guidelines in the European Community. Harmonization of requirements for influenza vaccine. (1992), pages 93- 98. Luxembourg: European Community Publication Office).

The humoral mucosal immune response (saliva) is shown in Table 3.

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The highest increases in IgA titer were measured in group A. These were significantly better than in other groups. Taking into account total IgA, GMTs in Group A were also the largest. The mucoconversion rate (four-fold rise in IgA titer) was again significantly highest in group A. However, in the case of intramuscular vaccination, only a very low mucoconversion rate was achieved.

Nasal mucosal swab cytology was performed in groups A, B, and C. The results are summarized in Figure 5. The numbers of nasal epithelial mucosa cells (oscillating and non-oscillating column cells, goblet cells and epithelial scale cells) and myelo / mono- and lymphopoiesis cells were determined. (lymphocytes, eosinophils, neutrophils and centroblasts). In group A, significant goblet cell hyperplasia was demonstrated on days 4 and 8 after vaccination. In addition, a strong increase in lymphocytes and centroblasts was observed on the same days. In addition, an increase in eosinophils and neutrophils was observed on day 8 after the initial vaccination. The number of column cells remained unchanged.

In Group B, there was only a slight increase in lymphocytes, neutrophils and eosinophilic granulocytes. There were no indications of activated lymphocytes in this group.

In Group C, even stronger goblet cell hyperplasia was found than in Group B. In addition, eosinophilic and neutrophil granulocytes rose most strongly in this group on days 4 and 8. The increase in lymphoblasts in this group was weaker than in group A. p <0.05. One month after the first vaccination, the original cell composition was reached in all groups.

Figure 5 relates to nasal cytology: quantification of different cell populations obtained by nasal smear technique (20 in each group) up to 29 days after three different types of intranasal vaccination. A marked increase in centroblasts was observed as indications of a local immune response in Group A as opposed to a significantly lower increase in Groups B and C (p <0.05).

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Example 8

Comparison of Clinical Efficacy of Intranasal Virosomal Influenza Vaccines with HTL or PCG in Human Volunteers by New Spray Device Described in the Present Invention

The immune response and tolerability of two antiinfluenza spray vaccines were examined in a double comparative study conducted with a total of 158 healthy Swiss volunteers aged 18 to 67 years.

The trivalent virosomal influenza vaccine (purified HA, formulated with phosphatidylcholine) is combined with the Heat Labile Toxin (HTL) E. coli to a format suitable for intranasal administration. The human dose contains 7.5 µg HA of each influenza strain and 2 µg HLT. A single dose was administered to each nostril on days 1 and 8 in groups of individuals aged 18 to 59 or> 60 years. Serum samples were collected about 4 weeks after immunization. Reactions were sparse and weak. The percentages of subjects (adults and older) who achieved the serum titer of anti-HA antibodies (> 40) were as follows: A / Bayern (92%, 91%), A / Wuhan (92%, 78%) and B / Beijing (59%, 50%). GMT after immunization and multiples of GMT increase were comparable between the two age groups. The percentage of subjects (adults or elderly) who did not reach the baseline protective titre but reached protective levels after immunization was as follows: A / Bayern (79%, 85%), A / Wuhan (86%, 56%) and B / Beijing (48%, 41%).

The second mucosal vaccine formulation contained 7.5 µg HA and 12 µg procholeragenoid (PCG). This formulation was equally well tolerated and proved less immunogenic than HLT. The percentages of subjects who achieved the serum titer of anti-HA antibodies were as follows: A / Bayern 94.2%, A / Wuhan 80.8% and B / Beijing 36.5%. The serum conversion rates of all groups are shown in Table 4.

These results indicate that the virosomal influenza vaccine administered by the intranasal route is more confident and more immunogenic in adults, including

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Example 9

Mucosal immune response in mice following intranasal use of virosomal influenza vaccine with HTL, with PCG or without additional mucosal adjuvant

Groups of 10 adult female Balb / c mice were intranasally vaccinated with 30 μί of either the influenza vaccine described in Example 1 or Example 2. Control groups of 10 mice received the virosomal vaccine formulation without additional adjuvant but with the same virosome composition. Half of each group received a second intranasal dose a week later. Three weeks later, nasal swabs (NW) and bronchoalveolar lavages (BAL) were performed. Table 5 to Example 9. Specific IgA determination was performed by known ELISA procedure. The results (GMT) are summarized in Table 6.

The highest GMT could be demonstrated for all three vaccine strains (A / Johannesburg, A / Nanching, B / Harbin) in the group that was twice vaccinated with the vaccine of Example 1. This group responded even with the highest IgA level in BAL after only one vaccination.

A satisfactory IgA response was also obtained in a group of mice that were vaccinated twice intranasally with the composition of Example 2. The control group receiving the virosome preparation without an additional mucosal adjuvant showed only a very weak mucosal immune response.

The results show that a virosomal influenza vaccine administered intranasally to mice that contains a mucosal adjuvant can induce a high mucosal antibody response.

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Preclinical study of viral influenza vaccine in mice: intranasal use (Table 5 to Example 9)

Skup. 1 Skup. 2 Skup. 3 Skup. 4 Skup. 5 Skup. 6 Day 0 Infl.Viros. + HLTi.n. Infl.Viros. + HLTi.n. Infl.Viros. + PCGi.n. Infl.Viros. + PCGi.n. Influenza virosome Influenza virosome Day 7 Infl.Viros. + HLTi.n. Infl.Viros. + PCGi.n. Infl.Viros. i.n. Day 28 S & BAL S & BAL S & BAL S & BAL S & BAL S & BAL

IgA influenza antibodies in mouse GMT reciprocal titer (ELISA) (Table to Example 9)

Group H1N1 NW H1N1 BAL H3N2 NW H3N2 BAL B NW B BAL 1 320 12800 380 3200 320 9220 2 neg. 140 neg. 24 neg. 1540 3 160 760 470 760 220 770 4 neg. neg. neg. neg. 10 neg. 5 4 neg. neg. neg. 4 neg. 6 neg. neg. neg. neg. neg. neg.

Example 10

Clinical evaluation of the mucosal vaccine Hepatitis-B- (HBs-)

The vaccine is made according to Example 4 and filled into a nasal spray applicator according to Example 5. The product is tested on 10 volunteers: 100 μΙ each is applied to each nostril on day 1 and repeated one week later. Blood samples are taken on day 1 (before vaccination) and then day 29. Anti-HBs antibodies were determined by RIA (Abbott). Geometrically the mean titer before vaccination is 7 IU / ml and on day 29 159 IU / ml.

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Example 11

Mucosal immune response in mice following intranasal use of a DNA plasmid that was encoded for the HN-antigen of the mumps inflammatory virus, introduced into an influenza virosome containing 10% cationic phospholipids and mixed with HLT (5 gg / 1 mL)

Groups of mice were immunized intranasally (a) without DNA encoding the mumps inflammation virus HN-antigen (group C), or (b) virosome-encapsulated DNA after pre-immunization with virosomes (group A) or (c) without pre-immunization (group B). Control group (B) was immunized with living Urabe mumps inflammation virus (Priorix, SKB Rixensart). As shown in Table 7, the geometric mean titer (GMT) of IgT in the group of mice receiving pre-immunization (A) is higher than that described in groups (B) and (C) (Lovell, GH: Proteosomes, hydrophobic anchors, iscoms and liposomes for improved presentation of peptide and protein vaccines in New Generation Vaccines (1990) (GC Woodrow and M. M. Levine, editors) Dekke, New York, pages 141-168; Cusi, MG and Gluck, R .: Intranasal immunization of mice with DNA mups entrapped into influenza virosomes (IBC with 4 ^th Annual Conference of Genetic Vaccines, October 25-27, 1998, Washington DC). A group of mice that had been immunized non-DNA developed a very low IgG level, whereas mice immunized with the mumps inflammation virus (group H) showed a good IgG response. Analysis of mucosal immunity revealed that all groups of mice except those immunized without DNA developed IgA. Only in nasal swabs (NW) of mice immunized with mumps inflammation virus increased IgA titer could be demonstrated.

Cytokine measurements were performed using primary spleen cells from spleen mice that were taken 12 days after immunization. Table 8 summarizes representative measurements obtained from two separate experiments. Cells from mice that had been previously (i.n.) vaccinated with DNA virosomes, stimulated with mumps inflammation virus antigens, induced IL-2 and IFN-gamma production. In addition, influenza-infected mice induced IL-4 production. Cells taken from

31704 / Γ ···························· Animals immunized with mumps inflammation virus produced IFN-gamma, IL-2, IL-4, and IL-10 mumps inflammation antigens following in vitro stimulation. DNA-virosome immunization as a control immunization with purified mumps inflammation antigens correlates with the Th-1 phenotype. In addition, considering the ratio between total IgG level and virus-specific IgG1 and IgG2a, the amount of IgGa-isotype dominated in group (A), suggesting a Th-2 response.

Table 7 - Geometric mean titer (GMT) of humoral IgG, IgA in bronchoalveolar wash (BAL) and IgA in nasal swab of mice

mice IgG BAL IgA NW IgA Group A 356 8 10 Group B 15 11 10 Group C 12 2 2 Group H 1585 2 20

Table 8 - Representative measurement of cytokine production in mice, obtained from two separate experiments

Group IL-2 IFN-gamma IL-4 IL-10 A 300 300 150 0 B 150 625 0 0 C 300 100 0 0 D 600 100 150 600

Example 12

Treatment of clogged nose in volunteers with influenza virosome with and without HLT with a new spray applicator

30 volunteers with cold symptoms were selected at the clinic

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and with an acute clogged nose. In all volunteers, the breathing intensity of each individual nostril was measured rhinomanometrically (in Pascals). Subsequently, the volunteers were randomized and divided into three groups of ten. Group A received one dose of vaccine (100 μΙ per nostril) according to Example (1), group B received the same dose but without mucosal adjuvant HLT, group C received 100 μΙ 0.9% NaCl each (saline) into each nostril. Air streams were measured 15 minutes, 30 minutes, 1 hour and 2 hours later.

The data are shown in Table 9. In both Group A and Group B, the values were significantly better than in Group C. A significant improvement in respiratory function was observed in both groups.

Table 9

Group Breathing intensity in Pascals 0 min 15 min 30 min 60 min 120 min A 490 693 712 681 521 B 497 687 705 672 538 C 488 497 513 521 501

Example 13

Treatment of mucosal lesions in volunteers with influenza virosomes and HLT

Volunteers were intranasally vaccinated as described in Example 7. As described, goblet cell diameters were determined 3, 7 and 28 days after intranasal vaccination. It has been possible to determine changes in goblet cell hyperplasia epithelium in cytological sterols (Gliick, U., Gebber, JO.). The goblet cells are protective

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function for the mucosal layer. One month after the first nasal vaccination, the mucosa cell state was significantly superior to the previous state.

This new vaccine can therefore be used as a therapeutic for treating the nasal mucosa in a damaged condition.

Example 14

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

The group of travelers visiting Tunis consisted of 38 people. After informed consent, all participants proceeded to the study. After randomization, 19 volunteers were vaccinated at 1 week intervals with the preparation of Example 1, with 19 subjects receiving no vaccine. Blood samples from all volunteers were taken 28 days after the first intranasal vaccination. 19 vaccinated subjects showed high serum IgG versus mucosal adjuvant (HLT). The control group remained anti-HLT negative. Before leaving Tunisia, one month later, a special medical event form was submitted to all participants. The group returned from Tunisia 20 days later and the completed medical incident forms were evaluated for diarrhoe disease. In the vaccine group, only two people reported diarrhoe problems, whereas in the non-vaccinated group, 9 people suffered from diarrhoe while in Tunisia. These data demonstrate that the vaccine is also effective in preventing diarrhea enterotoxic diseases of E. coli.

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Example 7

Table 1 - Clinical protocol

Group N male Middle age in years Number of vaccinations í (distance) The use Preparation in pg of complete HA vaccination per strain Preparation in pg of complete HLT vaccination A 20/14 39.7 2 1 week intranasally 15 2 B 20/14 35.5 2 2 weeks intranasally 15 - C 20/16 43.8 1 intranasally 15 2 D 20/14 41.2 1. intramuscularly 15 -

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Claims (42)

PATENT CLAIMS • ···· · · ·· ·· · · · • • · • · · · · • · · · • · ··· 9 · • • • • · · • · · · · • • • • · • · · · • · · ·· ·· · · · 1. Intranasal vaccine, consisting of: (a) proteins with an influential surface which are formulated liposomal (virosomes); (b) mucosal adjuvant of bacterial origin; (c) a specific spray applicator which is designed to be almost 100% of the sprayed dose can be applied throughout the volume to the nasal mucosa important for efficacy, The vaccine of claim 1, wherein the influenza surface protein is haemagglutinin HA. The vaccine of claim 1 or 2, wherein the influential surface proteins are hemagglutinin HA and neuraminidase NA. The vaccine of any one of claims 1 to 3, which comprises further antigens. The vaccine of claim 4, wherein the further antigens are bound to the surface of the virosomes. The vaccine of any one of claims 4 and 5, wherein the antigens are antigens of pathogenic organisms. The vaccine of claim 6, wherein the pathogenic organism is a virus, a bacterium, a fungus, or a parasite. The vaccine of claim 7, wherein the virus, bacterium, fungus or parasite is selected from the group consisting of: hepatitis A virus, hepatitis B virus, respiratory Syncitial virus (pneumovirus), parainfluenzavirus, mumps virus, mobilli virus, HIV, bacteria diphtheria (Corynebacterium diphtheríae), tetanus bacillus (Clostridium tetani), pneumococci, Haemophilus influenzae, E. coli, Candida albicans, Candida tropicalis, Candida pseudotropicalis, Candida parapsilosis, 31704 / T • ···· ·· ·· ·· • · · • · · • · · · · • ··· · · ··· · · • • • · · · • · · · · • • • · · • · · · • oz ·· ·· · · · Candida krusei, Aspergillus-arten, Trichomonas species, Trypanosoma species, Leishmania species, Toxoplasma gondii, Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale, Plasmodium malariae, Trematodes and Nematodes species. The vaccine of any one of claims 1 to 8, which is an influenza vaccine. The vaccine according to any one of claims 1 to 9, which can be used for the prevention and / or treatment of general infections / infectious diseases. A vaccine according to any one of claims 1 to 10 for the prevention and / or treatment of influenza diseases. The vaccine of claim 10 or 11 for preventing and / or treating a nasal congestion. A vaccine according to any one of claims 10, 11 or 12 for the treatment of damaged nasal mucous membranes. The vaccine according to any one of claims 1 to 13, wherein the hemagglutinin content per dose (100 µl) lies between 1 - 30 µg, preferably between 3 - 10 µg, and most preferably 3.75 µg. The vaccine of any one of claims 2 to 14, wherein the ratio of liposome-phospholipid to HA is between 1: 10 and 20: 1. The vaccine of claim 15, wherein the ratio of liposome-phospholipid to HA * is between 1: 1 and 10: 1. The vaccine of any one of claims 15 and / or 16, wherein the ratio is 3: 1. The vaccine of any one of claims 15 to 17, wherein the liposome phospholipid is selected from the group of neutral, cationic and anionic phospholipids. The vaccine of any one of claims 1 to 18, wherein the mucosal adjuvant is an active toxin, an inactive toxin, and / or a non-toxic derivative thereof. 31704 / T • ········· The vaccine of claim 19, wherein the toxin and / or non-toxic derivative thereof is Heat Labile Toxin (HLT), choleratoxin (CT) and / or procholeragenoid (PCG). Vaccine according to any one of claims 1 to 20, which comprises a Heat Labile Toxin (THL) and / or choleratoxin (CT) in a ratio of between 1: 2 to 20: 1, preferably between 1: 1 to 1: 10 and especially 7.5: 1. The vaccine of any one of claims 19 and 20, which comprises heat-inactivated procholeragenoid (PCG), recombinant technology-inactivated HLT and / or choleratoxin (CT) as a mucosal adjuvant, which is in a HA: PCG (and / or rHLT, and / or rHLT, and / or rCT) between 3: 1 and 1:20. The vaccine of claim 22, wherein the HA: PCG ratio (and / or rHLT, and / or rCT) is between 1: 1 and 1: 10. The vaccine of claim 23, wherein the HA: PCG ratio (and / or rHLT, and / or rCT) is 1: 2. The vaccine of any one of claims 1 to 24, further comprising DNS- and / or RNS-plasmids bound to and / or virosomes. The vaccine of any one of claims 1 to 25, which is an influenza vaccine. The mucosal adjuvant of any one of claims 1 to 26, which is a Heat Labile Toxin (HLT) that has been eluted from E. coli lactose buffer according to Example 2 and purified on a Lactosyl-Gel (Pharmacia) chromatography column. Cuff (24) with a coupling device (28) for placement on a metering device (2), in particular for spray administration of a pharmaceutical active ingredient according to any one of claims 1 to 27, and a support device (30), wherein the coupling device (28) and support device the device (30) being arranged to define together an optimum injection angle (a) for dispensing the substance from the dispensing device. The cuff (24) of claim 28, wherein the injection angle (α) is between 50 ° and 80 ° to the horizontal. 31704Π · ············ · ··· The cuff (24) of claim 28 or 29, wherein the coupling device (28) is a substantially cylindrical tube section. The cuff (24) of claim 30, wherein the tube section (28) can be displaced at least partially over a portion of the support portion (20) of the dispensing device (2). The cuff (24) of any one of claims 28 to 31, wherein the support device (30) is bent at the user-side end in such a manner that it substantially corresponds to the shape of the outer portion of the user's upper lip. Collar (24) according to any one of claims 28 to 32, wherein the collar (24) is secured to the metering device (2) so as to be secured against rotation. The sleeve (24) of claim 33, wherein the tube section (28) and the portion of the support portion (20) that can be moved along the tube section (28) are formed at least partially in a polygonal shape in order to secure the rotation. A dosing device (2), in particular for spraying the pharmaceutical active ingredient according to any one of claims 1 to 27, with a pumping device (6) and a finger cuff (10) for actuating the pumping device (6) with a connecting element (8) for sealingly engaging the container (4) and the carrier (16), which comprises a first section (18) and a second section (20), the two sections (18, 20) of the carrier (16) being sized such that the outlet ( 14) of the carrier portion (16) substantially directed into the main nasal cavity of the patient. The dispensing device (2) of claim 35, wherein at least the second section (20) of the support portion (16) is substantially cylindrical. Dosing device (2) according to claim 36, wherein the diameter of the second section (20) is between 3 mm and 10 mm, preferably about 7 mm. The metering device (2) according to any one of claims 35 to 37, wherein the second section (20) of the support portion (16) has a length of between 5 mm and 50 mm, preferably about 20 mm. 31704/1 « • 4 • 4444 4 ··· 4 • · · 44 e e · · 4 · 4 • 4 • • · · · 4 • 4 4 4 i · 4 • • 4 44 4 · 44 The metering device (2) according to any one of claims 35 to 37, wherein the first section (18) of the support portion (16) has a length between 10 mm and 40 mm, preferably about 30 mm. The metering device (2) according to any one of claims 35 to 39, wherein the distance between the finger collar (10) and the outlet opening (14) located at the tip is at least 30 mm, preferably about 45 mm. Dispensing device (2) according to any one of claims 35 to 40 with a collar (24) according to any one of claims 28 to 34. Dispensing device (2) according to claim 41, which is formed in one piece or in one piece with the collar (24).