MXPA00001171A - Aqueous aerosol preparations containing biologically active macromolecules and method for producing the corresponding aerosols - Google Patents

Abstract

The invention relates to aqueous aerosol preparations containing biologically active macromolecules for producing inhalable aerosols without propellant gases.

Description

Preparations of aqueous aerosols containing biologically active macromolecules and procedures for the generation of corresponding aerosols DESCRIPTION OF THE INVENTION The invention relates to a process for the generation of aerosols intended for the application by inhalation of proteins and other biologically active macromolecules, as well as to aqueous preparations for the generation of such aerosols, especially the invention relates to aqueous preparations of highly concentrated solutions of insulin for the application by inhalation in order to perform the treatment of diabetes.

The application of medicines in the form of aerosols suitable for inhalation, has been known for a long time. Such aerosols serve not only for the treatment of diseases of the respiratory tract such as asthma; They are also used when the lungs or nasal mucous membranes should serve as a resorption organ. Frequently, high blood levels of the active substance can be generated in this manner to also treat diseases in other body regions. Inhaled aerosols can also be used as vaccines.

For the preparation of aerosols, multiple procedures are applied in practice. Either suspensions or solutions of active substances are projected with the help of propellant gases or active substances are swirled in the form of micronized powders (reduced in size) REF. : 32623 micrometers) in the breathing air, or finally aqueous solutions are atomized with the aid of nebulizers.

However, in the case of complicated structured molecules, such as for example interferons, the nebulization of aqueous solutions can easily lead to a disturbing decrease in the activity of the active substances, presumably by shearing and heating forces. It is assumed that in this process plays a certain role for example the formation of Protein aggregates of lower activity. A. Y. Ip et al. Have described, in an article "Stability of recombinant consensus interferon to air-jet and ultrasonic nebulisation" J. Pharm. Sci. 84: 1210-1.214 [1995], examples of the formation of interferon aggregates after nebulization by ultrasound or through nozzles, with a concomitant loss of the biological activity of interferon. Even if the destruction of the biomolecule (taken between biologically active macromolecules) is not complete, the decrease in activity described here is important, since it leads to a higher consumption of the biomolecule, possibly expensive, and allows it to become inaccurate dosing of active medication by bolus. This decrease in the activity of complicated molecules during the generation of aerosols is limited not only to interferons, but also to a greater or lesser extent when converting aerosols of other proteins (see, for example, Niven et al., Pharm). Res 12: 53-59 [1995J) and biomolecules.

Along with the technical-scale preparation of the aerosol containing biomolecules, a second stage is needed for the biomolecules to be absorbed into the lungs. The lung of an adult human being offers a large surface area for absorption, but also presents several obstacles for lung absorption of biomolecules. After an inspiration through the nose or mouth, the air with the aerosol carried by it enters the trachea and then, through bronchi and bronchioles each time smaller, into the alveoli. The alveoli have a much larger surface than the trachea, bronchi and bronchioles considered together. These constitute the main absorption zone, not only for oxygen, but also for biologically active macromolecules. In order to get from the air to the bloodstream, the molecules must cross the alveolar epithelium, the capillary endothelium and the interstitial space that contains lymph, between these two cellular layers. This can take place through active or passive transport processes. The cells in these two cell layers are tightly located next to each other, so that most of the large biological macromolecules (such as proteins, for example) cross this barrier much more slowly than the smaller molecules. The process of crossing the alveolar epithelium and the capillary endothelium takes place in competition with other biological processes that lead to the destruction of the biomolecule. The bronchoalveolar fluid contains exoproteases (see for example Wall DA and Lanutti AT "High levéis of exopeptidase activity are present in rat and canine bronchoalveolar lavage fluid", Int. J. Pharm. 97: 171-181 (1993)]. also macrophages, which by phagocytosis remove the inhaled particles of proteins.These macrophages migrate towards the base of the bronchial tree, from where they arrive by means of the mechanism of mucociliary clearance from the lung, so they can migrate to the lymphatic channel. The macrophages can be influenced by the protein converted into an aerosol in their physiology, for example interferons can activate alveolar macrophages.The migration of activated macrophages constitutes an additional mechanism for the propagation of the systemic effect of an inhaled protein. process means that the results of experiments using aerosols with one type of protein they can be transferred only to another type of protein. Small differences between interferons may have, for example, a clear influence on their susceptibility to the degradation mechanisms that develop in the lung (see Bocci et al. "Pulmonary catabolism of interferons: alveolar absorption of -I labelled human interferon alpha is accompanied by partial loss of biological activity "Antiviral Research 4: 211-220 / 1984)].

Proteins and other biological macromolecules can certainly be nebulized, but this nebulization usually takes place accompanied by a loss of activity. The object of the present invention is to provide a method for the preparation of inhalable aerosols, with which biologically active macromolecules, especially proteins, can be nebulized without essential loss of activity.

A new generation of propellent gas-free nebulizers has been described in US Pat. 5,497,944, which is hereby referenced in its contents. The special advantage of the nebulizers described there is that the use of propellant gases, especially fluoro-chloro-hydrocarbons, is dispensed with. An improved embodiment of the nebulizers disclosed therein is disclosed in PCT Patent Document EP96 / 04351 = WO 97/12687. In relation to the present invention, expressly refers to Figure 6 (Respimat®) described therein, as well as to the corresponding parts of the specification of that application. The nebulizer described there can be advantageously used for the generation of the inhalable aerosols according to the invention of biologically active macromolecules. In particular, the described nebulizer can be used for the application by inhalation of insulin. By virtue of its manageable size, this device can be carried at any time by a patient. In the nebulizer described there, solutions containing active substance in defined volumes (preferably around 15 microliters) are projected through small nozzles with application of high pressures, whereby inhalable aerosols with an average particle size of between 3 and 3 are formed. and 10 micrometers. For the application by inhalation of insulin, nebulizers are suitable which can nebulize between 10 and 50 microliters of an aerosol preparation for each application to form inhalable droplets. Of special significance, for the preparation of the aerosols according to the invention, is the use of the nebulizer described in the Patent or in the Patent Application described above, for the atomization without propellant gases of solutions having a certain content of active substances , which contain proteins or other biologically active macromolecules. Essentially, the atomiser (nebulizer, with a size of about 10 cm) operable, disclosed therein, consists of an upper part of the housing, a pump housing, a nozzle, a blocking-tensioning mechanism, a spring housing, a spring and a reserve container, characterized by the pump housing, which is fixed to the upper part of the housing, and which at one end carries a nozzle body, with the nozzle or nozzle arrangement, a hollow plunger with valve body, a drive flange, to which the hollow plunger is fixed, and which is located in the upper part of the housing, a locking mechanism-tensioner, which is located in the upper part of the housing, a spring housing with the spring that is inside it, which is supported in a manner capable of rotating in the upper part of the housing by means of a rotation support, a lower part of the housing, which is fitted in axial direction on the spring housing. The hollow plunger with a valve body of document W097 / 12687 corresponds to one existing in the disclosed devices. This partially penetrates into the cylinder of the pump housing and is arranged axially displaceable in that cylinder.

Especially, reference is made to Figures 1-4, especially to Figure 3 and to corresponding parts of the specification. The hollow plunger with a valve body exerts on its part at high pressure, at the moment of firing and releasing the spring, a pressure of 5 to 60 MPa (approximately 50 to 500 bars), preferably 10 to 60 MPa (approximately 100 to 600 bars) on the fluid, this is the measured solution of active substance. The valve body is preferably positioned adjacent the end of the hollow plunger, which faces the nozzle body. The nozzle in the nozzle body is preferably microstructured, that is, produced by a micrometric technique. Microstructured nozzle bodies are disclosed, for example, in WO-94/07607; to this document reference is made by the present invention in its content. The nozzle body consists, for example, of two glass and / or silicon plates firmly connected to one another, of which at least one of the plates has one or more microstructured channels, which connect the inlet part of the nozzles with the outlet part of the nozzles. On the outlet side of the nozzles, at least one circular or non-circular orifice of less than or equal to 10 μm is arranged. The directions of the jets of the nozzles in the nozzle body may run parallel to each other or be inclined relative to one another. In the case of a nozzle body having at least two nozzle orifices at the outlet part, the directions of the jets may be inclined at an angle from 20 degrees to 160 degrees with respect to each other, preferably at an angle from 60 to 150 degrees. The directions of the jets coincide with one another in the area around the nozzle orifices. The locking-tensioning mechanism contains a spring, preferably a helical and cylindrical compression spring, as an accumulator of mechanical energy. The spring acts on the drive flange as a sharp jump piece, whose movement is determined by the position of a blocking member. The path of the drive flange is precisely limited by an upper stop and a lower stop. The spring is preferably tensioned by a force-converting transmission, for example a helical transmission of longitudinal feed, tensioned by an external torsional moment, which is generated by rotating the upper part of the housing with respect to the spring housing in the part bottom of the housing. In this case, the top part of the housing and the drive flange contain a single or multiple taper transmission.

The locking member with intercalatable locking surfaces is arranged in a ring shape around the drive flange. This consists, for example, of a ring based on a synthetic material or metal, elastically deformable in the radial direction. The ring is arranged in a plane perpendicular to the axis of the atomizer. After the spring has been tensioned, the blocking surfaces of the blocking member move in the path of the drive flange and prevent the spring from falling out. The blocking member is triggered by means of a key. The trigger key is attached or engaged with the locking member. To trigger the blocking-tensioning mechanism, the firing key is displaced parallel to the plane of the ring, and preferably preferably into the atomizer; in such a case, the deformable ring is deformed in the plane of the ring. The construction details of the blocking-tensioning mechanism are described in WO 97/20590. The lower part of the housing is displaced in the axial direction through the spring housing and covers the support, the spindle drive and the reservoir for the fluid. By actuating the atomizer, the upper part of the housing is rotated with respect to the lower part of the housing, whereby the lower part of the housing carries the spring housing with it. In such a case, the spring is compressed and tensioned through the helical transmission of longitudinal advance, and the locking mechanism automatically engages. The angle of rotation is preferably an integral fraction of 360 degrees, for example 180 degrees. Simultaneously with the tensioning of the spring, the driving part is displaced in the upper part of the housing by a pre-established path, the hollow piston is retracted inside the cylinder in the pump housing, whereby a partial quantity of the fluid is drawn from the reservoir towards the high-pressure space located in front of the nozzle. In the atomizer, several exchangeable reservoirs containing the fluid that has been atomized can be introduced and possibly used consecutively. The reservoir contains the aqueous aerosol preparation according to the invention. The atomization process is initiated by lightly pressing the firing key. In this case, the locking mechanism leaves the path free for the drive part. The tensioned spring moves the plunger inwards into the cylinder of the pump housing. The fluid leaves the nozzle of the atomizer in atomized form. Other construction details are disclosed in PCT Patent Applications WO 97/12683 and WO 97/20590, which are hereby referenced in their contents. The components of the atomizer (nebulizer) are made of an appropriate material, corresponding to its function. The atomizer housing and - as long as the function allows - also other parts are preferably made of a synthetic material, for example according to the injection mold method. Physiologically innocuous materials are used for medical purposes. The atomizer described in WO 97/12687 is used, for example, for the generation of medicinal aerosols without propellant gases. With this, an inhalable aerosol can be generated with an average droplet size of approximately 5 μm. In Figures 4 a / b, which are identical to Figures 6 a / b of WO 97/12687, the nebulizer (Respimat®) is disclosed, with which the aqueous aerosol preparations according to the invention can be advantageously inhaled. Figure 4 shows a longitudinal section through the atomizer when the spring is tensioned, Figure 4 b shows a longitudinal section through the atomizer when the spring is de-stressed. The upper part (51) of the housing contains the pump housing (52), at the end of which is placed the support (53) for the atomizing nozzle. In the support are the body (54) of nozzles and a filter (55). The hollow piston (57), fixed to the drive flange (56) of the blocking-tensioning mechanism, partially penetrates the cylinder of the pump housing. At its end, the hollow plunger supports the valve body (58). The hollow piston is sealed by means of the sealing gasket (59). Inside the upper part of the housing is the stop (69), on which the drive flange rests when the spring is released. Next to the actuating flange is the stop (61), on which the actuating flange rests when the spring is tensioned. After the spring has been tensioned, the blocking member (62) moves between the stop (61) and a support (63) existing in the upper part of the housing. The firing key (64) is in connection with the blocking member. The upper part of the housing ends in the mouth (65) and is closed with the protective cap (66) engageable thereon. The housing (67) of the spring, with a compression spring (68), is supported in a manner capable of rotating in the upper part of the housing by means of elastic jump appendages (69) and rotation supports. The lower part (70) of the housing is displaced through the spring housing. In the interior of the spring housing is the exchangeable reservoir (81) for the fluid (72) to be atomized. The reservoir is closed with the cap (73), through which the hollow piston penetrates into the reservoir and is immersed with its end in the fluid (reservoir of active substance solution).The spindle (74) for the mechanical counter is placed on the wrapping surface of the spring housing. Next to the end of the spindle, which is oriented towards the upper part of the housing, is the drive pinion (75). The slide (76) sits on the spindle. The nebulizer described above is suitable for nebulizing the aerosol preparations according to the invention in order to form an aerosol suitable for inhalation. The effectiveness of a nebulization device can be tested in an in vitro system, in which a protein solution is nebulized and the mist is collected in a so-called "trap" (see Figure 1). The activity of the protein in the reservoir (a) for the aerosol is compared with the activity in the collected liquid (b), for example: with the help of an immunoassay or with the aid of an analysis of the biological activity of the protein. This experiment allows an evaluation of the degree of destruction of the protein by nebulization. A second parameter of the quality of the aerosol is the so-called inhalable proportion, which here is defined as the proportion of the mist droplets having a median aerodynamic diameter in dimensions (MMAD) of less than 5.8 μm. The inhalable proportion can be measured by means of an "Andersen Impactor". It is important, for a good absorption of the protein, not only to achieve a nebulization without essential loss of activity, but also to generate an aerosol with a good inhalable proportion (approximately 60%). Aerosols with a MMAD less than 5.8 μm are clearly better suited to reach the alveoli, where their chances of being absorbed are manifestly higher. The effectiveness of a nebulization device can also be tested in an in vivo system, with a game in this case factors such as susceptibility to lung proteases. As an example of an in vivo assay system, a mist containing a protein can be administered to a dog through a tracheal tube. Blood samples are taken at adapted time intervals and thereafter the mvel of protein in the plasma is measured by immunological or biological methods. Appropriate nebulizers are described in U.S. Pat. 5,497,944 and in WO 97/12687 that have already been mentioned above, especially as described in Figures 6 a / b (now 4 a / b). A preferred arrangement of nozzles for the nebulization of the aqueous aerosol preparations according to the invention of biologically active macromolecules is shown in Figure 8 of US Pat. Surprisingly, it was discovered that the above-described nebulizer, free of propellant gas, which can atomize a predetermined amount - for example 15 microliters - of a high pressure aerosol preparation between 100 and 500 bar through at least one nozzle with a hydraulic diameter of 1 to 12 micrometers, so that inhalable droplets with an average particle size of less than 10 micrometers are formed, is well suited for the nebulization of liquid aerosol preparations of proteins and other macromolecules, since it can nebulize a wide range of proteins without loss of activity that are worthy of mention. In this case, a nozzle arrangement is preferred, as shown in Figure 8 of U.S. Pat. aforementioned. The capacity of nebulizers of this type of construction to nebulize interferons, which otherwise can be nebulized only with a considerable loss of activity, is especially pleasing. The especially high activity of interferon omega after nebulization with this device is additionally surprising, not only in experiments in vitro but also in in vivo experiments.

Another advantage of the claimed process is its amazing property, of being able to nebulize also very concentrated solutions of biologically active macromolecules, without essential loss of activity. The use of highly concentrated solutions makes it possible to use an apparatus that is small enough to be carried constantly in a jacket pocket or in a handbag. The nebulizer disclosed in Figure 4 fulfills these premises and makes possible the nebulization of highly concentrated solutions of biologically active molecules. For example, such apparatuses are especially suitable to enable diabetic patients self-treatment by inhalation with insulin. Preferably, highly concentrated aqueous solutions with a concentration of 20 to 90 mg / ml of insulin are used, solutions with 30 to 60 mg / ml are preferred and insulin solutions with 33 to 40 mg / ml of insulin are especially preferred. Depending on the size of the reservoir of the nebulizer made available, solutions containing insulin at a concentration greater than 25 mg / ml, preferably greater than 30 mg / ml, are suitable for being able to inhale a therapeutically active amount of insulin with a handheld device, such as the device described above. The application of insulin by inhalation makes possible a rapid initiation of the effect of the active substance, so that the patient can apply to himself the quantity he needs, for example shortly before meals. The small size of the Respimat®, for example, makes it possible for the patient to carry the device at all times. The Respimat® (Figure 6 of WO 97/12687) has a dosing chamber with constant volume, which makes it possible for the patient to determine the dose of insulin that is necessary by means of the number of strokes (puffs), and to inhale it. In addition to the number of strokes, the dosage of insulin is determined by the concentration of the insulin solution in the reservoir (72). This can amount, for example, to values between 25 and 90 mg / ml, more concentrated solutions are preferred from about 30 mg / ml and above. A method for the preparation of stable and highly concentrated solutions of insulin has been described, for example, in Patent Applications WO 83/00288 (PCT / DK82 / 00068) and 83/03054 (PCT / DK83 / 00024), to which refers to the content of the present invention. The aerosol preparations according to the invention, which contain insulin and which are applied with the device described above, must not exceed a dynamic viscosity of more than 100%, so that the inhalable proportion of the generated projection does not decrease below of an acceptable value. Preferred are insulin solutions having a limit viscosity of up to 1,200-10"6, especially preferably up to 1100-10" 6 Pa-s (Pascal-second). If necessary, solvent mixtures can be used instead of water as a solvent, in order to reduce the viscosity of the drug solution. This can be effected, for example, by the addition of ethanol. The proportion of ethanol in the aqueous formulation can be, for example, up to 50%, with a proportion of 30% being preferred. Preferably, the aerosol preparation shows a viscosity of up to 100 ° -10 ° Pa-s, wherein it is especially preferred that it be in the region of 900 up to no-10 ^ Pa-s. In addition, they prefer aerosol preparations, whose aqueous solutions have a viscosity between 900 and 10,000-2000, where aqueous solutions are especially preferred with a viscosity in a region between 950 and 500,000 Pa.s. s. It is also a task of the present invention to propose an aerosol preparation that is suitable for the application of the claimed process. The invention also provides aerosol preparations in the form of aqueous solutions, which contain biologically active macromolecules as active substances, in particular a protein or a peptide, in an amount between 3 mg / ml and 100 mg / ml, or between 25 mg / ml and 100 mg / ml. Surprisingly, it has been shown that, according to the process claimed according to the invention, more viscous solutions of macromolecules can also be projected to form inhalable droplets with an appropriate size. This makes it possible to apply greater amounts of active substance for each application and thereby increases the therapeutic activity of the macromolecules in the case of inhalation therapy. According to the process according to the invention, aqueous aerosol preparations containing macromolecules (for example albumin) can be used until they reach a viscosity of 1. eoo -lO "6 Pa-s (measured at 25 ° C). of 1,500-10"6 Pa-s an inhalable proportion of 32% was still determined. More viscous solutions of macromolecules are preferred which have a viscosity of up to 100 ° -10 ° -s. In the case of such solutions, an inhalable proportion of droplets containing active substance of about 60% is reached. the indicated viscosities were determined with an Ostwald viscometer according to the method known from the literature. For comparison, the viscosity of the water is located at 894-10"6Pa-s (measured at 25 ° C.) In order to illustrate the advantages of the method according to the invention, in vitro and in vivo experiments with a omega interferon solution In vitro experiment with Respimat® and interferon omega The reservoir of a Respimat (a) device was filled with a 5 mg / ml solution of omega interferon (formulated in 50-mM trisodium citrate, 150 mV NaCl, pH 5.5) The apparatus was activated, and nebulized a volume of approximately 12.9 μl (one stroke) in an air flow rate of 28 1 / min.

The nebulized solution was collected in a trap (Figure 1). The interferon omega was determined, in the solution of the deposit and in the solution collected in the trap, immunologically by ELISA and biologically by inhibiting the destruction of A549 cells infected by the encephalon-myocarditis virus. The immunological determination of interferon is relatively simple. Published studies with nebulized proteins are limited in several cases to immunological measurements. However, additional biological measurements are very important, since they are an especially sensitive and therapeutically relevant method for the quantification of protein destruction. These do not always give the same result by physical-chemical or immunological methods, since a molecule can lose biological capacities, without modifying its fixation to anticuefos. In three experiments, 84%, 77% and 98% of the immunologically identifiable interferon in the solution of the trap (b) were found again, based on the starting solution. Biological measurements with the same solutions gave renewed findings of 54%, 47% and 81% of the interferon biologically identified in the trap solution. This very high proportion shows that nebulization by the Respimat apparatus destroys only a relatively small part of interferon activity. The fog of a Respimat apparatus, as described above, was also conducted inside an Andersen impactor by means of an air current (flow 28 1 / min). The proportion of particles having a size less than 5.8 μm ("inhalable proportion") was measured. The inhalable proportion corresponds to 70% (in immunological measurements). Proteins, such as interferons, are frequently formulated with human serum albumin, in order to offer additional protection for sensitive interferons. A formulation similar to the previous one was also tested, but with an additional amount of human serum albumin (0.5%). In three experiments, 83%, 83% and 79% of the immunologically identifiable interferon in the solution of trap (b) were also recovered, based on the starting solution. The biological measurements with the same solutions showed values of 60%, 54% and 66% of the biologically active interferon in the solution of the trap. The inhalable proportion (immunological measurements) was 67%. In a further batch a concentrated solution of interferon omega with a concentration of 53 mg / ml was loaded into the Respimat apparatus reservoir and then nebulized. In four experiments it was found again, referring to the starting solution, i-oau, SSL, ß & and T? of the interferon immunologically identifiable in the solution of the trap (n). The biological measurements with the same solutions yielded a renewed finding respectively of 95%, 98%, 61% and 83% of the interferon biologically identifiable in the solution of the trap. This high renewed finding shows that by means of the Respimat device, concentrated solutions of proteins can also be nebulized, without this resulting in excessive losses of interferon activity. In vivo experiment with Respimat® and interferon omega. The omega interferon was applied by inhalation and intravenously in separate experiments on the same dog. The mvel in blood of interferon was measured at different times, immunologically and biologically. In addition, the mvel of neopterin in the blood was measured. Neopterin is a marker substance for immunoactivation; is released by macrophages after excitation by interferon [see Fuchs et al. "Neopterin, biochemistry and clinical use as a marker for cellular immune reactions" Int. Arch. Allergy Appl. Immunol. 101: 1-6 (1993)]. The measurement of the level of neopterin is used for the quantification of interferon activity. The application of interferon to the dog was effected by means of pentobarbitai narcosis after the previous basic sedation. The animal was intubated and artificially breathed (controlled respiration in terms of volume: volume per minute 4 1 min, frequency 10 strokes / min). In total, 20 strokes were delivered through the Respimat device. Each stroke was applied at the beginning of an inspiration. After the inspiration phase, there was a pause of 5 seconds before the expiration. Before the next application of interferon omega, the animal was allowed two respiratory cycles without influencing them. Blood was drawn to obtain serum and heparinized plasma before the application of interferon and at different times up to 14 days after the application of interferon. The omega interferon was determined in immunologically heparinized plasma by an ELISA and biologically by inhibiting the destruction of A549 cells infected by the encephalon-myocarditis virus. Neopterin in serum was determined immunologically. Figure 2 shows the omega interferon levels measured immunologically (figure 2a) and biologically (Figure 2b) after 20 strokes of omega interferon by the Respimat apparatus. Surprisingly, after an administration by inhalation, a very high level of neopterin in serum was measured. In the in vitro experiment, the measurement produced of solution after a bolus of the Respimat device was on average 12.8 mg / bolus. Therefore, it is expected that in the case of a solution of 5 mg / ml with 20 strokes of the Respimat, approximately 1.28 mg of interferon will be administered. Neopterin measurements after administration of this amount yielded clearly higher and longer-lasting levels than neopterin measurements after intravenous administration of 0.32 mg intravenously (i.v.). Figure 3 illustrates this result. The high levels of neopterin serve as a demonstration that the application of interferon by Respimat can lead to good biological activity. The advantages of the Respimat device for the nebulization of biologically active macromolecules are not limited only to interferons, as demonstrated by a second example. In vitro experiments with Respimat® v manganese-superoxide-dismutase. In apparatus for nebulization of the test substance and the corresponding trap were as shown in Figure 1. In this experiment, the deposit (a) of the Respimat apparatus was loaded with 3.3 mg / ml manganese-superoxide dismutase (MnSOD ) in a phosphate buffered saline solution (PBS). The apparatus was activated and nebulized im volume of approximately 13 μl (one stroke) in an air flow rate of 28 1 / min. The exactly nebulized amount was determined gravimetrically (measurements in three consecutive experiments were: 12.8, 13.7 and 14.3 mg). The nebulized solution was collected in a trap (b). The content of this trap was 20 ml of PBS. Additionally, 2 ml of 5% bovine serum albumin was added in order to stabilize the proteins in the trap. The MnSOD was determined, in the solution of the deposit and in the solution collected in the trap, immunologically by an ELISA and enzymatically by reducing the amount of superoxide after a reaction between xanthine and xanthine oxidase. In three experiments 78%, 89% and 83% of the immunologically identifiable MnSOD of the nebulized solution were measured in the solution of trap (b). There was no measurable loss of enzymatic activity after nebulization. The inhalable proportion (in immunological measurements) was 61%. In the following Example, the preparation of an aerosol preparation according to the invention containing insulin as an active substance is described.

Preparation of the insulin solution and filling of the pebulizer. 175 mg of bovine insulin (sodium salt) crystallized (corresponding to 4,462 U.I. according to the manufacturer's data) were dissolved in 3.5 ml of sterile purified water (Seralpur® water). Then, by means of a slight stirring, 8.5 μl of m-cresol (corresponding to 8.65 mg) and 7.53 mg of phenol, dissolved in 100 μl of purified and sterile water, were added thereto. To this solution were added 365 μl of a 5 mg / ml solution of ZnCl 2 (corresponding to a weight ratio of 0.5% zinc, based on the amount of insulin used) and the pH value was adjusted to 7.4 with NaOH 0.2 N. The volume of the mixture was made up to 5 ml with purified and sterile water, and filtered through a sterile Millipore filter (pore size 0.22 μm). 4.5 ml of the aerosol preparation was transferred to the reservoir (72) in Figure 4) of the nebulizer (Respimat). The container was closed with a cap and the device was provided with the container. The aerosol preparation thus produced has a concentration of approximately 35 mg / ml of insulin, the viscosity of this solution being approximately 1020 lO ^ Pas.

Experiment in vivo with the Respimat® v a very concentrated insulin solution. The application of insulin to a dog was made by narcosis with pentobarbital after previous basic sedation. The animal was intubated and artificially breathed as described above. In total, Respimat 6 strokes of an insulin solution were delivered through the device. Each stroke was applied at the beginning of an inspiration. Between the inspiration phase and the expiration there was a pause of 5 seconds. Before the next application of insulin, the animal was allowed two respiratory cycles without influencing them. Blood was drawn 1 hour before the application, simultaneously with the application and at different times over a period of 8 hours. The level of glucose in blood was measured in the blood recently extracted, according to the method of Trasch, Koller and Tritschler (Klein). Chem. 30: 969 [1984], with the aid of a Refletron® apparatus (Boehringer Mannheim). Surprisingly, even with this highly concentrated insulin solution, good biological activity was found (decrease in blood glucose level after administration of insulin by inhalation). Figure 5 illustrates this result. The aqueous aerosol preparations according to the invention can contain, if necessary, in addition to the active substance and water, other solvents - such as, for example, ethanol -. The amount of ethanol is limited, depending on the dissolving properties of the active substances, by the fact that in the case of too high concentrations the active substance can precipitate from the aerosol preparation. Additions are possible for the stabilization of the solution, such as, for example, pharmacologically safe preservatives, such as for example ethanol, phenol, cresol or paraben, pharmacologically compatible acids or bases, or buffers for adjusting the pH value, or agents surfactants. In addition, for the stabilization of the solution or for the improvement of the quality of the aerosol it is possible the addition of a chelating compound with metals - such as for example ADTA. In order to improve the solubility and / or the stability of the active substance in the aerosol preparation, amino acids may be added, such as aspartic acid, glutamic acid and especially proline. In addition to interferons, superoxide dismutase and insulin, the following active substances are preferred active substances of the drug preparation according to the invention: Antisense oligonucleotides Orexins Erythropoietin Necrosis factor of tumors-alpha Necrosis factor of tumors-beta G-CSF ("stimulating factor" of granulocyte colonies, "of granulocyte colony stimulating factor") GM-CSF ("granulocyte and macrophage colony stimulating factor" of granulocyte-macrophage colony stimulating factor ") Annexins Calcitonin Leptins Hormones for thyroid hormones Fragments of thyroid hormones Interleukins such as for example interleukin 2, interleukin 10 and interleukin 12 ICAM ("intercellular adhesion molecule", from '? Ntercellular adhesion molecule ") soluble Somatostatin Somatotropin tPA (" plasminogen activator ") of type tisultar ", of lítissue plasminogen activator") TNK-tPA Tumor-associated antigens (in the form of peptide, protein or DNA) Antagonists of the peptide bradykinin Urodilatin GHRH ("hormone releasing hormone of the growth", of "growth hormone releasing hormone") CRF ("corticotropin releasing factor", from "corticotropin releasing factor") EMAP? Heparin Receptors of soluble interleukin, such as the receptor for sIL-1 vaccines, such as the hepatitis vaccine or the measles vaccine Antisense polynucleotides Transcription factors.

It is noted that in relation to this date, the best method known by the applicant to carry out the aforementioned invention, is that which is clear from the present description of the invention.

Claims (25)

1. CLAIMS 1. Aqueous aerosol preparation for the application of inhalation, characterized by containing in a concentration of 25 to 100 mg / ml an active substance selected from the following group: antisense oligonucleotides, orexins, erythropoietin, tumor necrosis factor-alpha, necrosis factor of tumors-beta, G-CSF (granulocyte colony stimulating factor), GM-CSF (macrophage granulocyte colony stimulating factor), annexins, calcitonin, leptins, parathyroid hormones, fragments of parathyroid hormones, interleukins, such as p. Interleukin 2, interleukin 10 and interleukin 12, soluble ICAM (intercellular adhesion molecule), Somatostatin, Somatotropin, TPA (tissue-type plasminogen activator), TNK-tPA, tumor-associated antigens (in the form of peptide, protein or DNA), antagonists of the peptide bradykinin, urodilatin, GHRH (growth hormone releasing hormone), CRF (corticotropin releasing factor), EMAP II, heparin, soluble interleukin receptors, such as the sIL-1 receptor, vaccines , such as the hepatitis vaccine or the measles vaccine, antisense polynucleotides, transcription factors, the concentration of G-CSF (granulocyte colony stimulating factor) not being 25 mg / dl. Aqueous aerosol preparation according to claim 1, characterized in that insulin is used as the active substance.) 3. Aqueous aerosol preparation according to claim 2, characterized in that insulin is used as an active substance in a concentration greater than 30 mg / ml. 4. Aqueous aerosol preparation according to claim 1, characterized in that an superoxide disutta is used as the active substance. 5. Aqueous aerosol preparation according to claim 1, characterized in that an interferon is used as the active substance. 6. Aqueous aerosol preparation according to claim 1, characterized in that an omega interferon is used as the active substance. 7. Aqueous aerosol preparation according to one of claims 1 to 6, characterized in that they contain one or more auxiliary substances taken from the group of surface-active substances, such as surfactants, emulsifiers, stabilizers, penetrability enhancers and / or preservatives. 8. Aqueous aerosol preparation according to one of claims 1 to 7, characterized in that it contains an amino acid. 9. Aqueous aerosol preparation according to claim 8, characterized in that it contains proline, aspartic acid or glutamic acid to improve the solubility or stability of the active substance. 10. Aqueous aerosol preparation according to one of the preceding claims, characterized in that it is prepared from an aerosol because it has a viscosity up to 25 ° C of 1,600-10"6 Pa's. 11. Aqueous aerosol preparation according to one of the preceding claims, characterized in that it is prepared from an aerosol because it has at 25 ° C a viscosity comprised between 900-10"6 and 100%" 6 Pa's. 12. Aqueous spray preparation for the application by inhalation, characterized in that it has at 25 ° C a viscosity comprised between 900'10-6 and 1-600.10"6 Pa-s, and containing an active substance, which is selected from the following group : antisense oligonucleotides, orexins, erythropoietin, tumor necrosis factor-alpha, tumor necrosis factor-beta, G-CSF (granulocyte colony stimulating factor), GM-CSF (macrophage granulocyte colony stimulating factor), annexins, calcitonin, leptins, parathyroid hormones, parathyroid hormone fragments, interleukins, such as interleukin 2, interleukin 10 and interleukin 12, ICAM (intercellular adhesion molecule) soluble, somatostatin, somatotropin, tPA (plasminogen activator of tissue type), TNK-tPA, tumor-associated antigens (in the form of peptide, protein DNA), antagonists of the peptide bradykinin, urodilatin, GHRH (growth hormone releasing hormone), CRF (corticotropin releasing factor), EMAP II, heparin, soluble interleukin receptors, such as the sIL-1 receptor, vaccines , such as hepatitis vaccine or measles vaccine, antisense polynucleotides, transcription factors, the concentration of G-CSF (facto granulocyte colony stimulator) not being 25 mg / dl. 13. Aqueous aerosol preparation according to claim 12, characterized in that the aqueous solution has a viscosity at 25 ° C comprised between 950-10"6 and 1300-10 ~ 6 Pa-s. 14, Aqueous aerosol preparation according to one of claims 12 or 13, characterized in that the active substance is insulin. 15. Aqueous aerosol preparation according to one of claims 12 or 13, characterized in that a superoxide dismutase is used as the active substance. 16. Aqueous aerosol preparation according to one of claims 12 or 13, characterized in that the active substance is an interferon. 17. Aqueous aerosol preparation according to one of claims 12 or 13, characterized in that the active substance is interferon omega. 18. Procedure for the preparation of aerosol? intended for the application by inhalation of an aerosol preparation according to one of claims 1 to 17, characterized in that, in a propellant-free nebulizer, a therapeutically effective amount of a single dosage of aerosol preparation is measured in a measuring chamber. it is atomized under high pressure, between 100 and 500 bar, through at least one nozzle with a hydraulic diameter of 1 to 12 micrometers, to form inhalable droplets with an average particle size of less than 10 micrometers in the course of a period d time between one and two seconds. 19. Process according to claim 18, characterized in that the effective amount of the individual dosage is between 10 and 20 microliters. 20. Process according to claim 19, characterized in that the nebulizer has two nozzles, which are directed in such a way that both jets coincide in such a way that the aerosol preparation is nebulized. 21. Procedure for the nebulization of insulin for the treatment of diabetes, giving an appropriate aerosol for inhalation, characterized in that they are sprayed between 10 and 5 microliters of a solution, containing between 20 mg / ml and 9 mg / ml of insulin, using a nebulizer in a single application to form droplets suitable for inhalation. 22. Process according to claim 21, characterized in that between 10 and 20 microliters of a solution are inhaled, which contains between 25 and 60 mg / ml of insulin. 23. Use of a solution, - containing more than mg / ml of insulin for the treatment by inhalation of diabetes, in order to prepare an aerosol with an average particle size of less than 10 micrometers. 24. Use of a solution, which contains between -25 and mg / ml of insulin for the treatment by inhalation of diabetes, in order to prepare an aerosol with an average particle size of less than 10 micrometers. 25. Use 23 or 24, characterized in that the aerosol s is prepared from 10 to 50 microliters preferably 10 20 microliters of solution, using a nebulizer free of propellant gas.