EP2152235A2

EP2152235A2 - Aqueous aerosol preparations containing therapeutically active micro-organisms or parts of micro-organisms and method for producing corresponding aerosols

Info

Publication number: EP2152235A2
Application number: EP08749852A
Authority: EP
Inventors: Georg Boeck; Michael Spallek
Original assignee: Boehringer Ingelheim International GmbH
Current assignee: Boehringer Ingelheim International GmbH
Priority date: 2007-05-02
Filing date: 2008-04-29
Publication date: 2010-02-17
Also published as: WO2008135460A2; DE102007020578A1; WO2008135460A3; US20100116268A1

Abstract

The invention relates to aqueous aerosol preparations for inhalation, containing therapeutically active micro-organisms or parts of micro-organisms as the active ingredient.

Description

AQUEOUS AEROSOL PREPARATIONS CONTAINING THERAPEUTIC

EFFECTIVE MICROORGANISMS OR PARTS OF MICROORGANISMS

AND METHOD FOR GENERATING CORRESPONDING AEROSOLS

The invention relates to aqueous aerosol preparations for inhalation administration containing therapeutically active microorganisms or parts of microorganisms as active ingredient.

The use of drugs in the form of inhalable aerosols has long been known. Such aerosols are not only for the treatment of respiratory diseases such as asthma; they are also used when the lungs or nasal membranes are to serve as a resorption organ. Frequently, such high blood levels of the drug can be generated to treat diseases in other parts of the body. Inhalable aerosols can also serve as vaccines.

For the production of aerosols, several methods are used in practice. Either suspensions or solutions of active ingredients, i.a. with the help of propellant gases, sprayed or swirled active ingredients in the form of micronized powder in the air, or finally atomized aqueous solutions using nebulizers.

For complicated built molecules, such. As interferons, the nebulization of aqueous solutions can easily lead to a disturbing reduction in drug activity, probably by shear forces and warming. It is thought that, for example, the formation of inactive protein aggregates plays a role in this process. AY Ip and colleagues have in their article 'Stability of recombinant consensus interferon to air-jet and ultrasonic nebulisation', J. Pharm. Sei. 84: 1210-1214 [1995], examples of the formation of interferon aggregates after ultrasound or jet nebulisation with concomitant loss of the biological activity of interferon. Although the destruction of the biomolecule is not complete, the activity reduction described herein is important because it causes greater consumption of the potentially expensive biomolecule and makes the dosage of active drug per stroke inaccurate. This decrease in activity of complex molecules during aerosol generation, not only is it limited to interferons, it also occurs to a lesser or greater extent during the aerosolization of other proteins (see, eg, Niven et al, Pharm Res. 12: 53-59 [1995]) and biomolecules, especially of such macromolecules, in front.

It is also known to treat cystic fibrosis patients with sulfide-splitting enzymes by nebulizing these enzymes.

EP 1003478 describes aqueous aerosol preparations with biologically active macromolecules for the propellant-free generation of inhalable aerosols.

In addition to the technical preparation of the biomolecule-containing aerosol, a second step is necessary for the biomolecules to be absorbed into the lungs. The lung of the adult human provides a large absorption area, but it also has several obstacles to the pulmonary absorption of biomolecules. After inspiration through the nose or mouth, air (with the aerosol carried by it) goes into the trachea and then over ever smaller bronchi and bronchioles into the alveoli. The alveoli have a much larger area than trachea, bronchi and bronchioles together. They are the main absorption zone, not only for oxygen, but also for biologically active macromolecules. To enter the bloodstream from the air, molecules must traverse the alveolar epithelium, the capillary endothelium and the lymph-containing interstitial space between these two cell layers. This can take place via active or passive transport processes. The cells in these two cell layers are close together, so that most large biological macromolecules (such as proteins) traverse this barrier much more slowly than smaller molecules. The process of

Traversing the alveolar epithelium and the capillary endothelium competes with other biological processes that lead to the destruction of the biomolecule. The bronchoalveolar fluid contains exoproteases [see eg Wall DA and Lanutti, AT High levels of exopeptidase activity are present in rat and canine bronchoalveolar lavage fluid '. Int.J.Pharm. 97: 171-181 (1993)]. It also contains macrophages which eliminate inhaled protein particles by phagocytosis. These macrophages migrate to the base of the bronchial tree, from where they release by means of the mucociliary clearance mechanism get out of the lungs. You can then migrate to the lymphatics. Furthermore, the macrophages can be influenced by the aerosolized protein in their physiology, eg interferons can activate alveolar macrophages. The migration of activated macrophages provides another mechanism for the dissemination of the systemic effects of an inhaled protein. The complexity of this process means that results from aerosol experiments with one type of protein to another protein type are only limitedly transferable. For example, small differences between interferons may have a significant influence on their susceptibility to the degradation mechanisms in the lung [see Bocci et al., Pulmonary catabolism of interferons: alveolar absorption of 125'-labeled human interferon alpha antiviral research 4: 211-220 (1984)].

Although microorganisms, which are a form of biological macromolecules, can in principle be aerosolized, this nebulisation usually takes place with a loss of activity. In this context, microorganisms are defined as all single-cell microorganisms with a size of <200 μm. The microorganisms include in particular bacteria and fungi.

It is the object of the present invention to provide aqueous aerosol preparations which contain therapeutically effective microorganisms or parts of

Microorganisms, in particular bacteria, fungi or parts thereof as active ingredient and can be used for inhalation.

Surprisingly, it has been found that liquid preparations of therapeutically effective microorganisms or parts of microorganisms can be nebulised as an active ingredient without appreciable loss of activity.

Preferably, these are propellant-free nebulizers, which spray a predetermined amount of an aerosol preparation under high pressure between 100 and 500 bar through at least one nozzle with a hydraulic diameter of 1 to 12 microns, so that inhalable droplets having an average particle size less than 10 microns arise. In addition, highly concentrated solutions of therapeutically active microorganisms or parts of microorganisms can be nebulized. The use of highly concentrated solutions allows the use of a device with many single doses in a small reservoir.

The invention further aerosol preparations in the form of aqueous solutions containing as active ingredient therapeutically active microorganisms or parts of microorganisms, especially therapeutically active bacteria, fungi or parts of bacteria or fungi, in an amount between 3 mg / ml and 100 mg / ml, contain. Particularly preferred are the microorganisms of the genus Bacillus, Staphylococcus, Pseudomonas, Escherichia, Salmonella, Candida or Aspergillus or a mixture of these genera. Very particular preference is given to microorganisms of the species Bacillus subtilis, Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, Salmonella abony, Candida albicans, Aspergillus niger, or a mixture of these species.

Surprisingly, it has been shown that even more viscous solutions of therapeutically effective microorganisms or parts of microorganisms can be sprayed to form inhalable droplets of suitable droplet size.

This allows the application of larger amounts of active ingredient per application and thus increases the therapeutic efficacy of therapeutically effective microorganisms or parts of microorganisms during inhalation therapy.

For the aerosol according to the invention, therapeutically active microorganisms or parts of aqueous aerosol preparations containing microorganisms can be up to one

Intrinsic viscosity of 1600x10 '"Pascal be used.

Preference is given to higher viscous solutions of therapeutically active microorganisms or parts of microorganisms which have an intrinsic viscosity of up to 1100 × 10 -5 pascals. The specified intrinsic viscosities were measured using an Oswald viscometer

-A- determined by literature method. For comparison, the intrinsic viscosity of water is 90OxIO " ⁶ Pascal.

The aqueous aerosol preparation may also contain one or more auxiliary substances from the group of surfactants, emulsifiers, stabilizers,

Permeationsverbesserer and / or preservatives and an amino acid for improving the solubility / stability of the active ingredient, preferably proline.

A further object of the invention is the use of the aqueous aerosol preparation for the treatment of respiratory diseases, in particular for the treatment of chronic obstructive pulmonary disease (COPD) or for the immune treatment of humans and animals.

As a nebulizer, all commercially available devices can be used with or without propellant gas.

A new generation of propellant-free nebulizers is described in US Patent 5,497,944 and WO 97/12687, the contents of which are hereby incorporated by reference. A preferred nozzle arrangement for the nebulisation of the aqueous aerosol preparations of biologically active macromolecules according to the invention is shown in FIG. 8 of the US patent specification. The particular advantage of the nebulizers described there is that it dispenses with the use of propellant gases.

A further developed embodiment of the nebulizers described therein is in

PCT / EP96 / 04351 = WO 97/12687. With regard to the present invention, reference is expressly made to FIG. 6 described there and to the associated parts of the description of the application. The nebulizer described there can advantageously be used to generate the claimed inhalable aerosols of biologically active macromolecules. In the nebulizers described therein active ingredient-containing solutions of defined volumes using high pressures by small nozzles are sprayed so that inhalable aerosols with a mean particle size between 3 and 10 microns are formed.

Of particular importance is the use of the device described in the above-described patent or patent application for propellant-free atomization of the aerosol preparation according to the invention. Essentially, the atomizer (nebulizer) consists of the upper housing part, a pump housing, a nozzle, a locking mechanism, a spring housing, a spring and a reservoir, characterized by - a pump housing which is fixed in the upper housing part, and at its one end a nozzle body carries with the nozzle, a hollow piston with valve body, a Abtriebsflansch in which the hollow piston is fixed, and which is located in the upper housing part, - a locking mechanism, which is located in the upper housing part, a spring housing with the spring therein, the housing upper part by means of a Rotary bearing is rotatably mounted, a lower housing part, which is plugged onto the spring housing in the axial direction.

The hollow piston with valve body (WO 97/12687) corresponds to one of the above-mentioned devices. It partially protrudes into the cylinder of the pump housing and is arranged axially displaceably in the cylinder. The hollow piston with valve body exerts on its high pressure side at the time of release of the spring a pressure of 5 to 60 MPa (about 50 to 600 bar), preferably 10 to 60 MPa (about 100 to 600 bar) on the fluid.

The nozzle in the nozzle body is preferably microstructured, i. produced by microtechnology. Microstructured nozzle bodies are disclosed, for example, in WO-94/07607; This document is hereby incorporated by reference.

The nozzle body consists for example of two firmly interconnected plates of glass and / or silicon, of which at least one plate microstructured one or more Having channels connecting the nozzle inlet side to the nozzle outlet side. At least one round or non-round opening of less than or equal to 10 μm is mounted on the nozzle outlet side.

The jet directions of the nozzles in the nozzle body may be parallel to each other or inclined relative to each other. In a nozzle body having at least two nozzle openings on the outlet side, the beam directions may be inclined at an angle of 20 degrees to 160 degrees to each other, preferably at an angle of 60 to 150 degrees. The jet directions meet in the vicinity of the nozzle openings.

The valve body is preferably attached to the end of the hollow piston, which faces the nozzle body.

The locking mechanism includes a spring, preferably a cylindrical helical compression spring as a memory for the mechanical energy. The spring acts on the

Abriebsflansch as a jump piece whose movement is determined by the position of a locking member. The path of Abriebsflansches is precisely limited by an upper and a lower stop. The spring is preferably transmitted via a force translating gear, e.g. a Schraubschubgetriebe, stretched by an external torque that is generated when turning the upper housing part against the spring housing in the lower housing part. In this case, the upper housing part and the Abtriebsfiansch contain a single or multi-speed wedge gear.

The locking member with engaging locking surfaces is arranged annularly around the output flange. It consists for example of a radially elastically deformable ring made of plastic or metal. The ring is arranged in a plane perpendicular to the atomizer axis. After tensioning the spring, the Sperrfiächen push the locking member in the path of the output flange and prevent the relaxation of the spring. The locking member is triggered by a button. The release button is connected or coupled to the locking member. To trigger the locking mechanism, the shutter button is parallel to the ring plane, and preferably in the atomizer, moved; while the deformable ring is deformed in the ring plane. The lower housing part is pushed in the axial direction over the spring housing and covers the storage, the drive of the spindle and the reservoir for the fluid.

When actuating the atomizer, the upper housing part is rotated against the lower housing part, wherein the lower housing part entrains the spring housing. The spring is compressed and tensioned via the screw slide, and the lock engages automatically. The angle of rotation is preferably an integer fraction of 360 degrees, e.g. 180 degrees. Simultaneously with the tensioning of the spring, the driven part is displaced in the upper housing part by a predetermined path, the hollow piston is withdrawn within the cylinder in the pump housing, whereby a subset of the fluid from the reservoir is sucked into the high-pressure chamber in front of the nozzle.

If necessary, a plurality of interchangeable reservoirs containing the fluid to be atomized can be inserted and used successively in the atomizer. The storage container contains the aqueous aerosol preparation according to the invention.

The effectiveness of a nebuliser can be tested in an in vitro system where a protein solution is nebulized and the aerosol collected and analyzed. The activity of the protein in the nebulizing solution (a) is compared with the activity in the analyzed aerosol (b), e.g. Example by means of an immunoassay or by means of an assay for the biological activity of the protein. This experiment allows an assessment of the degree of destruction of the protein by the nebulization.

A second parameter for assessing aerosol quality is the so-called inhalable fraction, which is defined here as the fraction of mist droplets with a mass median aerodynamic diameter (MMAD) of less than 5.8 μm. The MMAD can z. B. by means of an "Andersen Cascade Impactor" are measured. For efficient protein absorption, it is important not only to achieve nebulization without significant loss of activity, but to generate an aerosol with a good (about 60%) inhalable content. Aerosols with an MMAD of less than 5.8 microns are much better suited to reach the alveoli, where due to the very large Absorption surface their chances of being absorbed are significantly greater. The effectiveness of a nebuliser can also be tested in an in vivo system, in which case factors such as susceptibility to lung proteases are involved. As an example of an in vivo assay system, a dog may be administered a protein-containing mist via a tracheal tube. Blood samples are taken at appropriate intervals and then plasma protein levels are measured by immunological or biological methods.

In order to illustrate advantages of the aerosol according to the invention, the following in vitro experiments are described.

In vitro experiments with the Soft Inhaler Respimat®

The reservoir of a Respimat device (a) was filled in each case with a suspension of various microorganisms in 50 mM trisodium citrate, 150 mM NaCl, pH 5.5. The following microorganisms were used: 1.) Bacillus subtilis ATCC 6633 2.) Staphylococcus aureus ATCC 6538 3.) Pseudomonas aeruginosa ATCC 9027 4.) Escherichia coli ATCC 8739

5.) Salmonella abony NCTC 6017 6.) Candida albicans ATCC 10231 7.) Aspergillus niger ATCC 16404

These strains are deposited with the American Tissue Culture Collection.

Several strokes, the total corresponded to a volume of about 0.5 ml of solution were triggered on the Respimat ^®. The resulting aerosol was collected in a sealed 1000 ml shake flask with 100 ml of a physiological buffer solution and 0.1% Tween 80. Thereafter, the piston was completely closed at its opening and the aerosol is taken up by the buffer solution in the flask by means of gentle shaking. The delivered amount of aerosol was determined by weighing the Respimat ^® inhaler. The subsequent microbiological tests were carried out according to the specifications of Ph. Eur. 3, 2000 (2.6.12) and USP 24: 20 ml of the buffer solution from the shake flask and an aliquot of 0.1 ml of the reservoir in the Respimat ^® Inhalers were separated filtered from each other by membrane filters. As a control, 0.1 ml of the corresponding suspension of the above-mentioned microorganisms are filtered in 20 ml of buffer solution.

The membrane filters through which the suspensions of the bacteria were filtered were placed on agar plates following filtration and incubated for 5 days at 33 ° C. The membrane filters through which the suspensions of the yeasts and fungi were filtered were placed on agar plates following filtration and incubated for 5 days at 25 ° C. Overall, three experiments are carried out with each microorganism.

Table 1, Tab. 3 and Tab. 5 show the results of the three experiments. By comparison, the colony-forming units per milliliter (cfu / ml) are shown from the aerosol, the reservoir and the control group. Percent survival and kill rates were calculated for the trapped aerosol relative to the reservoir suspension.

Tab. 2, Tab. 4 and Tab. 6 show the results for the amount of aerosol delivered by weighing.

Tab. 1 Results of the first experiment.

Tab. 2 Determination of the delivered aerosol quantity for the first test

Tab. 3 Results of the second experiment Tab. 4 Determination of the delivered aerosol quantity for the second test

Tab. 5 Results of the third experiment Tab. 6 Determination of the delivered aerosol quantity for the second test

Tab. 7 Survival statistics for all three experiments Tab. 8 Statistics of the kill rate over all three attempts

Table 7 shows the survival statistics. For Bacillus subtilis, Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli and Salmonella abony, more than 87% of surviving microorganisms were found. This means that between 87 and 95 percent of the microorganisms that were aerosolized were still able to divide and grow after nebulisation and aerosol processing. This is a sign that the microorganisms have survived the nebulization.

The rates of Candida albicans and Aspergillus niger are below 3%. Table 8 shows the corresponding kill rate. This means that between 97 and 100 percent of these microorganisms, which were aerosolized, were no longer able to divide and grow after nebulization and aerosol processing. This is an indication that the microorganisms either did not survive the nebulization or were retained in the Respimat ^® Inhaler due to their size by the filtering mechanisms. The results of the above experiments show that bacteria usually after their

Use and nebulization in the Respimat ® Inhaler show no loss of activity.

Very large microorganisms such as yeasts and fungi (Candida albicans, Aspergillus niger) are apparently retained in the Respimat due to their size. You can not pass the filters of the Respimat ^® Inhaler.

Due to the high variability in biological studies, it can be assumed that bacteria are virtually not killed or retained by filtration during their nebulization in the Respimat ^® Inhaler. The aerosolization of bacterial suspensions in the Respimat ^® Inhaler has no effect on the vitality of the microorganisms. Thus, bacteria or bacterial components can be efficiently transported into the human lung for curative purposes.

Claims

PATE CLAIMS

1) Aqueous aerosol preparation for the inhalative administration of microorganisms or parts of microorganisms as active ingredient, characterized in that the aqueous aerosol preparation contains the active ingredient in a therapeutically active form.

2) Aqueous aerosol preparation according to claim 1, characterized in that the aqueous aerosol preparation contains the active ingredient in a concentration between 3 mg / ml and 100 mg / ml.

3) Aqueous aerosol preparation according to claim 1, characterized in that microorganisms of the genus Bacillus, Staphylococcus, Pseudomonas, Escherichia, Salmonella, or a mixture of these genera is used as the active ingredient.

4) Aqueous aerosol formulation according to claim 1, characterized in that as the active ingredient microorganisms of the species Bacillus subtilis, Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, Salmonella abony or a mixture of these species is used.

5) Aqueous aerosol preparation according to one of claims 1 to 4, characterized in that it contains one or more auxiliaries from the group of surfactants, emulsifiers, stabilizers, Permeationsverbesserer and / or preservatives.

6) Aqueous aerosol preparation according to one of claims 1 to 5, characterized in that it contains an amino acid for improving the solubility / stability of the active ingredient.

7) Aqueous aerosol preparation according to claim 6, characterized in that it is used in a propellant-free nebulizer. 8) Aqueous aerosol formulation according to any one of the preceding claims, characterized in that the aerosol preparation has an intrinsic viscosity of up to lβOOxlO " ⁶ Pascal.

9) A process for the preparation of aerosols for the inhalation administration of therapeutically effective microorganisms or parts of microorganisms, in particular of therapeutically active bacteria or parts of bacteria, from an aerosol preparation containing the active ingredient, characterized in that in a propellant-free nebulizer, a therapeutically effective amount of a Metered single metering of the aerosol preparation in a measuring chamber and sprayed under high pressure between 100 and 500 bar through at least one nozzle with a hydraulic diameter of 1 to 12 microns to inhalable droplets with a particle size smaller than 10 microns within a time between one and two seconds.

10) A method according to claim 9, characterized in that the effective amount of the single dosage is between 10 and 20 microliters.

11) A method according to claim 9 or 10, characterized in that the nebulizer has two nozzles which are directed so that the two beams meet so that the aerosol preparation is nebulized.

12) Method according to one or more of claims 9 to 11, characterized in that an aqueous aerosol preparation according to any one of claims 1 to 8 is used.

13) Use of the aqueous aerosol preparation according to one or more of claims 1-8 for the treatment of respiratory diseases.

14) Use of the aqueous aerosol preparation according to one or more of claims 1-8 for the treatment of COPD. 5) Use of the aqueous aerosol preparation according to one or more of claims 1-8 for the immune treatment of humans or animals.