WO2010063714A1 - Method for producing an inhalation atmosphere for provoking allergens - Google Patents

Abstract

The present invention relates to a method for producing an allergen inhalation atmosphere in an exposure room, comprising the following steps: preparing a liquid spray formulation comprising a solvent, a carrier substance and an allergen, and producing an aerosol through spray-drying, wherein the aerosol particles comprise the carrier substance and the allergen; and distributing the aerosol in the room air of an exposure room, wherein the exposure room contains device elements for generating a temporally and spatially constant allergen concentration in the room.

Description

 Process for the preparation of an inhalation atmosphere for allergen provocation

The invention relates to a method for producing an allergen inhalation atmosphere in an exposure room. The method can uniformly control the exposure to various indoor and environmental allergens and simulate them under environmentally relevant conditions. Furthermore, the invention relates to a device for carrying out an inhaled allergen challenge as well as the use of the method for clinical studies, in which under controlled conditions in patients who are in the exposure room, the typical symptoms of allergic diseases to be induced.

Worldwide, several allergic dreams have been established and validated, in which controlled clinical provocation studies are mainly carried out in the indication area "allergic rhinitis" using natural allergen particles, such as allergenic pollen (grass pollen, ragweed pollen), mite fouling, mite fragments, etc. Exposure rooms have different

Ventilation concepts and different concepts of the introduction and mixing of the allergen test particles with the respiratory air. Depending on the concept, up to 100 or even more patients can be exposed at the same time. A common feature of all procedures is the use of natural allergen particles. The naturally occurring allergen particles are characterized by their diameter and antigen content. The allergenic substances are generally located in the interior of the carrier particles and make up only a very small mass fraction overall (see, for example, Schäppi et al., Clin Exp Allergy, 1999, 29: 633-641; Price et al., The Lancet , 1990, 336: 895-897). Grass pollen with a mass of 11.4 ng contain on the order of about 0.05 ng group 5-Leitallergene per pollen grain. This corresponds to 0.5% of its mass and 15% of the total protein content. The allergen content (The pϊ) in the mite material also moves in the per mil range. After deposition in the respiratory system, the allergenic substances become bioavailable by aqueous extraction (Vrtala et al., Int. Arch. Allergy Immunol., 1993, 102: 160-169). Since allergen content and bioavailability are subject to natural fluctuations, the allergy potential of the exposure atmospheres produced using the natural allergen particles also varies.

Another feature of some natural allergens is their potential occurrence in different particle size fractions. It is known, for example, that grass pollen, which are non-respirable with a diameter of about 30 μm, burst under certain conditions and release their allergens in the form of smaller respirable particles with diameters of less than 5 to 10 μm (Taylor et al., J Allergy Clin. Immunol., 2002, 109: 51-56). Thus, they may cause not only the symptoms of allergic rhinitis but also those of the respiratory allergic asthma manifesting in the airways of the lungs (Tobias et al., Thorax, 2003, 58: 708-710).

The preparation of the atmospheres from natural test particles is sometimes very expensive. It usually takes place by dispersing a mass of allergen particles with the aid of compressed air. The procedures must correspond to the respective, strongly varying physical properties of the allergen particles (various pollen, ground house dust, mite feces particles, crushed dead mites, etc.) be adjusted. Important properties in this context are, above all, size, distribution width and surface properties. A change of the test particles is usually associated with extensive process engineering work. Furthermore, the setting of a suitable size distribution can only take place with elaborate grinding and sorting methods, if these deviate from the natural one or if the complexity of the natural occurrence is to be reproduced.

In medicine, both in the diagnosis (prick test) and in therapy (desensitization) are widely used formulations based on

Allergen extracts used. These include a well-defined amount of a particular allergen or a combination of multiple antigens along with various adjuvants and stabilizers. It is desirable to ensure very good control of allergen composition and concentration even in the case of inhaled allergen challenge. If you had the continue

Possibility to independently adjust the particle size of the provocation atmosphere, so one would be able to more targeted and technically easier to adjust the provocation studies to be examined indications (asthma, rhinitis, etc.), as is the case with natural allergen particles.

It is therefore an object of the present invention to provide a method for generating an allergen inhalation atmosphere, which enables a realistic mapping of environmental exposure, site of action in the respiratory tract and bioavailability, has high flexibility with regard to the allergen or allergen spectrum to be investigated, and ensures good control of allergen spectrum, allergen concentration and particle size.

This object is achieved by providing a method for producing an allergen inhalation atmosphere in an exposure room, which comprises the following steps: Providing a liquid spray formulation containing a solvent, a vehicle and an allergen, and producing an aerosol by spray drying, wherein the aerosol particles contain the vehicle and the allergen, and distributing the aerosol in the ambient air of an exposure room, the

Exposure room has device elements for producing a temporally and spatially constant allergen room air concentration.

In a first step, a spray formulation is subjected to spray drying in the process according to the invention. In spray-drying, from each liquid drop of the spray after evaporation of the solvent (preferably water), an aerosol particle of reduced size consisting of the carrier material and the allergen homogeneously distributed therein is formed. This particle is the surrogate of the natural environmental allergen and can be adapted in size, allergen spectrum and allergen content to the natural conditions.

The relationship between drop diameter do and

Aerosol particle diameter dAe is given by the concentration C _{T of} the support material in the starting solution and can be described by the following formula:

If, for example, the concentration is 10%, the droplet shrinks by 54% after complete release of the solvent (ie usually water), with a 0.1% strength solution the degree of shrinkage is 90%. The mean particle size of the aerosol atmosphere can be determined both by the choice of the spray method and the associated droplet spectrum and by the concentration C _T. The antigen concentration can be adjusted completely independently. The spray drying process per se and suitable spray dryers are known to those skilled in the art.

For the production of the aerosol by spray-drying it is preferred to use a freeze-dried allergen extract of a natural allergen carrier or a recombinantly produced allergen, in particular a guide allergen.

Common solvents can be used for the liquid spray formulation. Preferably, an aqueous spray solution is used for the spray drying.

The erfϊndungsgemäße method is very flexible and can be used for a variety of different allergens. Preferably, the allergen is selected from the group consisting of mite allergens, especially the mite mite allergen, flower pollen allergens, pet allergens and house-dust allergens.

In general, the allergen concentration in the spray formulation is very small compared to the vehicle concentration. Conventional concentration levels C _{A of} the allergen in the spray formulation are preferably in the range of 0.1 μg / ml to 1000 μg / ml, more preferably in the range of 1 μg / ml to 10 μg / ml.

As already discussed above, the spray formulation contains a carrier substance that forms the aerosol particle after spray-drying. The carrier then effectively acts as a solid matrix in which the allergen is dispersed. The carrier substance is preferably selected from the group of approved excipients by inhaled medicaments, more preferably lactose, sodium chloride, or mixtures thereof.

As can be seen from the formula given above and known to the person skilled in the art, the median diameter dso of the aerosol particles can be determined by the concentration C _{T of} the Carrier material can be controlled in the spray formulation. Depending on the desired particle size, C _T can be varied over a wide range. By way of example, as the preferred concentration of the vehicle in the spray formulation, a range of 0.1% by weight to 20% by weight, more preferably 1% by weight to 20% by weight, may be cited.

For spray drying in the context of the process according to the invention known atomization units can be used. By way of example, ultrasonic atomisers, compressed air spray nozzles or atomizer disks may be mentioned at this point. By choosing the atomizing unit, the width of the

Drop diameter distribution can be influenced. Thus, e.g. a fairly narrow size distribution can be achieved with suitable ultrasonic atomizers, while the use of compressed air spray nozzles generates a broader size distribution. As will be discussed in more detail below, this allows targeted achievement of different allergen provocations, e.g. a nasal and / or bronchial provocation when using compressed air spray nozzles or a provocation of the respiratory tract when using a Ultraschallzerstäubers.

Suitable atomization units for carrying out the spraying process are known per se to the person skilled in the art. With regard to a suitable ultrasonic atomizer, reference may be made by way of example to those of the US series from Lechler, Metzingen. These generate e.g. depending on the operating frequency used droplet spectra with median diameters dso of 35, 27 or 20 microns and a geometric standard deviation of 1.8.

The median diameter dso of the aerosol particles can be selectively varied over a wide range. The dso value to be set also results from the desired type of allergen provocation (eg nasal provocation, bronchial challenge, etc.). In general, the aerosol particles preferably have a median diameter, more preferably a mass median diameter dso of 25 μm or less. This median diameter is determined from distribution measurements by means of a cascade impactor.

If, for example, a 27 μm spray head and lactose are used as the carrier substance with a carrier concentration Cτ = 20%, then the aerosol generated has a median diameter d 50 of 16 μm and is deposited predominantly in the nasal area with an efficiency of 93%. At a carrier concentration C _T of 4%, the median diameter d 50 of the allergen-carrying aerosol is 9.2 μm. Here more than 10% are deposited in the thoracic and pulmonary area. The mass size distribution of the aerosol at different carrier substance concentrations is shown in FIG.

In a preferred embodiment, the aerosol particles have a median diameter d.sub.50 of 10 .mu.m or less, preferably 5 to 10 .mu.m, in particular if a bronchial provocation is desired. Alternatively, in another preferred embodiment, the aerosol particles may have a median diameter d.sub.50 of more than 10 .mu.m, preferably 10 .mu.m <d.sub.so.ltoreq.25 .mu.m, especially if a nasal provocation is desired.

As stated above, the aerosol generated by spray-drying is distributed in the room air of an exposure room, the exposure room having apparatus elements for generating a temporally and spatially constant allergen room air concentration.

The exposure space preferably has a volume of space which is sufficiently large for the admission of at least one subject. In a preferred embodiment, the exposure space has a volume in the range of 3 m ³ to 300 m ³ , more preferably in the range of 10 m ³ to 150 m ³ . The device elements present in the exposure space for generating a temporally and spatially constant allergen room air concentration ensure that the aerosol in the room air is distributed as uniformly as possible. The uniform distribution of the aerosol in the room air preferably takes place by means of turbulent mixing ventilation or fresh air flow.

Preferably, the distribution of the aerosol is effected by one or more of the following device elements present in the exposure space to produce a temporally and spatially constant allergen ambient air concentration: by means of swirl outlets mounted in the air supply (i.e., fresh air inlet); by one or more fans, in particular ceiling fans, and / or by a floor heating in the exposure room.

The air supply preferably takes place via the ceiling of the exposure room. Depending on the size of the exposure room, up to 20 air inlets (fresh air inlets) may be installed in the ceiling. If there are swirl outlets in the air inlets, these can preferably have adjustable lamellae, as a result of which intensive air turbulence is generated.

The atomization of the spray formulation preferably takes place centrally below the ceiling fan. This is operated in one direction of rotation, that it sucks air from below and then distributed radially in the ceiling area. This causes the generated aerosol particles to be effectively and evenly distributed in the exposure space.

Therefore, it is preferable that at least one atomizing aggregate such as an ultrasonic atomizer, atomizer plate or compressed air spraying nozzles is mounted centrally below the ceiling fan in the exposure room. In a preferred embodiment, the spray formulation is fed to a metering device, for example a perfusor, for setting a suitable liquid mass flow and then forwarded to the atomization unit, for example an ultrasonic atomizer. The metering device can be mounted both inside and outside the exposure space. Thus, for example, the spray formulation to be sprayed can first be supplied to a metering device (eg a perfusor) which is present outside the exposure space and can then be forwarded via a hose to the atomization unit, which is then located within the exposure space. The appropriate liquid mass flow is adjusted at the perfusor.

An exemplary exposure space according to a preferred embodiment of the present invention is shown in FIG. The exposure space 1 has at least two air inlets 2, in which preferably at least one swirl outlet is attached in each case. At least one fan 3 is attached to the ceiling of the exposure room. This is operated in the opposite direction of rotation, so that the air is sucked from below and distributed radially outwards under the ceiling. Immediately below the fan 3 is a sputtering unit 4 such as e.g. an ultrasonic atomizer. This allows the spray to flow directly into the fan flow where it evaporates within a few tenths of a second and is evenly distributed in the room with the fan flow and the turbulent air flow. To support the uniform distribution of the allergen-laden aerosol, the exposure space preferably contains a floor heating 5.

As shown in FIG. 2, the spray formulation is preferably supplied to a perfusor 6 located outside the exposure space for setting a suitable liquid mass flow and subsequently forwarded to the ultrasonic atomizer 4 mounted below the ceiling fan 3. Furthermore, the exposure space contains an aerosol monitor 7. With this, the measurement of the mass concentration of the carrier substance and thus the determination the allergen concentration can be made high in time. The exposure space shown in Fig. 2 also includes a filter medium 8. By collecting an air sample on the filter medium and then quantifying the allergen with an associated ELISA, an allergen-specific off-line measurement can be performed.

Preferably, the air volume flow Q through the exposure space is in the range of 600 m ³ / h to 2000 m ³ / h. Furthermore, the exposure space is preferably operated at an air exchange rate in the range of 1000 to 1600.

As already discussed above, the inventive method is characterized by its high flexibility. For example, the mean allergen concentration in the exposure room can be varied over a wide range. The distribution of the aerosol preferably leads to an average allergen concentration C _A in the room air of the exposure space in the range from 0.01 μg / m ³ to 1 μg / m ³ , more preferably in the range from 0.04 μg / m ³ to 0.1 μg / m ³ .

The allergen concentration C _A in the room air of the exposure room results from the allergen concentration C _A in the spray formulation, the total liquid mass flow mp of all nebulization units used and the air volume flow Q through the exposure room:

The air concentration C _{T of} the carrier material is related to the air concentration of the allergen substance as are the concentrations of the two components in the starting formulation:

An environmentally relevant ambient air concentration of the pl mite allergen is, for example, C _A = O.05 μg / m. This can be done in a 10x air exchange rate operated exposure space at Q = 1200 m ³ / h can be achieved theoretically, if spraying a spray solution with C _A = IO ug / ml and Cτ = 200 mg / ml with a liquid mass flow of mp = 6 ml / h. The air concentration of the carrier material is then C _T = 1 mg / m ³ . In practice, however, the feed rate must be higher because of particle losses in the room.

If a grass pollen exposure of 4000 pollen / m ³ , which is frequently used in clinical studies, was to be reproduced by the guide allergens, this means an allergen concentration in the air of 4000 × 0.05 ng / m ³ = 0.2 μg / m ³ . This could be achieved with a formulation with C _A = 40 μg / ml and otherwise with the parameter values given above.

The measurement of the allergen can be carried out by measuring the mass concentration of the carrier substance, which can be carried out with high-resolution aerosol monitors. An aerosol monitor can exploit the light scattering properties in air suspended particles. Particles that are in an optically defined measurement volume are illuminated with laser light. The sum of the scattered light emitted by all particles at a certain angle is a measure of the aerosol concentration. A photoelement converts the collected stray light into a voltage. Alternatively, one can use a particle counter which counts every single particle in a defined intake volume flow and determines the size of the particle by means of a scattered light pulse height analysis. From particle size and number concentration, the mass concentration can then be calculated. The measurement is preferably carried out with an aerosol monitor.

An allergen-specific off-line measurement can be performed by collecting an air sample on a filter medium and then quantifying the allergen with an associated ELISA. With the method according to the invention, it is possible to keep the average allergen concentration C _A constant in the room air of the exposure room with a maximum deviation of 20% over a period of 1 hour to 5 hours (see FIG. 3). The time course of the carrier substance concentration in the exposure space is shown in FIG. 3.

As already discussed above, an aerosol monitor and / or a laser particle counter for determining the allergen concentration may be appropriate in the exposure space.

In another aspect, the present invention relates to an apparatus for performing an inhalant allergen provocation, comprising: an exposure space, the uniformity device elements

Distribution of an aerosol in the room air of the exposure room by turbulent mixing ventilation includes, - a spray drying device, at least partially in

Exposure room is attached.

With regard to the preferred embodiment of the exposure space can be made at this point to the above statements.

Thus, the elements mounted in the exposure space for the even distribution of the aerosol through turbulent mixing ventilation are preferably effected in the air supply attached swirl outlets, fans, in particular ceiling fans, and / or underfloor heating.

Also with regard to the spray-drying apparatus including atomizing unit and the spray formulation supplied to this apparatus, reference may be made to the statements made above. In another aspect, the present invention relates to the use of a liquid, preferably aqueous, spray formulation to produce a temporally and spatially constant allergen room air concentration in an exposure room for conducting clinical trials.

With regard to the preferred properties of the spray formulation, the spray-drying process, the spray-drying apparatus and the exposure space, reference may again be made at this point to the above statements in the discussion of the method according to the invention.

By choosing the atomization unit and the width of the droplet diameter distribution can be influenced. Thus, e.g. a fairly narrow size distribution can be achieved with suitable ultrasonic atomizers, while the use of compressed air spray nozzles generates a broader size distribution. This makes it possible to achieve different allergen provocations in a targeted manner, e.g. a nasal and / or bronchial provocation when using compressed air spray nozzles or a provocation of the respiratory tract when using a Ultraschallzerstäubers.

If the allergen provocation of the respiratory tract is targeted in the clinical trial, it may be preferred that the spray formulation be spray dried with an ultrasonic nebulizer.

If, on the other hand, the nasal and / or bronchial allergen provocation is targeted in the clinical study, it may be preferred for the spray formulation to be subjected to spray drying with one or more compressed-air spray nozzles. Taking into account the statements made above, it can be stated that the method according to the invention and the device according to the invention have, inter alia, the following advantages: realistic imaging of environmental exposure, site of action in the respiratory tract and bioavailability, high flexibility with regard to the allergen to be investigated or

Allergen spectrum, very good control of allergen spectrum, allergen concentration and

Particle size, - use of only one technical method of generation, monitoring,

Plant decontamination regardless of the selected allergen and

Compatibility with the provisions of the Medicines Act.

Claims

claims 1. A method for producing an allergen inhalation atmosphere in an exposure room comprising the steps of: Providing a liquid spray formulation containing a solvent, a vehicle and an allergen, and preparing an aerosol by spray-drying, wherein the aerosol particles contain the vehicle and the allergen, and Distribution of the aerosol in the room air of an exposure room, wherein the exposure room device elements for generating a temporally and spatially constant allergen room air concentration. 2. The method of claim 1, wherein for the preparation of the aerosol by spray drying, a freeze-dried allergen extract of a natural allergen carrier or a recombinantly produced allergen is used. 3. The method of claim 1 or 2, wherein for the spray drying, an aqueous spray solution is used. 4. The method according to any one of the preceding claims, wherein the allergen selected from the group consisting of mite allergens, in particular The pl Mite allergen, pollen allergen, pet allergen, house dust allergen, or mixtures thereof. 5. The method according to any of the preceding claims, wherein the allergen is present in the spray formulation in a concentration C _A of 0.1 // g / ml to 1000 ug / ml. 6. The method according to any one of the preceding claims, wherein the Carrier substance selected from the group consisting of lactose, sodium chloride, or mixtures thereof. 7. The method according to any one of the preceding claims, wherein the Carrier substance in the spray formulation in a concentration of 0.1% by weight to 20% by weight. 8. The method according to any one of the preceding claims, wherein the atomization of the spray formulation with at least one Atomizing unit, which is selected from an ultrasonic atomizer, a Zerstäuberteller and / or at least one compressed air spray nozzle, takes place. 9. The method according to any one of the preceding claims, wherein the Aerosol particles have a median diameter dso of 25 microns or less. 10. The method of claim 9, wherein the aerosol particles have a median diameter dso of 10 microns or less, preferably 5 to 10 microns. 11. The method according to claim 9, wherein the aerosol particles have a median diameter d 50 of more than 10 μm, preferably 10 μm ≦ d 50 ≦ 25 μm. 12. The method according to any one of the preceding claims, wherein the exposure space has a volume in the range of 3 m ³ to 300 m ³ . 13. The method according to any one of the preceding claims, wherein the uniform distribution of the aerosol takes place in the room air by turbulent mixing ventilation. 14. The method according to any one of the preceding claims, wherein the distribution of the aerosol is effected by mounted in the air supply swirl outlets, one or more fans, in particular ceiling fans, and / or underfloor heating in the exposure room. 15. The method of claim 1, wherein atomization of the spray formulation occurs below the ceiling fan and the aerosol particles formed are evenly distributed in the exposure space by the fan flow and the turbulent fresh air flow. 16. The method according to any one of the preceding claims, wherein at least one atomization unit is mounted centrally below the ceiling fan and / or in the immediate vicinity of a fresh air inlet, in particular a swirl outlet. 17. The method according to any one of the preceding claims, wherein the spray formulation is fed to a metering device for adjusting a suitable liquid mass flow and is then forwarded to the atomization unit. 18. The method according to any one of the preceding claims, wherein the air volume flow Q through the exposure space in the range of 600 m ³ / h to 2000 m ³ / h. 19. The method according to any one of the preceding claims, wherein the Exposure room is operated at an air exchange rate in the range of 1000 to 1600. 20. The method according to any one of the preceding claims, wherein the Distribution of the aerosol leads to an allergen concentration C _A in the room air of the exposure room in the range of 0.01 μg / m to 1 μg / m. 21. The method according to any one of the preceding claims, wherein the allergen concentration C _A in the room air of the exposure room with a maximum deviation of 20% over a period of 1 to 5 hours is kept constant. 22. The method according to any one of the preceding claims, wherein in the exposure space an aerosol monitor and / or a laser particle counter for Determination of the allergen concentration is appropriate / are. 23. An apparatus for carrying out an inhaled allergen challenge, comprising an exposure space, the device elements to uniform Distribution of an aerosol in the room air of the exposure room by turbulent mixing ventilation includes a spray-drying device, which is at least partially mounted in the exposure room. 24. Use of a liquid, preferably aqueous spray formulation for generating a temporally and spatially constant allergen indoor air concentration in an exposure room for conducting clinical trials. Use according to claim 24 wherein the spray formulation is spray dried with an ultrasonic nebulizer and the clinical study relates to respiratory allergen challenge. 26. Use according to claim 24, wherein the spray formulation is subjected to spray drying with one or more compressed air spray nozzles and the clinical study relates to nasal and / or bronchial allergen provocation.