EXPOSURE SYSTEM

Abstract

The invention relates to methodologies of obtaining a controlled exposure of an aerosol to a model material. The invention also provides an exposure cap and a system for studying or predicting the interaction between a model material, e.g. a cell, and an aerosolized agent. The controlled exposure of the aerosol to the model material, makes is possible to accurately calculate the mass-balance of the aerosol exposure.

Claims

1.-9. (canceled)

10. An exposure cap device for establishing a controlled exposure of a model material to an aerosol following its insertion into a model material container, said exposure cap having: an upper part adapted to receive an inlet flow of an aerosol; and a lower part, adapted to be aligned to the model material, to collect an aerosol stream following its exposure of the model material and to shield the model material container from the aerosol, wherein the generally cylindrical exposure cap comprises: (a) an inlet duct for receiving the aerosol in said upper part, wherein the inlet duct is provided with a tangentially located inlet orifice for generating a rotating flow around a central axis, from the upper part to the lower part; and (b) a single outlet duct for transporting remaining aerosol from the model material from the lower part to an outlet in the upper part.

11. The device according to claim 10, wherein the outlet duct is located essentially along a central axis longitudinally extending between the upper and lower parts.

12. The device according to claim 10 comprising an inner central body providing an annular duct for transporting the received aerosol to said lower part, wherein the inner body has an extension providing an annular slit in the lower part for transportation of aerosol from the annular duct along the model material surface to the outlet duct.

13. The device according to claim 12, wherein an outlet orifice of the outlet duct has a diameter less than the width of an annular duct diameter.

14. The device according to claim 12, wherein a lower part of the inner body comprises a funnel-shaped cavity extending from a peripheral lower end of the inner body to the outlet orifice of the inner duct, thereby defining a cavity height.

15. The device according to claim 10, being essentially cylindrical and adapted to be attached to a container comprising the model material in order to obtain a substantially sealed environment during an aerosol exposure, and wherein a cross-sectional area of the annular duct is less than of the surface of the model material.

16. The device according to claim 12, wherein the inner central body has a funnel-shaped extension towards an inner wall and so that that the annular duct has an essentially larger area than the annular slit

17. A system suitable for establishing a controlled exposure of a model material to an aerosol resembling the airway-blood-barrier in a respiratory tract, comprising an aerosol transporting arrangement for transporting the aerosol from an aerosol generating source to an exposure unit, wherein: (i) the exposure unit comprises: (a) the exposure cap device according to claim 10; (b) a container comprising a model material on a permeable membrane; (c) a perfusion tray for establishing an even perfusion fluid contact between a fluid inlet, and the underside of the permeable membrane of the container; and (d) a perfusion fluid outlet adapted to counteract drop formation at the outlet, and (ii) the aerosol transporting arrangement comprises an exposure flow line, comprising one or more branches connected to one or more inlet ducts of exposure caps transporting the aerosol from said exposure line past the aerosol inlets of the exposure caps and onto the end filter located downstream of the exposure branches.

18. The system according to claim 17, wherein the perfusion tray is connected to a control device for controlling a perfusion fluid pressure against the membrane.

19. The system according to claim 18, wherein the control device monitors and adjusts the level of perfusion fluid in a perfusion fluid container connected to the inlet.

20. The system according to claim 17, wherein the perfusion tray is essentially circular in cross section and wherein the inlet and outlet are located in diametrically opposite positions in a peripheral region of the tray between at least two parallel ridges.

Description

SHORT DESCRIPTION OF DRAWINGS

[0026] FIG. 1 shows a schematic drawing of a system for exposing cultured cells to an aerosol according to the present invention.

[0027] FIG. 2 shows schematic drawing of the exposure unit inserted in a conventional cell culture container, an exposure cap and a perfusion tray.

[0028] FIG. 3 shows an embodiment of exposure cap as inserted in cell culture container attached to a perfusion tray.

[0029] FIG. 4 schematically shows the perfusion tray.

DETAILED DESCRIPTION OF THE INVENTION

[0030] Before the invention is disclosed and described in detail, it is to be understood that this invention is not limited to particular compounds, configurations, method steps, substrates, and materials disclosed herein as such compounds, configurations, method steps, substrates, and materials may vary somewhat.

[0031] It is also to be understood that the terminology employed herein is used for the purpose of describing particular embodiments only and is not intended to be limiting since the scope of the present invention is limited only by the appended claims and equivalents thereof.

[0032] If nothing else is defined, any terms and scientific terminology used herein are intended to have the meanings commonly understood by those skilled in the art to which this invention pertains.

[0033] It must be noted that, as used in this specification and the appended claims, the singular forms a, an and the include plural referents unless the context clearly dictates otherwise.

[0034] The term about as used in connection with a numerical value throughout the description and the claims denotes an interval of accuracy, familiar and acceptable to a person skilled in the art. Said interval is 10%.

[0035] Other features and uses of the invention and their associated advantages will be evident to a person skilled in the art upon reading the description and the examples.

[0036] It is to be understood that this invention is not limited to the particular embodiments shown here. The following examples are provided for illustrative purposes and are not intended to limit the scope of the invention since the scope of the present invention is limited only by the appended claims and equivalents thereof.

[0037] FIG. 1 shows a schematic drawing of a device for studying controlled exposure of a model material i.e. a cell layer, in vitro to a test aerosol.

[0038] The system comprise an aerosol generator (not shown) situated on the left side of a light beam instrument (100), an exposure flow line (101) connected to an end filter (102), the exposure flow line contains three branching points (104), each connecting an exposure unit (105), respectively, to the exposure flow line (101). The exposure unit (105) include an exposure cap (106) which comprises an inlet orifice conduit (107) and an outlet orifice conduit (108), an exposure filter (109) connected to a rotameter. The exposure cap (106) is in contact with a cell culture container, not shown (See FIG. 2), that is in turn in contact with a perfusion tray (111) (FIG. 3) comprising an outlet conduit orifice (113) that may be connected to a fraction collector (114) (FIG. 1). The temperature of the exposure unit is controlled by using a heating block (120), with one or several bores adapted to receive one or several exposure units, respectively. The heating block (120) is preferably made of steel. In FIG. 1, the device is shown with three exposure units (106), each comprising a commercial culture container with cultured cells.

[0039] When operating the system of FIG. 1, the aerosol is generated and passes from an aerosol generator (not shown) via a light beam instrument (100) coupled to an exposure flow line (101) that contains three dividing branching points (104) that each is connected to an inlet conduit (107) located in the upper parts of respective exposure cap (106). In each exposure cap, a flow of aerosol rotates downwards around a central axis of the cap is generated so that essentially only the layer of cells is evenly exposed to controlled amounts respirable aerosol at a time. Each cap has an outlet conduit (108) for transporting remaining aerosol from lower part through an opening in its upper part, where an exposure filter (109) collects all remaining aerosol. A rotameter (not shown) is connected to each outlet conduit (109) generating a constant flow rate through each branch of the exposure system. In addition, the overall flow rate of the exposure line is accomplished with yet another rotameter or similar device located downstream of the end filter (102). The cell culture container comprises a layer of cells cultured on one or both sides of a permeable membrane and which is contacted by a perfusate fluid which resembles the blood circulation in the airway mucosa on the other side of the permeable membrane. The flow rate of the perfusate fluid will be kept at a rate to resemble the flow rate in the mucosa. When chosen in the range of 0.4 ml/min for a model membrane 12 mm in diameter the renewal rate will be similar to that in the mucosa.

[0040] The thickness of the perfused layer below the permeable membrane (see FIG. 2, 115) will be approximately 0.5 mm, exemplified by 0.2 mm, in a way similar to the distribution of the rich superficial network of capillaries of the airway mucosa. The perfusate fluid will be transported in single-pass mode by the membrane and then immediately fractionated into suitable vessels e.g. a 96 well plate for analysis in a mass spec. A special fraction collector has been developed where the fraction sizes are not dependent on drop formation and are only controlled by the flow rate and the desired time window for the sample withdrawal. In this way, up to three wells may be exposed and samples withdrawn in parallel from one aerosol exposure. It is only the cell layer at the bottom of the well that shall be exposed with a good surface evenness and not in the inner walls of the test material container (Transwell cup), which is the case with the Cultexsystem and similar systems. The aerosol will be slowly rotating downwards around the axially located aerosol outlet (108) in the upper part of the exposure cap, to be subsequently sucked out of the exposure unit via the aerosol outlet conduit (108) which is centered above the middle of the circular surface coated with cells.

[0041] Following exposures both the trans-epithelial absorption of dissolved substances and eventual biological reactions in the cells caused by the tested substances will be studied, as it will be manifested by the production and excretion different markers from the cells.

[0042] FIG. 2 shows a schematic drawing of an exposure unit (105) comprising a conventional cell culture container (Transwell cup). The dotted lines show where the exposure cap (106) and the perfusion tray (111), respectively, are connected to assemble the exposure unit included in the system of FIG. 1. The exposure cap (106) of FIG. 2 has an inlet duct (107) with a tangentially locate inlet orifice. The exposure cap covers the inside of the Transwell cup down to the edge of the cell-covered membrane (115). This is to prevent any aerosol exposure outside of the cell-coated surface, and a correct figure for cell-retained aerosol material will be obtained even if the entire Transwell unit is rinsed in solvent after the exposure cap and tray have been removed. The cells are cultured on a permeable membrane located at the bottom of the Transwell cup and are perfused by a thin layer of perfusate fluid that is pumped into the gap of the perfusate tray under the permeable membrane in a configuration chosen to resemble the blood circulation of the airway mucosa. FIG. 2 further shows the outlet conduit (108) centrally located in the exposure cap having an outlet orifice at a defined height above the model material, here represented by the cell covered membrane (115). In operation, an aerosol introduced through the tangential inlet orifice of the inlet conduit (107) and rotates downwards in the hollow exposure cap, between the inner wall and the centrally and symmetrically placed outlet conduit (108). The rotation speed is controlled by aerosol inlet flow rate, the diameter of the inlet orifice and the height between the outlet orifice and the model material. A flow with even flow shear rate and concentration of aerosol will now be distributed over the model material for the exposure and a funnel-shaped flow will generated in the direction of the outlet orifice.

[0043] FIG. 3, shows a crossectional view of a special embodiment of an exposure cap (106) with an inner body (120). The exposure cap (106) is inserted in a cell culture container (100) having viable cells culture on the membrane (115). The exposure cap shields the cell container inner wall during the exposure in this inserted position in order to minimize and control aerosol transportation losses. The exposure cap (106) is provided with inlet conduit for aerosol (107) having a tangentially located inlet orifice and an axially extending outlet duct (108) arranged centrally and symmetrically in the inner body (120). An annular duct (122) is admitted between the inner body (120) and the inner wall of the exposure cap for downwards aerosol transportation. The inner body (120) axially extends towards model material represented by the cells of the membrane (115) to admit an annular slit (124). The lower part of the inner body (120) has funnel-shaped cavity or recess (126) extending from the periphery to the centrally located inlet orifice (128) of the outlet duct (108). The embodiment depicted in FIG. 3, shows how the annular body (122) extends in the funnel shaped extension 130 that extends towards an inner wall admits the annular slit (124). By the configuration of the funnel shaped extension and the dimensions annular duct (124) to be essentially narrower than the annular duct concentration gradients in the aerosol can be avoided or counteracted and a homogeneous exposure of aerosol can be established. By the so configured exposure cap, a controlled and extended downwards rotating flow can be established in the upper part of the exposure, before the aerosol is transported axially downwards towards the exposure area and the membrane (115).

[0044] In operation the arranged of FIG. 3, an aerosol is introduced with the tangentially located orifice of the inlet duct (107) so a downwards rotating aerosol flow is obtained in the annular duct (122) as described to the annular slit (124) and distributed along the cell culture for an aerosol exposing period. Remaining aerosol is transported away from the cell culture through the cavity (126) and into the outlet duct (108) which is connected to a conventional flow generating means (not shown). The so described embodiment admits even flow shear rate of aerosol along the surface of the model material, providing an even and controlled deposition of aerosol particles to the cultured cells and thereby realizing the many advantages of the invention outlined in earlier discussions.

[0045] FIG. 3 also shows the perfusion tray (111) attached to cell culture container (110) with the inserted perfusion cap (106). FIG. 4 schematically shows the perfusion tray (111) with diametrically opposite inlet and outlet (112, 113) and two intermediary ridges or baffles (119,119) serving as perfusion flow distributors. The perfusion tray for the provided with means for attachment and sealing to the cell culture container so a gap of about 0.2 mm is provided between the tray and the permeable membrane. As previously explained the perfusion tray can be accommodated in a heating block. In operation, the model material of the system (e.g. cells) is in contact with the perfusion fluid. A perfusion fluid is flowing through the perfusion tray, in order to stimulate the dissolution and absorption into the circulating blood of solutes emanating from the particles in the model material. The perfusion flow will receive the agent dissolved or metabolized, and diffused through the model material and membrane and be transported away for subsequent analysis. The perfusion fluid is generally adapted to interact with the model material in a manner that resembles the natural blood flow in the mucosa with a high renewal rate. Typically, this would correspond to a flow rate of on the order of 0.4 ml/min in a 12 mm diameter container. More specifically the perfusion fluid should be a physiologically acceptable fluid compatible with the model material. The perfusate pressure against the insert membrane can preferentially be regulated by adjusting the level of the perfusate outlet tube from the inserts as to achieve a suitable pressure head with a negative or positive static pressure component acing against the insert membrane. It is then important to prevent droplet formation at the tube exit, because the perfusate surface tension in the recurrent drops will cause an undulating pressure against the insert membrane.

[0046] The described system can advantageously be adapted to provide a controlled exposure simultaneously to a plurality of containers with model material, e.g. culture containers with cells. The containers can be of a conventional type with the model material, e.g. a cell culture on membrane. The exposure cap preferably is adapted to cover the entire inside of the container except the model material membrane during an aerosol exposure. Advantageously, in an exposure with a test substance, rinsing the model container with solvent after removal of the exposure cap will only recover model-retained substance. The system will provide highly reliable conditions by establishing similar or essentially identical exposure conditions in each container. In order to establish a reliable control system, wherein rotameters or mass flow regulators control and monitor the flow rate in the outlet conduits from the exposure caps and will thereby serve to control inlet flows from the branches of the exposure flow line so each model material (cell culture) obtains a similar exposure condition.

[0047] The inventive system is equally useful for estimating or predicting a deposited dose of an aerosol when the cell retained fraction of a substance cannot be measured, or for further pharmacokinetic modeling with isolated cells or another model material. Dummy containers with similar model material can then be used in line with the study containers to estimate the deposited dose of aerosol.

[0048] One advantage of an embodiment is the low amount of test compound needed, e.g. active substance in a pharmaceutical composition. For example, less than 1 mg test compound is enough to simultaneously expose three units each comprising a 12 mm well, respectively. Accordingly, the present invention admits improved methodology of screening drug candidates or other substances of interest.

[0049] Further the present invention of controlled exposure makes it possible to easily control the mass balance in the system; i.e. the deposited dose of aerosol is calculated as the sum of the remaining substance in the model material, e.g. cells and the cumulative amount in the perfusate. Because only the model material surface of the material container is exposed to the test aerosol, the correct fraction of material-retained substance can be obtained, even if the entire model container is rinsed in the analysis solvent.

[0050] Another advantage is that the model material is continuously provided with fresh media, i.e. perfusate fluid, where the used perfusate fluid containing absorbed or metabolized test substance from the aerosol particles are collected and analyzed.

[0051] A further advantage is that the aerosol may be evenly distributed into a plurality of culture containers, preferably three culture containers at the same time (SD+/15%).

[0052] The interaction of the aerosol with the model material could be due to physical-chemical properties such as density of particle deposition, particle size, dissolution- and absorption rates, or biochemical properties such as metabolism or other effects.

[0053] In the context of the present invention, a deposited model material may be cultured cells, such as isolated primary cells, preferably isolated epithelial cells and possibly endothelial cells or other relevant cell types from the respiratory tract.

[0054] Suitable cells can be isolated primary cell, preferably isolated epithelial primary cells or COPD cells or cells isolated from a person suffering from asthma, it could also be a cell line, such as A 549 cells, preferably of epithelial origin and it could also be isolated stem cells, preferably stem cells that will differentiate into an epithelial cell type or any type of suitable cell. In one embodiment the cells are derived from the respiratory tract, more preferably isolated cells derived from pulmonary cells. In another embodiment the cells are co-cultured with another cell type, i.e., feeder cells.

[0055] The cell culture container may comprise one or several layers of cells on one or both sides of a model membrane, or cells cultured in a suspension.

[0056] The cells cultured may also be co-cultures, e.g. epithelial cells co-cultured with endothelial cells to resemble a wall of an organ with an inner part and outer part, respectively.

[0057] Advantageously, the system can be used when screening drug candidates based on their interaction properties with a model material resembling the air-blood-barrier. Firstly, a powdered dose of a drug candidate is aerosolized and the system is adapted to determine the predicted dose deposited in the material container (Mdep). Secondly, the predicted deposition of the powdered drug can now be used for running the system to expose a model material to the same aerosol for determining how the drug candidate interacts with the model material at the desired dose level. In other terms the determination of dose to be expected in the model material, admits that the system can be directly run with optimized exposure times and other operation conditions in subsequent tests with the model material when only low amounts of test material and few model material containers are available. The model material can be cells preferably human cells in a culture container/vessel. Preferably the transport aerosol flow is kept essentially at the same rate, wherein the flow is controlled by a rotameter or mass flow regulator. Preferably and advantageously, small amounts of drugs are used, in the mg range such as 0.1 to 3 mg.

[0058] By following these routines, the general advantages of the system in operating with optimized amounts of expensive material are further enhanced and quick, reliable discrimination between different drug candidates can effectively be performed at an early preclinical stage. Interaction studies made available with the method include not only absorption studies of the drug candidate, but is also equally feasible for studying a number of physiological effects including drug activity exerted in or via lung tissues, such as metabolism, pharmacological- and toxicological responses including onset of therapeutic action, as well as bioavailability and pharmacokinetic aspects. The presently invented system and the methodologies using the system will reduce and simplify the discovery and preclinical phases of drug development and thereby be of significant benefit to bring down the costs in an industry suffering from increasing burdens when attempting to find new original products for market approval. The system and the methods described are equally useful in diagnostic applications, wherein the described methodology can be applied of determining pulmonary deposition and determining airway sensitivity to an agent. The same benefits will be evident for a diagnostic application regarding low dosing and test accuracy. The present invention in the form of the earlier disclosed methods, exposure cap and system is also intended to be applicable for biological validation of drug formulations with commercially available inhalators or nebulizers as aerosol generators. In such applications, the present invention admits a convenient and reliable evaluation of aerosolized drug formulations biological performance in terms of particle dissolution and pharmacokinetic characteristics, as well as the suitability of the available administration tools to sufficiently perform a controlled pulmonary administration of selected drug formulations.