DESC:FIELD OF THE INVENTION
The present invention relates to the field of development and assessment of effective drug formulations as route of administration and target cells. In particular, the present invention relates to a method for in-vitro assessment of drug formulations using A549 cells. The method of the present invention is simple, effective, and non-invasive.
BACKGROUND OF THE INVENTION
The use of in vitro data generated using tests that demonstrate good in vitro-in vivo correlation is accepted as supporting means for establishing the bioequivalence of the dosage forms, e.g. immediate release tablets and capsules. The pharmaceutical regulatory authorities in most countries consider the submission of a truncated clinical testing package to support the approval process if the redesigned product is shown to be “bioequivalent” to the licensed medicine. Determining bioequivalence of inhalation products such as pressurized metered dose inhalers (pMDI) is difficult because the critical characteristics of such products are poorly defined. The complexity of inhalation dosage forms renders difficulty in mimicking and raises questions regarding definitions of similarities and those properties that must be controlled to guarantee both the quality and the efficacy of the product.
M. Haghi et al. European Journal of Pharmaceutics and Biopharmaceutics 86 (2014) 38–45 discloses two solution-based pressurised metered dose inhaler (pMDI) formulations, which were prepared such that they delivered aerosols with identical mass median aerodynamic diameters but contained either beclomethasone dipropionate (BDP) alone (glycerol-free formulation) or BDP and glycerol in a 1:1 mass ratio (glycerol-containing formulation). The two formulations were deposited onto Calu-3 respiratory epithelial cell layers cultured at an air interface. Forbes et al have also presented a study using Calu – 3 cells which are essentially bronchial epithelial cells. Mukherjee et al have investigated the interaction between inhaled drug and organic cation transporters in layers of human bronchial epithelial Calu-3 cells grown at an air–liquid interface. The vital difference between the prior arts and the present invention is the choice of the cell line for such studies. The alveolar epithelial cells, A549 offer to be a much better and suitable model for these studies as they form a confluent monolayer with Type II characteristic morphology and tannic acid staining for typical lamellar bodies. Type II cells makes potential target for drug delivery as established by Audus et al owing to its special characteristics which includes capability to produce surfactants stored in organelles, being the progenitor cells for Type I pulmonary epithelial cells. Apart from these they also possess isozymes which are functional with fluorescent resorufin assay.
Further, cells in bronchial region of the respiratory tract are mostly subjected to non-absorptive elimination to aid mucociliary clearance and thus studies conducted on them will not reflect the true intrinsic kinetic properties of lungs i.e., absorption/ permeability. Moreover, with the advancement in aerosol and formulation technologies, delivery systems effectively deposit drugs to the alveolar regions wherefrom they are available for systematic absorption.
The use of A549 cells have mostly been known in the domain of understanding cellular toxicity as described in Birchall et al. However, there are no studies, which suggests use of A549 in permeability studies. Festen et al have presented a study to evaluate the pulmonary bioavailability for inhaled (BDP) aerosols of different sizes.
Holz et al., discloses direct measurement of beclomethasone dipropionate (BDP) and its metabolite beclomethasone mono propionate (17-BMP) in airways and peripheral tissues from lung specimens after inhalation with an objective to compare the performance of hydrofluoroalkane-BDP (HFA-BDP) and chlorofluorocarbon-BDP (CFC-BDP) in patients with lung cancer undergoing surgery. However, the approach is invasive in nature and use of such approach is associated with several associated procedural and ethical complications.
Although several reports/studies have been conducted so far, however none of the prior arts provides a method which can mimic the in-vivo conditions and enable simultaneous quantification of active agents delivered by the inhalation route such as BDP and its metabolite to understand drug deposition and permeability in alveolar cells. Accordingly, the present invention provides a non-invasive method to assess the deposition and permeability of a drug formulation in such a way so that results closely mimic the in-vivo conditions.
Particularly, the present invention provides a simple, effective, and non-invasive method for evaluating deposition and permeability of an active agent of an inhalation formulation, particularly by evaluating delivery of the active agents by the inhalation route such as BDP and its metabolite 17-BMP using A549 cells.

OBJECT OF THE INVENTION
An object of the present invention is to develop a method for evaluating the effectiveness of an active agent of an inhalation formulation, using monolayer culture of A549 cells and by specifically actuating the active agent into A549 cells using twin stage impinger.
Another objective of the present invention is to evaluate efficiency of different inhalation formulations, particularly pressurized metered dose inhaler (pMDI) formulations.
Yet another objective of the present invention is to provide an in vitro method for determining bioequivalence of a reformulated (test) drug formulation with the reference drug formulation.

SUMMARY OF THE INVENTION
The present invention relates to the field of development and assessment of effective drug formulations as route of administration and target cells. In particular, the present invention relates to a method for in-vitro assessment of drug formulations using A549 cells.
In an aspect of the present invention, there is provided a method for evaluating permeability of an active agent of an inhalation formulation, the method comprising:
a) simulation of in vivo conditions in ex vivo set-up by producing a monolayer culture of A549 cells grown in an air-liquid interface in a transwell insert in a suitable basal growth medium for a suitable period,
b) optimization of atleast one cell culture parameter selected from cell seeding density, cell proliferation upon culture, cell viability, and cellular monolayer integrity by measuring TEER or quantification of zero permeability marker,
c) placing the transwell insert of step (a) in contact with a buffered medium,
d) actuating a pre-defined dose of the active agent from the formulation onto the A549 culture of step (c),
e) collecting the cell samples from the buffered medium at predefined time intervals, and
f) analyzing the collected cell samples from step (e) to determine the amount of the active agent.

In an embodiment of the present invention, there is provided a method for evaluating permeability of an active agent of an inhalation formulation, wherein the active agent is selected from albuterol sulfate, Salbutamol xinafoate, Levalbuterol tartrate, Formoterol fumarate, Ipratropium bromide, Sodium cromoglycate, Beclomethasone dipropionate, Ciclesonide, Flunisolide, Fluticasone propionate, Budesonide, Mometasone furoate, Terbutaline sulfate, Aclidinium bromide, Glycopyrronium bromide, Umeclidinium, vilanterol, Indacaterol, olodaterol and iotropium.

In a preferred embodiment of the present invention, there is provided a method for evaluating permeability of an active agent of an inhalation formulation, wherein the active agent is beclomethasone dipropionate (BDP).

In another embodiment of the present invention, there is provided a method for evaluating permeability of an active agent of an inhalation formulation, wherein the cell density is 2 x 105 cells /ml to 2.5 x 105 cells / ml.

In yet another embodiment of the present invention, there is provided a method for evaluating permeability of an active agent of an inhalation formulation, wherein the cell density is 2 x 105 cells /ml.

In still another embodiment of the present invention, there is provided a method for evaluating permeability of an active agent of an inhalation formulation, wherein the cell density is 2.5 x 105 cells /ml.

In another embodiment of the present invention, there is provided a method for evaluating permeability of an active agent of an inhalation formulation, wherein the monolayer culture of A549 cells in a transwell insert in a basal growth medium is formed between 5 days to 15 days.

In yet another embodiment of the present invention, there is provided a method for evaluating permeability of an active agent of an inhalation formulation, wherein the monolayer culture of A549 cells in a transwell insert in a basal growth medium is formed between 8 days to 10 days.

In still another embodiment of the present invention, there is provided a method for evaluating permeability of an active agent of an inhalation formulation, wherein the transepithelial electrical resistance (TEER) is greater than 350 Ocm2.

In another embodiment of the present invention, there is provided a method for evaluating permeability of an active agent of an inhalation formulation, wherein the transepithelial electrical resistance (TEER) is greater than 400 Ocm2.

In yet another embodiment of the present invention, there is provided a method for evaluating permeability of an active agent of an inhalation formulation, wherein the percentage (%) permeation of zero permeability marker is less than 2%.

In still another embodiment of the present invention, there is provided a method for evaluating permeability of an active agent of an inhalation formulation, wherein the zero permeability marker is sodium fluorescein.

In an embodiment of the present invention, there is provided a method for evaluating permeability of an active agent of an inhalation formulation, wherein the pre-defined dose of the active agent from the formulation is actuated onto the A549 culture of step (c) using a twin stage impinger.

In another embodiment of the present invention, there is provided a method for evaluating permeability of an active agent of an inhalation formulation, wherein the method further comprises calculating the apparent permeability of the inhalation formulation.

In yet another embodiment of the present invention, there is provided a method for evaluating permeability of an active agent of an inhalation formulation, wherein in step (c), the buffered medium is modified HBSS buffer.

In still another embodiment of the present invention, there is provided a method for evaluating permeability of an active agent of an inhalation formulation, wherein the analysis of the cell sample is carried out using standard techniques like LC-MS, LC-MS/MS or any other suitable standard techniques known in the art.

In another aspect of the present invention, there is provided a device comprising a cell culture vessel with transwell insert with permeable membrane to separate the vessel into upper apical chamber, and a lower basolateral chamber, and a monolayer culture of A549 cell over the permeable membrane.

These and other features, aspects, and advantages of the present subject matter will be better understood with reference to the following description. This summary is provided to introduce a selection of concepts in a simplified form.

BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWINGS
These and other features, aspects, and advantages of the present disclosure will become better understood when the following detailed description is read with reference to the accompanying drawings in which like characters represent like parts throughout the drawings, wherein:

FIGURE 1 illustrates schematic representation of the method for in-vitro evaluation of deposition and permeability of an active agent of an inhalation formulation.

DETAILED DESCRIPTION OF THE INVENTION
The present invention relates to the field of development and assessment of effective drug formulations as route of administration and target cells. In particular, the present invention relates to a method for in-vitro assessment of drug formulations using A549 cells. More particularly, the present invention provides a method to non-invasively assess the deposition and permeability of an active agent of an inhalation formulation.
While the invention is susceptible to various modifications and/or alternative processes and/or compositions, specific embodiment thereof has been shown in detail below. It should be understood, however that it is not intended to limit the invention to the processes and/or compositions disclosed, but on the contrary, the invention is to cover all modifications, equivalents, and alternative falling within the spirit and the scope of the invention as defined by the appended claims.
For the purpose of promoting an understanding of the principles of the invention, reference will now be made to the embodiments and specific language will be used to describe the same. It will nevertheless be understood that no limitation of the scope of the invention is thereby intended, such alterations and further modifications in the illustrated system, and such further applications of the principles of the invention as illustrated therein being contemplated as would normally occur to one skilled in the art to which the invention relates.
It will be understood by those skilled in the art that the foregoing general description and the following detailed description are explanatory of the invention and are not intended to be restrictive thereof.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The process, methods, and examples provided herein are illustrative only and not intended to be limiting.
The inventors of the present invention have developed a method to non-invasively assess the deposition and permeability of a drug formulation in such a way so that results obtained closely mimic the results obtained in in-vivo conditions. The method described herein, particularly focused on the non-aerodynamic properties of the aerosol particles emitted from inhalation dosage forms such as pMDI formulation products, which determine their biopharmaceutical differences in terms of deposition, particularly permeation, using A549 human lung adenocarcinoma cell line. The inventors have specifically selected A549 cells for the method as these cells form tight intracellular junctions along with the formation of surfactant containing lamellar bodies and have Type II characteristic morphology. Type II cells, while covering substantially less surface area of the alveolus, are more numerous, 3.7 billion as compared to 1.9 billion of type I cells. Type II cells are also believed to be the progenitor cells for Type I pulmonary epithelial cells. Apart from this A549 cells possess P450 1A1 and P450 2B6 as determined by Western blots. Both CYP1A1 and CYP2B6 P450 isozymes were determined to be functional with the fluorescent resorufin assay. As recent developments in aerosol and formulation technologies, delivery systems are now more efficient in depositing the drugs to the alveolar regions, from where, they are available for systemic absorption. Thus, use of A549 pulmonary epithelial cells with Type II morphology for evaluating aerosolic drug formulations is of particular significance. Alveolar cell morphology has tight junctions of multiprotein complexes composed of trans-membrane, cytosolic and cytoskeletal proteins that interact in a coordinated manner. These tight junctions control the transepithelial paracellular passage of molecules between the alveolar space and the interstitium compartment. Thus, particle capture and analysis technique based upon using A549 cells were employed to characterize the physicochemical and biopharmaceutical properties of the inhalation formulations comprising active agents such as beclomethasone dipropionate (BDP).
Thus, in accordance with the present invention, there is provided a method for evaluating permeability of an active agent of an inhalation formulation, the method comprising:
a) simulation of in vivo conditions in ex vivo set-up by producing a monolayer culture of A549 cells grown in an air-liquid interface in a transwell insert in a suitable basal growth medium for a suitable period,
b) optimization of atleast one cell culture parameter selected from cell seeding density, cell proliferation upon culture, cell viability, and cellular monolayer integrity by measuring TEER or quantification of zero permeability marker,
c) placing the transwell insert of step (a) in contact with a buffered medium,
d) actuating a pre-defined dose of the active agent from the formulation onto the A549 culture of step (c),
e) collecting the cell samples from the buffered medium at predefined time intervals, and
f) analyzing the collected cell samples from step (e) to determine the amount of the active agent.

In a preferred embodiment of the present invention, there is a method for evaluating permeability of an active agent of an inhalation formulation, the method comprising:
a) simulation of in vivo conditions in ex vivo set-up by producing a monolayer culture of A549 cells grown in an air-liquid interface in a transwell insert in a basal growth medium for 5-15days,
b) optimization of atleast one cell culture parameter selected from cell seeding density, cell proliferation upon culture, cell viability, and cellular monolayer integrity by measuring TEER or quantification of zero permeability marker,
c) placing the transwell insert of step (a) in contact with a buffered medium,
d) actuating a pre-defined dose of the active agent from the formulation onto the A549 culture of step (c),
e) collecting the cell samples from the buffered medium in a duration of 2-150 minutes, and
f) analyzing the collected cell samples from step (e) to determine the amount of the active agent.

In an embodiment of the present invention, the active agent of an inhalation formulations is anti-infective, vaccinating, systemically acting diagnostic agents, anti-leukotrienes, anti-proteases and the combination thereof.
In an embodiment of the present invention, there is provided a method for evaluating permeability of an active agent of an inhalation formulation, wherein the active agent is selected from albuterol sulfate, Salbutamol xinafoate, Levalbuterol tartrate, Formoterol fumarate, Ipratropium bromide, Sodium cromoglycate, Beclomethasone dipropionate, Ciclesonide, Flunisolide, Fluticasone propionate, Budesonide, Mometasone furoate, Terbutaline sulfate, Aclidinium bromide, Glycopyrronium bromide, Umeclidinium, vilanterol, Indacaterol, olodaterol and iotropium.

In a preferred embodiment of the present invention, there is provided a provided a method for evaluating permeability of an active agent of an inhalation formulation, wherein the active agent is beclomethasone dipropionate (BDP).

The bioassay method of the present invention was methodically validated for cell density, cell viability, incubation period and membrane layer integrity. Particularly, A549 cells were maintained in a suitable media supplemented with a suitable cell culture media supplement. For optimization of cell seeding density, the cells may be seeded in a cell plate at different concentrations. After seeding, the cell seeding plates may be cultured under appropriate conditions for CO2/Oxygen blanket with media change at appropriate time intervals. Cell morphology needs to be checked at regular intervals to optimize number of days required for appropriate degree of cell morphology.
The ability of the A549 cells to form tight junctions under the air liquid interface, cells at varying densities may be seeded to an apical side of transwell inserts and may be placed in carrier wells with complete media on the basal side. After an appropriate time duration of seeding, media from apical side may be removed and cultures would be allowed to grow under air-liquid interface. The cell culture layer integrity can be evaluated by measuring the Trans-epithelial electrical resistance (TEER). The TEER can be measured at regular time points to determine and optimize the time duration required to form an integral cell culture layer. Formation of integrated tight junctions can be evaluated by using a zero-permeation marker, sodium fluorescein.

The cell viability might be evaluated by harvesting with an appropriate agent like trypsin and viability may be assessed using trypan blue dye exclusion method. The cell culture monolayer with integral tight junctions would be then used for the actual permeability assay. The permeability assay method might be initiated by replacing the cell culture media with an appropriate modified buffer, and the plate might be incubated for a suitable time in the modified buffer prior to drug treatment. A twin stage impinger might be modified for drug deposition by accommodating the transwell insert in the second stage of the twin stage impinger. This assembled twin stage impinger might be connected to a rotary vein pump and the flow rate might be suitably adjusted. The device containing the active agent of the formulation might be connected to the twin stage impinger and could be actuated for a set number of times to allow the drug to be deposited on the cell layer. The transwell containing the deposited drug formulation might be removed and transferred onto a carrier plate. An appropriate amount of modified buffer might be added to the apical side and a sample might be collected as a 0 minute time point sample. An appropriate amount of the modified buffer might be added to the basal side of the well and the carrier plate might be transferred to an incubator shaker operating at a desired shaking speed and controlled temperature. Appropriate amount of samples may be collected at suitable time intervals from the basal side of the transwell and may be replenished with an equivalent amount of the modified buffer. The twin stage impinger would be washed and the aliquots would be collected and quantified for mass balance and recovery of actuated drug.

In an embodiment of the present invention, there is provided a method for evaluating permeability of an active agent of an inhalation formulation, wherein the basal growth medium is minimal essential medium (MEM) a media supplemented with 10% Fetal bovine serum (FBS).

In another embodiment of the present invention, there is provided a method for evaluating permeability of an active agent of an inhalation formulation, wherein in step (c), the buffered medium is modified HBSS buffer.

In an embodiment of the present invention, there is provided a method for evaluating permeability of an active agent of an inhalation formulation, wherein the cell density is 2 x 105 cells /ml to 2.5 x 105 cells / ml.

In another embodiment of the present invention, there is provided a method for evaluating permeability of an active agent of an inhalation formulation wherein the cell density is 2 x 105 cells /ml.

In yet another embodiment of the present invention, there is provided a method for evaluating permeability of an active agent of an inhalation formulation, wherein the cell density is 2.5 x 105 cells /ml.

In still another embodiment of the present invention, there is provided a method for evaluating permeability of an active agent of an inhalation formulation, wherein the monolayer culture of A549 cells in a transwell insert in a basal growth medium is formed between 5 days to 15 days.

In another embodiment of the present invention, there is provided a method for evaluating permeability of an active agent of an inhalation formulation, wherein the monolayer culture of A549 cells in a transwell insert in a basal growth medium is formed between 8 days to 10 days.

In yet another embodiment of the present invention, there is provided a method for evaluating permeability of an active agent of an inhalation formulation, wherein the transepithelial electrical resistance (TEER) is greater than 350 Ocm2.

In another embodiment of the present invention, there is provided a method for evaluating permeability of an active agent of an inhalation formulation, wherein the transepithelial electrical resistance (TEER) is greater than 400 Ocm2.

In still another embodiment of the present invention, there is provided a method for evaluating permeability of an active agent of an inhalation formulation, wherein the percentage (%) permeation of zero permeability marker is less than 2%.

In yet another embodiment of the present invention, there is provided a method for evaluating permeability of an active agent of an inhalation formulation, wherein the zero permeability marker is sodium fluorescein.

In another embodiment of the present invention, there is provided a method for evaluating permeability of an active agent of an inhalation formulation, wherein the predefined time interval for collection of samples from basal side of monolayer culture of A549 is 2 - 150 minutes.

In an embodiment of the present invention, there is provided a method for evaluating permeability of an active agent of an inhalation formulation, wherein the pre-defined dose of the active agent from the formulation is actuated onto the A549 culture of step (c) using a twin stage impinger.

In another embodiment of the present invention, there is provided a method for evaluating permeability of an active agent of an inhalation formulation, wherein the method further comprises calculating the apparent permeability of the inhalation formulation.

In an embodiment of the present invention, there is provided a method for evaluating permeability of an active agent of an inhalation formulation, wherein the analysis of cell sample is carried out using standard techniques like LC-MS, LC-MS/MS or any other suitable standard techniques known in the art.
In another aspect of the present invention, there is provided a device comprising a cell culture vessel with transwell insert with permeable membrane to separate the vessel into upper apical chamber, and a lower basolateral chamber, and a monolayer culture of A549 cell over the permeable membrane.

It will thus be seen that the objects set forth above, among those made apparent from the preceding description, are efficiently attained, and since certain changes may be made in the assay method and assay for the assessment of the drug deposition and permeability of test drugs set forth without departing from the spirit and scope of the invention, it is intended that all matter contained in the above description and shown in the accompanying drawings shall be interpreted as illustrative and not in a limiting sense. The invention has been described with reference to preferred and alternate embodiments. Modifications and alterations will become apparent to those skilled in the art upon reading and understanding the detailed discussion of the invention provided herein. This invention is intended to include all such modifications and alterations in so far as they come within the scope of the present invention. These and other modifications of the preferred embodiments as well as other embodiments of the invention will be obvious from the disclosure herein, whereby the foregoing descriptive matter is to be interpreted merely as illustrative of the invention and not as a limitation.
EXAMPLES
The present invention illustrated, with the help of following example, which is not intended to limit the scope of the invention and any such modification therein falls within the scope of this invention. It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are intended to provide further explanation of the subject matter.
Example 1
To demonstrate working of the method described herein, inventors have selected pressurized metered dose inhalers (pMDI) formulations of Beclomethasone dipropionate (BDP). BDP was solubilized in an assay buffer (Hanks Blank Salt Solution, HBSS) having different additives wherein the final composition used is HBSS buffer containing 1%v/v HEPES, 1% FBS, 0.1% w/v Vitamin E and 1% of 97% Ethanol.
Standardization of In Vitro Lung Permeability Model
A549 cells were maintained in MEM a media supplemented with 10% FBS at 37°C and 5% CO2. For optimization of the cell seeding density, the cells were seeded in 6-well plate at 1.5×105, 2×105, 2.5×105 and 3×105 cells/mL. After seeding, the plates were cultured at 37 °C, 5 % CO2 for 15 days with a media change every alternate day. The cell morphology was checked under a microscope starting from day 3 till day 15.
Additionally, to elucidate the ability of A549 cells to form tight junctions under air-liquid (A-L) interface, cells at varying densities were seeded to the apical side (A) of the transwell inserts and placed in carrier wells with complete media in the basal side (B). After 24 h of seeding, media from apical side was removed and the cultures were grown under A-L interface. TEER measurements were conducted on day 8, 10, 12 and 15. The TEER values were determined with MERX electrode (Merck, USA). The TEER readings were noted in ohms and expressed as follows: {TEER (?*cm2)} = TEER readings (?) * membrane area (cm2). A zero-permeation marker, sodium fluorescein was used to confirm the formation of integrated tight junctions.
Cell viability at 2.5x105 cells / mL was ensured on days 2, 4, 8, 12 and 15. For cell viability, the cells were harvested with trypsin and viability was assessed using trypan blue dye exclusion method.

The optimization of A549 cell seeding density using various parameters viz, cell enumeration, viability, and morphology was done on days 3, 5, 8, 10, 12 and 15. Morphological evaluation revealed the formation of monolayer at all the cell concentrations from day 3 onwards. Cell viability remained at = 95% for all cell densities from day 4 till day 15. Furthermore, the integrity of cellular monolayer was assessed by measuring TEER on days 8, 10, 12 and 15 and by measuring the permeability of zero permeable dye sodium fluorescein. The TEER values remained over 400 ?*cm2 for all the seeded densities at all the days tested. However, a significant drop in TEER was observed for 1 x 105 cells/ mL from day 10 onwards. The TEER values of 2 x 105 and 2.5 x 105 cells / mL remained stable between 8 to 10 days and dropped thereafter. Sodium fluorescein retention was maximum for 2 x 105 and 2.5 x 105 cells / mL on days 8, 10 and 12 and therefore these cell seeding densities were considered as optimal for performing permeation studies.
In Vitro Cytotoxicity
The toxicity of BDP and 17-BMP on A549 cells was checked by plating the cells in 96 well-plates (2000–5000 cells/well) followed by incubation at 37 °C, 5% CO2 overnight. Post 24 hours incubation, the cells were exposed to BDP, 17-BMP and placebo diluted in appropriate medium at specific concentrations for 2 hour and 4 hour. The exposure time of BDP and 17-BMP was based on time period selected for permeability assessment. After exposure, media containing the active agent was removed and replaced with fresh media. The cells were allowed to recover for 24, 48 and 72 hours. At the end of each recovery phase, cytotoxicity was assessed using MTS: PMS. The formation of coloured tetrazolium product was measured at 490 nm using a Spectramax iD5 (Molecular Device, US) plate reader.
The results demonstrate that the IC50 of BDP was > 5 µg / mL and that of 17-BMP was > 0.1 µg /mL. Maximum toxicity was observed at 5 µg / mL for BDP and 0.1 µg /mL for 17-BMP. However, significant recovery was seen upon incubating the cells for 48 and 72 hours.
Permeability Studies:
Exponentially growing A549 cells were seeded at a density of 2.5 × 105 cells/mL in 6-well transwell plate. Post 24 hours, the medium from the apical compartment was removed and the cells were cultured under A-L interface at 37 °C, 5% CO2 for 8-12 days with a media change every alternate day. TEER values were checked on day 10 and wells with = 400 ?*cm2 were used for permeation study.
For initiating the permeability assay, the media was replaced with modified HBSS buffer. The plate was incubated for 1 hour in the buffer prior to the active agent/drug treatment. For drug deposition, a twin stage impinger (TSI) was modified to accommodate the transwell insert in the second stage. Deposition of drug particles generated by each formulation onto the cell layers and measurement of transepithelial BDP flux was conducted. The assembled TSI was then connected to a rotary vein pump and the flow rate was adjusted to 60 L × min-1. The pressurized meter dose inhaler (pMDI) containing the drug formulations was connected to the TSI and was actuated set number of times (1 actuation / puff with 40 µg / actuation) to allow the equivalent drug deposition on the cell layer. After particle deposition, the transwell was removed and transferred onto a carrier plate. Further, 1 mL of modified HBSS buffer was added to the apical side and mixed by swirling the plate, immediately, 50 µL sample was collected from the apical side as a 0 min sample (A0) and replaced with 50 µL of HBSS buffer. To the basal side of well, 2 ml modified HBSS buffer was added, and the plate was transferred to an incubator shaker with 70-90 rpm at 37 °C. From basal side, 100 µL samples were collected at 5, 10, 15, 30, 45, 60, 90 and 120 mins (The samples were termed as B5, B10, B15, B30, B45, B60, B90 and B120 respectively). Post sample collection at each time point, 100 µL of HBSS buffer was replenished. For mass balance and recovery of actuated drug, acetonitrile wash from TSI apparatus was collected and quantified.
Sample Processing and Analysis by LC-MS/MS
The samples were processed as mentioned previously. The calibration curve was determined by calculation of regression parameters using linear regression and weighing factor of 1/X2. The final concentrations for the samples were obtained using regression equation from the software.
Apparent Permeability (P-app) Calculations
The permeability of the drugs (active agent) was calculated by multipoint method as follows.
P-app = VR x (dQ/dt)/ (A x C0)
Where, VR is the volume of the solution of the receiver well (2 mL for A to B), dQ/dt = (slope of graph of cumulative concentrations in the receiver compartment vs time) x 120 / 7200, A is the area of the cell monolayer (4.5 cm2), C0 (donor concentration) is the initial concentration of the dosing solution in µg.
The cumulative concentrations were determined as follows.
At each time point after withdrawing the samples, equal volume of fluid was replaced with the appropriate volume of the buffer. BDP and 17-BMP concentrations were determined for each sample. The concentrations measured at each time point were used to calculate the cumulative concentrations. The calculations of drugs for different time points in triplicates was done as follows:
The cumulative concentration (Cc) at each time point is equal to the sum of the concentration achieved at that time point (Ct) and 1/20 of the measured concentrations at previous time points, since 100 µL of the 2000 µL sample was withdrawn from the basal side and 100 µL was replaced with buffer at each time point for example.
Cc60 = Ct60
Cc90 = Ct90+Ct60 x 1/20
Cc120 = Ct120 + Ct90x 1/8 +Ct60 x 1/20
These concentrations were used to calculate the apparent permeability (P-app).
For conducting the permeability studies, A549 cells were seeded onto cell culture inserts at a density of 2.5 x 105 cells / mL and cultured under Air Liquid Interface for 8-10 days. Before initiating the assay, TEER measurements were done and only those wells showing values greater than 400 ?*cm2 were selected for the study. Deposition of BDP on the cell monolayer was achieved by placing the insert in the 2nd stage of a modified TSI followed by actuating the pMDI using rotatory pump set at 60 L × min-1. Post drug deposition, samples were collected from the basal compartment at different time points from 5 mins to 120 mins. The samples collected were quantified for both BDP and 17-BMP by a validated LC-MS/MS method. As an approach to ratify the model, the amount of drug deposited, and the rate of drug permeated were compared between the in-house drug-device combination (Test) against Reference. The rate of permeation was elucidated using multi-point method and was expressed as apparent permeability in nm/sec. Identical time vs concentration profile was seen for Test and Reference for BDP and 17-BMP. The average amount of drug deposited post actuation was 3.223 ± 0.26 µg / mL and 2.761 ± 0.39 µg / mL while the apparent permeability was 150.89 ± 23.94 nm/sec and 171.94 ± 12.76 nm/sec for Test and Reference respectively. Recovery of the drug actuated (40 µg / actuation) was confirmed by quantitating the drug deposited in both the stages of TSI (Table 7). Cellular monolayer integrity was confirmed to be above 400 ?*cm2 by TEER measurements post completion of sample collection at the last time point.

Table 1: Amount of dose deposited on the monolayer, drug recovered from the TSI and total percent drug recovery post actuation:
Set Dose deposited Acetonitrile wash Total % recovery
Test (µg) 1 3.51 31.38 34.90 87.25%
2 3.00 35.72 38.72 96.8%
3 3.15 36.81 39.97 99.92%
Reference (µg) 1 2.70 39.77 42.47 106.17%
2 3.17 32.28 35.46 88.65%
3 2.40 36.81 39.21 98.02%

The amount of BDP deposited post actuation was 3.22 ± 0.26 µg/mL and 2.76 ± 0.39 µg/mL and a comparable and linear time vs concentration permeation profile was obtained for BDP and 17-BMP for Test and Reference respectively. Importantly, the calculated apparent permeability of Test and Reference was also akin and was in the range of 150.89 ± 23.94 nm/sec and 171.94 ± 12.76 nm/sec respectively. Importantly, we also estimated the overall drug recovery post actuation by quantifying the drug accumulated in both the stages of the TSI. The recovery ranged from 87.25% to 99.92% for Test and 88.65 % to 106.17% for Reference. ,CLAIMS:1. A method for evaluating permeability of an active agent of an inhalation formulation, the method comprising:
a) simulation of in vivo conditions in ex vivo set-up by producing a monolayer culture of A549 cells grown in an air-liquid interface in a transwell insert in a suitable basal growth medium for a suitable period,
b) optimization of atleast one cell culture parameter selected from cell seeding density, cell proliferation upon culture, cell viability, and cellular monolayer integrity by measuring TEER or quantification of zero permeability marker,
c) placing the transwell insert of step (a) in contact with a buffered medium,
d) actuating a pre-defined dose of the active agent from the formulation onto the A549 culture of step (c),
e) collecting the samples from a buffered medium at predefined time intervals, and
f) analyzing the collected samples from step (e) to determine the amount of the active agent.

2. The method as claimed in claim 1, wherein the active agent is selected from albuterol sulfate, Salbutamol xinafoate, Levalbuterol tartrate, Formoterol fumarate, Ipratropium bromide, Sodium cromoglycate, Beclomethasone dipropionate, Ciclesonide, Flunisolide, Fluticasone propionate, Budesonide, Mometasone furoate, Terbutaline sulfate, Aclidinium bromide, Glycopyrronium bromide, Umeclidinium, vilanterol, Indacaterol, olodaterol and iotropium.

3. The method as claimed in claims 1-2, wherein the active agent is beclomethasone dipropionate (BDP).

4. The method as claimed in claim 1, wherein the cell density is 2 x 105 cells /ml to 2.5 x 105 cells / ml.

5. The method as claimed in claim 1, wherein the cell density is 2 x 105 cells /ml.

6. The method as claimed in claim 1, wherein the cell density is 2.5 x 105 cells /ml.

7. The method as claimed in claim 1, wherein the monolayer culture of A549 cells in a transwell insert in a basal growth medium is formed between 5 days to 15 days.

8. The method as claimed in claim 1, wherein the monolayer culture of A549 cells in a transwell insert in a basal growth medium is formed between 8 days to 10 days.

9. The method as claimed in claim 1, wherein the transepithelial electrical resistance (TEER) is greater than 350 Ocm2.

10. The method as claimed in claim 1, wherein the transepithelial electrical resistance (TEER) is greater than 400 Ocm2.

11. The method as claimed in claim 1, wherein the percentage (%) permeation of zero permeability marker is less than 2%.

12. The method as claimed in claim 1, wherein the zero permeability marker is sodium fluorescein.

13. The method as claimed in claim 1, wherein the pre-defined dose of the active agent from the formulation is actuated onto the A549 culture of step (c) using a twin stage impinger.

14. The method as claimed in claim 1, wherein the method further comprises calculating the apparent permeability of the inhalation formulation.

15. The method as claimed in claim 1, wherein in step (c), the buffered medium is modified HBSS buffer.