Claims:1. A pharmaceutical composition comprising inhalable microparticles comprising hydroxychloroquine (HCQ), aspirin (ASA), a copolymer and at least one surfactant.
2. The pharmaceutical composition as claimed in claim 1, wherein the copolymer is selected from the group comprising of poly(lactic-co-glycolic acid) (PLGA), chitosan, poly(glycolic) acid, poly(lactic) acid, poly(butylcyanoacrylate), polycaprolactone, sodium alginate, hydroxypropyl-ß-cyclodextrin, bovine serum albumin and combinations thereof.
3. The pharmaceutical composition as claimed in claim 1, wherein the at least one surfactant is selected from the group comprising of dipalmitoylphosphatidylcholine (DPPC), phosphatidylcholine (PC), cholesterol (CH), colfosceril palmitate, pumactant, lucinactant and combinations thereof.
4. The pharmaceutical composition as claimed in claim 1, wherein the at least one surfactant is dipalmitoylphosphatidylcholine (DPPC): phosphatidylcholine (PC): cholesterol (CH) in the weight ratio of 4.5:4.5:1.
5. The pharmaceutical composition as claimed in claim 1, wherein the sum of the weights of HCQ and ASA is in the ratio of 1:1.3 with respect to the weight of the copolymer.
6. The pharmaceutical composition as claimed in claim 1, wherein the ratio of HCQ to ASA is 1:15 to 15:1.
7. A method of preparation of a pharmaceutical composition comprising inhalable microparticles comprising hydroxychloroquine, aspirin, a copolymer and at least one surfactant by double emulsion solvent evaporation and freeze drying.
8. The method as claimed in claim 7, wherein the inhalable microparticles are prepared by the steps comprising:
i. dissolving hydroxychloroquine and aspirin in an aqueous solvent to generate solution A;
ii. dissolving the copolymer and the at least one surfactant in an organic solvent to generate solution B;
iii. adding the solution A to the solution B with constant stirring to produce a primary emulsion;
iv. adding the primary emulsion to polyvinyl alcohol with constant stirring to produce a secondary emulsion with dispersed microparticles; and
iv. freeze-drying the secondary emulsion to give the inhalable microparticles.
9. The method as claimed in claim 8, wherein the aqueous solvent is selected from water, ethanol, methanol, propanol, or isopropyl alcohol.
10. The method as claimed in claim 8, wherein the organic solvent is selected from dichloromethane, tetrahydrofuran, ethyl acetate, acetone, hexane, or acetonitrile.

Description:FIELD OF INVENTION
[0001] The present disclosure relates to inhalable pharmaceuticals. Specifically, the present disclosure relates to a composition comprising inhalable microparticles comprising hydroxychloroquine, aspirin, a copolymer and at least one surfactant. The present disclosure further relates to a method for preparing a composition comprising inhalable microparticles comprising hydroxychloroquine, aspirin, a copolymer and at least one surfactant.

BACKGROUND OF THE INVENTION
[0002] Background description includes information that may be useful in understanding the present invention. It is not an admission that any of the information provided herein is prior art or relevant to the presently claimed invention, or that any publication specifically or implicitly referenced is prior art.
[0003] Respiratory diseases have made a huge impact on human life in the past. This includes, SARS, MERS and the recent coronavirus. Coronavirus (CoV) is an enveloped single-stranded RNA virus that is diversely found in humans and wildlife. A total of six species have been identified to cause disease in humans. They are known to infect the neurological, respiratory, enteric, and hepatic systems of the body. The world is currently witnessing the emergence of an outbreak due to a new strain called the SARS-CoV-2 virus initially presented as pneumonia. SARS-CoV-2 is highly contagious and has resulted in a pandemic of COVID-19 (Corona virus disease-2019). As the number of cases continues to rise, it is clear that these viruses pose a threat to public health. COVID-19 is a public health emergency of international concern. Patients with severe COVID-19 present with symptoms like severe pneumonia, acute respiratory distress syndrome (ARDS), sepsis, or septic shock. At the time of filing this application, there is no known specific, effective and proven pharmacological treatment for the disease.
[0004] Lung is a highly absorptive tissue with a large surface area and limited proteolytic activity, sufficient capillaries, and rapid absorption rate. Inhalation therapy has been used in the past for several pulmonary ailments. It requires potentially lower total dose of medication compared to other treatments and consequently lowers the production costs. The ability to deliver the therapeutic agents directly to the site of action also improves the therapeutic efficacy for many specific diseases of the respiratory tract and has numerous advantages over other routes of administration. Traditional methods of preparation of the pharmaceuticals involve spray drying techniques. However, the production of such inhalable drugs may not always be simple. The particles have to be of suitable aerodynamic size, have good flow properties and must be stable.
[0005] Therefore, there is a need to develop inhalable pharmaceuticals that can overcome the deficiencies of the known arts and provide targeted delivery of the active ingredients.

OBJECTS OF THE INVENTION
[0006] An object of the present disclosure is to provide inhalable pharmaceuticals that satisfy the existing needs, as well as others, and generally overcome the deficiencies found in the prior art.
[0007] Another object of the present disclosure is to provide a pharmaceutical composition comprising inhalable microparticles comprising hydroxychloroquine, aspirin, a copolymer and at least one surfactant.
[0008] An object of the present disclosure is to provide a pharmaceutical composition suitable for management of acute respiratory disease and cardiovascular complications associated with COVID and SARS.
[0009] Yet another object of the present disclosure is to provide a method of preparation of a pharmaceutical composition comprising inhalable microparticles comprising hydroxychloroquine, aspirin, a copolymer and at least one surfactant.
[0010] Still another object of the present disclosure is to provide a pharmaceutical composition comprising inhalable microparticles with optimized parameters like aerodynamic size.
[0011] Another object of the present disclosure is to provide a pharmaceutical composition comprising inhalable microparticles and its quality-by-design optimization.
SUMMARY OF THE INVENTION
[0012] This summary is provided to introduce a selection of concepts in a simplified form that are further described below in Detailed Description section. This summary is not intended to identify key features or essential features of the claimed subject matter, nor is it intended to be used as an aid in determining the scope of the claimed subject matter.
[0013] The present disclosure relates to inhalable microparticles suitable for respiratory conditions and diseases like SARS and coronavirus.
[0014] In an aspect, the present disclosure relates to a pharmaceutical composition comprising inhalable microparticles comprising hydroxychloroquine (HCQ), aspirin (ASA), a copolymer and at least one surfactant.
[0015] In an embodiment, the at least one surfactant may be selected from the group comprising of dipalmitoylphosphatidylcholine (DPPC), phosphatidylcholine (PC), cholesterol (CH), colfosceril palmitate, pumactant, KL-4, Venticute, lucinactant and combinations thereof.
[0016] In an embodiment, the copolymer may be selected from the group comprising of poly(lactic-co-glycolic acid) (PLGA), chitosan, poly(glycolic) acid, poly(lactic) acid, poly(butylcyanoacrylate), polycaprolactone, sodium alginate, hydroxypropyl-ß-cyclodextrin, bovine serum albumin and combinations thereof.
[0017] In an embodiment, hydroxychloroquine and aspirin may be present in the weight ratio ranging from about 15:1 to about 1:15. Hydroxychloroquine and aspirin constitute active pharmaceuticals of the present invention.
[0018] In an embodiment, the active pharmaceuticals may be present in the weight ratio of 1:1.3 with respect to the copolymer i.e., the sum of the weights of HCQ and ASA is in the ratio of 1:1.3 with respect to the weight of the copolymer.
[0019] In another aspect, the present disclosure relates to a method of preparation of the inhalable microparticles via double emulsion solvent evaporation technique and freeze drying.
[0020] In yet another aspect, the present disclosure relates to a method of preparation of optimized freeze dried inhalable microparticles via central composite design (CCD) layout.
[0021] In another aspect, the present disclosure relates to a method of preparation of a pharmaceutical composition comprising inhalable microparticles comprising hydroxychloroquine, aspirin, a copolymer and at least one surfactant.
[0022] Various objects, features, aspects and advantages of the inventive subject matter will become more apparent from the following detailed description of preferred embodiments

BRIEF DESCRIPTION OF DRAWINGS OF THE INVENTION
[0023] The following drawings form part of the present specification and are included to further illustrate aspects of the present disclosure. The disclosure may be better understood by reference to the drawings in combination with the detailed description of the specific embodiments presented herein.
[0024] Figure 1 represents the contour plot (2D) showing the effect of independent variables- surfactant and API:PLGA-on Daero (µm) of freeze dried inhalable microparticles (Y1).
[0025] Figure 2 represents the contour plot (2D) showing the effect of independent variables- stirring speed and API:PLGA-on Daero (µm) of freeze dried inhalable microparticles (Y1).
[0026] Figure 3 represents the contour plot (2D) showing the effect of independent variables- stirring speed and surfactants- on Daero (µm) of freeze dried inhalable microparticles (Y1).
[0027] Figure 4 represents the response surface plot (3D) showing the effect of independent variables-surfactants and API:PLGA- on Daero (µm) of freeze dried inhalable microparticles (Y1).
[0028] Figure 5 represents the response surface plot (3D) showing the effect of independent variables-stirring speed and API:PLGA- on Daero (µm) of freeze dried inhalable microparticles (Y1).
[0029] Figure 6 represents the response surface plot (3D) showing the effect of independent variables-stirring speed and surfactants on Daero (µm) of freeze dried inhalable microparticles (Y1).
[0030] Figure 7 represents the contour plot (2D) showing the desirability function of freeze dried inhalable microparticles.
[0031] Figure 8 represents the response surface plot (3D) showing the desirability function of freeze dried inhalable microparticles.

DETAILED DESCRIPTION
[0032] The following is a detailed description of embodiments of the disclosure. The embodiments are in such detail as to clearly communicate the disclosure. However, the amount of detail offered is not intended to limit the anticipated variations of embodiments; on the contrary, the intention is to cover all modifications, equivalents, and alternatives falling within the spirit and scope of the present disclosure as defined by the appended claims.
[0033] All publications herein are incorporated by reference to the same extent as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference. Where a definition or use of a term in an incorporated reference is inconsistent or contrary to the definition of that term provided herein, the definition of that term provided herein applies and the definition of that term in the reference does not apply.
[0034] Reference throughout this specification to “one embodiment” or “an embodiment” means that a particular feature, structure or characteristic described in connection with the embodiment is included in at least one embodiment. Thus, the appearances of the phrases “in one embodiment” or “in an embodiment” in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.
[0035] In some embodiments, the numbers expressing quantities of ingredients, properties such as concentration, reaction conditions, and so forth, used to describe and claim certain embodiments of the invention are to be understood as being modified in some instances by the term “about.”Accordingly, in some embodiments, the numerical parameters set forth in the written description and attached claims are approximations that can vary depending upon the desired properties sought to be obtained by a particular embodiment. In some embodiments, the numerical parameters should be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of some embodiments of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as practicable. The numerical values presented in some embodiments of the invention may contain certain errors necessarily resulting from the standard deviation found in their respective testing measurements.
[0036] As used in the description herein and throughout the claims that follow, the meaning of “a,” “an,” and “the” includes plural reference unless the context clearly dictates otherwise. Also, as used in the description herein, the meaning of “in” includes “in” and “on” unless the context clearly dictates otherwise.
[0037] Unless the context requires otherwise, throughout the specification which follow, the word “comprise” and variations thereof, such as, “comprises” and “comprising” are to be construed in an open, inclusive sense that is as “including, but not limited to.”
[0038] The recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g. “such as”) provided with respect to certain embodiments herein is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the invention.
[0039] Groupings of alternative elements or embodiments of the invention disclosed herein are not to be construed as limitations. Each group member can be referred to and claimed individually or in any combination with other members of the group or other elements found herein. One or more members of a group can be included in, or deleted from, a group for reasons of convenience and/or patentability. When any such inclusion or deletion occurs, the specification is herein deemed to contain the group as modified thus fulfilling the written description of all Markush groups used in the appended claims.
[0040] The description that follows, and the embodiments described therein, is provided by way of illustration of an example, or examples, of particular embodiments of the principles and aspects of the present disclosure. These examples are provided for the purposes of explanation, and not of limitation, of those principles and of the disclosure.
[0041] It should also be appreciated that the present disclosure can be implementedin numerous ways, including as a system, a method or a device. In this specification, these implementations, or any other form that the invention may take, may be referred to as processes. In general, the order of the steps of the disclosed processes may be altered within the scope of the invention.
[0042] The headings and abstract of the invention provided herein are for convenience only and do not interpret the scope or meaning of the embodiments.
[0043] The following discussion provides many example embodiments of the inventive subject matter. Although each embodiment represents a single combination of inventive elements, the inventive subject matter is considered to include all possible combinations of the disclosed elements. Thus if one embodiment comprises elements A, B, and C, and a second embodiment comprises elements B and D, then the inventive subject matter is also considered to include other remaining combinations of A, B, C, or D, even if not explicitly disclosed.
[0044] Various terms as used herein are shown below. To the extent a term used in a claim is not defined below, it should be given the broadest definition persons in the pertinent art have given that term as reflected in printed publications and issued patents at the time of filing.
[0045] The ‘aerodynamic diameter (da)’ is the diameter of the unit density sphere that has the same settling velocity as the particle.
[0046] The present disclosure relates to inhalable microparticles suitable for respiratory conditions and diseases. The inhalable particles comprise hydroxychloroquine and aspirin as actives. Without being bound to theory, Hydroxychloroquine (HCQ) has been demonstrated to have an anti SARS-CoV activity in vitro. (Awadhesh Kumar Singh, et al 2020). HCQ inhibits receptor binding and membrane fusion and is likely to attenuate the severe progression of COVID-19 by inhibiting the cytokine storm by reducing CD154 expression in T-cells.
[0047] Aspirin (ASA) showed a considerable antiviral activity against CA9, HRV1A, HRV2 and substantial activity against FluA H1N1, HRV14 and HRV39 (Bernadette et al., 2017).
[0048] In an embodiment, the present disclosure relates to a pharmaceutical composition comprising inhalable microparticles comprising hydroxychloroquine (HCQ), aspirin(ASA), a copolymer and at least one surfactant.
[0049] In an embodiment, hydroxychloroquine and aspirin may be present in the ratio ranging from about 15:1 to about 1:15, preferably about 1:10. HCQ and ASA together are the active pharmaceuticals.
[0050] In an embodiment, the at least one surfactant may be selected from the group comprising of dipalmitoylphosphatidylcholine (DPPC), phosphatidylcholine (PC), cholesterol (CH), Colfosceril palmitate, Pumactant, KL-4, Venticute, Lucinactant and combinations thereof. Pulmonary surfactants are surface-active lipoprotein complexes (phospholipoprotein). As a medication, pulmonary surfactants are on the WHO Model List of Essential Medicines, the most important medications needed in a basic health system (WHO Model List of Essential Medicines, 2015).
[0051] In a preferred embodiment, the at least one surfactant may be a combination of dipalmitoylphosphatidylcholine (DPPC), phosphatidylcholine (PC), and cholesterol (CH). More preferably, the surfactants may be in a ratio of 4.5:4.5:1.
[0052] In an embodiment, the surfactant may be present in the weight by volume percentage of about 0.30 to about 3.00, preferably it may be present in the weight by volume percentage of 0.32.
[0053] In an embodiment, the copolymer may be selected from the group comprising of poly(lactic-co-glycolic acid) (PLGA), chitosan, poly(glycolic) acid, poly(lactic) acid, poly(butylcyanoacrylate), polycaprolactone, sodium alginate, hydroxypropyl-ß-cyclodextrin, bovine serum albumin and combinations thereof. Preferably the copolymer is PLGA. PLGA is biocompatible and biodegradable, has tunable mechanical properties and most importantly, is a FDA approved polymer.
[0054] In a preferred embodiment, the active pharmaceuticals may be present in the weight ratio of 1:1.3 with respect to the copolymer, i.e., the sum of the weights of HCQ and ASA may be in a ratio of 1:1.3 to the weight of the copolymer.
[0055] In an embodiment, the pharmaceutical composition may be in the dosage form, including but not limited to a tablet, capsule, powder, lozenge, semi-solid, liquid, suspension, solution, emulsion or aerosol.
[0056] In an embodiment, the pharmaceutical composition comprising inhalable microparticles is administered via inhalation through an inhaler. Specifically the inhaler may be a dry powder inhaler. DPIs provide an environmentally suitable drug delivery method. The drug is released at the direct site of action thereby providing better efficacy and evading the complications of bioavailability or dissolution in blood.
[0057] In an embodiment, lower aerodynamic diameter (aerodynamic size) gives deeper penetration. It is favored by increase in stirring speed, amount of surfactant and PLGA:API.
[0058] In an embodiment, the present disclosure relates to inhalable microparticles that require potentially lower total dose of medication compared to conventional treatments and consequently lowers the costs.
[0059] In another embodiment, the present disclosure relates to a method of preparation of the inhalable microparticles via double emulsion solvent evaporation and freeze drying techniques.
[0060] In an embodiment, the present disclosure relates to a method of preparation of optimized freeze dried inhalable microparticles via central composite design (CCD) layout. The freeze dried inhalable microparticles are porous particles with optimized aerodynamic size.
[0061] In an embodiment, the present disclosure relates to a method of preparation of a pharmaceutical composition comprising inhalable microparticles comprising hydroxychloroquine, aspirin, a copolymer and at least one surfactant.
[0062] In an embodiment, the method of preparation of the inhalable microparticles comprises:
i. dissolving hydroxychloroquine and aspirin in an aqueous solvent to generate solution A;
ii. dissolving copolymer and at least one surfactant in an organic solvent to generate solution B;
iii. adding the solution A to the solution B with constant stirring to produce a primary emulsion;
iv. adding the primary emulsion to polyvinyl alcohol with constant stirring to produce a secondary emulsion with dispersed microparticles; and
iv. freeze-drying the secondary emulsion to give the inhalable microparticles.
[0063] In an embodiment, the aqueous solvent may be selected from water, or simple alcohols like ethanol, methanol, propanol, isopropyl alcohol or combinations thereof. Preferably the aqueous solvent is a mixture of ethanol and water.
[0064] In an embodiment, the organic solvent may be selected from dichloromethane, tetrahydrofuran, ethyl acetate, acetone, hexane, or acetonitrile.
[0065] In an embodiment, stirring may be performed on a magnetic stirrer. Stirring may be performed at a speed of about 600 rpm to about 2300 rpm, preferably it may be performed at a speed of about 1762 rpm.
[0066] In an embodiment, the freeze-dried inhalable microparticles obtained from the process possess low density and aerodynamic size suitable for lung deposition and retention.
[0067] In an embodiment, the pharmaceutical composition of the present invention may be used in the treatment of patients with acute respiratory distress syndrome, cardiovascular complications associated with COVID and SARS or any other severe respiratory disease or condition.
[0068] In an embodiment, the present disclosure relates to a method of treatment, prophylaxis or amelioration of respiratory diseases or conditions in a subject by administering the pharmaceutical composition.
[0069] While the foregoing describes various embodiments of the disclosure, other and further embodiments of the disclosure may be devised without departing from the basic scope thereof. The scope of the invention is determined by the clainhalable microparticles that follow. The invention is not limited to the described embodiments, versions or examples, which are included to enable a person having ordinary skill in the art to make and use the invention when combined with information and knowledge available to the person having ordinary skill in the art.
[0070] The present invention is further explained in the form of following examples. However, it is to be understood that the following examples are merely illustrative and are not to be taken as limitations upon the scope of the invention.
Example 1: Preparation of freeze-dried microparticles
[0071] HCQ and ASA (1:10) were dissolved in a mixture of ethanol: distilled water (1:1) to give the aqueous phase. Organic phase consisted of PLGA and the combination of surfactants- dipalmitoylphosphatidylcholine (DPPC): phosphatidylcholine (PC): cholesterol (CH) (in the ratio of 4.5: 4.5: 1) dissolved in dichloromethane (DCM). Aqueous solution was slowly added through a syringe to organic phase with continuous magnetic stirring (REMI, India) to produce primary emulsion. Further, primary emulsion was slowly added to 0.5% aqueous polyvinyl alcohol (PVA) with continuous magnetic stirring for 2 hours to produce fine dispersion of microparticles succeeded by freeze drying at - 55oC and 0.5 kPa (vacuum) for 24 hrs using lactose as cryoprotectant and carrier for dry powder inhalation to generate fine, porous powder having very less density (0.25 g/cm3).
EXAMPLE 2: Composition and Central composite design (CCD) layout for production of freeze dried inhalable microparticles
Table 1: Independent and response variables for freeze dried inhalable microparticles
Independent variables -1.68 (Axial) -1 (Low) 0 (Medium) +1 (High) +1.68 (Axial)
X1= API: PLGA (w/w) 1:1.32 1:2 1:3 1:4 1:4.68
X2 = Surfactants (% w/v) 0.32 1 2 3 3.68
X3= Stirring speed (rpm) 660 1000 1500 2000 2340
Response variables Constraint Importance
Y1 = Daero (µm) Minimize +++++
wherein Daero is the aerodynamic diameter

Table 2: Central composite design (CCD) layout for freeze dried inhalable microparticles
Batch X1[API:PLGA (w/w)] X2[Surfactants (% w/v)] X3 [Stirring speed (rpm)]
1 -1 -1 -1
2 1 -1 -1
3 -1 1 -1
4 1 1 -1
5 -1 -1 1
6 1 -1 1
7 -1 1 1
8 1 1 1
9 -1.68 0 0
10 1.68 0 0
11 0 -1.68 0
12 0 1.68 0
13 0 0 -1.68
14 0 0 1.68
15 0 0 0
16 0 0 0
17 0 0 0
18 0 0 0
19 0 0 0
20 0 0 0

[0072] Determination of aerodynamic diameter (Daero) (Y1) of freeze dried inhalable microparticles (batch 1-20)
The performance of inhalable particles largely depends on aerodynamic diameter, a parameter which is based on the particle size and density. The particle size of the microparticles was determined using an optical microscopy method. Approximately 100 microparticles were taken on a glass slide and particle size measured using a calibrated optical microscope (Erma, 23 Tokyo, Japan) equipped under regular polarized light. The aerodynamic diameter (Daero) can be expressed as follows:

Where, ?1 = 1 g/cm3, ?=particle density, d = Stokes’s diameter

EXAMPLE 3: Statistical analysis of aerodynamic diameter (Daero) (µm) (Y1) of freeze dried inhalable microparticles using quality by design (QbD)
[0073] On the basis of regression coefficient (R²), F-value and p-value calculated by Design-Expert software (Trial Version 12.0.9.0, Stat-Ease Inc., MN), quadratic model was suggested for Y1. The R², F-value and p-value for Daero (µm) were 0.9763, 45.82 and < 0.0001 (Keyhaneh et al., 2016). Therefore, second order polynomial model was generated by multiple regression analysis using Design-Expert software.
Eq. (1)
Where, Y denotes observed response variable and ß0 is constant coefficient. ßi, ßii and ßij represents coefficients of linear, quadratic parameter and interaction parameters, respectively. Xi represents average outcome of changing one variable at a time from low to high, polynomial terms Xi2 was employed to assess non-linearity effect of variable Xi, interaction terms XiXj illustrated how the response transforms when two variables Xi and Xj were altered concurrently. Aerodynamic diameter (Daero) (µm) (Y1) of freeze dried inhalable microparticles was synergistically influenced by API: PLGA (w/w) (X1) whereas antagonistically affected by surfactants (X2) and stirring speed (X3) which can be elucidated by following polynomial quadratic equation:
Daero=3.33+1.04X1-0.479X2-0.497X3-0.096X1X2+0.376X1X3+0.344X2X3+0.14X1²-0.146X2²-0.42X3² Eq. (1)
Table 3: Model Summary Statistics
Source Std. Dev. R² Adjusted R² Predicted R² PRESS
Linear 0.6198 0.7756 0.7335 0.6040 10.84
2FI 0.5543 0.8542 0.7868 0.6600 9.31
Quadratic 0.2903 0.9692 0.9415 0.7979 5.53 Suggested
Cubic 0.1543 0.9948 0.9835 0.9770 0.6308 Aliased

Table 4: Lack of Fit Tests
Source Sum of Squares df Mean Square F-value p-value
Linear 6.00 11 0.5459 19.36 0.0021
2FI 3.85 8 0.4816 17.08 0.0031
Quadratic 0.7018 5 0.1404 4.98 0.0514 Suggested
Cubic 0.0019 1 0.0019 0.0689 0.8035 Aliased
Pure Error 0.1410 5 0.0282

Table 5: ANOVA for Quadratic model for Daero (µm) (Y1)
Source Sum of Squares df Mean Square F-value p-value
Model 26.54 9 2.95 34.99 < 0.0001 Significant
A-API: PLGA (w/w) 14.73 1 14.73 174.80 < 0.0001
B-Surfactants (% w/v) 3.13 1 3.13 37.18 0.0001
C-Stirring speed (rpm) 3.37 1 3.37 40.03 < 0.0001
AB 0.0741 1 0.0741 0.8795 0.3704
AC 1.13 1 1.13 13.44 0.0043
BC 0.9453 1 0.9453 11.22 0.0074
A² 0.2736 1 0.2736 3.25 0.1017
B² 0.3107 1 0.3107 3.69 0.0838
C² 2.47 1 2.47 29.28 0.0003
Residual 0.8427 10 0.0843
Lack of Fit 0.7018 5 0.1404 4.98 0.0514 not significant
Pure Error 0.1410 5 0.0282
Cor Total 27.38 19

[0074] Table No. 3 was obtained through statistical design expert software for selection of model in which aerodynamic size data of 20 batches of freeze dried inhalable microparticles gets best fitted on the basis of R2 values and PRESS. Table 4 was obtained through statistical design expert software for selection of model in which aerodynamic size data of 20 batches of freeze dried inhalable microparticles gets best fitted on the basis of lack of fit p-value (p-value should be >0.05). Table 4 was obtained through statistical design expert software for ANOVA for examining the quadratic model fitting and independent variables having significant effect on Y1 (Daero) (p<0.05).
[0075] The Model F-value of 34.99 implies the model is significant. There is only a 0.01% chance that an F-value this large could occur due to noise. P-values less than 0.0500 indicate model terms are significant. The lack of fit F-value of 4.98 and lack of fit P-value > 0.05 implies best fitting of model. The Predicted R² of 0.7979 is in reasonable agreement with the Adjusted R² of 0.9415; i.e. the difference is less than 0.2. Therefore, statistical analysis of Y1 can be used to navigate the design space.
Example 4: Response surface analysis of aerodynamic diameter (Daero) (µm) (Y1) of freeze dried inhalable microparticles using quality by design (QbD)
[0076] Figure 1 represents the contour plot (2D) showing the effect of independent variables- surfactant and API:PLGA-on Daero (µm) of freeze dried inhalable microparticles (Y1).
[0077] Figure 2 represents the contour plot (2D) showing the effect of independent variables- stirring speed and API:PLGA-on Daero (µm) of freeze dried inhalable microparticles (Y1).
[0078] Figure 3 represents the contour plot (2D) showing the effect of independent variables- stirring speed and surfactants- on Daero (µm) of freeze dried inhalable microparticles (Y1).
[0079] Figure 4 represents the response surface plot (3D) showing the effect of independent variables-surfactants and API:PLGA- on Daero (µm) of freeze dried inhalable microparticles (Y1).
[0080] Figure 5 represents the response surface plot (3D) showing the effect of independent variables-stirring speed and API:PLGA- on Daero (µm) of freeze dried inhalable microparticles (Y1).
[0081] Figure 6 represents the response surface plot (3D) showing the effect of independent variables-stirring speed and surfactants on Daero (µm) of freeze dried inhalable microparticles (Y1).
[0082] Figures 1 & 4 explain effect of surfactant and API: PLGA on Daero (µm) of freeze dried inhalable microparticles (Y1). As amount of surfactant increases, Daero (µm) decreases. As amount of API: PLGA increases, Daero (µm) increases.
[0083] Figures 2 & 5 explain effect of stirring speed and API: PLGA on Daero (µm) of freeze dried inhalable microparticles (Y1). As amount of stirring speed increases, Daero (µm) decreases. As amount of API: PLGA increases, Daero (µm) increases.
[0084] Figures 3 & 6 explain effect of stirring speed and surfactants on Daero (µm) of freeze dried inhalable microparticles (Y1). As amount of surfactants and stirring speed increases, Daero (µm) decreases.
EXAMPLE 5: Characterization and optimization of freeze dried inhalable microparticles
[0085] Optimized composition consists of API: PLGA (w/w) (1:1.32), surfactants (% w/v) (0.32) and stirring speed (rpm) (1762) having Daero (1.09 µm) which has desirability function of 0.837. Figure 7 represents the contour plot (2D) showing the desirability function of freeze dried inhalable microparticles and Figure 8 represents the response surface plot (3D) showing the desirability function of freeze dried inhalable microparticles.
Characterization of optimized freeze dried inhalable microparticles:
5.1 Moisture content:
[0086] The oven-drying process determined the moisture content of the freeze dried microparticles. Accurately measured samples were put in a 102oC heated hot air oven, and weighed up to constant weight on an analytical balance. Measurements of three weight loss were reported on average. The % moisture content of optimized freeze dried inhalable microparticles was found to be < 1%.
5.2 Carr’s index (%)
[0087] The bulk density (?b) and tapped density (?t) values were used to calculate the Carr’s compressibility index (CI) according to the following equation:

[0088] Bulk density (?b) of microparticles was assessed by filling dry microparticles into 5 ml graduated cylinder and the top was leveled. The weight (W) and volume (Vb) occupied by the powder was recorded.

[0089] The tapped density (?t) of the freeze dried powders was evaluated by tapping the cylinder onto a level surface at a height of about 2 cm, until no change in volume is observed. The resultant volume was recorded as Vt. Each measurement was taken in triplicate.

[0090] Carr’s index (%) of optimized freeze dried inhalable microparticles was found to be 11% which indicates its excellent flowability.
[0091] Thus, the inhalable microparticles obtained by the present invention were successfully synthesized and optimized using double emulsion solvent evaporation technique followed by lyophilization and determination of aerodynamic diameter (Daero). The moisture content and Carr’s index (%) of the optimized freeze dried inhalable microparticles were found to be < 1% and 11 %, respectively, indicating low moisture and excellent flowability.
[0092] The foregoing examples are merely illustrative and are not to be taken as limitations upon the scope of the invention. Various changes and modifications to the disclosed embodiments will be apparent to those skilled in the art. Such changes and modifications may be made without departing from the scope of the invention.
ADVANTAGES OF THE PRESENT INVENTION
[0093] The present disclosure provides a pharmaceutical composition comprising inhalable microparticles that satisfies the existing needs, as well as others, and generally overcomes the deficiencies found in the prior art.
[0094] The present disclosure provides a pharmaceutical composition comprising inhalable microparticles with excellent flowability and negligible moisture content.
[0095] The present disclosure provides a pharmaceutical composition comprising inhalable microparticles that has reduced aerodynamic diameter such that it improves lung deposition and retention.
[0096] The present disclosure provides a pharmaceutical composition comprising inhalable microparticles wherein the composition is optimized using centre composite design (CCD) layout to produce porous FDIMs.
[0097] The present disclosure provides a pharmaceutical composition comprising inhalable microparticles wherein the composition is suitable for management of acute respiratory disease and cardiovascular complications associated with COVID and SARS like respiratory diseases.
REFERENCES:
1. 19th WHO Model List of Essential Medicines (April 2015) (PDF). WHO. April 2015. Retrieved May 10, 2015
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3. https://en.wikipedia.org/wiki/Pulmonary_surfactant
4. Keyhaneh Karimi, Edina Pallagi, Piroska Szabó-Révész, Iildikó Csóka, Rita Ambrus, “Development of a microparticle-based dry powder inhalation formulation of ciprofloxacin hydrochloride applying the quality by design approach.” Drug Design, Development and Therapy 2016; 10: 3331-3343.
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