1. A polymeric nanoparticulate formulation of bromelain, comprising drug bromelain, Polymethylmethacrylate (PMMA), lecithin, a surfactant and a synthetic polymer wherein said formulation is capable of being used in the treatment of asthma.

2. The polymeric nanoparticulate formulation as claimed in claim 1 wherein, said formulation comprises: Ingredient Quantity (% Range) Bromelain 7 - 14 Lecithin 10 - 25 PMMA 67 - 81 Surfactant 0.5 Synthetic polymer 1

3. The polymeric nanoparticulate formulation as claimed in claim 1 wherein, said formulation comprises: Ingredient Quantity (% Optimum) Bromelain 10.71 Lecithin 17.86 PMMA 71.43 Surfactant 0.5 Synthetic polymer 1

4. The polymeric nanoparticulate formulation as claimed in claim 1 wherein said synthetic polymer is selected from a group comprising poly(methyl methacrylic acid) and its copolymers, poly(vinyl alcohol), or substituted celluloses such as hydroxyethylcellulose.

5. The polymeric nanoparticulate formulation as claimed in claim 4 wherein said synthetic polymer is poly(vinyl alcohol).

6. The polymeric nanoparticulate formulation as claimed in claim 1 wherein said surfactant is selected from anionic, cationic and non-ionic surfactant.

7. The polymeric nanoparticulate formulation as claimed in claim 6 wherein said surfactant is Sorbitan monooleate (Span 80).

8. The polymeric nanoparticulate formulation as claimed in claim 1 wherein said lecithin is soya lecithin.

9. The polymeric nanoparticulate formulation as claimed in claim 1 wherein an optimum bromelain-PMMA weight ratio is 1:.6.67.

10. The polymeric nanoparticulate formulation as claimed in claim 1 wherein optimum percentage of the soya lecithin in the formulation is 17.86 %.

11. The polymeric nanoparticulate formulation as claimed in claim 1 wherein size of said nanoparticulate in the formulation is in the range of 190.90 ± 7.51 nm.

12. The polymeric nanoparticulate formulation as claimed in claim 1 wherein said formulation has high entrapment efficiency of bromelain in the nanoparticulate being in the range of 81.64 ± 2.87 %.

13. The polymeric nanoparticulate formulation as claimed in claim 1 wherein zeta potential of the nanoparticulate is in the range of -25.30±2.08 mV.

14. The polymeric nanoparticulate formulation as claimed in claim 1 wherein polydispersity index of the nanoparticulate is in the range of 0.14±0.02.

15. The polymeric nanoparticulate formulation as claimed in claim 1 wherein said nanoparticles are analyzed till 12 months and showed retention of bromelain activity up to 97.06±2.94± % under cool temperature of 4 ± 2 ºC at 65 ± 5 % relative humidity (RH) and up to 95.36±3.84 % under room temperature of 25±2ºC at 60 ±5% RH.

16. The polymeric nanoparticulate formulation as claimed in claim 1 wherein said nanoparticles are analyzed till 6 months at accelerated temperature storage conditions of 40 ± 2 ºC at 75 ± 5 % RH and showed retention of bromelain activity of up to 94.77±5.26 %.

17. The polymeric nanoparticulate formulation as claimed in claim 1 wherein t90 of said nanoparticulate formulation is ~ 6.5-folds higher compared to pure drug at room temperature.

18. A process to prepare the polymeric nanoparticulate formulation as claimed in claim 1, said process comprises: (i) preparing an aqueous solution of bromelain and lecithin co-dissolved in Tris-HCl to obtain aqueous phase (W1), (ii) preparing an organic phase of PMMA and a surfactant dissolved in an organic solvent (O), (iii) pouring drop by drop the aqueous solution of step (i) (W1) in organic solvent of step (ii) (O) employing ultrasonic disruptor under predefined conditions to obtain a primary colloidal dispersion, (iv) preparing an aqueous solution of a synthetic polymer (W2), (v) emulsifying said primary colloidal dispersion of step (iii) with aqueous solution of step (iv) (W2) under probe sonication under predefined conditions to form nanoparticulate emulsion (W1/O/W2), (vi) removing said organic solvent by continuous stirring of said nanoparticulate emulsion to obtain organic solvent free double emulsion, (vii) centrifuging said organic solvent free double emulsion at predefined conditions to obtain pellets, (viii) dispersing the said pellets in water with subsequent addition of cryoprotectant to obtain a suspension, (ix) lyophilizing the said suspension under specific conditions by freezing followed by primary drying and secondary drying using lyophilizer to obtain said polymeric nanoparticulate formulation.

19. The process as claimed in claim 18 wherein said organic solvent in step (ii) is selected from the group comprising ethanol, methanol, acetone, tetrachloroethylene, toluene, methyl acetate, dichloromethane, ethyl acetate, cyclohexane, hexane, formamide, chloroform, acetonitrile, benzene and mixtures thereof.

20. The process as claimed in claim 18 wherein said organic solvent is dichloromethane.

21. The process as claimed in claim 18 wherein said surfactant in step (ii) is Span 80.

22. The process as claimed in claim 18 wherein said predefined conditions in step (iii) for emulsification by ultrasonic disruptor are disruption for 7 min over an ice bath.

23. The process as claimed in claim 18 wherein said water soluble synthetic polymer in step (iv) is selected from the group comprising poly(methylmethacrylic acid) and its copolymers, poly(vinyl alcohol), or substituted celluloses such as hydroxyethylcellulose or a combination thereof.

24. The process as claimed in claim 23 wherein said water soluble synthetic polymer is poly(vinyl alcohol) (PVA).

25. The process as claimed in claim 18 wherein said surfactant is selected from anionic, cationic and non-ionic surfactant, preferably the surfactant is a non-ionic surfactant.

26. The process as claimed in claim 18 wherein said predefined conditions in step (v) are emulsifying the primary emulsion for 15 min over an ice bath.

27. The process as claimed in claim 18 wherein said predefined conditions in step (vii) are centrifugation at 30,000 rpm for 25 min at 4 ºC.

28. The process as claimed in claim 18 wherein said cryoprotectant is mannitol 10% w/v.

29. The process as claimed in claim 18 wherein said specific conditions for lyophilization are freezing to -80 °C for 5 h followed by primary drying at -80 °C for 36 h at 0.07 mbar and secondary drying at 20 °C for 8 h at 0.07 mbar.

30. A composition of formulation comprising drug bromelain, Polymethylmethacrylate (PMMA), lecithin, a surfactant and a synthetic polymer with pharmaceutically acceptable excipients or surfactants or carriers or a combination thereof for use in treatment of asthma.

31. The composition of formulation as claimed in claim 30 wherein said composition is in dosage form suitable for oral drug delivery.

32. The composition of formulation as claimed in claim 31 wherein said dosage form is sustained release dosage form selected from tablet, capsules, sachets, powders, granules, pellets, orally dispersible films, ampoules, dispersions, suspension, semi-solids, soft gels. ,

Description:FIELD OF THE INVENTION
The present invention relates to polymeric nanoparticulate formulation of bromelain capable of being used in the treatment of asthma. The present invention also relates to a process of preparation of the polymeric nanoparticulate formulation of bromelain and pharmaceutical compositions comprising thereof.

BACKGROUND OF THE INVENTION
Asthma, a chronic inflammatory respiratory disorder is a polygenic multifactorial autoimmune disease with complex and heterogeneous etiology. It is characterized by multicellular inflammation, bronchospasm, airway obstruction and hyper-responsiveness of airways associated with episodes of wheezing and spasmodic coughing often worsening at night. It is frequently perpetuated in response to specific and nonspecific triggers. Most of pathophysiological changes associated with asthma are contributed by the acute activation of inflammatory cells and their accumulation, elevated expression of helper T cells, dysplasia of goblet cells and airway muscles and alteration in mucus production both quantitatively and qualitatively ensuing reversible or permanent changes in airway wall. The worsening of airways inflammation is also often escorted by excessive production of free radicals, allergen-specific immunoglobulin E (IgE) antibodies and increased infiltration of neutrophils, eosinophils, basophils, leucocytes and alveolar macrophages to release mediators like histamine, prostaglandins, cysteinyl leukotrienes and cytokines. Thus, a cascade of event occurring at molecular level generates oxidative environment which impairs antioxidant defense of lungs and facilitates destruction of macromolecules.

Corticosteroids are first line therapeutics recommended according to guidelines for asthma management due to their anti-inflammatory potential. However, prolonged corticosteroid therapy is associated with several systemic (osteoporosis, skin thinning, reduced bone growth in children, weight gain, upset stomach, cataracts and glaucoma) and local side effects (oropharyngeal candidiasis, dysphonia, reflex cough and pharyngitis). Therefore, search of novel effective alternative therapy is crucially desired for asthma in the clinic from medications spanning across synthetic molecules, molecular inventions and alternatives from natural sources. The growing interest in herbal treatments with fruitful results in experimental and clinical investigations has drawn significant attention of pharmaceutical researchers and scientists to reduce or eliminate the usage of synthetic drugs and inhalers for asthma management.

Bromelain is a mixture of cysteine proteases obtained from stem of pineapple (Ananas comosus). It exhibits numerous therapeutic benefits like mucolytic, wound healing, fibrinolytic, antiedematous, antithrombotic, anti-inflammatory, antioxidant, anticancer as well as immunomodulatory activity. Wider range of therapeutic activity and non-toxic nature of bromelain has increased its acceptance as phytomedicine among pharmaceutical scientists.

Bromelain downregulates COX-2 and PGE-2 expression and reduces cytokines like IL-1ß, IL-6 and TNF-a secretion from immune cells stimulated during inflammatory pathologies. Several reports convincingly demonstrated the efficacy of bromelain in reducing airway reactivity and susceptibility to irritants, decreased markers (CD19+ B cells, CD4+ and CD8+ T lymphocytes) of lung inflammation in ovalbumin induced model of allergic airway disease. The proteolytic activity of bromelain also controls inflammation by proteolytic degradation of advanced glycation end products receptor and cell surface markers regulating lymphocyte homing and migration to inflammatory site. Furthermore, bromelain harmonizes expression of transforming growth factor (TGF)-ß, a paramount regulator of inflammation. However, gastric instability and poor patient compliance due to high oral dose limits its therapeutic potential. The complete release of protein from orally administered carrier systems may aggravate the probability of protein degradation, denaturation or aggregation which can lead to unpredictable prophylactic hypersensitivity or toxic reactions along with loss of therapeutic effect. Henceforth for asthma management, need exist to develop stable and patient compliant oral bromelain therapy.

Eric R. Secor Jr ALTERNATIVE THERAPIES, sept/oct 2012, VOL. 18, NO. 5: 9-17 suggests that bromelain has a therapeutic effect in established allergic airway disease, which may translate into an effective adjunctive therapy in patients with similar conditions, such as allergic asthma, who have chosen to initiate treatment after the onset of symptoms.

Nowadays polymeric nanoparticles have gained more attention as protein carrier system due to their characteristic properties like nano-size and higher surface area facilitating rapid gastric emptying, site-specific controlled delivery, higher cellular uptake, improved bioavailability, reduced first pass metabolism, reduced adverse or toxic effects and improved patient compliance. However, protein delivery is challenging due to its intrinsic property i.e. high molecular weight, polarity and instability. Acrylate polymer like polymethylmethacrylate (PMMA) has gained success in pH dependent controlled delivery of biomolecules via oral route due to poor gastric solubility, higher biocompatibility and non-toxic behavior.

In order to obviate the drawbacks in the existing state of the art, the present invention provides nanoparticulate formulation of bromelain using PMMA as a polymer.

OBJECTS OF THE INVENTION
In order to obviate the drawbacks in the existing state of the art, the main object of the present invention is to provide nanoparticulate formulation of bromelain capable of being used in the treatment of asthma.

Yet another object of the invention is to provide polymeric nanoparticulate formulation of bromelain having site specific controlled delivery, higher cellular uptake and improved patient compliance.

Yet another object of the invention is to provide pharmaceutical compositions comprising the nanoparticulate formulation of bromelain showing sustained release, enhanced therapeutic efficacy, bioavailability and stability.

Yet another object of the invention is to provide a process for the preparation of polymeric nanoparticulate formulation of bromelain for the treatment of asthma.

SUMMARY OF THE INVENTION
It will nevertheless be understood that no limitation of the scope of the invention is thereby intended by way of embodiments and examples. Such alterations and further modifications in the present invention, and such further applications of the principles of the invention as would normally occur to those skilled in the art are to be construed as being within the scope of the present invention.

It will be understood by those skilled in the art that the summary of the invention provided herein is exemplary and 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 those skilled in the art to which this invention belongs. The composition, methods, and examples provided herein are only illustrative and not intended to be limiting.

The terms "comprises", "comprising", or any other variations thereof, are intended to cover a non-exclusive inclusion, such that a process or method that comprises a list of steps does not include only those steps but may include other steps not expressly listed or inherent to such a process or method. Similarly, one or more steps of method or components preceded by "comprises... a" does not, without more constraints, preclude the existence of other, steps or components. Appearances of the phrase "in a preferred embodiment”, “in an embodiment", “in another embodiment” and similar language throughout this specification may, but not necessarily do, all refer to the same embodiment.

The inventors of the present invention conducted strategic experimentation and have surprisingly found that polymeric nanoparticulate formulation of bromelain in acrylate polymer like polymethylmethacrylate (PMAA) provides stable and patient compliant oral therapy for asthma management. The present invention, therefore, provides cost-effective polymeric nanoparticulate formulation of bromelain, a process to prepare thereof and a composition thereof.

Accordingly, one of the aspects of the present invention is to provide polymeric nanoparticulate formulation capable of being used in the treatment of asthma. More specifically, the invention provides polymeric nanoparticulate formulation of bromelain having enhanced therapeutic efficacy, bioavailability and improved drug loading capacity. The formulation of the present invention provides site specific controlled delivery, higher cellular uptake with reduced adverse effects and improved patient compliance.

The polymeric nanoparticulate formulation of the present invention comprises bromelain, PMMA, lecithin, a surfactant and a synthetic polymer.

In a preferred embodiment of the invention, the active ingredient having therapeutic effect for the treatment of asthma is bromelain.

The nanoparticles of the present invention have particle size in the range of 190.90 ± 7.51 nm, zeta potential ranging from -25.30 ± 2.08 mV and drug loading capacity of 81.64 ± 2.87%.

In second aspect, the present invention provides a process for the preparation of polymeric nanoparticulate by double emulsion solvent evaporation method.

In a non-limiting embodiment, the invention provides a process for the preparation of polymeric nanoparticulate formulation comprising the steps of:
a) preparing an aqueous solution of bromelain and soya lecithin co-dissolved in Tris-HCl (W1);
b) preparing an organic phase comprising PMMA and a pharmaceutically acceptable surfactant dissolved in an organic solvent (O);
c) pouring drop by drop the aqueous solution of step (a)(W1) in organic phase of step (b) (O) to form a primary emulsion;
d) emulsifying the primary emulsion formed in step c) with water soluble synthetic polymer to obtain double emulsion (W1/O/W2).

The organic solvent suitable for the process is selected from the group comprising ethanol, methanol, acetone, tetrachloroethylene, toluene, methyl acetate, dichloromethane, ethyl acetate, cyclohexane, hexane, formamide, chloroform, acetonitrile, benzene and mixtures thereof.

The water soluble synthetic polymer suitable for the process is selected from a group comprising poly(meth)acrylic acid and its copolymers, poly(vinyl alcohol), or substituted celluloses such as hydroxyethylcellulose. In a preferred embodiment the water soluble synthetic polymer is poly(vinyl alcohol).

The surfactant suitable for the process is selected from anionic, cationic and non-ionic surfactant. In a preferred embodiment the surfactant is a non-ionic surfactant. In another preferred embodiment, the surfactant is Sorbitan monooleate (Span 80).

Third aspect of the present invention is to provide polymeric nanoparticulate compositions of the present invention comprising bromelain, PMMA, lecithin, a surfactant and a synthetic polymer such as polyvinyl alcohol (PVA) with pharmaceutically acceptable surfactants or excipients or carriers or any combination thereof.

In one embodiment, the pharmaceutical composition is in the dosage form suitable for oral drug delivery.

In another embodiment the dosage form is selected from tablet, capsules, sachets, powders, granules, pellets, orally dispersible films, ampoules, dispersions, suspension, semi-solids, soft gels, etc.

In another embodiment the dosage form is a sustained release dosage form.

Non-limiting Examples:
a) Preparation of bromelain nanoparticles
A mixture of bromelain and lecithin is co-dissolved in Tris-HCl buffer to obtain aqueous phase. The aqueous phase is emulsified with organic phase employing ultrasonic disruptor to obtain primary colloidal dispersion. The organic phase comprises PMMA, DCM and Span 80. The primary colloidal dispersion is further emulsified with PVA under probe sonication. The resultant double emulsion (W1/O/W2) was stirred to remove organic solvent and centrifuged to remove unencapsulated bromelain and unabsorbed surface-active agents. The purified pellets are dispersed in cryoprotectant, and the resultant suspension is lyophilized to obtain the bromelain nanoparticles.
b) Characterization of bromelain nanoparticles: Morphological evaluation of the nanoparticulate formulation is executed employing Field emission scanning electron microscopy.
The suspension is evaluated for key indicators, including but not limited to, particle size, poly dispersibility index (PDI), zeta potential, and entrapment efficiency.

BRIEF DESCRIPTION OF DRAWINGS
Figure 1: Representation of data (A) Scanning electron microscopy photograph of optimized formulation (A9), (B) ATR FTIR spectra, (C) XRD diffractogram and (D) DSC thermogram of bromelain, polymethyl methacrylate, soya lecithin and optimized formulation, respectively.
Figure 2: Fluorescence spectra of (A) pure bromelain and bromelain obtained after lysis of Br-LPHNs, respectively; (B) pure drug and bromelain recovered from Br-LPHNs incubated at pH 1.2 and 6.8, respectively.
Figure 3: In vitro cumulative release profile of bromelain from optimized Br-LPHNs in pH progressive dissolution media.
Figure 4. Effect of (A) different concentration (0.1 – 0.4 % w/w) of pure drug on viscosity of artificial mucous with progression of time; (B) pure drug (0.2 % w/w) and Br-LPHNs (equivalent to 0.2 % w/w bromelain) on mucous viscosity at different time interval respectively.
Figure 5: Plasma drug concentration time profile of pure drug and optimized formulation (A9).
Figure 6: Comparative biodistribution study of pure bromelain and optimized formulation in different organs lung, liver, intestine and kidney respectively at different time interval.
Figure 7: Effect of different treatments on histamine induced acute bronchospasm (A) Bronchospasm onset time, (B) Percentage protection, (C) Convulsion period, (D) Percent recovery time respectively and (E) Effect of different doses of formulation on bronchospasm onset and % protection respectively.
Figure 8. Effect of bromelain, standard drug (theophylline) and optimized formulation on level of oxidative stress markers i.e. (A) LPO level and (B) carbonylated protein content in lung, liver and trachea respectively induced by histamine exposure.
Figure 9: Effect of different treatments on hematological parameters like (A) Total leukocyte count, (B) Neutrophils and (C) Lymphocyte level in acute asthma model induced by histamine exposure.
Figure 10: Effect of different treatment on (A) Bronchospasm onset time, recovery time and convulsion period; and (B) Percentage protection provided in ovalbumin induced asthma model respectively.
Figure 11: Effect of different treatments on weight of lung of guinea pigs having ovalbumin induced asthma.
Figure 12: Effect of different treatments on hematological parameters like (A) Hemoglobin, (B) Total leukocyte count, (C) Eosinophil, (D) Neutrophil and lymphocyte level, respectively in ovalbumin induced asthma model.
Figure 13: Effect of different treatments on the level of oxidative stress markers in ovalbumin induced asthma model (A) LPO level, (B) Carbonylated protein content, (C) Myeloperoxide (MPO) level, (D) Nitric oxide (NO) (%), (E) Catalase (CAT) activity (%), (F) Glutathione (GSH) level (%) and (G) Superoxide dismutase level (SOD) in lung, liver, spleen, trachea and BALF respectively; (H) TNF – a, (I) IL-5, (J) Ig-G level in serum and BALF (K) Eosinophil count and (L) BAL total cell count.
Figure 14: Histopathological images of lung of (A) Naïve group, (B) ovalbumin sensitized group, (C) Pure drug treated, (D) Chlorpheniramine treated and (E) optimized formulation treated respectively.
Figure 15: Histopathological images of liver of (A) Naïve group, (B1 and B2) ovalbumin sensitized group, (C) Pure drug treated, (D) Chlorpheniramine treated and (E) optimized formulation treated guinea pigs respectively.
Figure 16: Histopathological images of guinea pig lung in (A) Naïve group (B) Bromelain treated (C) Formulation treated; and liver in (D) Naïve group (E) Bromelain treated (F) Formulation treated group during chronic toxicity studies.
Figure 17: Effect of bromelain and optimized formulation at equivalent dose of 40 mg/kg bromelain over biochemical markers (A) SGOT and SGPT (B) Urea (C) Creatinine during chronic toxicity studies.

DETAILED DESCRIPTION OF THE INVENTION WITH NON-LIMITING EMBODIMENTS AND ILLUSTRATIONS
Poly methyl (methacrylate) (100.12 g/mol), bromelain, casein, tyrosine and trichloro acetate (98.0%) (Himedia laboratory pvt. Ltd, Mumbai, India) were used as received. Poly vinyl alcohol and mannitol were purchased from S.D fine chem. Ltd Mumbai. Dichloromethane (DCM) was received from Merck, Germany. All other chemicals and solvents were of analytical grade and were used without further purification. Double distilled water was used throughout the study.

The present invention provides polymeric nanoparticulate formulation capable of being used in the treatment of asthma. The polymeric nanoparticulate formulation of the present invention comprises a drug bromelain, polymethylmethacrylate (PMMA), lecithin, a surfactant and a synthetic polymer. The formulation shows site specific controlled delivery, enhanced bioavailability, therapeutic efficacy and improved drug loading capacity.

The synthetic polymer is selected from the group comprising poly(meth)acrylic acid and its copolymers, poly(vinyl alcohol), or substituted celluloses such as hydroxyethylcellulose. In a preferred embodiment the water soluble synthetic polymer is poly(vinyl alcohol).

The present invention also provides polymeric nanoparticulate compositions comprising bromelain, PMMA, lecithin, a surfactant and a synthetic polymer with pharmaceutically acceptable surfactants or excipients or carriers or any combination thereof.

The lecithin used in the invention is soya lecithin. The surfactant suitable for the process is selected from anionic, cationic and non-ionic surfactant. In a preferred embodiment the surfactant is a non-ionic surfactant. In another preferred embodiment, the surfactant is Sorbitan monooleate (Span 80).

The composition of the present invention provides site specific controlled delivery, higher cellular uptake with reduced adverse effects and improved patient compliance. The composition is analyzed till 12 months and showed retention of bromelain activity up to 97.06±2.94 % under cool temperature of 4 ± 2 ºC at 65 ± 5 % relative humidity (RH) and up to 95.36±3.84 % under room temperature of 25±2ºC at 60 ±5% RH. The composition is analyzed till 6 months at accelerated temperature storage conditions of 40 ± 2 ºC at 75 ± 5 % RH and showed retention of bromelain activity of up to 94.77±5.26 %.

The present invention also provides a process to prepare polymeric nanoparticles of bromelain (Br-LPHNs). A mixture of bromelain and lecithin is co-dissolved in Tris-HCl to form aqueous phase (W1). The W1 is poured drop by drop with an organic phase (O) employing ultrasonic disruption under specific conditions to obtain primary colloidal dispersion. The organic phase (O) is prepared by dissolving PMMA, DCM and a surfactant in an organic solvent. The organic solvent is selected from the group comprising ethanol, methanol, acetone, tetrachloroethylene, toluene, methyl acetate, dichloromethane, ethyl acetate, cyclohexane, hexane, formamide, chloroform, acetonitrile, benzene and mixtures thereof. The organic solvent is preferably dichloromethane.
Said primary colloidal dispersion is further emulsified with aqueous solution of a synthetic polymer (W2) under probe sonication under specific conditions to obtain secondary emulsion (W1/O/W2). Said synthetic polymer is selected from the group comprising poly((meth)acrylic acid) and its copolymers, poly(vinyl alcohol), or substituted celluloses such as hydroxyethylcellulose. Said surfactant is selected from anionic, cationic and non-ionic surfactant.

In a preferred embodiment the surfactant is a non-ionic surfactant. Said surfactant is preferably Span-80.

The secondary emulsion is continuously stirred overnight to remove organic solvent, thereby obtaining organic solvent free double emulsion. The organic solvent free double emulsion is centrifuged under pre-determined conditions to obtain pellets. Said pre-determined conditions are centrifugation at 30,000 rpm for 25 min at 4 ºC. The pellets are dispersed in distilled water with subsequent addition of a cryoprotectant to obtain a suspension. The cryoprotectant is preferably mannitol 10% w/v. Said suspension is lyophilized by freezing to -80 °C for 5 h, followed by primary drying at -80 °C for 36 h at 0.07 mbar and secondary drying at 20 °C for 8 h at 0.07 mbar using lyophilizer to obtain the polymeric nanoparticulate formulation of the present invention. The formulation is characterized and stored at 4 ºC till further use.

Characterization of Br-LPHNs
Particle size and zeta potential analysis: Br-LPHNs nanoparticles’ mean particle size, polydispersity index and surface zeta potential have been evaluated utilizing dynamic light scattering analysis. Samples have been dispersed by vortexing for 1 min in distilled water prior to the estimations.
Entrapment efficiency: Nanoparticulate formulations have been evaluated for entrapment efficiency employing centrifugation method. Supernatants and pellets collected after centrifugation of nanoparticulate dispersion at 20,000 rpm for 20 minutes at 4 ºC have been analyzed employing UV/Visible spectrophotometer at 280 nm. Entrapment efficiency has been determined by formula:
Entrapment efficiency (%) =
Enzyme assay: Proteolytic potential of bromelain has been determined by modified casein digestion method of USP (XXVII). The assay has been performed by incubating suitably diluted bromelain solution with buffered casein substrate solution (1 % w/v, pH 7.0 ± 0.1) for 20 min at 37°C. Subsequently, trichloroacetic acid (30 % w/v) has been added to deactivate the proteolytic activity of bromelain and coagulate the undigested protein. After 30 min, supernatants collected, filtered and estimated using UV-VIS spectrophotometer at 280 nm. Proteolytic activity of bromelain has been demonstrated as casein digestion unit (CDU) elucidated as microgram of tyrosine formed in 1 min by 1 mg of enzyme during assay set-up.

Protein estimation: Lowry method with slight modifications was used to determine total protein content of bromelain sample. The reagent for protein analysis was prepared by mixing 48 parts of 2 % w/v sodium carbonate in 0.1 N sodium hydroxide solutions and 2 parts of 0.5 % w/v copper sulphate in 1% w/v sodium tartrate solution. Suitably diluted bromelain solution was incubated with above prepared reagent (5 ml) for 15 min in dark. Folins reagent (0.5 ml, 2N) was added to each sample tube with subsequent incubation of samples for 30 min in dark. Thereafter, samples were analyzed at 660 nm using UV/VIS spectrophotometer.

Solid state characterization of Br-LPHNs: Morphology of lyophilized samples was examined by Field emission scanning electron microscopy. The lyophilized samples mounted on aluminum stubs using carbon adhesive tape and coated in thin layer using a vacuum evaporator were observed at an acceleration voltage of 5 KV and 30 mm of working distance with subsequent capturing of images.
AT-FTIR spectra of bromelain, PMMA, soya lecithin and optimized formulation were recorded using a spectrophotometer. Spectra of samples were scanned in the region from 4000 to 600 cm-1 at a resolution of 2 cm-1. An advanced ATR correction was applied to all spectra and percentage transmittance was recorded.

The affirmation of crystalline structure of bromelain, optimized formulation and formulation excipients was executed using X ray diffractometer. High intensity CuKa radiation (45kV, 40 mA) generated by copper anode equipped in diffractometer provided diffraction pattern in the range of 5º to 80°, 2?.

Thermal behavior of the drug, PMMA, soya lecithin and optimized formulation was determined using diffractometer. Samples (5-10 mg) were sealed in aluminum pans and heated at a rate of 10 ºC /min over a temperature range of 30 - 300 ºC, under nitrogen flow rate of 60 ml /min.

Structural integrity: The conformational integrity of bromelain in freeze dried nanoparticles was analyzed qualitatively using fluorescence spectrometer. Freeze dried formulation equivalent to 1mg bromelain was dispersed in dichloromethane to lyse the vesicles and centrifuged at 20,000 rpm at 4 °C. Pellet collected was dissolved in distilled water and analyzed by fluorescence spectrometer to acquire the emission spectrum (250-500 nm) of samples at an excitation wavelength of 280 nm and a scan rate of 100 nm min-1. The correction in each protein spectrum was done by subtracting the spectrum of blank solution.

Stability studies under simulated gastro-intestinal pH: Lyophilized optimized formulation was subjected to different pH ambience i.e., pH 1.2 and 6.8 equivalent to stomach and small intestine respectively to envisage their firmness in gastro-intestinal tract. In detail, precisely weighed 50 mg Br-LPHNs were dispersed in HCl buffer pH 1.2 and phosphate buffer pH 6.8 respectively followed by incubation at 37 ± 0.5 °C and 100 shakes per minute for 2h. The substantial effect of respective condition was determined by evaluating their impact on quality attribute like particle size, zeta potential, polydispersity index, percentage drug retained in formulation and conformational integrity after incubation at respective condition.
Dissolution profile: Bromelain release pattern from optimized lyophilized formulation was studied utilizing dialysis bag diffusion method. In detail, Br-LPHNs (equivalent to 50 mg bromelain) placed in dialysis sac was immersed in pH progressive dissolution media (100 ml, HCl buffer pH 1.2 for 2 h followed by phosphate buffer pH 6.8) stirred at 100 rpm and maintained at 37 ± 2 °C. Aliquots (2.5 ml) were drawn out at predestined time intervals up to 24 h and replaced with equal volume of respective buffer after each withdrawal. Bromelain concentration in samples collected was analyzed at 280 nm using UV/Visible spectrophotometer.
Mucolytic activity: The artificial mucous having viscoelastic behavior similar to human airway mucus was prepared by mixing locust bean gum (1% w/v) slowly in preheated sodium nitrite solution (1% w/v) at 80 °C followed by continuous stirring at magnetic stirrer for 24 h. Losses in volume occurring due to evaporation were adjusted by adding sufficient amount of sodium nitrite solution whenever required. The galactomannan chains in locust gum bean solution were cross linked by addition of 0.1 M sodium tetraborate. The resultant mucous (2 gm) was incubated with different concentration of drug (0.1 % w/w to 0.4 % w/w) respectively for 4 h at 37 °C in an orbital shaker at 100 rpm to determine the effect of bromelain concentration on its mucolytic activity. The comparative evaluation of drug (0.2 % w/w) and Br-LPHNs dispersion (equivalent to 0.2 % w/w bromelain) on viscoelastic properties of artificial mucous was also determined at predetermined time interval at 2, 4, 6, 8, 24 and 26 h using Rheometer.
Stability studies: Lyophilized optimized formulations were subjected to stability studies according to ICH guidelines for zone III and IV to assess their suitable storage condition and shelf-life. Formulations sealed in airtight amber colored glass vials were stored at cool (4 ± 2 ºC / 65 ± 5 % RH) and room temperature (25 ± 2 °C / 60 ± 5 % RH) storage conditions for 12 months respectively while for 6 months at accelerated temperature (40 ± 2 ºC / 75 ± 5 % RH) storage conditions. At different time intervals (0, 1.5, 3, 6, 9, 10 and 12 month), the samples were withdrawn and evaluated for drug content. Formulation samples were also analyzed for colloidal properties at respective time interval while color of samples was accessed visually.
Biodistribution and Pharmacokinetic studies: In order to determine the distribution pattern of drug and formulation in blood and tissues, biodistribution study was conducted. Healthy wistar rats (42) were randomly distributed into two groups for quantitative biodistribution study of drug and formulation. Animals of respective group received drug solution (40 mg/Kg) and optimized formulation (equivalent to 40 mg/ Kg bromelain) by oral gavage. After an interval of 0.5, 1, 2, 4, 8, 12 and 24 h post dosing, three rats per group were sacrificed to collect blood by cardiac puncture and tissues (lung, liver, intestine and kidney). Blood was centrifuged at 3000 rpm for 5 min to collect plasma. Collected tissues were washed, blotted dry on a tissue paper, weighed and homogenized in phosphate buffer saline (PBS) pH 7.4. Plasma as well as clear supernatants obtained by centrifugation of tissue homogenates for 20 min at 25,000 rpm were subjected to measure bromelain’s proteolytic activity present respectively. Samples (200 µl/ well) were placed in microtiter plate followed by addition of casein substrate (200 µl, 0.1 mg/ ml casein in 0.1M phosphate buffer, 4mM EDTA and 8mM cysteine) per well and incubated for 1h at 37 °C to determine proteolytic activity. A trichloroacetic acid solution (100 µl) was added to each well to stop the reaction. The undigested casein was allowed to precipitate by incubating the microtiter plate at room temperature for 1 h. The clear supernatant obtained was transferred to noncoated opaque microtiter plates. Fluorescence intensity was estimated at an excitation wavelength of 280 nm and 330 nm emission wavelengths. Fluorescence intensity observed during proteolytic assay was proportional to amount of bromelain in the samples. Results of tissue distribution study were normalized to protein level.
Toxicity studies: Toxicity study of drug and optimized formulation was performed in healthy guinea pigs kept on standard diet and water. Guinea pigs were randomly distributed into 3 groups (n= 6). Group I animals received normal saline orally while group II and III animals received bromelain solution (40 mg/Kg) and optimized formulation (equivalent to 40 mg/Kg bromelain) orally for a period of 28 days respectively. Normal saline treated group served as control group in study. Animals belonging to each group were daily monitored for survival, behavioral changes, body weight change or any signs of toxicity. After completion of 28 days, animals were sacrificed. Blood was collected by cardiac puncture and assessed for biomedical markers of kidney and liver like plasma creatinine, blood urea nitrogen, SGOT, SGPT level. Organs like lung and liver were isolated, incised into small blocks (0.5 cm cubes), fixed in 10 % v/v neutral buffered formalin and processed followed by embedding in paraffin wax. Sections were cut, mounted on glass slides and processed for haematoxylin-eosin dye prior to capture images on microscope.

Pharmacodynamics studies
Histamine induced bronchospasm
In vivo anti-histaminic and bronchodilator activity of bromelain solution and optimized formulation was evaluated in guinea pigs using histamine induced bronchospasm model. Forty-eight overnight fasted animals were randomly divided into four groups: Group I: control group received PBS; group II, III and IV treated with standard drug (theophylline), bromelain solution and optimized formulation respectively. Before histamine challenge, normal saline, theophylline (10 mg/Kg), bromelain (10 mg/Kg) or formulation (equivalent to 10 mg/Kg bromelain) was administered orally to test animals of respective group. Animals (n=3) of each group were exposed with an aerosol of 1% histamine dihydrochloride using a nebulizer at a pressure of 300 mm Hg in an air tight Plexiglass chamber (24 × 14 × 24 cm) for 1 min after a predetermined interval (0.5, 1, 2 and 4 h) of respective treatment. Subsequently after histamine spray, animals were closely observed to measure onset of asphyxia i.e., bronchospasm onset and preconvulsion time for 9 minutes of respective group. The severity of spasm has been represented as mild (+), moderate (++) and severe (+++). Afterward, animals were placed in fresh air to fully recover and recovery time (RT) was measured. The animals were sacrificed to collect tissue and blood samples. Samples were stored at -80 ºC for further analysis.
The percentage protection offered against asphyxia was determined by utilizing formula:
Protection (%) =
Where T1= time of bronchospasm onset in control and T2 = time of bronchospasm onset after pretreatment with standard or test samples.

Similarly, recovery time was calculated by equation
Recovery time (%) =
RT2= Recovery time in control, RT1= Recovery time after pretreatment with standard or test samples.
Ovalbumin induced asthma : Sensitization and Antigen Challenge
Twenty male healthy guinea pigs (250-300 g) were sensitized by intraperitoneal injections of OVA solution (150 µg ovalbumin and 100 mg aluminium hydroxide emulsified in 1 ml of normal saline) on day 1 and 7 respectively. Sensitization phase was completed by injecting a booster dose of ovalbumin solution on day 14. Animals were randomly divided into four groups (n = 5) on completion of sensitization phase. Animals of group I received normal saline while group II, III and IV received standard drug (10 mg/Kg, chlorpheniramine maleate), bromelain solution (10 mg/ Kg) and optimized formulation (10 mg/Kg equivalent to bromelain) for 5 days. Naïve animals (n=5) were sham sensitized with 100 µl normal saline similar to OVA sensitized animals. Animals of respective group were challenged with 1% histamine dihydrochloride solution in a clear plexiglass histamine chamber after 5 days of consecutive treatment respectively. Animals were observed for survival and physiological responses during 20 days of study. The asphyxia (bronchospasm) onset time, convulsion time and time for recovery (RT) i.e., the time taken by the treated OVA-sensitized animals with asphyxia of respective group to recover when placed in fresh air was recorded and compared with saline treated group.
Subsequently, animals were sacrificed after histamine aerosol exposure to collect blood samples by cardiac puncture and organs like lung, liver, spleen and trachea. Whole blood collected was evaluated for hematological parameters like WBC, RBC, hemoglobin level, blood cell counts etc. Serum separated from whole blood was collected and stored at -80 ºC until used to measure immunological parameters like TNF-a, IL-5 and IgG level using ELISA kits as per the manufacturer’s guidelines (Bioassay technology laboratory, China). Consecutively, tissue homogenates prepared in potassium chloride solutions (10% w/v) were evaluated for antigen specific response by quantitating level of oxidative stress markers like lipid peroxidation, carbonyl content, myeloperoxidase activity, reduced glutathione, superoxide dismutase, catalase, eosinophil peroxidase and nitric oxide level.
Bronchoalveolar fluid analysis
Trachea of sacrificed animals immediately after blood collection was carefully exposed and cannulated. Bronchoalveolar lavage was executed five times by infusing normal saline (2 ml) into lungs via cannula and aspirated after gentle massage. Samples collected were pooled to determine total number of cells/ ml using Neubauer hemocytometer. Slides were stained for 15 min with Giemsa stain to count for minimum of 200 cells/ slide. Separated BAL fluid was stored at -80 ºC until used to measure oxidative stress markers and immunological parameters like TNF-a IL-5 and IgG level using ELISA kits as per the manufacturer’s guidelines.
FORMULATION OPTIMIZATION
The formulation was optimized through strategic experimentation by optimizing various formulation variables like lipid-polymer ratio, drug-polymer ratio, organic phase to external aqueous phase volume and type of surface-active agent. The process variable such as sonication time was also optimized. Both formulation as well as process variable were optimized taking into account average particle size, entrapment efficiency, PDI and zeta potential as paradigm framework. (Table 1).
Table 1: Optimization of various formulation and process variables

Formulation code Polymer Drug (%) Surfactant (%) Sonication time (min) during Ratio of Volume of organic phase to continuous phase
Poly- (methyl methacrylate) (%) Soya lecithin (%) Tween 80 Pluronic F - 68 Span 80
Primary emulsify-cation Secondary emulsify-cation
A1 81.63 10.21 8.16 - - 0.5 7 15 1:6

A2 74.07 18.52 7.41 - - 0.5 7 15 1:6
A3 67.8 25.42 6.78 - - 0.5 7 15 1:6
A4 71.43 17.86 10.71 - - 0.5 7 15 1:6
A5 68.97 17.24 13.79 - - 0.5 7 15 1:6
A6 71.43 17.86 10.71 - - 0.5 5 15 1:4
A7 71.43 17.86 10.71 - - 0.5 9 15 1:4
A8 71.43 17.86 10.71 - - 0.5 7 20 1:4
A9 71.43 17.86 10.71 - - 0.5 7 15 1:4
A10 71.43 17.86 10.71 - - 0.5 7 15 1:8
A11 71.43 17.86 10.71 0.5 - 0.5 7 15 1:4
A12 71.43 17.86 10.71 - 0.5 0.5 7 15 1:4

The optimum range of soya lecithin in the formulation is from 10.21 % to 18.52 % w/w to achieve high entrapment efficiency. Soya lecithin amount had direct effect on PDI owing to its influence on thickness of lipid layer. Higher lipid weight percentage showed higher PDI value owing to acceleration of aggregation due to suppression of their native surface charge as represented by zeta potential in Table 2.

The optimum drug-polymer weight ratio is 1:6.67 while keeping amount of lecithin fixed. The optimum drug-polymer ratio facilitates formation of nanoparticulate structure, delays solvent and nonsolvent counter diffusion and improves entrapment efficiency. The drug-polymer weight ratio 1:6.67 substantially improved entrapment efficiency by facilitating formation of larger Br-LPHNs with higher diffusion path length due to formation of interfacial viscous diffusion barrier restricting the drug leaching to the external aqueous phase (W2) compared to 1:5. Although, further increment of polymer load when drug-polymer weight ratio is 1:10 reduces entrapment efficiency due to lower actual drug load (Table 2). Higher polymer concentration also favors the genesis of coarse dispersion as represented by higher PDI owing to paucity of ample energy to overwhelm the viscous forces.

Among surfactants, Span-80 (0.5 % w/v) facilitates the formulation of nanoparticles with higher encapsulation efficiency, smaller particle size, higher zeta potential and good PDI (Table 2). The results confirmed that Span-80 facilitated formation of mechanically and thermodynamically stable primary emulsion by forming a uniform barrier at interface averting aggregation and coalescence of particles. Sonication time also affects desired particle size and entrapment efficiency. The optimum sonication time to obtain the primary colloidal dispersion and the secondary emulsion is 7 min and 15 min, respectively.

The optimum ratio of volume of organic phase to external aqueous phase is 1:4 to achieve small uniform sized particles, optimum PDI and high entrapment efficiency.

The A9 formulation (Table 2) showed high entrapment efficiency of 81.64 ± 1.87 % with particle size of 190.9 ± 7.51 nm, PDI between 0.14 ± 0.02 and zeta potential between -25.3 ± 2.08 mV.

Bromelain activity declined from 198.11 CDU to 182.23 CDU respectively throughout the production of Br-LPHNs. This might be attributed to denaturation of bromelain due to organic solvent system and stress experienced during formulation development.
Table 2. Effect of formulation variables on particle size, zeta potential and entrapment efficiency
Formulation code Particle Size (nm ± SD) Polydispersity index Zeta potential (mV ± SD) Entrapment efficiency (% ± SD)
A1 286.43±8.61 0.18±0.01 -10.46±1.87 64.10±2.31
A2 230.56±15.25 0.15±0.01 -20.06±1.90 71.04±4.09
A3 375.33±12.61 0.39±0.01 -8.36±1.45 66.52±5.31
A4 244.83±12.55 0.11±0.02 -21.67±1.11 74.64±3.11
A5 292.23±10.50 0.18±0.01 -14.16±3.66 71.38±2.31
A6 842.9±28.25 0.67±0.05 -15.78±2.00 32.92±3.29
A7 328.83±9.63 0.27±0.01 -19.33±1.20 65.12±3.66
A8 449.2±6.39 0.20±0.01 -11.73±1.37 52.43±1.63
A9 190.90±7.51 0.14±0.02 -25.30±2.08 81.64±2.87
A10 296.13±10.62 0.18±0.02 -18.50±2.33 22.48±3.65
A11 323.36±10.90 0.17±0.01 -10.11±1.02 52.80±4.54
A12 315.76±10.33 0.13±0.02 -15.10±3.29 61.76±2.09

CHARACTERISTICS OF OPTIMIZED NANOPARTICLE FORMULATION (A9) AND COMPOSITION THEREOF:
Solid state characterization of Br-LPHNs
SEM images manifested uniform spherical shape with smooth surface of Br-LPHNs along with nanometric size which corresponds by the results of photon correlation spectroscopy (Fig 1-A).
The infrared spectrum of bromelain, PMMA, soya lecithin and Br-LPHNs are presented in Fig 1-B. Bromelain spectrum showed distinctive band of N-H stretching vibrations (3258 cm-1), C-H stretching vibrations (2926 cm-1), S-H stretching (2360 cm-1), C=O stretching of –CHO and –NH2 group (1727 cm-1, 1634 cm-1), N-O stretching (1337 cm-1), C-N stretch of primary NH2 (1423 cm-1), C-H bending of tyrosine (874 cm-1) and tryptophan (900 cm-1), N-H wagging of secondary NH2 (749 cm-1), C-S stretch of sulphide and disulphide (700-600 cm-1). PMMA is a synthetic polymer exhibited functional group of C-H stretching vibration of methyl and ethyl group (2948 cm-1, 2826 cm-1), acrylate carboxyl group (1725 cm-1), C-H and CH3 bending (1520 cm-1), CH3 vibrations (1368 cm-1, 745 cm-1), C-O-C stretching (1228 cm-1, 1130 cm-1). While soya lecithin showed N-H stretching (3685 cm-1), O-H stretching of hydroxyl and amino group (3428 cm-1), C-H stretching (2976-2800 cm-1), C=O vibrations (1733 cm-1), PO2 group (1241 cm-1) and P-O-C (1156-980 cm-1). However, spectrum of bromelain loaded nanoparticles showed band of N-H stretching (3255 cm-1), C-H stretching (2928.7 cm-1), aldehyde group (1727 cm-1), sulphide and disulphide in C-S (700-600 cm-1), C-H bending of tyrosine and tryptophan (837cm-1, 931 cm-1) confirming the encapsulation of bromelain in nanoparticles.
Diffractograms of bromelain, PMMA, soya lecithin and optimized formulation is displayed in Fig 1-C. Diffractogram of PMMA showed diffused peaks at 18.37°, 27.78°, 31.87°, 42.48° (2O) and soya lecithin showed single halo peak at 20.12° (2O) indicating their amorphous form. Sharp intense peak of bromelain at 20.3°, 21.04° and 37.53° (2O) whereas low intensity peaks at 13.42°, 16.19° and 23.81° (2O) represents its semi crystalline nature. Conversely, the characteristic peaks of bromelain were absent in formulation diffractogram which displayed peaks at 20.12° and 31.87° (2O) in halo pattern. This further confirmed the amorphous nature of bromelain in formulation.
DSC thermogram of bromelain, PMMA, soya lecithin and optimized formulation are depicted in Fig 1-D. Bromelain showed distinctive sharp peak at 147.44 °C and 216.56 °C. PMMA and soya lecithin showed broad endothermic peak at 152 °C and 87.84 °C respectively. However, bromelain loaded nanoparticles showed endothermic peak at 179 °C, demonstrating the deficit of characteristic peak of bromelain.

Fluorescence spectroscopy
The conformational integrity of bromelain encapsulated in nanoparticles was evaluated by fluorescence spectroscopy. The emission spectrum of pure bromelain and bromelain released from optimized formulation revealed ?max at 330 nm corresponding to intense emission of tryptophan. Fluorescence spectrum of bromelain obtained after lysis of optimized formulation indicated that formulation parameters were suitably optimized to retain the tertiary structure of bromelain during encapsulation (Fig 2-A).
Stability at simulated gastrointestinal pH environment
Various colloidal parameters like particle size, dispersibility, zeta potential, percentage drug retained and conformational stability of drug loaded in formulation was determined in simulated gastrointestinal milieu. Increase in particle size (361.23 ± 96.35 nm) and polydispersity index (0.96 ± 0.05) with reduction in zeta potential (-6.89 ± 0.97 mV) was observed under simulated gastric milieu confirming aggregation of nanoparticulate formulation (Table 3).
The lyophilized formulation incubated at simulated intestinal pH 6.8 had not showed significant alteration in particle size, PDI as well as zeta potential (Table 3). Since pH> pKa, polymer remained ionized and electrostatic repulsive forces maintained the consistent dispersibility of nanoparticles at pH ambience similar to intestine. Higher zeta potential (-24.5 ± 2.33 mV) and PDI (< 0.3) also ensured that aggregates released from stomach would de-aggregate on reaching to small intestine. Around 11% and 38% drug release was observed at simulated gastric and intestinal pH in 2 h respectively. Lower drug release at simulated gastric conditions might be bestowed to insufficient coating and film defects arising during lyophilization since PMMA polymer remains unionized and is insoluble at gastric pH. However, ionization of PMMA polymer at intestinal pH 6.8 corresponded higher drug release by hampering aggregation of nanoparticles and furnishing higher effective surface area for solvent contact and drug diffusion.
Bromelain recovered after lysis of nanoparticulate formulation earlier incubated at simulated gastric pH 1.2 and intestinal pH 6.8 for 2 h showed emission ?max at 330 nm. On the contrary, ?max shifted to 340 nm for pure drug incubated at simulated gastric pH with significantly lower fluorescence intensity. The red shift in ?max for pure bromelain might be attributed to conformational changes in the vicinity of tryptophan surface indicating denaturation of bromelain in acidic environment. However, no change in aromatic chromophore of bromelain released from nanoparticles incubated under simulated gastrointestinal pH indicated the structural protection offered by vesicular system to maintain its tertiary structure as well as proteolytic activity (Fig 2-B).
Table 3: Effect of different pH (1.2 and 6.8) on particle size, zeta potential, PDI and drug release pattern of formulation after incubation of Br-LPHNs for 2 h.
pH Particle size (nm ± SD) Zeta potential (mV ± SD) PDI Drug release (% ± SD)
1.2 361.23 ± 96.35 -6.89 ± 0.97 0.96 ± 0.05 11.04 ± 1.23
6.8 190.90 ± 7.51 -24.5 ± 2.33 0.14 ± 0.02 38.21 ± 2.98

Drug release
Dissolution study was executed in pH progressive media to ascertain the prospective of Br-LPHNs in modulating drug release for extending bromelain’s action. At simulated gastric pH 1.2 optimized formulation showed ~ 11 % drug release in 2 h. Such peculiar initial drug release when polymer coat remained unionized might be likely contributed owing to prompt diffusion and desorption of surface adsorbed or weakly bound drug and partial film defects emerged during lyophilization. Afterwards, a brisk increase in drug release for 2 h followed by prolonged release upto 24 h was observed as pH of dissolution media was raised to pH 6.8 to mimic the pH of small intestine. Furthermore, dissolution data of Br-LPHNs was fitted to different release kinetic models to ascertain the drug release kinetics and mechanism. The best fit release model for optimized formulation was established by regression analysis. Highest R2 (0.978) value for Korsmeyer-Peppas model and numerical value of release exponent (n = 0.734) indicated the anomalous behavior of drug release from nanoparticles i.e. combination of both diffusion and dissolution phenomenon regulated the bromelain release (Fig 3).
Mucolytic activity
The remarkable decrease in viscosity of mucus was observed with increase in concentration of bromelain from 0.1 % w/w to 0.4 % w/w (Fig 4-A) due to irreversible breakdown of three-dimensional network of locust bean gum mucilage. Subsequently, remarkable decrease in viscosity was observed with increased duration of exposure facilitated by penetration of bromelain in inward layers of 3-D network of mucus due to disruption of glycosidic bonds and splitting of protein in smaller fragments. However, no significant change in mucus hydrolysis quantified as change in viscosity was observed after 4 h in samples treated with 0.2 % w/w, 0.3% w/w and 0.4 % w/w of bromelain respectively. Therefore, 0.2 % w/w bromelain was used for further studies.
The profound diffusion of the nanoparticles within mucus was contributed by the continuous release of bromelain from formulation. Results indicated significant reduction in viscosity with nanoparticulate formulation similar to pure drug. Although, optimized formulation initially manifested lower degree of mucus liquefaction followed by robust influence on viscosity of artificial mucus similar to pure drug after 4h (Fig 4-B). The mucolytic results complied with slow and progressive in vitro release behavior of formulation. Br-LPHNs were more effective in reducing mucus viscosity due to their deeper penetration across the microarchitecture and mesh spacing of artificial mucus as well as prevented end product inhibition by progressive release of bromelain despite of availability of whole amount as in case of pure bromelain.
Stability studies:
The tendency of Br-LPHNs to sustain colloidal properties, release behavior and percentage bromelain activity was analyzed till 12 months under cool (4 ± 2 ºC / 65 ± 5 % RH) and room temperature (25 ± 5 ºC / 60 ± 5 % RH) storage conditions whereas for 6 months at accelerated temperature storage conditions (40 ± 2 ºC / 75 ± 5 % RH). Simultaneously, effect of storage conditions on percentage activity of pure drug remaining was also determined (Table 4, 5). Pure drug under accelerated and room temperature storage conditions showed remarkable decrease in drug content compared to Br-LPHNs. However, pure drug showed ~96 % drug content under cool conditions indicating that proteolytic activity of drug was protected at lower temperature. Br-LPHNs showed no significant change in quality parameters like particle size, PDI, zeta potential and proteolytic activity with progression of time at different storage conditions. This indicated that formulation parameters were appropriately optimized to formulate a stable bromelain loaded nano-formulation. Bromelain followed first order degradation kinetics. Higher Kcal and lower t90 value for pure drug demonstrated faster degradation rate compared to Br-LPHNs under real time stability conditions. The calculated t90 for formulation was ~ 6.5-folds higher compared to pure drug which is stable for around 4 months at room temperature manifesting significantly better stability of formulation. The visual changes in color of pure drug from off white to brown was observed at both room and accelerated temperature storage condition respectively whereas no color change was observed for Br-LPHNs.
Table 4: Stability study of pure drug and optimized formulation (A9) stored at different time interval
Storage condition Sample Bromelain activity remaining (%)
0 M 1.5 M 3 M 6M 9M 12 M Kcal (days-1) T90 (days-1)
25±2ºC/60 ±5% RH
Bromelain 100.01±1.23
95.45±2.34
90.46±1.11
85.86±1.90
81.23±2.34 73.12±4.32
Control Bromelain Fluticasone furoate Formulation
8.39×10-4
125.82

Br-LPHNs 100.01±2.72
99.09±3.67
98.88±2.05
97.81±1.86
96.81±2.05
95.36±3.84
1.29× 10-4
813.95

4°C±2°C/65 ± 5 % RH
Bromelain 100.01±1.23
99.72±4.21
98.79±4.26
97.52
±3.06 96.81±1.92 96.21±7.12 1.17× 10-4
897.43
Br-LPHNs 100.01±2.72
99.96±2.72
99.47±2.43
98.40±4.06
97.29±3.51
97.06±2.94 0.96× 10-4
1093.75

40±2ºC/75 ±5% RH
Bromelain 100.01±1.23
89.45±2.90
85.90±2.50
74.18±1.79 1.67×10-3
62.76

Br-LPHNs 100.01±2.72
98.26±2.01
98.73±3.99
94.77±5.26
3.03×10-4
346.53
# Values are expressed as mean ± SD, Abbreviations: M= month; Kcalc = calculated first order degradation rate constant; t90 = time to reach 90% of initial drug concentration
Table 5: Effect of storage condition on colloidal properties of optimized formulation (A9) at different time interval
Storage condition Formulation (A9) 0 M 1.5 M 3 M 6M 9M 12 M
25±2ºC/60 ±5% RH Particle Size (nm ± SD) 190.90± 11.82
190.23± 20.91
193.74± 33.14
198.22± 19.60
192.59± 17.55
195.70± 16.72
PDI 0.14± 0.03
0.15± 0.01
0.15± 0.01
0.16± 0.02
0.15± 0.01
0.15± 0.02
Zeta potential (mV ± SD) -25.31± 3.12
-25.12± 1.45
-25.11± 3.33
-24.93± 1.10 -24.16± 1.77
-22.56 ±1.67
4°C±2°C/65 ± 5 % RH Particle Size (nm ± SD) 190.90± 11.82
190.01± 17.64
191.58± 32.6
192.71± 20.5
195.32± 18.5
197.69± 15.9
PDI 0.14± 0.01
0.14± 0.02
0.13± 0.02
0.16± 0.03
0.13± 0.01
0.15± 0.01
Zeta potential (mV ± SD) -25.30± 1.22
-25.41± 2.65
-25.75± 2.09
-25.97± 1.54
-25.33± 2.22
-24.56 ±2.55
40±2ºC/75 ±5% RH Particle Size (nm ± SD) 190.90± 11.81
190.55± 10.87
194.72± 21.93
198.37± 10.52

PDI 0.14± 0.03
0.15± 0.01
0.17± 0.01
0.18± 0.01

Zeta potential (mV ± SD) -25.31± 3.12
-25.19± 5.09
-24.36± 3.09
-23.82± 1.87

Pharmacokinetic studies
The plasma drug concentration time profile post oral administration of free drug and Br-LPHNs in wistar rats are represented in Table 6, Figure 5. Optimized formulation after single dose administration showed significantly higher Cmax (1.53-fold) compared to pure bromelain. Higher Cmax value might be contributed by absorption of Br-LPHNs through specific absorption mechanisms owing to their nano-size and hydrophobic surface facilitating their permeation across GIT. Tmax was also 2-fold delayed for Br-LPHNs compared to pure drug. This confirmed sustained in vivo bromelain release from optimized nanoparticulate formulation similar to in vitro drug release study. Pure drug plasma level declined after 2 h, indicating rapid systemic elimination of bromelain which was further evident with their low systemic mean residence time (MRT). Besides, t1/2 and MRT enhanced 5.42-fold and 4.59-fold respectively with Br-LPHNs owing to prolonged absorption and slow release of drug from Br-LPHNs into systemic circulation. Meanwhile AUC0-24h was also significantly enhanced about 5.41-fold for formulation compared to pure drug. Apparently loading bromelain in lipid polymer hybrid nanocarrier helped in bypassing the extensive gut wall metabolism due to intimate association of drug with lipid polymeric shell. The enhanced systemic absorption through specialized absorption mechanisms like paracellular and transcellular transport via endocytosis and M-cells of Payer’s Patches might have ensured an increase in bioavailability.
Table 6: Pharmacokinetic parameter of pure drug and optimized formation obtained after oral administration at an equivalent dose of 40 mg/kg bromelain.
PARAMETER Drug Formulation
Cmax (ng ml-1) 4.69 ± 1.77 7.18 ± 0.93
Tmax (h) 2.00 ± 0.11 4.00 ± 0.21
Ke (h-1) 0.43 ± 0.11 0.08 ± 0.01
t1/2 (h) 1.61 ± 0.42 8.75 ± 0.94
MRT (h) 1.93 ± 0.49 8.86 ± 0.78
AUC (ng h2 ml-1) 19.54 ± 4.22 105.8 ± 22.11
Relative bioavailability (%) 541.45

Values are expressed as mean ± SD, n=3; p<0.05 level of significant difference

Biodistribution studies:
Fig 6 depicts the biodistribution of bromelain and Br-LPHNs in lung, liver, intestine and kidney at different time intervals. Pure drug achieved higher drug concentration in lung and intestine in initial hours compared to Br-LPHNs after oral administration. However, with progression of time drug concentration declined due to drug metabolism. On contrary, formulation showed increase in drug concentration in all examined organs with respect to time compared to drug. This might be contributed by the controlled release of drug from nanoparticles as well as deposition of drug in these organs. The results indicated that Br-LPHNs could markedly promote enrichment and increase tissue distribution of bromelain, markedly in the liver and lung. Furthermore, higher drug level in lung suggested that Br-LPHNs in blood circulation could be recognized and phagocytized as extraneous materials by the mononuclear phagocyte system prevalent in lung. The formulation also protected the bromelain from degradation in acidic environment which was indicated by the increased concentration of drug in intestine.

Pharmacodynamics
Histamine induced acute bronchospasm
During present study frequent onset of bronchospasm, jerks, severe spasm as well as death was observed among control group animals. However, treated animals with theophylline, bromelain and optimized formulation showed significant delay in bronchospasm onset, reduced intensity and duration of jerks and immediate recovery after histamine exposure (Fig 7, Table 7). Treated groups showed no mortality throughout the study. The significant delay in bronchospasm induction and reduction in convulsion period was observed among theophylline treated group animals compared to drug and formulation during initial first hour of study (Fig 7). However, with progression of time faster onset of bronchospasm was observed in theophylline treated group due to short half-life of theophylline. Lower percentage protection, increase in convulsion period and slower recovery time in bromelain treated group might be attributed to degradation of drug in acidic environment leading to reduction in its therapeutic efficacy. However, optimized formulation showed significantly increased percentage protection as well as inhibition in convulsion with respect to time compared to pure bromelain and theophylline. The prolonged controlled drug release behavior of drug from optimized formulation might be significantly contributing to delay in bronchospasm induction and significantly faster recovery time after 2 h of treatment with formulation compared to pure and standard drug.
Increase in dose of optimized formulation (equivalent to 10 mg/kg to 40 mg/kg bromelain) significantly prolonged the bronchospasm induction time however no significant enhancement in percentage protection was observed at 4th hour of treatment (Fig 7). Therefore, further studies were carried out at a dose of optimized formulation equivalent to 10 mg/Kg bromelain.
Table 7: Effect of different treatments with respect to duration of treatment on severity of bronchospasm in histamine induced acute bronchospasm
Severity of bronchospasm
Time (h) 0.5 1 2 4
Control +++ +++ +++ +++
Bromelain +++ +++ +++ +++
Standard drug ++ ++ +++ +++
Formulation +++ +++ ++ +

Biochemical estimation
Oxidative burden experienced during bronchospasm induced by histamine exposure was estimated by determining lipid peroxidation and protein carbonylation content in lung, trachea and liver. Both pure drug as well as optimized formulation was found effective in diminishing the level of lipid peroxides and protein carbonyl groups compared to control group (p<0.05). Among the treatments, optimized formulation showed significantly higher reduction of lipid peroxide and protein carbonyl level in lung, liver as well as trachea after 4 h of treatment (Fig 8). The protection provided by lipid polymeric hybrid nanocarrier to bromelain in optimized formulation as well as slow and controlled release of drug might have contributed to improved therapeutic efficacy of formulation.

Serum analysis of histamine sensitized animals showed significant elevation in total leucocyte, lymphocytes and neutrophils count (Fig 9). The infiltration of these cells led to cascade of reactions including excessive mucus production, inflammatory reactions and oedematous conditions along with stimulation of profibrotic transformation of TGF-ß and contributed to bronchospasm. The pre-treatment with bromelain, theophylline and optimized formulation significantly decreased the lymphocytes and neutrophils level compared to control group asserting reduced inflammation of lungs. The significant reduction in total leucocytes count compared to pure drug and theophylline confirmed the higher anti-inflammatory potential of Br-LPHNs.

Ovalbumin induced asthma model
Airway hyperresponsiveness is one of the distinctive features of asthma. Histamine challenge has been widely used aid in prognosis of allergic respiratory disorders to assess airway responsiveness. During present study, OVA sensitization evoked a significant hyperresponsiveness to histamine challenge in guinea pigs. The effect of various treatments on airways responsiveness against exposure to histamine in OVA induced asthma model was evaluated by estimating clinical parameters like onset of bronchospasm, severity of spasm and recurrent episodes of jerk as well as difficulty in breathing in response to histamine exposure (Figure 10, table 8). Bronchial hyperresponsiveness in response to histamine in OVA-sensitized animals was significantly higher than normal saline treated animals (p<0.05). Remarkable elevation in duration for histamine induced bronchospasm by 11.14 %, 45.08 % and 157.37 % (from 1.00 ± 0.04 min to 1.08 ± 0.07 min, 1.29 ± 0.12 min and 2.57 ± 0.32 min; p<0.05) and decline in recovery time after bronchospasm induction by 47.73 %, 64.80 % and 82.57 % (from 2.87 ± 0.54 min to 1.5 ± 0.07 min, 1.01 ± 0.03 min and 0.5 ± 0.07 min; p<0.05) in animals treated with drug, chlorpheniramine and optimized formulation respectively compared with normal animals (non-sensitized, normal saline treated). The delay in bronchospasm induction and jerks were observed in drug, standard drug and formulation treated animals as compared to positive control (sensitized, saline treated). Br-LPHNs significantly suppressed bronchial hyperresponsiveness by delaying bronchospasm induction approximately 292.46 %, 120.69 % and 67.08 % respectively compared to asthmatic group, drug and standard (chlorpheniramine) drug treated group. The protective effect might be contributed by higher systemic bioavailability of drug due to protection provided by PMMA polymer to bromelain in acidic milieu of stomach and sustained release behavior of drug from optimized formulation. Bromelain loaded in nanoparticles nullified the histamine induced bronchoconstriction similar to chlorpheniramine indicating antihistaminic effect of bromelain.
Table 8: Effect of different treatments on severity of bronchospasm in ovalbumin induced asthma model.
Treatment Severity of bronchospasm
Control +++
Bromelain ++
Standard drug ++
Formulation +

The increase in the relative weight of lungs (53.60 ± 2.02 %) in OVA sensitized animals compared to control group was observed. The increased weight of lung suggested that provocation with OVA, an allergen induced elevated microvascular infiltration and oedema leading to the inflammation of lung. Significant decrease in relative weight of lung was observed on treatment with drug, chlorpheniramine and formulation (p<0.05) compared to OVA sensitized group (Fig. 11). The results indicated that bromelain decreased the tissue damage and oedema in lungs due to its antioxidant and anti-inflammatory activity.
Ovalbumin sensitized guinea pigs and healthy animals showed significant variation in their hematological profile. Serum of saline treated animals showed significant elevation of total leukocyte count (3.86-fold), eosinophils (2.25-fold), lymphocytes (1.74-fold), neutrophil (2.04-fold) and hemoglobin (1.46-fold), relative to naïve control animals (Fig. 12). Increased level of eosinophil count confirmed the allergic inflammation associated with bronchial hyperresponsiveness. In addition, elevated total leukocyte count and lymphocytes count confirmed the inflammatory state. Elevated total cell count (54-fold) and EPO (18-fold) in BAL fluid too confirmed the infiltration of neutrophils, lymphocytes and eosinophils in lungs contributing to inflammation, mucus production and oedema. Elevated level of WBCs induced excessive release of histamine in lungs of OVA sensitized animals and thus induced runny nose and wheezing during allergic time in OVA sensitized animals. Asthma related pulmonary hypoxia might have provoked the hemoglobin synthesis and increased serum hemoglobin level in control group. Although, Br-LPHNs and chlorpheniramine significantly (p<0.05) reduced total leukocyte count, lymphocyte, platelet count, neutrophils and eosinophils count and brought back near to normal level (Fig. 12) compared to pure drug.
Estimation of oxidative stress markers
The magnitude of oxidative stress markers (LPO, MPO and carbonylated protein) and antioxidants (GSH, SOD and catalase) level in distinct groups is represented in Fig 13. The results indicated significant elevation of LPO and carbonylated protein level in OVA sensitized control group with significant decline in antioxidant enzyme activity. LPO, MPO and carbonylated protein are salient markers of oxidative stress exhibiting a vital role in pathogenesis of asthma. Under normal physiological condition, endogenous enzymatic (SOD and CAT) and non-enzymatic (reduced glutathione) defense mechanism prevent and terminate the oxidative free radicals induced damage. The lowered levels of SOD, CAT and GSH while elevated level of LPO and carbonylated protein in OVA challenged animals represented imbalance between free radical production and antioxidant defense leading to oxidative cellular stress. Bromelain, chlorpheniramine as well as Br-LPHNs manifested remarkable mitigation of oxidative stress by depleting LPO and carbonylated protein level in lung, liver, trachea and spleen tissues respectively compared to OVA sensitized control (p <0.05) (Fig 13- A, B). The results indicated that Br-LPHNs were more effective in restoring SOD, catalase and GSH level compared to pure bromelain and chlorpheniramine (p < 0.05) (Fig 13). The marked suppression of oxidative stress markers and amplification of antioxidant defense confirmed the improved antioxidant potential of Br-LPHNs. Such remarkably improved antioxidant activity of Br-LPHNs might be attributed to enhanced lymphatic uptake of Br-LPHNs through phagocytosis and paracellular pathways along with preservation of bromelain’s activity through stomach and sustained diffusion of drug from formulation.
Elevated level of NO indicated the upregulation of inducible nitric oxide synthase (iNOS) and prostaglandins genesis in OVA sensitized control. Similarly, escalated TNF-a and IL-5 serum level stipulated promotion of neutrophil migration to tissues, NO synthesis and reactive oxygen species production. However, treatment with bromelain and Br-LPHNs showed significant reduction of WBC count, MPO, NO and cytokines level (Ig-G, IL-5 and TNF-a) respectively (Fig 13). Thus, it can be assumed that bromelain’s inhibitory effect on NFKB overexpression and iNOS along with improved expression of Nrf2 pathway had bestowed to its beneficial analgesic and anti-inflammatory effects for treatment of inflammatory conditions like asthma. The enhanced anti-inflammatory activity of Br-LPHNs compared to bromelain might be attributed to improved gastro-stability and sustained release behavior of Br-LPHNs contributing prolonged inhibitory effect on cytokine storm. The results also confirmed the prospective of LPHNs as a carrier for bromelain delivery.

Histopathological analysis of lung and liver tissue:
Fig 14 represents the histopathological microphotographs of lung of naïve, normal saline, bromelain, chlorpheniramine and Br-LPHNs treated animals respectively. Microphotographs of lung of naïve group showed clear alveolar sacs with no gathering of cells in the region of bronchioles (Fig 14-A). Although, histopathological evaluation of OVA sensitized lungs showed thick deposition of cells around the bronchioles, blood vessels and alveolar regions indicating the excessive infiltration of cells leading to constriction of alveolar sacs (Fig 14-B). Oxidative stress induced by OVA challenge might be responsible for inflammation and remodeling of lung tissues. Groups treated with bromelain and chlorpheniramine showed less accumulation of cells around the bronchioles as well as less thickening in alveolar septa (Fig 14 -C, D). However, formulation treated group (Fig 14-E) showed no thickening around the bronchioles with very less accumulation of cells depicting the almost similar structure with naïve animals.
Liver segment of naïve animals showed a normal architecture (Fig 15 -A) whereas ovalbumin sensitized animals showed infiltration of inflammatory cells leading to excessive tissue damage (Fig 15 -B1) as well as loss of cellular order (Fig 15 -B2) due to elevation of hepatic enzyme activities. These enzymes affect variety of inflammation, necrosis in hepatocytes and congestion in sinusoids. The bromelain (Fig 15 -C) and chlorpheniramine (Fig 15 -D) treated groups showed less infiltration of cells as compared to ovalbumin sensitized animals. However, liver section of formulation treated groups (Fig 15 -E) represented similarity of features as were found in naïve animals. This improved effect of formulation might be attributed by PMMA polymer which protects the proteolytic activity of bromelain.

Toxicity studies:
Oral toxicity studies conducted in guinea pig showed no significant change in weight, behavior as well as no mortality in drug and formulation treated group respectively. Similarly, no significant change was observed in histopathology of lung and liver as well as biomedical markers of kidney and liver compared to naïve group respectively (Fig 16, 17). Pure drug had not showed any toxicity due to inactivation of its proteolytic activity in gastric environment. Whereas, characteristic features of optimized formulation i.e., nanosize contributing to lymphatic uptake, protection against gastric milieu, sustained drug release behavior improved bromelains therapeutic efficacy and prevented any untoward effect. Thus, results confirmed the safety of optimized formulation for prolonged use in management of asthma.
Thus, bromelain loaded nanoparticles were successfully fabricated with high loading capacity by double emulsion solvent evaporation method. Hybridization of lipid core containing drug with polymer coat augmented higher drug load, improved gastric stability and facilitated prolonged drug release to achieve higher bioavailability and prolonged therapeutic effect. Br-LPHNs mitigated improved anti-inflammatory, antioxidant as well as anti-asthmatic activity indicating wider acceptability of formulation in treatment of asthma along with other disorders associated with oxidative stress and inflammation.

Claims:

We Claim:
1. A polymeric nanoparticulate formulation of bromelain, comprising drug bromelain, Polymethylmethacrylate (PMMA), lecithin, a surfactant and a synthetic polymer wherein said formulation is capable of being used in the treatment of asthma.
2. The polymeric nanoparticulate formulation as claimed in claim 1 wherein, said formulation comprises:
Ingredient Quantity (% Range)
Bromelain 7 - 14
Lecithin 10 - 25
PMMA 67 - 81
Surfactant 0.5
Synthetic polymer 1
3. The polymeric nanoparticulate formulation as claimed in claim 1 wherein, said formulation comprises:
Ingredient Quantity (% Optimum)
Bromelain 10.71
Lecithin 17.86
PMMA 71.43
Surfactant 0.5
Synthetic polymer 1
4. The polymeric nanoparticulate formulation as claimed in claim 1 wherein said synthetic polymer is selected from a group comprising poly(methyl methacrylic acid) and its copolymers, poly(vinyl alcohol), or substituted celluloses such as hydroxyethylcellulose.
5. The polymeric nanoparticulate formulation as claimed in claim 4 wherein said synthetic polymer is poly(vinyl alcohol).
6. The polymeric nanoparticulate formulation as claimed in claim 1 wherein said surfactant is selected from anionic, cationic and non-ionic surfactant.
7. The polymeric nanoparticulate formulation as claimed in claim 6 wherein said surfactant is Sorbitan monooleate (Span 80).
8. The polymeric nanoparticulate formulation as claimed in claim 1 wherein said lecithin is soya lecithin.
9. The polymeric nanoparticulate formulation as claimed in claim 1 wherein an optimum bromelain-PMMA weight ratio is 1:.6.67.
10. The polymeric nanoparticulate formulation as claimed in claim 1 wherein optimum percentage of the soya lecithin in the formulation is 17.86 %.
11. The polymeric nanoparticulate formulation as claimed in claim 1 wherein size of said nanoparticulate in the formulation is in the range of 190.90 ± 7.51 nm.
12. The polymeric nanoparticulate formulation as claimed in claim 1 wherein said formulation has high entrapment efficiency of bromelain in the nanoparticulate being in the range of 81.64 ± 2.87 %.
13. The polymeric nanoparticulate formulation as claimed in claim 1 wherein zeta potential of the nanoparticulate is in the range of -25.30±2.08 mV.
14. The polymeric nanoparticulate formulation as claimed in claim 1 wherein polydispersity index of the nanoparticulate is in the range of 0.14±0.02.
15. The polymeric nanoparticulate formulation as claimed in claim 1 wherein said nanoparticles are analyzed till 12 months and showed retention of bromelain activity up to 97.06±2.94± % under cool temperature of 4 ± 2 ºC at 65 ± 5 % relative humidity (RH) and up to 95.36±3.84 % under room temperature of 25±2ºC at 60 ±5% RH.
16. The polymeric nanoparticulate formulation as claimed in claim 1 wherein said nanoparticles are analyzed till 6 months at accelerated temperature storage conditions of 40 ± 2 ºC at 75 ± 5 % RH and showed retention of bromelain activity of up to 94.77±5.26 %.
17. The polymeric nanoparticulate formulation as claimed in claim 1 wherein t90 of said nanoparticulate formulation is ~ 6.5-folds higher compared to pure drug at room temperature.
18. A process to prepare the polymeric nanoparticulate formulation as claimed in claim 1, said process comprises:
(i) preparing an aqueous solution of bromelain and lecithin co-dissolved in Tris-HCl to obtain aqueous phase (W1),
(ii) preparing an organic phase of PMMA and a surfactant dissolved in an organic solvent (O),
(iii) pouring drop by drop the aqueous solution of step (i) (W1) in organic solvent of step (ii) (O) employing ultrasonic disruptor under predefined conditions to obtain a primary colloidal dispersion,
(iv) preparing an aqueous solution of a synthetic polymer (W2),
(v) emulsifying said primary colloidal dispersion of step (iii) with aqueous solution of step (iv) (W2) under probe sonication under predefined conditions to form nanoparticulate emulsion (W1/O/W2),
(vi) removing said organic solvent by continuous stirring of said nanoparticulate emulsion to obtain organic solvent free double emulsion,
(vii) centrifuging said organic solvent free double emulsion at predefined conditions to obtain pellets,
(viii) dispersing the said pellets in water with subsequent addition of cryoprotectant to obtain a suspension,
(ix) lyophilizing the said suspension under specific conditions by freezing followed by primary drying and secondary drying using lyophilizer to obtain said polymeric nanoparticulate formulation.
19. The process as claimed in claim 18 wherein said organic solvent in step (ii) is selected from the group comprising ethanol, methanol, acetone, tetrachloroethylene, toluene, methyl acetate, dichloromethane, ethyl acetate, cyclohexane, hexane, formamide, chloroform, acetonitrile, benzene and mixtures thereof.
20. The process as claimed in claim 18 wherein said organic solvent is dichloromethane.
21. The process as claimed in claim 18 wherein said surfactant in step (ii) is Span 80.
22. The process as claimed in claim 18 wherein said predefined conditions in step (iii) for emulsification by ultrasonic disruptor are disruption for 7 min over an ice bath.
23. The process as claimed in claim 18 wherein said water soluble synthetic polymer in step (iv) is selected from the group comprising poly(methylmethacrylic acid) and its copolymers, poly(vinyl alcohol), or substituted celluloses such as hydroxyethylcellulose or a combination thereof.
24. The process as claimed in claim 23 wherein said water soluble synthetic polymer is poly(vinyl alcohol) (PVA).
25. The process as claimed in claim 18 wherein said surfactant is selected from anionic, cationic and non-ionic surfactant, preferably the surfactant is a non-ionic surfactant.
26. The process as claimed in claim 18 wherein said predefined conditions in step (v) are emulsifying the primary emulsion for 15 min over an ice bath.
27. The process as claimed in claim 18 wherein said predefined conditions in step (vii) are centrifugation at 30,000 rpm for 25 min at 4 ºC.
28. The process as claimed in claim 18 wherein said cryoprotectant is mannitol 10% w/v.
29. The process as claimed in claim 18 wherein said specific conditions for lyophilization are freezing to -80 °C for 5 h followed by primary drying at -80 °C for 36 h at 0.07 mbar and secondary drying at 20 °C for 8 h at 0.07 mbar.
30. A composition of formulation comprising drug bromelain, Polymethylmethacrylate (PMMA), lecithin, a surfactant and a synthetic polymer with pharmaceutically acceptable excipients or surfactants or carriers or a combination thereof for use in treatment of asthma.
31. The composition of formulation as claimed in claim 30 wherein said composition is in dosage form suitable for oral drug delivery.
32. The composition of formulation as claimed in claim 31 wherein said dosage form is sustained release dosage form selected from tablet, capsules, sachets, powders, granules, pellets, orally dispersible films, ampoules, dispersions, suspension, semi-solids, soft gels. ,