1. Bromelain nanoparticle for management of asthma, said nanoparticle comprising bromelain, methacrylate ester copolymer having repeating units of formula I, a surfactant and a synthetic polymer, Formula I wherein R1 is selected from hydrogen and methyl, R2 is selected from methyl, and ethyl, R3 is methyl and R4 is ethyltrimethylammonium chloride.

2. The bromelain nanoparticle as claimed in claim 1, wherein R1 is hydrogen, R2 is methyl, R3 is methyl.

3. The bromelain nanoparticle as claimed in claim 1, wherein the methacrylate ester copolymer is Eudragit RL100 or Eudragit RS 100.

4. The bromelain nanoparticle as claimed in claim 1, wherein the ratio of bromelain and the methacrylate ester copolymer of formula I is in the range of 1: 1 to 1:7.5.

5. The bromelain nanoparticle as claimed in claim 4, wherein the ratio of bromelain and the methacrylate ester copolymer of formula I is in the range of 1: 3.

6. The bromelain nanoparticle as claimed in claim 1, wherein said synthetic polymer is selected from a group comprising poly(vinyl alcohol), or substituted celluloses such as hydroxyethylcellulose.

7. The bromelain nanoparticle as claimed in claim 6, wherein said synthetic polymer is poly(vinyl alcohol).

8. The bromelain nanoparticle as claimed in claim 1, wherein said surfactant is selected from anionic, cationic and non-ionic surfactant.

9. The bromelain nanoparticle as claimed in claim 8, wherein said surfactant is Sorbitan monooleate (Span 80).

10. The bromelain nanoparticle as claimed in claim 1, wherein size of said nanoparticle is in the range of 227.91 ± 26.01 to 625.63 ± 43.77 nm.

11. The Bromelain nanoparticle as claimed in claim 10, wherein size of said nanoparticle is in the range of 259.73 ± 16.51 nm.

12. The bromelain nanoparticle as claimed in claim 1, wherein bromelain entrapment efficiency of the nanoparticle ranges from 40± 8 to 90 ± 8 %.

13. The bromelain nanoparticle as claimed in claim 12, wherein bromelain entrapment efficiency of the nanoparticle is of 84.76 ± 1.49 %.

14. The bromelain nanoparticle as claimed in claim 1, wherein zeta potential of the nanoparticle ranges from 8.5 ±2 to 29 ±2 mV

15. The bromelain nanoparticle as claimed in claim 14, wherein zeta potential of the nanoparticle is 27.53 ± 1.66 mV.

16. The bromelain nanoparticle as claimed in claim 1, wherein polydispersity index of the nanoparticle is in the range of 0.143±0.016.

17. The bromelain nanoparticle as claimed in claim 1, wherein said nanoparticles are analyzed till 12 months and showed retention of bromelain activity up to 97.04±1.65 % under cool temperature of 4 ± 2 ºC at 65 ± 5 % relative humidity (RH) and up to 95.23± 1.25% under room temperature of 25±2ºC at 60 ±5% RH.

18. The bromelain nanoparticle 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 94.16±3.10 %.

19. The bromelain nanoparticle as claimed in claim 1, wherein t90 of said nanoparticulate formulation is ~ 5.99 folds higher compared to pure drug at room temperature.

20. A process to prepare the bromelain nanoparticle as claimed in claim 1, said process comprises: a. emulsifying an aqueous solution of bromelain (W1) with an organic phase (O) to obtain primary emulsion (W1/O) by ultra-sonication under pre-defined conditions, wherein said organic phase (O) is prepared by dissolving the copolymer of formula I in an organic solvent comprising surfactant, b. dispersing the primary emulsion of step (a) to an aqueous solution of synthetic polymer (W2) emulsifying by probe sonication under predefined conditions to form double emulsion (W1/O/W2), c. removing said organic solvent by continuous stirring of said double emulsion to obtain organic solvent free double emulsion to obtain bromelain nanoparticle dispersion, d. centrifuging said bromelain nanoparticle dispersion at predefined conditions to obtain pellets, e. dispersing the said pellets in water with subsequent addition of cryoprotectant to obtain a suspension, f. lyophilizing said suspension under specific conditions by freezing followed by primary drying and secondary drying using lyophilizer to obtain lyophilized bromelain nanoparticle.

21. The process as claimed in claim 20 wherein said organic solvent in step (a) 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.

22. The process as claimed in claim 21 wherein said organic solvent is dichloromethane.

23. The process as claimed in claim 20 wherein said surfactant in step (a) is Sorbitan monooleate (Span 80).

24. The process as claimed in claim 20 wherein said predefined conditions in step (a) for emulsification is disruption by ultrasonic disruptor for 5 min at 4 ?C.

25. The process as claimed in claim 20 wherein said synthetic polymer in step (b) is selected from the group comprising poly(vinyl alcohol), or substituted celluloses such as hydroxyethylcellulose or a combination thereof.

26. The process as claimed in claim 25 wherein said water soluble synthetic polymer is poly(vinyl alcohol) (PVA).

27. The process as claimed in claim 20 wherein said predefined conditions in step (c) are emulsifying the primary emulsion for 7 min at 4 ?C.

28. The process as claimed in claim 20 wherein said predefined conditions in step (e) are centrifugation at 20,000 rpm for 20 min at 4 ?C.

29. The process as claimed in claim 20 wherein said cryoprotectant in step (f) is mannitol 10% w/v.

30. The process as claimed in claim 20 wherein said specific conditions for lyophilization in step (g) are freezing to -80 °C at 0.05 mbar pressure for 48 h.

31. A mucoadhesive bromelain nanoparticle formulation for management of asthma, said formulation comprising bromelain nanoparticle of claim 1 with or without pharmaceutically acceptable surfactant and/or excipient.

32. The mucoadhesive bromelain nanoparticle as claimed in claim 31, wherein said formulation is a sustained drug release formulation.

33. The mucoadhesive bromelain nanoparticle as claimed in claim 31, wherein said formulation is administered via nasal route.

FIELD OF THE INVENTION

The present invention broadly relates to a bromelain nanoparticle and a mucoadhesive drug formulation thereof in allergic asthma management. More particularly, the present invention discloses an intranasal formulation comprising bromelain and methacrylate ester copolymer. The present invention also relates to a process of preparation of an intranasal nanoparticle comprising bromelain and methacrylate ester copolymer for allergic asthma management.

BACKGROUND OF THE INVENTION
Asthma is a polygenic multifactorial autoimmune chronic inflammatory respiratory disorder 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.
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 fruitful results in experimental and clinical investigations have drawn significant attention of pharmaceutical researchers and scientists for development of herbal treatments 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 by decreasing 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, the gastric instability and poor patient compliance due to high oral dose limits therapeutic potential of bromelain. 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 bromelain therapy.
Nanomedicine has gained enormous attention in development of novel therapies for biopharmaceuticals like proteins, peptides and enzymes to impel their nasal delivery realistic. Their unique properties like small size, high surface area, controlled drug delivery improves patient compliance by enhancing bioavailability and reducing adverse or toxic effects associated with drug.
US11119861 discloses improved preparation of Bromelain in which the Bromelain coated with an organic network polymer is constructed by cross-linkage between organic acids and polysaccharides by electron-beam irradiation. The Bromelain is embedded in an organic network polymer, which will protect Bromelain from degradation in acidic environments.
“Implications of designing a bromelain loaded enteric nanoformulation on its stability and antiinflammatory potential upon oral administration”, RSC Adv., 2018, 8, 2541 teaches to develop an enteric nano-formulation of bromelain to improve its stability and anti-inflammatory potential. Bromelain loaded nanoparticles (Br-NPs) were developed using a Eudragit L 100 polymer by a double emulsion solvent evaporation method to obtain gastro-resistant properties.
“Anti-cancer activity of bromelain nanoparticles by oral administration”; J Biomed Nanotechnol. 2014 Dec;10(12):3558-75 discloses that oral administration of anti-cancer drugs is an effective alternative to improve their efficacy and reduce undesired toxicity. Bromelain (BL) is known as an effective anti-cancer phyto-therapeutic agent, however, its activity is reduced upon oral administration. In addressing the issue, BL was encapsulated in Poly(lactic-co-glycolic acid) (PLGA) to formulate nanoparticles (NPs). Further, the NPs were coated with Eudragit L30D polymer to introduce stability against the gastric acidic conditions. Oral administration of NPs reduced the tumor burden of Ehrlich ascites carcinoma (EAC) in Swiss albino mice and also increased their life-span (160.0 ± 5.8%) when compared with free BL (24 ± 3.2%). The generation of reactive oxygen species, induction of apoptosis and impaired mitochondrial membrane potential in EAC cells treated with NPs confirmed the suitability of Eudragit coated BL-NPs as a promising candidate for oral chemotherapy.

“Development of membranes based on carboxymethyl cellulose/acetylated arrowroot starch containing bromelain extract carried on nanoparticles and liposomes”, J Pharm Sci. 2021 Mar 1, discloses four membrane formulations produced by solvent evaporation: the control, membranes containing free bromelain, bromelain-loaded nanoparticles (NPs) and bromelain-loaded liposomes (LIPs). The enzyme concentration was the same for all formulations. Transparent, flexible and intact films were obtained. The membranes containing free bromelain, bromelain-loaded NPs and bromelain-loaded LIPs had higher water content, lower water vapor permeability and maximum tensile strength, and greater elongation at rupture. The capacity to absorb simulated exudate was higher in samples containing free bromelain, and bioadhesion was reduced in the presence of free bromelain compared to the control. An in vivo assay was performed to verify the membranes' healing potential. Histological analysis revealed no edema on the 14th day in animals treated with membranes containing bromelain-loaded NPs and LIPs.
“Preparation and Characterization of Mucoadhesive Nanoparticles (NPs) Containing Quercetin and Eudragit® RS 100 for Nasal Drug Delivery”, Shweta R. Jawanjar et al., Sch Acad J Pharm, Feb, 2020; 9(2): 58-67 teaches development of Quercetin (QCT) nanoparticles in order to accomplish poor aqueous solubility of quercetin and enhancement of drug absorption rate to nasal mucosa by increasing drug retention time using single emulsion technique. For this purpose, Eudragit® RS 100 was chosen as mucoadhesive polymer and used at different drug/polymer ratios in the nanoparticles formulations and discloses that QCT NPs based on a Eudragit RS 100 may be a promising nasal delivery system improved permeation profile for longer period of time and thereby increasing the patience compliance.
However, there is no preparation suggested for nasal drug delivery of bromelain for effective asthma management. Mucociliary clearance reduces the residence time of drug-loaded nanoparticles on mucosal surface of the nose. The inventors of the present invention found that the mucoadhesive polymeric nanoparticulate system may be one of the useful approaches for controlled drug delivery at absorption site and specific site of inflammation.
Eudragid RL 100, a cationic biodegradable and non-toxic copolymer of poly (ethylacrylate, methyl-methacrylate and chlorotrimethyl-ammonioethyl methacrylate) is a polymer of choice for development of nanoengineered biopharmaceutical therapies. It exhibits mucoadhesive behavior by selectively interacting with negatively charged mucosal surface and improves residence time of drug at absorption site. Polymeric nano-carriers also protects biopharmaceuticals against physical aggregation and in vivo enzymatic inactivation. Thus, reduces therapeutic effective dose and risk of dose dependent toxicities. Moreover, fabrication of polymeric nano-carriers in powder form also facilitates loading into sachets or capsules for inhalation.
Therefore, the present invention aims to develop a non-irritant bromelain loaded methacrylate ester copolymer nano-carriers (Br-EuNPs) intended for the prophylaxis and management of asthma via nasal route. The invention proposes to develop mucoadhesive formulation of bromelain for nasal delivery owing improved shelf-life and therapeutic efficacy in allergic asthma management by attenuating the allergen induced inflammatory responses.

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 bromelain nanoparticles comprising mainly of bromelain loaded methacrylate ester copolymer (Br-EuNPs).

Another object of the present invention is to provide bromelain nanoparticles comprising mainly of bromelain and methacrylate ester copolymer (Br-EuNPs) showing improved shelf-life and therapeutic efficacy in allergic asthma management by attenuating the allergen induced inflammatory responses.

Yet another object of the present invention is to provide a mucoadhesive formulation comprising bromelain and methacrylate ester copolymer with or without pharmaceutically acceptable surfactant and/or excipient.

Yet another object of the present invention is to provide a mucoadhesive sustained release drug formulation of bromelain and methacrylate ester copolymer for nasal delivery.

Yet another object of the invention is to provide a process to obtain bromelain nanoparticles comprising mainly of bromelain loaded methacrylate ester copolymer nanoparticles (Br-EuNPs).

Yet another object of the invention is to provide a mucoadhesive sustained release drug formulation of bromelain nanoparticles comprising mainly of bromelain and methacrylate ester copolymer.

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 present invention is to provide bromelain nanoparticle capable of being used in the management of asthma. More specifically, the invention provides bromelain nanoparticle comprising mainly of bromelain loaded methacrylate ester copolymer (Br-EuNPs). The Br-EuNPs shows enhanced therapeutic efficacy, bioavailability and improved drug loading capacity.

The invention also provides a mucoadhesive nanoparticle formulation of Br-EuNPs capable of being administered via nasal route. The formulation shows sustained release controlled drug delivery, higher cellular uptake with reduced adverse effects and improved patient compliance.

In one aspect, the present invention provides Bromelain nanoparticle comprising bromelain and methacrylate ester copolymer having repeating units of formula I, a surfactant and a synthetic polymer.
Formula I
wherein R1 is selected from hydrogen and methyl, R2 is selected from methyl, and ethyl, R3 is methyl and R4 is ethyltrimethylammonium chloride.

In other aspect of the present disclosure, the nanoparticle comprises bromelain and methacrylate ester copolymer of formula I in a ratio ranging from 1:1 to 1:7.5.

In another aspect of the present invention, the nanoparticle comprises bromelain and methacrylate ester copolymer of formula I in a ratio of 1:3.

In yet another aspect of the present invention, the formulation comprises Br-EuNPs with or without pharmaceutically acceptable surfactants or excipients or carriers or any combination thereof. The formulation is suitable for nasal drug delivery.

The nanoparticles of the present invention have particle size in the range of 227.91 ± 26.01 to 625.63 ± 43.77 nm, zeta potential ranging from 8.5 ± 2 to 29 ± 2 mV and entrapment efficiency of 40 ± 8 to 90 ± 8 %.

In an aspect of the present invention, the nanoparticles of the present invention have particle size 259.73 ± 16.51 nm, zeta potential 27.53 ± 1.66 mV and entrapment efficiency of 84.76 ± 1.49 %.

In yet another aspect, the present invention provides a process for the preparation of bromelain nanoparticle comprising of bromelain and methacrylate ester copolymer having repeating units of formula I, a surfactant and a synthetic polymer.
Formula I
wherein R1 is selected from hydrogen and methyl, R2 is selected from methyl, and ethyl, R3 is methyl and R4 is ethyltrimethylammonium chloride.

In one embodiment, the formulation is in the dosage form suitable for nasal drug delivery.

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

BRIEF DESCRIPTION OF DRAWINGS
Figure 1 shows SEM images of developed optimized formulation (B3)
Figure 2 shows fluorescence spectra of pure bromelain and bromelain released from Br-EuNPs of the present invention.
Figure 3 shows (A) In vitro release of bromelain from Br-EuNPs in dissolution media (phosphate buffer pH 6.8) with different bromelain polymer ratios, (B) In vitro release of bromelain from optimized ratio (1:3).
Figure 4 shows effect on viscosity of artificial mucous on (A) varying bromelain concentration (B) comparing effect of pure bromelain solution and formulation.
Figure 5 shows Plasma drug profile of pure bromelain and Br-EuNPs administered nasally.
Figure 6 shows Comparative biodistribution study of pure bromelain and optimized Br-EuNPs in different organs (A) Lung (B) Liver (C) Intestine (D) Spleen respectively at different time interval.
Figure 7 shows Effect of treatment on ovalbumin induced asthma on (A) Bronchospasm onset time, recovery time and convulsion period (B) Percentage protection.
Figure 8 shows Effect of different treatments on edema in (A) Lung (B) Liver of ovalbumin induced asthmatic guinea pigs.
Figure 9 shows Assessment of alterations in haematological parameters (A) Haemoglobin (B) Total leukocyte count (C) Eosinophils (D) Neutrophils (E) Lymphocytes respectively on 21st day in asthma.
Figure 10 shows Evaluation of oxidative markers (A) LPO level (B) Carbonyl content (C) MPO level (D) NO (%) (E) % Catalase level (F) % Glutathione level (G) % SOD in lung, liver, spleen, trachea and BALF respectively. Immunological markers level in serum and BALF (H) TNF – a (I) IL-5 (J) Ig-G. BAL total cell count (K) and (L) Eosinophil count in BALF in asthma model.
Figure 11 shows Histopathological images of guinea pig’s lung of (A) Naïve control, (B, C) ovalbumin sensitized group (D) Pure bromelain treated (E) Fluticasone furoate treated (F) optimized Br-EuNPs treated.
Figure 12 shows Histopathological images of guinea pig’s nasal mucosa of (A) Naïve control, (B, C) ovalbumin sensitized group (D) Pure bromelain treated (E) Fluticasone furoate treated (F) optimized Br-EuNPs treated.

DETAILED DESCRIPTION OF THE INVENTION WITH NON-LIMITING EMBODIMENTS AND ILLUSTRATIONS
The present invention relates to a bromelain nanoparticle comprising mainly of bromelain and methacrylate ester copolymer having of formula I

Formula I
wherein R1 is selected from hydrogen and methyl, R2 is selected from methyl, and ethyl, R3 is methyl and R4 is ethyltrimethylammonium chloride.

The invention also relates to a process to obtain bromelain nanoparticle comprising mainly of bromelain loaded with methacrylate ester copolymer having of formula I.

The present invention relates to a bromelain nanoparticle comprising bromelain and methacrylate ester copolymer having of formula I, a surfactant, a synthetic polymer and an organic solvent,

Formula I
wherein R1 is selected from hydrogen and methyl, R2 is selected from methyl, and ethyl, R3 is methyl and R4 is ethyltrimethylammonium chloride. The invention also relates to a process to obtain bromelain nanoparticle comprising bromelain and methacrylate ester copolymer having of formula I and a mucoadhesive formulation thereof.

In an aspect of the present invention, the methacrylate ester copolymer is Eudragit RL 100 or Eudragit RS 100.
In another aspect of the present invention, the methacrylate ester copolymer is Eudragit RL 100.

The process to prepare bromelain nanoparticle (Br-EuNPs) is via modified double emulsion solvent evaporation technique. An aqueous phase of bromelain is emulsified with an organic phase employing ultrasonic disruption under specific conditions to obtain primary emulsion (w/o). The organic phase is constituted by dissolving methacrylate ester copolymer in an organic solvent containing a surfactant.

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.

The primary emulsion is dispersed and emulsified in aqueous solution of a synthetic polymer by probe sonication under specific conditions to obtain secondary emulsion (W1/O/W2). Said synthetic polymer is selected from the group comprising 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 at room temperature to facilitate complete evaporation of the organic solvent to obtain bromelain nanoparticle dispersion.

The organic solvent free double emulsion or bromelain nanoparticle dispersion is centrifuged at specific conditions to obtain pellets. The pellets are suspended in distilled water containing a cryoprotectant and frozen under specific freezing conditions and resultant suspension is lyophilized to obtain lyophilized bromelain nanoparticles. The nanoparticles are characterized and stored at 4 ºC till further use.

In an aspect of the present invention, the bromelain and copolymer ratio is ranging from 1: 1 to 1:7.5.

In another aspect of the present invention, the bromelain and copolymer ratio is 1:3.

In a preferred embodiment, an aqueous phase (0.5 ml) consisting of bromelain (50 mg) is emulsified with an organic phase under probe sonication at 100 W output power, 40% amplitude, PCI analytics for 5 min at 4 ?C in ice bath to form primary (w/o) emulsion. Methacrylate ester copolymer (150 mg) is dissolved in dichloromethane (5 ml) containing a surfactant (0.5 % w/v, span 80) constituted the organic phase. The primary emulsion (w/o) is added drop by drop to PVA (1% w/v) solution for further emulsification under probe sonication for 7 min at 4 ?C. So formed w/o/W emulsion is continuously stirred at 75 rpm for 16 h at room temperature to facilitate complete evaporation of organic solvent to obtain bromelain nanoparticles.

Nanoparticles are collected as a pellet by centrifuging the dispersion at 20,000 rpm for 20 min at 4 ?C. Subsequently, collected pellet are suspended in distilled water containing mannitol (10 % w/v) and frozen at -20 ?C for 2 h followed by freezing at -80 ?C. Frozen samples are lyophilized at -80 ºC and 0.05 mbar pressure for 48 h to obtain lyophilized mucoadhesive nanoparticles of bromelain.

In order to optimize the formulation and process parameters, various Br-EuNPs batches are prepared by varying drug- polymer ratio, sonication time, type and concentration of surfactant, external phase volume and amount of drug as presented in Table 1.
The Br-EuNPs has been optimized through strategic experimentation by optimizing various variables like drug-polymer ratio, organic phase to external aqueous phase volume and type of surface-active agent. The process variable such as sonication time has also been optimized. Both the Br-EuNPs as well as process variable have been optimized taking into account average particle size, entrapment efficiency, PDI and zeta potential as paradigm framework. (Table 1, Table 2).

Various batches of Br-EuNPs are referred in the experimentation with formulation codes as B1, B2, B3, …B19. The nanoparticles in B3 formulation shows highest entrapment efficiency (84.76 ± 1.49 %) with particle size (259.73 ± 16.51 nm), PDI (0.14 ± 0.02) and zeta potential (27.53 ± 1.66 mV).
Table 1: Composition of different batches prepared.
Formulation code Polymer (mg) Drug (mg) Surfactant Sonication time during emulsification (min) Disperse phase volume (ml) Continuous phase volume (ml)
Tween 80 Pluronic F - 68 Span 80
Primary Secondary
B1 50 50 - - 0.5 5 7.5 0.5 20
B2 100 50 - - 0.5 5 7.5 0.5 20
B3 150 50 - - 0.5 5 7.5 0.5 20
B4 200 50 - - 0.5 5 7.5 0.5 20
B5 150 25 - - 0.5 5 7.5 0.5 20
B6 150 75 - - - 5 7.5 0.5 20
B7 150 20 - - 0.5 5 7.5 0.75 20
B8 150 20 - - 0.5 5 7.5 1 20
B9 150 20 - - 0.5 5 7.5 0.5 30
B10 150 20 - - 0.5 5 7.5 0.5 40
B11 150 20 - 0.5 - 5 7.5 0.5 20
B12 150 20 0.5 - - 5 7.5 0.5 20
B13 150 20 - - 0.125 5 7.5 0.5 20
B14 150 20 - - 0.25 5 7.5 0.5 20
B15 150 20 - - 0.5 2.5 5 0.5 20
B16 150 20 - - 0.5 5 5 0.5 20
B17 150 20 - - 0.5 7.5 5 0.5 20
B18 150 20 - - 0.5 5 2.5 0.5 20
B19 150 20 - - 0.5 5 5 0.5 20

Table 2: Effect of different variables on particle size, zeta potential, polydispersity index and entrapment efficiency.
Formulation code Particle Size (nm ± SD) Polydispersity index (PDI ± SD) Zeta potential (mV ± SD) Entrapment efficiency (% ± SD)
B1 227.91±26.01 0.176±0.015 18.46±1.02 61.46±0.79
B2 266.86±27.16 0.180±0.017 22.83±1.68 75.25±1.25
B3 259.73±16.51 0.143±0.016 27.53±1.66 84.76±1.49
B4 395.23±8.87 0.201±0.008 27.93±1.45 85.98±2.40
B5 238.16±13.80 0.122±0.067 17.86±1.20 61.79±1.54
B6 345.71±7.38 0.262±0.032 21.66±2.51 85.19±2.29
B7 328.26±34.02 0.248±0.033 19.8±1.28 75.98±1.23
B8 474.43±22.71 0.191±0.076 20.4±0.80 45.96±7.58
B9 313.99±11.10 0.247±0.031 23.36±7.75 78.04±0.87
B10 294.93±17.62 0.174±0.061 22.4±2.98 66.13±2.11
B11 255.03±59.29 0.345±0.03 9.4±1.21 58.17±10.4
B12 338.36±26.85 0.225±0.017 16.76±1.20 77.26±10.1
B13 446.16±25.90 0.183±0.010 17.5±0.91 69.8±23.01
B14 328.86±9.84 0.208±0.032 16.46±2.40 70.9±0.68
B15 625.63±43.77 0.456±0.090 19.33±1.06 72.01±1.37
B16 360.16±23.86 0.249±0.028 20.73±1.30 75.18±1.76
B17 549.87±33.11 0.359±0.022 22.33±0.66 73.41±1.21
B18 374.91±21.75 0.372±0.010 23.43±1.45 73.20±1.03
B19 389.95±21.60 0.246±0.018 23.6±0.90 73.71±2.34

Thus, the present invention provides mucoadhesive formulation comprising bromelain, methacrylate ester copolymer of formula I, a synthetic polymer, and a pharmaceutically acceptable surfactants or excipients or carriers or any combination thereof. The formulation shows sustained release controlled drug delivery, enhanced bioavailability, therapeutic efficacy and improved drug loading capacity. The formulation is capable of being used in the management of asthma.

The mucoadhesive formulation of bromelain nanoparticle comprising bromelain in the range of 0.4 - 1.5% (w/v), methacrylate ester copolymer of formula I in the range of 1.0 – 4.0% (w/v), surfactant in the range of 0.125 – 0.5%(w/v) and synthetic polymer is 1.0% (w/v).

In a preferred aspect of the present invention, the mucoadhesive formulation of bromelain nanoparticle comprises of: bromelain 1% (w/v), methacrylate ester copolymer of formula I 3.0% (w/v), surfactant 0.5% (w/v) and synthetic polymer 1.0% (w/v).

The mucoadhesive formulation 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 97.04±1.65 % under cool temperature of 4 ± 2 ºC at 65 ± 5 % relative humidity (RH) and up to 95.23±1.25 % 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 94.16±3.10 %.

Characterization of Br-EuNPs
The enzymatic activity of the Br-EuNPs is determined by modified casein digestion method to determine the proteolytic potential of bromelain.
Colloidal property of the lyophilized Br-EuNPs like particle size, polydispersibility index (PDI) and zeta potential are determined employing Nanosizer. Lyophilized formulations are dispersed in distilled water via sonication (30 s) and suitably diluted before estimation.
Dispersion of Br-EuNPs is centrifuged at 20,000 rpm for 20 min at 4 °C. The clear supernatant obtained is assayed for protein content by lowery method using U.V./Vis spectrophotometer at 750 nm. Entrapment efficiency is calculated by formula

The drug release studies of the optimized Br-EuNPs are performed employing modified dialysis bag diffusion method. The Br-EuNPs equivalent to 25 mg bromelain kept in dialysis bag is immersed in phosphate buffer pH 6.8 (100 ml) stirred at 100 rpm and maintained at 37 ± 2 ºC. Aliquots withdrawn at different time intervals are replaced with equal volume of dissolution media and analyzed spectrophotometrically for protein content by lowery method.

Morphological evaluation of the lyophilized formulation is examined by field emission scanning electron microscopy. A sample of the lyophilized formulation is mounted on aluminum stubs using carbon adhesive tape and coated in thin layer using a vacuum evaporator. The sample is observed at an acceleration voltage of 5 KV and 30 mm of working distance to capture images.

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

The artificial mucous having viscoelastic behavior similar to human airway mucus is 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 for 24 h using magnetic stirrer. Losses in volume occurring due to evaporation are adjusted by adding sufficient amount of sodium nitrite solution whenever required. The galactomannan chains in locust gum bean solution are cross linked by addition of 0.1 M sodium tetraborate. The resultant mucous (2 gm) is 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 bromelain (0.2 % w/w) and Br-EuNPs dispersion (equivalent to 0.2 % w/w bromelain) on viscoelastic properties of artificial mucous is also determined at predetermined time interval at 2, 4, 6, 8, 24 and 26 h using Rheometer at 25 °C. Samples without enzyme served as reference.

Bromelain and the Br-EuNPs are bestowed to stability testing respectively to establish their ideal storage conditions and shelf life according to ICH guideline for zone III and IV. Samples of bromelain and the Br-EuNPs are loaded in amber colored glass vials and sealed before storing at accelerated storage condition (40 ± 2 ºC / 75± 5 % RH) for 6 months, room temperature (25 ± 2 ºC / 60 ± 5 % RH) and cool temperature storage condition (4 ± 2 ºC / 60 ± 5 % RH) for 12 months respectively. Samples are evaluated for percentage bromelain activity remaining and colloidal properties (particle size, zeta potential and PDI) after a suitable time interval of 0, 3, 6, 9 and 12 months respectively. Visual inspection of samples is also done after completion of stability study.

In vivo studies
In vivo studies are performed in wistar rats (150-200 g) and guinea pigs (250-300 g). Animals used in study are received from animal house of Pharmacy Department, Banasthali Vidyapith. Wistar rats and guinea pigs are housed in an air-conditioned room at an ambient temperature of 23 ± 2 °C, 50 ± 5 % RH with 12h dark/light cycles, food and water ad libitum. The experimental protocols are approved by Institutional Animal Ethics Committee of Banasthali Vidyapith, Rajasthan, India. All the experimental procedures are in obedience with CPCSEA guidelines for the safe use and care of experimental animals.

Pharmacokinetic and Biodistribution studies
The quantitative systemic availability of drug in wistar rats after nasal administration of drug and optimized formulation is evaluated. Wistar rats (42) have been randomly distributed into two groups. The first group received drug solution (10 mg/kg) while second group received optimized formulation (equivalent to 10 mg/ kg bromelain) by nasal route in each nostril with the help of micropipette with 0.1 mm internal diameter at delivery place. After an interval of 0.5, 1, 2, 4, 6, 8, 10 and 24 h post dosing, blood was collected. Blood was centrifuged at 3000 rpm for 5 min to collect plasma.

The quantitative biodistribution of drug and optimized formulation is studied in healthy wistar rats after nasal administration. Wistar rats (36) have been randomly distributed into two groups. The first group received drug solution (10 mg/kg) while second group received optimized formulation (equivalent to 10 mg/ kg bromelain) by nasal route in each nostril with the help of micropipette with 0.1 mm internal diameter at delivery place. After an interval of 0.5, 1, 2, 4, 6 and 12 h post dosing, three rats per group have been sacrificed to collect tissues (lung, liver, intestine and spleen). Collected tissues have been washed, blotted dry on a tissue paper, weighed and homogenized in PBS. Clear supernatants obtained by centrifugation of tissue homogenates for 20 min at 25,000 rpm is used for further study.

Plasma or clear supernatant samples (200 µl/ well) are placed in microtiter plate respectively 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) has been added to each well to stop the reaction. The undigested casein has been allowed to precipitate by incubating the microtiter plate at room temperature for 1 h. The clear supernatant obtained has been transferred to noncoated opaque microtiter plates. Fluorescence intensity has been estimated at an excitation wavelength of 280 nm and 330 nm emission wavelengths. Fluorescence intensity observed during proteolytic assay has been proportional to amount of bromelain in the samples. Results of tissue distribution study have been normalized to protein level.
Pharmacodynamics studies on Ovalbumin induced asthma
Sensitization and Antigen Challenge
Twenty male healthy guinea pigs (250-300 g) have been 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 have been 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 (nasally 0.5 mg/Kg, fluticasone furoate), bromelain solution (5 mg/ Kg) and optimized formulation (5 mg/Kg equivalent to bromelain) for 5 days. Naïve animals (n=5) have been sham sensitized with 100 µl normal saline similar to OVA sensitized animals. Animals of respective group have been challenged with 1% histamine dihydrochloride solution in a clear plexiglass histamine chamber after 5 days of consecutive treatment respectively. Animals have been 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 have been recorded and compared with saline treated group. The treatment offered percentage protection against asphyxia was calculated by using this formula:
% Protection =
Where T1= time of onset of asphyxia before drug treatment, T2 = time of onset of asphyxia after treatment
% Recovery time =
RT2= Recovery time before drug treatment, RT1= Recovery time after drug treatment
Subsequently, animals have been sacrificed after histamine aerosol exposure to collect blood samples by cardiac puncture and organs like lung, liver, spleen and trachea. Whole blood collected has been 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. Consecutively, tissue homogenates prepared in potassium chloride solutions (10% w/v) have been evaluated for antigen specific response by quantitating level of oxidative stress markers like lipid peroxidation, carbonyl content, myeloperoxidase activity, reduced glutathione, superoxide dismutase, catalase, EPO and nitric oxide level.

Trachea of sacrificed animals immediately after blood collection has been carefully exposed and cannulated. Bronchoalveolar lavage has been executed five times by infusing normal saline (2 ml) into lungs via cannula and aspirated after gentle massage. Samples collected have been pooled to determine total number of cells/ ml using Neubauer hemocytometer. Slides have been 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.
Isolated lung and nasal mucosa are 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
SEM images manifest uniform spherical shape with smooth surface of Br-EuNPs along with nanometric size ~250 nm which abide by the results of photon correlation spectroscopy (Figure 1).

Fluorescence spectra of pure bromelain and bromelain released from formulation showed ?max at 330 nm corresponding to intense emission of tryptophan. The results confirmed that no conformational changes in the tertiary structure of encapsulated bromelain in nanoparticles has occurred (Figure 2). Thus, it clearly designates that formulation parameters are properly optimized to retain the tertiary structure i.e., functional stability of bromelain during encapsulation.

In vitro release profile of bromelain from Br-EuNPs formulations is shown in figure 3. All formulations exhibited nonlinear burst release followed by sustained bromelain release in 24 h. Prolonged sustained drug release with smaller burst release has been observed from formulation with higher proportionate amount of polymer i.e., having drug-polymer ratio 1:4. Formulations with drug-polymer ratio 1:1 and 1:2 respectively showed faster initial drug release compared to 1:3. The proportionately higher amount of polymer in formulation with drug -polymer ratio 1:3 hinders the penetration of dissolution medium in matrix and promote prolonged release of drug from polymer matrix. Depending upon the drug release profile along with characteristic features of formulation like particle size, PDI and zeta potential, B3 formulation was used for further studies. Further, release data of B3 formulation showed best fit in Korsmeyer Peppas release model (R2 = 0.946). The numerical value of release exponent (n=0.692) indicated the anomalous drug release behavior i.e., combination of both diffusion and dissolution (Figure 3).

Artificial mucus composed of locust bean gum, a galactomannan polysaccharide having (1?4) linked ß-D-mannopyranosyl backbone partially substituted with single (1?6) linked a-D-galactopyranosyl side groups with around 5% protein content has been utilized to evaluate the bromelain’s mucolytic activity. The effect of concentration of bromelain on its mucolytic activity has been evaluated by estimating the viscosity of artificial mucus after different time intervals of exposure using rheometer. Consequently, remarkable decrease in viscosity of mucous has been observed with increase in concentration of bromelain from 0.1 % w/w to 0.4 % w/w (Figure 4A) due to irreversible breakdown of three-dimensional network of locust bean gum mucilage. Subsequently, remarkable decrease in viscosity has been 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 has been 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 nanoparticles within mucus has been contributed by the continuous release of bromelain from the 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 (Figure 4B). The mucolytic results have complied with slow and progressive in vitro release behavior of formulation. In addition, positive surface charge of nanoparticle at physiological pH too favors strong adhesion on negative charged mucus surface providing sufficient residence time to bromelain to act. Br-EuNPs are 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.

The ability of optimized Br-EuNPs to sustain colloidal properties, release behavior and percentage bromelain activity has been evaluated till 12 months under room (25 ± 5 ºC / 60 ± 5 % RH) and cool (4 ± 2 ºC / 65 ± 5 % RH) whereas for 6 months under accelerated temperature storage conditions (40 ± 2 ºC / 75 ± 5 % RH) as per the ICH guidelines. Furthermore, influence of storage conditions on percentage activity remaining of pure drug has also been analyzed (Table 3, 4). Pure drug under accelerated and room temperature storage conditions showed remarkable decrease in drug content compared to Br-EuNPs. However, pure drug showed ~98 % drug content under cool conditions indicating that proteolytic activity of drug was protected at lower temperature. The Br-EuNPs 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 are 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-EuNPs under real time stability conditions. The calculated t90 for formulation has been ~ 5.99-folds higher compared to pure drug at room temperature manifesting significantly better storage stability of formulation. The visual changes in color of pure drug from off white to brown has been observed at both room and accelerated temperature storage condition respectively whereas no color change has been observed for Br-EuNPs.

Table 3: Stability study of pure drug and optimized formulation (B3) 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 powder 100.00±1.23
95.45±2.34
90.46±1.11
85.86±1.90
81.23±2.34 73.12±4.32
8.39×10-4
125.14

B3 100.00±3.74 99.82±2.44
98.44±4.89
96.85±7.06
96.44±3.41
95.23±1.25
1.40×10-4
750.00

4°C±2°C/65 ± 5 % RH Bromelain powder 100.00±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
B3 100.00±1.76 99.82±2.38
98.80±3.50
97.72±5.01
97.26±2.71
97.04±1.65
9.2×10-5
1141.30

40±2ºC/75 ±5% RH Bromelain powder 100.00±1.23
89.45±2.90
85.90±2.50
74.18±1.79 1.67×10-3
62.76

B3 100.00±2.34
98.68±2.09
97.23±1.11
94.16±3.10
2.20×10-4
125.82

# 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 4: Effect of storage condition on colloidal properties of optimized formulation at different time interval
Storage condition Formulation (A8) 0 M 1.5 M 3 M 6M 9M 12M
25±2ºC/60 ± 5% RH Particle Size 259.70±16.50
260.24±11.22
273.73±10.31
269.22±19.65
297.20±11.32
298.75±15.34
PDI 0.14±0.03
0.15±0.02
0.16±0.01
0.17±0.01
0.18±0.02
0.19±0.01
Zeta potential 27.31±3.12
27.01±1.45
27.18±3.33
26.94±1.10 25.23±2.04
24.56±1.67
4°C±2°C/65 ± 5 % RH Particle Size 259.70±16.56

262.61±14.12
265.11±9.23 273.42±21.33
288.14±10.13
291.20±14.31

PDI 0.14±0.03
0.15±0.02
0.15±0.02
0.18±0.02
0.17±0.01
0.18±0.03
Zeta potential 27.31±1.66

27.47±2.67

27.03±1.54

25.96±2.99
24.21±2.04 24.54±1.67
40±2ºC/75 ±5% RH Particle Size 259.70±13.45 267.31±16.18
272.48±19.23
282.13±25.38

PDI 0.14±0.01
0.15±0.01
0.17±0.01
0.19±0.01

Zeta potential 27.31±3.44
25.14±5.09
24.38±3.09
23.89±1.87

Pharmacokinetic studies
Plasma drug concentration profile of pure bromelain solution and optimized formulation post nasal administration in wistar rats is depicted in table 5 and figure 5 respectively. Br-EuNPs after single dose of administration showed significantly elevated Cmax (1.13-fold), tmax (2-fold) and AUC0-24 (2.82-fold) as compared to pure drug. Higher Cmax and AUC0-24 might be attributed by mucoadhesion of nanoparticles in nasal mucosa facilitating enhanced absorption due to their nano-size and their hydrophobic surface accelerating rapid permeation across the nasal mucosa. Apparently loading bromelain into nanoparticles might have helped in bypassing extensive metabolism of bromelain via nasal enzymes. Higher Tmax confirmed the sustained bromelain release from the formulation. Nanoparticulate formulation extended elimination half-life (2.05-fold) and MRT (1.37-fold) depicting contributory effect of cationic copolymer which interacts with anionic mucin layer to increase the residence time of nanoparticles. The results of pharmacokinetic study revealed that both rate of elimination (Ke) and clearance have been remarkably lower for formulation compared to free bromelain. Increase in MRT for bromelain in formulation can be correlated to reduced Ke and clearance. The obtained pharmacokinetic result signifies that Br-EuNPs exhibit 2.48-fold increase in relative bioavailability compared to drug solution.

Table 5: Pharmacokinetic parameter of pure drug and optimized formation obtained after nasal administration at an equivalent dose of 10 mg/kg bromelain.
PARAMETER Bromelain solution Formulation
Cmax (ng ml-1) 4.38 ± 2.09 4.97 ± 1.88
Tmax (h) 1.00 ± 0.12 2.00 ± 0.11
Ke (h-1) 0.19 ± 0.08 0.09 ±0.10
t1/2 (h) 3.58 ± 0.98 7.33 ±1.01
MRT (h) 4.94 ± 0.77 6.77 ±0.87
AUC (ng h2 ml-1) 19.06 ± 3.21 53.80 ±4.21
Relative bioavailability (%) 248.50
Values are expressed as mean ± SD, n=3; * p<0.001 level of significant difference; ** p<0.05 level of significant difference

Tissue distribution studies of Bromelain in pure solution and in the formulation of present invention have been assessed in wistar rats after nasal administration. Figure 6 depicts distribution of pure drug and bromelain loaded nanoparticles in all organs at definite time period (0.5, 1, 2, 4, 6, 12 h). Peak drug concentration in lung, liver and spleen has been achieved in 1 h and 2 h after nasal administration of pure drug solution and formulation respectively. This might be due to rapid clearance of drug from the nasal cavity resulting in rapid systemic drug absorption. However, with progression of time drug concentration declined due to drug metabolism. Conversely, bromelain nanoparticles of the formulation showed increase in drug concentration in all organs with progression of time. This might be contributed by increase in drug residence time in nasal cavity, protection offered by formulation to bromelain from enzymatic degradation, sustained drug release behavior of formulation absorbed systemically through M cells in nasal associated lymphoid tissues. The results assured that Br-EuNPs could markedly amplify tissue distribution of bromelain apparently in liver and lung. Moreover, increase in bromelain level has been observed in lung (18.38 ±1.60 ng/gm), signifying that nanoparticle in systemic circulation could be renowned and phagocytized as extraneous substances by the mononuclear phagocyte system prevalent in lung.

Pharmacodynamics on Ovalbumin induced asthma model
During study various parameters like bronchospasm onset, intensity of spasm, persistent episodes of convulsions and trouble in breathing have been observed to assess the effect of treatment on airways hyperresponsiveness against histamine aerosol exposure. OVA-sensitized animals showed significantly higher airway responsiveness than normal saline treated animals. The significant increase in preconvulsive dyspnea time by 29.50%, 14.75% and 196.72% (from 1±0.04 min to between 1.15±0.07 min, 1.22±0.12 min and 3.01±0.32 min; p<0.05) and decrease in recovery time was observed after pre-convulsive dyspnea by 53.31%, 52.31% and 88.85% (from 2.87±0.54 min to 1.34±0.07 min, 1.36±0.03 min and 0.32 ±0.07 min; p<0.05) with drug, fluticasone and Br-EuNPs formulation compared to normal animals (Figure 7).

The delay in bronchospasm onset and reduction in severity of spasm as well as jerks were observed in drug and standard drug treated animals as compared to asthmatic control (Table 6). However, Br-EuNPs has extensively abolished bronchial hyperresponsiveness by delaying bronchospasm induction, inhibiting jerks and reducing severity of spasm approximately 351.37%, 129.11% and 158.57% respectively compared to asthmatic group, pure drug and fluticasone treated group respectively.

The relative increase in weight of lung (53.60 ± 2.02 %) and liver (47.04 ± 1.31%) has been observed in OVA-sensitized animals compared to naïve group. The increased weight of lung and liver indicated that allergen (OVA) stimulate high microvascular infiltration and edema leading to the inflammation of respective organ. Although significant reduction in relative weight of lung and liver has been observed in bromelain, fluticasone furoate and formulation (p<0.05) treated groups compared to OVA sensitized group (Figure 8). The results indicated that bromelain decreased the tissue damage and oedema in lungs due to its antioxidant and anti-inflammatory activity. Br-EuNPs showed remarkable reduction in weight of lung and liver in OVA sensitized animals by reducing microvascular infiltration and edema compared to bromelain.
Table 6: Effect of different treatments on severity of bronchospasm in ovalbumin induced asthma model.
Treatment Severity of bronchospasm
Control +++
Bromelain ++
Standard drug ++
Formulation +
Ovalbumin sensitized guinea pigs showed significant variation in hematological parameters compared to healthy animals. Elevation in hemoglobin (1.42-fold), total leukocyte count (3.86-fold), eosinophils (2.25-fold), lymphocytes (1.74fold), neutrophils (2.04-fold) was recorded in ovalbumin sensitized animals corresponding to naïve animals (Figure 9). Increased level of WBCs accounted for excessive release of histamine and inflammatory state of lungs. Moreover, high level of eosinophils in blood confirmed allergic inflammation associated with airway hyperresponsiveness contributing to the pathogenesis of airway disease. Elevated count of total cell in BAL fluid confirmed the infiltration of neutrophils, lymphocytes and eosinophils in lungs contributing to inflammation, mucus production and oedema in airways of lung. Intermittent pulmonary hypoxia associated with OVA exposure provoked the increment in RBC and haemoglobin in ovalbumin induced model. Br-EuNPs remarkably (p<0.05) reduced total leukocyte count, lymphocyte, platelet count, neutrophil and eosinophil count and fetched back near to normal level relative to pure drug by improving anti-inflammatory and antioxidant potential of bromelain (Figure 9).

Histamine challenge in OVA sensitized animals induced overproduction of oxidative markers i.e., LPO, MPO and protein carbonylation along with reduction of enzymatic (SOD and catalase) and non-enzymatic (GSH) antioxidant defense resulting in oxidant-antioxidant imbalance (Figure 10). This imbalance might lead to pathological alteration such as mucus hypersecretion, vascular permeability, smooth muscle contraction, epithelial detaching and nasal inflammation in asthma. However, treated groups (bromelain, fluticasone furoate and Br-EuNPs) showed remission in oxidative stress by diminishing LPO, MPO and carbonylated protein level in lung, liver, trachea, BAL fluid and spleen tissues respectively as compared to control group (p<0.05) (Figure 10). The results manifested that Br-EuNPs have been more efficient in rehabilitating catalase, SOD and GSH level as compared to pure bromelain and fluticasone furoate (p<0.05) (Figure 10). The noticeable repression of oxidative stress markers and augmentation of antioxidant defense confirmed the enhanced antioxidant potential of Br-EuNPs. Improved activity of Br-EuNPs might be attributed to mucoadhesive property of polymer which increased the adherence and residence time of drug on mucosal surface and facilitates slow release of drug. Moreover, enhanced lymphatic uptake of Br-EuNPs through phagocytosis and paracellular pathways, prolonged direct systemic absorption across nasal cavity, protection of drug from nasal enzymes as well as bypass of hepato-gastrointestinal first pass metabolism might have improved antioxidant potential of bromelain.

The significant elevation in nitric oxide level in control group signifies the upregulation of inducible iNOS and prostaglandin along with recruitment of the eosinophil and T lymphocyte at adjacent tissues. NO plays a vital role in pathogenesis of allergic asthma by enabling the production of cytokines (IL-5) essential for survival, differentiation and migration of eosinophils. Thus, cascade of events associated with elevation of NO increased mucous production in airways and promoted airway inflammation and nasal blockage in ovalbumin induced asthma model. Correspondingly increased level of Ig-G, TNF-a and IL-5 indicated increased oxidative burden due to overproduction of reactive oxygen species, nitric oxide synthesis as well as migration of neutrophils in tissues. However, treatment with bromelain, fluticasone and Br-EuNPs significantly reduced IgG, IL-5 and TNF-a. Bromelain’s inhibitory effect on NFKB overexpression and iNOS along with improved expression of Nrf2 pathway has bestowed its beneficial analgesic and anti-inflammatory effects for treatment of inflammatory conditions like asthma. Br-EuNPs have more pronounced effect in restoring the elevated level of NO, IgG, TNF-a and IL-5 to normal compared to bromelain (p<0.05). The enhanced antioxidant and anti-inflammatory action of Br-EuNPs might be attributed to improved nasal-stability and sustained release behavior of Br-EuNPs contributing extended restrained effect on cytokine storm. The results also confirmed the prospective of Br-EuNPs as a carrier for nasal bromelain delivery.
Histopathological analysis
Histopathological microphotographs of lung architecture in naïve, normal saline treated, bromelain treated, fluticasone treated and Br-EuNPs treated groups are represented in figure 11. Microphotographs of structural design of lung in naïve animals showed clear alveolar sacs with no gathering of cells in the region of bronchioles. However, OVA challenged group showed thick deposition of cells around the bronchioles, blood vessels and alveolar regions indicating the excessive infiltration of cells (eosinophil, lymphocyte and monocyte) leading to constriction of alveolar sacs as well as tissue damage (Figure 11). Oxidative burden induced by OVA exposure might be contributing to thickening of airway smooth muscles and basement membrane leading to remodeling of lungs. Consequently, groups treated with bromelain and fluticasone showed less accumulation of cells around the bronchioles as well as less thickening in alveolar septa (Figure 11). Structural design of lungs of Br-EuNPs treated animals showed no thickening around the bronchioles and no gathering of cells portraying the almost analogous configuration with naïve animals (Figure 11).

Ovalbumin challenge in sensitized guinea pigs elicited a noticeable infiltration of inflammatory cells in nasal mucosa which was confirmed by elevated level of EPO, IL-5, Ig-G and TNF-a in BAL fluid. The architecture of nasal mucosa in ovalbumin sensitized showed epithelial disruption with mucosa thickening, increased infiltrated inflammatory cells and hyperplasia of mucin producing goblet cells. Nasal administration of bromelain, Br-EuNPs and fluticasone showed reduction in infiltration of cells. However, formulation treated groups showed more remarkable inhibition of infiltrated cells and architecture similar to normal mucosal epithelium (Figure 12 ). The histopathological results are in accordance with hematological and immunological parameters.

Therefore, the present strategy focuses on the development of target-oriented delivery systems with certain degree of cationic nature and hydrophobicity that could interact with inflamed mucosal surface of pulmonary tract to alleviate macrophage stimulation and circumvent further toxic cascading events. This strategy would be advantageous for therapeutic intervention of allergic asthma by augmenting selective drug permeation to inflamed tissues, extending the time span of therapeutic effect at reduced effective dose and minimize dosing frequency.

We Claim:

1. Bromelain nanoparticle for management of asthma, said nanoparticle comprising bromelain, methacrylate ester copolymer having repeating units of formula I, a surfactant and a synthetic polymer,

Formula I
wherein R1 is selected from hydrogen and methyl, R2 is selected from methyl, and ethyl, R3 is methyl and R4 is ethyltrimethylammonium chloride.
2. The bromelain nanoparticle as claimed in claim 1, wherein R1 is hydrogen, R2 is methyl, R3 is methyl.
3. The bromelain nanoparticle as claimed in claim 1, wherein the methacrylate ester copolymer is Eudragit RL100 or Eudragit RS 100.
4. The bromelain nanoparticle as claimed in claim 1, wherein the ratio of bromelain and the methacrylate ester copolymer of formula I is in the range of 1: 1 to 1:7.5.
5. The bromelain nanoparticle as claimed in claim 4, wherein the ratio of bromelain and the methacrylate ester copolymer of formula I is in the range of 1: 3.
6. The bromelain nanoparticle as claimed in claim 1, wherein said synthetic polymer is selected from a group comprising poly(vinyl alcohol), or substituted celluloses such as hydroxyethylcellulose.
7. The bromelain nanoparticle as claimed in claim 6, wherein said synthetic polymer is poly(vinyl alcohol).
8. The bromelain nanoparticle as claimed in claim 1, wherein said surfactant is selected from anionic, cationic and non-ionic surfactant.
9. The bromelain nanoparticle as claimed in claim 8, wherein said surfactant is Sorbitan monooleate (Span 80).
10. The bromelain nanoparticle as claimed in claim 1, wherein size of said nanoparticle is in the range of 227.91 ± 26.01 to 625.63 ± 43.77 nm.
11. The Bromelain nanoparticle as claimed in claim 10, wherein size of said nanoparticle is in the range of 259.73 ± 16.51 nm.
12. The bromelain nanoparticle as claimed in claim 1, wherein bromelain entrapment efficiency of the nanoparticle ranges from 40± 8 to 90 ± 8 %.
13. The bromelain nanoparticle as claimed in claim 12, wherein bromelain entrapment efficiency of the nanoparticle is of 84.76 ± 1.49 %.
14. The bromelain nanoparticle as claimed in claim 1, wherein zeta potential of the nanoparticle ranges from 8.5 ±2 to 29 ±2 mV
15. The bromelain nanoparticle as claimed in claim 14, wherein zeta potential of the nanoparticle is 27.53 ± 1.66 mV.
16. The bromelain nanoparticle as claimed in claim 1, wherein polydispersity index of the nanoparticle is in the range of 0.143±0.016.
17. The bromelain nanoparticle as claimed in claim 1, wherein said nanoparticles are analyzed till 12 months and showed retention of bromelain activity up to 97.04±1.65 % under cool temperature of 4 ± 2 ºC at 65 ± 5 % relative humidity (RH) and up to 95.23± 1.25% under room temperature of 25±2ºC at 60 ±5% RH.
18. The bromelain nanoparticle 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 94.16±3.10 %.
19. The bromelain nanoparticle as claimed in claim 1, wherein t90 of said nanoparticulate formulation is ~ 5.99 folds higher compared to pure drug at room temperature.
20. A process to prepare the bromelain nanoparticle as claimed in claim 1, said process comprises:
a. emulsifying an aqueous solution of bromelain (W1) with an organic phase (O) to obtain primary emulsion (W1/O) by ultra-sonication under pre-defined conditions, wherein said organic phase (O) is prepared by dissolving the copolymer of formula I in an organic solvent comprising surfactant,
b. dispersing the primary emulsion of step (a) to an aqueous solution of synthetic polymer (W2) emulsifying by probe sonication under predefined conditions to form double emulsion (W1/O/W2),
c. removing said organic solvent by continuous stirring of said double emulsion to obtain organic solvent free double emulsion to obtain bromelain nanoparticle dispersion,
d. centrifuging said bromelain nanoparticle dispersion at predefined conditions to obtain pellets,
e. dispersing the said pellets in water with subsequent addition of cryoprotectant to obtain a suspension,
f. lyophilizing said suspension under specific conditions by freezing followed by primary drying and secondary drying using lyophilizer to obtain lyophilized bromelain nanoparticle.
21. The process as claimed in claim 20 wherein said organic solvent in step (a) 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.
22. The process as claimed in claim 21 wherein said organic solvent is dichloromethane.
23. The process as claimed in claim 20 wherein said surfactant in step (a) is Sorbitan monooleate (Span 80).
24. The process as claimed in claim 20 wherein said predefined conditions in step (a) for emulsification is disruption by ultrasonic disruptor for 5 min at 4 ?C.
25. The process as claimed in claim 20 wherein said synthetic polymer in step (b) is selected from the group comprising poly(vinyl alcohol), or substituted celluloses such as hydroxyethylcellulose or a combination thereof.
26. The process as claimed in claim 25 wherein said water soluble synthetic polymer is poly(vinyl alcohol) (PVA).
27. The process as claimed in claim 20 wherein said predefined conditions in step (c) are emulsifying the primary emulsion for 7 min at 4 ?C.
28. The process as claimed in claim 20 wherein said predefined conditions in step (e) are centrifugation at 20,000 rpm for 20 min at 4 ?C.
29. The process as claimed in claim 20 wherein said cryoprotectant in step (f) is mannitol 10% w/v.
30. The process as claimed in claim 20 wherein said specific conditions for lyophilization in step (g) are freezing to -80 °C at 0.05 mbar pressure for 48 h.
31. A mucoadhesive bromelain nanoparticle formulation for management of asthma, said formulation comprising bromelain nanoparticle of claim 1 with or without pharmaceutically acceptable surfactant and/or excipient.
32. The mucoadhesive bromelain nanoparticle as claimed in claim 31, wherein said formulation is a sustained drug release formulation.
33. The mucoadhesive bromelain nanoparticle as claimed in claim 31, wherein said formulation is administered via nasal route.