1. A self- emulsifying rutin loaded nasal drops formulation, wherein the formulation comprises an oil phase, an aqueous phase, wherein the oil phase comprises Rutin and eucalyptus oil in a specific ratio, and wherein the aqueous phase comprises surfactant and co-surfactant in a specific ratio, wherein said formulation is used for intranasal administration in chronic respiratory disorders such as asthma.

2. The formulation as claimed in claim 1,wherein said oil is eucalyptus oil, the surfactant is Tween 80, and said co-surfactant is PEG300.

3. The formulation as claimed in claim 1, wherein the oil phase comprises 30 mg Rutin in eucalyptus oil in the range of 10% to 30% (w/w).

4. The formulation as claimed in claim 1, wherein the ratio of surfactant and co-surfactant in the aqueous phase ranges from 80:10 to 30:60 (w/w).

5. The formulation as claimed in claim 1, wherein the ratio of oil phase and aqueous phase is 1:9 to 3:7.

6. The formulation as claimed in claim 1, comprises: (i) Rutin 30mg in 10% (w/w) eucalyptus oil, (ii) a mixture of a Tween 80 and PEG 300 at 60:30 ratio.

7. A process to obtain a self- emulsifying rutin loaded nasal drops formulation, said process comprises: (i) selecting suitable excipients comprising an oil, a surfactant, and a co-surfactant depending on the solubility of rutin, (ii) dissolving the rutin in the selected oil of step (i) to obtain an oil phase, (iii) mixing a surfactant and a co-surfactantat specific ratio to obtain an aqueous phase, and (iv) mixing the oil phase of step (ii) in the aqueous phase of step (iii) in specific ratio with gentle vortexing to obtain a self- emulsifying rutin loaded nasal drops formulation, wherein said formulation is used for intranasal administration in chronic respiratory disorders such as asthma.

8. The process to obtain a self- emulsifying rutin loaded nasal drops formulationas claimed in claim 7, said process comprises: (i) selecting suitable excipients comprising a eucalyptus oil, a surfactant Tween 80, and a co-surfactant PEG 300 depending on the solubility of rutin, (ii) dissolving the 30mg rutin in the selected eucalyptus oil at 10% (w/w) of step (i) to obtain an oil phase, (iii) mixing a surfactant Tween 80 and a co-surfactant PEG 300 at specific 60:30 ratio to obtain an aqueous phase, and (iv) Mixing the oil phase of step (ii) in the aqueous phase of step (iii) at specific 1: 9 ratio with gentle vortexing to obtain a self- emulsifying rutin loaded nasal drops formulation, wherein said formulation is used for intranasal administration in chronic respiratory disorders such as asthma.

Description:FIELD OF THE INVENTION:
The present invention relates to the field of medicine. Particularly, the present invention relates to self-emulsifying rutin loaded nasal drops formulation for intranasal administration and process thereof. More particularly, the invention relates to a rutin loaded self- emulsifying (R-SEDD) nasal drops formulation for intranasal administration and a process thereof. The formulation of the present invention is capable of being used in chronic respiratory disorders.

BACKGROUND OF THE INVENTION:
Chronic inflammatory disorder of the airways such as allergic asthma is characterized by recurring episodes of wheezing, coughing, chest tightness, and shortness of breath. It is a complex disease influenced by a combination of genetic and environmental factors. Inhaling allergens including dust mites, pollen, and pet dander can cause allergic asthma. The immune system's recognition of these allergens causes T helper 2 (Th2) cells to become activated and produces cytokines including interleukin-4 (IL-4) and interleukin-5 (IL-5). Immunoglobulin E (IgE) antibodies are produced in response to these cytokines and attach to the surface of basophils and mast cells. This series of events causes the bronchi to become hyperresponsive and narrow in response to a wide range of stimulants/ allergens.

Allergic asthma is often poorly managed due to the limited effectiveness and significant side effects of current treatment options. Asthma has no particular cure. Inhaled corticosteroids and bronchodialators are first line treatment for asthma management. However, these therapies are associated with several systemic (osteoporosis, diminished bone growth, weight put on, stomach distress, cataracts and glaucoma) and localized (oropharyngeal candidiasis, dysphonia, reflex cough and pharyngitis) side effects along with ineffectiveness in 5-10% of patients. Apart from these therapies, other treatments for moderate to severe asthma include short and long acting sympathomimetics, anti-histaminic, anti-inflammatory agents, anti-cholinergic and immuno-modulators. However, limited efficacy, dependence and withdrawal, cost and accessibility, unclear long-term benefits of available treatments has surged the need to find a new and efficient alternative treatment for asthma in clinics using both natural and synthetic therapeutically active moieties.
Lipid-based formulations have attracted a lot of attention from both academia and industry as a possible formulation technique for enhancing the oral bioavailability of medications that are poorly soluble in water. These formulations can enhance drug absorption through a variety of ancillary mechanisms, such as promoting lymphatic transport, which transports the drug straight to the systemic circulation without going via hepatic first-pass metabolism, increasing gastrointestinal (GI) membrane permeability, and inhibiting P-glycoprotein-mediated drug efflux and pre-absorptive metabolism by gut membrane-bound cytochrome enzymes. These formulations include multi-excipient self-emulsifying components or straightforward drug solutions in dietary oils. Formulations that self-emulsify are physically stable, isotropic combinations of oil, cosurfactant, surfactant, and solubilized medication that can be administered orally, topically or intranasally.

Cited prior art “Suppressive effects of rutin, quercitrin, and isoquercitrin on atypical allergic asthma in an animal model” published on Dec. 2021 suggests rutin and quercitrin have the potential for treating or preventing atypical allergic asthma.

Rutin (3,3',4',5,7-pentahydroxyflavone-3-rahmnoglucoside) is a bioactive phytochemical having potential therapeutic benefits in management of various chronic inflammatory diseases, including bronchial asthma. Rutin can suppress airway inflammation by inhibiting NF-?ß pathways leading to lower the levels of chemokines. However, clinical application of rutin in asthma management is hampered by its extremely low aqueous solubility, poor absorption and rapid metabolism resulting in low bioavailability. Additionally, rutin’s susceptibility to photodegradation reduces its self-life.

In recent decades, plenty of efforts have been made to enhance the bioavailability of rutin. Another cited prior art “Therapeutic Potential of Controlled Delivery Systems in Asthma: Preclinical Development of Flavonoid-Based Treatments” published on Dec. 2022 highlights that there are few studies developing pharmaceutical forms for controlled release systems using flavonoids including the different classes as active molecules for asthma treatment.

According to several studies, the efficiency of rutin was enhanced by the use of nano-drug delivery systems administered via oral route. However, self-emulsifying drug delivery systems (SEDDS) have received extensive attention benefiting from its broad source of medical auxiliary materials, low cost and ease of fabrication. SEDDS can spontaneously form oil-in-water globule with size less than 100nm or with slight agitation at 37°C. With good adaptability for oral/ nasal administration, they can enhance permeability across membranes and saturation concentration of poorly soluble drugs and thus can amend their bioavailability and in-vivo performance.

Thus, in view of the above, the present invention aims to provide novel drug delivery system for intranasal delivery of Rutin in the management of chronic respiratory disorders. The formulation of the present invention enhances solubility, bioavailability and therapeutic efficacy of Rutin for intranasal administration in asthma management and a process thereof for use in chronic inflammatory disorder such as asthma.

OBJECTS OF THE INVENTION:
Some of the objects of the present disclosure, which at least one embodiment herein satisfy are as follows:

The main object of the present invention is to provide a self- emulsifying rutin loaded nasal drops formulation for intranasal administration.

Another object of the present invention is to provide a self- emulsifying rutin loaded nasal drops formulation comprising pure rutin, oil, a mixture of surfactant and co-surfactant for intranasal administration.

Yet another object of the present invention is to provide a self- emulsifying rutin loaded nasal drops formulation comprising pure rutin, oil, a mixture of surfactant to co-surfactant for intranasal administration in chronic respiratory disorder such as Asthma.

Yet another object of the present invention is to provide a process of obtaining a self- emulsifying rutin loaded nasal drops formulation for intranasal administration in the chronic respiratory disorders such as asthma.
Yet another object of the present invention is to provide a self- emulsifying rutin loaded nasal drops formulation for intranasal administration in the Asthma management with improved drug delivery properties, such as enhanced solubility, bioavailability, therapeutic efficacy, stability and sustained release.
Other objects and advantages of the present disclosure will be more apparent from the following description when read in conjunction with the accompanying figures, which are not intended to limit the scope of the present disclosure.

SUMMARY OF THE INVENTION:
Accordingly, the present invention provides a self- emulsifying rutin loaded nasal drops formulation for intranasal administration for chronic respiratory disorders such as Asthma. The present invention also provides a process to obtain a self- emulsifying rutin loaded nasal drops formulation for intranasal administration.
In one of the embodiments, the present invention provides a self- emulsifying rutin loaded nasal drops formulation for intranasal administration for chronic respiratory disorders such as Asthma comprising oil phase with rutin in a specific ratio and an aqueous phase with a mixture of surfactant to co-surfactant (Smix) in a specific ratio.
In an embodiment of the present disclosure, the oil is eucalyptus oil.
In an embodiment of the present disclosure, the surfactant is Tween 80.
In an embodiment of the present disclosure, the co-surfactant is PEG 300.
In another embodiment, the present invention provides a self- emulsifying rutin loaded nasal drops formulation for intranasal administration for chronic respiratory disorders such as Asthma comprising oil phase with rutin in a specific ratio and a aqueous phase with a mixture of surfactant to co-surfactant in a specific ratio.
In another embodiment, the present invention provides a process to obtain a self- emulsifying rutin loaded nasal drops formulation, starts with selecting suitable excipients an oil, a surfactant, and a co-surfactant depending on the solubility of rutin. The pure rutin is then dissolved in the selected oil to obtain the oil phase. An aqueous phase is obtained by mixing the selected surfactant and co-surfactant at a specific ratio, there after the oil phase and the aqueous phase mix at a specific ratio to obtain a self- emulsifying rutin loaded nasal drops formulation of the present invention.
In another embodiment, the present invention provides a self- emulsifying rutin loaded nasal drops formulation and a process thereof for intranasal administration in the Asthma management with improved drug delivery properties, such as enhanced solubility, bioavailability, therapeutic efficacy, stability and sustained release.
These and other aspects herein will be better appreciated and understood when considered in conjunction with the following description and the accompanying drawings. It should be understood, however, that the following descriptions are given by way of illustration and not of limitation. Many changes and modifications may be made within the scope of the invention herein without departing from the spirit thereof.
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 10 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 process 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.
BRIEF DESCRIPTION OF THE FIGURES
Fig. 1 depicts solubility of rutin in different (A) oils and (B) surfactants.
Fig. 2 depicts optimised formulations composed of oil and Smix in 1:9 ratio.
Fig.3 (A) and (B) depicts pseudo-ternary-phase diagrams manifesting nanoemulsion area for Oil: Smix in 1:9 and 2:8 ratio respectively.
Fig. 4 depicts Transmission electron microscopic (TEM) images of optimized SE4 R-SEDDS.
Fig. 5 depicts dissolution profile of free rutin and optimised formulation respectively.
Fig. 6 (A), (B), (C), (D) and (E) depicts effect of room temperature storage conditions on particle size, PDI, zeta potential, % emulsion stability index and % drug content of optimized formulation respectively.
Fig. 7 (A), (B), (C), (D)and (E) depicts effect of accelerated storage conditions on particle size, zeta potential, PDI, % emulsion stability index and % drug content of optimized formulation respectively.
Fig. 8 depicts plasma concentration-time curve of rutin and optimised formulation respectively.
Fig. 9 (A), (B) and (C) depicts effect of eucalyptus oil (EO), rutin, standard drug, and optimised formulation on bronchospasm onset time, % recovery time and % protection respectively.
Fig. 10 depicts effect of eucalyptus oil (EO), rutin, standard drug and optimised R-SEDDS on wet/dry weight ratio of lung and liver respectively.
Fig. 11 (A), (B), (C) and (D) depicts effect of eucalyptus oil (EO), rutin, standard drug and optimized formulation on haematological parameters respectively.
Fig. 12 (A), (B), (C) and (D) depicts effectiveness of distinct treatments on proinflammatory cytokine (IL-6, IL-5, TNF-a) and Ig-E in serum and BALF respectively.

DETAILED DESCRIPTION OF THE INVENTION WITH NON-LIMITING EMBODIMENTS AND EXAMPLES

In the following detailed description of the invention, numerous specific details are set forth in order to provide a thorough understanding of the invention. However, it will be obvious to a person skilled in the art that the invention may be practiced with or without these specific details. In other instances, well known methods, procedures and components have not been described in details so as not to unnecessarily obscure aspects of the invention.

Furthermore, it will be clear that the invention is not limited to these alternatives only. Numerous modifications, changes, variations, substitutions and equivalents will be apparent to those skilled in the art, without parting from the scope of the invention.

Accordingly, the present invention provides a self- emulsifying rutin loaded nasal drops formulation for intranasal administration for chronic respiratory disorders such as Asthma. The present invention also provides a process to obtain a self- emulsifying rutin loaded nasal drops formulation for intranasal administration.

In one of the embodiments, the present invention provides a self- emulsifying rutin loaded nasal drops formulation for intranasal administration for chronic respiratory disorders such as Asthma comprising oil phase with rutin in a specific ratio and an aqueous phase with a mixture of surfactant to co-surfactant in a specific ratio.
The rutin is a pure rutin.
In an embodiment of the present disclosure, the oil is eucalyptus oil.
In an embodiment of the present disclosure, the surfactant is Tween 80.
In an embodiment of the present disclosure, the co-surfactant is PEG 300.
In an embodiment of the present invention, the oil phase comprises Rutin in eucalyptus oil in the range of 10% to 30% (w/w).
In an embodiment of the present invention, the ratio of surfactant and co-surfactant in the aqueous phase ranges from 80:10 to 30:60 (w/w).
In an embodiment of the present invention, the ratio of oil phase and aqueous phase is 1:9 to 3:7 in the rutin loaded self-emulsifying drug delivery system.
In a preferred embodiment, the self- emulsifying rutin loaded nasal drops formulation comprises oil phase of Rutin 30 mg in 10% (w/w) eucalyptus oil, and an aqueous phase of mixture of a Tween 80 and PEG 300 at 60:30 ratio.
In another embodiment, the present invention provides a process to obtain a self- emulsifying rutin loaded nasal drops formulation, starts with selecting suitable excipients an oil, a surfactant, and a co-surfactant depending on the solubility of rutin. The pure rutin is then dissolved in the selected oil to obtain the oil phase. An aqueous phase is obtained by mixing the selected surfactant and co-surfactant at a specific ratio, there after the oil phase and the aqueous phase mix at a specific ratio to obtain a Rutin loaded self-emulsifying drug delivery system.
In a preferred embodiment, the present invention provides a process to obtain a self- emulsifying rutin loaded nasal drops formulation, starts with selecting suitable excipients an eucalyptus oil, a Tween 80 surfactant, and a PEG 300 co-surfactant depending on the solubility of rutin. The 30 mg pure rutin is then dissolved in the selected eucalyptus oil at 10% (w/w) to obtain the oil phase. An aqueous phase is obtained by mixing the selected Tween 80 surfactant and PEG 300 co-surfactant at a specific ratio of 60:30, there after the oil phase and the aqueous phase mix at a specific ratio of 1:9 to obtain a self- emulsifying rutin loaded nasal drops formulation.
Experiments:

Excipients selection
The shake flask method was used to determine the solubility of rutin in various oils such as peppermint oil, eucalyptus oil, cinnamon oil, clove oil, linseed oil, hydrocarpus oil and surfactants such as tween 60, tween 80, span 80 and, polyethylene glycol 300 respectively. An excess amount of pure rutin was added to 2 g of each oil and surfactants respectively kept in separate vials. Samples were kept on an orbital isothermal shaker (Orbital incubator shaker MR/ICS/18) for 24 h at 25 °C, 100 rpm. Subsequently, each sample was centrifuged for 10 min at 12,000 rpm using a Thermo scientific Pico-21 centrifuge. The obtained supernatants were diluted with methanol and analysed using UV-visible spectrophotometer at 363 nm. The experiment was carried out in triplicate and results were expressed as the mean value ± SD (mg/ml).

Depending upon the solubility of rutin, suitable excipients i.e., oil and surface-active agents showing the highest solubility of rutin were selected to prepare rutin loaded self-emulsifying system to obtain the novel therapeutic formation of the present invention. Among the oils used, rutin showed highest solubility in eucalyptus oil and in PEG 300 followed by Tween 80 among surfactants tested as shown in the Fig. 1. Therefore, eucalyptus oil, PEG 300 and Tween 80 were selected for the preparation of a self-emulsifying formulation of rutin of the present invention.

The excipients selection is crucial to formulate the novel therapeutic formulation of the present invention. Therefore, the selection of surfactant/ co-surfactant was done depending on their HLB value in order to obtain fine uniform emulsion droplets. Usually, surfactants with HLB >10 are much more effective in furnishing small uniform emulsion globules which provide higher surface area for rapid and complete absorption. When the combined HLB of the surfactant/cosurfactant mixture closely matches the HLB value of the oil, it results in the least interfacial tension between the oil and water phases, showcasing system stability. The eucalyptus oil (HLB value 10) was the oil of choice combined with surfactant Tween-80 (HLB value 15) and PEG-300 as a co-surfactant (HLB value 14.9). The combination acts as a synergistic surfactant, and therefore, increases the stability and solubility of the hydrophobic drug. Thus, eucalyptus oil, Tween-80 and PEG-300 were selected to load rutin in a self-emulsifying drug delivery system to form a stable system.

The eucalyptus oil was selected as preliminary component in development of R-SEDDS depending on the rutin solubility in oil as well as due its anti-inflammatory and mast cell inhibitory potential useful for aid on effect of rutin in asthma management.
Preparation of R-SEDDS
The eucalyptus oil varying from 10 to 30% w/w was mixed with different blends of Tween 80 and PEG 300 in varying ratio respectively at 400 rpm for 30 min using magnetic stirrer (Remi Instruments Ltd, Mumbai, India). The blend of oil phase having dissolved rutin with a mixture of surfactant to co-surfactant (Smix) in different weight ratios i.e., 1:9, 2:8 and 3:7 w/w was further studied for emulsion stability index (ESI) as disclosed in the Table 1. 1 ml of each blend was introduced into 10 ml of distilled water and gently mixed using magnetic stirrer at 300 rpm for 15 min at 37 °C. The diluted products were inspected for clarity, spontaneous emulsification, phase separation and coalescence of droplets. The pseudo-ternary phase diagram was constructed using Origin Pro 2019b.

Formulation Oil: Smix Oil (% w/w) Smix (% w/w)
Ratio Eucalyptus oil Tween-80 PEG 300
SE1 1:9 10 90 0
SE2 80 10
SE3 70 20
SE4 60 30
SE5 50 40
SE6 40 50
SE7 30 60
SE8 2:8 20 80 0
SE9 70 10
SE10 60 20
SE11 50 30
SE12 40 40
SE13 30 50
SE14 3:7 30 70 0
SE15 60 10
SE16 50 20
SE17 40 30
SE18 30 40

Table 1: Composition of different batches of R-SEDDS prepared

R-SEDDS formulations were prepared by dissolving rutin solution (30 mg) in oil followed by blending with Smix with gentle vortexing. Subsequently, menthol (15 mg) and thymol (5 mg) were added to the optimized pre-concentrate batches and sonicated for 2 min to facilitate their ease of dissolution. The homogeneous pre-concentrate formed was subjected to further analysis like self-emulsification time, dispersibility study, average globule size analysis etc. Out of 18 formulations, a blend containing 10 % w/w of oil produced a clear and stable emulsion with high Smix ratios (SE4, SE5, SE6, SE7) as shown in Figure 2.

Characterisation of R-SEDDS
Optical clarity and phase separation
All the batches of R-SEDDS equivalent to 10 mg rutin were diluted with distilled water and kept undisturbed for 24 h. Afterwards, formulations were noticed visually for clarity and homogeneity. R-SEDDS showing clarity and no phase separation were selected as stable formulations. The results of visual investigation were validated by determining % transmittance of each emulsion utilizing UV-Visible spectrophotometer at 369 nm. Batches with the highest % transmittance were chosen as stable and homogeneous formulations.

Dispersibility index
Duration needed by pre-concentrate to disappear and form a homogeneous mixture was evaluated as dispersibility index. It was evaluated in a USP dissolution apparatus II (Electrolab, India) by adding 1 ml of pre-concentrate drop wise to 750 ml of distilled water maintained at 37 ± 0.5°C and agitated at 100 rpm. The dispersibility index was graded visually depending upon the grading system explained in Table 2.

GRADE COMMENTS
A Prompt formation of nano-emulsion within 1min with clear or bluish look
B Prompt formation of moderately clear nano-emulsion with white look
C Milky white emulsion formation within 2 min
D Dull white emulsion formation with oily look with lengthy emulsification time > 2min
E Unsatisfactory emulsion formation with bigger oil droplets existing on the surface

Table 2: Grades related to dispersibility of pre-concentrate

The grades of dispersibility index too indicated the ability of pre-concentrates to emulsify spontaneously i.e., grade A indicated rapid emulsification whereas ascending to grade B, C and D confirmed the poor dispersibility of pre-concentrates to self-emulsify. Among the different batches of pre-concentrate, SE4 and SE5 manifested the development of nanoemulsions with clear bluish white appearance within 1 min in all solvents. The outcomes of study revealed that SE4 and SE5 exhibited dispersibility index of grade A in all solvent systems i.e., distilled water, 0.1 N HCl and phosphate buffer pH 7.4 respectively. These observations indicated the need of sufficient amount of surfactant and co-surfactant blend to be adsorbed at oil/water interface for uniform emulsification. The presence of suitable surfactant / co-surfactant concentration might have reduced the free energy for emulsion formation and also prevented the coalescence of dispersed droplets resulting in stable nanoemulsion.

Out of 18 formulations, a blend containing 10 % w/w of oil produced a clear and stable emulsion with high Smix ratios (SE4, SE5, SE6, SE7). Meanwhile, 20 % w/w oil with a lower Smix ratio (SE12, SE13) resulted in a translucent emulsion followed by phase separation whereas SE10 and SE11 produced slight bluish transparent emulsions. The formulations containing higher oil content (30 % w/w) produced milky emulsions with oil droplets on the surface of water as shown in Table 3. SE4 and SE5 exhibited higher percentage transmittance i.e., 98.56±4.73 % and 98.33 ± 4.16 % respectively which is closer to 100 % indicating highest clarity of systems and homogeneity. This assured the formation of transparent and stable nanoemulsion without any phase separation.

F. code Oil: Smix Visual appearance Transmittance
(%±SD) Dispersibility Index
0.1N HCl Phosphate buffer pH 7.4 Distilled water
SE1 1:9 Not forming 67.46±3.73 D D D
SE2 Not Clear 84.578±4.46 D D D
SE3 Translucent 90.46±5.36 D D D
SE4 White, Transparent 98.56±4.73 A A A
SE5 White, Transparent 98.33±4.16 A A A
SE6 Slightly bluish, Transparent 91.47±3.55 A A A
SE7 Slightly bluish, Transparent 92.46±4.10 A A A
SE8 2:8 Not forming 54.64±2.03 C C C
SE9 Phase separation 44.76±4.44 C C C
SE10 Slightly bluish, Transparent 93.37±5.67 B B B
SE11 Slightly bluish, Transparent 93.23±3.44 A B B
SE12 Translucent 90.56±3.99 A B B
SE13 Translucent 88.55±3.73 A B B
SE14 3:7 Milky phase 81.46±6.36 C C C
SE 15 Milky phase 79.23±4.23 C C C
SE 16 Milky phase 83.45±4.82 C C C
SE 17 Clear, bluish 94.37±6.03 B B B
SE 18 Clear, bluish 95.91±6.88 B B B

Table 3: Effect of composition of various batches on clarity and dispersibility index
Optimized ratios of eucalyptus oil and Smix were selected from the phase diagrams. The construction of the ternary phase-diagram gives an idea about the nature of emulsifying regions and constantly assists in selecting an optimum portion of oil, surfactant and co-surfactant. The batches of pre-concentrate which produced uniform and transparent emulsions after mixing with water having oil, surfactant and co-surfactant in a certain ratio were plotted to obtain pseudo-ternary phase diagram as shown in Fig. 3(A) and (B) for Oil: Smix in 1:9 and 2:8 ratio respectively. The shaded region of the diagram corresponds to the formation of nanoemulsion at room temperature. Higher nanoemulsification region was achieved when the oil and Smix (surfactant and co-surfactant) ratio was 1:9. The blend of surfactant and co-surfactant chosen significantly lowered the interfacial tension between oil and aqueous phase and generated nanoemulsion.

Colloidal characterization and drug content estimation

Depending upon the pseudo ternary phase diagram, clarity and dispersibility index, SE4, SE5, SE6, and SE7 batches were studied for their colloidal characteristics after dispersion in distilled water and gently mixed. Average particle size, polydispersity index (PDI) and zeta potential of resultant emulsions were estimated utilising photon correlation spectroscopy (Nano ZS, Malvern, UK).

Drug content of formulations prepared was determined by dissolving 100 mg of R-SEDDS in methanol containing 0.1% Triton X-100. Aliquots were appropriately diluted and evaluated at 369 nm using a UV-visible spectrophotometer to evaluate drug content.

The average globule size and PDI of nanoemulsions was in the range of 93.54±8.16 nm to 142.63±10.41 nm and 0.15±0.02 to 0.28±0.04 respectively. The results indicated that decrease in concentration of Tween-80 in Smix had increased the average globule size and PDI without any significant effect on zeta potential as given in Table 4. The value of zeta potential close to ±30 mV indicated higher colloidal stability of nanoemulsion formed. Batches prepared achieved high percentage rutin content (95.21 ± 7.85 % to 99.01 ± 2.36 %) respectively.

F. code Size
(nm ± SD) PDI±SD Zeta potential (mV ± SD) Drug content (% ± SD)
SE4 93.54±8.16 0.15±0.02 -28.35± 2.05 99.01 ± 2.36
SE5 106.71±14.08 0.17±0.03 -28.10± 3.20 98.36 ± 4.36
SE6 136.24±12.64 0.20±0.03 -27.72± 5.3 95.64 ± 5.69
SE7 142.63±10.41 0.28±0.04 -21.85± 3.14 95.21 ± 7.85
Table 4: Effect of Smix composition on the particle size, PDI, zeta potential and % drug content respectively

Thermodynamic stability studies
The pre-concentrate was diluted with distilled water to 1:10 ratio and evaluated for thermal and stress tests to evaluate their thermodynamic stability. Optimised formulations (SE4 and SE5) were analysed for their thermodynamic stability utilizing stress tests like centrifugation, heating and cooling cycles and freeze–thaw cycles. After each cycle of heating-cooling and freezing-thawing, moderate changes were observed in droplet size and zeta potential as given in Table 5. It was also recorded that nanoemulsions formed from SE4 pre-concentrate remained clear and transparent. However, a significant rise in droplet size and reduction of zeta potential of nanoemulsions formed from SE5 was observed. In addition, a milky white appearance was observed in nanoemulsions of SE5 batches after thermal stress. Hence depending upon the higher stability of nanoemulsion, SE4 pre-concentrate was selected.

F. code Centri-fugation Heating & Cooling cycles Freeze-thaw cycle
Size (nm± SD) Zeta potential (mV± SD) Visual appearance Size (nm± SD) Zeta potential (mV± SD) Visual appearance
SE4 No phase separation 123.52 ± 13.13 -22.69± 4.22 Clear 131.11±12.15 -21.55± 4.83 Clear
SE5 No phase separation 187.66±
14.16 -17.72± 5.69 Milky white 213.71±4.81 -15.40± 4.22 Milky white

Table 5: Effect of accelerated stress conditions on colloidal characteristics of self-emulsified nanoemulsion and dispersibility index respectively.

Centrifugation study: Diluted batches (5ml) were centrifuged at 4000-5000 rpm for 15 min. Thereafter, samples were observed for phase separation and drug precipitation.

Heating and cooling cycle: Diluted formulations were subjected to 6 heating/cooling cycles between 4°C and 40°C respectively with storage at each temperature for 48 h. The formulation that was found stable at variable temperatures will pass for further analysis. This test was performed to observe the effect of temperature on stability.

Freeze-thaw cycle (accelerated aging): Diluted formulations (5 ml) were subjected to 3-5 freeze-thaw cycles, which included freezing at -80°C and +25ºC for not less than 48 h at each temperature followed by a thawing process. Observation of phase changes was made for each cycle. Subsequently, samples were subjected to a dispersibility index where formulation in 1:10 ratios was dispersed in distilled water under room temperature for particle size, zeta potential, emulsification grade and visual turbidity index. The resultant formulations were then checked for any instability problem, such as phase separation, creaming or coalescence.

Surface morphology
Diluted samples were evaluated under transmission electron microscopy (TEM, Tecnai G2 20 S-TWIN) to determine morphology of nanoemulsion droplets at an acceleration voltage of 200 kV under 0.14 nm resolution. Figure 4 depicts TEM images of rutin loaded formulation. The photomicrographs depicted homogeneously distributed spherical shape of globules with smooth surface.

In-vitro drug dissolution
In-vitro dissolution of optimized RSEDDS was performed using modified dialysis bag diffusion method. RSEDDS samples equivalent to 14 mg rutin was kept in dialysis bag and immersed in dissolution fluid (200 ml, phosphate buffer pH 6.8) maintained at 37 ± 0.5 ºC and 100 rpm respectively. Sink condition was maintained by adding tween 80 (0.1% w/v) to dissolution media. At predestined time intervals, aliquots 3 ml were withdrawn and replaced with fresh dissolution media. The collected samples were filtered through a 0.22µm membrane filter and analysed spectrophotometrically. Measurements were conducted in triplicate. The cumulative percentage drug release versus time was plotted and evaluated for determination of release kinetics model.
The release profile of pure rutin and optimised formulation are presented in Figure 5. Pure drug facilitated around 3.47 % drug release in 2 h with ~47% drug release in 24 h. On the contrary, R-SEDDS showed notably rapid drug release ~13% in 2 h and 98% in 24 h. The results indicated that dissolution of pure drug was restricted by its poor wetting and aqueous solubility. Conversely, self-emulsification facilitated formation of nanosized vesicles with increased surface area of contact to dissolution fluid which might have contributed to burst release of rutin. In addition, blend of surfactant and co-surfactant too instigate rutin to dissolve rapidly by reducing surface tension between its hydrophobic surface and dissolution fluid. The prolonged release of rutin from the matrix of nanoemulsion was directly related to partitioning of rutin from oil phase and its dissolution and diffusion into dissolution media. Drug release data of optimised formulation showed best fit to Korsmeyer-Peppas model on mathematical kinetic modelling which illustrated that drug release was diffusion and dissolution controlled.

Storage stability
Optimized formulations were subjected to stability study at real time (25 ± 2 °C / 60±5% RH) and accelerated (40 ± 2 °C / 75 ± 5 % RH) stability conditions for 12 months and 6 months respectively. Optimised pre-concentrate formulation was stored in amber colored glass vials sealed via screw caps. Specimens were estimated for particle size, zeta potential, PDI, emulsion stability index (ESI) and % drug content respectively. Fig.6 (A), (B), (C), (D) and (E) and Fig, 7 (A), (B), (C), (D) and (E) depicts effect of room temperature and accelerated storage conditions on particle size, PDI, zeta potential, % emulsion stability index and % drug content of optimized formulation respectively. Optimized formulation stored at room temperature and accelerated storage conditions manifested no significant variation in their colloidal quality parameters like particle size, zeta potential, PDI, % drug content and % ESI. No remarkable variation in colour of formulation was observed at different storage conditions. This indicated that formulation variables were suitably optimized to retain colloidal characteristics for effective long-term use.

In-vivo studies
Healthy wistar rats (180 - 250g) and guinea pigs (200-230g) obtained from the animal house of the Department of Pharmacy, Banasthali Vidyapith were kept under disease free standard laboratory conditions with maintenance of temperature (22-24°C), humidity (55± 2%) and light-dark cycle of 12h each. Animals were fed with standard chow diet and provided water ad libitum. The protocol of in vivo experiments was approved by Institutional Animal Ethical Committee (IAEC) of Banasthali Vidyapith, and performed in accordance with CPCSEA guidelines of the Ministry of Animal Welfare, Government of India for the use and care of experimental animals.

Pharmacokinetics study
Twelve healthy Wistar rats (180-250g) were kept on overnight fasting with free access to water before the start of experiments. Animals were randomly divided into two groups (n=6). Animals of group I and II were administered with free rutin (15 mg/Kg) and formulation equivalent to 15 mg/Kg rutin intranasally respectively. Then, 200 µl blood samples were withdrawn from the rat tail vein at predetermined time intervals (0.5, 1, 2, 4, 6, 8, 12 and 24h after drug administration) in heparinised tubes and immediately centrifuged at 5000 rpm for 10 min at 4 °C. Plasma was extracted with methanol: acetonitrile (2:1 v/v), centrifuged at 12,000 rpm for 10 min at 4 °C. Supernatant collected was evaluated for rutin content via HPLC analysis using stationary phase of Phenomenex C18 column (250 mm X 4.6mm, 5µm) at room temperature with mobile phase of methanol: acetonitrile: water (45:15:40 v/v/v). Sample volume of 50 µl was injected and system was run isocratically at 0.6ml/min to detect rutin at 359 nm. The assay was linear over the rutin concentration range of 1–10 ng/ml. The limits of detection (LOD) and of quantification (LOQ) of rutin were 0.1 ng/mL and 1 ng/mL, respectively. The coefficients of variation (CV) for the intra- and inter day assay were all within 5%. Graph pad Prism 5 via non-compartment analysis was utilized to determine pharmacokinetic parameters and relative bioavailability.

The plasma concentration-time profile and pharmacokinetic parameters of rutin and optimised formulation after administration of single dose equivalent to 15mg/Kg of rutin have been elucidated in Figure 8 and Table 6 respectively. The proportionately higher Cmax (3.17-fold) and AUC0-24h (2.24-fold) was observed with formulation compared to free rutin. Higher intrinsic solubility offered by R-SEDDS and prevention of rutin precipitation might be accounting for higher rate and extent of absorption compared to rutin dispersion. Significantly lower Tmax of R-SEDDS (4.0±0.14 h) than pure rutin (6.0±0.28 h) indicated faster rutin absorption. In addition, R-SEDDS attained higher plasma concentration throughout its 24 h profile in comparison to rutin dispersion as shown in Figure 8. Nanosize of self-emulsified system, lymphoidal paracellular and transcellular uptake might have contributed to extended half-life (25.82±0.08 h) and MRT (21.76±0.22 h) of R-SEDDS. Sustained drug release behaviour and phagocytic uptake of R-SEDDS might be contributing to higher plasma concentration and 2.24-fold higher AUC0-24h than rutin dispersion.

Parameter Rutin dispersion Optimized formulation
Cmax (ng/ml) 96.65±33.47 306.84±21.66
Tmax (h) 6.00±0.28 4.00±0.14
Ke (h-1) 0.07±0.06 0.02±0.02
t1/2 (h) 14.60±2.10 25.83±0.08
MRT (h) 15.84±0.29 21.76±0.22
AUC0-24h (ng.h/ml) 1353.09±83.61 3039.05±94.22
Relative bioavailability (%) - 246.8035

Table 6: Pharmacokinetic parameters of rutin dispersion and optimised formulation respectively.

Pharmacodynamics study
Fifteen healthy guinea pigs were sensitized by intraperitoneal injections of OVA and alum solution (150 µg ovalbumin and 100 mg aluminium hydroxide emulsified in 1ml of normal saline) on 1st and 7th day respectively. Subsequently on the 14th day, a booster dose of only OVA solution was injected to complete the duration of the sensitization challenge. Naïve animals (n=3) were sham sensitized with 100 µL normal saline similar to OVA-sensitized animals.

OVA-sensitized animals were randomly divided into five groups (n=3) respectively. Guinea pigs of group I were treated with normal saline (positive control), while group II, III, IV and V received eucalyptus oil, rutin dispersion (15mg/Kg), optimised formulation (equivalent to 15mg/Kg rutin) and chlorphenamine maleate (15mg/Kg) as standard for 14 days. Animals of respective group were exposed with 1% histamine dihydrochloride solution in a clear plexiglass histamine chamber. Animals were closely observed for survival as well as onset of bronchospasm and recovery time were recorded and compared with the saline-treated group. The physiological response throughout the 28 days of study was also observed. On 29th day, animals were sacrificed to collect blood samples via cardiac puncture and organs like lung and trachea. Serum was evaluated for oxidative stress markers. The left lung and liver were excised, blotted dry and immediately weighed using a precision balance, then re-weighed after being dried for 48h in an oven at 60°C. Samples were stored at -80°C until further analysis.
During present investigation, histamine exposure significantly reduced the bronchospasm onset time and enhanced the episodes of jerks, uneasiness in breathing and recovery time in OVA-sensitized guinea pigs compared to saline-sensitized animals (p<0.05). However, a remarkable delay in bronchospasm onset time and faster recovery was observed with eucalyptus oil, rutin, standard drug (chlorpheniramine maleate) and R-SEDDS treatment compared to control group (p<0.05) as shown in Fig. 9(A), (B) and (C) respectively. Severity of spasm as well as jerk induction was inhibited by both pure drug and the R-SEDDS. Higher bioavailability during pharmacokinetics study respectively indicated stronger protective impact of R-SEDDS than pure drug.

Bronchoalveolar lavage Fluid (BALF) analysis
BALF was collected immediately after blood collection by cannulating trachea by three successive aspirations of infused saline.Whole blood collected from animals of each group was evaluated for % neutrophile, % eosinophile, % lymphocyte and total leukocyte count. Serum was separated by centrifuging blood at 4 °C for the detection for IgE, IL-5, IL-6 and TNF-a concentration using suitable ELISA kit respectively.

Wet/Dry ratio
Allergen induced inflammatory condition and associated oedema in the body of sensitized animals can also serve as marker to severity of asthma. A considerably higher wet/dry ratio in the control group compared to eucalyptus oil, pure rutin and optimised R-SEDDS (p<0.05) was observed. The remarkably higher leukocyte and neutrophil infiltration into the lung and liver might be contributing to inflammatory and oedematous condition of lungs and liver respectively as shown in Figure 10. Optimised R-SEDDS substantially reduced wet/dry ratio indicating a noticeably better protective impact on lowering inflammatory cell buildup and pulmonary oedema compared to pure drug and eucalyptus oil (p<0.05).

Haematological evaluation
OVA exposure enhanced lymphocyte, neutrophile, and eosinophil counts coupled with an elevated total leukocyte count (TLC) in control group. Fig. 11 (A), (B), (C) and (D) depicts effect of eucalyptus oil (EO), rutin, standard drug and optimized formulation on haematological parameters respectively. Increased oxidative stress due to OVA exposure might have elevated the systemic inflammatory markers. Significant reduction in total leucocyte count was observed with formulation treatment compared to eucalyptus oil, pure drug and control group (p<0.05). Higher systemic bioavailability of rutin via formulation might be contributing to its higher anti-inflammatory and anti-oxidant activity and regulating blood cell count near to normal.
Immunological estimation
Treatment with pure drug, standard drug and optimised formulation showed significant reduction in level of cytokines (TNF-a, IL-5 and IL-6) and IgE respectively as shown in Fig.12 (A), (B), (C) and (D). Similarly, decrease in TNF-a, IL-6, IL-5 and IgE in BALF was observed with different treatments respectively. The results demonstrated reduction of progress of allergen induced asthma by reducing T cell-driven inflammation and inhibiting NFKB over expression and iNOS. However, enhanced antioxidant and anti-inflammatory property of rutin in formulation might be attributed to enhanced bioavailability of drug from formulation as well as due to synergistic effect of eucalyptus oil in suppressing the production of cytokines, chemokines, and lipid mediators in monocytes, alveolar macrophages, and basophils. The application of advanced drug carrier system along with use of pharmacologically active oil (eucalyptus oil) in development of formulation might have delayed the development of bronchospasm and facilitated faster recovery.

Statistical data
Data has been disclosed as mean ± SD for each group (n=3). Statistical analysis using one-way ANOVA followed by Bonferroni’s multiple comparison tests was performed utilizing GraphPad Prism version 5.0 (GraphPad Software, California, USA). p<0.05 was considered statistically significant in all studies.

In view of above outcomes observed during study, formulation R-SEDDS of the present invention exhibits superior colloidal stability, efficient drug loading, and spontaneous emulsification, resulting in a nano-sized droplet distribution. Pharmacokinetic and pharmacodynamic studies in animal models indicated a significant enhancement in bioavailability, sustained release, and targeted lung delivery of rutin via optimised formulation. The formulation of the present invention facilitated around 3.17-fold increase in peak plasma concentration compared to free rutin, showed significantly enhanced the absorption, and prolonged therapeutic effects compared to free rutin. The novel formulation of the present invention effectively reduced pro-inflammatory cytokines and immune cell infiltration showcasing substantial protective effects against allergen-induced asthma. Additionally, the formulation exhibits strong protective effects against allergen-induced asthma, reducing bronchospasm, pulmonary oedema, and level of pro-inflammatory markers.

Accordingly, the R-SEDD formulation of rutin for intranasal administration of the present invention exhibits improved drug delivery properties such as enhanced solubility, bioavailability, therapeutic efficacy, stability and sustained release in the Asthma management.

From the features as applied to various alternatives, it can be understood that various omissions, substitutions, and changes in the form and details of the present disclosure can be made without departing from the scope of the disclosure. As can be recognized, certain alternatives described herein can be embodied within a form that does not provide all of the features and benefits set forth herein, as some features can be used or practiced separately from others.
, Claims:I/We claim:
1. A self- emulsifying rutin loaded nasal drops formulation, wherein the formulation comprises an oil phase, an aqueous phase,
wherein the oil phase comprises Rutin and eucalyptus oil in a specific ratio, and wherein the aqueous phase comprises surfactant and co-surfactant in a specific ratio,
wherein said formulation is used for intranasal administration in chronic respiratory disorders such as asthma.

2. The formulation as claimed in claim 1,wherein said oil is eucalyptus oil, the surfactant is Tween 80, and said co-surfactant is PEG300.

3. The formulation as claimed in claim 1, wherein the oil phase comprises 30 mg Rutin in eucalyptus oil in the range of 10% to 30% (w/w).

4. The formulation as claimed in claim 1, wherein the ratio of surfactant and co-surfactant in the aqueous phase ranges from 80:10 to 30:60 (w/w).

5. The formulation as claimed in claim 1, wherein the ratio of oil phase and aqueous phase is 1:9 to 3:7.

6. The formulation as claimed in claim 1, comprises:
(i) Rutin 30mg in 10% (w/w) eucalyptus oil,
(ii) a mixture of a Tween 80 and PEG 300 at 60:30 ratio.

7. A process to obtain a self- emulsifying rutin loaded nasal drops formulation, said process comprises:
(i) selecting suitable excipients comprising an oil, a surfactant, and a co-surfactant depending on the solubility of rutin,
(ii) dissolving the rutin in the selected oil of step (i) to obtain an oil phase,
(iii) mixing a surfactant and a co-surfactantat specific ratio to obtain an aqueous phase, and
(iv) mixing the oil phase of step (ii) in the aqueous phase of step (iii) in specific ratio with gentle vortexing to obtain a self- emulsifying rutin loaded nasal drops formulation,
wherein said formulation is used for intranasal administration in chronic respiratory disorders such as asthma.

8. The process to obtain a self- emulsifying rutin loaded nasal drops formulationas claimed in claim 7, said process comprises:
(i) selecting suitable excipients comprising a eucalyptus oil, a surfactant Tween 80, and a co-surfactant PEG 300 depending on the solubility of rutin,
(ii) dissolving the 30mg rutin in the selected eucalyptus oil at 10% (w/w) of step (i) to obtain an oil phase,
(iii) mixing a surfactant Tween 80 and a co-surfactant PEG 300 at specific 60:30 ratio to obtain an aqueous phase, and
(iv) Mixing the oil phase of step (ii) in the aqueous phase of step (iii) at specific 1: 9 ratio with gentle vortexing to obtain a self- emulsifying rutin loaded nasal drops formulation,
wherein said formulation is used for intranasal administration in chronic respiratory disorders such as asthma.