INTRODUCTION:

Nasal mucosa has also been considered as a potential administration route to achieve faster and higher level of drug absorption because it is permeable to more compounds than the gastrointestinal tract due to lack of pancreatic and gastric enzymatic activity, neutral pH of the nasal mucus and less dilution by gastrointestinal contents.

The nasal epithelium is a highly permeable monolayer; the sub mucosa is highly vascular zed with large and fenestrated capillaries facilitating rapid absorption. Moreover, direct systemic absorption avoids hepatic first-pass metabolism. Azelastine hydrochloride is BCS class IV drug i.e. low solubility and low permeability. The mechanism of action Azelastine hydrochloride is relatively selective histamine H₁antagonist which inhibits the release of histamine and other mediators from cells (e.g. mast cells) involved in allergic response. It has some affinity to H₂receptors; based on invitro studies using human cell lines, inhibition of other mediators involved in allergic reactions has been demonstrated with Azelastine hydrochloride. It may also inhibit the accumulation and degranulation of eosinophils at the site of allergic inflammation. Azelastine HCL is 4-(4-chlorobenzyl)-2-[(4RS)-1-methylhexahydro-1H-azepin-4yl] phthalazin-1(2H)-one hydrochloride. It is sparingly soluble in water, soluble in methanol, ethanol and methylene chloride. It has absolute bioavailability 40% after nasal administration. Azelastine hydrochloride is a acidic and it’s pKa value is 8.88.which satisfies the criterion for selection of drug. The dose of Azelastine hydrochloride is 0.1% one or two sprays in each nostril twice daily.

MATERIALS:

Azelastine hydrochloride was obtained as a gift sample from Benzochemical Industries, Ltd. Mumbai, cremophor RH 40 (Polyoxyl 40 hydrogenated castor oil) was procured as a gratis sample from Research Lab Fine Chem. Mumbai. castor oil, hydrochloric acid, Benzalkonium chloride, Sodium chloride, polysorbate 80,methanol were procured as gratis samples from Research Lab. Fine Chem. Mumbai, India.

METHODS:

Physicochemical studies:

Drug solubility study Drug Solubility Study:

Azelastine hydrochloride is sparingly soluble in water, soluble in methanol and ethanol. The solubility of Azelastine hydrochloride in variety of solvents was carried. The amount of 10mg Azelastine hydrochloride was added to 10 ml various solvents. The dispersions were shaken in thermostatically controlled water bath shaker at 37±0.5ºC until equilibrium. Afterwards samples were withdrawn diluted with a solvent. Drug concentration was analyzed and the absorbance of solution was measured at 200-400 nm by using UV-Visible Spectrophotometer.

Determination of λ_max of the drug:

The UV spectrum of Azelastine Hydrochloride was obtained by using UV- Visible double beam spectrophotometer [JASCO V 630]. Stock solutions [100 μg/ml] of Azelastine Hydrochloride were prepared in methanol. This solution was further diluted with methanol to obtain the required concentration. The UV spectrums were recorded in the range 200-400 nm. The wavelength of maximum absorption [λmax] was determined.

Determination of Beers- Lamberts plot:

Standard calibration curve of Azelastine Hydrochloride in methanol:

Accurately weighed 10 mg Azelastine hydrochloride (figure no.1) and it was transferred to 100 ml volumetric flask. The volume was made up to 100 ml with methanol and sonicated for 5 min to produce stock solution of 100 μg/ml. Working standard solutions of strengths 5, 10, 15, 20, 30 μg/ml were made from the stock solution by appropriate dilutions. The above solutions were analyzed by UV spectrophotometer at λmax 288 nm. Methanol was used as blank during Spectrophotometric analysis. The standard calibration curve was obtained by plotting absorbance vs. concentration. The concentration range over which the drug obeyed Beer- Lamberts law was chosen as the analytical concentration range.

Table 5: Composition of Formulation Batches as Per 3² Factorial Design

Formulation code	F1	F2	F3	F4	F5	F6	F7	F8	F9
Azelastine Hydrochloride (mg)	50	50	50	50	50	50	50	50	50
Castor oil (ml)	0.05	0.15	0.25	0.05	0.15	0.25	0.05	0.15	0.25
Polysorbate 80(ml)	0.5	0.5	0.5	1	1	1	1.5	1.5	1.5
Cremophor RH 40(ml)	0.5	0.5	0.5	0.5	0.5	0.5	0.5	0.5	0.5
Sodium Chloride(gm)	0.42	0.42	0.42	0.42	0.42	0.42	0.42	0.42	0.42
Benzalkonium Chloride (ml)	0.005	0.005	0.005	0.005	0.005	0.005	0.005	0.005	0.005
Hydrochloric acid	0.01	0.01	0.01	0.01	0.01	0.01	0.01	0.01	0.01
Purified water (ml)	50	50	50	50	50	50	50	50	50

EVALUATION OF NANOEMULSION:

Physical appearance:

The prepared Azelastine hydrochloride nanoemulsion was inspected visually for their color, homogeneity, consistency.

pH :

pH of all formulations was determined by using pH meter (DIGITAL pH METER ). The pH meter was calibrated before each use with standard pH 4 and pH 7 buffer solutions. 20 ml of formulation was taken in suitable beaker and pH was measured.

Specific gravity:

Specific gravity bottle is used to check the density of the formulation prepared and in turn compared with the density of water. Weight of empty gravity bottle is taken as (M1), weight of specific gravity bottle containing the preparation is considered as (M2), weight of gravity bottle containing water is taken as (M3). Considering this data we can find-

Weight of preparation: (M2-M1)

Weight of purified water: (M3-M1)

Specific gravity

Particle size measurement:

Particle size distribution of nanoemulsion can be determined by photon correlation spectroscopy that analyzes fluctuations in light scattering due to Brownian motion of the particles, using Zetasizer 1000 HS [ Malvern Instruments, UK].

Poly- dispersity index:

It indicates the uniformity of droplet size in nanoemulsion. The higher the value of poly- dispersity, lower will be uniformity of droplet size of nanoemulsion. It can be defined as the ratio of standard deviation to mean droplet size. It is measured by a spectrophotometer.

Viscosity:

The viscosity of different formulation was determined at room temperature using Ostwald viscometer at laboratory scale.

Drug content determination:

Drug concentration in nanoemulsion of Azelastine hydrochloride was measured by spectrophotometer. Azelastine hydrochloride content in emulsion was measure and added with known quantity of in solvent [Methanol] and it gets miscible. Absorbance was measured after suitable dilution at 289 nm in UV/VIS spectrophotometer [JASCO V-630, Japan] and % drug content was calculated.

In -Vitro diffusion study:

In vitro diffusion was carried out by modified Franz diffusion cell. A glass cylinder with both ends open, 10 cm height, 3.7 cm outer diameter and 3.1 cm inner diameter was used as permeation cell. An egg membrane (soaked in phosphate buffer 24 hours before use) was fixed to one end of the cylinder with the aid of an adhesive to result as a permeation cell. 1 gm of medicated solution was taken in the cell (donor compartment) and cell was immersed in a beaker containing 100 ml of 7.4 pH phosphate buffer as receptor compartment. The entire surface of the cell was in contact with the receptor compartment which was agitated using magnetic stirrer and a temperature of 37±1°C was maintained. Sample 1 mL of the receptor compartment was taken at 1 hour interval of time over a period 3 hours with same amount replaced. The sample was analyzed for Azelastine hydrochloride at 289 nm against blank using UV Spectroscopy. Amount of Azelastine hydrochloride released at various time intervals was calculated with the help of calibration curve with phosphate buffer pH 7.4 and plotted against time.

Drug release kinetic study:

To study the kinetics of in vitro drug release, data was applied to kinetic models such as zero order, first order, Higuchi and Korsmeyer- Peppas

Zero order:

C = K₀t

Where K0 is the zero-order rate constant expressed in units of concentration/time and t is the time in hrs.

Plot a graph of cumulative % drug released Vs time.

First order:

Log C = LogC₀ – Kt / 2.303

Where C0 is the initial concentration of drug, K is the first order constant, and t is the time in hrs. Plot a graph of Log cumulative percent drug remaining Vs time.

Higuchi:

Qt = Kt1/2

Where Qt is the amount of the release drug in time t, K is the kinetic constant and t is time in hrs. Plot a graph of cumulative percent drug released Vs square root of time.

Korsmeyer-Peppas:

Mt/ M∞= Ktn

Where Mt represents amount of the released drug at time t, M∞ is the overall amount of the drug (whole dose) released after 12 hr K is the diffusional characteristic of drug/ polymer system constant and n is a diffusional or release exponent that characterizes the mechanism of release of drug. The value of n indicates the drug release mechanism related to the geometrical shape of the delivery system, if the exponent n = 0.5, then the drug release mechanism is Fickian diffusion. If n < 0.5 the mechanism is quasi-Fickian diffusion, and 0.5 < n < 1.0, then it is non-Fickian or anomalous diffusion and when n = 1.0 mechanism is non Fickian case II diffusion, n> 1.0 mechanism is non Fickian super case II. Here plot a graph of Log cumulative percent drug released Vs log time.

In- Vitro dissolution study:

In- vitro dissolution test were performed in USP apparatus type II using paddle method at rotation speed of 100 rpm. Dissolution was carried out in 900 ml phosphate buffer of pH 6.8 as a dissolution medium and maintained temperature 37 ±0.5⁰C. Accurately weighed bulk drug and nanoemulsions were dispersed in dissolution medium. 5 ml aliquots were removed at predetermined time intervals 0, 5, 10, 15, 20, 25, 30, 35, 40, 45, 60 min. from dissolution medium and replace with same buffer solution for maintain sink condition and the sample were analyzed for the drug release using UV spectrophotometer at 289 nm.

Thermodynamic stability studies:

Freeze-thaw cycle:

Freeze thaw cycle was performed for four weeks. Formulation was kept at 5⁰C for 1 week and later change over by keeping the same formulation at 40⁰C in next week followed alternately with temperature.

Heating cooling cycle:

This test was performed for 1 week and the temperature was changed every day in between 25⁰C- 40⁰C. Here 25⁰C was considered as room temperature and 40⁰C was set in oven. It was further evaluated for organoleptic properties.

Stress testing:

In this testing formulation was kept at 60⁰C for one month.

Stability studies:

Test conditions for stability study are shown in (Table 6).

Table 6: Test Conditions for Stability Study

Test Conditions
Duration of study:	3 months
Temperature conditions:	Room temperature 25⁰C ± 2⁰C
Relative humidity conditions:	60 ± 5%
Frequency of testing the samples:	30 Days

The formulations were evaluated mainly for their physical characteristics at the predetermined intervals of 30 Days like appearance/clarity, pH, viscosity and drug content.

Nasal ciliotoxicity studies:

The nasal mucosa of goat was treated with formulation to evaluate the toxic effects of excipients used in the formulation. For nasal ciliotoxicity studies freshly excised goat nasal mucosa except for the septum were collected from the slaughter house in saline and treated with 0.5 ml formulation for 6 hrs. The treated nasal mucosa was then fixed in 10% buffered formalin, routinely processed and embedded in paraffin. Sections were cut on glass slides and stained with hematoxylin and eosin. Sections were examined under a light microscope to detect damage to the tissue.

RESULT AND DISCUSSION:

PREFORMULATION STUDY:

Preformulation study of drug:

Organoleptic properties:

Azelastine hydrochloride was studies for its organoleptic character such as appearance, color and odor. The result shows the details of organoleptic properties of Azelastine hydrochloride were found to be similar as mentioned in literature.

Table no. 7: Organoleptic properties of Azelastine Hydrochloride.

Sr. no.	Characteristics	Result
1.	Appearance	Buff powder
2.	Color	White
3.	Odor	Odorless

Melting point:

The melting point of compound was measured and reported as follows:

Table no.8: Melting point of Azelastine hydrochloride.

Drug compound	Reported melting point	Obtained melting point
Azelastine Hydrochloride	224ºC-225ºC	224⁰C

All the physical properties of the drugs were within the limit of reported standards which assures the purity of the drug samples.

pH:

The pH of all the formulations from F1 to F9was found to be in the range of 5 to 7 pH values of formulations shown in (Table 9).

Table no. 9: pH Values of Formulations

Sr. No.	Formulation code	Observed pH (± S.D)
1.	F1	6.1± 0.013
2.	F2	6.2± 0.05
3.	F3	6.1± 0.01
4.	F4	6.2± 0.004
5.	F5	6.4± 0.001
6.	F6	6.2± 0.01
7.	F7	6.1± 0.01
8.	F8	6.4± 0.01
9.	F9	6.2± 0.01

Ideally, the nasal solutions should possess pH in the range of 5-7, so as to minimize discomfort or irritation due to acidic pH and microbial growth due to basic pH.

Specific gravity :-

Table no. 10: Density of Formulations

Sr. No.	Formulation Code	Observed Density (± S.D)
1.	F1	1.17 ± 0.05
2.	F2	1.16 ± 0.05
3.	F3	1.15 ± 0.01
4.	F4	1.15 ± 0.01
5.	F5	1.17 ± 0.05
6.	F6	1.16 ± 0.05
7.	F7	1.13 ± 0.01
8.	F8	1.17 ± 0.005
9.	F9	1.13 ± 0.03

Measurement of globule size, poly-dispersity index and zeta potential:

The nanoemulsions have the least globule size as compared to the coarse emulsion due to presence of stabilizers and surfactant which reduces the interfacial tension to an ultralow value. Poly- dispersity index is a measure of particle homogeneity and it varies from 0.0 to 1.0. If PDI value closer to 0.0 which indicates narrow size distribution of the formulation. PDI of optimized nanoemulsion was found to be 0.250 which remains stable and will not convert to the macro emulsion. Results of globular size and PDI shown in figure respectively. Larger value of zeta potential indicated stable nature of nanoemulsion formulation and thus no changes of aggregation of particles.

Table no.11: Particle size and Poly-dispersity index

Sr. No.	Formulation Code	Observed Particle size	Poly-dispersity index
1.	F1	119.4	0.386
2.	F2	120.9	0.262
3.	F3	135.6	0.415
4.	F4	118.9	0.362
5.	F5	110.2	0.260
6.	F6	121.2	0.325
7.	F7	131.5	0.263
8.	F8	115.3	0.324
9.	F9	142.6	0.455

From the above results the F5 batch was selected using factorial design because it has maximum particle size which gives good stability to that of nanoemulsion formulation.

Table no.12: Droplet size, zeta potential and poly- dispersity index of the selected nanoemulsion:

Formulation

(Batch no.)

Globule size

Poly- dispersity Index

Zeta potential

106.2

0.250

-50.03

Fig.no.5 : Particle size and Poly-dispersity index

Zeta potential :

Figure no. 6: Intensity of Size Distribution

Factorial models and three dimensional response surface analysis:

Based on the 3² factorial designs, the factor combinations resulted in different, Particle size and PDI. Various models, such as Linear, 2FI, Quadratic and Cubic, were fitted to the data for two responses simultaneously using Design Expert software 8.0.6 and adequacy and good fit of the model was tested using analysis of variance (ANOVA). Mathematical relationships generated for the studied response variables are expressed as equations. Positive or negative signs before a coefficient in quadratic models indicate a synergistic effect or an antagonistic effect for the factor.

ANOVA for response surface quadratic model Y₁ (Particle size):

The statistical evaluation was performed by ANOVA and results are shown in Table 12 . The Model F-value of 14.55 implies the model is significant. There is only a 2.58 % chance that a "Model F-Value" this large could occur due to noise. Values of "Prob > F" less than 0.0500 indicate model terms are significant. In this case A, B, A2, B2 are significant model terms. Values greater than 0.1000 indicate the model terms are not significant. If there are many insignificant model terms (not counting those required to support hierarchy), model reduction may improve the model.

Three dimensional and response surface plots are presented in Figures 8 showing that % CDR. The 3-D plot shows that the PDI increase it can be derived that the change in both independent variables had significant effect on response Y₂.

Figure.no.9. Three dimensional response surface plots for PDI and particle size

Viscosity:

Table no. 12: Viscosity of Formulations

Sr. No.	Formulation Code	Observed viscosity(cp) (± S.D)
1.	F1	28.15 ± 0.04
2.	F2	29.23 ± 0.1
3.	F3	29.17 ± 0.1
4.	F4	29.80 ± 0.06
5.	F5	28.47 ± 0.09
6.	F6	29.38 ± 0.1
7.	F7	29.56 ± 0.05
8.	F8	29.77 ± 0.04
9.	F9	29.93 ± 0.01

Drug content:

Table no.13 : Percent Drug Content of formulations

Sr. No.	Formulation Code	Observed Drug content (%) ± S.D
1.	F1	92.8 ± 0.06
2.	F2	96.2 ± 0.07
3.	F3	91.7 ± 0.07
4.	F4	95.9 ± 0.04
5.	F5	91.9 ± 0.03
6.	F6	88.1 ± 0.01
7.	F7	95.8 ± 0.04
8.	F8	96.4 ± 0.03
9.	F9	95.8 ± 0.02

In-Vitro Diffusion study :-

Table no.14: % Drug Permeation through egg membrane using Modified Franz Diffusion cell apparatus

Sr. no.	Time (hr)	% Permeation ± S.D
1.	1	33.61±0.005
2.	2	43.45±0.005
3.	3	57.05±0.007
4.	4	61.68±0.005
5.	5	77.63±0.003
6.	6	85.66±0.009
7.	7	94.34±0.007
8.	8	99.05±0.009

% Drug release Kinetic study :

Table no.15: R² Values and slope values for applied values

Sr. no	Models	R²Values	Slope Values
1.	Zero order	0.982	10.25
2.	First order	0.980	0.993
3.	Higuchi model	0.971	0.273
4.	Hixson-Crowell Model	0.971	0.012
5.	Peppas model	0.888	0.106

Zero order:

Figure.no.10

First Order:

Figure.no.11

Hixson-Crowell:

Figure.no.12

Higuchi Model :

Figure.no.13

Korsmeyer-peppas :

Figure.no.14

In-vitro Dissolution study :

Table no.16: % drug release by Dissolution

Time [ min]	% Release
5	97.7±0.005
10	97.8±0.006
15	98.00±0.001
20	98.2±0.005
25	98.3±0.005
30	98.6±0.003
35	98.9±0.005
40	99.1±0.003
45	99.4±0.001
60	99.7±0.005

% Drug release :

Zero order model log % CDR:

Figure.no.15

Thermodynamic stability studies:

1. Freeze thaw cycle:

This study was performed for 1 month.

Table no. 17: Observation while performing Freeze thaw cycle.

Weeks	Color	Phase separation	Drug content
1^st week	No change	No	98.5 ± 0.0011
2^nd week	No change	No	98.2 ± 0.005
3^rd week	No change	No	96.9 ± 0.0007
4^th week	No change	No	97.78± 0.0007

2 Temperature cycling:

It was performed for one week. The temperature was changed daily.

Table no.18: Observations got in temperature cycling.

Days	Temperature	Color	Phase separation	Drug content
1	25⁰C	No change	No	98.5 ± 0.0002
2	40⁰C	No change	No	98.1 ± 0.0001
3	25⁰C	No change	No	98.2 ± 0.0001
4	40⁰C	No change	No	98.1 ± 0.0003
5	25⁰C	No change	No	98.3 ± 0.0009
6	40⁰C	No change	No	98.2 ± 0.0007
7	25⁰C	No change	No	98.5 ± 0.0002

3. Stress testing:

The formulation batch was kept at 60⁰C for one month. There was no change in color and drug content was found to be 98.5± 0.00023%.

4.Stability studies:

Optimized formulations were subjected to stability studies as per ICH guidelines. Various parameters such as Physical appearance, drug content, were measured before and after 30 and 60 days of stability. Results of stability studies are shown in table no.19 Physical appearances of all formulations were unaffected or did not show any significant changes.

Results of stability studies showed that there is no significant change in above mentioned parameters after elevated temperature and humidity conditions during stability studies. Thus it can be proved from the stability studies that the prepared formulation is stable and not much affected by elevated humidity and temperature conditions.

Nasal ciliotoxicity studies:

This study was performed to evaluate the toxic effects of excipients present in the formulation.

Figure no.16. Section of nasal mucosa treated with nanoemulsion.

CONCLUSION :

In the present study, Polysorbate 80 and castor oil was used for the nasal drug delivery system of the antihistamine drug Azelastine hydrochloride owing to its increased viscosity after nanoemulsion and mucoadhesive characteristics the formulation displays prolonged nasal residence time .Among all formulations Azelastine, containing 1% polysorbate 80 and 0.5% castor oil, was found to be optimized formulation .It can be concluded that the optimized in nasal nanoemulsion of Azelastine HCL appears to be suitable in seasonal allergic rhinitis, with prolonged residence time.

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Received on 15.07.2016 Accepted on 14.10.2016

Research J. Topical and Cosmetic Sci. 2016; 7(2): 55-66.

DOI: 10.5958/2321-5844.2016.00009.1

Range [cm^-1]	Values [cm^-1]	Bond
1680-1640	1651.05	C=C stretching
1600-1585	1589.57	C-C stretching
1335-1250	1328.78	C-N stretching
1250-1020	1084.26	C-N stretching
1250-1020	1013.27	C-N stretching

Range [cm^-1]	Values [cm^-1]	Bond
3000-2850	2856.76	C-H stretching
1740-1720	1731.37	C=O stretching
1250-1020	1096.06	C-N stretching

Variables	Factors
Independent
X1	Polysorbate 80
X2	Castor oil
Dependent
Y1	In-vitro drug release
Y2	Zeta potential

Source	Sum of Squares	df	Mean Square	F Value	p-value Prob > F
Model	946.56	5	189.31	14.55	0.0258	Significant
A- Polysorbate 80	12.91	1	12.91	0.99	0.3927
B- Castor oil	57.04	1	57.04	4.38	0.1273
Residual	39.04	3	13.01
Cor Total	985.60	8

Source	Sum of Squares	Df	Mean Square	F Value	p-value Prob > F
Model	0.041	5	8.24	30.76	0.0088	Significant
A- Polysorbat	6.801	1	6.80	25.39	0.0151
B- Castor oil	3.553	1	3.553	13.26	0.0357
Residual	8.037	3	2.679
Cor Total	0.042	8

Sr. No.	Concentration [ppm]	Absorbance
1.	5	0.1216
2.	10	0.2091
3.	15	0.2871
4.	20	0.3682
5.	25	0.4491
6.	30	0.5274

Sr. no.	Observations	Before accelerated	After accelerated
			30 days	60 days
1.	% Drug content	98.5± 0.0002	97.78±0.0007	98.2 ± 0.0002
2.	Physical	White color	White color	White color