INTRODUCTION:

Since ancient time intranasal drug delivery has proved a safe and effective route for central nervous system. Now a days this delivery system have-became one of the most interested, safe and effective way to overcome the drawbacks related to oral and parenteral route. Intranasal delivery may be a better suited route of delivery for local and systemic action of a drug candidate having poor bioavailability, hindered by the blood brain barrier. This delivery system may combat the other problem related to oral and parenteral route. Structurally epithelium of nasal mucus is monolayer, larger surface area, highly vascularized which offer instant absorption of drug substances.¹

Now a day’s disease related to the CNS has become a global threatening in developing country, including India thus researchers working toward delivery to CNS have a golden opportunity.² Sometime depression may be mild symptomatic or asymptomatic thus it becomes more ignorable, but progression of diseases, causes other neurological degeneration and become life threatening. In present scenario selective serotonin reuptake inhibitors are the first-choice drug for depression, but as per study of American Psychiatric Association Monoamine oxidase inhibitor may be used as an antidepressant when other antidepressant trial on patient have been failed.³ Selegine is both MAO-A and MOA-B inhibitor (dose dependent). In situ nasal gel delivery is a safest and effective drug delivery system to CNS which combat the problem related to its absorption and bioavailability. In situ gel delivery Chitosan based in situ nasal gel is a pHdependent in situ gelling system which is sol at room temperature, but converted into a gel on increasing its pHof respective environment. Present delivery system is based on the chitosan and β-glycrophosphate. β-Glycerophosphate is used in form of di sodium salt which is a weakly basic compound which is naturally found in the body. Addition of β- Glycerophosphate to chitosan polymer causes conversion of pH sensitive chitosan to temperature sensitive.⁴ Addition of glycerophosphate also increases the viscosity of sol as well as gel. Developed formulations were evaluated for various parameters like gelling time, pH, mucoadhesive strength, gel strength, drug content, in vitro ex vivo release and in vivo release study. Gelling time, pH, viscosity (sol and gel both), mucoadhesive strength, gel strength, drug content, in vitro release study of an optimized formulation batch revealed 17.0±1.0 seconds, 6.4±0.1, 229.3±1.0 CP, 1433.3±1.2 cp, 3035.78±0.46 dyne/cm², 35.7±0.6 seconds, 99.45±0.4 % respectively. Histopathological studies of treated nasal mucosa of sheep revealed that selected formulation batch have no toxic effect on mucosal layer and no significant change was observed in mucosal structure. In vivo study of prepared optimized in situ gel was found more efficacious compare to marketed oral tablet of selegiline.

MATERIALS AND METHODS:

Material:

Selegiline was purchased from sigma Aldrich Pvt. Ltd. India. Chitosan and β-Glycerophosphate were purchased from Loba Chemie Pvt. Ltd. Mumbai. Benzalkonium chloride and sorbitol were purchased from CDH New Delhi.

Preparation of chitosan based in situ gel:

Accurately weighed chitosan was dissolved in 0.1 N HCl to get solution of chitosan. β-glycerophosphate was dispersed in distilled water and allowed to cool at 4ºC. Chilled β-glycerophosphate was added drop wise to chitosan solution by continuous stirring. After that selegiline was added in above polymeric dispersion.⁵ Benzalkonium chloride and sorbitol were added as preservative and isotonic agent. Preparation was stored in a proper closed glass container.

Optimization of formulation batches:

Optimization for formulation batches were carried out on the basis of dependent and independent variables. The level of independent variables was selected and evaluated for the gelling time, viscosity of sol and gel, drug, content, mucoadhesive strength, gel strength

Experimental results may expressed as second order polynomial equation-

Yi = b₀ + b₁X₁ + b₂X₂ + b₁₂X₁X₂+ b₁₁ X²₁ +b₂₂X²₂

Where Y is dependent variable (response), b₀is arithmetic means of nine formulation and b1, b2, b_12,b_11,b₂₂are linear coefficients.⁶ Analytical study of combined effect on different variable on the responses 3-D surface plots were generated by design expert software.

Gel characteristics, gelling temperature and time:

Gel characteristics are the qualitative strength of gel while gelling time is the minimum time required in the conversion of sol to gel on transition of temperature. Gelling temperature of all formulation batches were characterized by increasing temperature of sol up to nasal cavity temperature (34.4 ºC).⁷Gelling time is the time required to conversion of sol to gel. Tube inversion method was used for gelation study in which 0.5 ml of sample was transferred to vial of 2 ml which immersed in water bath at 20ºC temperature and temperature of water bath was increased at rate of 0.5/min. Temperature on which sol was converted into gel was recorded and time required to convert in gel was recorded as gelling time.^{8,
9, 10}

Viscosity:

Viscosity of formulation affect the residence time of sol or gel in nasal cavity as well as mucocilliary clearance. Viscosity of both sol and gel was measured by using brookfield viscometer (Model no.LVDVE) at room temperature and nasal temperature respectively. For conversion of sol to gel, temperature of sol was increased up to nasal temperature then viscosity of gel was determined.^{11, 12}

pH:

pH of all formulations was determined by using digital pH meter (Century CP-901). Firstly pH meter was calibrated using PH 4, 7 and 9 buffers then 20 ml of sol was placed under electrode and each reading was recorded in triplicate.^13,14

Drug content:

Drug content of formulation was determined by using double beam UV visible spectrophotometer (Analytical technologies, Spectro 2080). One ml of formulation sample was diluted with 50 ml distilled water. One ml of sol withdraws from the above diluted sample of formulation and again diluted with 10 ml of distilled water. Diluted sample was analyzed by UV spectrophotometer at 288nm.^{15, 16}

Gel strength:

Gel strength is measured in terms of time required to penetrate 5 cm distance on applying 35 gm of weight on a piston. For this study a modified vessel (Fig. 1.A) having piston was put on a thermostatically controlled water bath (33.5±0.9ºC). A 25 ml of sample was transferred in vessel which gelled on increment in temperature. A weight of 35 gm was applied on the piston and time required to penetrate the piston 5 cm distance in gel was noted as gel strength.^{17, 18}

Mucoadhesive strength:

Mucoadhesive strength is the quantitative measurement of detachment force required to separate the prepared in situ gel from the nasal mucosa. For this study modified balance method used (Fig. 1.B), in which left side pan is substituted with a thread tied to a glass vial adhere over another glass vial and both vials were covered with nasal mucosa. Sheep nasal mucosa was used for mucoadhesive strength which obtained from local slaughter house. Nasal mucosa of sheep was isolated and soaked in simulated nasal fluid (SNF) for 10 minute. Separated mucosal layer was tied on two glass vial of 2 cm diameter in which one is attached by thread to balance and another fixed-on beaker containing SNF at temperature of 33.5±0.9ºC. In situ gel sample of 0.5 ml was applied between two glass vial and weight required on another pan to detach both vial was noted.^{19, 20}

Detachment force was calculated by following formulae

Mucoadhesive forece (dyne / cm²) = mG/A

Where m is weight required to detachment

G- gravitation force

A- surface area of exposed mucosa

Fig. 1. A. Modified vessel for gel strength measurement

Fig. 1. B. Modified balance for mucoadhesive strength measurement

In vitro release study:

In vitro release study was carried out using franz diffusion cell which consist of two chamber namely called donor and receptor chamber. Donor compartment is separated from receptor chamber with dialysis membrane which is previously soaked in SNF for 24 hours. Temperature is maintained 33.5±0.9ºC throughout the donor compartment. Whole apparatus is maintained at sink condition. Sampling of sample was carried out at particular time interval. Release study was carried out in SNF. One ml of sample taken and same amount was transferred to maintain sink condition.^{21,
22, 23}

Ex vivo permeation study:

Fresh nasal cavity of sheep was obtained from local slaughter house. Carefully nasal mucosa was removed and soaked in SNF overnight. Presoaked nasal mucosa was tied in donor chamber of diffusion cell in such a way that it can separate the donor and receptor cell. Formulation equivalent to 0.5 mg was transferred in donor chamber at predetermined time interval. One ml of sample withdrawn from receptor chamber and same amount is transferred to receptor chamber to maintain sink condition.^24,
25

Histopathological evaluation:

For histopathological evaluation of nasal mucosa mucosal membrane was carefully removed and cut into two pieces one is treated with SNF as negative control another treated with iso propyl alcohol as positive control and third was treated with in situ gel which is carefully removed from diffusion cell used for ex-vivo study. All nasal tissues were stored in formalin and stained with eosin.²⁶

In vivo study (Behavioral activity):

In vivo study namely forced swim test and locomotor activity was accessed in mice for in vivo study. For both study three group of animal (each n=6) was selected namely control, standard and test control. Control group animal were treated with SNF, standard group with marketed selegiline tablet (Selgin 5 mg) and test group with formulated in situ gel. Marketed tablet and in situ gel dose is adjusted for mice (average weight 20 gm). For in situ gel and marketed tablet 2.5 µg dose is required and dosing of all grouped animal done till 13^th days for accuracy of result and avoiding lagging effect of antidepressant. All in vivo study was performed under CPCSEA guideline under CPCSEA No- KNIMT/PHAR/IAEC/18/02.

Forced swim test:

Forced swim test is carried out for behavioral studies like immobility, climbing and swimming. For this study a cylindrical tank of 30X20 cm was selected which filled with water and water level is maintained in such a manner that neither paw of mice nor tail can touch the bottom of tank. Water level should also be sufficient by which mice cannot jump out and temperature of water should be identical to room temperature. All selected animal should preconditioned before 24 hours for experimentation and again transferred to the respective cage. After dosing completion to 13^th days animal of each group were judged for immobility, climbing and swimming within 5 minute.²⁷

Locomotor activity:

Locomotor activity is the study of movement of animal from place to place. For this study open field study conducted in apparatus of 200X200 cm and 50cm height square arena having 30 uniforms square. Individual animal from each group was placed in apparatus and recorded for number of square crossed within 5 minute. Apparatus floor should be cleaned and free from residue after each study for which iso propyl alcohol was used to clean the floor.²⁸

Stability study:

For stability study ICH guideline was followed in which optimized selected batch of formulation was stored in a stability chamber (Icon India Sci Instrument) at 40±2ºC, 75±5 % RH, 25±2ºC and 4±1ºC for three month and evaluated for pH_,viscosity and drug content every month.^{29, 30}

RESULTS:

Optimization of formulation batches:

On the basis of preliminary study three levels of independent variable were found that is chitosan and Glycerophosphate. Thus a 3² full factorial design is applicable for preparation of the formulation batches that exhibit the adequate viscosity, mucoadhesive strength and in vitro drug release. As per factorial design nine formulation batches require to formulating the in situ gelling system⁶. Composition of optimized chitosan gum based selegiline in situ nasal gel was shown in Table 1.

Table 1: Composition of optimized chitosan based Selegiline in situ nasal gel

Ingredients	Batch Code (% W/V)
Ingredients	G₁C₁	G₂C₁	G₃C₁	G₁C₂	G₂C₂	G₃ C₂	G₁C₃	G₂ C₃	G₃ C₃
Selegiline	0.5	0.5	0.5	0.5	0.5	0.5	0.5	0.5	0.5
Chitosan	1.2	1.2	1.2	1.4	1.4	1.4	1.6	1.6	1.6
Glycerophosphate	10	12	14	10	12	14	10	12	14
Sorbitol	0.5	0.5	0.5	0.5	0.5	0.5	0.5	0.5	0.5
Benzalkonium chloride	0.01	0.01	0.01	0.01	0.01	0.01	0.01	0.01	0.01
Distilled water	100	100	100	100	100	100	100	100	100

Table 2: Evaluation of Chitosan based selegiline in situ nasal gel

Batch Code	Gel Characteristics	Gelling time(sec)	Gelling temp (◦C)	pH	Viscosity of Sol (cp)	Viscosity of Gel (at nasal temp. cp)	Drug Content	Mucoadhesive strength (dyne/cm²)	Gel strength (Sec)
G₁C₁	+	43.0±2.6	32.6±1.3	5.6±0.4	162.7±2.6	1216.2±2.3	97.04±1.2	2826.34±2.07	24.0±1.0
G₂C₁	+	34.0±2.0	34.2±1.2	5.8±0.4	170.6±1.3	1266.4±2.2	96.85±1.6	2888.55±2.03	25.0±1.0
G₃C₁	++	35.7±2.5	39.3±1.1	6.0±0.3	182.1±2.8	1309.0±2.8	97.82±0.9	2919.52±1.45	25.7±0.6
G₁C₂	+++	25.7±1.5	34.2±1.2	6.5±0.2	204.9±1.4	1368.8±1.3	98.08±0.9	3009.92±1.96	33.0±1.0
G₂C₂	+++	17.0±1.0	33.5±1.3	6.4±0.1	229.3±1.0	1433.3±1.2	99.45±0.4	3035.78±0.46	35.7±0.6
G₃C₂	+++	18.0±1.0	34.1±1.0	6.4±0.2	234.7±1.6	1490.2±2.1	98.39±1.2	3097.62±1.01	39.0±1.0
G₁C₃	+++	16.7±2.1	34.2±0.5	6.5±0.2	249.7±2.2	1558.0±2.6	98.56±0.2	3127.34±1.46	40.7±1.2
G₂C₃	+++	19.3±1.5	34.8±1.0	6.5±0.3	279.1±1.8	1592.9±2.4	97.43±1.5	3174.28±1.92	45.7±1.5
G₃C₃	+++	18.7±0.6	34.6±1.2	6.6±0.2	290.1±2.2	1613.5±1.5	98.02±0.7	3196.99±1.62	46.7±0.6

Gel characteristics, gelling temperature and time, Viscosity, pH, Drug content, Mucoadhesive strength, Gel Strength:

All formulated batches were evaluated for above mentioned parameter (Table 2). All reading was recorded in triplicate and expressed in mean±sd.

In vitro release study:

Franz diffusion cell was used for in vitro release study in SNF which revealed that on increasing concentration of chitosan and glycerophosphate increases drug release to an extent after which decreases (Fig.2.A). On the basis of in vitro release characterstics batch G₂C₂(98.94) selected for further evaluation process.

Ex-vivo release study:

Optimized selected formulation batch was studied for ex-vivo release study using sheep nasal mucosa. Optimized batch of formulation shows 98.16% drug release at the end of six hours (Fig.2.B).

Stability study:

Stability study of optimized selected batch G₂C₂was carried out in stability chamber at 40±2◦C/75±5 % RH, 25±2◦C and 4±1◦C for three months. pH_,viscosity, drug content and drug release were evaluated at end of every month for three months. Effect of temperature and humidity on optimized selected batch revealed that drug content remains constant throughout the study. No significant change is observed throughout the study. Viscosity of sample increases due to long time storage and loss of vehicle due to evaporation. There was no significant change observe in drug release of optimized batch (Fig.2.C). All related study was showed in Table 3 and expressed in mean±sd.

Table 3: Stability study of selected optimized batch

Formulation batch	Temperature	P_H	Viscosity of sol	Drug content
G₂C₂(Before study)	25±2◦C	6.4±0.1	229.3±1.0	99.45±0.4
G₂C₂(After study)	Temperature	P_H	Viscosity of sol	Drug content
One month	40±2◦C	6.4±0.1	246.0±2.2	97.22±1.1
	25±2◦C	6.5±0.1	296.8±1.5	98.01±0.5
	4±1◦C	6.5±0.2	298.8±1.3	97.75±0.5
Two months	40±2◦C	6.4±0.1	253.2±0.4	97.58±0.54
	25±2◦C	6.2±0.2	298.5±1.2	98.19±0.65
	4±1◦C	6.5±0.1	299.9±0.9	97.50±0.54
Three months	40±2◦C	6.3±0.2	257.9±1.0	97.43±0.4
	25±2◦C	6.4±0.2	297.9±1.7	97.45±1.0
	4±1◦C	6.5±0.2	300.1±0.6	97.80±0.6

Fig. 2. A. In vitro release study, B. Ex – vivo release and C. In vitro release before and after stability study

Histopathological evaluation:

Histopathological evaluation of all three treated group namely negative control (Fig.3.A), positive control (Fig.3.B) and in situ gel treated (Fig.3.C) membrane revealed that negative control and in situ gel treated have no significant damages in mucosal layer while positive control treated mucosal layer have significant damage on mucous layer which is ruptured or partially disappear in microscopy.

Fig. 3. Histopathological evaluation of sheep nasal mucosa representing A. Negative control, B. Positive control and C. In situ gel treated

In vivo study:

Forced swim test:

Optimized selected batch of formulation G₂C₂ as test control was compared to standard control and control group which revealed immobility is less in test control while climbing and swimming is more over standard and control group (Fig.4.A). p value, <0.0001 was found to be significant.

Locomotor activity:

Number of square crossed for each group was observed in 5 minute and found more in test control group compare to standard and control group (Fig.4.B).

A. Forced swim test

B. Locomotor activity

Fig. 4 In vivo test

DISCUSSION:

There are various chitosan and glycerophosphate based in situ gel but all related formulations are of drug other than selegiline. Selegiline in situ nasal gel reported by Sridhar V. was a thermosensitive nasal gel for treatment of Parkinson disease³¹. Previous reports describe use of intranasal route of delivery of selegiline to the brain for treatment of depression^32,
33. Report of intranasal Levodopa, Amantadine, Huperazine-A, Curcumin and Venlafaxine all favor the fact that intranasal route of administration is advantageous compared to the other routes^34-38. Nasal in situ gel of selegiline was developed and evaluated for various physical chemical and biological parameter. Delivery system was based on the chitosan which is a pH sensitive polymer and beta glycerophosphate which is a natural occurring compound in the body. Addition of beta glycerophosphate causes transformation of pH sensitive chitosan to thermosensitive thus whole of the developed formulation is a thermosensitive. Developed formulation was observed for gelling characterstics by transition of temperature from room temperature to nasal temperature and for extent of gelation denoted by Weak gelation (+), Good gelation (++), Excellent gelation (+++)³⁹. Gelation time was found within minute and gelling temperature was identical to nasal temperature. Viscosity of sol and gel both was evaluated by using brookfield viscometer which revealed that increment in concentration of glycerophosphate causes increment in viscosity. pH of all formulated batches was found identical to nasal mucosal pH. Drug content in all formulated batches were found 97.04 to 99.73% which shows polymer inclusion is reproducible for drug delivery. Mucoadhesive strength and gel strength of all formulated batches was found 2826.34 to 3196.99 dyne/cm²and 24.0 to 46.7 second respectively. In vitro release study revealed release of drug all formulation is approximately identical to ex vivo study but after a concentration extent of chitosan and glycerophosphate release decreases due to entrapment of drug in the inclusion of polymer. Histopathological study revealed that the optimized selected formulation batch (G₂C₂) have no corrosive and toxic effect on nasal mucosa. In vivo study revealed that optimized selected formulation batch (G₂C₂) was more acceptable and effective compare to oral marketed preparation of slegiline. Selected formulation batch was also found stable at 40±2◦C, 75±5 % RH, 25±2◦C and 4±1◦C for three month against evaluated parameter namely pH_,viscosity and drug content.

CONCLUSION:

As per discussion and result of formulation batches it was concluded that in situ nasal drug delivery of selegiline using chitosan is a best and promising carrier system for targeting the brain for antidepressant drugs. A major challenge observed in targeting brain is blood brain barrier but by using this delivery system this challenge can be combat successfully. Overall this delivery is non-invasive, cost effective system which may further commercialized for treatment different depressive disorders.

DECLARATION OF INTEREST:

The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article.

ACKNOWLEDGEMENT:

The correspondence author would like to thank KNIMT, Faculty of Pharmacy Sultanpur, UP, India for providing support to studying devotedly as the scholar of the project.

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Received on 02.01.2021 Modified on 24.03.2021

Research J. Pharm.and Tech 2022; 15(4):1424-1430.

DOI: 10.52711/0974-360X.2022.00236