INTRODUCTION:

The development of in situ gel systems has received considerable importance over the past few years. In situ gel forming drug delivery systems release drug in a sustained manner maintaining relatively constant plasma drug concentration. These polymeric solutions are liquid at room temperature but undergo gelation when in contact with body fluids or change in pH. Polymeric solution shows gelation in different conditions such as temperature dependent, pH dependent and cation induced gelation. In situ forming drug delivery systems are simple to manufacture, reduced frequency of administration, improved patient compliance and comfort as compared to conventional controlled release formulations.

Insitu gel forming drug delivery is a type of mucoadhesive drug delivery system. At the site of drug absorption they swell to form a strong gel that is capable of prolonging the residence time of the active substance. There are different types of polymers which are from natural and synthetic origin, both polymers can be used for the production of in situ gels .In situ gel formation occurs due to one or combination of various stimuli like pH change, temperature modulation and ionic cross- linking. In situ gels have made an irreplaceable space because of their unique characteristics.

MATERIALS AND METHODS:

The drug Tapentadol was obtained as gift sample. All the chemicals and reagents utilised in the current study were of analytical grade. Deacetylated Gellan Gum was procured from (CPKelco, USA), Carbopol 934P from (Lubrizol Inc., U.S.A), Poly ethylene glycol (Qualigens Ltd., Mumbai), Mannitol (Loba Chemie Ltd., Mumbai), Benzalkonium chloride (Optho remedies Ltd. New Delhi).

Formulation:

Preparation of Gellan gum in situ gel:

Aqueous dispersions of Gellan gum, 1% Polyethylene glycol (PEG), and Carbopol 934P (C934P) were prepared in various concentration as shown in the Table 1 using ultra pure water. The dispersions were then stirred for 20 minutes at 100°C in a water bath and then cooled to room temperature. Tapentadol (3%w/v) solubilized in minimum amount of water and 1% Polyethylene Glycol (PEG) 400 aqueous solution; was added on cooling. The solutions were allowed to equilibrate for at least 16h at 4-8°C temperature. Required quantity of Carbopol 934P (C934P) was added slowly with stirring. Sufficient amount of Mannitol and Benzalkonium chloride were also added simultaneously. pH of formulations was adjusted between 4.5 to 5.5; by using 0.1N HCl. The formulations were filled in 10-mL amber colored glass vials, capped with rubber closures and sealed with aluminum caps. Sterilized formulations were stored in a refrigerator (4–8°C) until use [13].

Table1. Composition of Tapentadol HCl in situ gelling systems.

Sr No.	Ingredient (%w.v)	K₁	K₂	K₃	K₄	K₅
1	Kelcogel	0.3	0.4	0.5	0.6	0.7
2	Carbapol934P	0.1	0.1	0.1	0.1	0.1
3	Tapentadol HCl	3	3	3	3	3
4	Mannitol	4	4	4	4	4
5	Benzalconium Chloride	0.001	0.001	0.001	0.001	0.001
6	Deionized water	q.s.	q.s.	q.s.	q.s.	q.s.

Gel strength determination:

It is expressed in terms of time (in seconds) required by a 35g piston for penetration of 5 cm distance, through the 50g gel formulation. Test was performed using ‘Gel strength apparatus’ modified at laboratory(Figure-1). Gellan carbopol solution (50g) was placed in a 100ml measuring cylinder and gelation was induced by SNF. The piston (weighing 35g) was then placed onto the gel. The gel strength was measured as the time (seconds) required to move the piston 5 cm down through the gel. In cases that took over 5 minutes to drop the apparatus into the gel, additional weights were placed on top of the apparatus and gel strength was described by the minimal weights that pushed the apparatus5 cm down through the gel. [9,15]

Figure 1: Gel strength measuring apparatus. (A)Weight; (B) Shaft; (C) measuring cylinder; (D) Polymer gel

Evaluation of the Mucoadhesive Strength:

The mucoadhesive potential of each formulation was determined by measuring the force required to detach the formulation from nasal mucosal tissue using a modified method.[17].

The probes were equilibrated and gelation was induced by means that of SNF. Probe with nasal tissue was down till the tissue contacted the surface of the sample. Immediately, a small force was applied for two minutes to make sure intimate contact between the tissues and the samples. The probe was then moved upwards at a constant speed of 0.15 mm/s. The bioadhesive force, expressed as the detachment stress in dyne/cm², was determined from the minimal weights that detached the tissues from the surface of each formulation using the following equation.

Drug content uniformity:

The vial (n = 3)containing the preparation were shaken for 2–3 minutes manually and 100 µL of the preparation was transferred to 25-mL volumetric flasks with a micropipette and the final volume was made up with Phosphate buffer pH 6.6.(IP) Tapentadol concentration was determined at 272 nm (UV-1700, Shimadzu) by using previously plotted standard calibration curve.[7]

In vitro diffusion studies:

In-vitro diffusion study of formulated in situ gels was carried out on Franz diffusion cell having 2.0 cm diameter and 16 mL capacity. Dialysis membrane (Himedia) having molecular weight cut off range 12000 – 14000 kDa was used as diffusion membrane. Pieces of dialysis membrane were soaked in phosphate buffer (PB) pH 6.6 for 24 hrs prior to experiment. Diffusion cell was filled with phosphate buffer pH 6.6; and the dialysis membrane was mounted on cell. The temperature was maintained at 34°C. After a pre-incubation time of 20 minutes, pure drug solution and formulation equivalent to 3mg of Tapentadol HCl was placed in the donor chamber. Gelation was induced using SNF.[4]

At predetermined time points, 0.5mL samples were withdrawn from the acceptor compartment, replacing the sampled volume with PB pH 6.6 after each sampling, for a period of 210 minutes. The samples withdrawn were filtered and used for analysis. Blank samples (without Tapentadol) were run simultaneously throughout the experiment to check for any interference. The amount of permeated drug was determined using a UV- spectrophotometer at 272 nm (Linearity range = 10 to 100 mcg/mL, R²= 0.997).

Diffusion equation and Korsmeyer- Peppas exponential equation as follows- Higuchi’s square root of time equation:

Q = K_Ht^1/2……………………………………………(2)

Korsmeyer - Peppas equation: Mt / M = ktⁿ……….(3)

Where

Q – Amount of drug released at time t

K_H- Higuchi’s square root of time release rate constant

Mt/M – Fraction of drug released at time t

M – Amount of drug released at infinite time ~ 100%

k – Release rate constant

n – Diffusion exponent indicating the release mechanism

Ex vivo permeation studies:

Fresh nasal tissues were carefully removed from the nasal cavity of sheep obtained from the local stockyard. Tissue samples were placed in Franz diffusion cells exhibiting a permeation area of 3.14 cm². 16 mL of PB pH 6.6 was added to the acceptor chamber. The temperature was maintained at 34°C. After a pre-incubation time of 20 minutes, formulation equivalent to 3mg of Tapentadol was placed in the donor chamber. At predetermined time points, 1-mL samples were withdrawn from the acceptor compartment, replacing the sampled volume with PBS pH 6.6 after each sampling, for a period of 5 hours.[3].

The samples withdrawn were filtered and used for analysis. Blank samples (without Tapentadol) were run at the same time throughout the experiment to envision for any interference. The amount of diffused drug was determined using a UV-visible spectrophotometer at 272 nm (Linearity range = 10 to 100 mcg/mL, R²= 0.997). Permeability coefficient (P) was calculated by following formula

dQ/dt

P = ––––––––––– [5]

C_o× A

Where,

dQ/dt: Flux or Permeability rate (mg/ hr),

C_o: Initial concentration in donor compartment,

A: Effective surface area of nasal mucosa.

Histopathological evaluation of mucosa:

Histopathological evaluation of tissue incubated in Phosphate buffer saline (PBS pH 6.6; IP) after collection it was compared with tissue incubated in the diffusion chamber with gel formulation. Tissue was fastened in 10% buffered formalin (pH 7.2), routinely processed and embedded in paraffin. Paraffin sections (7 µm) were cut on glass slides and stained with hematoxylin and eosin. Sections were examined under a light microscope, to detect any damage to the tissue during ex vivo permeation study. [11]

RESULTS AND DISCUSSION:

FTIR Spectroscopy:

IR spectra of drug free polymers showed no matching peaks with the drug spectra. The characteristic peaks of the drug (3170, 1588, 1449, 881, 795, 709, 682 cm¹) were also appeared in the spectra of all the drug- polymer combinations.

For the formulation of in situ gels, Gellan gum was selected for ion induced gelation, Carbopol 934P was combined with polymer as a mucoadhesive agent. The developed formulations were evaluated for clarity, pH, gelation properties, gel strength, viscosity, mucoadhesion, percent drug content, in vitro diffusion, ex-vivo permeation, stability study and finally histopathological evaluation. Formulations containing plain Gellan gum were clear while those having Carbopol in combination showed slight transparency pH of all formulations was observed in range of 5.4 to 6.2.

Gelation study:

Gelation studies were carried out using simulated nasal fluid. In these studies the gelling capacity (speed and extent of gelation) for all formulations were determined. After easy instillation in to nasal cavity the liquid polymeric solutions should undergo rapid sol to gel transition by means of ionic gelation. Thus the in situ formed gel should preserve its integrity without dissolving or eroding so as to localize the drug at absorption site for extended duration. As per the visual inspection the preformed gels were graded in following grades.

Table 2. Gelation study of in situ gel formulations.

Sr. No.	Formulation Code	Degree of gelation
1	K₁	+
2	K₂	+ +
3	K₃	+ + +
4	K₄	+ + + +
5	K₅	+ + + +

(-) No gelation, (+) weak gelation; dissolves rapidly, (+ +) Immediate gelation remains for few hrs (less stiff gel), (+ + +) Immediate gelation remains for extended period (stiff gel), (+ + + +) Very stiff gel.

Viscosity study:

The apparent viscosity values were measured for liquid and gel formulations using Brookfield viscometer DV-E with spindle no.63 at varying rpm. The marked decrease in viscosity of sol was observed than that of gel. (Figure 6)

In the liquid state formulations was observed to be exhibiting Newtonian flow in solution state while in the gel state it exhibits the pseudoplastic flow.

Figure 1: Viscosity plot (a – ol, b – gel)

Table 3. Viscosit data of in situ gel formulations.

Sr No.		Formulation code	Viscosity (cP) Sol	Viscosity (cP) Gel
	1	K₁	197	752
	2	K₂	262	1172
	3	K₃	295	1320
	4	K₄	416	2016
	5	K₅	528	3168

Evaluation of the Mucoadhesive Strength:

The reinforcement of the mucoadhesive forces within in the nasal in situ gels by the utilisation of mucoadhesive polymers could be explained by the actual fact that secondary bond forming groups (hydroxy, ethoxy and amine) are the principle source of mucoadhesion. The bioadhesive force is known to be dependent on the nature and concentration of bioadhesive polymers. The stronger the bioadhesive force more is the nasal residence time. But if the mucoadhesion is too strong the gel can damage to the mucosal membrane. The Mucoadhesive force was increased with respect to corresponding increasing concentration of Kelcogel from range K₁(0.3 % w/v) to K₅(0.7 % w/v). Combination of Carbopol with 0.5% w/v Kelcogel solution was found to show better mucoadhesion.

Table 4. Gel strength and mucoadhesive force of formulations

Sr No.	Formulation code	Gel strength(second)	Mucoadhesive force(dyne/cm²)
1	K₁	7.16 + 0.05	1975.66 + 0.41
2	K₂	16.53+ 0.05	2401.26 + 0.25
3	K₃	27.2 + 0.10	2970.1 + 0.10
4	K₄	37.46 + 0.11	3301.83 + 0.05
5	K₅	39.23 + 0.15	3912.58 + 0.32

Figure 2: Mucoadhesive strength determination plot.

Drug Content:

The drug content of the formulations was ranging from 93.85 % to 103.85 %.

Table 5. Percentage drug content of in situ gel formulations.

Sr No.	Formulation code	Drug content %
1	K₁	93.98664 +0.65
2	K₂	96.05 +0.18
3	K₃	98.2 +0.35
4	K₄	98.143 +0.55
5	K₅	101.99 +1.61

On the basis of gelation properties, gel strength, mucoadhesion, percent drug content, the optimum in situ gelling formulation of K₃was selected and subjected for further studies.

In vitro release study:

In vitro release studies of formulations were performed using the Franz diffusion cell with dialysis membrane. Phosphate buffer of pH 6.6 was used as diffusion media. The initial rates of drug release were very rapid due to incomplete gel formation, but as the time progresses the release rate decreases due to complete gel formation. With increase in concentration of Carbopol 934P the release rates were found to decrease gradually. The release profiles exhibited an inflection point, which indicated the gel formation in the donor compartment of diffusion cell. During gel formation, a small amount of drug might be loaded into the gel matrix, resulting in the cross linking of polymer which reduces the drug release rate.

Figure 3. In vitro drug release profile of in situ gel formulations K₃

The initial rapid release of Tapentadol HCl was may be due to formation of prehydrated matrix containing water filled pores due to presence of aqueous vehicle. The results showed that the formed gels had the ability to extend the release of Tapentadol HCl for the duration of about 300 minutes.[10]

Drug release kinetics:

To precisely know the drug release mechanisms from in situ gelling systems, the in vitro release data of formulation K₃was treated with Higuchi’s diffusion equation (Q= kt^1/2). The graph between percentage cumulative drug release and square root of time showed almost linear relationship after the initial period[6]. It was not possible to correlate the release in the early stages of drug release study, due to incomplete gel formation. After the complete gel formation, the release profiles were found to be linear with square root of time and followed the Higuchi’s equation (2). (Figure 6) The drug transport mechanism of the same formulations was determined by using the Korsmeyer- Peppas exponential equation [(Mt/M) = ktⁿ], from the plot of log (Mt/M); proportion of drug released at time‘t’ versus log of time.

The slope of the K - P plot of K₃formulation was observed to be 0.758, indicating the anomalous transport mechanism. (Table 7)

Figure 5. Ex vivo permeation study of in situ gel formulation K₃

Table 7. Drug release kinetics data from optimized in situ gel formulation.

Plot	R² value	N value
Zero order plot	0.976	-
First order plot	0.948	-
Higuchi plot	0.977	-
K-P plot	0.974	0.758

Ex vivo permeation study:

Formulation K₃was further subjected to ex vivo permeation studies using the sheep nasal mucosa. The percent drug permeated after 5 hours was found to be 87.51 % (Figure 10).

The permeability coefficient (P) was also calculated and found to be 0.07944 cm/hr.

Histopathological study:

Photomicrographs of sheep nasal mucosa after the permeation studies were observed for histopathological changes in comparison with the PBS treated mucosa.

Table 8. Histopathological inference of Photomicrographs

Histological findings	Formulations
Histological findings	PBS treated (A)	III(B)
Degeneration of mucosa	-	-
Erosion of mucosa	-	-

- : No change, +: Very slight, + +: Slight

The section of mucosa treated with formulation K₃showed no degeneration of nasal epithelium as well as no erosion of mucosa. There was increased vascularity in basal membrane and superficial part of submucosa as compared with PBS-treated mucosa. This might be the result of mucoadhesive and permeability enhancing property of Carbopol in the formulation. (Table 8) There was no sign of remarkable destructive effect of formulations on the treated nasal mucosa.

Stability study:

The stability studies carried out on optimized formulations K₃at 30±2°C temperature and 60±5% RH for 30 days. Formulation was showing good stability with no remarkable change in drug content, gelation property, gel strength and in vitro drug release profile.

Table 9. Stability study data of K₃formulation.

Sr. No.	Parameters	Storage period (Days) at 30±2°C temperature and 60±5% RH
Sr. No.	Parameters	0	30
1	Appearance	Clear	Clear
2	Drug content (%)	98.2 +0.356	97.83+ 0.25
3	Gelation study	+ + +	+ + +
4	Gel strength (s)	27.2 + 0.1	26.1 + 0.25
5	In vitro drug release (%) after 5 hrs	95.29	94.89

CONCLUSION:

Nasal mucoadhesive drug delivery system is designed with an aim to target the drug and to maintain the dosage form at its absorption site for an extended period of time. This will result in the enhancement of the absorption of the drug, which will in turn reduce the presystemic metabolism; increase the bioavailability of the drug, initiate rapid onset of action and thus will decrease the dosing frequency and dose related side effects of the drugs. For this purpose in situ gelling system is preferred as it has several advantages like ease of administration, less irritation, enhanced retention period. The present study was aimed towards formulating the nasal mucoadhesive in situ gels of Tapentadol HCl by ionic gelation using mucoadhesive polymer Carbopol 934P. The in situ gels so prepared were characterized for its gelation properties, viscosity, gel strength, mucoadhesion, drug content, drug release rate and for its histopathological studies. From the above evaluation studies, formulation K₃from ionic gelation technique was optimized and further subjected for stability studies carried out at 30± 2°C and 60 ± 5% RH for 30 days in order to know the influence of temperature and relative humidity on drug content and on drug release profile. Formulated Gellan gum- Carbopol ion based in situ gel K₃showed the effective gelation viscosity, gel strength and drug release properties along with good mucoadhesive strength. The drug release mechanism of the gel matrices was found to be anomalous and following the Higuchi equation. These formulations did not show any remarkable damage to nasal mucosa so it seems to be safe for preclinical use. The formulation also retained the good stability at accelerated conditions over the period of 30 days. Owing to these properties it can be used as an effective delivery system for the nasal route. These in situ gelling systems would be definitely useful for reliving neuronal pain. The present study was aimed towards formulating the nasal mucoadhesive in situ gels of Tapentadol HCl by ionic gelation using mucoadhesive polymer Carbopol 934P. Formulated Gellan gum- Carbopol ion based in situ gel showed the effective gelation viscosity, gel strength and drug release properties along with good mucoadhesive strength.

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Received on 19.04.2019 Modified on 23.05.2019

Research J. Pharm. and Tech. 2019; 12(10):4741-4746.

DOI: 10.5958/0974-360X.2019.00817.5