1. INTRODUCTION:

Eye is important sensory organs, convert light to recognize brain¹. Also, restrict the entry of all exogenous substance due to defense mechanisms². Eye may be infected by different diseases such as conjunctivitis, dry eye syndrome, glaucoma and keratitis³. Targeting drug to the ocular site with therapeutic dose is task⁴. Factors of nasolacrimal drainage, drugs binding to lachrymal protein, increased lachrymation, minimum availability of corneal area are reducing absorption of drug in ocular routes⁵. The conventional eye drops are having problem with blinking and results in 10 folds decrease in drug concentration within 4-20 minutes necessities regular dosing and bring about pulse kinetics of the drugs in the eye⁶.

Ophthalmic inserts, ointment, aqueous gel and nanosuspension are some of the traditional approaches exploited to make longer residence time and enhance bioavailability of instilled ophthalmic dose.

However, despite these advantages, the ocular delivery systems show shortcomings such as blurred vision (ointment) or low patient compliance (inserts) ^{7, 8}.

In recent times, in situ gel forming systems have been used as vehicles for controlled drug delivery as it has advantages like improved patient compliance, reduced frequency of administration, and ease of administration⁹. Moreover, polymeric solutions in this system on exposure to the physiological temperature, pH or ionic composition of the lachrymal fluid changes its physical state from solution to a gel thereby it improves the bioavailability and pre-corneal residence time of drugs. Based on the approach deployed to initiate sol-to-gel phase transition, it is put on as three types of system: pH triggered systems, temperature-dependent system and ion activated systems^10,
11.

Generally, pH sensitive polymers are polyelectrolytes having acid or basic groups that responses with pH changes in the local surrounding environment¹². One such polymer is Carbopol 934, a polyacrylic acid polymer with mucoadhesive property usually employed in the preparation of ophthalmic pH triggered gel systems^13-15. In solid-state the Carbopol 934 molecule is in strongly coiled spiral form, on hydration of this spiral form ends in gel formation thereby enhances the viscosity^[16]. Additionally, Carbopol 934 shows excellent organoleptic characteristics, compatibility and stability with many API’s, and sol-to-gel phase transition at low concentration¹⁷. Acidic nature of this polymer may cause eye irritation. Therefore, another polymer such as HPMC K4M was added as a viscosity enhancer to reduce the concentration of Carbopol 934¹⁸.

Cromolyn sodium (2% W/V) eye drop is used as mast cell stabilizer in the treatment of seasonal allergic conjunctivitis. The objective of the present investigation work was to formulate a pH triggered in situ gelling system of Cromolyn sodium for sustained ophthalmic delivery. The polymeric combination of Carbopol 934 and HPMC K4M was explored as a vehicle for the Cromolyn sodium (0.2% W/V) eye drops formulation and was characterized for gelling capacity, viscosity, in vitro release and its kinetic.

2. MATERIALS AND METHODS:

Cromolyn sodium was gifted sample from Aurolab private limited (Madurai, Tamilnadu), Carbopol 934 and HPMC K4M polymers were purchased from Hi media laboratories Pvt. Ltd (Mumbai). Benzalkonium chloride were obtained from Loba Chemie Pvt. Ltd (Mumbai).

2.1. Identification of Cromolyn sodium:

Before the development of pharmaceutical formulation, the drug’s intrinsic chemical and physical properties have been taken into account to provide the framework for drug’s combination with ingredients in the fabrication of dosage form. Preformulation studies serve as an important establishment tool every in the development of both API and drug products.

2.1.1. Description (appearance of drug):

Cromolyn sodium was observed visually to check the colour, odour and nature of the powder.

2.1.2. Melting point:

Melting point of cromolyn sodium was determined by the capillary tube method. The sample was filled into a capillary tube which was sealed at one side. Then, the tube was placed inside the melting point apparatus and the temperature at which drug was changed over to fluid was recorded as melting point^19,20.

2.1.3. Saturation solubility studies of Cromolyn sodium:

2.1.3.1. Preparation of simulated tear fluid:

Simulated tear fluid (STF) of pH 7.4: 0.68 g of sodium chloride, 0.20 g of sodium bicarbonate, 0.008 g calcium chloride dehydrate was weighed accurately, dissolved and made up to 100 ml with distilled water²¹.

2.1.3.2. Procedure:

The solubility of Cromolyn sodium was determined in different solvents like distilled water and simulated tear fluid, pH 7.4. The samples were added to each test tube containing 2 ml of different solvents with continuous shaking for 30 minutes to prepare a saturated solution. Then the saturated solution was transferred to 2 ml centrifuge tube and kept in centrifugation for 15 minutes at 3000 rpm. The samples were filtered through a Whatman filter and aliquots were suitably diluted. It was measured by UV spectrophotometer at 239 nm²².

2.1.4. Determination of lambda max:

From the stock solution of Cromolyn sodium (1000 µg/ml), an appropriate dilution was made to 10 µg / ml by using distilled water and it was scanned between 200-400 nm by using UV spectrophotometer. The peak showing maximum absorbance was noted as the lambda max of drug²¹.

2.1.5. Preparation of standard curve for Cromolyn sodium:

The standard stock solution of Cromolyn sodium was prepared by dissolving 50 mg of drug in 50 ml of simulated tear fluid (pH 7.4) having a concentration of 1000 µg/ml. Then secondary stock solution (100 μg/ml) was prepared from the primary stock solution by pipetting out 1ml and transferred into a 99ml of STF in 100 ml volumetric flask. From the secondary stock solution, the aliquots of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 ml were pipetted out in a 10ml volumetric flasks and the volume was made up to 10 ml with STF to get concentrations of 1 - 10 µg /ml. The absorbance of the sample solutions was measured spectrophotometrically at 239 nm by using distilled water as a blank. The calibration curve was plotted by using concentrations versus absorbance²³.

2.1.6. Drug-Excipient compatibility study:

Fourier transform infrared studies was performed to identify the interaction between drug and polymer. The FT-IR spectrum of Cromolyn sodium, polymer, physical mixture of Cromolyn sodium and polymer (1:1) were obtained by KBr pellet method which involves grinding samples with KBr powder and compressed into pellets^24,
25. The FT-IR spectra were estimated over the range of 4000-400cm^-1.

2.2. Preparation of in situ gel:

Preparation of in situ gel involves 2 phase, the first phase involves dissolving the required quantity of HPMC K4M in distilled water kept on magnetic stirrer to get a clear solution followed by addition of the desired amount of Carbopol 934 with continues stirring for overnight. The second phase involves dissolving the desired amount of drug in distilled water to get 2% w/v concentration followed by addition of disodium EDTA in 0.1% w/v and Benzalkonium chloride in 0.001% w/v concentration was added to the previously prepared first phase (polymeric solution) under constant stirring at room temperature to get a clear solution. Then volume was made up to 100 ml by adding distilled water²⁶.

Table 1: Composition of Cromolyn sodium in situ gel

INGREDIENTS	F-1 (W/V)	F-2 (W/V)	F-3 (W/V)
Cromoyln sodium	2%	2%	2%
Carbopol 934	0.1%	0.15%	0.2%
HPMC K4M	0.5%	0.5%	0.5%
Disodium EDTA	0.1%	0.1%	0.1%
Benzalkonium chloride	0.01%	0.01%	0.01%
water	100ml	100ml	100ml

2.3. Characterization of formulation:

2.3.1 Clarity:

Clarity test for all formulations was visually inspected against a white and black background to inspect the turbidity²⁷.

2.3.2. pH:

The pH of the prepared formulations was measured by a digital pH meter. This was previously calibrated by standard pH 4 and pH 7^{28, 29}.

2.3.3. Gelling capacity:

The gelling capacity study was performed by introduction of a drop of formulation in 2ml of simulated tear fluid (STF) that was equilibrated at 37^oC n a clear glass vial^{30, 31}. Then, it was visually assessed based on account of the time taken for gelation and the time taken for the gel formed to dissolve.

2.3.4. Drug content:

The drug content determination involves the sample preparation by diluting 1ml of the formulation to 100 ml with STF solution pH 7.4 from this 1 ml was withdrawn and further diluted to 10 ml with STF³². Then, it was spectroscopically determined at 239 nm by using UV-visible spectrometer against balnk of STF solution.

2.3.5. Viscosity study:

The developed in situ gel formulations were poured into the small adapter of Brook field viscometer and the angular velocity was increased gradually from 10 to 50 rpm. The optimum viscosity of the formulation should be in the range of 5 to 1000 m Pas before gelling and, 50 to 50,000 m Pas after the formation of gel ^{33, 34}.

2.4. In vitro drug release:

By dialysis bag diffusion method in vitro drug release study of Cromolyn sodium in situ gel formulations was performed. The formulated in situ gel (2 ml) dispersed in a dialysis bag was studied in a 200 ml of simulated tear fluid (pH 7.4) medium contained beaker placed over a magnetic stirrer at 100 rpm that was maintained at 37^oC±1^oC. At predetermined time intervals, 2 ml of samples were withdrawn and replaced it with equal amounts of fresh pH 7.4 simulated tear fluid and the collected samples were immediately analyzed by using UV-visible spectrometer at 239 nm. In vitro drug release profile of prepared formulation was compared with marketed eye drop^{35, 36}.

2.4.1. In vitro drug release kinetic studies:

The release kinetics of optimized Cromolyn sodium in situ gel formulation was assessed by considering four unique models including zero order, first order, Higuchi model, korsmeyer’s-peppas model^{37, 38}.

3. RESULT AND DISCUSSION:

3.1. Identification of Cromolyn sodium:

3.1.1. Description or appearance of drug:

The visually assessed appearance of the drug was found to be white colored, odorless, crystalline powder.

3.1.2. Melting point:

Melting point of drug was measured by capillary tube method. The result was found to be 241^oC-242^oC respectively, which confirms the identification of the drug.

3.1.3. Saturation solubility study:

The saturation solubility of Cromolyn sodium in distilled water and simulated tear fluid of pH 7.4 was determined and found that Cromolyn sodium was more soluble in distilled water and STF (pH 7.4).

3.1.4. Determination of lambda max:

The lambda max of the Cromolyn sodium was found to be 239 nm and their absorbance 0.265³⁹.

3.1.5. Standard curve:

Calibration curve of Cromolyn sodium was done in simulated tear fluid pH 7.4. The regression coefficient was discovered to be 0.998 which was shown in Figure 1.

Fig. 1: Standard graph of Cromolyn sodium in STF

3.1.6. Drug - Excipient compatibility studies:

The FT-IR spectra of pure drug, polymer, its physical mixture were obtained. Cromolyn sodium shows the broad characteristic peaks at 3393.86 cm^-1 (OH stretching), 1702.24 cm^-1 (-C=O stretching), 1475.59 cm^-1 (-C=C- stretching), 1051.24 cm^-1 (-C-O stretching), 951.90 cm^-1 (-C-H aromatic bending). All these peaks were present both in the physical mixture (drug and polymer). There was no interference of any functional groups in all the spectra. Thus, it showed that there is no specific physicochemical interaction between the drug and polymer used in the formulation.

3.2. Characterization of formulation:

3.2.1. Clarity:

The prepared in situ gel formulations was found to be clear without any turbidity and suspended particles.

3.2.2. pH:

The pH of all formulations from F-1 to F-3 was found to be in the range of 4.3 to 4.5 (non physiological condition).

3.2.3. Gelling capacity:

Gelling capacity of all in situ gel formulations was given in table 2. All the formulation showed better gelling capacity at physiological condition.

Table 2: Gelling capacity of in situ gel

FORMULATION	GELLING CAPACITY
F-1	+++
F-2	+++
F-3	+++

‘+++’ Gelation immediate, and for extended period

3.2.4. Drug content:

Drug content of all formulations was discovered to be in the range of 73-81%. Least % of drug content was observed in F-1, where the amount of Carbopol 934: HPMC K4M (1:5). The loading of drug was found to be increased, when the Carbopol 934 concentration in the formulation was enhanced, which may be attributed to the charge-based interaction between Cromolyn sodium and Carbopol 934⁴⁰.

3.2.5. Viscosity study:

The viscosity of in situ gel formulations was measured by Brookfield viscometer and the results were shown in table 5. F-1 formulation shows least viscosity and F-3 was more viscous. This says an increase in viscosity of the formulation when increasing the polymer concentration⁴¹.

The rheological studies of the optimum formulation were studied by plotting graph of shear rate versus viscosity which was shown in Figure 2. This showed that the viscosity of the formulation decreased with an increase in shear rate, which indicates the character of pseudoplastic fluids.

3.3. In vitro drug release studies:

In vitro drug release profile of Cromolyn sodium from the gels containing different concentration of Carbopol 934 along with HPMC K4M in the same ratio was tabulated in table 6. Formulation F-3 had shown the least drug release 70.48% in 8 hours compared to formulation F-1 that is 94.45% and formulation F-2 had shown drug release 83.26%.

Fig. 2: Rheological profile of the in situ gelling system

In vitro drug release study of marketed eye drop (conventional dosage form) demonstrated that around 99.4% medication discharges inside 4 hours.

Table 3: in vitro drug profile of Cromolyn sodium

Time (hours)	cumulative % drug release
Time (hours)	F-1	F-2	F-3
1	46.21	33.20	17.80
2	50.65	39.58	22.67
3	55.44	43.19	31.77
4	56.10	60.89	48.09
5	66.23	66.22	60.18
6	77.34	75.18	61.39
7	85.12	78.46	65.90
8	94.45	83.26	70.48

In vitro drug release study of marketed eye drop and in situ gel formulation as a function of time is plotted (figure 3). The result showed that when the polymer concentration decreases drug discharge increases when polymer concentration increases in vitro drug discharge from the formulation decreases. In situ gel formulations slowly release the drug compared with a marketed eye drop.

Fig.3: Market formulation compare with in situ gel

3.3.1. In vitro drug release kinetic studies:

The result of in vitro drug release of optimized in situ gel formulation was fitted to different kinetics models to discover the drug release mechanism, as shown in table 4.

The korsemeyer-peppas release exponent (n) was found to be 0.84 which was followed non-fickian diffusion (n value was found between 0.43-0.85) mechanism (combination of diffusion and erosion) and first order release pattern ^{42, 43}.

CONCLUSION:

Cromolyn sodium in situ gel was successfully prepared by using Carbopol 934 and HPMC K4M by pH triggered in situ gel technique. F-3 formulation evaluated here has potential in ophthalmic use for reason that it is easily administered and hence the pH triggered in situ gelling considered to be promising for improving the ocular residence time without irritating eyes and a viable alternative to marketed eye drops.

CONFLICTS OF INTEREST:

The author declares no conflict of interest with the data contained in the paper.

ACKNOWLEDGEMENTS:

I am thankful to my guide Dr. S. Subramanian, and teaching and non-teaching staffs, Department of Pharmaceutics from PSG College of Pharmacy for providing their innovative ideas and necessary research facilities for performing my work without any fail. I heartily thank my friend vignesh for support every stage.

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Received on 02.09.2020 Modified on 18.12.2020

Research J. Pharm. and Tech 2021; 14(12):6211-6215.

DOI: 10.52711/0974-360X.2021.01075

Formulation code	Mathematical model kinetics					Best fit model
	Zero order	First order	Higuchi model	Korsmeyer- peppas model
	R²	R²	R²	R²	n
F-3	0.93	0.96	0.85	0.95	0.84	First order