Glycerogelatin-based ocular inserts of aceclofenac: Physicochemical, drug release studies and efficacy against prostaglandin E₂-induced ocular inflammation

Abstract

An attempt has been made in the present study to formulate soluble ocular inserts of aceclofenac to facilitate the bioavailability of the drug into the eye, as no eye drop solution could be formulated. Glycero-gelatin ocular inserts/films were prepared and physicochemical parameters and drug release profiles of glycerol-gelatin films of aceclofenac were compared with surface cross-linked films of similar compositions. Ocular irritation of the developed formulation was also checked by HET-CAM test and efficacy of the developed formulation against prostaglandin-induced ocular inflammation in rabbit eye was determined. The non-cross-linked films showed poor mechanical, physicochemical properties, and very little potential of sustaining drug release, however cross-linking the films enhanced tensile strength by 70%, but elasticity decreased by 95%. The cross-linked ocular inserts showed less swelling than non-cross-linked. Formulation AF8 (20% gelatin and 70% glycerin, treated by cross-linker for 1 h) demonstrated the longest drug release for 24 h. As per the kinetic models all films showed a constant drug release with Higuchi diffusion mechanism. Formulation was found to be practically non-irritant. The optimized formulation was tested and compared with eye drops of aceclofenac for anti-inflammatory activity in rabbits against PGE₂-induced inflammation. In vivo studies with developed formulation indicated a significant inhibition of PGE₂-induced PMN migration as compared to eye drops. In conclusion, ocular inserts of aceclofenac was found promising as it achieved sustained drug release and better pharmacodynamic activity.

Keywords::

• Ocular drug delivery system
• Gelatin
• cross-linking
• controlled release
• PG-induced inflammation.

Introduction

Inflammatory conditions of the eye are designated according to the tissue affected. Conjunctiva and cornea are constantly exposed to various types of physical, chemical, microbial (bacteria, fungi, viruses), and allergic agents and hence prone to develop acute, sub-acute, and chronic inflammations. Inflammation of the conjunctiva (conjunctivitis) is classically defined as conjunctival hyperaemia associated with a discharge which may be watery, mucoid, mucopurulent, or purulent. Allergic conjunctivitis is a severe chronic ocular allergic disease, occurring throughout the year but increasing in intensity during spring and summer. Typical clinical symptoms are itching, tearing, foreign-body sensation, and severe photophobia. Ocular symptoms have been estimated to be present in 40–60% of the allergic population (Ono & Abelson, 2005). The disease ranges in severity from mild forms, which can still interfere significantly with quality-of-life, to severe cases characterized by potential impairment of visual function.

Often such ocular conditions are treated with medicated eye drops which are instilled frequently, sometimes up to every 4 h, to affect the therapy. A major problem in conventional ophthalmic drug delivery is low drug bioavailability due to ocular anatomical and physiological constraints, which include the poor permeability of cornea, nasolacrimal drainage effect, and short retention time in the pre-corneal area (Davies, 2000). Attempts to improve ocular bioavailability have been focused on overcoming pre-corneal constraints through improving corneal penetration and prolonging the pre-corneal retention (Kaur & Smitha, 2002). Besides this, the drug absorption also depends upon the chemical nature of the drugs (Grass & Robinson, 1984). Sometimes the drug is transported via the naso-lacrimal duct to the GI tract, where it may be absorbed, causing systemic side-effects. To avoid the above-mentioned side-effects and to increase effectiveness of the drug, a dosage form should be chosen which increases the contact time of the drug in the eye. This may increase bioavailability and reduce the need for frequent administration, leading to improved patient compliance (Rathore & Nema, 2009).

Ocular inserts are defined as preparations with a solid or semi-solid consistency, whose size and shape are especially designed for ophthalmic application (i.e. rods or shields)(Saettone 1993). These inserts are placed in the lower fornix and, less frequently, in the upper fornix or on the cornea. They are usually composed of a polymeric vehicle containing the drug and are mainly used for topical therapy (Gorle et al., 2009; Karthikeyan et al., 2008).

In recent years, an explosion in the level of research devoted to the development of new biodegradable materials has been seen (Orliac et al., 2003). Among other biopolymers, gelatin has been extensively studied due to its low cost, biodegradability, biocompatibility, and non-immunogenicity (Bigi et al., 2002). Gelatin has multiple functional properties, it is used as a coating agent; film-former; gelling agent; suspending agent; tablet binder; and viscosity-increasing agent. Gelatin swells in cold water and is completely soluble in hot water (Rowe et al., 2006). Although gelatin has some advantages, there were some problems with the use of gelatin as a biomaterial in ocular tissue as it dissolves in aqueous solution and has poor mechanical properties, which limits its possible applications as a biomaterial, especially for long-term applications (Bigi et al., 2001; 2002). On the other hand, gelatin contains a large number of functional side groups that readily undergo chemical cross-linking. Thus, cross-linking gelatin materials would improve the mechanical stability and decrease the solubility (Bigi et al., 2001; 2002; Vandelli et al., 2001). Chemical cross-linking usually is performed using cross-linking agents such as formaldehyde, glyoxal (De Carvalho & Grosso, 2004), glutaraldehyde (Bigi et al., 2001), and genipin (Bigi et al., 2002), as well as polyepoxy compounds (Nagura et al., 2002) and transglutaminase (De Carvalho & Grosso, 2004), were used to produce modified gelatin films.

Mundada and Shrikhande (2008) explored the cross-linked gelatin films as ocular drug delivery systems for ciprofloxacin hydrochloride. A sustained and constant ciprofloxacin drug release was observed from gelatin ocular inserts. The formulation was advocated as ‘once a day’ preparation.

As a class, NSAIDs have proven to be a safe, effective, and useful in decreasing ocular inflammation. The various ocular inserts of NSAIDs like Diclofenac (Sankar et al., 2006), Ketorolac (Jayaprakash et al., 2000), Indomethacin (Pandit et al., 2003; Patel et al., 2008), and Piroxicam (Gilhotra et al., 2009) had been developed to date.

Therefore, in the present study, an attempt is made to deliver aceclofenac; a non-steroidal anti-inflammatory drug by glycero-gelatin ocular inserts. Ocular polymeric film is attempted for delivery of aceclofenac with the rationale of prolonging the contact of the drug with ocular tissue and facilitating a constant concentration of the drug for better inhibition of the further inflammation in conjunctival tissue. Since no ocular formulation of aceclofenac has been reported in the literature before; therefore, in the present study, a novel ocular drug delivery system (NODDS) of aceclofenac is developed based on the glycerol–gelatin bioadhesive polymer matrix, with an objective to sustain the drug release, reduce frequency of dosing, and enhance ocular bioavailability of aceclofenac.

Materials and methods

Materials

Aceclofenac was obtained as gift from IPCA Laboratories (Ratlam, India). Gelatin and glutaraldehyde were purchased from Qualigens Fine Chemicals (Mumbai, India). All other chemicals used were of reagent grade.

Methods

Preparation of ocular inserts of aceclofenac

Ocular inserts of aceclofenac were prepared by solvent casting method using different concentrations of the gelatin (drug–polymer matrix) casted on the mercury surface (Mundada & Shrikhande, 2006; 2008). A required amount of gelatin was dissolved in purified water at 35°C, using a magnetic stirrer. To this, glycerin (70% w/w) as plasticizer and calculated amount of aceclofenac were added, and stirred until homogenous. The solution was poured over a glass ring on the mercury surface and covered with an inverted funnel to allow slow and uniform evaporation at room temperature for 24 h. The films so obtained were punched with the help of a sharp edged die having an area of 77.3 mm² and 2 mg drug in it. Cross-linking was done by dipping the cut inserts in 10% w/v solution of glutaraldehyde in isopropyl alcohol for three different time periods of 30, 45, and 60 min. After the prescribed time of cross-linking (hardening) at room temperature, the inserts were dipped in the 2% v/v solution of sodium metabisulfide to oxidize the excess unreacted glutaraldehyde from the surface of the inserts (Mundada & Shrikhande, 2008). Finally, the inserts were washed with an ample amount of distilled water. The formulations so prepared were designated as AF1, AF2, AF3, AF4, AF5, AF6, AF7, and AF8 (Table 1). These formulations were packed in polyethylene laminated aluminum foil and sterilized by 2.5 Mrads dose of gamma rays for 18 h (Khan et al., 2008). A test for sterility on the sterilized ocular inserts was performed according to IP.

Table 1.  Composition of aceclofenac ocular inserts.

Formulation code	Hardening/cross-linking time (min)	Gelatin concentration (w/v)	Glycerin concentration (w/v)
AF1	—	15%	70%
AF2	30
AF3	45
AF4	60
AF5	—	20%
AF6	30
AF7	45
AF8	60

Each insert had 2 mg of drug.

Interaction studies

Interaction studies were conducted to investigate any interaction between drug and polymer as well as to study the effect of gamma radiation on aceclofenac. Pure drug, unsterilized, and sterilized medicated ocular inserts were analysed by UV scanning (between 200–500 nm) and infrared analysis in the range of 400–4000 cm⁻¹ by KBr disc method (Shimadzu 84008, Tokyo, Japan).

Physicochemical evaluation

Mean thickness of insert

Thickness of the inserts (n = 3) was determined using a dead weight thickness gauge (Prolific, New Delhi, India) (Gilhotra & Mishra, 2008; Gilhotra et al., 2009). After intial settings, the foot was lifted with the help of the lifting lever fixed on the side of the dial gauge. The insert was placed on the anvil, such that the area where the thickness is to be measured lies below the foot. Readings of the dial gauge were recorded after gentle lowering of the foot and mean thickness was calculated.

Uniformity of weight

Inserts from each batch were randomly selected and weighed individually on an electronic balance. Mean weight of the inserts (n = 20) of each formulation was recorded.

Surface pH determination

Inserts were left to swell for 5 h on agar plate prepared by dissolving 2% (m/v) agar in warm simulated tear fluid (STF; sodium chloride: 0.670 g, sodium bicarbonate: 0.200 g, calcium chloride. 2H₂O: 0.008 g, and purified water q.s. 100 g) (Gilhotra & Mishra, 2008) of pH 7.4 under stirring and then pouring the solution into a Petri dish until gelling at room temperature. The surface pH was measured by means of a pH paper placed on the surface of the swollen patch.

Drug content uniformity

Uniformity of the drug contents was determined by assaying the individual insert. Each insert was grounded in a glass pestle mortar and up to 10 ml of STF was added to make a suspension. The suspension so obtained was filtered and the filtrate was assayed spectrophotometrically at 273 nm (UV- shimadzu 1601).

Folding endurance value

The folding endurance is expressed as the number of folds (number of times the insert is folded at the same place) either to break the specimen or to develop visible cracks. This test is important to check the ability of the sample to withstand folding. This also gives an indication of brittleness. The specimen was folded in the center, between the fingers and the thumb, and then opened. This was termed as one folding. The process was repeated till the insert showed breakage or cracks in center of insert. The total folding operations were named as folding endurance value (Khanna et al., 1997; Gilhotra & Mishra, 2008).

Mechanical strength

Ocular insert with good tensile strength and percent elongation would resist tearing due to stress generated by blinking action of the eye. The film was cut into strips (50 × 10 mm). Tensile strength and elongation at break was determined by modification of the reported method (Dandagi et al., 2004). The apparatus consisted of a base plate with a pulley aligned on it. The film was fixed in insert holder at one end of base plate and another end was fixed with the help of forceps having triangular end to keep the film straight during stretching. A thread was tied to the triangular end and passed over the pulley, to which a small pan was attached to hold weights. A small pointer was attached to the thread that travels over the graph paper affixed on the base plate. The weights were gradually added to the pan till the film was broken. The weight necessary to break the film was noted as break force and the simultaneous distance travelled by the pointer on the graph paper indicated the elongation at break:

1 2

Swelling index

To determine the swelling index of prepared films, initial weight of film was taken, and then it was placed on an agar gel plate (2% w/v agar in STF, pH 7.4) and incubated at 37 ± 1°C. For 5 h, film was removed from plate after every hour, surface water was removed with the help of filter paper, and the film was reweighed. The swelling index was calculated as follows (Wan et al., 1995).

3

where (Sw)% = equilibrium percent swelling, W_t = weight of swollen film after time t, and W₀ = original weight of film at zero time.

Fourier transform infrared spectroscopy

FTIR spectra were obtained of pure aceclofenac drug (powder form), a non-cross-linked drug-gelatin film, and cross-linked drug-gelatin film in potassium bromide (KBr) pellet. All spectra were obtained using a Fourier Transformed Infrared (FT-IR) spectrophotometer (Shimadzu, 84008) over a range of 4000–500 cm⁻¹ with a resolution of 4 cm⁻¹.

In vitro drug release studies

In vitro drug release study was carried out by using a biochemical donor-receptor compartment model (Sreenivas et al., 2006). The commercial semi-permeable cellophane membrane, pre-soaked overnight in the freshly prepared dissolution medium (STF pH7.4), was tied to one end of a cylinder (open at both the sides) which acted as a donor compartment. The ocular insert (n = 3) was placed inside the donor compartment in contact with the semi-permeable membrane. The donor compartment was attached to a stand and suspended in 25 ml of the dissolution medium maintained at 37 ± 1°C in the way that touches the receptor medium surface. The dissolution medium was stirred at a low speed using a magnetic stirrer. Aliquots of 5 ml were withdrawn at regular intervals for 24 h and replaced by an equal volume of dissolution medium every time. The samples were analyzed spectrophotometrically at 273 nm. (UV- Shimadzu 1601).

The release data were kinetically analyzed using different kinetic models (zero-order, first-order, Higuchi diffusion model and Korsmeyer-Peppas model) to determine the mechanism of aceclofenac release from the prepared ocular inserts. In order to determine the release model that best describes the pattern of drug release, the in vitro release data were fitted to zero-order, first-order, and diffusion-controlled release mechanisms according to the simplified Higuchi model and Kosermeyer-Pappas model. The equations used were as follows:

4 5 6

where C₀ is the initial drug concentration (released) at time t, Q is the amount of drug released/unit area, K₀ is the zero-order rate constant, K is the first-order rate constant, and D is the diffusion coefficient, and it was calculated according to the following equation:

7

The Kosermeyer-Pappas model is a more comprehensive model. The data were fitted according to Korsmeyer et al. equation (Korsmeyer et al., 1983; Ritger & Peppas, 1987) which is a simple empirical equation to describe drug release from polymeric systems which is called the power law.

8

where m_t/m_∞ = fraction of drug released, k = kinetic constant, t = release time, and n = diffusional exponent for drug release.

It has been stated that the above equation could adequately describe the release of solutes from slabs, spheres, cylinders, and discs regardless of the release mechanism. The value of n gives an indication of the release mechanism. When n = 1, the release rate is independent of time (zero-order) (case II transport); n = 0.5 is for fickian diffusion; and 0.5 < n < 1.0, means diffusion and non-fickian transport are implicated. Lastly, when n > 1.0 super case II transport is apparent, n is the slope value of log m_t/m_∞ vs log time curve. The drug release data were computed and graphed according to the Korsmeyer equation.

Ocular irritation test (HET-CAM test)

For the present study, a modified hen’s egg chorioallantoic membrane (HET-CAM) test as reported by Velpandian et al. (2006) was carried out. Briefly, fertilized hen’s eggs were obtained from a poultry farm. Three eggs for each formulation weighing between 50–60 g were selected and candled in order to discard the defective ones. These eggs were incubated in a humidified incubator at a temperature of 37 ± 0.5°C for 3 days. The trays containing eggs were rotated manually in a gentle manner after every 12 h. On day 3, egg albumin (3 ml) was removed by using sterile techniques from the pointed end of the egg. The hole was sealed and the eggs were kept in the equatorial position for the development of CAM away from the shell. The eggs were candled on the fifth day of incubation and everyday thereafter non-viable embryos were removed. On the tenth day, a window (2 × 2 cm) was made on the equator of the eggs through which formulations were instilled. A 0.9% NaCl solution was used as a control as it is reported to be practically non-irritant. The scores were recorded according to the scoring schemes as shown in Table 2.

Table 2.  Scoring chart for HET-CAM test.

Effect	Scores	Inference
No visible hemorrhage	0	Non-irritant
Just visible membrane discoloration	1	Mild irritant
Structures are covered partially due to membrane discoloration or hemorrhage	2	Moderately irritant
Structures are covered totally due to membrane discoloration or haemorrhages	3	Severe irritant

In vivo drug release studies

Two groups containing six healthy rabbits in each were used to study the in vivo drug release from formulation AF8, which showed the desired in vitro drug release. Each rabbit was kept in good hygienic condition in order to avoid vulnerability to any disease including ophthalmic type. The inserts sterilized using γ-radiations were used for in vivo drug release studies. A sterilized insert was placed in the cul-de-sac of each rabbit while the other eye served as a control. At periodic intervals (according to in vitro release time point), the inserts were taken out carefully from the cul-de-sac of each rabbit and analyzed for the remaining drug content. A cumulative percentage of drug released in vivo was calculated, and scatter diagrams were constructed to determine the in vitro and in vivo correlations (Charoo et al., 2003; Mundada & Shrikhande, 2008).

In vivo ocular anti-inflammatory study

The pge₂-induced ocular inflammation in rabbit was used to compare the efficacy of ocular inserts with eye drops. The experimental protocol was designed, and approval of the institutional animal ethics committee (IAEC) was taken.

Selection of animals

White albino rabbits (1–1.5 kg) of either sex were selected for the study. Rabbits were divided randomly into groups of five each. Animals were housed in an institutional animal room under standard conditions with free access to food and tap water.

Methodology

Eye drop (n = 5) was instilled into the left eye of rabbits of Group I and ocular insert (n = 5) was inserted into the left eye of rabbits serving as the control, treated with normal saline. After 10 min of administration of formulations in respective eyes, 50 µl of PGE₂ (1 µg/ml in normal saline) (Dinoprostone, Astra Zanacea India Ltd. Bangalore, India) was instilled in both eyes of all the rabbits. All eyes were then evaluated for parameters of inflammation (i.e. lid closure extent and polymorphonuclear leukocyte (PMN) migration) (Gupta et al., 2000; Malhotra & Majumdar, 2006).

Lid closure extent was scored as 0 for fully open, 1 for one-third closed, 2 for two-thirds closed, and 3 for a fully closed eye (Table 3). PMN migration was evaluated by counting PMN in tear fluid. Two drops of normal saline were instilled into the inferior cul-de-sac of the rabbit eye. After gentle mixing, 50 µl of the tear fluid was withdrawn with the help of micropipette at periodic intervals following PGE₂ instillation. Tear fluid so withdrawn was diluted with Turke’s fluid in a WBC pipette and the number of PMN was counted in Neubauer’s hemocytometer (Sood, 1999).

Table 3.  Lid closure scores for evaluation of in vivo ocular anti-inflammatory study done in rabbit eye model after PGE₂-induced inflammation.

Lid closure	Lid closure score
Fully open	0
One-third closed	1
Two-thirds closed	2
Fully closed	3

Statistical analysis

The results of mechanical strength, swelling, in vitro release, and in vivo studies were presented as mean ± SD. All the experimental data was subjected to statistical analysis, using one-way analysis of variance (ANOVA) followed by t-test; p < 0.05 was considered to be statistically significant.

Result and discussion

Table 1 shows the composition of the prepared films. The present investigation was undertaken with the objective of preparing a controlled release ocular insert of aceclofenac using gelatin as the matrix former. Glycerin was employed as a plasticizer in the preparation to get inserts with good elasticity. Film casting procedure was followed to prepare films that resulted in the preparation of uniform aceclofenac-gelatin inserts. The ocular insert was sterilized by γ-radiation. It was found that ocular inserts passed the test for sterility conducted as per I.P.

Interaction studies were carried out to ascertain any kind of interaction of the drug with the excipients used in the formulation of ocular inserts before and after sterilization. For this purpose, the optimized formulation AF8 (20% gelatin and 60 min cross-linking, cross-linked and non-cross-linked film) and the pure drug were subjected to the UV, IR, and TLC analyses. The principle spot in TLC obtained with the test solution was similar in position, color, and size to the size chromatogram obtained with the reference standard of the drug. The UV absorption maximum for the pure drug and the medicated formulations was found to be at 273 nm. The spectra recorded were taken as qualitative in order to assess the changes in peak, patterns of curve, etc. No major differences were observed in the IR spectra of the pure drug and the medicated formulations. Also, the results of the interaction studies indicated that there was no chemical interaction between the drug and the excipients in the ocular inserts before and after sterilization.

The prepared inserts were smooth in texture, uniform in appearance, thickness, and weight; and showed no visible crack or imperfection. Each ocular insert had an area of ∼ 77.3 mm². The film had a thickness varying from 0.188 (0.011–0.227 (0.014) mm and weight varying from 14.27 (0.13) to 23.54 (0.14) mg (Table 4). It was found that the thickness and weight of the inserts increased with increasing total polymer concentration. There was very little gain in weight and increase in thickness after cross-linking in the films. The films retained their integrity and appearance after cross-linking, but there was a considerable hardening of the film surface. The pH of the gelatin solution is almost neutral (7.01). Surface pH was within the range of 5–7 (Table 4). This shows prepared inserts would not cause irritation in the eye. The drug content was consistent in all batches and varied from 97.95 (1.46) to 99.25 (1.11)% (Table 4). The recorded folding endurance for all the films was greater than 300, which is considered satisfactory and reveals good film properties (Table 5). The reason behind the high folding endurance was high concentration of plasticizer (70% w/w) used in the formulations resulted in higher flexibility of the films.

Table 4.  Physicochemical parameters of ocular gelatin films.

Formulation code	Mean thickness(mm) (n = 3)	Mean weight(mg) (n = 20)	Surface pH	Mean drug content (mg)(n = 3)	% Drug content
AF1	0.188 ± 0.011	14.27 ± 0.13	6	1.975 ± 0.03	98.75 ± 1.45
AF2	0.192 ± 0.014	14.46 ± 0.11	6	1.958 ± 0.04	97.90 ± 2.08
AF3	0.194 ± 0.021	15.13 ± 0.15	5	1.962 ± 0.05	98.10 ± 2.56
AF4	0.195 ± 0.012	16.27 ± 0.11	7	1.939 ± 0.03	97.95 ± 1.46
AF5	0.224 ± 0.011	19.42 ± 0.14	5	1.985 ± 0.02	99.25 ± 1.11
AF6	0.225 ± 0.013	19.58 ± 0.10	6	1.953 ± 0.05	97.65 ± 2.28
AF7	0.227 ± 0.011	21.32 ± 0.13	7	1.943 ± 0.04	97.15 ± 1.56
AF8	0.227 ± 0.014	23.54 ± 0.14	6	1.947 ± 0.05	97.35 ± 2.34

Table 5.  Folding endurance, tensile strength, percentage elongation, and percentage equilibrium swelling of ocular films.

Formulation code	Folding endurance value	Tensile strength (g/mm^2) (n = 3)	(%) Elongation (n = 3)	(%) Equilibrium swelling (n = 3)
AF1	> 300	14.51 ± 0.06	189 ± 2.65	104.76 ± 0.21
AF2	> 300	21.81 ± 0.05	11.62 ± 0.012	55.54 ± 0.15
AF3	> 300	23.98 ± 0.07	9.30 ± 0.015	30.65 ± 0.37
AF4	> 300	25.40 ± 0.07	4.65 ± 0.012	18.43 ± 0.23
AF5	> 300	15.42 ± 0.04	246 ± 4.35	110.21 ± 0.12
AF6	> 300	22.16 ± 0.06	18.18 ± 0.011	47.32 ± 0.17
AF7	> 300	24.19 ± 0.04	13.63 ± 0.013	28.23 ± 0.33
AF8	> 300	25.60 ± 0.07	6.81 ± 0.015	14.14 ± 0.26

Tensile testing gives an indication of the strength and elasticity of the insert, reflected by the parameters tensile strength (TS) and elongation at break (E/B). A soft and weak insert is characterized by low TS and high E/B. A hard and brittle insert is defined by a moderate TS and low E/B. A soft and tough insert is characterized by moderate TS and high E/B, whereas a hard and tough insert is characterized by a high TS and E/B. Hence, it is suggested that a suitable ocular insert should have a relatively moderate TS and E/B. For ocular application, soft and tough inserts are preferred as these can withstand the continuous wear and tear of frequent blinking of the eye without irritating the ocular tissue and enhance patient compliance. The tensile strength of the inserts ranged between 14.51 (0.06)–25.60 (0.07) g/mm² and elongation at break was between 4.65 (0.012)–246 (4.35)% (Table 5). After cross-linking with glutaraldehyde (10% v/v), the tensile strength was increased up to 70% whereas elongation at break was decreased up to 95% as the cross-linking period was increased. It was observed by Talebian et al. (2007) that tensile strength of gelatin film increased ∼ 350% on cross-linking. On the contrary, elongation at break was decreased due to hardening of the insert with the cross-linking agent. Hence, the film with higher period of cross-linking showed better tensile strength and lower elongation at break on cross-linking. This study indicates that cross-linking significantly increased the mechanical strength and stiffness of the matrix film. Formulation AF4 showed a statistically significant difference (p < 0.05) in mechanical strength with respect to non-cross-linked formulation AF1 and cross-linked formulations AF2 and AF3 (in the 15% gelatin batch). Further, formulation AF8 also showed a statistically significant difference (p < 0.05) in mechanical strength with respect to non-cross-linked formulation AF5 and cross-linked formulations AF6 and AF7 (in the 20% gelatin batch).

Swelling behavior was assessed by measuring equilibrium degree of swelling by the weight method. Percent equilibrium swelling of all the formulations is shown in Table 5, which varied from 14.14 (0.26) to 110.21 (0.12)%. It was observed that un-cross-linked gelatin film AF5 showed a maximum degree of swelling, i.e. 110.21 (0.12)% as compared to any other formulation. On the comparison of AF1 and AF5 formulations, it was seen that formulation AF5 showed a higher degree of swelling (AF5 > AF1) owing to higher gelatin ratio. Swelling of the formulations significantly decreased on cross-linking. The swelling extent of the formulation decreased up to 95% after the cross-linking. This indicates that the swelling of the film has considerably decreased because of surface treatment. As the cross-linking extent increased, the surface pores might have decreased, leading to significantly (p < 0.05) decreased water uptake. This was demonstrated by Talebian et al. (2007) in SEM micrographs of un-cross-linked and cross-linked gelatin films. They also found swelling percentage of un-cross-linked gelatin film 390% higher than the cross-linked film. Formulation AF4 showed a statistically significant difference (p < 0.05) in swelling behavior with respect to non-cross-linked formulation AF1 and cross-linked formulations AF2 and AF3 in the 15% gelatin batch. Further, formulation AF8 also showed a statistically significant difference (p < 0.05) in swelling behavior with respect to non-cross-linked formulation AF5 and cross-linked formulations AF6 and AF7 in the 20% gelatin batch.

The FTIR spectra exhibited by non-cross-linked gelatin ocular film differ from those exhibited by cross-linked films specially in the amide I (∼ 1650 cm⁻¹), amide II (∼ 1550 cm⁻¹), and amide III (∼ 1240 cm⁻¹) regions. On comparing the spectra for un-cross-linked film, the cross-linked gelatin films show higher intensity amide I and amide II bands. It indicates that the extent of order, in glutaraldehyde cross-linked films, may be higher than that in un-cross-linked films. Furthermore, the intensity of the amide III band associated with triple helical structure, for the glutaraldehyde cross-linked films, is higher than that for the un-cross-linked films. FTIR data indicates higher intermolecular association in cross-linked films as compared to non-cross-linked films (Figure 1).

Figure 1:  FTIR spectra of A) Pure aceclofenac; B) Non cross linked gelatin film (AF5); C) Non Sterilized Cross linked gelatin film (AF8) D) Sterilized Cross linked gelatin film (AF8)

The cumulative percentage of aceclofenac released from polymeric films as a function of time is shown in Figure 2. Non-cross-linked formulations AF1 and AF5 sustained the drug release for 2–5 h. The surface cross-linked formulations AF2 and AF3 sustained the drug release for 10 h, AF4 and AF6 sustained the drug release for 12 h, while AF7 sustained the drug release for 18 h which was higher than their non-cross-linked films. Formulation AF8 showed a sustained release of 24 h. Hence, AF8 could be considered as an optimized ‘once a day’ formulation of aceclofenac. It is well known that gelatin has an excellent property of increasing viscosity as its concentration increases. Furthermore, gelatin swells upon imbibition of water molecules in it and retards/slows down the movement of drug molecules outside of the matrices. The sustaining action of the polymeric films could be explained by the cross-linking mechanism of gelatin and glutaraldehyde. Molecularly, gelatin is a mixture of purified protein fractions which consists almost entirely of amino acids joined together by amide linkages to form linear polymers (Rowe et al., 2006). Chemical cross-links can be introduced, to alter the gel properties, using transglutaminase to link lysine to glutmine residues or by use of glutaraldehyde to link lysine to lysine.

Figure 2:  In vitro drug release profiles of ocular inserts containing aceclofenac.

The high reactivity of glutaraldehyde toward proteins at around neutral pH is based on the presence of several reactive residues in proteins and molecular forms of glutaraldehyde in aqueous solution, leading to many different possible reaction mechanisms. Several studies have shown that commercially available glutaraldehyde represents multi-component mixtures. Glutaraldehyde can react with several functional groups of proteins, such as amine, thiol, phenol, and imidazole, because the most reactive amino acid side-chains are nucleophiles. The linkage formed by the reaction of glutaraldehyde with an amino group has shown exceptional stability at extreme pHs and temperatures. The most advocated mechanism of glutaraldehyde-gelatin cross-linking is aldol condensation, which was proposed by Richards and Knowles (1968).

Mundada and Shrikande (2008) developed ocular inserts of ciprofloxacin using gelatin and glutaraldehyde. They reported a prolonged and controlled drug release for cross-linked gelatin matrices.

The data obtained from in vitro studies of all eight formulations were subjected for kinetic treatment in order to know the order of release. Regression coefficient values obtained for each formulation were compared to understand the release kinetics (Table 6). Comparison of R² values obtained by Zero order, First order, and Higuchi kinetic equation revealed that in vitro drug release followed square root of time (Higuchi release) kinetics as the R² values obtained by Higuchi kinetic equation were nearer to unity. It can be interpreted from the in vitro drug release kinetics that diffusion was the primary mechanism of drug release from given formulations. Based on Korsmeyer-Pepaas semi-empirical model, the best fitting was obtained with n ≤ 0.5, indicating a fickian release mechanism. In swellable system, factors affecting the release kinetic are liquid diffusion rate and polymeric chain relaxation rate. When the liquid diffusion rate is slower than the relaxation rate of the polymeric chain, the diffusion is fickian; whereas when the relaxation process is very slow as compared to diffusion the case II transport occurs. When liquid diffusion rate and polymer relaxation rate are of the same order of magnitude, anomalous or non-fickian diffusion is observed. On the basis of these considerations, it is clear that the drug release from our formulation is controlled by polymer chain relaxation. The observations thus support the result obtained with Higuchi’s equation, which shows that the formulation released the drug by a diffusion dominated mechanism.

Table 6.  Kinetic modeling of drug release data from ocular inserts of aceclofenac.

Formulation code	Zero order		First order		Higuchi Kinetics		Korsmeyer-Peppas kinetics (n)
	Rate constant (K)	R^2	Rate constant (K)	R^2	Rate constant (K)	R^2
AF1	−0.0162	0.998	−0.6862	0.974	42.19	0.998	0.414
AF2	−0.0032	0.926	−0.1473	0.815	35.45	0.981	0.409
AF3	−0.0031	0.957	−0.1612	0.859	36.17	0.991	0.348
AF4	−0.0026	0.960	−0.1358	0.875	32.49	0.987	0.406
AF5	−0.0065	0.971	−0.2095	0.931	45.43	0.996	0.397
AF6	−0.0026	0.944	−0.1128	0.835	30.26	0.992	0.389
AF7	−0.0026	0.974	−0.1404	0.917	33.74	0.978	0.404
AF8	−0.0013	0.985	−0.0667	0.869	20.73	0.989	0.503

Statistical analysis (ANOVA) of the percent drug release showed that release of the drug from formulations AF4, AF7, and AF8 (Figure 2) was significantly lower (p < 0.05) than that of other inserts. Further, formulation AF8 released the drug significantly lower (p < 0.05) than formulations AF4 and AF7.

Ocular irritation of the developed formulation was checked by hen’s egg chorioallantoic membrane test (HET-CAM) which is a rapid, sensitive, and inexpensive test. Testing with incubated eggs is a borderline case between in vivo and in vitro systems and does not conflict with the ethical and legal obligations. The chorioallantoic membrane of the chick embryo is a complete tissue including veins, arteries, and capillaries, and is technically very easy to study. It responds to injury with a complete inflammatory process, a process similar to that induced in the conjunctival tissue of the rabbit eyes (Spielmann, 1997; Velpandian et al., 2006). Developed formulation was tested by this method and the result was compared with those obtained using normal saline, which was used as control that is supposed to be practically non-irritant (Table 7). The study shows that the formulation is non-irritant and is well tolerated as per the HET-CAM test. Therefore, ocular inserts of aceclofenac based on glycerogelatin matrices could be a potential dosage form for treatment of ocular inflammation conditions.

Table 7.  Scores obtained in HET-CAM test.

Formulation	Scores (time in min)
		0	5	15	30	60	120	240	720	1440
Control (normal saline)	Egg1	0	0	0	0	0	0	0	0	0
	Egg2	0	0	0	0	0	0	0	0	0
	Egg3	0	0	0	0	0	0	0	0	0
	Mean	0	0	0	0	0	0	0	0	0
Developed formulation	Egg1	0	0	0	0	0	0	0	0	0
	Egg2	0	0	0	0	0	0	0	0	0
	Egg3	0	0	0	0	0	0	0	0	1
	mean	0	0	0	0	0	0	0	0	0.33

The drug inserts were sterilized using γ-radiation before carrying out the in vivo drug release study. Formulation AF8 was chosen for in vivo drug release study. In vivo release studies were performed using rabbits. Ocular inserts were removed carefully at periodic intervals and analyzed for residual drug content. The drug remaining was subtracted from the initial drug content of the insert, which gave the amount of drug released in the rabbit eye. The in vivo drug release study from formulation AF8 was found to be in accordance with that of the in vitro drug release study (Figure 3). Hence, we tried to correlate in vivo results with the in vitro percentage drug release of AF8 (Charoo et al., 2003; Mundada & Shrikhande, 2008). The in vitro/in vivo correlation for formulation AF8 was found to be strong and productive. The strong in vitro and in vivo correlation (R² = 0.9976) revealed the efficacy of the formulation (Figure 4).

Figure 3:  In vivo drug release profiles of ocular inserts containing aceclofenac.

Figure 4:  Scatter diagram showing in vitro and in vivo correlations of ocular inserts containing aceclofenac (r = 0.9976).

On the basis of release rate data, inserts of formulation AF8 was chosen for the in vivo evaluation. Topical instillation of PGE₂ results in PMN migration in tears and closure of eyelids as a result of induced inflammation. Hence, lid closure score and PMN count in tears was used for evaluation of ocular inflammation. Table 8 shows lid closure scores and Figure 3 draws a comparison of PMN count in the tears of rabbits. The results show that the lid closure scores and PMN count was less in all the eyes treated with aceclofenac as compared to their respective controls. The lid closure scores in the eyes treated with ocular inserts were much less as compared to eyes treated with eye drops, indicating a higher inhibitory effect of aceclofenac inserts over the eye drops formulation against PGE₂-induced ocular inflammation. Ocular insert showed a statistically significant difference in (p < 0.05) lid closure score (for all values, at each time) with respect to eye drops.

Table 8.  Comparison of effect of the aceclofenac ocular film with eye drop of drug at the same concentration on PGE2-induced lid closure in rabbit eye. For each group of tested dosage form, the left eye was treated as test (either eye drop or ocular insert) and right eye as control (normal saline).

Time	Control(n = 5)	Eye drop of aceclofenac(n = 5)	Ocular film of aceclofenac (n = 5)
1h	2.000 ± 0.000	1.800 ± 0.447	1.600 ± 0.547
2h	1.400 ± 0.547	1.400 ± 0.547	1.200 ± 0.836
4h	1.000 ± 0.000	0.800 ± 0.447	0.800 ± 0.447
6h	0.600 ± 0.547	0.400 ± 0.547	0.200 ± 0.447
8h	0.200 ± 0.447	0.200 ± 0.447	0

The results of PMN migration (Figure 5) indicate that the inhibitory effect of eye drops was more prominent during the first hour. However, the ocular inserts produced a more prominent and longer lasting effect after the initial hour. The results from the in vivo study conclusively indicate effectiveness of inserts in sustaining the release of the drug. The eye drops, because of pre-corneal losses of the drug, were able to counteract the PGE₂-induced ocular inflammation only during the first hour. Similarly, a statistically significant lowering (p < 0.05) in PMN count (for all values, at each time) was noted with respect to the eye drops, indicating a better potential of ocular inserts over the eye drops in anti-inflammatory studies.

Figure 5:  Comparison of effect of Aceclofenac Ocular film with aqueous solution of drug on PGE₂ induced PMN migration in tears of rabbit. For each group of tested dosage form, left eye was treated as test (either eye drop or ocular insert) and right eye as control (normal saline)

Conclusion

Glycerogelatin-based aceclofenac ocular insert was successfully prepared. Cross-linked ocular insert proved to be significantly better in various evaluating parameters such as tensile strength, swelling potential, and drug release. The prepared formulation was non-irritant, prolonged the drug retention at the corneal site, and enhanced drug bioavailability, as indicated by ocular inflammatory conditions. The formulation was found suitable for sustained topical drug delivery to the eyes for rational drug therapy in the case of various ocular diseases.

Acknowledgement

The authors are grateful to IPCA Laboratories, Ratlam, India, for providing the gift sample of drug, and School of Pharmacy, Suresh Gyan Vihar University, Jaipur for providing the necessary research facilities.

Declaration of interest

The authors report no conflict of interest. The authors alone are responsible for the content and writing of the paper.