Trimetazidine dihydrochloride was collected as a free sample from Medley Research Centre (Mumbai, Maharashtra), Hydroxypropyl methylcellulose (HPMC K 15M) was purchased locally from Burgoyne and Co (Mumbai, India). Triethanolamine was purchased from HIMEDIA, Mumbai, India. All other chemical reagents used were of analytical grade.
Institutional animal ethics committee (IAEC) approval has been taken for this animal experiment [Approval No.IAEC/SPS/SOA/04/2020] for Control and Supervision of Experiments on Animals (CPCSEA) Guidelines, India. Westar male rats between 170-200 gm were taken and kept under constant environmental condition of 22 ± 1 °C (RH 50-60 %) with natural light. Animals were fed normal laboratory diet and water ad libitum. All the animals were healthy and free from clinically ocular abnormalities.
Fabrication of Trimetazidine Ocular Film
HPMC K 15M was swelled in the presence of an adequate amount of water (1 gm/40 ml) in refrigeration condition for 12 h. The swelled gel was stirred magnetically for 24 h. Trimetazidine and triethanolamine were dissolved in a small amount of water and poured into the HPMC dispersion with continuous stirring for 3-4 hour. Trimetazidine ocular film formulation was prepared by casting and solvent evaporation method on Petri plate (Tarsons, diameter: 90 mm). Drying was done at 40 °C in an incubator until the consecutive weighings were not varying. The thickness of the film formulation was measured by a digital micrometer (Mitutoyo, Japan). Folding endurance was conducted by repeated folding a piece of film at the same place until it breaks. The surface pH of the film was measured by dipping the pH meter’s glass electrode inside the swelled segment of the film formulation .
Infrared Spectroscopy Analysis
Infrared spectroscopy analysis of trimetazidine and the prepared TZ filmformulation was carried out in FTIR-Spectrophotometer (Model-JASCO FTIR-4100, Japan) by usingthe KBr pellet method.
Differential Scanning Calorimetry (DSC)
Differential scanning analysis of pure trimetazidine and the film wasperformed by differential calorimeter (DSC-1, Mettler Toledo; Software- Star E, SNR- 18289) within a range of 30-300 °C in a constant heat flow of 10 °C per minute in continuous nitrogen purging of 50 ml per min.
Assessment of the in vitro drugrelease was done by USP type-II (Paddle type) dissolution apparatus (Electrolab TDT067 Dissolution tester). Accurately weighed a suitable cut piece of film was glued properly onto a glass slide by using adhesive (cyanoacrylate) and merged completely inside the dissolution vessel containing 200 ml phosphate buffer (pH 7.4) as dissolution medium. Dissolution test was carried out at temperature 34.0 ± 0.5 °C with 50 rpm paddle rotation speed. Samples were withdrawn as per predetermined time intervals and analyzed spectrophotometrically at lmax 269 nm after filtering through a 0.45 µm syringe driven filter .
Ex-vivo corneal permeation study
Goat eyeballs were acquired from a local slaughterhouse within 1 h after sacrificing. These were properly rinsed with distilled water followed by phosphate buffer (pH 7.4). The cornea along with about 5 mm wide sceral ring was excised from ocular globe of goat and again rinsed with phosphate buffer solution. A pre-weighed circular cut piece of film was placed on the middle of the epithelial corneal surface and faced towards the donor compartment side of the diffusion chamber with an effective surface area of 1.56 cm². Endothelial side compartment was filled with phosphate buffer (pH 7.4, 200 ml) and ex vivo corneal permeation was carried out for six hour at 34 ± 2 °C under steady stirring speed of 50 rpm. At the regular time interval, 10 ml of samples were withdrawn after filtering through a 0.45 µm syringe driven filter. Absorbance was recorded at 269 nm using a Ultra-Violet visible spectrophotometer (JASCO V-630). The experiment was triplicated and mean ± SD were recorded .
Wistar male rats were divided into three groups containing 3 animals each.Group-I was served as a test group (a small piece trimetazidine film (2 mg/Kg dose) previously sterilized by UV exposure, followed by carrageenan injection after 1 h), Group-II was served as + ve control group (not drug-treated), and Group-III was served as a control group. Lipopolysaccharide (carrageenan solution 20 µl, 2 % w/v) was injected into the sub-conjunctiva (upper palpebral region) in the rat eye for induction of oxidative stress . Before commencing sub-conjunctival injection, the rats were anaesthetized with an intraperitoneal injection (0.1 ml/100 g of rat weight of ketamine-xylazine mixture at 91 and 9.1 mg/kg respectively) . The sign of reddening of the conjunctiva followed by tearing was visualized. The rats were euthanized within 1 h of carrageenan injection under a higher dose of thiopental sodium anaesthesia. Then the eyeballs were removed to carry out histology and Malondialdehyde (MDA) (a product of lipid peroxidation) assay.
Histological evaluation was performed by taking the rat eyes which were enucleated and firmly place in paraffin. The sections were obtained in the inferior portion and the vertical meridian of the eyeball. Each paraffin block was sectioned with 5 µm and staining was done using hematoxylin and eosin. The histopathology of the specimens was examinedusing a photo microscope Olympus make model Magnus MLX-TR with a digital camera.
Determination of lipid peroxidation
Lipid-peroxidation was carried out to measuring the MDA according to the Thiobarbituric acid (TBA) test described by Yeh et al. 1990 with the following modification . After treatment, the whole eyeballs were homogenized with a one-tenth lens weight of 6 % perchloric acid (HClO4) at 0 °C by using a glass homogenizer. The sample was then centrifuged for 15 minutes at 4 °C and 200 µl of supernatant was separated in a test tube. In the test tube 42 µl ofsodium acetate (5 mol/l), 200 µl of 8 % sodium lauryl sulphateand 200 µl of thiobarbituric acid (0.8 %) were added. The tube was then placed into a heating block at 95 °C for 50-60 min and cooled with tap water. Butanol and pyridine mixture (15:1) of 2.5 ml and distilled water 0.5 ml was added over the sample mixture and centrifuged at 4000 revolutions per min. At lmax of 532 nm the absorbance of the organic layer was measured and MDA level was estimated in nmol per g on wet tissue.
Fresh goat eyeballs were acquired from a local slaughter house and stored in refrigeration condition (about 4 °C). The extracapsular method was adapted for the removal of the lens then collected lens were incubated in simulated aqueous humour (NaHCO3 0.5 mM, KCl 5 mM, NaH (PO4)2 0.5 mM, CaCl2 0.4 mM, NaCl 140 mM, MgCl2 2 mM and Glucose 5.5 mM, pH 7.8) at room temperature for 4 h. Streptomycin 250 mg % and penicillin 32 mg % were combined with the culture media to avoid microbial growth . This healthy normal isolated lens was used as control (Group-I). A high concentration glucose of 55 mM was used to induce cataract of the isolated lens (Group-II) . In the drug-treated group trimetazidine solution was used in combination with glucose solution for examining the protective effect of cataract formation. Here, Group-III Group-IV were served as treatment group with trimetazidine (120 µg/ml, and 240 µg/ml) incubation of lens both in combination with glucose (55 mM) respectively.
Lens protein Content
The protein estimation of the samples was carried out as per Lowry et al.1951 technique, with bovine serum albumin serving as the standard .