5 mg of the TC was dissolved in the 5 ml of the methanol to form 1,000 µg/ml solution.

(stock I); Further, 0.5 ml of the stock I was diluted up to 5 ml with methanol to form the concentration of 100 µg/ml (stock II). Moreover, 0.5 ml of stock II was diluted up to 5 ml with methanol to form 10 µg/ml (stock III). Similarly, 0.5 ml of stock III was diluted up to 5 ml with methanol to form 1 µg/ml or 1,000 ng/ml (Stock IV) (11). The final spiking of the drug from stock IV in isolated plasma, as presented in Table 1.

After the dilution, each sample was mixed with an equal volume of organic solvent and vortexed for 10 min for liquid-liquid extraction. After 10 min, the organic layer was separated and evaporated to dryness. The samples were reconstituted in the 0.5 ml of mobile phase and, transferred into HPLC vials and subjected to the analysis (12). Figure 1 represents calibration curve of the TC by HPLC. Figure 2 represents HPLC chromatogram of (A) blank plasma and (B) plasma spiked with TC.

The chromatographic condition for the analysis of the TC has been presented in Table 2.

The developed method was validated by evaluating linearity, precision, accuracy, limit of detection (LOD), and limit of quantification (LOQ), which were done in acceptance of ICH and EMA guidelines.

The study was approved by the animal ethical committee NMIMS Shirpur campus, under reference no. SPTM/09/2023/IEAC/13. All protocols were followed according to the guidelines approved by the animal ethical committee. In-vivo studies were conducted with an appropriate number of animals, preferably in healthy Sprague Dawley rats.

The marketed or comparator formulation of the TC named as “Brilinta” used in this study was obtained from AstraZeneca. Healthy Sprague Dawley rats with an average weight of 250–350 g were used for the study. All the rats were subjected to standard food and environmental conditions. Rats were divided (Table 3) into 2 groups each containing 6 rats (n = 6).

The in-vivo pharmacokinetics study was performed in order to compare the plasma profile of the developed formulation and conventional formulation to establish the enhanced bioavailability of the developed formulation (16). All experiments were performed according to the guidelines approved by the animal ethical committee. In vivo, a pharmacokinetics study was conducted with an appropriate number of animals, preferably in healthy Sprague Dawley rats. The conventional TC and TC solid dispersion formulations corresponding to 10 mg/kg of TC were administrated to each animal via the oral route. The samples were dispersed in a carboxymethylcellulose solution prior to the administration. After the oral administration, blood sampling was done at 0.5, 1, 2, 4, 8, 12 and 24 h via retro-orbital plexus. Blood samples were centrifuged to collect the plasma. TC in the plasma samples was extracted with the help of ethyl acetate and was allowed for evaporation in distinct tubes; 300 μl of mobile phase was added to the dried tubes, then vortexed and subjected to centrifugation at 10,500 rpm for 15 min; 200 μl of the supernatant liquid was separated and transferred to HPLC vial inserts. The samples were analyzed by using developed HPLC analytical methods. The chromatographic condition includes the mobile phase of acetonitrile and water (65:35), 1 ml flow rate, 10 min run time, 5.8 retention time min, column C18 SHIMADZU (5 µm* 25 cm), photodiode array detector at 255 nm at λmax. The plasma samples of TC concentration were calculated by comparing them with the standard calibration curve of the TC (R² = 0.9992).

Male Wistar rats were maintained in a temperature-regulated environment under a 12 h light-dark cycle. The rats were housed two per cage and fed for 10 weeks with either a conventional chow diet or a high-fat diet to induce obesity. This study was carried out to understand the platelet aggregation prevention capability of the developed formulation of the ticagrelor. A healthy group of rats fed with a conventional chow diet and disease group rat fed with high-fat diet. Additionally, 10 mg/kg dose of ticagrelor comparator and SD1, were simultaneously given along with high-fat diet, separately. Rats were anesthetized with isoflurane, and blood was collected in a 1:9 (v/v) ratio of ACD-C anticoagulant (12.4 mM sodium citrate, 13 mM citric acid, 11 mM glucose). Platelet-rich plasma (PRP) was obtained by centrifuging whole blood at 200 g for 15 min at ambient temperature. 5 ml of PRP was combined with 7 ml of washing buffer and centrifuged at 800 g for 13 min. The pellet was resuspended in a washing buffer, and this process was repeated once. The platelets were then gently suspended in Krebs solution, and the platelet count was adjusted to 1.2 × 10⁸ platelets/ml with the addition of 1 mM CaCl₂. Platelet aggregation was measured using a dual-channel aggregometer at 37°C with stirring at 1,000 rpm. Aggregation was induced using ADP thrombin (100 mU/ml). Additionally, samples were observed under a brightfield microscope. The different groups were healthy control, disease control (high fat diet), Treatment group 1 (high fat diet + comparator), and Treatment group 2 (high fat diet + SD1).

All values in this study were presented as the Mean ± Standard Deviation (SD). The significant difference was evaluated with Prism 8 (GraphPad Software, CA, USA) according to student t-test) with the unpaired test. The level of the significance was denoted as; P > 0.05 (ns), P < 0.05 (Significant), P ≤ 0.01 (*), P ≤ 0.001 (**), P ≤ 0.0001 (***).

The calibration curve for TC was successfully prepared using the HPLC method under the specified experimental condition. A series of TC standard solutions with concentrations ranging from 100 ng/ml to 800 ng/ml were injected into the HPLC system in duplicate, and corresponding peak areas were recorded. The calibration was constructed by plotting the concentration of TC (x-axis) against the peak area (y-axis), as shown in Table 4.

These studies provide critical information about the drug's absorption, distribution, metabolism, and excretion (ADME) properties (21). Specific methods of statistical experimental sections were applied to evaluate the patterns and trends in data.

All values in this study were presented as the Mean ± Standard Deviation (SD). The significant difference was evaluated with Prism 8 (GraphPad Software, CA, USA) according to student t-test) with the unpaired test. The level of the significance was denoted as; P > 0.05 (ns)_, P < 0.05 (Significant), P ≤ 0.01(*), P ≤ 0.001(**), P ≤ 0.0001(***). These studies provide critical information about the drug's absorption, distribution, metabolism, and excretion. The specific results of in-vivo studies pharmacokinetic studies can vary on different parameters such as Tmax, Cmax, Half-life, AUC, Volume of distribution, and Relative bioavailability. The results of in-vivo studies are shown in Table 8.

The in-vivo pharmacokinetic study demonstrated that after the development of the solid dispersion formulation¹¹, there was a significant enhancement in C_max (P ≤ 0.001), AUC (P ≤ 0.0001), and relative bioavailability (P ≤ 0.001) compared to the conventional marketed Ticagrelor (TC) Tablet. Notably, the bioavailability of the developed TC amorphous solid dispersion formulation was improved by up to 64.33% (P < 0.0001). There was a significant reduction in Vd (P ≤ 0.001), which indicates reduced nonspecific delivery of the drug to lipid compartment of the body space and indirectly indicates enhancement in bioavailability. Whereas, there was no significant difference between T_1/2 and MRT (P ≥ 0.05). Figure 4 represents plasma concentration time profile of the comparator and SD1.

The pharmacokinetic profile Ticagrelor [Comparator Vs Amorphous Solid Dispersion] has been demonstrated in the Table 8.

Ticagrelor reduces platelet aggregation by antagonizing the P₂Y₁₂ receptor, which plays a key role in ADP-induced platelet activation and aggregation. In this, investigation, inhibition of the platelet's aggregation induced by ADP thrombin were evaluated for RLD and SD1 formulation. Platelet aggregation is one of the key steps in the formation of the clot; ticagrelor is supplied as a drug that inhibits the aggregation of the platelets and hence prevents clot formation.

In Figure 5IA, demonstration the platelets free from, as healthy control, whereas Figure 5IB, platelets were aggregated following treatment with ADP thrombin (disease control). The RLD-treated group showed fewer platelet aggregation and, which was further reduced in SD1 treated group. Additionally, data obtained from the platelet aggregometer (Figure 5II), depicted that SD1 treated group had a lower% of platelet aggregation compared to the RLD. Hence, our developed formulation (SD1) demonstrated better platelet aggregation protection compared to the marketed formulation of ticagrelor. Overall, it was observed that when ticagrelor formulation was given to the rats (orally), it reduced the plate aggregation by binding to the P₂Y₁₂ receptor.