Tropac rabbit dose dispenser of size 5 was purchased from Custom Capsules Pvt. Ltd. in Maharashtra (India). The gelatin capsule (size 5) was provided by Patco Pharmaceuticals Pvt Ltd., Mumbai (India). MTS was a generous gift from Torrent Pharmaceuticals Ltd., Gujrat (India). Sodium acetate was purchased from Sigma-Aldrich Pvt. Ltd. in Mumbai (India). Methanol and acetonitrile of HPLC quality were purchased from SD Fine-Chem Ltd., Mumbai (India). Wherever necessary, HPLC-grade water was used.

UHPLC (Agilent 1260, USA) equipped with an autosampler, quaternary pump, and PDA detector with ChemStation software was used for chromatographic analysis. Poroshell 120EC-C18 column (4.6 × 50 mm × 5.0 μm) was employed as the stationary phase.

The drug release characteristics of both the marketed formulation (Montek 10) and MTS ChrDDS were determined on a USP type II dissolution apparatus (Electrolab, TDT08L, India) at 37 ± 0.5 °C and 75 rpm. The study was performed with 0.1N HCl solution (pH 1.2) for 2 h, after that with acetate buffer (pH 4.5) for 1 h, then with pH 6.8 SLS solution (0.5% w/v) for the remaining time as dissolution medium. Samples were taken at predetermined time intervals, filtered with a 0.45-mm membrane filter, and subjected at 345 nm to a UV spectrophotometer for further analysis.

The stock solution of MTS was prepared by dissolving 100 mg of the drug in 100 ml methanol solution. 1 ml of prepared solution was taken and diluted up to 10 ml (100 μg/ml) to make a working solution. From the stock solution (1 mg/ml), series of dilutions were prepared in a range of 10–600 μg/ml to create a calibration curve. It will be used for validation of the developed bioanalytical method. The quantitative determination of the drug was performed with the mobile phase of a mixture of methanol: acetonitrile: 0.2 mM sodium acetate buffer (5:90:5) with a 0.5 ml/min flow rate. Analysis was performed at a wavelength of 285 nm [17]. To filter mobile phase glass vacuum-filtration microfilter (0.22 mm) and to remove entrapped air, degassing ultrasonic bath was used. The calibration curve in rabbit plasma was prepared by spiking 20 µl of MTS working solution (10, 20, 50, 100, 200, 500, 1000, 1500, and 2000 ng/ml) in the HPLC column. From the literature review, Zafirlukast was cast off as an internal standard with 5000 ng/ml plasma concentration [18]. It has an appropriate retention time and a good resolution from MTS under chromatographic conditions. No interference peaks were observed in the regions where the drug was eluted. Three quality control samples were prepared at 25, 250, and 1250 ng/ml by spiking the drug and internal standard into rabbit plasma. Before that drug-free plasma was tested to check any interferences in the peak of the drug and internal standard. The established method was verified for selectivity, specificity, accuracy, precision, robustness, and ruggedness.

Pharmacokinetic studies were performed on two groups of white New Zealand rabbits (n = 4). Rabbits fasted overnight with only a water supply. The developed dosage form was administered orally in a size 5 capsule by using a Torpac animal dose dispenser [19,20,21]. Ears were shaved before taking out blood samples. From the marginal ear vein, 100 µl of blood samples were collected in Eppendorf tubes at times 0, 2, 4, 6, 8, 10, 12, 18, and 24 h after administration. Due to the high log p (8.49) value of MTS, 10 µl of the internal standard was injected into the sample before being retrieved with 1.5 ml tertiary butyl methyl ether [22]. The prepared mixture was vortexed for 15 min then centrifuged for 10 min at 10,000 rpm. After centrifugation, the organic supernatant was put into a glass tube and evaporated for 5 min at 50 °C under a stream of Nitrogen Evaporator (Caliper Life Sciences, Hopkinton, MA, USA). The remaining residue after complete drying was reconstituted with mobile phase (150 μl) with vertexing for 1 min. The prepared drug sample was stored at − 70 °C till analysis.

By plotting a graph, Level A IVIVC was developed for optimized formulation. The graph was plotted between the fraction of drug release (FDR) from the in-vitro dissolution study and the fraction of drug absorbed (FDA) from the in-vivo dissolution study [23]. From the deconvolution approach of the Wagner–Nelson method, fraction of the absorbed drug was determined from AUC. It was calculated from the Plasma concentration profile by using concentration at time t (Ct), elimination rate constant (Ke), and area under the plasma concentration curve (AUC0−t & AUC0−∞).

$$} = \left( + K_}} *}_} } \right)/\left( }} *}_} } \right)$$

(1)

All the statistical analyses were performed by GraphPad Prism v 9.4.1 to assess the p values (statistical significance) between the groups.

To determine the mucoadhesive strength and time, ex-vivo studies were performed.

It was measured in grams which are required for the complete detachment of the tablet from the intestinal mucosa [24]. From a local slaughterhouse, the fatty acid-free freshly cut porcine intestine membrane was obtained and stored for 30 min in Krebb’s buffer solution at pH 6.8 and temperature 37 ± 0.5 °C. The experiment was conducted on a modified physical double beam balance where the right pan was replaced by a glass slide. The left pan was carrying weight to measure the mucoadhesive strength. A 2 × 2 × 2 cm wooden cubicle block tied with intestinal membrane was dipped in Krebb’s buffer solution till the upper layer of mucosa was. The tablet was adhered to the glass slide with cyanoacrylate glue and allowed to maintain contact with the membrane under a constant weight of 5 g. Mucoadhesive strength was determined for both T1F1 and T2F3 with n = 3 by estimating the force necessary to break the contact between the tablet surface and intestinal mucosa [25, 26].

This study was conducted for both coated and uncoated tablets of T1F1 and T2F3 similar to mucoadhesive strength [26]. The tablet was stuck on the wet intestinal membrane tied over the glass slide, and a stirring of 50 rpm was applied to simulate peristaltic movement. Time taken by a tablet to separate from mucosa was noted in triplicate and averaged as mucoadhesion time [27].

X-ray radiography was adopted to track the ChrDDS's time-dependent mobility after receiving approval from the College IEAC (Institutional Animal Ethics Committee). MTS (Montelukast sodium) of T1F1 and T2F3 of formulated ChrDDS was replaced by BaSO4 to take X-ray radiographs [14, 28]. Two white New Zealand rabbits fasted overnight and a size 5 capsule containing individual parts of ChrDDS was administered through a Tropac oral dispenser followed by 10–15 ml of water [14]. For the entire duration of the study, rabbits had free access to water only. To take X-ray radiographs, rabbits were anesthetized with ketamine hydrochloride (20 mg/kg dose) and exposed to the lateral and ventral abdominal region. Images were taken at 0 h (before administration), 2 h, and 6 h for T1F1 and 0 h (before administration), 2 h, 6 h, and 9 h for T2F3 by X-ray generator (Orvee medical solutions) at 100 mA, 100 KVP, 230 V, 50 Hz in a single phase [29]. The position of tablets was observed and noted.

Developed ChrDDS was kept for 6 months in a stability chamber (HICON® 216 Grover Enterprises, New Delhi, India) to conduct accelerated stability studies following ICH guideline Q1A (R2) at 40 ± 2 °C temperature and 75% relative humidity [16, 30]. Samples were evaluated at 0, 90, and 180 days for cumulative percentage release, drug content, and disintegration time. All data were investigated for similarity factors regarding the zero-minute release profile. Capsules were also monitored for physical appearance. Optimized formulations' in-vitro drug release was statistically analyzed using GraphPad Prism's 9.4.1 one-way ANOVA.