2.2.1 Validation of analytical method
The analytical method of ATZ employing high-performance liquid chromatography (HPLC) system was validated. The analytical method was regulated using 50 mM Potassium dihydrogen phosphate Buffer pH 4.5: ACN: Methanol::40:45:15 and 0.07% TFA (trifluoroacetic acid) as mobile phase at 249 nm λmax; 0.7 mL/min flow rate; Xbridge 3.5 µm, 4.6 x 150 mm analytical column. Further, this estimation of ATZ was validated for linearity (200 ng/mL- 100000 ng/mL), limit of detection (13.62 ng/mL), limit of quantification (45.40 ng/mL), Inter-assay and intra-assay Precision and Accuracy. The linearity equation (y = 72.26x + 3542; R² = 0.99, where ‘‘y’’ is the area under curve and ‘‘x’’ is the concentration in ng/mL) was then employed to obtain unknown ATZ concentration in various samples.
2.2.2 Equilibrium solubility of ATZ in various oils, surfactants and co-surfactants
The equilibrium solubility of ATZ was quantified in various natural oils, synthetic/semi synthetic oils, surfactants, and co-surfactants. For this purpose, an excess amount of ATZ was added to each capped glass vial formerly containing 1 g of surfactant and/or co-surfactant and/or oil and vortexed for 5 min after every 2 h for 24 h at a constant temperature (40± 0.5°C) in shaking incubator at 50 rpm until the attainment of equilibrium phase . An aliquot of 0.5 mL sample was withdrawn at 72 h and centrifuged at 2,000 rpm (~180 × g) for 10 minutes. The resultant supernatant was appropriately filtered, diluted with methanol and was examined by utilizing validated HPLC method.
2.2.3 Ternary phase diagram
In order to determine the levels of the material attributes (MA) for D-optimal mixture design, ternary phase diagram with selected surfactant and co-surfactant along with combination of oils (1:1) was constructed; each of them positioned at an apex of the triangle using PCP-Disso software (Poona College of Pharmacy, Pune, India). After equilibrium, the time of self-micro emulsification efficiency, dispersibility, appearance, percentage transmittance (at 560 nm) and flowability was observed [26, 27]. The clear isotropic mixture regions in the diagrams were plotted, and the wider region indicated the better self-emulsification efficiency was determined.
2.2.4 Systematic optimization of ATZ loaded isotropic mixture as per mixture design
D-Optimal mixture design (Design-Expert® software version 11; Stat-Ease, Inc., Minneapolis, MN) was used to optimize the composition of the critical material attributes (CMAs) of the isotropic mixture. The range of Corn oil: Oleic acid (1:1) (oil; X1), Tween 80 (surfactant; X2), and Propylene glycol (co-surfactant; X3) were set to 0.1-0.2 g, 0.5-0.7 g and 0.2-0.4 g respectively (Table 1). For each formulation, the total of the composition of X1, X2 and X3 was summed to 100% i.e. 1 g. Briefly, the ATZ loaded IM were prepared by simple admixture method. An amount of ATZ (50 mg) was dissolved in propylene glycol at 80˚C by stirring on a magnetic stirrer. Subsequently, the Tween 80 and corn oil: oleic acid (1:1) was further added as per the design matrix to form IM. Particle size (Y1; nm), PDI (Y2), Self emulsification time (Y3; sec), % Transmittance (Y4; %) and Drug content (Y5; %) were evaluated as the critical response variables (CRVs) to optimize the ATZ loaded IM by utilizing the desirability function. The design consisted of 16 experimental runs to find an appropriate polynomial model and to evaluate the effects of CMAs on the CRVs.
2.2.5 Determination of critical response variables (CRVs) as per D-optimal mixture design
The developed formulations were determined for various critical response variables (CRVs) namely particle size (Y1), Poly dispersity index (Y2), Percent transmittance (Y3), Drug Content (Y4) and Self emulsification time (Y5). The method for estimating Y1, Y2, Y3 and Y4 has been discussed in Supplementary data.
Self Emulsification Time (Y5)
The self-emulsification time of IM pre-concentrates were determined through aqueous dilution method using a standard US Pharmacopeia dissolution apparatus type II (Electrolab, Mumbai, India) . The time required by IM pre-concentrates to completely emulsify in the dilution media was noted.
2.2.6 Selection of polymer precipitation inhibitor (PPI)
(a) In-silico analysis
The 3D-structure of ligand (ATZ) and different PPIs like Poloxamer 188, Poloxamer 407, HPMC E15, HPMC-AS and PVP K17 were downloaded from freely available chemical structure database i.e. chemspider. Further, the 3D structure of ligand and polymers were clean up in Chem Draw Ultra 12.0 software. Each polymer containing one repeating unit was docked with ligand (ATZ) respectively by using the MGL tool (Autodock software version 1.5.6) (M/s GNU General Public Licence, CA, USA). For all docking calculations, the size of grids was set as 40 Å × 40 Å × 40 Å with grid spaces of 0.375 Å. Lamarkian genetic algorithms (LGA) was applied to probe the most favorable drug-excipient complex geometry. The other docking parameters were set to the default values [29-31].
After molecular docking study, the stability of the different ligand-PPIs complex was accessed by molecular dynamics simulations (MDS) for a period of 30 ns, using a freely available academic version of “Desmond” program. The solvent system was built around the ligand-PPIs complex using the TIP3P water model and the shape of the box was kept as orthorhombic with dimensions 10 x 10 x 10 Å. The neutralization of physiological pH of the system was done by adding the Na+ counterions and the salt concentration was set as 0.15 M. After the energy minimization, the constant temperature was maintained with Nose-Hoover chain thermostat to attain NPT equilibration at 310K, while, constant pressure was acquired using the MartyTobias-Klein barostat. Thereafter, RMSD was calculated using the desmond protocol [32-34].
(b) In-vitro supersaturation test
To prevent the ATZ loaded IM from precipitation in the intestinal condition, supersaturable IM (SP-IM) were prepared by adding different types of PPIs (i.e., Poloxamer 188, Poloxamer 407, HPMC E15, HPMC-AS and PVP K17) to the optimized IM. The SP-IM was prepared by adding different type of PPIs along with ATZ in propylene glycol. Subsequently, the Tween 80 and Corn oil: Oleic acid (1:1) was further added to form ATZ loaded SP-IM. PPIs are incorporated in an amount equivalent to 2.5% of the total weight of optimized IM. Further, the prepared formulations were subjected to in-vitro supersaturation test in simulated intestinal fluid (pH 6.8) under non-sink conditions. A 500 mL of dissolution media was added to a 1000 mL dissolution basket equipped with a rotating paddle (Electrolab, Mumbai, India). An aliquot of 3 mL was withdrawn from different formulations at specific time intervals without volume replacement and the amount of drug at each time interval was estimated by utilizing the validated in house HPLC method. The apparent drug concentration-time profile and the duration of the supersaturated state were subsequently determined [23, 24, 35, 36]. Further, the PPI with a higher degree of supersaturation was selected for further studies.
2.2.7 Evaluation of supersaturable ATZ-SP-IM
220.127.116.11 Determination of particle size (PS), poly dispersity index (PDI) and Zeta potential
The particle size, PDI and zeta potential of the ATZ-SP-IM were determined by dynamic light scattering technique using particle size analyzer (Nano ZS 90, M/s Malvern, Worcestershire, UK).
18.104.22.168 Transmission electron microscopy
The morphological and structural behavior of the microemulsion droplet of the ATZ-SP-IM was examined by using high-resolution transmission electron microscopy (JEM 2100 Plus, Jeol, Tokyo, Japan) .
22.214.171.124 Stability testing of ATZ-SP-IM
The storage stability of ATZ-SP-IM was assessed as per ICH Q1A (R2) guidelines under accelerated study conditions (40 ± 2˚C/75 ± 5%). The 10 g of ATZ-SP-IM sample was packed in an air tight culture tube and maintained in a stability chamber (Remi, SC-10 Plus, India). At specific time points, drug content, cloud point, signs of drug precipitation, self-emulsification time and % Transmittance were estimated to confer stability . In addition, the thermodynamic stability testing of ATZ-SP-IM was also assured by freeze thaw cycle and centrifugation test as per method reported by Kamboj et al. .
2.2.8 In-vitro drug dissolution study
Dissolution studies of ATZ-SP-IM and ATZ powder were conducted using USP apparatus II (Electrolab, Mumbai, India) employing 900 ml of 0.025N HCl and phosphate buffer (pH 6.8) as the dissolution medium maintained at 37 ±0.5 ˚C and 50 rpm [17, 38]. ATZ-SP-IM containing ATZ equivalent to 50 mg or ATZ powder (50 mg) loaded into hard gelatin capsule were subjected to dissolution testing. At pre-determined time intervals, an aliquot of 3 mL sample (each) was withdrawn with subsequent replacement of equivalent volumes of the medium. The percentage ATZ released at specific time intervals were determined by in house validated HPLC method.
2.2.9 Drug permeation study across rat’s everted intestinal sac
An everted rat gut sac experiment was performed as described in the literature reports [26, 39]. Cognizance was taken that the research work, involving drug permeation and pharmacokinetic study adheres to the guidelines for care and use of the laboratory animals. Thus, all the animal investigations were performed as per the requisite protocol approved by the Institutional Animal Ethics Committee (IAEC), Punjabi University, Patiala, India (107/99/ CPCSEA/2018-05).
Male Wistar rats weighing 200–250 g were used for this study. Prior to surgical procedure, the rats were fasted overnight with water ad libitum. Overnight-fasted rats were sacrificed by spinal dislocation. The excised medial jejunum segment (length ~6 cm; internal diameter ~0.3cm; area ~5.8 cm2) was immediately flushed with ice cold Kreb’s Ringer phosphate buffer (KRPB). After washing, one end of the tissue segment was then ligated with thread to one end of the thin glass rod and carefully everted on a glass rod. The prepared formulations equivalent to 50 mg of ATZ was transferred to donor compartment. The everted gut sac filled with 2 mL of KRPB solution was submerged inside the aerated (10-15 bubbles/min) bath (50 mL; 37±0.5 °C) containing ATZ-SP-IM or ATZ powder. An aliquot of drug solution was withdrawn from the serosal compartment at a predetermined time intervals up to 2 h and replaced with fresh KRPB solution. The amount of ATZ permeated was determined by analyzing samples using in-house validated HPLC method. The values of flux, apparent and relative permeability of different formulations were calculated.
2.2.10 In-vivo pharmacokinetic study
The in-vivo pharmacokinetic study involved five groups according to the two way cross over design. All animals were fasted overnight before dosing, water was provided ad libitum throughout the study. Four rats (Male Wistar, weighing 250-280 g) were randomly distributed amongst each group and received the following treatment:
Group A and Group B rats received ATZ-SP-IM (equivalent to 7 mg/kg of ATZ) and ATZ suspension (7 mg/kg) . While, the rats in Group C were pretreated with cycloheximide (3 mg/kg; i.p; 0.6 mg/mL solution in normal saline) one hour prior to the administration of ATZ-SP-IM . Further, rats in Group D and E were pretreated with famotidine (25 mg/kg; per oral; 5 mL/kg in 100mM phosphate buffer, pH 6.5) one hour prior to the administration of ATZ-SP-IM and ATZ suspension . All the animals received a dose equivalent to 7 mg/kg of atazanavir.
Aliquots of blood samples (0.5 mL) were periodically withdrawn from the retro-orbital plexus of the animals at predetermined time intervals. Plasma was then harvested by centrifugation at 6000 rpm for 20 min and stored at -20°C till further analysis. Extraction of ATZ was carried out by liquid-liquid extraction method . The drug content was analyzed by HPLC and standard non-compartmental pharmacokinetic parameters were calculated using in house pharmacokinetic program.