3.1 Equilibrium solubility of ATZ
Formation of stable and robust ATZ-IM without any precipitation depends upon the equilibrium solubility of ATZ in various oils, surfactants and co-surfactants. Solubility of ATZ in different oils, surfactants and co-surfactants has been illustrated in Fig. 1A & B. ATZ exhibited maximum solubility in oleic acid and corn oil. Oleic acid and corn oil chosen from the list of oils will assist in the lymphatic uptake of the atazanavir . In case of surfactants and co-surfactants, ATZ possesses higher solubility in Tween 80 and propylene glycol. Being a non-ionic surfactant, Tween 80 is considered to be safe, effective, and an inhibitor of the P-gp efflux of atazanavir after oral ingestion . In addition, propylene glycol being a co-surfactant is non volatile and compatible with gelatin capsules compared to other alcoholic co-surfactants. Based upon the equilibrium solubility studies of atazanavir, oleic acid, corn oil, Tween 80 and propylene glycol were chosen for delineating a stable and clear IM region during the ternary phase diagram studies.
3.2 Ternary Phase Diagram
Based on solubility study, ternary mixtures were firstly prepared by taking individual oil i.e. oleic acid or corn oil in combination with tween 80 and propylene glycol. It was observed that oleic acid and corn oil alone doesn’t able to form a clear and stable ternary mixture. Further, oleic acid: corn oil was used in a ratio of 1:1, 1:2 and 1:3 along with surfactant and co-surfactant to prepare a ternary mixture. In a ratio of 1:1, ternary mixture formed was clear and stable with a transmittance of more than 97%. Beyond that concentration, the ternary mixtures so prepared were turbid and undergoes phase separation after 24 h. So, the ternary phase diagram comprising of Oleic acid: Corn oil (1:1), Tween 80 and propylene glycol was constructed (Fig. 1C). It was observed that when oil phase was taken from 0.1–0.2 g, the IM formed after reconstitution was clear and stable with a particle size < 100 nm and transmittance approaches towards 100%. But, when the concentration of oil phase increased to 0.25 g, the particle size of IM was increased significantly. Beyond that, the IM was translucent having particle size more 200 nm and got turbid when left aside for some time. Hence, the ternary phase diagram showed that 0.1–0.2 g Oleic acid: Corn Oil (1:1), 0.5–0.8 g Tween 80 and 0.1–0.4 g Propylene glycol could be further used to optimize the ATZ-IM
3.3 Systematic optimization of ATZ-IM as per D-optimal mixture design
On the basis of phase diagram and screening studies (supplementary data), 16 IM were developed as per D-optimal mixture design each containing 50 mg of ATZ/g of IM. The developed formulations were further evaluated for various critical response variables (CRVs) namely particle size (Y1), PDI (Y2), Self Emulsification Time (Y3), % Transmittance (Y4) and Drug Content (Y5). Various CMAs and CRVs of D-optimal mixture design are summarized in Table 1. The CRVs obtained were given into Design Expert® 11 software for statistical analysis that generates polynomial equation for each respective CRVs. The magnitude of each estimated regression coefficient in the polynomial equation indicated the relative contribution of the corresponding independent variable to the response variable. Larger magnitude of the coefficient signifies that the CMAs have greater influence on the CRVs. A positive sign of the coefficient indicates a synergistic effect, while a negative sign indicates an antagonistic effect of the coefficient on the CRVs. The mathematical models used for all the CRVs were found to be appropriate and acceptable (Model p value > F is less than 0.01). The value of R2 for all mathematical models indicates the magnificent fit of the polynomials produced by statistical analysis to the response variables (p < 0.0001). The values of “lack of fit” were found to be insignificant in all the mathematical models, suggesting that the models are appropriate. Further, the closeness in the magnitude of predicted (Pred) and adjusted (Adj) R2 to the actual model R2 also affirms the magnificent fit to the data. The equation obtained after critical material attributes (CMAs) with critical response variables (CRVs) has been illustrated below:
P.S = + 60.23X1 + 15.51X2 + 17.14X3-27.44X1X2-76.69X1X3-5.51X2X3 (Eq. 3) R2 = 0.791; (model = Quadratic) (Eq. 1)
PDI = + 1.52X1 + 0.3196X2 + 0.1824X3-2.22X1X2-2.82X1X3-0.5241X2X3 (Eq. 4) R2 = 0.8680; (model = Quadratic) (Eq. 2)
% Transmittance = + 69.64X1 + 100.21X2 + 98.91X3 + 38.99X1X2 + 51.04X1X3 + 1.82X2X3-199.14X1X2X3 (Eq. 5) R2 = 0.8523; (model = Special Cubic) (Eq. 3)
Drug Content (%) = 388.33X1 + 32.53X2 + 444.18X3-289.10X1X2-1310.18X1X3-377.44X2X3 R2 = 0.9860; (model = Quadratic) (Eq. 4)
Self-emulsification time = + 8.61X1 + 20.76X2 + 23.79X3 + 52.15X1X2 + 19.91X1X3 + 3.45X2X3-24.91X1X2X3 (Eq. 5) R2 = 0.9130; (model = Special Cubic) (Eq. 5)
The results obtained from the polynomial equations were in concordance to the 3D response surface plot (Fig. S2).
Numerical optimization of ATZ loaded IM utilizing desirability approach with an aid of Design Expert 11 software was carried out to determine the optimized concentration of CMAs to generate a high-quality robust product. In the formulation design space, five critical response variables namely particle size (minimum), PDI (minimum), self-emulsification time (minimum), % transmittance (maximize), and drug content (maximize) with optimum goals were selected. The outcome of numerical optimization suggested formulation composition comprised of oleic acid: corn oil (0.1 g), Tween 80 (0.5 g), and propylene glycol (0.4 g) fulfilled all the requirements of an optimized formulation because of better regulation of critical response variables (Table 2). The desirability plot of the optimized ATZ loaded IM has been illustrated in Fig. S2; supplementary data. For the above optimized formulation composition, 0.763 desirability was obtained. Table 3 manifested the predicted and the observed value of CRVs after developing the optimized formulation (F1). The current findings revealed that not more than 5% biasness was observed when observed response values were correlated with predicted one. The optimized formulation was then subjected to further study.
3.4 Selection of PPI (polymer precipitation inhibitor)
(a) In-silico analysis
ATZ being a weakly basic drug undergoes drug precipitation in the intestinal pH. To prevent drug precipitation in the intestinal media, polymeric precipitation inhibitors (PPIs) were used to maintain the drug in the supersaturated state. ATZ being a ligand was docked with different types of PPIs via molecular docking study . In the present investigations, the lowest binding energy and the intermolecular H-bond formation were considered as important criteria for inhibiting the crystallization of ATZ . The H-bond surfaces between PPIs and ATZ were evident in Fig. 2. The results revealed that the intermolecular H-bonds were formed between the O-atom present in the glycosidic linkage of the PPI and H-atom of the ATZ. During molecular docking, the free energy (ΔG) is used to investigate the strength of interactions between ATZ and PPIs . The binding energies of all the ligand-PPIs complexes have been shown in Table 3. From the above results, it was observed that, ATZ-HPMC-AS complex possesses lowest binding energy (-2.98 Kcal/mol) and had a strong interaction with ATZ than any other PPIs in inhibiting the crystallization of ATZ and prolonging the stabilized supersaturation state in intestinal pH. However, the docking study was usually established for ligand-PPIs interaction in the absence of H2O molecules that would be a demerit for the theoretical verification of our results. This flaw of docking study was rectified by carrying out further dynamics simulations for the period of 30 ns in the build system.
While running the MD simulations, the root-mean-square deviation (RMSD) is usually used to study the stability of the ligand-PPIs complex . From Fig. S3; supplementary data, it was concluded that all the ATZ-PPIs complexes could arrive at the equilibrium state but ATZ-HPMC-AS complex was able to attain the stability at the earliest with a RMSD fluctuations less than 1.5Å. The solvent-accessible surface area (SASA) of solute was also calculated to compare the hydrophilic property of the ATZ-PPI complex . After carrying out the MD simulation, it was observed that all polymers except HPMC-AS were able to interact with the system constituting the H2O molecules. The existence of carbonyl and the ester moiety in the HPMC-AS exhibited a large hydrophobic area than other PPIs, which could help HPMC-AS to interact greatly with the hydrophobic moiety of the ATZ molecule . Thus, the above observations provide an insight into the potential of the HPMC-AS to inhibit the crystallization of ATZ in the intestinal media.
(b) In-vitro supersaturation test
To validate the results obtained from in-silico analysis, in-vitro supersaturation test was conducted. The in-vitro supersaturation test was conducted for optimized ATZ-IM containing different polymeric precipitation inhibitors (PPIs) in 500 mL of simulated intestinal fluid (pH 6.8) under non-sink conditions. The performance of PPIs was evaluated by comparing the apparent concentration of the drug v/s time, as illustrated in Fig. 3A. The initial drug concentration of ATZ was found out to be 0.1 mg/mL (based on the dilution factor of 500, i.e. 1 g of ATZ-IM containing 50 mg of ATZ dissolved in 500 mL of simulated intestinal fluid) before the commencement of supersaturation test. After initiating the experiment, at time t = 5 min, the apparent drug concentration was found out to be 0.034 mg/mL for HPMC E15, 0.086 mg/mL for HPMC-AS, 0.024 mg/mL for PVP K17, 0.028 mg/mL for Poloxamer 188 and 0.024 mg/mL for poloxamer 407, respectively. A rapid decline in the apparent drug concentration was observed in case of HPMC E15, PVP K17, Poloxamer 188, and Poloxamer 507 (0.004 mg/mL for all the PPIs), while the HPMC-AS maintains the supersaturated state (0.041 mg/mL) to a great extent. From the outcome of in-vitro supersaturation test, it was concluded that HPMC-AS was found successful in inhibiting the crystallization of ATZ in intestinal media. Hence, HPMC-AS was selected as PPIs in formulating ATZ-SP-IM for further investigation.
3.5 Evaluation of optimized ATZ-SP-IM
3.5.1 Determination of particle size (PS), poly dispersity index (PDI) and Zeta potential
The developed formulation was evaluated for particle size, PDI and zeta potential. During the particle size and PDI analysis, it was observed that ATZ-SP-IM exhibited a mean particle size of 14.42 ± 1.9 nm and a PDI of 0.186 ± 0.02 (Fig. 3B). No significant difference was observed in particle size and PDI when HPMC-AS was incorporated into ATZ-IM. The above results clearly vouch the presence of nano-sized globules in the IM with a monodisperse nature of their distribution, thus fulfilling the requisite of SP-IM as per the published literature reports. The zeta potential of the SP-IM was estimated to be -25.7 ± 2.4 mV (Fig. 3C). The negative zeta potential was ascribed to the presence of free fatty acids in the formulation . The above results suggested that ATZ-SP-IM generated a more stable system.
3.5.2 Transmission electron microscopy
The TEM image of reconstituted ATZ-SP-IM illustrated in Fig. 3D exhibited the presence of spherically shaped oil globules. In addition, no sign of drug precipitation was observed suggesting that ATZ is present in stable and solubilized form. Although, the TEM analysis also revealed that oil droplets retain their integrity in terms of morphology, uniformity in size and shape after reconstituting ATZ-SP-IM.
3.5.3 Stability testing of ATZ-SP-IM
The ATZ-SP-IM was subjected to accelerated stability studies for a time period of 3 months. The results of particle size analysis and PDI at different time intervals shows that no significant change in the values of particle size and PDI was observed when ATZ-SP-IM was kept in a stability chamber. No significant change in the values of drug content, cloud point, self-emulsification time and % Transmittance was observed during the study (Table 3). Hence, it can be concluded that ATZ-SP-IM exhibited good shelf life. During thermodynamic stability study, no signs of phase separation or drug precipitation were observed when ATZ-SP-IM was subjected to centrifugation test (Table 3). The freeze thaw cycles did not able to change the clarity and stability of ATZ-SP-IM. The above results suggested that the developed formulation is an isotropic mixture with high stability attributes.
3.6 In-vitro drug release study
To demonstrate the superiority of the SP-IM, dissolution profiles of ATZ from ATZ-SP-IM and ATZ powder loaded in hard gelatin capsule were determined in both 0.025N HCl (OGD media) and intestinal pH 6.8 (Fig. 4). Being a weakly basic drug, ATZ powder exhibited higher drug release in 0.025M HCl (Fig. 4A). It was observed that more than 90% of ATZ was release in 45 min. In contrast, ATZ-SP-IM exhibited similar behavior in 0.025N HCl but persistently at a faster release rate. The ATZ-SP-IM releases more than 90% drug and achieved a plateau state within 10 min (Fig. 4A). This predominantly occurs due to the presence to non-ionic surfactant (Tween 80) in ATZ-SP-IM. It has been reported that surfactants with higher HLB values shows higher emulsification ability and allows rapid dispersion of oil in the aqueous phase, producing nanosized oil-in-water emulsion. In case of intestinal pH, ATZ powder undergoes only 36% of drug release in 3 h of time course (Fig. 4B). The above results has been evident by the literature reports that ATZ solubility gets reduced to less than 1µg/mL when pH goes beyond 3 . On the other hand, the ATZ-SP-IM showed more than 80% of drug release in 15 min with a maximum of 93% within 90 min (Fig. 4B). The presence of HPMC-AS in the IM decelerates the crystallization of ATZ and maintaining the drug in the supersaturated state leading to the higher drug release in the intestinal pH. Overall, the above results clearly demonstrate the improved dissolution characteristics of ATZ-SP-IM.
3.7 Drug permeation study across rat’s everted intestinal sac
The ex-vivo performance of ATZ-SP-IM and ATZ powder was carried out by using rat’s everted intestinal sac. To compare the results, flux (µg/cm2/min), apparent permeability (cm/min) and relative permeability were calculated (Table 4). The rate of permeation (flux) of pure ATZ across intestinal wall was calculated to be 0.032 µg/cm2/min, suggested that ATZ belongs to BCS-Class II drug. While, the ATZ release from SP-IM was found to be 0.065 µg/cm2/min, which was calculated to be 2.03 fold enhancement in flux. The results also suggested a 3.63-fold enhancement in relative permeability of ATZ in case of ATZ-SP-IM formulation. The enhanced permeability of the can be attributed to the larger concentration gradient created by the increased solubility of the ATZ into the SP-IM. From the above results, it can be postulated that larger surface area of nanoparticulates provided by ATZ-SP-IM increases the adherence to the intestinal membrane which in turn enhances the intestinal transport of the drug .
3.8 In-vivo pharmacokinetic and lymphatic tissue distribution study
The in-vivo animal pharmacokinetic study employing two way crossover design was carried out to compare the pharmacokinetic profile of ATZ-SP-IM with pure ATZ. The observed plasma concentration-time profile after oral administration of ATZ in the form of pure drug and ATZ-SP-IM is depicted in Fig. 4C and the non-compartmental pharmacokinetic parameters have been summarized in Table 4. The peak plasma concentration (Cmax) value of atazanavir after a single dose of suspension as a reference product and SP-IM as a test formulation were found to be 14.46 ± 3.72 µg/mL and 28.76 ± 5.43 µg/mL, respectively. A significant enhancement in Cmax (1.98 fold) was evident, when ATZ-SP-IM was administered. The area under curve (AUC) was also found to be 1.8 fold enhanced for ATZ-SP-IM (95.47 ± 5.77 µg/mL) than for ATZ suspension (60.25 ± 4.01 µg/mL). The half-life (t1/2) of ATZ-SP-IM was found to be 1.47 fold increased as compared to pure ATZ. In line with the AUC values, the mean residence time (MRT) in case of ATZ-SP-IM was found to be increased when compared with pure ATZ. The improvement in the pharmacokinetic performance of ATZ could be attributed to the high solubility of ATZ into the SP-IM components and a large surface area of microemulsion formed after reconstitution with GI fluids may be responsible for promoting gastrointestinal absorption and improving the ATZ oral bioavailability.
The effect of histamine H2 receptor antagonist (famotidine) on the pharmacokinetic performance of ATZ-SP-IM and pure ATZ was also investigated. A sharp decline in the value of Cmax and AUC was observed when pure ATZ was administered to the rats pretreated with famotidine (Table 4). This sudden decrease in the pharmacokinetic parameters could be due to the steep decline in the solubility of ATZ with respect to the change in the gastric pH. At elevated pH, the solubility of ATZ decreases that may lead to insufficient dissolution in the stomach due to which the absorption of ATZ from the GIT decreased . However, the absorption of ATZ doesn’t get affected when drug was administered into SP-IM (Table 4 and Fig. 4D). Such type of behavior was ascribed to the presence of HPMC-AS in the SP-IM that induced and maintained the drug in the supersaturated state over a period of time till absorption. This augmentation in the rate and extent of drug bioavailability from SP-IM would eventually results in significant escalation in the intensity of therapeutic effect of atazanavir too.
The intestinal lymphatic transport of any formulation is elucidated by comparing the pharmacokinetic profiles in animals pretreated with cycloheximide, a chylomicron flow blocker. As compared to pure ATZ, the ATZ-SP-IM exhibited significant increase in the extent of oral bioavailability in rats (i.e., nearly 1.58 fold), evident from the corresponding values of the AUC (Table 4). However, a remarkable change in the value of AUC was observed when ATZ-SP-IM formulation was administered to rats group pretreated with cycloheximide over ATZ-SP-IM control group (Fig. 4E). A considerable 1.31 fold decrease in the value of AUC could be ascribed solely to the inhibition of the lymphatic pathways caused by blockage of chylomicron flow. Hence, it can be said that absorption of ATZ was highly affected by cycloheximide and intestinal lymphatic system was involved in the absorption of ATZ-SP-IM. The intestinal lymphatic uptake of ATZ-SP-IM occurs due to the presence of high concentration of surfactant and the long chain triglycerides in the formulation. In our case, presence of corn oil and oleic acid as long chain triglycerides assists in the lymphatic transport of ATZ . Besides, Tween 80 being a surfactant has been reported to inhibit P-glycoprotein (P-gp) activity which might be another benefit for enhanced permeability and intestinal lymphatic transport, resulting in enhanced oral bioavailability . To validate the results of chylomicron flow blocking approach in analyzing the drug transport to the lymphatics, concentration of ATZ in the lymph nodes was also estimated. For this, rats were administered orally with ATZ-SP-IM and ATZ suspension at a dose of 7 mg/kg. The concentration of ATZ in the lymph nodes at specific time intervals was estimated by excising the lymph nodes after cervical dislocation. It was observed that the rats administered with supersaturable preconcentrated isotropic mixture (ATZ-SP-IM) exhibited higher concentration of ATZ in the lymph nodes over that of putative form. The enhanced drug concentration in the lymph nodes could be ascribed to the presence of long chain fatty acids in the supersaturable formulation which assists in the transport of ATZ to the lymphatics. The improved distribution of ATZ to the lymphatics would potentially results in the site-specific targeted drug delivery, inhibition of the HIV and further prevents the suppression of biodistribution of the virus to the other vital organs.