Optimized self nano-emulsifying systems of ezetimibe with enhanced bioavailability potential using long chain and medium chain triglycerides

Colloids and Surfaces B: Biointerfaces 100 (2012) 50–61

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Optimized self nano-emulsifying systems of ezetimibe with enhanced bioavailability potential using long chain and medium chain triglycerides Shantanu Bandyopadhyay, O.P. Katare, Bhupinder Singh ∗ University Institute of Pharmaceutical Sciences (UGC Centre of Advanced Studies), Panjab University, Chandigarh 160014, India

a r t i c l e

i n f o

Article history: Received 19 April 2012 Accepted 12 May 2012 Available online 24 May 2012 Keywords: SNEDDS Formulation by Design (FbD) Intestinal perfusion Hyperlipidemia Triglycerides Nanoemulsion

a b s t r a c t The objective of the current work is to develop systematically optimized self-nanoemulsifying drug delivery systems (SNEDDS) using long chain triglycerides (LCT’s) and medium chain triglycerides (MCT’s) of ezetimibe employing Formulation by Design (FbD), and evaluate their in vitro and in vivo performance. Equilibrium solubility studies indicated the choice of Maisine 35-1 and Capryol 90 as lipids, and of Labrasol and Tween 80 as emulgents for formulating the LCT and MCT systems, respectively. Ternary phase diagrams were constructed to select the areas of nanoemulsion, and the amounts of lipid (X1 ) and emulgent (X2 ) as the critical factor variables. The SNEDDS were systematically optimized using 32 central composite design and the optimized formulations located using overlay plot. TEM studies on reconstituted SNEDDS demonstrated uniform shape and size of globules. The nanometer size range and high negative values of zeta potential depicted non-coalescent nature of the optimized SNEDDS. Thermodynamic studies, cloud point determination and accelerated stability studies ascertained the stability of optimized formulations. In situ perfusion (SPIP) studies in Sprague Dawley (SD) rats construed remarkable enhancement in the absorptivity and permeability parameters of SNEDDS vis-à-vis the conventional marketed product. In vivo pharmacodynamic studies in SD rats indicated significantly superior modification in plasma lipid levels of optimized SNEDDS vis-à-vis marketed product, inclusion complex and pure drug. The studies, therefore, indicate the successful formulation development of self-nanoemulsifying systems with distinctly improved bioavailability potential of ezetimibe. © 2012 Elsevier B.V. All rights reserved.

1. Introduction Self-nanoemulsifying drug delivery systems (SNEDDS) are newer and novel technological innovations with immense potential in oral bioavailability enhancement of lipophilic drugs. Being nano in size, such lipidic drug carrier systems are capable of surmounting the problems of low oral bioavailability of drug(s) caused owing to their poor aqueous solubility, hepatic first-pass effect, metabolism by cytochrome P450 family of enzymes present in the gut enterocytes and liver hepatocytes, P-glycoprotein (P-gp) efflux and/or restricted intestinal permeability [1]. Of the excipients employed for the formulation of SNEDDS, lipids have an immense role for the biological fate of drug. Studies have shown that the type of absorption pathway and subsequent transportation of drug is significantly influenced by the two types

∗ Corresponding author at: University Institute of Pharmaceutical Sciences, UGC Centre of Advanced Studies, Chandigarh 160 014, India. Tel.: +91 172 2534103; fax: +91 172 2543101. E-mail addresses: [email protected] (S. Bandyopadhyay), [email protected] (O.P. Katare), [email protected], [email protected] (B. Singh). 0927-7765/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.colsurfb.2012.05.019

of lipids viz. medium chain triglycerides (MCT’s) and long chain triglycerides (LCT’s) [2,3]. The MCT’s are directly transported by the portal blood to the systemic circulation, whereas the LCT’s are transported via the intestinal lymphatics. The LCT’s are likely to augment the lymphatic transport of a lipophilic drug substance leading to enhance oral bioavailability. Nevertheless, if the lipophilicity of the molecule is sufficiently high (i.e., log P ≥ 4.5) then the MCT-based systems are also likely to favour the lymphatic transportation. The drug, i.e., ezetimibe, chosen in the present study is a BCS class II hypolipidemic drug with poor water-solubility and high permeability (log P of 4.56) [4]. Besides these, it undergoes rapid first-pass metabolism and P-gp efflux, leading eventually to marked reduction in the drug oral bioavailable fraction (i.e., 35%) in humans and animals like dogs, rats, etc. [5,6]. To circumvent the afore-mentioned limitations various formulation approaches of ezetimibe have been reported like, nanocrystals, cyclodextrins inclusion complex, suspensions but all with limited fruition [7–9]. Systematic optimization of such isotropic delivery systems using design of experiments (DoE), on the other hand, offers numerous advantages including high degree of precision and prognosis, and economy in terms of time, effort and money [10]. Application of such DoE techniques for the development of optimized drug

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delivery products, lately termed as formulation by design (FbD), is known to provide an in-depth understanding and ability to explore and defend the ranges for varied formulation and processing factors [11]. Thus, the present research aims at studying the effect of LCT and MCT-SNEDDS of ezetimibe formulated employing systematic FbD for enhancement of its dissolution and bypassing the P-gp and firstpass effect, resulting consequently in improved oral bioavailability potential.

2. Material and methods Ezetimibe was provided ex-gratis by M/s Ranbaxy Laboratories Ltd., Gurgaon, India. LabrafacTM Lipophile WL 1349 (medium-chain triglycerides), CapryolTM 90 (propylene glycol monocaprylate), LauroglycolTM 90 (propylene glycol monolaurate), Labrasol® (caprylocaproyl macrogol-8 glycerides) and MaisineTM 35-1 (glycerol monolinoleate) were received as the gift samples from M/s Gattefosse, Saint-Priest, France. Cremophor® EL (polyoxyl 35 Castor Oil) was supplied ex-gratis by M/s BASF Mumbai, India. Captex® 200P (propylene Glycol Dicaprylate/Dicaprate), Capmul MCM® (medium chain fatty acids mixture, mainly of caprylic and capric) and Capmul® PG 8 (propylene glycol monocaprylate) were received as the gift samples from M/s Abitec Corp., Wisconsin, USA. Tween® 20, Tween® 60 and Tween® 80 were procured from M/s HIMEDIA Laboratories Pvt. Ltd., Mumbai, India. Ethyl Oleate and Olive Oil were procured from M/s Loba Chemie, Mumbai, India. Sesame Oil was obtained from M/s S K Oil Industries, Jalgaon, India. All other materials and chemicals procured for the studies were of analytical grade, and were used as such as obtained.

3. Initial studies for screening of excipients 3.1. Solubility studies Equilibrium solubility of ezetimibe was determined employing various MCT’s viz. CapryolTM 90, Capmul® PG 8, Captex® 200P, LabrafacTM Lipophile WL 1349 and Capmul MCM® , and various LCT’s, i.e., olive oil, sesame oil, LauroglycolTM 90, ethyl oleate, MaisineTM 35-1. An excess amount of ezetimibe was added to each of the selected lipids, and the mixture was stirred continuously for 72 h at 37 ± 1 ◦ C. Following attainment of equilibrium, the mixture was centrifuged at 1500 rpm (88.04 × g) for 20 min, and the obtained supernatant was filtered through a membrane filter having pore size of 0.45 ␮m (M/s mdi Membrane Technologies LLC, California, USA). Spectrophotometric absorbance of the filtrate was measured using UV 3000+ spectrophotometer (M/s Labindia, Mumbai, India) at a max of 232.5 nm. Analogously, equilibrium solubility studies were also conducted in emulgents viz. Labrasol® , Cremophor® EL, Tween® 20, Tween® 60 and Tween® 80. Drug content was determined using a previously constructed standard calibration plot, taking E11%cm as 363 and molar extinction coefficient as 14,861.22. 3.2. Ternary phase diagram Compositions with different oil-to-emulgent ratios, within the range of 1:9 and 9:1, were selected and titrated with water at ambient temperature. The mixtures were observed visually following equilibration. Subsequently a series of ternary phase diagrams were constructed using PCP Disso software ver 3.0 (M/s Pune College of Pharmacy, Pune, India). The phase diagrams were so constructed to delineate the boundaries of various phases, i.e., nano/microemulsion, emulsion, emulgel and microgel [12]. The

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generated samples, clear or slightly bluish in appearance, were taken as the nanoemulsions. 3.3. Preparation of SNEDDS as per experimental design The LCT and MCT-SNEDDS formulations were prepared by the standard admixture method, as reported by us earlier [13]. Drug was dissolved in the lipid(s) at 37 ◦ C and the emulgent, in the predetermined ratio, was added to the lipidic drug solution, while stirring at high speed using a magnetic stirrer (M/s Perfit, Ambala, India) maintained at 37 ◦ C. Lipidic and emulsifying excipients were chosen on the basis of the formation of maximal nanoemulsion region in the ternary phase diagram. For the preparation of LCT-SNEDDS, Maisine 35-1 as lipid (X1 ) and Labrasol as emulgent (X2 ), while for the preparation of MCT-SNEDDS, Capryol 90 as lipid (X1 ) and Tween 80 as emulgent (X2 ) were chosen and finally selected as the two critical influential factors for further formulation optimization work [10,14]. A CCD with ˛ = 1 was employed, where the amounts of oil and emulgent were studied at three levels each. Overall, a set of 13 experimental runs each were studied as per the experimental design matrix as depicted in Table 1. The formulation at the intermediate coded factor levels (i.e., 0,0) was studied in quintuplicate. The response variables considered for the current DoE optimization studies encompassed, amount permeated in 45 min (Perm45 min ), globule size (Dnm ) and %dissolution efficiency in 30 min (%DE30 min ). 4. Pre-optimization formulation characterization 4.1. Determination of globule size Aliquots (1 mL) of the samples, serially diluted 100-fold with purified water, were employed to assess the globule size [15]. The emulsion globule size was determined by dynamic light scattering technique using particle size analyser (ZS 90, M/s Malvern, Worcestershire, UK) [16]. 4.2. In vitro dissolution studies Drug release studies were carried out for both LCT and MCTSNEDDS formulation combinations, in triplicate, employing USP 31 Apparatus 2 (DS 8000A/S, M/s Labindia, Mumbai, India) using 500 mL of dissolution medium containing 0.5% (w/v) SLS as at 50 rpm and 37 ± 0.5 ◦ C. Aliquots of sample (5 mL each) were periodically withdrawn at suitable time intervals and replaced with fresh dissolution medium. The samples, after suitable dilution(s), were analysed spectrophotometrically at a max of 231 nm, taking E11%cm as 260 and molar extinction coefficient as 10,644.4. Dissolution study was also conducted on pure ezetimibe in an analogous manner. 4.3. Non-everted gut sac method Taking cognizance that the research work, involving gut sac method, in situ SPIP studies and the subsequent in vivo pharmadynamic studies, adheres to the guidelines for care and use of the laboratory animals, all the animal investigations were performed as per the requisite protocol approved by the Institutional Animal Ethics Committee of the Panjab University, Chandigarh, India [Letter no IAEC/54 dated 06/12/2010]. The Committee is duly approved for the purpose of control and supervision of experiments on the animals by the Government of India. Ex vivo permeation studies were performed using non-everted gut sac technique in female Sprague Dawley (SD) rats, weighing between 200 and 300 g, previously made to abstain from solid

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Table 1 Preparation of LCT and MCT-SEDDS as per the experimental design. Trial no.

Coded factor levels LCT-SEDDS

1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13.

MCT-SEDDS

Factor 1, lipid (mg)

Factor 2, surfactant (mg)

Factor 1, lipid (mg)

Factor 2, surfactant (mg)

−1 (100) −1 (100) −1 (100) 0 (250) 0 (250) 0 (250) 1 (400) 1 (400) 1 (400) 0 (250) 0 (250) 0 (250) 0 (250)

−1 (500) 0 (650) 1 (800) −1 (500) 0 (650) 1 (800) −1 (500) 0 (650) 1 (800) 0 (650) 0 (650) 0 (650) 0 (650)

−1 (250) −1 (250) −1 (250) 0 (500) 0 (500) 0 (500) 1 (750) 1 (750) 1 (750) 0 (500) 0 (500) 0 (500) 0 (500)

−1 (500) 0 (650) 1 (800) −1 (500) 0 (650) 1 (800) −1 (500) 0 (650) 1 (800) 0 (650) 0 (650) 0 (650) 0 (650)

food at least 12 h prior to the study. The studies were conducted for both the types of SNEDDS in triplicate, employing an in-house fabricated assembly. Rats were sacrificed by cervical dislocation, i.e., breaking the neck or snapping the spine. Following a midline incision in the abdomen, medial jejunum segment was cut for investigational purposes. The segment was washed six times with 5 mL each of Krebs ringer solution (KRB) along with continuous aeration. The segment was finally ligated with a thread to one end and fitted into the in-house fabricated assembly. A weight of 1 g was fixed and tied to the end of the non-everted gut segment to make an empty gut sac and to prevent peristaltic muscular contractions. The gut sac, filled with the SNEDDS formulations, was placed inside the bath containing 50 mL of KRB, continuously bubbled with atmospheric air at 10–15 bubbles per min. The gut sac bath was surrounded by an outer water jacket, thereby maintaining the temperature of the gut sac bath and its medium at 37 ± 0.5 ◦ C [17]. An aliquot of drug solution was withdrawn at different time points (i.e., 15, 30, 45, 60 min), and replaced each time with fresh KRB. The amount of ezetimibe was determined spectrophotomet1% as 350 and molar extinction rically at a max of 231 nm, taking E1cm coefficient as 14,329.0.

4.4. Optimization data analysis and validation of optimization model For the studied design, the multiple linear regression analysis (MLRA) was applied to fit full second-order polynomial equation with added interaction terms to correlate the studied responses with the examined variables using Design Expert software ver. 8.0 (M/s Stat-Ease, Minneapolis, USA). The polynomial regression results were demonstrated for the studied responses and their statistical significance discerned. Finally, the prognosis of optimum formulation was conducted using a two-stage technique. A feasible space was located initially and an exhaustive grid search was conducted subsequently to predict the possible solutions. Six formulations were selected as the confirmatory check-points to validate the application of FbD. Linear correlation plots between predicted and observed responses, and the corresponding residual graphs were constructed for the chosen check-point formulations. Overlay plot was obtained by superimposing the values of various response variables in order to locate the optimized formulation. The observed and predicted responses were critically compared and the percent bias (i.e., prediction error) was also calculated with respect to the observed responses.

5. Formulation characterization of optimized formulations 5.1. Determination of globule size and zeta potential Aliquots (1 mL) of the optimized SNEDDS, serially diluted 100fold with purified water, were employed to assess the globule size and zeta potential using particle size analyser (ZS 90, M/s Malvern, Worcestershire, UK).

5.2. Thermodynamic stability studies The optimized SNEDDS formulations were subjected to various thermodynamic studies in order to assess the phase separation and/or stability of the nanoemulsion [18,19].

5.2.1. Centrifugation study The optimized formulations were centrifuged at 5000 rpm (978.25 × g) for 30 min. The resultant formulations were then checked for any instability problem, such as phase separation, creaming or cracking.

5.2.2. Heating and cooling cycle The contents of the optimized formulations were subjected to six heating/cooling cycles between 4 ◦ C and 40 ◦ C with storage at each temperature for not less than 48 h. The resultant formulations were assessed for their physical instability like phase separation and precipitation.

5.3. Cloud point measurement The optimized SNEDDS formulations were diluted with distilled water in the ratio of 1:250. The diluted samples were placed in a water bath and its temperature was increased gradually. Cloud point was spectrophotmetrically determined as the temperature at which there was a sudden appearance of cloudiness [20].

5.4. Drug release comparison with marketed brand Drug release profiles of the optimized SNEDDS and HP-␤-CD inculsion complex of drug (CD) were compared with that of the conventional marketed brand, MKT (Ezedoc, M/s Lupin Ltd., Mumbai, India) and pure drug, each containing 10 mg of Ezetimibe.

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5.5. In vivo pharmacodynamic studies

5.8. Transmission electron microscopy (TEM)

Initially for ten days, all the rats were induced with hypercholesterolemia by administering them with high fat diet, i.e., 200 mg of cholesterol in coconut oil [21,22]. After the induction, the pharmacodynamic study was carried for 28 days in female SD rats weighing between 200 and 300 g. Thirty rats were divided randomly in five groups with six rats in each group. Group I control, wherein only hyperlipidemia was induced. Animals of Group II, III, IV and V received OPT-LCT, OPT-MCT, CD and MKT, respectively. Each group received their respective formulations consecutively as above, once daily, for a period of 28 days. Blood samples were withdrawn periodically under light ether anesthesia by retro-orbital puncture at weekly intervals, i.e., at day 0, 7, 14, 21 and 28 in anticoagulated (EDTA-treated) glass vials. The plasma was separated by centrifugation at 3000 rpm (352.17 × g) for 25 min and stored at 2 ◦ C until further used. The samples were analysed for percent reduction in the levels of triglycerides (TG) and LDL, and percent enhancement in HDL levels using in vitro diagnostic kit (M/s ERBA Diagnostics, Mannheim GmbH, Germany) [23]. Absorbance of the developed colour was determined spectrophotometrically at a max of 505 nm. The statistical analysis for the determination of the difference in the lipid levels of control and treatment groups was performed by two-way ANOVA using GraphPad Prism software ver 5.0 (M/s GraphPad Software Inc., California, USA). The results were confirmed by Bonferroni’s multiple comparison as a post hoc test [24,25].

The morphology of the optimized SNEDDS formulations was observed using transmission electron microscope (JEM-2100 F, M/s Jeol, Tokyo, Japan) with AMT image capture engine software. SNEDDS formulations were diluted with distilled water in 1:200 and mixed by gentle shaking. One drop of the diluted sample was deposited on a film coated copper grid, stained with one drop of phosphotungstic acid and allowed to dry before observation under the transmission microscope. The image was magnified and focused on a layer of photographic film [20].

5.6. In situ single pass intestinal perfusion (SPIP) studies Perfusion studies were performed in female SD rats, previously made to abstain from solid food at least 24 h prior to the study. The SPIP studies were carried out on the optimized SNEDDS, i.e., OPT-LCT, OPT-MCT, and MKT and pure drug each in triplicate, employing an in-house fabricated assembly [26]. The animals were anesthetized using thiopental sodium (50 mg/kg) injected intraperitoneally. Following mid-line incision of the abdomen, the proximal part of the jejnum, 2–5 cm below the Ligament of Trietz, was cannulated with a glass cannula and connected to a reservoir [27]. The second incision was made 10–15 cm below the first incision and connected with an outflow glass cannula. The intestinal segments were perfused with KRB until the perfusate was clear. The intestinal segments were subsequently perfused with respective formulations, maintained at 37 ± 1 ◦ C at a perfusion rate of 0.2 mL min−1 . Steady state was achieved within 30 min, after which aliquots of samples (1 mL each) were periodically withdrawn at a regular interval of 15 min each. Diethyl ether (4 mL) was added to each perfusate sample (1 mL), and the mixture was centrifuged at 5000 rpm (978.25 × g) for 20 min. Following centrifugation, 3 mL of the supernatant etheral fraction was collected and evaporated. Finally, 1.5 mL of perfusion solution was added to the ethereal extract and the drug concentration of the mixture was determined spectrophotometrically at a 1% as 350 and molar extinction coefficient max of 231 nm, taking E1cm as 14,329.0. 5.7. Rheological studies Rheological studies of optimized SNEDDS were performed using a rotational type rheometer (Rheolab QC, M/s Anton Paar GmbH, Vienna, Austria) attached with a water jacket (C-LTD80/QC) for maintaining a constant temperature of 25 ◦ C. Data analysis was carried out using Rheoplus/32 software ver 3.40. For observing the rheological behaviour of the samples, a spindle geometry (DG26) was used. The rheological measurements were subsequently carried out at 25 ◦ C.

5.9. Accelerated stability study The optimized SNEDDS formulations were subjected to accelerated stability studies, carried out at 40 ± 2 ◦ C/75% ± 5% RH, as per the ICH guidelines, Q1A(R2), for the climatic zone IV [28]. The formulations were kept in air-tight glass vials and were assayed for Q15 min , % transmittance, viscosity and emulsification time periodically at the time points of 0, 1, 3 and 6 months. Similarity factor (f2 ) was also calculated to investigate the analogy of the dissolution parameters during the stability studies [29]. 6. Results and discussion 6.1. Pre-optimization formulation characterization 6.1.1. Solubility studies Equilibrium solubility studies (Supplementary data 1A and B) were carried out to investigate the maximum soluble fraction of ezetimibe in different lipids and emulgents. For LCT’s, the maximum solubility of ezetimibe was observed in Maisine 351 (14.72 mg mL−1 ), while the minimum was found in sesame oil (1.20 mg mL−1 ). Among MCT’s, the highest solubility of ezetimibe was observed in Capryol 90 (46.30 mg mL−1 ), while the lowest solubility was seen in Labrafac lipophilic WL1349 (6.85 mg mL−1 ). The dose of ezetimibe being small (i.e., 10 mg), higher solubility of drug in any lipid would augment its drug loading potential [30]. Accordingly, on the basis of maximum solubilities, Maisine 35-1 and Capryol 90 were chosen as the lipidic constituent(s) for formulation of the LCT-SNEDDS and MCT-SNEDDS, respectively. Solubility of ezetimibe was found to be much higher in MCT’s visà-vis LCT’s. This can be attributed to the shorter chain length and better fluidity of MCT’s, in consonance with the literature reports [2,31]. Likewise among the emulgents, the maximum solubility of ezetimibe was observed in Labrasol (77.69 mg mL−1 ) and minimum solubility was found in Tween® 60 (32.18 mg mL−1 ). Albeit maximum solubility was found in Labrasol, the results were impressive with that of Tween 80 (51.92 mg mL−1 ) and Cremophor EL (52.18 mg mL−1 ) as well. It is widely reported that nonionic emulgents are generally considered safer than the ionic emulgents and are usually accepted for oral ingestion [32]. In addition, they can produce reversible changes in intestinal mucosa, thus leading to enhance permeability absorption of drug [33]. Hence, it was planned to explore the effect of all of the three emulgents for delineating a stable nanoemulsion region. 6.2. Ternary phase diagram To ensure the spontaneity of SNEDDS to form the nanoemulsion within the GI conditions, the construction of ternary plots is considered to be a vital exercise [34]. As indicated in Fig. 1A, the nanoemulsion region for LCT-SNEDDS is prominent with Labrasol, whereas it is minimal with Tween 80. Further, the ternary plot between Maisine 35-1 (LCT) and Labrasol yielded only two regions

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Fig. 1. (A) Ternary phase diagrams employing Maisine 35-1 with various emulgents. (B) Ternary phase diagrams employing and Capryol 90 with various emulgents.

(i.e., nanoemulsion and emulsion) vis-à-vis other plots where all the four regions are observed. Likewise, the nanoemulsion region for MCT-SNEDDS was markedly increased with Tween 80, while minimal effect was noticed with Labrasol. Further, the ternary plot between Capryol 90 (MCT) and Cremophor EL yielded a microgel region (Fig. 1B), i.e., usually considered undesirable for SNEDDS formulation. Although the ternary plots between Capryol 90 and other two emulgents depicted all the four regions, yet the nanoemulsion region was more pronounced with Tween 80. Hence, Maisine 351 and Labrasol were selected for the formulation of LCT-SNEDDS, while for MCT-SNEDDS Capryol 90 and Tween 80 were chosen. The

MCT’s tend to form larger nanoemulsion areas compared to LCT’s, ostensibly due to higher polarity and lower hydrophobicity of the former [35,36]. There are reports which clearly indicate that high HLB value of an emulgent facilitates in lowering the interfacial energy, leading to the formation of a stable nanoemulsion [37]. The emulgents used in the present study, i.e., Labrasol and Tween 80, have high HLB values viz. 14 and 15, respectively. Thus the HLB values and ternary plots constructed clearly indicate the synergism of the excipients in reducing the interfacial tension as well as formation of stable nanoemulsion region.

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Fig. 2. Response surface plots showing the influence of Maisine 35-1 (LCT) and Capryol 90 (MCT) with Labrasol and Tween 80, respectively on amount permeated in 45 min (Perm45 min ), globule size (Dnm ) and (%DE30 min ).

6.3. Determination of globule size During the pre-optimization studies, the globule size for LCTSNEDDS was found to range between 62.1 and 307.8 nm, whereas it was observed to be between 81 and 311.2 nm for MCT-SNEDDS. The results apparently showed that there is a remarkable diminution in globule size on increasing the levels of emulgent.

6.4. Response surface analyses The response surface plots were constructed to facilitate the understanding of contribution of formulation variables and their interactions. For both LCT and MCT-SNEDDS, a curvilinear response surface plot was observed for Perm45 min . The plot (Fig. 2A) shows increase in Perm45 min till intermediate levels of Maisine 35-1, where after a sharp decline was observed. The plot (Fig. 2B) depicts a similar trend with Capryol 90. With Labrasol, Perm45 min showed marginal reduction from lower to higher levels at lower levels of Maisine 35-1. However, Tween 80 showed an increase in Perm45 min from lower to higher levels at lower levels of Capryol 90. A similar

pattern of permeation was reported employing the above constituents for both LCT and MCT-SNEDDS [38,39]. The response surface of Dnm is shown in Fig. 2C and D. For LCT-SNEDDS, a sharp linear decrease in globule size was observed from lower to higher levels of Maisine 35-1. On varying the concentrations of Labrasol, relatively little effect was observed on globule size, esp. at the high levels of Labrasol. However, with MCTSNEDDS, an opposite trend was observed viz. a linear decrease in globule size from lower to intermediate levels following which there was drastic increase in globule size with higher levels of Capryol 90. While, varying the concentrations of Tween 80, similar trend was observed as that of LCT-SNEDDS. The effect of above excipients in the formation of nano-sized globules is quite analogous to the earlier reports [40,41]. As illustrated in Fig. 2E, %DE30 min increased till intermediate levels of Maisine 35-1, where after a sharp decline was observed at lower levels of Labrasol. However, at higher levels of Labrasol, %DE30 min decreased till intermediate levels of Maisine 35-1, after which a sharp increase was observed thereby suggesting an interaction between the excipients. The plot Fig. 2F portrayed an increase in %DE30 min till intermediate levels of Capryol 90 and thereafter

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a decline at lower levels of Tween 80. Though, at higher levels of Tween 80, a linear increase in %DE30 min was observed.

out the plausibility of coalescence. As such, the results clearly shows that phase separation did not occur which indicate the formation of stable SNEDDS [43,44].

6.5. DoE validation and selection of optimum formulations 7.2. In vitro drug release comparison with marketed formulation Upon comparison of the observed responses with those of the anticipated ones, the prediction error varied between −3.29% and 3.18% with overall mean ± SD as −0.88 ± 1.691%. Linear correlation plots drawn between the predicted and observed responses after forcing the line through the origin, also demonstrated high values of r, ranging between 0.9786 and 0.9914.The corresponding residual plots show nearly uniform and random scatter around the zero axis. The optimum formulation was selected by “trading off” various response variables and adopting the following criteria: Perm45 min > 9.3 mg; Dnm < 70 nm; %DE30 min > 32.5% for LCT-SEDDS and Perm45 min > 9.5 mg; Dnm < 80 nm; %DE30 min > 32.3% for MCTSEDDS. Upon comprehensive evaluation of grid searches, OPT-LCT (i.e., Maisine 35-1: 286.0 mg and Labrasol: 500.0 mg) and OPT-MCT (i.e., Capryol 90: 420.0 mg and Tween 80: 800.0 mg) were found to be fulfilling maximal criteria for optimal performance. OPT-LCT exhibited Perm45 min of 9.59 mg, Dnm of 52.68 nm and %DE30 min of 36.36%, while the formulation OPT-MCT exhibited Perm45 min of 9.92 mg, Dnm of 67.75 nm and %DE30 min of 32.54%. Selection of the optimized formulation was based upon “trading off” of various response variables, i.e., maximization of Perm45 min (i.e., indicating drug transport potential across GI tract) and %DE30 min (i.e., indicating adequate drug release), and minimization of Dnm (i.e., necessary for micellar absorption via lymphatic route). 7. Formulation characterization of optimized formulations 7.1. Determination of globule size and zeta potential The globule size of the optimized SNEDDS was found to be was 54.07 nm and 65.88 nm for OPT-LCT and OPT-MCT, respectively. The nano globule size is considered ideal to result in lower emulsification time, enhanced absorption through lymphatics and subsequent augmentation in the therapeutic efficacy of drugs [2,42]. The zeta potential of the OPT-LCT and OPT-MCT was found to be −38.76 mV and −34.98 mV, respectively. High values of zeta potential construe an increase in electrostatic repulsive forces, thus ruling

Marked improvement was observed in the drug release profiles (Fig. 3) of the formulations OPT-LCT and OPT-MCT as compared to that of CD, MKT and pure drug. Drug dissolution was nearly completed within 30 min in case of the optimized formulations, as compared to that of the CD, MKT and pure drug, wherein drug release was only 67.04%, 40.77% and 27.36%, respectively. This significant reduction in the values of dissolution time for OPT-LCT and OPT-MCT is quite indicative of higher bioavailability potential of ezetimibe, a BCS class II drug exhibiting dissolution-limited absorption. Analogous in vitro dissolution profiles from OPT-LCT and OPT-MCT construe that the lipidic chain length does not seem to affect the drug release from the SNEDDS significantly. 7.3. Thermodynamic stability studies Thermodynamic stability indicates the kinetic stability of a formulation and is employed to study the chemical reaction occurring between the excipients of a formulation. The SNEDDS system undergoes spontaneous in situ solubilisation in the GI lumen to form a nanoemulsion system. As such, formulation should possess considerable stability in order to prevent precipitation, creaming or cracking. Both the optimized formulations were subjected to centrifugation studies and, heating and cooling cycle exposure. The OPT-LCT and OPT-MCT SNEDDS did not show any signs of precipitation, creaming or cracking thereby establishing the kinetic stability of both the systems. 7.4. Cloud point measurement The cloud point for OPT-LCT and OPT-MCT was observed to be 58–61 ◦ C and 79–81 ◦ C, respectively. Determination of cloud point is considered an important factor in the SNEDDS formulation consisting of nonionic emulgents. At temperatures higher than the cloud point, an irreversible phase separation occurs due to dehydration of its ingredients, which may affect drug absorption [45,46]. Hence, to avoid this phenomenon, the cloud point for the SNEDDS

Fig. 3. Plot showing mean percent ezetimibe release from optimized formulations, i.e., OPT-LCT and OPT-MCT, inclusion complex, marketed formulation and pure drug. The inset shows the mean percent drug release in 30 min. (n = 6 ± SD).

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Table 2A Effect of various treatment groups on percent reduction of plasma triglyceride levels in comparison with the control group.a Treatment groups (n = 6)

Days of hypolipidemic activity 7th day

OPT-LCT OPT-MCT HP-␤-CD MKT a b c d

3.19 2.84 1.86 3.43

± ± ± ±

14th day 5.66 6.92 9.46 7.78

5.84 5.11 2.89 5.75

± ± ± ±

21st day

6.52c 7.45c 8.41b 5.03c

20.73 18.90 10.23 16.14

± ± ± ±

28th day 5.77d 7.30d 16.90d 6.18d

53.67 48.67 18.28 24.49

± ± ± ±

7.22d 6.28d 8.83d 6.00d

Data represented as percent mean ± SD. P < 0.05. P < 0.01. P < 0.001.

Table 2B Effect of various treatment groups on percent enhancement of plasma HDL levels in comparison with the control group.a Treatment groups (n = 6)

Days of hypolipidemic activity 7th day

OPT-LCT OPT-MCT HP-␤-CD MKT a b c d

−7.58 1.08 2.37 −0.01

14th day

± ± ± ±

3.49 4.58 6.42 5.29

20.81 6.61 8.29 10.80

± ± ± ±

21st day 5.43c 4.85c 6.67 5.15b

39.47 19.53 15.58 21.37

± ± ± ±

28th day 4.14d 5.51d 6.79c 5.37d

46.59 37.70 21.52 26.27

± ± ± ±

5.01d 6.58d 5.08d 6.25d

Data represented as percent mean ± SD. P < 0.05. P < 0.01. P < 0.001.

formulations should be above body temperature (i.e., 37 ◦ C). Much higher magnitudes of the cloud points for both the SNEDDS formulations were found to indicate high stability of the SNEDDS formulations without any risk of phase separation.

7.5. In vivo pharmacodynamic studies Following ten days of continuous treatment with high fat diet, both the optimized SNEDDS formulations (i.e., OPT-LCT and OPTMCT) significantly modified the plasma TG, HDL and LDL levels as compared to that of the control group as well as MKT and CD. All the formulations initiated their effect on reduction in TG on 14th day but CD showed little effect as compared to other formulations. As illustrated in Table 2A, from 21st till 28th day post-treatment, all the formulations showed significant reduction (P < 0.001) in the TG levels. Both optimized SNEDDS formulations were able to significantly reduce the plasma TG levels on 28th day by 53.67% and 48.67%, respectively as compared to MKT and CD. In case of plasma HDL, only optimized SNEDDS formulations started showing improvement in the levels from 14th day onwards. Though MKT showed a little less significant effect on 14th day but CD did not show any effect on HDL. All the formulations showed considerable increase in HDL from 21st to 28th day (P < 0.001) except CD which showed an improved effect on 28th day, as showed in Table 2B. The optimized SNEDDS formulations were able to considerably increase the plasma HDL levels by 46.59% and 37.70%, respectively vis-à-vis MKT and CD.

The results of %reduction in LDL (Table 2C) are quite analogous with the above ones. Except CD all the formulations were able to slightly reduce the LDL levels on 14th day. However, from 21st till 28th day, all the formulations, except CD, showed significant %reduction (P < 0.001) in LDL. In case of CD, it showed an improved effect only on 28th day. Both the optimized SNEDDS formulations were able to markedly reduce the plasma LDL levels by 41.22% and 52.70%, vis-à-vis MKT and CD. Thus, the OPT-LCT and OPT-MCT of ezetimibe were much better in controlling the plasma TG, HDL and LDL levels in animals. Significant improvement in plasma lipid levels observed with OPTLCT may be due to the Labrasol present in the formulation, as it is reported to enhance the paracellular transport of drug [41]. Further, the lipidic excipient, i.e., Maisine 35-1, employed for LCT-SNEDDS formulation have been reported to surmount the hepatic firstpass effect facilitating the drug transportation through lymphatic system and ultimately enhanced drug oral bioavailability [47]. Although MCT-SNEDDS formulations favour direct portal transportation of drug, yet Capryol 90 based MCT’s have been reported to considerably modify the TG and LDL levels too [21,48]. The results obtained in the present studies on MCT-SNEDDS encompassing Capryol 90 in modifying the levels of TG and LDL, are thus in accordance with literature. In general, HP-␤-CD is known to enhance the solubility profile of a poorly water soluble drug, leading eventually to improved drug dissolution profile. Markedly superior hypolipidemic activities observed with SNEDDS vis-àvis CD thus unequivocally indicate that the mechanisms other than the solubility enhancement are underway in explaining high

Table 2C Effect of various treatment groups on percent reduction of plasma LDL levels in comparison with the control group.a Treatment groups (n = 6)

OPT-LCT OPT-MCT HP-␤-CD MKT a c d

Data represented as percent mean ± SD. P < 0.01. P < 0.001.

Days of hypolipidemia activity 7th day

14th day

± ± ± ±

± ± ± ±

1.91 0.26 0.83 1.59

4.33 5.53 6.44 6.23

6.62 20.89 5.11 6.57

21st day c

3.85 4.16c 6.44 5.23c

26.06 42.28 11.86 15.48

± ± ± ±

28th day d

3.85 4.94d 6.44c 6.23d

41.22 52.70 18.12 23.85

± ± ± ±

3.85d 5.26d 6.44d 5.23d

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pharmacodynamic potential of SNEDDS. Already the SNEDDS are known to improve the oral bioavailability of drug(s) by surmounting P-gp efflux, hepatic first pass effect, etc. Better performance of the optimized SNEDDS formulations could be attributed to increased solubility of drug, leading to fast and complete absorption of drug. The results from drug release studies of both the optimized formulations also indicate a similar pattern. As ezetimibe is reported not to affect the absorption of triglycerides, the enhanced drug permeation may have led to the reduction in variability in the bioavailability, resulting in pronounced effect on reduction in the TG and LDL and enhancement of HDL levels [49]. Hence, the distinct pharmacodynamic performance of optimized ezetimibe SNEDDS formulations could be ascribed to the combined effect of different mechanisms, like the presentation of drug in solubilized form, large interfacial area for absorption, enhanced dissolution in the presence of emulgents, and increased cellular uptake of drug due to better permeation and probably due to inhibition of cellular efflux systems [31,50]. The intraluminal processing of the triglycerides (i.e., digestion) is well established to depend upon the chain length of the lipids [2]. Owing to the higher chain length of LCT’s, the solubilisation capacity and intestinal absorption is significantly improved, thus leading to augmented lymphatic transportation. This phenomenon is responsible for reduction in hepatic first-pass metabolism, and ultimately augmentation of oral drug bioavailability. Marked modification in the plasma lipid levels observed during the current studies, can thus be ascribed to the improved oral bioavailability of ezetimibe by LCT’s, in close agreement with the reports on danazol and halofantrine [47,51]. Grove et al. [35], on the other hand, observed insignificant difference in the oral bioavailability of seocalcitol as a function of the chain length of lipids. 7.6. In situ intestinal perfusion studies Having observed significant bioavailability enhancement potential of SNEDDS, it was envisaged to investigate the underlying biological mechanism(s) of bioavailability enhancement. The in situ SPIP studies, in this regard, are known to provide tangible inkling on the absorption and permeation potential of a drug when administered as self-nanoemulsifying systems [3]. In the present studies, much higher magnitudes of absorptivity parameters were observed with OPT-LCT and OPT-MCT vis-à-vis pure drug and MKT, in consonance with the reports, documenting the ability of LCT’s in promoting the absorption of drugs via lymphatic pathways [2,52,53]. The dimensionless parameter, absorption number (An), provides an idea of the amount of drug transferred across the intestine and eventually affecting the absorption process of drug [54]. In the current study (Fig. 4), OPT-LCT and OPT-MCT significantly improved the magnitude of An by 7.55-fold and 5.27-fold, respectively, with reference to the pure drug. Nearly identical values of An for the marketed product and pure drug suggest analogous absorption mechanisms. Another dimensionless absorption parameter, fraction absorbed (Fa), provides an estimate of the extent of drug absorption [55]. As depicted in Fig. 4, the marked increase in the magnitude of Fa in case of OPT-LCT (6.65-fold) and OPT-MCT (i.e., 4.78-fold) vis-à-vis pure drug indicates high bioavalaiblity improvement potential of the SNEDDS. Not only the SPIP studies construe the absorptivity parameters of a drug from various delivery systems, but these also furnish a definitive insight on differential transport mechanisms across the animal intestine [56,57]. Efflux by P-gp transporters in the gut wall has been documented as the major cause for reduced oral bioavailability of ezetimibe [6]. Results of the SPIP studies, accordingly, were employed to investigate the role of intestinal P-gp during in vivo absorption too [58,59]. The effective permeability

Fig. 4. Plots depicting the increase in the values of absorptivity parameters (fraction absorbed and absorption number), and effective permeability of optimized formulations, i.e., OPT-LCT and OPT-MCT vis-à-vis inclusion complex, marketed formulation and pure drug using in situ SPIP technique.

(Peff ) provides a direct measurement of absorption rate across the intestinal epithelium [56]. As shown in Fig. 4, the Peff for pure drug and marketed drug product were found to be 0.386 × 10−4 cm s−1 and 0.482 × 10−4 cm s−1 , respectively. For OPT-LCT and OPT-MCT, however, the values were observed to be 1.647 × 10−4 cm s−1 and 1.346 × 10−4 cm s−1 , respectively. Nearly 4.27-fold and 3.49-fold enhancement in the values of Peff of ezetimibe was noticeable upon formulating it as SNEDDS. Thus, the results clearly demonstrate that the optimized SNEDDS were able to augment drug absorption in rat primarily by inhibiting the P-gp efflux. Already, considerable number of literature reports documents the role of SEDDS in P-gp inhibition of drugs like vinpocetine and sirolimus [52,60]. 7.7. Rheological characterization Viscosity is a crucial parameter in determining the ability of the SNEDDS formulation to be filled in hard gelatin capsules [61–63]. Too low values of viscosity indicate the plausibility of contaminating the area of overlap between the capsule body and cap thus preventing the capsule from being effectively sealed, whereas too high viscosity may lead to problems of pourability [61]. Further, the viscosity values are also known to provide an inkling on whether the system is w/o or o/w type [62]. There are reports indicating that SNEDDS having lower viscosity tend to form o/w type of nanoemulsion system and follow Newtonian-type of flow behaviour [63]. A linear correlation (Supplementary data 2A and B) was observed between shear rate and shear stress over a wide range of 0–100 s−1 and 0–10 Pa, respectively, unequivocally inferring Newtonian type of flow characteristics of both the SNEDDS formulation. The viscosities of the undiluted OPT-LCT and OPT-MCT formulations at ambient temperature were found to be ∼0.10 Pa s and 0.12 Pa s, respectively. As the values are within the desirable

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59

Table 3 Effect on the parameters during the accelerated stability studies kept at 40 ◦ C ± 2 ◦ C/75% RH ± 5% RH. Time (month)

Q15 min

OPT-LCT 0 1 3 4 6

Viscosity (cp)

92.53 92.08 91.73 92.65 90.76

96.91 96.83 95.2 96.26 95.10

OPT-MCT 0 1 3 4 6

94.29 94.01 93.55 92.79 93.86

113.50 112.87 112.51 111.67 112.11

Emulsification time (s)

% Transmittance

f2 values

0.60 0.54 0.46 0.51 0.41

99.50 99.20 98.70 99.00 98.40

– 99.56 98.57 99.12 97.46

10.20 9.76 9.44 8.91 9.21

99.70 99.40 98.70 98.30 98.50

– 99.77 98.16 97.38 97.79

and OPT-MCT. As is evident from the figures, all the globules were of uniform shape, with globule size of most of them as less than 100 nm. The figures clearly illustrate that there are no signs of coalescence, indicating thereby the enhanced physical stability of the formulations. 7.9. Accelerated stability studies Table 3 shows miniscule variation in the values of Q15 min , % transmittance, viscosity and emulsification time during six months of their storage. The values of the similarity factor (f2 ) ranged between 99.56 and 97.46 for OPT-LCT, and 99.77 and 97.79 for OPT-MCT, respectively, vouching high robustness of the formulations even under stressful conditions of temperature and humidity. In conclusion, the studies could be judiciously extrapolated to develop SNEDDS as nano colloidal carriers providing suitable platform technology(ies) for augmenting the oral bioavailability of other BCS class II and IV drugs, particularly those undergoing extensive hepatic first-pass effect, P-gp efflux and/or restricted permeation. Acknowledgments Authors are thankful to M/s Ranbaxy Research Labs, Gurgaon, India for providing the gift sample of Ezetimibe. We appreciate the generosity and cooperation of M/s Abitec, New Jersey, USA; M/s Gattefosse, San-Priest, France, and M/s Associated Capsules, Mumbai, India, for the procurement of excipients and gelatin shell, ex gratis supply, employed during the current studies. Financial grants obtained from the University Grants Commission, New Delhi, India are gratefully acknowledged. Declaration of interest There are no conflicts of interest. Fig. 5. TEM images showing particle shape and size of (A) OPT-LCT and (B) OPT-MCT.

limits of viscosity, i.e., 0.1–1.0 Pa s at the temperature of dosing, the developed SNEDDS formulation fulfils the requisite rheological attributes for being filled in the hard gelatin capsules [64]. The moderate magnitudes of viscosity also construe excellent pourability in the capsules with negligible potential of leakage from the filled capsules. The outcome of the current rheological studies is in close agreement with that of the liquid SEDDS of several drugs like xibornol, atorvastatin, griseofulvin, lovastatin, fenofibrate, adefovir and lercanidipine [44,65–70]. 7.8. Electron microscopic examination Fig. 5 portrays the electron microscopic images, depicting the morphology of the reconstituted optimized formulations, OPT-LCT

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