Solid supersaturatable self-nanoemulsifying drug delivery systems for improved dissolution, absorption and pharmacodynamic effects of glipizide

Journal of Drug Delivery Science and Technology 28 (2015) 28e36

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Journal of Drug Delivery Science and Technology journal homepage: www.elsevier.com/locate/jddst

Original research

Solid supersaturatable self-nanoemulsifying drug delivery systems for improved dissolution, absorption and pharmacodynamic effects of glipizide Rajendra Narayan Dash a, *, Habibuddin Mohammed b, Touseef Humaira b, Atla Venkateshwara Reddy c a Alliance Institute of Advanced Pharmaceutical & Health Sciences, Plot No.64, Survey No.145, Sardar Patel Nagar, Kukatpally, Hyderabad 500072, Telangana, India b Adept Pharma and Bioscience Excellence Private Limited, Corporate Office: 10-3-561/3/A/102, Vijayanagar Colony, Hyderabad 500057, Telangana, India c Anwarul Uloom College of Pharmacy, New Mallepally, Hyderabad 500001, Telangana, India

a r t i c l e i n f o

a b s t r a c t

Article history: Received 13 March 2015 Received in revised form 2 May 2015 Accepted 3 May 2015 Available online 4 May 2015

The objective of this study was to prepare a solid supersaturatable self-nanoemulsifying drug delivery system (solid S-SNEDDS) to improve the dissolution, absorption and pharmacodynamic effects of a poorly water-soluble drug: glipizide. The liquid supersaturatable formulation (liquid S-SNEDDS) was prepared by adding a polymeric precipitation inhibitor (HPMC-E5 at 5% w/w) to a liquid SNEDDS. Dilution of the liquid S-SNEDDS generated a nanoemulsion with a mean droplet size of 28.0 nm. The liquid S-SNEDDS was transformed into a free-flowing powder (solid S-SNEDDS) by adsorption onto calcium carbonate and talc. The solid S-SNEDDS generated a higher glipizide concentration in comparison with the solid SNEDDS (without HPMC-E5) during an in-vitro supersaturation test. Moreover, glipizide precipitated in an amorphous form from the solid S-SNEDDS. SEM studies of solid S-SNEDDS indicated the existence of molecularly dissolved glipizide. The solid S-SNEDDS was found to be stable during accelerated stability studies. In-vivo pharmacokinetic studies showed a significant (p < 0.001) increase in Cmax (3.4-fold) and AUC0e12h (2.7-fold) of glipizide from solid S-SNEDDS as compared with the pure drug. Solid S-SNEDDS showed a significant (p < 0.001) decrease in the plasma glucose level by 1.3, 1.3, and 2.9-fold as compared with solid SNEDDS, the commercially available drug product and the pure drug, respectively. © 2015 Elsevier B.V. All rights reserved.

Keywords: Solid S-SNEDDS Solid SNEDDS Supersaturation Pharmacokinetic parameters Plasma glucose level

1. Introduction Glipizide, a sulfonyl urea oral hypoglycemic agent, used to decrease the blood-glucose level in individuals with Type II diabetes mellitus. Glipizide lowers blood glucose by stimulating insulin release from the functioning pancreatic beta cells [1]. Glipizide is a weakly acidic drug (pKa ¼ 5.9), practically insoluble in water, and exhibits better solubility at basic pH [2]. Owing to its poor water solubility, several formulation approaches have been explored to improve the solubility of glipizide, including solid dispersion [3], nanosuspension [4], cyclodextrin complex [5e7], bionanocomposites [8], micro particles [9], co-solvent assisted

* Corresponding author. E-mail address: [email protected] (R.N. Dash). http://dx.doi.org/10.1016/j.jddst.2015.05.004 1773-2247/© 2015 Elsevier B.V. All rights reserved.

solubilization [10], self-emulsifying drug-delivery system [11], and solid self-nanoemulsifying drug-delivery system [12]. Self-nanoemulsifying drug-delivery system (SNEDDS) is the technological advances of the self-emulsifying drug-delivery system that attracted significant attention to improve oral bioavailability of lipophilic drugs [13]. SNEDDS is a transparent, thermodynamically stable, anhydrous isotropic mixture of oil, surfactant, co-surfactant, and drug that forms oil-in-water nanoemulsion (usually droplet size less than 200 nm) when exposed to the aqueous media upon gentle agitation or digestive motility of gastrointestinal (GI) tract [14,15]. The large surface area that arises from the nano-range droplet size facilitates pancreatic lipase to hydrolyze more effectively forming mixed micelles, which promotes solubilization of the lipophilic drug in the intestinal aqueous environment [16]. As a dosage form, SNEDDS offers distinctive advantages for enhanced drug absorption by various mechanisms

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such as (i) improving intra-luminal drug solubilization, (ii) inhibiting P-glycoprotein (P-gp) mediated drug efflux, (iii) enhancing lymphatic transport of drug, (iv) avoiding hepatic first-pass metabolism, and (v) increasing GI membrane permeability [16e18]. Additional benefits of SNEDDS include, reduction in inter- and intra-subject variability, quick onset of action, reduction in drug's dose and ease of manufacturing along with scale-up [14]. However, low drug loading and risk of drug precipitation following dilution, neutralizes the advantage of formulating lipophilic drug candidate as SNEDDS [19]. In-vivo drug precipitation might pose a risk for drug absorption. To overcome such difficulties there has been an increasing focus on the application of supersaturatable SNEDDS (S-SNEDDS) [20,21]. S-SNEDDS contains a water-soluble polymeric precipitation inhibitor (PPI) in addition to the typical composition of SNEDDS. The PPI retards excessive drug precipitation following dilution and maintain a temporary supersaturated state [22]. Consequently, the generation and stabilization of intraluminal supersaturation can provide an efficient solution for the oral bioavailability enhancement of lipophilic drugs [23]. Further, it is important to have these liquid supersaturatable formulations as a solid dosage form having higher stability, better transportability, simple and cost effective manufacturing, and improved therapeutic success owing to the better patient compliance [12,24,25]. Thus, the present research work aims at developing a stable, solid S-SNEDDS of glipizide that could generate a supersaturated state by retarding the precipitation of solubilized drug. Simultaneously, it was hypothesized that the developed formulation would generate a nanoemulsion upon dilution, thereby providing a larger interfacial area for enhanced drug solubilization and dissolution. Hence, this combined approach would enhance the oral bioavailability and glucose lowering efficacy of glipizide due to its precipitation resistant as well as nanosized nature.

validated in-house. The method was linear (r2 ¼ 0.999) over the concentration range of 0.05e70 mg/ml. The relative standard deviations for inter-day and intra-day precision were less than 2%.

2. Material and methods

2.5. Preparation of solid S-SNEDDS

2.1. Materials

Adsorption studies were carried out to prepare solid S-SNEDDS with an extensively employed porous adsorbent such as calcium carbonate [26]. Briefly, 10 g of each liquid S-SNEDDS was poured onto calcium carbonate (15 g) placed in a mortar, mixed for 5 min to obtain a homogenous mass. Talc (2 g) (used as a lubricant) was added to the above mass, mixed gently, and passed through a mesh (250-mm). Correspondingly, solid SNEDDS was prepared by adsorbing the liquid SNEDDS onto above-mentioned excipients. Respective blank formulations of solid S-SNEDDS and solid SNEDDS were prepared using above excipients in the same proportion but without using glipizide. The drug contents in each formulation were determined by HPLC. Briefly, 100 mg of solid S-SNEDDS or solid SNEDDS were transferred to 10-ml volumetric flasks containing methanol (HPLC) and sonicated for 10 min to solubilize glipizide. The resulting solutions were filtered through a 0.22-mm nylon filter. The filtrate (2 ml) was diluted to 10 ml with mobile phase, mixed and injected six times into the HPLC system. Similarly, blank injections were made in the same way by using blank formulations. Powder of solid S-SNEDDS or solid SNEDDS equivalent to 5 mg of glipizide were filled into size “1” hard gelatin capsules (Capsugel, Mumbai, India) and stored in glass bottles at 25  C until used for the subsequent studies.

Pharmaceutical grade of glipizide was a generous gift from Alembic Ltd. (Vadodara, India). Medium chain tri-glycerides (Captex 355®) was supplied by Abitec Corp. (Janesville, USA). Poly-glycol mono and di-esters of 12-hydroxy stearic acid (Solutol HS15®) was provided by BASF SE (Ludwigshafen, Germany). Medium chain mono glycerides (Imwitor 988®) was obtained from Sasol (GmbH Germany). Five centipoise viscosity grade hydroxypropyl methylcellulose (HPMC-E5) was provided by Colorcon Asia Ltd. (Mumbai, India). Size “1” hard gelatin capsules were kindly gifted by Capsugel Health Care Ltd. (Mumbai, India). Acetonitrile (HPLC), Methanol (HPLC), Potassium dihydrogen phosphate (Chromatography), Hydrochloric acid (AR), and Sodium chloride (AR) were purchased from Merck Specialties Pvt. Ltd. (Mumbai, India). 18 MU Water (HPLC grade) was obtained in-house from a Direct Q-3 UV water purification system (Millipore India Pvt. Ltd., Bengaluru, India). Drug-excipients compatibility studies were carried out (data not shown) before selecting the excipients for subsequent studies. 2.2. Analytical methodology Chromatographic estimation of glipizide was achieved on a HPLC system (series 200, Perkin Elmer, USA) at a temperature of 30 ± 2  C. The analytical column used was Luna C8, 100  4.6 mm, 3 mm (Phenomenex, CA, USA). The mobile phase was a mixture of acetonitrile and potassium dihydrogen phosphate buffer (pH 4.5; 20 mM) (35:65 v/v). The injection volume, mobile phase flow rate, and detection wavelength were selected as 20 ml, 0.8 ml/min, and 226 nm, respectively. The method was stability-indicating and was

2.3. Preparation of liquid S-SNEDDS For the present study, a previously developed and characterized liquid SNEDDS was used for the preparation of liquid S-SNEDDS [12]. The liquid SNEDDS was an isotropic mixture of glipizide, and SNEDDS preconcentrate [Captex 355: Solutol HS15: Imwitor 988 (30:45:25% w/w)]. The concentration of glipizide in liquid SNEDDS was 4% w/v. For preparing liquid S-SNEDDS, variable amounts (0.5, 1, 3, 5, 7.5, and 10% w/w) of PPI (HPMC-E5) were added to a series of liquid SNEDDS (10 g) kept in dust-free glass vials. The mixtures were mixed for five minutes using a Cyclo-mixer (CM101, Remi, Mumbai, India) to obtain uniform suspensions.

2.4. Characterization of liquid S-SNEDDS 2.4.1. Measurement of droplet size and zeta potential Liquid S-SNEDDS (0.1 g) or liquid SNEDDS (0.1 g) were diluted to 50 ml with water (HPLC) in volumetric flasks and were gently mixed by inverting the flask. The flasks were allowed to stand for 12 h at room temperature [24]. The droplet size and zeta potential of the diluted liquid S-SNEDDS and diluted liquid SNEDDS were measured using dynamic light scattering techniques (at a 90 scattering angle). Measurement was done at 25  C using a Zeta potential/Particle sizer (Nanopartica SZ100, Horiba instrument, UK).

2.6. Characterization of solid S-SNEDDS 2.6.1. Micromeritic properties The micromeritic properties of solid S-SNEDDS were evaluated in terms of angle of repose, Carr's index and Hausner's ratio [27].

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2.6.2. In vitro supersaturation test and selection of PPI concentration In-vitro supersaturation tests were carried out on the final product (solid S-SNEDDS) with varying percentages of HPMC-E5 as described by Wei et al., 2012 [28]. A simulated gastric fluid containing 0.01 M HCl and 0.15 M NaCl (pH 2.0) was chosen as the invitro medium. The contents of solid SNEDDS capsule and solid SSNEDDS capsules (with varying amount of HPMC-E5) were placed in 100 ml of the test medium, maintained at 37  C, and stirred continuously at 100 rpm on a magnetic stirrer (2MLH, Remi, Mumbai, India). Samples (500 ml) were withdrawn without volume replacement at a particular time interval (2, 5, 15, 30, 60, and 120 min). The samples were filtered through a 0.45 mm filter and first 300 ml was discarded before collecting 200 ml filtrate. 100 ml of the collected filtrates were diluted up to 1 ml with the mobile phase and analyzed for glipizide content by HPLC. A control experiment was carried out using blank solid SNEDDS capsule (without HPMCE5 and glipizide). All experiments were performed in triplicates. The precipitates were collected (after 24 h of in-vitro supersaturation test) from the tested formulations and were subjected to the powder XRD measurement as mentioned in section 2.6.3. 2.6.3. Powder XRD studies Powder X-ray diffraction of glipizide, blank precipitates (collected from the control experiment), precipitates obtained from in-vitro supersaturation tests, physical mixtures (blank precipitates spiked with corresponding amount of glipizide present in the solid S-SNEDDS), and individual excipients (e.g. calcium carbonate, talc, and HPMC-E5) were evaluated by an X-ray diffractometer (D8 Advanced, Bruker AXS, Germany). The instrument uses a CuKa radiation generated at 40 kV with 40 mA current and operated over a 2q angle ranging from 6 to 50 at a step size of 0.1. 2.6.4. Scanning electron microscopy (SEM) Surface characteristics of glipizide, solid S-SNEDDS, solid SSNEDDS blank, and physical mixtures of glipizide with solid SSNEDDS blank were observed by a scanning electron microscope (S-4100, Hitachi, Japan). The photographs were taken at an accelerating voltage of 15 kV. 2.7. Comparative in vitro drug release studies In-vitro drug release studies of solid S-SNEDDS, solid SNEDDS, commercial tablet (Glucotrol®, Pfizer Inc. USA) and pure drug each containing 5 mg of glipizide were performed in 900 ml of pH 6.8 phosphate buffer. The in-vitro release studies were conducted at 37 ± 0.5  C using a USP dissolution type-I apparatus (TDT-08 L, Electrolab, Mumbai, India). The rotation of the baskets was adjusted to 75 rpm. An aliquot (five ml) of the samples was collected at predetermined time intervals and replaced by a fresh dissolution medium. The drug contents in each sample were determined by HPLC after diluting with the mobile phase. 2.8. In vivo pharmacokinetic studies in rabbit Himalayan rabbits (male) weighing between 1.25 and 1.50 kg were used for the pharmacokinetic studies. The animals were housed in separate cages, fed on standard animal chow with a free access to drinking water. Animal study was approved (Protocol Number: 1534/PO/A/11/CPCSEA/15) by the Institutional Ethical Animal Committee. The animals were fasted 12 h prior to the drug administration, but had free access to water. The animals were divided among four groups containing six animals per group. Each group received one of the following: solid S-SNEDDS, solid SNEDDS, Glucotrol®, and pure drug. The formulations and the pure drug (as a

suspension in 0.5% w/v carboxy methyl cellulose) were administrated (1 mg/kg body weight of the animal) orally with gastric intubation and subsequently flushed with 10 ml water. The D90 and D50 values of pure drug suspension were 25 mm and 15 mm respectively. Blood samples (2 ml) were collected from the marginal ear vein at time zero (pre-dosing) and 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 4.0, 6.0, 9.0, 12, and 24 h of post-dosing and transferred to 2-ml eppendorf tubes containing sodium citrate as an anticoagulant. Blood samples were centrifuged at 6000 rpm for 15 min (RM-12C, Remi, Mumbai, India) and the supernatant plasma were transferred to 1.5-ml eppendorf tube and kept at 20  C until analyzed. For determining glipizide concentration in rabbit plasma, an aliquot of plasma (200 ml) was added to 1.5-ml eppendorf tube containing acetonitrile (600 ml) and internal standard (10 mg/ml glimepiride in methanol) (200 ml), and mixed for 5 min using a Cyclo-mixer (CM101, Remi, Mumbai, India). The eppendorf tubes were centrifuged at 6000 rpm for 10 min (RM-12C, Remi, Mumbai, India). The collected supernatants were injected into the HPLC system as described under section 2.2 but using a different composition and flow rate of mobile phase. The mobile phase consisted of acetonitrile: potassium dihydrogen phosphate buffer (pH 3.5; 20 mM) (45:55, v/v). The flow rate of the mobile phase was kept at 0.8 ml/min. The method was linear (r2 ¼ 0.999) over the concentration range of 25e2000 ng/ml. Pharmacokinetic analysis was performed by non-compartmental analysis using PK solver (Version 2.0, an add-in program for Microsoft Excel). The plasma concentration-time curve from time (t ¼ 0) to the last measurable plasma concentration at a time “t” (AUC0-t) was measured by the linear trapezoidal rule. Rest parameters such as maximum plasma concentration (Cmax), and the time at which Cmax observed (tmax) were determined from the plasma concentration-time profile data. 2.9. Plasma glucose analysis The glucose concentrations in the collected plasma (section 2.8) were determined using a semi auto-analyzer (Optimas-Labindia, Mumbai, India). The data for each animal was expressed as the percentage decrease in plasma glucose level (PGL) at a time (t ¼ t) with respect to the initial plasma glucose level at the time (t ¼ 0) (Eq. (1)). Hence, each animal served as their own control.

% Decrease in PGL ¼

PGLðt ¼ 0Þ  PGLðt ¼ tÞ  100 PGLðt ¼ 0Þ

(1)

The area under the percentage decrease in PGL-time curve (AUC0e12h) was calculated using the linear trapezoidal rule. 2.10. Accelerated stability studies of solid S-SNEDDS Capsules are very sensitive to moisture, hence should be well packed in an impermeable container to protect it from moisture. Sensitivity to moisture or potential for solvent loss is not a concern for drug products packaged in impermeable containers that provide a permanent barrier to the passage of moisture or solvent. Thus, stability studies for products stored in impermeable containers can be conducted under any controlled or ambient humidity condition [29]. Three batches of solid S-SNEDDS capsules enclosed in glass bottles, sealed with aluminum caps were stored at 40 ± 2  C/75 ± 5% RH in a stability chamber (TH325S, Thermo lab, Mumbai, India). The stability was evaluated by monitoring timedependent changes (at 0, 3, and 6 months) of solid S-SNEDDS such as changes in appearance, mean droplet size after emulsification (nm), the percentage of drug released in 15 min (DR15min), similarity factor in dissolution profiles (f2), and drug content (by HPLC). For determining mean droplet size, contents of one capsule

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from each batch were reconstituted in 50 ml of water (HPLC) and treated as per the procedure mentioned under section 2.4.1. 2.11. Statistical analysis The pharmacokinetics parameters were compared by ANOVA followed by Tukey post-hoc test using Sigma-Plot® (Version.12, Systat software Inc., CA, USA). P value <0.05 considered to be significant. 3. Results and discussion 3.1. Characterization of liquid S-SNEDDS 3.1.1. Droplet size analysis and zeta potential analysis Diluted liquid S-SNEDDS exhibited mean droplet size within 27.2e35.4 nm. The mean droplet size of the diluted liquid SSNEDDS (containing 5% w/w HPMC-E5) was found to be 28 nm, and was smaller than the mean droplet size of diluted SNEDDS preconcentrate (35.6 nm) or diluted liquid SNEDDS (35.7 nm). This might be due to the presence of HPMC-E5 (in liquid S-SNEDDS) that forms a physical barrier around oil droplets, and prevented aggregation of droplets [28]. Further, these small droplets could provide a large interfacial area for pancreatic lipase to hydrolyze triglycerides and consequently, forming mixed micelles that promotes drug solubilization and absorption [16]. Zeta potentials of diluted liquid S-SNEDDS were found to be ranging from 35.0 mV to 35.8 mV, while the zeta potential of diluted liquid SNEDDS was found to be 35.0 mV. The negative value of zeta potential might be due to the presence of anionic groups of free fatty acids, and glycols present in the oil, surfactant and co-surfactant [30]. This in turn implies that the formulations were negatively charged, and sufficient repulsion exists among emulsion droplets to form an uncoagulated system, which is an indication of a kinetically stable system [31]. 3.2. Characterization of solid S-SNEDDS 3.2.1. Micromeritic properties Solid S-SNEDDS showed a good flow property having acceptable Carr's index (17.8e19.3), Hausner's ratio (1.21e1.24), and angle of repose (27e29 ). The results for Carr's index and Hausner's ratio were found to be in good agreement with each other. The results indicated that the solid S-SNEDDS displayed a good flow characteristics and could be filled into the hard gelatin capsule as a unit dosage form. 3.2.2. In vitro supersaturation test and selection of PPI concentration In-vitro supersaturation tests were designed to find out the apparent glipizide concentration following dilution of solid SSNEDDS. The total volume of the medium selected was 100 ml. This volume is equivalent to the combined volume of the residual stomach fluid (20e50 ml) in a fasted state, plus the amount of water (30e60 ml) administered during the clinical study that can provide a non sink condition for glipizide [32]. The total amount of glipizide in the media can be the summation of three states (1) free molecules in solution, (2) solubilized molecules partitioned into the nanoemulsion and (3) precipitated molecules as solid particles [33]. As the transfer of glipizide between state 1 and 2 was dynamic that rapidly changes over time, it was difficult to measure glipizide concentration separately as free molecules (state 1) or molecules partitioned into nanoemulsion (state 2) at a particular time. The use of 0.45 mm filter to process test samples was intended to exclude the precipitated glipizide (state 3), while allowing nanoemulsion

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globules and free glipizide molecules. Consequently, the measured glipizide concentration was the combination of state 1 and 2 rather than free glipizide (state 1) alone. The apparent concentration-time profile of glipizide is presented in Fig. 1a. It can be seen that the initial glipizide concentration (~50 mg/ml) dropped down rapidly to less than 40 mg/ml within the first 5 min from the solid S-SNEDDS containing 0e1% w/w of HPMC-E5. This may be due to the loss of solvent capacity of the formulation upon dilution [34]. Moreover, the precipitation augmented due to a weak acidic drug such as glipizide being exposed to the acidic pH (2.0) of the tested media. However, glipizide precipitation slowed down in the presence of HPMC-E5 (3% w/w), with a maximum retardation at 5% w/w of HPMC-E5. Further increase in HPMC-E5 concentration (>5% w/w) had an insignificant effect on concentration-time profile of glipizide. Accordingly, at 5% w/w level, HPMC-E5 was incorporated within the SNEDDS preconcentrate. It has been confirmed that, PPI such as HPMC at a small concentration, retards excessive drug precipitation, thus temporarily maintaining a supersaturated state [21,22,28,33,35]. As suggested by Gao et al., 2009 [22], the mechanism behind this phenomenon might be due to the adsorption of hydrophobic HPMC chain onto the molecular surface to forms a mechanical barrier for nucleation as well as crystal growth that required for crystallization process. This in turn inhibits precipitation of the drug out of solution. Further, this proposal supported the comparative precipitation inhibition efficiency among different series of HPMC polymers, where a hydrophobic E-type (29% methyl substitution) was found to be better PPI than their less hydrophobic K-type counterpart (22% methyl substitution). In contrast, a hydrophilic polymer like polyvinyl pyrrolidone remained an inactive PPI [22]. Moreover, among E-type HPMC polymers, the effectiveness of a lower viscous HPMC-E5 (5 mPa s) was found to be superior as compared with the higher viscous E-type HPMC polymers such as HPMC-E15 (15 mPa s), HPMC-E50 (50 mPa s), and HPMC-E4 M (4000 mPa s) [21,22]. 3.2.3. Powder XRD studies The crystalline structure of glipizide can be evident in its PXRD pattern (Fig. 2a), where glipizide exhibited several sharp peaks. This PXRD pattern of glipizide was found to be in line with the earlier report of Huang et al., 2013 [6]. The sharp diffraction peaks due to the glipizide and each excipient such as calcium carbonate (Fig. 2f) and talc (Fig. 2g) can be seen in the physical mixture (Fig. 2b). However, HPMC-E5 (Fig. 2h) showed a diffused spectrum having no prominent peaks. Calcium carbonate and talc showed several sharp peaks that appeared in the physical mixture (Fig. 2b), solid SNEDDS precipitates (Fig. 2c), solid S-SNEDDS precipitates (Fig. 2d), and blank precipitates (Fig. 2e). In case of precipitates obtained from solid S-SNEDDS and solid SNEDDS, the major peaks of glipizide were found to be of reduced intensity along with a complete absence of peaks at 18.0 and 18.7, indicating a reduction in crystalline property of glipizide. However, the precipitates obtained from the solid S-SNEDDS containing 5% w/w HPMC-E5 (Fig. 2d) was completely amorphous lacking major peaks of glipizide at 7.4 , 11.0 , and 15.7 in contrast to the partially amorphous precipitates obtained from the solid SNEDDS (Fig. 2c). This indicates that glipizide was precipitated in a complete amorphous state from the solid S-SNEDDS containing HPMC-E5 at 5% w/w level. 3.2.4. Scanning electron microscopy The scanning electron micrograph of glipizide appeared as smooth surfaced rectangular crystals (Fig. 3a). These glipizide crystals were noticed in the physical mixture (Fig. 3d). Whereas glipizide crystals were absent in the solid S-SNEDDS (Fig. 3b), indicating its incorporation into the matrix of solid S-SNEDDS. Further, solid S-SNEDDS surface was found to be in close

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Fig. 1. (a) Apparent glipizide concentration-time profile obtained from different concentration of HPMC-E5 during in-vitro supersaturation test; (b) In-vitro release profiles for the solid S-SNEDDS, solid SNEDDS, Glucotrol®, and glipizide (pure drug).

Fig. 2. Powdered XRD diffractogram of a, glipizide; b, physical mixture; c, precipitates obtained from solid SNEDDS; d, precipitates obtained from solid S-SNEDDS (5% w/w HPMCE5); e, blank precipitates obtained from control experiment; f, calcium carbonate; g, talc and h, HPMC-E5.

resemblance with the agglomerated surface of solid S-SNEDDS blank (Fig. 3c). This suggests that the glipizide was absorbed and dissolved in a molecular level within solid S-SNEDDS excipient's matrix. 3.3. Comparative in vitro drug release studies Comparative release studies indicated that there was significant (p < 0.001) release of glipizide from the solid S-SNEDDS

-vis solid SNEDDS (89.30%), Glucotrol® (DR15min ¼ 98.41%) vis-a (61.32%) and the pure drug (22.60%) (Fig. 1b). This enhancement of DR15min (>85%) in case of solid S-SNEDDS and solid SNEDDS were higher as compared with DR15min values of Glucotrol® and pure drug. Further, it indicates that self-emulsifying systems needs less free energy to form an emulsion due to the spontaneous formation of the interface between oil droplets and water. This in turn decreases the droplet size leading to immediate solubilization of drug in the dissolution medium [24]. Initially, 15e25% higher drug

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Fig. 3. Scanning electron microscope images of a, glipizide; b, solid S-SNEDDS; c, solid S-SNEDDS blank; d, physical mixture.

release was observed from the solid S-SNEDDS as compared with the solid SNEDDS. This might be due to the presence of HPMC-E5 that resisted the excessive precipitation of glipizide leading to higher apparent glipizide concentration. Some studies that did not use any type of PPI found that the drug precipitates (obtained during the in-vitro test) to be amorphous, suggesting re-dissolution of the precipitates during in-vitro release [36,37]. Though the mechanism behind this formation of amorphous precipitates is still not understood and opens up an area for further studies. Moreover, in the present study, the precipitates containing PPI (HPMC-E5 at 5% w/w level) were found to be completely amorphous than the partially amorphous precipitates obtained in an absence of PPI. Amorphous forms have higher internal energy and molecular motion over crystalline forms. This in turn requires less energy to dissolve leading to an increased dissolution rate [38]. As evident from the SEM results, as the drug is molecularly dissolved within the matrix of solid S-SNEDDS, a large surface area might have generated to improve the wettability and in-vitro release of drug [26]. The micro-porous surface of the solid S-SNEDDS matrix might have created channels for infiltration of dissolution media to facilitate dispersion of nanoemulsion [25].

of solid S-SNEDDS when compared with Glucotrol® (p < 0.05), and the pure drug (p < 0.001). However, insignificant difference was observed between tmax of solid S-SNEDDS and solid SNEDDS. The decrease in tmax was consistent with the differences in the in-vitro release pattern of different groups. Solid S-SNEDDS exhibited a rapid dissolution pattern, thus ensured a lowest tmax for glipizide. The above results suggest that both solid S-SNEDDS and solid SNEDDS exhibited better pharmacokinetic profiles than Glucotrol® (immediate release, uncoated tablet) and pure drug. This might have contributed to the marked increase in the absorption rate of glipizide due to increased rate of dissolution from the SNEDDS formulations (solid S-SNEDDS and solid SNEDDS) than the pure drug and Glucotrol®. Further, this may be explained by the spontaneous dispersion of solid S-SNEDDS and solid SNEDDS in the GI fluid to form a nanoemulsion, where the active components are present in a solubilized state (i.e. free molecule incorporated into the micelles or nanoemulsion droplets). In addition, the small droplet size contributes a large surface area for enhanced drug absorption [16,39]. Moreover, the better pharmacokinetic parameters that observed with solid S-SNEDDS in comparison with solid SNEDDS might be due to the higher glipizide concentration in GI lumen, resulting from the supersaturated state.

3.4. In vivo pharmacokinetic studies in rabbit 3.5. Plasma glucose analysis Though the study conducted up to 24 h, it was not possible to quantify glipizide at 24 h, due to the existence of glipizide below the LOQ level (25 ng/ml). The overlay chromatograms obtained at different time points from the solid S-SNEDDS administered group are shown in supplementary figure (Fig. S1). The mean glipizide plasma concentrationetime profiles of the tested groups are shown in Fig. 4a. The corresponding pharmacokinetic parameters are listed in Table 1. The results clearly depict that a significant increase in AUC0e12h was observed in case of solid S-SNEDDS as compared to solid SNEDDS (p < 0.05), Glucotrol® (p < 0.001), and pure drug (p < 0.001). The Cmax of solid S-SNEDDS was found to be significantly (p < 0.001) higher than that of solid SNEDDS, Glucotrol®, and pure drug. The tmax was found to be significantly shorter in the case

The observed plasma glucose concentrations of the individual group are shown in supplementary figure (Fig. S2). The percentage decrease in PGL versus time is shown in Fig. 4b. The mean AUC0e12h (percentage decrease in PGL. h) for solid S-SNEDDS was found to be 302.70, which was significantly (p < 0.001) higher than the AUC0e12h of solid SNEDDS (240.66), Glucotrol® (226.30), and pure drug (105.09). These results from the plasma glucose analysis were consistent with the in-vivo absorption pattern of the individual groups. AUC0e12h, a measure of therapeutic efficacy increased significantly (p < 0.001) by 1.3, 1.3, and 2.9-fold in the case of solid S-SNEDDS as compared to the solid SNEDDS, Glucotrol®, and the pure drug, respectively. The performance rank order of the studied

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Fig. 4. (a) Plasma concentration-time profile of glipizide in fasted rabbits after oral administration of glipizide, Glucotrol®, solid SNEDDS and solid S-SNEDDS; (b) The mean percentage decrease in plasma glucose in normal rabbit after oral administration of glipizide, Glucotrol®, solid SNEDDS and solid S-SNEDDS.

Table 1 Observed and predicted pharmacokinetic parameters for glipizide after oral administration of pure drug, Glucotrol®, solid-SNEDDS and solid S-SNEDDS to rabbits (n ¼ 6). Group Glipizide (Pure drug) Glucotrol®b Solid-SNEDDSc Solid S-SNEDDSd

AUC0e12h (ng h/ml) a

2957 6596 7272 7930

± ± ± ±

222 421a*** 582a*** 546a***,b***,c*

Cmax (ng/ml)

tmax (h)

562 ± 27 1399 ± 54a*** 1598 ± 51a*** 1925 ± 83a***,b***,c***

2.41 1.83 1.50 1.08

± ± ± ±

0.38 0.41 0.32a** 0.49a***,b*

Values are presented as Mean ± SD (n ¼ 6). Values are compared with ANOVA followed by Tukey post hoc test; significant at *P < 0.05; **P < 0.01; ***P < 0.001. a ¼Glipizide (Pure drug). b ¼Glucotrol ®. c ¼Solid SNEDDS. d ¼Solid S-SNEDDS.

groups in terms of in-vitro release, in-vivo absorption, and therapeutic efficacy can be illustrated as solid S-SNEDDS > solid SNEDDS > Glucotrol® > pure drug. Solid SNEDDS (without HPMC-E5) witnessed an enhanced invitro release (4.0-fold in DR15min), in-vivo absorption (2.8-fold in Cmax, 2.5-fold in AUC0e24h), and therapeutic activity (2.3-fold) of glipizide as compared with the pure drug. This might be attributed to the multi-process mechanism of SNEDDS, both physical and biological: (a) higher solubilization of drug in the GI milieu [16,32], (b) enhanced drug absorption by lymphatic transport [17,18], (c) enhanced mucosal permeability due to disruption of the lipid bilayer by surfactant [24], (d) enhanced drug absorption through P-gp inhibitory effect of the employed surfactant (Solutol-HS15) [40], and (e) reduction in cytochrome P-450 mediated metabolism [18,25]. Moreover, the in-vitro release, in-vivo absorption, and therapeutic efficacy of solid S-SNEDDS were found to be significantly (p < 0.05) higher than the solid SNEDDS. Hence, it can be concluded that the overall improved performance of solid SSNEDDS lies in its precipitation resistance nature in addition to the aforementioned classical property of SNEDDS formulations. This in turn might have led to the availability of higher free drug concentration in GI lumen, resulting from the supersaturated state

as confirmed from the in-vitro supersaturation test, where microscopic precipitation of glipizide was effectively suppressed [28]. In addition, glipizide precipitated in an amorphous state from the solid S-SNEDDS, which can be the driving force for its enhanced dissolution, absorption, and therapeutic activity. The drug precipitation involves two stages: nucleation and crystal growth. Several mechanisms have been put forth for HPMC induced precipitation inhibition [41]. Formation of hydrogen bond between HPMC and drug molecule that increases activation energy for nucleation, thus delaying nucleation was observed by Raghavan et al., 2001 [42]. For this criterion, the molecule must have an electronegative atom or groups containing the electronegative atoms to form hydrogen bonds with the hydroxyl group of HPMC. Hydrophobic interaction between HPMC and drug that inhibit nucleation as well as crystal growth was proposed by Gao et al., 2009 [22]. As per this proposal, the polymer gets adsorbed onto the hydrophobic molecular surfaces and act as a barrier for the nucleation process, subsequently inhibiting the formation and growth rate of crystals. A combination of hydrophobic and steric hindrance effect of high molecular weight HPMC (HPMC acetate succinate) upon enhancement and stabilization of itraconazole concentration was observed by DiNunzio et al., 2010 [43]. The

R.N. Dash et al. / Journal of Drug Delivery Science and Technology 28 (2015) 28e36

35

Table 2 Data obtained from the accelerated stability studies (at 40 ± 2  C/75 ± 5% RH) of solid S-SNEDDS. Time (months)

Assay (%)a

DR15min (%)a,b

Mean droplet size (nm)a

Similarity factor (f2)a

0 3 6

100.02 ± 0.39 97.78 ± 0.31 95.63 ± 0.46

98.42 ± 0.97 98.65 ± 0.86 99.32 ± 1.23

28.00 ± 0.11 29.21 ± 0.28 29.82 ± 0.14

e 92.22 ± 4.58 90.27 ± 2.97

a b

Data presented in Mean ± SD, n ¼ 3. Percentage of drug released at 15 min.

increase in solution viscosity due to incorporation of polymer may affect molecular mobility, thus is expected to inhibit crystallization. However, there are no such consistent results available to support the role of solution viscosity on precipitation inhibition [41], as this phenomenon is most likely due to the direct interference between the polymer and molecular nucleation and/or crystal growth process [23]. The exact mechanism of HPMC induced precipitation inhibition and formation of amorphous nuclei are still not understood and remained a challenging area. Glipizide being a hydrophobic drug and having electronegative atoms (O, N) within one sulfonyl, two carbonyl, and three amide groups (Fig. S3); both mechanisms such as hydrophobic interaction and hydrogen bond formation are proposed in this study. The formation of polar hydrogen bonds might have increased the solubility of glipizide during the in-vitro supersaturation test that generated a higher glipizide concentration. However, additional investigations are required in this direction to understand the mechanism behind the precipitation as well as the crystallization inhibiting effect of HPMC-E5. 3.6. Accelerated stability studies of solid S-SNEDDS The chromatograms obtained from the stability studies are shown in supplementary figure (Fig. S4). During accelerated stability studies, insignificant changes in mean droplet size and DR15min were observed over the period of six-month (Table 2). As per ICH Q1A (R2) guidelines, a significant change in drug content from the initial value (>5%) did not observe. The calculated f2 values (at three and six months) were found to be within the accepted limit (f2  50). The results indicated that the mean droplet size of the diluted solid S-SNEDDS remain unaffected by the solidification process of liquid S-SNEDDS. Therefore, the selfemulsification ability of liquid S-SNEDDS was preserved within the solid S-SNEDDS. Solid S-SNEDDS was found to be stable under the accelerated conditions, and glipizide remained chemically stable within it. 4. Conclusion In the present study, the generation of nanoemulsion following dilution of solid S-SNEDDS resulted in a large surface area for enhanced drug solubilization and dissolution. As indicated from invitro supersaturation test, the incorporation of HPMC-E5 into solid S-SNEDDS effectively inhibited drug precipitation and consequently, maintained a supersaturated state of glipizide at least for 60 min. As confirmed from the PXRD studies, solid S-SNEDDS precipitated glipizide in an absolute amorphous state, implying the presence of an optimal amount of HPMC-E5 effectively inhibited the nucleation process and subsequent crystallization of glipizide. Hence, this stable supersaturatable formulation substantially improved in-vitro release, in-vivo absorption and plasma glucose lowering efficacy of glipizide due to its nanosized and precipitation resistant nature, thus may offer a useful oral dosage form option for oral delivery of glipizide.

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