http://informahealthcare.com/drd ISSN: 1071-7544 (print), 1521-0464 (electronic) Drug Deliv, Early Online: 1–10 ! 2014 Informa Healthcare USA, Inc. DOI: 10.3109/10717544.2014.914986

RESEARCH ARTICLE

Candesartan cilexetil loaded solid lipid nanoparticles for oral delivery: characterization, pharmacokinetic and pharmacodynamic evaluation Narendar Dudhipala and Kishan Veerabrahma

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Department of Nanotechnology, University College of Pharmaceutical Sciences, Kakatiya University, Warangal, Andra Pradesh, India

Abstract

Keywords

Candesartan cilexetil (CC) is used in the treatment of hypertension and heart failure. It has poor aqueous solubility and low oral bioavailability. In this work, CC loaded solid lipid nanoparticles (CC-SLNs) were developed to improve the oral bioavailability. Components of the SLNs include either of trimyristin/tripalmitin/tristearin, and surfactants (Poloxamer 188 and egg lecithin E80). The CC loaded nanoparticles were prepared by hot homogenization followed by ultrasonication method. The physicochemical properties, morphology of CC-SLNs were characterized, the pharmacokinetic and pharmacodynamic behaviour of CC-SLNs were evaluated in rats. Stable CC-SLNs having a mean particle size of 180–220 nm with entrapment efficiency varying in between 91–96% were developed. The physical stability of optimized formulation was studied at refrigerated and room temperature for 3 months. Further, freeze drying was tried for improving the physical stability. DSC and XRD analyses indicated that the drug incorporated into SLN was in amorphous form but not in crystalline state. The SLN-morphology was found to be nearly spherical by electron microscopic studies. Pharmacokinetic results indicated that the oral bioavailability of CC was improved over 2.75-fold after incorporation into SLNs. Pharmacodynamic study of SLNs in hypertensive rats showed a decrease in systolic blood pressure for 48 h, while suspension showed a decrease in systolic blood pressure for only 2 h. Taken together, these effects are due to enhanced bioavailability coupled with sustained action of CC in SLN formulation. Thus, the results conclusively demonstrated the role of CC-SLNs for a significant enhancement in oral bioavailability along with improved pharmacodynamic effect.

Candesartan cilexetil, pharmacodynamics, pharmacokinetics, solid lipid nanoparticles, triglycerides

Introduction Candesartan cilexetil (CC) is an ester prodrug of candesartan, a non-peptide angiotensin II type 1 (AT1) receptor antagonist, used in the treatment of hypertension and heart failure. Candesartan cilexetil is rapidly and completely bioactivated by ester hydrolysis during absorption from gastro intestinal tract to candesartan. The major drawback in the therapeutic application and efficacy of Candesartan cilexetil as an oral dosage form is its very low aqueous solubility and first-pass metabolism. Based on its solubility in physiologically relevant pH conditions and absorption characteristics, Candesartan cilexetil was classified in the Biopharmaceutics Classification System (BCS) as a class II drug. To overcome hepatic first-pass metabolism and to enhance oral bioavailability, lipid–based drug delivery systems like solid lipid nanoparticles can be used. These systems enhance the lymphatic transport of the lipophilic drugs and therefore increase the bioavailability (Uner & Yener, 2007).

Address for correspondence: Prof. V. Kishan, Dept of Pharmaceutics, University College of Pharmaceutical Sciences, Kakatiya University, Warangal, Andhra Pradesh 506009, India. E-mail: [email protected]

History Received 10 February 2014 Revised 9 April 2014 Accepted 10 April 2014

SLNs were reported as an alternative drug delivery system to traditional polymeric nanoparticles (Mu¨hlen et al., 1998). Advantage of solid-lipid nanoparticles (SLNs) over polymeric nanoparticles is based on the lipid matrix, made from physiologically tolerated lipid components, which would decrease the potential for acute and chronic toxicity (Mehnert & Ma¨der, 2001, 2012). At room temperature the particles remained in the solid state (Schwarz et al., 1994). SLNs could combine advantages of polymeric nanoparticles, fat emulsions and liposomes (Schwarz, 1999), i.e. controlled release just like polymeric nanoparticles, large scale production and toxicologically acceptable compare with fat emulsions and liposomes. Controlled drug delivery, enhancement of bioavailability of entrapped drugs via modification of dissolution rate and/or improvement of tissue distribution and targeting of drugs were reported by using SLNs in various application routes (Torsten & Mu¨ller, 2005). So far, no reports were published describing the role of SLNs on pharmacodynamic effects. The main objective of the present investigation was to incorporate CC into triglycerides, to get solid lipid matrices to improve the oral bioavailability by exploiting the intestinal lymphatic transport. Further, to understand the pharmacodynamic effect of CC-SLNs in animal models.

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Accordingly, CC-SLNs were prepared by hot homogenization followed by ultrasonication method. Prepared SLNs were characterized and optimal system was evaluated for pharmacokinetic and pharmacodynamic effects in comparison to a suspension of CC in fructose induced hypertensive wistar rats.

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Materials Candesartan cilexetil was a kind gift sample from Dr. Reddy’s labs, India. Trimyristin (Dynasan-114), tripalmitin (Dynasan116) and tristearin (Dynasan-118) were purchased from Sigma-Aldrich Chemicals, Hyderabad, India. Egg Lecithin E-80 was a gift sample from Lipoid, Germany. Irbesartan (internal standard) and Poloxamer-188 were gift samples from Aurobindo Labs, India. Methanol, acetonitrile, chloroform were of HPLC grade (Merck, Mumbai, India). Centrisart filters (molecular weight cut off 20 000) were purchased from Sartorius, Goettingen, Germany.

Methods Preparation of CC- SLNs CC loaded SLNs were prepared by hot homogenization followed by the ultrasonication (Mu¨ller et al., 2000; Manjunath & Venkateswarlu, 2005). Candesartan cilexetil, lipid, and egg lecithin were dissolved in 5 mL of 1:1 mixture of chloroform and methanol. Organic solvents were completely removed using a rota evaporator (Heidolph, Schwabach, Germany). The drug embedded lipid layer was molten by heating to 5  C above melting point of the lipid. Aqueous phase was prepared by dissolving Poloxamer 188 in double distilled water and heated to same temperature (based on lipid melting point) of oil phase. Hot aqueous phase was added to the oil phase, and homogenization was carried out (at 12 000 rpm) using homogenizer (Diax900, Heidolph, Germany) for 4 min. The coarse hot oil in water emulsion so obtained was ultrasonicated using a 12 T probe sonicator (Vibracell, Sonics, CT, USA) for 20 min. CC loaded solid lipid nanoparticles were obtained by allowing hot nanoemulsion to cool to room temperature. The composition of various formulations is shown in Table 1.

Table 1. Composition of Candesartan cilexetil loaded solid lipid nanoparticles and suspension. Ingredients

Formulation code FC1 FC2 FC3 FC4 FC5 FC6 FC7*

ORGANIC PHASE CC (mg) 8 8 8 8 8 8 Dynasan-118 (mg) 100 200 – – – – Dynasan-116 (mg) – – 100 200 – – Dynasan 114 (mg) – – – – 100 200 Egg lecithin (E-80)(mg) 100 100 100 100 100 100 Chloroform: Methanol 10 10 10 10 10 10 (1:1) (mL) AQUEOUS PHASE CC (mg) – – – – – – Sodium carboxy methyl – – – – – – cellulose (mg) Poloxamer-188 (mg) 150 150 150 150 150 150 Double distilled water (mL) 10 10 10 10 10 10 *CC-Suspension formulation

– – – – – – 8 50 – 10

Characterization of solid lipid nanoparticles Measurement of Particle size, PDI and Zeta potential of SLN The size, polydispersity index and zeta potential (ZP) of the SLNs were measured by using a Zetasizer (Nano ZS90, Malvern, Worcestershire, UK). From the prepared SLN dispersion, 100 mL was diluted to 5 mL with double distilled water to get optimum kilo counts per second (Kcps) of 50–200 for measurements. Determination of entrapment efficiency Entrapment efficiency (EE) was determined by measuring the concentration of free drug (unentrapped) in aqueous medium as reported previously (Venkateswarlu & Manjunath, 2004). The aqueous medium was separated by ultra-filtration using centrisart tubes (Sartorius, Goettingen, Germany), which consisted of filter membrane (M.Wt. cut off 20 000 Da) at the base of the sample recovery chamber. About 2.5 mL of the formulation was placed in the outer chamber and sample recovery chamber was placed on top of the sample and centrifuged. The SLN along with encapsulated drug remained in the outer chamber and aqueous phase moved into the sample recovery chamber through filter membrane. The amount of CC in the aqueous phase was estimated by HPLC method (Vijaykumar et al., 2009). Determination of total drug content About 100 mL of the SLN formulation was dissolved in chloroform and methanol mixture (1:1) and then further dilutions were made with mobile phase. The diluted samples were injected onto the column of HPLC and the amount of CC in formulations was calculated by HPLC method (Manjunath & Venkateswarlu, 2005). In vitro drug release studies In vitro release studies were performed using dialysis method. Dialysis membrane (Himedia, Mumbai, India) having a pore size 2.4 nm and molecular weight cut-off between 12 000– 14 000 was used for the release studies. Dialysis membrane was soaked overnight in double distilled water prior to the release studies. Hydrochloric acid (0.1 N) and phosphate buffer pH 6.8 were used as release media. The experimental unit had donor and receptor compartments. Donor compartment consisted of a boiling tube which was cut open at one end and tied with dialysis membrane at the other end into which 1 mL of SLN dispersion was taken for release study. Receptor compartment consisted of a 250 mL beaker which was filled with 100 mL of release medium and the temperature was maintained at 37 ± 0.5  C. At 0.25, 0.5, 1, 2, 3, 4, 6, 8, 10, 12 and 24 hour time points, 2 mL samples were withdrawn from receiver compartment and replenished with the same volume of release medium. The collected samples were suitably diluted and analyzed by UV-Visible Spectrophotometer (SL-150, ELICO, Hyderabad, India) at 254 nm (Pradhan et al., 2011). Stability studies CC loaded solid lipid nanoparticles were stored at room temperature and refrigerated temperature for three months.

DOI: 10.3109/10717544.2014.914986

Candesartan cilexetil loaded solid lipid nanoparticles for oral delivery

The average size, PDI, ZP, assay and entrapment efficiency were determined periodically after 1st day, 15days, one month, two months and three months (Vinay Kumar et al., 2012).

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film; any excess solution was drained off with a filter paper. The grid was allowed to air dry, and samples were viewed under a transmission electron microscope (JEOL-100CX-II, Tokyo, Japan).

Lyophilization of SLNs Lyophilization was used for enhancement of stability of SLNs. The SLNs containing 10% w/v maltose were prepared and kept in deep freezer at 40  C (Sanyo, Tokyo, Japan) for overnight. The frozen samples were then transferred into freeze-dryer (Lyodel, Delvac Pumps Pvt. Ltd, Chennai, India). Vacuum was applied and sample was subjected to various drying phases for about 48 h to get powdered lyophilized product (Cavalli et al., 1997; Schwarz & Mehnert, 1997).

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Solid state characterization Drug-excipient compatibility studies by Differential scanning calorimeter (DSC) DSC analysis of CC, trimyristin (TM), tripalmitin (TP) and tristearin (TS), physical mixtures (PM in 1:1 ratio), and lyophilized CC-SLNs were performed using Universal V4 TA instruments, Chicago, IL, USA. The instrument was calibrated with indium. All the samples (10 mg) were heated in aluminium pans using dry nitrogen as the effluent gas. The analysis was performed within a heating range of 20–200  C and at a rate of 20  C/min (Arjun & Kishan, 2013). Characterization of crystallinity by powder X-ray diffractometry (PXRD) Powder X-ray diffractometer (Multiflex, M/s. Rigaku, Tokyo, Japan) was used for diffraction studies (Vinay Kumar et al., 2007). Powder XRD studies were performed on the samples by exposing them to nickel filtered CuKa radiation (40 kV, 30 mA) and scanned from 2 to 70 , 2y at a step size of 0.045 and step time of 0.5 s. Samples used for PXRD analysis were pure CC, pure lipids (Dynasan – 114, 116 & 118), physical mixtures of drug with each lipid (1:1ratio) and lyophilized CC loaded solid lipid nanoparticles. Morphology by Scanning electron microscopy (SEM) The morphology of nanoparticles was studied by Scanning Electron Microscope (SEM, Hitachi, Tokyo, Japan). Freeze dried solid lipid nanoparticles of CC were suitably diluted with double distilled water (1 in 100) and a drop of nanoparticle formulation was placed on sample holder and air dried. Then the sample was observed at accelerating voltage of 15 000 volts at various magnifications. Imaging was carried out in high vacuum (Vijaykumar et al., 2009). Morphology by Transmission electron microscopy (TEM) TEM observations were also performed to know the morphology of freeze dried CC-SLN following negative staining with sodium phosphotungstate solution (0.2% w/v) (Madhu et al., 2013). A thin film was made on a carbon-coated copper grid by placing a drop of SLN dispersion. Before the film was dried on the grid, it was negatively stained with phosphotungstic acid by adding a drop of the staining solution to the

Bioavailability study Study design and sampling schedule A single dose bioavailability study was designed in male wistar rats under fasting conditions. The oral bioavailabilities of the optimized SLN formulation FC3 and suspension FC7 were estimated by conducting bioavailability studies in male wistar rats with oral dose of 10 mg/kg body weight. All experimental procedures were reviewed and approved by the institutional animal ethical committee, University College of Pharmaceutical Sciences, Kakatiya University (Warangal, India). Male wistar rats weighing 200–250 g were taken for study (6 animals per group). Blood samples were withdrawn by retro-orbital venous plexus puncture at 0, 0.5, 1, 1.5, 2, 3, 4, 6, 8, 10, 12 and 24 h post dose. About 1.5 mL of blood samples were withdrawn in eppendorf tubes and centrifuged at 3000 rpm for 30 min. The serum was transferred to another eppendorf tube and stored at 20  C until analysis. HPLC analysis The HPLC column, Merck C18, (250  4.6 mm), was equilibrated with an eluent mixture of methanol – 20 mM potassium dihydrogen phosphate (pH 4.5) and triethanolamine with composition of (70:30:0.2 v: v) at a flow rate of 1 mL/min. The peaks were eluted at 254 nm wavelength without any interferences from serum (Ashok et al., 2010). LOD of CC was 20 ng/mL and LOQ was 50 ng/mL and linearity range was found to be 50–5000 ng/mL with regression value, r2 of 0.996. Extraction procedure To 0.1 mL of serum, 0.1 mL of internal standard irbesartan (3 mg/mL) and 0.4 mL of acetonitrile as precipitating agent were added. The solutions were vortex mixed for 2 min and centrifuged at 5000 rpm for 25 min using an Eltek, Model TC 650 D, Mumbai, India centrifugal device. The upper phase was filtered through 0.45 m filter and evaporated under nitrogen evaporator. The extracted sample was reconstituted in mobile phase and 20 mL of ultra filtrate was then injected. The retention times for CC and internal standard were 7.6 and 3.8 mins, respectively. Estimation of pharmacokinetic parameters and statistical significance The pharmacokinetic parameters such as peak serum concentration (Cmax), time for peak serum concentration (tmax), AUCtotal, biological half life (t½) and mean residence time (MRT) were calculated by using the Kinetica software (version 5.0). The values were expressed as mean ± SD. The statistical comparison of data of two samples was done with unpaired student t-test using Graph pad prism software (version 6.02.2013) and p50.01 was considered as statistically significant.

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Pharmacodynamic study

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Male wistar rats weighing 210–250 g were used and allowed free access to standard diet and drinking fluid. Drinking fluid consisted either of tap water or 10% fructose solution (Hwang et al., 1987; Dai & McNeill, 1995). The rats were trained to stay in the rat holder in a calm and non-aggressive state during BP measurement. Two weeks later, rats with a minimum mean systolic BP of 140–145 mm of Hg were selected. The hypertensive rats were divided into three groups, A, B & C each consisting six animals. Group C served as control, group A and B received SLN formulation, and a suspension of drug respectively at 10 mg/kg orally (Bhaskar et al., 2009). Using the tail-cuff method (NIBP, IITC, CA, USA), systolic blood pressure was measured at different time intervals (0, 1, 2, 4, 6, 12, 24, 36, 48 and 60 h) and simultaneously measured for normal rats also (group D). Tail-cuff method By using tail-cuff method, the systolic blood pressure and heart rate of rats at constant ambient temperature (33  C) were measured. Rats were kept in plastic holders and then placed in specially designed temperature regulated chambers. A cuff with a pneumatic pulse sensor was attached to the tail. Rats were allowed to habituate to this procedure for 2 days before experiments were performed. Systolic BP and HR values were recorded on a NIBP, IITC 59/29 model and were averaged from at least three consecutive readings obtained from each rat.

Results and discussion In this study, candesartan cilexetil loaded SLNs were prepared by hot homogenisation followed by ultrasonication method (Gasco, 1993; Mu¨ller et al., 1995; Manjunath & Venkateswarlu, 2005), using three lipids, each at two different concentrations. Based on the particle size and uniformity of dispersion, the homogenization time and sonication time were optimized to 4 and 20 min respectively. Beyond this point we could not notice any appreciable change in the particle size, because with higher stirring rates the particle size could not change significantly. Furthermore, the obtained polydispersity indices were within the acceptable limits (50.3) for all the SLN formulations and suggested that the particles formed were of uniform size (Mehnert & Ma¨der, 2001). Measurement of particle size, PDI and Zeta potential of SLN All the prepared formulations were analyzed in order to determine their particle size distribution, zeta potential and PDI values. The mean size of all the formulations was ranging

from 130.8 ± 9.8 nm to 227.2 ± 12.9 nm (Table 2). The PDI was ranging from 0.197 ± 0.01 to 0.400 ± 0.04, indicating the narrow size distribution. The particle size was dependent on the alkyl chain length of the triglyceride, longer the alkyl chain length greater the particle size. The SLN formulations exhibited negative surface charge with the inclusion of CC which clearly suggested the orientation of CC in the lipid matrix. The surface charge is a key factor for the stability of colloidal dispersion. In our case, the zeta potential values of SLN formulations were found to be in between 23.07 ± 2.84 mV to 29.83 ± 2.44 mV. It is currently admitted that zeta potential 30 mV is required for electrostatic stabilization (Thatipamula et al., 2011). However, many experiments demonstrated that not only electrostatic repulsion, but also the steric stabilizer could impart stability to the SLN dispersion. Poloxamer 188 was used in the formulation as surfactant. It is a non-ionic surfactant and decreased the electrostatic repulsion between the particles following sterical stabilization of the nanoparticles by forming a coat around their surface for maintaining the stability of SLN (Mu¨ller et al., 1996). A surfactant mixture, i.e., phosphatidylcholine and poloxamer 188 was employed in the formulations because SLNs stabilized by combination of surfactants were reported to have lower particle size and higher storage stability when compared to formulations stabilized with only one surfactant. From the results obtained, formulations containing Dynasan114 showed lower particle sizes but the PDI was higher and ZP was lower. Formulations containing Dynasan-118 showed lower PDI, but higher particle sizes and lower ZP. But formulations containing Dynasan-116 (FC3 and FC4) showed relatively better size, PDI and ZP when compared to other formulations (Table 2). Determination of entrapment efficiency and drug content Total drug content in the SLN formulations was determined and found to be ranging from 98.96 ± 0.04% to 99.82 ± 0.01%. Entrapment efficiency of the SLN formulations were found to be 91.86 ± 0.51% to 96.16 ± 0.19%. High lipophilicity of CC resulted in high entrapment efficiency of drug in triglyceride nanoparticles (Table 2). This might be because of the long-chain fatty acids attached to the glyceride resulting in increased accommodation of lipophilic drugs (Reddy & Murthy, 2005). The less ordered lipid matrix created imperfections leading to void spaces in which drug molecules could be entrapped (Rawat et al., 2011). In this method of preparation, drug was dissolved in molten lipid at temperature above the melting point of lipid and there was no drug leakage or precipitation of drug during the preparation. High encapsulation efficiency of drug in lipid nanoparticles can cause

Table 2. Size, PDI, Zeta potential, assay and entrapment efficiency of CC-SLNs (n ¼ 3). Formulation FC1 FC2 FC3 FC4 FC5 FC6

Size

PDI

ZP

Assay (%)

EE (%)

213 ± 17.4 227.2 ± 12.9 186.7 ± 4.2 215.5 ± 19.7 130.8 ± 9.8 223.3 ± 18.0

0.228 ± 0.01 0.197 ± 0.01 0.264 ± 0.08 0.400 ± 0.04 0.268 ± 0.03 0.290 ± 0.03

26.26 ± 2.59 25.90 ± 2.61 29.83 ± 2.44 25.53 ± 2.97 24.42 ± 2.83 23.07 ± 2.84

99.12 ± 0.08 99.09 ± 0.09 99.82 ± 0.01 98.96 ± 0.04 99.33 ± 0.04 99.54 ± 0.04

92.42 ± 0.39 95.23 ± 0.17 94.95 ± 0.43 96.16 ± 0.19 91.86 ± 0.51 92.21 ± 0.39

DOI: 10.3109/10717544.2014.914986

Candesartan cilexetil loaded solid lipid nanoparticles for oral delivery

high amount of drug to pass through the lymphatic transport, which in turn bypasses the first pass metabolism.

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In vitro drug release In vitro release of CC from CC-SLNs was studied in 0.1 N HCl (pH 1.2) and pH 6.8 phosphate buffer by dialysis method. In 0.1 N HCl, the cumulative percentage of release from formulations FC1-FC7 was 22.84%, 21.43%, 29.98%, 28.83%, 37.30%, 34.51%, and 48.87% respectively for a period of 24 hours (Figure 1). In pH 6.8 phosphate buffer, the cumulative % of release from formulations FC1-FC7 was 56.56%, 46.78%, 69.45%, 50.85%, 67.96%, 61.03% and 80.19% respectively in 24 hours. In general, the CC-SLNs released slowly in 0.1 N HCl medium when compared to that in pH 6.8 phosphate buffer, which could be due to the solubility difference of drug in these media. The release profiles of SLN formulations exhibited a typical biphasic pattern with an initial rapid phase followed by a slow phase in phosphate buffer (Figure 2). The initial rapid phase could be due to the burst release of drug. A possible explanation is a short diffusion path due to enrichment of drug in the outer region of Figure 1. In-vitro release of CC from CCSLNs in 0.1 N HCl (mean ± SD, n ¼ 3).

Figure 2. In-vitro release of CC from CC-SLNs in pH 6.8 phosphate buffer (mean ± SD, n ¼ 3).

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SLN or drug deposition on the solid surface (Mu¨hlen et al., 1998). Formulation FC3 showed maximum release of 69.45% in pH 6.8 phosphate buffer during 24 hours. In comparison, FC3 formulation exhibited reasonably good particle size, better PDI, high zeta potential value, and the higher entrapment efficiency with release of drug from the lipid matrix in pH 6.8 phosphate buffer, hence it was considered as the optimized formulation. Since the aqueous SLN dispersion is liable to physical and chemical stability problems, lyophilization was considered to increase the stability of SLN for extended period of time. Furthermore, transformation into solid form offered possibilities of incorporating SLN into pellets, tablets and capsules. The diluted SLN dispersions were reported to have high sublimation velocities and high specific surface areas (Pikal et al., 1990). In this regard a cryoprotectant was necessary to ensure ease of redispersion without any aggregation. They interact with the polar head groups of the surfactant and serve as a kind of ‘pseudo hydration shell’ (Mobley & Schreier, 1994). In this study the maltose (10% w/v) was selected as cryoprotectant.

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Figure 3. DSC thermograms of (a) drug, (b) Dynasan-114, (c) physical mixture (Dynasan-114:drug), (d) lyophilized SLN (FC5) formulation, (e) Dynasan-116, (f) physical mixture (Dynasan-116:drug), (g) lyophilized SLN (FC3) formulation, (h) Dynasan-118, (i) physical mixture (Dynasan-118:drug), and (j) lyophilized SLN (FC1) formulation.

Solid state characterization Drug-excipient compatibility studies by Differential scanning calorimeter (DSC) The compatibility status of the lipids in the SLN formulation was investigated by differential scanning calorimetry (DSC) and was based on the fact that different lipids possessed different melting points and enthalpies. DSC thermograms of pure drug, lipids, physical mixtures and lyophilized SLN formulation are shown in Figure 3. The DSC thermogram of pure CC showed a sharp endothermic peak at 164.90  C with high enthalphy. Physical mixtures of drug with Dynasan 114, Dynasan 116 and Dynasan 118 showed drug peaks at 168.56  C, 164.78  C and 165.72  C, respectively, however with less enthalphy (Bhaskar et al., 2009). In all the cases, melting endotherm of drug was well preserved with slight changes in terms of broadening or shifting in the temperature of the melt. It is known that the quantity of material used, especially in drugexcipient mixtures, could influence the peak shape and enthalpy. Thus, these minor changes in the melting endotherm of drug could be due to the mixing of drug and excipient, which lowered the purity of each component in the mixture and this, might not necessarily indicate potential

incompatibility. The absence of endotherm peak of drug in lyophilized SLN formulations unravels the conversion of native crystalline state of the drug to amorphous state. The phase transition temperatures of colloidal dispersions were always much lower than that of the anhydrous lipid mixtures (Montenegro et al., 2011). The melting points of colloidal systems were distinctly decreased by about 3–8  C (Choi et al., 2004). In accordance with the literature reports, the phase transition temperature and enthalpy of SLN formulation was significantly lower than the corresponding anhydrous physical mixture. As the crystal is more ordered, less space is available for dissimilar molecules. These molecules serve to disrupt the thermodynamically preferred crystal ordering and responsible for higher drug loading. Powder X-ray diffractometry Powder-XRD patterns of CC showed sharp peaks at 2y scattered angles of 9.9, 17.26, 18.7, 19.28, 20.32, 23.26, and 25.14 degrees, these were indicating the crystalline nature of drug (Figure 4). These characteristic peaks of CC existed in physical mixtures, and drug peaks were absent in the lyophilized sample. This indicated that the drug was not in crystalline form after lyophilization of SLN. Intensity of pure lipid peaks was also decreased in the lyophilized samples.

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Candesartan cilexetil loaded solid lipid nanoparticles for oral delivery

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Figure 4. X-ray diffraction patterns of (a) drug, (b) Dynasan-114, (c) physical mixture (Dynasan-114:drug), (d) lyophilized SLN (FC5) formulation, (e) Dynasan-116, (f) physical mixture (Dynasan-116:drug), (g) lyophilized SLN (FC3) formulation, (h) Dynasan-118, (i) physical mixture (Dynasan-118:drug), and (j) lyophilized SLN (FC1) formulation.

This reduced intensity indicated the decreased crystallinity of lipid. The change in crystallinity of lipid and drug would influence the release of CC from nanoparticles. This reduction in crystallinity was noticed in DSC analysis also. Morphology of SLN using Scanning electron microscopic and Transmission electron microscopic studies SLN formulation was studied for surface morphology at 10 k, 15 k and 20 k magnification times using SEM. The particles possessed smooth surface and spherical in shape with increasing in particle size due to lyophilisation process, the agglomeration phenomenon was increased as shown by Figure 5 (Choi et al., 2004; Vijaykumar et al., 2009). TEM studies demonstrated that particle shape was nearly sperical and in nanometer size (Figure 6).

221.3 ± 6.48 nm at 25  C; 0.264 ± 0.08 to 0.283 ± 0.06 at 4  C and 0.201 ± 0.08 to 0.419 ± 0.06 at 25  C; 29.8 ± 2.44 to 29.5 ± 6.3 mV at 4  C; 28 ± 2.84 to 28.2 ± 4.68 mV at 25  C, respectively. Assay and EE was found to be in the range of 99.12 ± 0.02 to 99.38 ± 0.06 at 4  C, 99.02 ± 0.02 to 98.99 ± 0.08 at 25  C; 91.94 ± 0.06 to 91.02 ± 0.06% at 4  C, 90.08 ± 0.06 to 88.01 ± 0.04% at 25  C, respectively. No drastic increase in particle size, PDI and ZP was observed when stored at refrigerated temperature and room temperature for a period of 3 months. However, relatively less change in size was noticed in samples stored at refrigerated temperature, no significant (p40.05) reduction in the assay and entrapment efficiency at refrigerated temperature and at room temperature was observed, this could be due to the polymorphic transition of lipid matrix from metastable to stable form resulting in expulsion of drug from the lipid matrix (Vinay Kumar et al., 2012).

Stability study The stability of the optimized SLN formulation (FC3) was ascertained by monitoring the physical appearance, particle size, PDI, ZP, assay and entrapment efficiency of CC after storage at refrigerated temperature and room temperature for a period of 3 months. At definite time intervals, we could not notice any signs of drug crystallization in SLN formulation. Size, PDI, ZP of the formulation was found to be in the range of 186.7 ± 4.42 to 204.8 ± 6.22 nm at 4  C, 181.9 ± 4.22 to

Lyophilization The optimized (FC3) SLN formulation was freeze dried with 10% maltose and resulted in SLN powder. Upon reconstitution, increase in size, PDI and zeta potential were noticed. Due to removal of water in freeze drying process, particle attractive forces would increase (Choi et al., 2004), this might be reason for increase in the particle size of the SLN formulation.

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Figure 5. SEM image of lyophilized SLN powder (FC3 formulation) at different magnifications.

Figure 6. TEM image of lyophilized SLN powder (FC3 formulation).

Figure 7. Pharmacokinetic profile of Candesartan cilexetil in rat serum following oral administration of SLN formulation and suspension formulation (mean ± SD; n ¼ 6).

Pharmacokinetic study CC has poor oral absorption due to low aqueous solubility. This drug is prone for first pass metabolism. CC nanosuspensions were explored for improving the oral bioavailability (Vijaykumar et al., 2009). This study was focused to investigate the feasibility of SLN for improved oral delivery of CC. The pharmacokinetic parameters of CC in individual rats for optimized SLN (FC3) formulation and suspension (FC7) were calculated by non-compartmental estimations using Kinetica 2000 software, Version 5.0, Innaphase Corporation, Philadelphia, USA. The pharmacokinetic parameters AUCtotal, Tmax. Cmax, MRT and t1/2 were calculated for optimized SLN formulation and compared with suspension. The statistical comparison of data was done by Student Unpaired t-test at a significance level of p value 50.01 using Graph pad prism (version 6.02. 2013, GraphPad software, San Diego, CA, USA). The serum concentration versus time profiles following single dose administration of CC-SLN formulation and suspension are shown in Figure 7. The pertinent pharmacokinetic parameters were calculated and shown in Table 3. From Figure 7, the higher Cmax for SLN formulation (4.33 ± 0.26 mg/mL) with respect to suspension

Table 3. Pharmacokinetic parameters of CC after oral administration of optimized SLN formulation (FC3) and Suspension (FC7) (mean ± SD, n ¼ 6). Parameter Cmax (mg/ml) tmax (hr) AUCtot (mg/ml hr) thalf (hr) MRT

SLN

Suspension

4.33 ± 0.26*** 5 ± 1.09 64.86 ± 5.41**** 14.71 ± 0.86*** 21.37 ± 1.32****

3.34 ± 0.20 4±0 22.72 ± 1.62 7.63 ± 0.44 10.42 ± 0.41

****p50.0001 and ***p50.001. Statistically significant at p50.01 in comparison to suspension.

(3.34 ± 0.20 mg/mL) was statistically significant at p50.001 was observed. However, the time to reach the peak concentration was comparable to that of control. The AUCtotal, which denote the extent of absorption, was also significantly (p50.0001) higher for SLN formulation (64.86 ± 5.41 mg/ mL.hr) when compared to suspension (22.72 ± 1.62 mg/ mL.hr). The biological half life and mean residence time were higher for SLN formulation, because of slower elimination rate of CC from SLN formulation. SLN formulation

Candesartan cilexetil loaded solid lipid nanoparticles for oral delivery

DOI: 10.3109/10717544.2014.914986

9

Table 4. Antihypertensive effect of CC-SLN and suspension formulation after oral administration (mean ± SD, n ¼ 6). Mean systolic blood pressure (mm of Hg) Group Treatment A B C D

SLN Suspension Control Normal

Initial

1h

2h

4h

6h

12 h

24 h

36 h

48 h

60 h

141.7 ± 3.2 140.8 ± 2.9 142.3 ± 2.48 93.87 ± 3.56

132.9 ± 2.5* 132.6 ± 2.3* 142.1 ± 2.92 92.24 ± 2.01

127.2 ± 2.8* 104.7 ± 3.3* 140.6 ± 2.29 92.18 ± 2.20

124.0 ± 2.8* 115.3 ± 3.4* 140.9 ± 2.74 94.95 ± 4.37

120.6 ± 3.7* 122.2 ± 1.3* 142.0 ± 2.29 93.88 ± 3.94

117.8 ± 4.2* 128.6 ± 1.0* 141.6 ± 2.42 95.78 ± 2.68

112.6 ± 2.8* 138.0 ± 1.6* 140.6 ± 1.74 96.15 ± 3.65

106.5 ± 2.6* 139.9 ± 2.9* 141.8 ± 1.95 97.12 ± 4.02

100.4 ± 3.4* 139.1 ± 2.4* 141.9 ± 2.13 95.64 ± 1.54

127.3 ± 2.3* 139.9 ± 2.9* 141.9 ± 2.4 93.57 ± 4.7

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All values expressed as (mean ± SD) (n ¼ 6). *p50.05, significant compared with control, normal.

showed 2.75 fold improvement in oral bioavailability when compared to a suspension. Several mechanisms (Arik & Amnon, 2008) either alone or in combination might have contributed for the increased bioavailability of CC from SLN formulations. They being: (i) the surface active property of the phosphatidylcholine could augment the absorption due to the altered GI membrane fluidity characteristics or increased affinity of lipid particles with GI membrane, (ii) the enormous effective surface area by virtue of the nanosize of the SLN might have resulted in increased rate of absorption, (iii) the small size of the SLN permit to adhere to GI tract and also to enter the intervillar spaces thus increasing the residence time for increased bioavailability, (iv) the presence of fatty acid favor the lymphatic transport and such an effect is dependent on the chain length of fatty acid used, higher the chain length greater the extent of lymphatic transport and, (v) the influence of surfactant on the preferential uptake of lipid particles by Peyer’s patches also result in improved bioavailability of CC due to the avoidance of first pass metabolism. It can be envisaged from the results that the SLNs offer potential advantages as a suitable carrier for the improved oral bioavailability of CC. Pharmacodynamic study The antihypertensive effect of SLN formulation was studied in comparison to suspension in the rat model. The hypertension was induced in rats by 10% oral fructose solution. The systolic blood pressure was measured with instrument (NIBP, IITC, CA, USA) and results are given in Table 4. The oral administration of CC suspension (FC7) significantly (p50.05) controlled the hypertension initially, with the maximum effect observed at 2 h, but after 2 h, the BP started rising gradually until it was the same as the initial value at 24 h. By contrast, the oral administration of CC-SLN (FC3) formulation resulted in a gradual decrease of BP, with the maximum effect observed at 24 h (p50.05) and the effect continued for 48 h. In control group, no decrease in the systolic blood pressure was observed upto 60 h after the hypertension induction due to effect of fructose. In normal rat group, normal systolic blood pressure was observed. From pharmacokinetic studies in rats increased AUCtotal, half-life and MRT of SLN (more than two times) in comparison to suspension were observed and this clearly indicated that the SLN had released the drug gradually over a period of 48 h. Oral CC suspension acted quickly (2 h) and drastically, but then its effect dropped off after 24 h, where as the SLN formulation could not decrease the BP greatly in the initial phase when compared with the suspension form. The

administration of CC-SLN resulted in sustained and continued drug release for 24 h and beyond. Thus, the designed SLNs were able to control the hypertension throughout 48 h period. Clearly, the prepared SLN formulation (FC3) was capable of surmounting the shortcomings of oral administration of CC, such as low bioavailability and high first-pass metabolism. Further, it becomes a clinical advantage in controlling the hypertension slowly, steadily and for extended period by designing the drugs in SLN formulation.

Conclusion Candesartan cilexetil loaded solid lipid nanoparticles were prepared and characterized. The solid state characterization revealed the transformation of crystalline state of the drug to amorphous state. The components were found to be compatible during DSC-excipient studies. Controlled release purposes have been accomplished by incorporating CC into the solid matrix of Dynasan 116-based lipid nanoparticles. The optimized formulation CC-SLN was lyophilized to improve the stability. The pharmacokinetic studies of optimized SLN were carried out in rats, which showed a 2.75-fold improvement in bioavailability and reduction in systolic blood pressure upto 48 h and confirmed the potential of SLN as suitable carrier for oral delivery of CC. Nevertheless, to extrapolate the findings, pharmacokinetic and pharmacodynamic studies in humans is necessary to confirm the improved oral delivery of CC.

Acknowledgements Mr. Narendar Dudhipala acknowledges the UGC, New Delhi, India for BSR fellowship to carry out this research work. Further, we thank Dr. P. Govardhan, Vaagdevi college of Pharmacy, Warangal for allowing to use the equipment, IITC 59/29, NIBP for BP estimation.

Declaration of interest The authors report no conflicts of interest. The authors are alone responsible for the content and writing of this paper.

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Candesartan cilexetil loaded solid lipid nanoparticles for oral delivery: characterization, pharmacokinetic and pharmacodynamic evaluation.

Candesartan cilexetil (CC) is used in the treatment of hypertension and heart failure. It has poor aqueous solubility and low oral bioavailability. In...
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