Enhanced intestinal absorption and bioavailability of raloxifene hydrochloride via lyophilized solid lipid nanoparticles

Advanced Powder Technology 24 (2013) 393–402

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Advanced Powder Technology journal homepage: www.elsevier.com/locate/apt

Original Research Paper

Enhanced intestinal absorption and bioavailability of raloxifene hydrochloride via lyophilized solid lipid nanoparticles Madhu Burra a, Raju Jukanti b,⇑, Karthik Yadav Janga b, Sharath Sunkavalli b, Ashok Velpula b, Srinivas Ampati a, K.N. Jayaveera c a b c

Kakatiya Institute of Pharmaceutical Sciences, Warangal, AP, India Department of Pharmaceutics, St. Peter’s Institute of Pharmaceutical Sciences, Warangal, AP, India Oil Technology Research Institute, Jawaharlal Nehru Technological University, Anantapur, AP, India

a r t i c l e

i n f o

Article history: Received 16 June 2012 Received in revised form 11 September 2012 Accepted 14 September 2012 Available online 16 October 2012 Keywords: Raloxifene HCl Tristearin Solid lipid nanoparticles Perfusion Bioavailability

a b s t r a c t The current oral therapy with raloxifene hydrochloride (RXH) is less effective due to its poor bioavailability (only 2%). Henceforth, an attempt was made to investigate the utility of triglyceride (trimyristin, tripalmitin and tristearin) based solid lipid nanoparticles (SLNs) for improved oral delivery of RXH. The SLN formulations prepared were evaluated for particle size, zeta potential and % entrapment and the optimized formulation was lyophilized. Solid state characterization studies unravel the transformation of RXH to amorphous or molecular state from the native crystalline form. Further the in situ perfusion studies carried out in rat intestine reveal the potential of SLN for enhanced permeation of raloxifene HCl across gastrointestinal barrier. To derive the conclusions, in vivo pharmacokinetic study was conducted in rats to assess the bioavailability of RXH from SLN formulation compared to drug suspension. Overall a twofold increase in bioavailability with SLN formulations confer their potential for improved oral delivery of RXH. Ó 2012 The Society of Powder Technology Japan. Published by Elsevier B.V. and The Society of Powder Technology Japan. All rights reserved.

1. Introduction Oral drug delivery remains to be the preferred route of administration because of convenience and ease of administration. Nevertheless, a number of factors which limit the rate and extent of absorption include stability and solubility of drug in gastrointestinal (GI) fluids, GI residence time, membrane permeability and presystemic metabolism. Since the rate and extent of absorption is often controlled by the dissolution rate of the drug in the gastrointestinal tract [1], improving the dissolution characteristics of insoluble drugs (Classes II and IV) continues to be a major task for the formulation scientists. In the past, several strategies have been adopted for enhancing the dissolution behavior of insoluble drugs by complexation, drug derivatization and solid state manipulation, inclusion of surfactants, increasing the surface area by micronization or nanonization, spray drying and microencapsulation [2–5]. Despite the improvement in the dissolution behavior, majority of the drugs pose bioavailability problems due to the barrier function of GI tract and first pass metabolism. Earlier reports suggest that the oral bioavailability of lipophilic drugs can be enhanced when co-administered with a meal rich in ⇑ Corresponding author. E-mail address: [email protected] (R. Jukanti).

fat [6,7]. The concept has made to develop the formulation of colloidal lipid carrier systems as a means to improve the drug solubilization and permeation across the GI barrier [8,9]. Among the various conventional and colloidal drug delivery systems, solid lipid nanoparticles (SLNs) confer distinct advantages like biocompatibility and scale up [10]. In addition, due to lymphatic transport the first pass metabolism is reduced with an increase in bioavailability. Such an effect seems to be due to the drain of SLN directly into the systemic circulation via thoracic duct circumventing the portal circulation [11]. Raloxifene HCl (RXH), a selective estrogen receptor modulator (SERM) is used to prevent the osteoporosis in post menopausal women and also used in long term female hormone replacement therapy. The current oral therapy with RXH is not so effective because of poor availability of the drug to the systemic circulation (only 2%) which is due to its low solubility and extensive first pass metabolism [12]. Earlier researchers have tried to improve the dissolution characteristics and bioavailability of RXH from inclusion complexes [13] and co-grinding with hydrophilic carriers [14] and bioadhesive microspheres [15]. To the best of our knowledge, no reports were available in literature on solid lipid nanoparticles and henceforth we made an attempt to formulate the triglyceride based solid lipid nanoparticles for improved oral delivery of RXH. The present systematic study encompasses the formulation and

0921-8831/$ - see front matter Ó 2012 The Society of Powder Technology Japan. Published by Elsevier B.V. and The Society of Powder Technology Japan. All rights reserved. http://dx.doi.org/10.1016/j.apt.2012.09.002

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characterization of solid lipid nanoparticles using different triglycerides (trimyristin, tripalmitin and tristearin). Further to derive the conclusions, the in situ permeation and pharmacokinetic studies have been carried out to assess the potential of SLN for improved oral bioavailability of RXH. 2. Materials and methods Raloxifene hydrochloride (RXH) was a kind gift sample from Dr. Reddy’s laboratories, Hyderabad, India. Dynasan 114 (Glyceryl trimyristate, TM), Dynasan 116 (Glyceryl tripalmitate, TP) and Dynasan 118 (Glyceryl tristearate, TS), were purchased from Sasol Germany GmbH, Germany. Soyphosphatidylcholine (Phospholipon 90G) was a generous gift from Lipoid, Ludwigshafen, Germany. Polysorbate 80 was procured from Merck, Mumbai, India. Dialysis membrane (DM-70) was obtained from Hi-media, Mumbai, India. Centrisart filters (mol. wt cut-off, 20,000 Da) were purchased from Sartorius, Goettingen, Germany. All other chemicals used were of analytical grade and solvents were of HPLC grade. Freshly collected double distilled water was used throughout the study. 2.1. Partitioning behavior of raloxifene HCl in triglycerides The partition coefficient of raloxifene HCl in the selected triglycerides (TM, TP and TS) was determined as described elsewhere [16]. In brief, RXH (10 mg) was added to the biphasic mixture comprising of 1 g of molten lipid in 1 mL of hot distilled water. The resultant dispersion was agitated for 30 min by maintaining in a hot water bath. The mixture was allowed to room temperature and the aqueous phase was separated by using centrisart filters and quantified for drug content by HPLC.

distilled water. Analysis was performed at 25 °C with an angle of detection of 90 °C. The zeta potential (ZP) of dispersions was determined using Zetasizer NanoZS90 (Malvern Instruments, Malvern, UK). The sample was diluted suitably with distilled water. Charge on SLN and their mean ZP values (±SD) were obtained from the instrument. The entrapment efficiency of the system was determined by earlier reported method [18]. In brief, ultra-filtration was carried out using centrisart (Sartorius AG, Gottingen, Germany) at 3500 rpm for 15 min, which consist of filter membrane (Molecular weight cut-off 20,000 Da) at the base of the sample recovery chamber. The amount of the RXH in the aqueous phase was quantified by HPLC. Entrapment efficiency was calculated from the difference between the initial amount of RXH added and that present in the unentrapped form and was expressed as a percentage of the total amount of RXH added. 2.3.2. In vitro drug release study The dialysis membrane (DM-70; MW cut off 12,000–14,000) was used for studying the release behavior of RXH from solid lipid nanoparticles. After soaking the dialysis membrane in the release medium (20% w/v propylene glycol in 0.1 N HCl was used to maintain sink condition) for 24 h, control (drug solution in 40% w/v propylene glycol) or SLN dispersions (2 mL equivalent to 4 mg of drug) were placed in the dialysis bag and kept in 50 mL of release medium which was stirred continuously at 200 rpm and maintained at 37 °C. At pre set time intervals, 1 mL of sample was withdrawn and replenished with equal volume of fresh medium to maintain constant volume. The samples were analyzed by HPLC and the obtained data was fitted into mathematical equations (zero order, first order, Higuchi and Korsmeyer Peppas models) [19] and regression analysis was carried out to describe the kinetics and mechanism of drug release from the SLN formulations.

2.2. Preparation of solid lipid nanoparticles Solid lipid nanoparticles were prepared by homogenization followed by ultrasonication method as described previously [17]. Briefly, the triglyceride (400 mg), raloxifene HCl (20 mg) and phosphatidylcholine 99% (100 mg) were dissolved in 10 mL of solvent mixture consisting of chloroform and methanol (9:1). After ensuring the formation of homogenous solution, the organic solvent was removed using rotary vacuum evaporator (Laborota 4000, Heidolph, Germany). The dried lipid layer was kept in vacuum dryer for 30 min to remove the traces of organic solvent if any. The drug embedded lipid layer was heated to 5 °C above the melting point of the lipid and emulsified with hot aqueous phase (sufficient to produce 10 ml of preparation) containing polysorbate 80 (3% w/v) using homogenizer (Diax 900, Heidolph, Germany) (the temperature was maintained at 5 °C above the melting point of lipid and the speed was set at 10,000 rpm for 5 min). The resultant hot coarse o/w emulsion was subjected to sonication using ultrasonicator (Vibracell, Sonics, USA) for 10 min on which power was set at 25% of maximum output. The hot o/w emulsion was allowed to cool down to room temperature to get RXH solid lipid nanoparticles. The preparations were finally stored in nitrogen purged vials and the formulations containing TM, TP and TS were coded as RXHTM, RXH-TP and RXH-TS respectively. 2.3. Characterization of solid lipid nanoparticles 2.3.1. Determination of size, zeta potential and entrapment efficiency The mean size and polydispersity index of the size distribution of SLN was determined by photon correlation spectroscopy using zetasizer NanoZS90 (Malvern Instruments, Malvern, UK). Each sample was diluted to a suitable concentration (1 in 100) with

2.3.3. Lyophilization The SLN were lyophilized using a programmable freeze–dryer (Lyodel, India). Before the freezing step, the formulation was transferred into glass vials and cryoprotectant (5% w/v mannitol) was added to the SLN dispersion. The glass vials were placed on the shelves in the freeze dryer followed by slow freezing at a shelf temperature of 40 °C). The samples were lyophilized for 24 h from 40 °C to 25 °C at an increasing rate of 5 °C/h. The reconstitution of lyophilized formulation was made using bath sonicator (Sonica, Italy). 2.4. Solid state characterization 2.4.1. Transmission Electron Microscopy (TEM) TEM observations were performed to know the morphology of SLN following negative staining with sodium phosphotungstate solution (0.2% w/v). A thin film was made on a carbon-coated copper grid by placing a drop of SLN dispersion. Before the film dried on the grid, it was negatively stained with phosphotungstic acid by adding a drop of the staining solution to the 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). 2.4.2. Differential scanning calorimetry The thermotropic properties and phase transition behavior of pure drug (RXH), lipid (TS), physical mixture and lyophilized SLN preparation was studied by using differential scanning calorimeter (Mettler DSC 821e, Mettler-Toledo, Switzerland). Average sample weight of 5 ± 2 mg were heated in hermitically sealed aluminum pan over a temperature range of 20–300 °C under a constant

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nitrogen gas flow of 30 mL/min at a heating rate of 10 °C/min. The instrument was calibrated with indium (calibration standard, purity > 99.9%) for melting point and heat of fusion. The degree of crystallinity (% Crystallinity index, CI) was calculated using the following equation [20]: DH SLN dispersion  100 % Crystallinity Index ðCIÞ ¼ DH Bulk material  Concentration lipid phase ð1Þ

where DH SLN and DH bulk material are the melting enthalpy (J g1) of SLN dispersion and bulk lipid respectively. 2.4.3. Powder X-ray Diffractometry (PXRD) The PXRD patterns of pure drug, tristearin and lyophilized SLN formulation were obtained using X-ray diffractometer (X’ Pert PRO PANalytical, USA). The measuring conditions were as follows: Cu Ka radiation, nickel filtered; graphite monochromator; 45 kV voltage; and 40 mA current with X’celerator detector. All samples were run at 1° (2h) min1 from 3° to 45° (2h). 2.4.4. Fourier transform infrared (FT-IR) spectroscopy Infrared spectra of RXH, tristearin and lyophilized SLN formulation were obtained using FT-IR spectrophotometer (Paragon 1000, Perkin Elmer, USA) by the conventional KBr pellet method. The scanning range was 4000–500 cm1 and the resolution was 4 cm1. 2.5. In situ single pass perfusion study The study was conducted with the prior approval of Institutional Animal Ethical Committee, St. Peter’s Institute of pharmaceutical sciences. Euthanasia and disposal of carcass was in accordance of the guidelines. Male wistar rats weighing between 180 and 200 gm used in the study were obtained from Mahaveera Enterprises (146-CPCSEA No.: 199; Hyderabad, India). The animals were housed in separate cages in a clean room and maintained under controlled condition of temperature and the rats had free access to food and water. The in situ single-pass perfusion studies were performed using established methods reported elsewhere [21]. The rats fasted overnight with free access to water were anesthetized by an intraperitoneal injection of thiopental sodium (60 mg/kg body weight) and placed on a thermostatic surface to maintain body temperature. Under anesthesia, an incision was made through a midline to expose the abdominal content. The lower part of the small intestine segment used for perfusion was exposed and semi-circular incisions were made on both ends and cannulated with PE tubing followed by ligation with silk suture. After cannulation the surgical area was covered with cotton soaked in physiological saline (37 °C). The intestine segment was flushed with phosphate buffered saline (PBS) (pH 7.4 at 37 °C) to remove the adhered intestinal contents and stabilized by perfusing the blank PBS for 15 min. The SLN dispersion (RXH-TS) and control (drug suspended in 20% w/v propylene glycol) equivalent to 3 mg of zaleplon containing phenol red (7.5 lg/mL) in PBS were passed at a steady flow rate of 0.2 mL/min (NE-1600, New Era Syringe Pumps, USA) for 90 min. The perfusate was collected for every 15 min and at the end of the perfusion the circumference and length of the perfused intestine was measured. The samples were stored at 20 °C until further analysis by HPLC. Prior to analysis, the perfusate samples were allowed to thaw, deproteinized with methanol, centrifuged and the drug content in the supernatant was quantified for RXH by HPLC. Each experiment was performed in triplicate.

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2.5.1. Data analysis The absorption rate constant (Ka) was calculated from the slope of the remaining amount of drug vs. time plot. The effective permeability coefficient was determined using the following equation:

Peff ðratÞ ¼ ½Q in  lnðC in =C out Þ=A

ð2Þ

where Qin is the rate of perfusion (0.2 mL/min), A is the surface area within the intestinal segment that is assumed to be the area of a cylinder (2prL) with the length (L) (measured at the end of the experiment) and radius (r) of 0.18 cm. Cin and Cout are the inlet and fluid-transport-corrected outlet solution concentrations respectively. The enhancement ratio (ER) was calculated by using the following equation: ER = Peff(rat) of SNEP-N formulation/Peff(rat) of control. The data from the rat permeability studies were extrapolated to predict the fraction dose absorbed in human (Fa) by fitting the data into Chapman type equation [21]:

F a ¼ 1  eð38450Peff ðratÞ Þ

ð3Þ

The predicted effective permeability coefficient in human was calculated from

Peff ðhumanÞ ¼ 11:304  Peff ðratÞ  0:0003

ð4Þ

2.6. Pharmacokinetic study 2.6.1. Study protocol The study was conducted with the prior approval of Institutional Animal Ethical Committee, St. Peter’s Institute of Pharmaceutical Sciences, Warangal, India. Male albino wistar rats (180–200 g) were selected for the study and had free access to food and water. Before dosing, the animals were kept for overnight fasting. The rats were divided into two groups containing six in each. Control group received an oral suspension of RXH (drug suspended in 0.5% w/v of CMC-Na) and the test group received the optimized SLN formulation (RXH-TS) at a dose of 20 mg/kg body weight. At predetermined time intervals, blood samples (500 lL) were collected from retro orbital plexus into micro-centrifuge tubes. The blood was allowed to clot and the serum was separated by centrifugation at 10,000 rpm for 10 min in a micro-centrifuge (Remi equipments, India) and stored at 20 °C until analysis. 2.6.2. Sample analysis Raloxifene HCl was quantitatively determined in serum by HPLC using 35:65 (v/v) acetonitrile and water containing 0.25% (v/v) triethylamine at pH 3.9 respectively as mobile phase at a flow rate of 1.0 mL/min equipped with LC-10 AT solvent delivery unit (Shimadzu, Japan). An octadecylsilane (C18) reverse phase stainless steel analytical column (250  4.6 mm) with 5 lm particle size was employed for chromatographic separation (Lichrospher, Merck, Germany). The column eluent was monitored at a wavelength of 230 nm using an SPD-10 AVP ultraviolet detector and the sensitivity was set at 0.005 AUFS at ambient temperature. The serum samples were processed as described earlier in reports [15]. Briefly, 100 lL of serum sample was treated with 100 lL of methanol, vortexed followed by addition of 300 lL of acetonitrile. The samples were centrifuged and 20 lL of supernatant layer was injected onto HPLC. The concentration vs. peak area plot was linear (r2 > 0.9987) over the concentration range of interest and the RXH content in samples was quantified using this plot. 2.6.3. Pharmacokinetic parameters The peak concentration (Cmax) and its time (Tmax) were obtained directly from the serum concentration vs. time profile. The area under the curve (AUC0t) was calculated by using trapezoidal rule method. The AUCt1 was determined by dividing the plasma

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Fig. 1. Chemical structures of (A) RXH, (B) trimyristin, (C) tristearin, and (D) tripalmitin.

Table 1 Mean size, zeta potential and percent entrapment of RXH loaded SLN formulations. Formulation

Size (nm)

PI

Zeta potential (mV)

% Entrapment

RXH-TM RXH-TP RXH-TS

65.5 ± 4.3 68.2 ± 6.0 70.5 ± 3.6

0.132 ± 0.03 0.121 ± 0.02 0.115 ± 0.04

22.3 ± 5.6 21.4 ± 7.2 26.5 ± 5.7

93.11 ± 1.2 95.25 ± 1.9 97.34 ± 1.2*

Each point is mean ± SD (n = 3); PI indicates polydispersity index. RXH-TM, RXH-TP and RXH-TP represent raloxifene HCl loaded solid lipid nanoparticles containing trimyristin, tripalmitin and tristearin respectively. * Significant difference at p < 0.05 against RXH-TM formulation.

concentration at last time point with elimination rate constant (K). Mean residence time (MRT) was obtained by dividing the area under first moment curve with area under curve. The relative bioavailability (F) was estimated by dividing the AUC01 of SLN formulation with control oral suspension.

between formulations was calculated by student–Newman–Keuls (compare all pairs) with Instat Graphpad prism software (version 4.00; GraphPad Software, San Diego California). The level of statistical significance was chosen as less than p < 0.05. 3. Results and discussion

2.7. Stability studies The formulations stored in glass vials covered with aluminum foil were kept in refrigerator (4 °C) for a period of 3 months. At definite time intervals (0, 30, 60 and 90 days), samples were withdrawn, reconstituted and observed for any sign of drug crystallization under optical microscope. Further the samples were also evaluated for particle size and % entrapment of RXH. 2.8. Statistical analysis The data obtained was subjected to student ‘t’ test and one way analysis of variance (ANOVA) and the significance of difference

3.1. Preparation and physicochemical characterization of RXH loaded solid lipid nanoparticles One of the important parameter to be considered in the formulation of solid lipid nanoparticles is to obtain sufficient drug loading and stable incorporation of the drug in the lipid matrix. Since the drug loading capacity of SLN depend on the solubility behavior of the drug in the lipid melt [22], the partition behavior of RXH in selected triglycerides was carried out for the screening of lipid (Fig. 1). The partition coefficient values of RXH in TM, TP and TS were 142 ± 9, 166 ± 12 and 193 ± 16 respectively. The higher solubility characteristics of RXH in TS can be attributable to the higher

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Fig. 3. TEM image of reconstituted lyophilized SLN powder (RXH-TS). Fig. 2. In vitro release profile of raloxifene hydrochloride from SLN formulations (mean ± SD; n = 3).

alkyl chain length of TS compared to TM and TP because higher the alkyl chain length of lipid more the lipophilic region for the drug to get embed within the lipid matrix [16]. Among the various methods used for the preparation of SLN [23–26], the homogenization followed by ultrasonication method was adapted in the present study because it is simple and reproducible method of preparation of SLN [16]. Based on the particle size and uniformity of dispersion, the homogenization and sonication time was optimized to 5 and 10 min respectively. Beyond this point we could not notice any appreciable change in the particle size because with higher stirring rates the particle size will not change significantly. Furthermore, the obtained polydispersity index values, a measure of uniformity in particle size distribution, are within the acceptable limits (<0.3) for all the SLN formulations suggest that the particles formed were of uniform size [27]. In our preliminary study, the concentrations of surfactant and drug load were varied in order to achieve the stable SLN formulation (data not shown). In order to yield successful SLN formation, at least 1% (w/v) of polysorbate 80 is required in the case of SLN [28]. The formation and particle size of SLN seems to be dependent on the amount of the surfactant used in the formulation, higher the surfactant concentration greater the incorporation into SLN reducing the crystallinity of the lipid particles [10]. In accordance, the surfactant concentration was varied and fixed at 3% (w/v) where we could obtain stable formulation with narrow particle size below 100 nm. Since the formulation is intended for oral administration it can be acceptable. In order to determine the drug loading capacity of the lipid, the drug concentration was varied keeping the surfactant concentration constant i.e. 3% (w/v). The preparations were stored at room temperature and observed for physical

changes and the preparations containing 20 mg of RXH seems to be stable without any signs of crystallization. We could notice crystallization with further increase in drug concentration which might be due to the expulsion of drug from the lipid matrix. This shows that only a definite quantity of drug can be accommodated in the lipid. The mean size of all the formulations was ranging from 60 to 75 nm (Table 1). The polydispersity index which indicates the size uniformity was within the acceptable limits (<0.3). The particle size was dependent on the alkyl chain length of the triglyceride, longer the alkyl chain length greater the particle size. The blank SLN formulations without RXH exhibited negative surface charge and with the inclusion of RXH the surface charge became positive which clearly suggest the orientation of RXH in the lipid matrix. The similar findings were observed for clozapine SLN [16]. 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 between 21 and 27 mV. It is currently admitted that zeta potential 30 mV is required for electrostatic stabilization [29]. However, many experiments demonstrated that not only electrostatic repulsion dominate the stability of nanoparticles rather the presence of steric stabilizer can also impart stability to the SLN dispersion. The surfactant i.e. polysorbate 80 used in the formulation can also offer steric stability for maintaining the stability of SLN [30]. Indeed a surfactant mixture i.e. phosphatidylcholine and polysorbate 80 were employed in the formulations because SLN stabilized by combination of surfactants have lower particle size and higher storage stability when compared to formulations with only one surfactant. The percent RXH entrapment in all the SLN formulations was in the range of 91–97% (Table 1). A marginal improvement in the entrapment efficiency in case of RXH-TS could be due to the favored partitioning of RXH into the lipid matrix. This can be explained based on the fact that higher the solubility of drug

Table 2 In vitro release kinetics of RXH from different SLN formulations. Formulation

RXH-TM RXH-TP RXH-TS

Regression coefficient (R2)

Release exponent ‘n’

Zero order

First order

Higuchi

Hixson crowell

0.872 0.851 0.860

0.657 0.642 0.640

0.985 0.979 0.984

0.522 0.517 0.513

0.449 0.457 0.428

RXH-TM, RXH-TP and RXH-TS represent raloxifene HCl loaded solid lipid nanoparticles containing trimyristin, tripalmitin and tristearin respectively.

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Fig. 4. DSC thermograms of (a) physical mixture, (b) raloxifene hydrochloride, (c) tristearin, and (d) SLN formulation (RXH-TS).

Table 3 Melting peak, enthalpy and crystallinity index of SLN formulation (RXH-TS) and their respective bulk and physical mixtures.

Bulk lipid Physical mixture SLN dispersion

Melting peak (°C)

Enthalpy (J g1)

CI (%)

66.84 63.39 60.15

515.39 48.27 35.40

100 93.15 68.82

CI indicate crystallinity index.

in lipid, greater the less ordered structure of the lipid matrix. The less order lipid matrix creates imperfections leading to void spaces in which drug molecules can be entrapped [31]. Drug solution shows a very rapid drug diffusion indicating permeability of the membrane to the drug whereas the release profiles of SLN formulations exhibit a typical biphasic pattern with an initial rapid phase followed by a slow phase (Fig. 2). The initial rapid phase as expected could be due to the burst release of drug. A possible explanation is a short diffusion path due to an enrichment of drug in the outer region of SLN or drug deposition on the solid surface [32]. Furthermore, the solubility of RXH in aqueous phase is increased at high temperatures used to maintain the lipid in molten state which becomes supersaturated at the re-crystallization temperature of the lipid matrix. During re-crystallization the solid lipid core is formed entrapping the drug present in solution. Further lowering of temperature reduces the solubility of RXH and the existing lipid core will not allow the drug molecules inside consequently the drug starts crystallizing and gets adsorbed on the surface of the lipid particle [10]. The in vitro release data subjected to mathematical modeling suggest that the drug release from SLN formulations follow zero order kinetics as indicated by higher R2 values (Table 2). The higuchi model and the release exponent ‘n’ obtained from Korsmeyer–Peppas plot (n < 0.5) suggest that the drug release was through fickian diffusion. Among the

formulations tested, the RXH release was comparatively less for RXH-TS formulation compared to RXH-TM and RXH-TP formulations (Fig. 2). The results suggest an inverse relationship between the partition co-efficient and drug release i.e. higher the partition coefficient of drug in lipid slower the drug release which might be due to the increased hydrophobic interactions [16]. In comparison, the particle size was less and the entrapment efficiency was higher with slow release of the drug from the lipid matrix for RXH-TS formulation, hence it was considered to be the optimized formulation. Since the aqueous SLN dispersion is pliable to physical and chemical stability problems, lyophilization seems to be the promising way to increase the stability of SLN for extended period of time. Furthermore transformation into solid form offers principal possibilities of incorporating SLN into pellets, tablets and capsules [27]. The diluted SLN dispersions have higher sublimation velocities and a higher specific surface area [33]. In this regard a cryoprotectant is 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’ [34]. In this study the mannitol (5% w/v) was selected as cryoprotectant. 3.2. Solid state characterization TEM image demonstrate that the particle shape is regular and is in nanometer size (Fig. 3). Differential scanning calorimetry (DSC) is widely used to investigate the status of the lipid in SLN formulations and uses the fact that different lipid modifications possess different melting points and enthalpies. In this study, DSC has been carried out and thermograms of pure drug, lipid (TS), physical mixture and lyophilized SLN formulation (RXH-TS) were depicted in (Fig. 4). The onset and melting temperatures, melting enthalpy and the crystallinity index was calculated and recorded in (Table 3). The prominent endothermic peak at 265.9 °C with melting

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399

Fig. 5. Powder X-ray diffraction patterns of (a) raloxifene hydrochloride, (b) tristearin, and (c) SLN formulation (RXH-TS).

Fig. 6. FT-IR spectra of (a) raloxifene hydrochloride, (b) tristearin, and (c) SLN formulation (RXH-TS).

enthalpy 208.13 J g1 corresponding to the melting temperature of RXH indicates the crystalline nature of pure RXH (Fig. 4b). In case of physical mixture the endothermic peak of RXH was observed however the melting enthalpy was less (Fig. 4a). The absence of endothermic peak within the melting range of RXH in SLN

formulation unravels the conversion of native crystalline state of the drug to amorphous state. However the peak at 164.5 °C is due to the mannitol used as cryoprotectant in the lyophilized formulation (Fig. 4d). The degree of crystallinity was calculated and were depicted in (Table 3) for SLN formulation from the enthalpies of

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Table 4 In situ parameters of RXH from SLN formulation (RXH-TS) across rat intestine (mean ± SD; n = 3). Formulation

Peff(rat) (cm/s)  106

Peff(human) (cm/s)  104

Fa(%)

Ka (h1)

ER

Control SMEP-N

3.08 ± 0.68 9.59 ± 1.26 

0.29 ± 0.16 1.03 ± 0.11 

10.9 ± 1.5 30.9 ± 10.6 

0.008 ± 0.001 0.019 ± 0.002 

– 3.11 ± 1.85

Peff(rat), Peff(human), Fa (%), Ka and ER represent effective permeability coefficient in rat, predicted effective permeability coefficient in human, % fraction oral dose absorbed in human, absorption rate constant, and enhancement ratio respectively. RXH-TS represent raloxifene HCl loaded solid lipid nanoparticles containing tristearin.   Indicates significant difference at p < 0.01 against control.

reveal that the presence of drug in the lipid particle has resulted in a mixed lipid matrix with less ordered structure hence the phase transition temperature of SLN is lowered when compared to anhydrous physical mixtures. The higher drug loading and slower release in these systems is related to less ordered lipid matrix in SLN. The pure RXH demonstrate numerous intense peaks at 2h of 13.4, 14.4, 15.7, 19.0, 20.9, 21.1, 22.6 and 25.9 (Fig. 5) which suggests the crystalline nature of the drug. The diffraction patterns of SLN formulation were comparable to the lipid, tristearin. The absence of typical RXH peaks in lyophilized SLN formulation confirms the amorphization of the drug in lipid matrix. This is in correlation with the DSC data. Fig. 6 illustrates the FT-IR spectra of RXH, tristearin and lyophilized RXH-TS formulation. The IR spectrum of RXH is characterized by the absorption peaks at 1643.41 cm1 (C@O stretching), 1597.33 cm1 (ACAOACA stretching), 1464.02 cm1 (AS-benzothiofuron), and 908.57 cm1 (benzene ring). The appearance of characteristic peaks of RXH in SLN formulation indicates the absence of chemical interaction between the lipid and drug. Fig. 7. Pharmacokinetic profile of raloxifene hydrochloride in serum following oral administration of SLN formulation (RXH-TS) (mean ± SD; n = 6).

SLN and bulk lipid. The lower crystallinity index (68.82%) for the lyophilized SLN formulation compared to physical mixture (93.15%) confers the embedment of drug in the lipid matrix. Ishigami and Machida, compared phase transition temperature data of anhydrous physical mixtures and their corresponding colloidal dispersions [35]. The phase transition temperature of colloidal dispersions were always much lower than the anhydrous lipid mixtures [18]. The melting points of colloidal systems were distinctly decreased by about 3–8 °C [28]. The melting point decrease of colloidal systems can be assigned to the colloidal dimensions of the particles in particular to their large surface to volume ratio. Nevertheless, the melting point reduction of the different formulations has no apparent relation to the particle size [36]. In accordance with the literature reports the phase transition temperature and enthalpy of SLN formulation was significantly lower than their corresponding anhydrous physical mixture. As the crystal more ordered, less space is available for dissimilar molecules that serve to disrupt the thermodynamically preferred crystal ordering. Our DSC data on SLN

3.3. In situ single pass perfusion study The in situ perfusion study facilitates to ascertain the potential of SLN formulation for improved absorption of RXH across the intestine. The effective permeability coefficient (Peff), absorption rate constant (Ka) and enhancement ratio which denote the enhancement in absorption were calculated and represented in (Table 4). The obtained effective permeability coefficient of RXH from SLN formulation (9.59 ± 1.26 cm/s (106 cm)) was significantly higher than control (p < 0.01). Obviously the absorption rate constant was also increased for SLN formulation in comparison with control (p < 0.01). The enhancement ratio above 1 indicates an enhanced permeation and in our case the ER was 3.11 ± 1.85 which is an indicative of the potential of SLN for improved oral delivery of RXH. The fraction oral dose absorbed in human (Fa) and human Peff values was predicted by correlating with the obtained rat Peff values [21]. The predicted human Peff value of RXH from SLN formulation was 1.03 ± 0.11x104 cm/s which is significantly higher in comparison with control (0.29 ± 0.16  104 cm/ s) (Table 4) (p < 0.01). Obviously the higher Fa (30.9 ± 10.6%) of RXH from SLN formulation with respect to control (10.9 ± 1.5%) confers their potential as a carrier for improved absorption of

Table 5 Pharmacokinetic parameters of RXH in rats following oral administration of SLN formulation (RXH-TS) and control oral suspension (mean ± SD; n = 6). Formulation

Control RXH-TS

Pharmacokinetic parameters Cmax (lg/ml)

Tmax (h)

T1/2 (h)

K (h1)

AUC01 (lg h ml1)

MRT01 (h)

F

3.43 ± 1.13 5.76 ± 1.86*

4 4

14.08 ± 1.81 15.42 ± 1.36

0.049 ± 0.016 0.044 ± 0.012***

116.3 ± 11.2 264.5 ± 20.3***

8.45 ± 1.12 11.24 ± 1.03**

– 2.1 ± 0.85*

K – Elimination rate constant; MRT – Mean residence time; AUC – Area under the curve; F – Relative bioavailability. RXH-TS represent raloxifene HCl loaded solid lipid nanoparticles containing tristearin. * Indicates significant difference at p < 0.05 against control. ** Indicates significant difference at p < 0.01 against control. *** Indicates significant difference at p < 0.001 against control.

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membrane (ii) the effective absorption from SLN can also be explicit due to the modulation of p-glycoprotein inhibitory function by the polysorbate 80 used as a primary surfactant, thus reducing the efflux of RXH from the intestinal membrane (iii) the enormous effective surface area by virtue of the nanosize of the SLN promote the dissolution characteristics of RXH and therefore prevalence of higher concentration gradient might have resulted in increased rate of absorption (iv) 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 (v) 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 (vi) the influence of surfactant on the preferential uptake of lipid particles by Peyer’s patches also result in improved bioavailability of RXH due to the avoidance of first pass metabolism. It can be envisaged from the results that the SLN offer potential advantages as a suitable carrier for the improved oral bioavailability of RXH. 3.5. Stability study Fig. 8. Stability profiles of lyophilized SLN formulation with respect to size and entrapment efficiency of raloxifene hydrochloride upon storage for 90 days at refrigerated temperature.

RXH. It is well established that along with passive diffusion and facilitated diffusion processes, active transport plays a significant role in drug absorption across the gastrointestinal membrane. Pglycoprotein, a multidrug resistance protein is considered to be one of the important active transport system expressed in many biological membrane barriers and is responsible for the drug efflux resulting in poor absorption of P-gp substrates. Literature evidence suggests that polysorbate 80 is known to modulate the membrane fluidity and in turn inhibit the P-gp function at low concentration [37]. In accordance with the research findings, the RXH absorption in human was increased by threefold with SLN formulation containing polysorbate 80 as a primary surfactant in contrary to the control.

3.4. Pharmacokinetic study The oral bioavailability of RXH is limited due to poor dissolution and extensive first pass metabolism. Earlier solid dispersions and bioadhesive microspheres have been explored for improving the bioavailability of RXH [14,15]. Keeping this in view the present study was focused to investigate the feasibility of SLN for improved oral delivery of RXH. The serum concentration vs. time profiles following single dose administration of SLN formulation and control were represented in (Fig. 7). The pertinent pharmacokinetic parameters were calculated and shown in (Table 5). Fig. 7 illustrates the higher Cmax for SLN formulation (5.76 ± 1.86 lg/mL) with respect to control (3.43 ± 1.13 lg/mL) and was statistically significant at p < 0.01. However the time to reach the peak concentration was comparable to that of control. The AUC which denote the extent of absorption was also significantly higher for SLN formulation compared to control (p < 0.001). The biological half life and mean residence time were higher for SLN formulation because of slower elimination rate of RXH from SLN formulation. Overall a twofold improvement in the bioavailability from SLN formulation confers their potential for improved oral delivery of RXH. Several mechanisms either alone or in combination might have contributed for the increased bioavailability of RXH form SLN formulations (i) the surface active property of the phosphatidylcholine can augment the absorption due to the altered GI membrane fluidity characteristics or increased affinity of lipid particles with GI

The stability of the SLN formulation was ascertained by monitoring the physical appearance, particle size and% retention of RXH after storage at refrigerated temperature for a period of 3 months. At definite time intervals, the lyophilized SLN powder was reconstituted to form SLN dispersion and we could not notice any signs of drug crystallization. Further, no dramatic increase in particle size was observed when stored at refrigerated temperature for a period of 3 months (Fig. 8). However a marginal reduction in the% retention of RXH was observed which is statistically insignificant (p > 0.05). The change in% retention could be due to the polymorphic transition of lipid matrix from metastable to stable from resulting in expulsion of drug from the lipid matrix [38]. 4. Conclusions Raloxifene hydrochloride loaded solid lipid nanoparticles were prepared and characterized. The optimized formulation RXH-TS was lyophilized to improve the stability. The solid state characterization unravels the transformation of crystalline state of the drug to amorphous or molecular state. In situ single pass perfusion studies demonstrate higher effective permeability coefficient and absorption rate constant for SLN compared to control. To derive the conclusions, the pharmacokinetic studies carried out in rats show a twofold improvement in bioavailability from SLN formulation which confers the potential of SLN as suitable carriers for oral delivery of RXH. Nevertheless to extrapolate the findings bioavailability studies has to be carried out in humans to check the feasibility of these systems for improved oral delivery of RXH. Declaration of interest The authors report no conflicts of interest. The authors are alone responsible for the content and writing of this paper. Acknowledgement One of the authors Mr. Madhu Burra thanks Director, Kakatiya Institute of Pharmaceutical Sciences for providing the necessary facilities. References [1] R. Lobenberg, G.L. Amidon, Modern bioavailability, bioequivalence and biopharmaceutics classification system. New scientific approaches to international regulatory standards, Eur. J. Pharm. Biopharm. 50 (2000) 3–12.

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[2] J.C. Chaumeil, Micronization: a method of improving the bioavailability of poorly soluble drugs, Methods Find, Exp. Clin. Pharmacol. 20 (1998) 211–215. [3] S.G. Kapsi, J.W. Ayres, Processing factors in development of solid solution formulation of itraconazole for enhancement of drug dissolution and bioavailability, Int. J. Pharm. 229 (2001) 193–203. [4] F. Veiga, C. Fernandes, P. Maincent, Influence of the preparation method on the physicochemical properties of tolbutamide/cyclodextrin binary systems, Drug Dev. Ind. Pharm. 27 (2001) 523–532. [5] T.L. Rogers, J. Hu, Z. Yu, K.P. Johnston, R.O. Williams III, A novel particle engineering technology: spray freezing into liquid, Int. J. Pharm. 242 (2002) 93–100. [6] W.N. Charman, C.J.H. Porter, S. Mithani, J.B. Dressman, Physicochemical and physiological mechanisms for the effects of food on drug absorption: the role of lipids and pH, J. Pharm. Sci. 86 (1997) 269–282. [7] V.H. Sunesen, R. Vedesdal, H.G. Kristensen, L. Christrup, A. Mullertz, Effect of liquid volume and food intake on the absolute bioavailability of danazol, a poorly soluble drug, Eur. J. Pharm. Sci. 24 (2005) 297–303. [8] C.W. Pouton, Formulation of poorly water-soluble drugs for oral administration: physicochemical and physiological issues and the lipid formulation classification system, Eur. J. Pharm. Sci. 29 (2006) 278–287. [9] C.J.H. Porter, N.L. Trevaskis, W.N. Charman, Lipids and lipid-based formulations: optimizing the oral delivery of lipophilic drugs, Nat. Rev. Drug Discov. 6 (2007) 231–248. [10] R.H. Muller, K. Mader, S. Gohla, Solid lipid nanoparticles (SLN) for controlled drug delivery – a review of the state of the art, Eur. J. Pharm. Biopharm. 50 (2000) 161–177. [11] C.J.H. Porter, W.N. Charman, Intestinal lymphatic drug transport: an update, Adv. Drug Deliv. Rev. 50 (2001) 61–80. [12] B.H. Mitlak, F.J. Cohen, In search of optimal long term female hormone replacement: the potential of selective estrogen receptor modulators, Horm. Res. 48 (1997) 155–163. [13] F.W. Michael, V.J. Wacher, K.M. Ruble, M.G. Ramsey, K.J. Edgar, N.L. Buchanan, Pharmacokinetics of raloxifene in male Wistar–Hannover rats: influence of complexation with hydroxybutenyl-beta-cyclodextrin, Int. J. Pharm. 346 (2008) 25–37. [14] B. Jagadish, R. Yelchuri, K. Bindu, H. Tangi, S. Maroju, V.U. Rao, Enhanced dissolution and bioavailability of raloxifene hydro-bioavailability enhancement of raloxifene hydrochloride chloride by co-grinding with different superdisintegrants, Chem. Pharm. Bull. 58 (2010) 293–300. [15] R.K. Jha, S. Tiwari, B. Mishra1, Bioadhesive microspheres for bioavailability enhancement of raloxifene hydrochloride: formulation and pharmacokinetic evaluation, AAPS PharmSciTech. (2011), http://dx.doi.org/10.1208/s12249011-9619-9. [16] K. Manjunath, V. Venkateswarlu, Preparation, characterization and in vitro release kinetics of clozapine solid lipid nanoparticles, J. Control. Rel. 95 (2004) 627–638. [17] V.V. Kumar, D. Chandrasekar, S. Ramakrishna, V. Kishan, Y.M. Rao, P.V. Diwan, Development and evaluation of nitrendipine loaded solid lipid nanoparticles: influence of wax and glyceride lipids on plasma pharmacokinetics, Int. J. Pharm. 335 (2007) 167–175. [18] R. Jukanti, G. Devaraj, R. Devaraj, S. Apte, Drug targeting to inflammation: studies on antioxidant surface loaded diclofenac liposomes, Int. J. Pharm. 414 (2011) 279–285. [19] R. Jukanti, S. Sheela, S. Bandari, P.R. Veerareddy, Enhanced bioavailability of exemestane via proliposomes based transdermal delivery, J. Pharm. Sci. 100 (2011) 3208–3222.

[20] V. Teeranachaideekul, E.B. Souto, R.H. Müller, V.B. Junyaprasert, Physicochemical characterization and in vitro release studies of ascorbylpalmitate-loaded semi-solid nanostructured lipid carriers (NLC gels), J. Microencapsul. 25 (2008) 111–120. [21] P. Zakeri-Milani, H. Valizadeh, H. Tajerzadeh, Y. Azarmi, Z. Islambolchilar, S. Barzegara, Predicting human intestinal permeability using single-pass intestinal perfusion in rat, J. Pharm. Pharm. Sci. 10 (2007) 368–379. [22] K. Westesen, Particles with Modified Physico-Chemical Properties, their Preparation and Uses, US Patent No. 6197349, 2000. [23] M.R. Gasco, Method for Producing Solid Lipid Microspheresc Having Narrow Size Distribution, US Patent 5 (1993) 250,236. [24] R.H. Muller, W. Mehenert, J.S. Lucks, A. Schwarz, M.H. Zur, C. Weyhers, Solid lipid nanoparticles (SLN)—an alternative colloidal carrier system for controlled drug delivery, Eur. J. Pharm. Biopharm. 41 (1995) 62–69. [25] A. Dingler, S. Gohla, Production of solid lipid nanoparticles (SLN): scaling up feasibilities, J. Microencapsul. 19 (2002) 11–16. [26] F.Q. Hu, H. Yuan, H.H. Zhang, M. Fang, Preparation of solid lipid nanoparticles with clobetasol propionate by a novel solvent diffusion method in aqueous system and physicochemical characterization, Int. J. Pharm. 239 (2002) 121– 128. [27] W. Mehnert, K. Mader, Solid lipid nanoparticles production, characterization and applications, Adv. Drug Deliv. Rev. 47 (2001) 165–196. [28] M.A. Schubert, C.C. Muller-Goymann, Characterisation of surface modified solid lipid nanoparticles (SLN): influence of lecithin and nonionic emulsifier, Eur. J. Pharm. Biopharm. 61 (2005) 77–86. [29] R.P. Thatipamula, C.R. Palem, R. Gannu, S. Mudragada, M.R. Yamsani, Formulation and in vitro characterization of domperidone loaded solid lipid nanoparticles and nanostructured lipid carriers, Daru 19 (2011) 23–32. [30] B.G. Muller, H. Leuenberger, T. Kissel, Albumin nanospheres as carriers for passive drug targeting: an optimized manufacturing technique, Pharm. Res. 13 (1996) 32–37. [31] M.K. Rawat, A. Jain, S. Singh, Studies on binary lipid matrix based solid lipid nanoparticles of repaglinide: in vitro and in vivo evaluation, J. Pharm. Sci. 100 (2011) 2366–2378. [32] A.Z. Muhlen, C. Schwarz, W. Mehnert, Solid lipid nanoparticles (SLN) for controlled release drug delivery – drug release and release mechanism, Eur. J. Pharm. Biopharm. 45 (1998) 149–155. [33] M.J. Pikal, S. Shah, M.L. Roy, R. Putman, The secondary drying stage of freeze drying: drying kinetics as a function of temperature and chamber pressure, Int. J. Pharm. 60 (1990) 203–217. [34] W.C. Mobley, H. Schreier, Phase transition temperature reduction and glass transformation in dehydroprotected lyophilized liposomes, J. Control. Rel. 31 (1994) 73–87. [35] Y. Ishigami, H. Machida, Vesicles from sucrose fatty acid esters, JOACS 66 (1989) 599–603. [36] K. Westesen, B. Siekmann, M.H.J. Koch, Investigations on the physical state of lipid nanoparticles by synchrotron radiation X-ray diffraction, Int. J. Pharm. 93 (1993) 189–199. [37] B.D. Rege, J.P. Kao, J.E. Polli, Effects of nonionic surfactants on membrane transporters in Caco-2 cell monolayers, Eur. J. Pharm. Sci. 16 (2002) 237–246. [38] Y. Luo, D. Chen, L. Ren, X. Zhao, J. Qin, Solid lipid nanoparticles for enhancing vinpocetine’s oral bioavailability, J. Control. Rel. 114 (2006) 53–59.