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8 April 2013 European Journal of Pharmaceutics and Biopharmaceutics xxx (2013) xxx–xxx 1
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European Journal of Pharmaceutics and Biopharmaceutics journal homepage: www.elsevier.com/locate/ejpb
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Research paper
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Increased dissolution and oral absorption of itraconazole/Soluplus extrudate compared with itraconazole nanosuspension
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Keru Zhang, Hongxia Yu, Qing Luo, Shenshen Yang, Xia Lin, Yu Zhang, Bin Tian, Xing Tang ⇑ School of Pharmacy, Shenyang Pharmaceutical University, China
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a r t i c l e
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Article history: Received 5 December 2012 Accepted in revised form 5 March 2013 Available online xxxx
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Keywords: Itraconazole Wet milling Nanocrystal Hot melt extrude Amorphous state Dissolution Bioavailability
a b s t r a c t The purpose of this article was to compare the in vitro and in vivo profiles of itraconazole (ITZ) extrudates and nanosuspension separately prepared by two different methods. And it was proved truly to form nanocrystalline and amorphous ITZ characterized by differential scanning calorimetry (DSC), X-ray powder diffraction (XRD) analysis, Fourier transform infrared spectrum (FTIR), transmission electron microscope (TEM), and scanning electron microscope (SEM). The release of ITZ/Soluplus solid dispersions with amorphous ITZ was almost complete while only 40% release was obtained with ITZ nanocrystals. The amorphous state need not to cross over the crystal lattice energy upon dissolution while the crystalline need to overcome it. In the in vivo assay, the AUC(0–t) and Cmax of ITZ/Soluplus were 6.9- and 11.6-time higher than those of pure ITZ. The formulation of the extrudate had an AUC(0–t) and Cmax similar to those of ITZ and also OH-ITZ compared with the commercial capsule (SporanoxÒ). The relative bioavailability values with their 95% confidence limit were calculated to be 98.3% (92.5–104.1%) and 101.3% (97.9– 104.1%), respectively. The results of this study showed increased dissolution and bioavailability of the solid dispersion of Soluplus-based carrier loading ITZ prepared by HME compared with the ITZ nanosuspension prepared by wet milling. Ó 2013 Published by Elsevier B.V.
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1. Introduction ITZ is a typical triazole antifungal agent with a broad spectrum of antimycotic activity. According to the biopharmaceutics classification system (BCS) [1], ITZ is a BCS II model drug with a poor water solubility and high permeability. The solubility of ITZ is about 4 lg/ml in hydrochloric acid at pH 1.0 while it is almost insoluble in pure water with a solubility of 1 ng/ml. ITZ is a weak base with a pKa of 3.7 and is lipophilic with a log P of 7.13 [2]. From the Noyes–Whitney equation, we can deduce some ways of improving the limited dissolution of poorly water soluble drugs. The main approach is to increase the particle surface area by reducing the particle size by milling; however, this may cause aggregation leading to an increased particle size and, therefore, what was an advantage is turned into a disadvantage. In this article, an ITZ nanosuspension was prepared as a control. Reducing the thickness of the boundary layer is an alternative approach. The reduced particle size also reduces the diffusion layer thickness. Last but not least is an improvement in the apparent solubility of the
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⇑ Corresponding author. School of Pharmacy, Shenyang Pharmaceutical Univer-
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sity, China. Tel.: +86 24 23986343; fax: +86 24 23911736. E-mail addresses:
[email protected] (K. Zhang), woaiwodexin.2009@163. com (H. Yu),
[email protected] (Q. Luo),
[email protected] (S. Yang),
[email protected] (X. Lin),
[email protected] (Y. Zhang), tianbin_1015@163. com (B. Tian),
[email protected] (X. Tang).
compound. Based on the lower solubility of ITZ, many approaches Q2 have been used to improve the apparent solubility and thereby the dissolution and, ultimately, the oral absorption, e.g. A cyclodextrin complex [3], addition of organic or inorganic surfactants [4], ordered mesoporous silica [5], self-emulsion [6], solid lipid nanoparticles, polymeric micelles [7], nanosuspensions [8] and solid dispersions. Nanoparticles can be obtained in two ways involving top-down and bottom-up approaches [9]. Generally, the top-down preparation methods for nanosuspension often consist of wet milling, high pressure homogenization, and microfluidization. Wet milling is a common method used to reduce the particle size to the sub-micron region to increase the dissolution velocity and thereby the bioavailability. Nanosized crystals are produced during the process of wet milling due to the production of a high shear force. The mechanisms for breakup of the drug particles include drug–media collision, drug–drug collision, and media–media collision [10]. Wet-milled nanosuspensions have an advantage in that they are less affected by food intake during oral administration [11] but are affected by stability issues including physical (e.g. Ostwald ripening and agglomeration) and chemical (e.g. hydrolyzation) stability. The reduction of drug crystals produces an increased surface area with a huge Gibbs free energy. So, aggregation occurs more readily leading to the formation of larger particles and so impaired dissolution and bioavailability. Hence, it is necessary to add a stabilizer to protect against ripening and aggregation. In the prep-
0939-6411/$ - see front matter Ó 2013 Published by Elsevier B.V. http://dx.doi.org/10.1016/j.ejpb.2013.03.002
Q1 Please cite this article in press as: K. Zhang et al., Increased dissolution and oral absorption of itraconazole/Soluplus extrudate compared with itraconazole nanosuspension, Eur. J. Pharm. Biopharm. (2013), http://dx.doi.org/10.1016/j.ejpb.2013.03.002
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aration of nanosuspensions, stabilizers consisting of ionic (sodium lauryl sulfate) surfactants, non-ionic (Tweens, poloxamer) surfactants, and polymers (cellulose derivatives, povidone, TPGS) act via steric and electronic stabilization. ITZ nanocrystals were obtained by the wet-milled nanosuspension technique. Solid dispersions play an important role in enhancing the solubility and dissolution by an amorphous drug dispersing in the carrier [12]. Conventionally, the preparation methods for solid dispersions include the melting method and the solvent method and a combination of these [13], and others like spray drying [14], co-precipitation, and co-milling [15,16]. One of the most popular methods to prepare solid solutions is HME which was first used on an industrial scale in the 1930s [17] and then, early in 1971, it was applied in the pharmaceutical field [12]. HME is a promising processing technique to prepare solid dispersions with amorphous active pharmaceutical ingredients (API) and the improved dissolution and bioavailability. The mixture of the API and hydrophilic carrier was melted at an elevated temperature, resulting in an amorphous state of API dispersed in the carrier. The process to prepare extrudates by HME is relatively economic and does not require organic solvents like some conventional methods. In addition, the HME process is relatively easy to scale up. ITZ has already been prepared combining a variety of polymeric hydrophilic excipients, such as HPMC, HPMCAS, PVP, PVPVA, and poloxamer, singly or in combination with solid dispersion. Young-Joon Park et al. prepared an ITZ-loaded solid dispersion using PVP and poloxamer without any crystalline change with improved bioavailability [18]. Geert Verreck et al. reported that melt extrusion of ITZ and HPMC 2910 5 mPa s formed an amorphous solid dispersion with enhanced in vitro dissolution [19]. This study prepared a ITZ-loaded solid dispersion by HME in which ITZ was in an amorphous state and, when the drug content was below 40%, it was found to be a stable system when stored at 40 °C/RH75% for 3 months. A novel solubility enhancing excipient, polyvinyl caprolactampolyvinyl acetate-polyethylene glycol graft copolymer (Soluplus), with amphiphilic properties was used in this study [20]. Unlike other polymeric carriers, Soluplus served not only as a matrix but also as a solubilizer. Polymers with a high glass transition temperature (Tg) are often not suitable for HME unless used with plasticizers or other additives. Soluplus has a wider extrusion temperature range because of its lower Tg of about 70 °C, so that it is suitable for many poorly water soluble drugs with high melting points. So, this ensures that the API and Soluplus are thermally stable at the processing temperature. In addition, good flowability and low hygroscopicity are also excellent properties for HME processing. Soluplus was proved to be able to improve the solubility of BCS II drug substances in solid dispersions [21]. Kalivoda et al. showed a significant increase in the dissolution rate of an extrudate of fenofibrate/Soluplus compared with that of commercial capsules [22]. The key objective of this study was to make a comparison between the nanocrystal and amorphous state regarding the in vitro dissolution and in vivo oral bioavailability properties. ITZ nanosuspensions and ITZ/Soluplus extrudates were prepared by wet media milling and HME, respectively. Simultaneously, they were characterized by PSD, TEM, DSC, XRD, FTIR, and SEM analysis methods to make sure that ITZ nanocrystals and an amorphous solid solution were formed.
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2. Materials and methods
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2.1. Materials
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ITZ was purchased from Tianjin Lisheng Pharmaceutical Company (Tianjin, China). Hydroxypropyl cellulose (HPC-L) was
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provided by Nisso (Nippon Chemical Co., Japan), and Soluplus was kindly provided by BASF (Beijing, China). SporanoxÒ pellets removed from their original capsules were obtained from Xian Janssen Pharmaceutical Ltd. (Xian, China). Itraconazole and the active metabolite, hydroxyitraconazole (purity more than 99%), were purchased from J&K Scientific Ltd. (Beijing, China). All other reagents were analytical or chromatographic grade. The rats used for the in vivo evaluation were purchased from the Experimental Animal Center (Shenyang Pharmaceutical University, Shenyang, China). The experimental protocol was evaluated and approved by the University Ethics Committee for the use of experimental animals and conformed to the Guide for Care and Use of Laboratory Animals.
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2.2. Sample preparation
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2.2.1. Preparation of the nanosuspension A Mini-easy-MEM015 nanocirculation grinding machine was used, and before milling, 20 g ITZ was dispersed in 200 ml 5% (W/V) HPC-L aqueous solution under magnetic stirring to form a coarse suspension. Then, the suspension was poured into the milling bowl and milled for 2 h with the rpm of 3500. A high shear force was generated during the milling process by the grinding media of yttrium-stabilized zirconium oxide beads (0.6–0.8 mm). To further characterize the ITZ nanocrystals, the nanosuspension was solidified by freeze drying.
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2.2.2. Preparation of solid dispersion Solid dispersions containing ITZ and Soluplus were prepared by HME using a co-rotating twin-screw extruder TE-20 32:1 (Coperion Keya Co., China). Physical mixtures were carried forward by the kneading screw set at 200 rpm. The five temperature zones were set at 140, 160, 160, 160, and 160 °C separately from feeder to die. The extrudates were collected after cooling at ambient temperature, milled using a laboratory cutting mill, and then sieved to exclude particles >380 lm. The concentration of drug in the dispersions was 15%, 20%, 30%, 40%, and 60% (w/w).
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2.3. Characterization in vitro
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2.3.1. Particle size determination The particle size was measured by laser light diffraction using a Coulter LS230 instrument (Beckman-Coulter Co. Ltd., USA). The nanosuspension was diluted with ITZ-saturated water, and this diluted solution was added to the sample holder until the required obscuration was obtained.
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2.3.2. Transmission electron microscopy The morphology of ITZ in the nanosuspension was observed by TEM. Samples were dropped on to film-coated copper grids and dried at room temperature.
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2.3.3. Differential scanning calorimetry DSC (METTLER TOLEDO) measurements were carried out to characterize the thermal properties of the crystalline drug ITZ, polymer, a physical mixture, powdered hot melt extrudates, and the freeze-dried powder (obtained from the nanosuspension using the freeze dry method). Each sample was heated over a temperature range of 10–200 °C at a linear heating rate of 10 °C/min in an atmosphere of nitrogen.
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2.3.4. X-ray powder diffraction analysis X-ray powder diffraction was performed at room temperature using a type D/Max-2400 diffractometer (Rigaku Instrument, Japan). The samples were exposed to Cu Ka radiation under
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Q1 Please cite this article in press as: K. Zhang et al., Increased dissolution and oral absorption of itraconazole/Soluplus extrudate compared with itraconazole nanosuspension, Eur. J. Pharm. Biopharm. (2013), http://dx.doi.org/10.1016/j.ejpb.2013.03.002
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K. Zhang et al. / European Journal of Pharmaceutics and Biopharmaceutics xxx (2013) xxx–xxx Table 1 Transition reactions of the analytes and internal standard.
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Molecule
Transition
Dwell (s)
Cone voltage (V)
Collision energy (eV)
ITZ OH-ITZ LD
705.4 ? 392.3 721.4 ? 408.2 383.1 ? 337.2
0.2 0.2 0.2
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56 kV and 182 mA over the 2 h range of 3–45° in increments of 0.5°/min. 2.3.5. Fourier transform infrared spectroscopy The KBr pressed disk technique was used to make infrared measurements. The wavelength ranged from 4000 to 400 cm1 with a resolution of 4 cm1. 2.3.6. Scanning electron microscopy A scanning electron microscope (SSX-500, Shimadzu, Japan) was used to obtain SEM micrographs of the extrudates of the different samples and the freeze-dried powder. An accelerating voltage of 15 kV was used.
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2.3.7. Dissolution test Dissolution tests were performed using a ZRS-8G dissolution apparatus according to dissolution test method 2 as described in the Chinese Pharmacopeia. In order to compare the dissolution properties of the powdered extrudates, physical mixtures, nanosuspension and pure ITZ, samples equivalent to 100 mg drug were directly added to 1000 ml dissolution media of simulated gastric fluid without pepsin at a temperature of 37 °C and a paddle rotation speed of 75 rpm. 5 ml samples were taken and immediately replaced with fresh dissolution medium after 5, 10, 20, 30, 45, and 60 min. These samples were passed through a 0.45 lm Millipore filter, and the first 2 ml was discarded, then diluted 2–10 ml with fresh dissolution medium, and analyzed using a UV–Visible spectrophotometer at a maximum absorption wavelength of 254 nm.
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2.4. In vivo studies
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2.4.1. Animal experiments Male wistar rats (200 ± 20 g) were kept in air-conditioned animal quarters at a temperature of 22 ± 2 °C and a relative humidity of 50 ± 10%. Access to water and laboratory food was given ad libitum. The rats were allowed to acclimatize for at least 7 days and then fasted but with free access to water for 12 h prior to the experiment. All rats received oral doses of 10 mg/kg including 40/60 ITZ/Soluplus solid dispersion powder, the milled marketed SporanoxÒ pellet (DSC analysis showed no ITZ endothermic peak), the ITZ nanosuspension, and crystalline ITZ. The formulations were dispersed in pure water and then immediately administered the rats by oral gavage according to the concentration of ITZ in the suspension. Six animals were used for each formulation.
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2.4.2. Sample determination by UPLC-ESI-MS/MS Chromatography was performed on an ACQUITYTM UPLC system (Waters Corp., Milford, MA, USA) with a column temperature of 40 °C and an autosampler temperature of 4 °C. 5 lL of sample was eluted with a mobile phase consisting of acetonitrile and 0.1% (w/w) methanolic acid solution (50:50, v/v) through an ACQUITY UPLCTM BEH C18 column (50 mm 2.1 mm i.d., 1.7 lm; Waters Corp. Milford, MA, USA). The ESI source was operated in positive ionization mode with the capillary voltage set at 3.5 kV and an extractor voltage of 45.0 V and an RF voltage of 0.1 V. The temperature of the source was 100 °C, and desolvation
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was carried out at 400 °C. Nitrogen was used as the desolvation gas (500 l/h) and cone gas (50 l/h), while argon was used as the collision gas (0.25 ml/min). The multiple reaction monitoring (MRM) mode was used for quantification. Transition reactions of the analytes and internal standard are given in Table 1. The scanning interval was 0.2 s. Validation of the analytical methods for both ITZ and OH-ITZ under selected conditions showed that the chosen method was of acceptable precision and accuracy with a linear response of 2–2500 ng/ml and 5–2500 ng/ml. The lower limit of quantification (LLOQ) of the two analytes was 2 ng/ml for ITZ and 5 ng/ml for OHITZ. The intra-day variation at three concentrations (10, 200, 2000 ng/ml) was within the limits (
