Development of novel solid dispersion of tranilast using amphiphilic block copolymer for improved oral bioavailability

International Journal of Pharmaceutics 452 (2013) 220–226

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Development of novel solid dispersion of tranilast using amphiphilic block copolymer for improved oral bioavailability Satomi Onoue a,∗ , Yoshiki Kojo a , Hiroki Suzuki a , Kayo Yuminoki b , Keitatsu Kou c , Yohei Kawabata a,d , Yukinori Yamauchi e , Naofumi Hashimoto b , Shizuo Yamada a a Department of Pharmacokinetics and Pharmacodynamics, School of Pharmaceutical Sciences, University of Shizuoka, 52-1 Yada, Suruga-ku, Shizuoka 422-8526, Japan b Department of Pharmaceutical Physicochemistry, Faculty of Pharmaceutical Sciences, Setsunan University, 45-1 Nagaotoge-cho, Hirakata, Osaka 573-0101, Japan c Laboratory of Electron Microscopy, Showa University School of Medicine, 1-5-8, Hatanodai, Shinagawa-ku, Tokyo 142-8555, Japan d Department of Chemistry, Manufacturing and Control, Kobe Pharma Research Institute, Nippon Boehringer Ingelheim Co., Ltd., 6-7-5, Minatojima-minamimachi, Chuo-ku, Kobe, Hyogo 650-0047, Japan e Department of Pharmaceutical Physical Chemistry, College of Pharmaceutical Sciences, Matsuyama University, 4-2, Bunkyo, Matsuyama, Ehime 790-8578, Japan

a r t i c l e

i n f o

Article history: Received 9 January 2013 Received in revised form 10 April 2013 Accepted 3 May 2013 Available online 18 May 2013 Keywords: Tranilast Solid dispersion Dissolution Bioavailability Crystallinity

a b s t r a c t The present study aimed to develop novel solid dispersion (SD) of tranilast (TL) using amphiphilic block copolymer, poly[MPC-co-BMA] (pMB), to improve the dissolution and pharmacokinetic behavior of TL. pMB-based SD of TL (pMB-SD/TL) with drug loading of 50% (w/w) was prepared by wet-mill technology, and the physicochemical properties were characterized in terms of morphology, crystallinity, dissolution, and hygroscopicity. Powder X-ray diffraction and polarized light microscopic experiments demonstrated high crystallinity of TL in pMB-SD/TL. The pMB-SD/TL exhibited immediate micellization when introduced to aqueous media, forming fine droplets with a mean diameter of ca. 122 nm. There was marked improvement in the dissolution behavior for the pMB-SD/TL even under acidic conditions, although the supersaturated TL concentration gradually decreased. NMR analyses demonstrated interaction between TL and pMB, as evidenced by the chemical shift drifting and line broadening. Pharmacokinetic behaviors of orally dosed TL formulations were evaluated in rats using UPLC/ESI-MS. After oral administration of pMB-SD/TL (10 mg TL/kg) in rats, enhanced TL exposure was observed with increases of Cmax and AUC by 125- and 52-fold, respectively, compared with those of crystalline TL. From these findings, pMB-based SD formulation approach might be an efficacious approach for enhancing the therapeutic potential of TL. © 2013 Elsevier B.V. All rights reserved.

1. Introduction

Abbreviations: ANOVA, analysis of variance; ASD, amorphous solid dispersion; AUC, area under the curve of blood concentration vs. time; BA, bioavailability; BCS, biopharmaceutics classification system; BMA, butyl methacrylate; Cmax , maximum concentration; CSD, crystalline solid dispersion; DLS, dynamic light scattering; DVS, dynamic vapor sorption; HPC, hydroxypropyl cellulose; MPC, 2methacryloyloxyethyl phosphorylcholine; NMR, nuclear magnetic resonance; PLM, polarized light microscopy; pMB, poly[MPC-co-BMA]; PXRD, powder X-ray diffraction; SEM, scanning electron microscopy; SD, solid dispersion; SIR, selected ion recording; TEM, transmission electron microscopy; TL, tranilast; T1/2 , half-life; Tmax , time to maximum concentration; UPLC/ESI-MS, ultra-performance liquid chromatography equipped with electrospray ionization mass spectrometry. ∗ Corresponding author. Tel.: +81 54 264 5633; fax: +81 54 264 5635. E-mail address: [email protected] (S. Onoue). 0378-5173/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ijpharm.2013.05.022

Recently, a number of new drugs and drug candidates have been identified to be poorly soluble in water, although drug release in aqueous media can be a crucial and limiting step for oral bioavailability, particularly for biopharmaceutics classification system (BCS) class II drugs with low gastrointestinal solubility and high permeability (Pinnamaneni et al., 2002). Both pharmacologic and toxic effects of drugs are proportional to bioavailability, and inter- and intra-subject variability in clinical outcomes might be magnified when bioavailability is very low. Therefore, strategic approaches to improve the solubility and release rate of drugs have been employed and are under constant and intense investigation in both academia and industry. Several types of strategy have been proposed to improve the aqueous solubility of BCS class II/IV drugs: in particular, solid dispersion (SD) approach can achieve dissolution and solubility enhancement by dispersing the poorly soluble drug

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in a solid carrier matrix (Chiou and Riegelman, 1971). Previously, Onoue and co-workers developed two SD systems of poorly soluble chemicals such as crystalline solid dispersion (CSD) employing wetmilling technologies (Kawabata et al., 2010) and amorphous solid dispersion (ASD) by a solvent evaporation method (Onoue et al., 2012). These SD formulations exhibited rapid dissolution behavior in water and improved oral bioavailability compared with active pharmaceutical ingredient itself. Numerous excipients including cellulose derivatives, polyethylene glycol, sugars, polyvinylpyrrolidone and sugar alcohols have been used as potential carriers for SD formulations (Kaushal et al., 2004). Selection of an ideal polymer with the correct structural and physicochemical features is essential for the performance of SD, since co-existing polymer could affect the drug releasing profile, supersaturation level and its duration, and stability during the manufacturing process and/or long-term storage. Recently, a co-polymer of hydrophilic 2-methacryloyloxyethyl phosphorylcholine (MPC) unit and hydrophobic butyl methacrylate (BMA) unit (poly[MPC-co-BMA], pMB) was newly prepared by radical copolymerization (Yusa et al., 2005). The pMB exhibited spontaneous micellization in aqueous media through a hydrophobic interaction (Konno et al., 2003), and then co-existing hydrophobic and water-insoluble chemicals could be solubilized. The pMB was also identified as blood-compatible biomaterial (Wada et al., 2007), and it was used as a potent solubilizer for the injectable form of paclitaxel. Use of the pMB as a SD carrier may lead to novel development of a pMB-based SD (pMB-SD) system with improved solubility and oral absorption; however, no information is available about its feasibility. The present study was undertaken to develop new pMB-SD formulation with the aim of enhancing the dissolution properties and oral bioavailability of BCS class II/IV drugs. In the present study, tranilast (TL), [N-(3,4-dimethoxycinnamoyl) anthranilic acid], an anti-allergic BSC class II drug (Koda et al., 1976; Suzawa et al., 1992), was employed as a model chemical, and pMB-SD formulation of TL (pMB-SD/TL) was prepared using pMB with wet-milling technologies (Onoue et al., 2009). The pMB-SD/TL was characterized by scanning/transmission electron microscopy (SEM/TEM) in terms of morphology, polarized light microscopy (PLM) and powder X-ray diffraction (PXRD) in terms of crystallinity, dynamic vapor sorption (DVS) in terms of hygroscopicity, dissolution testing, and dynamic light scattering in terms of particle size distribution. Possible interaction between TL and pMB was assessed by 1 H nuclear magnetic resonance (NMR) spectral analysis. Pharmacokinetic study was also carried out in rats after oral administration of pMB-SD/TL using ultra-performance liquid chromatography equipped with electrospray ionization mass spectrometry (UPLC/ESI-MS). 2. Materials and methods 2.1. Chemicals Crystalline TL was supplied by Kissei Pharmaceutical Co., Ltd. (Nagano, Japan), and poly[MPC-co-BMA] (PUREBRIGHT® MB-3750T) was provided by NOF Corporation (Tokyo, Japan). Diclofenac, hydrochloric acid (HCl), hydroxypropyl cellulose (HPC), and ammonium acetate were purchased from Wako Pure Chemical Industries (Osaka, Japan). Methanol (liquid chromatography grade) was purchased from Kanto Chemical (Tokyo, Japan).

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2009). Briefly, 100 mg of TL was weighed into the vessel of a rotation/revolution mixer (NP-100, Thinky Co., Ltd., Tokyo, Japan). 2.5 g of zirconia (zirconium oxide) balls with a diameter of 0.1 mm (Nikkato Co., Ltd., Osaka, Japan) were put into the vessel and the indicated volume of pMB solution (10 mg/mL) was added. TL suspension was nanosized by 2-step wet-milling with pulverizing conditions as follows: the first step, 2000 rpm for 2 min with 0.5 mL of pMB solution; and the second step, 400 rpm for 1 min after the addition of 9.5 mL of pMB solution (total volume: 10 mL). After nanosizing with wet-milling, the TL suspension, containing 100 mg of milled TL and 100 mg of pMB, in a 20 mL vial was frozen with liquid nitrogen and freeze-dried using an FD-81 freeze-dryer (Tokyo Rikakikai, Tokyo, Japan). The CSD/TL, composed of TL and HPCSL, was also prepared as we reported previously (Kawabata et al., 2010). 2.2.2. UPLC/ESI-MS analysis of TL The amount of TL in the obtained SD formulation was determined by an internal standard method using a Waters Acquity UPLC system (Waters, Milford, MA), which included binary solvent manager, sample manager, column compartment, and SQD connected with MassLynx software. An Acquity UPLC BEH C 18 column (particle size: 1.7 ␮m, column size: 2.1 mm × 50 mm; Waters) was used, and the column temperature was maintained at 50 ◦ C. The standard (diclofenac) and samples were separated using a gradient mobile phase consisting of methanol (A) and 5 mM ammonium acetate (B) with a flow rate of 0.25 mL/min. The gradient conditions of mobile phase were 0–0.5 min, 40% A; 0.5–3.5 min, 40–65% A; 3.5–4.0 min, 90% A; and 4.0–4.5 min, 40% A. Analysis was carried out using selected ion recording (SIR) for specific m/z 326 and 294 for TL [M−H]− and diclofenac [M−H]− , respectively. 2.3. Microscopic experiments 2.3.1. Scanning electron microscopy (SEM) Each sample was coated with platinum on HITACHI Ion Sputter E-1010 (Hitachi, Tokyo, Japan). Representative scanning electron microscopic images of crystalline TL and pMB-SD/TL were taken using a scanning electron microscope, VE-7800 (Keyence Corporation, Osaka, Japan). For the SEM observations, each sample was fixed on an aluminum sample holder using double-sided carbon tape. 2.3.2. Transmission electron microscopy (TEM) For transmission electron microscopy (TEM), an aliquot (2 ␮L) of water-suspended pMB-SD/TL was placed on a carbon-coated Formvar 200 mesh nickel grid, and the material excess was removed with a filter paper. A 2% (w/v) uranyl acetate solution was dropped onto the grid, and the excess of staining solution was removed with a filter paper in 30 s. The grid was examined under an H-7600 transmission electron microscope (Hitachi, Tokyo, Japan). 2.3.3. Polarized light microscopy (PLM) Representative PLM images of TL samples were taken using a CX41 microscope (Olympus Co. Ltd., Tokyo, Japan). TL samples were examined under various conditions including differential interference contrast, slightly uncrossed polars, and using a red wave compensator. 2.4. Powder X-ray diffraction (PXRD)

2.2. pMB-SD formulation of TL 2.2.1. Preparation The wet-milled TL formulation was prepared in accordance with a previous report with some modification (Onoue et al.,

X-ray diffraction measurement was carried out in the reflection mode on Mini Flex II (Rigaku Corporation, Tokyo, Japan) with Cu K␣ radiation generated at 15 mA and 30 kV. Data were obtained from 3◦ to 33◦ (2) at a step size of 0.1◦ and scanning speed of 4◦ /min.

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Back filled samples were prepared using ca. 20 mg of powder. Verification or standardization tests for the instrument were conducted using alumina reference standard. 2.5. Dynamic vapor sorption (DVS) Dynamic vapor sorption was recorded using an automated water sorption analyser, DVS Intrinsic (Surface Measurement Systems Ltd., Alperton, UK). The measurement temperature was maintained at 25.0 ± 0.1 ◦ C by enclosing the entire system in a temperature-controlled incubator. A mass of ca. 2 mg was loaded into a sample cup and placed in the system. The relative humidity (RH) was then increased from 0% to 90% in 10% steps, and then decreased in the opposite manner. For all RH steps, the instrument was run in a dm/dt mode (mass variation over time variation) to detect when equilibrium was reached. The “equilibrium” dm/dt value and minimum and maximum stage time were fixed to be 0.002%/min, 10 min and 6 h, respectively.

2.9.2. Pharmacokinetic study After intravenous administration of TL (0.5 mg/kg) dissolved in DMSO, or oral administration of TL samples (10 mg TL/kg) suspended in 1 mL of distilled water, blood samples were obtained at a volume of 400 ␮L from the tail vein of unanesthetized rats at the indicated times (0.25, 0.5, 1, 3, 6, 12, and 24 h). Each blood sample (400 ␮L) was centrifuged at 10,000 × g to prepare serum samples. The serum samples were kept frozen at below −20 ◦ C until they were analyzed. TL concentrations in serum were determined by UPLC/ESI-MS. In brief, 100 ␮L of methanol was added to 50 ␮L of serum sample, and the solution was centrifuged at 3000 rpm for 10 min. The supernatant was filtrated through a 0.2-␮m filter, and then the filtrate was analyzed by UPLC/ESI-MS, as described in Section 2.2.2. The pharmacokinetic parameters for TL were calculated by noncompartmental methods using the WinNonlin® program (Ver. 4.1, Pharsight Corporation, Mountain View, CA). 2.10. Statistical analysis

2.6. Dissolution test Dissolution testing on crystalline TL, CSD/TL, and pMB-SD/TL was carried out for 60 min in 900 mL of HCl solution (pH 1.2) with constant stirring of 50 rpm in a dissolution test apparatus, NTR 6100A (Toyama Sangyo, Osaka, Japan), at 37 ◦ C. Each powder sample (ca. 900 ␮g of TL) was weighed in the dissolution vessel (final concentration of TL: ca. 1.0 ␮g/mL). Samples (0.6 mL) were collected and filtered through a 0.2-␮m membrane filter (Millex LG, Millipore, Billerica, MA), and diluted with an equal volume of methanol. The concentrations of TL were determined by Waters UPLC/ESI-MS as described in Section 2.2.2. 2.7. Size and surface charge analysis The hydrodynamic size and the surface charge (␨-potential) of water-suspended pMB-SD/TL was characterized with a Zetasizer Nano ZS (Malvern Instruments Ltd., United Kingdom) utilizing dynamic light scattering (DLS) and electrophoretic light scattering (ELS), respectively. For measurement, 1 mg of pMB-SD/TL was dispersed in 10 mL of distilled water and dispersed homogeneously. The mean diameter was calculated using photon correlation from light scattering. All measurements were performed at 25 ◦ C at a measurement angle of 90◦ . The ␨-potential can also be calculated from the electrophoretic mobility using the Smoluchowski equation. 2.8.

1H

NMR spectroscopy

1 H NMR spectra of TL with or without pMB, using D O (99.95% D, 2

Wako Pure Chemical Industries) containing 100 mM sodium phosphate (pH 6.8) as a solvent, were recorded on a JEOL ECA 500 spectrometer (Nihon Denshi, Tokyo, Japan). 2.9. Pharmacokinetic studies 2.9.1. Animals Male Sprague–Dawley rats, weighing ca. 300 ± 50 g (8–9 weeks of age; Japan SLC, Shizuoka, Japan), were housed two per cage in the laboratory with free access to food and water, and maintained on a 12-h dark/light cycle in a room with controlled temperature (24 ± 1 ◦ C) and humidity (55 ± 5%). All procedures used in the present study were conducted in accordance with the guidelines approved by the Institutional Animal Care and Ethical Committee of the University of Shizuoka.

For statistical comparisons, one-way analysis of variance (ANOVA) with pairwise comparison by Fisher’s least significant difference procedure was used. A P-value of less than 0.05 was considered significant for all analyses. 3. Results and discussion 3.1. Preparation and physicochemical properties of pMB-SD formulation Theoretically, an amorphous formulation of poorly soluble chemicals tends to exceed its crystalline formulation markedly in terms of both aqueous solubility and dissolution behavior. In contrast, the solubility advantage driven by a high-energy amorphous form is often countered by its poor stability, favoring conversion to a more stable crystalline form with lower energy (Vasconcelos et al., 2007). In addition, our previous study demonstrated that ASD formulation of TL (ASD/TL) was highly sensitive to ultraviolet and visible light, resulting in photochemical degradation of TL; however, CSD formulation of TL (CSD/TL), as well as crystalline TL, was found to be less photoreactive (Kawabata et al., 2010). From these previous findings, the present study focused on the development of a nanocrystalline-loaded SD system using micelle-forming polymer with the aims of achieving improved biopharmaceutical behavior and high stability. To prepare a suspension of nanocrystalline TL in polymer solution, a wet-milling system employing zirconia beads was applied to crystalline TL particles in pMB solution. The resultant suspension of wet-milled crystalline TL was freeze-dried to provide a pMBSD/TL with an overall yield of over 90%, and degradation of TL was negligible after wet-milling and freeze-drying processes. SEM micrographs of crystalline TL (Fig. 1A–I) and pMB-SD/TL (Fig. 1AII) exhibited clear morphological changes of the powder particles after the wet-milling process. Crystalline TL particles were found to be predominantly dispersed and irregularly shaped, with sizes ranging from about 50 to 300 ␮m, whereas the solid dispersions appeared in the form of irregular particles with unhomogeneous size and shape. PLM micrographs of these samples demonstrated the high crystallinity of TL as evidenced by intense birefringence (Fig. 1B-I), whereas only slight birefringence was observed in the pMB-SD/TL (Fig. 1B-II), possibly due to marked reduction in particle size of crystalline TL down to the sub-micron range. The transition in PLM observations after wet-milling was in agreement with outcomes from a previous study on CSD formulation of wet-milled curcumin with a mean diameter of ca. 250 nm (Onoue et al., 2010).

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Fig. 1. Morphological observations of crystalline TL and pMB-SD/TL using (A) scanning electron microscopy and (B) polarized light microscopy. (I) Crystalline TL and (II) pMB-SD/TL. Black and white bars represent 100 and 50 ␮m, respectively.

Under the stress conditions generated during mechanical energy input, there is a possibility of physicochemical change in the crystalline solid, including polymorphic transitions or the formation of an amorphous phase (Crowley and Zografi, 2002), and the stable anhydrous crystal might be converted to the hydrate during aqueous wet granulation as previously observed in theophylline (Phadnis and Suryanarayanan, 1997). To clarify possible transition of the crystal form of wet-milled TL, PXRD analysis was carried out for crystalline TL, physical mixture of crystalline TL and pMB, and the pMB-SD/TL (Fig. 2A). Crystalline TL exhibited sharp and intense peaks specific for the most stable anhydrous crystalline form (␣-form) reported previously (Kawashima et al., 1991). The physical mixture also showed similar, but slightly broadened peaks, and almost identical PXRD patterns were seen in the pMB-SD/TL, regardless of the macroscopic morphology. The XRD diffractogram of pMB showed a halo pattern (data not shown), confirming that pMB is amorphous. Comparison of the PXRD patterns with those in the ␣-form of TL would indicate the absence of any polymorphic transformation or formation of hydrate during the wet-milling process, although the partial amorphization of wet-milled TL is still unclear. 3.2. Hygroscopic property of pMB-SD formulation Determination of the hygroscopic nature of a pharmaceutical product can be essential for better understanding of its physiochemical behaviors, such as microbiological activity and solid-state stability. Considering the solubilizing potency of the pMB, the hygroscopic property of pMB-SD/TL was evaluated by DVS (Fig. 2B). DVS is capable of measuring mass changes in a controlled environment with excellent accuracy, and moisture sorption isotherms obtained by DVS analysis are one of the simplest means of studying the interaction of water molecules with drug substances. The maximum water sorption in crystalline TL at 90% RH was less than 0.1%, which demonstrates that the ␣-form of crystalline TL is not

hygroscopic. In contrast, the representative DVS isotherm plot of pMB-SD/TL was indicative of its high hygroscopicity, depending on environmental humidity. No deliquescence was seen in the pMBSD/TL under current experimental conditions. There seemed to be virtually no hysteresis for each cycle considering the overall small scale of change in mass, and most likely it was a completely reversible adsorption mechanism. Since outcomes from the DVS experiments suggested high hygroscopicity of pMB-SD/TL, stability testing on the pMB-SD/TL was carried out under accelerated condition (40 ◦ C/75% RH) for 8 weeks. PXRD pattern of aged pMBSD/TL was almost identical to that of the pMB-SD/TL, and there were no significant changes in appearance and purity (data not shown). These findings suggested that the pMB-SD/TL would be stable even under high humidity. 3.3. Micelle-forming performance of pMB-SD formulation The micelle-forming performance of pMB-SD/TL was assessed by dissolution testing in acidic solution (pH 1.2) at 37 ◦ C (Fig. 3A) to simulate gastric environment, in comparison with crystalline TL and CSD/TL. The crystalline TL exhibited poor dissolution behavior in acidic solution, and the release of TL at 60 min was only less than 0.1%. Particle size reduction has also attracted considerable attention since these physical modifications lead to marked increases of the surface area, solubility, and/or wettability of the powder particles (Kawabata et al., 2011). Under the current experimental conditions, the CSD/TL exhibited accelerated drug releasing profile as ca. 20% of drug release could be achieved at 60 min. Interestingly, the pMB-SD/TL showed more rapid dispersion/dissolution, and release of TL from the pMB-SD/TL seemed to reach nearly the maximum level just after dispersion into aqueous medium, which subsequently decreased to ca. 60%. Although the wet-milling approach would be efficacious for the increase of effective surface area (Horter and Dressman, 2001) and the decrease of diffusion layer thickness (Mosharraf and Nystrom, 1999), the pMB-SD

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Fig. 2. Physicochemical properties of TL formulations. (A) Powder X-ray diffraction analysis on (i) crystalline TL, (ii) physical mixture of TL and pMB, and (iii) pMBSD/TL. (B) Dynamic vapor sorption analysis on crystalline TL (thick lines) and pMBSD/TL (thin lines). The sorption curves of water vapor are shown as the change in sample mass versus relative humidity. The absorptions are shown as solid lines and desorptions as dashed lines.

strategy employing pMB provided further improvement in the dissolution behavior of TL. The spontaneous micellization of pMB-SD/TL was also characterized by TEM observation (Fig. 3B) and DLS analysis (Fig. 3C). TEM experiment demonstrated evident formation of a number of spherical particles with particle sizes of less than 400 nm, whereas a small number of wet-milled TL particles were also seen just after dispersion into water. The formation of micelles would be attributable to improved solubility of TL, and thereby the residual TL particles might be eventually solubilized and incorporated into the pMB nanoparticles. This might explain in part the more rapid and extensive dissolution behavior of pMB-SD/TL compared with CSD/TL. According to the DLS analysis, the water-suspended pMB-SD/TL would form nanomicelles with an average particle size of 123 nm and a polydispersity index of 0.23, and the nanomicelle was found to be negatively charged as evidenced by a ␨-potential of ca. −18 mV. In DLS analysis on water-suspended pMB, mean particle size of pMB micelles was determined to be 141 nm (data not shown). These data would suggest no significant influence of inner

Fig. 3. Micelle-forming properties of pMB-SD/TL. (A) Dissolution profiles of TL formulations in acidic solution (pH 1.2). () Crystalline TL; () CSD/TL; and () pMB-SD/TL. Data represent mean ± SE of 3 independent experiments. (B) Transmission electron microscopic image of the pMB-SD/TL re-dispersed in distilled water. Arrowheads indicate unencapsulated particles of TL outside the micelles. Bar represents 500 nm. (C) Particle size distribution of the pMB-SD/TL re-dispersed in distilled water as determined by dynamic light scattering.

TL on the particle size of pMB micelles. Polydispersity index is a parameter to define the particle size distribution of nanoparticles obtained from photon correlation spectroscopic analysis, and the polydispersity index values obtained in this study further corroborate the observations made, with lower values being observed for relatively monodisperse systems. Previously, Konno et al. (2001) reported that a lightly anionic surface of pMB micelle, and inclusion of TL, a cationic drug, in micelles of pMB led to larger negative ␨-potential value. The negative ␨-potential value might contribute to good colloidal stability due to the electrostatic repulsive forces

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Fig. 4.

1

225

H NMR spectra of TL with or without pMB in D2 O containing 100 mM sodium phosphate (pH 6.8). (A) Full spectrum of TL and (B) aromatic proton peaks of pMB-SD/TL.

(Singh et al., 2005) and thereby high-energy barrier between particles (Mora-Huertas et al., 2010). From these observations, the pMB-SD approach might be a promising dosage option for oral use with improved dissolution property of TL and other BCS class II/IV drugs. 3.4. NMR spectroscopic analysis of molecular interaction between drug and polymer In the light of solubility enhancement in pMB-SD formulation, the molecular interaction between TL and the micelles was evaluated by NMR spectral analysis (Fig. 4). All 1 H NMR spectra of TL (500 ␮g) with or without pMB (500 ␮g) were recorded in D2 O containing 100 mM sodium phosphate (pH 6.8) at a sample temperature of 293 K. On the basis of the spectra recorded, most proton signals for TL with pMB micelles were shifted upfield relative to TL alone, although no chemical shift drifting was seen in aromatic proton peaks for H-3 (ı = 7.89 ppm) and H-6 (ı = 8.26 ppm). Thus, chemical shift drifting can be clearly observed with the addition of pMB, and these observations would indicate the changes in the microenvironment of TL through the interaction with pMB micelles. In addition, these shifts are accompanied by significant broadening of most TL resonances. In an aqueous environment, hydrophilic MPC units in pMB will face outward in contact with solvent, whereas hydrophobic BMA units will tend to aggregate in an inner layer. TL with ACD/log P value of 4.36 might be located in an inner layer through hydrophobic interaction, and then most resonances can theoretically display an increased line broadening upon incorporation into the micellar environment (Onoue et al., 2008; Yushmanov et al., 1994). This is most likely attributable to the reduced tumbling of the drug inside the micelles, providing evidence that the TL molecules are embedded in the micelles. Although the detailed orientation of TL in pMB micelles is still unclear, the interaction with polymer might be attributable to the improved dissolution behavior of TL and the stabilization of micelles.

(10 mg/kg), CSD/TL (10 mg-TL/kg), and pMB-SD/TL (10 mg-TL/kg) to clarify the possible improvement in oral bioavailability of TL. In the present study, after oral administration of TL formulations, the blood concentration of TL was determined by UPLC/ESI-MS analysis at various time points. As reported previously (Kawabata et al., 2010), there appeared to be slight systemic exposure of TL after oral administration of crystalline TL (Fig. 5), Cmax and AUC0−inf values of which were calculated to be 95 ± 48 ng/mL and 0.8 ± 0.4 ␮g h/mL, respectively (Table 1). In contrast, orally administered CSD/TL and pMB-SD/TL exhibited high systemic exposure with AUC0–inf values of 24.8 ± 2.4 ␮g h/mL and 41.2 ± 3.2 ␮g h/mL, respectively. On the basis of the AUC0–inf value (3.29 ␮g h/mL) of intravenously administered TL (0.5 mg/kg, data not shown), absolute bioavailabilities of crystalline TL, CSD/TL, and pMB-SD/TL were calculated to be 1.2%, 37.7%, and 62.6%, respectively. Thus, compared with crystalline TL, there appeared to be ca. 52-fold enhancement in the oral bioavailability of TL with the use of pMB-SD technology, and the pMB-SD/TL also exceeded the CSD/TL in both Cmax and oral bioavailability by 2.0- and 1.7-fold, respectively. These observations could be in agreement with their dissolution characteristics, and the micelleforming property of pMB-SD would contribute to both dissolution and pharmacokinetic behaviors.

3.5. Pharmacokinetic behavior of orally taken pMB-SD formulation The pMB-SD approach was found to be efficacious for improvement of the dissolution characteristics of TL, even under acidic conditions. In general, the oral absorption of BCS class II drugs tends to be rate-limited by its dissolution, so that even a small increase in dissolution rate can lead to a large increase in bioavailability (Lobenberg and Amidon, 2000). Herein, comparative pharmacokinetic study in rats was carried out for orally taken crystalline TL

Fig. 5. Blood TL concentrations in rats after oral administration of TL formulations. () Crystalline TL (10 mg/kg); () CSD/TL (10 mg-TL/kg); and () pMB-SD/TL (10 mgTL/kg). Data represent mean ± SE of 4–6 experiments.

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Table 1 Pharmacokinetic parameters of oral TL formulations.

Crystalline TL (10 mg/kg) CSD/TL (10 mg TL/kg) pMB-SD/TL (10 mg TL/kg)

Cmax (␮g/mL)

Tmax (h)

T1/2 (h)

AUC0–inf (␮g h/mL)a

BA (%)

0.1 ± 0.0 6.0 ± 1.3 11.9 ± 1.58

1.8 ± 0.7 2.5 ± 1.1 0.38 ± 0.1

2.9 ± 0.7 2.2 ± 0.5 3.3 ± 0.3

0.8 ± 0.4 (50%) 24.8 ± 2.4 (20%) 41.2 ± 3.2 (15%)

1.2 37.7 62.6

Cmax , maximum concentration; Tmax , time to maximum concentration; T1/2 , half-life; AUC0–inf , area under the curve of blood concentration vs. time from t = 0 to t = ∞ after administration; BA, oral bioavailability. The numbers in parentheses indicate coefficients of variation. Values are expressed as mean ± SE of 4–6 experiments.

After oral administration of TL formulations, the blood concentration of TL decreased gradually with a similar apparent elimination half-life of 2.2–3.3 h, although the Tmax for the pMBSD/TL was much shorter than that of crystalline TL or CSD/TL. These findings were not surprising considering the dissolution and micelle-forming characteristics of pMB-SD/TL, possibly leading to a higher rate and extent of absorption. The rapid absorption may offer rapid onset of action in clinical use, contributing to better clinical outcomes. Drugs with poor aqueous solubility sometimes exhibit a high degree of inter- and intra-individual variability in oral absorption (Jamei et al., 2009), which often causes lack of dose proportionality and therapeutic failure. In the present study, the coefficient of variation in AUC0–inf values for crystalline TL was as much as 50%, although those for CSD/TL and pMB-SD/TL were decreased to ca. 20% and 15%, respectively. Thus, there was a 70% reduction of inter-individual variation in AUC0–inf value for the pMB-SD/TL compared with that for crystalline TL. A marked increase in solubility of TL upon spontaneous micellization might be attributable to improved and consistent absorption with low variation in bioavailability, and the newly developed pMB-SD system may provide an attractive dosage option for other poorly soluble drugs. However, since the local and systemic toxicity of pMB in chronic use is still uncertain, further safety assessments may be needed before its clinical use. 4. Conclusion In the present study, a novel SD formulation of TL was designed with the use of amphipathic pMB polymer. There was marked improvement in the dissolution behavior of the pMB-SD/TL compared with those of crystalline TL and CSD/TL, although pMB-SD/TL was found to be hygroscopic in DVS analysis. The pMB-SD/TL formed nanoparticles in aqueous media, and NMR spectroscopic analysis was indicative of molecular interaction between TL and pMB. The pMB-SD/TL provided 52-fold increase in oral bioavailability compared with crystalline TL in rats, and its inter-individual variation was reduced by ca. 70%. From these findings, the newly developed pMB-SD formulation might be an efficacious dosage option for TL and other poorly water-soluble drugs to achieve improvements in dissolution and oral absorption. Acknowledgments The authors are grateful to Kissei Pharmaceutical Co., Ltd. (Nagano, Japan), for kindly providing tranilast. This work was supported in part by a Grant-in-Aid for Scientific Research (C) (No. 24590200; S. Onoue) from the Ministry of Education, Culture, Sports, Science and Technology. References Chiou, W.L., Riegelman, S., 1971. Pharmaceutical applications of solid dispersion systems. J. Pharm. Sci. 60, 1281–1302. Crowley, K.J., Zografi, G., 2002. Cryogenic grinding of indomethacin polymorphs and solvates: assessment of amorphous phase formation and amorphous phase physical stability. J. Pharm. Sci. 91, 492–507.

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