ARTICLE IN PRESS G Model IJP 13264 1–8 International Journal of Pharmaceutics xxx (2013) xxx–xxx Contents lists available at SciVerse ScienceDirect International Journal of Pharmaceutics journal homepage: www.elsevier.com/locate/ijpharm Pharmaceutical nanotechnology Physical properties and in vivo bioavailability in human volunteers of isradipine using controlled release matrix tablet containing self-emulsifying solid dispersion 10 11 Q1 Phuong Ha-Lien Tran a,1 , Thao Truong-Dinh Tran a,∗ , Zong Zhu Piao b,1 , Toi Van Vo a , Jun Bom Park b , Jisung Lim c , Kyung Teak Oh d , Yun-Seok Rhee e , Beom-Jin Lee b,∗ a International University, Vietnam National University, Ho Chi Minh City, Viet Nam College of Pharmacy, Ajou University, Suwon 443-749, Republic of Korea c College of Pharmacy, Kangwon National University, Chuncheon 200-701, Republic of Korea d College of Pharmacy, Chung-Ang University, Seoul 155-756, Republic of Korea e College of Pharmacy and Research Institute of Pharmaceutical Sciences, Gyeongsang National University, Jinju 660-751, Republic of Korea b 12 13 a r t i c l e i n f o a b s t r a c t 14 15 16 17 18 Article history: Received 11 November 2012 Received in revised form 24 February 2013 Accepted April 2013 Available online xxx 19 25 Keywords: Self-emulsifying solid dispersion Enhanced dissolution Controlled release tablet Physicochemical properties In vivo bioavailability 26 Introduction 20 21 22 23 24 Poorly water-soluble drug with a short half-life such as isradipine (IDP) offer challenges in the controlled release formulation because of low dissolution rate and poor bioavailability Self-emulsifying solid dispersions (SESD) of IDP consisted of surfactant and fatty acid in poloxamer 407 (POX 407) as a carrier and were manufactured by the melting method Then, controlled release HPMC matrix tablet containing SESD were prepared via direct compression The dissolution behaviors and in vivo bioavailability of controlled release matrix tablet in healthy human volunteers were investigated The physical properties of solid dispersion were also examined using differential scanning calorimetry (DSC), powder X-ray diffraction (PXRD) and scanning electron microscopy (SEM) It was shown that structure of IDP was amorphous in the solid dispersion The dissolution rate of IDP from SESD was markedly enhanced because of increased solubility and wetting effect Controlled release HPMC matrix tablets containing SESD released drug in a controlled manner and were stable during storage over months at 40 ◦ C/75% RH Furthermore, the tablet containing mg IDP SESD showed significantly increased oral bioavailability and extended plasma concentration compared with the marketed mg Dynacirc® capsule A combined method of solid dispersion and controlled release technology could provide versatile dosage formulations containing IDP with poor water solubility and short half-life © 2013 Published by Elsevier B.V Solubilization of poorly water-soluble drugs is very important to overcome rate-limiting dissolution, slow absorption and low 28 bioavailability of this drug type Various solubilization strategies 29 therefore, have been developed such as complexation, cosolvents, 30 micelles, microemulsions, self-microemulsifying drug delivery sys31 tems or self-nanoemulsifying drug delivery systems, or solid 32 dispersion (SD) techniques (Wong and Yuen, 2001; Pouton, 2006; 33 Tran et al., 2009) SD among those strategies has been considered as 34 one of common methods to enhance solubility, dissolution rate and 35 36Q2 bioavailability of various poorly water-soluble drugs (Vasconcelos et al., 2007; Tran et al., 2011a) 37 27 ∗ Corresponding authors Tel.: +82 31 219 3442; fax: +82 31 212 3653 E-mail addresses: ttdthao@hcmiu.edu.vn (T.T.-D Tran), beomjinlee@gmail.com (B.-J Lee) Equally contributed However, there are many difficulties associated with the preparation of SD dosage forms as follows: the use of unwanted organic solvent related to the environment in the solvent evaporation method, or the problem of drug stability related with elevated temperatures, and the soft and tacky physical state of the SD product to be hardly pulverized, leading to the use of more pharmaceutical excipients as well as complicated manufacturing procedures to compensate the poor flowing characteristics (Serajuddin, 1999) Self-emulsifying drug delivery systems, especially in the solid state obtained by the addition of some free-flowing adsorbents as one of preferable methods, are in current trends to be investigated due to their advantages over the liquid formulation for improving the bioavailability of hydrophobic drugs and good manufacturability (Serajuddin, 1999; Tang et al., 2008) It was recently reported that a SD utilizing a self-emulsifying carrier like Gelucire 44/14 as exposed to aqueous media could readily modify drug crystallinity and hence, improve drug dissolution rate of poorly water-soluble drug, aceclofenac (Tran et al., 2009) 0378-5173/$ – see front matter © 2013 Published by Elsevier B.V http://dx.doi.org/10.1016/j.ijpharm.2013.04.022 Please cite this article in press as: Tran, P.H.-L., et al., Physical properties and in vivo bioavailability in human volunteers of isradipine using controlled release matrix tablet containing self-emulsifying solid dispersion Int J Pharmaceut (2013), http://dx.doi.org/10.1016/j.ijpharm.2013.04.022 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 ARTICLE IN PRESS G Model IJP 13264 1–8 P.H.-L Tran et al / International Journal of Pharmaceutics xxx (2013) xxx–xxx Table Formulation compositions (weight basis) of SDs containing IDP (unit: mg) Code Drug PEG 6000 SD1 SD2 SD3 SD4 SD5 SD6 SD7 SD8 SD9 SD10 SD11 5 5 5 5 5 60 a 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 97 2.1 Materials 100 101 102 103 POX 407 Brij 98 5 5 5 5 10 60 60 60 50 40 30 60 60 60 60 We recently published that controlled release dosage forms containing self-emulsifying or nonself-emulsifying SDs of many poorly water-soluble drugs with short elimination half-life have been considered as effective drug delivery systems for the treatment of diseases over a longer period of time (Tran et al., 2010, 2011a, 2011b) Advanced controlled release of SDs can be achieved by a pertinent combination of pharmaceutical polymers (Wang et al., 1993) Most of all, HPMC-based hydrophilic matrix tablets offer several advantages in the development of oral sustained-release formulations such as flexibility of release modulation, simplicity of preparation, low production costs and ease to scalability (Cao et al., 2005) The release behavior of both water-soluble and waterinsoluble drugs is variable with the nature of the HPMC matrices as a consequence of the drug–polymer interaction via swelling, diffusion and erosion processes (Colombo et al., 1995; Velasco et al., 1999) In this study, isradipine (IDP), a calcium antagonist for treating hypertension was chosen as a model drug (Chrysant and Cohen, 1997) IDP is known to be poorly water-soluble in aqueous solution (less than 10 g/mL) (Verger et al., 1998) Moreover, IDP is also a good candidate for controlled release dosage form due to the short elimination half-life (Hafizullah et al., 2000) SESD of IDP was prepared using melting method and then loaded into HPMCbased hydrophilic matrix tablet for controlled release of IDP Here, poloxamer 407 (POX 407), a tri-block copolymer consisting of a central hydrophobic block of polypropylene glycol flanked by the two hydrophilic blocks of polyethylene glycol, was used as a carrier to prepare SESD due to its low melting point, good physical properties of facilitating the solubilization of many poorly watersoluble drugs as well as its stabilization (Shin and Cho, 1997; Chutimaworapan et al., 2000) The surface morphology and crystal structure of SESD were characterized using DSC, PXRD and SEM Thereafter, release characteristics of drug from SESDs and the HPMC matrix tablets were then evaluated in enzyme-free simulated intestinal fluid (pH 6.8) The stability of HPMC matrix tablets containing SESD was also investigated under various storage conditions Finally, the controlled released HPMC matrix tablet and the commercially available Dynacirc® capsule as a reference were compared for in vivo bioavailability studies Materials and methods 99 GUC 50/13 OA Triacetin Aerosil 200 BHT 3 3 3 3 0 5 5 5 0 0 17 17 17 17 17 17 17 17 17 17 17 0.1%a 0.1%a 0.1%a 0.1%a 0.1%a 0.1%a 0.1%a 0.1%a 0.1%a 0.1%a 0.1%a w/w percent value based on OA 96 98 PVP K30 IDP as a powder form was obtained from Daewoong Pharmaceutical Corp (Seoul, Korea) Oleic acid (OA), Brij-98, microcrystalline cellulose (Avicel® PH102), hydroxypropylmethylcellulose 4000 (HPMC-4000), polyvinyl pyrrolidone (PVP K30, Kollidon® 30) and poloxamer 407 (POX 407) were obtained from Seoul Pharmaceutical Corp (Seoul, Korea) Aerosil® 200 was purchased from Evonik (Seoul, Korea) Butylated hydroxyl toluene (BHT) was purchased from Sigma (Germany) Dynacirc® CR mg capsule (Daewoong Pharma, Korea) was chosen as a reference IDP formulation All other reagents were of reagent grade and used without further purification 2.2 Method 104 105 106 107 108 109 2.2.1 Solubility study The solubility of IDP was determined in various solvents, surfactants, co-surfactants and oils An excess amount of IDP was added to 1.5 mL snap-cap Eppendorf tube (Hamburg, Germany) containing various additives The resulting mixture was sufficiently mixed and then placed in a constant temperature water bath at 37 ◦ C for days Aliquots were centrifuged at 13,000 rpm for 10 (Hanil, Korea) The supernatant layer was carefully collected and then adjusted with a proper dilution The concentration of IDP was analyzed by a HPLC system as described below 2.2.2 Preparation of SESDs SESDs of IDP using various carriers were prepared by melting method The detailed formulation compositions of SESDs are shown in Table (Code: SD1-SD11) IDP, surfactant and fatty acid were homogenously mixed together based on the formulation compositions The resulting mixtures were slightly heated at various temperatures and sufficiently stirred Thereafter, the melted solution was added to adsorbent (Aerosil® 200) After sufficiently mixing, the mixtures were cooled at −38 ◦ C within h The solidified mass was pulverized thoroughly by a pestle and mortar and finally, passed through a 50 mesh sieve to obtain SESD powders 2.2.3 Preparation of controlled released tablet containing SESD The HPMC-based matrix tablets (150 mg) were prepared by the direct compression method Table shows compositions of the controlled release HPMC matrix tablets The SESD, HPMC polymer and the other excipients were mixed thoroughly with a pestle and mortar The resulting mixtures were directly compressed into tablet using a conventional tablet machine equipped with round punches (8 mm diameter) and a die The tablet hardness was in triplicate Table Formulation compositions (weight basis) for the preparation of HPMC-based controlled release matrix tablets containing SESD (unit: mg) No SESD HPMC 4000 Avicel® PH-102 Total weight T1 T2 T3 T4 T5 T6 90.03 90.03 90.03 90.03 90.03 90.03 7.5 15 30 37.5 45 60 52.5 45 30 22.5 15 150.03 150.03 150.03 150.03 150.03 150.03 Please cite this article in press as: Tran, P.H.-L., et al., Physical properties and in vivo bioavailability in human volunteers of isradipine using controlled release matrix tablet containing self-emulsifying solid dispersion Int J Pharmaceut (2013), http://dx.doi.org/10.1016/j.ijpharm.2013.04.022 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 131 132 133 134 135 136 137 138 G Model IJP 13264 1–8 ARTICLE IN PRESS P.H.-L Tran et al / International Journal of Pharmaceutics xxx (2013) xxx–xxx 139 140 141 142 143 144 145 146 147 148 149 150 151 152 153 154 155 156 157 158 159 160 161 162 163 164 165 166 167 168 169 170 171 172 173 174 175 176 177 178 179 180 181 182 183 184 185 186 187 188 189 190 191 192 193 194 measured using a hardness tester (Model SVM-12, Erweka GmbH, Heusenstamm, Germany) 2.2.4 HPLC analysis of IDP A reverse phase HPLC system was used for the analysis of IDP The HPLC system (Jasco, Tokyo, Japan) consisted of the pump (PU-980), the UV–visible spectrophotometric detector (UV-975), the autosampler (Jasco, AS-950-10), the degasser (DG-980-50), the reverse phase column (Luna m C18 100A, 150 × 4.6 mm) and integrator (Borwin 1.20 software) The concentration of IDP was determined at wavelength of UV 325 nm The mobile phase consisted of a mixture of methanol, deionized water and acetonitrile (7:3:5, v/v ratio) was degassed under vacuum for The flow rate of the mobile phase was mL/min A 20 l of the sample was injected into the HPLC system The stock solution was prepared by dissolving IDP in HPLC-grade ethanol (1 mg/10 mL) and then further diluted with the mobile phase to prepare standard solutions 2.2.5 In vitro dissolution study In vitro dissolution test of the SESD and tablet formulations equivalent to mg IDP was performed according to the USP dissolution II paddle method with a rotation speed of 50 rpm in 900 mL of the enzyme-free simulated intestinal fluid (pH 6.8 ± 0.1) at 37 ± 0.5 ◦ C using a dissolution tester (DCM1, Anyang, Korea) Dissolution samples were collected at 5, 15, 30, 60, 90 and 120 and 1, 2, 3, 4, 6, 8, 10, 12, 16, 20 and 24 h, respectively, with replacement of equal volume of temperature-equilibrated media The sinker was used for dissolution of the tablets The samples were instantly centrifuged at 10,000 rpm for 10 The supernatant of the centrifuged sample was diluted with the mobile phase The concentration of samples was determined by the HPLC system as described previously 2.2.6 Thermal analysis (DSC) The thermal behaviors of pure drug, POX and different SESD formulations were investigated using Dupont DSC (Dupont, USA) About mg of sample was weighed in a standard open aluminum pan; whereas an empty pan of same type was used as reference The samples were heated from 20 to 200 ◦ C at a heating rate of 10 ◦ C/min under purged dry nitrogen Calibration of temperature and heat flow was performed with indium 2.2.7 Powder X-ray diffraction (PXRD) Powder X-ray diffraction patterns were obtained for the samples of pure drug, POX and different SESD formulations using a D5005 (Bruker, Germany) with Cu-K radiation at 40 kV 50 mA The samples were scanned in steps of 0.02◦ from 3◦ to 40◦ with a rate of one second per step, using a zero background sample holder 2.2.8 Scanning electron microscope (SEM) Scanning electron microscopy was used to characterize the surface morphology and particle shape of the samples The samples were examined using a JSM-5410 (Jeol, Japan), at an acceleration voltage of 15 kV The samples were coated with a thin layer of gold for 10 2.2.9 Stability study The HPMC matrix tablets bearing SESD were stored for months in a plastic bottle with silica gel at 40 ◦ C/75% RH (relative humidity) The hardness and dissolution profiles for initial and stored samples were tested at the given period of time 2.3 In vivo comparative bioavailability in healthy human volunteers 2.3.1 Study design Eight healthy human volunteers aged 20–30 years old and weighing from 60 to 70 kg were participated in this study after submitting a written informed consent Document review and approval from a formally constituted Institutional Review Board in Kangwon National University were permitted The study was performed according to the revised declaration of Helsinki for biomedical research involving human subjects and the rules of Good Clinical Practice The in vivo bioavailability was carried out under the bioequivalence guidelines (KFDA 2008-25) according to the Korean Food & Drug Administration The eight volunteers were randomly divided into two groups The current controlled release tablets containing SESD and marketed Dynacicr® capsules equivalent to mg IDP were orally given to human volunteers with 250 mL of water for comparatibe bioavailability Food and drinks were withheld for at least h after dosing Standardized lunch and dinner were served h after dosing All subjects were prohibited from strenuous activity and consuming alcoholic drinks during the study Blood samples (10 mL) were withdrawn through an indwelling three-way catheter in the forearm and collected in heparin-loaded vacutainers at 0, 0.5, 1, 1.5, 2, 3, 4, 6, 8, 10, 12, 24 and 36 h after dosing The blood samples were centrifuged for 10 at 3000 rpm The collected samples were kept frozen at −70 ◦ C until analysis 2.3.2 Assay of IDP in human plasma The LC/MS/MS system was used for the analysis of IDP The LC/MS/MS system consisted of the HPLC (PerkinElmer Series 200, Boston, USA), the autosampler (CTC analytic SPA, Zwingen, Switzerland), the MS/MS (Applied Biosystems API 4000, Boston, USA), and the column (Capcell PAK UG120, 2.0 mm × 150 mm, 5.0 m pore size) The mobile phase consisted of 20% mM ammonium acetate and 80% acetonitrile (pH 6.0 with acetic acid) Felodipine was used as an internal standard The flow rate of the mobile phase was 0.2 mL/min For analysis of IDP in human plasma, 300 L of plasma, 50 L (20 ng/mL) of internal standard and 30 L of 10% ammonium hydroxide were put into test tube and mixed 10 s Two millliliters of ethyl ether was then added and mixed for 20 The resulting solution was centrifuged at 1500 rpm for The 20 L of the supernatant layer injected to the LC/MS/MS system 2.3.3 Pharmacokinetic analysis Non-compartmental pharmacokinetic analysis was performed The maximum plasma concentration of IDP (Cmax ) and time to reach Cmax (Tmax ) after the oral administration were directly determined from plasma concentration–time curves The area under the plasma concentration–time curve (AUC0–36 h ) from zero to 36 h was computed using the linear trapezoidal rule All data were expressed as mean ± S.D 2.3.4 Statistical analysis Logarithmically transformed or untransformed (arithmetic) AUC0–36 h and Cmax was used for statistical analysis of variance (ANOVA) using SPSS® for windows software and K-BE test® program, respectively The drug, period, group and subject nested within group were included in statistical model The Tmax was also analyzed as a reference All statistical calculations were performed at 5% significance level The confidence interval of pharmacokinetic parameters between the two preparations was allowed within 80–125% Please cite this article in press as: Tran, P.H.-L., et al., Physical properties and in vivo bioavailability in human volunteers of isradipine using controlled release matrix tablet containing self-emulsifying solid dispersion Int J Pharmaceut (2013), http://dx.doi.org/10.1016/j.ijpharm.2013.04.022 195 196 197 198 199 200 201 202 203 204 205 206 207 208 209 210 211 212 213 214 215 216 217 218 219 220 221 222 223 224 225 226 227 228 229 230 231 232 233 234 235 236 237 238 239 240 241 242 243 244 245 246 247 248 249 250 251 252 253 254 G Model IJP 13264 1–8 ARTICLE IN PRESS P.H.-L Tran et al / International Journal of Pharmaceutics xxx (2013) xxx–xxx 100 700 600 500 400 80 200 % Released solubility (ppm) 300 100 60 raw IDP powder SD1: PEG-6000 SD2: PVP K30 SD3: Gelucire 50/13 SD4: poloxamer 407 40 20 ter 1.2 6.8 -97- 9i8j- 5i8j- 35r ELH40SLSn-80n-60n-2c0etin600400000/19334pr407r18c8elinacidacidacidacid wapH pH BrijBrijB r Br ho r R ee ee ee ia G- G- e ol- me me ly ic ic lic ric p o Tw Tw Tw tr PE PElucirboploxaloxa g olienolaeprycap mooph l c Ge ca po po crreem c Fig The solubility of IDP in various pharmaceutical excipients 0 20 40 60 80 100 120 Time (min) 256 (logarithmic value) or 80–120% (arithmetic value) for bioequivalence, respectively 257 Results and discussion 258 3.1 Solubility study 255 259 260 261 262 263 264 265 266 267 268 269 270 271 272 273 274 275 276 277 278 279 280 281 282 283 284 285 286 287 288 289 The effects of pH, solubilizers and fatty acid on drug solubility were investigated at 37 ◦ C as shown in Fig The solubility of drug upon various pH conditions was very low On the other hand, all of the surfactants and co-surfactants in general had a tendency to enhance the drug solubility Especially, surfactants such as triacetin, Brij 98, SLS and POX 407 showed their potential capability of enhancing drug solubility remarkably Because solubility of IDP in POX 407 was about 1200 times higher than that in water, it was included in the SESD formulations as a good carrier Besides, other carriers such as PEG 6000, PVP K30 and GUC 50/13 were also compared in the SESD formulations 3.2 Effect of formulation compositions in SESDs on drug dissolution rate 3.2.1 Effect of carriers The effect of carrier types on drug dissolution rate in intestinal fluid (pH 6.8) is shown in Fig Dissolution rate of pure drug was very low (