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β-cyclodextrin inclusion complex
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International Journal of Pharmaceutics journal homepage: www.elsevier.com/locate/ijpharm
Physicochemical characterization and pharmacokinetics evaluation of -caryophyllene/-cyclodextrin inclusion complex
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Hua Liu a,1 , Guang Yang a,1 , Yuanjun Tang a , Di Cao a,b , Tian Qi a,c , Yunpeng Qi a,∗ , Guorong Fan a,∗ a Shanghai Key Laboratory for Pharmaceutical Metabolite Research, School of Pharmacy, Second Military Medical University, No. 325 Guohe Road, Shanghai 200433, PR China b Department of Pharmaceutical Analysis, School of Pharmacy, Guangdong Pharmaceutical University, Guangzhou 510006, PR China c Department of Pharmacognosy, School of Pharmacy, Anhui University of Traditional Chinese Medicine, No. 45 Shihe Road, Hefei 230031, PR China
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Article history: Received 4 January 2013 Received in revised form 26 March 2013 Accepted 9 April 2013 Available online xxx
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Keywords: Essential oil Caryophyllene GC–MS–MS Pharmacokinetics Cyclodextrin inclusion complex Bioavailability
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1. Introduction
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-Caryophyllene (BCP), a natural sesquiterpene existing in the essential oil of many plants, has exhibited a wide range of biological activities. However, its volatility and poor water-solubility limit its application in pharmaceutical field. -Cyclodextrin (-CD) has intrinsic ability to form specific inclusion complexes with different drugs to enhance their stability, solubility and bioavailability. The aim of this study is to investigate and compare the oral bioavailability and the pharmacokinetics of free BCP and BCP/-CD inclusion complex after a single oral dose of 50 mg/kg on rats. A simple, rapid, and sensitive gas chromatography–mass spectrometry method in selected ion monitoring (GC–MS/SIM) mode was developed on determination of BCP in rat plasma. The in vivo data showed that BCP/-CD inclusion complex displayed earlier Tmax , higher Cmax and the AUC0–12 h was approximately 2.6 times increase than those of free BCP. These results demonstrated that BCP/-CD inclusion complex has significantly increased the oral bioavailability of the drug in rats than free BCP. © 2013 Published by Elsevier B.V.
-Caryophyllene (BCP) (Fig. 1) is a bicyclic sesquiterpene compound existing in the essential oil of many plants, e.g. Eugenia caryophyllata (Fam. Myrtaceae), Piper nigrum L. (Fam. Piperaceae), and Zingiber nimmonii (J. Graham) Dalzell (Fam. Zinziberaceae). Due to its woody and spicy odour, BCP has been commonly used as a fragrance and flavouring agent since the 1930s (Di Sotto et al., 2010; Sabulal et al., 2006). In recent years, this compound has attracted considerable attention because of its extensive biological activities including antimicrobial (Sabulal et al., 2006), anticarcinogenic (Di Sotto et al., 2010), anti-inflammatory, anti-oxidant (Cho et al., 2007; Fernandes et al., 2007; Horváth et al., 2012), anxiolyticlike and local anaesthetic effects (Galdino et al., 2012). However, this compound is volatile, poorly water-soluble, and sensitive to light, oxygen, humidity and high temperature (Ponce Cevallos et al., 2010; Sköld et al., 2006; Wang et al., 2011). Among them, poor water-solubility may substantially decrease bioavailability of the
∗ Corresponding authors. Tel.: +86 21 8187 1260; fax: +86 21 8187 1260. E-mail addresses: [email protected] (Y. Qi), [email protected] (G. Fan). 1 These authors have equal contribution to this work. Both persons are the first authors.
drug and hence limit its further application in pharmaceutical field. Therefore, developing delivery system for drugs such as BCP to increase their solubility and dissolution rate so as to improve the bioavailability has been an important task in pharmaceutical development (Wempe et al., 2008). In recent years, cyclodextrin (CD) complexation has been successfully applied to improve the solubility, chemical stability, and bioavailability of a number of poorly soluble compounds (Hwang et al., 2012; Liu et al., 2006; Wempe et al., 2008). CDs are non-toxic macrocyclic oligosaccharides composed of 6, 7, 8 or 9 glucopyranose units (namely ␣-, -, ␥- or ␦-CD, respectively) with a relatively hydrophilic surface and hydrophobic central cavity. Through host–guest interactions with the organic molecules, CDdrug inclusion complex is formed so that the included drugs can be protected against hydrolyse, oxidation, photodecomposition and dehydration (Hwang et al., 2012). -CD as attractive materials for drug inclusion, giving prominence to its low biotoxicity and high biocompatibility, has been successfully used to increase the stability, solubility, and bioavailability of poorly soluble compounds in oral drug delivery (Gao et al., 2012; Hwang et al., 2012; Liao, 2010; Liu et al., 2006; Piette et al., 2006). The objective of the present study is to investigate and compare the pharmacokinetic characteristics of free BCP and its -CD inclusion complex in rat plasma after oral administration. The
0378-5173/$ – see front matter © 2013 Published by Elsevier B.V. http://dx.doi.org/10.1016/j.ijpharm.2013.04.013
Please cite this article in press as: Liu, H., et al., Physicochemical characterization and pharmacokinetics evaluation of -caryophyllene/cyclodextrin inclusion complex. Int J Pharmaceut (2013), http://dx.doi.org/10.1016/j.ijpharm.2013.04.013
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same temperature and rate. Then the solution was cooled to room temperature and maintained overnight at 4 ◦ C. Finally, the cold precipitated BCP/-CD inclusion complex was recovered by vacuum filtration. The precipitate was washed with light petroleum to remove BCP which was absorbed on the surface of -CD and then dried in baking oven at 40 ◦ C until the weight kept constant. The dried powder (3.2468 g) was stored in an airtight desiccator at room temperature. The recovery of inclusion complex was calculated according to Eq. (1):
Fig. 1. Chemical structure of -caryophyllene.
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inclusion complex of BCP/-CD in this study was prepared by the coprecipitation method and characterized by different analytical techniques including ultraviolet–visible spectrophotometry (UV–vis), differential thermal analysis (DTA) and fourier transform infrared spectroscopy (FT-IR) (Hwang et al., 2012; Liu et al., 2006; Ponce Cevallos et al., 2010; Wang et al., 2011). Moreover, its in vitro dissolution study was performed in the mediums of hydrochloric acid (0.1 mol/L) and phosphate buffer (pH 6.8) according to the Chinese Pharmacopoeia (Ch.P) and United States PharmacopoeiaNational Formulary (USP-NF) (Hwang et al., 2012; Liu et al., 2006; Wang et al., 2011; Wempe et al., 2008). GC (Dias et al., 2012; Liu et al., 2007, 2009; Sun et al., 2006; Wang et al., 2010) and GC–MS (Qin et al., 2007) methods which have been applied to analyse BCP in the essential oil of traditional Chinese medicine were used to determine BCP in rat plasma in our study. To the best of our knowledge, this is the first pharmacokinetic study on free BCP and BCP/-CD inclusion complex in rat plasma by a simple, rapid, and sensitive GC–MS/SIM method. The pharmacokinetic parameters of both formulations were analysed by non-compartmental model (Chu et al., 2008; Dong et al., 2007; Spichiger et al., 2004; Wang et al., 2007, 2008).
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2. Materials and methods
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2.1. Chemicals and reagents BCP (purity > 90%) was purchased from TCI Chemical Industry Development Co., Ltd (Shanghai, China). Eucalyptol (internal standard; purity ≥ 99.0%) was purchased from Sigma–Aldrich (Fluka Analytical, USA). -CD was purchased from Shandong Xinda Fine Chemical Co., Ltd (Shandong, China). High performance liquid chromatography (HPLC) grade ethyl acetate (Lot #54295; purity ≥ 99.8%) was purchased from Caledon Inc. (Canada). Anhydrous sodium sulfate (Na2 SO4 ), hydrochloric acid and spectrograde potassium bromide (KBr) were purchased from Sinopharm Chemical Reagent Co., Ltd (Shanghai, China). Analytical grade ethanol, potassium dihydrogen phosphate, sodium hydroxide, and light petroleum were purchased from Jiangsu Qiangsheng Chemical Co., Ltd (Jiangsu, China). The distilled water was prepared by Milli-Q academic water purification system. 2.2. Preparation and characterization of BCP/ˇ-CD inclusion complex 2.2.1. Preparation of BCP/ˇ-CD inclusion complex The BCP/-CD inclusion complex was prepared by the coprecipitation method. Briefly, -CD (3.0004 g) and 50 mL water were added into a round bottomed flask which was placed in a constant temperature magnetic stirring apparatus. After stirring for an hour at 50 ◦ C, the -CD saturated solution was obtained. 0.5 mL BCP (452.5 mg) dissolved in ethanol (0.5 mL) was decade to -CD saturated solution with continuous agitation for an hour at the
Recovery (%) =
mass of dried powder × 100 mass of added (-CD + BCP)
(1)
In order to characterize inclusion complex, the physical mixture of -CD and BCP was prepared by mixing -CD and BCP at a ratio of 6:1 (m/v) in a glass mortar until a homogeneous mixture. 2.2.2. Characterization of BCP/ˇ-CD inclusion complex using spectrometry 2.2.2.1. UV–vis spectrophotometry. The formation of BCP/-CD inclusion complex was demonstrated by a UV–vis spectrophotometer (Varian, USA). BCP (2 g/mL) was prepared in ethanol. The -CD, physical mixture and inclusion complex (30 mg) were added to ethanol (5 mL), respectively, and then the mixture was shaken for 10 min. The supernatant was separated by centrifugation and then diluted by 100 times in ethanol, and scanned in the range of 200–800 nm to obtain the UV–vis absorption spectrum. 2.2.2.2. Differential thermal analysis (DTA). DTA curves of -CD, BCP, their physical mixture and inclusion complex were measured with a Diamond TG/DTA instrument (Perkin Elmer, USA). Samples were accurately weighed and heated in a sealed aluminium pan at a rate of 5 ◦ C/min from 40 to 335 ◦ C under a nitrogen flow of 40 mL/min. An empty sealed pan was used as the reference. Duplicate determinations were carried out for each sample. 2.2.2.3. Fourier transform infrared spectroscopy (FT-IR). The FT-IR spectra of -CD, BCP, their physical mixture and inclusion complex were collected from 4000 to 400 cm−1 on a Nicolet 550-II FT-IR spectrophotometer (Nicolet, USA) with 32 scans at a resolution of 4 cm−1 . -CD, physical mixture and inclusion complex were respectively mixed with spectrograde KBr powder at a mass ratio of 1:100. Then they were ground and pressed to discs of diameters of 8 mm. A drop of BCP was spread on a piece of KBr window uniformly then nipped with another piece of KBr window. FT-IR spectra were analysed by the spectrophotometer software (OMNIC 5.2). 2.3. Determination of inclusion ratio of BCP in the inclusion complex
mass of recovered BCP × 100 mass of added BCP
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Steam distillation (SD) method (Lu et al., 2004; Xu et al., 2011) was used to determine BCP in the inclusion complex. 0.5 mL BCP (452.5 mg) and 150 mL water were added into a round bottomed flask heated and connected with essential oil extractor. BCP was gradually distilled until its volume kept constant. Then it was weighed (373.7 mg) after removing water with Na2 SO4 thereby blank recovery could be calculated as Eq. (2). Similarly, the amount of BCP in inclusion complex was obtained and the inclusion ratio was calculated as Eq. (3). Blank recovery (%) =
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(2)
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mass of recovered BCP in inclusion complex ×100 Inclusion ratio (%) = mass of added BCP in inclusion complex × blank recovery
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(3)
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Please cite this article in press as: Liu, H., et al., Physicochemical characterization and pharmacokinetics evaluation of -caryophyllene/cyclodextrin inclusion complex. Int J Pharmaceut (2013), http://dx.doi.org/10.1016/j.ijpharm.2013.04.013
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2.6. Method validation
Fig. 2. Full mass spectra of (A) eucalyptol, (B) BCP.
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Stock solutions of BCP (1.060 mg/mL) and eucalyptol (1.007 mg/mL) were prepared in ethyl acetate and stored at 4 ◦ C. The BCP stock solution was further diluted with ethyl acetate to obtain a set of standard working solutions with a concentration range of 0.53–53.0 g/mL. Internal standard (IS) solution was prepared by dilution of its stock solution to 5.035 g/mL with ethyl acetate. The specificity of the method was assessed by comparing the blank plasma spiked with BCP (0.053 g/mL) with blank plasma. BCP standard curve samples (0.053, 0.106, 0.212, 0.53, 1.06, 2.12, 5.30 g/mL) were prepared by the addition and complete mixing of 10 L standard working solution, 10 L IS solution and 90 L blank plasma. After being extracted, they were injected into GC–MS system and peak area ratio of BCP versus IS was calculated. Quality control samples (QCs) for evaluating the accuracy and precision of the method were prepared at low (0.106 g/mL), medium (0.53 g/mL), and high (4.24 g/mL) concentrations. Extraction yield was determined by the peak area ratio of BCP from extracted QCs and unextracted standards. Stability was investigated in room temperature, three successive freeze–thaw cycles and autosampler by three replicates of QCs. Analyte was considered to be stable if the peak area ratios of BCP versus IS of processed samples varied within ±15%.
2.4. In vitro dissolution study
2.7. In vivo experiments protocol and sample preparation
In vitro dissolution study of BCP/-CD inclusion complex was performed in triplicate according to Ch.P (2010 Edition, Part 2, Appendix XC. No.2 method) and USP 30-NF 25 (2007 Edition, Volume 1, <711> Apparatus 2) using a Rcz-602 Dissolution Apparatus (Shanghai Huanghai Medicine Checking Instrument Co., Ltd). However, according to the definition of dissolution, the dissolution study of free BCP was not necessary to be carried out because it was oleosus liquid. Briefly, 115.61 mg of BCP/-CD inclusion complex was added to 900 mL mediums of hydrochloric acid (0.1 mol/L) and phosphate buffer (pH 6.8) at 37 ± 0.5 ◦ C then stirred at 50 rpm. At predetermined time intervals, 5 mL of the medium was withdrawn and centrifuged at 12,000 rpm for 5 min. Then the drug content in the supernatant was assayed at 205 nm using UV spectrometry. The accumulative release rate was calculated at each time point.
SD rats (180–200 g, male and female half and half) were purchased from Shanghai Slac Laboratory Animal Co., Ltd (Shanghai, China), and were housed under standard conditions with free access to food and water. Before administration, rats were on overnight fast with free access to water. Then, they were randomly and equally divided into two groups (each consists of 6 rats). One group received free BCP and the other group received BCP/-CD inclusion complex. BCP and BCP/-CD inclusion complex were both dissolved in water and the rats were orally administrated at a dose of 50 mg/kg. Blood samples (0.5 mL) were collected from the orbit venous plexus into heparinized Eppendorf tubes at 0.167, 0.333, 0.5, 0.75, 1, 1.5, 2, 3, 4, 6, 8, 10 and 12 h after administration. The plasma was separated through centrifugation at 12,000 rpm for 10 min and stored at −20 ◦ C until analysis. After that, 100 L plasma sample, 10 L IS solution and 90 L ethyl acetate were mixed in 5 mL glass centrifuge tube. After vortexing for 5 min, the sample was centrifuged at 3200 rpm for 10 min and the supernatant (60 L) was transferred to a vial for GC–MS analysis.
2.5. GC–MS instrument and conditions The analysis was performed on a GC–MS system consists of an AS3000 autosampler, a Trace GC Ultra, and an ITQ1100 mass spectrometer (ThermoFisher Scientific, USA). A TR-5MS capillary column (30 m × 0.25 mm i.d.) coated with a 0.25 m thick film of 5% phenyl methyl siloxane (Thermo, USA) was used. The inlet temperature was set at 250 ◦ C. The oven temperature was initially held at 100 ◦ C, then was programmed to 140 ◦ C at 15 ◦ C/min, holding for 0.5 min, followed by 30 ◦ C/min to 280 ◦ C and held for 3 min. Helium (purity > 99.999%) was used as carrier gas at a constant flow rate of 1.0 mL/min. The sample volume was 1 L and the inlet was in the split mode with a split ratio of 10:1. Ionization was carried out in electron impact (EI) mode at 70 eV. The ion source temperature and transfer line temperature were 250 ◦ C and 280 ◦ C, respectively. Solvent was delayed for 2.2 min. The detection was operated in SIM mode: from 2.2 to 4.0 min m/z 93 (eucalyptol) and from 4.0 to 5.5 min m/z 91 (BCP) were respectively selected for quantitation as their relative abundance was the highest in individual full mass spectrum (Fig. 2). Data were collected and analysed by Xcalibur 2.0 workstation.
2.8. Pharmacokinetic analysis The pharmacokinetic parameters of free BCP and BCP/-CD inclusion complex in SD rats were calculated by BAPP software (China Pharmaceutical University, Nanjing, China) using noncompartment model analysis and their statistical comparisons were made by two-sample t-test (SPSS, Chicago), and p value less than 0.05 was considered to be statistically significant. All data were presented as mean value with its standard deviation (mean ± S.D.).
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3. Results and discussion
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3.1. Characterization of BCP/ˇ-CD inclusion complex
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3.1.1. UV–vis spectrophotometry The UV–vis absorption spectra were recorded for BCP, -CD, their physical mixture and inclusion complex. The spectra of the physical mixture (Fig. 3A) and BCP (2 g/mL) (Fig. 3B) were identical with maximum absorption wavelength (max ) at 205 nm, since -CD (Fig. 3C) in ethanol had no UV absorption. However, the
Please cite this article in press as: Liu, H., et al., Physicochemical characterization and pharmacokinetics evaluation of -caryophyllene/cyclodextrin inclusion complex. Int J Pharmaceut (2013), http://dx.doi.org/10.1016/j.ijpharm.2013.04.013
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Fig. 3. UV spectra of (A) physical mixture of -CD and BCP, (B) BCP (2 g/mL), (C) -CD, and (D) BCP/-CD inclusion complex.
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absorption peak was not observed in the spectrum of inclusion complex (Fig. 3D). These results indicated that BCP had formed inclusion complex with -CD. 3.1.2. DTA DTA is an important tool to study the formation of inclusion complex (Hwang et al., 2012; Liu et al., 2006; Ponce Cevallos et al., 2010; Wang et al., 2011). The thermal curves of BCP, -CD, their physical mixture and inclusion complex were shown in Fig. 4. A broad endothermic peak near 125 ◦ C appeared in the thermogram of BCP (Fig. 4A), corresponding to its boiling point. The thermogram of -CD (Fig. 4B) presented three endothermic peaks at 82, 273, and 310 ◦ C, respectively, which were likely to be related to the loss of water molecules originally binding to cyclodextrin molecules. The DTA curve of physical mixture (Fig. 4C) clearly exhibited superimposition of -CD and BCP thermograms. In Fig. 4D, however, the peak of BCP disappeared and the peaks originally in the -CD also changed, offering the proof for the formation of BCP/-CD inclusion complex. 3.1.3. FT-IR Fig. 5 presented the FT-IR spectra of BCP, -CD, their physical mixture and inclusion complex. The spectrum of BCP (Fig. 5A)
Fig. 5. FT-IR spectra of (A) BCP, (B) -CD, (C) physical mixture of -CD and BCP, and (D) BCP/-CD inclusion complex. The arrow points for the peak at 886 cm−1 .
consisted of the prominent absorption bands of stretching vibration (3067 cm−1 ) and scissoring vibration (1448 cm−1 ) of CH, asymmetrical stretching vibration (2926 cm−1 ) and symmetrical stretching vibration (2857 cm−1 ) of CH2 , and stretching vibration of C C (1633 cm−1 ). The double peaks (1382 and 1367 cm−1 ) of symmetrical deformation vibration of CH3 were ascribed to two methyls connecting to the same carbon atom. The very intense peak at 886 cm−1 was characteristic absorption band of BCP assigned to out-of-plane deformation vibration of CH. The spectrum of CD (Fig. 5B) displayed prominent absorption bands at 3384 cm−1 for O H stretching vibration, 2927 cm−1 for CH2 asymmetrical stretching vibration, 1648 cm−1 for H O H bending vibration, and 1157 cm−1 for C O C asymmetrical stretching vibration. The spectrum of physical mixture (Fig. 5C) presented the characteristic absorption band of BCP at 886 cm−1 , though its intensity was weak. However, there was not any feature similar to BCP when examining the spectrum of inclusion complex (Fig. 5D). These changes were related to the formation of intra-molecular hydrogen bonds between BCP and -CD. 3.2. Determination of inclusion ratio of BCP The recovery of inclusion complex and inclusion ratio of BCP are used as indexes to evaluate inclusion effect (Han and Zhang, 2011; Zhang and Liu, 2012). In our study, the blank recovery of BCP was 82.59%, and the content of recovered BCP in the BCP/CD inclusion complex was 231.84 mg. Using formulas (2) and (3), the recovery of inclusion complex and inclusion ratio of BCP were 94.03% and 62.04%, respectively, suggesting an acceptable inclusion ratio of BCP. 3.3. In vitro dissolution study
Fig. 4. DTA curves of (A) BCP, (B) -CD, (C) physical mixture of -CD and BCP, and (D) BCP/-CD inclusion complex.
The dissolution profiles of BCP/-CD inclusion complex were shown in Fig. 6. They were characterized by an initial fast release followed by a relatively slow release until the constant value. After 90 min, approximately 90% and 60% of BCP were released from inclusion complex into the mediums of hydrochloric acid (0.1 mol/L) and phosphate buffer (pH 6.8), respectively. The different dissolution of BCP in two mediums might be related to its physical property and the microenvironment around it and the rapid release of BCP was attributed to the fact that -CD had the ability to improve wettability of inclusion complex in dissolution mediums so that BCP was released more and faster from inclusion
Please cite this article in press as: Liu, H., et al., Physicochemical characterization and pharmacokinetics evaluation of -caryophyllene/cyclodextrin inclusion complex. Int J Pharmaceut (2013), http://dx.doi.org/10.1016/j.ijpharm.2013.04.013
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Table 1 Accuracy and precision results of BCP. Sample
Added (g/mL)
LQCa MQCb HQCc a b c d e f
Inter-dayf
Intra-day
0.106 0.53 4.24
Found ± S.D.
Accuracy
Precision
Found ± S.D.
Accuracy
Precision
0.110 ± 0.003 0.49 ± 0.02 4.28 ± 0.18
102.69 ± 3.24 91.73 ± 3.53 100.92 ± 4.32
3.16 3.85 4.28
0.105 ± 0.01 0.48 ± 0.02 4.11 ± 0.19
99.00 ± 4.90 90.65 ± 2.99 96.98 ± 4.49
4.95 3.30 4.63
d
e
LQC: low quality control. MQC: medium quality control. HQC: high quality control. Accuracy is given as recovery (%) ± S.D. Precision is given as percent of the relative standard deviation (R.S.D.%). Three days, five replicates per concentration per day.
3.4.2. Linearity The calibration curve showed good linearity within the range of 0.053–5.30 g/mL. Mean linear equation of calibration curves (n = 3) for BCP was (y = (0.000272267 ± 0.01) + (3.1421 ± 0.19)x, r2 = 0.9975, weighting: 1/x), where y was the peak area ratio of BCP versus IS and x was the concentration of BCP.
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Accumulative Release (%)
90 80 70 60
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50 40
hydrochloric acid (0.1 mol/L)
30
phosphate buffer (pH 6.8)
3.4.3. Accuracy and precision Table 1 showed that the method had good accuracy and precision. The relative recoveries for all samples were >90%. The intraand inter-day precision ranged from 3.16% to 4.95%.
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Time (min) Fig. 6. Mean dissolution profiles of BCP/-CD inclusion complex in the mediums of hydrochloric acid (0.1 mol/L) and phosphate buffer (pH 6.8) (n = 3).
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complex. The improved dissolution of BCP denoted increased oral bioavailability.
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3.4. Method validation
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3.4.1. Specificity Typical chromatograms of blank plasma, blank plasma spiked with BCP at the low limit of quantitation (LLOQ, 0.053 g/mL), and a real rat plasma sample were shown in Fig. 7. It was shown that no endogenous substances in plasma interfered with the assay of BCP.
3.4.4. Extraction yield and stability The extraction yields of BCP were 85.09 ± 3.14%, 83.52 ± 6.33%, and 80.87 ± 5.18% at the concentration of 0.106, 0.53, and 4.24 g/mL, respectively. Under room temperature for 3 h and being subjected to three freeze–thaw cycles, LQC and HQC samples remained stable. No instability of the extracted samples was observed for 24 h in autosampler. The stability results were listed in detail in Table 2. 3.5. Pharmacokinetics
Table 2 Summary of the stability of BCP. Initial mean area ratio
Found ± S.D.
Room temperature for 3 h LQC 0.106 4.24 HQC
0.324 13.218
0.332 ± 0.01 13.305 ± 0.24
2.69 ± 3.89 0.66 ± 1.85
Three freeze–thaw cycles LQC 0.106 4.24 HQC
0.335 13.400
0.338 ± 0.01 13.551 ± 0.23
0.95 ± 1.79 1.13 ± 1.74
Autosampler for 24 h 0.106 LQC 4.24 HQC
Fig. 7. Chromatograms of (A) blank plasma, (B) blank plasma spiked with BCP (0.053 g/mL) and IS, and (C) plasma sample at 0.5 h after oral administration of BCP/-CD inclusion complex (50 mg/kg); (1) IS, (2) BCP.
0.326 13.714
0.313 ± 0.003 13.654 ± 0.14
a
Added (g/mL)
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Mean plasma concentration versus time profiles of free BCP and BCP/-CD inclusion complex formulations were illustrated in Fig. 8. Pharmacokinetic parameters were summarized in Table 3. By comparing the results of two formulations, BCP/-CD inclusion complex had significant decrease at Tmax , T1/2 , MRT and increase at Cmax , AUC0–12 h and AUC0–∞ . Moreover, the AUC0–12 h after administration of BCP/-CD inclusion complex was about 2.6 times higher than that after administration of free BCP, suggesting that relative oral bioavailability of BCP in inclusion complex increased about 2.6 times. These results demonstrated that BCP/-CD inclusion complex significantly increased the oral bioavailability in rats comparing to free BCP.
Sample
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Stabilitya
−3.97 ± 1.06 −0.44 ± 1.04
Stability is given as relative error (%) ± S.D.
Please cite this article in press as: Liu, H., et al., Physicochemical characterization and pharmacokinetics evaluation of -caryophyllene/cyclodextrin inclusion complex. Int J Pharmaceut (2013), http://dx.doi.org/10.1016/j.ijpharm.2013.04.013
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Concentration (μg/mL)
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Acknowledgement
1.00 0.90 0.80 0.70 0.60 0.50 0.40 0.30 0.20 0.10 0.00
This study was financially supported by the National Natural Science Foundation of China (No. 81173019).
Free BCP BCP/β-CD
References
0
2
4
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8
10
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Time (h) Fig. 8. Mean (±S.D.) plasma concentration–time profiles of BCP after oral administration of free BCP or BCP/-CD inclusion complex at a dose of 50 mg/kg (n = 6).
Table 3 Summary of pharmacokinetic parameters. Free BCP a
Cmax (g/mL) Tmax (h)b T1/2 (h)c MRT (h)d AUC0–12 h (g h/mL)e AUC0–∞ (g h/mL) a b c d e *
364 365 366 367 368 369 370 371 372 373 374 375 376 377 378 379 380
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0.12 3.50 4.07 7.69 1.05 1.28
± ± ± ± ± ±
0.02 0.60 0.07 0.13 0.08 0.10
BCP/-CD 0.56 2.80 3.25 5.63 2.72 2.96
± ± ± ± ± ±
0.35 0.80 0.65 0.93 1.22 1.16
p-Value* 0.027 0.035 0.026 0.003 0.020 0.016
Cmax : maximum plasma concentration. Tmax : time to reach maximum plasma concentration. T1/2 : half-time. MRT: mean residence time. AUC: the area under the concentration–time curve. p < 0.05 is considered to be statistically significant.
The enhancement of oral bioavailability for BCP in the form of BCP/-CD inclusion complex may be ascribed to the increase of its dissolution and water-solubility since BCP could be embedded into the -CD cavity and -CD has hydrophilic surface (Gu et al., 2012; Liao et al., 2009). Stability constant of BCP in inclusion complex is relative small, thus it could be released rapidly from inclusion complex in vivo (Tang et al., 1992). Additionally, the high biocompatibility of -CD makes BCP being absorbed more in gastro-intestinal tract. Therefore, -CD is a safe vector to increase drug’s solubility and bioavailability, and the development of BCP delivery system can greatly improve its absorption. According to our results, the standard deviation of pharmacokinetic parameters of different rats after oral administration of inclusion complex were relatively large, which prompted that the individual difference of BCP absorption may exist. This would be a problem in determining the effective dose and hence still needs further study. 4. Conclusions In this study, BCP/-CD inclusion complex was successfully prepared by the coprecipitation method and characterized by spectrophotometry including UV–vis spectrophotometry, DTA, and FT-IR. Moreover, its in vitro dissolution study was performed and BCP was rapidly released from inclusion complex. For the first time a simple, rapid, and sensitive GC–MS/SIM method for determination of BCP in rat plasma was developed. Comparison on pharmacokinetic parameters of free BCP and its inclusion complex indicated that BCP/-CD inclusion complex had earlier Tmax , higher Cmax and better bioavailability. In conclusion, the results demonstrated that the formation of BCP/-CD inclusion complex could greatly improve the oral bioavailability of BCP.
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Contents lists available at SciVerse ScienceDirect
International Journal of Pharmaceutics journal homepage: www.elsevier.com/locate/ijpharm
Physicochemical characterization and pharmacokinetics evaluation of -caryophyllene/-cyclodextrin inclusion complex
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Hua Liu a,1 , Guang Yang a,1 , Yuanjun Tang a , Di Cao a,b , Tian Qi a,c , Yunpeng Qi a,∗ , Guorong Fan a,∗ a Shanghai Key Laboratory for Pharmaceutical Metabolite Research, School of Pharmacy, Second Military Medical University, No. 325 Guohe Road, Shanghai 200433, PR China b Department of Pharmaceutical Analysis, School of Pharmacy, Guangdong Pharmaceutical University, Guangzhou 510006, PR China c Department of Pharmacognosy, School of Pharmacy, Anhui University of Traditional Chinese Medicine, No. 45 Shihe Road, Hefei 230031, PR China
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Article history: Received 4 January 2013 Received in revised form 26 March 2013 Accepted 9 April 2013 Available online xxx
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Keywords: Essential oil Caryophyllene GC–MS–MS Pharmacokinetics Cyclodextrin inclusion complex Bioavailability
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1. Introduction
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-Caryophyllene (BCP), a natural sesquiterpene existing in the essential oil of many plants, has exhibited a wide range of biological activities. However, its volatility and poor water-solubility limit its application in pharmaceutical field. -Cyclodextrin (-CD) has intrinsic ability to form specific inclusion complexes with different drugs to enhance their stability, solubility and bioavailability. The aim of this study is to investigate and compare the oral bioavailability and the pharmacokinetics of free BCP and BCP/-CD inclusion complex after a single oral dose of 50 mg/kg on rats. A simple, rapid, and sensitive gas chromatography–mass spectrometry method in selected ion monitoring (GC–MS/SIM) mode was developed on determination of BCP in rat plasma. The in vivo data showed that BCP/-CD inclusion complex displayed earlier Tmax , higher Cmax and the AUC0–12 h was approximately 2.6 times increase than those of free BCP. These results demonstrated that BCP/-CD inclusion complex has significantly increased the oral bioavailability of the drug in rats than free BCP. © 2013 Published by Elsevier B.V.
-Caryophyllene (BCP) (Fig. 1) is a bicyclic sesquiterpene compound existing in the essential oil of many plants, e.g. Eugenia caryophyllata (Fam. Myrtaceae), Piper nigrum L. (Fam. Piperaceae), and Zingiber nimmonii (J. Graham) Dalzell (Fam. Zinziberaceae). Due to its woody and spicy odour, BCP has been commonly used as a fragrance and flavouring agent since the 1930s (Di Sotto et al., 2010; Sabulal et al., 2006). In recent years, this compound has attracted considerable attention because of its extensive biological activities including antimicrobial (Sabulal et al., 2006), anticarcinogenic (Di Sotto et al., 2010), anti-inflammatory, anti-oxidant (Cho et al., 2007; Fernandes et al., 2007; Horváth et al., 2012), anxiolyticlike and local anaesthetic effects (Galdino et al., 2012). However, this compound is volatile, poorly water-soluble, and sensitive to light, oxygen, humidity and high temperature (Ponce Cevallos et al., 2010; Sköld et al., 2006; Wang et al., 2011). Among them, poor water-solubility may substantially decrease bioavailability of the
∗ Corresponding authors. Tel.: +86 21 8187 1260; fax: +86 21 8187 1260. E-mail addresses: [email protected] (Y. Qi), [email protected] (G. Fan). 1 These authors have equal contribution to this work. Both persons are the first authors.
drug and hence limit its further application in pharmaceutical field. Therefore, developing delivery system for drugs such as BCP to increase their solubility and dissolution rate so as to improve the bioavailability has been an important task in pharmaceutical development (Wempe et al., 2008). In recent years, cyclodextrin (CD) complexation has been successfully applied to improve the solubility, chemical stability, and bioavailability of a number of poorly soluble compounds (Hwang et al., 2012; Liu et al., 2006; Wempe et al., 2008). CDs are non-toxic macrocyclic oligosaccharides composed of 6, 7, 8 or 9 glucopyranose units (namely ␣-, -, ␥- or ␦-CD, respectively) with a relatively hydrophilic surface and hydrophobic central cavity. Through host–guest interactions with the organic molecules, CDdrug inclusion complex is formed so that the included drugs can be protected against hydrolyse, oxidation, photodecomposition and dehydration (Hwang et al., 2012). -CD as attractive materials for drug inclusion, giving prominence to its low biotoxicity and high biocompatibility, has been successfully used to increase the stability, solubility, and bioavailability of poorly soluble compounds in oral drug delivery (Gao et al., 2012; Hwang et al., 2012; Liao, 2010; Liu et al., 2006; Piette et al., 2006). The objective of the present study is to investigate and compare the pharmacokinetic characteristics of free BCP and its -CD inclusion complex in rat plasma after oral administration. The
0378-5173/$ – see front matter © 2013 Published by Elsevier B.V. http://dx.doi.org/10.1016/j.ijpharm.2013.04.013
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same temperature and rate. Then the solution was cooled to room temperature and maintained overnight at 4 ◦ C. Finally, the cold precipitated BCP/-CD inclusion complex was recovered by vacuum filtration. The precipitate was washed with light petroleum to remove BCP which was absorbed on the surface of -CD and then dried in baking oven at 40 ◦ C until the weight kept constant. The dried powder (3.2468 g) was stored in an airtight desiccator at room temperature. The recovery of inclusion complex was calculated according to Eq. (1):
Fig. 1. Chemical structure of -caryophyllene.
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inclusion complex of BCP/-CD in this study was prepared by the coprecipitation method and characterized by different analytical techniques including ultraviolet–visible spectrophotometry (UV–vis), differential thermal analysis (DTA) and fourier transform infrared spectroscopy (FT-IR) (Hwang et al., 2012; Liu et al., 2006; Ponce Cevallos et al., 2010; Wang et al., 2011). Moreover, its in vitro dissolution study was performed in the mediums of hydrochloric acid (0.1 mol/L) and phosphate buffer (pH 6.8) according to the Chinese Pharmacopoeia (Ch.P) and United States PharmacopoeiaNational Formulary (USP-NF) (Hwang et al., 2012; Liu et al., 2006; Wang et al., 2011; Wempe et al., 2008). GC (Dias et al., 2012; Liu et al., 2007, 2009; Sun et al., 2006; Wang et al., 2010) and GC–MS (Qin et al., 2007) methods which have been applied to analyse BCP in the essential oil of traditional Chinese medicine were used to determine BCP in rat plasma in our study. To the best of our knowledge, this is the first pharmacokinetic study on free BCP and BCP/-CD inclusion complex in rat plasma by a simple, rapid, and sensitive GC–MS/SIM method. The pharmacokinetic parameters of both formulations were analysed by non-compartmental model (Chu et al., 2008; Dong et al., 2007; Spichiger et al., 2004; Wang et al., 2007, 2008).
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2. Materials and methods
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2.1. Chemicals and reagents BCP (purity > 90%) was purchased from TCI Chemical Industry Development Co., Ltd (Shanghai, China). Eucalyptol (internal standard; purity ≥ 99.0%) was purchased from Sigma–Aldrich (Fluka Analytical, USA). -CD was purchased from Shandong Xinda Fine Chemical Co., Ltd (Shandong, China). High performance liquid chromatography (HPLC) grade ethyl acetate (Lot #54295; purity ≥ 99.8%) was purchased from Caledon Inc. (Canada). Anhydrous sodium sulfate (Na2 SO4 ), hydrochloric acid and spectrograde potassium bromide (KBr) were purchased from Sinopharm Chemical Reagent Co., Ltd (Shanghai, China). Analytical grade ethanol, potassium dihydrogen phosphate, sodium hydroxide, and light petroleum were purchased from Jiangsu Qiangsheng Chemical Co., Ltd (Jiangsu, China). The distilled water was prepared by Milli-Q academic water purification system. 2.2. Preparation and characterization of BCP/ˇ-CD inclusion complex 2.2.1. Preparation of BCP/ˇ-CD inclusion complex The BCP/-CD inclusion complex was prepared by the coprecipitation method. Briefly, -CD (3.0004 g) and 50 mL water were added into a round bottomed flask which was placed in a constant temperature magnetic stirring apparatus. After stirring for an hour at 50 ◦ C, the -CD saturated solution was obtained. 0.5 mL BCP (452.5 mg) dissolved in ethanol (0.5 mL) was decade to -CD saturated solution with continuous agitation for an hour at the
Recovery (%) =
mass of dried powder × 100 mass of added (-CD + BCP)
(1)
In order to characterize inclusion complex, the physical mixture of -CD and BCP was prepared by mixing -CD and BCP at a ratio of 6:1 (m/v) in a glass mortar until a homogeneous mixture. 2.2.2. Characterization of BCP/ˇ-CD inclusion complex using spectrometry 2.2.2.1. UV–vis spectrophotometry. The formation of BCP/-CD inclusion complex was demonstrated by a UV–vis spectrophotometer (Varian, USA). BCP (2 g/mL) was prepared in ethanol. The -CD, physical mixture and inclusion complex (30 mg) were added to ethanol (5 mL), respectively, and then the mixture was shaken for 10 min. The supernatant was separated by centrifugation and then diluted by 100 times in ethanol, and scanned in the range of 200–800 nm to obtain the UV–vis absorption spectrum. 2.2.2.2. Differential thermal analysis (DTA). DTA curves of -CD, BCP, their physical mixture and inclusion complex were measured with a Diamond TG/DTA instrument (Perkin Elmer, USA). Samples were accurately weighed and heated in a sealed aluminium pan at a rate of 5 ◦ C/min from 40 to 335 ◦ C under a nitrogen flow of 40 mL/min. An empty sealed pan was used as the reference. Duplicate determinations were carried out for each sample. 2.2.2.3. Fourier transform infrared spectroscopy (FT-IR). The FT-IR spectra of -CD, BCP, their physical mixture and inclusion complex were collected from 4000 to 400 cm−1 on a Nicolet 550-II FT-IR spectrophotometer (Nicolet, USA) with 32 scans at a resolution of 4 cm−1 . -CD, physical mixture and inclusion complex were respectively mixed with spectrograde KBr powder at a mass ratio of 1:100. Then they were ground and pressed to discs of diameters of 8 mm. A drop of BCP was spread on a piece of KBr window uniformly then nipped with another piece of KBr window. FT-IR spectra were analysed by the spectrophotometer software (OMNIC 5.2). 2.3. Determination of inclusion ratio of BCP in the inclusion complex
mass of recovered BCP × 100 mass of added BCP
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Steam distillation (SD) method (Lu et al., 2004; Xu et al., 2011) was used to determine BCP in the inclusion complex. 0.5 mL BCP (452.5 mg) and 150 mL water were added into a round bottomed flask heated and connected with essential oil extractor. BCP was gradually distilled until its volume kept constant. Then it was weighed (373.7 mg) after removing water with Na2 SO4 thereby blank recovery could be calculated as Eq. (2). Similarly, the amount of BCP in inclusion complex was obtained and the inclusion ratio was calculated as Eq. (3). Blank recovery (%) =
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(3)
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2.6. Method validation
Fig. 2. Full mass spectra of (A) eucalyptol, (B) BCP.
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Stock solutions of BCP (1.060 mg/mL) and eucalyptol (1.007 mg/mL) were prepared in ethyl acetate and stored at 4 ◦ C. The BCP stock solution was further diluted with ethyl acetate to obtain a set of standard working solutions with a concentration range of 0.53–53.0 g/mL. Internal standard (IS) solution was prepared by dilution of its stock solution to 5.035 g/mL with ethyl acetate. The specificity of the method was assessed by comparing the blank plasma spiked with BCP (0.053 g/mL) with blank plasma. BCP standard curve samples (0.053, 0.106, 0.212, 0.53, 1.06, 2.12, 5.30 g/mL) were prepared by the addition and complete mixing of 10 L standard working solution, 10 L IS solution and 90 L blank plasma. After being extracted, they were injected into GC–MS system and peak area ratio of BCP versus IS was calculated. Quality control samples (QCs) for evaluating the accuracy and precision of the method were prepared at low (0.106 g/mL), medium (0.53 g/mL), and high (4.24 g/mL) concentrations. Extraction yield was determined by the peak area ratio of BCP from extracted QCs and unextracted standards. Stability was investigated in room temperature, three successive freeze–thaw cycles and autosampler by three replicates of QCs. Analyte was considered to be stable if the peak area ratios of BCP versus IS of processed samples varied within ±15%.
2.4. In vitro dissolution study
2.7. In vivo experiments protocol and sample preparation
In vitro dissolution study of BCP/-CD inclusion complex was performed in triplicate according to Ch.P (2010 Edition, Part 2, Appendix XC. No.2 method) and USP 30-NF 25 (2007 Edition, Volume 1, <711> Apparatus 2) using a Rcz-602 Dissolution Apparatus (Shanghai Huanghai Medicine Checking Instrument Co., Ltd). However, according to the definition of dissolution, the dissolution study of free BCP was not necessary to be carried out because it was oleosus liquid. Briefly, 115.61 mg of BCP/-CD inclusion complex was added to 900 mL mediums of hydrochloric acid (0.1 mol/L) and phosphate buffer (pH 6.8) at 37 ± 0.5 ◦ C then stirred at 50 rpm. At predetermined time intervals, 5 mL of the medium was withdrawn and centrifuged at 12,000 rpm for 5 min. Then the drug content in the supernatant was assayed at 205 nm using UV spectrometry. The accumulative release rate was calculated at each time point.
SD rats (180–200 g, male and female half and half) were purchased from Shanghai Slac Laboratory Animal Co., Ltd (Shanghai, China), and were housed under standard conditions with free access to food and water. Before administration, rats were on overnight fast with free access to water. Then, they were randomly and equally divided into two groups (each consists of 6 rats). One group received free BCP and the other group received BCP/-CD inclusion complex. BCP and BCP/-CD inclusion complex were both dissolved in water and the rats were orally administrated at a dose of 50 mg/kg. Blood samples (0.5 mL) were collected from the orbit venous plexus into heparinized Eppendorf tubes at 0.167, 0.333, 0.5, 0.75, 1, 1.5, 2, 3, 4, 6, 8, 10 and 12 h after administration. The plasma was separated through centrifugation at 12,000 rpm for 10 min and stored at −20 ◦ C until analysis. After that, 100 L plasma sample, 10 L IS solution and 90 L ethyl acetate were mixed in 5 mL glass centrifuge tube. After vortexing for 5 min, the sample was centrifuged at 3200 rpm for 10 min and the supernatant (60 L) was transferred to a vial for GC–MS analysis.
2.5. GC–MS instrument and conditions The analysis was performed on a GC–MS system consists of an AS3000 autosampler, a Trace GC Ultra, and an ITQ1100 mass spectrometer (ThermoFisher Scientific, USA). A TR-5MS capillary column (30 m × 0.25 mm i.d.) coated with a 0.25 m thick film of 5% phenyl methyl siloxane (Thermo, USA) was used. The inlet temperature was set at 250 ◦ C. The oven temperature was initially held at 100 ◦ C, then was programmed to 140 ◦ C at 15 ◦ C/min, holding for 0.5 min, followed by 30 ◦ C/min to 280 ◦ C and held for 3 min. Helium (purity > 99.999%) was used as carrier gas at a constant flow rate of 1.0 mL/min. The sample volume was 1 L and the inlet was in the split mode with a split ratio of 10:1. Ionization was carried out in electron impact (EI) mode at 70 eV. The ion source temperature and transfer line temperature were 250 ◦ C and 280 ◦ C, respectively. Solvent was delayed for 2.2 min. The detection was operated in SIM mode: from 2.2 to 4.0 min m/z 93 (eucalyptol) and from 4.0 to 5.5 min m/z 91 (BCP) were respectively selected for quantitation as their relative abundance was the highest in individual full mass spectrum (Fig. 2). Data were collected and analysed by Xcalibur 2.0 workstation.
2.8. Pharmacokinetic analysis The pharmacokinetic parameters of free BCP and BCP/-CD inclusion complex in SD rats were calculated by BAPP software (China Pharmaceutical University, Nanjing, China) using noncompartment model analysis and their statistical comparisons were made by two-sample t-test (SPSS, Chicago), and p value less than 0.05 was considered to be statistically significant. All data were presented as mean value with its standard deviation (mean ± S.D.).
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3.1.1. UV–vis spectrophotometry The UV–vis absorption spectra were recorded for BCP, -CD, their physical mixture and inclusion complex. The spectra of the physical mixture (Fig. 3A) and BCP (2 g/mL) (Fig. 3B) were identical with maximum absorption wavelength (max ) at 205 nm, since -CD (Fig. 3C) in ethanol had no UV absorption. However, the
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Fig. 3. UV spectra of (A) physical mixture of -CD and BCP, (B) BCP (2 g/mL), (C) -CD, and (D) BCP/-CD inclusion complex.
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absorption peak was not observed in the spectrum of inclusion complex (Fig. 3D). These results indicated that BCP had formed inclusion complex with -CD. 3.1.2. DTA DTA is an important tool to study the formation of inclusion complex (Hwang et al., 2012; Liu et al., 2006; Ponce Cevallos et al., 2010; Wang et al., 2011). The thermal curves of BCP, -CD, their physical mixture and inclusion complex were shown in Fig. 4. A broad endothermic peak near 125 ◦ C appeared in the thermogram of BCP (Fig. 4A), corresponding to its boiling point. The thermogram of -CD (Fig. 4B) presented three endothermic peaks at 82, 273, and 310 ◦ C, respectively, which were likely to be related to the loss of water molecules originally binding to cyclodextrin molecules. The DTA curve of physical mixture (Fig. 4C) clearly exhibited superimposition of -CD and BCP thermograms. In Fig. 4D, however, the peak of BCP disappeared and the peaks originally in the -CD also changed, offering the proof for the formation of BCP/-CD inclusion complex. 3.1.3. FT-IR Fig. 5 presented the FT-IR spectra of BCP, -CD, their physical mixture and inclusion complex. The spectrum of BCP (Fig. 5A)
Fig. 5. FT-IR spectra of (A) BCP, (B) -CD, (C) physical mixture of -CD and BCP, and (D) BCP/-CD inclusion complex. The arrow points for the peak at 886 cm−1 .
consisted of the prominent absorption bands of stretching vibration (3067 cm−1 ) and scissoring vibration (1448 cm−1 ) of CH, asymmetrical stretching vibration (2926 cm−1 ) and symmetrical stretching vibration (2857 cm−1 ) of CH2 , and stretching vibration of C C (1633 cm−1 ). The double peaks (1382 and 1367 cm−1 ) of symmetrical deformation vibration of CH3 were ascribed to two methyls connecting to the same carbon atom. The very intense peak at 886 cm−1 was characteristic absorption band of BCP assigned to out-of-plane deformation vibration of CH. The spectrum of CD (Fig. 5B) displayed prominent absorption bands at 3384 cm−1 for O H stretching vibration, 2927 cm−1 for CH2 asymmetrical stretching vibration, 1648 cm−1 for H O H bending vibration, and 1157 cm−1 for C O C asymmetrical stretching vibration. The spectrum of physical mixture (Fig. 5C) presented the characteristic absorption band of BCP at 886 cm−1 , though its intensity was weak. However, there was not any feature similar to BCP when examining the spectrum of inclusion complex (Fig. 5D). These changes were related to the formation of intra-molecular hydrogen bonds between BCP and -CD. 3.2. Determination of inclusion ratio of BCP The recovery of inclusion complex and inclusion ratio of BCP are used as indexes to evaluate inclusion effect (Han and Zhang, 2011; Zhang and Liu, 2012). In our study, the blank recovery of BCP was 82.59%, and the content of recovered BCP in the BCP/CD inclusion complex was 231.84 mg. Using formulas (2) and (3), the recovery of inclusion complex and inclusion ratio of BCP were 94.03% and 62.04%, respectively, suggesting an acceptable inclusion ratio of BCP. 3.3. In vitro dissolution study
Fig. 4. DTA curves of (A) BCP, (B) -CD, (C) physical mixture of -CD and BCP, and (D) BCP/-CD inclusion complex.
The dissolution profiles of BCP/-CD inclusion complex were shown in Fig. 6. They were characterized by an initial fast release followed by a relatively slow release until the constant value. After 90 min, approximately 90% and 60% of BCP were released from inclusion complex into the mediums of hydrochloric acid (0.1 mol/L) and phosphate buffer (pH 6.8), respectively. The different dissolution of BCP in two mediums might be related to its physical property and the microenvironment around it and the rapid release of BCP was attributed to the fact that -CD had the ability to improve wettability of inclusion complex in dissolution mediums so that BCP was released more and faster from inclusion
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Table 1 Accuracy and precision results of BCP. Sample
Added (g/mL)
LQCa MQCb HQCc a b c d e f
Inter-dayf
Intra-day
0.106 0.53 4.24
Found ± S.D.
Accuracy
Precision
Found ± S.D.
Accuracy
Precision
0.110 ± 0.003 0.49 ± 0.02 4.28 ± 0.18
102.69 ± 3.24 91.73 ± 3.53 100.92 ± 4.32
3.16 3.85 4.28
0.105 ± 0.01 0.48 ± 0.02 4.11 ± 0.19
99.00 ± 4.90 90.65 ± 2.99 96.98 ± 4.49
4.95 3.30 4.63
d
e
LQC: low quality control. MQC: medium quality control. HQC: high quality control. Accuracy is given as recovery (%) ± S.D. Precision is given as percent of the relative standard deviation (R.S.D.%). Three days, five replicates per concentration per day.
3.4.2. Linearity The calibration curve showed good linearity within the range of 0.053–5.30 g/mL. Mean linear equation of calibration curves (n = 3) for BCP was (y = (0.000272267 ± 0.01) + (3.1421 ± 0.19)x, r2 = 0.9975, weighting: 1/x), where y was the peak area ratio of BCP versus IS and x was the concentration of BCP.
100
Accumulative Release (%)
90 80 70 60
333 334 335 336 337 338
50 40
hydrochloric acid (0.1 mol/L)
30
phosphate buffer (pH 6.8)
3.4.3. Accuracy and precision Table 1 showed that the method had good accuracy and precision. The relative recoveries for all samples were >90%. The intraand inter-day precision ranged from 3.16% to 4.95%.
20
339 340 341 342
10 0 0
20
40
60
80
100
120
140
160
180
Time (min) Fig. 6. Mean dissolution profiles of BCP/-CD inclusion complex in the mediums of hydrochloric acid (0.1 mol/L) and phosphate buffer (pH 6.8) (n = 3).
325
complex. The improved dissolution of BCP denoted increased oral bioavailability.
326
3.4. Method validation
324
327 328 329 330 331 332
3.4.1. Specificity Typical chromatograms of blank plasma, blank plasma spiked with BCP at the low limit of quantitation (LLOQ, 0.053 g/mL), and a real rat plasma sample were shown in Fig. 7. It was shown that no endogenous substances in plasma interfered with the assay of BCP.
3.4.4. Extraction yield and stability The extraction yields of BCP were 85.09 ± 3.14%, 83.52 ± 6.33%, and 80.87 ± 5.18% at the concentration of 0.106, 0.53, and 4.24 g/mL, respectively. Under room temperature for 3 h and being subjected to three freeze–thaw cycles, LQC and HQC samples remained stable. No instability of the extracted samples was observed for 24 h in autosampler. The stability results were listed in detail in Table 2. 3.5. Pharmacokinetics
Table 2 Summary of the stability of BCP. Initial mean area ratio
Found ± S.D.
Room temperature for 3 h LQC 0.106 4.24 HQC
0.324 13.218
0.332 ± 0.01 13.305 ± 0.24
2.69 ± 3.89 0.66 ± 1.85
Three freeze–thaw cycles LQC 0.106 4.24 HQC
0.335 13.400
0.338 ± 0.01 13.551 ± 0.23
0.95 ± 1.79 1.13 ± 1.74
Autosampler for 24 h 0.106 LQC 4.24 HQC
Fig. 7. Chromatograms of (A) blank plasma, (B) blank plasma spiked with BCP (0.053 g/mL) and IS, and (C) plasma sample at 0.5 h after oral administration of BCP/-CD inclusion complex (50 mg/kg); (1) IS, (2) BCP.
0.326 13.714
0.313 ± 0.003 13.654 ± 0.14
a
Added (g/mL)
344 345 346 347 348 349 350
351
Mean plasma concentration versus time profiles of free BCP and BCP/-CD inclusion complex formulations were illustrated in Fig. 8. Pharmacokinetic parameters were summarized in Table 3. By comparing the results of two formulations, BCP/-CD inclusion complex had significant decrease at Tmax , T1/2 , MRT and increase at Cmax , AUC0–12 h and AUC0–∞ . Moreover, the AUC0–12 h after administration of BCP/-CD inclusion complex was about 2.6 times higher than that after administration of free BCP, suggesting that relative oral bioavailability of BCP in inclusion complex increased about 2.6 times. These results demonstrated that BCP/-CD inclusion complex significantly increased the oral bioavailability in rats comparing to free BCP.
Sample
343
Stabilitya
−3.97 ± 1.06 −0.44 ± 1.04
Stability is given as relative error (%) ± S.D.
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Concentration (μg/mL)
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Acknowledgement
1.00 0.90 0.80 0.70 0.60 0.50 0.40 0.30 0.20 0.10 0.00
This study was financially supported by the National Natural Science Foundation of China (No. 81173019).
Free BCP BCP/β-CD
References
0
2
4
6
8
10
12
Time (h) Fig. 8. Mean (±S.D.) plasma concentration–time profiles of BCP after oral administration of free BCP or BCP/-CD inclusion complex at a dose of 50 mg/kg (n = 6).
Table 3 Summary of pharmacokinetic parameters. Free BCP a
Cmax (g/mL) Tmax (h)b T1/2 (h)c MRT (h)d AUC0–12 h (g h/mL)e AUC0–∞ (g h/mL) a b c d e *
364 365 366 367 368 369 370 371 372 373 374 375 376 377 378 379 380
381
382 383 384 385 386 387 388 389 390 391 392 393 394
0.12 3.50 4.07 7.69 1.05 1.28
± ± ± ± ± ±
0.02 0.60 0.07 0.13 0.08 0.10
BCP/-CD 0.56 2.80 3.25 5.63 2.72 2.96
± ± ± ± ± ±
0.35 0.80 0.65 0.93 1.22 1.16
p-Value* 0.027 0.035 0.026 0.003 0.020 0.016
Cmax : maximum plasma concentration. Tmax : time to reach maximum plasma concentration. T1/2 : half-time. MRT: mean residence time. AUC: the area under the concentration–time curve. p < 0.05 is considered to be statistically significant.
The enhancement of oral bioavailability for BCP in the form of BCP/-CD inclusion complex may be ascribed to the increase of its dissolution and water-solubility since BCP could be embedded into the -CD cavity and -CD has hydrophilic surface (Gu et al., 2012; Liao et al., 2009). Stability constant of BCP in inclusion complex is relative small, thus it could be released rapidly from inclusion complex in vivo (Tang et al., 1992). Additionally, the high biocompatibility of -CD makes BCP being absorbed more in gastro-intestinal tract. Therefore, -CD is a safe vector to increase drug’s solubility and bioavailability, and the development of BCP delivery system can greatly improve its absorption. According to our results, the standard deviation of pharmacokinetic parameters of different rats after oral administration of inclusion complex were relatively large, which prompted that the individual difference of BCP absorption may exist. This would be a problem in determining the effective dose and hence still needs further study. 4. Conclusions In this study, BCP/-CD inclusion complex was successfully prepared by the coprecipitation method and characterized by spectrophotometry including UV–vis spectrophotometry, DTA, and FT-IR. Moreover, its in vitro dissolution study was performed and BCP was rapidly released from inclusion complex. For the first time a simple, rapid, and sensitive GC–MS/SIM method for determination of BCP in rat plasma was developed. Comparison on pharmacokinetic parameters of free BCP and its inclusion complex indicated that BCP/-CD inclusion complex had earlier Tmax , higher Cmax and better bioavailability. In conclusion, the results demonstrated that the formation of BCP/-CD inclusion complex could greatly improve the oral bioavailability of BCP.
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