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Investigation of inclusion complex of honokiol with sulfobutyl ether-β-cyclodextrin
Accepted Manuscript Title: Investigation of inclusion complex of honokiol with sulfobutyl ether--cyclodextrin Author: Caibing Xu Yalan Tang Wenjing Hu Rui Tian Yuntao Jia Ping Deng Liangke Zhang PII: DOI: Reference:
S0144-8617(14)00635-3 http://dx.doi.org/doi:10.1016/j.carbpol.2014.06.059 CARP 9024
To appear in: Received date: Revised date: Accepted date:
20-2-2014 1-6-2014 14-6-2014
Please cite this article as: Xu, C., Tang, Y., Hu, W., Tian, R., Jia, Y., Deng, P., and Zhang, L.,Investigation of inclusion complex of honokiol with sulfobutyl ether-rmbeta-cyclodextrin, Carbohydrate Polymers (2014), http://dx.doi.org/10.1016/j.carbpol.2014.06.059 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Investigation of inclusion complex of honokiol with sulfobutyl
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ether-β-cyclodextrin
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Caibing Xu a, Yalan Tang a, Wenjing Hu b, Rui Tian c, Yuntao Jia d, Ping Deng a,
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Liangke Zhang a,*
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a
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pharmacy, Chongqing Medical University, Chongqing 400016, P.R. China;
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b
Chongqingshi Shapingba District People's Hospital, Chongqing 400030, P.R. China;
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c
The Experimental Teaching Centre, Chongqing Medical University, Chongqing
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400016, P.R. China；
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Chongqing 400010, P.R. China;
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Department of Pharmacy, Children’s Hospital of Chongqing Medical University,
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Chongqing Key Laboratory of Biochemistry and Molecular Pharmacology, School of
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*Corresponding author: Liangke Zhang, School of Pharmacy, Chongqing Medical University, District of Yuzhong, Chongqing 400016, China. Tel: +86-23-6848-5161; fax: +86-23-6848-5161. E-mail address: [email protected](L. Zhang)
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Abstract This study aimed to prepare and characterize an inclusion complex of honokiol
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(HNK) with sulfobutyl ether-β-cyclodextrin (SB-β-CD). The inclusion complex
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(HNK/CD COMP) was prepared utilizing a freeze-drying method. Phase-solubility
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curves were employed to obtain stability constants and thermodynamic parameters.
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The phase-solubility diagram showed a typical AL-type, indicating that the 1:1 (HNK:
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SB-β-CD) inclusion complex was formed. The solid inclusion complex was then
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characterized by differential scanning calorimetry and Fourier transform infrared
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spectroscopy. Results showed that HNK/CD COMP exhibited a higher drug release
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rate than free HNK in vitro. A comparative study of the pharmacokinetics between
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HNK/CD COMP and free HNK was also performed in rats. In vivo results indicated
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that AUC0-t and Cmax of HNK/CD COMP increased by approximately 158% and
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123% compared with those of the free HNK, respectively. These results suggest that
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SB-β-CD will be potentially useful in the delivery of poorly soluble drugs, such as
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HNK.
Key words: Sulfobutyl ether-β-cyclodextrin; Honokiol; Inclusion complex; In
vitro release; Pharmacokinetic study
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1. Introduction Cyclodextrins (CDs) are a family of macrocyclic oligosaccharides mostly
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consisting of six, seven, or eight glucose units joined by α-1,4 glycosidic bonds
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(Brewster & Loftsson, 2007; Stella & He, 2008). The hydrophobic cavity of CDs
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provides these CDs the ability to form inclusion complexes with various compounds,
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including proteins, oligonucleotides, ions, and small molecules (Stella & He, 2008;
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Zhang & Ma, 2013). As a promising material with low toxicity and low
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immunogenicity, CDs have been extensively investigated in the field of biomedicine,
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particularly in pharmaceutical sciences and technologies. Related applications include
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the improvement of drug solubility and stability, increase in drug absorption and
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bioavailability, removal of odors and tastes, and decrease in local and systemic
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toxicity (Carrier, Miller & Ahmed, 2007; Garcia-Fernandez et al., 2013; Zhang & Ma,
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2013). CDs can be administered by oral, ocular, nasal, and rectal
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(Al-Rawashdeh, Al-Sadeh & Al-Bitar, 2010; Pahuja, Kashyap & Pawar, 2013; Singh,
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Kumar & Pathak, 2013; Tiwari, Tiwari & Rai, 2010). To improve these properties of natural CDs in terms of solubility, stability, inclusion capability, and toxicity, researchers synthesized various chemically modified CDs, such as amphiphilic, hydrophobic, and highly soluble derivatives.
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When new excipients are considered as drug carries, safety should be primary
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concern. Unmodified cyclodextrins, such as α- and β-cyclodextrin, were extensively
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investigated in pharmaceuticals. However, they were limited by hemolytic activity
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and nephrotoxicity because of their interaction with cholesterol and other lipid
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membrane components of cellular structures (Luke, Tomaszewski, Damle & Schlamm,
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2010). Sulfobutyl ether-β-cyclodextrin (SB-β-CD, Fig. 1A) is an excellent modified
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compound of β-cyclodextrin with improved water solubility and low toxicity.
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SB-β-CD does not undergo significant tubular reabsorption due to its ionized state
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after glomerular filtration and is concentrated in the urine for excretion, which
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attenuate the potential to harm the kidney(Albers & Muller, 1995; Irie & Uekama,
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1997; Uekama, 2004). SB-β-CD can form inclusion complexes with drug molecules.
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Thus, SB-β-CD has been utilized to improve the solubility and stability of drugs
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(Danel, Azaroual, Chavaria, Odou, Martel & Vaccher, 2013; Stella & He, 2008;
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Venuti et al., 2013).
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Honokiol (HNK, Fig. 1B) is a bioactive lignanoid extracted from the reed of
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Magnolia officinalis, which has been utilized in traditional Chinese medicine for a
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long time. Furthermore, HNK is extensively used in Japan (Cheng, 2012; Lee, Lee,
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Lee, Jung, Han & Hong, 2011). This compound exhibits several pharmacological properties, such as anti-cancer effects, anti-inflammatory effects, anti-oxidant actions, and anti-anxiety effects. However, the poor aqueous solubility of HNK has impeded clinical applications.
(Choi et al., 2002; Kang et al., 2008; Weeks, 2009; Yamaguchi,
Satoh-Yamaguchi & Ono, 2009).
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In this study, the inclusion complex of HNK and SB-β-CD (HNK/CD COMP) was
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prepared to improve the solubility and bioavailability of HNK using a freeze-drying
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method (Jadhav, Petkar, Pore, Kulkarni & Burade, 2013; Rajendiran & Siva, 2014;
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Zhang et al., 2013). Solid-state HNK/CD COMP was characterized by differential
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scanning calorimetry (DSC) and Fourier transform infrared spectroscopy (FT-IR). In a
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solution, solubility phase analysis was performed to estimate the enhanced solubility
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of HNK by SB-β-CD and the stoichiometry of the inclusion complex. In vitro release
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study and model fitting were systematically investigated and evaluated. Furthermore,
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the pharmacokinetic properties of HNK suspension and HNK/CD COMP in male
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Sprague-Dawley rats were analyzed.
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2. Materials and methods
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2.1. Materials
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Honokiol (purity ≥ 98%) was purchased from Xi’an Grass Plant Science and
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Technology CO., Ltd (Xi’an, China). SB-β-CD (DS=7.0) was purchased from
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Shandong Binzhou Zhiyuan Bio-Technology Co., Ltd (Shandong, China). All the
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other reagents were of analytical grade.
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2.2. Phase-solubility analysis
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Phase-solubility analysis was performed in a SHZ-88 thermostatic shaking water
bath (Jiangsu Taicang Medical Instrument Factory, China). The excess amount of HNK was added to volumetric flasks containing 10 mL of SB-β-CD solutions at various concentrations (0, 5, 10, 15, 20, 25, and 30 mM). The contents were then
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sonicated for 5 min. The flasks were sealed to prevent evaporation and shaken
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continuously at 100 rpm in a thermostatic bath at different temperatures (25, 37, and
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50 °C) for 48 h. After equilibrium was reached, suspensions were filtered using a 0.45
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µm syringe filter. The amount of drug was then analyzed at a wavelength of 292 nm
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by using a spectrophotometer (Shimazdu UV-3150, Japan). Experiments were
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performed in triplicate.
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2.3. Preparation of inclusion complex The inclusion complex was prepared using a freeze-dry method (Pralhad &
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Rajendrakumar, 2004; Ruz, Froeyen, Busson, Gonzalez, Baudemprez & Van den
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Mooter, 2012; Wang, Luo & Xiao, 2014). On the basis of the results of
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phase-solubility analysis, we dispersed 100 mg of HNK in 100 mL of water
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containing 1.68 g of SB-β-CD at 45 °C. After magnetically stirring for 2.5 h, we
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filtered the resultant solution with a 0.45 µm syringe filter and the filtrate was
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freeze-dried (ALPHA 1-2 LD plus, CHRIST, Germany) for 48 h.
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2.4. DSC
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DSC measurements were performed to confirm that the complex was formed.
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The powdered samples of HNK, SB-β-CD, HNK/CD COMP, and physical mixture
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(HNK/CD PM) were placed in an aluminum pan, heated to 70 °C for 5 min, and
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cooled to 20 °C for 5 min at the cooling rate of 10 °C ·min–1. And then the profiles were obtained by using a Netzsch STA449C thermal analyzer (Netzsch Corporation, Germany) from 20 °C to 300 °C (at a constant rate of 10 °C·min–1) under nitrogen gas flow (25 mL·min–1).
2.5. Fourier transform infrared spectroscopic (FT-IR) study
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The FT-IR spectra of the powdered samples of HNK, SB-β-CD, HNK/CD COMP,
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and HNK/CD PM were obtained using a spectrometer (Bruker-tensor 27, Germany)
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with the potassium bromide pellet method. The typical bands were recorded for
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different samples. Each spectrum was recorded in the range of 4000 cm–1 to 400 cm–1.
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The spectra were obtained under dry conditions at a resolution of 4 cm–1 to prevent
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contamination.
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2.6. Dissolution studies
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Dissolution studies were implemented using the dialysis membrane method.
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Three formulations, namely, HNK, HNK/CD PM, and HNK/CD COMP were
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transferred into a dialysis bag (molecular weight cutoff, 8-14kDa). The dialysis bag
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was then immersed in a container filled with 25 mL of 0.1 mM phosphate buffer
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solution (pH 7.4) containing sodium dodecyl sulfate (SDS, 0.5%) at 37 °C with
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constant shaking (100 rpm). The receptor compartments were closed to prevent the
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dissolution medium from evaporating. An aliquot (4.0 mL) of the incubation medium
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was withdrawn and replaced with the same volume of pre-warmed fresh medium at
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specific time intervals. The concentration of HNK was then determined using a UV
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spectrophotometer (Shimazdu UV-3150, Japan) after HNK was filtered using a 0.45
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µm membrane. The cumulative release percentage (Q) was calculated according to the following equation: Q (C
n
25
n 1
C i 4 ) / A 100 %
(1)
i 1
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where n and i are the sample numbers, Cn and Ci are the drug concentration at the point of n and i, respectively, and A is the total drug content in each sample.
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Similarity factor (f2) was first proposed by Moore and Flanner (Costa & Sousa
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Lobo, 2001; Shah, Tsong, Sathe & Liu, 1998) to compare release profiles. It was
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derived from Mean-Squared difference. Similarity factor (f2) was also recommended 7
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for release profile comparison in the FDA’s Guidances for Industry (FDA, 1997; Shah
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et al., 1997). In this study, f2 was used to evaluate the similarity between every two
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release profiles. f2 is defined by the following equation: f2
2
0 .5
100 (2)
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n 50 lg 1 1 / n R t T t t 1
where n is the number of sampling points, and Rt and Tt are HNK cumulative release
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percentages at time t of the two release curves, respectively. The result indicated that
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the similarity between the two release profiles was statistically significant at f2 > 50. A
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high similarity between two profiles is characterized by a high f2.
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2.7 Pharmacokinetic analysis in rats
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2.7.1 Animal handling and care
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Male Sprague-Dawley rats weighing approximately 200 ± 20 g were obtained
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from the Laboratory Animal Center of Chongqing Medical University and used for
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pharmacokinetic analysis. The animal experiment was approved by the Animal Ethics
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Committee of Chongqing Medical University (Chongqing, China), and their guidelines were followed throughout this study. Before the experiment was conducted, the rats were fasted overnight but provided free access to water. 2.7.2 Pharmacokinetic behavior of rats Eighteen rats were randomly divided into three groups, namely, A, B, and C (n = 6
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rats). Group A received a single oral dose of HNK/CD COMP (80 mg·kg–1 of HNK)
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and group B was orally administered a single dose of HNK suspension (80 mg·kg–1 of
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HNK suspended in 0.5% sodium carboxymethyl cellulose solution). The blood
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samples (0.5 mL) were collected at 0.083, 0.25, 0.5, 1, 2, 4, 6, 10, 12, 24, and 48 h 8
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post-treatment. Group C was injected with the HNK/CD COMP solution at a dose of 20 mg·kg–1
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of HNK in the tail vein. The blood samples (0.5 mL) were collected from the
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retro-orbital plexus of each rat and transferred into heparinized tubes at 0.033, 0.083,
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0.25, 0.5, 0.75, 1, 1.5, 2, 4, and 6 h post-administration. These samples were
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immediately centrifuged, and the plasma samples were stored at –20 °C for further
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studies.
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2.7.3 Preparation of plasma samples
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An aliquot of plasma (200 µL) was pre-treated with 20 µL of evodiamine
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methanol solution (20 µg·mL–1) as an internal standard and then vortexed for 5 min.
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The resulting sample was mixed with 1.5 mL of ethyl acetate, vortexed for another 5
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min, and centrifuged at 4000 rpm for 10 min. Afterward, the supernatant was
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evaporated to dryness in a vacuum, redissolved in 100 µL of methanol, and
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centrifuged at 12,000 rpm for 10 min. This sample solution (20 µL) was directly
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injected into a HPLC system (Agilent 1100, American) for analysis. 2.7.4 Measurement of HNK from plasma using RP-HPLC The plasma concentrations of HNK were determined by an Agilent 1100 HPLC
system (Agilent, American). A Lichrospher RP C18 column (4.6 mm × 150 mm, 5 µm,
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Hanbon Sci. & Tech., China) was used and the mobile phase was a mixture of
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methanol and water (80:20, v/v). The flow rate was 1.0 mL·min–1. All of the
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detections were performed at a wavelength of 292 nm at a constant temperature of
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30 °C.
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2.7.5 Pharmacokinetic analysis The valuable pharmacokinetic parameters, namely, AUC0-t (area under the plasma
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concentration versus time curve), Cmax (peak drug concentration), Tmax (time to reach
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the peak concentration), Vd (apparent volume of distribution), t1/2 (half-life), and CL
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(clearance) were collected by DAS2.1.1 (issued by the China Food and Drug
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Administration for pharmacokinetic study). All data were presented as the mean ±
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standard deviation (n = 6, x ± s).
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3. Results and discussion
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3.1 Phase solubility study
Phase-solubility study is a traditional and useful approach not only to determine
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the value of the stability constant, but also to give insight into the stoichiometry of the
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equilibrium. The practical and phenomenological implications of phase solubility
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study were developed by Higuchi and Connons (Higuchi.T, 1965). Based on the
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between HNK and SB-β-CD. The study infused the solubilizing ability of SB-β-CD to
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HNK and the stability constant of HNK/CD COMP according to the phase-solubility
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diagram. Figure 2 shows the phase solubility diagrams of HNK and SB-β-CD in three
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different temperatures. All of the apparent solubility curves of HNK with correlation
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profiles of the phase solubility study, several types of solubility behaviors can be classified. The phase solubility diagrams include two major types: A type( a soluble compound was formed ) and B type (a compound of limit solubility was formed) (Brewster & Loftsson, 2007; Thompson, 1997). In this study, phase-solubility study was performed to determine the interactions
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coefficient r > 0.9963 were regarded as straight lines. Moreover, all of them can be
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considered as AL-type diagrams, indicating that the observed increase in the solubility
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of HNK was due to the formation of a 1:1 molar ratio inclusion complex.
equation(Brewster & Loftsson, 2007; Thompson, 1997): K1:1 = Slope / S0(1-Slope)
(3)
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The inclusion stability constant K1:1 was calculated using the following
cr
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where S0 is the equilibrium solubility of HNK in water; slope is obtained from the
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phase-solubility diagram. K1:1 at 25, 37, and 50 °C was calculated from Equation 3.
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Thermodynamic parameters were calculated from Gibbs and Van’t Hoff equations:
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△G= -RTln K1:1
(4)
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ln K1:1=-△H/RT+△S/R
(5)
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Valuable parameters, including stability constant (K1:1), Gibbs free energy change
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(∆G), enthalpy change (∆H), and entropy change (∆S), are listed in Table 1. K1:1
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increased with rising temperature. ∆G of HNK was negative and decreased as
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temperature increased. ∆H was positive in this study. The thermodynamic parameters and K1:1 indicated that high temperatures were favorable for the inclusion process (Hu, Zhang, Song, Gu & Hu, 2012; Rajendiran & Siva, 2014). 3.2 DSC analysis
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The melting, boiling, and sublimation points of drug molecules either shift to
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different temperatures or disappear when these molecules are included in SB-β-CD
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cavities (Ascenso et al., 2011; de Azevedo Mde et al., 2011). The thermograms of
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HNK, SB-β-CD, HNK/CD PM, and HNK/CD COMP are shown in Fig. 3. HNK
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generated a sharp endothermic peak at 92 °C, indicating its melting point. The
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diagram of SB-β-CD exhibited an endothermic peak at 218 °C, due to the sample
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decomposition. In the case of HNK/CD PM, the melting peak of HNK at 92 °C and
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the endothermic peak of SB-β-CD were observed, indicating that there was no
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inclusion complex formed between HNK and SB-β-CD. However, no sharp peak was
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found in the thermogram of HNK/CD COMP at 92 °C, indicating the amorphous
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characteristic of HNK. The absence of the melting peak of HNK in the thermogram of
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HNK/CD COMP may have occurred because HNK was included in the SB-β-CD
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cavity.
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3.3 FT-IR analysis
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HNK/CD COMP was further analyzed by FT-IR spectroscopy. The spectra of
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HNK, SB-β-CD, HNK/CD PM, and HNK/CD COMP from 4000 cm–1 to 400 cm–1 are
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shown in Fig. 4. The HNK spectrum showed the bands in the 3100 cm–1 to 3000 cm–1
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wave number regions, indicating the presence of –CH and –OH groups. A band was
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found at 1638 cm–1, which can be assigned to alkene C=C present in HNK. The intense bands at 1498 cm-1 and 1430 cm-1 were assigned to the stretching vibrations from the aromatic ring of honokiol molecule. Moreover, other bands were located at 1217 and 908 cm–1 (C–O) as well as at 989 and 825 cm–1 (C–C). The spectrum of
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SB-β-CD was mainly characterized by the intense bands at 3800 cm–1 to 3100 cm–1
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caused by O–H stretching vibration. The band at 1643 cm-1 was ascribed to the
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δ-HOH bending of water molecules attached to SB-β-CD. The vibrations of –CH and
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–CH2 groups appeared in the 3000 cm–1 to 2800 cm–1 region. The sulfoxide stretched
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at 1046 cm–1 (Mahmoud, El-Feky, Kamel & Awad, 2011; Venuti et al., 2013). In the
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case of HNK/CD PM, the spectrum was basically composed of the overlapping peaks
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of HNK and SB-β-CD, and this spectrum showed a less intense absorption band at
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1638 cm–1 than at other regions because of the low ratio of HNK in the physical
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mixture. This demonstrates that there was no interaction between drug and SB-β-CD.
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Compared with the HNK/CD PM, the spectrum of HNK/CD COMP showed relevant
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changes in intensities and widths of the typical absorption peaks, indicating the
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formation of a new compound between HNK and SB-β-CD (Venuti et al., 2014). A
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less intense absorption band was also observed at 1638 cm–1 in the spectrum of
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HNK/CD COMP, indicating that no interaction occurred between the C=C of HNK
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molecule and that of SB-β-CD molecule. Thus, C=C was not included in the cavity of
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SB-β-CD molecule. Therefore, only a part of the HNK molecule was possibly
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included in the cavity and the C=C portion of HNK molecule was not included.
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3.4 In vitro release study
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<11% of the free HNK and 15% of HNK/CD PM. Furthermore, the cumulative
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release percentages of HNK/CD COMP and free HNK at 4 h were 82% and 28%,
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respectively.
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Figure 5 shows the release curves of the free HNK, HNK/CD PM, and HNK/CD
COMP in the 0.1 mM PBS (pH 7.4) containing SDS (0.5%). HNK/CD COMP exhibited a significantly higher release rate than that for the free HNK and HNK/CD PM. The cumulative release percentage of HNK/CD COMP at 0.5 h was >29% but
f2 between the two curves of HNK/CD COMP and free HNK was 19.0 (<50) and 13
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that of HNK/CD PM and free HNK was 54.1(>50). These values indicated that the
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cumulative release profile of HNK/CD COMP was significantly different from that of
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the free HNK. However, no significant difference was found in HNK/CD PM and free
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HNK.
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Different models, including Weibull, Higuchi (square root of time equation),
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Ritger-Peppas, Hixcon-Crowell model (cubic root law), zero-order, and first-order
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models, were utilized to fit the results of the mean release profiles. The model that
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fitted the release data more efficiently was selected on the basis of r, that is, a higher r
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corresponds to a better fit. For HNK/CD COMP, r of Weibull model (ln[1/1 – Q] =
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0.6872lnt + 0.7100, r = 0.9 914) was the highest, indicating that the release profile of
293
HNK/CD COMP best fitted the Weibull model among all of the models. The release
294
profile of HNK/CD PM and HNK fitted well to the Ritger-Peppas model (lnQ =
295
0.4023lnt – 1.5494, r = 0.9 870 and lnQ = 0.5012lnt – 1.9532, r = 0.9 953,
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respectively).
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3.5 Pharmacokinetic analysis of HNK in rats HNK/CD COMP and HNK suspensions were administered to the male
Sprague-Dawley rats to evaluate the enhanced bioavailability in vivo at post-oral administration. The validated HPLC method was employed in the pharmacokinetic
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analysis. A good linear relationship was obtained for HNK with concentration ranging
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from 0.0355 to 9.0909 µg·mL–1. The relative standard deviation of HNK for inter-day
303
and intra-day precision and accuracy at low, medium and high concentration (0.0450,
304
2.2727 and 9.0909 µg·mL–1) was below 8.02%. The recoveries of HNK at the up
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three concentrations were 112.91, 106.84%, 100.88%. The limit of quantification
306
(LOQ) for HNK was 0.0355 µg·mL–1. Satisfactory results were obtained after a single
307
oral or intravenous dose were administered.
after
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Non-compartmental pharmacokinetic analysis was performed to evaluate HNK/CD
311
COMP and HNK suspensions after these drugs were orally administered. The
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valuable pharmacokinetic parameters are listed in Table 2. The HNK/CD COMP
313
group exhibited a higher HNK maximum plasma concentration (Cmax) than that of
314
the HNK suspension group (Fig. 6A; Table 2). AUC0-t and Cmax of the HNK/CD
315
COMP group were 1.58 and 1.23 times higher than those of the HNK suspension
316
group, respectively. This result indicated that SB-β-CD significantly enhanced the
317
relative bioavailability of HNK (p < 0.01). The apparent volume of distribution (V) of
318
HNK/CD COMP and HNK suspensions were 267.57 ± 97.48 and 191.93 ± 67.29,
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HNK
suspensions
were
orally administered.
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COMP and
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HNK/CD
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Figure 6A shows the mean plasma concentration versus the time profiles of HNK
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respectively (Table 2). The body CL of HNK suspension was approximately three fold higher than that of HNK/CD COMP. Tmax and t1/2 of the HNK/CD COMP group displayed a statistically significant difference compared with those of the HNK suspension (p < 0.05). These results indicated that SB-β-CD significantly increased the absorption but delayed the elimination of HNK.
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As a traditional administration route in clinical therapy, intravenous injection
325
could work rapidly and prevent the first-pass effect. HNK could not be prepared into
326
the solution injection because of its limited solubility. Using SB-β-CD, we found that
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the apparent solubility of HNK was increased significantly. Therefore, the preparation
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of HNK solution injection is possible. Hence, the pharmacokinetics of HNK/CD
329
COMP after intravenous injection in rats was also investigated. The plasma
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concentration-time curve of HNK/CD COMP after administration to rats is shown in
331
Fig. 6B, and their key pharmacokinetic parameters are listed as follows: Cmax = 5.31 ±
332
0.84 mg/L, Tmax = 0.05 ± 0.02 h, and AUC0-t = 5.61 ± 1.08 mg h/L. These data are
333
valuable references for the clinical application of HNK.
334
4. Conclusion
an
us
cr
ip t
327
In this study, the inclusion complex of HNK and SB-β-CD was successfully
336
prepared using a freeze-drying method. The solubility enhancement of HNK was
337
investigated by the phase-solubility method, and the phase-solubility curves showed a
338
typical AL-type, indicating the formation of 1:1 (HNK: SB-β-CD) inclusion complex.
339
Moreover, the formation of the inclusion complex was characterized by FT-IR and
341 342 343
d
te
Ac ce p
340
M
335
DSC. The apparent water solubility of HNK was significantly improved after this molecule formed a complex with SB-β-CD. The HNK/CD COPM exhibited a higher drug release rate than that of the free HNK in vitro. A comparative study of the pharmacokinetics between HNK/CD COPM and free HNK was also performed in rats.
344
The in vivo results indicated that AUC0-t and Cmax of HNK/CD COPM resulted in an
345
approximately 158% and 123% increase compared with those of the free HNK,
346
respectively. These results suggest that SB-β-CD will be potentially useful in the
347
delivery of poorly soluble drugs, such as HNK.
16
Page 16 of 31
348 349
Acknowledgments This project was supported by Natural Science Foundation Project of CQ CSTC
351
(No. cstc2012jjA10021), the National Research Foundation for the Doctoral Program
352
of Higher Education of China (No. 20125503120003), Scientific and Technological
353
Research Program of Chongqing Municipal Education Commission (Grant No.
354
KJ110323 and KJ120307), Chongqing Board of Health Project (2013-2-060),
355
Chongqing Yuzhong District Science and Technology Project (No. 20100203),
356
Students' Research and Innovation Experimental Project of Chongqing Medical
357
University (201244, 201229, 201217) and Students' Scientific Research Fund of "star
358
of HIFU" (XS201309).
M
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362 363 364 365 366 367
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Tommasini, S., & Ventura, C. A. (2013). A characterization study of resveratrol/sulfobutyl
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Biointerfaces, 115C, 22-28.
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Venuti, V., Cannava, C., Cristiano, M. C., Fresta, M., Majolino, D., Paolino, D., Stancanelli, R.,
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ether-beta-cyclodextrin inclusion complex and in vitro anticancer activity. Colloids Surf B
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Biointerfaces, 115, 22-28.
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beta-cyclodextrin/soybean lecithin inclusion complex. Carbohydr Polym, 101, 1027-1032.
443
Weeks, B. S. (2009). Formulations of dietary supplements and herbal extracts for relaxation and
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anxiolytic action: Relarian. Med Sci Monit, 15(11), RA256-262.
445
Yamaguchi, N., Satoh-Yamaguchi, K., & Ono, M. (2009). In vitro evaluation of antibacterial,
446
anticollagenase, and antioxidant activities of hop components (Humulus lupulus) addressing acne
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Zhang, J., & Ma, P. X. (2013). Cyclodextrin-based supramolecular systems for drug delivery: recent
449
progress and future perspective. Adv Drug Deliv Rev, 65(9), 1215-1233.
450
Zhang, W., Gong, X., Cai, Y., Zhang, C., Yu, X., Fan, J., & Diao, G. (2013). Investigation of
451
Ac ce p
452 453 454 455
te
d
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us
cr
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water-soluble inclusion complex of hypericin with beta-cyclodextrin polymer. Carbohydr Polym, 95(1), 366-370.
20
Page 20 of 31
455 456 Fig.1. The chemical structure of HNK (A) and SB-β-CD (B).
ip t
457 458
Fig. 2. Phase solubility diagram of HNK and SB-β-CD at 25, 37, and 50 °C (n=3, error bars are
460
smaller than symbols)
us
cr
459
461
Fig. 3. Differential scanning calorimetry (DSC): A. HNK; B. HNK/CD PM; C. SB-β-CD; D.
463
HNK/CD COMP.
an
462
465
M
464
Fig.4. FT-IR spectra of HNK(A), SB-β-CD (B), HNK/CD PM(C) and HNK/CD COMP(D).
te
d
466
Fig. 5. Mean (n=3) release curves of HNK/CD COMP (cycle), HNK/CD PM (square) and HNK
468
(triangle) in 0.1mM PBS(pH 7.4 ).
469 470 471 472
Ac ce p
467
Fig. 6. Mean plasma concentration–time profiles of HNK in rats (n =6): (A) HNK suspension and HNK/CD COMP solution after oral administration, (B) HNK/CD COMP solution via intravenous.
473 474 475 476 21
Page 21 of 31
477 478
ip t
479
an
us
cr
480
484 485 486 487 488 489
d te
483
Fig. 1. The chemical structure of HNK (A) and SB-β-CD (B).
Ac ce p
482
M
481
490 491 492 493
22
Page 22 of 31
494 495
ip t
496
cr
20 15
25 ℃
10
37 ℃
5 0
0
5 10 15 20 25 Concentration of SB--CD (mM)
us
50 ℃
30
an
Solubility of HNK (mM)
25
497
Fig. 2. Phase solubility diagram of HNK and SB-β-CD at 25, 37, and 50 °C (n=3, error bars are
499
smaller than symbols)
502 503 504 505 506
d te
501
Ac ce p
500
M
498
507 508 509 510
23
Page 23 of 31
M
an
us
cr
ip t
511
512
te
d
513
Fig. 3. Differential scanning calorimetry (DSC): A. HNK; B. HNK/CD PM; C. SB-β-CD; D.
515
HNK/CD COMP.
516 517 518 519
Ac ce p
514
520 521 522 523
24
Page 24 of 31
524
526 527 528 529 530
Ac ce p
te
d
M
an
us
cr
ip t
525
Fig. 4. FT-IR spectra of HNK(A), SB-β-CD (B), HNK/CD PM(C) and HNK/CD
COMP(D).
531 532 533 534
25
Page 25 of 31
535 536
538
cr
HNK
80
HNK/CD PM
60
us
HNK/CD COMP
40 20
an
Cumulative release rate(%)
100
ip t
537
0 0
5
10
15
20
Time (h)
M
539
25
Fig. 5. Mean (n=3) release curves of HNK/CD COMP(cycle), HNK/CD PM (square) and HNK
541
(triangle) in 0.1mM PBS(pH 7.4 ).
544 545 546 547
te
543
Ac ce p
542
d
540
548 549 550 551 26
Page 26 of 31
552 553
ip t
554
an
us
cr
555
557 558 559 560 561
Ac ce p
te
d
M
556
Fig. 6. Mean plasma concentration–time profiles of HNK in rats (n =6): (A) HNK suspension and HNK/CD COMP solution after oral administration, (B) HNK/CD COMP solution via intravenous.
562 563
27
Page 27 of 31
563 564
566
Table 1. The main thermodynamic parameters of HNK/SBECD
cr
567
ip t
565
Table 2. The key non-compartmental parmacokinetic parameters of HK and HNK-SBECD (n=6,
569
x ±s)
us
568
an
570 571
M
572
576 577 578 579 580
te
575
Ac ce p
574
d
573
581 582 583 584
28
Page 28 of 31
585 586
Table 1. The main thermodynamic parameters of HNK/SBECD Regression equation
K1:1 -1
(L.mol )
（°C）
△G (KJ.mol-1)
y=0.580x-0.077,
3945
( r=0.9972) y=0.631x-0.144,
4407
M
37
y=0.717x+0.239,
5027
(KJ.mol-1)
(J.mol-1.k-1)
-21.636
7.765
94.86
-22.636
te
50
d
( r=0.9963)
△S
-20.525
an
25
△H
cr
Temperature
us
588
ip t
587
589 590 591 592 593
Ac ce p
(r =0.9979)
594 595 596 597
29
Page 29 of 31
598 599
ip t
600 601
cr
602
Table 2. The key non-compartmental parmacokinetic parameters of HK and HNK-SBECD (n=6,
604
x ±s) parameter
HNK (0.5%CMC-Na)
AUC0-t (mg h/L)
6.59±1.43
10.44±0.75
AUC0-∞ (mg h/L)
7.38±1.74
15.27±3.14
12.83±5.55
18.17±1.15
MRT0-∞ (h)
HNK-SBECD
an
M d
MRT0-t (h)
41.02±14.38
Cmax (mg/L)
0.70±0.21
0.86±0.16
Tmax (h)
0.47±0.15
0.14±0.08
t1/2 (h)
13.00±6.58
23.37±5.69
Vd (L/kg)
191.93±67.29
267. 57±97.48
CL (L/h/kg)
11.54±3.56
4.14±2.21
te
17.66±9.00
Ac ce p
605
us
603
606
30
Page 30 of 31
606 607
Highlight:
609
►Inclusion complex of honokiol and SB-β-CD was prepared and characterized.
610
►The inclusion complex exhibited a higher drug release rate than free honokiol in
611
vitro.
612
►The bioavailability of honokiol was significantly improved by complexation with
613
SB-β-CD.
us
cr
ip t
608
Ac ce p
te
d
M
an
614
31
Page 31 of 31
S0144-8617(14)00635-3 http://dx.doi.org/doi:10.1016/j.carbpol.2014.06.059 CARP 9024
To appear in: Received date: Revised date: Accepted date:
20-2-2014 1-6-2014 14-6-2014
Please cite this article as: Xu, C., Tang, Y., Hu, W., Tian, R., Jia, Y., Deng, P., and Zhang, L.,Investigation of inclusion complex of honokiol with sulfobutyl ether-rmbeta-cyclodextrin, Carbohydrate Polymers (2014), http://dx.doi.org/10.1016/j.carbpol.2014.06.059 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
1
Investigation of inclusion complex of honokiol with sulfobutyl
2
ether-β-cyclodextrin
3
Caibing Xu a, Yalan Tang a, Wenjing Hu b, Rui Tian c, Yuntao Jia d, Ping Deng a,
4
Liangke Zhang a,*
5
a
6
pharmacy, Chongqing Medical University, Chongqing 400016, P.R. China;
7
b
Chongqingshi Shapingba District People's Hospital, Chongqing 400030, P.R. China;
8
c
The Experimental Teaching Centre, Chongqing Medical University, Chongqing
9
400016, P.R. China；
ip t
d
11
Chongqing 400010, P.R. China;
14 15 16 17
te
d
Department of Pharmacy, Children’s Hospital of Chongqing Medical University,
Ac ce p
13
M
an
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cr
Chongqing Key Laboratory of Biochemistry and Molecular Pharmacology, School of
10
12
1
18
*Corresponding author: Liangke Zhang, School of Pharmacy, Chongqing Medical University, District of Yuzhong, Chongqing 400016, China. Tel: +86-23-6848-5161; fax: +86-23-6848-5161. E-mail address: [email protected](L. Zhang)
1
Page 1 of 31
19
Abstract This study aimed to prepare and characterize an inclusion complex of honokiol
21
(HNK) with sulfobutyl ether-β-cyclodextrin (SB-β-CD). The inclusion complex
22
(HNK/CD COMP) was prepared utilizing a freeze-drying method. Phase-solubility
23
curves were employed to obtain stability constants and thermodynamic parameters.
24
The phase-solubility diagram showed a typical AL-type, indicating that the 1:1 (HNK:
25
SB-β-CD) inclusion complex was formed. The solid inclusion complex was then
26
characterized by differential scanning calorimetry and Fourier transform infrared
27
spectroscopy. Results showed that HNK/CD COMP exhibited a higher drug release
28
rate than free HNK in vitro. A comparative study of the pharmacokinetics between
29
HNK/CD COMP and free HNK was also performed in rats. In vivo results indicated
30
that AUC0-t and Cmax of HNK/CD COMP increased by approximately 158% and
31
123% compared with those of the free HNK, respectively. These results suggest that
32
SB-β-CD will be potentially useful in the delivery of poorly soluble drugs, such as
34 35 36
cr
us
an
M
d
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Ac ce p
33
ip t
20
HNK.
Key words: Sulfobutyl ether-β-cyclodextrin; Honokiol; Inclusion complex; In
vitro release; Pharmacokinetic study
37 38 39 40
2
Page 2 of 31
41
1. Introduction Cyclodextrins (CDs) are a family of macrocyclic oligosaccharides mostly
43
consisting of six, seven, or eight glucose units joined by α-1,4 glycosidic bonds
44
(Brewster & Loftsson, 2007; Stella & He, 2008). The hydrophobic cavity of CDs
45
provides these CDs the ability to form inclusion complexes with various compounds,
46
including proteins, oligonucleotides, ions, and small molecules (Stella & He, 2008;
47
Zhang & Ma, 2013). As a promising material with low toxicity and low
48
immunogenicity, CDs have been extensively investigated in the field of biomedicine,
49
particularly in pharmaceutical sciences and technologies. Related applications include
50
the improvement of drug solubility and stability, increase in drug absorption and
51
bioavailability, removal of odors and tastes, and decrease in local and systemic
52
toxicity (Carrier, Miller & Ahmed, 2007; Garcia-Fernandez et al., 2013; Zhang & Ma,
53
2013). CDs can be administered by oral, ocular, nasal, and rectal
54
(Al-Rawashdeh, Al-Sadeh & Al-Bitar, 2010; Pahuja, Kashyap & Pawar, 2013; Singh,
56 57 58
cr
us
an
M
d
te
delivery
Ac ce p
55
ip t
42
Kumar & Pathak, 2013; Tiwari, Tiwari & Rai, 2010). To improve these properties of natural CDs in terms of solubility, stability, inclusion capability, and toxicity, researchers synthesized various chemically modified CDs, such as amphiphilic, hydrophobic, and highly soluble derivatives.
59
When new excipients are considered as drug carries, safety should be primary
60
concern. Unmodified cyclodextrins, such as α- and β-cyclodextrin, were extensively
61
investigated in pharmaceuticals. However, they were limited by hemolytic activity
62
and nephrotoxicity because of their interaction with cholesterol and other lipid
3
Page 3 of 31
63
membrane components of cellular structures (Luke, Tomaszewski, Damle & Schlamm,
64
2010). Sulfobutyl ether-β-cyclodextrin (SB-β-CD, Fig. 1A) is an excellent modified
66
compound of β-cyclodextrin with improved water solubility and low toxicity.
67
SB-β-CD does not undergo significant tubular reabsorption due to its ionized state
68
after glomerular filtration and is concentrated in the urine for excretion, which
69
attenuate the potential to harm the kidney(Albers & Muller, 1995; Irie & Uekama,
70
1997; Uekama, 2004). SB-β-CD can form inclusion complexes with drug molecules.
71
Thus, SB-β-CD has been utilized to improve the solubility and stability of drugs
72
(Danel, Azaroual, Chavaria, Odou, Martel & Vaccher, 2013; Stella & He, 2008;
73
Venuti et al., 2013).
M
an
us
cr
ip t
65
Honokiol (HNK, Fig. 1B) is a bioactive lignanoid extracted from the reed of
75
Magnolia officinalis, which has been utilized in traditional Chinese medicine for a
76
long time. Furthermore, HNK is extensively used in Japan (Cheng, 2012; Lee, Lee,
78 79 80 81
te
Ac ce p
77
d
74
Lee, Jung, Han & Hong, 2011). This compound exhibits several pharmacological properties, such as anti-cancer effects, anti-inflammatory effects, anti-oxidant actions, and anti-anxiety effects. However, the poor aqueous solubility of HNK has impeded clinical applications.
(Choi et al., 2002; Kang et al., 2008; Weeks, 2009; Yamaguchi,
Satoh-Yamaguchi & Ono, 2009).
82
In this study, the inclusion complex of HNK and SB-β-CD (HNK/CD COMP) was
83
prepared to improve the solubility and bioavailability of HNK using a freeze-drying
84
method (Jadhav, Petkar, Pore, Kulkarni & Burade, 2013; Rajendiran & Siva, 2014;
4
Page 4 of 31
Zhang et al., 2013). Solid-state HNK/CD COMP was characterized by differential
86
scanning calorimetry (DSC) and Fourier transform infrared spectroscopy (FT-IR). In a
87
solution, solubility phase analysis was performed to estimate the enhanced solubility
88
of HNK by SB-β-CD and the stoichiometry of the inclusion complex. In vitro release
89
study and model fitting were systematically investigated and evaluated. Furthermore,
90
the pharmacokinetic properties of HNK suspension and HNK/CD COMP in male
91
Sprague-Dawley rats were analyzed.
92
2. Materials and methods
93
2.1. Materials
an
us
cr
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85
Honokiol (purity ≥ 98%) was purchased from Xi’an Grass Plant Science and
95
Technology CO., Ltd (Xi’an, China). SB-β-CD (DS=7.0) was purchased from
96
Shandong Binzhou Zhiyuan Bio-Technology Co., Ltd (Shandong, China). All the
97
other reagents were of analytical grade.
98
2.2. Phase-solubility analysis
100 101 102
d
te
Ac ce p
99
M
94
Phase-solubility analysis was performed in a SHZ-88 thermostatic shaking water
bath (Jiangsu Taicang Medical Instrument Factory, China). The excess amount of HNK was added to volumetric flasks containing 10 mL of SB-β-CD solutions at various concentrations (0, 5, 10, 15, 20, 25, and 30 mM). The contents were then
103
sonicated for 5 min. The flasks were sealed to prevent evaporation and shaken
104
continuously at 100 rpm in a thermostatic bath at different temperatures (25, 37, and
105
50 °C) for 48 h. After equilibrium was reached, suspensions were filtered using a 0.45
106
µm syringe filter. The amount of drug was then analyzed at a wavelength of 292 nm
5
Page 5 of 31
107
by using a spectrophotometer (Shimazdu UV-3150, Japan). Experiments were
108
performed in triplicate.
109
2.3. Preparation of inclusion complex The inclusion complex was prepared using a freeze-dry method (Pralhad &
111
Rajendrakumar, 2004; Ruz, Froeyen, Busson, Gonzalez, Baudemprez & Van den
112
Mooter, 2012; Wang, Luo & Xiao, 2014). On the basis of the results of
113
phase-solubility analysis, we dispersed 100 mg of HNK in 100 mL of water
114
containing 1.68 g of SB-β-CD at 45 °C. After magnetically stirring for 2.5 h, we
115
filtered the resultant solution with a 0.45 µm syringe filter and the filtrate was
116
freeze-dried (ALPHA 1-2 LD plus, CHRIST, Germany) for 48 h.
117
2.4. DSC
M
an
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cr
ip t
110
DSC measurements were performed to confirm that the complex was formed.
119
The powdered samples of HNK, SB-β-CD, HNK/CD COMP, and physical mixture
120
(HNK/CD PM) were placed in an aluminum pan, heated to 70 °C for 5 min, and
122 123 124 125
te
Ac ce p
121
d
118
cooled to 20 °C for 5 min at the cooling rate of 10 °C ·min–1. And then the profiles were obtained by using a Netzsch STA449C thermal analyzer (Netzsch Corporation, Germany) from 20 °C to 300 °C (at a constant rate of 10 °C·min–1) under nitrogen gas flow (25 mL·min–1).
2.5. Fourier transform infrared spectroscopic (FT-IR) study
126
The FT-IR spectra of the powdered samples of HNK, SB-β-CD, HNK/CD COMP,
127
and HNK/CD PM were obtained using a spectrometer (Bruker-tensor 27, Germany)
128
with the potassium bromide pellet method. The typical bands were recorded for
6
Page 6 of 31
different samples. Each spectrum was recorded in the range of 4000 cm–1 to 400 cm–1.
130
The spectra were obtained under dry conditions at a resolution of 4 cm–1 to prevent
131
contamination.
132
2.6. Dissolution studies
ip t
129
Dissolution studies were implemented using the dialysis membrane method.
134
Three formulations, namely, HNK, HNK/CD PM, and HNK/CD COMP were
135
transferred into a dialysis bag (molecular weight cutoff, 8-14kDa). The dialysis bag
136
was then immersed in a container filled with 25 mL of 0.1 mM phosphate buffer
137
solution (pH 7.4) containing sodium dodecyl sulfate (SDS, 0.5%) at 37 °C with
138
constant shaking (100 rpm). The receptor compartments were closed to prevent the
139
dissolution medium from evaporating. An aliquot (4.0 mL) of the incubation medium
140
was withdrawn and replaced with the same volume of pre-warmed fresh medium at
141
specific time intervals. The concentration of HNK was then determined using a UV
142
spectrophotometer (Shimazdu UV-3150, Japan) after HNK was filtered using a 0.45
144 145
us
an
M
d
te
Ac ce p
143
cr
133
µm membrane. The cumulative release percentage (Q) was calculated according to the following equation: Q (C
n
25
n 1
C i 4 ) / A 100 %
(1)
i 1
146 147
where n and i are the sample numbers, Cn and Ci are the drug concentration at the point of n and i, respectively, and A is the total drug content in each sample.
148
Similarity factor (f2) was first proposed by Moore and Flanner (Costa & Sousa
149
Lobo, 2001; Shah, Tsong, Sathe & Liu, 1998) to compare release profiles. It was
150
derived from Mean-Squared difference. Similarity factor (f2) was also recommended 7
Page 7 of 31
151
for release profile comparison in the FDA’s Guidances for Industry (FDA, 1997; Shah
152
et al., 1997). In this study, f2 was used to evaluate the similarity between every two
153
release profiles. f2 is defined by the following equation: f2
2
0 .5
100 (2)
ip t
154
n 50 lg 1 1 / n R t T t t 1
where n is the number of sampling points, and Rt and Tt are HNK cumulative release
156
percentages at time t of the two release curves, respectively. The result indicated that
157
the similarity between the two release profiles was statistically significant at f2 > 50. A
158
high similarity between two profiles is characterized by a high f2.
159
2.7 Pharmacokinetic analysis in rats
160
2.7.1 Animal handling and care
M
an
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cr
155
Male Sprague-Dawley rats weighing approximately 200 ± 20 g were obtained
162
from the Laboratory Animal Center of Chongqing Medical University and used for
163
pharmacokinetic analysis. The animal experiment was approved by the Animal Ethics
165 166 167 168
te
Ac ce p
164
d
161
Committee of Chongqing Medical University (Chongqing, China), and their guidelines were followed throughout this study. Before the experiment was conducted, the rats were fasted overnight but provided free access to water. 2.7.2 Pharmacokinetic behavior of rats Eighteen rats were randomly divided into three groups, namely, A, B, and C (n = 6
169
rats). Group A received a single oral dose of HNK/CD COMP (80 mg·kg–1 of HNK)
170
and group B was orally administered a single dose of HNK suspension (80 mg·kg–1 of
171
HNK suspended in 0.5% sodium carboxymethyl cellulose solution). The blood
172
samples (0.5 mL) were collected at 0.083, 0.25, 0.5, 1, 2, 4, 6, 10, 12, 24, and 48 h 8
Page 8 of 31
173
post-treatment. Group C was injected with the HNK/CD COMP solution at a dose of 20 mg·kg–1
175
of HNK in the tail vein. The blood samples (0.5 mL) were collected from the
176
retro-orbital plexus of each rat and transferred into heparinized tubes at 0.033, 0.083,
177
0.25, 0.5, 0.75, 1, 1.5, 2, 4, and 6 h post-administration. These samples were
178
immediately centrifuged, and the plasma samples were stored at –20 °C for further
179
studies.
180
2.7.3 Preparation of plasma samples
an
us
cr
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174
An aliquot of plasma (200 µL) was pre-treated with 20 µL of evodiamine
182
methanol solution (20 µg·mL–1) as an internal standard and then vortexed for 5 min.
183
The resulting sample was mixed with 1.5 mL of ethyl acetate, vortexed for another 5
184
min, and centrifuged at 4000 rpm for 10 min. Afterward, the supernatant was
185
evaporated to dryness in a vacuum, redissolved in 100 µL of methanol, and
186
centrifuged at 12,000 rpm for 10 min. This sample solution (20 µL) was directly
188 189 190
d
te
Ac ce p
187
M
181
injected into a HPLC system (Agilent 1100, American) for analysis. 2.7.4 Measurement of HNK from plasma using RP-HPLC The plasma concentrations of HNK were determined by an Agilent 1100 HPLC
system (Agilent, American). A Lichrospher RP C18 column (4.6 mm × 150 mm, 5 µm,
191
Hanbon Sci. & Tech., China) was used and the mobile phase was a mixture of
192
methanol and water (80:20, v/v). The flow rate was 1.0 mL·min–1. All of the
193
detections were performed at a wavelength of 292 nm at a constant temperature of
194
30 °C.
9
Page 9 of 31
195
2.7.5 Pharmacokinetic analysis The valuable pharmacokinetic parameters, namely, AUC0-t (area under the plasma
197
concentration versus time curve), Cmax (peak drug concentration), Tmax (time to reach
198
the peak concentration), Vd (apparent volume of distribution), t1/2 (half-life), and CL
199
(clearance) were collected by DAS2.1.1 (issued by the China Food and Drug
200
Administration for pharmacokinetic study). All data were presented as the mean ±
201
standard deviation (n = 6, x ± s).
202
3. Results and discussion
203
3.1 Phase solubility study
Phase-solubility study is a traditional and useful approach not only to determine
205
the value of the stability constant, but also to give insight into the stoichiometry of the
206
equilibrium. The practical and phenomenological implications of phase solubility
207
study were developed by Higuchi and Connons (Higuchi.T, 1965). Based on the
208
212 213
between HNK and SB-β-CD. The study infused the solubilizing ability of SB-β-CD to
214
HNK and the stability constant of HNK/CD COMP according to the phase-solubility
215
diagram. Figure 2 shows the phase solubility diagrams of HNK and SB-β-CD in three
216
different temperatures. All of the apparent solubility curves of HNK with correlation
209 210 211
te
d
204
Ac ce p
M
an
us
cr
ip t
196
profiles of the phase solubility study, several types of solubility behaviors can be classified. The phase solubility diagrams include two major types: A type( a soluble compound was formed ) and B type (a compound of limit solubility was formed) (Brewster & Loftsson, 2007; Thompson, 1997). In this study, phase-solubility study was performed to determine the interactions
10
Page 10 of 31
217
coefficient r > 0.9963 were regarded as straight lines. Moreover, all of them can be
218
considered as AL-type diagrams, indicating that the observed increase in the solubility
219
of HNK was due to the formation of a 1:1 molar ratio inclusion complex.
equation(Brewster & Loftsson, 2007; Thompson, 1997): K1:1 = Slope / S0(1-Slope)
(3)
us
222
ip t
221
The inclusion stability constant K1:1 was calculated using the following
cr
220
where S0 is the equilibrium solubility of HNK in water; slope is obtained from the
224
phase-solubility diagram. K1:1 at 25, 37, and 50 °C was calculated from Equation 3.
225
Thermodynamic parameters were calculated from Gibbs and Van’t Hoff equations:
an
223
△G= -RTln K1:1
(4)
227
ln K1:1=-△H/RT+△S/R
(5)
M
226
Valuable parameters, including stability constant (K1:1), Gibbs free energy change
229
(∆G), enthalpy change (∆H), and entropy change (∆S), are listed in Table 1. K1:1
230
increased with rising temperature. ∆G of HNK was negative and decreased as
232 233 234
te
Ac ce p
231
d
228
temperature increased. ∆H was positive in this study. The thermodynamic parameters and K1:1 indicated that high temperatures were favorable for the inclusion process (Hu, Zhang, Song, Gu & Hu, 2012; Rajendiran & Siva, 2014). 3.2 DSC analysis
235
The melting, boiling, and sublimation points of drug molecules either shift to
236
different temperatures or disappear when these molecules are included in SB-β-CD
237
cavities (Ascenso et al., 2011; de Azevedo Mde et al., 2011). The thermograms of
238
HNK, SB-β-CD, HNK/CD PM, and HNK/CD COMP are shown in Fig. 3. HNK
11
Page 11 of 31
generated a sharp endothermic peak at 92 °C, indicating its melting point. The
240
diagram of SB-β-CD exhibited an endothermic peak at 218 °C, due to the sample
241
decomposition. In the case of HNK/CD PM, the melting peak of HNK at 92 °C and
242
the endothermic peak of SB-β-CD were observed, indicating that there was no
243
inclusion complex formed between HNK and SB-β-CD. However, no sharp peak was
244
found in the thermogram of HNK/CD COMP at 92 °C, indicating the amorphous
245
characteristic of HNK. The absence of the melting peak of HNK in the thermogram of
246
HNK/CD COMP may have occurred because HNK was included in the SB-β-CD
247
cavity.
248
3.3 FT-IR analysis
M
an
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cr
ip t
239
HNK/CD COMP was further analyzed by FT-IR spectroscopy. The spectra of
250
HNK, SB-β-CD, HNK/CD PM, and HNK/CD COMP from 4000 cm–1 to 400 cm–1 are
251
shown in Fig. 4. The HNK spectrum showed the bands in the 3100 cm–1 to 3000 cm–1
252
wave number regions, indicating the presence of –CH and –OH groups. A band was
254 255 256
te
Ac ce p
253
d
249
found at 1638 cm–1, which can be assigned to alkene C=C present in HNK. The intense bands at 1498 cm-1 and 1430 cm-1 were assigned to the stretching vibrations from the aromatic ring of honokiol molecule. Moreover, other bands were located at 1217 and 908 cm–1 (C–O) as well as at 989 and 825 cm–1 (C–C). The spectrum of
257
SB-β-CD was mainly characterized by the intense bands at 3800 cm–1 to 3100 cm–1
258
caused by O–H stretching vibration. The band at 1643 cm-1 was ascribed to the
259
δ-HOH bending of water molecules attached to SB-β-CD. The vibrations of –CH and
260
–CH2 groups appeared in the 3000 cm–1 to 2800 cm–1 region. The sulfoxide stretched
12
Page 12 of 31
at 1046 cm–1 (Mahmoud, El-Feky, Kamel & Awad, 2011; Venuti et al., 2013). In the
262
case of HNK/CD PM, the spectrum was basically composed of the overlapping peaks
263
of HNK and SB-β-CD, and this spectrum showed a less intense absorption band at
264
1638 cm–1 than at other regions because of the low ratio of HNK in the physical
265
mixture. This demonstrates that there was no interaction between drug and SB-β-CD.
266
Compared with the HNK/CD PM, the spectrum of HNK/CD COMP showed relevant
267
changes in intensities and widths of the typical absorption peaks, indicating the
268
formation of a new compound between HNK and SB-β-CD (Venuti et al., 2014). A
269
less intense absorption band was also observed at 1638 cm–1 in the spectrum of
270
HNK/CD COMP, indicating that no interaction occurred between the C=C of HNK
271
molecule and that of SB-β-CD molecule. Thus, C=C was not included in the cavity of
272
SB-β-CD molecule. Therefore, only a part of the HNK molecule was possibly
273
included in the cavity and the C=C portion of HNK molecule was not included.
274
3.4 In vitro release study
278
Ac ce p
te
d
M
an
us
cr
ip t
261
279
<11% of the free HNK and 15% of HNK/CD PM. Furthermore, the cumulative
280
release percentages of HNK/CD COMP and free HNK at 4 h were 82% and 28%,
281
respectively.
275 276 277
282
Figure 5 shows the release curves of the free HNK, HNK/CD PM, and HNK/CD
COMP in the 0.1 mM PBS (pH 7.4) containing SDS (0.5%). HNK/CD COMP exhibited a significantly higher release rate than that for the free HNK and HNK/CD PM. The cumulative release percentage of HNK/CD COMP at 0.5 h was >29% but
f2 between the two curves of HNK/CD COMP and free HNK was 19.0 (<50) and 13
Page 13 of 31
that of HNK/CD PM and free HNK was 54.1(>50). These values indicated that the
284
cumulative release profile of HNK/CD COMP was significantly different from that of
285
the free HNK. However, no significant difference was found in HNK/CD PM and free
286
HNK.
ip t
283
Different models, including Weibull, Higuchi (square root of time equation),
288
Ritger-Peppas, Hixcon-Crowell model (cubic root law), zero-order, and first-order
289
models, were utilized to fit the results of the mean release profiles. The model that
290
fitted the release data more efficiently was selected on the basis of r, that is, a higher r
291
corresponds to a better fit. For HNK/CD COMP, r of Weibull model (ln[1/1 – Q] =
292
0.6872lnt + 0.7100, r = 0.9 914) was the highest, indicating that the release profile of
293
HNK/CD COMP best fitted the Weibull model among all of the models. The release
294
profile of HNK/CD PM and HNK fitted well to the Ritger-Peppas model (lnQ =
295
0.4023lnt – 1.5494, r = 0.9 870 and lnQ = 0.5012lnt – 1.9532, r = 0.9 953,
296
respectively).
298 299 300
us
an
M
d
te
Ac ce p
297
cr
287
3.5 Pharmacokinetic analysis of HNK in rats HNK/CD COMP and HNK suspensions were administered to the male
Sprague-Dawley rats to evaluate the enhanced bioavailability in vivo at post-oral administration. The validated HPLC method was employed in the pharmacokinetic
301
analysis. A good linear relationship was obtained for HNK with concentration ranging
302
from 0.0355 to 9.0909 µg·mL–1. The relative standard deviation of HNK for inter-day
303
and intra-day precision and accuracy at low, medium and high concentration (0.0450,
304
2.2727 and 9.0909 µg·mL–1) was below 8.02%. The recoveries of HNK at the up
14
Page 14 of 31
305
three concentrations were 112.91, 106.84%, 100.88%. The limit of quantification
306
(LOQ) for HNK was 0.0355 µg·mL–1. Satisfactory results were obtained after a single
307
oral or intravenous dose were administered.
after
310
Non-compartmental pharmacokinetic analysis was performed to evaluate HNK/CD
311
COMP and HNK suspensions after these drugs were orally administered. The
312
valuable pharmacokinetic parameters are listed in Table 2. The HNK/CD COMP
313
group exhibited a higher HNK maximum plasma concentration (Cmax) than that of
314
the HNK suspension group (Fig. 6A; Table 2). AUC0-t and Cmax of the HNK/CD
315
COMP group were 1.58 and 1.23 times higher than those of the HNK suspension
316
group, respectively. This result indicated that SB-β-CD significantly enhanced the
317
relative bioavailability of HNK (p < 0.01). The apparent volume of distribution (V) of
318
HNK/CD COMP and HNK suspensions were 267.57 ± 97.48 and 191.93 ± 67.29,
321 322 323
HNK
suspensions
were
orally administered.
us
an
M
d
te
Ac ce p
320
COMP and
cr
309
319
HNK/CD
ip t
Figure 6A shows the mean plasma concentration versus the time profiles of HNK
308
respectively (Table 2). The body CL of HNK suspension was approximately three fold higher than that of HNK/CD COMP. Tmax and t1/2 of the HNK/CD COMP group displayed a statistically significant difference compared with those of the HNK suspension (p < 0.05). These results indicated that SB-β-CD significantly increased the absorption but delayed the elimination of HNK.
324
As a traditional administration route in clinical therapy, intravenous injection
325
could work rapidly and prevent the first-pass effect. HNK could not be prepared into
326
the solution injection because of its limited solubility. Using SB-β-CD, we found that
15
Page 15 of 31
the apparent solubility of HNK was increased significantly. Therefore, the preparation
328
of HNK solution injection is possible. Hence, the pharmacokinetics of HNK/CD
329
COMP after intravenous injection in rats was also investigated. The plasma
330
concentration-time curve of HNK/CD COMP after administration to rats is shown in
331
Fig. 6B, and their key pharmacokinetic parameters are listed as follows: Cmax = 5.31 ±
332
0.84 mg/L, Tmax = 0.05 ± 0.02 h, and AUC0-t = 5.61 ± 1.08 mg h/L. These data are
333
valuable references for the clinical application of HNK.
334
4. Conclusion
an
us
cr
ip t
327
In this study, the inclusion complex of HNK and SB-β-CD was successfully
336
prepared using a freeze-drying method. The solubility enhancement of HNK was
337
investigated by the phase-solubility method, and the phase-solubility curves showed a
338
typical AL-type, indicating the formation of 1:1 (HNK: SB-β-CD) inclusion complex.
339
Moreover, the formation of the inclusion complex was characterized by FT-IR and
341 342 343
d
te
Ac ce p
340
M
335
DSC. The apparent water solubility of HNK was significantly improved after this molecule formed a complex with SB-β-CD. The HNK/CD COPM exhibited a higher drug release rate than that of the free HNK in vitro. A comparative study of the pharmacokinetics between HNK/CD COPM and free HNK was also performed in rats.
344
The in vivo results indicated that AUC0-t and Cmax of HNK/CD COPM resulted in an
345
approximately 158% and 123% increase compared with those of the free HNK,
346
respectively. These results suggest that SB-β-CD will be potentially useful in the
347
delivery of poorly soluble drugs, such as HNK.
16
Page 16 of 31
348 349
Acknowledgments This project was supported by Natural Science Foundation Project of CQ CSTC
351
(No. cstc2012jjA10021), the National Research Foundation for the Doctoral Program
352
of Higher Education of China (No. 20125503120003), Scientific and Technological
353
Research Program of Chongqing Municipal Education Commission (Grant No.
354
KJ110323 and KJ120307), Chongqing Board of Health Project (2013-2-060),
355
Chongqing Yuzhong District Science and Technology Project (No. 20100203),
356
Students' Research and Innovation Experimental Project of Chongqing Medical
357
University (201244, 201229, 201217) and Students' Scientific Research Fund of "star
358
of HIFU" (XS201309).
M
an
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te
d
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362 363 364 365 366 367
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with cyclodextrines. Int J Pharm, 439(1-2), 275-285.
419
Shah, V., Lesko, L., Fan, J., Fleischer, N., Handerson, J., Malinowski, H., Makary, M., Ouderkirk, L.,
420
Roy, S., & Sathe, P. (1997). FDA guidance for industry: dissolution testing of immediate release solid
421
426
Ac ce p
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Thompson, D. O. (1997). Cyclodextrins--enabling excipients: their present and future use in
428
pharmaceuticals. Crit Rev Ther Drug Carrier Syst, 14(1), 1-104.
429
Tiwari, G., Tiwari, R., & Rai, A. K. (2010). Cyclodextrins in delivery systems: Applications. J Pharm
430
Bioallied Sci, 2(2), 72-79.
431
Uekama, K. (2004). Design and evaluation of cyclodextrin-based drug formulation. Chem Pharm Bull
432
(Tokyo), 52(8), 900-915.
422 423 424 425
te
d
M
an
us
cr
ip t
403
oral dosage forms. Dissolution Technol, 4, 15-22. Shah, V. P., Tsong, Y., Sathe, P., & Liu, J. P. (1998). In vitro dissolution profile comparison--statistics and analysis of the similarity factor, f2. Pharm Res, 15(6), 889-896. Singh, R. M., Kumar, A., & Pathak, K. (2013). Thermally triggered mucoadhesive in situ gel of loratadine: beta-cyclodextrin complex for nasal delivery. AAPS PharmSciTech, 14(1), 412-424. Stella, V. J., & He, Q. (2008). Cyclodextrins. Toxicol Pathol, 36(1), 30-42.
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Venuti, V., Cannava, C., Cristiano, M. C., Fresta, M., Majolino, D., Paolino, D., Stancanelli, R.,
434
Tommasini, S., & Ventura, C. A. (2013). A characterization study of resveratrol/sulfobutyl
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ether-beta-cyclodextrin inclusion complex and in vitro anticancer activity. Colloids Surf B
436
Biointerfaces, 115C, 22-28.
437
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Tommasini, S., & Ventura, C. A. (2014). A characterization study of resveratrol/sulfobutyl
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ether-beta-cyclodextrin inclusion complex and in vitro anticancer activity. Colloids Surf B
440
Biointerfaces, 115, 22-28.
441
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beta-cyclodextrin/soybean lecithin inclusion complex. Carbohydr Polym, 101, 1027-1032.
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Weeks, B. S. (2009). Formulations of dietary supplements and herbal extracts for relaxation and
444
anxiolytic action: Relarian. Med Sci Monit, 15(11), RA256-262.
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Yamaguchi, N., Satoh-Yamaguchi, K., & Ono, M. (2009). In vitro evaluation of antibacterial,
446
anticollagenase, and antioxidant activities of hop components (Humulus lupulus) addressing acne
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448
Zhang, J., & Ma, P. X. (2013). Cyclodextrin-based supramolecular systems for drug delivery: recent
449
progress and future perspective. Adv Drug Deliv Rev, 65(9), 1215-1233.
450
Zhang, W., Gong, X., Cai, Y., Zhang, C., Yu, X., Fan, J., & Diao, G. (2013). Investigation of
451
Ac ce p
452 453 454 455
te
d
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cr
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water-soluble inclusion complex of hypericin with beta-cyclodextrin polymer. Carbohydr Polym, 95(1), 366-370.
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455 456 Fig.1. The chemical structure of HNK (A) and SB-β-CD (B).
ip t
457 458
Fig. 2. Phase solubility diagram of HNK and SB-β-CD at 25, 37, and 50 °C (n=3, error bars are
460
smaller than symbols)
us
cr
459
461
Fig. 3. Differential scanning calorimetry (DSC): A. HNK; B. HNK/CD PM; C. SB-β-CD; D.
463
HNK/CD COMP.
an
462
465
M
464
Fig.4. FT-IR spectra of HNK(A), SB-β-CD (B), HNK/CD PM(C) and HNK/CD COMP(D).
te
d
466
Fig. 5. Mean (n=3) release curves of HNK/CD COMP (cycle), HNK/CD PM (square) and HNK
468
(triangle) in 0.1mM PBS(pH 7.4 ).
469 470 471 472
Ac ce p
467
Fig. 6. Mean plasma concentration–time profiles of HNK in rats (n =6): (A) HNK suspension and HNK/CD COMP solution after oral administration, (B) HNK/CD COMP solution via intravenous.
473 474 475 476 21
Page 21 of 31
477 478
ip t
479
an
us
cr
480
484 485 486 487 488 489
d te
483
Fig. 1. The chemical structure of HNK (A) and SB-β-CD (B).
Ac ce p
482
M
481
490 491 492 493
22
Page 22 of 31
494 495
ip t
496
cr
20 15
25 ℃
10
37 ℃
5 0
0
5 10 15 20 25 Concentration of SB--CD (mM)
us
50 ℃
30
an
Solubility of HNK (mM)
25
497
Fig. 2. Phase solubility diagram of HNK and SB-β-CD at 25, 37, and 50 °C (n=3, error bars are
499
smaller than symbols)
502 503 504 505 506
d te
501
Ac ce p
500
M
498
507 508 509 510
23
Page 23 of 31
M
an
us
cr
ip t
511
512
te
d
513
Fig. 3. Differential scanning calorimetry (DSC): A. HNK; B. HNK/CD PM; C. SB-β-CD; D.
515
HNK/CD COMP.
516 517 518 519
Ac ce p
514
520 521 522 523
24
Page 24 of 31
524
526 527 528 529 530
Ac ce p
te
d
M
an
us
cr
ip t
525
Fig. 4. FT-IR spectra of HNK(A), SB-β-CD (B), HNK/CD PM(C) and HNK/CD
COMP(D).
531 532 533 534
25
Page 25 of 31
535 536
538
cr
HNK
80
HNK/CD PM
60
us
HNK/CD COMP
40 20
an
Cumulative release rate(%)
100
ip t
537
0 0
5
10
15
20
Time (h)
M
539
25
Fig. 5. Mean (n=3) release curves of HNK/CD COMP(cycle), HNK/CD PM (square) and HNK
541
(triangle) in 0.1mM PBS(pH 7.4 ).
544 545 546 547
te
543
Ac ce p
542
d
540
548 549 550 551 26
Page 26 of 31
552 553
ip t
554
an
us
cr
555
557 558 559 560 561
Ac ce p
te
d
M
556
Fig. 6. Mean plasma concentration–time profiles of HNK in rats (n =6): (A) HNK suspension and HNK/CD COMP solution after oral administration, (B) HNK/CD COMP solution via intravenous.
562 563
27
Page 27 of 31
563 564
566
Table 1. The main thermodynamic parameters of HNK/SBECD
cr
567
ip t
565
Table 2. The key non-compartmental parmacokinetic parameters of HK and HNK-SBECD (n=6,
569
x ±s)
us
568
an
570 571
M
572
576 577 578 579 580
te
575
Ac ce p
574
d
573
581 582 583 584
28
Page 28 of 31
585 586
Table 1. The main thermodynamic parameters of HNK/SBECD Regression equation
K1:1 -1
(L.mol )
（°C）
△G (KJ.mol-1)
y=0.580x-0.077,
3945
( r=0.9972) y=0.631x-0.144,
4407
M
37
y=0.717x+0.239,
5027
(KJ.mol-1)
(J.mol-1.k-1)
-21.636
7.765
94.86
-22.636
te
50
d
( r=0.9963)
△S
-20.525
an
25
△H
cr
Temperature
us
588
ip t
587
589 590 591 592 593
Ac ce p
(r =0.9979)
594 595 596 597
29
Page 29 of 31
598 599
ip t
600 601
cr
602
Table 2. The key non-compartmental parmacokinetic parameters of HK and HNK-SBECD (n=6,
604
x ±s) parameter
HNK (0.5%CMC-Na)
AUC0-t (mg h/L)
6.59±1.43
10.44±0.75
AUC0-∞ (mg h/L)
7.38±1.74
15.27±3.14
12.83±5.55
18.17±1.15
MRT0-∞ (h)
HNK-SBECD
an
M d
MRT0-t (h)
41.02±14.38
Cmax (mg/L)
0.70±0.21
0.86±0.16
Tmax (h)
0.47±0.15
0.14±0.08
t1/2 (h)
13.00±6.58
23.37±5.69
Vd (L/kg)
191.93±67.29
267. 57±97.48
CL (L/h/kg)
11.54±3.56
4.14±2.21
te
17.66±9.00
Ac ce p
605
us
603
606
30
Page 30 of 31
606 607
Highlight:
609
►Inclusion complex of honokiol and SB-β-CD was prepared and characterized.
610
►The inclusion complex exhibited a higher drug release rate than free honokiol in
611
vitro.
612
►The bioavailability of honokiol was significantly improved by complexation with
613
SB-β-CD.
us
cr
ip t
608
Ac ce p
te
d
M
an
614
31
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