Journal of Pharmaceutical Sciences 105 (2016) 242e249
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Pharmaceutical Nanotechnology
Preparation and Physicochemical and Pharmacokinetic Characterization of Ginkgo Lactone Nanosuspensions for Antiplatelet Aggregation Tian-Qi Rui 1, 2, Liang Zhang 3, Hong-Zhi Qiao 1, 2, Ping Huang 1, 2, Shuai Qian 4, Jun-Song Li 1, 2, *, Zhi-Peng Chen 1, 2, Ting-Ming Fu 1, Liu-Qing Di 1, 2, Baochang Cai 1 1
College of Pharmacy, Nanjing University of Chinese Medicine, Nanjing, 210023, China Jiangsu Provincial TCM Engineering Technology Research Center of High Efficient Drug Delivery System (DDS), Nanjing University of Chinese Medicine, Nanjing, 210023, China 3 State Key Laboratory of Tea Plant Biology and Utilization, Anhui Agricultural University, Hefei, 230036, China 4 School of Traditional Chinese Medicine, China Pharmaceutical University, Nanjing, 210009, China 2
a r t i c l e i n f o
a b s t r a c t
Article history: Received 3 May 2015 Revised 1 August 2015 Accepted 13 October 2015 Available online 22 December 2015
The aim of this study was to investigate the potential of nanosuspensions (NSs) in improving the dissolution and absorption of poorly water-soluble ginkgo lactones (GLs), including ginkgolide A, ginkgolide B, and ginkgolide C. Liquid GL-NSs were prepared by a combined bottom-up and top-down approach with response surface methodology design, followed by freeze-drying solidification. Physicochemical characterization of the prepared freeze-dried GL-NSs was performed by photon correlation spectroscopy, scanning electron microscopy, powder X-ray diffraction, and differential scanning calorimetry. In vitro dissolution and in vivo bioavailability of ginkgolide A, ginkgolide B, and ginkgolide C in freeze-dried GL-NSs were evaluated with GLs coarse powder as control. Their inhibitory effects on platelet aggregation were also comparatively analyzed. GLs existed in an amorphous state in the prepared freeze-dried GL-NSs. The particle size, polydispersity index, zeta potential, and redispersibility index of freeze-dried GL-NSs were around 286 nm, 0.26, 25.19 mV, and 112%, respectively. The particle size reduction resulted in much more rapid and complete dissolution of ginkgolides from GL-NSs than coarse powder. Comparison with GLs coarse powder, freeze-dried GL-NSs showed a significant decreased Tmax, 2-fold higher peak concentration, and 2-fold higher area under plasma concentrations curve for 3 ginkgolides and exhibited significantly higher antiplatelet aggregation effect. © 2016 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.
Keywords: nanotechnology oral drug delivery drug transport dissolution physical characterization stabilization bioavailability oral absorption pharmacokinetics pharmacodynamics
Introduction Ginkgo lactones (GLs), including ginkgolide A (GA, PubChem CID: 6419993), ginkgolide B (GB, PubChem CID: 6324617), and ginkgolide C (GC, PubChem CID: 441295), are a unique group of natural diterpenoide lactones from the leaves of the Ginkgo biloba tree.1 These natural lactones could inhibit the platelet-activating factor (PAF) by binding to its membrane receptor and then producing anticoagulant effect. GLs have long been used to treat diseases of the central nervous system, such as degenerative dementia and neurosensory disorders.2 It was reported that GB could
The authors Tian-Qi Rui and Linang Zhang contributed equally. * Correspondence to: Jun-Song Li (Telephone: þ86-25-85811517; Fax: þ86-2585811517). E-mail address:
[email protected] (J.-S. Li).
improve memory and cognition,3,4 which may be due to its ability to scavenge free radicals and inhibit seryl and aspartyl proteases and, hence, protect against neural damage.5 In Europe and the United States, products containing extracts of GLs are top sellers in the growing market of herbal medicines.6 Nevertheless, GLs are poorly water-soluble compounds with low oral bioavailability, which greatly limits their formulation, development, and clinical application.7,8 For such hydrophobic compounds, poor solubility would result in a slow dissolution and may create delivery problems such as low oral bioavailability and erratic absorption. So far, several formulation strategies for GLs have been investigated, including liposomes,9 solid dispersion,10 and self-emulsifying drug delivery system.11 Although solubility and dissolution of GLs have been significantly enhanced by these formulation techniques, drugloading capacity and encapsulation efficiency were not sufficient especially for GLs with high clinical doses.
http://dx.doi.org/10.1016/j.xphs.2015.10.002 0022-3549/© 2016 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.
T.-Q. Rui et al. / Journal of Pharmaceutical Sciences 105 (2016) 242e249
Nanosuspensions (NSs) are carrier-free colloidal drug-delivery systems that contain drug particles and minimal stabilizers. The mean sizes of these drug particles are in the nanometer range, typically between 10 and 1000 nm. Nano-sized particles could significantly enhance solubility and dissolution rate of the drug. In addition, mucosal adhesive property of nano-sized particles prolongs the contact time of drugs to gastrointestinal tract epithelium. These factors could be involved in the oral bioavailability enhancement of poorly soluble drugs.12-14 NS can be prepared using 2 typical approaches: top-down and bottom-up technologies. Top-down processes reduce the size of large drug particles using wet-milling techniques, such as highpressure homogenization (HPH). The mean particle size of NS prepared by HPH is usually between 400 nm and 1000 nm.15,16 However, HPH is a high energyeconsuming and low-efficiency approach.17 It generates considerable heat so that it is inadequate for processing thermolabile materials. In bottom-up processes, the drug is dissolved in an organic solvent and then precipitated with an antisolvent in the presence of a stabilizer.18,19 This method usually makes NS formulation unstable through the generation of various unstable polymorphs, hydrates, or solvates. Needle-shaped particles usually formed owing to rapid growth in 1 direction, which influences the physical stability of the NS.17 Therefore, a combined bottom-up and top-down approach was studied to prepare an NS having narrow size distribution.20 During the physical modification (bottom up) process, first, the drugs and stabilizers were initially dissolved in different, suitable solvents. Second, the particle size was refined during crystallization using an antisolvent method, and a hydrophilic drug/stabilizer matrix (modified drug) was obtained. Third, HPH (a top-down process) was used to prepare an NS with a particle size 98%. Poloxamer 188 (P-188) and hydroxypropyl methyl cellulose (HPMC) were provided by BASF (Ludwigshafen, Germany). Methanol (HPLC-grade) was purchased from Merck (Darmstadt, Germany). Ethyl acetate, ammonium acetate, and other chemical reagents of analytic grade or better were obtained from Sinopharm Chemical Reagent Co. (Nanjing, China). The standardized GLs extract product containing GA (24%), GB (23%), and GC (48%) was provided by our laboratory. Animals Ninety-two Sprague Dawley male rats weighing 240 ± 20 g were supplied by the Animal Center of Nanjing Medical University (Nanjing, China). They were housed in controlled environmental conditions at 25 C ± 2 C and 50% ± 10% relative humidity under a 12-h dark-light cycle. The rats were kept with free access to food and water until 12 h before experiments. Animal welfare and experimental procedures were performed strictly in accordance with the Guide for the Care and Use of Laboratory Animals and the ethics regulations of Nanjing University of Chinese Medicine. Experimental Design RSM was used to investigate the influence of variables, namely the effect of the P-188/HPMC ratio, final homogenization pressures, and cycle numbers on the particle size and PI of the liquid GL-NSs. A BBD, with 3 factors each varied at 3 levels and with 3 center-point replications as well as with a second-order response surface, was used for the experimental design. The homogenization pressures (A), P-188/HPMC ratio (B), and cycle numbers (C) were used as 3 independent variables, whereas the particle size (R1) and PI (R2) were dependent variables. Table 1 shows the factors and the levels at which the experiments were carried out. All trials were carried out in triplicate. The experimental results obtained are expressed as mean ± SD. Preparation of GL-NSs GLs powder (600 mg) was dissolved in 10-mL of ethanol and then added drop-by-drop to 100-mL solution containing 5% P-188 and 5% HPMC(wt/vol) with a stirring speed of 600 rpm for 2 h
Table 1 Box-Behnken Design for Optimization of P-188/HPMC Ratio, Homogenization Pressures, and Cycle Number Runs
Homogenization Pressures (psi), (A)
P-188/HPMC Ratio (B)
Cycle Number (C)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
0 0 0 0 1 1 1 0 1 1 1 0 0 0 0 1 1
1 0 1 0 0 1 1 1 1 0 0 1 0 0 0 1 0
1 0 1 0 1 0 0 1 0 1 1 1 0 0 0 0 1
(14,500) (14,500) (14,500) (14,500) (17,400) (11,600) (11,600) (14,500) (17,400) (17,400) (11,600) (14,500) (14,500) (14,500) (14,500) (17,400) (11,600)
(1.5) (1) (1.5) (1) (1) (0.5) (1.5) (0.5) (0.5) (1) (1) (0.5) (1) (1) (1) (1.5) (1)
(8) (10) (12) (10) (8) (10) (10) (8) (10) (12) (8) (12) (10) (10) (10) (10) (12)
244
T.-Q. Rui et al. / Journal of Pharmaceutical Sciences 105 (2016) 242e249
under 40 C. The ethanol was then removed by vacuum evaporation, and the suspension was premilled by homogenization for 6 cycles at 3 continuous pressures of 4350 psi, 7250 psi, and 11600 psi by APV-2000 high-pressure homogenizer (SPX Flow Technology GmbH., Rheine, Germany). Finally, the liquid GL-NSs was obtained by further homogenizing the premilled suspension for 10 cycles at 14500 psi until an equilibrium particle size was reached. To enhance the chemical and physical stability, the liquid GLNSs were lyophilized before storage and subsequent use. Briefly, the liquid GL-NSs were rapidly cooled down with the addition of 5% (wt/vol) mannitol as the cryoprotectant at 80 C, then transferred to a LABCONCO 6L freeze-dryer (Labconco Corporation, Kansas) and dried at 30 C for 36 h with a vacuum degree of 1 psi to obtain a freeze-dried GL-NS powder.
Particle Size and Zeta Potential Analyses The particle size and PI of GL-NSs were determined by PCS using a Zetasizer (Malvern Instruments Ltd.). Samples of GL-NSs were diluted to an appropriate concentration and measured with a scattering angle of 90 at 25 C. The zeta potential values were assessed by determining the particle electrophoretic velocity using the same instrument. All measurements were repeated in triplicate, and the mean values are reported.
Redispersibility Index Redispersibility index (RDI) was used to evaluate the redispersibility of the freeze-dried NS powders and is calculated as follows:
RDI ¼ ðD=D0 Þ 100% where D represents the mean particle size of the redispersed suspensions, and D0 is the particle size of the liquid Ns before freeze drying. Freeze-dried NS powders were redispersed in distilled water and shaken gently for 10 s to yield redispersed suspensions with the same concentration as the liquid Ns before freeze drying. When the RDI value is close to 100%, the freeze-dried Ns powder is thought to be completely redispersed.25
Surface Morphology of Freeze-Dried GL-NSs The surface morphology of the GLs powder and freeze-dried GL-NSs was investigated using a scanning electron microscope (JSM-5610LV; Rigaku, Japan), and GLs coarse powder served as control. Samples were glued and mounted on metal sample plates. The samples were gold coated with a sputter coater using an electrical potential of 2.0 kV at 30 mA for 240 s. The surface morphology of the GLs powder and freeze-dried GL-NSs were examined by operating the SEM at 20 kV.
Differential Scanning Calorimeter A NETZSCH DSC-204 differential scanning calorimeter (Netzsch, Selb, Germany) was used to obtain DSC thermal profiles of the powder samples. Before measurement, samples of approximately 5 mg were accurately weighed into an aluminum pan and then sealed with a punched cover. Samples were heated from 50 C to 300 C at a rate of 10 C per minute in a nitrogen atmosphere.
In Vitro Dissolution Study A ZRS-8G dissolution apparatus (Tianjin Tianda Tianfa Technology Co. Ltd., Tianjin, China) operating at a rotation speed of 75 rpm was used to investigate in vitro dissolution of freeze-dried NS-GLs, and GLs coarse powder served as control. Saline solution with 0.5% SDS was used as the dissolution media. The volume and temperature of the dissolution medium were 250 mL and 37 C ± 0.5 C, respectively. At each predetermined sampling time, 2 mL of sample was withdrawn using a sampling port fitted with a 0.45-mm filter disc, and 2 mL of blank dissolution medium was added back into the vessels through the sampling port. Samples were centrifuged, and the resulting supernatant was diluted with an equivalent mobile phase; 20 mL was then injected into the HPLC for analysis. HPLC was performed using a Waters 2695 HPLC system (Waters, Milford, UK), equipped with an evaporative light-scattering detector. The analytes were separated on a Benetnach-C18 column (250 4.6 mm, 5 mm) maintained at 35 C. The mobile phase consisted of 36% methanol (A) and 64% water (B) using an isocratic elution (1.0 mL/min) for 20 min.
In Vivo Pharmacokinetic Study Twelve rats were randomly divided into 2 groups, a CLs-CS group and a freeze-dried GL-NSs group, with 6 animals in each group. Before the experiment, the rats were fasted for 12 h with free access to water. Before gavage administration to rats, GLs coarse powder and freeze-dried GL-NSs were dispersed homogeneously in 0.5% carboxymethyl cellulose sodium salt (CMC-Na) aqueous solution to form suspension. The same dose of GLs (equal to 60-mg GLs/kg) was given to rats in these 2 groups. Blood samples of approximately 0.3 mL were collected from the vein of the eyeground at 0, 5, 10, 15, 30, 60, 120, 240, 360, 480, 600, 720, and 1440 min after oral administration. The collected blood samples were placed in heparinized tubes and then separated immediately by centrifugation at 3000 rpm for 10 min. The obtained plasma was transferred into 1.5-mL Eppendorf tubes and then stored at 20 C
Table 2 Coefficients of the Quadratic Models and Their Corresponding p Values Factor
Powder X-Ray Diffraction The patterns of the GLs coarse powder, stabilizers, physical mixture of GLs coarse powder and stabilizers, and freeze-dried GL-NSs powder were analyzed by a powder X-ray diffractometer (D/Max-2500PC; Rigaku) with a Cu source of radiation. Measurements were performed at a voltage of 40 kV and 25 mA. The scanned angle was set from 2 2q 50 , and the scanning rate was 2 /min. The measurements were carried out in triplicate.
A B C AB AC BC A2 B2 C2
Particle Size (R1)
PI (R2)
F Value
p Value
F Value
p Value
63.98 154.29 13.71 1.1 5.88 0.057 7.6 188.03 9.54
