Article Cite This: Mol. Pharmaceutics XXXX, XXX, XXX−XXX
pubs.acs.org/molecularpharmaceutics
Oral Delivery of Honokiol Microparticles for Nonrapid Eye Movement Sleep Yang Yang,† Tianxiao Wang,† Juan Guan,† Juan Wang,† Junyi Chen,§ Xiaoqin Liu,∥ Jun Qian,§ Xinhong Xu,† Weimin Qu,*,†,⊥ Zhili Huang,*,†,⊥ and Changyou Zhan*,†,‡,§
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Department of Pharmacology, School of Basic Medical Sciences and ‡State Key Laboratory of Molecular Polymer Engineering, Fudan University, Shanghai 200032, China § School of Pharmacy and Key Laboratory of Smart Drug Delivery, Ministry of Education, Fudan University, Shanghai 201203, China ∥ Department of Pharmaceutical Engineering, Chongqing Chemical Industry Vocational College, Chongqing 401220, China ⊥ State Key Laboratory of Medical Neurobiology, and Institutes of Brain Science and Collaborative Innovation Center for Brain Science, Fudan University, Shanghai 200032, China ABSTRACT: Honokiol (HNK) is a small-molecule lignin extracted from Magnolia Of f icinalis, demonstrating high potency in promoting nonrapid eye movement (NREM) sleep by modulating the benzodiazepine site of the GABAA receptor. However, the clinical use of HNK in the treatment of insomnia is restricted by its extremely low oral bioavailability. In the present work, enhanced oral bioavailability of HNK was achieved by loading it into poly lactide− glycolide acid microparticles (HNK-MP). After oral administration, HNK-MP demonstrated 15-fold increase of AUC0−12 h in comparison to free HNK. The maximum blood concentration (Cmax) of HNK in HNK-MP-treated rats was 3.6 μg/mL at 2 h after oral administration, which was 6.5-fold of that in free HNK-treated rats. Oral administration of HNK-MP (20 mg/kg) efficiently increased NREM sleep by 60% by enhancing the transition from wakefulness to NREM sleep in rats. The biosafety of HNK-MP was assessed in vivo, and no damage occurred in the gastrointestinal tract. The present study provides a promising oral HNK formulation for the treatment of insomnia. KEYWORDS: honokiol, PLGA, microparticle, sleep, oral bioavailability
1. INTRODUCTION Insomnia is a neurological disorder characterized by early awakening and difficulty in falling asleep. It has become the direct or indirect cause of more than 80 diseases including depression1−3 and cardiovascular diseases.4,5 Aserinsky and Kleitman6,7 divided sleep into rapid eye movement (REM) sleep and nonrapid eye movement (non-REM, NREM) sleep based on characteristics of rapid eye movement. NREM sleep is the main episode of adult physiological-like sleep.8,9 In particular, deep NREM sleep plays important roles in maintaining immune functions,10−12 learning, and memory capabilities.13,14 Deep NREM sleep is increased in a compensatory manner in both animals and humans after sleep deprivation.15,16 Rechtschafffen and Kales divided NREM sleep into four phases based on the electroencephalogram (EEG) structure.17 Phases I and II of NREM sleep are light sleep and generally considered as the transition period to deep sleep, even though they account for 55% of total sleep time for the human adult. Phases III and IV of NREM sleep, characterized by the amount of delta waves, are deep NREM sleep. However, few compounds can promote sleep without changing the physiological-like sleep structure.17 Diazepam and zolpidem significantly prolong the total duration of NREM © XXXX American Chemical Society
sleep but also cause remarkable decreases of deep NREM sleep time.18,19 Honokiol (HNK) is a small-molecule lignin extracted from Magnolia Of f icinalis. It is among the few compounds that can promote NREM sleep by modulating the benzodiazepine site of the GABAA receptor. HNK induces physiological-like NREM sleep without affecting the EEG power density of NREM sleep.20 An ideal formulation for the treatment of insomnia should comply with at least the following rules: (1) Noninvasive administration is preferred, since multiple administrations are normally indispensable; (2) an appropriate duration (6−8 h) of biological effects in vivo is necessary to avoid early wakening or somnolence during the daytime. Even though HNK presents a good candidate for the treatment of insomnia without side effects, clinical use is impaired by its unfavorable physiochemical properties. HNK is highly hydrophobic, exhibiting very low oral bioavailability. An appropriate Received: September 29, 2018 Revised: December 20, 2018 Accepted: January 3, 2019
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DOI: 10.1021/acs.molpharmaceut.8b01016 Mol. Pharmaceutics XXXX, XXX, XXX−XXX
Article
Molecular Pharmaceutics
was sampled (0.5 mL) at the predetermined time points and replenished with 0.5 mL of blank medium. The concentration of HNK in medium was determined by HPLC, and cumulative release of HNK was calculated according to previous reports.28−30 2.4. Animals. Adult female Sprague−Dawley (SD) rats (Shanghai SLAC Laboratory Animal Co, Ltd.) weighing ∼250 g were housed in an insulated, sound-proof recording room with an ambient temperature of 22 ± 0.5 °C and relative humidity of 60 ± 2% as well as an automatically controlled 12 h light/12 h dark cycle (light on at 8:00 am). All rats had free access to food and water. All animal experiments were carried out in accordance with guidelines evaluated and approved by the ethics committee of Fudan University. 2.5. Pharmacokinetic Profiles of HNK-MP. HNK-MP (containing 50 mg of HNK) was dispersed in 5 mL of 0.5% CMC−Na. Animals were fasting for 24 h before experiments and randomly divided into two groups (n = 3). One group was treated with 40 mg/kg of HNK-MP, and the other group with 40 mg/kg of free HNK dispersed in 0.5% CMC−Na by intragastric administration (i.g.). Blood was sampled (0.5 mL) from the tail vein at the predetermined time points. Plasma was separated by centrifugation at 2800g for 8 min at 4 °C. The plasma was mixed with 100 μL of methanol and internal standard substance (magnolol, MNL, isomeride of honokiol) dissolved in 50 μL of methanol to precipitate plasma proteins. Chloroform (400 μL) was add into the mixture and vortexed for 2 min to extract HNK and MNL. After centrifugation at 9280g for 5 min at 4 °C, chloroform was collected and volatilized to dry. Acetonitrile/water (200 μL, 3/1 in volume) was used to dissolve the residual, and the concentration of HNK was determined by HPLC. 2.6. Retention Time and Biosafety of HNK-MP. Animals were administered with the rhodamine-B-labeled microparticles by i.g. and anesthetized by 10% chloral hydrate (4 mL/kg, intraperitoneal injection (i.p.)) at the predetermined time points. After heart perfusion, the stomach and small intestine were dissected and dipped in 4% paraformaldehyde (PFA). Fluorescence in the stomach and small intestine was detected and quantified using VISQUE in Vivo Elite (Vieworks, South Korea) to calculate the retention time of microparticles in gastrointestinal tract (GIT). After fluorescence detection, stomach and small intestine were digested, and the dye was extracted by DCM with ultrasonication (200 W, 10 min). The fluorescence intensity was detected at λem = 550 nm and λex = 580 nm (EnSpire Multimode Plate Reader, PerkinElmer) after dissolution in 90% acetonitrile and centrifugation (13 500g, 15 min) to remove the insoluble portion. The control group was administered with 0.9% saline. At 8 h after i.g. administration of HNK-MP, rats were sacrificed, and the stomach, small intestine, liver and kidney were dissected for hematoxylin and eosin (HE) staining. 2.7. EEG and EMG Recordings. Rats were anesthetized by 10% chloral hydrate (4 mL/kg, i.p.) for surgical implantation of electrodes for EEG and EMG recording. Two EEG electrodes were fixed in the skull with dental cement and four stainless steel screws for anchorage. Two stainless steel wire electrodes were placed into the trapezius for EMG recording. The rats were placed at warm pads to recover consciousness and autonomous motor ability after operation. After 10 days of recovery, rats were transferred to a soundproof recording chamber, where they were connected to an
formulation of HNK is extremely desirable for its clinical translation to benefit insomnia patients. We hypothesize that sustained release of HNK after oral administration would be favorable for increasing HNK bioavailability. Poly lactide−glycolide acid (PLGA) has been widely used to prepare sustained drug release systems,21−24 providing a potential carrier for HNK. In the present study, HNK-loaded PLGA microparticles (HNK-MP) were prepared by a single emulsion method,25 and the retention of HNK-MP in stomach and small intestine and the oral bioavailability of HNK were measured. Furthermore, the biosafety of HNK-MP was evaluated by histological staining, and pharmacodynamic effects of HNK-MP were investigated by EEG and electromyogram (EMG) after oral administration.
2. MATERIALS AND METHODS 2.1. Materials. Honokiol with >95% purity was from Yuansun Biological Technology Co. Ltd. (Xi’an, China). PLGA45000 (LA/GA = 50:50) was from Shenzhen Boli Biological Material Co. Ltd., China. Poly(vinyl alcohol) (PVA, average Mw of 13−23 kDa) was acquired from Sigma (St. Louis, MO). Carboxymethyl cellulose sodium (CMC− Na), dichloromethane (DCM), and methanol are of analytical grade and provided by Sinopharm Chemical Reagent Co. Ltd. (Shanghai, China). 2.2. Preparation and Characterization of Microparticles. HNK-loaded PLGA microparticles were prepared using a single emulsion method.26,27 HNK (60 mg) and PLGA45000 (240 mg) were codissolved in 1.5 mL of DCM, and the mixture was homogenized in an ice bath (1.55 kW, IKA T18 basic, Germany) in 25 mL of PVA (0.5%) for 1 min. The resultant suspension was decanted into 50 mL of PVA (0.05%) and stirred for 90 min to remove DCM. The turbid liquid was centrifuged at 920g for 3 min to remove any aggregates. HNKMP was collected by centrifugation at 9280g for 15 min, followed by thrice rinses with water. The fluorescence-labeled microparticles were prepared by the same method except for the addition of 5% (W/W) rhodamine B−PLGA15000 (λem = 550 nm, λex = 580 nm, Xi’an Ruixin Biological Technology CO. Ltd., China) as a probe. After freeze-drying, the morphology of HNK-MP was characterized by a scanning electron microscope (SEM, SU8000 II, Hitachi Ltd., Tokyo, Japan). Particle size in water was determined by dynamic light scattering (DLS, Mastersizer 2000, Malvern, UK). To determine the content of encapsulated HNK, 5 mg of HNK-MP was dissolved in 0.5 mL of acetone and diluted by 4.5 mL of 75% acetonitrile/water (3:1 in volume), and the concentration of HNK was measured using HPLC (Agilent 1260 Infinity II, Agilent Technologies, USA; WondaCract ODS-2 column, 5 μm, 4.6 × 150 mm, Japan; mobile phase: water/acetonitrile = 25:75; UV absorbance at 294 nm). HNK loading capacity was calculated using the following equation HNK loading capacity % =
weight of HNK × 100% weight of HNK − MP
2.3. Release of HNK from Microparticles in Vitro. After freeze-drying, microparticles (15 mg) were dispersed in 1 mL of 0.5% CMC−Na and placed in a dialysis bag (8−14 kDa, MYM Biological Technology Co. Ltd.) and incubated in 30 mL of phosphate-buffered saline containing 0.5% Tween-80 (pH 7.4 or 1.7) at 37 °C with continuous stirring. The medium B
DOI: 10.1021/acs.molpharmaceut.8b01016 Mol. Pharmaceutics XXXX, XXX, XXX−XXX
Article
Molecular Pharmaceutics
HNK resulted in bulky aggregation and decrease of loading capacity. HNK-MP prepared with a 1:4 HNK-to-PLGA ratio was used for the following studies. To study the release profile of HNK-MP in the gastrointestinal tract, microparticles were incubated in the artificial gastric juice (pH = 1.7) and intestinal juice (pH = 7.4),36−38 respectively. As shown in Figure 1D, HNK-MP demonstrated sustained release profiles in a pH-dependent manner. A low pH value induced relatively fast release of HNK from microparticles. The release curve was smooth, and no burst release occurred. The drug release from the PLGA-based drug delivery system generally contains two stages due to water penetration into the matrix and polymer degradation.39 In this case, HNK did not release from the microparticles explosively in the first 2 to 4 h, which may be explained by its poor water solubility.40 3.2. Pharmacokinetic Profile and Retention of HNKMP. To study the pharmacokinetic profiles, HNK-MP or free HNK suspended in 0.5% CMC−Na were intragastrically administered, and the plasma concentration of HNK was measured at the predetermined time points. As shown in Figure 2, the plasma concentrations of HNK in free HNK-
EEG/EMG recording cable for a 3 day period of habituation to the experimental conditions. The EEG/EMG of animals was recorded for 12 h at the natural awakening stage (19:00−7:00) after administration of HNK-MP (20 mg of HNK/kg, dispersed in 0.5% CMC−Na i.g.) or 0.5% CMC−Na (4 mL/kg, i.g.). Diazepam (6 mg/kg, i.g.), which is clinically used for insomnia treatment of depressed patients, was used as the positive control here. The vigilance states were automatically classified off-line by 10 s epochs, into wakefulness, NREM sleep, and REM sleep by SLEEPSING software, according to the standard criteria.31−34 As the final step, defined sleep−wake stages were visually examined, and corrected, if necessary. 2.8. Statistical Analysis. All data here are presented as means ± standard error of mean (SEM) and analyzed by Student’s t test using GraphPad Prism software 7.0. p < 0.05 was considered statistically significant.
3. RESULTS AND DISCUSSION 3.1. Characterization of HNK-MP. The HNK-loaded microparticles were prepared using a single emulsion method with an average particle size of 3.3 μm (Figure 1A). The size
Figure 2. Plasma concentration−time curve of HNK-MP and free HNK. The rats were intragastrically administered with 40 mg/kg of HNK-MP or 40 mg/kg of free HNK. Yellow and red dots represent profiles of plasma concentration of free HNK and HNK-MP after administration. Pharmacokinetic parameters are calculated using DAS 2.0 software. Data are means ± SEM (n = 3).
treated rats were close to the detection limitation (0.25 μg/ mL), while HNK in HNK-MP-treated rats clearly showed a Cmax of 3.6 μg/mL at 2 h after oral administration. The plasma HNK in HNK-MP-treated rats reached the baseline at 12 h (similar plasma concentration with that in free HNK-treated rats), indicating that HNK-MP demonstrated quick absorption and suitable elimination duration (2048 s) disappeared after HNK-MP administration, and medium-duration NREM sleep (−64 s) was increased by 52% in comparison to the vehicle-treated group. The stage transition number (Figure 8D) from wakefulness to NREM sleep increased by 36%. Both the episode numbers (Figure 8E) of NREM sleep and
Figure 6. Typical examples of polygraphic recordings and corresponding hypnograms of (A) vehicle and (B) HNK-MP administration. NREM sleep significantly increased during first 3 h after administration, and long-duration wakefulness disappeared.
Figure 7. Sleep−wake profiles produced by administration of HNK-MP in rats. (A) Changes in NREM, REM sleep, and wakefulness over time in rats treated with HNK-MP. Each circle represents the hourly mean amount of each stage. Yellow and red circles represent profiles of vehicle and HNK-MP treatments, respectively. Values are means ± SEM (n = 7−8). *P < 0.05 indicates significant differences compared to vehicle group as assessed by repeated measures ANOVA followed by the PLSD test. (B) Total amounts of NREM sleep, REM sleep, and wakefulness during the 3 h postadministration. Open and filled bars show the profiles of vehicle, HNK-MP, and free HNK or diazepam treatments, respectively. Values are the means ± SEM (n = 7−8). E
DOI: 10.1021/acs.molpharmaceut.8b01016 Mol. Pharmaceutics XXXX, XXX, XXX−XXX
Article
Molecular Pharmaceutics
Figure 8. Characteristics of sleep wake episodes and transitions during the 3 h after administration of 20 mg/kg of HNK-MP. (A−C) Number of NREM sleep, REM sleep, and Wake bouts; x-axis is the number of different durations, such as −32 means that the duration is ≥16 and
