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Oral Delivery of a Nanocrystal Formulation of Schisantherin A with Improved Bioavailability and Brain Delivery for the Treatment of Parkinson's Disease Tongkai Chen, Chuwen Li, Ye Li, Yi Xiang, Simon Ming Yuen Lee, and Ying Zheng Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.6b00644 • Publication Date (Web): 14 Oct 2016 Downloaded from http://pubs.acs.org on October 16, 2016

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Molecular Pharmaceutics

Oral Delivery of a Nanocrystal Formulation of Schisantherin A with Improved Bioavailability and Brain Delivery for the Treatment of Parkinson's Disease

Tongkai Chen1, Chuwen Li1, Ye Li1, Xiang Yi2, Simon Ming-Yuen Lee1, Ying Zheng1* 1

State Key Laboratory of Quality Research in Chinese Medicine, Institute of Chinese Medical Sciences, University of Macau, Macau, China

2

Division of Molecular Pharmaceutics, UNC Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, North Carolina, USA

*To whom correspondence should be addressed: Ying Zheng, Ph.D. Institute of Chinese Medical Sciences, University of Macau Tel: (853) 88224687; Fax: (853) 28841358 E-mail: [email protected]

ACS Paragon Plus Environment


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ABSTRACT

Schisantherin A (SA) is a promising anti-Parkinsonism Chinese herbal medicine but with poor water solubility and challenges to be delivered to the brain. We formulated SA as nanocrystals (SA-NC), aiming to improve its solubility, pharmacokinetic profile and thus provide a potential therapeutic agent for the treatment of Parkinson's disease (PD). The rod-shaped SA-NC had a particle size of ~160 nm with 33.3% drug loading, and the nanocrystals exhibited a fast dissolution rate in vitro. The intact drug nanocrystals could be internalized into Madin-Darby canine kidney (MDCK) cells, which were followed by rapid intracellular release, and most of the drug was transported to the basolateral side in its soluble form. Following oral administration of the SA-NC or an SA suspension, the accumulated concentration of the SA-NC in the plasma and brain was considerably higher than that observed for the SA suspension, but the drug targeting efficiency was similar. The SA-NC significantly

reversed

the

1-methyl-4-phenyl-1,

2,

3,

6-tetrahydropyridine

(MPTP)-induced dopaminergic (DA) neuronal loss and locomotion deficiency in zebrafish, as well as the 1-methyl-4-phenylpyridinium ion (MPP+)-induced damage of neuronal cell culture model. Further western blot analysis demonstrated that the stronger neuroprotective effect of SA-NC may be partially mediated by the activation of the protein kinase B (Akt)/glycogen synthase kinase-3β (Gsk3β) pathway. Taken together, these data provide solid evidence that the nanocrystal formulation has the potential to improve the bioavailability and brain concentration of this Biopharmaceutics Classification System (BCS) Class II compound, SA, for the


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treatment of PD. Keywords: Nanocrystals; Dissolution; Oral bioavailability; Brain delivery; Parkinson's disease (PD)

INTRODUCTION

Parkinson's disease (PD) is a disorder with a high prevalence, having an incidence rate in the populations of 4.5 to 19 per 100 000 per year.1 This disease causes vital medical and social problems all over the world. Effective prevention, reduction of morbidity and mortality, and improvements in the quality of life for survivors remain the major targets for developing new therapies for PD. However, poor oral bioavailability and limited drug penetration across the blood-brain barrier (BBB) prevent many drugs from having their therapeutic effects. Therefore, to fulfill this unmet clinical need, active components from herbal medicines that could provide prevention or treatment for PD have been studied as well.2, 3 Our previous studies have demonstrated that Schisantherin A (SA), which is a major dibenzocyclooctadience lignan that can be isolated from the fruit of Schisandra chinensis (Turcz.) Baill., provides significant protection against 6-hydroxydopamine (6-OHDA)- or 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP)-induced loss of tyrosine hydroxylase (TH)-positive dopaminergic (DA) neurons in zebrafish and mouse models of PD.4, 5 The action of this agent is primarily due to its abilities to regulate intracellular reactive oxygen species (ROS) accumulation, inhibit nitric oxide (NO) overproduction and activate mitogen-activated protein kinase (MAPK)-induced



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phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt)/glycogen synthase kinase-3β (Gsk3β) survival signaling.4,

5

Moreover, SA can translocate across the

BBB, as indicated by the presence of detectable concentration in the brain after intra-gastric administration to rat.6 However, the absolute bioavailability of SA in rats is only 4.3% after oral administration.7 According to a previous report, SA is a poorly water-soluble (0.01 mg/ml in water at room temperature) and highly permeable (Papp=3.44×10-5 cm/s in Caco-2 cells) compound

8

and can therefore be classified as

the Biopharmaceutics Classification System (BCS) Class II compound. The low oral bioavailability results in low concentration of SA in the blood and brain, which severely limits its clinical application for the treatment of PD. To address this problem, we aimed to develop SA nanocrystals (SA-NC) in the present study. Nanocrystals have been widely used to enhance oral absorption of BCS II compounds by dramatically increasing their saturated solubility, dissolution rates and adhesion to cell membrane.9, 10 Nanocrystals consist of pure drug crystal particles with small amounts of stabilizers absorbed onto the surfaces of the particles.11 These modifications produce a high level of drug loading that make nanocrystals capable of delivering a high dose and reaching a sufficiently high therapeutic concentration for their pharmacological effects.12 With the help of suitable stabilizers, nanocrystals can be stabilized to prevent significant aggregation or particle size growth.13 Moreover, the use of nanocrystal-based oral delivery systems has been found to reduce the variability of drug absorption as well as food-related effects.14 Techniques to produce nanocrystals can be categorized into top-down approaches (e.g., wet milling or high


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pressure homogenization) and bottom-up methods (e.g., anti-solvent precipitation). Top-down technologies start from large crystals in the millimeter range and reduce the sizes to the submicron range by breaking up the large crystals.13 However, it is difficult to achieve particle sizes below 200 nm with these methods.15 In contrast, in the bottom-up approaches, a poorly water-soluble drug is dissolved in an organic solvent and then rapidly introduced into a miscible anti-solvent (typically water), leading to the fast nucleation of small-sized nanocrystals. In addition, bottom-up technologies only require simple instrumentation, operate at low temperatures; therefore, they have advantages of lower cost and energy consumption.16 Thus small-sized nanocrystals were fabricated using the anti-solvent precipitation method in the present study. At present, many drug nanocrystal products, including Megace® ES, Abraxane®, Naprelan®, Triglide® and Theodur®, have been launched in the market.17 Despite the emerging applications of drug nanocrystals, the transport and fate of nanocrystals in cells are relatively poorly understood with respect to: a) whether dissolution or release occurs before cellular uptake; b) whether particles remain intact after uptake into cells; and c) whether particles are able to cross the epithelial barrier. To monitor the physical state of nanocrystals in cells, Madin-Darby canine kidney (MDCK) cells, which possess a relatively thin mucus layer and polarity and tight junctions similar to intestinal epithelium,18 were chosen as an in vitro model. In this study, we loaded a fluorescence

resonance

3’-dioctadecyloxacarbocyanine

energy perchlorate

transfer (DiO)

(FRET) as


the

pair donor

and

(3, 1,


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1’-dioctadecyl-3, 3, 3’, 3’-tetramethylindocarbocyanine perchlorate (DiI) as the acceptor) into SA-NC (DiO/DiI/SA-NC). The DiO/DiI/SA-NC were considered to be intact if a FRET signal was observed, clearly demonstrating that both fluorophores are present in a same nanocrystal. However, release or breakdown of DiO/DiI/SA-NC caused a loss in the FRET signal as the two fluorophores became dissociated. With the aim of fabricating SA-NC to improve the dissolution rate and oral bioavailability of SA and thus to increase the drug concentration in the brain for the treatment of PD, the optimized formulation was prepared using the anti-solvent precipitation method. The obtained SA-NC were then subjected to physiochemical characterization, in vitro cellular transport, intracellular integrity and real-time drug release monitoring, in vivo plasma and brain pharmacokinetic studies, and neuroprotective bioassays on zebrafish and cell culture models of PD.

MATERIALS AND METHODS

Materials

Schisantherin A (SA, purity> 99%) was purchased from Chengdu Preferred Biological Technology Co., LTD. (Chengdu, China). Bifendate was obtained from National Institutes for Food and Drug Control (Beijing, China). Hydroxypropyl methylcellulose E3 (HPMC E3) was a kind gift from Shanghai Colorcon Co., Ltd. (Shanghai, China). Pluronic F68 (F68) was provided by BASF Corp. (Parsippany, NJ, USA). DiO and DiI were purchased from Invitrogen (Carlsbad, CA, USA). Acetone of analytical reagent quality was purchased from Tianjin Fuchen Chemical Reagent


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Factory (Tianjin, China). High performance liquid chromatography (HPLC) grade acetonitrile and methanol were from Merck (Darmstadt, Germany). MDCK and SH-SY5Y cell lines were obtained from American Type Culture Collection (Manassas, VA, USA). 3-(4, 5-dimethyl-thiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT), polyvinylpyrrolidone K30 (PVP K30), MPTP, 1-methyl-4-phenylpyridinium ion (MPP+) and L-deprenyl (L-dep, Selegiline) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Dulbecco’s modified Eagle medium (DMEM) and fetal bovine serum (FBS) were purchased from Gibco (Carlsbad, CA, USA). Culture flasks (25 cm2 and 75 cm2 growth area), 96-well plates (0.32 cm2 growth area per well) and polycarbonate Transwell inserts (insert diameter 12 mm, pore size 0.4 µm, growth area 1.12 cm2) were procured from Corning Costar Corp. (Cambridge, MA, USA). The mouse monoclonal anti-TH antibody was obtained from Millipore (Billerica, MA, USA). The anti-Akt antibody, anti-phospho-Akt (Ser473) antibody, anti-Gsk3β antibody,

anti-phospho-Gsk3β

(Ser9)

antibody

anti-rabbit

IgG

and

anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody were purchased from Cell Signaling Technology (Danvers, MA, USA). Ultrapure water was pretreated with the Milli-Q® Plus System (Millipore Corporation, Bedford, MA, USA).

SA-NC preparation and characterization

The SA-NC were prepared using the anti-solvent precipitation method.19,

20

Briefly, 10 mg/ml of SA in 0.5 ml of acetone was injected into 10 ml of water containing 1 mg/ml of F68 as the stabilizer with stirring at the speed of 1000 r/min at



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room temperature. Presumably, the SA-NC were obtained at a final concentration of 0.5 mg/ml in water. To optimize the SA-NC formulation, SA-NC were also prepared under multiple conditions that involved varying the type of organic solvent, the kind and the concentration of the stabilizer, the stirring speed, the concentration of SA and the anti-solvent-to-solvent volume ratio. Based on the measurement of particle size and polydispersity index (PDI) obtained using the Malvern Zetasizer Nano-ZS system, an optimal SA-NC formulation using acetone as organic solvent and HPMC E3 as stabilizer was selected and further characterized in terms of its physicochemical properties including morphology by transmission electron microscope (TEM), thermal properties by differential scanning calorimetry (DSC), and crystalline patterns by powder X-ray diffraction (PXRD). A detailed description of the SA-NC characterization is provided in Supplementary material, section S1. This SA-NC formulation along with SA controls was used in the following in vitro and in vivo studies.

In vitro dissolution study

The dissolution of SA-NC was determined by the dialysis method. Briefly, 0.5 mg/ml of SA-NC, SA, or physical mixture of SA and HPMC E3 (SA-PM) in 1 ml was dialyzed through a dialysis bag with the molecular weight cut off 2000 (Biotopped, USA) into 50 ml of artificial gastric medium composed of 0.1 mol/l hydrochloric acid (HCl) and 0.1% (w/v) sodium dodecyl sulfate (SDS). While shaking at 37 °C and at a rate of 100 r/min, 1 ml of medium was collected from the 50 ml solution at various


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time points, followed by adding 1 ml of fresh medium to maintain the sink condition. The SA content was assessed using HPLC with a ZORBAX Eclipse Plus C18 (4.6×250 mm, 5 µm) analytical column in an Agilent 1200 series HPLC system (Santa Clara, USA). A sample of 10 µl was injected onto the column, eluted by methanol/water (83:17, v/v) at flow rate of 1 ml/min for 5 min, and detected using the UV absorption at 254 nm. The retention time for SA was 3.2 min; the detection limit was 110 ng/ml; and intra- and inter-day variability was below 1.5%. An SA standard curve was established to quantify the SA concentration in all samples.

In vitro cytotoxicity and transport study in MDCK cells

The effect of SA-NC on the cell viability was determined using the MTT assay. Full descriptions of the cell culture and in vitro cytotoxicity are detailed in Supplementary material, section S2. The permeability of SA-NC was assessed using MDCK cell monolayers. Full descriptions of the permeability assessment and the apparent permeability coefficient (Papp) are detailed in Supplementary material, section S2.

Intracellular integrity monitoring by FRET

The nanocrystals in cells were identified using the FRET technique.21 A FRET pair of DiO and DiI, was physically loaded into SA-NC (DiO/DiI/SA-NC) using a method similar to that used for the preparation of the SA-NC. The cells were incubated with DiO/DiI/SA-NC at the SA concentration of 20 µM at 37 °C for 1 h.



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After treatment, the incubation medium was removed, and the cells were rinsed three times with ice-cold phosphate-buffered saline (PBS), subsequently fixed in 4% paraformaldehyde and examined using a confocal laser scanning microscopy (CLSM) (Leica TCS SP8, Solms, Germany). Confocal images were acquired using an excitation wavelength of 488 nm and the emission wavelengths between 555 nm and 655 nm for DiI detection and between 500 nm and 530 nm for DiO detection. The extent to which FRET occurred was quantified using the FRET ratio IDiI/(IDiO + IDiI),22 where IDiO and IDiI were the fluorescence intensities of DiO at 505 and DiI at 565 nm, respectively using the excitation of 420 nm. The release of the drug from the nanocrystals in the cells was monitored by FRET. One hour after the cellular uptake of DiO/DiI/SA-NC at the SA concentration of 20 µM, the medium containing DiO/DiI/SA-NC was then replaced by fresh pH 7.4 PBS. The fluorescence spectra of the cells and the FRET ratios were determined by microplate reader at an excitation wavelength of 420 nm at 15 min, 30 min, 45 min and 60 min. The decrease of the FRET ratio indicated the release of the nanocrystals in cells. To identify whether nanocrystals had been transported across cell monolayers, we measured the fluorescence spectra of samples withdrawn from the basolateral compartment of the Transwell after 2 h of exposure of the apical compartment of the cell monolayers to DiO/DiI/SA-NC at the SA concentration of 20 µM.

In vivo pharmacokinetic study in rats

The animal experiments were approved by the Animal Ethics Committee of


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Institute of Chinese Medical Sciences, University of Macau (Macau SAR, China) and were in full compliance with national regulatory principles. All animals were housed individually in standard cages on a 12 h light-dark cycle, fed with standard animal chow daily, and provided with free access to drinking water. Male Sprague-Dawley (SD) rats (6 to 7 weeks) (Guangdong Medical Laboratory Animal Centre, Guangdong, China) were gavage with the SA-NC formulation, which was prepared as previously described. The SA dose was 4 mg/kg (cal. volume of 1.44 to 1.76 ml) or an equal dose of SA that was dispersed in 0.1% HPMC E3 and 0.5% CMC-Na solution, designated the SA suspension, as the control. Two separate experiments were conducted to collect the brain samples and serum samples. To collect the brain samples, at 0.5, 1, 2, 4, 8, 12 and 24 h following drug administration, the rats were anesthetized with an i.p. injection of 0.45 to 0.55 ml of 10% chloral hydrate (250 mg/kg, Melonepharma Co., LTD., Liaoning, China). The brains were perfused by opening the thorax, clamping the descending thoracic, severing both jugular veins, exposing the heart, and infusing into the left ventricle of the heart with 200 ml physiological saline over 60 min. Finally, the rat was decapitated, and the whole brain was removed, weighed, and homogenized in precooled saline at the ratio of 1:2 (w/v). Serum samples were collected by sampling 0.3 ml of blood from the tail veins of the same individual rats in each group (n=6 per group) at various time intervals; following centrifugation at 5000 r/min for 5 min, supernatant was collected. The SA concentration in the tissue samples were then determined by LC-MS/MS.8 Briefly, 200 µl of the homogenized brain samples or 200 µl of the serum supernatant were mixed with 190 µl acetonitrile



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and spiked with 10 µl acetonitrile containing 1 µg/ml of bifendate, an internal standard used for the LC-MS/MS signal quantitation. Following centrifugation at 10000 r/min for 10 min (for brain samples), or 20 min (for serum samples) at 4 °C, the supernatant was collected, filtered and subjected to the LC-MS/MS analysis. A detailed description of the LC-MS/MS analysis is provided in Supplementary material, section S3. The pharmacokinetic parameters, including terminal elimination half-life (T1/2), time to reach the maximum plasma concentration (Tmax), peak plasma concentration (Cmax), area under the plasma concentration-time curve from 0 to t (AUC0-t) and mean residence time (MRT0-t) were calculated using Drug and Statistics for Window (DAS, Version 2.0) system. A non-compartmental model was employed to estimate the pharmacokinetic parameters. The relative bioavailability (F) of SA-NC to control group was calculated according to the following Equation 1: F=

AUCSA − NC ×100% AUCcontrol group

1

The drug targeting efficiency (DTE) of the SA-NC and the control group was obtained using the Equation 2:23 DTE = ( AUCbrain / AUCplasma) ×100%

2

Neuroprotective effects on zebrafish

The neuroprotective effects of SA-NC were evaluated using MPTP treated zebrafish. Briefly, wild-type zebrafish were raised synchronously at 28.0 °C in embryo medium (13.7 mM NaCl, 540 µM KCl, 25µM Na2HPO4, 44 µM KH2PO4, 300


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µM CaCl2, 100 µM MgSO4, 420 µM NaHCO3, pH 7.4).24 The animal experiments were carried out in accordance with the approved guidelines of the Animal Research Ethics Committee, University of Macau and approved by the Animal Research Ethics Committee, University of Macau. MPTP intoxication was initiated 3 days post fertilization (dpf) by adding 150 µM MPTP to the embryo medium. After 2 days of incubation, the embryo medium was replaced with fresh embryo medium containing 10 µM MPTP and the SA-NC at various concentrations (1, 3, 10 µM), an equal dose of the unformulated SA, or L-dep (positive control, 20 µM). Three days after the drug treatment, the zebrafish were transferred to a 96-well plate (one fish per well) that was pre-installed in a Zebrafish box equipped with an automated video tracking system (Viewpoint, ZebrafishLab, LifeSciences) to record the movements of the zebrafish. The locomotion behavior was evaluated as the distance (in mm) that the zebrafish moved during 10 min. The immunohistochemistry study was carried out using zebrafish that had been exposed to MPTP (200 µM) in embryo medium at 1 dpf. SA-NC (1, 3, 10 µM), an equal dose of the unformulated SA, or L-dep (20 µM) was added to the medium at the same time, and the zebrafish were allowed to grow for another 2 days. The whole-mount anti-TH immunostaining was performed using a previously reported method.25

Akt/Gsk3β pathway in SH-SY5Y cells

To understand the neuroprotective mechanism of the SA-NC, the activity of the SA-NC was further studied in an in vitro experiment using human neuroblastoma



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SH-SY5Y cells, a neuron-like cell model that has been widely used to evaluate intracellular events linked to neurotoxic insults.26 A detailed description of the Akt/Gsk3β pathway in SH-SY5Y cells is provided in Supplementary material, section S5.

Statistical analysis

All values obtained are expressed as the means ± standard deviation (SD). The statistical significance of the results was analyzed using either a two-tailed independent sample t-test for two groups or a one-way analysis of variance (ANOVA) for more than two groups. A value of p

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