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Contents lists available at ScienceDirect
European Journal of Pharmaceutics and Biopharmaceutics journal homepage: www.elsevier.com/locate/ejpb
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Research paper
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Supermolecular evodiamine loaded water-in-oil nanoemulsions: Enhanced physicochemical and biological characteristics
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Medicine Engineering Research Center, Chongqing Key Laboratory of Biochemical & Molecular Pharmacology, Chongqing Medical University, Chongqing, People’s Republic of China Department of Health Statistics, College of Public Health, Chongqing Medical University, Chongqing, People’s Republic of China Department of Thoracic Surgery, Institute of Surgery Research, Daping Hospital, Third Military Medical University, Chongqing, People’s Republic of China
a r t i c l e
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Jiangbo Hu a,1, Lili Sun a,1, Dezhang Zhao a,1, Liangke Zhang a,1, Mengliang Ye b,1, Qunyou Tan c, Chunshu Fang c, Hong Wang a, Jingqing Zhang a,⇑
i n f o
Article history: Received 14 February 2014 Accepted in revised form 14 June 2014 Available online xxxx
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Keywords: Nanoemulsification Supermolecular evodiamine Brucea javanica oil Oral bioavailability Characteristics
a b s t r a c t The purpose of this study was to develop and evaluate the supermolecular evodiamine (EVO) loaded water-in-oil nanoemulsions containing brucea javanica oil (NESEB) with enhanced physicochemical and biological characteristics. NESEB was fabricated by applying supermolecular phytosome nanotechnology and nanoemulsification technology together, in addition to using synergistic plant essential oil as a basic composition. Preferred physicochemical and biological characteristics of NESEB were investigated and compared with free EVO and other nanoemulsive EVO carriers. The possible explanations for improved absorption and bioavailability were put forward here. NESEB had high absorption and bioavailability, for example: the absorption rate constants and permeabilities of NESEB in different intestinal segments were 3.65–6.76 times that of free EVO; the relative bioavailability of NESEB to free EVO was 846.97%. NESEB markedly improved the oral bioavailability of EVO, which was most likely due to the increased gastrointestinal absorption. The development of nanoemulsion-based supermolecular EVO nanocarriers provides valuable tactics in insoluble natural antitumor drug delivering. Ó 2014 Published by Elsevier B.V.
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1. Introduction In clinical practice, Evodia rutaecarpa or ‘‘Wu-zhu-yu’’ in Chinese has been traditionally administered orally together with some other traditional Chinese medicines to treat inflammatory diseases so far [2]. Evodiamine (EVO) is one of the most important active ingredients of E. rutaecarpa [1]. Recently, EVO has been drawing great interest of scientists due to its potent antitumor activities against a variety of tumors (such as gastric cancer and colorectal tumor) [3,4]. The action mechanism of EVO has been deeply investigated. On the other hand, in order to achieve the clinical translation of EVO, the primary consideration is to fabricate a suitable formulation with greatly enhanced oral bioavailability, since water insoluble EVO shows definite antitumor activity but simultaneously exhibits poor oral bioavailability. During the fabricating process, the biomaterials approved by national agency for drug administration are considered to be the first choice. These biomaterials are ⇑ Corresponding author. Room 802, Building 9, Shiyou Road 1, Yuzhong District, Chongqing Key Laboratory of Biochemical & Molecular Pharmacology, Medicine Engineering Research Center, Chongqing Medical University, Chongqing 400016, People’s Republic of China. Tel.: +86 13308300303. E-mail address: [email protected] (J. Zhang). 1 These authors contributed equally to this study.
expected to form new amazing nanocarriers by using cutting edge technologies, such as supermolecular phytosome nanotechnology and nanoemulsification technology. The development of such smart nanocarriers made of the approved biomaterials may ease pressure on developing new biomaterials for effectively delivering an insoluble EVO [5–8]. There has been progress in the development of supermolecular phytosome nanotechnology and nanoemulsification drug technology over the past decade. Supermolecular phytosomes refer to noncovalently bonded complexes of a natural active ingredient and phospholipids. A drug–phospholipid complex is one supermolecular drug. Distinguishing from ordinary emulsions, nanoemulsions refer to emulsive drug nanosystems with some distinctive physiochemical properties [9,10]. There are a few supermolecular phytosomal drug or emulsive drug nanosystem products currently on the market, including MerivaÒ (supermolecular phytosomal curcumin for the treatment of diabetes related oxidative stress) by Thorne Research Inc. (USA), SiliphosÒ (supermolecular phytosomal silybin for the treatment of hepatitis associated with reduction in serum ferritin) by Indena S.p.A. Inc. (Italy), NORVIR (emulsive ritonavir nanosystem for the treatment of HIV-1 infection) by Abbott Laboratories (USA), and NeoralÒ (emulsive ciclosporin nanosystem for suppressing the immune system) [11–14].
http://dx.doi.org/10.1016/j.ejpb.2014.06.007 0939-6411/Ó 2014 Published by Elsevier B.V.
Please cite this article in press as: J. Hu et al., Supermolecular evodiamine loaded water-in-oil nanoemulsions: Enhanced physicochemical and biological characteristics, Eur. J. Pharm. Biopharm. (2014), http://dx.doi.org/10.1016/j.ejpb.2014.06.007
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Supermolecular phytosomal EVO (SEVO, or EVO-phospholipid nanocomplex) had been developed in our lab [15]. The relative oral bioavailability of SEVO was increased to 218.82% compared with free EVO. Besides SEVO, only a few EVO delivery systems (such as microemulsion for transdermal delivery, superparamagnetic Fe3O4-loaded polymeric nanocarrier for intravenous delivery) have been reported so far [16,17]. Obviously, it is of great clinical significance to investigate some more effective EVO delivery systems with much higher bioavailabilities than EVO and SEVO. Since some drug–phospholipid complex based formulations, such as hydroxysafflor yellow A-phospholipid complex oil solution and salvianolic acid-phospholipid complex loaded nanoparticles, exhibited improved oral bioavailability over the simple drug–phospholipid complex [18,19], we would like to further package a SEVO in a nanoemulsive system to achieve the added effectiveness. The nanoemulsive and supermolecular phytosomal EVO was formulated by applying these two above-mentioned nanotechnologies simultaneously instead of separately. Furthermore, brucea javanica oil is a Chinese drug widely used in clinic to treat a broad spectrum of tumors, and is known for the effectiveness and safety of it plus other chemotherapy drugs [20,21]. Since oil phase is essential during the nanoemulsification process, it may be a good idea to entrap brucea javanica oil into the novel EVO delivery nanosystem, and then brucea javanica oil can provide a synergistic antitumor effect with EVO in addition to being the basic composition of the new nanoemulsive and supermolecular EVO nanocarrier. Although it is currently unavailable in either clinical practice or the research field, a nanoemulsified supermolecular drug nanocarriers composed of only biomaterials approved by national drug administration agency by applying supermolecular phytosome nanotechnology and nanoemulsification drug technology together, in addition to using synergistic plant essential oil as a basic composition, may be theoretically expected to deliver an insoluble natural drug effectively and exhibit greatly improved oral bioavailability. Here we reported some encouraging experimental data to support this hypothesis. In the experiments outlined below, a nanoemulsified supermolecular evodiamine containing brucea javanica oil (NESEB) only using biomaterials approved by national drug administration agency were designed and prepared, and then the in vivo pharmacokinetics and the in situ absorption of NESEB were evaluated. Compared with EVO, SEVO, conventional nanoemulsified evodiamine (NEE), nanoemulsified supermolecular evodiamine (NESE), and nano-emulsified evodiamine containing brucea javanica oil (NEEB), NESEB markedly improved the oral bioavailability of EVO, which was probably due to the increased gastrointestinal absorption. As far as we know, this was the first time to load a drug–phospholipid nanocomplex into a nanoemulsive delivering system. It was the first time that a preferred nanoemulsified supermolecular nanosystem with high oral bioavailability has been produced by combining phytosomal nanotechnology with nanoemulsifying technology, in addition to using synergistic antitumor oil as a basic composition, to deliver an insoluble antitumor natural drug. Our studies focus on the greatly improved oral bioavailability in vivo and the possible reason (obviously enhanced gastrointestinal absorption) of this delivery nanosystem. The development of such a novel nanosystem represented a valuable tactic in new medical application of commonly used biomaterials approved by national drug administration agency for effectively delivering insoluble antitumor natural drugs.
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2. Materials and methods
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2.1. Materials
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EVO was obtained from Yuancheng Technology Development Co., Ltd. (Wuhan, China), purity 99.13%. Ethyl oleate was purchased
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from Shanghai Chemical Reagent Co. (Shanghai, China), analytical grade. Polyethylene glycol 400 was purchased from Tianjin Guangfu Fine Chemical Co., Ltd. (Tianjin, China). Cremorphor EL 35 was purchased from BASF Corporation (Ludwigshafen, Germany). Brucea javanica oil was purchased from Shanghai Kexin Biology Engineering Co., Ltd. Soybean phospholipid (Lipoid S 75) was purchased from Phospholipid GmbH (Nattermannllee, Germany). All other chemicals and reagents used were of analytical or chromatographic grade. Male Sprague-Dawely rats obtained from the Animal Center of Chongqing Medical University (Chonqing, China) were all specific pathogen free animals. Their weights were 200–250 g. The animal studies were conducted in accordance with the protocol approved by the Laboratory Animal Committee, Chongqing Medical University. The animals were raised under controlled conditions with free access to diet and water, while fasted at least 18 h before drug administration in the gastrointestinal absorption study and 12 h in the pharmacokinetical study, respectively.
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2.2. Fabrication and characterization of NESEB
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Firstly, SEVO was produced using a modified solvent evaporation method [15]. Briefly, a mixture of phospholipid and EVO at a molar ratio of 2:1 was dissolved in 50 mL ethanol, and magnetically stirred at 60 °C for 3 h. The solvent was evaporated to dryness under hypobaric conditions; the resulting SEVO was stored until use. Secondly, SEVO was loaded into the NESEB using a nanoemulsifying technique. An appropriate amount of SEVO was added into an isotropic mixture of ethyl oleate, brucea javanica oil, cremorphor EL 35 and polyethylene glycol 400 (at a mass ratio of 24:12:13:10) and the resulting mixture was then magnetically stirred at 60 °C for 6 h, cooled to 30 °C, added dropwise by 5 mL of distilled water under continuous stirring with a magnetic stirrer. NEEB was prepared in a similar way to prepare NESEB, and the only difference was that EVO instead of SEVO was added in the preparing process. NESE was prepared in a similar way to prepare NESEB, and the only difference was that ethyl oleate instead of a blend oil (ethyl oleate and brucea javanica oil) was added in the preparing process, i.e., no brucea javanica oil was added in the preparing process. NEE was prepared in a similar way to prepare NESE, and the only difference was that EVO instead of SEVO was added in the preparing process. The conductivity of NESEB and its dilution (5 times with ethyl oleate) were determined at 25 °C by an electric conductivity analyzer (DDB-303A, Shanghai Precision & Scientific Instrument Co. Ltd, Shanghai, China), respectively. The size and zeta potential of NESEB were determined at 25 °C by dynamic light scattering (Zeta-Sizer Nano-ZS90, Malvern, UK). These studies were performed at refractive index of 1.45 because the refractive index for all formulation was around this value. The sample cell was made of quartz (Malvern, UK). The sample was prepared by diluting 4 mL of NESEB with 16 mL of ethyl oleate. In the in vitro release tests, NESEB was placed in a dialysis tube and then immersed into the release media of phosphate buffer system (PBS, pH 6.8). A HPLC method was used to determine the free EVO.
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2.3. Gastrointestinal absorption of NESEB
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The in situ gastric absorption test was carried out as previously described with some modifications [22,23]. Briefly, the experimental rats under intraperitoneal anesthesia with chloral hydrate were fixed supinely. After a 3 cm incision was made in the abdominal midline, the pylorus (the stomach outlet) was cannulated with a flexible 2 mm internal diameter tubing and then ligated. A small incision was made in the cardia (the stomach inlet). After the stomach was rinsed with the artificial gastric juice, the cardia was ligated
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and 4 mL of NESEB (or EVO, CNEE, NESE, and NEEB) at the same concentration of 100 lg/mL EVO was injected into stomach. The stomach was replaced in the abdomen. After 2 h, the solution remained in the stomach was taken out and mixed with the artificial gastric juice which was used to wash the stomach to make 25 mL. One volume of dilution was added with 3 volumes of methanol and 2 volumes of acetone. The resulting mixture was vortexed for 2 min, and then centrifuged at 12,000 rpm for 10 min. Aliquots of the supernatant were further subjected to HPLC analysis. The stationary phase (Lichrospher C18 column, 250 mm 4.6 mm, 5 lm) was kept at 35 °C, and the mobile phase (methanol:water = 75:25, v/v) was run at a flow rate of 1 mL/min. The effluent was monitored at 225 nm. The standard regression equation in the range of EVO concentration (C) 1–40 lg mL1 was linear (A = 16446C + 12550, r = 0.9994, n = 5; A means the peak area). Besides the linearity, the precision and accuracy (data not shown) of the HPLC method also met the requirements of application requirements. The surgical procedures for the in situ single pass intestinal perfusion (SPIP) test were conducted as previously described with some modifications [23,24]. After incision through medioventral line, enteric tracts of anesthetized rats were carefully exposed and cannulated separately with flexible tubing on two ends of four 10 cm long intestinal segments, i.e., duodenum (1 cm distal to the pyloric sphincter), jejunum (15 cm distal to the pyloric sphincter), ileum (20 cm proximal to the cecum), and colon (1 cm distal to the cecum). After rinsing with physiological saline, the chosen intestinal sections were attached to the perfusion assembly consisting of a BT100-1L peristaltic pump (Baoding Longer Precision Pump Co. Ltd., Baoding, China) and equilibrated with Krebs-Ringer’s solution (NaCl 118.1 mM; KCl 3.4 mM; CaCl2 2.5 mM; MgSO4 0.8 mM; KH2PO4 1.2 mM; NaHCO3 25.0 mM; Glucose 11.1 mM) at a flow rate of 0.4 mL min1 for 15 min. Next, the tested perfusate solution equivalent to 100 lg mL1 of EVO, prepared by displacing the tested drugs (NESEB, EVO, NEE, NESE and NEEB, respectively) into the Kerbs-Rings solution, was perfused through the segments at a flow rate of 0.2 mL min1 for 1 h. The remaining perfusate solution was mixed with the Kerbs-Rings solution which was used to wash the intestinal segment to make 25 mL. The resulting mixture was treated and measured according to the above method for determination of the EVO concentration in gastric perfusate solution. The gastrointestinal absorption rate constant (Ka) and absorption percentage (PA), as well as intestinal effective permeability (Peff) were described by the following equations:
K a ¼ ðX 0 X t Þ=C 0 t pr 2 l
ð1Þ
PA ð%Þ ¼ ðX 0 X t Þ=X 0 100%
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Peff ¼ R lnðX in =X out Þ=2prl
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where X0 and C0 were the drug amount and concentration at 0 h. Xt and Ct were the drug amount and concentration in the remaining perfusate solution, C0 was the drug concentration at 0 h, Ct was the remaining drug concentration in the perfusate solution, t was the perfusion time, R was the flow rate, Xin and Xout were the inlet and outlet drug amounts, and r and l were the radius and length of the perfused intestinal segment, respectively.
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2.4. Pharmacokinetic and bioavailability of NESEB
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All the experiments were approved by the Institutional Animal Care and Use Committee of Chongqing Medical University. Healthy male Sprague Dawley rats (weighing 200–250 g) were orally administered with NESEB, EVO, NEE, NESE, and NEEB at the same dose equivalent to 100 mg/kg EVO, respectively. Ophthalmic vein blood samples were drawn out at predetermined timepoints and centrifuged at 3000 rpm for 10 min immediately. The plasma samples were processed according to our previously reported
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procedure and analyzed with the HPLC method [15]. Honokiol was used as an internal standard. The standard regression equation in the range of EVO concentration (C) 0.01–3 lg mL1 was linear (R = 1.9818C + 0.0114, r = 0.9989, n = 5; R meant the peak area ratio of EVO to honokiol). Besides the linearity, the precision and accuracy (data not shown) of the HPLC method also met the requirements of application. Pharmacokinetic parameters, except maximum concentration (Cmax) and peak time (Tmax) which were directly determined by experiments, were calculated using DAS 2.1.1 software (Mathematical Pharmacology Professional Committee of China, Shanghai, China), while the relative bioavailability (RBA) of EVO formulation given orally could be calculated according to the Eq. (4) listed as follows:
RBA ð%Þ ¼ ðAUC F1 X F2 Þ=ðAUC F2 X F1 Þ
ð4Þ
where AUCF1 and XF1 were the area under the EVO concentration– time curve and the dose of EVO, respectively; AUCF2 and XF2 were the area under the EVO concentration–time curve and the dose of EVO, respectively. The absolute bioavailability (ABA) of EVO formulation given orally could be calculated according to the Eq. (5) listed as follows:
ABA ð%Þ ¼ ðAUC op;F1 X iv;EVO Þ=ðAUC iv;EVO X op;F1 Þ
ð5Þ
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where AUCop and Xop were the area under the EVO concentration– time curve and the dose of EVO when EVO formulation 1 was given orally; AUCiv and Xiv were the area under the concentration–time curve and dose when free EVO was given intravenously. Besides pharmacokinetic analysis, bioequivalence analysis between every two EVO formulation was also conducted using DAS software.
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2.5. Statistical analysis
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All data were shown as mean ± standard deviation unless particularly outlined. One-way ANOVA, Scheffe’s F-test and Student’s t test were used to calculate statistical difference. A statistical significance was established at P < 0.05. Pharmacokinetic and bioequivalence analyses were conducted using DAS software (Mathematical Pharmacology Professional Committee of China, Shanghai, China).
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3. Results
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3.1. Elementary configuration and characterization of NESEB
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The SEVO was produced by complexing EVO with phospholipid [15]. A circumscribed central composite design approach was chosen to properly formulate SEVO [15]. The NESEB loaded SEVO was further obtained using an emulsification method. The pseudo-ternary phase diagrams were constructed by Origin software (Version 7.5, Origin Lab Corp., Wellesley, USA) and the nanoemulsion formulation was optimized by a simplex lattix model (data not shown). The photos were taken by a C-60 ZOOM Olympus camera (Hongkong, China) and shown in Fig. 1. It was indicated that nanosized droplets of water were quite evenly dispersed in the light yellow–green translucent NESEB nanosystem. This NESEB nanosystem was a homogeneous (heterogeneous at molecular scale) and optically isotropic nanosystem. There was no significant difference between NESEB and NEEB in the appearance and color. However, although both NESE and NEE were translucent, there was a little difference from NESEB in color. NESE and NEE were light yellow and very light yellow, respectively. As seen in Table 1, the conductivity of NESEB was different from free EVO or other nanoemulsive carriers (NEE, NESE and NEEB). The
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Fig. 1. Schematic diagram of NESEB improving the oral bioavailability. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Table 1 The conductivity of NESEB and other EVO formulations. Formulation
Conductivity (ls/cm) Before dilution
Free EVO in water Free EVO in ethyl oleate Blank NEE NEE Blank NESE NESE Blank NEEB NEEB Blank NESEB NESEB
759.33 ± 8.50 0.37 ± 0.07 12.18 ± 0.31 12.98 ± 0.39 16.26 ± 0.53 16.22 ± 0.27 18.56 ± 0.39 18.27 ± 0.37 13.51 ± 0.42 13.61 ± 0.49
After dilution
0.23 ± 0.05 0.23 ± 0.05 0.11 ± 0.02 0.17 ± 0.05 0.32 ± 0.06 0.34 ± 0.06 0.43 ± 0.08 0.52 ± 0.06
Notes: A dilution consisted of 1 part formulation and 4 parts ethyl oleate. Data presented as mean ± standard deviation (n = 3).
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mean size and zeta potential of NESEB were 613.3 nm and 4.46 mV, respectively; for NEEB, 349.9 nm and 1.54 mV, respectively; for NESE, 554.1 nm and 8.16 mV, respectively; for NEE, 335.7 nm and 2.55 mV, respectively. The release rates of free EVO in the release media (pH 6.8 PBS) were (15.17 ± 1.25)% at 8 h and (16.02 ± 1.16)% at 12 h, respectively. In the case of NESEB, the EVO released from the NESEB in pH 6.8 PBS could not be detected by HPLC within 12 h, which suggested that NESEB provided a very slow release. The slow release of EVO from NESEB might be partly due to the barrier of oil phase (the outer phase of NESEB). Under our experimental conditions, NESEB was a controlled or sustained release dosage form. The release pattern and mechanism of drug release of NESEB were still unclear. The parameters (such as the appearance, color and conductivity) were investigated to evaluate the stability of the NESEB nanocarrier. The translucent NESEB system was light yellow–green all the time. No obvious changes were observed in NESEB after storage at 4 °C or 25 °C for 30 days. In our preliminary experiments, the formulation was stable against physical stress stability studies (placement at 60 °C for 10 days; illumination at (4500 lx ± 500 lx) for 10 days.
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NESEB was superior to free EVO or other EVO nanoemulsive systems (NEE, NESE and NEEB) in enhancement of gastrointestinal
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absorption. Comparison among different EVO delivery systems or gastrointestinal segments of gastrointestinal absorption rate constant (Ka) and absorption percentage (PA), and intestinal effective permeability (Peff) were presented in Figs. 2 and 3, respectively. As we know, stomach was seldom the major site for drug absorption. Here only a small amount of EVO (less than 5%) was absorbed from the stomach. The PA values of NEE, NESE and NEEB varied from 6.3% to 10%. NESEB had the highest absorption percentage (about 17%). The small intestine was, without question, the main area of oral absorption for EVO formulations. NESEB had the highest Ka (or Peff) values while free EVO had the least values among five EVO formulations (NESEB, NEEB, NESE, NEE and free EVO) in almost every corresponding intestinal segment. The Ka (or Peff) values of NESEB in the duodenum, jejunum, ileum, and colon were 3.74 (or 7.02), 5.06 (or 6.76), 4.82 (or 4.65), and 3.70 (or 3.65) times that of free EVO, respectively. Furthermore, the Ka (or Peff) value of NESEB in each intestinal segment was much higher than that of NEE, NESE and NEEB, respectively. The study on intestinal absorption of NESEB in different regions of rat intestine indicated that (1) the jejunum had the best absorption rate, followed by duodenum (5.68% less than the former), ileum, and colon in order; (2) the duodenum had the best permeability, followed by jejunum (3.73% less than the former), ileum, and colon in order. Besides NESEB, other EVO nanoemulsive formulations (NEE, NESE and NEEB) also altered the absorption rate and permeability of EVO in intestinal tract, albeit to different extents. Take NEE for example, the Ka (or Peff) values of NEE in the duodenum, jejunum, ileum, and colon were 2.72 (or 5.76), 3.56 (or 4.24), 4.43 (or 5.02), and 1.74 (or 1.74) times that of free EVO, respectively. As far as NESE and NEEB were concerned, following phenomena were worthy of notice: (1) the Ka and Peff values of NESE in colon were much higher (3.61-fold and 7.38-fold) than that of NEE; (2) the Ka and Peff values of NEEB in jejunum and colon were much higher (4.07-fold and 6.00-fold in jejunum; 2.77-fold and 3.75-fold in colon) than that of NEE.
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3.3. Pharmacokinetic and bioavailability of NESEB
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The plasma drug concentration–time curves for a single dose of oral administered EVO in deferent delivery systems were depicted in Fig. 4. Double peaks appeared in the plasma concentration–time
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Fig. 2. The gastrointestinal absorption constants, effective permeability coefficients and absorption percentages comparison among NESEB, NEEB, NESE, NEE and free EVO. Data presented as mean ± standard deviation (n = 6). P < 0.05, P < 0.01.
Fig. 3. The gastrointestinal absorption constants, effective permeability coefficients and absorption percentages comparison among five different gastrointestinal tracts. Data presented as mean ± standard deviation (n = 6). P < 0.05, P < 0.01.
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profile of free EVO, as well as the four nanoemulsive formulations. For free EVO, the first peak concentration was 75.87 ng/mL at 0.75 h and the second one was 72.46 ng/mL at 5 h, respectively. The double peaks of four nanoemulsive formulations occurred at 0.5 h and 1 h, respectively. Take NESEB for example, the first peak concentration was 581.65 ng/mL at 0.5 h and 616.66 ng/mL at 1 h,
respectively. EVO was detectable by HPLC until 12 h for free EVO and 48 h for other four EVO nanoemulsive formulations. The concentration–time data were subjected to compartmental pharmacokinetic analysis by DAS software. The results showed that both NESEB and free EVO could be best described with one-compartment models, while other three EVO nanoemulsive formulations
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Fig. 4. In vivo pharmacokinetic profiles (i.e., plasma concentration–time curves) and bioequivalence evaluation of NESEB and other EVO formulations after oral administration at the same EVO dose of 100 mg/kg. Data presented as mean ± standard deviation (n = 6). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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(NEE, NESE and NEEB) could be dealt with two-compartment models. The main pharmacokinetic parameters of free EVO and its four nanoemulsive formulations are depicted in Fig. 5. The area under concentration (AUC) values of free EVO, NEE, NESE, NEEB and NESEB increased in order. In the case of NESEB, the relative bioavailability (RBA) of NESEB to free EVO was 846.97%. The Cmax and Tmax values of NESEB were 7.54 and 0.31 times that of free EVO, respectively. The 90% confidence intervals of AUC and Cmax of NESEB and free EVO were considerably beyond the scope of 80–125% and 70–143%, and the Tmax values of NESEB and free EVO were significantly different (P < 0.05) when using the Wilcoxon rank sum test to compare them. These results indicated that NESEB and free EVO were definitely not bioequivalent (see Fig. 5 and Table 2). Compared to free EVO, different degrees of beneficial changes in the main pharmacokinetical parameters of the four EVO nanoemulsive systems occurred. Among these EVO nanoemulsive
systems, the one with the highest AUC and Ka values (3.44-fold and 38.5-fold that of free EVO, respectively), accompanying with the lowest Tmax and Cl values (three tenth and two tenth that of free EVO, respectively) was NESEB, the one with the highest MRT value (3.44-fold that of free EVO) was NESE, and the one with the highest Cmax value (8.27-fold that of free EVO, or 8.74% higher than that of NESEB) was NEEB, respectively. Besides, there had been a significant decrease in RBA values of NESEB (850%), NEEB (700%), NESE (600%), NEE (400%), SEVO (200%) (15). Bioequivalence analysis between every two EVO formulation conducted using DAS software further confirmed that NESEB and any other EVO nanoemulsive formulation mentioned above (NEE, NESE and NEEB) were not bioequivalent (see Table 2). Since the AUC value was documented as 349.83 lg h L1 when the free EVO was given intravenously at a dose of 2 mg/kg to the rats [25], the ABA values of EVO formulations given orally could be calculated by Eq. (5), there had been a significant decrease in ABA values of NESEB (40%), NEEB (33%), NESE (28%), NEE (17%), SEVO (8%) and EVO (4%).
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4. Discussion
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It takes a lot of time and effort evaluating the effectiveness and safety before a new biomaterial is finally approved by the national agency for drug administration. Obviously, it may ease pressure on developing new biomaterials to develop suitable system for delivering an insoluble drug if the new application of the biomaterials already officially approved can be explored successfully. Here, a novel nanoemulsive system (i.e., a nano-emulsified supermolecular evodiamine containing brucea javanica oil, NESEB) consisted of only biomaterial officially approved was elaborately designed to deliver an insoluble drug EVO. Elementary configuration of NESEB was described in Fig. 1. (1) The NESEB was essentially a water-in-oil nanoemulsion composed of oil phase (a blend of ethyl oleate and brucea javanica oil at a mass ratio of 2:1), water phase (distilled water), and two-phase interface (containing cremorphor EL 35, polyethylene glycol 400 and SEVO). The high phase volume ratio of oil phase to water phase (9:1, mL:mL) was beneficial to form stable water-in-oil nanoemulsions. The translucent NESEB system was light yellow. When NESEB was diluted with 4 times or 9 times volume of ethyl oleate (oil phase), it remained
447
Fig. 5. Pharmacokinetic parameters comparison among NESEB, NEEB, NESE, NEE and free EVO. Data presented as mean ± standard deviation (n = 6). P < 0.05,
P < 0.01.
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J. Hu et al. / European Journal of Pharmaceutics and Biopharmaceutics xxx (2014) xxx–xxx Table 2 Bioequivalence evaluation of NESEB and other EVO formulations after oral administration at the same EVO dose of 100 mg/kg. Formulation 1 and 2
Parameter
90% Confidential interval calculated
P value calculated
Bioequivalence standard
Bioequivalence
NESEB and EVO
AUC Cmax Tmax In all
2340.5–3447.8% 4.9–33.6% – –
– – 0.05 –
No No No No
NESEB and NEE
AUC Cmax Tmax In all
122.1–194.3% 96.9–105.8% – –
– – >0.05 –
80–125% 70–143% >0.05 –
No Yes Yes No
NESEB and NESE
AUC Cmax Tmax In all
89.2–138.2% 16.6–64.5% – –
– – >0.05 –
80–125% 70–143% >0.05 –
No No Yes No
NESEB and NEEB
AUC Cmax Tmax In all
86.6–130.8% 90.4–108.8% – –
No Yes Yes No
Notes: Data presented as mean ± standard deviation (n = 6). 466 467 468 469 470 471 472 473 474 475 476 477 478 479 480 481 482 483 484 485 486 487 488 489 490 491 492 493 494 495 496 497 498 499 500 501 502 503 504 505 506 507 508 509 510 511
translucent and kept one layer, which further suggested NESEB was a water-in-oil nanoemulsion. The average solubilities of EVO in ethyl oleate (oil phase) and cremophor EL 35 (surfactant) were determined by a HPLC method in our preliminary study. They were 50.66 lg/mL and 1968.95 lg/mL, respectively. Cremorphor EL 35 had both hydrophilic and hydrophobic groups and acted as an emulsifier (or a surfactant). Cremorphor EL 35 was a common excipient for injectable and oral use. In the material safety data sheet of BASF Corporation, it was recorded that the cremophor EL (CEL) was practically nontoxic. Polyethylene glycol 400 (soluble in water and many organic solvent such as ethyl oleate) acted as a coemulsifier or a cosurfactant. SEVO having both hydrophilic and hydrophobic groups might exist in the interface in a similar way to an amphiphilic emulsifier. (2) The fact that SEVO was formed by combining EVO and soybean phospholipid (Lipoid S 75, the phosphatide content was above 70%, w/w) through noncovalent bonding had been previously investigated by differential scanning calorimetry, ultraviolet spectroscopy, Fourier transformed infrared spectroscopy, H NMR spectroscopy, matrixassisted laser desorption/ionization time-of-flight spectroscopy [15]. EVO could be well complexed with Lipoid S 75, as evidenced by high average complexation rate of SEVO (above 95%). The complexation rate of SEVO was determined by the following formula: Complexation rate = m2/m1 100% = (m1 m3)/m1 100%, where ‘‘m1’’ is the total content of EVO added, ‘‘m2’’ is the content of EVO ‘‘present as a complex’’, and ‘‘m3’’ is the free EVO or non-complexed EVO. Lipoid S 75 was already approved to be used for oral and injectable administration by State Food and Drug Administration. Moreover, Lipoid S 75 was a low-cost pharmaceutical excipient which should be more suitable for industrial production from a cost perspective. The price of Lipoid S 75 (the phosphatide content was above 70%, w/w) was about 1/30 of egg phospholipid (the phosphatide content was above 98%, w/w). The conductivity of NESEB was much higher than that of free EVO in ethyl oleate (oil phase) and a litter higher than that of CNE, respectively. However, after dilution 5 times with ethyl oleate, the conductivity of NESEB increased to more than two times of NEE. The difference between the conductivities of NESEB and other EVO formulations indicated the different existing state of EVO. The reasons why NESEB was diluted with ethyl oleate before determination were listed as follows: NESEB was a water-in-oil nanoemulsive system embedding an evodiamine-phospholipid nanocomplex. The external phase was oil phase, i.e., ethyl oleate. When the NESEB (the translucent NESEB system was light yellow–green) was added with 4 times volume of ethyl oleate (oil phase), the mixture was still translucent.
The mean size and zeta potential of NESEB were 613.3 nm and 4.46 mV, respectively. The reason why the determined size values of the translucent NESEB systems were so big was not clear. On the other hand, there was no report on the size diameter of a waterin-oil nanoemulsive system used for oral delivery of hydrophobic drug so far, since not even a water-in-oil nanoemulsive system has been developed for oral delivery of hydrophobic drug so far. As far as we knew, only a few research papers about water-in-oil nanoemulsions were published, among them, two research papers mentioned the determination of the particle size of water-in-oil nanoemulsions. They were documented as follows: (1) there was one report on the size diameter of a water-in-oil nanoemulsive system used for intravesical delivery of hydrophobic drug (cisplatin) [26], the range of the mean size was 30–90 nm. The mean vesicle size of the nanoemulsion was measured by photon correlation spectroscopy (Malvern Nano ZSÒ 90, Worcestershire, UK) using a helium-neon laser with a wavelength of 633 nm. This formulation were diluted 5-fold with soybean oil (oil phase of formulation) before the measurement. (2) There was one report on the size diameter of a water-in-oil nanoemulsive system used for transdermal delivery of caffeine (a hydrophobic drug) [27], the mean droplet size of the caffeine nanoemulsion was found in the range of 20.14–105.25 nm. Droplet size distribution of the nanoemulsion was determined by photon correlation spectroscopy (PCS) using a Zeta sizer 1000 HS (Malvern Instruments, UK). The morphologies of NESEB were tried to be investigated by a transmission electron microscopy (TEM H-7500, Hitachi, Japan) and a scanning electron microscopy (E-1010, Hitachi, Japan), respectively, but the electron microscopy results of NEEPN were unavailable because the external phase was oil. As far as we knew, this was the first time to load a drug– phospholipid nanocomplex into a nanoemulsive system. The potential advantages of supermolecular lipophilic drug loaded nanoemulsive systems (containing synergistic bioactive oil) over free drug or conventional nanoemulsion might include agreeable appearance, higher bioavailability, biocompatibility, efficacy, thermodynamic and kinetic stability, the amazing biological features of the nanosystem were investigated in our further rat absorptive and pharmacokinetic studies. Compared to free EVO, the gastric absorption of EVO in nanoemulsive systems, showed the following increases: NESEB increased by 240%, NEE, NESE and NEEB increased by 20–100%. The increase of gastric absorption contributed to increase the oral bioavailability and cure gastric cancer. Different from stomach, intestinal tract was the major site for EVO absorption, regardless of the EVO delivery system type. Among free EVO and EVO
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nanoemulsive systems mentioned above, NESEB had the highest Ka (or Peff) values in almost every corresponding intestinal segment, thus it was undoubtedly the best intestinal absorption enhancement nanosystem. For NESEB, the absorption rate and permeability in duodenal and jejunal segments were almost the same, while they were higher than that in ileac and colonic segments. Other EVO nanoemulsive systems could improve the absorption rate and permeability of EVO, although to different extents. Particularly, compared to NEE, NESE greatly improved the absorption of EVO in colon, while NEEB in jejunum and colon (the improved colonic absorption might be favorable toward treating colorectal tumor). In short, as shown in Figs. 2 and 3, the percentage absorption of EVO had been obviously improved by nanoemulsifying EVO to form NEE; moreover, the effect of increasing absorption could be further enhanced by using supermolecular technique to form NESE alone or adding brucea javanica oil to form NEEB. As expected, the most effective way to enhance the percentage absorption of NEE was simultaneously using supermolecular technique and adding brucea javanica oil to form NESEB. In short, NESEB was highly favorable for EVO absorption, as evidenced by the presence of high Ka, Peff and PA values. The drug absorption rate referred to the drug absorption over a particular period of time. Factors affecting absorption included factors related to drugs (lipid water solubility, molecular size, particle size, degree of ionization, physical forms, chemical nature, dosage forms, formulation, concentration, etc.) and factors related to body (area of absorptive surface, vascularity, pH, presence of other substances, gastrointestinal motility, functional integrity of absorptive surface, diseases, etc.). Obviously, factors affecting EVO absorption in NESEB were not always the same in different gastrointestinal tracts, so there was significant difference of absorption rate for NESEB in different gastrointestinal tracts. The effective permeability was one of the factors affecting drug absorption. The effective permeability was mainly relative to the drug molecular, liposolubility, permeability glycoprotein (P-gp) and cytochrome P4503A (CYP3A). The effective permeability values of NESEB in different gastrointestinal tracts were the synthetic effects of all factors affecting the effective permeability, so it was reasonable that there was significant difference of effective permeability for NESEB in different gastrointestinal tracts.
Enhanced absorption of NESEB might ascribe to the following reasons: (1) solubilization of EVO by supermolecular state, mixed oil (or co-solvent), and nanoemulsive form; (2) protection from enzymatic oxidation (EVO was the substrate of CYP3A enzyme) [28] or p-glycoprotein (P-gp) mediated EVO efflux (lipophilic EVO was a P-gp substrate) by entrapping EVO inside the delivery nanosystem containing components such as the surfactants and oils which could act as P-gp/CYP450 inhibitors [29]; (3) high dispersibility of EVO in phospholipid nanocomplex and nanoemulsive form, surfactant-induced membrane fluidity and thus permeability improvement; (4) the occurrence of intestinal lymphatic transport [30,31]. As shown in Fig. 4, the pharmacokinetic behavior of NESEB was the most desirable for delivering EVO among free EVO and the four nanoemulsive formulations (NESEB, NEEB, NESE and NEE) when the same doses (equivalent to 100 mg/kg) of EVO were orally given to rats separately. According to experiments and data analysis (DAS software), one-compartment models best described the time course of the EVO concentration when NESEB was orally administered, indicating that NESEB could rapidly distribute to tissues; while two-compartment models suitable for other three EVO nanoemulsive formulations (NEE, NESE and NEEB), indicating that drug in these systems exhibited a slow equilibration with peripheral tissues. The phenomenon that the drug distribution of NESEB was faster in comparison with other nanoemulsive delivery systems might have some relevance to the existing state of EVO in different nanoemulsive formulations and need further study. Compared to free EVO, the smaller Tmax value of NESEB indicated a faster rate and a larger degree of gastrointestinal absorption; usually it also indicated a faster onset of action for patients in clinical practice. Moreover, the mean residence time (MRT) of NESEB rose by 2.73-fold from free EVO, while the clearance (Cl) dropped by 77.14%. NESEB demonstrated prolonged circulation in the bloodstream, which was beneficial to increase drug action time as well as effectiveness. The in vivo absorption rate constant (Ka) of NESEB was 38.51 times that of free EVO, suggesting that NESEB had better absorption than the latter. This situation was in accordance with the results obtained from the experiments of in situ gastrointestinal absorption.
Fig. 6. The effects of gastrointestinal segment and drug delivery system on the EVO absorption and pharmacokinetic behavior. Data presented as mean ± standard deviation (n = 6).
Please cite this article in press as: J. Hu et al., Supermolecular evodiamine loaded water-in-oil nanoemulsions: Enhanced physicochemical and biological characteristics, Eur. J. Pharm. Biopharm. (2014), http://dx.doi.org/10.1016/j.ejpb.2014.06.007
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The results (comparison of AUC, Cmax, Tmax between every two EVO delivery systems) obtained from the statistical bioequivalence analysis indicated that NESEB and free EVO (or NEE, NESE, NEEB) were not bioequivalent. The four EVO nanoemulsive systems reporting an increase in orders for the relative bioavailability over free EVO were: NEE, NESE, NEEB and NESEB. Favorably changes to the pharmacokinetic behavior of EVO took place in NESEB, so did in other three EVO nanoemulsive systems (NEE, NESE and NEEB) although to different extents. NESEB had the highest AUC and Ka values, besides the lowest Tmax and Cl values, while NESE had the highest MRT value and NEEB had the highest Cmax value. On the whole, NESEB was able to deliver EVO more effectively, although other EVO nanoemulsive systems also had some advantages for EVO delivery. What’s more, based on the above analysis, it was deduced that favorable pharmacokinetic consequence of NESEB might mainly be ascribed to the enhanced gastrointestinal absorption and partly to the decreased clearance from the systemic circulation in vivo (see Fig. 6).
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5. Conclusions
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A nano-emulsified supermolecular evodiamine coexisting with brucea javanica oil (NESEB), only composed of biomaterials approved by national agency for drug administration, i.e., a blend oil (ethyl oleate and brucea javanica oil), a surfactant (cremorphor EL 35), a cosurfactant (polyethylene glycol 400), a phospholipid (main constituent to form supermolecular EVO-phospholipid nanocomplex), and the distilled water, was designed and produced. It was found that NESEB markedly improved the oral bioavailability of EVO, which was likely due to the increased gastrointestinal absorption. NESEB might be a preferred alternative nanosystem for delivering evodiamine effectively via oral route, in comparison with free EVO and other three EVO nanoemulsive systems (conventional nano-emulsified evodiamine, nano-emulsified supermolecular evodiamine and nano-emulsified evodiamine containing brucea javanica oil). Our study supported our hypothesis that although it was unavailable in either clinical practice or research field, a novel nanosystem only consisted of biomaterials approved by national agency for drug administration and fabricated by combining phytosome nanotechnology with nanoemulsifying technology, in addition to using synergistic plant essential oil as a basic composition, was able to exhibit greatly improved oral bioavailability and deliver an insoluble natural drug more effectively. The development of such a novel nanosystem represented a valuable tactic in new medical application of common used biomaterials (or biomaterials approved by national agency for drug administration) for effective delivery of insoluble antitumor natural drugs.
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This research was partially supported by grants from Chongqing Natural Science Foundation (CSCT2012JJB10027), and Chongqing Education Committee Fund (the excellent university personnel financial aid plan, KJ120321).
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References
638 639 640 641 642 643 644 645 646 647 648 649 650 651 652 653 654
658 659 660 661 662 663 664 665 666 667 668 669 670 671 672 673 674 675 676 677 678 679 680 681 682
[4] [5]
[6]
[7]
[8]
[9] [10]
[11]
[12]
[13]
[14]
[15]
[16]
[17]
[18]
[19]
[20]
[21]
[22]
[23]
[24]
[25] 686 687
[26]
[27] [28]
690 691 692 693 694 695 696 697 698
[1] L. De Petrocellis, A. Schiano Moriello, G. Fontana, A. Sacchetti, D. Passarella, G. Appendino, V. Di Marzo, Effect of chirality and lipophilicity in the functional activity of evodiamine and its analogues at TRPV1 channels, Br. J. Pharmacol. (2013), http://dx.doi.org/10.1111/bph.12320. [2] J.F. Liao, W.F. Chiou, Y.C. Shen, G.J. Wang, C.F. Chen, Anti-inflammatory and anti-infectious effects of Evodia rutaecarpa (Wuzhuyu) and its major bioactive components, Chin. Med. 6 (2011) 6. [3] A.J. Liu, S.H. Wang, K.C. Chen, H.P. Kuei, Y.L. Shih, S.Y. Hou, W.T. Chiu, S.H. Hsiao, C.M. Shih, Evodiamine, a plant alkaloid, induces calcium/JNK-mediated
[29]
[30] [31]
9
autophagy and calcium/mitochondria-mediated apoptosis in human glioblastoma cells, Chem. Biol. Interact. 205 (2013) 20–28. H. Yu, H. Jin, W. Gong, Z. Wang, H. Liang, Pharmacological actions of multitarget-directed evodiamine, Molecules 18 (2013) 1826–1843. M.A. Alam, F.I. Al-Jenoobi, A.M. Al-Mohizea, Commercially bioavailable proprietary technologies and their marketed products, Drug Discov. Today 18 (2013) 936–949. F. Aqil, R. Munagala, J. Jeyabalan, M.V. Vadhanam, Bioavailability of phytochemicals and its enhancement by drug delivery systems, Cancer Lett. (2013) (pii: S0304-3835(13)00158-4). D. He, P. Deng, L. Yang, Q. Tan, J. Liu, M. Yang, J. Zhang, Molecular encapsulation of rifampicin as an inclusion complex of hydroxypropyl-b-cyclodextrin: design; characterization and in vitro dissolution, Colloids Surf. B: Biointerfaces 103C (2012) 580–585. Q. Tan, J. Wu, Y. Li, H. Mei, C. Zhao, J. Zhang, A supermolecular curcumin for enhanced antiproliferative and proapoptotic activities: molecular characteristics, computer modeling and in vivo pharmacokinetics, Nanotechnology 24 (2013) 03510. T. Kurita, Y. Makino, Novel curcumin oral delivery systems, Anticancer Res. 33 (2013) 2807–2821. D.J. McClements, Nanoemulsion-based oral delivery systems for lipophilic bioactive components: nutraceuticals and pharmaceuticals, Ther. Deliv. 4 (2013) 841–857. S. Ganta, D. Deshpande, A. Korde, M. Amiji, A review of multifunctional nanoemulsion systems to overcome oral and CNS drug delivery barriers, Mol. Membr. Biol. 27 (2010) 260–273. P.M. Kidd, Bioavailability and activity of phytosome complexes from botanical polyphenols: the silymarin, curcumin, green tea, and grape seed extracts, Altern. Med. Rev. 14 (2009) 226–246. S. Kotta, A.W. Khan, K. Pramod, S.H. Ansari, R.K. Sharma, J. Ali, Exploring oral nanoemulsions for bioavailability enhancement of poorly water-soluble drugs, Exp. Opin. Drug Deliv. 9 (2012) 585–598. A. Semalty, M. Semalty, M.S. Rawat, F. Franceschi, Supramolecular phospholipids–polyphenolics interactions: the phytosome strategy to improve the bioavailability of phytochemicals, Fitoterapia 81 (2010) 306–314. Q. Tan, S. Liu, X. Chen, M. Wu, H. Wang, H. Yin, D. He, H. Xiong, J. Zhang, Design and evaluation of a novel evodiamine-phospholipid complex for improved oral bioavailability, AAPS PharmSciTech. 13 (2012) 534–547. Y. Lv, G. Ding, J. Zhai, Y. Guo, G. Nie, L. Xu, A superparamagnetic Fe3O4-loaded polymeric nanocarrier for targeted delivery of evodiamine with enhanced antitumor efficacy, Colloids Surf. B: Biointerfaces 10 (2013) 411–418. Y.T. Zhang, Z.B. Huang, S.J. Zhang, J.H. Zhao, Z. Wang, Y. Liu, N.P. Feng, In vitro cellular uptake of evodiamine and rutaecarpine using a microemulsion, Int. J. Nanomed. 7 (2012) 2465–2472. Q. Peng, Z.R. Zhang, X. Sun, J. Zuo, D. Zhao, T. Gong, Mechanisms of phospholipid complex loaded nanoparticles enhancing the oral bioavailability, Mol. Pharm. 7 (2010) 565–575. S. Wang, M. Sun, Q. Ping, Enhancing effect of Labrafac Lipophile WL 1349 on oral bioavailability of hydroxysafflor yellow A in rats, Int. J. Pharm. 358 (2008) 198–204. Y.L. Nie, K.X. Liu, X.Y. Mao, Y.L. Li, J. Li, M.M. Zhang, Effect of injection of brucea javanica oil emulsion plus chemoradiotherapy for lung cancer: a review of clinical evidence, J. Evid. Based Med. 5 (2012) 216–225. Y.L. Yu, Y. Lu, X. Tang, F.D. Cui, Formulation, preparation and evaluation of an intravenous emulsion containing brucea javanica oil and coix seed oil for antitumor application, Biol. Pharm. Bull. 31 (2008) 673–680. L. Perioli, P. Mutascio, C. Pagano, Influence of the nanocomposite MgAl-HTlc on gastric absorption of drugs: in vitro and ex vivo studies, Pharm. Res. 30 (2013) 156–166. Q. Tan, N. Hu, G. Liu, H. Yin, L. Zhang, H. Wang, L.Y. Lu, J.Q. Zhang, Role of a novel pyridostigmine bromide-phospholipid nanocomplex in improving oral bioavailability, Arch. Pharm. Res. 35 (2012) 499–508. J.M. Zhang, W. Liao, Y.X. He, Y. He, D. Yan, C.M. Fu, Study on intestinal absorption and pharmacokinetic characterization of diester diterpenoid alkaloids in precipitation derived from Fuzi-Gancao herb-pair decoction for its potential interaction mechanism investigation, J. Ethnopharmacol. 147 (2013) 128–135. K.F. Jeng, Y.H. Lin, L.C. Lin, C.J. Chou, T.H. Tsai, C.F. Chen, High-performance liquid chromatographic determination of evodiamine in rat plasma: application to pharmacokinetic studies, J. Chromatogr. B: Biomed. Appl. 668 (1995) 343–345. T.L. Hwang, C.L. Fang, C.H. Chen, J.Y. Fang, Permeation enhancer-containing water-in-oil nanoemulsions as carriers for intravesical cisplatin delivery, Pharm. Res. 26 (2009) 2314–2323. F. Shakeel, W. Ramadan, Transdermal delivery of anticancer drug caffeine from water-in-oil nanoemulsions, Colloids Surf. B: Biointerfaces 75 (2010) 356–362. H.Z. Sun, Z.Z. Fang, Y.F. Cao, X.Y. Sun, M. Hong, Investigation of the in vitro metabolism of evodiamine: characterization of metabolites and involved cytochrome p450 isoforms, Phytother. Res. 27 (2013) 705–712. A.O. Nornoo, D.W. Osborne, D.S. Chow, Cremophor-free intravenous microemulsions for paclitaxel I: formulation, cytotoxicity and hemolysis, Int. J. Pharm. 349 (2008) 108–116. Y. Lu, J. Qi, W.Wu. Absorption, Disposition and pharmacokinetics of nanoemulsions, Curr. Drug Metab. 13 (2012) 396–417. J.Q. Zhang, J. Liu, X.L. Li, B.R. Jasti, Preparation and characterization of solid lipid nanoparticles containing silibinin, Drug Deliv. 14 (2007) 381–387.
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Contents lists available at ScienceDirect
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Research paper
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Supermolecular evodiamine loaded water-in-oil nanoemulsions: Enhanced physicochemical and biological characteristics
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Medicine Engineering Research Center, Chongqing Key Laboratory of Biochemical & Molecular Pharmacology, Chongqing Medical University, Chongqing, People’s Republic of China Department of Health Statistics, College of Public Health, Chongqing Medical University, Chongqing, People’s Republic of China Department of Thoracic Surgery, Institute of Surgery Research, Daping Hospital, Third Military Medical University, Chongqing, People’s Republic of China
a r t i c l e
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Jiangbo Hu a,1, Lili Sun a,1, Dezhang Zhao a,1, Liangke Zhang a,1, Mengliang Ye b,1, Qunyou Tan c, Chunshu Fang c, Hong Wang a, Jingqing Zhang a,⇑
i n f o
Article history: Received 14 February 2014 Accepted in revised form 14 June 2014 Available online xxxx
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Keywords: Nanoemulsification Supermolecular evodiamine Brucea javanica oil Oral bioavailability Characteristics
a b s t r a c t The purpose of this study was to develop and evaluate the supermolecular evodiamine (EVO) loaded water-in-oil nanoemulsions containing brucea javanica oil (NESEB) with enhanced physicochemical and biological characteristics. NESEB was fabricated by applying supermolecular phytosome nanotechnology and nanoemulsification technology together, in addition to using synergistic plant essential oil as a basic composition. Preferred physicochemical and biological characteristics of NESEB were investigated and compared with free EVO and other nanoemulsive EVO carriers. The possible explanations for improved absorption and bioavailability were put forward here. NESEB had high absorption and bioavailability, for example: the absorption rate constants and permeabilities of NESEB in different intestinal segments were 3.65–6.76 times that of free EVO; the relative bioavailability of NESEB to free EVO was 846.97%. NESEB markedly improved the oral bioavailability of EVO, which was most likely due to the increased gastrointestinal absorption. The development of nanoemulsion-based supermolecular EVO nanocarriers provides valuable tactics in insoluble natural antitumor drug delivering. Ó 2014 Published by Elsevier B.V.
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1. Introduction In clinical practice, Evodia rutaecarpa or ‘‘Wu-zhu-yu’’ in Chinese has been traditionally administered orally together with some other traditional Chinese medicines to treat inflammatory diseases so far [2]. Evodiamine (EVO) is one of the most important active ingredients of E. rutaecarpa [1]. Recently, EVO has been drawing great interest of scientists due to its potent antitumor activities against a variety of tumors (such as gastric cancer and colorectal tumor) [3,4]. The action mechanism of EVO has been deeply investigated. On the other hand, in order to achieve the clinical translation of EVO, the primary consideration is to fabricate a suitable formulation with greatly enhanced oral bioavailability, since water insoluble EVO shows definite antitumor activity but simultaneously exhibits poor oral bioavailability. During the fabricating process, the biomaterials approved by national agency for drug administration are considered to be the first choice. These biomaterials are ⇑ Corresponding author. Room 802, Building 9, Shiyou Road 1, Yuzhong District, Chongqing Key Laboratory of Biochemical & Molecular Pharmacology, Medicine Engineering Research Center, Chongqing Medical University, Chongqing 400016, People’s Republic of China. Tel.: +86 13308300303. E-mail address: [email protected] (J. Zhang). 1 These authors contributed equally to this study.
expected to form new amazing nanocarriers by using cutting edge technologies, such as supermolecular phytosome nanotechnology and nanoemulsification technology. The development of such smart nanocarriers made of the approved biomaterials may ease pressure on developing new biomaterials for effectively delivering an insoluble EVO [5–8]. There has been progress in the development of supermolecular phytosome nanotechnology and nanoemulsification drug technology over the past decade. Supermolecular phytosomes refer to noncovalently bonded complexes of a natural active ingredient and phospholipids. A drug–phospholipid complex is one supermolecular drug. Distinguishing from ordinary emulsions, nanoemulsions refer to emulsive drug nanosystems with some distinctive physiochemical properties [9,10]. There are a few supermolecular phytosomal drug or emulsive drug nanosystem products currently on the market, including MerivaÒ (supermolecular phytosomal curcumin for the treatment of diabetes related oxidative stress) by Thorne Research Inc. (USA), SiliphosÒ (supermolecular phytosomal silybin for the treatment of hepatitis associated with reduction in serum ferritin) by Indena S.p.A. Inc. (Italy), NORVIR (emulsive ritonavir nanosystem for the treatment of HIV-1 infection) by Abbott Laboratories (USA), and NeoralÒ (emulsive ciclosporin nanosystem for suppressing the immune system) [11–14].
http://dx.doi.org/10.1016/j.ejpb.2014.06.007 0939-6411/Ó 2014 Published by Elsevier B.V.
Please cite this article in press as: J. Hu et al., Supermolecular evodiamine loaded water-in-oil nanoemulsions: Enhanced physicochemical and biological characteristics, Eur. J. Pharm. Biopharm. (2014), http://dx.doi.org/10.1016/j.ejpb.2014.06.007
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Supermolecular phytosomal EVO (SEVO, or EVO-phospholipid nanocomplex) had been developed in our lab [15]. The relative oral bioavailability of SEVO was increased to 218.82% compared with free EVO. Besides SEVO, only a few EVO delivery systems (such as microemulsion for transdermal delivery, superparamagnetic Fe3O4-loaded polymeric nanocarrier for intravenous delivery) have been reported so far [16,17]. Obviously, it is of great clinical significance to investigate some more effective EVO delivery systems with much higher bioavailabilities than EVO and SEVO. Since some drug–phospholipid complex based formulations, such as hydroxysafflor yellow A-phospholipid complex oil solution and salvianolic acid-phospholipid complex loaded nanoparticles, exhibited improved oral bioavailability over the simple drug–phospholipid complex [18,19], we would like to further package a SEVO in a nanoemulsive system to achieve the added effectiveness. The nanoemulsive and supermolecular phytosomal EVO was formulated by applying these two above-mentioned nanotechnologies simultaneously instead of separately. Furthermore, brucea javanica oil is a Chinese drug widely used in clinic to treat a broad spectrum of tumors, and is known for the effectiveness and safety of it plus other chemotherapy drugs [20,21]. Since oil phase is essential during the nanoemulsification process, it may be a good idea to entrap brucea javanica oil into the novel EVO delivery nanosystem, and then brucea javanica oil can provide a synergistic antitumor effect with EVO in addition to being the basic composition of the new nanoemulsive and supermolecular EVO nanocarrier. Although it is currently unavailable in either clinical practice or the research field, a nanoemulsified supermolecular drug nanocarriers composed of only biomaterials approved by national drug administration agency by applying supermolecular phytosome nanotechnology and nanoemulsification drug technology together, in addition to using synergistic plant essential oil as a basic composition, may be theoretically expected to deliver an insoluble natural drug effectively and exhibit greatly improved oral bioavailability. Here we reported some encouraging experimental data to support this hypothesis. In the experiments outlined below, a nanoemulsified supermolecular evodiamine containing brucea javanica oil (NESEB) only using biomaterials approved by national drug administration agency were designed and prepared, and then the in vivo pharmacokinetics and the in situ absorption of NESEB were evaluated. Compared with EVO, SEVO, conventional nanoemulsified evodiamine (NEE), nanoemulsified supermolecular evodiamine (NESE), and nano-emulsified evodiamine containing brucea javanica oil (NEEB), NESEB markedly improved the oral bioavailability of EVO, which was probably due to the increased gastrointestinal absorption. As far as we know, this was the first time to load a drug–phospholipid nanocomplex into a nanoemulsive delivering system. It was the first time that a preferred nanoemulsified supermolecular nanosystem with high oral bioavailability has been produced by combining phytosomal nanotechnology with nanoemulsifying technology, in addition to using synergistic antitumor oil as a basic composition, to deliver an insoluble antitumor natural drug. Our studies focus on the greatly improved oral bioavailability in vivo and the possible reason (obviously enhanced gastrointestinal absorption) of this delivery nanosystem. The development of such a novel nanosystem represented a valuable tactic in new medical application of commonly used biomaterials approved by national drug administration agency for effectively delivering insoluble antitumor natural drugs.
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2. Materials and methods
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2.1. Materials
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EVO was obtained from Yuancheng Technology Development Co., Ltd. (Wuhan, China), purity 99.13%. Ethyl oleate was purchased
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from Shanghai Chemical Reagent Co. (Shanghai, China), analytical grade. Polyethylene glycol 400 was purchased from Tianjin Guangfu Fine Chemical Co., Ltd. (Tianjin, China). Cremorphor EL 35 was purchased from BASF Corporation (Ludwigshafen, Germany). Brucea javanica oil was purchased from Shanghai Kexin Biology Engineering Co., Ltd. Soybean phospholipid (Lipoid S 75) was purchased from Phospholipid GmbH (Nattermannllee, Germany). All other chemicals and reagents used were of analytical or chromatographic grade. Male Sprague-Dawely rats obtained from the Animal Center of Chongqing Medical University (Chonqing, China) were all specific pathogen free animals. Their weights were 200–250 g. The animal studies were conducted in accordance with the protocol approved by the Laboratory Animal Committee, Chongqing Medical University. The animals were raised under controlled conditions with free access to diet and water, while fasted at least 18 h before drug administration in the gastrointestinal absorption study and 12 h in the pharmacokinetical study, respectively.
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2.2. Fabrication and characterization of NESEB
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Firstly, SEVO was produced using a modified solvent evaporation method [15]. Briefly, a mixture of phospholipid and EVO at a molar ratio of 2:1 was dissolved in 50 mL ethanol, and magnetically stirred at 60 °C for 3 h. The solvent was evaporated to dryness under hypobaric conditions; the resulting SEVO was stored until use. Secondly, SEVO was loaded into the NESEB using a nanoemulsifying technique. An appropriate amount of SEVO was added into an isotropic mixture of ethyl oleate, brucea javanica oil, cremorphor EL 35 and polyethylene glycol 400 (at a mass ratio of 24:12:13:10) and the resulting mixture was then magnetically stirred at 60 °C for 6 h, cooled to 30 °C, added dropwise by 5 mL of distilled water under continuous stirring with a magnetic stirrer. NEEB was prepared in a similar way to prepare NESEB, and the only difference was that EVO instead of SEVO was added in the preparing process. NESE was prepared in a similar way to prepare NESEB, and the only difference was that ethyl oleate instead of a blend oil (ethyl oleate and brucea javanica oil) was added in the preparing process, i.e., no brucea javanica oil was added in the preparing process. NEE was prepared in a similar way to prepare NESE, and the only difference was that EVO instead of SEVO was added in the preparing process. The conductivity of NESEB and its dilution (5 times with ethyl oleate) were determined at 25 °C by an electric conductivity analyzer (DDB-303A, Shanghai Precision & Scientific Instrument Co. Ltd, Shanghai, China), respectively. The size and zeta potential of NESEB were determined at 25 °C by dynamic light scattering (Zeta-Sizer Nano-ZS90, Malvern, UK). These studies were performed at refractive index of 1.45 because the refractive index for all formulation was around this value. The sample cell was made of quartz (Malvern, UK). The sample was prepared by diluting 4 mL of NESEB with 16 mL of ethyl oleate. In the in vitro release tests, NESEB was placed in a dialysis tube and then immersed into the release media of phosphate buffer system (PBS, pH 6.8). A HPLC method was used to determine the free EVO.
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2.3. Gastrointestinal absorption of NESEB
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The in situ gastric absorption test was carried out as previously described with some modifications [22,23]. Briefly, the experimental rats under intraperitoneal anesthesia with chloral hydrate were fixed supinely. After a 3 cm incision was made in the abdominal midline, the pylorus (the stomach outlet) was cannulated with a flexible 2 mm internal diameter tubing and then ligated. A small incision was made in the cardia (the stomach inlet). After the stomach was rinsed with the artificial gastric juice, the cardia was ligated
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and 4 mL of NESEB (or EVO, CNEE, NESE, and NEEB) at the same concentration of 100 lg/mL EVO was injected into stomach. The stomach was replaced in the abdomen. After 2 h, the solution remained in the stomach was taken out and mixed with the artificial gastric juice which was used to wash the stomach to make 25 mL. One volume of dilution was added with 3 volumes of methanol and 2 volumes of acetone. The resulting mixture was vortexed for 2 min, and then centrifuged at 12,000 rpm for 10 min. Aliquots of the supernatant were further subjected to HPLC analysis. The stationary phase (Lichrospher C18 column, 250 mm 4.6 mm, 5 lm) was kept at 35 °C, and the mobile phase (methanol:water = 75:25, v/v) was run at a flow rate of 1 mL/min. The effluent was monitored at 225 nm. The standard regression equation in the range of EVO concentration (C) 1–40 lg mL1 was linear (A = 16446C + 12550, r = 0.9994, n = 5; A means the peak area). Besides the linearity, the precision and accuracy (data not shown) of the HPLC method also met the requirements of application requirements. The surgical procedures for the in situ single pass intestinal perfusion (SPIP) test were conducted as previously described with some modifications [23,24]. After incision through medioventral line, enteric tracts of anesthetized rats were carefully exposed and cannulated separately with flexible tubing on two ends of four 10 cm long intestinal segments, i.e., duodenum (1 cm distal to the pyloric sphincter), jejunum (15 cm distal to the pyloric sphincter), ileum (20 cm proximal to the cecum), and colon (1 cm distal to the cecum). After rinsing with physiological saline, the chosen intestinal sections were attached to the perfusion assembly consisting of a BT100-1L peristaltic pump (Baoding Longer Precision Pump Co. Ltd., Baoding, China) and equilibrated with Krebs-Ringer’s solution (NaCl 118.1 mM; KCl 3.4 mM; CaCl2 2.5 mM; MgSO4 0.8 mM; KH2PO4 1.2 mM; NaHCO3 25.0 mM; Glucose 11.1 mM) at a flow rate of 0.4 mL min1 for 15 min. Next, the tested perfusate solution equivalent to 100 lg mL1 of EVO, prepared by displacing the tested drugs (NESEB, EVO, NEE, NESE and NEEB, respectively) into the Kerbs-Rings solution, was perfused through the segments at a flow rate of 0.2 mL min1 for 1 h. The remaining perfusate solution was mixed with the Kerbs-Rings solution which was used to wash the intestinal segment to make 25 mL. The resulting mixture was treated and measured according to the above method for determination of the EVO concentration in gastric perfusate solution. The gastrointestinal absorption rate constant (Ka) and absorption percentage (PA), as well as intestinal effective permeability (Peff) were described by the following equations:
K a ¼ ðX 0 X t Þ=C 0 t pr 2 l
ð1Þ
PA ð%Þ ¼ ðX 0 X t Þ=X 0 100%
ð2Þ
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Peff ¼ R lnðX in =X out Þ=2prl
ð3Þ
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where X0 and C0 were the drug amount and concentration at 0 h. Xt and Ct were the drug amount and concentration in the remaining perfusate solution, C0 was the drug concentration at 0 h, Ct was the remaining drug concentration in the perfusate solution, t was the perfusion time, R was the flow rate, Xin and Xout were the inlet and outlet drug amounts, and r and l were the radius and length of the perfused intestinal segment, respectively.
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2.4. Pharmacokinetic and bioavailability of NESEB
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All the experiments were approved by the Institutional Animal Care and Use Committee of Chongqing Medical University. Healthy male Sprague Dawley rats (weighing 200–250 g) were orally administered with NESEB, EVO, NEE, NESE, and NEEB at the same dose equivalent to 100 mg/kg EVO, respectively. Ophthalmic vein blood samples were drawn out at predetermined timepoints and centrifuged at 3000 rpm for 10 min immediately. The plasma samples were processed according to our previously reported
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procedure and analyzed with the HPLC method [15]. Honokiol was used as an internal standard. The standard regression equation in the range of EVO concentration (C) 0.01–3 lg mL1 was linear (R = 1.9818C + 0.0114, r = 0.9989, n = 5; R meant the peak area ratio of EVO to honokiol). Besides the linearity, the precision and accuracy (data not shown) of the HPLC method also met the requirements of application. Pharmacokinetic parameters, except maximum concentration (Cmax) and peak time (Tmax) which were directly determined by experiments, were calculated using DAS 2.1.1 software (Mathematical Pharmacology Professional Committee of China, Shanghai, China), while the relative bioavailability (RBA) of EVO formulation given orally could be calculated according to the Eq. (4) listed as follows:
RBA ð%Þ ¼ ðAUC F1 X F2 Þ=ðAUC F2 X F1 Þ
ð4Þ
where AUCF1 and XF1 were the area under the EVO concentration– time curve and the dose of EVO, respectively; AUCF2 and XF2 were the area under the EVO concentration–time curve and the dose of EVO, respectively. The absolute bioavailability (ABA) of EVO formulation given orally could be calculated according to the Eq. (5) listed as follows:
ABA ð%Þ ¼ ðAUC op;F1 X iv;EVO Þ=ðAUC iv;EVO X op;F1 Þ
ð5Þ
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where AUCop and Xop were the area under the EVO concentration– time curve and the dose of EVO when EVO formulation 1 was given orally; AUCiv and Xiv were the area under the concentration–time curve and dose when free EVO was given intravenously. Besides pharmacokinetic analysis, bioequivalence analysis between every two EVO formulation was also conducted using DAS software.
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2.5. Statistical analysis
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All data were shown as mean ± standard deviation unless particularly outlined. One-way ANOVA, Scheffe’s F-test and Student’s t test were used to calculate statistical difference. A statistical significance was established at P < 0.05. Pharmacokinetic and bioequivalence analyses were conducted using DAS software (Mathematical Pharmacology Professional Committee of China, Shanghai, China).
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3. Results
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3.1. Elementary configuration and characterization of NESEB
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The SEVO was produced by complexing EVO with phospholipid [15]. A circumscribed central composite design approach was chosen to properly formulate SEVO [15]. The NESEB loaded SEVO was further obtained using an emulsification method. The pseudo-ternary phase diagrams were constructed by Origin software (Version 7.5, Origin Lab Corp., Wellesley, USA) and the nanoemulsion formulation was optimized by a simplex lattix model (data not shown). The photos were taken by a C-60 ZOOM Olympus camera (Hongkong, China) and shown in Fig. 1. It was indicated that nanosized droplets of water were quite evenly dispersed in the light yellow–green translucent NESEB nanosystem. This NESEB nanosystem was a homogeneous (heterogeneous at molecular scale) and optically isotropic nanosystem. There was no significant difference between NESEB and NEEB in the appearance and color. However, although both NESE and NEE were translucent, there was a little difference from NESEB in color. NESE and NEE were light yellow and very light yellow, respectively. As seen in Table 1, the conductivity of NESEB was different from free EVO or other nanoemulsive carriers (NEE, NESE and NEEB). The
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Fig. 1. Schematic diagram of NESEB improving the oral bioavailability. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Table 1 The conductivity of NESEB and other EVO formulations. Formulation
Conductivity (ls/cm) Before dilution
Free EVO in water Free EVO in ethyl oleate Blank NEE NEE Blank NESE NESE Blank NEEB NEEB Blank NESEB NESEB
759.33 ± 8.50 0.37 ± 0.07 12.18 ± 0.31 12.98 ± 0.39 16.26 ± 0.53 16.22 ± 0.27 18.56 ± 0.39 18.27 ± 0.37 13.51 ± 0.42 13.61 ± 0.49
After dilution
0.23 ± 0.05 0.23 ± 0.05 0.11 ± 0.02 0.17 ± 0.05 0.32 ± 0.06 0.34 ± 0.06 0.43 ± 0.08 0.52 ± 0.06
Notes: A dilution consisted of 1 part formulation and 4 parts ethyl oleate. Data presented as mean ± standard deviation (n = 3).
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mean size and zeta potential of NESEB were 613.3 nm and 4.46 mV, respectively; for NEEB, 349.9 nm and 1.54 mV, respectively; for NESE, 554.1 nm and 8.16 mV, respectively; for NEE, 335.7 nm and 2.55 mV, respectively. The release rates of free EVO in the release media (pH 6.8 PBS) were (15.17 ± 1.25)% at 8 h and (16.02 ± 1.16)% at 12 h, respectively. In the case of NESEB, the EVO released from the NESEB in pH 6.8 PBS could not be detected by HPLC within 12 h, which suggested that NESEB provided a very slow release. The slow release of EVO from NESEB might be partly due to the barrier of oil phase (the outer phase of NESEB). Under our experimental conditions, NESEB was a controlled or sustained release dosage form. The release pattern and mechanism of drug release of NESEB were still unclear. The parameters (such as the appearance, color and conductivity) were investigated to evaluate the stability of the NESEB nanocarrier. The translucent NESEB system was light yellow–green all the time. No obvious changes were observed in NESEB after storage at 4 °C or 25 °C for 30 days. In our preliminary experiments, the formulation was stable against physical stress stability studies (placement at 60 °C for 10 days; illumination at (4500 lx ± 500 lx) for 10 days.
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3.2. Gastrointestinal absorption of NESEB
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NESEB was superior to free EVO or other EVO nanoemulsive systems (NEE, NESE and NEEB) in enhancement of gastrointestinal
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absorption. Comparison among different EVO delivery systems or gastrointestinal segments of gastrointestinal absorption rate constant (Ka) and absorption percentage (PA), and intestinal effective permeability (Peff) were presented in Figs. 2 and 3, respectively. As we know, stomach was seldom the major site for drug absorption. Here only a small amount of EVO (less than 5%) was absorbed from the stomach. The PA values of NEE, NESE and NEEB varied from 6.3% to 10%. NESEB had the highest absorption percentage (about 17%). The small intestine was, without question, the main area of oral absorption for EVO formulations. NESEB had the highest Ka (or Peff) values while free EVO had the least values among five EVO formulations (NESEB, NEEB, NESE, NEE and free EVO) in almost every corresponding intestinal segment. The Ka (or Peff) values of NESEB in the duodenum, jejunum, ileum, and colon were 3.74 (or 7.02), 5.06 (or 6.76), 4.82 (or 4.65), and 3.70 (or 3.65) times that of free EVO, respectively. Furthermore, the Ka (or Peff) value of NESEB in each intestinal segment was much higher than that of NEE, NESE and NEEB, respectively. The study on intestinal absorption of NESEB in different regions of rat intestine indicated that (1) the jejunum had the best absorption rate, followed by duodenum (5.68% less than the former), ileum, and colon in order; (2) the duodenum had the best permeability, followed by jejunum (3.73% less than the former), ileum, and colon in order. Besides NESEB, other EVO nanoemulsive formulations (NEE, NESE and NEEB) also altered the absorption rate and permeability of EVO in intestinal tract, albeit to different extents. Take NEE for example, the Ka (or Peff) values of NEE in the duodenum, jejunum, ileum, and colon were 2.72 (or 5.76), 3.56 (or 4.24), 4.43 (or 5.02), and 1.74 (or 1.74) times that of free EVO, respectively. As far as NESE and NEEB were concerned, following phenomena were worthy of notice: (1) the Ka and Peff values of NESE in colon were much higher (3.61-fold and 7.38-fold) than that of NEE; (2) the Ka and Peff values of NEEB in jejunum and colon were much higher (4.07-fold and 6.00-fold in jejunum; 2.77-fold and 3.75-fold in colon) than that of NEE.
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3.3. Pharmacokinetic and bioavailability of NESEB
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The plasma drug concentration–time curves for a single dose of oral administered EVO in deferent delivery systems were depicted in Fig. 4. Double peaks appeared in the plasma concentration–time
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Fig. 2. The gastrointestinal absorption constants, effective permeability coefficients and absorption percentages comparison among NESEB, NEEB, NESE, NEE and free EVO. Data presented as mean ± standard deviation (n = 6). P < 0.05, P < 0.01.
Fig. 3. The gastrointestinal absorption constants, effective permeability coefficients and absorption percentages comparison among five different gastrointestinal tracts. Data presented as mean ± standard deviation (n = 6). P < 0.05, P < 0.01.
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profile of free EVO, as well as the four nanoemulsive formulations. For free EVO, the first peak concentration was 75.87 ng/mL at 0.75 h and the second one was 72.46 ng/mL at 5 h, respectively. The double peaks of four nanoemulsive formulations occurred at 0.5 h and 1 h, respectively. Take NESEB for example, the first peak concentration was 581.65 ng/mL at 0.5 h and 616.66 ng/mL at 1 h,
respectively. EVO was detectable by HPLC until 12 h for free EVO and 48 h for other four EVO nanoemulsive formulations. The concentration–time data were subjected to compartmental pharmacokinetic analysis by DAS software. The results showed that both NESEB and free EVO could be best described with one-compartment models, while other three EVO nanoemulsive formulations
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Fig. 4. In vivo pharmacokinetic profiles (i.e., plasma concentration–time curves) and bioequivalence evaluation of NESEB and other EVO formulations after oral administration at the same EVO dose of 100 mg/kg. Data presented as mean ± standard deviation (n = 6). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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(NEE, NESE and NEEB) could be dealt with two-compartment models. The main pharmacokinetic parameters of free EVO and its four nanoemulsive formulations are depicted in Fig. 5. The area under concentration (AUC) values of free EVO, NEE, NESE, NEEB and NESEB increased in order. In the case of NESEB, the relative bioavailability (RBA) of NESEB to free EVO was 846.97%. The Cmax and Tmax values of NESEB were 7.54 and 0.31 times that of free EVO, respectively. The 90% confidence intervals of AUC and Cmax of NESEB and free EVO were considerably beyond the scope of 80–125% and 70–143%, and the Tmax values of NESEB and free EVO were significantly different (P < 0.05) when using the Wilcoxon rank sum test to compare them. These results indicated that NESEB and free EVO were definitely not bioequivalent (see Fig. 5 and Table 2). Compared to free EVO, different degrees of beneficial changes in the main pharmacokinetical parameters of the four EVO nanoemulsive systems occurred. Among these EVO nanoemulsive
systems, the one with the highest AUC and Ka values (3.44-fold and 38.5-fold that of free EVO, respectively), accompanying with the lowest Tmax and Cl values (three tenth and two tenth that of free EVO, respectively) was NESEB, the one with the highest MRT value (3.44-fold that of free EVO) was NESE, and the one with the highest Cmax value (8.27-fold that of free EVO, or 8.74% higher than that of NESEB) was NEEB, respectively. Besides, there had been a significant decrease in RBA values of NESEB (850%), NEEB (700%), NESE (600%), NEE (400%), SEVO (200%) (15). Bioequivalence analysis between every two EVO formulation conducted using DAS software further confirmed that NESEB and any other EVO nanoemulsive formulation mentioned above (NEE, NESE and NEEB) were not bioequivalent (see Table 2). Since the AUC value was documented as 349.83 lg h L1 when the free EVO was given intravenously at a dose of 2 mg/kg to the rats [25], the ABA values of EVO formulations given orally could be calculated by Eq. (5), there had been a significant decrease in ABA values of NESEB (40%), NEEB (33%), NESE (28%), NEE (17%), SEVO (8%) and EVO (4%).
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4. Discussion
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It takes a lot of time and effort evaluating the effectiveness and safety before a new biomaterial is finally approved by the national agency for drug administration. Obviously, it may ease pressure on developing new biomaterials to develop suitable system for delivering an insoluble drug if the new application of the biomaterials already officially approved can be explored successfully. Here, a novel nanoemulsive system (i.e., a nano-emulsified supermolecular evodiamine containing brucea javanica oil, NESEB) consisted of only biomaterial officially approved was elaborately designed to deliver an insoluble drug EVO. Elementary configuration of NESEB was described in Fig. 1. (1) The NESEB was essentially a water-in-oil nanoemulsion composed of oil phase (a blend of ethyl oleate and brucea javanica oil at a mass ratio of 2:1), water phase (distilled water), and two-phase interface (containing cremorphor EL 35, polyethylene glycol 400 and SEVO). The high phase volume ratio of oil phase to water phase (9:1, mL:mL) was beneficial to form stable water-in-oil nanoemulsions. The translucent NESEB system was light yellow. When NESEB was diluted with 4 times or 9 times volume of ethyl oleate (oil phase), it remained
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Fig. 5. Pharmacokinetic parameters comparison among NESEB, NEEB, NESE, NEE and free EVO. Data presented as mean ± standard deviation (n = 6). P < 0.05,
P < 0.01.
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J. Hu et al. / European Journal of Pharmaceutics and Biopharmaceutics xxx (2014) xxx–xxx Table 2 Bioequivalence evaluation of NESEB and other EVO formulations after oral administration at the same EVO dose of 100 mg/kg. Formulation 1 and 2
Parameter
90% Confidential interval calculated
P value calculated
Bioequivalence standard
Bioequivalence
NESEB and EVO
AUC Cmax Tmax In all
2340.5–3447.8% 4.9–33.6% – –
– – 0.05 –
No No No No
NESEB and NEE
AUC Cmax Tmax In all
122.1–194.3% 96.9–105.8% – –
– – >0.05 –
80–125% 70–143% >0.05 –
No Yes Yes No
NESEB and NESE
AUC Cmax Tmax In all
89.2–138.2% 16.6–64.5% – –
– – >0.05 –
80–125% 70–143% >0.05 –
No No Yes No
NESEB and NEEB
AUC Cmax Tmax In all
86.6–130.8% 90.4–108.8% – –
No Yes Yes No
Notes: Data presented as mean ± standard deviation (n = 6). 466 467 468 469 470 471 472 473 474 475 476 477 478 479 480 481 482 483 484 485 486 487 488 489 490 491 492 493 494 495 496 497 498 499 500 501 502 503 504 505 506 507 508 509 510 511
translucent and kept one layer, which further suggested NESEB was a water-in-oil nanoemulsion. The average solubilities of EVO in ethyl oleate (oil phase) and cremophor EL 35 (surfactant) were determined by a HPLC method in our preliminary study. They were 50.66 lg/mL and 1968.95 lg/mL, respectively. Cremorphor EL 35 had both hydrophilic and hydrophobic groups and acted as an emulsifier (or a surfactant). Cremorphor EL 35 was a common excipient for injectable and oral use. In the material safety data sheet of BASF Corporation, it was recorded that the cremophor EL (CEL) was practically nontoxic. Polyethylene glycol 400 (soluble in water and many organic solvent such as ethyl oleate) acted as a coemulsifier or a cosurfactant. SEVO having both hydrophilic and hydrophobic groups might exist in the interface in a similar way to an amphiphilic emulsifier. (2) The fact that SEVO was formed by combining EVO and soybean phospholipid (Lipoid S 75, the phosphatide content was above 70%, w/w) through noncovalent bonding had been previously investigated by differential scanning calorimetry, ultraviolet spectroscopy, Fourier transformed infrared spectroscopy, H NMR spectroscopy, matrixassisted laser desorption/ionization time-of-flight spectroscopy [15]. EVO could be well complexed with Lipoid S 75, as evidenced by high average complexation rate of SEVO (above 95%). The complexation rate of SEVO was determined by the following formula: Complexation rate = m2/m1 100% = (m1 m3)/m1 100%, where ‘‘m1’’ is the total content of EVO added, ‘‘m2’’ is the content of EVO ‘‘present as a complex’’, and ‘‘m3’’ is the free EVO or non-complexed EVO. Lipoid S 75 was already approved to be used for oral and injectable administration by State Food and Drug Administration. Moreover, Lipoid S 75 was a low-cost pharmaceutical excipient which should be more suitable for industrial production from a cost perspective. The price of Lipoid S 75 (the phosphatide content was above 70%, w/w) was about 1/30 of egg phospholipid (the phosphatide content was above 98%, w/w). The conductivity of NESEB was much higher than that of free EVO in ethyl oleate (oil phase) and a litter higher than that of CNE, respectively. However, after dilution 5 times with ethyl oleate, the conductivity of NESEB increased to more than two times of NEE. The difference between the conductivities of NESEB and other EVO formulations indicated the different existing state of EVO. The reasons why NESEB was diluted with ethyl oleate before determination were listed as follows: NESEB was a water-in-oil nanoemulsive system embedding an evodiamine-phospholipid nanocomplex. The external phase was oil phase, i.e., ethyl oleate. When the NESEB (the translucent NESEB system was light yellow–green) was added with 4 times volume of ethyl oleate (oil phase), the mixture was still translucent.
The mean size and zeta potential of NESEB were 613.3 nm and 4.46 mV, respectively. The reason why the determined size values of the translucent NESEB systems were so big was not clear. On the other hand, there was no report on the size diameter of a waterin-oil nanoemulsive system used for oral delivery of hydrophobic drug so far, since not even a water-in-oil nanoemulsive system has been developed for oral delivery of hydrophobic drug so far. As far as we knew, only a few research papers about water-in-oil nanoemulsions were published, among them, two research papers mentioned the determination of the particle size of water-in-oil nanoemulsions. They were documented as follows: (1) there was one report on the size diameter of a water-in-oil nanoemulsive system used for intravesical delivery of hydrophobic drug (cisplatin) [26], the range of the mean size was 30–90 nm. The mean vesicle size of the nanoemulsion was measured by photon correlation spectroscopy (Malvern Nano ZSÒ 90, Worcestershire, UK) using a helium-neon laser with a wavelength of 633 nm. This formulation were diluted 5-fold with soybean oil (oil phase of formulation) before the measurement. (2) There was one report on the size diameter of a water-in-oil nanoemulsive system used for transdermal delivery of caffeine (a hydrophobic drug) [27], the mean droplet size of the caffeine nanoemulsion was found in the range of 20.14–105.25 nm. Droplet size distribution of the nanoemulsion was determined by photon correlation spectroscopy (PCS) using a Zeta sizer 1000 HS (Malvern Instruments, UK). The morphologies of NESEB were tried to be investigated by a transmission electron microscopy (TEM H-7500, Hitachi, Japan) and a scanning electron microscopy (E-1010, Hitachi, Japan), respectively, but the electron microscopy results of NEEPN were unavailable because the external phase was oil. As far as we knew, this was the first time to load a drug– phospholipid nanocomplex into a nanoemulsive system. The potential advantages of supermolecular lipophilic drug loaded nanoemulsive systems (containing synergistic bioactive oil) over free drug or conventional nanoemulsion might include agreeable appearance, higher bioavailability, biocompatibility, efficacy, thermodynamic and kinetic stability, the amazing biological features of the nanosystem were investigated in our further rat absorptive and pharmacokinetic studies. Compared to free EVO, the gastric absorption of EVO in nanoemulsive systems, showed the following increases: NESEB increased by 240%, NEE, NESE and NEEB increased by 20–100%. The increase of gastric absorption contributed to increase the oral bioavailability and cure gastric cancer. Different from stomach, intestinal tract was the major site for EVO absorption, regardless of the EVO delivery system type. Among free EVO and EVO
Please cite this article in press as: J. Hu et al., Supermolecular evodiamine loaded water-in-oil nanoemulsions: Enhanced physicochemical and biological characteristics, Eur. J. Pharm. Biopharm. (2014), http://dx.doi.org/10.1016/j.ejpb.2014.06.007
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nanoemulsive systems mentioned above, NESEB had the highest Ka (or Peff) values in almost every corresponding intestinal segment, thus it was undoubtedly the best intestinal absorption enhancement nanosystem. For NESEB, the absorption rate and permeability in duodenal and jejunal segments were almost the same, while they were higher than that in ileac and colonic segments. Other EVO nanoemulsive systems could improve the absorption rate and permeability of EVO, although to different extents. Particularly, compared to NEE, NESE greatly improved the absorption of EVO in colon, while NEEB in jejunum and colon (the improved colonic absorption might be favorable toward treating colorectal tumor). In short, as shown in Figs. 2 and 3, the percentage absorption of EVO had been obviously improved by nanoemulsifying EVO to form NEE; moreover, the effect of increasing absorption could be further enhanced by using supermolecular technique to form NESE alone or adding brucea javanica oil to form NEEB. As expected, the most effective way to enhance the percentage absorption of NEE was simultaneously using supermolecular technique and adding brucea javanica oil to form NESEB. In short, NESEB was highly favorable for EVO absorption, as evidenced by the presence of high Ka, Peff and PA values. The drug absorption rate referred to the drug absorption over a particular period of time. Factors affecting absorption included factors related to drugs (lipid water solubility, molecular size, particle size, degree of ionization, physical forms, chemical nature, dosage forms, formulation, concentration, etc.) and factors related to body (area of absorptive surface, vascularity, pH, presence of other substances, gastrointestinal motility, functional integrity of absorptive surface, diseases, etc.). Obviously, factors affecting EVO absorption in NESEB were not always the same in different gastrointestinal tracts, so there was significant difference of absorption rate for NESEB in different gastrointestinal tracts. The effective permeability was one of the factors affecting drug absorption. The effective permeability was mainly relative to the drug molecular, liposolubility, permeability glycoprotein (P-gp) and cytochrome P4503A (CYP3A). The effective permeability values of NESEB in different gastrointestinal tracts were the synthetic effects of all factors affecting the effective permeability, so it was reasonable that there was significant difference of effective permeability for NESEB in different gastrointestinal tracts.
Enhanced absorption of NESEB might ascribe to the following reasons: (1) solubilization of EVO by supermolecular state, mixed oil (or co-solvent), and nanoemulsive form; (2) protection from enzymatic oxidation (EVO was the substrate of CYP3A enzyme) [28] or p-glycoprotein (P-gp) mediated EVO efflux (lipophilic EVO was a P-gp substrate) by entrapping EVO inside the delivery nanosystem containing components such as the surfactants and oils which could act as P-gp/CYP450 inhibitors [29]; (3) high dispersibility of EVO in phospholipid nanocomplex and nanoemulsive form, surfactant-induced membrane fluidity and thus permeability improvement; (4) the occurrence of intestinal lymphatic transport [30,31]. As shown in Fig. 4, the pharmacokinetic behavior of NESEB was the most desirable for delivering EVO among free EVO and the four nanoemulsive formulations (NESEB, NEEB, NESE and NEE) when the same doses (equivalent to 100 mg/kg) of EVO were orally given to rats separately. According to experiments and data analysis (DAS software), one-compartment models best described the time course of the EVO concentration when NESEB was orally administered, indicating that NESEB could rapidly distribute to tissues; while two-compartment models suitable for other three EVO nanoemulsive formulations (NEE, NESE and NEEB), indicating that drug in these systems exhibited a slow equilibration with peripheral tissues. The phenomenon that the drug distribution of NESEB was faster in comparison with other nanoemulsive delivery systems might have some relevance to the existing state of EVO in different nanoemulsive formulations and need further study. Compared to free EVO, the smaller Tmax value of NESEB indicated a faster rate and a larger degree of gastrointestinal absorption; usually it also indicated a faster onset of action for patients in clinical practice. Moreover, the mean residence time (MRT) of NESEB rose by 2.73-fold from free EVO, while the clearance (Cl) dropped by 77.14%. NESEB demonstrated prolonged circulation in the bloodstream, which was beneficial to increase drug action time as well as effectiveness. The in vivo absorption rate constant (Ka) of NESEB was 38.51 times that of free EVO, suggesting that NESEB had better absorption than the latter. This situation was in accordance with the results obtained from the experiments of in situ gastrointestinal absorption.
Fig. 6. The effects of gastrointestinal segment and drug delivery system on the EVO absorption and pharmacokinetic behavior. Data presented as mean ± standard deviation (n = 6).
Please cite this article in press as: J. Hu et al., Supermolecular evodiamine loaded water-in-oil nanoemulsions: Enhanced physicochemical and biological characteristics, Eur. J. Pharm. Biopharm. (2014), http://dx.doi.org/10.1016/j.ejpb.2014.06.007
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The results (comparison of AUC, Cmax, Tmax between every two EVO delivery systems) obtained from the statistical bioequivalence analysis indicated that NESEB and free EVO (or NEE, NESE, NEEB) were not bioequivalent. The four EVO nanoemulsive systems reporting an increase in orders for the relative bioavailability over free EVO were: NEE, NESE, NEEB and NESEB. Favorably changes to the pharmacokinetic behavior of EVO took place in NESEB, so did in other three EVO nanoemulsive systems (NEE, NESE and NEEB) although to different extents. NESEB had the highest AUC and Ka values, besides the lowest Tmax and Cl values, while NESE had the highest MRT value and NEEB had the highest Cmax value. On the whole, NESEB was able to deliver EVO more effectively, although other EVO nanoemulsive systems also had some advantages for EVO delivery. What’s more, based on the above analysis, it was deduced that favorable pharmacokinetic consequence of NESEB might mainly be ascribed to the enhanced gastrointestinal absorption and partly to the decreased clearance from the systemic circulation in vivo (see Fig. 6).
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A nano-emulsified supermolecular evodiamine coexisting with brucea javanica oil (NESEB), only composed of biomaterials approved by national agency for drug administration, i.e., a blend oil (ethyl oleate and brucea javanica oil), a surfactant (cremorphor EL 35), a cosurfactant (polyethylene glycol 400), a phospholipid (main constituent to form supermolecular EVO-phospholipid nanocomplex), and the distilled water, was designed and produced. It was found that NESEB markedly improved the oral bioavailability of EVO, which was likely due to the increased gastrointestinal absorption. NESEB might be a preferred alternative nanosystem for delivering evodiamine effectively via oral route, in comparison with free EVO and other three EVO nanoemulsive systems (conventional nano-emulsified evodiamine, nano-emulsified supermolecular evodiamine and nano-emulsified evodiamine containing brucea javanica oil). Our study supported our hypothesis that although it was unavailable in either clinical practice or research field, a novel nanosystem only consisted of biomaterials approved by national agency for drug administration and fabricated by combining phytosome nanotechnology with nanoemulsifying technology, in addition to using synergistic plant essential oil as a basic composition, was able to exhibit greatly improved oral bioavailability and deliver an insoluble natural drug more effectively. The development of such a novel nanosystem represented a valuable tactic in new medical application of common used biomaterials (or biomaterials approved by national agency for drug administration) for effective delivery of insoluble antitumor natural drugs.
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Acknowledgements
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This research was partially supported by grants from Chongqing Natural Science Foundation (CSCT2012JJB10027), and Chongqing Education Committee Fund (the excellent university personnel financial aid plan, KJ120321).
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References
638 639 640 641 642 643 644 645 646 647 648 649 650 651 652 653 654
658 659 660 661 662 663 664 665 666 667 668 669 670 671 672 673 674 675 676 677 678 679 680 681 682
[4] [5]
[6]
[7]
[8]
[9] [10]
[11]
[12]
[13]
[14]
[15]
[16]
[17]
[18]
[19]
[20]
[21]
[22]
[23]
[24]
[25] 686 687
[26]
[27] [28]
690 691 692 693 694 695 696 697 698
[1] L. De Petrocellis, A. Schiano Moriello, G. Fontana, A. Sacchetti, D. Passarella, G. Appendino, V. Di Marzo, Effect of chirality and lipophilicity in the functional activity of evodiamine and its analogues at TRPV1 channels, Br. J. Pharmacol. (2013), http://dx.doi.org/10.1111/bph.12320. [2] J.F. Liao, W.F. Chiou, Y.C. Shen, G.J. Wang, C.F. Chen, Anti-inflammatory and anti-infectious effects of Evodia rutaecarpa (Wuzhuyu) and its major bioactive components, Chin. Med. 6 (2011) 6. [3] A.J. Liu, S.H. Wang, K.C. Chen, H.P. Kuei, Y.L. Shih, S.Y. Hou, W.T. Chiu, S.H. Hsiao, C.M. Shih, Evodiamine, a plant alkaloid, induces calcium/JNK-mediated
[29]
[30] [31]
9
autophagy and calcium/mitochondria-mediated apoptosis in human glioblastoma cells, Chem. Biol. Interact. 205 (2013) 20–28. H. Yu, H. Jin, W. Gong, Z. Wang, H. Liang, Pharmacological actions of multitarget-directed evodiamine, Molecules 18 (2013) 1826–1843. M.A. Alam, F.I. Al-Jenoobi, A.M. Al-Mohizea, Commercially bioavailable proprietary technologies and their marketed products, Drug Discov. Today 18 (2013) 936–949. F. Aqil, R. Munagala, J. Jeyabalan, M.V. Vadhanam, Bioavailability of phytochemicals and its enhancement by drug delivery systems, Cancer Lett. (2013) (pii: S0304-3835(13)00158-4). D. He, P. Deng, L. Yang, Q. Tan, J. Liu, M. Yang, J. Zhang, Molecular encapsulation of rifampicin as an inclusion complex of hydroxypropyl-b-cyclodextrin: design; characterization and in vitro dissolution, Colloids Surf. B: Biointerfaces 103C (2012) 580–585. Q. Tan, J. Wu, Y. Li, H. Mei, C. Zhao, J. Zhang, A supermolecular curcumin for enhanced antiproliferative and proapoptotic activities: molecular characteristics, computer modeling and in vivo pharmacokinetics, Nanotechnology 24 (2013) 03510. T. Kurita, Y. Makino, Novel curcumin oral delivery systems, Anticancer Res. 33 (2013) 2807–2821. D.J. McClements, Nanoemulsion-based oral delivery systems for lipophilic bioactive components: nutraceuticals and pharmaceuticals, Ther. Deliv. 4 (2013) 841–857. S. Ganta, D. Deshpande, A. Korde, M. Amiji, A review of multifunctional nanoemulsion systems to overcome oral and CNS drug delivery barriers, Mol. Membr. Biol. 27 (2010) 260–273. P.M. Kidd, Bioavailability and activity of phytosome complexes from botanical polyphenols: the silymarin, curcumin, green tea, and grape seed extracts, Altern. Med. Rev. 14 (2009) 226–246. S. Kotta, A.W. Khan, K. Pramod, S.H. Ansari, R.K. Sharma, J. Ali, Exploring oral nanoemulsions for bioavailability enhancement of poorly water-soluble drugs, Exp. Opin. Drug Deliv. 9 (2012) 585–598. A. Semalty, M. Semalty, M.S. Rawat, F. Franceschi, Supramolecular phospholipids–polyphenolics interactions: the phytosome strategy to improve the bioavailability of phytochemicals, Fitoterapia 81 (2010) 306–314. Q. Tan, S. Liu, X. Chen, M. Wu, H. Wang, H. Yin, D. He, H. Xiong, J. Zhang, Design and evaluation of a novel evodiamine-phospholipid complex for improved oral bioavailability, AAPS PharmSciTech. 13 (2012) 534–547. Y. Lv, G. Ding, J. Zhai, Y. Guo, G. Nie, L. Xu, A superparamagnetic Fe3O4-loaded polymeric nanocarrier for targeted delivery of evodiamine with enhanced antitumor efficacy, Colloids Surf. B: Biointerfaces 10 (2013) 411–418. Y.T. Zhang, Z.B. Huang, S.J. Zhang, J.H. Zhao, Z. Wang, Y. Liu, N.P. Feng, In vitro cellular uptake of evodiamine and rutaecarpine using a microemulsion, Int. J. Nanomed. 7 (2012) 2465–2472. Q. Peng, Z.R. Zhang, X. Sun, J. Zuo, D. Zhao, T. Gong, Mechanisms of phospholipid complex loaded nanoparticles enhancing the oral bioavailability, Mol. Pharm. 7 (2010) 565–575. S. Wang, M. Sun, Q. Ping, Enhancing effect of Labrafac Lipophile WL 1349 on oral bioavailability of hydroxysafflor yellow A in rats, Int. J. Pharm. 358 (2008) 198–204. Y.L. Nie, K.X. Liu, X.Y. Mao, Y.L. Li, J. Li, M.M. Zhang, Effect of injection of brucea javanica oil emulsion plus chemoradiotherapy for lung cancer: a review of clinical evidence, J. Evid. Based Med. 5 (2012) 216–225. Y.L. Yu, Y. Lu, X. Tang, F.D. Cui, Formulation, preparation and evaluation of an intravenous emulsion containing brucea javanica oil and coix seed oil for antitumor application, Biol. Pharm. Bull. 31 (2008) 673–680. L. Perioli, P. Mutascio, C. Pagano, Influence of the nanocomposite MgAl-HTlc on gastric absorption of drugs: in vitro and ex vivo studies, Pharm. Res. 30 (2013) 156–166. Q. Tan, N. Hu, G. Liu, H. Yin, L. Zhang, H. Wang, L.Y. Lu, J.Q. Zhang, Role of a novel pyridostigmine bromide-phospholipid nanocomplex in improving oral bioavailability, Arch. Pharm. Res. 35 (2012) 499–508. J.M. Zhang, W. Liao, Y.X. He, Y. He, D. Yan, C.M. Fu, Study on intestinal absorption and pharmacokinetic characterization of diester diterpenoid alkaloids in precipitation derived from Fuzi-Gancao herb-pair decoction for its potential interaction mechanism investigation, J. Ethnopharmacol. 147 (2013) 128–135. K.F. Jeng, Y.H. Lin, L.C. Lin, C.J. Chou, T.H. Tsai, C.F. Chen, High-performance liquid chromatographic determination of evodiamine in rat plasma: application to pharmacokinetic studies, J. Chromatogr. B: Biomed. Appl. 668 (1995) 343–345. T.L. Hwang, C.L. Fang, C.H. Chen, J.Y. Fang, Permeation enhancer-containing water-in-oil nanoemulsions as carriers for intravesical cisplatin delivery, Pharm. Res. 26 (2009) 2314–2323. F. Shakeel, W. Ramadan, Transdermal delivery of anticancer drug caffeine from water-in-oil nanoemulsions, Colloids Surf. B: Biointerfaces 75 (2010) 356–362. H.Z. Sun, Z.Z. Fang, Y.F. Cao, X.Y. Sun, M. Hong, Investigation of the in vitro metabolism of evodiamine: characterization of metabolites and involved cytochrome p450 isoforms, Phytother. Res. 27 (2013) 705–712. A.O. Nornoo, D.W. Osborne, D.S. Chow, Cremophor-free intravenous microemulsions for paclitaxel I: formulation, cytotoxicity and hemolysis, Int. J. Pharm. 349 (2008) 108–116. Y. Lu, J. Qi, W.Wu. Absorption, Disposition and pharmacokinetics of nanoemulsions, Curr. Drug Metab. 13 (2012) 396–417. J.Q. Zhang, J. Liu, X.L. Li, B.R. Jasti, Preparation and characterization of solid lipid nanoparticles containing silibinin, Drug Deliv. 14 (2007) 381–387.
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