Cinnamaldehyde in a Novel Intravenous Submicrometer Emulsion

Jun 29, 2015 - All of the pharmacokinetic data of CA were processed by the DAS 2.0 software supplied by the Anhui Provincial Center for Drug Clinical ...
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Cinnamaldehyde in a Novel Intravenous Submicrometer Emulsion: Pharmacokinetics, Tissue Distribution, Antitumor Efficacy, and Toxicity Hang Zhao,†,‡ Jiani Yuan,†,‡ Qian Yang,†,‡ Yanhua Xie,†,‡ Wei Cao,*,†,‡ and Siwang Wang*,†,‡ †

Department of Natural Medicine & Institute of Materia Medica, School of Pharmacy, Fourth Military Medical University, Xi’an 710032, China ‡ The Cultivation Project of Collaborative Innovation Center for Chinese Medicine in QinBa Mountains, Xi’an 710032, China S Supporting Information *

ABSTRACT: The purpose of our research is to find a new lipid emulsion to deliver a low water-soluble compound, cinnamaldehyde (CA). Its characteristics, pharmacokinetics, antitumor efficacy, and toxicity were evaluated. The mean particle size, zeta potential, and encapsulation efficiency of the submicromemter emulsion of CA (SME-CA) were 130 ± 5.92 nm, −25.7 ± 6.00 mV, and 99.5 ± 0.25%, respectively. The area under the curve from 0 h to termination time (AUC0−t) of SME-CA showed a significantly higher value than that of CA (589 ± 59.2 vs 375 ± 83.5 ng h/L, P < 0.01). Tissue distribution study showed various changes; among them, a 27% higher concentration was found in brain tissue when using SME-CA at 15 min after administration. For the efficacy evaluation, SME-CA exhibited 8- and 11-fold antitumor activity in the depression of HeLa and A549 cell lines with the IC50 decreasing to 0.003 and 0.001 mmol/L, respectively. The LD50 values of CA and SME-CA in mice were 74.8 and 125 mg/kg, suggesting increased safety from the new formulation. The new formulation exhibited lower toxicity, higher antitumor activity, and a more satisfactory pharmacokinetic property, which displayed great potential for future pharmacological application. KEYWORDS: cinnamaldehyde, pharmacokinetics, submicrometer emulsion, intravenous injection, antitumor efficacy



INTRODUCTION Cinnamaldehyde (CA; 3-phenylprop-2-enal), a primary ingredient extracted from Cortex Cinnamomi, has been demonstrated to possess several biological and pharmacological activities such as antimicrobial,1,2 anticarcinogenic,3−5 antithrombic,6 and antiinflammatory activities.7,8 Our group also found that CA had obvious therapeutic effects on viral myocarditis, thrombus, and myocardial ischemia.9−11 A large amount of CA is also used in the food industry; approximately 500,000 kg/year of CA was used as a flavoring or antimicrobial agent in the food industry.12 Although CA has many potential effects in the pharmacological area and great consumption in the food area, the application of CA as a potential drug is at a standstill for various reasons including its low oral bioavailability and poor water solubility. In addition, the aldehyde part of CA, which is mainly responsible for its pharmacological activity, is easily oxidized when it is exposed in the blood.13 Our previous study also showed that CA maintained a very low concentration in plasma by the oral route, which might restrict its application.14 In this case, we believe intravenous injection of CA is an alternative way to enhance the blood concentration and reach target organs rapidly without its first passing through hepatic metabolism. However, invasiveness and potential toxicity of the intravenous route can be an obstacle to the development of an intravenous formulation. Thus, our group aimed to develop an intravenous formulation with low toxicity and superior efficacy to promote the application of CA. During the past decades, several drug delivery systems, such as nanoparticle, liposome, microencapsulation, and formulation © 2015 American Chemical Society

of complexes with cyclodextrins, were utilized to optimize the solubility, pharmacological activity, and bioavailability of CA.15−19 Among them submicrometer emulsions attracted our attention due to their several advantages for use as intravenous delivery systems in this study. The emulsion system can control the oxidation of CA and lower venous irritation because the emulsion interface avoids direct contact with the blood and vascular wall.20,21 Furthermore, the small droplet of drug provides a large interfacial surface area with target cells, which results in the enhancement of pharmacological activities such as anticancer effect.22 Thus, the emulsion system is a preferred drug delivery system to control the release of CA in the blood, reduce the toxicity of CA, and enhance the pharmacological effect. However, the complex components and complicated production process of submicrometer emulsions of CA (SME-CA) remained unknown. Therefore, it is highly desired to develop a convenient and stable process for making SME-CA. In the present study, we prepared a novel intravenous submicrometer emulsion with lower toxicity and higher antitumor efficacy. The emulsion delivery system not only enhanced the solubility and absorption but is also suited for future mass production.23 Several tumor cells were chosen to verify the anticancer effect of the optimal formulation. LD50 Received: Revised: Accepted: Published: 6386

April 14, 2015 June 14, 2015 June 29, 2015 June 29, 2015 DOI: 10.1021/acs.jafc.5b01883 J. Agric. Food Chem. 2015, 63, 6386−6392

Article

Journal of Agricultural and Food Chemistry

and weighed and then homogenized in a 2-fold weight saline solution (0.9%) for all tissue samples by a T10 Basic homogenizer (Staufen, Germany). Then the obtained homogenates were stored at −80 °C until analysis. Sample Preparation. A liquid−liquid preparation method was utilized to extract target compounds and eliminate the interference of endogenous protein. We found that acetonitrile enhanced the sensitivity and selectivity, especially in minimizing endogenous interference and improving extraction recovery. Consequently, we chose acetonitrile as the optimized solvent for sample preparation. Fifty microliters of propiophenone as internal standard solution (2500 ng/mL) and 500 μL acetonitrile were sequentially added in every 200 μL of homogenate and plasma samples. Then, the solution was thoroughly vortex-mixed for 120 s. After centrifugation at 12000g for 10 min, 1 μL of supernatant was injected into GC-MS for analysis. GC-MS Analysis. Chromatography was performed on a Trace ISQ Ultra (Thermo Fisher Scientific, Waltham, MA, USA) system combined with a Triplus atuosampler and injector. A DB-5 ms capillary column (30 m × 0.25 mm, 0.25 μm thickness, Agilent Technologies, Santa Clara, CA, USA) was used for separation. The initial oven temperature was set at 50 °C, held for 1 min, then increased to 160 °C at 10 °C/min for 1 min, then increased to 280 at 20 °C/min, and held at the final temperature for 1 min. High-purity helium was used as carrier gas at a constant flow rate of 1.0 mL/min. The transfer line and ion source of the MS system were both operated at 250 °C. MS analysis was operated in the selected ion monitoring (SIM) mode with the ionization energy being 70 eV. Quantifying ion masses (m/z 131, 105, and 92) were selected for simultaneously investigating IS, CA, methyl cinnamate, and cinnamyl alcohol. The mass spectra of all analytes were analyzed by the NIST library (Shimadzu, Kyoto, Japan) and are shown in the Supporting Information. Pharmacokinetics and Statistical Analysis. All of the pharmacokinetic data of CA were processed by the DAS 2.0 software supplied by the Anhui Provincial Center for Drug Clinical Evaluation. The different pharmacokinetic features of CA and SME-CA were assessed using the following parameters: area under the curve to termination time (AUC0−t), mean residence time (MRT0−t), half-life (t1/2), time at maximum plasma concentration (tmax), maximum plasma concentration (Cmax), plasma clearance (CLz), and volume of distribution (Vz). The data are presented as the mean ± SD, and Student’s t test was used to analyze the difference between groups. A P value of