Novel Composite Drug Delivery System for Honokiol Delivery: Self

Jul 2, 2009 - This study aims to develop a novel composite drug delivery system (CDDS) for hydrophobic honokiol delivery: honokiol loaded micelles in ...
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J. Phys. Chem. B 2009, 113, 10183–10188

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Novel Composite Drug Delivery System for Honokiol Delivery: Self-Assembled Poly(ethylene glycol)-Poly(ε-caprolactone)-Poly(ethylene glycol) Micelles in Thermosensitive Poly(ethylene glycol)-Poly(ε-caprolactone)-Poly(ethylene glycol) Hydrogel ChangYang Gong, Shuai Shi, XiuHong Wang, YuJun Wang, ShaoZhi Fu, PengWei Dong, LiJuan Chen, Xia Zhao, YuQuan Wei, and ZhiYong Qian* State Key Laboratory of Biotherapy, West China Hospital, and School of Life Sciences, Sichuan UniVersity, Chengdu, 610041, P. R. China ReceiVed: March 25, 2009; ReVised Manuscript ReceiVed: May 24, 2009

This study aims to develop a novel composite drug delivery system (CDDS) for hydrophobic honokiol delivery: honokiol loaded micelles in thermosensitive hydrogel (honokiol micelles/hydrogel) based on biodegradable poly(ethylene glycol)-poly(ε-caprolactone)-poly(ethylene glycol) (PEG-PCL-PEG, PECE) copolymers. In our work, we found that PECE copolymers with different molecular weight and PEG/PCL ratios could be administired to form micelles or thermosensitive hydrogel, respectively. Honokiol loaded PECE micelles (honokiol micelles) were prepared by self-assembly of biodegradable PECE copolymer (PEG5000-PCL5000PEG5000) triggered by its amphiphilic characteristic assisted by ultrasonication without using any organic solvents and surfactants. Meanwhile, biodegradable and injectable thermosensitive PECE hydrogel (PEG550PCL2400-PEG550) with a lower sol-gel transition temperature at around physiological temperature was also prepared successfully. Furthermore, the obtained honokiol micelles/hydrogel CDDS was a free-flowing sol at ambient temperature and became a nonflowing gel at body temperature. The cytotoxicity results showed that the CDDS was a safe carrier and the encapsulated honokiol retained its potent antitumor effect. In addition, the in vitro release profile demonstrated a significant difference between rapid release of free honokiol and much slower and sustained release of honokiol micelles/hydrogel. The results suggested that the CDDS might have great potential applications in cancer chemotherapy. Introduction Cancer is one of the most severe diseases in the world, and it causes millions of death each year. More than 1 million new cancer cases and 500 000 deaths from cancer are projected to occur in 2008 in the U.S.A.1 Honokiol is an active component isolated and purified from the traditional Chinese herb magnolia which has been demonstrated as a promising chemotherapy agent.2-10 Although honokiol shows great antitumor activity, the high hydrophobicity makes its formulation difficult, if not impossible, and therefore greatly restrains its further application in cancer chemotherapy. In our previous contributions, honokiol loaded poly(ε-caprolactone)-poly(ethylene glycol)-poly(ε-caprolactone) (PCLPEG-PCL, PCEC) nanoparticles (honokiol nanoparticles) were prepared by emulsion solvent evaporation method to enhance the solubility of honokiol.11 The obtained honokiol nanoparticles could be well dispersed in aqueous solutions, but organic solvent (ethyl acetate) and surfactant (Pluronic F127) were used, both of which were usually not desired. Also, thermosensitive Pluronic F127 hydrogel was employed as in situ honokiol delivery system by our group before.11 However, Pluronic copolymer formed a fast-eroding gel and could not persist longer than a few hours; therefore, all the encapsulated drug would be released in relatively short period, which could not meet the requirements of sustained release. Furthermore, Pluronic was found to induce the toxic enhancement of plasma cholesterol and triglycerol because * To whom correspondence should be addressed. Tel.: 86-28-85164063. Fax: 86-28-85164060. E-mail: [email protected].

it was nonbiodegradable and could be accumulated in the body.12 Thus, a more suitable drug delivery system is extremely important for the further clinical application of honokiol. In this study, we aim to develop a novel composite drug delivery system (CDDS) for honokiol delivery: honokiol loaded micelles in thermosensitive hydrogel (honokiol micelles/hydrogel) based on biodegradable poly(ethylene glycol)-poly(ε-caprolactone)-poly(ethylene glycol) (PEGPCL-PEG, PECE) copolymers. It is very interesting that by simply varying the molecular weight and PEG/PCL ratio of the PECE copolymers, they could be administrated to form micelles or thermosensitive hydrogel respectively. Biodegradable honokiol loaded PECE micelles (honokiol micelles) were prepared from self-assembly of triblock PECE copolymer (PEG5000-PCL5000-PEG5000) assisted by ultrasonication without using any organic solvents and surfactants. In addition, the biodegradable and injectable thermosensitive hydrogel based on PECE copolymer (PEG550-PCL2400-PEG550) was also fabricated successfully.13-19 Furthermore, a novel CDDS, honokiol micelles in hydrogel, was prepared by encapsulating honokiol micelles in thermosensitive hydrogel to form a homogeneous and stable solution. The obtained CDDS is an injectable flowing solution (sol) at low temperature and forms a nonflowing gel at body temperature working as a sustained drug delivery depot in vivo. Due to the absence of any organic solvent and surfactants, biodegradability, and thermosensitivity, the CDDS, honokiol micelles/hydrogel, might have great potential clinical application as a novel dosage form.

10.1021/jp902697d CCC: $40.75  2009 American Chemical Society Published on Web 07/02/2009

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Experimental Section Materials. Poly(ethylene glycol) methyl ether (MPEG, Mn ) 550 and 5000, Fluka, U.S.A.), ε-caprolactone (ε-CL, Alfa Aesar, U.S.A.), stannous octoate (Sn(Oct)2, Sigma, U.S.A.), hexamethylene diisocyanate (HMDI, Aldrich, U.S.A.), Roswell Park Memorial Institute (RPMI) 1640 medium (Sigma, U.S.A.), and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT, Sigma, U.S.A.) were used without further purification. Honokiol were isolated and purified in our lab. All the materials used in this article were analytic reagent (AR) grade and used as received. Synthesis and Characterization of PECE Copolymer. PECE copolymers were synthesized and purified as reported previously.13,14 Briefly, PEG-PCL diblock copolymers were prepared by ring-opening polymerization of ε-CL initiated by MPEG using stannous octoate as catalyst; PEG-PCL-PEG triblock copolymers were synthesized by coupling PEG-PCL diblock copolymers using HMDI as the coupling agent.13-15 The obtained PECE copolymers were characterized by Fourier transform infrared (FTIR; NICOLET 200SXV, Nicolet, U.S.A.), 1 H nuclear magnetic resonance (NMR; Varian 400 spectrometer, Varian, U.S.A.), and gel permeation chromatography (GPC; Agilent 110 HPLC, U.S.A.). In this paper, the copolymers were denoted as PEGYPCLX-PEGY, where X and Y represented the number average molecular weight (Mn) of the PCL and PEG blocks, respectively. Preparation and Characterization of Blank or Honokiol Micelles. Calculated amount of PECE copolymer (PEG5000PCL5000-PEG5000) was dissolved in 1 mL of distilled water at 50 °C, and a few minutes later, thermal induced self-assembled blank micelles were obtained. Honokiol micelles were prepared by direct dissolution method assisted by ultrasonication. As a model drug, honokiol was added into a blank micelles solution at 37 °C under ultrasonication (JY92-2D, Ningbo Scientz Biotechnology Co., China). Thirty minutes later, the suspension was ultracentrifuged and filtered with a syringe filter (pore size: 220 nm) (Millex-LG, Millipore Co., U.S.A.) to remove the insoluble drugs. Then, the blank or honokiol micelles were cooled to room temperature for future application and characterization. The particle size and zeta potential of prepared blank or honokiol micelles were determined by a Malvern Nano-ZS 90 laser particle size analyzer after equilibration for 10 min. All results were the means of three test runs, and all data were expressed as the mean ( standard deviation (SD). The morphological characteristics of honokiol micelles were examined by transmission electron microscope (TEM, H-6009IV, Hitachi, Japan). Honokiol micelles were diluted with distilled water and placed on a copper grid covered with nitrocellulose. The sample was negatively stained with phosphotungstic acid and dried at room temperature. High Performance Liquid Chromatography (HPLC). The concentration of honokiol was determined by an HPLC Instrument (Waters Alliance 2695), and the sample was diluted before measurement. The solvent delivery system was equipped with a column heater and a plus autosampler. Detection was taken on a Waters 2996 detector. Chromatographic separations were performed on a reversed phase C18 column (4.6 mm × 150 mm, 5µm, Sunfire Analysis column). And, the colume temperature was kept at 28 °C. Acetonitrile/water (60/40, v/v) was used as eluent at a flow rate of 1 mL/min. The standard curve equation is H ) 89 700X - 506 (H area of peak; X concentration of honokiol), and the correlation coefficient is 0.999 982.

Gong et al. Incorporating Honokiol Micelles into PECE Hydrogel. First, PECE copolymers (PEG550-PCL2400-PEG550) was welldissolved in water at certain temperature and cooled to 4 °C to form a solution (sol). Then, honokiol micelles with different concentrations were mixed into the hydrogel to form a homogeneous solution. Sol-Gel-Sol Phase Transition Behavior Study. The thermosensitive sol-gel-sol phase transition of honokiol micelles/ hydrogel was studied using the test tube-inverting method with a 4 mL tightly screw-capped vial with inner diameter of 10 mm.20,21 The sol-gel-sol transition was visually observed by inverting the vials, and conditions of gel and solution (sol) were defined as “no flow” and “flow” in 1 min, respectively. The volume of the hydrogel samples containing different amount of honokiol micelles was kept at 1 mL in total regardless of the micelles content. The hydrogel samples were slowly heated at a heating rate of 1 °C/min, from 4 °C to the temperature when precipitation occurred. Cell Culture and Cell Viability Assay. L929 cells were purchased from the American type Culture Collection (ATCC; Rockville, MD) and grew in RPMI 1640 supplement with 10% fetal bovine serum (FBS). The cell culture was maintained in a 37 °C incubator with a humidified 5% CO2 atmosphere. The cells were plated at a density of 1 × 104 cells per well in 100 µL of medium in 96-well plates and grown for 48 h. The cells were then exposed to a series of blank micelles or blank hydrogels at different concentrations for 48 h, respectively. Then, the viability of cells was measured using the methylthiazoletetrazolium (MTT) method. Briefly, the mean percentage of cell survival relative to that of untreated cells was estimated from data of six individual experiments, and all data were expressed as the mean ( SD. In vitro Drug Release Behavior. In vitro release behavior of honokiol from honokiol micelles or honokiol micelles/ hydrogel was determined by a modified dialysis method as follows. A 500 µL portion of honokiol micelles solution or honokiol micelles/hydrogel solution (30 wt %) was placed in a dialysis tube (molecular mass cutoff is 3.5 kDa, and the dialysis area is 1 cm2), and 500 µL of honokiol solution in dimethyl sulfoxide (DMSO; 1 mg/mL) was used as control. The dialysis tubes were incubated in 10 mL of phosphate-buffered saline (PBS: prewarmed to 37 °C, pH ) 7.4) containing Tween80 (0.5 wt %) at 37 °C with gentle shaking (100 rpm), and the media were displaced by prewarmed fresh PBS at a predetermined time. After centrifugation at 13 000 rpm for 10 min, the supernatants of the removed release media were collected and stored at -20 °C until analysis. The released drug was quantified using HPLC. All results were the means of three test runs, and all data were expressed as the mean ( SD. In vitro Tumor Cell Growth Inhibition Assay. Murine melanoma cell line B16 was also obtained from ATCC and grew in RPMI 1640 medium with 10% FBS. The tumor cells were plated at a density of 1 × 104 cells per well in 100 µL of medium in 96-well plates and grown for 48 h. For comparison, honokiol loaded PCEC nanoparticles (honokiol nanoparticles) was prepared by an emulsion solvent evaporation method as described before.11 The cells were then exposed to a series of honokiol micelles or honokiol nanoparticles at different concentrations for 48 h respectively, and honokiol solution in DMSO at the same concentration was used as control. Then, the viability of cells was measured using the MTT method. The data were estimated from six individual experiments, and all data were expressed as the mean ( SD. The concentration of samples at

System of Honokiol-PECE Micelles in Hydrogel

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Figure 2. Morphology of prepared honokiol micelles. (A) Honokiol crystal. (B) Biodegradable PECE triblock copolymer. (C) Prepared blank (left) and honokiol (right) micelles. (D) TEM image of prepared honokiol micelles.

Figure 1. Preparation scheme of honokiol micelles. (A) Chemical structure of honokiol. (B) Synthesis of PECE triblock copolymer. (C) Biodegradable honokiol micelles prepared by self-assembly assisted with ultrasonication.

which cell killing was 50% (IC50) was calculated by curve fitting using SPSS software. Results Synthesis and Characterization of PECE Copolymers. The biodegradable PECE copolymers were successfully synthesized by ring-opening polymerization and coupling reaction according to Figure 1B. The molecular weight of prepared PECE triblock copolymers for micelles and hydrogel calculated from 1H NMR spectra were 14809 (PEG/PCL ratio: 9675/5134) and 3408 (PEG/PCL ratio: 960/2448), respectively. The molecular weight and polydispersity (PDI) of the two PECE triblock copolymers determined by GPC were 15228/1.41 and 4391/1.30, respectively. In this paper, the PECE copolymer for preparation of micelles was denoted as PEG5000-PCL5000-PEG5000, and the PECE copolymer for preparation of hydrogels was denoted as PEG550-PCL2400-PEG550 for simplicity. Preparation and Characterization of Honokiol Micelles. We incorporated honokiol (Figure 1A) into PECE micelles using a self-assembly procedure assisted with ultrasonication (shown schematically in Figure 1C). The appearance of the honokiol crystal, PECE copolymer (PEG5000-PCL5000-PEG5000), and prepared honokiol micelles suspension was shown in Figure 2, and the stable and homogeneous solution of blank (Figure 2C left) or honokiol micelles (Figure 2C right) could be observed.

Figure 2D showed the TEM image of the prepared honokiol micelle. The TEM image revealed that both blank (data not shown) and honokiol micelle formed with PECE copolymer solutions are monodisperse, thus confirming the spherical shape of micelles in aqueous solution. The average particle size and PDI of obtained blank and honokiol micelles were 83.47 nm/0.274 and 58.75 nm/0.266, respectively (Figure 3A and C). Incorporation of honokiol in the micelles architecture increased the hydrophobicity of the PCL core, thus, making the hydrophobic core more compact and decreasing the particle size of honokiol micelles. Besides, the zeta potential of prepared blank and honokiol micelles was -0.413 and -0.385 mV, respectively (Figure 3B and D), which implied that the surface charge could be regarded as neutral. The diameters of the polymeric micelles observed by TEM were in good agreement with the determination of the particle size. The nanostructure of the micelles observed by TEM, in combination with the particle size analysis, determined that the prepared micelles is stable and could be well-dispersed in water. Preparation of Honokiol Micelles/Hydrogel CDDS. Owing to the combination of the hydrophilic PEG block and hydrophobic PCL block, the PECE triblock copolymer is amphiphilic in nature. PECE hydrogel based on central PCL blocks (Mn ) 2400) and end PEG blocks (Mn ) 550) showed temperaturedependent sol-gel transition (lower transition) and gel-sol transition (upper transition). The hydrogel (30 wt %) flowed freely at lower temperature, but became an opaque gel at body temperatures of about 37 °C. After honokiol micelles were well-dispersed into the hydrogel, the injectable homogeneous honokiol micelles/hydrogel was obtained. With increase of temperature, the honokiol micelles/ hydrogel transformed to a gel state at about 37 °C, which implied the potential application of the demonstrated CDDS in local delivery of a hydrophobic drug.

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Figure 3. Particles size and zeta potential distribution spectrum of prepared blank micelles (A, B) and honokiol loaded micelles (C, D) at 37 °C, respectively.

Figure 4. Effect of the honokiol micelles content on the sol-gel-sol transition diagram of the composite system.

Figure 5. Cytotoxicity of PECE micelles and PECE hydrogel at different concentrations on L929 cells. Error bars represent the standard deviation (n ) 6).

Figure 4 presented the sol-gel-sol transition phase diagram of the honokiol micelles/hydrogel. The system had a concentration-dependent critical gelation concentration (CGC), lower critical gelation temperature (LCGT), and upper critical gelation temperature (UCGT). When different amounts of honokiol micelles were incorporated into the hydrogel, the effect of the amount of micelles on the sol-gel transition temperature was investigated, and the result was presented in Figure 4. With an increase in the amount of micelles, the LCGT decreased slightly and the UCGT dramatically increased, which meant that the gelation window became wider, whereas the CGC of hydrogels with or without micelles was kept constant. Cytotoxicity of Blank Micelles and Blank Hydrogel. The cytotoxicities of blank micelles and blank hydrogel were evaluated by a cell viability assay using L929 cells for 48 h. As shown in Figure 5, with an increase in the micelles or hydrogel concentration, the viability of L929 cells decreased accordingly. The cell viability was still higher than 31% or 27%,

Figure 6. In vitro drug release profiles of free honokiol, honokiol micelles, and honokiol micelles/hydrogel in PBS solution at pH 7.4. Error bars represent the standard deviation (n ) 3).

even when the input copolymer concentration was 5 mg/mL. The cell viability study implied that the blank micelles and blank hydrogels prepared in this paper were biocompatible with very low cell cytotoxicity. Therefore, the honokiol micelles/hydrogel could be regarded as a safe drug delivery system. In vitro Release Behavior. Figure 6 presented the in vitro release profiles of free honokiol, honokiol micelles, and honokiol micelles/hydrogel in PBS. Compared with the release profile of free honokiol or honokiol micelles, the culmulative release rate of honokiol from honokiol micelles/hydrogel is much slower followed by a sustained release for up to 2 weeks. It was observed that only 11% of honokiol was released from honokiol micelles/hydrogel within 72 h, whereas free honokiol or honokiol micelles released approximately 91% or 44% into the outside media, respectively. In comparison with free honokiol, the much slower release rate of honokiol from honokiol micelles/ hydrogel can be attributed to the internal structural characteristics of polymeric hydrogel. During the process, honokiol micelles were first released from the hydrogel, and then honokiol diffused out from the micelle, eventually, into the incubation medium. This delay of drug release indicated their potential applicability in drug carrier to minimize the exposure of healthy tissues while increasing the accumulation of therapeutic drug in the tumor site. In vitro Tumor Cell Growth Inhibition Assay. The MTT assay was performed to evaluate the toxicity of honokiol micelles, honokiol nanoparticles, and free honokiol to investigate whether micelles influenced the cytotoxity of honokiol. Honokiol micelles, honokiol nanoparticles, and free honokiol at various

System of Honokiol-PECE Micelles in Hydrogel

Figure 7. In vitro tumor cell growth inhibition assay of murine melanoma cell line B16. B16 cells were exposed to different concentration of free honokiol, honokiol nanoparticles, or honokiol micelles for 48 h, respectively. Error bars represent the standard deviation (n ) 6).

concentrations significantly inhibited the growth of B16 cells in a dose-dependent manner. Figure 7 presented the influence of drug concentration on cell viability of tumor cells. The IC50 values of free honokiol, nonokiol nanoparticles, and honokiol micelles were 5.357, 6.274, and 6.746 µg/mL, respectively. The result indicated that the cytotoxicity of the honokiol micelles was lower than that of free honokiol, which was attributed to the sustained release behavior of honokiol from honokiol micelles. Besides, compared with honokiol nanoparticles, the cytotoxicity of honokiol micelles was a little lower, which might be due to the absence of organic solvent and surfactant in the honokiol micelles. Discussion Chemotherapy, as conventional therapy for cancer, has been proven to be effective, which has wide applications in clinical study. Chemotherapeutic agents may prolong the survival time of the patients; however, most of them have severe side effects, which would decrease the quality of life for the patients. Most conventional methods for chemotherapeutic agent delivery, including intravenous injection and oral ingestion, fail to achieve therapeutic concentrations of chemotherapeutic agents, despite reaching toxic systemic levels. Currently, novel controlled drug delivery systems are designed to deliver drugs in predefined amounts for predetermined periods at the target site, which could be used to overcome the shortcomings of conventional drug formulations, which could therefore diminish the side effects and improve the quality of life of the patients.22,23 Since Nobel Laureate Richard Feynman predicted the emergence of a new science called nanotechnology in 1959,24 nanotechnology has been accelerating the identification and evaluation of drug candidates. Nanotechnology provides an important method to overcome the problems of hydrophobic drugs.25-28 After hydrophobic drugs are manipulated to form nanoscale particles, they could be well-dispersed in aqueous solution to form stable and homogeneous suspensions, therefore meeting the requirements of clinical administration. Drugs encapsulated into polymer matrix exhibit improved drug delivery behaviors and have more advantages compared to conventional dosage forms. Their therapeutic value has been enormously increased in the form of nanoparticles by improving the therapeutic effect, prolonging the biological activity, controlling the drug release rate, and decreasing the administration frequency. Amphiphilic materials are the most studied examples of nanocarriers for hydrophobic drugs.29-31 The solvent diffusion method,32 cosolvent evaporation method,33 dialysis method,33 emulsion polymerization method,34 interfacial polymer disposition method,35 and nanoprecipitation method36 were intensively studied to form polymeric nanocarriers. However during the

J. Phys. Chem. B, Vol. 113, No. 30, 2009 10187 formation, organic solvents, like EtAc11 and acetone,37 are widely used as the solvent for both drugs and materials, eventually followed by a removal by freeze-drying or evaporating. Furthermore, surfactants, such as Pluronic F127 and poly(vinyl alcohol) (PVA),11 were also extensively used in the above-mentioned methods, which are not desirable and could not always be removed completely. To the best of our knowledge, little work has been done to prepare polymeric micelles by a direct dissolving method in distilled water without using any organic solvents and surfactants. This method could be highly welcomed in preparation of micelles. Thus, formulation of polymeric micelles using this method could ensure the safety of micelles both in the formation process and further clinical use. Several observations were made in the present study concerning honokiol micelles/hydrogel based on biodegradable PECE copolymers. Honokiol micelles have been successfully prepared with small particle size, stable loading capacity, and the ability for sustained drug release by self-assembly. And, we also prepared the CDDS successfully, with only honokiol and PECE copolymers. Moreover, findings in cytotoxicity revealed some properties of honokiol micelles as well. The explanations for the comparable cytotoxicity of prepared honokiol micelles and free honokiol might involve two conflicting effects. After honokiol was manipulated to form nanoscale micelles, the celluar uptake of honokiol micelles might be enhanced, which would increase the cytotoxicity of honokiol micelles, whereas the extended release behavior by polymeric micelles would keep honokiol in a relatively low concentration compared with free honokiol in the first 48 h. In comparison with free honokiol, the much slower release of honokiol from micelles can be attributed to the molecular structural characteristics of polymeric micelles as drug carriers. This delay of drug release indicates their potential applicability in drug carriers to minimize the exposure of healthy tissues while increasing the accumulation of the drug in the tumor area. Conclusions We have successfully prepared a novel biodegradable and injectable drug delivery system based on honokiol micelles/ hydrogel. The CDDS is an injectable free-flowing sol at ambient temperature and changes into a nonflowing gel at body temperature. Moreover, the system could release drugs for an extended period. The CDDS demonstrated in this paper might have great potential clinic application for local administration of hydrophobic drugs such as honokiol. Acknowledgment. This work was financially supported by National Natural Science Foundation of China (NSFC20704027), National 863 project (2007AA021902, 2007AA021804, and 2006AA03Z356), Specialized Research Fund for the Doctoral Program of Higher Education (200806100065), Sichuan Key Project of Science and Technology (2007SGY019), New Century Excellent Talents in University (NCET-08-0371), and Chinese Key Basic Research Program (2004CB518807). References and Notes (1) Jemal, A.; Siegel, R.; Ward, E.; Hao, Y.; Xu, J.; Murray, T.; Thun, M. J. CA Cancer J. Clin. 2008, 58, 71. (2) Chen, L.; Zhang, Q.; Yang, G.; Fan, L.; Tang, J.; Garrard, I.; Ignatova, S.; Fisher, D.; Sutherland, L. A. J. Chromatogr. A 2007, 1142, 115. (3) Xu, D.; Lu, Q.; Hu, X. Cancer Lett. 2006, 243, 274. (4) Konoshima, T.; Kozuka, M.; Tokuda, H.; Nishino, H.; Iwashima, A.; Haruna, M.; Ito, K.; Tanabe, M. J. Nat. Prod. 1991, 54, 816.

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