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Soluplus® based 9-nitrocamptothecin solid dispersion for peroral administration: Preparation, characterization, in vitro and in vivo evaluation
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IJP 14423 1–9 International Journal of Pharmaceutics xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
International Journal of Pharmaceutics journal homepage: www.elsevier.com/locate/ijpharm
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Soluplus1 based 9-nitrocamptothecin solid dispersion for peroral administration: Preparation, characterization, in vitro and in vivo evaluation Xianghong Lian, Jianxia Dong, Jinjie Zhang, Yanwei Teng, Qing Lin, Yao Fu 1, *, Tao Gong 2, * Key Laboratory of Drug Targeting, Ministry of Education, Sichuan University, Chengdu, China
A R T I C L E I N F O
A B S T R A C T
Article history: Received 20 August 2014 Received in revised form 17 October 2014 Accepted 26 October 2014 Available online xxx
Our study aimed to develop an amorphous 9-nitrocamptothecin solid dispersion (9-NC-SD) using polyvinyl caprolactam–polyvinyl acetate–polyethylene glycol graft copolymer (Soluplus1) for improving its oral bioavailability and antitumor efficacy in vivo. Freeze-dried 9-NC-SD with an optimized drug/ polymer ratio at 1:15 (w/w) was characterized by powder X-ray diffraction, scanning electron microscopy and Fourier transform infrared spectroscopy. The amorphous form of 9-NC was obtained by freeze-drying and the aqueous solubility of 9-NC was increased to 1.42 mg/mL. Upon dilution, 9-NC-SD was proven to form micellar structures with an average size distribution around 58 nm 5 nm (PDI = 0.107 0.016). Moreover, 9-NC-SD showed significantly increased intracellular uptake efficiency in Caco-2 cells compared to free 9-NC. Furthermore, the AUC0–8 h of 9-NC-SD following oral administration showed a 2.68-fold increase in the lactone form of 9-NC compared to that of free 9-NC in Sprague-Dawley rats. The 9-NC-SD did not show obvious inflammatory responses and gastrointestinal toxicity following oral administration as demonstrated by the histological analysis of the rat intestinal sections. Thus, 9-NCSD represents a promising approach for improving the solubility and oral bioavailability of drugs with poor solubility. ã 2014 Published by Elsevier B.V.
Keywords: 9-Nitrocamptothecin Soluplus1 Solid dispersion Oral bioavailability Antitumor effect
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1. Introduction
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9-nitrocamptothecin (9-NC), an analog of camptothecin (CPT), is a potent chemotherapeutic compound for the treatment of pancreatic cancer and other solid tumors (Haglof et al., 2006). As a second-generation topoisomerase-I inhibitor, pharmacological studies found that 9-NC displayed better antitumor activity than CPT and other CPT analogs in xenografted human tumors in nude mice (Pantazis et al., 1993). The antitumor efficacy of 9-NC is structure-specific: the pentacyclic structure of 9-NC with an a-hydroxy-d-lactone moiety shows high antitumor activity (Fig. 1A) (Saha et al., 2013). However, under physiological conditions, 9-NC may undergo ring opening hydrolysis which leads to the formation of the inactive carboxylate form (Fig. 1B)
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* Corresponding authors at: Key Laboratory of Drug Targeting, Ministry of Education, Sichuan University, No. 17, Section 3, Southern Renmin Road, Chengdu 610041, China. E-mail addresses: [email protected] (Y. Fu), [email protected] (T. Gong). 1 Tel.: +86 28 8550 3798. 2 Tel.: +86 28 8550 1615.
(Gao et al., 2008). Thus, maintaining 9-NC in its active lactone form represents the primary goal for formulation development. Despite the antitumor potency of 9-NC, the oral bioavailability of 9-NC remained extremely low mainly due to its poor water solubility (<5 mg/mL in distilled water at 25 C) (Verschraegen et al., 1998), and low permeability (Sha and Fang, 2004), thus resulting in poor therapeutic efficacy and various adverse effects such as neutropenia, thrombocytopenia and gastrointestinal reactions (Venditto and Simanek, 2010). To improve the solubility and bioavailability of 9-NC, oil and glycol solutions (Burcham et al., 1997), self-emulsifying drug delivery systems (Lu et al., 2008) and liposomes (Knight et al., 1999) were explored during the past decade, which inevitably involved the usage of oils and surfactants such as Tween 80, Cremophor EL, which were proven to induce hypersensitive reactions in humans (Tong et al., 2012). Recently, a liposomal aerosol formulation has been successfully developed and already subjected to preclinical and clinical studies (Knight et al., 2000). However, the aerosol formulations must require special devices such as inhalers for administration which is less user-friendly and cost-effective compared to oral formulations (Smith and Parry-Billings, 2003). Thereby, solid dispersions, i.e.,
http://dx.doi.org/10.1016/j.ijpharm.2014.10.055 0378-5173/ ã 2014 Published by Elsevier B.V.
Please cite this article in press as: Lian, X., et al., Soluplus1 based 9-nitrocamptothecin solid dispersion for peroral administration: Preparation, characterization, in vitro and in vivo evaluation. Int J Pharmaceut (2014), http://dx.doi.org/10.1016/j.ijpharm.2014.10.055
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drug compounds that are dispersed amorphously in a hydrophilic matrix material, appear to be an attractive alternative for enhancing drug solubility and bioavailability (Chiou and Riegelman, 1971). In addition, solid dispersions can be further developed into dosage forms for oral administration such as tablets and capsules, which are highly acceptable in patients. Soluplus1, a polyvinyl caprolactam–polyvinyl acetate–polyethylene glycol (57/ 30/13) graft copolymer, has been extensively used in the fourthgeneration of solid dispersions such as itraconazole (Zhang et al., 2013), carvedilol (Shamma and Basha, 2013), and CPT (Thakral et al., 2012). As a solubility enhancing excipient, Soluplus1 carries multiple advantages such as minimum toxicity, low hygroscopicity and amphiphilicity. Compared with matrix materials such as Solutol1 HS 15, Cremophor1 RH 40 and Tween1 80 (Strickley, 2004), Soluplus1 can serve either as a solubilizer to form micellar structures in the aqueous medium or as a matrix material for solid dispersions. Moreover, Soluplus1 based solid dispersions were demonstrated to significantly improve the solubility or oral bioavailability of poorly water-soluble drugs (Kawabata et al., 2011). In this study, the Soluplus1-based solid dispersion system has been applied for the first time to 9-NC. The present study aimed to develop and characterize the optimal formulation of Soluplus1 based solid dispersions for 9-NC via freeze-drying, and to assess its oral bioavailability, antitumor activity and gastrointesetinal safety compared with free 9-NC suspensions and 9-NC/Soluplus1 physical mixtures in animals. In addition, a preliminary study was conducted to explore the absorption mechanism of 9-NC-SD using Caco-2 cell monolayers in the current study.
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2. Materials and methods
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2.1. Materials
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Soluplus1 was kindly gifted by BASF SE (Ludwigshafen, Germany). 9-NC (purity >98.0%) was purchased from Chengdu Lanbei Plant & Chemical Technology Co., Ltd. (China). Dulbecco’s modified eagle’s medium (DMEM), fetal bovine serum (FBS), Hank ’s balanced salt solution (HBSS), L-glutamine, penicillin–streptomycin, trypsin–EDTA and non-essential amino acids were obtained from Gibco Laboratory (Invitrogen Co., Grand Island, NY, USA). Transwell permeable membrane inserts (0.4 mm pore size, 1.12 cm2 membrane area, polycarbonate) were purchased from Corning Incorporated Life Science (Lowell, MA, USA). All other chemical reagents were of HPLC grade.
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2.2. Cell culture
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Caco-2 cells were obtained from American Type Culture Collection (Manassas, CA, USA). Caco-2 cells (passages: 40–50) were cultured under the same condition as previously reported (Gao et al., 2011). For cellular uptake studies, cells were seeded in 12-well plates at a density of 50,000 cells/cm2 and cultured for
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14 days. For transport experiments, cells were seeded on transwell permeable membrane inserts at a density of 50,000 cells/cm2 in 12-well plates and cultivated for 21 days. Monolayers with transepithelial electrical resistance (TEER) value higher than 600 V cm2 were used in the transport assay.
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2.3. Animals
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Healthy male Sprague-Dawley rats were obtained from the Experimental Animal Center of Sichuan University (Chengdu, China). ICR mice were supplied by Chengdu Dossy Biological Technology Co., Ltd. (Chengdu, China). All animal experiments were approved by Sichuan University Animal Ethical Experimentation Committee (Chengdu, China), according to the requirements of the National Act on the use of experimental animals (People’s Republic of China).
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2.4. LC-MS/MS
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A HPLC tandem triple quadrupole mass spectrometry (LC-MS/ MS, 6410B, Agilent Technologies, Santa Clara, CA, USA) was used to analyze 9-NC. A diamonsil column (ODS, 50 4.6 mm, 1.8 mm) was used. The column was maintained at 30 C. The mobile phase consisted of 0.02% formic acid and methanol (55:45, v/v). The flow rate was set at 0.4 mL/min. The injection volume was 3 mL. The quantitative analysis was performed using multiple reaction monitoring (MRM) mode. General mass spectrometer conditions were listed as follows: 9-NC transition (m/z), 394–350; fragmentor, 169 eV; collision energy, 24 eV; gas temperature, 350 C; nebulizer pressure, 35 psi and spray voltage, 4000 V.
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2.5. Sample preparation and solubility test
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The 9-NC-SD was prepared by freeze-drying. Mixtures of 9-NC and Soluplus1 with varying w/w ratios such as 1:5, 1:10, 1:15, 1:20 and 1:25 were dissolved in dimethylsulfoxide (DMSO). The solutions were frozen at 40 C for 8 h, and then lyophilized for 24 h at 45 C using Allegra X-22R freeze-dryer (Beckman Coulter, USA). Physcial mixtures of 9-NC and Soluplus1 (9-NC-PM) of same w/w ratios were obtained by simple mixing in a mortar with pestle using geometric dilutions. An excess amount of free 9-NC, 9-NC-PM and 9-NC-SD were placed in 5 mL of distilled water, and incubated in a shaking water bath at 37 0.5 C for 48 h. The supernatant was collected and filtered through a 0.45 mm membrane filter, and diluted appropriately with methanol. The concentration of 9-NC was then determined by LC-MS/MS.
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2.6. Characterization of physicochemical properties
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X-ray diffraction (X-RD) patterns of 9-NC, Soluplus1, 9-NC-PM, and 9-NC-SD were recorded by an X-ray diffractometer (X’Pert Pro MPD, The Netherlands Philips), using nickel-filtered Cu Ka
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Please cite this article in press as: Lian, X., et al., Soluplus1 based 9-nitrocamptothecin solid dispersion for peroral administration: Preparation, characterization, in vitro and in vivo evaluation. Int J Pharmaceut (2014), http://dx.doi.org/10.1016/j.ijpharm.2014.10.055
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radiation generated at 40 kV and 25 mA and scanning rate of 2 / min over a 2u range of 10–50 . The Fourier transform infrared (FITR) spectra (Nicolet 6700, Thermo Electron Corporation, USA) was used to characterize the possible interactions between the 9-NC and Soluplus1 in 9-NC-SD. 5 mg of each sample was lightly grounded and mixed with KBr at range 4000–500 cm1 with a resolution of 2 cm1. The surface morphology of 9-NC and 9-NC-SD were observed under a scanning electron microscope (SEM) (JSM-7500F scanning microscope, Tokyo, Japan) operated at 5.0 kV. The samples were mounted on a glass stub with double-sided adhesive tape and coated under vacuum with gold in an argon atmosphere prior to observation. The lyophilized 9-NC-SD product was dispersed in ddH2O to form homogeneous micellar solution. The particle size and polydispersity index of the 9-NC-SD micelles were measured by dynamic light scattering (Malvern Zetasizer Nano ZS90, Malvern Instruments Ltd. Malvern, UK) at 25 C. 5 mg/mL of sample solution was measured accordingly with no further dilution. The morphology of 9-NC-SD micelles was observed under a transmission electron microscope (TEM) (Tecnai G2F20, FEI, Eindhoven, The Netherlands). The sample was placed on copper grids and stained with 2% (w/v) phosphotungstic acid for 30 s. After the excess solution was drawn off, the sample was dried and then subjected to TEM observation. Dissolution profiles of free 9-NC and 9-NC-SD were evaluated by the paddle method at a rotation speed of 75 rpm (Jiang et al., 2010). Briefly, 1 mg of free 9-NC and equivalent amount of 9-NC-SD were added to 0.9 L dissolution media (0.1 M hydrochloric acid with 0.05% Tween 80) at 37 0.5 C. At predetermined times, 2 mL of dissolution media was withdrawn and replaced by equal volume of fresh media. The samples were filtered through 0.45 mm membrane filter, diluted with methanol (1:1, v/v) and analyzed by LC-MS/MS. 2.7. Cellular uptake of 9-NC, 9-NC-PM and 9-NC-SD To evaluate the cell uptake efficiency of 9-NC-SD micelles, Caco2 cells were incubated with 9-NC, 9-NC-PM or 9-NC-SD micelles (5 mg/mL) at 37 C. For energy depletion, the cellular uptake were performed at 4 C and 37 C for 1 h. Next, the cells were subjected to three freeze-thawing cycles following detachment from plates. 20 mL of 0.1 M glacial acetic acid and 200 mL of ice-cold methanol/ acetonitrile (1:2, v/v) were added to 100 mL of detachment cells sample. The mixture was vortexed for 1 min and centrifuged at 12,500 rpm for 10 min. The supernatant was collected and filtrated to 0.22 mm membrane filter to LC-MS/MS analysis. The cell uptake amount was analyzed using LC-MS/MS. To explore the mechanism of 9-NC-SD micelle mediated internalization, Caco-2 cells were pre-incubated with indicated inhibitors for 1 h and then incubated with 9-NC-SD for another 30 min. Chlorpromazine (Chlo, 10 mg/mL) was an inhibitor of clathrin-mediated endocytosis (Mahmud and Lavasanifar, 2005). Nystatin (25 mM) was an inhibitor of cholesterol disrupted caveolae-mediated endocytosis (Nabi and Le, 2003). MbCD (10 mg/mL) with 1 mg/mL simvastatin (MS), an inhibitor of de novo synthesis of cholesterol, inhibited both caveolae and clathrinmediated pathways by cholesterol depletion (Mayor and Pagano, 2007). Dimethyl amiloride (DMA, 5 mg/mL) was an indicator of macropinocytosis (Sun et al., 2010). The procedures were kept the same as mentioned above. 2.8. Transport across Caco-2 monolayers
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Caco-2 monolayers were developed to investigate the transport behavior of 9-NC and 9-NC-SD across the intestinal membrane as
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described previously (Sha and Fang, 2004). Before the experiment, the cell monolayers were washed twice with HBSS followed by incubating at 37 C for 20 min. HBSS solution on both sides of the cell monolayers was then removed. For the measurement of the apical side (AP) to basolateral side (BL) transport, 0.5 mL of HBSS (pH 6.0) containing: (i) the pure 9-NC solution (5 mg/mL), (ii) the 9NC-SD micelles (5 mg/mL) were added on the AP side, and 1.5 mL of HBSS (pH 7.4) without 9-NC was added to the BL side. The monolayers were incubated at 37 C and shaken at 50 rpm in a shaker. Samples were taken from the BL side at 15, 30, 45, 60, 90, 120 min and replaced by an equal volume of prewarmed fresh HBSS. Samples were then diluted in a 1:1 ratio with methanol before LC-MS/MS analysis. The transepithelial electrical resistance (TEER) of monolayer was measured using Millicell electrical resistance system (Millipore, USA) to evaluate the formation of the monolayer and its integrity during the experiment. To observe how 9-NC-SD micelles were transported across the cell monolayer, cell monolayers were incubated with HBSS as a control and with 9-NC-SD micelles at 37 C for 2 h, respectively. The basolateral medium was then collected, treated with uranyl acetate as a contrasting agent which fixes preferentially the periphery of the particles, and observed under a transmission electron microscopy (TEM).
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2.9. Pharmacokinetics
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Male Sprague-Dawley rats (180 10 g) were fasted for 12 h before drug administration with free access to water. 9-NC and 9NC-PM suspension was prepared by adding 9-NC or 9-NC-PM (1:15, w/w) into 0.5% carboxymethylcellulose sodium (CMC-Na) solution and then ultrasonicated for several minutes to obtain homogenous suspensions. 9-NC-SD was dissolved in diH2O. Three groups of rats were orally administrated with 9-NC suspension, 9NC-PM, and 9-NC-SD at an equivalent dose of 6 mg/kg 9-NC. The blood samples (about 0.3 mL) were collected via orbital venous at predetermined time intervals (5, 15, 30, 45, 60, 90, 120, 180 min). The samples were then centrifuged at 5000 rpm for 5 min to obtain the plasma. 20 mL of 0.1 M glacial acetic acid and 200 mL of ice-cold methanol/acetonitrile (1:2, v/v) were added to 100 mL of plasma sample. The mixture was vortexed for 1 min and centrifuged at 12,500 rpm for 10 min. The supernatant was collected and filtrated to 0.22 mm membrane filter to LC-MS/MS analysis.
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2.10. Antitumor effects in sarcoma 180 tumor-bearing mice
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In vivo antitumor activity was evaluated in sarcoma 180 (S180) tumor bearing mice (Jiang et al., 2011). S180 cells were transplanted to the intraperitoneal cavity of ICR mice, and then collected from the mice at fixed time points. The S180 cells (1 106 cells/ 0.2 mL) were inoculated subcutaneously in ICR mice (20 2 g) at the right armpit. To simulate the early stage of tumor growth, the dose schedule was started at 24 h after transplantation. The animals were randomly divided into four groups with five mice in each group. The negative control group received saline (0.9% NaCl) and blank Soluplus1 solution (0.75%). Mice were treated with 9-NC and 9-NC-SD at an equivalent dose of 4 mg/kg 9-NC on day 1 and day 4 after transplantation. All mice were sacrificed on day 11 to collect tumor tissues, which were washed with saline and accurately weighed. The inhibitory rate of tumor (IRT%) was calculated according to the following equation:
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IRT% ¼
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W control W treated 100% W control
Please cite this article in press as: Lian, X., et al., Soluplus1 based 9-nitrocamptothecin solid dispersion for peroral administration: Preparation, characterization, in vitro and in vivo evaluation. Int J Pharmaceut (2014), http://dx.doi.org/10.1016/j.ijpharm.2014.10.055
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2.11. Gastrointestinal toxicity
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Male Sprague-Dawley rats (200 20 g) were fasted for 12 h before drug administration, and were randomized into four groups. The control group received saline, blank Soluplus1 (0.75%); treatment groups were orally administered with 9-NC suspension and 9-NC-SD at an equivalent dose of 6 mg/kg 9-NC for 2 continuous days. 48 h after the last administration, all rats were sacrificed. Afterwards, the duodenum, jejunum, ileum and colon were collected and then cleaned. The samples were fixed with 4% polyoxymethylene for at least 48 h and then embedded in paraffin. Tissue sections of 5 mm thickness were stained with hematoxylin and eosin (H&E). Injuries such as villus atrophy, bleeding, hyperaemia, glandular destruction, interstitial edema and inflammatory cell infiltration were carefully examined, and photographed under light microscope (Axiovert 40CFL, Germany).
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2.12. Statistical analysis
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The pharmacokinetic parameters including area under the concentration–time curve (AUC), time to peak plasma concentration (Tmax) and biological half life (T1/2) were calculated using DAS 3.0 (BioGuider Co., Shanghai, China). Data represent mean standard deviation (SD). The statistical analysis was performed using Student’s t-test for comparison between two groups and one-way ANOVA for comparison among multiple groups. A p-value less than 0.05 was considered as significantly different.
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3. Results
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3.1. Physicochemical properties and dissolution
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Table 1 shows the water solubility of 9-NC in 9-NC-PM and 9NC-SD, respectively. For 9-NC-SD, the solubility of 9-NC increased slightly with the increasing amount of Soluplus1 mainly due to the micelle solubilization effect of Soluplus1. 9-NC in different 9-NCSD formulations displayed water solubilities over 1.3 mg/mL, which were significantly higher than in corresponding 9-NC-PM of the same 9-NC to Soluplus1 weight ratio (p < 0.01). In addition, 9-NC-SD formulation with 1:15 (w/w) 9-NC/Soluplus1 achieved a saturated solubility of 1.42 mg/mL and appeared to reach saturation in the solubility test, where the solubility of 9-NC no longer increased when increasing the percentage of Soluplus1. Thus, 9NC-SD with a drug/Soluplus1 ratio (w/w) of 1:15 was selected for the following study. 9-NC is a highly crystalline molecule with main characteristic crystalline peaks at 7.50, 20.97, 22.63, 25.70 and 27.41-u degrees (Fig. 2A). In comparison, the characteristic peaks of 9-NC were absent in 9-NC-SD, suggesting a conversion of the crystalline form of 9-NC to its amorphous form. To investigate the effect of freeze-drying, FTIR was used to elucidate possible interactions between 9-NC and Soluplus1. Interactions between active pharmaceutical ingredient and excipients may be relevant to stabilizing 9-NC-SD in the highenergy amorphous state. CPT was reported to form hydrogen bonds with Soluplus1 (Thakral et al., 2012), and as a derivative of CPT, 9-NC was considered to interact with Soluplus1 by forming
Table 1 Solubility of 9-NC, 9-NC-PM, and 9-NC-SD in water at 25 C. Data represent mean S.D. (n = 5). 9-NC/Soluplus (w/w)
9-NC-PM (mg/mL)
9-NC-SD (mg/mL)
1:10 1:15 1:20 1:25
39.70 2.13 66.77 8.54 54.32 6.12 85.14 12.12
1322.47 65.14 1480.30 50.12 1399.15 48.56 1482.67 60.38
intermolecular hydrogen bonds. As shown in the FTIR spectrum (Fig. 2B), 9-NC displayed —NO2 stretching at 1511 cm1 and 1388 cm1,—OH stretching at 3410 cm1, and carbonyl stretching for cyclic ester (lactone) at 1736 cm1. Soluplus1 showed intermolecularly hydrogen bonded —OH stretching in the range of 3342–3350 cm1, ester carbonyl stretching 1729–1635 cm1, and C¼O stretching for tertiary amide at 1729 cm1. No spectral shift was found in the 9-NC-PM, which was a simple superposition of 9NC and Soluplus1, indicating no interactions between drug and polymer. However, in the FTIR spectrum of 9-NC-SD, —NO2 stretching peak was covered by numerous small minor peaks, and —OH stretching peaks of Soluplus1 appeared at 3424 cm1 and became slightly broader, indicating that Soluplus1 might interact with 9-NC by forming intermolecular hydrogen bonds. As for the surface morphology, the SEM image of bulk 9-NC presented large, inerratic clavate crystals (Fig. 2C). In contrast, 9-NC-SD displayed in the flake form and no 9-NC crystals were observed (Fig. 2C). Within 10 h, the cumulative dissolution percentage of 9-NC-SD was significantly higher than that of free 9-NC at predetermined time points (Fig. 2D). In addition, the amount dissolved from free 9-NC was less than 58% in the first 90 min, while nearly 92% achieved cumulative dissolution from 9-NC-SD within 90 min.
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3.2. Particle size, morphology and endocytosis of 9-NC-SD after dispersion in water
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After dispersion in water, 9-NC-SD would assemble into micellar structures with the mean particle size of about 58 nm 5 nm with a polydispersity index of 0.107 0.016 (Fig. 3A) as determined by DLS measurement. As shown in TEM images, 9-NCSD micelles displayed near spherical shapes and a uniform morphology with an average particle size of around 60 nm (Fig. 3B). During 30 min incubation, cellular uptake of 9-NC-SD micelles increased significantly compared with that of free 9-NC and 9-NCPM (Fig. 4A). The cellular uptake efficiency of 9-NC-SD micelles increased to 146 ng/mL at 30 min, which was about 1.44 and 1.38 times that of free 9-NC and 9-NC-PM, respectively. However, the physical mixture of 9-NC and blank Soluplus1 did not show significant impact on cellular uptake efficiency of 9-NC (Fig. 4A). Nevertheless, incubating Caco-2 cells at 4 C significantly inhibited the cell uptake efficiency of 9-NC-SD micelles compared with 37 C (Fig. 4B), suggesting an energy-dependent absorption behavior. To elucidate the possible endocytic mechanism of 9-NCSD micelles, an in-depth cell uptake study was conducted using various endocytosis inhibitors (Fig. 4C). Nystatin did not affect the cell uptake of 9-NC-SD micelles indicating the endocytic pathway is not caveolae-mediated. Chlorpromazine, an inhibitor of clathrinmediated endocytosis, resulted in a 15.0% reduction in cell uptake, indicating that clathrin other than caveolae was involved in the internalization of 9-NC-SD micelles. MbCD (10 mg/mL) with 1 mg/ mL simvastatin, which inhibited both caveolae and clathrinmediated pathways by cholesterol depletion, decreased the cell uptake by 63.58%, indicating that other cholesterol-dependent endocytosis pathways such as macropinocytosis, may also be involved in the cell entry of 9-NC-SD micelles. A 34.46% decrease in the cell uptake of 9-NC-SD micelles was observed while inhibiting macropinocytosis by dimethyl amiloride, suggesting that the transport of 9-NC-SD micelles was mainly driven via macropinocytosis. Taken together, 9-NC-SD micelles was likely internalized into enterocytes via macropinocytosis and clathrindependent pathways.
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3.3. Transport across Caco-2 monolayers
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The crystalline form of free 9-NC showed a flux of 2.5 ng/min/ cm2 (Fig. 4D), while 9-NC-SD showed a relatively higher transport rate of 8.0 ng/min/cm2 across Caco-2 cells, which was increased by 3.2 times compared to free 9-NC. Besides, the cumulative absorption amount of 9-NC in 9-NC-SD at 2 h were significantly increased by 2.94-fold compared to free 9-NC. Fig. 3C shows the TEM images of the apical and basolateral side media at 2 h after applying 9-NC-SD micelles or blank HBSS to Caco-2 cell monolayers. After applying blank HBSS, fragments of 9NC crystals were observed on both the apical and basolateral sides. After applying 9-NC-SD micelles to the apical side, near spherical particles of approximately 50–70 nm were observed on the basolateral side, which were similar to those observed at the apical side. Based upon above results, 9-NC-SD micelles were likely endocytosed and exocytosed across the Caco-2 monolayers in the micellar form, which remains to be further elucidated.
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3.4. Pharmacokinetics
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The plasma concentration–time profiles of lactone and total ( lactone + carboxylate) of 9-NC in healthy Sprague-Dawley rats following oral administration of 9-NC, 9-NC-PM, and 9-NC-SD were shown in Fig. 5. 9-NC-SD displayed a pharmacokinetic profile dramatically different from that of free 9-NC or 9-NC-PM. Specifically, 9-NC-SD showed significantly higher Cmax values than 9-NC and 9-NC-PM (p < 0.01) (Table 2). As for the total form of 9NC, 9-NC-SD also displayed significantly higher AUC0–8 h values than 9-NC and 9-NC-PM (p < 0.01) (Table 2). Approximately 1.91and 1.93-fold increases in AUC0–8 h values for the total content were observed for 9-NC-SD as compared to the 9-NC suspension and 9NC-PM (p < 0.01), respectively. For the lactone form of 9-NC, the AUC0–8 h values were 745.48 18.42 ng h/mL for 9-NC-SD,
278 27.42 ng h/mL for free 9-NC suspension and 230.63 13.56 ng h/mL for 9-NC-PM. Thus, 9-NC-SD displayed approximately 2.68-fold and 3.23-fold increases of AUC0–8 hlactone compared to the 9-NC suspension and 9-NC-PM (p < 0.01). Importantly, compared to free 9-NC, A 2.68-fold increase in AUC0–8 h for the lactone content and a 1.93-fold increase in AUC0–8 h for the total content were observed for 9-NC-SD.
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3.5. Antitumor activity
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9-NC is an active chemotherapeutic compound with a broad spectrum of antitumor activity, especially for pancreatic cancers (Konstadoulakis et al., 2001). In the current study, three formulations were orally administered at a 3-day interval from the day after S180 inoculation. The photographs and the growth inhibitory rate (%) of tumors were shown in Fig. 6, respectively. The Soluplus1 alone showed no obvious inhibitory effect on tumor growth compared to the saline group. In contrast, free 9-NC suspension and 9-NC-SD groups showed comparable strong inhibitory effect against tumor growth compared to the saline and Soluplus1 groups. Moreover, 9-NC-SD showed significantly higher tumor growth inhibitory rate (IRT%) than 9-NC suspension (p < 0.05), which can be attributed to the higher oral bioavailability of 9-NC-SD than that of free 9-NC.
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3.6. Gastrointestinal toxicity
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Gastrointestinal toxicity has been one of the most commonly observed side effects of 9-NC in clinic (Garcia-Carbonero and Supko, 2002). In this regard, histological analysis of the gastrointestinal tract in rats was performed to gain further insight into the impact of 9-NC-SD. In the free 9-NC treatment group, the duodenum, jejunum and ileum showed severe intestinal villus atrophy and glandular destruction (Fig. 7). Besides, moderate
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Please cite this article in press as: Lian, X., et al., Soluplus1 based 9-nitrocamptothecin solid dispersion for peroral administration: Preparation, characterization, in vitro and in vivo evaluation. Int J Pharmaceut (2014), http://dx.doi.org/10.1016/j.ijpharm.2014.10.055
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Fig. 3.
Fig. 4.
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Fig. 5. Table 2 Pharmacokinetic parameters of lactone, and total 9-NC after oral administration of free 9-NC, 9-NC-PM, and 9-NC-SD to rats. Data represent mean S.D. (n = 5). Parameters
Cmax (ng/ml) Tmax (h) MRT (h) T1/2 (h) AUC0–8 h (ng/mL h) *
Lactone of 9-NC
Total of 9-NC
Free 9-NC
9-NC-PM
9-NC-SD
Free 9-NC
9-NC-PM
9-NC-SD
147.4 60.31 0.5 0.01 1.6 0.16 1.5 0.21 278 63.48
144.25 72.6 0.75 0.16 1.3 0.28 1.7 0.02 230.63 166
668.92 175.54* 0.3 0.13 0.98 0.43* 1.15 0.22 745.48 166*
225.02 64.01 0.5 0.12 1.6 0.15 1.42 0.31 471.14 131
255.85 114.35 0.95 0.13 1.5 0.25 1.81 0.12 467.96 197
1097 171.42* 0.25 0.01 0.85 0.16* 0.91 0.33 903.03 175*
p < 0.01 vs. free 9-NC.
Fig. 6. 423
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interstitial edema and mucosal congestion and inflammatory cell infiltration were also observed in the free 9-NC treatment group (Fig. 7). In comparison, 9-NC-SD displayed a slight extent of intestinal epithelial cell degeneration (Fig. 7). Moreover, 9-NC-SD and free 9-NC did not show obvious pathological symptoms in the stomach and colon, which is probably because colon and stomach were not the primary absorption site for 9-NC-SD. Additionally, no significant defects were detected in the Soluplus1 and saline group indicating the matrix material Soluplus1 is biocompatible and could be a suitable candidate for orally administered dosage forms.
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4. Discussions
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Hot-melt extrusion (HME) technique has been one of the most popular methods to prepare solid dispersions, which was first established on an industrial scale in 1930 s (Rauwendaal, 2014) and has gained extensive application in pharmaceutical field since 1971 (Chiou and Riegelman, 1971). HME method was commonly used when the drug and the matrix material display close melting
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points. However, HME cannot be used to prepare the solid dispersion of 9-NC in Soluplus1, because 9-NC has a relative high melting temperature of 219.58 C while Soluplus1 has a relative lower melting point around 70 C (Sambath et al., 2013). Thus, freeze-drying method was selected which is more suitable for drugs and excipients of dramatically different melting points. Also, the nucleation of drug crystals was minimized or completely prevented during lyophilization thus forming amorphous and highly porous product (Yu, 2001). 9-NC was poorly water soluble (<5 mg/mL in distilled water at 25 C), while 9-NC-SD with 1:15 (w/w) of 9-NC/Soluplus1 achieved a saturated 9-NC solubility of 1.42 mg/mL, which was dramatically higher than the reported solubility achieved by hydroxypropyl-bcyclodextrin (0.52 mg/mL) (Jiang et al., 2010). As demonstrated by XRD, SEM and FTIR analysis, 9-NC and Soluplus1 is likely to form a molecular dispersion thus resulting in greatly increased solubility of 9-NC. As an amphiphilic polymer with a hydrophilic PEG block, Soluplus1 was proven a good solubilizer for water-insoluble drugs such as itraconazole (Zhang et al., 2013), carvedilol (Shamma and
Please cite this article in press as: Lian, X., et al., Soluplus1 based 9-nitrocamptothecin solid dispersion for peroral administration: Preparation, characterization, in vitro and in vivo evaluation. Int J Pharmaceut (2014), http://dx.doi.org/10.1016/j.ijpharm.2014.10.055
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Basha, 2013), and CPT (Thakral et al., 2012). Moreover, the hydrophobic vinylcaprolactam/vinylvacetate block might help stabilize the lactone structure of 9-NC to avoid hydrolysis. Interestingly, 9-NC-SD formed homogeneous micelles with an average size diameter of 58 nm after dispersed in water, which significantly increased the intracellular uptake of 9-NC in Caco2 cells. Caco-2 cells were reported to express multiple efflux transporters, e.g., P-gp or MRP2 which might be involved in inhibiting the transport of 9-NC across the cell monolayer (Sha and Fang, 2004). However, the 9-NC-PM did not show significant effect on cell uptake (Fig. 4A), which was consistent with that low concentrations of Soluplus1 had a negligible impact on P-gp mediated drug efflux (Yu et al., 2013). In addition, to elucidate the possible endocytic mechanism of 9NC-SD micelles, an in-depth cell uptake study was conducted using various endocytosis inhibitors (Fig. 4C), which demonstrated that 9-NC-SD micelles could be internalized into enterocytes via clathrin-mediated endocytosis and macropinocytosis. Two possible routes were proposed for the transport of micelles across the Caco-2 cell monolayer, i.e., paracellular and/or transcellular transport. No obvious change in the TEER values of the monolayer during the period of transport experiments (data not shown) indicated that the 9-NC-SD micelles were not transported across the monolayer by paracellular route. Thus, 9-NC-SD may be transported across the Caco-2 monolayer via the transcellular route. Specifically, 9-NC-SD could be uptaken by Caco-2 cells at the apical side of the monolayer via clathrin-dependent pathway and macropinocytosis, and then exocytosed from the basolateral side. Moreover, 9-NC-SD displayed a pharmacokinetic profile with dramatically increased AUC0–8 h compared with free 9-NC suspension and 9-NC-PM. The high-energy solid dispersions might form supersaturated solutions in the intestine, and the polymeric carrier could act as a stabilizer of supersaturation upon drug release (Brouwers et al., 2009). Due to the higher absorption of 9-NC-SD, 9NC-SD showed significantly higher tumor growth inhibitory rate (IRT%) in the animal study. Furthermore, antitumor drugs frequently induce serious side effects such as neutropenia, thrombocytopenia, diarrhea and hemorrhagic cystitis (GarciaCarbonero and Supko, 2002). According to previous studies, the
carboxylic salt form of 9-NC resulting from hydrolysis was shown to induce various adverse effects (Giovanella et al., 1991). In our study, animals treated with free 9-NC exhibited severe gastrointestinal reactions such as intestinal villus atrophy and glandular destruction. In contrast, 9-NC-SD did not show obvious gastrointestinal reactions. As previously discussed, 9-NC-SD greatly improved the absorption of lactone of 9-NC in the intestine and reduced the accumulation of carboxylic acid salts in the intestine thus resulting in the minimum gastrointestinal toxicity. In addition, the reduced Tmax and MRT (p < 0.05) of 9-NC-SD (Table 2), indicated 9-NC-SD was rapidly absorbed, thereby alleviating the contact of carboxylic acid salt form to the intestinal tracts.
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5. Conclusion
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We have successfully developed Soluplus1-based 9-NC-SD using a simple and highly reproducible freeze-drying method. The water solubility of 9-NC in the solid dispersion was dramatically improved by 291 times compared to free 9-NC thus resulting in greatly increased dissolution rates and improved transport performance across the Caco-2 cell monolayer compared to free 9-NC. In vitro cell uptake studies demonstrated that 9-NC-SD were internalized into enterocytes in a cholesterol and energy-dependent manner via clathrin-mediated endocytosis and macropinocytosis. The pharmacokinetic study demonstrated that 9-NC-SD displayed much higher oral bioavailability compared to free 9-NC in rats. Moreover, 9-NC-SD was shown to have better therapeutic efficacy in S180 tumor-bearing mice and minimum gastrointestinal toxicity in rats. Thus, Soluplus1-based solid dispersion system showed a great potential in improving the solubility, oral bioavailability, antitumor effects and safety of poorly water soluble drugs for oral administration.
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Authors’ contributions
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Yao Fu,Tao Gong: conceived and designed the study. Xianghong Lian: carried out trials, generated laboratory data, preliminary analysis of the data and writing the initial article. Jianxia Dong: have a hand in the antitumor activity and gastrointestinal toxicity
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Please cite this article in press as: Lian, X., et al., Soluplus1 based 9-nitrocamptothecin solid dispersion for peroral administration: Preparation, characterization, in vitro and in vivo evaluation. Int J Pharmaceut (2014), http://dx.doi.org/10.1016/j.ijpharm.2014.10.055
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test. Jinjie Zhang, Qin Lin: data analysis, manuscript writing and modification. Yanwei Teng: collected plasma samples. All authors read and approved the final manuscript.
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Acknowledgements
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We are grateful for the financial support of the National Basic Research Program of China (973 program, No.: 2013CB932504) and the National S&T Major Project of China (Grant No.: 2012ZX09304004).
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IJP 14423 1–9 International Journal of Pharmaceutics xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
International Journal of Pharmaceutics journal homepage: www.elsevier.com/locate/ijpharm
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Soluplus1 based 9-nitrocamptothecin solid dispersion for peroral administration: Preparation, characterization, in vitro and in vivo evaluation Xianghong Lian, Jianxia Dong, Jinjie Zhang, Yanwei Teng, Qing Lin, Yao Fu 1, *, Tao Gong 2, * Key Laboratory of Drug Targeting, Ministry of Education, Sichuan University, Chengdu, China
A R T I C L E I N F O
A B S T R A C T
Article history: Received 20 August 2014 Received in revised form 17 October 2014 Accepted 26 October 2014 Available online xxx
Our study aimed to develop an amorphous 9-nitrocamptothecin solid dispersion (9-NC-SD) using polyvinyl caprolactam–polyvinyl acetate–polyethylene glycol graft copolymer (Soluplus1) for improving its oral bioavailability and antitumor efficacy in vivo. Freeze-dried 9-NC-SD with an optimized drug/ polymer ratio at 1:15 (w/w) was characterized by powder X-ray diffraction, scanning electron microscopy and Fourier transform infrared spectroscopy. The amorphous form of 9-NC was obtained by freeze-drying and the aqueous solubility of 9-NC was increased to 1.42 mg/mL. Upon dilution, 9-NC-SD was proven to form micellar structures with an average size distribution around 58 nm 5 nm (PDI = 0.107 0.016). Moreover, 9-NC-SD showed significantly increased intracellular uptake efficiency in Caco-2 cells compared to free 9-NC. Furthermore, the AUC0–8 h of 9-NC-SD following oral administration showed a 2.68-fold increase in the lactone form of 9-NC compared to that of free 9-NC in Sprague-Dawley rats. The 9-NC-SD did not show obvious inflammatory responses and gastrointestinal toxicity following oral administration as demonstrated by the histological analysis of the rat intestinal sections. Thus, 9-NCSD represents a promising approach for improving the solubility and oral bioavailability of drugs with poor solubility. ã 2014 Published by Elsevier B.V.
Keywords: 9-Nitrocamptothecin Soluplus1 Solid dispersion Oral bioavailability Antitumor effect
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1. Introduction
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9-nitrocamptothecin (9-NC), an analog of camptothecin (CPT), is a potent chemotherapeutic compound for the treatment of pancreatic cancer and other solid tumors (Haglof et al., 2006). As a second-generation topoisomerase-I inhibitor, pharmacological studies found that 9-NC displayed better antitumor activity than CPT and other CPT analogs in xenografted human tumors in nude mice (Pantazis et al., 1993). The antitumor efficacy of 9-NC is structure-specific: the pentacyclic structure of 9-NC with an a-hydroxy-d-lactone moiety shows high antitumor activity (Fig. 1A) (Saha et al., 2013). However, under physiological conditions, 9-NC may undergo ring opening hydrolysis which leads to the formation of the inactive carboxylate form (Fig. 1B)
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* Corresponding authors at: Key Laboratory of Drug Targeting, Ministry of Education, Sichuan University, No. 17, Section 3, Southern Renmin Road, Chengdu 610041, China. E-mail addresses: [email protected] (Y. Fu), [email protected] (T. Gong). 1 Tel.: +86 28 8550 3798. 2 Tel.: +86 28 8550 1615.
(Gao et al., 2008). Thus, maintaining 9-NC in its active lactone form represents the primary goal for formulation development. Despite the antitumor potency of 9-NC, the oral bioavailability of 9-NC remained extremely low mainly due to its poor water solubility (<5 mg/mL in distilled water at 25 C) (Verschraegen et al., 1998), and low permeability (Sha and Fang, 2004), thus resulting in poor therapeutic efficacy and various adverse effects such as neutropenia, thrombocytopenia and gastrointestinal reactions (Venditto and Simanek, 2010). To improve the solubility and bioavailability of 9-NC, oil and glycol solutions (Burcham et al., 1997), self-emulsifying drug delivery systems (Lu et al., 2008) and liposomes (Knight et al., 1999) were explored during the past decade, which inevitably involved the usage of oils and surfactants such as Tween 80, Cremophor EL, which were proven to induce hypersensitive reactions in humans (Tong et al., 2012). Recently, a liposomal aerosol formulation has been successfully developed and already subjected to preclinical and clinical studies (Knight et al., 2000). However, the aerosol formulations must require special devices such as inhalers for administration which is less user-friendly and cost-effective compared to oral formulations (Smith and Parry-Billings, 2003). Thereby, solid dispersions, i.e.,
http://dx.doi.org/10.1016/j.ijpharm.2014.10.055 0378-5173/ ã 2014 Published by Elsevier B.V.
Please cite this article in press as: Lian, X., et al., Soluplus1 based 9-nitrocamptothecin solid dispersion for peroral administration: Preparation, characterization, in vitro and in vivo evaluation. Int J Pharmaceut (2014), http://dx.doi.org/10.1016/j.ijpharm.2014.10.055
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drug compounds that are dispersed amorphously in a hydrophilic matrix material, appear to be an attractive alternative for enhancing drug solubility and bioavailability (Chiou and Riegelman, 1971). In addition, solid dispersions can be further developed into dosage forms for oral administration such as tablets and capsules, which are highly acceptable in patients. Soluplus1, a polyvinyl caprolactam–polyvinyl acetate–polyethylene glycol (57/ 30/13) graft copolymer, has been extensively used in the fourthgeneration of solid dispersions such as itraconazole (Zhang et al., 2013), carvedilol (Shamma and Basha, 2013), and CPT (Thakral et al., 2012). As a solubility enhancing excipient, Soluplus1 carries multiple advantages such as minimum toxicity, low hygroscopicity and amphiphilicity. Compared with matrix materials such as Solutol1 HS 15, Cremophor1 RH 40 and Tween1 80 (Strickley, 2004), Soluplus1 can serve either as a solubilizer to form micellar structures in the aqueous medium or as a matrix material for solid dispersions. Moreover, Soluplus1 based solid dispersions were demonstrated to significantly improve the solubility or oral bioavailability of poorly water-soluble drugs (Kawabata et al., 2011). In this study, the Soluplus1-based solid dispersion system has been applied for the first time to 9-NC. The present study aimed to develop and characterize the optimal formulation of Soluplus1 based solid dispersions for 9-NC via freeze-drying, and to assess its oral bioavailability, antitumor activity and gastrointesetinal safety compared with free 9-NC suspensions and 9-NC/Soluplus1 physical mixtures in animals. In addition, a preliminary study was conducted to explore the absorption mechanism of 9-NC-SD using Caco-2 cell monolayers in the current study.
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2. Materials and methods
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2.1. Materials
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Soluplus1 was kindly gifted by BASF SE (Ludwigshafen, Germany). 9-NC (purity >98.0%) was purchased from Chengdu Lanbei Plant & Chemical Technology Co., Ltd. (China). Dulbecco’s modified eagle’s medium (DMEM), fetal bovine serum (FBS), Hank ’s balanced salt solution (HBSS), L-glutamine, penicillin–streptomycin, trypsin–EDTA and non-essential amino acids were obtained from Gibco Laboratory (Invitrogen Co., Grand Island, NY, USA). Transwell permeable membrane inserts (0.4 mm pore size, 1.12 cm2 membrane area, polycarbonate) were purchased from Corning Incorporated Life Science (Lowell, MA, USA). All other chemical reagents were of HPLC grade.
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2.2. Cell culture
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Caco-2 cells were obtained from American Type Culture Collection (Manassas, CA, USA). Caco-2 cells (passages: 40–50) were cultured under the same condition as previously reported (Gao et al., 2011). For cellular uptake studies, cells were seeded in 12-well plates at a density of 50,000 cells/cm2 and cultured for
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14 days. For transport experiments, cells were seeded on transwell permeable membrane inserts at a density of 50,000 cells/cm2 in 12-well plates and cultivated for 21 days. Monolayers with transepithelial electrical resistance (TEER) value higher than 600 V cm2 were used in the transport assay.
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2.3. Animals
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Healthy male Sprague-Dawley rats were obtained from the Experimental Animal Center of Sichuan University (Chengdu, China). ICR mice were supplied by Chengdu Dossy Biological Technology Co., Ltd. (Chengdu, China). All animal experiments were approved by Sichuan University Animal Ethical Experimentation Committee (Chengdu, China), according to the requirements of the National Act on the use of experimental animals (People’s Republic of China).
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2.4. LC-MS/MS
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A HPLC tandem triple quadrupole mass spectrometry (LC-MS/ MS, 6410B, Agilent Technologies, Santa Clara, CA, USA) was used to analyze 9-NC. A diamonsil column (ODS, 50 4.6 mm, 1.8 mm) was used. The column was maintained at 30 C. The mobile phase consisted of 0.02% formic acid and methanol (55:45, v/v). The flow rate was set at 0.4 mL/min. The injection volume was 3 mL. The quantitative analysis was performed using multiple reaction monitoring (MRM) mode. General mass spectrometer conditions were listed as follows: 9-NC transition (m/z), 394–350; fragmentor, 169 eV; collision energy, 24 eV; gas temperature, 350 C; nebulizer pressure, 35 psi and spray voltage, 4000 V.
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2.5. Sample preparation and solubility test
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The 9-NC-SD was prepared by freeze-drying. Mixtures of 9-NC and Soluplus1 with varying w/w ratios such as 1:5, 1:10, 1:15, 1:20 and 1:25 were dissolved in dimethylsulfoxide (DMSO). The solutions were frozen at 40 C for 8 h, and then lyophilized for 24 h at 45 C using Allegra X-22R freeze-dryer (Beckman Coulter, USA). Physcial mixtures of 9-NC and Soluplus1 (9-NC-PM) of same w/w ratios were obtained by simple mixing in a mortar with pestle using geometric dilutions. An excess amount of free 9-NC, 9-NC-PM and 9-NC-SD were placed in 5 mL of distilled water, and incubated in a shaking water bath at 37 0.5 C for 48 h. The supernatant was collected and filtered through a 0.45 mm membrane filter, and diluted appropriately with methanol. The concentration of 9-NC was then determined by LC-MS/MS.
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2.6. Characterization of physicochemical properties
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X-ray diffraction (X-RD) patterns of 9-NC, Soluplus1, 9-NC-PM, and 9-NC-SD were recorded by an X-ray diffractometer (X’Pert Pro MPD, The Netherlands Philips), using nickel-filtered Cu Ka
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radiation generated at 40 kV and 25 mA and scanning rate of 2 / min over a 2u range of 10–50 . The Fourier transform infrared (FITR) spectra (Nicolet 6700, Thermo Electron Corporation, USA) was used to characterize the possible interactions between the 9-NC and Soluplus1 in 9-NC-SD. 5 mg of each sample was lightly grounded and mixed with KBr at range 4000–500 cm1 with a resolution of 2 cm1. The surface morphology of 9-NC and 9-NC-SD were observed under a scanning electron microscope (SEM) (JSM-7500F scanning microscope, Tokyo, Japan) operated at 5.0 kV. The samples were mounted on a glass stub with double-sided adhesive tape and coated under vacuum with gold in an argon atmosphere prior to observation. The lyophilized 9-NC-SD product was dispersed in ddH2O to form homogeneous micellar solution. The particle size and polydispersity index of the 9-NC-SD micelles were measured by dynamic light scattering (Malvern Zetasizer Nano ZS90, Malvern Instruments Ltd. Malvern, UK) at 25 C. 5 mg/mL of sample solution was measured accordingly with no further dilution. The morphology of 9-NC-SD micelles was observed under a transmission electron microscope (TEM) (Tecnai G2F20, FEI, Eindhoven, The Netherlands). The sample was placed on copper grids and stained with 2% (w/v) phosphotungstic acid for 30 s. After the excess solution was drawn off, the sample was dried and then subjected to TEM observation. Dissolution profiles of free 9-NC and 9-NC-SD were evaluated by the paddle method at a rotation speed of 75 rpm (Jiang et al., 2010). Briefly, 1 mg of free 9-NC and equivalent amount of 9-NC-SD were added to 0.9 L dissolution media (0.1 M hydrochloric acid with 0.05% Tween 80) at 37 0.5 C. At predetermined times, 2 mL of dissolution media was withdrawn and replaced by equal volume of fresh media. The samples were filtered through 0.45 mm membrane filter, diluted with methanol (1:1, v/v) and analyzed by LC-MS/MS. 2.7. Cellular uptake of 9-NC, 9-NC-PM and 9-NC-SD To evaluate the cell uptake efficiency of 9-NC-SD micelles, Caco2 cells were incubated with 9-NC, 9-NC-PM or 9-NC-SD micelles (5 mg/mL) at 37 C. For energy depletion, the cellular uptake were performed at 4 C and 37 C for 1 h. Next, the cells were subjected to three freeze-thawing cycles following detachment from plates. 20 mL of 0.1 M glacial acetic acid and 200 mL of ice-cold methanol/ acetonitrile (1:2, v/v) were added to 100 mL of detachment cells sample. The mixture was vortexed for 1 min and centrifuged at 12,500 rpm for 10 min. The supernatant was collected and filtrated to 0.22 mm membrane filter to LC-MS/MS analysis. The cell uptake amount was analyzed using LC-MS/MS. To explore the mechanism of 9-NC-SD micelle mediated internalization, Caco-2 cells were pre-incubated with indicated inhibitors for 1 h and then incubated with 9-NC-SD for another 30 min. Chlorpromazine (Chlo, 10 mg/mL) was an inhibitor of clathrin-mediated endocytosis (Mahmud and Lavasanifar, 2005). Nystatin (25 mM) was an inhibitor of cholesterol disrupted caveolae-mediated endocytosis (Nabi and Le, 2003). MbCD (10 mg/mL) with 1 mg/mL simvastatin (MS), an inhibitor of de novo synthesis of cholesterol, inhibited both caveolae and clathrinmediated pathways by cholesterol depletion (Mayor and Pagano, 2007). Dimethyl amiloride (DMA, 5 mg/mL) was an indicator of macropinocytosis (Sun et al., 2010). The procedures were kept the same as mentioned above. 2.8. Transport across Caco-2 monolayers
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Caco-2 monolayers were developed to investigate the transport behavior of 9-NC and 9-NC-SD across the intestinal membrane as
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described previously (Sha and Fang, 2004). Before the experiment, the cell monolayers were washed twice with HBSS followed by incubating at 37 C for 20 min. HBSS solution on both sides of the cell monolayers was then removed. For the measurement of the apical side (AP) to basolateral side (BL) transport, 0.5 mL of HBSS (pH 6.0) containing: (i) the pure 9-NC solution (5 mg/mL), (ii) the 9NC-SD micelles (5 mg/mL) were added on the AP side, and 1.5 mL of HBSS (pH 7.4) without 9-NC was added to the BL side. The monolayers were incubated at 37 C and shaken at 50 rpm in a shaker. Samples were taken from the BL side at 15, 30, 45, 60, 90, 120 min and replaced by an equal volume of prewarmed fresh HBSS. Samples were then diluted in a 1:1 ratio with methanol before LC-MS/MS analysis. The transepithelial electrical resistance (TEER) of monolayer was measured using Millicell electrical resistance system (Millipore, USA) to evaluate the formation of the monolayer and its integrity during the experiment. To observe how 9-NC-SD micelles were transported across the cell monolayer, cell monolayers were incubated with HBSS as a control and with 9-NC-SD micelles at 37 C for 2 h, respectively. The basolateral medium was then collected, treated with uranyl acetate as a contrasting agent which fixes preferentially the periphery of the particles, and observed under a transmission electron microscopy (TEM).
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2.9. Pharmacokinetics
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Male Sprague-Dawley rats (180 10 g) were fasted for 12 h before drug administration with free access to water. 9-NC and 9NC-PM suspension was prepared by adding 9-NC or 9-NC-PM (1:15, w/w) into 0.5% carboxymethylcellulose sodium (CMC-Na) solution and then ultrasonicated for several minutes to obtain homogenous suspensions. 9-NC-SD was dissolved in diH2O. Three groups of rats were orally administrated with 9-NC suspension, 9NC-PM, and 9-NC-SD at an equivalent dose of 6 mg/kg 9-NC. The blood samples (about 0.3 mL) were collected via orbital venous at predetermined time intervals (5, 15, 30, 45, 60, 90, 120, 180 min). The samples were then centrifuged at 5000 rpm for 5 min to obtain the plasma. 20 mL of 0.1 M glacial acetic acid and 200 mL of ice-cold methanol/acetonitrile (1:2, v/v) were added to 100 mL of plasma sample. The mixture was vortexed for 1 min and centrifuged at 12,500 rpm for 10 min. The supernatant was collected and filtrated to 0.22 mm membrane filter to LC-MS/MS analysis.
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2.10. Antitumor effects in sarcoma 180 tumor-bearing mice
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In vivo antitumor activity was evaluated in sarcoma 180 (S180) tumor bearing mice (Jiang et al., 2011). S180 cells were transplanted to the intraperitoneal cavity of ICR mice, and then collected from the mice at fixed time points. The S180 cells (1 106 cells/ 0.2 mL) were inoculated subcutaneously in ICR mice (20 2 g) at the right armpit. To simulate the early stage of tumor growth, the dose schedule was started at 24 h after transplantation. The animals were randomly divided into four groups with five mice in each group. The negative control group received saline (0.9% NaCl) and blank Soluplus1 solution (0.75%). Mice were treated with 9-NC and 9-NC-SD at an equivalent dose of 4 mg/kg 9-NC on day 1 and day 4 after transplantation. All mice were sacrificed on day 11 to collect tumor tissues, which were washed with saline and accurately weighed. The inhibitory rate of tumor (IRT%) was calculated according to the following equation:
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IRT% ¼
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3
W control W treated 100% W control
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2.11. Gastrointestinal toxicity
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Male Sprague-Dawley rats (200 20 g) were fasted for 12 h before drug administration, and were randomized into four groups. The control group received saline, blank Soluplus1 (0.75%); treatment groups were orally administered with 9-NC suspension and 9-NC-SD at an equivalent dose of 6 mg/kg 9-NC for 2 continuous days. 48 h after the last administration, all rats were sacrificed. Afterwards, the duodenum, jejunum, ileum and colon were collected and then cleaned. The samples were fixed with 4% polyoxymethylene for at least 48 h and then embedded in paraffin. Tissue sections of 5 mm thickness were stained with hematoxylin and eosin (H&E). Injuries such as villus atrophy, bleeding, hyperaemia, glandular destruction, interstitial edema and inflammatory cell infiltration were carefully examined, and photographed under light microscope (Axiovert 40CFL, Germany).
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2.12. Statistical analysis
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The pharmacokinetic parameters including area under the concentration–time curve (AUC), time to peak plasma concentration (Tmax) and biological half life (T1/2) were calculated using DAS 3.0 (BioGuider Co., Shanghai, China). Data represent mean standard deviation (SD). The statistical analysis was performed using Student’s t-test for comparison between two groups and one-way ANOVA for comparison among multiple groups. A p-value less than 0.05 was considered as significantly different.
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3. Results
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3.1. Physicochemical properties and dissolution
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Table 1 shows the water solubility of 9-NC in 9-NC-PM and 9NC-SD, respectively. For 9-NC-SD, the solubility of 9-NC increased slightly with the increasing amount of Soluplus1 mainly due to the micelle solubilization effect of Soluplus1. 9-NC in different 9-NCSD formulations displayed water solubilities over 1.3 mg/mL, which were significantly higher than in corresponding 9-NC-PM of the same 9-NC to Soluplus1 weight ratio (p < 0.01). In addition, 9-NC-SD formulation with 1:15 (w/w) 9-NC/Soluplus1 achieved a saturated solubility of 1.42 mg/mL and appeared to reach saturation in the solubility test, where the solubility of 9-NC no longer increased when increasing the percentage of Soluplus1. Thus, 9NC-SD with a drug/Soluplus1 ratio (w/w) of 1:15 was selected for the following study. 9-NC is a highly crystalline molecule with main characteristic crystalline peaks at 7.50, 20.97, 22.63, 25.70 and 27.41-u degrees (Fig. 2A). In comparison, the characteristic peaks of 9-NC were absent in 9-NC-SD, suggesting a conversion of the crystalline form of 9-NC to its amorphous form. To investigate the effect of freeze-drying, FTIR was used to elucidate possible interactions between 9-NC and Soluplus1. Interactions between active pharmaceutical ingredient and excipients may be relevant to stabilizing 9-NC-SD in the highenergy amorphous state. CPT was reported to form hydrogen bonds with Soluplus1 (Thakral et al., 2012), and as a derivative of CPT, 9-NC was considered to interact with Soluplus1 by forming
Table 1 Solubility of 9-NC, 9-NC-PM, and 9-NC-SD in water at 25 C. Data represent mean S.D. (n = 5). 9-NC/Soluplus (w/w)
9-NC-PM (mg/mL)
9-NC-SD (mg/mL)
1:10 1:15 1:20 1:25
39.70 2.13 66.77 8.54 54.32 6.12 85.14 12.12
1322.47 65.14 1480.30 50.12 1399.15 48.56 1482.67 60.38
intermolecular hydrogen bonds. As shown in the FTIR spectrum (Fig. 2B), 9-NC displayed —NO2 stretching at 1511 cm1 and 1388 cm1,—OH stretching at 3410 cm1, and carbonyl stretching for cyclic ester (lactone) at 1736 cm1. Soluplus1 showed intermolecularly hydrogen bonded —OH stretching in the range of 3342–3350 cm1, ester carbonyl stretching 1729–1635 cm1, and C¼O stretching for tertiary amide at 1729 cm1. No spectral shift was found in the 9-NC-PM, which was a simple superposition of 9NC and Soluplus1, indicating no interactions between drug and polymer. However, in the FTIR spectrum of 9-NC-SD, —NO2 stretching peak was covered by numerous small minor peaks, and —OH stretching peaks of Soluplus1 appeared at 3424 cm1 and became slightly broader, indicating that Soluplus1 might interact with 9-NC by forming intermolecular hydrogen bonds. As for the surface morphology, the SEM image of bulk 9-NC presented large, inerratic clavate crystals (Fig. 2C). In contrast, 9-NC-SD displayed in the flake form and no 9-NC crystals were observed (Fig. 2C). Within 10 h, the cumulative dissolution percentage of 9-NC-SD was significantly higher than that of free 9-NC at predetermined time points (Fig. 2D). In addition, the amount dissolved from free 9-NC was less than 58% in the first 90 min, while nearly 92% achieved cumulative dissolution from 9-NC-SD within 90 min.
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3.2. Particle size, morphology and endocytosis of 9-NC-SD after dispersion in water
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After dispersion in water, 9-NC-SD would assemble into micellar structures with the mean particle size of about 58 nm 5 nm with a polydispersity index of 0.107 0.016 (Fig. 3A) as determined by DLS measurement. As shown in TEM images, 9-NCSD micelles displayed near spherical shapes and a uniform morphology with an average particle size of around 60 nm (Fig. 3B). During 30 min incubation, cellular uptake of 9-NC-SD micelles increased significantly compared with that of free 9-NC and 9-NCPM (Fig. 4A). The cellular uptake efficiency of 9-NC-SD micelles increased to 146 ng/mL at 30 min, which was about 1.44 and 1.38 times that of free 9-NC and 9-NC-PM, respectively. However, the physical mixture of 9-NC and blank Soluplus1 did not show significant impact on cellular uptake efficiency of 9-NC (Fig. 4A). Nevertheless, incubating Caco-2 cells at 4 C significantly inhibited the cell uptake efficiency of 9-NC-SD micelles compared with 37 C (Fig. 4B), suggesting an energy-dependent absorption behavior. To elucidate the possible endocytic mechanism of 9-NCSD micelles, an in-depth cell uptake study was conducted using various endocytosis inhibitors (Fig. 4C). Nystatin did not affect the cell uptake of 9-NC-SD micelles indicating the endocytic pathway is not caveolae-mediated. Chlorpromazine, an inhibitor of clathrinmediated endocytosis, resulted in a 15.0% reduction in cell uptake, indicating that clathrin other than caveolae was involved in the internalization of 9-NC-SD micelles. MbCD (10 mg/mL) with 1 mg/ mL simvastatin, which inhibited both caveolae and clathrinmediated pathways by cholesterol depletion, decreased the cell uptake by 63.58%, indicating that other cholesterol-dependent endocytosis pathways such as macropinocytosis, may also be involved in the cell entry of 9-NC-SD micelles. A 34.46% decrease in the cell uptake of 9-NC-SD micelles was observed while inhibiting macropinocytosis by dimethyl amiloride, suggesting that the transport of 9-NC-SD micelles was mainly driven via macropinocytosis. Taken together, 9-NC-SD micelles was likely internalized into enterocytes via macropinocytosis and clathrindependent pathways.
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Fig. 2. 362
3.3. Transport across Caco-2 monolayers
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The crystalline form of free 9-NC showed a flux of 2.5 ng/min/ cm2 (Fig. 4D), while 9-NC-SD showed a relatively higher transport rate of 8.0 ng/min/cm2 across Caco-2 cells, which was increased by 3.2 times compared to free 9-NC. Besides, the cumulative absorption amount of 9-NC in 9-NC-SD at 2 h were significantly increased by 2.94-fold compared to free 9-NC. Fig. 3C shows the TEM images of the apical and basolateral side media at 2 h after applying 9-NC-SD micelles or blank HBSS to Caco-2 cell monolayers. After applying blank HBSS, fragments of 9NC crystals were observed on both the apical and basolateral sides. After applying 9-NC-SD micelles to the apical side, near spherical particles of approximately 50–70 nm were observed on the basolateral side, which were similar to those observed at the apical side. Based upon above results, 9-NC-SD micelles were likely endocytosed and exocytosed across the Caco-2 monolayers in the micellar form, which remains to be further elucidated.
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3.4. Pharmacokinetics
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The plasma concentration–time profiles of lactone and total ( lactone + carboxylate) of 9-NC in healthy Sprague-Dawley rats following oral administration of 9-NC, 9-NC-PM, and 9-NC-SD were shown in Fig. 5. 9-NC-SD displayed a pharmacokinetic profile dramatically different from that of free 9-NC or 9-NC-PM. Specifically, 9-NC-SD showed significantly higher Cmax values than 9-NC and 9-NC-PM (p < 0.01) (Table 2). As for the total form of 9NC, 9-NC-SD also displayed significantly higher AUC0–8 h values than 9-NC and 9-NC-PM (p < 0.01) (Table 2). Approximately 1.91and 1.93-fold increases in AUC0–8 h values for the total content were observed for 9-NC-SD as compared to the 9-NC suspension and 9NC-PM (p < 0.01), respectively. For the lactone form of 9-NC, the AUC0–8 h values were 745.48 18.42 ng h/mL for 9-NC-SD,
278 27.42 ng h/mL for free 9-NC suspension and 230.63 13.56 ng h/mL for 9-NC-PM. Thus, 9-NC-SD displayed approximately 2.68-fold and 3.23-fold increases of AUC0–8 hlactone compared to the 9-NC suspension and 9-NC-PM (p < 0.01). Importantly, compared to free 9-NC, A 2.68-fold increase in AUC0–8 h for the lactone content and a 1.93-fold increase in AUC0–8 h for the total content were observed for 9-NC-SD.
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3.5. Antitumor activity
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9-NC is an active chemotherapeutic compound with a broad spectrum of antitumor activity, especially for pancreatic cancers (Konstadoulakis et al., 2001). In the current study, three formulations were orally administered at a 3-day interval from the day after S180 inoculation. The photographs and the growth inhibitory rate (%) of tumors were shown in Fig. 6, respectively. The Soluplus1 alone showed no obvious inhibitory effect on tumor growth compared to the saline group. In contrast, free 9-NC suspension and 9-NC-SD groups showed comparable strong inhibitory effect against tumor growth compared to the saline and Soluplus1 groups. Moreover, 9-NC-SD showed significantly higher tumor growth inhibitory rate (IRT%) than 9-NC suspension (p < 0.05), which can be attributed to the higher oral bioavailability of 9-NC-SD than that of free 9-NC.
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3.6. Gastrointestinal toxicity
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Gastrointestinal toxicity has been one of the most commonly observed side effects of 9-NC in clinic (Garcia-Carbonero and Supko, 2002). In this regard, histological analysis of the gastrointestinal tract in rats was performed to gain further insight into the impact of 9-NC-SD. In the free 9-NC treatment group, the duodenum, jejunum and ileum showed severe intestinal villus atrophy and glandular destruction (Fig. 7). Besides, moderate
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Fig. 3.
Fig. 4.
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Fig. 5. Table 2 Pharmacokinetic parameters of lactone, and total 9-NC after oral administration of free 9-NC, 9-NC-PM, and 9-NC-SD to rats. Data represent mean S.D. (n = 5). Parameters
Cmax (ng/ml) Tmax (h) MRT (h) T1/2 (h) AUC0–8 h (ng/mL h) *
Lactone of 9-NC
Total of 9-NC
Free 9-NC
9-NC-PM
9-NC-SD
Free 9-NC
9-NC-PM
9-NC-SD
147.4 60.31 0.5 0.01 1.6 0.16 1.5 0.21 278 63.48
144.25 72.6 0.75 0.16 1.3 0.28 1.7 0.02 230.63 166
668.92 175.54* 0.3 0.13 0.98 0.43* 1.15 0.22 745.48 166*
225.02 64.01 0.5 0.12 1.6 0.15 1.42 0.31 471.14 131
255.85 114.35 0.95 0.13 1.5 0.25 1.81 0.12 467.96 197
1097 171.42* 0.25 0.01 0.85 0.16* 0.91 0.33 903.03 175*
p < 0.01 vs. free 9-NC.
Fig. 6. 423
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interstitial edema and mucosal congestion and inflammatory cell infiltration were also observed in the free 9-NC treatment group (Fig. 7). In comparison, 9-NC-SD displayed a slight extent of intestinal epithelial cell degeneration (Fig. 7). Moreover, 9-NC-SD and free 9-NC did not show obvious pathological symptoms in the stomach and colon, which is probably because colon and stomach were not the primary absorption site for 9-NC-SD. Additionally, no significant defects were detected in the Soluplus1 and saline group indicating the matrix material Soluplus1 is biocompatible and could be a suitable candidate for orally administered dosage forms.
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4. Discussions
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Hot-melt extrusion (HME) technique has been one of the most popular methods to prepare solid dispersions, which was first established on an industrial scale in 1930 s (Rauwendaal, 2014) and has gained extensive application in pharmaceutical field since 1971 (Chiou and Riegelman, 1971). HME method was commonly used when the drug and the matrix material display close melting
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points. However, HME cannot be used to prepare the solid dispersion of 9-NC in Soluplus1, because 9-NC has a relative high melting temperature of 219.58 C while Soluplus1 has a relative lower melting point around 70 C (Sambath et al., 2013). Thus, freeze-drying method was selected which is more suitable for drugs and excipients of dramatically different melting points. Also, the nucleation of drug crystals was minimized or completely prevented during lyophilization thus forming amorphous and highly porous product (Yu, 2001). 9-NC was poorly water soluble (<5 mg/mL in distilled water at 25 C), while 9-NC-SD with 1:15 (w/w) of 9-NC/Soluplus1 achieved a saturated 9-NC solubility of 1.42 mg/mL, which was dramatically higher than the reported solubility achieved by hydroxypropyl-bcyclodextrin (0.52 mg/mL) (Jiang et al., 2010). As demonstrated by XRD, SEM and FTIR analysis, 9-NC and Soluplus1 is likely to form a molecular dispersion thus resulting in greatly increased solubility of 9-NC. As an amphiphilic polymer with a hydrophilic PEG block, Soluplus1 was proven a good solubilizer for water-insoluble drugs such as itraconazole (Zhang et al., 2013), carvedilol (Shamma and
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Basha, 2013), and CPT (Thakral et al., 2012). Moreover, the hydrophobic vinylcaprolactam/vinylvacetate block might help stabilize the lactone structure of 9-NC to avoid hydrolysis. Interestingly, 9-NC-SD formed homogeneous micelles with an average size diameter of 58 nm after dispersed in water, which significantly increased the intracellular uptake of 9-NC in Caco2 cells. Caco-2 cells were reported to express multiple efflux transporters, e.g., P-gp or MRP2 which might be involved in inhibiting the transport of 9-NC across the cell monolayer (Sha and Fang, 2004). However, the 9-NC-PM did not show significant effect on cell uptake (Fig. 4A), which was consistent with that low concentrations of Soluplus1 had a negligible impact on P-gp mediated drug efflux (Yu et al., 2013). In addition, to elucidate the possible endocytic mechanism of 9NC-SD micelles, an in-depth cell uptake study was conducted using various endocytosis inhibitors (Fig. 4C), which demonstrated that 9-NC-SD micelles could be internalized into enterocytes via clathrin-mediated endocytosis and macropinocytosis. Two possible routes were proposed for the transport of micelles across the Caco-2 cell monolayer, i.e., paracellular and/or transcellular transport. No obvious change in the TEER values of the monolayer during the period of transport experiments (data not shown) indicated that the 9-NC-SD micelles were not transported across the monolayer by paracellular route. Thus, 9-NC-SD may be transported across the Caco-2 monolayer via the transcellular route. Specifically, 9-NC-SD could be uptaken by Caco-2 cells at the apical side of the monolayer via clathrin-dependent pathway and macropinocytosis, and then exocytosed from the basolateral side. Moreover, 9-NC-SD displayed a pharmacokinetic profile with dramatically increased AUC0–8 h compared with free 9-NC suspension and 9-NC-PM. The high-energy solid dispersions might form supersaturated solutions in the intestine, and the polymeric carrier could act as a stabilizer of supersaturation upon drug release (Brouwers et al., 2009). Due to the higher absorption of 9-NC-SD, 9NC-SD showed significantly higher tumor growth inhibitory rate (IRT%) in the animal study. Furthermore, antitumor drugs frequently induce serious side effects such as neutropenia, thrombocytopenia, diarrhea and hemorrhagic cystitis (GarciaCarbonero and Supko, 2002). According to previous studies, the
carboxylic salt form of 9-NC resulting from hydrolysis was shown to induce various adverse effects (Giovanella et al., 1991). In our study, animals treated with free 9-NC exhibited severe gastrointestinal reactions such as intestinal villus atrophy and glandular destruction. In contrast, 9-NC-SD did not show obvious gastrointestinal reactions. As previously discussed, 9-NC-SD greatly improved the absorption of lactone of 9-NC in the intestine and reduced the accumulation of carboxylic acid salts in the intestine thus resulting in the minimum gastrointestinal toxicity. In addition, the reduced Tmax and MRT (p < 0.05) of 9-NC-SD (Table 2), indicated 9-NC-SD was rapidly absorbed, thereby alleviating the contact of carboxylic acid salt form to the intestinal tracts.
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5. Conclusion
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We have successfully developed Soluplus1-based 9-NC-SD using a simple and highly reproducible freeze-drying method. The water solubility of 9-NC in the solid dispersion was dramatically improved by 291 times compared to free 9-NC thus resulting in greatly increased dissolution rates and improved transport performance across the Caco-2 cell monolayer compared to free 9-NC. In vitro cell uptake studies demonstrated that 9-NC-SD were internalized into enterocytes in a cholesterol and energy-dependent manner via clathrin-mediated endocytosis and macropinocytosis. The pharmacokinetic study demonstrated that 9-NC-SD displayed much higher oral bioavailability compared to free 9-NC in rats. Moreover, 9-NC-SD was shown to have better therapeutic efficacy in S180 tumor-bearing mice and minimum gastrointestinal toxicity in rats. Thus, Soluplus1-based solid dispersion system showed a great potential in improving the solubility, oral bioavailability, antitumor effects and safety of poorly water soluble drugs for oral administration.
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Authors’ contributions
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Yao Fu,Tao Gong: conceived and designed the study. Xianghong Lian: carried out trials, generated laboratory data, preliminary analysis of the data and writing the initial article. Jianxia Dong: have a hand in the antitumor activity and gastrointestinal toxicity
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Please cite this article in press as: Lian, X., et al., Soluplus1 based 9-nitrocamptothecin solid dispersion for peroral administration: Preparation, characterization, in vitro and in vivo evaluation. Int J Pharmaceut (2014), http://dx.doi.org/10.1016/j.ijpharm.2014.10.055
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test. Jinjie Zhang, Qin Lin: data analysis, manuscript writing and modification. Yanwei Teng: collected plasma samples. All authors read and approved the final manuscript.
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Acknowledgements
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We are grateful for the financial support of the National Basic Research Program of China (973 program, No.: 2013CB932504) and the National S&T Major Project of China (Grant No.: 2012ZX09304004).
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Please cite this article in press as: Lian, X., et al., Soluplus1 based 9-nitrocamptothecin solid dispersion for peroral administration: Preparation, characterization, in vitro and in vivo evaluation. Int J Pharmaceut (2014), http://dx.doi.org/10.1016/j.ijpharm.2014.10.055
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