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IJP 13888 1–7 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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A novel oral delivery system consisting in ‘‘drug-in cyclodextrin-in nanostructured lipid carriers” for poorly water-soluble drug: Vinpocetine [TD$FIRSNAME]Congcong [TD$FIRSNAME.]ULin[TD$SURNAME.] a , [TD$FIRSNAME]Fen [TD$FIRSNAME.]UChen[TD$SURNAME.] a , [TD$FIRSNAME]Tiantian [TD$FIRSNAME.]UYe[TD$SURNAME.] a , [TD$FIRSNAME]Lina [TD$FIRSNAME.]UZhang[TD$SURNAME.] a , [TD$FIRSNAME]Wenji [TD$FIRSNAME.]UZhang[TD$SURNAME.] a , [TD$FIRSNAME]Dandan [TD$FIRSNAME.]ULiu[TD$SURNAME.] b , [TD$FIRSNAME]Wei [TD$FIRSNAME.]UXiong[TD$SURNAME.] a , [TD$FIRSNAME]Xinggang [TD$FIRSNAME.]UYang[TD$SURNAME.] a , [TD$FIRSNAME]Weisan [TD$FIRSNAME.]UPan[TD$SURNAME.] a, * a b
Department of Pharmaceutics, School of Pharmacy, Shenyang Pharmaceutical University, 103 Wenhua Road, Shenyang 10016, China School of Biomedical & Chemical Engineering, Liaoning Institute of Science and Technology, 176 Xianghuai Road, Benxi 117004, China
A R T I C L E I N F O
A B S T R A C T
Article history: Received 6 November 2013 Received in revised form 25 December 2013 Accepted 8 February 2014 Available online xxx
The purpose of this study was to develop a new delivery system based on drug cyclodextrin (CD) complexation and loading into nanostructured lipid carriers (NLC) to improve the oral bioavailability of vinpocetine (VP). Three different CDs and three different methods to obtain solid vinpocetine– cyclodextrin–tartaric acid complexes (VP–CD–TA) were contrasted. The co-evaporation vinpocetineb-cyclodextrin-tartaric acid loaded NLC (VP–b-CD–TA COE-loaded NLC) was obtained by emulsification ultrasonic dispersion method. VP–b-CD–TA COE-loaded NLC was suitably characterized for particle size, polydispersity index, zeta potential, entrapment efficiency and the morphology. The crystallization of drug in VP–CD–TA and NLC was investigated by differential scanning calorimetry (DSC). The in vitro release study was carried out at pH 1.2, pH 6.8 and pH 7.4 medium. New Zealand rabbits were applied to investigate the pharmacokinetic behavior in vivo. The VP–b-CD–TA COE-loaded NLC presented a superior physicochemical property and selected to further study. In the in vitro release study, VP–b-CD–TA COEloaded NLC exhibited a higher dissolution rate in the pH 6.8 and pH 7.4 medium than VP suspension and VP–NLC. The relative bioavailability of VP–b-CD–TA COE-loaded NLC was 592% compared with VP suspension and 92% higher than VP–NLC. In conclusion, the new formulation significantly improved bioavailability of VP for oral delivery, demonstrated a perspective way for oral delivery of poorly water-soluble drugs. ã 2014 Published by Elsevier B.V.
Keywords: Nanostructured lipid carriers (NLC) Cyclodextrins Vinpocetine Oral bioavailability Poorly water-soluble drugs
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1. Introduction
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Vinpocetine (ethyl-apovincamine-22-oate, VP), a derivative of the Vinca minor alkaloid vincamine, brought to the market in 1978 with the trade name Cavinton, which is well-known for the treatment of ischemic stroke and other cerebrovascular disease (Fig. 1) (Szaboet al. 1983). Its beneficial effects have been proved by many experimental studies (Bonoczk et al., 2000), the results showed that advantageous rheological effects can be maintained by per os vinpocetine treatment on chronic ischemic cerebrovascular patients. The oral drug therapy preserved the beneficial changes of the impaired red blood cell aggregation and plasma and whole blood viscosity values (Gergely et al., 2009). However, VP is a sparingly water soluble drug (the water solubility 5 mg/ml, pKa value of 7.31), has a low and irregular dissolution rate in the
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* Corresponding author. Tel.: +86 024 23986313; fax.: +86 024 23953241. E-mail address: [email protected] (W. Pan).
gastrointestinal tract because of the pH dependency and a remarkable first pass-effect , which also resulting in its low oral bioavailability about 7% in human that largely restricts its clinical use (Szakács et al., 2001). Therefore, to overcome these drawbacks, there is a need to design a novel delivery system possible to improve the oral absorption and bioavailability of VP. Lipid-based drug delivery systems are expected as promising oral carriers because of their potential to increase the solubility and improve oral bioavailability of poorly water-soluble and lipophilic drugs (O’Driscoll and Griffin, 2008). Nanostructure lipid carriers (NLCs) which developed from solid lipid nanoparticles (SLNs), consisting of a biocompatible solid lipid matrix entrapping a liquid lipid (oil) as nanocompartments, are attracting major attention as alternative colloidal drug carriers. Such a particular structure brings about distinct superiority comparing with SLN, such as improved drug loading ability, increased physical stability, reduced risks of drug leakage during storage(Müller et al., 2002; Souto and Müller, 2006). Furthermore, NLC particles could avoid the liver metabolism by forming micelles with bile salts in small intestine
http://dx.doi.org/10.1016/j.ijpharm.2014.02.013 0378-5173/$ – see front matter ã 2014 Published by Elsevier B.V.
Please cite this article in press as: Lin, C., et al. Int J Pharmaceut (2014), http://dx.doi.org/10.1016/j.aca.2013.12.001
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Fig. 1. Chemical structure of vinpocetine and b-cyclodextrin. 41
kindly donated by Roquette (France). Compritol 888 ATO (glyceryl behenate) was a gift from Gattefosse (Paris, France). Solutol HS-15 (poly-oxyethylene esters of 12-hydroxystearic acid) was sampled from BASF (Lud-wigshafen, Germany). Tartaric acid (TA) was product of Bo di chemical Co., LTD. (Tianjin, China). Soybean phosphatidylcholine (PC) that contained approximately 80% of the phosphatidyl was purchased from Aikang Chemical Co. Ltd. (Shanghai, China). Other chemicals and reagents were of analytical grade.
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2.2. Preparation and characterization of VP–CD–TA solid systems
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Physical mixtures (PMs) containing a VP:TA ratio of 2:1 (w/w) and VP with b-CD, HP–b-CD and SBE–b-CD were prepared by homogeneous blending in a mortar at a molar ratio of 1:2 (L.S.S. Ribeiro et al., 2003). Kneaded (KE) products were prepared by kneading thoroughly with least amount of water to obtain a paste which was then dried under vacuum at room temperature. Co-evaporation (COE) products were obtained by dissolving formulated amounts of CDs in water and VP in 3% (w/v) of TA solution respectively, and then adding the latter dropwise to the former. The resulting solution mixture was stirred at 100 rpm and a temperature of 60 C for 4 h. Furthermore, the obtained clear solution was evaporated under vacuum at 60 C in a rotatory evaporator. All solid residues were further dried at 40 C for 24 h and collected through 80 mesh sieve. For co-lyophilized (COL) products, a molar ratio of 2:1 of CDs and VP was dispersed in water or in 3% (w/ v) of TA solution, separately. The obtained solution mixture was sonicated for 2 h at 60 C.After equilibrium for 24 h at room temperature, the resultant solution was frozen by cryogenic refrigerator at 70 C and then the frozen solution was lyophilized in a freeze–dryer (FD-1 apparatus, Labconco) for 24 h. The thermal properties of pure components and their solid combinations were surveyed by DSC-60 differential scanning calorimeter (Shimadzu, Japan). The samples were scanned at a heating rate of 10 C/min over a temperature range of 30–250 C with a nitrogen purge of 0.2 ml/min in aluminum pan and sealed hermetically, using aluminum oxide as the reference.
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after entering in the gastrointestinal (GI) tract, which will enter the lymph circulation. Cyclodextrins (CDs), naturally available water-soluble cyclic oligosaccharides composed of a-1,4-linked-glucopyranose units (Fig. 1), have been used to modify the time of drug release during GI transit and decrease local irritation, may also alter oral absorption of drugs through mucosal membrane permeation enhancement (Stella and Rajeuski, 1997). Unfortunately, the complexation efficiency of CDs is rather low and consequently significant amounts of CDs are needed to solubilize small amounts of water-insoluble compounds. However, enhanced complexation can be obtained by formation of ternary complexes between a drug molecule, CD molecule and a third component (Loftsson and Brewster, 1996). Inclusion complexes with cyclodextrins have been reported as an effective way to increase bioavailability through improving the solubility and dissolution rate of VP. In view of all these premises, we thought it worthy of interest to investigate the feasibility of developing a novel oral drug delivery system for vinpocetine based on a combined strategy which exploits both cyclodextrin complexation, to boost drug solubility and dissolution properties, and loading of the complex into NLC, to avoid significant first-pass effect. In the shell frame of this project, considering the special chemical properties of VP itself (alkalescence), the ternary inclusion compound was prepared for the drug: b-cyclodextrin (b-CD), hydroxypropyl–b-cyclodextrin (HP–b-CD) and sulfobutylether–b-cyclodextrin (SBE–b-CD) have been selected to prepare multicomponent complexation in the presence of tartaric acid (TA, as an acidifier of the complexation medium), and the effectiveness of three different techniques (co-grinding, coevaporating and co-lyophilization) to obtain solid vinpocetine– cyclodextrin–tartaric acid (VP–CD–TA) complexes with improved drug dissolution properties has been investigated .The most effective VP–CD–TA system was screened for loading into NLC obtained by melt emulsification combined with ultra-sonication technique (Luo et al., 2011). Both empty and loaded NLC were suitably characterized for particle size, zeta potential and entrapment efficiency, evaluated on the drug release properties in vitro and oral bioavailability in vivo.
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2. Materials and methods
2.3. NLC preparation and characterization
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2.1. Materials
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Vinpocetine, the purity of which was over 99.2%, was kindly supplied by northeast general pharmaceutical Co., Ltd. (Shenyang, China). b-cyclodextrin (b-CD) and sulfobutylether–b-cyclodextrin (SBE–b-CD) were obtained from Xinda Fine Chemical Co., LTD. (Shandong, China). Hydroxypropyl–b-cyclodextrin (HP–b-CD) was
NLC were produced according to the procedure of Luo et al. (Luo et al., 2011), which was slightly altered. In brief, the solid lipid (Compritol 888 ATO 0.097 g) and Miglyol 812N (0.024 g) together were blended and melted at 85 C to form a uniform oil phase. Meanwhile, the aqueous phase was prepared by dispersing PC (0.077 g) and the surfactant (Solutol HS-15 0.127 g) in 20 mL of purified water and heated up to 85 C. Then the hot aqueous phase
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was added drop wisely to the oil phase at the same temperature with magnetic stirring at 600 rpm for 10 min. The obtained preemulsion was ultrasonified 5 min (active every 3 s for 3 s duration; 400 W) by probe-ultrasonic cell disruptor (JY-92-II; Xinzhi, Ningbo, China). Finally, the dispersion was rapidly cooled to solidify in ice bath (0 C) to form NLC. Pure drug (5 mg) or VP–CD–TA loaded NLC systems (equivalent to 5 mg VP) were performed, respectively, by adding VP or VP–CD–TA to the melted lipid phase. The mean particle size (PS), polydispersity index (PDI), and zeta potential (ZP) were determined by photon correlation spectroscopy (PCS) using a Zeta-sizer Nano (Malvern Instruments, UK) at room temperature. Avoiding multiscattering phenomena, NLC dispersions were diluted properly with purified water. Each measurement was performed in triplicate. The morphology of co-evaporation vinpocetine–b-cyclodextrin–tartaric acid loaded NLC (VP–b-CD–TA COE-loaded NLC) was observed by transmission electron microscopy (TEM, JEM-1200EX JEOL, Tokyo, Japan). Then the surface morphology of VP–b-CD COEloaded NLC was viewed and photographed by scanning electron microscopy (SSX-550, Shimadzu, Kyoto, Japan). The sample was prepared by fixing the lyophilized nanoparticles on a double-sided sticky tape and coating with gold in an argon atmosphere. The interaction of VP–b-CD–TA with lipids and the association of VP–b-CD–TA in the NLC nanoparticle formulation were evaluated by DSC-60. Accurately weighed samples (VP–b-CD–TA, Compritol 888 ATO, physical mixture and lyophilized VP–b-CD–TA COE-loaded NLC) were scanned under the same parameters as inclusion complexes.
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2.4. Determination of entrapment efficiency
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Entrapment efficiency (EE) of NLC was evaluated indirectly using ultrafiltration technique. 4 mL drug-loaded NLC colloidal solution was placed in the upper chamber of a membrane concentrator (MWCO 10 kDa, Amicon ultra, Millipore Co., USA) and centrifuged for 15 min at 4000 rpm. The unencapsulated drug in the lower chamber of the filtrate was determined by HPLC at 268 nm. The total drug concentration was determined as follows: 1 ml drug-loaded NLC dispersion were dissolved appropriately with methanol and chloroform (1:1) to disrupt the nanoparticles after 10 min ultrasonic in water bath at 60 C, then the obtained suspension diluted properly was filtered through 0.45 mm membrane filters, and the drug content was also detected following the same procedure. The percentage of drug encapsulation could be calculated by the following equations:
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EE% ¼ 173
WT WF 100 WT
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where the W T is the amount of the initial drug added in NLC and the WF is the amount of unencapsulated drug in the filtrate.
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2.5. Drug release studies
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The VP–b-CD–TA COE-loaded NLC release studies were carried out by the dialysis technique. One milliliter of NLC dispersion (equivalent to 0.5 mg VP) was filled in a pre-soaked dialysis bag (8000–12,000 molecular weight cut-off) and immersed in the dissolution flask containing 200 mL of 0.1 M HCL, a pH 6.8 or pH 7.4 phosphate buffer, stirred at 50 rpm and hold at a temperature of 37 0.5 C. At regular time intervals for 4 h or 12 h, 0.5 mL of the sample was collected and replaced with an equal volume of the dissolution medium. And the amount of VP released into the medium was determined by HPLC. The release of VP–NLC containing equivalent amount of drug was conducted with the same procedure as VP–b-CD–TA COE-loaded NLC. All the results were the mean values of three runs.
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2.6. In vivo evaluation
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Eighteen New Zealand white male rabbits, weighing between 2.50 0.20 kg were supplied by the Animal Centre of the Shenyang Pharmaceutical University and all studies were conducted in accordance with the principles of Laboratory Animal Care. The rabbits were fasted overnight with free access to water. Animals were randomly assigned to three groups with six rabbits each. The rabbits were oral administered using polyethylene tube of 10 mL VP–b-CD–TA COE-loaded NLC or an equivalent dose of VP–NLC or VP suspension, respectively. The VP suspension was prepared by dissolving commercial VP tablets in a 0.5% CMC–Na solution with 10 min ultrasonic (Zhuang et al., 2010). Blood samples (0.5 mL) of each animal were sampled via the marginal ear vein at 0, 0.083, 0.25, 0.5, 1, 1.5, 2, 3, 4, 5, 6, 8, 12, 24 h. The samples were collected in tubes with heparin and centrifuged at 4000 rpm for 5 min and stored at 20 C pending analysis. The concentrations of VP in rabbits were detected by HPLC (Elbary et al., 2002). The stationary phase, ultimate XB-C18 column (250 4.6 mm, 5 mm). The mobile phase was a mixture of methanol: 0.01 M (NH4)2 CO3 distilled water solution (80:20, V/V); the flow rate was 1.0 ml/min and the wavelength was 268 nm. Two hundred microliter plasma was injected into a 10 mL tapered centrifuging tube, then mixed with 50 mL of an internal standard (diazepam, 1000 ng/mL) and 100 mL NaOH (0.5 M) by swirling for 5 min.Then the VP in the plasma was extracted by adding 4 mL of aether in the mixture above and swirling for 5 min. The upper aether layer solution was transferred to another tapered centrifuging tube and evaporated under nitrogen at 48 C after centrifugation at 4000 rpm for 5 min. The residues were reconstituted with 150 mL mobile phase and swirled for 5 min in a vortex. All samples were filtered with a 0.45 mm cellulose nitrate membrane before HPLC determination. Pharmacokinetic program DAS 2.0 was used for the analysis of the pharmacokinetic parameters (Cmax, Tmax, t1/2z and AUC). The relative bioavailability of tested formulations was fitted using the expression below:
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Fr% ¼
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AUCT DR 100 AUCT DT
where Fr was the relative bioavailability, AUC was the area under the plasma concentration-time plot, D was the dose administrated, T was the test formulation, R was the reference formulation.
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3. Results and discussion
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3.1. Preparation and characterization of VP–CD–TA solid systems
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Equimolar solid systems of the drug with three CDs were prepared by three different techniques, i.e. kneading, co-evaporation and co-lyophilization so that we could investigate the influence of the preparation method on the physicochemical properties of the final product. Differential scanning calorimetry is an effective method to investigate the drug crystallinity in compounds by determining the variation of temperature and energy at phase transition (Baocheng et al., 2011). Fig. 2 showed the thermal curves of VP alone, physical mixtures (PMs) of TA with three different CD respectively, ternary physical mixtures and its ternary systems with TA and b-CD, HP–b-CD, SBE–b-CD obtained by the different methods. A sharp endothermic peak of VP at 151 C followed by decomposition and indicated its crystalline nature (Fig. 2a) and there was not an endothermic peak around for the case of physical mixtures of TA with three different CD (Fig. 2b–d). As shown, for the ternary physical mixtures (Fig. 2e–g) and kneading products (Fig. 2h–j), a progressive lowering of drug onset and melting peak temperature were observed. Concerning the ternary
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Fig. 2. DSC profiles of (a) VP; (b) b-CD–TA PM; (c) HP–b-CD–TA PM; (d) SBE–b-CD– TA PM; (e) VP–b-CD–TA PM; (f) VP–HP–b-CD–TA PM; (g) VP–SBE–b-CD–TA PM; (h) VP–b-CD–TA KE; (i) VP–HP–b-CD–TA KE; (j) VP–SBE–b-CD–TA KE; (k) VP–b-CD–TA COE; (l) VP–HP–b-CD–TA COE; (m) VP–SBE–b-CD–TA COE; (n) VP–b-CD–TA COL; (o) VP–HP–b-CD–TA COL; (p) VP–SBE–b-CD–TA COL.
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PMs, these little changes relative to the peak of pure VP may suggest a weak interaction between the components by mechanical activation during the mixing or heating for DSC scanning (Dollo et al., 1999; Mura et al., 2001). As far as the kneading method was concerned, the small peak correspondent to the melting of free drug suggests that, as for the ternary PMs, there was no inclusion compound formed in either system even though drug–CD interaction was expressive. In the case of co-evaporated (Fig. 2k–m) and co-lyophilized (Fig. 2n–p), no melting peaks of VP around 151 C were detected, irrespective of the cyclodextrin used. As the disappearance of an endothermic peak may be attributed to an amorphous state or to an inclusion complexation (Kurazumi et al.,1975), these results implied that VP–CD–TA COE and COL products can be considered as inclusion complexes, which was differ from simple PMs and kneading method. Therefore, VP– CD–TA systems prepared by COE and COL were chosen to loading into NLC.
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3.2. Preparation and characterization of drug-loaded NLC
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In the present study, we had used triglycerides such as Compritol and Miglyol to form the NLC formulation, in which Compritol is a mixture of mono-, di- and triglycerides of behenic acid (C22) and makes up the solid outer shell. Miglyol 812N (C8–C12 triglyceride) was used as the liquid lipid of the matrix, known to
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enhance the encapsulation of lipophilic drugs in the nanoparticles (Patlolla et al., 2010). Solutol HS-15 (poly-oxyethylene esters of 12-hydroxystearic acid) as a non-toxic, non-ionic surfactant was used in combination with lecithin. The lipophilic segments of Solutol HS-15 partially inserted into lipid core whereas the hydrophilic PEG chains protruded towards the external water phase to form stereospecific blockade (Zhuang et al., 2010). The lecithin was added to adjust the high HLB of Solutol HS-15 so that the co-surfactant can enhance the ability to emulsify the lipid and stability of the system. Particle size, polydispersity index, zeta potential and entrapment efficiency of the prepared NLCs were collected in Table 1. Empty NLC, drug alone or in combination with CDs loaded NLC, only slightly modified the particle size in nanometric range. As indicated by the polydispersity index values, NLCs containing the coevaporated products were found to be more homogenously. Whereas, a wider particle size distribution was found for NLC loaded with the co-lyophilized products, which were ranged between 0.3 and 0.4. Zeta potential (ZP) refers to the surface charge of the particles. The zeta potential of empty NLC and VP alone loaded NLC estimated to be 12 mV. Interestingly, the ternary solid system loaded NLC displayed positive ZP values ranged between 5.4 mV and 12.8 mV. The reason for this different result may due to the addition of tartaric. In a recent study by electric ion spray, proved the ternary clathrate exist the ‘‘outer-sphere interaction”. Organic acid reacted with the active nitrogen of amino drug by electrostatic interaction and connected with then cyclodextrine cavity by hydrogen bonding and gathered outside the cavity. Therefore, the surface charge of the particles showed the positive charge of tartaric. Entrapment efficiency of the obtained NLCs was varied between 59.4 and 85.7%. The satisfying results of entrapment efficiency attributed to the particular inner structure of NLC, where the use of liquid lipid gives rise to a less compact structure compare with SLN, able to accommodate larger amounts of drug (Souto et al., 2004). And the entrapment efficiency was the highest, arriving up to 85.7%, when NLC prepared with VP–b-CD–TA by co-evaporated method. In conclusion, the VP–b-CD–TA COE-loaded NLC system, which had good ability to encapsulate drug and displayed favorable physicochemical characteristics, was selected for further morphology and crystal form study. TEM picture was taken to obtain the morphology information of the prepared VP–b-CD–TA COE-loaded NLC (Fig. 3). It could be seen that the particles were almost spherical and uniform shapes and did not stick to each other. Fig. 4 represents the scanning electron microscopy (SEM) image of VP–b-CD–TA COE-loaded NLC after freeze-drying. The particles displayed a spherical shape. Surface morphology of VP–b-CD–TA COE-loaded NLC was smooth and the crystalline structures of VP were absent in the SEM images. Fig. 5 depicted the DSC profile of VP–b-CD–TA COE, Compritol 888 ATO, their physical mixture and VP–b-CD–TA COE-loaded NLC lyophilized powder. Compritol 888 ATO (Fig. 5a) showed a characteristic crystalline form melting peak at 74 C and there was no obvious melting peak of VP–b-CD–TA COE (Fig. 5c) powder.
Table 1 Mean particle size (PS, n = 3), polydispersity index (PDI), zeta potential (ZP) and entrapment efficiency (%EE) of empty NLC, NLC loaded with vinpocetine (VP) alone or co-evaporated (COE) or co-lyophilized (COL) product with b-CD, HP–b-CD, SBE–b-CD. Sample
PS (nm SD)
PDI (SD)
ZP (mVSD)
%EE (SD)
Empty NLC VP–loaded NLC VP–b-CD–TA COE-loaded NLC VP–HP–b-CD–TA COE-loaded NLC VP–SBE–b-CD–TA COE-loaded NLC VP–b-CD–TA COL-loaded NLC VP–HP–b-CD–TA COL-loaded NLC VP–SBE–b-CD–TA COL-loaded NLC
134 16 148 28 89 21 133 34 152 63 99 32 141 25 148 41
0.18 0.02 0.19 0.03 0.15 0.02 0.14 0.01 0.17 0.02 0.38 0.04 0.35 0.02 0.44 0.05
12.1 0.1 12.5 0.3 11.4 0.1 12.8 0.2 5.4 0.5 11.9 0.4 13.1 0.1 6.4 0.6
– 72.7 0.1 85.7 0.3 75.7 0.4 65.5 0.2 74.9 0.3 64.3 0.7 59.4 0.8
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Fig. 3. TEM image of VP–b-CD–TA COE loaded NLC. 323
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The melting process also took place in the curve of the physical mixture (Fig. 5b) with maximum peak at 74 C. However, the endothermic peak around 74 C was disappeared in the thermograms of the lyophilized NLC (Fig. 5d), which suggested reduced crystallinity of the lipid matrix in NLC. All the results illustrated that the drug was existed in NLC in amorphous states.
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3.3. Drug release studies from NLC
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The VP release curves in 0.1 M HCL, pH 6.8 PBS and pH 7.4 PBS were plotted in Fig. 6, Fig. 7 and Fig. 8, respectively. In 0.1 M HCL, an
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Fig. 5. DSC profiles of (a) Compritol 888 ATO; (b) physical mixture; (c) VP–b-CD–TA COE; (d)VP–b-CD–TA COE loaded NLC.
[(Fig._4)TD$IG]
obvious burst release occurs at the initial stage of the drug suspension, because of the fraction of drug in solution was immediately ready to diffuse. In the case of VP–NLC and VP– b-CD–TA COE loaded NLC, the initial fast release rate may be due to diffusion of unencapsulated and VP entrapped in surfactant micelles. VP suspension in 0.1 M HCL tended to release completely (up to 100%) in 4 h due to the faintly alkaline of VP while that was less than 45% in VP–NLC and VP–b-CD–TA COE loaded NLC. The results suggested that the matrix of NLC could protect the VP from the strong acidic environment of the stomach. Thus, the major content of VP in VP–NLC and VP–b-CD–TA COE loaded NLC could be uptake by intestinal cells and enter the body circulation, which
[(Fig._6)TD$IG]
Fig. 6. In vitro release profiles of VP from different vehicles in 0.1 M HCL (n = 3).
[(Fig._7)TD$IG]
Fig. 4. SEM image of VP–b-CD–TA COE loaded NLC(200 SE in A, 2.00k SE in B).
Fig. 7. In vitro release profiles of VP from different vehicles at pH 6.8 (n = 3).
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C. Lin et al. / International Journal of Pharmaceutics xxx (2014) xxx–xxx Table 2 The main pharmacokinetic parameters of VP–b-CD–TA loaded NLC, VP–NLC and VP suspension in New Zealand rabbits.
Fig. 8. In vitro release profiles of VP from different vehicles at pH 7.4 (n = 3). 344
showed great advantage compared with VP suspension. At pH 6.8, the drug release rate of VP–b-CD–TA COE loaded NLC (64.7%) was much higher, about 5-fold of VP tablets (12.1%) and 39% more than VP alone loaded NLC (25.2%). VP–b-CD–TA COE loaded NLC and VP– NLC in pH 7.4 showed approximate release curves as the case in pH 6.8 which may be attributed to the existence of NLC matrix. However, VP suspension in pH 7.4 displayed a much lower release percentage (2.7%) as its faintly alkaline property. The improvement in the drug dissolution registered for VP–b-CD–TA COE loaded NLC could be interpreted as a solubilizing effect and the consequence of ternary complexes, in which TA could increase the drug release slowly by supplying a slightly acidic environment.
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3.4. In vivo evaluation
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The concentration–time plots, in New Zealand rabbits after administration of test formulations (VP–b-CD–TA COE-loaded NLC, VP–NLC and VP suspension) were shown in Fig. 9 and the main pharmacokinetic parameters were tabulated in Table 2. The Tmax was 0.5 h, the Cmax was 355.178 29.161 ng/ml after oral administration of VP suspension and the drug was undetectable 6 h after treated with VP suspension. However, the Tmax of VP was delayed by 0.5 h and 1 h in the case of VP–b-CD–TA COE-loaded NLC and VP– NLC, respectively. The Cmax of VP–b-CD–TA COE-loaded NLC was 816.236 40.121 ng/ml which was remarkably higher than results
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[(Fig._9)TD$IG]
Fig. 9. The concentration–time curves of VP–b-CD–TA loaded NLC, VP–NLC and VP suspension (n = 6).
Parameters
VP–b-CD–TA loaded NLC
VP–NLC
Suspension
Cmax (ng/ml) Tmax (h) t1/2z (h) AUC(0–t) (ng/ml h) AUC(0–1) (ng/ml h)
816.236 40.121 1 13.242 7.533 4804.758 515.770
445.85 22.972 1.5 10.161 4.950 2505.657 92.638
355.178 29.161 0.5 2.208 0.369 811.475 92.650
6260.461 1264.337
2990.184 460.126
948.97 112.642
of VP suspension and VP–NLC. The AUC(0–1) of VP–b-CD–TA COEloaded NLC was 6260.461 1264.337 ng/ml h, which was 6.60 folds higher than AUC(0–1) of VP suspension and 2.09 times more than VP–NLC, clearly defining performance superiority of VP–b-CD–TA COE-loaded NLC over VP suspension and VP–NLC. The obvious difference of Tmax demonstrated that the rates of absorption of tested formulations were not the same. VP in suspension was immediately dissolved in the intestinal tract and absorbed directly into systemic circulation. In the case of NLCs, which were composed of solid and liquids lipids, the lipids could induce bile secretion in the small intestinal and the VP–b-CD–TA COE-loaded NLC or VP–NLC were interacted with bile salt to form mixed micelles which helped the intact NLCs get into the lymphatic vessels and avoid the liver first-pass metabolism (Plain and Wilson, 1984; Yang et al., 1999; Jacobs et al., 2000). For VP–b-CD–TA COEloaded NLC, the VP released rate was marked increased from the lipid matrix as a result of drug complexation. Therefore, the VP reached the peaked concentration at 1 h in the case of VP–b-CD–TA COE-loaded NLC which was quickly than VP–NLC. Meanwhile, the prominent increasing of the Cmax of VP–b-CD–TA COE-loaded NLC may also due to the uptake and lymphatic transport of NLCs, as well as to a solubilizing effect and the quicker release rate from the lipid matrix than VP–NLC, as the consequence of drug complexation. The results manifested that systemic absorption of VP was significantly boosted by the strategy of loading the cyclodextrin complex into NLC compared to VP suspension and VP–NLC. The drug-in cyclodextrin-in nanostructured lipid carriers revealed a promising potential for enhancing oral bioavailability of low water-soluble drugs.
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4. Conclusions
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This study developed a novel oral delivery system consisting in ‘‘drug-in cyclodextrin-in nanostructured lipid carriers” for poorly water-soluble drug: vinpocetine. The VP–b-CD–TA COE-loaded NLC obtained showed small and homogeneous particle size with high encapsulation efficiency. The in vitro release study indicated that VP–b-CD–TA COE-loaded NLC exhibited a higher dissolution rate in the pH 6.8 and pH 7.4 medium than VP suspension and VP– NLC. The pharmacokinetic study demonstrated that the relative bioavailability of VP–b-CD–TA COE-loaded NLC was 592% compared with VP suspension and 92% higher than VP–NLC in New Zealand rabbits after oral administration. The present investigation also showed the importance of the careful selection of the most suitable preparation method and of the valid CD derivative for obtaining effective solid drug–CD systems. The innovative delivery system enhanced the profits of both the applied carriers (CD and NLC), with respect to their potential for improving the oral bioavailability, permission of an improvement in the dissolution and avoiding the first-pass effect of VP. Future pathway and mechanism of the cellular uptake of VP–b-CD–TA COE-loaded NLC will be carried out.
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Uncited references
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Tian et al. (2011).
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Acknowledgements
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This work is supported by State Key Laboratory of Long-acting and Targeting Drug Delivery System and by special construction projects fund which belongs to ‘‘Taishan Scholar-Pharmacy Specially Recruited Experts”.
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References
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Bonoczk, P., Gulyas, B., Adam-Vizi, V., 2000. Role of sodium channel inhibition in neuroprotection: effect of vinpocetin. Brain Research Bulletin 53, 245–254. Zhuang, C.-Y., Li, N., Wang, M., 2010. Preparation and characterization of vinpocetine loaded nanostructured lipid carriers (NLC) for improved oral bioavailability. International Journal of Pharmaceutics 394, 179–185. Dollo, G., Le Corre, P., Chollet, M., Chevanne, F., Bertault, M., Burgot, J.-L., Verge, R., 1999. Improvement in solubility and dissolution rate of 1,2-dithiole-3thiones upon complexation with b-cyclodextrin and its hydroxypropyl and sulfobuthyl ether derivatives. Journal of Pharmaceutical Sciences 88, 889– 895. Souto, E.B., Müller, R.H., 2006. Polymorphic behaviour of Compritol 888 ATO as bulk lipid and as SLN and NLC. Journal of Microencapsulation 23, 377–388. Souto, E.B., Wissing, S.A., Barbosa, C.M., Müller, R.H., 2004. Development of a controlled release formulation based on SLN and NLC for topical clotrimazole delivery. International Journal of Pharmaceutics 278, 1–7. Elbary, A., Foda, N., El-Gazayerly, O., Khatib, M.E., 2002. Reversed phase liquid chromatographic determination of vinpocetine in human plasma and its pharmacokinetic application. Analytical Letters 35, 1041–1054. Gergely, F., Katalin, K., Gabor, K., 2009. Effect of parenteral or oral vinpocetine on the hemorheological parameters of patients with chronic cerebrovascular diseases. Phytomedicine 16, 111–117. Kurazumi, M., Nambu, N., Nagai, T., 1975. Pharmaceutical interactions in dosage forms and processing. 4. Inclusion compounds of non-steroidal anti-inflammatory and other slightly water-soluble drugs withb-cyclodextrins and
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b-cyclodextrins in powdered form. Chemical and Pharmaceutical Bulletin 23, 3062–3068. Ribeiro, L.S.S., Ferreira, D.C., Veiga, F.J.B., 2003. Physicochemical investigation of the effects of water-soluble polymers on vinpocetine complexation with b-cyclodextrin and its sulfobutyl ether derivative in solution and solid state. European Journal of Pharmaceutical Sciences 20, 253–266. Loftsson, T., Brewster, M.E., 1996. Pharmaceutical applications of cyclodextrins. 1. Drug solubilization and stabilization. Journal of Pharmaceutical Sciences 85, 1017–1025. Luo, Q., Zhao, J., Zhang, X., Pan, W., 2011. Nanostructured lipid carrier (NLC) coated with chitosan oligosaccharides and its potential use in ocular drug delivery system. International Journal of Pharmaceutics 403, 185–191. Mura, P., Faucci, M.T., Bettinetti, G.P., 2001. The influence of polyvinylpyrrolidone on naproxen complexation with hydroxypropyl-b-cyclodextrin. European Journal of Pharmaceutical Sciences 13, 187–194. Szabo, L., Sapi, J., Kalaus Gy, Argay, G., Kalman, A., Baitz-Gacs, E., Tamas, J., Szantay, C., 1983. New route to the stereoselective synthesis of (b)-vincamine, (b)-vincamone, and (b)-apovincaminic acid esters. Tetrahedron Letters 316, 629–638. O’Driscoll, C.M., Griffin, B.T., 2008. Biopharmaceutical challenges associated with drugs with low aqueous solubility’the potential impact of lipid-based formulations. Advanced Drug Delivery Reviews 60, 617–624. Plain, K.J., Wilson, C.G., 1984. The effect of different oilson the absorption of probucol in the rat. Journal of Pharmacy and Pharmacology 36, 641–643. Patlolla, R.R., Chougule, M., Patel, A.R., Jackson, T., Tata, P.N.V., Singh, M., 2010. Formulation, characterization and pulmonary deposition of nebulized celecoxib encapsulated nanostructured lipid carriers. Journal of Controlled Release 144, 233–241. Müller, R.H., Radtke, M., Wissing, S.A., 2002. Nanostructured lipid matrices for improved microencapsulation of drug. International Journal of Pharmaceutics 242, 121–128. Szakács, T., Veres, Z., Vereczkey, L., 2001. In vitro–in vivo correlation of the pharmacokinetics of vinpocetine. Polish Journal of Pharmacology 53, 623–628. Tian, B., Luo, Q., Song, S., et al., 2011. Novel surface-modified nanostructured lipid carriers with partially deacetylated water-soluble chitosan for efficient ocular delivery. Journal of Pharmaceutical Sciences 101, 1040–1049. Stella, V.J., Rajeuski, R.A., 1997. Cyclodextrins: their future in drug formulation and delivery. Pharmaceutical Research 14, NO.5. Yang, S., Zhu, J., Lu, Y., Liang, B., Yang, C.,1999. Body distribution of camptothecin solid lipid nanoparticles after oral administration. Pharmaceutical Research 16, 751– 757.
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Contents lists available at ScienceDirect
International Journal of Pharmaceutics journal homepage: www.elsevier.com/locate/ijpharm
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A novel oral delivery system consisting in ‘‘drug-in cyclodextrin-in nanostructured lipid carriers” for poorly water-soluble drug: Vinpocetine [TD$FIRSNAME]Congcong [TD$FIRSNAME.]ULin[TD$SURNAME.] a , [TD$FIRSNAME]Fen [TD$FIRSNAME.]UChen[TD$SURNAME.] a , [TD$FIRSNAME]Tiantian [TD$FIRSNAME.]UYe[TD$SURNAME.] a , [TD$FIRSNAME]Lina [TD$FIRSNAME.]UZhang[TD$SURNAME.] a , [TD$FIRSNAME]Wenji [TD$FIRSNAME.]UZhang[TD$SURNAME.] a , [TD$FIRSNAME]Dandan [TD$FIRSNAME.]ULiu[TD$SURNAME.] b , [TD$FIRSNAME]Wei [TD$FIRSNAME.]UXiong[TD$SURNAME.] a , [TD$FIRSNAME]Xinggang [TD$FIRSNAME.]UYang[TD$SURNAME.] a , [TD$FIRSNAME]Weisan [TD$FIRSNAME.]UPan[TD$SURNAME.] a, * a b
Department of Pharmaceutics, School of Pharmacy, Shenyang Pharmaceutical University, 103 Wenhua Road, Shenyang 10016, China School of Biomedical & Chemical Engineering, Liaoning Institute of Science and Technology, 176 Xianghuai Road, Benxi 117004, China
A R T I C L E I N F O
A B S T R A C T
Article history: Received 6 November 2013 Received in revised form 25 December 2013 Accepted 8 February 2014 Available online xxx
The purpose of this study was to develop a new delivery system based on drug cyclodextrin (CD) complexation and loading into nanostructured lipid carriers (NLC) to improve the oral bioavailability of vinpocetine (VP). Three different CDs and three different methods to obtain solid vinpocetine– cyclodextrin–tartaric acid complexes (VP–CD–TA) were contrasted. The co-evaporation vinpocetineb-cyclodextrin-tartaric acid loaded NLC (VP–b-CD–TA COE-loaded NLC) was obtained by emulsification ultrasonic dispersion method. VP–b-CD–TA COE-loaded NLC was suitably characterized for particle size, polydispersity index, zeta potential, entrapment efficiency and the morphology. The crystallization of drug in VP–CD–TA and NLC was investigated by differential scanning calorimetry (DSC). The in vitro release study was carried out at pH 1.2, pH 6.8 and pH 7.4 medium. New Zealand rabbits were applied to investigate the pharmacokinetic behavior in vivo. The VP–b-CD–TA COE-loaded NLC presented a superior physicochemical property and selected to further study. In the in vitro release study, VP–b-CD–TA COEloaded NLC exhibited a higher dissolution rate in the pH 6.8 and pH 7.4 medium than VP suspension and VP–NLC. The relative bioavailability of VP–b-CD–TA COE-loaded NLC was 592% compared with VP suspension and 92% higher than VP–NLC. In conclusion, the new formulation significantly improved bioavailability of VP for oral delivery, demonstrated a perspective way for oral delivery of poorly water-soluble drugs. ã 2014 Published by Elsevier B.V.
Keywords: Nanostructured lipid carriers (NLC) Cyclodextrins Vinpocetine Oral bioavailability Poorly water-soluble drugs
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1. Introduction
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Vinpocetine (ethyl-apovincamine-22-oate, VP), a derivative of the Vinca minor alkaloid vincamine, brought to the market in 1978 with the trade name Cavinton, which is well-known for the treatment of ischemic stroke and other cerebrovascular disease (Fig. 1) (Szaboet al. 1983). Its beneficial effects have been proved by many experimental studies (Bonoczk et al., 2000), the results showed that advantageous rheological effects can be maintained by per os vinpocetine treatment on chronic ischemic cerebrovascular patients. The oral drug therapy preserved the beneficial changes of the impaired red blood cell aggregation and plasma and whole blood viscosity values (Gergely et al., 2009). However, VP is a sparingly water soluble drug (the water solubility 5 mg/ml, pKa value of 7.31), has a low and irregular dissolution rate in the
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* Corresponding author. Tel.: +86 024 23986313; fax.: +86 024 23953241. E-mail address: [email protected] (W. Pan).
gastrointestinal tract because of the pH dependency and a remarkable first pass-effect , which also resulting in its low oral bioavailability about 7% in human that largely restricts its clinical use (Szakács et al., 2001). Therefore, to overcome these drawbacks, there is a need to design a novel delivery system possible to improve the oral absorption and bioavailability of VP. Lipid-based drug delivery systems are expected as promising oral carriers because of their potential to increase the solubility and improve oral bioavailability of poorly water-soluble and lipophilic drugs (O’Driscoll and Griffin, 2008). Nanostructure lipid carriers (NLCs) which developed from solid lipid nanoparticles (SLNs), consisting of a biocompatible solid lipid matrix entrapping a liquid lipid (oil) as nanocompartments, are attracting major attention as alternative colloidal drug carriers. Such a particular structure brings about distinct superiority comparing with SLN, such as improved drug loading ability, increased physical stability, reduced risks of drug leakage during storage(Müller et al., 2002; Souto and Müller, 2006). Furthermore, NLC particles could avoid the liver metabolism by forming micelles with bile salts in small intestine
http://dx.doi.org/10.1016/j.ijpharm.2014.02.013 0378-5173/$ – see front matter ã 2014 Published by Elsevier B.V.
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Fig. 1. Chemical structure of vinpocetine and b-cyclodextrin. 41
kindly donated by Roquette (France). Compritol 888 ATO (glyceryl behenate) was a gift from Gattefosse (Paris, France). Solutol HS-15 (poly-oxyethylene esters of 12-hydroxystearic acid) was sampled from BASF (Lud-wigshafen, Germany). Tartaric acid (TA) was product of Bo di chemical Co., LTD. (Tianjin, China). Soybean phosphatidylcholine (PC) that contained approximately 80% of the phosphatidyl was purchased from Aikang Chemical Co. Ltd. (Shanghai, China). Other chemicals and reagents were of analytical grade.
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2.2. Preparation and characterization of VP–CD–TA solid systems
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Physical mixtures (PMs) containing a VP:TA ratio of 2:1 (w/w) and VP with b-CD, HP–b-CD and SBE–b-CD were prepared by homogeneous blending in a mortar at a molar ratio of 1:2 (L.S.S. Ribeiro et al., 2003). Kneaded (KE) products were prepared by kneading thoroughly with least amount of water to obtain a paste which was then dried under vacuum at room temperature. Co-evaporation (COE) products were obtained by dissolving formulated amounts of CDs in water and VP in 3% (w/v) of TA solution respectively, and then adding the latter dropwise to the former. The resulting solution mixture was stirred at 100 rpm and a temperature of 60 C for 4 h. Furthermore, the obtained clear solution was evaporated under vacuum at 60 C in a rotatory evaporator. All solid residues were further dried at 40 C for 24 h and collected through 80 mesh sieve. For co-lyophilized (COL) products, a molar ratio of 2:1 of CDs and VP was dispersed in water or in 3% (w/ v) of TA solution, separately. The obtained solution mixture was sonicated for 2 h at 60 C.After equilibrium for 24 h at room temperature, the resultant solution was frozen by cryogenic refrigerator at 70 C and then the frozen solution was lyophilized in a freeze–dryer (FD-1 apparatus, Labconco) for 24 h. The thermal properties of pure components and their solid combinations were surveyed by DSC-60 differential scanning calorimeter (Shimadzu, Japan). The samples were scanned at a heating rate of 10 C/min over a temperature range of 30–250 C with a nitrogen purge of 0.2 ml/min in aluminum pan and sealed hermetically, using aluminum oxide as the reference.
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after entering in the gastrointestinal (GI) tract, which will enter the lymph circulation. Cyclodextrins (CDs), naturally available water-soluble cyclic oligosaccharides composed of a-1,4-linked-glucopyranose units (Fig. 1), have been used to modify the time of drug release during GI transit and decrease local irritation, may also alter oral absorption of drugs through mucosal membrane permeation enhancement (Stella and Rajeuski, 1997). Unfortunately, the complexation efficiency of CDs is rather low and consequently significant amounts of CDs are needed to solubilize small amounts of water-insoluble compounds. However, enhanced complexation can be obtained by formation of ternary complexes between a drug molecule, CD molecule and a third component (Loftsson and Brewster, 1996). Inclusion complexes with cyclodextrins have been reported as an effective way to increase bioavailability through improving the solubility and dissolution rate of VP. In view of all these premises, we thought it worthy of interest to investigate the feasibility of developing a novel oral drug delivery system for vinpocetine based on a combined strategy which exploits both cyclodextrin complexation, to boost drug solubility and dissolution properties, and loading of the complex into NLC, to avoid significant first-pass effect. In the shell frame of this project, considering the special chemical properties of VP itself (alkalescence), the ternary inclusion compound was prepared for the drug: b-cyclodextrin (b-CD), hydroxypropyl–b-cyclodextrin (HP–b-CD) and sulfobutylether–b-cyclodextrin (SBE–b-CD) have been selected to prepare multicomponent complexation in the presence of tartaric acid (TA, as an acidifier of the complexation medium), and the effectiveness of three different techniques (co-grinding, coevaporating and co-lyophilization) to obtain solid vinpocetine– cyclodextrin–tartaric acid (VP–CD–TA) complexes with improved drug dissolution properties has been investigated .The most effective VP–CD–TA system was screened for loading into NLC obtained by melt emulsification combined with ultra-sonication technique (Luo et al., 2011). Both empty and loaded NLC were suitably characterized for particle size, zeta potential and entrapment efficiency, evaluated on the drug release properties in vitro and oral bioavailability in vivo.
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2. Materials and methods
2.3. NLC preparation and characterization
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2.1. Materials
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Vinpocetine, the purity of which was over 99.2%, was kindly supplied by northeast general pharmaceutical Co., Ltd. (Shenyang, China). b-cyclodextrin (b-CD) and sulfobutylether–b-cyclodextrin (SBE–b-CD) were obtained from Xinda Fine Chemical Co., LTD. (Shandong, China). Hydroxypropyl–b-cyclodextrin (HP–b-CD) was
NLC were produced according to the procedure of Luo et al. (Luo et al., 2011), which was slightly altered. In brief, the solid lipid (Compritol 888 ATO 0.097 g) and Miglyol 812N (0.024 g) together were blended and melted at 85 C to form a uniform oil phase. Meanwhile, the aqueous phase was prepared by dispersing PC (0.077 g) and the surfactant (Solutol HS-15 0.127 g) in 20 mL of purified water and heated up to 85 C. Then the hot aqueous phase
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was added drop wisely to the oil phase at the same temperature with magnetic stirring at 600 rpm for 10 min. The obtained preemulsion was ultrasonified 5 min (active every 3 s for 3 s duration; 400 W) by probe-ultrasonic cell disruptor (JY-92-II; Xinzhi, Ningbo, China). Finally, the dispersion was rapidly cooled to solidify in ice bath (0 C) to form NLC. Pure drug (5 mg) or VP–CD–TA loaded NLC systems (equivalent to 5 mg VP) were performed, respectively, by adding VP or VP–CD–TA to the melted lipid phase. The mean particle size (PS), polydispersity index (PDI), and zeta potential (ZP) were determined by photon correlation spectroscopy (PCS) using a Zeta-sizer Nano (Malvern Instruments, UK) at room temperature. Avoiding multiscattering phenomena, NLC dispersions were diluted properly with purified water. Each measurement was performed in triplicate. The morphology of co-evaporation vinpocetine–b-cyclodextrin–tartaric acid loaded NLC (VP–b-CD–TA COE-loaded NLC) was observed by transmission electron microscopy (TEM, JEM-1200EX JEOL, Tokyo, Japan). Then the surface morphology of VP–b-CD COEloaded NLC was viewed and photographed by scanning electron microscopy (SSX-550, Shimadzu, Kyoto, Japan). The sample was prepared by fixing the lyophilized nanoparticles on a double-sided sticky tape and coating with gold in an argon atmosphere. The interaction of VP–b-CD–TA with lipids and the association of VP–b-CD–TA in the NLC nanoparticle formulation were evaluated by DSC-60. Accurately weighed samples (VP–b-CD–TA, Compritol 888 ATO, physical mixture and lyophilized VP–b-CD–TA COE-loaded NLC) were scanned under the same parameters as inclusion complexes.
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2.4. Determination of entrapment efficiency
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Entrapment efficiency (EE) of NLC was evaluated indirectly using ultrafiltration technique. 4 mL drug-loaded NLC colloidal solution was placed in the upper chamber of a membrane concentrator (MWCO 10 kDa, Amicon ultra, Millipore Co., USA) and centrifuged for 15 min at 4000 rpm. The unencapsulated drug in the lower chamber of the filtrate was determined by HPLC at 268 nm. The total drug concentration was determined as follows: 1 ml drug-loaded NLC dispersion were dissolved appropriately with methanol and chloroform (1:1) to disrupt the nanoparticles after 10 min ultrasonic in water bath at 60 C, then the obtained suspension diluted properly was filtered through 0.45 mm membrane filters, and the drug content was also detected following the same procedure. The percentage of drug encapsulation could be calculated by the following equations:
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EE% ¼ 173
WT WF 100 WT
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where the W T is the amount of the initial drug added in NLC and the WF is the amount of unencapsulated drug in the filtrate.
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2.5. Drug release studies
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The VP–b-CD–TA COE-loaded NLC release studies were carried out by the dialysis technique. One milliliter of NLC dispersion (equivalent to 0.5 mg VP) was filled in a pre-soaked dialysis bag (8000–12,000 molecular weight cut-off) and immersed in the dissolution flask containing 200 mL of 0.1 M HCL, a pH 6.8 or pH 7.4 phosphate buffer, stirred at 50 rpm and hold at a temperature of 37 0.5 C. At regular time intervals for 4 h or 12 h, 0.5 mL of the sample was collected and replaced with an equal volume of the dissolution medium. And the amount of VP released into the medium was determined by HPLC. The release of VP–NLC containing equivalent amount of drug was conducted with the same procedure as VP–b-CD–TA COE-loaded NLC. All the results were the mean values of three runs.
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2.6. In vivo evaluation
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Eighteen New Zealand white male rabbits, weighing between 2.50 0.20 kg were supplied by the Animal Centre of the Shenyang Pharmaceutical University and all studies were conducted in accordance with the principles of Laboratory Animal Care. The rabbits were fasted overnight with free access to water. Animals were randomly assigned to three groups with six rabbits each. The rabbits were oral administered using polyethylene tube of 10 mL VP–b-CD–TA COE-loaded NLC or an equivalent dose of VP–NLC or VP suspension, respectively. The VP suspension was prepared by dissolving commercial VP tablets in a 0.5% CMC–Na solution with 10 min ultrasonic (Zhuang et al., 2010). Blood samples (0.5 mL) of each animal were sampled via the marginal ear vein at 0, 0.083, 0.25, 0.5, 1, 1.5, 2, 3, 4, 5, 6, 8, 12, 24 h. The samples were collected in tubes with heparin and centrifuged at 4000 rpm for 5 min and stored at 20 C pending analysis. The concentrations of VP in rabbits were detected by HPLC (Elbary et al., 2002). The stationary phase, ultimate XB-C18 column (250 4.6 mm, 5 mm). The mobile phase was a mixture of methanol: 0.01 M (NH4)2 CO3 distilled water solution (80:20, V/V); the flow rate was 1.0 ml/min and the wavelength was 268 nm. Two hundred microliter plasma was injected into a 10 mL tapered centrifuging tube, then mixed with 50 mL of an internal standard (diazepam, 1000 ng/mL) and 100 mL NaOH (0.5 M) by swirling for 5 min.Then the VP in the plasma was extracted by adding 4 mL of aether in the mixture above and swirling for 5 min. The upper aether layer solution was transferred to another tapered centrifuging tube and evaporated under nitrogen at 48 C after centrifugation at 4000 rpm for 5 min. The residues were reconstituted with 150 mL mobile phase and swirled for 5 min in a vortex. All samples were filtered with a 0.45 mm cellulose nitrate membrane before HPLC determination. Pharmacokinetic program DAS 2.0 was used for the analysis of the pharmacokinetic parameters (Cmax, Tmax, t1/2z and AUC). The relative bioavailability of tested formulations was fitted using the expression below:
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Fr% ¼
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AUCT DR 100 AUCT DT
where Fr was the relative bioavailability, AUC was the area under the plasma concentration-time plot, D was the dose administrated, T was the test formulation, R was the reference formulation.
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3. Results and discussion
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3.1. Preparation and characterization of VP–CD–TA solid systems
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Equimolar solid systems of the drug with three CDs were prepared by three different techniques, i.e. kneading, co-evaporation and co-lyophilization so that we could investigate the influence of the preparation method on the physicochemical properties of the final product. Differential scanning calorimetry is an effective method to investigate the drug crystallinity in compounds by determining the variation of temperature and energy at phase transition (Baocheng et al., 2011). Fig. 2 showed the thermal curves of VP alone, physical mixtures (PMs) of TA with three different CD respectively, ternary physical mixtures and its ternary systems with TA and b-CD, HP–b-CD, SBE–b-CD obtained by the different methods. A sharp endothermic peak of VP at 151 C followed by decomposition and indicated its crystalline nature (Fig. 2a) and there was not an endothermic peak around for the case of physical mixtures of TA with three different CD (Fig. 2b–d). As shown, for the ternary physical mixtures (Fig. 2e–g) and kneading products (Fig. 2h–j), a progressive lowering of drug onset and melting peak temperature were observed. Concerning the ternary
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Fig. 2. DSC profiles of (a) VP; (b) b-CD–TA PM; (c) HP–b-CD–TA PM; (d) SBE–b-CD– TA PM; (e) VP–b-CD–TA PM; (f) VP–HP–b-CD–TA PM; (g) VP–SBE–b-CD–TA PM; (h) VP–b-CD–TA KE; (i) VP–HP–b-CD–TA KE; (j) VP–SBE–b-CD–TA KE; (k) VP–b-CD–TA COE; (l) VP–HP–b-CD–TA COE; (m) VP–SBE–b-CD–TA COE; (n) VP–b-CD–TA COL; (o) VP–HP–b-CD–TA COL; (p) VP–SBE–b-CD–TA COL.
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PMs, these little changes relative to the peak of pure VP may suggest a weak interaction between the components by mechanical activation during the mixing or heating for DSC scanning (Dollo et al., 1999; Mura et al., 2001). As far as the kneading method was concerned, the small peak correspondent to the melting of free drug suggests that, as for the ternary PMs, there was no inclusion compound formed in either system even though drug–CD interaction was expressive. In the case of co-evaporated (Fig. 2k–m) and co-lyophilized (Fig. 2n–p), no melting peaks of VP around 151 C were detected, irrespective of the cyclodextrin used. As the disappearance of an endothermic peak may be attributed to an amorphous state or to an inclusion complexation (Kurazumi et al.,1975), these results implied that VP–CD–TA COE and COL products can be considered as inclusion complexes, which was differ from simple PMs and kneading method. Therefore, VP– CD–TA systems prepared by COE and COL were chosen to loading into NLC.
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3.2. Preparation and characterization of drug-loaded NLC
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In the present study, we had used triglycerides such as Compritol and Miglyol to form the NLC formulation, in which Compritol is a mixture of mono-, di- and triglycerides of behenic acid (C22) and makes up the solid outer shell. Miglyol 812N (C8–C12 triglyceride) was used as the liquid lipid of the matrix, known to
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enhance the encapsulation of lipophilic drugs in the nanoparticles (Patlolla et al., 2010). Solutol HS-15 (poly-oxyethylene esters of 12-hydroxystearic acid) as a non-toxic, non-ionic surfactant was used in combination with lecithin. The lipophilic segments of Solutol HS-15 partially inserted into lipid core whereas the hydrophilic PEG chains protruded towards the external water phase to form stereospecific blockade (Zhuang et al., 2010). The lecithin was added to adjust the high HLB of Solutol HS-15 so that the co-surfactant can enhance the ability to emulsify the lipid and stability of the system. Particle size, polydispersity index, zeta potential and entrapment efficiency of the prepared NLCs were collected in Table 1. Empty NLC, drug alone or in combination with CDs loaded NLC, only slightly modified the particle size in nanometric range. As indicated by the polydispersity index values, NLCs containing the coevaporated products were found to be more homogenously. Whereas, a wider particle size distribution was found for NLC loaded with the co-lyophilized products, which were ranged between 0.3 and 0.4. Zeta potential (ZP) refers to the surface charge of the particles. The zeta potential of empty NLC and VP alone loaded NLC estimated to be 12 mV. Interestingly, the ternary solid system loaded NLC displayed positive ZP values ranged between 5.4 mV and 12.8 mV. The reason for this different result may due to the addition of tartaric. In a recent study by electric ion spray, proved the ternary clathrate exist the ‘‘outer-sphere interaction”. Organic acid reacted with the active nitrogen of amino drug by electrostatic interaction and connected with then cyclodextrine cavity by hydrogen bonding and gathered outside the cavity. Therefore, the surface charge of the particles showed the positive charge of tartaric. Entrapment efficiency of the obtained NLCs was varied between 59.4 and 85.7%. The satisfying results of entrapment efficiency attributed to the particular inner structure of NLC, where the use of liquid lipid gives rise to a less compact structure compare with SLN, able to accommodate larger amounts of drug (Souto et al., 2004). And the entrapment efficiency was the highest, arriving up to 85.7%, when NLC prepared with VP–b-CD–TA by co-evaporated method. In conclusion, the VP–b-CD–TA COE-loaded NLC system, which had good ability to encapsulate drug and displayed favorable physicochemical characteristics, was selected for further morphology and crystal form study. TEM picture was taken to obtain the morphology information of the prepared VP–b-CD–TA COE-loaded NLC (Fig. 3). It could be seen that the particles were almost spherical and uniform shapes and did not stick to each other. Fig. 4 represents the scanning electron microscopy (SEM) image of VP–b-CD–TA COE-loaded NLC after freeze-drying. The particles displayed a spherical shape. Surface morphology of VP–b-CD–TA COE-loaded NLC was smooth and the crystalline structures of VP were absent in the SEM images. Fig. 5 depicted the DSC profile of VP–b-CD–TA COE, Compritol 888 ATO, their physical mixture and VP–b-CD–TA COE-loaded NLC lyophilized powder. Compritol 888 ATO (Fig. 5a) showed a characteristic crystalline form melting peak at 74 C and there was no obvious melting peak of VP–b-CD–TA COE (Fig. 5c) powder.
Table 1 Mean particle size (PS, n = 3), polydispersity index (PDI), zeta potential (ZP) and entrapment efficiency (%EE) of empty NLC, NLC loaded with vinpocetine (VP) alone or co-evaporated (COE) or co-lyophilized (COL) product with b-CD, HP–b-CD, SBE–b-CD. Sample
PS (nm SD)
PDI (SD)
ZP (mVSD)
%EE (SD)
Empty NLC VP–loaded NLC VP–b-CD–TA COE-loaded NLC VP–HP–b-CD–TA COE-loaded NLC VP–SBE–b-CD–TA COE-loaded NLC VP–b-CD–TA COL-loaded NLC VP–HP–b-CD–TA COL-loaded NLC VP–SBE–b-CD–TA COL-loaded NLC
134 16 148 28 89 21 133 34 152 63 99 32 141 25 148 41
0.18 0.02 0.19 0.03 0.15 0.02 0.14 0.01 0.17 0.02 0.38 0.04 0.35 0.02 0.44 0.05
12.1 0.1 12.5 0.3 11.4 0.1 12.8 0.2 5.4 0.5 11.9 0.4 13.1 0.1 6.4 0.6
– 72.7 0.1 85.7 0.3 75.7 0.4 65.5 0.2 74.9 0.3 64.3 0.7 59.4 0.8
Please cite this article in press as: Lin, C., et al. Int J Pharmaceut (2014), http://dx.doi.org/10.1016/j.aca.2013.12.001
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[(Fig._3)TD$IG]
[(Fig._5)TD$IG]
Fig. 3. TEM image of VP–b-CD–TA COE loaded NLC. 323
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The melting process also took place in the curve of the physical mixture (Fig. 5b) with maximum peak at 74 C. However, the endothermic peak around 74 C was disappeared in the thermograms of the lyophilized NLC (Fig. 5d), which suggested reduced crystallinity of the lipid matrix in NLC. All the results illustrated that the drug was existed in NLC in amorphous states.
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3.3. Drug release studies from NLC
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The VP release curves in 0.1 M HCL, pH 6.8 PBS and pH 7.4 PBS were plotted in Fig. 6, Fig. 7 and Fig. 8, respectively. In 0.1 M HCL, an
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Fig. 5. DSC profiles of (a) Compritol 888 ATO; (b) physical mixture; (c) VP–b-CD–TA COE; (d)VP–b-CD–TA COE loaded NLC.
[(Fig._4)TD$IG]
obvious burst release occurs at the initial stage of the drug suspension, because of the fraction of drug in solution was immediately ready to diffuse. In the case of VP–NLC and VP– b-CD–TA COE loaded NLC, the initial fast release rate may be due to diffusion of unencapsulated and VP entrapped in surfactant micelles. VP suspension in 0.1 M HCL tended to release completely (up to 100%) in 4 h due to the faintly alkaline of VP while that was less than 45% in VP–NLC and VP–b-CD–TA COE loaded NLC. The results suggested that the matrix of NLC could protect the VP from the strong acidic environment of the stomach. Thus, the major content of VP in VP–NLC and VP–b-CD–TA COE loaded NLC could be uptake by intestinal cells and enter the body circulation, which
[(Fig._6)TD$IG]
Fig. 6. In vitro release profiles of VP from different vehicles in 0.1 M HCL (n = 3).
[(Fig._7)TD$IG]
Fig. 4. SEM image of VP–b-CD–TA COE loaded NLC(200 SE in A, 2.00k SE in B).
Fig. 7. In vitro release profiles of VP from different vehicles at pH 6.8 (n = 3).
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IJP 13888 1–7 6
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C. Lin et al. / International Journal of Pharmaceutics xxx (2014) xxx–xxx Table 2 The main pharmacokinetic parameters of VP–b-CD–TA loaded NLC, VP–NLC and VP suspension in New Zealand rabbits.
Fig. 8. In vitro release profiles of VP from different vehicles at pH 7.4 (n = 3). 344
showed great advantage compared with VP suspension. At pH 6.8, the drug release rate of VP–b-CD–TA COE loaded NLC (64.7%) was much higher, about 5-fold of VP tablets (12.1%) and 39% more than VP alone loaded NLC (25.2%). VP–b-CD–TA COE loaded NLC and VP– NLC in pH 7.4 showed approximate release curves as the case in pH 6.8 which may be attributed to the existence of NLC matrix. However, VP suspension in pH 7.4 displayed a much lower release percentage (2.7%) as its faintly alkaline property. The improvement in the drug dissolution registered for VP–b-CD–TA COE loaded NLC could be interpreted as a solubilizing effect and the consequence of ternary complexes, in which TA could increase the drug release slowly by supplying a slightly acidic environment.
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3.4. In vivo evaluation
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The concentration–time plots, in New Zealand rabbits after administration of test formulations (VP–b-CD–TA COE-loaded NLC, VP–NLC and VP suspension) were shown in Fig. 9 and the main pharmacokinetic parameters were tabulated in Table 2. The Tmax was 0.5 h, the Cmax was 355.178 29.161 ng/ml after oral administration of VP suspension and the drug was undetectable 6 h after treated with VP suspension. However, the Tmax of VP was delayed by 0.5 h and 1 h in the case of VP–b-CD–TA COE-loaded NLC and VP– NLC, respectively. The Cmax of VP–b-CD–TA COE-loaded NLC was 816.236 40.121 ng/ml which was remarkably higher than results
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[(Fig._9)TD$IG]
Fig. 9. The concentration–time curves of VP–b-CD–TA loaded NLC, VP–NLC and VP suspension (n = 6).
Parameters
VP–b-CD–TA loaded NLC
VP–NLC
Suspension
Cmax (ng/ml) Tmax (h) t1/2z (h) AUC(0–t) (ng/ml h) AUC(0–1) (ng/ml h)
816.236 40.121 1 13.242 7.533 4804.758 515.770
445.85 22.972 1.5 10.161 4.950 2505.657 92.638
355.178 29.161 0.5 2.208 0.369 811.475 92.650
6260.461 1264.337
2990.184 460.126
948.97 112.642
of VP suspension and VP–NLC. The AUC(0–1) of VP–b-CD–TA COEloaded NLC was 6260.461 1264.337 ng/ml h, which was 6.60 folds higher than AUC(0–1) of VP suspension and 2.09 times more than VP–NLC, clearly defining performance superiority of VP–b-CD–TA COE-loaded NLC over VP suspension and VP–NLC. The obvious difference of Tmax demonstrated that the rates of absorption of tested formulations were not the same. VP in suspension was immediately dissolved in the intestinal tract and absorbed directly into systemic circulation. In the case of NLCs, which were composed of solid and liquids lipids, the lipids could induce bile secretion in the small intestinal and the VP–b-CD–TA COE-loaded NLC or VP–NLC were interacted with bile salt to form mixed micelles which helped the intact NLCs get into the lymphatic vessels and avoid the liver first-pass metabolism (Plain and Wilson, 1984; Yang et al., 1999; Jacobs et al., 2000). For VP–b-CD–TA COEloaded NLC, the VP released rate was marked increased from the lipid matrix as a result of drug complexation. Therefore, the VP reached the peaked concentration at 1 h in the case of VP–b-CD–TA COE-loaded NLC which was quickly than VP–NLC. Meanwhile, the prominent increasing of the Cmax of VP–b-CD–TA COE-loaded NLC may also due to the uptake and lymphatic transport of NLCs, as well as to a solubilizing effect and the quicker release rate from the lipid matrix than VP–NLC, as the consequence of drug complexation. The results manifested that systemic absorption of VP was significantly boosted by the strategy of loading the cyclodextrin complex into NLC compared to VP suspension and VP–NLC. The drug-in cyclodextrin-in nanostructured lipid carriers revealed a promising potential for enhancing oral bioavailability of low water-soluble drugs.
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Q3 382 383 384 385 386 387 388 389 390 391 392 393 394 395
4. Conclusions
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This study developed a novel oral delivery system consisting in ‘‘drug-in cyclodextrin-in nanostructured lipid carriers” for poorly water-soluble drug: vinpocetine. The VP–b-CD–TA COE-loaded NLC obtained showed small and homogeneous particle size with high encapsulation efficiency. The in vitro release study indicated that VP–b-CD–TA COE-loaded NLC exhibited a higher dissolution rate in the pH 6.8 and pH 7.4 medium than VP suspension and VP– NLC. The pharmacokinetic study demonstrated that the relative bioavailability of VP–b-CD–TA COE-loaded NLC was 592% compared with VP suspension and 92% higher than VP–NLC in New Zealand rabbits after oral administration. The present investigation also showed the importance of the careful selection of the most suitable preparation method and of the valid CD derivative for obtaining effective solid drug–CD systems. The innovative delivery system enhanced the profits of both the applied carriers (CD and NLC), with respect to their potential for improving the oral bioavailability, permission of an improvement in the dissolution and avoiding the first-pass effect of VP. Future pathway and mechanism of the cellular uptake of VP–b-CD–TA COE-loaded NLC will be carried out.
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Uncited references
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Tian et al. (2011).
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Acknowledgements
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This work is supported by State Key Laboratory of Long-acting and Targeting Drug Delivery System and by special construction projects fund which belongs to ‘‘Taishan Scholar-Pharmacy Specially Recruited Experts”.
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References
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