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Preparation and evaluation of liposome-encapsulated codrug LMX
International Journal of Pharmaceutics 438 (2012) 240–248
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Preparation and evaluation of liposome-encapsulated codrug LMX Yan Zhong a , Jing Wang b , Yao Wang b , Bin Wu b,∗ a b
School of Chemistry and Chemical Engineering, Southeast University, Nanjing 210096, PR China School of Pharmacy, Nanjing Medical University, Nanjing 210029, PR China
a r t i c l e
i n f o
Article history: Received 27 June 2012 Received in revised form 1 August 2012 Accepted 29 August 2012 Available online 4 September 2012 Keywords: Liposomes Codrug LMX Bioavailability Lamivudine, Ursolic acid
a b s t r a c t A novel codrug (LMX) consisting of Lamivudine and Ursolic acid has been shown to possess the dual action of anti-hepatitis B virus activity and hepatoprotective effects against acute liver injury in vivo. Because of the limited water solubility of LMX, our aims were to design and optimize a liposomal formulation that could facilitate its in vivo administration, and to estimate the potential of LMX-loaded liposomes as oral or intravenous delivery system. In this work, LMX-loaded liposomes were prepared by the thin film hydration method coupled with sonication. LMX-loaded liposomes showed spherical morphology under transmission electron microscope (TEM) analysis. The mean particle size of liposomes was about 210 nm, and the drug entrapment efficiency was more than 90%. Stability data indicated that lyophilized liposomes were stable for at least 6 months at 4 ◦ C. In vitro drug release profile of LMX-loaded liposomes showed a sustained release profile of LMX and an initial mild burst was observed. The relative bioavailability of LMX-loaded liposomes was 1074.8% compared with LMX suspension after oral administration, and 135.2% relative to 50% alcohol solution after intravenous (i.v.) administration. These results indicated that LMX-loaded liposomes were valued to develop as a practical preparation for oral or i.v. administration. © 2012 Elsevier B.V. All rights reserved.
1. Introduction Hepatitis B virus (HBV) infection is a serious public health problem worldwide and is a major cause of chronic hepatitis, cirrhosis, and hepatocellular carcinoma (HCC) (Cha and Dematteo, 2005). At least 2 billion people are infected with HBV and among them 350–400 million are chronic HBV carriers. An estimated 1 million people die each year from acute and chronic sequelae secondary to HBV infection (Ganem and Prince, 2004). Approximately 4.5 million new cases of HBV infection occur worldwide each year, and 25% of these cases progress to liver disease (Goldstein et al., 2005). Antiviral medication plays an important role in the treatment of chronic hepatitis B (CHB). Anti-HBV therapy can postpone the spread of the disease, enhance patient quality of life, and prolong the patient’s life span (Chen and Ju, 2011). One type of antiviral medication, the nucleotide analogues, is becoming increasingly popular, such as Lamivudine (LMV). However, incomplete viral suppression and emergence of drug resistance is a major concern (Zoulim and Locarnini, 2012). Moreover, drug rechallenge can lead to serious or fatal liver injury. A broad spectrum of medications including LMV is also associated with liver injury (Julie et al., 2009; Bjornesson and Olsson, 2006). Ursolic acid (UA) is the major component of extracts of the Chinese herb, Souyang, which has been shown a variety of
∗ Corresponding author. Tel.: +86 25 86862763; fax: +86 25 86863165. E-mail address: [email protected] (B. Wu). 0378-5173/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ijpharm.2012.08.051
biological effects, such as anti-inflammatory, anti-tumor, hepatoprotective and immunomodulatory effects. Among the biological effects of UA, hepatoprotection is one of the more notable effects (Liu, 1995; Shih et al., 2004). Previous research of our team has reported a novel codrug, LMX, consisting of LMV and UA coupled with ethyl chloroacetate via an amide and ester linkage (Zhong et al., 2012). Pharmacological studies indicated that LMX had the dual action of anti-hepatitis B virus activity and hepatoprotective effects against acute liver injury. This design offers a novel strategy for chronic hepatitis B treatment. However, the water solubility of LMX is low (<10 g/mL), which would hinder its clinical applications. Therefore, it is essential to design a valuable formulation to facilitate its in vivo administration. Liposomes are well-recognized drug delivery vehicles. It has been shown that liposomes could act as biocompatible, biodegradable, non-immunogenic drug carriers, the advantages of liposomal-encapsulated drugs are prolonged duration of exposure, selective delivery of entrapped drug to the site of action (Zamboni, 2005), improved therapeutic index, and potentially overcoming resistance. In addition, liposomes as drug delivery systems have the potential for providing controlled release of the administered drug and increasing the stability of labile drugs (Gregoriadis, 1991), such as LMX. Oral liposomes may provide increased solubility of their load and protection from the hostile environment in the gastrointestinal tract (Ariën et al., 1993, 1994). In trial experiment LMX-loaded liposomes showed significantly improved dissolution (1.0–1.5 mg/mL), which indicated an enhanced oral bioavailability.
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Most importantly, the similarity between liposomal lipid bilayers and biomembranes and the suitable size of liposomes significantly facilitate oral absorption (Aungust, 1993). More recently, for poorly water-soluble drugs, substantial enhancement in bioavailability or in vivo efficacy has been observed following liposomal encapsulation (Guo et al., 2001; Xiao et al., 2006). The objective of the present study was to synthesize LMX, and develop a liposomal formulation of it for oral and intravenous administration. LMX-loaded liposomes were prepared by the film dispersion–sonication techniques, and the physicochemical characteristics, stabilities as well as the drug release characteristics of lipsomes were investigated in detail. Furthermore, in vivo pharmacokinetic characteristics and bioavailabilities were evaluated in rats after oral or intravenous administration of LMX-loaded liposomes.
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evaporator until a thin lipid film was formed. Solvent traces were eliminated by drying the film at 5 mbar overnight. The resulting thin film was hydrated in double-distilled water (methylene chloride to water, 0.75:1, v/v) to obtain a crude dispersion of liposomes by rotation of the flask which contained small glass beads. The liposome dispersion was then sonicated for 20 min by intermittent probe sonication (JYD-650, Shanghai, China) under cooling in a water bath, and finally extruded 3 times through polycarbonate membrane (Millipore Billerica, MA) with the size of 800 nm. An opalescent dispersion of liposomes was obtained. Sufficient quantum of sucrose was added into the homogeneous liposomes suspension (ratio of sucrose to lipids, 1.3:1–2:1, w/w), and lyophilized (LGJ0.5, Beijing Four-Ring Scientific Instrument Co., China) (Glavas-Dodov et al., 2005). The lyophilized powders were kept in a refrigerator at 4 ◦ C until use.
2. Materials and methods 2.1. Materials and animals Injectable soya lecithin was provided by Shanghai Taiwei Pharmaceutical Co., Ltd. (Shanghai, China). Cholesterol was obtained from Shanghai Medical Chemical Reagent Co., Ltd. (Shanghai, China). LMV was obtained from Nordhuns Chemical Technology Co., Ltd. (Beijing, China). UA was purchased from Huisheng Medicament Technology Co., Ltd. (Xi’an, Shanxi, China). Dialysis bags were provided from Sigma (molecular weight cutoff, MWCO, 12,000–14,000, USA). All the other chemicals and reagents used were of analytical purity grade or higher, obtained commercially. Sprague-Dawley (SD) rats (200 ± 20) g were supplied by Centre of Laboratory Animal of Nanjing Medical University. The animals were used following the National Act on the use of experimental animals. 2.2. Synthesis of LMX LMX was prepared according to the method reported by our previous research (Zhong et al., 2012). In brief, LMX was obtained from LMV and UA through an amide and ester linkage, respectively, by using ethyl chloroacetate as a coupling group (Fig. 1). LMX was efficiently purified by silica gel column chromatography and carefully characterized by 1 H NMR, 13 C NMR, IR and HRMS. 2.3. Preparation of LMX-loaded liposomes The thin film hydration method was selected to prepare the crude liposomes suspension (Pupo et al., 2005). The parameters such as weight ratio of soya lecithin to cholesterol (SL/Cho ratios), weight ratio of drug to lipids (lecithin and cholesterol) (D/L ratios) and the volume ratio of methylene chloride to double-distilled water (O/W ratios) were optimized each at four levels taking the entrapment efficiency as index. When one factor was under investigated, the other two were fixed and the fixed parameters (SL/Cho ratios, D/L ratios and O/W ratios) were 4:1, 3:14 and 1:1, respectively. The LMX-loaded liposomes were obtained from the crude liposomes suspension by sonication and extrusion (Xiao et al., 2010; Bhardwaj and Burgess, 2010). The crude liposomes were sonicated at 40 ◦ C (180 W, 20 min). Next, the suspension was extruded 3 times through polycrbonate membrane (Millipore Billerica, MA) with the size of 800 nm. The formulations and the manufacturing parameters were optimized concerning drug capsulation efficiency, particle size and drug loading. The optimal formulation was as follows: soya lecithin, cholesterol (4:1, w/w) and LMX (drug-to-lipids ratio, w/w of 3:14) were dissolved in methylene chloride (the concentration of lipids was 9.3 mg/mL), the mixture was warmed to 40 ◦ C in a round-bottomed flask, and the solvent was evaporated under vacuum in a rotary
2.4. Determination of drug encapsulation efficiency and drug loading 2.4.1. HPLC analysis A Shimadzu LC-2010 C system (Shimadzu, Kyoto, Japan) with built-in UV detector (SPD-10A, Shimadzu, Japan) was used to perform all the analyses. Chromatographic software Class VP 6.12 was used for data collection and processing. A HYPERSIL C18 column (10 m particle size, 250 mm × 4.6 mm) was used for the separation. Mobile phase was composed of methanol and water containing 0.5% ammonium acetate (95:5, v/v); the flow rate and column temperature were set at 1 mL/min and 25 ◦ C, respectively. The UV absorbance was determined at 245 nm. The sample was prepared by dissolving the lyophilized powder in double distilled water and being diluted by methanol (to destroy liposome and dissolve LMX) and 20 L of the sample was injected. The calibration curve of various LMX concentrations (C, 10–150 g/mL) versus integrated area (A, mAUs) was A = 18210C–385.9, with a correlation coefficient of 0.9999. 2.4.2. Drug encapsulation efficiency and drug loading Encapsulation efficiency (EE) of LMX-loaded liposomes was calculated by determining the amount of free drug using ultrafiltration technique. 1 mL LMX-loaded liposomes colloidal solution was placed in the upper chamber of a centrifuge tube matched with an ultrafilter (Amicon Ultra, Millipore Co., USA, MWCO 10 kDa) and centrifuged for 10 min at 4000 rpm. The ultrafiltrate containing the unencapsulated drug was determined by HPLC. The total drug content in liposomes was determined as follows: aliquots of 1 mL liposomes dispersion were dissolved and diluted appropriately by methanol to dissolve the liposomes and then the obtained suspension was allowed to filter through 0.45 m membrane filters. The resulting solution was analyzed by HPLC. The drug loading (DL) was the ratio of entrapped drug to lipid (w/w). The EE and DL could be calculated by the following equations: EE% =
Wtotal − Wfree × 100 Wtotal
DL% =
Wtotal − Wfree × 100 Wlipid
where Wtotal , Wfree , Wlipid were the weight of total drug in liposomes dispersions; the weight of unentrapped drug in ultrafiltrate and the weight of lipid added in system, respectively. 2.5. Characterization of LMX-loaded liposomes 2.5.1. Morphology The morphology of LMX-loaded liposomes was observed by transmission electron microscopy (TEM) (JEM-1200EX, Jeol, Japan).
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Fig. 1. Structure of codrug LMX, UA and LMV.
Fresh prepared liposomes were diluted with distilled water and dropped on a formvar-coated copper grid and air-dried for 1 min at room temperature after removing the excessive sample with filter paper. The air-dried samples were then directly examined under the TEM. 2.5.2. Particle size and zeta potential The lyophilized powders were reconstituted with 5% dextrose injection solution. The average diameter, polydispersity index and zeta potential were measured using a Malvern Zetasizer 3000 system (Malvern Instruments Ltd., Malvern, UK). All of the dynamic light scattering (DLS) measurements were performed at 25 ◦ C and at a scattering angle of 90◦ . The zeta potential values were calculated using the Smoluchowski equation. 2.6. Stability studies The freeze-dried LMX-loaded liposomes were stored at 4 ◦ C for 6 months under a sealed condition. The mean particle size and drug entrapment efficiency were determined at fixed time intervals, respectively. 2.7. The drug release experiment in vitro In vitro release of LMX from liposomes was performed using the dialysis bag diffusion technique (Avgoustakis et al., 2002). The dialysis bags (MWCO, 12,000–14,000, Sigma) were soaked in deionized water for 12 h before use. In this study, 20% ethanol–water was used as dissolution medium. 14 mg of the freeze-dried powder with LMX (equivalent to 1.5 mg LMX) rehydrated in 1 mL of 5% glucose solution or 1 mL LMX solution (equivalent to 1.5 mg LMX) were placed in dialysis bags. Both ends of the bag were clamped and each bag was then individually immersed in a beaker containing 70 mL of the dissolution medium. All beakers were placed in a water bath maintained at 37 ± 0.5 ◦ C throughout the release study.
The constant shaking was at 90 strokes/min. The release medium was changed every 3 h to ensure sink conditions and the stability of LMX. The sampling time intervals were 0, 3, 6, 9, 12, . . ., 80 h. A sample of 70 mL at each time point was withdrawn, and the beaker was replenished with fresh 70 mL of 20% C2 H5 OH H2 O (Nornoo and Chow, 2008; Alani et al., 2010). Drug concentrations in the dissolution medium were finally analyzed using the HPLC method as described above (Section 2.4.1). The release experiments were carried out in triplicates and the results were expressed as means ± standard deviation. 2.8. Pharmacokinetic studies in rats 2.8.1. Chromatographic analysis conditions in vivo A Waters HPLC system (Milford, Massachusetts) was used to quantify LMX and LMV in plasma samples, using a HYPERSIL C18 column (10 m particle size, 250 mm × 4.6 mm). The mobile phase for gradient elution consisted of two solvent systems: solvent A, methanol; solvent B, 0.5% (g/v) ammonium acetate solution adjusted to pH 3.56 with glacial acetic acid. A + B = 100%. A gradient elution was carried out as follows: 93% (v/v) solvent A was used during 0–3 min, then solvent A percentage was linearly decreased to 50% in the 4th min and maintained during 4–13 min, solvent A percentage was linearly increased to 93% in the 14th min and maintained for another 6 min. The total run time was 20 min including equilibration of the system. The flow-rate was 1 mL/min. The eluent was monitored by a UV detector, and the detection wavelength was set at 245 nm for LMX and 270 nm for LMV. The sample injection volume was 20 L and the column temperature was 40 ◦ C. Calibration curves were constructed by plotting the peak-area ratios of each analyte/internal standard R (Ai /Ais ) versus analyte concentration in plasma. The linearity of LMX and LMV was observed over the concentration range of 1500–3.0 g/mL and 750–1.5 g/mL, respectively. The calibration curves of LMX and LMV were as follows: R = (7.00E−05)C − 0.002 (r2 = 0.995, n = 6) and
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R = 0.003C+0.035 (r2 = 0.996, n = 6), respectively. The limits of detection in rat plasma for LMX and LMV were 3.0 g/mL and 1.5 g/mL, respectively. Accuracy of the determination of LMX and LMV in rat plasma (n = 6) is 100.42 ± 2.70% and 100.40 ± 1.01%, respectively. Within-day and between-day precisions of LMX and LMV were all below 4.72%. Extraction recovery of LMX and LMV in rat plasma (n = 6) was above 80.16 ± 3.46%. 2.8.2. Drug extraction Liquid–liquid double extraction was performed prior to analysis by HPLC. Briefly, a 300 L of rat plasma was mixed with 30 L Edaravone (internal standard, 100 g/mL) solution and vortexed for 1 min. Then the mixture was extracted with 4 mL of methylene chloride vortexing for 3 min. Following centrifugation at 3000 rpm for 10 min, the organic phase 3 mL was transferred to a glass tube and the solvent was evaporated under nitrogen stream at 35 ◦ C. The resulting residue was reconstituted with 100 L of mobile phase by vortexing for 4 min and 20 L of the solution was injected into the column. HPLC conditions were described above. 2.8.3. Pharmacokinetic study Pharmacokinetics studies were performed as described elsewhere (Gao et al., 2006). The bioavailability studies were complied with the requirements of the National Act on the use of experimental animals (People’s Republic of China). The rats used for this study were housed individually under normal conditions, and fasted overnight before experiment with free access to water. Rats were randomly divided into four groups (5 per group). Rats in group 1 were orally administered with LMX suspension (equivalent to 32 mg/kg of LMX, 1.5 mL/rats) and the group 2 was orally administered with LMX-loaded liposomes suspensions (equivalent to 32 mg/kg of LMX, 1.5 mL/rats). Similarly, LMX 50% alcohol solution (equal to 32 mg/kg of LMX, 1 mL/rats) and LMX-loaded liposomes (equal to 32 mg/kg of LMX, 1 mL/rats) were injected intravenously into the other two groups, respectively. Blood samples were collected from the postorbital venous plexuses of rats and placed into heparinized test tubes according to the designed time interval (Gutierro et al., 2002). The plasma samples were harvested after centrifugation (15 min, 4000 rpm) and stored at −20 ◦ C until analysis. The main pharmacokinetic parameters were acquired with the help of a pharmacokinetic program DAS 2.0 (Mathematical Pharmacology Professional Committee, China). The values of maximum concentration (Cmax ) and time of maximum concentration (Tmax ) were obtained directly from the concentration–time plotting. The area under the concentration–time curve (AUC) was calculated by linear trapezoidal method. 2.9. Statistical analysis Statistical analysis was performed with Student’s t-test for two groups and one-way ANOVA for multiple groups. All results were expressed as mean ± S.D. unless otherwise noted. P < 0.05 was considered statistically significant.
Fig. 2. Transmission electron micrographs of LMX-loaded liposomes.
145.58, 138.04, 126.31, 96.14, 88.24, 79.05, 62.86, 55.22, 52.89, 48.53, 47.56, 42.12, 39.55, 39.16, 38.76, 38.59, 37.01, 36.74, 32.90, 30.54, 28.16, 27.27, 24.51, 23.60, 23.23, 21.11, 18.32, 17.06. ESIHRMS calculated for C40 H59 N3 O7 NaS: 748.3971. Found 748.3952 (MNa+). IR (KBr): 3439 (N H), 1732 (C O), 1658 (C O). 3.2. Preparation of LMX-loaded liposomes The effects of the three influential factors, SL/Cho ratios (X1 ), D/L ratios (X2 ), O/W ratios (X3 ), on encapsulation efficiency (EE) of liposomes were shown in Table 1. The results showed that these three factors had little influence on the particle size (data not shown) and the maximal value of EE could be reached when SL/Cho ratio was set at 4:1. Concerning drug loading, the optimized D/L ratio and O/W ratio were 3:14 and 0.75:1, respectively. The drug loading could be enhanced with the increase of D/L ratio, while a higher lipids ratio improved EE in this study. Moreover, EE value increased when SL/Cho ratio increased until SL/Cho ratio reached 4:1. Certain concentration of cholesterol could reduce the bilayer permeability, increase the stability of liposomes and cause high EE (Gregoriadis, 1993), while beyond a certain ratio, cholesterol could disrupt the regular structure of the liposomal membrane, result in lower EE (El-Samaligy et al., 2006). In our case, the O/W ratio (0.75:1) caused higher EE and better stability in vitro. Since the entrapment capacity of liposomes for drugs strongly diminishes with decrease of liposome size, larger liposomes were considered more promising pharmaceutical carriers (Torchilin et al., 2001). The optimized formulation was repeated in triplicates. The average entrapment efficiency and the average drug loading of LMX-loaded liposomes were (91.28 ± 3.25%) and (12.59 ± 0.19%), respectively.
3. Results and discussion 3.3. Characterization of LMX-loaded liposomes 3.1. Synthesis of LMX LMX was obtained from LMV and UA. LMX is a white solid. m.p.: 174.5–175.1 ◦ C. 1 H NMR (300 MHz, CDCl3 ): 8.55–8.57 (d, 1H, J = 3.6 Hz), 7.39–7.42 (d, 1H, J = 7.3 Hz), 6.34 (s, 1H,), 5.38 (s, 2H), 4.74–4.79 (m, 1H), 4.56–4.62 (m, 2H), 4.17–4.21 (m, 1H), 4.09–4.16 (m, 1H), 3.96–4.01 (m, 1H), 3.63–3.69 (m, 1H), 3.19–3.30 (m, 2H), 0.71–2.28 (m, 45H). 13 C NMR (75 MHz, CDCl3 ): 176.11, 161.53,
TEM was conducted to investigate the morphology of liposomes, showing that the nanoparticles had near spherical shapes. Vesicular structure was discernable (Fig. 2). The particle size and polydispersity index of LMX-loaded liposomes were estimated by DLS. The sizes of LMX-loaded liposomes (200–220 nm) were significantly bigger than those of blank liposomes (about 170 nm) (P < 0.05). It is also worth noting that the
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Table 1 The levels of experimental factors, the liposomes entrapment efficiencies and drug loading.
1 2 3 4
X1 (w/w)
EE (%)
LD (%)
X2 (w/w)
EE (%)
LD (%)
X3 (V/V)
EE (%)
LD (%)
1:1 2.5:1 4:1 6:1
29.13 84.64 88.89 79.71
3.72 11.49 10.87 9.82
1:14 2:14 3:14 4:14
93.5 87.98 84.64 63.40
3.58 8.69 11.49 11.17
0.5:1 0.75:1 1:1 1.5:1
88.22 91.2 90.13 78.84
11.23 12.59 12.16 10.70
X1 : weight ratio of soya lecithin to cholesterol (SL/Cho ratios). X2 : weight ratio of drug to lipids (D/L ratios). X3 : volume ratio of methylene chloride to water (O/W ratios).
size of the drug-loaded liposomes was not significantly affected by three influential factors (data not shown). The polydispersity index of LMX-loaded liposomes, estimated by the cumulant method, was low (<0.220), which suggested a narrow size distribution. Zeta potential or particle surface charge is an important parameter indicating to the stability of nanocarrier systems. A relatively high surface charge may provide a repelling force between the particles, thus increasing the stability of the solution (Kwon et al., 2003). From the date obtained, all the LMX-loaded liposomes had relatively high negative zeta potentials of around −40 mV. It is reasonable to conclude that the charged particles may repel each other and prevent aggregation or precipitation happening, thus resulting in good stability. The negative or neutral liposomes provide more effective barrier to plasma macromolecular protein adsorption and are easy to resuspend in blood (Mobed and Chang, 1998). 3.4. The effects of lyophilization The process of freeze-drying did not affect the particle size of rehydrated LMX-loaded liposomes containing sucrose as cryoprotectant (ratio of sucrose to lipids, 1.3:1–2:1, w/w), the mean diameter was 213.7 ± 6.12 nm before lyophilization and 217.7 ± 3.14 nm after lyophilization/rehydration. Those values are not different from each other (P > 0.05). On the other side, formulations prepared without sucrose as cryoprotectant showed an increase in vesicle size after lyophilization (272.7 ± 4.73 nm). The process of lyophilization was harmful for the liposome integrity also, as freezing caused a pronounced decrease in the encapsulation efficiency. EE% of LMX-loaded liposomes was only 42.79% after lyophilization without cryoprotectant, while only a small decrease in the encapsulation efficiency was observed by addition of sucrose as cryoprotectant. In addition, a mixture of mannitol and sucrose was also found to be suitable cryoprotectant at the weight ratio of mannitol to sucrose to lipid of 3:2:1 (Fig. 3). Freeze-drying tended to destroy the membrane function of the phospholipid bilayer (Ozer and Talsma, 1989), and lead to leakage of encapsulated drug. Particle size increase after freezedrying was in good agreement with the drug leakage (Stensrud et al., 2000). Sugars are well-known cryoprotective agents (Li et al., 2000). Their protective effect has been related to their ability to interact with the polar head groups of the phospholipids and to stabilize the membranes when the bilayer stabilizing water is removed by sublimation (Stevens and Lee, 2003).
Fig. 3. Encapsulation efficiency of LMX liposome before and after freeze-drying process. (a) Samples before lyophilization; (b) samples after lyophilization without cryoprotectant; (c) samples after lyophilization with cryoprotectant (sucrose:lipid = 1.3:1); (d) samples after lyophilization with cryoprotectant (sucrose:lipid = 2:1); and (e) samples after lyophilization with cryoprotectant (mannitol:sucrose:lipid = 3:2:1).
20% ethanol for 80 h. LMX was continuously released from liposomes and the cumulative release amount of LMX in 80 h was around 52%. As shown in Fig. 4, there was an initial mild burst for the first 2.5 h as the drug molecules adsorbed on the surface were released, followed by a period of moderate release, which was believed that the entrapped drug slowly diffused out into the surrounding medium. This diffusion release phase then flattened out, and a subsequent increase in release rate after 47 h corresponded to an increase in the erosion rate of the liposome, which finally tapered off again as 52% or more of the drug has been released (Jabara et al.,
3.5. Stability studies The stability test of the freeze-dried LMX-loaded liposomes at 4 ◦ C indicated that the mean particle size and drug entrapment efficiency had no significant change during 6 months (Table 2). 3.6. In vitro drug release The release experiment was conducted under sink conditions and the dynamic dialysis was employed for separation of free drug from LMX-loaded liposomes. Fig. 4 shows the cumulative LMX release profile from liposomes in the aqueous medium containing
Fig. 4. Cumulative release of LMX from liposome and solution into 20% ethanol–water at 37 ◦ C. The error bars represent the S.D. of 3 samples.
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Table 2 Physical stability of the freeze-dried LMX-loaded liposomes (n = 3). Time (days)
EE (%) r (nm)
0
1
7
15
30
90
180
89.5 ± 2.15 215.3 ± 3.22
90.04 ± 2.72 215.8 ± 5.09
89.2 ± 3.12 215.6 ± 4.21
89.0 ± 1.68 216.7 ± 6.33
88.1 ± 3.39 217.5 ± 5.18
86.8 ± 2.18 218.8 ± 4.89
84.7 ± 2.66 220 ± 5.21
Table 3 Fitted degradation kinetic equations of LMX liposome and solutions 1 and 2. Time (h) LMX liposomes
0–47.5 47.5–80
LMX solution 1 LMX solution 2
0–4 0–7
Models
Fitted kinetic equations
r2
First-order Hixcon–Crowell
ln(1 − Q) = −0.006t − 0.094 Q = 0.001t3 − 0.342t2 + 22.60t − 448.7
0.991 0.992
First-order First-order
ln(1 − Q) = −1.085t − 0.06 ln(1 − Q) = −0.383t − 0.018
0.993 0.992
2008; Vlasta et al., 2009). The release was a complex process consisting of two phases: during 0–47.5 h, the release of LMX from LMX-loaded liposomes followed the First-order equation; during 47.5–80 h, the release followed the Hixcon–Crowell equation. The delayed release might be attributed to the lipophilic LMX that was held by the small fragment of the liposomal membrane and the drug encapsulated in lipid membrane that released mainly through dissolution and diffusion from the lipid bilayer (Cócera et al., 2000). In contrast, the release of LMX from LMX solution was much fast. In solution 1 (20% ethanol–water) approximately 100% of the drug had been dissolved after 4 h. In solution 2 (50% acetonitrile–water), approximately 85% of the drug had been dissolved after 4 h. These may be due to the different dissolution/diffusion rates of the drug in different solvents. The release dynamics characteristics were fitted to first-order kinetics model in solutions 1 and 2. The fitted degradation kinetic equations of LMX liposomes and solutions 1 and 2 in 20% C2 H5 OH H2 O were shown in Table 3. Maintaining a good sink condition for poorly water-soluble drugs has been one of the difficulties in designing in vitro release experiments. Continuous flow methods to provide infinite water to the release media have been designed to mimic a perfect sink condition (Washington, 1990; Soo et al., 2002). In this method, the release media is removed and replaced infinitely rapidly and the instantaneous release rate is measured. However, this method is sometimes not useful for poorly soluble drugs because of limitations in measurement of the drug concentration due to their low concentrations in the aqueous media (Cho et al., 2004). A hydrotropic agent provides an alternative tool for studying release
of poorly soluble drugs from liposomes in aqueous solutions. In trial experiment, we researched the accumulative release amount of LMX liposomes in other media, such as phosphate buffer (PBS, pH 7.4), HCl (0.1 mol/L), 0.5% Tween 80–water and 0.4% sodium lauryl sulfate (SDS)–water. We found that sink conditions could not be obtained in buffers and HCl (0.1 mol/L) because of the low solubility and instability of LMX (with time prolonging LMV was observed from the analysis of HPLC), and in 0.4% SDS the release of drug from liposomes was so slow that less than 12% of the drug was released in 80 h. The release of LMX from liposomes in 0.5% Tween 80 was little faster than that in 0.4% SDS. Thus, we selected 20% ethanol–water as dissolution medium, which was changed every 3 h to ensure sink conditions and the stability of LMX. From the result of the test, the addition of ethanol significantly increased the drug release rate. In this study, one potential drawback of the in vitro assay is that the media contained 20% ethanol, 0.5% Tween 80 or 0.4% SDS, thus may not faithfully mimic in vivo situation. Furthermore, the in vitro assay failed to take into account the possible disruption of liposomal vesicular structure in physiological conditions. Thus, the results of in vitro release test cannot completely reflect the in vivo factual characteristics. 3.7. Pharmacokinetic studies in rats In the pharmacokinetic experiment, the method of HPLC and Edaravone as internal standard were used. The chromatograms showed stable baselines, as well as displaying good resolution among LMX, Edaravone and endogenous material in plasma. The
Fig. 5. Profile of mean plasma concentration–time of LMV (left) and LMX (right) after intravenous administration of LMX 50% alcohol solution and LMX-loaded liposomes in rats (mean ± S.D., n = 5).
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Fig. 6. Profile of mean plasma concentration–time of LMV (left) and LMX (right) after oral administration of LMX suspension and LMX-loaded liposomes in rats (mean ± S.D., n = 5).
Table 4 Mean pharmacokinetic parameters of LMX after intravenous administration of LMX 50% alcohol solution and LMX-loaded liposomes or oral administration of LMX suspension and LMX-loaded liposomes in rats (mean ± S.D., n = 5). Parameters
Unit
AUC(0→t) MRT(0→t) t1/2 Tmax CL/F Cmax
mg/L h h h h L/h/kg mg/L
i.v.
Oral
LMX 50% alcohol solution 2567.42 5.34 5.14 0.083 0.012 691.78
± ± ± ± ± ±
LMX-loaded liposomes
141.56 0.33 0.04 0.0013 0.005 34.66
3472.2 4.70 2.74 0.10 0.009 1094.34
± ± ± ± ± ±
98.23* 0.27 0.03* 0.02 0.0006 43.22*
LMX suspension 987.94 10.10 4.96 8.13 0.05 113.16
± ± ± ± ± ±
34.68 0.48 0.04 0.45 0.004 15.11
LMX-loaded liposomes 10618.4 11.73 2.14 10.03 0.003 1315.59
± ± ± ± ± ±
892.11## 0.59# 0.03 0.68# 0.0004# 110.90#
Each data was from five rats. * P < 0.05 vs. LMX 50% alcohol solution. # P < 0.05 vs. LMX suspension. ## P < 0.01 vs. LMX suspension.
concentrations of LMX and LMV in the samples were determined by comparing the peak area ratios of the samples versus a calibration curve obtained from spiking known amounts of LMX and LMV in pooled rats plasma. LMX and LMV plasma concentrations, pharmacokinetic parameters, half-life and clearance, in the control group (suspension or 50% alcohol solution) were compared with those of the liposome treated groups. Pharmacokinetic analysis was performed using non-compartmental methods with DAS (2.0) program. Results were presented as mean ± S.D. Figs. 5 and 6 show the mean concentration in plasma versus time profiles of LMX and LMV after oral or i.v. administration of liposomes and suspension (or 50% alcohol solution) at a dose of 32 mg/kg to rats, respectively, and the pharmacokinetic parameters of LMX and LMV were summarized in Tables 4 and 5, respectively.
Oral liposomal administration showed a higher Cmax compared with the LMX suspension (P < 0.05). The AUC(0→24) h following oral liposomal administration was significantly higher than LMX suspension (P < 0.01), and the relative bioavailability was 1074.8%. Compared with the LMX suspension, liposomes exhibited a higher mean residence time (MRT) (11.73 h) (P < 0.05), while CL decreased by 16 times (Table 4). These could be explained by the increased affinity between LMX-loaded liposomes and the gut liquid, which caused better dispersibility of LMX in the gastrointestinal tract, prolonged the drug residence time in the absorption sites, and resulted in better absorption effects (Vasir et al., 2003). LMX-loaded liposomes showed a delayed Tmax and higher MRT (P < 0.05) compared with the LMX suspension, which indicated that the liposomes after oral administration retarded the clearance and exhibited the property of sustained release. The prolonged drug circulation was
Table 5 Mean pharmacokinetic parameters of LMV after intravenous administration of LMX 50% alcohol solution and LMX-loaded liposomes or oral administration of LMX suspension and LMX-loaded liposomes in rats (mean ± S.D., n = 5). Parameters
Unit
AUC(0→t) MRT(0→t) t1/2 Tmax CL/F Cmax
mg/L h h h h L/h/kg mg/L
i.v.
Oral
LMX 50% alcohol solution
Each data was from five rats. * P < 0.05 vs. LMX 50% alcohol solution. # P < 0.05 vs. LMX suspension.
319.98 7.91 4.54 6.0 0.095 40.53
± ± ± ± ± ±
45.21 0.89 0.05 0.45 0.004 3.32
LMX-loaded liposomes 932.5 6.25 2.51 5.0 0.034 105.7
± ± ± ± ± ±
39.1* 0.76 0.03 0.56 0.002* 4.01*
LMX suspension 348.50 11.0 1.99 10.22 0.091 41.64
± ± ± ± ± ±
27.54 0.88 0.02 0.86 0.005 3.44
LMX-loaded liposomes 421.9 12.52 3.50 12.98 0.074 30.24
± ± ± ± ± ±
38.63# 0.94# 0.04 1.02# 0.005# 2.98
Y. Zhong et al. / International Journal of Pharmaceutics 438 (2012) 240–248
consistent with the results obtained from the release in vitro. Compared with the i.v. administration of the LMX 50% alcohol solution, the relative bioavailability of LMX-loaded liposomes after i.v. administration was 135.2%, which confirmed the better absorption effects of LMX-loaded liposomes. No significant difference of the MRT was noted between liposomes and 50% alcohol solution group (P > 0.05) after i.v. administration. Both the rapid uptake of liposomes by the organs of the reticuloendothelial system (RES) and the slow release due to the reservoir effect might be the reasons (Klibanov and Huang, 1992). Liposomes as circulating reservoirs in tissues probably enter the blood again, and repeatedly, achieve dynamic balance eventually. Similarly, LMV pharmacokinetic parameters of LMX-loaded liposomes formulation displayed higher AUC(0→t) and MRT(0→t) , with lower CL, in comparison with LMX suspension after oral administration (Table 5). After i.v. administration, LMV had a higher AUC(0→t) and Cmax , with lower CL, compared with LMX 50% alcohol solution. 4. Conclusion In this study, a novel codrug of LMX was successfully synthesized, and incorporated into liposomes for applying to oral and intravenous administration. The liposomes formulation did enhance the gastrointestinal absorption of LMX by oral administration, about 10.75-fold of relative bioavailability comparing to LMX suspension was observed, and had significantly prolonged circulation duration, which was consistent with the results obtained from the release tests in vitro. LMX-loaded liposomes exhibited a 1.35fold increase in bioavailability relative to LMX 50% alcohol solution after i.v. administrated. These results provided an important reference for the use of LMX in the field of the treatment of hepatitis. Further studies are needed to focus on the safety and efficiency of LMX-loaded liposomes to evaluate the potential clinical application value. Acknowledgements The authors thank the financial support provided by the Natural Science Foundation of Jiangsu Province (No. BK2010538). The authors wish to thank Dr. Rui Li (School of Pharmacy, Nanjing Medical University, China) for helpful suggestions and discussion for the paper. References Alani, A.W.G., Bae, Y., Rao, D.A., Kwon, G.S., 2010. Polymeric micelles for the pHdependent controlled, continuous low dose release of paclitaxel. Biomaterials 31, 1765–1772. Ariën, A., Goigoux, C., Baquey, C., Dupuy, B., 1993. Study of in vitro and in vivo stability of liposomes loaded with calcitonin or indium in the gastrointestinal tract. Life Sci. 53, 1279–1290. Ariën, A., Henry-Toulmé, N., Dupuy, B., 1994. Calcitonin-loaded liposomes: stability under acidic conditions and bile salts-induced disruption resulting in calcitonin–phospholipid complex formation. Biochim. Biophys. Acta 1193, 93–100. Aungust, B.J., 1993. Novel formulation strategies for improving oral bioavailability of drugs with poor membrane permeation or presystemic metabolism. J. Pharm. Sci. 82, 979–987. Avgoustakis, K., Beletsi, A., Panagi, Z., 2002. PLGA–mPEG nanoparticles of cisplatin: in vitro nanoparticle degradation, in vitro drug release and in vivo drug residence in blood properties. J. Control. Release 79, 123–135. Bhardwaj, U., Burgess, D.J., 2010. Physicochemical properties of extruded and nonextruded liposomes containing the hydrophobic drug dexamethasone. Int. J. Pharm. 388, 181–189. Bjornesson, E., Olsson, R., 2006. Suspected drug-induced liver fatalities reported to the WHO database. Dig. Liver Dis. 38, 33–38. Cha, C., Dematteo, R.P., 2005. Molecular mechanisms in hepatocellular carcinoma development. Best Pract. Res. Clin. Gastroenterol. 19, 25–37. Chen, Y.F., Ju, T., 2011. Comparative meta-analysis of adefovir dipivoxil monotherapy and combination therapy of adefovir dipivoxil and lamivudine for lamivudineresistant chronic hepatitis B. Int. J. Infect. Dis. 16, e152–e158.
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International Journal of Pharmaceutics journal homepage: www.elsevier.com/locate/ijpharm
Preparation and evaluation of liposome-encapsulated codrug LMX Yan Zhong a , Jing Wang b , Yao Wang b , Bin Wu b,∗ a b
School of Chemistry and Chemical Engineering, Southeast University, Nanjing 210096, PR China School of Pharmacy, Nanjing Medical University, Nanjing 210029, PR China
a r t i c l e
i n f o
Article history: Received 27 June 2012 Received in revised form 1 August 2012 Accepted 29 August 2012 Available online 4 September 2012 Keywords: Liposomes Codrug LMX Bioavailability Lamivudine, Ursolic acid
a b s t r a c t A novel codrug (LMX) consisting of Lamivudine and Ursolic acid has been shown to possess the dual action of anti-hepatitis B virus activity and hepatoprotective effects against acute liver injury in vivo. Because of the limited water solubility of LMX, our aims were to design and optimize a liposomal formulation that could facilitate its in vivo administration, and to estimate the potential of LMX-loaded liposomes as oral or intravenous delivery system. In this work, LMX-loaded liposomes were prepared by the thin film hydration method coupled with sonication. LMX-loaded liposomes showed spherical morphology under transmission electron microscope (TEM) analysis. The mean particle size of liposomes was about 210 nm, and the drug entrapment efficiency was more than 90%. Stability data indicated that lyophilized liposomes were stable for at least 6 months at 4 ◦ C. In vitro drug release profile of LMX-loaded liposomes showed a sustained release profile of LMX and an initial mild burst was observed. The relative bioavailability of LMX-loaded liposomes was 1074.8% compared with LMX suspension after oral administration, and 135.2% relative to 50% alcohol solution after intravenous (i.v.) administration. These results indicated that LMX-loaded liposomes were valued to develop as a practical preparation for oral or i.v. administration. © 2012 Elsevier B.V. All rights reserved.
1. Introduction Hepatitis B virus (HBV) infection is a serious public health problem worldwide and is a major cause of chronic hepatitis, cirrhosis, and hepatocellular carcinoma (HCC) (Cha and Dematteo, 2005). At least 2 billion people are infected with HBV and among them 350–400 million are chronic HBV carriers. An estimated 1 million people die each year from acute and chronic sequelae secondary to HBV infection (Ganem and Prince, 2004). Approximately 4.5 million new cases of HBV infection occur worldwide each year, and 25% of these cases progress to liver disease (Goldstein et al., 2005). Antiviral medication plays an important role in the treatment of chronic hepatitis B (CHB). Anti-HBV therapy can postpone the spread of the disease, enhance patient quality of life, and prolong the patient’s life span (Chen and Ju, 2011). One type of antiviral medication, the nucleotide analogues, is becoming increasingly popular, such as Lamivudine (LMV). However, incomplete viral suppression and emergence of drug resistance is a major concern (Zoulim and Locarnini, 2012). Moreover, drug rechallenge can lead to serious or fatal liver injury. A broad spectrum of medications including LMV is also associated with liver injury (Julie et al., 2009; Bjornesson and Olsson, 2006). Ursolic acid (UA) is the major component of extracts of the Chinese herb, Souyang, which has been shown a variety of
∗ Corresponding author. Tel.: +86 25 86862763; fax: +86 25 86863165. E-mail address: [email protected] (B. Wu). 0378-5173/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ijpharm.2012.08.051
biological effects, such as anti-inflammatory, anti-tumor, hepatoprotective and immunomodulatory effects. Among the biological effects of UA, hepatoprotection is one of the more notable effects (Liu, 1995; Shih et al., 2004). Previous research of our team has reported a novel codrug, LMX, consisting of LMV and UA coupled with ethyl chloroacetate via an amide and ester linkage (Zhong et al., 2012). Pharmacological studies indicated that LMX had the dual action of anti-hepatitis B virus activity and hepatoprotective effects against acute liver injury. This design offers a novel strategy for chronic hepatitis B treatment. However, the water solubility of LMX is low (<10 g/mL), which would hinder its clinical applications. Therefore, it is essential to design a valuable formulation to facilitate its in vivo administration. Liposomes are well-recognized drug delivery vehicles. It has been shown that liposomes could act as biocompatible, biodegradable, non-immunogenic drug carriers, the advantages of liposomal-encapsulated drugs are prolonged duration of exposure, selective delivery of entrapped drug to the site of action (Zamboni, 2005), improved therapeutic index, and potentially overcoming resistance. In addition, liposomes as drug delivery systems have the potential for providing controlled release of the administered drug and increasing the stability of labile drugs (Gregoriadis, 1991), such as LMX. Oral liposomes may provide increased solubility of their load and protection from the hostile environment in the gastrointestinal tract (Ariën et al., 1993, 1994). In trial experiment LMX-loaded liposomes showed significantly improved dissolution (1.0–1.5 mg/mL), which indicated an enhanced oral bioavailability.
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Most importantly, the similarity between liposomal lipid bilayers and biomembranes and the suitable size of liposomes significantly facilitate oral absorption (Aungust, 1993). More recently, for poorly water-soluble drugs, substantial enhancement in bioavailability or in vivo efficacy has been observed following liposomal encapsulation (Guo et al., 2001; Xiao et al., 2006). The objective of the present study was to synthesize LMX, and develop a liposomal formulation of it for oral and intravenous administration. LMX-loaded liposomes were prepared by the film dispersion–sonication techniques, and the physicochemical characteristics, stabilities as well as the drug release characteristics of lipsomes were investigated in detail. Furthermore, in vivo pharmacokinetic characteristics and bioavailabilities were evaluated in rats after oral or intravenous administration of LMX-loaded liposomes.
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evaporator until a thin lipid film was formed. Solvent traces were eliminated by drying the film at 5 mbar overnight. The resulting thin film was hydrated in double-distilled water (methylene chloride to water, 0.75:1, v/v) to obtain a crude dispersion of liposomes by rotation of the flask which contained small glass beads. The liposome dispersion was then sonicated for 20 min by intermittent probe sonication (JYD-650, Shanghai, China) under cooling in a water bath, and finally extruded 3 times through polycarbonate membrane (Millipore Billerica, MA) with the size of 800 nm. An opalescent dispersion of liposomes was obtained. Sufficient quantum of sucrose was added into the homogeneous liposomes suspension (ratio of sucrose to lipids, 1.3:1–2:1, w/w), and lyophilized (LGJ0.5, Beijing Four-Ring Scientific Instrument Co., China) (Glavas-Dodov et al., 2005). The lyophilized powders were kept in a refrigerator at 4 ◦ C until use.
2. Materials and methods 2.1. Materials and animals Injectable soya lecithin was provided by Shanghai Taiwei Pharmaceutical Co., Ltd. (Shanghai, China). Cholesterol was obtained from Shanghai Medical Chemical Reagent Co., Ltd. (Shanghai, China). LMV was obtained from Nordhuns Chemical Technology Co., Ltd. (Beijing, China). UA was purchased from Huisheng Medicament Technology Co., Ltd. (Xi’an, Shanxi, China). Dialysis bags were provided from Sigma (molecular weight cutoff, MWCO, 12,000–14,000, USA). All the other chemicals and reagents used were of analytical purity grade or higher, obtained commercially. Sprague-Dawley (SD) rats (200 ± 20) g were supplied by Centre of Laboratory Animal of Nanjing Medical University. The animals were used following the National Act on the use of experimental animals. 2.2. Synthesis of LMX LMX was prepared according to the method reported by our previous research (Zhong et al., 2012). In brief, LMX was obtained from LMV and UA through an amide and ester linkage, respectively, by using ethyl chloroacetate as a coupling group (Fig. 1). LMX was efficiently purified by silica gel column chromatography and carefully characterized by 1 H NMR, 13 C NMR, IR and HRMS. 2.3. Preparation of LMX-loaded liposomes The thin film hydration method was selected to prepare the crude liposomes suspension (Pupo et al., 2005). The parameters such as weight ratio of soya lecithin to cholesterol (SL/Cho ratios), weight ratio of drug to lipids (lecithin and cholesterol) (D/L ratios) and the volume ratio of methylene chloride to double-distilled water (O/W ratios) were optimized each at four levels taking the entrapment efficiency as index. When one factor was under investigated, the other two were fixed and the fixed parameters (SL/Cho ratios, D/L ratios and O/W ratios) were 4:1, 3:14 and 1:1, respectively. The LMX-loaded liposomes were obtained from the crude liposomes suspension by sonication and extrusion (Xiao et al., 2010; Bhardwaj and Burgess, 2010). The crude liposomes were sonicated at 40 ◦ C (180 W, 20 min). Next, the suspension was extruded 3 times through polycrbonate membrane (Millipore Billerica, MA) with the size of 800 nm. The formulations and the manufacturing parameters were optimized concerning drug capsulation efficiency, particle size and drug loading. The optimal formulation was as follows: soya lecithin, cholesterol (4:1, w/w) and LMX (drug-to-lipids ratio, w/w of 3:14) were dissolved in methylene chloride (the concentration of lipids was 9.3 mg/mL), the mixture was warmed to 40 ◦ C in a round-bottomed flask, and the solvent was evaporated under vacuum in a rotary
2.4. Determination of drug encapsulation efficiency and drug loading 2.4.1. HPLC analysis A Shimadzu LC-2010 C system (Shimadzu, Kyoto, Japan) with built-in UV detector (SPD-10A, Shimadzu, Japan) was used to perform all the analyses. Chromatographic software Class VP 6.12 was used for data collection and processing. A HYPERSIL C18 column (10 m particle size, 250 mm × 4.6 mm) was used for the separation. Mobile phase was composed of methanol and water containing 0.5% ammonium acetate (95:5, v/v); the flow rate and column temperature were set at 1 mL/min and 25 ◦ C, respectively. The UV absorbance was determined at 245 nm. The sample was prepared by dissolving the lyophilized powder in double distilled water and being diluted by methanol (to destroy liposome and dissolve LMX) and 20 L of the sample was injected. The calibration curve of various LMX concentrations (C, 10–150 g/mL) versus integrated area (A, mAUs) was A = 18210C–385.9, with a correlation coefficient of 0.9999. 2.4.2. Drug encapsulation efficiency and drug loading Encapsulation efficiency (EE) of LMX-loaded liposomes was calculated by determining the amount of free drug using ultrafiltration technique. 1 mL LMX-loaded liposomes colloidal solution was placed in the upper chamber of a centrifuge tube matched with an ultrafilter (Amicon Ultra, Millipore Co., USA, MWCO 10 kDa) and centrifuged for 10 min at 4000 rpm. The ultrafiltrate containing the unencapsulated drug was determined by HPLC. The total drug content in liposomes was determined as follows: aliquots of 1 mL liposomes dispersion were dissolved and diluted appropriately by methanol to dissolve the liposomes and then the obtained suspension was allowed to filter through 0.45 m membrane filters. The resulting solution was analyzed by HPLC. The drug loading (DL) was the ratio of entrapped drug to lipid (w/w). The EE and DL could be calculated by the following equations: EE% =
Wtotal − Wfree × 100 Wtotal
DL% =
Wtotal − Wfree × 100 Wlipid
where Wtotal , Wfree , Wlipid were the weight of total drug in liposomes dispersions; the weight of unentrapped drug in ultrafiltrate and the weight of lipid added in system, respectively. 2.5. Characterization of LMX-loaded liposomes 2.5.1. Morphology The morphology of LMX-loaded liposomes was observed by transmission electron microscopy (TEM) (JEM-1200EX, Jeol, Japan).
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Fig. 1. Structure of codrug LMX, UA and LMV.
Fresh prepared liposomes were diluted with distilled water and dropped on a formvar-coated copper grid and air-dried for 1 min at room temperature after removing the excessive sample with filter paper. The air-dried samples were then directly examined under the TEM. 2.5.2. Particle size and zeta potential The lyophilized powders were reconstituted with 5% dextrose injection solution. The average diameter, polydispersity index and zeta potential were measured using a Malvern Zetasizer 3000 system (Malvern Instruments Ltd., Malvern, UK). All of the dynamic light scattering (DLS) measurements were performed at 25 ◦ C and at a scattering angle of 90◦ . The zeta potential values were calculated using the Smoluchowski equation. 2.6. Stability studies The freeze-dried LMX-loaded liposomes were stored at 4 ◦ C for 6 months under a sealed condition. The mean particle size and drug entrapment efficiency were determined at fixed time intervals, respectively. 2.7. The drug release experiment in vitro In vitro release of LMX from liposomes was performed using the dialysis bag diffusion technique (Avgoustakis et al., 2002). The dialysis bags (MWCO, 12,000–14,000, Sigma) were soaked in deionized water for 12 h before use. In this study, 20% ethanol–water was used as dissolution medium. 14 mg of the freeze-dried powder with LMX (equivalent to 1.5 mg LMX) rehydrated in 1 mL of 5% glucose solution or 1 mL LMX solution (equivalent to 1.5 mg LMX) were placed in dialysis bags. Both ends of the bag were clamped and each bag was then individually immersed in a beaker containing 70 mL of the dissolution medium. All beakers were placed in a water bath maintained at 37 ± 0.5 ◦ C throughout the release study.
The constant shaking was at 90 strokes/min. The release medium was changed every 3 h to ensure sink conditions and the stability of LMX. The sampling time intervals were 0, 3, 6, 9, 12, . . ., 80 h. A sample of 70 mL at each time point was withdrawn, and the beaker was replenished with fresh 70 mL of 20% C2 H5 OH H2 O (Nornoo and Chow, 2008; Alani et al., 2010). Drug concentrations in the dissolution medium were finally analyzed using the HPLC method as described above (Section 2.4.1). The release experiments were carried out in triplicates and the results were expressed as means ± standard deviation. 2.8. Pharmacokinetic studies in rats 2.8.1. Chromatographic analysis conditions in vivo A Waters HPLC system (Milford, Massachusetts) was used to quantify LMX and LMV in plasma samples, using a HYPERSIL C18 column (10 m particle size, 250 mm × 4.6 mm). The mobile phase for gradient elution consisted of two solvent systems: solvent A, methanol; solvent B, 0.5% (g/v) ammonium acetate solution adjusted to pH 3.56 with glacial acetic acid. A + B = 100%. A gradient elution was carried out as follows: 93% (v/v) solvent A was used during 0–3 min, then solvent A percentage was linearly decreased to 50% in the 4th min and maintained during 4–13 min, solvent A percentage was linearly increased to 93% in the 14th min and maintained for another 6 min. The total run time was 20 min including equilibration of the system. The flow-rate was 1 mL/min. The eluent was monitored by a UV detector, and the detection wavelength was set at 245 nm for LMX and 270 nm for LMV. The sample injection volume was 20 L and the column temperature was 40 ◦ C. Calibration curves were constructed by plotting the peak-area ratios of each analyte/internal standard R (Ai /Ais ) versus analyte concentration in plasma. The linearity of LMX and LMV was observed over the concentration range of 1500–3.0 g/mL and 750–1.5 g/mL, respectively. The calibration curves of LMX and LMV were as follows: R = (7.00E−05)C − 0.002 (r2 = 0.995, n = 6) and
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R = 0.003C+0.035 (r2 = 0.996, n = 6), respectively. The limits of detection in rat plasma for LMX and LMV were 3.0 g/mL and 1.5 g/mL, respectively. Accuracy of the determination of LMX and LMV in rat plasma (n = 6) is 100.42 ± 2.70% and 100.40 ± 1.01%, respectively. Within-day and between-day precisions of LMX and LMV were all below 4.72%. Extraction recovery of LMX and LMV in rat plasma (n = 6) was above 80.16 ± 3.46%. 2.8.2. Drug extraction Liquid–liquid double extraction was performed prior to analysis by HPLC. Briefly, a 300 L of rat plasma was mixed with 30 L Edaravone (internal standard, 100 g/mL) solution and vortexed for 1 min. Then the mixture was extracted with 4 mL of methylene chloride vortexing for 3 min. Following centrifugation at 3000 rpm for 10 min, the organic phase 3 mL was transferred to a glass tube and the solvent was evaporated under nitrogen stream at 35 ◦ C. The resulting residue was reconstituted with 100 L of mobile phase by vortexing for 4 min and 20 L of the solution was injected into the column. HPLC conditions were described above. 2.8.3. Pharmacokinetic study Pharmacokinetics studies were performed as described elsewhere (Gao et al., 2006). The bioavailability studies were complied with the requirements of the National Act on the use of experimental animals (People’s Republic of China). The rats used for this study were housed individually under normal conditions, and fasted overnight before experiment with free access to water. Rats were randomly divided into four groups (5 per group). Rats in group 1 were orally administered with LMX suspension (equivalent to 32 mg/kg of LMX, 1.5 mL/rats) and the group 2 was orally administered with LMX-loaded liposomes suspensions (equivalent to 32 mg/kg of LMX, 1.5 mL/rats). Similarly, LMX 50% alcohol solution (equal to 32 mg/kg of LMX, 1 mL/rats) and LMX-loaded liposomes (equal to 32 mg/kg of LMX, 1 mL/rats) were injected intravenously into the other two groups, respectively. Blood samples were collected from the postorbital venous plexuses of rats and placed into heparinized test tubes according to the designed time interval (Gutierro et al., 2002). The plasma samples were harvested after centrifugation (15 min, 4000 rpm) and stored at −20 ◦ C until analysis. The main pharmacokinetic parameters were acquired with the help of a pharmacokinetic program DAS 2.0 (Mathematical Pharmacology Professional Committee, China). The values of maximum concentration (Cmax ) and time of maximum concentration (Tmax ) were obtained directly from the concentration–time plotting. The area under the concentration–time curve (AUC) was calculated by linear trapezoidal method. 2.9. Statistical analysis Statistical analysis was performed with Student’s t-test for two groups and one-way ANOVA for multiple groups. All results were expressed as mean ± S.D. unless otherwise noted. P < 0.05 was considered statistically significant.
Fig. 2. Transmission electron micrographs of LMX-loaded liposomes.
145.58, 138.04, 126.31, 96.14, 88.24, 79.05, 62.86, 55.22, 52.89, 48.53, 47.56, 42.12, 39.55, 39.16, 38.76, 38.59, 37.01, 36.74, 32.90, 30.54, 28.16, 27.27, 24.51, 23.60, 23.23, 21.11, 18.32, 17.06. ESIHRMS calculated for C40 H59 N3 O7 NaS: 748.3971. Found 748.3952 (MNa+). IR (KBr): 3439 (N H), 1732 (C O), 1658 (C O). 3.2. Preparation of LMX-loaded liposomes The effects of the three influential factors, SL/Cho ratios (X1 ), D/L ratios (X2 ), O/W ratios (X3 ), on encapsulation efficiency (EE) of liposomes were shown in Table 1. The results showed that these three factors had little influence on the particle size (data not shown) and the maximal value of EE could be reached when SL/Cho ratio was set at 4:1. Concerning drug loading, the optimized D/L ratio and O/W ratio were 3:14 and 0.75:1, respectively. The drug loading could be enhanced with the increase of D/L ratio, while a higher lipids ratio improved EE in this study. Moreover, EE value increased when SL/Cho ratio increased until SL/Cho ratio reached 4:1. Certain concentration of cholesterol could reduce the bilayer permeability, increase the stability of liposomes and cause high EE (Gregoriadis, 1993), while beyond a certain ratio, cholesterol could disrupt the regular structure of the liposomal membrane, result in lower EE (El-Samaligy et al., 2006). In our case, the O/W ratio (0.75:1) caused higher EE and better stability in vitro. Since the entrapment capacity of liposomes for drugs strongly diminishes with decrease of liposome size, larger liposomes were considered more promising pharmaceutical carriers (Torchilin et al., 2001). The optimized formulation was repeated in triplicates. The average entrapment efficiency and the average drug loading of LMX-loaded liposomes were (91.28 ± 3.25%) and (12.59 ± 0.19%), respectively.
3. Results and discussion 3.3. Characterization of LMX-loaded liposomes 3.1. Synthesis of LMX LMX was obtained from LMV and UA. LMX is a white solid. m.p.: 174.5–175.1 ◦ C. 1 H NMR (300 MHz, CDCl3 ): 8.55–8.57 (d, 1H, J = 3.6 Hz), 7.39–7.42 (d, 1H, J = 7.3 Hz), 6.34 (s, 1H,), 5.38 (s, 2H), 4.74–4.79 (m, 1H), 4.56–4.62 (m, 2H), 4.17–4.21 (m, 1H), 4.09–4.16 (m, 1H), 3.96–4.01 (m, 1H), 3.63–3.69 (m, 1H), 3.19–3.30 (m, 2H), 0.71–2.28 (m, 45H). 13 C NMR (75 MHz, CDCl3 ): 176.11, 161.53,
TEM was conducted to investigate the morphology of liposomes, showing that the nanoparticles had near spherical shapes. Vesicular structure was discernable (Fig. 2). The particle size and polydispersity index of LMX-loaded liposomes were estimated by DLS. The sizes of LMX-loaded liposomes (200–220 nm) were significantly bigger than those of blank liposomes (about 170 nm) (P < 0.05). It is also worth noting that the
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Table 1 The levels of experimental factors, the liposomes entrapment efficiencies and drug loading.
1 2 3 4
X1 (w/w)
EE (%)
LD (%)
X2 (w/w)
EE (%)
LD (%)
X3 (V/V)
EE (%)
LD (%)
1:1 2.5:1 4:1 6:1
29.13 84.64 88.89 79.71
3.72 11.49 10.87 9.82
1:14 2:14 3:14 4:14
93.5 87.98 84.64 63.40
3.58 8.69 11.49 11.17
0.5:1 0.75:1 1:1 1.5:1
88.22 91.2 90.13 78.84
11.23 12.59 12.16 10.70
X1 : weight ratio of soya lecithin to cholesterol (SL/Cho ratios). X2 : weight ratio of drug to lipids (D/L ratios). X3 : volume ratio of methylene chloride to water (O/W ratios).
size of the drug-loaded liposomes was not significantly affected by three influential factors (data not shown). The polydispersity index of LMX-loaded liposomes, estimated by the cumulant method, was low (<0.220), which suggested a narrow size distribution. Zeta potential or particle surface charge is an important parameter indicating to the stability of nanocarrier systems. A relatively high surface charge may provide a repelling force between the particles, thus increasing the stability of the solution (Kwon et al., 2003). From the date obtained, all the LMX-loaded liposomes had relatively high negative zeta potentials of around −40 mV. It is reasonable to conclude that the charged particles may repel each other and prevent aggregation or precipitation happening, thus resulting in good stability. The negative or neutral liposomes provide more effective barrier to plasma macromolecular protein adsorption and are easy to resuspend in blood (Mobed and Chang, 1998). 3.4. The effects of lyophilization The process of freeze-drying did not affect the particle size of rehydrated LMX-loaded liposomes containing sucrose as cryoprotectant (ratio of sucrose to lipids, 1.3:1–2:1, w/w), the mean diameter was 213.7 ± 6.12 nm before lyophilization and 217.7 ± 3.14 nm after lyophilization/rehydration. Those values are not different from each other (P > 0.05). On the other side, formulations prepared without sucrose as cryoprotectant showed an increase in vesicle size after lyophilization (272.7 ± 4.73 nm). The process of lyophilization was harmful for the liposome integrity also, as freezing caused a pronounced decrease in the encapsulation efficiency. EE% of LMX-loaded liposomes was only 42.79% after lyophilization without cryoprotectant, while only a small decrease in the encapsulation efficiency was observed by addition of sucrose as cryoprotectant. In addition, a mixture of mannitol and sucrose was also found to be suitable cryoprotectant at the weight ratio of mannitol to sucrose to lipid of 3:2:1 (Fig. 3). Freeze-drying tended to destroy the membrane function of the phospholipid bilayer (Ozer and Talsma, 1989), and lead to leakage of encapsulated drug. Particle size increase after freezedrying was in good agreement with the drug leakage (Stensrud et al., 2000). Sugars are well-known cryoprotective agents (Li et al., 2000). Their protective effect has been related to their ability to interact with the polar head groups of the phospholipids and to stabilize the membranes when the bilayer stabilizing water is removed by sublimation (Stevens and Lee, 2003).
Fig. 3. Encapsulation efficiency of LMX liposome before and after freeze-drying process. (a) Samples before lyophilization; (b) samples after lyophilization without cryoprotectant; (c) samples after lyophilization with cryoprotectant (sucrose:lipid = 1.3:1); (d) samples after lyophilization with cryoprotectant (sucrose:lipid = 2:1); and (e) samples after lyophilization with cryoprotectant (mannitol:sucrose:lipid = 3:2:1).
20% ethanol for 80 h. LMX was continuously released from liposomes and the cumulative release amount of LMX in 80 h was around 52%. As shown in Fig. 4, there was an initial mild burst for the first 2.5 h as the drug molecules adsorbed on the surface were released, followed by a period of moderate release, which was believed that the entrapped drug slowly diffused out into the surrounding medium. This diffusion release phase then flattened out, and a subsequent increase in release rate after 47 h corresponded to an increase in the erosion rate of the liposome, which finally tapered off again as 52% or more of the drug has been released (Jabara et al.,
3.5. Stability studies The stability test of the freeze-dried LMX-loaded liposomes at 4 ◦ C indicated that the mean particle size and drug entrapment efficiency had no significant change during 6 months (Table 2). 3.6. In vitro drug release The release experiment was conducted under sink conditions and the dynamic dialysis was employed for separation of free drug from LMX-loaded liposomes. Fig. 4 shows the cumulative LMX release profile from liposomes in the aqueous medium containing
Fig. 4. Cumulative release of LMX from liposome and solution into 20% ethanol–water at 37 ◦ C. The error bars represent the S.D. of 3 samples.
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Table 2 Physical stability of the freeze-dried LMX-loaded liposomes (n = 3). Time (days)
EE (%) r (nm)
0
1
7
15
30
90
180
89.5 ± 2.15 215.3 ± 3.22
90.04 ± 2.72 215.8 ± 5.09
89.2 ± 3.12 215.6 ± 4.21
89.0 ± 1.68 216.7 ± 6.33
88.1 ± 3.39 217.5 ± 5.18
86.8 ± 2.18 218.8 ± 4.89
84.7 ± 2.66 220 ± 5.21
Table 3 Fitted degradation kinetic equations of LMX liposome and solutions 1 and 2. Time (h) LMX liposomes
0–47.5 47.5–80
LMX solution 1 LMX solution 2
0–4 0–7
Models
Fitted kinetic equations
r2
First-order Hixcon–Crowell
ln(1 − Q) = −0.006t − 0.094 Q = 0.001t3 − 0.342t2 + 22.60t − 448.7
0.991 0.992
First-order First-order
ln(1 − Q) = −1.085t − 0.06 ln(1 − Q) = −0.383t − 0.018
0.993 0.992
2008; Vlasta et al., 2009). The release was a complex process consisting of two phases: during 0–47.5 h, the release of LMX from LMX-loaded liposomes followed the First-order equation; during 47.5–80 h, the release followed the Hixcon–Crowell equation. The delayed release might be attributed to the lipophilic LMX that was held by the small fragment of the liposomal membrane and the drug encapsulated in lipid membrane that released mainly through dissolution and diffusion from the lipid bilayer (Cócera et al., 2000). In contrast, the release of LMX from LMX solution was much fast. In solution 1 (20% ethanol–water) approximately 100% of the drug had been dissolved after 4 h. In solution 2 (50% acetonitrile–water), approximately 85% of the drug had been dissolved after 4 h. These may be due to the different dissolution/diffusion rates of the drug in different solvents. The release dynamics characteristics were fitted to first-order kinetics model in solutions 1 and 2. The fitted degradation kinetic equations of LMX liposomes and solutions 1 and 2 in 20% C2 H5 OH H2 O were shown in Table 3. Maintaining a good sink condition for poorly water-soluble drugs has been one of the difficulties in designing in vitro release experiments. Continuous flow methods to provide infinite water to the release media have been designed to mimic a perfect sink condition (Washington, 1990; Soo et al., 2002). In this method, the release media is removed and replaced infinitely rapidly and the instantaneous release rate is measured. However, this method is sometimes not useful for poorly soluble drugs because of limitations in measurement of the drug concentration due to their low concentrations in the aqueous media (Cho et al., 2004). A hydrotropic agent provides an alternative tool for studying release
of poorly soluble drugs from liposomes in aqueous solutions. In trial experiment, we researched the accumulative release amount of LMX liposomes in other media, such as phosphate buffer (PBS, pH 7.4), HCl (0.1 mol/L), 0.5% Tween 80–water and 0.4% sodium lauryl sulfate (SDS)–water. We found that sink conditions could not be obtained in buffers and HCl (0.1 mol/L) because of the low solubility and instability of LMX (with time prolonging LMV was observed from the analysis of HPLC), and in 0.4% SDS the release of drug from liposomes was so slow that less than 12% of the drug was released in 80 h. The release of LMX from liposomes in 0.5% Tween 80 was little faster than that in 0.4% SDS. Thus, we selected 20% ethanol–water as dissolution medium, which was changed every 3 h to ensure sink conditions and the stability of LMX. From the result of the test, the addition of ethanol significantly increased the drug release rate. In this study, one potential drawback of the in vitro assay is that the media contained 20% ethanol, 0.5% Tween 80 or 0.4% SDS, thus may not faithfully mimic in vivo situation. Furthermore, the in vitro assay failed to take into account the possible disruption of liposomal vesicular structure in physiological conditions. Thus, the results of in vitro release test cannot completely reflect the in vivo factual characteristics. 3.7. Pharmacokinetic studies in rats In the pharmacokinetic experiment, the method of HPLC and Edaravone as internal standard were used. The chromatograms showed stable baselines, as well as displaying good resolution among LMX, Edaravone and endogenous material in plasma. The
Fig. 5. Profile of mean plasma concentration–time of LMV (left) and LMX (right) after intravenous administration of LMX 50% alcohol solution and LMX-loaded liposomes in rats (mean ± S.D., n = 5).
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Fig. 6. Profile of mean plasma concentration–time of LMV (left) and LMX (right) after oral administration of LMX suspension and LMX-loaded liposomes in rats (mean ± S.D., n = 5).
Table 4 Mean pharmacokinetic parameters of LMX after intravenous administration of LMX 50% alcohol solution and LMX-loaded liposomes or oral administration of LMX suspension and LMX-loaded liposomes in rats (mean ± S.D., n = 5). Parameters
Unit
AUC(0→t) MRT(0→t) t1/2 Tmax CL/F Cmax
mg/L h h h h L/h/kg mg/L
i.v.
Oral
LMX 50% alcohol solution 2567.42 5.34 5.14 0.083 0.012 691.78
± ± ± ± ± ±
LMX-loaded liposomes
141.56 0.33 0.04 0.0013 0.005 34.66
3472.2 4.70 2.74 0.10 0.009 1094.34
± ± ± ± ± ±
98.23* 0.27 0.03* 0.02 0.0006 43.22*
LMX suspension 987.94 10.10 4.96 8.13 0.05 113.16
± ± ± ± ± ±
34.68 0.48 0.04 0.45 0.004 15.11
LMX-loaded liposomes 10618.4 11.73 2.14 10.03 0.003 1315.59
± ± ± ± ± ±
892.11## 0.59# 0.03 0.68# 0.0004# 110.90#
Each data was from five rats. * P < 0.05 vs. LMX 50% alcohol solution. # P < 0.05 vs. LMX suspension. ## P < 0.01 vs. LMX suspension.
concentrations of LMX and LMV in the samples were determined by comparing the peak area ratios of the samples versus a calibration curve obtained from spiking known amounts of LMX and LMV in pooled rats plasma. LMX and LMV plasma concentrations, pharmacokinetic parameters, half-life and clearance, in the control group (suspension or 50% alcohol solution) were compared with those of the liposome treated groups. Pharmacokinetic analysis was performed using non-compartmental methods with DAS (2.0) program. Results were presented as mean ± S.D. Figs. 5 and 6 show the mean concentration in plasma versus time profiles of LMX and LMV after oral or i.v. administration of liposomes and suspension (or 50% alcohol solution) at a dose of 32 mg/kg to rats, respectively, and the pharmacokinetic parameters of LMX and LMV were summarized in Tables 4 and 5, respectively.
Oral liposomal administration showed a higher Cmax compared with the LMX suspension (P < 0.05). The AUC(0→24) h following oral liposomal administration was significantly higher than LMX suspension (P < 0.01), and the relative bioavailability was 1074.8%. Compared with the LMX suspension, liposomes exhibited a higher mean residence time (MRT) (11.73 h) (P < 0.05), while CL decreased by 16 times (Table 4). These could be explained by the increased affinity between LMX-loaded liposomes and the gut liquid, which caused better dispersibility of LMX in the gastrointestinal tract, prolonged the drug residence time in the absorption sites, and resulted in better absorption effects (Vasir et al., 2003). LMX-loaded liposomes showed a delayed Tmax and higher MRT (P < 0.05) compared with the LMX suspension, which indicated that the liposomes after oral administration retarded the clearance and exhibited the property of sustained release. The prolonged drug circulation was
Table 5 Mean pharmacokinetic parameters of LMV after intravenous administration of LMX 50% alcohol solution and LMX-loaded liposomes or oral administration of LMX suspension and LMX-loaded liposomes in rats (mean ± S.D., n = 5). Parameters
Unit
AUC(0→t) MRT(0→t) t1/2 Tmax CL/F Cmax
mg/L h h h h L/h/kg mg/L
i.v.
Oral
LMX 50% alcohol solution
Each data was from five rats. * P < 0.05 vs. LMX 50% alcohol solution. # P < 0.05 vs. LMX suspension.
319.98 7.91 4.54 6.0 0.095 40.53
± ± ± ± ± ±
45.21 0.89 0.05 0.45 0.004 3.32
LMX-loaded liposomes 932.5 6.25 2.51 5.0 0.034 105.7
± ± ± ± ± ±
39.1* 0.76 0.03 0.56 0.002* 4.01*
LMX suspension 348.50 11.0 1.99 10.22 0.091 41.64
± ± ± ± ± ±
27.54 0.88 0.02 0.86 0.005 3.44
LMX-loaded liposomes 421.9 12.52 3.50 12.98 0.074 30.24
± ± ± ± ± ±
38.63# 0.94# 0.04 1.02# 0.005# 2.98
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consistent with the results obtained from the release in vitro. Compared with the i.v. administration of the LMX 50% alcohol solution, the relative bioavailability of LMX-loaded liposomes after i.v. administration was 135.2%, which confirmed the better absorption effects of LMX-loaded liposomes. No significant difference of the MRT was noted between liposomes and 50% alcohol solution group (P > 0.05) after i.v. administration. Both the rapid uptake of liposomes by the organs of the reticuloendothelial system (RES) and the slow release due to the reservoir effect might be the reasons (Klibanov and Huang, 1992). Liposomes as circulating reservoirs in tissues probably enter the blood again, and repeatedly, achieve dynamic balance eventually. Similarly, LMV pharmacokinetic parameters of LMX-loaded liposomes formulation displayed higher AUC(0→t) and MRT(0→t) , with lower CL, in comparison with LMX suspension after oral administration (Table 5). After i.v. administration, LMV had a higher AUC(0→t) and Cmax , with lower CL, compared with LMX 50% alcohol solution. 4. Conclusion In this study, a novel codrug of LMX was successfully synthesized, and incorporated into liposomes for applying to oral and intravenous administration. The liposomes formulation did enhance the gastrointestinal absorption of LMX by oral administration, about 10.75-fold of relative bioavailability comparing to LMX suspension was observed, and had significantly prolonged circulation duration, which was consistent with the results obtained from the release tests in vitro. LMX-loaded liposomes exhibited a 1.35fold increase in bioavailability relative to LMX 50% alcohol solution after i.v. administrated. These results provided an important reference for the use of LMX in the field of the treatment of hepatitis. Further studies are needed to focus on the safety and efficiency of LMX-loaded liposomes to evaluate the potential clinical application value. Acknowledgements The authors thank the financial support provided by the Natural Science Foundation of Jiangsu Province (No. BK2010538). The authors wish to thank Dr. Rui Li (School of Pharmacy, Nanjing Medical University, China) for helpful suggestions and discussion for the paper. References Alani, A.W.G., Bae, Y., Rao, D.A., Kwon, G.S., 2010. Polymeric micelles for the pHdependent controlled, continuous low dose release of paclitaxel. Biomaterials 31, 1765–1772. Ariën, A., Goigoux, C., Baquey, C., Dupuy, B., 1993. Study of in vitro and in vivo stability of liposomes loaded with calcitonin or indium in the gastrointestinal tract. Life Sci. 53, 1279–1290. Ariën, A., Henry-Toulmé, N., Dupuy, B., 1994. Calcitonin-loaded liposomes: stability under acidic conditions and bile salts-induced disruption resulting in calcitonin–phospholipid complex formation. Biochim. Biophys. Acta 1193, 93–100. Aungust, B.J., 1993. Novel formulation strategies for improving oral bioavailability of drugs with poor membrane permeation or presystemic metabolism. J. Pharm. Sci. 82, 979–987. Avgoustakis, K., Beletsi, A., Panagi, Z., 2002. PLGA–mPEG nanoparticles of cisplatin: in vitro nanoparticle degradation, in vitro drug release and in vivo drug residence in blood properties. J. Control. Release 79, 123–135. Bhardwaj, U., Burgess, D.J., 2010. Physicochemical properties of extruded and nonextruded liposomes containing the hydrophobic drug dexamethasone. Int. J. Pharm. 388, 181–189. Bjornesson, E., Olsson, R., 2006. Suspected drug-induced liver fatalities reported to the WHO database. Dig. Liver Dis. 38, 33–38. Cha, C., Dematteo, R.P., 2005. Molecular mechanisms in hepatocellular carcinoma development. Best Pract. Res. Clin. Gastroenterol. 19, 25–37. Chen, Y.F., Ju, T., 2011. Comparative meta-analysis of adefovir dipivoxil monotherapy and combination therapy of adefovir dipivoxil and lamivudine for lamivudineresistant chronic hepatitis B. Int. J. Infect. Dis. 16, e152–e158.
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