International Journal of Pharmaceutics 490 (2015) 265–272

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International Journal of Pharmaceutics journal homepage: www.elsevier.com/locate/ijpharm

An injectable liquid crystal system for sustained delivery of entecavir Jong-Lae Lim a,b , Min-Hyo Ki c, Min Kyung Joo c, Sung-Won An c, Kyu-Mok Hwang b , Eun-Seok Park b, * a

CKD Research Institute, Chong Kun Dang Pharm Corp., 315-20, Dongbaekjukjeon-daero, Giheung-gu, Yongin-si, Gyeonggi-do 446-916, Republic of Korea School of Pharmacy, Sungkyunkwan University, 300 Cheoncheon-dong, Jangan-gu, Suwon 440-746, Republic of Korea DDS Research Lab., CKD Research Institute, Chong Kun Dang Pharm Corp., 315-20, Dongbaekjukjeon-daero, Giheung-gu, Yongin-si, Gyeonggi-do 446-916, Republic of Korea b c

A R T I C L E I N F O

A B S T R A C T

Article history: Received 9 January 2015 Received in revised form 28 April 2015 Accepted 17 May 2015 Available online 21 May 2015

Liquid crystal (LC) technology has attracted much interest for new injectable sustained-release (SR) formulations. In this study, an injectable liquid crystal-forming system (LCFS) including entecavir was prepared for the treatment of hepatitis B. In particular, an anchoring effect was introduced because LCFSs are relatively hydrophobic while entecavir is a slightly charged drug. The physicochemical properties of LCFSs were investigated by cryo-transmission electron microscopy (cryo-TEM), polarized optical microscopy, and small-angle X-ray scattering (SAXS), showing typical characteristics of the liquid crystalline phase, which was classified as the hexagonal phase. A pharmacokinetic study in rats showed sustained release of entecavir for 3–5 days with a basic LCFS formulation composed of sorbitan monooleate (SMO), phosphatidyl choline (PC), and tocopherol acetate (TA) as the main LC components. 1,2-Dipalmitoyl-sn-glycero-3-phosphatidic acid (DPPA), an anionic phospholipid, was added to increase the anchoring effect between the cationic entecavir and the anionic DPPA, which resulted in a 1.5-times increase in half-life in rats. In addition, anchoring was strengthened by optimizing the pH to 2.5–4.5, increasing the half-life in the rat and dog. Also, due to the increasing terminal half-life from rat to dog resulting from species differences, LCFS produced one week delivery of entecavir in rat and two weeks delivery in dog. Therefore, LCFS injection using the anchoring effect for entecavir can potentially be used to deliver the drug over more than 2 weeks or even 1 month for the treatment of hepatitis B. ã2015 Elsevier B.V. All rights reserved.

Keywords: Liquid crystal Entecavir Sustained release injection Pharmacokinetics Anchoring effect

1. Introduction Hepatitis B is an infectious disease of the liver caused by hepatitis B virus (HBV), which requires constant treatment. Entecavir is a drug for the treatment of hepatitis virus B with a low drug tolerance less than 1% even after 4 years administration compared to the existing drugs. Other drugs show 30–70% increases in drug tolerance within 2 years. For the efficient therapy of hepatitis B using entecavir, the drug must be taken consistently every day and the disease can re-occur if the drug is discontinued (Palumbo, 2009; Honkoop and de Man, 2003). Therefore, the development of an injectable sustained-delivery system for the treatment of hepatitis B can be a solution to overcome these disadvantages.

* Corresponding author. Tel.: +82 31 290 7755; fax: +82 31 290 7729. E-mail address: [email protected] (E.-S. Park). http://dx.doi.org/10.1016/j.ijpharm.2015.05.049 0378-5173/ ã 2015 Elsevier B.V. All rights reserved.

In the past decade, injectable PLGA microspheres or implants have been developed as injectable sustained release systems through various studies (Teutonico et al., 2012; Astaneh et al., 2007; Wilson et al., 2007; Svenson and Tomalia, 2012; Appel et al., 2012). Though PLGA microspheres are available for clinical application, they are difficult to prepare and are known to decrease the stability of protein drugs (Okada, 1997; Namur et al., 2004; Weert et al., 2000). Unfortunately, no alternative polymers are yet reported as safe excipients despite many research efforts (He et al., 2008). Recently, a novel injectable SR formulation based on liquid crystal (LC) technology has been developed for drug delivery (Guo et al., 2010; Shah et al., 2001). Many drugs, including lowmolecular-weight chemicals and macromolecular drugs like proteins, peptides and nucleic acids were delivered using the reversed hexagonal phase (H2) and the reversed cubic phase (Q2) of the lyotropic LC system (Angelova et al., 2011). The LC mesophase is spontaneously formed from the liquid crystal-forming system (LCFS) in an aqueous fluid. The formed tortuous networks of

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aqueous nano-channels in the mesophases or mesophase particles play important roles as passageways for the sustained release of drugs from LCs (Guo et al., 2010; Shah et al., 2001; Angelova et al., 2011; Fong et al., 2010). Various amphiphilic liquid crystal-forming materials (LCFMs), such as glycerol monooleate (GMO), glycerol dioleate (GDO), glycerol oleyl ether, oleyl glycerate, phytanyl glycerate, and phytantriol, have been reported (Boyd et al., 2006, 2009; Qiu and Caffrey, 2000; Barauskas et al., 2010). Lately, a new LCFS was developed that uses sorbitan monooleate (SMO) (also known as Span 80) as a new injectable LCFM for injection, and its use as a clinically available SR formulation has been evaluated (Ki et al., 2014). The LCFS containing SMO showed sustained release of leuprolide for 1 month, indicating the possibility to replace conventional depot injections in terms of safety, ease of preparation, and sustained release properties. Functionalization of LCs to enhance their loading and release properties has been investigated for several years (Clogston et al., 2005; Spicer et al., 2003). Adding anionic phospholipids or cationic surfactants as an anchoring material were studied previously. The charge interaction between a charged drug and an oppositely charged anchoring material showed enhanced loading and prolonged release properties (Lindell et al., 1998; Lynch et al., 2003). These in vitro studies provided theoretical demonstrations of functionalizing LCs. In this study, SMO-based LCFS was primally prepared for the sustained delivery of entecavir. However, the LCFS showed limitations in the sustained delivery of hydrophilic charged drugs like entecavir. Therefore, the concept of anchoring effect was introduced for the in vivo sustained delivery using an anionic phospholipid as an anchoring material. The sustained release properties by the anchoring effect in LC were practically investigated through in vivo pharmacokinetic studies with or without pH optimization to provide circumstances at which the anionic phospholipid species and the cationic drug species existed simultaneously. 2. Materials and methods 2.1. Materials Entecavir was obtained from Jeil Pharmaceutical Co., Ltd. (Seoul, Korea). SMO and tocopherol acetate (TA) were purchased from Seppic (Puteaux, France) and DSM Nutritional Products Limited (Sisseln, Switzerland), respectively. Cholesterol and Tween 80 were purchased from Croda (Edison, NJ, USA). PC and 1, 2-dipalmitoyl-sn-glycero-3-phosphatidic acid (DPPA) were purchased from Lipoid GmBH (Ludwigshafen, Germany). All the other chemicals were of analytical grade. 2.2. Methods 2.2.1. Animals Male Sprague-Dawley (SD) rats (8 weeks of age, about 300 g body weight) were obtained from Orient Bio Inc. (Seongnam, Korea). The rats were acclimated for approximately 7 days before dosing and had free access to food and water. Male beagle dogs (5–6 months old; about 10 kg body weight) were purchased from Beijing Marshall Biotechnology (Beijing, China). Beagle dogs were acclimated for approximately 14 days before dosing, and were fed approximately 300 g dog diet once daily and municipal water was supplied ad libitum. The temperatures and relative humidities of the two separate animal rooms (rats and beagle dogs) were generally kept at 23
C and 55  15%, respectively. The number of changes of air was approximately 10–20 per hour. Light was provided by fluorescent lamp fittings or light bulbs regulated to give 12 h each of daylight and darkness (from 8 a.m. to 8 p.m.). All

animals were cared for in accordance with OECD guidelines for the testing of chemicals (Standards of toxicity study for medicinal products; No. 2013-121). 2.2.2. Preparation of LCFS and its formation test The LCFS was prepared by mixing SMO, PC, TA, Tween 80, and ethanol (31:42:13:2:12, w/w). SMO, PC, and TA were used as core components for LCFS. Tween 80 and ethanol were used to prevent the separation of components, thus making a homogenized solution. The LCFS was prepared by mixing and dissolving freeze-dried entecavir liposomes in remaining LCFS components. To prepare entecavir-encapsulated liposomes, an entecavir solution in water (2.8 mg/ml) was mixed with a one-third volume of a PC solution, a one-third volume of SMO, and Tween 80. A PC solution was prepared by dissolving PC in ethanol (0.5 mg/ml) in advance. Then, the water and ethanol were removed by freezedrying. When DPPA was added (as an anionic phospholipid), it was dissolved in an entecavir solution. After the entecavir-encapsulated liposome solution was freeze-dried over 72 h, the dried liposomes were finally mixed with the remaining LCFS components. The final LCFS was prepared to contain entecavir 14.0 mg in 650 ml LCFS (ETV1), entecavir 14.0 mg in 675 ml LCFS (ETV2), entecavir 14.0 mg in 700 ml LCFS (ETV3), and entecavir 21.0 mg in 700 ml LCFS (ETV4, ETV5, ETV6). The total volumes of the final LCFSs differed slightly, because of the added amounts of DPPA. Additionally the control was prepared by dissolving entecavir in water to a 1.00 mg/ml solution. 2.2.3. Cryo-transmission electron microscopy (Cryo-TEM) and polarized optical microscopy Cryo-TEM (Tecnai G2 F20Cryo-TEM, FEI Company, Hillsboro, OR, USA) was used to observe the inner structure of the LC that was formed with exposure to water (Kuntsche et al., 2011; Gustafsson et al., 1997; Sagalowicz et al., 2006). For convenient observation of the liquid crystalline phase, 25 ml of the LCFS in the oil phase was added to 5 ml of triple distilled water and dispersed using a probe sonicator (CL-334, Qsonica, LLC, Newtown, CT, USA) to form mesophase particles. The dispersed liquid crystalline phase was placed on the holey carboncoated grid (Quantifoil Micro Tools GmbH, Jena, Germany) like a water film and quickly frozen at 170
C. The frozen grid was fixed in the cryo-holder and moved to the cryo-TEM at 170
C. The sample was observed at the 80–200 kV dose. Polarized optical microscopy (ECLIPSE Ci POL, Nikon, Tokyo, Japan) was also used to investigate the structure of the liquid crystalline phase (Amar-Yuli and Garti, 2005; Pindzola et al., 2003; Gurfinkel et al., 2011; Farkas et al., 2007). LCFS (15 ml) was dropped and thinly spread on the glass slide. The glass slide was placed in a petri dish that contained 10 ml of triple distilled water for 15 min to form the liquid crystalline phase on the glass. The cover glass was placed slowly on the glass slide so as not to form air bubbles, and then the cover glass was sealed with silicone grease to prevent water evaporation at 25
C. All the photographs were taken under the condition of the cross-polarizer. 2.2.4. Small-angle X-ray scattering (SAXS) measurements Small-angle X-ray scattering spectroscopy (SAXSess mc2; Anton Paar, Graz, Austria) was used to analyze the LCFS; 15 ml samples were dropped onto slides, thinly spread, and watered to form the liquid crystalline phase at room temperature before measurement. Membranes (10  40 mm) were placed on the sample holder. The X-ray wavelength was l 1.542 Å, and the distance between the sample and detector was 25 cm. The exposure time was 15 min. 2.2.5. Pharmacokinetic study in rats and dogs LCFSs containing entecavir (ETV1, ETV2, ETV3: 5.6 mg/kg; ETV4, ETV5, ETV6: 8.4 mg/kg) were subcutaneously injected into the

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back of each rat (four animals per group). For the control, entecavir solution (0.4 mg/kg) was injected as a fourteenth dose of LCFS group in the same manner, because about half a month sustained delivery was hypothesized. Blood was collected from the tail vein of each animal at 0 and 1 h, and at 1, 2, 3, 4, 5, 6, and 7 days after LCFS injection; or at 0, 0.25, 0.5, 0.75, 1, 1.5, 2, 3, 5, 8, and 12 h in the control group after injection. Each blood sample (0.75 ml) was loaded into a tube containing 10.0 ml EDTA (0.5 M), and the tubes were centrifuged at 4
C and 4000 rpm for 20 min. The centrifuged plasma samples (100 ml) were aliquoted and stored at –80
C before analysis. LCFSs containing entecavir (ETV4 and ETV5: 0.7 mg/kg) were subcutaneously injected into the back of beagle dogs (five animals per group). A blood sample was collected from the cephalic vein of each animal at 0, 1, 3, 6 and 12 h, and at 1, 2, 3, 4, 5, 6, 7, 9, 11, and 14 days after the injection. Each blood sample (2 ml) was loaded in a tube that contained 20.0 ml EDTA (0.5 M), and the tubes were centrifuged at 4
C and 4000 rpm for 20 min. The centrifuged plasma samples (250 ml) were aliquoted and stored at –80
C until analysis. Drug concentrations in the plasma of the rats and beagle dogs were determined by the ultra-high-performance liquid chromatography tandem-mass spectroscopy (UPLC–MS/MS) method. A stock solution of entecavir (50.0 mg/ml in 50% (v/v) methanol) was prepared. A working solution was prepared by diluting a stock solution to 5.0, 2.0, 1.0, 0.5, 0.1, 0.05, 0.02 and 0.01 mg entecavir/ml using 50% (v/v) methanol. An internal standard solution was prepared by dissolving entecavir-13C2,15N to 10.0 mg/ml in 50% (v/v) methanol, and diluting it to 0.100 mg/ml with 50% (v/v) methanol. Sequentially, a series of calibration standards was prepared by diluting 10 ml working standard solution with 0.99 ml of plasma. The final concentrations of the calibration standards were 50.0, 20.0, 10.0, 5.0, 1.0, 0.5, 0.2, and 0.1 ng/ml. Then the plasma samples (50 ml) and the calibration standards were mixed with the internal standard solution (10 ml; methanol was used as the reagent blank and the blank matrix) and centrifuged for 1 min at 13,000 rpm after vortexing for 1 min. Trifluoroacetic acid (10 ml) was added to each sample and centrifuged for 6 min followed after vortexing for 1 min. Each supernatant was separated by centrifugation for 6 min. Samples of all supernatants (50 ml volumes) was put in wells of a 96-well round-well plate and 350 ml 0.05% (w/v) ammonium formate in water was added. The plate was centrifuged at 4000 rpm and room temperature for 1 min, after mixing at 1500 rpm for 3 min. The prepared samples (20 ml volumes) were injected into an ultra high-performance tandem mass spectroscopy system consisting of the Waters ACQUITY UPLC System (Waters, Milford, MA, USA) and the Waters XEVO TQ-S (Waters). Chromatographic separation was achieved with the aid of an ACQUITY UPLC1 BEH C18 column (2.1 mm  50 mm; internal diameter 1.7 mm; Waters) at a column temperature of 35
C. The mobile phase consisted of 0.05% (w/v) ammonium formate in water (MP-A) and 0.05% (w/v) ammonium formate in methanol (MP-B), and the flow rate was 0.3 ml/min. The gradient elution program of the mobile phase was as follows: 95% MP-A, 5% MP-B: 0.00–1.00 min; 95–90% MP-A, 5–10% MP-B: 1.00–1.10 min; 90–85% MP-A, 10–15% MP-B: 1.10–3.00 min; 85–20% MP-A, 15–80% MP-B: 3.00–3.10 min; 20% MP-A, 80% MP-B: 3.10–4.00 min; 20–95% MP-A, 80–5% MP-B: 4.00–4.10 min; and 95% MP-A, 5% MP-B: 4.10–4.50 min. The UPLC–MS/MS system was interfaced with an electrospray ionization (ESI) probe. The ion source and desolvation temperatures were set at 150
C and 500
C, respectively. The flow rates of the cone gas and the desolvation gas were kept at 50 l/h and 800 l/h, respectively. The voltages were set at 28 V for the entrance potential and at 18 V for the collision energy voltage. The ions for selective monitoring were chosen via positive scanning from 100 to 800 m/z; and for the quantification, the product ions

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were monitored. The protonated molecules of entecavir were monitored at 278.14 m/z [M + H]+ for the precursor ions and at 152.10 m/z [M + H]+ for the product ions. The protonated molecule of entecavir-13C2,15N was monitored at 281.21 m/z [M + H]+ for the precursor ions and at 155.09 m/z [M + H]+ for the product ions. Using the above UPLC–MS/MS analysis method afforded good linearity of the calibration curve at the tested concentration ranges of 0.1–50.0 ng/ml for entecavir in rat and dog plasmas as demonstrated by the high correlation coefficient (R2 > 0.99; Fig. S1). The developed assay exhibited acceptable precision and accuracy, and the lower limit of quantification was 0.05 ng/ml in both rat and dog plasma. 2.2.6. Pharmacokinetic analysis and statistical analysis The maximum plasma concentration (Cmax) of entecavir and the time to reach the maximum (Tmax) were determined directly from the data. Non-compartmental pharmacokinetic analysis was performed on the plasma concentration-vs.-time curves using commercially available pharmacokinetic software. The area under the plasma concentration time curve (AUC) from time zero to the last measurable concentration (AUClast), the area under the curve from time zero to infinity (AUCinf), and the terminal half-life of the drug (T1/2) were calculated by WinNonlin 5.3 (Pharsight Corporation, Mountain View, CA, USA). Statistical analyses were carried out using Student’s t test or analysis of variance (ANOVA), followed by post hoc Duncan’s or Dunnett’s T3 test with a statistical software package (SPSS1 release 21.0, IBM, Chicago, IL, USA). Significance was tested at the 0.05 level of probability. 3. Results and discussion 3.1. Liquid crystal formulations with various LCFSs A LCFS was developed for the subcutaneous long-term delivery of entecavir. However, the polar entecavir is poorly miscible with or soluble in the non-polar LCFS. Therefore, entecavir was encapsulated in liposomes and freeze-dried to enhance the superficial area of entecavir, to allow it to be easily dissolved in the LCFS. A series of various LCFS formulations were prepared. A basic LCFS formulation for sustained delivery of entecavir (ETV1) was modified by addition of DPPA as an anionic phospholipid, designed to afford an anchoring effect (ETV2, ETV3). Formulations with a 1.5 times increased amount of entecavir (ETV4), and in which the pH was optimized to maximize the anchoring effect (ETV5, ETV6), were also prepared to increase overall plasma concentration in pharmacokinetic profiles. All LCFS formulations turned into gel-like mesophases, via formation of lyotropic LCs, 5 min after the LCFSs contacted PBS as shown in the photographs inserted in Table 1. Representative formulations; namely, the basic formulation (ETV1), the phospholipid-added formulation (ETV3), and the pH-optimized formulation with the anionic phospholipid (ETV5) were investigated by cryo-TEM, polarized optical microscopy, and SAXS. All formulations were tested in pharmacokinetic studies in rats or beagle dogs. 3.2. Cryo-TEM and polarized optical microscopy The crystallographic structures of the mesophases of ETV1, ETV3, and ETV5 were determined via cryo-TEM. The crystalline phase formed by LCFS was observed to have a mesophase with striation textures or beehive-like lattice structures (Fig. 1). The striation textures showed the curved longitudinal axis of one direction or many duplicated directions and lattice structures showed hexagonal or cubosomal symmetry. It has already been reported that non-lamellar phases like LCs, which have no separation between the internal and external layers, have a lattice

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Table 1 Various formulations of LCFSs containing entecavir.

Entecavir (mg) DPPA (mg) pHa

a

Control

ETV1

ETV2

ETV3

ETV4

ETV5

ETV6

1.0 – –

14.0 – –

14.0 25.0 –

14.0 50.0 –

21.0 50.0 –

21.0 50.0 3.5–4.5

21.0 50.0 2.5–3.5

ETV5 and ETV6 were adjusted at pH 3.5–4.5 or 2.5–3.5 by adding HCl to the entecavir-encapsulated liposome solution.

structure that is filled with many nano-sized unit structures (Kuntsche et al., 2011; Gustafsson et al., 1997; Sagalowicz et al., 2006). Therefore, based on this study, we can predict that the LCFSs ETV1, ETV3, and ETV5 are of the non-lamellar phase type, which is the major characteristic of the liquid crystalline phase. Polarized optical microscopy was also performed, because it is difficult to distinguish the hexagonal phase from the cubic phase based on mesophase morphology. ETV1, ETV3, and ETV5 showed that the liquid-crystalline phase had an angular or anisotropic texture in polarized optical microscopy (Fig. 2). It has already been reported that the angular texture is observed mostly in the hexagonal liquid-crystalline phase (Amar-Yuli and Garti, 2005; Pindzola et al., 2003), and also that anisotropic texture is evidence of the hexagonal phase, while no angular or anisotropic textures were detected in cubic phase (Gurfinkel et al., 2011; Farkas et al., 2007). Therefore, polarized optical microscopy revealed that ETV1, ETV3, and ETV5 had the typical hexagonal phase. All of the results from cryo-TEM and polarized optical microscopy proved that the mesophase from the LCFSs with or without DPPA addition exhibited characteristics typical of the liquid crystalline phase, classified as the hexagonal phase. 3.3. SAXS measurement SAXS analysis was carried out to investigate the inner structures p of ETV1, ETV3, and ETV5. The typical spacing ratio 1: 3:2 is present in the reverse hexagonal (H2) liquid crystalline structure (Nguyen et al., 2011; Hyde, 2001). Each formulation revealed three

typical peaks: ETV1 at approximately q = 0.11, 0.21, and 0.25 Å1; ETV3 at q = 0.10, 0.19, and 0.22 Å1; and ETV5 at q = 0.12, 0.21 and 0.24 Å1, respectively (Fig. 3). Calculation of spacing in all cases p showed a ratio of about 1: 3:2 indicating the hexagonal liquid crystalline structure. Based on these results, all LCFSs were of typical hexagonal structure, which was supported by the results of cryo-TEM and polarized microscopy. 3.4. In vivo pharmacokinetics in rats (Part I) Entecavir can be administered orally to patients up to 1.0 mg per day, so 14.0 mg is needed for about 2 weeks (14 days) of treatment, which is 0.233 mg/kg considering the human body weight as 60.0 kg. When calculating the dose for the experiment in rats, the dose per weight was increased about six times to 1.4 mg/kg after application of the human-equivalent-dose (HED) conversion factor. In the pharmacokinetic experiment in rats we increased this dose four-fold, to 5.6 mg/kg, because the injection volume of 1.4 mg/kg (about 20 ml per rat) was too small to be exactly measured and injected. The experiment was conducted during 1 week, because the plasma concentration fell enough to determine T1/2 after that time in preliminary experiments. ETV1 showed a promising sustained drug delivery pattern with a plasma concentration of over 4.0 ng/ml during 5 days. Anti-virus activity of entecavir was known to be maintained and adequate, when the concentration is over 1.0 ng/ml (Yan et al., 2006; Innaimo et al., 1997). Because the dose injected into rats was four-fold human equivalent dose, a SR formulation maintaining the plasma

Fig. 1. Cryo-TEM micrographs of the inner structures of mesophase and dispersed mesophase particles formed from LCFSs.

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Fig. 2. Polarized optical microscope images of mesophases formed from LCFSs. All images were taken at 200 magnification and the scale bar is 100 mm.

0.35 mg/kg. So the experimental injection dose for the rat was determined to be 8.4 mg/kg in the same manner as the doses of ETV1, ETV2, and ETV3 were calculated (1.5-fold greater than 5.6 mg/kg). ETV4, with the increased amount of only entecavir in the formula, showed a slightly higher plasma drug concentration compared to that of ETV3 (Fig. 4B). However, ETV4 revealed a slight decrease in half-life, possibly caused by a relatively lower ratio of LCFS vehicle per entecavir amount than that of ETV3. To improve the sustained delivery property, another attempt was made. By

Fig. 3. SAXS diffraction patterns of LCFSs. Notations 1, positions for the hexagonal nanostructure.

p

3, and 2 indicate the peak

drug concentration at greater than 4.0 ng/ml in the last in vivo timepoint was considered optimal. ETV1 showed a reduced Cmax value that was 28.3  6.7 ng/ml which was less than the control value (89.7  12.4 ng/ml), although the LCFSs contained 14 times entecavir than the control. Also, the T1/2 was increased from 2.0  0.4 h (control) to 31.4  3.2 h (ETV1; Fig. 4A, Table 2). To improve the sustained release pattern of the LCFS, DPPA was added to the LCFS. The DPPA is likely to be present as an anionic species at neutral pH conditions due to its pKa (Lee et al., 2008; Khiati et al., 2009). On the contrary, entecavir is a slightly cationic drug that can form charge interactions with anionic species (Gao et al., 2014). Therefore, the charge interaction between entecavir and DPPA was expected to produce an anchoring effect in LCs (Fig. 5). To utilize this anchoring effect, ETV2 and ETV3 were prepared with 25.0 mg and 50.0 mg DPPA, respectively (Table 1), because more than 50.0 mg DPPA induced precipitation of itself in LCFSs. ETV2 and ETV3 showed prolonged half-lives of 47.9  9.0 and 50.5  16.0 h, respectively (Table 2), and both formulas maintained the plasma drug concentrations at over 4.0 ng/ml for nearly 7 days. These results indicated that DPPA efficiently retained the release of entecavir from the LC via charge interaction. Therefore, further studies were carried out using a formulation based on LCFS with 50.0 mg DPPA. 3.5. In vivo pharmacokinetics in rats (Part II) At the steady state, the AUC of entecavir is proportional to the dose (Yan et al., 2006). To increase the overall plasma drug concentration during the in vivo experimental period, the amount of injected entecavir was increased from 14.0 mg to 21.0 mg as a unit dose (ETV4, ETV5, ETV6). Namely, the human doses of ETV4, ETV5, and ETV6 were increased 1.5 times, from 0.233 mg/kg to

Fig. 4. Plasma concentration of entecavir after subcutaneous injection of control and LCFS in rats. Data are presented as mean  SD (n = 4).

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Table 2 Pharmacokinetic parameters of entecavir after subcutaneous injection into rats.

Dose (mg/kg) Cmax (ng/ml) Tmax (h) AUClast (h ng/ml) AUCinf (h ng/ml) T1/2 (h)

Control

ETV1

ETV2

ETV3

ETV4

ETV5

ETV6

0.4 89.7  12.4 0.3  0.0 158.5  43.0 164.9  49.6 2.0  0.4

5.6 28.3  6.7a,b,c,d 1.0  0.0 1587.1  231.0a,c,d 1649.6  246.0a,c,d 31.4  3.2a,c,d

5.6 23.3  7.4a,b,c,d 18.25  11.5 1507.4  140.2a,b,c,d 1639.6  121.4a,c,d 47.9  9.0a,d

5.6 20.4  10.2a,b,c,d 18.75  35.5 1673.9  318.2a,c,d 1943.6  396.0a,c,d 50.5  16.0d

8.4 42.1  5.0a,d 1.0  0.0 2155.7  156.0a 2312.2  189.3a,d 40.3  8.9a,d

8.4 49.0  9.2a,d 2.5  1.0 2394.1  598.4a 2808.8  777.1a 59.1  8.4a,d

8.4 76.2  8.7a 1.5  1.0 2324.5  576.9a 3212.6  490.5a 81.9  23.4a

Data are presented as mean  SD (n = 4). a P < 0.05 vs. control. One-way ANOVA with Dunnett’s T3 test was used. b P < 0.05 vs. ETV4. One-way ANOVA with Duncan’s test was used. c P < 0.05 vs. ETV5. One-way ANOVA with Duncan’s test was used. d P < 0.05 vs. ETV6. One-way ANOVA with Duncan’s test was used.

adjusting the pH, enhancement of charge interaction between the cationic species and anionic species was expected to be strengthened (Bergers et al., 1993). Entecavir has values of pKa1 2.8 and pKa2 9.6, and DPPA has values of pKa1 3.5–4.0 and pKa2 9.0–9.5, and respectively (Zakanda et al., 2011; Desai et al., 2013). Therefore, to increase the anchoring effect between entacavir and DPPA, the pH was optimized by providing circumstances at which the anionic phospholipid species and the cationic drug species existed simultaneously (Fig. 6). The formulas of ETV5 and ETV6 were adjusted at pH 3.5–4.5 and pH 2.5–3.5, respectively. ETV5 showed an increased sustained release pattern; the plasma drug concentration was maintained at about 5.0 ng/ml for 7 days, although the Cmax value (49.0  9.2 ng/ml) was similar to the Cmax of ETV4. Meanwhile, ETV6 was estimated theoretically to have the strongest charge interaction between entecavir and DPPA, because the pH 2.5–3.5 is located between the pKa1 of entecavir and the pKa1 of DPPA. ETV6 showed a well-maintained plasma drug concentration of 7.0 ng/ml over 7 days. However, the Cmax value was relatively high with a value of 76.2  8.7 ng/ml. Also, by adjusting the pH to prepare ETV5 and ETV6, the half-lives could be prolonged to 59.1 8.4 h and 81.9  23.4 h, respectively, compared to the 40.3  8.9 h half-life of ETV4 (Fig. 4B, Table 2). Therefore, use of DPPA and pH adjustment to maximize anchoring between entecavir and DPPA improved sustained delivery and produced a generally higher plasma drug concentration during the experimental period.

3.6. In vivo pharmacokinetics in beagle dogs (Part III) Entecavir shows a significant difference among species in terminal half-life, which differs among the rat, dog, and human. After oral drug administration, the T1/2 values were 2.2 h in rats, 22.7 h in dogs, 27.4 h in monkeys, and approximately 135 h in humans (Baraclude Scientific Discussion, 2006). Based on the species difference, the terminal half-life in the beagle dog was expected to be much longer than in the rat after subcutaneous injection. Therefore, sustained delivery systems were designed to measure plasma concentrations over 2 weeks. Because the human doses of ETV4, ETV5, and ETV6 would be 0.35 mg/kg, application of the HED conversion factor (about a two-fold increase) suggested that the subcutaneous injection dose for beagle dogs should be increased to 0.7 mg/kg. The in vivo experiment in beagle dogs was carried out using ETV4 and ETV5 with the anchor effects, without (ETV4) or with (ETV5) pH optimization. Although ETV6 was optimized to the pH condition for the strongest charge interaction, it was not tested in dogs due to the possible pain by low pH. The plasma concentrations of both LCFSs were maintained for 2 weeks, especially with more than 1.00 ng/ml of drug level in plasma. As discussed in terms of the anti-virus activity in Section 3.5, an adequate concentration for a therapeutic effect was considered to be over 1.00 ng/ml when the human-equivalent dose (0.7 mg/kg) was administered in the dog (Fig. 7). The AUClast values of ETV4 and ETV5 for 14 days were 1614.5  438.9 and 2391.9  1087.4 ng/ml, and the Cmax values of

Fig. 5. Schematic presentation of release of neutral drug (A) without anchoring effect, and cationic drug (B) with anchoring effect from liquid crystalline mesophases.

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effect to the system improved the LC technology for the sustained delivery of polar drugs. 4. Conclusion

Fig. 6. Schematic presentation of pH optimization for charge interaction between entecavir and DPPA. Dotted square is optimal pH area for the charge interaction.

A novel LCFS was used for sustained delivery of entecavir. CryoTEM, polarized microscopy, and SAXS revealed that all LCFSs showed typical characteristics of the liquid crystalline mesophase, which was classified as hexagonal phase. Several LCFSs using SMO as a LCFM exhibited sustained drug release over 1 week in rats and 2 weeks in beagle dogs. In particular, the addition of DPPA as an anchoring material prolonged sustained delivery of entecavir. Moreover, by optimizing the pH of the LCFS, the anchoring effect was strengthened and the sustained delivery pattern was improved. The promising pharmacokinetic data in the rat and beagle dog suggest that the LCFS using SMO could serve as at least a 2-week drug delivery system for the treatment of hepatitis B. Based on the successful delivery of entecavir, the SMO-based LCFS with the anchoring effect can be used as a promising platform technology for the sustained delivery of polar drugs. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j. ijpharm.2015.05.049. References

Fig. 7. Plasma concentrations of entecavir after subcutaneous injections of ETV4 and ETV5 in beagle dogs. Data are presented as mean  SD (n = 5).

ETV4 and ETV5 were 20.2  7.4 ng/ml and 40.0  22.0 ng/ml, respectively (Table 3). ETV4 showed a 1.7 times-increased T1/2 value (72.6  15.1 h) and ETV5 showed a 1.5 times-increased T1/2 value (92.6  30.0 h) compared with the T1/2 value of each formulation in rats. These results supported the hypothesis that T1/2 values would increase significantly when the species changed from rat to beagle dog. Therefore, although the Cmax value of ETV5 was rather high, overall the sustained delivery patterns of ETV4 or ETV5 suggested that the use of the LCFS injection in humans with hepatitis B would afford at least a 2-week therapeutic period. The pharmacokinetics in the rat and beagle dog promises that the LCFS using SMO can be used as a novel SR injection system for entecavir. In particular, adding the anchoring

Table 3 Pharmacokinetic parameters of entecavir after subcutaneous injections in beagle dogs.

Dose (mg/kg) Cmax (ng/ml) Tmax (h) AUClast (h ng/ml) AUCinf (h ng/ml) T1/2 (h)

ETV4

ETV5

0.7 20.2  7.4 1.0  0.0a 1614.5  438.9 1710.1  432.5 72.6  15.1

0.7 40.0  22.0 15.6  11.5 2391.9  1087.4 2510.0  1068.8 92.6  30.0

Data are presented as mean  SD (n = 5). a P < 0.05 vs. ETV5. Student’s t test was used.

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