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Development of direct compression entecavir 0.5 mg-loaded tablet exhibiting enhanced content uniformity
Powder Technology 267 (2014) 302–308
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Development of direct compression entecavir 0.5 mg-loaded tablet exhibiting enhanced content uniformity Abid Mehmood Yousaf a,1, Jun-Pil Jee b,1, Seung Rim Hwang b, Han-Joo Maeng d, Young-Joon Park e, Jong Oh Kim c, Chul Soon Yong c, Han-Gon Choi a, Kwan Hyung Cho d,⁎ a
College of Pharmacy & Institute of Pharmaceutical Science and Technology, Hanyang University, 55 Hanyangdaehak-ro, Ansan 426-791, South Korea College of Pharmacy, Chosun University, 309 Pilmun-daero, Gwangju 501-759, South Korea College of Pharmacy, Yeungnam University, 280 Daehak-ro, Gyoungsan 712-749, South Korea d College of Pharmacy, Inje University, 197 Inje-ro, Gimhae 621-749, South Korea e College of Pharmacy, Ajou University, 206 World cup-ro, Suwon 443-749, South Korea b c
a r t i c l e
i n f o
Article history: Received 26 May 2014 Received in revised form 25 July 2014 Accepted 26 July 2014 Available online xxxx Keywords: Entecavir Direct compression Content uniformity Particle size Bioequivalent
a b s t r a c t The aim of the present research was to develop direct compression entecavir 0.5 mg-loaded tablet (DCET) providing enhanced content uniformity. Various compositions and preblending methods were tested at labscale, and the optimum composition and method were applied to pilot-scale production for further confirmation of the entire process. The content uniformity, physical properties and dissolution behavior of the final film-coated DCET were compared to the commercial product. In lab-scale preparation, the method involving preblending, micronization of API (d0.5 = 5.13 μm), addition of a larger quantity of colloidal silicon dioxide (1%) and sieving through smaller pores (300 μm) yielded an excellent acceptance value (AV) in the content uniformity criteria compared to a control method and composition (AV 1.0 vs. 9.8). In pilot-scale production, the film-coated DCET provided better content uniformity than the commercial product (AV 1.3 vs. 3.8). Furthermore, both products exhibited similar dissolution profiles in various media. Thus, direct compression entecavir 0.5 mg-loaded tablet developed in this study would be a promising dosage form with excellent content uniformity that may be bioequivalent to the commercial product. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Hepatitis B virus (HBV) annually affects approximately 400 million people across the globe [1,2] and can lead to hepatic cirrhosis, hepatic failure and hepatocellular carcinomas [3]. These complications result in about 1 million deaths per year [1]. In long-term therapy of hepatitis B, one way is to inhibit replication of HBV [4]. Entecavir potentially and selectively inhibits HBV reverse transcriptase resulting in suppression of its DNA replication [5,6]. Entecavir at a dose of 0.5 mg once daily has resulted in remarkable reduction in DNA replication of HBV [7]. Baraclude® 0.5 mg tablet (Bristol-Myers Squibb, New York, USA) is the commercial product of entecavir which has been frequently prescribed for the treatment of HBV [8,9].
⁎ Corresponding author. Tel.: +82 55 320 3883; fax: +82 55 320 3940. E-mail address: [email protected] (K.H. Cho). 1 Two authors contributed equally to this work.
http://dx.doi.org/10.1016/j.powtec.2014.07.041 0032-5910/© 2014 Elsevier B.V. All rights reserved.
In low-dose tablet formulations, content uniformity, which is an essential quality criteria to produce safe, effective unit products consistently, remains a critical challenge [10]. To overcome the problems of the content uniformity, numerous approaches have been described [11–14]. Previously, the content uniformity for lowdose formulations has been achieved by homogeneous mixing with starch 1500 [14], sugar [15], maltose and dextrose [16]. The direct compression is the simplest method to prepare tablets [14,15]. The main procedures of direct compression method generally consist of blending and tableting, which can give the advantages such as cost effectiveness, stability, faster dissolution, and simplified process validation [14,15]. However, with decreasing doses of the drug, achievement of proper content uniformity also decreases [17]. Accordingly, special care is required for direct compression of formulations containing potent active pharmaceutical ingredients (APIs) [18]. The purpose of this study was to develop direct compression entecavir 0.5 mg-loaded tablet with improved content uniformity. The optimization of formulation and method was performed at lab-scale.
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Then, the formulation and method exhibiting the optimum content uniformity at lab-scale were chosen for making direct compression tablets at pilot-scale. The content uniformity, physical properties and dissolution of the final tablet were assessed in comparison with the commercial product. 2. Materials and methods 2.1. Materials Entecavir monohydrate was obtained from Cipla Ltd. (Mumbai, ® Maharashtra, India). Aerosil® 200 (colloidal silicon dioxide), Kollidon ® ® VA64 (copovidone), Kollidon CL (crospovidone), Pharmatose DCL11 ® (lactose monohydrate), Pharmatose 200M (lactose monohydrate), ® CeolusTM PH102 (microcrystalline cellulose), and Pruv (sodium stearyl fumarate) supplied by Whawon Pharm. Co., Ltd. (Seoul, Kangnam, South ® Korea) were of USP grade. Commercial entecavir tablets (Baraclude 0.5 mg) were from Bristol-Myers Squibb (New York, USA). All other chemicals were of reagent grade and were used without further purification. 2.2. Particle size reduction of API The drug was micronized using a Micro-Jet™ Size Reduction System (Fluid Energy Processing and Equipment Co., Telford, PA, USA) with nitrogen gas at a pressure of about 100 psi. The milled drug lot was compared with the non-treated lot in the particle-size distribution, microscopic examination and thermal analysis. The detailed characterization is described below. 2.3. Particle-size distribution of API The particle size-distribution was determined using laser scattering particle size analyzer (Master sizer 2000®, Malvern Instruments Ltd., Malvern, UK) at an air pressure of 3 bar. The particle-size distribution was evaluated by cumulative distribution data (d0.1, d0.5 and d0.9 measurements). The median diameter (d0.5) was considered for comparing particle-size of the milled and non-treated API. 2.4. Thermal characterization Thermal analysis was performed using differential scanning calorimetry (DSC-Q10, TA Instruments; New Castle, Delaware, USA) and thermal gravimetric analysis (TGA-Q50, TA Instruments; New Castle, Delaware, USA). For DSC, about 4 mg of each sample was sealed in an aluminum pan and heated from 25 to 250 °C at a rate of 10 °C/min. For TGA, about 4 mg of each sample was heated up to 800 °C at a rate of 10 °C/min. 2.5. Microscopic examination Polarized microscopic images of jet-milled drug and non-treated API were captured at 400-fold magnification using an optical microscope (Olympus BX51, Olympus Optical Co., Tokyo, Japan) to examine particle size and homogeneity.
303
Table 1 Compositions of entecavir 0.5 mg-loaded core tablet in the divided four steps. Ingredients in each step
F1
F2
F3
Amount (mg) Preblending I Entecavir monohydrate Aerosil® 200 (colloidal silicon dioxide) Pharmatose® 200M (lactose monohydrate) Preblending II Pharmatose® DCL11 (lactose monohydrate) Blending Ceolus™ PH102 (microcrystalline cellulose) Kollidon® VA64 (copovidone) Kollidon® CL (crospovidone) Final blending Pruv® (sodium stearyl fumarate) Total (mg/tablet)
0.503 0.000 20.000
0.503 0.500 20.000
0.503 1.000 20.000
99.497
98.997
98.497
65.000 5.000 8.000
65.000 5.000 8.000
65.000 5.000 8.000
2.000 200.000
2.000 200.000
2.000 200.000
2.7. Lab-scale preparation of DCET The composition of each formulation and the characteristics of each batch are summarized in Tables 1 and 2, respectively. The batch size for lab-scale preparations was 1000 tablets. The entire blending method for B2–B8 consisted of four steps: preblending I, preblending II, blending and final blending. However, B1 was prepared using only the latter two steps. In step 1 (preblending I), entecavir monohydrate and Pharmatose® 200M, with or without Aerosil® 200, were mixed together in a cube mixer (AR403 equipped with KB15, Erweka, GmbH, Frankfurt, Germany) for 5 min at 60 rpm (Table 1). Subsequently, the mixture was sieved through a specific screen diameter (Table 2) and poured into the cube mixer again. In step 2 (preblending II), Pharmatose® DCL11 was added to each preceding mixture as shown in Table 1 and mixed for 5 min at 60 rpm. The sieving after preblending II was performed for B3, B5, B6, B7 and B8 only (Table 2). In step 3 (blending), Ceolus™ PH102, Kollidon® VA64 and Kollidon® CL were added, and further mixed for 20 min at 60 rpm. In step 4 (final blending), Pruv® pre-sieved through 50 mesh was incorporated into the blended mixture and further blended for 3 min at 60 rpm. For the control batch (B1), all constituents (except Pruv®) were mixed in the cube mixer for 20 min at 60 rpm, and Pruv® was added and mixed for further 3 min at 60 rpm. The entecavir 0.5 mg-loaded core tablets with a triangular shape and 5–6 KP hardness were directly compressed with the above-mentioned each final blend using ERWEKA tablet machine (GmbH, Frankfurt, Germany) [19]. 2.8. Pilot-scale production of film-coated DCET At pilot-scale production, batch size was 10,000 tablets. The composition of the formulation was exactly the same as F3 (Table 1). Moreover, the preparation method was the same as for B8 (Table 2).
Table 2 Summary of preparation characteristics for the lab-scale batches.
2.6. High performance liquid chromatography (HPLC) assay
Batch number
Compositiona API particle size (d0.5, μm)
Screen-hole diameter (μm)
Sieving times
For the HPLC analysis of entecavir in the standard and sample solutions, Agilent 1200 HPLC system (Agilent Technologies, Santa Clara, CA, USA) outfitted with a C18 column (Symmetry®, Waters, 5 μm, 100 mm × 4.6 mm i.d.) and UV detector (Model L-7450, Agilent Technologies, Santa Clara, CA, USA) was used. The injection volume was 75 μl. The mobile phase (water/acetonitrile, 92/8, v/v) eluted at the rate of 1.0 ml/min was monitored at 254 nm for entecavir concentration measurement.
B1 B2 B3 B4 B5 B6 B7 B8
F1 F1 F1 F2 F2 F3 F3 F3
N/A 600 600 600 600 600 600 300
N/A 1 2 1 2 2 2 2
a
17.72 17.72 17.72 17.72 17.72 17.72 5.13 5.13
The composition given in Table 1.
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Table 3 Criteria for USP b905 N content uniformity. (A) General formula AV ¼ ⌊M−X⌋þks; X ¼
n 1 n ∑ j¼1 x j ;
sffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi 2 n ∑ j¼1 x j −X ; RSD ¼ 100s=X s¼ n−1
(B) Reference value M I. T ≤ 101.5 i. XN101:5; then M ¼ 101:5 ii. Xb98:5; then M ¼ 98:5 iii. 98:5≤ X ≤101:5; then M ¼ X II. T N 101.5 i. XNT; then M ¼ T
Then, the core tablets were coated with 10% (w/v) aqueous dispersion of Opadry® (03B64610, Colorcon Inc., PA, USA) using a Hi-Coater® (LDCS-5, Freund-Vector Corporation, IA, USA). The spraying rate of the coating dispersion was 40 g/min. The rotation speed of the coating pan was 8 ± 2 rpm. The inlet and outlet temperatures of air-flow were 75 ± 5 °C and 35 ± 5 °C, respectively. The physical features including the hardness and weight variation were determined using hardness tester (PTB 111E, Pharma Test Apparatebau AG, Hainburg, Germany) and the balance (MS-204, Mettler-Toledo, OH, USA), respectively. 2.9. Determination of content uniformity
ii. Xb98:5; then M ¼ 98:5 iii. 98:5≤ X ≤T; then M ¼ X (C) Acceptance criteria Stage 1 (n = 10, k = 2.4) I. AV ≤ L1 i. xmax ≤ (1 + L2 × 0.01)M ii. xmin N (1 − L2 × 0.01)M (D) Notation AV: acceptance value; X: mean; s: sample standard deviation; RSD: relative standard deviation T: target content percentage unit, expressed as the percentage of the label claim, unless otherwise state, T is 100.0%. xmax: maximal individual value; xmin: minimal individual value LC: label claim L1 = 15 and L2 = 25 unless otherwise specified in the individual monograph
However, double cone mixer (AR403 equipped with DKM, Erweka, Germany) was used for all mixing steps. During the preblending II and blending steps, the mixture was sampled randomly (n = 10 sampling locations) via a thief probe at the specified time points [20]. These samples were assayed by the HPLC method as described above for evaluation of mixing homogeneity. The entecavir 0.5 mg-loaded core tablets were prepared by using the rotary tablet machine (Model D-8, Riva S.A., Buenos Aires, Argentina) with the same tablet size and hardness as in the lab-scale preparation.
The 10 dosage unit samples from the lab-scale batches and pilotscale batch, as well as the commercial product and the mixture samples taken from pilot-scale mixing, were assayed for drug content using the HPLC method as described above. The mean drug content (%) and acceptance value (AV) were calculated in accordance with the formula from USP b905 N content uniformity of dosage units as described in Table 3. 2.10. Dissolution The dissolution test of the film-coated DCET was completed using USP dissolution apparatus II (Shinseang Instrument Co., South Korea) with 900 ml water, 0.1 N HCl solution (pH 1.2), acetate buffer solution (pH 4.0) or phosphate buffer solution (pH 6.8) as a dissolution medium at 37 ± 0.5 °C. The speed of the paddle rotation was 50 rpm. The dissolution test was performed in comparison with the commercial product. At particular time points, 1 ml dissolution medium was withdrawn, filtered (0.45 μm) and assayed for entecavir by the HPLC method as described above. The similarity factor ( f2) and difference factor ( f1) were calculated in accordance with Moore and Flanner's method to compare the dissolution profiles of the film-coated DCET and the commercial product [21]. The similarity factor ( f2) ≥ 50 represents an appropriately comparable dissolution profile [22].
Fig. 1. Particle size distribution of entecavir monohydrate: (A), non-treated; (B), air jet milled.
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A)
305
B)
Fig. 2. Polarized microscopic images of entecavir monohydrate: (A), non-treated; (B), air jet milled.
3. Results and discussion Entecavir (0.5 mg once daily dose) remarkably inhibits replication of HBV [4]. Baraclude® 0.5 mg tablet (Bristol-Myers Squibb, New York, USA) is the commonly prescribed commercial product of entecavir [8, 9]. The content uniformity is a critical issue of the potent drugs [10]. Suitable content uniformity is very important for the safety and efficacy
of a product containing a low-dose API [11–14]. The present research is an endeavor to promote the content uniformity of entecavir in tablet dosage form. As compared to non-treated API, the air jet milled API exhibited 3-times smaller in median particle size (Fig. 1). Moreover, the cumulative particle-size distribution data proved that d0.1, d0.5 and d0.9 of the milled vs. non-treated API were 0.93 μm vs. 4.86 μm, 5.13 μm vs.
Fig. 3. Thermal analysis of entecavir monohydrate: (A), differential scanning calorimetric thermograms; (B), thermal gravimetric analysis. The continuous line represents non-treated drug and dotted line represents air jet milled drug.
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12 Mean content Acceptance value
Mean content (%) S.D.
10
9.8
102 100
7.7
8
7.6 6.7
98
6.2
6 5.4
96
4 94
2.7
Acceptance value (AV)
104
2
92 1.0
90
B1
B2
B3
B4
B5
B6
B7
B8
0
Batch number Fig. 4. Entecavir content (mean % ± S.D.) and acceptance value (AV) elicited from labscale batches (B1–B8) according to the USP b905N content uniformity (n = 10).
17.72 μm and 10.15 μm vs. 36.48 μm, respectively. Furthermore, the polarized microscopic examination also affirmed reduced particle size in milled API as compared to non-treated API sample (Fig. 2). Micronization may substantially alter the physical properties such as crystallinity and surface features of a drug substance [23]. Accordingly, investigation of change in the crystalline property from crystalline state to the amorphous form should be carried out [24,25]. In this study, DSC and TGA techniques were used. In DSC analysis, the air jet milled API and non-treated API generated in common an intrinsic peak at 165.2 °C (Fig. 3A). Moreover, other endotherms also appeared in both cases, suggesting that the drug was not converted into the amorphous state. The TGA thermograms of milled API and non-treated API are shown in Fig. 3B. The loss in weight, during heating up to 100 °C, was about 6.04% in both cases, which was very close to the theoretical moisture content (6.09%) of entecavir monohydrate. Thus, DSC results suggested that the drug did not change into the amorphous form in the milling process. Further, TGA results confirmed that entecavir monohydrate retained its water of hydration even after the milling process. In lab-scale preparation, the entecavir 0.5 mg-loaded tablets were obtained via direct compression method using three compositions, F1, F2 and F3, carrying 0%, 0.5% and 1.0% Aerosil® 200 (CSD), respectively (Table 1). Moreover, eight batches (B1–B8) of 1000 tablets each were prepared from these compositions (Table 2). The preparation characteristics from B1 to B8 varied in preblending method, particle-size of API, quantity of CSD (%), number of sieving times and the screen-hole diameter. The content uniformity of each batch was evaluated by the drug content (%) and acceptance value (AV) (Fig. 4). The AV was calculated according to USP b905 N content uniformity criteria given in Table 3. The drug content values for B1, B2, B3, B4, B5, B6, B7 and B8 were 96.31 ± 3.57, 98.51 ± 3.22, 99.92 ± 2.79, 99.41 ± 2.81, 99.02 ± 2.59, 98.54 ± 2.26, 99.21 ± 1.12, and 99.33 ± 0.43%, respectively. And, the AV was 9.8, 7.7, 7.6, 6.7, 6.2, 5.4, 2.7, and 1.0, respectively. The batches
B1, B2 and B3 were prepared according to formulation F1 (Table 1). In the preparation of B1, preblending and sieving were excluded. The batch B2 provided better mean drug content (n = 10) and AV than those of B1 (Fig. 4). This suggested that preblending and sieving improved the content uniformity. Likewise, the mean drug content and AV for B3 were slightly improved as compared to B2 (Fig. 4). This suggested that sieving again after preblending II also contributed in enhancing the content uniformity. The batches B4 and B5 were prepared according to formulation F2 (Table 1). In these formulations, 0.5% CSD was used as a glidant to improve the flowability of the powder mixtures [26,27]. As compared with the AV of B2 and B3, the improved AV of B4 and B5 was due to the addition of 0.5% CSD in the formulations. Moreover, the AV of B5 was better than that of B4 (Fig. 4). This proved that the number of times of sieving was also important in improving the content uniformity due to further homogeneity [28,29]. The batches B6, B7 and B8 were prepared in accordance with formulation F3 (Table 1). In these batches, 1.0% CSD was used as a glidant to further improve the flow property of the powder mixtures [28,29]. The acceptance values of these batches were better than AV of B5 and B6 (Fig. 4). As compared to B5, the batch B6 showed better AV. This suggested that increased quantity of CSD (0.5% vs. 1.0%) further improved the content uniformity. The batches B6 and B7 demonstrated the influence of particle size of the API on enhancing the content uniformity. The API with median particle size of 17.72 μm and 5.13 μm was used in batches B6 and B7, respectively. The batch B7 gave about 2-times better AV than that of batch B6. This revealed that the smaller particle size of the drug improved the content uniformity owing to improved mixing [30,31]. The batches B7 and B8 exhibited the role of screen-hole diameter in increased content uniformity. The AV of B8 was approximately 3-times better than that of B7 (Fig. 4). This was because of sieving B8 through the screen having comparatively smaller hole-size (300 μm vs. 600 μm). This suggested that exclusion of larger particles considerably improved the AV of B8. Thus, the batch B8 showed the excellent AV (1.0) among all the batches and was chosen for pilot-scale production of entecavir 0.5 mg-loaded direct compression tablets. The pilot-scale production was conducted in order to establish the reliability of the whole process employed in the lab-scale preparation of B8. The composition and preparation method of this batch were exactly the same as B8 (Tables 1, 2). However, the batch size of pilotscale production was 10-times larger than that of the lab-scale preparation (10,000 vs. 1000 tablets). Moreover, a double cone mixer was used in the pilot-scale production instead of a cube mixer. The tablet compression was accomplished with a rotary tablet machine. The optimum mixing time in both preblending II and blending cases was 30 min (Fig. 5). At the optimum mixing time, the mixture samples presented the best AV of 1.74 and 2.05, respectively. The comparison of quality control characteristics of the film-coated pilot-scale DCET such as weight variation, hardness, drug content and
Table 4 General properties of film-coated DCET and commercial product. Properties
Film-coated DCET
Commercial tablet
Weight (mg)a Hardness (KP)a Content (%)a Content uniformity (AV)b Content difference % highest–lowestc
205.2 ± 1.58 9.3 ± 1.10 99.35 ± 0.54 1.3 1.9
205.7 ± 2.04 8.5 ± 1.60 100.43 ± 1.58 3.8 5.5
a
Each value represents the mean ± S.D. (n = 10). Calculated using the formula in Table 3. Range between the highest content % of unit tablet and the lowest content % of unit tablet. b c
Fig. 5. Acceptance value (AV) of powder mixtures sampled in the preblending I and blending step (n = 10).
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307
Fig. 6. Dissolution profiles of drug from the entecavir 0.5 mg-loaded film-coated tablet and commercial product: (A), pH 1.2; (B), pH 4.0; (C), pH 6.8; (D), water. Each value denotes the mean ± S.D. (n = 6).
AV with those of the commercial product is summarized in Table 4. The weight variation, hardness and drug content were not significantly different (P N 0.05) from each other (n = 10). The acceptance value (AV) of the film-coated pilot-scale DCET was 3-times better than that of the commercial product (1.3 vs. 3.8). Likewise, the content difference % highest–lowest for the film-coated DCET was approximately 3-times narrower than that of the commercial product (1.9 vs. 5.5%). As a result, the film-coated pilot-scale DCET showed enhanced content uniformity. The enhanced content uniformity of this tablet might be attributed to the excellent homogeneity of the final mixture obtained from micronization of API, preblending I, preblending II and sieving through smaller screen-hole diameter [32]. The dissolution of film-coated DCET in pH 1.2, 4.0, 6.8 and water is compared to that of the commercial product in Fig. 6. In all dissolution media, about 90% of the incorporated entecavir dissolved from both products within 15 min, with comparable dissolution profiles. The similarity ( f2) and difference factors ( f1) calculated from the dissolution profiles in each medium are given in Table 5 [21]. A similarity factor ( f2) ≥ 50 designates a suitably similar dissolution profile [22]. In the present research, all the dissolution profiles resulted in similarity factor ( f2) N 50. Accordingly, the dissolution behavior of the film-coated DCET
Table 5 Dissolution comparison of film-coated DCET and commercial product. Dissolution medium
Similarity factor ( f2) Difference factor ( f1)
pH 1.2
pH 4.0
pH 6.8
H2O
78 2
89 1
77 3
60 6
was considered as similar to that of the commercial product. This suggested that the film-coated tablet might be bioequivalent to the commercial product. 4. Conclusions In lab-scale preparation, entecavir 0.5 mg-loaded tablets of batch B8 formulated with F3 composition furnished the excellent content uniformity (AV = 1.0). In pilot-scale production, the optimized composition and process demonstrated consistent content uniformity. The content uniformity of the film-coated DCET was improved as compared to the commercial product (1.3 vs. 3.8). Moreover, the dissolution profile of film-coated DCET was similar to that of the commercial product in all dissolution media ( f2 N 50). Thus, the film-coated DCET with enhanced content uniformity might be bioequivalent to the commercial product. Acknowledgment This work was supported by the 2014 Inje University research grant. References [1] W.M. Lee, Hepatitis B virus infection, N. Engl. J. Med. 337 (1997) 1733–1745. [2] T. Poynard, M.-F. Yuen, V. Ratzin, C.L. Lai, Viral hepatitis C, Lancet 362 (2003) 2095–2100. [3] M. Sherman, C. Yurdaydin, J. Sollano, M. Silva, Y.F. Liaw, J. Cianciara, A. BoronKaczmarska, A. Martin, Z. Goodman, R. Colonno, Entecavir for treatment of lamivudine-refractory, HBeAg-positive chronic hepatitis B, Gastroenterology 130 (2006) 2039–2049. [4] Y.-F. Liaw, N. Leung, J.-H. Kao, T. Piratvisuth, E. Gane, K.-H. Han, R. Guan, G.K. Lau, S. Locarnini, Asian-Pacific consensus statement on the management of chronic hepatitis B: a 2008 update, Hepatol. Int. 2 (2008) 263–283.
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Contents lists available at ScienceDirect
Powder Technology journal homepage: www.elsevier.com/locate/powtec
Development of direct compression entecavir 0.5 mg-loaded tablet exhibiting enhanced content uniformity Abid Mehmood Yousaf a,1, Jun-Pil Jee b,1, Seung Rim Hwang b, Han-Joo Maeng d, Young-Joon Park e, Jong Oh Kim c, Chul Soon Yong c, Han-Gon Choi a, Kwan Hyung Cho d,⁎ a
College of Pharmacy & Institute of Pharmaceutical Science and Technology, Hanyang University, 55 Hanyangdaehak-ro, Ansan 426-791, South Korea College of Pharmacy, Chosun University, 309 Pilmun-daero, Gwangju 501-759, South Korea College of Pharmacy, Yeungnam University, 280 Daehak-ro, Gyoungsan 712-749, South Korea d College of Pharmacy, Inje University, 197 Inje-ro, Gimhae 621-749, South Korea e College of Pharmacy, Ajou University, 206 World cup-ro, Suwon 443-749, South Korea b c
a r t i c l e
i n f o
Article history: Received 26 May 2014 Received in revised form 25 July 2014 Accepted 26 July 2014 Available online xxxx Keywords: Entecavir Direct compression Content uniformity Particle size Bioequivalent
a b s t r a c t The aim of the present research was to develop direct compression entecavir 0.5 mg-loaded tablet (DCET) providing enhanced content uniformity. Various compositions and preblending methods were tested at labscale, and the optimum composition and method were applied to pilot-scale production for further confirmation of the entire process. The content uniformity, physical properties and dissolution behavior of the final film-coated DCET were compared to the commercial product. In lab-scale preparation, the method involving preblending, micronization of API (d0.5 = 5.13 μm), addition of a larger quantity of colloidal silicon dioxide (1%) and sieving through smaller pores (300 μm) yielded an excellent acceptance value (AV) in the content uniformity criteria compared to a control method and composition (AV 1.0 vs. 9.8). In pilot-scale production, the film-coated DCET provided better content uniformity than the commercial product (AV 1.3 vs. 3.8). Furthermore, both products exhibited similar dissolution profiles in various media. Thus, direct compression entecavir 0.5 mg-loaded tablet developed in this study would be a promising dosage form with excellent content uniformity that may be bioequivalent to the commercial product. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Hepatitis B virus (HBV) annually affects approximately 400 million people across the globe [1,2] and can lead to hepatic cirrhosis, hepatic failure and hepatocellular carcinomas [3]. These complications result in about 1 million deaths per year [1]. In long-term therapy of hepatitis B, one way is to inhibit replication of HBV [4]. Entecavir potentially and selectively inhibits HBV reverse transcriptase resulting in suppression of its DNA replication [5,6]. Entecavir at a dose of 0.5 mg once daily has resulted in remarkable reduction in DNA replication of HBV [7]. Baraclude® 0.5 mg tablet (Bristol-Myers Squibb, New York, USA) is the commercial product of entecavir which has been frequently prescribed for the treatment of HBV [8,9].
⁎ Corresponding author. Tel.: +82 55 320 3883; fax: +82 55 320 3940. E-mail address: [email protected] (K.H. Cho). 1 Two authors contributed equally to this work.
http://dx.doi.org/10.1016/j.powtec.2014.07.041 0032-5910/© 2014 Elsevier B.V. All rights reserved.
In low-dose tablet formulations, content uniformity, which is an essential quality criteria to produce safe, effective unit products consistently, remains a critical challenge [10]. To overcome the problems of the content uniformity, numerous approaches have been described [11–14]. Previously, the content uniformity for lowdose formulations has been achieved by homogeneous mixing with starch 1500 [14], sugar [15], maltose and dextrose [16]. The direct compression is the simplest method to prepare tablets [14,15]. The main procedures of direct compression method generally consist of blending and tableting, which can give the advantages such as cost effectiveness, stability, faster dissolution, and simplified process validation [14,15]. However, with decreasing doses of the drug, achievement of proper content uniformity also decreases [17]. Accordingly, special care is required for direct compression of formulations containing potent active pharmaceutical ingredients (APIs) [18]. The purpose of this study was to develop direct compression entecavir 0.5 mg-loaded tablet with improved content uniformity. The optimization of formulation and method was performed at lab-scale.
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Then, the formulation and method exhibiting the optimum content uniformity at lab-scale were chosen for making direct compression tablets at pilot-scale. The content uniformity, physical properties and dissolution of the final tablet were assessed in comparison with the commercial product. 2. Materials and methods 2.1. Materials Entecavir monohydrate was obtained from Cipla Ltd. (Mumbai, ® Maharashtra, India). Aerosil® 200 (colloidal silicon dioxide), Kollidon ® ® VA64 (copovidone), Kollidon CL (crospovidone), Pharmatose DCL11 ® (lactose monohydrate), Pharmatose 200M (lactose monohydrate), ® CeolusTM PH102 (microcrystalline cellulose), and Pruv (sodium stearyl fumarate) supplied by Whawon Pharm. Co., Ltd. (Seoul, Kangnam, South ® Korea) were of USP grade. Commercial entecavir tablets (Baraclude 0.5 mg) were from Bristol-Myers Squibb (New York, USA). All other chemicals were of reagent grade and were used without further purification. 2.2. Particle size reduction of API The drug was micronized using a Micro-Jet™ Size Reduction System (Fluid Energy Processing and Equipment Co., Telford, PA, USA) with nitrogen gas at a pressure of about 100 psi. The milled drug lot was compared with the non-treated lot in the particle-size distribution, microscopic examination and thermal analysis. The detailed characterization is described below. 2.3. Particle-size distribution of API The particle size-distribution was determined using laser scattering particle size analyzer (Master sizer 2000®, Malvern Instruments Ltd., Malvern, UK) at an air pressure of 3 bar. The particle-size distribution was evaluated by cumulative distribution data (d0.1, d0.5 and d0.9 measurements). The median diameter (d0.5) was considered for comparing particle-size of the milled and non-treated API. 2.4. Thermal characterization Thermal analysis was performed using differential scanning calorimetry (DSC-Q10, TA Instruments; New Castle, Delaware, USA) and thermal gravimetric analysis (TGA-Q50, TA Instruments; New Castle, Delaware, USA). For DSC, about 4 mg of each sample was sealed in an aluminum pan and heated from 25 to 250 °C at a rate of 10 °C/min. For TGA, about 4 mg of each sample was heated up to 800 °C at a rate of 10 °C/min. 2.5. Microscopic examination Polarized microscopic images of jet-milled drug and non-treated API were captured at 400-fold magnification using an optical microscope (Olympus BX51, Olympus Optical Co., Tokyo, Japan) to examine particle size and homogeneity.
303
Table 1 Compositions of entecavir 0.5 mg-loaded core tablet in the divided four steps. Ingredients in each step
F1
F2
F3
Amount (mg) Preblending I Entecavir monohydrate Aerosil® 200 (colloidal silicon dioxide) Pharmatose® 200M (lactose monohydrate) Preblending II Pharmatose® DCL11 (lactose monohydrate) Blending Ceolus™ PH102 (microcrystalline cellulose) Kollidon® VA64 (copovidone) Kollidon® CL (crospovidone) Final blending Pruv® (sodium stearyl fumarate) Total (mg/tablet)
0.503 0.000 20.000
0.503 0.500 20.000
0.503 1.000 20.000
99.497
98.997
98.497
65.000 5.000 8.000
65.000 5.000 8.000
65.000 5.000 8.000
2.000 200.000
2.000 200.000
2.000 200.000
2.7. Lab-scale preparation of DCET The composition of each formulation and the characteristics of each batch are summarized in Tables 1 and 2, respectively. The batch size for lab-scale preparations was 1000 tablets. The entire blending method for B2–B8 consisted of four steps: preblending I, preblending II, blending and final blending. However, B1 was prepared using only the latter two steps. In step 1 (preblending I), entecavir monohydrate and Pharmatose® 200M, with or without Aerosil® 200, were mixed together in a cube mixer (AR403 equipped with KB15, Erweka, GmbH, Frankfurt, Germany) for 5 min at 60 rpm (Table 1). Subsequently, the mixture was sieved through a specific screen diameter (Table 2) and poured into the cube mixer again. In step 2 (preblending II), Pharmatose® DCL11 was added to each preceding mixture as shown in Table 1 and mixed for 5 min at 60 rpm. The sieving after preblending II was performed for B3, B5, B6, B7 and B8 only (Table 2). In step 3 (blending), Ceolus™ PH102, Kollidon® VA64 and Kollidon® CL were added, and further mixed for 20 min at 60 rpm. In step 4 (final blending), Pruv® pre-sieved through 50 mesh was incorporated into the blended mixture and further blended for 3 min at 60 rpm. For the control batch (B1), all constituents (except Pruv®) were mixed in the cube mixer for 20 min at 60 rpm, and Pruv® was added and mixed for further 3 min at 60 rpm. The entecavir 0.5 mg-loaded core tablets with a triangular shape and 5–6 KP hardness were directly compressed with the above-mentioned each final blend using ERWEKA tablet machine (GmbH, Frankfurt, Germany) [19]. 2.8. Pilot-scale production of film-coated DCET At pilot-scale production, batch size was 10,000 tablets. The composition of the formulation was exactly the same as F3 (Table 1). Moreover, the preparation method was the same as for B8 (Table 2).
Table 2 Summary of preparation characteristics for the lab-scale batches.
2.6. High performance liquid chromatography (HPLC) assay
Batch number
Compositiona API particle size (d0.5, μm)
Screen-hole diameter (μm)
Sieving times
For the HPLC analysis of entecavir in the standard and sample solutions, Agilent 1200 HPLC system (Agilent Technologies, Santa Clara, CA, USA) outfitted with a C18 column (Symmetry®, Waters, 5 μm, 100 mm × 4.6 mm i.d.) and UV detector (Model L-7450, Agilent Technologies, Santa Clara, CA, USA) was used. The injection volume was 75 μl. The mobile phase (water/acetonitrile, 92/8, v/v) eluted at the rate of 1.0 ml/min was monitored at 254 nm for entecavir concentration measurement.
B1 B2 B3 B4 B5 B6 B7 B8
F1 F1 F1 F2 F2 F3 F3 F3
N/A 600 600 600 600 600 600 300
N/A 1 2 1 2 2 2 2
a
17.72 17.72 17.72 17.72 17.72 17.72 5.13 5.13
The composition given in Table 1.
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Table 3 Criteria for USP b905 N content uniformity. (A) General formula AV ¼ ⌊M−X⌋þks; X ¼
n 1 n ∑ j¼1 x j ;
sffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi 2 n ∑ j¼1 x j −X ; RSD ¼ 100s=X s¼ n−1
(B) Reference value M I. T ≤ 101.5 i. XN101:5; then M ¼ 101:5 ii. Xb98:5; then M ¼ 98:5 iii. 98:5≤ X ≤101:5; then M ¼ X II. T N 101.5 i. XNT; then M ¼ T
Then, the core tablets were coated with 10% (w/v) aqueous dispersion of Opadry® (03B64610, Colorcon Inc., PA, USA) using a Hi-Coater® (LDCS-5, Freund-Vector Corporation, IA, USA). The spraying rate of the coating dispersion was 40 g/min. The rotation speed of the coating pan was 8 ± 2 rpm. The inlet and outlet temperatures of air-flow were 75 ± 5 °C and 35 ± 5 °C, respectively. The physical features including the hardness and weight variation were determined using hardness tester (PTB 111E, Pharma Test Apparatebau AG, Hainburg, Germany) and the balance (MS-204, Mettler-Toledo, OH, USA), respectively. 2.9. Determination of content uniformity
ii. Xb98:5; then M ¼ 98:5 iii. 98:5≤ X ≤T; then M ¼ X (C) Acceptance criteria Stage 1 (n = 10, k = 2.4) I. AV ≤ L1 i. xmax ≤ (1 + L2 × 0.01)M ii. xmin N (1 − L2 × 0.01)M (D) Notation AV: acceptance value; X: mean; s: sample standard deviation; RSD: relative standard deviation T: target content percentage unit, expressed as the percentage of the label claim, unless otherwise state, T is 100.0%. xmax: maximal individual value; xmin: minimal individual value LC: label claim L1 = 15 and L2 = 25 unless otherwise specified in the individual monograph
However, double cone mixer (AR403 equipped with DKM, Erweka, Germany) was used for all mixing steps. During the preblending II and blending steps, the mixture was sampled randomly (n = 10 sampling locations) via a thief probe at the specified time points [20]. These samples were assayed by the HPLC method as described above for evaluation of mixing homogeneity. The entecavir 0.5 mg-loaded core tablets were prepared by using the rotary tablet machine (Model D-8, Riva S.A., Buenos Aires, Argentina) with the same tablet size and hardness as in the lab-scale preparation.
The 10 dosage unit samples from the lab-scale batches and pilotscale batch, as well as the commercial product and the mixture samples taken from pilot-scale mixing, were assayed for drug content using the HPLC method as described above. The mean drug content (%) and acceptance value (AV) were calculated in accordance with the formula from USP b905 N content uniformity of dosage units as described in Table 3. 2.10. Dissolution The dissolution test of the film-coated DCET was completed using USP dissolution apparatus II (Shinseang Instrument Co., South Korea) with 900 ml water, 0.1 N HCl solution (pH 1.2), acetate buffer solution (pH 4.0) or phosphate buffer solution (pH 6.8) as a dissolution medium at 37 ± 0.5 °C. The speed of the paddle rotation was 50 rpm. The dissolution test was performed in comparison with the commercial product. At particular time points, 1 ml dissolution medium was withdrawn, filtered (0.45 μm) and assayed for entecavir by the HPLC method as described above. The similarity factor ( f2) and difference factor ( f1) were calculated in accordance with Moore and Flanner's method to compare the dissolution profiles of the film-coated DCET and the commercial product [21]. The similarity factor ( f2) ≥ 50 represents an appropriately comparable dissolution profile [22].
Fig. 1. Particle size distribution of entecavir monohydrate: (A), non-treated; (B), air jet milled.
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A)
305
B)
Fig. 2. Polarized microscopic images of entecavir monohydrate: (A), non-treated; (B), air jet milled.
3. Results and discussion Entecavir (0.5 mg once daily dose) remarkably inhibits replication of HBV [4]. Baraclude® 0.5 mg tablet (Bristol-Myers Squibb, New York, USA) is the commonly prescribed commercial product of entecavir [8, 9]. The content uniformity is a critical issue of the potent drugs [10]. Suitable content uniformity is very important for the safety and efficacy
of a product containing a low-dose API [11–14]. The present research is an endeavor to promote the content uniformity of entecavir in tablet dosage form. As compared to non-treated API, the air jet milled API exhibited 3-times smaller in median particle size (Fig. 1). Moreover, the cumulative particle-size distribution data proved that d0.1, d0.5 and d0.9 of the milled vs. non-treated API were 0.93 μm vs. 4.86 μm, 5.13 μm vs.
Fig. 3. Thermal analysis of entecavir monohydrate: (A), differential scanning calorimetric thermograms; (B), thermal gravimetric analysis. The continuous line represents non-treated drug and dotted line represents air jet milled drug.
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12 Mean content Acceptance value
Mean content (%) S.D.
10
9.8
102 100
7.7
8
7.6 6.7
98
6.2
6 5.4
96
4 94
2.7
Acceptance value (AV)
104
2
92 1.0
90
B1
B2
B3
B4
B5
B6
B7
B8
0
Batch number Fig. 4. Entecavir content (mean % ± S.D.) and acceptance value (AV) elicited from labscale batches (B1–B8) according to the USP b905N content uniformity (n = 10).
17.72 μm and 10.15 μm vs. 36.48 μm, respectively. Furthermore, the polarized microscopic examination also affirmed reduced particle size in milled API as compared to non-treated API sample (Fig. 2). Micronization may substantially alter the physical properties such as crystallinity and surface features of a drug substance [23]. Accordingly, investigation of change in the crystalline property from crystalline state to the amorphous form should be carried out [24,25]. In this study, DSC and TGA techniques were used. In DSC analysis, the air jet milled API and non-treated API generated in common an intrinsic peak at 165.2 °C (Fig. 3A). Moreover, other endotherms also appeared in both cases, suggesting that the drug was not converted into the amorphous state. The TGA thermograms of milled API and non-treated API are shown in Fig. 3B. The loss in weight, during heating up to 100 °C, was about 6.04% in both cases, which was very close to the theoretical moisture content (6.09%) of entecavir monohydrate. Thus, DSC results suggested that the drug did not change into the amorphous form in the milling process. Further, TGA results confirmed that entecavir monohydrate retained its water of hydration even after the milling process. In lab-scale preparation, the entecavir 0.5 mg-loaded tablets were obtained via direct compression method using three compositions, F1, F2 and F3, carrying 0%, 0.5% and 1.0% Aerosil® 200 (CSD), respectively (Table 1). Moreover, eight batches (B1–B8) of 1000 tablets each were prepared from these compositions (Table 2). The preparation characteristics from B1 to B8 varied in preblending method, particle-size of API, quantity of CSD (%), number of sieving times and the screen-hole diameter. The content uniformity of each batch was evaluated by the drug content (%) and acceptance value (AV) (Fig. 4). The AV was calculated according to USP b905 N content uniformity criteria given in Table 3. The drug content values for B1, B2, B3, B4, B5, B6, B7 and B8 were 96.31 ± 3.57, 98.51 ± 3.22, 99.92 ± 2.79, 99.41 ± 2.81, 99.02 ± 2.59, 98.54 ± 2.26, 99.21 ± 1.12, and 99.33 ± 0.43%, respectively. And, the AV was 9.8, 7.7, 7.6, 6.7, 6.2, 5.4, 2.7, and 1.0, respectively. The batches
B1, B2 and B3 were prepared according to formulation F1 (Table 1). In the preparation of B1, preblending and sieving were excluded. The batch B2 provided better mean drug content (n = 10) and AV than those of B1 (Fig. 4). This suggested that preblending and sieving improved the content uniformity. Likewise, the mean drug content and AV for B3 were slightly improved as compared to B2 (Fig. 4). This suggested that sieving again after preblending II also contributed in enhancing the content uniformity. The batches B4 and B5 were prepared according to formulation F2 (Table 1). In these formulations, 0.5% CSD was used as a glidant to improve the flowability of the powder mixtures [26,27]. As compared with the AV of B2 and B3, the improved AV of B4 and B5 was due to the addition of 0.5% CSD in the formulations. Moreover, the AV of B5 was better than that of B4 (Fig. 4). This proved that the number of times of sieving was also important in improving the content uniformity due to further homogeneity [28,29]. The batches B6, B7 and B8 were prepared in accordance with formulation F3 (Table 1). In these batches, 1.0% CSD was used as a glidant to further improve the flow property of the powder mixtures [28,29]. The acceptance values of these batches were better than AV of B5 and B6 (Fig. 4). As compared to B5, the batch B6 showed better AV. This suggested that increased quantity of CSD (0.5% vs. 1.0%) further improved the content uniformity. The batches B6 and B7 demonstrated the influence of particle size of the API on enhancing the content uniformity. The API with median particle size of 17.72 μm and 5.13 μm was used in batches B6 and B7, respectively. The batch B7 gave about 2-times better AV than that of batch B6. This revealed that the smaller particle size of the drug improved the content uniformity owing to improved mixing [30,31]. The batches B7 and B8 exhibited the role of screen-hole diameter in increased content uniformity. The AV of B8 was approximately 3-times better than that of B7 (Fig. 4). This was because of sieving B8 through the screen having comparatively smaller hole-size (300 μm vs. 600 μm). This suggested that exclusion of larger particles considerably improved the AV of B8. Thus, the batch B8 showed the excellent AV (1.0) among all the batches and was chosen for pilot-scale production of entecavir 0.5 mg-loaded direct compression tablets. The pilot-scale production was conducted in order to establish the reliability of the whole process employed in the lab-scale preparation of B8. The composition and preparation method of this batch were exactly the same as B8 (Tables 1, 2). However, the batch size of pilotscale production was 10-times larger than that of the lab-scale preparation (10,000 vs. 1000 tablets). Moreover, a double cone mixer was used in the pilot-scale production instead of a cube mixer. The tablet compression was accomplished with a rotary tablet machine. The optimum mixing time in both preblending II and blending cases was 30 min (Fig. 5). At the optimum mixing time, the mixture samples presented the best AV of 1.74 and 2.05, respectively. The comparison of quality control characteristics of the film-coated pilot-scale DCET such as weight variation, hardness, drug content and
Table 4 General properties of film-coated DCET and commercial product. Properties
Film-coated DCET
Commercial tablet
Weight (mg)a Hardness (KP)a Content (%)a Content uniformity (AV)b Content difference % highest–lowestc
205.2 ± 1.58 9.3 ± 1.10 99.35 ± 0.54 1.3 1.9
205.7 ± 2.04 8.5 ± 1.60 100.43 ± 1.58 3.8 5.5
a
Each value represents the mean ± S.D. (n = 10). Calculated using the formula in Table 3. Range between the highest content % of unit tablet and the lowest content % of unit tablet. b c
Fig. 5. Acceptance value (AV) of powder mixtures sampled in the preblending I and blending step (n = 10).
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307
Fig. 6. Dissolution profiles of drug from the entecavir 0.5 mg-loaded film-coated tablet and commercial product: (A), pH 1.2; (B), pH 4.0; (C), pH 6.8; (D), water. Each value denotes the mean ± S.D. (n = 6).
AV with those of the commercial product is summarized in Table 4. The weight variation, hardness and drug content were not significantly different (P N 0.05) from each other (n = 10). The acceptance value (AV) of the film-coated pilot-scale DCET was 3-times better than that of the commercial product (1.3 vs. 3.8). Likewise, the content difference % highest–lowest for the film-coated DCET was approximately 3-times narrower than that of the commercial product (1.9 vs. 5.5%). As a result, the film-coated pilot-scale DCET showed enhanced content uniformity. The enhanced content uniformity of this tablet might be attributed to the excellent homogeneity of the final mixture obtained from micronization of API, preblending I, preblending II and sieving through smaller screen-hole diameter [32]. The dissolution of film-coated DCET in pH 1.2, 4.0, 6.8 and water is compared to that of the commercial product in Fig. 6. In all dissolution media, about 90% of the incorporated entecavir dissolved from both products within 15 min, with comparable dissolution profiles. The similarity ( f2) and difference factors ( f1) calculated from the dissolution profiles in each medium are given in Table 5 [21]. A similarity factor ( f2) ≥ 50 designates a suitably similar dissolution profile [22]. In the present research, all the dissolution profiles resulted in similarity factor ( f2) N 50. Accordingly, the dissolution behavior of the film-coated DCET
Table 5 Dissolution comparison of film-coated DCET and commercial product. Dissolution medium
Similarity factor ( f2) Difference factor ( f1)
pH 1.2
pH 4.0
pH 6.8
H2O
78 2
89 1
77 3
60 6
was considered as similar to that of the commercial product. This suggested that the film-coated tablet might be bioequivalent to the commercial product. 4. Conclusions In lab-scale preparation, entecavir 0.5 mg-loaded tablets of batch B8 formulated with F3 composition furnished the excellent content uniformity (AV = 1.0). In pilot-scale production, the optimized composition and process demonstrated consistent content uniformity. The content uniformity of the film-coated DCET was improved as compared to the commercial product (1.3 vs. 3.8). Moreover, the dissolution profile of film-coated DCET was similar to that of the commercial product in all dissolution media ( f2 N 50). Thus, the film-coated DCET with enhanced content uniformity might be bioequivalent to the commercial product. Acknowledgment This work was supported by the 2014 Inje University research grant. References [1] W.M. Lee, Hepatitis B virus infection, N. Engl. J. Med. 337 (1997) 1733–1745. [2] T. Poynard, M.-F. Yuen, V. Ratzin, C.L. Lai, Viral hepatitis C, Lancet 362 (2003) 2095–2100. [3] M. Sherman, C. Yurdaydin, J. Sollano, M. Silva, Y.F. Liaw, J. Cianciara, A. BoronKaczmarska, A. Martin, Z. Goodman, R. Colonno, Entecavir for treatment of lamivudine-refractory, HBeAg-positive chronic hepatitis B, Gastroenterology 130 (2006) 2039–2049. [4] Y.-F. Liaw, N. Leung, J.-H. Kao, T. Piratvisuth, E. Gane, K.-H. Han, R. Guan, G.K. Lau, S. Locarnini, Asian-Pacific consensus statement on the management of chronic hepatitis B: a 2008 update, Hepatol. Int. 2 (2008) 263–283.
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