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Release Behavior of Tanshinone IIA Sustained-Release Pellets Based on Crack Formation Theory
Release Behavior of Tanshinone IIA Sustained-Release Pellets Based on Crack Formation Theory PAN LIU, JIN LI, JIANPING LIU, JIKUN YANG, YONGQING FAN Department of Pharmaceutics, China Pharmaceutical University, Nanjing 210009, China Received 12 January 2012; revised 29 March 2012; accepted 27 April 2012 Published online 18 May 2012 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/jps.23199 ABSTRACT: The objective of this study was to investigate the drug release mechanism and in vivo performance of Tanshinone IIA sustained-release pellets, coated with blends of polyvinyl acetate (PVAc) and poly(vinyl alcohol)–poly(ethylene glycol) (PVA–PEG) graft copolymer. A formulation screening study showed that pellets coated with PVAc–PVA–PEG at a ratio of 70:30 (w/w) succeeded in achieving a 24 h sustained release, irrespective of the coating weight (from 2% to 10%). Both the microscopic observation and mathematical model gave further insight into the underlying release mechanism, indicating that diffusion through water-filled cracks was dominant for the control of drug release. In vivo test showed that the maximum plasma concentration of sustained-release pellets was decreased from 82.13 ± 17.05 to 40.50 ± 11.72 ng mL as that of quick-release pellets. The time of maximum concentration, half time, and mean residence time were all prolonged from 3.80 ± 0.40 to 8.02 ± 0.81 h, 4.28 ± 1.21 to 8.18 ± 2.06 h, and 8.60 ± 1.59 to 17.50 ± 2.78 h, compared with uncoated preparations. A good in vitro–in vivo correlation was characterized by a high coefficient of determination (r = 0.9772). In conclusion, pellets coated with PVAc–PVA–PEG could achieve a satisfactory sustained-release behavior based on crack formation theory. © 2012 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 101:2811–2820, 2012 Keywords: crack formation; oral drug delivery; coating; sustained release; pellets; Kollicoat; microscopic observation; mathematical model; pharmacokinetics; in vitro–in vivo correlations
INTRODUCTION Tanshinone IIA (TA), one of the main bioactive components isolated from the root of Salvia miltiorrhiza Bunge, has been widely used in the treatment of cardiovascular disease for its vasorelaxation and cardioprotective effects.1–3 In our previous studies, TA ternary solid dispersion (tSD) pellets were prepared to improve the dissolution rate and oral bioavailability of the drug.4 However, many cardiovascular diseases such as coronary artery disease and angina pectoris may need long-term therapy.5 The less frequent administration and plasma drug concentration fluctuations are accepted to reduce side effects and improve patient compliance.6,7 Consequently, developing from our previous studies, TA tSDs immediate-release pellets were encased in a sustained-release layer by uti-
Correspondence to: Jianping Liu (Telephone: +86-25-83271293; Fax: +86-25-83271293; E-mail: [email protected]) Pan Liu and Jin Li contributed equally to this work. Journal of Pharmaceutical Sciences, Vol. 101, 2811–2820 (2012) © 2012 Wiley Periodicals, Inc. and the American Pharmacists Association
lizing fluid-bed coating technology to form once-a-day preparations. Polymeric film coating of pellets is a process commonly performed in order to modify drug release characteristics.8,9 In recent years, the influence of the coating composition on drug release was intensively investigated in a majority of publications,10–13 whereby blends of polymers with different permeability have become a focus of interest.14–16 In this study, the polyvinyl acetate (PVAc) aqueous disperR SR30D), an insoluble film sion (trade name Kollicoat coating polymer for sustained-release applications,17 as well as poly(vinyl alcohol)–poly(ethylene glycol) R (PVA–PEG) graft copolymer (trade name Kollicoat IR), a soluble film coating polymer for immediaterelease applications, were taken into account.18 Coating composition with the blend of PVAc and PVA–PEG have demonstrated several advantages, such as a drug release independent of pH and ionic strength, a high resistance to mechanical stress, and a flexible adjustment of drug release by varying blend ratios.19,20 Numerous studies concerning on the drug release characteristic and mechanism of PVAc–PVA–PEG
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film-coated pellets have been carried out. Research work by Ensslin et al.21 reported the blending ratio and the film coat thickness had a major impact on drug release, and showed that the water penetration was the rate-limiting step in drug release progress. In contrast to this viewpoint, Ho et al.22 observed that coating thickness did not influence drug release rate, which indicated that the driving force of drug diffusion induced by the saturated drug solution at the inner face of coating film was strong enough to surpass the effect of the diffusion path length on the drug release. Drug release from a coated formulation could occur by diffusion through intact macromolecular membrane or water-filled paths in the film. These paths might be pores produced in the coating by the leaching of water-soluble polymer, or cracks created in the coating by the build-up of the hydrostatic pressure inside the pellet23 (when the mechanical stability of the polymeric membrane is insufficient to withstand the pressure, stressing the coating). The hydrostatic pressure generated upon water penetration into the pellet core and the mechanical stability of film determined whether or not crack formation in the polymeric membrane was the predominant release mechanism. In our published paper, the model drug of TA, combined with Polyvinylpyrrolidone (PVP)–poloxamer 188 as dispersing carries, has been layered onto starter cores to form the TA tSDs pellets. Considering the hydrophily and tackiness of PVP,24 TA tSDs sustained-release pellets coated with PVAc–PVA–PEG may exhibit different release characteristics from the reported results before. The aim of the current study was to explore the underlying drug release mechanism from PVAc– PVA–PEG film-coated TA tSDs pellets using both the microscopic observation and mathematical modeling methods. Besides, the in vivo performance and in vitro–in vivo correlation (IVIVC) were carried out to evaluate the final preparation.
MATERIALS AND METHODS Materials Tanshinone IIA (98.63%) was purchased from Xi’an Honson Biotechnology Company, Ltd. (Xi’an, Shaanxi, China) TA standard was purchased from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). Sugar spheres (0.75–0.85 mm) were from JRS Pharma (Rosenberg, Germany). PVP-K29/32 was kindly donated from China Division, ISP Chemicals Company R II was supplied by Col(Shanghai, China). Opadry orcon Coating Technology, Ltd. (Shanghai, China). R R F68), PVAc (Kollicoat Poloxamer 188 (Pluronic R SR30D), and PVA–PEG (Kollicoat IR) graft copolyJOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 101, NO. 8, AUGUST 2012
mer were received from BASF (Ludwigshafen, Germany). Talc (1200 mesh) was delivered from MerckSchuchardt (Hohenbrunn, Germany). 1,2-Propylene glycol was obtained from Shanghai Chemical Agent Company, Ltd. (Shanghai, China). All reagents were of analytical grade except methanol and acetonitrile, which were of chromatographic grade. Animals Healthy male New Zealand rabbits (body weight 2.0 ± 0.9 kg) were purchased from Experimental Animal Center of China Pharmaceutical University (Nanjing, China). Prior to the experiments, the rabbits were housed in a temperature and humidity controlled room (23◦ C, 55% air humidity) with free access to water and standard rabbit chow. The rabbits were acclimated for at least 5 days and fasted overnight but supplied with water ad libitum before the experiments. All experiments were approved by the Institutional Animal Care and Use Committee of China Pharmaceutical University. Preparation of TA Sustained-Release Pellets
TA Solid Dispersion-Layered Pellets Deposition of TA–PVP–poloxamer 188 on the sugar cores was performed in a fluid-bed granulator and coater (JHQ-100; Shenyang, China). First, a fixed ratio of TA–PVP–poloxamer 188 (1:4:1) was dissolved in a solvent mixture of ethyl acetate–anhydrous ethanol (5:1, v/v) under continuous stirring till a homogeneous solution was obtained. The solution was then sprayed onto the sugar cores (50 g) from a nozzle attached to a peristaltic pump (HL-2; Shanghai, China) under the condition of 35◦ C–37◦ C coating temperature, 1.0–1.2 mL min−1 spray rate, 1.5–1.6 bar atomization pressure, 100–150 mL min−1 the rate of air blow, and 1.0 mm nozzle diameter. After drug/carriers layering, the pellets were dried for a further 15 min at 40◦ C in the coating chamber. The resulting pellets were kept in the container until further processing.
Film-Coated Pellets The water dispersion of Opadry II with 20% solid content was sprayed onto TA tSDs pellets as isolated coating film. For membrane-controlled coating, R SR30D [30% the PVAc aqueous dispersion Kollicoat (w/w) solids content, referred to the total dispersion mass] was plasticized overnight with 1,2-propylene glycol [2.5% (w/w) based on the polymer weight]. The talcum [25% (w/w) based on the polymer weight] suspension was then stirred into the polymer suspension. Then, various amounts of the aqueous PVA–PEG R IR were added to prepare graft copolymer Kollicoat a suspension of 15% (w/w) total solid content. Pellets of 50 g were used for each coating operation in a fluid-bed granulator and coater (JHQ-100; DOI 10.1002/jps
TANSHINONE IIA SUSTAINED-RELEASE PELLETS BASED ON CRACK FORMATION THEORY
Shenyang, China). Before the film coating procedure, the blend solution was sieved with an 80 mesh. During the entire spraying process, the spray suspension was continuously stirred. The coating parameters were as follows: coating temperature 38◦ C–40◦ C, spray rate 0.6–0.8 mL min−1 , atomization pressure 0.8–1.2 bar, the rate of air blow 120–180 mL min−1 , and nozzle diameter 0.8 mm. After coating, the pellets were further fluidized for 10 min and subsequently cured for 24 h at 60◦ C in an oven. A series of coated preparations were produced with different PVAc– PVA–PEG blend ratios (90:10, 85:15, 70:30, 60:40, and 50:50) and coating weights (from 2% to 10%). The samples were accurately weighted before (Wa ) and after (Wb ) coating; the coating weight was then calculated using equation F = (Wa − Wb )/Wb × 100%. The drug’s content in the pellets after coating was 3.68%.
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Analysis of Release Data Difference in the release profiles was assessed using the model-independent approach based on the similarity factor (f2 ), a criterion for dissolution comparison recommended by US Food and Drug Administration. Thef2 equation was shown as follows: ⎧ ⎫ −0.5 n ⎨ ⎬ 1 f 2 = 50 log 1+ (Rt − Tt)2 × 100 (1) ⎩ ⎭ n t=1
where n is the number of time points, Rt and Tt are the reference and test dissolution values at time point. Thef2 is a popular measurement used to compare two drug dissolution curves, which were evaluated similarity on the values of 50 or above. Release Mechanism Study
In Vitro Release Study
Microscopic Observation
Quantitative Analysis of TA
Drug release from single TA sustained-release pellet was observed at 37 ± 0.5◦ C under a sink condition in 6 mL of distilled water containing 0.5% sodium dodecyl sulfate in agitated glass vial (80 rpm, horizontal shaker, SHY-2A; Jiangsu, China).15 At predetermined time intervals, glass vial was taken out of water bath onto the viewing platform of the optical inverted microscope (Nikon Eclipse Ti-S; Nikon, Tokyo, Japan) equipped with a Nikon DS-Ri1 camera. The diameter variations and morphology of the coated pellet were monitored in real time to elucidate the underlying drug release mechanism.
The amounts of TA in dissolution medium were quantified by high-pressure liquid chromatography (HPLC) system. The system consisted of a Shimadzu LC-20AB pump, a Shimadzu SIL-20AC autosampler, a Shimadzu SPD-M20A diode array detector (Kyoto, Japan), and a RP C18 column (5 :m, 150 × 4.6 mm2 ; Kromasil, Akzo Nobel, Bohus, Sweden) connected with a C18 security guard column (5 :m, 10 × 4.6 mm2 ; Kromasil, Akzo Nobel, Bohus, Sweden). The mobile phase was methanol–water (90:10, v/v) with a flow rate of 1.0 mL min. The injection volume was 20 :L. The detection wavelength was set at 268 nm.25 The linearity of the method was studied in the concentration range of 0.5–5.0 :g mL−1 (r = 0.9997). The relative standard deviation (RSD) of the intra-day and inter-day precision for TA was less than 2%. The recovery rates for TA were in the range of 98%–102%, and the RSD was below 2%.
Drug Release Test Release experiments were performed using USP34 Apparatus I (ZRS-8G; Tianjin, China; basket method) at 37 ± 0.5◦ C medium temperature and 100 rpm rotation speed (n = 3). The dissolution medium was 900 mL of distilled water containing 0.5% sodium dodecyl sulfate, and perfect sink conditions were maintained throughout the release experiments. Samples containing 2.5 mg of TA were sealed in hard gelatin capsules (Suzhou Capsugel Ltd., Suzhou, China) with a manual capsule-filling machine (CapsulCN, Zhejiang, China). At predetermined time intervals, 5 mL samples were withdrawn and replaced by fresh medium. The samples were filtered through 0.22 :m filter and analyzed by HPLC for TA as described above. DOI 10.1002/jps
Mathematical Modeling The modeling was used to identify an appropriate mathematical model and quantitatively predict the effects of best selected coating formulation (30% PVA–PEG) on the resulting drug release kinetics from the investigated pellets. Drug release data were analyzed according to zero-order (2), firstorder (3), Higuchi26 (4), Korsmeyer–Peppas27 (5), Peppas–Sahlin28 (6), and Baker–Lonsdale29 (7) equations: Mt/M∞ = k0 t
(2)
Mt/M∞ = 1 − exp(−k1 t)
(3)
Mt/M∞ = kH t1/2
(4)
Mt/M∞ = kKP tn
(5)
Mt/M∞ = kd tm + kr t2m
(6)
3/2[1 − (1 − Mt/M∞ )2/3 ] − Mt/M∞ = kBL t
(7)
In these equations, Mt /M∞ is the drug released fraction at time t (M∞ is the amount of drug release after JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 101, NO. 8, AUGUST 2012
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infinite time); M0 is the dose of the drug incorporated in the delivery system. k0 is the zero-order release rate constant on Eq. 2, k1 is the first-order release rate constant on Eq 3, kH is the rate constant for Higuchi model on Eq. 4, kKP is the Korsmeyer–Peppas’s kinetic constant and n is the diffusional exponent or release exponent on Eq. 5, kd is the diffusional rate constant and kr is the relaxational rate constant, m is the purely Fickian diffusion exponent on Eq. 6, and kBL is the Baker–Lonsdale’s release rate constant on Eq. 7. The optimum values for the parameters present in each equation were determined by use of DDSolver software.30 The determination coefficient (r) was used to test the applicability of the release models.
In Vivo Study Animal Experiment Healthy male New Zealand rabbits (2.0 ± 0.9 kg) were randomly divided into two groups with six rabbits in each group. Uncoated and coated pellets were filled into size 0 hard gelatin capsules (Suzhou Capsugel Ltd.) with a manual capsule-filling machine (CapsulCN). Capsules were orally administered with a dose of 30 mg kg to the rabbits, which were fasted overnight with water freely available. At each time point, 1.5 mL of blood was collected into heparinized tubes. Plasma samples were obtained after centrifugation at 600 g for 10 min and stored at −20◦ C until analysis.
Plasma Sample Preparation and Method Validation A single-step precipitation protein procedure was adopted to extract TA from rabbit plasma. Rabbit plasma (200 :L) was thoroughly mixed with 400 :L acetonitrile and then vortexed for 3 min to fully precipitate protein. The mixture was centrifuged at 600 g for 10 min. The supernate was taken and evaporated to dryness under a stream of nitrogen at 40◦ C water bath. Then, the residue was resuspended in 200 :L methanol and centrifuged at 9600 g for 10 min. Aliquots (20 :L) of the supernate were injected into the HPLC system for analysis.
Data Analysis and Statistics All pharmacokinetic parameters, including maximum plasma concentration (Cmax ), time of maximum concentration (Tmax ), half time (t1/2 ), mean residence time (MRT) and area under the plasma concentrationtime curve (AUC0–t and AUC0–∞ ) were calculated by compartmental analysis using the software program PKSolver.31,32 Comparison between TA sustainedrelease pellets and immediate-release pellets were made with two-tailed Student’s t-tests. Statistical significance was determined at p < 0.05 level. The relaJOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 101, NO. 8, AUGUST 2012
Figure 1. Diagram showing the structure of TA sustained-release pellets.
tive bioavailability was evaluated using equation F = AUCtext /AUCreference × 100%.
In Vitro–In Vivo Correlation In vitro–in vivo correlation model has been widely applied to describe the relationship between in vitro release data and in vivo pharmacokinetics.33 In current study, a multiple level C correlation34 was investigated to relate the mean percent released at several time points (4, 6, 8, 10, 12, and 24 h) as the in vitro parameters and corresponding AUC0–t values as the in vivo parameters. The time points above were chosen as representative of the early, middle, and late stages of the release profile.
RESULTS AND DISCUSSION Structure of TA Sustained-Release Pellets As illustrated in Figure 1, TA sustained-released pellets from inner to outer were composed of three layers: sugar starter cores were used as substrate to load innermost TA tSDs layer; the middle layer using Opadry II as an isolated coating film (5% coating weight) was applied to prevent drug migration from the interior of pellets to the polymer films; and the outermost layer was PVAc–PVA–PEG polymer blends, which could adjust the drug release flexibly by varying blend ratios and coating weight. Effect of PVAc–PVA–PEG Blend Ratio Release behaviors of TA sustained-release pellets coated with different blend ratios of PVAc–PVA–PEG at the same coating weight of 5% were evaluated in vitro. As shown in Figure 2, the release rate of TA from pellets increased with the ratio of PVAc to PVA–PEG until 85:15. But interestingly, progressive enlargement of the proportion of PVA–PEG made the release profiles consistent, which was confirmed by the similar factor f2 analysis that the release DOI 10.1002/jps
TANSHINONE IIA SUSTAINED-RELEASE PELLETS BASED ON CRACK FORMATION THEORY
Figure 2. Release profiles of TA pellets coated with different blend ratios of PVAc/PVA-PEG at the same coating weight of 5% (n = 3).
behaviors of 60:40 and 50:50 were similar to that of 70:30 with f2 of 56.66 and 58.65. The similar release behavior of PVAc–PVA–PEG film-coated pellets described above (upon 70:30) was different with published results of other research in this area.21,35 There were two reasons given as follows: First, when the amounts of higher permeable PVA–PEG were greater than 30%, the dramatically increased rate and extent of water uptake through film coating promoted the water content of lower permeable PVAc beyond its polymer-specific water concentration. A resulting “polymer chain relaxation” phenomenon would be observed and remarkably enhance the mobility of polymer macromolecules.36,37 This decreasing mechanical stability of the polymer film facilitated the formation of cracks and flaws within the coating layer through which the drug could rapidly diffuse out. Second, in our study, a high hydrophilic polymer PVP was used as the carrier of TA tSDs deposited onto the sugar cores. After contacting with dissolution medium, the significant hydrostatic pressure of PVP24 built-up within the pellet could also stress the relaxed polymer film from inside. When the mechanical stability of polymer chain was insufficient to withstand the inner pressure, pellet coating began to rupture and diffusion through cracks gradually governed drug release process.15,38,39 The mechanical stability of the film coatings and the hydrostatic pressure generated upon water penetration into the pellet core determined whether or not crack formation in the polymeric membrane occurs, which has been investigated by Muschert et al.14,15 studies. Consequently, the “polymer chain relaxation” phenomenon combined with the highly soluble PVP was responsible for the drug release that weekly influenced by the DOI 10.1002/jps
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blend ratio when PVA–PEG content was above 30% proportion. As it could be seen in Figure 2, at 90:10 and 85:15 blend ratios of PVAc–PVA–PEG, pellets exhibited a slow increasing tendency with only approximate 50% drug release at 24 h. More time was probably required for the dissolution medium to diffuse through the coating due to the relatively low amounts of PVA–PEG in coating formulation. Meanwhile, polymeric membrane with a higher level of PVAc was more flexible40 to prevent formation of breakage or cracks in the coating film. As a result, membrane diffusion-controlled release was speculated to be the major rate-limiting process in drug release. In addition, the adhesive strength between the TA-tSDs layer and the inner face of coating film may be an obstacle to the diffusion of dissolved drug due to the tackiness of PVP,24 resulting in an incomplete drug release profile at 24 h. When PVA–PEG content increased over a critical point (30% proportion), the main release mechanism could be assumed to “crack formation” substitute for “membrane diffusion,” followed by a sustained and complete release. Researchers had found that the presence of small amounts of PVA–PEG in aqueous dispersion essentially decreased further polymer particle coalescence during long-term storage and effectively improved film formation owing to its ability to hold water.16,41,42 In this study, although the PVAc–PVA–PEG blend ratio (60:40 and 50:50) exhibited similar release behaviors to 70:30, the much higher water reserve might inversely influence the long-term stability of pellets. Taking this consideration into account, 30% PVA–PEG was the best selection for film coating composition. Effect of Film Coating Thickness Pellets coated with 90:10, 85:15, and 70:30 blend ratios of PVAc–PVA–PEG at different coating weights were investigated respectively. As shown in Figure 3, the increase in the coating weight from 3% to 10% (w/w) resulted in a significant decrease in the release rate of pellets coating with 90:10 and 85:15 blend ratios. A coating level of 3% was still not allowed for around 80% drug release within the final 24 h. This result indicated that the increment in polymer film thickness would create a longer length for dissolved drug to diffuse into the external dissolution medium, which was typical for the characteristic of membrane diffusion-limited transport.43 Although at 70:30 blend ratio of PVAc–PVA–PEG, the release behaviors of pellets at 2%, 3%, 4%, and 10% coating weight were similar to that of 5%, with f2 about 59.54, 60.71, 54.86, and 65.32, respectively (Fig. 4). The release process appeared irrelative with the variation in coating weight, showing that the JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 101, NO. 8, AUGUST 2012
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Figure 4. Effects of the coating weight on drug release from pellets coated with PVAc/PVA-PEG on 70/30 (n = 3).
Figure 3. Effects of the coating weight on drug release from pellets coated with PVAc/PVA-PEG on (a) 90/10 and (b) 85/15 (n = 3).
difference in diffusion path length had no obvious effects on drug release, which cannot be explained purely by the theory of “membrane diffusion” transport. “Crack formation,” therefore, was further confirmed to be the dominant process for TA sustained release and majority of dissolved drug released through the production of cracks within the polymer film. The specific process would be further investigated both in the microscopic observation and mathematical modeling. Microscopic Observation In order to further demonstrate the theory of “crack formation” as the dominant mechanism in drug release, the diameter variations and surface morphology of film-coating pellets were monitored during the release process. As seen in Figure 5, a remarkable diameter increase was detected during the first 2 h. This JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 101, NO. 8, AUGUST 2012
could be attributed to the significant hydrostatic pressure of PVP built-up within the coating film, which made the polymer chains expand perpendicular to the surface and generate an increase in its thickness (diameter). The immediate observation of TA (orange color) release process of pellet was achieved according to color change effects by optical microscopic observation. Judging from the optical micrograph in Figure 6a, a relatively intact surface of polymer film was observed in coated pellet and no obvious drug release occurred in the dissolution medium at the beginning of 2 h. However, the drug release was not delayed in the first 2 h as demonstrated in Figure 4, which implied that the diffusion of dissolved drug through the polymer layer may appear to exist during this period. At 6 h, crack formation could be seen apparently from the arrow direction (Fig. 6b), and majority of dissolved drug released rapidly from the film crack channel. And then, during the release process from 6 to 12 h, with increasing hydrostatic pressure to polymer layer, the crack progressively expanded and induced approximately 80% content (obtained from release data) of TA were released from pellet (Fig. 6c). Finally at 24 h, almost all the residual TA were released to the dissolution medium, and only some fragments of polymer film were remained (Fig. 6d). Mathematical Modeling On the basis of confirmed drug release mechanism, the modeling was applied to indirectly describe the crack formation theory, which was proved adequately through the formulation screening studies and the microscopic observation. As illustrated in Table 1, the value of the Korsmeyer–Peppas release exponent is 0.47, which is between 0.43 and 0.85, indicating an anomalous transport mechanism (non-Fickian DOI 10.1002/jps
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Table 1. Mathematical Modeling and Drug Release Kinetics from Pellets Equation
ra
Qb = 5.28t Qb = 1 − exp (− 0.165t) Qb = 22.798t0.5 Qb = 24.287t0.47 Qb = 20.417t0.72 − 1.05t1.44 3/2[1 − (1 − Qb )2/3 ] − Qb = 0.014t
0.4968 0.9868 0.9588 0.9608 0.9899 0.9546
Mathematical Model Zero order First order Higuchi Korsmeyer-Peppas Peppas-Sahlin Baker-Lonsdale a Determination b Fraction
coefficient. released (Mt /M∞ ).
In Vivo Pharmacokinetic Study Figure 5. Diameter variations of TA pellet coated with 70/30 PVAc/PVA-PEG ratio at 5% coating weight during the first 2 h (n = 6).
diffusion). Furthermore, Peppas–Sahlin equation provided an appropriate contribution of the coexistence between diffusional and relaxational mechanisms in the release process about pellets.28 The determination coefficient of nearly 0.99 suggested that both Peppas–Sahlin and first-order models were appropriate to quantitatively predict the drug release kinetics. As kd is 20 times larger than kr , the diffusional mechanism predominated and relaxation gave a small contribution in the release process.
Tanshinone IIA in plasma could be completely separated under analytical conditions and no significant matrix effect was observed for the analytes in the plasma samples. The standard curve was found to be linear y = 175264x + 324.5 (n = 3, r = 0.9976, where x is concentration of TA and y is the corresponding peak area in the 0.005–0.5 :g mL−1 range). The limit of quantification was 10 ng mL−1 . Intra-day and inter-day variabilities were below 10%. The results attained from the relative recoveries of high, middle, and low concentrations were 107.31 ± 22.56%, 104.63 ± 10.74%, and 98.29 ± 5.35%, respectively. All of the absolute recoveries were above 80%, with all RSD less than 10%, which were within the acceptable limits to meet the guidelines for bioanalytical methods.
Figure 6. Optical micrographs of TA pellet coated with 70/30 PVAc/PVA-PEG ratio at 5% coating weight release in 2 h (a), 6 h (b), 12 h (c), and 24 h (d). DOI 10.1002/jps
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Figure 7. Mean plasma concentration–time curves of TA immediate-release pellets and TA sustained-release pellets in rabbits after oral administration (n = 6).
The pharmacokinetic data of TA immediate-release pellets and TA sustained-release pellets were processed following the open one-compartment model. The concentration versus time profile after oral administration of the two formulations was shown in Figure 7. The main pharmacokinetic parameters were listed in Table 2. Compared with the immediate-release pellets, TA sustained-release pellets had a relatively flatter plasma concentration–time curve and lower degree of concentration fluctuation. There was a statistically significant difference for Cmax and Tmax values between coated and uncoated pellets (p < 0.05). Uncoated pellets resulted in a mean Tmax value of 3.80 h, with 82.13 ng mL serum TA concentration, whereas the coated ones gave a mean Tmax value of 8.02 h, with a TA concentration of 40.50 ng mL. Meanwhile, t1/2 and MRT differed from each other significantly (p < 0.05). The mean t1/2 and MRT values of coated preparations were both prolonged from 4.28 to 8.18 h and from 8.60 to 17.50 h separately, as compared with the uncoated pellets. Furthermore, no significant change in the AUC values between the groups was detected and the relative bioavailability value was 101.5% (p <
0.05). The above results indicated that the favorable TA sustained-release behavior from PVAc–PVA–PEG coated pellets was achieved in vivo.
In Vitro–In Vivo Correlation The IVIVC correlations of TA sustained-release pellets were obtained from the linear regression analysis between AUC0–t values and the mean percentage released. On the basis of the observed relationship for 4, 6, 8, 10, 12, and 24 h time points, a good linear regression relationship between the percent in vitro release and in vivo absorption was constructed with a correlation coefficient r of 0.9772. The high correlations suggested that the conditions for the in vitro release test seemed to mimic very closely the physiological conditions in vivo gastrointestinal tract. The result also indicated that TA release is the main ratelimiting step in its absorption.
CONCLUSION In this study, it has been possible to explain the release behavior of TA sustained-release pellets coated with PVAc–PVA–PEG aqueous dispersions based on
Table 2. Pharmacokinetics Parameters of TA After Oral Administration of Uncoated and Coated Pellets in Rabbits Plasma (n = 6) in a Dose of 30 mg/kg Parameter Cmax (ng/mL) Tmax (h) t1/2 (h) MRT (h) AUC0–t [ng/(h mL)] AUC0–∞ [ng/(h mL)] Relative bioavailability (%)
Immediate Release (Uncoated) Sustained Release (Coated) 82.13 ± 17.05a 3.80 ± 0.40a 4.28 ± 1.21a 8.60 ± 1.59a 899.02 ± 142.39 929.30 ± 150.53 –
40.50 ± 11.72 8.02 ± 0.81 8.18 ± 2.06 17.50 ± 2.78 912.45 ± 144.18 943.53 ± 153.72 101.5
a p < 0.05 are statistical significances with TA sustained-release pellets versus TA immediate-release pellets. Data are expressed as mean ±SD (n = 6).
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DOI 10.1002/jps
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the diffusion through water-filled cracks. The dissolved drug released continuously through the production of cracks and flaws within the polymer film, irrespective of the blend ratio (above 70:30) and the coating weight (in the investigated range). It has been appreciated that the combination of “polymer chain relaxation” phenomenon and the hydrophilic properties of PVP made essential contribution on the formation of breakage or cracks in the coating film, which was confirmed by the results of the microscopic observation and mathematical modeling. In vivo test showed that TA tSDs-coated pellets exhibited a lower Cmax , prolonged Tmax , t1/2 , and MRT compared with uncoated preparations. IVIVC model showed a good linear regression relationship between the percent in vitro release and in vivo absorption.
ACKNOWLEDGMENTS This study is financially supported by the major project of National Science and Technology of China for new drugs development (No.2009ZX09310-004) and Jiangsu province ordinary college and university innovative research programs (No.CX10B-374Z). The authors thank JRS, ISP, Colorcon, and BASF Corporations for providing the excipients and starter cores.
REFERENCES 1. Cheng TO. 2007. Cardiovascular effects of Danshen. Int J Cardiol 121:9–22. 2. Sun DD, Wang HC, Wang XB, Luo Y, Jin ZX, Li ZC, Li GR, Dong MQ. 2008. Tanshinone IIA: A new activator of human cardiac KCNQ1/KCNE1 (IKs) potassium channels. Eur J Pharmacol 590:317–321. 3. Wu GB, Zhou EX, Qing DX. 2009. Tanshinone IIA elicited vasodilation in rat coronary arteriole: Roles of nitric oxide and potassium channels. Eur J Pharmacol 617:102– 107. 4. Li J, Liu P, Liu JP, Zhang WL, Yang JK, Fan YQ. 2012. Novel Tanshinone II A ternary solid dispersion pellets prepared by a single-step technique: In vitro and in vivo evaluation. Eur J Pharm Biopharm 80:426–432. 5. Spertus J. 2001. Testing the effectiveness of converting patients to long-acting antianginal medications: The quality of life in Angina research trial (QUART). Am Heart J 141:550–558. 6. Varum FJO, Merchant HA, Basit AW. 2010. Oral modifiedrelease formulations in motion: The relationship between gastrointestinal transit and drug absorption. Int J Pharm 395:26–36. 7. Shamma RN, Basalious EB, Shoukri RA. 2011. Development and optimization of a multiple-unit controlled release formulation of a freely water soluble drug for once-daily administration. Int J Pharm 405:102–112. 8. McGinity JW. 2008. Aqueous polymeric coatings for pharmaceutical dosage forms. 2nd ed. New York: Marcel Dekker, pp 1997. 9. Borgquist P, Zackrisson G, Nilsson B, Axelsson A. 2002. Simulation and parametric study of a film-coated controlled-release pharmaceutical. J Control Release 80:229–245. DOI 10.1002/jps
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10. Siepmann F, Siepmann J, Walther M, MacRae RJ, Bodmeier R. 2008. Polymer blends for controlled release coatings. J Control Release 125:1–15. 11. Lecomte F, Siepmann J, Walther M, MacRae RJ, Bodmeier R. 2004. Polymer blends used for the coating of multiparticulates: Comparison of aqueous and organic coating techniques. Pharm Res 21:882–890. 12. Lecomte F, Siepmann J, Walther M, MacRae RJ, Bodmeier R. 2004. Polymer blends used for the aqueous coating of solid dosage forms: Importance of the type of plasticizer. J Control Release 99:1–13. 13. Siepmann F, Siepmann J, Walther M, MacRae RJ, Bodmeier R. 2005. Blends of aqueous polymer dispersions used for pellet coating: Importance of the particle size. J Control Release 105:226–239. 14. Muschert S, Siepmann F, Leclercq B, Carlin B, Siepmann J. 2009. Prediction of drug release from ethylcellulose coated pellets. J Control Release 135:71–79. 15. Muschert S, Siepmann F, Leclercq B, Carlin B, Siepmann J. 2009. Drug release mechanisms from ethylcellulose: PVAPEG graft copolymer-coated pellets. Eur J Pharm Biopharm 72:130–137. 16. Muschert S, Siepmann F, Cuppok Y, Leclercq B, Carlin B, Siepmann J. 2009. Improved long term stability of aqueous ethylcellulose film coatings: Importance of the type of drug and starter core. Int J Pharm 368:138–145. 17. Dashevsky A, Wagner K, Kolter K, Bodmeier R. 2005. Physicochemical and release properties of pellets coated with R Kollicoat SR 30 D, a new aqueous polyvinyl acetate dispersion for extended release. Int J Pharm 290:15–23. R ¨ 18. Buhler V. 2007. Kollicoat grades. Functional polymers for the pharmaceutical industry. Ludwigshafen, Germany: BASF Pharma Solutions, pp 15–65. 19. Mies S, Meyer K, Kolter K. 2004. Correlation of drug permeation through isolated films and coated dosage forms based on Kollicoat SR 30D/IR. AAPS Annual Meeting and Exposition: Baltimore, Maryland, pp 7–11. 20. Meyer K, Kolter K. 2004. Reliability of drug release from R an innovative single unit Kollicoat drug delivery system.International Symposium on Controlled Release of Bioactive Materials: Honolulu, Hawaii, pp 12–16. ¨ 21. Ensslin S, Moll KP, Metz H, Otz M, Mader K. 2009. Modulating pH-independent release from coated pellets: Effect of coating composition on solubilization processes and drug release. Eur J Pharm Biopharm 72:111–118. 22. Ho L, Cuppok Y, Muschert S, Gordon KC, Pepper M, Shen Y, Siepmann F, Siepmann J, Taday PF, Rades T. 2009. Effects of film coating thickness and drug layer uniformity on in vitro drug release from sustained-release coated pellets: A case study using terahertz pulsed imaging. Int J Pharm 382:151–159. 23. Marucci M, Ragnarsson G, Nilsson B, Axelsson A. 2010. Osmotic pumping release from ethyl-hydroxypropyl-cellulosecoated pellets: A new mechanistic model. J Control Release 142:53–60. 24. Rowe RC, Sheskey PJ, Owen SC, Association AP, Library R. 2003. Handbook of pharmaceutical excipients. Pharmaceutical Press: London, pp 584–589. 25. Zhang WL, Liu JP, Liu XX, Chen ZQ. 2009. Stealth tanshinone IIA-loaded solid lipid nanoparticles: Effects of poloxamer 188 coating on in vitro phagocytosis and in vivo pharmacokinetics in rats. Acta Pharmaceutica Sinica 44:1421–1428. 26. Higuchi T. 1963. Mechanism of sustained-action medication. Theoretical analysis of rate of release of solid drugs dispersed in solid matrices. J Pharm Sci 52:1145–1149. 27. Korsmeyer RW, Gurny R, Doelker E, Buri P, Peppas NA. 1983. Mechanisms of solute release from porous hydrophilic polymers. Int J Pharm 15:25–35. JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 101, NO. 8, AUGUST 2012
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28. Peppas NA, Sahlin JJ. 1989. A simple equation for the description of solute release. III. Coupling of diffusion and relaxation. Int J Pharm 57:169–172. 29. Baker R, Lonsdale H. 1974. Controlled release of biologically active agents. Plenum: New York, pp 15–71. 30. Zhang Y, Huo M, Zhou J, Zou A, Li W, Yao C, Xie S. 2010. DDSolver: An add-in program for modeling and comparison of drug dissolution profiles. AAPS J 12:263–271. 31. Zhang Y, Huo M, Zhou J, Xie S. 2010. PKSolver: An add-in program for pharmacokinetic and pharmacodynamic data analysis in Microsoft Excel. Comput Methods Programs Biomed 99:306–314. 32. Yong Z. 2007. A data analysis program in pharmacokinetics base on microsoft excel—Development and validation of PKSlover 1.0. J Math Med 20:58–61. 33. US FDA Guidance for Industry. 1997. Extended release oral dosage forms: Development, evaluation, and application of in vitro/in -vivo correlations. US Department of Health and Human Services, Food and Drug Administration, Center for Drug Evaluation and Research. 34. Emami J. 2006. In vitro–in vivo correlation: From theory to applications. J Pharm Pharm Sci 9:169–189. ¨ 35. Ensslin S, Moll KP, Paulus K, Mader K. 2008. New insight into modified release pellets—Internal structure and drug release mechanism. J Control Release 128:149–156.
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36. Siepmann J, Siepmann F. 2008. Mathematical modeling of drug delivery. Int J Pharm 364:328–343. 37. Siepmann J, Lecomte F, Bodmeier R. 1999. Diffusioncontrolled drug delivery systems: Calculation of the required composition to achieve desired release profiles. J Control Release 60:379–389. 38. Lecomte F, Siepmann J, Walther M, MacRae RJ, Bodmeier R. 2003. Blends of enteric and GIT-insoluble polymers used for film coating: Physicochemical characterization and drug release patterns. J Control Release 89:457–471. 39. Gunder W, Lippold B, Lippold B. 1995. Release of drugs from ethyl cellulose microcapsules (diffusion pellets) with pore formers and pore fusion. Eur J Pharm Sci 3:203–214. 40. Dashevsky A, Kolter K, Bodmeier R. 2004. Compression of pellets coated with various aqueous polymer dispersions. Int J Pharm 279:19–26. 41. Siepmann F, Hoffmann A, Leclercq B, Carlin B, Siepmann J. 2007. How to adjust desired drug release patterns from ethylcellulose-coated dosage forms. J Control Release 119:182–189. 42. Siepmann F, Muschert S, Leclercq B, Carlin B, Siepmann J. 2008. How to improve the storage stability of aqueous polymeric film coatings. J Control Release 126:26–33. 43. Rhodes C, Porter S. 1998. Coatings for controlled-release drug delivery systems. Drug Dev Ind Pharm 24:1139–1154.
DOI 10.1002/jps
INTRODUCTION Tanshinone IIA (TA), one of the main bioactive components isolated from the root of Salvia miltiorrhiza Bunge, has been widely used in the treatment of cardiovascular disease for its vasorelaxation and cardioprotective effects.1–3 In our previous studies, TA ternary solid dispersion (tSD) pellets were prepared to improve the dissolution rate and oral bioavailability of the drug.4 However, many cardiovascular diseases such as coronary artery disease and angina pectoris may need long-term therapy.5 The less frequent administration and plasma drug concentration fluctuations are accepted to reduce side effects and improve patient compliance.6,7 Consequently, developing from our previous studies, TA tSDs immediate-release pellets were encased in a sustained-release layer by uti-
Correspondence to: Jianping Liu (Telephone: +86-25-83271293; Fax: +86-25-83271293; E-mail: [email protected]) Pan Liu and Jin Li contributed equally to this work. Journal of Pharmaceutical Sciences, Vol. 101, 2811–2820 (2012) © 2012 Wiley Periodicals, Inc. and the American Pharmacists Association
lizing fluid-bed coating technology to form once-a-day preparations. Polymeric film coating of pellets is a process commonly performed in order to modify drug release characteristics.8,9 In recent years, the influence of the coating composition on drug release was intensively investigated in a majority of publications,10–13 whereby blends of polymers with different permeability have become a focus of interest.14–16 In this study, the polyvinyl acetate (PVAc) aqueous disperR SR30D), an insoluble film sion (trade name Kollicoat coating polymer for sustained-release applications,17 as well as poly(vinyl alcohol)–poly(ethylene glycol) R (PVA–PEG) graft copolymer (trade name Kollicoat IR), a soluble film coating polymer for immediaterelease applications, were taken into account.18 Coating composition with the blend of PVAc and PVA–PEG have demonstrated several advantages, such as a drug release independent of pH and ionic strength, a high resistance to mechanical stress, and a flexible adjustment of drug release by varying blend ratios.19,20 Numerous studies concerning on the drug release characteristic and mechanism of PVAc–PVA–PEG
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film-coated pellets have been carried out. Research work by Ensslin et al.21 reported the blending ratio and the film coat thickness had a major impact on drug release, and showed that the water penetration was the rate-limiting step in drug release progress. In contrast to this viewpoint, Ho et al.22 observed that coating thickness did not influence drug release rate, which indicated that the driving force of drug diffusion induced by the saturated drug solution at the inner face of coating film was strong enough to surpass the effect of the diffusion path length on the drug release. Drug release from a coated formulation could occur by diffusion through intact macromolecular membrane or water-filled paths in the film. These paths might be pores produced in the coating by the leaching of water-soluble polymer, or cracks created in the coating by the build-up of the hydrostatic pressure inside the pellet23 (when the mechanical stability of the polymeric membrane is insufficient to withstand the pressure, stressing the coating). The hydrostatic pressure generated upon water penetration into the pellet core and the mechanical stability of film determined whether or not crack formation in the polymeric membrane was the predominant release mechanism. In our published paper, the model drug of TA, combined with Polyvinylpyrrolidone (PVP)–poloxamer 188 as dispersing carries, has been layered onto starter cores to form the TA tSDs pellets. Considering the hydrophily and tackiness of PVP,24 TA tSDs sustained-release pellets coated with PVAc–PVA–PEG may exhibit different release characteristics from the reported results before. The aim of the current study was to explore the underlying drug release mechanism from PVAc– PVA–PEG film-coated TA tSDs pellets using both the microscopic observation and mathematical modeling methods. Besides, the in vivo performance and in vitro–in vivo correlation (IVIVC) were carried out to evaluate the final preparation.
MATERIALS AND METHODS Materials Tanshinone IIA (98.63%) was purchased from Xi’an Honson Biotechnology Company, Ltd. (Xi’an, Shaanxi, China) TA standard was purchased from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). Sugar spheres (0.75–0.85 mm) were from JRS Pharma (Rosenberg, Germany). PVP-K29/32 was kindly donated from China Division, ISP Chemicals Company R II was supplied by Col(Shanghai, China). Opadry orcon Coating Technology, Ltd. (Shanghai, China). R R F68), PVAc (Kollicoat Poloxamer 188 (Pluronic R SR30D), and PVA–PEG (Kollicoat IR) graft copolyJOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 101, NO. 8, AUGUST 2012
mer were received from BASF (Ludwigshafen, Germany). Talc (1200 mesh) was delivered from MerckSchuchardt (Hohenbrunn, Germany). 1,2-Propylene glycol was obtained from Shanghai Chemical Agent Company, Ltd. (Shanghai, China). All reagents were of analytical grade except methanol and acetonitrile, which were of chromatographic grade. Animals Healthy male New Zealand rabbits (body weight 2.0 ± 0.9 kg) were purchased from Experimental Animal Center of China Pharmaceutical University (Nanjing, China). Prior to the experiments, the rabbits were housed in a temperature and humidity controlled room (23◦ C, 55% air humidity) with free access to water and standard rabbit chow. The rabbits were acclimated for at least 5 days and fasted overnight but supplied with water ad libitum before the experiments. All experiments were approved by the Institutional Animal Care and Use Committee of China Pharmaceutical University. Preparation of TA Sustained-Release Pellets
TA Solid Dispersion-Layered Pellets Deposition of TA–PVP–poloxamer 188 on the sugar cores was performed in a fluid-bed granulator and coater (JHQ-100; Shenyang, China). First, a fixed ratio of TA–PVP–poloxamer 188 (1:4:1) was dissolved in a solvent mixture of ethyl acetate–anhydrous ethanol (5:1, v/v) under continuous stirring till a homogeneous solution was obtained. The solution was then sprayed onto the sugar cores (50 g) from a nozzle attached to a peristaltic pump (HL-2; Shanghai, China) under the condition of 35◦ C–37◦ C coating temperature, 1.0–1.2 mL min−1 spray rate, 1.5–1.6 bar atomization pressure, 100–150 mL min−1 the rate of air blow, and 1.0 mm nozzle diameter. After drug/carriers layering, the pellets were dried for a further 15 min at 40◦ C in the coating chamber. The resulting pellets were kept in the container until further processing.
Film-Coated Pellets The water dispersion of Opadry II with 20% solid content was sprayed onto TA tSDs pellets as isolated coating film. For membrane-controlled coating, R SR30D [30% the PVAc aqueous dispersion Kollicoat (w/w) solids content, referred to the total dispersion mass] was plasticized overnight with 1,2-propylene glycol [2.5% (w/w) based on the polymer weight]. The talcum [25% (w/w) based on the polymer weight] suspension was then stirred into the polymer suspension. Then, various amounts of the aqueous PVA–PEG R IR were added to prepare graft copolymer Kollicoat a suspension of 15% (w/w) total solid content. Pellets of 50 g were used for each coating operation in a fluid-bed granulator and coater (JHQ-100; DOI 10.1002/jps
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Shenyang, China). Before the film coating procedure, the blend solution was sieved with an 80 mesh. During the entire spraying process, the spray suspension was continuously stirred. The coating parameters were as follows: coating temperature 38◦ C–40◦ C, spray rate 0.6–0.8 mL min−1 , atomization pressure 0.8–1.2 bar, the rate of air blow 120–180 mL min−1 , and nozzle diameter 0.8 mm. After coating, the pellets were further fluidized for 10 min and subsequently cured for 24 h at 60◦ C in an oven. A series of coated preparations were produced with different PVAc– PVA–PEG blend ratios (90:10, 85:15, 70:30, 60:40, and 50:50) and coating weights (from 2% to 10%). The samples were accurately weighted before (Wa ) and after (Wb ) coating; the coating weight was then calculated using equation F = (Wa − Wb )/Wb × 100%. The drug’s content in the pellets after coating was 3.68%.
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Analysis of Release Data Difference in the release profiles was assessed using the model-independent approach based on the similarity factor (f2 ), a criterion for dissolution comparison recommended by US Food and Drug Administration. Thef2 equation was shown as follows: ⎧ ⎫ −0.5 n ⎨ ⎬ 1 f 2 = 50 log 1+ (Rt − Tt)2 × 100 (1) ⎩ ⎭ n t=1
where n is the number of time points, Rt and Tt are the reference and test dissolution values at time point. Thef2 is a popular measurement used to compare two drug dissolution curves, which were evaluated similarity on the values of 50 or above. Release Mechanism Study
In Vitro Release Study
Microscopic Observation
Quantitative Analysis of TA
Drug release from single TA sustained-release pellet was observed at 37 ± 0.5◦ C under a sink condition in 6 mL of distilled water containing 0.5% sodium dodecyl sulfate in agitated glass vial (80 rpm, horizontal shaker, SHY-2A; Jiangsu, China).15 At predetermined time intervals, glass vial was taken out of water bath onto the viewing platform of the optical inverted microscope (Nikon Eclipse Ti-S; Nikon, Tokyo, Japan) equipped with a Nikon DS-Ri1 camera. The diameter variations and morphology of the coated pellet were monitored in real time to elucidate the underlying drug release mechanism.
The amounts of TA in dissolution medium were quantified by high-pressure liquid chromatography (HPLC) system. The system consisted of a Shimadzu LC-20AB pump, a Shimadzu SIL-20AC autosampler, a Shimadzu SPD-M20A diode array detector (Kyoto, Japan), and a RP C18 column (5 :m, 150 × 4.6 mm2 ; Kromasil, Akzo Nobel, Bohus, Sweden) connected with a C18 security guard column (5 :m, 10 × 4.6 mm2 ; Kromasil, Akzo Nobel, Bohus, Sweden). The mobile phase was methanol–water (90:10, v/v) with a flow rate of 1.0 mL min. The injection volume was 20 :L. The detection wavelength was set at 268 nm.25 The linearity of the method was studied in the concentration range of 0.5–5.0 :g mL−1 (r = 0.9997). The relative standard deviation (RSD) of the intra-day and inter-day precision for TA was less than 2%. The recovery rates for TA were in the range of 98%–102%, and the RSD was below 2%.
Drug Release Test Release experiments were performed using USP34 Apparatus I (ZRS-8G; Tianjin, China; basket method) at 37 ± 0.5◦ C medium temperature and 100 rpm rotation speed (n = 3). The dissolution medium was 900 mL of distilled water containing 0.5% sodium dodecyl sulfate, and perfect sink conditions were maintained throughout the release experiments. Samples containing 2.5 mg of TA were sealed in hard gelatin capsules (Suzhou Capsugel Ltd., Suzhou, China) with a manual capsule-filling machine (CapsulCN, Zhejiang, China). At predetermined time intervals, 5 mL samples were withdrawn and replaced by fresh medium. The samples were filtered through 0.22 :m filter and analyzed by HPLC for TA as described above. DOI 10.1002/jps
Mathematical Modeling The modeling was used to identify an appropriate mathematical model and quantitatively predict the effects of best selected coating formulation (30% PVA–PEG) on the resulting drug release kinetics from the investigated pellets. Drug release data were analyzed according to zero-order (2), firstorder (3), Higuchi26 (4), Korsmeyer–Peppas27 (5), Peppas–Sahlin28 (6), and Baker–Lonsdale29 (7) equations: Mt/M∞ = k0 t
(2)
Mt/M∞ = 1 − exp(−k1 t)
(3)
Mt/M∞ = kH t1/2
(4)
Mt/M∞ = kKP tn
(5)
Mt/M∞ = kd tm + kr t2m
(6)
3/2[1 − (1 − Mt/M∞ )2/3 ] − Mt/M∞ = kBL t
(7)
In these equations, Mt /M∞ is the drug released fraction at time t (M∞ is the amount of drug release after JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 101, NO. 8, AUGUST 2012
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infinite time); M0 is the dose of the drug incorporated in the delivery system. k0 is the zero-order release rate constant on Eq. 2, k1 is the first-order release rate constant on Eq 3, kH is the rate constant for Higuchi model on Eq. 4, kKP is the Korsmeyer–Peppas’s kinetic constant and n is the diffusional exponent or release exponent on Eq. 5, kd is the diffusional rate constant and kr is the relaxational rate constant, m is the purely Fickian diffusion exponent on Eq. 6, and kBL is the Baker–Lonsdale’s release rate constant on Eq. 7. The optimum values for the parameters present in each equation were determined by use of DDSolver software.30 The determination coefficient (r) was used to test the applicability of the release models.
In Vivo Study Animal Experiment Healthy male New Zealand rabbits (2.0 ± 0.9 kg) were randomly divided into two groups with six rabbits in each group. Uncoated and coated pellets were filled into size 0 hard gelatin capsules (Suzhou Capsugel Ltd.) with a manual capsule-filling machine (CapsulCN). Capsules were orally administered with a dose of 30 mg kg to the rabbits, which were fasted overnight with water freely available. At each time point, 1.5 mL of blood was collected into heparinized tubes. Plasma samples were obtained after centrifugation at 600 g for 10 min and stored at −20◦ C until analysis.
Plasma Sample Preparation and Method Validation A single-step precipitation protein procedure was adopted to extract TA from rabbit plasma. Rabbit plasma (200 :L) was thoroughly mixed with 400 :L acetonitrile and then vortexed for 3 min to fully precipitate protein. The mixture was centrifuged at 600 g for 10 min. The supernate was taken and evaporated to dryness under a stream of nitrogen at 40◦ C water bath. Then, the residue was resuspended in 200 :L methanol and centrifuged at 9600 g for 10 min. Aliquots (20 :L) of the supernate were injected into the HPLC system for analysis.
Data Analysis and Statistics All pharmacokinetic parameters, including maximum plasma concentration (Cmax ), time of maximum concentration (Tmax ), half time (t1/2 ), mean residence time (MRT) and area under the plasma concentrationtime curve (AUC0–t and AUC0–∞ ) were calculated by compartmental analysis using the software program PKSolver.31,32 Comparison between TA sustainedrelease pellets and immediate-release pellets were made with two-tailed Student’s t-tests. Statistical significance was determined at p < 0.05 level. The relaJOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 101, NO. 8, AUGUST 2012
Figure 1. Diagram showing the structure of TA sustained-release pellets.
tive bioavailability was evaluated using equation F = AUCtext /AUCreference × 100%.
In Vitro–In Vivo Correlation In vitro–in vivo correlation model has been widely applied to describe the relationship between in vitro release data and in vivo pharmacokinetics.33 In current study, a multiple level C correlation34 was investigated to relate the mean percent released at several time points (4, 6, 8, 10, 12, and 24 h) as the in vitro parameters and corresponding AUC0–t values as the in vivo parameters. The time points above were chosen as representative of the early, middle, and late stages of the release profile.
RESULTS AND DISCUSSION Structure of TA Sustained-Release Pellets As illustrated in Figure 1, TA sustained-released pellets from inner to outer were composed of three layers: sugar starter cores were used as substrate to load innermost TA tSDs layer; the middle layer using Opadry II as an isolated coating film (5% coating weight) was applied to prevent drug migration from the interior of pellets to the polymer films; and the outermost layer was PVAc–PVA–PEG polymer blends, which could adjust the drug release flexibly by varying blend ratios and coating weight. Effect of PVAc–PVA–PEG Blend Ratio Release behaviors of TA sustained-release pellets coated with different blend ratios of PVAc–PVA–PEG at the same coating weight of 5% were evaluated in vitro. As shown in Figure 2, the release rate of TA from pellets increased with the ratio of PVAc to PVA–PEG until 85:15. But interestingly, progressive enlargement of the proportion of PVA–PEG made the release profiles consistent, which was confirmed by the similar factor f2 analysis that the release DOI 10.1002/jps
TANSHINONE IIA SUSTAINED-RELEASE PELLETS BASED ON CRACK FORMATION THEORY
Figure 2. Release profiles of TA pellets coated with different blend ratios of PVAc/PVA-PEG at the same coating weight of 5% (n = 3).
behaviors of 60:40 and 50:50 were similar to that of 70:30 with f2 of 56.66 and 58.65. The similar release behavior of PVAc–PVA–PEG film-coated pellets described above (upon 70:30) was different with published results of other research in this area.21,35 There were two reasons given as follows: First, when the amounts of higher permeable PVA–PEG were greater than 30%, the dramatically increased rate and extent of water uptake through film coating promoted the water content of lower permeable PVAc beyond its polymer-specific water concentration. A resulting “polymer chain relaxation” phenomenon would be observed and remarkably enhance the mobility of polymer macromolecules.36,37 This decreasing mechanical stability of the polymer film facilitated the formation of cracks and flaws within the coating layer through which the drug could rapidly diffuse out. Second, in our study, a high hydrophilic polymer PVP was used as the carrier of TA tSDs deposited onto the sugar cores. After contacting with dissolution medium, the significant hydrostatic pressure of PVP24 built-up within the pellet could also stress the relaxed polymer film from inside. When the mechanical stability of polymer chain was insufficient to withstand the inner pressure, pellet coating began to rupture and diffusion through cracks gradually governed drug release process.15,38,39 The mechanical stability of the film coatings and the hydrostatic pressure generated upon water penetration into the pellet core determined whether or not crack formation in the polymeric membrane occurs, which has been investigated by Muschert et al.14,15 studies. Consequently, the “polymer chain relaxation” phenomenon combined with the highly soluble PVP was responsible for the drug release that weekly influenced by the DOI 10.1002/jps
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blend ratio when PVA–PEG content was above 30% proportion. As it could be seen in Figure 2, at 90:10 and 85:15 blend ratios of PVAc–PVA–PEG, pellets exhibited a slow increasing tendency with only approximate 50% drug release at 24 h. More time was probably required for the dissolution medium to diffuse through the coating due to the relatively low amounts of PVA–PEG in coating formulation. Meanwhile, polymeric membrane with a higher level of PVAc was more flexible40 to prevent formation of breakage or cracks in the coating film. As a result, membrane diffusion-controlled release was speculated to be the major rate-limiting process in drug release. In addition, the adhesive strength between the TA-tSDs layer and the inner face of coating film may be an obstacle to the diffusion of dissolved drug due to the tackiness of PVP,24 resulting in an incomplete drug release profile at 24 h. When PVA–PEG content increased over a critical point (30% proportion), the main release mechanism could be assumed to “crack formation” substitute for “membrane diffusion,” followed by a sustained and complete release. Researchers had found that the presence of small amounts of PVA–PEG in aqueous dispersion essentially decreased further polymer particle coalescence during long-term storage and effectively improved film formation owing to its ability to hold water.16,41,42 In this study, although the PVAc–PVA–PEG blend ratio (60:40 and 50:50) exhibited similar release behaviors to 70:30, the much higher water reserve might inversely influence the long-term stability of pellets. Taking this consideration into account, 30% PVA–PEG was the best selection for film coating composition. Effect of Film Coating Thickness Pellets coated with 90:10, 85:15, and 70:30 blend ratios of PVAc–PVA–PEG at different coating weights were investigated respectively. As shown in Figure 3, the increase in the coating weight from 3% to 10% (w/w) resulted in a significant decrease in the release rate of pellets coating with 90:10 and 85:15 blend ratios. A coating level of 3% was still not allowed for around 80% drug release within the final 24 h. This result indicated that the increment in polymer film thickness would create a longer length for dissolved drug to diffuse into the external dissolution medium, which was typical for the characteristic of membrane diffusion-limited transport.43 Although at 70:30 blend ratio of PVAc–PVA–PEG, the release behaviors of pellets at 2%, 3%, 4%, and 10% coating weight were similar to that of 5%, with f2 about 59.54, 60.71, 54.86, and 65.32, respectively (Fig. 4). The release process appeared irrelative with the variation in coating weight, showing that the JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 101, NO. 8, AUGUST 2012
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Figure 4. Effects of the coating weight on drug release from pellets coated with PVAc/PVA-PEG on 70/30 (n = 3).
Figure 3. Effects of the coating weight on drug release from pellets coated with PVAc/PVA-PEG on (a) 90/10 and (b) 85/15 (n = 3).
difference in diffusion path length had no obvious effects on drug release, which cannot be explained purely by the theory of “membrane diffusion” transport. “Crack formation,” therefore, was further confirmed to be the dominant process for TA sustained release and majority of dissolved drug released through the production of cracks within the polymer film. The specific process would be further investigated both in the microscopic observation and mathematical modeling. Microscopic Observation In order to further demonstrate the theory of “crack formation” as the dominant mechanism in drug release, the diameter variations and surface morphology of film-coating pellets were monitored during the release process. As seen in Figure 5, a remarkable diameter increase was detected during the first 2 h. This JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 101, NO. 8, AUGUST 2012
could be attributed to the significant hydrostatic pressure of PVP built-up within the coating film, which made the polymer chains expand perpendicular to the surface and generate an increase in its thickness (diameter). The immediate observation of TA (orange color) release process of pellet was achieved according to color change effects by optical microscopic observation. Judging from the optical micrograph in Figure 6a, a relatively intact surface of polymer film was observed in coated pellet and no obvious drug release occurred in the dissolution medium at the beginning of 2 h. However, the drug release was not delayed in the first 2 h as demonstrated in Figure 4, which implied that the diffusion of dissolved drug through the polymer layer may appear to exist during this period. At 6 h, crack formation could be seen apparently from the arrow direction (Fig. 6b), and majority of dissolved drug released rapidly from the film crack channel. And then, during the release process from 6 to 12 h, with increasing hydrostatic pressure to polymer layer, the crack progressively expanded and induced approximately 80% content (obtained from release data) of TA were released from pellet (Fig. 6c). Finally at 24 h, almost all the residual TA were released to the dissolution medium, and only some fragments of polymer film were remained (Fig. 6d). Mathematical Modeling On the basis of confirmed drug release mechanism, the modeling was applied to indirectly describe the crack formation theory, which was proved adequately through the formulation screening studies and the microscopic observation. As illustrated in Table 1, the value of the Korsmeyer–Peppas release exponent is 0.47, which is between 0.43 and 0.85, indicating an anomalous transport mechanism (non-Fickian DOI 10.1002/jps
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Table 1. Mathematical Modeling and Drug Release Kinetics from Pellets Equation
ra
Qb = 5.28t Qb = 1 − exp (− 0.165t) Qb = 22.798t0.5 Qb = 24.287t0.47 Qb = 20.417t0.72 − 1.05t1.44 3/2[1 − (1 − Qb )2/3 ] − Qb = 0.014t
0.4968 0.9868 0.9588 0.9608 0.9899 0.9546
Mathematical Model Zero order First order Higuchi Korsmeyer-Peppas Peppas-Sahlin Baker-Lonsdale a Determination b Fraction
coefficient. released (Mt /M∞ ).
In Vivo Pharmacokinetic Study Figure 5. Diameter variations of TA pellet coated with 70/30 PVAc/PVA-PEG ratio at 5% coating weight during the first 2 h (n = 6).
diffusion). Furthermore, Peppas–Sahlin equation provided an appropriate contribution of the coexistence between diffusional and relaxational mechanisms in the release process about pellets.28 The determination coefficient of nearly 0.99 suggested that both Peppas–Sahlin and first-order models were appropriate to quantitatively predict the drug release kinetics. As kd is 20 times larger than kr , the diffusional mechanism predominated and relaxation gave a small contribution in the release process.
Tanshinone IIA in plasma could be completely separated under analytical conditions and no significant matrix effect was observed for the analytes in the plasma samples. The standard curve was found to be linear y = 175264x + 324.5 (n = 3, r = 0.9976, where x is concentration of TA and y is the corresponding peak area in the 0.005–0.5 :g mL−1 range). The limit of quantification was 10 ng mL−1 . Intra-day and inter-day variabilities were below 10%. The results attained from the relative recoveries of high, middle, and low concentrations were 107.31 ± 22.56%, 104.63 ± 10.74%, and 98.29 ± 5.35%, respectively. All of the absolute recoveries were above 80%, with all RSD less than 10%, which were within the acceptable limits to meet the guidelines for bioanalytical methods.
Figure 6. Optical micrographs of TA pellet coated with 70/30 PVAc/PVA-PEG ratio at 5% coating weight release in 2 h (a), 6 h (b), 12 h (c), and 24 h (d). DOI 10.1002/jps
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Figure 7. Mean plasma concentration–time curves of TA immediate-release pellets and TA sustained-release pellets in rabbits after oral administration (n = 6).
The pharmacokinetic data of TA immediate-release pellets and TA sustained-release pellets were processed following the open one-compartment model. The concentration versus time profile after oral administration of the two formulations was shown in Figure 7. The main pharmacokinetic parameters were listed in Table 2. Compared with the immediate-release pellets, TA sustained-release pellets had a relatively flatter plasma concentration–time curve and lower degree of concentration fluctuation. There was a statistically significant difference for Cmax and Tmax values between coated and uncoated pellets (p < 0.05). Uncoated pellets resulted in a mean Tmax value of 3.80 h, with 82.13 ng mL serum TA concentration, whereas the coated ones gave a mean Tmax value of 8.02 h, with a TA concentration of 40.50 ng mL. Meanwhile, t1/2 and MRT differed from each other significantly (p < 0.05). The mean t1/2 and MRT values of coated preparations were both prolonged from 4.28 to 8.18 h and from 8.60 to 17.50 h separately, as compared with the uncoated pellets. Furthermore, no significant change in the AUC values between the groups was detected and the relative bioavailability value was 101.5% (p <
0.05). The above results indicated that the favorable TA sustained-release behavior from PVAc–PVA–PEG coated pellets was achieved in vivo.
In Vitro–In Vivo Correlation The IVIVC correlations of TA sustained-release pellets were obtained from the linear regression analysis between AUC0–t values and the mean percentage released. On the basis of the observed relationship for 4, 6, 8, 10, 12, and 24 h time points, a good linear regression relationship between the percent in vitro release and in vivo absorption was constructed with a correlation coefficient r of 0.9772. The high correlations suggested that the conditions for the in vitro release test seemed to mimic very closely the physiological conditions in vivo gastrointestinal tract. The result also indicated that TA release is the main ratelimiting step in its absorption.
CONCLUSION In this study, it has been possible to explain the release behavior of TA sustained-release pellets coated with PVAc–PVA–PEG aqueous dispersions based on
Table 2. Pharmacokinetics Parameters of TA After Oral Administration of Uncoated and Coated Pellets in Rabbits Plasma (n = 6) in a Dose of 30 mg/kg Parameter Cmax (ng/mL) Tmax (h) t1/2 (h) MRT (h) AUC0–t [ng/(h mL)] AUC0–∞ [ng/(h mL)] Relative bioavailability (%)
Immediate Release (Uncoated) Sustained Release (Coated) 82.13 ± 17.05a 3.80 ± 0.40a 4.28 ± 1.21a 8.60 ± 1.59a 899.02 ± 142.39 929.30 ± 150.53 –
40.50 ± 11.72 8.02 ± 0.81 8.18 ± 2.06 17.50 ± 2.78 912.45 ± 144.18 943.53 ± 153.72 101.5
a p < 0.05 are statistical significances with TA sustained-release pellets versus TA immediate-release pellets. Data are expressed as mean ±SD (n = 6).
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DOI 10.1002/jps
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the diffusion through water-filled cracks. The dissolved drug released continuously through the production of cracks and flaws within the polymer film, irrespective of the blend ratio (above 70:30) and the coating weight (in the investigated range). It has been appreciated that the combination of “polymer chain relaxation” phenomenon and the hydrophilic properties of PVP made essential contribution on the formation of breakage or cracks in the coating film, which was confirmed by the results of the microscopic observation and mathematical modeling. In vivo test showed that TA tSDs-coated pellets exhibited a lower Cmax , prolonged Tmax , t1/2 , and MRT compared with uncoated preparations. IVIVC model showed a good linear regression relationship between the percent in vitro release and in vivo absorption.
ACKNOWLEDGMENTS This study is financially supported by the major project of National Science and Technology of China for new drugs development (No.2009ZX09310-004) and Jiangsu province ordinary college and university innovative research programs (No.CX10B-374Z). The authors thank JRS, ISP, Colorcon, and BASF Corporations for providing the excipients and starter cores.
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DOI 10.1002/jps