International Journal of Pharmaceutics 462 (2014) 66–73
Contents lists available at ScienceDirect
International Journal of Pharmaceutics journal homepage: www.elsevier.com/locate/ijpharm
Personalised Medicine
A novel pulsatile drug delivery system based on the physiochemical reaction between acrylic copolymer and organic acid: In vitro and in vivo evaluation Ziwei Zhang a,1 , Xiaole Qi a,1 , Xiangbo Li a , Jiayu Xing a , Xuehua Zhu a , Zhenghong Wu a,b,∗ a b
Key Laboratory of Modern Chinese Medicines, China Pharmaceutical University, Nanjing 210009, PR China Yangtze River Pharmaceutical Group, State Key Laboratory for Advanced Formulation Technologies, Taizhou, PR China
a r t i c l e
i n f o
Article history: Received 27 September 2013 Received in revised form 18 November 2013 Accepted 15 December 2013 Available online 22 December 2013 Keywords: Pulsatile pellets Organic acid Acrylic copolymer Enalapril Maleate Film coating
a b s t r a c t Multilayer-coating technology is the traditional method to achieve pulsatile drug release with the drawbacks of time consuming, more materials demanding and lack of efficiency. The purpose of this study was to design a novel pulsatile drug delivery system based on the physiochemical interaction between acrylic copolymer and organic acid with relatively simpler formulation and manufacturing process. The Enalapril Maleate (EM) pulsatile release pellets were prepared using extruding granulation, spheronization and fluid-bed coating technology. The ion-exchange experiment, hydration study and determination of glass transition temperature were conducted to explore the related drug release mechanism. Bioavailability experiment was carried out by administering the pulsatile release pellets to rats compared with marketed rapid release tablets Yisu® . An obvious 4 h lag time period and rapid drug release was observed from in vitro dissolution profiles. The release mechanism was a combination of both disassociated and undisassociated forms of succinic acid physiochemically interacting with Eudragit® RS. The AUC0- of the EM pulsatile pellets and the market tablets was 702.384 ± 96.891 h ng/mL and 810.817 ± 67.712 h ng/mL, while the relative bioavailability was 86.62%. These studies demonstrate this novel pulsatile release concept may be a promising strategy for oral pulsatile delivery system. © 2013 Elsevier B.V. All rights reserved.
1. Introduction A variety of diseases including hypertension, angina pectoris, epilepsy, asthma, diabetes, hyperchlorhydria and arthritis exhibit circadian variation (Hermida et al., 2007b; Lemmer, 1999; Miyamoto et al., 2004; Roy and Shahiwala, 2009a). With the development of chronotherapy, which refers to a clinical practice of drug delivery consistent with the body’s circadian rhythm (Hermida et al., 2007a; Innominato et al., 2010; Ohdo, 2010; Portaluppi and Lemmer, 2007; Roy and Shahiwala, 2009b) to produce maximum health benefit and minimum harmful effects (Roy and Shahiwala, 2009a), time-controlled release systems for comparative drug efficacy have gained intensive attention worldwide since first introduced. And pulsatile drug delivery system is an important and desirable part among time-controlled release systems. Pulsatile
∗ Corresponding author at: Key Laboratory of Modern Chinese Medicines, China Pharmaceutical University, Nanjing 210009, PR China. Tel.: +86 15062208341; fax: +86 025 83179703. E-mail address: [email protected] (Z. Wu). 1 These authors contributed equally to this work. 0378-5173/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ijpharm.2013.12.026
drug delivery system is characterized by a predetermined lag time period in the starting stage followed by a drug release phase in the drug release profiles (Karavas et al., 2006; Kashyap et al., 2007; Lin et al., 2008; Sungthongjeen et al., 2004; Yadav et al., 2011). Pulsatile release has the advantages of avoiding drug tolerance, matching the release of specific peptides or hormones, and control of tissue (Iskakov et al., 2002) and drug release may be controlled by time, by site or a combination of the two parameters (Yadav et al., 2011). Devices which show pulsatile release upon applying an external trigger such as pH (Déjugnat et al., 2005; Lynn et al., 2001), electric field (Kiser et al., 1998, 2000), IR-light (Angelatos et al., 2005; Radt et al., 2004; Skirtach et al., 2005), etc. have been described. But generally all manufacturing approaches work on the same basic principles of swelling and rupturing (Bussemer and Bodmeier, 2003; Bussemer et al., 2003; Sungthongjeen et al., 2004), erosion (Gazzaniga et al., 1994; McConville et al., 2005) or dissolution, and systems based on changes in membrane permeability (Roy and Shahiwala, 2009a). As to the coating technology, multilayer-coating is the most popular method to achieve pulsatile drug release. However, this method has drawbacks of incomplete drug release and more materials demanding. Moreover, multilayer-coating is time consuming, tedious operating and lack
Z. Zhang et al. / International Journal of Pharmaceutics 462 (2014) 66–73
of efficiency, leading to technical hurdles in commercial scale process. Eudragit® RS is a kind of acrylic copolymer synthesized from acrylic and methacrylic acid esters with a low content of quaternary ammonium groups in the form of chloride. The coating film formed by Eudragit® RS is insoluble in water and has a low permeability but swells in water by incorporating H2 O molecules into its hydrophilic groups. Researchers found that some organic acid solutions can induce the structural change of Eudragit® RS by physiochemical interaction, leading to the changing membrane permeability (Narisawa et al., 1994; Wagner and McGinity, 2002). In order to overcome the drawbacks of conventional multi-layer coating technology for pulsatile pellets, we proposed a novel pulsatile drug delivery system by incorporating organic acid in the core pellets and coating with Eudragit® RS copolymer based on the above principle. Various kinds and amount of organic acid were screened, respectively. Ideally, when water slowly penetrates across Eudragit® RS, organic acid in core pellets interacting with the low amount of quaternary ammonium groups of Eudragit RS copolymer is responsible for changes of the film surface properties, which contributes to drug release after a predetermined lag time period due to its low water permeability. Enalapril Maleate (EM) is an angiotensin-converting enzyme inhibitor and used as an antihypertensive agent. Nowadays, marketed dosage forms of EM are mainly oral rapid release tablets. However, blood pressure in human body presents circadian rhythms – a dramatic morning rise and obvious bedtime decline, both of which act as a push for the chronotherapy of hypertension (Hermida et al., 2007b; Portaluppi and Lemmer, 2007). Therefore, EM was chosen as the model drug. Then patients can take the designated EM dosage form at a proper time in the evening and drug therapeutic concentrations will be achieved the next day in the occurrence of blood pressure morning peak. A novel single-layer coated pulsatile release system, which is meant to contain organic acid in the core and coated with acrylic copolymer, was designed with a relatively simpler formulation and manufacturing process compared to the traditional multilayer coated explosive pulsatile drug release system. In vitro and in vivo evaluation was conducted in contrast with marketed rapid release tablets of Yisu® . The drug release mechanism was demonstrated and investigated through a string of experiment, such as ion-exchange study, hydration research and determination of glass transition temperature. 2. Materials and methods 2.1. Materials Enalapril Maleate (EM) was obtained from Yangtze Pharmaceutical Co., Ltd. (Taizhou, China). Eudragit® RS 30D using as the coating material was supplied by Röhm Pharma (Darmstadt, Germany). Lactose (GranuLac® 200) was obtained from Meggle (Wasserburg, Germany). Five organic acids including succinic acid, dl-tartaric acid, citric acid, malic acid and acetic acid, plus with all other chemicals were used as received and were of standard pharmaceutical grade: HPMC (E5, Colorcon, Orpington, UK), Microcrystalline Cellulose (MCC; Avicel® PH 101, Asahi Kasei. Co., Ltd., Tokyo, Japan) and triethyl citrate (TEC; Aladdin Chemistry Co., Ltd., Shanghai, China). The marketed rapid release tablets Yisu® (Yangtze Pharmaceutical Co., Ltd., Taizhou, China) were used as the reference. 2.2. Methods 2.2.1. Demonstration of feasibility of the mechanism The components of EM, MCC and lactose (10:80:10) were passed through a 200 m sieve to obtain a well-dispersed mixture and wet
67
Table 1 The core and coating layer composition of the Erudragit® RS coated pulsatile release pellets of EM. Core
Coating layer
Continents
Added amount
Continents
Added amount
EM MCC Succinic acid Lactose
10% 42% 38% 10%
Eudragit® RS 30D TEC Talc Water
20% 2% 2% 76%
massed with 3% HPMC aqueous solution as a binder. The soft material was made into strip granules with 1 mm screen by means of an extruding granulator (JBZ-300, New Drug Research Institute of Liaoning Yilian, China), which were then broken into smaller cylindrical rods and rounded into spherical pellets using a high-speed rotating friction machine (JBZ-300, New Drug Research Institute of Liaoning Yilian, China). The extruding and rotating speed were both 385 × g. Then the pellets were dried for 3 h at 40 ◦ C in the oven (DHG-9245A, Shanghai Huiyi Technology Co., Ltd, China). After drying, the core pellets were passed through two sieves of 800 m and 1000 m to remove fine, large and agglomerate particles. The pellets were then coated by spraying with a mixture of Eudragit® RS 30D, talc, TEC, water (20:2:2:76) in a fluid-bed coater (Werner Glatt, Germany). The coating conditions were as follows: spray air pressure, 0.14 MPa; spray solution speed, 1.0 mL/min; inlet temperature, 35 ◦ C; outlet temperature, 30 ◦ C. The coating level was 50%, which can be determined from the following equation (Lemmer, 1999) L (%) =
Mafter − Mbefore Mbefore
× 100
where L represents the gaining weight of coating in terms of percentage, Mbefore and Mafter are the weight of pellets before and after coating, respectively. Release study of the acid unloaded pellets was performed using USP 32 apparatus 1 (basket) with 900 mL of 0.5 mol/L various testing fluid at 37 ◦ C with the basket speed of 100 rpm using a dissolution tester (ZRS-8G, Tianda Tianfa Techenology Co., Ltd., Tianjin, China). Five organic acids including succinic acid, dl-tartaric acid, citric acid, malic acid and acetic acid solution (each 0.5 M) were selected as dissolution medium to demonstrate the feasibility of the principle and find suitable additives using release rate as the evaluation index. Samples through a 45 m filter were taken at preparatory time interval and measured by HPLC (Shimadzu, Kyoto, Japan) with a reverse-phase column (Inertsil ODS-3, 4.6 × 250 mm, i.d. 5 m, GL Sciences, Japan), and UV detection at 207 nm. An acidified (pH 2.2, adjusting with phosphoric acid) aqueous solution of potassium dihydrogen phosphate was used as the mobile phase at a flow rate of 1.0 mL/min. Methodological studies, such as linearity, specificity, precision of with-in and between days were also demonstrated to satisfy the requirements of the methodology. A linear detector response (r = 0.9999) was observed over the concentration range of 1–20 g/mL and blank solvent did not interfere with the determination of EM. 2.2.2. Preparation of pulsatile release pellets The formula of the core pellets and coating solution are shown in Table 1. The experimental conditions and preparing method were referred to organic acid unloaded pellets in Section 2.2.1. For one thing, to optimize extrusion – spheronization process and get the desired particle size range with adequate friability and flow properties, blank (without drug) pellets were prepared with MCC alone initially. Then the optimized process was applied to the
68
Z. Zhang et al. / International Journal of Pharmaceutics 462 (2014) 66–73
preparation of EM loaded pellets. Besides, a progressive set-up of the operating conditions was well required during the coating process (Yadav et al., 2011). The mentioned parameters were varied sequentially on the basis of their influences on film coating in optimization period. Plus, the proportion of components in the coating solution was adjusted so as to balance the desired high coating efficiency and the pellets’ adhesion problem while coating. 2.2.3. In vitro release characteristics of EM pulsatile pellets The release characteristics of EM pulsatile pellets were determined using the USP 32 apparatus I (basket) at a rotation speed of 100 rpm in 900 mL of purified water at 37 ◦ C. After the dissolution samples (10 mL) were selected at predetermined time intervals, a similar volume of fresh dissolution medium was added to maintain the volume in the vessel constant. Collected samples were filtered through a 0.45 m Millipore filter. The concentration of EM in samples was assayed as mentioned in Section 2.2.1. The cumulative release percentage of drug from the tablets was calculated and plotted as a function of time. The marketed Yisu® was assayed under the same conditions. All the dissolution studies were performed in triplicate to obtain mean and standard deviation. 2.2.4. Investigation of the release mechanism 2.2.4.1. Scanning electron microscopy (SEM). The morphology of the surface of designed pulsatile pellets before and after in vitro dissolution was characterized using SEM. The samples were mounted on Al stub, sputter-coated with a thin layer of Pt using sputter coater (Polaron, Japan) under Argon atmosphere, and then examined using SEM (S-3000N, HITACHI Company, Japan). 2.2.4.2. Ion-exchange experiment. 2 g of Eudragit RS PO was added in 100 mL of various concentrations of succinic aqueous solutions (5 × 10−4 , 1 × 10−3 , 5 × 10−3 , 1 × 10−2 and 5 × 10−2 M). After stirring for 4 h with 100 rpm at 37 ◦ C, the solutions should be settled for 1 h and then filtered with 0.45 m film. Chloride concentrations in the mentioned solutions were assayed by means of titration method using silver nitrate after evaporation and concentration. On the other hand, similar experiments were conducted in citric acid and tartaric acid using the operating methods as above. 2.2.4.3. Hydration study of the free film. The 95% coating solutions were slowly spreading on a sheet glass then dried for a week at 25 ◦ C and oven cured at 40 ◦ C for 24 h. After being weighed precisely, each 3 pieces of free film (10 mm × 10 mm) were immersed into purified water, 0.5 mol/L tartaric acid, malic acid and citric acid solutions, plus with succinic acid aqueous solutions at 37 ◦ C for 24 h. Then the wet film should be quickly bolted to remove the excess surface water and weighed. The hydration percentage (Hm ) can be obtained from the following equation Hm (%) =
Ww − Wd × 100 Wd
where Ww and Wd represent the wet and dry weight of the free film, respectively. The hydration velocity was assayed in the similar operation but for the thermal insulation time is 1 h, and can also be calculated from the above equation. 2.2.4.4. Determination of glass transition temperature (Tg ). The obtained Eudragit RS 100 free films (10 mm × 10 mm) were immersed into 50 mL of purified water, 0.5 M succinic acid and monosodium succinate aqueous solutions separately for 24 h at 37 ◦ C and dried for another 24 h at 25 ◦ C. Tg of the free film was determined by DSC machine (Q 200, TA instruments, America) before and after immersion, with a differential scanning
calorimeter at a scanning speed of 10 ◦ C/min in the temperature range of 0–100 ◦ C under nitrogen gas flow. 2.2.5. In vivo evaluation of the EM pulsatile release pellets All studies were conducted in accordance with the principles of Laboratory Animal Care and were approved by the Department of Laboratory Animal Research in China Pharmaceutical University. Rats were supplied by the Qinglong Mountain Animal Center. 2.2.5.1. Animals. 12 Male Wistar rats (6–7 weeks old) were weighting ranged from 180 to 200 g. They were separated randomly into two groups. All animals were housed singly in standard cages and had free access to tap water and pelleted diet. Rats were deprived of food 12 h before the experiment and food was reoffered 4 h after administration. 2.2.5.2. Dosing and blood sampling. Before the intragastric administration, EM pulsatile release pellets were suspended with purified water to a concentration of 4 mg/mL. At time zero, 20 mg/kg of samples were given to rats by gavage (n = 6). Yisu® suspension as a control, which contained the same concentration of EM, was also given to rats by intragastric administration. 0.5, 1, 1.5, 2, 3, 4, 6, 8, 10, 12, 24 h after intragastric administration, the rats were anesthetized with ether. Blood samples (0.5 mL) were collected from the eye ground vein of one eye of each rat in heparinized plastic centrifuge tubes using 0.8–1.1 mm capillary glass tube. Individual plasma samples were prepared by centrifugation at 2600 × g for 15 min in an Nr.12154 rotor (Sigma 3K30). 200 L of plasma was taken by pipettor (Eppendorf, Hamburg, Germany) and mixed with 50 L carbamazepine solution (50 g/mL) as internal standard, and 600 L acetonitrile to precipitate proteins. The mixture was stirred on a vortex mixer for 90 s and centrifuged at 7500 × g for 10 min. Then, 20 L of supernatant was analyzed by HPLC using the LC-10AT Liquid Chromatograph, with acetonitrile:methanol:purified water:phosphoric acid:triethylamine (50:230:275:0.3:0.1) as mobile phase at a flow rate of 1.0 mL/min. The column was a reversed-phase column (Inertsil ODS-3, 4.6 × 250 mm, i.d. 5 m, GL Sciences, Japan), and UV detection was performed at 207 nm. Methodological studies, such as linearity, specificity, precision of with-in and between days were demonstrated to satisfy the requirements of the methodology. 2.2.6. Data statistical analysis The observed maximum plasma concentration (Cmax ), time of its occurrence (Tmax ) and the lag time were determined directly from the individual plasma concentration–time profiles. The area under the plasma concentration–time curve (AUC) was calculated by linear trapezoidal method from time zero to the last sampling point. Statistical analysis of the Cmax and AUC values of the designed and marketed forms was performed in Excel using a one-way analysis of variance (ANOVA) and the t-test and p < 0.05 was considered statistically significant by Graph Pad Instat Software-1.13 (Graph Pad Software, San Diego, CA, USA). 3. Results and discussion 3.1. Demonstration of feasibility of the mechanism Uncoated EM loaded pellets were coated with an aqueous dispersion of Eudragit® RS 30D containing TEC as a plasticizer and talc as an anti-block agent. Results of drug release study in 0.5 M of different organic acid aqueous solutions and purified water at 37 ◦ C are shown in Fig. 1. Because of the thick coating, drugs were almost not seen to release in purified water after long hours during dissolution period. However, in various kinds of organic acid dissolution fluid, EM was dramatically released to different extents depending on the
Z. Zhang et al. / International Journal of Pharmaceutics 462 (2014) 66–73
Fig. 1. Dissolution profiles of EM releasing from Eudragit® RS coated pellets (coating level of 40%) in 0.5 M of different organic acid aqueous solutions (acetic acid, succinic acid, tartaric acid, malic acid and citric acid) and purified water at 37 ◦ C (Mean ± SD, n = 6).
acid used, indicating the feasibility of acid–copolymer interaction mechanism. Among all the organic acids, succinic acid exhibited the most obvious enhancement in drug release compared with other solid organic acids, which prompts an idea to add succinic acid in the core to achieve a changed permeability of the copolymer as water penetrates through the film. But further investigation will still be necessary to clarify the issue.
69
Fig. 3. Comparison of EM releasing from Eudragit® RS coated pellets (coating level of 50%) as a function of succinic acid added in purified water at 37 ◦ C (Mean ± SD, n = 6).
changes of the film surface properties, which contributes to drug release after a predetermined lag time. 10% of succinic acid showed an obvious low release rate and incomplete release tendency. On the other hand, the ratio over 40% will lead to pellets’ adhesion in the process of extrusion and spheronization due to the aqueous binder. In this paper, a pulsatile release system could be achieved by adding 38% (w/w) of succinic acid in the core.
3.2. Effects of manufacturing coated pellets
3.3. In vitro dissolution study
Different coating levels can cause flexible lag time in the drug release profiles as shown in Fig. 2. Thicker coating layer lead to longer lag time but the velocity of drug release was remained almost constant (with the slope of 24.0, 24.3 and 24.7 in each profile). Thus, different appropriate lag time for treating various diseases can be achieved by adjusting the coating levels. The ratio of succinic acid added in the core was researched and it was found that drug release rate was suppressed more as the ratio decreased. The Eudragit® RS coated EM pellets were prepared by altering the weight ratio of succinic acid added in the core and their dissolution behaviors were determined (Fig. 3). As shown in Fig. 3, when water slowly penetrates across Eudragit® RS, organic acid in core pellets interacting with the low amount of quaternary ammonium groups of Eudragit® RS copolymer is responsible for
Three batches of designated EM pulsatile release coated pellets were manufactured and their in vitro dissolution curves are shown in Fig. 4. A significant 4 h lag time period in the early stage and an evident drug release phase could be observed. The pH independent Eudragit® RS was insoluble and had a low water permeability, leading to the predetermined lag time by adjusting the coating levels. While the medium penetrates through gradually, it swells in water by incorporating H2 O molecules into its hydrophilic groups and also has interaction with anions of succinic acid in the core. The appearance of Eudragit® RS coated pellets after dissolution period remained complete and no flaws but mini pores were observed (Fig. 5), but the volume increased obviously compared to that before dissolution, meaning it was the film structural change that leads to drug release but not film rupture. Additionally, as the dissolution results demonstrated, the designed Eudragit® RS coated EM pulsatile pellets showed relatively complete release.
Fig. 2. Dissolution profiles of EM releasing from Eudragit® RS coated pellets (38% (w/w) succinic acid in the core) with different coating levels (30%, 50% and 60%) under the condition of purified water at 37 ◦ C (Mean ± SD, n = 6).
Fig. 4. Release profiles of EM loaded Eudragit® RS coated pellets (coating level of 50% and 38% (w/w) succinic acid in the core) of three batches under the condition of purified water at 37 ◦ C (Mean ± SD, n = 6).
70
Z. Zhang et al. / International Journal of Pharmaceutics 462 (2014) 66–73
Fig. 6. Relationship between chloride ions released and succinate anions in different concentrations of succinic acid aqueous solutions (5 × 10−4 , 1 × 10−3 , 5 × 10−3 , 1 × 10−2 and 5 × 10−2 M) at 37 ◦ C.
method should be conducted before titration assay. As Eudragit® RS suspended in the succinic acid aqueous solution, chloride ion was detected and its concentration was found to increase linearly with that of succinic acid environment. The relationship between chloride ion released and succinic anion of different concentrations is shown in Fig. 6, and the equation of linear regression was CCl− = 8.1 × 10−3 Csuc. + 5.639 × 10−4 ,
Fig. 5. Scanning electron microscope of EM pulsatile release pellets with coating level of 50% and 38% (w/w) succinic acid in the core. (A) Eudragit® RS coated pellets before dissolution, (B) Eudragit® RS coated pellets after dissolution at the time of 12 h under the condition of purified water at 37 ◦ C.
3.4. Investigation of the drug release mechanism 3.4.1. Ion-exchange experiment There’s a small amount of quaternary ammonium groups in the form of chloride in Eudragit® RS copolymer, which might have an ion-exchange interaction with the succinic acid despite of the small exchange capacity (Kaur and Kim, 2009). Thus evaporation
r 2 = 0.9627
where CCl− and Csuc. represent the concentration of chloride ion and succinic acid, respectively. Similar phenomena were found in other organic acids, and the exchange capacity decreased in the order of succinic acid, dl-tartaric acid, malic acid and citric acid. Eudragit® RS coated pellets exhibited pulsatile drug release profiles in the above organic acid aqueous solutions, which may indicate a close relationship between the ion-exchange interaction and the appearance of our designated unique pulsatile drug release behavior. From the EM release profiles in Fig. 2, the time of 50% drug released (T50 ) can be calculated. To examine the relationship between drug release enhancing effect and the acidity of various kinds of organic acid quantitatively, T50 was plotted against pKa of each organic acid in Fig. 7A. Fig. 7A shows an inverse correlation
Fig. 7. Relationship between T50 and pKa of various kinds of organic acids (A) and relationship between T50 and Hm of free Eudragit® RS film (B). The organic acids are citric acid (a), tartaric acid (b), malic acid (c), and succinic acid (d).
Z. Zhang et al. / International Journal of Pharmaceutics 462 (2014) 66–73
71
Table 2 Results of hydration study of Erudragit® RS free film in different organic acid solutions (n = 3).
Hm (%)
Water
Citric acid
Tartaric acid
Succinic acid
Malic acid
34 ± 1.32
45 ± 2.14
49 ± 2.03
92 ± 4.15
58 ± 3.85
Table 3 Concentration of succinate anion, pH values and EM release rate in different succinic acid and monosodium succinate aqueous solutions at 37 ◦ C. The release rate is calculated from the linear part of drug release curves. Substance
Concentration (M)
pH
Concentration of succinate anion (M)
Release rate (%/h)
Succinic acid
5 × 10−1 1 × 10−1 5 × 10−2 1 × 10−2 5 × 10−3
2.26 2.50 2.65 2.98 3.19
5.61 × 10−3 1.94 × 10−3 1.37 × 10−3 5.89 × 10−4 4.77 × 10−4
69.2 53.7 24.2 17.1 6.8
Monosodium succinate
5 × 10−1 1 × 10−1 1 × 10−2 1 × 10−3 1 × 10−4 1 × 10−5
4.46 4.53 4.68 4.75 4.82 5.27
2.34 × 10−1 6.82 × 10−2 5.82 × 10−3 7.82 × 10−4 8.01 × 10−5 9.02 × 10−6
7.1 15.6 24.6 36.3 20.5 10.2
between T50 and pKa , indicating a more effective impact of weaker acid. 3.4.2. Hydration study Table 2 shows Hm of free Eudragit® RS film in various kinds of organic acids was significant higher than in purified water, suggesting that organic acids’ release-increasing effect was close related to their enhancement on the hydration of free film (Okor, 1982). From Fig. 7B it was found that the T50 value increased with the decreasing Hm , which further demonstrated the above suggestion. The “free volume” concept has been proposed to explain the positive relationship between the hydration effect and film permeability (Yasuda and Lamaze, 1971). 3.4.3. A further study of undisassociated form of succinic acid Differential scanning calorimetry (DSC) (Fig. 8) shows the Tg before and after immersing into water, succinic acid and monosodium succinate aqueous solutions. The value of Tg had an evident decline in succinic acid compared with that in monosodium succinate aqueous solution, meaning that the flexibility of Eudragit® RS film was strengthened. When the disassociated form concentration coexisted in each test fluid is comparable,
undisassociated form also has a large enhancing effect (Table 3). For example, release rate in 5 × 10−1 and 1 × 10−1 M succinic acid solutions was much faster than that in 1 × 10−2 and 1 × 10−3 M monosodium succinate solutions, although the succinate anion concentration is almost the equal. Meanwhile, Hm of the free film in succinic acid solutions was dramatically higher than in monosodium succinate solutions, which indicated that succinic acid molecules increased the drug release in another special way. A possible explanation is related to the effect of cross-linking: when copolymer is acting as the drug delivery system, the permeability plays an important role in drug release. Structural changes and physical states, such as environmental temperature and flexibility of chains will definitely effect on their permeability. Chains or groups of copolymers will have more spacious room as flexibility increases, leading to an improvement in permeability. When lipophilic succinic acid molecules distribute into hydrophobic structure, the relative longer chains make a contribution to a better flexibility of copolymers. Thus the repelling force against hydration of insoluble Eudragit® RS is distinctly weaken and permeability is obviously increased. To conclude, the possible release mechanism was a combination of both disassociated and undisassociated forms of succinic acid physiochemically interacting with Eudragit® RS (see Fig. 9). 3.5. In vivo pharmacokinetic study To evaluate the in vivo performance, the pulsatile pellets and the marketed Yisu® were administered to rats. Fig. 10 shows the mean plasma EM concentration versus time profiles following oral administration, and the main pharmacokinetic parameters were shown in Table 4. The EM pulsatile release pellets exhibited an evident drug release after a lag time period
Table 4 Pharmacokinetic parameters obtained following oral administration of marketed Yisu® and the pulsatile release pellets containing EM to rats.
Fig. 8. Differential scanning calorimetry (DSC) curves of dried free Eudragit® RS film after being immersed in purified water 0.5 M succinic acid and monosodium succinate aqueous solutions for 24 h at 37 ◦ C.
Parameters
Marketed tablets
Pulsatile pellets
Cmax (ng/mL) Tmax (h) Tlag (h) AUC0− (h ng/mL) MRT0− (h) Relative bioavailability
170.333 ± 96.090 1.667 ± 0.289 0 810.817 ± 67.712 5.007 ± 0.441 100
75.322 ± 17.059 12.010 ± 0.320 4.534 ± 0.113 702.384 ± 96.891 11.717 ± 0.703 86.62
72
Z. Zhang et al. / International Journal of Pharmaceutics 462 (2014) 66–73
4. Conclusion Cured pulsatile release EM pellets coated with Eudragit® RS polymers were found to be an applicable form for antihypertension due to the relatively simple formula and manufacturing process. The drug release curves exhibited a clear lag time period in the early stage followed by a drug release phase. The release mechanism of pulsatile pellets was a combination of both disassociated and undisassociated forms of organic acid physiochemically interacting with Eudragit® RS. In vivo pharmacokinetic evaluation showed the relative bioavailability of designed EM pulsatile pellets was 86.62% and can be considered bioequivalence with marketed Yisu® .
Acknowledgements This work was financially supported by the Major State Basic Research Development Program of the National Science and Technology of China for new drugs development (Program No. 2012CB724002) and Natural Science Foundation of Jiangsu Province (No. BK20130663).
Fig. 9. Possible mechanism of acid- Eudragit® RS copolymers interactions in EM loaded pulsatile pellets.
of 4.534 ± 0.113 h (different with 4 h in vitro dissolution) and the mean value of Cmax was 75.322 ± 17.059 ng/mL and the Tmax was 12 ± 0.000 h after oral administration. However, conventional marketed tablets did not present any lag time before the drug release, with Cmax value of 170.333 ± 96.090 ng/mL and Tmax of 1.667 ± 0.289 h. The parameter AUC0− of the Eudragit® RS coated pellets and the market tablets was 702.384 ± 96.891 h ng/mL and 810.817 ± 67.712 h ng/mL (p > 0.05). The relative bioavailability of EM calculated from the ratio AUC ((testing products/reference products) × 100) was 86.62%. While the range of bioequivalence is 80–125%, the designed pulsatile pellets can be considered as bioequivalence with the marketed tablets (p > 0.05). The pulsatile pellets having lower AUC in contrast with Yisu® may be caused by the decreased solubility of drug in vivo microenvironment, leading to an absorption-reducing of EM.
Fig. 10. Mean plasma concentration-time profiles of EM from the pulsatile release pellets with coating level of 50% and 38% (w/w) succinic acid in the core and the marketed tablets following oral administration (Mean ± SD, n = 6).
References Angelatos, A.S., Radt, B., Caruso, F., 2005. Light-responsive polyelectrolyte/gold nanoparticle microcapsules. J. Phys. Chem. B 109, 3071–3076. Bussemer, T., Bodmeier, R., 2003. Formulation parameters affecting the performance of coated gelatin capsules with pulsatile release profiles. Int. J. Pharm. 267, 59–68. Bussemer, T., Dashevsky, A., Bodmeier, R., 2003. A pulsatile drug delivery system based on rupturable coated hard gelatin capsules. J. Control. Rel. 93, 331–339. Déjugnat, C., Haloˇzan, D., Sukhorukov, G.B., 2005. Defined picogram dose inclusion and release of macromolecules using polyelectrolyte microcapsules. Macromol. Rapid Commun. 26, 961–967. Gazzaniga, A., Sangalli, M.E., Giordano, F., 1994. Oral Chronotopic® drug delivery systems: achievement of time and/or site specificity. Eur. J. Pharm. Biopharm. 40, 246–250. Hermida, R.C., Ayala, D.E., Calvo, C., Portaluppi, F., Smolensky, M.H., 2007a. Chronotherapy of hypertension: administration-time-dependent effects of treatment on the circadian pattern of blood pressure. Adv. Drug Del. Rev. 59, 923–939. Hermida, R.C., Ayala, D.E., Portaluppi, F., 2007b. Circadian variation of blood pressure: the basis for the chronotherapy of hypertension. Adv. Drug Del. Rev. 59, 904–922. Innominato, P.F., Lévi, F.A., Bjarnason, G.A., 2010. Chronotherapy and the molecular clock: clinical implications in oncology. Adv. Drug Del. Rev. 62, 979–1001. Iskakov, R.M., Kikuchi, A., Okano, T., 2002. Time-programmed pulsatile release of dextran from calcium-alginate gel beads coated with carboxyn-propylacrylamide copolymers. J. Control. Rel. 80, 57–68. Karavas, E., Georgarakis, E., Bikiaris, D., 2006. Application of PVP/HPMC miscible blends with enhanced mucoadhesive properties for adjusting drug release in predictable pulsatile chronotherapeutics. Eur. J. Pharm. Biopharm. 64, 115–126. Kashyap, N., Viswanad, B., Sharma, G., Bhardwaj, V., Ramarao, P., Ravi Kumar, M., 2007. Design and evaluation of biodegradable, biosensitive in situ gelling system for pulsatile delivery of insulin. Biomaterials 28, 2051–2060. Kaur, K., Kim, K., 2009. Studies of chitosan/organic acid/Eudragit® RS/RL-coated system for colonic delivery. Int. J. Pharm. 366, 140–148. Kiser, P.F., Wilson, G., Needham, D., 1998. A synthetic mimic of the secretory granule for drug delivery. Nature 394, 459–462. Kiser, P.F., Wilson, G., Needham, D., 2000. Lipid-coated microgels for the triggered release of doxorubicin. J. Control. Rel. 68, 9–22. Lemmer, B., 1999. Chronopharmacokinetics: implications for drug treatment. J. Pharm. Pharmacol. 51, 887–890. Lin, H.-L., Lin, S.-Y., Lin, Y.-K., Ho, H.-O., Lo, Y.-W., Sheu, M.-T., 2008. Release characteristics and in vitro–in vivo correlation of pulsatile pattern for a pulsatile drug delivery system activated by membrane rupture via osmotic pressure and swelling. Eur. J. Pharm. Biopharm. 70, 289–301. Lynn, D.M., Amiji, M.M., Langer, R., 2001. pH-responsive polymer microspheres: rapid release of encapsulated material within the range of intracellular pH. Angew. Chem. Int. Ed. 40, 1707–1710. McConville, J.T., Ross, A.C., Florence, A.J., Stevens, H.N., 2005. Erosion characteristics of an erodible tablet incorporated in a time-delayed capsule device. Drug Dev. Ind. Pharm. 31, 79–89. Miyamoto, S., Fujita, M., Tambara, K., Sekiguchi, H., Eiho, S., Hasegawa, K., Tamaki, S.-i., 2004. Circadian variation of cardiac autonomic nervous activity is well preserved in patients with mild to moderate chronic heart failure: effect of patient position. Int. J. Cardiol. 93, 247–252.
Z. Zhang et al. / International Journal of Pharmaceutics 462 (2014) 66–73 Narisawa, S., Nagata, M., Danyoshi, C., Yoshino, H., Murata, K., Hirakawa, Y., Noda, K., 1994. An organic acid-induced sigmoidal release system for oral controlledrelease preparations. Pharm. Res. 11, 111–116. Ohdo, S., 2010. Chronotherapeutic strategy: rhythm monitoring, manipulation and disruption. Adv. Drug Delivery Rev. 62, 859–875. Okor, R., 1982. Effect of polymer cation content on certain film properties. J. Pharm. Pharmacol. 34, 83–86. Portaluppi, F., Lemmer, B., 2007. Chronobiology and chronotherapy of ischemic heart disease. Adv. Drug Del. Rev. 59, 952–965. Radt, B., Smith, T.A., Caruso, F., 2004. Optically addressable nanostructured capsules. Adv. Mater. 16, 2184–2189. Roy, P., Shahiwala, A., 2009a. Multiparticulate formulation approach to pulsatile drug delivery: current perspectives. J. Control. Rel. 134, 74–80. Roy, P., Shahiwala, A., 2009b. Statistical optimization of ranitidine HCl floating pulsatile delivery system for chronotherapy of nocturnal acid breakthrough. Eur. J. Pharm. Sci. 37, 363–369.
73
Skirtach, A.G., Dejugnat, C., Braun, D., Susha, A.S., Rogach, A.L., Parak, W.J., Möhwald, H., Sukhorukov, G.B., 2005. The role of metal nanoparticles in remote release of encapsulated materials. Nano Lett. 5, 1371–1377. Sungthongjeen, S., Puttipipatkhachorn, S., Paeratakul, O., Dashevsky, A., Bodmeier, R., 2004. Development of pulsatile release tablets with swelling and rupturable layers. J. Control. Rel. 95, 147–159. Wagner, K.G., McGinity, J.W., 2002. Influence of chloride ion exchange on the permeability and drug release of Eudragit RS 30 D films. J. Control. Rel. 82, 385–397. Yadav, D., Survase, S., Kumar, N., 2011. Dual coating of swellable and rupturable polymers on Glipizide loaded MCC pellets for pulsatile delivery: formulation design and in vitro evaluation. Int. J. Pharm. 419, 121–130. Yasuda, H., Lamaze, C., 1971. Permselectivity of solutes in homogeneous water-swollen polymer membranes. J. Macromol. Sci. Part B: Phys. 5, 111–134.
Contents lists available at ScienceDirect
International Journal of Pharmaceutics journal homepage: www.elsevier.com/locate/ijpharm
Personalised Medicine
A novel pulsatile drug delivery system based on the physiochemical reaction between acrylic copolymer and organic acid: In vitro and in vivo evaluation Ziwei Zhang a,1 , Xiaole Qi a,1 , Xiangbo Li a , Jiayu Xing a , Xuehua Zhu a , Zhenghong Wu a,b,∗ a b
Key Laboratory of Modern Chinese Medicines, China Pharmaceutical University, Nanjing 210009, PR China Yangtze River Pharmaceutical Group, State Key Laboratory for Advanced Formulation Technologies, Taizhou, PR China
a r t i c l e
i n f o
Article history: Received 27 September 2013 Received in revised form 18 November 2013 Accepted 15 December 2013 Available online 22 December 2013 Keywords: Pulsatile pellets Organic acid Acrylic copolymer Enalapril Maleate Film coating
a b s t r a c t Multilayer-coating technology is the traditional method to achieve pulsatile drug release with the drawbacks of time consuming, more materials demanding and lack of efficiency. The purpose of this study was to design a novel pulsatile drug delivery system based on the physiochemical interaction between acrylic copolymer and organic acid with relatively simpler formulation and manufacturing process. The Enalapril Maleate (EM) pulsatile release pellets were prepared using extruding granulation, spheronization and fluid-bed coating technology. The ion-exchange experiment, hydration study and determination of glass transition temperature were conducted to explore the related drug release mechanism. Bioavailability experiment was carried out by administering the pulsatile release pellets to rats compared with marketed rapid release tablets Yisu® . An obvious 4 h lag time period and rapid drug release was observed from in vitro dissolution profiles. The release mechanism was a combination of both disassociated and undisassociated forms of succinic acid physiochemically interacting with Eudragit® RS. The AUC0- of the EM pulsatile pellets and the market tablets was 702.384 ± 96.891 h ng/mL and 810.817 ± 67.712 h ng/mL, while the relative bioavailability was 86.62%. These studies demonstrate this novel pulsatile release concept may be a promising strategy for oral pulsatile delivery system. © 2013 Elsevier B.V. All rights reserved.
1. Introduction A variety of diseases including hypertension, angina pectoris, epilepsy, asthma, diabetes, hyperchlorhydria and arthritis exhibit circadian variation (Hermida et al., 2007b; Lemmer, 1999; Miyamoto et al., 2004; Roy and Shahiwala, 2009a). With the development of chronotherapy, which refers to a clinical practice of drug delivery consistent with the body’s circadian rhythm (Hermida et al., 2007a; Innominato et al., 2010; Ohdo, 2010; Portaluppi and Lemmer, 2007; Roy and Shahiwala, 2009b) to produce maximum health benefit and minimum harmful effects (Roy and Shahiwala, 2009a), time-controlled release systems for comparative drug efficacy have gained intensive attention worldwide since first introduced. And pulsatile drug delivery system is an important and desirable part among time-controlled release systems. Pulsatile
∗ Corresponding author at: Key Laboratory of Modern Chinese Medicines, China Pharmaceutical University, Nanjing 210009, PR China. Tel.: +86 15062208341; fax: +86 025 83179703. E-mail address: [email protected] (Z. Wu). 1 These authors contributed equally to this work. 0378-5173/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ijpharm.2013.12.026
drug delivery system is characterized by a predetermined lag time period in the starting stage followed by a drug release phase in the drug release profiles (Karavas et al., 2006; Kashyap et al., 2007; Lin et al., 2008; Sungthongjeen et al., 2004; Yadav et al., 2011). Pulsatile release has the advantages of avoiding drug tolerance, matching the release of specific peptides or hormones, and control of tissue (Iskakov et al., 2002) and drug release may be controlled by time, by site or a combination of the two parameters (Yadav et al., 2011). Devices which show pulsatile release upon applying an external trigger such as pH (Déjugnat et al., 2005; Lynn et al., 2001), electric field (Kiser et al., 1998, 2000), IR-light (Angelatos et al., 2005; Radt et al., 2004; Skirtach et al., 2005), etc. have been described. But generally all manufacturing approaches work on the same basic principles of swelling and rupturing (Bussemer and Bodmeier, 2003; Bussemer et al., 2003; Sungthongjeen et al., 2004), erosion (Gazzaniga et al., 1994; McConville et al., 2005) or dissolution, and systems based on changes in membrane permeability (Roy and Shahiwala, 2009a). As to the coating technology, multilayer-coating is the most popular method to achieve pulsatile drug release. However, this method has drawbacks of incomplete drug release and more materials demanding. Moreover, multilayer-coating is time consuming, tedious operating and lack
Z. Zhang et al. / International Journal of Pharmaceutics 462 (2014) 66–73
of efficiency, leading to technical hurdles in commercial scale process. Eudragit® RS is a kind of acrylic copolymer synthesized from acrylic and methacrylic acid esters with a low content of quaternary ammonium groups in the form of chloride. The coating film formed by Eudragit® RS is insoluble in water and has a low permeability but swells in water by incorporating H2 O molecules into its hydrophilic groups. Researchers found that some organic acid solutions can induce the structural change of Eudragit® RS by physiochemical interaction, leading to the changing membrane permeability (Narisawa et al., 1994; Wagner and McGinity, 2002). In order to overcome the drawbacks of conventional multi-layer coating technology for pulsatile pellets, we proposed a novel pulsatile drug delivery system by incorporating organic acid in the core pellets and coating with Eudragit® RS copolymer based on the above principle. Various kinds and amount of organic acid were screened, respectively. Ideally, when water slowly penetrates across Eudragit® RS, organic acid in core pellets interacting with the low amount of quaternary ammonium groups of Eudragit RS copolymer is responsible for changes of the film surface properties, which contributes to drug release after a predetermined lag time period due to its low water permeability. Enalapril Maleate (EM) is an angiotensin-converting enzyme inhibitor and used as an antihypertensive agent. Nowadays, marketed dosage forms of EM are mainly oral rapid release tablets. However, blood pressure in human body presents circadian rhythms – a dramatic morning rise and obvious bedtime decline, both of which act as a push for the chronotherapy of hypertension (Hermida et al., 2007b; Portaluppi and Lemmer, 2007). Therefore, EM was chosen as the model drug. Then patients can take the designated EM dosage form at a proper time in the evening and drug therapeutic concentrations will be achieved the next day in the occurrence of blood pressure morning peak. A novel single-layer coated pulsatile release system, which is meant to contain organic acid in the core and coated with acrylic copolymer, was designed with a relatively simpler formulation and manufacturing process compared to the traditional multilayer coated explosive pulsatile drug release system. In vitro and in vivo evaluation was conducted in contrast with marketed rapid release tablets of Yisu® . The drug release mechanism was demonstrated and investigated through a string of experiment, such as ion-exchange study, hydration research and determination of glass transition temperature. 2. Materials and methods 2.1. Materials Enalapril Maleate (EM) was obtained from Yangtze Pharmaceutical Co., Ltd. (Taizhou, China). Eudragit® RS 30D using as the coating material was supplied by Röhm Pharma (Darmstadt, Germany). Lactose (GranuLac® 200) was obtained from Meggle (Wasserburg, Germany). Five organic acids including succinic acid, dl-tartaric acid, citric acid, malic acid and acetic acid, plus with all other chemicals were used as received and were of standard pharmaceutical grade: HPMC (E5, Colorcon, Orpington, UK), Microcrystalline Cellulose (MCC; Avicel® PH 101, Asahi Kasei. Co., Ltd., Tokyo, Japan) and triethyl citrate (TEC; Aladdin Chemistry Co., Ltd., Shanghai, China). The marketed rapid release tablets Yisu® (Yangtze Pharmaceutical Co., Ltd., Taizhou, China) were used as the reference. 2.2. Methods 2.2.1. Demonstration of feasibility of the mechanism The components of EM, MCC and lactose (10:80:10) were passed through a 200 m sieve to obtain a well-dispersed mixture and wet
67
Table 1 The core and coating layer composition of the Erudragit® RS coated pulsatile release pellets of EM. Core
Coating layer
Continents
Added amount
Continents
Added amount
EM MCC Succinic acid Lactose
10% 42% 38% 10%
Eudragit® RS 30D TEC Talc Water
20% 2% 2% 76%
massed with 3% HPMC aqueous solution as a binder. The soft material was made into strip granules with 1 mm screen by means of an extruding granulator (JBZ-300, New Drug Research Institute of Liaoning Yilian, China), which were then broken into smaller cylindrical rods and rounded into spherical pellets using a high-speed rotating friction machine (JBZ-300, New Drug Research Institute of Liaoning Yilian, China). The extruding and rotating speed were both 385 × g. Then the pellets were dried for 3 h at 40 ◦ C in the oven (DHG-9245A, Shanghai Huiyi Technology Co., Ltd, China). After drying, the core pellets were passed through two sieves of 800 m and 1000 m to remove fine, large and agglomerate particles. The pellets were then coated by spraying with a mixture of Eudragit® RS 30D, talc, TEC, water (20:2:2:76) in a fluid-bed coater (Werner Glatt, Germany). The coating conditions were as follows: spray air pressure, 0.14 MPa; spray solution speed, 1.0 mL/min; inlet temperature, 35 ◦ C; outlet temperature, 30 ◦ C. The coating level was 50%, which can be determined from the following equation (Lemmer, 1999) L (%) =
Mafter − Mbefore Mbefore
× 100
where L represents the gaining weight of coating in terms of percentage, Mbefore and Mafter are the weight of pellets before and after coating, respectively. Release study of the acid unloaded pellets was performed using USP 32 apparatus 1 (basket) with 900 mL of 0.5 mol/L various testing fluid at 37 ◦ C with the basket speed of 100 rpm using a dissolution tester (ZRS-8G, Tianda Tianfa Techenology Co., Ltd., Tianjin, China). Five organic acids including succinic acid, dl-tartaric acid, citric acid, malic acid and acetic acid solution (each 0.5 M) were selected as dissolution medium to demonstrate the feasibility of the principle and find suitable additives using release rate as the evaluation index. Samples through a 45 m filter were taken at preparatory time interval and measured by HPLC (Shimadzu, Kyoto, Japan) with a reverse-phase column (Inertsil ODS-3, 4.6 × 250 mm, i.d. 5 m, GL Sciences, Japan), and UV detection at 207 nm. An acidified (pH 2.2, adjusting with phosphoric acid) aqueous solution of potassium dihydrogen phosphate was used as the mobile phase at a flow rate of 1.0 mL/min. Methodological studies, such as linearity, specificity, precision of with-in and between days were also demonstrated to satisfy the requirements of the methodology. A linear detector response (r = 0.9999) was observed over the concentration range of 1–20 g/mL and blank solvent did not interfere with the determination of EM. 2.2.2. Preparation of pulsatile release pellets The formula of the core pellets and coating solution are shown in Table 1. The experimental conditions and preparing method were referred to organic acid unloaded pellets in Section 2.2.1. For one thing, to optimize extrusion – spheronization process and get the desired particle size range with adequate friability and flow properties, blank (without drug) pellets were prepared with MCC alone initially. Then the optimized process was applied to the
68
Z. Zhang et al. / International Journal of Pharmaceutics 462 (2014) 66–73
preparation of EM loaded pellets. Besides, a progressive set-up of the operating conditions was well required during the coating process (Yadav et al., 2011). The mentioned parameters were varied sequentially on the basis of their influences on film coating in optimization period. Plus, the proportion of components in the coating solution was adjusted so as to balance the desired high coating efficiency and the pellets’ adhesion problem while coating. 2.2.3. In vitro release characteristics of EM pulsatile pellets The release characteristics of EM pulsatile pellets were determined using the USP 32 apparatus I (basket) at a rotation speed of 100 rpm in 900 mL of purified water at 37 ◦ C. After the dissolution samples (10 mL) were selected at predetermined time intervals, a similar volume of fresh dissolution medium was added to maintain the volume in the vessel constant. Collected samples were filtered through a 0.45 m Millipore filter. The concentration of EM in samples was assayed as mentioned in Section 2.2.1. The cumulative release percentage of drug from the tablets was calculated and plotted as a function of time. The marketed Yisu® was assayed under the same conditions. All the dissolution studies were performed in triplicate to obtain mean and standard deviation. 2.2.4. Investigation of the release mechanism 2.2.4.1. Scanning electron microscopy (SEM). The morphology of the surface of designed pulsatile pellets before and after in vitro dissolution was characterized using SEM. The samples were mounted on Al stub, sputter-coated with a thin layer of Pt using sputter coater (Polaron, Japan) under Argon atmosphere, and then examined using SEM (S-3000N, HITACHI Company, Japan). 2.2.4.2. Ion-exchange experiment. 2 g of Eudragit RS PO was added in 100 mL of various concentrations of succinic aqueous solutions (5 × 10−4 , 1 × 10−3 , 5 × 10−3 , 1 × 10−2 and 5 × 10−2 M). After stirring for 4 h with 100 rpm at 37 ◦ C, the solutions should be settled for 1 h and then filtered with 0.45 m film. Chloride concentrations in the mentioned solutions were assayed by means of titration method using silver nitrate after evaporation and concentration. On the other hand, similar experiments were conducted in citric acid and tartaric acid using the operating methods as above. 2.2.4.3. Hydration study of the free film. The 95% coating solutions were slowly spreading on a sheet glass then dried for a week at 25 ◦ C and oven cured at 40 ◦ C for 24 h. After being weighed precisely, each 3 pieces of free film (10 mm × 10 mm) were immersed into purified water, 0.5 mol/L tartaric acid, malic acid and citric acid solutions, plus with succinic acid aqueous solutions at 37 ◦ C for 24 h. Then the wet film should be quickly bolted to remove the excess surface water and weighed. The hydration percentage (Hm ) can be obtained from the following equation Hm (%) =
Ww − Wd × 100 Wd
where Ww and Wd represent the wet and dry weight of the free film, respectively. The hydration velocity was assayed in the similar operation but for the thermal insulation time is 1 h, and can also be calculated from the above equation. 2.2.4.4. Determination of glass transition temperature (Tg ). The obtained Eudragit RS 100 free films (10 mm × 10 mm) were immersed into 50 mL of purified water, 0.5 M succinic acid and monosodium succinate aqueous solutions separately for 24 h at 37 ◦ C and dried for another 24 h at 25 ◦ C. Tg of the free film was determined by DSC machine (Q 200, TA instruments, America) before and after immersion, with a differential scanning
calorimeter at a scanning speed of 10 ◦ C/min in the temperature range of 0–100 ◦ C under nitrogen gas flow. 2.2.5. In vivo evaluation of the EM pulsatile release pellets All studies were conducted in accordance with the principles of Laboratory Animal Care and were approved by the Department of Laboratory Animal Research in China Pharmaceutical University. Rats were supplied by the Qinglong Mountain Animal Center. 2.2.5.1. Animals. 12 Male Wistar rats (6–7 weeks old) were weighting ranged from 180 to 200 g. They were separated randomly into two groups. All animals were housed singly in standard cages and had free access to tap water and pelleted diet. Rats were deprived of food 12 h before the experiment and food was reoffered 4 h after administration. 2.2.5.2. Dosing and blood sampling. Before the intragastric administration, EM pulsatile release pellets were suspended with purified water to a concentration of 4 mg/mL. At time zero, 20 mg/kg of samples were given to rats by gavage (n = 6). Yisu® suspension as a control, which contained the same concentration of EM, was also given to rats by intragastric administration. 0.5, 1, 1.5, 2, 3, 4, 6, 8, 10, 12, 24 h after intragastric administration, the rats were anesthetized with ether. Blood samples (0.5 mL) were collected from the eye ground vein of one eye of each rat in heparinized plastic centrifuge tubes using 0.8–1.1 mm capillary glass tube. Individual plasma samples were prepared by centrifugation at 2600 × g for 15 min in an Nr.12154 rotor (Sigma 3K30). 200 L of plasma was taken by pipettor (Eppendorf, Hamburg, Germany) and mixed with 50 L carbamazepine solution (50 g/mL) as internal standard, and 600 L acetonitrile to precipitate proteins. The mixture was stirred on a vortex mixer for 90 s and centrifuged at 7500 × g for 10 min. Then, 20 L of supernatant was analyzed by HPLC using the LC-10AT Liquid Chromatograph, with acetonitrile:methanol:purified water:phosphoric acid:triethylamine (50:230:275:0.3:0.1) as mobile phase at a flow rate of 1.0 mL/min. The column was a reversed-phase column (Inertsil ODS-3, 4.6 × 250 mm, i.d. 5 m, GL Sciences, Japan), and UV detection was performed at 207 nm. Methodological studies, such as linearity, specificity, precision of with-in and between days were demonstrated to satisfy the requirements of the methodology. 2.2.6. Data statistical analysis The observed maximum plasma concentration (Cmax ), time of its occurrence (Tmax ) and the lag time were determined directly from the individual plasma concentration–time profiles. The area under the plasma concentration–time curve (AUC) was calculated by linear trapezoidal method from time zero to the last sampling point. Statistical analysis of the Cmax and AUC values of the designed and marketed forms was performed in Excel using a one-way analysis of variance (ANOVA) and the t-test and p < 0.05 was considered statistically significant by Graph Pad Instat Software-1.13 (Graph Pad Software, San Diego, CA, USA). 3. Results and discussion 3.1. Demonstration of feasibility of the mechanism Uncoated EM loaded pellets were coated with an aqueous dispersion of Eudragit® RS 30D containing TEC as a plasticizer and talc as an anti-block agent. Results of drug release study in 0.5 M of different organic acid aqueous solutions and purified water at 37 ◦ C are shown in Fig. 1. Because of the thick coating, drugs were almost not seen to release in purified water after long hours during dissolution period. However, in various kinds of organic acid dissolution fluid, EM was dramatically released to different extents depending on the
Z. Zhang et al. / International Journal of Pharmaceutics 462 (2014) 66–73
Fig. 1. Dissolution profiles of EM releasing from Eudragit® RS coated pellets (coating level of 40%) in 0.5 M of different organic acid aqueous solutions (acetic acid, succinic acid, tartaric acid, malic acid and citric acid) and purified water at 37 ◦ C (Mean ± SD, n = 6).
acid used, indicating the feasibility of acid–copolymer interaction mechanism. Among all the organic acids, succinic acid exhibited the most obvious enhancement in drug release compared with other solid organic acids, which prompts an idea to add succinic acid in the core to achieve a changed permeability of the copolymer as water penetrates through the film. But further investigation will still be necessary to clarify the issue.
69
Fig. 3. Comparison of EM releasing from Eudragit® RS coated pellets (coating level of 50%) as a function of succinic acid added in purified water at 37 ◦ C (Mean ± SD, n = 6).
changes of the film surface properties, which contributes to drug release after a predetermined lag time. 10% of succinic acid showed an obvious low release rate and incomplete release tendency. On the other hand, the ratio over 40% will lead to pellets’ adhesion in the process of extrusion and spheronization due to the aqueous binder. In this paper, a pulsatile release system could be achieved by adding 38% (w/w) of succinic acid in the core.
3.2. Effects of manufacturing coated pellets
3.3. In vitro dissolution study
Different coating levels can cause flexible lag time in the drug release profiles as shown in Fig. 2. Thicker coating layer lead to longer lag time but the velocity of drug release was remained almost constant (with the slope of 24.0, 24.3 and 24.7 in each profile). Thus, different appropriate lag time for treating various diseases can be achieved by adjusting the coating levels. The ratio of succinic acid added in the core was researched and it was found that drug release rate was suppressed more as the ratio decreased. The Eudragit® RS coated EM pellets were prepared by altering the weight ratio of succinic acid added in the core and their dissolution behaviors were determined (Fig. 3). As shown in Fig. 3, when water slowly penetrates across Eudragit® RS, organic acid in core pellets interacting with the low amount of quaternary ammonium groups of Eudragit® RS copolymer is responsible for
Three batches of designated EM pulsatile release coated pellets were manufactured and their in vitro dissolution curves are shown in Fig. 4. A significant 4 h lag time period in the early stage and an evident drug release phase could be observed. The pH independent Eudragit® RS was insoluble and had a low water permeability, leading to the predetermined lag time by adjusting the coating levels. While the medium penetrates through gradually, it swells in water by incorporating H2 O molecules into its hydrophilic groups and also has interaction with anions of succinic acid in the core. The appearance of Eudragit® RS coated pellets after dissolution period remained complete and no flaws but mini pores were observed (Fig. 5), but the volume increased obviously compared to that before dissolution, meaning it was the film structural change that leads to drug release but not film rupture. Additionally, as the dissolution results demonstrated, the designed Eudragit® RS coated EM pulsatile pellets showed relatively complete release.
Fig. 2. Dissolution profiles of EM releasing from Eudragit® RS coated pellets (38% (w/w) succinic acid in the core) with different coating levels (30%, 50% and 60%) under the condition of purified water at 37 ◦ C (Mean ± SD, n = 6).
Fig. 4. Release profiles of EM loaded Eudragit® RS coated pellets (coating level of 50% and 38% (w/w) succinic acid in the core) of three batches under the condition of purified water at 37 ◦ C (Mean ± SD, n = 6).
70
Z. Zhang et al. / International Journal of Pharmaceutics 462 (2014) 66–73
Fig. 6. Relationship between chloride ions released and succinate anions in different concentrations of succinic acid aqueous solutions (5 × 10−4 , 1 × 10−3 , 5 × 10−3 , 1 × 10−2 and 5 × 10−2 M) at 37 ◦ C.
method should be conducted before titration assay. As Eudragit® RS suspended in the succinic acid aqueous solution, chloride ion was detected and its concentration was found to increase linearly with that of succinic acid environment. The relationship between chloride ion released and succinic anion of different concentrations is shown in Fig. 6, and the equation of linear regression was CCl− = 8.1 × 10−3 Csuc. + 5.639 × 10−4 ,
Fig. 5. Scanning electron microscope of EM pulsatile release pellets with coating level of 50% and 38% (w/w) succinic acid in the core. (A) Eudragit® RS coated pellets before dissolution, (B) Eudragit® RS coated pellets after dissolution at the time of 12 h under the condition of purified water at 37 ◦ C.
3.4. Investigation of the drug release mechanism 3.4.1. Ion-exchange experiment There’s a small amount of quaternary ammonium groups in the form of chloride in Eudragit® RS copolymer, which might have an ion-exchange interaction with the succinic acid despite of the small exchange capacity (Kaur and Kim, 2009). Thus evaporation
r 2 = 0.9627
where CCl− and Csuc. represent the concentration of chloride ion and succinic acid, respectively. Similar phenomena were found in other organic acids, and the exchange capacity decreased in the order of succinic acid, dl-tartaric acid, malic acid and citric acid. Eudragit® RS coated pellets exhibited pulsatile drug release profiles in the above organic acid aqueous solutions, which may indicate a close relationship between the ion-exchange interaction and the appearance of our designated unique pulsatile drug release behavior. From the EM release profiles in Fig. 2, the time of 50% drug released (T50 ) can be calculated. To examine the relationship between drug release enhancing effect and the acidity of various kinds of organic acid quantitatively, T50 was plotted against pKa of each organic acid in Fig. 7A. Fig. 7A shows an inverse correlation
Fig. 7. Relationship between T50 and pKa of various kinds of organic acids (A) and relationship between T50 and Hm of free Eudragit® RS film (B). The organic acids are citric acid (a), tartaric acid (b), malic acid (c), and succinic acid (d).
Z. Zhang et al. / International Journal of Pharmaceutics 462 (2014) 66–73
71
Table 2 Results of hydration study of Erudragit® RS free film in different organic acid solutions (n = 3).
Hm (%)
Water
Citric acid
Tartaric acid
Succinic acid
Malic acid
34 ± 1.32
45 ± 2.14
49 ± 2.03
92 ± 4.15
58 ± 3.85
Table 3 Concentration of succinate anion, pH values and EM release rate in different succinic acid and monosodium succinate aqueous solutions at 37 ◦ C. The release rate is calculated from the linear part of drug release curves. Substance
Concentration (M)
pH
Concentration of succinate anion (M)
Release rate (%/h)
Succinic acid
5 × 10−1 1 × 10−1 5 × 10−2 1 × 10−2 5 × 10−3
2.26 2.50 2.65 2.98 3.19
5.61 × 10−3 1.94 × 10−3 1.37 × 10−3 5.89 × 10−4 4.77 × 10−4
69.2 53.7 24.2 17.1 6.8
Monosodium succinate
5 × 10−1 1 × 10−1 1 × 10−2 1 × 10−3 1 × 10−4 1 × 10−5
4.46 4.53 4.68 4.75 4.82 5.27
2.34 × 10−1 6.82 × 10−2 5.82 × 10−3 7.82 × 10−4 8.01 × 10−5 9.02 × 10−6
7.1 15.6 24.6 36.3 20.5 10.2
between T50 and pKa , indicating a more effective impact of weaker acid. 3.4.2. Hydration study Table 2 shows Hm of free Eudragit® RS film in various kinds of organic acids was significant higher than in purified water, suggesting that organic acids’ release-increasing effect was close related to their enhancement on the hydration of free film (Okor, 1982). From Fig. 7B it was found that the T50 value increased with the decreasing Hm , which further demonstrated the above suggestion. The “free volume” concept has been proposed to explain the positive relationship between the hydration effect and film permeability (Yasuda and Lamaze, 1971). 3.4.3. A further study of undisassociated form of succinic acid Differential scanning calorimetry (DSC) (Fig. 8) shows the Tg before and after immersing into water, succinic acid and monosodium succinate aqueous solutions. The value of Tg had an evident decline in succinic acid compared with that in monosodium succinate aqueous solution, meaning that the flexibility of Eudragit® RS film was strengthened. When the disassociated form concentration coexisted in each test fluid is comparable,
undisassociated form also has a large enhancing effect (Table 3). For example, release rate in 5 × 10−1 and 1 × 10−1 M succinic acid solutions was much faster than that in 1 × 10−2 and 1 × 10−3 M monosodium succinate solutions, although the succinate anion concentration is almost the equal. Meanwhile, Hm of the free film in succinic acid solutions was dramatically higher than in monosodium succinate solutions, which indicated that succinic acid molecules increased the drug release in another special way. A possible explanation is related to the effect of cross-linking: when copolymer is acting as the drug delivery system, the permeability plays an important role in drug release. Structural changes and physical states, such as environmental temperature and flexibility of chains will definitely effect on their permeability. Chains or groups of copolymers will have more spacious room as flexibility increases, leading to an improvement in permeability. When lipophilic succinic acid molecules distribute into hydrophobic structure, the relative longer chains make a contribution to a better flexibility of copolymers. Thus the repelling force against hydration of insoluble Eudragit® RS is distinctly weaken and permeability is obviously increased. To conclude, the possible release mechanism was a combination of both disassociated and undisassociated forms of succinic acid physiochemically interacting with Eudragit® RS (see Fig. 9). 3.5. In vivo pharmacokinetic study To evaluate the in vivo performance, the pulsatile pellets and the marketed Yisu® were administered to rats. Fig. 10 shows the mean plasma EM concentration versus time profiles following oral administration, and the main pharmacokinetic parameters were shown in Table 4. The EM pulsatile release pellets exhibited an evident drug release after a lag time period
Table 4 Pharmacokinetic parameters obtained following oral administration of marketed Yisu® and the pulsatile release pellets containing EM to rats.
Fig. 8. Differential scanning calorimetry (DSC) curves of dried free Eudragit® RS film after being immersed in purified water 0.5 M succinic acid and monosodium succinate aqueous solutions for 24 h at 37 ◦ C.
Parameters
Marketed tablets
Pulsatile pellets
Cmax (ng/mL) Tmax (h) Tlag (h) AUC0− (h ng/mL) MRT0− (h) Relative bioavailability
170.333 ± 96.090 1.667 ± 0.289 0 810.817 ± 67.712 5.007 ± 0.441 100
75.322 ± 17.059 12.010 ± 0.320 4.534 ± 0.113 702.384 ± 96.891 11.717 ± 0.703 86.62
72
Z. Zhang et al. / International Journal of Pharmaceutics 462 (2014) 66–73
4. Conclusion Cured pulsatile release EM pellets coated with Eudragit® RS polymers were found to be an applicable form for antihypertension due to the relatively simple formula and manufacturing process. The drug release curves exhibited a clear lag time period in the early stage followed by a drug release phase. The release mechanism of pulsatile pellets was a combination of both disassociated and undisassociated forms of organic acid physiochemically interacting with Eudragit® RS. In vivo pharmacokinetic evaluation showed the relative bioavailability of designed EM pulsatile pellets was 86.62% and can be considered bioequivalence with marketed Yisu® .
Acknowledgements This work was financially supported by the Major State Basic Research Development Program of the National Science and Technology of China for new drugs development (Program No. 2012CB724002) and Natural Science Foundation of Jiangsu Province (No. BK20130663).
Fig. 9. Possible mechanism of acid- Eudragit® RS copolymers interactions in EM loaded pulsatile pellets.
of 4.534 ± 0.113 h (different with 4 h in vitro dissolution) and the mean value of Cmax was 75.322 ± 17.059 ng/mL and the Tmax was 12 ± 0.000 h after oral administration. However, conventional marketed tablets did not present any lag time before the drug release, with Cmax value of 170.333 ± 96.090 ng/mL and Tmax of 1.667 ± 0.289 h. The parameter AUC0− of the Eudragit® RS coated pellets and the market tablets was 702.384 ± 96.891 h ng/mL and 810.817 ± 67.712 h ng/mL (p > 0.05). The relative bioavailability of EM calculated from the ratio AUC ((testing products/reference products) × 100) was 86.62%. While the range of bioequivalence is 80–125%, the designed pulsatile pellets can be considered as bioequivalence with the marketed tablets (p > 0.05). The pulsatile pellets having lower AUC in contrast with Yisu® may be caused by the decreased solubility of drug in vivo microenvironment, leading to an absorption-reducing of EM.
Fig. 10. Mean plasma concentration-time profiles of EM from the pulsatile release pellets with coating level of 50% and 38% (w/w) succinic acid in the core and the marketed tablets following oral administration (Mean ± SD, n = 6).
References Angelatos, A.S., Radt, B., Caruso, F., 2005. Light-responsive polyelectrolyte/gold nanoparticle microcapsules. J. Phys. Chem. B 109, 3071–3076. Bussemer, T., Bodmeier, R., 2003. Formulation parameters affecting the performance of coated gelatin capsules with pulsatile release profiles. Int. J. Pharm. 267, 59–68. Bussemer, T., Dashevsky, A., Bodmeier, R., 2003. A pulsatile drug delivery system based on rupturable coated hard gelatin capsules. J. Control. Rel. 93, 331–339. Déjugnat, C., Haloˇzan, D., Sukhorukov, G.B., 2005. Defined picogram dose inclusion and release of macromolecules using polyelectrolyte microcapsules. Macromol. Rapid Commun. 26, 961–967. Gazzaniga, A., Sangalli, M.E., Giordano, F., 1994. Oral Chronotopic® drug delivery systems: achievement of time and/or site specificity. Eur. J. Pharm. Biopharm. 40, 246–250. Hermida, R.C., Ayala, D.E., Calvo, C., Portaluppi, F., Smolensky, M.H., 2007a. Chronotherapy of hypertension: administration-time-dependent effects of treatment on the circadian pattern of blood pressure. Adv. Drug Del. Rev. 59, 923–939. Hermida, R.C., Ayala, D.E., Portaluppi, F., 2007b. Circadian variation of blood pressure: the basis for the chronotherapy of hypertension. Adv. Drug Del. Rev. 59, 904–922. Innominato, P.F., Lévi, F.A., Bjarnason, G.A., 2010. Chronotherapy and the molecular clock: clinical implications in oncology. Adv. Drug Del. Rev. 62, 979–1001. Iskakov, R.M., Kikuchi, A., Okano, T., 2002. Time-programmed pulsatile release of dextran from calcium-alginate gel beads coated with carboxyn-propylacrylamide copolymers. J. Control. Rel. 80, 57–68. Karavas, E., Georgarakis, E., Bikiaris, D., 2006. Application of PVP/HPMC miscible blends with enhanced mucoadhesive properties for adjusting drug release in predictable pulsatile chronotherapeutics. Eur. J. Pharm. Biopharm. 64, 115–126. Kashyap, N., Viswanad, B., Sharma, G., Bhardwaj, V., Ramarao, P., Ravi Kumar, M., 2007. Design and evaluation of biodegradable, biosensitive in situ gelling system for pulsatile delivery of insulin. Biomaterials 28, 2051–2060. Kaur, K., Kim, K., 2009. Studies of chitosan/organic acid/Eudragit® RS/RL-coated system for colonic delivery. Int. J. Pharm. 366, 140–148. Kiser, P.F., Wilson, G., Needham, D., 1998. A synthetic mimic of the secretory granule for drug delivery. Nature 394, 459–462. Kiser, P.F., Wilson, G., Needham, D., 2000. Lipid-coated microgels for the triggered release of doxorubicin. J. Control. Rel. 68, 9–22. Lemmer, B., 1999. Chronopharmacokinetics: implications for drug treatment. J. Pharm. Pharmacol. 51, 887–890. Lin, H.-L., Lin, S.-Y., Lin, Y.-K., Ho, H.-O., Lo, Y.-W., Sheu, M.-T., 2008. Release characteristics and in vitro–in vivo correlation of pulsatile pattern for a pulsatile drug delivery system activated by membrane rupture via osmotic pressure and swelling. Eur. J. Pharm. Biopharm. 70, 289–301. Lynn, D.M., Amiji, M.M., Langer, R., 2001. pH-responsive polymer microspheres: rapid release of encapsulated material within the range of intracellular pH. Angew. Chem. Int. Ed. 40, 1707–1710. McConville, J.T., Ross, A.C., Florence, A.J., Stevens, H.N., 2005. Erosion characteristics of an erodible tablet incorporated in a time-delayed capsule device. Drug Dev. Ind. Pharm. 31, 79–89. Miyamoto, S., Fujita, M., Tambara, K., Sekiguchi, H., Eiho, S., Hasegawa, K., Tamaki, S.-i., 2004. Circadian variation of cardiac autonomic nervous activity is well preserved in patients with mild to moderate chronic heart failure: effect of patient position. Int. J. Cardiol. 93, 247–252.
Z. Zhang et al. / International Journal of Pharmaceutics 462 (2014) 66–73 Narisawa, S., Nagata, M., Danyoshi, C., Yoshino, H., Murata, K., Hirakawa, Y., Noda, K., 1994. An organic acid-induced sigmoidal release system for oral controlledrelease preparations. Pharm. Res. 11, 111–116. Ohdo, S., 2010. Chronotherapeutic strategy: rhythm monitoring, manipulation and disruption. Adv. Drug Delivery Rev. 62, 859–875. Okor, R., 1982. Effect of polymer cation content on certain film properties. J. Pharm. Pharmacol. 34, 83–86. Portaluppi, F., Lemmer, B., 2007. Chronobiology and chronotherapy of ischemic heart disease. Adv. Drug Del. Rev. 59, 952–965. Radt, B., Smith, T.A., Caruso, F., 2004. Optically addressable nanostructured capsules. Adv. Mater. 16, 2184–2189. Roy, P., Shahiwala, A., 2009a. Multiparticulate formulation approach to pulsatile drug delivery: current perspectives. J. Control. Rel. 134, 74–80. Roy, P., Shahiwala, A., 2009b. Statistical optimization of ranitidine HCl floating pulsatile delivery system for chronotherapy of nocturnal acid breakthrough. Eur. J. Pharm. Sci. 37, 363–369.
73
Skirtach, A.G., Dejugnat, C., Braun, D., Susha, A.S., Rogach, A.L., Parak, W.J., Möhwald, H., Sukhorukov, G.B., 2005. The role of metal nanoparticles in remote release of encapsulated materials. Nano Lett. 5, 1371–1377. Sungthongjeen, S., Puttipipatkhachorn, S., Paeratakul, O., Dashevsky, A., Bodmeier, R., 2004. Development of pulsatile release tablets with swelling and rupturable layers. J. Control. Rel. 95, 147–159. Wagner, K.G., McGinity, J.W., 2002. Influence of chloride ion exchange on the permeability and drug release of Eudragit RS 30 D films. J. Control. Rel. 82, 385–397. Yadav, D., Survase, S., Kumar, N., 2011. Dual coating of swellable and rupturable polymers on Glipizide loaded MCC pellets for pulsatile delivery: formulation design and in vitro evaluation. Int. J. Pharm. 419, 121–130. Yasuda, H., Lamaze, C., 1971. Permselectivity of solutes in homogeneous water-swollen polymer membranes. J. Macromol. Sci. Part B: Phys. 5, 111–134.
