Controlled delivery of carvedilol nanosuspension from osmotic pump capsule: In vitro and in vivo evaluation

Accepted Manuscript Title: Controlled delivery of carvedilol nanosuspension from osmotic pump capsule: In vitro and in vivo evaluation Author: Dandan Liu Shihui Yu Zhihong Zhu Chunyang Lyu Chunping Bai Huiqi Ge Xinggang Yang Weisan Pan PII: DOI: Reference:

S0378-5173(14)00651-6 http://dx.doi.org/doi:10.1016/j.ijpharm.2014.09.008 IJP 14317

To appear in:

International Journal of Pharmaceutics

Received date: Revised date: Accepted date:

29-5-2014 18-8-2014 6-9-2014

Please cite this article as: Liu, Dandan, Yu, Shihui, Zhu, Chunyang, Bai, Chunping, Ge, Huiqi, Yang, Xinggang, Controlled delivery of carvedilol nanosuspension from osmotic In vitro and in vivo evaluation.International Journal of http://dx.doi.org/10.1016/j.ijpharm.2014.09.008

Zhihong, Lyu, Pan, Weisan, pump capsule: Pharmaceutics

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Controlled delivery of carvedilol nanosuspension from osmotic pump capsule: In vitro and in vivo evaluation

Dandan Liu1,2, Shihui Yu1, Zhihong Zhu1, Chunyang Lyu1,Chunping Bai2, Huiqi Ge2, Xinggang Yang1, Weisan Pan1,*

School of Pharmacy, Shenyang Pharmaceutical University, Shenyang 110016, PR

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Technology, Benxi 117004, PR China

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School of Biomedical & Chemical Engineering, Liaoning Institute of Science and

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China

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∗ Corresponding author at: School of Pharmacy, Shenyang Pharmaceutical

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University, PO Box No. 122, 103 Wenhua Road, Shenyang 110016, PR China.

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Tel.: +86 24 23986313; fax: +86 24 23953241.

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E-mail addresses: [email protected] (D. Liu), [email protected] (W. Pan).

Graphical abstract

Abstract This study intended to develop a novel controlled delivery osmotic pump capsule of carvedilol nanosuspension. The capsule is assembled using a semi-permeable

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capsule shell with contents including nanosuspension drying powder, mannitol and Plasdone S-630. The physical characteristics of semi-permeable capsule walls were

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compared among different coating solutions under different temperature. The

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composition of the coating solution and drying temperature appeared to be important

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for the formation of the shells. Carvedilol nanosuspension was prepared by

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precipitation-ultrasonication technique and was further lyophilized. Response

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surface methodology was used to investigate the influence of factors on the

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responses. The optimized formulation displayed complete drug delivery and

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zero-order release rate. The TEM and particle size analysis indicated that the

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morphology of the resultant nanoparticle in the capsule was spherical shaped with a

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mean size of 252 ± 19 nm. The in vivo test in beagle dogs demonstrated that the relative bioavailability of the novel system was 203.5% in comparison to that of the

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marketed preparation. The capsule successfully controlled the release of carvedilol and the fluctuation of plasma concentration was minimized. The system is a promising strategy to improve the oral bioavailability for poorly soluble drugs and preparing it into elementary osmotic pump conveniently.

Keywords: Carvedilol; Nanosuspensions; Osmotic pump; Central composite design; Oral bioavailability; Poorly water-soluble drugs

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1. Introduction Recently, about 40% of active pharmaceutical ingredients (API) identified by

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pharmaceutical companies are poorly soluble in water, which greatly hinders their

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clinical application (Chen et al., 2010). Low aqueous solubility puts a large burden

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on the oral bioavailability and absorption of these agents. Consequently, a variety of

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new drug delivery systems have been investigated to overcome these limitations,

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such as nanosuspensions, nanoemulsions, solid lipid nanoparticles, solid dispersions,

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2011; Xu et al., 2009).

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and liposomes (Fu et al., 2013; Gao et al., 2011a; Gao et al., 2011b; Planinsek et al.,

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Among these new formulation approaches, nanosuspensions are considered to

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be the most promising candidate for poor water-soluble drugs due to their non-specific applicability. The resulting drug nanoparticles have an increased

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surface area and an enhanced solubility which in turn could result in substantial increases in drug dissolution rate and oral bioavailability (Sinha et al., 2013). However, this will give rise to pronounced fluctuations in plasma concentration and it is definitely harmful to patients with specific disease such as hypertension and

diabetes. Therefore, it is necessary to develop a controlled delivery system of nanosuspensions to solve the problem. It is well known that osmotic drug delivery systems (OPS) utilize osmotic pressure as energy source and driving force for delivery of drugs. It has been extensively used for controlled drug delivery in oral administration. Drug

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dissolution from OPS exhibits zero-order release kinetics irrespective of media pH, presence of food and other physiological factors (Abrahamsson et al., 1998; Liu and

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Che, 2006). In the 1970s, elementary osmotic pump (EOP) was developed

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(Theeuwes, 1975). As known to all, EOP is very simple to prepare. However, it is

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only suitable for water soluble drugs. For those poor water-soluble drugs, since they

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could hardly dissolve in water, they could not generate osmotic pressure by

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themselves. To overcome this limitation, push–pull osmotic pump (PPOP)

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(Theeuwes, 1978) was specially designed. However, the disadvantage of PPOP, side

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identification and double compression, exists until today, which makes the

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production procedure complex and increases the chances of defective goods.

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Osmotic pump capsules (Guan et al., 2009; Philip and Pathak, 2007; Thombre et al., 1999a; Thombre et al., 1999b; Wang et al., 2005) have also been developed using

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controlled porosity from asymmetric membranes to solve the problem. But they can only deliver drugs with sufficient solubility. Carvedilol is a non-selective β-blocking agent (Frishman WH, 1998). It has been used extensively in patients with essential hypertension, heart failure, and

patients with systolic dysfunction after myocardial infarction. Its oral bioavailability in humans is quite low (30% or less) due to its poor water solubility and extensive first-pass metabolism in the liver (Chen and Chow, 1997). Our previous studies have proved that carvedilol nanosuspension could improve drug bioavailability after oral administration. But it showed fast drug release behavior and extensive fluctuations

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in plasma concentration (Liu et al., 2012). To avoid the limitations mentioned above and to propose a new solution for

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poorly water soluble drugs to be prepared into EOP, a novel controlled delivery

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osmotic pump capsule of drug nanosuspenion was designed in the present work. The

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capsule consists of semi-permeable capsule shell with contents including drug

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nanosuspension drying powder, penetration enhancers and suspending agents.

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Carvedilol nanosuspension drying powder was obtained by lyophilisation.The

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capsule shell was produced from cellulose acetate (CA) by perfusion method. The

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formulation and preparation factors which affect the characterization of the capsules

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were investigated. Central composite design (CCD) was used to optimize the

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formulation of the capsule. Transmission electron microscope (TEM) and particle size analysis were performed to investigate the characterization of the resulting

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particle in the capsule. Finally, the pharmacokinetics study was carried out in beagle dogs. 2. Materials and methods 2.1. Materials Carvedilol was purchased from Shandong Qilu Pharmaceutical Co., Ltd.

(Shandong, China). Carvedilol commercially available tablet (Luode® , 10 mg/tablet) was purchased from Beijing Juneng Pharmaceutical Co. Ltd. (Beijing, China). VES (alpha-tocopherol succinate) was obtained from Jiangsu Xixin Vitamin Co., Ltd. (Jiangsu, China). SDS (sodium dodecyl sulfate) and PEG 400 were obtained from TianJin Bodi Chemical Holding Co., Ltd. (Tianjin, China). Propranolol was purchased from Changzhou Yabang Pharmaceutical Co., Ltd. (Jiangsu, China). Copovidone (Plasdone S-630) were gifted from International

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Specialty Products (ISP) Co. (New Jersey, U.S.A.). Cellulose acetate (CA, 54.5 –

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56.0 wt. % acetyl content) was from Sinopharm Chemical Reagent Co. (Shanghai, China). Acetone, ethyl alcohol, diethyl ether and diethyl phthalate (DEP) were from

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Yuwang Chemical Reagent Co. (Shandong, China). All other chemicals and reagents

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used were of analytical grade or better.

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2.2. Preparation of carvedilol nanosuspension drying powder

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Carvedilol nanosuspensions were prepared by anti-solvent

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precipitation-ultrasonication technique. First, the organic phase was prepared by

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adding carvedilol (296 mg/mL) and VES (195mg/mL) to acetone and stirred until

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complete dissolution. The solution was then passed through a 0.45 μm filter

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(Shanghai Huan’ao Trading Company, Shanghai, China) to remove the possible impurities. Meanwhile, the anti-solvent phase was prepared by dissolving SDS

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(0.47%, w/v) in distilled water. At 10°C, 1 ml of the organic solution was quickly injected by syringe into 50 ml of anti-solvent using a B25 high shear homogenizer (BRT Equipment Technology Co. Ltd., Shanghai, China) for 1 min at 10,000 rpm. The obtained premix was treated with an Ultrasonic Processor (20–25 kHz, Ningbo

Scientz Biotechnology Co. Ltd., China) for 15 min (active every 3 s for a 3 s duration, 400 W) to form homogenous nanoparticles. During the process, the temperature was controlled at 4~8 °C using an ice-water bath. For the downstream processing of the preparation, the obtained nanosuspensions were freeze-dried with cryoprotectant (i.e. maltose) at the concentration of 3% w/v. The nanosuspensions

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were pre-frozen in the refrigerator at -75 °C for 12 h and subsequently freeze-dried in a FD-1C-50 freeze-drier (Boyikang Laboratory Instruments Co. Ltd., China) at

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−25 °C for 12 h, followed by a secondary drying phase at 20 °C for 4 h.

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2.3. Preparation of CA capsule shells

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The coating solution was prepared by dissolving CA, PEG 400 and DEP in 100

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mL of acetone. This solution was first filled into Coni-Snap® hard gelatin capsule

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shells (Size 00el, CAPSUGEL, Suzhou, China), which were used as the molds. Then

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the filled shells were dried at a fixed temperature. After volatilizing the acetone, CA

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semi-permeable capsule shells were formed and were withdrawn from the hard

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gelatin capsule shells.

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2.4. Preparation of osmotic pump capsules Freeze-dried carvedilol nanosuspensions powder and other excipients were

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passed through a 60 mesh screen respectively and precisely weighed using an electronic balance (Shanghai Minqiao Precise Science Instrument Co., Shanghai, China) as well as mixed artificially with a plastic bottle. After the drug-excipient mixture was filled into the CA capsules in the form of powders, the capsule shells

were trimmed to size with a razor blade and the two halves were joined and sealed with the coating solution. Finally, a drug release orifice with the size of 0.4 mm was drilled with a micro drill on either side of the capsule. 2.5. Particle size analysis and zeta potential measurement Particle size and size distribution of nanosuspensions were determined by laser

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diffraction using a Coulter LS 230 analyzer (Beckman-Coulter Co. Ltd., USA) at room temperature. The zeta potential was measured using a Malvern Zeta Sizer

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(Malvern Instruments, UK).

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2.6. TEM analysis

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TEM analysis was performed to evaluate the morphology of the resulting

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nanosuspension in the capsule. The content of the capsule was placed in 100 mL

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distilled water. After evenly dispersion, the obtained solution was filtered through a

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0.8 μm filter to remove the insoluble materials. Then the filtered solution was

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observed by a Hitachi H-600 transmission electron microscope (Hitachi

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High-Technologies Co., Ltd., Tokyo, Japan). Samples for TEM analysis were

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prepared by drying a dispersion of the particles on a copper grid coated with amorphous carbon film and then negative staining with 1% (w/v) phosphotungstic

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acid.

2.7. In Vitro release test In vitro dissolution test was conducted in a dissolution apparatus (ZRS-6G, TiandaTianfa Technology Co., Ltd, Tianjin, China) according to the USP paddle

method. The capsules (containing 12.5 mg of carvedilol) were placed in stainless steel sedimentation baskets. The test was carried out under a stirring rate of 100 rpm in 500 mL artificial simulated gastric fluid (pH 1.0) thermostatically maintained at 37 ± 0.5 °C. Samples of 5 mL were drawn, and the same volume of fresh dissolution medium was added at 2, 4, 6, 8, 10 and 12 h, respectively. Then, the samples were

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filtered through a 0.1 μm syringe filter (Shanghai Huan’ao Trading Company,

determined with a UV spectrophotometer at 242 nm.

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2.8. Experimental design and data analyzing

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Shanghai, China) immediately before dilution, when necessary. Drug content was

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According to the results of preliminary experiments, response surface

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methodology based on Central Composite Design (CCD) was utilized to evaluate

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the formulation factors that affect the ultimate cumulative release in 12 h (Y1) and

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correlation coefficient of drug release profile (Y2), i.e., content of Plasdone S-630

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(X1), content of mannitol (X2) and content of PEG 400 (X3) in the coating solution,

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respectively. The experiments were designed by DesignExpert® software. Table1

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shows the corresponding CCD in the present study and the experiments were completely randomized.

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The two responses obtained for this study were well modeled using the

Design-Expert® software. The second-order polynomial model was given as below. Yi   0  1 X 1   2 X 2   3 X 12   4 X 22   5 X 1 X 2

(1)

whereYi represents the predicted response, X represents the independent variable, and β represents the coefficient. F-test was used to evaluate lack-of-fit. The nominal of which p>0.05 were selectively deleted for model simplifying within each equation. Graphs of surface responses were plotted using Origin 8.0 software according to the equation.

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2.9. In Vivo pharmacokinetic study in beagle dog The experimental protocol was conducted in accordance with the Ethical

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Guidelines for Investigations in Laboratory Animals and was approved by the Ethics

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Review Committee for Animal Experimentation of Shenyang Pharmaceutical

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University (Shenyang, China). Eight healthy male beagle dogs weighing 10~13 kg,

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were divided into two groups comprising four animals in each. A randomized,

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two-period crossover single-dose study was conducted and the washout period was

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one week. The dogs were fasted overnight with free access to water. Two types of

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carvedilol formulations at a dose of 50 mg/body weight were orally administrated to

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two groups of dogs, i.e. carvedilol nanosuspension osmotic pump capsules (the test

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preparation) and commercial tablets (the reference preparation). Blood samples (3 mL) were collected before administration and at 0.25, 0.5, 0.75, 1, 1.5, 2, 3, 4, 6, 8,

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10, 12, 14 and 24 h after administration. Plasma samples were obtained after centrifugation (4,000×g) for 10 min and stored at −20 °C until determination. The concentration of carvedilol in plasma was determined using a validated HPLC assay.

The HPLC conditions for carvedilol analysis conformed to the former researches (Liu et al., 2012). Pharmacokinetic data analysis was carried out using DAS 2.0 software (Mathematical Pharmacology Professional Committee of China, Shanghai, China). The various pharmacokinetic parameters that were analyzed including maximum

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peak concentration of the drug in plasma (Cmax), the time to reach maximum concentration (Tmax), and the area under the plasma concentration-time curve

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(AUC0-t). All results were presented as mean ± S.D. values. Student’s t tests and

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ANOVA were performed to determine the significance of any differences. The level

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of significance was defined as p value <0.05 using the statistical package for social

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3. RESULTS AND DISCUSSION

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science (SPSS, version 12.0).

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3.1 Characterization of the lyophilized nanosuspensions

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In this study, prepared nanosuspensions were used to generate orally controlled

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release osmotic pump capsules. Therefore, drying of nanosuspensions should be

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considered as an important step. Freeze-drying was selected as the drying method. In the process of lyophilisation, maltose was added to the nanosuspensions to protect

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them from freezing damage and to minimise the particle size growth. The mean particle size and zeta potential of the nanoparticles were determined before and after lyophilisation. As it can be seen in Table 2, the lyophilisation process little influences the characterization of the particles.

3.2 Factors affecting the physical characteristics of CA capsule shells Factors influencing the physical characterization of the shells, i.e., the uniformity, flexibility, and the color of the shells were investigated by changing the formulation of the coating solution and the drying temperature. The physical characteristics of capsules so obtained are shown in Table 3. From the formulation

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of F1, F2 and F3, it can be observed that the uniformity of the shells firstly enhanced then worsened with an increase in CA level. CA is the membrane forming material.

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The amount of it affects the viscosity of the coating solution significantly. When the

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concentration of it was low (F1), the coating solution tended to accumulate at the

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bottom of the mold during the formation of the shells due to the low viscosity of the

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solution. However, an excessive amount of CA would make the viscosity of the

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solution too high (F3). The perfusion of the solution was found to be difficult and

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the homogeneity of shells became worse. As shown in formulation F2, F4, F5 and

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F6, when DEP was added, the formability of the shell was much improved. DEP acts

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as a plasticizer and is essential to maintain the flexibility and strength of the film. If

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the DEP/CA mass ratio falls below the limit (F5), the capsules will become brittle. PEG 400 with dual functionality of plasticizer and pore former is also an important

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factor that influences the appearance of the shells. Therefore the effect of PEG 400 on the final properties of capsule shells was evaluated in formulation F2, F7, F8 and F9. When the ratio of PEG 400/CA reached 36% (F9), the shells would become white. Therefore, only an appropriate ratio of PEG 400/CA makes good capsule

appearance. From the formulation of F2, F10 and F11, it can be seen that the properties of the shells were significantly influenced by drying temperature. The evaporation rate of acetone was directly influenced by manufacturing temperature. When the temperature was low, acetone could not leave the capsule effectively leaving ununiformed capsule shell and elongating the producing time. When the

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temperature was high, the acetone evaporate too fast leading to the transformation, bodiness and bubbling of the shell. Therefore, the capsule shells were prepared at

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4~8 °C.

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3.3 Selection of suspending agent

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It was reported that Poiseuille’s law of laminar flow could be employed to

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describe drug release from monolithic osmotic pump system as described in Eq. (2)

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(Lu et al., 2003)

dM  C R 4 P1  P2 = dt 8  h

(2)

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where dM/dt is the drug release rate, C is the drug concentration in the capsule

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nanosuspension, r is the radius of the orifice, η is the viscosity of the

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nanosuspension, (P1−P2) is the pressure difference between the inside and outside of

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the capsule shells, and h is the capsule shell thickness. From Eq.(2), the drug release rate is inversely proportional to η. It was found that the type of suspending agent affected the overall release rate greatly (data not shown). Plasdone S-630 is a synthetic, 60:40, linear, random copolymer of N-vinyl-2-pyrrolidone and vinyl acetate. The addition of vinyl acetate to the vinylpyrrolidone polymer chain reduces

hydrophilicity and glass transition temperature of the polymer relative to PVP homopolymer. As a result, Plasdone S-630 has a certain surface activity. It is commonly used to improve the dissolution of drug and prevent amorphous drug recrystallization (Hong et al., 2011). In our study, the presence of Plasdone S-630 created a stable environment that assured the proper viscosity of the content within

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the capsule and effectively avoided the particle settlement inside the capsule on nanosuspension delivery.

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3.4 Central composite design

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A total of 20 experiments were conducted to evaluate the influence of

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parameters on the two responses. Observed response data for all experimental runs

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of CCD are listed in Table 4. The data was fitted to a quadratic polynomial model

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and the equations are shown below in terms of coded factors:

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Y1  90.49-2.72X 1  1.28 X 3  1.15 X 22

(4)

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Y2  0.99+0.015X 1  4.455  103 X 2 +4.867  103 X 1 X 2  7.785  103 X 12

(3)

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Each obtained model was validated by ANOVA to determine the significance of the variable effects and their interactions. The values of the determination coefficient

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(R2) were 0.8823 and 0.9677, which implied that 88.23% and 96.77% of the sample

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variation in the two responses was attributed to the independent variables. Accordingly, the fitted equations can predict the best formulation for the capsules. 3.4.1 Influence of factors on the ultimate cumulative release in 12 h (Y1) and the correlation coefficient of drug release profile (Y2)

As can be seen from (a), (b), (d) and (e) of Fig.1,Y1 decreases as X1 increases in a qualified range, while Y2 decreases after increasing with an increase of X1. This could be explained that when the Plasdone S-630 of low percentage, the viscosity of the nanosuspension containing in the pumps was low, an initial burst release with subsequent emptying of the pumps and high variability in release behavior among

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capsules was observed. Therefore the capsules exhibited a release deviating from the expected zero order. More Plasdone will provide appropriate viscosity and the

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release behavior differences among capsules were reduced. However, if the Plasdone

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content is high, it will impede the drug release due to its high inherent viscosity. As

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shown in (a), (c), (d) and (f) of Fig.1, Y1 increases and Y2 decreases with an increase

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of X2. Mannitol, the osmotic agent, played an important role in speeding up both

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hydration rate and swelling rate of core. More mannitol will provide better osmotic

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effects. Hence, Plasdone is hydrated sufficiently and more drug will be released.

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However, if the mannitol content is high, it could cause higher osmotic pressure

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which could introduce more medium influx into the membrane leading to an earlier

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and faster drug release. From (b), (c), (e) and (f) of Fig.1, when X3 raises, Y1 increases and Y2 shows no significant changes. PEG400 was used as pore-forming

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agent as well as plasticizers in the capsule shells. The more PEG400 in the shells, the permeability of the membrane increased, the faster and completely drug released from the capsules. 3.4.2 Formulation optimization

The optimum ranges for each factor were found to generate capsules with maximum Y1 and Y2. The optional formulation of the capsules is not only predicted by the equation but also responds in response surface. The predicted formulation was that Plasdone S-630: 200 mg, mannitol: 94 mg, and PEG400: 2.34g. Using the formulation predicted above, the predicted as well as actual values of the two

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responses are shown in Table 5. The biases for the formulation were no more than 1%, which proved the model was effective. The release profile of the capsule

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prepared using the optimized formulation was shown in Fig.2. The ultimate

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cumulative release and correlation coefficient of drug release profile of the

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optimized formulation indicate that this novel osmotic pump capsule is able to

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deliver drug completely and performs a zero-order release rate based on the

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mechanism of osmotic pump.

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3.5 Characterization of the capsule content

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The morphology of the resulting nanoparticle in the capsule were examined.

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TEM images (Fig.3) showed that the particles obtained had a nearly spherical shape.

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The resultant nanosuspension for TEM analysis was also used for the particle size measurement. The average diameter of the nanoparticle was found to be 252 ± 19

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nm. This further indicate that nanosuspension was formed in the capsule. 3.6 In vivo studies The main pharmacokinetic parameters and the plasma concentration–time profiles of carvedilol resulted from the oral administration of the novel osmotic

pump capsule of nanosuspension and the reference formulation in beagle dogs are presented in Table 6 and Fig. 4, respectively. As expected, peak plasma concentration (Cmax) for the novel capsules is 706.59 ng/mL against carvedilol conventional tablet of 1062.61 ng/mL. Similarly, time to reach peak plasma concentration (tmax) is extended from 2.75 h for conventional tablet to 5.75 h for the

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capsule, and the elimination half-life (t1/2) is also elongated from 4.17 to 6.42 h as in case of the capsule. These findings clearly indicate that the developed novel capsule

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successfully controlled the release of carvedilol and the fluctuation of plasma

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concentration was minimized. The results confirm that the pharmacokinetic behavior

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of the drug was modified and the side effects of drugs will be reduced. What is more

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important, the area under the curve (AUC0-∞) for the capsule is 9013.95 ng•h/mL,

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compared with 4657.86 ng•h/mL for the commercial tablet. The relative

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bioavailability is about 203.5% in comparison to that of the marketed preparation.

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The possible reason for the improved absorption of the drug might be that the drug

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released from the capsule in the form of nanoparticles. The small particle size

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provided a large interfacial surface area for drug absorption. Meanwhile, the nanoparticles could be direct absorbed by lymph, thus avoid the first-pass

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metabolism effect, which is primarily responsible for the low bioavailability of carvedilol. 3.7 Release Mechanism As illustrated by Fig.5, the capsule is composed of semi-permeable capsule

shell with contents including drug nanosuspension, penetration enhancers and suspending agents. After administration, water entered the capsule through the semi-permeable shell, and cause a hydration and expansion of Plasdone, the solution of mannitol, and the dispersion of the nanoparticles. A homogeneous solution of drug nanoparticles was formed inside the capsule shell. As Theeuwes’s study

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(Theeuwes, 1975), the drug release rate from an elementary osmotic pump system

dM A  Lp C   p  dt h

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can be described by the following equation:

(5)

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where dM/dt is the volume influx rate of water across the semipermeable membranes,

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A is the membrane area of the capsule shell, h is the wall thickness, Lp is the filtration

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coefficient, C is the concentration of drug in the dispersed fluids, σ is the reflection

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coefficient, Δπ and Δp are the osmotic and hydrostatic pressure differences,

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respectively.

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When the osmotic pressure of the formulation is much more great compared

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with the hydrostatic pressure of the environment, the value of (σΔπ– Δp) can be

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substituted by that of σΔπ. When the formulation and manufacturing parameters were determined, the area and thickness of the membrane were fixed, then the Eq.(5) can

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be reduced to a simpler expression in which constant K replaces the product of (A/h)Lpσ. After simplification, the following equation is obtained: dM  K  C dt

(6)

As for the novel osmotic pump capsule, Δπ was generated by the osmotic effect of mannitol (Δo) and the saturated solution of drug nanoparticles (Δn), and the swelling pressure of Plasdone (Δs). Then Eq.(6) can be changed to the following equation: dM  K( s+o+n) C dt

(7)

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Therefore, a constant release of drug from the systems would be realized as long as the three kinds of materials above are at a appropriate ratio in the core to maintain

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both total pressure and concentration at a constant level. The drug released at a

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constant rate in the form of nanoparticles until the content of the capsule dissolved

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completely. After that the pressure difference between the inside and outside of the

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capsule shells decreased gradually, the drug release rate would slow down.

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4. Conclusion

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The present study developed a novel osmotic pump capsule which can be

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applied to controlled delivery of drug nanosuspensions. The capsule is assembled

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using a semi-permeable capsule shell with contents including drug nanosuspension

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drying powder, penetration enhancers and suspending agents. The physical characteristics of the capsule shells were compared by dissolving CA using different

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ratios of PEG 400 and DEP under different temperature. The uniformity, flexibility, and the color of the shells was highly dependent on the composition of the coating solution and drying temperature. The content of Plasdone S-630, mannitol, and the concentration of PEG 400 in the coating solution were found to have an important

effect on the drug release behavior. The formulation optimization was carried out by central composite design response surface methodology. The actual responses for the optimum formulation were in close agreement with the predicted values, indicating the excellent predictability of the optimization procedure. With the optimized formulation, the novel capsule displayed a complete drug delivery and zero-order

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release rate in the medium. The TEM morphology and particle size analysis manifested that spherical nanoparticle was formed in the capsule and the mean

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particle size was 252 ± 19 nm. The in vivo test in beagle dogs demonstrated that the

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novel system could improve the bioavailability of carvedilol significantly and the

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plasma concentrations were more stable than that of the marketed tablets. In

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summary, the novel controlled delivery nanosuspension system combined the

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advantages of nanosuspension and osmotic pump system. It was a promising

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strategy in improving the oral bioavailability, minimizing the frequency of drug

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administration and lower the average peak plasma concentration of poorly soluble

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drugs.

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Acknowledgements This work was supported by State Key Laboratory of Long-acting and

Targeting Drug Delivery System, by NSFC for Talents of Basic Sciences-Undergraduate Project for Research Training (No.J1103606), and by a

special construction project which belongs to “Taishan Scholar-Pharmacy Specially Recruited Experts”.

References

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Abrahamsson, B., Alpsten, M., Bake, B., Jonsson, U.E., Eriksson-Lepkowska, M., Larsson, A., 1998. Drug absorption from nifedipine hydrophilic matrix

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extended-release (ER) tablet-comparison with an osmotic pump tablet and effect of

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food. J. Controlled Release 52, 301-310.

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Chen, B., Chow, M., 1997. Focus on carvedilol: a novel beta-adrenergic blocking

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agent for the treatment of congestive heart failure. Formulary 32.

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Chen, H., Khemtong, C., Yang, X., Chang, X., Gao, J., 2010. Nanonization

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strategies for poorly water-soluble drugs. Drug Discovery Today

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Frishman WH, 1998. Carvedilol. N Engl J Med. 339, 1759-1765.

Fu, Q., Sun, J., Zhang, D., Li, M., Wang, Y., Ling, G., Liu, X., Sun, Y., Sui, X., Luo, C., Sun, L., Han, X., Lian, H., Zhu, M., Wang, S., He, Z., 2013. Nimodipine nanocrystals for oral bioavailability improvement (part I): Preparation,

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Gao, F., Bu, H., Zhang, Z., Xiao, J., Li, Y., 2011a. Solid lipid nanoparticles loading candesartan cilexetil enhance oral bioavailability: in vitro characteristics and absorption mechanism in rats. Nanomedicine: Nanotechnology, Biology and

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Medicine.

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Gao, F., Zhang, Z., Bu, H., Huang, Y., Gao, Z., Shen, J., Zhao, C., Li, Y., 2011b.

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Nanoemulsion improves the oral absorption of candesartan cilexetil in rats:

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Controlled Release Society 149, 168-174.

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Performance and mechanism. Journal of controlled release : official journal of the

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Guan, J., Zhou, L., Pan, Y., Han, H., Xu, H., Pan, W., 2009. A Novel

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Gastro-Retentive Osmotic Pump Capsule Using Asymmetric Membrane Technology:

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In Vitro and In Vivo Evaluation. Pharm. Res. 27, 105-114.

Hong, S.W., Lee, B.S., Park, S.J., Jeon, H.R., Moon, K.Y., Kang, M.H., Park, S.H.,

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Choi, S.U., Song, W.H., Lee, J., Choi, Y.W., 2011. Solid dispersion formulations of megestrol acetate with copovidone for enhanced dissolution and oral bioavailability. Arch Pharm Res 34, 127-135.

Liu, D., Xu, H., Tian, B., Yuan, K., Pan, H., Ma, S., Yang, X., Pan, W., 2012. Fabrication of carvedilol nanosuspensions through the anti-solvent precipitation-ultrasonication method for the improvement of dissolution rate and oral bioavailability. AAPS PharmSciTech 13, 295-304.

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the indented core tablet. Eur. J. Pharm. Biopharm. 64, 180-184.

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Liu, L., Che, B., 2006. Preparation of monolithic osmotic pump system by coating

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Lu, E.-X., Jiang, Z.-Q., Zhang, Q.-Z., Jiang, X.-G., 2003. A water-insoluble drug

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monolithic osmotic tablet system utilizing gum arabic as an osmotic, suspending and

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expanding agent. J. Controlled Release 92, 375-382.

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Philip, A., Pathak, K., 2007. In situ-formed asymmetric membrane capsule for

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varying degrees of solubility. Google Patents.

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Thombre, A., Cardinal, J., DeNoto, A., Gibbes, D., 1999a. Asymmetric membrane

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capsules for osmotic drug delivery II. In vitro and in vivo drug release performance.

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Thombre, A., Cardinal, J., DeNoto, A., Herbig, S., Smith, K., 1999b. Asymmetric

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membrane capsules for osmotic drug delivery: I. Development of a manufacturing

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process. J. Controlled Release 57, 55-64.

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Figure captions

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Fig. 1. Response surfaces for ultimate cumulative release in 12 h (Y1) and

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correlation coefficient of drug release profile (Y2) as functions of two factors.

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Fig. 2. In vitro cumulative release profiles of carvedilol nanosuspension osmotic

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pump capsules prepared with optimized formulation (mean ± S.D., n=6).

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Fig. 3. TEM morphology of the resulting nanosuspension in the capsule, bar = 300

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nm.

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Fig. 4. In vivo pharmacokinetics profiles of carvedilol in beagle dogs from the novel

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osmotic pump capsule and the commercial tablet (n=8).

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Fig. 5. Schematic diagram describing the process of drug release from the osmotic capsule system containing drug nanosuspension.

Table 1 Independent variables and their levels investigated in the central composite design Range and levels Variables

Symbols -1.682

-1

0

1

1.682

X1

150

170

200

230

250

Mannitol (mg)

X2

75

85

100

115

125

PEG 400 (g)

X3

2.08

2.14

2.24

2.34

2.40

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Plasdone S-630 (mg)

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Table 2 Particle size and zeta potential of carvedilol nanosuspension before and after lyophilisation (Mean ± S.D., n =3)

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Size distribution (nm) Zeta potential (mV) D10 D50 D90 Before lyophilisation 177 ± 7 212 ± 12 255 ± 15 -42 ± 3 After lyophilisation 172 ± 13 224 ± 9 271 ± 6 -39 ± 2

Table 3 Formulations and characterization of capsule shells Physical characterization

F1 F2 F3 F4 F5 F6 F7 F8 F9 F10 F11

12 12 12 6 16 12 12 12 12 12

6 8 10 8 8 8 8 8 8 8 8

4~8 4~8 4~8 4~8 4~8 4~8 4~8 4~8 4~8 -20 25

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(++) good; (+) moderate; (–) poor

Uniformity

Flexibility

Color

+ ++ + + ++ ++ ++ + -

++ ++ + + ++ ++ ++ + + +

Transp. Transp. Opag. Transp. Transp. Transp. Transp. Transp. Opag. Opag. Opag.

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DEP/CA (% w/w)

Drying temperature (°C)

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Code CA (g)

PEG 400/CA (% w/w) 28 28 28 28 28 28 20 32 36 28 28

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Formulation

Table 4 Factor levels and observed responses for CCD Levels of independent factors

Response

No. X2 (mg)

X3 (g)

Y1 (%)

Y2

1

200

100

2.40

90.15

0.9935

2

170

115

2.34

91.58

0.9471

3

250

100

2.24

85.39

0.9930

4

200

100

2.24

89.79

0.9839

5

150

100

2.24

95.24

0.9407

6

230

85

2.34

88.28

7

230

115

2.14

86.20

8

200

100

2.24

93.33

0.9894

9

200

75

2.24

85.69

10

200

125

2.24

87.75

0.9818

11

200

100

2.24

12

170

85

2.34

13

200

100

14

200

100

15

230

16

200

17

200

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0.9737

2.24

89.59

0.9869

2.24

87.81

0.9905

115

2.34

88.98

0.9945

100

2.08

86.08

0.9930

100

2.24

90.82

0.9849

170

85

2.14

89.85

0.9798

170

115

2.14

92.36

0.9654

230

85

2.14

82.96

0.9960

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0.9920

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20

0.9935

91.75

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19

0.9940

0.9925

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18

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X1 (mg)

factors—X1: content of Plasdone S-630, X2 : content of mannitol, X3: content of PEG 400; responses—Y1 : ultimate cumulative release in 12 h (%), Y2: correlation coefficient of drug release profile

Table 5 Comparision of the predicted value and observed value Response

Predicted value

Observed value

Errors (%)

Y1 (%)

90.99

91.09

-0.11

Y2

0.9901

0.9960

-0.60

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Bias (%)=(Actual value-Predicted value)/ Predicted value×100; responses—Y1: ultimate cumulative release in 12 h (%), Y2:correlation coefficient of drug release profile; response values: avg., n=6

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Table 6 In vivo parameters of the novel capsules and the commercial tablets after oral administration, . Novel capsules

Cmax (ng/mL)

706.59±187.71a

Tmax (h)

5.75±1.67a

T1/2 (h)

6.42±2.86a

4.17±3.14

7987.54±2023.87a

4498.89±1655.72

9013.95±2460.78a

4657.86±1727.24

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AUC0-t (ng•h/mL)

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Parameters

1062.61±416.26 2.75±0.71

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AUC0-∞ (ng•h/mL)

Commercial tablets

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All the data was presented in the form of mean±SD.

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Statistically significant compared with the commercial tablet (p<0.05)

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FIG 1 .

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FIG 2 .

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A FIG 3 .

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FIG 4 .

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FIG 5 .