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Development and characterization of an orodispersible film containing drug nanoparticles
European Journal of Pharmaceutics and Biopharmaceutics xxx (2013) xxx–xxx
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Research paper
Development and characterization of an orodispersible film containing drug nanoparticles Bao-de Shen a,b,1, Cheng-ying Shen a,c,1, Xu-dong Yuan d, Jin-xia Bai a,c, Qing-yuan Lv a, He Xu a,c, Ling Dai a,c, Chao Yu a,b, Jin Han a,⇑, Hai-long Yuan a,⇑ a
302 Hospital of PLA&PLA Institute of Chinese Materia Medica, Beijing, China Key Lab of Modern Preparation of TCM, Ministry of Education, Jiangxi University of Traditional Chinese Medicine, Nanchang, China Pharmacy College, Chengdu University of Traditional Chinese Medicine, Chengdu, China d School of Science, Monmouth University, West Long Branch, NJ, USA b c
a r t i c l e
i n f o
Article history: Received 30 May 2013 Accepted in revised form 28 September 2013 Available online xxxx Keywords: Nanosuspensions High pressure homogenization Orodispersible films Film casting
a b s t r a c t In this study, a novel orodispersible film (ODF) containing drug nanoparticles was developed with the goal of transforming drug nanosuspensions into a solid dosage form and enhancing oral bioavailability of drugs with poor water solubility. Nanosuspensions were prepared by high pressure homogenization and then transformed into ODF containing drug nanoparticles by mixing with hydroxypropyl methylcellulose solution containing microcrystalline cellulose, low substituted hydroxypropylcellulose and PEG400 followed by film casting and drying. Herpetrione, a novel and potent antiviral agent with poor water solubility that extracted from Herpetospermum caudigerum, was chosen as a model drug and studied systematically. The uniformity of dosage units of the preparation was acceptable according to the criteria of Japanese Pharmacopoeia 15. The ODF was disintegrated in water within 30 s with reconstituted nanosuspensions particle size of 280 ± 11 nm, which was similar to that of drug nanosuspensions, indicating a good redispersibility of the fast dissolving film. Result of X-ray diffraction showed that HPE in the ODF was in the amorphous state. In the in vitro dissolution test, the ODF containing HPE nanoparticles showed an increased dissolution velocity markedly. In the pharmacokinetics study in rats, compared to HPE coarse suspensions, the ODF containing HPE nanoparticles exhibited significant increase in AUC0–24h, Cmax and decrease in Tmax, MRT. The result revealed that the ODF containing drug nanoparticles may provide a potential opportunity in transforming drug nanosuspensions into a solid dosage form as well as enhancing the dissolution rate and oral bioavailability of poorly water-soluble drugs. Ó 2013 Elsevier B.V. All rights reserved.
1. Introduction At present nearly 40% of all newly developed drugs are poorly soluble in water, resulting in poor oral bioavailability [1], and thus limits their application. The main focus in the pharmaceutical development is to explore new technological approaches for enhancing water solubility of drugs with poor water solubility. Particle size reduction to the nanometer range has been reported to be a very promising approach for dissolution enhancement not only because of increased surface area but also because of increased saturation solubility as described by Ostwald–Freundlich equation [2]. Therefore, the poor drug solubility may be overcome by particle size reduction using nanoscience approaches [3]. In recent years, nanosuspensions as one of the nanoscience approaches has ⇑ Corresponding authors. 302 Hospital of PLA&PLA Institute of Chinese Materia Medica, Beijing 100039, China. Tel.: +86 1066933367; fax: +86 1063879589. E-mail addresses: [email protected] (J. Han), [email protected] (H.-l. Yuan). 1 Bao-de Shen and Cheng-ying Shen contributed equally to this work as first author.
become a promising and serious development tool for improving the water solubility, dissolution rate and oral bioavailability of poorly water soluble drugs by reducing the particle size in the pharmaceutical industry [4,5]. Nanosuspensions of drugs are a carrier-free sub-micron colloidal dispersion of drug particles in a dispersion medium with a mean particle size in the nanometer range, typically between 10 and 1000 nm, which are stabilized by polymers, surfactants or a mixture of both [6–8]. It can be produced either by top-down process (breaking of large drug particles as in milling and homogenization) or by bottom-up process (building up nanoparticles from drug molecules via precipitation) [9], as well as a combination of the two methods mentioned [10,11]. Apart from enhancing solubility or dissolution rate of poorly water-soluble drugs, nanosuspensions have some other advantages such as high drug loading, low incidence of side effects by the excipients, and low cost [6,12]. However, nanosuspension is a thermodynamic unstable system and the stability is one of the critical aspects in ensuring safety and efficacy of drug nanosuspensions. Storage and shipping of the drug
0939-6411/$ - see front matter Ó 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ejpb.2013.09.019
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nanosuspensions may also bring about a variety of stability problems such as sedimentation, agglomeration and crystal growth [13,14] and nanosuspensions are liquid formulations that are not convenient to take and carry, which seriously restricted the application and promotion of the nanosuspension delivery system [15]. So it is essential to convert the nanosuspension into a dry powder form for further physical stability and/or patient convenience reasons [2,16,17]. The solidification of nanosuspensions can be achieved by various methods such as freeze drying, spray drying and vacuum drying [18–21]. But the drying process is often accompanied by the irreversible aggregation of the nanoparticles, which then decreases the dissolution rate of drugs [5,22]. The redispersibility of drying drug nanoparticles is key issues that convert nanosuspension into solid nanoparticles. The drying drugs nanoparticles must have the ability to return to their original nanosuspension state upon reconstitution in water so that it could improve the oral bioavailability of poorly water-soluble drugs. Therefore, it is a great challenge to develop solid nanoparticles that have a good redispersibility for the enhancement of oral bioavailability of poorly water-soluble drugs. Orodispersible films (ODFs), a relatively new dosage form for oral route of administration, are postage stamp-sized strips of thin polymeric films formulated to disintegrate or dissolve almost instantaneously when placed onto the tongue [23,24]. They are used in case of patients who have difficulty in swallowing such as elderly, pediatric patients, and others suffering from mental illness and developmental disorders [25–27]. The drug in ODFs is absorbed through the oral mucosa, which make drugs enter the systemic circulation without undergoing first-pass hepatic metabolism [23,28,29]. More recently, ODFs are gaining interest as an alternative to fast dissolving tablets to definitely eliminate patients’ fear of chocking and improve patient compliance [30]. In addition, ODFs overcome the drawbacks of oral disintergrating tablets that they are fragile and brittle, which needs special package for protection during storage and transportation, and prepared by using the expensive lyophillization process [26,31]. However, ODFs are not suitable for the delivery of drugs with poor water solubility because the drugs with poor water solubility have poorly oral mucosa drug absorption. Self-microemulsifying mouth dissolving films were developed to improve the water solubility of poorly water-soluble drugs [32–34], but the self-microemulsifying microemulsion requires a huge amount of oils. Hydroxypropyl-b-cyclodextrin (HP-b-CD) and poloxamer 407 were also used to improve the poor solubility of drugs in the preparation of ODFs [35], but the application of HP-b-CD demands an appropriate molecular size. Moreover, they lack universal applicability to all drugs. Therefore, the drugs with poor water solubility are a real challenge in the development of ODFs. Upon consideration, ODFs seem to be a good choice for the solidification of drug nanosuspensions. It not only transforms drug nanosuspensions into solid products, but also overcomes the problem that ODFs are not suitable for the delivery of drugs with poor water solubility. In this study, a novel ODFs containing drug nanoparticles was developed. Herpetrione (HPE, Fig. 1), a novel and potent antiviral agent with poor water solubility that extracted from Herpetospermum caudigerum [36,37], was chosen as a model drug. HPE nanosuspensions were prepared by high pressure homogenization and then transformed into ODFs containing drug nanoparticles using casting method. The particle size distribution of HPE nanosuspensions and HPE nanoparticles re-dispersed from ODFs were investigated by photon correlation spectroscopy (PCS). The prepared ODFs were characterized using scanning electron microscopy (SEM) and X-ray diffraction (XRD). The content uniformity and stability were also tested. To verify the advantages of the ODFs containing HPE nanoparticles, the dissolution behavior and the pharmacokinetic profiles were investigated.
Fig. 1. Chemical structure of HPE.
2. Materials and methods 2.1. Materials Herpetrione (the purity is up to 98%, determined by area normalization method) was prepared in the laboratory of Dr. Yuan (302 Military Hospital of China, Beijing). Sodium dodecyl sulfate (SDS) and KollidonÒ 30 (PVP K-30) were obtained from the BASF Corp. (Ludwigshafen, Germany). Hydroxypropyl methylcellulose (HPMC, E50), 5% low substituted hydroxypropylcellulose (L-HPC) and microcrystalline cellulose (PH101, MCC) were purchased from Xi’an Fine Chemical Co., Ltd. (Xi’an, China). PEG-400 was obtained from Guangdong Guanghua Sci-Tech Co., Ltd. (Guangzhou, China). HPLC grade acetonitrile was purchased from Fisher, USA. All other chemicals were of analytical grade. 2.2. Preparation of HPE coarse suspensions HPE coarse suspensions were prepared by dispersing herpetrione in a SDS and PVP K-30 bidistilled water solution and then stirred by a magnetic stirrer (SH-2, Beijing Jinbeide Industrial And Trading Co., Ltd., China) to obtain a uniform suspension. Coarse suspensions were prepared using the herpetrione, SDS, PVP K-30 ratio of 1:0.2:0.3 (w/w/w). 2.3. Preparation of HPE nanosuspensions HPE nanosuspensions were prepared by trituration and ultraturrax homogenization, followed by a high pressure homogenization technique according to the literature method [38]. Briefly, the HPE powder (1%, w/v) was dispersed in an aqueous surfactant solution, containing SDS (0.2%, w/v) and PVP K-30 (0.3%, w/v) under trituration. Then the dispersion was pre-mixed using a high shear homogenizer (Ultra-TurraxÒT25, IKA, Germany) at 12,000 rpm for 5 min to obtain a uniform system. The obtained pre-mixture was homogenized at high pressure using a pistongap high pressure homogenizer (EmulsiFlex-C3, Avestin Inc., Ottawa, Canada). Firstly, 500 bar with 5 cycles as a kind of pre-milling was applied, and then 20 cycles at 1000 bar were run to obtain the nanosuspensions. 2.4. Preparation of orodispersible films containing HPE particles and nanoparticles The ODFs containing HPE nanoparticles were prepared by casting method [39] with a modification. Fig. 2 shows a schematic of the process used for film formation. In the optimized formulation
Please cite this article in press as: B.-d. Shen et al., Development and characterization of an orodispersible film containing drug nanoparticles, Eur. J. Pharm. Biopharm. (2013), http://dx.doi.org/10.1016/j.ejpb.2013.09.019
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[40], the constituents of the basic materials were HPMC (5%, w/v), L-HPC (0.5%, w/v), MCC (0.12%, w/v), PEG-400 (5%, v/v) and mannitol (10%, w/v). The bases of the film preparation dispersed or dissolved in an appropriate amount of water were then added into the HPE nanosuspensions under the condition of stirring at 800 rpm using a magnetic stirrer (SH-2, Beijing Jinbeide Industrial And Trading Co., Ltd., China) and mixed moderately. The mixture was poured onto the plastic substrate and then dried overnight by vacuum at 40 °C to obtain smooth and uniform film. The resultant film was cut into the quadrate of 2 2 cm2 in size. The preparative method of the blank ODFs (without HPE) and the ODFs containing HPE particles (with HPE coarse suspension) was the same as the ODFs containing HPE nanoparticles.
3
2.6.2. Uniformity of dosage units of the films [41] The uniformity of dosage units of the films was measured using 20 pieces of films, and the content of HPE was determined by HPLC as described in Section 2.12. The acceptance value (AV) of the preparation is less than 15.0, according to the Japanese Pharmacopoeia 15 (JP15). AV for CP2010 was calculated according to the following equations:
AV ¼ jM Xj þ ks where M is relative content of label claim (100%), X is the average (%) of individual contents, k is the acceptability constant (2.2), s is the standard deviation. In USP27, the contents of major component in the preparation should be within a range between 85% and 115% and the relative standard deviation should be less than or equal to 6.0%.
2.5. Particle size and zeta potential analysis The average diameter and polydispersity index (PI) of HPE nanosuspensions were analyzed by photon correlation spectroscopy (PCS) with a laser particle size analyzer (Winner 801, Jinan Winner Particle Instrument Stock Co., Ltd., China). Prior to measurement, HPE nanosuspensions were diluted with distilled water to achieve the suitable concentration for analysis. The samples were measured at a fixed angle of 90° at 25 °C. The zeta potential was analyzed by the method of electrophoretic mobility using a Zeta-sizer (3000SH, Malvern Instruments Ltd., UK) at 25 °C after dilution with distilled water. The same methods were used to measure the particles size and zeta potential of HPE nanoparticles re-dispersed from ODFs. For assessment of the redispersibility, quadrate films with an area of 2 2 cm2 were placed into an appropriate amount of distilled water and shaken gently for film disintegration to form a fine nanosuspension. The measurement was performed in triplicate and the average value was used.
2.6. Properties of orodispersible films 2.6.1. Film thickness Film thickness was measured in 3 different locations of the film using a micrometer screw gauge (Mitutoyo, Kawasaki, Japan). Three pieces of films were measured and the average thickness was used.
2.6.3. Disintegration test Disintegration test was performed according to the specifications of the Ph. Eur. 5.4 ed. (2.9.1) using a disintegration tester (ZBS-6E, Tianda Tianfa technology Co., Inc., China). The time required for the film to disintegrate was recorded and the results are expressed as a maximum of 6 determinations. 2.7. Morphology of HPE nanoparticles-loaded films The morphology of HPE coarse suspensions and HPE nanosuspension and the distribution of HPE particles and nanoparticles loaded films were observed using scanning electron microscopy (SEM) (S-4800, Hitachi Technologies Corporation, Japan). The film was directly fixed on a metal stub using a double sided adhesive tape, while HPE coarse suspensions or nanosuspensions were dropped onto a slice of silicon and left overnight to dry under vacuum, which was also fixed on the metal stub using a double sided adhesive tape. Samples were gold coated at 10 mA for 20 s and repeated six times in vacuum by a sputter coater (SBC-12, KYKY technology Co., Ltd., China) before analysis. Surface morphologies were obtained at 15 kV and 5 kV, respectively. 2.8. X-ray diffractograms of films The X-ray diffractograms (XRD) of HPE raw material, the blank films, and the films containing HPE particles or nanoparticles were recorded by an X-ray diffractometer (D/Max-2500PC, Rigaku,
Fig. 2. Process schematic for preparation of orodispersible film containing drug nanoparticles.
Please cite this article in press as: B.-d. Shen et al., Development and characterization of an orodispersible film containing drug nanoparticles, Eur. J. Pharm. Biopharm. (2013), http://dx.doi.org/10.1016/j.ejpb.2013.09.019
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Japan) with a Cu line as the source of radiation. The XRD were performed at 25 mA and 40 kV over the range 2h = 3° to 60° at rate of 2°/min.
was reconstituted with 100 lL absolute methanol under vortex mixing for 5 min. The suspension was centrifuged at 8000 rpm for 10 min and then 20 lL supernatant was directly injected into the HPLC system for analysis as described in Section 2.12.
2.9. In vitro dissolution studies In vitro dissolution studies were performed in a Ph. Eur. 5.4 ed. paddle dissolution apparatus (RC-8, Tianjin Guoming Medicine and Equipment Co., Inc., China) according the literature [30]. The dissolution medium was 900 mL of freshly deionized water kept at 37 ± 1 °C and stirred at 100 rpm. The samples of HPE raw material (10 mg) were sealed in gelatin capsules for dissolution test, while the HPE particles and nanoparticles loaded films (2 2 cm2), containing 10 mg HPE, were used for dissolution test. Samples of HPE nanoparticles-loaded films were exactly weighed in order to assure the sink condition. At preselected time intervals (1, 2.5, 5, 10, 20, 30, 45, 60 min), 1 mL samples were withdrawn and filtered through 0.45 lm Millipore filter immediately. Simultaneously, equal blank medium was compensated immediately after the withdrawal. The amount of dissolved HPE in the sample solution was determined using HPLC method as described in Section 2.12. Dissolution was performed three times for each formulation to calculate the drug-release profile. 2.10. Stability study The ODFs were stored in aluminum package at room temperature. The HPE content in the films was determined after storage for 10, 20, 30, 60, 90 and 120 days to evaluate their physical stability using the same method as determination of HPE content in the film. The particle size and PI of HPE nanoparticles re-dispersed from ODFs were also investigated.
2.12. HPLC assay The HPE content and concentrations of HPE in dissolution media and plasma were measured by the same HPLC method using a Prominence LC system (Shimadzu Corporation, Kyoto, Japan) equipped with a quaternary pump (LC-20AT), a diode array detector (SPD-M20A), an auto sampler (SIL-20A), a degassing unit (DGU20A5R) and Shimadzu CBM-20A station for data analysis. Analyses were performed on an Alltima C18 column (250 mm 4.6 mm, 5 lm, Alltech). The mobile phase was a mixture of 0.1% phosphoric acid and acetonitrile (75:25, v/v) with a flow rate of 1.0 mL/min. The samples were put into HPLC vials and 20 lL automatically injected into the HPLC system. The effluent is monitored for UV absorption at 240 nm.
2.13. Statistical analysis Pharmacokinetic analysis was performed using statistics software (DASÒ 2.0, Boying Corporation, China). Data are expressed as the mean ± standard deviation (SD). Statistical analysis was performed using one-way analysis of variance (ANOVA) followed by the least significant difference (LSD) post hoc test. Differences between experimental groups were considered significant at P < 0.05.
3. Result and discussion 2.11. Pharmacokinetics study in rats 3.1. Particle size and zeta potential analysis Wistar rats (adult male, 200–220 g) used in the experiments were provided by the Experimental Animal Center of Military Medical Sciences (Beijing, China). They received care in compliance with the Principles of Laboratory Animal Care and the Guide for the Care and Use of Laboratory Animals. Experiments followed protocol approved by 302 Military Hospital of China Institutional Animal Care and Use Committee. They were randomly divided into three groups with six animals in each group; the rats were fasted for 12 h with free access to water prior to experiments. The film containing HPE nanoparticles, HPE nanosuspensions and HPE coarse suspensions, all corresponding to a dose of 50 mg/kg, were administered to rats in the three groups. For the administration of films, 50 lL aliquot of distilled water was dropped into the rat oral cavity under light ether anesthesia [42,43], then a piece of film containing 10 mg HPE was cut into two pieces about 1 2 cm2 and applied to the buccal cavity bilaterally. For oral administration, rats were given 1 mL of HPE nanosuspensions or HPE coarse susupensions containing 10 mg HPE under light ether anesthesia. About 0.5 mL blood samples were taken in heparinized tube via the orbit vein at 0.083, 0.25, 0.5, 1, 2, 3, 4, 6, 8, 10, 12 and 24 h after drug administration. The blood samples were separated immediately by centrifugation (H 2050R, Xiang Yi Centrifuge Instrument Co., Ltd., Hunan, China) at 8000 rpm for 10 min, then plasma was taken in a polyethylene tube and stored at 20 °C until analysis. The plasma (200 lL) was put into 1.5 mL centrifuge tube and mixed with 400 lL absolute methanol under vortex mixing (MS3, IKAÒ, Germany) for 5 min. After centrifuging at 8000 rpm for 5 min at 4 °C, the supernatant was transferred to another tube and dried under a light stream of nitrogen at 40 °C. The residue
Particle size of HPE nanosuspensions and HPE nanoparticles redispersed from ODFs was determined by PCS. As shown in Fig. 3, the average particle size of HPE nanosuspensions was 260 ± 5 nm with a PI value of 0.108 ± 0.024 (Fig. 3A). Following redispersion of the ODFs, the mean particle size of HPE nanoparticles was 280 ± 11 nm with a PI value of 0.196 ± 0.032 (Fig. 3B). The result showed that there was a slight increase in particle size and PI of HPE nanoparticles re-dispersed from ODFs compared with that of HPE nanosuspensions. A PI value of 0.1–0.25 indicates a fairly narrow size distribution whereas PI value greater than 0.5 indicates a very broad distribution [44]. The PI value of HPE nanosuspensions and HPE nanoparticles re-dispersed from ODFs were 0.108 ± 0.024 and 0.196 ± 0.073 respectively, which indicated a narrow particle size distribution [45]. Zeta potential gives certain information about the surface charge properties and further the long-term physical stability of the nanosuspensions [46]. In order to obtain a nanosuspension exhibiting good stability, for an electrostatically stabilized nanosuspension a minimum zeta potential of ±30 mV is required whereas in the case of a combined electrostatic and steric stabilization, a minimum zeta potential of ±20 mV is desirable [44]. The zeta potential of HPE nanosuspensions and HPE nanoparticles redispersed from ODFs was 31.7 ± 1.6 mV and 29.5 ± 2.9 mV respectively, which provided a guarantee for the stability of HPE nanosuspensions and HPE nanoparticles re-dispersed from ODFs. The result of particle size analysis showed that the film did not have a conspicuous influence on the particle size of HPE nanosuspension, indicating a good redispersibility of the ODF containing HPE nanoparticles.
Please cite this article in press as: B.-d. Shen et al., Development and characterization of an orodispersible film containing drug nanoparticles, Eur. J. Pharm. Biopharm. (2013), http://dx.doi.org/10.1016/j.ejpb.2013.09.019
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Fig. 3. Particle size distribution of HPE nanosuspensions (A) and HPE nanoparticles re-dispersed from the orodispersible film (B).
3.2. Properties of orodispersible films The prepared ODFs were opaque and yellow in appearance (Fig. 4), which was similar to that of HPE. The thickness of films was 112 ± 5 lm. The AV of the films was 8.0 and less than 15.0, satisfying the requirements of uniformity of dosage units of the preparation in JP15. Each film with an area of 2 2 cm2 contains 10.0 mg HPE. The relative drug content was found to be 100.9 ± 3.3%, which suggested that the drug content was within the range between 85% and 115% as specified by the USP27. The relative standard deviation in respect of the HPE content was 3.3%, indicating that the preparation met the criteria of USP specification for drug content (66.0%). Each film was disintegrated within 30 s after immersion into water and formed nanosuspensions.
Fig. 4. Appearance of orodispersible films containing HPE nanoparticles.
3.4. X-ray diffractograms of orodispersible films
3.3. Morphology of HPE nanoparticles in orodispersible films In order to characterize the morphology of the surface of the films, SEM imaging was performed. As shown in Fig. 5, SEM images revealed apparent differences in the morphologies of the samples. HPE coarse suspensions showed irregular shape with a great deal of angularities (Fig. 5A), which was similar to HPE particles in the films (Fig. 5C). HPE nanosuspensions had a granular shape with obvious adhesion to each other after dried by vacuum (Fig. 5B), while well-dispersed HPE nanoparticles with slight adhesion to each other were observed in the films (Fig. 5D). Compared with the particle size of HPE coarse suspensions (5–50 lm, Fig. 5A and C), the particle size of HPE nanosuspension and HPE nanoparticles in the films was markedly reduced (0.1–0.5 lm, Fig. 5B and D). This can be attributed to the high pressure homogenization process of the HPE coarse suspensions. During high pressure homogenization process, cavitation effect, shear force and collisions between the drug particles easily disintegrated large drug particles into nanoparticles [47].
X-ray diffractograms (XRD) could be used to investigate potential changes in the internal structure of drug crystals. To assess the crystallinity of HPE nanoparticles incorporated in films, XRD was performed. As displayed in Fig. 6, there were no characteristic peaks of HPE raw material, which indicated HPE was amorphous state. No crystalline diffraction peaks were observed for the film containing HPE particles and nanoparticles, indicating amorphous structure. No apparent differences were found between HPE particles loaded films and HPE nanoparticles loaded films, indicating that the crystalline state of HPE in the film was unaltered following the homogenization, which may be expected to enhance the dissolution and bioavailability of the water-insoluble drug [48].
3.5. In vitro dissolution studies In order to prove the advantages of the ODF containing HPE nanoparticles, the dissolution behavior of the film containing HPE nanoparticles was compared to the film containing HPE particles and HPE raw material. In the process of dissolution, HPE raw
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Fig. 5. SEM images of HPE coarse suspensions (A), HPE nanosuspensions (B), HPE particles in the orodispersible film (C), HPE nanoparticles in the orodispersible film (D) and blank orodispersible film (E).
5000
4000
3000
2000
1000
0
3
10
20
30
40
50
60
2-Theta - Scale Fig. 6. X-ray diffractograms for HPE raw material, the blank orodispersible films, the orodispersible films containing HPE particles and the orodispersible films containing HPE nanoparticles.
material was submerged rapidly in the medium and sediments of particles were also observed after the film containing HPE particles disintegrated fleetly. Difference from the film containing HPE particles and HPE raw material, the film containing HPE nanoparticles
dispersed homogeneously quickly. As displayed in Fig. 7, dissolution rate of the film containing HPE nanoparticles was distinctly superior compared to those of the film containing HPE particles and HPE raw material. Approximate 90% of HPE was released from
Please cite this article in press as: B.-d. Shen et al., Development and characterization of an orodispersible film containing drug nanoparticles, Eur. J. Pharm. Biopharm. (2013), http://dx.doi.org/10.1016/j.ejpb.2013.09.019
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film containing HPE nanoparticles within 10 min. In the same interval of 10 min, the dissolution rate of the film containing HPE particles was only about 30% and the rate of HPE raw material was less than 20%. This can be attributed to its amorphous structure and nanosuspensions performance. Significant particle size reduction of nanosuspensions provides a strong increase in the surface area, resulting in the enhancement in dissolution velocity of the drug [49].
HPE relative content (%)
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3.6. Stability of orodispersible films
3.7. Pharmacokinetic parameters in rats
Fig. 8. HPE content of orodispersible films containing HPE nanoparticles after storage at room temperature (mean ± SD, n = 3).
400
Particle size
0.4
PI 300
200
0.2
PI
To estimate the characteristic of the ODF containing HPE nanoparticles in vivo and allow evaluation of formulation effects on the proportion of drug absorbed, the pharmacokinetic profiles of the ODF containing HPE nanoparticles, HPE nanosuspension and HPE coarse suspensions were determined and compared. Fig. 10 shows differences in mean plasma concentration of HPE after administration of the ODF containing HPE nanoparticles, HPE nanosuspensions and HPE coarse suspensions to rats with the same dose of 50 mg/kg. The pharmacokinetic parameters are listed in Table 1. As shown in these data, HPE coarse suspensions, HPE nanosuspensions and the ODF containing HPE nanoparticles were different from each other in the corresponding parameters. Compared to the HPE coarse suspension, the Tmax from HPE nanosuspension and the ODF containing HPE nanoparticles were lower significantly (P < 0.05) and the AUC0–24h from HPE nanosuspension and the ODF containing HPE nanoparticles were increased by 66% and 85%, respectively. It could be observed that the MRT from HPE nanosuspension (3.38 h) and the ODF containing HPE nanoparticles (4.09 h) were statistically different with the HPE coarse suspension (5.79 h) (P < 0.05). The mean value of Cmax from HPE nanosuspension (1.54 lg/mL) and the ODF containing HPE nanoparticles (1.75 lg/mL) was increased by 133% and 165%, respectively. There were no difference in the corresponding phar-
Time (day)
Particle size (nm)
When the ODF preparations was stored in aluminum package at room temperature for 10–120 days, no apparent change in the HPE content was observed (P > 0.05). The contents of HPE were fairly stable ranging from 97.2% to 104.4% during 120 days after storage at room temperature (Fig. 8). Moreover, the mean particle size and PI of HPE nanoparticles re-dispersed from fast dissolving films vary slightly during storage (Fig. 9, P > 0.05). In conclusion, all obtained data suggested that the ODFs are stable when they are stored at room temperature.
100
0
0
Time (day) Fig. 9. Particle size and PI of the reconstituted nanosuspensions from orodispersible films containing HPE nanoparticles after storage at room temperature (mean ± SD, n = 3).
The film containing HPE nanoparticles HPE coarse suspension HPE nanosuspension
Dissolution (%)
Fig. 10. Mean plasma concentration–time curve of HPE administered with orodispersible films containing HPE nanoparticles, HPE nanosuspension and HPE suspension in rats (mean ± SD, n = 6).
Time (min) Fig. 7. Dissolution profiles of the orodispersible films containing HPE nanoparticles ( ), the orodispersible films containing HPE particles ( ) and HPE raw material ( ) (mean ± SD, n = 3).
macokinetic parameters between HPE nanosuspension and the ODF containing HPE nanoparticles. All obtained data indicated that the ODF containing HPE nanoparticles was easier to be absorbed in vivo and had a certain degree of increase in bioavailability. The ODF containing HPE nanoparticles was absorbed easily, which led to increased AUC0–24h, Cmax and decreased Tmax, MRT. These phenomena were probably caused by the absorption pattern of HPE and particle size reduction. When the ODF is administered into the mouth, it is instantly wetted by saliva and then rapidly disintegrates and dissolves to release the drug. The drug was partly absorbed through the cavity mucosa, which makes drugs enter the systemic circulation without undergoing first-pass hepatic metabolism [28], thus improving the bioavailability in vivo. On the other
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Table 1 Main pharmacokinetic parameters of HPE coarse suspension (HPE CS), HPE nanosuspension (HPE NS) and the films containing HPE nanoparticles (HPE NPs) after oral administration in rats (mean ± SD, n = 6).
* **
Parameters
HPE CS
HPE NS
The films containing HPE NP
Cmax (lg/mL1) AUC0–t (lg h/mL1) Tmax (h) MRT (h)
0.66 ± 0.06 3.58 ± 0.84
1.54 ± 0.11** 5.96 ± 0.92**
1.75 ± 0.09** 6.62 ± 0.62**
1.10 ± 0.10 5.79 ± 1.05
0.70 ± 0.17* 3.38 ± 0.81**
0.51 ± 0.05* 4.09 ± 0.78*
P < 0.05 vs. the parameters of HPE CS. P < 0.01 vs. the parameters of HPE CS.
hand, particle size reduction to nanometer range enhances the dissolution rate through increasing surface area and saturation solubility, which lead to the improvement of bioavailability [50]. However, there exists a fact that there is an uncertainty how much of the HPE is absorbed buccally and how much from intestine after unintentional swallowing of HPE containing saliva, which is needed to further study.
4. Conclusions In this study, a novel ODFs containing drug nanoparticles was successfully prepared by incorporating drug nanosuspensions with solid film carriers mainly composed of HPMC, MCC and L-HPC, which provides a new route of transforming nanosuspensions of poorly water-soluble drugs into a solid dosage as well as overcome the shortcoming that ODFs are not suitable for the delivery of poorly water-soluble drugs. The ODFs achieves the criteria in the dosage uniformity test for JP15. The fast dissolving film was disintegrated in water within 30 s with reconstituted nanosuspensions particle size of 280.69 ± 11.57 nm, indicating a good redispersibility of the ODFs containing HPE nanoparticles. The XRD analysis suggested that HPE in the ODFs was in the amorphous state. The resulting ODFs exhibit much faster dissolution rates compared to the ODFs containing HPE particles and HPE raw material. In vivo pharmacokinetic study showed that the ODFs containing HPE nanoparticles significantly increased the oral bioavailability of HPE compared to HPE coarse suspensions. From these results above, it was suggested that the ODFs containing drug nanoparticles may provide a potential opportunity in transforming drug nanosuspensions into a solid dosage form as well as improving oral bioavailability of poorly water-soluble drugs. Acknowledgments The authors would like to acknowledge the financial support from the Natural Science Found of Beijing city of China (No. 7122176), the Scientific Research Foundation for the Returned Overseas Chinese Scholars, State Education Ministry (No. 20101561) and the National Key New Drugs Innovation Foundation (No. 2013ZX09J13109-06C). Reference [1] C.A. Lipinski, Drug-like properties and the causes of poor solubility and poor permeability, J. Pharmacol. Toxicol. Methods 44 (1) (2000) 235–249. [2] F. Kesisoglou, S. Panmai, Y. Wu, Nanosizing-oral formulation development and biopharmaceutical evaluation, Adv. Drug Delivery Rev. 59 (7) (2007) 631–644. [3] T. Io, T. Fukami, K. Yamamoto, T. Suzuki, J.D. Xu, K. Tomono, A. Ramamoorthy, Homogeneous nanoparticles to enhance the efficiency of a hydrophobic drug, antihyperlipidemic probucol, characterized by solid-state NMR, Mol. Pharm. 7 (1) (2010) 299–305. [4] J. Salazar, O. Heinzerling, R.H. Müller, J.P. Möschwitzer, Process optimization of a novel production method for nanosuspensions using design of experiments (DoE), Int. J. Pharm. 420 (2) (2011) 395–403.
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Research paper
Development and characterization of an orodispersible film containing drug nanoparticles Bao-de Shen a,b,1, Cheng-ying Shen a,c,1, Xu-dong Yuan d, Jin-xia Bai a,c, Qing-yuan Lv a, He Xu a,c, Ling Dai a,c, Chao Yu a,b, Jin Han a,⇑, Hai-long Yuan a,⇑ a
302 Hospital of PLA&PLA Institute of Chinese Materia Medica, Beijing, China Key Lab of Modern Preparation of TCM, Ministry of Education, Jiangxi University of Traditional Chinese Medicine, Nanchang, China Pharmacy College, Chengdu University of Traditional Chinese Medicine, Chengdu, China d School of Science, Monmouth University, West Long Branch, NJ, USA b c
a r t i c l e
i n f o
Article history: Received 30 May 2013 Accepted in revised form 28 September 2013 Available online xxxx Keywords: Nanosuspensions High pressure homogenization Orodispersible films Film casting
a b s t r a c t In this study, a novel orodispersible film (ODF) containing drug nanoparticles was developed with the goal of transforming drug nanosuspensions into a solid dosage form and enhancing oral bioavailability of drugs with poor water solubility. Nanosuspensions were prepared by high pressure homogenization and then transformed into ODF containing drug nanoparticles by mixing with hydroxypropyl methylcellulose solution containing microcrystalline cellulose, low substituted hydroxypropylcellulose and PEG400 followed by film casting and drying. Herpetrione, a novel and potent antiviral agent with poor water solubility that extracted from Herpetospermum caudigerum, was chosen as a model drug and studied systematically. The uniformity of dosage units of the preparation was acceptable according to the criteria of Japanese Pharmacopoeia 15. The ODF was disintegrated in water within 30 s with reconstituted nanosuspensions particle size of 280 ± 11 nm, which was similar to that of drug nanosuspensions, indicating a good redispersibility of the fast dissolving film. Result of X-ray diffraction showed that HPE in the ODF was in the amorphous state. In the in vitro dissolution test, the ODF containing HPE nanoparticles showed an increased dissolution velocity markedly. In the pharmacokinetics study in rats, compared to HPE coarse suspensions, the ODF containing HPE nanoparticles exhibited significant increase in AUC0–24h, Cmax and decrease in Tmax, MRT. The result revealed that the ODF containing drug nanoparticles may provide a potential opportunity in transforming drug nanosuspensions into a solid dosage form as well as enhancing the dissolution rate and oral bioavailability of poorly water-soluble drugs. Ó 2013 Elsevier B.V. All rights reserved.
1. Introduction At present nearly 40% of all newly developed drugs are poorly soluble in water, resulting in poor oral bioavailability [1], and thus limits their application. The main focus in the pharmaceutical development is to explore new technological approaches for enhancing water solubility of drugs with poor water solubility. Particle size reduction to the nanometer range has been reported to be a very promising approach for dissolution enhancement not only because of increased surface area but also because of increased saturation solubility as described by Ostwald–Freundlich equation [2]. Therefore, the poor drug solubility may be overcome by particle size reduction using nanoscience approaches [3]. In recent years, nanosuspensions as one of the nanoscience approaches has ⇑ Corresponding authors. 302 Hospital of PLA&PLA Institute of Chinese Materia Medica, Beijing 100039, China. Tel.: +86 1066933367; fax: +86 1063879589. E-mail addresses: [email protected] (J. Han), [email protected] (H.-l. Yuan). 1 Bao-de Shen and Cheng-ying Shen contributed equally to this work as first author.
become a promising and serious development tool for improving the water solubility, dissolution rate and oral bioavailability of poorly water soluble drugs by reducing the particle size in the pharmaceutical industry [4,5]. Nanosuspensions of drugs are a carrier-free sub-micron colloidal dispersion of drug particles in a dispersion medium with a mean particle size in the nanometer range, typically between 10 and 1000 nm, which are stabilized by polymers, surfactants or a mixture of both [6–8]. It can be produced either by top-down process (breaking of large drug particles as in milling and homogenization) or by bottom-up process (building up nanoparticles from drug molecules via precipitation) [9], as well as a combination of the two methods mentioned [10,11]. Apart from enhancing solubility or dissolution rate of poorly water-soluble drugs, nanosuspensions have some other advantages such as high drug loading, low incidence of side effects by the excipients, and low cost [6,12]. However, nanosuspension is a thermodynamic unstable system and the stability is one of the critical aspects in ensuring safety and efficacy of drug nanosuspensions. Storage and shipping of the drug
0939-6411/$ - see front matter Ó 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ejpb.2013.09.019
Please cite this article in press as: B.-d. Shen et al., Development and characterization of an orodispersible film containing drug nanoparticles, Eur. J. Pharm. Biopharm. (2013), http://dx.doi.org/10.1016/j.ejpb.2013.09.019
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B.-d. Shen et al. / European Journal of Pharmaceutics and Biopharmaceutics xxx (2013) xxx–xxx
nanosuspensions may also bring about a variety of stability problems such as sedimentation, agglomeration and crystal growth [13,14] and nanosuspensions are liquid formulations that are not convenient to take and carry, which seriously restricted the application and promotion of the nanosuspension delivery system [15]. So it is essential to convert the nanosuspension into a dry powder form for further physical stability and/or patient convenience reasons [2,16,17]. The solidification of nanosuspensions can be achieved by various methods such as freeze drying, spray drying and vacuum drying [18–21]. But the drying process is often accompanied by the irreversible aggregation of the nanoparticles, which then decreases the dissolution rate of drugs [5,22]. The redispersibility of drying drug nanoparticles is key issues that convert nanosuspension into solid nanoparticles. The drying drugs nanoparticles must have the ability to return to their original nanosuspension state upon reconstitution in water so that it could improve the oral bioavailability of poorly water-soluble drugs. Therefore, it is a great challenge to develop solid nanoparticles that have a good redispersibility for the enhancement of oral bioavailability of poorly water-soluble drugs. Orodispersible films (ODFs), a relatively new dosage form for oral route of administration, are postage stamp-sized strips of thin polymeric films formulated to disintegrate or dissolve almost instantaneously when placed onto the tongue [23,24]. They are used in case of patients who have difficulty in swallowing such as elderly, pediatric patients, and others suffering from mental illness and developmental disorders [25–27]. The drug in ODFs is absorbed through the oral mucosa, which make drugs enter the systemic circulation without undergoing first-pass hepatic metabolism [23,28,29]. More recently, ODFs are gaining interest as an alternative to fast dissolving tablets to definitely eliminate patients’ fear of chocking and improve patient compliance [30]. In addition, ODFs overcome the drawbacks of oral disintergrating tablets that they are fragile and brittle, which needs special package for protection during storage and transportation, and prepared by using the expensive lyophillization process [26,31]. However, ODFs are not suitable for the delivery of drugs with poor water solubility because the drugs with poor water solubility have poorly oral mucosa drug absorption. Self-microemulsifying mouth dissolving films were developed to improve the water solubility of poorly water-soluble drugs [32–34], but the self-microemulsifying microemulsion requires a huge amount of oils. Hydroxypropyl-b-cyclodextrin (HP-b-CD) and poloxamer 407 were also used to improve the poor solubility of drugs in the preparation of ODFs [35], but the application of HP-b-CD demands an appropriate molecular size. Moreover, they lack universal applicability to all drugs. Therefore, the drugs with poor water solubility are a real challenge in the development of ODFs. Upon consideration, ODFs seem to be a good choice for the solidification of drug nanosuspensions. It not only transforms drug nanosuspensions into solid products, but also overcomes the problem that ODFs are not suitable for the delivery of drugs with poor water solubility. In this study, a novel ODFs containing drug nanoparticles was developed. Herpetrione (HPE, Fig. 1), a novel and potent antiviral agent with poor water solubility that extracted from Herpetospermum caudigerum [36,37], was chosen as a model drug. HPE nanosuspensions were prepared by high pressure homogenization and then transformed into ODFs containing drug nanoparticles using casting method. The particle size distribution of HPE nanosuspensions and HPE nanoparticles re-dispersed from ODFs were investigated by photon correlation spectroscopy (PCS). The prepared ODFs were characterized using scanning electron microscopy (SEM) and X-ray diffraction (XRD). The content uniformity and stability were also tested. To verify the advantages of the ODFs containing HPE nanoparticles, the dissolution behavior and the pharmacokinetic profiles were investigated.
Fig. 1. Chemical structure of HPE.
2. Materials and methods 2.1. Materials Herpetrione (the purity is up to 98%, determined by area normalization method) was prepared in the laboratory of Dr. Yuan (302 Military Hospital of China, Beijing). Sodium dodecyl sulfate (SDS) and KollidonÒ 30 (PVP K-30) were obtained from the BASF Corp. (Ludwigshafen, Germany). Hydroxypropyl methylcellulose (HPMC, E50), 5% low substituted hydroxypropylcellulose (L-HPC) and microcrystalline cellulose (PH101, MCC) were purchased from Xi’an Fine Chemical Co., Ltd. (Xi’an, China). PEG-400 was obtained from Guangdong Guanghua Sci-Tech Co., Ltd. (Guangzhou, China). HPLC grade acetonitrile was purchased from Fisher, USA. All other chemicals were of analytical grade. 2.2. Preparation of HPE coarse suspensions HPE coarse suspensions were prepared by dispersing herpetrione in a SDS and PVP K-30 bidistilled water solution and then stirred by a magnetic stirrer (SH-2, Beijing Jinbeide Industrial And Trading Co., Ltd., China) to obtain a uniform suspension. Coarse suspensions were prepared using the herpetrione, SDS, PVP K-30 ratio of 1:0.2:0.3 (w/w/w). 2.3. Preparation of HPE nanosuspensions HPE nanosuspensions were prepared by trituration and ultraturrax homogenization, followed by a high pressure homogenization technique according to the literature method [38]. Briefly, the HPE powder (1%, w/v) was dispersed in an aqueous surfactant solution, containing SDS (0.2%, w/v) and PVP K-30 (0.3%, w/v) under trituration. Then the dispersion was pre-mixed using a high shear homogenizer (Ultra-TurraxÒT25, IKA, Germany) at 12,000 rpm for 5 min to obtain a uniform system. The obtained pre-mixture was homogenized at high pressure using a pistongap high pressure homogenizer (EmulsiFlex-C3, Avestin Inc., Ottawa, Canada). Firstly, 500 bar with 5 cycles as a kind of pre-milling was applied, and then 20 cycles at 1000 bar were run to obtain the nanosuspensions. 2.4. Preparation of orodispersible films containing HPE particles and nanoparticles The ODFs containing HPE nanoparticles were prepared by casting method [39] with a modification. Fig. 2 shows a schematic of the process used for film formation. In the optimized formulation
Please cite this article in press as: B.-d. Shen et al., Development and characterization of an orodispersible film containing drug nanoparticles, Eur. J. Pharm. Biopharm. (2013), http://dx.doi.org/10.1016/j.ejpb.2013.09.019
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[40], the constituents of the basic materials were HPMC (5%, w/v), L-HPC (0.5%, w/v), MCC (0.12%, w/v), PEG-400 (5%, v/v) and mannitol (10%, w/v). The bases of the film preparation dispersed or dissolved in an appropriate amount of water were then added into the HPE nanosuspensions under the condition of stirring at 800 rpm using a magnetic stirrer (SH-2, Beijing Jinbeide Industrial And Trading Co., Ltd., China) and mixed moderately. The mixture was poured onto the plastic substrate and then dried overnight by vacuum at 40 °C to obtain smooth and uniform film. The resultant film was cut into the quadrate of 2 2 cm2 in size. The preparative method of the blank ODFs (without HPE) and the ODFs containing HPE particles (with HPE coarse suspension) was the same as the ODFs containing HPE nanoparticles.
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2.6.2. Uniformity of dosage units of the films [41] The uniformity of dosage units of the films was measured using 20 pieces of films, and the content of HPE was determined by HPLC as described in Section 2.12. The acceptance value (AV) of the preparation is less than 15.0, according to the Japanese Pharmacopoeia 15 (JP15). AV for CP2010 was calculated according to the following equations:
AV ¼ jM Xj þ ks where M is relative content of label claim (100%), X is the average (%) of individual contents, k is the acceptability constant (2.2), s is the standard deviation. In USP27, the contents of major component in the preparation should be within a range between 85% and 115% and the relative standard deviation should be less than or equal to 6.0%.
2.5. Particle size and zeta potential analysis The average diameter and polydispersity index (PI) of HPE nanosuspensions were analyzed by photon correlation spectroscopy (PCS) with a laser particle size analyzer (Winner 801, Jinan Winner Particle Instrument Stock Co., Ltd., China). Prior to measurement, HPE nanosuspensions were diluted with distilled water to achieve the suitable concentration for analysis. The samples were measured at a fixed angle of 90° at 25 °C. The zeta potential was analyzed by the method of electrophoretic mobility using a Zeta-sizer (3000SH, Malvern Instruments Ltd., UK) at 25 °C after dilution with distilled water. The same methods were used to measure the particles size and zeta potential of HPE nanoparticles re-dispersed from ODFs. For assessment of the redispersibility, quadrate films with an area of 2 2 cm2 were placed into an appropriate amount of distilled water and shaken gently for film disintegration to form a fine nanosuspension. The measurement was performed in triplicate and the average value was used.
2.6. Properties of orodispersible films 2.6.1. Film thickness Film thickness was measured in 3 different locations of the film using a micrometer screw gauge (Mitutoyo, Kawasaki, Japan). Three pieces of films were measured and the average thickness was used.
2.6.3. Disintegration test Disintegration test was performed according to the specifications of the Ph. Eur. 5.4 ed. (2.9.1) using a disintegration tester (ZBS-6E, Tianda Tianfa technology Co., Inc., China). The time required for the film to disintegrate was recorded and the results are expressed as a maximum of 6 determinations. 2.7. Morphology of HPE nanoparticles-loaded films The morphology of HPE coarse suspensions and HPE nanosuspension and the distribution of HPE particles and nanoparticles loaded films were observed using scanning electron microscopy (SEM) (S-4800, Hitachi Technologies Corporation, Japan). The film was directly fixed on a metal stub using a double sided adhesive tape, while HPE coarse suspensions or nanosuspensions were dropped onto a slice of silicon and left overnight to dry under vacuum, which was also fixed on the metal stub using a double sided adhesive tape. Samples were gold coated at 10 mA for 20 s and repeated six times in vacuum by a sputter coater (SBC-12, KYKY technology Co., Ltd., China) before analysis. Surface morphologies were obtained at 15 kV and 5 kV, respectively. 2.8. X-ray diffractograms of films The X-ray diffractograms (XRD) of HPE raw material, the blank films, and the films containing HPE particles or nanoparticles were recorded by an X-ray diffractometer (D/Max-2500PC, Rigaku,
Fig. 2. Process schematic for preparation of orodispersible film containing drug nanoparticles.
Please cite this article in press as: B.-d. Shen et al., Development and characterization of an orodispersible film containing drug nanoparticles, Eur. J. Pharm. Biopharm. (2013), http://dx.doi.org/10.1016/j.ejpb.2013.09.019
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Japan) with a Cu line as the source of radiation. The XRD were performed at 25 mA and 40 kV over the range 2h = 3° to 60° at rate of 2°/min.
was reconstituted with 100 lL absolute methanol under vortex mixing for 5 min. The suspension was centrifuged at 8000 rpm for 10 min and then 20 lL supernatant was directly injected into the HPLC system for analysis as described in Section 2.12.
2.9. In vitro dissolution studies In vitro dissolution studies were performed in a Ph. Eur. 5.4 ed. paddle dissolution apparatus (RC-8, Tianjin Guoming Medicine and Equipment Co., Inc., China) according the literature [30]. The dissolution medium was 900 mL of freshly deionized water kept at 37 ± 1 °C and stirred at 100 rpm. The samples of HPE raw material (10 mg) were sealed in gelatin capsules for dissolution test, while the HPE particles and nanoparticles loaded films (2 2 cm2), containing 10 mg HPE, were used for dissolution test. Samples of HPE nanoparticles-loaded films were exactly weighed in order to assure the sink condition. At preselected time intervals (1, 2.5, 5, 10, 20, 30, 45, 60 min), 1 mL samples were withdrawn and filtered through 0.45 lm Millipore filter immediately. Simultaneously, equal blank medium was compensated immediately after the withdrawal. The amount of dissolved HPE in the sample solution was determined using HPLC method as described in Section 2.12. Dissolution was performed three times for each formulation to calculate the drug-release profile. 2.10. Stability study The ODFs were stored in aluminum package at room temperature. The HPE content in the films was determined after storage for 10, 20, 30, 60, 90 and 120 days to evaluate their physical stability using the same method as determination of HPE content in the film. The particle size and PI of HPE nanoparticles re-dispersed from ODFs were also investigated.
2.12. HPLC assay The HPE content and concentrations of HPE in dissolution media and plasma were measured by the same HPLC method using a Prominence LC system (Shimadzu Corporation, Kyoto, Japan) equipped with a quaternary pump (LC-20AT), a diode array detector (SPD-M20A), an auto sampler (SIL-20A), a degassing unit (DGU20A5R) and Shimadzu CBM-20A station for data analysis. Analyses were performed on an Alltima C18 column (250 mm 4.6 mm, 5 lm, Alltech). The mobile phase was a mixture of 0.1% phosphoric acid and acetonitrile (75:25, v/v) with a flow rate of 1.0 mL/min. The samples were put into HPLC vials and 20 lL automatically injected into the HPLC system. The effluent is monitored for UV absorption at 240 nm.
2.13. Statistical analysis Pharmacokinetic analysis was performed using statistics software (DASÒ 2.0, Boying Corporation, China). Data are expressed as the mean ± standard deviation (SD). Statistical analysis was performed using one-way analysis of variance (ANOVA) followed by the least significant difference (LSD) post hoc test. Differences between experimental groups were considered significant at P < 0.05.
3. Result and discussion 2.11. Pharmacokinetics study in rats 3.1. Particle size and zeta potential analysis Wistar rats (adult male, 200–220 g) used in the experiments were provided by the Experimental Animal Center of Military Medical Sciences (Beijing, China). They received care in compliance with the Principles of Laboratory Animal Care and the Guide for the Care and Use of Laboratory Animals. Experiments followed protocol approved by 302 Military Hospital of China Institutional Animal Care and Use Committee. They were randomly divided into three groups with six animals in each group; the rats were fasted for 12 h with free access to water prior to experiments. The film containing HPE nanoparticles, HPE nanosuspensions and HPE coarse suspensions, all corresponding to a dose of 50 mg/kg, were administered to rats in the three groups. For the administration of films, 50 lL aliquot of distilled water was dropped into the rat oral cavity under light ether anesthesia [42,43], then a piece of film containing 10 mg HPE was cut into two pieces about 1 2 cm2 and applied to the buccal cavity bilaterally. For oral administration, rats were given 1 mL of HPE nanosuspensions or HPE coarse susupensions containing 10 mg HPE under light ether anesthesia. About 0.5 mL blood samples were taken in heparinized tube via the orbit vein at 0.083, 0.25, 0.5, 1, 2, 3, 4, 6, 8, 10, 12 and 24 h after drug administration. The blood samples were separated immediately by centrifugation (H 2050R, Xiang Yi Centrifuge Instrument Co., Ltd., Hunan, China) at 8000 rpm for 10 min, then plasma was taken in a polyethylene tube and stored at 20 °C until analysis. The plasma (200 lL) was put into 1.5 mL centrifuge tube and mixed with 400 lL absolute methanol under vortex mixing (MS3, IKAÒ, Germany) for 5 min. After centrifuging at 8000 rpm for 5 min at 4 °C, the supernatant was transferred to another tube and dried under a light stream of nitrogen at 40 °C. The residue
Particle size of HPE nanosuspensions and HPE nanoparticles redispersed from ODFs was determined by PCS. As shown in Fig. 3, the average particle size of HPE nanosuspensions was 260 ± 5 nm with a PI value of 0.108 ± 0.024 (Fig. 3A). Following redispersion of the ODFs, the mean particle size of HPE nanoparticles was 280 ± 11 nm with a PI value of 0.196 ± 0.032 (Fig. 3B). The result showed that there was a slight increase in particle size and PI of HPE nanoparticles re-dispersed from ODFs compared with that of HPE nanosuspensions. A PI value of 0.1–0.25 indicates a fairly narrow size distribution whereas PI value greater than 0.5 indicates a very broad distribution [44]. The PI value of HPE nanosuspensions and HPE nanoparticles re-dispersed from ODFs were 0.108 ± 0.024 and 0.196 ± 0.073 respectively, which indicated a narrow particle size distribution [45]. Zeta potential gives certain information about the surface charge properties and further the long-term physical stability of the nanosuspensions [46]. In order to obtain a nanosuspension exhibiting good stability, for an electrostatically stabilized nanosuspension a minimum zeta potential of ±30 mV is required whereas in the case of a combined electrostatic and steric stabilization, a minimum zeta potential of ±20 mV is desirable [44]. The zeta potential of HPE nanosuspensions and HPE nanoparticles redispersed from ODFs was 31.7 ± 1.6 mV and 29.5 ± 2.9 mV respectively, which provided a guarantee for the stability of HPE nanosuspensions and HPE nanoparticles re-dispersed from ODFs. The result of particle size analysis showed that the film did not have a conspicuous influence on the particle size of HPE nanosuspension, indicating a good redispersibility of the ODF containing HPE nanoparticles.
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Fig. 3. Particle size distribution of HPE nanosuspensions (A) and HPE nanoparticles re-dispersed from the orodispersible film (B).
3.2. Properties of orodispersible films The prepared ODFs were opaque and yellow in appearance (Fig. 4), which was similar to that of HPE. The thickness of films was 112 ± 5 lm. The AV of the films was 8.0 and less than 15.0, satisfying the requirements of uniformity of dosage units of the preparation in JP15. Each film with an area of 2 2 cm2 contains 10.0 mg HPE. The relative drug content was found to be 100.9 ± 3.3%, which suggested that the drug content was within the range between 85% and 115% as specified by the USP27. The relative standard deviation in respect of the HPE content was 3.3%, indicating that the preparation met the criteria of USP specification for drug content (66.0%). Each film was disintegrated within 30 s after immersion into water and formed nanosuspensions.
Fig. 4. Appearance of orodispersible films containing HPE nanoparticles.
3.4. X-ray diffractograms of orodispersible films
3.3. Morphology of HPE nanoparticles in orodispersible films In order to characterize the morphology of the surface of the films, SEM imaging was performed. As shown in Fig. 5, SEM images revealed apparent differences in the morphologies of the samples. HPE coarse suspensions showed irregular shape with a great deal of angularities (Fig. 5A), which was similar to HPE particles in the films (Fig. 5C). HPE nanosuspensions had a granular shape with obvious adhesion to each other after dried by vacuum (Fig. 5B), while well-dispersed HPE nanoparticles with slight adhesion to each other were observed in the films (Fig. 5D). Compared with the particle size of HPE coarse suspensions (5–50 lm, Fig. 5A and C), the particle size of HPE nanosuspension and HPE nanoparticles in the films was markedly reduced (0.1–0.5 lm, Fig. 5B and D). This can be attributed to the high pressure homogenization process of the HPE coarse suspensions. During high pressure homogenization process, cavitation effect, shear force and collisions between the drug particles easily disintegrated large drug particles into nanoparticles [47].
X-ray diffractograms (XRD) could be used to investigate potential changes in the internal structure of drug crystals. To assess the crystallinity of HPE nanoparticles incorporated in films, XRD was performed. As displayed in Fig. 6, there were no characteristic peaks of HPE raw material, which indicated HPE was amorphous state. No crystalline diffraction peaks were observed for the film containing HPE particles and nanoparticles, indicating amorphous structure. No apparent differences were found between HPE particles loaded films and HPE nanoparticles loaded films, indicating that the crystalline state of HPE in the film was unaltered following the homogenization, which may be expected to enhance the dissolution and bioavailability of the water-insoluble drug [48].
3.5. In vitro dissolution studies In order to prove the advantages of the ODF containing HPE nanoparticles, the dissolution behavior of the film containing HPE nanoparticles was compared to the film containing HPE particles and HPE raw material. In the process of dissolution, HPE raw
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B.-d. Shen et al. / European Journal of Pharmaceutics and Biopharmaceutics xxx (2013) xxx–xxx
Fig. 5. SEM images of HPE coarse suspensions (A), HPE nanosuspensions (B), HPE particles in the orodispersible film (C), HPE nanoparticles in the orodispersible film (D) and blank orodispersible film (E).
5000
4000
3000
2000
1000
0
3
10
20
30
40
50
60
2-Theta - Scale Fig. 6. X-ray diffractograms for HPE raw material, the blank orodispersible films, the orodispersible films containing HPE particles and the orodispersible films containing HPE nanoparticles.
material was submerged rapidly in the medium and sediments of particles were also observed after the film containing HPE particles disintegrated fleetly. Difference from the film containing HPE particles and HPE raw material, the film containing HPE nanoparticles
dispersed homogeneously quickly. As displayed in Fig. 7, dissolution rate of the film containing HPE nanoparticles was distinctly superior compared to those of the film containing HPE particles and HPE raw material. Approximate 90% of HPE was released from
Please cite this article in press as: B.-d. Shen et al., Development and characterization of an orodispersible film containing drug nanoparticles, Eur. J. Pharm. Biopharm. (2013), http://dx.doi.org/10.1016/j.ejpb.2013.09.019
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film containing HPE nanoparticles within 10 min. In the same interval of 10 min, the dissolution rate of the film containing HPE particles was only about 30% and the rate of HPE raw material was less than 20%. This can be attributed to its amorphous structure and nanosuspensions performance. Significant particle size reduction of nanosuspensions provides a strong increase in the surface area, resulting in the enhancement in dissolution velocity of the drug [49].
HPE relative content (%)
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3.6. Stability of orodispersible films
3.7. Pharmacokinetic parameters in rats
Fig. 8. HPE content of orodispersible films containing HPE nanoparticles after storage at room temperature (mean ± SD, n = 3).
400
Particle size
0.4
PI 300
200
0.2
PI
To estimate the characteristic of the ODF containing HPE nanoparticles in vivo and allow evaluation of formulation effects on the proportion of drug absorbed, the pharmacokinetic profiles of the ODF containing HPE nanoparticles, HPE nanosuspension and HPE coarse suspensions were determined and compared. Fig. 10 shows differences in mean plasma concentration of HPE after administration of the ODF containing HPE nanoparticles, HPE nanosuspensions and HPE coarse suspensions to rats with the same dose of 50 mg/kg. The pharmacokinetic parameters are listed in Table 1. As shown in these data, HPE coarse suspensions, HPE nanosuspensions and the ODF containing HPE nanoparticles were different from each other in the corresponding parameters. Compared to the HPE coarse suspension, the Tmax from HPE nanosuspension and the ODF containing HPE nanoparticles were lower significantly (P < 0.05) and the AUC0–24h from HPE nanosuspension and the ODF containing HPE nanoparticles were increased by 66% and 85%, respectively. It could be observed that the MRT from HPE nanosuspension (3.38 h) and the ODF containing HPE nanoparticles (4.09 h) were statistically different with the HPE coarse suspension (5.79 h) (P < 0.05). The mean value of Cmax from HPE nanosuspension (1.54 lg/mL) and the ODF containing HPE nanoparticles (1.75 lg/mL) was increased by 133% and 165%, respectively. There were no difference in the corresponding phar-
Time (day)
Particle size (nm)
When the ODF preparations was stored in aluminum package at room temperature for 10–120 days, no apparent change in the HPE content was observed (P > 0.05). The contents of HPE were fairly stable ranging from 97.2% to 104.4% during 120 days after storage at room temperature (Fig. 8). Moreover, the mean particle size and PI of HPE nanoparticles re-dispersed from fast dissolving films vary slightly during storage (Fig. 9, P > 0.05). In conclusion, all obtained data suggested that the ODFs are stable when they are stored at room temperature.
100
0
0
Time (day) Fig. 9. Particle size and PI of the reconstituted nanosuspensions from orodispersible films containing HPE nanoparticles after storage at room temperature (mean ± SD, n = 3).
The film containing HPE nanoparticles HPE coarse suspension HPE nanosuspension
Dissolution (%)
Fig. 10. Mean plasma concentration–time curve of HPE administered with orodispersible films containing HPE nanoparticles, HPE nanosuspension and HPE suspension in rats (mean ± SD, n = 6).
Time (min) Fig. 7. Dissolution profiles of the orodispersible films containing HPE nanoparticles ( ), the orodispersible films containing HPE particles ( ) and HPE raw material ( ) (mean ± SD, n = 3).
macokinetic parameters between HPE nanosuspension and the ODF containing HPE nanoparticles. All obtained data indicated that the ODF containing HPE nanoparticles was easier to be absorbed in vivo and had a certain degree of increase in bioavailability. The ODF containing HPE nanoparticles was absorbed easily, which led to increased AUC0–24h, Cmax and decreased Tmax, MRT. These phenomena were probably caused by the absorption pattern of HPE and particle size reduction. When the ODF is administered into the mouth, it is instantly wetted by saliva and then rapidly disintegrates and dissolves to release the drug. The drug was partly absorbed through the cavity mucosa, which makes drugs enter the systemic circulation without undergoing first-pass hepatic metabolism [28], thus improving the bioavailability in vivo. On the other
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Table 1 Main pharmacokinetic parameters of HPE coarse suspension (HPE CS), HPE nanosuspension (HPE NS) and the films containing HPE nanoparticles (HPE NPs) after oral administration in rats (mean ± SD, n = 6).
* **
Parameters
HPE CS
HPE NS
The films containing HPE NP
Cmax (lg/mL1) AUC0–t (lg h/mL1) Tmax (h) MRT (h)
0.66 ± 0.06 3.58 ± 0.84
1.54 ± 0.11** 5.96 ± 0.92**
1.75 ± 0.09** 6.62 ± 0.62**
1.10 ± 0.10 5.79 ± 1.05
0.70 ± 0.17* 3.38 ± 0.81**
0.51 ± 0.05* 4.09 ± 0.78*
P < 0.05 vs. the parameters of HPE CS. P < 0.01 vs. the parameters of HPE CS.
hand, particle size reduction to nanometer range enhances the dissolution rate through increasing surface area and saturation solubility, which lead to the improvement of bioavailability [50]. However, there exists a fact that there is an uncertainty how much of the HPE is absorbed buccally and how much from intestine after unintentional swallowing of HPE containing saliva, which is needed to further study.
4. Conclusions In this study, a novel ODFs containing drug nanoparticles was successfully prepared by incorporating drug nanosuspensions with solid film carriers mainly composed of HPMC, MCC and L-HPC, which provides a new route of transforming nanosuspensions of poorly water-soluble drugs into a solid dosage as well as overcome the shortcoming that ODFs are not suitable for the delivery of poorly water-soluble drugs. The ODFs achieves the criteria in the dosage uniformity test for JP15. The fast dissolving film was disintegrated in water within 30 s with reconstituted nanosuspensions particle size of 280.69 ± 11.57 nm, indicating a good redispersibility of the ODFs containing HPE nanoparticles. The XRD analysis suggested that HPE in the ODFs was in the amorphous state. The resulting ODFs exhibit much faster dissolution rates compared to the ODFs containing HPE particles and HPE raw material. In vivo pharmacokinetic study showed that the ODFs containing HPE nanoparticles significantly increased the oral bioavailability of HPE compared to HPE coarse suspensions. From these results above, it was suggested that the ODFs containing drug nanoparticles may provide a potential opportunity in transforming drug nanosuspensions into a solid dosage form as well as improving oral bioavailability of poorly water-soluble drugs. Acknowledgments The authors would like to acknowledge the financial support from the Natural Science Found of Beijing city of China (No. 7122176), the Scientific Research Foundation for the Returned Overseas Chinese Scholars, State Education Ministry (No. 20101561) and the National Key New Drugs Innovation Foundation (No. 2013ZX09J13109-06C). Reference [1] C.A. Lipinski, Drug-like properties and the causes of poor solubility and poor permeability, J. Pharmacol. Toxicol. Methods 44 (1) (2000) 235–249. [2] F. Kesisoglou, S. Panmai, Y. Wu, Nanosizing-oral formulation development and biopharmaceutical evaluation, Adv. Drug Delivery Rev. 59 (7) (2007) 631–644. [3] T. Io, T. Fukami, K. Yamamoto, T. Suzuki, J.D. Xu, K. Tomono, A. Ramamoorthy, Homogeneous nanoparticles to enhance the efficiency of a hydrophobic drug, antihyperlipidemic probucol, characterized by solid-state NMR, Mol. Pharm. 7 (1) (2010) 299–305. [4] J. Salazar, O. Heinzerling, R.H. Müller, J.P. Möschwitzer, Process optimization of a novel production method for nanosuspensions using design of experiments (DoE), Int. J. Pharm. 420 (2) (2011) 395–403.
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Please cite this article in press as: B.-d. Shen et al., Development and characterization of an orodispersible film containing drug nanoparticles, Eur. J. Pharm. Biopharm. (2013), http://dx.doi.org/10.1016/j.ejpb.2013.09.019