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Dissolution improvement of fenofibrate by melting inclusion in mesoporous silica
a s i a n j o u r n a l o f p h a r m a c e u t i c a l s c i e n c e s 8 ( 2 0 1 3 ) 3 2 9 e3 3 5
Available online at www.sciencedirect.com
ScienceDirect journal homepage: http://ees.elsevier.com/ajps/default.asp
Original Research Paper
Dissolution improvement of fenofibrate by melting inclusion in mesoporous silica Fumiaki Uejo a,b,*, Waree Limwikrant c, Kunikazu Moribe a, Keiji Yamamoto a a
Graduate School of Pharmaceutical Sciences, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba 260-8675, Japan Analytical Research Labs., Astellas Pharma Inc., 180 Ozumi, Yaizu, Shizuoka 425-0072, Japan c Department of Manufacturing Pharmacy, Faculty of Pharmacy, Mahidol University, 447 Sri Ayudhya Road, Ratchatewi, Bangkok 10400, Thailand b
article info
abstract
Article history:
In this study, using mesoporous silica for the solubility enhancement of poorly water-
Received 9 September 2013
soluble drug was investigated. Although the incorporating drug into mesoporous silica is
Received in revised form
generally performed through the solvent method, the new melting method was proposed
24 October 2013
in the present study. Fenofibrate, a poorly water-soluble drug, was incorporated into
Accepted 30 October 2013
mesoporous silica by solvent method and melting method. The obtained samples were observed by SEM and their physicochemical properties were evaluated by PXRD and DSC measurement. The dissolution and supersaturated property were also investigated.
Keywords: Mesoporous silica
The results from SEM, PXRD and DSC measurement showed that drug could be loaded
Poorly water-soluble drugs
into pore via the melting method as well as by the solvent method. The drug loaded
Fenofibrate
quantity depended on the pore volume. Drug up to 33% could be incorporated into mes-
Melting method
oporous silica and existed in amorphous state. When drug was overloaded or difficulty in
Dissolution improvement
incorporation into pore was found, recrystallization of drug occurred at the outer surface of mesoporous silica. From the dissolution test, samples prepared by solvent method and melting method gave the supersaturated drug concentration which sample from melting method showed superior dissolution to the one from solvent method. From this study, drug was efficiently incorporated into mesoporous silica by the melting method which is a simple and solvent-free process, and the aqueous solubility enhancement of poorly watersoluble drug was achieved. ª 2013 Shenyang Pharmaceutical University. Production and hosting by Elsevier B.V. All rights reserved.
* Corresponding author. Analytical Research Labs., Astellas Pharma Inc., 180 Ozumi, Yaizu, Shizuoka 425-0072, Japan. Tel.: þ81 54 627 9024; fax: þ81 54 627 9821. E-mail address: [email protected] (F. Uejo). Peer review under responsibility of Shenyang Pharmaceutical University
Production and hosting by Elsevier 1818-0876/$ e see front matter ª 2013 Shenyang Pharmaceutical University. Production and hosting by Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ajps.2013.11.001
330
1.
a s i a n j o u r n a l o f p h a r m a c e u t i c a l s c i e n c e s 8 ( 2 0 1 3 ) 3 2 9 e3 3 5
Introduction
Using mesoporous silica has recently gained attention as one of alternative methods to improve the drug solubility. Mesoporous silica is a surfactant template with synthesized meso pore sizes approximately 2e50 nm. Due to its unique structural characteristics, i.e. uniform pore diameter and vast surface area [1,2], mesoporous silica has been applied to various fields such as separation, catalysis, and synthesis [3e5]. Size and molecular arrangement of drug in the pore of mesoporous silica can be controlled simultaneously with the drug amorphization and size reduction to nano level [6e9]. This will lead to the significant difference in dissolution property compared to the formulation containing bulk crystalline drug. However, to date the method used to incorporate drug into mesoporous silica is the solvent method where solvent is used and the drying step is required to remove solvent in the final step. This results in the more complicated inclusion process and problems when applied to pharmaceutical production. The purpose of this study was to establish the simple and effective method for incorporating drug into mesoporous silica. In addition to a conventional solvent method, drug in molten state was included in mesoporous silica by melting method. Fenofibrate was used as a model of poorly water-soluble drug. Several methods have been studied to enhance its poor aqueous solubility such as micronization and solid dispersion [10,11]. Fenofibrate loading into mesoporous silica by solvent method and melting method were investigated at various ratios. Moreover, inclusion drug into the macromolecule used as precursor for mesoporous silica was performed. Molecular state of drug in the prepared samples was evaluated using differential scanning calorimetry and powder X-ray diffractometry. Dissolution of fenofibrate loaded in the mesoporous silica by different methods and their supersaturated condition were studied.
2.
Materials and methods
2.1.
Materials
4.00 g of Pluronic 123 was added to 120 g of 2.00 N HCl solution and stirred until fully dissolved. Sodium silicate solution (10.2 g) was diluted with 30 g of deionized water. The latter solution was poured into the Pluronic 123 solution while stirring at 35 C. Then, the solution was kept under static condition at 35 C for 24 h. The mixture was transferred to a Teflon-lined autoclave and aged at 90 C for 48 h. The mixture was cooled to room temperature and filtered through a 0.45 mm membrane filter. Subsequently, obtained white powder was washed with deionized water and dried at 45 C for 60 h. The silicaePluronic 123 composite sample was referred to as “Pre MPS”. The Pre MPS solid was calcined at 550 C for 8 h to remove the Pluronic 123 template. This synthesized mesoporous silica was called “MPS”.
2.2.2.
Drug loading by solvent method
MPS or Pre MPS sample was soaked into 50 mg/ml of fenofibrate in dichloromethane. The moist powder was stirred using a spatula for pre-drying. Then, dichloromethane was completely evaporated in a water bath at 50 C. The samples obtained by the solvent method were named as “Solv-MPS (FF: X%)” or “Solv-Pre MPS (FF: X%)”. X% represented the weight percent of loaded fenofibrate (FF) in fenofibrateeMPS or fenofibrateePre MPS composite.
2.2.3.
Drug loading by melting method
MPS or Pre MPS sample was mixed with fenofibrate in a mortar to obtain a physical mixture, PM. PM was transferred to a stainless steel container and heated at 100 C for 1 h to melt fenofibrate. The samples obtained by the melting method were named as “Melt-MPS (FF: X%)” or “Melt-Pre MPS (FF: X%)”. X% represented the weight percent of loaded fenofibrate (FF) in fenofibrateeMPS or fenofibrateePre MPS composite.
2.2.4.
Scanning electron microscopy (SEM)
The morphology of prepared samples was observed using field emission scanning electron microscopy (JSM-6700F, JEOL Inc.). The samples were fixed on aluminum stabs using doublesided adhesive tape and coated with Au through a sputtercoater. SEM micrographs were obtained at an accelerated voltage of 3 kV.
Fenofibrate was purchased from SigmaeAldrich. Its chemical structure is shown in Fig. 1. Mesoporous silica samples were prepared by using Pluronic 123 [(EO)20(PO)70(EO)20 triblock copolymer] and sodium silicate solution purchased from SigmaeAldrich. All other chemicals and solvents were of reagent or HPLC grade and purchased from Kanto Chemicals (Japan).
2.2.5.
Powder X-ray diffractometry (PXRD)
2.2.
Methods
Crystallinity of the sample was analyzed using powder X-ray diffractometer (RINT-TTRIII, Rigaku) with Cu Ka radiation source. The sample was applied on a glass plate. The measurement was performed at a voltage of 50 kV and a current of 300 mA. The observed range was from 3 to 40 (2q) with a step size of 0.02 .
2.2.1.
Preparation of mesoporous silica
2.2.6.
Mesoporous silica was synthesized by the previous reported method [7e12] using Pluronic 123 as a template. An amount of O O
Cl
O
O
Fig. 1 e Chemical structure of fenofibrate.
Differential scanning calorimetry (DSC)
Thermal analysis was performed using differential scanning calorimetry (DSC Q1000, TA instrument). Approximately 2 mg of sample was weighed in a crimped aluminum pan and measured under nitrogen purge with a heating rate of 10 C/ min over the temperature range of 50 Ce200 C.
2.2.7.
Dissolution test
Dissolution test was carried out by using a USP apparatus II (DT-810, JASCO) with a paddle speed of 75 rpm at 37 C. A
a s i a n j o u r n a l o f p h a r m a c e u t i c a l s c i e n c e s 8 ( 2 0 1 3 ) 3 2 9 e3 3 5
331
Fig. 2 e SEM micrographs of Pre MPS and MPS; (A) Pre MPS, (B) and (C) MPS. 900 ml of water containing 0.10% sodium lauryl sulfate was used as dissolution medium to evaluate a dissolution profile under a supersaturated condition. The sample corresponding to 20.0 mg of fenofibrate was added into the vessel. Sampling time was at 5, 10, 15, 30, 45, 60 and 120 min. At each sampling, 10 ml of sample solution was drawn and filtered through a 0.45 mm membrane filter. Then, 1.0 ml of the filtrate was diluted with 0.50 ml of acetonitrile and analyzed by HPLC.
2.2.8.
HPLC analysis
The amount of dissolved fenofibrate in dissolution medium was determined by HPLC analysis (Agilent 1100, Agilent). A modified USP method was used. The mobile phase consisted of methanol and phosphate buffer (pH 2.9) in a volume ratio of 4/1 and the flow rate was maintained at 1.0 ml/min. For a HPLC column, a C18 column (X Bridge C18, 5 mm, 4.6 mm i.d. 15 cm, Waters) was used at 40 C. The detection wavelength was set to 285 nm. The injection volume was 20 ml.
3.
Results and discussion
3.1.
Morphology of fenofibrate-loaded MPS and Pre MPS
Fig. 2 shows SEM images of Pre MPS and MPS samples. Pre MPS (Fig. 2A) had a fiber-like structure with hundreds of micrometer in length and ca. 10 mm in width. Morphology of MPS (Fig. 2B) was similar to that of Pre MPS, indicating that the macroscopic structure of MPS was thermally stable and able to maintain after calcination at 550 C. Magnified image (Fig. 2C) revealed that several rod-like units with ca. 1 mm in width aggregated and formed the fiber-like structure.
Fig. 3 shows SEM images of fenofibrate-loaded samples. In the SEM image of PM sample (Fig. 3A), both bulk fenofibrate powder and fiber-like MPS structure were found. On the other hand, fenofibrate-loaded samples prepared by solvent (Fig. 3B) or melting (Fig. 3C and 3D) methods showed only fiber-like structure. This demonstrated that fenofibrate molecules interacted with Pre MPS/MPS during the drug loading process and changed into different dispersion state comparing to that in the physical mixture. It was considered that micro pores of Pre MPS/MPS were important factor for this specific dispersion state. Furthermore, the morphology of the sample with percent of loaded fenofibrate up to 66% showed the particles that could be considered as fenofibrate adsorbed onto outer surfaces of MPS fiber-like structure (Fig. 3E and 3F).
3.2. Physicochemical characterization of fenofibrateloaded MPS and Pre MPS PXRD patterns and DSC curves of samples prepared by the solvent method are shown in Fig. 4 and Fig. 5. In the PXRD analysis, crystalline fenofibrate showed its characteristic diffraction peaks. In contrast, the fenofibrate-loaded MPS at 20% and 33% gave halo patterns and diffraction peaks of crystalline fenofibrate disappeared. This was confirmed by the DSC analysis. DSC thermogram of crystalline fenofibrate showed an endothermic melting peak at 81 C. Melting peak of fenofibrate was absent in these fenofibrate-loaded MPS. These results suggested that fenofibrate was loaded into MPS with dichloromethane as a vehicle and remained in an amorphous state after removal of dichloromethane. For Pre MPS where triblock copolymer Pluronic 123 still remained in MPS pores, fenofibrate-loaded Pre MPS at 10%
332
a s i a n j o u r n a l o f p h a r m a c e u t i c a l s c i e n c e s 8 ( 2 0 1 3 ) 3 2 9 e3 3 5
Fig. 3 e SEM micrographs of fenofibrate-loaded MPS and Pre MPS prepared by solvent method and meting method; (A) Physical mixture of MPS (FF: 33%), (B) Solv-MPS (FF: 33%), (C) Melt-Pre MPS (FF: 20%), (D) Melt-MPS (FF: 33%), (E) and (F) MeltMPS (FF: 66%).
exhibited a halo pattern, whereas samples with loading over 20% showed diffraction peaks of crystalline fenofibrate. This is consistent with the DSC results that the melting peak of fenofibrate was observed in drug-loaded Pre MPS at 20% and 33%. Since the Pre MPS pores were filled with triblock copolymer Pluronic 123 and incorporating fenofibrates into MPS pores was more difficult, it could be concluded that fenofibrate molecules aggregated on outer surface of the Pre MPS and crystallized as bulk fenofibrate. PXRD patterns and DSC curves of samples prepared by the melting method are shown in Fig. 6 and Fig. 7. Like the samples prepared by the solvent method, the PXRD patterns of fenofibrate-loaded MPS at 20% and 33% exhibited halo patterns and their DSC curves showed no endothermic peaks of fenofibrate, indicating that fenofibrate loaded into MPS was in an amorphous state. In the melting process, fenofibrate should be in a molten state and be confined in the pore of MPS as amorphous state. On the contrary, the MPS samples loaded
up to 50% and 66% showed crystalline peaks of fenofibrate in both PXRD and DSC results and their peak intensities became stronger depending on a ratio of loaded fenofibrate. As shown in SEM observation (Fig. 3E and 3F), it indicated that MPS pores were saturated with fenofibrate and then excess fenofibrate molecules adsorbed and crystallized onto the outer surface of MPS. The PXRD measurement of Pre MPS loaded at 10% gave halo pattern. However crystalline peaks were observed in Pre MPS loaded over 20%. This agreed with the result of samples prepared by the solvent method. Crystallization of fenofibrate could be due to prefilling of Pluronic 123 in micro pores of Pre MPS. The results obtained from PXRD and DSC experiments demonstrated that by using the melting method which was simple and solvent-free, fenofibrate molecules were loaded into MPS as an amorphous state as same as sample prepared by the solvent method. The drug loading capacity depended
a s i a n j o u r n a l o f p h a r m a c e u t i c a l s c i e n c e s 8 ( 2 0 1 3 ) 3 2 9 e3 3 5
Fig. 4 e PXRD patterns of fenofibrate-loaded MPS and Pre MPS by solvent method; (a) Crystalline FF, (b) MPS, (c) SolvMPS (FF: 20%), (d) Solv-MPS (FF: 33%), (e) Pre MPS, (f) SolvPre MPS (FF: 10%), (g) Solv-Pre MPS (FF: 20%), (h) Solv-Pre MPS (FF: 33%).
on the volume of MPS pores and its maximum capacity for loading fenofibrate molecules as an amorphous state was ca. 33%. It was proved that when overloading fenofibrate or blocking micro pores with packed polymers like Pre MPS, incorporating drug molecule into pores became physically difficult and drug molecules aggregated on outer surface of MPS resulting in crystallization at the outside.
333
Fig. 6 e PXRD patterns of fenofibrate-loaded MPS and Pre MPS prepared by melting method; (a) Crystalline FF, (b) MPS, (c) Melt-MPS (FF: 20%), (d) Melt-MPS (FF: 33%), (e) MeltMPS (FF: 50%), (f) Melt-MPS (FF: 66%), (g) Pre MPS, (h) MeltPre MPS (FF: 10%), (i) Melt-Pre MPS (FF: 20%), (j) Melt-Pre MPS (FF: 33%).
Fig. 8 shows the dissolution profiles of samples prepared by the solvent method. For fenofibrate-loaded MPS at 20% and 33%, fenofibrate was rapidly dissolved within the first 15 min. The highly supersaturated concentration of fenofibrate was achieved comparing to the dissolution of crystalline fenofibrate. As confirmed by PXRD and DSC measurement, drug incorporated in the nanostructure of mesoporous silica was
not in crystalline form but in amorphous form. The supersaturated condition was facilitated by the drug molecules in amorphous state. For Pre MPS, drug-loaded Pre MPS at 10% showed the supersaturated condition within 15 min and remained constant till 120 min. The increase in drug solubility and its supersaturated concentration could be explained by that drug in amorphous state and Pluronic, a surfactant, were existed in the drug-loaded Pre MPS sample. In drug-loaded Pre MPS at 20%, its supersaturated concentration was reached but decreased rapidly. Moreover, no supersaturated condition was observed in the drug-loaded Pre MPS at 33%. It suggested that crystallization of drug at the outer surface of pore caused the decrease in supersaturated condition and dissolution rate. Fig. 9 shows the dissolution profiles of samples prepared by the melting method. The dissolution profiles of fenofibrate-
Fig. 5 e DSC thermograms of fenofibrate-loaded MPS and Pre MPS prepared by solvent method; (a) MPS, (b) Solv-MPS (FF: 20%), (c) Solv-MPS (FF: 33%), (d) Pre MPS, (e) Solv-Pre MPS (FF: 10%), (f) Solv-Pre MPS (FF: 20%), (g) Solv-Pre MPS (FF: 33%), (h) Crystalline FF.
Fig. 7 e DSC thermograms of fenofibrate-loaded MPS and Pre MPS by melting method; (a) MPS, (b) Melt-MPS (FF: 20%), (c) Melt-MPS (FF: 33%), (d) Melt-MPS (FF: 50%), (e) Melt-MPS (FF: 66%), (f) Pre MPS, (g) Melt-Pre MPS (FF: 20%), (h) Melt-Pre MPS (FF: 33%), (i) Crystalline FF.
3.3.
Dissolution test
334
a s i a n j o u r n a l o f p h a r m a c e u t i c a l s c i e n c e s 8 ( 2 0 1 3 ) 3 2 9 e3 3 5
A
2500
B
800
2000
Dissolution (ng/mL)
Dissolution (ng/mL)
700
1500 1000 500
600 500 400 300 200 100
0
0 0
20
40
60 80 Time (min)
100
120
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Solv-MPS (FF:33%)
Crystalline FF
PM-MPS (FF:20%)
0
20
40
60 80 Time (min)
Solv-Pre MPS (FF:10%) Solv-Pre MPS (FF:33%) PM-MPS (FF:20%)
100
120
Solv-Pre MPS (FF:20%) Crystalline FF
Fig. 8 e Dissolution profiles of fenofibrate-loaded MPS prepared by solvent method; (A) Solv-MPS, (B) Solv-Pre MPS.
2500
800
A
B
2000
Dissolution (ng/mL)
Dissolution (ng/mL)
700
1500
1000
500
600 500 400 300 200 100
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Melt-MPS(FF:20%) Melt-MPS(FF:50%) Crystalline FF
100
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40
60 80 Time (min)
Melt-Pre MPS(FF:10%) Melt-Pre MPS(FF:33%) PM-MPS (FF:20%)
Melt-MPS(FF:33%) Melt-MPS(FF:66%) PM-MPS (FF:20%)
100
120
Melt-Pre MPS(FF:20%) Crystalline FF
Fig. 9 e Dissolution profiles of fenofibrate-loaded MPS prepared by melting method; (A) Melt-MPS, (B) Melt-Pre MPS.
loaded MPS at 20% and 33% were similar to those of samples prepared by the solvent method but the supersaturated concentration was higher. Samples with high percent drug loading (50% and 66%) did not show any supersaturated condition and drug crystallization in vessel was observed during the dissolution test. It indicated that small amount of drug may crystallize during the melting method, became nuclei and accelerated the drug crystallization during the dissolution test. For Pre MPS, the supersaturated condition was achieved in Pre MPS (FF: 10%) whereas the dissolved drug concentration and dissolution rate decreased in samples with drug loading at 20% and 33%. Although no crystallization out of solution was observed during the dissolution test, results from PXRD and DSC measurement showed that crystallization happened during sample preparation by melting method. This may affect their dissolution properties. For solvent and melting methods, the most important factor affecting the solubilization of fenofibrate was the quantity of drug loaded into the nanopore of mesoporous silica.
From the above results, drug in amorphous state could be incorporated in the pore. The enhancement of solubility and dissolution rate of fenofibrate, a poorly water-soluble drug, was succeeded by using the nanosized pore of mesoporous silica. Supersaturation of drug was obtained from samples prepared by solvent and melting methods. Although drug loading by the melting method tended to have a higher supersaturated concentration than that by the solvent method.
4.
Conclusion
The results from SEM, PXRD and DSC measurement showed that fenofibrate in amorphous form could be incorporated into mesoporous silica by both solvent and melting methods. The drug-loaded quantity depended on the volume of pores. Amorphous drug up to 33% could be loaded into the pore. When drug was overloaded or pores were occupied by macromolecule as in Pre MPS, the drug incorporation was difficult and recrystallization occurred. For the dissolution test
a s i a n j o u r n a l o f p h a r m a c e u t i c a l s c i e n c e s 8 ( 2 0 1 3 ) 3 2 9 e3 3 5
of fenofibrate-loaded MPS, samples prepared from both solvent and melting methods could reach the supersaturated condition. While the sample prepared from melting method gave a higher supersaturated drug concentration. The melting method is a simple preparation process without using a solvent. From this study, drug could be efficiently loaded into MPS by melting method. It is easy to enhance the solubility and dissolution rate of poorly water-soluble drug by loading into mesoporous silica using the proposed melting method.
references
[1] Beck JS, Vartuli JC, Roth WJ, et al. A new family of mesoporous molecular-sieves prepared with liquid-crystal templates. J Am Chem Soc 1992;114:10834e10843. [2] Zhao DY, Feng JL, Huo QS, et al. Triblock copolymer syntheses of mesoporous silica with periodic 50 to 300 angstrom pores. Science 1998;279:548e552. [3] Shin YS, Liu J, Wang LQ, et al. Ordered hierarchical porous materials: towards tunable size- and shape-selective microcavities in nanoporous channels. Angew Chem Int Ed 2000;39:2702e2707. [4] Zhou WZ, Thomas JM, Shephard DS, et al. Ordering of ruthenium cluster carbonyls in mesoporous silica. Science 1998;280:705e708.
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[5] Kageyama K, Tamazawa J, Aida T. Extrusion polymerization: catalyzed synthesis of crystalline linear polyethylene nanofibers within a mesoporous silica. Science 1999;285:2113e2115. [6] Heikkila T, Salonen J, Tuura J, et al. Evaluation of mesoporous TCPSi, MCM-41, SBA-15, and TUD-1 materials as API carriers for oral drug delivery. Drug Deliv 2007;14:337e347. [7] Van Speybroeck M, Barillaro V, Thi TD, et al. Ordered mesoporous silica material SBA-15: a broad-spectrum formulation platform for poorly soluble drugs. J Pharm Sci 2009;98:2648e2658. [8] Zhang YZ, Zhang JH, Jiang TY, et al. Inclusion of the poorly water-soluble drug simvastatin in mesocellular foam nanoparticles: drug loading and release properties. Int J Pharm 2011;410:118e124. [9] Nishiwaki A, Watanabe A, Higashi K, et al. Molecular states of prednisolone dispersed in folded sheet mesoporous silica (FSM-16). Int J Pharm 2009;378:17e22. [10] Vogt M, Kunath K, Dressman JB. Dissolution enhancement of fenofibrate by micronization, cogrinding and spray-drying: comparison with commercial preparations. Eur J Pharma Biopharm 2008;68:283e288. [11] Kalivoda A, Fischbach M, Kleinebudde P. Application of mixtures of polymeric carriers for dissolution enhancement of fenofibrate using hot-melt extrusion. Int J Pharm 2012;429:58e68. [12] Kosuge K, Sato T, Kikukawa N, et al. Morphological control of rod- and fiberlike SBA-15 type mesoporous silica using water-soluble sodium silicate. Chem Mater 2004;16:899e905.
Available online at www.sciencedirect.com
ScienceDirect journal homepage: http://ees.elsevier.com/ajps/default.asp
Original Research Paper
Dissolution improvement of fenofibrate by melting inclusion in mesoporous silica Fumiaki Uejo a,b,*, Waree Limwikrant c, Kunikazu Moribe a, Keiji Yamamoto a a
Graduate School of Pharmaceutical Sciences, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba 260-8675, Japan Analytical Research Labs., Astellas Pharma Inc., 180 Ozumi, Yaizu, Shizuoka 425-0072, Japan c Department of Manufacturing Pharmacy, Faculty of Pharmacy, Mahidol University, 447 Sri Ayudhya Road, Ratchatewi, Bangkok 10400, Thailand b
article info
abstract
Article history:
In this study, using mesoporous silica for the solubility enhancement of poorly water-
Received 9 September 2013
soluble drug was investigated. Although the incorporating drug into mesoporous silica is
Received in revised form
generally performed through the solvent method, the new melting method was proposed
24 October 2013
in the present study. Fenofibrate, a poorly water-soluble drug, was incorporated into
Accepted 30 October 2013
mesoporous silica by solvent method and melting method. The obtained samples were observed by SEM and their physicochemical properties were evaluated by PXRD and DSC measurement. The dissolution and supersaturated property were also investigated.
Keywords: Mesoporous silica
The results from SEM, PXRD and DSC measurement showed that drug could be loaded
Poorly water-soluble drugs
into pore via the melting method as well as by the solvent method. The drug loaded
Fenofibrate
quantity depended on the pore volume. Drug up to 33% could be incorporated into mes-
Melting method
oporous silica and existed in amorphous state. When drug was overloaded or difficulty in
Dissolution improvement
incorporation into pore was found, recrystallization of drug occurred at the outer surface of mesoporous silica. From the dissolution test, samples prepared by solvent method and melting method gave the supersaturated drug concentration which sample from melting method showed superior dissolution to the one from solvent method. From this study, drug was efficiently incorporated into mesoporous silica by the melting method which is a simple and solvent-free process, and the aqueous solubility enhancement of poorly watersoluble drug was achieved. ª 2013 Shenyang Pharmaceutical University. Production and hosting by Elsevier B.V. All rights reserved.
* Corresponding author. Analytical Research Labs., Astellas Pharma Inc., 180 Ozumi, Yaizu, Shizuoka 425-0072, Japan. Tel.: þ81 54 627 9024; fax: þ81 54 627 9821. E-mail address: [email protected] (F. Uejo). Peer review under responsibility of Shenyang Pharmaceutical University
Production and hosting by Elsevier 1818-0876/$ e see front matter ª 2013 Shenyang Pharmaceutical University. Production and hosting by Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ajps.2013.11.001
330
1.
a s i a n j o u r n a l o f p h a r m a c e u t i c a l s c i e n c e s 8 ( 2 0 1 3 ) 3 2 9 e3 3 5
Introduction
Using mesoporous silica has recently gained attention as one of alternative methods to improve the drug solubility. Mesoporous silica is a surfactant template with synthesized meso pore sizes approximately 2e50 nm. Due to its unique structural characteristics, i.e. uniform pore diameter and vast surface area [1,2], mesoporous silica has been applied to various fields such as separation, catalysis, and synthesis [3e5]. Size and molecular arrangement of drug in the pore of mesoporous silica can be controlled simultaneously with the drug amorphization and size reduction to nano level [6e9]. This will lead to the significant difference in dissolution property compared to the formulation containing bulk crystalline drug. However, to date the method used to incorporate drug into mesoporous silica is the solvent method where solvent is used and the drying step is required to remove solvent in the final step. This results in the more complicated inclusion process and problems when applied to pharmaceutical production. The purpose of this study was to establish the simple and effective method for incorporating drug into mesoporous silica. In addition to a conventional solvent method, drug in molten state was included in mesoporous silica by melting method. Fenofibrate was used as a model of poorly water-soluble drug. Several methods have been studied to enhance its poor aqueous solubility such as micronization and solid dispersion [10,11]. Fenofibrate loading into mesoporous silica by solvent method and melting method were investigated at various ratios. Moreover, inclusion drug into the macromolecule used as precursor for mesoporous silica was performed. Molecular state of drug in the prepared samples was evaluated using differential scanning calorimetry and powder X-ray diffractometry. Dissolution of fenofibrate loaded in the mesoporous silica by different methods and their supersaturated condition were studied.
2.
Materials and methods
2.1.
Materials
4.00 g of Pluronic 123 was added to 120 g of 2.00 N HCl solution and stirred until fully dissolved. Sodium silicate solution (10.2 g) was diluted with 30 g of deionized water. The latter solution was poured into the Pluronic 123 solution while stirring at 35 C. Then, the solution was kept under static condition at 35 C for 24 h. The mixture was transferred to a Teflon-lined autoclave and aged at 90 C for 48 h. The mixture was cooled to room temperature and filtered through a 0.45 mm membrane filter. Subsequently, obtained white powder was washed with deionized water and dried at 45 C for 60 h. The silicaePluronic 123 composite sample was referred to as “Pre MPS”. The Pre MPS solid was calcined at 550 C for 8 h to remove the Pluronic 123 template. This synthesized mesoporous silica was called “MPS”.
2.2.2.
Drug loading by solvent method
MPS or Pre MPS sample was soaked into 50 mg/ml of fenofibrate in dichloromethane. The moist powder was stirred using a spatula for pre-drying. Then, dichloromethane was completely evaporated in a water bath at 50 C. The samples obtained by the solvent method were named as “Solv-MPS (FF: X%)” or “Solv-Pre MPS (FF: X%)”. X% represented the weight percent of loaded fenofibrate (FF) in fenofibrateeMPS or fenofibrateePre MPS composite.
2.2.3.
Drug loading by melting method
MPS or Pre MPS sample was mixed with fenofibrate in a mortar to obtain a physical mixture, PM. PM was transferred to a stainless steel container and heated at 100 C for 1 h to melt fenofibrate. The samples obtained by the melting method were named as “Melt-MPS (FF: X%)” or “Melt-Pre MPS (FF: X%)”. X% represented the weight percent of loaded fenofibrate (FF) in fenofibrateeMPS or fenofibrateePre MPS composite.
2.2.4.
Scanning electron microscopy (SEM)
The morphology of prepared samples was observed using field emission scanning electron microscopy (JSM-6700F, JEOL Inc.). The samples were fixed on aluminum stabs using doublesided adhesive tape and coated with Au through a sputtercoater. SEM micrographs were obtained at an accelerated voltage of 3 kV.
Fenofibrate was purchased from SigmaeAldrich. Its chemical structure is shown in Fig. 1. Mesoporous silica samples were prepared by using Pluronic 123 [(EO)20(PO)70(EO)20 triblock copolymer] and sodium silicate solution purchased from SigmaeAldrich. All other chemicals and solvents were of reagent or HPLC grade and purchased from Kanto Chemicals (Japan).
2.2.5.
Powder X-ray diffractometry (PXRD)
2.2.
Methods
Crystallinity of the sample was analyzed using powder X-ray diffractometer (RINT-TTRIII, Rigaku) with Cu Ka radiation source. The sample was applied on a glass plate. The measurement was performed at a voltage of 50 kV and a current of 300 mA. The observed range was from 3 to 40 (2q) with a step size of 0.02 .
2.2.1.
Preparation of mesoporous silica
2.2.6.
Mesoporous silica was synthesized by the previous reported method [7e12] using Pluronic 123 as a template. An amount of O O
Cl
O
O
Fig. 1 e Chemical structure of fenofibrate.
Differential scanning calorimetry (DSC)
Thermal analysis was performed using differential scanning calorimetry (DSC Q1000, TA instrument). Approximately 2 mg of sample was weighed in a crimped aluminum pan and measured under nitrogen purge with a heating rate of 10 C/ min over the temperature range of 50 Ce200 C.
2.2.7.
Dissolution test
Dissolution test was carried out by using a USP apparatus II (DT-810, JASCO) with a paddle speed of 75 rpm at 37 C. A
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Fig. 2 e SEM micrographs of Pre MPS and MPS; (A) Pre MPS, (B) and (C) MPS. 900 ml of water containing 0.10% sodium lauryl sulfate was used as dissolution medium to evaluate a dissolution profile under a supersaturated condition. The sample corresponding to 20.0 mg of fenofibrate was added into the vessel. Sampling time was at 5, 10, 15, 30, 45, 60 and 120 min. At each sampling, 10 ml of sample solution was drawn and filtered through a 0.45 mm membrane filter. Then, 1.0 ml of the filtrate was diluted with 0.50 ml of acetonitrile and analyzed by HPLC.
2.2.8.
HPLC analysis
The amount of dissolved fenofibrate in dissolution medium was determined by HPLC analysis (Agilent 1100, Agilent). A modified USP method was used. The mobile phase consisted of methanol and phosphate buffer (pH 2.9) in a volume ratio of 4/1 and the flow rate was maintained at 1.0 ml/min. For a HPLC column, a C18 column (X Bridge C18, 5 mm, 4.6 mm i.d. 15 cm, Waters) was used at 40 C. The detection wavelength was set to 285 nm. The injection volume was 20 ml.
3.
Results and discussion
3.1.
Morphology of fenofibrate-loaded MPS and Pre MPS
Fig. 2 shows SEM images of Pre MPS and MPS samples. Pre MPS (Fig. 2A) had a fiber-like structure with hundreds of micrometer in length and ca. 10 mm in width. Morphology of MPS (Fig. 2B) was similar to that of Pre MPS, indicating that the macroscopic structure of MPS was thermally stable and able to maintain after calcination at 550 C. Magnified image (Fig. 2C) revealed that several rod-like units with ca. 1 mm in width aggregated and formed the fiber-like structure.
Fig. 3 shows SEM images of fenofibrate-loaded samples. In the SEM image of PM sample (Fig. 3A), both bulk fenofibrate powder and fiber-like MPS structure were found. On the other hand, fenofibrate-loaded samples prepared by solvent (Fig. 3B) or melting (Fig. 3C and 3D) methods showed only fiber-like structure. This demonstrated that fenofibrate molecules interacted with Pre MPS/MPS during the drug loading process and changed into different dispersion state comparing to that in the physical mixture. It was considered that micro pores of Pre MPS/MPS were important factor for this specific dispersion state. Furthermore, the morphology of the sample with percent of loaded fenofibrate up to 66% showed the particles that could be considered as fenofibrate adsorbed onto outer surfaces of MPS fiber-like structure (Fig. 3E and 3F).
3.2. Physicochemical characterization of fenofibrateloaded MPS and Pre MPS PXRD patterns and DSC curves of samples prepared by the solvent method are shown in Fig. 4 and Fig. 5. In the PXRD analysis, crystalline fenofibrate showed its characteristic diffraction peaks. In contrast, the fenofibrate-loaded MPS at 20% and 33% gave halo patterns and diffraction peaks of crystalline fenofibrate disappeared. This was confirmed by the DSC analysis. DSC thermogram of crystalline fenofibrate showed an endothermic melting peak at 81 C. Melting peak of fenofibrate was absent in these fenofibrate-loaded MPS. These results suggested that fenofibrate was loaded into MPS with dichloromethane as a vehicle and remained in an amorphous state after removal of dichloromethane. For Pre MPS where triblock copolymer Pluronic 123 still remained in MPS pores, fenofibrate-loaded Pre MPS at 10%
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Fig. 3 e SEM micrographs of fenofibrate-loaded MPS and Pre MPS prepared by solvent method and meting method; (A) Physical mixture of MPS (FF: 33%), (B) Solv-MPS (FF: 33%), (C) Melt-Pre MPS (FF: 20%), (D) Melt-MPS (FF: 33%), (E) and (F) MeltMPS (FF: 66%).
exhibited a halo pattern, whereas samples with loading over 20% showed diffraction peaks of crystalline fenofibrate. This is consistent with the DSC results that the melting peak of fenofibrate was observed in drug-loaded Pre MPS at 20% and 33%. Since the Pre MPS pores were filled with triblock copolymer Pluronic 123 and incorporating fenofibrates into MPS pores was more difficult, it could be concluded that fenofibrate molecules aggregated on outer surface of the Pre MPS and crystallized as bulk fenofibrate. PXRD patterns and DSC curves of samples prepared by the melting method are shown in Fig. 6 and Fig. 7. Like the samples prepared by the solvent method, the PXRD patterns of fenofibrate-loaded MPS at 20% and 33% exhibited halo patterns and their DSC curves showed no endothermic peaks of fenofibrate, indicating that fenofibrate loaded into MPS was in an amorphous state. In the melting process, fenofibrate should be in a molten state and be confined in the pore of MPS as amorphous state. On the contrary, the MPS samples loaded
up to 50% and 66% showed crystalline peaks of fenofibrate in both PXRD and DSC results and their peak intensities became stronger depending on a ratio of loaded fenofibrate. As shown in SEM observation (Fig. 3E and 3F), it indicated that MPS pores were saturated with fenofibrate and then excess fenofibrate molecules adsorbed and crystallized onto the outer surface of MPS. The PXRD measurement of Pre MPS loaded at 10% gave halo pattern. However crystalline peaks were observed in Pre MPS loaded over 20%. This agreed with the result of samples prepared by the solvent method. Crystallization of fenofibrate could be due to prefilling of Pluronic 123 in micro pores of Pre MPS. The results obtained from PXRD and DSC experiments demonstrated that by using the melting method which was simple and solvent-free, fenofibrate molecules were loaded into MPS as an amorphous state as same as sample prepared by the solvent method. The drug loading capacity depended
a s i a n j o u r n a l o f p h a r m a c e u t i c a l s c i e n c e s 8 ( 2 0 1 3 ) 3 2 9 e3 3 5
Fig. 4 e PXRD patterns of fenofibrate-loaded MPS and Pre MPS by solvent method; (a) Crystalline FF, (b) MPS, (c) SolvMPS (FF: 20%), (d) Solv-MPS (FF: 33%), (e) Pre MPS, (f) SolvPre MPS (FF: 10%), (g) Solv-Pre MPS (FF: 20%), (h) Solv-Pre MPS (FF: 33%).
on the volume of MPS pores and its maximum capacity for loading fenofibrate molecules as an amorphous state was ca. 33%. It was proved that when overloading fenofibrate or blocking micro pores with packed polymers like Pre MPS, incorporating drug molecule into pores became physically difficult and drug molecules aggregated on outer surface of MPS resulting in crystallization at the outside.
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Fig. 6 e PXRD patterns of fenofibrate-loaded MPS and Pre MPS prepared by melting method; (a) Crystalline FF, (b) MPS, (c) Melt-MPS (FF: 20%), (d) Melt-MPS (FF: 33%), (e) MeltMPS (FF: 50%), (f) Melt-MPS (FF: 66%), (g) Pre MPS, (h) MeltPre MPS (FF: 10%), (i) Melt-Pre MPS (FF: 20%), (j) Melt-Pre MPS (FF: 33%).
Fig. 8 shows the dissolution profiles of samples prepared by the solvent method. For fenofibrate-loaded MPS at 20% and 33%, fenofibrate was rapidly dissolved within the first 15 min. The highly supersaturated concentration of fenofibrate was achieved comparing to the dissolution of crystalline fenofibrate. As confirmed by PXRD and DSC measurement, drug incorporated in the nanostructure of mesoporous silica was
not in crystalline form but in amorphous form. The supersaturated condition was facilitated by the drug molecules in amorphous state. For Pre MPS, drug-loaded Pre MPS at 10% showed the supersaturated condition within 15 min and remained constant till 120 min. The increase in drug solubility and its supersaturated concentration could be explained by that drug in amorphous state and Pluronic, a surfactant, were existed in the drug-loaded Pre MPS sample. In drug-loaded Pre MPS at 20%, its supersaturated concentration was reached but decreased rapidly. Moreover, no supersaturated condition was observed in the drug-loaded Pre MPS at 33%. It suggested that crystallization of drug at the outer surface of pore caused the decrease in supersaturated condition and dissolution rate. Fig. 9 shows the dissolution profiles of samples prepared by the melting method. The dissolution profiles of fenofibrate-
Fig. 5 e DSC thermograms of fenofibrate-loaded MPS and Pre MPS prepared by solvent method; (a) MPS, (b) Solv-MPS (FF: 20%), (c) Solv-MPS (FF: 33%), (d) Pre MPS, (e) Solv-Pre MPS (FF: 10%), (f) Solv-Pre MPS (FF: 20%), (g) Solv-Pre MPS (FF: 33%), (h) Crystalline FF.
Fig. 7 e DSC thermograms of fenofibrate-loaded MPS and Pre MPS by melting method; (a) MPS, (b) Melt-MPS (FF: 20%), (c) Melt-MPS (FF: 33%), (d) Melt-MPS (FF: 50%), (e) Melt-MPS (FF: 66%), (f) Pre MPS, (g) Melt-Pre MPS (FF: 20%), (h) Melt-Pre MPS (FF: 33%), (i) Crystalline FF.
3.3.
Dissolution test
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Fig. 8 e Dissolution profiles of fenofibrate-loaded MPS prepared by solvent method; (A) Solv-MPS, (B) Solv-Pre MPS.
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Fig. 9 e Dissolution profiles of fenofibrate-loaded MPS prepared by melting method; (A) Melt-MPS, (B) Melt-Pre MPS.
loaded MPS at 20% and 33% were similar to those of samples prepared by the solvent method but the supersaturated concentration was higher. Samples with high percent drug loading (50% and 66%) did not show any supersaturated condition and drug crystallization in vessel was observed during the dissolution test. It indicated that small amount of drug may crystallize during the melting method, became nuclei and accelerated the drug crystallization during the dissolution test. For Pre MPS, the supersaturated condition was achieved in Pre MPS (FF: 10%) whereas the dissolved drug concentration and dissolution rate decreased in samples with drug loading at 20% and 33%. Although no crystallization out of solution was observed during the dissolution test, results from PXRD and DSC measurement showed that crystallization happened during sample preparation by melting method. This may affect their dissolution properties. For solvent and melting methods, the most important factor affecting the solubilization of fenofibrate was the quantity of drug loaded into the nanopore of mesoporous silica.
From the above results, drug in amorphous state could be incorporated in the pore. The enhancement of solubility and dissolution rate of fenofibrate, a poorly water-soluble drug, was succeeded by using the nanosized pore of mesoporous silica. Supersaturation of drug was obtained from samples prepared by solvent and melting methods. Although drug loading by the melting method tended to have a higher supersaturated concentration than that by the solvent method.
4.
Conclusion
The results from SEM, PXRD and DSC measurement showed that fenofibrate in amorphous form could be incorporated into mesoporous silica by both solvent and melting methods. The drug-loaded quantity depended on the volume of pores. Amorphous drug up to 33% could be loaded into the pore. When drug was overloaded or pores were occupied by macromolecule as in Pre MPS, the drug incorporation was difficult and recrystallization occurred. For the dissolution test
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of fenofibrate-loaded MPS, samples prepared from both solvent and melting methods could reach the supersaturated condition. While the sample prepared from melting method gave a higher supersaturated drug concentration. The melting method is a simple preparation process without using a solvent. From this study, drug could be efficiently loaded into MPS by melting method. It is easy to enhance the solubility and dissolution rate of poorly water-soluble drug by loading into mesoporous silica using the proposed melting method.
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