RESEARCH ARTICLE – Pharmaceutical Nanotechnology
Preparation and Evaluation of High Dispersion Stable Nanocrystal Formulation of Poorly Water-Soluble Compounds by Using Povacoat KAYO YUMINOKI,1 FUKO SEKO,1 SHOTA HORII,1 HARUKA TAKEUCHI,1 KATSUYA TERAMOTO,1 YUICHIRO NAKADA,2 NAOFUMI HASHIMOTO1 1 2
Faculty of Pharmaceutical Sciences, Setsunan University, Hirakata, Osaka 573-0101, Japan Quality Management Group, Quality Compliance Division, Santen Pharmaceutical Company Ltd., Kita-ku, Osaka 530-8552, Japan
Received 26 May 2014; revised 5 August 2014; accepted 7 August 2014 Published online 10 September 2014 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/jps.24147 ABSTRACT: In this study, we reported the application of Povacoat , a hydrophilic polyvinylalcohol copolymer, as a dispersion stabilizer of nanoparticles of poorly water-soluble compounds. In addition, the influence of aggregation of the nanoparticles on their solubility and oral absorption was studied. Griseofulvin (GF) was used as a model compound with poor water solubility and was milled to nanoparticles by wet bead milling. The dispersion stability of GF milled with Povacoat or the generally used polymers (polyvinylalcohol, hydroxypropylcellulose SSL, and polyvinylpyrrolidone K30) was compared. Milled GF suspended in Povacoat aqueous solution with D-mannitol, added to improve the disintegration rate of freeze-dried GF, exhibited high dispersion stability without aggregation (D90 = ca. 0.220 m), whereas milled GF suspended in aqueous solutions of the other polymers aggregated (D90 > 5 m). Milled GF with Povacoat showed improved aqueous solubility and bioavailability compared with the other polymers. The aggregation of nanoparticles had significant impact on the solubility and bioavailability of GF. Povacoat also prevented the aggregation of the various milled poorly water-soluble compounds (hydrochlorothiazide and tolbutamide, etc.) more effectively than the other polymers. These results showed C 2014 Wiley that Povacoat could have wide applicability to the development of nanoformulations of poorly water-soluble compounds. Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 103:3772–3781, 2014 Keywords: nanoparticles; milling; nanotechnology; poorly water-soluble drugs; polymers; freeze-drying: oral absorption R
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INTRODUCTION Advances in combinatorial chemistry and high-throughput screening have resulted in an increase in the number of drug candidates with high pharmacological activity currently in pharmaceutical development. However, most of these candidates have low solubility in water and low bioavailability.1–3 It is known that milling poorly water-soluble compounds to nanoparticles enhances their solubility.4–6 Reducing the particle size of a compound increases its surface area and its dissolution rate according to the Nernst–Brunner and Levich modifications of the Noyes–Whitney equation for dissolution.7,8 The enhancement of solubility by reducing the particle size to the order of nanometers is also described by the Ostwald– Freundlich equation.9 In addition to enhanced solubility, it is reported that milling a compound to nanoparticles increases its bioavailability, reduces fed/fasted effects, and also has the potential to realize intravenous sustained release.4,10,11 However, a compound milled to nanoparticles tends to aggregate because it has a greater surface energy than the original compound. It is reported that the aggregation of nanoparticles cause a decrease in the dissolution rate and solubility.12 It is predicted that the aggregation of nanoparticles had influence on the oral absorption as well. Therefore, dispersion stabilizers are essential for preventing nanoparticles from aggregating. Nanoparticles of a poorly water-soluble compound can be
maintained as a well-dispersed aqueous suspension by the addition of a water-soluble polymer such as hydroxypropylcellulose SSL (HPC), polyvinylpyrrolidone K30 (PVP), or polyvinylalcohol (PVA). These polymers exhibit both hydrophobic and hydrophilic properties, so the surface of a poorly water-soluble compound can be properly wetted and then stabilized in a liquid medium. Many oral formulations are in a solid form, but it is difficult to maintain the physicochemical stability of nanosized compounds. Nanoparticles prepared by wet bead milling were obtained as suspension, so a drying process is needed to get powders including nanoparticles. Freeze-drying is widely used to obtain powders including nanoparticles, but it can be difficult to maintain the physical stability of nanosuspension due to the stresses imparted by the freeze-dry process.13–15 It is well known that polymers, sugars, and sugar alcohols can prevent the aggregation of nanoparticles during freeze-drying and improve their redispersion.14,16 Therefore, it is important to select a polymer appropriate for each poorly water-soluble compound to obtain nanoparticles with high dispersion and redispersion stability. However, there are many poorly watersoluble compounds that cannot be dispersed using the generally used polymers for this purpose. Povacoat is a new aqueous PVA copolymer (Fig. 1) with grafted poly acrylic acid (PAA) and poly methyl methacrylate (PMMA) groups. Povacoat was developed as a film-coating polymer with good film-forming ability without the need for plasticizers. Povacoat has been used to prepare oil-resistant and oxygen impermeable hard capsules, and wet granulation binder materials.17 Povacoat has also been used to prepare a solid dispersion by hot-melt extrusion.18 As shown in Figure 1, R
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Correspondence to: Kayo Yuminoki (Telephone: +81-72-866-3153; Fax: +8172-866-3153; E-mail: [email protected])
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Journal of Pharmaceutical Sciences, Vol. 103, 3772–3781 (2014) C 2014 Wiley Periodicals, Inc. and the American Pharmacists Association
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RESEARCH ARTICLE – Pharmaceutical Nanotechnology
Figure 1. The chemical structures of water-soluble polymer as dispersion stabilizer used in the manuscript. PovacoatR (a), PVA (b), HPC (c), and PVP (d).
Povacoat comprises three parts: PVA, PAA, and PMMA. PVA, PAA, and PMMA can be used as dispersion stabilizers for poorly water-soluble compounds.19–21 Therefore, Povacoat has potential to effectively prevent the aggregation of nanoparticles of poorly water-soluble compounds. In this study, we used Povacoat as a dispersion stabilizer for several poorly water-soluble compounds milled to nanoparticles. The dispersion and redispersion stabilities of the milled compounds with Povacoat were compared with the milled compounds with HPC, PVP, and PVA. The effects of aggregation of the nanoparticles on the solubility and oral absorption of the compounds were also studied using formulations with high and low dispersion characters. R
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MATERIALS AND METHODS Chemicals Griseofulvin (GF), hydrochlorothiazide (HYD), tolbutamide (TOL), acyclovir, indomethacin, diprydamole, naproxen, piroxicam, and phenytoin were purchased from Wako Pure Chemical Industries Company, Ltd. (Osaka, Japan). Povacoat Type F [POVA, molecular weight (MW): 40,000] was supplied by Daido Chemical Corporation (Osaka, Japan). HPC (MW: 10,000– 30,000), PVP (MW: 40,000), and D-mannitol (MAN) were purchased from Wako Pure Chemical Industries Company, Ltd. PVA (MW: ca. 25,000) was purchased from Nacalai Tesque Inc. (Kyoto, Japan). Zirconia (zirconium oxide) beads (beads) with diameters of 0.1 and 0.3 mm were purchased from Nikkato Company, Ltd. (Osaka, Japan). All other reagents were analytical-grade commercial products. R
Preparation of Suspensions and Powders Including Milled Poorly Water-Soluble Compounds We previously reported methods for milling poorly watersoluble compounds and compounds with low melting points to nanoparticles using a rotation/revolution pulverizer (NP-100; Thinky Corporation, Tokyo, Japan).22,23 Compounds (100 mg) were weighed into the NP-100 zirconia vessel, and then 0.1 mm beads (2.5 g) and an appropriate volume of polymer aqueDOI 10.1002/jps.24147
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ous solution were added. The following three steps were used to prepare the suspensions: (1) milling at 2000 rpm for 2 min with 0.5 mL of polymer aqueous solution; (2) dispersion at 400 rpm for 1 min after adding 9.5 mL of polymer aqueous solution [total volume: 10 mL, the concentration of suspension: 1% (w/v)]; (3) removal of the 0.1 mm zirconia beads from the suspension. The suspensions [1% (w/v)] of other compounds except TOL were milled by the same method as above described. TOL could not be milled to nanoparticles by the same milling method used for the other compounds because the particle size of the original TOL [the diameters at 90% of the population distribution (D90) = ca. 200 :m] was larger than that of the other poorly watersoluble compounds [ex. GF (D90 = ca. 20 :m] and HYD (D90 = ca. 50 :m). Consequently, TOL was milled at 2000 rpm for 10 min (5 min × 2) with 0.3 mm beads (2.5 g) and polymer aqueous solution (0.5 mL × 2). After milling, the TOL suspension [1% (w/v)] was prepared by the same method as for the other poorly water-soluble compounds. 5% and 10% (w/v) GF suspension were prepared to evaluate the dispersion stability of the higher concentration of GF suspension. Five hundred milligrams of GF was milled at 2000 rpm for 5 min with 0.1 mm beads (2.5 g) and 2.5 mL polymer aqueous solution. Thousand milligrams of GF was milled at 2000 rpm for 5 min with 0.1 mm bead (5.0 g) and 5.0 mL polymer aqueous solution. After milling, the same polymer solution was added to prepare 5% and 10% (w/v) GF suspension. The powders including the milled compounds were obtained by freeze-drying the suspension. The suspension was frozen with liquid nitrogen and freeze-dried for 48 h using an FD-81 freeze-dryer (Tokyo Rikakikai, Tokyo, Japan). Redispersion Test of the Powders Including the Milled Compounds The powders including the milled compounds were redispersed as the same concentration of suspension before freeze-drying. In case of 1% (w/v) suspension of the milled compound, the milled powders (20 mg of compounds) were weighed into vials, and then 2 mL of distilled water was added. In case of 5% and 10% (w/v) GF suspension, the milled GF powder (100 and 200 mg of GF) was weighted into vials, then 2 mL of distilled water was added. The powders of compounds was redispersed using a rotator (radius of rotation: 9 cm, rotation speed: 25 rpm) for 1 min. Particle Size Distribution The particle size distribution of the milled compounds in suspension was analyzed using a laser diffractometer (Mastersizer 2000; Malvern Instruments, Worcestershire, UK) with a smallvolume dispersing unit (Hydro 2000 :P; Malvern Instruments). In this study, all suspensions were not handled by sonication before the measurement of their particle size distribution. The particle size distribution was expressed in terms of D90 and D50 (the diameters at 50% of the population distribution) values. The particles size distribution of the milled GF suspension after the storage for a week at 25◦ C was measured to evaluate the physical stability. Morphology of Milled GF by Scanning Electron Microscopy A scanning electron microscope (JSM-840; JEOL Ltd., Tokyo, Japan) operating at 15 keV was used to analyze the morphology of the milled GF powders. Prior to scanning electron microscopy (SEM) analysis, each powder was dispersed onto a carbon-tapecoated aluminum stub and then sputtered with gold. Yuminoki et al., JOURNAL OF PHARMACEUTICAL SCIENCES 103:3772–3781, 2014
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Viscosity of the Milled GF Suspension with POVA, HPC, PVA, and PVP The viscosity of the GF suspension was performed using the Brookfield viscometer, BLIImodel with the BL adaptor (Rotor) and a small sample vessel (volume: 20 mL) (Toki Sangyo Company, Ltd., Tokyo, Japan). The viscosity of GF suspension (20 mL) was measured at 30 rpm of rotor speed and at 25◦ C. Powder X-Ray Diffraction Powder X-ray diffraction (PXRD) patterns were obtained using RINT (Rigaku Corporation, Tokyo, Japan) with Cu radiation generated at 40 mA and 40 kV. Data were obtained from 10◦ to 40◦ (22) at a scanning speed of 5◦ /min. HPLC Analysis A HPLC system (Shimadzu Corporation, Kyoto, Japan) equipped with a SIL-20AC auto-injector, a LC-20AD solvent delivery pump, a DGU-20AC degasser, and a SPD-M20A photodiode array detector with a detection wavelength of 293 nm was used. The column was a ZORBAX XRB-C18 (particle size: 5.0 :m; column size: I.D. 2.1 × 50 mm2 ; Agilent Technologies, Inc., CA, US); the column temperature was maintained at 30◦ C. The mobile phase consisted of 20 mM sodium di-hydrogen phosphate aqueous solution–acetonitrile [70:30 (v/v)] and the flow rate was 0.5 mL/min.
milled GF with POVA, or PVA was studied. For oral administration, three GF samples (original GF, GF/POVA, and GF/PVA) were suspended in 1 mL/kg body weight of distilled water. Rats were fasted for 18 h before being orally administered the original GF (50 mg/kg body weight), GF/POVA, and GF/PVA (50 mg GF/kg). Plasma Concentration of GF Blood samples (200 :L) were taken from the cannulated jugular vein at 0.5, 1.0, 2.0, 4.0, and 8.0 h after oral administration. The blood samples were centrifuged at 2330g for 5 min (4◦ C) to prepare plasma samples, then the plasma samples (75 :L) were mixed with acetonitrile (75 :L) and centrifuged at 12,100g for 5 min. The supernatants were collected in a glass tube and were kept frozen at −80◦ C until analysis. The plasma concentration of GF was determined by HPLC-DAD as described in section HPLC Analysis. The pharmacokinetic parameters for GF were calculated by 1-compartmental methods using Gastroplus program (Ver. 8.5; Simulations Plus, Inc., California). R
Statistical Analysis The p values were calculated using the F-test and t-test. Values less than 0.05 were considered significant.
RESULTS AND DISCUSSION
Dissolution Test
Preparation of Suspensions and Powders of Milled GF
Dissolution tests were carried out for 60 min in 900 mL of phosphate buffer solution (pH 6.8) with constant stirring at 100 rpm in a dissolution test apparatus (NTR-V6S; Toyama Sangyo, Osaka, Japan) at 37◦ C. Each milled GF powder (including 25 mg of GF) was weighed in the dissolution vessel after drug assay in the milled GF powders. Samples were collected and centrifuged at 12,100g for 5 min. The amount of dissolved GF in the supernatant was determined using the HPLC-DAD system as described in section HPLC Analysis.
Optimization of the Ratio of GF to POVA
Pharmacokinetic Studies Animals and Drug Administration Male Sprague–Dawley rats (Japan SLC, Shizuoka, Japan), weighing 281.6 ± 33.9 g, were housed three per cage in the laboratory with free access to food and water, and maintained on a 12 h dark/light cycle at controlled temperature (24 ± 1◦ C) and humidity (55 ± 5%). All the procedures used in the present study were conducted according to the guidelines approved by the Institutional Animal Care and Ethical Committee of Setsunan University. The pharmacokinetics of the original GF, the Table 1.
Formulation of GF Suspension at Milling Process and after Dispersion Process
Drug (mg)
Conc. (%, w/v)
Vol. (mL)
Concentration of GF suspension at milling (%, w/v)
100 100 100 100 500 1000
1 3 5 10 5 5
0.5 0.5 0.5 0.5 2.5 5.0
20 20 20 20 20 20
Polymer (Milling) No. 1 2 3 4 5 6
It was important to optimize the ratio of the compound to polymer to obtain a nanocrystal formulation which had small particle size, high dispersion stability, and redispersion stability. The optimum ratio of GF to POVA was studied by changing the concentration of the POVA aqueous solution during preparation of the GF nanoparticle suspensions (Table 1). When a higher concentration of polymer aqueous solution was used, the polymer prevented the aggregation of the nanoparticles. However, when the concentration of the polymer aqueous solution was too high, rotation/revolution pulverizer could not mill GF effectively because the high viscosity of the polymer aqueous solution decreased the speed of the zirconia beads, hampering milling. Therefore, the concentration of POVA aqueous solution was varied from 1% to 10% (w/v) (Table 2). When the concentration of POVA aqueous solution was above 3% (w/v), a nanoparticle suspension of GF was obtained. The smallest particles (before freeze-drying: D90= 0.224 :m) was obtained by using 5% (w/v) POVA aqueous solution. Table 2 also showed the redispersion stability of the freeze-dried powder of the milled GF (the GF
Vol. (mL)
Final concentration of GF suspension (%, w/v)
The mass ratio of GF to Polymer (GF:Polymer)
9.5 9.5 9.5 9.5 7.5 5.0
1 1 1 1 5 10
1:1 1:3 1:5 1:10 1:1 1:0.5
Polymer (Dispersion) Conc. (%, w/v) 1 3 5 10 5 5
The volume of GF suspension was 10 mL. Yuminoki et al., JOURNAL OF PHARMACEUTICAL SCIENCES 103:3772–3781, 2014
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Table 2.
Effect of POVA on the Dispersion Stability of the GF Suspension Before Freeze-Drying (:m)
No. 1 2 3 4 5 6
D50 0.159 0.127 0.121 0.134 0.121 0.121
± ± ± ± ± ±
The Storage of GF Suspension at 25 ◦ C for a Week (:m)
D90
0.012 0.002 0.004 0.002 0.003 0.002
1.201 0.276 0.224 0.295 0.229 0.234
± ± ± ± ± ±
1.044 0.001 0.003 0.004 0.008 0.005
D50 0.168 0.127 0.115 0.137 0.127 0.125
± ± ± ± ± ±
D90
0.017 0.002 0.000 0.009 0.000 0.000
1.708 0.261 0.220 0.289 0.241 0.234
± ± ± ± ± ±
After Freeze-Drying (:m) D50
1.506 0.002 0.001 0.016 0.001 0.000
± ± ± ± ± ±
0.171 0.121 0.115 0.140 0.123 0.121
D90
0.027 0.002 0.001 0.001 0.000 0.002
5.438 0.234 0.196 0.322 0.236 0.236
± ± ± ± ± ±
3.245 0.014 0.002 0.006 0.002 0.010
GF Contents (%, w/w) 50.38 26.04 17.24 8.49 49.06 67.15
± ± ± ± ± ±
4.23 1.33 1.73 1.32 1.03 3.99
Data represent mean ± SD of three determinations. Number in Table 2 corresponded to Number in Table 1.
powder). The redispersion method was used by rotator and was simulated like gently shaking because it was described that the freeze dried powder that could be redispersed by simple shaking without mechanical, sonic, and thermal energy were ideal.24 When the concentration of POVA aqueous solution was above 3%, the redispersion of the milled GF after freeze-drying had almost the same particle size as before freeze-drying, but the GF powder could not disintegrate completely by mixing for 1 min. In addition, the dispersion stability of higher concentration of GF suspension (5% and 10% (w/v)) was evaluated (Table 2). 5% and 10% (w/v) GF suspension formulated with 5% (w/v) POVA had high dispersion stability as well as 1% (w/v) GF suspension. 1% (w/v) GF suspension and 5% (w/v) POVA were used in following studies because the particle size distribution after redispersion was the smallest.
when the concentration of MAN was below 1% (w/v), the GF powder did not completely disintegrated by mixing for 15 min. It was reported that the disintegration rate of the GF powder influenced on the dissolution rate of GF.32 In section Dissolution Test, the effect of MAN on the dissolution behavior of the milled GF powder was evaluated. An optimum concentration of 2.5% (w/v) MAN was chosen since the disintegration rate of the GF powders decreased with increasing amount of MAN. Comparison of the Dispersion Stability and Redispersion Stability of Milled GF with Various Polymers Milled GF with the smallest particle size and redispersion stability could be prepared by using 5% (w/v) POVA aqueous solution containing 2.5% (w/v) MAN (GF/POVA/MAN mass ratio of 1:5:2.5). GF was also milled by using 5% (w/v) of the other polymer aqueous solutions (HPC, PVP, and PVA) containing 2.5% (w/v) MAN to compare the effect of the polymer on the dispersion stability of the milled GF suspension (Fig. 2a). As shown in Figure 2a, milled GF suspended in POVA aqueous solution (GF/POVA) had high dispersion stability without aggregation (D90: 0.222 :m), whereas milled GF suspended in HPC (GF/HPC), PVP (GF/PVP), and PVA (GF/PVA) aqueous solution did not (D90; GF/HPC: 16.345 :m, GF/PVP: 5.772 :m, GF/PVA: 12.929 :m). The viscosity of GF suspension with each polymer was measured (GF/POVA: 5.40 ± 0.07 mPas, GF/PVA: 9.40 ± 0.21 mPas, GF/PVP: 3.77 ± 0.08 mPas, GF/HPC: 6.95 ± 0.11 mPas). However, there were no relationship between the viscosity and the dispersion stability. It was reported that GF powders obtained by freeze-drying the milled GF suspension were redispersed in water, and then the redispersion stability of the GF powders was evaluated by measuring the particle size distribution (Fig. 2b). GF/POVA powder disintegrated rapidly and had the same particle size distribution as before freeze-drying. GF/HPC, GF/PVP, and GF/PVA powders
Optimization of the Amount of MAN Many oral formulations are in a solid form, so it is necessary that the nanosized compound should be in powder form. Freeze-drying is a common process for drying nanosuspensions.13,25,26 Sugars and sugar alcohols can inhibit the aggregation of nanoparticles during freeze-drying and improve the redispersion of freeze-dried nanoparticles.14,16 MAN is widely used to improve the redispersion stability of freeze dried products.13,27,28 It was reported that 1%–5% (w/v) sugars or sugar alcohols were required to obtain freeze-dry nanoparticles successfully.29–31 Therefore, MAN was added to POVA aqueous solutions at concentrations between 0% and 5% (w/v). As shown in Table 3, MAN had little effect on the particle size distribution of GF before and after freeze-drying, but significantly influenced the disintegration rate of the GF powder. When the concentration of MAN was above 2.5% (w/v), the GF powder completely disintegrated by mixing for 1 min and the GF powder was completely suspended. On the other hand, Table 3.
Effect of MAN on the Redispersion Stability of the GF Powder
Concentration of MAN Aqueous Solution (%, w/v) 0 1.0 2.5 5.0
Ratio of GF, POVA to MAN (GF:POVA:MAN) (w/w) 1:5:0 1:5:1 1:5:2.5 1:5:5
Before Freeze-Drying (Mean ± SD) (:m) D50 0.121 0.120 0.121 0.124
± ± ± ±
0.004 0.002 0.003 0.005
After Freeze-Drying (Mean ± SD) (:m)
D90 0.224 0.225 0.222 0.258
± ± ± ±
0.003 0.007 0.002 0.025
D50 0.115 0.119 0.117 0.110
± ± ± ±
0.001 0.002 0.001 0.003
D90 0.196 0.217 0.200 0.188
± ± ± ±
0.002 0.010 0.002 0.007
GF Contents (%, w/w) 17.24 15.62 11.63 10.45
± ± ± ±
1.73 1.03 0.70 0.97
Data represent mean ± SD of three determinations. DOI 10.1002/jps.24147
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Figure 2. Particle size distributions of the original GF and the milled GF suspension before (a) and after freeze-drying (b). The original GF was suspended in water and the milled GF suspensions were prepared with 5% (w/v) polymer aqueous solution containing 2.5% (w/v) MAN. Original GF (×), GF/POVA (), GF/PVA (), GF/HPC (), and GF/PVP (•).
Figure 3. SEM micrographs of milled GF: GF/POVA (a), GF/PVA (b), GF/HPC (c), and GF/PVP (d).
disintegrated by rotating for 1 min, but their particle size distribution after redispersion was larger than that of GF/POVA. In this study, GF was milled using the same conditions (the amount and diameter of beads, and the rotation/revolution speed) for all four polymers. This milling condition was also applied for some poorly water-soluble drugs.22,33,34 So, it seemed likely that these conditions would generate enough collision energy to mill GF to nanoparticles. To confirm whether or not the nanoparticles aggregated, the morphology of the GF powder with each polymer was analyzed by SEM (Fig. 3). The results showed that GF could be milled to nanoparticles using each polymer aqueous solution, but GF/HPC, GF/PVP, and GF/PVA suspension and powder did not show high dispersion stability and redispersion stability due to aggregation of the GF nanoparticles. In previous study, it was reported that GF could be milled to nanoparticle by using the comYuminoki et al., JOURNAL OF PHARMACEUTICAL SCIENCES 103:3772–3781, 2014
bination of Polymer [HPC and hydroxyl propyl methyl cellulose and sodium lauryl sulfate (SDS)].35–39 It was also reported that the even when the concentration of HPC was 15% (w/v) without SDS, the milled GF aggregated.36 SDS played an important role to prevent aggregation of the milled GF. Bhakay et al.32 also reported the GF nanoparticles without surfactant using the combination of HPC and croscarmellose sodium (CCS) without surfactant. However, the preparation and evaluation of GF/HPC/CCS formulation were more difficult than those of GF/HPC/SDS formulation. In addition, the redispersibility and the dissolution rate of GF/HPC/SDS formulation were superior to those of GF/HPC/CCS formulation. POVA could disperse the nanosized GF without surfactant and was more excellent stabilizer than HPC, PVP, and PVA. Most water-soluble polymers used to disperse nanoparticles of poorly water-soluble compounds comprise DOI 10.1002/jps.24147
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both hydrophilic and hydrophobic parts. Nonionic polymers were used in this study. The mechanism by which these polymers prevent the aggregation of nanoparticles involves the hydrophobic parts of the polymer adsorbing onto the surface of the poorly water-soluble compound nanoparticles,40,41 while the hydrophilic part is hydrated and forms a hydration layer which acts as a steric barrier. Van Eerdenbrugh et al.42 used D-"-tocopherol polyethylene glycol 1000 succinate as dispersion stabilizer for drug nanoparticles and reported that the hydrophobic parts of the dispersion stabilizer could be expected to associate with the surface of the drug nanoparticles. Lee et al.43 reported that the mole fraction of hydrophobic moieties of amino acid copolymers needed to at least 15 mol % to obtain the dispersion stable nanosuspension of naproxen. Therefore, it is important to select a polymer that has both hydrophilic and hydrophobic parts at an appropriate ratio to obtain high dispersion stable nanoparticles of poorly water-soluble compounds. POVA is a PVA derivative grafted with PAA and PMMA (Fig. 1a). A comparison of the structures of POVA, PVA, and PVP suggested that the hydrophobicity of POVA is higher than that of the other polymers due to the presence of PMMA; consequently, POVA strongly adsorbed onto the surface of nanoparticles. In addition, due to the presence of PAA, POVA provides a larger hydration layer that acts as a steric barrier. It seemed likely that POVA acted as a good dispersion stabilizer for the milled GF because POVA has PAA and PMMA. Crystallinity Crystallinity is an important factor affecting solubility and physicochemical stability. Therefore, it is necessary to confirm the crystallinity of the compound after milling. The GF powders were analyzed by PXRD to investigate their crystallinity after milling (Fig. 4). Figure 4a showed the PXRD pattern of the GF powder containing MAN. There was little difference in the PXRD patterns between the physical mixture (PM) and the GF powder with each polymer and MAN. However, the PXRD pattern of MAN was so strong that it was difficult to check the crystallinity of the milled GF. Figure 4b showed the PXRD pattern of the GF powder without MAN
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and indicates that the PXRD pattern of the GF powder with each polymer was similar to that of the original GF. The PXRD results therefore show that milled GF maintained its crystallinity. Dissolution Behavior The dissolution rate and solubility of compounds are important factors affecting oral absorption. Dissolution rate and solubility can be improved by decreasing the particle size. Wet bead milling is an important method for decreasing the particle size of poorly water-soluble compounds. In this study, dissolution tests of the original GF, PM, and the milled GF with each polymer, were carried out in phosphate buffer (pH 6.8) at 37◦ C to clarify the influence of particle size on dissolution behavior (Fig. 5). First, the influence of disintegration property on the dissolution behavior of the GF powder was evaluated by comparing the GF/POVA powder and GF/POVA powder without MAN (Fig. 5a). Figure 5a showed that the dissolution of GF was improved by milling to nanoparticles. In addition, the dissolution behavior of the GF/POVA was more rapid and higher than that of the GF/POVA without MAN. These GF/POVA powder had almost the same particles size distribution (Table 3), but, the disintegration property was different. The GF/POVA powder without MAN did not completely disintegrate by mixing for 15 min in the redispersion test. On the other hand, the GF/POVA powder disintegrated rapidly and was suspended completely by mixing for 1 min. Therefore, the disintegration property was influenced on the dissolution rate of the milled GF. Then, the influence of the particles size and aggregation of nanoparticles on the dissolution profiles was evaluated by using GF/POVA, GF/PVA, GF/HPC, and GF/PVP (Fig. 5b). The concentration of the original GF in the buffer was ca. 8 :g/mL during the experimental period (60 min). In contrast, the milled GF exhibited a significant increase in solubility. In particular, GF/POVA showed more superior dissolution behavior (the maximum concentration: ca. 24 :g/mL) than that of the milled GF with the other polymers: its dissolved GF was ca. threefold and twofold higher than that of the original GF and the PM of GF/POVA, respectively. As for GF/POVA, the supersaturated
Figure 4. PXRD patterns of the original GF and the milled GF with each polymer (POVA, HPC, PVA, and PVP); the milled GF including MAN (a) and the milled GF without MAN (b). DOI 10.1002/jps.24147
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Figure 5. Dissolution profiles of the original GF, the physical mixture, and the milled GF in pH 6.8 phosphate buffer: (a) the effect of disintegration property of GF powder on the dissolution profile with GF/POVA powder and GF/POVA powder without MAN and (b) the effect of aggregation of GF nanoparticle on the dissolution profile with the GF powder with each polymer (POVA, PVA, HPC, and PVP). (Drug contents; GF/POVA: 11.63 ± 0.70%, GF/POVA without MAN: 17.24 ± 1.73%, GF/PVA: 11.71 ± 0.55%, GF/HPC: 11.74 ± 0.84%, GF/PVP: 11.27 ± 0.14%).
concentration of GF did not decrease for at least 6 h (data not shown). These observations indicate that the aggregation of nanoparticles influenced on the dissolution behavior. It was important to select the optimum polymer for poorly water-soluble compound, which could disperse nanoparticle without aggregation to improve the dissolution behavior as high as possible. Pharmacokinetic Behavior in Rats A pharmacokinetic study in rats was carried out using the original GF (50 mg/kg) and GF/POVA and GF/PVA (50 mg GF/kg) to obtain the biopharmaceutical characteristics of GF/POVA and GF/PVA (Fig. 6). Milled GF aggregated in PVA aqueous solution. GF/PVA (50 mg GF/kg) was administrated to rats to study the influence of aggregation of GF on oral absorption. GF/POVA showed higher exposure than GF/PVA, with Cmax and AUC0–8 values of 6.35 ± 0.98 :g/mL and 19.96 ± 1.12 :g h/mL, respectively (Table 4). GF/POVA provided ca. eightfold enhancement of oral bioavailability of GF in comparison with the original GF. The Cmax and AUC0–8 values of GF/ POVA were significantly higher than those of GF/PVA (p < 0.05). The results showed that the aggregation of nanoparticles influenced oral absorption.
Yuminoki et al., JOURNAL OF PHARMACEUTICAL SCIENCES 103:3772–3781, 2014
Figure 6. Plasma concentrations of GF after oral administration of the original GF and milled GF with POVA or PVA to rats. Original GF suspension (50 mg/kg) (×), GF/PVA suspension (50 mg GF/kg) (), and GF/POVA suspension (50 mg GF/kg) (). Data represent mean ± SD of three independent experiments.
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Table 4. Pharmacokinetic Parameters of GF Following Oral Administration to Rats AUC0–8 (:g h/mL)
Cmax (:g/mL)
Tmax (h)
2.52 ± 0.63 9.33 ± 1.79 19.96 ± 1.12b
0.44 ± 0.12 3.43 ± 0.92 6.35 ± 0.98b
5.05 ± 1.99 0.52 ± 0.16 1.03 ± 0.11a
Original PVA (aggregated) POVA
AUC0–8 , area under the curve of blood concentration versus time from t = 0 to t = 8 after administration; Cmax , maximum concentration; Tmax , time to maximum concentration. Data represent mean ± SD of three determinations. a p < 0.05 between the original GF and the pulverized GF with POVA. b p < 0.01 between the original GF and the pulverized GF with POVA.
The Application of POVA to Other Poorly Water-Soluble Compounds The above results showed that POVA was a good dispersion stabilizer for milled GF particles. Therefore, POVA was also applied to two other poorly water-soluble compounds (HYD and TOL) (Fig. 7). POVA could disperse and redisperse the milled HYD and TOL more effectively than the other polymer. We also confirmed that POVA could efficiently disperse various other poorly water-soluble compounds (acyclovir, indomethacin, diprydamole, naproxen, piroxicam, and phenytoin) milled to nanoparticles (Table 5). Taken together, the results
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Table 5. Particle Sizes (D90 Values) of Various Poorly Water-Soluble Compounds Pulverized with POVA The Particle Size (D90) (:m) Mean ± SD Compound Acyclovir Indomethacin Diprydamole Griseofulvin Hydrochlorothiazide Naproxen Piroxicam Phenytoin Tolbutamide
Before Freeze-Drying
After Freeze-Drying
0.257 ± 0.005 0.189 ± 0.003 0.266 ± 0.030 0.222 ± 0.002 0.203 ± 0.002 0.223 ± 0.014 0.172 ± 0.018 0.176 ± 0.000 0.243 ± 0.005
0.231 ± 0.051 0.184 ± 0.013 0.251 ± 0.013 0.200 ± 0.002 0.255 ± 0.066 0.210 ± 0.007 0.199 ± 0.010 0.179 ± 0.012 0.291 ± 0.049
Data represent mean ± SD of three determinations.
showed that POVA was widely applicable as a dispersion stabilizer of various poorly water-soluble compounds.
CONCLUSIONS We found that milled GF with POVA showed higher dispersion stability and redispersion stability, and improved solubility and oral absorption in comparison with other polymers
Figure 7. Particle size distribution of the original and milled HYD and TOL suspension before (a) and after freeze-drying (b). The original HYD and TOL was suspended in water and the milled HYD and TOL suspensions were prepared with 5% (w/v) polymer aqueous solution containing 2.5% (w/v) MAN. Original HYD and TOL (×), milled HYD and TOL with POVA (), PVA (), HPC (), and PVP (•). DOI 10.1002/jps.24147
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(PVA, HPC, and PVP). The aggregation of nanoparticles influenced their solubility and oral absorption clearly. In this study, the dissolution profile and oral absorption of the nanosized GF (GF:Polymer:MAN = 1:5:2.5) was evaluated because the particle size of the nanosized GF was the smallest at 1% (w/v) GF suspension including 5% (w/v) POVA and 2.5% (w/v) MAN. Compared with a Nanocrystal suspension formulated with 4% HPC-SL, 0.08% SDS, and 20% sucrose,44 the amount of dispersion stabilizers (POVA and MAN) was not so much, but the GF loading was low. However, it was possible to improve the GF loading because 5% (w/v) and 10% (w/v) GF suspensions formulated with 5% (w/v) POVA had the high dispersion stability (Table 2). It was found that POVA could be widely applicable for nanocrystal formulations of poorly water-soluble compounds since various poorly water-soluble compounds could be milled to nanoparticles with high dispersion and redispersion stability using POVA. These studies will contribute to the development of poorly water-soluble compounds using nanocrystal formulations. R
ACKNOWLEDGMENT The authors are grateful to Daido Chemical Corporation (Osaka, Japan) for kindly providing POVA.
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33. Onoue S, Kojo Y, Suzuki H, Yuminoki K, Kou K, Kawabata Y, Yamauchi Y, Hashimoto N, Yamada S. 2013. Development of novel solid dispersion of tranilast using amphiphilic block copolymer for improved oral bioavailability. Int J Pharm 452:220–226. 34. Onoue S, Uchida A, Takahashi H, Seto Y, Kawabata Y, Ogawa K, Yuminoki K, Hashimoto N, Yamada S. 2011. Development of highenergy amorphous solid dispersion of nanosized nobiletin, a citrus polymethoxylated flavone, with improved oral bioavailability. J Pharm Sci 100(9):3793–3801. 35. Bhakay A, Azad M, Bilgili E, Dave R. 2014. Redispersible fast dissolving nanocomposite microparticles of poorly water-soluble drugs. Int J Pharm 461(1–2):367–379. ˜ 36. Sievens-Figueroa L, Bhakay A, Jerez-Rozo JI, Pandya N, Romanach RJ, Michniak-Kohn B, Iqbal Z, Bilgili E, Dav´e RN. 2012. Preparation and characterization of hydroxypropyl methyl cellulose films containing stable BCS Class II drug nanoparticles for pharmaceutical applications. Int J Pharm 423(2):496–508. 37. Bilgili E, Afolabi A. 2012. A combined microhydrodynamicspolymer adsorption analysis for elucidation of the roles of stabilizers in wet stirred media milling. Int J Pharm 439(1–2):193–206. 38. Monteiro A, Afolabi A, Bilgili E. 2013. Continuous production of drug nanoparticle suspensions via wet stirred media milling: A
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fresh look at the rehbinder effect. Drug Dev Ind Pharm 39(2):266– 283. 39. Bhakay A, Dave R, Bilgili E. 2013. Recovery of BCS class II drugs during aqueous redispersion of core-shell type nanocomposite particles produced via fluidized bed coating. Powder Technology 236:221–234. 40. Van Eerdenbrugh B, Van den Mooter G, Augustijns P. 2008. Topdown production of drug nanocrystals: Nanosuspension stabilization, miniaturization and transformation into solid products. Int J Pharm 364:64–75. 41. Choia JY, Yooa JY, Kwakb HS, Namc BU, Lee J. 2005. Role of polymeric stabilizers for drug nanocrystal dispersion. Curr Appl Phys 5:472–474. 42. Van Eerdenbrugh B, Vermant J, Martens JA, Froyen L, Van Humbeeck J, Augustijns P, Van den Mooter G. 2009. A screening study of surface stabilization during the production of drug nanocrystals. J Pharm Sci 98:2091–2103. 43. Lee J, Lee SJ, Choi JY, Yoo JY, Ahn CH. 2005. Amphiphilic amino acid copolymers as stabilizers for the preparation of nanocrystal dispersion. Eur J Pharm Sci 24:441–449. 44. Kesisoglou F, Mitra A. 2012. Crystalline nanosuspensions as potential toxicology and clinical oral formulations for BCS II/IV compounds. AAPS J. 14(4):677–687.
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Preparation and Evaluation of High Dispersion Stable Nanocrystal Formulation of Poorly Water-Soluble Compounds by Using Povacoat KAYO YUMINOKI,1 FUKO SEKO,1 SHOTA HORII,1 HARUKA TAKEUCHI,1 KATSUYA TERAMOTO,1 YUICHIRO NAKADA,2 NAOFUMI HASHIMOTO1 1 2
Faculty of Pharmaceutical Sciences, Setsunan University, Hirakata, Osaka 573-0101, Japan Quality Management Group, Quality Compliance Division, Santen Pharmaceutical Company Ltd., Kita-ku, Osaka 530-8552, Japan
Received 26 May 2014; revised 5 August 2014; accepted 7 August 2014 Published online 10 September 2014 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/jps.24147 ABSTRACT: In this study, we reported the application of Povacoat , a hydrophilic polyvinylalcohol copolymer, as a dispersion stabilizer of nanoparticles of poorly water-soluble compounds. In addition, the influence of aggregation of the nanoparticles on their solubility and oral absorption was studied. Griseofulvin (GF) was used as a model compound with poor water solubility and was milled to nanoparticles by wet bead milling. The dispersion stability of GF milled with Povacoat or the generally used polymers (polyvinylalcohol, hydroxypropylcellulose SSL, and polyvinylpyrrolidone K30) was compared. Milled GF suspended in Povacoat aqueous solution with D-mannitol, added to improve the disintegration rate of freeze-dried GF, exhibited high dispersion stability without aggregation (D90 = ca. 0.220 m), whereas milled GF suspended in aqueous solutions of the other polymers aggregated (D90 > 5 m). Milled GF with Povacoat showed improved aqueous solubility and bioavailability compared with the other polymers. The aggregation of nanoparticles had significant impact on the solubility and bioavailability of GF. Povacoat also prevented the aggregation of the various milled poorly water-soluble compounds (hydrochlorothiazide and tolbutamide, etc.) more effectively than the other polymers. These results showed C 2014 Wiley that Povacoat could have wide applicability to the development of nanoformulations of poorly water-soluble compounds. Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 103:3772–3781, 2014 Keywords: nanoparticles; milling; nanotechnology; poorly water-soluble drugs; polymers; freeze-drying: oral absorption R
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INTRODUCTION Advances in combinatorial chemistry and high-throughput screening have resulted in an increase in the number of drug candidates with high pharmacological activity currently in pharmaceutical development. However, most of these candidates have low solubility in water and low bioavailability.1–3 It is known that milling poorly water-soluble compounds to nanoparticles enhances their solubility.4–6 Reducing the particle size of a compound increases its surface area and its dissolution rate according to the Nernst–Brunner and Levich modifications of the Noyes–Whitney equation for dissolution.7,8 The enhancement of solubility by reducing the particle size to the order of nanometers is also described by the Ostwald– Freundlich equation.9 In addition to enhanced solubility, it is reported that milling a compound to nanoparticles increases its bioavailability, reduces fed/fasted effects, and also has the potential to realize intravenous sustained release.4,10,11 However, a compound milled to nanoparticles tends to aggregate because it has a greater surface energy than the original compound. It is reported that the aggregation of nanoparticles cause a decrease in the dissolution rate and solubility.12 It is predicted that the aggregation of nanoparticles had influence on the oral absorption as well. Therefore, dispersion stabilizers are essential for preventing nanoparticles from aggregating. Nanoparticles of a poorly water-soluble compound can be
maintained as a well-dispersed aqueous suspension by the addition of a water-soluble polymer such as hydroxypropylcellulose SSL (HPC), polyvinylpyrrolidone K30 (PVP), or polyvinylalcohol (PVA). These polymers exhibit both hydrophobic and hydrophilic properties, so the surface of a poorly water-soluble compound can be properly wetted and then stabilized in a liquid medium. Many oral formulations are in a solid form, but it is difficult to maintain the physicochemical stability of nanosized compounds. Nanoparticles prepared by wet bead milling were obtained as suspension, so a drying process is needed to get powders including nanoparticles. Freeze-drying is widely used to obtain powders including nanoparticles, but it can be difficult to maintain the physical stability of nanosuspension due to the stresses imparted by the freeze-dry process.13–15 It is well known that polymers, sugars, and sugar alcohols can prevent the aggregation of nanoparticles during freeze-drying and improve their redispersion.14,16 Therefore, it is important to select a polymer appropriate for each poorly water-soluble compound to obtain nanoparticles with high dispersion and redispersion stability. However, there are many poorly watersoluble compounds that cannot be dispersed using the generally used polymers for this purpose. Povacoat is a new aqueous PVA copolymer (Fig. 1) with grafted poly acrylic acid (PAA) and poly methyl methacrylate (PMMA) groups. Povacoat was developed as a film-coating polymer with good film-forming ability without the need for plasticizers. Povacoat has been used to prepare oil-resistant and oxygen impermeable hard capsules, and wet granulation binder materials.17 Povacoat has also been used to prepare a solid dispersion by hot-melt extrusion.18 As shown in Figure 1, R
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Correspondence to: Kayo Yuminoki (Telephone: +81-72-866-3153; Fax: +8172-866-3153; E-mail: [email protected])
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Journal of Pharmaceutical Sciences, Vol. 103, 3772–3781 (2014) C 2014 Wiley Periodicals, Inc. and the American Pharmacists Association
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Figure 1. The chemical structures of water-soluble polymer as dispersion stabilizer used in the manuscript. PovacoatR (a), PVA (b), HPC (c), and PVP (d).
Povacoat comprises three parts: PVA, PAA, and PMMA. PVA, PAA, and PMMA can be used as dispersion stabilizers for poorly water-soluble compounds.19–21 Therefore, Povacoat has potential to effectively prevent the aggregation of nanoparticles of poorly water-soluble compounds. In this study, we used Povacoat as a dispersion stabilizer for several poorly water-soluble compounds milled to nanoparticles. The dispersion and redispersion stabilities of the milled compounds with Povacoat were compared with the milled compounds with HPC, PVP, and PVA. The effects of aggregation of the nanoparticles on the solubility and oral absorption of the compounds were also studied using formulations with high and low dispersion characters. R
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MATERIALS AND METHODS Chemicals Griseofulvin (GF), hydrochlorothiazide (HYD), tolbutamide (TOL), acyclovir, indomethacin, diprydamole, naproxen, piroxicam, and phenytoin were purchased from Wako Pure Chemical Industries Company, Ltd. (Osaka, Japan). Povacoat Type F [POVA, molecular weight (MW): 40,000] was supplied by Daido Chemical Corporation (Osaka, Japan). HPC (MW: 10,000– 30,000), PVP (MW: 40,000), and D-mannitol (MAN) were purchased from Wako Pure Chemical Industries Company, Ltd. PVA (MW: ca. 25,000) was purchased from Nacalai Tesque Inc. (Kyoto, Japan). Zirconia (zirconium oxide) beads (beads) with diameters of 0.1 and 0.3 mm were purchased from Nikkato Company, Ltd. (Osaka, Japan). All other reagents were analytical-grade commercial products. R
Preparation of Suspensions and Powders Including Milled Poorly Water-Soluble Compounds We previously reported methods for milling poorly watersoluble compounds and compounds with low melting points to nanoparticles using a rotation/revolution pulverizer (NP-100; Thinky Corporation, Tokyo, Japan).22,23 Compounds (100 mg) were weighed into the NP-100 zirconia vessel, and then 0.1 mm beads (2.5 g) and an appropriate volume of polymer aqueDOI 10.1002/jps.24147
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ous solution were added. The following three steps were used to prepare the suspensions: (1) milling at 2000 rpm for 2 min with 0.5 mL of polymer aqueous solution; (2) dispersion at 400 rpm for 1 min after adding 9.5 mL of polymer aqueous solution [total volume: 10 mL, the concentration of suspension: 1% (w/v)]; (3) removal of the 0.1 mm zirconia beads from the suspension. The suspensions [1% (w/v)] of other compounds except TOL were milled by the same method as above described. TOL could not be milled to nanoparticles by the same milling method used for the other compounds because the particle size of the original TOL [the diameters at 90% of the population distribution (D90) = ca. 200 :m] was larger than that of the other poorly watersoluble compounds [ex. GF (D90 = ca. 20 :m] and HYD (D90 = ca. 50 :m). Consequently, TOL was milled at 2000 rpm for 10 min (5 min × 2) with 0.3 mm beads (2.5 g) and polymer aqueous solution (0.5 mL × 2). After milling, the TOL suspension [1% (w/v)] was prepared by the same method as for the other poorly water-soluble compounds. 5% and 10% (w/v) GF suspension were prepared to evaluate the dispersion stability of the higher concentration of GF suspension. Five hundred milligrams of GF was milled at 2000 rpm for 5 min with 0.1 mm beads (2.5 g) and 2.5 mL polymer aqueous solution. Thousand milligrams of GF was milled at 2000 rpm for 5 min with 0.1 mm bead (5.0 g) and 5.0 mL polymer aqueous solution. After milling, the same polymer solution was added to prepare 5% and 10% (w/v) GF suspension. The powders including the milled compounds were obtained by freeze-drying the suspension. The suspension was frozen with liquid nitrogen and freeze-dried for 48 h using an FD-81 freeze-dryer (Tokyo Rikakikai, Tokyo, Japan). Redispersion Test of the Powders Including the Milled Compounds The powders including the milled compounds were redispersed as the same concentration of suspension before freeze-drying. In case of 1% (w/v) suspension of the milled compound, the milled powders (20 mg of compounds) were weighed into vials, and then 2 mL of distilled water was added. In case of 5% and 10% (w/v) GF suspension, the milled GF powder (100 and 200 mg of GF) was weighted into vials, then 2 mL of distilled water was added. The powders of compounds was redispersed using a rotator (radius of rotation: 9 cm, rotation speed: 25 rpm) for 1 min. Particle Size Distribution The particle size distribution of the milled compounds in suspension was analyzed using a laser diffractometer (Mastersizer 2000; Malvern Instruments, Worcestershire, UK) with a smallvolume dispersing unit (Hydro 2000 :P; Malvern Instruments). In this study, all suspensions were not handled by sonication before the measurement of their particle size distribution. The particle size distribution was expressed in terms of D90 and D50 (the diameters at 50% of the population distribution) values. The particles size distribution of the milled GF suspension after the storage for a week at 25◦ C was measured to evaluate the physical stability. Morphology of Milled GF by Scanning Electron Microscopy A scanning electron microscope (JSM-840; JEOL Ltd., Tokyo, Japan) operating at 15 keV was used to analyze the morphology of the milled GF powders. Prior to scanning electron microscopy (SEM) analysis, each powder was dispersed onto a carbon-tapecoated aluminum stub and then sputtered with gold. Yuminoki et al., JOURNAL OF PHARMACEUTICAL SCIENCES 103:3772–3781, 2014
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Viscosity of the Milled GF Suspension with POVA, HPC, PVA, and PVP The viscosity of the GF suspension was performed using the Brookfield viscometer, BLIImodel with the BL adaptor (Rotor) and a small sample vessel (volume: 20 mL) (Toki Sangyo Company, Ltd., Tokyo, Japan). The viscosity of GF suspension (20 mL) was measured at 30 rpm of rotor speed and at 25◦ C. Powder X-Ray Diffraction Powder X-ray diffraction (PXRD) patterns were obtained using RINT (Rigaku Corporation, Tokyo, Japan) with Cu radiation generated at 40 mA and 40 kV. Data were obtained from 10◦ to 40◦ (22) at a scanning speed of 5◦ /min. HPLC Analysis A HPLC system (Shimadzu Corporation, Kyoto, Japan) equipped with a SIL-20AC auto-injector, a LC-20AD solvent delivery pump, a DGU-20AC degasser, and a SPD-M20A photodiode array detector with a detection wavelength of 293 nm was used. The column was a ZORBAX XRB-C18 (particle size: 5.0 :m; column size: I.D. 2.1 × 50 mm2 ; Agilent Technologies, Inc., CA, US); the column temperature was maintained at 30◦ C. The mobile phase consisted of 20 mM sodium di-hydrogen phosphate aqueous solution–acetonitrile [70:30 (v/v)] and the flow rate was 0.5 mL/min.
milled GF with POVA, or PVA was studied. For oral administration, three GF samples (original GF, GF/POVA, and GF/PVA) were suspended in 1 mL/kg body weight of distilled water. Rats were fasted for 18 h before being orally administered the original GF (50 mg/kg body weight), GF/POVA, and GF/PVA (50 mg GF/kg). Plasma Concentration of GF Blood samples (200 :L) were taken from the cannulated jugular vein at 0.5, 1.0, 2.0, 4.0, and 8.0 h after oral administration. The blood samples were centrifuged at 2330g for 5 min (4◦ C) to prepare plasma samples, then the plasma samples (75 :L) were mixed with acetonitrile (75 :L) and centrifuged at 12,100g for 5 min. The supernatants were collected in a glass tube and were kept frozen at −80◦ C until analysis. The plasma concentration of GF was determined by HPLC-DAD as described in section HPLC Analysis. The pharmacokinetic parameters for GF were calculated by 1-compartmental methods using Gastroplus program (Ver. 8.5; Simulations Plus, Inc., California). R
Statistical Analysis The p values were calculated using the F-test and t-test. Values less than 0.05 were considered significant.
RESULTS AND DISCUSSION
Dissolution Test
Preparation of Suspensions and Powders of Milled GF
Dissolution tests were carried out for 60 min in 900 mL of phosphate buffer solution (pH 6.8) with constant stirring at 100 rpm in a dissolution test apparatus (NTR-V6S; Toyama Sangyo, Osaka, Japan) at 37◦ C. Each milled GF powder (including 25 mg of GF) was weighed in the dissolution vessel after drug assay in the milled GF powders. Samples were collected and centrifuged at 12,100g for 5 min. The amount of dissolved GF in the supernatant was determined using the HPLC-DAD system as described in section HPLC Analysis.
Optimization of the Ratio of GF to POVA
Pharmacokinetic Studies Animals and Drug Administration Male Sprague–Dawley rats (Japan SLC, Shizuoka, Japan), weighing 281.6 ± 33.9 g, were housed three per cage in the laboratory with free access to food and water, and maintained on a 12 h dark/light cycle at controlled temperature (24 ± 1◦ C) and humidity (55 ± 5%). All the procedures used in the present study were conducted according to the guidelines approved by the Institutional Animal Care and Ethical Committee of Setsunan University. The pharmacokinetics of the original GF, the Table 1.
Formulation of GF Suspension at Milling Process and after Dispersion Process
Drug (mg)
Conc. (%, w/v)
Vol. (mL)
Concentration of GF suspension at milling (%, w/v)
100 100 100 100 500 1000
1 3 5 10 5 5
0.5 0.5 0.5 0.5 2.5 5.0
20 20 20 20 20 20
Polymer (Milling) No. 1 2 3 4 5 6
It was important to optimize the ratio of the compound to polymer to obtain a nanocrystal formulation which had small particle size, high dispersion stability, and redispersion stability. The optimum ratio of GF to POVA was studied by changing the concentration of the POVA aqueous solution during preparation of the GF nanoparticle suspensions (Table 1). When a higher concentration of polymer aqueous solution was used, the polymer prevented the aggregation of the nanoparticles. However, when the concentration of the polymer aqueous solution was too high, rotation/revolution pulverizer could not mill GF effectively because the high viscosity of the polymer aqueous solution decreased the speed of the zirconia beads, hampering milling. Therefore, the concentration of POVA aqueous solution was varied from 1% to 10% (w/v) (Table 2). When the concentration of POVA aqueous solution was above 3% (w/v), a nanoparticle suspension of GF was obtained. The smallest particles (before freeze-drying: D90= 0.224 :m) was obtained by using 5% (w/v) POVA aqueous solution. Table 2 also showed the redispersion stability of the freeze-dried powder of the milled GF (the GF
Vol. (mL)
Final concentration of GF suspension (%, w/v)
The mass ratio of GF to Polymer (GF:Polymer)
9.5 9.5 9.5 9.5 7.5 5.0
1 1 1 1 5 10
1:1 1:3 1:5 1:10 1:1 1:0.5
Polymer (Dispersion) Conc. (%, w/v) 1 3 5 10 5 5
The volume of GF suspension was 10 mL. Yuminoki et al., JOURNAL OF PHARMACEUTICAL SCIENCES 103:3772–3781, 2014
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Table 2.
Effect of POVA on the Dispersion Stability of the GF Suspension Before Freeze-Drying (:m)
No. 1 2 3 4 5 6
D50 0.159 0.127 0.121 0.134 0.121 0.121
± ± ± ± ± ±
The Storage of GF Suspension at 25 ◦ C for a Week (:m)
D90
0.012 0.002 0.004 0.002 0.003 0.002
1.201 0.276 0.224 0.295 0.229 0.234
± ± ± ± ± ±
1.044 0.001 0.003 0.004 0.008 0.005
D50 0.168 0.127 0.115 0.137 0.127 0.125
± ± ± ± ± ±
D90
0.017 0.002 0.000 0.009 0.000 0.000
1.708 0.261 0.220 0.289 0.241 0.234
± ± ± ± ± ±
After Freeze-Drying (:m) D50
1.506 0.002 0.001 0.016 0.001 0.000
± ± ± ± ± ±
0.171 0.121 0.115 0.140 0.123 0.121
D90
0.027 0.002 0.001 0.001 0.000 0.002
5.438 0.234 0.196 0.322 0.236 0.236
± ± ± ± ± ±
3.245 0.014 0.002 0.006 0.002 0.010
GF Contents (%, w/w) 50.38 26.04 17.24 8.49 49.06 67.15
± ± ± ± ± ±
4.23 1.33 1.73 1.32 1.03 3.99
Data represent mean ± SD of three determinations. Number in Table 2 corresponded to Number in Table 1.
powder). The redispersion method was used by rotator and was simulated like gently shaking because it was described that the freeze dried powder that could be redispersed by simple shaking without mechanical, sonic, and thermal energy were ideal.24 When the concentration of POVA aqueous solution was above 3%, the redispersion of the milled GF after freeze-drying had almost the same particle size as before freeze-drying, but the GF powder could not disintegrate completely by mixing for 1 min. In addition, the dispersion stability of higher concentration of GF suspension (5% and 10% (w/v)) was evaluated (Table 2). 5% and 10% (w/v) GF suspension formulated with 5% (w/v) POVA had high dispersion stability as well as 1% (w/v) GF suspension. 1% (w/v) GF suspension and 5% (w/v) POVA were used in following studies because the particle size distribution after redispersion was the smallest.
when the concentration of MAN was below 1% (w/v), the GF powder did not completely disintegrated by mixing for 15 min. It was reported that the disintegration rate of the GF powder influenced on the dissolution rate of GF.32 In section Dissolution Test, the effect of MAN on the dissolution behavior of the milled GF powder was evaluated. An optimum concentration of 2.5% (w/v) MAN was chosen since the disintegration rate of the GF powders decreased with increasing amount of MAN. Comparison of the Dispersion Stability and Redispersion Stability of Milled GF with Various Polymers Milled GF with the smallest particle size and redispersion stability could be prepared by using 5% (w/v) POVA aqueous solution containing 2.5% (w/v) MAN (GF/POVA/MAN mass ratio of 1:5:2.5). GF was also milled by using 5% (w/v) of the other polymer aqueous solutions (HPC, PVP, and PVA) containing 2.5% (w/v) MAN to compare the effect of the polymer on the dispersion stability of the milled GF suspension (Fig. 2a). As shown in Figure 2a, milled GF suspended in POVA aqueous solution (GF/POVA) had high dispersion stability without aggregation (D90: 0.222 :m), whereas milled GF suspended in HPC (GF/HPC), PVP (GF/PVP), and PVA (GF/PVA) aqueous solution did not (D90; GF/HPC: 16.345 :m, GF/PVP: 5.772 :m, GF/PVA: 12.929 :m). The viscosity of GF suspension with each polymer was measured (GF/POVA: 5.40 ± 0.07 mPas, GF/PVA: 9.40 ± 0.21 mPas, GF/PVP: 3.77 ± 0.08 mPas, GF/HPC: 6.95 ± 0.11 mPas). However, there were no relationship between the viscosity and the dispersion stability. It was reported that GF powders obtained by freeze-drying the milled GF suspension were redispersed in water, and then the redispersion stability of the GF powders was evaluated by measuring the particle size distribution (Fig. 2b). GF/POVA powder disintegrated rapidly and had the same particle size distribution as before freeze-drying. GF/HPC, GF/PVP, and GF/PVA powders
Optimization of the Amount of MAN Many oral formulations are in a solid form, so it is necessary that the nanosized compound should be in powder form. Freeze-drying is a common process for drying nanosuspensions.13,25,26 Sugars and sugar alcohols can inhibit the aggregation of nanoparticles during freeze-drying and improve the redispersion of freeze-dried nanoparticles.14,16 MAN is widely used to improve the redispersion stability of freeze dried products.13,27,28 It was reported that 1%–5% (w/v) sugars or sugar alcohols were required to obtain freeze-dry nanoparticles successfully.29–31 Therefore, MAN was added to POVA aqueous solutions at concentrations between 0% and 5% (w/v). As shown in Table 3, MAN had little effect on the particle size distribution of GF before and after freeze-drying, but significantly influenced the disintegration rate of the GF powder. When the concentration of MAN was above 2.5% (w/v), the GF powder completely disintegrated by mixing for 1 min and the GF powder was completely suspended. On the other hand, Table 3.
Effect of MAN on the Redispersion Stability of the GF Powder
Concentration of MAN Aqueous Solution (%, w/v) 0 1.0 2.5 5.0
Ratio of GF, POVA to MAN (GF:POVA:MAN) (w/w) 1:5:0 1:5:1 1:5:2.5 1:5:5
Before Freeze-Drying (Mean ± SD) (:m) D50 0.121 0.120 0.121 0.124
± ± ± ±
0.004 0.002 0.003 0.005
After Freeze-Drying (Mean ± SD) (:m)
D90 0.224 0.225 0.222 0.258
± ± ± ±
0.003 0.007 0.002 0.025
D50 0.115 0.119 0.117 0.110
± ± ± ±
0.001 0.002 0.001 0.003
D90 0.196 0.217 0.200 0.188
± ± ± ±
0.002 0.010 0.002 0.007
GF Contents (%, w/w) 17.24 15.62 11.63 10.45
± ± ± ±
1.73 1.03 0.70 0.97
Data represent mean ± SD of three determinations. DOI 10.1002/jps.24147
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Figure 2. Particle size distributions of the original GF and the milled GF suspension before (a) and after freeze-drying (b). The original GF was suspended in water and the milled GF suspensions were prepared with 5% (w/v) polymer aqueous solution containing 2.5% (w/v) MAN. Original GF (×), GF/POVA (), GF/PVA (), GF/HPC (), and GF/PVP (•).
Figure 3. SEM micrographs of milled GF: GF/POVA (a), GF/PVA (b), GF/HPC (c), and GF/PVP (d).
disintegrated by rotating for 1 min, but their particle size distribution after redispersion was larger than that of GF/POVA. In this study, GF was milled using the same conditions (the amount and diameter of beads, and the rotation/revolution speed) for all four polymers. This milling condition was also applied for some poorly water-soluble drugs.22,33,34 So, it seemed likely that these conditions would generate enough collision energy to mill GF to nanoparticles. To confirm whether or not the nanoparticles aggregated, the morphology of the GF powder with each polymer was analyzed by SEM (Fig. 3). The results showed that GF could be milled to nanoparticles using each polymer aqueous solution, but GF/HPC, GF/PVP, and GF/PVA suspension and powder did not show high dispersion stability and redispersion stability due to aggregation of the GF nanoparticles. In previous study, it was reported that GF could be milled to nanoparticle by using the comYuminoki et al., JOURNAL OF PHARMACEUTICAL SCIENCES 103:3772–3781, 2014
bination of Polymer [HPC and hydroxyl propyl methyl cellulose and sodium lauryl sulfate (SDS)].35–39 It was also reported that the even when the concentration of HPC was 15% (w/v) without SDS, the milled GF aggregated.36 SDS played an important role to prevent aggregation of the milled GF. Bhakay et al.32 also reported the GF nanoparticles without surfactant using the combination of HPC and croscarmellose sodium (CCS) without surfactant. However, the preparation and evaluation of GF/HPC/CCS formulation were more difficult than those of GF/HPC/SDS formulation. In addition, the redispersibility and the dissolution rate of GF/HPC/SDS formulation were superior to those of GF/HPC/CCS formulation. POVA could disperse the nanosized GF without surfactant and was more excellent stabilizer than HPC, PVP, and PVA. Most water-soluble polymers used to disperse nanoparticles of poorly water-soluble compounds comprise DOI 10.1002/jps.24147
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both hydrophilic and hydrophobic parts. Nonionic polymers were used in this study. The mechanism by which these polymers prevent the aggregation of nanoparticles involves the hydrophobic parts of the polymer adsorbing onto the surface of the poorly water-soluble compound nanoparticles,40,41 while the hydrophilic part is hydrated and forms a hydration layer which acts as a steric barrier. Van Eerdenbrugh et al.42 used D-"-tocopherol polyethylene glycol 1000 succinate as dispersion stabilizer for drug nanoparticles and reported that the hydrophobic parts of the dispersion stabilizer could be expected to associate with the surface of the drug nanoparticles. Lee et al.43 reported that the mole fraction of hydrophobic moieties of amino acid copolymers needed to at least 15 mol % to obtain the dispersion stable nanosuspension of naproxen. Therefore, it is important to select a polymer that has both hydrophilic and hydrophobic parts at an appropriate ratio to obtain high dispersion stable nanoparticles of poorly water-soluble compounds. POVA is a PVA derivative grafted with PAA and PMMA (Fig. 1a). A comparison of the structures of POVA, PVA, and PVP suggested that the hydrophobicity of POVA is higher than that of the other polymers due to the presence of PMMA; consequently, POVA strongly adsorbed onto the surface of nanoparticles. In addition, due to the presence of PAA, POVA provides a larger hydration layer that acts as a steric barrier. It seemed likely that POVA acted as a good dispersion stabilizer for the milled GF because POVA has PAA and PMMA. Crystallinity Crystallinity is an important factor affecting solubility and physicochemical stability. Therefore, it is necessary to confirm the crystallinity of the compound after milling. The GF powders were analyzed by PXRD to investigate their crystallinity after milling (Fig. 4). Figure 4a showed the PXRD pattern of the GF powder containing MAN. There was little difference in the PXRD patterns between the physical mixture (PM) and the GF powder with each polymer and MAN. However, the PXRD pattern of MAN was so strong that it was difficult to check the crystallinity of the milled GF. Figure 4b showed the PXRD pattern of the GF powder without MAN
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and indicates that the PXRD pattern of the GF powder with each polymer was similar to that of the original GF. The PXRD results therefore show that milled GF maintained its crystallinity. Dissolution Behavior The dissolution rate and solubility of compounds are important factors affecting oral absorption. Dissolution rate and solubility can be improved by decreasing the particle size. Wet bead milling is an important method for decreasing the particle size of poorly water-soluble compounds. In this study, dissolution tests of the original GF, PM, and the milled GF with each polymer, were carried out in phosphate buffer (pH 6.8) at 37◦ C to clarify the influence of particle size on dissolution behavior (Fig. 5). First, the influence of disintegration property on the dissolution behavior of the GF powder was evaluated by comparing the GF/POVA powder and GF/POVA powder without MAN (Fig. 5a). Figure 5a showed that the dissolution of GF was improved by milling to nanoparticles. In addition, the dissolution behavior of the GF/POVA was more rapid and higher than that of the GF/POVA without MAN. These GF/POVA powder had almost the same particles size distribution (Table 3), but, the disintegration property was different. The GF/POVA powder without MAN did not completely disintegrate by mixing for 15 min in the redispersion test. On the other hand, the GF/POVA powder disintegrated rapidly and was suspended completely by mixing for 1 min. Therefore, the disintegration property was influenced on the dissolution rate of the milled GF. Then, the influence of the particles size and aggregation of nanoparticles on the dissolution profiles was evaluated by using GF/POVA, GF/PVA, GF/HPC, and GF/PVP (Fig. 5b). The concentration of the original GF in the buffer was ca. 8 :g/mL during the experimental period (60 min). In contrast, the milled GF exhibited a significant increase in solubility. In particular, GF/POVA showed more superior dissolution behavior (the maximum concentration: ca. 24 :g/mL) than that of the milled GF with the other polymers: its dissolved GF was ca. threefold and twofold higher than that of the original GF and the PM of GF/POVA, respectively. As for GF/POVA, the supersaturated
Figure 4. PXRD patterns of the original GF and the milled GF with each polymer (POVA, HPC, PVA, and PVP); the milled GF including MAN (a) and the milled GF without MAN (b). DOI 10.1002/jps.24147
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Figure 5. Dissolution profiles of the original GF, the physical mixture, and the milled GF in pH 6.8 phosphate buffer: (a) the effect of disintegration property of GF powder on the dissolution profile with GF/POVA powder and GF/POVA powder without MAN and (b) the effect of aggregation of GF nanoparticle on the dissolution profile with the GF powder with each polymer (POVA, PVA, HPC, and PVP). (Drug contents; GF/POVA: 11.63 ± 0.70%, GF/POVA without MAN: 17.24 ± 1.73%, GF/PVA: 11.71 ± 0.55%, GF/HPC: 11.74 ± 0.84%, GF/PVP: 11.27 ± 0.14%).
concentration of GF did not decrease for at least 6 h (data not shown). These observations indicate that the aggregation of nanoparticles influenced on the dissolution behavior. It was important to select the optimum polymer for poorly water-soluble compound, which could disperse nanoparticle without aggregation to improve the dissolution behavior as high as possible. Pharmacokinetic Behavior in Rats A pharmacokinetic study in rats was carried out using the original GF (50 mg/kg) and GF/POVA and GF/PVA (50 mg GF/kg) to obtain the biopharmaceutical characteristics of GF/POVA and GF/PVA (Fig. 6). Milled GF aggregated in PVA aqueous solution. GF/PVA (50 mg GF/kg) was administrated to rats to study the influence of aggregation of GF on oral absorption. GF/POVA showed higher exposure than GF/PVA, with Cmax and AUC0–8 values of 6.35 ± 0.98 :g/mL and 19.96 ± 1.12 :g h/mL, respectively (Table 4). GF/POVA provided ca. eightfold enhancement of oral bioavailability of GF in comparison with the original GF. The Cmax and AUC0–8 values of GF/ POVA were significantly higher than those of GF/PVA (p < 0.05). The results showed that the aggregation of nanoparticles influenced oral absorption.
Yuminoki et al., JOURNAL OF PHARMACEUTICAL SCIENCES 103:3772–3781, 2014
Figure 6. Plasma concentrations of GF after oral administration of the original GF and milled GF with POVA or PVA to rats. Original GF suspension (50 mg/kg) (×), GF/PVA suspension (50 mg GF/kg) (), and GF/POVA suspension (50 mg GF/kg) (). Data represent mean ± SD of three independent experiments.
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Table 4. Pharmacokinetic Parameters of GF Following Oral Administration to Rats AUC0–8 (:g h/mL)
Cmax (:g/mL)
Tmax (h)
2.52 ± 0.63 9.33 ± 1.79 19.96 ± 1.12b
0.44 ± 0.12 3.43 ± 0.92 6.35 ± 0.98b
5.05 ± 1.99 0.52 ± 0.16 1.03 ± 0.11a
Original PVA (aggregated) POVA
AUC0–8 , area under the curve of blood concentration versus time from t = 0 to t = 8 after administration; Cmax , maximum concentration; Tmax , time to maximum concentration. Data represent mean ± SD of three determinations. a p < 0.05 between the original GF and the pulverized GF with POVA. b p < 0.01 between the original GF and the pulverized GF with POVA.
The Application of POVA to Other Poorly Water-Soluble Compounds The above results showed that POVA was a good dispersion stabilizer for milled GF particles. Therefore, POVA was also applied to two other poorly water-soluble compounds (HYD and TOL) (Fig. 7). POVA could disperse and redisperse the milled HYD and TOL more effectively than the other polymer. We also confirmed that POVA could efficiently disperse various other poorly water-soluble compounds (acyclovir, indomethacin, diprydamole, naproxen, piroxicam, and phenytoin) milled to nanoparticles (Table 5). Taken together, the results
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Table 5. Particle Sizes (D90 Values) of Various Poorly Water-Soluble Compounds Pulverized with POVA The Particle Size (D90) (:m) Mean ± SD Compound Acyclovir Indomethacin Diprydamole Griseofulvin Hydrochlorothiazide Naproxen Piroxicam Phenytoin Tolbutamide
Before Freeze-Drying
After Freeze-Drying
0.257 ± 0.005 0.189 ± 0.003 0.266 ± 0.030 0.222 ± 0.002 0.203 ± 0.002 0.223 ± 0.014 0.172 ± 0.018 0.176 ± 0.000 0.243 ± 0.005
0.231 ± 0.051 0.184 ± 0.013 0.251 ± 0.013 0.200 ± 0.002 0.255 ± 0.066 0.210 ± 0.007 0.199 ± 0.010 0.179 ± 0.012 0.291 ± 0.049
Data represent mean ± SD of three determinations.
showed that POVA was widely applicable as a dispersion stabilizer of various poorly water-soluble compounds.
CONCLUSIONS We found that milled GF with POVA showed higher dispersion stability and redispersion stability, and improved solubility and oral absorption in comparison with other polymers
Figure 7. Particle size distribution of the original and milled HYD and TOL suspension before (a) and after freeze-drying (b). The original HYD and TOL was suspended in water and the milled HYD and TOL suspensions were prepared with 5% (w/v) polymer aqueous solution containing 2.5% (w/v) MAN. Original HYD and TOL (×), milled HYD and TOL with POVA (), PVA (), HPC (), and PVP (•). DOI 10.1002/jps.24147
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(PVA, HPC, and PVP). The aggregation of nanoparticles influenced their solubility and oral absorption clearly. In this study, the dissolution profile and oral absorption of the nanosized GF (GF:Polymer:MAN = 1:5:2.5) was evaluated because the particle size of the nanosized GF was the smallest at 1% (w/v) GF suspension including 5% (w/v) POVA and 2.5% (w/v) MAN. Compared with a Nanocrystal suspension formulated with 4% HPC-SL, 0.08% SDS, and 20% sucrose,44 the amount of dispersion stabilizers (POVA and MAN) was not so much, but the GF loading was low. However, it was possible to improve the GF loading because 5% (w/v) and 10% (w/v) GF suspensions formulated with 5% (w/v) POVA had the high dispersion stability (Table 2). It was found that POVA could be widely applicable for nanocrystal formulations of poorly water-soluble compounds since various poorly water-soluble compounds could be milled to nanoparticles with high dispersion and redispersion stability using POVA. These studies will contribute to the development of poorly water-soluble compounds using nanocrystal formulations. R
ACKNOWLEDGMENT The authors are grateful to Daido Chemical Corporation (Osaka, Japan) for kindly providing POVA.
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