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Formulation of poorly water-soluble Gemfibrozil applying power ultrasound
Ultrasonics Sonochemistry 19 (2012) 286–291
Contents lists available at ScienceDirect
Ultrasonics Sonochemistry journal homepage: www.elsevier.com/locate/ultsonch
Formulation of poorly water-soluble Gemfibrozil applying power ultrasound R. Ambrus ⇑, N. Naghipour Amirzadi, Z. Aigner, P. Szabó-Révész Department of Pharmaceutical Technology, University of Szeged, Eötvös u. 6, H-6725 Szeged, Hungary
a r t i c l e
i n f o
Article history: Received 24 January 2011 Received in revised form 7 July 2011 Accepted 7 July 2011 Available online 22 July 2011 Keywords: Power ultrasound Sonocrystallization Particle size reduction Dissolution
a b s t r a c t The dissolution properties of a drug and its release from the dosage form have a basic impact on its bioavailability. Solubility problems are a major challenge for the pharmaceutical industry as concerns the development of new pharmaceutical products. Formulation problems may possibly be overcome by modification of particle size and morphology. The application of power ultrasound is a novel possibility in drug formulation. This article reports on solvent diffusion and melt emulsification, as new methods supplemented with drying in the field of sonocrystallization of poorly water-soluble Gemfibrozil. During thermoanalytical characterization, a modified structure was detected. The specific surface area of the drug was increased following particle size reduction and the poor wettability properties could also be improved. The dissolution rate was therefore significantly increased. Ó 2011 Elsevier B.V. All rights reserved.
1. Introduction The solubility and dissolution behavior and permeability of drugs are key factors determining their bioavailability. An increasing number of newly developed drugs exhibit poor water-solubility, and overcoming this is of great importance in drug formulation [1]. Different strategies are available to solve such problems, including salt formation, solubilization, complexation with cyclodextrins, milling, crystallization, amorphization [2,3], etc. Particle engineering approaches, which can potentially be applied to a wide range of crystalline drugs, can offer alternatives for improvement of the solubility, dissolution rate, permeability and subsequent bioavailability of poorly-soluble drugs [4]. Ultrasound irradiation is commonly known to induce acoustic streaming, micro streaming and highly localized changes in temperature and pressure within a fluid, these effects offering considerable benefits to crystallization processes on the use of a sonotrode. Ultrasound energy has been used to induce nucleation at moderate supersaturation during the crystallization process, or as terminal treatment to achieve deagglomeration and the desired crystal habit. These effects lend considerable advantages to the crystallization process, such as the rapid induction of primary nucleation, a reduction of crystal size, inhibition of agglomeration and the manipulation of crystal size distribution. Recrystallization of poorly-soluble materials through the use of liquid solvents and antisolvents has also been employed successfully to reduce particle size [5–8]. A novel approach for particle size reduction on the basis of crystallization from the melt and solution by the application of ⇑ Corresponding author. Tel.: +36 62 545575; fax: +36 62 545571. E-mail address: [email protected] (R. Ambrus). 1350-4177/$ - see front matter Ó 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.ultsonch.2011.07.002
ultrasound is sonocrystallization [9]. Sonocrystallization utilizes ultrasound power characterized by the frequency range 20– 100 kHz to induce crystallization. Most applications make use of ultrasound in the range 20 kHz–5 MHz [10]. In some cases, ultrasound has been utilized for size reduction and crystallization, but very few reports are available which specify the use of ultrasound for pharmaceutical formulations [11–13]. During formulation, the role of the excipient as carrier and stabilizer is also important. In the course of sonocrystallization, solvent diffusion and melt emulsification can be applied as novel techniques to change the crystal size, distribution and structure, as shown in our previous study [9]. In both cases, two liquid phases were present: involving the dissolved or melted state of the drug, and the other an aqueous solution of additives. Gemfibrozil (GEM) is a fibric acid antilipaemic agent used to treat hyperlipoproteinaemia and as second-line therapy for type IIb hypercholesterolaemia [14]. We chose GEM as a model crystalline drug, because of its poor water-solubility and low melting point (61 °C). The objective of the current work was to study the effects of power ultrasound on the physico-chemical and structural properties and on in vitro dissolution of GEM, which could offer a novel possibility in the field of pharmaceutical formulation. 2. Experimental part 2.1. Materials Gemfibrozil (GEM) [2,2-dimethyl-5-(2,5-xylyloxy)valeric acid] was from Plantex Chemical Israel, API Division Teva group; ethyl acetate was from Merck, Germany; b-D-mannitol was from
R. Ambrus et al. / Ultrasonics Sonochemistry 19 (2012) 286–291
Hungaropharma Rt., Hungary; and PVP K-25 (polyvinylpyrrolidone) was from BASF Germany. Simulated gastric medium (SGM) (pH = 1.1 ± 0.1; 470.0 g of 1 M HCl, 1.75 g of NaCl, and 5000 ml with distilled water) and simulated intestinal medium (SIM) (pH = 6.7 ± 0.1; 34.0 g of Na2HPO42H2O, and 385 ml of 0.20 M NaOH and 5000 ml with distilled water) were prepared. 2.2. Sonocrystallization procedures A high-intensity ultrasound device (Hielscher UP 200S Ultrasonic processor, Germany) was applied as energy input for sample preparation. Our previous work revealed the effects of the preparation methods and drying on GEM crystals, as concerns size, morphology and structure. After preliminary experiments, an amplitude intensity of 30% was applied via a 200 W model ultrasound device for 7.5 min during both procedures (Fig. 1) [9]. In the first step of the bottom-up procedure, power ultrasound was used to decrease the size of precipitated GEM particles before drying. 2.2.1. Sonocrystallization from solution In the solvent diffusion method, 500 mg of GEM was dissolved in 2 ml of ethyl acetate. This solution was then dropped into 15 ml of a 0.5% aqueous solution of PVP-K25, a transparent nanoemulsion first being prepared by means of sonication where nanosized ethyl acetate drops were the internal phase. PVP as additive was used to promote stabilization and wetting of the system. After dilution of the system with 33 ml of water, the ethyl acetate diffused into the water and crystals precipitated. The temperature of the mother liquid was controlled at around 55 °C during the procedure. In the drying, 2 g of mannitol was applied as a carrier in all cases, to facilate homogeneous distribution of the drug. Two types of drying were used in this method: lyophilization (MOM freeze-drying system, Hungary), with the product referred to below; as Solv.diff.lio; and spray-drying, with the product referred to below as Solv.diff.spd. In the drying, 2 g of mannitol was applied as a carrier under continuous mechanical stirring, using a Büchi B-191 Laboratory Spray-dryer (Büchi Co., Flawil, Switzerland) with a standard 0.7 mm nozzle. The particles were separated in the novel highperformance cyclone, with a high separation and recovery rate. Spray-drying was carried out under the same conditions: at an air flow of 11.6 1 min1, a pressure of 5 bar, and a pump flow rate of 1.06 ml min1. The inlet temperature was set to 110 °C, and the outlet temperature was varied in the range 64 ± 2 °C.
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2.2.2. Sonocrystallization from the melt Melt emulsification was applied, which is organic solvent-free technology. In this method, 500 mg of GEM was melted at 75 °C in 50 ml of a 0.5% aqueous solution of PVP K-25, and the droplet size of the drug was decreased by using sonication. On fast cooling in an ice bath, applying magnetic stirring at 1000 rpm for 2 min, the drug particles precipitated. It should be mentioned that during the procedure the temperature should be kept above the melting point of GEM. In this protocol, only spray-drying was used as a drying step because lyophilization was not desirable. The product will be referred to below as Melt.emulsif.spd. 2.3. Particle size, size distribution and morphology To compare the size and size distribution of raw GEM with those in the products, the samples were dispersed in water by using an ultrasonic bath for 2 min. Sonication is necessary in order to separate the particles from each other. The individual sizes of the resulting particles were determined as reported by PrigoCapote [15]. The volume particle size distribution was measured by laser (Mastersizer S, Malvern Instrument Ltd., UK), using a 300 RF lens small volume dispersion unit (1000 rpm) and 3 PAD presentations for measurements. During the investigation, water was applied as dispersion phase, so that the carrier and additives could wash out from the products, and therefore only GEM particles were detected by the instrument. The average size volume distribution, D[0.5], surface mean diameter (Sauter diameter), D[3.2], and volume mean diameter D[4.3] were determined and evaluated. The morphology of the particles was examined with a scanning electron microscope (SEM) (Hitachi S4700, Hitachi Scientific Ltd., Japan). A sputter coating apparatus (Bio-Rad SC 502, VG Microtech, England) was applied to induce electric conductivity on the surface of the samples. The air pressure was 1.3–13.0 mPa. 2.4. Structural analysis DSC measurements were carried out with a Mettler Toledo DSC 821e thermal analysis system with the STARe thermal analysis program V6.0 (Mettler Inc., Schwerzenbach, Switzerland). Approximately 3–5 mg of pure drug or product was examined in the temperature range between 25 and 300 °C. The heating rate was 5 °C min1. Argon was used as carrier gas at a flow rate of 10 l h1 during the DSC investigations. The physical state of GEM in the different samples was evaluated by X-ray powder diffraction (XRPD). XRPD patterns were produced with an X-ray Diffractometer Miniflex II (Rigaku 0Co. Tokyo, Japan), where the tube anode was Cu with Ka = 1.5405 Å A. The pattern was collected with a tube voltage of 30 kV and a tube current of 15 mA in step scan mode (4° min1). The instrument was calibrated by using Si. 2.5. Investigation of contact angle and polarity
Fig. 1. Protocol of sample preparation.
The OCA Contact Angle System (Dataphysics OCA 20, Dataphysics Inc., GmbH, Germany) was used for studies of the wettability of the products containing GEM. 0.15 g of powder was compressed under a pressure of 1 ton by a Specac hydraulic press (Specac Inc., USA). The wetting angles of the pressings were determined after 4.3 ll of distilled water had been dropped onto the surface of the pressings. The change in the wetting angle was registered from 1 to 25 s (a minimum of five parallel numbers), using the circle-fitting method of the OCA System. The method of Wu was applied, in which two liquids with known polar (cpl ) and dispersion (cdl ) components are used for measurement. The solid surface free
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energy is the sum of the polar (cp) and non-polar (cd) components, and is calculated according to the following equation:
3. Results and discussion 3.1. Particle size, size distribution and morphology
4ðcd cd Þ 4ðcps cp Þ ð1 þ cos HÞcl ¼ d s l d þ p l p cs þ cl cs þ cl Where H is the contact angle, cs is the solid surface free energy and cl is the liquid surface tension. The percentage polarity can be calculated from the cp and c values: (cp/c) 100. The liquids used for our contact angle measurements were bidistilled water (cp = 50.2 mN m1, cd = 22.6 mN m1) and diiodomethane p 1 d (c = 1.8 mN m , c = 49 mN m1) [16,17]. 2.6. In vitro dissolution testing The GEM contents in the dried samples were determined by dissolving 100 mg of dried sample in 100 ml of phosphate buffer solution (pH 6.8 ± 0.1), stirring the solution with a magnetic stirrer (400 rpm) at room temperature for 24 h, filtering and analyzing spectrophotometrically at 362 nm. Each sample was prepared and analysed in triplicate. The modified paddle method with the USP dissolution apparatus (USP rotating-basket dissolution apparatus, Pharma Test, Heinburg, Germany) was used to examine 66.7 mg samples of raw GEM or products containing 66.7 mg of drug dissolved in 100 ml of SGM or SIM. The basket was rotated at 100 rpm and sampling was performed up to 120 min (sample volume 5.0 ml). After filtration and dilution, the GEM contents of samples were determined by spectrophotometry at 276 nm.
Table 1 Particle size analyses. Sample name
D[V, 3.2] lm
D[V, 0.5] lm
D[V, 4.3] lm
Gemfibrozil Solv.diff.lio Solv.diff.spd Melt.emulsif.spd
46.87 26.28 2.57 10.92
64.53 38.30 2.89 21.68
79.80 52.23 3.04 32.50
Modification of the particle size and morphology overcame the formulation problems. Table 1 demonstrates that the methods used can help decrease the particle size relative to that of pure GEM. The most important parameters, D[V, 0.5], D[V, 3.2] and D[V, 4.3], revealed the average size of the resulting particles. The pure drug is comprised almost only of large particles. In all cases, the precipitated GEM particles in the samples were significantly smaller than those in the pure drug. There were differences depending on the preparation methods and drying type. The difference in size when the solvent diffusion method was used was due to the drying procedures. In the solvent diffusion method involving lyophilization, we obtained larger particles because the drying process was longer (8–12 h) than that in the solvent diffusion method involving spray-drying, where the solvent evaporation was fast. Sonocrystallization from solution with spray-drying resulted in the smallest particles (20 times smaller than those of pure GEM). Melt emulsification also produced particles smaller those than of GEM, but after drying micronization was not observed. Fig. 2 presents the patterns of particle size distribution, which were monodisperse in all cases. The SEM pictures (Fig. 3) provided an overview of the morphology of the modified particles. The crystal habit of pure GEM changed significantly. The raw GEM (Fig. 3A) consisted mainly of columnar crystals with a broad focal size distribution. The edges of the columnar crystals were rounded. The freeze-dried product (Fig. 3B) comprised needle-shaped crystals. Fig. 3C and D depict amorphous particles, while Fig. 3B and D present the differences in morphology depending on the drying and preparation methods. It may be seen that solvent diffusion with spray-drying resulted in small, spherical particles. 3.2. Structure (DSC and XRPD) The DSC curves of pure GEM gave a sharp endothermic peak at 61 °C, which is its melting point, revealing its crystalline structure.
Fig. 2. Size distribution of GEM and products.
R. Ambrus et al. / Ultrasonics Sonochemistry 19 (2012) 286–291
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Fig. 3. SEM pictures of GEM (A), Solv.diff.lio (B), Solv.diff.spd (C) and Melt.emulsif.spd (D).
Fig. 4. DSC curves of GEM, mannitol and products.
With mannitol as carrier, the DSC curves exhibited a sharp endothermic peak at 166 °C, its melting point. Mannitol is a polymorphic material. Depending on the physico-chemical parameters applied, b-mannitol can be transformed into a and d forms (Fig. 4). The DSC curves in Fig. 4 show that the physico-chemical properties of GEM changed during the technological procedures. With the solvent diffusion method and lyophilization, a small endothermic peak was observed at 59.53 °C, close to the GEM melting point, indicating that the crystallinity of the drug was retained in the lyophilized product. The GEM present in Solv.diff.spd and Melt.emulsif.spd was amorphous because the characteristic sharp endothermic peak for the model drug was not detected. After
the solvent diffusion method, the GEM was still crystalline, but the melt emulsification process resulted in amorphous particles. The spray-drying procedures always furnished amorphous GEM particles. The XRPD patterns for pure GEM demonstrate its crystalline structure, as expected. The characteristic patterns involve the following 2h data: 12.04, 16.77 and 24.12. The XRPD patterns indicate mainly the b form for pure mannitol, according to the following points: 14.7, 18.82 and 23.48. The melt emulsification method resulted in an amorphous structure of the GEM particles. With the solvent diffusion method involving the use of spray-drying, the patterns likewise showed the amorphous character of the GEM
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Fig. 5. XRPD pattern of GEM, mannitol and products.
and the a form of mannitol with 2h intensity at 17.12, 18.57, 19.64, 20.24 and 21.16. On use of the solvent diffusion method with lyophilization, we obtained a semicrystalline drug and the d form, as reflected by the value of 9.52 for mannitol. Finally, we can conclude that amorphous or crystalline GEM could be formed in the different preparation and drying methods, and the carrier mannitol varied from b to a and d modifications (Fig. 5).
the decrease was from 71° to 11°. The contact angle of GEM (71°) was relatively high, showing its poor hydrophilicity. The values for mannitol and PVP K-25 were very low, as an indication that they are good wetting agents with high polarity values (26° and 20°). Excipients can help stabilize the decreased size and improve the wettability too. The large surface is hydrophilized by adsorbed stabilizers, as shown by the decreased contact angle. Tables 1 and 2 show a tendency to a correlation between the size decrease, the contact angles and the polarity. Solv.diff.spd resulted in microparticles with the largest surface, which means that this product had the highest surface free energy and polarity values. We observed that for the other two samples these data were improved as compared with those for raw GEM. Moreover, the reduction in size and
3.3. Contact angle and polarity tests The wettability study (Table 2) indicated that the products had a more hydrophilic character as compared with GEM. Significantly lower contact angles with water were measured for all samples:
Table 2 Contact angle and polarity measurement. Materials
Hwater ()
Hdiiodomethane ()
cd (mN m1)
cp (mN m1)
c (mN m1)
Polarity (%)
GEM PVP K-25 Mannitol Solv.diff.lio Solv.diff.spd Melt.emulsif.spd
71.05 ± 2.55 25.95 ± 2.96 20.13 ± 4.98 34.05 ± 4.03 11.8 ± 2.16 22.1 ± 2.31
17.05 ± 1.25 19.3 ± 5.65 24.87 ± 5.13 27.30 ± 1.02 23.4 ± 0.85 20.6 ± 0.07
44.41 43.20 41.64 40.88 42.08 42.92
11.16 32.49 35.06 29.87 37.03 33.52
55.57 75.69 76.70 70.75 79.12 76.45
20.08 42.93 45.71 41.79 46.80 43.84
Fig. 6. In vitro dissolution of GEM at pH 1.2.
R. Ambrus et al. / Ultrasonics Sonochemistry 19 (2012) 286–291
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Fig. 7. In vitro dissolution of GEM at pH 6.8.
the excipients applied influenced the surface free energy (cl) and the polarity of the material. 3.4. In vitro dissolution UV analysis of the drug content in the dried products confirmed that GEM could be found at a level of 96.5–102% of the theoretically added amount. The dissolution of raw GEM was very slow, as expected because of its low solubility. At pH 1.2, only Solv.diff.spd showed higher dissolution (Fig. 6), a larger amount of product dissolving in the first 10–20 min in comparison with GEM and the other product. During dissolution, some amorphous GEM presumably returned to the crystalline state and because of this the dissolution was around 70% instead of 100%. The dissolution rate for Melt.emulsif.spd was slightly higher that than for Solv.diff.lio because the lyophilized product was crystalline and had larger particles (38.3 lm) than those of Melt.emulsif.spd (21.68 lm). At pH 6.8, pure GEM dissolved better than in acidic media. Increased dissolution rates and higher dissolution were observed for all samples in comparison with the raw drug. The rate of dissolution increased proportionally to the degree of particle size reduction and the applied drying method. Melt.emulsif.spd and Solv.diff.lio showed the same dissolution after 2 h. For Solv.diff.spd, 100% of the drug was released during the first 10 min (Fig. 7). 4. Conclusions Our study has demonstrated the important role of power ultrasound in drug formulation. Sonocrystallization, involving a particle size reduction and a change in crystal habit, may solve the problem of the in vitro drug release of GEM. With the applied preparation method and drying procedure, we could achieve differences in size distribution, wettability, surface free energy and structure. In our work, power ultrasound served an important role in the first part of the preparation. The solvent diffusion method can help block precipitated crystal growth and in the case of melt emulsification can decrease the droplet size. The engineering of drug particles with power ultrasound is a major research area in view of the limitations of conventional particle formation and pretreatment processes. In this way, the particle size reduction and change in crystal habit attained may solve solubility/permeability problems and also facilitate generic formulation.
Acknowledgments ‘‘TÁMOP-4.2.1/B-09/1/KONV-2010-0005. Creating the Center of Excellence at the University of Szeged’’ is supported by the European Union and co-financed by the European Regional Development Fund. References [1] M. Lindenberg, S. Kopp, J.B. Dressman, Classification of orally administered drugs on world health organization model list of essential medicines according to biopharmaceutics classification systems, Eur. J. Pharm. Biopharm. 58 (2004) 265–278. [2] R. Ambrus, Z. Aigner, L. Catenacci, G. Bettinetti, P. Szabó-Révész, M. Sorrenti, Physico-chemical characterization and dissolution properties of nifluminic acid–cyclodextrin–PVP ternary systems, J. Therm. Anal. Chalorim. (2010) (doi: 10.1007/s10973-010-1069-1). [3] R. Ambrus, Z. Aigner, C. Dehelean, C. Soica, P. Szabó-Révész, Physicochemical studies on solid dispersions of nifluminic acid prepared with PVP, Rev. Chim. 58 (2007) 60–64. [4] R.G. Iacocca, C.L. Burcham, L.R. Hilden, Particle engineering: a strategy for establishing drug substance physical property specifications during small molecule development, J. Pharm. Sci. 99 (2010) 51–75. [5] M. Manish, J. Harshal, P. Anant, Melt sonocrystallization of ibuprofen: effect on crystal properties, Eur. J. Pharm. Sci. 25 (2005) 41–48. [6] A. Kordylla, S. Koch, F. Tumakaka, G. Schembecker, Towards an optimized crystallization with ultrasound: effect of solvent properties and ultrasonic process parameters, J. Crystal Growth 310 (2008) 4177–4184. [7] J.M. Asua, Miniemulsion polymerization, Prog. Polym. Sci. 27 (2002) 1283– 1346. [8] R.H. Muller, C.M. Keck, Challenges and solutions for the delivery of biotech drugs – A review of drug nanocrystal technology and lipid nanoparticles, J. Biotech. 113 (2004) 151–170. [9] R. Ambrus, N.N. Amirzadi, P. Sipos, P. Szabó-Révész, Effect of sonocrystallization on the habit and structure of Gemfibrozil crystals, Chem. Eng. Technol. 33 (2010) 827–832. [10] D.K. Sandilya, A. Kannan, Effect of ultrasound on the solubility limit of sparingly soluble solid, Ultrason. Sonochem. 17 (2010) 427–434. [11] N. Blagden, M. de Matas, P.T. Gavan, P. York, Crystal engineering of active pharmaceutical ingredients to improve solubility and dissolution rates, Adv. Drug Deliv. Rev. 59 (2007) 617–630. [12] R.K. Bund, A.B. Pandit, Sonocrystallization: effect on lactose recovery and crystal habit, Ultrason. Sonochem. 14 (2007) 143–152. [13] M.R. Abu Bakar, Z.K. Nagy, A.N. Saleemi, C.D. Rielly, The impact of direct nucleation control on crystal size distribution in pharmaceutical crystallization processes, Crystal Growth Des. 9 (2009) 1378–1384. [14] S. Budavari, The Merck Index, Gemfibrozil, 11th ed., Merck & Co., Inc., Rahway, 1989. [15] F. Priego-Capote, M.D. Luque de Castro, Ultrasound in analytical chemistry, Anal. Bioanal. Chem. 387 (2007) 249–257. [16] S. Wu, Calculation of interfacial tension in polymer systems, J. Polym. Sci. 34 (1971) 19–30. [17] E. Oh, P.E. Luner, Surface free energy of ethylcellulose films and the influence of plasticizers, Int. J. Pharm. 188 (1999) 203–219.
Contents lists available at ScienceDirect
Ultrasonics Sonochemistry journal homepage: www.elsevier.com/locate/ultsonch
Formulation of poorly water-soluble Gemfibrozil applying power ultrasound R. Ambrus ⇑, N. Naghipour Amirzadi, Z. Aigner, P. Szabó-Révész Department of Pharmaceutical Technology, University of Szeged, Eötvös u. 6, H-6725 Szeged, Hungary
a r t i c l e
i n f o
Article history: Received 24 January 2011 Received in revised form 7 July 2011 Accepted 7 July 2011 Available online 22 July 2011 Keywords: Power ultrasound Sonocrystallization Particle size reduction Dissolution
a b s t r a c t The dissolution properties of a drug and its release from the dosage form have a basic impact on its bioavailability. Solubility problems are a major challenge for the pharmaceutical industry as concerns the development of new pharmaceutical products. Formulation problems may possibly be overcome by modification of particle size and morphology. The application of power ultrasound is a novel possibility in drug formulation. This article reports on solvent diffusion and melt emulsification, as new methods supplemented with drying in the field of sonocrystallization of poorly water-soluble Gemfibrozil. During thermoanalytical characterization, a modified structure was detected. The specific surface area of the drug was increased following particle size reduction and the poor wettability properties could also be improved. The dissolution rate was therefore significantly increased. Ó 2011 Elsevier B.V. All rights reserved.
1. Introduction The solubility and dissolution behavior and permeability of drugs are key factors determining their bioavailability. An increasing number of newly developed drugs exhibit poor water-solubility, and overcoming this is of great importance in drug formulation [1]. Different strategies are available to solve such problems, including salt formation, solubilization, complexation with cyclodextrins, milling, crystallization, amorphization [2,3], etc. Particle engineering approaches, which can potentially be applied to a wide range of crystalline drugs, can offer alternatives for improvement of the solubility, dissolution rate, permeability and subsequent bioavailability of poorly-soluble drugs [4]. Ultrasound irradiation is commonly known to induce acoustic streaming, micro streaming and highly localized changes in temperature and pressure within a fluid, these effects offering considerable benefits to crystallization processes on the use of a sonotrode. Ultrasound energy has been used to induce nucleation at moderate supersaturation during the crystallization process, or as terminal treatment to achieve deagglomeration and the desired crystal habit. These effects lend considerable advantages to the crystallization process, such as the rapid induction of primary nucleation, a reduction of crystal size, inhibition of agglomeration and the manipulation of crystal size distribution. Recrystallization of poorly-soluble materials through the use of liquid solvents and antisolvents has also been employed successfully to reduce particle size [5–8]. A novel approach for particle size reduction on the basis of crystallization from the melt and solution by the application of ⇑ Corresponding author. Tel.: +36 62 545575; fax: +36 62 545571. E-mail address: [email protected] (R. Ambrus). 1350-4177/$ - see front matter Ó 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.ultsonch.2011.07.002
ultrasound is sonocrystallization [9]. Sonocrystallization utilizes ultrasound power characterized by the frequency range 20– 100 kHz to induce crystallization. Most applications make use of ultrasound in the range 20 kHz–5 MHz [10]. In some cases, ultrasound has been utilized for size reduction and crystallization, but very few reports are available which specify the use of ultrasound for pharmaceutical formulations [11–13]. During formulation, the role of the excipient as carrier and stabilizer is also important. In the course of sonocrystallization, solvent diffusion and melt emulsification can be applied as novel techniques to change the crystal size, distribution and structure, as shown in our previous study [9]. In both cases, two liquid phases were present: involving the dissolved or melted state of the drug, and the other an aqueous solution of additives. Gemfibrozil (GEM) is a fibric acid antilipaemic agent used to treat hyperlipoproteinaemia and as second-line therapy for type IIb hypercholesterolaemia [14]. We chose GEM as a model crystalline drug, because of its poor water-solubility and low melting point (61 °C). The objective of the current work was to study the effects of power ultrasound on the physico-chemical and structural properties and on in vitro dissolution of GEM, which could offer a novel possibility in the field of pharmaceutical formulation. 2. Experimental part 2.1. Materials Gemfibrozil (GEM) [2,2-dimethyl-5-(2,5-xylyloxy)valeric acid] was from Plantex Chemical Israel, API Division Teva group; ethyl acetate was from Merck, Germany; b-D-mannitol was from
R. Ambrus et al. / Ultrasonics Sonochemistry 19 (2012) 286–291
Hungaropharma Rt., Hungary; and PVP K-25 (polyvinylpyrrolidone) was from BASF Germany. Simulated gastric medium (SGM) (pH = 1.1 ± 0.1; 470.0 g of 1 M HCl, 1.75 g of NaCl, and 5000 ml with distilled water) and simulated intestinal medium (SIM) (pH = 6.7 ± 0.1; 34.0 g of Na2HPO42H2O, and 385 ml of 0.20 M NaOH and 5000 ml with distilled water) were prepared. 2.2. Sonocrystallization procedures A high-intensity ultrasound device (Hielscher UP 200S Ultrasonic processor, Germany) was applied as energy input for sample preparation. Our previous work revealed the effects of the preparation methods and drying on GEM crystals, as concerns size, morphology and structure. After preliminary experiments, an amplitude intensity of 30% was applied via a 200 W model ultrasound device for 7.5 min during both procedures (Fig. 1) [9]. In the first step of the bottom-up procedure, power ultrasound was used to decrease the size of precipitated GEM particles before drying. 2.2.1. Sonocrystallization from solution In the solvent diffusion method, 500 mg of GEM was dissolved in 2 ml of ethyl acetate. This solution was then dropped into 15 ml of a 0.5% aqueous solution of PVP-K25, a transparent nanoemulsion first being prepared by means of sonication where nanosized ethyl acetate drops were the internal phase. PVP as additive was used to promote stabilization and wetting of the system. After dilution of the system with 33 ml of water, the ethyl acetate diffused into the water and crystals precipitated. The temperature of the mother liquid was controlled at around 55 °C during the procedure. In the drying, 2 g of mannitol was applied as a carrier in all cases, to facilate homogeneous distribution of the drug. Two types of drying were used in this method: lyophilization (MOM freeze-drying system, Hungary), with the product referred to below; as Solv.diff.lio; and spray-drying, with the product referred to below as Solv.diff.spd. In the drying, 2 g of mannitol was applied as a carrier under continuous mechanical stirring, using a Büchi B-191 Laboratory Spray-dryer (Büchi Co., Flawil, Switzerland) with a standard 0.7 mm nozzle. The particles were separated in the novel highperformance cyclone, with a high separation and recovery rate. Spray-drying was carried out under the same conditions: at an air flow of 11.6 1 min1, a pressure of 5 bar, and a pump flow rate of 1.06 ml min1. The inlet temperature was set to 110 °C, and the outlet temperature was varied in the range 64 ± 2 °C.
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2.2.2. Sonocrystallization from the melt Melt emulsification was applied, which is organic solvent-free technology. In this method, 500 mg of GEM was melted at 75 °C in 50 ml of a 0.5% aqueous solution of PVP K-25, and the droplet size of the drug was decreased by using sonication. On fast cooling in an ice bath, applying magnetic stirring at 1000 rpm for 2 min, the drug particles precipitated. It should be mentioned that during the procedure the temperature should be kept above the melting point of GEM. In this protocol, only spray-drying was used as a drying step because lyophilization was not desirable. The product will be referred to below as Melt.emulsif.spd. 2.3. Particle size, size distribution and morphology To compare the size and size distribution of raw GEM with those in the products, the samples were dispersed in water by using an ultrasonic bath for 2 min. Sonication is necessary in order to separate the particles from each other. The individual sizes of the resulting particles were determined as reported by PrigoCapote [15]. The volume particle size distribution was measured by laser (Mastersizer S, Malvern Instrument Ltd., UK), using a 300 RF lens small volume dispersion unit (1000 rpm) and 3 PAD presentations for measurements. During the investigation, water was applied as dispersion phase, so that the carrier and additives could wash out from the products, and therefore only GEM particles were detected by the instrument. The average size volume distribution, D[0.5], surface mean diameter (Sauter diameter), D[3.2], and volume mean diameter D[4.3] were determined and evaluated. The morphology of the particles was examined with a scanning electron microscope (SEM) (Hitachi S4700, Hitachi Scientific Ltd., Japan). A sputter coating apparatus (Bio-Rad SC 502, VG Microtech, England) was applied to induce electric conductivity on the surface of the samples. The air pressure was 1.3–13.0 mPa. 2.4. Structural analysis DSC measurements were carried out with a Mettler Toledo DSC 821e thermal analysis system with the STARe thermal analysis program V6.0 (Mettler Inc., Schwerzenbach, Switzerland). Approximately 3–5 mg of pure drug or product was examined in the temperature range between 25 and 300 °C. The heating rate was 5 °C min1. Argon was used as carrier gas at a flow rate of 10 l h1 during the DSC investigations. The physical state of GEM in the different samples was evaluated by X-ray powder diffraction (XRPD). XRPD patterns were produced with an X-ray Diffractometer Miniflex II (Rigaku 0Co. Tokyo, Japan), where the tube anode was Cu with Ka = 1.5405 Å A. The pattern was collected with a tube voltage of 30 kV and a tube current of 15 mA in step scan mode (4° min1). The instrument was calibrated by using Si. 2.5. Investigation of contact angle and polarity
Fig. 1. Protocol of sample preparation.
The OCA Contact Angle System (Dataphysics OCA 20, Dataphysics Inc., GmbH, Germany) was used for studies of the wettability of the products containing GEM. 0.15 g of powder was compressed under a pressure of 1 ton by a Specac hydraulic press (Specac Inc., USA). The wetting angles of the pressings were determined after 4.3 ll of distilled water had been dropped onto the surface of the pressings. The change in the wetting angle was registered from 1 to 25 s (a minimum of five parallel numbers), using the circle-fitting method of the OCA System. The method of Wu was applied, in which two liquids with known polar (cpl ) and dispersion (cdl ) components are used for measurement. The solid surface free
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energy is the sum of the polar (cp) and non-polar (cd) components, and is calculated according to the following equation:
3. Results and discussion 3.1. Particle size, size distribution and morphology
4ðcd cd Þ 4ðcps cp Þ ð1 þ cos HÞcl ¼ d s l d þ p l p cs þ cl cs þ cl Where H is the contact angle, cs is the solid surface free energy and cl is the liquid surface tension. The percentage polarity can be calculated from the cp and c values: (cp/c) 100. The liquids used for our contact angle measurements were bidistilled water (cp = 50.2 mN m1, cd = 22.6 mN m1) and diiodomethane p 1 d (c = 1.8 mN m , c = 49 mN m1) [16,17]. 2.6. In vitro dissolution testing The GEM contents in the dried samples were determined by dissolving 100 mg of dried sample in 100 ml of phosphate buffer solution (pH 6.8 ± 0.1), stirring the solution with a magnetic stirrer (400 rpm) at room temperature for 24 h, filtering and analyzing spectrophotometrically at 362 nm. Each sample was prepared and analysed in triplicate. The modified paddle method with the USP dissolution apparatus (USP rotating-basket dissolution apparatus, Pharma Test, Heinburg, Germany) was used to examine 66.7 mg samples of raw GEM or products containing 66.7 mg of drug dissolved in 100 ml of SGM or SIM. The basket was rotated at 100 rpm and sampling was performed up to 120 min (sample volume 5.0 ml). After filtration and dilution, the GEM contents of samples were determined by spectrophotometry at 276 nm.
Table 1 Particle size analyses. Sample name
D[V, 3.2] lm
D[V, 0.5] lm
D[V, 4.3] lm
Gemfibrozil Solv.diff.lio Solv.diff.spd Melt.emulsif.spd
46.87 26.28 2.57 10.92
64.53 38.30 2.89 21.68
79.80 52.23 3.04 32.50
Modification of the particle size and morphology overcame the formulation problems. Table 1 demonstrates that the methods used can help decrease the particle size relative to that of pure GEM. The most important parameters, D[V, 0.5], D[V, 3.2] and D[V, 4.3], revealed the average size of the resulting particles. The pure drug is comprised almost only of large particles. In all cases, the precipitated GEM particles in the samples were significantly smaller than those in the pure drug. There were differences depending on the preparation methods and drying type. The difference in size when the solvent diffusion method was used was due to the drying procedures. In the solvent diffusion method involving lyophilization, we obtained larger particles because the drying process was longer (8–12 h) than that in the solvent diffusion method involving spray-drying, where the solvent evaporation was fast. Sonocrystallization from solution with spray-drying resulted in the smallest particles (20 times smaller than those of pure GEM). Melt emulsification also produced particles smaller those than of GEM, but after drying micronization was not observed. Fig. 2 presents the patterns of particle size distribution, which were monodisperse in all cases. The SEM pictures (Fig. 3) provided an overview of the morphology of the modified particles. The crystal habit of pure GEM changed significantly. The raw GEM (Fig. 3A) consisted mainly of columnar crystals with a broad focal size distribution. The edges of the columnar crystals were rounded. The freeze-dried product (Fig. 3B) comprised needle-shaped crystals. Fig. 3C and D depict amorphous particles, while Fig. 3B and D present the differences in morphology depending on the drying and preparation methods. It may be seen that solvent diffusion with spray-drying resulted in small, spherical particles. 3.2. Structure (DSC and XRPD) The DSC curves of pure GEM gave a sharp endothermic peak at 61 °C, which is its melting point, revealing its crystalline structure.
Fig. 2. Size distribution of GEM and products.
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Fig. 3. SEM pictures of GEM (A), Solv.diff.lio (B), Solv.diff.spd (C) and Melt.emulsif.spd (D).
Fig. 4. DSC curves of GEM, mannitol and products.
With mannitol as carrier, the DSC curves exhibited a sharp endothermic peak at 166 °C, its melting point. Mannitol is a polymorphic material. Depending on the physico-chemical parameters applied, b-mannitol can be transformed into a and d forms (Fig. 4). The DSC curves in Fig. 4 show that the physico-chemical properties of GEM changed during the technological procedures. With the solvent diffusion method and lyophilization, a small endothermic peak was observed at 59.53 °C, close to the GEM melting point, indicating that the crystallinity of the drug was retained in the lyophilized product. The GEM present in Solv.diff.spd and Melt.emulsif.spd was amorphous because the characteristic sharp endothermic peak for the model drug was not detected. After
the solvent diffusion method, the GEM was still crystalline, but the melt emulsification process resulted in amorphous particles. The spray-drying procedures always furnished amorphous GEM particles. The XRPD patterns for pure GEM demonstrate its crystalline structure, as expected. The characteristic patterns involve the following 2h data: 12.04, 16.77 and 24.12. The XRPD patterns indicate mainly the b form for pure mannitol, according to the following points: 14.7, 18.82 and 23.48. The melt emulsification method resulted in an amorphous structure of the GEM particles. With the solvent diffusion method involving the use of spray-drying, the patterns likewise showed the amorphous character of the GEM
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Fig. 5. XRPD pattern of GEM, mannitol and products.
and the a form of mannitol with 2h intensity at 17.12, 18.57, 19.64, 20.24 and 21.16. On use of the solvent diffusion method with lyophilization, we obtained a semicrystalline drug and the d form, as reflected by the value of 9.52 for mannitol. Finally, we can conclude that amorphous or crystalline GEM could be formed in the different preparation and drying methods, and the carrier mannitol varied from b to a and d modifications (Fig. 5).
the decrease was from 71° to 11°. The contact angle of GEM (71°) was relatively high, showing its poor hydrophilicity. The values for mannitol and PVP K-25 were very low, as an indication that they are good wetting agents with high polarity values (26° and 20°). Excipients can help stabilize the decreased size and improve the wettability too. The large surface is hydrophilized by adsorbed stabilizers, as shown by the decreased contact angle. Tables 1 and 2 show a tendency to a correlation between the size decrease, the contact angles and the polarity. Solv.diff.spd resulted in microparticles with the largest surface, which means that this product had the highest surface free energy and polarity values. We observed that for the other two samples these data were improved as compared with those for raw GEM. Moreover, the reduction in size and
3.3. Contact angle and polarity tests The wettability study (Table 2) indicated that the products had a more hydrophilic character as compared with GEM. Significantly lower contact angles with water were measured for all samples:
Table 2 Contact angle and polarity measurement. Materials
Hwater ()
Hdiiodomethane ()
cd (mN m1)
cp (mN m1)
c (mN m1)
Polarity (%)
GEM PVP K-25 Mannitol Solv.diff.lio Solv.diff.spd Melt.emulsif.spd
71.05 ± 2.55 25.95 ± 2.96 20.13 ± 4.98 34.05 ± 4.03 11.8 ± 2.16 22.1 ± 2.31
17.05 ± 1.25 19.3 ± 5.65 24.87 ± 5.13 27.30 ± 1.02 23.4 ± 0.85 20.6 ± 0.07
44.41 43.20 41.64 40.88 42.08 42.92
11.16 32.49 35.06 29.87 37.03 33.52
55.57 75.69 76.70 70.75 79.12 76.45
20.08 42.93 45.71 41.79 46.80 43.84
Fig. 6. In vitro dissolution of GEM at pH 1.2.
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Fig. 7. In vitro dissolution of GEM at pH 6.8.
the excipients applied influenced the surface free energy (cl) and the polarity of the material. 3.4. In vitro dissolution UV analysis of the drug content in the dried products confirmed that GEM could be found at a level of 96.5–102% of the theoretically added amount. The dissolution of raw GEM was very slow, as expected because of its low solubility. At pH 1.2, only Solv.diff.spd showed higher dissolution (Fig. 6), a larger amount of product dissolving in the first 10–20 min in comparison with GEM and the other product. During dissolution, some amorphous GEM presumably returned to the crystalline state and because of this the dissolution was around 70% instead of 100%. The dissolution rate for Melt.emulsif.spd was slightly higher that than for Solv.diff.lio because the lyophilized product was crystalline and had larger particles (38.3 lm) than those of Melt.emulsif.spd (21.68 lm). At pH 6.8, pure GEM dissolved better than in acidic media. Increased dissolution rates and higher dissolution were observed for all samples in comparison with the raw drug. The rate of dissolution increased proportionally to the degree of particle size reduction and the applied drying method. Melt.emulsif.spd and Solv.diff.lio showed the same dissolution after 2 h. For Solv.diff.spd, 100% of the drug was released during the first 10 min (Fig. 7). 4. Conclusions Our study has demonstrated the important role of power ultrasound in drug formulation. Sonocrystallization, involving a particle size reduction and a change in crystal habit, may solve the problem of the in vitro drug release of GEM. With the applied preparation method and drying procedure, we could achieve differences in size distribution, wettability, surface free energy and structure. In our work, power ultrasound served an important role in the first part of the preparation. The solvent diffusion method can help block precipitated crystal growth and in the case of melt emulsification can decrease the droplet size. The engineering of drug particles with power ultrasound is a major research area in view of the limitations of conventional particle formation and pretreatment processes. In this way, the particle size reduction and change in crystal habit attained may solve solubility/permeability problems and also facilitate generic formulation.
Acknowledgments ‘‘TÁMOP-4.2.1/B-09/1/KONV-2010-0005. Creating the Center of Excellence at the University of Szeged’’ is supported by the European Union and co-financed by the European Regional Development Fund. References [1] M. Lindenberg, S. Kopp, J.B. Dressman, Classification of orally administered drugs on world health organization model list of essential medicines according to biopharmaceutics classification systems, Eur. J. Pharm. Biopharm. 58 (2004) 265–278. [2] R. Ambrus, Z. Aigner, L. Catenacci, G. Bettinetti, P. Szabó-Révész, M. Sorrenti, Physico-chemical characterization and dissolution properties of nifluminic acid–cyclodextrin–PVP ternary systems, J. Therm. Anal. Chalorim. (2010) (doi: 10.1007/s10973-010-1069-1). [3] R. Ambrus, Z. Aigner, C. Dehelean, C. Soica, P. Szabó-Révész, Physicochemical studies on solid dispersions of nifluminic acid prepared with PVP, Rev. Chim. 58 (2007) 60–64. [4] R.G. Iacocca, C.L. Burcham, L.R. Hilden, Particle engineering: a strategy for establishing drug substance physical property specifications during small molecule development, J. Pharm. Sci. 99 (2010) 51–75. [5] M. Manish, J. Harshal, P. Anant, Melt sonocrystallization of ibuprofen: effect on crystal properties, Eur. J. Pharm. Sci. 25 (2005) 41–48. [6] A. Kordylla, S. Koch, F. Tumakaka, G. Schembecker, Towards an optimized crystallization with ultrasound: effect of solvent properties and ultrasonic process parameters, J. Crystal Growth 310 (2008) 4177–4184. [7] J.M. Asua, Miniemulsion polymerization, Prog. Polym. Sci. 27 (2002) 1283– 1346. [8] R.H. Muller, C.M. Keck, Challenges and solutions for the delivery of biotech drugs – A review of drug nanocrystal technology and lipid nanoparticles, J. Biotech. 113 (2004) 151–170. [9] R. Ambrus, N.N. Amirzadi, P. Sipos, P. Szabó-Révész, Effect of sonocrystallization on the habit and structure of Gemfibrozil crystals, Chem. Eng. Technol. 33 (2010) 827–832. [10] D.K. Sandilya, A. Kannan, Effect of ultrasound on the solubility limit of sparingly soluble solid, Ultrason. Sonochem. 17 (2010) 427–434. [11] N. Blagden, M. de Matas, P.T. Gavan, P. York, Crystal engineering of active pharmaceutical ingredients to improve solubility and dissolution rates, Adv. Drug Deliv. Rev. 59 (2007) 617–630. [12] R.K. Bund, A.B. Pandit, Sonocrystallization: effect on lactose recovery and crystal habit, Ultrason. Sonochem. 14 (2007) 143–152. [13] M.R. Abu Bakar, Z.K. Nagy, A.N. Saleemi, C.D. Rielly, The impact of direct nucleation control on crystal size distribution in pharmaceutical crystallization processes, Crystal Growth Des. 9 (2009) 1378–1384. [14] S. Budavari, The Merck Index, Gemfibrozil, 11th ed., Merck & Co., Inc., Rahway, 1989. [15] F. Priego-Capote, M.D. Luque de Castro, Ultrasound in analytical chemistry, Anal. Bioanal. Chem. 387 (2007) 249–257. [16] S. Wu, Calculation of interfacial tension in polymer systems, J. Polym. Sci. 34 (1971) 19–30. [17] E. Oh, P.E. Luner, Surface free energy of ethylcellulose films and the influence of plasticizers, Int. J. Pharm. 188 (1999) 203–219.