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Dissolution study of nanocrystal powders of a poorly soluble drug by UV imaging and channel flow methods
European Journal of Pharmaceutical Sciences 50 (2013) 511–519
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Dissolution study of nanocrystal powders of a poorly soluble drug by UV imaging and channel flow methods Annika Sarnes a,⇑, Jesper Østergaard b, Sabrine Smedegaard Jensen b, Jaakko Aaltonen a, Jukka Rantanen b, Jouni Hirvonen a, Leena Peltonen a a b
Division of Pharmaceutical Technology, Faculty of Pharmacy, University of Helsinki, P.O. Box 56, 00014 Helsinki, Finland Department of Pharmacy, Faculty of Health and Medical Sciences, University of Copenhagen, Universitetsparken 2, 2100 Copenhagen, Denmark
a r t i c l e
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Article history: Received 11 April 2013 Received in revised form 20 August 2013 Accepted 20 August 2013 Available online 30 August 2013 Keywords: Channel flow method Dissolution rate Nanocrystals Poloxamer Solubility UV imaging
a b s t r a c t Application of drug nanocrystals provides advantageous options for the pharmaceutical formulation development of poorly soluble drugs. The objective of this study was to investigate the dissolution behavior improving effects of differently sized nanocrystals of a poorly soluble model drug, indomethacin. Nanocrystal suspensions were prepared using a top-down wet milling technique with three stabilizers: poloxamer F68, poloxamer F127 and polysorbate 80. The dissolution of the differently sized indomethacin nanocrystals were investigated using a channel flow dissolution method and by UV imaging. Unmilled bulk indomethacin and physical mixtures were used as references. According to both the dissolution methods, the dissolution properties of indomethacin were improved by the particle size reduction. UV imaging was used for the first time as a dissolution testing method for fast dissolving nanoscale material. The technique provided new information about the concentration of the dissolved drug next to the sample surface; with the smallest nanocrystals (580 nm) the indomethacin concentration next to the particle surface exceeded five-fold the thermodynamic saturated indomethacin solution concentration. Thus the solubility improvement itself, not only the increased surface area for dissolution, may have an important role in the higher dissolution rates of nanocrystal formulations. Poloxamer F68 was the most optimal stabilizer in the preparation of the indomethacin nanocrystal suspensions and in the solubility and dissolution enhancement as well. Ó 2013 Elsevier B.V. All rights reserved.
1. Introduction In vitro drug release and dissolution testing play an important role in pharmaceutical formulation development and quality control. Poorly soluble candidate molecules constitute a major challenge for the formulation development since the low solubility and low dissolution rate often lead to poor bioavailability (Keck and Müller, 2006; Kipp, 2004). It is necessary to develop new analytical methods in order to understand better the complex dissolution processes and overcome the difficulties related to dissolution (Amidon and Hawley, 2010). New in vitro methods that could exclude ‘impossible’ molecules as early as possible are thus vital to secure streamlined formulation development. The long-term goal of dissolution research is to be able to predict the in vivo behavior of the formulation based on in vitro dissolution studies. This may be achieved by an adequately discriminative and robust in vitro method enabling the establishment of an in vitro–in vivo correlation (IVIVC). ⇑ Corresponding author. Tel.: +358 9 191 59159; fax: +358 9 191 59144. E-mail address: annika.sarnes@helsinki.fi (A. Sarnes). 0928-0987/$ - see front matter Ó 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ejps.2013.08.030
Nanotechnology, especially nanocrystals, is currently offering promising possibilities to address the solubility problems of poorly soluble candidates (Van Eerdenbrugh et al., 2008b). Nanocrystallization techniques are used to decrease the particle size and thus increase the dissolution rate and to improve the bioavailability (Peltonen and Hirvonen, 2010). A drug nanocrystal is a nano-sized crystalline particle containing 100% drug without any matrix material (Keck and Müller, 2006). In the field of pharmacy the definition of nanocrystals covers particles below one micron (Merisko-Liversidge et al., 2003). In the production process of nanocrystals by a top-down wet milling technique, a nanocrystal suspension is formed. It is a colloidal dispersion of drug nanoparticles, where the particles have been stabilized by coating them with surface active agents, polymers or a mixture of polymers (Merisko-Liversidge et al., 2003; Merisko-Liversidge and Liversidge, 2011). Nanocrystals possess an increased surface area/volume-ratio, which facilitates a very fast dissolution rate (Kocbek et al., 2006). With nanocrystal formulations a high drug load, low incidence of side effects due to the excipients, versatile administration routes, improvement of bioavailability, and an overall improvement of efficiency and safety can be achieved.
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When considering the existing traditional, material and time consuming, pharmacopeial dissolution methods (USP, European and Japanese Pharmacopoeia) that were mainly developed for quality control purposes, they are time to time regarded inadequate and not evolved enough to correspond the needs of modern dissolution study (Dokoumetzidis and Macheras, 2006; McAllister, 2010). The traditional methods, e.g. paddle method combined with UV spectroscopy or HPLC, do not provide real-time, spatially and temporally resolved information of the dissolution process, since the monitoring of the process immediately next to the surface of the dosage form is impossible with those techniques (Greco and Bogner, 2011; Windbergs et al., 2009). However a number of recent methods to study the dissolution of solid dosage forms are based on imaging, e.g., UV, FT-IR, NIR, and MRI imaging, CARS and Raman spectroscopy in order to cover those missing aspects (Aaltonen et al., 2006; Kazarian and van der Weerd, 2008; Kowalczuk and Tritt-Goc, 2011; Metz and Mader, 2008; Nott, 2010; Ostergaard et al., 2010; van der Weerd and Kazarian, 2005). In this study, the dissolution and solubility properties of a poorly soluble model drug, indomethacin (IND), were improved by preparing nanocrystal suspensions using a top-down, wet milling technique (Liu et al., 2011; Rabinow, 2004; Van Eerdenbrugh et al., 2008a). The dissolution and solubility behavior of the different particle size fractions of the dried IND nanocrystal suspensions were investigated using a channel flow dissolution method and UV imaging (Ostergaard et al., 2010; Peltonen et al., 2003; Peltonen and Hirvonen, 2010). The aim of the study was to investigate both the real-time, spatially and temporally resolved, dissolution behavior of the nanocrystals with different size fractions for the first time using UV imaging, as well as the critical particle size limit, which still benefits the dissolution process. Additionally the creation of possible supersaturated states, as compared to the thermodynamic IND solubility value, when the particle size was decreased from the bulk material (tens of lm) to 500 nm, was examined. In order to eliminate the effect of increased surface area on the dissolution testing, all the tests were performed from a flat surface of compressed nanocrystalline powders. 2. Materials and methods 2.1. Materials In this study, indomethacin (IND, Hawkins Pharmaceutical Group, Minneapolis, USA) was used as the model drug. Poloxamer 188 and 407 (LutrolÒ F68 and F127, BASF Co., Ludwigshafen, Germany) and polysorbate 80 (TweenÒ 80, Sigma–Aldrich Chemie GmbH, Steinheim, Germany) were used as stabilizers. Lactose (particle size 30–150 lm, PharmatoseÒ 200 M, DMV International, Veghel, The Netherlands) was used to prevent particle aggregation during freeze-drying and microcrystalline cellulose (MCC, Avicel PH-102, FMC International, Cork, Irland) as an excipient in sample compaction (see Section 2.7.1). Ethanol (96%, v/v, Altia Oyj., Rajamäki, Finland) and acetate buffer (pH 5.0, NaC2H3O23 H2O and CH3COOH, Riedel-de Haën Laborchemikalien GmbH & Co. KG, Seelze, Germany or pH 5.0, CH3COONa3 H2O, and CH3COOH, Merck KGaA, Darmstadt, Germany) were used for the dissolution studies. Agarose (type I, Sigma–Aldrich Chemie GmbH, Steinheim, Germany) was used as gel former. The water used was ultrapurified Milli-QÒ-water (Millipore SAS, Molsheim, France or MilliporeÒ, Bedford, MA, USA).
or surfactant, 1.2 g) was dissolved in 5 ml of water and, thereafter, 2 g of IND was dispersed in the aqueous stabilizer solution. Three types of stabilizers were used: polysorbate 80 and poloxamers F68 and F127. The stabilizer:drug ratio was kept constant (0.6:1) in all the tests. The suspension was inserted into the milling bowl containing varying amounts of milling pearls (both bowl and pearls were made of zirconium oxide), depending of the size of the pearls used (Ø 1, 5 or 10 mm, being equivalent to 70 g, 180 pcs. or 18 pcs. of pearls, respectively). By varying the used milling pearl size, different particle sizes were effectively produced. Additional 5 ml of water was used to collect the residual suspension from the beaker into the milling bowl. The milling bowl was placed in a planetary ball mill (Pulverisette 7 Premium, Fritsch Co., Idar-Oberstein, Germany), and the milling experiment was performed in ten 3-min milling cycles. After each cycle there was a 15 min pause for the system to cool down to 32 °C. The grinding speed was adjusted according to the milling pearl size (Ø 1 mm, 5 mm or 10 mm corresponded to 1100 rpm, 1000 rpm or 850 rpm, respectively). After milling, the nanocrystal suspension was separated from the pearls by pipetting. All the nanocrystal suspensions were freeze dried (72 h, LyoPro 3000, Heto-Holten A/S, Allerød, Denmark). Prior to freeze-drying, lactose, dissolved in water (0.2 g, 10 w/w% in relation to the drug amount), was added to the nanocrystal suspension to prevent particle aggregation during the drying process. The dried samples were stored in closed vials protected from light at room temperature (19–22 °C). 2.3. Particle size The mean particle sizes and polydispersity indexes (PI) of the nanocrystal suspensions were analyzed by photon correlation spectroscopy (PCS) using a Malvern Zetasizer 3000HS (Malvern Instruments, Malvern, UK). Measurements were performed from the fresh nanocrystal suspensions, as well as from the dried and redispersed nanocrystal suspensions. All the samples were measured in triplicate. 2.4. Solid state properties The solid state of IND was assessed with differential scanning calorimetry (DSC 823e, Mettler Toledo Inc., Columbia, USA) after milling and freeze-drying. Lactose was excluded from the DSC analyzed samples. The dried nanocrystal suspension samples were placed (2–5 mg) in an aluminum pan closed with a perforated lid (Liu et al., 2011). Heating rate of 10 °C/min was used between 25 °C and 200 °C. Nitrogen purge of 50 ml/min was used during the measurements. The data was analyzed with STARe software (Mettler Toledo, Columbia, USA). Bulk materials (IND, F68, F127) were as well measured. 2.5. Morphology of nanocrystals The morphological evaluation of the dried nanocrystal suspensions was performed using scanning electron microscopy (SEM, JSM-7500F, JEOL Ltd., Japan). The samples were prepared for the analysis by assembling a small amount of nanocrystal suspension onto a carbon-coated tape and allowed to dry at room temperature, after which the samples were sputter-coated with platin (Q150T Quomm, Turbo-Pumped Sputter Coater, China). The coated samples were imaged.
2.2. Preparation of dried nanocrystal samples
2.6. Effect of stabilizer on drug solubility
The nanocrystal suspensions were prepared using a top-down, wet-milling technique (Liu et al., 2011). The stabilizer (polymer
The solubility of the drug in aqueous stabilizer solutions was determined with the traditional shake-flask method (Wang et al.,
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2007). Acetate buffer (pH 5.0) was used as medium. Solubility determinations were made from the physical mixtures of bulk IND and stabilizers. The ratio of stabilizer and drug was kept constant (0.6:1), the total amount of polymer being 0.5 mg/ml.
Table 1 The effect of stabilizer (stabilizer:drug ratio 0.6:1, the total concentration of stabilizer being 0.5 mg/ml) on the apparent solubility of bulk IND in acetate buffer (pH 5.0, after 12 h of shaking, n = 3). Composition
2.7. Dissolution studies 2.7.1. Channel flow method Dissolution properties of the compacted nanocrystal surfaces were studied by the channel flow dissolution method (Peltonen et al., 2003), in which one face of the compacted sample was exposed to the dissolution medium. The dried nanocrystal suspensions were manually compacted (150 mg) with a single-punch tablet press (Korsch, EK-0, Korsch, Germany). First, MCC powder (50 mg) was inserted to the bottom of the mold as supportive material in order to facilitate the compaction. Then nanocrystalline drug (100 mg) was put on the top of MCC and the compression was performed. The dissolution was studied from the nanocrystalline tablet surface, which was adjusted to be thick enough to remain during the experiment. Flat-faced punches with a diameter of 9 mm were used in order to achieve a flat and constant surface area for dissolution. The tablet press was instrumented and the compression force of the upper press was monitored (SingleStation DAAS Measure, software version 1.2) and kept as even as possible (1000 N). Acetate buffer (pH 5.0, V = 500 ml, room temperature) was used as the dissolution medium. The medium circulated by a peristaltic pump (Watson-Marlow, Cornwall, UK) through the channel flow cell, medium reservoir and UV–Vis spectrophotometer with a flow-through cuvette (UV-1600PC, VWR International, Leuven, Belgium). The parts of the system were interconnected in a closed-loop fashion using silicone tubings. The optimal pumping speed was found to be 50 rpm (corresponding to a flow rate of 8.1 ml/min). UV–Vis data was collected and analyzed with M. Wave Professional software (v 1.0, VWR International, Leuven, Belgium). The analytical wavelength was 318 nm (European Pharmacopoeia, 2011). All the measurements were performed at room temperature (19–22 °C). Dissolution rate results were calculated as a released drug amount in time unit per constant area (Peltonen et al., 2003). 2.7.2. UV imaging Dissolution studies of dried IND nanocrystal suspensions were also performed using an Actipix SDI300 dissolution imaging system (Paraytec Ltd., York, UK) with an Actipix flow-through type dissolution cartridge (CADISS-3) (Boetker et al., 2011; Ostergaard et al., 2010; Ye et al., 2011; Østergaard et al., 2011). The total detection area of the imager is 9 mm 7 mm (1280 1024 pixels); however, the selected imaging area was 3.64 mm 8.12 mm. The pixels (7 lm 7 lm) were binned 4 4. A pulsed Xe lamp was used as the light source. The quartz flow cell had a light path of 4.0 mm. Images were recorded (2.6 images per s) and analyzed with Actipix D100 software version 1.3 (Paraytec Ltd., York, UK). All experiments were carried out at ambient temperature (19– 22 °C). Based on initial experiments an acetate buffer (pH 5.0) and 265 nm were selected as dissolution medium and detection wavelength, respectively. Compacts for the UV imaging were prepared by weighing 6 mg of the sample into a stainless steel cylinder (inner diameter: 2 mm) held in a manual press (Actipress, Paraytec Ltd., York, UK) (Boetker et al., 2011). A torque screwdriver (Quickset MINOR, Torqueleader, M.H.H. Engineering Co., Ltd., England) was used to compact the nanocrystal powders at constant torque of 110 cN m. The general procedure for UV imaging has been presented previously (Boetker et al., 2011; Ostergaard et al., 2010; Ye et al., 2011; Østergaard et al., 2011). The dissolution of the compacted nanocrystals was studied both with a solution phase (acetate buffer,
IND/F68 IND/F127 IND/polysorbate 80 IND
Solubility
lg/ml
mM
6.43 ± 0.06 4.80 ± 0.00 10.9 ± 1.54 4.89 ± 0.18
0.018 ± 0.00 0.013 ± 0.00 0.030 ± 0.004 0.014 ± 0.001
pH 5.0; absence and presence of flow: 0.5 ml/min to 0.1 ml/min) and a gel matrix (agarose/acetate buffer, pH 5.0) as the dissolution medium. The gel matrix was prepared as follows: 10 mg agarose was mixed with acetate buffer (V = 1 ml, pH 5.0) in an Eppendorf-tube and heated in a water bath (90 °C) for 0.5 h, after which the hot polymer solution was pipetted into the quartz cell. The gel matrix was allowed to cool and settle for 2 h prior to dissolution testing. After measurements, the pixel intensities were converted into absorbance values using the Actipix software (Ostergaard et al., 2010), allowing the determination of the apparent IND concentration within the imaging area as a function of position and time. The absorbance maps were collected with the Actipix software for each sample using the same detection area (V = 118.23 ll) in order to get comparable results. The dissolution rates were determined during the first 10 min, from the linear region of the dissolution profile, during which the sample remained in the detection area. 3. Results and discussion 3.1. Effect of the stabilizer on the solubility of IND Surface active agents may have solubility-improving effects, and for that reason the apparent solubility of bulk IND was measured in the presence of the stabilizers (Table 1). F127 did not alter the solubility, but F68 improved the solubility of bulk IND by 34%. Polysorbate 80 improved the solubility even more, by 127%, because of the micelle formation (Hagan et al., 1996). There were no micelles present in the poloxamer systems (F68 and F127), since the stabilizer concentrations were below the critical micelle forming concentrations (CMC) (Hagan et al., 1996; Maskarinec et al., 2002). The solubilizing effect of the poloxamers depends on the block structure, chain lengths and interactions of the hydrophobic and hydrophilic parts, which finally cause a decrease in the interfacial tension between the drug and dissolution medium and, hence, improve the wetting (BASF, 2004; Lan et al., 2013; Prasanthi et al., 2011). 3.2. Particle sizes of the nanocrystals IND nanocrystal suspensions were successfully prepared in varying particle sizes by changing the size of the used milling pearls (values presented in Table 2). Along the increasing particle sizes the PIs were also detected to increase, implying about more heterogeneous material. A low PI value (<0.2) indicates monodisperse particles (Liu et al., 2011). When the PI value lies around 0.5–0.7 and higher, the particle size distribution is considered broad (Muller and Jacobs, 2002; Yadav et al., 2012). Polydisperse particles have PI-values larger than 0.7. Aggregation of the nanocrystals during the freeze-drying process was prevented by optimizing the amount of lactose. Thus the particle size was kept constant. The stabilizing agents, poloxamers F68 and F127, were successfully applied for the preparation
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Table 2 The average dissolution rate of IND from the nanocrystal compacts in acetate buffer (pH 5.0) as determined using the channel flow dissolution method (n = 2). The corresponding particle size and polydispersity index of the nanocrystals are also shown (n = 3). Sample
Dissolution rate (lg/min/mm2)
Particle size (nm) (PI)
F68 F68 F68 F127 F127 F127 Polysorbate 80 Physical mixtureb
0.50 0.35 0.14 0.31 0.16 0.08 0.05 0.05
580 ± 30 (0.4 ± 0.1) 950 ± 190 (0.8 ± 0.1) ND (micronsized)a 580 ± 20 (0.6 ± 0.06) 740 ± 30 (0.7 ± 0.2) ND (micronsized)a NDa 80 lm
a
ND = not determined, above the detection limit of PCS (<10 lm). Physical mixture of IND and lactose, particle size of IND announced by the manufacturer. b
of nanocrystal suspensions (Table 2). Both the poloxamers are linear triblock polymers including hydrophilic and hydrophobic segments (Liu et al., 2011). Together the hydrophilic and hydrophobic regions provide effective steric hindrance against aggregation. Polysorbate 80 was a problematic material in the milling process. The decrease of the surface tension resulted in a leakage of the suspension out of the sealed bowl during the milling. Regardless of several attempts, only one particle size with polysorbate 80 as the stabilizer could be prepared, using the smallest milling pearls (Ø 1 mm). The resulting particle sizes were not as small as with F68 and F127 and the prepared material was not homogenous. Polysorbate 80 is a hydrophilic, low molecular weight polymer that forms a thin adsorption layer, thus offering weak steric stabilization when approaching the particles (Sepassi et al., 2007). In addition, the freeze dried nanocrystal suspensions having polysorbate 80 as the stabilizer were extremely sticky and difficult to handle. 3.3. Solid state properties
Fig. 1. The DSC thermograms of IND nanocrystal suspension (NPs) stabilized with F68 and F127 compared to the bulk materials (IND, F68, F127).
The solid state analysis using DSC was performed for all the samples: nanocrystal suspensions stabilized with F68, F127 and bulk materials (IND, F68, F127, polysorbate 80) (Fig. 1). The consistency of the polysorbate 80 was too paste-like to be able to perform the thermal analysis. The obtained DSC thermograms of the nanocrystal suspensions showed endothermic melting peaks at approx. 160 °C corresponding to the melting point of pure, unmilled bulk IND. Milling and freeze-drying did not induce formation of amorphous IND, supported by the absence of glass transition or recrystallization events (Chamarthy and Pinal, 2008). Additionally, the rapid wet milling technique, using the same protocol, is demonstrated to provide crystalline material, which was confirmed by both DSC and X-ray analyses (Liu et al., 2011). The lower peak intensities of the nanosamples derive from the presence of the stabilizers surrounding the particles after milling (Hecq et al., 2005). Poloxamers were separated from the drug based on their melting
Fig. 2. SEM images of the freeze dried indomethacin nanocrystal suspensions. (A) and (B) Present F68 samples (scale bar: 10 lm and 5 lm). (C) and (D) Are produced using F127 (scale bar: 10 lm) and Tween 80 (scale bar: 10 lm), respectively. All the figures are taken from the smallest particle size samples.
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points. The melting points of poloxamers F68 (50 °C) and F127 (53–57 °C) and polysorbate 80 (21 °C) are clearly lower than the melting point of IND (162 °C), which made the interpretation of the thermograms straightforward (BASF, 2004). 3.4. Morphology of nanocrystals The morphology of the nanocrystals was studied using SEM (Fig. 2). All the freeze dried samples showed a porous structure. The individual particles were highlighted by changing the magnification. The grey mass between the particles mainly consists of the stabilizing agent (F68/F127/polysorbate 80), because lactose was used at a relatively low concentration (10 w/w%). The porous structure is formed when water is evaporated during the drying process. The poloxamer samples showed a uniform structure where the single particles could be detected, whereas when looking at the surface of polysorbate 80 samples, a mixed surface formed by the drug crystals and the medium was observed. 3.5. Channel flow method After compacting the dried IND nanocrystals, dissolution experiments were carried out using the channel flow method (Fig. 3). Particle sizes of the samples were analyzed prior to the experiments using PCS. Factors that were optimized before the channel-flow dissolution experiments were the selection of the dissolution medium and the optimal flow rate. An acidic medium was chosen, because IND (pKa 4.5) has low solubility in this pH which is to enhance the differences in dissolution behavior. With
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regards to the pumping speed, if it was too high (e.g. 90 rpm), detection of the signal was difficult because the sample did not have time to dissolve at such a high flow rate. On the other hand, a too slow flow rate resulted in saturated solutions near the surface of the sample, and thus limited the possibility of higher dissolution rates skewing the results. The optimal speed was obtained by both visual and absorbance monitorings. Results of the dissolution tests showed that an increase in the particle size caused a decrease in the dissolution rate. In the measurements done with the channel flow dissolution method, the effect of particle size was ruled out by preparing compacts with a constant surface area. Thus, according to the Noyes–Whitney equation, the dissolution rate was affected only by the concentration gradient. The different particle sizes and stabilizing agents can be distinguished clearly in the experiments with the channel flow dissolution method (Table 2). Differences between the dissolution rates of the samples were at highest ten-fold. The largest differences were found out between the nanocrystal samples and physical mixture of indomethacin and lactose. The samples were set in order as predicted according to their particle size and the used stabilizer. For example, the dissolution rates of the samples containing F127 were doubled, when the particle size was decreased from 740 nm to 580 nm. Interestingly, the IND dissolution rates from the F68 samples were faster than from the F127 in every particle size fraction, although the actual particle sizes were slightly higher in the F68 samples. This may be related to the higher drug solubility observed in the presence of F68 as compared to F127. Overall the dissolution studies with the channel flow method suggested that the poloxamer F68 was the most optimal choice for both the preparation of the nanocrystals, and to enhance indomethacin solubility and dissolution. However, as demonstrated, the comparison of the dissolution behavior is here performed within each stabilizer group, since the stabilizer has some influence on the dissolution. Additionally, it must be taken into account that the concentration of the stabilizer does not remain constant throughout the experiment. That is, there may exist local concentration differences of the stabilizer during dissolution, especially at the diffusion front, which may lead to higher solubilities of the drug. In conclusion, the results from the bulk IND solubility tests cannot be directly applied to these circumstances since the measurements were performed only at a constant concentration level. 3.6. UV imaging
Fig. 3. Channel flow method (n = 2, all the measurements are shown (gray and black) in the figure): dissolution of indomethacin from the nanocrystal compacts. Micronsized bulk IND samples were used as corresponding references.
In the dissolution studies done with UV imaging, the objective was to identify local concentration changes near the compact surfaces, and reveal possible supersaturation states of indomethacin.
Fig. 4. Dissolution of F68/Ø 580 ± 30 nm compact, using UV imaging with a flow-through system (contour interval 200 mAU, contour offset 20 mAU) and acetate buffer as dissolution medium (pH 5.0). (A) The dissolution medium flow rate: 4 min 1 ml/min, at wavelength 265 nm. (B) Control study with the same sample using flow rate: 4 min 1 ml/min and 5 min 0.1 ml/min, at wavelength 550 nm. Image (B) is taken 2 min after the flow is stopped, in the absence of flow.
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Fig. 5. UV imaging using the agarose gel matrix prepared in acetate buffer (pH 5.0) (contour interval 200 mAU, contour offset 20 mAU). Bulk-IND after 14 h of dissolution at the wavelength (A) 265 nm and (B) 550 nm. F68/micronsized particles sample after 0.5 h of dissolution at the wavelength (C) 265 nm and (D) 550 nm.
The initial UV imaging setup investigated was a flow-through system. During preliminary testing, however, it was noticed that the samples induced an exceptionally strong signal immediately upon initiation of the flow-through dissolution experiment which was significantly different from the dissolution maps expected based on previous flow-through imaging studies (Fig. 4a) (Boetker et al., 2011; Gordon et al., 2012). The phenomenon was examined by changing the wavelength from 265 nm to 550 nm, where the dissolved IND does not absorb light. Again strong absorbance was observed which indicated that the solid nanocrystals dissociated from the compact surface and carried downstream by the flow caused absorbance at 550 nm (Fig. 4b). Light scattering from particles has been confirmed also previously (Van Eerdenbrugh et al., 2011). The effluent was however clear, presumably due to the rapid dissolution of the nanocrystals. An attempt to prevent the erosion of the sample by increasing the compaction force was
unsuccessful. The undissolved nanocrystals left the surface even in the experiments performed in the absence of flow. Thus, UV imaging provided valuable information on the dissolution mechanism of compacted IND nanocrystals. The method revealed that nanoparticulate material dissociated from the compact surface. In order to focus on molecular IND dissolution, an agarose-based hydrogel matrix (1%) was used as a dissolution matrix instead of the buffer solution. The adjustments, high torque (110 cN m) in compact preparation and gel matrix, prevented the nanocrystals from escaping the compact surface. Also bulk-IND and a physical mixture of IND and lactose were investigated. Nanocrystal samples containing polysorbate 80 were impossible to study with UV imaging since proper compacts were not possible to prepare. The dissolution behavior of IND nanocrystal samples and bulk IND into agarose matrix is compared in the absorbance maps shown in Fig. 5.
Table 3 Released IND (after 10 min) and dissolution rates of the nanocrystals studied with UV imaging (n = 3). In addition, the concentration (lg/ml) as a function of the distance (0, 2 and 3 mm) from the compact surface after 10 min is given. Sample (size/nm)
Released IND ± STD (lg) (RSD%)
Dissolution rate ± STD (lg/min/mm2) (RSD%)
Concentration (lg/ml) (distance/mm)
F68(580 ± 30)
0.59 ± 0.16 (27.07)
0.019 ± 0.006 (28.87)
28.69 (0) 11.71 (2) 5.84 (3)
F68(micronsized)
0.52 ± 0.13 (24.43)
0.016 ± 0.00 (0.00)
11.35 (0) 4.04 (2) 02.57 (3)
F127(580 ± 20)
0.65 ± 0.16 (25.51)
0.019 ± 0.00 (0.00)
22.08 (0) 9.37 (2) 4.31 (3)
F127(micronsized)
0.63 ± 0.10 (15.91)
0.019 ± 0.003 (16.67)
17.08 (0) 7.61 (2) 3.97 (3)
Bulk IND (80 lm)
0.13 ± 0.05 (39.73)
0.004 ± 0.002 (43.30)
2.07 (0) 0.26 (2) 0.00 (3)
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Fig. 6. Apparent concentration-distance profiles obtained from UV imaging of IND nanoparticles at time points 5, 15 and 30 min: (A) F68/580 ± 30 nm, (B) F68/micronsized particles, (C) F127/580 ± 20 nm, (D) F127/micronsized particles and (E) bulk IND/tens of lm.
One of the aims of this study was to determine, if supersaturated states of IND can be achieved next to the compact surface, and if the achieved concentrations show significant difference between the different particle sizes (Table 3 and Fig. 6). Difference in the dissolution rates between the nanocrystals and the unmilled bulk IND was noticed by UV imaging, similarly to the dissolution results obtained by the channel flow method. However, dissolution rates among the nanoparticles in the gel matrix were similar irrespective of the particle sizes; the dissolution-enhancing effect observed in the solubility tests might have diminished the differences in the dissolution rate. Compared to the channel flow method, the UV imaging could not separate the nanocrystal samples that well. The gel matrix has likely impacted on the dissolution. The use of agarose gel provided a highly symmetric absorbance maps indicating that natural convective effects due to the density gradients had been efficiently supressed (Østergaard et al., 2011). All the measurements were carried out using agarose gel (acetate
buffer, pH = 5) as dissolution medium and the analytical wavelength of 265 nm. The absorbances were converted into the amount of drug released, and the dissolution rate determined (Ye et al., 2011). The determined dissolution rates are depicted in Table 3. The released drug amount is determined after 10 min imaging which is an adequate time to study the dissolution of the fast dissolving nanocrystals (Liu et al., 2011). Apparent IND concentrations determined by UV imaging (Table 3) were compared to the earlier concentrations of saturated IND solutions (Table 1). Each stabilizer group was compared separately because of the different solubilizing effects of the stabilizers. It must be noticed, like in the case of channel flow method, that there exist differences in drug concentration during the entire dissolution process; high concentrations of surfactant may exist at specific locations in the hydrogel leading to very high local solubilities of the drug. Thus no direct conclusions about the impact of the stabilizer can be withdrawn based only on the bulk drug solubility
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tests performed at constant concentration level. Fig. 6 shows that with the studied nanocrystals at least a saturated, and in majority of the cases a supersaturated system was achieved, e.g. the thermodynamic solubility was exceeded. The thermodynamic solubility was exceeded almost five-fold for the F127/580 ± 20 nm sample. By preparing nanocrystals from a poorly soluble drug, the dissolution of the material is strongly improved. Difference in the IND concentration as a function of distance was achieved between all the particle size fractions. The released drug amount was greater with the small particles (580 ± 30 nm, 580 ± 20 nm) than with the micronsized particles – at each measurement timepoint (5, 15 and 30 min). With micronsized particles it took 30 min to reach a concentration that was achieved with the smaller particles in 15 min (Fig. 6). Bulk IND was used as a reference, and it was altogether measured for 15 h. In this time, the released drug content was still low even compared to the micronsized crystals. Overall, UV imaging provides insight into the behavior of the nanocrystals during the dissolution process. With small particle size, much higher concentration as a function of distance from the surface is achieved in comparison with the large particle sizes in the gel matrixes. The thermodynamic solubility was exceeded even five-fold, which is a remarkable result when considering the fact that in this experimental set-up the effect of the dissolution area was excluded. The effect of the particle size is even more pronounced when the nanocrystals are allowed to perform in particulate form. Compared to UV imaging, the channel flow dissolution method gives substantially higher dissolution rates (lg/min/ mm2) and differentiates the samples clearer at the conditions investigated. UV imaging provided valuable information about the concentrations profiles. The sample escaping from the compact surface in the channel flow method was not a problem. The compression force used in the sample preparation was 1000 N in the channel flow method, while in the UV imaging the used torque was 110 cN m. In addition, the fast dissolving nature of the nanocrystals ensured that all the material in the set-up was fully dissolved at the time reaching the spectrophotometer. The use of gel matrix and stationary mode in the UV imaging, versus the flowing liquid medium in the channel flow method, at least partially explains the differences between the two dissolution methods. Interestingly, the results indicate the possibility that the difference in IND nanocrystal particle sizes between 500 and 1000 nanometers is not necessarily critical, because both of the studied size fractions were several times smaller particle size than the unmilled indomethacin (80 lm). In principle, it is possible that particles around 1000 nanometers are sufficiently small for substantial improvement of the dissolution properties of the poorly soluble indomethacin, but problems with further formulation of a potential drug product from these nanocrystals related to the small size could be partly avoided. As a whole, UV imaging offered a flexible experimental setup, allowing non-intrusive real-time studies of the dissolution process. This technique enables, similar to other imaging methods (e.g. FTIR and NMR), dissolution studies with benefits, such as excluding radioactive or fluorescent labeling (Coutts-Lendon et al., 2003; Dahlberg et al., 2007; Kazarian and Chan, 2006; Kazarian and van der Weerd, 2008; Kowalczuk and Tritt-Goc, 2011; van der Weerd and Kazarian, 2004, 2005; van der Weerd et al., 2004). The first experiments with UV imaging made here indicate that it may be an interesting approach to study dissolution behavior of nanoscale materials.
4. Conclusions Dissolution properties of a poorly soluble drug, indomethacin, were improved by preparing different sized nanocrystals using ra-
pid wet milling technique with three different stabilizers, poloxamers F68 and F127 and polysorbate 80. The dissolution properties of the freeze dried and compacted nanocrystals were studied using the channel flow dissolution method and by novel UV imaging. UV imaging was used in this study for the first time in the dissolution testing of fast-dissolving nanoscale samples. Both methods ranked the samples in decreasing dissolution rate order according to the increasing particle size, and the results supported well each other. Each stabilizer group was handed individually due to the possible impact of the stabilizer to the drug solubility. The use of stationary gel matrix versus flowing liquid as dissolution medium explains the differences between the methods. Even though the effect of the variation in the area available for dissolution was eliminated by studying even, constant surfaces instead of particulate samples, the differences between particle sizes, which will be increased even further in actual drug formulation, were evident. UV imaging provided valuable new information about the concentration of the dissolved drug next to the sample surface: with the smallest nanocrystals the concentration next to the particle surface exceeded fivefold the thermodynamic solubility. This indicates that the solubility improvement itself, and not only the increased dissolution area, has a crucial role in higher dissolution rates of nanoparticle formulations. UV imaging is a promising technique to analyze the dissolution properties of nanoscale formulations in detail. Acknowledgment Financial support from NordForsk (Project No. 43703, Future performance testing of pharmaceuticals) is acknowledged. References Aaltonen, J., Heinänen, P., Peltonen, L., Kortejärvi, H., Tanninen, V.P., Christiansen, L., Hirvonen, J., Yliruusi, J., Rantanen, J., 2006. In situ measurement of solventmediated phase transformations during dissolution testing. J. Pharm. Sci. 95, 2730–2737. Amidon, G.E., Hawley, M., 2010. Oral bioperformance and 21st century dissolution. Mol. Pharm.. BASF, 2004. PluronicÒ F68 ja F127, blockpolymer, surfactant.
A. Sarnes et al. / European Journal of Pharmaceutical Sciences 50 (2013) 511–519 Keck, C.M., Müller, R.H., 2006. Drug nanocrystals of poorly soluble drugs produced by high pressure homogenisation. Eur. J. Pharm. Biopharm. 62, 3–16. Kipp, J.E., 2004. The role of solid nanoparticle technology in the parenteral delivery of poorly water-soluble drugs. Int. J. Pharm. 284, 109–122. Kocbek, P., Baumgartner, S., Kristl, J., 2006. Preparation and evaluation of nanosuspensions for enhancing the dissolution of poorly soluble drugs Int. J. Pharm. 312, 179–186. Kowalczuk, J., Tritt-Goc, J., 2011. A possible application of magnetic resonance imaging for pharmaceutical research. Eur. J. Pharm. Sci. 42, 354–364. Lan, Y., Shaukat, A., Langley, N., 2013. Poloxamers as Solubilizing Agents in Solid Dispersions. Liu, P., Rong, X., Laru, J., van Veen, B., Kiesvaara, J., Hirvonen, J., Laaksonen, T., Peltonen, L., 2011. Nanosuspensions of poorly soluble drugs: Preparation and development by wet milling. Int. J. Pharm. 411, 215–222. Maskarinec, S.A., Hannig, J., Lee, R.C., Lee, K.Y.C., 2002. Direct observation of poloxamer 188 insertion into lipid monolayers. Biophys. J. 82, 1453–1459. McAllister, M., 2010. Dynamic dissolution: a step closer to predictive dissolution testing? Mol. Pharm.. Merisko-Liversidge, E., Liversidge, G.G., 2011. Nanosizing for oral and parenteral drug delivery: a perspective on formulating poorly-water soluble compounds using wet media milling technology. Adv. Drug Deliv. Rev. 63, 427–440. Merisko-Liversidge, E., Liversidge, G.G., Cooper, E.R., 2003. Nanosizing: a formulation approach for poorly-water-soluble compounds. Eur. J. Pharm. Sci. 18, 113–120. Metz, H., Mader, K., 2008. Benchtop-NMR and MRI – a new analytical tool in drug delivery research. Int. J. Pharm. 364, 170–175. Muller, R.H., Jacobs, C., 2002. Buparvaquone mucoadhesive nanosuspension: preparation, optimisation and long-term stability Int. J. Pharm. 237, 151–161. Nott, K.P., 2010. Magnetic resonance imaging of tablet dissolution. Eur. J. Pharm. Biopharm. 74, 78–83. Ostergaard, J., Meng-Lund, E., Larsen, S.W., Larsen, C., Petersson, K., Lenke, J., Jensen, H., 2010. Real-time UV imaging of nicotine release from transdermal patch. Pharm. Res.. Østergaard, J., Ye, F., Rantanen, J., Yaghmur, A., Larsen, S.W., Larsen, C., Jensen, H., 2011. Monitoring lidocaine single-crystal dissolution by ultraviolet imaging. J. Pharm. Sci., 100. Peltonen, L., Hirvonen, J., 2010. Pharmaceutical nanocrystals by nanomilling: critical process parameters, particle fracturing and stabilization methods. J. Pharm. Pharmacol. 62, 1569–1579. Peltonen, L., Liljeroth, P., Heikkilä, T., Kontturi, K., Hirvonen, J., 2003. Dissolution testing of acetylsalicylic acid by a channel flow method—correlation to USP basket and intrinsic dissolution methods. Eur. J. Pharm. Sci. 19, 395–401.
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Prasanthi, N.L., Manikiran, S.S., Sowmya, S., Anusha, B., Rama Rao, N., 2011. Effect of poloxamer 188 on in vitro dissolution properties of solid dispersions. Int. J. Pharm. Sci. Rev. Res. 2, 27–32. Rabinow, B.E., 2004. Nanosuspensions in drug delivery. Nat. Rev. Drug Discov. 3, 785–796. Sepassi, S., Goodwin, D.J., Drake, A.F., Holland, S., Leonard, G., Martini, L., Lawrence, M.J., 2007. Effect of polymer molecular weight on the production of drug nanoparticles. J. Pharm. Sci. 96, 2655–2666. van der Weerd, J., Kazarian, S.G., 2004. Combined approach of FTIR imaging and conventional dissolution tests applied to drug release. J. Control. Release 98, 295–305. van der Weerd, J., Andrew Chan, K.L., Kazarian, S.G., 2004. An innovative design of compaction cell for in situ FT-IR imaging of tablet dissolution. Vib. Spectrosc. 35, 9–13. van der Weerd, J., Kazarian, S.G., 2005. Release of poorly soluble drugs from HPMC tablets studied by FTIR imaging and flow-through dissolution tests. J. Pharm. Sci. 94, 2096–2109. Van Eerdenbrugh, B., Alonzo, D.E., Taylor, L.S., 2011. Influence of particle size on the ultraviolet spectrum of particulate-containing solutions: implications for insitu concentration monitoring using UV/Vis fiber-optic probes. Pharm. Res. 28, 1643–1652. Van Eerdenbrugh, B., Froyen, L., Van Humbeeck, J., Martens, J.A., Augustijns, P., Van den Mooter, G., 2008a. Drying of crystalline drug nanosuspensions-the importance of surface hydrophobicity on dissolution behavior upon redispersion. Eur. J. Pharm. Sci. 35, 127–135. Van Eerdenbrugh, B., Van den Mooter, G., Augustijns, P., 2008b. Top-down production of drug nanocrystals: nanosuspension stabilization, miniaturization and transformation into solid products. Int. J. Pharm. 364, 64–75. Wang, J., Urban, L., Bojanic, D., 2007. Maximising use of in vitro ADMET tools to predict in vivo bioavailability and safety Expert Opin. Drug Metab. Toxicol. 3, 641–665. Windbergs, M., Jurna, M., Offerhaus, H.L., Herek, J.L., Kleinebudde, P., Strachan, C.J., 2009. Chemical imaging of oral solid dosage forms and changes upon dissolution using coherent anti-stokes Raman scattering microscopy. Anal. Chem. 81, 2085–2091. Yadav, S.K., Mishra, S., Mishra, B., 2012. Eudragit-based nanosuspension of poorly water-soluble drug: formulation and in vitro-in vivo evaluation AAPS. PharmSciTech. Ye, F., Yaghmur, A., Jensen, H., Larsen, S.W., Larsen, C., Østergaard, J., 2011. Real-time UV imaging of drug diffusion and release from Pluronic F127 hydrogels. Eur. J. Pharm. Sci. 43, 236–243.
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European Journal of Pharmaceutical Sciences journal homepage: www.elsevier.com/locate/ejps
Dissolution study of nanocrystal powders of a poorly soluble drug by UV imaging and channel flow methods Annika Sarnes a,⇑, Jesper Østergaard b, Sabrine Smedegaard Jensen b, Jaakko Aaltonen a, Jukka Rantanen b, Jouni Hirvonen a, Leena Peltonen a a b
Division of Pharmaceutical Technology, Faculty of Pharmacy, University of Helsinki, P.O. Box 56, 00014 Helsinki, Finland Department of Pharmacy, Faculty of Health and Medical Sciences, University of Copenhagen, Universitetsparken 2, 2100 Copenhagen, Denmark
a r t i c l e
i n f o
Article history: Received 11 April 2013 Received in revised form 20 August 2013 Accepted 20 August 2013 Available online 30 August 2013 Keywords: Channel flow method Dissolution rate Nanocrystals Poloxamer Solubility UV imaging
a b s t r a c t Application of drug nanocrystals provides advantageous options for the pharmaceutical formulation development of poorly soluble drugs. The objective of this study was to investigate the dissolution behavior improving effects of differently sized nanocrystals of a poorly soluble model drug, indomethacin. Nanocrystal suspensions were prepared using a top-down wet milling technique with three stabilizers: poloxamer F68, poloxamer F127 and polysorbate 80. The dissolution of the differently sized indomethacin nanocrystals were investigated using a channel flow dissolution method and by UV imaging. Unmilled bulk indomethacin and physical mixtures were used as references. According to both the dissolution methods, the dissolution properties of indomethacin were improved by the particle size reduction. UV imaging was used for the first time as a dissolution testing method for fast dissolving nanoscale material. The technique provided new information about the concentration of the dissolved drug next to the sample surface; with the smallest nanocrystals (580 nm) the indomethacin concentration next to the particle surface exceeded five-fold the thermodynamic saturated indomethacin solution concentration. Thus the solubility improvement itself, not only the increased surface area for dissolution, may have an important role in the higher dissolution rates of nanocrystal formulations. Poloxamer F68 was the most optimal stabilizer in the preparation of the indomethacin nanocrystal suspensions and in the solubility and dissolution enhancement as well. Ó 2013 Elsevier B.V. All rights reserved.
1. Introduction In vitro drug release and dissolution testing play an important role in pharmaceutical formulation development and quality control. Poorly soluble candidate molecules constitute a major challenge for the formulation development since the low solubility and low dissolution rate often lead to poor bioavailability (Keck and Müller, 2006; Kipp, 2004). It is necessary to develop new analytical methods in order to understand better the complex dissolution processes and overcome the difficulties related to dissolution (Amidon and Hawley, 2010). New in vitro methods that could exclude ‘impossible’ molecules as early as possible are thus vital to secure streamlined formulation development. The long-term goal of dissolution research is to be able to predict the in vivo behavior of the formulation based on in vitro dissolution studies. This may be achieved by an adequately discriminative and robust in vitro method enabling the establishment of an in vitro–in vivo correlation (IVIVC). ⇑ Corresponding author. Tel.: +358 9 191 59159; fax: +358 9 191 59144. E-mail address: annika.sarnes@helsinki.fi (A. Sarnes). 0928-0987/$ - see front matter Ó 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ejps.2013.08.030
Nanotechnology, especially nanocrystals, is currently offering promising possibilities to address the solubility problems of poorly soluble candidates (Van Eerdenbrugh et al., 2008b). Nanocrystallization techniques are used to decrease the particle size and thus increase the dissolution rate and to improve the bioavailability (Peltonen and Hirvonen, 2010). A drug nanocrystal is a nano-sized crystalline particle containing 100% drug without any matrix material (Keck and Müller, 2006). In the field of pharmacy the definition of nanocrystals covers particles below one micron (Merisko-Liversidge et al., 2003). In the production process of nanocrystals by a top-down wet milling technique, a nanocrystal suspension is formed. It is a colloidal dispersion of drug nanoparticles, where the particles have been stabilized by coating them with surface active agents, polymers or a mixture of polymers (Merisko-Liversidge et al., 2003; Merisko-Liversidge and Liversidge, 2011). Nanocrystals possess an increased surface area/volume-ratio, which facilitates a very fast dissolution rate (Kocbek et al., 2006). With nanocrystal formulations a high drug load, low incidence of side effects due to the excipients, versatile administration routes, improvement of bioavailability, and an overall improvement of efficiency and safety can be achieved.
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When considering the existing traditional, material and time consuming, pharmacopeial dissolution methods (USP, European and Japanese Pharmacopoeia) that were mainly developed for quality control purposes, they are time to time regarded inadequate and not evolved enough to correspond the needs of modern dissolution study (Dokoumetzidis and Macheras, 2006; McAllister, 2010). The traditional methods, e.g. paddle method combined with UV spectroscopy or HPLC, do not provide real-time, spatially and temporally resolved information of the dissolution process, since the monitoring of the process immediately next to the surface of the dosage form is impossible with those techniques (Greco and Bogner, 2011; Windbergs et al., 2009). However a number of recent methods to study the dissolution of solid dosage forms are based on imaging, e.g., UV, FT-IR, NIR, and MRI imaging, CARS and Raman spectroscopy in order to cover those missing aspects (Aaltonen et al., 2006; Kazarian and van der Weerd, 2008; Kowalczuk and Tritt-Goc, 2011; Metz and Mader, 2008; Nott, 2010; Ostergaard et al., 2010; van der Weerd and Kazarian, 2005). In this study, the dissolution and solubility properties of a poorly soluble model drug, indomethacin (IND), were improved by preparing nanocrystal suspensions using a top-down, wet milling technique (Liu et al., 2011; Rabinow, 2004; Van Eerdenbrugh et al., 2008a). The dissolution and solubility behavior of the different particle size fractions of the dried IND nanocrystal suspensions were investigated using a channel flow dissolution method and UV imaging (Ostergaard et al., 2010; Peltonen et al., 2003; Peltonen and Hirvonen, 2010). The aim of the study was to investigate both the real-time, spatially and temporally resolved, dissolution behavior of the nanocrystals with different size fractions for the first time using UV imaging, as well as the critical particle size limit, which still benefits the dissolution process. Additionally the creation of possible supersaturated states, as compared to the thermodynamic IND solubility value, when the particle size was decreased from the bulk material (tens of lm) to 500 nm, was examined. In order to eliminate the effect of increased surface area on the dissolution testing, all the tests were performed from a flat surface of compressed nanocrystalline powders. 2. Materials and methods 2.1. Materials In this study, indomethacin (IND, Hawkins Pharmaceutical Group, Minneapolis, USA) was used as the model drug. Poloxamer 188 and 407 (LutrolÒ F68 and F127, BASF Co., Ludwigshafen, Germany) and polysorbate 80 (TweenÒ 80, Sigma–Aldrich Chemie GmbH, Steinheim, Germany) were used as stabilizers. Lactose (particle size 30–150 lm, PharmatoseÒ 200 M, DMV International, Veghel, The Netherlands) was used to prevent particle aggregation during freeze-drying and microcrystalline cellulose (MCC, Avicel PH-102, FMC International, Cork, Irland) as an excipient in sample compaction (see Section 2.7.1). Ethanol (96%, v/v, Altia Oyj., Rajamäki, Finland) and acetate buffer (pH 5.0, NaC2H3O23 H2O and CH3COOH, Riedel-de Haën Laborchemikalien GmbH & Co. KG, Seelze, Germany or pH 5.0, CH3COONa3 H2O, and CH3COOH, Merck KGaA, Darmstadt, Germany) were used for the dissolution studies. Agarose (type I, Sigma–Aldrich Chemie GmbH, Steinheim, Germany) was used as gel former. The water used was ultrapurified Milli-QÒ-water (Millipore SAS, Molsheim, France or MilliporeÒ, Bedford, MA, USA).
or surfactant, 1.2 g) was dissolved in 5 ml of water and, thereafter, 2 g of IND was dispersed in the aqueous stabilizer solution. Three types of stabilizers were used: polysorbate 80 and poloxamers F68 and F127. The stabilizer:drug ratio was kept constant (0.6:1) in all the tests. The suspension was inserted into the milling bowl containing varying amounts of milling pearls (both bowl and pearls were made of zirconium oxide), depending of the size of the pearls used (Ø 1, 5 or 10 mm, being equivalent to 70 g, 180 pcs. or 18 pcs. of pearls, respectively). By varying the used milling pearl size, different particle sizes were effectively produced. Additional 5 ml of water was used to collect the residual suspension from the beaker into the milling bowl. The milling bowl was placed in a planetary ball mill (Pulverisette 7 Premium, Fritsch Co., Idar-Oberstein, Germany), and the milling experiment was performed in ten 3-min milling cycles. After each cycle there was a 15 min pause for the system to cool down to 32 °C. The grinding speed was adjusted according to the milling pearl size (Ø 1 mm, 5 mm or 10 mm corresponded to 1100 rpm, 1000 rpm or 850 rpm, respectively). After milling, the nanocrystal suspension was separated from the pearls by pipetting. All the nanocrystal suspensions were freeze dried (72 h, LyoPro 3000, Heto-Holten A/S, Allerød, Denmark). Prior to freeze-drying, lactose, dissolved in water (0.2 g, 10 w/w% in relation to the drug amount), was added to the nanocrystal suspension to prevent particle aggregation during the drying process. The dried samples were stored in closed vials protected from light at room temperature (19–22 °C). 2.3. Particle size The mean particle sizes and polydispersity indexes (PI) of the nanocrystal suspensions were analyzed by photon correlation spectroscopy (PCS) using a Malvern Zetasizer 3000HS (Malvern Instruments, Malvern, UK). Measurements were performed from the fresh nanocrystal suspensions, as well as from the dried and redispersed nanocrystal suspensions. All the samples were measured in triplicate. 2.4. Solid state properties The solid state of IND was assessed with differential scanning calorimetry (DSC 823e, Mettler Toledo Inc., Columbia, USA) after milling and freeze-drying. Lactose was excluded from the DSC analyzed samples. The dried nanocrystal suspension samples were placed (2–5 mg) in an aluminum pan closed with a perforated lid (Liu et al., 2011). Heating rate of 10 °C/min was used between 25 °C and 200 °C. Nitrogen purge of 50 ml/min was used during the measurements. The data was analyzed with STARe software (Mettler Toledo, Columbia, USA). Bulk materials (IND, F68, F127) were as well measured. 2.5. Morphology of nanocrystals The morphological evaluation of the dried nanocrystal suspensions was performed using scanning electron microscopy (SEM, JSM-7500F, JEOL Ltd., Japan). The samples were prepared for the analysis by assembling a small amount of nanocrystal suspension onto a carbon-coated tape and allowed to dry at room temperature, after which the samples were sputter-coated with platin (Q150T Quomm, Turbo-Pumped Sputter Coater, China). The coated samples were imaged.
2.2. Preparation of dried nanocrystal samples
2.6. Effect of stabilizer on drug solubility
The nanocrystal suspensions were prepared using a top-down, wet-milling technique (Liu et al., 2011). The stabilizer (polymer
The solubility of the drug in aqueous stabilizer solutions was determined with the traditional shake-flask method (Wang et al.,
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2007). Acetate buffer (pH 5.0) was used as medium. Solubility determinations were made from the physical mixtures of bulk IND and stabilizers. The ratio of stabilizer and drug was kept constant (0.6:1), the total amount of polymer being 0.5 mg/ml.
Table 1 The effect of stabilizer (stabilizer:drug ratio 0.6:1, the total concentration of stabilizer being 0.5 mg/ml) on the apparent solubility of bulk IND in acetate buffer (pH 5.0, after 12 h of shaking, n = 3). Composition
2.7. Dissolution studies 2.7.1. Channel flow method Dissolution properties of the compacted nanocrystal surfaces were studied by the channel flow dissolution method (Peltonen et al., 2003), in which one face of the compacted sample was exposed to the dissolution medium. The dried nanocrystal suspensions were manually compacted (150 mg) with a single-punch tablet press (Korsch, EK-0, Korsch, Germany). First, MCC powder (50 mg) was inserted to the bottom of the mold as supportive material in order to facilitate the compaction. Then nanocrystalline drug (100 mg) was put on the top of MCC and the compression was performed. The dissolution was studied from the nanocrystalline tablet surface, which was adjusted to be thick enough to remain during the experiment. Flat-faced punches with a diameter of 9 mm were used in order to achieve a flat and constant surface area for dissolution. The tablet press was instrumented and the compression force of the upper press was monitored (SingleStation DAAS Measure, software version 1.2) and kept as even as possible (1000 N). Acetate buffer (pH 5.0, V = 500 ml, room temperature) was used as the dissolution medium. The medium circulated by a peristaltic pump (Watson-Marlow, Cornwall, UK) through the channel flow cell, medium reservoir and UV–Vis spectrophotometer with a flow-through cuvette (UV-1600PC, VWR International, Leuven, Belgium). The parts of the system were interconnected in a closed-loop fashion using silicone tubings. The optimal pumping speed was found to be 50 rpm (corresponding to a flow rate of 8.1 ml/min). UV–Vis data was collected and analyzed with M. Wave Professional software (v 1.0, VWR International, Leuven, Belgium). The analytical wavelength was 318 nm (European Pharmacopoeia, 2011). All the measurements were performed at room temperature (19–22 °C). Dissolution rate results were calculated as a released drug amount in time unit per constant area (Peltonen et al., 2003). 2.7.2. UV imaging Dissolution studies of dried IND nanocrystal suspensions were also performed using an Actipix SDI300 dissolution imaging system (Paraytec Ltd., York, UK) with an Actipix flow-through type dissolution cartridge (CADISS-3) (Boetker et al., 2011; Ostergaard et al., 2010; Ye et al., 2011; Østergaard et al., 2011). The total detection area of the imager is 9 mm 7 mm (1280 1024 pixels); however, the selected imaging area was 3.64 mm 8.12 mm. The pixels (7 lm 7 lm) were binned 4 4. A pulsed Xe lamp was used as the light source. The quartz flow cell had a light path of 4.0 mm. Images were recorded (2.6 images per s) and analyzed with Actipix D100 software version 1.3 (Paraytec Ltd., York, UK). All experiments were carried out at ambient temperature (19– 22 °C). Based on initial experiments an acetate buffer (pH 5.0) and 265 nm were selected as dissolution medium and detection wavelength, respectively. Compacts for the UV imaging were prepared by weighing 6 mg of the sample into a stainless steel cylinder (inner diameter: 2 mm) held in a manual press (Actipress, Paraytec Ltd., York, UK) (Boetker et al., 2011). A torque screwdriver (Quickset MINOR, Torqueleader, M.H.H. Engineering Co., Ltd., England) was used to compact the nanocrystal powders at constant torque of 110 cN m. The general procedure for UV imaging has been presented previously (Boetker et al., 2011; Ostergaard et al., 2010; Ye et al., 2011; Østergaard et al., 2011). The dissolution of the compacted nanocrystals was studied both with a solution phase (acetate buffer,
IND/F68 IND/F127 IND/polysorbate 80 IND
Solubility
lg/ml
mM
6.43 ± 0.06 4.80 ± 0.00 10.9 ± 1.54 4.89 ± 0.18
0.018 ± 0.00 0.013 ± 0.00 0.030 ± 0.004 0.014 ± 0.001
pH 5.0; absence and presence of flow: 0.5 ml/min to 0.1 ml/min) and a gel matrix (agarose/acetate buffer, pH 5.0) as the dissolution medium. The gel matrix was prepared as follows: 10 mg agarose was mixed with acetate buffer (V = 1 ml, pH 5.0) in an Eppendorf-tube and heated in a water bath (90 °C) for 0.5 h, after which the hot polymer solution was pipetted into the quartz cell. The gel matrix was allowed to cool and settle for 2 h prior to dissolution testing. After measurements, the pixel intensities were converted into absorbance values using the Actipix software (Ostergaard et al., 2010), allowing the determination of the apparent IND concentration within the imaging area as a function of position and time. The absorbance maps were collected with the Actipix software for each sample using the same detection area (V = 118.23 ll) in order to get comparable results. The dissolution rates were determined during the first 10 min, from the linear region of the dissolution profile, during which the sample remained in the detection area. 3. Results and discussion 3.1. Effect of the stabilizer on the solubility of IND Surface active agents may have solubility-improving effects, and for that reason the apparent solubility of bulk IND was measured in the presence of the stabilizers (Table 1). F127 did not alter the solubility, but F68 improved the solubility of bulk IND by 34%. Polysorbate 80 improved the solubility even more, by 127%, because of the micelle formation (Hagan et al., 1996). There were no micelles present in the poloxamer systems (F68 and F127), since the stabilizer concentrations were below the critical micelle forming concentrations (CMC) (Hagan et al., 1996; Maskarinec et al., 2002). The solubilizing effect of the poloxamers depends on the block structure, chain lengths and interactions of the hydrophobic and hydrophilic parts, which finally cause a decrease in the interfacial tension between the drug and dissolution medium and, hence, improve the wetting (BASF, 2004; Lan et al., 2013; Prasanthi et al., 2011). 3.2. Particle sizes of the nanocrystals IND nanocrystal suspensions were successfully prepared in varying particle sizes by changing the size of the used milling pearls (values presented in Table 2). Along the increasing particle sizes the PIs were also detected to increase, implying about more heterogeneous material. A low PI value (<0.2) indicates monodisperse particles (Liu et al., 2011). When the PI value lies around 0.5–0.7 and higher, the particle size distribution is considered broad (Muller and Jacobs, 2002; Yadav et al., 2012). Polydisperse particles have PI-values larger than 0.7. Aggregation of the nanocrystals during the freeze-drying process was prevented by optimizing the amount of lactose. Thus the particle size was kept constant. The stabilizing agents, poloxamers F68 and F127, were successfully applied for the preparation
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Table 2 The average dissolution rate of IND from the nanocrystal compacts in acetate buffer (pH 5.0) as determined using the channel flow dissolution method (n = 2). The corresponding particle size and polydispersity index of the nanocrystals are also shown (n = 3). Sample
Dissolution rate (lg/min/mm2)
Particle size (nm) (PI)
F68 F68 F68 F127 F127 F127 Polysorbate 80 Physical mixtureb
0.50 0.35 0.14 0.31 0.16 0.08 0.05 0.05
580 ± 30 (0.4 ± 0.1) 950 ± 190 (0.8 ± 0.1) ND (micronsized)a 580 ± 20 (0.6 ± 0.06) 740 ± 30 (0.7 ± 0.2) ND (micronsized)a NDa 80 lm
a
ND = not determined, above the detection limit of PCS (<10 lm). Physical mixture of IND and lactose, particle size of IND announced by the manufacturer. b
of nanocrystal suspensions (Table 2). Both the poloxamers are linear triblock polymers including hydrophilic and hydrophobic segments (Liu et al., 2011). Together the hydrophilic and hydrophobic regions provide effective steric hindrance against aggregation. Polysorbate 80 was a problematic material in the milling process. The decrease of the surface tension resulted in a leakage of the suspension out of the sealed bowl during the milling. Regardless of several attempts, only one particle size with polysorbate 80 as the stabilizer could be prepared, using the smallest milling pearls (Ø 1 mm). The resulting particle sizes were not as small as with F68 and F127 and the prepared material was not homogenous. Polysorbate 80 is a hydrophilic, low molecular weight polymer that forms a thin adsorption layer, thus offering weak steric stabilization when approaching the particles (Sepassi et al., 2007). In addition, the freeze dried nanocrystal suspensions having polysorbate 80 as the stabilizer were extremely sticky and difficult to handle. 3.3. Solid state properties
Fig. 1. The DSC thermograms of IND nanocrystal suspension (NPs) stabilized with F68 and F127 compared to the bulk materials (IND, F68, F127).
The solid state analysis using DSC was performed for all the samples: nanocrystal suspensions stabilized with F68, F127 and bulk materials (IND, F68, F127, polysorbate 80) (Fig. 1). The consistency of the polysorbate 80 was too paste-like to be able to perform the thermal analysis. The obtained DSC thermograms of the nanocrystal suspensions showed endothermic melting peaks at approx. 160 °C corresponding to the melting point of pure, unmilled bulk IND. Milling and freeze-drying did not induce formation of amorphous IND, supported by the absence of glass transition or recrystallization events (Chamarthy and Pinal, 2008). Additionally, the rapid wet milling technique, using the same protocol, is demonstrated to provide crystalline material, which was confirmed by both DSC and X-ray analyses (Liu et al., 2011). The lower peak intensities of the nanosamples derive from the presence of the stabilizers surrounding the particles after milling (Hecq et al., 2005). Poloxamers were separated from the drug based on their melting
Fig. 2. SEM images of the freeze dried indomethacin nanocrystal suspensions. (A) and (B) Present F68 samples (scale bar: 10 lm and 5 lm). (C) and (D) Are produced using F127 (scale bar: 10 lm) and Tween 80 (scale bar: 10 lm), respectively. All the figures are taken from the smallest particle size samples.
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points. The melting points of poloxamers F68 (50 °C) and F127 (53–57 °C) and polysorbate 80 (21 °C) are clearly lower than the melting point of IND (162 °C), which made the interpretation of the thermograms straightforward (BASF, 2004). 3.4. Morphology of nanocrystals The morphology of the nanocrystals was studied using SEM (Fig. 2). All the freeze dried samples showed a porous structure. The individual particles were highlighted by changing the magnification. The grey mass between the particles mainly consists of the stabilizing agent (F68/F127/polysorbate 80), because lactose was used at a relatively low concentration (10 w/w%). The porous structure is formed when water is evaporated during the drying process. The poloxamer samples showed a uniform structure where the single particles could be detected, whereas when looking at the surface of polysorbate 80 samples, a mixed surface formed by the drug crystals and the medium was observed. 3.5. Channel flow method After compacting the dried IND nanocrystals, dissolution experiments were carried out using the channel flow method (Fig. 3). Particle sizes of the samples were analyzed prior to the experiments using PCS. Factors that were optimized before the channel-flow dissolution experiments were the selection of the dissolution medium and the optimal flow rate. An acidic medium was chosen, because IND (pKa 4.5) has low solubility in this pH which is to enhance the differences in dissolution behavior. With
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regards to the pumping speed, if it was too high (e.g. 90 rpm), detection of the signal was difficult because the sample did not have time to dissolve at such a high flow rate. On the other hand, a too slow flow rate resulted in saturated solutions near the surface of the sample, and thus limited the possibility of higher dissolution rates skewing the results. The optimal speed was obtained by both visual and absorbance monitorings. Results of the dissolution tests showed that an increase in the particle size caused a decrease in the dissolution rate. In the measurements done with the channel flow dissolution method, the effect of particle size was ruled out by preparing compacts with a constant surface area. Thus, according to the Noyes–Whitney equation, the dissolution rate was affected only by the concentration gradient. The different particle sizes and stabilizing agents can be distinguished clearly in the experiments with the channel flow dissolution method (Table 2). Differences between the dissolution rates of the samples were at highest ten-fold. The largest differences were found out between the nanocrystal samples and physical mixture of indomethacin and lactose. The samples were set in order as predicted according to their particle size and the used stabilizer. For example, the dissolution rates of the samples containing F127 were doubled, when the particle size was decreased from 740 nm to 580 nm. Interestingly, the IND dissolution rates from the F68 samples were faster than from the F127 in every particle size fraction, although the actual particle sizes were slightly higher in the F68 samples. This may be related to the higher drug solubility observed in the presence of F68 as compared to F127. Overall the dissolution studies with the channel flow method suggested that the poloxamer F68 was the most optimal choice for both the preparation of the nanocrystals, and to enhance indomethacin solubility and dissolution. However, as demonstrated, the comparison of the dissolution behavior is here performed within each stabilizer group, since the stabilizer has some influence on the dissolution. Additionally, it must be taken into account that the concentration of the stabilizer does not remain constant throughout the experiment. That is, there may exist local concentration differences of the stabilizer during dissolution, especially at the diffusion front, which may lead to higher solubilities of the drug. In conclusion, the results from the bulk IND solubility tests cannot be directly applied to these circumstances since the measurements were performed only at a constant concentration level. 3.6. UV imaging
Fig. 3. Channel flow method (n = 2, all the measurements are shown (gray and black) in the figure): dissolution of indomethacin from the nanocrystal compacts. Micronsized bulk IND samples were used as corresponding references.
In the dissolution studies done with UV imaging, the objective was to identify local concentration changes near the compact surfaces, and reveal possible supersaturation states of indomethacin.
Fig. 4. Dissolution of F68/Ø 580 ± 30 nm compact, using UV imaging with a flow-through system (contour interval 200 mAU, contour offset 20 mAU) and acetate buffer as dissolution medium (pH 5.0). (A) The dissolution medium flow rate: 4 min 1 ml/min, at wavelength 265 nm. (B) Control study with the same sample using flow rate: 4 min 1 ml/min and 5 min 0.1 ml/min, at wavelength 550 nm. Image (B) is taken 2 min after the flow is stopped, in the absence of flow.
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Fig. 5. UV imaging using the agarose gel matrix prepared in acetate buffer (pH 5.0) (contour interval 200 mAU, contour offset 20 mAU). Bulk-IND after 14 h of dissolution at the wavelength (A) 265 nm and (B) 550 nm. F68/micronsized particles sample after 0.5 h of dissolution at the wavelength (C) 265 nm and (D) 550 nm.
The initial UV imaging setup investigated was a flow-through system. During preliminary testing, however, it was noticed that the samples induced an exceptionally strong signal immediately upon initiation of the flow-through dissolution experiment which was significantly different from the dissolution maps expected based on previous flow-through imaging studies (Fig. 4a) (Boetker et al., 2011; Gordon et al., 2012). The phenomenon was examined by changing the wavelength from 265 nm to 550 nm, where the dissolved IND does not absorb light. Again strong absorbance was observed which indicated that the solid nanocrystals dissociated from the compact surface and carried downstream by the flow caused absorbance at 550 nm (Fig. 4b). Light scattering from particles has been confirmed also previously (Van Eerdenbrugh et al., 2011). The effluent was however clear, presumably due to the rapid dissolution of the nanocrystals. An attempt to prevent the erosion of the sample by increasing the compaction force was
unsuccessful. The undissolved nanocrystals left the surface even in the experiments performed in the absence of flow. Thus, UV imaging provided valuable information on the dissolution mechanism of compacted IND nanocrystals. The method revealed that nanoparticulate material dissociated from the compact surface. In order to focus on molecular IND dissolution, an agarose-based hydrogel matrix (1%) was used as a dissolution matrix instead of the buffer solution. The adjustments, high torque (110 cN m) in compact preparation and gel matrix, prevented the nanocrystals from escaping the compact surface. Also bulk-IND and a physical mixture of IND and lactose were investigated. Nanocrystal samples containing polysorbate 80 were impossible to study with UV imaging since proper compacts were not possible to prepare. The dissolution behavior of IND nanocrystal samples and bulk IND into agarose matrix is compared in the absorbance maps shown in Fig. 5.
Table 3 Released IND (after 10 min) and dissolution rates of the nanocrystals studied with UV imaging (n = 3). In addition, the concentration (lg/ml) as a function of the distance (0, 2 and 3 mm) from the compact surface after 10 min is given. Sample (size/nm)
Released IND ± STD (lg) (RSD%)
Dissolution rate ± STD (lg/min/mm2) (RSD%)
Concentration (lg/ml) (distance/mm)
F68(580 ± 30)
0.59 ± 0.16 (27.07)
0.019 ± 0.006 (28.87)
28.69 (0) 11.71 (2) 5.84 (3)
F68(micronsized)
0.52 ± 0.13 (24.43)
0.016 ± 0.00 (0.00)
11.35 (0) 4.04 (2) 02.57 (3)
F127(580 ± 20)
0.65 ± 0.16 (25.51)
0.019 ± 0.00 (0.00)
22.08 (0) 9.37 (2) 4.31 (3)
F127(micronsized)
0.63 ± 0.10 (15.91)
0.019 ± 0.003 (16.67)
17.08 (0) 7.61 (2) 3.97 (3)
Bulk IND (80 lm)
0.13 ± 0.05 (39.73)
0.004 ± 0.002 (43.30)
2.07 (0) 0.26 (2) 0.00 (3)
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Fig. 6. Apparent concentration-distance profiles obtained from UV imaging of IND nanoparticles at time points 5, 15 and 30 min: (A) F68/580 ± 30 nm, (B) F68/micronsized particles, (C) F127/580 ± 20 nm, (D) F127/micronsized particles and (E) bulk IND/tens of lm.
One of the aims of this study was to determine, if supersaturated states of IND can be achieved next to the compact surface, and if the achieved concentrations show significant difference between the different particle sizes (Table 3 and Fig. 6). Difference in the dissolution rates between the nanocrystals and the unmilled bulk IND was noticed by UV imaging, similarly to the dissolution results obtained by the channel flow method. However, dissolution rates among the nanoparticles in the gel matrix were similar irrespective of the particle sizes; the dissolution-enhancing effect observed in the solubility tests might have diminished the differences in the dissolution rate. Compared to the channel flow method, the UV imaging could not separate the nanocrystal samples that well. The gel matrix has likely impacted on the dissolution. The use of agarose gel provided a highly symmetric absorbance maps indicating that natural convective effects due to the density gradients had been efficiently supressed (Østergaard et al., 2011). All the measurements were carried out using agarose gel (acetate
buffer, pH = 5) as dissolution medium and the analytical wavelength of 265 nm. The absorbances were converted into the amount of drug released, and the dissolution rate determined (Ye et al., 2011). The determined dissolution rates are depicted in Table 3. The released drug amount is determined after 10 min imaging which is an adequate time to study the dissolution of the fast dissolving nanocrystals (Liu et al., 2011). Apparent IND concentrations determined by UV imaging (Table 3) were compared to the earlier concentrations of saturated IND solutions (Table 1). Each stabilizer group was compared separately because of the different solubilizing effects of the stabilizers. It must be noticed, like in the case of channel flow method, that there exist differences in drug concentration during the entire dissolution process; high concentrations of surfactant may exist at specific locations in the hydrogel leading to very high local solubilities of the drug. Thus no direct conclusions about the impact of the stabilizer can be withdrawn based only on the bulk drug solubility
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tests performed at constant concentration level. Fig. 6 shows that with the studied nanocrystals at least a saturated, and in majority of the cases a supersaturated system was achieved, e.g. the thermodynamic solubility was exceeded. The thermodynamic solubility was exceeded almost five-fold for the F127/580 ± 20 nm sample. By preparing nanocrystals from a poorly soluble drug, the dissolution of the material is strongly improved. Difference in the IND concentration as a function of distance was achieved between all the particle size fractions. The released drug amount was greater with the small particles (580 ± 30 nm, 580 ± 20 nm) than with the micronsized particles – at each measurement timepoint (5, 15 and 30 min). With micronsized particles it took 30 min to reach a concentration that was achieved with the smaller particles in 15 min (Fig. 6). Bulk IND was used as a reference, and it was altogether measured for 15 h. In this time, the released drug content was still low even compared to the micronsized crystals. Overall, UV imaging provides insight into the behavior of the nanocrystals during the dissolution process. With small particle size, much higher concentration as a function of distance from the surface is achieved in comparison with the large particle sizes in the gel matrixes. The thermodynamic solubility was exceeded even five-fold, which is a remarkable result when considering the fact that in this experimental set-up the effect of the dissolution area was excluded. The effect of the particle size is even more pronounced when the nanocrystals are allowed to perform in particulate form. Compared to UV imaging, the channel flow dissolution method gives substantially higher dissolution rates (lg/min/ mm2) and differentiates the samples clearer at the conditions investigated. UV imaging provided valuable information about the concentrations profiles. The sample escaping from the compact surface in the channel flow method was not a problem. The compression force used in the sample preparation was 1000 N in the channel flow method, while in the UV imaging the used torque was 110 cN m. In addition, the fast dissolving nature of the nanocrystals ensured that all the material in the set-up was fully dissolved at the time reaching the spectrophotometer. The use of gel matrix and stationary mode in the UV imaging, versus the flowing liquid medium in the channel flow method, at least partially explains the differences between the two dissolution methods. Interestingly, the results indicate the possibility that the difference in IND nanocrystal particle sizes between 500 and 1000 nanometers is not necessarily critical, because both of the studied size fractions were several times smaller particle size than the unmilled indomethacin (80 lm). In principle, it is possible that particles around 1000 nanometers are sufficiently small for substantial improvement of the dissolution properties of the poorly soluble indomethacin, but problems with further formulation of a potential drug product from these nanocrystals related to the small size could be partly avoided. As a whole, UV imaging offered a flexible experimental setup, allowing non-intrusive real-time studies of the dissolution process. This technique enables, similar to other imaging methods (e.g. FTIR and NMR), dissolution studies with benefits, such as excluding radioactive or fluorescent labeling (Coutts-Lendon et al., 2003; Dahlberg et al., 2007; Kazarian and Chan, 2006; Kazarian and van der Weerd, 2008; Kowalczuk and Tritt-Goc, 2011; van der Weerd and Kazarian, 2004, 2005; van der Weerd et al., 2004). The first experiments with UV imaging made here indicate that it may be an interesting approach to study dissolution behavior of nanoscale materials.
4. Conclusions Dissolution properties of a poorly soluble drug, indomethacin, were improved by preparing different sized nanocrystals using ra-
pid wet milling technique with three different stabilizers, poloxamers F68 and F127 and polysorbate 80. The dissolution properties of the freeze dried and compacted nanocrystals were studied using the channel flow dissolution method and by novel UV imaging. UV imaging was used in this study for the first time in the dissolution testing of fast-dissolving nanoscale samples. Both methods ranked the samples in decreasing dissolution rate order according to the increasing particle size, and the results supported well each other. Each stabilizer group was handed individually due to the possible impact of the stabilizer to the drug solubility. The use of stationary gel matrix versus flowing liquid as dissolution medium explains the differences between the methods. Even though the effect of the variation in the area available for dissolution was eliminated by studying even, constant surfaces instead of particulate samples, the differences between particle sizes, which will be increased even further in actual drug formulation, were evident. UV imaging provided valuable new information about the concentration of the dissolved drug next to the sample surface: with the smallest nanocrystals the concentration next to the particle surface exceeded fivefold the thermodynamic solubility. This indicates that the solubility improvement itself, and not only the increased dissolution area, has a crucial role in higher dissolution rates of nanoparticle formulations. UV imaging is a promising technique to analyze the dissolution properties of nanoscale formulations in detail. Acknowledgment Financial support from NordForsk (Project No. 43703, Future performance testing of pharmaceuticals) is acknowledged. References Aaltonen, J., Heinänen, P., Peltonen, L., Kortejärvi, H., Tanninen, V.P., Christiansen, L., Hirvonen, J., Yliruusi, J., Rantanen, J., 2006. In situ measurement of solventmediated phase transformations during dissolution testing. J. Pharm. Sci. 95, 2730–2737. Amidon, G.E., Hawley, M., 2010. Oral bioperformance and 21st century dissolution. Mol. Pharm.. BASF, 2004. PluronicÒ F68 ja F127, blockpolymer, surfactant.
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