Enhancement of the apparent solubility and bioavailability of Tadalafil nanoparticles via antisolvent precipitation

European Journal of Pharmaceutical Sciences 128 (2019) 222–231

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European Journal of Pharmaceutical Sciences journal homepage: www.elsevier.com/locate/ejps

Enhancement of the apparent solubility and bioavailability of Tadalafil nanoparticles via antisolvent precipitation

T

Qiuhong Raoa,1, Zhenwen Qiua,1, Deen Huangb, Tiejun Luc, Zhenyu Jason Zhangc, Dandong Luoa, ⁎ ⁎ Piaopiao Panb, Lei Zhangb, Yingyan Liud, Shixia Guanb, , Qingguo Lib, a

First Clinical Medical College, Guangzhou University of Chinese Medicine, Guangzhou 510405, PR China School of Pharmaceutical Sciences, Guangzhou University of Chinese Medicine, Guangzhou 510006, PR China c School of Chemical Engineering, University of Birmingham, Edgbaston, Birmingham B15 2TT, UK d Department of Laboratory, Dangyang People's Hospital, Dangyang 444100, PR China b

A R T I C LE I N FO

A B S T R A C T

Keywords: Tadalafil Antisolvent precipitation Amorphous solid dispersion Apparent solubility Bioavailability

The ability to increase the bioavailability and dissolution of poorly soluble hydrophobic drugs has been a major challenge for pharmaceutical development. This study shows that the dissolution rate, apparent solubility and oral bioavailability of tadalafil (Td) can be improved by nano-sized amorphous particles prepared by using antisolvent precipitation. Acetone and an acetone-water solution (v:v, 9:1) were selected as solvents, with deionized water as the antisolvent. The antisolvent precipitation process was conducted at a constant drug concentration of 10 mg/ml, at temperatures of 5, 10 and 15 °C and at volume ratios of antisolvent to solvent (AS/ S) of 5, 8 and 10. Solid dispersion was achieved by dissolving the polymer in the antisolvent prior to the precipitation and by spray drying the suspension after the antisolvent precipitation process. The selected polymers were HPMC, VA64, and PVPK30 at concentrations of 33, 100 and 300 mg per 100 ml of water (equivalent to weight ratios of drug-to-polymer of 1:3, 1:1 and 3:1, respectively). The solid dispersions were characterized by scanning electron microscopy (SEM), X-ray powder diffraction (XRPD), differential scanning calorimetry (DSC) and fourier transform infrared spectroscopy (FT-IR). The improvements in the dissolution rate, equilibrium solubility, apparent solubility and bioavailability were tested and compared with unprocessed Td. Td particles in the suspension (before spray drying) were 200 nm, and the obtained Td solid dispersion had a size of approximately 5–10 μm. The XRPD, DSC and FT-IR analyses confirmed that the prepared Td particles in the solid dispersions were amorphous. The solid dispersion obtained using the optimized process conditions exhibited 8.5 times faster dissolution rates in the first minute of dissolution, 22 times greater apparent solubility at 10 min and a 3.67-fold increase in oral bioavailability than the as-received Td. The present work demonstrated that low temperature antisolvent precipitation technique has excellent potential to prepare nano-sized amorphous particles with a faster release and a higher bioavailability.

1. Introduction Approximately 40% of drugs are categorised as class II (low solubility-high permeability) and class IV (low solubility-low permeability), according to Biopharmaceutics Classification System (BCS), because of their poor solubility and permeability (Lipinski, 2000; Vandecruys et al., 2007). Over the past few decades, various formulations and processes have been implemented to improve the delivery efficiency and effectiveness of these compounds. To enhance the solubility and dissolution rate of low water-soluble molecules, one of the promising

approaches is to reduce the particle size, which consequently increases the surface-area-to-volume ratio (Leuner and Dressman, 2000; Singh and Van den Mooter, 2016). Additionally, organic particles possessing amorphous form would have a greater solubility than those in crystalline form due to its thermodynamic properties. Therefore, antisolvent precipitation, a process generating amorphous solid dispersion with nano-sized drug particles, has been demonstrated with dual beneficial effects. A typical antisolvent precipitation process involves precipitating drug particles from a supersaturated solution, resulting multiple

⁎

Corresponding authors at: School of Pharmaceutical Sciences, Guangzhou University of Chinese Medicine, 232 University City Ring Road East, Panyu District, Guangzhou 510006, PR China. E-mail addresses: [email protected] (S. Guan), [email protected] (Q. Li). 1 QR and ZQ contributed equally to this paper. https://doi.org/10.1016/j.ejps.2018.12.005 Received 22 September 2018; Received in revised form 6 December 2018; Accepted 9 December 2018 Available online 13 December 2018 0928-0987/ © 2018 Published by Elsevier B.V.

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2. Materials and methodology

advantages such as narrow size distribution and better control of particle properties, such as size, morphology and crystallinity (Aghajani et al., 2011; D'Addio and Prud'Homme, 2011; Pu et al., 2017). This process has been applied to several drugs, including beclomethasone dipropionate (Wang et al., 2007), budesonide (Rasenack et al., 2003), griseofulvin (Beck et al., 2013), and ketoprofen (Belkacem et al., 2015). The solvent-antisolvent process has a range of competitive edges in comparison with conventional procedures: operation at ambient temperature and pressure; rapid and direct process; easily scalable; simple and less expensive instruments are required, which enables it particularly suitable for thermolabile drugs (Zhong et al., 2005). Under certain circumstance, such as contamination by surfactants absorbed to the surface of the drug particles, there will be low yield and thermal degradation (Chow et al., 2007; Rogers et al., 2002). Organic solvents possessing the highest solubility for the drug molecules are often selected, despite that the solvent-solvate interaction has to be taken into account (Dong et al., 2009; Wang et al., 2005; Zhang et al., 2006). Various factors such as the volume ratio between antisolvent and solvent, temperature of the antisolvent, and drug concentration in the solvent phase, can affect the outcome of the precipitation process. Tadalafil (Td), a phosphodiesterase type 5 (PDE5) inhibitor used in the treatment of erectile dysfunction, is a BCS class II drug substance (Galiè et al., 2009). Despite its good permeability, its bioavailability is limited by the poor solubility and dissolution rate. Using antisolvent process to produce Td suspension has been reported previously. Wlodarski and co-workers (Wlodarski et al., 2014) compared the apparent solubility and dissolution rate of crystalline and amorphous Td produced by different processes, including ball milling, cryogenic grinding, spray drying, freeze-drying and antisolvent precipitation. A less noticeable improvement with the apparent solubility of Td was observed for all of the approaches tested except the antisolvent precipitation method. This work demonstrates the significant benefits could be introduced by the antisolvent process. Furthermore, effects of polymers and their weight ratios on the apparent solubility and dissolution rate of antisolvent precipitated tadalafil were systemically investigated (Wlodarski et al., 2015). However, the effects of processing parameters, such as temperature, ratio of AS/S, solvent, polymer dosage, and antisolvent process parameters, on the quality of the final dispersion have not been examined fully. In the present work, we studied the effects of process parameters (temperature, ratio of AS/S, polymer type, solvent and polymer dosage) on tadalafil solid dispersion to provide an enhanced dissolution rate and increased bioavailability. The particles obtained were characterized via particle size analysis, dissolution rate tests, equilibrium and apparent solubility tests, SEM, XRPD, DSC, FT-IR, and bioavailability studies.

2.1. Materials Tadalafil (series 20,151,001) was kindly donated by Gansu Haotian Pharmatech Co. Ltd. (Lanzhou, Gansu Province, China). Tadalafil and sulfamethoxazole (Internal Standard, IS) standard substances were purchased from the National Institutes for Food and Drug Control (Beijing, China). Hydroxypropylmethyl cellulose K15M (HPMC K15M) was donated by The Dow Chemical Co. (Midland, Michigan, USA). Kollidon VA64 and Kollidon 30 (PVPK30) were kindly donated by BASF SE (Germany). HPLC-grade acetonitrile was purchased from Merck (Darmstadt, Germany). Formic acid was purchased from Fisher (San Jose, USA). Ultrapure water was produced by a Milli-Q system (Merck, Darmstadt, Germany). Acetone and sodium dodecyl sulfate (SDS) were purchased from Damao Co. (Tianjin Province, China). All reagents were analytical grade chemicals and were used without further purification. 2.2. Antisolvent precipitation and spray drying procedures Tadalafil (used as received) was dissolved in the solvent (acetonewater (9:1) or acetone) to produce a Td concentration of 10 mg/ml. Deionized water was selected as the antisolvent, in which a specific quantity (300, 100 or 33 mg) of polymer was dissolved in 100 ml water aliquots. The antisolvent phase was brought to and maintained at the process temperature. At the desired temperature, the Td solution was quickly poured into the antisolvent with vigorous agitation by magnetic stirring at 1100 rpm. Particles precipitation from the liquid mixture was observed immediately, with a milk-like suspension formed simultaneously. After stirring for 30 s, the suspension was processed by spray drying to obtain the Td solid dispersion. Spray drying was performed using a Mini Spray Dryer B-290 (BUCHI, Switzerland) equipped with a standard atomization nozzle with a 1.5 mm cap and a 0.7 mm tip. The spray dryer was operated in a closed system with nitrogen as the drying and atomizing gas. The atomization gas flow was 6.83 l/min. The pump speed was set to 6 ml/min, and the aspirator was operated at 100%. The inlet temperature was set to 190 °C and resulted in an outlet temperature of approximately 110 °C. 2.3. Optimization of the antisolvent process All studies were performed by varying one parameter and keeping all others constant. The experimental variables and their ranges are summarized in Table 1. The effects of temperature, ratio of AS/S, polymer type, solvent and polymer dosage on the sizes of the particles

Table 1 List of process parameters for the antisolvent precipitation, the measured Td size with standard deviation in liquid suspension and the solubility of Td in antisolvent relative to the antisolvent precipitation conditions. Sample number

Tadalafil

T

AS/S

Polymer

Solvent

Polymer mass

Particle size (nm ± SD)

(mg/100 ml)

10 min

Equilibrium solubility

Acetone-Water

1 2 3 4 5 6 7 8 9 10

(total mg)

(°C)

(V:V)

100 100 100 125 200 100 100 100 100 100

15 10 5 5 5 5 5 5 5 5

100:10 100:10 100:10 40:12.5 100:20 100:10 100:10 100:10 100:10 100:10

(V:V) VA64 VA64 VA64 VA64 VA64 HPMC PVPK30 VA64 VA64 VA64

9:1 9:1 9:1 9:1 9:1 9:1 9:1 10:0 9:1 9:1

300 300 300 300 300 300 300 300 100 33

192 186 202 204 242 207 195 204 235

± ± ± ± ± – ± ± ± ±

30 min 3.2 2.3 5.9 2.0 4.6

751 522 194 201 244

2.3 3.5 2.2 2.9

201 194 213 234

± ± ± ± ± – ± ± ± ±

60 min 3.9 4.6 1.3 4.3 2.6

198 203 238

3.0 4.2 2.0 3.2

200 193 212 233

“–” indicates that the particle size was larger than 1000 nm and could not be accurately measured by the available instrument. 223

– – ± ± ± – ± ± ± ±

(μg/ml ± SD)

4.5 2.5 3.2 2.6 2.6 3.1 2.8

23.17 ± 0.56 17.15 ± 0.28 14.31 ± 0.73 17.05 ± 0.94 33.81 ± 2.01 7.01 ± 1.88 11.22 ± 0.94 14.13 ± 0.42 11.83 ± 1.07 11.40 ± 1.41

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mean and standard deviation values were reported.

in the solution, coating efficiency of solid dispersion (actual drug loading divided by theoretical drug loading), and its dissolution and apparent solubility were investigated and were selected as factors to assess the process and to evaluate the optimal operation conditions within the experimental ranges. In Table 1, the effect of temperature was tested at 15, 10 and 5 °C (samples 1 to 3 in Table 1). The effect of volume ratio of AS/S was determined at 10, 8 and 5 (samples 3 to 5 in Table 1). The effect of polymer types was investigated using VA64, HPMC and PVPK30 (samples 3, 6 and 7 in Table 1). The effect of different solvents was determined by using acetone-water (9:1) and acetone (samples 3 and 8 in Table 1). Finally, the effect of polymer amount in the antisolvent was investigated at VA64 concentrations of 33, 100 and 300 mg in 100 ml of water (samples 3, 9 and 10, respectively, in Table 1). Samples prepared using optimized parameters (samples 3 and 7) were subjected to SEM, XRD, DSC and FT-IR analysis.

2.4.5. Apparent solubility investigation The spray-dried sample powder, which was equivalent to containing approximately 8 mg of Td, was suspended in 50 ml of ultrapure water at 37 °C in a 100 ml conical flask. The flask was shaken (70 rpm) for 48 h in a water bath shaker at 37 ± 0.5 °C. At specified times of 5, 10, 20, 30, and 60 min and 2, 4, 24 and 48 h, 1 ml of suspension sample was taken and replaced by 1 ml of dissolution medium. The extracted solution was filtered through a 0.45 μm nylon syringe filter. The filtered solution was quantitatively diluted with acetonitrile (1:1, v/v) to prevent possible precipitation; then, the amount of dissolved Td was determined by the same procedure described in Section 2.4.1. The apparent solubility of each sample was determined in triplicate. 2.4.6. Scanning electron microscope (SEM) The antisolvent precipitated Td suspension described in Section 2.2 was dropped on a glass slide, dried with an infrared lamp, then sputtered with gold for 200 s at a pressure of < 0.5 mbar before analysis. Solid dispersion powder was deposited onto double-sided tape and then sputtered with gold. The microstructures of the examined samples were observed using a ZEISS EVO 18 electron microscope (ZEISS Co., Germany) at 5 to 10 kV and 10 mA.

2.4. Particle characterization 2.4.1. Quantification of the drug content The sample, containing approximately 5 mg of Td, was dissolved in 10 ml of acetonitrile and analysed by HPLC. The analysis was performed using an ACCELA UHPLC (Thermo Scientific Inc., San Jose, USA) and a Hypersil BDS C18 column (50 × 2.1 mm, 2.4 μm, Thermo Scientific, San Jose, USA) operating in a reverse phase (RP) system with an acetonitrile and water mobile phase (38:62 v/v) at a flow rate of 0.4 ml/min. The detection wavelength was 220 nm, and the injection volume was 5 μl. The run time and the retention time of Td were 2.99 and 1 min, respectively. Calibration curves of Td were prepared at 7 concentrations between 0.1 and 10 μg/ml (A = 837.373 + 61,884.7C, R2 = 0.9993).

2.4.7. X-ray powder diffraction (XRPD) XRPD patterns were recorded on an X-ray powder diffraction system (D8 ADVANCE, BRUKER, Germany) with Cu Kα radiation. Samples were examined over the most informative range of 2θ: 3–30°. The generator tension (voltage) and generator current were maintained at 40 kV and 30 mA, respectively. 2.4.8. Differential scanning calorimetry (DSC) Thermograms were obtained using a DSC-60 system (Shimadzu Co., Japan). Approximately 3 mg samples were hermetically sealed in aluminium pans and heated from 50 to 350 °C at 10 °C/min under a nitrogen atmosphere.

2.4.2. Particle size analysis After the antisolvent process, the suspension was stored at the same temperature as the process temperature for size analysis. Approximately 2 ml of the suspension was taken at a specific time (10, 30 and 60 min) and placed in a plastic cuvette to measure the particle size by a Zetasizer Nano ZS90 (Malvern, UK) at 25 °C. The measurements were performed in triplicate to determine the Z-average size of the particles.

2.4.9. Fourier transform infrared spectroscopy (FT-IR) FT-IR spectra were recorded using a Spectrum 100 FT-IR Spectrometer (PerkinElmer, USA). The raw data was processed by smoothing of the spectra and baseline correlation. Approximately 3 mg of sample and 100 mg potassium bromide were pulverized in a mortar and pressed into a disk. The FTIR measurements were performed in the scanning range of 4000–400 cm−1 at ambient temperature.

2.4.3. In vitro dissolution of tadalafil solid dispersions The tests were conducted following the CP (China Pharmacopoeia) Apparatus 2 (paddle) method using the ZRS-8G dissolution tester. The dissolution medium consisted of 1000 ml of 0.2% sodium dodecyl sulfate (SDS) aqueous solution. The prepared Td solid dispersions were weighed to be equivalent to 3 mg of Td. The stirring speed and temperature were set at 75 rpm and 37.0 ± 0.5 °C, respectively. At sampling times of 1, 3, 5, 10, 20, 30, 45 and 60 min, 1 ml aliquots were removed and replaced by 1 ml of dissolution medium. All solutions prepared for analysis were filtered through a 0.45 μm nylon membrane before analysis. The filtered solutions were quantitatively diluted with acetonitrile (1:1, v/v) to prevent possible precipitation. The content of Td in the filtrate was analysed by ultra-high-performance liquid chromatography (ACCELA, Thermo Scientific Inc., San Jose, USA), as described in Section 2.4.1. Each measurement was repeated three times, and the average was recorded.

2.5. Bioavailability study All animal experiments were approved by the Institutional Animal Care and Use Committee of Guangzhou University of Chinese Medicine (Guangzhou, China). Animal experiments were conducted following institution guidelines and were approved by the Ethics Review Committee for Animal Experimentation of the Guangzhou University of Chinese Medicine. Male Sprague-Dawley rats weighing 180–220 g were supplied by the Guangzhou University of Chinese Medicine Experimental Animal Centre (Guangzhou, China). In total, 24 rats were divided randomly into three groups of 8 rats each. The three groups of rats were given either raw Td or prepared product samples of 3 or 7 by gavage suspension (dispersed in water) at doses of 1.8 mg/kg (according to Td calculation), respectively. After ether anaesthesia, blood samples was taken from the posterior orbital venous plexus at 0.25, 0.5, 1, 2, 4, 8, 12 and 24 h, respectively. The samples were placed in centrifuge tubes containing heparin and centrifuged at 12000 rpm for 5 min. The plasma was separated and stored at −80 °C for further analysis. The plasma from the blood sample was then processed by adding 100 μl of each plasma sample, 10 μl of acetonitrile and 10 μl of 2.5 μg/

2.4.4. Equilibrium solubility investigation The Td equilibrium solubility in the antisolvent for samples 1 to 10 was measured at the same compositions in accordance with Table 1. An appropriate dose of tadalafil, polymer, solvent and antisolvent were added to a conical flask and subjected to ultrasound for 30 min. The conical flasks were maintained at the appropriate temperature, and the resulting suspensions were removed after 72 h for analysis by HPLC. The solubility of each sample was determined in triplicate, and the 224

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The following reasons may be responsible for these observations. Firstly, at low temperature, the drug solubility decreases. The solubility measurements confirmed that when the temperature changed from 15 to 5 °C, the equilibrium solubility decreased from 23 to 14 μg/ml, respectively. The metastability range became narrow; thus, it easily reached a high supersaturation when the solution was infused in the antisolvent (Zhang et al., 2006). Secondly, a lower temperature can inhibit particle growth (Cushing et al., 2004). And finally, temperature influences the crystal growth rate, which can be expressed as dl/ dt = Kg(Ci − C∗)b (Chen et al., 2004), where Kg is the crystal growth rate constant. The value of b was usually in the range of 1 to 3 and decreased as the temperature decreased. To restrict growth of the drug particle size, the optimal precipitation temperature was therefore set to 5 °C.

ml sulfamethoxazole (internal standard) in a 1.5 ml centrifuge tube and vibrated for 2 min using a vortex mixer. This mixture was then vortexmixed with 1 ml of ethyl acetate to precipitate the proteins and then centrifuged for 10 min at 12000 rpm. Then, 900 μl of the supernatant was removed and concentrated to dry under nitrogen; then, it was dissolved in the mobile phase (acetonitrile and ultrapure water containing 0.1% formic acid (30:70 v/v)) for analysis via ACCELA LC-MS (Thermo Scientific Inc., San Jose, USA). Pharmacokinetic parameters, including the time to reach the maximum plasma concentration (Tmax), half-life (t1/2), maximum plasma concentration (Cmax), and area under the curve (AUC0-∞), were calculated from the plasma-concentration time using PKsolver (V 2.0) software. 2.6. Statistical analysis

3.1.2. Effect of AS/S volume ratio on particle size The results are shown in Fig. 1 for samples 3, 4 and 5, which have AS/S volume ratios of 10, 8 and 5, respectively. As the AS/S volume ratio decreased from 10 to 5, the particle size of Td in the suspensions slightly increased from approximately 200 to 240 nm. Mitali Kakranet et al. reported that a decrease in the AS/S volume ratio will increase the aggregation between the particles during precipitation, once the nuclei are formed, simultaneous growth occurs. For subsequent growth, a high antisolvent-to-solvent ratio increases the diffusion distance for species growth; consequently diffusion becomes the limiting step for nuclei growth (Kakran et al., 2012).

The results were expressed as the mean ± standard deviation (SD) or mean ± standard error (SE). The statistical significance was determined by one-way and two-way analyses of variance, followed by the Tukey test (comparison of the solid dispersions, physical mixture and the raw tadalafil sample) using GraphPad Prism 5 (Graphpad Inc., USA). The difference in P values < 0.05 were considered significant. 3. Results and discussion As shown in Table 2, the coating efficiencies of all solid dispersion samples (3 to 12) are similar and > 85%, which indicates that the Td is highly dispersed in the polymer. The effect of each processing parameters on the aforementioned factors presented below.

3.1.3. Effect of the polymer on the Td particle size The theory of stabilizing a nano-suspension by simultaneous coprecipitation with a polymer has been outlined by Thorat and Dalvi (Thorat and Dalvi, 2012). According to their observations, the adsorption strength of the stabilizer on the drug surface depends on two factors: (a) the amount of stabilizer on the surface, which is inversely proportional to its solubility in the liquid phase, and (b) the stabilizerparticle interaction strength should be more than the stabilizer-solution interaction strength (Deshpande et al., 2015). The polymers, PVPK30, VA64 and HPMC K15M, were screened to stabilize the antisolvent precipitated Td. The effect of these polymers on the particle size is shown in Fig. 1 (samples 3, 6 and 7). For up to 1 h of standing, the particle size remained constant at approximately 200 nm in both PVPK30 and VA64 polymer-stabilized suspensions. The particle size in the HPMC K15M suspension increased to > 1 μm after 10 min of standing and could not be measured by the available instrument.

3.1. The effect of antisolvent process parameters on the drug size in the liquid suspension 3.1.1. Effect of temperature Temperature is a crucial factor for the solubility and crystallization rate (Zhang et al., 2017). Sizes of Td drug acquired in the liquid suspension are shown in Fig. 1. The effect of the precipitation temperature and on maintaining a constant temperature on the particle size was studied from 5 to 15 °C at 5 °C intervals (samples 1 to 3). As shown in Fig. 1, after precipitation at the three different temperatures, the particle sizes in the Td suspensions for samples 1 to 3 were found similar approximately 193 nm within the first 10 min. The particle sizes then substantially increased to 522 nm and 751 nm in the suspensions kept at 10 and 15 °C for 30 min respectively, and above 1000 nm at 60 min. On the contrary, the particle size remained unchanged up to 60 min for the suspension kept at 5 °C (sample 3). The result suggests that the process temperature plays a key role in controlling the particle size.

3.1.4. Effect of solvent on particle size The effect of the solvent used in the antisolvent process was

Table 2 Dissolution and apparent solubility results for the Td solid dispersion samples prepared under optimal antisolvent process conditions (the drug content, coating efficiency, dissolution and apparent solubility for various dissolution times). Sample

3 4 5 6 7 8 9 10 11# 12#

Drug content (%)

25 29 40 25 25 25 50 75 100 25

Coating efficiency (%)

95.8 95.6 86.6 85 95.4 85 90.4 96.7 100 97.6

Dissolution

Dissolution

Dissolution

In 1 min (% ± SD)

In 10 min (% ± SD)

In 60 min (% ± SD)

85.6 ± 0.6 c 55.3 ± 1.6 c 35.0 ± 1.2 c 3.7 ± 2.3 a 51.8 ± 7.3 c 45.9 ± 2.0 c 37.9 ± 2.0 c 11.3 ± 5.8 10.2 ± 1.6 15.6 ± 3.2

99.3 ± 3.5 c 77.9 ± 13.0 b 74.1 ± 7.7 c 19.3 ± 6.3 93.1 ± 2.3 c 88.3 ± 0.6 c 76.4 ± 0.5 c 62.1 ± 1.7 c 27.1 ± 2.0 33.9 ± 4.3

99.7 ± 4.8 c 97.8 ± 10.0 b 94.0 ± 5.4 b 63.2 ± 13.9 98.3 ± 8.9 b 95.3 ± 3.6 c 92.1 ± 2.9 c 80.2 ± 0.6 b 67.7 ± 2.7 66.6 ± 13.6

Apparent solubility max conc. (μg/ml ± SD)

Apparent solubility in 1 h conc. (μg/ml ± SD)

60.4 54.1 51.3 27.9 56.8 33.4 47.5 39.6 3.6 5.2

± ± ± ± ± ± ± ± ± ±

8.9 c 2.4 c 2.8 c 1.2 c 9.4 c 2.6 c 3.2 c 7.0 c 0.0 0.0

27.3 36.3 28.0 21.2 10.6 12.8 27.5 14.3 3.5 4.8

± 4.4 c ± 5.2 c ± 8.4 b ± 4.7 b ± 3.3 a ± 2.7 b ± 5.1 c ± 3.6 b ± 0.1 ± 0.2

# For comparison, sample 11: as-received Td, sample 12: physical mixture of Td and VA64 (drug content of 25%). aP < 0.05, bP < 0.01, cP < 0.001 compared to sample 11 (Td) at the same time. 225

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Fig. 1. Particle sizes for 10 samples dispersed in water at 10, 30 and 60 min after precipitation. Data for 30 and 60 min from sample 6 were not recorded due to being out of the instrumental range (n = 3).

produced from the acetone-water solution had a higher dissolution rate than the sample made using pure acetone. At 10 min, dissolution reached 88.3% for acetone and 99.3% for acetone-water.

investigated by selecting either acetone or an acetone-water (9:1) solution as the solvent. The results are shown in Fig. 1 (samples 3 and 8). The Td suspension particle size was similar when the solvent changed from acetone-water (9:1) to acetone.

3.2.4. Effect of VA64 amount on in vitro dissolution The effect of VA64 amount on the dissolution of the produced solid dispersion was investigated. Three VA64 concentrations in the antisolvent solution (300, 100 and 33 mg/100 ml) were used to prepare Td solid dispersions (samples 3, 9 and 10) to produce polymer-drug composition ratios of 3:1, 1:1 and 1:3, which when converted to drug loadings, these equated to 25, 50, and 75%, respectively. The results of these dissolution tests are shown in Fig. 2(d). Clearly, the dissolution rate increases with an increase in polymer amount and gave dissolved drug percentages of 99.3, 76.4 and 62.1% for 25, 50, and 75% drug contents, respectively, in 10 min. The results suggest that introducing and increasing the wettability of the component during drug formation led to an increase in the drug's dissolution rate.

3.1.5. Effect of VA64 amount on particle size Three VA64 concentrations in the antisolvent solution (300, 100 and 33 mg/100 ml) were used to prepare the Td solid dispersions. The effect of VA64 amount on the precipitated particle size is shown in Fig. 1 (samples 3, 9 and 10). As the polymer dosage decreased, the drug particle size slightly increased from 200 nm to 230 nm. The experimental results indicate that within the tested range, VA64 could effectively coat the drug to inhibit its growth (Zu et al., 2014). 3.2. Effect of process parameters on the in vitro dissolution of the solid dispersion products For poorly soluble drugs, dissolution is usually the rate-limiting step of drug absorption. When in vitro drug dissolution is fast, it can achieve high concentrations in the gastrointestinal fluid and then easily permeate through the membrane. The in vitro dissolution results for the solid dispersions and two untreated API samples (for the purpose of comparison) are shown in Table 2. The effect of the process parameters on the in vitro dissolution are shown in Fig. 2 and discussed in the following section.

3.3. Effect of the process parameters on the equilibrium solubility and apparent solubility of the solid dispersions The results of the equilibrium solubility tests are shown in Table 1. The results of the apparent solubility tests are shown in Table 2 and Fig. 3. The effects of the individual process parameters on the equilibrium and apparent solubilities are discussed in the following sections.

3.2.1. Effect of the antisolvent-to-solvent volume ratio (AS/S) on in vitro dissolution The results shown in Fig. 2(a) show that for the three antisolvent-tosolvent volume ratios of 10, 8 and 5 (samples 3, 4 and 5 in Table 2), the Td solid dispersions with the higher AS/S volume ratios led to more rapid dissolution rates. All processed samples had higher dissolution rates than the as-received API. At 1 min, the dissolution for AS/S volume ratios of 10, 8 and 5 reached 85.6%, 55.3% and 35.0%, respectively, whereas the as-received API reached only 10.2%.

3.3.1. Effect of AS/S volume ratio on the equilibrium solubility and apparent solubility The effect of AS/S volume ratio on the equilibrium solubility is shown in Table 1. Upon increasing the AS/S volume ratio, the equilibrium solubility increased, which is not conducive to drug nucleation. The effect of the AS/S volume ratio on the apparent solubility is presented in Fig. 3(a). The apparent solubility Td concentrations of different samples produced from AS/S volume ratios of 10, 8 and 5 at 10 min were 60.4, 54.1 and 51.3 μg/ml, respectively. Thus, an increase of the AS/S volume ratio decreased the amount of ultrafine particles per unit volume of liquid and reduced the collision and agglomeration between particles in the suspension, which are both conducive for small particle formation (Kakran et al., 2012) and better apparent solubility of the solid dispersion. According to the results for the particle size, dissolution and apparent solubilities, the optimal ratio of antisolventto-solvent is therefore 10.

3.2.2. Effect of polymer type on in vitro dissolution The effect of polymer on in vitro dissolution is shown in Fig. 2(b). Dissolution profiles for samples 3 (VA64) and 7 (PVPK30) exhibited an increase in dissolution rate; however, sample 6 (HPMC-K15M) showed a decrease compared with the as-received Td. The results show that the polymer type affected the dissolution of the end product. 3.2.3. Effect of solvent type on in vitro dissolution Pure acetone and an acetone solution of 9:1 v/v acetone-water were used as solvents in the antisolvent precipitation. The effects of the two solvents on in vitro dissolution are shown in Fig. 2(c). The sample

3.3.2. Effect of polymer type on the equilibrium solubility and apparent solubility The effect of polymer type on the Td equilibrium solubility is shown 226

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Fig. 2. Effect of factors on in vitro dissolution. (a) AS/S volume ratio; (b) polymer type; (c) solvent type; (d) polymer dosage. Values represent mean tadalafil dissolution ± SD (n = 3).

Fig. 3. Effect of each factor on the apparent solubility. (a) volume ratio of AS/S; (b) polymer type; (c) solvent; (d) polymer dosage. Values represent mean tadalafil concentration ± SD (n = 3). 227

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acetone, the max apparent solubility of the solid dispersions produced by the two solvents were 60.4 and 33.4 μg/ml, respectively. Although there were no significant differences in particle size (Section 3.1.4) and dissolution (Section 3.2.3), the apparent solubility of acetone-water (9:1) was relatively larger than that of acetone.

in Table 1. The equilibrium solubility of HPMC K15M (sample 6) was the lowest for all three polymers, which implies that its ability to improve Td solubility is not sufficient. The solubilization of PVPK30 (sample 7) and VA64 (sample 3) were both higher than sample 6; however, they had similar equilibrium solubilities. The effect of polymer on the apparent solubility is presented in Fig. 3(b). The results indicate that the apparent solubility increased for all Td solid dispersions, especially within the first 10 min. Clearly, PVPK30 and VA64 had different impacts on the level and duration of drug supersaturation in the apparent solubility test. The Td solid dispersion with VA64 exhibited the greatest (22-fold compared with the API) increase in Td apparent solubility (60.4 μg/ml) at 10 min, whereas Td solid dispersions with PVPK30 and HPMC-K15 M had values of 56.8 and 22.6 μg/ml, respectively. Similar solubility studies were conducted on the as-received Td and its physical mixture with VA64 (Td-VA64PM, drug loading is 25%). The physical mixture increased the Td concentration to 4–5 μg/ml (not shown in the Figure); however, this was still close to the intrinsic solubility (3.21 μg/ml (Park et al., 2014)) of the as-received Td and much lower than the corresponding solid dispersions. The drug on the polymer surface and the amorphous nature of the nanoparticle led to a rapid release, which caused the apparent solubility curve to reach a peak at 10 min and presents a “Spring and Parachute” effect as shown in Fig. 3(b). This is a phenomenon of amorphous solid dispersions, which caused by the amorphous drug very quickly dispersing in a liquid to achieve a peak concentration of supersaturation. Then, the drug begins to re-crystallize, and the concentration steeply decreases (spring). The polymer can prevent or delay the re-crystallization process and maintain a high concentration to produce a flat curve (parachute) (Babu and Nangia, 2011). After 1 h, possibly due to re-crystallization, the solubility decreased. The concentration decline of the Td-VA64 solid dispersion compared to TdPVPK30 was relatively slow, indicating that the VA64 polymer is better than PVPK30 at inhibiting the amorphous-to-crystalline phase transformation. Amorphization is an approach that can be used to change the crystalline drug to amorphous. The rationale behind this approach can be understood by the following equation. Amorphization is an approach that can be used to change the crystalline drug to amorphous. The rationale behind this approach can be understood by the following equation.

3.3.4. Effect of VA64 amount on the equilibrium solubility and apparent solubility The Td equilibrium solubility decreased from 14 μg/ml to 11 μg/ml when the amount of VA64 decreased from 300 mg to 33 mg. The solubilization slightly decreased when using less polymer. The effect of VA64 amount on the apparent solubility of Td is presented in Fig. 3(d). An increase in drug loading or a decrease in amount of the VA64 polymer resulted in a decrease in apparent solubility from 60.4 μg/ml (for 25% loading) to 47.5 μg/ml (for 50% loading) and then to 39.6 μg/ml (for 75% loading) at 10 min. The physical mixture of VA64 and as-received Td did not result in a significant increase in Td concentration in the apparent solubility test. A drug loading of 25% exhibited a higher dissolution and apparent solubility than the others. Further reduction of the drug loading was not conducive to the production process and will increase the required pill dosage and reduce patient compliance. Thus, 300 mg of VA64 in 100 ml of water was chosen as the polymer amount. 3.4. SEM morphology SEM images of the as-received Td and processed Td samples are shown in Fig. 4. Fig. 4(a) and (b) show the as-received Td, which appear as needle-like irregular-shaped crystals that are large in size. Fig. 4(c) shows particles in a liquid suspension obtained from sample 3, which was immediately dried using an infrared lamp after the anti-solvent precipitation process. The suspensions had a particle size of approximately 200 nm, and the particles had a high density. After spray drying, a micron-sized and shrivelled spherical structure for the Td solid dispersion was observed as shown in Fig. 4(d). The particle size of the solid dispersion was approximately 5–10 μm, which was attributed to agglomeration during spray drying. Similar samples for the liquid suspension and spray-dried powder prepared from sample 7 are shown in Fig. 4(e) and (f). 3.5. X-ray power diffraction (XRPD)

Amorphous

σ ∆GTo Amorphous, Crystalline = −RTln ⎜⎛ TCrystalline ⎞⎟. Here, ΔGTo Amorphous, σT ⎠ ⎝ Crystalline is the energy difference between the crystalline and the amorphous state, R is the gas constant, T is the absolute temperature of

concern and

σTAmorphous σTCrystalline

The XRPD patterns for the Td solid dispersions prepared from different samples are displayed in Fig. 5. The X-ray diffraction pattern of the as-received Td showed a sharp peak at 2θ values of 7.20, 14.46, 18.43 and 21.66°, which are diagnostic peaks for the crystalline nature of Td (Vyas et al., 2009). The results for the processed Td samples showed no obvious peaks but a broad diffraction pattern for all the dispersions. These results confirmed that the processed Td solid dispersions are amorphous materials. It is known that transforming the physical state of a drug to amorphous will lead to a high-energy state and to a high disorder, resulting in an enhanced dissolution rate (Zhang et al., 2009).

is the solubility ratio of the two forms. It follows

from above equation that the amorphous form has a higher theoretical solubility compared with the crystalline form due to its excess thermodynamic properties. The amorphous state requires no energy to break the crystal lattice structure; thus, the drug molecules can interact with solvent molecules via intermolecular interactions and become soluble. Therefore, the polymer acts as a solubilizer to increase the saturation solubility of the drug. The polymer also as a stabilizer that is absorbed on the surface of the drug, resulting in the drug being difficult to aggregate to form a crystal precipitate. Additionally, the polymer can form hydrogen bonds with the drug, which has a delayed effect on drug nucleation.

3.6. Differential scanning calorimetry (DSC) As presented in Fig. 6, Td sample shows a strong and sharp melting point peaks at approximately 290 °C, whilst sample 3 has a weak endothermic peak at 300 °C, and sample 7 had a crystallization peak at 200 °C, with no endothermic peak. DSC data confirms that both samples 3 and 7 possess an amorphous nature.

3.3.3. Effect of solvent type on the equilibrium solubility and apparent solubility The effect of solvent type on the Td equilibrium solubility is shown in Table 1. The equilibrium solubility of acetone-water (9:1) and acetone are similar because the solvent system only increased by 1 ml of acetone in 110 ml of total volume. The effect of solvent type on the apparent solubility is shown in Fig. 3(c). For the two samples produced using acetone-water (9:1) and

3.7. Fourier transform infrared spectroscopy (FT-IR) FTIR spectra of Td and other solid dispersions studied are shown in Fig. 7. The solid dispersions broadly shifted the absorption band of API 228

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Fig. 4. SEM images of Td API, Td liquid suspension and spray-dried powder. Images: (a): untreated Td API, 1000× and (b): 5000×. (c): Td particles in liquid suspension (sample 3 in Table 2, 10,000×. (d): Td spray-dried powder (sample 3 in Table 2) from antisolvent precipitation, 5000×. (e): Td particles in liquid suspension (sample 7 in Table 2, 10,000×. (f): Td spray-dried powder (sample 7 in Table 2) made via antisolvent precipitation and then spray drying, 5000×.

Fig. 5. X-ray diffraction patterns of the API and samples 3 to 10. Fig. 6. DSC thermograms of the API, sample 3 (VA64) and sample 7 (PVPK30).

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of the Td solid dispersions was also higher than that of the as-received drug group. The Tmax is slightly later that of the as-received Td. The drug concentration for the Td solid dispersions and the as-received Td in the plasma of rats reached maximum values of 84 ng/ml (sample 7), 102 ng/ml (sample 3) and 58 ng/ml (as-received Td), respectively. The Cmax values for the Td solid dispersions were higher than that of the asreceived drug (Table 3) because of the enhanced solubility and faster dissolution rate of Td from the Td solid dispersions. The AUC0-∞ values for samples 7 and 3 were 3.4 and 3.7 times higher than that of the asreceived Td, suggesting that good absorption occurred after the faster dissolution of Td from the solid dispersion in the gastrointestinal tract. The plasma concentrations from the Td solid dispersion group tests were maintained at a high level for a long time, resulting in an improvement in the bioavailability (Fig. 8). The result shows that the bioavailability of sample 3 was the best; its relative bioavailability to tadalafil API reached 3.7, which obviously benefited from the higher dissolution rate and solubility of the processed Td over that of the asreceived API in the gastrointestinal fluid.

Fig. 7. FT-IR spectra of the API, sample 3 (VA64) and sample 7 (PVPK30).

4. Conclusion The key parameters for an antisolvent process producing Td solid dispersion powders were investigated. The studied process can enhance the aqueous solubility of Td and its oral bioavailability by changing its crystal form and producing nano-particles. Using a single factor design of a 10 mg/ml Td solution, within the test range, the optimal antisolvent precipitation operating conditions were as follows: solvent, acetone-water (9:1) mixture; precipitation temperature, 5 °C; antisolvent, polymer water solution; polymer, VA64; volume ratio of antisolvent-to-solvent, 10; and polymer dosage, 300 mg/100 ml. The XRPD, DSC and FT-IR results indicated that the Td solid dispersions were amorphous. The dissolution and apparent solubility tests showed that the Td solid dispersions in vitro exhibited enhanced dissolution rates and solubilities compared with the as-received Td. The oral bioavailability of the Td solid dispersion improved significantly compared with that of the as-received Td. This work provides further understanding of the physico-chemical parameters that can influence anti-solvent precipitation of particles and their effect on dissolution and solubility. Without using mechanical size reduction, the antisolvent precipitation and spray-drying technique in the presence of protective hydrophilic polymers prepared a drug powder with a considerably enhanced drug dissolution. This technique offers a relatively easy way to produce nano-sized drugs that have a high dissolution and solubility.

Fig. 8. The plasma concentrations of as-received Td and the Td solid dispersions (n = 8). Values are shown as the means ± SE.

at 3326 cm−1 to 3316 cm−1, which corresponded to the Td secondary amine stretching vibration. Additionally, the signal for the carbonyl group at 1665 cm−1 was distorted, suggesting the presence of hydrogen bonds between both groups (Wlodarski et al., 2015). A shift in the stretching vibrations at 2910 cm−1 and 1851 cm−1 also indicated the formation of an amorphous solid dispersion. 3.8. Bioavailability analysis

Acknowledgements After the Td solid dispersion properties were investigated, the samples from tests 3 and 7, which gave better dissolutions, apparent solubilities and different crystallization inhibition effect, were selected for the bioavailability study. Fig. 8 shows the average plasma concentration-time profiles, and the pharmacokinetic parameters are summarized in Table 3. The relative bioavailability value was determined by dividing the AUC value of the prepared solid dispersion by the AUC of the API. The absorption of Td from the solid dispersions was faster than that of the as-received Td in vivo, and the drug concentration

The authors acknowledge financial support from the Science Program for Overseas Scholars of Guangzhou University of Chinese Medicine (Torch program), the Guangdong Science and Technology Program (2017ZC0140; 2017ZC0157), the School of Chemical Engineering, University of Birmingham, and the School of Pharmaceutical Sciences, Guangzhou University of Chinese Medicine. The SEM and XRPD used in this research were obtained at South China University of Technology. The authors would like to thank Prof.

Table 3 Pharmacokinetic parameters of as-received Td and the Td solid dispersions (n = 8). Values are shown as the means ± SE. a P < 0.05, bP < 0.01 compared to Td. Pharmacokinetic parameters

Td

Sample 7 (PVPK30)

Sample 3 (VA64)

t1/2 (h) Tmax (h) Cmax (ng/ml) AUC0-∞ (ng/ml * h) Relative bioavailability

4.47 ± 0.35 2.00 ± 0.45 58.00 ± 6.57 503.28 ± 80.30 1

8.38 ± 2.93 2.12 ± 0.33 83.65 ± 7.06 1695.31 ± 108.42 3.369

7.50 ± 1.06 2.31 ± 0.28 102.32 ± 8.65 1861.62 ± 225.24 3.699

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b

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Jianfeng Hu, Dr. Yan Cai, and Dr. Qian Sun of South China University of Technology for providing technical guidance with SEM and XRPD. We also acknowledge Dr. Qiuping Guo of Guangzhou General Pharmaceutical Institute for providing experimental guidance.

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