Preparation of apigenin nanocrystals using supercritical antisolvent process for dissolution and bioavailability enhancement

European Journal of Pharmaceutical Sciences 48 (2013) 740–747

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Preparation of apigenin nanocrystals using supercritical antisolvent process for dissolution and bioavailability enhancement Jianjun Zhang a, Yanting Huang a, Dapeng Liu a, Yuan Gao b,⇑, Shuai Qian c,⇑ a

School of Pharmacy, China Pharmaceutical University, Nanjing 210009, PR China School of Traditional Chinese Medicine, China Pharmaceutical University, Nanjing 210009, PR China c School of Pharmacy, The Chinese University of Hong Kong, Shatin, N.T., Hong Kong, PR China b

a r t i c l e

i n f o

Article history: Received 12 October 2012 Received in revised form 22 December 2012 Accepted 23 December 2012 Available online 7 January 2013 Keywords: Apigenin Nanocrystals Supercritical antisolvent process Dissolution Bioavailability

a b s t r a c t The aim of the study was to investigate the potential of nanocrystals to enhance the oral bioavailability of apigenin (AP), a bioactive flavonoid with various pharmacological activities but poor aqueous solubility. In the present investigation, the AP nanocrystals were prepared by the supercritical antisolvent process. In vitro characterization was performed by scanning electron microscopy, FT-IR, differential scanning calorimetry, X-ray powder diffractometry. In vitro dissolution of prepared nanocrystals was studied and compared with untreated coarse powder. In addition, the pharmacokinetic study of AP nanocrystals, in comparison to coarse powder, was also performed in rats after a single oral dose. The prepared AP nanocrystals, without change in crystalline structure, appeared in spherical shape with particle size of about 400– 800 nm. The reduction of particle size resulted in a more rapid dissolution of AP from nanocrystals than from coarse powder. In comparison to coarse powder, AP nanocrystals exhibited a significantly decreased tmax, a 3.6-fold higher peak plasma concentration (Cmax) and 3.4-fold higher area under the curve (AUC). Ó 2013 Elsevier B.V. All rights reserved.

1. Introduction Nowadays, nanotechnology has been employed in drug delivery field for solubilization and dissolution rate enhancement of poorly soluble drugs, and tipped as the next revolutionary step forward. Several drug delivery systems based on nanotechnology have been developed, for example, nanomicelles (Wang and Grayson, 2012), nanoemulsion (Ammar et al., 2009), polymeric nanoparticles (Liu et al., 2008; Patel et al., 2012), solid lipid nanoparticles (Blasi et al., 2007; Manjunath et al., 2005), nanogel (Chacko et al., 2012), and nanosuspensions (Rabinow, 2004). Different from other nanocarriers, the nanosuspension is a carrier-free sub-micron colloidal dispersion of pure drug particles in crystalline state (Mauludin et al., 2009) or amorphous state (Gao et al., 2010), which are stabilized by a minimum amount of surfactants. When the drug particles exist in crystalline state, such nanosuspension could be also called as nanocrystals. The pharmaceutical benefits of nanocrystals include improvement in saturation solubility, dissolution rate, oral bioavailability as well as reproducibility of oral absorption (Rabinow, 2004; Zhang et al., 2011).

⇑ Corresponding authors. Address: School of Traditional Chinese Medicine, China Pharmaceutical University, Nanjing 210009, PR China (Y. Gao), School of Pharmacy, The Chinese University of Hong Kong, Shatin, N.T., Hong Kong, PR China (S.Qian). Tel./fax: +86 25 83379418. E-mail addresses: [email protected] (Y. Gao), [email protected] (S. Qian). 0928-0987/$ - see front matter Ó 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ejps.2012.12.026

Two basic approaches are involved in production of nanocrystals, the top-down process (size reduction of large particles into nanosized particles) and the bottom-up process (growth of small particles from individual molecules). Various production techniques have been developed based on these two basic approaches, including wet milling (Liversidge and Conzentino, 1995), high pressure homogenization (Müller and Peters, 1998), microfluidization (Verma et al., 2009), controlled precipitation (Auweter et al., 1998), hydrosols technique (List and Sucker, 1988), controlled crystallization during freeze-drying (CCDF) (de Waard et al., 2008) and supercritical fluid technologies (Jung and Perrut, 2001). Supercritical antisolvent (SAS) process, as one kind of supercritical fluid technologies, is a new recrystallization technology developed in the recent years. In this process, a drug solution in organic solvent is injected into the supercritical fluid (usually supercritical CO2, acting as an antisolvent) and the solvent gets extracted by the supercritical fluid, thereby the drug solution gets supersaturated and consequently precipitates the drug as fine crystals. Nanoparticles of several drugs with poor aqueous solubilities (e.g. sirolimus (Kim et al., 2011), 10-hydroxycamptothecin (Zhao et al., 2011), and griseofulvin (Chattopadhyay and Gupta, 2001)) were successfully prepared by such SAS process. Apigenin (AP), 5,7-dihydroxy-2-(4-hydroxyphenyl)-4H-1ben-zopyran-4-one (Fig. 1), is one of the active ingredients found naturally in a variety of fruits, plants and vegetables, which is usually marketed as diet supplements (Peterson and Dwyer, 1998).

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carboxymethyl cellulose (CMC-Na), pentobarbital sodium and dimethyl sulfoxide (DMSO, purity P 99.5%) and polysorbate 80 were purchased from Guoyao Chemical Co., Ltd. (Shanghai, China). Heparin sodium salt (180 IU/mg) was purchased from Sigma Chemical Co., Ltd. (St. Louis, USA). HPLC grade water was produced by a Milli-Q water purification system (Millipore Co. Ltd., Bedford, USA). Other chemicals were of HPLC or analytical grade. Fig. 1. Chemical structure of apigenin (molecular weight: 270.24).

2.2. Preparation of AP nanocrystals Many pharmacological activities of AP have been recognized, including anti-inflammatory effects (Funakoshi-Tago et al., 2011), free radical scavenging effects (Horvathova et al., 2003), growth inhibitory properties in several cancer lines (Tong et al., 2007; Wang et al., 2000; Yin et al., 2001) and so on. In our previous study, AP was classified as BCS class II drug with poor aqueous solubility and high permeability in the whole intestine (Zhang et al., 2012). For such hydrophobic compound, poor solubility would result in a slow dissolution and may create delivery problems such as low oral bioavailability and erratic absorption. So far, several formulation strategies for AP have been investigated, including self-microemulsion (Zhao et al., in press), liposome (Arsic et al., 2011), and nanocrystals fabricated by high pressure homogenization (Al Shaal et al., 2010, 2011). In vitro performances including solubility and dissolution as well as in vitro activity of AP (Al Shaal et al., 2011) have been significantly enhanced by these formulation techniques, however, there is no report about the in vivo pharmacokinetic performance of AP in the nano-drug delivery systems till now. In the present study, we attempted to improve dissolution rate and increase the bioavailability of AP by preparing nanocrystals via SAS process. The physiochemical and pharmacokinetic properties of prepared AP nanocrystals were to be investigated in comparison to untreated AP coarse powder. 2. Materials and methods

A schematic diagram of SAS equipment used in this study is depicted in Fig. 2. The SAS process was performed using the experimental equipment as previously described (Shikhar et al., 2011; Tien et al., 2010; Zhao et al., 2010; Zordi et al., 2010). Firstly, AP coarse powder was dissolved in DMSO (20 mg/ml). Secondly, CO2 from the storage tank (1) was liquefied by the low temperature thermostat bath (2) and compressed by the high pressure pump (3). After preheating by CO2 preheater (4), CO2 was delivered to the crystallization vessel (7) through metal filter screen (8). After the pressure and temperature had equilibrated under the supercritical conditions of CO2 (14.5 MPa, 35 °C), the prepared AP DMSO solution was pumped into the crystallization vessel (7) by means of the high pressure infusion pump (6) at the rate of 0.5 ml/min. The organic solvent DMSO was recovered in solvent recovery kettle (10) after pressure reduction and temperature cooling down, while CO2 was exhausted out from the system through rotameter (11), the outlet flow rate of CO2 was adjusted to 4.0 l/min by fine-tuning valve (9). The AP nanocrystals were prepared under such dynamic equilibrium conditions. At washing step, additional supercritical CO2 continued to flow into the crystallization vessel (7) to wash out the residual organic solvent. After that, the crystallization vessel (7) was slowly depressurized to atmospheric pressure. Finally, the generated particles were collected from both the inner wall and the metal frit (mean pore diameter 0.5 lm) of the crystallization vessel for further analysis.

2.1. Materials 2.3. Scanning electron microscopy (SEM) Apigenin (purity > 99.0% by HPLC) was provided by Nanjing Zelang Medical Technology Co., Ltd. (Nanjing, China). Silybin standard was purchased from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). Sodium

Morphological evaluation of the AP nanocrystals was performed by scanning electron microscope (SEM) (Hitachi X650, Tokyo, Japan). All samples were examined on a brass stub using carbon

6

5

P

P1

2 9

11

7 3 10 8 1 4 Fig. 2. The equipment diagram of supercritical CO2 antisolvent process. 1 – CO2 storage tank, 2 – low temperature thermostat bath, 3 – high pressure pump, 4 – CO2 preheater, 5 – liquid storage tank, 6 – high pressure infusion pump, 7 – crystallization vessel, 8 – metal filter screen, 9 – fine-tuning valve, 10 – solvent recovery kettle, and 11 – rotameter.

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double-sided tape. Powder samples were glued and mounted on metal sample plates. The samples were gold coated (thickness  15–20 nm) with a sputter coater (Fison Instruments, UK) using an electrical potential of 2.0 kV at 25 mA for 10 min. An excitation voltage of 20 kV was used in the experiments. 2.4. X-ray powder diffraction (XRPD) X-ray powder diffraction patterns of the AP nanocrystals were investigated using a Bruker D8 Advance X-ray powder diffractometer (Karlsruhe, Germany) with Cu Ka radiation (1.5406 Å). The samples were gently consolidated in an aluminum holder and scanned at 40 kV and 30 mA from 5° to 45° 2h using a scanning speed of 2 °/min with a step size of 0.05°. 2.5. Differential scanning calorimetry (DSC) The status of crystallinity of the AP nanocrystals was characterized with DSC on a Netzsch DSC 204 F1 Phoenix Differential Scanning Calorimeter (Germany) which was calibrated for temperature and cell constants using indium. Samples were placed on non-hermetic aluminum pans. The sample cell was equilibrated at 25 °C and then heated at a rate of 20 °C/min in a range of 30–500 °C. Data analysis was performed using NETZSCH-Proteus software (version 4.2). 2.6. FT-IR analysis A thermo Nicolet Impact 410 FT-IR Spectrophotometer (Thermo Fisher Scientific Inc., Waltham, MA, USA) was employed in KBr diffuse reflectance mode for recording the IR spectra of AP coarse powder and SAS prepared AP nanocrystals. 2 mg of each sample was mixed with 200 mg KBr and compressed into tablets. A total of 64 scans were performed (with a spectral resolution of 4 cm 1) over the range of 490–4000 cm 1. Data were collected and analyzed by Nicolet Omnic software (version 8.0). 2.7. In vitro dissolution studies Ten milligram AP coarse powder and 10 mg AP nanocrystals were accurately weighed and put into the capsule (gelatin shell size #2), respectively. In vitro dissolution profiles of AP from capsules containing its coarse powder or prepared nanocrystals were generated using the USP II dissolution apparatus (Sotax A7 Dissolution Apparatus: Sotax Ltd, London, UK) with the rotary basket rotating at 100 rpm. 0.1 M hydrochloric acid (HCl), 0.1 M phosphate buffer (pH 6.8) with/without 0.5% Polysorbate 80, and 0.1 M phosphate buffer (pH 7.4) were employed as the dissolution medium (37 °C, V = 900 ml). Samples (5 ml) were collected and filtered through a membrane filter (0.22 lm) for analysis at predetermined time points (5, 10, 15, 20, 30, 45, 60, and 120 min). The filtered samples were analyzed by a validated HPLC/UV method we reported previously (Zhang et al., 2012). 2.8. In vivo absorption study 2.8.1. Animals Male Sprague–Dawley rats (230–270 g) were obtained from the Laboratory Animal Center, China Pharmaceutical University (Nanjing, China). Animals were housed in standard cages on a 12 h light–dark cycles, fed with standard animal chow and tap water daily. All animals used in this study were handled in accordance with the guidelines of the Principles of Laboratory Animal Care (State Council, revised 1988). The study was approved by the Ethical Committee of China Pharmaceutical University.

One day before the pharmacokinetic study, each animal was operated with a cannula insert into the right jugular vein under anesthesia by intraperitoneal injection of pentobarbital sodium (50 mg/kg). A surgical incision was made on the ventral side of the neck of rats to expose the jugular vein. The jugular vein was then cannulated with a polyethylene tubing (0.5 mm ID, 1 mm OD, Portex Ltd., Hythe, Kent, England) that was led under the skin and exteriorized at the back of the neck for blood sampling. 50 IU/ ml of heparin sodium in normal saline was filled into the catheter to prevent the blood clotting. After the exposed areas were surgically sutured, the rats were placed individually in standard cages. The animals were allowed to recover for 24 h and were fasted overnight prior to administration (Qian et al., 2012). 2.8.2. Drug administration Before gavage administration to the rats, AP coarse powder and the prepared AP nanocrystals were dispersed homogeneously in 0.5% CMC-Na aqueous solution to form suspensions. To prepare AP intravenous injection of 0.8 mg/ml, 8 mg AP was dissolved in 1 ml ethanol and diluted to 10 ml using 0.5% polysorbate 80 aqueous solution. Rats were randomly divided into three groups (six rats in each group) and administrated orally with fresh prepared suspensions at dose of 50 mg/kg as AP or tail vein injection at dose of 2 mg/kg. Around 200 ll of blood was collected from the jugular vein into heparinized centrifuge tube at predetermined time points (20, 40, 60, 90, 120, 180, 240, 300, 360, 420, 540, and 720 min for oral formulations and 5, 10, 15, 30, 45, 60, 90, 120, 180, and 240 min for intravenous AP) post dosing. Plasma was immediately separated by centrifugation of blood at 10,000g for 5 min (refrigerated centrifuge, Sigma 1–15 K, Sigma, Germany) and stored at 20 °C until analysis. Rats were allowed for free access to food and water 4 h after drug administration. 2.8.3. Analysis of AP in rat plasma cIn this study, a modified HPLC/UV method was employed to determine the concentration of AP in rat plasma using a reversed phase HPLC (Shimadzu LC 10AD, Shimadzu Corporation, Kyoto, Japan) (Lou et al., 2009). Chromatographic separation of AP was performed on a C18 column (Kromasil ODS column, 150 mm  4.6 mm, 5 lm) guarded with a C18 precolumn (Shimadzu). Mobile phase consisting of methanol, acetonitrile and 0.1% phosphoric acid aqueous solution in a volume ratio of 32/18/50 was run at 1.0 ml/min and monitored at 337 nm with the column temperature at 30 °C. Frozen plasma samples were thawed at room temperature just before sample preparation. 200 ll of rat plasma was mixed with 600 ll of silybin solution (internal standard, 13.3 lg/ml in methanol) and vortex-mixed for 5 min. After centrifugation for 5 min (10 °C, 10,000g), the supernatant was collected. The rest of residue was extracted with another 300 ll of methanol by vortex-mixing for 5 min and centrifuged for 5 min (10 °C, 10,000g) again, and the obtained supernatant was pooled together with the previous supernatant. Then, the total supernatant was dried under a nitrogen flow at 35 °C and the obtained residues were reconstituted with 200 ll of methanol. After centrifugation for another 5 min (10 °C, 10,000g), an aliquot of 20 ll supernatant was injected into the HPLC system for analysis. 2.9. Calculation of pharmacokinetic parameters Pharmacokinetic parameters were calculated using DAS 2.0 software (issued by the State Food and Drug Administration of China for pharmacokinetic study) employing a non-compartmental model approach. The maximum plasma concentration (Cmax) and the time to reach Cmax (tmax) were directly obtained from plasma data. The area under the plasma concentration–time curve (AUC0 t) was calculated using the trapezoidal method.

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nanocrystals. The particle size was determined 562.5 ± 56 nm with a PI value of 0.92 ± 0.21.

2.10. Statistical analysis Statistical data analyses were performed using one-way analysis of variance (ANOVA) with p < 0.05 as the criterion of significance. 3. Results and discussion 3.1. Scanning electron microscopy (SEM) Morphology assessment of particles using SEM helped to exhibit the morphological changes of AP after SAS process. As seen from Fig. 3, SAS process obviously modified the shape and size of AP. The AP coarse powder showed irregular spherical shape (Fig. 3a). A laser diffraction instrument (Malvern Mastersizer 2000, Malvern Instruments, Malvern, UK) was utilized for measurement of the particle size and distribution after dispersing the AP coarse powder in water. The AP coarse powder had the volume mean diameter (D[4, 3]) of 19.184 lm with a span of 3.005 (d(0.1) = 2.691 lm, d(0.5) = 13.733 lm and d(0.9) = 43.957 lm). After the SAS process, the AP nanocrystals presented the regular spherical shape with smooth surfaces and the particle size of about 400–800 nm (Fig. 3b). Photon correlation spectroscopy (Zetasizer 3000HSA, Malvern Instruments Ltd., Malvern, UK) was used to measure the particle size of AP nanocrystals. PCS yields the volume weighted mean particle size and the polydispersity index (PI) of the redispersed

to

be

3.2. X-ray powder diffractometry (XRPD) XRPD analysis was performed to analyze whether any potential changes happened in the inner structure of the AP nanocrystals in comparison to the AP coarse powder. The XRPD patterns for the AP coarse powder and the AP nanocrystals are presented in Fig. 4. It can be observed that the characteristic peaks in X-ray diffraction pattern of the AP nanocrystals are the same as that of the AP, indicating the same crystalline modification. The identical 2h peaks at 7.12°, 10.21°, 11.52°, 14.34°, 15.03°, 16.58°, and 17.86° appeared at both XRPD. However, the degree of crystallinity, representing as intensity in XRPD diagram, was significantly decreased after preparation of AP nanocrystals by SAS process. This could be attributed to so-called ‘‘particle size broadening’’ phenomenon in the XRPD analysis of crystalline materials less than 1 lm (Jenkins and Snyder, 1996) and the partially amorphous property of the AP nanocrystals. 3.3. Differential scanning calorimetry (DSC) DSC curves of the AP coarse powder and the AP nanocrystals were studied to determine the potential change in the crystalline state of AP after the SAS process. As shown in Fig. 5, DSC curves

Fig. 3. SEM images of apigenin coarse powder (a) and apigenin nanocrystals (b).

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1000 800

Apigenin nanocrystals

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Intensity (Counts)

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Apigenin coarse powder

800 600 400 200 0 0

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Two-Theta (deg) Fig. 4. XPRD patterns of apigenin coarse powder and the apigenin nanocrystals.

SAS process, which could be beneficial to physicochemical stability.

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Apigenin nanocrystals

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3.4. FT-IR analysis

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Heat Flow ( W⋅g-1)

0 -6

364°C

-12 0

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From the FT-IR patterns for the AP coarse powder and the AP nanocrystals (figure was not shown), it can be observed that the characteristic FT-IR peaks of the AP nanocrystals were very similar with that of the AP coarse powder, indicating that AP was chemically stable during SAS process.

5

3.5. In vitro dissolution studies

0 -5 -10

Apigenin coarse powder

-15 -20

367°C

-25 0

100

200

300

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Temperature (°C) Fig. 5. DSC curves of apigenin coarse powder and prepared nanocrystals.

for the AP coarse powder and the AP nanocrystals were quite identical, with a single sharp endothermic peak, attributing to the melting point of AP at 367 °C and 364 °C. The small shift to lower melting point after SAS process may attribute to the reduction of particle size to nanometer range (Lai et al., 1996). By reduction of dimensions of particles from micron range or even bigger down to nano range, the surface-to-volume ratio increases significantly and the surface energy substantially effects the interior ‘‘bulk’’ properties of the material. In other words, the nanosized small particles have a higher proportion of surface molecules with fewer nearest neighbors than larger particles, and thus are more weakly bound and less constrained in their thermal motion than molecules in the body of crystals, which is supposed to be responsible for the decrease of the melting point (Schmidt et al., 1998). Considering the DSC and XRPD patterns, it can be demonstrated that the crystalline state was apparently unaltered by the use of

In vitro dissolution profiles of AP coarse powder and prepared nanocrystals in capsule are shown in Fig. 6. Under non-sink conditions (0.1 M HCl, 0.1 M PBS 6.8 and 0.1 M PBS 7.4), both AP coarse powder and nanocrystals achieved a plateau of cumulative dissolution at 45 min, but AP nanocrystals exhibited more rapid dissolution velocity with much higher cumulative amount of dissolved AP. The higher dissolution platform of AP nanocrystals could be attributed to the enhanced saturated solubility which was due to its significant decrease of particle size (Gibbs–Kelvin relation (Buckton and Beezer, 1992; Hammond et al., 2007)). The reduced crystallinity of AP nanocrystals might lead to extra and valuable contribution to a faster dissolution rate (Xiao, 2009). Under sink condition (0.1 M PBS 6.8 with 0.5% Polysorbate 80), only about 40% of the drug dissolved from the AP coarse powder during the 120 min study, which indicated that the dissolution rate might be the limiting step for AP to be absorbed in vivo. However, the prepared AP nanocrystals demonstrated more than 90% of cumulative dissolution within only 20 min. Different from nanosuspensions prepared by top-down technologies (wet milling (Liversidge and Conzentino, 1995)), high pressure homogenization (Gao et al., 2010; Mauludin et al., 2009) and combined technology (Al Shaal et al., 2010, 2011)), no surfactants and/or other soluble excipients were used in AP nanocrystals prepared by bottom-up precipitation technology (SAS process used here). Although no soluble excipients surrounding the spherical particle, which may dissolve rapidly to form a hydrophilic wetting environment and to cause the rapid dispersion of nanosuspension

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Fig. 6. Dissolution profiles for apigenin coarse powder (j), apigenin nanocrystals (N) in 0.1 M hydrochloric acid (dot line), pH 6.8 phosphate buffer (solid line), pH 7.4 phosphate buffer (dash dot line) and pH 6.8 phosphate buffer containing 0.5% polysorbate 80 (dash line). Each value represents mean from six experiments.

in media (Gao et al., 2010), AP nanocrystals still demonstrated a good performance in in vitro dissolution behavior. 3.6. In vivo absorption study A HPLC–UV method was developed and validated for the determination of AP in rat plasma samples. AP could be baseline separated from internal standard silybin with no interference from endogenous materials in rat plasma. The retention time for AP and silybin were 5.3 and 8.1 min, respectively. Good linearity was obtained over AP concentration range of 0.25–10.2 lg/ml (r2 = 0.9943, n = 6). At concentrations of 0.25, 2.5 and 10.2 lg/ml, spiked recoveries of AP from rat plasma were 86.78%, 84.59% and 92.34%, respectively (n = 3); the %RSD (relative standard deviation) of both inter-day and intra-day precision was below 5%; and the accuracy was within the range of 4.1% to 6.4% RE (relative error). After storage for 15 days at 20 °C and freeze-thawing for three cycles, AP was found to be stable in rat plasma.

To evaluate the effect of nanocrystals formulation in improving the oral bioavailability of AP, an in vivo absorption study was carried out in rats via gavage administration of suspension of AP coarse powder or prepared nanocrystals. The mean plasma concentrations of AP versus time profiles of two oral formulations and intravenous AP are shown in Fig. 7. Mean pharmacokinetic parameters for AP coarse powder and AP nanocrystals are listed in Table 1. Plasma AP concentrations after intravenous administration declined sharply with time and followed a conventional biphasic pattern due to the inchoative distribution to body tissues and subsequent metabolism/excretion of the drug. The plasma concentrations of intravenous AP before 20 min were significantly higher than those of two oral AP formulations, generating an area under the curve (AUC0–4h) of 875 lg min/ml. 2 mg/kg of AP given i.v. in rats gave a maximum plasma AP level of 46.2 lg/mL, whereas a 25-fold higher AP dose administered orally as coarse powders gave only 1.5 lg/mL maximum plasma level in rat. Curcumin, another active ingredient found naturally from the rhizome of the herb

50

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Time (min) Fig. 7. Plasma drug concentration-time curves for apigenin coarse powder (j) and apigenin nanocrystals (N) after oral administration at the equivalent dose (50 mg/kg) to rats, and intravenous apigenin (d) (2 mg/kg) to rats (mean ± SD, n = 6).

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J. Zhang et al. / European Journal of Pharmaceutical Sciences 48 (2013) 740–747 Table 1 Pharmacokinetic parameters in rats after oral administration of AP coarse powder and nanocrystals (dose 50 mg/kg, n = 6, mean ± SD).

*

Formulation

Cmax (lg/ml)

tmax (min)

t1/2 (min)

AUC0

AP coarse powder AP nanocrystals

1.5 ± 0.2 5.4 ± 0.6*

120 ± 16 90 ± 14*

80.0 ± 13.0 90.7 ± 13.0

445 ± 45 1509 ± 196*

12h

(lg min/ml)

MRT (min) 222 ± 33 221 ± 31

p < 0.05.

Curcuma longa, was reported to have the similar pharmacokinetic profile, with a maximum serum curcumin level of 0.36 lg/mL (10 mg/kg of curcumin given i.v.) and 0.06 lg/mL (500 mg/kg of curcumin administered orally) in rat (Yang et al., 2007). The difference between two oral AP formulations was also evident. After oral administration of AP nanocrystals, the AP plasma concentrations in rats were significantly higher than that administered with AP coarse powder. Cmax and AUC0–12h of AP after oral administration of AP nanocrystals were enhanced by 3.6-fold and 3.4-fold higher than those of AP coarse powder, respectively. The absolute bioavailability was enhanced from 2.0% of AP coarse powder to 6.9% by nanocrystal formation. In addition, tmax was significantly decreased after oral administration of AP nanocrystals, indicating the more rapid onset of AP. Several factors could be involved in the improvement of oral AP bioavailability. The decreased particle size could increase the dissolution rate by increasing the surface area of AP as elaborated in the Noyes–Whitney equation (Tong, 2000). It was reported that reducing the particle size from 20–30 lm to 270 nm led to a 4-fold faster absorption of naproxen in rats (Liversidge and Conzentino, 1995). In the present investigation, the in vitro dissolution study demonstrated that AP could dissolve much faster and more completely from the prepared nanocrystals than that from the coarse powder, which finally contributed in the higher Cmax and AUC obtained after oral administration of AP nanocrystals. In addition, decreased particle size and increased surface area can lead to the increased muco-adhesion of AP, which can increase gastrointestinal transit time of AP and subsequently lead to its bioavailability enhancement (Rabinow, 2004). It was found that even orally administered AP nanocrystals had low absolute bioavailability. The causes for low bioavailability of orally administered drugs within the body include absorption problems due to low solubility/dissolution rate, insufficient time for absorption in the GI tract, or poor membrane permeation, presystemic degradation problems, metabolism problems such as intestinal wall/liver first-pass metabolism, and rapid elimination and clearance problems from the body (Aungst, 1993). The classification of AP as BCS class II drug due to its low solubility and high permeability (Zhang et al., 2012) in our previous study and the significantly improved dissolution by nanocrystals in the present study explained some problems associated with absorption. However, very limited information related to metabolism and excretion of AP had been disclosed over the past decades. These urge us to further investigate the other possible causes that severely curtail AP bioavailability, thus facilitating the farther development of strategies for bioavailability improvement. 4. Conclusion In the present investigation, AP nanocrystals were prepared by the supercritical antisolvent process. No substantial change in crystalline structure but the decreased particle size with smooth surface in spherical shape was observed after nanocrystals formation. The prepared AP nanocrystals exhibited much more rapid dissolution profiles than AP coarse powder in different dissolution media. The improved dissolution rate of nanocrystals might be the key factor responsible for bioavailability enhancement of such BCS

II compound AP. This study provides a great potential to utilize oral nanocrystals as the effective formulation strategy for drugs with solubility/dissolution as the rate-limiting step to absorption from the gastrointestinal tract.

Acknowledgements This research was funded by the Important National Science & Technology Specific Projects (No. 2011ZX09201-101-02), Peak of six major talents in Jiangsu Province (2010, level A) and Fundamental Research Funds for the Central Universities (Program No. JKP2011015).

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