http://informahealthcare.com/drd ISSN: 1071-7544 (print), 1521-0464 (electronic) Drug Deliv, 2014; 21(8): 588–594 ! 2014 Informa Healthcare USA, Inc. DOI: 10.3109/10717544.2013.865815
ORIGINAL ARTICLE
Pharmaceutical and pharmacokinetic characteristics of different types of fenofibrate nanocrystals prepared by different bottom-up approaches Drug Delivery Downloaded from informahealthcare.com by Biblioteka Uniwersytetu Warszawskiego on 01/09/15 For personal use only.
Hua Zhang1, Yuan Meng1, Xueqing Wang1, Wenbing Dai1, Xinglin Wang2, and Qiang Zhang1 1
State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University, Beijing, China and 2State Key Laboratory of Drug Delivery Technology and Pharmacokinetics, Tianjin Institute of Pharmaceutical Research, Tianjin, China Abstract
Keywords
Low dissolution rate of a poorly water soluble drug often leads to low and variable oral bioavailability. Formulating drugs as nanocrystals can help to overcome these problems by increasing the solubility and dissolution velocity. But different preparation approaches may result in different nanocrystals with different characteristics. In this study, three types of fenofibrate nanocrystals (FNT-NCs) were prepared by bottom-up methods, antisolvent and thermal precipitation under different conditions. These FNT-NCs were characterized by scanning electron micrography, dissolution testing, differential scanning calorimetry and powder X-ray diffractometry. A significant increase of dissolution rate was observed in the drug nanocrystals compared to the crude FNT powder (from 20% to 80% in 5 min). The crystallinity of the FNT-NCs prepared by antisolvent precipitation increased slightly, while that by thermal precipitation decreased. The oral bioavailability of two types of FNT-NCs prepared by antisolvent precipitation in rats increased notably compared to that of the crude powder (5.5folds and 5.0-folds, respectively). However, the oral absorption of FNT-NCs prepared by thermal precipitation did not increase, although its dissolution rate was higher than that of the crude powder. In conclusion, different bottom-up methods produce different FNT-NCs with different crystallinity, which results in different oral bioavailability. Namely, a careful study and rational choice on preparation approaches are significant for the nanocrystal techniques.
Bioavailability, fenofibrate, nanocrystals, pharmaceutical characterization
Introduction Oral administration is the main delivery route for drugs due to its convenience and good compliance. The greatest obstacle for oral administration is the poor aqueous solubility of drugs or low dissolution rate, resulting in low or variable oral bioavailability. Poorly soluble drugs are considered class II or class IV drugs according to the Biopharmaceutics Classification System (BCS) (Amidon et al., 1995). Class II drugs are poorly water soluble, but their gastrointestinal permeability is higher, meaning that these drugs can be absorbed easily when they are dissolved after oral administration. Pharmaceutical scientists have worked to develop strategies to improve the solubility of class II drugs and increase the oral bioavailability. These strategies include chemical modification, such as structural changes (pro-drugs) (Stella & Nti-Addae, 2007) and salt formation(Serajuddin, 2007), the use of physical approaches, such as the addition of co-solvent or solubilizing agents and particle size reduction (Rabinow, 2004) and the use of a variety of formulation methods, such as lipid formulation (emulsion or liposome),
Address for correspondence: Qiang Zhang, PhD. Tel: +86-10-82802791. Fax: +86-10-82802791. Email: [email protected]
History Received 12 September 2013 Revised 8 November 2013 Accepted 11 November 2013
incorporation in cyclodextrins, solid dispersion and the use of nanocrystals (O’Dricoll & Griffin, 2008). Drug nanocrystals were invented over 20 years ago, and several commercial products have been marketed (Mo¨schwitzer, 2013). The advantages of nanocrystals include high drug loading and increased dissolution rate, which result in enhanced and consistent oral bioavailability. The nanocrystal formulations can avoid the use of additional excipients that may introduce adverse or toxic reactions to the human body. Two approaches for the production nanocrystals have been used: top-down and bottom-up (Shegoker & Mu¨ller, 2010). The top-down approach has been widely employed for industrial-scale production, and almost all the nanocrystal products in the market are prepared by this method (Mu¨ller & Keck, 2012). The larger crystalline particles are broken into nanocrystals by media milling or high-pressure homogenization in the top-down approach (Van Eerdenbrugh et al., 2008). In contrast, the molecular compound can be used to form nanocrystals by precipitation or crystallization in a bottom-up approach. Many studies have been carried out to produce nanocrystals by bottom-up approaches, such as common antisolvent precipitation, controlled solvent evaporation, antisolvent precipitation under ultrasound or supercritical fluid, spray-drying and freeze-drying and so on (Chan &
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DOI: 10.3109/10717544.2013.865815
Kwok, 2011). Although the industrial scale-up is easy to obtain in top-down method, the defects are not neglectable. For example, the nanoparticles with size below 100 nm is not easy to form, the processes are very time consuming and sometimes require much energy. Furthermore, these methods may induce contamination from milling media or homogenization chamber (Thorat & Dalvi, 2012). Fenofibrate (FNT) has been used for the treatment of hypertriglyceridemia, mixed dyslipidemia and hypercholesterolemia for many years. As a BCS class II drug, FNT is virtually insoluble (50.5 mg/ml) and highly lipophilic (Vogt et al., 2008). The oral bioavailability of FNT is approximately 30% in humans, and the individual variation in the bioavailability is large. Pharmaceutical scientists have developed different formulations to increase the solubility and oral bioavailability of FNT. These formulations include, for example, liposomes (Chen et al., 2009), nanomatrix system (Jia et al., 2011), solid dispersion (Zhang et al., 2012), solid lipid nanoparticles (Hanafy et al., 2007), solid solution (Linn et al., 2012), nanosuspension (de Waard et al., 2008; de Waard et al., 2009; Hu et al., 2011a; Ige et al., 2013; Zuo et al., 2013), incorporation into mesoporous silica (Van Speybroeck et al., 2010) and nanoemulsion (Hu et al., 2011b). Two types of FNT-NCs have been marketed. One is TricorÕ , which is produced using media milling technology, and the other is TriglideÕ , which is produced using high-pressure homogenization technology. As both products are produced by top-down technologies, in order to overcome the disadvantages associating with the top-down approach, we attempted to provide a bottom-up approach to prepare FNT-NCs in this study. The nanocrystals were characterized by scanning electron micrography (SEM), dissolution testing, differential scanning calorimetry (DSC) and powder X-ray diffractometry (PXRD). The in vivo pharmacokinetic behavior of the nanocrystals was also verified in rats after oral administration.
Materials and methods Materials FNT was purchased from Kaifeng Pharmaceutical Factory (He Nan, China); fenofibric acid was purchased from Yinhe Chemical Co. Ltd. (Wu Han, China); tragacanth (the viscosity of 1% tragacanth solution is 400 mPa.s) was purchased from Beijing Pharmaceuticals Company (Beijing, China); HPLC-grade methanol and acetonitrile were obtained from Promptar (Elk Grove, CA). All other chemicals, such as sodium dodecyl sulfate (SDS), ethanol and inorganic salt, were of analytical grade. Male Sprague-Dawley (SD) rats weighing 190–210 g were obtained from the Experimental Animal Center of Peking University (Beijing, China). All of the animal experiments complied with the principles of care and use of laboratory animals and were approved by the Institutional Animal Care and Use Committee of Peking University Health Science Center.
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anti-solvent precipitation augmented by sonication, 3 ml of a 30 mg/ml FNT in an acetone solution was injected into 30 ml of 0.18% tragacanth solution under probe sonication for 1 min (formulation 1; F1) or under rapid stirring and intense waterbath sonication followed by stirring at 50 C for 60 min (formulation 2; F2). For the thermal precipitation method, 3 ml of a 30 mg/ml FNT in an acetone solution was injected into 30 ml of 0.18% tragacanth solution at 90 C. The system was maintained at 90 C for 30 min, and then it was cooled at room temperature (formulation 3; F3). The final product was filtered through a 50-nm polycarbonate membrane filter to remove the material that was dissolved in the solution, and pure nanocrystals were obtained. HPLC analysis of FNT in vitro The HPLC system consisted of a Shimadzu LC-10 A pump and SPD-10A UV/VIS detector set at 286 nm. The chromatographic column was a Phenomenex C18 (5 mm, 250 4.6 mm), and the temperature of the column was set at 30 C. The mobile phase was a mixture of methanol:water (90:10, v/v) at a flow rate of 1.0 ml/min. The calibration curve was liner with a correlation coefficient of 0.9995 over the range of 1–30 mg/ml. Solubility studies The solubility of FNT in different formulations was determined by shake-flask method. An excess amount of drug was added into 8 ml of 1% SDS solution. Then the suspensions were continuously shaken in a THZ-82B gas bath shaker (Zhejiang, China) up to 24 h and maintained at 37 C 0.5 C. Samples were taken out and filtered through a 0.22 mm membrane filter prior to analysis. The concentration of FNT was analyzed using HPLC method described in ‘‘HPLC analysis of FNT in vitro’’. All solubility studies were performed in triplicate. In vitro dissolution study The dissolution study was conducted using a ZRS-8G dissolution tester (Tianjin, China) based on Chinese Pharmacopoeia Method II (the paddle method). A sample containing 30 mg of the FNT-NCs or crude FNT powder was placed in 1000 ml of deionized water containing 1% SDS at 37 C, and the paddle speed was set at 100 rpm. At the appropriate time intervals, 1 ml of release media was withdrawn and immediately filtered using a 0.22-mm syringe filter. The filtrate was submitted to HPLC analysis. Meanwhile, an equal volume of fresh dissolution media at the same temperature was added to maintain a constant volume. Differential scanning calorimetry Thermal analysis of the FNT-NCs and crude FNT powder was carried out with a DSC Q100 V9.8 Build 296 calorimeter (Thermal Analysis Co., New Castle, DE). Approximately 4 mg of the sample was weighed in an open aluminum sample pan and analyzed with a heating rate of 10 C/min over the temperature range of 20–160 C.
Preparation of FNT-NCs
Powder X-ray diffraction
The FNT-NCs were prepared by anti-solvent precipitation under sonication or by thermal precipitation. For the
The crystallinity of the FNT nanocrystals and crude powder was analyzed using the D/MAX 2000 X-ray diffractometer
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(Rigaku Co., Tokyo, Japan) equipped with a Cu Ka radiation source at a 40 kV voltage and 40 mA current. The samples were scanned from 5 to 40 (2) with a 4 /min scan speed. Scanning electron micrography
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The shape and surface morphology of the FNT nanocrystals and crude powder were studied using a Hitachi S-4800 coldfield emission scanning electron microscope (Hitachi, Tokyo, Japan). Prior to imaging, the samples were filtered with a 50-nm polycarbonate membrane and then air-dried. The samples were attached to a SEM sample holder and sputter coated with a conductive layer of gold palladium (Au/Pd) for 3 min by an EIKO Id3 Ion Coater (EIKO Engineering Co. Ltd., Ibaragi, Japan). In vivo bioavailability study Animals and dosing Twenty-four Male SD rats (200 10 g) were randomly divided into four groups (n ¼ 6). The rats were fasted for 12 h before the experiment, and water was given freely. Samples containing 6.6 mg of the FNT nanocrystals or crude powder suspended in 1 ml saline were given orally to each animal at a dose of 33 mg/kg. Then, 0.5 ml blood samples were taken at time points of 0.5, 1, 2, 3, 4, 6, 8, 10, 12, 24 and 36 h post-dose and collected into heparin-wetted tubes. The plasma samples were obtained by centrifugation (10 000 rpm, 4 C, 10 min) and frozen at 20 C until analysis. Plasma processing and HPLC analysis FNT is metabolized to the main active metabolite fenofibric acid by plasma and tissue esterases. In this study, all plasma samples were quantified for fenofibric acid using the HPLC method. Plasma processing and HPLC analysis were performed according to the literature (Jia et al., 2011). The calibration curves were linear over the concentration range of 0.2–100 mg/ml. The intraday variability for 5, 10 and 50 mg/ml standard solutions did not exceed 5%, and the interday variability of the same standards was less than 10%. The recovery of the method was within the range of 97–99%. Data analysis The pharmacokinetic analysis was performed by a noncompartmental method using the pharmacokinetic program WinNonlin (Pharsight Corporation, Mountain View, CA). The significant differences were assessed using one-way analysis of variance, and the results were considered statistically significant at p50.05.
Results and discussion The preparation of FNT-NCs For the anti-solvent precipitation method, the stability problem of the nanocrystals was the greatest drawback (Thorat & Dalvi, 2012). The size of the FNT crystals was much larger when they were prepared in pure water, and aggregation occurred quickly. Therefore, different types and concentrations of surfactants or macromolecular compounds were used
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to reduce the size of the crystals and increase the stability of the system. We compared the effect of polyethylene glycol (MW ¼ 12 000, 0.2% and 0.5%), polyvinyl alcohol (0.5% and 1.0%), polyvinylpyrrolidine (PVP, k30, 0.2% and 0.5%), gelatin (0.5% and 1.0%), polysorbate 80 (0.5% and 2.0%) and tragacanth (0.045%, 0.09%, 0.18% and 0.36%) solution on the size and stability of FNT-NCs. For all three preparation methods, the tragacanth (0.18%) solution showed the best stabilizing effect and was chosen as the stabilizer in our study. The drug content is almost 100% in three formulations after filter, and the percent yield of the three formulations are large than 95%. The crystal size is the most important parameter in drug nanocrystals, and the nucleation step clearly plays a crucial role in bottom-up approach. In order to produce crystals of suitable size, a large number of nuclei have to be generated. Ultrasound waves have been shown capable of enhancing nucleation by creating acoustic cavitation in solution and subsequently reducing the induction time (Guo et al., 2005). Ultrasound waves are also helpful to disperse the drug solution quickly and resulting in a large number of nuclei generated. Although FNT-NCs was produced with probe sonication method (F1), the drawbacks of probe need to be considered. One of the disadvantages of probe sonication is the occurrence of metal particles in the formulation, so it must be always avoided for industrial production. The scanning electron microscope (SEM) images of FNTNCs are shown in Figure 1. As observed from the SEM images, the size of the majority of the precipitated FNT particles in all three nanocrystal formulations was less than 2 mm, and some of the particles were less than 1 mm. Importantly, it was found under SEM that, although the major axis of our FNT crystals was more than 1 mm, the minor axis was much less than 1 mm, so these crystals would show nano-properties. However, the particle size in the crude FNT powder exceeded 10 mm. There were some irregular masses in F3 prepared by thermal precipitation, and these masses might be amorphous particles, which need to be validated by the PXRD and DSC experiments. The size and shape of the particles in F1 and F2 were very similar, indicating that antisolvent precipitation of FNT under probe-based or water bath sonication did not affect the characteristics of the nanocrystals. Solubility studies Ostwald–Freundlich equation is used widely to describe the relationship between particle size and saturation concentration (Kaptay, 2012). According to Ostwald–Freundlich equation, the saturation concentration at the surface of small particles is higher than the saturation concentration at the surface of large particles. Simultaneously, the surface area can increase significantly by reducing the particle size of the drugs. In the three formulations of FNT-NCs, the particle size decreased, so the surface area increased and resulted in higher saturation concentration. The solubility of our various FNT formulations was found to be 387.45 6.01, 252.25 9.40, 266.40 0.14 and 180.30 3.54 mg/mL for F1, F2, F3 and F4, respectively. It was clear that solubility increased with the particle size decreasing.
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DOI: 10.3109/10717544.2013.865815
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Figure 1. SEM pictures of fenofibrate crystals. F1: FNT-NCs prepared by probe sonication; F2: FNT-NCs prepared by stirring with sonication; F3: FNT-NCs prepared by thermal precipitation; and F4: crude FNT powder (the bars represent 10.0 mm).
In vitro dissolution study According to the Noyes–Whitney equation, a faster rate of dissolution can be achieved by increasing the surface area of drugs (Salazar et al., 2012). Nanocrystals can decrease the size of crude drug and lead to a faster dissolution rate. The release of FNT from the different formulations is illustrated in Figure 2. As expected, the crude drug displayed the smallest dissolution rate under the same conditions. Only 20% of the drug was released within 5 min for the crude FNT powder, but more than 80% of the drug was dissolved for three FNT nanocrystal formulations in the same period. Less than 50% of the FNT was dissolved from the crude FNT powder after 45 min, and almost all the drug had dissolved from the nanocrystal formulations after 45 min. The order of the dissolution rate was as follows: crude FNT powder5FNTNCs prepared by probe sonication5FNT-NCs prepared by stirring with sonication5FNT-NCs prepared by thermal precipitation. Differential scanning calorimetry The DSC thermographs of the various formulations are shown in Figure 3, and the corresponding values are summarized in Table 1. The melting points of the FNT formulations were very similar, and the value was approximately 80 C, which matched to the melting point of the crystalline FNT form I reported by Heinz et al. (2009), demonstrating that crystalline FNT form I were existed in the FNT formulations.
Figure 2. Dissolution of fenofibrate from different formulations in deionized water containing 1% sodium dodecyl sulfate (SDS) at 37 C. F1: FNT-NCs prepared by probe sonication; F2: FNT-NCs prepared by stirring with sonication; F3: FNT-NCs prepared by thermal precipitation; and F4: crude FNT powder.
However, the heat of fusion was very different, particularly for F3, which had a value that was much lower than that of the other three formulations. The beginning melting temperature of F3 was also the lowest of all four formulations, indicating that the crystallinity of F3 was lower than that of the other three formulations. Using crude FNT as the 100% crystalline
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reference, the degree of crystallinity of FNT in the F1, F2 and F3 was calculated to be approximately 125%, 122% and 49%, respectively. The results suggest that the crystallinity of FNT in F1 and F2 is high, and it is low in F3. This finding might be explained by the rapid achievement of the precipitation conditions during the precipitation process; compact crystalline structures cannot be formed when insufficient time is available for crystal growth (Thorat & Dalvi, 2012).
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Powder X-ray diffraction Figure 4 displays the PXRD patterns of the different formulations investigated. FNT-NCs (F1 and F2) and crude powder showed obvious diffraction peaks at 12 (2), 14.5 (2), 16.2 (2), 16.8 (2) and 22.4 (2), which fit the data reported by Heinz et al. (2009). The diffractive intensities of peak in F1 and F2 at 16.2 (2) and 16.8 (2) were higher than that of the FNT crude powder, but the diffractive intensity of the peak for the FNT crude powder at 22.4 (2) was much higher than that of the FNT-NCs. The number of diffraction peaks in F3 was much less than that of the other three formulations, and the diffractive intensity of the peak in F3 was also much lower than that of the other three formulations, indicating that the crystallinity of F3 was the lowest in all formulations, and some amorphous particles were enveloped in the F3 formulation. This phenomenon agreed well with the DSC results and the dissolution test because the disordered amorphous state can increase the dissolution rate (Zhang et al., 2008).
In vivo bioavailability study The oral bioavailability of the FNT-NCs prepared by different approaches in rats was compared with that of FNT crude powder. Plots of the mean plasma fenofibric acid concentration versus time for four formulations are presented in Figure 5, and the pharmacokinetic parameters obtained by the non-compartmental method are shown in Table 2. As shown in Figure 5, the FNT-NCs prepared by probe sonication (F1) and by stirring with sonication (F2) showed much higher drug concentrations in the plasma than the crude FNT powder (fivefold increase in Cmax). The relative bioavailability of these two formulations was 552% and 495% in comparison with FNT crude powder, respectively. The FNT-NCs prepared by thermal precipitation showed slow absorption, and the AUC0–36 was also the lowest among the four formulations. This result matched the trend of heat of fusion, but it countered the results of the dissolution study, in which F3 exhibited the fastest dissolution rate among the four formulations. This phenomenon was also reported in another study reported by Van Speybroeck et al. (2010). For the nanocrystals prepared by thermal precipitation (F3), the crystallinity was the lowest among all four formulations according the
Figure 4. XRD profiles of the different fenofibrate formulations. F1: FNT-NCs prepared by probe sonication; F2: FNT-NCs prepared by stirring with sonication; F3: FNT-NCs prepared by thermal precipitation; and F4: crude FNT powder.
Figure 3. DSC curves for the different fenofibrate formulations. F1: FNT-NCs prepared by probe sonication; F2: FNT-NCs prepared by stirring with sonication; F3: FNT-NCs prepared by thermal precipitation; and F4: crude FNT powder.
Table 1. The thermoanalysis parameters of the fenofibrate formulations measured by DSC.
Formulations F1 F2 F3 F4
Melting point ( C)
Start melting temperature ( C)
Enthalpy of fusion (J/g)
80.3 80.4 80.2 80.7
79.1 79.2 78.0 79.5
94.2 91.9 37.0 75.3
F1: FNT-NCs prepared by probe sonication; F2: FNT-NCs prepared by stirring with sonication; F3: FNT-NCs prepared by thermal precipitation; and F4: crude FNT powder.
Figure 5. Average plasma concentration of fenofibric acid following a single dose of orally administration to SD rats (data presented are means SE, n ¼ 6, dose is 33 mg/kg). F1: FNT-NCs prepared by probe sonication; F2: FNT-NCs prepared by stirring with sonication; F3: FNTNCs prepared by thermal precipitation; and F4: crude FNT powder.
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Table 2. Pharmacokinetic parameters of the fenofibrate formulations in SD rats (dose 33 mg/kg, n ¼ 6, means SD). Tmax (h)
Cmax (mg/ml)
AUC0–36 (hmg/ml)
MRT0–36 (h)
13.7 8.3 5.0 3.2 10.5 7.3 8.7 1.6
51.9 13.9a,b 54.7 13.9a,b 11.5 3.6 10.1 6.1
935.8 245.3a,b 792.3 188.2a,b 120.5 17.4 160.4 107.9
16.7 4.5 9.9 0.9 13.0 5.6 14.7 3.2
Formulation F1 F2 F3 F4 a
p50.001 versus F3. p50.001 versus F4. F1: FNT-NCs prepared by probe sonication; F2: FNT-NCs prepared by stirring with sonication; F3: FNT-NCs prepared by thermal precipitation; and F4: crude FNT powder.
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b
DSC and PXRD data. We inferred that some amorphous particles were present, causing the sample to dissolve rapidly. These amorphous particles might be recrystallized or suspended particles formed in the gastrointestinal tract of rats in a fasting state, resulting in a very low bioavailability. There are several approaches to prepare FNT-NCs. Most of the approaches are top-down methods (Ige et al., 2013; Zuo et al., 2013), and a few approaches are bottom-up methods (de Waard et al., 2009; Hu et al., 2011a), but the in vivo behavior of FNT-NCs prepared by bottom-up methods was seldom reported. In this study, we prepared three types of FNT-NCs by three different bottom-up methods and compared their pharmaceutical characterization and oral bioavailability. The results showed that different bottom-up methods produced different FNT-NCs with different crystallinity, resulting in different oral bioavailability in rats.
Conclusions In this study, we prepared nanocrystals of insoluble FNT for oral administration using bottom-up approaches. The physicochemical characteristics of the nanocrystals were investigated, and the drug absorption of FNT-NCs was also studied in rats. The dissolution rate and the oral bioavailability of the FNT nanocrystals prepared by antisolvent precipitation increased significantly compared to the FNT crude powder. Although the dissolution rate of FNT nanocrystals prepared by thermal precipitation was also obviously enhanced, the oral bioavailability did not increase coincidentally. The in vitro drug release and in vivo drug absorption were not always equal. In summary, FNT-NCs can be produced by simple bottomup methods, and the solubility and bioavailability of the poorly water-soluble FNT could be enhanced. Our study may allow for the improvement of the biopharmaceutical performance of orally administered drugs with lower water solubility.
Declaration of interest The authors declare that they have no conflicts of interest to disclose. This study was supported by the National Natural Science Foundation of China (No. 81202470) and the National Basic Research Program of China (No. 2009CB930300 and No. 2012CB724002).
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ORIGINAL ARTICLE
Pharmaceutical and pharmacokinetic characteristics of different types of fenofibrate nanocrystals prepared by different bottom-up approaches Drug Delivery Downloaded from informahealthcare.com by Biblioteka Uniwersytetu Warszawskiego on 01/09/15 For personal use only.
Hua Zhang1, Yuan Meng1, Xueqing Wang1, Wenbing Dai1, Xinglin Wang2, and Qiang Zhang1 1
State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University, Beijing, China and 2State Key Laboratory of Drug Delivery Technology and Pharmacokinetics, Tianjin Institute of Pharmaceutical Research, Tianjin, China Abstract
Keywords
Low dissolution rate of a poorly water soluble drug often leads to low and variable oral bioavailability. Formulating drugs as nanocrystals can help to overcome these problems by increasing the solubility and dissolution velocity. But different preparation approaches may result in different nanocrystals with different characteristics. In this study, three types of fenofibrate nanocrystals (FNT-NCs) were prepared by bottom-up methods, antisolvent and thermal precipitation under different conditions. These FNT-NCs were characterized by scanning electron micrography, dissolution testing, differential scanning calorimetry and powder X-ray diffractometry. A significant increase of dissolution rate was observed in the drug nanocrystals compared to the crude FNT powder (from 20% to 80% in 5 min). The crystallinity of the FNT-NCs prepared by antisolvent precipitation increased slightly, while that by thermal precipitation decreased. The oral bioavailability of two types of FNT-NCs prepared by antisolvent precipitation in rats increased notably compared to that of the crude powder (5.5folds and 5.0-folds, respectively). However, the oral absorption of FNT-NCs prepared by thermal precipitation did not increase, although its dissolution rate was higher than that of the crude powder. In conclusion, different bottom-up methods produce different FNT-NCs with different crystallinity, which results in different oral bioavailability. Namely, a careful study and rational choice on preparation approaches are significant for the nanocrystal techniques.
Bioavailability, fenofibrate, nanocrystals, pharmaceutical characterization
Introduction Oral administration is the main delivery route for drugs due to its convenience and good compliance. The greatest obstacle for oral administration is the poor aqueous solubility of drugs or low dissolution rate, resulting in low or variable oral bioavailability. Poorly soluble drugs are considered class II or class IV drugs according to the Biopharmaceutics Classification System (BCS) (Amidon et al., 1995). Class II drugs are poorly water soluble, but their gastrointestinal permeability is higher, meaning that these drugs can be absorbed easily when they are dissolved after oral administration. Pharmaceutical scientists have worked to develop strategies to improve the solubility of class II drugs and increase the oral bioavailability. These strategies include chemical modification, such as structural changes (pro-drugs) (Stella & Nti-Addae, 2007) and salt formation(Serajuddin, 2007), the use of physical approaches, such as the addition of co-solvent or solubilizing agents and particle size reduction (Rabinow, 2004) and the use of a variety of formulation methods, such as lipid formulation (emulsion or liposome),
Address for correspondence: Qiang Zhang, PhD. Tel: +86-10-82802791. Fax: +86-10-82802791. Email: [email protected]
History Received 12 September 2013 Revised 8 November 2013 Accepted 11 November 2013
incorporation in cyclodextrins, solid dispersion and the use of nanocrystals (O’Dricoll & Griffin, 2008). Drug nanocrystals were invented over 20 years ago, and several commercial products have been marketed (Mo¨schwitzer, 2013). The advantages of nanocrystals include high drug loading and increased dissolution rate, which result in enhanced and consistent oral bioavailability. The nanocrystal formulations can avoid the use of additional excipients that may introduce adverse or toxic reactions to the human body. Two approaches for the production nanocrystals have been used: top-down and bottom-up (Shegoker & Mu¨ller, 2010). The top-down approach has been widely employed for industrial-scale production, and almost all the nanocrystal products in the market are prepared by this method (Mu¨ller & Keck, 2012). The larger crystalline particles are broken into nanocrystals by media milling or high-pressure homogenization in the top-down approach (Van Eerdenbrugh et al., 2008). In contrast, the molecular compound can be used to form nanocrystals by precipitation or crystallization in a bottom-up approach. Many studies have been carried out to produce nanocrystals by bottom-up approaches, such as common antisolvent precipitation, controlled solvent evaporation, antisolvent precipitation under ultrasound or supercritical fluid, spray-drying and freeze-drying and so on (Chan &
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DOI: 10.3109/10717544.2013.865815
Kwok, 2011). Although the industrial scale-up is easy to obtain in top-down method, the defects are not neglectable. For example, the nanoparticles with size below 100 nm is not easy to form, the processes are very time consuming and sometimes require much energy. Furthermore, these methods may induce contamination from milling media or homogenization chamber (Thorat & Dalvi, 2012). Fenofibrate (FNT) has been used for the treatment of hypertriglyceridemia, mixed dyslipidemia and hypercholesterolemia for many years. As a BCS class II drug, FNT is virtually insoluble (50.5 mg/ml) and highly lipophilic (Vogt et al., 2008). The oral bioavailability of FNT is approximately 30% in humans, and the individual variation in the bioavailability is large. Pharmaceutical scientists have developed different formulations to increase the solubility and oral bioavailability of FNT. These formulations include, for example, liposomes (Chen et al., 2009), nanomatrix system (Jia et al., 2011), solid dispersion (Zhang et al., 2012), solid lipid nanoparticles (Hanafy et al., 2007), solid solution (Linn et al., 2012), nanosuspension (de Waard et al., 2008; de Waard et al., 2009; Hu et al., 2011a; Ige et al., 2013; Zuo et al., 2013), incorporation into mesoporous silica (Van Speybroeck et al., 2010) and nanoemulsion (Hu et al., 2011b). Two types of FNT-NCs have been marketed. One is TricorÕ , which is produced using media milling technology, and the other is TriglideÕ , which is produced using high-pressure homogenization technology. As both products are produced by top-down technologies, in order to overcome the disadvantages associating with the top-down approach, we attempted to provide a bottom-up approach to prepare FNT-NCs in this study. The nanocrystals were characterized by scanning electron micrography (SEM), dissolution testing, differential scanning calorimetry (DSC) and powder X-ray diffractometry (PXRD). The in vivo pharmacokinetic behavior of the nanocrystals was also verified in rats after oral administration.
Materials and methods Materials FNT was purchased from Kaifeng Pharmaceutical Factory (He Nan, China); fenofibric acid was purchased from Yinhe Chemical Co. Ltd. (Wu Han, China); tragacanth (the viscosity of 1% tragacanth solution is 400 mPa.s) was purchased from Beijing Pharmaceuticals Company (Beijing, China); HPLC-grade methanol and acetonitrile were obtained from Promptar (Elk Grove, CA). All other chemicals, such as sodium dodecyl sulfate (SDS), ethanol and inorganic salt, were of analytical grade. Male Sprague-Dawley (SD) rats weighing 190–210 g were obtained from the Experimental Animal Center of Peking University (Beijing, China). All of the animal experiments complied with the principles of care and use of laboratory animals and were approved by the Institutional Animal Care and Use Committee of Peking University Health Science Center.
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anti-solvent precipitation augmented by sonication, 3 ml of a 30 mg/ml FNT in an acetone solution was injected into 30 ml of 0.18% tragacanth solution under probe sonication for 1 min (formulation 1; F1) or under rapid stirring and intense waterbath sonication followed by stirring at 50 C for 60 min (formulation 2; F2). For the thermal precipitation method, 3 ml of a 30 mg/ml FNT in an acetone solution was injected into 30 ml of 0.18% tragacanth solution at 90 C. The system was maintained at 90 C for 30 min, and then it was cooled at room temperature (formulation 3; F3). The final product was filtered through a 50-nm polycarbonate membrane filter to remove the material that was dissolved in the solution, and pure nanocrystals were obtained. HPLC analysis of FNT in vitro The HPLC system consisted of a Shimadzu LC-10 A pump and SPD-10A UV/VIS detector set at 286 nm. The chromatographic column was a Phenomenex C18 (5 mm, 250 4.6 mm), and the temperature of the column was set at 30 C. The mobile phase was a mixture of methanol:water (90:10, v/v) at a flow rate of 1.0 ml/min. The calibration curve was liner with a correlation coefficient of 0.9995 over the range of 1–30 mg/ml. Solubility studies The solubility of FNT in different formulations was determined by shake-flask method. An excess amount of drug was added into 8 ml of 1% SDS solution. Then the suspensions were continuously shaken in a THZ-82B gas bath shaker (Zhejiang, China) up to 24 h and maintained at 37 C 0.5 C. Samples were taken out and filtered through a 0.22 mm membrane filter prior to analysis. The concentration of FNT was analyzed using HPLC method described in ‘‘HPLC analysis of FNT in vitro’’. All solubility studies were performed in triplicate. In vitro dissolution study The dissolution study was conducted using a ZRS-8G dissolution tester (Tianjin, China) based on Chinese Pharmacopoeia Method II (the paddle method). A sample containing 30 mg of the FNT-NCs or crude FNT powder was placed in 1000 ml of deionized water containing 1% SDS at 37 C, and the paddle speed was set at 100 rpm. At the appropriate time intervals, 1 ml of release media was withdrawn and immediately filtered using a 0.22-mm syringe filter. The filtrate was submitted to HPLC analysis. Meanwhile, an equal volume of fresh dissolution media at the same temperature was added to maintain a constant volume. Differential scanning calorimetry Thermal analysis of the FNT-NCs and crude FNT powder was carried out with a DSC Q100 V9.8 Build 296 calorimeter (Thermal Analysis Co., New Castle, DE). Approximately 4 mg of the sample was weighed in an open aluminum sample pan and analyzed with a heating rate of 10 C/min over the temperature range of 20–160 C.
Preparation of FNT-NCs
Powder X-ray diffraction
The FNT-NCs were prepared by anti-solvent precipitation under sonication or by thermal precipitation. For the
The crystallinity of the FNT nanocrystals and crude powder was analyzed using the D/MAX 2000 X-ray diffractometer
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(Rigaku Co., Tokyo, Japan) equipped with a Cu Ka radiation source at a 40 kV voltage and 40 mA current. The samples were scanned from 5 to 40 (2) with a 4 /min scan speed. Scanning electron micrography
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The shape and surface morphology of the FNT nanocrystals and crude powder were studied using a Hitachi S-4800 coldfield emission scanning electron microscope (Hitachi, Tokyo, Japan). Prior to imaging, the samples were filtered with a 50-nm polycarbonate membrane and then air-dried. The samples were attached to a SEM sample holder and sputter coated with a conductive layer of gold palladium (Au/Pd) for 3 min by an EIKO Id3 Ion Coater (EIKO Engineering Co. Ltd., Ibaragi, Japan). In vivo bioavailability study Animals and dosing Twenty-four Male SD rats (200 10 g) were randomly divided into four groups (n ¼ 6). The rats were fasted for 12 h before the experiment, and water was given freely. Samples containing 6.6 mg of the FNT nanocrystals or crude powder suspended in 1 ml saline were given orally to each animal at a dose of 33 mg/kg. Then, 0.5 ml blood samples were taken at time points of 0.5, 1, 2, 3, 4, 6, 8, 10, 12, 24 and 36 h post-dose and collected into heparin-wetted tubes. The plasma samples were obtained by centrifugation (10 000 rpm, 4 C, 10 min) and frozen at 20 C until analysis. Plasma processing and HPLC analysis FNT is metabolized to the main active metabolite fenofibric acid by plasma and tissue esterases. In this study, all plasma samples were quantified for fenofibric acid using the HPLC method. Plasma processing and HPLC analysis were performed according to the literature (Jia et al., 2011). The calibration curves were linear over the concentration range of 0.2–100 mg/ml. The intraday variability for 5, 10 and 50 mg/ml standard solutions did not exceed 5%, and the interday variability of the same standards was less than 10%. The recovery of the method was within the range of 97–99%. Data analysis The pharmacokinetic analysis was performed by a noncompartmental method using the pharmacokinetic program WinNonlin (Pharsight Corporation, Mountain View, CA). The significant differences were assessed using one-way analysis of variance, and the results were considered statistically significant at p50.05.
Results and discussion The preparation of FNT-NCs For the anti-solvent precipitation method, the stability problem of the nanocrystals was the greatest drawback (Thorat & Dalvi, 2012). The size of the FNT crystals was much larger when they were prepared in pure water, and aggregation occurred quickly. Therefore, different types and concentrations of surfactants or macromolecular compounds were used
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to reduce the size of the crystals and increase the stability of the system. We compared the effect of polyethylene glycol (MW ¼ 12 000, 0.2% and 0.5%), polyvinyl alcohol (0.5% and 1.0%), polyvinylpyrrolidine (PVP, k30, 0.2% and 0.5%), gelatin (0.5% and 1.0%), polysorbate 80 (0.5% and 2.0%) and tragacanth (0.045%, 0.09%, 0.18% and 0.36%) solution on the size and stability of FNT-NCs. For all three preparation methods, the tragacanth (0.18%) solution showed the best stabilizing effect and was chosen as the stabilizer in our study. The drug content is almost 100% in three formulations after filter, and the percent yield of the three formulations are large than 95%. The crystal size is the most important parameter in drug nanocrystals, and the nucleation step clearly plays a crucial role in bottom-up approach. In order to produce crystals of suitable size, a large number of nuclei have to be generated. Ultrasound waves have been shown capable of enhancing nucleation by creating acoustic cavitation in solution and subsequently reducing the induction time (Guo et al., 2005). Ultrasound waves are also helpful to disperse the drug solution quickly and resulting in a large number of nuclei generated. Although FNT-NCs was produced with probe sonication method (F1), the drawbacks of probe need to be considered. One of the disadvantages of probe sonication is the occurrence of metal particles in the formulation, so it must be always avoided for industrial production. The scanning electron microscope (SEM) images of FNTNCs are shown in Figure 1. As observed from the SEM images, the size of the majority of the precipitated FNT particles in all three nanocrystal formulations was less than 2 mm, and some of the particles were less than 1 mm. Importantly, it was found under SEM that, although the major axis of our FNT crystals was more than 1 mm, the minor axis was much less than 1 mm, so these crystals would show nano-properties. However, the particle size in the crude FNT powder exceeded 10 mm. There were some irregular masses in F3 prepared by thermal precipitation, and these masses might be amorphous particles, which need to be validated by the PXRD and DSC experiments. The size and shape of the particles in F1 and F2 were very similar, indicating that antisolvent precipitation of FNT under probe-based or water bath sonication did not affect the characteristics of the nanocrystals. Solubility studies Ostwald–Freundlich equation is used widely to describe the relationship between particle size and saturation concentration (Kaptay, 2012). According to Ostwald–Freundlich equation, the saturation concentration at the surface of small particles is higher than the saturation concentration at the surface of large particles. Simultaneously, the surface area can increase significantly by reducing the particle size of the drugs. In the three formulations of FNT-NCs, the particle size decreased, so the surface area increased and resulted in higher saturation concentration. The solubility of our various FNT formulations was found to be 387.45 6.01, 252.25 9.40, 266.40 0.14 and 180.30 3.54 mg/mL for F1, F2, F3 and F4, respectively. It was clear that solubility increased with the particle size decreasing.
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DOI: 10.3109/10717544.2013.865815
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Figure 1. SEM pictures of fenofibrate crystals. F1: FNT-NCs prepared by probe sonication; F2: FNT-NCs prepared by stirring with sonication; F3: FNT-NCs prepared by thermal precipitation; and F4: crude FNT powder (the bars represent 10.0 mm).
In vitro dissolution study According to the Noyes–Whitney equation, a faster rate of dissolution can be achieved by increasing the surface area of drugs (Salazar et al., 2012). Nanocrystals can decrease the size of crude drug and lead to a faster dissolution rate. The release of FNT from the different formulations is illustrated in Figure 2. As expected, the crude drug displayed the smallest dissolution rate under the same conditions. Only 20% of the drug was released within 5 min for the crude FNT powder, but more than 80% of the drug was dissolved for three FNT nanocrystal formulations in the same period. Less than 50% of the FNT was dissolved from the crude FNT powder after 45 min, and almost all the drug had dissolved from the nanocrystal formulations after 45 min. The order of the dissolution rate was as follows: crude FNT powder5FNTNCs prepared by probe sonication5FNT-NCs prepared by stirring with sonication5FNT-NCs prepared by thermal precipitation. Differential scanning calorimetry The DSC thermographs of the various formulations are shown in Figure 3, and the corresponding values are summarized in Table 1. The melting points of the FNT formulations were very similar, and the value was approximately 80 C, which matched to the melting point of the crystalline FNT form I reported by Heinz et al. (2009), demonstrating that crystalline FNT form I were existed in the FNT formulations.
Figure 2. Dissolution of fenofibrate from different formulations in deionized water containing 1% sodium dodecyl sulfate (SDS) at 37 C. F1: FNT-NCs prepared by probe sonication; F2: FNT-NCs prepared by stirring with sonication; F3: FNT-NCs prepared by thermal precipitation; and F4: crude FNT powder.
However, the heat of fusion was very different, particularly for F3, which had a value that was much lower than that of the other three formulations. The beginning melting temperature of F3 was also the lowest of all four formulations, indicating that the crystallinity of F3 was lower than that of the other three formulations. Using crude FNT as the 100% crystalline
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reference, the degree of crystallinity of FNT in the F1, F2 and F3 was calculated to be approximately 125%, 122% and 49%, respectively. The results suggest that the crystallinity of FNT in F1 and F2 is high, and it is low in F3. This finding might be explained by the rapid achievement of the precipitation conditions during the precipitation process; compact crystalline structures cannot be formed when insufficient time is available for crystal growth (Thorat & Dalvi, 2012).
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Powder X-ray diffraction Figure 4 displays the PXRD patterns of the different formulations investigated. FNT-NCs (F1 and F2) and crude powder showed obvious diffraction peaks at 12 (2), 14.5 (2), 16.2 (2), 16.8 (2) and 22.4 (2), which fit the data reported by Heinz et al. (2009). The diffractive intensities of peak in F1 and F2 at 16.2 (2) and 16.8 (2) were higher than that of the FNT crude powder, but the diffractive intensity of the peak for the FNT crude powder at 22.4 (2) was much higher than that of the FNT-NCs. The number of diffraction peaks in F3 was much less than that of the other three formulations, and the diffractive intensity of the peak in F3 was also much lower than that of the other three formulations, indicating that the crystallinity of F3 was the lowest in all formulations, and some amorphous particles were enveloped in the F3 formulation. This phenomenon agreed well with the DSC results and the dissolution test because the disordered amorphous state can increase the dissolution rate (Zhang et al., 2008).
In vivo bioavailability study The oral bioavailability of the FNT-NCs prepared by different approaches in rats was compared with that of FNT crude powder. Plots of the mean plasma fenofibric acid concentration versus time for four formulations are presented in Figure 5, and the pharmacokinetic parameters obtained by the non-compartmental method are shown in Table 2. As shown in Figure 5, the FNT-NCs prepared by probe sonication (F1) and by stirring with sonication (F2) showed much higher drug concentrations in the plasma than the crude FNT powder (fivefold increase in Cmax). The relative bioavailability of these two formulations was 552% and 495% in comparison with FNT crude powder, respectively. The FNT-NCs prepared by thermal precipitation showed slow absorption, and the AUC0–36 was also the lowest among the four formulations. This result matched the trend of heat of fusion, but it countered the results of the dissolution study, in which F3 exhibited the fastest dissolution rate among the four formulations. This phenomenon was also reported in another study reported by Van Speybroeck et al. (2010). For the nanocrystals prepared by thermal precipitation (F3), the crystallinity was the lowest among all four formulations according the
Figure 4. XRD profiles of the different fenofibrate formulations. F1: FNT-NCs prepared by probe sonication; F2: FNT-NCs prepared by stirring with sonication; F3: FNT-NCs prepared by thermal precipitation; and F4: crude FNT powder.
Figure 3. DSC curves for the different fenofibrate formulations. F1: FNT-NCs prepared by probe sonication; F2: FNT-NCs prepared by stirring with sonication; F3: FNT-NCs prepared by thermal precipitation; and F4: crude FNT powder.
Table 1. The thermoanalysis parameters of the fenofibrate formulations measured by DSC.
Formulations F1 F2 F3 F4
Melting point ( C)
Start melting temperature ( C)
Enthalpy of fusion (J/g)
80.3 80.4 80.2 80.7
79.1 79.2 78.0 79.5
94.2 91.9 37.0 75.3
F1: FNT-NCs prepared by probe sonication; F2: FNT-NCs prepared by stirring with sonication; F3: FNT-NCs prepared by thermal precipitation; and F4: crude FNT powder.
Figure 5. Average plasma concentration of fenofibric acid following a single dose of orally administration to SD rats (data presented are means SE, n ¼ 6, dose is 33 mg/kg). F1: FNT-NCs prepared by probe sonication; F2: FNT-NCs prepared by stirring with sonication; F3: FNTNCs prepared by thermal precipitation; and F4: crude FNT powder.
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Table 2. Pharmacokinetic parameters of the fenofibrate formulations in SD rats (dose 33 mg/kg, n ¼ 6, means SD). Tmax (h)
Cmax (mg/ml)
AUC0–36 (hmg/ml)
MRT0–36 (h)
13.7 8.3 5.0 3.2 10.5 7.3 8.7 1.6
51.9 13.9a,b 54.7 13.9a,b 11.5 3.6 10.1 6.1
935.8 245.3a,b 792.3 188.2a,b 120.5 17.4 160.4 107.9
16.7 4.5 9.9 0.9 13.0 5.6 14.7 3.2
Formulation F1 F2 F3 F4 a
p50.001 versus F3. p50.001 versus F4. F1: FNT-NCs prepared by probe sonication; F2: FNT-NCs prepared by stirring with sonication; F3: FNT-NCs prepared by thermal precipitation; and F4: crude FNT powder.
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b
DSC and PXRD data. We inferred that some amorphous particles were present, causing the sample to dissolve rapidly. These amorphous particles might be recrystallized or suspended particles formed in the gastrointestinal tract of rats in a fasting state, resulting in a very low bioavailability. There are several approaches to prepare FNT-NCs. Most of the approaches are top-down methods (Ige et al., 2013; Zuo et al., 2013), and a few approaches are bottom-up methods (de Waard et al., 2009; Hu et al., 2011a), but the in vivo behavior of FNT-NCs prepared by bottom-up methods was seldom reported. In this study, we prepared three types of FNT-NCs by three different bottom-up methods and compared their pharmaceutical characterization and oral bioavailability. The results showed that different bottom-up methods produced different FNT-NCs with different crystallinity, resulting in different oral bioavailability in rats.
Conclusions In this study, we prepared nanocrystals of insoluble FNT for oral administration using bottom-up approaches. The physicochemical characteristics of the nanocrystals were investigated, and the drug absorption of FNT-NCs was also studied in rats. The dissolution rate and the oral bioavailability of the FNT nanocrystals prepared by antisolvent precipitation increased significantly compared to the FNT crude powder. Although the dissolution rate of FNT nanocrystals prepared by thermal precipitation was also obviously enhanced, the oral bioavailability did not increase coincidentally. The in vitro drug release and in vivo drug absorption were not always equal. In summary, FNT-NCs can be produced by simple bottomup methods, and the solubility and bioavailability of the poorly water-soluble FNT could be enhanced. Our study may allow for the improvement of the biopharmaceutical performance of orally administered drugs with lower water solubility.
Declaration of interest The authors declare that they have no conflicts of interest to disclose. This study was supported by the National Natural Science Foundation of China (No. 81202470) and the National Basic Research Program of China (No. 2009CB930300 and No. 2012CB724002).
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