Development of omega-3 phospholipid-based solid dispersion of fenofibrate for the enhancement of oral bioavailability

Accepted Manuscript Development of Omega-3 Phospholipid-based Solid Dispersion of Fenofibrate for the Enhancement of Oral Bioavailability Liang Yang, Yating Shao, Hyo-Kyung Han PII: DOI: Reference:

S0928-0987(15)00328-0 http://dx.doi.org/10.1016/j.ejps.2015.07.007 PHASCI 3317

To appear in:

European Journal of Pharmaceutical Sciences

Received Date: Revised Date: Accepted Date:

31 March 2015 9 July 2015 9 July 2015

Please cite this article as: Yang, L., Shao, Y., Han, H-K., Development of Omega-3 Phospholipid-based Solid Dispersion of Fenofibrate for the Enhancement of Oral Bioavailability, European Journal of Pharmaceutical Sciences (2015), doi: http://dx.doi.org/10.1016/j.ejps.2015.07.007

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Development of Omega-3 Phospholipid-based Solid Dispersion of Fenofibrate for the Enhancement of Oral Bioavailability

Liang Yang, Yating Shao, Hyo-Kyung Han * BK plus project team, College of Pharmacy, Dongguk University-Seoul, Dongguk-ro-32, Ilsan-Donggu, Goyang, Korea

*Corresponding Author

Hyo-Kyung Han, Ph.D.

College of Pharmacy, Dongguk University-Seoul, Dongguk-ro-32, Insan-Donggu, Goyang, Korea

E-mail: [email protected]

Tel.: +82-31-961-5217

Fax: +82-31-961-5206

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Abstract This research aimed to develop the omega-3 phospholipids based solid dispersion to improve the oral bioavailability of fenofibrate. The omega-3 phospholipids based solid dispersion formulation (OPSD) was prepared by an antisolvent precipitation with immediate freeze-drying and the optimal composition of the formulation was determined as the ratios of sucrose to krill oil of 5:1 (w/w), krill oil to fenofibrate of 1.5:1 (w/w), and antisolvent to solvent of 6:4 (v/v). The developed OPSD formulation was characterized by using scanning electron microscopy (SEM), X-ray powder diffraction (XRPD), and differential scanning calorimetry (DSC), which indicated the crystalline state of fenofibrate in the OPSD. The drug release profiles were also examined at different pHs. The OPSD achieved almost complete dissolution within 15 min, while the untreated powder and physical mixture exhibited minimal dissolution (less than 10% even after 2 hr). Furthermore, this formulation effectively increased the oral drug exposure in rats, as the Cmax and AUC of fenofibric acid (an active metabolite) were enhanced by approximately 6-7 folds. These results suggest that the OPSD formulation should be promising for improving the oral bioavailability of fenofibrate.

Keywords Antisolvent, Omega-3 phospholipids, Fenofibrate, Dissolution, Bioavailability

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1. Introduction Cardiovascular disease is one of the most prominent causes of morbidity and mortality in the elderly (Wissler and Robert, 1996). Considering the effect of aging on the incidence of cardiovascular disease, the effective control of cardiovascular diseases is important to reduce the cost of health care as well as improve the quality of life. Among the medicines available for controlling cardiovascular diseases, fenofibrate is a potent lipid-regulating agent that is effective in reducing total cholesterol, low-density lipoprotein

(LDL)

cholesterol,

apolipoprotein

B,

total

triglycerides

and

triglyceride-rich lipoprotein (Chapman, 2003; Fazio and Linton, 2004; Vakkilainen et al., 2003). However, as a BCS class II drug (low solubility and high permeability), poor water solubility limits the oral absorption of fenofibrate which results in low bioavailability and high variability in systemic exposure (Guay, 2002; Guivarc'h et al., 2004). Therefore, the effective solubilization of fenofibrate is critical to improving the oral bioavailability and dose-proportionality, thereby reducing the intra- and inter-subject variability. Among the various oral formulations, reducing the particle size can lead to an increase in the dissolution rate of poorly soluble drugs and frequently to higher bioavailability (Kesisoglou et al., 2007; Sauron et al., 2006). In addition to particle size reduction, phospholipids based formulation should be one of promising approaches to enhance the oral absorption of poorly soluble drugs (Fricker et al., 2010). Phospholipids have a unique amphiphilic property and effectively enhance the solubilization of lipophilic drugs. Dong et al. (2013) have used sodium

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deoxycholate/phospholipid mixed micelles as a carrier for improving oral absorption of fenofibrate. Krill oil is rich in phospholipids carrying long-chain omega-3 polyunsaturated fatty acids (mainly docosahexaenoic acid (DHA) and eicosapentanoic acid (EPA)) (Bunea et al., 2004; Maki et al., 2009; Werner et al., 2004). Omega-3 phospholipids are the key building blocks of cellular life and the association between phospholipids and long-chain omega-3 fatty acids highly facilitates the intestinal penetration of fatty acid molecules, increasing the bioavailability of omega-3 polyunsaturated fatty acids (Maki et al., 2009; Werner et al., 2004). Accordingly, previous studies have demonstrated that krill oil was effective for the management of hyperlipidemia by lowering total cholesterol, low-density lipoprotein (LDL) and triglycerides, while slightly increasing the high-density lipoprotein (HDL) levels as well (Backes and Howard, 2014; Bunea et al., 2004; Werner et al., 2004; Zhu et al., 2008). For example, in the clinical studies reported by Bunea et al. (2004), 1-3 g/day of krill oil was more effective in lowering total cholesterol, triglycerides, and LDL compared to both fish oil and placebo. In addition, a maintenance dose of 500 mg/day krill oil was significantly effective for long-term regulation of blood lipid (Bunea et al., 2004). Krill oil also contains various potent antioxidants including provitamin E, flavonoids and vitamin A (Bunea et al., 2004). Therefore, if krill oil is used in the formulation of fenofibrate, it might be beneficial to control serum lipid levels as well as to enhance the drug absorption. Furthermore, for better stability and downstream processability,

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the present study aimed to develop a solid crystalline formulation by coupling antisolvent precipitation with an immediate freeze-drying process. This process is based on a technique developed to produce nanocrystals (de Waard et al., 2009; De Waard et al., 2008). In order to stabilize the nanocrystals produced by antisolvent precipitation against particle growth (“Ostwald ripening” effect) and aggregation, antisolvent precipitation was followed by an immediate freeze-drying process to solidify the nanocrystal suspension (Kesisoglou et al., 2007). Omega-3 phospholipids in krill oil could play a role as a stabilizer in antisolvent precipitation, with sucrose functioning as a cryoprotectant in freeze-drying. The present study optimized the composition of an omega-3 phospholipids based solid dispersion and evaluated the in vitro and in vivo characteristics of the developed formulation.

2. Materials and methods 2.1. Materials Fenofibrate, sucrose, polyoxyethylene (20)-sorbitan monooleate (Tween® 80), sodium taurodeoxycholate (NaTDC) and pancreatin (8 x USP specifications) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Lecithin (ca. 99.2% soybean-phosphatidylcholine) was purchased from Avanti lipid (USA). Superba™ Krill Oil was kindly supplied by Aker BioMarine AS (Oslo, Norway). Tertiary butyl alcohol (TBA), sodium chloride, hydrochloric acid, potassium dihydrogen phosphate,

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sodium hydrogen phosphate and phosphoric acid were obtained from Junsei Chemical Co. Ltd. (Tokyo, Japan). Acetonitrile was HPLC grade from Merck (Darmstadt, Germany).

2.2. Preparation of omega-3 phospholipids based solid dispersion Omega-3 phospholipids based solid dispersion (OPSD) was prepared by antisolvent precipitation with immediate freeze-drying process. The experimental processes for the preparation are illustrated in Fig. 1. In this process, two separate solutions were prepared: one was sucrose in water and the other was krill oil and fenofibrate in t-butyl alcohol (TBA). The ratios of sucrose/krill oil, krill oil/fenofibrate, and water/TBA were varied, while the drug amount and the total volume of the solution were fixed as 40 mg and 10 ml, respectively (Table 1). The aqueous solution was mixed with the TBA solution in glass vials under vigorous stirring. Immediately after mixing, the solution was rapidly frozen at -80°C and subsequently lyophilized. Freeze drying was performed using a lyophilizer (LYOPH-PRIDE 10R, Ilshin BioBase Co. Ltd, South Korea). The frozen solutions were lyophilized at a shelf temperature of -39°C and a condenser temperature of -65°C for 24 hr. Thereafter, the shelf temperature was gradually raised to 20°C over another 24 hr. Physical mixtures were prepared using a spatula and a mortar.

2.3. X-ray powder diffraction (XRPD)

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XRPD was performed using CuKα radiation with a wavelength of 1.5405 Å at 40 kV and 40 mA from an X'Pert PRO MPD diffractometer (PANalytical, Almelo, Netherlands). Samples were scanned over 7-45° (2θ) with a step size of 0.02° and a time per step of 1 sec. The sample powders were placed on a zero-background silicon holder. XRPD experiments were conducted at the Korea Basic Science Institute (Daegu Center, Korea).

2.4. Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) The morphological characteristics of OPSD powder and OPSD suspension in water was determined using scanning electron microscopy (SEM) and transmission electron microscopy (TEM), respectively. For SEM analysis, OPSD powder was spread on specimen stub using double-sided sticky tape, coated with platinum, and analyzed by a field-emission scanning electron microscope (FE–SEM: S-4700; Hitachi, Tokyo, Japan). For TEM picture, sample was prepared by dispersing OPSD powder in distilled water, placing a drop over the carbon-coated copper grid, stained with phosphotungstic acid (1% w/v) solution, and analyzed using TEM (Tecnai F30st, FEI, Netherland). Both SEM and TEM images were obtained at the Korea Basic Science Institute (Seoul Center, Korea).

2.5. Differential scanning calorimetry (DSC)

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The DSC measurements were performed using a DSC Q2000 (TA Instruments, Ghent, Belgium) equipped with an intercooler to determine the degree of crystallinity of the formulation. The samples were accurately weighed (5-15 mg) and placed into a hermetically closed aluminum pan. The thermograms were obtained at the scanning rate of 2°C/min over a temperature range of -40~200°C under an inert atmosphere flushed with nitrogen at a rate of 30 mL/min. The degree of crystallinity in each formulation was estimated based on the following equation (Chen and Hwang, 1995): Degree of crystallinity = ∆Hsample/∆Hdrug×1/(drug load) where ΔHsample and ΔHdrug are the measured heat of fusion from each formulation and from untreated drug (100% crystalline), respectively. ΔHsample was calculated from the peak area of the melting endotherm in each formulation. ΔHdrug was measured in a separate experiment of untreated fenofibrate.

2.6. Equilibrium solubility The equilibrium solubility of fenofibrate from each formulation was determined in water using the shake-flask method. An excess amount of each formulation was added into 8 mL of water and then the suspension was equilibrated for 72 hr on a mechanical shaker at 37°C. Samples were filtered through PTFE syringe filters (0.2 μm) and analyzed by a UPLC assay, as described in section 2.11.

2.7. Dissolution study

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Dissolution studies were conducted using the USP paddle method with 50 rpm at 37 ± 0.5°C in the dissolution tester DT 1420 (ERWEKA, Heusenstamm, Germany). For the dissolution studies to select the optimal formulation, dissolution tests were carried out in 900 ml of aqueous media and 0.5% Tween-80 was added into the medium to keep the sink condition. Each tested formulation contained drug amount equivalent to 10 mg of fenofibrate and was filled in hard gelatin capsules. In the dissolution studies for the characterization of the optimal OPSD formulation, sample was filled in hard gelatin capsules and 5% L-HPC (Low-Substituted Hydroxypropyl Cellulose) was included as disintegrant. Drug amount in OPSD formulation was equivalent to 40 mg of fenofibrate to reflect the commercially available dose strength. The corresponding physical mixture and untreated fenofibrate were also prepared for comparison. Each formulation filled in capsules was exposed to the dissolution medium (pH 1.2, pH 6.8 buffer and water, with 2% tween-80) for 2 hr. One ml of each sample was collected at the predetermined time points (5, 15, 30, 45, 60, 90, 120 min) and filtered through PTFE syringe filters (0.2 μm). After sample collection, 1 ml of fresh medium was added to the vessel to maintain the constant volume of dissolution media. Each filtrate was appropriately diluted with mobile phase and the released drug amount was analyzed by a UPLC assay.

2.8. Drug release study in simulated intestinal fluid Drug release profile of OPSD formulation was examined in FaSSIF (fasted state

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simulated intestinal fluid) with/without pancreatin. FaSSIF with/without pancreatin was prepared as described by the previous reports (Jantratid et al., 2008; Klein and Shah, 2008). Prewarmed FaSSIF (100 ml, 37°C) was added to 250 ml glass beaker containing OPSD powder (10 mg of fenofibrate). The dose of fenofibrate in the release medium corresponded to the theoretical concentration of 100 μg/ml. In FaSSIF, 100 μg/ml represents a supersaturated state as drug solubility in FaSSIF was 13.7 μg/ml) (Vogt et al., 2008). The medium was constantly stirred at 37°C by 50 rpm and 1 ml of each sample was withdrawn at the predetermined time points (5, 15, 30, 60, 120, 180, 240 min). Each sample was filtered through PTFE syringe filters (0.2 μm). All samples were diluted appropriately with mobile phase and analyzed by a UPLC assay.

2.9. Stability study To evaluate the stability of the OPSD formulation, samples were placed in airtight vials and stored at 4°C or 25°C. At the predetermined time points (1, 2, and 3 months), samples were collected and subjected to the dissolution studies for 2 hr in order to examine whether the dissolution behavior of the OPSD formulation was changed after storage.

2.10. Animal studies The animal studies were carried out in accordance with the ‘Guiding Principles in

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the Use of Animals in Toxicology’ adopted by the Society of Toxicology (USA) and the study protocol was approved by the review committee of Dongguk University (IACUC-2013-007). Male Sprague-Dawley rats (220–250 g) were supplied by Samtako bio Co., Ltd (Osan, Korea). All rats were given free access to tap water and a normal standard chow diet (Superfeed Company, Wonju, Korea). Before experiments all rats were fasted for 18 hr and given free access to tap water. On the day of the experiment, rats were divided into 3 groups (6 rats per group). Each formulation (untreated fenofibrate, physical mixture, or OPSD) was administered to rats via oral gavage at the dose equivalent to 20 mg/kg of fenofibrate. All the samples were suspended in 0.5% aqueous methylcellulose solution. Blood samples were collected from the femoral artery at 0.25, 0.5, 1, 2, 3, 4, 8, 12, and 24-hr post dose and centrifuged at 9900× g for 10 min. The obtained plasma samples were stored at -80°C until analyzed.

2.11.

Analytical assay

In vitro samples: Drug concentration was measured by a UPLC assay modified from previously reported methods (Fei et al., 2013; Van Speybroeck et al., 2010). The UPLC assay was conducted by using a Waters® ACQUITY UPLC® System and a reversed-phase C18 column (Kinetex 1.7µ XB-C18 100A, 50 mm × 2.10 mm; Phenomenex, Torrance, CA, USA) with an injection volume of 5 µl. The mobile phase consisted of acetonitrile:water (80:20, v/v, pH adjusted to 3.5 with phosphoric acid).

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The flow-rate was 0.2 ml/min and the UV wavelength set at 287 nm. Calibration curves were linear over the concentration range of 0.1-10 µg/ml (R2=0.999). The intra-day and inter-day variability was below 5%. In vivo samples: The plasma concentration of fenofibric acid was measured by an HPLC method reported in previous studies (Zhang et al., 2012). The HPLC system (Flexar, Perkin Elmer, USA) consisted of a UV detector, an automatic injector, and two solvent delivery pumps. An octadecylsilane column (Gemini C18, 4.6 × 150 mm, 5μm ; Phenomenex, Torrance, CA, USA) was eluted with a mobile phase consisting of acetonitrile:0.2% phosphoric acid solution (50/50, v/v). The flow rate was 1.0 ml/min at 40°C and the detection wavelength was 286 nm. Plasma sample (150 μl) was mixed with 15 μl of ketoprofen (100 μg/ml) as an internal standard and vortexed for 5 min. Then, 415 μl of acetonitrile was added and mixed vigorously, followed by centrifugation at 10,000 × g for 10 min. The supernatant was evaporated under vacuum and the residue was reconstituted with 150 μl of the mobile phase. After vigorous mixing for 10 min, 20 μl of the supernatant was injected into the HPLC system. Calibration curves were linear over the concentration range of 0.05-100 µg/ml (R2=0.9976). At three different concentration levels (0.1, 5.0 and 80 µg/ml), the intra-day and inter-day variability was less than 9% and 12%, respectively.

2.12.

Pharmacokinetic analysis and statistical analysis

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The plasma concentration data were analyzed by noncompartment analysis. The peak plasma concentration (Cmax) and the time to reach the peak plasma concentration (Tmax) were observed values from the experimental data. The area under the plasma concentration-time curve (AUC) was calculated using the linear trapezoidal rule. All the means were presented with their standard deviations. Statistical analysis was conducted using a one-way ANOVA followed by Dunnet’s correction. A value of p < 0.05 was considered as a statistically significant difference.

3. Results and Discussion 3.1. Effects of formulation variables on drug dissolution Single factor analysis was utilized to optimize the formulation parameters for the preparation of the OPSD formulation. The preliminary experiments indicated that the particle size of the OPSD formulation in aqueous solution could not be easily measured because of interference between phospholipids and drug particles. Therefore, dissolution characteristics were used for the selection of optimal formulations. Three formulation variables and their compositions are summarized in Table 1. The weight ratio of sucrose to krill oil: As shown in Fig. 2(a), the dissolution rate increased when the amount of sucrose increased. This may be due to the presence of smaller drug crystals in the formulation which have a high content of sucrose, a cryoprotectant as similarly observed by De Waard et al. (2008). The drug particles

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enveloped by the phospholipids was inter-dispersed and stabilized in the continuous solid matrix of the sucrose to prevent aggregation during lyophilization (Miyajima, 1997; Strauss et al., 1986). Consequently, the high content of sucrose could minimize particle aggregation and inhibit the particle growth. Therefore, the optimal weight ratio of sucrose to krill oil was set at 5:1 (w/w). The weight ratio of krill oil to fenofibrate: Fig. 2(b) shows the effects of the weight ratio of fenofibrate to krill oil on the drug dissolution. Omega-3 phospholipids in krill oil could work as a surfactant during antisolvent crystallization. When the weight ratio of krill oil to fenofibrate increased from 0.5:1 to 1.5:1, the drug dissolution rate increased significantly. As the weight ratio of krill oil to fenofibrate further increased to 3:1, the dissolution rate increased slightly but the difference was not statistically significant. This result may be explained by the fact that omega-3 phospholipids could effectively coat the surface of drug particles and inhibit the growth of particles, leading to the smaller particles of fenofibrate (Zu et al., 2014a; Zu et al., 2014b). Therefore, the weight ratio of krill oil to fenofibrate of 1.5:1 was selected in the final formulation. The volume ratio of antisolvent water to solvent TBA: Fig. 2(c) illustrates the effects of the volume ratio of water to TBA on the drug dissolution. When the water:TBA ratio of the solution increased from 4:6 (v/v) to 6:4 (v/v), the dissolution rate increased accordingly. However, a further increase of the water:TBA ratio to 8:2 (v/v) resulted in the decrease of dissolution rate. As reported previously (Zu et al.,

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2014a; Zu et al., 2014b), the solubility of fenofibrate in the mixture of solvent and antisolvent becomes smaller and smaller with an increasing proportion of antisolvent in the system. This caused an increase in the degree of supersaturation, leading to the smaller particle size during crystallization and subsequently higher dissolution rate. However, when the water:TBA ratio of the solution was further increased to 8:2 (v/v), the agglomeration of particles may occur due to the high nucleus concentration of drug, thereby resulting in a lower drug dissolution. Therefore, the optimal volume ratio of antisolvent to solvent was selected as 6:4 (v/v). Taking together all the results of the single factor experiments, the optimal composition of the OPSD formulation was determined as the ratios of sucrose to krill oil of 5:1 (w/w), krill oil to fenofibrate of 1.5:1 (w/w), and antisolvent to solvent of 6:4 (v/v).

3.2. Structural and morphological characteristics XRPD and DSC profiles were examined to evaluate the crystalline state of the drug in the OPSD formulation. As shown in Fig. 3, characteristic peaks of fenofibrate were clearly observed in the diffraction patterns of the untreated drug powder, as well as in the OPSD formulation. This result suggests that the crystalline form of fenofibrate is present in the OPSD formulation. However, the peaks observed in the diffraction patterns of sucrose disappeared in the OPSD formulation, implying the change of crystal state of

15

sucrose to an amorphous state during the freeze-drying process (Te Booy et al., 1992). The low amorphous humps in the diffraction patterns of the OPSD formulation, as compared to those of untreated drug, are likely attributed to the amorphous sucrose after lyophilization. As shown in Fig. 4, DSC thermographs of pure drug (untreated powder formulation) exhibited a sharp endothermic peak with the melting point of 81°C. The thermal curve of fenofibrate in OPSD exhibited an endothermic peak at 65°C, which had shifted backwards approximately 16°C with the reduced peak intensity, while becoming much broader. It may be explained by the presence of moisture content and polarity of phospholipids (Ige et al., 2013; Sakellariou et al., 1985). Partial solubilization into the krill oil phase upon heating and smaller particle size may also explain this observation as reported by Van Eerdenbrugh et al. (2007). The subsequent exothermic peak in the thermal curve of the OPSD may be due to the recrystallization of amorphous sucrose (Van Eerdenbrugh et al., 2007). The degree of crystallinity of fenofibrate in the OPSD was calculated to be approximately 90%, using untreated fenofibrate as the 100% crystalline reference. DSC data was in good agreement with the XRPD results and indicated the crystalline nature of fenofibrate in the OPSD. The morphological characteristics of the OPSD formulation were also investigated by SEM and TEM. As shown in Fig. 5A, it exhibited a homogeneous blend of all ternary components in the coarse, long, irregular-shaped particles. The size of drug particles could be estimated from TEM image of re-dispersed OPSD

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formulation in water. As shown in Fig. 5B, the drug particle size was about 200 nm, indicating that fine drug crystal was formed by an antisolvent precipitation coupled with immediate freeze-drying.

3.3. Equilibrium solubility The equilibrium solubility of fenofibrate from the untreated powder formulation and the OPSD formulation was almost same in water, which was determined as 0.34 ± 0.03 μg/mL, agreeing well with other studies (Guay, 2002; Hanafy et al., 2007; Vogt et al., 2008). At the initial stage, the drug existed in a supersaturated state temporarily (data not shown) but due to the instability, the solubilized drug molecules precipitated slowly and achieved similar equilibrium solubility as the untreated powder formulation after 72 hr.

3.4. Dissolution of the optimized OPSD formulation The dissolution profiles of fenofibrate from the OPSD formulation were evaluated in different dissolution media (pH 1.2 and 6.8 buffers, water, with 2% Tween 80) and compared to those from the untreated powder formulation and the physical mixture. As shown in Fig. 6, the OPSD formulation dramatically improved drug dissolution rates as well as the extent of drug release at all the tested conditions. The OPSD formulation achieved almost complete dissolution within 15 min, while the untreated drug and the physical mixture exhibited minimal drug release (less than 10 %) even

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after 2 hr. Consequently, drug dissolution increased by about 10 fold via OPSD formulation. In principle, antisolvent precipitation produces the finely dispersed drug nanocrystals and the phospholipids in krill oil can act as short-term stabilizer to retard the growing process of the drug nanocrystals. Furthermore, prolonged stabilization is achieved by the immediate freeze-drying in the presence of sucrose after the antisolvent precipitation, which minimizes the phenomenon called “Ostwald ripening” and particle aggregation (Kesisoglou et al., 2007). Therefore, enhanced drug dissolution from the OPSD formulation may be explained by the reduced particle size and increased surface area. The improved surface-wetting by the phospholipids in the formulation may also contribute to further enhancement in drug dissolution. Drug release profiles of OPSD formulation were also examined in FaSSIF with/without pancreatin for assessing the impact of lipolysis on the formulation. As shown in Fig. 7, in both media, supersaturation was achieved with maximum drug release at 30 min, which was sustained up to 1 hr and followed by a steady decline (Fig. 7). Also, addition of pancreatin lowered the drug release. This may be explained by, at least in part, that the solubilization capacity of lipid-based formulation decreases on digestion as lipid digestion products are usually more polar and more water-soluble than the undigested materials (Williams et al., 2013). While in vitro experiments lack an absorptive barrier, drug absorption process in gastrointestinal tract may attenuate drug precipitation from supersaturated solutions by lowering drug concentrations (Bevernage et al. 2012; Kataoka et al., 2012). Bevernage et al. (2012) also suggest

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that degree of supersaturation over the precipitation threshold in vivo is higher than that obtained from in vitro experiments. Particularly, for highly permeable drugs, the high degree of supersaturation may be beneficial if absorption is sufficiently rapid to precede drug precipitation (Williams et al., 2013). Therefore, highly permeable drugs such as fenofibrate may require relatively short periods of supersaturation to support absorption and the assessment of drug release at earlier time-points may better assist in formulation discrimination (Williams et al., 2013). This hypothesis is also supported by the recent publications reporting that extensive precipitation observed in vitro did not adversely impact drug absorption of lipid-based formulation containing fenofibrate in vivo (Griffin et al., 2014; Stillhart et al., 2014; Thomas et al., 2014).

3.5. Stability test As summarized in Table 2, the dissolution behavior of the OPSD was unchanged after 3 months of storage at both 4°C and 25°C. While amorphous solid dispersion often suffers from recrystallization during storage and subsequent changes in drug dissolution behavior, the drug in the OPSD formulation is in stable crystalline state, which can prevent undesired recrystallization during storage. Also, the stability study suggests that there was no particle growth (“Ostwald ripening” effect) and aggregation in the solid state.

3.6. In vivo pharmacokinetics

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Fenofibrate is completely metabolized to fenofibric acid, an active metabolite after oral administration (Hanafy et al., 2007). Therefore, the oral pharmacokinetics of fenofibrate should be evaluated based on the plasma concentration of fenofibric acid. The plasma concentration–time profiles of fenofibric acid after the oral administration of fenofibrate in different formulations (untreated drug, physical mixture, OPSD) are illustrated in Fig. 8. The pharmacokinetic parameters are also summarized in Table 3. In the case of the untreated powder formulation, the drug was slowly absorbed with a Tmax of about 3 hr and the systemic exposure was very low, resulting in Cmax and AUC of 4.42±1.53 μg/mL and 56.7±16.5 µg*hr/ml, respectively. Physical mixtures also exhibited similar profiles with the untreated drug. In contrast, the OPSD formulation significantly (p<0.05) increased Cmax and AUC (29.8±11.4 μg/mL and 378.5±66.6 µg*hr/ml, respectively). Consequently, the OPSD formulation achieved an approximately 6-7 fold higher oral exposure of fenofibric acid compared to the untreated drug. In addition, more rapid drug absorption was achieved with the OPSD formulation. These results were consistent with the observation from the in vitro dissolution studies. Rapid and enhanced drug dissolution via the OPSD formulation resulted in the rapid drug absorption in GI tract. Furthermore, the phospholipid component in the OPSD formulation has an amphiphilic character, which may promote the drug absorption of poorly soluble drugs (Fricker et al., 2010). As OPSD formulation appeared to be effective in improving the oral absorption of fenofibrate, process optimization for cost-effective scale-up should be necessary in

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future studies for industrial production. Also, for inclusion of omega-3 phospholipids as much as possible to take advantage of its health benefits, it may need to attempt minimizing the amount of non-lipid components included in the formulation while maintaining good dispersion characteristics.

4. Conclusions Omega-3 phospholipids based solid dispersion formulation (OPSD) of fenofibrate was prepared by an antisolvent precipitation coupled with immediate freeze-drying. The optimal composition of the formulation was determined as the ratios of sucrose to krill oil of 5:1 (w/w), krill oil to fenofibrate of 1.5:1 (w/w), and antisolvent to solvent of 6:4 (v/v). Drug in the OPSD formulation was present in crystalline form and the particle size was about 200 nm. The OPSD formulation of fenofibrate significantly increased the drug dissolution rate as well as the extent of drug release. In addition, this formulation was effective in improving the oral bioavailability of fenofibrate in rats.

5. Acknowledgement This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (NRF-2013R1A1A2061289).

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1653-1663. Fazio, S., Linton, M.F., 2004. The role of fibrates in managing hyperlipidemia: mechanisms of action and clinical efficacy. Curr. Atheroscler. Rep. 6, 148-157. Fei, Y., Kostewicz, E.S., Sheu, M.-T., Dressman, J.B., 2013. Analysis of the enhanced oral bioavailability of fenofibrate lipid formulations in fasted humans using an in vitro–in silico–in vivo approach. Eur. J. Pharm. Biopharm. 85, 1274-1284. Fricker, G., Kromp, T., Wendel, A., Blume, A., Zirkel, J., Rebmann, H., Setzer, C., Quinkert, R.-O., Martin, F., Müller-Goymann, C., 2010. Phospholipids and lipid-based formulations in oral drug delivery. Pharm. Res. 27, 1469-1486. Griffin, B.T., Kuentz, M., Vertzoni, M., Kostewicz, E.S., Fei, Y., Faisal, W., Stillhart, C., O'Driscoll, C.M., Reppas, C., Dressman, J.B., 2014. Comparison of in vitro tests at various levels of complexity for

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Table 1. Process and formulation parameters for single factor analysis

Group

1

2

3

sucrose / krill oil (w/w)

krill oil/fenofibrate (w/w)

water/TBA (v/v)

5:1

3:1

6:4

3:1

3:1

6:4

2:1

3:1

6:4

5:1

3:1

6:4

5:1

1.5:1

6:4

5:1

0.5:1

6:4

5:1

1.5:1

4:6

5:1

1.5:1

6:4

5:1

1.5:1

8:2

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Table 2. Dissolution behavior of OPSD after the storage at 4°C and 25°C (Mean ± S.D., n = 3).

Dissolution (%) Temperature 1 month

2 month

3 month

4 °C

104 ± 6.76

99.6 ± 12.4

97.0±9.59

25 °C

102 ± 6.59

99.2 ± 1.52

98.6±9.68

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Table 3. Pharmacokinetic parameters of fenofibric acid after an oral administration of different formulations (OPSD, physical mixture, untreated drug) in rats (Mean ± SD, n=6). Formulation

AUC (µg*hr/ml)

Cmax (µg/ml)

Tmax (hr)

Untreated drug

56.7±16.5

4.42±1.53

3.0±0.7

Physical mixture

31.6±19.4

3.01±0.91

3.0±1.3

OPSD

379±66.6*

29.8±11.4*

2.3±0.6*

*p<0.05, compared to untreated drug Dose equivalent to 20 mg/kg of fenofibrate

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Figure Legends

Fig. 1. Experimental process to prepare the Omega-3 phospholipids based solid dispersion. Fig. 2. The effect of formulation variables on the dissolution of fenofibrate in water with 0.5% Tween 80 (Mean±SD, n=3). (a) the weight ratio of sucrose to krill oil; (b) the weight ratio of krill oil to fenofibrate; (c) the volume ratio of antisolvent water to solvent TBA. Fig. 3. XRPD diffractograms of sucrose, fenofibrate and OPSD formulation. Fig. 4. DSC thermograms. Fig. 5. SEM image of OPSD powder (a) and TEM image of re-dispersed OPSD formulation in distilled water (b). Fig. 6. In vitro dissolution profiles of fenofibrate in different formulations at pH 1.2 (a), pH 6.8 (b) and in water (c) (Mean±SD, n=6). Each dissolution medium included 2% Tween 80. Fig. 7. In vitro drug release profiles of OPSD formulation in FaSSIF with/without pancreatin (Mean±SD, n=3). Fig. 8. Plasma concentration-time profiles of fenofibric acid after an oral administration of different formulations (OPSD, physical mixture and untreated drug) in SD rats (Mean ± SD, n=6). Dose was equivalent to 20 mg/kg of fenofibrate.

31

Fig. 1. Experimental process to prepare the Omega-3 phospholipids based solid dispersion

32

(a)

(b)

(c)

Fig. 2. The effect of formulation variables on the dissolution of fenofibrate in water with 0.5% Tween 80 (Mean±SD, n=3). (a) the weight ratio of sucrose to krill oil; (b) the weight ratio of krill oil to fenofibrate; (c) the volume ratio of antisolvent water to solvent TBA.

33

Fig. 3. XRPD diffractograms of sucrose, fenofibrate and OPSD formulation.

34

Fig. 4. DSC thermograms

35

(a)

(b)

Fig. 5. SEM image of OPSD powder (a) and TEM image of re-dispersed OPSD formulation in distilled water (b).

36

(a)

(b)

(c)

Fig. 6. In vitro dissolution profiles of fenofibrate in different formulations at pH 1.2 (a), pH 6.8 (b) and in water (c) (Mean±SD, n=6). Each dissolution medium included 2% Tween 80. 37

7 In vitroo drrug releasse prof p filess off OP PSD D form mulatioon in FaSSIF F with//wiithoout Fig. 7. paancrreattin (M Meann±S SD, n= =3).

38 8

Fig. 8. Plasma concentration-time profiles of fenofibric acid after an oral administration of different formulations (OPSD, physical mixture and untreated drug) in SD rats (Mean ± SD, n=6). Dose was equivalent to 20 mg/kg of fenofibrate.

39

Development of Omega-3 Phospholipid-based Solid Dispersion of Fenofibrate for the Enhancement of Oral bioavailability Liang Yang, Yating Shao, Hyo-Kyung Han * BK plus project team, College of Pharmacy, Dongguk University-Seoul, Dongguk-ro 32, Insan-Donggu, Goyang, Korea

Drug/surfactant in organic solution

Aqueous solution

Precipitation

Freeze-drying

40