http://informahealthcare.com/mnc ISSN: 0265-2048 (print), 1464-5246 (electronic) J Microencapsul, Early Online: 1–8 ! 2015 Informa UK Ltd. DOI: 10.3109/02652048.2015.1057252
RESEARCH ARTICLE
Solid self-nanoemulsifying drug delivery system (SNEDDS) for enhanced oral bioavailability of poorly water-soluble tacrolimus: physicochemical characterisation and pharmacokinetics Journal of Microencapsulation Downloaded from informahealthcare.com by Nyu Medical Center on 06/21/15 For personal use only.
Youn Gee Seo1, Dong Wuk Kim2, Abid Mehmood Yousaf2, Jong Hyuck Park2, Pahn-Shick Chang3, Hyung Hee Baek4, Soo-Jeong Lim5, Jong Oh Kim1, Chul Soon Yong1, and Han-Gon Choi2 1
College of Pharmacy, Yeungnam University, Gyongsan, South Korea, 2College of Pharmacy & Institute of Pharmaceutical Science and Technology, Hanyang University, Ansan, South Korea, 3Department of Agricultural Biotechnology, Seoul National University, Seoul, South Korea, 4Department of Food Engineering, Dankook University, Cheonan, South Korea, and 5Department of Bioscience and Biotechnology of Life Sciences and Biotechnology, Sejong University, Seoul, South Korea Abstract
Keywords
To develop a novel self-nanoemulsifying drug delivery system (solid SNEDDS) with better oral bioavailability of tacrolimus, the solid SNEDDS was obtained by spray-drying the solutions containing the liquid SNEDDS and colloidal silica. Its reconstitution properties were determined and correlated to solid state characterisation of the powder. Moreover, the dissolution and pharmacokinetics in rats was done in comparison to the commercial product. Among the liquid SNEDDS formulations tested, the liquid SNEDDS comprised of Capryol PGMC, Transcutol HP and Labrasol (10:15:75, v/v/v) presented the highest dissolution rate. In the solid SNEDDS, this liquid SNEDDS was absorbed in the pores and attached onto the surface of the colloidal silica. Drug was present in the amorphous state in it. The solid SNEDDS with 5% w/v tacrolimus produced the nanoemulsions and improved the oral bioavailability of tacrolimus in rats. Therefore, this solid SNEDDS would be a potential candidate for enhancing the oral bioavailability of tacrolimus.
Bioavailability, colloidal silica, self-nanoemulsifying drug delivery system, solid carrier, tacrolimus
Introduction The oral route is the safest and most frequently used way to administer drugs for the treatment of various diseases. However, more than 50% of the active pharmaceutical compounds exhibit very low aqueous solubility after oral administration, which results in poor oral bioavailability, high intra-subject and intersubject differences and a lack of dose proportionality. Tacrolimus, a 23-member macrolide lactone obtained from Streptomyces tsukubaensis, is an effective immunosuppressive drug that has been therapeutically employed for avoiding rejection after solid organ transplantation (Shapiro, 1999; Spencer et al., 1997). However, its efficacy is profoundly affected due to its poor solubility in aqueous media (lower than 5 mg/ml) (Joe et al., 2010) and pre-systemic biotransformation by cytochrome P450 3A containing enzymes and P-glycoprotein efflux (Shimomura et al., 2002). Numerous formulations of tacrolimus for oral administration such as nanoparticles (Nassar et al., 2008; Address for correspondence: Prof. Dr Han-Gon Choi, College of Pharmacy & Institute of Pharmaceutical Science and Technology, Hanyang University, 55 Hanyangdaehak-ro, Sangnok-gu, Ansan 426791, South Korea. Tel: +82-31-400-5802. Fax: +82-31-400-5958. E-mail: [email protected] Prof. Dr Chul Soon Yong, College of Pharmacy, Yeungnam University, 214-1, Dae-Dong, Gyongsan 712-749, South Korea. Tel: +82-53-8102812. Fax: +82-53-810-4654. E-mail: [email protected]
History Received 11 November 2014 Revised 20 April 2015 Accepted 4 May 2015 Published online 16 June 2015
Sinswat et al., 2008; Shin et al., 2010), prodrugs with poly(ethylene glycol) esters (Chung and Cho, 2004), inclusion complexes (Gao et al., 2012), microemulsion (Borhade et al., 2008; Wang et al., 2011) and solid dispersion (Park et al., 2009; Watts et al., 2009, 2010; Joe et al., 2010; Yoshida et al., 2012) have been used to avoid such complications. In recent years, considerable attention has been paid to develop lipid-based pharmaceutical preparations that amend the aqueous solubility and oral bioavailability of poorly water-soluble drugs. Self-nanoemulsifying drug delivery system (SNEDDS), a lipidbased formulation is defined as isotropic mixtures of natural or synthetic oils, surfactants/solvents and co-surfactants/co-solvents. SNEDDS is a promising technology that conveniently develops emulsion with gentle agitation, presents a high surface area for interaction between the formulation and the gastrointestinal fluid, offers a large solubilisation capacity and produces a small droplet size, which could facilitate permeation across the GI membrane (Rane and Anderson, 2008). SNEDDS is normally a liquid preparation encapsulated in soft gelatine capsules, which might be sensitive to humidity and results in high production cost. Furthermore, the liquid preparation might cause compatibility issues with the shell of the soft gelatine capsule and capsules that are inconvenient to the patient (Tuleu et al., 2004). The conversion of liquid SNEDDS into solid SNEDDS provides the advantages of solid SNEDDS and simultaneously eliminates the disadvantages of liquid SNEDDS (Kang et al., 2012).
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Spray drying is the one of the processes used to get dry emulsion. It is defined as a method by which a liquid solution is sprayed into a hot air chamber to evaporate the volatile fraction, i.e. the organic solvent or water in the microemulsion. Moreover, the spray-drying technique could resolve the stability issues pertaining to traditional emulsions during storage and deliver the dry product without injurious or deleterious organic solvent (Balakrishnan et al., 2009a). Colloidal silica is extensively used in the preparation of solid SNEDDS (Oh et al., 2012). In this research, a novel tacrolimus-loaded solid SNEDDS showing enhanced oral bioavailability of tacrolimus was prepared by spray-drying the solutions containing liquid SNEDDS and colloidal silica. Reconstitution aspects of the solid SNEDDS formulations were assessed as well. The solid state characterisation of the solid SNEDDS powder was achieved using scanning electron microscopy (SEM), differential scanning calorimetry (DSC) and powder X-ray diffraction (PXRD). In addition, dissolution study and pharmacokinetic investigations in rats were conducted compared to the commercial product.
Materials and methods Materials Tacrolimus was purchased from Shanghai Qiao Chemical Science Co. (Shanghai, China). Polyglycolysed glycerides, such as Capryol 90, Capryol PGMC, Labrafac CC, Labrafac LIPO WL 1349, Labrasol, Transcutol HP, Lauroglycol FCC and Peceol, were donated by Gattefosse Co. (St. Priest, France). Castor oil was provided by Sigma (St. Louis, MO). Sorbitan monolaurate 20 (Span 20) and sorbitan monooleate 80 (Span 80), polysorbate 20 (Tween 20) and polysorbate 80 (Tween 80) were bought from the DC Chemical Co. (Seoul, South Korea). AerosilÕ 200 was obtained from Degussa (Frankfurt, Germany). The commercial product (PrografÕ , hard capsule form) was procured from the Astellas Korea Pharm. Co. (Seoul, South Korea). All other chemicals were of reagent grade and used without further purification. Solubility The solubility test was carried out in order to select appropriate constituents for poorly water-soluble tacrolimus-loaded SNEDDS formulations (Yan et al., 2012; Lee et al., 2013). Solubility tests were executed by pouring an excess amount of tacrolimus (approximately 600 mg) in a 2 ml microtube (Axygen MCT-200, Union City, CA) carrying 1 ml of the vehicle (Table 1). After vortex-mixing, this mixture was loaded on the shaker in a water Table 1. Solubility of tacrolimus in various vehicles.
Vehicle Water Oils Capryol 90 Capryol PGMC Castor oil Peceol Surfactants Transcutol HP Labrasol Lauroglycol FCC Span 20 Span 80 Tween 20 Tween 80
Solubility of tacrolimus (mg/ml) 5.00 ± 0.12 ( 103) 46.77 ± 8.07 44.85 ± 4.72 1.69 ± 0.48 3.58 ± 1.17 59.15 ± 2.15 24.62 ± 3.20 23.72 ± 3.19 2.86 ± 2.37 1.33 ± 0.14 11.44 ± 4.35 10.34 ± 3.00
Note: Each value represents the mean ± S.D. (n¼3).
bath for 5 days at ambient temperature to attain equilibrium. The equilibrated sample was centrifuged at 10 000g for 15 min (Eppendorf, Hauppauge, NY) and the supernatant was filtered through a membrane filter (0.45 mm) to eliminate undissolved tacrolimus. Then, the filtrate was diluted with ethanol and analysed by HPLC for the quantification of tacrolimus (Hitachi, Tokyo, Japan). The HPLC system consisted of a pump (L-2130, Hitachi), an Inertsil ODS-2 column (5 mm, 250 4.6 mm) and a Phenomenex TS-130 column oven (Torrance, CA). As reported by a previous study, the column temperature was maintained at 60 C to generate the sharp and single peak of tacrolimus. The mobile phase, a mixture of acetonitrile and water (75:25, v/v), was filtered through a 0.45 mm membrane filter and was eluted at a flow rate of 1 ml/min. The eluent was monitored at a wavelength of 210 nm by the UV detector (L-2400, Hitachi, Tokyo, Japan) for tacrolimus concentration detection. Construction of the ternary phase diagram The pseudo-ternary phase diagram was constructed without tacrolimus to recognise the maximum self-emulsifying domain existence and to specify the optimal ratio of oil, surfactant and cosurfactant for the SNEDDS formulations (Balakrishnan et al., 2009a; Kang et al., 2012). The procedure described by Craig et al. (1995) was amended and applied in this investigation. A series of self-emulsifying compositions were formed for each of the four systems (Figure 1) with various volume percentages of surfactant (Labrasol) from 35 to 90% (v/v), different oils (Capryol 90 and Capryol PGMC) from 10 to 65% (v/v) and co-surfactants (Transcutol HP and Lauroglycol FCC) from 0 to 60% (v/v). For each mixture, the sum of individual percentages of the constituents was fixed to 100% (v/v) (Table 2). The ternary mixture (0.3 ml) was mildly agitated with 300 ml of distilled water in a glass beaker using a magnetic stirrer at 37 C. The tendency to emulsify spontaneously and the propagation of emulsion droplets were visually observed. The emulsion was referred to as ‘‘good’’ when oil globules dispersed evenly in water and developed a fine emulsion. On the other hand, the emulsion with coarse droplets or temporary emulsion exhibiting coalescence or creaming on terminating stirring was considered ‘‘bad’’ (Craig et al., 1995). All tests were performed in triplicate. To examine the influence of a drug on the self-emulsifying efficiency of SNEDDS, tacrolimus (5%, w/v) was incorporated into the boundary formulations of the self-emulsifying domain of the ternary phase diagrams. Preparation of liquid and solid SNEDDS Tacrolimus (50 mg) was added to 1 ml of each of the mixtures shown in Table 2 and vortexed until a transparent solution was achieved, leading to tacrolimus-loaded liquid SNEDDS. This formulation was visually observed for turbidity or phase separation before self-emulsification and particle size determination. Then, colloidal silica (1 g) was uniformly suspended in 400 ml ethanol by magnetic stirring. Subsequently, 4 g of liquid SNEDDS was poured into it with continuous stirring at ambient temperature for 15 min for excellent suspensions or emulsions. With constant stirring, the resultant liquid mixture was spray-dried using Bu¨chi mini spray dryer B-190 (Bu¨chi, Flawil, Switzerland) under the following spray-drying conditions: inlet temperature, 62 C; outlet temperature, 35 C; aspiration, 85%; drying air flow, 500 NL/h; feeding rate, 5 ml/min. Physicochemical characterisation Morphology The shape and surface features of the solid SNEDDS were determined by a scanning electron microscope
Developing a novel tacrolimus-loaded SNEDDS
DOI: 10.3109/02652048.2015.1057252
(S-4100, Hitachi, Tokyo, Japan) with an image analysis system (ImageInside Ver 2.32; Hitachi, Tokyo, Japan). The samples were secured on a brass specimen club using double-side adhesive tape and made electrically conductive by coating in vacuum (6 Pa) with platinum (6 nm/min) using Hitachi Iron Sputter (E-1030, Tokyo, Japan) for 180 s at 15 mA.
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scattering particle size analyser at a wavelength of 635 nm and a scattering angle of 90 at 25 C. Liquid or solid SNEDDS (equivalent to 10 mg tacrolimus) was added to 25 ml of distilled water and agitated slightly to get a fine emulsion; this was retained for 12 h at room temperature. The values of z-average diameters derived from cumulated analysis by Automeasure software (Malvern Instruments, Malvern, UK) were used.
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Crystallinity The thermal properties of tacrolimus-loaded solid SNEDDS were examined using a differential scanning calorimeter (DSC Q200 v24.2 build 107, TA Instruments; New Castle, DE). The samples (about 2 mg) sealed in aluminium pans were heated under a nitrogen flow (25 ml/min) at a heating rate of 10 C/min from 50 C to 2000 C. Moreover, the crystallinity of the solid SNEDDS formulations was tested by powder X-ray diffraction (D/Max-2500, Rigacu, Akishima, Japan), carried out at room temperature using monochromatic Cu-Ka-radiation (k ¼ 1.5418 ˚ ) at 40 mA and 40 kV in the region of 2.5 º 2y 40 with an A angular increment of 0.02 º/s. Emulsion particle size The particle size of the emulsion was analysed by a Zetasizer Nano ZS (Malvern Instruments, Malvern, UK) dynamic light
Dissolution Dissolution studies were completed using modified USP XXIII Dissolution Apparatus II (paddle; Shinseang Instrument Co., Hwasung, South Korea). The dissolution medium consisted of 0.005% hydroxypropyl cellulose solution (500 ml) adjusted to pH Table 2. Compositions formulations. Composition Tacrolimus (g) Capryol 90 (ml) Transcutol HP (ml) Lauroglycol FCC (ml) Labrasol (ml)
of
tacrolimus-loaded
liquid
SNEDDS
I
II
III
IV
5 – 15 – 75
5 10 15 – 75
5 – – 15 75
5 10 – 15 75
Figure 1. Pseudo-ternary phase diagram of formulation I to VI. (A), formulation I; (B), formulation II; (C), formulation III; (D), formulation IV. Black region shows the domain of efficient self-emulsification.
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4.5 by phosphoric acid at 36.5 C. The paddles were revolved at 50 rpm. The solid SNEDDS formulations and the commercial product equivalent to 1 mg of tacrolimus were filled in hard gelatine capsules. Each capsule was inserted into a sinker and dropped into the dissolution tester (Shinseang Instrument Co., Hwasung, South Korea). At fixed time-points, 1 ml medium was withdrawn with syringe and passed through a membrane filter (0.45 lm). After that, the titre of tacrolimus in the filtrate (100 ll) was quantified by HPLC as mentioned above.
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In vivo study Male Sprague-Dawley rats (7–9 weeks old, weighing 270–330 g) were acquired from the Charles River Company Korea (Orient, Seoul, Korea) and placed at a temperature of 20–23 C and the relative humidity of 50 ± 5%. They were fasted for 24–36 h prior to pharmacokinetic investigations but were allowed free access to water. All animal care and experimentations were done in accordance with the Guiding Principles in the Use of Animals in Toxicology, as accepted in 1989, reviewed in 1999, and modified in 2008 by the Society of Toxicology (SOT, 2008). The criteria for the use of animals in research were also permitted by the Institute of Laboratory Animal Resources of Yeungnam University. All examinations were commenced at 10 AM in order to avoid circadian-dependent variations in the pharmacokinetic behaviour of tacrolimus (Park et al., 2007). The solid SNEDDS formulations and tacrolimus powder were placed in small hard colloidal silica capsules (#9, Suheung Capsule Co., Seoul, Korea), respectively. The rats, divided into two groups, were orally given tacrolimus-loaded solid SNEDDS preparations and the commercial product at a drug dose of 3 mg/kg, respectively. Under light anaesthesia (induced with diethyl ether), 100 ml of blood was sampled from the right subclavian vein into heparin-treated tube at pre-set time-points and centrifuged at 3000g for 15 min using a 5415C centrifuge (Eppendorf, Hauppauge, NY). The titre of tacrolimus in each sample was detected by the Pro-TracÔ II FK 506 Enzyme-Linked Immunosorbent Assay (ELISA) kit (Diasorin Inc., Stilwater, MN) (Watts et al., 2010).
Results and discussion The purpose of the solubility study was to select appropriate constituents for SNEDDS formulations. The solubility of tacrolimus in various vehicles is shown in Table 1. The aqueous solubility of tacrolimus was about 5 lg/ml, advocating that the drug was poorly water-soluble (Joe et al., 2010). In our study, all of the vehicles enhanced the drug solubility. Capryol 90 and Capryol PGMC demonstrated higher drug solubility than other oil candidates. Therefore, they were chosen as oily phase owing to more hydrophilic nature, good drug solubility and emulsion forming ability. Amongst the surfactants tested, Labrasol was selected as a surfactant because it exhibited relatively higher drug solubility and was reported to improve the absorption of drugs from the intestine (Balakrishnan et al., 2009b). Likewise, Lauroglycol FCC and Transcutol HP were designated as co-surfactants because they improved drug solubility more compared to the others. In addition, they were likely to improve the drug loading capacity and form the excellent fine emulsion spontaneously. A number of SNEDDS formulations were prepared and their self-emulsification aspects were witnessed visually. Pseudo-ternary phase diagram was constructed to identify the optimum concentration of oil, surfactant and co-surfactant in the SNEDDS formulation. In this research, Capryol 90 and Capryol PGMC were nominated as oily vehicles, Labrasol as surfactant, Lauroglycol FCC or Transcutol HP as co-surfactants. All of these showed a higher solubility of tacrolimus and physically well mixed without any chemical interaction, leading to excellent liquid SNEDDS.
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Generally, in the development of pharmaceutical products, drug and ingredients should not be interacted for drug stability (Cho et al., 2010). Each phase diagram of four formulations is revealed in Figure 1. It was stated that the emulsification was not efficient with less than 60% of surfactant ratio. In this condition, the emulsion domain exhibited immediate coalescence and creaming of emulsion. It was observed that an increased concentration of the co-surfactant (Transcutol HP or Lauroglycol FCC) resulted in an increased tendency of the spontaneous self-emulsification process. Addition of the co-surfactant to the compositions further reduced the interfacial tension between the oil and water. Pouton (1985) reported that drugs incorporated in the SNEDDS might present some interference on the self-emulsification. However, in this study, no considerable changes were noticed in self-emulsifying performance when compared with the corresponding formulations loaded with 5% w/v tacrolimus. Droplet size is one of the most imperative factors that might influence the dissolution and bioavailability of drugs. Larger particle size delivers lower solubility and dissolution rates in gastrointestinal tracts and, in turn, lowers absorption and bioavailability. This is particularly significant for drugs whose dissolution is the rate-limiting factor in absorption (Han et al., 2006). It is shown that increasing the surfactant concentration (Labrasol with Capryol PGMC or Capryol 90) from 40 to 85% in the SNEDDS formulation decreased z-average particle size of emulsion, but that above 75% of Labrasol, the z-average particle size rapidly increased (Figure 2A). In general, as the concentration of surfactant was increased, the particle size was decreased (Balakrishnan et al., 2009a). In addition, the effect of the ratio of oil and co-surfactants (Transcutol HP and Lauroglycol FCC) (from 5/15 to 20/0) on the z-average particle size in the SNEDDS formula with 75% surfactant was investigated (Figure 2B). In all four mixed compositions, with up to 15% co-surfactant, the z-average particle sizes were gradually reduced. However, with 20% co-surfactant, the z-average particle size was slightly increased (Figure 2B). Hence, four compositions of liquid SNEDDS were chosen for further study, as mentioned in Table 2. The 5% tacrolimus-loaded solid SNEDDS formulation was obtained by spray-drying liquid mixture containing colloidal silica and liquid SNEDDS (Table 2) (Marasini et al., 2013). Colloidal silica has been used as a drug carrier and absorbent in the pharmaceutical industry because it is a porous material and can be physically attached to the liquid material without chemical interaction (Oh et al., 2012). The z-average particle size of the liquid and solid SNEDDS are illustrated in Figure 3. Both z-average particle sizes of liquid and solid SNEDDS were less than 300 nm, except formulation IV (all PDI50.3; data not shown). Furthermore, there were no considerable variances in z-average particle size between liquid and solid SNEDDS. The dissolution rate of the drug from each solid SNEDDS preparation was compared to that of the commercial product (Figure 4). Capryol PGMC bettered the dissolution of tacrolimus more than Capryol 90 (formulation I vs. II; III vs. IV). Moreover, the solid SNEDDS preparations formulated with Transcutol HP enhanced the dissolution rate more compared with those with Lauroglycol FCC (formulation I vs. III; II vs. IV). Among the solid SNEDDS preparations tested, only formulation I prepared with Capryol PGMC and Transcutol HP showed remarkably faster dissolution rates at all time-points compared to the commercial product. In particular, formulation I gave approximately 3.5-fold and 1.5-fold dissolution rates of drug compared to the commercial product at 5 and 30 min (67.6 ± 5.6 vs. 19.2 ± 3.5%; 87.4 ± 11.0% vs. 60.3 ± 5.7%), respectively. Accordingly, Capryol PGMC/ Transcutol HP/Labrasol (10:15:75%) with 5% w/v tacrolimus was selected as a tacrolimus-loaded solid SNEDDS formulation for further investigation.
Developing a novel tacrolimus-loaded SNEDDS
DOI: 10.3109/02652048.2015.1057252
(A) 1600
(B)
Transcutol HP/Capryol PGMC Transcutol HP/Capryol 90 Lauroglycol FCC/Capryol PGMC Lauroglycol FCC/Capryol 90
Z - average particle size (nm)
Z- average particle size (nm)
1000
1200
800
400 Capryol PGMC Capryol 90 20
40
60
800
600
400
200
0 80
5 5/1
100
Labrasol (%, v/v)
10/
10
5 15/
20/
0
Co-surfactant/Oil (%, v/v)
600
100
Liquid SNEDDS Solid SNEDDS
80 Tacrolimus dissolved (%)
500 Z- average particle size (nm)
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Figure 2. Effect of surfactant (A) and co-surfactant/oil ratio (B) on the Z-average particle size of liquid SNEDDS. Notes: These liquid SNEDDS formulations were composed of a 0.1 ml mixture of surfactant/co-surfactant/oil and 100 ml water. In (B), Labrasol percentage volume was fixed to 80%. Each value represents the mean ± S.D. (n ¼ 3).
5
400
300
200
60
40 I II III IV Commercial product
20
100 0
0
0 I
II
III
IV
10
20
30
Time (min)
Figure 3. Mean z-average particle size of liquid and solid SNEDDS formulations. Notes: Each value represents the mean ± S.D. (n ¼ 3). Each liquid SNEDDS formulation was seen in Table 2. Each solid SNEDDS formulation was composed of each corresponding liquid SNEDDS and colloidal silica at the weight ratio of 1:4.
Figure 4. Dissolution profiles of solid SNEDDS formulations and the commercial product in water. Notes: Each value represents the mean ± S.D. (n ¼ 6). Each liquid SNEDDS formulation was seen in Table 2. Each solid SNEDDS formulation was composed of each corresponding liquid SNEDDS and colloidal silica at the weight ratio of 1:4.
The scanning electron micrographs of tacrolimus powder, colloidal silica and the solid SNEDDS are shown in Figure 5. Tacrolimus powder (Figure 5A) appeared as smooth-surfaced rectangular-shaped crystals (Oh et al., 2011). Figure 5(B) shows the characteristic features of colloidal silica with an extremely coarse and porous surface, which might permit the percolation of the liquid phase into the matrix. Likewise, the solid SNEDDS had a relatively rough surface (Figure 5C). Our results suggest that the tacrolimus-loaded liquid SNEDDS might be absorbed in the pores of solid carriers and adsorbed onto their surface, resulting in the tacrolimus-loaded solid SNEDDS after spray-drying (Kang et al., 2012). The thermal features of the drug powder, colloidal silica and solid SNEDDS are displayed in Figure 6(a). The physical mixtures were made by simply mixing the carriers and drug.
Our DSC results proved that tacrolimus had an exclusive endothermic peak at about 115 C, representing its melting point and crystalline nature (Figure 6a-A). Furthermore, a relatively minute endothermic peak corresponding to the melting point of the drug was also noticed in the DSC curve of physical mixture (Figure 6a-B). Conversely, colloidal silica (Figure 6a-C) and solid SNEDDS (Figure 6a-D) had no intrinsic peak. Our DSC results confirmed that tacrolimus exists in an amorphous form in the solid SNEDDS (Kim et al., 2011; Oh et al., 2011). The powder X-ray diffraction patterns are presented in Figure 6(b). Tacrolimus generated typical crystalline sharp peaks at diffraction angles (Figure 6b-A). Colloidal silica (Figure 6b-C) gave no intrinsic peaks. All of the chief distinctive crystalline peaks pertaining to the drug and colloidal silica were witnessed in
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Figure 6. Differential scanning calorimetric thermogram (a) and Powder X-ray diffractogram (b): (A) tacrolimus; (B) physical mixture; (C) colloidal silica; (D) solid SNEDDS. 40
Plasma concentration (ng/mL)
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Figure 5. Scanning electron micrographics: (A) tacrolimus powder (5000); (B) colloidal silica (3000); (C) solid SNEDDS (5000).
30
* *
Commercial product Solid SNEDDS
** * * *
20
* 10
*
0 0
3
6
9 12 Time (h)
24
Figure 7. Plasma concentration-time profiles of tacrolimus after oral administration of the commercial product and solid SNEDDS formulation in rats. Notes: Each value represents the mean ± S.D. (n ¼ 6). *p50.05 compared with the commercial product. The solid SNEDDS formulation composed of liquid SNEDDS [tacrolimus/Capryol PGMC/Transcutol HP/ Labrasol (5:10:15:75, w/v/v/v)] and colloidal silica at the weight ratio of 1:4.
the physical mixture (Figure 6b-B). The representative crystalline peaks of tacrolimus were not witnessed in the PXRD pattern of SNEDDS, confirming the amorphous state of drug in the SNEDDS (Figure 6b-D) (Kang et al., 2012; Lee et al., 2013).
According to our results, the drug-loaded liquid SNEDDS was spray-dried together with colloidal silica, resulting in the amorphous state of the drug in the solid SNEDDS formulations. In this tacrolimus-loaded solid SNEDDS, the liquid SNEDDS was physically and not chemically absorbed in the pores of colloidal silica and adsorbed onto its surface. On reconstituting in aqueous solution, the liquid SNEDDS leaked out from the solid carriers in the solid SNEDDS, was in contact with water, and spontaneously formed the nanoemulsion in aqueous solution. Moreover, the drug loaded in the solid SNEDDS was quickly dissolved, diffused and dispersed in the dissolution medium. Thus, this SNEDDS formulation resulted in the spontaneous formation of an interface between the oil droplets and water, ultimately decreasing the particle size of the droplets (Balakrishnan et al., 2009a; Kang et al., 2012). In the self-emulsifying compositions, because the amount of free energy required to form an emulsion is very low, the spontaneous formation of an interface between oil droplets and water was allowed (Balakrishnan et al., 2009b). This suggested that the oil/surfactant phase and water phase swelled effectively, reduced the size of the oil droplets and consequently improved the drug release rate. The change in the mean plasma concentration of tacrolimus after oral administration of the solid SNEDDS and the commercial formulation to rats is shown in Figure 7. Furthermore, the pharmacokinetic parameters are displayed in Table 3. The solid SNEDDS offered markedly higher plasma levels of tacrolimus at every time point compared to the commercial product (Figure 7). The solid SNEDDS furnished considerably higher AUC and Cmax and shorter Tmax value than those of the commercial product (p50.05). In particular, the AUC value of solid SNEDDS was approximately 2-fold greater than that of the commercial product. Our results suggested that the solid SNEDDS greatly promoted
Developing a novel tacrolimus-loaded SNEDDS
DOI: 10.3109/02652048.2015.1057252
Table 3. Pharmacokinetic parameters. Parameter Tmax (h) Cmax (ng/ml) T1/2 (h) Ke (h1) AUC (hng/ml)
Commercial product
Solid SNEDDS
0.57 ± 0.17 10.72 ± 4.82 7.32 ± 3.16 0.12 ± 0.07 166.60 ± 82.48
0.30 ± 0.09* 35.42 ± 5.20* 9.27 ± 1.97 0.08 ± 0.02 357.41 ± 79.05*
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Note: Each value represents the mean ± S.D. (n ¼ 6). *p50.05 compared with the commercial product.
the oral bioavailability of tacrolimus owing to faster and better absorption because of the increased dissolution of the drug (Yan et al., 2012; Tran et al., 2013). However, there was no considerable difference between Kel values and t1/2 values corresponding to the solid SNEDDS formulation and the commercial product.
Conclusion In the tacrolimus-loaded solid SNEDDS formulated with colloidal silica, the liquid SNEDDS composed of Capryol PGMC, Transcutol HP and Labrasol (10:15:75, v/v/v) was adsorbed in the pores and onto the surface of the solid carrier. Moreover, the drug was in the amorphous state in the solid SNEDDS. The solid SNEDDS with 5% w/v tacrolimus produced nanoemulsions with about 300 nm droplet size, and augmented the dissolution rate and the oral bioavailability of tacrolimus in rats due to the swift spontaneous emulsion formation without chemical interaction and the reduced droplet size. Therefore, this solid SNEDDS would be a potential candidate with enhanced oral bioavailability of tacrolimus.
Declaration of interest The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper. This work was supported by the National Research Foundation of Korea (NRF) grant supported by the Korean government (MEST) (No. 2012R1A2A2A01045658) and the High value-added Food Technology Development Program 313021-3 by the Ministry of Agriculture, Food and Rural Affairs in South Korea.
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Han J, Beeton A, Long P, Wong I, Tuleu C. Physical and microbiological stability of an extemporaneous tacrolimus suspension for paediatric use. J Clin Pharm Ther, 2006;31:167–72. Joe JH, Lee WM, Park YJ, Joe KH, Oh DH, Seo YG, Woo JS, Yong CS, Choi HG. Effect of the solid-dispersion method on the solubility and crystalline property of tacrolimus. Int J Pharm, 2010;395:161–6. Kang JH, Oh DH, Oh YK, Yong CS, Choi HG. Effects of solid carriers on the crystalline properties, dissolution and bioavailability of flurbiprofen in solid self-nanoemulsifying drug delivery system (solid SNEDDS). Eur J Pharm Biopharm, 2012;80(2):289–97. Kim YI, Kim KS, Suh KH, Shanmugam S, Woo JS, Yong CS, Choi HG. New clopidogrel napadisilate salt and its solid dispersion with improved stability and bioequivalence to the commercial clopidogrel bisulphate salt in beagle dogs. Int J Pharm, 2011;415:129–39. Lee SN, Poudel BK, Tran TH, Marasini N, Pradhan R, Lee YI, Lee DW, Woo JS, Choi HG, Yong CS, Kim JO. A novel surface-attached carvedilol solid dispersion with enhanced solubility and dissolution. Arch Pharm Res, 2013;36(1):79–85. Marasini N, Tran TH, Poudel BK, Cho HJ, Choi YK, Chi SC, Choi HG, Yong CS, Kim JO. Fabrication and evaluation of pH-modulated solid dispersion for telmisartan by spray-drying technique. Int J Pharm, 2013;441:424–32. Nassar T, Rom A, Nyska A, Benita S. A novel nanocapsule delivery system to overcome intestinal degradation and drug transport limited absorption of P-glycoprotein substrate drugs. Pharm Res, 2008;25: 2019–29. Oh DH, Balakrishnan P, Oh YK, Kim DD, Yong CS, Choi HG. Effect of process parameters on nanoemulsion droplet size and distribution in SPG membrane emulsification. Int J Pharm, 2011;14:191–7. Oh DH, Kang JH, Kim DW, Lee BJ, Kim JO, Yong CS, Choi HG. Comparison of solid self-microemulsifying drug delivery system (solid SMEDDS) prepared with hydrophilic and hydrophobic solid carrier. Int J Pharm, 2012;420:412–18. Park SI, Felipe CR, Pinheiro-Machado PG, Garcia R, Tedesco-Silva Jr H, Medina-Pestana JO. Circadian and time-dependent variability in tacrolimus pharmacokinetics. Fundam Clin Pharmacol, 2007;21: 191–7. Park YJ, Ryu DS, Li DX, Quan QZ, Oh DH, Kim JO, Seo YG, Lee YI, Yong CS, Woo JS, Choi HG. Physicochemical characterisation of tacrolimus-loaded solid dispersion with sodium carboxylmethyl cellulose and sodium lauryl sulphate. Arch Pharm Res, 2009;32(6):893–8. Pouton C. Self-emulsifying drug delivery systems: assessment of the efficiency of emulsification. Int J Pharm, 1985;27:335–48. Rane SS, Anderson BD. What determines drug solubility in lipid vehicles: Is it predictable? Adv Drug Deliv Rev, 2008;60:638–56. Shapiro R. Tacrolimus in solid organ transplantation: An update. Transplant Proc, 1999;31:2203–5. Shimomura M, Masuda S, Saito H, Sakamoto S, Uemoto S, Tanaka K, Inui K. Roles of the jejunum and ileum in the first-pass effect as absorptive barriers for orally administered tacrolimus. J Surg Res, 2002;103:215–22. Shin SB, Cho HY, Kim DD, Choi HG, Lee YB. Preparation and evaluation of tacrolimus-loaded nanoparticles for lymphatic delivery. Eur J Pharm Biopharm, 2010;74:164–71. Sinswat P, Overhoff KA, McConville JT, Johnston KP, Williams 3rd RO. Nebulisation of nanoparticulate amorphous or crystalline tacrolimussingle-dose pharmacokinetics study in mice. Eur J Pharm Biopharm, 2008;69(3):1057–66. Society of Toxicology (SOT). 2008. Guiding principles in the use of animals in toxicology. Available at www.toxicology.org/AI/FA/guidingprinciples.pdf. Accessed December. Spencer CM, Goa KL, Gillis JC. Tacrolimus. An update of its pharmacology and clinical efficacy in the management of organ transplantation. Drugs, 1997;54:925–75. Tran TH, Poudel BK, Marasini N, Chi SC, Choi HG, Yong CS, Kim JO. Preparation and evaluation of raloxifene-loaded solid dispersion nanoparticle by spray-drying technique without an organic solvent. Int J Pharm, 2013;443:50–7. Tuleu C, Newton M, Rose J, Euler D, Saklatvala R, Clarke A, Booth S. Comparative bioavailability study in dogs of a self-emulsifying formulation of progesterone presented in a pellet and liquid form compared with an aqueous suspension of progesterone. J Pharm Sci, 2004;93:1495–502. Wang Y, Sun J, Zhang T, Liu H, He F, He Z. Enhanced oral bioavailability of tacrolimus in rats by self-microemulsifying drug delivery systems. Drug Dev Ind Pharm, 2011;37(10):1225–30.
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Yan YD, Sung JH, Kim KK, Kim DW, Kim JO, Lee BJ, Yong CS, Choi HG. Novel valsartan-loaded solid dispersion with enhanced bioavailability and no crystalline changes. Int J Pharm, 2012;422:202–10. Yoshida T, Kurimoto I, Yoshihara K, Umejima H, Ito N, Watanabe S, Sako K, Kikuchi A. Aminoalkyl methacrylate copolymers for improving the solubility of tacrolimus. I: Evaluation of solid dispersion formulations. Int J Pharm, 2012;428:18–24.
RESEARCH ARTICLE
Solid self-nanoemulsifying drug delivery system (SNEDDS) for enhanced oral bioavailability of poorly water-soluble tacrolimus: physicochemical characterisation and pharmacokinetics Journal of Microencapsulation Downloaded from informahealthcare.com by Nyu Medical Center on 06/21/15 For personal use only.
Youn Gee Seo1, Dong Wuk Kim2, Abid Mehmood Yousaf2, Jong Hyuck Park2, Pahn-Shick Chang3, Hyung Hee Baek4, Soo-Jeong Lim5, Jong Oh Kim1, Chul Soon Yong1, and Han-Gon Choi2 1
College of Pharmacy, Yeungnam University, Gyongsan, South Korea, 2College of Pharmacy & Institute of Pharmaceutical Science and Technology, Hanyang University, Ansan, South Korea, 3Department of Agricultural Biotechnology, Seoul National University, Seoul, South Korea, 4Department of Food Engineering, Dankook University, Cheonan, South Korea, and 5Department of Bioscience and Biotechnology of Life Sciences and Biotechnology, Sejong University, Seoul, South Korea Abstract
Keywords
To develop a novel self-nanoemulsifying drug delivery system (solid SNEDDS) with better oral bioavailability of tacrolimus, the solid SNEDDS was obtained by spray-drying the solutions containing the liquid SNEDDS and colloidal silica. Its reconstitution properties were determined and correlated to solid state characterisation of the powder. Moreover, the dissolution and pharmacokinetics in rats was done in comparison to the commercial product. Among the liquid SNEDDS formulations tested, the liquid SNEDDS comprised of Capryol PGMC, Transcutol HP and Labrasol (10:15:75, v/v/v) presented the highest dissolution rate. In the solid SNEDDS, this liquid SNEDDS was absorbed in the pores and attached onto the surface of the colloidal silica. Drug was present in the amorphous state in it. The solid SNEDDS with 5% w/v tacrolimus produced the nanoemulsions and improved the oral bioavailability of tacrolimus in rats. Therefore, this solid SNEDDS would be a potential candidate for enhancing the oral bioavailability of tacrolimus.
Bioavailability, colloidal silica, self-nanoemulsifying drug delivery system, solid carrier, tacrolimus
Introduction The oral route is the safest and most frequently used way to administer drugs for the treatment of various diseases. However, more than 50% of the active pharmaceutical compounds exhibit very low aqueous solubility after oral administration, which results in poor oral bioavailability, high intra-subject and intersubject differences and a lack of dose proportionality. Tacrolimus, a 23-member macrolide lactone obtained from Streptomyces tsukubaensis, is an effective immunosuppressive drug that has been therapeutically employed for avoiding rejection after solid organ transplantation (Shapiro, 1999; Spencer et al., 1997). However, its efficacy is profoundly affected due to its poor solubility in aqueous media (lower than 5 mg/ml) (Joe et al., 2010) and pre-systemic biotransformation by cytochrome P450 3A containing enzymes and P-glycoprotein efflux (Shimomura et al., 2002). Numerous formulations of tacrolimus for oral administration such as nanoparticles (Nassar et al., 2008; Address for correspondence: Prof. Dr Han-Gon Choi, College of Pharmacy & Institute of Pharmaceutical Science and Technology, Hanyang University, 55 Hanyangdaehak-ro, Sangnok-gu, Ansan 426791, South Korea. Tel: +82-31-400-5802. Fax: +82-31-400-5958. E-mail: [email protected] Prof. Dr Chul Soon Yong, College of Pharmacy, Yeungnam University, 214-1, Dae-Dong, Gyongsan 712-749, South Korea. Tel: +82-53-8102812. Fax: +82-53-810-4654. E-mail: [email protected]
History Received 11 November 2014 Revised 20 April 2015 Accepted 4 May 2015 Published online 16 June 2015
Sinswat et al., 2008; Shin et al., 2010), prodrugs with poly(ethylene glycol) esters (Chung and Cho, 2004), inclusion complexes (Gao et al., 2012), microemulsion (Borhade et al., 2008; Wang et al., 2011) and solid dispersion (Park et al., 2009; Watts et al., 2009, 2010; Joe et al., 2010; Yoshida et al., 2012) have been used to avoid such complications. In recent years, considerable attention has been paid to develop lipid-based pharmaceutical preparations that amend the aqueous solubility and oral bioavailability of poorly water-soluble drugs. Self-nanoemulsifying drug delivery system (SNEDDS), a lipidbased formulation is defined as isotropic mixtures of natural or synthetic oils, surfactants/solvents and co-surfactants/co-solvents. SNEDDS is a promising technology that conveniently develops emulsion with gentle agitation, presents a high surface area for interaction between the formulation and the gastrointestinal fluid, offers a large solubilisation capacity and produces a small droplet size, which could facilitate permeation across the GI membrane (Rane and Anderson, 2008). SNEDDS is normally a liquid preparation encapsulated in soft gelatine capsules, which might be sensitive to humidity and results in high production cost. Furthermore, the liquid preparation might cause compatibility issues with the shell of the soft gelatine capsule and capsules that are inconvenient to the patient (Tuleu et al., 2004). The conversion of liquid SNEDDS into solid SNEDDS provides the advantages of solid SNEDDS and simultaneously eliminates the disadvantages of liquid SNEDDS (Kang et al., 2012).
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Spray drying is the one of the processes used to get dry emulsion. It is defined as a method by which a liquid solution is sprayed into a hot air chamber to evaporate the volatile fraction, i.e. the organic solvent or water in the microemulsion. Moreover, the spray-drying technique could resolve the stability issues pertaining to traditional emulsions during storage and deliver the dry product without injurious or deleterious organic solvent (Balakrishnan et al., 2009a). Colloidal silica is extensively used in the preparation of solid SNEDDS (Oh et al., 2012). In this research, a novel tacrolimus-loaded solid SNEDDS showing enhanced oral bioavailability of tacrolimus was prepared by spray-drying the solutions containing liquid SNEDDS and colloidal silica. Reconstitution aspects of the solid SNEDDS formulations were assessed as well. The solid state characterisation of the solid SNEDDS powder was achieved using scanning electron microscopy (SEM), differential scanning calorimetry (DSC) and powder X-ray diffraction (PXRD). In addition, dissolution study and pharmacokinetic investigations in rats were conducted compared to the commercial product.
Materials and methods Materials Tacrolimus was purchased from Shanghai Qiao Chemical Science Co. (Shanghai, China). Polyglycolysed glycerides, such as Capryol 90, Capryol PGMC, Labrafac CC, Labrafac LIPO WL 1349, Labrasol, Transcutol HP, Lauroglycol FCC and Peceol, were donated by Gattefosse Co. (St. Priest, France). Castor oil was provided by Sigma (St. Louis, MO). Sorbitan monolaurate 20 (Span 20) and sorbitan monooleate 80 (Span 80), polysorbate 20 (Tween 20) and polysorbate 80 (Tween 80) were bought from the DC Chemical Co. (Seoul, South Korea). AerosilÕ 200 was obtained from Degussa (Frankfurt, Germany). The commercial product (PrografÕ , hard capsule form) was procured from the Astellas Korea Pharm. Co. (Seoul, South Korea). All other chemicals were of reagent grade and used without further purification. Solubility The solubility test was carried out in order to select appropriate constituents for poorly water-soluble tacrolimus-loaded SNEDDS formulations (Yan et al., 2012; Lee et al., 2013). Solubility tests were executed by pouring an excess amount of tacrolimus (approximately 600 mg) in a 2 ml microtube (Axygen MCT-200, Union City, CA) carrying 1 ml of the vehicle (Table 1). After vortex-mixing, this mixture was loaded on the shaker in a water Table 1. Solubility of tacrolimus in various vehicles.
Vehicle Water Oils Capryol 90 Capryol PGMC Castor oil Peceol Surfactants Transcutol HP Labrasol Lauroglycol FCC Span 20 Span 80 Tween 20 Tween 80
Solubility of tacrolimus (mg/ml) 5.00 ± 0.12 ( 103) 46.77 ± 8.07 44.85 ± 4.72 1.69 ± 0.48 3.58 ± 1.17 59.15 ± 2.15 24.62 ± 3.20 23.72 ± 3.19 2.86 ± 2.37 1.33 ± 0.14 11.44 ± 4.35 10.34 ± 3.00
Note: Each value represents the mean ± S.D. (n¼3).
bath for 5 days at ambient temperature to attain equilibrium. The equilibrated sample was centrifuged at 10 000g for 15 min (Eppendorf, Hauppauge, NY) and the supernatant was filtered through a membrane filter (0.45 mm) to eliminate undissolved tacrolimus. Then, the filtrate was diluted with ethanol and analysed by HPLC for the quantification of tacrolimus (Hitachi, Tokyo, Japan). The HPLC system consisted of a pump (L-2130, Hitachi), an Inertsil ODS-2 column (5 mm, 250 4.6 mm) and a Phenomenex TS-130 column oven (Torrance, CA). As reported by a previous study, the column temperature was maintained at 60 C to generate the sharp and single peak of tacrolimus. The mobile phase, a mixture of acetonitrile and water (75:25, v/v), was filtered through a 0.45 mm membrane filter and was eluted at a flow rate of 1 ml/min. The eluent was monitored at a wavelength of 210 nm by the UV detector (L-2400, Hitachi, Tokyo, Japan) for tacrolimus concentration detection. Construction of the ternary phase diagram The pseudo-ternary phase diagram was constructed without tacrolimus to recognise the maximum self-emulsifying domain existence and to specify the optimal ratio of oil, surfactant and cosurfactant for the SNEDDS formulations (Balakrishnan et al., 2009a; Kang et al., 2012). The procedure described by Craig et al. (1995) was amended and applied in this investigation. A series of self-emulsifying compositions were formed for each of the four systems (Figure 1) with various volume percentages of surfactant (Labrasol) from 35 to 90% (v/v), different oils (Capryol 90 and Capryol PGMC) from 10 to 65% (v/v) and co-surfactants (Transcutol HP and Lauroglycol FCC) from 0 to 60% (v/v). For each mixture, the sum of individual percentages of the constituents was fixed to 100% (v/v) (Table 2). The ternary mixture (0.3 ml) was mildly agitated with 300 ml of distilled water in a glass beaker using a magnetic stirrer at 37 C. The tendency to emulsify spontaneously and the propagation of emulsion droplets were visually observed. The emulsion was referred to as ‘‘good’’ when oil globules dispersed evenly in water and developed a fine emulsion. On the other hand, the emulsion with coarse droplets or temporary emulsion exhibiting coalescence or creaming on terminating stirring was considered ‘‘bad’’ (Craig et al., 1995). All tests were performed in triplicate. To examine the influence of a drug on the self-emulsifying efficiency of SNEDDS, tacrolimus (5%, w/v) was incorporated into the boundary formulations of the self-emulsifying domain of the ternary phase diagrams. Preparation of liquid and solid SNEDDS Tacrolimus (50 mg) was added to 1 ml of each of the mixtures shown in Table 2 and vortexed until a transparent solution was achieved, leading to tacrolimus-loaded liquid SNEDDS. This formulation was visually observed for turbidity or phase separation before self-emulsification and particle size determination. Then, colloidal silica (1 g) was uniformly suspended in 400 ml ethanol by magnetic stirring. Subsequently, 4 g of liquid SNEDDS was poured into it with continuous stirring at ambient temperature for 15 min for excellent suspensions or emulsions. With constant stirring, the resultant liquid mixture was spray-dried using Bu¨chi mini spray dryer B-190 (Bu¨chi, Flawil, Switzerland) under the following spray-drying conditions: inlet temperature, 62 C; outlet temperature, 35 C; aspiration, 85%; drying air flow, 500 NL/h; feeding rate, 5 ml/min. Physicochemical characterisation Morphology The shape and surface features of the solid SNEDDS were determined by a scanning electron microscope
Developing a novel tacrolimus-loaded SNEDDS
DOI: 10.3109/02652048.2015.1057252
(S-4100, Hitachi, Tokyo, Japan) with an image analysis system (ImageInside Ver 2.32; Hitachi, Tokyo, Japan). The samples were secured on a brass specimen club using double-side adhesive tape and made electrically conductive by coating in vacuum (6 Pa) with platinum (6 nm/min) using Hitachi Iron Sputter (E-1030, Tokyo, Japan) for 180 s at 15 mA.
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scattering particle size analyser at a wavelength of 635 nm and a scattering angle of 90 at 25 C. Liquid or solid SNEDDS (equivalent to 10 mg tacrolimus) was added to 25 ml of distilled water and agitated slightly to get a fine emulsion; this was retained for 12 h at room temperature. The values of z-average diameters derived from cumulated analysis by Automeasure software (Malvern Instruments, Malvern, UK) were used.
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Crystallinity The thermal properties of tacrolimus-loaded solid SNEDDS were examined using a differential scanning calorimeter (DSC Q200 v24.2 build 107, TA Instruments; New Castle, DE). The samples (about 2 mg) sealed in aluminium pans were heated under a nitrogen flow (25 ml/min) at a heating rate of 10 C/min from 50 C to 2000 C. Moreover, the crystallinity of the solid SNEDDS formulations was tested by powder X-ray diffraction (D/Max-2500, Rigacu, Akishima, Japan), carried out at room temperature using monochromatic Cu-Ka-radiation (k ¼ 1.5418 ˚ ) at 40 mA and 40 kV in the region of 2.5 º 2y 40 with an A angular increment of 0.02 º/s. Emulsion particle size The particle size of the emulsion was analysed by a Zetasizer Nano ZS (Malvern Instruments, Malvern, UK) dynamic light
Dissolution Dissolution studies were completed using modified USP XXIII Dissolution Apparatus II (paddle; Shinseang Instrument Co., Hwasung, South Korea). The dissolution medium consisted of 0.005% hydroxypropyl cellulose solution (500 ml) adjusted to pH Table 2. Compositions formulations. Composition Tacrolimus (g) Capryol 90 (ml) Transcutol HP (ml) Lauroglycol FCC (ml) Labrasol (ml)
of
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5 10 15 – 75
5 – – 15 75
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Figure 1. Pseudo-ternary phase diagram of formulation I to VI. (A), formulation I; (B), formulation II; (C), formulation III; (D), formulation IV. Black region shows the domain of efficient self-emulsification.
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4.5 by phosphoric acid at 36.5 C. The paddles were revolved at 50 rpm. The solid SNEDDS formulations and the commercial product equivalent to 1 mg of tacrolimus were filled in hard gelatine capsules. Each capsule was inserted into a sinker and dropped into the dissolution tester (Shinseang Instrument Co., Hwasung, South Korea). At fixed time-points, 1 ml medium was withdrawn with syringe and passed through a membrane filter (0.45 lm). After that, the titre of tacrolimus in the filtrate (100 ll) was quantified by HPLC as mentioned above.
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In vivo study Male Sprague-Dawley rats (7–9 weeks old, weighing 270–330 g) were acquired from the Charles River Company Korea (Orient, Seoul, Korea) and placed at a temperature of 20–23 C and the relative humidity of 50 ± 5%. They were fasted for 24–36 h prior to pharmacokinetic investigations but were allowed free access to water. All animal care and experimentations were done in accordance with the Guiding Principles in the Use of Animals in Toxicology, as accepted in 1989, reviewed in 1999, and modified in 2008 by the Society of Toxicology (SOT, 2008). The criteria for the use of animals in research were also permitted by the Institute of Laboratory Animal Resources of Yeungnam University. All examinations were commenced at 10 AM in order to avoid circadian-dependent variations in the pharmacokinetic behaviour of tacrolimus (Park et al., 2007). The solid SNEDDS formulations and tacrolimus powder were placed in small hard colloidal silica capsules (#9, Suheung Capsule Co., Seoul, Korea), respectively. The rats, divided into two groups, were orally given tacrolimus-loaded solid SNEDDS preparations and the commercial product at a drug dose of 3 mg/kg, respectively. Under light anaesthesia (induced with diethyl ether), 100 ml of blood was sampled from the right subclavian vein into heparin-treated tube at pre-set time-points and centrifuged at 3000g for 15 min using a 5415C centrifuge (Eppendorf, Hauppauge, NY). The titre of tacrolimus in each sample was detected by the Pro-TracÔ II FK 506 Enzyme-Linked Immunosorbent Assay (ELISA) kit (Diasorin Inc., Stilwater, MN) (Watts et al., 2010).
Results and discussion The purpose of the solubility study was to select appropriate constituents for SNEDDS formulations. The solubility of tacrolimus in various vehicles is shown in Table 1. The aqueous solubility of tacrolimus was about 5 lg/ml, advocating that the drug was poorly water-soluble (Joe et al., 2010). In our study, all of the vehicles enhanced the drug solubility. Capryol 90 and Capryol PGMC demonstrated higher drug solubility than other oil candidates. Therefore, they were chosen as oily phase owing to more hydrophilic nature, good drug solubility and emulsion forming ability. Amongst the surfactants tested, Labrasol was selected as a surfactant because it exhibited relatively higher drug solubility and was reported to improve the absorption of drugs from the intestine (Balakrishnan et al., 2009b). Likewise, Lauroglycol FCC and Transcutol HP were designated as co-surfactants because they improved drug solubility more compared to the others. In addition, they were likely to improve the drug loading capacity and form the excellent fine emulsion spontaneously. A number of SNEDDS formulations were prepared and their self-emulsification aspects were witnessed visually. Pseudo-ternary phase diagram was constructed to identify the optimum concentration of oil, surfactant and co-surfactant in the SNEDDS formulation. In this research, Capryol 90 and Capryol PGMC were nominated as oily vehicles, Labrasol as surfactant, Lauroglycol FCC or Transcutol HP as co-surfactants. All of these showed a higher solubility of tacrolimus and physically well mixed without any chemical interaction, leading to excellent liquid SNEDDS.
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Generally, in the development of pharmaceutical products, drug and ingredients should not be interacted for drug stability (Cho et al., 2010). Each phase diagram of four formulations is revealed in Figure 1. It was stated that the emulsification was not efficient with less than 60% of surfactant ratio. In this condition, the emulsion domain exhibited immediate coalescence and creaming of emulsion. It was observed that an increased concentration of the co-surfactant (Transcutol HP or Lauroglycol FCC) resulted in an increased tendency of the spontaneous self-emulsification process. Addition of the co-surfactant to the compositions further reduced the interfacial tension between the oil and water. Pouton (1985) reported that drugs incorporated in the SNEDDS might present some interference on the self-emulsification. However, in this study, no considerable changes were noticed in self-emulsifying performance when compared with the corresponding formulations loaded with 5% w/v tacrolimus. Droplet size is one of the most imperative factors that might influence the dissolution and bioavailability of drugs. Larger particle size delivers lower solubility and dissolution rates in gastrointestinal tracts and, in turn, lowers absorption and bioavailability. This is particularly significant for drugs whose dissolution is the rate-limiting factor in absorption (Han et al., 2006). It is shown that increasing the surfactant concentration (Labrasol with Capryol PGMC or Capryol 90) from 40 to 85% in the SNEDDS formulation decreased z-average particle size of emulsion, but that above 75% of Labrasol, the z-average particle size rapidly increased (Figure 2A). In general, as the concentration of surfactant was increased, the particle size was decreased (Balakrishnan et al., 2009a). In addition, the effect of the ratio of oil and co-surfactants (Transcutol HP and Lauroglycol FCC) (from 5/15 to 20/0) on the z-average particle size in the SNEDDS formula with 75% surfactant was investigated (Figure 2B). In all four mixed compositions, with up to 15% co-surfactant, the z-average particle sizes were gradually reduced. However, with 20% co-surfactant, the z-average particle size was slightly increased (Figure 2B). Hence, four compositions of liquid SNEDDS were chosen for further study, as mentioned in Table 2. The 5% tacrolimus-loaded solid SNEDDS formulation was obtained by spray-drying liquid mixture containing colloidal silica and liquid SNEDDS (Table 2) (Marasini et al., 2013). Colloidal silica has been used as a drug carrier and absorbent in the pharmaceutical industry because it is a porous material and can be physically attached to the liquid material without chemical interaction (Oh et al., 2012). The z-average particle size of the liquid and solid SNEDDS are illustrated in Figure 3. Both z-average particle sizes of liquid and solid SNEDDS were less than 300 nm, except formulation IV (all PDI50.3; data not shown). Furthermore, there were no considerable variances in z-average particle size between liquid and solid SNEDDS. The dissolution rate of the drug from each solid SNEDDS preparation was compared to that of the commercial product (Figure 4). Capryol PGMC bettered the dissolution of tacrolimus more than Capryol 90 (formulation I vs. II; III vs. IV). Moreover, the solid SNEDDS preparations formulated with Transcutol HP enhanced the dissolution rate more compared with those with Lauroglycol FCC (formulation I vs. III; II vs. IV). Among the solid SNEDDS preparations tested, only formulation I prepared with Capryol PGMC and Transcutol HP showed remarkably faster dissolution rates at all time-points compared to the commercial product. In particular, formulation I gave approximately 3.5-fold and 1.5-fold dissolution rates of drug compared to the commercial product at 5 and 30 min (67.6 ± 5.6 vs. 19.2 ± 3.5%; 87.4 ± 11.0% vs. 60.3 ± 5.7%), respectively. Accordingly, Capryol PGMC/ Transcutol HP/Labrasol (10:15:75%) with 5% w/v tacrolimus was selected as a tacrolimus-loaded solid SNEDDS formulation for further investigation.
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DOI: 10.3109/02652048.2015.1057252
(A) 1600
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Figure 2. Effect of surfactant (A) and co-surfactant/oil ratio (B) on the Z-average particle size of liquid SNEDDS. Notes: These liquid SNEDDS formulations were composed of a 0.1 ml mixture of surfactant/co-surfactant/oil and 100 ml water. In (B), Labrasol percentage volume was fixed to 80%. Each value represents the mean ± S.D. (n ¼ 3).
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Figure 3. Mean z-average particle size of liquid and solid SNEDDS formulations. Notes: Each value represents the mean ± S.D. (n ¼ 3). Each liquid SNEDDS formulation was seen in Table 2. Each solid SNEDDS formulation was composed of each corresponding liquid SNEDDS and colloidal silica at the weight ratio of 1:4.
Figure 4. Dissolution profiles of solid SNEDDS formulations and the commercial product in water. Notes: Each value represents the mean ± S.D. (n ¼ 6). Each liquid SNEDDS formulation was seen in Table 2. Each solid SNEDDS formulation was composed of each corresponding liquid SNEDDS and colloidal silica at the weight ratio of 1:4.
The scanning electron micrographs of tacrolimus powder, colloidal silica and the solid SNEDDS are shown in Figure 5. Tacrolimus powder (Figure 5A) appeared as smooth-surfaced rectangular-shaped crystals (Oh et al., 2011). Figure 5(B) shows the characteristic features of colloidal silica with an extremely coarse and porous surface, which might permit the percolation of the liquid phase into the matrix. Likewise, the solid SNEDDS had a relatively rough surface (Figure 5C). Our results suggest that the tacrolimus-loaded liquid SNEDDS might be absorbed in the pores of solid carriers and adsorbed onto their surface, resulting in the tacrolimus-loaded solid SNEDDS after spray-drying (Kang et al., 2012). The thermal features of the drug powder, colloidal silica and solid SNEDDS are displayed in Figure 6(a). The physical mixtures were made by simply mixing the carriers and drug.
Our DSC results proved that tacrolimus had an exclusive endothermic peak at about 115 C, representing its melting point and crystalline nature (Figure 6a-A). Furthermore, a relatively minute endothermic peak corresponding to the melting point of the drug was also noticed in the DSC curve of physical mixture (Figure 6a-B). Conversely, colloidal silica (Figure 6a-C) and solid SNEDDS (Figure 6a-D) had no intrinsic peak. Our DSC results confirmed that tacrolimus exists in an amorphous form in the solid SNEDDS (Kim et al., 2011; Oh et al., 2011). The powder X-ray diffraction patterns are presented in Figure 6(b). Tacrolimus generated typical crystalline sharp peaks at diffraction angles (Figure 6b-A). Colloidal silica (Figure 6b-C) gave no intrinsic peaks. All of the chief distinctive crystalline peaks pertaining to the drug and colloidal silica were witnessed in
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Figure 6. Differential scanning calorimetric thermogram (a) and Powder X-ray diffractogram (b): (A) tacrolimus; (B) physical mixture; (C) colloidal silica; (D) solid SNEDDS. 40
Plasma concentration (ng/mL)
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Figure 5. Scanning electron micrographics: (A) tacrolimus powder (5000); (B) colloidal silica (3000); (C) solid SNEDDS (5000).
30
* *
Commercial product Solid SNEDDS
** * * *
20
* 10
*
0 0
3
6
9 12 Time (h)
24
Figure 7. Plasma concentration-time profiles of tacrolimus after oral administration of the commercial product and solid SNEDDS formulation in rats. Notes: Each value represents the mean ± S.D. (n ¼ 6). *p50.05 compared with the commercial product. The solid SNEDDS formulation composed of liquid SNEDDS [tacrolimus/Capryol PGMC/Transcutol HP/ Labrasol (5:10:15:75, w/v/v/v)] and colloidal silica at the weight ratio of 1:4.
the physical mixture (Figure 6b-B). The representative crystalline peaks of tacrolimus were not witnessed in the PXRD pattern of SNEDDS, confirming the amorphous state of drug in the SNEDDS (Figure 6b-D) (Kang et al., 2012; Lee et al., 2013).
According to our results, the drug-loaded liquid SNEDDS was spray-dried together with colloidal silica, resulting in the amorphous state of the drug in the solid SNEDDS formulations. In this tacrolimus-loaded solid SNEDDS, the liquid SNEDDS was physically and not chemically absorbed in the pores of colloidal silica and adsorbed onto its surface. On reconstituting in aqueous solution, the liquid SNEDDS leaked out from the solid carriers in the solid SNEDDS, was in contact with water, and spontaneously formed the nanoemulsion in aqueous solution. Moreover, the drug loaded in the solid SNEDDS was quickly dissolved, diffused and dispersed in the dissolution medium. Thus, this SNEDDS formulation resulted in the spontaneous formation of an interface between the oil droplets and water, ultimately decreasing the particle size of the droplets (Balakrishnan et al., 2009a; Kang et al., 2012). In the self-emulsifying compositions, because the amount of free energy required to form an emulsion is very low, the spontaneous formation of an interface between oil droplets and water was allowed (Balakrishnan et al., 2009b). This suggested that the oil/surfactant phase and water phase swelled effectively, reduced the size of the oil droplets and consequently improved the drug release rate. The change in the mean plasma concentration of tacrolimus after oral administration of the solid SNEDDS and the commercial formulation to rats is shown in Figure 7. Furthermore, the pharmacokinetic parameters are displayed in Table 3. The solid SNEDDS offered markedly higher plasma levels of tacrolimus at every time point compared to the commercial product (Figure 7). The solid SNEDDS furnished considerably higher AUC and Cmax and shorter Tmax value than those of the commercial product (p50.05). In particular, the AUC value of solid SNEDDS was approximately 2-fold greater than that of the commercial product. Our results suggested that the solid SNEDDS greatly promoted
Developing a novel tacrolimus-loaded SNEDDS
DOI: 10.3109/02652048.2015.1057252
Table 3. Pharmacokinetic parameters. Parameter Tmax (h) Cmax (ng/ml) T1/2 (h) Ke (h1) AUC (hng/ml)
Commercial product
Solid SNEDDS
0.57 ± 0.17 10.72 ± 4.82 7.32 ± 3.16 0.12 ± 0.07 166.60 ± 82.48
0.30 ± 0.09* 35.42 ± 5.20* 9.27 ± 1.97 0.08 ± 0.02 357.41 ± 79.05*
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Note: Each value represents the mean ± S.D. (n ¼ 6). *p50.05 compared with the commercial product.
the oral bioavailability of tacrolimus owing to faster and better absorption because of the increased dissolution of the drug (Yan et al., 2012; Tran et al., 2013). However, there was no considerable difference between Kel values and t1/2 values corresponding to the solid SNEDDS formulation and the commercial product.
Conclusion In the tacrolimus-loaded solid SNEDDS formulated with colloidal silica, the liquid SNEDDS composed of Capryol PGMC, Transcutol HP and Labrasol (10:15:75, v/v/v) was adsorbed in the pores and onto the surface of the solid carrier. Moreover, the drug was in the amorphous state in the solid SNEDDS. The solid SNEDDS with 5% w/v tacrolimus produced nanoemulsions with about 300 nm droplet size, and augmented the dissolution rate and the oral bioavailability of tacrolimus in rats due to the swift spontaneous emulsion formation without chemical interaction and the reduced droplet size. Therefore, this solid SNEDDS would be a potential candidate with enhanced oral bioavailability of tacrolimus.
Declaration of interest The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper. This work was supported by the National Research Foundation of Korea (NRF) grant supported by the Korean government (MEST) (No. 2012R1A2A2A01045658) and the High value-added Food Technology Development Program 313021-3 by the Ministry of Agriculture, Food and Rural Affairs in South Korea.
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