Improved oral absorption of tacrolimus by a solid dispersion with hypromellose and sodium lauryl sulfate

International Journal of Biological Macromolecules 83 (2016) 282–287

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Improved oral absorption of tacrolimus by a solid dispersion with hypromellose and sodium lauryl sulfate Hyuck Jun Jung, Hye In Ahn, Ji Yeon Park, Myoung Jin Ho, Dae Ro Lee, Ha Ra Cho, Jun Seo Park, Yong Seok Choi, Myung Joo Kang ∗ College of Pharmacy, Dankook University, 119 Dandae-ro, Dongnam-gu, Cheonan, Chungnam 330-714, South Korea

a r t i c l e

i n f o

Article history: Received 10 October 2015 Received in revised form 6 November 2015 Accepted 24 November 2015 Available online 28 November 2015 Keywords: Tacrolimus Solid dispersion Hydroxypropyl methylcellulose Sodium lauryl sulfate Dissolution Bioavailability

a b s t r a c t A novel surfactant-incorporated hydroxypropyl methylcellulose (HPMC) solid dispersion (SD) system was constructed in order to facilitate the release rate and oral absorption of tacrolimus (FK506), a poorly water-soluble immunosuppressant. Several emulsifiers including sodium lauryl sulfate (SLS), as drug release promotors, were employed with HPMC to fabricate SD using the solvent wetting method. The solid state characteristics using differential scanning calorimetry and X-ray powder diffraction, revealed that FK506 was molecularly distributed within all dispersions in amorphous form. The dissolution rates of FK506 in SLS-incorporated SDs were much higher than those in SDs prepared with HPMC alone, and even with stearoyl polyoxyl-32 glycerides or tocopheryl polyethylene glycol 1000 succinate. In particular, the greatest dissolution enhancement was obtained from the SD consisting of the drug, HPMC, and SLS in a weight ratio of 1:1:3, providing a 50-fold higher drug concentration within 15 min, compared with HPMC SD. In vivo absorption study in rats demonstrates that the optimized formula remarkably increased the oral absorption of FK506, providing about 4.0-fold greater bioavailability (p < 0.05) compared with the marketed product (Prograf® , Astellas Pharma). These data suggest that a novel SLS/HPMC SD may be an advantageous dosage form of FK506, boosting the dissolution and absorption in gastrointestinal tract. © 2015 Elsevier B.V. All rights reserved.

1. Introduction Tacrolimus (FK506), an immunosuppressant, has been clinically used in the prevention of organ rejection following hepatic and renal transplantation. It inhibits calcineurin by forming a complex with the FK506-binding protein, and was clinically demonstrated to be effective in the prevention of persistent refractory rejections in patients [1,2]. However, the oral therapy of FK506 has been relatively challenging due to its low and erratic intestinal absorption. Its poor water solubility (∼5 ␮g/ml), pre-systemic metabolism by cytochrome P450, and P-glycoprotein-mediated efflux in the gastrointestinal tract, are predominantly attributed to its low and variable oral bioavailability (BA) [3,4]. To boost the dissolution rate and oral absorption of FK506, the originator formulated a solid dispersion (SD) system with hydroxypropyl methylcellulose (HPMC) (Prograf® , Astellas Pharma, US). The HPMC-based SD system increased the dissolution rate of the calcineurin inhibitor by reducing the drug particle size at the molecular level, thus

∗ Corresponding author at: College of Pharmacy, Dankook University, 119 Dandaero, Dongnam-gu, Cheonan, Chungnam 330-714, South Korea. E-mail address: [email protected] (M.J. Kang). http://dx.doi.org/10.1016/j.ijbiomac.2015.11.063 0141-8130/© 2015 Elsevier B.V. All rights reserved.

changing the drug crystallinity to an amorphous state and hindering drug crystallization in aqueous medium [5–7]. However, the dissolution pattern and oral BA of the marketed product are still unsatisfactory, taking over 1 h for complete drug release and exhibiting a low BA (approximately 21%) with large intra- and interindividual variability (4–89%) [8]. When considering the favorable absorbency of the compound in upper intestinal regions (e.g., highest in the jejunum, intermediate in the ileum, and lowest in the colon) [3,4], a more rapid and profound drug release from the solid dosage form is desired in order to improve the oral absorption of FK506. Over the last decade, amphiphilic materials including surfactants such as sodium lauryl sulfate (SLS), polysorbates, and d-alpha tocopheryl polyethylene glycol 1000 succinate (TPGS), have been introduced as carriers and/or additives in combination with polymeric materials for the preparation of SD systems to improve not only the physical stability of active compounds in the dispersions but also to boost the dissolution rate of poorly water-soluble compounds [9–11]. These surfactants with an amphiphilic character aid the physical miscibility of hydrophobic drugs with hydrophilic polymers, and reduce drug recrystallization in the dispersions. Moreover, these emulsifiers have been reported to improve drug wettability and prevent drug precipitation in the aqueous medium,

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by absorbing into the outer layer of drug particles and/or forming drug-loaded micelles [12]. SLS, a substance generally recognized as safe (GRAS), has been widely used as a solubilizer or co-carrier in solid systems to improve the solubility and release rate of Biopharmaceutical Classification System (BCS) II compounds, and the flow-ability of dispersion [13,14]. Previous study revealed that the incorporation of SLS into the sugar glass-based SD formula helped to preserve the high drug concentration in aqueous medium during the dissolution process, by preventing drug precipitation [15]. The aim of the present study was to formulate a novel surfactant-incorporated HPMC SD system to improve the dissolution rate and oral absorption of FK506. A supersaturable SD formula of FK506 was prepared with different kinds of surfactants including SLS using the solvent wetting technique. Each SD powder was evaluated for its physicochemical characteristics such as morphology, drug crystallinity, and in vitro dissolution profile under non-sink conditions. The pharmacokinetic profile of the optimized SD was comparatively evaluated with that of the marketed product (Prograf® ) in rats, using a validated LC–MS/MS method. 2. Experimental 2.1. Materials FK506 monohydrate was kindly provided by Chong Kun Dang Pharm (Seoul, Korea, purity over 99.0% w/w). Ascomycin (purity over 98% w/w), used as an internal standard (IS) for LC–MS/MS analysis, and SLS (purity over 98% w/w) were acquired from Sigma Chemical Co. (St. Louis, MO, USA). HPMC (Pharmacoat 606) was kindly provided from Shin-Etsu Chemical Co Ltd. (Tokyo, Japan). Poloxamer 188, Soluplus (polyvinyl caprolactam-polyvinyl acetate-polyethylene glycol graft copolymer), and Compritol 888 ATO (Glyceryl behenate) were kindly provided by BASF Co. (Ludwigshafen, Germany). Vit E TPGS was supplied by Abitec Co (Janesville, WI, USA). Gelucire 44/14 (Lauroyl polyoxyl-32 glycerides), 50/13 (Stearoyl polyoxyl-32 glycerides), Peceol (Glyceryl monooleate) and Transcutol P (diethylene glycol monoethyl ether) were kindly provided by Gattefosse (Saint Priest, France). Acetonitrile and methanol of HPLC grade were purchased from J.T. Baker (Phillipsburg, NJ, USA). All other chemicals were of analytical grade. 2.2. Aqueous solubility of FK506 in the presence of surfactants The equilibrium solubility of FK506 in aqueous medium was determined by the shaking flask method, in the presence of different kinds of surfactants at a concentration of 1% (w/v). An excess of FK506 (5 mg) was added to a capped glass vial containing 3 ml distilled water with 10 mg/ml pharmaceutical excipients. Mixtures were vortexed for 5 min and then incubated for 24 h in a shaking incubator at 37 ◦ C. Each vial was subsequently centrifuged at 13,000 rpm for 10 min. The supernatant was diluted with acetonitrile, and the drug concentration was analyzed by HPLC. The quantitative FK506 analysis was performed using a Waters HPLC system (Model 515 pump, Model 717 plus auto sampler, Model 486 UV detector) equipped with a 4.6 mm × 150 mm ODS column (TSK-Gel ODS 80TM, Tosoh, Tokyo). The mobile phase consisted of distilled water, isopropyl alcohol, and tetrahydrofuran, at a volume ratio of 5:2:2. The flow rate was 1.0 ml/min, and the eluent was monitored at 220 nm. The peak of FK506 was identified at a retention time of 7.5 min. 2.3. Preparation of SD formulations by the solvent wetting technique FK506 (100 mg) was dissolved in an appropriate quantity of ethanol. The amount of ethanol used was 2-fold higher than

283

Table 1 Compositions of the surfactant-incorporated HPMC SD formulations.

FK506 (mg) HPMC (mg) SLS (mg) Gelucire 50/13 (mg) Vit E TPGS (mg)

F1

F2

F3

F4

F5

F6

F7

F8

F9

5 5 5 – –

5 5 10 – –

5 5 15 – –

5 5 – 5 –

5 5 – 10 –

5 5 – 15 –

5 5 – – 5

5 5 – – 10

5 5 – – 15

the total weight of the carrier (HPMC and each surfactant). The drug-containing ethanolic solutions were dropped onto different amounts of HPMC/surfactant mixture (100, 200, 300, and 400 mg) as described in Table 1. Solvents were then removed under a vacuum at 30 ◦ C for 2 h. The prepared SD powders were pulverized for 10 min and then passed through a 200 ␮m microplate sieve. 2.4. Morphological and physical characterization of SD formulations 2.4.1. Scanning electron microscopy (SEM) The appearance of the drug powder, the physical mixture of the carrier materials, and the SD formula were observed using SEM (JSM-6510, Japan). The samples were placed on a copper grid using double-sided tape and coated with a thin layer of gold and palladium using an automatic magnetron sputter coater system (108Auto, Cressington Scientific Instruments Ltd., UK). The samples were then observed at an acceleration voltage of 10.0 kV. 2.4.2. X-ray powder diffraction (XRD) Powder X-ray diffraction patterns of SD formulations were performed to verify the crystallinity state of FK506 in the formulations using an X-ray diffractometer (Ultima IV, Rigaku Corporation, USA) at room temperature. The diffraction pattern was measured over the most informative 2Â range, from 5◦ to 50◦ , using a step size of 0.02◦ and a scanning speed of 2 s/step. 2.4.3. Differential scanning calorimetry (DSC) Thermal analyses were conducted using a DSC unit (DSC 50, Shimadzu Scientific Instruments, MD). Approximately 2 mg each sample was placed in aluminum pans and progressively heated at a scanning rate of 10 ◦ C/min from 0 to 300 ◦ C. Indium was used to calibrate the temperature scale and enthalpic response. A nitrogen flow rate of 20 ml/min was used for each differential scanning calorimetry run. 2.5. Dissolution studies The dissolution profiles of each formulation under non-sink conditions were evaluated according to the USP apparatus II (paddle) method equipped with a dissolution testing station and a heater (Hanson, USA). Each formulation, containing 50 mg FK506, was located in the glass vessel containing 500 ml dissolution medium (simulated gastric juice, intestinal fluid, and distilled water) maintained at 37.0 ± 0.5 ◦ C and stirred at 50 rpm. Approximately 5 ml dissolution medium was withdrawn and replaced with fresh and pre-warmed dissolution medium. Withdrawn aliquots were then centrifuged at 13,000 rpm for 10 min and the supernatants were diluted with mobile phase for HPLC analysis. 2.6. In vivo oral absorption study 2.6.1. Animals and drug administration Sprague-Dawley rats (male; 200–230 g; 6–7 weeks) purchased from Orient Bio (Kyungki-do, Korea) were housed in

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specific pathogen-free conditions and maintained on a light cycle (light/dark: 12 h) at a controlled temperature (23 ± 1 ◦ C). Pelleted chow and tap water were freely available ad libitum. The animal experiment was performed in accordance with the NIH “Principles of laboratory animal care” guidelines, after approval by Dankook University Institutional Animal Care and Use Committee in Cheonan, Chungnam, Korea. Acclimated rats were randomly divided into two groups (n = 5) and deprived of food for 12 h prior to the experiment. Powdered marketed product (Prograf® ) and optimized SD formula were dispersed in distilled water at a drug concentration of 1 mg/ml, prior to dosing. The drugs were administered to rats through a syringe fitted with a flexible oral-zoned needle at a dose of 5 mg/kg. Blood samples were withdrawn from the retro-orbital plexus using EDTA-treated syringes at 0, 0.25, 0.5, 1, 2, 4, 6, and 12 h following drug dosing. The blood samples were kept below −20 ◦ C until analysis by LC–MS/MS.

2.6.2. LC–MS/MS assay for FK506 in rat whole blood The preparation of a blood sample and its LC–MS/MS analysis was carried out by following Park et al.’s method validated according to the FDA guidelines [16]. Briefly, a whole blood sample (90 ␮l) was mixed with a 150 ng/ml IS in methanol solution (10 ␮l) and a 10% (v/v) aqueous methanol solution (100 ␮l) by the help of mild temperature ultrasonication for 30 min. Then, a 0.05 mol/l aqueous hydrochloric acid (10 ␮l) and methyl tert-butyl ether (1 ml) were added, and the supernatant from the centrifugation of the resulting mixture was taken. After drying the supernatant under mild nitrogen flow, the residue was resuspended by 50 ␮l of a 50% (v/v) aqueous acetonitrile solution. After centrifugation, 20 ␮l of the supernatant was analyzed by a LC–MS/MS system composed of a LC-20 Prominence HPLC system (Shimadzu, Tokyo, Japan) and an API 2000 triple quadrupole mass spectrometer (AB/SCIEX, Poster City, CA, USA). Sample separation was carried out with Phenomenex Luna C8 column (2.0 × 150 mm, 5 ␮m) under the isocratic mobile phase condition of 95% (v/v) aqueous acetonitrile solution including 2 mmol/l ammonium acetate and 0.1% (v/v) formic acid at the flow rate of 0.25 ml/min. Both the target and IS were detected at about 2.3 min. Electrospray ionization (ESI) in positive ion mode was employed for the interface between the HPLC system and the mass spectrometer: spray voltage at 5300 V, spray temperature at 400 ◦ C, gas 1 at 35, gas 2 at 35, collision gas at 6, curtain gas at 16, and declustering potential at 70 V. For multiple reaction monitoring (MRM), screening transitions (precursor ion, m/z/product ion, m/z/collision energy, V) for the target and IS were 821.1/768.0/29 and 809.1/756.1/29, respectively. For confirmatory purpose, the transitions of 821.1/575.9/29 and 809.1/564.1/29 were used for the target and IS, respectively (confirmatory transitions). For data acquisition and analysis, Analyst software (version 1.5.2, AB/SCIEX) was used and peak area ratios of the target to IS were calculated for the quantitation purpose.

Table 2 Solubility of FK506 in various surfactant solutions (1% w/v). Surfactants

Solubilitya

SLS Vit E TPGS Gelucire 50/13 Gelucire 44/14 Compritol 888 ATO Poloxamer 188 Glyceryl mono oleate Soluplus

1904.0 199.7 111.9 106.0 22.9 19.1 10.4 6.9

a

± ± ± ± ± ± ± ±

42.4 2.4 10.5 2.3 3.9 3.5 0.2 0.7

Data are expressed as the mean ± SD (n = 3).

2.6.3. Pharmacokinetic analysis Maximum blood concentration (Cmax ) and the time taken to reach the peak blood concentration (Tmax ) were directly obtained from the mean blood concentration–time curve. The area under the curve (AUC0–12 h ) of the blood concentration–time profile of FK506 was estimated using the linear trapezoidal rule of the BA Calc. 2007 pharmacokinetic analysis program (Korean Food & Drug Administration). Data are expressed as the mean ± SD, and a statistically significant difference was demonstrated using a Student’s t-test with a threshold of P < 0.05. 3. Results and discussion 3.1. Effect of surfactant carriers on drug aqueous solubility The equilibration solubility of FK506 in the different surfactant solutions (10 mg/ml) is shown in Table 2. The solubility test aimed to choose suitable surfactant carriers that possess a good solubilizing capacity for FK506. We assumed that an excellent solubilizing effect could improve drug wettability and retard drug precipitation in aqueous medium. FK506 showed high solubility in SLS, Vit E TPGS, and Gelucire 50/13 and 44/14. In particular, SLS solubilized the maximum amount of compound (1900 ␮g/ml), providing approximately a 380-fold higher solubility than that of distilled water (∼5 ␮g/ml, data not shown). On the other hand, Poloxamer 188, Soluplus, Compritol 888 ATO, and Peceol were not effective for FK506 solubilization. Taken together, three different kinds of surfactant carriers (SLS, Vit E TPGS, and Gelucire 50/13) were employed to formulate surfactant-incorporated HPMC SD systems of FK506. 3.2. Development and physical characteristics of SD formulations Three different kinds of surfactant carriers (SLS, Vit E TPGS, and Gelucire 50/13) were employed to formulate supersaturable SD formulations in combination with HPMC, using the solvent wetting technique. The weight ratio of drug to HPMC was predetermined to be 1:1, which is the same as the marketed product (Prograf® , Astellas Pharma, US). The composition has been demonstrated to

Fig. 1. Representative SEM images of (a) drug powder, (b) HPMC and SLS mixture, and (c) SD powder (F3), prepared by the solvent wetting technique (200× magnification).

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285

Fig. 2. DSC thermograms of DRN, the individual polymers, and the corresponding SDs in a weight ratio of 1:1. (A) (a) drug powder, (b) HPMC, (c) SLS, and (d) SD with SLS/HPMC; (B) (a) drug powder, (b) HPMC, (c) Gelucire 50/13, and d SD with Gelucire 50/13/HPMC; and (C) (a) drug powder, (b) HPMC, (c) TPGS, and (d) SD with TPGS/HPMC.

100

Conc. (μg/ml)

80 60 40 20 0 0

1

2

3

4

5

6

Time (h) Fig. 3. Representative XRD patterns of (a) drug powder, (b) HPMC, (c) SLS, and (d) SD with SLS/HPMC (F3).

provide not only physicochemical stability of FK506 during the storage period, but also to improve the dissolution rate of the drug in aqueous medium [17]. The amount of surfactant carrier used in the SD formulations was varied from 5 to 15 mg, which is the minimal quantity for improving drug wettability and maintaining a supersaturated state in aqueous medium, while minimizing gastrointestinal irritation by surfactants. An ethanol-based solvent wetting technique was utilized to fabricate SD formulations, which proved to be effective in the preparation of the HPMC SD system of poorly water-soluble compounds including FK506 [17,18]. It has been reported in monkeys that the oral BA of FK506 is significantly enhanced by the granulated composition, providing an approximately 10-times higher oral BA in comparison with the crystalline powder [17]. Fig. 1 shows the representative SEM images of the intact drug powder (Fig. 1a), the SLS/HPMC carrier mixture (Fig. 1b), and the SD powder (F3) prepared by the solvent wetting method (Fig. 1c). The drug crystals were slightly prismatic in shape, with sizes ranging from 10 to 50 ␮m (Fig. 1a). On the other hand, it was difficult to find the drug crystals in the SD formula (Fig. 1c), suggesting that the drug compound was uniformly dispersed in the swollen SLS/HPMC matrix. FK506 and the anionic surfactant appeared to be adsorbed onto the swollen fibrous polymer, forming a granular structure with sizes ranging from 30 to 150 ␮m. The appearance of SDs with different kinds of surfactants was analogous to that of F3 as shown in Fig. 1c (data not shown). The crystallinity of FK506 in surfactant/HPMC dispersions was assessed using DSC (Fig. 2) and XRD (Fig. 3) tools. The DSC thermograms of the drug powder, carrier ingredients, and corresponding SDs are shown in Fig. 2. An endothermic peak appeared at 127 ◦ C, which represents the melting point of the active compound. Conversely, there was no noticeable peak at 127 ◦ C in any of the surfactant/HPMC SD formulations prepared with SLS (Fig. 2A), Gelucire 50/13 (Fig. 2B), or Vit E TPGS (Fig. 2C) as the co-carrier, indicating that the immunosuppressant was molecularly dispersed

Fig. 4. Dissolution profiles of FK506 from the HPMC-based SD formulation in the different pH media; gastric juice (pH 1.2, ), intestinal fluid (pH 6.8, ), and distilled water (䊐). Note: Data are expressed as the mean ± SD (n = 3).

in the SD systems in an amorphous form. To check the crystallinity of FK506 in the surfactant/HPMC SD systems, the X-ray diffraction pattern of FK506 in the SD formulations was further investigated. The diffraction spectrum of FK506 powder showed that the drug is a highly crystalline powder possessing sharp peaks at 2Â equal to 7.3◦ , 13.5◦ , 15.4◦ , 21.1◦ , 21.3◦ , and 25.7◦ (Fig. 3). On the other hand, the XRD patterns of the SDs were analogous to the patterns of the carrier materials, with no characteristic diffraction peaks corresponding to FK506. These XRD results are in accordance with previous DSC data, indicating that the drug was completely transformed from a crystalline structure to the amorphous form in all the surfactant/HPMC SD formulations by the solvent wetting method. 3.3. In vitro dissolution study In order to distinctively evaluate the formulation variables of the dissolution profile and the degree of supersaturation of FK506, an in vitro dissolution test was performed under non-sink conditions. The aqueous solubility of FK506 has been reported to be approximately 5 ␮g/ml at 25 ◦ C, regardless of the pH conditions [1]. Thus, the volume of medium (500 ml) does not guarantee the sink conditions for the amount of FK506 initially loaded (50 mg). The SD system prepared with HPMC alone provided a gradual increase in the extent of drug released, exhibiting a concentration in aqueous medium of over 20 ␮g/ml at 1 h (Fig. 4). The release rate of the calcineurin inhibitor was pH-independent, yielding a supersaturation state over the solubility of drug crystalline (about 5 ␮g/ml) in media at pH 1.2, pH 6.8, and in distilled water. The dissolution enhancement by HPMC dispersion generally mirrored the previous report that the high concentration of FK506 in a supersaturated state was maintained up to 24 h in the case of the SD system with HPMC, by increasing the dispersibility as well as inhibiting the precipitation progression of the drug in aqueous medium [17]. However, the release rate of FK506 was delayed by the low

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100

100

80

80

Conc. (μg/ml)

Conc. (μg/ml)

286

60 40

60 40 20

20

0 0

0 0

1

2

3

4

5

1

2

6

Time (h) Fig. 5. Dissolution profiles of FK506 from the SLS/HPMC SD formulations in simulated gastric fluid with different amounts of SLS added; 5 mg (F1, ); 10 mg (F2, ); and 15 mg (F3, ), and those from F3 in intestinal fluid (pH 6.8, •), and distilled water (). Note: Data are expressed as the mean ± SD (n = 3).

3

4

5

6

Time (h) Fig. 6. Dissolution profiles of FK506 from the Gelucire 50/13/HPMC SD formulations in simulated gastric fluid with different amounts of surfactant added; 5 mg (F4, ); 10 mg (F5, ); and 15 mg (F6, ). Note: Data are expressed as the mean ± SD (n = 3).

100 80

Conc. (μg/ml)

solubility and viscous hydrogel-forming property of the cellulose derivatives in the medium. Interestingly, the incorporation of a relatively small amount of SLS (5, 10, or 15 mg) into HPMC-based SD formulations (F1-F3) dramatically elevated the initial dissolution rate and extent to over 4 h (Fig. 5). Dissolution took place rapidly upon contact with aqueous medium and displayed a drug concentration of more than 50 ␮g/ml within 15 min, while that of the reference was 2 ␮g/ml. However, in the case of F1 and F2, the drug concentration in simulated gastric fluid was rapidly decreased after 1 h of supersaturation, and decreased to below 20 ␮g/ml at 6 h. On the other hand, the F3 formula, an SD prepared with SLS and HPMC in a weight ratio of 3:1, achieved a drug concentration of about 100 ␮g/ml within 15 min, which was upheld to over 60 ␮g/ml after 6 h, although the concentration of FK506 in simulated gastric fluid gradually decreased as a function of time. It is assumed that the anionic surfactant with a high hydrophilicity–lipophilicity balance value over 40 could drastically enhance drug wettability and decrease surface tension of the HPMC-based SD powder and/or drug particles in dissolution medium, triggering drug release from the dispersion. Subsequently, HPMC, and likely also some fraction of the anionic surfactant, may thermodynamically or kinetically extend the supersaturated state of FK506 in aqueous medium by reducing the rate of drug nucleation and crystal growth, through physical interactions or by adsorbing to the outer surface of drug particles. This result mirrors a previous report that a small quantity of the anionic surfactant (5, 20, or 33% w/w) added into a polyvinylpyrroline-based SD could facilitate dissolution rates compared with SD prepared with amorphous polymer alone and pure sulfathiazole [19]. Moreover, Mura et al. (2005) revealed that the dissolution profile of ketoprofen from a polyethylene glycol-based SD was significantly boosted when surfactants were incorporated to the binary system [20]. On the other hand, the release rate of the calcineurin inhibitor from the F3 formula was pH-independent, with more than 60 ␮g/ml of drug concentration after 6 h in media at pH 1.2, pH 6.8, and in distilled water (Fig. 5). In contrast, the incorporation of Gelucire 50/13 (F4-F6) or Vit E TPGS (F7-F9) to the HPMC-based SD systems did not increase the extent of drug released, but instead, slightly decreased the accumulated dissolution rate during the test period (Figs. 6 and 7). In an in vitro dissolution test under non-sink conditions, the drug concentration from F4-F9 peaked at 20 ␮g/ml within 2 h, and decreased to below 8 ␮g/ml at 6 h. The solubility enhancement via micellar solubilization effect of surfactants would be negligible, because the concentration of these surfactants in the medium was below those critical micelle concentrations (CMCs), except F9 formula. The CMC value of Vit E TPGS reported is 0.02% (w/w) in aqueous medium

60 40 20 0 0

1

2

3

4

5

6

Time (h) Fig. 7. Dissolution profiles of FK506 from the TPGS/HPMC SD formulations in simulated gastric fluid with different amounts of surfactant added; 5 mg (F7, ); 10 mg (F8, ); and 15 mg (F9, ). Note: Data are expressed as the mean ± SD (n = 3).

[21]. Inversely, the surface active agents maybe interfered physical interactions between the drug and the amorphous polymer, by adsorbing onto the drug particles and/or altering the conformational structure of the polymer, and thereby accelerating the precipitation progression of the drug in aqueous medium. Taken together, a novel SD system consisting of the drug, HPMC, and SLS at a ratio of 1:1:3 (F3), providing a rapid and higher dissolution rate of FK506, was established as the final formula for further in vivo absorption studies. 3.4. In vivo absorption study in rats Following oral administration, FK506 is known to be absorbable from the duodenum to the colon segments, possessing a preferential permeability in the upper small intestinal region including the duodenum [3,4]. Thus, in order to improve the oral absorption of the BCS class II compound possessing limited absorption sites within the gastrointestinal tract, we constructed a novel SD system displaying both a rapid and profound release profile under non-sink conditions. Subsequently, the pharmacokinetic profile of FK506 following oral administration of the optimized SD system was evaluated in rats with comparison to that of the marketed product. Fig. 8 shows the mean blood concentration–time profiles of FK506 following administration of either the marketed product or the optimized SD (F3) at a dose of 5 mg/kg in rats. In both formulations, the immunosuppressant was rapidly absorbed, exhibiting a maximum drug concentration within 1 h following oral administration. However, the mean FK506 concentrations in blood as a function of time obtained from the F3 formula were much higher compared with those obtained with the marketed product, due to the rapid and high dissolution rate as demonstrated through the

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Blood Conc. (ng/ml)

150 120 90 60 30 0 0

4

8

12

16

20

24

Time (h) Fig. 8. Blood concentration-time profiles of FK506 in rats following oral administration of the marketed product (Prograf® , ♦), and the optimized SLS/HPMC SD (F3, ) at a dose of 5 mg/kg. Note: Data are expressed as the mean ± SD (n = 5). Table 3 Pharmacokinetic parameters of FK506 following oral administration of the marketed product (Prograf® ) and the optimized SLS/HPMC SD (F3) at a dose of 5 mg/kg in fasted rats. Parameters

Marketed product

F3

AUC0–12 h (ng h/ml)a Cmax (ng/ml)a Tmax (h) t1/2 (h)a

236.6 ± 98.2 47.8 ± 21.9 0.5 9.5 ± 5.2

940.9 ± 289.2 85.6 ± 29.9 0.5 20.0 ± 4.1

a

Values represent the mean ± SD (n = 5).

in vitro dissolution test. The pharmacokinetic parameters of the calcinuerin inhibitor such as the AUC0–12 h , Cmax , and Tmax of each formulation are represented in Table 3. The AUC0–12 h and Cmax values of the F3 formula were 940.9 ng h/ml and 85.6 ng/ml, respectively, which are 4.0-fold (p < 0.05) and 1.8-fold (p < 0.05) greater than those of the marketed product. Therefore, we concluded that our in vivo pharmacokinetic data demonstrates that the accelerated and enhanced initial drug release by the incorporation of SLS into the HPMC SD drastically contributed to the significant improvement in drug absorption in the gastrointestinal tract. 4. Conclusions A novel SD formulation of FK506 was successfully fabricated by incorporating SLS as a drug release promotor to the amorphous

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polymeric carrier using the solvent wetting technique. The optimized SD system consisted of the drug, HPMC, and SLS at a weight ratio of 1:1:3. The physical characterizations using SEM, DSC, and XRD show that the active compound was molecularly distributed in the surfactant-incorporated SD system in a stable amorphous form. The dissolution rate of FK506 from the novel formula was markedly faster and higher than that of the marketed product in simulated gastric fluid, intestinal fluid, and distilled water. The in vivo pharmacokinetic study in rats reveals that the SLS/HPMC formulation significantly improved the oral absorption of FK506, providing 4fold greater BA compared with the marketed product. Upon these observations, we conclude that the SLS/HPMC SD dispersion may be a rational formula for the improvement of intestinal absorption of the calcineurin inhibitor. References [1] T. Kino, H. Hatanaka, S. Miyata, N. Inamura, M. Nishiyama, T. Yajima, T. Goto, M. Okuhara, M. Kohsaka, H. Aoki, T. Ochiai, J. Antibiot. 40 (1987) 1256–1265. [2] C.M. Spencer, K.L. Goa, J.C. Gillis, Drugs 54 (1997) 925–975. [3] R. Venkataramanan, A. Swaminathan, T. Prasad, A. Jain, S. Zuckerman, V. Warty, J. McMichael, J. Lever, G. Burckart, T. Starzl, Clin. Pharmacokinet. 29 (1995) 404–430. [4] S. Tamura, A. Ohike, R. Ibuki, G.L. Amidon, S. Yamashita, J. Pharm. Sci. 91 (2002) 719–729. [5] L. Shargel, Applied Biopharmaceutics and Pharmacokinetics, 2nd ed., Appleton & Lange, Norwalk, CT, 1993. [6] C. Leuner, J. Dressman, Eur. J. Pharm. Biopharm. 50 (2000) 47–60. [7] D.Q.M. Craig, Int. J. Pharm. 231 (2002) 131–144. [8] E. Christine, E. Susan, Clin. Pharmacokinet. 43 (2004) 623–653. [9] H.N. Joshi, R.W. Tejwani, M. Davidovich, V.P. Sahasrabudhe, M. Jemal, M.S. Bathala, S.A. Varia, A. Serajuddin, Int. J. Pharm. 269 (2004) 251–258. [10] C. Goddeeris, T. Willems, K. Houthoofd, J. Martens, G. Van den Mooter, Eur. J. Pharm. Biopharm. 70 (2008) 861–868. [11] J. Moes, S. Koolen, A. Huitema, J. Schellens, J. Beijnen, B. Nuijen, Int. J. Pharm. 420 (2011) 244–250. [12] C.L. Vo, C. Park, B.J. Lee, Eur. J. Pharm. Biopharm. 85 (2013) 799–813. [13] L.K. Ghosh, N.C. Ghosh, M. Chatterjee, B.K. Gupta, Drug Dev. Ind. Pharm. 24 (1998) 473–477. [14] E.J. Lee, S.W. Lee, H.G. Choi, C.K. Kim, Int. J. Pharm. 218 (2001) 125–131. [15] H. De Waard, W. Hinrichs, M. Visser, C. Bologna, H. Frijlink, Int. J. Pharm. 349 (2008) 66–73. [16] J.S. Park, H.R. Cho, M.J. Kang, Y.S. Choi, Arch. Pharm. Res. (2015), Accepted. [17] K. Yamashita, T. Nakate, K. Okimoto, A. Ohike, Y. Tokunaga, R. Ibuki, K. Higaki, T. Kimura, Int. J. Pharm. 267 (2003) 79–91. [18] E.J. Kim, M.K. Chun, J.S. Jang, I.H. Lee, K.R. Lee, H.K. Choi, Eur. J. Pharm. Biopharm. 64 (2006) 200–205. [19] R.H. Dave, H.H. Patel, E. Donahue, A.D. Patel, Drug Dev. Ind. Pharm. 39 (2013) 1562–1572. [20] P. Mura, J.R. Moyano, M.L. Gonzalez-Rodriguez, A.M. Rabasco-Alvarez, M. Cirri, F. Maestrelli, Drug Dev. Ind. Pharm. 31 (2005) 425–434. [21] S.H.W. Wu, W.K. Hopkins, Pharm. Tech. 23 (1999) 52–60.