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International Journal of Pharmaceutics 438 (2012) 123– 133

Contents lists available at SciVerse ScienceDirect

International Journal of Pharmaceutics

jo ur nal homep a ge: www.elsev ier .com/ locate / i jpharm

harmaceutical Nanotechnology

irolimus solid self-microemulsifying pellets: Formulation development,haracterization and bioavailability evaluation

iongwei Hua,b, Chen Linc, Dingxiong Chenc, Jing Zhanga, Zhihong Liua, Wei Wub, Hongtao Songa,∗

Department of Pharmacy, Fuzhou General Hospital of Nanjing Military Region, Fuzhou 350025, ChinaSchool of Pharmacy, Fudan University, Shanghai 201203,ChinaDepartment of Chemistry, Fujian Provincial Institute for Drug Control, Fuzhou 350001, China

r t i c l e i n f o

rticle history:eceived 3 May 2012eceived in revised form 14 July 2012ccepted 24 July 2012vailable online 28 July 2012

eywords:irolimuself-microemulsifying drug delivery systemolidificationellets

a b s t r a c t

To enhance the dissolution and oral absorption of water insoluble drug sirolimus (SRL), self-microemulsifying pellets of SRL were developed and evaluated. Solubility test, self-emulsifying gradingtest, ternary phase diagrams and central composite design were adopted to screen and optimize the com-position of liquid SRL-SMEDDS. The selected liquid SRL-SMEDDS formulations were prepared into pelletsby extrusion–spheronization method and the optimal formulation of 1 mg SRL-SMEDDS pellets capsule(1.0, 22.4, 38.4, 19.2, 121.6, 30.4 and 8.0 mg of SRL, Labrafil M1944CS, Cremophor EL, Transcutol P, MCC,Lactose and CMS-Na, respectively) was finally determinated by the feasibility of the preparing processand redispersibility. The optimal SRL-SMEDDS pellets showed a significant quicker redispersion rate thanthe dissolution rate of commercial SRL tablets Rapamune® in water. The droplet size and polydispersityindex of the reconstituted microemulsion was almost unchanged after solidification, and pellet size and

edispersibilityral bioavailiability

friability were all qualified. Visual observation and scanning electron microscopic analysis confirmedgood appearance of the solid pellets. DSC, XRPD, and IR analysis confirmed that there was no crystallinesirolimus in the pellets. Pharmacokinetic study in beagle dogs showed the oral relative bioavailabilityof SRL-SMEDDS pellets to the commercial SRL tablets Rapamune® was about 136.9%. In conclusion, thesolid SMEDDS pellets might be an encouraging strategy to improve the oral absorption of SRL and theextrusion–spheronization method was a feasible technology for the solidification of liquid SMEDDS.

. Introduction

Sirolimus (SRL), previously known as rapamycin, is an immuno-uppressive agent which is widely used for anti-rejection therapyn organ transplantation (del Carmen Rial et al., 2010; Toso et al.,010). But its water insolubility and high lipophilicity made it hardo be formulated into either an intravenous or oral dosage form. In999, the first commercially available product of SRL was an oralolution Rapamune® (Myeth, USA) with a concentration of 1 mg/mln Phosal 50 PG (a dispersion of 50% phosphatidylcholine in a pro-ylene glycol/ethanol carrier) and polysorbate 80 (Simamora et al.,001). The taste, requirement for refrigerator storage, protectionrom light, and disposal of the oral syringe after a single use make

he oily solution an inconvenient dosage form and its oral bioavail-bility is only 14% (Vasquez, 2000). In 2002, an oral tablet of SRLas launched under the same product name Rapamune® using

∗ Corresponding author at: Department of Pharmacy, Fuzhou General Hospital ofanjing Military Region, 156 West Second-Ring Road, Fuzhou 350025, China.el.: +86 591 2285 9459; fax: +86 591 8371 2298.

E-mail address: sohoto@vip.sohu.com (H. Song).

378-5173/$ – see front matter © 2012 Elsevier B.V. All rights reserved.ttp://dx.doi.org/10.1016/j.ijpharm.2012.07.055

© 2012 Elsevier B.V. All rights reserved.

NanoCrystal® technology acquired by Elan Corporation (Rosen andAbribat, 2005) with greater palatability and convenience of admin-istration and storage (at 20–25 ◦C). Although its oral bioavailabilitywas increased to about 17% (Shen and Wu, 2007), it is still lowerthan most oral preparation, and the requirement of special pro-duction equipment and huge energy consuming of NanoCrystal®

technology limits its wide application.To improve the solubility of SRL, various solubilization strate-

gies have been explored, such as inclusion complexes (Abdur Roufet al., 2011), liposomes (Alemdar et al., 2004), and solid dispersions(Kim et al., 2011; Preetham and Satish, 2011). Self microemulsify-ing drug delivery system (SMEDDS) as an effective bioavailabilityenhancement pharmaceutical technology has been widely usedduring the recent years (Chen et al., 2008; Mezghrani et al., 2011;Singh et al., 2009; Wu et al., 2006) and have some successful prod-ucts on the market (e.g. Neoral®, Norvir® and Fortovase®). SMEDDSis an isotropic mixtures of oil, surfactant and co-surfactant, whichcan form fine O/W microemulsion sized below 100 nm in aqueous

phase of gastro-intestinal tract (GIT) upon gentle agitation offeredby the peristalsis of stomach and intestine (Kohli et al., 2010; Luet al., 2012). Most of the SMEDDS formulations are encapsulatedin either hard or soft gelatin capsule. Lipid formulations however

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24 X. Hu et al. / International Journa

ay interact with the capsule shell resulting in either brittlenessr softness of it, to avoid this limitation, lipid formulations could beransformed into solid dosage forms by loading them on a suitableolid carrier. The solid SMEDDS system combines the advantagesf liquid SMEDDS with those of solid dosage form, which can over-ome the limitations of liquid formulations and improve the storagetability and patient compliance (Lei et al., 2011, 2012).

In this study, we developed a novel SRL-loaded SMEDDS pel-ets using extrusion–spheronization method. The key principle ofhis method was using the liquid SRL-SMEDDS as main moist-ning agents or adhesives to prepare the paste for extrusion. Anfficient SRL-SMEDDS formulation was screened by solubility test,elf-emulsifying grading test, phase diagrams, and central com-osite design and finally determinated by the feasibility of thereparing process and redispersibility. Finally, the optimum pel-

ets were characterized by scanning electron microscopy (SEM),ifferential scanning calorimetry (DSC), X-ray powder diffractionXRPD), infrared spectroscopy (IR) and reconstitute test to investi-ate the existent state of SRL in pellets and the changes of dropletize and polydispersity index of reconstituted microemulsion afterolidification. Furthermore, an oral bioavailability study in beagleogs was carried out to evaluate the absorption of SRL-SMEDDSellets vs. commercial SRL tablets.

. Materials and methods

.1. Materials

Sirolimus and Ascomycin were purchased from Fujian Keruiharmaceutical Co., Ltd. (Fuzhou, China). Crodamol EO, CrodamolTCC, Oleic acid and Tween-80 were purchased from Croda (Cow-

ck Hall, England). Labrafil M1944CS, Maisine35-1, Labrasol andranscutol P were kindly gifted by Gattefossé (Brittany, France).remophor EL, Cremophor RH40 and Solutol HS15 were purchasedrom BASF (Ludwigshafen, Germany). Propylene glycol, PEG400 andlycerin were purchased from Sinopharm Chemical Reagent Co.,td. (Shanghai, China) Silicon dioxide (SYLOID AL-1P and SYLOID44FP) were purchased from Grace Davison (Shanghai, China).icrocrystalline cellulose (Avicel PH-101) was purchased from

MC (Philadelphia, USA). Lactose (Foremost 315WG) was pur-hased from Kerry Group (Tralee, Ireland). Polyvinylpyrrolidone30, Dextrin, Low substitution hydroxypropylcellulose, Sodiumarboxymethyl starch, and crosslinking polyvinylpyrrolidone wereurchased from Chineway Pharmaceutical Co., Ltd. (Shanghai,hina). Commercial sirolimus tablet Rapamune® (1 mg) was sup-lied by Fuzhou general hospital (Fuzhou, China). High-pressure

iquid chromatographic grade methanol and acetonitrile were pur-hased from Sigma–Aldrich (Shanghai) Trading Co., Ltd. (Shanghai,hina). All other reagents were of analytical grade.

.2. Determination of sirolimus

Sirolimus was determined by high-pressure liquid chromatog-aphy (HPLC) on an Agilent 1200 HPLC system with an ultravioletetector set at wave-length of 278 nm (French et al., 2001),

column heater set at 50 ◦C, and an automatic injector withnjection volume setting at 20 �l. The mobile phase (acetoni-rile/methanol/water, 45/34/21, v/v/v) was online mixed andumped by a quaternary pump at a flow rate of 1.0 ml/min.irolimus was separated by a C18 column (Eclipse XDB-C18 col-

mn, 4.6 mm × 150 mm, 5 �m; Agilent, USA) and retention timeas ∼8 min. The linearity ranged from 0.1 to 10 �g/mL, with a cor-

elation coefficient of 0.9997. Intraday and interday precisions werell below 2%.

armaceutics 438 (2012) 123– 133

2.3. Solubility of sirolimus

The solubility of SRL in various oils, surfactants, and cosurfac-tants was determined. 1 ml of each of the selected vehicles wasadded to each cap vial containing an excess of SRL. After sealing,the mixture was heated at 40 ◦C in a water bath and treated withultrasonic for 5 min to facilitate solubilization. These mixtures wereshaken at 25 ◦C for 72 h. After reaching equilibrium, each vial wascentrifuged at 12,000 rpm for 10 min (25 ◦C). The supernatant wasfiltered through a 0.45 �m syringe filter membrane and the fil-trate was diluted with methanol for quantification by HPLC methoddescribed above. Solubility studies were carried out in triplicate.

2.4. Self-emulsifying grading test

All the optional oils, surfactants, and cosurfactants were homog-enized at different ratios (2:6:2, 2:5:3, 2:4:4, w/w/w) in tubes byvortexing for 5 min. After equilibrated at room temperature for24 h, the mixtures were examined visually after centrifugation at3500 r min−1 for 10 min. Then put 1 ml of the mixture that has atransparent appearance into 100 ml water (37 ◦C) with magneticstirring (50 rpm) to observe the emulsion forming process and finalappearance, which had been divided into four grades using a visualgrading system (Khoo et al., 1998):

A: denoting a rapidly forming (within 1 min) microemulsionthat was clear or slightly bluish in appearance.

B: denoting a rapidly forming, slightly less clear emulsion whichhad a bluish white appearance.

C: denoting a bright white emulsion (similar in appearance tomilk) that formed within 2 min.

D: denoting a dull, grayish white emulsion with a slightly oilyappearance that was slow to emulsify (longer than 2 min).

2.5. Construction of ternary phase diagram

Many previous studies used pseudoternary phase diagrams(Parmar et al., 2011; Zhuang et al., 2011) which admits water asone phase to investigate the forming process of microemulsion,the influences of water proportion on the phase behavior wasincluded in these diagrams. However, our study only constructedternary phase diagrams with oil, surfactant and cosurfactants underfixed volume ratio of water to liquid SMEDDS (100:1), in viewof that the basic volume of gastric juice (10–100 ml) is hundredstimes larger than the total amount of SRL-SMEDDS compositionin our unit preparation. Thus, the composition of oil, surfactantand cosurfactant was fixed as the three vertices of triangle phasediagram. Different oil-surfactant-cosurfactants mixtures were pre-pared according to the proportion of each point in it. Then eachmixture was dispersed into water follow the methods in Section2.4 to get a visual grading and the Grade A and B points were joinedtogether to figure out the microemulsion region. 1% SRL was addedinto the mixtures on the edge of microemulsion region to observeits influence on microemulsion forming.

2.6. Formulation optimization of SMEDDS

Central composite design was adopted to optimize the selectedSMEDDS system. Set two factors (X1 = the weight percent of oil,ranges from 20% to 40%; X2 = Km = surfactant/co-surfactant, rangingfrom 1 to 5) and five levels to arrange thirteen batches of tests(Table 3 and Table 4). Samples of each test was prepared and dis-persed follow the methods in section 2.4, then droplet size (DS)

and droplet size polydispersity index (PI) of forming microemul-sion were determined by a particle sizing system (Nicomp380, PSSInc, USA). Self-emulsifying time (t) was determined by recordingthe time from viscous SMEDDS mixtures contacted with water to

X. Hu et al. / International Journal of Ph

Table 1Solubility of SRL in various oily vehicles (mean ± SD, n = 3).

Vehicles Compositons Solubility (mg/g)

OilsMaisine 35–1 Glyceryl mono linoleate 5.42 ± 0.12Labrafil M1944CS Oleoyl polyoxyl glycerides 4.41 ± 0.09Crodamol EO Oleic acid ethyl ester 3.42 ± 0.08Crodamol GTCC Middle chain triglyceride 1.79 ± 0.04Oleic acid Long chain fatty acid 1.19 ± 0.04SurfactantsLabrasol Caprylocaproyl macrogol-8

glycerides22.22 ± 0.48

Tween-80 Polyoxyethylene sorbitanmonooleate

5.58 ± 0.11

Solutol HS15 Polyoxyethylene esters of12-hydroxystearic acid

4.60 ± 0.07

Cremophor EL Polyoxyethylene castor oilderivatives

4.07 ± 0.06

Cremophor RH40 Polyoxyethylene castor oilderivatives

1.49 ± 0.09

Co-surfactantsTranscutol P Diethylene glycol monoethyl ether 88.53 ± 1.45Propylene glycol 1,2-Propanediol 9.75 ± 0.37

drmctwt2

2

2

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PEG-400 Polyethylene glycol 400 2.75 ± 0.11Glycerin Propanetriol 0.54 ± 0.08

isappear using a timer. Different models were selected to fit theesponses of DS, PI and t to the variables of X1 and X2 using a polyno-ial fitting software Expert Curve 1.4. The model that had largest

orrelation coefficient (r) of the regression was adopted to drawhe contour lines, from which, some fine formulations of SMEDDSere finally selected for solidification study. The solubility of SRL in

hese formulations was determined follow the methods in Section.3 to provide basis for drug loading design of the final preparation.

.7. Development of SRL-SMEDDS pellets

.7.1. Preparation of SRL-SMEDDS pelletsVarious microporous excipients that have high-surface-area

e.g. silica, silicates, magnesium trisilicate, magnesium hydrox-de, talcum, crospovidone, and cross-linked sodium carboxymethylellulose etc.) were reported to absorb the liquid SEDDS andultiple techniques (e.g. spray drying, melt granulation, and

xtrusion–spheronization etc.) were adopted to prepare them intoolid dosage forms (Agarwal et al., 2009; Tang et al., 2008). In ourtudy, inorganic microporous silicon dioxide and other commonsed macromolecule materials were pre-screened by evaluatinghe redispersibility of the liquid–solid mixture, which was prepared

y blending liquid SMEDDS formulation with the solid absorbentst a weight ratio of 1:2. The solid absorbents that had little impactn the redispersibility of the liquid SRL-SMEDDS they absorbedere selected as solid matrix excipients of pellets.

able 2isual grading results of compatibility study with oils, surfactants and co-surfactantst different ratios.

Oils Oils:SA:Co-SA(w/w/w)

Surfactants

Labrasol Tween-80 Solutol HS15 Cremophor EL

Maisine35–1

2:6:2 C C C C2:5:3 C C C C2:4:4 C C C C

LabrafilM1944CS

2:6:2 C A B B2:5:3 C A B B2:4:4 C A B B

CrodamolEO

2:6:2 C B B A2:5:3 C B B B2:4:4 C C B B

armaceutics 438 (2012) 123– 133 125

The selected liquid SRL-SMEDDS formulations in Section 2.6were used for formulating solid pellets. Different formulations weredesigned to optimize the composition of solid pellets. First, SRLwas dissolved in the liquid blank SMEDDS formulations with anultrasound aid until the solution was clear (SRL-SMEDDS). Sepa-rately, the solid excipients were homogeneously mixed. Then thesolid mixture was wetted by gradual addition of the liquid SRL-SMEDDS and proper amount of distilled water (approximately 30%of the solid excipients w/w) to get a damp mass. The wet mass wasthen extruded and spheronized by an extrusion–spheronizationmachine (JBZ-300, Shenyang yilian, China) with screen pore0.8 mm, extrusion frequency 22 Hz, spheronization frequency20 Hz for 2 min. The moist pellets were dried to constant weightat 40 ◦C in an oven (DHG-9123A, Shanghai jinghong, China), thedrying process lasted about 2 h.

2.7.2. Redispersibility studyThis study was performed in a dissolution tester (RCZ-2B6,

Shanghai huanghai, China) according to the Chinese Pharma-copoeia Appendix Method III (the small glass method). Theredispersing medium was 250 ml of distilled water, 0.4% sodiumdodecyl sulfate (SDS) solution, pH 1.0 hydrochloride acid solutionor pH 6.8 phosphate buffer solution maintained at 37.0 ± 0.5 ◦Cand stirred at a revolution speed of 100 rpm, separately. The SRL-SMEDDS pellets were sealed into hard gelatin capsule shells of sizeII (contains 1 mg SRL per capsule) and each capsule was locked intoa stainless steel sink baskets before immersed it into the medium.5 ml of the medium was withdrawn at 5, 10, 20, 30, 45 and 60 minwith replacement by an equal volume of temperature-equivalentblank medium. The withdrawn samples were filtered through amembrane filter (0.45 �m), and the filtrate was diluted with iso-volumetric methanol, and the concentration of SRL in the diluentwas assayed by HPLC as described in Section 2.2. Three separatereplicate studies were conducted for each of the formulations.

2.8. Characterization of SRL-SMEDDS pellets

2.8.1. Pellet size distribution and friabilitySize distribution of pellets was determined by sieving method.

50 g produced SRL-SMEDDS pellets were screened through a set ofChinese Standard Sieves with pore size of 0.8, 1.0, 1.2 and 2.0 mm,and the weight percent of the retained pellets in each sieve wascalculated. Pellet friability was conducted on 5 g of the pellets com-bined with 5 g of glass beads (2 mm diameter) using a CJY-300Cfriabilator (Huanghai, Shanghai, China). The drum was rotated at25 rpm for 4 min. Loss of pellet weight with respect to the initialvalue was then calculated as percent friability.

2.8.2. Scanning electron microscopy (SEM)The morphological features of solid SRL-SMEDDS pellets were

observed by a scanning electron microscope (Nova Nano SEM 230,FEI, USA). The samples were fixed on a brass specimen club usingdouble-sided sticky tape and made electrically conductive by coat-ing in a vacuum (6 Pa) with platinum (5 nm/min) using an ironsputter (E-1030, Hitachi, Japan) for 30 s at 15 mA. The photographswere taken at an excitation voltage of 10 kV.

2.8.3. Differential scanning calorimetry (DSC)Sample preparation: SRL-SMEDDS pellets and blank SMEDDS

pellets without SRL loaded were prepared and grinded to powdersrespectively. Physical mixture was prepared by mixing SRL withblank SMEDDS pellets powder according to the proportion of SRL-

SMEDDS pellets.

Thermal investigations were performed using DSC (STA449 C,Netzsch, Germany). The temperature ranged from 25 ◦C to 300 ◦Cwith a heating rate of 10 ◦C/min. Experiments were carried out in

1 l of Pharmaceutics 438 (2012) 123– 133

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Table 3The level code of each factor and operation value of each test.

Factor Level

−1.414 −1 0 1 1.414

26 X. Hu et al. / International Journa

luminum pans with a pierced lid. 10 mg sample was placed inton aluminum pan and the pan was sealed. An empty aluminum panith its lid was used as a control. The samples were purged withure dry nitrogen at a flow rate of 50 ml/min.

.8.4. X-ray powder diffraction (XRPD)The physical states of samples mentioned in Section 2.8.2 were

valuated by XRPD. Patterns were obtained using a diffractometerystem (X’pert PRO MPD, Philips, Holland) with a CuK� radiationource, all samples were scanned over the 5–95◦2� range at a stepize of 0.01◦, the tube voltage was 40 kV and tube current was0 mA.

.8.5. Infrared (IR) spectroscopySamples in Section 2.8.2 were also characterized by IR

pectroscopy. Infrared spectra were recorded on an infraredpectrophotometer (Avatar330, Nicolet, USA) by using potas-ium bromide pressed disc method. The scanning range was00–4000 cm−1.

.8.6. Reconstitution studyIn a 20 ml vial, 100 �l of liquid SRL-SMEDDS and 300 mg of

he SRL-SMEDDS powders were dispersed in 10 ml distilled watery vortex mixing for 30 s, respectively. The resulting microemul-ion was incubated for 30 min at room temperature, then theupernatant were withdrawn for droplet size (DS) and polydis-ersity index (PI) determination using a particle sizing systemNicomp380, PSS Inc., USA).

.9. Pharmacokinetic study

.9.1. Experimental designSix healthy female beagle dogs (10–12 kg), fasted but free access

o water for 12 h prior to the experiment, were used in this study.hey were random allocated to two treatment groups and admin-stered orally hard capsules containing the optimal SRL-SMEDDSellets and commercial SRL tablets in a crossover design with 2eeks washout between dosing. The dose of SRL administered toogs was 2.0 mg. The animal studies were approved by the Fuzhoueneral Hospital Animal Care and Use Committee.

Blood 2.0 ml samples were collected from the leg veins with aeparinized vein indwelling needle at 0, 0.25, 0.5, 0.75, 1, 1.5, 2,.5, 3, 4, 6, 8, 9, 9.5, 10, 10.5, 11, 12, 14, 18, 24 and 48 h post-dosing.amples were kept frozen at −20 ◦C until analysis.

.9.2. Quantitative analysis of SRL in bloodThe SRL concentrations of blood were determined by rapid

esolution liquid chromatography tandem mass spectrometryRRLC-MS/MS) (Agilent, USA) with a lower limit of quantitationf 0.2 ng/ml. Ascomycin (FK520) was used as internal standard,ample pretreatment utilized a simple liquid–liquid extraction. A0 �l acetonitrile solution of internal standard (1000 ng/ml) and00 �l sodium acetate buffer solution (pH 4.6) were added to 500 �llood. After vortex-mixing for 30 s, 2 ml tert-butylmethylether wasdded and mixing for 20 min in a swing mixer. After centrifugationt 5000 rpm for 3 min, the organic layer was collected and driednder nitrogen gas at 40 ◦C. The residue was reconstituted with00 �l mobile phase. The separation was carried out on an AgilentOBAX C18 column (50 mm × 2.1 mm, 1.8 �m; Agilent, USA) withater and acetonitrile (1:9) as the mobile phase at a flow rate of

.3 ml/min and column temperature 50 ◦C.The detection was performed by an Agilent triple quadrupole

etector (G6410, Agilent, USA). The mass spectrometer was oper-ted with an electrospray ionization (ESI) interface in positiveonization mode and with multiple reaction monitoring mode. Theelected reaction monitoring (SRM) of SRL and internal standard

X1 20.00 22.93 30.00 37.07 40.00X2 1.00 1.59 3.00 4.41 5.00

were m/z 937.2 → 409.3 and m/z 814.4 → 604.2, respectively. Theconcentration of SRL was determined by standard linear calibrationcurve in the concentration range of 0.5–32 ng/ml.

2.9.3. Pharmacokinetic data analysisThe peak blood concentration (Cmax) and the time for their

occurrence (Tmax) were obtained directly from the individualplasma concentration versus time profiles. The area under theconcentration–time curve (AUC0−t) was estimated according to thelinear trapezoidal rule. The ANONA and two one-side t test wereused to compare the pharmacokinetic parameters. P-values < 0.05was considered statistically significant. The relative bioavailability(Fr) of SRL-SMEDDS pellets (T) to the commercial SRL tablets (R)was calculated using the following equation:

Fr = (AUC0→t)T

(AUC0→t)R× 100%

3. Results and discussion

3.1. Solubility of sirolimus

SMEDDS system is generally consists of oil, surfactant and cosur-factant, so the drug solubilizing potential of these vehicles is thepremise of optimum drug loading while maintaining an excellentemulsifying performance (Kim et al., 2011). The apparent solubil-ity of SRL in various oily vehicles is shown in Table 1. Of the oils,Maisine 35-1, Labrafil M1944CS and Crodamol EO had higher solu-bility of SRL than the other two, so they were selected as oil phasefor further investigations. Similarly, Labrasol showed highest solu-bilization capacity for SRL among all the surfactants, but Tween-80,Solutol HS15 and Cremophor EL which had relative lower apparentsolubility of SRL could not be abandoned, because the variabil-ity of emulsifying ability among different surfactants might playa more important role than solubility in preparing SRL-SMEDDS.Transcutol P was selected directly as cosurfactant due to the highestsolubility of SRL in it. Volatile vehicles that might be removed fromthe formulation during preparation and storage were not includedin our test.

3.2. Self-emulsifying grading test

All the mixtures of oil, surfactant and cosurfactant in this testhad a homogeneous and transparent appearance after equilibriumand centrifugation, which revealed good compatibility to form anisotropic mixture. The visual grading results of microemultionsformed by different SMEDDS mixtures are shown in Table 2 withTranscutol P as fixed cosurfactant. From the results, oil phase Mai-sine 35-1 mixed with all the observed surfactants and surfactantLabrasol mixed with all the oils showed poor microemulsion form-ing ability, which only got a C grade. However, Labrafil M1944CSand Crodamol EO of oils mixed with Tween-80, Solutol HS15 orCremophor EL of surfactants seemed to form fine microemulsion,most of which got visual grade A or B except Crodamol EO with

Tween-80 at the ratio of 2:4. Labrafil M1944CS had a higher solu-bility of SRL than Crodamol EO (Table 1), and it is also a potentialbioavailability enhancer due to its long chain triglyceride compo-sition which could facilitate the lymphatic transport of lipophilic

X. Hu et al. / International Journal of Pharmaceutics 438 (2012) 123– 133 127

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ig. 1. Ternary phase diagrams composed of oil phase (Labrafil M1944CS), surfactTranscutol P).

rugs (Porter et al., 2007), So Labrafil M1944CS was selected as oilhase, Tween 80, Solutol HS15 and Cremophor EL were roughlyelected as surfactant for further screen.

.3. Ternary phase diagrams

The ternary phase diagrams of oil (Labrafil M1944CS), surfac-ants (Tween-80, Solutol HS15 or Cremophor EL) and cosurfactantTranscutol P) were constructed (Fig. 1A–C). The largest microemul-ion region was observed when Cremophor EL was used asurfactant (Fig. 1C), and there was little change on the result-ng microemulsion’s appearance when 1% SRL was added into theormulations on the edge of microemlsion region. So the Labrafil

1944CS-Cremophor EL-Transcutol P mixture was selected as liq-id SMEDDS system for SRL and the optimum proportion of themeed further study.

.4. Formulation optimization of SMEDDS

All the tested parameters (DS, PI and t) of different SMEDDSormulations are reported in Table 4 (columns 4–6). The poly-omial fitting correlation coefficient (r) of DS, PI and t for firstrder polynomial was 0.8249, 0.7107 and 0.8862; for secondrder polynomial was 0.8696, 0.8623 and 0.9824; for third orderolynomial was 0.9391, 0.9441 and 0.9962, respectively. And thetting significance value P was all less than 0.05. Obviously, theelationship between Y (DS, PI and t) and X (weight percent ofil and Km) fitted best with the third order polynomial model.

hus, the contour maps were drawn according to the fitted thirdrder equations (YDS = −30.2632 + 5.3617X1 − 4.5965X2 − 0.105421 + 6.8443 X2

2 − 1.3680X1X2 + 0.0426 X21 X2 − 0.2044X1 X2

2 ;

PI = −1.2621 + 0.1208X1 + 0.2321X2 − 0.0022 X21 + 0.0773 X2

2

able 4est design and results of each test (n = 3).

No. X1 X2 Y1 (DS) Y2 (PI) Y3 (t)

1 37.07 4.41 21.6 0.100 15.332 37.07 1.59 25.0 0.134 1.033 22.93 4.41 17.4 0.052 3.504 22.93 1.59 19.2 0.082 0.405 40.00 3.00 32.0 0.193 5.256 20.00 3.00 16.8 0.048 0.837 30.00 5.00 18.9 0.068 10.508 30.00 1.00 31.1 0.279 0.279 30.00 3.00 21.1 0.057 3.75

10 30.00 3.00 20.0 0.066 3.5011 30.00 3.00 19.5 0.064 3.7212 30.00 3.00 19.6 0.059 3.8713 30.00 3.00 20.9 0.061 4.10

s follows: Tween-80 (A), Solutol HS15 (B) and Cremophor EL(C) and co-surfactant

− 0.0386X1X2 + 0.0008 X21 X2 − 0.0019X1 X2

2 ; Yt = −41.8776 +2.4640X1 + 20.4778X2 − 0.0349 X2

1 − 2.8078 X22 − 1.0152X1X2 +

0.0106 X21 X2 + 0.1105X1 X2

2 ) by software Origin pro 8 (Fig. 2).From Fig. 2, the contour map of DS was similar to that of PI, whenthe weight percent of oil phase was lower, both DS and PI firstdecreased and then increased with the increase of Km, maybebecause the emulsifying ability and the viscosity of the systemincreased with the increasingly percentage of surfactant in theformulations, the enhanced emulsifying ability predominatedin the microemulsion forming process when the viscosity waslower at first, so DS and PI decreased, however, when the viscosityincreased to a higher extent, the increase of emulsifying ability wasinhibited by the high viscosity, so DS and PI increased again. Whenthe weight percent of oil phase was higher, the DS and PI decreasedwith the increase of Km, maybe because the inherent low viscosityof oil phase was magnified, so the viscosity of the whole SMEDDSsystem was not a significant factor that influence the emulsifyingeffect, DS and PI decreased with the increasingly emulsifyingability. The self-microemulsifying time (t) increased either withthe increase of Km or the weight percent of oil phase, maybebecause the increase of viscosity and decrease of emulsifyingability (relative decreased percent of surfactant and cosurfactantaccompany with the increasing percent of oil phase).

However, DS of the resulting microemulsion from all the investi-gated formulations were less than 50 nm, which was small enoughfor an ideal microemulsion. So the contour maps of PI and t wereoverlapped to form the optimized region of final formulation with-out concerning DS (Fig. 3), the formulations in which should formmicroemulsions with DS smaller than 50 nm, PI lower than 0.1and t less than 3 min. To get the maximum drug loading, liquidSMEDDS formulations A–C that had as higher percent of cosurfac-tants as possible while oil percent varying were selected out fromthe shadow region of Fig. 3 for further solidification study. Formu-lations A–C had an oil phase percent of 32%, 28% and 22%, Km of2.5, 2.0 and 1.5, respectively. The DS, PI and t of these three for-mulations were determined (observed value) and compared withthe results calculated from the fitted third order equation above(predicted value), all errors are lower than 5%, which indicated anexactly predictability of the model (Table 5).

The solubility of SRL in formulation A, B and C were about14.93 ± 1.32, 16.73 ± 1.65 and 15.25 ± 1.44 mg/g, respectively. So,at least 67 mg of these formulations was needed to dissolve 1 mgSRL. However, a size II empty hard gelatin capsule shell only had acapacity of 250 mg prepared SRL-SMEDDS pellets, and the pellets

could be processed only the weight of solid excipients was twiceas much of the liquid SMEDDS. So, the amount of liquid SMEDDSused in each capsule was set at 80 mg to balance the solubility andcapsule shell capacity.

128 X. Hu et al. / International Journal of Pharmaceutics 438 (2012) 123– 133

Fig. 2. Contour maps of droplet size (A), polydipersit

Fmo

3

3

hepapolqffiD

TP

beause it could improve the detection sensitivity of SRL in our smalldose (1 mg/capsule) preparation, moreover, the medium volume of250 ml was more close to the basic volume of human gastric juice.

ig. 3. The overlapping contour maps of polydipersity index (full line) and selficroemulsifying time (dotted line). The gray shadow represents optimum region

f SRL-SMEDDS formulations.

.5. Development of SRL-SMEDDS pellets

.5.1. Preparation of SRL-SMEDDS pelletsThe ideal solid matrix excipients of SMEDDS pellets should

ave great adsorbability and appropriate formability, which couldnsure a larger liquid SMEDDS loading amount and facilitate thereparation of pellets. However, as shown in Fig. 4, strongerbsorbents like Silicon dioxide (SYLOID AL-1 and 244FP) led to aoor redispersibility of SRL-SMEDDS, so they were excluded fromur formulation. Among other solid excipients investigated, MCC,actose, and disintegrants (CMS-Na, L-HPC, PVPP) had a relativeuickly and completely redispersibility of SRL-SEMDDS, and dif-

erent SRL-SMEDDS pellets were prepared with these materialsollowing the formulations in Table 6, which had fixed drug load-ng and relative proportion of liquid SMEDDS to solid excipients.uring the preparing process, the extrudate of F1 had a rough

able 5redicted and observed values of optimized formulations of SRL-SMEDDS.

Factors Formulations Predictedvalues

Observedvalues

Error %

Particlesize(nm)

A 23.4 24.5 −4.49B 21.7 20.9 3.83C 20.5 19.7 4.06

Polydispersityindex

A 0.102 0.099 3.03B 0.101 0.105 −3.81C 0.093 0.089 4.49

Selfemulsifyingtime (min)

A 2.82 2.94 −4.08B 1.88 1.91 −1.57C 1.14 1.11 2.70

y index (B) and self microemulsifying time (C).

surface and was likely to be crushed to powder during the processof spheronization. However, using lactose alone as solid matrix wastoo sticky to prepare a damp mass. It was found that adding somelactose into MCC could improve the viscoelasticity of the extrudate,F2 and F3 could be well processed and the resulting pellets hadfine appearance and roundness, but with the increasing amount oflactose in formulations, conglutination and combination of pelletswere observed, which was serious in F4. Formulations contain dif-ferent disintegrants or different liquid SMEDDS formulations werealso prepared into pellets successfully (F5–F11) with fixed lactoseproportion.

3.6. Redispersibility of SRL-SMEDDS pellets

Solid SRL-SMEDDS pellets should retain the ability of liq-uid SRL-SMEDDS to redisperse quickly into microemulsion inaqueous medium. So the above prepared SRL-SEMDDS pelletswere further screened by redispersibility test. Similar studies hadbeen performed under “dissolution” or “in vitro release” items(Setthacheewakul et al., 2010; Yao and Li, 2011), however, itwould be better to use “Redispersibility” rather than “dissolution”,because those tests only quantitated the total drug in dissolutionmedium rather than free drug. In our study, we assumed that SRLwas associated with reconstituted microemulsion after redispers-ing, the redispersibility could be evaluated by determining SRLin the redispersing medium, because SRL was almost insolublein water, so the contribution of free drug to the redispersibilityprofiles could be negligible. Small glass method was adopted just

Fig. 4. Redispersion profiles of mixtures of liquid SRL-SMEDDS with different solidabsorbents (Data are expressed as mean, n = 3, where relative SD < 10%).

X. Hu et al. / International Journal of Pharmaceutics 438 (2012) 123– 133 129

Table 6Composition of SRL-SMEDDS pellets.

Compositions (mg/capsule) Formulations (mg/capsule)

F1 F2 F3 F4 F5 F6 F7 F8 F9 F10 F11

Sirolimus 1 1 1 1 1 1 1 1 1 1 1Labrafil M1944CS 22.4 22.4 22.4 22.4 22.4 22.4 22.4 22.4 22.4 25.6 17.6Cremophor EL 38.4 38.4 38.4 38.4 38.4 38.4 38.4 38.4 38.4 38.86 37.44Transcutol P 19.2 19.2 19.2 19.2 19.2 19.2 19.2 19.2 19.2 15.54 24.96MCC 160 144 128 112 121.6 121.6 121.6 125.44 117.76 121.6 121.6Lactose 16 32 48 30.4 30.4 30.4 31.36 29.44 30.4 30.4L-HPC 8CMS-Na 8 3.2 12.8 8 8

A ylcelld

wlu2siwdlCuwftdpSruilomziFrtbba0oaatscTmw

3

3

tai

PVPP

bbreviations: MCC, microcrystalline cellulose; L-HPC, low substitution hydroxypropone.

As shown in Fig. 5A, the redispersion of SRL-SMEDDS pelletsas improved by the increasingly adding of lactose into formu-

ations, and no significant improvement was observed when thesing amount of lactose in solid excipients mixture increased from0% (F3) to 30% (F4), so 20% lactose was preferred. Lactose is wateroluble excipients, when pellets contact with water, the lactose int dissolves quickly and accelerates the hydration of SRL-SEMDDS,

hich might be the reason of the lactose effects. The using ofisintegrants improved the general redispersion of all the formu-

ations in Fig. 5B compared to Fig. 5A, the improving effect ofMS-Na was more significant than PVPP and L-HPC at the samesing amount of 5%. However, little improvement was observedhen 2% CMS-Na was used, and the redispersion of 8% CMS-Na

ormulation (F9) was not distinguished with that of 5% (F8) whenhese disintergrants containing pellets contacted with water, theisintergrants expanded quickly and cracked the matrix into microieces, which enlarged the specific surface for redispersion. TheMEDDS composition of formulation F10, F6 and F11 were cor-esponding to formulation A, B, and C in Section 3.4, with fixedsing amount of 20% lactose and 5% CMS-Na. According to profiles

n Fig. 5C, redispersion of all the investigated SRL-SMEDDS pel-ets in distilled water were obviously better than the dissolutionf commercial SRL tablets, because 250 ml distilled water cannoteet the sink condition need of SRL while SMEDDS is a solubili-

ation strategy itself. Among F10, F6 and F11, redispersing rate wasn the order of F11 > F6 > F10 and redispersing extent order was6 > F11 > F10, a complete redispersion is more important than aapid redispersing rate, so F6 was selected as optimum formula-ion of SRL-SMEDDS pellets for further characterization and oralioavailability study. However, there was no significant differenceetween the redispersing profiles of different SRL-SMEDDS pelletsnd the dissolution profile of commercial SRL tablets (Fig. 5D) in.4% SDS solution, because this medium fulfills the sink conditionf SRL. It was interesting to observe that SRL in SMEDDS pelletsnd the commercial tablets almost could not be determined 30 minfter putting in pH 1.0 Hydrochloric acid solution (Fig. 5E), and allhe redispersing or dissolution profiles in pH 6.8 phosphate bufferolution (Fig. 5F) were similar to that in water (Fig. 5C) exceptumulative dispersion or dissolution extent were a little lower.hese results might due to the poor stability of SRL in acid environ-ent and relatively more stable in nearly neutral pH environment,hich were consistent with that reported earlier (Sun et al., 2011).

.7. Characterization of SRL-SMEDDS pellets

.7.1. Pellets size distribution and friability

Three batches of SRL-SMEDDS pellets were prepared with

he composition of F6. The pellet size distribution were 0%bove 2.0 mm, 9.6% in 1.2–2.0 mm, 57.4% in 1.0–1.2 mm, 22.8%n 0.8–1.0 mm and 10.2 below 0.8 mm. Obviously, modal fraction

8

ulose; CMS-Na, sodium carboxymethyl starch; PVPP, cross linking polyvinylpyrroli-

was 1.0–1.2 mm and more than 80% of the pellet size was in0.8–1.2 mm. The reduction in mass of the pellets in friability testwas 0.64 ± 0.12%, which meets the quality standards of ChinesePharmacopoeia for pellets.

3.7.2. Scanning electron microscopy (SEM)The surfaces and cross-sections of pellets were studied by scan-

ning electron microscope. SEM photographs of SRL-SMEDDS pellets(F6) reveal that the pellets had a spherical shape (Fig. 6A) SRL-SMEDDS was uniformly dispersed on the surface and entrappedin the solid matrix of the pellets (Fig. 6B and C). The microporouson surface and in the matrix might form channels for water toinfiltrate, which could facilitate the microemulsion forming anddispersion.

3.7.3. Differential scanning calorimetry (DSC)DSC curves of SRL powder, blank SMEDDS pellets powder, phys-

ical mixture and SRL-SMEDDS pellets powder are shown in Fig. 7.The thermogram of pure SRL exhibited an endothermic peak atabout 185 ◦C (Fig. 7A), corresponding to its melting point. BlankSRL-SMEDDS pellets powder showed no specific peaks from 25 ◦Cto 300 ◦C (Fig. 7B). However, an obtuse endothermic peak withreduced intensity was observed in physical mixture at about 185 ◦Ctoo (Fig. 7C). This might be caused by the melting of SRL crys-tals that existed in the mixture, and the reduced concentration ofSRL in the mixture and dissolving of SRL in blank SMEDDS duringheating might lead to an obtuse shape with reduced intensity ofthe endothermic peak relative to pure SRL. In SRL-SMEDDS pelletspowder, no endothermic peak of SRL was observed (Fig. 7D). There-fore, it could be concluded that SRL was still dissolved in SMEDDSformulations after the SRL-SMEDDS was prepared into solid pellets.

3.7.4. X-ray powder diffraction (XRPD)The physical state of SRL in solid SRL-SMEDDS pellets was inves-

tigated by XRPD, which could provide information on crystallinityand crystal orientation. Fig. 8A demonstrated typical diffractionpeaks ranging from 5◦ to 50◦ (2�) and three high intensity peaksamong them were labeled as a–c, which were also shown in thephysical mixture with a reduced intensity due to reduced concen-tration of SRL in the mixture (Fig. 8C). But a–c peaks were not shownat the similar position in blank SMEDDS pellets powder (Fig. 8B) andthe SRL-SMEDDS pellets powder (Fig. 8D). As expected, there wasno crystalline SRL in the final solid SRL-SMEDDS pellets and SRLmight be well dissolved in the SMEDDS formulation.

3.7.5. Infrared (IR) spectroscopy

IR spectra of SRL powder, blank SMEDDS pellets powder, phys-

ical mixture and SRL-SMEDDS pellets powder are presented inFig. 9. SRL has six C O double bonds and four C C double bondsin its chemical structure, so the characteristic C C stretch peak

130 X. Hu et al. / International Journal of Pharmaceutics 438 (2012) 123– 133

F les ofh n = 3).

(sosSapo1

ig. 5. Redispersion profiles of different SRL-SMEDDS pellets or the dissolution profiydrochloric acid solution (E) or pH 6.8 phosphate buffer solution (F) (mean ± S.D.,

1680–1640 cm−1) and C O stretch peak (1760–1670 cm−1) werehown in pure SRL spectra (Fig. 9A), which could also be clearlybserved in the physical mixture (Fig. 9C), but the peaks at theame wave number in blank SMEDDS pellets powder (Fig. 9B) andRL-SMEDDS pellets powder (Fig. 9D) were very weak and alike,

lthough we could not confirm they must be the C C or C O stretcheaks of SRL, but the sharply decreased IR absorption strengthf these peaks was the fact. Additionally, some atypical peaks at100–1500 cm−1 shown in Fig. 9A and C were also weakened or

commercial SRL tablets, in distilled water (A–C), in 0.4% SDS solution (D), in pH 1.0

disappeared in Fig. 9B and D. All of the above indicated an interac-tion of SRL with the carrier, which might be hydrogen bonds resultsfrom the dissolving of SRL in SMEDDS excipients.

3.7.6. Reconstitution properties of SRL-SMEDDS pellets

The droplet size (DS) and polydispersity index (PI) of the recon-

stituted microemulsion were 25.8 ± 9.8 nm and 0.095 ± 0.009,which were not significantly different (P > 0.05) from the22.0 ± 7.1 nm and 0.094 ± 0.011 of liquid SRL-SMEDDS. The results

X. Hu et al. / International Journal of Pharmaceutics 438 (2012) 123– 133 131

Fig. 6. SEM micrographs of SRL-SMEDDS pellets (F6). (A) (200×) and (B) (2000×) surface of the pellets; (C) (2000×) cross-section of the pellets.

Fig. 7. DSC thermograms of SRL powder (A), blank SMEDDS pellets powder (B),physical mixture(C) and SRL-SMEDDS powder (D).

Fig. 8. XRPD graphs of SRL powder (A), blank SMEDDS pellets powder (B), physicalmixture (C) and SRL-SMEDDS powder (D) (the typical diffraction peaks of SRL waslabeled with a–c).

Fig. 9. Infrared spectroscopy of SRL powder (A), blank SMEDDS pellets powder (B),physical mixture (C) and SRL-SMEDDS powder (D).

132 X. Hu et al. / International Journal of Ph

Fmn

insS

iermodi

3

co

aaicoep(frSHiwo

TPa

ig. 10. Blood concentration–time profiles of SRL after oral administration of opti-al SRL-SMEDDS pellets (F6) and commercial SRL tablets to beagle dogs (mean ± SD,

= 6).

ndicated that the solidification process did not led to sig-ificant variation in the droplet size of microemulsion. Theelf-microemulsifying ability was well maintained in the solid SRL-MEDDS pellets.

In general, the in vitro redispersiblity study and character-zation confirmed that the SRL-SMEDDS pellets prepared byxtrusion–spheronization had good quality. The drug content andelated substances were monitored during the process of experi-ent, and no significant content lose and drug degradation were

bserved (data were not shown), but the storage stability underifferent conditions are still unknown, which will be investigated

n the next stage of our research.

.8. Pharmacokinetic study

Bioavailability studies of optimum SRL-SMEDDS pellets (F6)ompared with commercial SRL tablets were investigated followingral administration of 2 mg SRL to six healthy beagle dogs.

The profiles of the mean plasma concentrations of SRL vs. timend the main pharmacokinetic parameters are shown in Fig. 10nd Table 7, respectively. Fig. 10 indicated significantly greatermprovement of drug absorption for SRL-SMEDDS pellets than theommercial SRL tablets. In the curves, there was a little increasef blood concentration at about 10 h, which might be caused bynterohepatic circulation. From Table 7, the Cmax of SRL-SMEDDSellets was about 45% higher than that of the commercial tabletsP < 0.01), and Tmax seemed shorter but was not significantly dif-erent (P > 0.05), suggesting that the SMEDDS could improve drugelease and absorption in GIT. The relative bioavailability of theRL-SMEDDS pellets to commercial tablets was about 136.89%.

owever, another earlier liquid SRL-SMEDDS was reported and

ts oral relative bioavailability to SRL oral solution (Rapamune®)as 215.04% in rats, but we think it was not comparable with

ur results that got from beagle dogs (Sun et al., 2010). Without

able 7harmacokinetic parameters after oral administration of commercial SRL tabletsnd SRL-SMEDDS pellets to beagle dogs (mean ± SD, n = 6).

Parameters Commercial SRL tablets SRL-SMEDDS pellets

Tmax (h) 1.04 ± 0.25 0.88 ± 0.21Cmax (ng/ml) 12.72 ± 0.36 18.44 ± 1.97*

AUC0→48 (ng h/ml) 96.17 ± 8.45 131.65 ± 7.81*

t1/2 (h) 12.18 ± 2.12 12.28 ± 2.53Relative bioavailability (%) – 136.89

* P < 0.01 versus commercial SRL tablets group by the ANOVA test.

armaceutics 438 (2012) 123– 133

a doubt, all of these studies indicated that the adsorption of SRLcould be evidently improved after it was loaded in SMEDDS for-mulations with a dissolved state. Therefore, for poorly solubledrugs such as SRL, SMEDDS can improve their solubility and main-tain them as a dissolved form. Once the SMEDDS enters the GIT,the spontaneous formation of microemulsion can improve drugrelease significantly and be beneficial to enhance absorption. Fur-thermore, the solid SMEDDS pellets, combining the advantages ofSMEDDS and solid dosage form, will enlarge the application scopeof advanced SMEDDS technology.

4. Conclusion

Sirolimus is a representative water insoluble drug with poororal bioavailability. The marketed formulation Rapamune® tabletshad overcome its problems of dissolution and erratic absorptionto some extent due to the inherent advantages of nanomedicine,but the oral bioavailability was still low. In this study, we suc-cessfully prepared SRL-SMEDDS and solidified it into pellets, aSRL-microemulsion sized below 50 nm was formed and dispersedrapidly when the pellets contacted with the water in the GIT, thesmall droplet size of microemulsion and dissolving state of SRL init (certified by phase characterization) could greatly facilitate theabsorption of SRL, finally a relative oral availability of 136.9% tothe Rapamune® tablets was achieved as our results. Furthermore,the oil phase and surfactant used in this system might have someactivity of facilitating the lymphatic transport and inhibiting thefirst-pass effect via intestinal and hepatic metabolism and effluxtransporter P-glycoprotein, the solid pellets also might improve thestability of liquid SRL-SMEDDS, which need further study. Basedon the above, SRL-SMEDDS pellets might be a useful system forthe oral delivery of drugs with poorly water solubility as SRL andthe extrusion–spheronization technology is conducive to industrialproduction.

Acknowledgement

This work was supported by the Natural Science Foundation ofFujian Province, China (Grant No. 2010J01218).

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