ORIGINAL ARTICLE

Enhanced Solubility and Oral Bioavailability of c-TocotrienolUsing a Self-Emulsifying Drug Delivery System (SEDDS)

Saeed Alqahtani • Alaadin Alayoubi •

Sami Nazzal • Paul W. Sylvester • Amal Kaddoumi

Received: 16 March 2014 / Accepted: 31 May 2014

� AOCS 2014

Abstract The aim of this study was to evaluate the

in vitro and in vivo performance of c-tocotrienol (c-T3)

incorporated in a self-emulsifying drug delivery system

(SEDDS) and to compare its enhanced performance to a

commercially available product, namely Tocovid Supra-

bioTM (hereafter Tocovid), containing tocotrienols. The

solubilization of c-T3 was tested in a dynamic in vitro

lipolysis model followed by in vitro cellular uptake study

for the lipolysis products. In addition, in vitro uptake

studies using Caco2 cells were conducted at different

concentrations of c-T3 prepared as SEDDS, Tocovid, or

mixed micelles. c-T3 incorporated in SEDDS or Tocovid

was orally administered to rats at different doses and

absolute oral bioavailability from both formulations were

determined. The dynamic in vitro lipolysis experiment

showed about two fold increase in the solubilization of c-

T3 prepared as SEDDS compared to Tocovid, which cor-

related with higher cellular uptake in the subsequent uptake

studies. In vitro cellular uptake and in vivo oral bioavail-

ability studies have shown a twofold increase in the cellular

uptake and oral bioavailability of c-T3 incorporated in

SEDDS compared to Tocovid as a result of improvement in

its solubility and passive uptake as confirmed by in vitro

studies. In conclusion, incorporation of c-T3 in SEDDS

formulation enhanced c-T3 solubilization and passive

permeability, thus its cellular uptake and oral bioavail-

ability when compared to Tocovid.

Keywords c-Tocotrienol � SEDDS � In vitro lipolysis �Oral bioavailability � Solubility � Non-linear kinetics

Abbreviations

MM Mixed micelles

NPC1L1 Niemann-pick C1-like 1

SEDDS Self-emulsifying drug delivery system

TRF Tocotrienols rich fraction

d-T3 Delta-tocotrienol

c-T3 Gamma-tocotrienol

Introduction

c-Tocotrienol (c-T3) is a member of the tocotrienols sub-

family of vitamin E natural products. Recently, several

studies have been conducted to explore c-T3 biological

activities. c-T3 exhibited an anticancer activity in various

types of cancer such as breast, leukemic, prostate, and

gastric cancers [1–5]. Other studies have reported that c-T3

works synergistically with other chemotherapeutic agents

to enhance their anticancer activities and reduce drug

resistance [6–8]. In addition, c-T3 exhibited other biolog-

ical activities including antioxidant, neuroprotective, and

anti-osteoporosis activities [9–11].

c-T3 is lipophilic in nature and has poor water solubility

and miscibility, thus, demonstrating low bioavailability

after oral administration [12, 13]. At 10 mg/kg dose, c-T3

oral bioavailability in rats was very low approaching 9 %

[14]. Recently, in situ permeability studies in our labora-

tory have shown that in addition to its low solubility, c-T3

intestinal permeability is also low [15]. Findings of these

studies revealed that c-T3 intestinal absorption is concen-

tration dependent and saturable process that is mediated by

S. Alqahtani � A. Alayoubi � S. Nazzal �P. W. Sylvester � A. Kaddoumi (&)

Department of Basic Pharmaceutical Sciences, College of

Pharmacy, University of Louisiana at Monroe, 1800 Bienville

Dr., Monroe, LA 71201, USA

e-mail: kaddoumi@ulm.edu

123

Lipids

DOI 10.1007/s11745-014-3923-6

the transport protein Niemann-Pick C1-like 1 (NPC1L1)

[15], which could explain its low oral bioavailability when

administered at therapeutic doses that saturate its own

uptake by NPC1L1. To overcome such limitation and

enhance its oral bioavailability, a delivery system that

enhances c-T3 permeability is essential.

One of the promising strategies to enhance the absorp-

tion of poor water soluble drugs is the utilization of lipid-

based delivery systems. Lipid-based formulations enhance

the oral bioavailability by several mechanisms including

increase in drug solubilization, increase drug incorporation

into lipoproteins which facilitate lymphatic absorption, and

bypass the first past metabolism effect by utilizing the

lymphatic absorption [16–22]. Self-emulsifying drug

delivery systems (SEDDS) have been widely used as a

successful example of lipid-based formulations. SEDDS

are composed of oils (synthetic or natural), surfactants, and

co-solvents [16, 23, 24]. Upon exposure to GI fluids with

mild agitation, SEDDS will form oil-in-water (o/w)

emulsions with droplet size ranging from 100 to 300 nm

[25, 26]. These small droplets provide a large surface area

to load more amounts of drug molecules and increase drug

partitioning through the intestinal wall [27]. Thus, for poor

water-soluble drugs that exhibit poor dissolution in the GI

tract and limited absorption, SEDDS should provide an

excellent improvement in absorption and oral bioavail-

ability. The selection of SEDDS formulation components is

governed by several criteria including: chemical stability,

purity, compatibility with drug, solvent capacity, digest-

ibility, and miscibility [28].

Several approaches have been utilized to improve the

oral bioavailability of c-T3 including SEDDS, in addition

to oil solutions and solid-lipid nanoparticles [29–31]. Most

of these formulations investigated tocotrienols rich fraction

(TRF). In addition, in a recent work, we have studied the

effect of SEDDS as a delivery system on the oral bio-

availability of a mixture of c-T3 and d-T3 (10:90 %) [32].

Findings from this work showed enhancement in the bio-

availability of d-T3, whereas, the effect of SEDDS on the

bioavailability of c-T3 was unclear due to the very low

doses present in conjunction with d-T3. Thus, in the current

study we aimed first, to further characterize the in vivo

bioavailability, and in vitro lipolysis and uptake of c-T3

incorporated into SEDDS at doses more relevant to its

potential clinical uses. Second, to compare the results to

those obtained from commercially available soft-gelatin

capsules named Tocovid SupraBioTM. To our knowledge,

Tocovid is the only commercially available product that

contains a formulation to enhance the absorption of to-

cotrienols. Tocovid soft-gelatin capsule contains TRF with

c-T3 as the major tocotrienol encapsulated in SupraBio

delivery system which is a patented bioenhanced self-

emulsifying formulation [33].

Materials and Methods

Materials

c-T3 (purity 96 %) was provided by First Tech Interna-

tional Co., Ltd. (Hong Kong). The commercially available

Tocovid capsules were purchased from a local drug store;

each capsule contains 200 mg of mixed tocotrienols divi-

ded as 61.52, 112.8, and 25.68 mg for a, c, and d-tocot-

rienols, respectively, 91.6 IU D-a-tocopherol, 51.28 mg

plant squalene, 20.48 mg phytosterol complex and 360 lg

phytocarotenoid complex [34]. Sodium taurocholate and

phosphatidylcholine were purchased from Avanti Polar

Lipids, Inc. (Alabaster, AL). d-Tocopherol, 4-bromophen-

ylboronic acid (4-BPBA), pancreatin from porcine pan-

creas, bile salts (BS) and Trizma� maleate were purchased

from Sigma (St Louis, MO). Polyoxyethylated castor oil

(Cremophor� EL), triglycerides of caprylic/capric acid

(Captex� 355), and C8/C10 polyglycolyzed glycerides

from coconut oil (Labrasol�) were provided by BASF

(Mount Olive, NJ, USA), Abitec Corporation (Janesville,

WI, USA), and Gattefosse (Saint-Priest, Cedex, France),

respectively. Ethyl alcohol USP was purchased from

AAPER Alcohol and Chemical Co. (Shelbyville, KY,

USA). Supplies of the cell culture were obtained from the

American Type Cell Culture Collection (ATCC; Manassas,

VA). Other chemicals and reagents were obtained from

VWR Scientific (West Chester, PA). All chemicals were

used as supplied without further modifications.

Animals

Male Sprague–Dawley rats weighing 250–350 g were

acquired from Harlan Laboratories (Houston, TX). All

animal experiments were approved by the Institutional

Animal Care and Use Committee (IACUC) of the Uni-

versity of Louisiana at Monroe, and all the surgical and

treatment procedures were consistent with the Institutional

Animal Care and Use Committee policies and procedures.

The rats were maintained on a 12 h light/dark cycle before

the study and had free access to food and water ad libitum

before each experiment.

SEDDS and Mixed Micelles Preparations

c-T3 loaded SEDDS were prepared as previously described

using cremophor EL (40.7 % w/w) as the primary surfac-

tant, labrasol (40.7 % w/w) as a co-surfactant, captex 355

(7.2 % w/w) as an oil, and ethanol (11.4 % w/w) as a co-

solvent [32, 35]. Mixed micelles (MM) were prepared as

previously reported [32, 36], and were formulated to con-

tain: taurocholate 300 lM, phosphatidylcholine 150 lM,

oleic acid 500 lM, and the required c-T3 concentrations in

Lipids

123

each experiment. MM were used as a control formulation

as they represent the end product of ingested oil in the

intestinal lumen that is readily available for absorption [29,

37].

Characterization of SEDDS Formulation

SEDDS characterization included intensity-weighed mean

particle size, population distribution (Polydispersity Index,

PI) which is a measure of homogeneity and width of the

size distribution, and Zeta potential (n) measured by photon

correlation spectroscopy (PCS) at 23 �C and a fixed angle

of 90� using a NicompTM 380 ZLS submicron particle size

analyzer (PSS Inc., Santa Barbara, CA). Before analysis,

SEDDS were reconstituted in transport buffer to obtain a

final tocotrienols concentration of 1 mg/ml. When needed,

samples for size analysis were diluted further with 0.2 ml-

filtered and deionized water in order to minimize multiple-

particle scattering and to achieve an optimal scattering

intensity of 300 kHz. The size was recorded for 3 min with

the viscosity and the dielectric constant of the medium set

to 1.33 and 78.5, respectively. The intensity-weighted

mean diameter of the particles was calculated based on the

Stokes–Einstein law by curve fitting of the correlation

function. The Zeta potential of the SEDDS was measured

using NicompTM 380 ZLS under zeta mode, and the Zeta

potential was determined using the Helmholz-Smolu-

chowsky equation. The stability of the SEDDS formulation

was also observed by measuring the particle size after

4 months of storage in refrigerated conditions (4–8 �C) to

monitor any change in particle size over the period of the

experiment. Analyses were performed in triplicates unless

otherwise specified.

In Vitro Lipolysis Studies

In vitro lipolysis studies were performed to measure and

compare released and solubilized c-T3 into the aqueous

phase from both SEDDS formulations. The procedure was

designed to mimic the dynamic physiological conditions as

previously reported [14, 38], where upon agitation, SEDDS

will form emulsions and disperse in the digestion medium.

The released lipophilic drug molecule will then be able to

form MM in the aqueous phase that will be ready for

absorption by enterocytes of the small intestine.

The model is built around a temperature-controlled

(37 �C) vessel that contains 100 ml of digestion buffer

containing bile salts (5 mM), phosphatidyl choline

(1.25 mM), Trizma� maleate (2 mM), sodium chloride

(150 mM), calcium chloride (0.5 mM), and pancreatic

lipase (800 USP units/ml) [14, 38, 39]. The pH was

adjusted to 6.8 with 1 M NaOH solution. The medium was

stirred continuously (400 rpm) and maintained at 37 �C

using a water-jacketed reaction vessel connected to a

thermostatically controlled water bath (Haake, Germany).

One gram of SEDDS formulation and Tocovid (each

containing 20 mg c-T3) was prepared by stirring with

gentle heating for 30 min before the experiment, and was

dispersed and stirred for 10 min in the digestion buffer to

prepare a 50 lM concentration in 100 ml buffer. Fresh

pancreatic extract was prepared by adding 2.2 g of porcine

pancreatin powder to 2 ml digestion buffer, and stirred and

homogenized for 2 min (T10 basic Ultra-Turax homoge-

nizer; IKA Works, Inc., Wilmington, NC). The pancreatin

suspension was then warmed in a water bath for 20 min to

allow the activation of the lipase enzyme. A pancreatin

suspension (800 IU/ml, 1 ml) was inserted into the med-

ium to initiate the enzymatic digestion of the formulations.

A pH titrator unit (Radiometer Analytical SAS, France)

was used to maintain the pH at 6.8 throughout the exper-

iment in order to achieve maximum simulation of the

physiological conditions. This is important because during

the lipolysis process, triglycerides are hydrolyzed to release

free fatty acids that will decrease the pH of the medium.

The experiment was continued for 120 min. Samples of

10 ml were withdrawn at 0, 5, 10, 15, 30, 60, and 120 min

and the sampled volume was replaced by blank (non-drug

containing) digestion buffer. Lipolysis was inhibited with

20 ll of 1 M 4-BPBA. Each sample was transferred to

glass centrifuge tubes and centrifuged at 75,000 rpm

(5.4 9 105g) for 35 min at 10 �C in a Beckmann-Coulter

OptimaTM MAX Ultracentrifuge with a 90-Ti rotor (Ful-

lerton, CA). Centrifugation resulted in separation of the

end product solution into three phases: an aqueous phase, a

lipid phase and sediment. The aqueous phase containing

the solubilized drug, bile salts, and fatty acids was sampled

and analyzed by HPLC to determine the concentrations of

solubilized c-T3.

In Vitro Studies

Cell Uptake Studies of c-T3 from Lipolysis Test

Products

Caco2 cells were cultured in Dulbecco’s Modified Eagle

Medium (DMEM) supplemented with 20 % fetal bovine

serum (FBS) and 2.5 % antibiotics (10,000 I.U. penicillin

and 10 mg streptomycin per ml) in a humidified incubator

with 5 % CO2 at 37 �C. For the experiments, cells were

seeded onto a 48-well plate at a density of 50,000 cells/well.

When confluent, uptake studies were performed as descri-

bed below. All experiments were conducted in triplicate.

In order to attain maximal simulation of the physio-

logical absorption process, 1 ml of the in vitro lipolysis

product at each time point (0, 5, 10, 15, 30, 60, and

Lipids

123

120 min) of the lipolysis process was diluted in transport

buffer to prepare 5 lM, and then directly transferred to the

consecutive uptake study. Two hundred microliters from

each time point of the diluted lipolysis products of the

SEDDS formulation and Tocovid were added and incu-

bated for 45 min. At the end of the experiment, cells were

washed twice with ice cold PBS and lysed with 100 ll lysis

buffer overnight at 4 �C. Cell lysates were then subjected

to extraction and analysis by HPLC as described below.

Cell Uptake Studies for SEDDS, Tocovid, and MM

c-T3 loaded SEDDS, MM and Tocovid were diluted in

transport buffer to prepare different concentrations ranging

from 1 to 50 lM of c-T3. Two hundred microliters from

each concentration of SEDDS, MM, and Tocovid were

added and incubated for 45 min. At the end of the exper-

iment, cells were washed twice with ice cold PBS and

lysed with 100 ll lysis buffer overnight at 4 �C followed

by extraction and analysis by HPLC as described below.

Passive Uptake Studies

Passive uptake studies of SEDDS formulation and Tocovid

were performed at 4 �C. Different concentrations of c-T3

(1, 5, 10, 25, 50 lM) loaded SEDDS or Tocovid were

added to the cells at 4 or 37 �C for 45 min. After the

treatment period, cells were washed, lysed and analyzed by

HPLC as described below.

In Vivo Oral Bioavailability Studies

c-T3 prepared as SEDDS or Tocovid were orally adminis-

tered to fed rats via oral gavage at 1, 2.5, 10, 25 and 50 mg/

kg doses. Rats were anesthetized with intraperitoneal

injection of 1 g/kg urethane dissolved in normal saline.

Blood samples were withdrawn from the femoral vein at 1,

2, 3, 4, 5, 6, 8, 10, 12 h. Blood samples were collected in

heparinized Eppendorf tubes. The samples were then cen-

trifuged at 13,000 rpm for 10 min. The separated plasma

was stored at -20 �C until analysis by HPLC. Three rats

were used for each dose. Plasma levels versus time profiles

were plotted and fitted using a PK-Plus module in Gastro-

plusTM (Simulation Plus Inc., Lancaster, CA). The AUC

from time 0 to lat time point (12 h) was calculated by the

trapezoidal rule method. Pharmacokinetic parameters were

calculated using non-compartmental analysis.

HPLC Analysis of c-T3

c-T3 quantification in cell lysate and plasma samples was

performed as described previously [40]. The method is

fully validated and achieved using an isocratic Prominence

Shimadzu HPLC system (Columbia, MD). The system

consisted of a SIL 20-AHT autosampler, fluorescence

detector (Shimadzu, RF10A XL) and an LC-20AB pump

connected to a Dgu-20A3 degasser. Data acquisition was

achieved by LC Solution software version 1.22 SP1. The

chromatographic conditions were a XDB-C18 Column

(5 lm, 150 9 4.6 mm i.d.; Agilent, CA, USA), the mobile

phase consisted of methanol, ethanol and acetonitrile

(85:7.5:7.5, v/v/v) delivered at a 1.0 ml/min flow rate. The

c-T3 in plasma and cell lysate was detected by a fluores-

cence detection set at 298 nm excitation and 325 nm

emission. The total run time was 7 min with retention

times of 4.8 min for c-T3 and 6.5 min for the internal

standard. The injection volume was 20 ll. The extraction

of c-T3 from the plasma was performed as described pre-

viously [15]. In brief, 50 ll plasma and 5 ll ethanol con-

taining 1 % ascorbic acid and 1 lg/ml d-tocopherol

(internal standard) were put into a glass tube and vortex

mixed, followed by the addition of 1.0 ml hexane. The

mixture was vortexed for 90 s before centrifugation for

10 min at 5,000 rpm. Eight hundred microliters of the

supernatant was then transferred to a glass tube and

evaporated to dryness with a centrifugal evaporator (Cen-

trivap concentrator, Kansas City, MO, USA) followed by

reconstitution with 100 ll methanol before a 20-ll injec-

tion onto the HPLC column. For the extraction of c-T3

from the cell lysate, 1:1 precipitation with acetonitrile

followed by centrifugation at 13,000 rpm for 10 min was

performed before injection into the HPLC.

Data Analysis

GraphPad Prism, version 6.00 (GraphPad Software, San

Diego, CA, USA) was used to perform all statistical anal-

yses. The observed maximum concentration (Cmax) and the

time of peak (Tmax) were obtained directly from the indi-

vidual plasma concentration versus time profiles. The area

under the curve (AUC) was calculated using the trapezoidal

rule method. The Michaelis -Menten parameters Vmax and

Km were determined from non-linear regression of the

concentration versus cellular uptake curves by GraphPad

Prism. The results were presented as means ± SD. p val-

ues \ 0.05 were considered statistically significant.

Results

SEDDS Characterization

The mean droplet size, PI, and Zeta potential values of the

dispersion generated by SEDDS formulation in transport

buffer used in the cellular studies were 117 ± 4 nm,

0.5 ± 0.01, and -14 ± 3, respectively. The SEDDS

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123

formulation proved to be stable for 4 months when stored at

4 �C, where their dispersion in transport buffer following

storage demonstrated mean droplet size, PI, and Zeta

potential values comparable to the original values (Table 1).

In Vitro Lipolysis Studies

The solubilization behavior of c-T3 prepared as SEDDS or

Tocovid was tested in a biorelevant dissolution system

[38], followed by HPLC analysis for the fraction solubi-

lized in the aqueous phase for c-T3. As shown in Fig. 1, the

release performance of c-T3 from SEDDS formulation was

significantly improved compared to Tocovid. The solubi-

lization rate and the fraction dissolved for c-T3 prepared as

SEDDS was relatively higher and reached 60 % compared

to Tocovid that was less than 20 % at the end of the

experiment (120 min). The concentrations observed at zero

time represent the solubilization of c-T3 as a result of its

intrinsic solubility before micelle formation due to lipolysis

(Fig. 1).

Cell Uptake Studies of c-T3 from Lipolysis Products

Two hundred microliters of the diluted lipolysis products at

each time point of 0, 5, 10, 15, 30, 60, and 120 min of the

SEDDS formulation and Tocovid (each containing 5 lM c-

T3, confirmed by HPLC analysis) were incubated with

Caco2 cells for 45 min. As shown in Fig. 2, the cellular

uptake of c-T3 prepared as SEDDS after the lipolysis test

was significantly higher compared to the same time point

from Tocovid (p \ 0.001 at all-time points). These findings

demonstrate that incorporation of c-T3 into SEDDS for-

mulation significantly increased the solubilization and as a

result the cellular uptakes after the lipolysis test.

Cell Uptake Studies for SEDDS, Tocovid, and MM

Different concentrations of c-T3 (1, 5, 10, 25, and 50 lM)

prepared as SEDDS, Tocovid or MM were incubated with

Caco2 cells for 45 min. MM were used as the control

formulation as they represent the end product of ingested

oil in the intestinal lumen that is readily available for

absorption [29, 37]. Viability of the cells following treat-

ment with different concentrations of different formula-

tions was confirmed by visual inspection under the

microscope and the treated cells looked healthy at the end

of experiment. As illustrated in Fig. 3, the cellular uptake

of c-T3 prepared as SEDDS were significantly higher

compared to the same concentrations from Tocovid or MM

(p \ 0.001). Vmax and KM for c-T3 uptake from SEDDS,

Tocovid and MM were calculated and showed higher

values with SEDDS compared to the other two formula-

tions. The Vmax value for c-T3 from SEDDS was signifi-

cantly higher with 310 ± 22 ng/mg protein/45 min

(p \ 0.001), compared to 159.6 ± 7 and 184.4 ± 15 ng/

mg protein/45 min from Tocovid and MM, respectively.

The KM values for c-T3 from SEDDS, Tocovid and MM

were not significantly different with the values of

36.4 ± 4.8, 30 ± 3 and 45.2 ± 7 lM, respectively

(p [ 0.05). These uptake results demonstrated that incor-

poration of c-T3 into the SEDDS formulation significantly

increased overall cellular uptake at high and low

concentrations.

Table 1 Physical characterization of SEDDS formulation, particle

size, polydispersity index, and Zeta potential

Storage time Particle size

(nm)

Polydispersity

index (PI)

Zeta potential (n)

Freshly prepared 117 ± 4 0.50 ± 0.01 -14 ± 3

After 4 months 124 ± 5 0.45 ± 0.04 -15 ± 2

Fig. 1 Release rate (%) of c-T3 from 20 mg c-T3/g SEDDS and

Tocovid at different time points into the aqueous phase. Each value

represents the mean ± SD of n = 4 for each formulation

Fig. 2 Cellular uptake of c-T3 in ng/mg protein following incubation

with lipolysis products obtained from the lipolysis test at different

time points. Each value represents the mean ± SD from three

independent experiments. ***Indicates significantly different

between SEDDS and Tocovid (p \ 0.001)

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123

Passive Uptake Studies

In order to examine the passive uptake contribution to the

total cellular uptake, we conducted an uptake study at 4 �C.

Previous studies showed that at 4 �C active carrier medi-

ated transport and pinocytic/endocytic uptake of molecules

are usually stalled [41]. Data obtained following cells

treatment with SEDDS or Tocovid loaded with different

concentrations of c-T3 (1–50 lM) at 37 and 4 �C are

presented in Fig. 4. At 4 �C the cellular uptake of c-T3

loaded in SEDDS delivery system or Tocovid was reduced

significantly when compared to the cellular uptake at 37 �C

with 40–50 % reduction (p \ 0.0001). Although the cel-

lular uptake of c-T3 from SEDDS was reduced signifi-

cantly at 4 �C, it remained at twofold higher than the

cellular uptake of c-T3 from the Tocovid at 4 �C

(p \ 0.0001). These findings demonstrate that SEDDS

formulation enhanced the passive uptake of c-T3.

In Vivo Oral Bioavailability Studies

The mean plasma concentrations versus time profiles fol-

lowing oral administration of 1, 2.5, 10, 25, and 50 mg/kg

of c-T3 as SEDDS or Tocovid to fed rats are shown in

Fig. 5. The oral pharmacokinetic parameters of c-T3 fol-

lowing administration of both formulations are listed in

Table 2. Peak concentration (Cmax) and time of peak con-

centration (Tmax) were obtained directly from individual

plasma concentration–time profile of each rat. The AUC

following intravenous administration of 10 mg/kg of c-T3

to the rats was obtained from previous work [14]. The AUC

from time zero to time t was calculated and the absolute

bioavailability for each dose of c-T3 from SEDDS and

Tocovid was calculated using the following Eq:

F ¼ AUCoral=AUCivð Þ � Doseiv=Doseoralð Þ½ �:

As illustrated in Fig. 5, plasma concentration of c-T3 at 10,

25, and 50 mg/kg doses are significantly higher for rats

administered with SEDDS formulation compared to

Tocovid (p \ 0.001), whereas plasma concentration pro-

files at 1 and 2.5 mg/kg for both formulations were the

same (p [ 0.05). Furthermore, the AUC values of 10, 25,

and 50 mg/kg of c-T3 from SEDDS were significantly

higher compared to Tocovid (p \ 0.001), whereas, the

AUC of 1 and 2.5 mg/kg from SEDDS were not signifi-

cantly different compared to Tocovid (p [ 0.05). The AUC

values for 1, 2.5, 10, 25, and 50 mg/kg of c-T3 from

SEDDS were 317 ± 66, 765 ± 67, 2,016 ± 178,

5,218 ± 372 and 5,191 ± 291 ng.h/ml, respectively,

compared to 338 ± 98, 748 ± 114, 1,190 ± 78,

2,607 ± 99 and 2,620 ± 385 ng.h/ml, respectively, from

Tocovid. The bioavailability of c-T3 was two times higher

at 10, 25 and 50 mg/kg doses administered as SEDDS

compared to the same doses from Tocovid (Table 2). These

findings demonstrated that the SEDDS formulation was

able to significantly improve the oral bioavailability of c-

T3 at doses [ 2.5 mg/kg compared to Tocovid.

Discussion

Despite all promising biological activities, c-T3 is a lipo-

philic compound with very limited aqueous solubility but

displays high solubility in organic solvents (about 10 mg/

ml) (CAS registry numbers 14,101-61-2). Due to its poor

water solubility, c-T3 has limited absorption and low

bioavailability. Additionally, our previous work has shown

that the intestinal absorption of c-T3 is mediated by

NPC1L1 transport protein, which becomes saturated in the

presence of high c-T3 concentrations resulting in limited

permeability and bioavailability [15, 32]. Thus, in the

Fig. 3 Cellular uptake of c-T3 in ng/mg protein/45 min following

incubation with various concentrations of c-T3 ranging from 1 to

50 lM prepared as SEDDS, Tocovid or MM. Each value represents

the mean ± SD from three independent experiments

Fig. 4 Cellular uptake of c-T3 in ng/mg protein following incubation

with various concentrations of c-T3 loaded as SEDDS or Tocovid at

37 and 4 �C. Each value represents the mean ± SD from three

independent experiments

Lipids

123

current study we hypothesized that the oral bioavailability

of c-T3 will be improved by SEDDS formulations due to

enhanced solubility and passive permeability. While there

are a number of potential mechanisms whereby SEDDS

formulations may increase the oral bioavailability,

enhancing the solubility and passive permeability are

considered the key. SEDDS formulations have been

reported to promote solubility and passive permeability of

highly lipophilic drugs [25].

In the present work, c-T3 was incorporated in a SEDDS

formulation, then investigated for its in vitro and in vivo

performance at different doses and was compared to a

commercially available product known as Tocovid

SuprabioTM which also contains a self-emulsifying drug

delivery system [33]. Solubilization and drug release are

considered among the most important factors that impact

the bioavailability and the absorption of orally adminis-

tered compounds. Therefore, in vitro lipolysis and solubi-

lization studies for c-T3 loaded in SEDDS formulation or

in Tocovid were conducted to understand and predict the

fate of this compound upon its dilution and dispersion in

the GI media. Findings from these studies showed that the

fraction of c-T3 released into the aqueous phase was

twofold higher from SEDDS formulation compared to

Tocovid (Fig. 1). The presence of more drug molecules

solubilized in the aqueous phase of the lipolysis medium

Fig. 5 Plasma concentrations of c-T3 in ng/ml versus time (h) profiles following oral administration of c-T3 a 1 mg/kg, b 2.5 mg/kg, c 10 mg/

kg, d 25 mg/kg, and e 50 mg/kg loaded in SEDDS or Tocovid in rats. Each value represents the mean ± SD for n = 3 rats/dose/formulation

Lipids

123

increases the chance for more absorption; on the other

hand, drug molecules that remain in the oil phase will not

be available for absorption. Following the completion of

the lipolysis process and before centrifugation, 200 ll of

the diluted lipolysis products were incubated with Caco2 in

order to achieve maximal simulation of the physiological

absorption process and to demonstrate the effect of

enhanced drug solubilization on cellular uptake. Lipolysis

products media contained the digestion buffer, the drug

molecules in the different states, solubilized in the aqueous

phase or unsolubilized in the oil phase, and the lipolytic

products liberated from the tested lipidic vehicle following

the lipolysis process [38]. Findings of this study revealed

that the cellular uptake of c-T3 was significantly higher

from SEDDS lipolysis products compared to Tocovid

(Fig. 2).

Furthermore, in vitro uptake studies were conducted to

compare the cellular uptake of SEDDS and Tocovid to MM

as the most readily available form for intestinal absorption.

SEDDS formulation improved the in vitro cellular uptake

of c-T3 compared to Tocovid and MM formulations

(Fig. 3). The cellular uptake of c-T3 loaded in SEDDS at

different concentrations was approximately twofold higher

than its cellular uptake from Tocovid and MM at all con-

centrations tested. Additionally, in vitro studies have been

conducted to explain enhanced c-T3 cellular uptake from

SEDDS compared to Tocovid. At 4 �C, the active transport

processes are essentially halted and only the passive pro-

cesses are functioning [32, 41]. The cellular uptake of c-T3

at 4 �C was significantly reduced when compared to the

cellular uptake at 37 �C for both formulations (Fig. 4).

However, the passive uptake of SEDDS containing c-T3

was twofold higher compared to Tocovid. These results

suggested that SEDDS formulation enhanced the cellular

uptake through passive permeability. A possible explana-

tion for the increased passive permeability is SEDDS ex-

cipients that can act to modulate membrane fluidity and/or

the ability of the SEDDS formulation to hide the drug

molecules from being recognized by transport systems

[29].

Consistent with the in vitro data, SEDDS formulation

improved the in vivo oral bioavailability of c-T3 compared

to Tocovid. The SEDDS formulation showed significantly

higher oral bioavailability at 10, 25, and 50 mg/kg doses

with a twofold increase, but not at 1 and 2.5 mg/kg doses

where no improvement was observed compared to Tocovid

(Table 2). The improvement in oral bioavailability goes

along with improvement in AUC and Cmax of c-T3 pre-

pared as SEDDS compared to Tocovid (Fig. 5). The AUC

and bioavailability of 2.5 mg/kg dose prepared as SEDDS

were similar to the AUC and bioavailability values for the

same dose in our previous study [32]. In the present work,

c-T3 showed different behavior when it is incorporated inTa

ble

2C

om

par

ativ

ep

har

mac

ok

inet

icp

aram

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hen

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edas

SE

DD

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vid

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91

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±1

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22

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Lipids

123

SEDDS compared to d-T3 of our previous work [32]. The

bioavailability of c-T3 prepared as SEDDS at higher doses

were significantly enhanced compared to Tocovid, a find-

ing which is different from that observed with d-T3 pre-

pared in SEDDS where d-T3 showed reduced

bioavailability in the range 0.5–25 mg/kg [32]. The in vitro

uptake studies for d-T3 and c-T3 showed that d-T3 has a

slightly higher uptake affinity (KM = 26.6 lM) compared

to c-T3 (KM = 36.4 lM), but c-T3 has a much higher

uptake rate, thus the intrinsic uptake (Vmax/KM) of c-T3

was 1.6-fold higher than d-T3 (0.45 and 0.28 ll/min/mg

protein for c-T3 and d-T3, respectively), which could

explain the different in vivo bioavailability and behavior of

c-T3 compared to d-T3.

The in vitro lipolysis model provides an efficient tool to

simulate the digestion of lipid-based formulations in the

small intestine and offers important information in this

regard. However, this model has limitations in predicting

the in vivo performance of lipid-based formulation, where

some of loaded lipophilic molecules suffer from the limited

permeability in addition to their solubility problems. From

our SEDDS lipolysis and consequent in vitro uptake studies

of the lipolysis products, a positive correlation was

observed between the percent of c-T3 solubilized in the

aqueous phase of the lipolysis medium and the amount of

the cellular uptake indicating an association between the

enhanced solubility of c-T3 and the higher cellular uptake

(Fig. 6). However and as demonstrated in Fig. 6, this

association ends at a certain point where the permeability

as a barrier becomes evident at high solubilized concen-

trations due to saturation of the active uptake process.

These results are also in alignment with the in vivo data

(Fig. 7) where the increase in the percent of soluble c-T3

from SEDDS formulation caused a twofold increase in the

AUC values of c-T3 compared to Tocovid (Fig. 7), and

similar to the in vitro data, this increase was to a certain

point due uptake saturation in case of SEDDS, and

solubility and possible saturation in the case of Tocovid. At

low doses (1 and 2.5 mg/kg), however, there were no dif-

ferences in c-T3 bioavailability when compared to Toco-

vid. A possible explanation for this result could be related

to the efficient function of NPC1L1 in c-T3 transport in the

presence of small amounts of c-T3 (much lower than its

KM) in the GI tract from both formulations. Collectively,

the current SEDDS formulation increased the solubility

(from lipolysis studies) and passive uptake (from Caco2

uptake studies) of c-T3, thus enhanced its bioavailability at

the investigated doses, however, compared to Tocovid, c-

T3 bioavailability from SEDDS increased only at[2.5 mg/

kg doses.

In the present study, the SEDDS formulation was loaded

with c-T3 only, while Tocovid capsules contain a mixture

of tocotrienols, a-tocopherol, plant squalene, phytosterol,

and phytocarotenoid complex. It is highly possible, espe-

cially when given at high doses, that presence of these

compounds beside c-T3 have negatively affected c-T3

absorption and bioavailability by competing for the same

transport protein. For example, d-T3 and a-tocopherol have

already been established as substrates for NPC1L1 trans-

port protein [32, 36], and their coexistence is expected to

limit c-T3 transport thus absorption and bioavailability. In

addition, the possibility of the other components to com-

pete with c-T3 cannot be excluded. Collectively, the

human daily dose, one capsule per day, of c-T3 provided

by Tocovid is about 1.9 mg/kg (for a 60 kg person) which

is equivalent to 10–15 mg/kg in rats calculated using the

body surface area (BSA) normalization method [42]. Our

SEDDS formulation, on the other hand, was able to

enhance the oral bioavailability of c-T3 in rats at doses

higher than 2.5 mg/kg by twofold, providing an excellent

solution to achieve the same plasma level of c-T3 with

lower intake amount compared to Tocovid.

In summary, the results of this study demonstrated that

the SEDDS formulation has successfully enhanced the

Fig. 6 Correlation between the percentage of c-T3 solubilized in the

aqueous phase of SEDDS lipolysis medium and the in vitro cellular

uptake of c-T3 from the lipolysis medium at each time point

Fig. 7 The in vivo AUC values following oral administration of

different doses of c-T3 loaded in SEDDS or Tocovid. Each value

represents the mean ± SD for n = 3 rats/formulation. **Indicates

significantly different between SEDDS and Tocovid (P \ 0.01)

Lipids

123

solubility and passive permeability, thus the oral bio-

availability, of c-T3. This study showed the capability of

the SEDDS formulation to enhance the drug release into

the aqueous phase and increase drug solubilization as well.

In addition, SEDDS formulation components were able to

enhance c-T3 intestinal permeability acting as permeation

enhancers. The present study showed the significance of

the information obtained from the in vitro lipolysis model.

Furthermore, the consecutive uptake study for lipolysis

products achieve maximal simulation of the physiological

absorption process and provide a valuable tool to predict

the in vivo performance of lipid-based formulations.

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