Absorption of the novel artemisinin derivatives artemisone and artemiside: Potential application of Pheroid technology

Article abstract of DOI:10.1016/j.ijpharm.2011.05.003

Artemisinins have low aqueous solubility that results in poor and erratic absorption upon oral administration. The poor solubility and erratic absorption usually translate to low bioavailability. Artemisinin-based monotherapy and combination therapies are

Full text of DOI:10.1016/j.ijpharm.2011.05.003

International Journal of Pharmaceutics 414 (2011) 260–266
Contents lists available at ScienceDirect
International Journal of Pharmaceutics
journal homepage: www.elsevier.com/locate/ijpharm
Pharmaceutical Nanotechnology
Absorption of the novel artemisinin derivatives artemisone and artemiside:
TM
Potential application of Pheroid technology
a,∗
b
a
a
J. Dewald Steyn , Lubbe Wiesner , Lissinda H. du Plessis , Anne F. Grobler ,
b
c
c
a
Peter J. Smith , Wing-Chi Chan , Richard K. Haynes , Awie F. Kotzé
Unit for Drug Research and Development, North-West University, Potchefstroom 2531, South Africa
Division of Clinical Pharmacology, University of Cape Town, Medical School Observatory, 7925 Cape Town, South Africa
Department of Chemistry, Open Laboratory of Chemical Biology, Institute of Molecular Technology for Drug Discovery and Synthesis,
a
b
c
The Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Artemisinins have low aqueous solubility that results in poor and erratic absorption upon oral administra-
tion. The poor solubility and erratic absorption usually translate to low bioavailability. Artemisinin-based
monotherapy and combination therapies are essential for the management and treatment of uncompli-
cated as well as cerebral malaria. Artemisone and artemiside are novel artemisinin derivatives that have
Received 20 January 2011
Received in revised form 26 April 2011
Accepted 2 May 2011
Available online 7 May 2011
TM
very good antimalarial activities. Pheroid technology is a patented drug delivery system which has the
ability to entrap, transport and deliver pharmacologically active compounds. Pharmacokinetic models
were constructed for artemisone and artemiside in Pheroid vesicle formulations. The compounds were
Keywords:
Malaria
TM
administered at a dose of 50.0 mg/kg bodyweight to C57 BL/6 mice via an oral gavage tube and blood
samples were collected by means of tail-bleeding. Drug concentrations in the samples were determined
using an LC/MS/MS method. There was 4.57 times more artemisone in the blood when the drug was
Artemisone
Artemiside
TM
Pheroid technology
TM
Pharmacokinetic analysis
entrapped in Pheroid vesicles in comparison to the drug only formulation (p < 0.0001). The absorption
TM
of artemiside was not dramatically enhanced by the Pheroid delivery system.
©
2011 Elsevier B.V. All rights reserved.
1
. Introduction
malarials. Artemisinin-based combination therapy is the treatment
of choice and it is of great importance that the efficacy of those
therapeutic regimens is maintained (Dondorp et al., 2009). There
is presently no other effective alternative to surmount the ever
increasing problem of drug resistance. It is thus essential to focus
all efforts on the research and development of novel antimalar-
ial compounds and the effective delivery thereof (Baird, 2005;
Mutabingwa, 2005).
Artemisinins are based on the natural product artemisinin
that was first isolated in China in the early 1970s. Artemether,
an artemisinin derivative, is known to be as effective as qui-
nine for the treatment of severe P. falciparum malaria (Meshnick
et al., 1996). Other artemisinin derivatives include dihydro-
artemisinin (DHA) and artesunate. One key advantage of these
agents is the fact that they are active against all of the red
blood cell stages of P. falciparum (Krishna et al., 2004). At this
stage, there is limited resistance to these agents (Dondorp et al.,
2009). Due to the short elimination half-life of artemisinin-based
drugs it is recommended that they are used in combination with
other drugs such as mefloquine, lumefantrine or amodiaquine as
first-line therapies for the treatment of uncomplicated malaria
Malaria is an infectious disease caused by parasites of the
Plasmodium genus. The parasites are primarily hosted by female
Anopheles mosquitoes, which act as vectors that transmit the pro-
tozoan organisms to humans when feeding. There are four known
species that infect humans: Plasmodium falciparum, Plasmodium
vivax, Plasmodium ovale and Plasmodium malariae. However, P. fal-
ciparum can be held liable for the majority of malaria infections
(
Hay et al., 2004; Gkrania-Klotsas and Lever, 2006; WHO, 2009).
Drug-resistant malaria is a huge threat and stringent measures
must be taken to ensure the preservation of effectiveness of current
antimalarial drugs. Drug-resistant malaria materializes with evo-
lutionary, single or multiple, point-mutations in the Plasmodium
genome rendering parasites insensitive to drugs (White, 2004). The
emergence and spread of this phenomenon has greatly affected the
control and treatment of malaria in endemic countries, specifically
concerning P. falciparum infections (Hay et al., 2004; WHO, 2006).
Combination drug therapy is currently the mainstay approach in
preventing the development of further resistance to current anti-
(White, 2004). New artemisinin derivatives such as artemisone
∗ Corresponding author. Tel.: +27 18 299 2102; fax: +27 18 299 2101.
E-mail address: dewald.steyn@nwu.ac.za (J.D. Steyn).
and artemiside are reported to be much more potent than the
existing derivatives and it would be of great value to optimize
0
378-5173/$ – see front matter © 2011 Elsevier B.V. All rights reserved.
doi:10.1016/j.ijpharm.2011.05.003

J.D. Steyn et al. / International Journal of Pharmaceutics 414 (2011) 260–266
261
Table 1
CH3
H
Aqueous solubility and octanol–water partition coefficients.
CH3
H
H
Artemiside
Artemisone
Solubility [mg/L]
Log P
Log P (calculated)
<2
4.97
3.98
89
2.49
2.08
O
O
O
O
H
O
Solubility = aqueous solubility at pH 7.2.
O
Log P = octanol–water partition coefficient for the neutral compound determined at
pH 7.4, except artesunate which was determined by HPLC at pH 2.
Log P (calculated) = calculated log P value (Haynes et al., 2006).
O
CH
O
N
amine nucleophile (Fig. 2, route a). The yield of the two com-
pounds ranged between 40 and 60%. Oxidation of artemiside (12)
produced artemisone (14) and also a sulfoxide (13). An easier
route to follow involves the treatment of DHA (2) with a mix-
ture of NaBr and then with TMSCI in toluene followed by the
amine (Fig. 2, route b). Both artemiside (12) and artemisone (14)
were isolated by crystallization of the crude product mixtures and
are subsequently relatively accessible and produced as isomeri-
cally pure, air-stable, substances (Haynes et al., 2006). The aqueous
solubility and octanol–water partition coefficients of artemisone
and artemiside are given in Table 1. Vitamin F ethyl ester was
obtained from Chemimpo (South Africa) and Cremaphor® EL was
obtained from BASF (South Africa). Polyethylene glycol (PEG 400),
d-␣-tocopherol and butylated hydroxyanisole (BHA) were pur-
chased from Chempure (South Africa). Analytical grade artemisinin
and dimethyl sulfoxide (DMSO) were obtained from Merck (South
Africa).
N
S
O
O
S
Artemisone
Artemiside
Fig. 1. Structures of artemisone and artemiside.
the delivery of these compounds by using novel drug delivery
systems (Woodrow et al., 2005; Haynes et al., 2006).
Alternative drug delivery options, such as the Pheroid
ery system, may play a key role in ensuring effective delivery
TM
deliv-
and enhanced absorption of these novel antimalarial compounds.
TM
Pheroid
technology is a patented, novel, colloidal type drug
delivery system. It primarily consists of the ethyl esters of essen-
2.2. Reference formulations
tial fatty acids and nitrous oxide (N O)-water. It is postulated
2
that the role of N O is to stabilize the moving autofluores-
Oral reference formulations were prepared (0.0625 g drug
in 10 ml of the final formulation) and administered at a
2
TM
cent particles surrounding the Pheroid
vesicles and that it is
essential for ensuring stability of the formulation and efficient
delivery of test compounds. The stabilization can be microscop-
ically observed through the tracking of the movement of the
autofluorescent or fluorescently labeled particles representing the
ethyl fatty acids. Additional studies are, however, still being con-
ducted. The PheroidTM delivery system is known to remain stable
and structurally intact for a period of at least 24 months at room
temperature.
dose of 50.0 mg/kg bodyweight for each compound to com-
TM
pare against the Pheroid
vesicle formulations. The reference
formulations were prepared immediately before administration
by dissolving the appropriate amount of each drug in ana-
lytical grade DMSO. Purified water was then added until the
desired volume was reached. The final DMSO to water ratio
was 1:9 v/v. Both of the reference formulations formed a micro-
suspension after the addition of water to the DMSO-drug
solution.
TM
The Pheroid delivery system is superior to most other delivery
systems and is able to improve the delivery of dynamic com-
plexes, reduce the time to onset of action, decrease the minimal
effective drug concentration and enhance therapeutic efficacy. It
is also able to indirectly decrease the cytotoxicity of therapeutic
compounds and to infiltrate virtually all known barriers in the
body. The system is further capable of targeting specific treat-
ment areas, to transport genetic material to the cellular nucleus
and to decrease drug resistance (Grobler et al., 2008). The aim of
this study was to evaluate the possibility to enhance the absorp-
.3. PheroidTM formulations
2
PheroidTM vesicles were prepared by heating and mixing
vitamin F ethyl ester (66.2 g), Cremaphor EL (27.6 g) and d-
®
␣-tocopherol (1.0 g). PEG 400 was then added (5.0 g) together
with BHA (0.2 g) and mixed thoroughly. This mixture consti-
tuted the oil-phase of the Pheroid
oxide water was prepared by saturating purified water with N O
under high pressure. The required amount of drug (0.0625 g)
was added to 1.0 ml of the prepared oil-phase (at room tem-
perature). This mixture was then agitated for a period of 2 min
until all the drug particles had dissolved. The prepared nitrous
oxide water was then added to the oil-phase to achieve a total
volume of 10.0 ml (oil to water ratio, 1:9 v/v). The mixture was
homogenised with a Heidolph Diax 600 homogeniser (Labotec,
TM
vesicle formulations. Nitrous
2
TM
tion of artemisone and artemiside with the aid of Pheroid -based
formulations.
2
. Materials and methods
2.1. Materials
Artemiside is the thiomorpholine precursor of artemisone
Fig. 1). This was prepared from thiomorpholine and DHA, and then
South Africa) at 8000 rpm for 1 min (Jonker et al., 2002). The
TM
(
size of the Pheroid
vesicles was measured with a Malvern
converted into artemisone according to the methods described in
the literature from a multigram-scale reaction. In short, Artemiside
Mastersizer. The average size of the vesicles, prior to drug load-
ing, was 3.81 ␮m and the median was 2.85 ␮m. Obscuration
values of between 10 and 20% were used and the span was
1.94. All experimental formulations were kept in amber glass
bottles.
(
12) and artemisone (14), were obtained from DHA ␣-trimethylsilyl
ether (7) and trimethylsilyl bromide (TMSBr), followed by treat-
ment of the intermediate bromide (8), formed in situ, with the

2
62
J.D. Steyn et al. / International Journal of Pharmaceutics 414 (2011) 260–266
CH3
H
TMSCl
O
O
Et N
3
H C
3
CH Cl2
O
2
H
13
14
O
CH3
a
OTMS
12
12
7
mCPBA (1 equiv)
NMO (3 equiv)
TPAP (cat.)
CH Cl
Et O
2
2
(DHA)
0 °C
TMSBr
CH Cl2
2
2
20 °C
a
2
0
°C
CH3
b
CH3
H
H
O
1° / 2° amine
Et N
TMSCl
NaBr
H C
3
O
O
O
3
H C
3
O
O
O
Toluene
CH Cl
2 2
O
H
0
°C
or toluene
- 20 °C
H
0
CH3
10
Br
R''
8
6
CH3
N
CH3
N
CH3
CH3
N
CH3
N
CH3
R'' =
N
N
N
N
N
N
O
S
S
S
O
O
O
12
13
14
10
11
9
Fig. 2. Preparation of 10-alkylamino derivatives. Artemisone (14) is obtained by either treatment of (8) with thiomorpholine-5,5-dioxide or by oxidation of artemiside (12).
Artemiside is obtained from (8) and thiomorpholine. Oxidation of artemiside also produces a sulfoxide (13) (Haynes et al., 2006).
2.4. Drug administration
2.4.1. Artemisone and artemiside
Reference group (p.o.) (N = 10): Artemisone/artemiside was
administered orally at a dose of 50.0 mg/kg in DMSO/water
(1:9 v/v). The total volume per administration was 200 ␮l. Blood
samples (50 ␮l) were collected via tail-bleeding at 5, 10, 30,
60 and 120 min for artemisone and at 10, 30, 60, 120 and
180 min after drug administration for artemiside. The sample
collection times vary between the two compounds because a
pilot study indicated that artemiside had a longer half-life than
artemisone. The five sample limit per test subject was also taken
into consideration. The samples were centrifuged at 2000 rcf and
The absorption of artemisone and artemiside were evaluated
in a mouse model. The drugs were tested in both a reference for-
TM
mulation and a Pheroid vesicle formulation. The animals utilized
were male C57 BL/6 mice, weighing approximately 25 g each (Basco
et al., 1999; Ojo-Amaize et al., 2007). The study and all procedures
were approved by the Ethics Committee of the University of Cape
Town, approval number 009/034. The drugs were administered via
the oral route. Test animals were randomly allocated to each group.
The exact procedure is described below.

J.D. Steyn et al. / International Journal of Pharmaceutics 414 (2011) 260–266
263
Table 2
ESI settings.
acetic acid) (75:25) and was delivered at 0.5 ml/min for 3 min. The
column was kept in a column compartment at 35 C. An autosam-
◦
Curtain gas
Collision gas
Ionspray voltage (V)
Source temperature ( C)
Gas 1 (psi)
20
5
5000
500
50
pler injected 10 ␮l (artemisone) and 5 ␮l (artemiside) into the HPLC
column. The injection needle was rinsed with mobile phase before
each injection for 10 s using the flush port wash station. The sam-
◦
◦
ples were cooled to 5 C while awaiting injection.
Gas 2 (psi)
60
2.5.5. Liquid–liquid extraction
1
5 ␮l of plasma were collected from each sample and stored
The extraction procedure was performed on ice using
◦
at −20 C.
polypropylene test tubes. The plasma samples were thawed on ice
and briefly vortexed. Twenty-five microlitres of a universal Britton
Robinson buffer (pH 9) was aliquotted into clean polypropylene
tubes and 15 ␮l of the plasma sample was added. The ISTD was
spiked at an appropriate concentration into the universal buffer
and 25 ␮l was subsequently added to the extraction tubes. 1-
Chlorobutane (350 ␮l) was also added to each tube to function
as an organic solvent. The samples were vortexed for 1.5 min and
centrifuged at 7000 rcf for 5 min. The organic phase (300 ␮l) was
transferred to clean polypropylene tubes and evaporated under
TM
Pheroid
group (p.o.) (N = 10): Artemisone/artemiside,
TM
entrapped in Pheroid
5
reference group.
vesicles, was administered orally at
0.0 mg/kg. The same protocol was used as described for the oral
2
2
.5. Measurement of drug content
.5.1. Instrumentation
A sensitive and selective LC/MS/MS method was developed to
◦
determine the drug concentrations in the collected plasma samples.
An Agilent 1200 series HPLC system and an Applied Biosystems API
3
vacuum in a rotor evaporation system at 30 C for 45 min. Mobile
phase (50 ␮l) was added to the dry samples. The samples were then
vortexed for 30 s and transferred to 96 well polypropylene plates.
Five or ten microlitres of each sample was then injected into the
HPLC column and analyzed.
200 triple quadrupole mass spectrometer were used.
2.5.2. Calibration standards
Stock solutions of artemisone and artemiside were prepared in
ethanol at a concentration of 1 mg/ml. Mouse plasma (1240 ␮l) was
spikedwith the stock solution (10 ␮l) to obtain STD 1 at a concentra-
tion of 8 ␮g/ml. Serial dilution with mouse plasma resulted in STD 2
2
.6. Statistical evaluation
The recovered experimental data was evaluated in terms of the
(
4 ␮g/ml), STD 3 (2 ␮g/ml), STD 4 (1 ␮g/ml), STD 5 (0.5 ␮g/ml), STD
(0.25 ␮g/ml), STD 7 (0.125 ␮g/ml), STD 8 (0.0625 ␮g/ml), STD 9
0.0313 ␮g/ml) and STD 10 (0.0156 ␮g/ml). Artemisinin was added
drug plasma concentration versus time. The following parameters
were calculated:
6
(
to act as an internal standard (ISTD). The calibration standards were
briefly vortexed, aliquotted into labeled polypropylene tubes and
stored at −20 C.
◦
•
•
•
the peak drug plasma concentration (Cmax) (Cp ) in ng/ml;
time to peak plasma concentration (Tmax);
apparent elimination half-life (T1/2);
◦
2.5.3. Mass spectrometry
• area under the plasma concentration–time curve between time
The detection of artemisone, artemiside and artemisinin (ISTD)
zero and the time of last sample collection (AUC0
and the
) in ng h/ml
-last
was performed on an AB Sciex API 3200 mass spectrometer (ESI
in the positive ion mode, MRM). The settings on the apparatus are
summarized in Tables 2 and 3.
• area under the plasma concentration–time curve from time zero
to infinity (AUC0 ) in ng h/ml.
-inf
•
results are reported as mean ± SEM.
2.5.4. Chromatography
Chromatography was performed on a Phenomenex Gemini-NX
Noncompartmental analysis was used to calculate the param-
(
5 ␮m, C18, 110A, 50 mm × 2 mm) analytical column using an Agi-
eters for artemisone and artemiside (WinNonlin version 5.2,
Pharsight Corporation, CA, USA). Linear interpolation was used to
determine the area under the concentration time curve. AUC0-last
is defined as the AUC computed from time zero to the time of the
last Y-value above the lower limit of quantitation of the assay. All
values below this limit were treated as “missing”. AUC0-inf was cal-
culated by extrapolating the concentration time curve from time
zero to infinity, using the last three concentration time points
to estimate the elimination rate constant (ꢀz). This constant was
also used to determine the observed elimination half-life (T1/2) of
the compounds. The summary statistics and Mann–Whitney non-
parametric test were performed using Prism version 4 (GraphPad
Software Inc., CA, USA).
lent 1200 series HPLC. For artemisone the mobile phase consisted
of methanol and ammonium acetate (10 mM with 0.1% acetic acid)
(
60:40) and was delivered at 0.5 ml/min for 3 min. The organic
phase was increased after 3 min to 95% for another 2 min to clean
the column and was then brought back to 60% organic phase for
min to equilibrate the column. For artemiside the mobile phase
consisted of methanol and ammonium acetate (10 mM with 0.1%
3
Table 3
MS/MS settings.
Setting
Artemisone
ISTD
Artemiside
Q1 mass [M+H]+
Q3 mass
402.2
163.2
150
46
2
25
283.2
151.1
150
26
9.5
21
370.2
163.2
150
26
5
25
The relative absorption was determined by using the calculated
arithmetic mean of the area under the curve (AUC0-last) values of
Dwell time (ms)
Declustering potential (V)
Entrance potential (V)
Collision energy (V)
Collision cell exit potential (V)
Scan type
TM
both the oral reference formulation and the oral Pheroid vesicle
formulation data. Eq. (1) was used to calculate the relative absorp-
tion:
4
4
4
MRM
Positive
5
MRM
Positive
5
MRM
Positive
5
Polarity
Pause time (ms)
[AUC]A
Relative absorption (RA) =
(1)
[
AUC]B

2
64
J.D. Steyn et al. / International Journal of Pharmaceutics 414 (2011) 260–266
2
1
1
00
50
00
formulation extended the T1/2 to more than 60 min (T1/2 = 1.10 h)
(p < 0.005). The difference amounts to an increase of more than
3-times that of the reference formulation.
Artemiside
reference (p.o.)
Artemiside
Pheroid (p.o.)
The area under the concentration time curves (AUC) was
determined. There was a significant difference between the
TM
two values (reference, AUC0-last = 458.7 ng h/ml and Pheroid
,
AUC0-last = 2219.0 ng h/ml) (p < 0.0001). The AUC0-inf of the
reference formulation was calculated as 604.6 ng h/ml and
TM
3
094.0 ng h/ml for the Pheroid
vesicle formulation (p < 0.0001).
TM
The relative absorption (RA) of the Pheroid
vesicle formulation
5
0
was RA = 4.57 in comparison to the reference formulation which
was represented by RA = 1.00. These calculated values imply that
TM
the Pheroid vesicle formulation was absorbed 4.57 times better
0
than the reference formulation, or when converted to percentage
values, reasoning that the reference is represented by 100%, the
Pheroid vesicle formulation was 357% better absorbed.
0
30
60
90
120
150
180
210
Time (minutes)
Fig. 3. Mean concentration versus time graph overlay of the oral artemisone refer-
ence and Pheroid vesicle formulations.
TM
3.2. Oral artemiside formulations
The parameters of the oral reference formulation and the oral
Pheroid vesicle formulation are presented in Table 4. The results
3
. Results
TM
are graphically presented in the form of an overlay of the mean
concentration versus time graphs in Fig. 4.
Calibration standards for artemisone and artemiside were ana-
lyzed in duplicate during each study sample batch. The calibration
range was between 31.3 ng/ml and 4000 ng/ml for artemisone,
and between 15.6 ng/ml and 4000 ng/ml for artemiside. There-
fore, the limit of quantification for artemisone was 31.3 ng/ml, and
5.6 ng/ml for artemiside. Quadratic (1/x, weighting) regressions
were used to construct calibration curves. The accuracies (%Nom)
for the artemisone calibration standards were between 94.1% and
TM
The incorporation of artemiside in a Pheroid vesicle formula-
tion produced less promising results. The Tmax of artemiside, when
TM
incorporated in a Pheroid vesicle formulation, was not extended
as was the case with artemisone. When comparing the Tmax val-
ues of the two data sets, the reference formulation had a Tmax of
1
TM
approximately 30 min (0.55 h) while the Pheroid vesicle formu-
lation delayed the Tmax to approximately 35 min (0.57 h) (p > 0.05).
1
03.3% and the precision (%CV) for the artemisone calibration
TM
The Cmax was also not greatly increased by the Pheroid
vesicle
standards was between 0.1% and 15.1% for the entire calibration
range. The accuracies for the artemiside calibration standards were
between 88.3% and 113.4% and the precision for the artemiside
calibration standards was between 1.2% and 11.6% for the entire
calibration range. No peaks were observed in the double blank sam-
ples (without analytes and internal standard), and no peaks were
observed for the analytes in the blank sample (without analytes,
but with internal standard). The methods performed well during
all the sample batches and reproducible calibration curves could
be constructed.
formulation, the reference formulation had a Cmax of 116.4 ng/ml
while the Pheroid vesicle formulation had a Cmax of 137.7 ng/ml
TM
(
p > 0.05). The difference amounts to an increase in artemiside’s
TM
Cmax by approximately 18% with the aid of the Pheroid
delivery
system. The data generated from this study did not lend itself to the
accurate determination of either the T1/2 or the AUCo-inf. The reason
for this may be due to the manner in which the elimination rate con-
stant (ꢀz) was calculated. A least squares regression was calculated
using the last few points of the concentration curve, assuming that
the elimination phase had been reached in the mouse. The slope
of that regression then represents ꢀz. Due to the fact that so few
subjects were sampled, and due to some of the detected concen-
trations which did not return to zero, the estimation for certain
subjects were exaggerated. The elimination rate constant for cer-
tain subjects could not be calculated due to that occurrence. For
3.1. Oral artemisone formulations
The parameters of the oral reference and the oral PheroidTM
vesicle group were calculated and are given in Table 4. The results
are graphically presented in the form of an overlay of the mean
concentration versus time graphs in Fig. 3.
TM
The incorporation of artemisone in a Pheroid
vesicle formu-
2000
Artemisone
lation produced promising results. The Tmax of artemisone, when
vesicles, was increased dramatically.
When comparing the Tmax of the two data sets, the reference for-
reference (p.o.)
TM
incorporated in Pheroid
Artemisone
Pheroid (p.o.)
1
1
500
000
mulation rendered a Tmax of just 7 min (0.12 h) while a time delay
TM
was observed with the Pheroid
vesicle formulation for which
the Tmax was approximately 30 min (0.55 h) (p < 0.0001). The Tmax
of artemisone, when incorporated in the PheroidTM vesicle for-
mulation, was delayed by a time-factor of 4 in comparison to
that of the reference formulation. The Cmax was greatly increased
by the PheroidTM vesicle formulation, the reference formulation
500
0
TM
produced a Cmax of 809.5 ng/ml while the Pheroid
vesicle for-
mulation gave a Cmax of 1550.0 ng/ml (p < 0.005). That amounts
to an increase in artemisone’s Cmax by approximately 90% with
TM
0
30
60
90
120
150
the aid of the Pheroid
artemisone in a Pheroid
delivery system. The incorporation of
vesicle formulation also had a major
TM
Time (minutes)
impact on the T1/2 of the drug. The reference formulation had a T
of approximately 20 min (T1/2 = 0.36 h) while the Pheroid vesicle
1/2
Fig. 4. Mean concentration versus time graph overlay of the oral artemiside refer-
ence and Pheroid vesicle formulations.
TM
TM

J.D. Steyn et al. / International Journal of Pharmaceutics 414 (2011) 260–266
265
Table 4
Summary of the pharmacokinetic parameters.
Formulation: 50.0 mg/kg p.o. and 5.0 mg/kg IV
Mean ± SEM
◦
Tmax (h)
Cmax/Cp (ng/ml)
T1/2 (h)
AUC0-last (ng h/ml)
AUC0-inf (ng h/ml)
Artemisone (reference, p.o.)
Artemisone (Pheroid , p.o.)
0.12 ± 0.01
0.55 ± 0.05
0.55 ± 0.19
0.57 ± 0.09
809.50 ± 98.12
1550.00 ± 105.40
116.40 ± 14.03
137.70 ± 18.44
0.36 ± 0.06
1.10 ± 0.26
7.52 ± 4.84
6.50 ± 4.46
485.70 ± 106.30
2219.00 ± 122.70
174.20 ± 27.47
211.20 ± 30.80
604.60 ± 143.80
3094.00 ± 392.10
749.80 ± 387.40
554.20 ± 212.4
TM
Artemiside (reference, p.o.)
TM
Artemiside (Pheroid , p.o.)
example, the concentrations detected in the last three samples col-
Various studies concerning the interaction of lipophilic com-
pounds with intestinal fatty acid-binding protein (I-FABP) suggest
that the binding of these lipophilic entities (Pheroid vesicles) to
TM
lected from subject 2 (Pheroid
vesicle formulation) were 36.0,
TM
3
6.0 and 34.0 ng/ml respectively. The values would not translate to
the calculation of a useful T1/2 or AUC0-inf value and consequently
I-FABP may lead to an increase in the cytosolic solubility of those
entities (Velkov et al., 2005). Thus increased cytosolic solubility
may also subsequently facilitate the transport of the Pheroid
resulted in grossly inflated estimations. Nevertheless, the incor-
TM
TM
poration of artemiside in Pheroid
vesicles did not have a major
impact on the calculated T1/2 of the drug, the reference formula-
vesicles from the intestinal lumen across the enterocytes to sites
tion had a calculated T1 of approximately 450 min (T1/2 = 7.52 h)
of drug distribution. This occurrence, in conjunction with the
/2
while the PheroidTM vesicle formulation had a T1/2 of approximately
improved solubility of artemisone in the Pheroid
TM
vesicles, may
4
00 min (T1/2 = 6.49 h) (p > 0.05). The AUC0-inf of the reference for-
explain the observed increase in the Cmax value. The increase in
mulation was calculated as 749.80 ng h/ml and 554.20 ng h/ml for
Cmax and Tmax values explain why the T1/2 had more than doubled
TM
TM
the Pheroid vesicle formulation (p > 0.05).
and the absorption increased with the use of Pheroid
technol-
TM
The relative absorption of the Pheroid vesicle formulation was
ogy. This did not hold true for the reference formulation which was
not able to completely dissolve the drug nor was it able to provide
RA = 1.21 in comparison to the reference formulation which was
represented by RA = 1.00. These calculated values imply that the
TM
the same, well defined, lipophilic characteristics as the Pheroid
TM
Pheroid vesicle formulation was not significantly more absorbed
system to promote an interaction with I-FABP.
when compared to the reference formulation. A summary of the
results is given in Table 5.
The results obtained with artemiside, when compared to
artemisone, did not indicate any increase in absorption. Artemiside
is more hydrophobic than artemisone, and the reference formula-
tion also formed a precipitate with the addition of water to the
DMSO-drug solution. The precipitate also had a more flaky appear-
ance than the artemisone reference. The obtained data is the result
of the poor solubility characteristics of the drug. The poor solubility
probably caused erratic absorption which in turn led to irregular-
ities in the concentration versus time curve which had relatively
large SEM-values. Fig. 4 (reference graph) did not show a represen-
tative drug concentration value during the absorption phase. This
may be attributed to the fact that the first samples were collected
at 10 min instead of 5 min. Since no previous studies have been
conducted on this drug in a mouse model which could otherwise
be used as a reference, the sample intervals were chosen based on
the relatively short T1/2 history of the drug class. Another considera-
tion was the small blood volume of the test animals. Only 5 samples
could be collected from each animal. Thereafter, the reduction in
blood volume may start to have a concentrating effect of the drug
in the plasma due to the decrease in the blood volume as a result of
the sample collection. The sample intervals were chosen to ensure
the best possible utilization of the limited amount of samples. For
future studies, the first samples should be collected at 2–5 min post
administration rather than at 10 min. However, the Tmax and Cmax
values in the reference formulation curve (Fig. 4) are still in relative
concordance with the calculated values.
4
. Discussion
The results acquired with artemisone are very promising. The
concentration versus time graphs obtained from the reference for-
TM
mulation and the Pheroid
vesicle formulation data, in terms of
the basic configuration and the well defined data points obtained
during both the absorption and elimination phases of the drug, are
robust. The Cmax and Tmax are also well defined in these graphs. The
calculated Cmax and Tmax values correlate very well with the appar-
ent corresponding points on the concentration versus time curve
TM
of both the reference and Pheroid formulations.
The reference formulation produced significantly lower Cmax
TM
and Tmax values than the Pheroid
formulation. The difference
may be due to the less favourable absorption characteristics of
the reference formulation. The reference formulation appeared to
form a micro-suspension after the addition of water to the DMSO-
drug solution. It was previously reported that most artemisinins
are known for their low aqueous solubility and resultant poor
and erratic absorption upon oral administration (Wong and Yuen,
TM
2
001). It is possible that the Pheroid system was able to improve
the solubility of artemisone and subsequently enhance the other-
wise more erratic absorption characteristics associated with this
drug class by encapsulating the drug in a lipophilic layer. This
The drug concentration versus time curve of the artemiside
was achieved by first dissolving artemisone in the oil-phase of
TM
Pheroid vesicle formulation was more favourable in terms of the
TM
the Pheroid
system and then adding the appropriate amount
basic configuration with definitive data points obtained during both
the absorption and elimination phases of the drug. The Cmax and
Tmax were both well defined on the corresponding graph. The calcu-
lated Cmax and Tmax values correlated well with the corresponding
of nitrous oxide water to promote the formation of a lipophilic
TM
Pheroid /drug complex (Velkov et al., 2005).
TM
points on the concentration versus time curve of the Pheroid for-
Table 5
mulation. Artemiside was encapsulated by first dissolving the drug
Summary of the absorption results.
TM
in the oil-phase of the Pheroid system and then adding the appro-
Relative absorption (RA)
priate amount of nitrous oxide water to promote the formation of a
lipophilic Pheroid /drug complex (Velkov et al., 2005). Although
a small improvement was observed in the results with the aid of
this formulation in terms of the Cmax and Tmax over that of the ref-
erence there was not a very apparent increase in the absorption of
TM
Artemisone
Artemiside
Reference
Pheroid
1.00
4.57
1.00
1.21
TM
Reference
TM
Pheroid

2
66
J.D. Steyn et al. / International Journal of Pharmaceutics 414 (2011) 260–266
artemiside when administered in conjunction with PheroidTM tech-
nology. The PheroidTM vesicle formulation, however, took longer
to reach its Cmax in comparison to the reference formulation which
made it possible to record definite data points during the absorp-
tion phase of the curve. This may probably be explained by the
usually rapid conversion of most artemisinin derivatives to DHA or
other metabolites when it is not protected against this rapid con-
version processes (Haynes et al., 2006). It is very likely that the
in the Tmax and Cmax values with no added benefit to the T1/2 of
the drug. The contrast between artemisone and artemiside may
be attributed to varying solubility characteristics and to different
metabolic pathways of the drugs.
References
Baird, J.K., 2005. Effectiveness of antimalarial drugs. N. Engl. J. Med. 352, 1565–1577.
Basco, L.K., Ringwald, P., Franetich, J.F., Mazier, D., 1999. Assessment of pyronaridine
activity in vivo and in vitro against the hepatic stages of malaria in laboratory
mice. Trans. R. Soc. Trop. Med. Hyg. 93, 651–652.
Dondorp, A.M., Nosten, F., Yi, P., Das, D., Phyo, A.P., Tarning, J., Lwin, K.M., Ariey, F.,
Hanpithakpong, W., Lee, S.J., Ringwald, P., Silamut, K., Imwong, M., Chitovanich,
K., Lim, P., Herdman, T., An, S.S., Yeung, S., Singhasivanon, P., Day, N.P.J., Linde-
gardh, N., Socheat, D., White, N.J., 2009. Artemisinin resistance in Plasmodium
falciparum malaria. N. Engl. J. Med. 361, 455–467.
TM
lipophilic attributes of the Pheroid system were able to partially
protect the drug against this rapid metabolic conversion. This in
turn would explain the increase in the Tmax and Cmax of artemi-
side, the first samples were collected at 10 min and represented
the Tmax for the reference formulation while the same samples of
TM
the Pheroid
vesicle formulation represented a point during the
Gkrania-Klotsas, E., Lever, A.M.L., 2006. An update on malaria prevention, diagnosis
and treatment for the returning traveller. Blood Rev. 21, 73–87.
absorption phase, thus suggesting a slight delay in absorption.
When comparing the drug absorption results of artemisone to
that of artemiside the differences become very apparent. Both com-
pounds were orally administered at a dose of 50.0 mg/kg and yet the
artemisone PheroidTM vesicle formulation provided a peak plasma
Grobler, A., Kotzé, A.F., Du Plessis, J., 2008. The design of a skin-friendly carrier for
TM
cosmetic compounds using Pheroid
technology. In: Wiechers, J. (Ed.), Sci-
ence and Applications of Skin Delivery Systems. Allured Publishing Corporation,
Wheaton, IL.
Hay, S.I., Guerra, C.A., Tatem, A.J., Noor, A.M., Snow, R.W., 2004. The global distribu-
tion and population at risk of malaria: past, present, and future. Lancet: Infect.
Dis. 4, 327–336.
Haynes, R.K., Fugmann, B., Stetter, J., Rieckmann, K., Heilmann, H.D., Chan, H.W.,
Cheung, M.K., Lam, W.L., Wong, H.N., Croft, S.L., Vivas, L., Rattray, L., Stewart, L.,
Peters, W., Robinson, B.L., Edstein, M.D., Kotecka, B., Kyle, D.E., Beckermann, B.,
Gerisch, M., Radtke, M., Schmuck, G., Steinke, W., Wollborn, U., Schmeer, K., Röh-
mer, A., 2006. Artemisone – a highly active antimalarial drug of the artemisinin
class. Angew. Chem. Int. Ed. 45, 2082–2088.
Jonker, C., Hamman, J.H., Kotzé, A.F., 2002. Intestinal paracellular permeation
enhancement with quaternised chitosan: in situ and in vitro evaluation. Int.
J. Pharm. 238, 205–213.
Krishna, S., Uhlemann, A.C., Haynes, R.K., 2004. Artemisinins: mechanisms of action
and potential for resistance. Drug Resist. Updat. 7, 233–244.
Meshnick, S.R., Taylor, T.E., Kamchonwonpaisan, S., 1996. Artemisinin and the
antimalarial endoperoxides: from herbal remedy to targeted chemotherapy.
Microbiol. Rev. 60, 301–315.
Mutabingwa, T.K., 2005. Artemisinin-based combination therapies (ACTs): best hope
for malaria treatment but inaccessible to the needy. Acta Trop. 95, 305–315.
Ojo-Amaize, E.A., Nchekwube, E.J., Cottam, H.B., Oyemade, O.A., Adesomoju, A.A.,
Okogun, J.I., 2007. Plasmodium berghei: antiparasitic effects of orally adminis-
tered hypoestoxide in mice. Exp. Parasitol. 117, 218–221.
Velkov, T., Chuang, S., Wielens, J., Sakkelaris, H., Charman, W.N., Porter, C.J.H.,
Scanlon, M.J., 2005. The interaction of lipophilic drugs with intestinal fatty acid-
binding protein. J. Biol. Chem. 280, 17769–17776.
White, N.J., 2004. Antimalarial drug resistance. J. Clin. Invest. 113, 1084–1092.
Wong, J.W., Yuen, K.H., 2001. Improved oral bioavailability of artemisinin through
inclusion complexation with ␤- and ␥-cyclodextrins. Int. J. Pharm. 227,
concentration of 1500.0 ng/ml in contrast to the 137.0 ng/ml of the
artemiside PheroidTM vesicle formulation. This may be due to the
differences in the metabolic conversion and degradation of these
drugs. Most artemisinin class drugs are very quickly converted
into DHA or other metabolites after oral administration while
artemisone, in contrast to other artemisinins, is not converted into
DHA. Artemisone is metabolized to the products M1–M5 (Haynes
et al., 2006). The differences in metabolic products and the varied
rapidity of the conversion rates may shed some light on the differ-
ences encountered in the results of the two compounds. However,
further studies are required to fully elucidate the results.
5
. Conclusion
Artemisone was proven to be relatively well absorbed. The
results provide compelling evidence in favour of the ability of the
TM
Pheroid
delivery system to further enhance the absorption of
artemisone. The experiments indicated a very dramatic improve-
ment in the Cmax and T1/2 of the drug and a time delay in Tmax
when administered in a PheroidTM vesicle formulation. This effec-
tively translates to a scenario where the drug concentration could
be significantly decreased and still achieve therapeutic drug plasma
177–185.
Woodrow, C.J., Haynes, R.K., Krishna, S., 2005. Artemisinins. Postgrad. Med. J. 81,
71–78.
World Health Organization (WHO), 2006. Guidelines for the Treatment of Malaria,
TM
concentrations. The combination of artemisone and Pheroid
technology should prove to be an essential component in anti-
266 pp.
malarial combination therapy regimens in the very near future.
World Health Organization (WHO), 2009. International Travel and Health. Sit-
uation as on January 2009, 250 pp. http://www.scribd.com/doc/19637506/
International-travel-and-health (accessed on 16.11.09).
TM
The Pheroid
delivery system did not produce such promising
results with artemiside. Only a marginal increase was observed