Original Papers 259

Transepithelial Transport of a Natural Cholinesterase

Inhibitor, Huperzine A, along the Gastrointestinal Tract:
the Role of Ionization on Absorption Mechanism

Authors Gregory Burshtein, Michael Friedman, Sarit Greenberg, Amnon Hoffman

Affiliation Institute for Drug Research, Faculty of Medicine, The Hebrew University of Jerusalem, Jerusalem, Israel

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Key words Abstract cellular diffusion. Addition of the paracellular
l
" huperzine A
! permeability modulator palmitoylcarnitine in
l
" permeability
During recent years there has been increasing in- the Caco-2 model led to significant enhancement
l
" PAMPA
terest in the Lycopodium alkaloid huperzine A as a in the permeability of the ionized huperzine A
l
" Caco‑2

potential therapeutic agent for neurodegenera- fraction. No evidence of active transport of huper-
l
" Ussing chamber

" Huperzia serrate

l tive diseases. This study aimed to characterize hu-
l
" Lycopodiaceae perzine Aʼs permeability across the enterocyte
barrier along the gastrointestinal tract with an
emphasis on the effect of ionization on the drug
absorption. Intestinal permeability of huperzine
A was evaluated by in vitro Caco-2 and parallel ar-
tificial membrane permeation assay models and
by the ex vivo Ussing chamber model. The perme-
ability rate was strongly dependent on the degree
of ionization and increased with elevation of the
donor medium pH in all studied models. The
transport of the unionized fraction was similar to
the permeability of the markers for passive trans-
zine A was detected in this study. The Ussing
chamber model experiments showed similar drug
permeability along the entire rat intestine. In con-
clusion, huperzine A permeates the intestinal
border mainly by passive transcellular diffusion
whereas some fraction, dependent on the degree
of huperzine A ionization, is absorbed by the par-
acellular route. Huperzine Aʼs permeability char-
acteristics pave the way to the development of its
oral extended release dosage form. The specific
population of the potential users of huperzine A
and the high potency of this molecule support
the rationale for such a delivery.

Introduction form of AChE (G4 form), which exists predomi-

! nantly in the mammalian brains [4]. In addition
received May 20, 2012
Huperzine A (HupA) is a natural alkaloid derived to its acetylcholinesterase inhibitory effect, HupA
revised Dec. 4, 2012 from the pteridophytic firmoss Huperzia serrata also demonstrated neuroprotective properties,
accepted Dec. 7, 2012 (Lycopodiaceae) (Lycopodium serrata) (l " Fig. 1). specifically against damage that can be caused by
The average content of HupA in the plant is about hydrogen peroxide, β‑amyloid protein, glutamate,
Bibliography
DOI http://dx.doi.org/ 0.01 % [1]. The compound also can be found in and ischemia [5]. Antagonizing effects of HupA on
10.1055/s-0032-1328128 varying quantities in other Huperzia species, in- N-methyl-D-aspartate (NMDA) receptors and po-
Published online January 23, cluding H. elmeri, H. carinat, and H. aqualupian tassium currents may contribute to this neuro-
2013 [2]. The extracts of QianCeng Ta (Huperzia serra- protective effect [6].
Planta Med 2013; 79: 259–265
ta) have been used in traditional Chinese medi- Numerous clinical trials have shown the efficacy
© Georg Thieme Verlag KG
Stuttgart · New York · cine for centuries for the treatment of contusions, of HupA in the treatment of AD and dementia,
ISSN 0032‑0943 swelling, and schizophrenia [3]. HupA is a specif- with minimal adverse cholinergic effects [7]. En-
ic, reversible, and potent acetylcholinesterase couraging results of the multiple preclinical and
Correspondence
Amnon Hoffman (AChE) inhibitor that has unique chemophysical clinical studies along with the accumulating data
Institute for Drug Research properties in comparison to other AChE inhib- regarding various pharmacological mechanisms
Faculty of Medicine itors. In vitro and in vivo comparison studies with emphasize HupAʼs potential to be used as a treat-
The Hebrew University of
Jerusalem respect to AChE inhibition showed that the po- ment for central nervous system (CNS)-related
Jerusalem 91120 tency of HupA was similar or superior to the in- disorders.
Israel hibitors currently being used in Alzheimerʼs Dis- Although significant progress in the understand-
Phone: + 97 2 26 75 86 64
Fax: + 97 2 26 75 72 46 ease (AD) treatment [3]. This is probably due to ing of HupAʼs pharmacology has been made, only
amnonh@ekmd.huji.ac.il HupAʼs preferential inhibition of the tetrameric

Burshtein G et al. Transepithelial Transport of … Planta Med 2013; 79: 259–265

260 Original Papers
Fig. 1 The chemical
structure of huperzine
A.
limited and fragmented data exist about the drugʼs biopharma-
ceutical characteristics.
Chinese scientists reported good bioavailability of this molecule
in comparison to other acethylcholine esterase inhibitors [3]. Ad-
ditionally, rapid HupA absorption and high bioavailability was
mentioned in dogs and rats [8, 9]. However, there is no data re-
garding the transepithelial transport of HupA. These data are of
great interest due to the fact that the pKa of HupA is about 7, im-
plying possible absorption dependence on the varying intestinal
pH. An understanding of HupAʼs absorption rate and mechanism
is critical for further development of the optimal route of the
drug delivery, particularly extended-release formulations. The
advantages of extended delivery may be relevant for HupA, since
it is a very potent AChE inhibitor with a typical concentration-de-
pendent adverse effect profile. Moreover, the specific population
of the potential users of the drug will particularly benefit from
the decrease in the frequency of administrations.
The objective of the current study was to characterize HupAʼs
permeability via the enterocyte barrier along the gastrointestinal
(GI) tract. Emphasis was placed on identifying the drug carrier-
mediated transport via biologic membranes, pH dependence of
the drugʼs absorption, and regional differences in the permeabil-
ity of the molecule along the GI tract. Additionally, the study
aimed to verify the feasibility of controlled release administra-
tion of HupA.
There are a number of well-established methods to evaluate sub-
stance permeability via the intestinal barrier. Human intestinal
adenocarcinoma cells (Caco-2), parallel artificial membrane per-
meation assay (PAMPA), and the side by side diffusion chamber
(Ussing chamber) model were used in this study. We have com-
pared the HupA permeability to known transcellular markers
with similar lipophilicity (i.e., log P values 1.78, 1.22, 1.8 for Hup-
A, antipyrine, and metoprolol, respectively) and either atenolol or
mannitol as paracellular markers.
Materials and Methods
! were 25 and 20 µg/mL, respectively (i.e., 103 and 106 µM). The
Materials
HupA (±) (98.9 %) was purchased from Shaghai Tauto Biotech.
Antipyrine (100 %), atenolol (100 %), and metoprolol (100 %) were
purchased from Sigma-Aldrich. Radioactive-labeled mannitol
was produced by GE Healthcare. All other materials and reagents
were standard items from reputable commercial sources. All sol-
vents used were of analytical grade.
Permeability through PAMPA model
The model consists of a hydrophobic filter (polyvinylidene fluo-
ride) coated with a mixture of lecithin phospholipids (2%) dis-
solved in an inert organic solvent (dodecane), creating an artifi-
cial membrane barrier (5 µL/well). PAMPA permeability of HupA
Burshtein G et al. Transepithelial Transport of … Planta Med 2013; 79: 259–265
at different donor pH conditions and of the known permeability
markers antypirine, metoprolol, and atenolol was determined.
The pH-values of the donor solutions were adjusted to 5.6, 6.5,
6.8, and 7.4 with 1 M HCl. The initial concentration of the donor
solutions of HupA, metoprolol, and antipyrine was 25 µg/mL (i.e.,
103 µM, 94 µM, and 133 µM, respectively) and for atenolol 50 µg/
mL (188 µM). These solutions were prepared in PBS containing
5 % DMSO. The assay was carried out in a 96-well MultiScreen
permeability plate (Millipore). After a 16 hr incubation period at
25 °C, samples were taken from the donor and receiver wells and
analyzed. The permeability values of the tested drugs were deter-
mined from the flux factors between donor and acceptor com-
partments based on the Wohnsland et al. equations [10].
Caco-2 cell culture model
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Caco-2 cells were obtained from ATCC. The growth/preparation
of the cells and the permeability experiments were performed
as previously described [11]. Transport studies were performed
when the cells were fully differentiated and the TEER (transepi-
thelial electrical resistance) values were stable (300–500 ohm/
cm2). Experiments were done in 24 transwell plates, 12 mm (Co-
star™) where a polycarbonate membrane (pore size − 0.4 µm)
separates between the apical and the basolateral side of the in-
serts. Over the course of the experiment, the cells were incubated
at 37 °C with shaking. The initial donor concentration of HupA
and antipyrine was 20 µg/mL (i.e., 83 µM and 106 µM, respective-
ly) and mannitol 2µci/mL. The Papp of the drugs was evaluated at
two apical pH values (6.5 and 7.4), whereas basolateral pH was
7.4 in all the experiments.
Side by side diffusion chamber (Ussing chamber) model
Ex vivo permeability experiments were performed in a modified
Ussing chamber system (Physiological Instruments Inc.) as previ-
ously described [12]. Male Wistar rats (Harlan), 275–325 g in
weight, were used. All surgical and experimental procedures
were reviewed and approved by the Animal Experimentation
Ethics Committee of the Hebrew University Hadassah Medical
School Jerusalem (MD-09-11834-1; 01/02/09). Rat intestine seg-
ments were obtained and the underlying muscularis was re-
moved from the serosal side of the tissue before mounting. The
permeability experiments continued for 150 min at 37 °C (main-
tained by constant circulation), and samples were withdrawn at
predetermined times. The sampled volume was replaced by
blank buffer to maintain sink conditions.
The integrity of the epithelial tissue was monitored by measuring
the TEER throughout the experiment. Generally, TEER values
were 70–130 Ωcm2 and remained steady throughout the experi-
ment. The initial donor drug solutions of HupA and antipyrine
Papp of the drugs was evaluated at three different apical pH val-
ues (5, 6.8, and 7.5), whereas basolateral pH was 7.5 in all the ex-
periments.
Caco-2 and Ussing chamber models data analysis
Cumulative corrections were made at each time point for the pre-
viously removed samples. The apparent permeability coefficient
(Papp) for each compound was calculated from the linear plot of
drug accumulated vs. time, using the following equation:
Papp ¼ dQ
dt
 ðCoA
1
Þ
 Original Papers 261

Table 1 Permeability data of HupA and passive transcellular absorption markers in three in vitro models at different donor medium pH values. Data are presented
as Papp for CACO-2 and Ussing chamber models and as Peff for the PAMPA model. Permeability coefficients are presented as mean ± SD × 106(cm/sec). HupA
concentration was 103 µM in PAMPA and Ussing chamber models, and 83 µM in Caco-2 assay. Antipyrine concentration was 106 µM in Ussing chamber and Ca-
co-2 assays and 133 µM in PAMPA model. Metoprolol concentration in PAMPA model was 94 µM.

Drug CACO-2 PAMPA Ussing chamber

pH 6.8 pH 7.4 pH 5.6 pH 6.5 pH 6.8 pH 7.4 pH 5 pH 6.8 pH 7.5
HupA 16 ± 4a 31 ± 1a 0.05 ± 0.01a 0.19 ± 0.03a 0.27 ± 0.02a 0.52 ± 0.06a 4.1 ± 2.1b 5.9 ± 2.6 7.3 ± 1.7 c
antipyrine 39 ± 4 39.4 ± 1.9 ND ND ND 0.29 ± 0.01 14.4 ± 2.9 14.7 ± 4.3 14.9 ± 1.7
metoprolol ND ND ND ND ND 0.59 ± 0.04 ND ND ND

ND – not determined; a significantly different from all other pH values in the same model; b significantly different from pH 7.5 in the same model; c significantly different from pH 5
in the same model

Where dQ/dt is steady state appearance rate of the drug on the

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receiver side, C0 is the initial concentration of the drug on the do-
nor compartment, and A is the exposed tissue/cells surface area.

Analytical procedures
HupA and antipyrine were detected using a Waters 2695 Separa-
tion Module HPLC system with Waters 2475 fluorescence detec-
tor and Waters 2996 photodiode array detector (Waters Corpora-
tion). The separation was achieved by a Hypersil GOLD C8 column
(5 µm, 4.6 × 150 mm) at 40 °C. The mobile phase consisted of
methanol : water, adjusted to pH 10 with triethylamine (45 :
55 v/v). The flow was set to 1 mL/min. HupA was detected with a Fig. 2 HupA (83 µM), antipyrine (106 µM), and mannitol (2 µci/mL) per-
λ excitation of 310 nm and λ emission of 370 nm, and retention meability via CACO-2 cell monolayer presented as apparent permeability
time was 4.7 min. The calibration curve for HupA was linear be- coefficient (Papp). Permeability in the A–B direction is presented by black
bars and in the B–A direction by white bars. When the Papp of HupA was
tween 2 and 50 000 ng/mL. Antipyrine was detected by a UV de-
evaluated at two apical pH values (6.5 and 7.4), about two fold greater
tector at 243 nm wavelength, and its calibration curve was linear
permeability was detected at the higher pH value (p < 0.05). The basolat-
between 5 and 50 000 ng/mL. Samples containing [C14]-mannitol eral pH was 7.4 in all the experiments. Each bar represents the mean ± SD
were analyzed using a liquid scintillation counter. (n = 6).
Atenolol and metoprolol were separated by a Nova-Pak C-18 col-
umn (4 µm) at 35 °C and a flow rate of 0.5 mL/min. The mobile
phase consisted of methanol (25%): 50 mM sodium acetate buffer
(pH 3.3, 75 %) for atenolol and of methanol (50%): 50 mM sodium
acetate buffer (pH 3.3, 50%) for metoprolol. Both molecules were
detected with a λ excitation of 276 nm and λ emission of 296 nm.
Retention times were 5.87 min for atenolol and 6.15 min for me-
toprolol. The calibration curves for atenolol and metoprolol were
linear between 10 and 100 000 ng/mL.

Statistical analysis
Statistical analyses of differences between two values were per-
formed using Studentʼs t-test. The comparison of results from
multiple group experiments was performed by a one-way analy-
sis of variance followed by Tukeyʼs test. A p value < 0.05 was con-
sidered to be statistically significant.
Fig. 3 Permeability of HupA at different apical buffer concentrations (in
the range of 2 µM – 100 µM) across the CACO-2 cell monolayer. No statisti-
cally significant differences (p = 0.99) were detected between the Papp
values of the drug at different concentrations in the tested range. Each bar
represents the mean ± SD (n = 3).

Results
!
The permeability of HupA across the Caco-2 monolayer was eval-
uated and compared to antipyrine and mannitol, acceptable stan-
dards of transcellular and paracellular transport, respectively [13,
14] (l" Table 1). Additionally, the effect of donor medium pH on
The transport rate was determined in both apical-to-basolateral
and basolateral-to-apical directions in order to detect polarized
transport in the absorptive or secretory direction. As presented
in l" Fig. 2, in pH-gradient conditions the absorption of HupA in

drug permeability was evaluated (l " Table 1). Permeability rates basolateral-to-apical direction was more than 2 fold greater as
of the marker molecules were not affected by the donor medium compared to apical-to-basolateral permeability, whereas in iso-
pH and matched the previously reported data in the same model pH conditions HupA permeated the Caco-2 monolayer identically
[15]. The Papp of HupA increased from 1.6E‑05 cm/sec to in the opposite directions.
3.1E‑05 cm/sec with the elevation in donor medium pH from 6.5 The effect of donor concentration (ranging from 2 µM to 100 µM)
to 7.4 (l
" Table 1). on the permeability rate of the drug across Caco-2 monolayer

Burshtein G et al. Transepithelial Transport of … Planta Med 2013; 79: 259–265

262 Original Papers
was evaluated. As can be seen from l " Fig. 3, drug permeability
stayed constant and was independent of the donor concentration
in the tested range.
The involvement of the paracellular pathway in HupAʼs transepi-
thelial absorption was evaluated by the specific modulation of
tight junctions by palmitoylcarnitine (PC) (0.3 mM), a potent par-
acellular permeability enhancer [16, 17]. Additionally, the effect
of the different apical medium pH values on HupA paracellular
transport was evaluated.
As presented in l " Fig. 4 A, PC increased the mannitol permeabili-
ty via Caco-2 monolayer by 760%, while antipyrine Papp was not
affected at all (l" Fig. 4 B). There was a significant increase in
HupA permeability (37% increase in Papp value) via Caco-2 cells
after the tight junction modulation when the apical pH was 6.8.
Addition of PC to the apical buffer at pH 7.4 led only to a slight,
statistically insignificant increase in the drug permeability
(l" Fig. 4 C).
In order to isolate the drugʼs passive transcellular transport,
PAMPA experiments were performed. Known permeability stan-
dards were used to confirm the integrity of the phospholipid
membrane. Antipyrine and metoprolol were used as markers of
transcellular transport, whereas atenolol was used as a standard
for paracellular permeability [18, 19]. The permeability results of
the marker molecules supported the existence of the integral
phospholipid barrier (l " Fig. 5 A). Similarly to the Caco-2 model
results, HupA permeability dependence on the donor pH was ob-
tained also in the artificial membrane model (l " Fig. 5). Drug per-
meability increased dramatically with the elevation of the donor
medium pH. Above pH 6.8, HupA permeability across artificial
phospholipid membrane was similar or even greater than the
permeability of antipyrine and metoprolol (l " Fig. 5 A). As ex-
pected, HupA permeability via the phospholipid barrier in-
Burshtein G et al. Transepithelial Transport of … Planta Med 2013; 79: 259–265
Fig. 4 Effect of the paracellular transit modulator,
palmitoylcarnitine (PC, 0.3 mM) (black bars) on
mannitol, antipyrine (106 µM), and HupA (83 µM)
permeability via the CACO-2 cell monolayer. The
Papp of mannitol was substantially increased (A),
whereas the Papp of antipyrine was not changed
(B), indicating the specific modulation of the para-
cellular pathway by PC. C The effect of PC on the
permeability of HupA was tested in different apical
pH values (6.5 vs. 7.4), while the basolateral me-
dium pH remained constant (7.4). At an apical pH
of 6.5, HupAʼs permeability was significantly en-
hanced by PC (p < 0.05), whereas at pH 7.4 only a
slight, statistically insignificant increase was
achieved. Each bar represents the mean ± SD
(n = 3).
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creased along with the increase in the drug unionized fraction
(l" Fig. 5 B).
HupAʼs permeability across rat intestine segments was studied in
the side by side diffusion chamber in order to test the drug per-
meability in conditions more similar to the physiological state
and to evaluate its absorption from the different segments of rat
GI tract. Antipyrine was added to the donor medium of each of
the diffusion cells to serve as an internal standard for the trans-
cellular permeability.
l" Fig. 6 demonstrates Papp values of the tested compounds
found in the different regions of the rat gastrointestinal tract. No
statistically significant differences were found in HupA absorp-
tion rates via the duodenum, the jejunum, and the colon tissues
(l" Fig. 6 A). The drug permeability profile across different seg-
ments of the GI tract was similar to the permeability profile of
antipyrine (l " Fig. 6 B).
In order to evaluate the presence of carrier-mediated transport,
HupA permeability via rat jejunum was tested from the mucosal
to serosal and from the serosal to mucosal side of the tissue. In
the pH-gradient conditions (mucosal pH 5 and serosal pH 7.5),
the permeability constant from mucosal to serosal side of the je-
junum was 1.8 fold smaller than the Papp of the drug placed on
the serosal side (l " Fig. 7). However, when the mucosal pH went
up from 5 to 7.5 (iso-pH conditions), no polarity in the tissue per-
meability was detected, similary to the Caco-2 results (l " Fig. 7).
Discussion
!
It is of considerable importance to understand the rate and the
mechanism of the moleculeʼs absorption from the GI tract into
the blood in order to allow development of the optimal route of

Fig. 6 HupA (103 µM) (A) and antipyrine (106 µM) (B) permeability via rat
duodenum (black bar), jejunum (lined bar), and colon (white bar) in the
side by side diffusion chamber model. Apical pH was 6.8, and basolateral
was 7.4 in all the experiments. Each bar represents the mean ± SD of 4 an-
imals (3 intestinal segments from each animal for each tested group).
the drugʼs delivery. Moreover, knowledge of the permeability
mechanisms via biological membranes can increase and deepen
our understanding of the moleculeʼs tissue disposition, entrance
to CNS, and potential interaction with other drugs.
HupA is a slightly alkaline molecule with a pKa of 6.97 [20]. This
pKa value falls in the range of the varying intestinal pH, implying
possible drug absorption dependence on the anatomical location
in the GI tract and on the fed or fasted state. According to the
Henderson-Hasselbalch equation, in pH 6.5 the unionized frac-
tion of HupA available for the transcellular absorption is 24 %. At
Original Papers 263
Fig. 5 A Effective permeability coefficient of
HupA and permeability standards (metoprolol,
antipyrine, and atenolol) (103 µM, 94 µM, 133 µM,
and 188 µM, respectively) in the PAMPA model.
Donor medium pH effect on the permeability of
HupA was evaluated. Acceptor medium pH was 7.4
in all the experiments, and the permeability of the
transport markers was evaluated in iso-pH condi-
tions (pH = 7.4). In the tested range, HupAʼs phos-
pholipid membrane permeability was highly af-
fected by the donor medium pH (p < 0.01). Each
point represents the mean ± SD (n = 4). B HupAʼs
effective permeability coefficients in the PAMPA
model as a function of the unionized fraction of the
drug molecules.
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Fig. 7 Coefficients of HupA (103 µM) and antipyrine (106 µM) permeabil-
ity via rat jejunum in the side by side diffusion chamber model. Permeabil-
ity in the A–B direction is presented by black bars and in the B–A direction
by white bars. The Papp was obtained in two different donor pH values, 5
and 7.5. The absorption polarity (p < 0.05) that existed in pH gradient con-
ditions disappeared in iso-pH conditions, indicating that HupAʼs absorption
is a pH dependent process. The absorption of antipiryne was independent
of the apical pH. The basolateral pH was 7.5 in all the experiments. Each bar
represents the mean ± SD of 4 animals (3 intestinal segments from each
animal for each tested group).
pH 7.4 this fraction increases three fold. This can explain the sig-
nificant increase of HupA Papp in the higher donor pH values in
Caco-2 and PAMPA permeability assays (l " Figs. 2 and 5). In the
Ussing chamber model, the effect of different mucosal pH on the
Burshtein G et al. Transepithelial Transport of … Planta Med 2013; 79: 259–265
264 Original Papers
tissue permeability was also seen but to a lesser extent (l " Fig. 7).
The reason for this diminished effect could be that the mucus
layer, present only in the Ussing Chamber model, creates a micro
environment with a different pH compared to the donor medium
and therefore neutralizes the mediumʼs influence on the passage
of the molecule across the intestinal barrier.
In the Caco-2 experiments performed at higher donor medium
pH, where the major part of HupA molecules were unionized,
the permeability of the drug was very close to the permeability
of the transcellular marker antipyrine (l " Fig. 3). These findings
can be supported by the results of the PAMPA model. In the case
of the donor pH of 7.4, HupA permeability was very close to the
permeability of the transcellular marker metoprolol and greater
than the permeability of antipyrine (l " Fig. 5 A). In addition, a lin-
ear correlation was found between the drugʼs unionized fraction
and the phospholipid membrane permeability (l " Fig. 5 B), indi-
cating that the unionized fraction of the drug absorbs passively
across the phospholipid cell membrane. However, even when
the unionized fraction was only 24 %, the HupA permeability co-
efficient in the Caco-2 model was much closer to the Papp of the
transcellular marker rather than to the Papp of the paracellularly
permeable compound (l " Table 1). These results imply that the
predominant pathway of HupA absorption in the intestinal pH
range is passive transcellular diffusion.
To determine the involvement of the paracellular pathway in the
drugʼs absorption, palmitoylcarnitine was added to the apical
buffer of the Caco-2 cells. As expected, the permeability of man-
nitol was highly affected by palmitoylcarnitine, whereas the Papp
of antipyrine was not influenced at all (l " Fig. 4). The significant
increase in HupA permeability, caused by palmitoylcarnitine, at
a donor pH 6.8 (l " Fig. 4 C) could be explained by the fact that
60 % of the drug molecules are ionized in this pH. The ionized
fraction is more likely to permeate via the paracellular route
[21]. These data indicate that in the physiological environment
some fraction of the drug can be absorbed by the paracellular
pathway. This assumption can be supported by the fact that the
paracellular pathway is more expressed in vivo in comparison to
the Caco-2 model [22–24]. Nonetheless, comparison between the
effect of palmitoylcarnitine on the permeability of mannitol and
its effect on HupA clarifies that the major fraction of HupA still
enters the body by the transcellular pathway.
No evidence of carrier mediated transport of HupA was detected
in this study. This can be seen firstly by the absence of HupA
transport polarity across the intestinal barrier in the Caco-2 and
Ussing chamber models (l " Fig. 2). In addition, when concentra-
tion dependency of HupAʼs permeability was evaluated, the drug
transport across the Caco-2 monolayer remained identical
(l" Fig. 3), indicating that the drug is transported passively in the
range of concentrations to which the intestinal tissue is likely to
be exposed on the basis of the drugʼs therapeutic dose.
Moreover, in order to verify the absence of carrier-mediated-
transport, HupAʼs permeability profile across different segments
of rat intestine was compared to antipyrine, the marker for pas-
sive transcellular diffusion. A slight decrease in Papp between the
small intestine and the colon occurred in both drugs (l " Fig. 6 -
A, B), indicating the same mechanism of transepithelial drug
transport. Influx and efflux transporters are frequently heteroge-
neously distributed along the rat GI tract [21, 25, 26]. Hence, a
substrate of such transporters would be absorbed differently
from the colon in comparison to the proximal section of the small
intestine.
Burshtein G et al. Transepithelial Transport of … Planta Med 2013; 79: 259–265
In summary, the results of the current study clarify that HupA
crosses the intestinal barrier by the passive route with strong de-
pendence on the pH of the surrounding medium. The predomi-
nant fraction is absorbed via the enterocytes, whereas the minor
fraction is absorbed between the cells. The increase in the union-
ized fraction of HupA, caused by the increase in the pH of the do-
nor medium, elevated the predominant, passive transcellular ab-
sorption of the drug, whereas reduction in the medium pH led to
an increase in the paracellular transit. No signs of active absorp-
tive or secretory transport in the relevant concentration range
were detected.
HupA permeability in pH conditions near and above the pKa val-
ue is comparable to the permeability of markers of good intesti-
nal absorption (antipyrine and metoprolol). Because a significant
part of the GI tract has a neutral or slightly alkaline pH, and as
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clinically relevant dosages of the drug are low, full absorption of
HupA can be expected. This conclusion is corroborated by the
good HupA bioavailability reported in a few publications [3, 8].
However, a limited permeability in lower pH conditions may im-
ply food effect on the absorption profile of the drug. The pH of the
small intestine decreases below 6 in response to a meal with the
arrival of acidic chime from the stomach and is later reestab-
lished by pancreatic bicarbonate output [27]. Hence, the Tmax
and Cmax of the drug can differ between the fed and fasted state.
In addition, based on the current study findings, some pharma-
cokinetic parameters of the drug can be predicted. Good transcel-
lular absorption in physiologic pH values can contribute to a large
volume of distribution and to a good blood brain barrier perme-
ability of the drug.
Focusing on the drug delivery, good passive transcellular perme-
ability of HupA enables the oral extended-release drug delivery.
Efficient, homogeneous absorption from all parts of the GI tract
is a critical condition for this route of administration [28, 29].
Moreover, pH dependent permeability which grows in higher
pH values (l " Table 1) may contribute to the drugʼs complete ab-
sorption from the colon, supporting the rationale of HupA ex-
tended-release administration.
Acknowledgements
!
This research was partially supported by the “NOFAR” program of
the Israel Ministry of Industry, Trade and Labor. Prof. Amnon
Hoffman and Prof. Michael Friedman are affiliated with the David
R. Bloom Center for Pharmacy at The Hebrew University of Jeru-
salem.
Conflict of Interest
!
The authors declare that they have no conflict of interest with re-
spect to this work.
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