Conjugated chitosan as a novel platform for oral delivery of paclitaxel

Article abstract of DOI:10.1021/jm800767c

A new platform for oral delivery of paclitaxel (PTX) was developed through chemical conjugation of PTX to a low molecular weight chitosan (LMWC). The LMWC-PTX conjugate contained ～12 wt % PTX and showed greatly enhanced water solubility (> 1 mg/mL) as com

Full text of DOI:10.1021/jm800767c

6
442
J. Med. Chem. 2008, 51, 6442–6449
Conjugated Chitosan as a Novel Platform for Oral Delivery of Paclitaxel
†
†
†
†
†
‡
,†,×
Eunhye Lee, Jinju Lee, In-Hyun Lee, Mikyung Yu, Hyungjun Kim, Su Young Chae, and Sangyong Jon*
Cell Dynamics Research Center, Department of Life Science, Gwangju Institute of Science and Technology (GIST),
1
Oryong-dong, Gwangju 500-712, South Korea, College of Pharmacy, SungKyunKwan UniVersity, 300 Chenchen-dong,
Suwon, South Korea, and AnyGen Corp., Gwangju TechnoPark, Daechon-dong, Gwangju 500-706, South Korea
ReceiVed June 23, 2008
A new platform for oral delivery of paclitaxel (PTX) was developed through chemical conjugation of PTX
to a low molecular weight chitosan (LMWC). The LMWC-PTX conjugate contained ∼12 wt % PTX and
showed greatly enhanced water solubility (>1 mg/mL) as compared to native PTX. The conjugate showed
comparable IC50 values to that of the parent PTX against human cancer cell lines. The pharmacokinetic
data revealed ∼42% of bioavailability after oral administration of 5 mg PTX/kg of the conjugate. When the
conjugate (10 mg/kg based on PTX content) was administered orally to mice bearing xenograft or allograft
tumors, the conjugate-treated group showed significant inhibition of tumor growth, which was comparable
to that seen with PTX of the clinically available injected form, formulated in cremophor EL/ethanol (iv) but
125
with much lower toxicity. Tracking I -labeled conjugate showed that LMWC-PTX was likely to be absorbed
mainly from the ileum and reach the blood as the intact conjugate.
Introduction
proaches appear promising in terms of increasing the oral
2
7-29
a
bioavailability of PTX to clinically useful levels.
One
Paclitaxel (PTX ) has shown promise as an effective anti-
neoplastic agent against a broad spectrum of human malignan-
approach is based on the concomitant use of PTX and P-gp
inhibitors such as cyclosporine A and Valspodar, which greatly
1
,2
cies, including breast and ovarian cancers. But because of its
extremely poor aqueous solubility, the most widely used form
of PTX is formulated in a vesicle containing ethanol and
cremophor EL. When administered by intravenous (iv) infusion,
this formulation frequently triggers acute hypersensitivity reac-
tions, characterized by dyspnea, flushing, rash, chest pain,
tachycardia, hypotension, angioedema, and generalized urti-
2
7,30
enhances bioavailability and is currently in clinical study.
Despite these promising results, there is a concern about the
potential side effects of P-gp inhibitors, in that P-gp is known
to protect the GI tract, brain, and excretory organs from
3
1
xenotoxins and drugs.
Chitosan has been widely used in the pharmaceutical field
because it is known to be biocompatible, biodegradable,
3
caria. In addition, this formulation contributes substantially to
3
2,33
the nonlinear pharmacokinetic behavior of PTX observed in
mucoadhesive, and nontoxic.
In addition, low molecular
4
humans. It would thus be highly desirable to develop an
weight chitosan (LMWC) has recently emerged with even more
favorable characteristics than the conventional high molecular
weight equivalents (HMWC), including greater water solubility,
improved delivery platform for PTX that could, perhaps, also
be used for other poorly water-soluble drugs.
Various attempts have been made to overcome the drawbacks
of current PTX therapy. These have included developing a
micellular formulation,
macromolecules,
efforts, moreover, an increasing amount of research has focused
on development of an oral delivery system for PTX. Oral
administration is the preferred regimen for treatment of chronic
diseases such as cancer because it is convenient for patients
and it eliminates the need to frequently visit a hospital for
3
4
lower toxicity, and a narrower molecular weight distribution.
What’s more, one recent study showed that LMWC (average
MW < 10 kDa) could reversibly open the tight junctions
between intestinal epithelial cells in a Caco-2 cell model and
that subsequent recovery of initial levels of transepithelial
5
-12
conjugation with water-soluble
and prodrug approaches.
1
3-18
19-24
With these
3
4
resistance was much faster than was seen with HMWC. This
ability of LMWC to quickly and reversibly open tight junctions
could be a useful characteristic for a carrier of drug molecules,
especially for oral delivery.
2
5
intravenous infusion. A PTX formulation suitable for oral
administration has not been available in large part because the
overexpression of P-glycoprotein (P-gp) in gastrointestinal (GI)
epithelial cells and first-pass metabolism by CYP450-dependent
enzymes in the GI tract and liver severely limit the drug’s
Herein we describe a LMWC-based conjugate system de-
veloped as a new delivery platform for hydrophobic anticancer
drugs, especially PTX. Several favorable characteristics are
anticipated with this conjugate system: (1) improved water
solubility of PTX due to the presence of chitosan; (2) prolonged
retention of the conjugate in the GI tract due to the mucoad-
hesive property of chitosan, which could result in enhanced
overall uptake; (3) an ability to bypass the P-gp-mediated barrier
(efflux pump), as the opening of tight junctions may enable the
conjugate to be transported via a paracellular pathway; and (4)
an ability to bypass CYP450-dependent metabolism because,
once conjugated, PTX will no longer be a substrate of those
enzymes. In addition, it is noteworthy that the conjugate is
designed to release parent PTX through cleavage of a linker
2
6
bioavailability. Nonetheless, several recently reported ap-
*
To whom correspondence should be addressed. Phone: + 82 62 970
2
504. Fax: + 82 62 970 2484. E-mail: syjon@gist.ac.kr.
†
Gwangju Institute of Science and Technology.
AnyGen Corp.
SungKyunKwan University.
Abbreviations: BHR, Bolton-Hunter reagent; CYP, cytochrome; DMF,
×
‡
a
N,N-dimethylformamide; EDC, 1-ethyl-3-(3-dimethylaminopropyl) carbo-
diimide hydrochloride; GI, gastrointestinal; HMWC, high molecular weight
chitosan; iv, intravenous; LMWC, low molecular weight chitosan; NHS,
N-hydroxysuccinimide; P-gp, P-glycoprotein; po, per oral; PTX, paclitaxel;
SGF, simulated gastric fluid; SIF, simulated intestinal fluid.
3
5
under physiological conditions so that the conjugate can be
considered to be a prodrug. Furthermore, we used LMWC with
1
0.1021/jm800767c CCC: $40.75

2008 American Chemical Society
Published on Web 10/01/2008

Conjugated Chitosan for Oral DeliVery of Paclitaxel
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 20 6443
Scheme 1. Schematic Diagram (A) and Synthetic Scheme (B) of LMWC-PTX; Arrow Indicates the Cleavable Bond between LMWC
and PTX
a narrow distribution of molecular weights so as to minimize
fluctuation in the absorption and pharmacokinetic profiles of
the conjugates. Within this context, we have examined the
feasibility of using this conjugate system as a platform for oral
delivery of PTX. In this report, we describe the synthesis,
characterization, in vitro anticancer activity, pharmacokinetics,
biodistribution, in vivo antitumor activity, and absorption
mechanism of the LMWC-PTX conjugate.
obtained in a 50/50 (v/v) mixture of acetonitrile and water
revealed that the conjugate was 12 ( 0.5 wt % PTX (see
Supporting Information, Figure S2) and showed much greater
mw aL t )e .r solubility (∼1 mg/mL) than the native PTX (∼0.03 mg/
To verify that the linker was cleavable, we tested the stability
of LMWC-PTX by incubating it in PBS (pH 7.4), simulated
gastric fluid (SGF, pH 1.2), simulated intestinal fluid (SIF, pH
7
1
.5), rat plasma, and cell culture medium (10% FBS) (Figure
). While only ∼2.4% and ∼9.3% of PTX was released from
Results and Discussion
the conjugate at 4 h post incubation in PBS and SGF,
respectively, much higher amounts of drug release were
observed for SIF (∼17%), rat plasma (∼25%), and cell culture
medium (∼51%). Considering that the transit time in the
stomach is less than 4 h, the cleavage of the conjugate in SGF
would seem insubstantial. Overall, this cleavage data implies
that the conjugate would withstand the gastric environment but
may be cleaved during or after uptake by the intestine and
transport to the blood capillary, where the release of parent drug
molecules mainly takes place.
Synthesis and Characterization of LMWC-PTX. A sche-
matic illustration of the synthesis of LMWC-PTX conjugate is
shown in Scheme 1. The drug molecules were covalently linked
to LMWC through a succinate linker that is known to be cleaved
to release a parent PTX drug under physiological conditions
35
with hours of half-life. LMWC-PTX was synthesized in a two-
step process as seen in Scheme 1B: (i) modification of PTX
3
6
with a succinic acid (the cleavable linker) and (ii) coupling
of the activated ester form of the succinic acid-modified PTX
to the amine groups of LMWC under conditions. The cleavable
bond was indicated by an arrow in Scheme 1B. The formation
of the conjugate was confirmed by H NMR, which showed
peaks corresponding to both LMWC and PTX (see Supporting
Information, Figure S1). The UV spectrum of the conjugate
37
In Vitro Cytotoxicity of LMWC-PTX. We next compared
the in vitro anticancer activity of LMWC-PTX with that of
parent PTX against human nonsmall cell lung cancer (NCI-
H358), ovarian cancer (SK-OV-3), and breast cancer (MDA-
1

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444 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 20
Lee et al.
concentrations found in tumors 30 min after administration were
0
(
.5 ( 0.1% and 2.4 ( 0.6% for PTX (iv) and LMWC-PTX
po), respectively, indicating a ∼5-fold higher PTX uptake in
the latter. Moreover, very similar PTX concentrations (2.24 (
.37%) were seen in tumors 24 h after administration of LMWC-
0
PTX, a time when no PTX was detected in the tumors of mice
administered PTX (iv). On the other hand, PTX (iv) showed
much greater uptake by the liver and intestine than the conjugate,
presumably because PTX is known to pass through the
enterohepatic circulation and undergo first pass metabolism in
those organs. Taken together, these results suggest that lower
toxicity and higher antitumor efficacy can be anticipated with
LMWC-PTX (po) than with PTX (iv).
In Vivo Antitumor Activity of LMWC-PTX. Encouraged
by the high bioavailability and large tumor uptake of LMWC-
PTX after oral administration, we next examined its in vivo
antitumor efficacy using mice bearing murine melanomas
Figure 1. Stability assessment of LMWC-PTX with times upon
incubation in PBS (pH 7.4), SIF (simulated intestinal fluid, pH 7.5),
SGF (simulated gastric fluid, pH 1.2), rat plasma, and cell culture
medium (10% FBS); bars, SE (n ) 3). *P < 0.05, **P < 0.01, ***P
<
0.001 vs PBS treated group.
(B16F10) or human nonsmall cell lung carcinomas (NCI-H358)
as allograft and xenograft models, respectively (Figure 3). In
the allograft model, we allowed the tumors to grow until their
volumes were >50 mm , after which the mice (n ) 8) were
orally administered one of the following at the scheduled times:
MB-231) cells (see Supporting Information, Figure S3). We
found that the two were similarly cytotoxic: the IC50 values
against NCI-H358, SK-OV-3, and MDA-MB-231 were 3.8, 4.0,
and 3.9 µg/mL for parent PTX and 5.1, 5.0, and 4.8 µg/mL for
LMWC-PTX, respectively. We speculate that the similar
cytotoxicity is attributed to a parent, active form of PTX released
from the conjugate during the assay through cleavage of the
succinate linker between the chitosan and PTX, as the half-
life of drug release from the conjugate in cell culture medium
was about 4 h (Figure 1).
3
2
5 mg/kg PTX, LMWC-PTX (25 mg PTX/kg, 184 mg/kg
LMWC), 184 mg/kg LMWC, or saline (Figure 3A,B). Both the
PTX and LMWC groups showed no statistically meaningful
antitumor effects as compared to the control group (saline). By
contrast, tumor growth was effectively suppressed in the
LMWC-PTX group, resulting in ∼86% inhibition relative to
control (Figure 3A). This is likely indicative of the greater
bioavailability and tumor uptake of the conjugate. We also
measured body weights as an index of drug toxicity. The
LMWC-PTX group, like the other groups, showed no ap-
preciable loss in body weight for up to 23 d, indicating
conjugation caused no additional toxicity.
3
5
Pharmacokinetics and Biodistribution of LMWC-PTX. To
access the pharmacokinetics of LMWC-PTX in vivo, we
administered PTX (iv) formulated in cremophor EL/ethanol or
LMWC-PTX (po) to normal ICR mice. The time-dependent
clearance of LMWC-PTX from plasma is shown in Figure 2A,B.
While PTX had a relatively short half-life in the plasma (t1/2 ≈
In the xenograft tumor model, tumors were allowed to grow
1
h), the apparent half-life of LMWC-PTX was significantly
3
to an average volume of 150 mm before treatment with one of
longer (t1/2 ) 32 h). The apparent bioavailability of LMWC-
the followings: PTX (iv) (formulated in cremophor EL/ethanol;
PTX at a dose of 10 mg and 5 mg PTX/kg was ∼27% and
1
0 mg PTX/kg), which served as a positive control; LMWC-
4
2% in comparison to that of 10 mg/kg PTX (iv), respectively;
PTX (po, 10 mg PTX/kg), LMWC (po, 73 mg/kg); and saline
the value was ∼54% and 86% in comparison to that of 5 mg/
kg PTX (iv), respectively. It is still unclear, but this nonlinear
relationship between bioavailability and dose is often found in
the pharmacokinetics of PTX. To address this issue, we are
planning to carry out more detailed and thorough pharmacoki-
netic studies using other animal species such as rats and dogs
in the future preclinical development. We found that the
pharmacokinetic profile of LMWC-PTX was similar to that
obtained with 24 h of continuous iv infusion of PTX in
(
(
po), which served as a negative control (Figure 3C,D). PTX
iv) and LMWC-PTX effectively suppressed tumor growth by
∼
80% and 66%, respectively, whereas LMWC itself showed
no antitumor activity. Considering that equivalent doses of PTX
10 mg/kg) were used in the conjugate and PTX (iv), the
(
observed antitumor efficacy of the conjugate can be regarded
as sufficiently high to warrant a clinical study. Furthermore, it
should be noted that, unlike the PTX (iv) group, which showed
a rapid decrease in the body weight, the LMWC-PTX group
did not show an appreciable reduction in body weight, as
compared to control (Figure 3D). This suggests that orally
administered LMWC-PTX likely has advantages over the current
clinical formulation of PTX, as it would eliminate the use of
Cremophor EL, increase patient compliance, and may be more
cost-effective in that it eliminates the need for hospitalization.
3
8
humans. It is also noteworthy that, unlike PTX (iv), the
concentration of PTX remained above the required minimum
3
9
therapeutic concentration (85.3 ng/mL) over 48 h after
administration of the conjugate (Figure 2A), which suggests that
the present conjugate system could improve the therapeutic
efficacy of cancer treatments with cell cycle-dependent drugs
such as PTX. We speculate that these results reflect the transport
of the intact conjugate into the blood stream and subsequent
sustained release of the drug molecules through cleavage of the
linker between LMWC and PTX.
Mechanism of LMWC-PTX Absorption. To better under-
stand the mechanism by which LMWC-PTX is absorbed, we
first determined where the conjugate adheres within the GI tract
by synthesizing a rhodamine-labeled LMWC conjugate (LMWC-
Rhodamine), which we assumed would have characteristics
similar to those of LMWC-PTX. Figure 4A shows an overlay
of transmission and fluorescence images of ileum taken after
oral administration of saline, LMWC, rhodamine, or LMWC-
Rhodamine to ICR mice. While the rhodamine dye itself showed
some nonspecific absorption throughout the small intestine,
We next used murine melanoma-bearing B16F10 mice to
examine the uptake of orally administered LMWC-PTX by the
tumor and compared the efficiency with that of intravenously
injected PTX (iv). We found that the apparent PTX concentra-
tions in tumor tissues were much higher in mice administered
LMW-PTX than in those receiving PTX (iv), irrespective of
the time of measurement (Figure 2C). For example, the PTX

Conjugated Chitosan for Oral DeliVery of Paclitaxel
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 20 6445
Figure 2. Dose-dependent plasma concentration of PTX (A), pharmacokinetic parameters (B), and tissue distribution of PTX (C) after administration
of formulated PTX (iv) and LMWC-PTX (po), respectively; bars, SE (n ) 5).
including the duodenum, jejunum (data not shown), and ileum,
the fluorescent signal from LMWC-Rhodamine was seen
exclusively in the ileum. In addition, unlike rhodamine,
significant amounts of LMWC-Rhodamine were observed on
both the surface of microvilli (lumenal side) and on the serosal
side of the ileal epithelium. This suggests that the conjugate
was able to adhere within the GI tract, particularly in the ileum,
and then be transported across the epithelial layers.
Moreover, radioactivity levels were much higher in the ileum
than duodenum or jejunum, which is in good agreement with
the results obtained with the LMWC-Rhodamine. We also found
it interesting that little radioactivity was detected in any other
organs, including the lung, heart, spleen, and stomach, although
low levels were detected in the liver. On the other hand,
substantially decreased radioactivity was detected in blood with
125
I
-labeled LMWC compared to the conjugate (∼17% vs 53%).
To further verify that LMWC-PTX is absorbed from the GI
This fact together with the pharmacokinetics of the conjugate
suggests that LMWC-PTX is absorbed mostly in its intact
conjugate form until it reaches the blood stream, where parent
PTX is released.
1
25
tract, we prepared I -labeled LMWC-PTX and administered
it to ICR mice. Figure 4B shows the time-dependent changes
in the level of radioactivity detected in blood and several major
organs after oral administration of the radiolabeled conjugate.
Surprisingly, over half (∼53% relative radioactivity) of the initial
dose was detected in blood within 30 min after administration,
and the remainder was found exclusively in the small intestine.
We next investigated the effect of P-gp, which represents a
major barrier to effective oral absorption of a variety of drugs,
including PTX, and the effects of two major drug metabolic
enzymes (Figure 4C,D) on LMWC-PTX absorption. The effect

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446 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 20
Lee et al.
Figure 3. Antitumor efficacy of LMWC-PTX in mice bearing B16F10 murine melanoma (A,B) or NCI-H358 human nonsmall cell lung cancer
C,D). (A) Drugs were administered orally on the indicted schedules (solid lines); bars, SE (n ) 8). **P < 0.01 vs saline control. (B) Body weight
(
changes in B16F10 tumor-bearing mice after oral administration; bars, SE (n ) 8). (C) PTX was injected iv, and saline, LMWC, and LMWC-PTX
were administered orally at the indicated times (arrowheads); bars, SE (n ) 8-12). **P < 0.01 vs saline control. (D) Body weight changes in
NCI-H358 tumor-bearing mice after drug treatment; bars, SE (n ) 8-12). ***P < 0.001 vs saline control.
of P-gp on the absorption of PTX and LMWC-PTX was
evaluated in pharmacokinetic studies after oral administration
of each compound with or without cyclosporine A, a commonly
used P-gp inhibitor. As expected, coadministration of PTX (20
mg/kg) with cyclosporine A significantly increased the plasma
PTX concentration, thereby increasing the bioavailability ∼9-
fold, from 0.23% to 2.1%. By contrast, we observed a slight
reduction in the bioavailability of PTX, from 37% to 29%, when
LMWC-PTX (20 mg PTX/kg) was administered together with
cyclosporine A. This suggests LMWC-PTX is absorbed in a
P-gp-independent manner, avoiding the undesired P-gp-mediated
efflux of the drug in GI tract. In addition, the metabolic stability
of LMWC-PTX and free PTX were compared using competitive
inhibition assay kits (Figure 4D). PTX and positive controls
both competitively inhibited the binding of a fluorescent
substrate to CYP2C8, whereas LMWC-PTX had a much higher
IC50. In other words, the conjugate had a much lower affinity
for the enzyme, suggesting it may bypass CYP-dependent drug
metabolism, leading to greater oral bioavailability.
based conjugate system may also be a useful tool for oral
delivery of other poorly water-soluble drugs.
Experimental Section
Chemicals, Cells, and Animals. Paclitaxel (PTX) was purchased
from Samyang Genex (Seoul, Korea). Low molecular weight
chitosan (LMWC, average MW: 6 kDa) was from KITTOLIFE
(
Seoul, Korea) and was further purified by ultrafiltration. N-
125
succinimidyl 3-(4-hydroxy, 5-[ I] iodophenyl)-propionate (Bolton
& Hunter reagent) was from Amersham Biosciences (Uppsala,
Sweden). All other solvents and reagents were from Sigma
Chemical (St. Louis, MO), were of reagent-grade, and were used
as received. Cell lines were obtained from Korean Cell Line Bank
(
KCLB, Seoul, Korea) and were cultured according to the instruc-
tions from KCLB. All animals were obtained from Orient Bio Inc.
Seoul, Korea) and handled in accordance with the guidelines of
(
the Animal Care and Use Committee in Gwangju Institute of
Science and Technology.
Synthesis and Characterization of LMWC-PTX. A 2′-
hemisuccinate derivative (PTX-Suc) of PTX was prepared by the
37
In summary, we have developed a new platform for oral
delivery of PTX using a chemical conjugate system comprised
of PTX and LMWC with a narrow molecular weight distribution
previously reported method with some modifications. Briefly, PTX
(300 mg, 0.351 mmol) and succinic anhydride (46 mg, 1.3 equiv)
2 2
were dissolved in CH Cl (14 mL) at ambient temperature. After
pyridine (39 µL, 1.37 equiv) was added, the mixture was vigorously
stirred for 3 days at ambient temperature. The reaction mixture was
concentrated in vacuum and loaded on silica gel column chroma-
tography for purification. The purified PTX-Suc was obtained as
white solid in 78% yield.
PTX-Suc (100 mg, 0.1 mmol) dissolved in anhydrous DMF (2
mL) was activated by N-hydroxy succiimide (NHS, 5 equiv) and
(
average MW: 6 kDa). This prodrug form of PTX is absorbed
in the small intestine after oral administration and effectively
inhibits tumor growth with an efficacy comparable to the
clinically available injected form, but with much lower toxicity.
The strong antitumor activity of LMWC-PTX after oral
administration may be attributable to its greater water solubility,
prolonged retention in the GI tract, and ability to bypass P-gp
efflux pumps in the GI tract and CYP 450-dependent metabolism
in intestine and liver. Taken together, we suggest this LMWC-
1
-ethyl-3-(3-dimethylaminopropyl carbodiimide hydrochloride) (EDC,
1.1 equiv) for 4 h at room temperature to afford an NHS ester form
of PTX-Suc. Subsequently, the resulting mixture was added to a

Conjugated Chitosan for Oral DeliVery of Paclitaxel
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 20 6447
Figure 4. Mechanism of LMWC-PTX absorption. (A) Confocal laser scanning microscopy images of LMWC-Rhodamine. (B) Biodistribution of
1
25
I
-labeled LMWC-PTX (10 mg/kg PTX) after oral administration; bars, SE (n ) 3). (C) Effect of P-gp inhibition (cyclosporin A, 15 mg/kg) after
oral administration of 20 mg/kg PTX or LMWC-PTX (20 mg/kg PTX); bars, SE (n ) 4-5). (D) Metabolic stability of LMWC-PTX in the presence
of CYP450-dependent enzymes.
LMWC (157 mg, 0.025 equiv) in borate buffer (pH 10). After
stirring for 12 h at room temperature, the reaction mixture was
diluted by distilled water (5×) and extracted by ethyl ether (3 times)
to remove unreacted PTX. The aqueous layer was dialyzed against
distilled water using a dialysis membrane (MW cut off size: 3000),
after which LMWC-PTX was lyophilized into white solid.
for 20 min to remove the undissolved conjugate. The supernatant
solution was analyzed by H NMR spectroscopy to confirm the
presence of the conjugate and then was further analyzed by UV-vis
spectroscopy to obtain the concentration of the conjugate.
1
Stability Study of LMWC-PTX. Stability studies were carried
out in PBS (pH 7.4), simulated intestinal fluid containing 1%
pancreatin (SIF, pH 7.5), simulated gastric fluid containing 0.32%
pepsin (SGF, pH 1.2), rat plasma, and cell culture medium (10%
FBS) at 37 °C for up to 12 h. The conjugate (5 mg) was dissolved
in 2.5 mL of each fluid, after which 100 µL of samples were taken
at given times. The free PTX was then extracted by using 1 mL of
ethyl acetate. Released free PTX was measured by reverse-phase
high-performance liquid chromatography (HPLC; Shimadzu, Kyoto,
Japan) on a C18 column (300 mm × 3.9 mm Nova-Pak Waters)
with acetonitrile/water. The significance of differences in the uptake
levels between groups was assessed using Student’s t-test.
1
17
PTX-Suc: H NMR (Xeol, 300 MHz, CDCl
3
): δ1.11 [s, CH
3
],
1
6
19
18
1
[
4
5
[
.20 [s, CH
3
], 1.69 [s, CH
3
], 1.9 [s, CH
CH -COO-TXL], 3.78 [d, CH],
], 4.46 [dd, CH], 4.96 [d, CH],
3
], 2.2 [m, OAc], 2.4
3
m, OAc], 2.5-2.8 [m, HOOC-CH
2
2
2
0
20
7
5
.17 [d, CH
2
], 4.3 [d, CH
2
2
′
2
3′
13
.50 [d, CH], 5.67 [d, CH], 5.98 [dd, CH], 6.22 [t, CH], 6.27
1
0
s, CH], 7.09 [d, NH], 7.25 [s, 3′-Ph], 7.4 [m, 3′-NBz], 7.5 [m,
2
-OBz], 7.73 [d, 3′-NBz], 8.1 [d, 2-OBz].
Measurement of Solubility of LMWC-PTX. Water solubility
36
of LMWC-PTX conjugate was measured as described elsewhere.
Briefly, the conjugate (16.6 mg) was dissolved in 1.0 mL of D
2
O
with sonication for 15 min. The resulting mixture was centrifuged

6
448 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 20
Pharmacokinetic and Biodistribution Studies. Normal female
Lee et al.
sections were then prepared by using a microtome (Leica RM2245,
Wetzlar, Germany), and the distributions of the rhodamine deriva-
tive and LMWC-Rhodamine were assessed using a fluorescence
microscope (Axiovert 200M, ZEISS, Germany).
ICR mice (25-30 g) were administered 10 mg/kg of PTX (iv)
formulated in cremophor EL/ethanol via the tail vein. Mice used
for oral administration were fasted for 12 h and then administered
a single oral dose of LMWC-PTX (5 or 10 mg of equivalent PTX/
kg) through a blunt needle that was passed down the esophagus
into the stomach. The LMWC-PTX solution was prepared in a 10%
DMSO solution, and the total volume of the administered LMWC-
PTX solution was 300 µL. For subsequent analyses, blood (450
µL) was collected from a capillary in the retro-orbital plexus and
mixed with 50 µL of sodium citrate (3.8% solution), after which
the sample was immediately centrifuged at 800g for 20 min. The
PTX was then extracted from 100 µL of plasma using 1 mL of
ethyl acetate. The extraction efficiency for PTX was 95%. The
samples were then centrifuged for 10 min at 35000g, after which
the supernatant was separated and dried. The dried extract was
reconstituted with 1 mL of HPLC mobile phase, and a 50-µL aliquot
was injected into the HPLC for determination of free PTX.
Pharmacokinetic parameters were analyzed based on a noncom-
125
Biodistribution Study Using I -Labeled LMWC-PTX. LM-
1
25
WC-PTX was radiolabeled using I -labeled Bolton-Hunter
reagent (BHR). Briefly, LMWC-PTX (12 µg, 2 nmol) dissolved in
1
25
a borate buffer (6 µL) was added to a solution of I -labeled
Bolton-Hunter reagent (0.2 nmol, 80 µL, 400 µCi) and the reaction
mixture was stirred for 3 h at room temperature. After reaction,
the unbound BHR was removed by extraction using ethyl acetate
(
×3) and the radiolabeled conjugate was purified by dialysis using
125
a cellulose membrane of MWCO 1000 for 6 h. The content of I
in the conjugate was measured by using a gamma counter (1480
125
Wizard3, PerkinElmer, Inc.).
I-labeled LMWC-PTX (38 nCi)
was then orally administered to ICR mice (25-30 g). Then 0.5, 2,
and 6 h after administration, blood was collected, mice were
sacrificed, and the liver, stomach, intestine, colon, spleen, kidney,
lung, heart, and tumor were excised and the tissue-associated
radioactivity was determined using a gamma counter. The radio-
activity in the mouse tissues was expressed as a percentage of
administered doses. Uptake levels were expressed as % administered-
dose per each organ.
Effect of a P-gp Inhibitor on LMWC-PTX Absorption. To
estimate effects of P-gp on absorption, ICR mice (25-30 g) were
fasted for 12 h before oral administration of 10 mg/kg PTX or
LMWC-PTX (10 mg/kg PTX equivalent) using a blunt needle via
the esophagus into the stomach. When administering a P-gp
inhibitor, the oral solution also contained 15 mg/kg cyclosporine
A. Blood samples were subsequently collected from the retro-orbital
vein and centrifuged for 20 min at 3000g, after which plasma PTX
concentrations were determined by HPLC.
4
0
partmental model using Microsoft Excel.
In a separate experiment, C57BL6 mice bearing B16F10 murine
3
melanomas (100 mm ) were administered 10 mg PTX/kg of
formulated PTX (iv) via the tail vein or LMWC-PTX (po). Then
3
, 6, 9, 12, and 24 h after injection or oral administration, mice
were sacrificed and the liver, stomach, intestine, colon, spleen,
kidney, lung, heart, and tumor were excised. After weighting each
tissue, 5 mL of acetonitrile was added and the tissue was
homogenized. The homogenates were centrifuged at 20000g for
1
0 min, after which 500 µL of the supernatant was collected and
added to 500 µL of water. Aliquots (50 µL) of the reconstituted
samples were then injected into an HPLC for determination of free
PTX.
In Vivo Antitumor Activity. To estimate the antitumor effect
To assess metabolism by CYP450-dependent enzymes, CYP450
inhibition assays were conducted using a High Throughput Inhibitor
Screening Kit (HTS Kit, BD GENTEST). These assays enabled
of LMWC-PTX against allografts, tumors were induced in female
C57BL6 mice (20-25 g) by sc inoculation of approximately 1 ×
5
41
1
0 murine melanoma (B16F10) cells into their backs. When the
3
tumors were at least 50 mm in size, PTX or LMWC-PTX was
administered orally at 25 mg/kg/day for two 5-day cycles separated
by a 2-day interval.
us to monitor, via fluorescence detection, metabolite formation
following incubation of the CYP enzyme with a specific substrate.
PTX and LMWC-PTX were tested with CYP3A4 and CYP2C8,
as these enzymes had been shown to exhibit inhibition. Reactions
were run in 96-well plates at 37 °C. Inhibition of metabolic product
formation from the test compound was tested for each enzyme in
the absence and presence of the test compound. The amount of
metabolic product formed was quantified using a fluorescence plate
reader with excitation and emission filters that were optimized for
detection of each metabolite.
To assess the antitumor effect of LMWC-PTX against xenografts,
tumors were induced in female athymic nude (NCR nu/nu) mice
6
(
20-25 g) by sc inoculation of approximately 6 × 10 human
nonsmall cell lung cancer (NCI-H358) cells. When the tumors were
3
at least 150 mm in size, the mice were administered 10 mg/kg/
day formulated PTX (iv) or LMWC-PTX (10 mg PTX/kg/day) (po)
every other day until six doses had been given. Control mice were
administered saline, LMWC solution. Tumor volumes were mea-
1
sured using calipers and calculated using the equation ( /
2
) (L ×
Acknowledgment. This work was supported by the Cell
Dynamics Research Center, Korean Ministry of Science and
Technology.
2
W ). The significance of the difference in uptake levels between
groups was assessed using Student’s t-test.
Assessment of the Site of Conjugate Absorption in GI
Tract Using LMWC-Rhodamine. LMWC-Rhodamine was syn-
thesized from LMWC and 5(6)-carboxytetramethylrhodamine N-
succinimidyl ester (BioChemiac) under similar conditions as
LMWC-PTX. Briefly, the rhodamine derivative (8.4 mg, 15.9 µmol)
dissolved in DMF (1 mL) was added to a solution of LMWC (47.7
mg, 7.6 µmol) in water (0.8 mL)/DMF (1.6 mL) and the reaction
mixture was stirred for 8 h at room temperature. Then unbound
rhodamine was eliminated by extraction using ethyl acetate (×3)
and the conjugate was further purified by dialysis using a cellulose
membrane with MWCO 1000 for 1 day. The content of rhodamine
in the conjugate was quantified by fluorescence measurement using
a fluorescence spectrometer (RF-5301PC, Shimadzu, Japan) at an
excitation and emission wavelength of 543 and 576 nm, respectively.
Absorption experiment of LMWC-Rhodamine was carried out
as follows. A rhodamine derivative (20 mg/kg), LMWC (184 mg/
kg), and LMWC-Rhodamine (204 mg/kg) were orally administered
to ICR mice (25-30 g). After 30 min and 1 h, mice were euthanized
and then duodenum, jejunum, and ileum of each mouse were
collected. Several parts of the collected tissues were laid onto the
OCT in the mold for fixation and stored at -70 °C. Thin tissue
1
Supporting Information Available: H NMR, UV spectra, and
cytotoxicity data in vitro of the LMWC-PTX conjugate. This
material is available free of charge via the Internet at http://
pubs.acs.org.
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