The development of orally administrable gemcitabine prodrugs with d-enantiomer amino acids: Enhanced membrane permeability and enzymatic stability

Article abstract of DOI:10.1016/j.ejpb.2013.12.009

Gemcitabine prodrugs with d- and l-configuration amino acids were synthesized and their chemical stability in buffers, resistance to glycosidic bond metabolism, enzymatic activation, permeability in Caco-2 cells and mouse intestinal membrane, anti-prolife

Full text of DOI:10.1016/j.ejpb.2013.12.009

European Journal of Pharmaceutics and Biopharmaceutics xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
European Journal of Pharmaceutics and Biopharmaceutics
journal homepage: www.elsevier.com/locate/ejpb
Research paper
The development of orally administrable gemcitabine prodrugs
with D-enantiomer amino acids: Enhanced membrane permeability
and enzymatic stability
Yasuhiro Tsume a, Tuba Incecayir b, Xueqin Song c, John M. Hilfinger d, Gordon L. Amidon a,
⇑
a Department of Pharmaceutical Science, University of Michigan, Ann Arbor, MI, USA
b Department of Pharmaceutical Technology, Gazi University, Etiler-Ankara, Turkey
c Hospira, Inc., Lake Forest, IL, USA
d TSRL, Inc., Ann Arbor, MI, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 18 June 2013
Accepted in revised form 12 December 2013
Available online xxxx
Gemcitabine prodrugs with
bility in buffers, resistance to glycosidic bond metabolism, enzymatic activation, permeability in Caco-2
cells and mouse intestinal membrane, anti-proliferation activity in cancer cell were determined and com-
D
- and
L
-configuration amino acids were synthesized and their chemical sta-
pared to that of parent drug, gemcitabine. Prodrugs containing
matically more stable than ones with -configuration amino acids. The activation of all gemcitabine
prodrugs was 1.3–17.6-fold faster in cancer cell homogenate than their hydrolysis in buffer, suggesting
enzymatic action. The enzymatic activation of amino acid monoester prodrugs containing -configuration
amino acids in cell homogenates was 2.2–10.9-fold slower than one of amino acid monoester prodrugs
with -configuration amino acids. All prodrugs exhibited enhanced resistance to glycosidic bond metab-
D-configuration amino acids were enzy-
L
Keywords:
Monoester gemcitabine prodrugs
Mouse in situ perfusion
Unnatural form
Amino acid
D
L
olism by thymidine phosphorylase compared to parent gemcitabine. Gemcitabine prodrugs showed
superior the effective permeability in mouse jejunum to gemcitabine. More importantly, the high plasma
Stability
Permeability
concentration of D-amino acid gemcitabine prodrugs was observed more than one of L-amino acid gem-
citabine prodrugs. In general, the 50-mono-amino acid monoester gemcitabine prodrugs exhibited higher
permeability and uptake than their parent drug, gemcitabine. Cell proliferation assays in AsPC-1 pancre-
atic ductal cell line indicated that gemcitabine prodrugs were more potent than their parent drug,
gemcitabine. The transport and enzymatic profiles of 50- -valyl-gemcitabine and 50-
D D-phenylalanyl-gem-
citabine suggest their potential for increased oral uptake and delayed enzymatic bioconversion as well as
enhanced uptake and cytotoxic activity in cancer cells, would facilitate the development of oral dosage
form for anti-cancer agents and, hence, improve the quality of life for the cancer patients.
Ó 2014 Published by Elsevier B.V.
1. Introduction
Prodrug strategies have been utilized to overcome undesirable
physicochemical properties of the drug, to improve oral bioavail-
The anti-cancer agent 2,20-difluoro-20-deoxyuridine or GemzarÒ
(gemcitabine), one of the nucleoside analogs, has been used to
treat pancreatic and non-small-cell lung cancers as the first-line
therapy [1,2]. However, the adverse effects associated with chemo-
therapeutics are still unresolved and many efforts have been made
to minimize side-effects and maximize therapeutic efficacy.
ability. A majority of the efforts have focused on anti-viral and
anti-cancer drugs to develop oral alternatives. Amino acid ester
prodrugs of poorly permeable anti-cancer and anti-viral drugs
have been designed for targeted delivery via specific transporters
to improve their oral bioavailability and metabolic disposition
[2–11].
Amino acid ester anti-cancer prodrugs have been synthesized
and tested for potential improvement of oral drug delivery
[5,10–15]. It has been reported that amino acid ester prodrugs
are recognized as substrates for intake transporters such as PEPT1,
PEPT2, and ATB0,+, and this carrier-mediated mechanism improves
their oral bioavailability [10,13,16–21]. PEPT1 is predominantly
Abbreviations: Gly-Pro, Glycine-Proline; Paap, apparent permeability; Peff, effec-
tive permeability.
Corresponding author. College of Pharmacy, The University of Michigan, 428
Church Street, Ann Arbor, MI 48109-1065, USA. Tel.: +1 734 764 2440; fax: +1 734
763 6423.
⇑
E-mail address: glamidon@umich.edu (G.L. Amidon).
http://dx.doi.org/10.1016/j.ejpb.2013.12.009
0939-6411/Ó 2014 Published by Elsevier B.V.
Please cite this article in press as: Y. Tsume et al., The development of orally administrable gemcitabine prodrugs with
D-enantiomer amino acids: En-
hanced membrane permeability and enzymatic stability, Eur. J. Pharm. Biopharm. (2014), http://dx.doi.org/10.1016/j.ejpb.2013.12.009

2
Y. Tsume et al. / European Journal of Pharmaceutics and Biopharmaceutics xxx (2014) xxx–xxx
expressed in the small intestine and can transport dipeptides, trip-
eptides, amino acid monoester prodrugs and b-lactam antibiotics
[10,11,16,22–28]. PEPT1 has broad substrate specificity and recog-
and DMAP (0.1 mmol) were allowed to react with gemcitabine
(1 mmol) in 7 mL of dry DMF for 24 h. The reaction progress was
monitored by thin layer chromatography (TLC) (ethyl acetate).
The reaction mixture was filtered and dichloromethane (DCM)
was removed under vacuum at 40 °C. The residue was extracted
with ethyl acetate (30 mL) and washed with water (2 ꢀ 20 mL),
and saturated NaCl (20 mL). The organic layer was dried over
MgSO4 and concentrated under vacuum. The reaction yielded a
mixture of gemcitabine Boc-protected prodrugs. The three spots
observed on TLC were separated and purified using column chro-
matography (dichloromethane/methanol, 20:1). Fractions from
each spot (the first spot: 50-monoester gemcitabine prodrug, the
second spot: 30-monoester gemcitabine prodrug, and the third
spot: 30,50-diester gemcitabine prodrug) were concentrated under
vacuum separately. The Boc group was cleaved by treating the res-
idues with 5 mL TFA:DCM (1:1). After 4 h, the solvent was removed
and the residues were reconstituted with water and lyophilized.
The TFA salts of amino acid prodrugs of gemcitabine were obtained
as white fluffy solids. The yield of 50-mono-amino acid gemcitabine
prodrug was 12–20%. HPLC was used to evaluate the prodrug pur-
ity. Prodrugs were between 90–99% pure. These prodrugs were
easily separated from parent drug by HPLC. Electrospray ionization
mass spectra (ESI-MS) were obtained on a Micromass LCT ESI-MS.
The observed molecular weights of all prodrugs were found to be
consistent with that required by their structure. The structural
identity of the prodrugs was then confirmed using proton nuclear
magnetic resonance spectra (1H NMR). 1H NMR spectra were
obtained on a 300 MHz Bruker DPX-300 NMR spectrometer.
nizes
PEPT1 is stereoselective and exhibits greater affinity for
mers of amino acids than -enantiomers [10,29,30]. Amino acid es-
D
-enantiomers of amino acid as a substrate even though
L
-enantio-
D
ter prodrugs may facilitate enhanced delivery to pancreatic cancer
cells such as AsPC-1 due to the high expression of oligopeptide
transporters [31].
The mechanism of action for anti-cancer nucleoside analogs
such as 5-Fluorouracil (5-FU), floxuridine, and gemcitabine is well
investigated and understood [32–35]. Most of anti-cancer drugs
including nucleoside analogs are intravenously administered due
to their low oral bioavailability and stability issues [36,37]. More-
over, nucleoside analogs are enzymatically converted to pyrimi-
dine structure in many tissues including the liver [37,38]. As a
consequence, higher doses of chemotherapeutic agents are
required to assure clinical efficacy, leading to greater toxicity. Oral
anti-cancer therapy obviously improves the quality of life for can-
cer patients compared to intravenous therapy because of its conve-
nience and, eventually, the reduction in insurance costs [39].
Improving the chemical and enzymatic stabilities and membrane
permeability of gemcitabine may enhance its therapeutic efficacy
at low doses and obviate toxicity concerns with orally administra-
ble chemotherapeutic drugs.
In this report, we describe the stability and permeability of (
D-/
L-)amino acid monoester prodrugs of gemcitabine, as well as their
antiproliferative activity. Uptake studies were conducted with
Caco-2 and AsPC-1 cells and permeability studies were conducted
with Caco-2 cell monolayer and in situ mouse jejunal perfusion.
Furthermore, the feasibility of developing orally administrable
chemotherapeutic agents was assessed by measuring the drug con-
centration and drug species in plasma after the perfusion study.
The chemical stability at physiological pH and the enzymatic acti-
vation of the prodrugs in Caco-2, and AsPC-1 cell homogenates as
well as thymidine phosphorylase were also evaluated to determine
the effects of the amino acid configuration on enzyme-mediated
activation. The antiproliferative action of amino acid gemcitabine
prodrugs and their parent drug, gemcitabine, was explored using
pancreatic ductal cancer cell, AsPC-1.
2.3. Cell culture
AsPC-1 cells (passages 25–38) from American type Culture Col-
lection (Rockville, MD) were routinely maintained in RPMI-1640
containing 10% fetal bovine serum. Caco-2 cells (passages 28–31)
from American type Culture Collection (Rockville, MD) were rou-
tinely maintained in DMEM containing 10% fetal bovine serum.
Cells were grown at 37 °C at 5% CO2 and 90% relative humidity in
antibiotic-free media to avoid the possible transport interference
by antibiotics.
2.4. Hydrolysis studies
2. Materials and methods
2.4.1. Enzymatic stability
Confluent Caco-2 and AsPC-1cells were rinsed twice with phos-
phate buffered saline (PBS). The cells were lysed with phosphate
buffer (pH 7.4) by ultrasonication (Micro ultrasonic cell disrupter
Model KT40, Kontes, Vineland, NJ, USA), and pelleted by centrifuga-
tion for 5 min at 1000g. The protein amount was quantified with
the Bio-Rad (Hercules, CA, USA) DC Protein Assay using bovine ser-
um albumin as a standard. The amount of protein was adjusted to
2.1. Materials
Gemcitabine was extracted from the lyophilized powder (Gem-
zar) supplied by Eli Lilly Pharmaceuticals (Indianapolis, IN). The
tert-butyloxycarbonyl (Boc) protected amino acids Boc-
L-valine,
Boc- -valine, Boc- -phenylalanine, and Boc- -phenylalanine were
D
L
D
obtained from Chem-Impex (Wood Dale, IL). High-performance li-
quid chromatography (HPLC) grade acetonitrile was obtained from
Fisher Scientific (St. Louis, MO). N,N-dicyclohexylcarbodiimide
(DCC), N,N-dimethylaminopyridine (DMAP), trifluoroacetic acid
(TFA), and all other reagents and solvents were purchased from Al-
drich Chemical Co. (Milwaukee, WI). Cell culture reagents were ob-
tained from Invitrogen (Carlsbad, CA) and cell culture supplies
were obtained from Corning (Corning, NY) and Falcon (Lincoln
Park, NJ). All chemicals were of either analytical or HPLC grade.
500
plates (Corning, NY, USA). Caco-2 and AsPC-1 cell suspensions
(250 L) were placed in triplicate wells, the reactions were started
with the addition of substrate, and cells were incubated at 37 °C for
120 min. At the desired time point, sample aliquots (35 L) were
removed and added to acetonitrile (ACN, 150 L) with 0.1% TFA.
The mixtures were filtered with a 0.45 m filter membrane at
lg/mL and hydrolysis reactions were carried out in 96-well
l
l
l
l
1000g for 10 min at 4 °C. The filtrate was then analyzed via
reverse-phase HPLC.
2.2. Gemcitabine prodrug synthesis
2.4.2. Chemical stability
The nonenzymatic hydrolysis of the prodrugs was determined as
described above, except that each well contained pH 7.4 phosphate
buffers (10 mmol/L) instead of cell homogenate. Also, the chemical
stability of the prodrugs was determined in simulated gastric fluid
(SGF) pH 1.2 and simulated intestinal fluid (SIF) pH 6.8.
The synthesis and characterization of 50-mono-amino acid ester
prodrugs of gemcitabine have been reported previously [5]. Briefly,
Boc-protected amino acid, Boc-
L-valine, Boc-D-valine, Boc-L-phen-
ylalanine, or Boc- -phenylalanine, (1.1 mmol), DCC (1.1 mmol),
D
Please cite this article in press as: Y. Tsume et al., The development of orally administrable gemcitabine prodrugs with
D-enantiomer amino acids: En-
hanced membrane permeability and enzymatic stability, Eur. J. Pharm. Biopharm. (2014), http://dx.doi.org/10.1016/j.ejpb.2013.12.009

Y. Tsume et al. / European Journal of Pharmaceutics and Biopharmaceutics xxx (2014) xxx–xxx
3
2.4.3. Microsomal stability
The assay was performed in triplicate containing substrates,
500 lg/mL of microsomal proteins diluted in PBS (pH 7.4) and
16.7 mg/mL of b-nicotinamide-adenine dinucleotide phosphate
(NADPH). After applying NADPH solution into the microsomal sus-
of Animals (UCUCA). Female BALB/c mice (Charles River, IN)
weighing 20–25 g were used for all perfusion studies. Prior to each
experiment, mice were fasted overnight with free access to water.
The procedure for the in situ single-pass intestinal perfusion is
previously reported [40]. Briefly, mice were anesthetized with an
i.m. injection of ketamine-xylazine mixture (ketamine: 80–
120 mg/kg, xylazine: 5–10 mg/kg) and placed on a heated pad
maintained at 37 °C. The abdomen was opened by a midline inci-
sion and a jejunal segment (approximately 10 cm) was carefully
exposed to cannulate both ends with flexible polyvinyl chloride
(PVC) tubing (2.06 mm i.d., Fisher Scientific Inc., Pittsburgh, PA,
USA). All solutions were incubated in a 37 °C water-bath. The iso-
lated segment was rinsed with blank perfusion buffer to clean out
any residual debris.
pension (250
ately taken as t = 0. The sample mixtures were incubated at 37 °C
for 120 min. At the desired time point, sample aliquots (30 L)
were removed and added to acetonitrile (ACN, 150 L) with 0.1%
TFA. The mixtures were filtered with a 0.45 m filter membrane
lL) containing substrates, the sample was immedi-
l
l
l
at 1000g for 10 min at 4 °C. The filtrate was then analyzed via re-
verse-phase HPLC.
2.5. Uptake studies
At the start of the study, the test compound (100 lM) in perfu-
Caco-2 and AsPC-1 cells were grown on a 6-well plate for 18 and
6 days, respectively. Wells were rinsed with MES (pH 6.0) buffer
twice. Fresh MES buffer was reapplied to each well and incubated
at 37 °C for 15 min. Each drug was individually tested from freshly
prepared solutions in MES buffer (0.1 mM, total 0.3 mL) with both
the presence and the absence of 10 mM glycyl-proline (Gly-Pro).
Additionally, uptake studies with the lower drug concentration,
0.05 mM, in AsPC-1 cells were carried out to determine the dose lin-
earity. The solution was placed in each well and incubated at 37 °C
for 60 min. Drug solution was removed and 3 mL of ice-cold PBS
was immediately placed in each well. Each well was rinsed with
3 mL of cold-PBS twice and 0.5 mL of methanol:H2O (1:1) containing
0.1% TFA was placed in each well. The cell suspension was collected
and transferred to a new tube. Those tubes were spun at 1000g at
4 °C for 5 min. The supernatant was mixed with an equal amount
of water for HPLC analysis. The cell pullets were used to determine
the protein amount with the Bio-Rad (Hercules, CA, USA) DC Protein
Assay using bovine serum albumin as a standard.
sion buffer (pH 6.0) including phenol red was perfused through the
intestinal segment (Watson-Marlow Pumps 323S, Watson-Marlow
Bredel Inc., Wilimington, MA), at the flow rate of 0.08 mL/min. The
perfusion buffer was perfused for 30 min to assure steady-state
and samples were taken every 10 min for 90 min. At the end of per-
fusion, blood samples were collected by cardiac puncture. Blood
samples with heparin were centrifuged at 1000g for 10 min at
4 °C to collect plasma samples for LC–MS analysis. Following the
termination of the experiment, the length of each perfused intesti-
nal segment was measured.
2.9. Cell proliferation assays
Cell proliferation studies were conducted with AsPC-1 cells. The
cells were seeded into 96-well plates at 125,000 cells per well and
allowed to attach/grow for 24 h before drug solutions were added.
The culture medium (RPMI-1640 + 10% fetal bovine serum) was re-
moved and the cells were gently washed once with sterile pH 6.0
uptake buffer. Gemcitabine and gemcitabine prodrugs were 2-fold
serially diluted in pH 6.0 uptake buffer from 5 to 0.078 mmol/L.
Buffer alone was used as a 100% viability control. The wash buffer
2.6. Caco-2 cell monolayer permeability studies
Caco-2 cell monolayers were grown on collagen-coated polytet-
rafluoroethylene membrane for 21–24 days. Transepithelial
electrical resistance (TEER) was monitored and values of 550–
was removed and 30 lL drug solution per well was added and
incubated at 37 °C for 2 h in the cell incubator. After this time,
the drug solutions were removed and the cells were again gently
washed twice with sterile uptake buffer. The culture medium
was then added to each well after washing. The cells were allowed
to recover for 24 h before evaluating cell viability via 2,3-bis[2-
methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide
600
X
cm2 (total growth area was 1.12 cm2) were used in the
study. Apical and basolateral sides of transwell inserts were
washed with MES (pH 6.0) and HEPES (pH 7.4), respectively. Fresh
MES and HEPES buffers were reapplied to transwell inserts and
incubated at 37 °C for 15 min. Each drug was individually tested
from freshly prepared solutions in MES buffer (0.1 mM, total
0.5 mL) with both the presence and the absence of 10 mM Gly-
Pro. The solution was placed in the donor chamber, while the recei-
ver chamber was filled with HEPES buffer (total 1.5 mL). Sampling
from the receiver chamber was conducted up to a period of 2 h at
time intervals of 15, 30, 45, 60, 75, 90, and 120 min, at 37 °C and
replaced with an equal volume of fresh HEPES buffer to maintain
sink conditions in the receiver chamber. All samples were immedi-
ately acidified with 0.1% TFA and analyzed by HPLC.
inner salt (XTT) assays. A mixture (30 lL) containing XTT (1 mg/
mL) in sterile RPMI-1640 without phenol red and phenazine
methosulfate (N-methyldibenzopyrazine methyl sulfate in sterile
PBS, 0.383 mg/mL) reagents were added to the cells and incubated
at 37 °C. The absorbance at 450 nm was read within 1 h. The con-
centrations required to inhibit cell growth by 50% (GI50) were cal-
culated using GraphPad Prism version 3.0 by nonlinear data fitting.
2.10. Data analysis
The net water flux in the mouse perfusion studies was deter-
mined using phenol red (14 lM), a non-absorbed and non-metab-
2.7. Solution for single-pass intestinal perfusion
olized marker. The measured Cout/Cin ratio of test compound was
corrected for water flux according to the following equation:
The perfusion buffer (pH 6.0) consisted of 145 mM NaCl,
0.5 mM MgCl2, 1 NaH2PO4, 1 mM CaCl2, 3 mM KCl, 5 mM glucose,
and 5 mM MES. The pH of the buffer was adjusted to pH 6.0. This
C0out Cout Cin phonol red
¼
ꢀ
C0in
perfusion buffer also contained phenol-red (14
absorbable marker for water flux measurements.
lM) as a non-
Cin Cout phenol red
where Cin phenol red and Cout phenol red are equal to the concentration
of phenol red in the inlet and the outlet samples, respectively. The
effective permeability (Peff; cm/s) through the mouse intestinal wall
in the single-pass intestinal perfusion studies was determined
according to the following equation:
2.8. Single-pass intestinal perfusion studies in mice
All animal experiments were conducted using protocols
approved by the University of Michigan Committee of Use and Care
Please cite this article in press as: Y. Tsume et al., The development of orally administrable gemcitabine prodrugs with
D-enantiomer amino acids: En-
hanced membrane permeability and enzymatic stability, Eur. J. Pharm. Biopharm. (2014), http://dx.doi.org/10.1016/j.ejpb.2013.12.009

4
Y. Tsume et al. / European Journal of Pharmaceutics and Biopharmaceutics xxx (2014) xxx–xxx
ꢀ
ꢁ
C0out
C0in
ꢁQln
Peff
¼
2
p
RL
where Q is the perfusion buffer flow rate (0.08 mL/min), C0out=Ci0n is
the ratio of the outlet/inlet concentration of test compound that is
adjusted for water transport, R is the radius of the intestinal seg-
ment (set to 0.1 cm), and L is the length of the perfused intestinal
segment.
2.11. HPLC analysis
The stability samples, cell permeated amounts of prodrugs and
their metabolites, and perfusion samples were determined on an
Agilent HPLC system (Agilent Technologies, Santa Clara, CA). The
HPLC system consisted of Agilent pumps (1100 series), an Agilent
autosampler (1200 series), and an Agilent UV–Vis detector (1100
series) controlled by ChemstationÒ 32 software (version B.01.03).
Samples were resolved in an Agilent Eclipse Plus C18 reverse-
Fig. 1. Amino acid monoester prodrugs of gemcitabine.
have been described in the previous report [5]. The structures
and analytical data of those prodrugs are shown in Fig. 1 and
Table 1.
phase column (3.5
lm, 4.6 ꢀ 75 mm) equipped with a guard
column. The mobile phase consisted of 0.1% TFA/water (Solvent
A) and 0.1% TFA/acetonitrile (Solvent B) with the solvent B gradient
changing from 0% to 56% at a rate of 2%/min during a 15 min run
for gemcitabine and gemcitabine prodrugs. Standard curves gener-
ated for each prodrug and their parent drug were utilized for quan-
titation of integrated area under peaks. The detection wavelength
was 254 nm and spectra were acquired in the 220–380 nm wave-
length range. The detection wavelength for phenol red was
430 nm.
3.2. The stability of gemcitabine and gemcitabine prodrugs in SGF, SIF
(pH 6.5), phosphate buffer (pH 7.4), Caco-2 and AsPC-1 cell
homogenates, human liver microsomes and thymidine phosphorylase
enzyme
The experiments concerning prodrug stability were performed
at 37 °C in pH 7.4 phosphate buffer. The estimated half-lives (t1/2
)
obtained from linear regression of pseudo-first-order plots of pro-
drug concentration vs. time for gemcitabine prodrugs in SGF, SIF
(pH 6.5), pH 7.4 phosphate buffers alone, human liver microsomes,
thymidine phosphorylase enzyme and in Caco-2 and AsPC-1 cell
homogenates are listed in Table 2. Prodrug metabolites such as
gemcitabine, 20-deoxy-20,20-difluorouridine, uracil, and cytosine
were monitored along with prodrug disappearance in this experi-
ment. However, the mass balance could not be established because
cytosine and uracil were metabolized even further and those
metabolites could not be quantified by HPLC. All tested prodrugs
and their parent drug did not show the significant degradation in
SGF and SIF and exhibited more than 2 h of half-lives in pH 7.5
2.12. LC–MS analysis
The LC–MS analytical method of 50-mono-amino acid ester pro-
drugs of floxuridine has been reported previously [41]. The LC–MS
analysis of amino acid monoester prodrugs of gemcitabine was
modified and performed in a similar manner. Briefly, LC–MS anal-
ysis of the uptake drug amount was performed in triplicate on
LCMS-2010EV (Shimadzu Scientific Instruments, Kyoto, Japan)
equipped with an ESI (electrospray ionization) source. The Shima-
dzu LC–MS system consisting of Shimadzu LC-20AD pumps with
DGU-20A in-line vacuum degasser units, and SIL-20A HT autosam-
phosphate buffer. 50-
D-phenylalanyl-gemcitabine exhibited the
pler with a Hypercarb column, 2.1 ꢀ 100 mm, 5
lm particle size
highest stability for prodrugs and gemcitabine exhibited no signif-
icant degradation in SGF, SIF, and pH 7.4 phosphate buffer. All pro-
drugs and gemcitabine exhibited 1.1–60.0-fold shorter half-lives in
cell homogenates than in pH 7.4 phosphate buffer suggesting
enzyme-catalyzed hydrolysis. The composition of the amino acids
(Thermo Scientific) was used for the separation and the effluent
from the column was introduced directly to the ionization source.
The system was controlled by Shimadzu LCMS solution software
(version 3) to collect and process data. All samples were run with
Solvent A and Solvent B (described earlier) with Solvent B gradient
changing from 0% to 90% at a rate of 13.8%/min over a 22 min run.
The ESI probe was operated with a detector voltage of 1.5 kV, CDL
temperature of 250 °C, heat block of 200 °C, and nebulizing gas
flow of 1.2 mL/min in positive mode for gemcitabine prodrugs
and their metabolites. The drying gas was N2 delivered at 0.1 MPa.
(
L
- and
ity of the ester bond. The stability of 50-
drug was 2.2- and 6.3-fold better enzymatically in cell
homogenates than one of 50-
-valyl-gemcitabine prodrug, while
the stability of 50-
-phenylalanyl-gemcitabine prodrug was also
9.4- and 10.9-fold better enzymatically in cell homogenates than
one of 50-
-phenylalanyl-gemcitabine prodrug. In human liver
microsomes, 50-
-phenylalanyl-gemcitabine prodrug exhibited
3.7-fold better stability than one of 50-
-phenylalanyl-gemcitabine
D
-) in the promoiety exerted a profound effect on the stabil-
D
-valyl-gemcitabine pro-
L
D
L
2.13. Statistical analysis
D
L
Statistical analysis was performed by Student’s t-test for two
groups. All results were expressed as the mean standard
deviation (SD). A probability (p) of less than 0.05 is considered sta-
tistically significant.
prodrug, while valyl-gemcitabine prodrugs did not exhibit much
difference. All prodrugs exhibited the resistance against the thymi-
dine phosphorylase, while their parent drug, gemcitabine, did not
display the resistance to this enzyme (Table 2).
3. Results
3.3. Uptake study of gemcitabine and gemcitabine prodrugs in Caco-2
and AsPC-1 cells
3.1. Gemcitabine prodrugs
The uptake of mono-amino acid monoester prodrugs of gemcit-
abine and parent gemcitabine was determined with both the
presence and the absence of 10 mM Gly-Pro at 37 °C in Caco-2
The synthesis of gemcitabine prodrugs, the evaluation of pro-
drug purity by HPLC, NMR condition, and their characterization
Please cite this article in press as: Y. Tsume et al., The development of orally administrable gemcitabine prodrugs with
D-enantiomer amino acids: En-
hanced membrane permeability and enzymatic stability, Eur. J. Pharm. Biopharm. (2014), http://dx.doi.org/10.1016/j.ejpb.2013.12.009

Y. Tsume et al. / European Journal of Pharmaceutics and Biopharmaceutics xxx (2014) xxx–xxx
5
Table 1
Analytical data for amino acid ester prodrugs of gemcitabine.
Prodrug
Purity (%) (HPLC)
ESI-MS (M+H)+
Required
CLogPa
Observed
Gemcitabine
95.3
98.2
97.4
98.1
98.6
263.2
411.4
411.4
363.3
363.3
263.9
411.8
411.8
364.0
364.0
ꢁ0.71
ꢁ0.36
ꢁ0.36
ꢁ0.16
ꢁ0.16
50-
50-
50-
50-
L
-Phenylalanyl-gemcitabine (50-
L
-Phe-Gem)
-Phe-Gem)
-Val-Gem)
-Valyl-gemcitabine (50-
D-Phe-Gem)
D
-Phenylalanyl-gemcitabine (50-
-Valyl-gemcitabine (50-
D
L
L
D
a
Calculated using BioLoom.
Table 2
Stability of gemcitabine and gemcitabine prodrugs in pH 7.4 Buffer and biological media (mean SD, n = 3).
Prodrug
SGF t1/2
(min)
SIF (pH 6.5)
t1/2 (min)
Buffer pH 7.4
t1/2 (min)
Caco-2 cell
homogenates
t1/2 (min)
Human liver
microsome
t1/2 (min)
Thymidine
phosphorylase
t1/2 (min)
AsPC-1 cell
homogenates
t1/2 (min)
Gemcitabine
>120
>120
>120
>120
>120
>120
>120
>120
>120
>120
>120
>120
>120
>120
>120
105.0 6.1
2.0 0.3
21.7 0.2
19.5 1.9
42.0 17.5
45.3 3.3
1.8 0.2
6.6 1.3
27.8 13.6
19.5 4.4
6.0 1.8
31.9 5.0
>120
>120
>120
33.7 14.5
6.8 2.0
63.9 11.2
13.8 3.3
87.2 13.5
50-
50-
50-
50-
L
-Phenylalanyl-gemcitabine
-Phenylalanyl-gemcitabine
-Valyl-gemcitabine
D
L
D
-Valyl-gemcitabine
w/o Gly-Pro
w/ Gly-Pro
*
**
8
250
200
150
100
50
(A)
**
*
*
6
4
2
0
*
*
**
*
0
Fig. 2. The amount of gemcitabine and gemcitabine prodrugs found in Caco-2 cells
with 1 h incubation. Each column represents total amount of gemcitabine prodrug,
8
6
4
2
0
(B)
gemcitabine, and cytosine. Data are expressed as the amount,
lM/mg of protein,
mean SD, n = 3. ꢂp < 0.05, the uptake amount of gemcitabine prodrug is compared
with gemcitabine’s. §p < 0.05, the uptake amount of gemcitabine prodrug is
compared with one of the same prodrug with the presence of Gly-Pro.
*
Table 3
Composition of gemcitabine and gemcitabine prodrugs in Caco-2 cell uptake studies
without Gly-Pro (mean SD, n = 3).
Compound
Cytosine
(%)
Gemcitabine
(%)
Prodrug
(%)
Gemcitabine
50
0
50
18
50-
L-Phenylalanyl-
82
69
gemcitabine
50-
D-Phenylalanyl-
0
31
gemcitabine
50-
50-
L-Valyl-gemcitabine
0
0
15
10
85
90
D-Valyl-gemcitabine
Fig. 3. The amount of gemcitabine and gemcitabine prodrugs found in AsPC-1 with
1 h incubation; (A) 100 M drug solution and (B) 50 M drug solution. Each column
represents the total amount of gemcitabine prodrug, gemcitabine, and cytosine.
Data are expressed as the amount,
M/mg of protein, mean SD, n = 3. ꢂp < 0.05, the
l
l
l
uptake amount of gemcitabine prodrug is compared with gemcitabine’s.
and AsPC-1 cells. Fig. 2 and Table 3 show the uptake amounts with
observed compound species and the uptake difference with both
the presence and the absence of 10 mM Gly-Pro in Caco-2 cell sys-
tem. Fig. 3 shows the uptake amounts with two different drug con-
centrations and Table 4 shows observed compound species in
AsPC-1, a ductal pancreatic cancer cell line, cell system. All ester
prodrugs of gemcitabine exhibited 1.2–18.1-fold higher uptake
amount than gemcitabine in Caco-2 and AsPC-1 cells except the
uptake study with the lower concentration of 50-
D
-phenylalanyl-
gemcitabine prodrug in AsPC-1 cells, which did not show the
Please cite this article in press as: Y. Tsume et al., The development of orally administrable gemcitabine prodrugs with
D-enantiomer amino acids: En-
hanced membrane permeability and enzymatic stability, Eur. J. Pharm. Biopharm. (2014), http://dx.doi.org/10.1016/j.ejpb.2013.12.009

6
Y. Tsume et al. / European Journal of Pharmaceutics and Biopharmaceutics xxx (2014) xxx–xxx
Table 4
Table 5
Composition of gemcitabine and gemcitabine prodrugs (100
uptake studies (mean SD, n = 3).
l
M) in AsPC-1 cell
Apparent (Papp) and effective (Peff) permeability coefficients of gemcitabine and its
amino acid ester prodrugs in the apical-to-basolateral direction across Caco-2
monolayer and in situ perfusion study in mouse (mean SD, n = 3).
Compound
Cytosine
(%)
Gemcitabine
(%)
Prodrug
(%)
Prodrug/drug
Papp, Caco-2
Peff, mouse perfusion
(ꢀ10ꢁ5 cm/s)
(ꢀ10ꢁ6 cm/s)
Gemcitabine
10
10
90
84
50-
L-Phenylalanyl-
6
Gemcitabine
1.0 0.2
3.8 0.9ꢂ
4.5 0.9ꢂ
4.0 0.1ꢂ
3.9 0.1ꢂ
2.0 0.2
33.8 3.4ꢂ
21.6 4.1ꢂ
35.8 9.2ꢂ
19.1 6.1ꢂ
gemcitabine
50-
50-
50-
50-
L
-Phenylalanyl-gemcitabine
-Phenylalanyl-gemcitabine
-Valyl-gemcitabine
D-Valyl-gemcitabine
50-
D-Phenylalanyl-
1
87
12
D
gemcitabine
L
50-
50-
L-Valyl-gemcitabine
20
4
11
77
69
19
D-Valyl-gemcitabine
ꢂ
p < 0.05, Compared with gemcitabine.
enhancement of uptake to gemcitabine. The uptake amounts of 50-
-valyl-gemcitabine prodrug and 50-
-phenylalanyl-gemcitabine
prodrug were 3.1-fold and 3.3-fold higher in AsPC-1 cells than ones
of 50- -valyl-gemcitabine prodrug and 50-
-phenylalanyl-gemcita-
bine prodrug with 100 M drug solution (Fig. 3 A). The uptake
studies in AsPC-1 cells with lower drug solution (50 M) exhibited
3.8–4.5-fold higher permeability than gemcitabine in Caco-2 cells.
Gemcitabine did not exhibit the difference in the membrane per-
meability with the presence and the absence of 10 mM Gly-Pro.
The permeability of gemcitabine prodrugs across Caco-2 monolay-
ers was 1.6–6.5-fold lower with the presence of 10 mM Gly-Pro
than one without 10 mM Gly-Pro, suggesting that the improved
permeability of gemcitabine prodrugs is attributed to transporters
such as a PEPT1 transporter at Caco-2 monolayers.
D
D
L
L
l
l
the same trend in valyl-gemcitabine prodrugs but not in phenylal-
anyl-gemcitabine prodrug (Fig. 3B). The dose linearity of gemcita-
bine prodrug uptake in AsPC-1 cells was not clear (R2 = 0.15) even
though 50-
D
-valyl-gemcitabine prodrug exhibited the dose linearity
in uptake studies (Fig. 4). Gemcitabine and gemcitabine prodrugs
with -configuration amino acids exhibited the similar uptake
amounts regardless of starting drug concentration for the study.
The uptake amounts of 50- -valyl-gemcitabine prodrug and 50-
phenylalanyl-gemcitabine prodrug were 46% and 70% lower in
Caco-2 cells than ones of 50- -valyl-gemcitabine prodrug and 50-
3.5. In situ permeability in the single-pass intestinal perfusion study
and the drug concentration in plasma in mice
L
The effective permeability (Peff) values obtained for gemcitabine
prodrugs and gemcitabine in small intestinal segments in mice at
physiological pH are presented in Table 5. The in situ permeability
of parent drug, gemcitabine, exhibited 2.0 ꢀ 10ꢁ6 cm/s in the
mouse jejunum. On the other hand, the in situ permeability of
D
D-
L
L-
phenylalanyl-gemcitabine prodrug. Gemcitabine prodrugs exhib-
ited 5.4–18.1-fold higher uptake amounts in Caco-2 cells than
gemcitabine. Gemcitabine did not exhibit the difference in the up-
take amount in Caco-2 cells with the presence and the absence of
10 mM Gly-Pro. The uptake amounts of gemcitabine prodrugs in
Caco-2 cells were reduced 1.2–10.6-fold with the presence of
10 mM Gly-Pro compared with ones with the absence 10 mM
Gly-Pro, suggesting that the improved cellular uptake of gemcita-
bine prodrugs attributes to transporters at Caco-2 cells.
(
D
-/
9.6–17.9-fold higher membrane permeability than their parent
drug, gemcitabine. Gemcitabine prodrugs with -amino acid exhib-
ited superior membrane permeability to gemcitabine prodrugs
with -phen-
-amino acid. 50- -valyl-gemcitabine prodrug and 50-
ylalanyl-gemcitabine prodrug exhibited 87% and 56% better
membrane permeability than 50-
-valyl-gemcitabine prodrug and
50-
-phenylalanyl-gemcitabine prodrug, respectively. In plasma
L-) amino acid monoester prodrugs of gemcitabine exhibited
L
D
L
L
D
D
samples following in situ perfusion, all prodrugs exhibited 1.3–
9.8-fold higher concentration of prodrugs including their
metabolites, gemcitabine and cytosine, than their parent drug,
3.4. Caco-2 cell permeability of gemcitabine and gemcitabine prodrugs
gemcitabine. 50-
D
-valyl-gemcitabine prodrug and 50-
D-phenylala-
The apical-to-basolateral permeability of mono-amino acid
monoester prodrugs of gemcitabine and parent gemcitabine was
determined with the presence and the absence of 10 mM Gly-Pro
at 37 °C in Caco-2 cell monolayers. Table 5 and Fig. 5 show the
permeability values and the permeability difference in this
cell system. All monoester prodrugs of gemcitabine exhibited
nyl-gemcitabine prodrug exhibited 2.3-fold and 2.0-fold higher
w/o Gly-Pro
w/ Gly-Pro
*
6
5
4
3
2
1
0
**
**
*
*
*
7.5
R2 = 0.15
5.0
2.5
0.0
0
50
100
Fig. 5. Apparent permeability coefficients (Papp) of gemcitabine and its amino acid
ester prodrugs in the apical-to-basolateral direction across Caco-2 monolayer with
the presence and the absence of Gly-Pro (mean SD, n = 3). ꢂp < 0.05, the apparent
permeability coefficient of gemcitabine prodrugs is compared with gemcitabine.
ꢂꢂp < 0.05, the apparent permeability coefficient of gemcitabine and gemcitabine
prodrugs is compared with one of the same drug with the presence of Gly-Pro.
Drug Starting Concentration (µM)
Fig. 4. The correlation of uptake amount for gemcitabine and gemcitabine prodrugs
in AsPC-1 cells between the starting concentration of 50 M and the starting
concentration of 100 M. Values are the mean SD, n = 3.
l
l
Please cite this article in press as: Y. Tsume et al., The development of orally administrable gemcitabine prodrugs with
D-enantiomer amino acids: En-
hanced membrane permeability and enzymatic stability, Eur. J. Pharm. Biopharm. (2014), http://dx.doi.org/10.1016/j.ejpb.2013.12.009

Y. Tsume et al. / European Journal of Pharmaceutics and Biopharmaceutics xxx (2014) xxx–xxx
7
concentration in the plasma than 50-
and 50-
-phenylalanyl-gemcitabine prodrug, even though the per-
meability numbers of gemcitabine prodrugs with -form amino
acids were lower than ones with -form amino acid (Fig. 6).
Furthermore, 50- -valyl-gemcitabine prodrug and 50-
-phenylala-
nyl-gemcitabine prodrug appeared more prodrug forms in the
plasma than 50- -valyl-gemcitabine prodrug and 50-
-phenylala-
nyl-gemcitabine prodrug, suggesting that the -amino acid mono-
L-valyl-gemcitabine prodrug
Table 6
Cell growth inhibition in AsPC-1 cells (mean SD, n = 3–6).
L
D
Prodrug/drug
Gemcitabine
GI50 AsPC-1 (mM)
L
10.2 1.6
4.3 1.5ꢂ
5.6 1.1ꢂ
2.0 0.8ꢂ
2.2 0.9ꢂ
50-
50-
50-
50-
L
-Phenylalanyl-gemcitabine
-Phenylalanyl-gemcitabine
-Valyl-gemcitabine
-Valyl-gemcitabine
D
D
D
L
L
L
D
D
ꢂ
p < 0.05, Compared with gemcitabine.
ester prodrugs of gemcitabine improved their stability toward
metabolic enzymes.
revealed that mono-amino acid/dipeptide ester prodrugs in gen-
eral enhance the PEPT1-mediated transport and glycosidic bond
resistance to metabolic enzymes such as thymidine phosphorylase
and cytidine deaminase. In this report, we describe the chemical
and enzymatic stability and bioactivation of mono-amino acid
monoester prodrugs of gemcitabine in buffer, Caco-2 cells, a surro-
gate for intestine, AsPC-1 cells, a surrogate for tumor, liver micro-
somes and the enzyme, thymidine phosphorylase. We conducted
uptake studies in Caco-2 and AsPC-1 cells to evaluate the transport.
We also performed transepithelial studies with Caco-2 monolayers
and in situ perfusion studies in mice to calculate the membrane
permeability along with the drug concentration in plasma for the
evaluation of oral drug delivery for pancreatic cancer treatment.
The mono-amino acid prodrugs of gemcitabine appeared to be
relatively stable in pH 7.4 buffers and exhibited more stable in
acidic pH, SGF and SIF (pH 6.5), which agreed with our previous re-
3.6. Cell proliferation assay
GI50 values for gemcitabine and gemcitabine prodrugs, 50-amino
acid monoester prodrugs of gemcitabine, determined in cell prolif-
eration studies with the pancreatic ductal cell line, AsPC-1, are
shown in Table 6. All prodrugs exhibited 1.8–5.1-fold enhanced
antiproliferative activity in the cancer cell line compared to their
parent, gemcitabine. Valyl-gemcitabine (
2.8-fold better growth inhibitory effects than phenylalanyl-gem-
citabine ( - and -). Thus, the GI50 values of all gemcitabine
L- and D-) exhibited 2.0–
L
D
prodrugs were in the range of 2.0–5.6 mM in AsPC-1 cell as
opposed to GI50 value of 10.2 mM for gemcitabine in AsPC-1 cell.
D
-Form amino acid attached gemcitabine prodrugs exhibited a
slightly inferior antiproliferative activity to
attached gemcitabine.
L-form amino acid
sults [30]. The enzymatic stability of 50-
bine and 50-
-valyl-gemcitabine was significantly enhanced
compared to ones of 50- -phenylalanyl-gemcitabine and 50-
-va-
D-phenylalanyl-gemcita-
4. Discussion
D
L
L
Amino acid ester prodrugs have been widely employed to im-
prove intestinal absorption of poorly permeable drugs. The anti-
virals valacyclovir and valganciclovir are early examples for the
success of amino acid ester prodrug strategies [21,42]. The im-
proved oral bioavailability of these anti-virals has been attributed
to their enhanced affinity to transporters [3,4,43–45]. A variety of
amino acid, dipeptide and tripeptide have been investigated to
improve the oral absorption via intake transporters as a part of
prodrug approaches [16,17,22–24,46–49]. We have synthesized
mono-amino acid and dipeptide prodrugs of floxuridine, gemcita-
bine and 2-bromo-5,6- dichloro-1-(beta-d-ribofuranosyl) benz-
imidazole (BDCRM) and characterized their stability and
membrane permeability [5,6,9–11,13,14,30,50]. These studies
lyl-gemcitabine suggesting that the recognition of enzymes to their
substrates is specific and, hence, the unnatural form of amino acids
such as
lyzed hydrolysis of the ester linkage. The stability profiles of 50-
phenylalanyl-gemcitabine and 50-
-valyl-gemcitabine in cell
homogenates, Caco-2 and AsPC-1, liver microsomes, and thymi-
dine phosphorylase suggest that activation of -amino acid mono-
ester prodrug to the parent drug following transport would be
much slower than activation of -amino acid monoester prodrugs,
50- -valyl-gemcitabine and 50-
-phenylalanyl-gemcitabine. The
D-valine and D-phenylalanine decelerates enzyme-cata-
D-
D
D
L
L
L
improved stability in biological surrogate media would facilitate
prolonged systemic circulation of intact prodrugs and, hence, tar-
get delivery to the cancer sites for enhanced therapeutic action.
The plasma concentration of
D-amino acid monoester prodrug
1200
1000
800
600
400
200
0
forms of gemcitabine after their in situ perfusion in mice was high-
prodrug
gemcitabine
cytosine
*
er than one of
bine (Fig. 6).
L-amino acid monoester prodrug forms of gemcita-
The results of the uptake studies of the amino acid monoester
prodrugs in Caco-2 and AsPC-1 are shown in Figs. 2 and 3 and
Tables 3 and 4. The uptake amounts of gemcitabine prodrugs were
consistently lower in AsPC-1 cells compared to their corresponding
values in Caco-2 cells. Gemcitabine was degraded almost three
times faster in AsPC-1 cells than in Caco-2 cells. Those indicate that
prodrug stability and enzyme upregulation in AsPC-1 cells would
be largely attributed to the observed uptake amount for gemcita-
bine prodrugs and their metabolites. The noticeable difference be-
tween those results was the trend of uptake amount. Gemcitabine
**
prodrugs with
Caco-2 cells than ones with
citabine prodrugs with -amino acid exhibited higher uptake
amount in AsPC-1 cells than ones with -amino acid. Those uptake
results exhibited different pattern of drug species, gemcitabine
prodrug, gemcitabine, and cytosine, in those two cell lines.
The uptake of gemcitabine prodrugs in Caco-2 uptake exhibited
69–90% of prodrug form, 10–31% of gemcitabine and 0% of cyto-
sine, while the uptake of gemcitabine prodrugs in AsPC-1 exhibited
L
-amino acid exhibited higher uptake amount in
D-amino acid. On the other hand, gem-
D
Fig. 6. The concentration of gemcitabine and gemcitabine prodrugs found in
plasma at 2 h following in situ single-pass intestinal perfusion to the mouse
jejunum. Each column represents gemcitabine prodrug, gemcitabine, and cytosine.
Data are expressed as the concentration, ng/mL, mean SD, n = 3. Error bars are
L
shown for the total concentration. ꢂp < 0.05, the drug concentration of 50-
D
-Val-Gem
-enantio-
D-enantiomers of amino acid
ꢂꢂ
is compared with gemcitabine. p < 0.05, the drug concentration of 50-
L
mers of amino acid prodrug is compared with one of
prodrug.
Please cite this article in press as: Y. Tsume et al., The development of orally administrable gemcitabine prodrugs with
D-enantiomer amino acids: En-
hanced membrane permeability and enzymatic stability, Eur. J. Pharm. Biopharm. (2014), http://dx.doi.org/10.1016/j.ejpb.2013.12.009

8
Y. Tsume et al. / European Journal of Pharmaceutics and Biopharmaceutics xxx (2014) xxx–xxx
6–69% of prodrug form, 11–87% of gemcitabine and 1–20% of
cytosine. Those results suggest that the expression level of metab-
olizing enzymes for gemcitabine prodrugs and gemcitabine in
those cells was different, the substrate recognition of enzymes
was specific depending on the configuration amino acid, and, as a
result, the bioactivation rate of those prodrugs was different. Thus,
250
200
150
100
50
R2 = 0.87
attachment of the
D-configuration amino acid to gemcitabine
yielded 2–10-fold improved stability for enzymatic degradation
even though it lowered the affinity to transporters. The intake
transporter, PEPT1, has a broad substrate specificity with a low-
0
1
2
3
4
5
-50
affinity (Km of 200
lM to 15 mM) and high capacity, and recog-
in situmouse permeability
(x 10-5 cm/s)
nizes -configuration amino acid promoiety as
D
a
substrate
[27,51]. The interactions between transporters and prodrugs and
between activation enzymes and prodrugs are different as each
prodrug possesses unique binding affinity and rates to those trans-
porters and enzymes [52]. With the complexity of those processes,
the meaningful dose linearity (R2 = 0.15) in uptake studies for gem-
citabine and gemcitabine prodrugs in AsPC-1 cells was not
observed even though those studies were carried out below their
Km values (Fig. 4).
Fig. 7. The correlation between the amount of gemcitabine and gemcitabine
prodrugs in Caco-2 cells and the membrane permeabilities of gemcitabine and
gemcitabine prodrugs in mouse jejunum. Values are the mean SD, n = 3.
drugs of gemcitabine exhibited better GI50 values compared to one
of gemcitabine. The GI50 values of prodrugs did not exhibit any dis-
cernible correlations with their uptake values in AsPC-1 cells. Since
the bioconversion rate of prodrugs will be various according to
The apical-to-basolateral permeability of mono-amino acid
monoester prodrugs of gemcitabine and their parent drug, gemcit-
abine, were determined at 37 °C in Caco-2 cell monolayer with
both the presence and the absence of Gly-Pro (Fig. 5). The prodrug
permeability across Caco-2 cell monolayer was reduced with the
presence of Gly-Pro, while the permeability of gemcitabine did
not change with the presence and the absence of Gly-Pro. Thus,
the improved permeability of the mono-amino acid monoester
prodrugs across Caco-2 cell monolayer indicates enhanced trans-
porter-mediated transport of gemcitabine prodrugs. The detection
of only cytosine in the basolateral receiver compartment following
transport of gemcitabine across Caco-2 monolayers suggests the
instability of the glycosidic bond. The conversion of prodrugs to
cytosine following transport was substantially lower in Caco-2
their different (D-/L-) amino acid promoieties, the time after trans-
port into cancer cells to reach their maximum antiproliferative
activity will be different [52]. Therefore, it would be difficult to dis-
tinguish a significant correlation between GI50 values and prodrug
permeabilities with a limited experimental time course.
5. Conclusion
Intracellular anabolism of gemcitabine prodrugs may illustrate
that transported drugs are converted to gemcitabine and cytosine
via a sequential enzymatic pathway including the high expression
of thymidine phosphorylase in tumor cells (Tables 3 and 4). The
cells, especially for 50-
D
-valyl-gemcitabine and 50-
D
-phenylalanyl-
gemcitabine prodrugs with D-configuration amino acids demon-
gemcitabine. The average percent cytosine in the basolateral
compartment was observed 76% (the range 61–92%) in Caco-2
monolayer studies (data not shown). In general, conversion of
strated the superior stability against metabolic enzymes. As a re-
sult, those prodrugs would exhibit higher concentration of cancer
drugs in systemic circulation after in situ perfusion of mouse small
intestine even though those prodrugs have inferior affinity for
gemcitabine prodrugs with
transport across the monolayers was about 1.4-fold lower than
that observed with -amino acid ester prodrugs of gemcitabine.
D-amino acid to cytosine following
transporters compared to gemcitabine prodrugs with
tion amino acids. Even though those gemcitabine prodrugs with
D-configuration amino acids showed some potential to develop
L-configura-
L
The results are consistent with stability profiles of gemcitabine
prodrugs in the presence of thymidine phosphorylase, an enzyme
involved in the in vivo for phosphorytic cleavage of gemcitabine
[53]. Peff values of gemcitabine prodrugs were 9- and 18-fold high-
er than one of gemcitabine (Table 5). The effective permeability
(Peff) values of the gemcitabine prodrugs as well as parent gemcit-
abine in mouse jejunal intestine are consistent with the trends ob-
served in uptake studies in Caco-2 cells and the excellent
correlation between those two was observed (R2 = 0.87) (Fig. 7).
In the previous studies with mono-amino acid prodrugs of floxur-
idine, our group has demonstrated the excellent linear correlations
between Caco-2 permeability and PEPT1-mediated transport in
HeLa/PEPT1 cells [54].
Gemcitabine was rapidly cleaved by thymidine phosphorylase
but all amino acid ester prodrugs examined in this study were
more stable toward thymidine phosphorylase (Table 2). The ester-
ification of 50-hydroxyl groups would protect the glycosidic bond
cleavage by thymidine phosphorylase, which has been discussed
in previous studies [9,13].
the oral dosage forms for cancer, oral bioavailability of those pro-
drugs has to be determined by another set of experiment to evalu-
ate their potentials. Our results indicate that (D-/L-) amino acid
monoester prodrugs of gemcitabine exhibit significantly higher
permeability in Caco-2 cell monolayer and mouse intestinal mem-
brane than their parent drug, gemcitabine, suggesting their poten-
tial for the development of oral dosage form for anti-cancer
nucleoside analogs. Especially, gemcitabine prodrugs with D-con-
figuration amino acids exhibited higher prodrug concentration in
systemic circulation after in situ perfusion of mouse small intes-
tine. Therefore, prodrugs with
possess advantages over prodrugs with
acids for the development of oral dosage form. Gemcitabine pro-
drug, 50-
-valyl-gemcitabine, displayed significantly higher perme-
D
-configuration amino acids might
L-configuration amino
D
ability in Caco-2 cell monolayer and mouse intestinal membrane
and highest prodrug concentration in blood with improved
antiproliferative activity against pancreatic ductal cancer cells,
AsPC-1. The enhanced metabolic resistance, the higher prodrug
concentration in plasma after oral absorption, and the improved
anti-cancer effect may facilitate the development of oral dosage
form for pyrimidine analogues such as gemcitabine and floxuridine
to treat cancer. The successful development of oral dosage form for
cancer therapeutic agents will vastly improve the quality of life for
cancer patients due to its convenient chemotherapy.
The cell proliferation studies in the pancreatic ductal cancer cell
line confirmed the enhanced potency of the amino acid ester pro-
drugs compared to their parent gemcitabine. The results of uptake
study in AsPC-1 cell suggested that the enhanced antiproliferative
effect on cancer cells attributed the improved membrane perme-
ability of gemcitabine prodrugs. Mono-amino acid monoester pro-
Please cite this article in press as: Y. Tsume et al., The development of orally administrable gemcitabine prodrugs with
D-enantiomer amino acids: En-
hanced membrane permeability and enzymatic stability, Eur. J. Pharm. Biopharm. (2014), http://dx.doi.org/10.1016/j.ejpb.2013.12.009

Y. Tsume et al. / European Journal of Pharmaceutics and Biopharmaceutics xxx (2014) xxx–xxx
9
[24] C.U. Nielsen, R. Andersen, B. Brodin, S. Frokjaer, M.E. Taub, B. Steffansen,
Dipeptide model prodrugs for the intestinal oligopeptide transporter. Affinity
for and transport via hPepT1 in the human intestinal Caco-2 cell line, J.
Control. Release 76 (2001) 129–138.
Acknowledgment
This work was supported by Grants NIGMD-2R01GM037188.
[25] M. Satake, M. Enjoh, Y. Nakamura, T. Takano, Y. Kawamura, S. Arai, M. Shimizu,
Transepithelial transport of the bioactive tripeptide, Val-Pro-Pro, in human
intestinal Caco-2 cell monolayers, Biosci. Biotechnol. Biochem. 66 (2002) 378–
384.
[26] N. Surendran, K.M. Covitz, H. Han, W. Sadee, D.M. Oh, G.L. Amidon, R.M.
Williamson, C.F. Bigge, B.H. Stewart, Evidence for overlapping substrate
specificity between large neutral amino acid (LNAA) and dipeptide (hPEPT1)
transporters for PD 158473, an NMDA antagonist, Pharm. Res. 16 (1999) 391–
395.
[27] U. Wenzel, I. Gebert, H. Weintraut, W.M. Weber, W. Clauss, H. Daniel,
Transport characteristics of differently charged cephalosporin antibiotics in
oocytes expressing the cloned intestinal peptide transporter PepT1 and in
human intestinal Caco-2 cells, J. Pharmacol. Exp. Therap. 277 (1996) 831–839.
[28] U. Wenzel, D.T. Thwaites, H. Daniel, Stereoselective uptake of beta-lactam
antibiotics by the intestinal peptide transporter, Br. J. Pharmacol. 116 (1995)
3021–3027.
[29] I. Rubio-Aliaga, H. Daniel, Mammalian peptide transporters as targets for drug
delivery, Trends Pharmacol. Sci. 23 (2002) 434–440.
[30] Y. Tsume, J.M. Hilfinger, G.L. Amidon, Potential of amino acid/dipeptide
monoester prodrugs of floxuridine in facilitating enhanced delivery of active
drug to interior sites of tumors: a two-tier monolayer in vitro study, Pharm.
Res. 28 (2011) 2575–2588.
[31] D.E. Gonzalez, K.M. Covitz, W. Sadee, R.J. Mrsny, An oligopeptide transporter is
expressed at high levels in the pancreatic carcinoma cell lines AsPc-1 and
Capan-2, Cancer Res. 58 (1998) 519–525.
[32] J. Garcia-Manteiga, M. Molina-Arcas, F.J. Casado, A. Mazo, M. Pastor-Anglada,
Nucleoside transporter profiles in human pancreatic cancer cells: role of
hCNT1 in 20,20-difluorodeoxycytidine- induced cytotoxicity, Clin. Cancer Res.:
An Off. J. Am. Assoc. Cancer Res. 9 (2003) 5000–5008.
[33] J.L. Grem, 5-Fluorouracil: forty-plus and still ticking. A review of its preclinical
and clinical development, Invest. New Drugs 18 (2000) 299–313.
[34] O. Kahramanogullari, G. Fantaccini, P. Lecca, D. Morpurgo, C. Priami,
Algorithmic modeling quantifies the complementary contribution of
metabolic inhibitions to gemcitabine efficacy, PLoS ONE 7 (2012) e50176.
[35] S.A. Veltkamp, D. Pluim, M.A. van Eijndhoven, M.J. Bolijn, F.H. Ong, R.
Govindarajan, J.D. Unadkat, J.H. Beijnen, J.H. Schellens, New insights into the
pharmacology and cytotoxicity of gemcitabine and 20,20-difluorodeoxyuridine,
Mol. Cancer Ther. 7 (2008) 2415–2425.
[36] V.J. O’Neill, C.J. Twelves, Oral cancer treatment: developments in
chemotherapy and beyond, Br. J. Cancer 87 (2002) 933–937.
[37] F.E. Stuurman, B. Nuijen, J.H. Beijnen, J.H. Schellens, Oral anticancer drugs:
mechanisms of low bioavailability and strategies for improvement, Clin.
Pharmacokinet. 52 (2013) 399–414.
[38] G.D. Birnie, H. Kroeger, C. Heidelberger, Studies of fluorinated pyrimidines.
Xviii. The degradation of 5-fluoro-20-deoxyuridine and related compounds by
nucleoside phosphorylase, Biochemistry 2 (1963) 566–572.
[39] J. Bonastre, P. Jan, Y. Barthe, S. Koscielny, Metastatic breast cancer: we do need
primary cost data, Breast 21 (2012) 384–388.
[40] T. Incecayir, Y. Tsume, G.L. Amidon, Comparison of the permeability of
metoprolol and labetalol in rat, mouse, and Caco-2 cells: use as a reference
standard for BCS classification, Mol. Pharm. (2013).
[41] Y. Tsume, C.J. Provoda, G.L. Amidon, The achievement of mass balance by
simultaneous quantification of floxuridine prodrug, floxuridine, 5-fluorouracil,
References
[1] C.J. van Moorsel, G.J. Peters, H.M. Pinedo, Gemcitabine: future prospects of
single-agent and combination studies, Oncologist 2 (1997) 127–134.
[2] Y. Zhang, W.Y. Kim, L. Huang, Systemic delivery of gemcitabine triphosphate
via LCP nanoparticles for NSCLC and pancreatic cancer therapy, Biomaterials
34 (2013) 3447–3458.
[3] H. Han, R.L. de Vrueh, J.K. Rhie, K.M. Covitz, P.L. Smith, C.P. Lee, D.M. Oh, W.
Sadee, G.L. Amidon, 50-Amino acid esters of antiviral nucleosides, acyclovir,
and AZT are absorbed by the intestinal PEPT1 peptide transporter, Pharm. Res.
15 (1998) 1154–1159.
[4] H.K. Han, D.M. Oh, G.L. Amidon, Cellular uptake mechanism of amino acid ester
prodrugs in Caco-2/hPEPT1 cells overexpressing a human peptide transporter,
Pharm. Res. 15 (1998) 1382–1386.
[5] X. Song, P.L. Lorenzi, C.P. Landowski, B.S. Vig, J.M. Hilfinger, G.L. Amidon, Amino
acid ester prodrugs of the anticancer agent gemcitabine: synthesis,
bioconversion, metabolic bioevasion, and hPEPT1-mediated transport, Mol.
Pharm. 2 (2005) 157–167.
[6] X. Song, B.S. Vig, P.L. Lorenzi, J.C. Drach, L.B. Townsend, G.L. Amidon, Amino
acid ester prodrugs of the antiviral agent 2-bromo-5,6-dichloro-1-(beta-D-
ribofuranosyl)benzimidazole as potential substrates of hPEPT1 transporter, J.
Med. Chem. 48 (2005) 1274–1277.
[7] R.S. Talluri, S.K. Samanta, R. Gaudana, A.K. Mitra, Synthesis, metabolism and
cellular permeability of enzymatically stable dipeptide prodrugs of acyclovir,
Int. J. Pharm. 361 (2008) 118–124.
[8] S. Tolle-Sander, K.A. Lentz, D.Y. Maeda, A. Coop, J.E. Polli, Increased acyclovir
oral bioavailability via a bile acid conjugate, Mol. Pharm. 1 (2004) 40–48.
[9] Y. Tsume, J.M. Hilfinger, G.L. Amidon, Enhanced cancer cell growth inhibition
by dipeptide prodrugs of floxuridine: increased transporter affinity and
metabolic stability, Mol. Pharm. 5 (2008) 717–727.
[10] Y. Tsume, B.S. Vig, J. Sun, C.P. Landowski, J.M. Hilfinger, C. Ramachandran, G.L.
Amidon, Enhanced absorption and growth inhibition with amino acid
monoester prodrugs of floxuridine by targeting hPEPT1 transporters,
Molecules 13 (2008) 1441–1454.
[11] B.S. Vig, P.J. Lorenzi, S. Mittal, C.P. Landowski, H.C. Shin, H.I. Mosberg, J.M.
Hilfinger, G.L. Amidon, Amino acid ester prodrugs of floxuridine: synthesis and
effects of structure, stereochemistry, and site of esterification on the rate of
hydrolysis, Pharm. Res. 20 (2003) 1381–1388.
[12] T. Kawaguchi, M. Saito, Y. Suzuki, N. Nambu, T. Nagai, Specificity of esterases
and structure of prodrug esters. II. Hydrolytic regeneration behavior of 5-
fluoro-20-deoxyuridine (FUdR) from 30,50-diesters of FUdR with rat tissue
homogenates and plasma in relation to their antitumor activity, Chem. Pharm.
Bull. (Tokyo) 33 (1985) 1652–1659.
[13] C.P. Landowski, X. Song, P.L. Lorenzi, J.M. Hilfinger, G.L. Amidon, Floxuridine
amino acid ester prodrugs: enhancing Caco-2 permeability and resistance to
glycosidic bond metabolism, Pharm. Res. 22 (2005) 1510–1518.
[14] C.P. Landowski, B.S. Vig, X. Song, G.L. Amidon, Targeted delivery to PEPT1-
overexpressing cells: acidic, basic, and secondary floxuridine amino acid ester
prodrugs, Mol. Cancer Ther. 4 (2005) 659–667.
[15] Y. Nishizawa, J.E. Casida, 30,50-Diesters of 5-fluoro-20-deoxyuridine: synthesis
and biological activity, Biochem. Pharmacol. 14 (1965) 1605–1619.
[16] B.S. Anand, J. Patel, A.K. Mitra, Interactions of the dipeptide ester prodrugs of
acyclovir with the intestinal oligopeptide transporter: competitive inhibition
of glycylsarcosine transport in human intestinal cell line-Caco-2, J. Pharmacol.
Exp. Therap. 304 (2003) 781–791.
5-dihydrouracil,
alpha-fluoro-beta-ureidopropionate,
alpha-fluoro-beta-
alanine using LC–MS, J. Chromatogr. B: Anal. Technol. Biomed. Life Sci. 879
(2011) 915–920.
[42] B.S. Anand, S. Dey, A.K. Mitra, Current prodrug strategies via membrane
transporters/receptors, Expert Opin. Biol. Ther. 2 (2002) 607–620.
[43] M.E. Ganapathy, W. Huang, H. Wang, V. Ganapathy, F.H. Leibach, Valacyclovir:
a substrate for the intestinal and renal peptide transporters PEPT1 and PEPT2,
Biochem. Biophys. Res. Commun. 246 (1998) 470–475.
[44] H. Steingrimsdottir, A. Gruber, C. Palm, G. Grimfors, M. Kalin, S. Eksborg,
Bioavailability of aciclovir after oral administration of aciclovir and its prodrug
valaciclovir to patients with leukopenia after chemotherapy, Antimicrob.
Agents Chemother. 44 (2000) 207–209.
[45] S. Weller, M.R. Blum, M. Doucette, T. Burnette, D.M. Cederberg, P. de Miranda,
M.L. Smiley, Pharmacokinetics of the acyclovir pro-drug valaciclovir after
escalating single- and multiple-dose administration to normal volunteers,
Clin. Pharmacol. Ther. 54 (1993) 595–605.
[46] A.H. Eriksson, P.L. Elm, M. Begtrup, R. Nielsen, B. Steffansen, B. Brodin, HPEPT1
affinity and translocation of selected Gln-Sar and Glu-Sar dipeptide
derivatives, Mol. Pharm. 2 (2005) 242–249.
[47] J. Li, K. Tamura, C.P. Lee, P.L. Smith, R.T. Borchardt, I.J. Hidalgo, Structure–
affinity relationships of Val-Val and Val-Val-Val stereoisomers with the apical
oligopeptide transporter in human intestinal Caco-2 cells, J. Drug Target. 5
(1998) 317–327.
[48] K. Tamura, P.K. Bhatnagar, J.S. Takata, C.P. Lee, P.L. Smith, R.T. Borchardt,
Metabolism, uptake, and transepithelial transport of the diastereomers of Val-
Val in the human intestinal cell line, Caco-2, Pharm. Res. 13 (1996) 1213–
1218.
[17] G.M. Friedrichsen, W. Chen, M. Begtrup, C.P. Lee, P.L. Smith, R.T. Borchardt,
Synthesis of analogs of L-valacyclovir and determination of their substrate
activity for the oligopeptide transporter in Caco-2 cells, Eur. J. Pharm. Sci. 16
(2002) 1–13.
[18] A. Guo, P. Hu, P.V. Balimane, F.H. Leibach, P.J. Sinko, Interactions of
a
nonpeptidic drug, valacyclovir, with the human intestinal peptide
transporter (hPEPT1) expressed in a mammalian cell line, J. Pharmacol. Exp.
Therap. 289 (1999) 448–454.
[19] T. Hatanaka, M. Haramura, Y.J. Fei, S. Miyauchi, C.C. Bridges, P.S. Ganapathy,
S.B. Smith, V. Ganapathy, M.E. Ganapathy, Transport of amino acid-based
prodrugs by the Na+- and Cl(ꢁ)-coupled amino acid transporter ATB0,+ and
expression of the transporter in tissues amenable for drug delivery, J.
Pharmacol. Exp. Therap. 308 (2004) 1138–1147.
[20] D.D. Phan, P. Chin-Hong, E.T. Lin, P. Anderle, W. Sadee, B.J. Guglielmo, Intra-
and interindividual variabilities of valacyclovir oral bioavailability and effect
of coadministration of an hPEPT1 inhibitor, Antimicrob. Agents Chemother. 47
(2003) 2351–2353.
[21] N.S. Umapathy, V. Ganapathy, M.E. Ganapathy, Transport of amino acid esters
and the amino-acid-based prodrug valganciclovir by the amino acid
transporter ATB(0,+), Pharm. Res. 21 (2004) 1303–1310.
[22] B.S. Anand, S. Katragadda, A.K. Mitra, Pharmacokinetics of novel dipeptide
ester prodrugs of acyclovir after oral administration: intestinal absorption and
liver metabolism, J. Pharmacol. Exp. Therap. 311 (2004) 659–667.
[23] D. Meredith, C.S. Temple, N. Guha, C.J. Sword, C.A. Boyd, I.D. Collier, K.M.
Morgan, P.D. Bailey, Modified amino acids and peptides as substrates for the
intestinal peptide transporter PepT1, Eur. J. Biochem. 267 (2000) 3723–3728.
[49] J. Vabeno, T. Lejon, C.U. Nielsen, B. Steffansen, W. Chen, H. Ouyang, R.T.
Borchardt, K. Luthman, Phe-Gly dipeptidomimetics designed for the di-/
Please cite this article in press as: Y. Tsume et al., The development of orally administrable gemcitabine prodrugs with
D-enantiomer amino acids: En-
hanced membrane permeability and enzymatic stability, Eur. J. Pharm. Biopharm. (2014), http://dx.doi.org/10.1016/j.ejpb.2013.12.009

10
Y. Tsume et al. / European Journal of Pharmaceutics and Biopharmaceutics xxx (2014) xxx–xxx
tripeptide transporters PEPT1 and PEPT2: synthesis and biological
investigations, J. Med. Chem. 47 (2004) 1060–1069.
[50] P.L. Lorenzi, C.P. Landowski, X. Song, K.Z. Borysko, J.M. Breitenbach, J.S. Kim,
J.M. Hilfinger, L.B. Townsend, J.C. Drach, G.L. Amidon, Amino acid ester
[52] Y. Tsume, G.L. Amidon, The feasibility of enzyme targeted activation for amino
acid/dipeptide monoester prodrugs of floxuridine; cathepsin D as a potential
targeted enzyme, Molecules 17 (2012) 3672–3689.
[53] P.W. Woodman, A.M. Sarrif, C. Heidelberger, Specificity of pyrimidine
nucleoside phosphorylases and the phosphorolysis of 5-fluoro-20-
deoxyuridine, Cancer Res. 40 (1980) 507–511.
[54] C.P. Landowski, P.L. Lorenzi, X. Song, G.L. Amidon, Nucleoside ester prodrug
substrate specificity of liver carboxylesterase, J. Pharmacol. Exp. Therap. 316
(2006) 572–580.
prodrugs of 2-bromo-5,6-dichloro-1-(beta-D-ribofuranosyl)benzimidazole
enhance metabolic stability in vitro and in vivo, J. Pharmacol. Exp. Therap.
314 (2005) 883–890.
[51] F. Cao, Y. Gao, Q. Ping, Advances in research of PepT1-targeted prodrug, Asian J.
Pharm. Sci. 7 (2012) 110–122.
Please cite this article in press as: Y. Tsume et al., The development of orally administrable gemcitabine prodrugs with
D-enantiomer amino acids: En-
hanced membrane permeability and enzymatic stability, Eur. J. Pharm. Biopharm. (2014), http://dx.doi.org/10.1016/j.ejpb.2013.12.009