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Introduction
The clinical utility of ara-C, one of the most effective
drugs against the acute myeloid leukemia, acute
lymphoblastic leukemia and other hematological
malignancies[1,2], is severely limited by rapid deamination primarily in the liver,
spleen and gastrointestinal mucosa[3,4]. Consequently,
ara-C has a very short plasma half-life as well as low systemic
exposure, and must be administered in continuous infusion
or on a complex schedule to provide maximum therapeutic
efficacy[3,4]. Therefore, considerable efforts have been
directed at an enhancement of the therapeutic efficacy of
ara-C, and many prodrug strategies have been explored for the oral
delivery of ara-C with varied degrees of
success[5_8]. However, few have led to an approved product and thus, there
continues to be a great need for improved methods to deliver
pharmacologically active ara-C.
Recently, our group synthesized the L-valyl-ara-C by
masking the N4-amino group of the cytosine ring in ara-C
with L-valine in order to enhance the intestinal absorption of
ara-C, and evaluated the cellular uptake characteristics of
L-valyl-ara-C in Caco-2 cells[9]. Compared to ara-C,
L-valyl-ara-C appeared to be stable in the intestinal lumen and
exhibited 5 fold higher cellular uptake via the carrier-mediated
transport pathways. Those results indicated that
L-valyl-ara-C could be a promising candidate for the oral delivery of ara-C.
Therefore, the present study aimed to examine the
pharmacokinetic characteristics of L-valyl-ara-C in rats and
evaluate its utility as a potential oral delivery system of ara-C
in vivo.
Materials and methods
Materials Ara-C, acyclovir and 5-bromo-2'-deoxyuridine
were purchased from Sigma Chemical Co (St Louis, MO, USA).
Acetonitrile and methanol were obtained from Merck Co
(Darmstadt, Germany). All other chemicals were of reagent
grade and all solvents were of HPLC grade. AML2 and L1210
cells were purchased from Korean Cell Line Bank (Seoul,
Korea).
Cells The L1210 cells were routinely maintained in
RPMI-1640 medium supplemented with 10% fetal bovine serum and
penicillin (100 U/mL)/streptomycin (50 mg/mL). The AML-2
cells were grown in DMEM supplemented with 10% FBS and
penicillin (100 U/mL)/streptomycin (50 mg/mL). All of the
cells were maintained in an atmosphere of 5%
CO2 and 90% relative humidity at 37 ºC.
Synthesis of L-valyl-ara-C Prodrug was synthesized as
previously described by Cheon et
al[9], and identified by the
1H-NMR, 13C-NMR and EI-MS spectrometers.
In vitro stability study Stabilities of
L-valyl-ara-C were evaluated at 37 °C by incubating a drug solution (10 µmol/L)
in the fresh plasma and cell homogenates. Enzymatic
cleavage of amide bond between valine and ara-C was also
measured in the presence of plasmin. Plasmin 30 µL (10 Cu/mL)
was added to the thermostated buffer composed of 50
mmol/L Tris-HCl, pH 7.4 and 110 mmol/L NaCl, followed by the
addition of the drug solution (10 µmol/L) to a final volume of 1.0
mL at 37 °C. At each time point, the metabolic reaction was
stopped by adding ice-cold acetonitrile followed by
vigorous mixing. The mixture was then centrifuged at 3000 r/min
for 10 min at 4 °C and the supernatant was filtered through a
membrane filter (0.45 μm) and analyzed by HPLC. The
chemical stability of L-valyl-ara-C was also examined in aqueous
solutions of different pH (2.0, 7.4 and 10).
Antiproliferative assays Cells were resuspended at a
density of 1×105 cells/mL in growth medium containing
serial dilutions of test drugs. Cell viability was determined
after 96 h at 37 °C by the
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) method, as previously
described by Pauwels et al[10]. Cell growth at each drug
solution was expressed as percentage of untreated controls,
and IC50 was determined as the concentration resulting in
50% growth inhibition.
Pharmacokinetic studies in rats Male Sprague-Dawley
rats (270_300 g) were purchased from Dae Han Laboratory
Animal Research and Co (Choongbuk, Korea), and given a
normal standard chow diet (No 322-7-1) purchased from
Superfeed Co (Gangwon, Korea) and tap water ad libitum.
All animal studies were performed in accordance with the
Principles for Biomedical Research Involving Animals
developed by the Council for International Organizations of
Medical Sciences, and the experimental protocols were
approved by the animal care committee of Chosun University.
The animals were kept in these facilities for at least 1 week
before the experiment and fasted for 24 h prior to the
experiment. At the experiment, the rats (n=4 per each
treatment) were given 10 mg/kg of L-valyl-ara-C orally or 2
mg/kg of ara-C intravenously. The drugs were dissolved in
saline and the dosing volume was 1 mL for each animal. Blood
samples were collected from the right femoral artery at 0,
0.083, 0.25, 0.5, 1, 2, 4, 8, 12, and 24 h post-dose and then
centrifuged at 3000 r/min for 10 min to obtain the plasma for
the HPLC assay. All samples were stored at -70 °C until
analyzed.
HPLC assay Drug concentrations were determined by a
HPLC assay described as follows: acyclovir and
5-bromo-2'-deoxyuridine were used as the internal standard for the
assay of ara-C and L-valyl-ara-C, respectively. The
chromatographic system, consisting of a pump (LC-10AD), an
automatic injector (SIL-10A) and a UV detector (SPD-10A;
Shimadzu Scientific Instruments, Japan), was set at 272 nm.
An octadecylsilane column (Gemini C18, 4.6 mm×250 mm, 5
µm; Phenomenex, Torrance, CA, USA) was eluted with a
mobile phase at a flow rate of 1.0 mL/min. The mobile phase
consisted of 0.01 mol/L ammonium acetate buffer (pH
6.5):acetonitrile (93:7, v/v ) for L-valyl-ara-C and 0.01 mol/L
ammonium acetate buffer (pH 4.5):acetonitrile (99:1,
v/v) for ara-C. The calibration curve from the standard samples was
linear over the concentration range of 0.01 µg/mL to 5
µg/mL. The limit of detection was 0.01 µg/mL.
Pharmacokinetic analysis Non-compartmental
pharmacokinetic analysis was performed using WinNonlin®
(Pharsight Corp, CA, USA). The area under the plasma
concentration-time curve (AUC) was calculated using the linear
trapezoidal method. The maximum plasma concentration
(Cmax) and the time to reach the maximum plasma
concentration (Tmax) were observed values from the experimental data.
The terminal elimination rate constant (lz) was estimated
from the slope of the terminal phase of the log plasma
concentration-time points fitted by the method of least-squares,
and then the terminal elimination half-life
(T1/2) was calculated as 0.693/lz. Additional estimated parameters using
noncompartmental pharmacokinetic analysis were systemic
plasma clearance (CL) and the volume of distribution
(Vdss).
Statistical analysis All the means are presented with
their standard deviation. An unpaired Student's
t-test was used to determine the significant difference between
treat-ments. A P value <0.05 was considered statistically
signi-ficant.
Results
In vitro metabolic stability studies The kinetics of
L-valyl-ara-C degradation were studied in various conditions.
As shown in Figure 1, L-valyl-ara-C appeared to be stable in
fresh rat plasma and leukemia cell homogenates. The
degradation of L-valyl-ara-C was negligible in fresh rat plasma
and leukemia cell homogenates over the 2 h incubation
period. In addition, the metabolic stability of
L-valyl-ara-C was assessed using pure serine protease plasmin, and the
results indicated that L-valyl-ara-C was resistant to the
degradation by plasmin. The chemical stability of
L-valyl-ara-C was also examined in aqueous solutions of different pH (2.0,
7.4, and 10). While L-valyl-ara-C was stable in aqueous
solutions of pH 7.4 and pH 10, the hydrolysis of amide bond
appeared to be rather accelerated in acidic pH and the
disappearance half-life of L-valyl-ara-C was approximately 4 h in
the aqueous solution of pH 2.0 (Figure 1).
Antiproliferative assays The antiproliferative activity
of ara-C and L-valyl-ara-C was evaluated against leukemia
cells such as AML-2 and L1210. The
IC50 values were 3.89±
1.11 and 606.0±87.3 µmol/L for ara-C and
L-valyl-ara-C,
respectively in the AML-2 cells. In the case of the L1210
cells, the IC50 values were 0.25±0.03 and 37.5±6.69 µmol/L for
ara-C and L-valyl-ara-C, respectively. In both types of cells,
the antiproliferative activity of L-valyl-ara-C was
approximately 150 fold less potent than ara-C.
Pharmacokinetic studies The plasma pharmacokinetic
profiles of L-valyl-ara-C and ara-C were determined in rats
and summarized in Table 1 and Figure 2. After an oral
administration of L-valyl-ara-C, the release of ara-C from the prodrug
was observed in the plasma although the systemic exposure
of the prodrug was much higher than that of ara-C. The
bioavailability of ara-C was about 4% via the prodrug
administration in the rats.
Discussion
The development of oral alternatives to intravenous
administration of ara-C would not only avert the high costs
dictated by hospital treatments, but also be more patient
friendly. In addition, it may also be possible to incorporate
prodrug strategies that could reduce undesirable side
effects such as toxicity, and improve therapeutic action into
such oral alternatives. In our previous studies,
L-valyl-ara-C appeared to be beneficial in enhancing the cellular uptake of
ara-C in Caco-2 cells[9]. However, in the present study,
prodrug itself exhibited far less antiproliferative activity than
ara-C in AML-2 and L1210 cells and thus, the rate of
conversion of the prodrug to the pharmacologically-active parent
drug after membrane transport would determine the
therapeutic effectiveness of L-valyl-ara-C.
If the prodrug is stable in plasma and hydrolyzed mainly
near the target cells, this should result in a continuous and
relatively high concentration of the active agent around
target cells. Many types of malignant cells and human tumors
display increased concentrations of the protease
plasminogen activators that convert plasminogen to the
highly-active protease, plasmin[11_13]. Leukemic cells also secrete these
enzymes[14]. Since plasmin rapidly cleaves various low
molecular weight compounds coupled to appropriate peptide
specifiers, the coupling of such peptide specifiers to
anticancer drugs might create `prodrugs' which would be
locally activated by tumor-associated plasmin and
consequently would be less toxic to normal cells. For example, Carl
et al[15] reported that the peptide prodrugs of several
anticancer agents designed to be specific plasmin substrates
showed selective cytotoxicity. Since L-valyl-ara-C is a
pepti-domimetic prodrug and appears to be stable in plasma or
leukemia cell homogenates, the potential bioconversion of
L-valyl-ara-C to ara-C in the surroundings of the leukemia
cells was examined using pure plasmin. As shown in Figure
1, L-valyl-ara-C exhibited high resistance to the degradation
by plasmin, implying that L-valyl-ara-C may not be a
substrate of plasmin. Overall, in vitro stability studies have
indicated that L-valyl-ara-C is metabolically stable in plasma
and leukemia cells. However, in the case of chemical stability,
the hydrolysis of prodrug appeared to be rather accelerated
in acidic pH. Considering that the tumor pH is on average
lower than the pH of normal tissues[16], chemical hydrolysis
of L-valyl-ara-C may be more favorable in tumor cells.
As summarized in Table 1, following an intravenous
administration of ara-C to rats, ara-C showed a short plasma
half-life of 1.5 h, and its volume of distribution was greater
than total body water in the rats[17]. The pharmacokinetic
parameters of ara-C obtained from the present study appeared
to be comparable to those from previous
studies[18_20]. Following an oral administration of
L-valyl-ara-C to the rats, the appearance of ara-C was observed in the plasma, implying
that the prodrug conversion to the parent could occur by
intestinal or hepatic metabolism (Figure 2).
L-valyl-ara-C is a peptidyl derivative of ara-C and thus, during intestinal
absorption, it may interact with peptidases which catalyze
the hydrolysis of peptide bonds in peptidyl derivatives or
the hydrolysis of various simple
amides[21]. However, as implicated by the results from the
in vitro stability study of L-valyl-ara-C, the metabolic conversion of
L-valyl-ara-C appeared to be minimal in the rats. Consequently, the systemic
exposure (AUC) of the prodrug was much higher than that
of ara-C, and the bioavailability of ara-C was low (about 4%)
via the prodrug administration. Therefore, the metabolic
conversion of the prodrug to the parent did not appear to be
sufficient to ensure therapeutic effectiveness in the
treatment of tumors, although L-valyl-ara-C could prevent the
rapid deamination of ara-C by masking the N4-amino group
of the cytosine ring. Further studies should be required for
the considerable tuning of the metabolic stability of prodrugs
by varying its amino acid component.
In conclusion, the amide bond of L-valyl-ara-C was stable
against the enzymatic hydrolysis, and the utility of
L-valyl-ara-C as an oral delivery system of ara-C appeared to be
limited by its low metabolic conversion to ara-C in the rats.
Acknowledgement
The authors wish to express their appreciation to Mr
Ming-ji JIN for his help with the animal experiment.
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