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ABSORPTION, DISTRIBUTION, METABOLISM, AND EXCRETION
Division of Pharmaceutical Sciences, School of Pharmacy, University of Missouri-Kansas City, Kansas City, Missouri
Received September 13, 2002; accepted October 25, 2002.
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Abstract |
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 Top Abstract Materials and MethodsResultsDiscussionReferences |
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-lactam antibiotics are
known substrates for intestinal PEPT1
(Dantzig and Bergin, 1990
;
Hashimoto et al., 1994;
Ganapathy et al., 1995;
Han et al., 1998a;
Inui et al., 2000). Strategies
have been used to design prodrugs of various poorly absorbed drugs targeted
toward receptors/transporters for improved bioavailability
(Lupia et al., 1993;
Weller et al., 1993;
Guo and Lee, 1999;
Sakaeda et al., 2001;
Anand et al., 2002a;
Manfredini et al., 2002).
Valacyclovir (VACV) is such a prodrug, which is derived from acyclovir
(ACV) by esterifying 3'-hydroxyl group of ACV with L-valine. Acyclovir,
an antiviral nucleoside, possesses activity against human herpesviruses. Owing
to its limited bioavailability, the drug has shown moderate antiviral efficacy
after oral (Steingrimsdottir et al.,
2000) and topical administration
(Sanitato et al., 1984). After
oral and topical administration, VACV is rapidly and completely converted in
vivo by enzymatic hydrolysis to acyclovir, the active parent drug. VACV has
been reported to increase the oral bioavailability of acyclovir 3- to 5-fold
in humans (Beauchamp et al.,
1992; Lupia et al.,
1993; Weller et al.,
1993). Enhanced oral (Balimane
et al., 1998; de Vrueh et al.,
1998; Han et al.,
1998b) and ocular (Anand and
Mitra, 2002) absorption of acyclovir after administration of
valacyclovir have been attributed to the hPEPT1-mediated translocation of the
amino acid prodrug
Valacyclovir has been indicated in the treatment of genital herpes, the
incidence of which has increased significantly in the past 20 years
(Fleming et al., 1997).
Although genital herpes is self-limiting in healthy adults, the disease is
painful and distressing, with severe psychosocial impact
(Manne and Sandler, 1984;
Goldmeier et al., 1988). On
the other hand, herpes simplex virus (HSV) keratitis is the leading cause of
blindness in the United States (Green and
Dunkel, 1985) and the most frequent cause of corneal opacities in
developed countries (Easty,
1985). Currently available therapy for HSV keratitis involves the
topical instillation of trifluorothymidine, idoxuridine, and vidarabine.
However, one of the major problems associated with these drugs is their poor
ocular absorption, cytotoxicity, and mutagenicity, restricting their use in
long-term treatment. Although the utility of valacyclovir against oral and
genital herpes infections is well established and well documented
(Fleming et al., 1997), it has
not been used for topical application against ocular herpes infection, herpes
simplex virus keratitis, probably due to a short half-life of 
72 h in pH
5.6 (Anand et al., 2002b).
Various lipophilic prodrugs of nucleoside analog acyclovir have been studied
for improved ocular absorption (Hughes and
Mitra, 1993) to cure HSV keratitis. These prodrugs, although
exhibiting an increase in permeability across the cornea, lacked aqueous
solubility, thereby restricting their formulation into 1 to 3% eyedrops.
A series of novel water-soluble dipeptide ester prodrugs of acyclovir (U.S.
patent pending) were thus synthesized (Y. E. Nashed, B. S. Anand, and A. K.
Mitra, manuscript submitted for publication) to target the peptide transporter
on the cornea and intestinal epithelial cells for improved ocular and oral
bioavailability of acyclovir, respectively. In comparison with VACV, the
dipeptide prodrugs exhibit increased solution stability in the pH range
studied with no measurable degradation in pH 5.6 during a 7-day experiment,
thereby rendering the aqueous formulation to be stable for a period of 2 to 3
years (Anand et al., 2002b).
These prodrugs once transported across or influxed into the target cells would
undergo chemical as well as enzymatic hydrolysis to release the active parent
drug, ACV. In this report, we have discussed the application of these prodrugs
for improved oral bioavailability by assessing their hydrolysis and affinity
toward hPEPT1 using the well characterized human intestinal Caco-2 cell line
(Hidalgo et al., 1989). Caco-2
cells have been shown to express the human di/tripeptide transporter hPEPT1
and have been used to characterize various peptidomimetics and other
substrates that are recognized by the peptide transporter
(Nielsen et al., 2001) The
affinities of these prodrugs toward the hPEPT1 transporter present on Caco-2
cells were determined. The transport characteristics of one of the dipeptide
prodrugs, Gly-Val-ACV (GVACV), across Caco-2 monolayer were compared with
those of VACV to establish whether these compounds may be transported across
cell membranes owing to their recognition by the peptide transporter.
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Materials and Methods |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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Cell Culture
All cultures were maintained in humidified incubator at 37°C with a 5%
carbon dioxide in air atmosphere. Caco-2 cells were obtained at passage 25
from American Type Culture Collection and grown in plastic tissue culture
flasks. Conventional culture medium containing Dulbecco's modified Eagle's
medium, 10% FBS (heat-inactivated), 1% nonessential amino acids, 4 mM
L-glutamine, 100 IU/ml penicillin, 100 µg/ml streptomycin, and 14 mM HEPES
at pH 7.4 was used as per the protocol established in our laboratory for
maintaining the cell line. When 80% confluent, these cells were removed by
treating them with trypsin/EDTA and plated at a density of 100,000
cells/cm2 on collagen-coated plastic dishes containing clear
polyester membranes (0.636 cm2, 3.0-µm mean pore size) or
12-well tissue culture treated plastic plates. Cells were then grown in medium
containing 10% FBS (heat-inactivated). Caco-2 cells used in our studies were
grown for 21 to 23 days. [14C]Mannitol transport was determined as
a marker of cellular integrity, which was <0.3% per hour in representative
cell monolayers.
Metabolism Studies in Cell Suspensions
Confluent Caco-2 cells, grown in tissue culture flasks were isolated with
the aid of mechanical scraper and washed thrice with Dulbecco's
phosphate-buffered saline (DPBS). The cells were resuspended in DPBS, pH 7.4,
at a concentration of 1.0 x 106 cells/ml, and 800 µl of
the cell suspension was incubated with 200 µl of 1 mM solutions of prodrugs
at 37°C in a shaking water bath for the length of the study.
Hundred-microliter samples were withdrawn at predetermined time intervals, and
the sample was purified by precipitating the cellular proteins into the
organic solvent mixture and stored at -80°C until further analysis. The
protein content of the cell suspension was determined by the method of
Bradford (1976) using bovine
serum albumin as the standard (protein estimation kit; Bio-Rad, Hercules, CA).
Apparent first order rate constants were calculated and corrected for any
chemical hydrolysis observed with the control.
Transport Studies
Transport experiments were done using side-by-side diffusion cells (type
VSC-1; Crown Glass Company, Inc., Somerville, NJ) and Transwell inserts.
Before the experiment with Gly-Sar, Caco-2 cell monolayers grown on the clear
polyester membranes and Transwell inserts were washed with DPBS (pH 6.0) and
incubated at 37°C. Freshly prepared drug solutions in DPBS (pH 6.0) was
placed in the donor chamber and the receiver chamber was filled with DPBS. The
volumes of donor and receptor chambers were 3 ml each for side-by-side
diffusion cells and 0.5 and 1.5 ml, respectively, for Transwell inserts.
Sampling from the receiver chamber was done up to a period of 3 h at time
intervals of 15, 30, 45, 60, 90, 120, 150, and 180 min, and fresh DPBS
solution was replaced to maintain sink conditions in receiver chamber. The
samples were stored at -80°C until analyzed HPLC. All experiments were
performed at 37°C. Transport studies with the prodrugs were also carried
out using side-by-side diffusion cells. The pH-dependent transport of VACV and
GVACV was assessed at pHs 6.0 and 7.4 at a concentration of 1 mM. Transport
inhibition experiments of prodrugs with Gly-Sar were carried out at pH 6.0
because it has been reported as the pH of maximal transport for the
prototypical oligopeptide transporter substrate Gly-Sar
(Guo et al., 1999).
Concentration-dependent transport of GVACV was also determined at varying
concentrations (0.1–10 mM) and Michaelis-Menten parameters
Km and Jmax were calculated.
Uptake Studies
In typical uptake experiments, cell monolayers were incubated with the
prodrug solutions prepared in DPBS (pH 6.0) for 10 min, except for time-course
studies. The concentration-dependent uptake of glycylsarcosine was studied
using [3H]Gly-Sar along with varied concentrations (0.25–20
mM) of unlabeled Gly-Sar (pH 6.0). For affinity studies, prodrugs (10 mM) were
incubated along with radiolabeled and unlabeled Gly-Sar for 10 min. For Dixon
plots and dose-response studies, [3H]Gly-Sar was incubated along
with increasing concentrations (0.25–20 mM) of unlabeled Gly-Sar and
Val-Val-ACV. After incubation, the cell monolayers were washed three times
with ice-cold HEPES buffer to terminate the uptake experiment. After the
washings, cells were lysed overnight using 1 ml 0.1% (w/v) Triton X-100 in 0.3
N NaOH at room temperature. Aliquots (500 µl) from each well were then
transferred to scintillation vials containing 5 ml of scintillation cocktail
(Fisher Scientific Co.). Samples were then analyzed by the liquid
scintillation spectrophotometry using scintillation counter (model LS-6500;
Beckman Coulter, Inc., Fullerton, CA) and the rate of uptake was normalized to
the protein content of each well. The amount of protein in the cell lysate was
measured by the protein estimation kit using bovine serum albumin as standard
(Bio-Rad).
Analytical Procedures
All samples were assayed using HPLC. The system comprised of a Rainin
Dynamax pump SD-200; Rainin Dynamax UV detector UV-C at 254 nm; a Hewlett
Packard 1100 Series fluorescence detector at excitation 
= 285 nm,
emission = 370 nm; and an Alcott autosampler (model 718 AL HPLC). The
column used was a C18 Luna column (4.6 x 250 mm; Phenomenex, Torrance,
CA). The mobile phase consisted of a mixture of buffer and an organic
modifier. The percentage of organic phase was varied to elute compounds of
interest. This method gave rapid and reproducible results. HPLC conditions for
the various compounds have been summarized in
Table 1.
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Data Analysis
Permeability Measurements across Caco-2 Monolayers. Steady-state
fluxes were determined from the slope of the cumulative amount of drug
transported versus time graph and expressed per unit of cross-sectional
surface area of the membrane as described by eq. 1. The cumulative amount of
drug transported is the sum of the receptor cell prodrug and the regenerated
drug.
 | (1) |
 | (2) |
Affinity Calculations. The concentration-dependent uptake of
[3H]glycylsarcosine was fitted to the modified Michaelis-Menten
equation described in eq. 3.
 | (3) |
All the prodrugs inhibited the uptake of [3H]glycylsarcosine in
a competitive manner, and the kinetics can be expressed according to eq. 4.
 | (4) |
 | (5) |
The affinity of VVACV was also calculated from Dixon transformation of eq.
4, which yields eq. 6.
 | (6) |
 | (7) |
) in which
Ki is equivalent to IC50/(1 +
C/Km). The affinity of cephalexin, which was used
as a positive control, was also evaluated to compare the
Ki values of the dipeptide prodrugs.
Statistical Analysis
All experiments were conducted at least in triplicate and the results are
expressed as mean ± S.D. except in the case of Michaelis-Menten
parameters Km, Vmax,
Kd, and the affinities KI, where the
values are presented as mean ± S.E. Student's t test was used
to detect statistical significance between the affinities of the prodrugs and
VACV and p < 0.05 was considered to be statistically significant.
Statistical significance was also tested by t test between the
affinities of the prodrugs and cephalexin. Statistical comparisons between the
affinities of various prodrugs were performed using the analysis of variance
(SPSS for Windows, release 10.0.7; SPSS, Inc., Chicago, IL).
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Results |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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Caco-2 Metabolism Studies. The prodrugs hydrolyzed to yield the parent drug ACV in Caco-2 homogenates. The percentage remaining of the intact prodrugs after a 10-min period ranged from 38 to 97%. The prodrugs hydrolyzed to the active parent drug, ACV. The dipeptide prodrug GGACV was rapidly hydrolyzed (no intact drug detected within 1 min) after incubation with the cell suspension and was therefore not used for further inhibition experiments (Table 2).
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Uptake Experiments. All the prodrugs at a concentration of 10 mM
were found to significantly inhibit the uptake of Gly-Sar. The amino acid
prodrugs tyrosine-ACV and glycine-ACV and the parent drug acyclovir alone did
not inhibit the uptake of [3H]Gly-Sar, whereas unlabeled
glycylsarcosine and the dipeptide val-val significantly inhibited (p
< 0.05) the uptake of [3H]Gly-Sar
(Fig. 4). Lineweaver-Burk
transformations of the Michaelis-Menten data showed that the prodrugs
inhibited the uptake of glycylsarcosine in a competitive manner.
Ki values of the prodrugs except YGACV
(Table 3) were higher than that
of cephalexin (p < 0.05). The IC50 values of Gly-Sar
and VVACV from dose-response curves (Fig.
5) were estimated by fitting the data to the nonlinear equation
E/E0 = 1/(1 + [I]/IC50) and
were found to be 2.78 ± 0.34 and 3.46 ± 0.21 mM, respectively.
Ki values were calculated by the method of Cheng and
Prusoff (1973) and were
estimated to be 2.49 and 3.18 mM for Gly-Sar and VVACV, respectively.
Ki values for Gly-Sar and VVACV calculated by different
approaches were in agreement (Table
3).
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Transport Experiments. The transport of [3H]Gly-Sar in
presence of 10 mM VACV, VVACV, and VYACV was also studied using Transwell
inserts. The dipeptide prodrugs of ACV significantly inhibited (p
< 0.05) the transepithelial transport of Gly-Sar
(Fig. 6). VACV, VVACV, and
VYACV had a similar effect on the inhibition of the transport of Gly-Sar. The
transepithelial transport of 1 mM VACV and GVACV was also studied across
Caco-2 monolayers. Cumulative amount of drug transported (the sum of the
prodrug and the regenerated parent drug) was plotted as a function of time
(Fig. 7). Apparent
permeabilities (Papp) were determined from the linear
portion of the cumulative amount versus time plot. The results indicated that
the permeabilities of VACV (5.67 ± 1.13 x
10-6 cm/s) and GVACV (5.23 ± 0.57 x
10-6 cm/s) at pH 7.4 across Caco-2 monolayers were
comparable. Moreover, the transport of VACV and GVACV was found to be
pH-dependent with a Papp of 3.01 ± 0.21 x
10-6 cm/s at pH 6.0 compared with 5.67 ± 1.13
x 10-6 cm/s at pH 7.4 for VACV and a
Papp of 2.99 ± 0.59 x
10-6 cm/s at pH 6.0 compared with 5.23 ± 0.57
x 10-6 cm/s at pH 7.4 for GVACV
(Fig. 8). Similar results for
uptake and transport of VACV in Chinese hamster ovary cells
(Guo et al., 1999) and intact
rabbit cornea (Anand and Mitra,
2002), respectively, have been reported, wherein the pH of maximum
transport was found to be 7.4. Also the transport of VACV and GVACV was
significantly inhibited in the presence of 10 mM concentration of Gly-Sar
(Table 4). The inhibition in
transport of VACV (47% inhibition) and GVACV (63% inhibition) in presence of
Gly-Sar indicates the involvement of the oligopeptide transporter in the
absorption of the amino acid and the dipeptide prodrug of acyclovir. The
concentration-dependent transport of GVACV comprised a saturable component
with a Km of 3.16 ± 0.31 mM and
Vmax of 0.014 ± 0.00058 nmol
cm-2 min-1
(Fig. 9). Transformation of the
data from the transport of GCACV resulted in a Woolf-Augustinsson-Hofstee plot
(R2 = 0.935) (Fig.
9, inset). The kinetics of GVACV transport matched a single,
saturable carrier model.
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Discussion |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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). The Caco-2 cell suspension hydrolysis studies were carried out to evaluate the regeneration characteristics of the prodrugs to the parent drug. All the prodrugs hydrolyzed to regenerate the active parent drug, ACV. The half-lives of the prodrugs ranged from 6.92 to 250.4 min (Table 2), demonstrating varied susceptibility of the prodrugs to the intestinal cellular enzymes.
Lineweaver-Burk transformations of the uptake of Gly-Sar in presence of various prodrugs were of a competitive type, revealing that the prodrugs shared a common enzyme site as that of Gly-Sar. Therefore, it can be speculated that these prodrugs would be transported by the peptide transporter. The Vmax values of Gly-Sar in presence of all the prodrugs did not change compared with control, whereas the Km values were different (Table 5), confirming that the dipeptide prodrugs inhibited the uptake of Gly-Sar in a competitive manner.
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The affinities of the prodrugs were evaluated as a measure of inhibition of
uptake of [3H]Gly-Sar across Caco-2 cells
(Fig. 4). Cephalexin, a
-lactam antibiotic, has been reported to be a substrate for PEPT1 with a
Ki of 10.9 ± 1.31 mM
(Sawada et al., 1999). In our
studies, we calculated the Ki of cephalexin to be close to
8 mM (Table 3), which is
in agreement with the previous reports. Also the calculated value of
Ki for VACV (1.41 mM) toward PEPT1 from our studies
(Table 3) was very similar to
the reported value of 2.7 mM (Nielsen et
al., 2001). The Ki values of the dipeptide
prodrugs were compared with VACV and cephalexin. Cephalexin and VACV indicated
the lower limit and the higher limit of affinity for the prodrugs,
respectively.
The affinities, Ki, of all the prodrugs (except YGACV)
were significantly higher (p < 0.05) than that of cephalexin. The
affinities of VACV, GVACV, and VVACV were significantly (p < 0.05)
higher (lower Ki) than VYACV, GYACV, YVACV, and YGACV.
Moreover the Ki values of all the prodrugs were
significantly different (p < 0.05) from that of YGACV, indicating
a higher affinity for hPEPT1 than YGACV
(Table 3). Recently, Bailey et
al. (2000) proposed a general
template for substrate specificity for peptide transporter PEPT1. According to
this model, which identifies 10 key features for substrate binding to PEPT1,
the side chain of the dipeptides may interact with the hydrophobic pocket in
the PEPT1 protein. All dipeptide prodrugs studied here differ from each other
by the amino acid residues attached to the parent drug, ACV
(Fig. 1). A comparative account
of hydrophobicity scales of various amino acids has been reported previously
(Janin, 1979;
Kyte and Doolittle, 1982).
According to both scales, the order of hydrophobicity decreases from valine to
glycine to tyrosine. Although speculative, we can suggest that the high
affinity observed for VACV, GVACV, and VVACV could be due to the high and
moderate hydrophobicity of the valine and glycine moiety, respectively, with
probably a major contribution from the valine moiety. On the other hand, the
moderate affinity of prodrugs with valine-tyrosine, glycinetyrosine, and
tyrosine-valine as the amino acid residues in comparison with VACV, VVACV, and
GVACV could be explained by the rank order of hydrophobicity values of the
three amino acid residues, i.e., valine > glycine > tyrosine and also
due to a less favorable orientation of the molecule due to the presence of the
tyrosine residue. The low affinity observed for YGACV can be explained by the
short half-life for YGACV and the presence of two amino acids, glycine and
tyrosine, having lower hydrophobicity values than valine. These observations
need to be ultimately confirmed by structure activity relationships.
From studies on the in vitro antiviral efficacy against HSV-1 and HSV-2, it
was observed that VVACV was more effective than ACV, VACV, and also the other
dipeptide prodrugs, thereby making it the most effective prodrug against
ocular HSV keratitis (Anand et al.,
2002b). Therefore, the mechanism of inhibition of Gly-Sar by VVACV
was further examined by dose-response curve
(Fig. 5) and Dixon plots.
Dose-response curve (Fig. 5)
and Dixon plot for radiolabeled Gly-Sar in presence of unlabeled Gly-Sar were
also studied to compare the affinities of Gly-Sar and VVACV.
Ki of VVACV calculated from all the approaches
(Table 3) was in agreement and
was best estimated to be 2.38 mM, which was very similar to the calculated
Ki value of 2.42 mM approximately for Gly-Sar
(Table 3). It was observed
during the hydrolysis studies that the dipeptide prodrugs cleaved to the
parent drug via the formation of the amino acid intermediate. Therefore, the
uptake of Gly-Sar in presence of the amino acid prodrugs YACV and GACV and ACV
was also studied. It was observed that ACV, YACV, and GACV did not inhibit the
uptake of Gly-Sar (Fig. 4).
Therefore, it can be concluded that the dipeptide prodrugs cleaving to YACV or
GACV inhibited the uptake of Gly-Sar, owing to the interaction of the intact
prodrug with hPEPT1. In addition to the uptake inhibition experiments,
transport of [3H]Gly-Sar in presence of VACV, VVACV, and VYACV was
also studied. VYACV was chosen as it showed the longest half-life of 250.4 min
(Table 1). Results indicated
that VYACV caused a similar inhibition of transport as VACV and VVACV
(Fig. 6), suggesting that the
dipeptide prodrugs of ACV interact with hPEPT1.
Generally speaking, inhibition studies may not be a good predictor for the
actual cellular uptake of drug candidates, because the substrates might only
bind to the transporter without being translocated. Hence, the affinity of
these prodrugs for hPEPT1 may not be translated into hPEPT1 mediated
transepithelial transport and oral delivery. Therefore, transport experiments
with VACV and GVACV in absence and presence of Gly-Sar were carried out. Once
the prodrug traverses the apical membrane, the transport across the
basolateral membrane might be mediated by another peptide transporter present
on the surface of the basolateral membrane, which might be distinct from the
one on the apical membrane. The transport of GVACV was compared with that of
VACV because VACV has been shown to be transported across Caco-2 by the
intestinal peptide transporter (Han et
al., 1998b). GVACV was chosen for these studies because it showed
a comparable affinity to that of VACV and had a half-life of approximately 108
min. The transport of VACV and GVACV was found to be pH-dependent
(Fig. 8), which is consistent
with the report on the interaction of positively charged dipeptides with PEPT1
(Temple et al., 1996;
Amasheh et al., 1997;
Lister et al., 1997). VACV and
GVACV have three pKa values (1.90, 7.47, and 9.43) and
exist as a mixture of cationic and neutral species at pH 7.4, which is the pH
of maximal transport among the range studied. Similar findings have been
reported wherein the transport of VACV was maximum at pH 7.4 due to the
presence of a mixture of cationic and neutral species at pH 7.4
(Guo et al., 1999;
Anand and Mitra, 2002). The
permeability (at pH 6.0) of GVACV, 2.99 ± 0.59 x
10-6 cm/s, was similar to a permeability of 3.01
± 0.21 x 10-6 cm/s for VACV
(Fig. 8). Inhibition of
transport of both VACV and GVACV by Gly-Sar lends direct support to the
probable interaction of the newly synthesized dipeptide prodrugs with the
oligopeptide transporter.
In addition to the inhibition data the concentration-dependent transport of GVACV was found to be saturable at higher concentrations (Km of 3.16 ± 0.31 mM; Vmax of 0.014 ± 0.00058 nmol cm-2 min-1) (Fig. 9). Transformation of the data resulted in a linear Woolf-Augustinsson-Hofstee plot (R2 = 0.935) (Fig. 9, inset) and therefore the kinetics of GVACV transport matched a single, saturable carrier model. The Ki and Km of GVACV were found to be very similar further confirming the sharing of the same binding site on the transporter.
In conclusion, the results of the present study indicate that the dipeptide prodrugs of ACV, a poorly absorbed antiviral nucleoside, exhibit high affinity toward the intestinal oligopeptide transporter. The uptake of these prodrugs was efficiently mediated by hPEPT1 because they significantly inhibit the uptake of glycylsarcosine. These prodrugs hydrolyze readily to regenerate the active parent drug, acyclovir, thereby fulfilling the basic requirement of a prodrug. These prodrugs owing to their high affinity, excellent solution stability, and in vitro antiviral activity against herpes infections are promising drug candidates against oral and ocular herpes infections.
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Acknowledgements |
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Footnotes |
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ABBREVIATIONS: hPEPT1, human intestinal peptide transporter; VACV, valacyclovir; ACV, acyclovir; HSV, herpes simplex virus; GVACV, glycine-valine acyclovir; Gly-Sar, glycylsarcosine; FBS, fetal bovine serum; VVACV, valine-valine acyclovir; YGACV, tyrosine-glycine acyclovir; GYACV, glycine-tyrosine acyclovir; GGACV, glycine-glycine-acyclovir; VYACV, valine-tyrosine acyclovir; YVACV, tyrosine-valine acyclovir; DPBS, Dulbecco's phosphate-buffered saline; HPLC, high-performance liquid chromatography.
Address correspondence to: Dr. Ashim K. Mitra, School of Pharmacy, University of Missouri-Kansas City, 5005 Rockhill Rd., Kansas City, MO 64110-2499. E-mail: mitraa{at}umkc.edu
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References |
|---|
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
Amasheh S, Wenzel U, Boll M, Dorn D, Weber W, Clauss W, and Daniel H (1997) Transport of charged dipeptides by the intestinal H+/peptide symporter PepT1 expressed in Xenopus laevis oocytes. J Membr Biol 155: 247–256.[CrossRef][Medline]
Anand BS, Dey S, and Mitra AK (2002a) Current prodrug strategies via membrane transporters/receptors. Expert Opin Biol Ther 2: 607–620.[CrossRef][Medline]
Anand BS, Nashed YE, and Mitra AK (2002b) Novel dipeptide prodrugs of acyclovir for ocular herpes infections: bioreversion, antiviral activity, and transport across rabbit cornea. Curr Eye Res, in press.
Anand BS and Mitra AK (2002) Mechanism of corneal permeation of L-valyl ester of acyclovir: targeting the oligopeptide transporter on the rabbit cornea. Pharm Res 19: 1194–1202.[CrossRef][Medline]
Bailey PD, Boyd CA, Bronk JR, Collier ID, Meredith D, Morgan KM, and Temple CS (2000) How to make drugs orally active: a substrate template for peptide transporter PepT1. Angew Chem Int Ed Engl 39: 505–508.[CrossRef][Medline]
Balimane PV, Tamai I, Guo A, Nakanishi T, Kitada H, Leibach FH, Tsuji A, and Sinko PJ (1998) Direct evidence for peptide transporter (PepT1)-mediated uptake of a nonpeptide prodrug, valacyclovir. Biochem Biophys Res Commun 250: 246–251.[CrossRef][Medline]
Beauchamp LM, Orr GF, de Miranda P, Doucette M, Burnette T, and Krenitsky TA (1992) Amino acid ester prodrugs of acyclovir. Antiviral Chem Chemother 3: 157–164.
Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72: 248–254.[CrossRef][Medline]
Cheng Y and Prusoff WH (1973) Relationship between the inhibition constant (Ki) and the concentration of inhibitor which causes 50 per cent inhibition (IC50) of an enzymatic reaction. Biochem Pharmacol 22: 3099–3108.[CrossRef][Medline]
Dantzig AH and Bergin L (1990) Uptake of the cephalosporin, cephalexin, by a dipeptide transport carrier in the human intestinal cell line, Caco-2. Biochim Biophys Acta 1027: 211–217.[Medline]
de Vrueh RL, Smith PL, and Lee CP (1998) Transport of
L-valine-acyclovir via the oligopeptide transporter in the human intestinal
cell line, Caco-2. J Pharmacol Exp Ther
286:
1166–1170.
Easty DL (1985) Clinical Aspects of Ocular Herpes Simplex Virus Infection. Chicago Year Book Medical Publishers, Chicago.
Fleming DT, McQuillan GM, Johnson RE, Nahmias AJ, Aral SO, Lee FK,
and St Louis ME (1997) Herpes simplex virus type 2 in the United
States, 1976 to 1994. N Engl J Med
337:
1105–1111.
Ganapathy ME, Brandsch M, Prasad PD, Ganapathy V, and Leibach FH
(1995) Differential recognition of -lactam antibiotics by
intestinal and renal peptide transporters, PEPT 1 and PEPT 2. J
Biol Chem 270:
25672–25677.
Goldmeier D, Johnson A, Byrne M, and Barton S (1988) Psychosocial implications of recurrent genital herpes simplex virus infection. Genitourin Med 64: 327–330.[Medline]
Green MT and Dunkel E (1985) Herpes Simplex Virus Infections. Lea & Febiger, Philadelphia, PA.
Guo A, Hu P, Balimane PV, Leibach FH, and Sinko PJ
(1999) Interactions of a nonpeptidic drug, valacyclovir, with the
human intestinal peptide transporter (hPEPT1) expressed in a mammalian cell
line. J Pharmacol Exp Ther
289:
448–454.
Guo W and Lee RL (1999) Receptor-targeted gene delivery via folate-conjugated polyethylenimine. AAPS PharmSci 1: E19.[CrossRef][Medline]
Han H, de Vrueh RL, Rhie JK, Covitz KM, Smith PL, Lee CP, Oh DM, Sadee W, and Amidon GL (1998a) 5'-Amino acid esters of antiviral nucleosides, acyclovir and AZT are absorbed by the intestinal PEPT1 peptide transporter. Pharm Res 15: 1154–1159.[CrossRef][Medline]
Han HK, Oh DM, and Amidon GL (1998b) Cellular uptake mechanism of amino acid ester prodrugs in Caco-2/hPEPT1 cells overexpressing a human peptide transporter. Pharm Res 15: 1382–1386.[CrossRef][Medline]
Hashimoto N, Fujioka T, Toyoda T, Muranushi N, and Hirano K (1994) Renin inhibitor: transport mechanism in rat small intestinal brush-border membrane vesicles. Pharm Res 11: 1448–1451.[CrossRef][Medline]
Hidalgo IJ, Raub TJ, and Borchardt RT (1989) Characterization of the human colon carcinoma cell line (Caco-2) as a model system for intestinal epithelial permeability. Gastroenterology 96: 736–749.[Medline]
Hughes PM and Mitra AK (1993) Effect of acylation on the ocular disposition of acyclovir. II: Corneal permeability and anti-HSV 1 activity of 2'-esters in rabbit epithelial keratitis. J Ocul Pharmacol 9: 299–309.[Medline]
Inui K, Terada T, Masuda S, and Saito H (2000)
Physiological and pharmacological implications of peptide transporters, PEPT1
and PEPT2. Nephrol Dial Transplant
15:
11–13.
Janin J (1979) Surface and inside volumes in globular proteins. Nature (Lond) 277: 491–492.[CrossRef][Medline]
Kyte J and Doolittle RF (1982) A simple method for displaying the hydropathic character of a protein. J Mol Biol 157: 105–132.[CrossRef][Medline]
Lister N, Bailey PD, Collier ID, Boyd CA, and Bronk JR (1997) The influence of luminal pH on transport of neutral and charged dipeptides by rat small intestine, in vitro. Biochim Biophys Acta 1324: 245–250.[Medline]
Lupia RH, Ferencz N, Lertora JJ, Aggarwal SK, George WJ, and
Agrawal KC (1993) Comparative pharmacokinetics of two prodrugs of
zidovudine in rabbits: enhanced levels of zidovudine in brain tissue.
Antimicrob Agents Chemother
37:
818–824.
Manfredini S, Pavan B, Vertuani S, Scaglianti M, Compagnone D, Biondi C, Scatturin A, Tanganelli S, Ferraro L, Prasad P, et al. (2002) Design, synthesis and activity of ascorbic acid prodrugs of nipecotic, kynurenic and diclophenamic acids, liable to increase neurotropic activity. J Med Chem 45: 559–562.[CrossRef][Medline]
Manne S and Sandler I (1984) Coping and adjustment to genital herpes. J Behav Med 7: 391–410.[CrossRef][Medline]
Nielsen CU, Andersen R, Brodin B, Frokjaer S, Taub ME, and Steffansen B (2001) 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: 129–138.[CrossRef][Medline]
Sakaeda T, Tada Y, Sugawara T, Ryu T, Hirose F, Yoshikawa T, Hirano K, Kupczyk-Subotkowska L, Siahaan TJ, Audus KL, et al. (2001) Conjugation with L-glutamate for in vivo brain drug delivery. J Drug Target 9: 23–37.[Medline]
Sanitato JJ, Asbell PA, Varnell ED, Kissling GE, and Kaufman HE (1984) Acyclovir in the treatment of herpetic stromal disease. Am J Ophthalmol 98: 537–547.[Medline]
Sawada K, Terada T, Saito H, Hashimoto Y, and Inui KI
(1999) Recognition of L-amino acid ester compounds by rat peptide
transporters PEPT1 and PEPT2. J Pharmacol Exp Ther
291:
705–709.
Steingrimsdottir H, Gruber A, Palm C, Grimfors G, Kalin M, and
Eksborg S (2000) Bioavailability of aciclovir after oral
administration of aciclovir and its prodrug valaciclovir to patients with
leukopenia after chemotherapy. Antimicrob Agents
Chemother 44:
207–209.
Temple CS, Bailey PD, Bronk JR, and Boyd CA (1996) A model for the kinetics of neutral and anionic dipeptide-proton cotransport by the apical membrane of rat kidney cortex. J Physiol (Lond) 494: 795–808.[Medline]
Weller S, Blum MR, Doucette M, Burnette T, Cederberg DM, de Miranda
P, and Smiley ML (1993) Pharmacokinetics of the acyclovir
pro-drug valaciclovir after escalating single- and multiple-dose
administration to normal volunteers. Clin Pharmacol
Ther 54:
595–605.[Medline]
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, [3H]Gly-Sar with 5 mM
unlabeled Gly-Sar. Inset, comparison of uptake of [3H]Gly-Sar in
presence of 5 mM Gly-Sar at various time intervals.
).
,[3H]Gly-Sar with VVACV; and X, [3H]Gly-Sar with
VYACV.
, intact
GVACV; and •, cumulative amount of Gly-Val-ACV (R2 =
0.98). Values are mean ± S.D. (n = 3).
