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ABSORPTION, DISTRIBUTION, METABOLISM, AND EXCRETION
College of Pharmacy, University of Michigan, Ann Arbor, Michigan (C.P.L., D.S., D.R.F., S.S.M., L.S.W., C.R., G.L.A.); Exploratory Biopharmaceutics and Stability, Drug Delivery Enablement, Pharmaceutical Research Institute, Bristol-Myers Squibb Company, New Brunswick, New Jersey (D.S.); Department of Pharmacy Practice, School of Pharmacy and Pharmacal Sciences, Purdue University, West Lafayette, Indiana (D.R.F.); Department of Internal Medicine, Division of Gastroenterology, University of Michigan Medical Center, Ann Arbor, Michigan (J.L.B.); Division of Gastroenterology, St. Joseph Mercy Hospital, Ann Arbor, Michigan (J.L.B.); and Department of Pharmacy, University Hospital, Ann Arbor, Michigan (L.S.W.)
Received February 27, 2003; accepted May 1, 2003.
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Abstract |
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 Top Abstract Materials and MethodsResultsDiscussionReferences |
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;
Beauchamp and Krenitsky, 1993;
Purifoy et al., 1993). The
L-valacyclovir transport mechanism has since been extensively
examined in animal models and in cell culture systems
(Smith et al., 1993;
Balimane et al., 1998; Han et
al.,
1998a,b;
Sinko and Balimane, 1998).
These studies revealed that valacyclovir absorption may be facilitated by
several carrier-mediated transporters in the intestine. Thus, in addition to
the intestinal proton-dependent oligopeptide transporter PEPT1, it has been
suggested that organic anion (OATs) and organic cation (OCT) transporters may
also play a role in intestinal valacyclovir uptake
(Sinko and Balimane, 1998).
The relative contribution of oligopeptide and other transporters in overall
valacyclovir uptake in humans has not yet been reported, however.
Over the past few years the dramatic increase in prodrug strategies to
improve both oral absorption as well as efficacy and safety considerations can
be directly attributed to a growing emphasis to better understand the role and
importance of carrier-mediated transport in the human intestine
(Shin et al., 2003). The wide
use of cell culture systems and/or animal models as surrogates for the human
intestine has contributed enormously to this endeavor. Nevertheless, such
reliance on model systems may also lead to the implication of multiple
transporters contributing simultaneously to overall intestinal transport.
Furthermore, such model systems cannot address the important issues of
relevancy to the role of human intestinal transporters in vivo, and when
relevant, the relative contributions of these transporters in vivo. Thus, it
is essential to investigate the role and importance of multiple transporters
in the in vivo transport of prodrugs across the human intestine. With the
emergence of genomics and advances in microarray technology, thousands of
genes from tissues or cells can be simultaneously analyzed to acquire the mRNA
expression levels. This technology is of particular importance to in vivo
studies with humans since it not only obviates the need to assay gene
expression levels one at a time but also facilitates the global assessment of
the role of several thousands of genes in the modulation of biopharmaceutical
processes and parameters of interest. Thus, it would be possible to generate a
global database of gene expression in intestinal tissues from a group of
healthy human volunteers. If the absorption of an orally administered drug is
also determined in the same group of volunteers, the global database of gene
expression can then be examined to identify plausible putative or novel
mediators of drug transport and oral drug absorption in the intestinal
tissues.
In this article, we describe the results of a multiphase study in humans designed specifically to identify genes that mediate the oral absorption of valacyclovir and acyclovir in healthy humans. Thus, duodenal tissue biopsies were first obtained from all subjects in phase 1 of the study. Subsequently, in phases 2 and 3 of the study, pharmacokinetic studies were conducted in the same subjects to monitor acyclovir absorption following valacyclovir and acyclovir oral administration, respectively. The gene expression profiles determined from duodenal tissue biopsies were then compared with acyclovir pharmacokinetic parameters obtained for each individual to elicit correlations. Gene expression was analyzed using microarray expression technology (Affymetrix GeneChip, Affymetrix, Inc., Santa Clara, CA) that contains 12,559 gene transcripts. The possible involvement of relevant transporters in determining overall valacyclovir absorption, as indicated by significant positive correlations with expression levels, was then examined with functionality tests in cell culture constructs in vitro. These studies are expected not only to provide a more thorough understanding of valacyclovir transport in humans in vivo but also reveal general indications regarding the importance of various transporters in the intestinal transport of amino acid ester prodrugs and peptidomimetics.
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Materials and Methods |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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Human Study Protocol. Eleven healthy subjects (seven males and four females) gave written informed consent to participate in the study. This investigation complied with tenets of the Declaration of Helsinki promulgated in 1964 and was approved by the University of Michigan Institutional Review Board. The subjects were 21 to 36 years of age (29.0 ± 5.8 years) and were within 20% of their ideal body weight (75.7 ± 15.7 kg). Subjects were deemed healthy based on medical history, physical examination, and complete blood count and serum chemistries. Persons with a history of renal, hepatic, gastrointestinal, cardiovascular, or psychiatric disease were excluded from the study, as were subjects with a history of clinical illness within 2 weeks of the start of their participation in the study. In addition, all subjects were medication free, including over-the-counter agents, for at least 3 days before the study (hormonal contraceptive medications were permitted). This crossover study consisted of three phases, and each subject participated in all three phases. Phase I was always conducted first with each subject, and the sequence of phase II and phase III studies were conducted in a randomized manner. Female subjects had to test negative in pregnancy tests before participation in each phase of the study. A washout period of at least 5 days was allowed between each phase of the study.
Phase I. The duodenal biopsy samples for the measurement of mRNA
expression levels for subsequent gene correlations were obtained in this
phase. The studies in phase I also involved the estimation of jejunal
valacyclovir and acyclovir permeability using a regional perfusion technique,
the results of which will be reported elsewhere. Briefly, following a 10-h
overnight fast, subjects were admitted to the General Clinic Research Center
at the University of Michigan Medical Center at 7 AM on the day of the study
and fed a standard breakfast over the next half-hour. The subjects remained
fasted for the duration of the study, approximately 14 h. The intubation and
placement of the perfusion tube in the upper jejunum was performed according
to the procedure described previously
(Takamatsu et al., 1997).
Briefly, esophago-gastroduodenoscopy was performed to facilitate the passage
of a fiberoptic endoscope to the upper duodenum of the small intestine. Ten
biopsy samples of approximately 5 mg each were then obtained from the duodenal
mucosa using the forceps at the tip of the endoscope. The biopsy specimens
were snap frozen in liquid nitrogen and stored at –80°C until RNA
was processed for microarray analysis.
Phases II and III. Phases II and III of the study involved the estimation of acyclovir pharmacokinetics following oral valacyclovir and acyclovir administration, respectively. Briefly, following a 10-h overnight fast, subjects were admitted to the General Clinic Research Center at the University of Michigan Medical Center on the day of the study at 7 am. Subsequently, a single dose of either 500 mg of Valtrex (phase II) or 400 mg of Zovirax (phase III) was orally administered with 180 ml of water. Blood samples for measurement of acyclovir and valacyclovir plasma concentrations were obtained at specified times. The subjects were fed a standard meal 4 and 10 h following drug administration.
Collection of Blood Samples and Drug Analysis. Blood samples were obtained through a forearm venous catheter for multiple blood draws and placed in heparinized Vacutainer vials (BD Biosciences). In phase II studies, 10-ml samples were withdrawn at 0, 0.25, 0.5, 0.75, 1, 1.5, 2, 2.5, 3, and 4 h, followed by 5-ml samples at 6, 8, 10, and 12 h. In phase III studies, 5-ml samples were obtained at 0, 0.25, 0.5, 1, 1.5, 2, 2.5, 4, 6, 8, 10, and 12 h. The blood samples were immediately centrifuged at 3000 rpm for 5 to 10 min at 4°C. Plasma was removed, snap frozen in liquid nitrogen, and immediately stored at –80°C until further analysis.
The acyclovir and valacyclovir concentrations in plasma samples were
simultaneously assayed by HPLC. The HPLC system consisted of a Waters
interface module system, a Waters WISP 712 Autosampler, a Waters 996
photodiode array detector, and a Waters HPLC 515 pump (Waters, Milford, MA).
The reversed-phase column used was an Ultrasphere ODS-1 (5 µm, 250 x
4.6 mm; Beckman Coulter, Inc., Fullerton, CA) column equipped with a guard
column. The mobile phase used was 25 mM sodium acetate buffer, pH 3.5,
containing 4.5% (v/v) acetonitrile. The flow rate used was 1 ml/min, and the
UV detection wavelength was set at 254 nm. The HPLC system was controlled with
Waters Millennium software (Version 3.0.1; Waters). Assays of plasma samples
were carried out as follows. In a typical assay, plasma samples were thawed at
room temperature and 0.5 ml of 20% (v/v) trifluoroacetic acid in water was
added to 1 ml of plasma in an Eppendorf tube. The mixture was vortexed for 1
min and centrifuged at 12,500 rpm and 4°C for 15 min. The supernatant was
filtered using a 0.45-µm filter cartridge, and 100 µl of the filtered
supernatant was injected directly onto the column for HPLC analyses. The
retention times were 
5 and 10 min for acyclovir and valacyclovir,
respectively. Standard curves using solutions of acyclovir and valacyclovir in
distilled water were constructed over the concentration range of 0.3 to 300
µM and were found to be linear (r2 > 0.999).
Additionally, plasma blanks spiked with known acyclovir and valacyclovir
standards were subjected to the extraction procedure described above and
assayed to determine extraction efficiency. The recovery was greater than 98%
over the concentration range of 0.3 to 80 µM for both acyclovir and
valacyclovir. All samples were assayed in triplicate. The limit of
quantitation was set at the lowest concentration of 0.3 µM (0.07
µg/ml) used in the standard curve. The limit of detection was 0.1
µM (0.02 µg/ml).
GeneChip Analysis. The human duodenal samples were prepared as
described earlier (Sun et al.,
2002). Briefly, the tissue samples were homogenized in TRIzol, and
total RNA was isolated. From the total RNA, cDNA was made and then converted
back to biotin labeled cRNA. The biotin-labeled cRNA was fragmented and
hybridized along with controls (Bio B, C, D, and Cre) to the U95A GeneChip
(Affymetrix). The GeneChip® was then washed and stained with streptavidin
phycoerythrin solution. After washing, the GeneChip was scanned with a laser
scanner (Affymetrix). The gene expression profiles were analyzed by Affymetrix
Microarray Suite and Data Mining Tool software.
Semiquantitative RT-PCR Analysis. For RT-PCR, total RNA from the
tissue and Caco-2 cell samples was purified using TRIzol reagent. One
microgram of total RNA from each sample was subjected to RT-PCR (PCR access
system; Promega, Madison, WI) using PEPT1- and HPT1-specific primers. The
PEPT1 RT-PCR assay was performed as described previously
(Sun et al., 2002). The HPT1
assay was done using the forward primer (CATAGAAGTGAAGGACA) and the reverse
primer (GATGGGGATCTGATCATTG). The first-strand cDNA was synthesized using
avian myeloblastosis virus reverse transcriptase at 48°C for 45 min. This
was followed by a 2-min cycle at 94°C to inactivate avian myeloblastosis
virus reverse transcriptase and to denature the primers and cDNA. The PCR was
performed for 25 cycles of 94°C for 30 s, primer annealing for 1 min at
55°C, extension at 68°C for 1 min, and a final extension at 68°C
for 7 min. The conditions were established to obtain linear amplification of
PCR product. The expected HPT1 PCR fragment was 1 kilobase. The reaction
mixture was separated on a 4 to 20% Tris borate-EDTA-polyacrylamide gel
(Invitrogen) and visualized with SYBR Green nucleic acid gel stain (Molecular
Probes, Eugene, OR).
Transfection of HPT1 into HeLa Cells. HeLa cells were cultured in Dulbecco's modified Eagle's medium with high glucose supplemented with 1% nonessential amino acid, 1% L-glutamine, 1% sodium pyruvate, and 10% fetal bovine serum. Cells were plated onto a 12-well plate (Falcon, Cowley, UK) for 24 h before transfection. Transfection was performed after the cells reached 50 to 70% confluence. The HPT1/pcDNA3.0 construct (a gift of Eli Lilly and Company, Indianapolis, IN) was transfected into the cells using Fugene reagent (Roche, Indianapolis, IN) after incubating the cells with Fugene/DNA complex (3:1) in Dulbecco modified Eagle medium with 10% fetal bovine serum for 48 h before functional assay.
[3H]Valacyclovir Uptake Studies in HPT1 Transfected HeLa Cells. After a 48-h transfection, cells were washed twice with transport buffer (pH 6, 1 mM CaCl2, 1 mol/l MgCl2, 150 mM NaCl, 3 mM KCl, 1 mM NaH2PO4, 5 mM D-Glucose, 5 mM MES) and incubated with 10 µM valacyclovir (9.80 µM valacyclovir and 0.20 µM [3H]valacyclovir) in 1 ml transport buffer for 30 min at room temperature. After 30 min, the uptake was stopped by the addition of 0.5 ml of ice-cold transport buffer. Cells were washed 3 times with ice-cold transporter buffer, collected in 0.5 ml of 1.5% Triton X-100, and sonicated 3 times for 10 s. Sonicated cell suspension (200 µl) was used for scintillation counting, and the remaining sample was saved for protein assay.
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Results |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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Intestinal mRNA Expression and Variability of Selected Genes. The
mRNA expression data for 12,559 gene sequences from biopsy samples of the 10
subjects (excluding subject 5,115), assayed using GeneChip expression analysis
and reported previously (Sun et al.,
2002), were used in this correlation study. A narrower list of 281
transporters, channels, and metabolizing enzymes was selected based on the
expression levels in the tissues. The variability in expression levels with
the 10 subjects observed with the 281 genes was in the range of 5 to 148%,
with an average of 37%. The average variability in expression levels of
various classes of genes were as follows: transporters only, 33%;
channels/exchangers only, 33%; and enzymes only, 38%. The mRNA expression
intensities and variabilities of selected transporters (peptide, amino acid,
and nucleoside), ion exchangers, and intestinally related genes are shown in
Fig. 1. Of the intestinal
peptide transporters, the average HPT1 expression level in human duodenum was
4.5-fold higher than the average PEPT1 expression level. Furthermore, the
variability in PEPT1 expression levels (25%) was lower than the average
variability in expression levels of all transporters in the set (33%), whereas
the variability in HPT1 expression levels was even lower (14%). The highest
variability in expression levels of solute transporters expressed in the
duodenum was found with the purine nucleoside transporter CNT2 (54%), whereas
the sodium/glucose cotransporter SGLT1 exhibited the lowest variability
(11%).
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Correlations of Gene Expression Levels in Duodenal Biopsies with Acyclovir Pharmacokinetic Parameters following Oral Administration of Valacyclovir and Acyclovir. To identify transporters that may potentially contribute to valacyclovir or acyclovir absorption, linear correlations between the microarray expression profiles determined from duodenal biopsy samples and the corresponding pharmacokinetic parameters from the same individual were determined. The correlation parameters are summarized in a cluster diagram (Fig. 2). The areas in red denote the existence of positive correlations, whereas those in green represent negative correlations. Areas in black in the cluster diagram indicate lack of any correlation between the two parameters of interest. Positive correlations (red areas) between valacyclovir-associated pharmacokinetic parameters and several solute transporters are evident (Fig. 2). Curiously, the PEPT1 expression levels correlated poorly and negatively with valacyclovir pharmacokinetic parameters (r = –0.147, p = 0.710 with AUC0–last; r = –0.132, p = 0.740 with AUC0–inf; r = –0.589, p = 0.095 with Cmax). On the other hand, positive and significant correlations were observed between AUC values following valacyclovir oral administration and the expression levels of HPT1 peptide transporter (r = 0.794, p = 0.011 with AUC0–last; r = 0.766, p = 0.016 with AUC0–inf). The linear correlations of HPT1 and PEPT1 expression levels with AUC0–last following oral valacyclovir administration are shown in Fig. 3. The highest positive linear correlations of valacyclovir parameters were observed with the expression levels of 4F2hc, a membrane glycoprotein (r = 0.875, p = 0.002 with AUC0–inf), and with proline transporter, an amino acid transporter (r = 0.857, p = 0.003 with AUC0–last). A linear correlation plot of 4F2hc expression levels with AUC0–last after valacyclovir oral administration is shown in Fig. 4. No positive correlations were found involving the valacyclovir-associated pharmacokinetic parameters and organic cation transporter (OCT1 and OCT2) expression levels or with a variety of organic anion transporters.
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Positive correlations were observed between valacyclovir-related
pharmacokinetic parameters and ion channel and exchanger expression levels.
Although such genes are not expected to be involved in direct valacyclovir
transport, the ion gradients generated could potentially influence ion-coupled
transporters. Figure 5 shows
the linear correlations in a cluster diagram. It was found that the expression
levels of the Na+/H+ exchanger gene, NHE-1, exhibited a
better positive correlation with AUC0–last following
valacyclovir administration (r = 0.680, p = 0.044)
(Fig. 4) compared with that
with AUC0–last (r = 0.230, p = 0.050)
following acyclovir oral administration. A relatively high positive
correlation was also observed between expression levels of SLC4A2, an ion
exchange protein, and valacyclovir related pharmacokinetic parameters
(r = 0.785, p = 0.012, with Cmax and
r = 0.600, p = 0.088 with AUC0–inf). There
were a few other Na+/H+ exchanger protein genes such as
SLC9A3R2 that also exhibited similar correlations. It was also found that
Na+/K+-ATPase proteins on the basolateral membrane may
potentially be involved as well. Thus, the
Na+/K+-ATPase
1 (ATP11) and 2
(ATP12) subunit expression levels, especially the 1 subunit,
exhibited a positive correlation with valacyclovir pharmacokinetic parameters.
The highest significant positive correlation observed was between the
ATP6V11 proton transporting ATPase expression levels and
AUC0–last following valacyclovir oral administration
(r = 0.780, p = 0.013) and is shown in
Fig. 4.
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The linear correlations of pharmacokinetic parameters following valacyclovir and acyclovir oral administration with expression levels of select metabolizing enzymes are shown as a cluster diagram in Fig. 6. High negative correlations were obtained with expression levels of efflux proteins such as MDR1 and MRP2 (cMOAT) with pharmacokinetic parameters following either acyclovir or valacyclovir oral administration (Fig. 7). Similar high negative correlations were also observed with the cytochrome P450 IIIA subfamily metabolism enzymes (r values ranging from –0.6 to –0.8).
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Linear correlations of pharmacokinetic parameters with expression levels of junction proteins and other intestinal proteins were also determined. The best positive correlations were between expression levels of the tight junction protein claudin-7, with AUC0–last (r = 0.788, p = 0.012), AUC0–inf (r = 0.708, p = 0.033), and Cmax (r = 0.544, p = 0.130) following valacyclovir oral administration. There also appears to be a weak relationship of these valacyclovir-related pharmacokinetic parameters with the mucin protein secreted in the intestine.
The prominent positive correlation coefficients of gene expression levels with acyclovir pharmacokinetic parameters AUC0–last, AUC0–inf, and Cmax following oral valacyclovir administration are summarized in Table 3. There were few significant positive correlations between the acyclovir pharmacokinetic parameters and transporter expression. The moderate positive correlation of CNT2 (purine transporter) expression levels with AUC0–inf (r = 0.602, p = 0.065) may be of interest.
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RT-PCR Analysis. The duodenal mRNA expression profiles obtained for PEPT1 and HPT1 from microarray data analyses were validated using semiquantitative RT-PCR. PEPT1 mRNA expression in the individual biopsies determined by RT-PCR exhibited a pattern similar to that observed with the microarray data (r2 = 0.89). HPT1 mRNA expression determined by RT-PCR was also found to parallel the expression pattern in the microarray data (r2 = 0.80).
[3H]Valacyclovir Uptake by HPT1. The
[3H]valacyclovir uptake in transiently HPT1-expressing HeLa cells
was compared with uptake in normal HeLa cells. The HPT1 mRNA expression in the
transfected cells was enhanced compared with control HeLa cells (data not
shown). The uptake experiment results are shown in
Fig. 8. It is seen from
Fig. 8 that the uptake of
[3H]valacyclovir after a 30-min incubation period was 1.8-fold
higher (p < 0.05) than that obtained with control HeLa cells.
Figure 8 also shows the
[3H]valacyclovir uptake results obtained with HeLa cells
overexpressing PEPT1. The valacyclovir uptake was found to be 1.6-fold
(p < 0.05) greater in HeLa cells overexpressing PEPT1 compared
with that obtained in normal HeLa cells. The valacyclovir uptake in HeLa/HPT1
cells and in HeLa/PEPT1 cells was not statistically different (p =
0.296).
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Discussion |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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;
Soul-Lawton et al., 1995). The
absence of detectable amounts of valacyclovir in plasma also suggests rapid
conversion of valacyclovir to acyclovir following transport.
The significant positive linear correlations of absorption parameters
following valacyclovir oral administration with expression levels of 4F2hc, a
membrane glycoprotein, PROT, a proline transporter, and HPT1, a less widely
examined peptide transporter that has been reported to be present in the human
intestine (Dantzig et al.,
1994; Yang, 1998;
Yang et al., 1999), suggests
their possible involvement in valacyclovir transport. The lack of positive
linear correlations between valacyclovir pharmacokinetic parameters and PEPT1
expression levels (Figs. 2 and
3B) is rather surprising in
light of previous studies that demonstrated dipeptide and valacyclovir
transport by this oligopeptide transporter (Han et al.,
1998a,b;
Oh et al., 1999;
Chu et al., 2001;
Shin et al., 2003). The
absence of significant positive correlations of valacyclovir absorption
parameters with organic cation, organic anion, and nucleoside transporters
strongly suggests that conclusions based on rat perfusion studies may not be
tenable in humans. Although no direct evidence of valacyclovir transport by
organic cation, organic anion, and nucleoside transporters has been reported,
it appears that the contribution of these transporters and PEPT1 to
valacyclovir transport and subsequent absorption may be negligible in vivo
compared with that from HPT1.
HPT1 is an intestinal peptide transporter that was identified from Caco-2
membrane proteins and reported almost simultaneously with the discovery of
rabbit PEPT1 (Dantzig et al.,
1994; Fei et al.,
1994). HPT1, containing 832 amino acids with a reported mass
120 ± 10 kDa, is apically expressed in Caco-2 cells and may
contain one to six transmembrane domains
(Hoffman and Stoffel, 1993;
Dantzig et al., 1994). HPT1 and
PEPT1 exhibit only 16% identity and 41% similarity in their amino acid
sequences (Liang et al.,
1995). The PEPT1 transporter has been extensively studied for its
role in transporting a variety of peptides and peptidomimetic compounds
(Oh and Amidon, 1999;
Oh et al., 1999). This 708
amino acid transporter has been functionally expressed in a variety of cell
systems including Chinese hamster ovary cells and HeLa cells
(Covitz et al., 1996;
Han et al., 1999;
Surendran et al., 1999;
Chu et al., 2001;
Sun et al., 2001). It was
demonstrated that PEPT1 in overexpressed cells transported several di- and
tripeptides as well as a few peptidomimetic compounds but was not capable of
transporting amino acids. These studies have established that PEPT1 is a
proton-coupled, low-affinity, high-capacity transporter, with substrate
Km values in the millimolar range.
The expression of several oligopeptide transporters in human and rat
gastrointestinal tracts and in Caco-2 cells obtained using RT-PCR and Southern
blot analysis has recently been reported
(Herrera-Ruiz et al., 2001).
The authors found that PEPT1 was predominantly expressed in the human
duodenum, with minimal expression in the jejunum and ileum. HPT1 expression,
however, was significant in all regions of the gastrointestinal tract. In
contrast, the authors found that the rat isoforms of PEPT1 and HPT1 were
widely expressed throughout the rat gastrointestinal tract. The results
reported by Herrera-Ruiz et al.
(2001) are consistent with an
earlier report of the discovery of rPEPT1 and rPT1 in rat intestine that were
found to be evenly distributed in various small intestine regions
(Erickson et al., 1995).
Furthermore, Erickson et al.
(1995) found that a
high-protein diet induced a 1.5- to 2-fold increase in rPEPT1 and rPT1 mRNA
expression in the mid and distal regions of intestine suggesting a role for
the two transporters in peptide transport. Dantzig et al.
(1994) also detected HPT1
protein along the entire human gastrointestinal tract. Sun et al.
(2002) compared PEPT1 and HPT1
expression levels in Caco-2 cells with that in human duodenum using microarray
analyses. These microarray results indicated that in differentiated Caco-2
cells PEPT1 expression levels were 45-fold lower than HPT1 expression levels.
Interestingly, HPT1 levels in differentiated Caco-2 cells and in human
duodenum were similar (Sun et al.,
2002). The findings of Herrera-Ruiz et al.
(2001) and of Dantzig et al.
(1994) suggest that HPT1 may
play an important role in peptide and peptidomimetic transport. Indeed, in
Dantzig's pioneering study (Dantzig et al.,
1994), up to 90% of cephalexin uptake in Caco-2 cells was
attributed to HPT1. Additionally, the uptake of bestatin into Chinese hamster
ovary/HPT1 cells has also been demonstrated
(Dantzig et al., 1994). The
HPT1 mediated uptake in the two cell systems was found to be proton dependent
and inhibited by dipeptides. The active transport of cephalexin and
p-hydroxyloracarbef into liposomes reconstituted with purified HPT1
protein further supports its capacity to transport peptidomimetic substrates
independent of regulatory factors (Yang,
1998).
The PEPT1 and HPT1 expression levels in the human biopsy samples obtained
from microarray data were validated with RT-PCR. The excellent correlation
between the microarray and RT-PCR mRNA patterns indicates the reliability of
the microarray analyses. The positive correlation observed between HPT1
expression and valacyclovir-related pharmacokinetics suggested that
valacyclovir might be a HPT1 substrate. We therefore investigated this
previously unreported relationship in vitro. The functionality of the HPT1
transporter in facilitating valacyclovir uptake was examined using HeLa cells
that were transfected with a HPT1/pcDNA3.0 construct. Enhanced mRNA expression
in the transfected cells compared with normal HeLa cells confirmed HPT1
overexpression in the transfected cells. The 1.8-fold enhancement of
[3H]valacyclovir uptake in HeLa/HPT1 cells compared with the
controls suggests quite clearly the ability of HPT1 to transport valacyclovir.
The ability of PEPT1 to transport valacyclovir was determined as a positive
control. The observed 1.6-fold enhancement of [3H]valacyclovir
uptake in HeLa cells overexpressing PEPT1 was comparable to that reported by
Balimane et al. (1998). These
results demonstrate that valacyclovir is a substrate for both transporters and
that they appear to have similar valacyclovir transport abilities. Therefore,
it is quite likely that in vivo, the much higher expression levels of HPT1
compared with PEPT1 may determine its predominance in valacyclovir
transport.
Recently, the nonlinear absorption of valacyclovir as a function of dose
was simulated using ACAT (GastroPlus)
(Bolger et al., 2003). The
authors found that a uniform transporter distribution predicted absorption
better than one whose expression decreased aborally in the intestine. These
modeling results also point to the possibility that valacyclovir absorption in
humans might be influenced by HPT1. Besides PEPT1 and HPT1, the peptide
transporters PTR3 and PHT1 are also known to be expressed in human intestine
(Herrera-Ruiz et al., 2001).
The expression levels of PTR3 and PHT1 were not determined in this study and
their possible involvement in valacyclovir transport cannot be ruled out. The
combined results presented here are consistent with suggestions that more than
one peptide transporter may be involved in facilitating transport of peptides
and peptidomimetics (Grauland and Sadee,
1997; Botka et al.,
2000; Herrera-Ruiz et al.,
2001).
In evaluating other potential factors that may contribute to valacyclovir
absorption, we investigated the role of channels, exchangers, and metabolizing
enzymes. The positive correlations observed between pharmacokinetic parameters
and proton and ion exchanger expression levels may be the result of their
modulating effects on the proton-dependence of the oligopeptide transporters.
Thus, enhanced expression of Na+/H+ exchangers such as
NHE-1 and NHE-3 that reside on the apical enterocyte membrane could produce a
larger proton gradient across the intestinal membrane and contribute to more
active peptide transport (Thwaites et al.,
2002). Similarly, ion channels and exchanger proteins that may not
be directly involved in valacyclovir transport may contribute to ion gradient
generation that could potentially influence the ion coupled transporters. For
instance, oligopeptide transporters are proton cotransporters and require a
proton gradient that is maintained by Na+/H+ exchangers
on the luminal membrane, whereas the Na+/K+-ATPases on
the basolateral membrane regulate the cellular Na+ concentration.
The significant negative correlations of pharmacokinetic parameters with
expression levels of MDR1, MRP2 (cMOAT), and the cytochrome P450 IIIA
subfamily member genes may indicate that these genes are involved in
valacyclovir efflux and metabolism
(Sandusky et al., 2002;
Dantzig et al., 2003).
The overall absorption parameters of valacyclovir and acyclovir following oral administration undoubtedly are determined by several interdependent processes such as intestinal transport, gut, and liver metabolism, efflux, as well as secondary effects such as ion and pH gradients, and regulatory and transcription factors. The simple univariate correlation results of microarray expression analyses of human duodenal biopsies with absorption parameters following oral valacyclovir and acyclovir administration presented in this study are a first step toward understanding the roles and interdependence of these factors.
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Acknowledgements |
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ABBREVIATIONS: OAT, organic anion transporter; OCT, organic cation transporter; HPLC, high-performance liquid chromatography; RT-PCR, reverse transcription-polymerase chain reaction; PEPT1, oligopeptide transporter; HPT1, human oligopeptide transporter; MES, 4-morpholineethanesulfonic acid; AUC, area under the curve; CNT2, concentrative purine nucleoside transporter.
Address correspondence to: Gordon L. Amidon, College of Pharmacy, University of Michigan, Ann Arbor, MI 48109-1065. E-mail: glamidon{at}umich.edu
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