Download effect of antimetabolite immunosuppressants on flaviviridae

0041-1337/04/7704-562/0
TRANSPLANTATION
Copyright © 2004 by Lippincott Williams & Wilkins, Inc .
Vol . 77, 562-567, No . 4, February 27, 2004
Printed in U.S.A .
EFFECT OF ANTIMETABOLITE IMMUNOSUPPRESSANTS ON
FLAVIVIRIDAE, INCLUDING HEPATITIS C VIRUS
JASON
R.
STANGL, KATHLEEN
Background. Recurrence of hepatitis C virus (HCV)
after liver transplantation is almost universal and decreases both graft and patient survival . Medications
that alter nucleic acid metabolism, including some
common immunosuppressants used in HCV-infected
patients, may affect viral replication .
Methods . Bovine viral diarrhea virus (BVDV) is in
the Flaviviridae family and is closely related to HCV .
We measured the effect of two immunosuppressants,
azathioprine (AZA) and mycophenolate acid (MPA), on
both BVDV replication by plaque assay and host-cell
replication by flow cytometry. We also compared the
effect of ribavirin and AZA on the level of HCV replicon RNA by real-time reverse-transcriptase polymerase chain reaction.
Results. At doses that achieved similar cytotoxicity,
AZA decreased BVDV replication 10 times more than
MPA. The inhibition of BVDV by AZA occurred at
lower doses than the cellular cytotoxicity and did not
depend on cytotoxicity. A two-log reduction in viral
titers occurred despite blocking the cytotoxicity of
AZA by inhibiting ribonucleotide reductase with high
concentrations of thymidine . A metabolite of AZA,
6-mercaptopurine, still possessed this antiviral effect,
but a metabolite further downstream, 6-thioguanine,
did not, even though 6-thioguanine is the metabolite
responsible for cellular toxicity . The effect of AZA on a
HCV replicon was at least as large as that of ribavirin .
Conclusions. This report suggests that AZA is a more
potent antiviral than MPA for Flaviviridae and may
exert a specific antiviral effect on HCV . Additional
clinical studies to investigate this previously unanticipated antiviral effect of AZA on HCV in the posttransplant setting are indicated.
Hepatitis C virus (HCV) is the most common indication for
liver transplantation in developed countries . Reinfection of
liver allografts by HCV is virtually universal . Moreover, the
natural history of HCV is accelerated after liver transplantation, and there are reports that mortality after liver transplant for HCV is increasing recently (1) . The presence of
active HCV infection decreases patient survival and liverallograft survival in recipients of orthotopic liver transplantation (2) . Hepatitis C viremia usually exceeds pretransplantation levels (3), and it is unknown whether new specific viral
variants are selected after transplantation . In addition, the
L.
CARROLL, MITCHELL ILLICHMANN, AND ROBERT STRIKER
amount each individual immunosuppressant drug contributes to the acceleration of the natural history of HCV is
controversial (4) . Answering these questions is difficult given
the lack of cell-culture models for HCV . This requires a study
of HCV in the context of closely related viruses or with HCV
replicons, which consist of RNA elements coding for nonstructural proteins of HCV that can autonomously replicate
in Huh7 hepatoma cells (5) .
Ribavirin has been shown to enhance the activity of interferon in the treatment of HCV and increase the percentage of
sustained responders (6) . The exact mechanism of this enhancement is unknown, although at high concentrations,
ribavirin can be incorporated into RNA viral genomes and
decrease replication (7) . Antimetabolite immunosuppressants have some commonalties with ribavirin . Both ribavirin
and mycophenolate acid (MPA) inhibit inosine monophosphate dehydrogenase (IMPDH) (8, 9) . This and other data
have led some to quantify the antiviral effect of MPA and
other IMPDH inhibitors (9) . At the same time, ribavirin and
three metabolites of azathioprine (AZA) are all processed to
monophosphate nucleotides by IMPDH intracellularly (8,
10), which then compete with endogenous nucleotide pools .
Clinical data investigating potential antiviral effects of either AZA or MPA and whether different immunosuppressive
regimens are associated with different rates of HCV recurrence are conflicting (11-15) . These trials are also difficult to
compare given a number of variables, such as genotypically
different viruses, different immunosuppressive regimens,
and different patient populations . To address the limited
question of whether MPA or AZA have specific antiviral
effects because of their similarities to ribavirin, we turned to
a virus closely related to HCV and in the same viral family,
bovine viral diarrhea virus (BVDV), which can be grown in
cell culture . Using BVDV as a surrogate for HCV, we directly
measured the antiviral effect of antimetabolite immunosuppressants independent of their effects on the adaptive immune system . In this report, we demonstrate that AZA has
significantly more specific antiviral activity than MPA . In
addition, we show that the antiviral activity of AZA is comparable with that of ribavirin on an HCV replicon .
RS received support from IRG-58-011-45-03 from the American
Cancer Society and holds a basic science grant from the American
Society of Transplantation, and JS is supported by an NIH fellowship T32 CA09614-15 . There are no conflicts of interest to report .
Department of Medicine, University of Wisconsin-Madison, Madison, Wisconsin.
Address correspondence to : Dr . Robert Striker, University of Wisconsin Medical School, 1300 University Avenue, Room 4638 MSC,
Madison, WI 53706-1532 . E -mail : rtstriker@wisc .edu .
Received 11 July 2003 . Accepted 16 September 2003 .
562
DOI : 10 .1097/01 .TP .0000114610 .40412 .C6
MATERIALS AND METHODS
Madin-Darby bovine kidney (MDBK) cells (ATCC CCL22, Manassas, VA) were grown in Dulbecco's modified Eagle's medium-F12
(Cellgro, Herndon, VA) supplemented with 10% heat-inactivated
bovine serum (Atlanta Biologicals lot #k0041, Norcross, GA) that
was demonstrated to be free of cytopathic and noncytopathic BVDV
by enzyme-linked immunoadsorbent assay and antibodies to BVDV
type 1 strains by serum neutralization assay . The cells were also
tested for BVDV contamination by reverse-transcriptase polymerase
chain reaction (RT-PCR) (16) . Cytopathic pNADL BVDV viral stock
that had been extensively passed in this media was kindly provided
by Ron Schultz, University of Wisconsin . AZA, MPA, 6-methyl mercaptopurine, 6-thioguanine, and thymidine were purchased from
February 27, 2004
STANGL ET AL .
Sigma (St . Louis, MO) . 6-mercaptopurine and ribavirin were purchased from ICN (Costa Mesa, CA) .
563
al. (17) . Samples were analyzed with the ABI 7700 Sequencer (Langenfeld, Germany) and the OOCt calculated according to Stuyver et
al. (18) .
Plaque Assays
Unless otherwise noted, freshly seeded MDBK cell monolayers
(1x10 5 cells in a 100 mm dish) were seeded in the presence of
varying concentrations of antimetabolite drugs and incubated for 3
hours at 37°C, 5% C02 . Then, a low multiplicity inoculum (approximately 0 .01 pfu/cell of cp BVDV) was added, and cells with virus
were further incubated for 72 hours, after which the supernatant
was collected . Mock-infected plates with the same drug exposure for
the same amount of time were trypsinized and counted in triplicate
with a flow cytometer . Serial dilutions of each supernatant were
added to 4-hour old, newly seeded monolayers without any drug . One
hour after infection, the inoculum was removed, and MDBK cell
medium containing 1% methylcellulose was added to the monolayers . Plaques were counted 96 hours postinfection . Dilutions that
gave approximately 25 to 75 plaques per plate were repeated in
triplicate .
Real-Time RT-PCR
HCV 1bN replicon with no adaptive mutations transfected into
Huh7 cells (clone 1) (5) was kindly supplied by Stanley Lemon,
University of Texas, Galveston . Subconfluent cells were incubated
for 72 hours in the presence of media containing 1 mM thymidine
alone or thymidine with either 100 µM ribavirin or 100 µM AZA .
RNA was isolated with Trizol (Invitrogen, Carlsbad, CA) according to
manufacturers instructions, and 50 ng of total RNA was used per
replicate of the real-time PCR assay . Primers and probes for the
HCV 5' untranslated region as well as cellular glyceraldehydes-3phosphate dehydrogenase were identical to that used by Cheney et
RESULTS
Comparison of Azathioprine and Mycophenolate on Bovine
Viral Diarrhea Virus
To begin to look for selective pressure that altered nucleotide pools may place on RNA viruses, we measured BVDV
growth in MDBK cells exposed to MPA or AZA . When cells
were actively replicating (subconfluent cells), both MPA, and
to a much lesser extent AZA, had cytostatic and toxic effects
in addition to any potential specific antiviral effects (data not
shown) . The cytostatic and toxic effects of MPA, presumably
attributable to decreased de novo purine synthesis from IMPDH inhibition, were such that even very low concentrations
of MPA killed all the cells when the cells were rapidly dividing . By allowing cells to reach confluency and a slower
growth rate before exposing cells to drug and virus, the effect
of the drug on viral replication on an intact cell monolayer
could be evaluated (Fig. 1) . Under these conditions, the antiviral effect of AZA was larger than that of MPA, with only
12% of the virus produced per living cell grown in AZA
compared with the amount of virus produced per living cell
grown in MPA when concentrations of both drugs caused an
approximately similar (approximately 50%) decrease in cell
growth .
1000 -
"'
a
100
L
L
O
L
d
a
E
c
- +
10
AZA cells
-MPA cells
--0--AZA virus
- fl--MPA virus
v
w
0
R
L
e
0
0
2x
6x
18x
Drug (uM)
FIGURE 1 . Azathioprine (AZA) has a larger specific antiviral effect than mycophenolic acid (MPA) on bovine viral diarrhea
virus (BVDV) in confluent cells . Confluent Madin-Darbin bovine kidney (MDBK) cells were grown in varying concentrations
of AZA (circles) or MPA (squares), with (open symbols) and without (closed symbols) virus . After 72 hours, host cells were
counted by flow cytometry, and virus was titered by plaque assay . Because of the cytotoxicity of MPA, confluent cells (closed
symbols) were exposed to doses of AZA and MPA that allowed similar amounts of cellular growth (x=0 .1 for MPA and 1 for
AZA) . The amount of BVDV virus (open symbols) generated was significantly less in cells exposed to AZA than MPA .
564
Vol . 77, No . 4
TRANSPLANTATION
Effect of Azathioprine on Bovine Viral Diarrhea Virus does
not Depend on Cytotoxic Effects
an appreciable effect on cell growth, similar to the prodrug
AZA itself. However, 6-mercaptopurine still caused a two-log
The cytostatic and toxic effects of AZA are more modest decrease in BVDV replication, whereas 6-thioguanine had no
than MPA and thus allowed the addition of AZA to rapidly effect .
dividing cells, which in turn allowed more robust viral proHCV Replicon is More Sensitive to Azathioprine than
duction . With increasing concentrations of AZA, viral producRibavirin at Equivalent Doses
tion was significantly curtailed, even at concentrations
where no detectable decrease in cell growth occurred (Fig . 2) .
Cell confluency affects the amount of HCV replicon per cell
The decrease in cell growth caused by AZA at higher concen(18, 20), and, because high concentrations of AZA without
trations can be prevented by high concentrations of thymi- thymidine does affect Huh7 cell confluency (data not shown),
dine (1 mM) . Under these conditions, DNA synthesis is lim- we also tested the HCV replicon in the presence of thymidine .
ited, so there should be no production and incorporation of Huh7 cells bearing the 1bN replicon were grown in the presthe toxic metabolite of AZA, 6-thioguanosine triphosphate, ence of 1 mM thymidine with no other drug, AZA, or ribaviinto cellular DNA (10) (Fig . 3) and therefore no cytotoxicity. rin . After 72 hours, total RNA was isolated, and equivalent
The viral suppression (Fig . 2) still occurred, even though, amounts were assayed by real-time RT-PCR with probes to
under these conditions, AZA caused no change in host cell the viral 5' untranslated region and a cellular housekeeping
growth . Because only a small minority of hepatocytes in a gene to normalize the results . AZA reliably produced almost
diseased liver (and even smaller minority in a normal liver) a one-cycle increase in the number of cycles required to reach
are actively dividing (19), this cellular state of high thymi- the critical threshold (C t ) compared with no drug, which
dine causing low cell turnover may mimic liver tissue better corresponds to only approximately 50% as much replicon in
than rapidly dividing cells in a tissue culture dish .
cells exposed to AZA (Fig . 5) . Although this change was small
compared with the effect of AZA on BVDV, it was of similar
6-Mercaptopurine, but not the Downstream Metabolite 6magnitude or larger than the change on the HCV replicon
Thioguanine, is the Likely Mediator of Azathioprine's
because of equimolar amounts of ribavirin .
Antiviral Effect
To begin to determine the mechanism of the antiviral effect
DISCUSSION
of AZA, we examined whether any of the metabolites of AZA
(Fig. 3) also had antiviral activity . 6-methyl mercaptopurine,
AZA has been used in liver transplantation for more than
6-mercaptopurine, and 6-thioguanine were each individually 30 years . It is a prodrug that is converted to 6-mercaptopuadded to MDBK cells with or without BVDV . 6-methyl mer- rine and eventually into 6-thioinosine and 6-thioguanosine
captopurine had no effect on cell growth or viral yield, triphosphate (Fig . 3) . The triphosphate of 6-thioguanosine is
whereas both 6-mercaptopurine and 6-thioguanine decreased converted to deoxy6-thioguanosine and incorporated into celcell growth and viral yield (data not shown) . To isolate the lular DNA (21) . The thioribonucleotides are also available for
antiviral effect, cells and virus were grown in the presence of inhibition or incorporation by viral enzymes including the
thymidine with either 6-mercaptopurine or 6-thioguanine RNA polymerase . Therefore, AZA could share some proposed
(Fig . 4) . In the presence of thymidine, neither metabolite had mechanisms with ribavirin that are dependent upon both
1000 --AZA inhibition of
RNA viral replication is more
profound than the inhibition
of cell growth. MDBK cells
were grown in varying concentrations of AZA with (dashed
lines) and without (solid lines)
virus and with (open symbols)
and without (closed symbols)
thymidine (T) . After 72 hours,
host cells were counted by flow
cytometry, and virus was titered by plaque assay . With increasing AZA, viral replication
is inhibited 10- to 100-fold
more than cell growth . In the
presence of thymidine, no decrease in cell survival was
seen, but the majority of the
antiviral effect was maintained. This shows the antiviral effect of AZA occurs in the
absence of cell death .
FIGURE 2 .
w
°
L
100
°
~~
v
10 -
-k-cells
- --virus
cells with T
o-- virus with T
\~
-
y
~~
V
~~
`o
0
0
10
20
30
AZATHIOPRINE (uM)
40
50
February 27, 2004
STANGL ET AL .
H,C
\ N~\\
Azathioprine
NO,
NH
N
S
N ~ ~~ H
H
N_:'~
6-mercaptopurine
~l
6-thioguanine
6-methyl mercaptopurine
MeMPR
~-
MPR
time
MetIMP
MetlOP
MetITP
TGR
tGMP
~,
tIDP
ttrP
tGDP
tGTP
tdGTP_ .
DNA
Thiopurine metabolism . AZA is a prodrug that is
metabolized to 6-mercaptopurine . 6-mercaptopurine is converted to 6-methyl mercaptopurine (MeMP) or 6-mercaptopurive riboside (MPR) . In turn, MPR is converted to mono-, di-,
and triphosphate derivatives of thioinosine (tIM(D,T)P),
methyl thioinosine (MetIM(D,T)P), and thioguanosine (tGM(D,T)P) . Any of these metabolites could be responsible for the
antiviral effect, and any triphosphate could potentially be
incorporated into the viral genome . Only 6-thioguanosine is
processed by ribonucleotide reductase into a deoxyribonucleotide (tdGTP) and incorporated into cellular DNA, and
this step is blocked by 1 mM thymidine. Incorporation of
above thiopurine nucleotides into cellular RNA has not been
observed (10) . Metabolites of AZA decrease purine synthesis
through effects on glutamine-5-phosphoribosylpyrophosphate amidotransferase, whereas both mycophenolate acid
(MPA) and ribavirin decrease GTP synthesis by inhibiting
inosine monophosphate dehydrogenase .
FIGURE 3.
drugs being triphosphorylated and recognized by the viral
replication machinery .
The effect of ribavirin and many other antivirals as monotherapy is quite small and, at least in the case of HIV, the
effect of nucleoside analogs is quickly obscured by the selection of viral resistance . In certain situations, the selection of
less-fit HIV viruses with mutant polymerases can be clinically preferable to wild-type virulent HIV (22) . A similar
detailed understanding regarding the selection of mutant
HCV viruses is lacking even though the HCV polymerase is
the likely target of ribavirin and possibly an indirect target of
other antimetabolite drugs, including IMPDH inhibitors .
565
Ribavirin monophosphate is a competitive inhibitor of IMPDH and is further phosphorylated to a triphosphate nucleotide analog which, at least in vitro, is a substrate for the
HCV genotype lb polymerase (23) . Metabolites of AZA also
decrease purine synthesis through a different mechanism
(inhibition of glutamine-5-phosphoribosylpyrophosphate
aminotransferase) (21), whereas MPA and ribavirin both inhibit guanosine triphosphate (GTP) synthesis by inhibiting
IMPDH . Because 6-mercaptopurine has an antiviral effect
and 6-thioguanine does not, but both decrease purine synthesis, the indirect effects of AZA on purine synthesis do not
seem to be sufficient for the antiviral activity . These results
are consistent with two recent studies that show ribavirin
has weak antiviral (hepatitis GB) (24) and anti-HCV replicon
activity (25), but MPA has none . Furthermore, MPA has been
shown to lack clinically significant antiviral activity as monotherapy in nontransplant HCV patients (26) . Ours is the first
study to demonstrate antiviral activity of AZA and compare
its magnitude with that of MPA and ribavirin .
Mycophenolate mofetil, the prodrug of MPA, clearly decreases the risk of rejection relative to AZA in clinical trials
(11, 12) . Yet, even in the face of more rejection, AZA has been
associated with a variable amount of HCV recurrence . One
study showed less recurrence with an AZA-containing regimen versus a non-AZA-containing regimen (13), whereas
another found more HCV recurrence in patients with higher
doses of AZA and corticosteroids (14) . Meanwhile, mycophenolate mofetil has also been associated with less HCV recurrence than AZA (11), no benefit compared with a regimen
without mycophenolate mofetil or AZA (12), or an increased
risk of graft failure (15) .
Given the molecular effect demonstrated in this report,
several issues need to be considered that might explain the
disparities in clinical trials . First, the degree of sensitivity to
AZA may depend on the specific strain of HCV . Second,
immunosuppressive regimens in transplantation are rapidly
evolving multicomponent cocktails that are difficult to compare between, and sometimes within, one study population .
Third, thiopurine metabolism differs between patients because of polymorphisms in thiopurine methyltransferase and
possibly other loci, which may lead to variability in thiopuring levels after drug administration . Bergan et al . (27)
showed significant variability in 6-mercaptopurine plasma
levels after oral AZA dosing in renal-transplant patients,
with plasma concentrations up to approximately 1 µM . Although this represents the lower range of 6-mercaptopurineused in our study, previous data indicates that thiopurine
efficacy correlates more with intracellular nucleotide concentrations as opposed to plasma levels (28) . Furthermore, studies in mice suggest that hepatic tissue levels of thiopurine
metabolites are approximately fourfold greater than plasma
levels (29) .
In conclusion, we present evidence that the antiviral effect
of AZA is mediated by a thioinosine metabolite, perhaps by
being incorporated into the viral genome, as has been suggested to occur with ribavirin . If it is incorporated, the antiviral effect could be mediated either through the induction of
mutations (5) or by altering RNA structure (18), which, in
turn, may affect enzyme processivity, ribosome translation,
or other properties of an RNA genome . Whether this will
prove to be clinically useful depends in part upon how large
the antiviral effect is relative to its immunosuppressive effect
566
Vol. 77, No . 4
TRANSPLANTATION
1000 -
-------------
100 g
a
o
\
\
-~ 6MP cells
_
-a-6TG cells
\
u
o
o
°
o
\
10-
\
\
--0--6MP virus
\
--0--6TG virus
\
\
O
---------------------
0
1
1
0
10
20
30
40
50
Drug (uM)
4.
Antiviral effect of AZA is mediated through 6-mercaptopurine but not 6-thioguanine . Viral plaque-forming units
and cell numbers were determined as a function of drug equivalent thiopurines . The addition of 6-mercaptopurine (open
circles, dashed line) significantly reduced the amount of virus produced in a dose-dependent manner, but 6-thioguanine (open
squares, dashed line) did not . Their effects on the cells, however, are similar .
FIGURE
and obviously requires validation in more clinical trials . Case AZA causes the overall effect to be proviral, the antiviral
reports exist of patients with both autoimmune hepatitis and effect may still select for specific viral mutants that are
hepatitis C treated with AZA . However, the effect of AZA on synergistic (or antagonistic) to other antivirals . With this in
HCV viral load in this setting is uncertain and is potentially mind, AZA's role in liver transplantation should be reevaluconfounded by the concomitant use of corticosteroids (30) .
ated to determine whether it affects viral load or selects for
Theoretically, even if the immunosuppressive property of AZA-resistant HCV . In a broader sense, the studies described
here indicate an important line of future inquiry into the
effects of HCV and other viruses on allograft integrity . They
.4
1
show that with appropriate models, it should be possible to
parse the conflicting actions of immunosuppressants on viro7 .2
logic behavior through their direct actions, without the confounding effects of altered adaptive immunity .
Acknowledgments. The authors thank Ron Schultz for providing
0 .8
guidance on BVDV free serum and viral stocks and Michael Lucey
AACt
for helpful discussions .
0 .6
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0 .4
0 .2 .
0 -AZA
RIB
5.
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DOCt is the difference of ACt with drug minus ACt without
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