Immunopharmacology 47 Ž2000.

85–118
www.elsevier.comrlocaterimmpharm

Review
Mycophenolate mofetil and its mechanisms of action
Anthony C. Allison a,b,) , Elsie M. Eugui a,b
a
SurroMed Incorporated, 1060 E. Meadow Circle, Palo Alto, CA 94303, USA
b
Roche Bioscience, Palo Alto, CA 94303, USA
Received 8 December 1999; accepted 8 December 1999

Abstract

Mycophenolate mofetil ŽMMF, CellCept w . is a prodrug of mycophenolic acid ŽMPA., an inhibitor of inosine
monophosphate dehydrogenase ŽIMPDH.. This is the rate-limiting enzyme in de novo synthesis of guanosine nucleotides. T-
and B-lymphocytes are more dependent on this pathway than other cell types are. Moreover, MPA is a fivefold more potent
inhibitor of the type II isoform of IMPDH, which is expressed in activated lymphocytes, than of the type I isoform of
IMPDH, which is expressed in most cell types. MPA has therefore a more potent cytostatic effect on lymphocytes than on
other cell types. This is the principal mechanism by which MPA exerts immunosuppressive effects.
Three other mechanisms may also contribute to the efficacy of MPA in preventing allograft rejection and other
applications. First, MPA can induce apoptosis of activated T-lymphocytes, which may eliminate clones of cells responding
to antigenic stimulation. Second, by depleting guanosine nucleotides, MPA suppresses glycosylation and the expression of
some adhesion molecules, thereby decreasing the recruitment of lymphocytes and monocytes into sites of inflammation and
graft rejection. Third, by depleting guanosine nucleotides MPA also depletes tetrahydrobiopterin, a co-factor for the
inducible form of nitric oxide synthase ŽiNOS.. MPA therefore suppresses the production by iNOS of NO, and consequent
tissue damage mediated by peroxynitrite.
CellCept w suppresses T-lymphocytic responses to allogeneic cells and other antigens. The drug also suppresses primary,
but not secondary, antibody responses. The efficacy of regimes including CellCept w in preventing allograft rejection, and in
the treatment of rejection, is now firmly established. CellCept w is also efficacious in several experimental animal models of
chronic rejection, and it is hoped that the drug will have the same effect in humans. q 2000 Elsevier Science B.V. All rights
reserved.

Keywords: CellCept w ; Mycophenolate mofetil; Mycophenolic acid; Inosine monophosphate dehydrogenase; Immunosuppression; Adhesion
molecules; Nitric oxide; Chronic rejection

AbbreÕiations: ADA — adenosine deaminase; ADP — adenosine diphosphate; AMP — adenosine monophosphate; APP — adenosine
triphosphate; AZA — azathioprine; CsA — cyclosporin A; dATP — deoxyadenosine triphosphate; dGTP — deoxyguanosine triphosphate;
GDP — guanosine diphosphate; GMP — guanosine monophosphate; GTP — guanosine triphosphate; HGPRTase — hypoxanthine-guanine
phosphoribosyl transferase; IMPDH — inosine monophosphate dehydrogenase; iNOS — inducible nitric oxide synthase; MMF —
mycophenolate mofetil; MPA — mycophenolic acid; PHA — phytohemagglutinin; PRPP — phosphoribosyl pyrophosphate; PWM —
pokeweed mitogen; XMP — xanthosine monophosphate
)
Corresponding author. Tel.: q1-650-592-0233; fax: q1-650-594-1210.

0162-3109r00r$ - see front matter q 2000 Elsevier Science B.V. All rights reserved.
PII: S 0 1 6 2 - 3 1 0 9 Ž 0 0 . 0 0 1 8 8 - 0
86 A.C. Allison, E.M. Euguir Immunopharmacology 47 (2000) 85–118
1. Introduction
In 1982 our research program on immunosuppres-
sive drugs at Syntex, Palo Alto, was initiated. This
program had as a background our own experience of
cyclosporin A ŽCsA. and of purine synthesis in
lymphocytes, as well as the experience of Syntex
chemists in the synthesis of anti-inflammatory and
immunosuppressive compounds such as fluorinated
glucocorticoids. Soon after the discovery by Borel et
al. Ž1976. that CsA inhibits responses of T-lympho-
cytes in rodents, our group showed that CsA also
suppresses human T-lymphocytic responses ŽLeoni
et al., 1978.. By oral administration of CsA to
rabbits we were able to establish donor-specific tol-
erance to kidney allografts ŽGreen and Allison, 1978;
Green et al., 1979.. We were the first to use CsA for
bone marrow transplantation, establishing long-term
blood cell chimerism after termination of treatment
ŽTutschka et al., 1979.. Concurrent studies in the
laboratories of Calne et al. established the potency of
CsA in preventing organ graft rejection in several
experimental animal models. These led to clinical
trials and the development of a triple regime of CsA,
azathioprine ŽAZA. and a glucocorticoid in trans-
plantation.
In the decade that followed, this regime greatly
improved the outcome of organ transplantation.
However, using a nephrotoxic drug for kidney trans-
plantation was not ideal, and the incidence of rejec-
tion episodes was still unacceptably high Žaround
40%.. Rejection episodes require escalation of steroid
dosage or administration of anti-lymphocytic serum,
which increases the risk of infections and other
complications.
For all these reasons there seemed to be a need
for a new immunosuppressive drug with reversible
cytostatic effects which are more potent on lympho-
cytes than on other cell types, including hemato-
poietic cells, and which is free of hepatotoxicity,
nephrotoxicity, mutagenicity and other limiting side
effects. We approached the design of such a drug by
identifying a metabolic pathway more susceptible to
inhibition in human T- and B-lymphocytes than in
other cell types. Our observations on purine
metabolism in human lymphocytes, and on children
with inherited defects of purine metabolism, pro-
vided a lead. The immunosuppressive effects of AZA
also suggested that purine synthesis would be a good
target. However, AZA has several metabolites which
inhibit many enzymes ŽWolberg, 1988.. The histori-
cal background leading to our selection of inosine
monophosphate dehydrogenase ŽIMPDH. as a target,
and mycophenolic acid ŽMPA. as a drug, has been
reviewed elsewhere ŽAllison and Eugui, 1993.. When
MPA was found at Syntex to be a stronger inhibitor
of the isoform of IMPDH expressed in activated
human T- and B-lymphocytes than of the housekeep-
ing isoform of the enzyme, the validity of the selec-
tion was confirmed.
That MPA is a more potent inhibitor of the prolif-
eration of lymphocytes than of other cell types is
now widely accepted. Less well known are our
observations that MPA inhibits the glycosylation,
expression and function of adhesion molecules ŽAl-
lison et al., 1993., as well as the activity of inducible
nitric oxide synthase ŽiNOS. ŽSenda et al., 1995.. In
this paper, evidence from other laboratories confirm-
ing these observations in experimental animals and
humans is reviewed, and their relevance to transplan-
tation and other applications is discussed. More re-
cently we have found that MPA can induce apoptosis
of human T-lymphocytes ŽCohn et al., 1999a,b.,
which has interesting implications for tolerance in-
duction. Observations from several laboratories
showing that mycophenolate mofetil ŽMMF. can in-
hibit chronic allograft rejection in various experi-
mental animal models are summarized. Activity of
MMF in experimental animal models of kidney dis-
eases, autoimmune uveoretinitis, experimental au-
toimmune encephalomyelitis and adjuvant arthritis
suggest possible new clinical applications. MPA has
long been known to have antimicrobial effects; re-
cent evidence indicates that activity against Pneumo-
cystis carinii, hepatitis C virus ŽHCV. and human
immunodeficiency virus ŽHIV. may be clinically
useful.
2. Purine metabolism in lymphocytes and genetic
defects
2.1. Background
There are two major pathways of purine synthesis
ŽFig. 1.; details can be found in textbooks of bio-
 A.C. Allison, E.M. Euguir Immunopharmacology 47 (2000) 85–118 87

Fig. 1. Pathways of purine biosynthesis, showing the central position of inosine monophosphate ŽIMP.. Mycophenolic acid inhibits IMP
dehydrogenase, thereby depleting GMP, GTP and dGTP. Two rate-limiting enzymes in lymphocytes are activated by guanosine
ribonucleotides and dGTP, but inhibited by AMP, ADP and by dATP, respectively.

chemistry. In the de novo pathway, the ribose phos-
phate portion of purine nucleotides is derived from
5-phosphoribosyl-1-pyrophosphate ŽPRPP., which is
synthesized from ATP and ribose-5-phosphate, a
product of the pentose phosphate pathway. The purine
ring is assembled on ribose phosphate by a series of
steps, the first of which is catalyzed by glutamine-
PRPP aminotransferase. PRPP is also used by the
purine salvage pathways, including the major path-
way catalyzed by hypoxanthine-guanine phosphori-
bosyltransferase ŽHGPRTase.. Production of an
adequate level of PRPP is, therefore, essential for
synthesis of purine ribonucleotides by either path-
way. Ribonucleotide diphosphates ŽADP and GDP.
are converted by ribonucleotide diphosphate reduc-
tase into the corresponding deoxyribonucleotide
triphosphates ŽdADP and dGDP., which are phos-
phorylated to produce dATP and dGTP, substrates
for DNA polymerase.
2.2. Allosteric regulation of enzymes by nucleotide
concentrations
Activities of key, rate-limiting enzymes, PRPP
synthetase and ribonucleotide reductase, are allosteri-
cally regulated by nucleotides ŽFig. 1.. In bacteria
both adenosine and guanosine nucleotides, signalling
an abundance of purine nucleotides, inhibit PRPP
synthetase ŽStryer, 1988.. However, we found that
this is not the case with PRPP synthetase from
human lymphocytes ŽGarcia et al., 1977.. The en-
zyme is inhibited by adenosine nucleotides ŽAMP
and ADP. but activated by guanosine nucleotides
ŽGMP, GDP and GTP, Fig. 2.. Such regulation is a
critical factor in the therapeutic strategy discussed in
88 A.C. Allison, E.M. Euguir Immunopharmacology 47 (2000) 85–118
Fig. 2. Two experiments showing effects of added nucleotides on
the activity of 5-phosphoribosyl-1-pyrophosphate ŽPRPP. syn-
thetase from human lymphocytes ŽGarcia et al., 1977.. The en-
zyme is activated by guanosine ribonucleotides and inhibited by
AMP and ADP.
this review. The overall catalytic activity of ribonu-
cleotide reductase is decreased by binding of dATP,
whereas binding of dGTP stimulates ADP reduction
ŽStryer, 1988.. It follows that an excess of adenosine
nucleotides andror depletion of guanosine nu-
cleotides can decrease the pool of PRPP, and that an
excess of dATP andror depletion of dGTP can
inhibit ribonucleotide reductase activity, thereby de-
creasing the pool of substrates required for DNA
polymerase activity. In other words, adequate levels
of guanosine and deoxyguanosine nucleotides are
required for proliferative responses of lymphocytes
to antigenic and mitogenic stimulation, whereas an
excess of adenosine or deoxyadenosine nucleotides
would be expected to inhibit proliferation. As shown
in Fig. 1, IMP is at the branch point in purine
nucleotide synthesis, since it can be converted to
AMP or GMP. Two enzymes decrease levels of
adenosine nucleotides relative to those of guanosine
nucleotides: adenosine deaminase ŽADA. and ino-
sine 5X-monophosphate dehydrogenase ŽIMPDH.,
which limits the rate of conversion of IMP to XMP.
From the arguments just presented, it would be
Table 1
Effects of genetic defects of enzymes catalyzing purine metabolism on the functions of different cell types in humans
Enzyme defect T-lymphocytes
Adenosine deaminase x x
Hypoxanthine-guanine phosphoribosyl transferase q q
Fig. 3. Classification of cell types according to the relative
importance of de novo purine synthesis and the major purine
salvage pathway catalyzed by hypoxanthine-guanine phosphoribo-
syltransferase ŽHGPRTase..
expected that both of these enzymes would be re-
quired for lymphocyte proliferation.
2.3. Genetic defects of purine metabolism
Giblett et al. Ž1972. showed that children with
inherited ADA deficiency have a selective decrease
in the numbers and functions of T- and B-lympho-
cytes in the presence of normal numbers of neu-
trophils, erythrocytes and platelets and normal brain
function. Shortly afterwards we showed ŽAllison et
al., 1975. that children lacking HGPRTase ŽLesch–
Nyhan syndrome. have essentially normal numbers
and functions of T- and B-lymphocytes ŽTable 1..
These findings led us to postulate ŽAllison et al.,
1975, 1977. that de novo purine synthesis is cru-
cially important for proliferative responses of human
T- and B-lymphocytes to mitogens, whereas the
major salvage pathway catalyzed by HGPRTase is
not required for lymphocyte proliferation. Children
with the Lesch–Nyhan syndrome have mental defi-
ciency and compulsive self mutilation, showing the
importance of the salvage pathway controlled by
HGPRTase for brain cells. The level of this enzyme
in brain is higher than in any other tissue, whereas
the activity of glutamine-PRPP aminotransferase,
which catalyzes the committed step in the de novo
pathway, is low in brain ŽStryer, 1988.. Thus, cell
types and tissues can be arranged according to their
B-lymphocytes Neurons Refs.
q Giblett et al. Ž1972.
x Allison et al. Ž1975.
 A.C. Allison, E.M. Euguir Immunopharmacology 47 (2000) 85–118 89

dependence on the de novo and salvage pathways of of IMPDH per cell must be increased. The impor-
purine synthesis ŽFig. 3., with lymphocytes at one tance of IMPDH in the function of lymphoid cells is
extreme, brain cells at the other and most cell types, reflected in the tissue distribution of the enzyme
able to use both pathways, occupying an intermedi- ŽTable 2.. The relative amount of IMPDH is greater
ate position. in the thymus and spleen than in other tissues, even
those that are rapidly dividing, such as the testis and
2.4. Increased de noÕo purine synthesis accompanies bone marrow. From these considerations it was rea-
lymphocyte transformation sonable to postulate that inhibiting IMPDH might
have more potent cytostatic effects on lymphocytes
The requirement of adequate PRPP levels and of than on other cell types.
de novo purine synthesis in lymphocytes responding
to antigenic and mitogenic stimulation is clear. We
found a rapid and sustained increase in PRPP con- 3. Inhibiting de novo synthesis of GMP
centrations in human lymphocytes following mito-
genic stimulation ŽHovi et al., 1975.. Elevation of 3.1. MPA
PRPP concentrations in human lymphocytes after
mitogenic stimulation has likewise been reported by From several possible IMPDH inhibitors we se-
Peters et al. Ž1982., who also found suppression of lected for detailed study MPA ŽFig. 4., a fermenta-
that elevation by addition of adenosine, as expected tion product of several Penicillium species. MPA
from Fig. 2. We also found that following polyclonal was preferred to nucleoside analogues such as mi-
activation of human lymphocytes the rate of de novo zoribine, bredinin, ribavirin and tiazofurin because
purine synthesis is increased, as shown by incorpora- the latter must be phosphorylated to inhibit IMPDH,
tion of w14 Cx-glycine into purine nucleotides ŽHovi et and because of the side effects of this class of
al., 1977.. This observation has been repeatedly con- compounds. The efficiency of nucleoside phosphory-
firmed ŽFairbanks et al., 1995.. lation can vary in different cell types ŽRichman et
For reasons already discussed, proliferative re- al., 1987., and the relevance of target cells in addi-
sponses of activated lymphocytes require that the tion to lymphocytes Že.g., cells of the monocyte-
pools of guanosine nucleotides are increased as well macrophage lineage, endothelial cells and smooth
as those of adenosine nucleotides. Hence, the activity muscle cells. is described below. Nucleoside analogs
frequently have undesirable effects, such as inhibit-
Table 2 ing DNA repair enzymes and producing chromo-
Tissue distribution of IMPDH activity
Observations of Jackson et al. Ž1977. and Hirai et al. Ž1977. for
some breaks. MPA is not a nucleoside, and is a
the rat; compiled by Wu Ž1994.. potent, non-competitive, reversible inhibitor of eu-
Tissue IMPDH Relative amount
karyotic but not prokaryotic IMP dehydrogenases
ŽmUrmg. ŽFranklin and Cook, 1969; Verham et al., 1987; Carr
Thymus 347 15.5
et al., 1993.. Contrary to early reports, MPA does
Spleen 264 11.8 not inhibit GMP synthetase, the enzyme catalysing
Testis 181 8.1 the conversion of XMP to GMP. Because of the
Ovary 159 7.1 importance of guanosine and deoxyguanosine nu-
Bone marrow 127 5.7 cleotides in activating PRPP synthetase and ribonu-
Lung 111 5.0
Adipose tissue 78 3.5
cleotide reductase, respectively, we postulated that
Brain 73 3.3 depletion of GMP Žand consequently GTP and dGTP.
Intestine 67 3.0 by inhibiting IMPDH would have antiproliferative
Liver 46 2.0 effects; furthermore, since lymphocytes rely on de
Kidney 44 2.0 novo purine synthesis, whereas other cell types do
Heart 44 2.0
Peripheral leukocytes 23 1.0
not, the cytostatic effects produced in this way would
Skeletal muscle 22 1.0 be more potent on lymphocytes than on other cell
types. Another factor increases the selectivity of
90 A.C. Allison, E.M. Euguir Immunopharmacology 47 (2000) 85–118

Fig. 4. Structure of the morpholinoethyl ester of mycophenolic acid Žmycophenolate mofetil., mycophenolic acid and its glucuronide, and
sites of interconversion and excretion.

action of MPA, namely the existence of isoforms of
the target enzyme.
4. Isoforms of IMPDH
Natsumeda et al. Ž1990. isolated two distinct
cDNA clones Žtypes I and II. encoding IMPDH from
a human spleen cDNA library. The clones encode
closely related proteins of 514 residues showing 84%
sequence correspondence. Northern hybridization
analysis of polyŽA.q RNA from normal human
leukocytes showed expression of the type I enzyme,
whereas leukemic cells and ovarian tumor cells ex-
pressed predominantly the type II gene. Subse-
quent studies ŽKonno et al., 1991; Nagai et al., 1992.

Table 3
Inhibition constants for types I and II IMPDH
Observations of Carr et al. Ž1993..
K i ŽmM.
XMP NADH MPA
Isoform Type I 80 102 0.033
Type II 0.007
Ratio ŽIrII. 0.9 1.1 4.8
Kinetics vs. substrate IMP competitive uncompetitive uncompetitive
NAD mixed noncompetitive uncompetitive
 A.C. Allison, E.M. Euguir Immunopharmacology 47 (2000) 85–118
showed predominant expression of the type I gene in
resting human lymphocytes; when the cells were
stimulated by PHA ŽT cells. or transformed by Ep-
stein–Barr virus ŽB cells. the type II gene was
strongly expressed. Both isoforms of the enzyme
have been overexpressed as nonfusion forms in E.
coli and purified ŽCarr et al., 1993.. The type II
isoform is nearly five times more sensitive to inhibi-
tion by MPA than is the type I isoform ŽTable 3..
The mechanism by which MPA inhibits IMPDH has
been discussed by Wu Ž1994. and in an accompany-
ing paper in this journal ŽSintchak and Nimmesgern,
2000..
5. Development of MMF
The strategy of chemically modifying antibiotics
to improve their activity is well known, e.g. modifi-
cations of penicillin, cephalosporin and rifamycin.
Early in the research program, synthetic effort was
focused on obtaining a derivative of MPA with
improved oral bioavailability. The morpholinoethyl
ester of MPA ŽMMF, Fig. 4. was found to have
improved bioavailability in primates as compared
with MPA ŽLee et al., 1990.. The ester is rapidly
hydrolysed to yield MPA, both in human peripheral
blood mononuclear cell cultures and in vivo. MMF
proved suitable for pharmaceutical formulation and
was selected for development. Most of our in vivo
studies were made with MMF, although comparative
studies with MPA were often included.
The in vivo metabolism of MMF is beyond the
scope of this article. MMF is rapidly converted to
MPA, which is detectable in peripheral blood. MPA
is converted to the glucuronide, biliary secretion of
which gives significant enterohepatic recycling ŽFig.
4..
6. Preferential inhibition of lymphocyte prolifera-
tion by MPA
Concentrations of MPA that are readily attainable
therapeutically inhibit the proliferative responses of
human peripheral blood mononuclear cells to phyto-
hemagglutinin Ža T-cell mitogen., pokeweed mitogen
Ža T-dependent B-cell mitogen. and Staphylococcus
91
protein A sepharose Ža B-cell mitogen. ŽEugui et al.,
1991a.. Mixed lymphocyte responses are also inhib-
ited by MMF and MPA. In all cases the IC 50 is less
than 100 nM, a concentration of drug having no
cytostatic effect on fibroblasts or endothelial cells
ŽFig. 5.. Thus the drug has more potent in vitro
cytostatic effects on lymphocytes than on other cell
types, as predicted. Nevertheless, clinically attain-
able concentrations of MPA inhibit the multiplica-
tion of human smooth muscle cells and mesangial
cells, which is relevant to effects on proliferative
arteriopathy and glomerulopathy, as discussed in
Sections 17 and 19.
When added to ongoing mixed lymphocyte reac-
tions 72 h after initiation, MPA was still found to be
inhibitory, showing that it blocks a late event in
lymphocyte responses. Flow cytometric analyses
show that in the presence of MPA, cells pass through
the G1 stage and are blocked in the S phase ŽCohn et
al., 1999a,b.. MPA and MMF also strongly inhibit
proliferation of all the human T- and B-lymphocytic
Fig. 5. Potent inhibition by mycophenolic acid ŽMPA. of the
proliferation of human peripheral lymphocytes ŽPBL. responding
to stimulation by a T-cell mitogen Žphytohemagglutinin; PHA.,
pokeweed mitogen ŽPWM. and a B-cell mitogen Žstaphylococcal
protein A-sepharose, Eugui et al., 1991a.. Higher concentrations
of MPA are required to inhibit the proliferation of human dermal
fibroblasts ŽFIB. in response to IL-1b or human umbilical vein
endothelial cells ŽEC. in response to basic fibroblast growth factor
ŽFGF.. B ECrFGF; I FIBrIL-1; v FIBrFGF; ^ PBLrPHA;
% PBLrPW; e PBLr5PAS.
92 A.C. Allison, E.M. Euguir Immunopharmacology 47 (2000) 85–118
Fig. 6. Treatment of human T-lymphocytic cells ŽCEM. with 1
mM MPA for 6 h depletes GTP and dGTP pools. Adding back
guanine in the presence of MPA restores GTP and dGTP to levels
higher than observed in the absence of MPA Ž hatched . ŽAllison et
al., 1991..
cell lines tested ŽEugui et al., 1991a.. One of these
lines lacks HGPRTase, which was experimentally
convenient.
7. Intracellular pools of GTP and dGTP
It has been postulated that G-proteins may be
involved in the transduction of mitogenic signals to
Fig. 7. Incubation of human peripheral blood lectin-stimulated lymphocytes and monocytes with 10 mM MPA significantly depletes GTP
levels whereas there is no depletion in neutrophils. G GTP; B ATP ŽAllison and Eugui, 1993..
human T-lymphocytes; depletion of GTP might
therefore affect such transduction systems. To ascer-
tain whether the antiproliferative effects of MPA are
due to depletion of GTP or of dGTP, pools of
nucleotides were measured in mitogen-activated hu-
man peripheral blood mononuclear cells and human
T-lymphocytic cell lines in the presence or absence
of MPA, and in the presence of MPA when Guo,
Gua or dGuo were added back to the culture medium
ŽFig. 6.. As predicted, MPA was found to deplete
GTP and dGTP; Gua or Guo efficiently reverse
depletion of GTP and less efficiently restore dGTP;
dGuo efficiently reverses depletion of dGTP and less
efficiently restores GTP ŽAllison et al., 1991.. In the
MPA-treated HGPRTase-deficient T-cell line, BUC-
7, dGuo was found to restore dGTP but not GTP, as
expected from metabolic pathways. Clinically attain-
able concentrations of MPA significantly deplete
GTP in human lymphocytes and monocytes but not
in neutrophils, showing the cellular selectivity of the
inhibition of IMPDH ŽAllison and Eugui, 1993; Fig.
7..
MPA or MMF Ž1 mM. was found to suppress
completely DNA synthesis in PHA-stimulated pe-
ripheral blood cells. Adding back 10 mM dGuo or 50
mM Guo restored DNA synthesis to control levels
observed in the absence of the drug, as shown by
 A.C. Allison, E.M. Euguir Immunopharmacology 47 (2000) 85–118
3
H-wTdRx incorporation ŽFig. 8.. Neither Ado nor
dAdo in any concentration tested restored DNA syn-
thesis in MPA-treated cells or had significant effects
in the absence of the drug. In HGPRTase-deficient
cells treated with MPA dGuo partially restored DNA
synthesis whereas Gua or Guo did not; the lack of
complete restoration of DNA synthesis may be due
to other effects, e.g. on membrane glyco-protein
synthesis. In polyclonally stimulated human periph-
eral blood lymphocytes, higher concentrations of
Guo than of dGuo were needed to restore DNA
synthesis ŽFig. 8., in keeping with the requirement
for conversion to dGTP. Thus the inhibition of DNA
synthesis in MPA-treated lymphocytes is primarily
due to depletion of dGTP. The findings show the
Fig. 8. MPA Ž1 mM. inhibits to baseline levels DNA synthesis in
human PBL stimulated by PHA. Addition of deoxyguanosine, or a
higher concentration of guanosine, restores DNA synthesis to
levels observed in the absence of MPA. No restoration is observed
following addition of adenosine or deoxyadenosine. Excess de-
oxyguanosine in the presence or absence of MPA decreases DNA
synthesis ŽEugui et al., 1991a.. -I- guanosine; -v- deoxyguano-
sine; -B- adenosine; -`- deoxyadenosine.
93
metabolic selectivity of action of MPA: if the drug
were acting on other enzymes or metabolic func-
tions, or on TdR transport, it would not have been
possible to restore proliferation with Guo or dGuo.
Excess dGuo inhibits DNA synthesis in the presence
or absence of MPA ŽFig. 8., showing that the dGTP
pool must be kept within a rather narrow optimal
range.
MPA was found also to deplete GTP selectively
in lymphoid cells in vivo ŽByars et al., 1995.. Rats
were immunized and boosted, and lymph nodes of
the drainage chain recovered. In rats receiving 20
mgrkg MPA twice daily for 4 days, GTP pools in
lymph node cells were significantly decreased. GTP
pools in liver, testis and bone marrow, and ATP
pools in these organs and lymph nodes, were unaf-
fected by MPA.
8. Cytokine production
CsA and FK-506 inhibit early stages of lympho-
cyte activation, including the production of IL-2. In
contrast, MMF or MPA in concentrations up to 1
mM had no detectable effect on IL-2 production in
mitogen-activated human peripheral blood lympho-
cytes ŽEugui et al., 1991a.. Unlike CsA, MPA does
not inhibit IL-2 gene expression in activated T-cells
ŽThomson et al., 1993.. Thus MPA does not inhibit
early responses to antigenic or mitogenic stimulation,
but blocks DNA synthesis in the S phase of the cell
cycle ŽCohn et al., 1999a..
Chang et al. Ž1993. stimulated a human Th0 and a
Th2 cell clone with Con A for 24 h. Production by
the former of IFN-g, GM-CSF, IL-2, IL-4 and IL-5,
and by the latter of IL-4 and IL-5, was blocked by
CsA but not by MPA Žup to 10 mM.. Nagy et al.
Ž1993. studied the effect of MPA on cytokine pro-
duction by human lymphocytes polyclonally acti-
vated by the superantigen staphylococcal enterotoxin
A. Cells were fixed and permeabilized, and stained
with antibodies to several cytokines. In stimulated
cells incubated for 24 h in the presence of MPA,
GM-CSF production was reduced, but that of other
cytokines was not. However, by 48 h after stimula-
tion, MPA inhibited the production of all cytokines
94 A.C. Allison, E.M. Euguir Immunopharmacology 47 (2000) 85–118
studied, including IL-2, IL-3, IL-4, IL-5, IL-6, IL-10,
IFN-g and TNF-a. Thus treatment of patients for
several days would be expected to decrease the
production of lymphocyte-derived cytokines, such as
IFN-g and TNF-a. Nagy et al. Ž1993. found that
CsA suppressed the production of cytokines in lym-
phocytes polyclonally activated by phorbol ester and
ionomycin, whereas MPA did not, with the excep-
tion of some late suppression of IL-3 production.
These findings emphasize the major differences be-
tween effects of MPA and CsA on lymphocytes.
8.1. Monocytes and macrophages
While the major effects of MPA and MMF are on
lymphocytes, they significantly deplete GTP levels
in monocytes ŽFig. 7.. This accelerates the differenti-
ation of human promonocytic cells, and augments
expression of the IL-1 receptor antagonist ŽWaters et
al., 1993.. Production of cytokines by monocytes
isolated from the peripheral blood of patients with
long-term stable kidney graft function and healthy
controls, stimulated by lipopolysaccharide, was com-
pared by Weimer et al. Ž1999.. In MMF and CsA-
treated patients, LPS-stimulated production of IL-1b,
TNF-a, IL-6 and IL-10 was significantly lower than
in the control group. In patients receiving MMFrCsA
therapy but no steroids even stronger suppression of
cytokine production was observed. Thus one effect
of long-term MMF therapy may be to decrease pro-
duction of pro-inflammatory cytokines and increase
production of the IL-1 receptor antagonist. This could
be a factor in the reduced expression of adhesion
molecules observed in treated patients.
9. Antibody formation in vitro
Antibody formation by polyclonally activated hu-
man B-lymphocytes was almost completely inhibited
by 100 nM MPA ŽAllison et al., 1991, Fig. 9.. In
another laboratory, therapeutically attainable doses
of MPA were found to inhibit secondary responses
of human spleen cells to tetanus toxoid; CsA did not
inhibit ongoing antibody responses ŽGrailer et al.,
1991.. Chang et al. Ž1993. found that MPA Ž1 mM.
strongly suppressed IgG4 and IgE synthesis by hu-
man B-lymphocytes activated by a monoclonal anti-
Fig. 9. Inhibition by MPA of antibody formation by human
peripheral blood lymphocytes polyclonally activated by staphylo-
coccal protein A-sepharose ŽAllison et al., 1991..
body against CD40 and IL-4 or IL-13. In vivo, MMF
inhibits primary humoral responses efficiently, but
not secondary responses ŽSections 15 and 24..
10. Induction by MPA of apoptosis in activated
T-lymphocytes
Although the principal mode of action of MPA on
lymphocytes is cytostatic, it can also induce apopto-
sis of polyclonally activated human T-lymphocytes
and of human T-lymphocytic cell lines ŽCohn et al.,
1999a,b.. Cells were stained with Hoechst 33342,
examined by epifluorescence microscopy and apop-
totic cells quantified by nuclear morphology. Elec-
tron microscopy confirmed the presence of typical
apoptotic cells. Human peripheral blood leukocytes
activated by anti-CD3 and cultured for 72 h showed
24% apoptotic cells; in the presence of 0.5 mM MPA
this was increased to 67%; and in the presence of
MPA and 20 mM Guo it was 25%. A human T-
lymphocytic cell line ŽMOLT-4., incubated with 1
mM MPA for 72 h, showed 82–98% apoptotic cells
as compared with 12% in the absence of MPA. A
lesser degree of apoptosis Ž27%. was observed in
U937 human promonocytic cells incubated with
MPA.
These findings suggest that MPA, in addition to
exerting cytostatic effects, can eliminate T-cells re-
sponding to T-cell-receptor activation and, presum-
ably, antigenic stimulation. This may be one factor
 A.C. Allison, E.M. Euguir Immunopharmacology 47 (2000) 85–118
in the induction of tolerance against alloantigens
observed in some experimental models. It may also
contribute to the successful use of MMF in human
allograft recipients. A regime for optimizing this
effect is discussed in an accompanying article ŽAl-
lison, 2000..
The induction of apoptosis by MPA may be selec-
tive for lymphocytes and monocyte–macrophage lin-
eage cells. In patients treated with MMF, the number
of apoptotic cells in renal tubular epithelium was
found to be less than in those receiving AZA, CsA
and steroids ŽPardo-Mindan ´ et al., 1999..
11. Inhibition of the glycosylation and expression
of adhesion molecules
If depletion of GTP in lymphocytes by MPA does
not impair early signal transduction in these cells, the
question arises whether it has any other important
metabolic consequences. The answer to that question
is yes: we have defined two effects which are likely
to be important in vivo, and there may be others.
Firstly, MPA-mediated depletion of GTP inhibits the
transfer of fucose and mannose to glycoproteins,
some of which are adhesion molecules facilitating
the attachment of leukocytes to endothelial cells
ŽAllison et al., 1993.. MPA also downregulates ex-
pression of adhesion molecules, for reasons that are
not fully understood. Passage of newly synthesized
proteins through Golgi and secretory vesicles is often
accompanied by glycosylation; inhibition of glyco-
sylation may impair this movement. The passage of
vesicles through secretory pathways is regulated by
small GTPases, and GTP depletion may impede this
process. By antagonizing the induced expression and
function of adhesion molecules MPA decreases the
recruitment of lymphocytes and monocytes into sites
of chronic inflammation, including those in sites of
vascularized organ graft rejection.
Surface expression of a group of adhesion
molecules, the selectins, plays a major role in the
initial interaction between leukocytes and endothelial
cells responsible for rolling; this interaction is a
prerequisite for integrin-mediated sticking ŽLaski,
1992.. Selectins are so termed because of their
lectin-like structures and properties. They share
amino acid sequences with lectins, and their comple-
95
Table 4
Adhesion molecules on leukocytes and complementary ligands on
endothelial cells
References in Laski Ž1992..
Leukocyte Endothelial cell
Lacto-N-fucapentaose P-selectin ŽGMP-140, CD62P.
III ŽCD15.
a Ž2,3.-Sialyl-a Ž1,3.- E-selectin ŽELAM-1.
fucosyl-lactosaminoglycan
L-selectin ŽLECAM-1, Sialylated fucosyl oligosaccharide
MEL14.
VLA-4 VCAM-1
mentary ligands are fucose-containing oligosaccha-
rides ŽTable 4..
While most attention has been given to comple-
mentary interactions of adhesion molecules in leuko-
cytes and endothelial cells, it is clear that adhesion
molecules also participate in the initiation and effec-
tor phases of immune responses. Interactions be-
tween antigen-presenting cells and lymphocytes re-
quire complementary binding of adhesion molecules
ŽAltmann et al., 1989., and the same is true of
interactions of effector lymphocytes with target cells
ŽGregory et al., 1988.. It follows that blocking the
interactions between complementary adhesion
molecules could exert immunosuppressive and anti-
inflammatory activity. An example is the administra-
tion of monoclonal antibodies against ICAM-1 and
its complementary ligand LFA-1 to mice that have
received cardiac allografts. Treatment with these an-
tibodies for 6 days after transplantation allows long-
term acceptance of the alloantigens ŽIsobe et al.,
1992..
Glycosylation of proteins and lipids occurs
through nucleotide intermediates ŽFig. 10.. Glucose,
galactose and their amines are transferred to dolichol
phosphate and then to proteins through uridine-di-
phospho intermediates, whereas fucose and mannose
are transferred through guanosine-diphospho inter-
mediates ŽKornfeld and Kornfeld, 1980.. We found
that, in activated human peripheral blood lympho-
cytes, treatment with MPA suppresses the transfer
of mannose to dolichol phosphate and to mem-
brane glycoproteins ŽAllison et al., 1993.. This was
shown by following labelled mannose, by measuring
the expression of mannose on the surface of the
cells, using a specific lectin for terminal mannose
96 A.C. Allison, E.M. Euguir Immunopharmacology 47 (2000) 85–118

T-lymphocyte subsets and their penetration rates

through endothelial cells in a dose-dependent fash-
ion. The ID50 values for cellular adhesion were 0.03
mM ŽCD4. and 1.0 mM ŽCD8.. The ID50 values for
cellular penetration were 1.2 mM ŽCD4. and 1.1 mM
ŽCD8.. MMF also inhibited the induced expression
of adhesion molecules on endothelial cells measured
by marked antibodies and scanning fluorimetry. IL-1
was used to induce expression of E-selectin, VCAM-
1 and ICAM-1, and prostaglandin E 2 was used to
induce expression of P-selectin. MMF was found to
suppress strongly the expression of VCAM-1, E-
selectin and P-selectin, whereas that of ICAM-1 was
Fig. 10. The role of sugar nucleotides and dolichol phosphate in only slightly affected. The optimal dose of MMF
the transfer of N-acetylglucosamine and mannose to asparagine required to suppress VCAM-1 expression was 100
residues of membrane glycoproteins. Depletion of UTP andror nM. This may be the in vitro counterpart of the
GTP inhibits glycosylation.
strong inhibition of VCAM expression reported in
patients treated with MPA ŽLi et al., 1998, Section
23.. MMF treatment of CD4 and CD8 cells sup-
ŽFig. 11., and by measuring the terminal mannose
content of different membrane glycoproteins. Im-
munoprecipitation studies showed that one of the
lymphocyte glycoproteins affected is VLA-4, the
ligand for VCAM-1 on activated endothelial cells.
Treatment of either T cells or IL-1-activated en-
dothelial cells with MPA in therapeutically attainable
doses Ž1–10 mM. decreased lymphocyte attachment
and, when both cell types were treated with MPA,
the attachment was further inhibited ŽFig. 12.. This
finding is presumably due to decreased expression of
ligands for adhesion molecules on interacting cells.
Paul et al. Ž1998. studied the induced expression
on rat endothelial cells of saccharides with terminal
mannose able to bind Galanthus niÕalis agglutinin,
measured by flow cytometry. Only 7% of unstimu-
lated endothelial cells showed lectin binding; treat-
ment with IL-1 for 42 to 72 h increased the propor-
tion of binding cells to 30% and 75% respectively.
MPA inhibited this induced expression in a dose-re-
lated fashion.
Effects of MMF have been analysed further by
Blaheta et al. Ž1999., using assays for attachment of
human peripheral blood lymphocytes to cultured Fig. 11. Flow cytometric measurements of the binding to human
human umbilical vein endothelial cells ŽHUVEC. peripheral blood lymphocytes of a labeled lectin wfluorochrome 1,
and penetration through the allogeneic endothelial Galanthus niÕalis agglutinin selective for terminal, a Ž1–3.-linked
mannosex. The lymphocytes were stimulated for 48 h with con-
cell layer. CsA does not affect attachment or canavalin A in the presence or absence of MPA added to the
transendothelial migration of lymphocytes. MMF culture medium for the last 8 h. MPA markedly decreases man-
treatment was found to inhibit both the adhesion of nose available on the cells for lectin binding ŽAllison et al., 1993..
 A.C. Allison, E.M. Euguir Immunopharmacology 47 (2000) 85–118
Fig. 12. Binding of human T cells to human umbilical vein
endothelial cells stimulated by IL-1a Ž100 ngrml. is decreased
when either the T cells or the endothelial cells are treated with
MPA. The inhibition is strongest when both cell types are treated
ŽAllison et al., 1993.. T cells: I no MPA; G 1 mM MPA; Z 10
mM MPA. On the X-axis is the concentration of MPA in endothe-
lial cell cultures.
pressed binding to receptor constructs ŽIg chimeras.
in the following order: P-selectin ) VCAM-1 )
ICAM-1) E-selectin. MMF in the doses used had
no effect on the viability of lymphocytes or endothe-
lial cells, or on esterase activity. The authors suggest
that MMF can modulate the immune system through
suppression of lymphocyte attachment and penetra-
tion through endothelial cells as well as by cytostatic
effects ŽBlaheta et al., 1999..
Treatment of human monocytes with MPA Ž10
mM. was found to decrease their attachment to
endothelial cells and to laminin, but not to type 1
collagen or fibronectin ŽLaurent et al., 1996.. How-
ever, Hauser et al. Ž1997a. reported that MPA aug-
ments TNFa-induced expression of ICAM-1 and
E-selectin in HUVEC and the binding to them of
U937 cells Ža human promonocytic cell line, not
treated with MPA.. The discrepancy between these
observations and the inhibition by MMF of E-selec-
tin expression found by Blaheta et al. Ž1999. remains
unexplained, as is the reason why the effects of MPA
reported by Hauser et al. were not reversed by
guanosine.
Effects of MMF on the expression of adhesion
molecules, and the recruitment of lymphocytes and
monocytes into rat renal allografts and diseased kid-
97
neys, are discussed in Section 17. Effects of MMF in
ischemia–reperfusion injury ŽSection 20. may be
due, at least in part, to decreased leukocyte adhesion.
MPA also inhibits the degranulation of rat peri-
toneal mast cells ŽMulkins et al., 1992.. The amount
of w3 Hx-5-HT released from granules was decreased
by 44% with 10 mM MPA. The drug had no effect
on IgE receptor-mediated production of PGD 2 . The
results suggested that depletion of GTP can decrease
the efficiency of signaling between IgE receptors and
secretory granules.
12. Effects of iNOS
Nitric oxide ŽNO., a multifunctional biological
mediator, is synthesized from the guanidino nitrogen
of L-arginine by nitric oxide synthases in several
mammalian cell types ŽMoncada et al., 1991.. The
activity of constitutive NO synthases is regulated by
Ca2q and calmodulin; the enzymes produce small
amounts of NO for short periods following stimula-
tion, mediating vasodilation and modulating neuro-
transmission. Inhibition of constitutive NO synthases
can result in hypertension, impaired blood flow in
allografts and other undesirable side effects. The
production of a different enzyme, iNOS, is regulated
at the level of transcription, and its activity is con-
trolled primarily by the concentration of a cofactor,
tetrahydrobiopterin ŽBH 4 ., which is derived from
GTP ŽFig. 13.. This enzyme is induced by cytokines,
such as IFN-g and TNF-a, and produces larger
amounts of NO over a longer period than do the
constitutive enzymes. In sites of inflammation NO is
often produced by iNOS in parallel with superoxide
ŽOy 2 ; these two products combine rapidly to gener-
.
ate peroxynitrite, a highly reactive molecule which
can have cytotoxic effects. A reaction product, ni-
trotyrosine, can be identified by specific antibodies,
which are used to demonstrate the presence in tissues
of a protein nitrosylation adduct. NO can also alter
the balance of factors regulating apoptosis ŽKoglin et
al., 1999.. NO production through iNOS augments
expression of p53 and Bax and decreases the expres-
sion of Bcl-2 and Bcl-X1, which can activate apopto-
sis. In the absence of iNOS apoptosis is prevented.
Evidence is accumulating that induction of iNOS
and peroxynitrite formation contribute to tissue dam-
age in immunologically driven inflammatory reac-
98 A.C. Allison, E.M. Euguir Immunopharmacology 47 (2000) 85–118

Fig. 13. Proposed regulation of tetrahydrobiopterin ŽBH 4 . synthesis and NO production by IMP dehydrogenase ŽSenda et al., 1995.. BH 4 is
a rate-limiting cofactor for inducible nitric oxide synthase ŽNOS.. Cytokines induce both GTP cyclohydrolase I and iNOS transcriptionally
Žrepresented by dashed arrows.. The open arrows show inhibitory effects; MPA suppresses NO production by iNOS.

tions. For example, iNOS mRNA and nitrotyrosine
are demonstrable in the brains of animals with exper-
imental allergic encephalomyelitis ŽEAE., but not in
the brains of normal animals. In the rat EAE is
inhibited by aminoguanidine, an inhibitor of NOS.
Messenger RNA for iNOS and nitrotyrosine were
also found in brain lesions in patients with multiple
sclerosis ŽMS. but not in the brains of patients dying
without neuropathologies ŽBagasra et al., 1995.. The
iNOS mRNA was detected by in situ hybridization
in the cytoplasm of cells that also expressed the
ligand recognized by Ricinus communis agglutinin 1,
a marker of cells of monocytermacrophage lineage.
While there has been some doubt about the induction
 A.C. Allison, E.M. Euguir Immunopharmacology 47 (2000) 85–118
of iNOS by human cells of this lineage, it is now
clear that induction does occur when the right com-
bination of cytokines is used, corresponding to what
is observed in vivo.
Evidence has recently accumulated that, during
acute rejection of allografts, transplanted organs be-
come infiltrated by inflammatory cells strongly ex-
pressing iNOS. Acute rejection is accompanied by
increased NO production, reflected by measurements
of NO bound to proteins, or of the NO metabolites
nitrate and nitrate in plasma or serum. Nitrotyrosine
is demonstrable in parenchymal cells of grafted or-
gans, and increased apoptosis of these cells during
rejection is attributable, at least in part, to induced
NO production. The same mechanism can result in
dysfunction of graft parenchymal cells. Thus induced
NO production is one of several mechanisms con-
tributing to allograft rejection. However, in some
situations constitutive NO production can have the
opposite effect, favoring allograft survival.
A detailed discussion of this already complex
subject is beyond the scope of this review. However,
the main points can be illustrated by examples.
During the course of rat liver allograft rejection,
Goto et al. Ž1997. observed intense iNOS expression
in cells with monocytermacrophage markers infil-
trating the graft. In the same model Yamaguchi et al.
Ž1999. observed intense nitrotyrosine staining of liver
parenchymal cells,. In rat cardiac allografts Worrall
et al. Ž1999. found iNOS expression and increased
NO production during acute rejection; the iNOS was
localized to infiltrating inflammatory cells but not
allograft parenchymal cells. In acute rejection of rat
heart allografts Szabolcs et al. Ž1996. observed that
apoptosis of myocardial cells parallels the expression
of iNOS, suggesting that apoptosis may be triggered
by NO and peroxynitrite. To ascertain whether iNOS
augments apoptosis in rejecting heterotopic heart
allografts, Koglin et al. Ž1999. used recipient mice
with targeted deletion of the NOS2 gene, which
encodes iNOS. In these mice, apoptosis was signifi-
cantly reduced, and the histologic outcome was im-
proved, in comparison to grafts in NOS2q rq
recipients. The authors concluded that iNOS-media-
ted pathways can promote acute rejection, at least in
part, by inducing apoptotic cell death. Szabolcs et al.
Ž1998. presented evidence that apoptosis is a major
form of myocyte death during human cardiac allo-
99
graft rejection. Cardiac myocyte apoptosis is closely
associated with expression of iNOS in macrophages
and with nitration of myocyte proteins by peroxyni-
trite.
Regarding dysfunction, heart transplants exhibit
depressed contractile function during acute rejection.
Ziolo et al. Ž1998. reported that treatment with
aminoguanidine, an inhibitor of NO synthase, pre-
vented this effect. They concluded that overstimula-
tion of cardiac cGMP synthase by NO production is
a major factor responsible for cardiac depression
associated with acute rejection of transplanted hearts.
In contrast, survival of rat renal allografts
ŽStojanovic et al., 1996. and rat cardiac allografts
ŽPaul et al., 1996. can be decreased by nonselective
inhibitors of NO synthase. The authors postulate that
ischemic graft necrosis is secondary to unopposed
vasoconstriction. This would be an argument for
inhibiting iNOS but not endothelial cNOS, which is
a major regulator of arterial and arteriolar wall tonus.
There are also indications that prevention of NO
production may favor chronic rejection. Koglin et al.
Ž1998. reported that in mice with targeted inactiva-
tion of iNOS acute cardiac allograft rejection was
ameliorated but chronic rejection was aggravated. In
the latter situation leukocytic infiltration of the graft
was greater in mice lacking iNOS. Shears et al.
Ž1997. found that iNOS was expressed in rat aortic
allografts. Inhibiting NO production increased inti-
mal thickening, whereas transduction of iNOS with
an adenoviral vector decreased it. The mechanisms
underlying these effects are not established. Produc-
tion of peroxynitrite can kill leukocytes, and may
thereby prevent their accumulation over time. In the
rat aortic allograft model, MMF decreases intimal
thickening ŽSection 17., so the effect of the drug on
iNOS production is presumably counterbalanced by
inhibition of the expression of adhesion molecules.
In chronic renal allografts MMF also decreases
leukocytic infiltration and intimal thickening ŽAzuma
et al. 1995..
13. Selective inhibition by MPA of inducible NO
synthase
From the observations summarized above it is
clear that useful additional activity of a drug in organ
graft recipients could be inhibition of the induction
100 A.C. Allison, E.M. Euguir Immunopharmacology 47 (2000) 85–118
andror function of iNOS. The suppression by MPA
and MMF of cytokine production by lymphocytes
and monocytes ŽSection 8. should result in decreased
production of IFN-g and TNF-a, inducers of iNOS.
Moreover, the depletion by MMF of levels of GTP
in human monocytermacrophage lineage cells ŽFig.
7. should also lower the concentration of BH 4 , the
rate-limiting cofactor for iNOS activity ŽFig. 13..
We have demonstrated the expected effect of
MPA on iNOS activity in mouse and rat vascular
endothelial cells ŽSenda et al., 1995.. The enzyme
was induced by a combination of IFN-g and TNF-a.
The 50% inhibitory concentration of MPA was in the
range of 0.5–1 mM, which is readily attainable in
treated humans. However, MPA had no effect on
basal NO production, mediated by constitutive en-
zymes, in these cells. Sepiapterin reversed the in-
hibitory effect of MPA on induced NO production,
confirming that the effect was due to BH 4 depletion.
Observations showing that MMF suppresses cy-
tokine-induced production of NO in humans are
summarized below. These findings could have rele-
vance for preventing allograft rejection. Inhibition of
induced NO production may be one of the mecha-
nisms by which MPA and MMF efficiently prevent
experimental allergic encephalomyelitis and uveore-
tinitis.
14. Antimicrobial activity of MPA and MMF
MPA is a much more potent inhibitor of the
IMPDH of eukaryotes than that of prokaryotes
ŽVerham et al., 1987.. Hence, the compound would
be expected to inhibit weakly the replication of
bacteria, but might have greater activity against pro-
tozoa and fungi. By depleting dGTP, MPA also have
synergistic activity with inhibitors of viral DNA
polymerases and reverse transcription.
14.1. Parasites
Activity of MPA has been reported against Leish-
mania tropica in macrophages ŽBerman and Web-
ster, 1982., against Trichomonas foetus ŽWang et al.,
1984. and against Eimeria tenella ŽHupe et al.,
1986.. Some forms of systemic leishmaniasis are
complicated by splenomegaly and immunopathology,
and in such cases MMF might have clinical utility.
14.2. Pneumocystis carinii
O’Gara et al. Ž1997. cloned IMPDH from
P. carinii and expressed it in Escherichia coli. The
sequence of IMPDH from P. carinii showed greater
homology with the fungal and protozoal enzymes
than with bacterial IMPDH, supporting other genetic
evidence of the relatedness of P. carinii to eukary-
otes. P. carinii IMPDH was moderately sensitive to
inhibition by MPA. Oz and Hughes Ž1997. compared
effects of several immunosuppressive drugs with
those of dexamethasone in provoking P. carinii
pneumonitis in virus-free rats. In rats injected with
tacrolimus P. carinii infections were activated to the
point of severe pneumonitis. Following sirolimus
treatment, 30% of rats had P. carinii infections.
None of the animals treated with MMF alone or in
combination with dexamethasone had detectable P.
carinii infections. The authors conclude that MMF is
unique because of its dual activity as an immunosup-
pressant and as an agent with antimicrobial action
against P. carinii. In human patients receiving MMF
no P. carinii pneumonia was observed, whereas
these pneumonias were seen in patients receiving
CsA alone or in combination with AZA ŽKeown et
al., 1996..
14.3. HIV
This virus replicates in lymphocytes and mono-
cytes, cells in which MPA depletes GTP and dGTP
ŽFigs. 6 and 7.. Activation of the provirus in lym-
phocytes is correlated with their proliferation, so the
cytostatic effect of MPA might be expected to exert
some anti-HIV activity. Ichimura and Levy Ž1995.
reported that MPA in clinically attainable concentra-
tions Ž1–10 mM. suppresses HIV replication. Margo-
lis et al. Ž1999. recently found that MPA has a
synergistic anti-HIV effect with abacavir, the first
clinically available guanosine analog reverse tran-
scriptase ŽRT. inhibitor. Abacavir is converted into a
2X-deoxyguanosine triphosphate analog within cells,
and MPA-depleted depletion of dGTP Žwith which it
competes. would be expected to increase the effi-
ciency of RT inhibition by the abacavir metabolite.
Margolis et al. Ž1999. propose combination therapy
of abacavir and MMF in patients with HIV strains
 A.C. Allison, E.M. Euguir Immunopharmacology 47 (2000) 85–118
that are resistant to triple nucleoside antiretroviral
therapies.
14.4. Herpes simplex Õirus (HSV)
MPA has weak activity against HSV, but potenti-
ates the anti-HSV activities of acyclovir, ganciclovir
and penciclovir in vitro and in experimental animals
ŽNeyts et al., 1998a.. MPA also has synergistic
activity against HSV with the investigational guanine
analog H2G ŽNeyts et al., 1998b..
14.5. Epstein–Barr Õirus (EBV)
This is an important virus infection in transplant
recipients. Transformation of B-lymphocytes by EBV
is associated with expression of the type II isoform
of IMPDH, which is efficiently inhibited by MPA
ŽTable 3.. We investigated effects of MPA on the
interactions of EBV with human B-lymphocytes ŽAl-
fieri et al., 1994.. The drug did not inhibit events
associated with cell transformation or EBV lytic
cycle gene expression, but did block the proliferation
of newly infected or established EBV-transformed
cell lines. In groups of renal transplant recipients on
steroid-free regimens, those treated with MMF and
CsA had a significantly lower Ž p - 0.00005. rate of
EBV infections than those receiving CsA but no
MMF ŽBirkeland, 1988..
There is a direct correlation between the load of
EBV-infected lymphocytes in the peripheral blood of
transplant recipients and the risk of lymphoprolifera-
tive disease ŽSavoie et al., 1994.. CsA does not
inhibit outgrowth of EBV-transformed B-lympho-
cytes, but suppresses the T-cell-mediated immune
response that normally restricts that process ŽBird,
1982.. It might therefore be expected that patients
treated with MMF for long periods would have less
risk of developing EBV-related lymphoproliferative
disease than those treated with CsA. However, lym-
phoproliferative disorders have been observed in pa-
tients receiving MMF. Further observations are
needed on the cumulative incidence of such disor-
ders and whether they are related to EBV.
14.6. HCV
Chronic HCV infection, with hepatocyte damage
and cirrhosis, is a common indication for liver trans-
101
plantation. In all recipients HCV infection recurs,
and it produces hepatitis in the majority ŽPlatz et al.,
1998.. Chronic HCV infection is also a risk factor
for the development of primary hepatocellular carci-
noma. It would therefore be useful to identify a drug
that provides maintenance immunosuppression in
liver allograft recipients and at the same time inhibits
the replication of HCV, as well as the associated
hepatitis.
Evidence is accumulating that T-cell-mediated
immunopathology contributes to the pathogenesis of
hepatitis associated with HCV infection ŽAndo et al.,
1997., and MMF would be expected to suppress
such responses. In addition, MPA has been found to
inhibit the replication of yellow fever virus in mon-
key kidney cells ŽNeyts et al., 1996.. This virus is
related to, and considered a model for, HCV. An-
other IMPDH inhibitor, Ribavirin, is approved for
treatment of HCV infections, often administered with
interferon.
In a preliminary report, Platz et al. Ž1998. de-
scribed the use of MMF together with CsA or
Tacrolimus in 11 liver transplant recipients with
recurrent HCV hepatitis, with or without signs of
acute or chronic rejection. At the time of writing, all
recipients of MMF were in good condition. If these
results are confirmed, MMF may become a preferred
drug for use in HCV-infected liver allograft recipi-
ents. The drug may also be useful in the treatment of
symptomatic HCV infections that have not re-
sponded to Ribavirin and interferon.
15. Effects of MPA and MMF in experimental
animals
15.1. Lymphocyte-selectiÕe, reÕersible antiprolifera-
tiÕe effects
To ascertain whether the antiproliferative effects
of MPA and MMF are also lymphocyte-selective in
vivo, we injected mice subcutaneously with an anti-
gen Žovalbumin. in adjuvant, which stimulates DNA
synthesis in lymph nodes of the drainage chain:
while a secondary response to antigen was in
progress, the mice were injected intraperitoneally
with w3 Hx-thymidine. One group of mice was given
MPA Ž100 mgrkg per day orally. while a control
102 A.C. Allison, E.M. Euguir Immunopharmacology 47 (2000) 85–118
group received vehicle. Incorporation of w3 Hx-
thymidine into DNA was measured in draining lymph
nodes, spleen and testis ŽEugui et al., 1991b.. As
shown in Table 5, the orally administered drug sig-
nificantly inhibited DNA synthesis in lymph nodes
but had no detectable effect on DNA synthesis in
germinal cells of the testis or basal epithelial cells of
the small intestine Ždemonstrated by autoradiog-
raphy.. Effects on the spleen were intermediate, in
keeping with the presence in the mouse spleen of
haematopoeitic cells as well as lymphocytes. Doses
of MMF required to prevent allograft rejection did
not affect the production of neutrophils or platelets
in any species. In the rat a reversible hypoplastic
anaemia was observed. These observations show that
the cytostatic effects of MPA and MMF are more
potent on lymphocytes than on other cell types in
vivo. This appears to be true also in humans: in
humans treated with CellCept the frequency of pro-
liferating cells in renal tubular epithelium and
glomeruli was not decreased when compared with
that in normal control kidneys ŽPardo-Mindan ´ et al.,
1999..
The cytostatic effects of MPA on lymphocytes are
rapidly reversible: peripheral blood mononuclear cells
separated from the plasma of patients treated with
MMF respond normally to mitogenic stimulation,
even when the plasma is strongly suppressive. How-
ever, in MMF-treated experimental animals and hu-
mans some clones of T-lymphocytes responding to
antigenic stimulation may be lost through induction
of apoptosis ŽSection 10.. Infections other than cy-
tomegalovirus have not been a serious problem in
patients treated with MMF. However, if virus or
other infections do occur, the drug can be with-
Table 5
Effect of MPA w3 Hx-TdR incorporation into DNA in different tissues
Tissue Control, cpmr
samplea Ž n s 7.
Inguinal lymph node c 8765 " 793
Spleend 2727 " 524
Testis c 7435 " 536
a
Values represent mean " SE of the number of replicate samples indicated Žfrom Eugui et al., 1991b..
b
50 mgrkgrb.i.d. for 2 days.
c
10 7 cells.
d
10 7 mg tissue.
drawn, the immunosuppressive effects disappear
within a few days and recovery is rapid.
Assessed the effect of MMF on in vivo induction
of cytokines and histocompatibility antigens in trans-
planted mouse kidneys. MMF suppressed IFN-g pro-
duction in response to an allogeneic stimulus, and
consequent MHC class I and II induction, whereas
MMF had less effect on the induction of IFN-g by a
nonimmune stimulus, bacterial LPS. MMF was
thought to have the former effect by limiting clonal
expansion.
15.2. Inhibition of cell-mediated immune responses
to allogeneic cells
Cytotoxic T-lymphocytes play an important role
in allograft rejection ŽRosenberg and Singer, 1992..
We used a classical model ŽBrunner et al., 1988. to
ascertain the effects of MPA and MMF on cytotoxic
T-lymphocytic responses to allogeneic cells ŽEugui
et al., 1991b.. Tumour cells ŽP815, H-2 d . were
injected intraperitoneally into C57Bl mice ŽH-2 b . to
immunize them. Recipient mice were orally dosed
with MPA, MMF or vehicle and spleens were re-
moved on day 10 or 11 for in vitro cytotoxicity
studies using 51 Cr-labelled P815 target cells. This
lysis is genetically restricted and largely due to
cytoxic T-cells. As shown in Fig. 14, MPA inhibited
in a dose-related fashion the induction of a cytotoxic
T-lymphocyte response to allogeneic cells.
Allogeneic tumour cells in the peritoneal cavity
can be quantified and their viability assessed by
capacity to grow as colonies in agar. In vehicle-
treated animals, the tumour cells were found to
increase rapidly for the first 4 days and thereafter
decrease as the host immune response became effec-
MPA-treatedb , cpmr Percent P values
sample Ž n s 8. inhibition
2614 " 204 70.2 0.000
1070 " 94 60.7 0.019
7043 " 950 5.3 0.726
 A.C. Allison, E.M. Euguir Immunopharmacology 47 (2000) 85–118
tive; by the 8th day no viable tumour cells could be
recovered. In MPA-treated mice, the tumour cells
continued to increase and remain viable until the
animals were killed for humane reasons ŽEugui et al.,
1991b.. This is a convenient model because cyto-
toxic T-lymphocytes can be analysed without com-
plicating effects on the vasculature of allografts.
These investigations show that MMF and MPA
inhibit the generation of cytotoxic T-cells and the
rejection of allogeneic cells. Together with our stud-
ies on inhibition of antibody formation and of the
expression of adhesion molecules, they provided the
theoretical justification for use of the drug in organ
transplantation.
15.3. Inhibition of antibody formation
Antibody responses of rats and mice to sheep
erythrocytes were analysed by the Jerne plaque as-
say. Administration of MMF inhibited the formation
of antibodies in a dose-dependent manner ŽAllison et
al., 1991; Eugui et al., 1991b.; oral administration 30
mgrkg per day to rats virtually abolished the forma-
tion of antibodies against xenogeneic cells ŽFig. 15..
Eugui et al. Ž1995. ascertained the effect of oral
doses of MPA Ž80 mgrkg per day. on antibody
responses of mice to influenza virus hemagglutinin
ŽHA.. The isotypes of anti-HA were measured by
ELISA. MPA administered following primary immu-
nization inhibited significantly anti-HA responses of
IgG2a, IgG2b and IgG3 isotypes, with less effect on
IgG1 responses. In contrast, MPA administered after
Fig. 14. Dose-dependent inhibition of the generation of cytotoxic
T lymphocytes in mice treated with MPA ŽEugui et al., 1991b..
The hatching in the histograms represents doses of MPA: 0, 50
mg and 100 mgrkg per day.
103
Fig. 15. Dose-dependent inhibition of the number of antibody-for-
ming cells in the spleens of rats immunized with sheep red blood
cells and dosed with MPA ŽEugui et al., 1991b..
an antigen boost Ždays 21–30. slightly increased
anti-HA responses of all isotypes. These observa-
tions suggest that MPA suppresses primary humoral
responses, especially of isotypes dependent on IFN-g
production, but does not suppress secondary anti-
body responses.
15.4. Suppression of contact hypersensitiÕity
Shoji Ž1994. reported that a topical formulation of
MPA suppresses dermal hypersensitivity induced in
guinea pigs by dinitrofluorobenzene. This type of
allergic reaction is mediated by T-lymphocytes.
16. Prevention by MMF of allograft rejection
Following our basic observations on the immuno-
suppressive effects of MMF, collaborations were
established with transplantation immunologists to as-
certain whether the drug can prevent the rejection of
tissue and organ allografts.
16.1. Rat hearts
After preliminary studies in the mouse, Dr. Ran-
dall Morris et al. at Stanford systematically investi-
gated effects of MMF in Lewis rat recipients of
heterotopically grafted BN hearts ŽMorris et al.,
1990.. Monotherapy with MMF Ž30–40 mgrkg per
day. was found to prevent rejection of the grafts; if
treatment was discontinued after 50 days, all of the
104 A.C. Allison, E.M. Euguir Immunopharmacology 47 (2000) 85–118
hearts in the animals receiving the higher dose sur-
vived in good functional condition indefinitely. When
such animals were challenged in the absence of
further immunosuppression with donor-strain atrial
tissue beneath the renal capsule, it continued to beat
indefinitely, whereas atrial tissue from a third-party
strain ŽACI. was promptly rejected ŽMorris et al.,
1991.. Thus short-term treatment with MMF could
induce a state of donor-specific tolerance. In these
animals mixed lymphocyte responses of recipient to
donor-strain lymphocytes were significantly lower
than in untreated recipients, whereas responses to
third-party cells were normal.
The second finding was that when MMF and CsA
were used together, their effects were at least addi-
tive without any demonstrable increase in toxicity
ŽMorris et al., 1990.. Since the drugs act by different
mechanisms this was not surprising. The third find-
ing was that if the first dose of MMF was delayed
until the fifth day following transplantation, by which
time there is a marked mononuclear cell infiltrate
into the graft and oedema, the heart could still be
preserved in good functional condition indefinitely
ŽMorris et al., 1990.. When AZA or CsA was given
under comparable conditions it could not prevent
rejection. This finding suggested that MMF might be
efficacious for the treatment of rejection crises.
16.2. Mouse pancreatic islets
Dr. Kevin Lafferty et al. in the University of
Colorado, Denver, studied pancreatic islet allografts
in streptozoticin-treated diabetic mice and rats.
BALBrc recipients of C57Blr6 islets were orally
dosed with MMF Ž80 mgrkg per day. for 30 days;
this regime prevented graft rejection and following
cessation of therapy allowed indefinite graft survival
in 55% of mice. Animals carrying stable islet allo-
grafts exhibited a state of donor-specific tolerance,
resistant to subsequent challenge with donor spleen
cells in the absence of drug ŽHao et al., 1990..
Combined treatment of graft recipients for the first
30 days with cyclosporin A increased the proportion
of animals with long-term graft survival following
cessation of treatment to 89%, but decreased the
proportion of those surviving challenge with donor-
strain spleen cells ŽHao et al., 1990; 1992.. In this
model the initial advantage of combined drug ther-
apy was accompanied by the long-term disadvantage
of less stable tolerance induction during the period of
therapy.
16.3. Canine kidney
Some immunosuppressive drugs preventing allo-
graft rejection in rodents have failed to do so in large
animal models and man. Activity in dogs has proven
to be a useful predictor of efficacy in humans. We
were therefore pleased when studies in the laboratory
of Dr. Hans Sollinger, University of Wisconsin,
showed that MMF was effective in the dog renal
allograft model ŽPlatz et al., 1990.. Monotherapy
with MMF Ž40 mgrkg per day. markedly prolonged
graft survival, but combined therapy ŽMMF 20
mgrkg per day, cyclosporin A 5 mgrkg per day and
methylprednisolone 0.1 mgrkg per day. was more
efficacious than either modality alone. There was no
nephrotoxicity, hepatotoxicity or bone marrow sup-
pression. The only serious toxicity in the dog was in
the small intestine, predicting the most troublesome
side effect of MMF in humans. Later studies showed
that MMF can reverse acute renal allograft rejection
in dogs ŽPlatz et al., 1991.. While treatment with
steroid bolus therapy could only temporarily halt
rejection in some dogs, MMF reversed rejection and
prevented subsequent rejection episodes.
These three studies in experimental animals
showed the efficacy of MMF in preventing and
treating allograft rejection without limiting toxicity
or increased susceptibility to infections. They pro-
vided the experimental basis on which studies in
human organ transplant recipients were undertaken.
Reports by other investigators confirmed the utility
of MMF for preventing rejection of various allo-
grafts in several species of experimental animals.
The literature on this subject is already too extensive
to review here, and is easily accessible.
17. Effects of MMF in experimental animal mod-
els of chronic rejection
17.1. Background
Now that acute rejection can be reasonably well
controlled, chronic rejection is emerging as the major
 A.C. Allison, E.M. Euguir Immunopharmacology 47 (2000) 85–118 105

limitation of long-term allograft survival and func- nomenon is discussed further in the introductory
tion. A major objective of transplantation immunolo- review ŽAllison, 2000..
gists is to define a therapy that prevents chronic as
well as acute rejection. The two are related: chronic 17.2. MPA suppresses the proliferation of arterial
rejection is more likely to occur when there have smooth muscle cells and fibroblasts
been episodes of acute rejection. Chronic rejection is
associated with a proliferative and obliterative arteri- As shown in Figs. 5 and 16, MPA, in clinically
opathy, attributed to recruitment of mononuclear cells attainable concentrations Ž1–10 mM. suppresses the
and induced proliferation of smooth muscle cells proliferation of arterial smooth muscle cells and
first observed in small and medium-sized arteries fibroblasts. This has been shown for human smooth
and later throughout the arterial tree of transplanted muscle cells ŽAllison and Eugui, 1993; Gregory et
¨
hearts, kidneys and livers ŽHayry et al., 1993.. The ¨ ¨
al., 1993. and rat smooth muscle cells ŽRaisanen-
lesions in graft recipients are usually generalized and Sokolowski et al., 1995.. The suppression by MPA
the neointimal thickening is concentric, whereas in of fibroblast proliferation ŽEugui et al., 1991a. is
the general population atherosclerotic lesions tend to relevant to preventing interstitial fibrosis in the kid-
be focal and asymmetric and foam cells are more ney and elsewhere. In clinically attainable concentra-
prominent. Chronic rejection of kidney allografts is tions, CsA does not inhibit the proliferation of smooth
associated not only with vascular obliteration, but muscle cells and fibroblasts.
also with glomerulosclerosis, tubular atrophy and
interstitial fibrosis. 17.3. Rat carotid denudation
The pathogenesis of proliferative arteriopathy in
grafts is complex: some authorities believe that it is To ascertain whether drugs can suppress the pro-
predominantly mediated by T-lymphocytes, others liferation of arterial smooth muscle cells in response
that antibodies against donor endothelial cell and to a non-immunological in vivo stimulus, the rat
other antigens play a pathogenetic role; probably carotid denudation model has been used. When the
both humoral and cellular mechanisms are involved. intima is stripped, platelets are deposited and
During chronic rejection, genes for several growth macrophages accumulate; platelet-derived and other
¨
factors are expressed in arterial walls ŽHayry et al., growth factors stimulate the proliferation of smooth
1993.; these factors could stimulate proliferation of muscle cells in the neointima. This process con-
smooth muscle cells in the neointima. It is therefore
a better therapeutic strategy to inhibit the end result,
proliferation of arterial smooth muscle cells, than to
attempt inhibition of the production or effects of
individual cytokines.
While there is some deposition of collagen in
arterial walls in chronic allograft rejection, this pro-
cess is more prominent in interstitial sites in the
kidney and other organs. Collagen formation is asso-
ciated with the proliferation of fibroblasts and in-
duced expression of procollagen genes. Cytokines
such as TGF-b are inducers of procollagen gene
expression. Suppressing the proliferation of fibrob-
lasts and the expression of TGF-b in allografts is
desirable. CsA augments the formation of TGF-b by
several cell types ŽKhanna, 1999., and recent evi-
dence shows that tacrolimus and rapamycin increase Fig. 16. Effect of mycophenolic acid ŽMPA. and cyclosporin A
the formation of TGF-b by human lymphocytes ŽCsA. on the proliferation of human arterial smooth muscle cells
ŽDodge et al., 1999; Khanna, 1999.. This phe- in culture ŽAllison and Eugui, 1993..
106 A.C. Allison, E.M. Euguir Immunopharmacology 47 (2000) 85–118
tributes to restenosis following angioplasty. In sev-
eral laboratories MMF has been found to reduce
restenosis following rat carotid denudation ŽFraser-
¨ ¨
Smith et al., 1995; Gregory et al., 1995; Raisanen-
Sokolowski et al., 1995.. MMF may be useful in
combined therapy with an inhibitor of platelet activa-
tion to prevent restenosis following angioplasty in
humans.
17.4. Rat heart allografts
In collaboration with Morris et al. Ž1991. the
possibility that MMF might be superior to currently
used therapies for the prevention of chronic rejection
was explored, using rat heterotropic heart allografts.
In this model moderate doses of CsA did not prevent
arteriopathy, and low or moderate doses of FK506
prevented mononuclear cell infiltration of the my-
ocardium but not graft coronary disease. Only a
highly toxic dose of AZA was able to prevent arteri-
opathy in grafts examined one month after transplan-
tation. Even in long-term recipients of MMF the
incidence and severity of proliferative arteriopathy
was low compared to that in recipients of other
drugs.
17.5. Rat aortic allografts
A rat aortic allograft model was studied in collab-
oration with Sollinger et al. ŽSteele et al., 1993..
Three months after aortic allografting, the intima
showed marked proliferation, which was not seen
when syngeneic aortas were grafted. MMF signifi-
cantly decreased neointimal proliferation, whereas
CsA and Brequinar did not.
This model was studied further in the laboratory
¨
of Hayry, ¨ ¨
who introduced it ŽRaisanen-Sokolowski et
al., 1995.. MMF Ž20 mgrkg per day. was adminis-
tered orally from the day of transplantation, and the
animals were killed 1 to 12 months later. MMF was
found to suppress all major histological manifesta-
tions of allograft arteriosclerosis, namely adventitial
inflammation, medial necrosis and neointimal thick-
ening and cellularity. There was a significant de-
crease in the replication rate Ž3 H-thymidine incorpo-
ration. of inflammatory cells in the adventitia and of
smooth muscle cells in the media.
17.6. Primate cardiac xenografts
O’Hair et al. Ž1994. compared two immuno-
suppressive regimens for preventing primate cardiac
xenograft rejection. Hearts from cynomolgus mon-
keys Ž Macaca fascicularis. were transplanted hetero-
topically into nine baboons Ž Papio anubis .. In one
group of six recipients receiving CsA, steroid and
AZA, the mean graft survival was 3 months, whereas
in a second group of three recipients receiving CsA,
steroid and MMF, the mean graft survival was 10
months. In 29 biopsy specimens from the group
receiving AZA, histological evidence of vascular
rejection was found in 16; in 21 biopsy specimens
from the group receiving MMF only two showed
signs of vascular rejection. In grafts surviving for 1
year, strong intimal proliferation was seen in coro-
nary arteries from the group receiving AZA, whereas
the coronary vessels were normal in the group re-
ceiving MMF.
17.7. Rat kidney allografts
A rat model of chronic kidney allograft rejection
has been informative ŽAzuma et al., 1995; Heemann
et al., 1996.. When a short regimen of CsA is used
to overcome acute rejection, chronic rejection starts
12 weeks after grafting. This is manifested by
mononuclear cell infiltration, proteinuria and
glomerulosclerosis. A relatively low dose of MMF
Ž15 mgrkg per day., starting 8 weeks after trans-
plantation, allowed the grafts to function normally
throughout the follow-up period. In MMF-treated
animals, expression of ICAM-1 on endothelial cells
and of VLA-4 on mononuclear cells was signifi-
cantly suppressed. Infiltration of cells with lympho-
cyte and monocyte–macrophage markers into the
allografts was strongly suppressed, as was binding of
normal mononuclear cells to sections of the kidneys.
Thus the in vitro observations that MPA inhibits
expression of adhesion molecules and adhesion of
lymphocytes and monocytes to endothelial cells
ŽSection 11. can be extrapolated to relevant in vivo
situations.
In the same rat model of chronic rejection, Nadeau
et al. Ž1996. examined the sequential expression in
long-surviving renal allografts of cytokine genes,
using reverse transcription PCR. In renal allografts
 A.C. Allison, E.M. Euguir Immunopharmacology 47 (2000) 85–118
of animals on long-term CsA therapy, marked in-
creases of TGF-b, Hsp70 and endothelin expression
were observed as compared with control animals.
Conversely, IL-2 receptor, IFN-g and TNF-a were
expressed at lower levels in animals maintained on
CsA than in those receiving CsA for only 10 days
after transplantation. Morphologically, the long-term
CsA-treated kidneys showed more extensive arterial
obliterative changes and glomerulosclerosis after 24
weeks than observed in control isografts; these
changes were attributed by the authors to the pro-
gressive effects of chronic rejection superimposed on
the nephrotoxicity of the drug. CsA is known to
augment expression in several cell types of TGF-b,
which is a major inducer of collagen synthesis by
fibroblasts; and endothelins can produce hyper-
tension. In contrast MMF inhibited the production of
all lymphocyte- and macrophage-derived cytokines
throughout the entire follow-up period. Allograft kid-
neys in MMF recipients showed no late morphologi-
cal abnormalities.
If all these findings can be extrapolated to human
recipients, the utility of the drug for prevention of
chronic rejection will be established.
18. Prevention by MMF of graft-versus-host dis-
ease (GVHD)
18.1. Intestinal transplantation in the rat
GVHD occurs following transplantation of the
lymphoid tissue-rich small intestine. Sonnino Ž1982.
and Shaffer et al. Ž1992; 1993. studied the effects of
MPA on allotransplantation of small intestine in a rat
model. In Shaffer’s experiments parental ŽLewis.
strain intestinal allotransplants were made into F1
ŽLewis X BN. recipients. In a group of 11 untreated
recipients, the mean survival time was 15.7 " 2.8
days. Oral administration of MPA Ž30 mgrkg per
day., on days 0 through 6 resulted in indefinite
survival of all recipients. Administration of MPA on
days 7 to 20 was ineffective.
18.2. Transfer of allogeneic spleen cells in mice
Following an early report of Shaffer et al. Ž1993.,
the efficacy of MMF in prevention of GVHD was
investigated in nonlethal and lethal murine models
107
ŽMirkovich and Eugui, 1995.. In the first, injection
of spleen cells from parental into nonirradiated F1
recipients induced splenomegaly, increased NK ac-
tivity, and decreased responses of spleen cells to
mitogens. Oral administration of MMF in doses of
50 mgrkg per day or higher inhibited all manifesta-
tions of GVHD. In the lethal model, lethally irradi-
ated hosts ŽC57rB1r6, H-2 d . were reconstituted
with allogeneic bone marrow and spleen cells
ŽBALBrc, H-2 b .. Oral administration of MMF 50
mgrkg per day increased mean survival from 7.6 "
0.7 to 52.2 " 5.3 days Ž p - 0.0001.. In other experi-
ments, MMF and CsA together increased mean sur-
vival time further. In five mice surviving more than
252 days, flow cytometric analysis showed that 98%
of nucleated spleen cells were of donor origin,
demonstrating that chimerism had been established.
In conclusion, MMF alone, or in combination with
CsA, prevents the development of GVHD in non-
lethal and lethal rodent models.
18.3. GVHD in dogs following bone marrow trans-
plantation
Yu et al. Ž1998. evaluated MMF administered
alone or in combination with CsA for preventing
GVHD in dogs given 9.2 Gy total body irradiation
and DLA-nonidentical unrelated bone marrow grafts.
Marrow autograft studies showed intestinal toxicity
to be the dose-limiting side effect of MMF. Dogs
given MMF or CsA alone survived longer than
untreated controls. However, administration of MMF
together with CsA gave the best results. The authors
conclude that there was synergism between MMF
and CsA as evidenced by stable graft–host tolerance
in the majority of dogs.
19. Inhibition by MMF of nephritis in experimen-
tal animal models
19.1. ActiÕe Heymann nephritis (AHN)
This rat model of human idiopathic membranous
nephropathy is induced with renal antigen Fx1A in
complete Freund’s adjuvant ŽCFA.. Penny et al.
Ž1998. found that MMF administration Ž30 mgrkg
per day, for 4 weeks after immunization. prevents
the induction of AHN. In treated animals, serum
108 A.C. Allison, E.M. Euguir Immunopharmacology 47 (2000) 85–118
antibodies against Fx1A were suppressed, as was
glomerular Ig deposition. The drug also prevented
interstitial infiltration of abTCRq, CD4q and CD8q
T-cells, natural killer cells and macrophages. Re-
verse transcription-PCR showed increased expres-
sion of Th1 and Th2 cytokines ŽIFN-g and lympho-
toxin; and IL-4. as well as TNF-a in kidneys of
immunized rats. The increased expression of cy-
tokine genes was prevented by MMF treatment.
In lymph nodes draining sites of treatment MMF
limited both the enlargement and the increased pro-
portion of CD3q, CD4q and CD8q T-cells observed
in HN and CFA controls. MMF suppressed the
expression of the Th2 cytokine IL-4 but not the Th1
cytokine IFN-g and lymphotoxin mRNAs in lymph
nodes. These findings suggest that MMF may prefer-
entially suppress the production of Th2 cytokines, at
least with this regimen of immunization in the rat.
Treatment with MMF from 4–8, 6–12 or 10–14
weeks did not prevent the formation of serum anti-
bodies against Fx1A, glomerular Ig deposition or
proteinuria when compared to untreated AHN ani-
mals. These observations are consistent with others
showing that MMF suppresses primary humoral re-
sponses more effectively than ongoing responses.
The authors suggest that MMF therapy may prove
useful in human idiopathic membranous nephropa-
thy.
19.2. PassiÕe Heymann nephritis (PHN)
This is produced by antibodies against renal tubu-
lar antigens in the rat, and is regarded as a model of
the human nephrotic syndrome. CsA has become
standard therapy for the latter, but has nephrotoxic
effects, so alternatives are being explored. Heering et
al. Ž1998. found that MMF Ž25 mgrkg. significantly
decreased proteinuria in rats with PHN, to a greater
extent than CsA did. In MMF-treated rats no impair-
ment of renal function, or abnormality in prosta-
glandins, was observed, whereas these were dis-
turbed in CsA-treated animals. The authors suggest
that the administration of MMF to patients with the
nephrotic syndrome deserves exploration.
19.3. Renal ablation
In the rat removal of five-sixths of the kidney
results in hyperfiltration, endothelial cell activation,
infiltration of mononuclear cells and progressive
sclerosis. Mueller et al. Ž1998. found that MMF Ž20
mgrkg per day. reduces expression of ICAM-1 and
infiltration of mononuclear cells in rat remnant kid-
neys; tacrolimus did not have these effects. Noronha
et al. Ž1998. and Fujihara et al. Ž1998. reported that
in rat remnant kidneys, macrophages and cells ex-
pressing angiotensin II and TGF-b are observed in
ischemic, perinecrotic areas and in areas of intersti-
tial expansion. MMF decreased the interstitial ex-
pression of TGF-b and attenuated renal injury.
Romero et al. Ž1999. found that MMF reduces ex-
pression of CD18 and CD11b adhesion molecules,
decreases monocytic and lymphocytic infiltration,
and suppresses interstitial fibrosis in rat remnant
kidneys.
19.4. Rat mesangial proliferatiÕe nephritis
In vitro, MMF inhibits the proliferation of rat
kidney mesangial cells, whereas CsA and FK506 do
not ŽHauser et al., 1997b.. Mesangial proliferative
nephritis can be induced in rats by administration of
an antibody against Thy-1. Hauser et al. Ž1997b.,
Huang et al. Ž1998. and Ziswiler et al. Ž1998. re-
ported that MMF administration prevents the induc-
tion in rats of mesangial proliferative glomerulo-
nephritis by this procedure.
19.5. Nephritis in mice
McMurray et al. Ž1998. and Corna et al. Ž1997.
reported that MMF prevents lupus nephritis and mor-
tality in the female ŽNZBxNZW. F1 mouse model of
systemic lupus erythematosus. Van Bruggen et al.
Ž1998. reported that MMF delays the onset of lupus
nephritis in MRLrLPR mice.
20. Attenuation by MMF of ischemia–reperfusion
injury
When graft function is delayed, effects of is-
chemia and reperfusion become manifest. These in-
clude increased expression of adhesion molecules
and attachment of leukocytes to endothelial cells.
Extending their in vitro findings ŽSection 8. to an in
vivo situation, Paul et al. Ž1998. pretreated rats with
 A.C. Allison, E.M. Euguir Immunopharmacology 47 (2000) 85–118
MMF Ž20 mgrkg per day for 1 week., recovered
their hearts, stressed them by ischemia and trans-
planted them. It was found that MMF inhibits ex-
pression of endothelial cell adhesion molecules,
binding leukocytes to endothelium and protects
against ischemia-induced graft failure. Wang et al.
Ž1999. and Wei et al. Ž1999. reported that MPA
inhibits the over-expression of ICAM and osteopon-
tin in ischemiarreperfusion injury. The human coun-
terpart may be the finding that MMF prevents acute
rejection and ensures high 1-year graft survival even
in patients with delayed graft function ŽMaurizio et
al., 1998..
21. Inhibition by MPA of autoimmune and other
disorders in experimental animals
21.1. AdjuÕant arthritis
Injection into rats of Freund’s complete adjuvant
elicits an arthritis characterized by inflammatory
swelling of the distal joints of the feet and erosion of
cartilage and bone. Adjuvant arthritis can be inhib-
ited by immunosuppressive agents as well as anti-in-
flammatory agents such as inhibitors of prosta-
glandin synthesis. It is regarded as a model for drugs
potentially useful for treating rheumatoid arthritis.
When MPA was orally administered to rats in doses
of 10, 20 and 30 mgrkg per day, it was found to
inhibit the signs of adjuvant arthritis in a dose-de-
pendent fashion relative to vehicle-treated animals
ŽSyntex Research Report, 22 April 1988..
21.2. Osteopenia
Post-transplantation bone disease is a well-known
phenomenon. Glucocorticoids depress osteoblastic
function, as manifested by low serum osteocalcin,
and produce low-turnover osteopenia and osteoporo-
sis Žmostly in trabecular bone.. CsA produces a
high-turnover osteopenia affecting predominantly
trabecular bone. These osteopenic changes appear in
treated rats within 1 month. Dissanayake et al. Ž1998.
found that administration of MMF to rats Ž30 mgrkg
per day for 28 days. did not cause osteopenia, al-
though there was some decrease in circulating osteo-
calcin levels.
109
21.3. Experimental allergic encephalomyelitis
Rats receiving an intradermal injection of syn-
geneic spinal cord homogenate in Freund’s complete
adjuvant develop an autoimmune response to myelin
basic protein and other antigens. There is T-
lymphocyte-mediated damage to nerve fibers ini-
tially in the lumbar region of the spinal cord. This is
manifested by paralysis of hind limbs beginning
10–13 days after immunization, and loss of body
weight. Immunosuppressive drugs prevent these ef-
fects. Oral administration of MPA in the dose range
5–30 mgrkg per day for 16 days after immunization
significantly attenuated the manifestations of EAE in
a dose-related fashion ŽSyntex Research Report, 22
April 1988..
21.4. Experimental autoimmune uÕeoretinitis (EAU)
Immune-mediated inflammatory eye disorders
comprise a major group of sight-threatening condi-
tions ŽNussenblatt and Palestine, 1989.. They can be
treated by CsA and other immunosuppressive drugs,
but side effects are limiting ŽWhitcup and Nussen-
blatt, 1993.. A model is EAU induced in rats by
administration of retinal S-antigen in Freund’s com-
plete adjuvant. In collaboration with Nussenblatt’s
laboratory, MMF Ž30 mgrkg per day, days 0–13.
was found to suppress cellular and humoral re-
sponses to SAg and to prevent completely the devel-
opment of EAU in the majority of rats ŽChanaud et
al., 1995.. MMF almost completely suppressed the
development of EAU in most rats adoptively trans-
ferred by SAg-sensitized lymphocytes, suggesting
that the drug also has activity against the efferent
limb of the immune response. The authors suggest
that MMF may be useful to treat immune-mediated
uveitic disorders in humans. Preliminary observa-
tions suggest that this may be the case ŽSection 25..
21.5. Diabetes in bio-breeding (BB) rats
BB rats are genetically predisposed to develop
autoimmune diabetes mellitus. Hao et al. Ž1993.
found that administration of MMF Ž20 mgrkg per
day from 40 days of age. prevented the development
of diabetes as long as it was continued; when dosage
of the drug stopped, diabetes developed. In this
110 A.C. Allison, E.M. Euguir Immunopharmacology 47 (2000) 85–118
model a short period of administration of MMF does
not establish tolerance to the relevant autoantigens.
21.6. Colitis in rats
Zeeh et al. Ž1998. induced colitis by rectal instal-
lation of denitrofluorobenzene in rats. Intraperitoneal
administration of MMF significantly decreased tissue
damage in this model of experimental colitis.
22. NO production in human allograft recipients
and suppression by MMF
Evidence is summarized in Section 9 that, in
acutely rejecting allografts, induced NO synthase is
strongly expressed and that NO production con-
tributes to parenchymal cell death and dysfunction.
MPA inhibits NO production by iNOS but not by the
constitutive enzymes that regulate vascular tone and
neural functions ŽSection 10..
It is now widely accepted that in humans with
acutely rejecting heart allografts ŽBenvenuti et al.,
1996. and renal allografts ŽWatarai et al., 1999.,
levels of the NO metabolites nitrate and nitrite in the
circulation rise. Successful treatment of rejection is
accompanied by a fall in circulating NO metabolites
before creatinine does. The authors suggest that mea-
surement of nitrate in serum may have predictive
value in monitoring transplant patients. However, a
rise in serum nitrate also occurs during infections, at
least in lung transplants, so the assay does not dis-
criminate between rejection and infection ŽWang et
al., 1998..
Because serum nitraternitrite levels may be influ-
enced by renal function, Winklhofer et al. Ž1999.
measured NO bound to plasma proteins. The cy-
tokine release syndrome following antithymocyte
globulin ŽATG. administration was associated with a
marked increase in levels of serum NO. Treatment of
patients with MMF effectively blocked this rise,
whereas AZA treatment did not. Basal levels of NO
were lower in patients receiving MMF Žwithout ATG.
than in patients receiving AZA. The authors con-
clude that the immunosuppressive effects of MMF
include inhibition of iNOS-mediated responses,
known to be involved in acute rejection as well as in
the cytokine release syndrome. By inhibiting cy-
tokine production MMF can decrease iNOS expres-
sion, and by inhibiting selectively iNOS activity the
drug can reduce damage mediated by NO and perox-
ynitrite. In renal allograft recipients treated with
MMF the frequency of apoptotic cells in tubular
epithelium is significantly less than in those treated
with CsA, AZA and steroids ŽPardo-Mindan ´ et al.,
1999..
23. Suppression by MMF of the expression of
adhesion molecules in humans
23.1. Renal biopsy specimens
The induced expression of VCAM-1 on cultured
human endothelial cells is strongly suppressed by
MMF ŽBlaheta et al., 1999; Section 11.. Li et al.
Ž1998. reported that in humans with diffuse prolifer-
ative lupus nephritis VCAM expression in renal
glomeruli was high. MMF treatment Ž1.5–2 grday.
was followed by clinical improvement and a dra-
matic decrease in VCAM expression. Four of the
five cases studied showed intense staining before
treatment; this was markedly decreased in intensity
in MMF-treated patients, the staining in two cases
becoming essentially negative. The number of
glomerular CD68q cells Žmonocytesrmacrophages.
was also reduced dramatically in MMF-treated pa-
tients. Treatment with cytoxan or prednisone had not
decreased VCAM expression. If these findings are
confirmed in other patients and conditions, decreased
expression of adhesion molecules such as VCAM
must be regarded as a major effect of MMF treat-
ment.
24. Effect of MMF on antibody formation in
humans
Kimball et al. Ž1998. compared IgG antibodies
against equine polyclonal anti-thymocyte antibody
ŽATGAM. in renal transplant recipients receiving
MMF Ž2–3 grday. and AZA. The incidence and
titer of antibodies was significantly lower in the
MMF treated group. Al-Akash et al. Ž1998. exam-
ined the effect of MMF, humanized anti-IL-2 recep-
 A.C. Allison, E.M. Euguir Immunopharmacology 47 (2000) 85–118
tor antibody ŽZenapax., cyclosporin A ŽCsA. and
prednisone on responses of children to influenza
vaccination. The authors conclude that: Ž1. children
who were immunologically primed before vaccina-
tion and treatment were more likely to develop a
protective Ab titer to viral antigens; Ž2. children
vaccinated during and up to 2 months after Zenapax
treatment are unlikely to mount an Ab response; Ž3.
patients receiving a relatively high dose of CsA are
less likely to produce an Ab response than those on a
lower dose of CsA; and Ž4. MMF when administered
with CsA may further suppress the response to the
vaccine.
These findings support the interpretation that
MMF suppresses primary Ab responses more effi-
ciently than secondary responses. They also suggest
that potential transplant recipients should be vacci-
nated before the event, since immunosuppressive
drugs, and especially Zenapax, are likely to inhibit
the humoral response to vaccines administered later.
25. Preliminary reports of the efficacy of MMF in
various human disorders
While the principal application of MMF is in
transplantation, the mechanisms of action of the drug
suggest that it may also be useful in immunologi-
cally driven inflammatory disorders. Preliminary
reports suggest that this may be the case, but con-
trolled studies are needed in each disorder to estab-
lish efficacy.
25.1. Rheumatoid arthritis (RA)
Two double-blind placebo-controlled studies in-
volving about 600 patients with RA suggested that 2
grday dose of MMF was efficacious and fairly well
tolerated ŽGoldblum, 1993.. Improvement was seen
in some patients who had been refractory to treat-
ment with several disease-modifying anti-rheumatic
drugs. MMF reduced titers of rheumatoid factor,
immunoglobulin levels and the total number of T-
cells ŽCD62q. in peripheral blood. Adverse effects
were gastrointestinal: no clinically significant
nephrotoxicity, hepatotoxicity or bone marrow toxic-
ity was attributable to MMF. Schiff and Leishman
Ž1998. reported the results of a randomized, double-
111
blind trial in RA patients comparing 1 g bid and 2 g
bid. Marked efficacy was seen by week 4, with a
peak effect around weeks 8 to 12, which was sus-
tained to week 36. The authors concluded that MMF
1 g bid is as effective as 2 g bid, and the lower dose
has fewer gastrointestinal side effects.
25.2. Myasthenia graÕis
Hauser et al. Ž1998. reported the successful treat-
ment of a patient with severe refractory myasthenia
gravis with MMF.
25.3. Skin diseases
The first clinical application of MPA was for the
treatment of psoriasis ŽEpinette et al., 1987.. Re-
cently, efficacy of MMF in the treatment of psoriasis
has been confirmed ŽGeilen et al.,1998; Haufs et al.,
1998.. Dishidrotic eczema has also been treated suc-
cessfully with MMF ŽPickenacker et al., 1998.. Evi-
dence has accumulated suggesting that MMF may be
a therapeutic option for the treatment of blistering
autoimmune diseases. Successful treatment of bul-
lous pemphigus by MMF has been reported by Bohm ¨
et al. Ž1997., Nousari et al. Ž1998a . and
Grundmann-Kollmann et al. Ž1999.. Efficacy of
MMF in pemphigus vulgaris has been described by
Enk and Knop Ž1999. and Grundmann-Kollmann et
al. Ž1999.. Successful treatment of three cases of
relapsing idiopathic nodular panniculitis ŽPfeiffer–
Weber–Christian disease. by MMF has been re-
ported ŽEnk and Knop, 1998.. A combination of
MMF and CsA was used successfully in recalcitrant
pyoderma gangrenosum ŽHohenleutner et al., 1997;
Nousari et al., 1998b..
25.4. Renal disease
As reviewed by Hauser and Bernd Sterzel Ž1999.,
current treatment of immune-mediated and inflam-
matory diseases of the kidney is unsatisfactory, being
associated with unacceptable toxicity, a poor clinical
response, or both. Briggs et al. Ž1998. and Zimmer-
man et al. Ž1998. reported the results of MMF the-
rapy in three patients with membranous nephropathy,
two patients with minimal change disease, one pa-
tient with focal and segmental sclerosis and two
112 A.C. Allison, E.M. Euguir Immunopharmacology 47 (2000) 85–118
patients with lupus nephritis. MMF treatment for 12
months suppressed proteinuria as much as CsA or
more; this resulted in less toxicity and steroid spar-
ing. Nowack et al. Ž1997. reported that four patients
with anti-neutrophil cytoplasmic antibody-positive
vasculitis could be switched from cyclophosphamide
to less toxic MMF, without relapse. The same au-
thors found that MMF treatment of two patients with
IgA nephritis decreased proteinuria and creatinine,
and less steroid side effects such as diabetes mellitus.
Patients with lupus nephritis resistant to cy-
closphamide have also been treated successfully with
MMF ŽNachman et al., 1997; Glinklich and Acharya
1998; Li et al., 1998.. Hauser and Bernd Sterzel
Ž1999. state that controlled prospective studies are
under way to clarify the potential advantage of MMF
over other treatments of kidney diseases.
25.5. Ocular inflammation
Reis et al. Ž1998. reported the use of MMF in a
patient with ocular cicatrical pemphigoid, the switch
from CsA therapy following high-risk keratoplasty
due to CsA allergy, and combination MMF and CsA
therapy in a patient following high-risk keratoplasty
in whom CsA alone was insufficient to prevent
allograft rejection. In these three cases MMF proved
to be safe and effective. A second pilot study also
suggests that MMF may be useful for controlling
other types of ocular inflammation with minimal side
effects ŽLarkin and Lightman, 1999.. MMF Ž1 g
twice daily. was administered with steroids, as an
additional agent with CsA, or instead of CsA or
AZA. Ten of eleven patients showed a favorable
response, leading to improvement of symptoms and
the ability to reduce the dose of prednisone. Further
studies and long-term follow-ups are needed, but
MMF may be useful in combination therapy with
CsA following high-risk keratoplasty, and with CsA
or other agents in autoimmune uveitis. Preventing
blindness with minimal side effects is a worthwhile
objective.
25.6. Autoimmune hemolytic anemia
The use of MMF for treatment of autoimmune
hemolytic anemia has been described by Zimmer-
Molsberger et al. Ž1997..
25.7. GVHD
Treatment of high-risk chronic GVHD with
FK506, MMF and methylprednisolone is described
by Wolff et al. Ž1998..
25.8. Inflammatory bowel disease (IBD)
The use of MMF in refractory IBD has been
proposed ŽNehme et al., 1998; Neurath et al., 1998..
The possible benefits of the drug have to be weighed
against its major side effects on the gastrointestinal
tract.
26. The future
As outlined in this chapter, MMF has several
mechanisms of action, all of which may contribute in
some degree to prevent allograft rejection. These
mechanisms are related: MMF does not suppress
IL-2 gene expression, but blocks the replication of
activated T-lymphocytes at the S-phase, thereby fa-
voring the induction of apoptosis. Depletion of GTP
in lymphocytes and monocytes by MMF suppresses
the expression of adhesion molecules and the pro-
duction of NO by iNOS. It would be academically
interesting to know the relative importance of all
these mechanisms in transplantation and other appli-
cations, but because of their interactions it will not
be easy to disentangle their roles.
An earlier goal is using MMF even more effi-
ciently to reduce the incidence of acute allograft
rejection. A regime by which that might be achieved
is discussed in an accompanying article ŽAllison,
2000.. Preventing acute rejection should also de-
crease the likelihood of chronic rejection, and MMF
has shown superiority to other immunosuppressive
drugs in models of chronic rejection, as reviewed in
this article, and in relevant cell biological assays
ŽAllison, 2000.. Under favorable conditions which
include good lipid profiles, human kidney grafts can
continue to function for at least 15 years ŽViklicky et
al., 1999., so the possibility of organ graft function
for decades exists. As the data unfold, it will be
interesting to learn whether the promise of long-term
benefits for MMF treatment of graft recipients is
fulfilled in practice.
 A.C. Allison, E.M. Euguir Immunopharmacology 47 (2000) 85–118
In this review several other possible applications
of MMF are outlined. To mention only a few, the
preliminary findings in renal disease and liver graft
recipients with HCV deserve to be followed up
systematically. Resistance of HIV to currently used
drugs is spreading, and the combination of MMF
with Abacavir deserves exploration. Treatment of
ocular inflammation and of patients with high-risk
keratoplasty could prevent blindness in groups of
patients.
The first decade of this century should consolidate
the use of MMF in transplantation and establish
whether other applications are useful. That was our
hope in initiating a research program on inhibitors of
IMPDH.
References
Al-Akash, S., Ettenger, R., Gales, B. et al., 1998. Influenza
vaccine antibody response in pediatric renal transplant patients
treated with newer immunosuppressive agents. J. Am. Soc.
Nephrol. 9, 663A, ŽAbstr..
Alfieri, C., Allison, A.C., Kieff, E., 1994. Effect of mycophenolic
acid on Epstein–Barr virus infection of human B-lymphocytes.
Antimicro. Agents Chemother. 38, 126–129.
Allison, A.C., 2000. Immunosuppressive drugs: the first 50 years
and a glance forward. Immunopharmacology 47, 63–83.
Allison, A.C., Almquist, S.J., Muller, C.D., Eugui, E.M., 1991. In
vitro immunosuppressive effects of mycophenolic acid and an
ester prodrug, RS-61443. Transplant. Proc. 23 ŽSuppl. 2.,
10–14.
Allison, A.C., Eugui, E.M., 1993. The design and development of
an immunosuppressive drug, mycophenolate mofetil. Springer
Semin. Immunopathol. 14, 353–380.
Allison, A.C., Hovi, T., Watts, R.W.E., Webster, A.D.B., 1975.
Immunological observations on patients with the Lesch–Nyhan
syndrome, and on the role of de novo purine synthesis in
lymphocyte transformation. Lancet II, 1179–1183.
Allison, A.C., Hovi, T., Watts, R.W.E., Webster, A.D.B., 1977.
The role of de novo purine synthesis in lymphocyte transfor-
mation. Ciba Found. Symp. 48, 207–223.
Allison, A.C., Kowalski, W.J., Muller, C.J. et al., 1993. Mycophe-
nolic acid and brequinar, inhibitors of purine and pyrimidine
synthesis, block the glycosylation of adhesion molecules.
Transplant. Proc. 25 ŽSuppl. 2., 67–70.
Altmann, D.M., Hogg, N., Trowsdale, J., Wilkinson, D., 1989.
Cotransfection of ICAM-1 and HLA-DR reconstitutes human
antigen-presenting function in mouse L cells. Nature 338,
512–514.
Ando, K., Hiroishi, K., Kaneko, T. et al., 1997. Perforin, FasrFas
ligand and TNF-a pathways as specific and bystander killing
mechanisms of hepatitis C virus-specific human CTL. J. Im-
munol. 158, 5283–5291.
113
Azuma, H., Binder, J., Heeman, U. et al., 1995. Effects of
RS61443 on functional and morphological changes in chroni-
cally rejecting rat kidney allografts. Transplantation 59, 460–
466.
Bagasra, D., Michaels, F.H., Zheng, Y.M. et al., 1995. Activation
of the inducible nitric oxide synthase in brains of patients with
multiple sclerosis. Proc. Natl. Acad. Sci. U. S. A. 92, 12041–
12045.
Benvenuti, C., Bories, P.N., Loisance, D., 1996. Increased serum
nitrate concentration in cardiac transplant patients. A marker
for acute allograft cellular rejection. Transplantation 61, 745–
749.
Berman, J.D., Webster, H.K., 1982. In vitro effects of mycophe-
nolic acid and allopurinol against Leishmania tropica in hu-
man macrophages. Antimicro. Agents Chemother. 21, 887–
891.
Bird, A.G., 1982. Cyclosporin A, lymphomata and Epstein-Barr
virus. In: White, D.J. ŽEd.., Cyclosporin A. Elsevier, Amster-
dam, pp. 307–315.
Birkeland, S.A., 1988. Steroid-free immunosuppression after kid-
ney transplantation with antithymocyte globulin induction and
cyclosporine and mycophenolate mofetil maintenance therapy.
Transplantation 66, 1207–1210.
Blaheta, R.A., Leckel, K., Wittig, B. et al., 1999. Mycophenolate
mofetil impairs transendothelial migration of allogeneic CD4
and CD8 T-cells. Transplant. Proc. 31, 1250–1252.
¨
Bohm, M., Beissert, S., Schwartz, T. et al., 1997. Bullous pem-
phigoid treated with mycophenolate mofetil. Lancet, 349–351.
Borel, J.F., Feurer, C., Gubler, H.V. et al., 1976. Biological
effects of cyclosporein A: a new anti-lymphocyte agent. Agents
Actions 6, 468–475.
Briggs, W.A., Choi, M.J., Scheel, P.J. Jr., 1998. Successful
mycophenolate mofetil treatment of glomerular disease. Am. J.
Kidney Dis. 31, 213–217.
Brunner, K.T., Mauel, J., Cerrotini, J.C., Chapuis, B., 1988.
Quantitative assay of the lytic interaction of murine lymphoma
cells on 51 Cr-labelled allogeneic target cells in vitro; inhibition
by isoantibody and drugs. J. Immunol. 14, 181–196.
Byars, N.E., Nakano, G.M., Kang, R., Allison, A.C., 1995. Oral
administration of mycophenolic acid selectively depletes GTP
in lymph node cells of immunized rats. In: 9th Internat. Congr.
Immunology, Abstract 855.
Carr, S.F., Papp, E., Wu, J.C., Natsumeda, Y., 1993. Characteriza-
tion of human type I and type II IMP dehydrogenases. J. Biol.
Chem. 268, 27286–272902.
Chanaud, N.P. III, Vistica, B.P., Eugui, E. et al., 1995. Inhibition
of experimental autoimmune uveoretinitis by mycophenolate
mofetil, an inhibitor of purine metabolism. Exp. Eye Res. 61,
429–434.
Chang, C.-C.J., Aversa, G., Punnonen, J., Yssel, H., de Vries, J.,
1993. Brequinar sodium, mycophenolic acid, and cyclosporin
A inhibit different stages of IL-4 or IL-3-induced human IgG4
and IgE production in vitro. Ann. N. Y. Acad. Sci. 696,
108–122.
Cohn, R.G., Mirkovich, A., Caulfield, J., Eugui, E.M., 1999a.
Apoptosis of human activated peripheral T-cells and T-
lymphocytic and promonocytic cell lines induced by mycophe-
114 A.C. Allison, E.M. Euguir Immunopharmacology 47 (2000) 85–118
nolic acid, the active metabolite of CellCept. In: The Sixth
Basic Sciences Symposium of the Transplantation Society,
Monterey, CA. p. 173.
Cohn, R.G., Mirkovich, A., Dunlap, B. et al., 1996b. Mycopheno-
lic acid increases apoptosis, lysosomes and lipid droplets in
human lymphoid and monocytic cell lines. Transplantation 68,
411–418.
Corna, D., Morigi, M., Facchinetti et al., 1997. Mycophenolate
mofetil limits renal damage and prolongs life in murine lupus
autoimmune disease. Kidney Int. 51, 1583–1589.
Dissanayake, I.R., Goodman, G.R., Bowman, A.R. et al., 1998.
Mycophenolate mofetil: a promising new immunosuppressant
that does not cause bone loss in the rat. Transplantation 65,
275–278.
Dodge, I.L., Li, X.C., Strom, ¨ T.B., 1999. Rapamycin induces
TGF-b production in lymphocytes. Transplantation 67, S50.
Enk, A.H., Knop, J., 1998. Treatment of relapsing idiopathic
nodular panniculitis ŽPfeiffer–Weber–Christian disease. with
mycophenolate mofetil. J. Am. Acad. Dermatol. 39, 508–509.
Enk, A.H., Knop, J., 1999. Mycophenolate mofetil is effective in
the treatment of pemphigus vulgaris. Arch. Dermatol. 135,
54–56.
Eugui, E.M., Almquist, S., Muller, C.D., Allison, A.C., 1991a.
Lymphocyte-selective cytostatic and immunosuppressive ef-
fects of mycophenolic acid in vitro: role of deoxyguanosine
nucleotide depletion. Scand. J. Immunol. 33, 161–173.
Eugui, E.M., Mirkovich, A., Allison, A.C., 1991b. Lymphocyte-
selective antiproliferative and immunosuppressive effects of
mycophenolic acid in mice. Scand. J. Immunol. 33, 175–183.
Eugui, E.M., Nakano, G.M., Byars, N.E., 1995. Effect of my-
cophenolic acid on antibody titers and isotypes in mice vacci-
nated with influenza virus hemagglutinin. In: 9th Internat.
Congr. Immunology, Abstracts 5073. .
Fairbanks, L.D., Bofill, M., Ruckemann, K., Simmonds, H.A.,
1995. Importance of ribonucleotide availability to proliferating
T-lymphocytes from healthy humans. Disproportionate expan-
sion of pyrimidine pools and contrasting effects of de novo
synthesis inhibitors. J. Biol. Chem. 270, 29682–29689.
Franklin, T.J., Cook, J.M., 1969. The inhibition of nucleic acid
synthesis by mycophenolic acid. Biochem. J. 113, 515–524.
Fraser-Smith, E.B., Rosette, J.D., Schatzman, R.C., 1995. Sup-
pression by mycophenolate mofetil of the neointimal thicken-
ing caused by vascular injury in a rat arterial stenosis model. J.
Pharmacol. Exp. Ther. 275, 1204–1208.
Fujihara, C.K., Malheiros, D.M., Zatz, R., Noronha, I.D., 1998.
Mycophenolate mofetil attenuates renal injury in the rat rem-
nant kidney. Kidney Int. 45, 1510–1519.
Garcia, R.C., Leoni, P., Allison, A.C., 1977. Control of phospho-
ribosyl pyrophosphate synthesis in human lymphocytes.
Biochem. Biophys. Res. Commun. 77, 1067–1073.
Geilen, C.C., Tebbe, B., Garcia-Bartels, C., Krengel, S., Orfanos,
C.E., 1998. Successful treatment of erythrodermic psoriasis
with mycophenolate mofetil. Br. J. Dermatol. 138, 1101–1102.
Giblett, E.R., Anderson, J.E., Cohen, F., Meuwissen, H.J., 1972.
Adenosine deaminase deficiency in two patients with severely
impaired cellular immunity. Lancet II, 1067–1069.
Glinklich, D., Acharya, A., 1998. Mycophenolate mofetil therapy
for lupus nephritis refractory to intravenous cyclophospha-
mide. Am. J. Kidney Dis. 32, 318–322.
Goldblum, R., 1993. Therapy of rheumatoid arthritis with my-
cophenolate mofetil. Clin. Exp. Rheumatol. 11, S117–119,
ŽSuppl...
Goto, M., Yamaguchi, Y., Ichiguchi, O. et al., 1997. Phenotype
and localization of macrophages expressing inducible nitric
oxide synthase in rat hepatic allograft rejection. Transplanta-
tion 64, 303–310.
Grailer, A., Nichols, J., Hullett, D., Sollinger, H.W., Burlingham,
W.J., 1991. Inhibition of human B cell responses in vitro by
RS-61443, cyclosporine A and FK506. Transplant. Proc. 23,
314–315.
Green, C.J., Allison, A.C., 1978. Extensive prolongation of rabbit
kidney allograft survival after short-term cyclosporin A treat-
ment. Lancet ii, 1182–1184.
Green, C.J., Allison, A.C., Precious, S., 1979. Induction of spe-
cific tolerance in rabbits by kidney allografting and short
period of cyclosporin A therapy. Lancet ii, 123–125.
Gregory, C.R., Huang, X. et al., 1995. Treatment with rapamycin
and mycophenolic acid reduces arterial intimal thickening
produced by mechanical injury and allows endothelial replace-
ment. Transplantation 59, 655–661.
Gregory, C.D., Murray, R.J., Edwards, C.F., Rickinson, A.D.,
1988. Down regulation of cell adhesion molecules LFA-3 and
ICAM-1 in Epstein–Barr virus-positive Burkitt’s lymphoma
underlies tumor cell escape from virus-specific T cell surveil-
lance. J. Exp. Med. 167, 1811–1824.
Gregory, C.R., Pratt, R.E., Huie, P. et al., 1993. Effects of
treatment with cyclosporine, FK-506, rapamycin, mycopheno-
lic acid, or dexoyspergualin on vascular muscle proliferation
in vitro and in vivo. Transplant Proc. 25, 770–771.
Grundmann-Kollman, M., Korting, H.C. et al., 1999. Mycopheno-
late mofetil: a new therapeutic option in the treatment of
blistering autoimmune diseases. J. Am. Acad. Dermatol. 40,
957–960.
Hao, L., Calcinaro, F., Gill, R.G. et al., 1992. Facilitation of
tolerance induction in adult mice by RS-61443. Transplanta-
tion 53, 590–595.
Hao, L.M., Chan, S.M., Lafferty, K.J., 1993. Mycophenolate
mofetil can prevent the development of diabetes in BB rats.
Ann. N. Y. Acad. Sci. 696, 328–332.
Hao, L., Lafferty, K.J., Allison, A.C., Eugui, E.M., 1990. RS-
61443 allows islet allografting and specific tolerance induction
in adult mice. Transplant Proc. 22, 876–879.
Haufs, M.G., Beissert, S., Grabbe, S., Schutte, B., Luger, T.A.,
1998. Psoriasis vulgaris treated successfully with mycopheno-
late mofetil. Br. J. Dermatol. 138, 179–181.
Hauser, I.A., Bernd Sterzel, R., 1999. Mycophenolate mofetil:
therapeutic applications in kidney transplantation and
immune-mediated renal disease. Curr. Opin. Nephrol. Hyper-
tens. 8, 1–6.
Hauser, R.A., Malek, A.R., Rosen, R., 1998. Successful treatment
of a patient with severe refractory myasthenia gravis using
mycophenolate mofetil. Neurology 51, 912–913.
´
Hauser, I.A., Johnson, D.R., Thevenod, ¨
F., Goppelt-Strube, M.,
1997a. Effect of mycophenolic acid on TNF alpha-induced
 A.C. Allison, E.M. Euguir Immunopharmacology 47 (2000) 85–118
expression of cell adhesion molecules in human venous en-
dothelial cells in vitro. Br. J. Pharmacol. 122, 1315–1322.
Hauser, I.A., Renders, L., Merkel, C., Goppelt-Struebe, M., 1997b.
Mycophenolate mofetil, but not cyclosporin A or FK506,
inhibits rat mesangial cell proliferation in vitro. J. Am. Soc.
Nephrol. 9, 496A, ŽAbstr..
¨
Hayry, P., Isoniemi, H., Yilmaz, S. et al., 1993. Chronic allograft
rejection. Immunol. Rev. 134, 33–81.
Heemann, U., Azuma, H., Hamar, P. et al., 1996. Mycophenolate
mofetil inhibits lymphocyte binding and the upregulation of
adhesion molecules in acute rejection of rat kidney allografts.
Transplant. Immunol. 4, 64–67.
Heering, P., Heise, G., Hess, A., 1998. MMF reduces proteinuria
in passive Heymann nephritis. J. Am. Soc. Nephrol. 9, 456A–
457A, ŽAbstr..
Hohenleutner, U., Mohr, V.D., Michel, S., Landthaler, M., 1997.
Mycophenolate mofetil and cyclosporin treatment for recalci-
trant pyoderma gangrenosum. Lancet 350, 1748.
Hovi, T., Allison, A.C., Allsop, J., 1975. Rapid increase of
phosphoribosyl pyrophosphate concentration after mitogenic
stimulation of lymphocytes. FEBS Lett. 55, 291–293.
Hovi, T., Allison, A.C., Raivio, K.O., Vaheri, A., 1977. Purine
metabolism and control of cell proliferation. Ciba Found.
Symp. 48, 225–248.
Huang, Q.-Y., Ye, W.-L., Zheng, F.-L., 1998. Effect of mycophe-
nolate mofetil on anti-Thy-1 mesangial proliferative glomer-
ulonephritis. J. Am. Soc. Nephrol. 9, 496A.
Hupe, D.J., Azzolina, B.A., Behrens, N.D., 1986. IMP dehydroge-
nase from the intracellular parasitic protozoan Eimeria tenella
and its inhibition by mycophenolic acid. J. Biol. Chem. 261,
8363–8369.
Ichimura, H., Levy, J.A., 1995. Polymerase substrate depletion: a
novel strategy for inhibiting the replication of the human
immunodeficiency virus. Virology 211, 554–560.
Isobe, M., Yagita, H., Okumura, K., Ihara, A., 1992. Specific
acceptance of cardiac allograft after treatment with antibodies
to ICAM-1 and LFA-1. Science 255, 1125–1127.
Jackson, R.C., Morris, H.P., Weber, G., 1977. Partial purification,
X
properties and regulation of inosine 5 -phosphate dehydroge-
nase in normal and malignant rat tissues. Biochem. J. 166,
1–10.
Keown, P.A. et al., 1996. A blinded, randomized clinical trial of
mycophenolate mofetil for the prevention of acute rejection in
cadaveric renal transplantation. Transplantation 61, 1029–
1037.
Khanna, A., 1999. TGF-beta provides the rationale for the syner-
gistic immunosuppression with rapamycin ŽRAPA., cy-
closporine ŽCsA. and tacrolimus ŽTAC. calcineurin inhibitors
sparing immunosuppression protocol. Transplantation 67, S58.
Koglin, J., Granville, D.J., Glysing-Jensen, T. et al., 1999. Attenu-
ated acute cardiac rejection in NOS2yry recipients. Circula-
tion 99, 836–842.
Koglin, J., Glysing-Jensen, T., Mudgett, S.S., Russell, M.E., 1998.
NOS2 mediates opposing effects in models of acute and
chronic cardiac rejection: insights from NOS2-knockout mice.
Am. J. Pathol. 153, 1371–1376.
Konno, Y., Natsumeda, Y., Nagai, M. et al., 1991. Expression of
IMP dehydrogenase types I and II in Escherichia coli and
115
distribution in normal human lymphocytes and leukemic cell
lines. J. Biol. Chem. 266, 506–509.
Kornfeld, R., Kornfeld, S., 1980. Structure of glycoproteins and
their oligosaccharide units. In: Lennarz, W.J. ŽEd.., Biochem-
istry Glycoproteins and Proteoglycans. Plenum, New York,
pp. 1–137.
Larkin, G., Lightman, S., 1999. Mycophenolate mofetil. A useful
immunosuppressive in inflammatory eye disease. Ophthomol-
ogy 106, 370–374.
Laski, L.A., 1992. Selectins: interpreters of cell-specific carbo-
hydrate information during inflammation. Science 258, 964–
969.
Laurent, A.F., Dumont, S., Poindron, P., Muller, C.D., 1996.
Mycophenolic acid suppresses protein N-linked glycosylation
in human monocytes and their adhesion to endothelial cells
and some substrates. Exp. Hematol. 24, 59–67.
Lee, W.A., Gu, L., Miksztal, A.R., Chu, N. et al., 1990. Bioavail-
ability improvement of mycophenolic acid through amino
ester derivitization. Pharm. Res. 7, 161–166.
Leoni, P., Garcia, R.C., Allison, A.C., 1978. Effects of cy-
closporin A on human lymphocytes in culture. J. Lab. Clin.
Immunol. 1, 67–75.
Li, L.-S., Liu, Z.-H., Hong, Z., Hu, W.-X., 1998. Mycophenolate
mofetil showed inhibitory effect on VCAM expression during
successful treatment of diffuse proliferative lupus nephritis
ŽSLE-DPGN. with vascular lesions. J. Am. Soc. Nephrol. 9,
153A.
Margolis, D., Heredia, A., Gaywee, J., 1999. Abacavir and my-
cophenolic acid, an inhibitor of inosine monophosphate dehy-
drogenase, have profound and synergistic anti-HIV activity. J.
AIDS, Žin press..
Maurizio, S., Bertoni, E., Rosati, A. et al., 1998. Mycophenolate
mofetil ŽMMF. prevents acute rejection and assures high
one-year graft survival also in the case of delayed graft
function. J. Am. Soc. Nephrol. 9, 696A, ŽAbstr..
McMurray, R.W., Ellbourne, K., Lagoo, A. et al., 1998. My-
cophenolate mofetil suppresses autoimmunity and mortality in
the female ŽNZB=NZW.F1 mouse model of systemic lupus
erythematosis. J. Rheumatol. 25, 2364–2370.
Mirkovich, A., Eugui, E.M., 1995. Prevention of murine graft-
versus-host disease ŽGVHD. by mycophenolate mofetil. In:
9th Internat. Congr. Immunol. ŽAbstr.. p. 2523.
Moncada, S., Palmer, R.M.J., Higgs, E.A., 1991. Nitric oxide:
physiology, pathophysiology and pharmacology. Pharmacol.
Rev. 43, 109–142.
Morris, R.E., Hoyt, E.G., Murphy, M.P., Eugui, E.M., Allison,
A.C., 1990. Mycophenolic acid morpholinoethylester ŽRS-
61443. is a new immunosuppressant that prevents and halts
heart allograft rejection by selective inhibition of T and B cell
purine synthesis. Transplant. Proc. 22, 1659–1662.
Morris, R.E., Wang, J., Blum, J.R. et al., 1991. Immunosuppres-
sive effects of the morpholinoethyl ester of mycophenolic acid
ŽRS-61443. in rat and nonhuman primate recipients of heart
allografts. Transplant. Proc. 23 ŽSuppl. 2., 19–25.
Mueller, V., Hamar, P., Szabo, A. et al., 1998. Effect of mycophe-
nolate mofetil on the in vivo infiltration of lymphocytes in the
rat remnant kidney. Transplant. Proc. 30, 982.
Mulkins, M.A., Ng, M., Lewis, R.G., 1992. Mycophenolic acid
116 A.C. Allison, E.M. Euguir Immunopharmacology 47 (2000) 85–118
inhibits the degranulation of rat peritoneal mast cells. Cell.
Immunol. 141, 508–517.
Nachman, P.H., Dooley, M.A., Hogan, S.L. et al., 1997. My-
cophenolate mofetil therapy in patients with cyclophospha-
mide-ŽCyP. resistant or relapsing diffuse proliferative lupus
nephritis ŽSLE-DPGN.. J. Am. Soc. Nephrol. 9, 94A.
Nadeau, K.C., Azuma, H., Tilney, N.L., 1996. Sequential cytokine
expression in renal allografts in rats immunosuppressed with
maintenance cyclosporine or mycophenolate mofetil. Trans-
plantation 62, 1363–1366.
Nagai, M., Natsumeda, Y., Weber, G., 1992. Proliferation-linked
regulation of the type II IMP dehydrogenase gene in human
normal lymphocytes and HL-60 leukemic cells. Cancer Res.
52, 258–261.
Nagy, S.E., Andersson, J.P., Andersson, U.G., 1993. Effect of
mycophenolate mofetil ŽRS-61443. on cytokine production:
inhibition of superantigen-induced cytokines. Immunopharma-
cology 26, 11–20.
Natsumeda, Y., Ohno, S., Kawasaki, H. et al., 1990. Two distinct
cDNAs for human IMP dehydrogenase. J. Biol. Chem. 265,
5292–5295.
Nehme, O.S., Overley, C.A., O’Brien, J.J., 1998. The role of
mycophenolate mofetil in the management of refractory in-
flammatory bowel disease ŽIBD.. Gastroenterology 114 Ž4 ŽPt.
2.., A1049.
Neurath, M.F., Wanitschke, R., Peters, M. et al., 1998. Random-
ized trial of mycophenolate mofetil versus azathioprine for
treatment of chronic active Crohn’s disease. Gastroenterology
114 Ž4 ŽPt. 2.., A1050.
Neyts, J., Andrei, G., De Clercq, E., 1998a. The novel immuno-
suppressive agent mycophenolate mofetil markedly potentiates
the antiherpesvirus activities of acyclovir, ganciclovir, and
penciclovir in vitro and in vivo. Antimicro. Agents Chemother.
42, 216–222.
Neyts, J., Andrei, G., De Clercq, E., 1998b. The antiherpesvirus
activity of H2G is markedly enhanced by the novel immuno-
suppressive agent mycophenolate mofetil. Antimicro. Agents
Chemother. 42, 3285–3289.
Neyts, J., Meerbach, A., McKenna, P., De Chercq, E., 1996. Use
of the yellow fever virus vaccine strain 17D for the study of
strategies for the treatment of yellow fever virus infection.
Antiviral Res. 30, 125–132.
Noronha, I.L., Fujihara, C.K., Oliviera, I.B., Leitao, T.G., Zatz,
R., 1998. Enhanced interstitial expression of angiotensin II and
TGF-b in the renal ablation model: effect of mycophenolate
mofetil. J. Am. Soc. Nephrol. 9, 618A, ŽAbstr..
Nousari, H.C., Griffin, W.A., Anhalt, G.J., 1998a. Successful
therapy for bullous pemphigoid with mycophenolate mofetil.
J. Am. Acad. Dermatol. 39, 497–498.
Nousari, H.C., Lynch, W., Anhalt, G.J., Petri, M., 1998b. The
effectiveness of mycophenolate mofetil in refractory pyoderma
gangrenosum. Arch. Dermatol. 134, 1509–1511.
Nowack, R., Birch, R., Van de Woude, F.J., 1997. Mycophenolate
mofetil for systemic vasculitis and IgA nephropathy. Lancet
349, 774.
Nussenblatt, R.B., Palestine, A.G., 1989. Uveitis: Fundamentals
and Clinical Practice. Year Book Medical Publishers, Chicago,
USA.
O’Gara, M.J., Lee, C.H., Weinberg, G.A., Nott, J.M., Queener,
S.F., 1997. IMP dehydrogenase from Pneumocystis carinii as
a potential drug target. Antimicro. Agents Chemother. 41,
40–48.
O’Hair, D.P., McManus, R.P., Komorowski, R., 1994. Inhibition
of chronic vascular rejection in primate cardiac xenografts
using mycophenolate mofetil. Ann. Thorac. Surg. 58, 1311–
1315.
Ozchgfhj, H.S., Hughes, W.T., 1997. Novel anti-Pneumocystis
carinii effects of the immunosuppressant mycophenolate
mofetil in contrast to the provocative effects of tacrolimus,
sirolimus and dexamethasone. J. Infect. Dis. 175, 901–904.
Pardo-Mindan,´ F.J., Errasti, P., Panizo, A. et al., 1999. Decrease
of apoptosis rate in patients treated with mycophenolate
mofetil. Nephron 82, 232–237.
Paul, L.C., Muzaffar, S., Valentin, J.-F., 1998. Donor pretreatment
with mycophenolate mofetil protects against ischemia–reper-
fusion injury. Transplantation 65, 1193–1195.
¨
Paul, L.C., Myllarniemi, M., Muzaffar, S., Benediktsson, H.,
1996. Nitric oxide synthase inhibition is associated with de-
creased survival of cardiac allografts in the rat. Transplanta-
tion 65, 1193–1195.
Penny, M.J., Boyd, R.A., Hall, B.M., 1998. Mycophenolate mofetil
prevents the induction of active Heymann nephritis: associa-
tion with Th2 cytokine inhibition. J. Am. Soc. Nephrol. 9,
2272–2282.
Peters, G.J., Oosterhof, A., Verkamp, J.H., 1982. Effects of
adenosine and deoxyadenosine on PHA stimulation of lym-
phocytes of man, horse and pig. Int. J. Biochem. 14, 377–385.
Pickenacker, A., Luger, T.A., Schwarz, T., 1998. Dyshidrotic
eczema treated with mycophenolate mofetil. Arch. Dermatol.
134, 378–379.
Platz, K.P., Eckhoff, D., Bechstein, W.O. et al., 1991. RS-61443
for reversal of acute rejection in canine renal allografts. Surgery
110, 736–741.
Platz, K.P., Mueller, A.R., Willimeki, C. et al., 1998. Indications
for mycophenolate mofetil therapy in hepatitis C patients
undergoing liver transplantation. Transplant. Proc. 30, 1468–
1469.
Platz, K.P., Sollinger, H.W., Hullett, D.A. et al., 1990. RS-61443,
a new, potent immuno-suppressive agent. Transplantation 51,
27–31.
¨ ¨
Raisanen-Sokolowski, A., Vuoristo, P. et al., 1995. Mycopheno-
late mofetil ŽMMF, RS-61443. inhibits proliferation of rat
aortic allografts. Transplant. Immunol. 3, 342–351.
Reis, A., Reinhard, T., Sundmacher, R. et al., 1998. Mycopheno-
late mofetil in ocular immunological disorders. A survey of
the literature with 3 case reports. Klin. Monatsbl. Augen-
heilkd. 213, 257–261.
Richman, D.D., Kornbluth, R.S., Carson, P.A., 1987. Failure of
dideoxynucleosides to inhibit HIV replication in cultured hu-
man macrophages. J. Exp. Med. 166, 1144–1149.
Romero, F., Rodriguez-Iturbe, B., Parra, G. et al., 1999. My-
cophenolate mofetil prevents the progressive renal failure in-
duced by 5r6 renal ablation in rats. Kidney Int. 55, 945–955.
Rosenberg, A.S., Singer, A., 1992. Cellular basis of skin graft
rejection: an in vivo model of immune-mediated tissue de-
struction. Annu. Rev. Immunol. 10, 333–358.
 A.C. Allison, E.M. Euguir Immunopharmacology 47 (2000) 85–118
Schiff, M.H., Leishman, B., 1998. CellCept Žmycophenolate
mofetil-MMF., a new treatment for RA: a 12-week, double-
blind, randomized, placebo-controlled withdrawal trial. Arthri-
tis Rheum. 41 ŽSuppl. 9., S364.
Senda, M., DeLustro, B., Eugui, E., Natsumeda, Y., 1995. My-
cophenolic acid, an inhibitor of IMP dehydrogenase that is
also an immunosuppressive agent, suppresses the cytokine-in-
duced nitric oxide production in mouse and rat vascular
endothelial cells. Transplantation 60, 1143–1148.
Shaffer, D., Blakely, M.L., Gottschalk, R., Monaco, A.P., 1992.
Small bowel transplantation in rats using RS-61443: effect on
GVHD and rejections. Transplant. Proc. 24, 1159–1160.
Shaffer, D., Muanza, T., Blakely, M.L. et al., 1993. Prevention of
graft-versus-host disease by RS-61443 in two different rodent
models. Transplantation 55, 221–223.
Shears, L.L., Kawaharada, N., Tzeng, E. et al., 1997. Inducible
nitric oxide synthase suppresses the development of allograft
arteriosclerosis. J. Clin. Invest. 100, 2035–2042.
Shoji, Y., 1994. Effect of topical preparation of mycophenolic
acid on experimental allergic contact dermatitis of guinea-pigs
induced by dinitrofluorobenzene. J. Pharm. Pharmacol. 46,
643–646.
Sintchak, M.D., Nimmesgern, E., 2000. The structure of inosine
monophosphate dehydrogenase and the design of novel in-
hibitors. Immunopharmacology 47, 163–184.
Steele, D.M., Hullett, D.A., Bechstein, W.O. et al., 1993. Effects
of immunosuppressive therapy on the rat aortic allograft model.
Transplant. Proc. 25, 754–755.
Stojanovic, T., Grave, H.J., Gieseler, R.K. et al., 1996. Enhanced
renal allograft rejection by inhibitors of alloreactivity. Lab.
Invest. 74, 496–512.
Stryer, L., 1988. Biosynthesis of nucleotides. In: Biochemistry.
3rd edn. Freeman, New York, pp. 602–626.
Szabolcs, M., Michler, R.E., Yang, X. et al., 1996. Apoptosis of
cardiac myocytes during cardiac allograft rejection. Relation to
inhibition of nitric oxide synthase. Circulation 94, 1665–1673.
Szabolcs, M.J., Ravalli, S., Minanov, O. et al., 1998. Apoptosis
and increased expression of inducible nitric oxide synthase in
human allograft rejection. Transplantation 65, 804–812.
Thomson, A.W., Woo, J., Yao, G.Z. et al., 1993. Effects of
combined administration of FK-506 and the purine biosynthe-
sis inhibitors mizoribine or mycophenolic acid on lymphocyte
DNA synthesis and T-cell activation molecule expression in
human mixed lymphocyte cultures. Transplant. Immunol. 1,
146–150.
Tutschka, P.J., Beschorner, W.E., Allison, A.C. et al., 1979. Use
of cyclosporin A in allogeneic bone marrow transplantation in
the rat. Nature 280, 148–150.
Van Bruggen, M.C.J., Walgreen, B., Riike, T.P.M., Berden,
J.H.M., 1998. Attenuation of murine lupus nephritis by my-
cophenolate mofetil. J. Am. Soc. Nephrol. 9, 1407–1415.
Verham, R., Meek, T.D., Hedstrom, L., Wang, C.C., 1987. Purifi-
cation, characterization and kinetic analysis of inosine-
X
5 monophosphate dehydrogenase of Trichomonas foetus. Mol.
Biochem. Parasitol. 24, 1–12.
´
Viklicky, O., Hubacek, ´
J.A., Lacha, J. et al., 1999. Long-term
graft survival and ACE gene polymorphism. In: The Sixty
117
Basic Sciences Symposium of the Transplantation Society,
Monterey, CA. p. 186.
Wang, X., Lewis, D.A., Kim, H.K. et al., 1998. Alterations in
mRNA for inducible endothelial nitric oxide synthase and
plasma nitric oxide with rejection andror infection of allo-
transplanted lungs. Transplantation 66, 567–572.
Wang, W., Tong, M.K.H., Lee, S.H., Chan, L., 1999. Mycopheno-
late mofetil protects against renal ischemia–reperfusion injury
by inhibiting osteopontin and ICAM-1 expression. Transplan-
tation 67, 537, ŽAbstr..
Wang, C.C., Verham, R., Cheng, H.W. et al., 1984. Differential
effects of inhibitors of purine metabolism on two trichomonad
species. Biochem. Pharmacol. 33, 1323–1329.
Watarai, Y., Takeuchi, I., Togashi, H., Yoshioka, M. et al., 1999.
Nitric oxide production in renal transplant recipients and iNOS
expression in renal allografts. Transplantation 67, S9, ŽAbstr..
Waters, R., Webster, D., Allison, A.C., 1993. Mycophenolic acid
and some antioxidants induce differentiation of monocytic
lineage cells and augment production of the IL-1 receptor
antagonist. Ann. N. Y. Acad. Sci. 696, 185–196.
Wei, W., Tong, M.K.H., Chan, L., 1999. Mycophenolic acid
ŽMPA. inhibits the over-expression of osteopontin and ICAM-1
proteins in ischemiarreperfusion injury. J. Am. Soc. Nephrol.
9, 660A, ŽAbstr..
Weimer, R., Melk, A., Daniel, V. et al., 1999. Monocyte re-
sponses in patients with chronic renal transplant rejection:
beneficial effects of MMF-based immunosuppression. Trans-
plantation 67, 27, ŽAbstr..
Whitcup, S.M., Nussenblatt, R.B., 1993. Treatment of autoim-
mune uveitis. Am. NY Acad. Sci. 696, 307–318.
Winklhofer, F., Dai, F.-X., Cowley, B.D. Jr. et al., 1999. My-
cophenolate inhibits nitric oxide ŽNO. production via iNOS in
renal allograft recipients. Transplantation 67, S218.
Wolberg, G., 1988. Antipurines and purine metabolism. Handb.
Exp. Pharmacol. 85, 517–550.
Wolff, D., Kuerschner, D., Skibbe, T. et al., 1998. Treatment of
high risk chronic GVHD with FK506, mycophenolate mofetil
ŽMMF. and methylprednisolone ŽMP.. Br. J. Haematol. 102,
210.
Worrall, N.K., Misko, P.P., Botney, M.D. et al., 1999. Time
course and cellular localization of indicible nitric oxide syn-
thases expression during cardiac allograft rejection. Ann. Tho-
rac. Surg. 67, 716–722.
Wu, J.C., 1994. Mycophenolate mofetil: mechanisms of action.
Perspect. Drug Discov. Design 2, 185–204.
Yamaguchi, Y., Olsabe, K., Matsumura, F. et al., 1999. Peroxyni-
trite formation during rat hepatic allograft rejection. Hepatol-
ogy 29, 777–784.
Yu, C., Seidel, K., Nash, R.A., Deeg, H.J., Sandmaier, B.M.,
Barsoukov, A., 1998. Synergism between mycophenolate
mofetil and cyclosporine in preventing graft-versus-host dis-
ease among lethally irradiated dogs given DLA-nonidentical
marrow grafts. Blood 91, 2581–2587.
Zeeh, J.M., Zorlu, I., Riley, N.E., Goebell, H., Dignass, A.U.,
1998. Mycophenolate mofetil reduces tissue damage in an
experimental model of colitis in rats. Gastroenterology 114 Ž4
ŽPt. 2.., A1122.
118 A.C. Allison, E.M. Euguir Immunopharmacology 47 (2000) 85–118

Zimmer-Molsberger, B., Knauf, W., Thiel, E., 1997. Mycopheno-
late mofetil for severe autoimmune haemolytic anaemia. Lancet
350, 1003–1004.
Zimmerman, R.F., Miller, G., Radhakrishnan, J., Appel, G.B.,
1998. Mycophenolate mofetil ŽMMF. treatment of idiopathic
membranous nephropathy ŽIMN.. J. Am. Soc. Nephrol. 9,
103A.
Ziolo, M.T., Bollinger, S.S., Wabler, G.M., 1998. Myocytes iso-
lated from rejecting transplanted hearts exhibit reduced basal
shortening which is reversible by aminoguanidine. J. Mol. Cell
Cardiol. 30, 1009–1017.
Ziswiler, R., Steinman-Niggli, K., Kappeler, A. et al., 1998.
Mycophenolic acid: a new approach to the therapy of experi-
mental mesangial proliferative glomerulonephritis. J. Am. Soc.
Nephrol. 9, 2055–2066.