Review
Mycophenolate mofetil and its mechanisms of action

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Abstract

Mycophenolate mofetil (MMF, CellCept®) is a prodrug of mycophenolic acid (MPA), an inhibitor of inosine monophosphate dehydrogenase (IMPDH). 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 (iNOS). MPA therefore suppresses the production by iNOS of NO, and consequent tissue damage mediated by peroxynitrite.

CellCept® 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® in preventing allograft rejection, and in the treatment of rejection, is now firmly established. CellCept® 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.

Introduction

In 1982 our research program on immunosuppressive drugs at Syntex, Palo Alto, was initiated. This program had as a background our own experience of cyclosporin A (CsA) 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. (1976) that CsA inhibits responses of T-lymphocytes in rodents, our group showed that CsA also suppresses human T-lymphocytic responses (Leoni et al., 1978). By oral administration of CsA to rabbits we were able to establish donor-specific tolerance to kidney allografts Green 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 (Tutschka 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 (AZA) and a glucocorticoid in transplantation.

In the decade that followed, this regime greatly improved the outcome of organ transplantation. However, using a nephrotoxic drug for kidney transplantation was not ideal, and the incidence of rejection episodes was still unacceptably high (around 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 lymphocytes than on other cell types, including hematopoietic 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, provided 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 (Wolberg, 1988). The historical background leading to our selection of inosine monophosphate dehydrogenase (IMPDH) as a target, and mycophenolic acid (MPA) as a drug, has been reviewed elsewhere (Allison 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 housekeeping isoform of the enzyme, the validity of the selection was confirmed.

That MPA is a more potent inhibitor of the proliferation 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 (Allison et al., 1993), as well as the activity of inducible nitric oxide synthase (iNOS) (Senda et al., 1995). In this paper, evidence from other laboratories confirming these observations in experimental animals and humans is reviewed, and their relevance to transplantation and other applications is discussed. More recently we have found that MPA can induce apoptosis of human T-lymphocytes Cohn et al., 1999a, Cohn et al., 1996b, which has interesting implications for tolerance induction. Observations from several laboratories showing that mycophenolate mofetil (MMF) can inhibit chronic allograft rejection in various experimental animal models are summarized. Activity of MMF in experimental animal models of kidney diseases, autoimmune uveoretinitis, experimental autoimmune encephalomyelitis and adjuvant arthritis suggest possible new clinical applications. MPA has long been known to have antimicrobial effects; recent evidence indicates that activity against Pneumocystis carinii, hepatitis C virus (HCV) and human immunodeficiency virus (HIV) may be clinically useful.

Section snippets

Background

There are two major pathways of purine synthesis (Fig. 1); details can be found in textbooks of biochemistry. In the de novo pathway, the ribose phosphate portion of purine nucleotides is derived from 5-phosphoribosyl-1-pyrophosphate (PRPP), 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

MPA

From several possible IMPDH inhibitors we selected for detailed study MPA (Fig. 4), a fermentation product of several Penicillium species. MPA was preferred to nucleoside analogues such as mizoribine, bredinin, ribavirin and tiazofurin because the latter must be phosphorylated to inhibit IMPDH, and because of the side effects of this class of compounds. The efficiency of nucleoside phosphorylation can vary in different cell types (Richman et al., 1987), and the relevance of target cells in

Isoforms of IMPDH

Natsumeda et al. (1990) isolated two distinct cDNA clones (types 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 poly(A)+ RNA from normal human leukocytes showed expression of the type I enzyme, whereas leukemic cells and ovarian tumor cells expressed predominantly the type II gene. Subsequent studies Konno et al., 1991, Nagai et al., 1992 showed

Development of MMF

The strategy of chemically modifying antibiotics to improve their activity is well known, e.g. modifications 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 (MMF, Fig. 4) was found to have improved bioavailability in primates as compared with MPA (Lee et al., 1990). The ester is rapidly hydrolysed to yield MPA, both in human peripheral

Preferential inhibition of lymphocyte proliferation by MPA

Concentrations of MPA that are readily attainable therapeutically inhibit the proliferative responses of human peripheral blood mononuclear cells to phytohemagglutinin (a T-cell mitogen), pokeweed mitogen (a T-dependent B-cell mitogen) and Staphylococcus protein A sepharose (a B-cell mitogen) (Eugui et al., 1991a). Mixed lymphocyte responses are also inhibited by MMF and MPA. In all cases the IC50 is less than 100 nM, a concentration of drug having no cytostatic effect on fibroblasts or

Intracellular pools of GTP and dGTP

It has been postulated that G-proteins may be involved in the transduction of mitogenic signals to human T-lymphocytes; depletion of GTP might therefore affect such transduction systems. To ascertain whether the antiproliferative effects of MPA are due to depletion of GTP or of dGTP, pools of nucleotides were measured in mitogen-activated human 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

Cytokine production

CsA and FK-506 inhibit early stages of lymphocyte activation, including the production of IL-2. In contrast, MMF or MPA in concentrations up to 1 μM had no detectable effect on IL-2 production in mitogen-activated human peripheral blood lymphocytes (Eugui et al., 1991a). Unlike CsA, MPA does not inhibit IL-2 gene expression in activated T-cells (Thomson 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

Antibody formation in vitro

Antibody formation by polyclonally activated human B-lymphocytes was almost completely inhibited by 100 nM MPA (Allison 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 (Grailer et al., 1991). Chang et al. (1993) found that MPA (1 μM) strongly suppressed IgG4 and IgE synthesis by human B-lymphocytes activated by a monoclonal

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 apoptosis of polyclonally activated human T-lymphocytes and of human T-lymphocytic cell lines Cohn et al., 1999a, Cohn et al., 1996b. Cells were stained with Hoechst 33342, examined by epifluorescence microscopy and apoptotic cells quantified by nuclear morphology. Electron microscopy confirmed the presence of typical apoptotic cells. Human peripheral blood leukocytes activated by anti-CD3 and cultured

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

Effects of iNOS

Nitric oxide (NO), a multifunctional biological mediator, is synthesized from the guanidino nitrogen of l-arginine by nitric oxide synthases in several mammalian cell types (Moncada et al., 1991). The activity of constitutive NO synthases is regulated by Ca2+ and calmodulin; the enzymes produce small amounts of NO for short periods following stimulation, mediating vasodilation and modulating neurotransmission. Inhibition of constitutive NO synthases can result in hypertension, impaired blood

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 and/or function of iNOS. The suppression by MPA and MMF of cytokine production by lymphocytes and monocytes (Section 8) should result in decreased production of IFN-γ and TNF-α, inducers of iNOS. Moreover, the depletion by MMF of levels of GTP in human monocyte/macrophage lineage cells (Fig. 7) should also lower the concentration of BH4, the

Antimicrobial activity of MPA and MMF

MPA is a much more potent inhibitor of the IMPDH of eukaryotes than that of prokaryotes (Verham et al., 1987). Hence, the compound would be expected to inhibit weakly the replication of bacteria, but might have greater activity against protozoa and fungi. By depleting dGTP, MPA also have synergistic activity with inhibitors of viral DNA polymerases and reverse transcription.

Lymphocyte-selective, reversible antiproliferative effects

To ascertain whether the antiproliferative effects of MPA and MMF are also lymphocyte-selective in vivo, we injected mice subcutaneously with an antigen (ovalbumin) 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 [3H]-thymidine. One group of mice was given MPA (100 mg/kg per day orally) while a control group received vehicle. Incorporation of [3H]-thymidine into

Prevention by MMF of allograft rejection

Following our basic observations on the immunosuppressive effects of MMF, collaborations were established with transplantation immunologists to ascertain whether the drug can prevent the rejection of tissue and organ allografts.

Background

Now that acute rejection can be reasonably well controlled, chronic rejection is emerging as the major limitation of long-term allograft survival and function. A major objective of transplantation immunologists is to define a therapy that prevents chronic as well as acute rejection. The two are related: chronic rejection is more likely to occur when there have been episodes of acute rejection. Chronic rejection is associated with a proliferative and obliterative arteriopathy, attributed to

Intestinal transplantation in the rat

GVHD occurs following transplantation of the lymphoid tissue-rich small intestine. Sonnino (1982) and Shaffer et al., 1992, Shaffer et al., 1993 studied the effects of MPA on allotransplantation of small intestine in a rat model. In Shaffer's experiments parental (Lewis) strain intestinal allotransplants were made into F1 (Lewis X BN) recipients. In a group of 11 untreated recipients, the mean survival time was 15.7±2.8 days. Oral administration of MPA (30 mg/kg per day), on days 0 through 6

Active Heymann nephritis (AHN)

This rat model of human idiopathic membranous nephropathy is induced with renal antigen Fx1A in complete Freund's adjuvant (CFA). Penny et al. (1998) found that MMF administration (30 mg/kg per day, for 4 weeks after immunization) prevents the induction of AHN. In treated animals, serum antibodies against Fx1A were suppressed, as was glomerular Ig deposition. The drug also prevented interstitial infiltration of αβTCR+, CD4+ and CD8+ T-cells, natural killer cells and macrophages. Reverse

Attenuation by MMF of ischemia–reperfusion injury

When graft function is delayed, effects of ischemia and reperfusion become manifest. These include increased expression of adhesion molecules and attachment of leukocytes to endothelial cells. Extending their in vitro findings (Section 8) to an in vivo situation, Paul et al. (1998) pretreated rats with MMF (20 mg/kg per day for 1 week), recovered their hearts, stressed them by ischemia and transplanted them. It was found that MMF inhibits expression of endothelial cell adhesion molecules,

Adjuvant 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 inhibited by immunosuppressive agents as well as anti-inflammatory agents such as inhibitors of prostaglandin 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 mg/kg per day, it

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 contributes 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 (Section 10).

It is now widely accepted that in humans with acutely rejecting heart allografts (Benvenuti et al., 1996) and renal allografts (Watarai et al., 1999), levels of the NO

Renal biopsy specimens

The induced expression of VCAM-1 on cultured human endothelial cells is strongly suppressed by MMF (Blaheta et al., 1999; Section 11). Li et al. (1998) reported that in humans with diffuse proliferative lupus nephritis VCAM expression in renal glomeruli was high. MMF treatment (1.5–2 g/day) was followed by clinical improvement and a dramatic decrease in VCAM expression. Four of the five cases studied showed intense staining before treatment; this was markedly decreased in intensity in

Effect of MMF on antibody formation in humans

Kimball et al. (1998) compared IgG antibodies against equine polyclonal anti-thymocyte antibody (ATGAM) in renal transplant recipients receiving MMF (2–3 g/day) and AZA. The incidence and titer of antibodies was significantly lower in the MMF treated group. Al-Akash et al. (1998) examined the effect of MMF, humanized anti-IL-2 receptor antibody (Zenapax), cyclosporin A (CsA) and prednisone on responses of children to influenza vaccination. The authors conclude that: (1) children who were

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 immunologically driven inflammatory disorders. Preliminary reports suggest that this may be the case, but controlled studies are needed in each disorder to establish efficacy.

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 favoring the induction of apoptosis. Depletion of GTP in lymphocytes and monocytes by MMF suppresses the expression of adhesion molecules and the production of NO by iNOS. It would be academically

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