Elsie M. Eugui

Immunosuppressive and other effects of mycophenolic acid and an ester prodrug, mycophenolate mofetil.

The advantages and limitations of currently used immunosuppressive drugs are well known. Each drug has specific side effects: for example, cyclophosphamide can produce hemorrhagic cystitis (Levine & Jarrad 1993); cyclosporin can cause nephrotoxicity and hypertension (Bennett & Pulliam 1983); and methotrexate may lead to liver damage, pneumonitis and bone marrow suppression (Jolivet et al. 1983). In addition, there are short-term and long-term effects common to many immunosuppressive drugs. In the short term, immunosuppressive drugs can increase susceptibility to herpesvirus and other infections; it is desirable that when treatment is terminated immune function is rapidly restored, so that recovery from infections is facilitated. A long-term risk of immunosuppressive therapy is the development of lymphomas. This is well known in organ graft recipients (Penn 1988), but is observed also in patients with rheumatoid arthritis (Baker et al. 1987). As discussed more fully below, two factors predispose to lymphoma development. One is inhibition of T-cell-mediated surveillance against outgrowth of Epsten-Barr virus-transformed B lymphocytes (Rickinson et al. 1984). The second is chromosomal translocations, which can convert polyclonal B-cell proliferation into frank malignancy (Klein & Klein 1989). Mutagenic effects of immunosuppressive drugs are obviously undesirable. Cyclosporin inhibits surveillance against EBV-transformed lymphocytes (Rickinson et al. 1984). Cyclophosphamide and azathioprine have several active metabolites and mechanisms of action (Clarke & Waxman 1989. Elion 1989). Metabolites of cyclophosphamide are alkylating agents, and their effects on lymphocytes are not rapidly reversible. Cyclophosphamide is mutagenic and carcinogenic (De Neve et al. 1989). Azathio-

RS-61443 : A NEW, POTENT IMMUNOSUPPRESSIVE AGENT

The immunosuppressive activity of RS-61443, a semisynthetic derivative of mycophenolic acid, was examined in 33 canine renal allografts. Initial studies established that triple therapy consisting of 20 mg/kg RS-61443 in combination with 5 mg/kg cyclosporine and 0.1 mg/kg methylprednisolone was the optimal combination to prevent graft rejection. The median survival time was 8.1 +/- 1.2 days in dogs without treatment (n = 5), 8.5 +/- 1.7 days in the treatment control group (CsA 5 mg/kg, MP 0.1 mg/kg; n = 6), 36.0 +/- 9.6 days with RS-61443 monotherapy (40 mg/kg; n = 6); and 122.4 +/- 38.75 days with triple therapy (n = 16). Graft prolongation was statistically significant when compared with controls (P less than 0.05 and 0.002, respectively). Six recipients in the triple therapy group survived over 150 days without major adverse effects. Long-term administration of RS-61443 (20 mg/kg/day) did not cause nephrotoxicity, hematotoxicity, or hepatotoxicity, with the exception of a slight elevation of the alkaline phosphatase levels. Gastrointestinal symptoms including gastritis, diarrhea, and anorexia were common, especially under 40 mg/kg RS-61443 monotherapy, and appeared to be dose-related. Despite its immunosuppressive activity, an increased susceptibility to bacterial or viral infections was not observed. Histological studies of the kidney grafts revealed slight interstitial cell infiltration without vascular or glomerular damage.

The design and development of an immunosuppressive drug, mycophenolate mofetil

Immunosuppressive agents have traditionally been identified in two ways. Some drugs developed for cancer chemotherapy were incidentally found to have immunosuppressive activity (cyclophosphamide, methotrexate, azathioprine), whereas screening of fermentation products revealed the immunosuppressive activity of cyclosporins, FK 506 and rapamycin. Establishing the mode of action of these compounds lagged behind the demonstration of their immunosuppressive activity, and information on this subject is still incomplete. Cyclophosphamide and azathioprine have several active metabolites and mechanisms of action [18, 25]. Many years after the demonstration that cyclosporin A is immunosuppressive [16] the drug was found to bind prolyl cis-trans isomerase and inhibit the activity of the enzyme [29, 84]. All of the currently used immunosuppressive drugs have limitations. Patients with diseases such as rheumatoid arthritis and organ transplant recipients are treated with immunosuppressive agents for years. Genotoxic effects of these drugs and their metabolites can increase the risk of malignancy [54, 57]. Immunosuppressive therapy also increases susceptibility to viral and other infections, and it is desirable that when such a drug is withdrawn immune function can soon recover to control the infections. The effects of cyclophosphamide on lymphocytes are not rapidly reversible, and the drug produces hemorrhagic cystitis, as well as being mutagenic and carcinogenic [12, 22]. The most serious side effect of cyclosporin A is, of course, nephrotoxicity [13]. Methotrexate, which inhibits synthesis of thymidylate as well as de novo purine synthesis [52], has non-selective antiproliferative effects, which are no more potent on lymphocytes than on other cell types, including hemopoietic precursors, fibroblasts and endothelial cells [47, 52]. The toxicities associated with long-term methotrexate therapy include liver damage, pneumonitis and bone marrow suppression [1].

Mycophenolate mofetil (RS-61443): Mechanisms of action and effects in transplantation

W hereas cyclosporine and glucocorticoids have had a major impact in transplantation, their side effects are well known. Cyclosporine efficiently suppresses T-cell-mediated immune responses and acute rejection of allografts. Nevertheless, many human organ graft recipients who have been given combination therapy with cyclosporine and steroids, with or without azathioprine, have episodes of rejection. Often these are controlled by high-dose bolus steroid and/or OKT3 therapy. However, such treatments may increase susceptibility to infections and the eventual development of lymphomas. The first requirement is for a drug that can be used in combination therapy with well-tolerated doses of cyclosporine to decrease the incidence of rejection episodes in allograft recipients. The second is for a drug that can reverse ongoing rejection without increasing susceptibility to infections or causing other limiting side effects. Now that grafts have better survival rates, other problems are emerging. The problem of chronic rejection and its effects on the ~asculature of grafted organs is becoming recognized as a major limitation of long-term graft survival. A third and major requirement in transplantation is for a drug that can prevent chronic rejection when used over long periods. With these considerations in mind, we and other investigators in the laboratories at Syntex and the University of Wisconsin developed a novel immunosuppressive drug, demonstrated efficacy in experimental animal models of transplantation, and conducted the first human trials in allograft recipients. Our strategy was to define a biochemical pathway that is especially important for proliferation of human T and B lymphocytes and, by inhibiting that pathway, to suppress the proliferation of clones of lymphocytes responding to antigenic stimulation.

IL‐6 Is the Principal Factor Produced by Synovia of Patients with Rheumatoid Arthritis That Induces B‐Lymphocytes to Secrete Immunoglobulins

First, IL-6 is produced by synovial tissue of patients with rheumatoid arthritis (RA) and is the principal mediator produced by that tissue inducing differentiation of B-lymphocytes into antibody-forming cells. The Leu-1+ subset of B-lymphocytes is induced by IL-6 to secrete rheumatoid factor (IgM with anti-Fc gamma specificity). Second, the main cell types producing IL-6 in cells dissociated from RA synovial tissue are mononuclear leukocytes. Connective tissue type cells (synoviocytes) cultured from RA synovial tissue produce IL-6 in response to IL-1 beta, and IL-6 formation is increased by TGF-beta. Glucocorticoids strongly inhibit and PGE2 slightly inhibits IL-1-induced IL-6 mRNA expression in synoviocytes. Production of IL-6 increases when undissociated RA synovial tissue is maintained in culture, thus suggesting release from inhibition by a factor or factors not yet identified. Third, the major known local effect of IL-6 in RA synovial tissue is the augmentation of antibody formation and the major known systemic effect is the induction of the synthesis by the liver of acute-phase proteins, especially C-reactive protein. Levels of circulating C-reactive protein are reported to decrease in RA patients receiving long-acting antirheumatic drugs, which would be consistent with the interpretation that immature monocyte-derived macrophages are major producers of IL-6 in these patients.

Facilitation of specific tolerance induction in adult mice by RS-61443.

RS-61443 is an immunosuppressive agent that facilitates pancreatic islet allograft acceptance in two mouse strain combinations (BALB/c----CBA and C57Bl/6J----BALB/c). A remarkable feature of this agent is its ability to facilitate long-term graft acceptance after a short (30 days) period of treatment; following withdrawal of the agent 40-70% of islet allografts are maintained for an indefinite period. This long-term graft acceptance has been shown to result from specific tolerance induction in the recipient animal. The state of specific tolerance is an active rather than a passive form of tolerance, such as clonal deletion or anergy. Active tolerance induction is cyclosporine-sensitive, although cyclosporine enhances graft acceptance when used in combination therapy with RS-61443, this agent inhibits tolerance development under the influence of RS-61443.

NATURAL CELL-MEDIATED IMMUNITY AND INTERFERON IN MALARIA AND BABESIA INFECTIONS

Publisher Summary This chapter examines natural cell-mediated immunity and interferon in malaria and Babesia infections. The investigation described in the chapter began with an analysis of the susceptibility of different mouse strains to Plasmodium chabaudi (P. chabaudi) and Babesia microti (B. microti). It was found that strain A/He mice are highly susceptible to both infections; in this strain, P. chabaudi is nearly always lethal and the mice do not recover from B. microti infections. Investigations of congenic strains suggested that susceptibility does not correlate with H-2 haplotype. The F-1 offspring of susceptible A and resistant BIO.A mice are resistant. The observations in the study also suggest that sensitized T-cells, reacting with parasite antigens, recruit and activate macrophages and natural killer (NK) cells. These effector cells may, in turn, liberate factors that can inhibit the replication of parasites in circulating erythrocytes, leading to degenerate crisis forms. Effector cells appear to be NK cells and/or macrophages. The chapter discusses the possibility of these cell types being of the same lineage. It also highlights the role that they may play in immunity and immunopathology in these infections.

Activated human langerhans cells express mRNA for IL-1α and IL-1β and produce these cytokines but do not secrete them

Abstract Human Langerhans cells (LC) were isolated from epidermal cell preparations by panning with mouse anti-CD1 monoclonal antibody. RNA was prepared and probed for the presence of mRNAs for various cytokines using radiolabeled cDNAs. After stimulation with phorbol myristate acetate LC express RNA for interleukin 1α (IL-1α) and interleukin 1β (IL-1β) and produce proteins but do not secrete them at detectable levels. LC-associated IL-1, particularly IL-1α, may play a role in antigen presentation. PMA did not induce IL-6 expression in LC. The addition of lipopolysaccharide, a muramyl dipeptide analog, ionomycin, IL-1α, tumor necrosis factor-α, insulin-like growth factor-1 or IL-6 did not induce IL-1 mRNA in LC. UVB augmented IL-1β mRNA expression. Glucocorticoids did not detectably affect IL-1α or IL-1β mRNA levels following PMA induction, however, staurosporin inhibited IL-1β mRNA synthesis. Thus the inducers and regulators of IL-1 formation in human LC and monocytes are not identical.

Prolongation of murine thyroid allografts by interleukin 2 (DAB486)-toxin and RS-61443.

We evaluated the efficacy of interleukin 2 (DAB486)-toxin (IL-2-diphtheria toxin fusion protein; IL-2-toxin) in combination with RS-61443 to prolong murine thyroid allograft survival. B10.BR thyroid allografts were transplanted beneath the renal subcapsule in recipient (C57BL/10 mice and graft survival determined 21 days later. Treatment with IL-2-toxin (25 microg/day for 14 days) was unable to prolong graft survival significantly. RS-61443 treatment (21 days) achieved significant graft prolongation only at doses of 300 mg/kg daily or greater. When both drugs were used in combination (IL-2-toxin, 25 microg/day for 14 days RS-61443 200 mg/kg daily for 21 days), statistically significant (P < 0.0001) graft prolongation was obtained. Our results suggest that IL-2-toxin in combination with subtherapeutic RS-61443 levels significantly prolongs murine thyroid allograft survival. IL-2-toxin and RS-61443, because of their unique and complementary mechanisms, hold promise for more selective immunosuppression.