The history, pharmacokinetics, mechanisms of action, and experimental as well as clinical data on the immunosuppressive potential of the novel drugs tacrolimus (FK506), sirolimus (rapamycin), mycophenolic acid (mycophenolate mofetil), and leflunomide (and its malononitriloamide analogues) are provided. Novel approaches with the following conventional immunosuppressants are outlined: methotrexate, aerosolized immunosuppression and the implementation of steroid taper. Total lymphoid irradiation and photopheresis for treatment of recurrent rejection are also discussed.

Graft Rejection↗

PubMed1997

Theoretical benefits of mitogen applications for HIV-1 infections.

Ideal treatment of HIV-1 infections should include an agent that can reverse the capacity of the virus to evade destruction by hiding in sanctuaries and by frequently mutating the epitopes it displays. The rapid proliferation of virions during the years of symptomatic quiescence obligates rapid replacement of CD4+ lymphocytes that leads to a gradual attrition of the T lymphocytes needed to control infections. In vitro evidences suggest that, given systematically, certain mitogenic lectins would interfere with HIV-1 invasion of CD4+ cells by blocking gp120 molecules on the viral membrane before activating T lymphocytes subsequent to binding with their Ti/CD3 molecules. The nonspecific nature of antiviral effector cells generated by this activation should circumvent HIV-1 mutations at the same time it reconstitutes depleted T lymphocytes, stimulates myelopoiesis, and reinforces resistance to malignancies and infections prevalent with the immunodeficiency state. Properly coordinating these effects with appropriate combinations of reverse transcriptase and protease inhibitors could theoretically expedite complete elimination of HIV in a timely fashion that shorten the required treatment duration and excludes the detrimental effects of virus mutations. The proper sequence of this treatment should be maximum reduction of the HIV-1 load with drug combinations, control of complicating infection by other means to reduce mitogen-induced tissue necrosis, and addition of systemic PHA-L4 administration regulated to maintain a 5-10 micrograms/mL serum concentration. The antiviral regimen should be continued an undetermined time beyond when HIV-1 is no longer detectable, and systemic L4 administration until satisfactory immunologic and hematologic competences are re-established. Partially-matched mitogen-activated adoptive leukocyte therapy might be additionally helpful.

Acquired Immunodeficiency Syndrome↗

PubMed1997

A universal post-embedding protocol for immunogold labelling of osmium-fixed, epoxy resin-embedded tissue.

The authors have found that double etching of epoxy-embedded, ultrathin sections with 3% sodium metaperiodate rendered epitope expression comparable to that obtained with either saturated or half-saturated sodium metaperiodate solutions. In contrast to either of the two more concentrated oxidizing solutions, double etching with 3% sodium metaperiodate neither bleached the specimens nor generated holes in the plastic resin.

Animals↗

PubMed1997

Leflunomide and malononitrilamides.

Leflunomide is a new immunomodulatory drug that is effective in experimental models of autoimmune diseases and in allo or xenotransplantation. In a phase II clinical trial, leflunomide showed high tolerability and efficacy in patients with advanced rheumatoid arthritis. The immunomodulatory activity of leflunomide is attributed to its primary metabolite A77 1726, which is a malononitrilamide. The in vitro and in vivo mechanisms of action of this class of compounds are not defined completely. Several malononitrilamide analogues and A77 1726 inhibit T- and B-cell proliferation, suppress immunoglobulin production, and interfere with cell adhesion. Although no central molecular mechanism of action has been proposed to explain all the effects of the malononitrilamides, the inhibition of de novo pyrimidine biosynthesis and of cytokine- and growth factor receptor-associated tyrosine kinase activity are leading hypotheses for the effects of A77 1726 on T- and B-cell proliferation and function. Leflunomide is effective when administered in daily dosages of 10 mg and 25 mg to patients with active rheumatoid arthritis. The improved efficacy of a 25 mg dose is associated with a higher incidence of adverse effects (gastrointestinal symptoms, weight loss, allergic reactions, skin rash, and reversible alopecia). Because of the long plasma half-life of A77 1726 (11 to 16 days), loading doses are necessary to achieve steady state concentrations. Phase III randomized, placebo-controlled trials that use daily dosages of 10 mg or 20 mg are under way in the United States and Europe to confirm and extend the results of the phase II study. Malononitrilamide analogues of A77 1726 are being evaluated for immunosuppressive efficacy in preclinical models of transplantation. If these analogues show efficacies and therapeutic indexes that are similar to leflunomide in these models and that have shorter half-lives than A77 1726 in phase I trials, the preclinical and phase I data will be used to select the analogues for phase II trials in organ transplant recipients.

Aniline Compounds↗

PubMed1997

Azurophilic granules of human neutrophils contain CD14.

CD14, the leukocyte receptor for lipopolysaccharide (LPS), is important in the response of human polymorphonuclear leukocytes (PMNs) to infection with gram-negative bacteria. The level of CD14 on the PMN surface increases after exposure to some inflammatory stimuli such as N-formyl-methionyl-leucyl-phenylalanine (fMLP). These newly expressed CD14 molecules probably come from an intracellular pool of preformed receptors. We sought to further characterize PMN CD14 expression, upregulation, and shedding and to define the intracellular location of CD14 molecules. Our results demonstrate that both LPS and fMLP significantly increased CD14 cell surface expression; however, neither phorbol myristate acetate (PMA) or A23187 increased receptor levels on the PMN surface. Neither fMLP, PMA, or A23187 stimulated the release of soluble CD14 from PMNs. Intracellular CD14 was observed in >90% of PMNs examined by flow cytometry and confocal microscopy. Additional analyses using CD14 enzyme-linked immunosorbent assays and electron microscopy studies, examining PMN granules separated by discontinuous sucrose or Percoll gradients, showed that CD14 was present in both the plasma membrane-secretory vesicle fractions and azurophilic granules.

Calcimycin↗

PubMed1997

New immunosuppressive regimens in lung transplantation.

Survival after lung transplantation is less than 50% after 5 yrs and is limited by infection and obliterative bronchiolitis. There is, therefore, a need for new immunosuppressive regimens if we are to attempt to improve long-term survival. Several trials in lung transplantation of new immunosuppressive agents are in the planning stages. In this article, we review the experience with a new monoclonal agent (interleukin 2 (IL2) receptor antagonist) in kidney transplantation, together with the pharmacokinetic (PK) and pharmacodynamic properties and experience in transplantation in general, of the more promising of the new xenobiotic compounds (cyclosporine microemulsion, mycophenolate mofetil, tacrolimus and sirolimus). Recent novel approaches to the vexing problem of resistant lung rejection and obliterative bronchiolitis, such as the use of aerosolized cyclosporine, methotrexate, total lymphoid irradiation and phototherapy, are discussed. Finally an immunosuppressive regimen, using these new drugs in lung transplantation is suggested.

Bronchiolitis Obliterans↗

PubMed1997

Leflunomide and malononitriloamides.

Leflunomide is a new immunomodulatory drug effective in experimental models of autoimmune diseases and allo- or xenotransplantation. In a Phase II clinical trial leflunomide has shown high tolerability and efficacy in patients with advanced rheumatoid arthritis. The immunomodulatory activity of leflunomide is attributed to its primary metabolite, A77 1726, a malononitriloamide. The in vitro and in vivo mechanisms of action of this class of compounds remain to be completely defined. A77 1726 and several malononitriloamide analogues inhibit T- and B-cell proliferation, suppress immunoglobulin production, and interfere with cell adhesion. While no one central molecular mechanism of action has been proposed to explain all the effects of the malononitriloamides, inhibition of de novo pyrimidine biosynthesis and inhibition of cytokine- and growth factor-receptor associated tyrosine kinase activity are leading hypotheses for the effects of A77 1726 on T- and B-cell proliferation and function. Leflunomide is effective when administered at daily doses of 10 and 25 mg to patients with active rheumatoid arthritis. The improved efficacy at the 25 mg dose is associated with a higher incidence of adverse effects (gastrointestinal symptoms, weight loss, allergic reactions, skin rash, and reversible alopecia). Due to the long plasma half-life of A77 1726 (11-16 days), loading doses are required to achieve steady-state concentrations. Phase III randomised, placebo-controlled trials using daily doses of 10 or 20 mg are underway in the US and Europe to confirm and extend the results of the Phase II study. Malononitriloamide analogues of A77 1726 are being evaluated for immunosuppressive efficacy in preclinical models of transplantation, because these compounds have a shorter half-life in animals than A77 1726. If these analogues show efficacies similar to leflunomide in these models and have shorter half-lives than A77 1726 in Phase I trials, the preclinical and Phase I data will be used to select the analogues for Phase II trials in organ transplant recipients.

Journal Article↗

PubMed1996

In Situ Single-Crystal X-ray Diffraction Study of Crystallization Kinetics in Clathrasil Dodecasil-3C.

The formation of single crystals of the clathrasil dodecasil-3C from a solvothermal synthesis has been followed by in situ diffraction techniques using synchrotron radiation and an image-plate area detector. The high intensity of the X-ray beam, coupled with the ability to record time-resolved two-dimensional data using the image plates, allowed the crystallization kinetics to be studied and rate expressions to be fitted to the crystallization curves.

Journal Article↗

PubMed1996

Effects of leflunomide and cyclosporine on myocutaneous allograft survival in the rat.

The immunosuppressive effects of leflunomide and cyclosporine were evaluated in a rat neurovascularized myocutaneous allograft model. Inbred Brown-Norway and Lewis rats were served as donors and recipients, respectively. All recipients were observed for 60 days or until allograft rejection occurred. All isograft controls (Lewis to Lewis, n=6) survived uneventfully. All control allografts (n=6) were rejected within 6 days. Allograft recipients (n=6) administered leflunomide (10 mg/kg/24 hr) rejected their allografts in 28.50+/-6.12 days, and allograft recipients (n=6), administered cyclosporine (5 mg/kg/24 hr) rejected their allografts in 24.33+/-10.48 days. When allograft recipients were administered a combination of leflunomide and cyclosporine (10 mg/kg/24 hr and 5 mg/kg/24 hr, respectively), all allografts survived to 60 days with only partial rejection of the skin of one graft. The neuromuscular function of the allografts of the rats receiving combination therapy was comparable to that of the isografts. The combination of leflunomide and cyclosporine controlled myocutaneous allorejection despite a strong immunological challenge.

Animals↗

PubMed1996

Long-term survival of solid organ allografts by brief anti-lymphocyte function-associated antigen-1 monoclonal antibody monotherapy.

Strategies targeting lymphocyte function-associated antigen-1 (LFA-1, CD11a/CD18) and intercellular adhesion molecule-1 (ICAM-1) have previously been shown to produce long-term survival of solid organ allografts in animals only when both CD11a and ICAM-1 are targeted for a brief (6-7 days) time or when extended (14 weeks) treatment with anti-CD11a monoclonal antibody (mAb) is administered. We show that recipient pretreatment followed by a brief (13 days) treatment course with high-dose anti-CD11a mAb alone produces long-term survival of cardiac allografts in the rigorous, nonprimarily vascularized heart allograft model in mice. This treatment regimen induces specific unresponsiveness in our model. In recipients bearing long-term beating cardiac grafts after treatment with anti-CD11a mAb, there still exists a high frequency of potentially antigen-reactive T cells in isolated peripheral blood lymphocyte fractions. Therefore, clonal deletion does not appear to explain the induction of specific unresponsiveness by treatment with anti-CD11a mAb in this model. These findings support the further investigation of the use of high-dose anti-LFA-1 mAb monotherapy in the pre- and early postoperative period to promote solid organ allograft survival.

Animals↗

PubMed1996

Altered surfactant function and structure in SP-A gene targeted mice.

The surfactant protein A (SP-A) gene was disrupted by homologous recombination in embryonic stem cells that were used to generate homozygous SP-A-deficient mice. SP-A mRNA and protein were not detectable in the lungs of SP-A(-/-) mice, and perinatal survival of SP-A(-/-) mice was not altered compared with wild-type mice. Lung morphology, surfactant proteins B-D, lung tissue, alveolar phospholipid pool sizes and composition, and lung compliance in SP-A(-/-) mice were unaltered. At the highest concentration tested, surfactant from SP-A(-/-) mice produced the same surface tension as (+/+) mice. At lower concentrations, minimum surface tensions were higher for SP-A(-/-) mice. At the ultrastructural level, type II cell morphology was the same in SP-A(+/+) and (-/-) mice. While alveolar phospholipid pool sizes were unperturbed, tubular myelin figures were decreased in the lungs of SP-A(-/-) mice. A null mutation of the murine SP-A gene interferes with the formation of tubular myelin without detectably altering postnatal survival or pulmonary function.

Animals↗

PubMed1996

Stanford experience with obliterative bronchiolitis after lung and heart-lung transplantation.

BACKGROUND: Obliterative bronchiolitis (OB) is the main chronic complication after heart-lung (HLTx) and lung transplantation (LTx), limiting the long-term success of both transplant procedures. METHODS: Since 1981, 135 HLTxs and 61 isolated LTxs were performed in 184 patients at Stanford University. RESULTS: The overall prevalence of OB in patients surviving longer than 3 months postoperatively was 64% after HLTx and 68% after LTx. The actuarial freedom from OB was 72%, 51%, 44%, and 29% at 1, 2, 3, and 5 years, respectively, after HLTx and LTx. An analysis of potential risk factors revealed that the frequency and severity of acute rejection episodes (p < 0.001) and the appearance of lymphocytic bronchiolitis on biopsy (p < 0.05) were significantly associated with the development of OB. With regard to diagnosis of OB, pulmonary function tests show early reductions of the forced expiratory flow between 25% and 75% of the forced vital capacity with subsequent decreases in the forced expiratory volume in 1 second. The sensitivity of transbronchial biopsies has increased to 71% since 1993. Current treatment consists of augmented immunosuppression. Concurrent acute rejection episodes or active OB on biopsy have been treated aggressively with high-dose steroid pulses. Analysis of data from 73 patients with OB after HLTx and LTx revealed actuarial 1-, 3-, 5-, and 10-year survival of 89%, 71%, 44%, and 17% versus 86%, 77%, 63% and 56% in patients without OB (p < 0.05 by log-rank analysis). The main complication and cause of death in patients with OB was superimposed respiratory tract infection, which was treated aggressively. CONCLUSIONS: Early diagnosis of OB using pulmonary function tests or transbronchial biopsy is possible and important, because immediate treatment initiation has led to acceptable survival rates, with nearly 50% of affected patients still alive 5 years after transplantation. Current experimental research on OB suggests that immune injury is the main pathogenetic event of airway obliteration in animal models; rapamycin and leflunomide are new immunosuppressive agents that may have the potential to prevent and treat airway obliteration.

Actuarial Analysis↗

PubMed1996

Immunosuppressants: cellular and molecular mechanisms of action.

The basic immunosuppressive protocol used in most transplant centers involves the use of multiple drugs, each directed at a discrete site in the T-cell activation cascade and each with distinct side effects. Cyclosporine, azathioprine, corticosteroids, FK506 (tacrolimus), and RS61443 (mycophenolate mofetil) have been approved by the Food and Drug Administration, and the clinical efficacy of rapamycin (sirolimus), mizoribine, 15-deoxyspergualin, and leflunomide is being explored. Based on their primary site of action, the immunosuppressants can be classified as inhibitors of transcription (cyclosporine, tacrolimus), inhibitors of nucleotide synthesis (azathioprine, mycophenolate mofetil, mizoribine, leflunomide), inhibitors of growth factor signal transduction (sirolimus, leflunomide), and inhibitors of differentiation (15-deoxyspergualin). Polyclonal antilymphocyte antibodies, monoclonal antibodies directed at the T-cell antigen receptor complex (OKT3, TIOB9), and monoclonal antibodies directed at additional cell surface antigens, including interleukin-2 receptor alpha, afford cell-specific regulation of the immune response and are being used in the clinical setting as induction therapy and/or antirejection drugs. Clearly, the transplant clinician now has a greater choice in the selection and application of immunosuppressants in the clinic for the fine regulation of the antiallograft repertory. The prevailing paradigm regarding the mechanisms of action of immunosuppressants is that they all function to prevent allograft rejection by preventing/inhibiting cell activation, cytokine production, differentiation, and/or proliferation. One hypothesis, albeit provocative, is that some of the immunosuppressants might function by stimulating the expression of immunosuppressive molecules and/or cells.

Humans↗

PubMed1996

Molecular mechanisms of action of new xenobiotic immunosuppressive drugs: tacrolimus (FK506), sirolimus (rapamycin), mycophenolate mofetil and leflunomide.

Among all the new immunosuppressive molecules being investigated either preclinically or clinically, four stand out: tacrolimus (FK506), sirolimus (rapamycin), mycophenolate mofetil and leflunomide (and its malononitriloamide analogs). Each drug has distinct mechanisms of immunosuppressive action, and in the past year significant advances have been made in our understanding of the actions of these drugs at the molecular and even atomic levels. Data from recent clinical trials demonstrate that these drugs very effectively suppress graft rejection or autoimmune diseases, validating the pivotal role played by each of their distinct molecular targets in the normal functioning of immune cells.

Animals↗