Eur J Pediatr (1992) 151 [Suppl 1] : $9-$12
European Journal of
Pediatrics
9 Springer-Verlag1992
New immunosuppressive drugs: needs in and applications to pediatric transplantation B. D. Kahan Division of Immunologyand Organ Transplant, Department of Surgery, The Universityof Texas MedicalSchool at Houston, 6431 Fannin, Suite 6.240 MSB, Houston, TX 77030, USA
Abstract. The evolution of immunosuppressive therapy toward synergistic drug combinations seeks to minimize toxicity while potentiating efficacy. Median effect analysis discerns synergistic drug combinations that may be suitable for in vivo experiments in animals and for subsequent clinical trials. These studies suggest that two drugs rapamycin (RAPA) and brequinar (BQR) display synergistic effects in combination with cyclosporine. This combination must be evaluated for relative toxicity versus efficacy. Clinical trials to assess the individual toxicities of RAPA and BQR are presently underway in order to discern appropriate doses for randomized trials of clinical efficacy.
suppressive agents are under clinical trial to substitute for, or to complement the action(s) of, the presently available agents: FK506 [26], rapamycin [16] (RAPA), mizorbine [28], RS-61443 [23], and quinoline carboxylic acid [3]. Each of these agents not only prolongs allograft survival in preclinical models but also produces substantial toxicity when relied on as monotherapy for rejection prophylaxis. In the fashion that cancer chemotherapeutic agents have been combined in order to potentiate efficacy but mitigate toxicity, individually subtherapeutic, but subtoxic doses of immunosuppressive drugs may overcome the limitations of currently available regmines.
Key words: Immunosuppression - Drug interactions Cyclosporine
Analysis of dose-response relations of immunosuppressive drugs
Introduction Pediatric patients present a special challenge to the transplant physician for the design of an appropriate immunosuppressive regimen. On the one hand pediatric patients, particularly under the age of 10 years, display an inordinately vigorous allorejection response [5]. On the other hand because of the unique requirements for excellent allograft function in order to achieve growth and development in children, the optimal immunosuppressive regimen must be minimally toxic to kidneys, liver and bone. Existent immunosuppressive regimens are not only suboptimally effective, but also display a narrow therapeutic window. The pleiotropic renal, vascular, hepatic, bone and endocrine toxicities of cyclosporine (CSA) preclude the use of optimally immunosuppressive doses of this drug [12]. In order to potentiate immunosuppression pediatricians frequently combine azathioprine and corticosteroids with CsA, even though three randomized trials failed to document any benefit of the triple compared with the double (CsA/prednisone) drug regimen [1, 20, 24]. Contrariwise, it proffers the possibility of synergistic hepatic injury. Several new immuno-
Abbreviations: BMA=Behringwerke monoclonal antibody; BQR = brequinar; CI = combinationindex; CsA = cyclosporine; I1 = interleukin; MAb = monoclonal antibodies; NFAT = nuclear factor of activated T-cell; RAPA = rapamycin
Since various immunosuppressive drugs produce different patterns of inhibition of immunologic responses, it is necessary to utilize mathematical models to rigorously analyze dose-response relations, in order to discern synergistic, additive or antagonistic interactions. The median effect equation [2] has been applied to analyze the interactions between cancer chemotherapeutic and between anti-viral agents: ?a
=
(o)m
where fa is the percent inhibition (fraction affected), fu is the percent unaffected (100-fraction affected) by the agent used at dose D. Dm is the 50% inhibitory concentration and m is a slope coefficient of the relationship. The Dm is precisely determined by the abscissa of the log/log plot of: log (lf_~a/a) = m l o g D - m l o g D m The degree to which the data fit the median effect model is confirmed by a linear regression coefficient r > 0.75. The slope of this plot yields the "m" value. The log/log plot provides a ready estimate of the dose Dx to achieve a given effect level of x % inhibition. In order to assess interactions one performs an immunologic assay in the presence of each agent alone or of drug combinations. The combination index at x % inhibition is expressed as the ratios of the concentrations
$10 required to produce that effect level in the presence of second agent versus that when the drug is used alone. CIx = dose drug~ with drug2 + dose drug2 with drug1 + dose drug1 alone dose drug2 alone [dose drug1 with drug2] [dose drugs with drug1] [dose drug1 alone] [dose drug2 alone] Combination index (CI) values equal to 1.0 document additive interactions; less than 1.0, drug synergism; and greater than 1.0, drug antagonism. Several factors affect the CI [14]. First, the ratio of the molar concentrations of the two drugs tested in the combination. A range of ratios, using drug concentrations relevant for clinical application must be tested, in order to ascertain the optimal ratio to achieve synergistic effects. Second, a variety of immunologic assays should be tested, since each performance system may reveal different values. In general immunosuppressive drugs tend to be more synergistic in assys reflecting T-lymphocyte activation via the T-cell receptor, namely anti-CD3 stimulation and mixed lymphocyte reactions, than those triggered by polyclonal mitogens such as phytohemagglutinin or by phorbol myristate acetate plus ionomycin. Third, a variety of volunteers must be tested, since there are considerable inter-individual differences among combination indices observed in human subjects. Although the CI values among a group of ten volunteers may span 2 - 4 log values, they usually all show a similar type of interaction. One important index of a significant synergistic interaction is a consistency of values less than 1.0 among a large population sample.
Agents affecting reception of the T-cell activation signal Activation of the T-helper cell depends upon antigenic recognition via the T-cell receptor accompanied by a second, humoral, co-stimulator signal. Corticosteroids inhibit the generation of messenger RNA for Interleukin (IL)-1[3, a co-stimulatory lymphokine. However, the multitude of side-effects from corticosteroids, particularly on growth in the pediatric population, contraindicate their longterm use. The antigen signal reception step can be interrupted with monoclonal antibodies (MAb). The IgG2b MAb Behringwerke monoclonal antibody (BMA) 031 directed against the common region of the T-cell receptor interrupts T-cell sensitization and delays the onset of allograft rejection without significant side-effects [19]. In contradistinction to the therapeutic agent, OKT3 an antiCD3 IgG2a MAb, BMA 031 does not trigger cytokine release. However, the clinical application of BMA 031 is limited by the rapid, almost uniform appearance of antiidiotypic antibodies that limit the length of the treatment course to less than 5 days.
Drug inhibiting lymphokine synthesis Two structurally unrelated compounds inhibit the transduction of the cell surface signal into a cytoplasmic mes-
sage that eventuates in transcription of m-RNA for lymphokine humoral mediators. This generation of the antigen-activation signal through inositol phosphates increases intracellular calcium concentrations, augments oncogene products, and leads to the generation of regulatory proteins. The exact sequence of chemical events distal to the increase in calcium is uncertain. Ullman and colleagues [29] suggest that the initial regulatory protein, the nuclear factor of activated T-cells (NFAT), is assembled from a cytoplasmic and a nuclear fraction. After the binding of NFAT, additional regulatory proteins are assembled in the enhancer region controlling IL-2 transcription, thereby triggering RNA Polymerase activity. CsA (molecular weight 1202 daltons) is a cyclic fungal undecapeptide bearing at position 1, a unique nine carbon amino acid containing a double bond. FK506 (molecular weight 750 daltons) is a macrocyclic molecule, containing a critical a-keto-homoprolyl subunit. These two compounds bind intracellular proteins that have cis-trans prolylisomerase activity [8]: CsA to cyclophilin [10], FK506 to fujiphilin [30]. Liu and colleagues [21] suggest that the binding of drug alters the substrate specificity of the isomerase. Although its native substrate is not known, the immunophilin-drug complex inhibits the enzymatic action of calcineurins A and B, two calcium- and calmodulin-dependent phosphatases. This inhibition may prevent calcineurin from cleaving the phosphate form the cytoplasmic fraction of NFAT, an event necessary for NFAT to penetrate the nuclear membrane, and consequently assemble with the nuclear fraction into the full NFAT moiety that promotes IL-2 gene transcription. Alternatively, the enzymatic action of the isomerase may convert the administered "pro"-drug into an active moiety. Wtithrich et al. [32] found that when CsA binds to cyclophilin, it undergoes a cis- to trans-conformational change by inverting of the polar surface of amide protons and carbonyl oxygens to the outside and the hydrophobic exterior edges of N-methyl groups of the inside, thereby rendering the drug more hydrophilic. Similarly, Jorgenson [11] described a conformational change of FK506 due to cis-trans isomerization of the amide bond, thereby repositioning the homoproline structure with inward movement of the pyranose ring toward the macrocycle. Contrariwise, the related macrolide, R A P A (see below) does not undergo a structure change upon binding to the isomerase protein. The isomerase changes thus may confer these drugs' unique inhibition of the lymphocyte activation cascade, thereby interrupting lymphokine synthesis and preventing cytotoxic T-cell generation. In spite of this common effect, the two drugs produce unresponsiveness by distinctive mechanisms. CsA promotes the generation of T-suppressor cells, either of the CD4 § [9] or CD8 + [19] phenotype; FK506 does not produce this effect [22]. The pleiotropic side-effect profile of CsA displays nephrotoxic > hepatotoxic >> neurotoxic side-effects [12]. A similar spectrum of side-effects beclouds FK506 which is about 10- to 50-fold more potent than CsA on a molar basis, but shows a profile of neurotoxic > nephrotoxic >> hepatotoxic side-effects [26]. At therapeutic
$11 immunosuppressive doses there appears to be little difference between CsA and FK506 not only in the incidence of side-effects, but also in their efficacy to prevent human renal allograft rejection [27]. Presently, rational therapeutic application of both CsA and FK506 is precluded due to marked inter-individual pharmacokinetic variations between transplant recipients, both in gastrointestinal absorption and in drug clearance rates [13]. Children especially present unique problems due to their more rapid clearance rates and poorer absorption of CsA than adults.
Agents blocking lymphokine action Lymphokines bind to surface receptors that transduce the activation signal via a calcium-independent pathway that results in proliferation and cellular differentiation. There are several opportunities to interfere with the action of lymphokines. Anti-lymphokine antibodies, namely anti-y-interferon [4], potentiate the immunosuppressive activity of CsA. There are two other ways to decrease effective lymphokine concentrations. One approach delivers soluble constructs of the extra-membranous portion of IL-1 [6] or IL-4 [7] lymphokine receptors. The second approach administers lymphokine antagonists that bind to receptor without triggering the activation event. IL-1 antagonists are presently under trial for treatment of rheumatoid arthritis. A similar effect may be produced by antibodies that cover lymphokine receptors. Recent clinical trials using an anti-IL-2 receptor (IL-2R) MAb revealed a delayed onset of rejection with no difference in graft survival rates [25]. The most potent pharmacologic agent inhibiting receptor-mediated events is R A P A (molecular weight 950 daltons) [16]. While the exact mode of R A P A action on lymphocyte activation is uncertain, the drug does not interfere with the binding of radiolabelled IL-2 lymphokine to IL-2R, with the phosphorylation of the 75 kDa [3 chain of the IL-2R, or with changes in cyclic AMP. Although R A P A inhibits the endocytosis of the IL-2/ IL-2R complex, this effect does not totally explain the drug's inhibitory action. The synergistic interaction between CsA and R A P A is potentially of great clinical import, for it may permit the use of subtherapeutic doses of each drug in an effective combination [17]. R A P A does not interfere with the induction of suppressor T-cells by CsA; additionally R A P A contributes a unique action to induce a humoral immunosuppressive principle. The R A P A plus CsA combination synergistically prolongs the survival of rat cardiac, renal, and small bowel grafts, of heterotopic nonvascularized murine cardiac allografts, and of mongrel canine renal transplants [16]. The clinical evaluation of R A P A is at a preliminary stage. Since the R A P A doses to be used in combination with CsA will be low (0.05-0.5 mg/kg per day), it is unlikely that the gastro-intestinal mucositis and vasculitis previously observed in dogs receiving R A P A monotherapy will be encountered in patients under a CsA-RAPA dual drug regimen.
Agents inhibiting lymphocyte proliferation The immunosuppressive anti-proliferative chemotherapeutic agents have a common mechanism of action to inhibit nucleic acid synthesis. Three agents inhibit the purine salvage pathwy: 6-mercaptopurine, mizorbine, and mycophenolic acid. The first drug 6-mercaptopurine, whose imidazole derivative azathioprine has been in clinical use for over 30 years, has only a modest clinical immunosuppressive effect, due to its poor therapeutic index. Azathioprine tends to more effectively inhibit the proliferation of bone marrow elements than lymphocytes. Unfortunately, Azathioprine acts only additively with CsA [30]. Therefore it is not unexpected that there was no evident clinical benefit of this combination in randomized trials [1, 20, 24]. Although mizorbine has been in clinical use for over a decade in Japan, it has never been compared with 6-mercaptopurine in controlled, randomized trials. Mycophenolic acid and its analog RS-61443 also inhibit purine salvage pathways. RS-61443 is presently undergoing multicenter phase III efficacy trials. The fourth antiproliferative agent, brequinar (BQR, quinoline carboxylic acid) inhibits de novo synthesis of pyrimidines. This agent more effectively inhibits proliferative responses in mixed lymphocyte reactions than in mitogen stimulation assays. Oral administration of 2.0mg/kg per day B Q R prolongs rat renal and cardiac allograft survival [3]. Interestingly B Q R acts synergistically with CsA in vitro [15]. Indeed the three drug combination CsA: R A P A : B Q R (0.5:0.01:0.5 mg/kg per day) produces permanent survival of allogeneic Buffalo (RT-1 b) heart transplants in Wistar Furth (RT-1 u) rats.
Acknowledgement. This work was supported by NIH/NIDDK award 38016-05.
References 1. Brinker KR, Dickerman R, Gonwa T (1990) A randomized trial comparing double drug and triple drug therapy in primary cadaveric renal transplantation. Transplantation 50 : 43 2. Chou TC, Talaly P (1984) Quantitative analysis of dose-effect relationships: the combined effects of multiple drugs or enzyme inhibitors. Adv Enzyme Regul 22 : 27 3. Cramer D, Chapman FA, Jaffee BD, Jones EA, Knoop M, Eiras Hreha G, Makowka L (1992) The fact of a new immnnosuppressive drug, brequinar sodium, on heart, liver and kidney allograft rejection in the rat. Transplantation (in press) 4. Didlake RH, Kim EK, Sheehan K, Screiber RD, Kahan BD (1988) Effect of combined antigamma interferon antibody and cyclosporine therapy on cardiac allograft survival in the rat. Transplantation 45 : 353 5. Drachman R, Schlesinger M, Shapira H, Drukker A (9189) The immune status of uremic children/adolescents with chronic renal failure and renal replacement therapy. Pediatr Nephrol 3 : 305-308 6. Fanslow WC, Sims JE, Sassenfeld H, Morrisey P, Gillis S, Dower S, Widmer M (1990) Regulation of alloreactivity in vivo by a soluble form of the interleukin-1 receptor. Science 147: 739 7. Fanslow WC, Clifford K, Park I, Rubin A, Voice R, Beckmann M, Widmer M (1991) Regulating alloreactivity in vivo by IL-4 and the soluble IL-4 receptor. J Immunol 147 : 535
$12 8. Fischer G, Wittmann-Liebold B, Lary K, Kiefhaber T, Schmid F (1989) Cyciophilin and peptidyl cis-trans isomerase are probably identical proteins. Nature 337 : 476 9. Hall BM, Jelbest ME, Dorsch SE (1984) Suppressor T cells in rats prolonged cardiac allograft survival after treatment with cyclosporine. Transplantation 37: 595 10. Handschumacher R, Harding M, Rice J, Drugge R, Speicher D (1984) Cyclophilin: a specific cytosolic binding protein for Cyclosporine A. Science 226 : 544 11. Jorgenson W (1991) Rusting of the lock and key model for protein-ligand binding. Science 254 : 954 12. Kahan BD (1981) Drug therapy: cyclosporine. N Engl J Med 321 : 1725 13. Kahan BD (1985) Overview: individualization of cyclosporine therapy using pharmacokinetic and pharmacodynamic parameters. Transplantation 40 : 457 14. Kahan BD (1991) Synergism, how assessed and how achieved. Clin Transplant 5 : 534 15. Kahan BD (1992) Optimization of cyclosporine therapy with combinations of immunosuppressive agents. Transplant Proc (in press) 16. Kahan BD, Chang JY, Sehgal SN (1991) Preclinical evaluation of a new potent immunosuppressive agent, rapamycin. Transplantation 52 : 185 17. Kahan BD, Gibbons S, Tejpal N, Stepkowski S, Chou TC (1991) Synergistic interactions of cyclosporine and rapamycin to inhibit immune performances of normal human peripheral blood lymphocytes in vitro. Transplantation 51:232 18. Kupiec-Weglinski JW, Filho MA, Strom TB, Tilney NL (1984) Sparing of suppressor cells: a critical action of cyclosporine. Transplantation 38 : 97 19. Kurrle R, Seyfert W, Trautwein A, Seiler F (1985) Cellular mechanisms of T cell activation by modulation of the T3 antigen complex. Transplant Proc 17 : 880 20. Lindholm A, Albrechtsen D, Tufveson G, Karlberg I, Persson N, Groth C (1992) A randomized trial of cyclosporine and prednisolone versus cyclosporine, azathioprine and prednisolone in primary cadaveric transplantation. Transplantation 53 (in press)
21. Liu J, Farmer J, Friedman J, Weissman I, Screiber S (1991) Calcineurin is a common target of cyclophilin - Cyclosporin A and FKBP-FK50136 complexes. Cell 66: 807 22. Miyagawa S, Stepkowski SM, Kahan BD (1991) Mechanism of unresponsiveness in rats induced by a short course of FK 506 or CyA. Transplant Proc 23 : 334 23. Platz KP, Sollinger HW, Hullett DA, et al (1991) RS-61443 a new, potent immunosuppressive agent. Transplantation 51: 27 24. Ponticelli C, Tarantino A, Montagnino G (1988) A randomized trial comparing triple drug and double drug therapy in renal transplantation. Transplantation 45 : 913 25. Soullilou JP, Cantarovich D, LeMauff B (1990) Randomized controlled trial of a monoclonal antibody against the interleukin-2 receptor (33B3.1) as compared with rabbit antithymocyte globulin for prophylaxis against rejection of renal allografts. N Engl J Med 322 : 1175 26. Starzl TE, Todo S, Fung J, et al (1989) FK506 for human liver, kidney and pancreas transplantation. Lancet II: 1000 27. Starzl TE, Fung J, Jordan M, et al (1990) Kidney transplantation under FK506. JAMA 264 : 63 28. Turka LA, Dayton J, Sinclair G, Thompson CB, Mitchell BS (1991) Guanine ribonucleotide depletion inhibits T cell activation: mechanism of action of the immunosuppressive drug mizorbine. J Clin Invest 87 : 940-948 29. Ullman KS, Flanagan WM, Cothesy B, Kuo P, Northrop JP, Crabtree GR (1991) Site of action of cyclosporine and FK506 in the pathways of communication between the T-lymphocyte antigen receptor and the early activation genes. Transplant Proc 23 : 2845 30. Van Duyne G, Standaert R, Karplus P, Screiber S, Clardy J (1991) Atomic structure of FKBP-FK506, an immunophilinimmunosuppressant complex. Science 252: 839 31. Vathsala A, Chou TC, Kahan BD (1990) Analysis of the interaction of immunosuppressive drugs with cyclosporine in inhibiting DNA proliferation. Transplantation 49 : 436 32. Wtithrich K, Freyberg B, Weber C, Wider G, Traber R, Widmer H, Braun W (1991) Receptor-induced conformation change of the immunosuppressant cyclosporin A. Science 254 : 953
European Journal of
Pediatrics
9 Springer-Verlag1992
New immunosuppressive drugs: needs in and applications to pediatric transplantation B. D. Kahan Division of Immunologyand Organ Transplant, Department of Surgery, The Universityof Texas MedicalSchool at Houston, 6431 Fannin, Suite 6.240 MSB, Houston, TX 77030, USA
Abstract. The evolution of immunosuppressive therapy toward synergistic drug combinations seeks to minimize toxicity while potentiating efficacy. Median effect analysis discerns synergistic drug combinations that may be suitable for in vivo experiments in animals and for subsequent clinical trials. These studies suggest that two drugs rapamycin (RAPA) and brequinar (BQR) display synergistic effects in combination with cyclosporine. This combination must be evaluated for relative toxicity versus efficacy. Clinical trials to assess the individual toxicities of RAPA and BQR are presently underway in order to discern appropriate doses for randomized trials of clinical efficacy.
suppressive agents are under clinical trial to substitute for, or to complement the action(s) of, the presently available agents: FK506 [26], rapamycin [16] (RAPA), mizorbine [28], RS-61443 [23], and quinoline carboxylic acid [3]. Each of these agents not only prolongs allograft survival in preclinical models but also produces substantial toxicity when relied on as monotherapy for rejection prophylaxis. In the fashion that cancer chemotherapeutic agents have been combined in order to potentiate efficacy but mitigate toxicity, individually subtherapeutic, but subtoxic doses of immunosuppressive drugs may overcome the limitations of currently available regmines.
Key words: Immunosuppression - Drug interactions Cyclosporine
Analysis of dose-response relations of immunosuppressive drugs
Introduction Pediatric patients present a special challenge to the transplant physician for the design of an appropriate immunosuppressive regimen. On the one hand pediatric patients, particularly under the age of 10 years, display an inordinately vigorous allorejection response [5]. On the other hand because of the unique requirements for excellent allograft function in order to achieve growth and development in children, the optimal immunosuppressive regimen must be minimally toxic to kidneys, liver and bone. Existent immunosuppressive regimens are not only suboptimally effective, but also display a narrow therapeutic window. The pleiotropic renal, vascular, hepatic, bone and endocrine toxicities of cyclosporine (CSA) preclude the use of optimally immunosuppressive doses of this drug [12]. In order to potentiate immunosuppression pediatricians frequently combine azathioprine and corticosteroids with CsA, even though three randomized trials failed to document any benefit of the triple compared with the double (CsA/prednisone) drug regimen [1, 20, 24]. Contrariwise, it proffers the possibility of synergistic hepatic injury. Several new immuno-
Abbreviations: BMA=Behringwerke monoclonal antibody; BQR = brequinar; CI = combinationindex; CsA = cyclosporine; I1 = interleukin; MAb = monoclonal antibodies; NFAT = nuclear factor of activated T-cell; RAPA = rapamycin
Since various immunosuppressive drugs produce different patterns of inhibition of immunologic responses, it is necessary to utilize mathematical models to rigorously analyze dose-response relations, in order to discern synergistic, additive or antagonistic interactions. The median effect equation [2] has been applied to analyze the interactions between cancer chemotherapeutic and between anti-viral agents: ?a
=
(o)m
where fa is the percent inhibition (fraction affected), fu is the percent unaffected (100-fraction affected) by the agent used at dose D. Dm is the 50% inhibitory concentration and m is a slope coefficient of the relationship. The Dm is precisely determined by the abscissa of the log/log plot of: log (lf_~a/a) = m l o g D - m l o g D m The degree to which the data fit the median effect model is confirmed by a linear regression coefficient r > 0.75. The slope of this plot yields the "m" value. The log/log plot provides a ready estimate of the dose Dx to achieve a given effect level of x % inhibition. In order to assess interactions one performs an immunologic assay in the presence of each agent alone or of drug combinations. The combination index at x % inhibition is expressed as the ratios of the concentrations
$10 required to produce that effect level in the presence of second agent versus that when the drug is used alone. CIx = dose drug~ with drug2 + dose drug2 with drug1 + dose drug1 alone dose drug2 alone [dose drug1 with drug2] [dose drugs with drug1] [dose drug1 alone] [dose drug2 alone] Combination index (CI) values equal to 1.0 document additive interactions; less than 1.0, drug synergism; and greater than 1.0, drug antagonism. Several factors affect the CI [14]. First, the ratio of the molar concentrations of the two drugs tested in the combination. A range of ratios, using drug concentrations relevant for clinical application must be tested, in order to ascertain the optimal ratio to achieve synergistic effects. Second, a variety of immunologic assays should be tested, since each performance system may reveal different values. In general immunosuppressive drugs tend to be more synergistic in assys reflecting T-lymphocyte activation via the T-cell receptor, namely anti-CD3 stimulation and mixed lymphocyte reactions, than those triggered by polyclonal mitogens such as phytohemagglutinin or by phorbol myristate acetate plus ionomycin. Third, a variety of volunteers must be tested, since there are considerable inter-individual differences among combination indices observed in human subjects. Although the CI values among a group of ten volunteers may span 2 - 4 log values, they usually all show a similar type of interaction. One important index of a significant synergistic interaction is a consistency of values less than 1.0 among a large population sample.
Agents affecting reception of the T-cell activation signal Activation of the T-helper cell depends upon antigenic recognition via the T-cell receptor accompanied by a second, humoral, co-stimulator signal. Corticosteroids inhibit the generation of messenger RNA for Interleukin (IL)-1[3, a co-stimulatory lymphokine. However, the multitude of side-effects from corticosteroids, particularly on growth in the pediatric population, contraindicate their longterm use. The antigen signal reception step can be interrupted with monoclonal antibodies (MAb). The IgG2b MAb Behringwerke monoclonal antibody (BMA) 031 directed against the common region of the T-cell receptor interrupts T-cell sensitization and delays the onset of allograft rejection without significant side-effects [19]. In contradistinction to the therapeutic agent, OKT3 an antiCD3 IgG2a MAb, BMA 031 does not trigger cytokine release. However, the clinical application of BMA 031 is limited by the rapid, almost uniform appearance of antiidiotypic antibodies that limit the length of the treatment course to less than 5 days.
Drug inhibiting lymphokine synthesis Two structurally unrelated compounds inhibit the transduction of the cell surface signal into a cytoplasmic mes-
sage that eventuates in transcription of m-RNA for lymphokine humoral mediators. This generation of the antigen-activation signal through inositol phosphates increases intracellular calcium concentrations, augments oncogene products, and leads to the generation of regulatory proteins. The exact sequence of chemical events distal to the increase in calcium is uncertain. Ullman and colleagues [29] suggest that the initial regulatory protein, the nuclear factor of activated T-cells (NFAT), is assembled from a cytoplasmic and a nuclear fraction. After the binding of NFAT, additional regulatory proteins are assembled in the enhancer region controlling IL-2 transcription, thereby triggering RNA Polymerase activity. CsA (molecular weight 1202 daltons) is a cyclic fungal undecapeptide bearing at position 1, a unique nine carbon amino acid containing a double bond. FK506 (molecular weight 750 daltons) is a macrocyclic molecule, containing a critical a-keto-homoprolyl subunit. These two compounds bind intracellular proteins that have cis-trans prolylisomerase activity [8]: CsA to cyclophilin [10], FK506 to fujiphilin [30]. Liu and colleagues [21] suggest that the binding of drug alters the substrate specificity of the isomerase. Although its native substrate is not known, the immunophilin-drug complex inhibits the enzymatic action of calcineurins A and B, two calcium- and calmodulin-dependent phosphatases. This inhibition may prevent calcineurin from cleaving the phosphate form the cytoplasmic fraction of NFAT, an event necessary for NFAT to penetrate the nuclear membrane, and consequently assemble with the nuclear fraction into the full NFAT moiety that promotes IL-2 gene transcription. Alternatively, the enzymatic action of the isomerase may convert the administered "pro"-drug into an active moiety. Wtithrich et al. [32] found that when CsA binds to cyclophilin, it undergoes a cis- to trans-conformational change by inverting of the polar surface of amide protons and carbonyl oxygens to the outside and the hydrophobic exterior edges of N-methyl groups of the inside, thereby rendering the drug more hydrophilic. Similarly, Jorgenson [11] described a conformational change of FK506 due to cis-trans isomerization of the amide bond, thereby repositioning the homoproline structure with inward movement of the pyranose ring toward the macrocycle. Contrariwise, the related macrolide, R A P A (see below) does not undergo a structure change upon binding to the isomerase protein. The isomerase changes thus may confer these drugs' unique inhibition of the lymphocyte activation cascade, thereby interrupting lymphokine synthesis and preventing cytotoxic T-cell generation. In spite of this common effect, the two drugs produce unresponsiveness by distinctive mechanisms. CsA promotes the generation of T-suppressor cells, either of the CD4 § [9] or CD8 + [19] phenotype; FK506 does not produce this effect [22]. The pleiotropic side-effect profile of CsA displays nephrotoxic > hepatotoxic >> neurotoxic side-effects [12]. A similar spectrum of side-effects beclouds FK506 which is about 10- to 50-fold more potent than CsA on a molar basis, but shows a profile of neurotoxic > nephrotoxic >> hepatotoxic side-effects [26]. At therapeutic
$11 immunosuppressive doses there appears to be little difference between CsA and FK506 not only in the incidence of side-effects, but also in their efficacy to prevent human renal allograft rejection [27]. Presently, rational therapeutic application of both CsA and FK506 is precluded due to marked inter-individual pharmacokinetic variations between transplant recipients, both in gastrointestinal absorption and in drug clearance rates [13]. Children especially present unique problems due to their more rapid clearance rates and poorer absorption of CsA than adults.
Agents blocking lymphokine action Lymphokines bind to surface receptors that transduce the activation signal via a calcium-independent pathway that results in proliferation and cellular differentiation. There are several opportunities to interfere with the action of lymphokines. Anti-lymphokine antibodies, namely anti-y-interferon [4], potentiate the immunosuppressive activity of CsA. There are two other ways to decrease effective lymphokine concentrations. One approach delivers soluble constructs of the extra-membranous portion of IL-1 [6] or IL-4 [7] lymphokine receptors. The second approach administers lymphokine antagonists that bind to receptor without triggering the activation event. IL-1 antagonists are presently under trial for treatment of rheumatoid arthritis. A similar effect may be produced by antibodies that cover lymphokine receptors. Recent clinical trials using an anti-IL-2 receptor (IL-2R) MAb revealed a delayed onset of rejection with no difference in graft survival rates [25]. The most potent pharmacologic agent inhibiting receptor-mediated events is R A P A (molecular weight 950 daltons) [16]. While the exact mode of R A P A action on lymphocyte activation is uncertain, the drug does not interfere with the binding of radiolabelled IL-2 lymphokine to IL-2R, with the phosphorylation of the 75 kDa [3 chain of the IL-2R, or with changes in cyclic AMP. Although R A P A inhibits the endocytosis of the IL-2/ IL-2R complex, this effect does not totally explain the drug's inhibitory action. The synergistic interaction between CsA and R A P A is potentially of great clinical import, for it may permit the use of subtherapeutic doses of each drug in an effective combination [17]. R A P A does not interfere with the induction of suppressor T-cells by CsA; additionally R A P A contributes a unique action to induce a humoral immunosuppressive principle. The R A P A plus CsA combination synergistically prolongs the survival of rat cardiac, renal, and small bowel grafts, of heterotopic nonvascularized murine cardiac allografts, and of mongrel canine renal transplants [16]. The clinical evaluation of R A P A is at a preliminary stage. Since the R A P A doses to be used in combination with CsA will be low (0.05-0.5 mg/kg per day), it is unlikely that the gastro-intestinal mucositis and vasculitis previously observed in dogs receiving R A P A monotherapy will be encountered in patients under a CsA-RAPA dual drug regimen.
Agents inhibiting lymphocyte proliferation The immunosuppressive anti-proliferative chemotherapeutic agents have a common mechanism of action to inhibit nucleic acid synthesis. Three agents inhibit the purine salvage pathwy: 6-mercaptopurine, mizorbine, and mycophenolic acid. The first drug 6-mercaptopurine, whose imidazole derivative azathioprine has been in clinical use for over 30 years, has only a modest clinical immunosuppressive effect, due to its poor therapeutic index. Azathioprine tends to more effectively inhibit the proliferation of bone marrow elements than lymphocytes. Unfortunately, Azathioprine acts only additively with CsA [30]. Therefore it is not unexpected that there was no evident clinical benefit of this combination in randomized trials [1, 20, 24]. Although mizorbine has been in clinical use for over a decade in Japan, it has never been compared with 6-mercaptopurine in controlled, randomized trials. Mycophenolic acid and its analog RS-61443 also inhibit purine salvage pathways. RS-61443 is presently undergoing multicenter phase III efficacy trials. The fourth antiproliferative agent, brequinar (BQR, quinoline carboxylic acid) inhibits de novo synthesis of pyrimidines. This agent more effectively inhibits proliferative responses in mixed lymphocyte reactions than in mitogen stimulation assays. Oral administration of 2.0mg/kg per day B Q R prolongs rat renal and cardiac allograft survival [3]. Interestingly B Q R acts synergistically with CsA in vitro [15]. Indeed the three drug combination CsA: R A P A : B Q R (0.5:0.01:0.5 mg/kg per day) produces permanent survival of allogeneic Buffalo (RT-1 b) heart transplants in Wistar Furth (RT-1 u) rats.
Acknowledgement. This work was supported by NIH/NIDDK award 38016-05.
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