Duquesnoy, RJ

Fung, JJ

Starzl, TE

Todo, S

Venkataramanan, R

Yao, GZ

Zeevi, A

English

PubMed

D-Scholarship@Pitt

.; -. lit:}/' Comparative In Vitro Studies on the Immunosuppressive Effects of Purine and Pyrimidine Synthesis Inhibitors A. Zeevi, G.- Z. Yao, R. Venkataramanan, R.J. Duquesnoy, S. Todo, J.J. Fung, and T.E. Starzl OVER the past few decades the implementation of new immunosuppressive regimens has remarkably im-proved the success of clin!cal transplantation. Immuno-suppressive drugs that can inhibit both cell-mediated and humoral immune responses have an important role in controlling acute cellular rejection and chronic rejection. In this report we compare the inhibitory effects of four antiproliferative drugs: two purine synthesis inhibitors, mycophenolic acid (MPA)l and mizorbine (MZR),2 and two pyrimidine synthesis inhibitors, brequinar sodium (BQR)3 and N-phosphonacetyl-L-aspartic acid (PALA).4 A variety of in vitro culture systems have been used to determine the potency of these drugs including T-cell activation via calcium (Ca)-dependent (A23187) and Ca-independent (lnterleukin-2 [IL-2] and Phorbol myristic acetate [PMAD pathways. Transformed T and non-T cells were also used to test the effect of these antiproliferative drugs. MATERIALS AND METHODS Cells Peripheral blood lymphocytes were isolated by Ficoll-Hypaque gradient centrifugation of heparinized blood from healthy donors. Cells were resuspended in RPMI 1640 tissue culture medium (TCM) supplemented with 2S nmoUL Hepes buffer and 100 UlmL gentamicin and 5% normal human serum. Reagents FK 506 and cyclosporin A (CyA) (Sandoz, Basel, Switzerland) were dissolved in methanol (l mg/mL). The MPA (Sigma Chem-ical Co, St Louis, Mo), and MZR were dissolved in ethanol and water, respectively, before use. The BQR and PALA were suspended in distilled water. Proliferative Responses of Lymphocytes Mononuclear cells were cultured in TCM at lOS cells/well in the presence of A23187 (ll-'8lmL) or PMA (0.1 ~miF in a volume of 200 ~ for 3 days. Interleukin-2-induced proliferation was mea-sured by culturing alloreactive T cells (2 x lit cells/well) in the presence of 30 IU of IL-2 for 3 days. Mixed lymphocyte reaction (MLR assays) were established with equal numbers of responder cells (5 x lit cells/well) and irradiated stimulator cells in TCM and incubated for 6 days. For the secondary proliferative response (PLTI, alloreactive T cells (2 x I <t cells/well) were incubated with irradiated stimulator cells for 3 days. Proliferative Responses of Various Cell Lines T-Iymphoma cell lines (DND4I, Peer), Epstein-Barr (EB) virus-transformed lymphoblastoid cell lines, a B-Iymphoma cell line (RPMI-1788), an erythroleukemia cell line (KS62), and a promy-elocytic cell line (HL-60) were maintained in 10% TCM with fetal calf serum. Proliferation was measured after 72 hours of incuba-tion by 3H-thymidine incorporation. Drug Inhibition Assays The inhibitory effects of various drugs alone or in combination were measured at different concentrations. The percent inhibition was calculated as follows: % inhibition = (I - {cpm with drugicpm without}) x 100. RESULTS Table 1 summarizes the drug dose required to obtain 50% inhibition (IC50) for five immunosuppressive drugs. Both BQR and MPA showed similar IC50 (80 to 100 nmoUL) in both Ca-dependent and Ca-independent T-cell prolifera-tion. The IC50 for MZR was about 100-fold higher than that observed for MPA or BQR. FK 506 and CyA exhibited significant inhibition of Ca-ionophore stimulation (IC50 0.18 nmoUL and 23 nmoUL, respectively) but had no effect on IL-2 and PMA stimulation. These data suggest that BQR and MPA have similar in vitro potency for suppress-ing both Ca-dependent and Ca-independent T-cell activa-tion pathways. The PALA, which was also tested in Table 1. Inhibitory Effects of Immunosuppressive Drugs on T-cell Proliferation IC 50 nmollL' FK506 CyA SOR MPA MZR Mitogen A23187 0.18 23 88 81 NT IL-2 No Inhibition 75 119 7,637 PMA No Inhibition 100 81 14,861 Allostimulation Primary (MLR) 0.1 10 50 103 14,706 Secondary (PLT) 0.17 29 18 163 28,761 'Concentration of drugs required to obtain 50% Inhibition 1C5O. From the Divisions of Transplantation Pathology (A.Z., G.-Z.Y., and R.J.D.), and Surgery (A.Z., R.J.D., S.T., J.J.F., and T.E.S.), and the Department of Pharmacology (R.V.), University of Pitts-burgh Medical Center, Pittsburgh, Pennsylvania. Supported by the Pathology Education and Research Founda-tion and NIH Grant No. DK29961-12. Address reprint requests to Adriana Zeevi, PhD, University of Pittsburgh Medical Center, Biomedical Science Tower, Room W1551, Tothrop and Terrace Sts, Pittsburgh, PA 15261. ~ 1993 by Appleton & Lange 0041-1345/931$3.001 +0 Transplantation Proceedings, Vol 25, No 1 (February), 1993: pp 781-783 781 s 782 various T-cell activation assays, had minimal inhibitory effect even at a high dose of 10 /-Lg/mL (data not shown). All five immunosuppressive drugs inhibited the primary MLR and the secondary PL T proliferation of alloreactive T cells. In these assays BQR was two- to fourfold more potent than MPA whereas MZR and PALA were about 1<Y- and 104-fold, respectively, less active than BQR (Table O. Combinations of low doses of BQR, MPA, or MZR with FK 506 showed, at best, an additive effect in the suppression of PLT responses (data not shown). The MPA uniformly inhibited all cell lines tested at drug concentrations of 90 to 365 nmoVL, whereas BQR ap-peared to have a more restricted pattern of inhibition. Proliferation of EB virus B-celliines and HL-60 cells were inhibited by BQR at a concentration of 250 nmoUL, whereas T-Iymphoma cell lines were resistant even at high doses of BQR (100 !J.g/mL). All cell lines tested were resistant to the inhibitory effects of PALA. DISCUSSION This study demonstrates that BQR, MPA, and MZR inhibit the proliferative responses of normal T cells to mitogenic and allogeneic stimulation, whereas P ALA has a minimal effect. The proliferative responses of T cells induced by T-cell receptor, IL-2, and protein kinase C are equally affected by BQR, MPA, and MZR, since these drugs block DNA synthesis. l -4 In contrast, FK 506 and CyA are more efficient in blocking signal transduction via T-cell receptors by inhibiting the transcription of early activation genes.5 •7 T and B cells lack the salvage pathway found in other types of cells and depend solely on the de novo pathway of purine and pyrimidine synthesis.8,9 Although both BQR and P ALA efficiently inhibit the de novo pyrimidine syn-thesis, T cells are 103 to lW times more sensitive to the immunosuppressive effects of BQR than PALA. The BQR, an anticancer drug, exerts its antiproliferative activ-ity by noncompetitively inhibiting the activity of de hydro-orotate dehydrogenase, the fourth enzyme in the de novo pyrimidine biosynthetic pathway. 3,9 The P ALA inhibits DNA synthesis by blocking the second enzyme aspartate transcarbamylase in the de novo pyrimidine pathway.4 Growth inhibition of P ALA can be easily reversed by carbamyl-L-asparate, whereas BQR inhibitory activity is not affected by the addition of dihydroorotic acid. 10.11 The BQR inhibits the. growth of a broad spectrum of murine and human solid tumors, whereas P ALA is effective against a limited number of solid tumors. 12 Consistent with these findings are our observations that BQR blocks the proliferative responses of many lymphoid and nonlym-phoid cell lines, while PALA, even at 100 !J.g/mL, had only a minimal inhibitory effect on nonlymphoid cell lines. Other antiproliferative drugs such as MPA and MZR have also been shown to have both antitumor and immu-nosuppressive activities, 13.14 The MPA, an antibiotic, in-hibits reversibly and noncompetitively only inosine mono-ZEEVI, YAO, VENKATARAMANAN ET AL phosphate dehydrogenase (IMPDH), an enzyme in the de novo purine biosynthetic pathway. Mizorbine, which is an imidazole nucleoside, also inhibits IMPDH but is 102_ to 103-fold less active in blocking T-cell proliferation than MPA or BQR, respectively. All anti-DNA synthesis drugs tested act at a late stage of the cell activation pathway by blocking the movement of cells from Gl to S, whereas FK 506 and CyA act at an early stage of T-cell activation by inhibiting cell division cycle at GO/G 1 interface. Since T cells and B cells depend on de novo nucleotide synthesis, both humoral responses mediated primarily by B cells and cellular immune re-sponses governed by T cells could be significantly im-paired in the presence of purine and pyrimidine inhibi-tors. 15,16 The MPA and its acid derivative RS 61443 have shown promising results in preventing acute cellular rejec-tion in an experimental canine renal allograft model and in suppressing chronic rejection associated with vasculitis in a rat heart allograft model. I7 Also BQR is effective in suppressing the development of contact sensitivity and adjuvant arthritis in rodent models and preventing kidney, heart, and liver allograft rejection in rats. 16 Although no synergism between BQR, MPA, or MZR with FK 506 was observed in inhibition of allogeneic T cell proliferation, an additive effect was seen. 18 Thus, the antiproliferative drugs may offer an alternative regimen when used in combination with other immunosuppressive drugs in treatment of acute and chronic cellular rejection. However, the most important benefit from the introduc-tion of the experimental antimetabolite drugs or the estab-lished anti-DNA synthesis drug cyclophosphamide, is the ability to break down the antibody barrier to xenotrans-plantation. 19 Murase et al l9 have recently shown that the combination of FK 506 with either one of the anti-DNA synthesis inhibitors significantly prolongs the survival of hamster to rat heart or liver xenotransplantation by sup-pressing the anti-hamster antibody response. ACKNOWLEDGMENT FK 506 was a gift from Fujisawa Pharmaceuticals, Osaka, Japan; MZR was a gift from Asahi Chemical Industry Co, Ltd, Tokyo, Japan; BQR was donated by DuPont Medical Products, Wilming-ton, Del; PALA was a gift from Dr Steve Veach, Bioscience, West Conshohocken, Penn. REFERENCES I. Franklin TJ, Cook JM: J Biochem 113:515, 1969 2. Mizuno K, Tsugino M, Takada M, et al: J Antibiot (Tokyo) 24:775, 1974 3. Chen SF, Ruben RL, Dexter DL: Cancer Res 46:5014, 1986 4. Collins KD, Stark GR: J Bioi Chem 244:6599, 1969 5. Sawada S, Suzuki C, Kawase Y, et al: J Immunol 139:797, 1987 6. Tocci MJ, Matkovich OM, Collier KA. et al: J Immunol 143:718. 1989 7. Thomson AW, Woo J. Zeevi A. et al: In Brossard C (ed): Handbook of Immune Response Modifiers. Berlin Lavopisieri: Springer-Verlag,I991 PURINE AND PYRIMIDINE SYNTHESIS INHIBITORS 8. Allison AC. Hovi T, Watts RWE: Lancet 2:1174, 1975 9. Peters GJ, Sharma SL. Laurensse E, et al: Invest New Drugs 5:235. 1987 10. Johnson RK: Biochem Pharmacol 26:81, 1977 Il. Moyer 10. Handschumacher RE: Cancer Res 39:3089, 1979 12. Peters GJ, Nadal JC, Laurensse E, et al: Biochem Pharma-col 39:135. 1990 13. Eugui EM. Sjalquist C, Muller CD, et al: Scand J Immunol 33:940, 1991 14. Turka LA, Dayton J. Sinclair G. et al: J Clin Invest 87:940. 1991 -783 15. Burlingham WJ. Grailer AP. Hullett DA. et al: Transplan-tation 51 :546. 1990 16. Craver DV. Chapman FA. Jalfe BD. et al: Transplantation 53:303. 1992 17. Morris RE. WangJ, Blum JR. et al: Transplant Proc 23:19. 1991 18. Zeevi A. Woan M. Yao GZ. et al: Transplant Proc 23:2928. 1991 19. Murase N. Starzl TE. Demetris AI. et al: Transplantation, 1992 (in press) 

Comparative in vitro studies on the immunosuppressive effects of purine and pyrimidine synthesis inhibitors.

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Comparative in vitro studies on the immunosuppressive effects of purine and pyrimidine synthesis inhibitors

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Comparative in vitro studies on the immunosuppressive effects of purine and pyrimidine synthesis inhibitors.

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