Mycophenolate Mofetil and FK506: Two Novel Immunosuppressants in Murine Corneal Transplantation A. Reis, T. Reinhard, R. Sundmacher, S. Braunstein, and E. Godehardt I MMUNOLOGIC transplant rejection represents one of the main reasons for transplant failure among patients with negative prognostic factors (e.g., corneal neovascular- ization, previous transplantation). 1 New immunosuppres- sive agents that more specifically target the immune system would be of great benefit in treating allograft rejection. FK506, like CsA, is a macrolide antibiotic derived from a fungus, Streptomyces tsukubaensis 2,3 discovered in the area of Tsukuba in Japan. On the molecular level, FK506 once bound to its intracellular binding protein (FKBP), is be- lieved to form a drug–isomerase complex with calcineurin, and reduce its phosphatase activity. 4 As a result, transloca- tion of nuclear factor of activated T cells (NF-AT) is prevented which ultimately results in a reduction of the transcription of IL-2. In vitro studies have shown that, even in concentrations 40 to 200 times lower that CsA, FK506 possesses extremely powerful immunosuppressive effective- ness. 5,6 The immunosuppressive effect of FK506 has also been successfully shown in allogenic 7 and xenogenic 8 trans- plantation models, including two allogenic corneal trans- plantation models in the rat. 9,10 The dose–response curves investigated by Nishi et al 9 were the basis for the FK506 concentrations used in this study. Another very promising immunomodulating substance, whose immunosuppressive effect in combination with CsA following kidney transplan- tation has already been proven in several clinical studies, is mycophenolatemofetil (MMF, the morpholinoethylester of mycophenolic acid). 11–13 Olsen et al 14 have constructed a dose–response curve for this agent in the murine corneal transplant model. The MMF concentrations used in our study were chosen according to their results. MMF is an inhibitor of an enzyme which controls purine synthesis at a central point. Mycophenolic acid reversibly inhibits the formation of guanosine nucleotides. 15 As T and B cells are predominantly dependent on a de novo synthesis of guanosine nucleotides, the purine biosynthesis of these cells is selectively inhibited. 16 Due to their distinct molecular targets in immune cells, a synergistic effect in combating corneal allograft rejection should be expected when com- bining FK506 and MMF. The purpose of this study was to investigate the suitability of MMF and FK506 as well as their combination for suppressing allograft rejection after corneal transplantation. METHODS The strains used in this experiment were Lewis (Rt le ) and Brown Norway (Rt ln ), (Janvier, France). The animals were obtained and cared for in accordance with the Directives of the European Community as well as with the recommendations of the NIH Guide for the Care and Use of Laboratory Animals, National Institutes of Health Publication No. 85-23 (revised 1985). Operative Technique We conducted an orthotopic perforating keratoplasty according to the technique of Herbort et al. 17 Mydriasis in the eyes of both the donors and the recipients was achieved by the topical application of phenylephrine 10% eyedrops. These eyedrops were administered three times at intervals of 10 minutes before the operation. During an inhalation anaesthesia with diethyl ether the corneas of the donor rats (Brown Norway, BN) were obtained by the use of a 3.5 mm trephane. Until their implantation, the donor buttons were stored in a conservation medium for corneas (Likorol). The recipient animals (Lewis) were pretreated in the same way and, after a brief inhalation anesthesia with diethyl ether, were anesthetized with an intraperitoneal mixed injection of ketamine hydrochloride 100 mg/kg BW, midazolam 0.5 mg/kg BW, and atropine 0.5 mg/kg BW, and fixed in a dextral lateral position. After the left host cornea had been removed with a 3.0 mm trephane, the donor cornea was transplanted. The transplant was sewn in with 8 interrupted sutures (Ethicon 11.0). The anterior eye chamber was restored at the end of the operation by the instillation of balanced saline solution. To protect the transplant, a blepharorrhaphy was attached by means of 2 interrupted sutures (Prolene 6.0) and remained in place for 3 days. Refobacin (gentamicin) was applied in the palpebral fissure. The groups were divided up as follows. Group 1 (6 animals), Lewis/Lewis (syngenic transplant, control 1); group 2 (8 animals), BN/Lewis (allogenic transplant, no therapy, control 2); group 3 (7 animals), BN/Lewis (MMF, 40 mg/kg bw/d orally, according to the dose–response curves by Olsen et al. 14 ); group 4 (7 animals), BN/Lewis (FK506, 0.3 mg/kg bw/d i.p., according to the dose– response curves by Nishi et al. 9 ); and group 5 (6 animals), BN/Lewis (MMF 40 mg/kg, FK506 0.3 mg/kg). Medication in the From the Eye Hospital (A.R., T.R., R.S.) Department of Pathol- ogy (S.B.) and Department for Biometry in the Clinic of Cardiac and Thoracic Surgery (E.G.), Heinrich Heine University, Duessel- dorf, Germany. Address reprint requests to Dr Alexander Reis, Eye Hospital, Heinrich-Heine-University, Moorenstrasse 5, 40225 Duessel- dorf, Germany. 0041-1345/98/$19.00 © 1998 by Elsevier Science Inc. PII S0041-1345(98)01434-1 655 Avenue of the Americas, New York, NY 10010 4344 Transplantation Proceedings, 30, 4344–4347 (1998)