The 4-Amino Analogue of Tetrahydrobiopterin Efficiently Prolongs Murine Cardiac- Allograft Survival Gerald Brandacher, MD, a Yiping Zou, MD, a,b Peter Obrist, MD, c Wolfgang Steurer, MD, c Gabriele Werner-Felmayer, PhD, d Raimund Margreiter, MD, a and Ernst R. Werner, DSc d We tested the 4-amino analogue of tetrahydrobiopterin (H 4 aminobiopterin), a novel pterin-based inhibitor of nitric oxide synthases, for its efficacy in a murine cardiac- transplant model employing an improved cuff technique. We treated groups of 5 animals each for the first 7 post-operative days with various doses of H 4 aminobiopterin, with Cyclosporin A (15 mg/kg/day), or no treatment. H 4 aminobiopterin (3 times 50 mg/ kg/day) proved to be as efficient as high-dose Cyclosporin A (15 mg/kg/day) in prolonging allograft survival and in suppressing histologic changes caused by the immunoreaction. Surprisingly, the doses of H 4 aminobiopterin effective in prolonging allograft survival did not change the plasma nitrite plus nitrate, or the expression of inducible nitric oxide synthase, interferon-, tumor necrosis factor–, and B7-1 (CD80), indicating that H 4 aminobiopterin may act through a novel, yet undiscovered mechanism. J Heart Lung Transplant 2001;20:747–749. Inducible nitric oxide synthase (iNOS) is an essen- tial part of the armature of cytotoxic responses of cells, in particular of macrophages. 1 Inducible NOS is expressed during allograft rejection in animal models 2 as well as in humans. 3 The role of this induction in the rejection process, however, is not clear, as elimination or inhibition of NOS has not shown consistent effects on allograft rejection. 4,5 The 4-amino analogue of tetrahydrobiopterin (H 4 aminobiopterin) has been developed to inhibit potential recycling reactions inside the active NOS dimer. 6 In tissue culture, the compound selectively inhibits the inducible isoform of NOS, with little effect on other pterin-dependent enzymes. 7 MATERIALS AND METHODS C57BL/10 (H-2b) donor hearts were transplated to the neck of C3H/He (H-2k) recipients using a modified cuff technique for revascularization. 8 Ani- mals were obtained from Harlan-Winkelmann (Borchen, Germany). All animals received humane care in compliance with the Principles of Laboratory Animal Care, formulated by the National Society for Medical Research, and the Guide for the Care and Use of Laboratory Animals, prepared by the Institute of Laboratory Animal Resources and published by the National Institute of Health (NIH Pub. No. 86-23, revised 1985). We used male animals weigh- ing 25 to 30 g for the experiments. During the first 7 post-operative days, recipient animals received IM H 4 aminobiopterin (20 to 200 mg/kg/day, or 50 mg/kg every 8 hours) or Cyclosporin A (15 mg/kg/day). Control animals received no treatment. We moni- tored the function of the transplanted heart by palpation. Tissue samples were fixed in 7% formal- dehyde, embedded in paraffin, and stained with From the Department of Transplant Surgery, a University Hospi- tal Innsbruck; Institute of Pathology, c and Institute for Medical Chemistry and Biochemistry, d University of Innsbruck, Inns- bruck, Austria; and Department of Hepatobiliary Surgery, b Bejing 309 Hospital, Bejing, Peoples Republic of China. Submitted June 6, 2000; accepted November 13, 2000. Reprint requests: Ernst R. Werner, Institute for Medical Chem- istry and Biochemistry, University of Innsbruck, Fritz-Pregl- Str. 3, A-6020 Innsbruck, Austria. Telephone: 43-512-507-3517. Fax: 43-512-507-2865. E-mail: ernst.r.werner@uibk.ac.at. Copyright © 2001 by the International Society for Heart and Lung Transplantation. 1053-2498/01/$–see front matter PII S1053-2498(00)00329-6 747