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The 4-amino analogue of tetrahydrobiopterin efficiently prolongs murine cardiac-allograft survival
The 4-Amino Analogue of Tetrahydrobiopterin Efficiently Prolongs Murine CardiacAllograft 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, DScd We tested the 4-amino analogue of tetrahydrobiopterin (H4aminobiopterin), a novel pterin-based inhibitor of nitric oxide synthases, for its efficacy in a murine cardiactransplant model employing an improved cuff technique. We treated groups of 5 animals each for the first 7 post-operative days with various doses of H4aminobiopterin, with Cyclosporin A (15 mg/kg/day), or no treatment. H4aminobiopterin (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 H4aminobiopterin 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 H4aminobiopterin may act through a novel, yet undiscovered mechanism. J Heart Lung Transplant 2001;20:747–749.
I
nducible nitric oxide synthase (iNOS) is an essential part of the armature of cytotoxic responses of cells, in particular of macrophages.1 Inducible NOS is expressed during allograft rejection in animal models2 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 (H4aminobiopterin) has been developed to inhibit potential recycling reactions inside the active NOS dimer.6 In tissue culture, the compound selectively From the Department of Transplant Surgery,a University Hospital Innsbruck; Institute of Pathology,c and Institute for Medical Chemistry and Biochemistry,d University of Innsbruck, Innsbruck, 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 Chemistry and Biochemistry, University of Innsbruck, Fritz-PreglStr. 3, A-6020 Innsbruck, Austria. Telephone: 43-512-507-3517. Fax: 43-512-507-2865. E-mail: [email protected]. Copyright © 2001 by the International Society for Heart and Lung Transplantation. 1053-2498/01/$–see front matter PII S1053-2498(00)00329-6
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 Animals 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 weighing 25 to 30 g for the experiments. During the first 7 post-operative days, recipient animals received IM H4aminobiopterin (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 monitored the function of the transplanted heart by palpation. Tissue samples were fixed in 7% formaldehyde, embedded in paraffin, and stained with 747
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The Journal of Heart and Lung Transplantation July 2001
FIGURE 1 Levels of H4aminobiopterin and
Cyclosporin A in the course of treatment. Animals were grafted with an additional heart at Day 0 and treated for 7 days with H4aminobiopterin (3 ⫻ 50/mg/kg/day) or Cyclosporin A (15 mg/kg/day). At the indicated time points, the animals were sacrificed. Levels of H4aminobiopterin were monitored in liver and kidney, levels of Cyclosporin A in plasma as described in the Materials and Methods section. Full squares, full line show H4aminobiopterin (liver); full circles, full line show H4aminobiopterin (kidney); open circles, dashed lined show Cyclosporin A (plasma). The figure shows the mean of 2 determinations ⫾ range.
hematoxylin-eosin. Classification and grading of cardiac rejection was based on the International Society for Heart and Lung Transplantation working formulation.9 H4aminobiopterin and nitrite plus nitrate were determined by high-performance liquid chromatography as previously described.7 Cyclosporin A in plasma was determined using fluorescence polarization immunoassay (Abbott; Abbott Park, IL).
RESULTS First we investigated pharmacokinetics of H4aminobiopterin. After a single dose of 20 mg/kg and 100 mg/kg IM, respectively, H4aminobiopterin appeared within 5 minutes in plasma and all tissues investigated, which were heart, liver, thymus, and kidney, with a half-life of 1 to 2 hours. In the dose regimen most effective in prolonging graft survival, 50 mg/kg every 8 hours, we saw a significant buildup of H4aminobiopterin concentration during treatment (Figure 1). At Day 8, however, H4aminobiopterin was already below the detection limit, whereas Cyclosporin A concentrations remained high because of the drug’s longer half-life (Figure 1). Figure 2 shows dependence of allograft survival on the dose of H4aminobiopterin used.
FIGURE 2 Survival of allografted hearts depending on
treatment. Hearts were grafted under the skin and the animals (groups of 5 per regimen) treated for 7 days with the following dose regimens: A, untreated control; B, H4aminobiopterin 20 mg/kg/day; C, H4aminobiopterin 100 mg/kg/day; D, H4aminobiopterin 200 mg/kg/day; E, H4aminobiopterin 3 ⫻ 50 mg/kg/day; F, Cyclosporin A (15 mg/kg/day). Allograft survival was monitored visually by observing the beats of the grafted heart.
Graft survival steadily increased with increasing dose of H4aminobiopterin. A dose of 50 mg/kg H4aminobiopterin applied every 8 hours (survival 14.8 ⫾ 2.1 days) produced results comparable to high-dose Cyclosporin A treatment (15 mg/kg/day; survival, 15.0 ⫾ 1.9 days). The survival in all treated groups was significantly different from controls (Mann-Whitney statitics, p ⬍ 0.037 for 20 mg/kg/day H4aminobiopterin, p ⬍ 0.025 for all other groups). We saw evidence of attenuated allograft rejection by H4aminobiopterin in function of the rejected heart and on histologic sections of the grafted hearts at Day 8 post-transplantation. Although the untreated animals all showed Grade 4 rejection with no palpable heart action, H4aminobiopterin-treated animals showed decreased mononuclear cell infiltrate, decreased mycoyte necrosis, and less interstitial edema as well as improved contractile function. Rejection ranged only from Grade 1B (25%), 2 (20%), 3A (20%), to 3b (40%) in animals treated with 3 ⫻ 50 mg/kg/day H4aminobiopterin, which again is comparable to the histologic observations in animals treated with 15 mg/kg/day Cyclosporin A (Grade 1B, 33%; 2B, 66%). When we compared plasma nitrite-plus-nitrate levels at the peak of its time course (Day 6), we found that nitrite-plusnitrate formation in H4aminobiopterin-treated animals (79.2 ⫾ 23.5 M, mean ⫾ SD, n ⫽ 5) was not
The Journal of Heart and Lung Transplantation Volume 20, Number 7
significantly lower than in untreated controls (93.7 ⫾ 26.3 M, mean ⫾ SD, n ⫽ 5). The expression of iNOS, interferon-␥, tumor necrosis factor-␣, and B7-1 in the cardiac-allograft tissue at Day 6 also was not lower in H4aminobiopterin-treated animals when compared with untreated control allografts, as assayed by Northern blots (data not shown), whereas the Cyclosporin A–treated animals showed significant decrease in expression of interferon-␥.
DISCUSSION Here we demonstrate efficacy of the 4-amino analogue of tetrahydrobiopterin in prolonging murine cardiac-allograft survival, comparable to that of high-dose (15 mg/kg/day) Cyclosporin A, although the interval of drug action is 1 day shorter with H4aminobiopterin. This action is superiour to other NOS inhibitors, which were tested in rat heterotopic cardiac transplantation models and used in much higher doses (500 to 600 mg/kg/day). NG-monomethyl L-arginine could only produce a small increase in allograft survival (from 5.1 to 6.3 days).10 Aminoguanidine, a compound that selectively inhibits the inducible isoform of NOS, could prolong survival from 10.1 to 15.0 days11 when administered at 600 mg/kg/day. In contrast to the present study, which discontinues treatment at Day 7, aminoguanidine was administered continuously until rejection. Thus, aminoguanidine is as active as low-dose Cyclosporin A in preventing rejection,11 whereas the efficacy of H4aminobiopterin used in our study compares to high-dose Cyclosporin A treatment. The authors are indebted to Bettina Fritz and Marion Amort for expert technical assistance. The probe for murine iNOS was a kind gift from C. Nathan and X. W. Xie, from the Cornell University Medical College, New York. This work was supported by Grant 13793 MOB
Brandacher et al.
749
from the Austrian research funds “zur Fo ¨rderung der wissenschaftlichen Forschung.”
REFERENCES 1. MacMicking J, Xie QW, Nathan C. Nitric oxide and macrophage function. Annu Rev Immunol 1997;15:323–50. 2. Yang XC, Chowdhury N, Cai BL, et al. Induction of myocardial nitric oxide synthase by cardiac allograft rejection. J Clin Invest 1994;94:714 –21. 3. Szabolcs MJ, Ravalli S, Minanov O, Sciacca RR, Michler RE, Cannon PJ. Apoptosis and increased expression of inducible nitric oxide synthase in human allograft rejection. Transplantation 1998;65:804 –12. 4. Casey JJ, Wei XQ, Orr DJ, et al. Skin allograft rejection in mice lacking inducible nitric oxide synthase. Transplantation 1997;64:589 –93. 5. Paul LC, Myllarniemi M, Muzaffar S, Benediktsson H. Nitric oxide synthase inhibition is associated with decreased survival of cardiac allografts in the rat. Transplantation 1996;62: 1193–5. 6. Werner ER, Pitters E, Schmidt K, Wachter H, WernerFelmayer G, Mayer B. Identification of the 4-amino analogue of tetrahydrobiopterin as a dihydropteridine reductase inhibitor and potent pteridine antagonist of rat neuronal nitric oxide synthase. Biochem J 1996;320:193– 6. 7. Schmidt K, Werner-Felmayer G, Mayer B, Werner ER. Preferential inhibition of inducible nitric oxide synthase by the 4-amino analogue of tetrahydrobiopterin in cultured cells. Eur J Biochem 1999;259:25–31. 8. Matsuura A, Abe T, Yasuura K. Simplified mouse cervial heart transplantation using a cuff technique. Transplantation 1991;51:896 –900. 9. Billingham ME, Cary NR, Hammond ME, et al. A working formulation for the standardization of nomenclature in the diagnosis of heart and lung rejection. J Heart Transplant 1990;9:587– 601. 10. Winlaw DS, Schyvens CG, Smythe GA, et al. Selective inhibition of nitric oxide production during cardiac allograft rejection causes a small increase in graft survival. Transplantation 1995;60:924 – 6. 11. Worall NK, Lazenby WD, Misko LP, et al. Modulation of in vivo alloreactivity by inhibition of inducible nitric oxide synthase. J Exp Med 1995;181:63–70.
I
nducible nitric oxide synthase (iNOS) is an essential part of the armature of cytotoxic responses of cells, in particular of macrophages.1 Inducible NOS is expressed during allograft rejection in animal models2 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 (H4aminobiopterin) has been developed to inhibit potential recycling reactions inside the active NOS dimer.6 In tissue culture, the compound selectively From the Department of Transplant Surgery,a University Hospital Innsbruck; Institute of Pathology,c and Institute for Medical Chemistry and Biochemistry,d University of Innsbruck, Innsbruck, 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 Chemistry and Biochemistry, University of Innsbruck, Fritz-PreglStr. 3, A-6020 Innsbruck, Austria. Telephone: 43-512-507-3517. Fax: 43-512-507-2865. E-mail: [email protected]. Copyright © 2001 by the International Society for Heart and Lung Transplantation. 1053-2498/01/$–see front matter PII S1053-2498(00)00329-6
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 Animals 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 weighing 25 to 30 g for the experiments. During the first 7 post-operative days, recipient animals received IM H4aminobiopterin (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 monitored the function of the transplanted heart by palpation. Tissue samples were fixed in 7% formaldehyde, embedded in paraffin, and stained with 747
748
Brandacher et al.
The Journal of Heart and Lung Transplantation July 2001
FIGURE 1 Levels of H4aminobiopterin and
Cyclosporin A in the course of treatment. Animals were grafted with an additional heart at Day 0 and treated for 7 days with H4aminobiopterin (3 ⫻ 50/mg/kg/day) or Cyclosporin A (15 mg/kg/day). At the indicated time points, the animals were sacrificed. Levels of H4aminobiopterin were monitored in liver and kidney, levels of Cyclosporin A in plasma as described in the Materials and Methods section. Full squares, full line show H4aminobiopterin (liver); full circles, full line show H4aminobiopterin (kidney); open circles, dashed lined show Cyclosporin A (plasma). The figure shows the mean of 2 determinations ⫾ range.
hematoxylin-eosin. Classification and grading of cardiac rejection was based on the International Society for Heart and Lung Transplantation working formulation.9 H4aminobiopterin and nitrite plus nitrate were determined by high-performance liquid chromatography as previously described.7 Cyclosporin A in plasma was determined using fluorescence polarization immunoassay (Abbott; Abbott Park, IL).
RESULTS First we investigated pharmacokinetics of H4aminobiopterin. After a single dose of 20 mg/kg and 100 mg/kg IM, respectively, H4aminobiopterin appeared within 5 minutes in plasma and all tissues investigated, which were heart, liver, thymus, and kidney, with a half-life of 1 to 2 hours. In the dose regimen most effective in prolonging graft survival, 50 mg/kg every 8 hours, we saw a significant buildup of H4aminobiopterin concentration during treatment (Figure 1). At Day 8, however, H4aminobiopterin was already below the detection limit, whereas Cyclosporin A concentrations remained high because of the drug’s longer half-life (Figure 1). Figure 2 shows dependence of allograft survival on the dose of H4aminobiopterin used.
FIGURE 2 Survival of allografted hearts depending on
treatment. Hearts were grafted under the skin and the animals (groups of 5 per regimen) treated for 7 days with the following dose regimens: A, untreated control; B, H4aminobiopterin 20 mg/kg/day; C, H4aminobiopterin 100 mg/kg/day; D, H4aminobiopterin 200 mg/kg/day; E, H4aminobiopterin 3 ⫻ 50 mg/kg/day; F, Cyclosporin A (15 mg/kg/day). Allograft survival was monitored visually by observing the beats of the grafted heart.
Graft survival steadily increased with increasing dose of H4aminobiopterin. A dose of 50 mg/kg H4aminobiopterin applied every 8 hours (survival 14.8 ⫾ 2.1 days) produced results comparable to high-dose Cyclosporin A treatment (15 mg/kg/day; survival, 15.0 ⫾ 1.9 days). The survival in all treated groups was significantly different from controls (Mann-Whitney statitics, p ⬍ 0.037 for 20 mg/kg/day H4aminobiopterin, p ⬍ 0.025 for all other groups). We saw evidence of attenuated allograft rejection by H4aminobiopterin in function of the rejected heart and on histologic sections of the grafted hearts at Day 8 post-transplantation. Although the untreated animals all showed Grade 4 rejection with no palpable heart action, H4aminobiopterin-treated animals showed decreased mononuclear cell infiltrate, decreased mycoyte necrosis, and less interstitial edema as well as improved contractile function. Rejection ranged only from Grade 1B (25%), 2 (20%), 3A (20%), to 3b (40%) in animals treated with 3 ⫻ 50 mg/kg/day H4aminobiopterin, which again is comparable to the histologic observations in animals treated with 15 mg/kg/day Cyclosporin A (Grade 1B, 33%; 2B, 66%). When we compared plasma nitrite-plus-nitrate levels at the peak of its time course (Day 6), we found that nitrite-plusnitrate formation in H4aminobiopterin-treated animals (79.2 ⫾ 23.5 M, mean ⫾ SD, n ⫽ 5) was not
The Journal of Heart and Lung Transplantation Volume 20, Number 7
significantly lower than in untreated controls (93.7 ⫾ 26.3 M, mean ⫾ SD, n ⫽ 5). The expression of iNOS, interferon-␥, tumor necrosis factor-␣, and B7-1 in the cardiac-allograft tissue at Day 6 also was not lower in H4aminobiopterin-treated animals when compared with untreated control allografts, as assayed by Northern blots (data not shown), whereas the Cyclosporin A–treated animals showed significant decrease in expression of interferon-␥.
DISCUSSION Here we demonstrate efficacy of the 4-amino analogue of tetrahydrobiopterin in prolonging murine cardiac-allograft survival, comparable to that of high-dose (15 mg/kg/day) Cyclosporin A, although the interval of drug action is 1 day shorter with H4aminobiopterin. This action is superiour to other NOS inhibitors, which were tested in rat heterotopic cardiac transplantation models and used in much higher doses (500 to 600 mg/kg/day). NG-monomethyl L-arginine could only produce a small increase in allograft survival (from 5.1 to 6.3 days).10 Aminoguanidine, a compound that selectively inhibits the inducible isoform of NOS, could prolong survival from 10.1 to 15.0 days11 when administered at 600 mg/kg/day. In contrast to the present study, which discontinues treatment at Day 7, aminoguanidine was administered continuously until rejection. Thus, aminoguanidine is as active as low-dose Cyclosporin A in preventing rejection,11 whereas the efficacy of H4aminobiopterin used in our study compares to high-dose Cyclosporin A treatment. The authors are indebted to Bettina Fritz and Marion Amort for expert technical assistance. The probe for murine iNOS was a kind gift from C. Nathan and X. W. Xie, from the Cornell University Medical College, New York. This work was supported by Grant 13793 MOB
Brandacher et al.
749
from the Austrian research funds “zur Fo ¨rderung der wissenschaftlichen Forschung.”
REFERENCES 1. MacMicking J, Xie QW, Nathan C. Nitric oxide and macrophage function. Annu Rev Immunol 1997;15:323–50. 2. Yang XC, Chowdhury N, Cai BL, et al. Induction of myocardial nitric oxide synthase by cardiac allograft rejection. J Clin Invest 1994;94:714 –21. 3. Szabolcs MJ, Ravalli S, Minanov O, Sciacca RR, Michler RE, Cannon PJ. Apoptosis and increased expression of inducible nitric oxide synthase in human allograft rejection. Transplantation 1998;65:804 –12. 4. Casey JJ, Wei XQ, Orr DJ, et al. Skin allograft rejection in mice lacking inducible nitric oxide synthase. Transplantation 1997;64:589 –93. 5. Paul LC, Myllarniemi M, Muzaffar S, Benediktsson H. Nitric oxide synthase inhibition is associated with decreased survival of cardiac allografts in the rat. Transplantation 1996;62: 1193–5. 6. Werner ER, Pitters E, Schmidt K, Wachter H, WernerFelmayer G, Mayer B. Identification of the 4-amino analogue of tetrahydrobiopterin as a dihydropteridine reductase inhibitor and potent pteridine antagonist of rat neuronal nitric oxide synthase. Biochem J 1996;320:193– 6. 7. Schmidt K, Werner-Felmayer G, Mayer B, Werner ER. Preferential inhibition of inducible nitric oxide synthase by the 4-amino analogue of tetrahydrobiopterin in cultured cells. Eur J Biochem 1999;259:25–31. 8. Matsuura A, Abe T, Yasuura K. Simplified mouse cervial heart transplantation using a cuff technique. Transplantation 1991;51:896 –900. 9. Billingham ME, Cary NR, Hammond ME, et al. A working formulation for the standardization of nomenclature in the diagnosis of heart and lung rejection. J Heart Transplant 1990;9:587– 601. 10. Winlaw DS, Schyvens CG, Smythe GA, et al. Selective inhibition of nitric oxide production during cardiac allograft rejection causes a small increase in graft survival. Transplantation 1995;60:924 – 6. 11. Worall NK, Lazenby WD, Misko LP, et al. Modulation of in vivo alloreactivity by inhibition of inducible nitric oxide synthase. J Exp Med 1995;181:63–70.