Clinical Biochemistry, Vol. 31, No. 5, 369 –373, 1998
`Copyright © 1998 The Canadian Society of Clinical Chemists
`Printed in the USA. All rights reserved
`0009-9120/98 $19.00 1 .00
`
`PII S0009-9120(98)00043-5
`
`Preclinical Evaluation of a New Immunosuppressive
`Agent, FTY720
`
`PABLO TRONCOSO and BARRY D. KAHAN
`
`Division of Immunology and Organ Transplantation, Department of Surgery, University of Texas
`Medical School at Houston, 6431 Fannin Street, Houston, Texas 77030, USA
`
`Introduction
`
`IN VIVO
`
`FTY720 is a synthetic analog of the drug Myriocin
`
`(ISP-1), which is present in culture filtrates of
`Isaria sinclairii (1), an ascomycete. Ascomycetes are
`mycelial forms of Fungi Imperfecti, which are charac-
`terized by asexual spore phases and are usually para-
`sitic on insects or plants. Although Isaria sinclairii has
`been widely used in Chinese traditional medicine,
`toxicology studies have shown that ISP-1, which is a
`structural analog of sphingosine, produces severe di-
`gestive disorders, resulting in the death of experimen-
`tal animals (2). Because of the potent immunosuppres-
`sive activity of ISP-1 in vitro, synthetic modifications
`have been performed to generate less toxic and more
`active compounds (2,3). One of these new compounds
`is FTY720, 2 amino-2-(2-[4-octylphenyl]ethyl)-1,3-pro-
`panediol hydrochloride (Figure 1), which was devel-
`oped by Prof. T. Fujita (Taito Co. Ltd.) in collaboration
`with Yoshitomi Pharmaceuticals Ltd.; hence the
`name, FTY720.
`
`Immunosuppressive effects
`
`IN VITRO
`
`In mouse models, ISP-1 and related compounds
`have been shown to inhibit allogenic mixed lympho-
`cyte reactions (MLR) (2) and interleukin-2 (IL-2)-
`dependent proliferation (4) of the mouse cell line
`CTLL-2 in a dose-dependent manner. The drug’s
`potency is 10- to 100-fold greater than that of cyclo-
`sporine (CsA). Unlike ISP-1, exogenous addition of
`FTY720 in doses up to 1000 nM does not inhibit
`MLR proliferation, or IL-2 production by antigen- or
`mitogen-stimulated T cells (5).
`
`Correspondence: Barry D. Kahan, Ph.D., M.D., Univer-
`sity of Texas Medical School at Houston, Department of
`Surgery, Division of Organ Transplantation, 6431 Fannin,
`Suite 6.240, Houston, TX 77030. E-mail bkahan@orgtx71.
`med.uth.tmc.edu
`Manuscript received January 9, 1998; received and
`accepted April 17, 1998.
`
`Autoimmune and inflammatory models
`
`FTY720 administered to mice at doses greater
`than 0.03 mg/kg/d has been shown to inhibit induc-
`tion of delayed-type hypersensitivity responses by
`human albumin in a dose-dependent manner. Using
`a model of adjuvant arthritis in rats, researchers
`showed that low doses (0.1 mg/kg) of the drug
`completely inhibited joint destruction and paw
`edema. In the same study, FTY720 also effectively
`ameliorated T cell-mediated autoimmune responses
`in rat models of collagen-induced arthritis and aller-
`gic encephalomyelitis (6).
`
`Transplant models
`
`ISP-1 has been shown to reduce the number of
`plaque-forming cells generated in response to sheep
`red blood cells, and to reduce the frequency of alloreac-
`tive cytotoxic T lymphocytes (1). It has been shown
`that FTY720 remarkably prolongs the survival of skin,
`heart, or liver allografts in MHC-incompatible and
`-compatible rat strain combinations in dose-dependent
`fashion over the range of 0.1 to 10 mg/kg (Table 1)
`(7,8). FTY720 potentiated the immunosuppressive ef-
`fects of subtherapeutic doses of CsA (5,7,9) and/or
`rapamycin (10). In addition, administration of FTY720
`(5 mg/kg) to rats on days 3 and 4 post-grafting signif-
`icantly prolonged allograft survival, suggesting that
`the drug can reverse ongoing rejection (9). In a model
`of graft-versus-host disease in rats,
`low doses of
`FTY720 (0.1 to 0.3 mg/kg) induced long-lasting unre-
`sponsiveness (11). In addition, 10 mg/kg doses of
`FTY720 delayed acute rejection after canine kidney
`transplantation (9,12,13) and potentiated the effects of
`subtherapeutic doses of CsA (7,9). Finally, a brief
`pretransplant 2-day course of FTY720 prolonged rat
`liver and kidney dog allograft survival (9). Our own
`preliminary results show that FTY720 doses of 0.3 and
`1 mg/kg prolong monkey kidney transplant survival.
`Remarkably, the drug has shown no toxic effects in
`
`CLINICAL BIOCHEMISTRY, VOLUME 31, JULY 1998
`
`369
`
`Apotex v. Novartis
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`
`

`

`TRONCOSO AND KAHAN
`
`Figure 1 — Molecular structure of Myriocin (ISP-1) and FTY720.
`
`preclinical studies when used either alone or in com-
`bination with other immunosuppressive agents.
`Preliminary data suggest that trough blood level
`concentrations of unchanged drug display a linear
`correlation with dose (manufacturer information).
`In rats and dogs, FTY720 seems to be metabolized
`predominantly by oxidation to produce carboxylic
`acid derivates, primarily by T-oxidization at the
`terminal position of the side chain. None of the
`identified metabolites seems to display immunosup-
`pressive activity, suggesting that only the parent
`compound is active. Metabolites have been identi-
`fied in the urine and in the feces at ratios of 40 to
`50% and 20 to 50%, respectively. Under the rigorous
`median effect analysis, FTY720 displays a high
`degree of synergism with CsA and rapamycin ad-
`ministered either separately or in combination (10).
`
`Mechanism of action
`
`IN VITRO
`
`High doses of FTY720 inhibit the proliferation of
`human mononuclear cells in response to stimulation
`with phytohemagglutinin (PHA) or anti-CD3 mono-
`clonal antibody (OKT3) (10). The inhibitory activity
`of ISP-1 on (CTLL-2) apparently depends upon in-
`hibition of the activity of serine palmitoyl tranferase
`
`(4), the enzyme that catalyzes the first step in the
`biosynthetic pathway of mammalian sphingolipids:
`namely, the condensation of serine and palmitoyl
`CoA into ketodihydrosphingosine. The sphingolipid
`pathway has been associated with various steps in
`signal transduction, differentiation, and apoptosis
`(14). In theory, FTY720 might also act on the sphin-
`golipid during the early events of T cell activation.
`In vitro, however, FTY720 has no inhibitory effect on
`serine palmitoyl tranferase (4). Unlike CsA or ta-
`crolimus, FTY720 has not been shown to inhibit the
`production of IL-2 or the induction of IL-2 mRNA
`expression by alloantigen (5). Therefore, FTY720
`must act on a distinctive pathway.
`Colorimetric assays suggested a dose-dependent
`reduction in cell viability, and genomic DNA analy-
`ses suggested apoptosis when human lymphocytes
`were incubated with FTY720 (15). Addition of
`FTY720 (2 to 10 3 106 M) to rat spleen cells
`produced an increased percentage of stained dead
`cells. On electron microscopy, these cells displayed
`features characteristic of apoptosis, including the
`absence of surface microvilli, condensation of chro-
`matin, and formation of apoptotic bodies (9). Aga-
`rose gel electrophoresis revealed fragmentation of
`chromosomal DNA. All of these phenomena were
`consistent with apoptotic cell death.
`Because FTY720 treatment produces a dose-de-
`
`370
`
`CLINICAL BIOCHEMISTRY, VOLUME 31, JULY 1998
`
`

`

`PRECLINICAL EVALUATION OF FTY720
`
`TABLE 1
`Effects of FTY720 on Graft Survival in Different Models of Animal Transplantation
`
`Species
`
`Type of graft
`
`Rat
`
`Skin
`
`Heart
`
`Liver
`
`Small bowel
`
`Heart
`
`Islet
`
`Kidney
`Kidney
`
`Mouse
`
`Dog
`Monkey
`
`FTY720
`(mg/kg/d)
`
`0.1
`1
`3
`0.1
`1
`10
`1
`2
`2
`8
`2
`8
`2
`4
`10
`0.1
`0.3
`1
`
`MST 6 SD
`
`23 6 2
`44 6 6
`59 6 10
`20.0 6 4.5
`22.3 6 5.7
`72.2 6 38.2
`57.6 6 29.8
`74.4 6 35.1
`12.2 6 2.3
`23.5 6 4
`11.4 6 2.8
`20 6 3.7
`58.6 6 28.8
`71.7 6 28.4
`27.7 6 7.6
`70.7 6 34.5
`67 6 15.9
`50 6 25.5
`
`% Above
`control
`
`Reference
`
`155
`388
`555
`78
`83
`545
`350
`481
`15
`122
`39
`143
`113
`406
`147
`732
`688
`488
`
`(3)
`
`(7)
`
`(10)
`
`(8)
`
`(13)
`(a)
`
`a Stepkowski S, unpublished data, 1998.
`
`pendent reduction of cell viability of Fas-negative
`mutant
`thymocytes, FTY720-induced apoptosis
`must occur via a mechanism distinct from Fas-
`mediated cell death (16). A study suggested that
`human lymphocytes treated with FTY720 displayed
`a reduced ratio of Bcl-2 to Bax (15,17), thereby
`changing the intracellular ratio of the Ced-9 related
`protein products of a multigene family of cell death
`regulators. This change favors the generation of
`apoptotic stimuli leading to cell death (18). In addi-
`tion, Jurkat lymphoma cells transfected with Bcl-2
`genes were shown to be resistant to FTY720.
`
`IN VIVO
`
`Some in vivo evidence from our unpublished ex-
`periments is not consistent with the hypothesis that
`an effect of FTY720 to cause apoptotic cell death is
`the mechanism of prolonged graft survival. First,
`thymidine deoxynucleotide kinase staining of the
`lymph node lymphocytes from rats that displayed
`unresponsiveness toward heterotopic heart trans-
`plants showed the same frequency of apoptotic ele-
`ments as those from normal hosts. Second, the
`frequency of specific memory cytotoxic T cells on
`limiting dilution analysis was also the same as in
`naı¨ve animals; there was no evidence of deletion of
`alloreactive lymphocyte clones. A similar response
`was observed in animals rendered tolerant by com-
`bined treatment with FTY720 and allochimeric class
`I MHC antigens (19). In contrast, a study showed
`that FTY720 therapy was highly effective in induc-
`ing lymphocyte apoptosis in vivo (16). Finally, high-
`dose FTY720 treatment did not block the capacity of
`alloreactive cells to mediate accelerated rejection in
`pre-sensitized hosts. In order to analyze the in vivo
`effects of FTY720, methodologies must be applied to
`
`detect specific apoptotic events induced in lympho-
`cytes and other immune cells by a variety of physi-
`ological stimuli, including glucocorticoids, antigen
`receptor engagement, tumor necrosis factor, or an-
`tibodies to the APO-1/Fas surface antigen (20). In
`order to elucidate the mechanism of in vivo action of
`FTY720, the relative significance of altered lympho-
`cyte circulation compared to that of augmented
`lymphocyte apoptosis and other mechanisms must
`be discerned.
`Studies have consistently shown that the immu-
`nosuppressive effects of FTY720 are related to a
`decrease in the number of circulating lymphocytes
`(5,9). In vivo administration of FTY720 to normal
`rats rapidly reduces the number of peripheral blood
`lymphocytes to less than 3% of the control value
`during the first 3 days of treatment (21) without
`altering either the total number or the percentage of
`lymphocytes of various subpopulations in the thy-
`mus, spleen, or lymph nodes (9,21). The number of
`peripheral blood lymphocytes
`significantly de-
`creased within 3 to 6 h after oral administration of
`FTY720, while the number of polymorphonuclear
`leukocytes is increased. The number of monocytes
`was not affected. In vivo, the number of CD41 T cells
`seems to be sensitive to FTY720, while B cells are
`resistant to its effect (9,21).
`treatment with
`It has been suggested that
`FTY720 may increase the expression, affinity, or
`avidity of a4-integrin (VLA-4) expression based upon
`the effect of pre-incubation of treated lymphocytes
`with an anti-VLA-4 monoclonal antibody to prevent
`the lymphodepletion effect of FTY720. If FTY720
`alters VLA-4 expression, it would “home” lympho-
`cytes to high endothelial venules in lymph nodes
`and Peyer’s patches and thus away from the allo-
`graft.
`
`CLINICAL BIOCHEMISTRY, VOLUME 31, JULY 1998
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`371
`
`

`

`Pharmacokinetics
`
`Acknowledgement
`
`TRONCOSO AND KAHAN
`
`low molecular weight
`A stable compound of
`(343.94 daltons), FTY720 (C19H33NO2; HCl) is solu-
`ble in water and ethanol. The oral bioavailability of
`FTY720 exceeds 60% in dogs, 80% in rats, and 40%
`in subhuman primates. It is more highly distributed
`in blood cells than in plasma, with ratios in rats or
`dogs of 7.8 to 17.6 or 4.0 to 8.0, respectively, follow-
`ing oral administration of 3 mg/kg FTY720. The
`ratios after intravenous administration of 1 mg/kg to
`rats and dogs are 5.3 to 20.8 and 4.8 to 8.3. The
`nature and extent of binding of FTY720 to plasma
`proteins is unclear. After administration of 3 mg/kg
`FTY720 orally to rats or dogs, the drug reaches a
`maximum concentration at 8 or 9 hours, respec-
`tively, and shows an elimination half-life (t1/2) of
`unchanged drug in the blood of 12 and 29 h, respec-
`tively, after oral dosing, and 12 and 25 h, respec-
`tively, after intravenous administration (Murakami,
`unpublished data, 1997).
`Concomitant administration of CsA and FTY720
`to animals does not appear to affect the blood con-
`centrations of either drug (9).
`
`Toxicity
`
`In rats, the LD50 value after a single oral admin-
`istration is 300 to 600 mg/kg. No deaths were re-
`ported in dogs that received doses of up to 200
`mg/kg. Vomiting, diarrhea, and anorexia were the
`most conspicuous pre-morbid symptoms. Autopsies
`on animals treated with supralethal doses showed
`gastric ulceration, vacuolation of medullary cells in
`the adrenal and peripheral nerves, and mononuclear
`cell infiltration into the brain and around the spinal
`vessels. Upon chronic administration of FTY720 to
`animals for 4 to 6 months, the lethal dose was more
`than 10 mg/kg. No toxic effects were observed at
`doses of 3 mg/kg in monkeys or 0.3 mg/kg in rats.
`Although FTY720 seems to increase fetal mortal-
`ity in rats, there has not been a study of mutagenic-
`ity, fertility, or embryo toxicity. Unlike CsA and
`tacrolimus, FTY720 does not produce renal, hepatic,
`pancreatic, or bone marrow toxicities or an in-
`creased incidence of neoplasms in animals.
`
`Summary
`
`FTY720 is a synthetic analog of a fungal metabo-
`lite that shows potent immunosuppressive activity
`in vitro and in vivo with little apparent toxicity. The
`drug displays marked synergistic effects in vivo with
`CsA and/or rapamycin. Therefore, this drug may
`improve the therapeutic window of agents that tar-
`get cytokine synthesis or signal transduction. Be-
`cause of these promising findings, the agent is likely
`to be tested in humans as an adjunct to clinical
`immunosuppressive regimens.
`
`This work was supported by a grant from the National
`Institute of Diabetes and Digestive and Kidney Diseases
`(NIDDK 38016-11).
`
`References
`
`1. Fujita T, Inoue K, Yamamoto S, et al. Fungal metab-
`olites. Part 11. A potent immunosuppressive activity
`found in Isaria sinclairii metabolite. J Antibiotics
`1994; 47: 208–15.
`2. Fujita T, Yoneta M, Hirose R, et al. Simple com-
`pounds, 2-alkyl-2-amino-1,3-propanediols have potent
`immunosuppressive activity. Bio Med Chem Lett
`1995; 5: 847–52.
`3. Adachi K, Kahora T, Nakao N, et al. Design, synthe-
`sis, and structure-activity relationships of 2-substitut-
`ed-2-amino-1,3-propanediols: discovery of a novel im-
`munosuppressant, FTY720. Bio Med Chem Lett 1995;
`5: 853–6.
`4. Miyake Y, Kozutsumi Y, Nakamura S, Fujita T, Ka-
`wasaki T. Serine palmitoyltransferase is the primary
`target of a sphingosine-like immunosuppressant, ISP-
`I/Myriocin. Biochem Biophys Research Comm 1995;
`211: 396–403.
`5. Chiba K, Hoshino Y, Suzuki C, et al. FTY720, a novel
`immunosuppressant, processing unique mechanisms.
`I. Prolongation of skin allograft survival and synergis-
`tic effect in combination with cyclosporine in rats.
`Transplant Proc 1996; 28: 1056–9.
`6. Teshima K, Imayoshi T, Matsura M, Yamaguchi S,
`Chiba K, Terasawa M. FTY720, a novel immunosup-
`pressant, possessing unique mechanisms. III. Phar-
`macological activities in several autoimmune and in-
`flammatory models. Abstracts of the 9th International
`Congress of Immunology. San Francisco: 1995; 5127.
`7. Hoshino Y, Suzuki M, Ohtsuki M, Masubuchi Y,
`Amano Y, Chiba K. FTY720, a novel immunosuppres-
`sant possessing unique mechanisms. II. Long-term
`graft survival induction in rat heterotopic cardiac
`allografts and synergistic effect in combination with
`cyclosporine A. Transplant Proc 1996; 28: 1060–1.
`8. Katz SM, Stepkowski S, Pham T, Wang M-E, Kahan
`BD. Long-term murine islet allograft survival with
`FTY720. ASTP 16th Annual Meeting, Chicago. 1997.
`9. Suzuki S, Enosawa S, Kakefuda T, et al. A novel
`immunosuppressant, FTY720, with a unique mecha-
`nism of action, induces long-term graft acceptance in
`rat and dog allotransplantation. Transplantation
`1996; 61: 200–5.
`10. Wang ME, Tegpal N, Qu X, et al. Immunosuppressive
`effects of FTY720 alone or in combination with cyclo-
`sporine or sirolimus. Transplantation 1998 65: 899–
`905.
`11. Masubuchi Y, Kawaguchi T, Ohtsuki M, et al.
`FTY720, a novel
`immunosuppressant, possessing
`unique mechanisms. IV. Prevention of graft versus
`host rejection in rats. Transplant Proc 1996; 28:
`1064–5.
`12. Kawaguchi T, Hoshino Y, Rahman F, et al. FTY720, a
`novel immunosuppressant, possessing unique mecha-
`nisms. III. Synergistic prolongation of canine renal
`allograft survival in combination with cyclosporine A.
`Transplant Proc 1996; 28: 1062–3.
`13. Suzuki S, Enosawa S, Kakefuda T, Amemiya H,
`Hoshino Y, Chiba K. Long-term graft acceptance in
`allografted rats and dogs by treatment with a novel
`
`372
`
`CLINICAL BIOCHEMISTRY, VOLUME 31, JULY 1998
`
`

`

`PRECLINICAL EVALUATION OF FTY720
`
`immunosuppressant, FTY720. Transplant Proc 1996;
`28: 1375–6.
`14. Hale AJ, Smith CA, Sutherland LC, et al. Apoptosis:
`molecular regulation of cell death. Eur J Biochem
`1996; 236: 1–26.
`15. Li XK, Shinomiya T, Enosawa S, Kakefuda T,
`Amemiya H. Induction of lymphocyte apoptosis by a
`novel
`immunosuppressant, FTY720: Relation with
`FAS, Bcl-2 and Bax expression. Transplant Proc 1997;
`29: 1267–8.
`16. Suzuki S, Li XK, Shinomiya T, et al. The in vivo
`induction of
`lymphocyte apoptosis in MRL-lpr/lpr
`mice treated with FTY720. Clin Exp Immunol 1997;
`107: 103–11.
`17. Suzuki S, Li XK, Shinomiya T, et al. Induction of
`lymphocyte apoptosis and prolongation of allograft sur-
`vival by FTY720. Transplant Proc 1996; 28: 2049–50.
`
`18. McDonnell T, Beham A, Sarkiss M, Andersen M, Lo P.
`Importance of the Bcl-2 family in cell death regula-
`tion. Experientia 1996; 52: 1008–17.
`19. Chueh S-C, Tian L, Wang M-E, Stepkowski S, Kahan
`BD. Induction of tolerance toward heterotopic cardiac
`transplantation in rats by treatment with allochi-
`meric class I antigen and FTY720. ASTP 16th Annual
`Meeting, Chicago 1997; 678.
`20. Orrenius S. Apoptosis: molecular mechanisms and
`implications for human disease. J Inter Med 1995;
`237: 529–36.
`21. Enosawa S, Suzuki S, Kakefuda T, Li XK, Amemiya
`H. Induction of selective cell death targeting on ma-
`ture T-lymphocytes in rats by a novel immunosup-
`pressant, FTY720.
`Immunopharmacol 1996; 34:
`171–9.
`
`CLINICAL BIOCHEMISTRY, VOLUME 31, JULY 1998
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