Pharmacology & Therapeutics 108 (2005) 308 – 319
`
`Associate editor: M. Endoh
`
`www.elsevier.com/locate/pharmthera
`
`FTY720, a new class of immunomodulator, inhibits lymphocyte egress
`from secondary lymphoid tissues and thymus by agonistic activity at
`sphingosine 1-phosphate receptors
`
`Research Laboratory III (Immunology), Pharmaceuticals Research Unit, Research and Development Division, Mitsubishi Pharma Corporation, Japan
`
`Kenji Chiba*
`
`Abstract
`
`FTY720 is the first of a new immunomodulator class: sphingosine 1-phosphate (S1P) receptor agonist. In 1994, an immunosuppressive
`natural product, ISP-I (myriocin), was isolated from the culture broth of Isaria sinclairii, a type of vegetative wasp. The chemical
`modification of ISP-I yielded a new compound, FTY720, which has more potent immunosuppressive activity and less toxicity than ISP-I
`does. FTY720 has been shown to be highly effective in experimental allotransplantation models and autoimmune disease models. A striking
`feature of FTY720 is the induction of a marked decrease in peripheral blood T- and B-cells at doses that show immunosuppressive activity in
`these models. Reportedly, FTY720 is rapidly converted to FTY720-phosphate (FTY720-P) by sphingosine kinase 2 in vivo, and FTY720-P
`acts as a potent agonist at S1P receptors. Recently, it has been suggested that FTY720-P internalizes S1P1 on lymphocytes and thereby
`inhibits the migration of lymphocytes toward S1P. Thus, it is likely that the reduction of circulating lymphocytes by FTY720 is due to the
`inhibition of S1P/S1P1-dependent lymphocyte egress from secondary lymphoid tissues and thymus. Because FTY720 displays a novel
`mechanism of action that has not been observed with other immunosuppressive agents and shows a synergism with cyclosporin A (CsA) and
`tacrolimus, it is presumed that FTY720 provides a useful tool for the prevention of transplant rejection and a new therapeutic approach for
`autoimmune diseases including multiple sclerosis and rheumatoid arthritis.
`D 2005 Elsevier Inc. All rights reserved.
`
`Keywords: FTY720; Sphingosine 1-phosphate; S1P1; Immunosuppression; Lymphocyte egress
`
`Abbreviations: AZ, azathioprine; CI, combination index; CsA, cyclosporin A; EAE, experimental autoimmune encephalomyelitis; FK506, tacrolimus;
`FTY720, 2-amino-2-[2-(4-octylphenyl)ethyl]propane-1,3-diol hydrochloride; FTY720-P, FTY720-phosphate; GvHR, graft versus host reaction; IL-2,
`interleukin 2; IFN-g, interferon-g; MHC, major histocompatibility complex antigen; MMF, mycophenolate mofetil; MST, median survival time; S1P,
`sphingosine 1-phosphate; S1P1, sphingosine 1-phosphate receptor type 1; (S)-FTY720-P, (S)-enantiomer of FTY720-phosphate.
`
`Contents
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`1.
`2.
`3.
`4.
`5.
`6.
`7.
`8.
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`Introduction .
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`Effect of FTY720 in experimental allograft models .
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`Synergistic effect of FTY720 in combination with calcineurin inhibitors .
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`Effect of FTY720 on graft versus host disease models .
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`Effect of FTY720 on experimental autoimmune disease models.
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`FTY720 sequesters circulating lymphocytes into secondary lymphoid tissues .
`FTY720 decreases T-cell infiltration into inflammatory sites .
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`FTY720-phosphate converted from FTY720 acts as an agonist at S1P
`receptors .
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`* 1000, Kamoshida-cho, Aoba-ku, Yokohama 227-0033, Japan. Tel.: +81 45 963 4527; fax: +81 45 963 3977.
`E-mail address: Chiba.Kenji@mk.m-pharma.co.jp.
`
`0163-7258/$ - see front matter D 2005 Elsevier Inc. All rights reserved.
`doi:10.1016/j.pharmthera.2005.05.002
`
`APOTEX - EXHIBIT 1022
`
`

`

`K. Chiba / Pharmacology & Therapeutics 108 (2005) 308 – 319
`
`309
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`9.
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`FTY720-phosphate down-regulates sphingosine 1-phosphate receptor type 1
`and inhibits lymphocyte egress from secondary lymphoid tissues and thymus.
`10. Clinical trails of FTY720.
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`11. Conclusion .
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`References .
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`315
`317
`317
`317
`
`1. Introduction
`
`It has been previously reported that a potent immunosup-
`pressive natural product, ISP-I (myriocin), and its derivative,
`mycestericins, can be isolated from a culture broth of Isaria
`sinclairii, a kind of vegetative wasp that is an ‘‘eternal youth’’
`nostrum in traditional Chinese herbal medicine (Fujita et al.,
`1994a, 1994b; Sasaki et al., 1994). The chemical modifica-
`tion of ISP-I led to a novel synthetic compound, 2-amino-2-
`[2-(4-octylphenyl)ethyl]propane-1,3-diol hydrochloride
`(FTY720), which has more potent
`immunosuppressive
`activity and less toxicity than ISP-I (Adachi et al., 1995;
`Fujita et al., 1995, 1996; Hirose et al., 1996; Kiuchi et al.,
`2000). FTY720, at 0.1 mg/kg or higher doses, significantly
`prolongs skin or cardiac allograft survival and host survival in
`lethal graft versus host reaction (GvHR) in rats (Chiba et al.,
`1996; Hoshino et al., 1996; Masubuchi et al., 1996; Chiba &
`Adachi, 1997). In addition, combination treatment with
`FTY720 and a subtherapeutic dose of cyclosporin A (CsA)
`or tacrolimus (FK506) results in a synergistic effect on canine
`renal allograft as well as rat skin or cardiac allografts (Chiba
`et al., 1996; Hoshino et al., 1996, 1999; Kawaguchi et al.,
`
`O
`
`HO
`
`H2N
`
`COOH
`OH
`
`ISP-I (myriocin)
`
`OH
`
`OH
`
`H2N
`
`OH
`
`
`
`HCl
`
`FTY720
`
`OH
`
`H2N
`
`O
`
`OH
`OH
`
`P
`O
`
`H2N
`
`OH
`
`H2N
`
`OH
`
`OH
`
`O
`
`OH
`OH
`
`P
`O
`
`FTY720-P
`
`Sphingosine
`
`S1P
`
`Fig. 1. The chemical structures of ISP-I, FTY720, FTY720-P, sphingosine,
`and S1P.
`
`1996; Suzuki et al., 1996a, 1996b; Chiba & Adachi, 1997;
`Yanagawa et al., 1998a, 1998b). A striking feature of
`FTY720 is the induction of a marked decrease in the number
`of peripheral blood lymphocytes, especially T-cells, at doses
`that prolong allograft survival (Chiba et al., 1996, 1998;
`Hoshino et al., 1996). FTY720 does not impair lymphocyte
`function, including T-cell activation, but instead induces the
`sequestration of circulating mature lymphocytes into the
`secondary lymphoid tissues and decreases T-cell infiltration
`into grafted organs (Chiba et al., 1998, 1999; Yanagawa et al.,
`1998a, 1998b; Brinkmann et al., 2000). FTY720, unlike ISP-
`I, does not inhibit serine-palmitoyl-transferase (Fujita et al.,
`1996), the first enzyme in sphingolipid biosynthesis, but both
`molecules are structurally similar to sphingosine. Recently, it
`has been reported that FTY720 is effectively phosphorylated
`by sphingosine kinase 2 and that FTY720-phosphate
`(FTY720-P) is a high affinity agonist for sphingosine 1-
`phosphate (S1P) receptors (Brinkmann et al., 2002; Mandala
`et al., 2002; Paugh et al., 2003). Fig. 1 shows the chemical
`structures of ISP-1, FTY720, FTY720-P, sphingosine, and
`S1P. Moreover,
`it has been suggested that FTY720-P
`internalizes S1P receptor type 1 (S1P1) on lymphocytes and
`inhibits S1P/S1P1-dependent lymphocyte egress from sec-
`ondary lymphoid tissues and thymus (Matloubian et al.,
`2004; Lo et al., 2005). This paper summarizes the current
`understanding of the pharmacological actions and the
`mechanism of action of FTY720.
`
`2. Effect of FTY720 in experimental allograft models
`
`FTY720 has been shown to be highly effective in
`prolonging allograft survival
`in various experimental
`allotransplantation models (Chiba & Adachi, 1997; Brink-
`mann et al., 2000). To clarify the efficacy and potency of the
`immunosuppressive activity of FTY720,
`the prolonging
`effect of FTY720, CsA, FK506, mycophenolate mofetil
`(MMF), and azathioprine (AZ) on rat skin allograft survival
`was compared in major histocompatibility complex antigen
`(MHC)-incompatible rat strains of WKAH donors and F344
`recipients (Chiba et al., 1996, 1998, 2005; Fig. 2A). The
`immunosuppressants were administered orally for 14 days
`from the day of the transplantation. FTY720 at 0.03 mg/kg
`or higher doses significantly prolongs allograft survival in a
`dose-dependent manner. CsA and FK506 are also effective
`at doses of 3 mg/kg or more and 0.3 mg/kg or more,
`respectively, in this model. MMF and AZ show a marginal
`immunosuppressive effect only at high doses, and all
`
`

`

`310
`
`K. Chiba / Pharmacology & Therapeutics 108 (2005) 308 – 319
`
`LEW F344
`
`B
`
`FTY720
`CsA
`FK506
`MMF
`AZ
`
`*
`
`*
`
`*
`
`*
`
`*
`*
`
`*
`
`*
`
`*
`
`*
`
`*
`
`*
`Control
`
`*
`
`*
`
`*
`
`45
`45
`
`40
`40
`
`35
`35
`
`30
`30
`
`25
`25
`
`20
`20
`
`15
`15
`
`10
`10
`
`05
`05
`
`WKAH
`
`F344
`*
`
`A
`
`*
`
`*
`
`*
`*
`
`*
`
`*
`
`*
`*
`
`*
`
`*
`
`*
`
`*
`
`*
`
`*
`
`*
`*
`Control
`
`
`
`3030
`
`
`
`2525
`
`
`
`2020
`
`
`
`1515
`
`10
`10
`
`05
`05
`
`Mean survival time (Day)
`
`0.01
`
`10
`1
`0.1
`Dose (mg/kg p.o.)
`
`100
`
`0.01
`
`10
`1
`0.1
`Dose (mg/kg p.o.)
`
`100
`
`Fig. 2. Dose – response relationship between FTY720, CsA, FK506, MMF, and AZ and skin allograft survival in MHC-incompatible and MHC-compatible rat
`strain systems. (A) MHC-incompatible rat skin allograft was performed using WKAH rats (RT1k) as donors and F344 rats (RT1lv1) as recipients. (B) MHC-
`compatible rat skin allograft was performed using LEW rats (RT1l) as donors and F344 rats (RT1lv1) as recipients. Full-thickness skin grafts (2.0 2.0 cm2)
`from donor rats were transplanted to the lateral thorax of the recipient rats. The grafts were inspected daily until rejection, which was defined as more than 90%
`necrosis of the graft epithelium. FTY720, CsA, FK506, MMF, and AZ were orally administered to the grafted recipients for 14 days after the transplantation.
`T
`Each symbol represents the mean
`SE of the 8 animals. The statistical differences in allograft survival time compared with the vehicle-treated control group
`were calculated by generalized Wilcoxon test with Hommel’s multiple comparison test (*P < 0.05).
`
`MMF with CsA on acute rejection in rat skin allograft
`models (Chiba et al., 1996, 1998, 2005; Hoshino et al.,
`1999). As shown in Table 1, the combination therapy of
`FTY720 with CsA or FK506 has a marked prolonging effect
`on allograft survival as compared with the monotherapy of
`FTY720, CsA, or FK506. The concomitant effect of
`
`Table 1
`Concomitant effect of FTY720 and calcineurin inhibitors in rat skin
`allograft
`
`Treatment
`
`animals died in a group given AZ at 100 mg/kg. In MHC-
`compatible rat strains of LEW donor and F344 recipient,
`data are similar (Fig. 2B; Chiba et al., 1996, 1999, 2005).
`The effect of FTY720 on heterotopic cardiac allograft
`survival was compared with those of CsA and FK506 in
`MHC-incompatible rat strains of WKAH donors and ACI
`recipients (Hoshino et al., 1996, 1999). All cardiac allografts
`in control group are rejected within 14 days after the
`transplantation: median survival time (MST) is 12.0 days.
`Treatment with FTY720 at 0.1 mg/kg p.o. or higher doses for
`14 days significantly prolonged the cardiac allograft survival
`in a dose-dependent manner. The MST of FTY720 admin-
`istration with 0.1, 0.3, 1, 3, and 10 mg/kg for 14 days is 20.0,
`21.0, 25.5, 29.5, and 58.5 days, respectively. FTY720 at 10
`mg/kg induces long-term graft survival for more than 100
`days in 3 out of 8 recipient rats. CsA and FK506 also
`significantly prolong the cardiac allograft survival at doses of
`10 mg/kg or more and 1 mg/kg or more, respectively. These
`results indicate that FTY720 possesses more potent immu-
`nosuppressive activity than do other immunosuppressive
`drugs on graft rejection in rat allograft models.
`
`3. Synergistic effect of FTY720 in
`combination with calcineurin inhibitors
`
`In clinical organ transplantations, CsA- or FK506-based
`combination therapy with prednisolone or other immuno-
`suppressants is widely used to reduce the side effects of
`individual drugs. Therefore, it is important to investigate
`whether combined use of both FTY720 and CsA or FK506
`produces a synergistic effect on experimental allograft
`models. We evaluated the concomitant effect of FTY720
`with CsA or FK506 in comparison with those of AZ or
`
`WKAH to F344 control
`(vehicle)
`FTY720, 0.1 mg/kg p.o. 10.5
`FTY720, 1 mg/kg p.o.
`19.4
`CsA, 3 mg/kg p.o.
`8.4
`FTY720, 0.1 mg/kg p.o.
`20.8
`+ CsA, 3 mg/kg p.o.
`FK506, 1 mg/kg p.o.
`FTY720, 0.1 mg/kg p.o.
`+ FK506, 1 mg/kg
`AZ, 30 mg/kg p.o.
`AZ, 30 mg/kg p.o.
`+ CsA, 3 mg/kg p.o.
`MMF, 100 mg/kg p.o.
`MMF, 100 mg/kg p.o.
`+ CsA, 3 mg/kg p.o.
`LEW to F344 control
`(vehicle)
`FTY720, 0.1 mg/kg p.o. 18.5
`CsA, 3 mg/kg p.o.
`11.4
`FTY720, 0.1 mg/kg p.o.
`35.1
`+ CsA, 3 mg/kg p.o.
`
`Mean survival time P value
`T
`
`6.6
`
`0.2
`
`T
`T
`T
`T
`
`T
`T
`
`T
`T
`
`T
`T
`
`T
`
`T
`T
`T
`
`0.3
`0.4
`0.2
`2.0
`
`0.3
`2.8
`
`0.2
`0.3
`
`0.6
`0.8
`
`0.3
`
`0.7
`0.3
`2.4
`
`< 0.05 vs. control
`< 0.05 vs. control
`< 0.05 vs. control
`< 0.05 vs. CsA alone
`
`< 0.05 vs. control
`< 0.05 vs. FK506 alone
`
`< 0.05 vs. control
`< 0.05 vs. CsA alone
`
`< 0.05 vs. control
`< 0.05 vs. CsA alone
`
`< 0.05 vs. control
`< 0.05 vs. control
`< 0.05 vs. CsA alone
`
`11.3
`28.8
`
`9.6
`10.8
`
`14.6
`16.4
`
`8.8
`
`Rat skin allograft was performed between WKAH or LEW donors and
`F344 recipients. FTY720, CsA, FK506, AZ, and MMF were administered
`orally for 14 days from the day of transplantation. The results were
`T
`expressed as mean
`SE of 8 animals, and statistical significance was
`calculated by generalized Wilcoxon test.
`
`

`

`K. Chiba / Pharmacology & Therapeutics 108 (2005) 308 – 319
`
`311
`
`FTY720 with CsA or FK506 is stronger than that of AZ
`with CsA or MMF with CsA. To clarify the concomitant
`effects,
`the combination index (CI) values of
`these
`combination therapy groups were calculated according to
`the method of median effect analysis (Hoshino et al., 1999;
`Chiba et al., 2005). Since the CI values are less than 0.2 in
`the concomitant groups with FTY720 and CsA or FK506, it
`confirms that the combination therapy with FTY720 and
`CsA or FK506, exerts a synergistic effect. On the other
`hand, the concomitant treatment of AZ and CsA or MMF
`and CsA shows only an additive effect, because the CI
`values of these groups are 0.9 to 1.1. In MHC-compatible
`rat strains of LEW donors and F344 recipients, FTY720 at
`an oral dose of 0.1 mg/kg significantly prolongs the
`allograft survival and shows a synergistic effect in combi-
`nation with CsA at 3 mg/kg (Table 1).
`In rat heterotopic cardiac allograft model using WKAH
`donors and ACI recipients, the combination therapy with
`FTY720 and CsA or FK506 shows a more marked
`prolonging effect compared with that in concomitant treat-
`ment with AZ and CsA (Table 2; Hoshino et al., 1996, 1999;
`Chiba et al., 2005). The CI values are less than 0.2 in the
`concomitant group with FTY720 and CsA or FK506,
`indicating a synergistic effect, whereas those in the group
`with AZ plus CsA are 0.5 to 0.9.
`Canine renal allograft survival is significantly prolonged
`by combination therapy of FTY720 at 0.03 and 1 mg/kg
`with CsA at 10 mg/kg compared with the monotherapy with
`FTY720 or CsA (Table 3; Kawaguchi et al., 1996; Suzuki et
`al., 1998,;Chiba et al., 2005). With combination treatment of
`FTY720 and CsA, there is no severe toxicity in kidney and
`liver functions and the blood concentration of FTY720 or
`CsA did not affect each other. On the contrary, there is no
`clear effect
`in the group given AZ at 1 mg/kg in
`combination with CsA at 10 mg/kg compared with the
`CsA monotherapy. The combination therapy with AZ at 2.5
`mg/kg and CsA at 10 mg/kg has a significant prolonging
`
`Table 2
`Concomitant effect of FTY720 and calcineurin inhibitors in rat heterotopic
`cardiac allograft
`
`Mean survival time P value
`T
`T
`T
`T
`
`11.3
`30.6
`26.4
`63.7
`
`0.8
`10.9
`11.4
`14.7
`
`< 0.05 vs. control
`< 0.05 vs. control
`< 0.05 vs. CsA alone
`
`Treatment
`
`Control (vehicle)
`FTY720, 0.1 mg/kg p.o.
`CsA, 3 mg/kg p.o.
`FTY720, 0.1 mg/kg p.o.
`+ CsA, 3 mg/kg p.o.
`FK506, 1 mg/kg p.o.
`FTY720, 0.1 mg/kg
`+ FK506, 1 mg/kg p.o.
`AZ, 10 mg/kg p.o.
`AZ, 10 mg/kg p.o.
`+ CsA, 3 mg/kg p.o.
`
`20.2
`47.9
`
`12.3
`36.1
`
`T
`T
`
`T
`T
`
`0.8
`12.2
`
`0.4
`11.4
`
`< 0.05 vs. control
`< 0.05 vs. FK506 alone
`
`NS vs. control
`NS vs. CsA alone
`
`Rat heterotopic cardiac allograft was performed between WKAH donors
`and ACI recipients. FTY720, CsA, FK506, and AZ were administered
`orally for 14 days from the day of transplantation. The results were
`T
`expressed as mean
`SE of 8 animals, and statistical significance was
`calculated by generalized Wilcoxon test. NS: not significant.
`
`Table 3
`Concomitant effect of FTY720 and CsA in canine renal allograft
`
`Treatment
`
`Control (vehicle)
`FTY720, 3 mg/kg p.o.
`CsA, 10 mg/kg p.o.
`FTY720, 0.03 mg/kg p.o.
`+ CsA, 10 mg/kg p.o.
`FTY720, 1 mg/kg p.o.
`+ CsA, 10 mg/kg p.o.
`AZ, 1 mg/kg p.o.
`+ CsA, 10 mg/kg p.o.
`AZ, 2.5 mg/kg p.o.
`+ CsA, 10 mg/kg p.o.
`
`Mean survival time
`T
`T
`T
`T
`
`7.3
`11.7
`10.2
`59.6
`
`0.4
`1.9
`1.0
`11.8
`
`T
`
`T
`
`T
`
`59.8
`
`11.5
`
`37.3
`
`13.2
`
`0.7
`
`16.4
`
`P value
`
`NS vs. control
`NS vs. control
`< 0.05 vs. CsA alone
`
`< 0.05 vs. CsA alone
`
`NS vs. CsA alone
`
`< 0.05 vs. CsA alone
`
`Canine renal allograft was performed between mongrel donors and beagle
`recipients. FTY720 and AZ were orally administered from 1 day before
`transplantation and CsA orally from the day of transplantation. These
`agents were given daily until the death of the recipient dogs. The results
`T
`were expressed as mean
`SE of 5 to 8 animals, and statistical significance
`was calculated by generalized Wilcoxon test. NS: not significant.
`
`effect on renal allograft survival; however, 2 dogs died
`during the administration period without showing increased
`serum creatinine and blood urea nitrogen, suggesting AZ
`toxicity. Moreover, it was previously reported that FTY720,
`in combination with a subtherapeutic dose of CsA, displays
`a synergistic effect on the prolongation of renal allograft
`survival in cynomolgus monkeys (Troncoso et al., 1999).
`From these results, it is presumed that the combination
`therapy with FTY720 and calcineurin inhibitors provides a
`more beneficial
`tool
`for human organ transplantation
`compared with the conventional combination therapies,
`including calcineurin inhibitors plus AZ or MMF.
`
`4. Effect of FTY720 on graft versus host disease models
`
`When spleen cells from LEW rats are injected into the
`foot pad of (LEW BN)F1 rats,
`local graft versus host
`reaction (GvHR) is induced and the popliteal lymph node
`increases to its maximum weight after 7 days. FTY720
`significantly inhibits the popliteal lymph node enlargement
`at doses of 0.1 mg/kg or more in a dose-dependent manner
`(Masubuchi et al., 1996). To examine the effect of FTY720
`in preventing lethal GvHR, splenic lymphocytes from LEW
`donor rats were injected intravenously into cyclophospha-
`mide-pretreated (LEW BN)F1 recipients. In the control
`group, all rats develop severe GvHR-associated symptoms,
`including redness of skin and hair loss, within 15 days after
`the injection of LEW spleen cells and die with a MST of
`22.0 days (Masubuchi et al., 1996). The oral administration
`of FTY720 at 0.1 mg/kg p.o. for 30 days prevents the
`development of GvHR-associated symptoms and prolongs
`host survival significantly (MST: 50.0 days). Treatment with
`FTY720 at a dose of 0.3 mg/kg induces survival for more
`than 60 days in 4 out of 5 rats without GvHR-associated
`symptoms. Thus, FTY720 induces long-lasting unrespon-
`siveness by treatment with low doses (0.1 to 0.3 mg/kg) in a
`
`

`

`312
`
`K. Chiba / Pharmacology & Therapeutics 108 (2005) 308 – 319
`
`lethal GvHR model. Similar results are obtained in
`rat
`mouse lethal GvHR model using C57BL/6 donors and
`(C57BL/6 DBA/2)F1 recipients (Kataoka et al., 2005).
`
`5. Effect of FTY720 on
`experimental autoimmune disease models
`
`FTY720 at 0.1 mg/kg p.o. or higher doses almost
`completely prevents paralysis in experimental autoimmune
`encephalomyelitis (EAE) induced by myelin basic protein in
`LEW rats (Teshima et al., 1995; Chiba & Adachi, 1997;
`Fujino et al., 2003). Therapeutic treatment with FTY720
`inhibits EAE relapse induced by myelin proteolipid protein
`immunization in SJL mice (Brinkmann et al., 2002; Kataoka
`et al., 2004). The therapeutic potential of FTY720 is more
`markedly compared with recombinant mouse interferon-h
`(rm-IFN-h; Fig. 3A) and the area of demyelination and the
`infiltration of CD4+ T-cells into the spinal cord are reduced
`by FTY720 treatment (Kataoka et al., 2004). In the same
`dose range, FTY720 almost completely inhibits joint
`destruction as well as paw edema in adjuvant arthritis and
`type II collagen-induced arthritis in LEW rats (Chiba &
`
`Adachi, 1997; Matsuura et al., 2000a, 2000b). Moreover,
`FTY720 shows a marked prophylactic and therapeutic effect
`on lupus nephritis in autoimmune MRL/lpr mice (Okazaki
`et al., 2002; Sugahara et al., 2004). Fig. 3B shows the
`therapeutic effect of FTY720 on proteinuria in aged MRL/
`lpr mice with lupus nephritis. With only 4 weeks of
`FTY720 treatment at low doses, long-term improvement of
`lupus nephritis is observed in this autoimmune model.
`Moreover, therapeutic FTY720 administration decreases the
`number of CD4+ Th1 cells infiltrated into lupus kidney.
`Based on these findings, it is presumed that FTY720 can
`provide a new approach not only for the prevention of
`transplant rejection but also for the therapy of autoimmune
`diseases including multiple sclerosis, rheumatoid arthritis,
`and systemic lupus erythematosus.
`
`6. FTY720 sequesters circulating
`lymphocytes into secondary lymphoid tissues
`
`A striking feature of FTY720 is the induction of a
`marked decrease in the number of peripheral blood
`lymphocytes (lymphopenia) at doses that display an
`
`A
`
`EAE in SJL mice
`
`EAE control
`IFN-β β 10000 U
`FTY720 0.3 mg/kg
`FTY720 1 mg/kg
`
`#
`
`0
`
`*
`*
`*
`*
`*
`*
`*
`14
`12
`10
`8
`6
`4
`2
`Days after therapeutic treatment
`
`*
`
`*
`
`*
`16
`
`Lupus nephritis in MRL/lpr mice
`
`B
`
`**
`* **
`
`*
`
`*
`
`MRL/lpr control
`FTY720 0.03 mg/kg
`FTY720 0.1 mg/kg
`FTY720 0.3 mg/kg
`FTY720 1 mg/kg
`
`*
`**
`
`*
`
`**
`**
`
`**
`
`**
`
`**
`****
`**
`**
`**
`
`**
`**
`
`**
`
`**
`**
`
`****
`
`
`
`1616
`
`**
`**
`
`****
`
`2020
`
`3.0
`
`2.5
`
`2.0
`
`1.5
`
`1.0
`
`0.5
`
`0.0
`
`Clinical disease score
`
`of EAE (mean± SE)
`
`2.75
`2.75
`2.50
`2.50
`2.25
`2.25
`2.00
`2.00
`1.75
`1.75
`1.50
`1.50
`1.25
`1.25
`1.00
`1.00
`0.75
`0.75
`
`Score of proteinuria
`
`(mean ± SE)
`
`
`
`2424
`
`
`
`2828
`
`
`
`3232
`
`
`3636
`
`
`4040
`4444
`Age (weeks)
`
`
`
`4848
`
`Administration of FTY720
`
`Fig. 3. Therapeutic effect of FTY720 on EAE in SJL mice and lupus nephritis in MRL/lpr mice. (A) EAE was induced by the immunization of myelin
`proteolipid protein and Freund’s complete adjuvant in SJL mice. EAE-developed mice were pooled and evaluated for the relapse of EAE. FTY720 was orally
`T
`administered daily and rm-IFN-h at 10,000 IU was given intraperitoneally 3 times a week to EAE-developed mice. Each symbol represents the mean
`SE of 6
`animals. Statistical significance was calculated by nonparametric Dunnett’s test (FTY720 vs. control, *P < 0.05) and Mann – Whitney U test (rm-IFN-h vs.
`control, #P < 0.05). (B) FTY720 was orally administrated to 16-week-old, proteinuria-developed MRL/lpr mice for 4 weeks, and the urinary protein levels were
`T
`assessed weekly. Each symbol represents the mean
`SE of 10 animals. The statistical significance was calculated by nonparametric Dunnett’s test as compared
`with the control (*P < 0.05, **P < 0.01).
`
`

`

`K. Chiba / Pharmacology & Therapeutics 108 (2005) 308 – 319
`
`313
`
`0
`
`50
`
`100
`
`150
`
`200
`
`T cells
`B cells
`Other cells
`
`FTY720
`(mg/kg)
`-
`0.1
`
`1-0
`
`.1
`
`1
`-
`0.1
`
`1-0
`
`.1
`
`1
`
`-
`0.1
`1
`
`A
`
`Blood
`
`Thoracic duct
`
`Spleen
`
`Lymph nodes
`
`Peyer’s patches
`
`immunosuppressive effect in experimental allograft models
`and autoimmune disease models. In rats, the number of
`lymphocytes (T-cells and B-cells)
`in peripheral blood
`decreases dramatically within 6 hr after oral administration
`of FTY720 at 0.1 to 1 mg/kg (Chiba et al., 1996, 1998,
`1999). In particular,
`the reduction in T-cell numbers is
`remarkable. Fig. 4 shows the relationship between the
`number of peripheral blood lymphocytes and blood con-
`centration after a single oral administration of FTY720 in
`rats. FTY720-induced lymphopenia is highly dependent on
`the blood concentration of FTY720. In mice, dogs, and
`cynomolgus monkeys, marked lymphopenia is also induced
`by FTY720 administration (Chiba & Adachi, 1997; Suzuki
`et al., 1998; Li et al., 2002; Yagi et al., 2000). Reportedly,
`FTY720 at 4 AM (1.4 Ag/mL) or more induces apoptosis of
`rat spleen cells and human peripheral blood cells; however,
`the blood concentration range of FTY720 is lower than 100
`ng/mL when given to rats at 0.1 to 1 mg/kg (Fig. 4). Thus,
`the hypothesis concerning FTY720-induced apoptosis is
`insufficient
`to explain the intrinsic mechanism of
`the
`decreasing effect on peripheral blood lymphocyte number
`by FTY720, because it is clearly impossible for FTY720 to
`induce apoptotic cell death of lymphocytes at a dose range
`of 0.1 to 1 mg/kg in vivo (Suzuki et al., 1996a, 1996b).
`The immunologically mature lymphocytes are known to
`continuously recirculate in the blood, spleen, lymph nodes,
`Payer’s patches, and lymphatic vessels. To clarify the
`mechanism of FTY720-induced lymphopenia, the lympho-
`
`A
`
`**
`
`**
`**
`
`**
`
`**
`
`**
`
`**
`
`24
`
`**
`
`**
`
`**
`
`**
`
`
`
`00
`
`B
`
`*
`
`**
`
`**
`
`**
`
`48
`
`72
`
`96
`
`120
`
`144
`
`168
`
`Control (Vehicle)
`FTY720 0.1 mg/kg p.o.
`FTY720 1 mg/kg p.o.
`
`
`
`00
`
`24
`
`48
`
`72
`
`96
`
`120
`
`144
`
`168
`
`Hours after administration
`
`5000
`5000
`
`4000
`4000
`
`3000
`3000
`
`2000
`2000
`
`1000
`1000
`
`
`
`00
`
`
`
`100100
`
`
`
`1010
`
`1
`
`0.1
`
`Number of peripheral blood
`
`lymphocytes (cells/µl)
`
`of FTY720 (ng/ml)
`
`Blood concentration
`
`Fig. 4. The relationship between the number of peripheral blood
`lymphocytes and blood concentration after a single oral administration of
`FTY720 in rats. (A) The number of peripheral blood lymphocytes was
`T
`determined by flow cytometry. Each symbol represents the mean
`SE of 6
`animals. The statistical significance was calculated by Dunnett’s test as
`compared with the control
`(*P < 0.05, **P < 0.01).
`(B) The blood
`concentration of FTY720 was measured by gas chromatography-mass
`spectrometry.
`
`0
`50
`100
`150
`200
`Number of cells (% of control)
`
`B
`
`0
`
`50
`
`100
`
`150
`
`200
`
`250
`
`300
`
`Lymph nodes
`
`Peyer’s patches
`
`Spleen
`
`Blood
`
`**
`
`**
`
`**
`
`**
`
`0.1 mg/kg
`1 mg/kg
`
`0
`
`250
`200
`150
`100
`50
`Number of fluorescein-labeled
`lymphocytes (% of control)
`
`300
`
`Fig. 5. Effect of FTY720 on the distribution of lymphocytes in blood and
`lymphoid tissues in rats. (A) The number of T-cells and B-cells in the
`blood, thoracic duct lymph, spleen, lymph nodes, and Peyer’s patches in
`F344 rats was determined by flow cytometry using fluorescein-isothio-
`cyanide (FITC)-conjugated antirat CD3 and phycoerythrin-conjugated
`antirat CD45RA/B monoclonal antibodies, 12 hr after a single oral
`administration of FTY720. Each column represents the mean of 4 animals.
`(B) The fluorescein-labeled lymphocytes (5 107 cells) were transfused
`intravenously into F344 rats 2.5 hr after
`the oral administration of
`FTY720. The numbers of fluorescein-labeled lymphocytes in the blood,
`spleen,
`lymph nodes, and Peyer’s patches were determined by flow
`T
`cytometry. Each column represents the mean
`SE of 4 animals. Statistical
`significance was calculated by Dunnett’s test (**P < 0.01 vs. vehicle-
`treated control group).
`
`cyte distribution in blood, lymph, and various lymphoid
`tissues were analyzed after FTY720 administration in rats
`(Chiba et al., 1998; Yanagawa et al., 1998b; Chiba et al.,
`1999). Within 3 to 24 hr after a single oral administration
`of FTY720 at 0.1 to 1 mg/kg, the number of lymphocytes
`in rats decreased markedly in the peripheral blood, as well
`as in thoracic duct
`lymph, and partially in spleen. In
`contrast, at the same time, the number of lymphocytes in
`peripheral
`lymph nodes, mesenteric lymph nodes, and
`Peyer’s patches increases significantly (Fig. 5A). Intra-
`venous transfusion of fluorescein-labeled rat lymphocytes
`into rats reveals that the labeled lymphocytes accumulate
`in lymph nodes and Peyer’s patches with FTY720
`administration (Fig. 5B). These data suggest
`that
`FTY720-induced sequestration of circulating mature lym-
`
`

`

`314
`
`K. Chiba / Pharmacology & Therapeutics 108 (2005) 308 – 319
`
`phocytes into secondary lymphoid tissues, such as lymph
`nodes and Peyer’s patches, decreases the number of
`lymphocytes in peripheral blood,
`thoracic duct
`lymph,
`and spleen. Thus, the sequestration of circulating mature
`lymphocytes is presumed to be the main mechanism of
`FTY720 immunosuppressive activity.
`Moreover, FTY720 reportedly inhibits mature thymo-
`cyte emigration from the thymus to the periphery in mice
`(Yagi et al., 2000).
`In the thymus,
`long-term daily
`FTY720 administration causes a 3- to 4-fold increase in
`the population of mature medullary thymocytes
`
`
`(CD4+CD8
`CD8+) as well as a slight decrease
`and CD4
`in the double-positive immature thymocyte (CD4+CD8+)
`ratio. An intrathymic fluorescein-labeling technique con-
`firms that only 1/4 of the labeled cells are detected in the
`lymph nodes and in the spleen of FTY720-treated mice
`compared with the control mice. These results suggest that
`the immunosuppressive activity of FTY720, at
`least
`in
`part, could be due to its
`inhibitory effect on the
`emigration of mature thymocytes
`from the thymic
`medullar to the periphery.
`
`7. FTY720 decreases T-cell
`infiltration into inflammatory sites
`
`It is well known that calcineurin inhibitors, CsA and
`FK506, inhibit Th1-associated cytokines such as interleu-
`kin 2 (IL-2) and interferon-g (IFN-g)
`in alloantigen-
`stimulated T-cells. Unlike calcineurin inhibitors, FTY720
`up to 1000 nM does not affect T-cell proliferation and Th1-
`associated cytokine production induced by antigen stim-
`ulation (Chiba et al., 1996, 1998; Brinkmann et al., 2000).
`To elucidate the mechanism of the synergistic effect of
`FTY720 in combination with CsA, we analyzed mRNA
`expression of IL-2 and IFN-g and that of CD3, which
`reflects T-cell infiltration, in rat skin allograft (Yanagawa et
`al., 1998a). In the skin allograft, mRNA levels of IL-2,
`
`IFN-g, and CD3 increase, peaking on days 4 to 5 after
`transplantation. CsA at 10 mg/kg significantly inhibits the
`elevation of IL-2 and IFN-g mRNA. On the contrary,
`FTY720 at 0.1 mg/kg markedly inhibits the elevation of
`CD3 mRNA, while slightly inhibiting those of IL-2 and
`IFN-g mRNA. FTY720 combined with CsA almost
`completely suppresses the intragraft expression of mRNA
`for IL-2, IFN-g, and CD3 (Fig. 6). Immunohistochemical
`staining and flow cytometric analysis also confirmed that
`FTY720 decreases T-cell
`infiltration into the allograft
`(Yanagawa et al., 1998a, 1999, 2000). These findings
`suggest that unlike calcineurin inhibitors, FTY720 prolongs
`allograft survival by decreasing the T-cell infiltration into
`grafts but not Th1-associated cytokine production.
`In
`several autoimmune disease models, the reduction of T-
`cell infiltration into inflammatory sites is observed with
`FTY720 treatment (Teshima et al., 1995; Fujino et al.,
`2003; Kataoka et al., 2004; Sugahara et al., 2004). It is
`highly probable that the decreasing effect of FTY720 on T-
`cell infiltration into inflammatory sites is due to reduction
`in the number of circulating T-cells by sequestration of
`lymphocytes into secondary lymphoid tissues. Thus, it is
`presumed that the synergistic effect of FTY720 combined
`with calcineurin inhibitors on prolongation of allograft
`survival
`is based on the respective inhibitions of T-cell
`infiltration and cytokine production in allografts.
`
`8. FTY720-phosphate converted from
`FTY720 acts as an agonist at S1P receptors
`
`Two reports demonstrated that a phosphorylated form of
`FTY720 acts as an agonist at S1P receptors (Brinkmann et
`al., 2002; Mandala et al., 2002). S1P, a pleiotropic
`lysophospholipid mediator,
`is converted primarily by
`phosphorylation of sphingosine by sphingosine kinase 1
`and stimulates multiple signaling pathways, resulting in
`calcium mobilization from intracellular stores, polymer-
`
`IL-2
`
`IFN-γγ
`
`CD3
`
`HPRT
`
`Control
`
`FTY720 0.1 mg/kg
`
`CsA 10 mg/kg
`
`FTY720 0.1 mg/kg
`+ CsA 10 mg/kg
`
`4 5 6 7
`
`4 5 6 76
`
`4 5 6 7
`
`4 5 6 76 7
`
`Days after transplantation
`
`Fig. 6. Effects of FTY720, CsA, and both on IL-2, IFN-g, and CD3 mRNA levels in rat skin allografts between WKAH donors and F344 recipients. FTY720 at
`0.1 mg/kg and CsA at 10 mg/kg was given orally for consecutive days following transplantation. Levels of IL-2, IFN- g, and CD3 mRNA in rat skin allografts
`of the recipients treated with FTY720, CsA, or both were analyzed using the polymerase chain reaction method. As a control, those of vehicle-treated recipients
`were also analyzed.
`
`

`

`K. Chiba / Pharmacology & Therapeutics 108 (2005) 308 – 319
`
`315
`
`ization of actin, chemotaxis/migration, and escape from
`apoptosis (Pyne & Pyne, 2000; Hla et al., 2001). S1P is
`released by platelets during inflammatory processes and is
`found in significant amount (100 to 400 nM) in the serum
`(Kimura et al., 2001). S1P binds with nanomolar (nM)
`affinities to 5 related G-protein-coupled receptors (GPCRs),
`termed S1P1 – 5 (formerly Edg-1, -5, -3, -6, and-8). It is
`clear that FTY720 is a substrate for recombinant