当前位置：文档库 › FTY720 review 2005 phamocology

FTY720 review 2005 phamocology

Associate editor:M.Endoh

FTY720,a new class of

immunomodulator,inhibits lymphocyte egress from secondary lymphoid tissues and thymus by agonistic activity at

sphingosine 1-phosphate receptors

Kenji Chiba *

Research Laboratory III (Immunology),Pharmaceuticals Research Unit,Research and Development Division,Mitsubishi Pharma Corporation,Japan

Abstract

FTY720is 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.FTY720has been shown to be highly effective in experimental allotransplantation models and autoimmune disease models.A striking feature of FTY720is the induction of a marked decrease in peripheral blood T-and B-cells at doses that show immunosuppressive activity in these models.Reportedly,FTY720is rapidly converted to FTY720-phosphate (FTY720-P)by sphingosine kinase 2in vivo,and FTY720-P acts as a potent agonist at S1P receptors.Recently,it has been suggested that FTY720-P internalizes S1P 1on lymphocytes and thereby inhibits the migration of lymphocytes toward S1P.Thus,it is likely that the reduction of circulating lymphocytes by FTY720is due to the inhibition of S1P/S1P 1-dependent lymphocyte egress from secondary lymphoid tissues and thymus.Because FTY720displays 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 FTY720provides a useful tool for the prevention of transplant rejection and a new therapeutic approach for autoimmune diseases including multiple sclerosis and rheumatoid arthritis.D 2005Elsevier Inc.All rights reserved.

Keywords:FTY720;Sphingosine 1-phosphate;S1P 1;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;S1P 1,sphingosine 1-phosphate receptor type 1;(S )-FTY720-P,(S )-enantiomer of FTY720-phosphate.

Contents 1.Introduction ........................................3092.Effect of FTY720in experimental allograft models ....................3093.Synergistic effect of FTY720in combination with calcineurin inhibitors .........3104.Effect of FTY720on graft versus host disease models ..................3115.Effect of FTY720on experimental autoimmune disease models..............3126.FTY720sequesters circulating lymphocytes into secondary lymphoid tissues.......3127.FTY720decreases T-cell infiltration into inflammatory sites ...............

3148.

FTY720-phosphate converted from FTY720acts as an agonist at S1P receptors ....................................

......

314

*1000,Kamoshida-cho,Aoba-ku,Yokohama 227-0033,Japan.Tel.:+81459634527;fax:+81459633977.E-mail address:Chiba.Kenji@mk.m-pharma.co.jp.

Pharmacology &Therapeutics 108(2005)308–319

https://www.wendangku.net/doc/947447238.html,/locate/pharmthera

FTY720-phosphate down-regulates sphingosine 1-phosphate receptor type 1and inhibits lymphocyte egress from secondary lymphoid tissues and thymus.......31510.Clinical trails of FTY720..................................31711.Conclusion .........................................317References ......................................

(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 0.1mg/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 FTY720and 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.,

1996;Suzuki et al.,1996a,1996b;Chiba &Adachi,1997;Yanagawa et al.,1998a,1998b ).A striking feature of FTY720is 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).FTY720does 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 FTY720is effectively phosphorylated by sphingosine kinase 2and 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.1shows 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(S1P 1)on lymphocytes and inhibits S1P/S1P 1-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 FTY720in experimental allograft models FTY720has 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 F344recipients (Chiba et al.,1996,1998,2005;Fig.2A).The immunosuppressants were administered orally for 14days from the day of the transplantation.FTY720at 0.03mg/kg or higher doses significantly prolongs allograft survival in a dose-dependent manner.CsA and FK506are also effective at doses of 3mg/kg or more and 0.3mg/kg or more,respectively,in this model.MMF and AZ show a marginal immunosuppressive effect only at high doses,and all

O P

OH O

H 2N

OH FTY720

FTY720-P

Sphingosine

O P

OH O

OH OH

HCl

Fig.1.The chemical structures of ISP-I,FTY720,FTY720-P,sphingosine,and S1P.

K.Chiba /Pharmacology &Therapeutics 108(2005)308–319309

animals died in a group given AZ at 100mg/kg.In MHC-compatible rat strains of LEW donor and F344recipient,data are similar (Fig.2B;Chiba et al.,1996,1999,2005).The effect of FTY720on heterotopic cardiac allograft survival was compared with those of CsA and FK506in MHC-incompatible rat strains of WKAH donors and ACI recipients (Hoshino et al.,1996,1999).All cardiac allografts in control group are rejected within 14days after the transplantation:median survival time (MST)is 12.0days.Treatment with FTY720at 0.1mg/kg p.o.or higher doses for 14days significantly prolonged the cardiac allograft survival in a dose-dependent manner.The MST of FTY720admin-istration with 0.1,0.3,1,3,and 10mg/kg for 14days is 20.0,21.0,25.5,29.5,and 58.5days,respectively.FTY720at 10mg/kg induces long-term graft survival for more than 100days in 3out of 8recipient rats.CsA and FK506also significantly prolong the cardiac allograft survival at doses of 10mg/kg or more and 1mg/kg or more,respectively.These results indicate that FTY720possesses more potent immu-nosuppressive activity than do other immunosuppressive drugs on graft rejection in rat allograft models.

3.Synergistic effect of FTY720in

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 FTY720and CsA or FK506produces a synergistic effect on experimental allograft models.We evaluated the concomitant effect of FTY720with CsA or FK506in comparison with those of AZ or

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 FTY720with CsA or FK506has a marked prolonging effect on allograft survival as compared with the monotherapy of FTY720,CsA,or FK506.The concomitant effect of

51015202530

FTY720CsA FK506MMF AZ

M e a n s u r v i v a l t i m e (D a y )

Dose (mg/kg p.o.)0

51015202530354045

Dose (mg/kg p.o.)

Control

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 F344rats (RT1lv1)as recipients.(B )MHC-compatible rat skin allograft was performed using LEW rats (RT1l )as donors and F344rats (RT1lv1)as recipients.Full-thickness skin grafts (2.0?2.0cm 2)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 14days after the transplantation.Each symbol represents the mean T SE of the 8animals.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).

Table 1

Concomitant effect of FTY720and calcineurin inhibitors in rat skin allograft Treatment

Mean survival time P value WKAH to F344control (vehicle)

6.6T 0.2FTY720,0.1mg/kg p.o.10.5T 0.3<0.05vs.control FTY720,1mg/kg p.o.19.4T 0.4<0.05vs.control CsA,3mg/kg p.o.

8.4T 0.2<0.05vs.control FTY720,0.1mg/kg p.o.+CsA,3mg/kg p.o.20.8T 2.0<0.05

vs.

CsA alone

FK506,1mg/kg p.o.11.3T 0.3<0.05vs.control

FTY720,0.1mg/kg p.o.+FK506,1mg/kg 28.8T 2.8<0.05vs.FK506alone AZ,30mg/kg p.o.9.6T 0.2<0.05vs.control AZ,30mg/kg p.o.+CsA,3mg/kg p.o.10.8T 0.3<0.05vs.CsA alone MMF,100mg/kg p.o.14.6T 0.6<0.05vs.control MMF,100mg/kg p.o.+CsA,3mg/kg p.o.16.4T 0.8<0.05vs.CsA alone

LEW to F344control (vehicle)

8.8T 0.3FTY720,0.1mg/kg p.o.18.5T 0.7<0.05vs.control CsA,3mg/kg p.o.

11.4T 0.3<0.05vs.control FTY720,0.1mg/kg p.o.+CsA,3mg/kg p.o.

35.1T 2.4

<0.05vs.CsA alone Rat skin allograft was performed between WKAH or LEW donors and F344recipients.FTY720,CsA,FK506,AZ,and MMF were administered orally for 14days from the day of transplantation.The results were expressed as mean T SE of 8animals,and statistical significance was calculated by generalized Wilcoxon test.

K.Chiba /Pharmacology &Therapeutics 108(2005)308–319

310

FTY720with CsA or FK506is 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 than0.2in the concomitant groups with FTY720and CsA or FK506,it confirms that the combination therapy with FTY720and 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 are0.9to1.1.In MHC-compatible rat strains of LEW donors and F344recipients,FTY720at an oral dose of0.1mg/kg significantly prolongs the allograft survival and shows a synergistic effect in combi-nation with CsA at3mg/kg(Table1).

In rat heterotopic cardiac allograft model using WKAH donors and ACI recipients,the combination therapy with FTY720and CsA or FK506shows a more marked

prolonging effect compared with that in concomitant treat-ment with AZ and CsA(Table2;Hoshino et al.,1996,1999; Chiba et al.,2005).The CI values are less than0.2in the concomitant group with FTY720and CsA or FK506, indicating a synergistic effect,whereas those in the group with AZ plus CsA are0.5to0.9.

Canine renal allograft survival is significantly prolonged by combination therapy of FTY720at0.03and1mg/kg with CsA at10mg/kg compared with the monotherapy with FTY720or CsA(Table3;Kawaguchi et al.,1996;Suzuki et al.,1998,;Chiba et al.,2005).With combination treatment of FTY720and CsA,there is no severe toxicity in kidney and liver functions and the blood concentration of FTY720or CsA did not affect each other.On the contrary,there is no clear effect in the group given AZ at1mg/kg in combination with CsA at10mg/kg compared with the CsA monotherapy.The combination therapy with AZ at2.5 mg/kg and CsA at10mg/kg has a significant prolonging effect on renal allograft survival;however,2dogs 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 FTY720and 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 FTY720on graft versus host disease models

When spleen cells from LEW rats are injected into the foot pad of(LEW?BN)F1rats,local graft versus host reaction(GvHR)is induced and the popliteal lymph node increases to its maximum weight after7days.FTY720 significantly inhibits the popliteal lymph node enlargement at doses of0.1mg/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)F1recipients.In the control group,all rats develop severe GvHR-associated symptoms, including redness of skin and hair loss,within15days after the injection of LEW spleen cells and die with a MST of 22.0days(Masubuchi et al.,1996).The oral administration of FTY720at0.1mg/kg p.o.for30days prevents the development of GvHR-associated symptoms and prolongs host survival significantly(MST:50.0days).Treatment with FTY720at a dose of0.3mg/kg induces survival for more than60days in4out of5rats without GvHR-associated symptoms.Thus,FTY720induces long-lasting unrespon-siveness by treatment with low doses(0.1to0.3mg/kg)in a

Table2

Concomitant effect of FTY720and calcineurin inhibitors in rat heterotopic cardiac allograft

Treatment Mean survival time P value

Control(vehicle)11.3T0.8

FTY720,0.1mg/kg p.o.30.6T10.9<0.05vs.control CsA,3mg/kg p.o.26.4T11.4<0.05vs.control

FTY720,0.1mg/kg p.o.

+CsA,3mg/kg p.o.

63.7T14.7<0.05vs.CsA alone FK506,1mg/kg p.o.20.2T0.8<0.05vs.control

FTY720,0.1mg/kg

+FK506,1mg/kg p.o.

47.9T12.2<0.05vs.FK506alone AZ,10mg/kg p.o.12.3T0.4NS vs.control

AZ,10mg/kg p.o.

+CsA,3mg/kg p.o.

36.1T11.4NS vs.CsA alone

Rat heterotopic cardiac allograft was performed between WKAH donors and ACI recipients.FTY720,CsA,FK506,and AZ were administered orally for14days from the day of transplantation.The results were expressed as mean T SE of8animals,and statistical significance was calculated by generalized Wilcoxon test.NS:not significant.Table3

Concomitant effect of FTY720and CsA in canine renal allograft Treatment Mean survival time P value

Control(vehicle)7.3T0.4

FTY720,3mg/kg p.o.11.7T1.9NS vs.control CsA,10mg/kg p.o.10.2T1.0NS vs.control

FTY720,0.03mg/kg p.o.

+CsA,10mg/kg p.o.

59.6T11.8<0.05vs.CsA alone

FTY720,1mg/kg p.o.

+CsA,10mg/kg p.o.

59.8T13.2<0.05vs.CsA alone AZ,1mg/kg p.o.

+CsA,10mg/kg p.o.

11.5T0.7NS vs.CsA alone

AZ,2.5mg/kg p.o.

+CsA,10mg/kg p.o.

37.3T16.4<0.05vs.CsA alone

Canine renal allograft was performed between mongrel donors and beagle recipients.FTY720and AZ were orally administered from1day before transplantation and CsA orally from the day of transplantation.These agents were given daily until the death of the recipient dogs.The results were expressed as mean T SE of5to8animals,and statistical significance was calculated by generalized Wilcoxon test.NS:not significant.

K.Chiba/Pharmacology&Therapeutics108(2005)308–319311

rat lethal GvHR model.Similar results are obtained in mouse lethal GvHR model using C57BL/6donors and (C57BL/6?DBA/2)F 1recipients (Kataoka et al.,2005).5.Effect of FTY720on

experimental autoimmune disease models

FTY720at 0.1mg/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 FTY720inhibits EAE relapse induced by myelin proteolipid protein immunization in SJL mice (Brinkmann et al.,2002;Kataoka et al.,2004).The therapeutic potential of FTY720is 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 FTY720treatment (Kataoka et al.,2004).In the same dose range,FTY720almost 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,FTY720shows 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 FTY720on proteinuria in aged MRL/lpr mice with lupus nephritis.With only 4weeks of FTY720treatment at low doses,long-term improvement of lupus nephritis is observed in this autoimmune model.Moreover,therapeutic FTY720administration decreases the number of CD4+Th1cells infiltrated into lupus kidney.Based on these findings,it is presumed that FTY720can 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.FTY720sequesters circulating

lymphocytes into secondary lymphoid tissues

A striking feature of FTY720is the induction of a marked decrease in the number of peripheral blood lymphocytes (lymphopenia)at doses that display an

S c o r e o f p r o t e i n u r i a

(m e a n ± S E )

Age (weeks)

1620242832

36404448

0.75

1.001.251.501.75

2.002.252.50

2.75

Administration of FTY720

0.0

0.51.01.52.02.53.0

β 10000 U C l i n i c a l d i s e a s e s c o r e o f E A E (m e a n ± S E )

Days after therapeutic treatment

lpr control Fig.3.Therapeutic effect of FTY720on 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.FTY720was orally administered daily and rm-IFN-h at 10,000IU was given intraperitoneally 3times a week to EAE-developed mice.Each symbol represents the mean T SE of 6animals.Statistical significance was calculated by nonparametric Dunnett’s test (FTY720vs.control,*P <0.05)and Mann–Whitney U test (rm-IFN-h vs.control,#P <0.05).(B )FTY720was orally administrated to 16-week-old,proteinuria-developed MRL/lpr mice for 4weeks,and the urinary protein levels were assessed weekly.Each symbol represents the mean T SE of 10animals.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

312

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 6hr after oral administration of FTY720at 0.1to 1mg/kg (Chiba et al.,1996,1998,1999).In particular,the reduction in T-cell numbers is remarkable.Fig.4shows the relationship between the number of peripheral blood lymphocytes and blood con-centration after a single oral administration of FTY720in 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 FTY720administration (Chiba &Adachi,1997;Suzuki et al.,1998;Li et al.,2002;Yagi et al.,2000).Reportedly,FTY720at 4A M (1.4A g/mL)or more induces apoptosis of rat spleen cells and human peripheral blood cells;however,the blood concentration range of FTY720is lower than 100ng/mL when given to rats at 0.1to 1mg/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 FTY720to induce apoptotic cell death of lymphocytes at a dose range of 0.1to 1mg/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-cyte distribution in blood,lymph,and various lymphoid tissues were analyzed after FTY720administration in rats (Chiba et al.,1998;Yanagawa et al.,1998b;Chiba et al.,1999).Within 3to 24hr after a single oral administration of FTY720at 0.1to 1mg/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 FTY720administration (Fig.5B).These data suggest that FTY720-induced sequestration of circulating mature lym-

01000

20003000

4000

5000N u m b e r o f p e r i p h e r a l b l o o d l y m p h o c y t e s (c e l l s /μl )

Hours after administration

10.1

100B l o o d c o n c e n t r a t i o n o f F T Y 720 (n g /m l )

24487296120144168

0Fig. 4.The relationship between the number of peripheral blood lymphocytes and blood concentration after a single oral administration of FTY720in rats.(A )The number of peripheral blood lymphocytes was determined by flow cytometry.Each symbol represents the mean T SE of 6animals.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 FTY720was measured by gas chromatography-mass spectrometry.

50100150200

Number of cells (% of control)

Blood

Thoracic duct

Spleen

Lymph n odes Peyer’s patches -0.11-0.11-0.11-0.1

1-0.1

050100150200

FTY720(mg/kg)T cells B cells Other cells

Lymph nodes Peyer’s patches

Spleen Blood

50100150200250300

Number of fluorescein-labeled lymphocytes (% of control)

0100150200250300

50Fig.5.Effect of FTY720on 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 F344rats was determined by flow cytometry using fluorescein-isothio-cyanide (FITC)-conjugated antirat CD3and phycoerythrin-conjugated antirat CD45RA/B monoclonal antibodies,12hr after a single oral administration of FTY720.Each column represents the mean of 4animals.(B )The fluorescein-labeled lymphocytes (5?107cells)were transfused intravenously into F344rats 2.5hr 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 cytometry.Each column represents the mean T SE of 4animals.Statistical significance was calculated by Dunnett’s test (**P <0.01vs.vehicle-treated control group).

K.Chiba /Pharmacology &Therapeutics 108(2005)308–319313

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 FTY720immunosuppressive activity.

Moreover,FTY720reportedly inhibits mature thymo-cyte emigration from the thymus to the periphery in mice (Yagi et al.,2000).In the thymus,long-term daily FTY720administration causes a3-to4-fold increase in the population of mature medullary thymocytes (CD4+CD8àand CD4àCD8+)as well as a slight decrease in the double-positive immature thymocyte(CD4+CD8+) ratio.An intrathymic fluorescein-labeling technique con-firms that only1/4of 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.FTY720decreases T-cell

infiltration into inflammatory sites

It is well known that calcineurin inhibitors,CsA and FK506,inhibit Th1-associated cytokines such as interleu-kin2(IL-2)and interferon-g(IFN-g)in alloantigen-stimulated T-cells.Unlike calcineurin inhibitors,FTY720 up to1000nM 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 FTY720in combination with CsA,we analyzed mRNA expression of IL-2and 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 CD3increase,peaking on days4to5after transplantation.CsA at10mg/kg significantly inhibits the elevation of IL-2and IFN-g mRNA.On the contrary, FTY720at0.1mg/kg markedly inhibits the elevation of CD3mRNA,while slightly inhibiting those of IL-2and IFN-g mRNA.FTY720combined 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 FTY720decreases T-cell infiltration into the allograft (Yanagawa et al.,1998a,1999,2000).These findings suggest that unlike calcineurin inhibitors,FTY720prolongs 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 FTY720treatment(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 FTY720on 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 FTY720combined 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

FTY720acts as an agonist at S1P receptors

Two reports demonstrated that a phosphorylated form of FTY720acts 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 kinase1 and stimulates multiple signaling pathways,resulting in calcium mobilization from intracellular stores,

polymer-

FTY720 0.1 mg/kg

+ CsA 10 mg/kg

CsA 10 mg/kg

FTY720 0.1 mg/kg

Control

Days after transplantation

4567

IL-2IFN-γCD3HPRT

4567

45674567

Fig.6.Effects of FTY720,CsA,and both on IL-2,IFN-g,and CD3mRNA levels in rat skin allografts between WKAH donors and F344recipients.FTY720at 0.1mg/kg and CsA at10mg/kg was given orally for consecutive days following transplantation.Levels of IL-2,IFN-g,and CD3mRNA 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&Therapeutics108(2005)308–319

314

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 (100to 400nM)in the serum (Kimura et al.,2001).S1P binds with nanomolar (nM)affinities to 5related G-protein-coupled receptors (GPCRs),termed S1P 1–5(formerly Edg-1,-5,-3,-6,and-8).It is clear that FTY720is a substrate for recombinant sphingo-sine kinase 1a and is rapidly phosphorylated in vivo (Brinkmann &Lynch,2002;Brinkmann et al.,2002;Mandala et al.,2002).After oral or intravenous FTY720administration,the plasma concentration of FTY720-P was 2to 6times higher than FTY720and FTY720-P is a high affinity agonist at 4out of 5S1P receptors (Brinkmann et al.,2002;Mandala et al.,2002).

Recently,it has been reported that S1P 1is essential for lymphocyte recirculation and that S1P 1regulates lympho-cyte egress from thymus and secondary lymphoid tissues (Brinkmann et al.,2004;Matloubian et al.,2004;Lo et al.,2005).In mice whose hematopoietic cells lack a single S1P receptor,S1P 1,there are no T-cells in the periphery because mature T-cells are unable to exit the thymus and secondary lymphoid tissues (Matloubian et al.,2004).Moreover,S1P 1-dependent chemotactic responsiveness is strongly up-regu-lated in T-cell development before exit from the thymus,whereas S1P 1is down-regulated during peripheral lympho-cyte activation,and this is associated with retention in lymphoid tissues (Graler &Goetzl,2004;Matloubian et al.,2004).FTY720treatment down-regulates S1P 1,creating a temporary pharmacological S1P 1-null state in lymphocytes,providing an explanation for the mechanism of FTY720-induced lymphocyte sequestration.

9.FTY720-phosphate

down-regulates sphingosine 1-phosphate receptor type 1and inhibits lymphocyte

egress from secondary lymphoid tissues and thymus After administration to rats,FTY720is metabolized by omega-oxidation of the octyl side chain,and subsequent beta-oxidation,or phosphorylation to FTY720-phosphate (FTY720-P)by sphingosine kinase 2rather than sphingo-sine kinase 1(Paugh et al.,2003;Chiba et al.,2005).We then clarified the contribution of FTY720metabolites including (S )-enantiomer of FTY720-P ((S)-FTY720-P)to the induction of lymphopenia and immunosuppression by FTY720in vivo,because only (S )-FTY720-P was detected in blood from rats administered FTY720.We successfully established an effective method for asymmetric synthesis of both (S )-and (R )-enantiomers of FTY720-P with respective high eantio-selectivity and confirmed that (S )-FTY720-P binds to (S1P 1,3,4,5),but not S1P 2,at nanomolar concentrations (Kiuchi et al.,2005;Fig.7).On the contrary,the binding affinities of (R )-enantiomer of FTY720-P for S1P receptors are more than 100-fold weaker than those of (S )-FTY720-P.In addition,omega-and beta-oxidized 4metabolites (M1,M2,M3,and M4)as well as FTY720up to 10,000nM do not bind S1P receptors.In MHC-incompatible rat skin allograft with WKAH donors and F344recipients,(S )-FTY720-P at 0.1and 1mg/kg i.v.induces a marked lymphopenia and significantly prolonged the allograft survival (Chiba et al.,2005;Table 4).A similar effect of (S )-FTY720-P was observed in MHC-compatible rat skin allograft with LEW donors and F344recipients (Table 4).On the other hand,M1,M2,M3,and M4show

0.1110100

1000

S1P 1S1P 2S1P 3S1P 4S1P 5

S )-FTY720-P (nM)

Concentration of (0.1

100

1000

20406080100

% I n h i b i t i o n o f [32P ]S 1P B i n d i n g

Concentration of S1P (nM)020406080S1P

S )-FTY720-P

(

P OH O OH

S )-FTY720-P

(Fig.7.Binding affinities of S1P and (S )-FTY720-P for human S1P receptors were determined by inhibition of [32P]S1P to CHO cells stably expressing S1P receptors in the presence or absence of various concentration of S1P (A )or (S )-FTY720-P (B ).

K.Chiba /Pharmacology &Therapeutics 108(2005)308–319315

neither lymphopenia nor immunosuppression at an intra-venous dose of 10mg/kg in the rat skin allograft model.These results suggest that the lymphopenia and the immunosuppression induced by FTY720administration are due to the agonistic activity against S1P receptors of the active metabolite,(S )-FTY720-P.

We also confirmed that (S )-FTY720-P shows agonist activity for

S1P 1at nanomolar concentrations using extracellular signal regulated kinase 1/2(ERK1/2)phos-phorylation assay and subsequently induces long-term down-regulation of S1P 1in Chinese hamster ovary (CHO)cells stably expressing human S1P 1.The down-regulation of S1P 1by (S )-FTY720-P appeared to be maintained longer than that by S1P (Fig.8).Although both S1P and (S )-FTY720-P act as agonist at S1P 1,it is still unclear why (S )-FTY720-P can induce long-term down-regulation of S1P 1compared with S1P.Further investigations are necessary to clarify the different actions of (S )-FTY720-P and S1P in S1P 1down-regulation.As shown in Fig.9A and B,S1P at

concentrations of 10to 100nM induces migration of lymph node CD4+T-cells and CD-single positive thymocytes in mice.Moreover,(S )-FTY720-P effectively inhibits the migration of lymph node CD4+T-cells and CD-single positive thymocytes toward S1P at 100nM (Kiuchi et al.,2005;Fig.9C and D).Therefore,it is presumed that (S )-FTY720-P converted from FTY720acts as an agonist at

Fig.8.Effect of (S )-FTY720-P and S1P on the surface expression of S1P 1on transfected CHO cells.Hemagglutinin (HA)-tagged human S1P 1stably expressed in transfected CHO cells was visualized by FITC-conjugated anti-HA antibody under fluorescence microscopy.(A )Confocal microscopy of HA-S1P 1control;(B )(S )-FTY720-P 100nM for 30min;(C )(S )-FTY720-P 100nM for 8hr;(D )S1P at 100nM for 30min;(E )S1P at 100nM for 3hr.

1101001000

1.0

1.5

2.02.5

3.03.5--0.11

2.52.0

1.51.0

3.03.5-+

--0.11S1P (100 nM)

-+++

(S )-FTY720-P (nM)10M i g r a t i o n t o w a r d S 1P (% o f c o n t r o l )

-Lymph node CD4SP thymocytes

M i g r a t i o n t o w a r d S 1P (% o f c o n t r o l )

Fig.9.The migration of CD4+T-cells and CD4-single positive (SP)thymocytes toward S1P was inhibited by (S )-FTY720-P.Lymph node CD4T-cells or CD4SP thymocytes (5?105cells)were added to the upper wells of 5-A m pore polycarbonate tissue culture inserts with S1P dilution in bottom wells.Migration toward S1P was performed at 37-C for 180min,and migrated cells were counted by flow cytometry (A and B ).(S )-FTY720-P was added to the upper well just before the migration assay toward S1P at 100nM (C and D ).Each column represents the mean T SE of triplicate determination.Statistical significance was calculated by Dunnett’s test compared with control migration toward S1P (**P <0.01).

Egress of lymphocytes from secondary lymphoid

tissues and thymus Inhibition of

S1P/S1P 1-dependent lymphocyte egress

Fig.10.(S )-FTY720-P down-regulates S1P 1on lymphocytes and inhibits S1P/S1P 1-dependent lymphocyte egress from secondary lymphoid tissues and thymus.

Table 4

Effect of (S )-FTY720-P on skin allograft survival in rats Donor Recipient Treatment Mean survival time P value WKAH

F344

Control (vehicle) 6.8T 0.2(S )-FTY720-P,0.1mg/kg i.v.12.1T 0.4<0.05(S )-FTY720-P,1mg/kg i.v.15.2T 0.9<0.05

LEW F344

Control (vehicle)8.8T 0.3(S )-FTY720-P,0.1mg/kg i.v.24.3T 4.9<0.05(S )-FTY720-P,1mg/kg i.v.

31.0T 3.1

<0.05

Rat skin allograft was performed between WKAH or LEW donors and F344recipients.(S )-FTY720-P was administered intravenously for 14days from the day of transplantation.The results were expressed as mean T SE of 8animals,and the statistical significance was calculated as compared with vehicle-treated control group by generalized Wilcoxon test.

K.Chiba /Pharmacology &Therapeutics 108(2005)308–319

316

S1P1,induces the down-regulation of S1P1on lymphocytes, and shows immunosuppressive activity by inhibition of S1P/S1P1-dependent lymphocyte egress from secondary lymphoid tissues and thymic medullar(Figs.10and11).

10.Clinical trails of FTY720

Clinical trials of FTY20have been performed.In phase 1a study,the administration of single oral doses of FTY720,ranging from0.25to3.5mg/kg,to stable renal transplant patients maintained on a regimen of CsA and corticosteroid,causes a dose-dependent,although transient, reduction in peripheral blood T-cells and B-cells(Budde et al.,2002).At doses greater than1.0mg,the mean nadir counts are30%to60%below the baseline values.In addition,the coadministration of single FTY720doses does not affect CsA blood concentration.In phase1b study on pharmacodynamics,pharmacokinetics,and safety of multiple FTY720doses in stable renal transplant patients, FTY720at 1.0mg/day or greater significantly reduces peripheral blood lymphocyte count by up to85%,which reverses within3days after the discontinuation of the study medication(Budde et al.,2003;Kahan et al.,2003). Compared with placebo-treated patients,FTY720subjects show no major increase in adverse events or change in renal function.Pharmacokinetic measurements reveal that FTY720displays linear relationship between doses and concentrations over a wide range.

The phase2a,multicenter,open-label,dose-finding study was performed to evaluate the efficacy and safety of FTY720 compared with MMF in combination with CsA and cortico-steroid in de novo renal transplant patients(Tedesco-Silva et al.,2004).The incidence of biopsy-confirmed acute rejection is23.3%,34.9%,17.5%,and9.8%,respectively,with FTY720at doses of0.25,0.5,1.0,and2.5mg,versus 17.1%with MMF.Safety is comparable between the FTY720 and MMF groups.Thus,FTY720at2.5mg is as effective as MMF in combination with CsA for the prevention of acute rejection after renal transplantation.FTY720is well tolerated and not associated with the side effects commonly observed with immunosuppressant therapy.

11.Conclusion

Recent published data suggest that FTY720,after phosphorylation,acts as an agonist at the S1P1receptor, down-regulates S1P1on lymphocytes,and inhibits S1P/ S1P1-dependent lymphocyte egress from secondary lym-phoid tissues and thymus.Thus,FTY720causes the sequestration of circulating mature lymphocytes into lym-phoid tissues and modulates the recirculation of lympho-cytes between blood and lymphoid tissues.Consequently,it is presumed that FTY720decreases the trafficking and the infiltration of antigen-specific T-cells into grafted organs or inflammatory sites in autoimmune diseases,thereby exerting powerful immunosuppressive activity.Since FTY720pos-sesses a completely new mechanism of action,FTY720may be a useful tool for the prevention of transplant rejection and a new therapeutic approach for autoimmune diseases, including multiple sclerosis,rheumatoid arthritis,and systemic lupus erythematosus.

References

Adachi,K.,Kohara,T.,Nakao,N.,Arita,M.,Chiba,K.,Mishina,T.,et al.

(1995).Design,synthesis and structure–activity relationships of2-substituted-2-amino-1.3-propanediols:discovery of a novel immuno-suppressant,FTY720.Biomed Chem Lett5,853–856.

T cells

Fig.11.(S)-FTY720-P converted from FTY720inhibits lymphocyte egress from secondary lymphoid tissues and thymus.

K.Chiba/Pharmacology&Therapeutics108(2005)308–319317

Brinkmann,V.,&Lynch,K.R.(2002).FTY720:target G-protein-coupled receptors for sphingosine1-phosphate in transplantation and auto-immunity.Curr Opin Immunol14,569–575.

Brinkmann,V.,Pinschewer,D.,Chiba,K.,&Feng,L.(2000).FTY720:a novel transplantation drug that modulates lymphocyte traffic rather than activation.Trends Pharmacol Sci21,49–52.

Brinkmann,V.,Davis,M.D.,Heise,C.E.,Albert,R.,Cottens,S.,Hof,R., et al.(2002).The immune modulator FTY720targets sphingosine1-phosphate receptors.J Biol Chem277,21453–21457. Brinkmann,V.,Cyster,J.G.,&Hla,T.(2004).FTY720:sphingosine1-phosphate receptor-1in the control of lymphocyte egress and endotherial barrier function.Am J Transplant4,1019–1025. Budde,K.,Schmouder,R.L.,Brunkhorst,R.,Nashan,B.,Luker,P.W., Mayer,T.,et al.(2002).Fuman first trail of FTY720,a novel immunomodulator,in stable renal transplant patients.J Am Soc Nephrol 13,1073–1083.

Budde,K.,Schmouder,R.L.,Nashan,B.,Brunkhorst,R.,Luker,P.W., Mayer,T.,et al.(2003).Pharmacodynamics of single doses of the novel immunosuppressant FTY720in stable renal transplant patients.Am J Transplant3,846–854.

Chiba,K.,&Adachi,K.(1997).FTY720,immunosuppressant.Drugs Future22,18–22.

Chiba,K.,Hoshino,Y.,Suzuki,C.,Masubuchi,Y.,Yanagawa,Y.,Ohtsuki, M.,et al.(1996).FTY720,a novel immunosuppressant possessing unique mechanisms:I.Prolongation of skin allograft survival and synergistic effect in combination with cyclosporin A in rats.Transplant Proc28,1056–1059.

Chiba,K.,Yanagawa,Y.,Masubuchi,Y.,Kataoka,H.,Kawaguchi,T., Ohtsuki,M.,et al.(1998).FTY720,a novel immunosuppressant, induces sequestration of circulating mature-lymphocytes by acceleration of lymphocyte homing in rats:I.FTY720selectively decreases the number of circulating mature lymphocytes by acceleration of lympho-cyte homing.J Immunol160,5037–5044.

Chiba,K.,Yanagawa,Y.,Kataoka,H.,Kawaguchi,T.,Ohtsuki,M.,& Hoshino,Y.(1999).FTY720,a novel immunosuppressant,induces sequestration of circulating lymphocytes by acceleration of lymphocyte homing.Transplant Proc31,1230–1233.

Chiba,K.,Hoshino,Y.,Ohtsuki,M.,Kataoka,H.,Maeda,Y.,Matsuyuki, K.,et al.(2005).Immunosuppressive activity of FTY720,sphingosine 1-phosphate receptor agonist:I.Prevention of allograft rejection in rats and dogs by FTY720and FTY720-phosphate.Transplant Proc37, 102–106.

Fujino,M.,Funeshima,N.,Kitazawa,Y.,Kimura,H.,Amemiya,H., Suzuki,S.,et al.(2003).Amelioration of experimental autoimmune encephalomyelitis in Lewis rats by FTY720treatment.J Pharmacol Exp Ther305,70–77.

Fujita,T.,Inoue,K.,Yamamoto,S.,Ikumoto,T.,Sasaki,S.,Toyama,R., et al.(1994a).Fungal metabolites:Part11.A potent immunosup-pressive activity found in Isaria sinclairii metabolite.J Antibiotics47, 208–215.

Fujita,T.,Inoue,K.,Yamamoto,S.,Ikumoto,T.,Sasaki,S.,Toyama,R., et al.(1994b).Fungal metabolites:Part12.Potent immunosuppres-sant,14-deoxomyruiocin,(2S,3R,4R)-(E)-2-amino-3,4-dihydroxy-2-hydroxymethyleicos-6-enoic acid and structure–activity relationships of myriocin derivatives.J Antibiotics47,216–224.

Fujita,T.,Yoneta,M.,Hirose,R.,Sasaki,S.,Inoue,K.,Kiuchi,M.,et al.

(1995).Simple compounds,2-alkyl-2-amino-1,3-propane-diols have potent immunosuppressive activity.Biomed Chem Lett5,847–852. Fujita,T.,Hirose,R.,Yoneta,M.,Sasaki,S.,Inoue,K.,Kiuchi,M.,et al.

(1996).Potent immunosuppressants,2-alkyl-2-aminopropane-1,3-diols.J Med Chem39,4451–4459.

Graler,M.H.,&Goetzl,E.J.(2004).The immunosuppressant FTY720 down-regulates sphingosine1-phosphate G-protein-coupled receptors.

F ASEB J18,551–553.

Hirose,R.,Hamamichi,N.,Kitao,Y.,Matsuzaki,T.,Chiba,K.,&Fujita,T.

(1996).2-Aminoalcohol immunosuppressants:structure–activity rela-tionships.Biomed Chem Lett6,2647–2650.Hla,T.,Lee,M.J.,Ancellin,N.,Paik,J.H.,&Kluk,M.J.(2001).

Lysophospholipids-receptor revelations.Science294,1875–1878. Hoshino,Y.,Suzuki,C.,Ohtsuki,M.,Masubuchi,Y.,Amano,Y.,&Chiba, K.(1996).FTY720,a novel immunosuppressant possessing unique mechanisms:II.Long-term graft survival induction in rat hetrotopic cardiac allograft and synergistic effect in combination with cyclosporin

A.Transplant Proc28,1060–1061.

Hoshino,Y.,Yanagawa,Y.,Ohtsuki,M.,Nakayama,S.,Hashimoto,T.,& Chiba,K.(1999).FTY720,a novel immunosuppressant,shows a synergistic effect in combination with FK506in rat allograft models.

Transplant Proc31,1224–1226.

Kahan, B. D.,Karlix,J.L.,Ferguson,R.M.,Leichtman, A. B., Mulgaonkar,S.,Gonwa,T. A.,et al.(2003).Pharmacodynamics, pharmacokinetics,and safety of multiple doses of FTY720in stable renal transplant patients:a multicenter,randomized,placebo-controlled, phase I study.Transplantation76,1079–1084.

Kataoka,H.,Sugahara,K.,Tanaka,H.,Ohtsuki,M.,Maeda,Y.,Murata, M.,et al.(2004).The therapeutic effect of FTY720on experimental autoimmune encephalomyelitis in mice.Immunology2004,87–89. Kataoka,H.,Ohtsuki,M.,Shimano,K.,Mochizuki,S.,Oshita,K.,Murata, M.,et al.(2005).Immunosuppressive activity of FTY720,sphingosine 1-phosphate receptor agonist:II.Effect of FTY720and FTY720-phosphate on host-versus-graft and graft-versus-host reaction in mice.

Transplant Proc37,107–109.

Kawaguchi,T.,Hoshino,Y.,Rahman,F.,Amano,Y.,Higashi,H.,Kataoka,

H.,et al.(1996).FTY720,a novel immunosuppressant possessing

unique mechanisms:III.Synergistic prolongation of canine renal allograft survival in combination with cyclosporin A.Transplant Proc 28,1062–1063.

Kimura,Y.,Sato,K.,Kuwabara,A.,Tomura,H.,Ishikawa,M.,Kobayashi,

I.,et al.(2001).Sphingosine1-phosphate may be a major component of

plasma lipoproteins responsible for the cytoprotective actions in human umbilical vein endotherial cells.J Biol Chem276,31780–31785. Kiuchi,M.,Adachi,K.,Kohara,T.,Minoguchi,M.,Hanano,T.,Aoki,Y., et al.(2000).Synthesis and immunosuppressive activity of2-substituted2-aminopropane-1,3-diols and2-aminoethanols.J Med Chem43,2946–2961.

Kiuchi,M.,Adachi,K.,Tomatsu,A.,Chino,M.,Takeda,S.,Tanaka,Y., et al.(2005).Asymmetric synthesis and biological evaluation of the enantiomeric isomer of immunosuppressive FTY720-phosphate.Bioorg Med Chem13,425–432.

Li,H.,Meno-Tetang,G.L.,Chiba,K.,Arima,N.,Heinig,P.,&Jusko, W.L.(2002).Pharmacokinetics and cell trafficking dynamics of2-amino-2-[2-(4-octylphenyl)ethyl]propane-1,3-diol hydrochloride (FTY720)in cynomolgus monkeys after single oral and intravenous doses.J Pharmacol Exp Ther301,519–526.

Lo,C.G.,Xu,Y.,Proia,R.,&Cyster,J.G.(2005).Cyclical modulation of sphingosine-1-phosphate receptor1surface expression during lympho-cyte recirculation and relationship to lymphoid organ transit.J Exp Med 201,291–301.

Mandala,S.,Hajdu,R.,Bergstrom,J.,Quackenbush,E.,Xie,J.,Milligan, J.,et al.(2002).Alteration of lymphocyte trafficking by sphingosine-1-phosphate receptor agonists.Science296,346–349.

Masubuchi,Y.,Kawaguchi,T.,Ohtsuki,M.,Suzuki, C.,Amano,Y., Hoshino,Y.,et al.(1996).FTY720,a novel immunosuppressant possessing unique mechanisms:IV.Prevention of graft versus host reactions in rats.Transplant Proc28,1064–1065.

Matloubian,M.,Lo, C.G.,Cinamon,G.,Lesneski,M.J.,Xu,Y., Brinkmann,V.,et al.(2004).Lymphocyte egress from thymus and peripheral lymphoid organs is dependent on S1P receptor1.Nature427, 355–360.

Matsuura,M.,Imayoshi,T.,Chiba,K.,&Okumoto,T.(2000a).Effect of FTY720,a novel immunosuppressant,on adjuvant-induced arthritis in rats.Inflamm Res49,404–410.

Matsuura,M.,Imayoshi,T.,&Okumoto,T.(2000b).Effect of FTY720,a novel imunosuppressant,on adjuvant-and collagen-induced arthritis in rats.Int J Immunopharmacol22,323–331.

K.Chiba/Pharmacology&Therapeutics108(2005)308–319 318

Okazaki,H.,Hirata,D.,Kamimura,T.,Sato,H.,Iwamoto,M.,Yoshio,T., et al.(2002).Effect of FTY720in MRL-lpr/lpr mice:therapeutic potential in systemic lupus erythematosus.J Rheumatol29,707–716. Paugh,S.W.,Payne,S.G.,Barbour,S.E.,Milstien,S.,&Spiegel,S.

(2003).The immunosuppressant FTY720is phosphorylated by sphin-gosine kinase type2.FEBS Lett554,189–193.

Pyne,S.,&Pyne,N.(2000).Sphingosine1phosphate signaling via the endotherial differentiation gene family of G-protein-coupled receptors.

Pharmacol Ther88,115–131.

Sasaki,S.,Hashimoto,R.,Kiuchi,M.,Inoue,K.,Ikumoto,T.,Hirose,R., et al.(1994).Fungal metabolites:Part14.Novel potent immunosup-pressants,mycestericins,produced by Mycelia sterilia.J Antibiot47, 420–433.

Sugahara,K.,Kataoka,H.,Ohtsuki,M.,Hoshino,Y.,&Chiba,K.(2004).

The therapeutic effect of FTY720on lupus nephritis in aoutoimmune MRL/lpr mice.Immunology2004,271–274.

Suzuki,S.,Enosawa,S.,Kakefuda,T.,Shinomiya,T.,Amari,M.,Naoe,S., et al.(1996a).A novel immunosuppressant,FTY720,having an unique mechanism of action induces long-term graft acceptance in rat and dog allotransplantation.Transplantation61,200–205.

Suzuki,S.,Li,X.-K.,Enosawa,S.,&Shinomiya,T.(1996b).A new immunosuppressant,FTY720induces bcl-2-associated apoptotic cell death in human lymphocytes.Immunology89,518.

Suzuki,S.,Kakefuda,T.,Amemiya,H.,Chiba,K.,Hoshino,Y., Kawaguchi,T.,et al.(1998).An immunosuppressive regime using FTY720combined with cyclosporin in canine kidney transplantation.

Transpl Int11,95–101.

Tedesco-Silva,H.,Mourad,G.,Kahan,B.D.,Boira,J.G.,Weimar,W., Mulgaonkar,S.,et al.(2004).FTY720,a novel immunomodulator: efficacy and safety results from the first phase2a study in de novo renal transplantation.Transplantation77,1826–1833.Teshima,K.,Imayoshi,T.,Matsuura,M.,Yamaguchi,S.,Chiba,K.,& Terasawa,M.(1995).FTY720,a novel immunosuppressant,possessing unique mechanisms:III.Pharmacological activities in several auto-immune and inflammatory models.Int Congr Immunol9,864 (No.5126).

Troncoso,P.,Stepkowski,S.M.,Wang,M.-E.,Qu,X.,Chueh,S.-C., Clark,J.,et al.(1999).Prophylaxis of acute renal allograft rejection using FTY720in combination with subtherapeutic doses of cyclo-sporine.Transplantation67,145–151.

Yagi,H.,Kamba,R.,Chiba,K.,Soga,H.,Yaguchi,K.,Nakamura,M.,et al.

(2000).Immunosuppressant FTY720inhibits thymocyte emigration.

Eur J Immunol30,1435–1444.

Yanagawa,Y.,Sugahara,K.,Kataoka,H.,Kawaguchi,T.,Masubuchi,Y.,& Chiba,K.(1998a).FTY720,a novel immunosuppressant,induces sequestration of circulating mature lymphocytes by acceleration of lymphocyte homing in rats:II.FTY720prolongs skin allograft survival by preventing infiltration of T cells into the grafts,but not production of cytokines in vivo.J Immunol160,5493–5499.

Yanagawa,Y.,Masubuchi,Y.,&Chiba,K.(1998b).FTY720,a novel immunosuppressant,induces sequestration of circulating mature lym-phocytes by acceleration of lymphocyte homing in rats:III.Increase in frequency of CD62L-positive T cells in Peyer’s patches by FTY720-induced lymphocyte homing.Immunology95,591–594. Yanagawa,Y.,Hoshino,Y.,Kataoka,H.,Kawaguchi,T.,Ohtsuki,M., Sugahara,K.,et al.(1999).FTY720,a novel immunosuppressant, prolongs rat skin allograft survival by decreasing T cell infiltration into grafts.Transplant Proc31,1227–1229.

Yanagawa,Y.,Hoshino,Y.,&Chiba,K.(2000).The significance of timing of FTY720administration on the immunosuppressive effect to prolong rat skin allograft survival.Int J Immunopharmacol22, 597–602.

K.Chiba/Pharmacology&Therapeutics108(2005)308–319319