Cellular & Molecular Immunology 439
`
`
`
`Article
`FTY720, Sphingosine 1-Phosphate Receptor Modulator, Ameliorates
`Experimental Autoimmune Encephalomyelitis by Inhibition of T
`Cell Infiltration
`
`
`Hirotoshi Kataoka1, Kunio Sugahara1, Kyoko Shimano1, Koji Teshima2, Mamoru Koyama3, Atsushi
`Fukunari3 and Kenji Chiba1, 4
`
`FTY720, a sphingosine 1-phosphate receptor modulator, induces a marked decrease in the number of peripheral
`blood lymphocytes and exerts immunomodulating activity in various experimental allograft and autoimmune
`disease models. In this study, we evaluated the effect of FTY720 and its active metabolite, (S)-enantiomer of
`FTY720-phosphate [(S)-FTY720-P] on experimental autoimmune encephalomyelitis (EAE) in rats and mice.
`Prophylactic administration of FTY720 at 0.1 to 1 mg/kg almost completely prevented the development of EAE,
`and therapeutic treatment with FTY720 significantly inhibited the progression of EAE and EAE-associated
`histological change in the spinal cords of LEW rats induced by immunization with myelin basic protein. Consistent
`with rat EAE, the development of proteolipid protein-induced EAE in SJL/J mice was almost completely prevented
`and infiltration of CD4+ T cells into spinal cord was decreased by prophylactic treatment with FTY720 and
`(S)-FTY720-P. When FTY720 or (S)-FTY720-P was given after establishment of EAE in SJL/J mice, the relapse of
`EAE was markedly inhibited as compared with interferon-β, and the area of demyelination and the infiltration of
`CD4+ T cells were decreased in spinal cords of EAE mice. Similar therapeutic effect by FTY720 was obtained in
`myelin oligodendrocyte glycoprotein-induced EAE in C57BL/6 mice. These results indicate that FTY720 exhibits
`not only a prophylactic but also a therapeutic effect on EAE in rats and mice, and that the effect of FTY720 on
`EAE appears to be due to a reduction of the infiltration of myelin antigen-specific CD4+ T cells into the
`inflammation site. Cellular & Molecular Immunology. 2005;2(6):439-448.
`
`Key Words: FTY720, S1P receptor modulator, lymphopenia, EAE, T cell infiltration
`
`
`Introduction
`
`2-Amino-2-[2-(4-octylphenyl)ethyl]propane-1,3-diol hydrochlo-
`ride (FTY720), a new class of immunomodulator, sequesters
`circulating mature lymphocyte into secondary lymphoid
`
`tissues and thymus by long-term down-regulation of S1P
`receptor type 1 (S1P1) on lymphocytes, and exerts immuno-
`modulating effect (1-6). It has been previously reported that a
`potent immunosuppressive natural product, ISP-I can be
`isolated from a culture broth of Isaria scinclairii, a kind of
`vegetative wasp (7-9). The chemical modification of ISP-I
`led to a novel synthetic compound, FTY720 that has more
`potent immunomodulating activity and less toxicity than
`ISP-I (10-13). FTY720 has been shown to be highly effective
`in prolonging allograft survival in various experimental
`allograft models (1-4, 14, 15). 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 (1-4). FTY720-induced
`
`Abbreviations: EAE, experimental autoimmune encephalomyelitis; FTY720,
`2-amino-2-[2-(4-octylphenyl)ethyl]propane-1,3-diol hydrochloride; FTY720-P,
`FTY720-phosphate; IFN, interferon; MBP, myelin basic protein; MOG,
`myelin oligodendrocyte glycoprotein; MS, multiple sclerosis; PLP,
`proteolipid protein; S1P, sphingosine 1-phosphate; S1P1, sphingosine
`1-phosphate receptor type 1; (S)-FTY720-P, (S)-enantiomer of FTY720-
`phosphate.
`
`1Research Laboratory III (Immunology), Pharmaceutical Research Unit,
`Research and Development Division, Mitsubishi Pharma Corporation,
`Yokohama, Japan;
`2Research Laboratory I (CNS), Pharmaceutical Research Unit, Research and
`Development Division, Mitsubishi Pharma Corporation, Yokohama, Japan;
`3Discovery Technology Laboratory I, Pharmaceutical Research Unit,
`Research and Development Division, Mitsubishi Pharma Corporation,
`Yokohama, Japan;
`4Corresponding to: Dr. Kenji Chiba, Research Laboratory III (Immunology),
`Pharmaceutical Research Unit, Research and Development Division,
`Mitsubishi Pharma Corporation, 1000 Kamoshida-cho, Aoba-ku, Yokohama
`227-0033, Japan. Tel: +81-45-963-4527, Fax: +81-45-963-3977, E-mail:
`Chiba.Kenji@mk.m-pharma.co.jp.
`Received Nov 9, 2005. Accepted Dec 1, 2005.
`
`Copyright © 2005 by The Chinese Society of Immunology
`
`Volume 2 Number 6 December 2005
`
`SUN - IPR2017-01929, Ex. 1029, 1 of 10
`
`

`

`440 FTY720 Ameliorates EAE
`
`lymphopenia is mainly caused by sequestration of circulating
`mature lymphocytes into secondary lymphoid tissues such as
`lymph nodes and Peyer’s patches (2). Recently, it has been
`reported that FTY720 is effectively phosphorylated to
`FTY720-phosphate (FTY720-P) by sphingosine kinase 2 and
`that FTY720-P is a high affinity agonist at S1P receptors (16,
`17). Moreover it has been suggested that FTY720-P induces
`long-term down-regulation of S1P1 on lymphocytes and
`lymphocyte egress
`from
`inhibits S1P/S1P1-dependent
`secondary lymphoid tissues and thymus (5, 6).
`Mutiple screrosis (MS) is a common and often disabling
`disease of the central nervous system (CNS). Early active MS
`lesions are characterized by the presence of infiltrated
`mononuclear cells around venules and small veins, followed
`by myeline breakdown and astrogliosis,
`resulting
`in
`irreversible disability (18, 19). The etiology of MS remains
`unknown, but is widely considered to involve the organ-
`specific autoimmune destruction of CNS myelin mediated by
`myelin-specific T cells (20, 21). Immunomodulating therapy
`using cyclophosphamide, interferon-β (IFN-β), or glatiramer
`acetate is widely used for the treatment of MS (22, 23).
`Experimental autoimmune encephalomyelitis (EAE), an animal
`model of human MS, is a demyelinating, inflammatory
`disease of the CNS and is induced by the immunization of
`susceptible strains of rats or mice with myelin antigens
`combined with adjuvant (24). Myelin antigen-specific,
`autoreactive Th1 cells can be found in the blood, lymphoid
`tissues and CNS in EAE and MS, and it is highly likely that
`trafficking and infiltration of myelin antigen-specific Th1
`cells into CNS play an important role in the development and
`progression of EAE and MS (25-27).
`As reported previously, immunohistochemical staining and
`flow cytomeric analysis confirmed that FTY720 decreases T
`cell infiltration into the allograft at doses that show
`lymphopenia and a prolonging effect on allograft survival (3,
`28, 29). These findings strongly suggest that FTY720 exerts
`immunomodulating activity by decreasing T cell infiltration
`into inflammatory sites. In this paper, we evaluate the
`prophylactic and therapeutic effect of FTY720 and its active
`metabolite, (S)-enantiomer of FTY720-P [(S)-FTY720-P] (30,
`31) on EAE in rats and mice using myelin basic protein
`(MBP), myelin proteolipid protein
`(PLP) or myelin
`oligodendrocyte glycoprotein (MOG) as myelin antigens. We
`also demonstrate the relationship between the magnitude of
`improvement for EAE and the decrease in T cell infiltration
`into the CNS by FTY720 in these EAE models.
`
`Materials and Methods
`
`Animals and agents
`Male LEW rats were purchased from Kyudo Co., Ltd.
`(Yoshitomi, Fukuoka, Japan), and female SJL/J and C57BL/6
`mice were purchased from Charles River Japan Inc.
`(Yokohama, Japan). All animals were used at 8 to 12 weeks
`of age. PLP139-151 (HSLGKWLGHPDKF) and MOG35-55
`(MEVGWYRSPFSRVVHLYRNGK) were obtained from
`Bachem AG (Bubendorf, Switzerland) and Peptide Institute
`
`(Osaka, Japan), respectively. FTY720 and (S)-FTY720-P
`were synthesized according
`to
`the methods described
`previously (11, 30). FTY720 dissolved in distilled water was
`given orally. (S)-FTY720-P was dissolved in 10% hydro-
`xypropyl-β-cyclodextrin solution and administered intra-
`peritoneally. Recombinant mouse IFN-β (rm-IFN-β) and
`prednisolone (Predonine® for injection) were purchased from
`Calbiochem (La Jolla, CA) and Shionogi & Co., Ltd. (Osaka,
`Japan), respectively. All monoclonal antibodies (mAbs) used
`in this paper were obtained from BD Biosciences (San Jose,
`CA).
`
`Induction and scoring of EAE
`MBP was extracted and purified from the spinal cord of
`guinea pigs according to the methods described previously
`(32). For the induction of EAE in LEW rats, guinea pig MBP
`saline solution (2 mg/ml) was emulsified with an equal
`volume of Freund’s complete adjuvant containing Myco-
`bacterium tuberculosis H37Ra (Difco Laboratories, Detroit,
`MI). LEW rats were immunized by subcutaneous injection
`into the right and left hind footpads with the emulsion
`containing guinea pig MBP at 100 μg/rat. PLP139-151 and
`MOG35-55 were used as antigens for the induction of EAE in
`SJL/J mice and C57BL/6 mice, respectively. A saline solution
`of PLP139-151 at 0.5 mg/mL or MOG35-55 at 2 mg/ml was
`emulsified with an equal volume of Freund’s complete
`adjuvant containing Mycobacterium tuberculosis H37Ra.
`SJL/J mice and C57BL/6 mice were immunized by sub-
`cutaneous injection into the flank regions with the emulsion
`containing PLP139-151 at 50 μg/mouse and MOG35-55 at 200
`μg/mouse, respectively. In addition, MOG35-55-immunized
`C57BL/6 mice were given 200 ng of pertussis toxin by
`intravenous injection twice on the day of immunization and 2
`days after. Individual animals were examined daily for
`clinical signs of neurological deficits scored on a 0 to 5 scale
`as follows: 0, no abnormality; 0.5, stiff tail; 1, limp tail; 1.5,
`limp tail with inability to right; 2, paralysis of one limb; 2.5,
`paralysis of one limb and weakness of one other limb; 3,
`complete paralysis of both hind limbs; 4, moribund; 5, death.
`
`Histology of spinal cords in EAE
`The spinal cords of EAE animals were obtained, fixed in 4%
`formalin
`in PBS, and embedded
`in paraffin. Six
`μm-thickness sections were prepared and then stained with
`hematoxylin and eosin. In rat EAE, the histological score was
`evaluated as follows: 0, no lesions; 1, solitary lesions with
`cell infiltrates of low cellular density in the entire section; 2,
`a few lesions with moderate cell infiltration in each of a few
`fields; 3, many lesions in almost all fields with massive cell
`infiltration and accompanying edema (33).
`
`Immunohistochemical staining of spinal cords in mouse EAE
`The spinal cords in EAE mice were removed and frozen in
`isopenthane prechilled in liquid nitrogen using embedding
`medium for frozen tissue specimens (Tissue-Tek OCT
`compound; Sakura Fine Technical, Tokyo, Japan). Six
`μm-thickness frozen sections were immediately fixed in
`
`Volume 2 Number 6 December 2005
`
`SUN - IPR2017-01929, Ex. 1029, 2 of 10
`
`

`

`Cellular & Molecular Immunology 441
`
`B
`
`*
`
`**
`
`-
`
`**
`0.1 0.3 1
`FTY720 (mg/kg)
`
`D
`
`*
`
`**
`
`**
`0.1 0.3 1
`FTY720 (mg/kg)
`
`-
`
`G
`
`3.0
`3.0
`2.5
`2.5
`2.0
`2.0
`1.5
`1.5
`1.0
`1.0
`0.5
`0.5
`0
`0
`
`3.0
`3.0
`2.5
`2.5
`2.0
`2.0
`1.5
`1.5
`1.0
`1.0
`0.5
`0.5
`0
`0
`
`Histological score　
`
`Histological score　
`
`A
`
`Prophylactic
`
`
`
`00
`
`C
`
`
`
`00
`
`** *
`**
`**
`**
`**
`**
`**
`**
`****** ******
`77
`
`
`1414
`** **
`Days after immunization
`
`
`
`2121
`
`Therapeutic
`
`*
`
`* * * *
`****** * * *
`** ****
`* * *
`77
`
`
`1414
`Days after immunization
`
`**
`**
`
`
`
`2121
`
`F
`
`012345
`012345
`
`012345
`012345
`
`EAE score　
`
`EAE score　
`
`E
`
`Control
`
`FTY720 0.1 mg/kg
`
`FTY720 1 mg/kg
`
`
`Figure 1. Effect of FTY720 on MBP-induced EAE in LEW rats.
`LEW rats were immunized with guinea pig MBP at 100 μg/rat in
`the presence of Freund’s complete adjuvant. FTY720 was
`administered orally to MBP-immunized LEW rats every day from
`the day of immunization (prophylactic treatment) or the day of
`onset (therapeutic treatment) until day 19. Rats in the control group
`were administered vehicle only. The spinal cords in EAE rats were
`obtained on day 20 after immunization, and hematoxylin and eosin
`(HE) staining and histological scoring were performed. ○, Control;
`●, FTY720 0.1 mg/kg; ▲, FTY720 0.3 mg/kg; ■, FTY720 1
`mg/kg. (A) EAE scores in prophylactic treatment; (B) Histological
`scores in prophylactic treatment; (C) EAE scores in therapeutic
`treatment; (D) Histological scores in therapeutic treatment; HE
`staining of a spinal cord in (E) control EAE rat, (F) FTY720 0.1
`mg/kg therapeutically, (G) FTY720 1 mg/kg therapeutically. The
`results were expressed as the mean ± SE of 8 animals and statistical
`differences were calculated by Steel’s test (* p < 0.05; ** p < 0.01).
`
`
`treated group, the statistical differences of EAE score were
`calculated by the Mann-Whitney U test. The number of
`lymphocytes (T cells, CD4+ T cells, CD8+ T cells, and B
`cells), body weight, and the ratio of IFN-γ/GAPDH mRNA
`levels were analyzed by Dunnett’s multi-comparison test.
`Differences between groups were considered significant
`when p < 0.05.
`
`Results
`
`Prophylactic and therapeutic effects of FTY720 on MBP-
`induced EAE in rats
`All of the LEW rats in the vehicle-treated control group
`developed EAE-associated clinical symptoms 10 days after
`
`cold-acetone and were incubated with rat anti-mouse CD3
`mAb (clone: 17A2), rat anti-mouse CD4 mAb (clone:
`GK1.5), or rat anti-mouse CD8a mAb (clone: 53-6.7). The
`sections were then incubated with appropriate secondary
`antibodies conjugated with amino acid polymer and
`peroxidase (Histofine® Simple Stain MAX-PO kit; Nichirei,
`Tokyo, Japan), colorized with diaminobenzidine in the
`presence of hydrogen peroxide, and counterstained with
`hematoxylin.
`
`Flow cytometry
`Peripheral blood lymphocytes obtained from EAE mice were
`stained with FITC-conjugated rat anti-hamster CD3ε mAb
`(clone: 145-2C11) and PE-conjugated
`rat anti-mouse
`CD45R/B220 mAb (clone: RA3-6B2), and the number of T
`cells and B cells was determined by 2-color flow cytometry
`using CytomicsTM FC500 (Beckman Coulter Inc., Fullerton,
`CA). In the analysis of T cell subsets, the numbers of CD4+ T
`cells and CD8+ T cells were determined by 3-color flow
`cytometry with FITC-conjugated hamster anti-mouse CD3ε
`mAb (clone: 145-2C11), PE-conjugated rat anti-mouse CD4
`mAb (clone: RM4-5), and PE-Cy5-conjugated rat anti-mouse
`CD8a mAb (clone: 53-6.7).
`
`Measurement of IFN-γ and GAPDH mRNA levels by
`TaqMan® PCR
`Total RNA was extracted from the spinal cords of EAE mice
`using RNA isolation reagent (Nippon Gene, Tokyo, Japan)
`according to the manufacturer’s protocol and then the
`concentration of total RNA was measured spectrophoto-
`metrically. An aliquot (0.5 μg) of total RNA from the spinal
`cord was reverse-transcribed to cDNA in a 50 μl volume at
`25°C for 10 min, 48°C for 30 min and 95°C for 10 min with
`TaqMan® reverse transcription reagents using a thermal
`cycler, Gene Amp® PCR System 9700 (Applied Biosystems,
`Branchburg, NJ). The levels of mRNA for IFN-γ and
`GAPDH were determined using the TaqMan® polymerase
`chain reaction (PCR). Five microliters of cDNA were
`amplified with IFN-γ TaqMan® probe (6-carboxy-fluorescein
`label)/primer, GAPDH TaqMan® probe (VICTM label) /primer,
`and TaqMan® Universal PCR Master Mix in an ABI
`PRISMTM 7900 Sequence Detection System (Applied
`Biosystems). The reaction was incubated for 2 min at 50°C,
`denatured for 10 min at 95°C and subjected to 40 two-step
`amplification cycles with annealing/extension at 60°C for 1
`min followed by denaturation at 95°C for 15 sec. The
`detection of PCR product was monitored by measuring the
`increase in fluorescence caused by degradation of the probe.
`For every sample, the level of IFN-γ mRNA was normalized
`by calculating the ratio of IFN-γ/GAPDH levels.
`
`Statistical analysis
`All values are expressed as mean and standard error mean
`(SEM) in each group. The statistical differences of EAE
`scores and histology scores in FTY720- or FTY720-P-treated
`groups were calculated by Steel’s test and compared with
`vehicle-treated control group. In the IFN-β- or prednisolone-
`
`Volume 2 Number 6 December 2005
`
`SUN - IPR2017-01929, Ex. 1029, 3 of 10
`
`

`

`442 FTY720 Ameliorates EAE
`
`B
`
`**
`
`**
`
`**
`
`**
`
`D
`
`**
`
`**
`
`**
`
`**
`
`-
`
`IFN-β
`
`0.1 1
`0.1 1
`FTY720 (S)-FTY720-P
`
`25
`25
`
`20
`20
`
`15
`15
`
`10
`10
`1000
`1000
`
`750
`750
`
`500
`500
`
`250
`250
`
`0
`0
`
`Body weight (g)
`
`B cells (cells/μL)
`
`A
`
`**
`
`C
`
`**
`
`**
`
`**
`
`-
`
`IFN-β
`
`*
`*
`*
`*
`0.1 1
`0.1 1
`FTY720 (S)-FTY720-P
`
`01234
`01234
`
`5000
`5000
`
`4000
`4000
`
`3000
`3000
`
`2000
`2000
`
`1000
`1000
`
`0
`0
`
`EAE score
`
`T cells (cells/μL)
`
`
`Figure 3. Prophylactic effects of FTY720, (S)-FTY720-P and
`rm-IFN-β on PLP139-151-induced EAE in SJL/J mice. SJL/J mice
`were immunized with PLP139-151 at 50 μg/mouse in the presence of
`Freund’s complete adjuvant. FTY720 (0.1 and 1 mg/kg) was
`administered orally (p.o.) to PLP139-151-immunized SJL/J mice every
`day. (S)-FTY720-P (0.1 and 1 mg/kg) was administered intra-
`peritoneally (i.p.) every day and rm-IFN-β (10,000 IU/mouse) were
`given three times a week i.p., respectively. Mice in the control
`groups were administered vehicle only. EAE scores, body weights,
`and the numbers of T and B cells in peripheral blood were
`determined at day 14 after immunization. The results were
`expressed as the mean ± SE of 6 animals. Statistical differences in
`EAE scores (A) were calculated by Steel’s test, and those in body
`weights (B), number of T cells (C), and B cells (D) were done by
`Dunnett’s multi-comparison test (* p < 0.05; ** p < 0.01).
`
`
`
`in a
`resulted
`therapeutic administration of FTY720
`significant decrease in the histological score of EAE (Figure
`1D). The infiltration of inflammatory cells into the spinal
`cords of EAE rats was inhibited in the FTY720-treated group
`(Figures 1E, 1F, 1G). A marked decrease in the number of
`peripheral lymphocytes was observed in all of the FTY720
`administration schedules (data not shown). These data
`indicate that FTY720 not only has a prophylactic but also a
`therapeutic potential in treating MBP-induced EAE in LEW
`rats.
`
`Prophylactic and therapeutic effects of FTY720 on PLP-
`induced EAE in SJL/J mice
`In human MS, neurological symptoms relapse over several
`years; however MBP-induced EAE in LEW rats was
`monophasic with no relapse. To clarify the therapeutic
`potential of FTY720 in human MS more precisely, we
`evaluated the effect of FTY720 and its active metabolite
`(S)-FTY720-P on relapsing EAE in SJL/J mice induced by
`immunization. SJL/J mice
`immunized with
`PLP139-151
`PLP139-151 emulsified in Freund’s complete adjuvant resulted
`in the development of EAE-associated clinical symptoms and
`a decrease in body weight 11 days after immunization. EAE
`scores were rapidly elevated and reached a maximal level on
`day 15. The first phase of EAE remitted with a low EAE
`score on day 20 and spontaneously relapsed thereafter
`(Figure 2A). The elevation of EAE-associated clinical score
`and the loss of body weight were prevented in groups given
`
`3.5
`3.5
`3.0
`3.0
`2.5
`2.5
`2.0
`2.0
`1.5
`1.5
`1.0
`1.0
`0.5
`0.5
`0
`0
`
`23
`23
`22
`22
`21
`21
`20
`20
`19
`19
`18
`18
`17
`17
`16
`16
`15
`15
`
`A
`
`EAE score
`
`B
`
`Body weight (g)
`
`
`
`00
`
`
`
`77
`
`* * *
`**
`* *
`**
`*
`************
`
`* * * ** * ** ** * * * * * * * * * *
`
`
`
`
`
`1414
`2121
`2828
`3535
`4242
`Days after immunization
`
`**
`
`
`
`00
`
`
`
`77
`
`**************************
`***** * * * * * * * *****
`**
`**** ** * **************
`* * * * *
`**
`**
`* *
`**
`
`*
`
`* * * *
`
`2828
`2121
`1414
`
`
`
`Days after immunization
`
`
`
`3535
`
`
`
`4242
`
`
`
`Figure 2. Prophylactic effect of FTY720 on PLP139-151-induced
`EAE in SJL/J mice. SJL/J mice were immunized with PLP139-151 at
`50 μg/mouse in the presence of Freund’s complete adjuvant.
`FTY720 was administered orally to PLP139-151-immunized SJL/J
`mice every day from the day of immunization for 6 weeks. Mice in
`the control groups were administered vehicle only. ○, Control; ●,
`FTY720 0.1 mg/kg; ▲, FTY720 0.3 mg/kg. The results were
`expressed as the mean ± SE of 7 animals and statistical differences
`in EAE scores (A) and body weights (B) were calculated by Steel’s
`test and Dunnett’s multi-comparison (hyphenation) test, respectively
`(*, p < 0.05; **, p < 0.01).
`
`
`
`immunization with guinea pig MBP, reaching a maximal
`level on day 13 followed by a gradual decline (Figure 1A).
`Elevation of EAE scores was significantly inhibited in groups
`given FTY720 prophylactically at oral doses of 0.1, 0.3 and 1
`mg/kg for 20 days from the day of MBP immunization.
`Consistent with EAE-associated symptoms, the histological
`scores in FTY720-treated groups were decreased significantly
`and dose-dependently as compared with the vehicle-treated
`control group (Figure 1B). Significantly, there was no
`increase in the EAE and histological scores in the group
`treated with FTY720 at 1 mg/kg, indicating a complete
`prevention of EAE development in MBP-immunized LEW
`rats.
`To evaluate the therapeutic potential of FTY720 in
`MBP-induced EAE in LEW rats, the administration was
`started from the day of EAE onset (Figure 1C). In the
`vehicle-treated control group, EAE had developed by day 9
`after immunization and reached a maximal level on day 11 to
`13. Thereafter, the mean of EAE scores remained within the
`2 to 3 range until day 20, because 4 out of 8 EAE rats died in
`the control group. Therapeutic administration of FTY720
`from the day of EAE onset significantly decreased post-peak
`EAE-associated clinical signs (Figure 1C). Moreover, the
`
`Volume 2 Number 6 December 2005
`
`SUN - IPR2017-01929, Ex. 1029, 4 of 10
`
`

`

`Cellular & Molecular Immunology 443
`
`
`
`4242
`
`* *
`
`****
`*
`**
`* *
`* *
`**
`*
`**
`**
`3535
`2121
`
`
`
`2828
`Days after immunization
`
`
`
`1414
`
`3.5
`3.5
`3.0
`3.0
`2.5
`2.5
`2.0
`2.0
`1.5
`1.5
`1.0
`1.0
`0.5
`0.5
`0
`0
`
`A
`
`EAE score
`
`#
`
`
`
`1414
`
`
`
`2121
`
`*
`
`*** * *
`**** *
`* **
`*
`
`*** * *
`**** *
`* **
`2828
`
`
`
`
`3535
`4242
`4949
`Days after immunization
`
`* ****
`* ****
`
`5656
`
`*
`*
`
`
`
`6363
`
`B
`
`Control
`(S)-FTY720-P
`0.1 mg/kg ip
`(S)-FTY720-P
`1 mg/kg ip
`
`**
`**
`**
`**
`B cells
`T cells
`
`
`500500
`10001000
`
`15001500
`
`
`01000 2000 3000 40001000 2000 3000 4000 0
`
`00
`Number of cells in blood (cells/μL)
`
`
`
`
`Figure 5. Therapeutic effect of (S)-FTY720-P on PLP139-151-
`induced EAE in SJL/J mice. SJL/J mice were immunized with
`PLP139-151 at 50 μg/mouse in the presence of Freund’s complete
`adjuvant. EAE-developed mice were pooled, divided into 3 groups
`and therapeutic administration of (S)-FTY720-P was started from
`day 17 after the immunization for 20 days. Mice in the control
`groups were administered vehicle only. The numbers of T and B
`cells in peripheral blood were determined at day 38 after
`immunization. ○, Control; ●, (S)-FTY720-P 0.1 mg/kg i.p. daily ▲,
`(S)-FTY720-P 0.3 mg/kg i.p. daily. The results are expressed as the
`mean ± SE of 7 animals. Statistical differences in EAE scores (A)
`were calculated by Steel’s test, and those in numbers of T and B
`cells (B) were done by Dunnett’s multi-comparison test. (*p < 0.05,
`** p < 0.01).
`
`
`
`To evaluate the therapeutic effect of FTY720 on
`PLP139–151-induced, relapsing EAE in SJL/J mice, EAE-
`developed mice were pooled, divided
`into 4 groups
`consisting of six mice, and administration of FTY720 was
`started 17 days after immunization. EAE-associated clinical
`signs were decreased rapidly by day 21, and thereafter,
`relapse of EAE occurred in the vehicle-treated control group
`(Figure 4A). By contrast, the relapse of EAE was markedly
`inhibited and no EAE-associated clinical signs were observed
`from day 32 to 59 in groups given FTY720 at 0.3 and 1
`mg/kg therapeutically, indicating complete inhibition of EAE
`relapse. In the group given rm-IFN-β at 10,000 IU three
`times a week
`intraperitoneally,
`the EAE score was
`significantly lowered at day 24, and relapse was delayed;
`however rm-IFN-β failed to inhibit the relapse of EAE. In
`another experiment, it was confirmed that the magnitude of
`the therapeutic effect of FTY720 is almost equal to that of
`prednisolone (Figure 4B). We also evaluated the therapeutic
`effect of (S)-FTY720-P on relapsing EAE in SJL/J mice
`induced by PLP139-151. (S)-FTY720-P was administered
`intraperitonelly to mice developed EAE from day 17 after
`immunization. In results similar to those seen in FTY720
`treatment, the relapse of EAE was markedly inhibited in
`
`
`3.53.5
`
`3.03.0
`
`2.52.5
`
`2.02.0
`
`1.51.5
`
`1.01.0
`
`0.50.5
`
`00
`
`
`3.53.5
`
`3.03.0
`
`2.52.5
`
`2.02.0
`
`1.51.5
`
`1.01.0
`
`0.50.5
`
`00
`
`A
`
`EAE score
`
`B
`
`EAE score
`
`#
`
`#
`
`*
`#
`## # # # # # #
`# #
`*
`**
`* *
`* ***
`*
`*
`* ** *
`* *
`*
`**
`*
`* ** ****** * ***** *
`**
`*
`*
`* ****
`*
`**
`**
`
`
`
`
`2828
`3535
`4242
`4949
`
`5656
`Days after immunization
`
`*
`*
`
`
`
`6363
`
`
`
`
`
`1414
`
`
`
`2121
`
`
`Figure 4. Therapeutic effect of FTY720 on PLP139-151-induced
`EAE in SJL/J mice. SJL/J mice were immunized with PLP139-151 at
`50 μg/mouse in the presence of Freund’s complete adjuvant.
`EAE-developed mice were pooled, divided into 4 groups and
`administrations of FTY720, rm-IFN-β and prednisolone were
`started from day 17 after immunization for 60 days. Mice in the
`control groups were administered vehicle only. (A) ○, Control;
`, △
`rm-IFN-β 10000 IU/mice i.p. 3 times a week; ▲, FTY720 0.3
`mg/kg p.o. daily;
`p.o. daily; (B) ○, Control;
`, FTY720 1 mg/kg
`◆
`p.o. daily; ●, FTY720 0.1 mg/kg p.o.
`, prednisolone 1 mg/kg
`◇
`daily ▲, FTY720 0.3 mg/kg p.o. daily. The results are expressed as
`the mean ± SE of 7 animals and statistical differences in EAE
`scores of FTY720 groups were calculated by Steel’s test (*p < 0.05;
`**p < 0.01), and those in rm-IFN-β or prednisolone were done by
`Mann Whitney U test (# p < 0.05).
`
`
`
`FTY720 prophylactically at oral doses of 0.1 and 0.3 mg/kg
`for 42 days from the day of immunization (Figures 2A, 2B).
`Consistent with MBP-induced EAE in LEW rats, there was
`no increase in EAE score in the group treated with FTY720
`at 1 mg/kg, indicating a complete prevention of PLP139-151-
`induced EAE in SJL/J mice. The prophylactic effects of
`FTY720, (S)-FTY720-P and rm-IFN-β were compared in
`PLP139-151-induced EAE in SJL/J mice, and the results on day
`17 are shown in Figure 3. Oral administration of FTY720 at
`0.1 and 1 mg/kg resulted in a markedly preventing effect on
`EAE score elevation and body weight loss, and induced a
`marked decrease in the number of T cells and B cells in
`peripheral blood. Almost the same prophylactic effect was
`observed when (S)-FTY720-P at 0.1 and 1 mg/kg was
`administered intraperitoneally to PLP139-151-immunized SJL
`mice
`from
`the day of
`immunization. By contrast,
`prophylactic treatment with rm-IFN-β at 10,000 IU three
`times a week intraperitoneally showed no clear effect or no
`lymphopenia.
`
`Volume 2 Number 6 December 2005
`
`SUN - IPR2017-01929, Ex. 1029, 5 of 10
`
`

`

`444 FTY720 Ameliorates EAE
`
`A
`
`B
`
`C
`
`50 μm
`
`100 μm
`Control CD3
`
`Control CD3
`
`Control CD8
`
`D
`
`100 μm
`G
`
`E
`
`H
`
`50 μm
`
`F
`
`I
`
`Control CD4
`
`FTY720 1 mg/kg
`CD4
`
`(S)-FTY720-P 1 mg/kg
`CD4
`
`
`Figure 7. Prophylactic administration of FTY720 and (S)-
`FTY720-P decreased the infiltration of CD4+ T cells in the
`spinal cord of PLP139-151-induced EAE mice. SJL/J mice were
`immunized with PLP139-151 at 50 μg/mouse in the presence of
`Freund’s complete adjuvant and administered FTY720 at 1 mg/kg
`p.o. and (S)-FTY720-P at 1 mg/kg i.p. prophylactically. The spinal
`cords of EAE-developed mice were obtained at day 17 after
`immunization and immunohistochemical staining were performed.
`Control: (A) CD3+ T cells (× 100), (B) CD3+ T cells (× 400), (C)
`CD8+ T cells (× 400), (D) CD4+ T cells (× 100), (G) CD4+ T cells
`(× 400); FTY720 1 mg/kg p.o.: (E) CD4+ T cells (× 100), (H) CD4+
`T cells (× 400); (S)-FTY720 1 mg/kg i.p.: (F) CD4+ T cells (× 100),
`(I) CD4+ T cells (× 400).
`
`
`
`A
`
`100 μm
`D
`
`B
`
`E
`
`50 μm
`
`C
`
`F
`
`Control
`
`FTY720 1 mg/kg
`
`(S)-FTY720-P 1 mg/kg
`
`
`Figure 6. Prophylactic administration of FTY720 and (S)-
`FTY720-P decreased the infiltration of inflammatory cells in the
`spinal cord of PLP139-151-induced EAE mice. SJL/J mice were
`immunized with PLP139-151 at 50 μg/mouse in the presence of
`Freund’s complete adjuvant and administered FTY720 at 1 mg/kg
`p.o. and (S)-FTY720-P at 1 mg/kg i.p. prophylactically. The spinal
`cords of EAE-developed mice at day 17 after immunization were
`obtained, and HE staining were performed. Control: (A) (× 100),
`(D) (× 400); FTY720 1 mg/kg p.o.: (B) (× 100), (E) (× 400); (S)-
`FTY720- P 1 mg/kg i.p.: (C) (× 100), (F) (× 400).
`
`
`
`(S)-FTY720-P at 0.1 and 1 mg/kg
`groups given
`therapeutically (Figure 5A). All the (S)-FTY720-P-treated
`groups showed a significant decrease in the number of T cells
`and B cells in peripheral blood on the day following the final
`administration (Figure 5B).
`The infiltration of inflammatory cells was observed in the
`spinal cord of SJL/J mice 17 days after immunization with
`PLP139-151. Prophylactic administration of FTY720 at 1
`mg/kg orally and (S)-FTY720-P at 1 mg/kg intraperitoneally
`resulted in a marked reduction of the infiltration of
`the spinal cord of PLP139–151-
`inflammatory cells
`in
`immunized SJL/J mice (Figure 6). Immunohistochemical
`staining analysis using anti-T cell subset mAbs revealed the
`infiltration of CD4+ T cells rather than CD8+ T cells into
`spinal cords, especially the perivascular area and funiculus
`dorsalis in white matter under pia mater, of PLP139–151-
`immunized SJL/J mice with developed EAE (Figure 7).
`Prophylactic administration of FTY720 at 1 mg/kg orally and
`(S)-FTY720-P at 1 mg/kg intraperitoneally markedly decreased
`the infiltration of CD4+ T cells into the spinal cord as
`compared with the vehicle-treated control EAE group. In
`addition to the infiltration of CD4+ T cells, demyelination
`was confirmed predominantly in the funiculus dorsalis in
`white matter under pia mater in another experiment (Figure
`8). In the group given FTY720 at 1 mg/kg therapeutically,
`the area of demyelination and the infiltration of CD4+ T cells
`into the spinal cord were decreased as compared with the
`vehicle-treated control group.
`
`Therapeutic effect of FTY720 on MOG-induced EAE in
`C57BL/6 mice
`
`EAE was developed 12 days after immunization of MOG35-55
`to C57BL/6 mice, and EAE-established mice were divided
`into 3 groups consisting of eleven mice 17 days after the
`immunization (Figure 9A). EAE-associated symptoms were
`maintained during administration period in the vehicle-
`treated control group of MOG35-55-immunized C57BL/6 mice.
`The EAE score was significantly decreased when FTY720
`was administered therapeutically at 0.1 and 0.3 mg/kg. The
`level of IFN-γ mRNA expression in the spinal cords of
`MOG35-55-immunized EAE mice was markedly elevated
`compared to that of normal mice. The elevation of IFN-γ
`mRNA level was significantly inhibited in groups given
`FTY720 therapeutically at 28 days after immunization
`(Figure 9B). Demyelination and infiltration of lymphocytes
`were observed
`in
`the spinal cords
`in
`the control
`MOG35-55-induced EAE mice at 42 days after immunization.
`The infiltration of CD4+ T cells was more markedly than that
`of CD8+ T cells in the spinal cord of MOG35-55-induced EAE
`mice. In FTY720-treated groups, the area of demyelination
`and the infiltration of CD4+ T cells and CD8+ T cells in the
`spinal cord were decreased as compared with the vehicle-
`
`Volume 2 Number 6 December 2005
`
`SUN - IPR2017-01929, Ex. 1029, 6 of 10
`
`

`

`Cellular & Molecular Immunology 445
`
`B
`
`D
`
`F
`
`A
`
`HE
`
`100 μm
`C
`
`100 μm
`E
`
`50 μm
`
`CD4
`
`CD4
`
`Control
`
`FTY720 1 mg/kg
`
`
`
`
`Figure 8. Therapeutic administration of FTY720 decreased the
`infiltration of CD4+ T cells into the spinal cords of PLP139-151-
`induced EAE mice. SJL/J mice were immunized with PLP139-151 at
`50 μg/mouse in the presence of Freund’s complete adjuvant and
`administered FTY720 at 1 mg/kg p.o. therapeutically from day 17.
`The spinal cords of EAE-developed mice were obtained at day 28,
`and HE and immunohistochmical staining were performed. Control:
`(A) HE (× 100), (C) CD4+ T cells (× 100), (E) CD4+ T cells (× 400);
`FTY720 1 mg/kg: (B) HE (× 100), (D) CD4+ T cells (× 100), (F)
`CD4+ T cells (× 400).
`
`
`
`treated control group (Figure 10).
`
`Discussion
`
`MS is a chronic inflammatory and autoimmune disease
`characterized by discrete areas of
`inflammation and
`demyelination that can occur in multipl