United States Patent [19J
`Chiba et al.
`
`[54] COMPOSITIONS AND METHODS OF USING
`COMPOSITIONS WITH ACCELERATED
`LYMPHOCYTE HOMING
`IMMUNOSUPPRESSIVE PROPERTIES
`
`[75]
`
`Inventors: Kenji Chiba; Kunitomo Adachi, both
`of Fukuoka, Japan
`
`[73] Assignee: Yoshitomi Pharmaceutical Industries,
`Ltd., Osaka, Japan
`
`[21] Appl. No.: 08/933,738
`
`[22] Filed:
`
`Sep. 23, 1997
`
`[30]
`
`Foreign Application Priority Data
`
`Sep. 2, 1997
`
`[JP]
`
`Japan .................................... 9-237273
`
`Int. Cl.6
`................................................... A61K 31/135
`[51]
`[52] U.S. Cl. ....................... 424/278.1; 514/487; 514/546;
`514/653; 560/29; 560/163; 564/223; 564/355
`[58] Field of Search ..................................... 564/223, 355;
`560/29, 163; 514/653, 487, 546; 424/278.1
`
`[56]
`
`References Cited
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`2/1997 Fujita et a!. .
`11/1997 Okumoto ................................ 514/383
`2/1998 Fugita ..................................... 514/440
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`[45] Date of Patent:
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`6,004,565
`Dec. 21, 1999
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`
`(List continued on next page.)
`
`Primary Examiner-David Saunders
`Assistant Examiner-Mary Beth Tung
`Attorney, Agent, or Firm---Evenson, McKeown Edwards &
`Lenahan P.L.L. C.
`
`[57]
`
`ABSTRACT
`
`The methods and compositions of the invention and the
`compounds used in the invention involve a novel immuno(cid:173)
`suppression mechanism, accelerated lymphocyte homing
`immunosuppression (ALH-immunosuppression). For
`example, the compound FTY720 specifically directs lym(cid:173)
`phocytes to the peripheral lymph nodes, mesenteric lymph
`nodes, and Peyer's patches. By reversibly sequestering
`lymphocytes in these tissues, the compounds can inhibit an
`immune response in a mammal. Understanding these
`mechanisms provides a novel immunosuppression therapy
`that can synergistically interact with other immunosuppres(cid:173)
`sive compounds. Screening methods for identifying similar
`ALH-immunosuppression compounds are also described.
`The invention allows better treatments and therapies wher(cid:173)
`ever an immunosuppression regimen is desired.
`
`6 Claims, 11 Drawing Sheets
`
`SUN - IPR2017-01929, Ex. 1006, p. 1 of 30
`
`

`

`6,004,565
`Page 2
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`cularised Organ Grafts: Analysis Using Semiquantitative
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`J. Wang et al., "Local Hormone Networks and Intestinal T
`Cell Homeostasis," Science, vol. 275, pp. 1937-1939 (Mar.
`28, 1997).
`European FK506 Multicentre Liver Study Group, "Ran(cid:173)
`domised
`trial
`comparing
`tacrolimus
`(FK506)
`and
`cyclosporin in prevention of liver allograft rejection", The
`Lancet, vol. 344, pp. 423-428 (Aug. 13, 1994).
`T. Tanaka et al., "Characterization of a CD3-like Rat T Cell
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`53, No. 1, pp. 87-90 (Jan. 1992).
`T. Tamatani et al., Characterization of rat LECAM-1 (L-se(cid:173)
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`monoclonal antibodies," Eur. J. Immunol., vol. 21, pp.
`627-633 (1991).
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`ment for quantitative PCR analysis of cytokine gene expres(cid:173)
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`al.,
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`Compounds,
`"Simple
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`pp. 847-852 (1995).
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`Fujita
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`39, pp. 4451-4459 (1996).
`Kunitomo Adachi et al., "Design, Synthesis, And Structure(cid:173)
`-Activity Relationships of 2-substituted-2-amino-1,3-pro(cid:173)
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`(1995).
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`2, pp. 1056-1059 (1996).
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`Possessing Unique Mechanisms. II. Long-Term Graft Sur(cid:173)
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`T. Kawaguchi et al., "FTY720,ANovel Immunosuppressant
`Possessing Unique Mechanisms. III. Synergistic Prolonga(cid:173)
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`1062-1063 (1996).
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`FTY720, With A Unique Mechanism of Action, Induces
`Long-Term Graft Acceptance in Rat and Dog Allotransplan(cid:173)
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`in
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`
`

`

`6,004,565
`Page 3
`
`F. Shanahan, "A Gut Reaction: Lymphoepithelial Commu(cid:173)
`nication in the Intestine", Science, vol. 275, pp. 1897-1898
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`al., Bioorgan. Med. Chern. Let.
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`26, 1997.
`
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`1996.
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`1996.
`
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`
`M. Slapak et al., "The Use of Low-Dose Cyclosporine in
`Combination With Azathioprine and Steriods in Renal
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`No. 1, Feb. 1985, pp. 1222-1226.
`
`SUN - IPR2017-01929, Ex. 1006, p. 3 of 30
`
`

`

`U.S. Patent
`
`Dec. 21, 1999
`
`Sheet 1 of 11
`
`6,004,565
`
`__, i 100
`
`~ 80
`t:;: 60
`~
`40
`20
`0
`0
`
`<.:)
`
`L&..
`0
`1--:z
`
`L&.J
`(..)
`0::
`L&.J a.
`
`V')
`
`__,
`?i 100
`>
`~ 80
`~ 60
`40
`20
`0
`0
`
`<.:)
`
`L&..
`0
`1--:z
`
`L&.J
`(..)
`0::
`L&.J a.
`
`-
`
`CONTROL
`FlY720 0.1 mg/kg •
`FlY720 0.3 mg/kg •
`- ·- FlY720 1 mg/kg •
`fTY720 3 mg/kg •
`FlY720 1 0 mg/kg •
`
`60
`
`70
`
`(n=B)
`
`CONTROL
`-
`- CsA 3 mg/kg •
`- -
`----- CsA 10 mg/kg •
`--- CsA 30 mg/kg •
`--·- CsA 100 mg/kg •
`
`10
`
`40
`50
`20
`30
`DAYS AFTER TRANSPLANTATION
`FIG. 1 A
`·-n
`~ : . 1
`
`~ :
`
`10
`
`40
`50
`20
`30
`DAYS AFTER TRANSPLANTATION
`FIG. 1 8
`
`60
`
`70
`
`{n=8}
`
`•
`
`~ . .
`~
`
`CONTROL
`-
`TRL 0.3 mg/kg *
`- - -
`----· TRL 1 mg/kg *
`TRL 3 mg/kg *
`TRL 1 0 mg/kg •
`
`{n=B)
`
`SUN - IPR2017-01929, Ex. 1006, p. 4 of 30
`
`

`

`U.S. Patent
`
`Dec. 21, 1999
`
`Sheet 2 of 11
`
`6,004,565
`
`CsA 3 mg/kg
`-
`- F1Y720 0.1 mg/kg + CsA 3 mg/kg •
`- -
`----- FIY720 0.3 mg/kg + CsA 3 mg/kg •
`--- FIY720 1 mg/kg + CsA 3 mg/kg •
`---- FIY720 3 mg/kg + CsA 3 mg/kg •
`FIY720 10 mg/kg + CsA 3 mg/kg •
`-
`(n=8}
`
`1 0
`
`50
`40
`30
`20
`DAYS AFTER TRANSPLANTATION
`
`FIG. 2A
`
`60
`
`70
`
`CsA 3 mg/kg
`-
`- - - FIY720 0.1 mg/kg + CsA 10 mg/kg •
`----- FIY720 0.3 mg/kg + CsA 10 mg/kg •
`--- FTY720 1 mg/kg + CsA 10 mg/kg •
`---- FTY720 3 mg/kg + CsA 10 mg/kg •
`FTY720 10 mg/kg + CsA 10 mg/kg •
`-
`~~~----~~------~-
`(n=B}
`
`U')
`
`~ 100
`~ 80
`i 60
`(..!) 40
`l.L-
`0
`20
`t -::z:
`u..J 0
`(..)
`0
`0:::
`u..J a..
`
`_ J
`
`U')
`
`~ 100
`~ 80
`i 60
`(..!) 40
`l.L-
`0
`20
`1-::z:
`u..J 0
`(..)
`0
`0:::
`u..J a..
`
`10
`
`50
`40
`30
`20
`DAYS AFTER TRANSPLANTATION
`
`FIG. 28
`
`60
`
`70
`
`l
`
`-----.-------.
`
`l_ \'1__,
`
`L-.,
`L--,
`
`-
`
`TRL 3 mg/kg
`
`---FTY720 0.1 mg/kg + TRL 3 mg/kg •
`
`----- FTY720 1 mg/kg + TRL 3 mg/kg •
`
`L1
`L---
`
`60
`
`70
`
`10
`
`I
`
`50
`40
`30
`20
`DAYS AFTER TRANSPLANTATION
`
`FIG. 2C
`
`~:~
`
`SUN - IPR2017-01929, Ex. 1006, p. 5 of 30
`
`

`

`U.S. Patent
`
`Dec. 21, 1999
`
`Sheet 3 of 11
`
`6,004,565
`
`CONTROl
`-
`- FlY720 0.1 mg/kg •
`- -
`----- FlY720 0.3 mg/kg •
`- ·- F1Y720 1 mg/kg •
`---- F1Y720 3 mg/kg •
`-
`FlY720 10 mg/kg •
`
`(n=B)
`
`0
`20
`~
`~ 0
`0
`L.s..l
`a...
`
`1
`L
`~-.------~~-=
`10 20 30
`40 50 60 70 80
`90 100
`DAYS NlER TRANSPlANTATION
`FIG. 38
`
`~ 100 .,..--,.L.,--·n_
`l "\_
`~ 80
`J_~ -------,
`~ 60
`~ 40
`
`L.-,
`
`CONTROL
`-
`- CsA 3 mg/kg
`- -
`----- CsA 10 mg/kg •
`--- CsA 30 mg/kg •
`{n=8)
`
`CONTROl
`-
`- - - TRL 0.3 mg/kg
`----- TRL 1 mg/kg •
`--- TRL 3 mg/kg •
`{n=B)
`
`SUN - IPR2017-01929, Ex. 1006, p. 6 of 30
`
`

`

`U.S. Patent
`
`Dec. 21, 1999
`
`Sheet 4 of 11
`
`6,004,565
`
`;t
`~100
`~ 80
`~ 60
`<...:> 40
`u...
`0
`20
`...... :z
`....... 0
`0
`....... c..
`
`(...)
`0::
`
`-
`CsA 3 mg/kg
`nY720 0.03 mg/kg + CsA 3 mg/kg
`-
`- FTY720 0.1 mg/kg + CsA mg/kg •
`- -
`----- FTY720 0.3 mg/kg + CsA 3 mg/kg•
`--- F1Y720 1 mg/kg + CsA 3 mg/kg •
`-,~
`T~ -·-··
`---- FTY720 3 mg/kg + CsA 3 mgfkg •
`(n=B)
`----- =t__
`------------
`
`10
`
`50
`40
`30
`20
`DAYS AFTER TRANSPLANTATION
`
`FIG. 4A
`
`60
`
`70
`
`i 100
`
`V')
`
`~ 80
`t: 60
`5 40
`u...
`0
`20
`...... :z
`....... 0
`~ .......
`0
`c..
`
`TRL1 mg/kg
`-
`- FTY720 0.1 mg/kg + TRL 1 mg/kg
`- -
`----- nY720 1 mg/kg + TRL 1 mg/kg •
`(n=S.-.8)
`
`---il
`L~--------------------
`
`1
`
`10
`
`50
`40
`30
`20
`OAYS AFTER TRANSPLANTATION
`
`FIG. 48
`
`60
`
`70
`
`SUN - IPR2017-01929, Ex. 1006, p. 7 of 30
`
`

`

`U.S. Patent
`
`Dec. 21, 1999
`
`Sheet 5 of 11
`
`6,004,565
`
`0'
`
`-:;;;- 15
`....._
`..§.. 10
`LA.J
`:z:
`z
`~
`~ 0
`u
`0
`
`5
`
`-:;;;- 15
`....._
`..§.. 10
`LA.J
`:z:
`z
`3 ~ 0
`
`0'
`
`u
`
`5
`
`0
`
`-:;;;- 15
`....._
`..§.. 10
`La...l
`:z:
`z
`8 c::: 0
`
`0'
`
`u
`
`5
`
`0
`
`0'
`
`~ 15
`....._
`.§.. 10
`.....
`:z:
`z
`5
`~ c::: 0
`0
`
`u
`
`CONTROL {n=3)
`
`20
`
`60
`40
`DAYS AFTER TRANSPLANTATION
`
`80
`
`100
`
`FIG.5A
`
`CsA 10mg/kg {n=3)
`
`20
`
`60
`40
`DAYS AFTER TRANSPLANTATION
`
`80
`
`100
`
`FIG.58
`
`FTY720 5 mg/kg {n=3)
`
`20
`
`60
`40
`DAYS AFTER TRANSPLANTATION
`
`80
`
`100
`
`FIG.5C
`
`F1Y720 5 mg/kg + CsA 10 mg/kg {n=5)
`
`20
`
`60
`40
`DAYS AFTER TRANSPLANTATION
`
`80
`
`100
`
`FIG. 50
`
`SUN - IPR2017-01929, Ex. 1006, p. 8 of 30
`
`

`

`U.S. Patent
`
`Dec. 21, 1999
`
`Sheet 6 of 11
`
`6,004,565
`
`~ 100
`~
`6:
`::>
`U')
`
`CONTROL
`CsA 10 mg/kg
`FTY720 5 mg/kg
`FTY720 5 mg/kg + CsA 10 mg/kg •
`
`80
`~ 60
`40
`
`(.!)
`
`L.o...
`C>
`1--::z:
`LA.J
`u
`a:::
`LA.J
`0...
`
`20
`
`0
`0
`
`20
`
`60
`40
`DAYS AfTER TRANSPLANTATION
`
`80
`
`100
`
`FIG.6
`
`100
`
`80
`
`~
`5:
`::>
`U')
`
`CsA 10 mg/kg
`fTY720 0.1 mg/kg + CsA 10 mg/kg •
`fTY720 0.3 mg/kg + CsA 10 mg/kg •
`FTY720 1 mg/kg + CsA 10 mg/kg •
`- - fTY720 3 mg/kg + CsA 10 mg/kg •
`
`~ 60
`40
`
`1--
`
`(.!)
`
`L.o...
`C>
`1--::z:
`LA.J
`u
`0::
`LA.J
`0...
`
`L - - - - - J,. --'--+--_,.,
`L---L__..._--n-
`
`I
`
`I
`
`'
`
`20
`
`0
`0
`
`20
`
`60
`40
`DAYS AFTER TRANSPLANTATION
`
`80
`
`100
`
`FIG. 7
`
`SUN - IPR2017-01929, Ex. 1006, p. 9 of 30
`
`

`

`U.S. Patent
`
`Dec. 21, 1999
`
`Sheet 7 of 11
`
`6,004,565
`
`-o-- FTY720
`•
`CsA
`
`*:p<0.05, **:p<0.01
`(DUNNETT'S TEST VS CONTROL)
`n=5, MEAN ± S.E.
`
`0.01
`
`t
`0.1
`DOSE (mg/kg p.o.)
`
`tO
`
`100
`
`FIG.8
`
`------------·-----·-r;----,
`! 1
`. I
`I
`t_ ___ _
`~ L l
`L __
`
`~
`•
`~
`
`CONTROL
`F1Y720 0. t mg/kg •
`F1Y720 0.3 mg/kg *
`CsA
`1 0 mg/kg •
`
`(n=5)
`
`60
`
`~- 100
`:::J~ 80
`o..E
`0 ' - "
`a..,_
`_,:I:
`oS2
`a:: Lo.J
`z==
`0
`L&..l 40
`uo
`0
`~:z
`,_:X: 20
`:z:O..
`tj~
`a::
`L&..l
`0..
`
`0
`
`100
`
`I 80
`
`~ 60
`Vl
`L.&...
`0
`,_
`:z
`t..J u
`a::
`t..J
`0..
`
`40
`
`20
`
`0
`0
`
`1 0
`
`50
`40
`30
`20
`DAYS AFTER TRANSPLANTATION
`
`60
`
`FIG.9
`
`SUN - IPR2017-01929, Ex. 1006, p. 10 of 30
`
`

`

`U.S. Patent
`
`Dec. 21, 1999
`
`Sheet 8 of 11
`
`6,004,565
`
`CONTROL
`
`FTY120 0. 1 mg/kg
`
`FTY720 1 mg/kg
`
`FlY720 1 0 mgkg
`
`~----~--~------~
`
`...__.........._ __ __,
`
`~--
`
`0
`
`8000
`6000
`4000
`2000
`NUMBER Of CELLS (CELLS/#J)
`CJ CD3-POSITIVE T CELLS
`CJ CD45RA OR A/B-POSITNE B CELLS
`C1 CD3. C045RA OR A/8-00UBlE NEGATIVE CEllS
`
`10000
`
`FIG. 11
`
`SUN - IPR2017-01929, Ex. 1006, p. 11 of 30
`
`

`

`U.S. Patent
`
`Dec. 21, 1999
`
`Sheet 9 of 11
`
`6,004,565
`
`PERIPHERAL BLOOD
`
`THORACIC DUCT
`
`SPLEEN
`
`MESENTERIC LYMPH NODE
`
`AXILLARY lYMPH NODE
`
`POPLITEAL LYMPH NODE
`
`FlY720
`-
`FlY720
`FlY720
`
`FlY720
`
`T CELLS
`-
`E3 8 CELLS
`D OTHER CELLS
`
`0
`
`50
`
`150
`100
`% OF CONTROL
`
`200
`
`250
`
`FIG. 12
`
`SUN - IPR2017-01929, Ex. 1006, p. 12 of 30
`
`

`

`U.S. Patent
`
`Dec. 21, 1999
`
`Sheet 10 of 11
`
`6,004,565
`
`PERIPHERAL LYMPH NODE
`
`CONTROL
`
`FTY720 0.1 mg/kg
`
`FlY720
`
`1 mg/kg
`
`•
`
`~~~~~~~~~~
`
`0.10
`
`0
`
`0.08
`
`0.06
`0.04
`0.02
`LYMPHOCYTE HOMING
`(~ OF MALE/FEMALE DNA)
`FIG. 13A
`
`CONTROL
`
`f1Y720 0.1 mg/kg
`
`f1Y720
`
`1 mg/kg
`
`PEYER'S PATCH
`
`•
`
`0
`
`0.02 0.04 0.06 0.08 0.10 0.12 0.14 0.16
`LYMPHOCYTE HOMING
`(~ OF MALE/fEMALE DNA)
`FIG. 138
`
`SUN - IPR2017-01929, Ex. 1006, p. 13 of 30
`
`

`

`U.S. Patent
`
`Dec. 21, 1999
`
`Sheet 11 of 11
`
`6,004,565
`
`MESENTERIC LYMPH NODE
`
`•
`
`CONTROL
`
`F1Y720 0.1 mg/kg
`
`FTY720
`
`1 mg/kg
`
`~~~~~~~~~~
`0
`0.02 0.04 0.06 0.08 0.10 0.12 0.14
`lYMPHOCYTE HOMING
`{% OF UAL.E/FEMALE DNA)
`FIG. 13C
`
`SPLEEN
`
`CONTROL
`
`FTY720 0.1 mg/kg
`
`FTY720
`
`1 mg/kg
`
`0
`
`0.8
`
`1.0
`
`0.2
`
`0.6
`0.4
`LYMPHOCYTE HOMING
`(% Of MALE/FEMALE DNA}
`FIG. 130
`
`SUN - IPR2017-01929, Ex. 1006, p. 14 of 30
`
`

`

`6,004,565
`
`1
`COMPOSITIONS AND METHODS OF USING
`COMPOSITIONS WITH ACCELERATED
`LYMPHOCYTE HOMING
`IMMUNOSUPPRESSIVE PROPERTIES
`
`BACKGROUND OF THE INVENTION
`
`1. Field of the Invention
`This invention relates to the chemistry and biology of
`compounds with immunosuppressive and lymphocyte hom(cid:173)
`ing activities and, more specifically, this invention relates to
`methods and comprises compositions for accelerating lym(cid:173)
`phocyte homing in a mammal.
`2. Description of Related Art
`In general, compounds used to suppress the immune
`response attack certain immune cells. By either removing
`these cells from the immune system or hampering their
`ability to respond to chemical messengers, the number of
`cells participating in any immune response decreases. With
`fewer cells responding, the immune system cannot mount 20
`the same response reaction. The result is immunosuppres-
`SlOn.
`The use of these compounds follows directly from our
`understanding of the immune response and the function of
`immune cells. Numerous publications in the art describe the 25
`molecular and cellular aspects of the immune response.
`Generally, the immune system responds to an antigen first by
`processing and presenting the antigen through antigen pre(cid:173)
`senting cells (APCs). Over the last decade, intensive
`research has resulted in a deep and detailed knowledge of 30
`this process at the molecular level (1-3). Following the
`APCs action are the T lymphocytes or T cells. Activated by
`a process involving antigen presentation by the APC, T cells
`then initiate the development of a variety of effector immune
`cells. The activities of phagocytes, natural killer cells, cyto- 35
`toxic T cells, and B cells and other effector cells each arise
`from the cytokines secreted from activated T cells. The
`cytokines, then, are the chemical messengers that trigger the
`immune response mechanisms that the effector cells per-
`form.
`By killing or modifying the cells or messengers of the
`immune system, certain compounds can be used as treat(cid:173)
`ments and therapies for suppressing the immune response.
`Our understanding of the immune response has led to two 45
`general groups of immunosuppressive compounds, those
`compounds effecting cytokine action and those directly
`effecting immune cell metabolism and activity.
`In the first group are cyclosporin A (CsA), tacrolimus
`(TRL) and rapamycin (4-5). CsA, a cyclic peptide, is pro- 50
`duced from the Trichoderma polysporum fungus (6-7). TRL,
`or FK-506, is a macrolide from Streptomyces tsukubaensis
`(8-10). These compounds cut out the cytokine messengers
`of the immune response by preventing their synthesis. The
`immune effector cells, therefore, cannot be recruited to 55
`complete the immune response. Rapamycin, on the other
`hand, blocks the cytokine signal from effecting the immune
`cells (34).
`More specifically, CsA suppresses the immune response
`by inhibiting production of the cytokine interleukin 2 (IL-2)
`in antigen-stimulated helper T cells, a subset ofT cells. TRL
`inhibits antigen-induced T cell proliferation by inhibiting
`IL-2 production in helper T cells. CsA and TRL act by
`binding to two different proteins (11), called cyclophilin and
`FKBP respectively. After the binding, both of the CsN
`cyclophilin and TRUFKBP complexes inhibit the phos(cid:173)
`phatase activity of a protein called calcineurin, which acti-
`
`5
`
`2
`vates nuclear factor (NF-AT) in activated T cells. NF-AT
`promotes IL-2 gene transcription and thus IL-2 production.
`However, when the CsNcyclophilin and TRLUFKBP com(cid:173)
`plexes inhibit activation of NF-AT, production of IL-2 is also
`inhibited.
`Since CsA and TRL have almost the same mechanism of
`action, these drugs also show quite similar side effects, such
`as renal and liver toxicity (12). Multiple drug therapies with
`either CsA or TRL, using steroids or other immunosuppres-
`10 sants such as azathioprine and mizoribine (13-14), were
`widely used in order to reduce the side effects of individual
`immunosuppressants. However, the similar side effects of
`CsA and TRL prohibits their use together. New immuno(cid:173)
`suppressant compounds should not only be highly safe but
`15 should also possess a mechanism of action distinct from CsA
`and TRL so that they can be concomitantly administered.
`Compounds from the second group of similarly-acting
`chemicals each interfere with nucleoside synthesis in the
`immune cells, arresting their metabolism and their immune
`activity. The group includes azathioprine (35), mizoribine
`(36), mycophenolic acid (37), and brequinar sodium (38).
`These compounds can also result in toxic side effects.
`Researchers and clinicians use these compounds in human
`therapies. Those employing CsA have made great contribu(cid:173)
`tions to the prevention of acute rejection in human organ
`transplantation. Immunosuppressants are also used to treat
`autoimmune diseases, such as rheumatoid arthritis, and
`diseases such as psoriasis, atopic dermatitis, bronchial
`asthma, and pollinosis. However, because of the toxic side
`effects of the currently used compounds, new, more effective
`and less toxic methods to suppress the immune response are
`needed in the art.
`
`SUMMARY OF THE INVENTION
`
`40
`
`The instant invention involves compositions and methods
`that suppress the immune response in mammals in a novel
`way. This immunosuppression results from accelerating
`lymphocyte homing, for example to any of the mesenteric or
`peripheral lymph tissues or Peyer's patches. This new
`activity, accelerated lymphocyte homing immunosuppres(cid:173)
`sion (ALH-immunosuppression), can be used in conjunction
`with other immunosuppressive therapies or compounds
`while avoiding dangerous or toxic side effects. The present
`invention provides new and useful methods, therapies,
`treatments, and compositions wherever immunosuppression
`is desired or manipulating lymphocyte populations is
`desired. For example, the invention can be used in therapies
`or treatments for preventing rejection in organ or cell
`transplantation, genetically modified cell therapy, ex vivo
`gene therapy, or other cell therapy methods. Research and
`development may provide additional or related uses directed
`to the intestinal immune system and the maintenance or
`manipulation of intestinal intraepithelial lymphocyte func(cid:173)
`tion. Thus, the ALH-immunosuppressive compositions of
`the invention can be used to direct or redirect lymphocytes
`within a mammal. Such uses do not necessarily require an
`immunosuppressive action.
`In one embodiment, the invention provides a method of
`60 suppressing the immune response by accelerating lympho(cid:173)
`cyte homing to any of the mesenteric or peripheral lymph
`tissues or Peyer's patches. This embodiment can be used to
`suppress the immune response in a mammal and comprises
`administering an ALH-immunosuppressive compound. The
`65 ALH-immunosuppressive compounds of this invention
`functionally act by directing lymphocytes to specific loca(cid:173)
`tions or lymphoid tissues. This lymphocyte homing activity
`
`SUN - IPR2017-01929, Ex. 1006, p. 15 of 30
`
`

`

`6,004,565
`
`3
`can be reversible, so that suspending treatment restores
`normal lymphocyte populations. The compounds may also
`act to selectively decrease populations of certain lympho(cid:173)
`cytes in blood or lymph tissue, such as specifically decreas(cid:173)
`ing populations of circulating lymphocytes or spleen lym-
`phocytes.
`Structurally, the class of ALH-immunosuppressive com(cid:173)
`pounds derives from myriocin or ISP-1, a natural product of
`Isaria sinclairii (15). Myriocin is depicted below.
`
`5
`
`4
`selected from the group consisting of an alkyl, hydroxy,
`an alkoxy, an acyloxy, amino, an akylamino, an
`acylamino, oxo, a haloalkyl, a halogen, and a phenyl,
`which may have a substituent, and wherein the phenyl,
`which may have a substituent, may have 1 to 3 sub(cid:173)
`stituents selected from the group consisting of an alkyl,
`hydroxy, an alkoxy, an acyl, an acyloxy, amino, an
`alkylamino, an acylamino, a holalkyl, and a halogen;
`
`~0
`HO-O OH
`
`OH
`
`0
`
`Numerous homologs, analogs or derivatives of these com(cid:173)
`pounds can be prepared by methods known in the art, such
`as described in the references, particularly (17), which is
`specifically incorporated herein by reference. In general, for 25
`this invention, the ALH-immunosuppressive compounds
`can be 2-aminopropane-1,3-diol compounds, according to
`the following formula:
`
`30
`
`35
`
`wherein R is an optionally substituted straight or branched
`carbon chain, an optionally substituted aryl, an option(cid:173)
`ally substituted cycloalkyl or the like;
`and R2, R3, R4, and R5 are the same or different and each
`is a hydrogen, an alkyl, an acyl, or an alkoxycarbonyl, 40
`or R4 and R5 may be bonded to form an alkylene chain,
`which may be substituted by alkyl, aryl, or an alkoxy(cid:173)
`carbonyl.
`Also, for this invention, the ALH-immunosuppressive
`compounds can be bezene compounds, of the formula:
`
`Y is hydrogen, an alkyl, hydroxy, an alkoxy, an acyl, an
`acyloxy, amino, an alkylamino, an acylamino, a
`haloalkyl, or a halogen;
`
`Z is a single bond or a straight-chain alkylene having
`carbon atoms in the number of q;
`
`p and q are the same or different and each is an integer of
`1 to 20, with the proviso that 6~p+q~23;
`
`m is 1, 2, or 3;
`
`n is 2 or 3;
`
`R1 and R2 are the same of different and each is hydrogen,
`an alkyl or an acyl;
`
`R3 is hydrogen, an alkyl or an acyl;
`
`and R4 is hydrogen, an alkyl or an acyl, where the
`benzene compounds can be optically active isomers of
`the above and salts of the compounds.
`A disclosure of specific compounds, substituent groups,
`and variations included in the ALH-immunosuppressive
`compounds of this invention can be found in U.S. Pat. No.
`45 5,604,229, copending U.S. application Ser. No. 08/801,390,
`filed Feb. 20, 1997, and PCT application PCT/JP95/01654,
`filed Aug. 22, 1995. These documents also describe methods
`to produce and isolate specific compounds that can be used
`according to
`this invention. Also, the references
`50 (particularly, 18-20) describe methods for producing these
`compounds. The entire contents of each of these patent
`documents and references are specifically incorporated by
`reference into this disclosure and can be relied on to make
`or isolate the compounds and practice this invention. The
`homo logs, analogs, or derivatives prepared can be tested, by
`one skilled in the art, to ensure that they possess ALH(cid:173)
`immunosuppressive activity, as described below.
`Furthermore, the compounds can be prepared or isolated as
`any of a number of pharmaceutically or physiologically
`acceptable salts or be prepared as optically active isomers of
`any of the described compounds.
`One preferred structural embodiment of the ALH(cid:173)
`immunosuppressive compounds used in the invention is the
`synthetic product FTY720, 2-amino-2[2-( 4-octylphenyl)
`ethyl]propane-1,3-diol hydrochloride, shown below.
`
`wherein W is hydrogen; a straight or branched chain alkyl
`having 1 to 6 carbon atoms; a straight or branched chain
`alkenyl having 2 to 6 carbon atoms; a straight or branch 55
`chain alkynyl having 2 to 6 carbon atoms; a phenyl,
`which may be substituted by hydroxy; R4(CH2)n; or a
`straight or branched chain C1-C6 alkyl substituted by
`1 to 3 substituents selected from the group consisting of
`a halogen, a cycloalkyl, and a phenyl, which may be 60
`substituted by hydroxy;
`X is hydrogen, a straight-chain alkyl having carbon atoms
`in the number of p or a straight-chain alkoxy having
`carbon atoms in the number of (p-1), wherein the
`straight-chain alkyl having carbon atoms in the number 65
`of p and the straight-chain alkoxy having carbon atoms
`in the number of (p-1) may have 1 to 3 substituents
`
`SUN - IPR2017-01929, Ex. 1006, p. 16 of 30
`
`

`

`6,004,565
`
`5
`
`•HCl
`
`5
`
`6
`culating blood versus lymphocytes in peripheral or mesen(cid:173)
`teric lymph nodes, before and after treatment, any ALH(cid:173)
`immunosuppressive activity can be identified. In these
`methods, rodents such as rats and mice, may be used and
`transplanted tissue or cells can be heart, kidney, or skin
`tissue. Also, for this aspect of the invention, populations of
`pre-labeled lymphocytes can be introduced into a mammal
`and detected following administration of a sample. Certain
`patterns of lymphocyte homing can result when the sample
`1