U.S. patent application number 14/060239 was filed with the patent office on 2014-07-03 for glycolipids and analogues thereof as antigens for nkt cells.
This patent application is currently assigned to THE SCRIPPS RESEARCH INSTITUTE. The applicant listed for this patent is THE ROCKEFELLER UNIVERSITY, THE SCRIPPS RESEARCH INSTITUTE. Invention is credited to Masakazu FUJIO, David D. HO, Xiangming LI, Moriya TSUJI, Chi-Huey WONG, Douglass WU.
Application Number | 20140186378 14/060239 |
Document ID | / |
Family ID | 39671771 |
Filed Date | 2014-07-03 |
United States Patent
Application |
20140186378 |
Kind Code |
A1 |
TSUJI; Moriya ; et
al. |
July 3, 2014 |
GLYCOLIPIDS AND ANALOGUES THEREOF AS ANTIGENS FOR NKT CELLS
Abstract
This invention relates to immunogenic compounds which serve as
ligands for NKT (natural killer T) cells and to methods of use
thereof in modulating immune responses.
Inventors: |
TSUJI; Moriya; (New York,
NY) ; HO; David D.; (New York, NY) ; WONG;
Chi-Huey; (Rancho Santa Fe, CA) ; WU; Douglass;
(San Diego, CA) ; FUJIO; Masakazu; (San Diego,
CA) ; LI; Xiangming; (Elmhurst, NY) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
THE SCRIPPS RESEARCH INSTITUTE
THE ROCKEFELLER UNIVERSITY |
La Jolla
New York |
CA
NY |
US
US |
|
|
Assignee: |
THE SCRIPPS RESEARCH
INSTITUTE
La Jolla
CA
THE ROCKEFELLER UNIVERSITY
New York
NY
|
Family ID: |
39671771 |
Appl. No.: |
14/060239 |
Filed: |
October 22, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
13420738 |
Mar 15, 2012 |
8586051 |
|
|
14060239 |
|
|
|
|
12958996 |
Dec 2, 2010 |
8163290 |
|
|
13420738 |
|
|
|
|
11735313 |
Apr 13, 2007 |
7923013 |
|
|
12958996 |
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|
11317900 |
Dec 27, 2005 |
7534434 |
|
|
11735313 |
|
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60639408 |
Dec 28, 2004 |
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Current U.S.
Class: |
424/184.1 ;
424/283.1 |
Current CPC
Class: |
A61P 37/06 20180101;
A61P 37/02 20180101; A61P 37/04 20180101; C07H 15/10 20130101; C07H
15/04 20130101; A61P 37/00 20180101; A61K 39/39 20130101 |
Class at
Publication: |
424/184.1 ;
424/283.1 |
International
Class: |
C07H 15/04 20060101
C07H015/04; A61K 39/39 20060101 A61K039/39 |
Goverment Interests
STATEMENT OF FEDERALLY SPONSORED RESEARCH
[0002] This invention was made in whole or in part with government
support under grant number GM44154, awarded by the National
Institute of Health. The government may have certain rights in the
invention.
Claims
1-40. (canceled)
41. A method for stimulating an immune response in a subject in
need of said stimulating of the immune response, the method
comprising: (a) administering to the subject a therapeutically
effective amount of a compound, composition, or vaccine designed to
stimulate an immune response, wherein the compound, composition, or
vaccine, comprises an adjuvant, wherein the adjuvant comprises a
compound represented by the structure of formula 1: ##STR00157##
wherein, R=COOR.sub.1 or CH.sub.2OR.sub.1; R.sub.1=H or an alkyl
group; R.sub.2=H or SO.sub.3.sup.-; R.sub.3=H or OH; R.sub.3'=H or
OH; R.sub.4 and R.sub.4' are independently chosen from (a) H, (b)
alkyl; (c) alkenyl; and (d) oxaalkyl; R.sub.5=OH, acetamido or a
halogen atom; or a pharmaceutically acceptable salt thereof,
wherein if R=CH.sub.2OR.sub.1 where R.sub.1 is H, then
R.sub.2=SO.sub.3.sup.-, and wherein if R=CH.sub.2OR.sub.1,
R.sub.2=H, R.sub.3 is OH and R.sub.3' is H, then R.sub.5=acetamido
or a halogen atom; or a compound represented by the structure of
formula (17): ##STR00158## wherein R.sub.4 is selected from: (a)
alkyl, (b) alkenyl, (c) alkyl terminating in aryl, substituted
aryl, heteroaryl or substituted heteroaryl; and (d) alkenyl
terminating in aryl, substituted aryl, heteroaryl or substituted
heteroaryl, and R.sub.6 is ##STR00159## X is an alkyl chain; and
R.sub.7 is chosen from halogen, H, phenyl, alkyl, alkoxy, nitro,
and CF.sub.3.
42. The method according to claim 41, wherein R.sub.4 and X of the
structure of formula (17) are saturated alkyl groups containing
from 1 to 30 carbons.
43. The method according to claim 42, wherein R.sub.4 and X of the
structure of formula (17) are saturated alkyl groups containing
from 5 to 25 carbons.
44. The method according to claim 43, wherein R.sub.7 of the
structure of formula (17) is H.
45. The method according to claim 44, wherein the compound is
selected from the group consisting of: ##STR00160##
##STR00161##
46. The method according to claim 43, wherein R.sub.7 of the
structure of formula (17) is halogen.
47. The method according to claim 41, wherein the compound is
selected from the group consisting of: ##STR00162##
48. The method according to claim 43, wherein R.sub.7 of the
structure of formula (17) is alkoxy.
49. The method according to claim 48, wherein the compound is
selected from the group consisting of: ##STR00163##
50. The method according to claim 43, wherein R.sub.7 of the
structure of formula (17) is CF.sub.3.
51. The method according to claim 50, wherein the compound is
selected from the group consisting of: ##STR00164##
52. The method according to claim 43, wherein R.sub.7 of the
structure of formula (17) is phenyl.
53. The method according to claim 52, wherein the compound is
selected from the group consisting of: ##STR00165##
54. The method according to claim 41, wherein the structure of
formula (17): R.sub.4 is ##STR00166## X is an alkyl chain; and
R.sub.7 is chosen from halogen, H, phenyl, alkyl, alkoxy, nitro,
and CF.sub.3.
55. The method according to claim 54, wherein R.sub.6 and X are
saturated alkyl groups containing from 1 to 30 carbons.
56. The method according to claim 55, wherein R.sub.6 and X are
substituted alkyl groups containing from 2 to 10 carbons.
57. The method according to claim 56, wherein R.sub.7 is H.
58. The method according to claim 57, wherein the compound is
selected from the group consisting of: ##STR00167##
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This Application is a continuation of U.S. patent
application Ser. No. 13/420,738, filed Mar. 15, 2012, now U.S. Pat.
No. 8,586,051, issued Nov. 19, 2013, which is a continuation of
U.S. patent application Ser. No. 12/958,996, filed Dec. 2, 2010,
now U.S. Pat. No. 8,163,290, issued Apr. 24, 2012, which is a
continuation of U.S. patent application Ser. No. 11/735,313, filed
Apr. 13, 2007, now U.S. Pat. No. 7,923,013, issued Apr. 12, 2011,
which is a continuation-in-part of U.S. patent application Ser. No.
11/317,900, filed Dec. 27, 2005, now U.S. Pat. No. 7,534,434,
issued May 19, 2009, which claims the benefit of U.S. Provisional
Patent Application Ser. No. 60/639,408, filed Dec. 28, 2004, each
of which is incorporated by reference herein in its entirety.
FIELD OF THE INVENTION
[0003] This invention relates to immunogenic compounds that serve
as ligands for natural killer T (NK T) cells and to methods of use
thereof in modulating immune responses.
BACKGROUND OF THE INVENTION
[0004] CD1 molecules are a family of highly conserved antigen
presenting proteins that are similar in function to classical MHC
molecules. While classical MHC molecules present peptides, CD1
proteins bind and display a variety of lipids and glycolipids to T
lymphocytes. The five known isoforms are classified into two
groups, group I (CD1a, CD1b, CD1c, and CD1e in humans) and group II
(CD1d in humans and mice) based on similarities between nucleotide
and amino acid sequences.
[0005] A great diversity of lipids and glycolipids has been shown
to bind specifically to each of the four isoforms. The first
crystal structure of murine (m) CD1d revealed that it adopts a
folded conformation closely related to major histocompatibility
complex (MHC) class I proteins. It further revealed that mCD1d
could accommodate long lipid tails in two hydrophobic pockets,
designated A' and F', located in the binding groove. Two additional
structures of hCD1b and hCD1a confirmed this model by demonstrating
that CD1, when loaded with antigenic glycolipids, binds the lipid
portion in a hydrophobic groove while making available the
hydrophilic sugar moiety to make contact with the T-cell
receptor.
[0006] Mammalian and mycobacterial lipids are known to be presented
by human CD1a, CD1b, CD1c and CD1d [Porcelli, S. A. & Modlin,
R. L. (1999) Annu. Rev. Immunol. 17, 297-329].
.alpha.-Galactosylceramide (.alpha.-GalCer), a lipid found in the
marine sponge Agelas mauritianus, has been, to date, the most
extensively studied ligand for CD1d. .alpha.-GalCer, when bound to
CD1d, stimulates rapid Th1 and Th2 cytokine production by
V.alpha.14i natural killer T (V.alpha.14i NKT) cells in mice and
the human homologue V.alpha.24i NKT cells. However, its
physiological significance in mammals remains unclear as it is
enigmatic why an .alpha.-galactosyl ceramide of marine origin is
such a potent agonist. Other known ligands, such as a bacterial
phospholipid (PIM.sub.4), a tumor derived ganglioside GD3, a
C-linked analog of .alpha.-GalCer, .alpha.-GalCer analogues with
different lipid chain lengths and a phosphoethanolamine, found in
human tumor extracts, stimulate only relatively small populations
of CD1d-restricted NKT cells.
[0007] Natural Killer (NK) cells typically comprise approximately
10 to 15% of the mononuclear cell fraction in normal peripheral
blood. Historically, NK cells were first identified by their
ability to lyse certain tumor cells without prior immunization or
activation. NK cells also serve a critical role in cytokine
production, which are thought to be involved in controlling cancer,
infection and fetal implantation.
[0008] In view of the above, it would be desirable to elucidate
additional glycolipid compounds and/or analogues of known
glycolipids, such as .alpha.-GalCer, that enhance cell mediated
immune responses and that are useful for the development of
glycolipid-containing adjuvants and vaccines and for any
immunotherapy designed to stimulate NKT cells.
SUMMARY OF THE INVENTION
[0009] The present invention is based in part on the discovery of
novel glycolipid analogues that enhance cell mediated immune
responses and that are useful for developing vaccines for treating
a variety of diseases and disorders requiring immunotherapy and
stimulation of NK T cells.
[0010] This invention provides, in one embodiment, a compound
represented by the structure of formula 1:
##STR00001##
[0011] wherein, [0012] R=COOR.sub.1 or CH.sub.2OR.sub.1; [0013]
R.sub.1=H or an alkyl group; [0014] R.sub.2=H or SO.sub.3.sup.-;
[0015] R.sub.3=H or OH; [0016] R.sub.3'=H or OH; [0017] R.sub.4 and
R.sub.4' are independently chosen from [0018] (a) H, [0019] (b)
alkyl; [0020] (c) alkenyl; and [0021] (d) oxaalkyl; [0022]
R.sub.5=OH, acetamido or a halogen atom; [0023] or a
pharmaceutically acceptable salt thereof, [0024] wherein if
R=CH.sub.2OR.sub.1, R.sub.2=H, R.sub.3 is OH and R.sub.3' is H,
then one of the following is true: [0025] (i) R.sub.5=acetamido,
halogen atom or OH in an axial position; [0026] (ii) R.sub.4=H, or
unsaturated or saturated alkyl chain having 9 carbon atoms or
fewer; or [0027] (iii) R.sub.4=H, or unsaturated or saturated alkyl
chain having 20 carbon atoms or fewer.
[0028] In another embodiment, this invention provides a compound
represented by the structure of formula 2:
##STR00002## [0029] wherein [0030] R=COOR.sub.1 or
CH.sub.2OR.sub.1; [0031] R.sub.1=H or an alkyl group; [0032]
R.sub.2=H or SO.sub.3.sup.-; [0033] R.sub.3=H or OH; [0034]
R.sub.3'=H or OH; and [0035] R.sub.4=H, unsaturated or saturated,
alkyl group; and [0036] R.sub.4'=H, unsaturated or saturated, alkyl
group; [0037] or a pharmaceutically acceptable salt thereof, [0038]
wherein if R=CH.sub.2OR.sub.1, R.sub.2=H, R.sub.3 is OH and
R.sub.3' is H, then R.sub.4=H, unsaturated or saturated, alkyl
chain having 9 carbon atoms or fewer, or R.sub.4'=H, unsaturated or
saturated, alkyl chain having 20 carbon atoms or fewer.
[0039] In another embodiment, this invention provides a compound
represented by the structure of formula 3:
##STR00003##
[0040] wherein, [0041] R=COOR.sub.1 or CH.sub.2OR.sub.1; [0042]
R.sub.1=H or an alkyl group; [0043] R.sub.2=SO.sub.3.sup.-; and
[0044] n=integer; [0045] or a pharmaceutically acceptable salt
thereof.
[0046] In another embodiment, this invention provides a compound
represented by the structure of formula 4:
##STR00004##
[0047] or a pharmaceutically acceptable salt thereof.
[0048] In another embodiment, the salt may be, inter alia, a sodium
salt.
[0049] In another embodiment, this invention provides a compound
represented by the structure of formula 5:
##STR00005##
[0050] In another embodiment, this invention provides a compound
represented by the structure of formula 6:
##STR00006##
[0051] In another embodiment, this invention provides a compound
represented by the structure of formula 7:
##STR00007##
[0052] In another embodiment, this invention provides a compound
represented by the structure of formula 8:
##STR00008##
[0053] In one embodiment, this invention provides a compound
represented by the structure of formula 9:
##STR00009##
[0054] wherein, [0055] R=COOR.sub.1 or CH.sub.2OR.sub.1 [0056]
R.sub.1=H or an alkyl group; [0057] R.sub.2=H or SO.sub.3.sup.-;
[0058] R.sub.3=OH; [0059] R.sub.3'=H or OH; and [0060] R.sub.4=H,
unsaturated or saturated, alkyl group; and [0061] R.sub.4'=H,
unsaturated or saturated, alkyl group; [0062] or a pharmaceutically
acceptable salt thereof, [0063] wherein if R=CH.sub.2OR.sub.1,
R.sub.2=H, R.sub.3 is OH and R.sub.3' is H, then R.sub.4=H,
unsaturated or saturated, alkyl chain having 9 carbon atoms or
fewer, or R.sub.4=H, unsaturated or saturated, alkyl chain having
20 carbon atoms or fewer.
[0064] In another embodiment, this invention provides a compound
represented by the structure of formula 10:
##STR00010##
[0065] or a pharmaceutically acceptable salt thereof. In another
embodiment, the salt may be, inter alia, a sodium salt.
[0066] This invention provides, in one embodiment, a compound
represented by the structure of formula 11:
##STR00011## [0067] wherein, [0068] R=COOR.sub.1 or
CH.sub.2OR.sub.1; [0069] R.sub.1=H or an alkyl group; [0070]
R.sub.2=H or SO.sub.3.sup.-; [0071] R.sub.3=H or OH; [0072]
R.sub.4=H, unsaturated or saturated, alkyl group; [0073]
R.sub.5=OH, acetamido or a halogen atom; and [0074] R.sub.6=X-A
[0075] A= [0076] dialkyl phenyl;
[0076] ##STR00012## [0077] X alkyl, alkenyl, alkoxy, thioalkoxy,
substituted furan, or unsubstituted furan; [0078] Y=N or C [0079]
R.sub.7=halogen, H, phenyl, alkyl, alkoxy, nitro or CF3; and [0080]
R.sub.8=methyl or H; [0081] or a pharmaceutically acceptable salt
thereof.
[0082] In another embodiment, this invention provides a compound
represented by the structure of formula 12:
##STR00013## [0083] or a pharmaceutically acceptable salt
thereof.
[0084] In another embodiment, this invention provides a compound
represented by the structure of formula 13:
##STR00014## [0085] or a pharmaceutically acceptable salt
thereof.
[0086] In another embodiment, this invention provides a compound
represented by the structure of formula 14:
##STR00015## [0087] or a pharmaceutically acceptable salt
thereof
[0088] In another embodiment, the salt may be, inter alia, a sodium
salt.
[0089] In another embodiment, this invention provides a compound
represented by the structure of formula 15:
##STR00016##
[0090] In another embodiment, this invention provides a compound
represented by the structure of formula 16:
##STR00017##
[0091] In still another embodiment, this invention provides a
compound represented by the structure of formula 17:
##STR00018##
[0092] wherein
[0093] R.sub.4 and R.sub.6 are independently selected from: [0094]
(a) alkyl, [0095] (b) alkenyl, [0096] (c) alkyl terminating in
aryl, substituted aryl, heteroaryl or substituted heteroaryl; and
[0097] (d) alkenyl terminating in aryl, substituted aryl,
heteroaryl or substituted heteroaryl.
[0098] In certain embodiments of compounds of formula 17, [0099]
R.sub.6 is
[0099] ##STR00019## [0100] X is an alkyl chain; and [0101] R.sub.7
is chosen from halogen, H, phenyl, alkyl, alkoxy, nitro, and
CF.sub.3.
[0102] In other embodiments of compounds of formula 17, [0103]
R.sub.4 is
[0103] ##STR00020## [0104] X is an alkyl chain; and [0105] R.sub.7
is chosen from halogen, H, phenyl, alkyl, alkoxy, nitro, and
CF.sub.3.
[0106] In one embodiment, any one of the compounds of the invention
may be a ligand for an NKT (natural killer T) cell. In another
embodiment, the ligand may be in a complex with a CD1 molecule. In
another embodiment, the CD1 molecule is a CD1d molecule. In another
embodiment, the ligand stimulates NKT cells, which express a CD161+
NK marker as well as an invariant T cell antigen receptor (TCR) on
the surface thereof.
[0107] In another embodiment, the invention provides a composition
or vaccine including, inter alia, any one of the compounds of the
invention. In another embodiment, the composition or vaccine may
include, inter alia, at least one cell population. In another
embodiment, the cell population may include, inter alia, NKT cells,
antigen presenting cells, or a combination thereof.
[0108] In another embodiment, the invention provides a method for
stimulating NKT cells, the method includes, inter alia, contacting
an NKT cell with any one of the compounds of the invention.
[0109] In another embodiment, the invention provides a cell
population obtained by any one of the methods of the invention.
[0110] In another embodiment, the invention provides a method for
stimulating, inhibiting, suppressing or modulating an immune
response in a subject, the method includes, inter alia, the step of
contacting an NKT cell in the subject with any one of the compounds
of the invention.
[0111] In another embodiment, the compound according to the
invention may be in a complex with a CD1 molecule. In another
embodiment, the CD1 molecule may be CD1d. In another embodiment,
the complex may be displayed on a dendritic cell. In another
embodiment, the complex may be displayed on any antigen presenting
cell.
[0112] In one embodiment of the invention, the NKT cells secrete a
cytokine. In another embodiment the NKT cell may be a
V.alpha.24iNKT cell in humans. In another embodiment the NKT cell
may be a V.alpha.14i NKT cell in mice.
[0113] In one embodiment of the invention, the subject may be
immunocompromised. In another embodiment, the subject is infected.
In another embodiment, the subject is infected with HIV. In another
embodiment, the subject is infected with mycobacteria. In another
embodiment, the subject is infected with malaria. In another
embodiment, the subject is infected with HIV, mycobacteria, or
malaria.
[0114] In one embodiment of the invention, the subject is afflicted
with cancer. In one embodiment of the invention, the subject is at
an elevated risk for cancer. In one embodiment of the invention,
the subject has precancerous precursors.
[0115] In one embodiment of the invention, the immune response is
biased toward Th1 or Th2. In another embodiment, the subject
suffers from, or is at an elevated risk for an autoimmune disease.
In another embodiment, the biasing of the immune response results
in the suppression, inhibition or abrogation of the autoimmune
disease. In another embodiment, the subject has an inappropriate or
undesirable immune response. In another embodiment, the response is
inflammatory. In another embodiment, the inappropriate or
undesirable response exacerbates an infection, disease or symptom
in the subject.
[0116] In another embodiment, the invention provides an adjuvant
including, inter alia, any one of the compounds according to the
invention.
[0117] In another embodiment, the invention provides a method of
enhancing immunogenicity of a compound, composition, or vaccine in
a subject, the method includes, inter alia, administering to the
subject a compound, composition or vaccine further comprising an
adjuvant of according to the invention, wherein the adjuvant
enhances the immunogenicity of the compound, composition or
vaccine.
[0118] In another embodiment, the invention provides a method of
stimulating or enhancing cytokine production in a subject, the
method includes, inter alia, administering to the subject any one
of the compounds of the invention, whereby an NKT cell in the
subject secretes a cytokine following contact with the compound. In
another embodiment, the cytokine may be interferon-.gamma. or
Interleukin-4.
[0119] Furthermore, in one embodiment, the invention provides a
process for the preparation of a compound represented by the
structure of formula (4)
##STR00021## [0120] or a pharmaceutically salt thereof, the process
includes, inter alia, the step of: [0121] removing the benzyldiene
protecting group and hydrogenating of the compound represented by
the structure of formula (4a),
##STR00022##
[0121] or a salt thereof, wherein PG is a hydroxy protecting group.
In another embodiment, the hydroxy protecting group may be
benzyl.
[0122] In one embodiment of the invention, the compound of formula
(4a) may be obtained by a process that includes, inter alia, the
step of: [0123] conducting a selective sulfation of the 3'' OH of
the galactose moiety of the compound represented by the structure
of formula (4b):
##STR00023##
[0123] wherein PG is a hydroxy protecting group and R is H. In
another embodiment, the hydroxy protecting group may be benzyl.
[0124] In one embodiment of the invention, the compound of formula
(4b) wherein R is H, may be obtained by a process including, inter
alia, the step of removing the levulinyl protecting group of a
compound of formula (4b) wherein R is levulinyl, thereby obtaining
a compound of formula (4b) wherein R is H.
[0125] In one embodiment of the invention, the compound of formula
(4b) wherein R is levulinyl may be obtained by a process including,
inter alia, the step of: [0126] reacting a compound represented by
the structure of formula (4c):
##STR00024##
[0126] wherein R is H or levulinyl with hexacosanoic acid, thereby
obtaining the compound of formula (4b) wherein R is levulinyl.
[0127] In one embodiment of the invention, the compound of formula
(4c), wherein R is H or levulinyl, may be obtained by a process
including, inter alia, the step of: [0128] reducing the azide group
of a compound represented by the structure of formula (4d):
[0128] ##STR00025## [0129] wherein R is levulinyl, thereby
obtaining a compound of formula (4c) wherein R is H or
levulinyl.
[0130] In one embodiment of the invention, the compound of formula
(4d) wherein R is levulinyl, may be obtained by a process
including, inter alia, the step of: [0131] reacting a compound
represented by the structure of formula (4e)
[0131] ##STR00026## [0132] wherein PG is a hydroxy protecting
group, LG is a leaving group and R is levulinyl, [0133] with a
compound represented by the structure of formula (4f)
[0133] ##STR00027## [0134] wherein PG is a hydroxy protecting
group, [0135] to form an alpha glycosidic bond, thereby obtaining
the compound of formula (4d) wherein R is levulinyl. In another
embodiment, the leaving group may be, inter alia,
##STR00028##
[0136] In one embodiment, the invention provides a process for the
preparation of a compound represented by the structure of formula
(10)
##STR00029## [0137] or a pharmaceutically salt thereof, including,
inter alia, the step of: [0138] conducting a selective sulfation of
the 3'' OH of the galactose moiety of the compound represented by
the structure of formula (10a):
[0138] ##STR00030## [0139] In another embodiment, the sulfation may
be conducted in the presence of Bu.sub.2SnO.
[0140] In one embodiment of the invention, the compound of formula
(10a) may be obtained by the process including, inter alia, the
step of: [0141] removing the hydroxy protecting groups and
hydrogenating the compound represented by the structure of formula
(10b):
##STR00031##
[0141] wherein PG and PG.sub.1 are hydroxy protecting groups,
thereby obtaining the compound of formula (10a). In another
embodiment, the PG may be, inter alia, benzyl. In another
embodiment, the PG1 may be, inter alia, benzoyl. In one embodiment
of the invention, the compound of formula (10b) may be obtained by
a process including, inter alia, the step of: [0142] reacting a
compound represented by the structure of formula (10c):
[0142] ##STR00032## [0143] wherein PG is a hydroxy protecting
group, [0144] with a compound represented by the structure of
formula (10d):
##STR00033##
[0144] wherein PG.sub.1 is a hydroxy protecting group and LG is a
leaving group, thereby obtaining the compound of formula (10b). In
another embodiment, the leaving group may be, inter alia,
##STR00034##
[0145] In one embodiment of the invention, the compound of formula
(10c) may be obtained by a process including, inter alia, the steps
of: [0146] reducing the azide of a compound represented by the
structure of formula (10e):
[0146] ##STR00035## [0147] wherein PG and PG.sub.2 are hydroxy
protecting groups; [0148] reacting the resulting amine with
hexacosanoic acid; and removing the hydroxy protecting group
PG.sub.2, thereby obtaining the compound of formula (10c). In
another embodiment, the PG.sub.2 may be, inter alia, TIPS.
[0149] In one embodiment, the invention provides a process for the
preparation of a compound represented by the structure of formula
(18):
##STR00036## [0150] or a pharmaceutically salt thereof, including,
inter alia, the step of: [0151] conducting a selective sulfation of
the 3'' OH of the galactose moiety of the compound represented by
the structure of formula (18a):
##STR00037##
[0151] thereby obtaining the a compound represented by the
structure of formula (18). In another embodiment, the sulfation may
be conducted in the presence of Bu.sub.2SnO.
[0152] In one embodiment of the invention, the compound of formula
(18a) may be obtained by the process including, inter alia, the
step of: [0153] removing the hydroxy protecting groups of the
compound represented by the structure of formula (18b):
##STR00038##
[0153] wherein PG and PG.sub.1 are hydroxy protecting groups,
thereby obtaining the compound of formula (18a). In another
embodiment, PG may be, inter alia, benzoyl. In another embodiment,
PG.sub.1 may be, inter alia, benzoyl. In one embodiment of the
invention, the compound of formula (18b) may be obtained by a
process including, inter alia, the step of: [0154] deprotecting the
amine of a compound represented by the structure of formula
(18c):
[0154] ##STR00039## [0155] wherein PG and PG.sub.1 are hydroxy
protecting groups, and [0156] PG.sub.3 is an amino protecting
group, [0157] and reacting with nervonic acid, thereby obtaining
the compound of formula (18b). In another embodiment, the amino
protecting group may be, inter alia, tBoc.
[0158] Furthermore, in one embodiment, the invention provides a
process for the preparation of a compound represented by the
structure of formula (13)
##STR00040## [0159] or a pharmaceutically salt thereof, wherein R
is CH.sub.2OH and R.sub.2 is H, the process including, inter alia,
the step of: [0160] removing the benzyldiol protecting group and
hydrogenating of the compound represented by the structure of
formula (13a),
##STR00041##
[0160] or a salt thereof, wherein PG is a hydroxy protecting group,
R.sub.2 is H. In another embodiment, the hydroxy protecting group
may be benzyl.
[0161] In one embodiment of the invention, the compound of formula
(13a) wherein R.sub.2O is SO.sub.3.sup.-, may be obtained by a
process including, inter alia, the step of conducting a selective
sulfation of the 3'' OH of the galactose moiety of the compound
represented by the structure of formula (13a).
[0162] In one embodiment of the invention, the compound of formula
(13a) wherein R.sub.2 is H, may be obtained by a process including,
inter alia, the step of removing the levulinyl protecting group of
a compound of formula (13b) wherein R.sub.2 is levulinyl, thereby
obtaining a compound of formula (13a) wherein R.sub.2 is H.
[0163] In one embodiment of the invention, the compound of formula
(13b) wherein R.sub.2 is levulinyl may be obtained by a process
including, inter alia, the step of: [0164] reacting a compound
represented by the structure of formula (13c):
##STR00042##
[0164] wherein R.sub.2 is levulinyl with an acid form of R.sub.6,
thereby obtaining the compound of formula (13b) wherein R.sub.2 is
levulinyl.
[0165] In one embodiment of the invention, the compound of formula
(13c), wherein R.sub.2 is levulinyl, may be obtained by a process
including, inter alia, the step of: [0166] reducing the azide group
of a compound represented by the structure of formula (13d):
[0166] ##STR00043## [0167] wherein R.sub.2 is levulinyl, thereby
obtaining a compound of formula (13c).
[0168] In one embodiment of the invention, the compound of formula
(13d) wherein R.sub.2 is levulinyl, may be obtained by a process
including, inter alia, the step of: [0169] reacting a compound
represented by the structure of formula (13e)
[0169] ##STR00044## [0170] wherein PG is a hydroxy protecting
group, LG is a leaving group and R.sub.2 is levulinyl, [0171] with
a compound represented by the structure of formula (13f)
[0171] ##STR00045## [0172] wherein PG is a hydroxy protecting
group, [0173] to form an alpha glycosidic bond, thereby obtaining
the compound of formula (13d) wherein R.sub.2 is levulinyl. In
another embodiment, the leaving group may be, inter alia,
##STR00046##
[0174] In one embodiment, the invention provides a process for the
preparation of a compound represented by the structure of formula
(14)
##STR00047## [0175] or a pharmaceutically salt thereof, including,
inter alia, the step of: [0176] conducting a selective sulfation of
the 3'' OH of the galactose moiety of the compound represented by
the structure of formula (13):
[0176] ##STR00048## [0177] Wherein R.sub.2 is H and R is
CH.sub.2OH. [0178] In another embodiment, the sulfation may be
conducted in the presence of Bu.sub.2SnO.
[0179] In one embodiment of the invention, the compound of formula
(13) may be obtained by the process including, inter alia, the step
of: [0180] removing the hydroxy protecting groups and hydrogenating
the compound represented by the structure of formula (13 g):
##STR00049##
[0180] wherein PG and PG.sub.1 are hydroxy protecting groups,
thereby obtaining the compound of formula (13), wherein R.sub.2 is
H and R is CH.sub.2OH. In another embodiment, the PG may be, inter
alia, benzyl. In another embodiment, the PG1 may be, inter alia,
benzoyl. In one embodiment of the invention, the compound of
formula (13 g) may be obtained by a process including, inter alia,
the step of: [0181] reacting a compound represented by the
structure of formula (13 h):
[0181] ##STR00050## [0182] wherein PG is a hydroxy protecting
group, [0183] with a compound represented by the structure of
formula (13i):
##STR00051##
[0183] wherein PG.sub.1 is a hydroxy protecting group and LG is a
leaving group, thereby obtaining the compound of formula (13g). In
another embodiment, the leaving group may be, inter alia,
##STR00052##
[0184] In one embodiment of the invention, the compound of formula
(13 h) may be obtained by a process comprising the steps of: [0185]
reducing the azide of a compound represented by the structure of
formula (13j):
[0185] ##STR00053## [0186] wherein PG and PG.sub.2 are hydroxy
protecting groups; [0187] reacting the resulting amine with an acid
form of R.sub.6; and removing the hydroxy protecting group
PG.sub.2, thereby obtaining the compound of formula (13 h). In
another embodiment, the PG.sub.2 may be, inter alia, TIPS.
[0188] In one embodiment of the invention, the compound of formula
(13 g) may be obtained by a process including, inter alia, the step
of: [0189] deprotecting the amine of a compound represented by the
structure of formula (13k):
[0189] ##STR00054## [0190] wherein PG and PG.sub.1 are hydroxy
protecting groups, and [0191] PG.sub.3 is an amino protecting
group, [0192] and reacting with an acid form of R.sub.6, thereby
obtaining the compound of formula (13 g). In another embodiment,
the amino protecting group may be, inter alia, tBoc.
[0193] In one embodiment of the invention, an "alkyl" group refers
to a saturated aliphatic hydrocarbon, including straight-chain,
branched-chain and cyclic alkyl groups. In one embodiment, the
alkyl group has 1-30 carbons. In another embodiment, the alkyl
group has 1-25 carbons. In another embodiment, the alkyl group has
1-20 carbons. In another embodiment, the alkyl group has 1-10
carbons. In another embodiment, the alkyl group has 1-5 carbons. In
another embodiment, the alkyl group has 10-25 carbons. In another
embodiment, the alkyl group has 15-25 carbons. The alkyl group may
be unsubstituted or substituted by one or more groups selected from
halogen, hydroxy, alkoxy carbonyl, amido, alkylamido, dialkylamido,
nitro, amino, alkylamino, dialkylamino, carboxyl, thio and
thioalkyl.
[0194] The term alkyl as used throughout the specification and
claims includes both "unsubstituted alkyls" and/or "substituted
alkyls", the latter of which may refer to alkyl moieties having
substituents replacing a hydrogen on one or more carbons of the
hydrocarbon backbone. Such substituents may include, for example, a
halogen, a hydroxyl, an alkoxyl, a silyloxy, a carbonyl, and ester,
a phosphoryl, an amine, an amide, an imine, a thiol, a thioether, a
thioester, a sulfonyl, an amino, a nitro, or an organometallic
moiety. It will be understood by those skilled in the art that the
moieties substituted on the hydrocarbon chain may themselves be
substituted, if appropriate. For instance, the substituents of a
substituted alkyl may include substituted and unsubstituted forms
of amines, imines, amides, phosphoryls (including phosphonates and
phosphines), sulfonyls (including sulfates and sulfonates), and
silyl groups, as well as ethers, thioethers, selenoethers,
carbonyls (including ketones, aldehydes, carboxylates, and esters),
--CF.sub.3, and --CN. Of course other substituents may be applied.
In another embodiment, cycloalkyls may be further substituted with
alkyls, alkenyls, alkoxys, thioalkyls, aminoalkyls,
carbonyl-substituted alkyls, CF.sub.3, and CN. Of course other
substituents may be applied. In certain embodiments, the alkyl will
be otherwise unsubstituted but will terminate in aryl, substituted
aryl, heteroaryl or substituted heteroaryl. The term "unsaturated
alkyl" will appear herein interchangeably with the term "alkenyl";
both refer to the same thing. Thus the residue
--CH.dbd.CH(CH.sub.2).sub.10CH.sub.3 can be called either
"unsaturated alkyl" or "alkenyl".
[0195] Alkoxy or alkoxyl refers to groups of from 1 to 8 carbon
atoms of a straight, branched, cyclic configuration and
combinations thereof attached to the parent structure through an
oxygen. Examples include methoxy, ethoxy, propoxy, isopropoxy,
cyclopropyloxy, cyclohexyloxy and the like. Lower-alkoxy refers to
groups containing one to four carbons. Lower-alkoxy refers to
groups containing one to four carbons. Methoxy is preferred. For
the purpose of this application, alkoxy and lower alkoxy include
methylenedioxy and ethylenedioxy.
[0196] Oxaalkyl refers to alkyl residues in which one or more
carbons (and their associated hydrogens) have been replaced by
oxygen. Examples include methoxypropoxy, 3,6,9-trioxadecyl and the
like. The term oxaalkyl is intended as it is understood in the art
[see Naming and Indexing of Chemical Substances for Chemical
Abstracts, published by the American Chemical Society, 196, but
without the restriction of 127(a)], i.e. it refers to compounds in
which the oxygen is bonded via a single bond to its adjacent atoms
(forming ether bonds); it does not refer to doubly bonded oxygen,
as would be found in carbonyl groups.
[0197] Acyl refers to groups of from 1 to 8 carbon atoms of a
straight, branched, cyclic configuration, saturated, unsaturated
and aromatic and combinations thereof, attached to the parent
structure through an carbonyl functionality. One or more carbons in
the acyl residue may be replaced by nitrogen, oxygen or sulfur as
long as the point of attachment to the parent remains at the
carbonyl. Examples include acetyl, benzoyl, propionyl, isobutyryl,
t-butoxycarbonyl, benzyloxycarbonyl and the like. Lower-acyl refers
to groups containing one to four carbons.
[0198] Aryl and heteroaryl mean a 5- or 6-membered aromatic or
heteroaromatic ring containing 0-3 heteroatoms selected from O, N,
or S; a bicyclic 9- or 10-membered aromatic or heteroaromatic ring
system containing 0-3 heteroatoms selected from O, N, or S; or a
tricyclic 13- or 14-membered aromatic or heteroaromatic ring system
containing 0-3 heteroatoms selected from O, N, or S. The aromatic
6- to 14-membered carbocyclic rings include, e.g., benzene,
naphthalene, indane, tetralin, and fluorene and the 5- to
10-membered aromatic heterocyclic rings include, e.g., imidazole,
pyridine, indole, thiophene, benzopyranone, thiazole, furan,
benzimidazole, quinoline, isoquinoline, quinoxaline, pyrimidine,
pyrazine, tetrazole and pyrazole.
[0199] The term "halogen" means fluorine, chlorine, bromine or
iodine.
[0200] Any numerical values recited herein include all values from
the lower value to the upper value in increments of one unit
provided that there is a separation of at least 2 units between any
lower value and any higher value. As an example, if it is stated
that the amount of a component or a value of a process variable
such as, for example, temperature, pressure, time and the like is,
for example, from 1 to 90, preferably from 20 to 80, more
preferably from 30 to 70, it is intended that values such as 15 to
85, 22 to 68, 43 to 51, 30 to 32 etc. are expressly enumerated in
this specification. For values which are less than one, one unit is
considered to be 0.0001, 0.001, 0.01 or 0.1 as appropriate. These
are only examples of what is specifically intended and all possible
combinations of numerical values between the lowest value and the
highest value enumerated are to be considered to be expressly
stated in this application in a similar manner.
[0201] The abbreviations Me, Et, Ph, Tf, Ts and Ms represent
methyl, ethyl, phenyl, trifluoromethanesulfonyl, toluensulfonyl and
methanesulfonyl respectively. A comprehensive list of abbreviations
utilized by organic chemists (i.e. persons of ordinary skill in the
art) appears in the first issue of each volume of the Journal of
Organic Chemistry. The list, which is typically presented in a
table entitled "Standard List of Abbreviations" is incorporated
herein by reference.
[0202] As used herein, and as would be understood by the person of
skill in the art, the recitation of "a compound" is intended to
include salts, solvates and inclusion complexes of that compound.
For example, the sulfonate (SO.sub.3.sup.-) residue will often
appear in formulae herein. As will be understood by the person of
skill in the art, there is an implication of a counterion, which
can be H.sup.+ (i.e. the acid form) or any cation discussed below.
Cations of alkali metals and alkaline earth metals, as well as
ammonium cations, are common. Similarly the carboxylate may be
written COOH, but the salt form COO.sup.-M.sup.+ is included within
the scope of the invention.
[0203] Unless chirality is specifically indicated, claims to a
compound include any stereoisomeric form, or a mixture of any such
forms of that compound in any ratio. The term "solvate" refers to a
compound in the solid state, wherein molecules of a suitable
solvent are incorporated in the crystal lattice. A suitable solvent
for therapeutic administration is physiologically tolerable at the
dosage administered. Examples of suitable solvents for therapeutic
administration are ethanol and water. When water is the solvent,
the solvate is referred to as a hydrate. In general, solvates are
formed by dissolving the compound in the appropriate solvent and
isolating the solvate by cooling or using an antisolvent. The
solvate is typically dried or azeotroped under ambient conditions.
Inclusion complexes are described in Remington: The Science and
Practice of Pharmacy 19th Ed. (1995) volume 1, page 176-177, which
is incorporated herein by reference. The most commonly employed
inclusion complexes are those with cyclodextrins, and all
cyclodextrin complexes, natural and synthetic, are specifically
encompassed within the claims.
[0204] The term "pharmaceutically acceptable salt" refers to salts
prepared from pharmaceutically acceptable non-toxic acids or bases
including inorganic acids and bases and organic acids and bases.
When the compounds contain an acidic function (e.g. a carboxylic
acid or sulfonic acid residue, suitable pharmaceutically acceptable
base addition salts for the compounds of the present invention
include metallic salts made from aluminum, calcium, lithium,
magnesium, potassium, sodium and zinc, or organic salts made from
lysine, N,N'-dibenzylethylenediamine, chloroprocaine, choline,
diethanolamine, ethylenediamine, meglumine (N-methylglucamine) and
procaine.
[0205] While it may be possible for the compounds of formula (I) to
be administered as the raw chemical, it is preferable to present
them as a pharmaceutical composition. According to a further
aspect, the present invention provides a pharmaceutical composition
comprising a compound of formula (I) or a pharmaceutically
acceptable salt or solvate thereof, together with one or more
pharmaceutically carriers thereof and optionally one or more other
therapeutic ingredients. The carrier(s) must be "acceptable" in the
sense of being compatible with the other ingredients of the
formulation and not deleterious to the recipient thereof.
[0206] The formulations include those suitable for oral, parenteral
(including subcutaneous, intradermal, intramuscular, intravenous
and intraarticular), rectal and topical (including dermal, buccal,
sublingual and intraocular) administration. The most suitable route
may depend upon the condition and disorder of the recipient. The
formulations may conveniently be presented in unit dosage form and
may be prepared by any of the methods well known in the art of
pharmacy. All methods include the step of bringing into association
a compound of formula (I) or a pharmaceutically acceptable salt or
solvate thereof ("active ingredient") with the carrier which
constitutes one or more accessory ingredients.
[0207] Formulations for parenteral administration include aqueous
and non-aqueous sterile injection solutions which may contain
anti-oxidants, buffers, bacteriostats and solutes which render the
formulation isotonic with the blood of the intended recipient.
Formulations for parenteral administration also include aqueous and
non-aqueous sterile suspensions, which may include suspending
agents and thickening agents. The formulations may be presented in
unit-dose of multi-dose containers, for example sealed ampoules and
vials, and may be stored in a freeze-dried (lyophilized) condition
requiring only the addition of a sterile liquid carrier, for
example saline, phosphate-buffered saline (PBS) or the like,
immediately prior to use. Extemporaneous injection solutions and
suspensions may be prepared from sterile powders, granules and
tablets of the kind previously described.
[0208] According to embodiments of the invention, the phrase
"protecting group" as used herein means temporary modifications of
a potentially reactive functional group which protect it from
undesired chemical transformations. Examples of such protecting
groups include esters of carboxylic acids, silyl ethers of
alcohols, and acetals and ketals of aldehydes and ketones,
respectively. Of course other appropriate protecting grouped may be
used.
[0209] In one embodiment of the invention, the protecting group may
be, inter alia, a hydroxy protecting group. In one embodiment of
the invention, the hydroxy protecting group may be, inter alia, an
alkyl, aryl, aralkyl, silyl or acyl radical. In another embodiment,
the protecting group may be, inter alia, trimethylsilyl,
triethylsilyl, tert-butyldimethylsilyl (TBS), triisopropylsilyl
(TIPS), or tert-butyldiphenylsilyl. Of course, any other
appropriate protecting group may be used. In one embodiment, the
aralkyl may be unsubstituted or substituted. In another embodiment,
the aralkyl may be, inter alia, arylmethyl. In another embodiment,
the protecting group may be, inter alia, benzyl. In another
embodiment, the protecting group may be, inter alia, methoxybenzyl.
In another embodiment, the methoxybenzyl may be, inter alia,
para-methoxybenzyl.
[0210] In one embodiment of the invention, the amino protective
group may be any of amino protective group (see for example
"Protection for the amino group" in T. W. Green & P. G. M.
Wuts, Protective groups in organic synthesis, 3rd Ed., 1999,
494-653).
[0211] In one embodiment of the invention, the protecting group may
be, inter alia, an amino protecting group. In one embodiment of the
invention, the amino protecting group may be, inter alia,
carbamate, an amide or an N-sulfonylamide. In another embodiment,
the amino protecting group may be, inter alia, benzyloxycarbonyl
(Cbz), 9-fluorenylmethyloxycarbonyl (Fmoc), t-butyloxycarbonyl,
(tBoc), biphenylisopropyloxycarbonyl, t-amyloxycarbonyl,
isobornyloxycarbonyl, alpha-dimethyl-3,5-dimethoxybenzyloxycarbonyl
or 2-cyano-t-butyloxycarbonyl. In another embodiment, the amino
protecting group (PG) may be, inter alia, benzyloxycarbonyl
(Cbz).
[0212] Furthermore, in one embodiment, the invention provides a
pharmaceutical composition including, inter alia, any one of the
compounds of this invention or any combination thereof, together
with one or more pharmaceutically acceptable excipients.
[0213] Furthermore, in one embodiment, the invention provides a
method for stimulating, inhibiting, suppressing or modulating an
immune response in a subject, the method may include, inter alia,
administering to a subject any one of the compounds of this
invention or any combination thereof.
[0214] Furthermore, in one embodiment, the invention provides a
method for stimulating, inhibiting, suppressing or modulating an
immune response in a subject, the method includes, inter alia,
administering to a subject a pharmaceutical composition including,
inter alia, any one of the compounds of this invention or any
combination thereof, together with one or more pharmaceutically
acceptable excipients.
[0215] Furthermore, in one embodiment, "pharmaceutical composition"
can mean a therapeutically effective amount of one or more
compounds of the present invention together with suitable
excipients and/or carriers useful for stimulating, inhibiting,
suppressing or modulating an immune response in a subject. In one
embodiment, "therapeutically effective amount" may refer to that
amount that provides a therapeutic effect for a given condition and
administration regimen. In one embodiment, such compositions can be
administered by any method known in the art.
[0216] In one embodiment, the compositions of the present invention
are formulated as oral or parenteral dosage forms, such as uncoated
tablets, coated tablets, pills, capsules, powders, granulates,
dispersions or suspensions. In another embodiment, the compositions
of the present invention are formulated for intravenous
administration. In another embodiment, the compounds of the present
invention are formulated in ointment, cream or gel form for
transdermal administration. In another embodiment, the compounds of
the present invention are formulated as an aerosol or spray for
nasal application. In another embodiment, the compositions of the
present invention are formulated in a liquid dosage form. Examples
of suitable liquid dosage forms include solutions or suspensions in
water, pharmaceutically acceptable fats and oils, alcohols or other
organic solvents, including esters, emulsions, syrups or elixirs,
solutions and/or suspensions.
[0217] Suitable excipients and carriers may be, according to
embodiments of the invention, solid or liquid and the type is
generally chosen based on the type of administration being used.
Liposomes may also be used to deliver the composition. Examples of
suitable solid carriers include lactose, sucrose, gelatin and agar.
Oral dosage forms may contain suitable binders, lubricants,
diluents, disintegrating agents, coloring agents, flavoring agents,
flow-inducing agents, and melting agents. Liquid dosage forms may
contain, for example, suitable solvents, preservatives, emulsifying
agents, suspending agents, diluents, sweeteners, thickeners, and
melting agents Parenteral and intravenous forms should also include
minerals and other materials to make them compatible with the type
of injection or delivery system chosen. Of course, other excipients
may also be used.
BRIEF DESCRIPTION OF THE DRAWINGS
[0218] FIG. 1 demonstrates structures of
.alpha.-galactosylceramide, sulfatide and
3-O-sulfo-.alpha./.beta.-galactosylceramides 10, 24, according to
embodiments of the invention.
[0219] FIGS. 2A and 2B demonstrate the preparation of (a) compound
IV (FIG. 2A), according to embodiments of the invention; (b) the
preparation of compound 4 (FIG. 2B), according to embodiments of
the invention.
[0220] FIG. 3 demonstrates the preparation of compound XV,
according to embodiments of the invention.
[0221] FIGS. 4A and 4B demonstrate (a) the preparation of compound
XVIII (FIG. 4A), according to embodiments of the invention; (b) the
preparation of compound 10 (FIG. 4B), according to embodiments of
the invention.
[0222] FIGS. 5A, 5B, and 5C depict structures of glycolipids and
analogues thereof, according to embodiments of the invention.
[0223] FIG. 6 schematically depicts the synthesis of sphingosine,
according to embodiments of the invention, as conducted herein.
Reagents and conditions; a) C2H3MgBr, THF, Anti:Syn 3.5:1 61%; b)
(i) Grubbs catalyst 2nd generation, CH2Cl2, Pentadecene, 71%; (ii)
BzC1, pyridine, 90%; (iii) Amberlyst 15 H+ form, MeOH 70%.
[0224] FIGS. 7A and 7B schematically depict the synthesis of some
glycolipids, according to embodiments of the invention, as
conducted herein. Reagents and conditions; a) 32, TMSOTf, 67%; b)
(i) TFA, DCM, (ii) HBTU, myristic acid or 2-(S)-hydroxy myristic
acid, n-Morpholine, .apprxeq.92% 2 steps; c) H.sub.2, 20% Pd(OH)2,
(ii) LiOH, H2O:THF:MeOH, 38%, 2 steps; d) 36, TMSOTf, 60%; e) (i)
TFA, DCM, (ii) HBTU, myristic acid or 2-(S)-hydroxy myristic acid,
n-Morpholine, .apprxeq.92% 2 steps; f) (i) NaOMe, MeOH, (ii) Pd/C,
H.sub.2, EtOH, 90% 2 steps; g) 40, TMSOTf, 62%. h) (i) TFA, DCM,
(ii) HBTU, nervonic acid, n-Morpholine, 60% 2 steps; i) (i) NaOMe,
MeOH, quant. (ii) Bu.sub.2SnO, MeOH, (iii) Me.sub.3N. SO.sub.3,
THF, 95%; j-k) (i) LDA, TMSOOTMS, (ii) H+, MeOH, 30%, 2 steps; then
LiOH, H.sub.2O:MeOH:THF, 81%; 1) Novozyme 435, CH.sub.2.dbd.CHOAc,
54% based on S isomer.
[0225] FIGS. 8A and 8B demonstrate the IL-2 secretion profiles
obtained with glycolipids, according to embodiments of the
invention. FIG. 8A: IL-2 secretion profile obtained with
3-O-sulfo-GalCer, as compared to .alpha.-GalCer and analogues. FIG.
8B: Dose dependent secretion of IL-2 by Sphingomonas GSLs and
analogues.
[0226] FIGS. 9A, 9B, 9C, and 9D demonstrate human NKT cell
responses to glycolipids, according to embodiments of the
invention. Human V.alpha.24i NKT cells respond to synthetic
Sphingomonas and sulfatide glycolipids, in terms of IFN-.gamma.
(FIG. 9A) and IL-4 (FIG. 9B) release after culture with
4.times.10.sup.5 autologous immature CD14+ dendritic cells pulsed
with the indicated glycolipid antigens at 10 .mu.g/ml. Negative
controls included similar numbers of NKT cells and dendritic cells,
cultured without added glycolipid. Data represent mean.+-.S.D. of
duplicate well; FIG. 9C, in vitro INF-.gamma. secretion by human
CD161.sup.+ NK+NKT cells (2.times.10.sup.5/well) in the presence of
CD14.sup.+ DCs (4.times.10.sup.5/well) and 20 .mu.g/ml of various
glycolipids; FIG. 9D, in vitro IL-4 secretion by human CD161.sup.+
NK+NKT cells (2.times.10.sup.5/well) in the presence of
CD14.sup.+DCs (4.times.10.sup.5/well) and 20 .mu.g/ml of various
glycolipids.
[0227] FIG. 10 demonstrates a flow cytometric analysis of a human
V.alpha.24i human NKT cell line with human CD1d dimers that were
unloaded or loaded with 10M of the indicated glycolipid antigen,
according to embodiments of the invention. The cells were also
stained with anti-human CD3-PerCP.
[0228] FIG. 11 depicts a computer-generated model of GSL-1 docked
to the crystal structure of mCD1d, according to embodiments of the
invention. The two acyl tails fit nicely into the hydrophobic
pockets of the protein allowing for the sugar head group to be
presented for NKT cell recognition.
[0229] FIG. 12 demonstrates IL-2 secretion profiles obtained from
murine NKT cells presented with the glycolipids as indicated,
according to embodiments of the invention.
[0230] FIG. 13 demonstrates IL-2 secretion profiles obtained from
murine NKT cells presented with other glycolipids as indicated,
according to embodiments of the invention.
[0231] FIGS. 14, 15 and 16 demonstrate IFN-.gamma. secretion from
human NKT cells presented with the glycolipids as indicated,
supplied at the indicated concentration.
[0232] FIGS. 17 and 18 demonstrate IFN-.gamma. secretion from human
NKT cells presented with the glycolipids as indicated, supplied at
higher concentration.
[0233] FIGS. 19A, 19B, and 19C demonstrate similar IFN-.gamma.
secretion from human NKT cells presented with the glycolipids, at
the indicated concentration, in the context of Hela cells
transfected with CD1d.
[0234] FIGS. 20A and 20B demonstrate IL-4 secretion from human NKT
cells presented with the glycolipids, at the indicated
concentration in the context of dendritic cells (FIG. 20A) or
transfected Hela cells (FIG. 20B).
[0235] FIGS. 21A, 21B, 21C, 21D, 21E, and 21F demonstrate
multi-cytokine secretion from human NKT cells presented with the
glycolipids, at the indicated concentration in the context of
dendritic cells: (FIG. 21A)/(FIG. 21B) 1010A and 1010B NKT
cells--IFN-.gamma.; (FIG. 21C)/(FIG. 21D) 1010A and 1010B NKT
cells--IL-12; (FIG. 21E)/(FIG. 21F) 1010A and 1010B NKT
cells--GMCSF.
[0236] FIGS. 22A, 22B, and 22C demonstrate in vivo secretion from
B6 mice presented with indicated glycolipids: FIG. 22A,
IFN-.gamma.; FIG. 22B, IL-4; FIG. 22C, IL-12.
[0237] FIG. 23A depicts the superimposition of docking results of
compound 84 from Example 10 (yellow) with the crystal structure of
.alpha.-GalCer (green)/hCD1d complex. The .alpha.2 helix is removed
for clarity. The overall binding motif of the docked compound did
not notably deviate from the crystallized structure. The terminal
phenyl group 84 is within distance to interact with the aromatic
ring of Tyr73; FIG. 23B depicts the hCD1d docking model of analogue
compounds 83-85.
DETAILED DESCRIPTION OF THE PRESENT INVENTION
[0238] This invention provides, in one embodiment, a compound
represented by the structure of formula 1:
##STR00055## [0239] wherein, [0240] R=COOR.sub.1 or
CH.sub.2OR.sub.1; [0241] R.sub.1=H or an alkyl group; [0242]
R.sub.2=H or SO.sub.3.sup.-; [0243] R.sub.3=H or OH; [0244]
R.sub.3'=H or OH; [0245] R.sub.4 and R.sub.4' are independently
chosen from [0246] (a) H, [0247] (b) alkyl; [0248] (c) alkenyl; and
[0249] (d) oxaalkyl; [0250] R.sub.5=OH, acetamido or a halogen
atom; [0251] or a pharmaceutically acceptable salt thereof, [0252]
wherein if R=CH.sub.2OR.sub.1, R.sub.2=H, R.sub.3 is OH and
R.sub.3' is H, then one of the following is true: [0253] (i)
R.sub.5=acetamido, halogen atom or OH in an axial position; [0254]
(ii) R.sub.4=H, or unsaturated or saturated alkyl chain having 9
carbon atoms or fewer; or [0255] (iii) R.sub.4=H, or unsaturated or
saturated alkyl chain having 20 carbon atoms or fewer.
[0256] In another embodiment, this invention provides a compound
represented by the structure of formula 2:
##STR00056## [0257] wherein [0258] R=COOR.sub.1 or
CH.sub.2OR.sub.1; [0259] R.sub.1=H or an alkyl group; [0260]
R.sub.2=H or SO.sub.3.sup.-; [0261] R.sub.3=H or OH; [0262]
R.sub.3'=H or OH; and [0263] R.sub.4=H, unsaturated or saturated,
alkyl group; and [0264] R.sub.4'=H, unsaturated or saturated, alkyl
group; [0265] or a pharmaceutically acceptable salt thereof,
wherein if R=CH.sub.2OR.sub.1, R.sub.2=H, R.sub.3 is OH and
R.sub.3' is H, then one of the following is true:
[0266] (i) R.sub.4=H, or unsaturated or saturated alkyl chain
having 9 carbon atoms or fewer;
[0267] or (ii) R.sub.4=H, or unsaturated or saturated alkyl chain
having 20 carbon atoms or fewer.
[0268] In one embodiment, the alkyl chain of R.sub.4 has 1 carbon
atom, in another embodiment, the alkyl chain of R.sub.4 has between
1-5, or in another embodiment, 2-6, or in another embodiment, 3-7,
or in another embodiment, 4-8, or in another embodiment 5-9 carbon
atoms. In one embodiment, the alkyl chain of R.sub.4 has 10-25
carbon atom, in another embodiment, the alkyl chain of R.sub.4 has
between 10-15 carbon atoms.
[0269] In another embodiment, the alkyl chain of R.sub.4' has 1
carbon atom, in another embodiment, the alkyl chain of R.sub.4' has
between 1-10, or in another embodiment, 10-15, or in another
embodiment, 5-13, or in another embodiment, 8-15, or in another
embodiment 10-25 carbon atoms or, in another embodiment, between
20-30 carbon atoms.
[0270] In another embodiment, this invention provides a compound
represented by the structure of formula 3:
##STR00057##
[0271] wherein, [0272] R=COOR.sub.1 or CH.sub.2OR.sub.1; [0273]
R.sub.1=H or an alkyl group; [0274] R.sub.2=SO.sub.3.sup.-; and
[0275] n=integer; [0276] or a pharmaceutically acceptable salt
thereof.
[0277] In another embodiment, n is an integer ranging from 1-5, or,
in another embodiment, between 5-10, or in another embodiment,
10-15, or in another embodiment, 10-20, or in another embodiment,
1-15, or in another embodiment 15-25 carbon atoms or, in another
embodiment, between 10-30.
[0278] In another embodiment, this invention provides a compound
represented by the structure of formula 4:
##STR00058##
[0279] or a pharmaceutically acceptable salt thereof
[0280] In another embodiment, the salt may be, inter alia, a sodium
salt.
[0281] In another embodiment, this invention provides a compound
represented by the structure of formula 5:
##STR00059##
[0282] In another embodiment, this invention provides a compound
represented by the structure of formula 6:
##STR00060##
[0283] In another embodiment, this invention provides a compound
represented by the structure of formula 7:
##STR00061##
[0284] In another embodiment, this invention provides a compound
represented by the structure of formula 8:
##STR00062##
[0285] In one embodiment, this invention provides a compound
represented by the structure of formula 9:
##STR00063##
[0286] wherein, [0287] R=COOR.sub.1 or CH.sub.2OR.sub.1 [0288]
R.sub.1=H or an alkyl group; [0289] R.sub.2=H or SO.sub.3.sup.-;
[0290] R.sub.3=OH; [0291] R.sub.3'=H or OH; and [0292] R.sub.4=H,
unsaturated or saturated, alkyl group; and [0293] R.sub.4'=H,
unsaturated or saturated, alkyl group; [0294] or a pharmaceutically
acceptable salt thereof, wherein if R=CH.sub.2OR.sub.1, R.sub.2=H,
R.sub.3 is OH and R.sub.3' is H, then one of the following is
true:
[0295] (i) R.sub.4=H, or unsaturated or saturated alkyl chain
having 9 carbon atoms or fewer; or
[0296] (ii) R.sub.4=H, or unsaturated or saturated alkyl chain
having 20 carbon atoms or fewer.
[0297] In another embodiment, this invention provides a compound
represented by the structure of formula 10:
##STR00064##
[0298] or a pharmaceutically acceptable salt thereof. In another
embodiment, the salt may be, inter alia, a sodium salt.
[0299] In still another embodiment, this invention provides a
compound represented by the structure of formula 17:
##STR00065##
[0300] wherein
[0301] R.sub.4 and R.sub.6 are independently selected from: [0302]
(a) alkyl, [0303] (b) alkenyl, [0304] (c) alkyl terminating in
aryl, substituted aryl, heteroaryl or substituted heteroaryl; and
[0305] (d) alkenyl terminating in aryl, substituted aryl,
heteroaryl or substituted heteroaryl.
[0306] In certain embodiments of compounds of formula 17, [0307]
R.sub.6 is
[0307] ##STR00066## [0308] X is an alkyl chain; and [0309] R.sub.7
is chosen from halogen, H, phenyl, alkyl, alkoxy, nitro, and
CF.sub.3.
[0310] In other embodiments of compounds of formula 17,
[0311] R.sub.4 is
##STR00067##
[0312] X is an alkyl chain; and
[0313] R.sub.7 is chosen from halogen, H, phenyl, alkyl, alkoxy,
nitro, and CF.sub.3.
[0314] Furthermore, in one embodiment, the invention provides a
process for the preparation of a compound represented by the
structure of formula (4)
##STR00068## [0315] or a pharmaceutically salt thereof, the process
including, inter alia, the step of: [0316] removing the benzyldiene
protecting group and hydrogenating of the compound represented by
the structure of formula (4a),
##STR00069##
[0316] or a salt thereof, wherein PG is a hydroxy protecting group.
In another embodiment, the hydroxy protecting group may be
benzyl.
[0317] In one embodiment of the invention, the compound of formula
(4a) may be obtained by a process including, inter alia, the step
of: [0318] conducting a selective sulfation of the 3'' OH of the
galactose moiety of the compound represented by the structure of
formula (4b):
##STR00070##
[0318] wherein PG is a hydroxy protecting group and R is H. In
another embodiment, the hydroxy protecting group may be benzyl.
[0319] In one embodiment of the invention, the compound of formula
(4b) wherein R is H, may be obtained by a process including, inter
alia, the step of removing the levulinyl protecting group of a
compound of formula (4b) wherein R is levulinyl, thereby obtaining
a compound of formula (4b) wherein R is H.
[0320] In one embodiment of the invention, the compound of formula
(4b) wherein R is levulinyl may be obtained by a process including,
inter alia, the step of: [0321] reacting a compound represented by
the structure of formula (4c):
##STR00071##
[0321] wherein R is H or levulinyl with hexacosanoic acid, thereby
obtaining the compound of formula (4b) wherein R is levulinyl.
[0322] In one embodiment of the invention, the compound of formula
(4c), wherein R is H or levulinyl, may be obtained by a process
including, inter alia, the step of: [0323] reducing the azide group
of a compound represented by the structure of formula (4d):
[0323] ##STR00072## [0324] wherein R is levulinyl, thereby
obtaining a compound of formula (4c) wherein R is H or
levulinyl.
[0325] In one embodiment of the invention, the compound of formula
(4d) wherein R is levulinyl, may be obtained by a process
including, inter alia, the step of: [0326] reacting a compound
represented by the structure of formula (4e)
[0326] ##STR00073## [0327] wherein PG is a hydroxy protecting
group, LG is a leaving group and R is levulinyl, [0328] with a
compound represented by the structure of formula (4f)
[0328] ##STR00074## [0329] wherein PG is a hydroxy protecting
group, [0330] to form an alpha glycosidic bond, thereby obtaining
the compound of formula (4d) wherein R is levulinyl. In another
embodiment, the leaving group may be, inter alia,
##STR00075##
[0331] In one embodiment, the invention provides a process for the
preparation of a compound represented by the structure of formula
(10)
##STR00076## [0332] or a pharmaceutically salt thereof, including,
inter alia, the step of: [0333] conducting a selective sulfation of
the 3'' OH of the galactose moiety of the compound represented by
the structure of formula (10a):
[0333] ##STR00077## [0334] In another embodiment, the sulfation may
be conducted in the presence of Bu.sub.2SnO.
[0335] In one embodiment of the invention, the compound of formula
(10a) may be obtained by the process including, inter alia, the
step of: [0336] removing the hydroxy protecting groups and
hydrogenating the compound represented by the structure of formula
(10b):
##STR00078##
[0336] wherein PG and PG.sub.1 are hydroxy protecting groups,
thereby obtaining the compound of formula (10a). In another
embodiment, the PG may be, inter alia, benzyl. In another
embodiment, the PG1 may be, inter alia, benzoyl. In one embodiment
of the invention, the compound of formula (10b) may be obtained by
a process including, inter alia, the step of: [0337] reacting a
compound represented by the structure of formula (10c):
[0337] ##STR00079## [0338] wherein PG is a hydroxy protecting
group, [0339] with a compound represented by the structure of
formula (10d):
##STR00080##
[0339] wherein PG.sub.1 is a hydroxy protecting group and LG is a
leaving group, thereby obtaining the compound of formula (10b). In
another embodiment, the leaving group may be, inter alia,
##STR00081##
[0340] In one embodiment of the invention, the compound of formula
(10c) may be obtained by a process comprising the steps of: [0341]
reducing the azide of a compound represented by the structure of
formula (10e):
[0341] ##STR00082## [0342] wherein PG and PG.sub.2 are hydroxy
protecting groups; [0343] reacting the resulting amine with
hexacosanoic acid; and removing the hydroxy protecting group
PG.sub.2, thereby obtaining the compound of formula (10c). In
another embodiment, the PG.sub.2 may be, inter alia, TIPS.
[0344] In one embodiment, the invention provides a process for the
preparation of a compound represented by the structure of formula
(11):
##STR00083## [0345] or a pharmaceutically salt thereof, including,
inter alia, the step of: [0346] conducting a selective sulfation of
the 3'' OH of the galactose moiety of the compound represented by
the structure of formula (11a):
##STR00084##
[0346] thereby obtaining the a compound represented by the
structure of formula (10). In another embodiment, the sulfation may
be conducted in the presence of Bu.sub.2SnO.
[0347] In one embodiment of the invention, the compound of formula
(11a) may be obtained by the process including, inter alia, the
step of: [0348] removing the hydroxy protecting groups of the
compound represented by the structure of formula (11b):
##STR00085##
[0348] wherein PG and PG.sub.1 are hydroxy protecting groups,
thereby obtaining the compound of formula (11a). In another
embodiment, PG may be, inter alia, benzoyl. In another embodiment,
PG.sub.1 may be, inter alia, benzoyl.
[0349] In one embodiment of the invention, the compound of formula
(10b) may be obtained by a process including, inter alia, the step
of: [0350] deprotecting the amine of a compound represented by the
structure of formula (11c):
[0350] ##STR00086## [0351] wherein PG and PG.sub.1 are hydroxy
protecting groups, and [0352] PG.sub.3 is an amino protecting
group, [0353] and reacting with nervonic acid, thereby obtaining
the compound of formula (11b). In another embodiment, the amino
protecting group may be, inter alia, tBoc.
[0354] In one embodiment, any one of the compounds of the invention
may be a ligand for an NKT (natural killer T) cell. In another
embodiment, the ligand may be in a complex with a CD1 molecule. In
another embodiment, the CD1 molecule is a CD1d molecule. In another
embodiment, the ligand stimulates NKT cells, which express a CD161+
NK marker as well as an invariant T cell antigen receptor (TCR) on
the surface thereof.
[0355] In another embodiment, the invention provides a composition
or vaccine including, inter alia, any one of the compounds of the
invention. In another embodiment, the composition or vaccine may
include, inter alia, at least one cell population. In another
embodiment, the cell population may include, inter alia, NKT cells,
antigen presenting cells, or a combination thereof.
[0356] In another embodiment, the invention provides a method for
stimulating NKT cells, the method including, inter alia, contacting
an NKT cell with any one of the compounds of the invention.
[0357] In another embodiment, the invention provides a cell
population obtained by any one of the methods of the invention.
[0358] In another embodiment, the invention provides a method for
stimulating, inhibiting, suppressing or modulating an immune
response in a subject, the method includes, inter alia, the step of
contacting an NKT cell in the subject with any one of the compounds
of the invention.
[0359] In another embodiment, the compound according to the
invention may be in a complex with a CD1 molecule. In another
embodiment, the CD1 molecule may be CD1d. In another embodiment,
the complex may be displayed on a dendritic cell. In another
embodiment, the complex may be displayed on any antigen presenting
cell.
[0360] In one embodiment of the invention, the NKT cells secrete a
cytokine. In another embodiment the NKT cell may be a
V.alpha.24iNKT cell in humans. In another embodiment the NKT cell
may be a V.alpha.14i NKT cell in mice.
[0361] In one embodiment of the invention, the subject may be
immunocompromised. In another embodiment, the subject is infected.
In another embodiment, the subject is infected with HIV. In another
embodiment, the subject is infected with mycobacteria. In another
embodiment, the subject is infected with malaria. In another
embodiment, the subject is infected with HIV, mycobacteria, or
malaria.
[0362] In one embodiment of the invention, the subject is afflicted
with cancer. In one embodiment of the invention, the subject is at
an elevated risk for cancer. In one embodiment of the invention,
the subject has precancerous precursors.
[0363] In one embodiment of the invention, the immune response is
biased toward Th1 or Th2. In another embodiment, the subject
suffers from, or is at an elevated risk for an autoimmune disease.
In another embodiment, the biasing of the immune response results
in the suppression, inhibition or abrogation of the autoimmune
disease. In another embodiment, the subject has an inappropriate or
undesirable immune response. In another embodiment, the response is
inflammatory. In another embodiment, the inappropriate or
undesirable response exacerbates an infection, disease or symptom
in the subject.
[0364] In another embodiment, the invention provides an adjuvant
including, inter alia, any one of the compounds according to the
invention.
[0365] In another embodiment, the invention provides a method of
enhancing immunogenicity of a compound, composition, or vaccine in
a subject, the method includes, inter alia, administering to the
subject a compound, composition or vaccine further comprising an
adjuvant of according to the invention, wherein the adjuvant
enhances the immunogenicity of the compound, composition or
vaccine.
[0366] In another embodiment, the invention provides a method of
stimulating or enhancing cytokine production in a subject, the
method includes, inter alia, administering to the subject any one
of the compounds of the invention, whereby an NKT cell in the
subject secretes a cytokine following contact with the compound. In
another embodiment, the cytokine may be interferon-.gamma. or
Interleukin-4
[0367] In another embodiment, this invention provides an NK T cell
obtained via contacting an NK T cell with a compound of this
invention. In one embodiment, such contact is in the presence of an
antigen presenting cell, which in another embodiment expresses a
CD1 molecule, wherein the compound, or a fragment thereof, is
displayed in the context of the CD1 molecule.
[0368] In one embodiment, the phrase "NKT cell" or "Natural Killer
cell", refers to a T cell population that causes, stimulates or
contributes to cytokine production, and/or in another embodiment,
is cytotoxic. In one embodiment, the NKT cells are a homogenous
population, or in another embodiment, a heterogeneous
population.
[0369] NKT cells are an exceptional subset of mature lymphocytes
that bear both NK and T cell receptors. Murine NKT cells express
NK1.1 and TCR.alpha..beta. receptors and are especially dense in
the bone marrow and liver. The cells may express a very limited TCR
repertoire, which may include an invariant .alpha.-chain. The
ligand for NKT cells may be non-polymorphic, and a non-classical
MHC class I molecule may present a specific antigen processed via a
TAP (transporter associated with antigen processing)-independent
pathway.
[0370] In one embodiment, the antigen is presented in the context
of a CD1 molecule, which in one embodiment is CD1d. Activated NK T
cells may display an NK-like perforin-dependent cytotoxicity
against various cells, including tumor cells or cell lines and
inhibit tumor metastasis, among other applications, as is described
further hereinbelow, and representing embodiments of the methods of
this invention.
[0371] The T cells of this invention may express CD161 and
V.alpha.24i TCR on their cell surface. In one embodiment, the T
cells may be classified as CD161.sup.high expressors, or in another
embodiment, the T cells may be classified as CD161.sup.low
expressors, or in another embodiment, a combination thereof.
[0372] It is to be understood that the NK T cells of this
invention, and those obtained via the methods of this invention,
may express any number or combination of cell surface markers, as
described herein, and as is well known in the art, and are to be
considered as part of this invention.
[0373] In one embodiment, the T cell subpopulation, are "invariant
NK T cells," which may represent a major fraction of the mature T
cells in thymus, the major T cell subpopulation in murine liver,
and/or up to 5% of splenic T cells.
[0374] In another embodiment, the T cell subpopulation may be
"non-invariant NK T cells", which may comprise human and mouse bone
marrow and human liver T cell populations that are, for example,
CD1d-reactive noninvariant T cells which express diverse TCRs, and
which can also produce a large amount of IL-4 and IFN-.gamma..
[0375] In one embodiment, the NKT cells of this invention are
obtained by positive selection for expression of CD161 and
V.alpha.24i TCR, and in another embodiment, the T cells may be
obtained via negative selection procedures, as are well known in
the art.
[0376] In one embodiment, the NK T cells of this invention may be
obtained from in vivo sources, such as, for example, peripheral
blood, leukopheresis blood product, apheresis blood product,
peripheral lymph nodes, gut associated lymphoid tissue, spleen,
thymus, cord blood, mesenteric lymph nodes, liver, sites of
immunologic lesions, e.g. synovial fluid, pancreas, cerebrospinal
fluid, tumor samples, granulomatous tissue, or any other source
where such cells may be obtained. In one embodiment, the NK T cells
are obtained from human sources, which may be, in another
embodiment, from human fetal, neonatal, child, or adult sources. In
another embodiment, the NK T cells of this invention may be
obtained from animal sources, such as, for example, porcine or
simian, or any other animal of interest. In another embodiment, the
NK T cells of this invention may be obtained from subjects that are
normal, or in another embodiment, diseased, or in another
embodiment, susceptible to a disease of interest.
[0377] In one embodiment, the T cells and/or cells, as described
further hereinbelow, of this invention are isolated from tissue,
and, in another embodiment, an appropriate solution may be used for
dispersion or suspension, toward this end. In another embodiment, T
cells and/or cells, as described further hereinbelow, of this
invention may be cultured in solution.
[0378] Such a solution may be, in another embodiment, a balanced
salt solution, such as normal saline, PBS, or Hank's balanced salt
solution, or others, each of which represents another embodiment of
this invention. The solution may be supplemented, in other
embodiment, with fetal calf serum, bovine serum albumin (BSA),
normal goat serum, or other naturally occurring factors, and, in
another embodiment, may be supplied in conjunction with an
acceptable buffer. The buffer may be, in other embodiments, HEPES,
phosphate buffers, lactate buffers, or the like, as will be known
to one skilled in the art.
[0379] In another embodiment, the solution in which the T cells or
cells of this invention may be placed is in medium is which is
serum-free, which may be, in another embodiment, commercially
available, such as, for example, animal protein-free base media
such as X-VIVO 10.TM. or X-VIVO 15.TM. (BioWhittaker, Walkersville,
Md.), Hematopoietic Stem Cell-SFM media (GibcoBRL, Grand Island,
N.Y.) or any formulation which promotes or sustains cell viability.
Serum-free media used, may, in another embodiment, be as those
described in the following patent documents: WO 95/00632; U.S. Pat.
No. 5,405,772; PCT US94/09622. The serum-free base medium may, in
another embodiment, contain clinical grade bovine serum albumin,
which may be, in another embodiment, at a concentration of about
0.5-5%, or, in another embodiment, about 1.0% (w/v). Clinical grade
albumin derived from human serum, such as Buminate.RTM. (Baxter
Hyland, Glendale, Calif.), may be used, in another embodiment.
[0380] In another embodiment, the T cells of this invention may be
separated via affinity-based separation methods. Techniques for
affinity separation may include, in other embodiments, magnetic
separation, using antibody-coated magnetic beads, affinity
chromatography, cytotoxic agents joined to a monoclonal antibody or
use in conjunction with a monoclonal antibody, for example,
complement and cytotoxins, and "panning" with an antibody attached
to a solid matrix, such as a plate, or any other convenient
technique. In other embodiment, separation techniques may also
include the use of fluorescence activated cell sorters, which can
have varying degrees of sophistication, such as multiple color
channels, low angle and obtuse light scattering detecting channels,
impedance channels, etc. It is to be understood that any technique,
which enables separation of the NK T cells of this invention may be
employed, and is to be considered as part of this invention.
[0381] In another embodiment, the affinity reagents employed in the
separation methods may be specific receptors or ligands for the
cell surface molecules indicated hereinabove.
[0382] In another embodiment, the antibodies utilized herein may be
conjugated to a label, which may, in another embodiment, be used
for separation. Labels may include, in other embodiments, magnetic
beads, which allow for direct separation, biotin, which may be
removed with avidin or streptavidin bound to, for example, a
support, fluorochromes, which may be used with a fluorescence
activated cell sorter, or the like, to allow for ease of
separation, and others, as is well known in the art. Fluorochromes
may include, in one embodiment, phycobiliproteins, such as, for
example, phycoerythrin, allophycocyanins, fluorescein, Texas red,
or combinations thereof.
[0383] In one embodiment, cell separations utilizing antibodies
will entail the addition of an antibody to a suspension of cells,
for a period of time sufficient to bind the available cell surface
antigens. The incubation may be for a varied period of time, such
as in one embodiment, for 5 minutes, or in another embodiment, 15
minutes, or in another embodiment, 30 minutes, or in another
embodiment, 45 minutes, or in another embodiment, 60 minutes, or in
another embodiment, 90 minutes. Any length of time which results in
specific labeling with the antibody, with minimal non-specific
binding is to be considered envisioned for this aspect of the
invention.
[0384] Any length of time which results in specific labeling with
the antibody, with minimal non-specific binding is to be considered
envisioned for this aspect of the invention.
[0385] In another embodiment, the staining intensity of the cells
can be monitored by flow cytometry, where lasers detect the
quantitative levels of fluorochrome (which is proportional to the
amount of cell surface antigen bound by the antibodies). Flow
cytometry, or FACS, can also be used, in another embodiment, to
separate cell populations based on the intensity of antibody
staining, as well as other parameters such as cell size and light
scatter.
[0386] In another embodiment, the labeled cells are separated based
on their expression of CD161 and V.alpha.24i TCR. The separated
cells may be collected in any appropriate medium that maintains
cell viability, and may, in another embodiment, comprise a cushion
of serum at the bottom of the collection tube.
[0387] In another embodiment, the culture containing the T cells of
this invention may contain other cytokines or growth factors to
which the cells are responsive. In one embodiment, the cytokines or
growth factors promote survival, growth, function, or a combination
thereof of the NK T cells. Cytokines and growth factors may
include, in other embodiment, polypeptides and non-polypeptide
factors.
[0388] In one embodiment, the NK T cell populations of this
invention are antigen specific.
[0389] In one embodiment, the term "antigen specific" refers to a
property of the population such that supply of a particular
antigen, or in another embodiment, a fragment of the antigen,
results, in one embodiment, in specific cell proliferation, when
presented the antigen, which in one embodiment, is in the context
of CD1. In one embodiment, the antigen is any compound of this
invention.
[0390] In another embodiment, supply of the antigen or fragment
thereof, results in NK T cell production of interleukin 2, or in
another embodiment, interferon-.gamma., or in another embodiment,
interleukin-4, or in another embodiment, a combination thereof. In
one embodiment, the NK T cell population expresses a monoclonal T
cell receptor. In another embodiment, the NK T cell population
expresses polyclonal T cell receptors.
[0391] In one embodiment, the T cells will be of one or more
specificities, and may include, in another embodiment, those that
recognize a mixture of antigens derived from an antigenic source.
In one embodiment, a mixture of the compounds of this invention may
be used to simulate a NK T cells of varying specificity.
[0392] In one embodiment, the NK T cell population suppresses an
autoimmune response. In one embodiment, the term "autoimmune
response" refers to an immune response directed against an auto- or
self-antigen. In one embodiment, the autoimmune response is
rheumatoid arthritis, multiple sclerosis, diabetes mellitus,
myasthenia gravis, pernicious anemia, Addison's disease, lupus
erythematosus, Reiter's syndrome, atopic dermatitis or Graves
disease. In one embodiment, the autoimmune disease caused in the
subject is a result of self-reactive T cells, which recognize
multiple self-antigens.
[0393] In another embodiment, the NK T cell population suppresses
an inflammatory response. In one embodiment, the term "inflammatory
response" refers to any response that is, in one embodiment, caused
by inflammation or, in another embodiment, whose symptoms include
inflammation. By way of example, an inflammatory response may be a
result of septic shock, or, in another embodiment, a function of
rheumatoid arthritis. The inflammatory response may be a part of an
overall inflammatory disorder in a subject, and may comprise, in
another embodiment, cardiovascular disease, rheumatoid arthritis,
multiple sclerosis, Crohn's disease, inflammatory bowel disease,
systemic lupus erythematosis, polymyositis, septic shock, graft
versus host disease, host versus graft disease, asthma, rhinitis,
psoriasis, cachexia associated with cancer, or eczema. In one
embodiment, as described hereinabove, the inflammation in the
subject may be a result of T cells, which recognize multiple
antigens in the subject. In one embodiment, the NK T cells of this
invention may be specific for a single antigen where multiple
antigens are recognized, yet the NK T cell population effectively
suppresses inflammation in the subject. In one embodiment,
suppression of inflammation is via modulating an immune response as
a result of production of a particular cytokine profile. In one
embodiment, the NK T cells produce cytokines which serve to
downmodulate the inflammatory response.
[0394] In another embodiment, the NK T cell populations of this
invention suppresses an allergic response. In one embodiment, the
term "allergic response" refers to an immune system attack against
a generally harmless, innocuous antigen or allergen. Allergies may
in one embodiment include, but are not limited to, hay fever,
asthma, atopic eczema as well as allergies to poison oak and ivy,
house dust mites, bee pollen, nuts, shellfish, penicillin or other
medications, or any other compound or compounds which induce an
allergic response. In one embodiment, multiple allergens elicit an
allergic response, and the antigen recognized by the NK T cells of
this invention may be any antigen thereof. In one embodiment,
suppression of allergic responses is via modulating an immune
response as a result of production of a particular cytokine
profile. In one embodiment, the NK T cells produce cytokines which
serve to downmodulate the allergic response.
[0395] In another embodiment, the NK T cells of the present
invention are utilized, in circumstances wherein eliciting a "Th1"
response is beneficial in a subject, wherein the subject has a
disease where a so-called "Th2" type response has developed.
Introduction of the NK T cells, in one embodiment, results in a
shift toward a Th1 type response, in response to the cytokine
profile produced from the NK T cells.
[0396] In one embodiment, the term "Th2 type response" refers to a
pattern of cytokine expression, elicited by T Helper cells as part
of the adaptive immune response, which support the development of a
robust antibody response. Typically Th2 type responses are
beneficial in helminth infections in a subject, for example.
Typically Th2 type responses are recognized by the production of
interleukin-4 or interleukin 10, for example.
[0397] In one embodiment, the term "Th1 type response" refers to a
pattern of cytokine expression, elicited by T Helper cells as part
of the adaptive immune response, which support the development of
robust cell-mediated immunity. Typically Th1 type responses are
beneficial in intracellular infections in a subject, for example.
Typically Th1 type responses are recognized by the production of
interleukin-2 or interferon .gamma., for example.
[0398] In another embodiment, the reverse occurs, where a Th1 type
response has developed, when Th2 type responses provide a more
beneficial outcome to a subject, where introduction of the NK T
cells, vaccines or compositions of the present invention provides a
shift to the more beneficial cytokine profile. One example would be
in leprosy, where the NK T cells, vaccines or compositions of the
present invention stimulates a Th1 cytokine shift, resulting in
tuberculoid leprosy, as opposed to lepromatous leprosy, a much more
severe form of the disease, associated with Th2 type responses.
[0399] In another embodiment, the NK T cells of this invention, and
obtained via the methods of this invention, may be a part of a
vaccine or composition. Such vaccines and/or compositions may be
used in any applicable method of this invention, and represents an
embodiment thereof.
[0400] For example, in one embodiment, the methods of this
invention for stimulating, inhibiting, suppressing or modulating an
immune response in a subject, which comprise contacting an NKT cell
in a subject with a compound of the invention, may also comprise
contacting the NKT cell with a compound in a composition, or in
another embodiment, contacting the NKT cell with a vaccine
comprising at least one compound of the invention.
[0401] It is to be understood that any use of the NK T cells,
vaccines or compositions of the present invention for methods of
enhancing immunogenicity, such as, for example, for purposes of
immunizing a subject to prevent disease, and/or ameliorate disease,
and/or alter disease progression are to be considered as part of
this invention.
[0402] Examples of infectious virus to which stimulation of a
protective immune response is desirable, which may be accomplished
via the methods of this invention, or utilizing the NK T cells,
vaccines or compositions of the present invention include:
Retroviridae (e.g., human immunodeficiency viruses, such as HIV-1
(also referred to as HTLV-III, LAV or HTLV-III/LAV, or HIV-III; and
other isolates, such as HIV-LP; Picornaviridae (e.g., polio
viruses, hepatitis A virus; enteroviruses, human coxsackie viruses,
rhinoviruses, echoviruses); Calciviridae (e.g., strains that cause
gastroenteritis); Togaviridae (e.g., equine encephalitis viruses,
rubella viruses); Flaviridae (e.g., dengue viruses, encephalitis
viruses, yellow fever viruses); Coronaviridae (e.g.,
coronaviruses); Rhabdoviridae (e.g., vesicular stomatitis viruses,
rabies viruses); Filoviridae (e.g., ebola viruses); Paramyxoviridae
(e.g., parainfluenza viruses, mumps virus, measles virus,
respiratory syncytial virus); Orthomyxoviridae (e.g. influenza
viruses); Bungaviridae (e.g., Hantaan viruses, bunga viruses,
phleboviruses and Nairo viruses); Arena viridae (hemorrhagic fever
viruses); Reoviridae (erg., reoviruses, orbiviurses and
rotaviruses); Birnaviridae; Hepadnaviridae (Hepatitis B virus);
Parvoviridae (parvoviruses); Papovaviridae (papilloma viruses,
polyoma viruses); Adenoviridae (most adenoviruses); Herpesviridae
(herpes simplex virus (HSV) 1 and 2, varicella zoster virus,
cytomegalovirus (CMV), herpes viruses'); Poxviridae (variola
viruses, vaccinia viruses, pox viruses); and Iridoviridae (e.g.
African swine fever virus); and unclassified viruses (e.g., the
etiological agents of Spongiform encephalopathies, the agent of
delta hepatities (thought to be a defective satellite of hepatitis
B virus), the agents of non-A, non-B hepatitis (class 1=internally
transmitted; class 2=parenterally transmitted (i.e., Hepatitis C);
Norwalk and related viruses, and astroviruses).
[0403] Examples of infectious bacteria to which stimulation of a
protective immune response is desirable, which may be accomplished
via the methods of this invention, or utilizing the NK T cells,
vaccines or compositions of the present invention include:
Helicobacter pylori, Borellia burgdorferi, Legionella pneumophilia,
Mycobacteria sps (e.g. M. tuberculosis, M. avium, M.
intracellulare, M. kansaii, M. gordonae), Staphylococcus aureus,
Neisseria gonorrhoeae, Neisseria meningitidis, Listeria
monocytogenes, Streptococcus pyogenes (Group A Streptococcus),
Streptococcus agalactiae (Group B Streptococcus), Streptococcus
(viridans group), Streptococcus faecalis, Streptococcus bovis,
Streptococcus (anaerobic sps.), Streptococcus pneumoniae,
pathogenic Campylobactersp., Enterococcus sp., Chlamidia sp.,
Haemophilus influenzae, Bacillus antracis, corynebacterium
diphtheriae, corynebacterium sp., Erysipelothrix rhusiopathiae,
Clostridium perfringers, Clostridium tetani, Enterobacter
aerogenes, Klebsiella pneumoniae, Pasturella multocida, Bacteroides
sp., Fusobacterium nucleatum, Streptobacillus moniliformis,
Treponema pallidium, Treponema pertenue, Leptospira, Actinomyces
israelli and Francisella tularensis.
[0404] Examples of infectious fungi to which stimulation of a
protective immune response is desirable, which may be accomplished
via the methods of this invention, or utilizing the NK T cells,
vaccines or compositions of the present invention include:
Cryptococcus neoformans, Histoplasma capsulatum, Coccidioides
immitis, Blastomyces dermatitidis, Chlamydia trachomatis, Candida
albicans. Other infectious organisms (i.e., protists) include:
Plasmodium sp., Leishmania sp., Schistosoma sp. and Toxoplasma
sp.
[0405] It is to be understood that the modulation of any immune
response, via the use of the NK T cell populations, vaccines or
compositions of this invention are to be considered as part of this
invention, and an embodiment thereof.
[0406] In another embodiment, the NK T cell populations of this
invention may be isolated, culture-expanded, or otherwise
manipulated, as will be understood by one skilled in the art. In
one embodiment, the NK T cells as derived by the methods of this
invention, may be further engineered to express substances of
interest. In one embodiment, the NK T cell populations may be
engineered to express particular adhesion molecules, or other
targeting molecules, which, when the cells are provided to a
subject, facilitate targeting of the NK T cell populations to a
site of interest. For example, when NK T cell activity is desired
to modulate an immune response at a mucosal surface, the isolated
NK T cell populations of this invention may be further engineered
to express the .alpha..sub.e.beta..sub.7 adhesion molecule, which
has been shown to play a role in mucosal homing. The cells can be
engineered to express other targeting molecules, such as, for
example, an antibody specific for a protein expressed at a
particular site in a tissue, or, in another embodiment, expressed
on a particular cell located at a site of interest, etc. Numerous
methods are well known in the art for engineering the cells, and
may comprise the use of a vector, or naked DNA, wherein a nucleic
acid coding for the targeting molecule of interest is introduced
via any number of methods well described.
[0407] A nucleic acid sequence of interest may be subcloned within
a particular vector, depending upon the desired method of
introduction of the sequence within cells. Once the nucleic acid
segment is subcloned into a particular vector it thereby becomes a
recombinant vector. Polynucleotide segments encoding sequences of
interest can be ligated into commercially available expression
vector systems suitable for transducing/transforming mammalian
cells and for directing the expression of recombinant products
within the transduced cells. It will be appreciated that such
commercially available vector systems can easily be modified via
commonly used recombinant techniques in order to replace, duplicate
or mutate existing promoter or enhancer sequences and/or introduce
any additional polynucleotide sequences such as for example,
sequences encoding additional selection markers or sequences
encoding reporter polypeptides.
[0408] There are a number of techniques known in the art for
introducing the above described recombinant vectors into cells,
such as, but not limited to: direct DNA uptake techniques, and
virus, plasmid, linear DNA or liposome mediated transduction,
receptor-mediated uptake and magnetoporation methods employing
calcium-phosphate mediated and DEAE-dextran mediated methods of
introduction, electroporation, liposome-mediated transfection,
direct injection, and receptor-mediated uptake (for further detail
see, for example, "Methods in Enzymology" Vol. 1-317, Academic
Press, Current Protocols in Molecular Biology, Ausubel F. M. et al.
(eds.) Greene Publishing Associates, (1989) and in Molecular
Cloning: A Laboratory Manual, 2nd Edition, Sambrook et al. Cold
Spring Harbor Laboratory Press, (1989), or other standard
laboratory manuals). Bombardment with nucleic acid coated particles
is also envisaged.
[0409] The efficacy of a particular expression vector system and
method of introducing nucleic acid into a cell can be assessed by
standard approaches routinely used in the art. For example, DNA
introduced into a cell can be detected by a filter hybridization
technique (e.g., Southern blotting) and RNA produced by
transcription of introduced DNA can be detected, for example, by
Northern blotting, RNase protection or reverse
transcriptase-polymerase chain reaction (RT-PCR). The gene product
can be detected by an appropriate assay, for example by
immunological detection of a produced protein, such as with a
specific antibody, or by a functional assay to detect a functional
activity of the gene product, such as an enzymatic assay. If the
gene product of interest to be expressed by a cell is not readily
assayable, an expression system can first be optimized using a
reporter gene linked to the regulatory elements and vector to be
used. The reporter gene encodes a gene product, which is easily
detectable and, thus, can be used to evaluate efficacy of the
system. Standard reporter genes used in the art include genes
encoding .beta.-galactosidase, chloramphenicol acetyl transferase,
luciferase and human growth hormone, or any of the marker proteins
listed herein.
[0410] In another embodiment, this invention provides a method for
producing an isolated, culture-expanded NK T cell population,
comprising contacting V.alpha.14i, or V.alpha.24i T cells with
dendritic cells and a compound of this invention, for a period of
time resulting in antigen-specific T cell expansion and isolating
the expanded T cells thus obtained, thereby producing an isolated,
culture-expanded NK T cell population.
[0411] In one embodiment, the method for producing an isolated
culture-expanded NK T cell population, further comprises the step
of adding a cytokine or growth factor to the dendritic cell, NK T
cell culture. In one embodiment, NK T cells secretion of
interleukin-2, interferon-.gamma. or interleukin-4 is detected, at
which time the NK T cells are used in the methods of this
invention.
[0412] Dendritic cells stimulated NK T cell cytokine production,
when presenting a compound of this invention, in the context of
CD1. In another embodiment of this invention, the stimulated NK T
cells may induce maturation of the dendritic cells, which may be
mediated via TCR and CD1d/glycolipid interactions, and engagement
of the CD40/CD4OL interaction. This in turn, in another embodiment,
may promote IL-12 secretion by the dendritic cells, and/or
upregulation of, inter-alia, MHC molecules, DEC-205, or
costimulatory molecules such as the B7 family. Dendritic cell
maturation as a result of this interaction, may, in another
embodiment, lead to enhanced adaptive immune responses, which in
another embodiment, includes adjuvant activity of the compounds of
this invention.
[0413] In one embodiment, the term "dendritic cell" (DC) refers to
antigen-presenting cells, which are capable of presenting antigen
to T cells, in the context of CD1. In one embodiment, the dendritic
cells utilized in the methods of this invention may be of any of
several DC subsets, which differentiate from, in one embodiment,
lymphoid or, in another embodiment, myeloid bone marrow
progenitors. In one embodiment, DC development may be stimulated
via the use of granulocyte-macrophage colony-stimulating-factor
(GM-CSF), or in another embodiment, interleukin (IL)-3, which may,
in another embodiment, enhance DC survival.
[0414] In another embodiment, DCs may be generated from
proliferating progenitors isolated from bone marrow. In another
embodiment, DCs may be isolated from CD34+ progenitors as described
by Caux and Banchereau in Nature in 1992, or from monocytes, as
described by Romani et al, J. Exp. Med. 180: 83-93 '94 and Bender
et al, J. Immunol. Methods, 196: 121-135, '96 1996. In another
embodiment, the DCs are isolated from blood, as described for
example, in O'Doherty et al, J. Exp. Med. 178: 1067-1078 1993 and
Immunology 82: 487-493 1994, all methods of which are incorporated
fully herewith by reference.
[0415] In one embodiment, the DCs utilized in the methods of this
invention may express myeloid markers, such as, for example, CD11c
or, in another embodiment, an IL-3 receptor-.alpha. (IL-3R.alpha.)
chain (CD123). In another embodiment, the DCs may produce type I
interferons (IFNs). In one embodiment, the DCs utilized in the
methods of this invention express costimulatory molecules. In
another embodiment, the DCs utilized in the methods of this
invention may express additional adhesion molecules, which may, in
one embodiment, serve as additional costimulatory molecules, or in
another embodiment, serve to target the DCs to particular sites in
vivo, when delivered via the methods of this invention, as
described further hereinbelow.
[0416] In one embodiment, the DCs may be obtained from in vivo
sources, such as, for example, most solid tissues in the body,
peripheral blood, lymph nodes, gut associated lymphoid tissue,
spleen, thymus, skin, sites of immunologic lesions, e.g. synovial
fluid, pancreas, cerebrospinal fluid, tumor samples, granulomatous
tissue, or any other source where such cells may be obtained. In
one embodiment, the dendritic cells are obtained from human
sources, which may be, in another embodiment, from human fetal,
neonatal, child, or adult sources. In another embodiment, the
dendritic cells used in the methods of this invention may be
obtained from animal sources, such as, for example, porcine or
simian, or any other animal of interest. In another embodiment,
dendritic cells used in the methods of this invention may be
obtained from subjects that are normal, or in another embodiment,
diseased, or in another embodiment, susceptible to a disease of
interest.
[0417] Dendritic cell separation may accomplished in another
embodiment, via any of the separation methods as described herein.
In one embodiment, positive and/or negative affinity based
selections are conducted. In one embodiment, positive selection is
based on CD86 expression, and negative selection is based on GRI
expression.
[0418] In another embodiment, the dendritic cells used in the
methods of this invention may be generated in vitro by culturing
monocytes in presence of GM-CSF and IL-4.
[0419] In one embodiment, the dendritic cells used in the methods
of this invention may express CD83, an endocytic receptor to
increase uptake of the autoantigen such as DEC-205/CD205 in one
embodiment, or DC-LAMP (CD208) cell surface markers, or, in another
embodiment, varying levels of the antigen presenting MHC class I
and II products, or in another embodiment, accessory (adhesion and
co-stimulatory) molecules including CD40, CD54, CD58 or CD86, or
any combination thereof. In another embodiment, the dendritic cells
may express varying levels of CD115, CD14, CD68 or CD32.
[0420] In one embodiment, mature dendritic cells are used for the
methods of this invention. In one embodiment, the term "mature
dendritic cells" refers to a population of dendritic cells with
diminished CD115, CD14, CD68 or CD32 expression, or in another
embodiment, a population of cells with enhanced CD86 expression, or
a combination thereof. In another embodiment, mature dendritic
cells will exhibit increased expression of one or more of p55,
CD83, CD40 or CD86 or a combination thereof. In another embodiment,
the dendritic cells used in the methods of this invention will
express the DEC-205 receptor on their surface. In another
embodiment, maturation of the DCs may be accomplished via, for
example, CD40 ligation, CpG oligodeoxyribonucleotide addition,
ligation of the IL-1, TNF.alpha. or TOLL like receptor ligand,
bacterial lipoglycan or polysaccharide addition or activation of an
intracellular pathway such as TRAF-6 or NF-.kappa.B.
[0421] In one embodiment, inducing DC maturation may be in
combination with endocytic receptor delivery of a preselected
antigen. In one embodiment, endocytic receptor delivery of antigen
may be via the use of the DEC-205 receptor.
[0422] In one embodiment, the maturation status of the dendritic
may be confirmed, for example, by detecting either one or more of
1) an increase expression of one or more of p55, CD83, CD40 or CD86
antigens; 2) loss of CD115, CD14, CD32 or CD68 antigen; or 3)
reversion to a macrophage phenotype characterized by increased
adhesion and loss of veils following the removal of cytokines which
promote maturation of PBMCs to the immature dendritic cells, by
methods well known in the art, such as, for example,
immunohistochemistry, FACS analysis, and others.
[0423] In one embodiment, the dendritic cells used for the methods
of this invention may express, or in another embodiment, may be
engineered to express a costimulatory molecule. In one embodiment,
dendritic cells used for the methods of this invention are enriched
for CD86.sup.high or CD80.sup.high expression.
[0424] In another embodiment, the dendritic cells used in the
methods of this invention are selected for their capacity to expand
antigen-specific NK T cells. In one embodiment, the DCs are
isolated from progenitors or from blood for this purpose. In
another embodiment, dendritic cells expressing high amounts of
DEC-205/CD205 are used for this purpose.
[0425] NK T cell expansion, in one embodiment, is antigen-specific.
In one embodiment, a compound of this invention is supplied in the
culture simultaneously with dendritic cell contact with the NK T
cells. In another embodiment, dendritic cells, which have already
processed antigen are contacted with the NK T cells.
[0426] In one embodiment, the term "contacting a target cell"
refers herein to both direct and indirect exposure of cell to the
indicated item. In one embodiment, contact of NK T cells with a
compound of this invention, a cytokine, growth factor, dendritic
cell, or combination thereof, is direct or indirect. In one
embodiment, contacting a cell may comprise direct injection of the
cell through any means well known in the art, such as
microinjection. It is also envisaged, in another embodiment, that
supply to the cell is indirect, such as via provision in a culture
medium that surrounds the cell, or administration to a subject, via
any route well known in the art, and as described hereinbelow.
[0427] Methods for priming dendritic cells with antigen are well
known to one skilled in the art, and may be effected, as described
for example Hsu et al., Nature Med. 2:52-58 (1996); or Steinman et
al. International application PCT/US93/03141.
[0428] In one embodiment, a compound of this invention is added to
a culture of dendritic cells prior to contact of the dendritic
cells with NK T cells. In one embodiment, a compound of this
invention is used at a concentration of between about 0.1 to about
200 .mu.g/ml. In one embodiment, 10-50 .mu.g/ml is used. The
dendritic cells are, in one embodiment, cultured in the presence of
the antigen for a sufficient time to allow for uptake and
presentation, prior to, or in another embodiment, concurrent with
culture with NK T cells. In another embodiment, the compound is
administered to the subject, and, in another embodiment, is
targeted to the dendritic cell, wherein uptake occurs in vivo, for
methods as described hereinbelow.
[0429] Antigenic uptake and processing, in one embodiment, can
occur within 24 hours, or in another embodiment, longer periods of
time may be necessary, such as, for example, up to and including 4
days or, in another embodiment, shorter periods of time may be
necessary, such as, for example, about 1-2 hour periods.
[0430] In one embodiment, NK T cells may be cultured with dendritic
cells with a dendritic cell to T cell ratio of 10:1 to 1:1 to 1:10,
which ratio, in some embodiments is dependent upon the purity of
the NKT cell population used. In one embodiment, about
20,000-100,000 cells/well (96-well flat bottom plate) of a NKT cell
line, or 5 million per ml T cells, or in another embodiment,
200,000-400,000 cells/well of enriched NKT are administered to a
subject, for some of the methods of this invention.
[0431] In one embodiment, about 5 million T cells are administered
to a subject, for some of the methods of this invention.
[0432] In another embodiment, the NK T cells expanded by the
dendritic cells in the methods of this invention are autologous,
syngeneic or allogeneic, with respect to the dendritic cells.
[0433] In another embodiment, the dendritic cells used in the
methods of this invention are isolated from a subject suffering
from an autoimmune disease or disorder, cancer, an infection, which
in one embodiment, is HIV, mycobacterial or malarial infection.
[0434] In another embodiment, the dendritic cells used in the
methods of this invention are isolated from a subject with an
inappropriate or undesirable immune response, or in another
embodiment, the dendritic cells used in the methods of this
invention are isolated from a subject with an allergic
response.
[0435] In one embodiment, the NK T cells can be used to modulate an
immune response, in a disease-specific manner. It is to be
understood that any immune response, wherein it is desired to
enhance cytokine production, or elicit a particular cytokine
profile, including interferon-.gamma., interleukin-2 and/or
interleukin-4, the NK T cells of this invention may be thus
utilized, and represents an embodiment of this invention.
[0436] In another embodiment, the methods of this invention may
further comprise the step of culturing previously isolated, NK T
cells with additional dendritic cells, and a compound of this
invention, for a period of time resulting in further NK T cell
expansion, cytokine production, or a combination thereof.
[0437] In another embodiment, this invention provides a method for
delaying onset, reducing incidence or suppressing a disease in a
subject, comprising the steps of contacting in a culture NK T cells
with dendritic cells and a compound of this invention, for a period
of time resulting in NK T cell expansion, cytokine production or a
combination thereof, and administering NK T cells thus obtained to
the subject, wherein the NK T cells delay onset, reduce incidence
or suppress a disease in the subject, thereby delaying onset,
reducing incidence or suppressing a disease in the subject.
[0438] In one embodiment, cells for administration to a subject in
this invention may be provided in a composition. These compositions
may, in one embodiment, be administered parenterally or
intravenously. The compositions for administration may be, in one
embodiment, sterile solutions, or in other embodiments, aqueous or
non-aqueous, suspensions or emulsions. In one embodiment, the
compositions may comprise propylene glycol, polyethylene glycol,
injectable organic esters, for example ethyl oleate, or
cyclodextrins. In another embodiment, compositions may also
comprise wetting, emulsifying and/or dispersing agents. In another
embodiment, the compositions may also comprise sterile water or any
other sterile injectable medium. In another embodiment, the
compositions may comprise adjuvants, which are well known to a
person skilled in the art (for example, vitamin C, antioxidant
agents, etc.) for some of the methods as described herein, wherein
stimulation of an immune response is desired, as described further
hereinbelow.
[0439] In one embodiment, the compounds, cells, vaccines or
compositions of this invention may be administered to a subject via
injection. In one embodiment, injection may be via any means known
in the art, and may include, for example, intra-lymphoidal, or
subcutaneous injection.
[0440] In another embodiment, the NK T cells and dendritic cells
for administration in this invention may express adhesion molecules
for targeting to particular sites. In one embodiment, NK T cells
and/or dendritic cells may be engineered to express desired
molecules, or, in another embodiment, may be stimulated to express
the same. In one embodiment, the DC cells for administration in
this invention may further express chemokine receptors, in addition
to adhesion molecules, and in another embodiment, expression of the
same may serve to attract the DC to secondary lymphoid organs for
priming. In another embodiment, targeting of DCs to these sites may
be accomplished via injecting the DCs directly to secondary lympoid
organs through intralymphatic or intranodal injection.
[0441] In one embodiment, the antigen is delivered to dendritic
cells in vivo in the steady state, which, in another embodiment,
leads to expansion of disease specific NK T cells. Antigen delivery
in the steady state can be accomplished, in one embodiment, as
described (Bonifaz, et al. (2002) Journal of Experimental Medicine
196: 1627-1638; Manavalan et al. (2003) Transpl Immunol. 11:
245-58).
[0442] In another embodiment, select types of dendritic cells in
vivo function to prime the NK T cells.
[0443] In one embodiment, this invention provides a method for
modulating an immune response, which is an inappropriate or
undesirable response. In one embodiment, the immune response is
marked by a cytokine profile which is deleterious to the host.
[0444] In one embodiment, the NK T cells of this invention may be
administered to a recipient contemporaneously with treatment for a
particular disease, such as, for example, contemporaneous with
standard anti-cancer therapy, to serve as adjunct treatment for a
given cancer. In another embodiment, the NK T cells of this
invention may be administered prior to the administration of the
other treatment.
[0445] In another embodiment, this invention provides a method for
modulating an immune response, which is directed to infection with
a pathogen, and the immune response is not protective to the
subject.
[0446] In one embodiment, the pathogen may mimic the subject, and
initiate an autoimmune response. In another embodiment, infection
with the pathogen results in inflammation, which damages the host.
In one embodiment, the response results in inflammatory bowel
disease, or in another embodiment, gastritis, which may be a
result, in another embodiment, of H. pylori infection.
[0447] In another embodiment, the immune response results in a
cytokine profile, which is not beneficial to the host. In one
embodiment, the cytokine profile exacerbates disease. In one
embodiment, a Th2 response is initiated when a Th1 response is
beneficial to the host, such as for example, in lepromatous
leprosy. In another embodiment, a Th1 response is initiated, and
persists in the subject, such as for example, responses to the egg
antigen is schistosomiasis.
[0448] According to this aspect, and in one embodiment,
administration of the NK T cells alters the immune response
initiated in the subject, was not beneficial to the subject. In
another embodiment, the method may further comprise the step of
administering an agent to the subject, which if further associated
with protection from the pathogen.
[0449] In one embodiment, the term "modulating" refers to
initiation, augmentation, prolongation, inhibition, suppression or
prevention of a particular immune response, as is desired in a
particular situation. In one embodiment, modulating results in
diminished cytokine expression, which provides for diminished
immune responses, or their prevention. In another embodiment,
modulation results in the production of specific cytokines which
have a suppressive activity on immune responses, or, in another
embodiment, inflammatory responses in particular. In another
embodiment, modulating results in enhanced cytokine expression,
which provides for enhanced immune responses, or their stimulation.
In another embodiment, modulation results in the production of
specific cytokines which have a stimulatory activity on immune
responses, or, in another embodiment, responses to infection, or
neoplasia, in particular.
[0450] In one embodiment, this invention provides a method for
modulating an immune response in a subject, comprising the steps of
contacting a dendritic cell population in vivo with compound of
this invention, whereby the dendritic cell population contacts NK T
cells in the subject, wherein NK T cell contact promotes cytokine
production from the NK T cell population, thereby modulating an
immune response in a subject.
[0451] In one embodiment, the term "modulating" refers to
stimulating, enhancing or altering the immune response. In one
embodiment, the term "enhancing an immune response" refers to any
improvement in an immune response that has already been mounted by
a mammal. In another embodiment, the term "stimulating an immune
response" refers to the initiation of an immune response against an
antigen of interest in a mammal in which an immune response against
the antigen of interest has not already been initiated. It is to be
understood that reference to modulation of the immune response may,
in another embodiment, involve both the humoral and cell-mediated
arms of the immune system, which is accompanied by the presence of
Th2 and Th1 T helper cells, respectively, or in another embodiment,
each arm individually. For further discussion of immune responses,
see, e.g., Abbas et al. Cellular and Molecular Immunology, 3rd Ed.,
W. B. Saunders Co., Philadelphia, Pa. (1997).
[0452] Modulation of an immune response can be determined, in one
embodiment, by measuring changes or enhancements in production of
specific cytokines and/or chemokines for either or both arms of the
immune system. In one embodiment, modulation of the immune response
resulting in the stimulation or enhancement of the humoral immune
response, may be reflected by an increase in IL-6, which can be
determined by any number of means well known in the art, such as,
for example, by ELISA or RIA. In another embodiment, modulation of
the immune response resulting in the stimulation or enhancement of
the cell-mediated immune response, may be reflected by an increase
in IFN-.gamma. or IL-12, or both, which may be similarly
determined.
[0453] In one embodiment, stimulating, enhancing or altering the
immune response is associated with a change in cytokine profile. In
another embodiment stimulating, enhancing or altering the immune
response is associated with a change in cytokine expression. Such
changes may be readily measured by any number of means well known
in the art, including as described herein, ELISA, RIA, Western Blot
analysis, Northern blot analysis, PCR analysis, RNase protection
assays, and others.
[0454] In one embodiment, the infection is a latent infection.
[0455] In another embodiment, the immune response inhibits disease
progression in the subject, or in another embodiment, the immune
response inhibits or prevents neoplastic transformation in the
subject.
[0456] In one embodiment, inhibition or prevention of neoplastic
transformation according to the methods of this invention may be
effected via the use of tumor specific antigens, in addition to the
compounds of this invention. In one embodiment, a tumor specific
antigen may be, for example, mutated proteins which are expressed
as a result of a neoplastic, or preneoplastic events. In one
embodiment, the antigen is a molecule associated with malignant
tumor cells, such as, for example altered ras. Non-limiting
examples of tumors for which tumor specific antigens have been
identified include melanoma, B cell lymphoma, uterine or cervical
cancer.
[0457] In one embodiment, a melanoma antigen such as the human
melanoma specific antigen gp75 antigen may be used, or, in another
embodiment, in cervical cancer, papilloma virus antigens may be
used for the methods of this invention. Tumor specific idiotypic
protein derived from B cell lymphomas, or in another embodiment,
antigenic peptide or protein is derived from the Epstein-Bass
virus, which causes lymphomas may be used, as well.
[0458] In another embodiment, the antigenic peptide or protein is
derived from HER2/neu or chorio-embryonic antigen (CEA) for
suppression/inhibition of cancers of the breast, ovary, pancreas,
colon, prostate, and lung, which express these antigens. Similarly,
mucin-type antigens such as MUC-1 can be used against various
carcinomas; the MAGE, BAGE, and Mart-1 antigens can be used against
melanomas. In one embodiment, the methods may be tailored to a
specific cancer patient, such that the choice of antigenic peptide
or protein is based on which antigen(s) are expressed in the
patient's cancer cells, which may be predetermined by, in other
embodiments, surgical biopsy or blood cell sample followed by
immunohistochemistry.
[0459] In one embodiment, the subject being treated via a method of
this invention has a precancerous precursor, and/or is at an
elevated risk for cancer. Such elements are well known in the art,
and may comprise inappropriate expression of a given surface marker
or oncoprotein, the presence of hyperplastic cells, or the subject
may have at least one family member afflicted with a given cancer,
or have a lifestyle associated with enhanced risk for the incidence
of cancer, such as, for example, exposure to radiation, certain
viral infections, smoking tobacco products, and others, as will be
appreciated by one skilled in the art.
[0460] It is to be understood that any disease, disorder or
condition, whereby such disease, disorder or condition may be
positively affected by the production of a given cytokine profile,
or in another embodiment, is positively affected by the presence of
NK T cells, and may be so positively affected via a method of this
invention, is to be considered as part of this invention.
[0461] The following non-limiting examples may help to illustrate
some embodiments of the invention.
EXAMPLES
[0462] A number of glycolipids were synthesized (FIG. 5) and tested
them for NKT cell activation. These included glycolipids of
bacterial origin (compounds 5, 6, 17, and 18), .alpha.-GalCer
analogues modified on the galactose moiety and acyl group, and
variations of sulfatide, the only known promiscuous ligand for CD1.
The bacterial glycolipids include those isolated from the outer
membrane of Sphingomonas wittichii [Kawahara, K., Kubota, M., Sato,
N., Tsuge, K. & Seto, Y. (2002) FEMS Microbiol. Lett. 214,
289-294] and glycolipids from Borrelia burgdorferi, the Lyme
disease spirochete. CD1d-deficient (CD1d 1) mice were shown to have
impaired host resistance to infection by B. burgdorferi making its
glycolipids attractive compounds for further study as possible
natural CD1d antigens [Kumar, H., Belperron, A., Barthold, S. W.
& Bockenstedt, L. K. (2000) J. Immunol. 165, 4797-4801]. The
structures of its two major glycolipids were recently elucidated as
cholesteryl 6-O-acyl-.beta.-D-galactopyranoside 5 (B. burgdorferi
glycolipid 1, BbGL-I) and
1,2-di-O-acyl-3-O-.alpha.-D-galactopyranosyl-sn-glycerol 6
(BbGL-II). The Sphingomonas glycolipids, two new .alpha.-linked
glycosphingolipids 5 and 6, (GSL-1 and GSL-2 respectively) differ
most significantly from .alpha.-GalCer in the carbohydrate moiety
as they contain galactosyluronic acids as the polar head group
[Ben-Menachem, G., Kubler-Kielb, J., Coxon, B., Yergey, A. &
Schneerson, R. (2003) Proc. Natl. Acad. Sci. USA 100, 7913-7918].
However, they are more physiologically relevant as natural ligands
for CD1d-mediated NKT-cell activation since they originate from
bacteria. Biological experiments further show that galactouronic
sphingolipids stimulate IL-2 secretion in 1.2 (V.alpha.14
V.beta.8.2 DN3A4) NKT cell hybridomas. An .alpha.-GalCer analogue
4,3-O-sulfo-galactosylceramide (3-O-sulfo-GalCer) also caused
significant IL-2 secretion demonstrating that V.alpha.14i NKT cell
response is less sensitive to modification at the 3-OH position of
galactose. By contrast, any modification made at the 2-OH position
of galactose abolished all biological activity. Most other
synthetic analogues, however, were active. In addition, reactivity
of human V.alpha.24i NKT cells to GSL-1 and GSL-2 and sulfatides
were conserved.
Example 1
Synthesis of Analogues of glycolipid .alpha.-galactosyl ceramide:
3-O-sulfo-.alpha.-galactosyl ceramide
Preparation of Reagents
Reagents
[0463] All chemicals were purchased as reagent grade and used
without further purification. Dichloromethane (CH.sub.2Cl.sub.2,
DCM) was distilled over calcium hydride and tetrahydrofuran (THF)
over sodium/benzophenone. Anhydrous methanol (MeOH) and pyridine
(Py) were purchased from a commercial source.
General Assay Information:
[0464] Reactions were monitored with analytical thin-layer
chromatography (TLC) on silica gel 60 F.sub.254 glass plates and
visualized under UV (254 nm) and/or by staining with acidic ceric
ammonium molybdate. Flash column chromatography was performed on
silica gel 60 Geduran (35-75 .mu.m EM Science). .sup.1H NMR spectra
were recorded on a 400-500- or 600-Hz NMR spectrometer at
20.degree. C. Chemical shift (in ppm) was determined relative to
tetramethylsilane (.delta.0 ppm) in deuterated solvents. Coupling
constant(s) in hertz (Hz) were measured from one-dimensional
spectra. .sup.13C Attached Proton Test (C-Apt) spectra were
obtained with the NMR-400, 500 or 600 spectrometer (100, 125 or 150
Hz) and were calibrated with either CDCl.sub.3 (.delta. 77.23 ppm)
or Py-d.sub.5 (.delta. 123.87 ppm).
p-Methylphenyl
2-O-benzyl-4,6-O-benzylidene-3-O-levulinyl-1-thio-D-galactopyranoside
(II)
[0465] 3 grams of I (6.45 mmol) was dissolved in DCM. LevOH (0.9
ml, 1.35 eq), EDC (1.6 g, 1.3 eq) and DMAP (197 mg, 0.25 eq) were
added. The reaction was allowed to proceed overnight while covered
in foil. The reaction was then diluted with DCM, washed with water,
saturated sodium bicarbonate solution, brine and dried over sodium
sulfate. After removal of the solvent the mixture was purified by
column chromatography (Hexanes:EtOAc:DCM 3:1:1) to give 2.83 g of
II in 78% yield.
[0466] .sup.1H(CDCl.sub.3 500 MHz) .delta.=7.61-7.03 (m, 14H), 5.48
(s, 1H), 4.98 (dd, J=3.7 Hz, J=9.6 Hz, 1H), 4.77 (d, J=11.0 Hz,
1H), 4.63 (d, J=9.5 Hz, 1H), 4.51 (d, J=11 Hz, 1H), 4.36-4.32 (m,
2H), 3.99-3.97 (m, 1H), 3.90-3.86 (m, 1H), 3.51 (s, 1H), 2.56-2.50
(m, 2H), 2.46-2.40 (m, 2H), 2.31 (s, 3H), 2.09, (s, 3H); .sup.13C
NMR (125 MHz, CDCl.sub.3) .delta.=206.05, 172.09, 138.18, 137.76,
137.64, 133.11, 129.61, 128.98, 128.57, 128.22, 128.01, 127.68,
127.57, 126.45, 100.83, 86.53, 75.41, 75.05, 73.77, 73.71, 69.09,
37.70, 29.60, 27.99; HRMS (MALDI-FTMS) calcd. for
C.sub.32H.sub.34O.sub.7SNa [M+Na].sup.+ 585.1923. found
585.1900.
2-O-benzyl-4,6-O-benzylidene-3-O-levulinyl-D-galactopyranoside
(III)
[0467] II (600 mg, 1.07 mmol) was dissolved in 50 mL of acetone.
The reaction mixture was cooled to 0.degree. C., and NBS (228 mg,
1.28 mmol, 1.2 equiv) was added. The reaction mixture turned orange
immediately. After 10 min the reaction was quenched by addition of
solid NH.sub.4Cl. The mixture was diluted with water and ethyl
acetate, and the aqueous layer was extracted with ethyl acetate (3
x). The combined organic layer was extracted with brine, dried over
sodium sulfate, and evaporated. The residue was subjected to column
chromatography (hexanes:ethyl EtOAc:DCM 1:1:1) to give 442 mg (91%)
of 7.
[0468] .sup.1H(CDCl.sub.3 500 MHz) .delta.=7.50-7.25 (m, 10H), 5.48
(d, J=4.8, 1H), 5.38 (s, 1H), 5.32 (dd, J=3.7 Hz, J=10.3 Hz, 1H),
4.94-4.90 (m, 1H), 4.73-4.62 (m, 3H), 4.36 (d, J=3.3 Hz, 1H), 4.05
(dd, J=3.3 Hz, 10.3 Hz, 1H), 4.00-3.98 (m, 2H), 3.93, (s, 1H),
3.52-3.51 (m, 1H), 2.71-2.53 (m, 4H), 2.08 (s, 3H); .sup.13C NMR
(125 MHz, CDCl.sub.3) .delta.=206.43, 177.73, 172.35, 172.24,
138.41, 137.78, 137.63, 137.57, 128.89, 128.85, 128.38, 128.21,
128.03, 127.75, 127.67, 127.51, 126.15, 126.12, 100.61, 97.50,
91.98, 77.57, 74.68, 74.10, 73.82, 73.56, 73.38, 73.28, 70.55,
69.17, 68.93, 66.24, 62.18, 37.82, 37.79, 29.67, 289.38, 28.11,
28.04; HRMS (MALDI-FTMS) calcd. for C.sub.25H.sub.29O.sub.8
[M+H].sup.+ 457.1862 found 457.1856.
O-(2-O-benzyl-4,6-O-benzylidene-3-O-levulinyl-D-galactopyranosyl)
Trichloroacetimidate (IV)
[0469] To a solution of III (188.5 mg, 0.46 mmol) dissolved in 4 ml
of DCM was added CCl.sub.3CN (0.46 ml, 4.62 mmol) and DBU (31
.mu.l, 0.21 mmol). After 2 hours at room temperature, the dark
solution was concentrated and then purified by flash chromatography
Hexanes:EtOAc (2:1) and 1% triethylamine to yield 8 (211 mg,
77%).
[0470] .sup.1H(CDCl.sub.3 500 MHz) .delta.=7.59-7.34 (m, 10H), 5.61
(s, 1H), 5.45 (dd, J=3.2 Hz, 10.7 Hz, 1H), 4.80-4.72 (m, 2H), 4.60
(d, J=3.3 Hz, 2H), 4.38-4.33 (m, 2H), 4.13-4.10 (dd, J=1.8 Hz, 12.5
Hz, 1H), 4.05 (s, 1H), 2.79-2.72 (m, 2H), 2.65 (m, 2H), 2.16 (s,
3H); .sup.13C NMR (125 MHz, CDCl.sub.3) .delta.=206.43, 177.73,
172.35, 172.27, 138.41, 137.78, 137.63, 137.57, 128.89, 128.85,
128.38, 128.21, 128.03, 127.86, 127.75, 127.67, 127.51, 126.15,
126.12, 100.61, 97.50, 91.98, 77.57, 74.68, 74.10, 73.56, 73.38,
73.28, 70.55, 69.17, 68.93, 66.24, 6218, 37.82 37.79, 29.67, 29.38,
28.11, 28.04.
2-Azido-3,4-di-O-benzyl-1-O-(2-O-benzyl-4,6-O-benzylidene-3-O-levulinyl-.a-
lpha.-D-galactopyranosyl)-D-ribo-octadeca-6-ene-1-ol (VI)
[0471] A solution of trichloroacetimidate IV (150 mg, 0.25 mmol,
1.5 equiv)) and sphingosine derivative V (86 mg, 0.16 mmol) in 2.5
mL of anhydrous THF was added over freshly dried powdered AW-300
molecular sieves and cooled to -20.degree. C. TMSOTf (23 .mu.L, 0.8
equiv) was slowly added to the solution, and the mixture was warmed
up to 0.degree. C. in 2.5 hours. The reaction was quenched by
addition of Et.sub.3N (0.1 mL), and the mixture was diluted with
EtOAc and filtered through Celite. The organic layer was washed
with saturated aqueous NaHCO.sub.3 and brine, dried
(Na.sub.2SO.sub.4), and concentrated. The residue was purified by
column chromatography on silica gel (hexanes:EtOAc 6:1) to furnish
VI (57 mg, 46% based on consumed acceptor V) as a syrup, and
recover V (18 mg).
[0472] .sup.1H NMR (CDCl.sub.3, 400 MHz): .delta.=7.49-7.23 (m
20H), 5.56-5.45 (m 3H), 5.32 (dd, 1H, J=3.5 Hz, 10.5 Hz), 4.98 (d,
1H, J=3.1 Hz), 4.70-4.51 (m, 6H), 4.38 (m, 1H), 4.13-3.82 (m, 5H),
3.71-3.62 (m, 4H), 2.75-2.40 (m, 6H), 2.08 (s, 3H), 2.06-1.97 (m,
2H), 1.25 (bs, 18H), 0.88 (t, 3H, J=7.0 Hz); .sup.13C NMR (125 MHz,
CDCl.sub.3) .delta.=206.30, 172.25, 138.21, 137.93, 137.67, 132.60,
128.89, 128.37, 128.35, 128.33, 129.29, 128.08, 127.27, 128.08,
127.78, 127.73, 127.69, 127.63, 127.60, 127.17, 124.69, 100.65,
98.61, 79.41, 78.95, 74.06, 73.65, 73.41, 73.10, 71.94, 70.79,
69.02, 68.21, 62.41, 61.97, 37.93, 31.89, 29.71-29.32, 28.19,
27.58, 22.66, 14.10; ESI-MS (positive-ion mode): m/z 982.4
[M+Na].sup.+.
3,4-Di-O-benzyl-1-O-(2-O-benzyl-4,6-O-benzylidene-.alpha.-D-galactopyranos-
yl)-2-hexacosylamino-D-ribo-octadeca-6-ene-1-ol (X)
[0473] The azide VI (57 mg, 0.059 mmol) was dissolved in 2.0 mL of
anhydrous THF and cooled to 0.degree. C. PMe.sub.3 (0.4 mL of 1.0 M
in toluene, 0.4 mmol) was added to the solution, and the reaction
was warmed up to room temperature and stirred over night. After
almost disappearance of the starting material, 0.8 mL of aq 1 M
NaOH was added to the mixture and stirred for 5 hours.
CH.sub.2Cl.sub.2 was then added to the solution, and the mixture
was washed with brine, dried over Na.sub.2SO.sub.4, and
concentrated. The residue was used for the next step without
further purification. Hexacosanoic acid (35 mg, 0.088 mmol, 1.5 eq)
was suspended in CH.sub.2Cl.sub.2 (2.0 ml), and then DEPBT (26 mg
0.087 mmol, 1.5 eq) and DIEA (15 .mu.L, 1.5 eq) were added. The
mixture was vigorously shaken for 1 h to give a clear light yellow
solution in which above crude amine mixture VIIIa and VIIIb was
added subsequently. The solution was stirred over night at room
temperature and then diluted with EtOAc and washed with saturated
NaHCO.sub.3 and brine. The organic phase was dried over
Na.sub.2SO.sub.4 and concentrated to afford a solid (IXa and IXb,
57 mg), which was dissolved in 2 mL Py-HOAc solution (3:1 v/v,
contains 0.30M NH.sub.2NH.sub.2.HOAc) and stirred for 1.5 h at room
temperature. After the usual workup similarly as above, the residue
was purified by column chromatography on silica gel (hexanes:EtOAc
2:1) to furnish X (40 mg, 56% over 3 steps) as a solid.
[0474] .sup.1H NMR (CDCl.sub.3, 400 MHz) .delta.=7.47-7.23 (m 20H),
5.67 (d, 1H, J=8.6 Hz), 5.51-5.44 (m, 3H), 4.95 (d, 1H, J=2.7 Hz),
4.77-4.49 (m, 6H), 4.40 (m, 1H), 4.21 (d, 1H, J=2.7 Hz), 4.12-4.07
(m, 2H), 3.94-3.58 (m, 8H), 2.45 (m, 2H), 2.08-1.88 (m, 4H), 1.49
(m, 2H), 1.25 (bs, 62H), 0.88 (t, 6H, J=7.0 Hz); .sup.13C NMR
(CDCl.sub.3, 100 MHz): .delta.=173.14, 138.50, 138.33, 137.74,
132.57, 129.32, 128.62, 128.40, 128.08, 127.95, 127.85, 126.45,
125.20, 101.37, 99.04, 79.97, 79.22, 76.29, 73.48, 73.41, 71.79,
69.50, 68.86, 68.19, 62.94, 50.26, 36.96, 32.13, 29.91-29.56,
28.14, 27.78, 25.93, 22.90, 14.34; HRMS (MALDI-FTMS) calcd for
C.sub.78H.sub.119NO.sub.9Na [M+Na].sup.+ 1236.8777. found
1236.8741.
3,4-Di-O-benzyl-1-O-(2-O-benzyl-4,6-O-benzylidene-3-O-sulfo-.alpha.-D-gala-
ctopyranosyl)-2-hexacosylamino-D-ribo-octadeca-6-ene-1-ol, sodium
salt (XI)
[0475] To a solution of X (40 mg, 0.033 mmol) in Py (2.5 mL) was
added SO.sub.3.Py complex (79 mg, 0.5 mmol, 15 eq). The mixture was
stirred at room temperature for 2.5 hours. Water solution (2.5 mL)
of NaHCO.sub.3 (62 mg) was added to quench the reaction. The
reaction mixture was diluted with CH.sub.2Cl.sub.2, and washed with
brine, dried (Na.sub.2SO.sub.4), and concentrated. The residue was
purified by column chromatography on silica gel
(CH.sub.2Cl.sub.2:MeOH 15:1) to give XI (39 mg, 90%) as a
solid.
[0476] .sup.1H NMR (CDCl.sub.3/CD.sub.3OD 1:1, 400 MHz)
.delta.=7.87 (d, 1H, J=8.9 Hz), 7.58-7.17 (m, 20H), 5.59 (s, 1H),
5.43 (m, 2H), 4.96 (m, 3H), 4.82 (m, 1H), 4.73 (d, 1H, J=2.3 Hz),
4.62-4.58 (m, 2H), 4.52-4.44 (m, 2H), 4.19-3.99 (m, 5H), 3.78 (bs,
2H), 3.66 (bs, 1H), 3.56 (m, 1H), 2.47 (m, 1H), 2.34 (m, 1H), 2.13
(t, 2H, J=7.0 Hz), 2.01 (m, 2H), 1.54 (bs, 2H), 1.27 (bs, 62H),
0.89 (t, 6H, J=7.0 Hz); .sup.13C NMR (CDCl.sub.3/CD.sub.3OD 1:1,
100 MHz): .delta.=173.92, 138.30, 137.66, 137.57, 131.42, 128.43,
128.01-127.03, 125.95, 125.66, 100.59, 98.99, 80.16, 79.75, 75.04,
74.84, 73.97, 73.60, 73.49, 71.09, 68.74, 67.00, 62.70, 49.71,
49.62, 31.56, 29.29-29.02, 27.01, 25.59, 22.27, 13.44; HRMS
(MALDI-FTMS) calcd for C.sub.78H.sub.118NO.sub.12SNaK [M+K].sup.+
1354.7909. found 1354.7933.
2-Hexacosylamino-1-O-(3-O-sulfo-.alpha.-D-galactopyranosyl)-D-ribo-1,3,4-o-
ctadecantriol, sodium salt (4)
[0477] XI (39 mg, 0.030 mmol) was dissolved in HOAc-MeOH (1:1 v/v,
6 mL). 80 mg of palladium black was added and the reaction solution
was saturated with hydrogen by a balloon. After stirring at room
temperature for 20 hours, the catalyst was removed by filtration
over Celite and washed with CH.sub.2Cl.sub.2/MeOH (1:1) thoroughly.
Evaporation of the solvent gave a residue which was dissolved in
CH.sub.2Cl.sub.2/MeOH (1:1) mixed solvent again and then saturated
NaHCO.sub.3 (3 mL) was added to stir at room temperature for half
an hour. After removal of the solvent, the residue was purified by
column chromatography on silica gel (CH.sub.2Cl.sub.2:MeOH 6:1) to
give 4 (24 mg, 83%) as a light yellow solid.
[0478] .sup.1H NMR (CDCl.sub.3/CD.sub.3OD 1:1, 400 MHz)
.delta.=4.95 (d, 1H, J=3.5 Hz), 4.49 (dd, 1H, J=2.7 Hz, 10.2 Hz),
4.35 (m, 1H), 4.17 (m, 1H), 4.02 (dd, 1H, J=2.7 Hz, 9.8 Hz),
3.88-3.85 (m, 2H), 3.80-3.72 (m, 4H), 3.69-3.65 (m, 2H), 3.61-3.57
(m, 1H), 2.24 (t, 2H, J=7.4 Hz), 1.59 (m, 4H), 1.27 (bs, 68H), 0.89
(t, 6H, J=7.0 Hz); .sup.13C NMR (CDCl.sub.3/CD.sub.3OD 1:1, 100
MHz): 6=174.31, 99.08, 77.57, 73.42, 71.64, 70.44, 67.81, 67.19,
66.48, 61.28, 49.90, 35.89, 31.51, 31.32, 29.29-28.94, 25.53,
22.22, 13.34; HRMS (MALDI-FTMS) calcd for
C.sub.50H.sub.98NO.sub.12SNa.sub.2 [M+Na].sup.+ 982.6599. found
982.6585.
Synthesis Scheme
[0479] Sulfatide and .alpha.-galactosyl ceramide have similar
structures and possess immunostimulatory and immunomodulatory
activity, when presented to T cells via CD1. In order to determine
whether hybrid molecules of sulfatide and .alpha.-galactosyl
ceramide, which are sulfate derivatives
3-O-sulfo-.alpha./.beta.-galactosylceramides 10 and 4 (FIG. 1),
have comparable activity, the molecules were synthesized and
evaluated for immunostimulatory activity.
[0480] For the synthesis of 3-O-sulfo-.alpha.-galactosylceramides
4, selective sulfation at 3'' OH of the galactose moiety is a key
step. Typically, regioselective sulfation of the 3-hydroxyl of the
sugar ring utilizes dibutylstannylene acetals as activated
intermediates, however, this method can only be applied to
.beta.-galactosides; for .alpha.-galactosides, the
dibutylstannylene acetal can form a complex between the 2-hydroxyl
and the anomeric oxygen to give the 2''-O-derivative by reaction
with an electrophile.
[0481] In order to address this, a 3''-lev and
2''-benzyl-4'',6''-benzylidene protected trichloroacetimidate donor
IV (FIG. 2). The temporary protecting group Lev, can be selectively
removed after glycosylation in the presence of hydrazine. The
benzyl and benzylidene groups at 2,4,6 positions direct the next
.alpha.-glycosidic bond formation (Figueroa-Perez, S. et al
Carbohydrate Res. 2000, 328, 95; Plettenburg, O. et al. J. Org.
Chem. 2002, 67, 4559).
[0482] As shown in FIG. 2A, the preparation of IV started with the
known thioglycoside I in 50% yield over three steps. The
sphingosine building block V was employed in this synthesis, with
donor IV coupled to acceptor V in the presence of TMSOTf, used as
promoter to give .alpha.-glycoside VI, in a moderate yield.
[0483] A staudinger reduction of VI with PMe.sub.3, in NaOH
solution was used to hydrolyze the imino-phosphorane intermediate
VII, however, the Lev group cannot survive under this conditions
and approximate 50% of the Lev group was cleaved to give an amine
mixture of VIIIa and VIIIb (1:1) determined by .sup.1H NMR. Since
VIIIa possesses a free C-3 hydroxyl, it is crucial to choose a
selective coupling reagent in the condensation between amine VIIIa
and the fatty acid.
[0484] Since DEPBT
[3-(diethoxyphosphoryloxy)-(1,2,3)-benzotriazin-4(3H)-one] can
selectively form an amide bond in the presence of unprotected
hydroxyl groups, it was used in the reaction mixture with VIIIa,
VIIIb, and hexacosanoic acid to give IXa and IXb, followed by
deprotection of the remaining Lev groups using hydrazine to provide
the desired galactosyl ceramide X in 56% yield over 3 steps.
Treating the 3''-OH free glycolipid X with Py.SO3 led to the
sulfate derivative XI in high yield, which gave compound 4 upon
hydrogenation with palladium black and neutralization with
NaHCO.sub.3 (aqueous solution) in 78% yield (FIG. 2B).
Example 2
Synthesis of Analogues of Glycolipid .alpha.-Galactosyl
Ceramide
3-O-sulfo-.beta.-galactosylceramide
[0485] For the synthesis of 10, perbenzoylated trichloroacetimidate
donor 40 is used in the glycosylation of the sphingosine acceptor V
to yield a .beta.-galactosyl ceramide derivative XII (FIG. 3).
After the Staudinger reduction of XII, a complex mixture was
produced, with no isolation of the amine XV. Since the
perbenzoylated galactosyl ceramide is sensitive to basic
conditions, a NaHSO.sub.4 solution instead of a NaOH solution was
used for the reduction work-up procedure to decompose the
imino-phosphorane intermediate XIV. However, hydrolyzation of XIV
into XV was very slow, and in turn, the longer reaction time led to
the degradation of glycosidic bond which attributed to complicated
product formation.
Example 3
Synthesis of analogues of glycolipid .alpha.-galactosyl
ceramide
3-O-sulfo-.beta.-galactosylceramide
[0486] Another synthetic strategy was used to synthesize
3-O-sulfo-.beta.-galactosylceramide. In this strategy, the azide
was first reduced, and the fatty acid coupled, prior to the
glycosylation step (FIG. 4A). Compound XVIII was prepared from the
sphingosine derivative XVI (Plettenburg, O. et al. J. Org. Chem.
2002, 67, 4559) in 54% yield over 2 steps.
[0487] Using TMSOTf as a promoter, the ceramide acceptor XVIII was
reacted with donor 40 to give the .beta.-glycoside XIX in 54%
yield. After debenzyolation and hydrogenation of XIX, the
.beta.-galactosyl ceramide XX was obtained in quantitative yield.
XX was finally sulfated by Bu.sub.2SnO/Me.sub.3N.SO.sub.3 and
subsequently neutralized by NaHCO.sub.3 to give the product 10, in
80% yield (FIG. 4B) (Compostella, F et al. Tetrahedron 2002, 58,
8703).
Example 4
Recognition of glycolipids by the human NKT cell line results in
cytokine secretion
Materials and Methods
Glycolipids
[0488] .alpha.-GalCer was obtained as described [Plettenburg, O.,
et al. (2002) J. Org. Chem. 67, 4559-64]. The intermediates 29, 36
and 40 (FIGS. 3 and 4), were obtained as described [Plettenburg,
O., et al. (2002) supra; Williams, L., et al. (1996) Tetrahedron
52, 11673-11694; Deng, S. Y., B et al. (1999) J. Org. Chem. 64,
7265-7266]. The compounds 5, 6, 19, 30, 33, 37, and 41 (FIGS. 3 and
4) were obtained as described hereinbelow. The remaining compounds,
except 19, 10 and 4, and their intermediates were obtained as
described hereinabove.
Sphingosine Acceptor
[0489] The synthesis scheme for the sphingosine acceptor (30) is
shown in FIG. 6. Compound 29 (3.31 g, 13.5 mmol) (Williams, L., et
al. supra) was dissolved in 70 ml of dry THF. The solution was
cooled to -40.degree. C. and vinyl grignard solution (31 ml of a 1
M solution in THF) was added via a dropping funnel over a period of
1 hr. The temperature was kept between -20.degree. C. and
-40.degree. C. The reaction mixture was allowed to warm to room
temperature and stirred for another hr. The reaction was quenched
by addition of 60 ml of saturated (NH.sub.4).sub.2SO.sub.4 solution
and evaporated to dryness. The residue was diluted with water and
extracted with ethyl acetate (3.times.). The combined organic layer
was extracted with brine, dried over MgSO.sub.4 and evaporated to
give a yellow oil. Column chromatography (Hex: EtOAc 3:1) yielded
the syn diastereomer (2.11 g, 8.2 mmol, anti/syn=3.5:1) in 61%
yield. Then the syn diastereomer (300 mg, 1.16 mmol) was dissolved
in 1 ml of dry dichloromethane in a two-necked flask equipped with
a reflux condenser under argon. 486 mg (3.48 mmol) of pentadecene
was added via a syringe. A solution of 20 mg (2 mol %) of Grubb's
second generation catalyst (purchased from Strem Chemicals) in 1 ml
of dichloromethane was added and the solution was heated under
rapid reflux for 40 hr. The reaction mixture was evaporated and
then directly chormatographed (Hex: EtOAc 6:1) which yielded (0.82
mmol, 71%) of the desired product.
Synthesis of Glycolipids
[0490] The synthesis scheme is shown in FIG. 4. A solution of
trichloroacetimidate 32 (160.4 mg, 0.258 mmol) and sphingosine
acceptor 31 (100 mg, 0.198 mmol) in 4 ml of anhydrous Et.sub.2O and
2 ml of anhydrous THF was added over freshly dried 4 .ANG.
molecular sieves and cooled to -50.degree. C. Trimethylsilylmethyl
trifluoromethanesulfonate (TMSOTf) (3.33 mg, 0.0198 mmol) was added
and the mixture stirred at -50.degree. C. for 1 hour. The mixture
was allowed to warm to -20.degree. C. and another 3.33 mg of TMSOTf
was added. The mixture was then slowly allowed to warm to room
temperature and stirred for 3 hour. The solution was then diluted
with EtOAc and filtered over celite. The organic layer was washed
with saturated aqueous NaHCO.sub.3 and brine, dried (MgSO.sub.4),
and concentrated. The residue was purified by column chromatography
on silica gel (toluene:EtOAc 12:1) to give 128 mg (67.5%, 0.134
mmol) of 33.
[0491] Compound 34 (36 mg, 0.03 mmol), dissolved in 6 ml of EtOAc,
was added to 36 mg of 20 wt % palladium hydroxide in 1 ml of EtOAc
and saturated with hydrogen. The reaction vessel was purged with
hydrogen, and the mixture was stirred at room temperature
overnight. The reaction mixture was filtered and the solvent was
evaporated. The above hydrogenated compound was dissolved in 2 ml
THF, 1 ml water, and 1 ml methanol. LiOH (9 mg, 0.14 mmol) was
added to the solution and the reaction was stirred at room
temperature for four hours. 100 mg of Na.sub.2CO.sub.3 was added
and the mixture stirred for 30 minutes. The solvent was evaporated
and the remaining residue was purified on silica gel by column
chromatography (CH.sub.2Cl.sub.2:MeOH 4:1) to give 7.8 mg of 1
(38%, 0.0114 mmol, 2 steps).
[0492] After deprotection of compound 42 (14 mg, 0.017 mmol),
Bu.sub.2SnO (6.5 mg, 0.0259 mmol) dissolved in 1 ml of MeOH was
added. The mixture was refluxed under argon for 2 h. After
evaporation of the solvent, Me.sub.3N.SO.sub.3 (5 mg, 0.035 mmol)
dissolved in 1 ml THF was added and the reaction was allowed to
proceed at room temperature for 6 hours. The solvent was then
removed under reduce pressure and the residue dissolved in
CHCl.sub.3/MeOH 1:1 (1 mL) and loaded onto an ion exchange column
(Dowex 50X8 Na+ form). After elution with CHCl.sub.3/MeOH 1:1, the
mixture was concentrated and purified by column chromatography
(CH.sub.2Cl.sub.2:MeOH 5:1) to give 18 (14.4 mg, 95%).
1.2 Hybridoma Assay
[0493] CD1d reactive T cell hybridomas with an invariant
V.alpha.14i T cell antigen receptor a chain were used, as described
(Sidobre, S., et al. (2004) Proc. Natl. Acad. Sci. USA 101,
12254-12259). T cell hybridomas were stimulated with the indicated
glycolipids that were added either to plates coated with soluble
CD1d, or with CD1d transfected A20 B lymphoma cells, as described
(Elewaut, D., et al. (2003) J. Exp. Med. 198, 1133-1146). As a
measure of T cell activation, IL-2 release into the tissue culture
medium was measured after 16 hours culture by an ELISA assay.
Generation of V.alpha.24i Human NKT Cell Line
[0494] Human NKT cell lines, expressing the V.alpha.24i T cell
receptor as well as CD161, were generated as follows: Anti-CD161
monoclonal antibodies, and anti-CD14 monoclonal antibodies, each
coupled to magnetic beads (Miltenyi biotec, Auburn, Calif.), were
used sequentially to isolate CD161+ cells and CD14.sup.+ cells from
leukopaks. Immature dendritic cells were generated from the CD14+
cells after a two-day incubation in the presence of 300 U/ml GM-CSF
(R&D systems, Minneapolis, Minn.) and 100 U/ml IL-4 (R&D
systems, Minneapolis, Minn.). Following irradiation with 2000 rads,
the immature dendritic cells were co-cultured with syngeneic
CD161.sup.+ cells in the presence of 100 ng/ml of
alpha-galactosylceramide and 10 IU/ml of IL-2 (Invitrogen, Carlsbad
Calif.) for 10 to 14 days. After stimulating the CD161+ cells a
second time with alpha-galactosylceramide-pulsed, irradiated
immature dendritic cells, NKT cell lines were shown by flow
cytometry to express both CD161.sup.+ and V 24i TCR (99%
purity).
In Vitro Cytokine Secretion Assay Using Human NKT Cell Lines
[0495] IFN-.gamma. and IL-4 secretion by the V.alpha. 24i human NKT
cell line was determined by ELISA (BD Pharmingen, San Diego,
Calif.) after culture for 16 hours. For these assays,
1.times.10.sup.5 V.alpha. 24i human NKT cells were co-cultured with
4.times.10.sup.5 irradiated, immature CD14.sup.+ dendritic cells,
in the presence of the glycolipid compounds at 10 .mu.g/ml in a
96-well flat-bottom plate.
Results
[0496] In order to test whether glycolipids of bacterial origin (5,
6, 8, 17) (represented in FIG. 5), or analogues thereof, which
comprise structures similar to .alpha.-GalCer either at the sugar
or lipid moiety, activate NKT cells through CD1d, the glycolipids
were synthesized and assayed. Analogues 7 and 8 (FIG. 5) were
prepared, and used to probe the effect of the carboxyl group on the
sugar and the .alpha.-hydroxyl group on the lipid. Compounds 19, 10
and 4 contain a 3'-sulfate group with an .alpha. or
.beta.-glycosidic linkage. 20-23 were prepared to probe the effect
of the 2'-modification of .alpha.-GalCer. Analogues of
.alpha.-GalCer with modification of the lipid moiety were also
prepared to probe their interaction with CD1d and the subsequent
effect on NKT cell activation.
[0497] Mouse V.alpha.14i NKT cells immortalized by cell fusion
provided a convenient method for assaying the ability of the
synthetic glycolipids to activate T cells. As shown in FIG. 8a, the
3-O-sulfo-.alpha.-GalCer, 4, stimulated significant IL-2 release
from the hybridomas when used at 10 .mu.g/ml. Dose response curves
indicated, however, that this compound was somewhat less active
than .alpha.-GalCer (data not shown) in this model. By contrast,
every modification of the 2 OH position of the galactose (compounds
10-13) that were tested abolished all biological activity. These
data indicate that the V.alpha.14i NKT cell response to glycolipids
apparently is more sensitive to modifications of the 2 than to the
3 position.
[0498] B. burgdorferi glycolipids (17-18) and compounds 24 and 25
were moderately active in the 1.2 hybridoma assay. However IL-2
secretion could only be detected when large quantities of the
glycolipids were used to stimulate the hybridoma cells.
[0499] CD1d coated plates were used to assay response of the
hybridomas to the Sphingomonas glycolipids (FIG. 8b). A substantial
level of IL-2 secretion can be observed for all compounds. The
structure of the sugar head group significantly affected the
activation of the hybridomas. .alpha.-GalCer and the galactose
analogue 7, consistently solicited greater IL-2 secretion when
compared to the galacturonic acid derivatives. Also affecting
activity was the (S)-2-hydroxy of the fatty amide tail. A fully
saturated tail was more greatly favored, suggesting that the
.alpha.-hydroxyl group is not optimal. In fact the (S)-2-hydroxy
appeared to have a greater affect on activity as compound to
compound 8, a galactose analogue, that was less able to activate
IL-2 secretion when compared with 5, the galacturonic acid compound
without the .alpha.-hydroxyl fatty amide. Though 7 and 8 are not
known to be natural products, both could be precursors to compounds
5 and 6.
[0500] IFN-.gamma. and IL-4 secretion from a V.alpha.24i NKT cell
line were assessed, after stimulation with irradiated, syngeneic
CD14.sup.+ immature dendritic cells in the presence of 10 .mu.g/ml
of the glycolipids and 10 IU/ml of IL-2 (FIG. 9a). Stimulation of
the NKT cell line by each glycolipid compound resulted in
significant IFN-.gamma. and IL-4 secretion, when compared to the
negative control. While greater IFN-.gamma. and IL-4 secretion was
observed after stimulation by the potent NKT cell agonist,
.alpha.-GalCer, secretion of IFN-.gamma. and IL-4 by NKT cells
stimulated by 1-10 .mu.g/ml of 3-O-sulfo-.alpha.-galactosylceramide
was approximately half that of .alpha.-GalCer, but twice that
induced by the other glycolipids. .beta.-linked sulfatides 19 and
10 were also observed to elicit both IFN-.gamma. and IL-4
production. In fact, the level of cytokine secretion was comparable
to the GSLs.
[0501] As illustrated in FIGS. 8C and 8D,
interferon-.gamma.secretion by human NKT cells in response to
glycolipid presentation by CD14.sup.+ DCs, was superior when the
glycolipid was 3-sulfo-.alpha.-GalCer 4, as compared to
.alpha.-GalCer, at a concentration of 10-20 .mu.g/mL. Compound 4
efficiently stimulated IL-4 and IFN-.gamma. secretion, indicating
that the modification of the 3''-OH position of the galactose
moiety with sulfate was useful in NKT cells stimulation.
[0502] NKT cells activation was sensitive to the configuration of
the anomeric carbon of glycolipid antigen molecules.
3-sulfo-.beta.-GalCer 10 had minimal to no affinity for NKT
lymphocytes due to the .beta.-linkage of glycosidic bond,
indicating that the .alpha.-linkage of the glycoside was essential
for CD1 antigen binding.
[0503] Other .alpha.-GalCer analogues with an acetyl side chain or
a shortened backbone were also tested and some activity was also
observed (FIGS. 8C and 8D).
Example 5
Human NKT Cell Lines Bind to Glycolipids in the Context of CD1d
Materials and Methods
[0504] In Vitro CD1d-Dimer Assay Using a Human NKT Cell Line
[0505] One mg of soluble divalent human CD1d-IgG1 fusion protein
(human CD1d-IgG1 dimers, BD Pharmingen) were incubated overnight
with 10 M of each glycolipid at 32.degree. C. and at neutral pH
according to the manufacturer's protocol. The glycolipid-loaded
CD1d-IgG1 dimers were incubated with human NKT cells at 4.degree.
C. for 60 minutes, followed by incubation with PE-coupled
anti-mouse IgG1 mAb (A85-1). The cells were also surface stained
with a PerCP-coupled anti-CD3 mAb (BD Pharmingen, San Diego,
Calif.).
Results
[0506] Although glycolipids stimulated the NKT cell line, it does
not necessarily follow that the glycolipids were presented by CD
molecules and were capable of triggering the V.alpha.24i T cell
receptor expressed by the NKT cells. Therefore, in order to
demonstrate glycolipid antigen reactivity to the V.alpha.24i T cell
receptor at the single cell level, a human NKT cell line with human
CD1d dimers loaded with different glycolipids was stained, and
unloaded CD1d dimers were used as a negative control. Each
glycoplid-loaded dimer nearly universally stained the human NKT
cells, while the unloaded dimer did not stain these cells (FIG.
10).
Example 6
Computer Modeling of GSL Complexed to mCD1d
Materials and Methods
Model Generation
[0507] A model of GSL 1 complexed with the crystal structure of
mCD1d (Zeng, Z., et al. (1997) Science 277, 339-45) was generated
by Autodock 3.0 (Morris, G. M., et al. (1998) J. Comput. Chem. 19,
1639-1662).
Results
[0508] To further understand the interaction of bacterial
glycolipid 1 with CD1d, a model of GSL 1 complexed with mCD1d was
generated, and is shown in FIG. 11. According to the model, the
fatty acyl chain extended into the F' pocket and the sphingosine
chain toward the A' pocket. This allowed for the polar head group
to be oriented such that it was exposed for recognition by a T cell
antigen receptor. Numerous favorable contacts could be observed
between mCD1d and the glycosphingolipid. Among them, possible
hydrogen bonding included interactions between the carboxylate of
the sugar and the backbone carbonyl of Asp80, and the amide
nitrogen of the fatty acid tail with the Asp80 sidechain.
[0509] While it was thought that mCD1d to be somewhat accommodating
in terms of lipid tail length on NKT cell reactivity, changes in
the lipid length, composition, or addition of an .alpha.-hydroxyl
group on the fatty acid, as seen in FIG. 11, could cause a slight
shift in orientation of the sugar and thereby affect
CD1d/glycolipid complex recognition by the T-cell receptor.
Substitution of galacturonic acid for galactose may produce similar
results. The perturbation caused by having the 6-OH oxidized to a
carboxylic acid caused only moderate changes in NKT cell
reactivity, thus the model provides an effective means for
designing additional ligands.
Example 7
Synthesis of Analogues of Glycolipid .alpha.-Galactosyl
Ceramide
[0510] A number of glycolipids were synthesized and tested for NKT
cell activation. A synthetic scheme is provided in scheme 1
below:
##STR00087##
[0511] wherein R is selected from Table 1 below to provide the
corresponding compound.
TABLE-US-00001 TABLE 1 Compound No. R = 58 ##STR00088## 59
##STR00089## 60 ##STR00090## 61 ##STR00091## 62 ##STR00092## 63
##STR00093## 64 ##STR00094## 65 ##STR00095## 66 ##STR00096## 67
##STR00097## 68 ##STR00098## 69 ##STR00099## 70 ##STR00100## 71
##STR00101## 72 ##STR00102## 73 ##STR00103## 74 ##STR00104## 75
##STR00105## 76 ##STR00106## 77 ##STR00107## 78 ##STR00108## 79
##STR00109## 80 ##STR00110## 81 ##STR00111## 82 ##STR00112## 83
##STR00113## 84 ##STR00114## 85 ##STR00115## 86 ##STR00116## 87
##STR00117## 88 ##STR00118## 89 ##STR00119## 90 ##STR00120## 91
##STR00121## 92 ##STR00122## 93 ##STR00123## 94 ##STR00124## 95
##STR00125## 96 ##STR00126## 97 ##STR00127## 98 ##STR00128## 99
##STR00129## 100 ##STR00130## 101 ##STR00131## 102 ##STR00132## 103
##STR00133## 104 ##STR00134## 105 ##STR00135## 106 ##STR00136## 107
##STR00137## 108 ##STR00138## 109 ##STR00139## 110 ##STR00140## 111
##STR00141##
##STR00142##
[0512] Other compounds were synthesized as described in Xing G W et
al. Bioorg Med Chem. 2005 Apr. 15; 13(8):2907-16; and Wu D. et al.,
Proc Natl Acad Sci USA. 2005 February 1; 102(5):1351-6.
[0513] All chemicals were purchased as reagent grade and used
without further purification. Dichloromethane (CH.sub.2Cl.sub.2)
were distilled over calcium hydride. Tetrahydrofuran (THF) and
ether were distilled over sodium metal/benzophenone ketyl.
Anhydrous N,N-dimethylformamide (DMF) was purchased from Aldrich.
Molecular sieves (MS) for glycosylation were AW300 (Aldrich) and
activated by flame. Reactions were monitored with analytical thin
layer chromatography (TLC) in EM silica gel 60 F254 plates and
visualized under UV (254 nm) and/or staining with acidic ceric
ammonium molybdate or ninhydrin. Flash column chromatography was
performed on silica gel 60 Geduran (35-75 um, EM Science). .sup.1H
NMR spectra were recorded on a Bruker DRX-500 (500 MHz)
spectrometer or a Bruker DRX-600 (600 MHz) spectrometer at
20.degree. C. Chemical shifts (6 ppm) were assigned according to
the internal standard signal of tetramethylsilane in CDCl.sub.3
(.delta.=0 ppm). .sup.13C NMR spectra were obtained using Attached
Proton Test (APT) on a Bruker DRX-500 (125 MHz) spectrometer Bruker
DRX-600 (150 MHz) spectrometer and were reported in 6 ppm scale
using the signal of CDCl.sub.3 (.delta.=77.00 ppm) for calibration.
Coupling constants (J) are reported in Hz. Splitting patterns are
described by using the following abbreviations: s, singlet; brs,
broad singlet; d, doublet; t, triplet; q, quartet; m, multiplet.
.sup.1H NMR spectra are reported in this order: chemical shift;
number(s) of proton; multiplicity; coupling constant(s).
##STR00143##
[0514] To a stirred solution of Wittig reagent a (6.1 g, 11 mmol),
prepared from 1-bromopentadecane and triphenylphosphine refluxed in
toluene for 5 days, in THF (30 mL) was added n-BuLi (1.6 mol/L in
hexane, 6.4 mL, 10 mmol) dropwise at -78.degree. C., then the
solution was stirred for 1 h at room temperature. After 1 h the
solution was cooled to -78.degree. C. and Garner's aldehyde A (2.1
g, 9.2 mmol) in THF (20 mL) was added. After stirring for 1 h at
room temperature, the solution was poured into ice-water and
extracted with AcOEt. The organic layer was washed with brine,
dried with MgSO.sub.4, and evaporated to dryness. The residue was
purified by flash column chromatography on silica gel (toluene
100%) to give B (2.6 g, 66%) as a pale yellow oil. .sup.1H NMR (600
MHz, CDCl.sub.3) .delta. 5.36-5.52 (2H, m), 4.51-4.75 (1H, m), 4.05
(1H, dd, J=6.3 Hz, 8.6 Hz), 3.63 (1H, dd, J=3.3 Hz, 8.6 Hz),
1.94-2.21 (2H, m), 1.20-1.66 (39H, m), 0.88 (1H, t, J=6.9 Hz).
.sup.13C NMR (150 MHz, CDCl.sub.3) .delta. 151.97, 131.98 (brs),
130.70 (brs), 130.28 (brs), 129.36 (brs), 93.94 (brs), 93.38 (brs),
79.69 (brs), 69.04, 54.55, 31.90, 29.72, 29.67, 29.65, 29.63,
29.59, 29.49, 29.33, 29.29, 28.46, 22.66, 14.08. HRMS (ESI-TOF) for
C.sub.26H.sub.49NO.sub.3Na.sup.+ [M+Na].sup.+ calcd 446.3604. found
446.3602.
Synthesis of Compound C
##STR00144##
[0516] To a stirred solution of B (2.6 g, 6.0 mmol) and
1-methylmorpholine N-oxide (1.1 g, 9.0 mmol) in BAH and H.sub.2O
(1:1, 30 mL), OsO.sub.4 (2.5 w/v in BAH, 3.1 mL) was added at room
temperature. The solution was stirred overnight and quenched with
Na.sub.2SO.sub.3 aq. The solution was extracted 2 times with AcOEt,
washed with brine, dried with MgSO.sub.4, and evaporated to
dryness. The residue was purified by flash column chromatography on
silica gel (CHCl.sub.3 to CHCl.sub.3:MeOH 20:1) to give C (1.6 g,
56%) as a white solid. .sup.1H NMR (600 MHz, CDCl.sub.3) .delta.
4.13-4.21 (2H, m), 3.95-4.03 (1H, m), 3.55-3.65 (1H, m), 3.29-3.42
(2H, m), 1.38-1.68 (17H, m), 1.21-1.38 (24H, m), 0.88 (3H, t, J=7.1
Hz). .sup.13C NMR (125 MHz, CDCl.sub.3) .quadrature..delta.:
153.93, 93.97, 81.35, 74.89, 73.85, 65.26, 59.41, 32.27, 31.87,
29.65, 29.63, 29.59, 29.31, 28.31, 26.80, 26.18, 23.94, 22.64,
14.07. HRMS (ESI-TOF) for C.sub.26H.sub.51NO.sub.5Na.sup.+
[M+Na].sup.+ calcd 480.3659. found 480.3659.
Synthesis of Compound D
##STR00145##
[0518] To a stirred solution of C (328 mg, 0.72 mmol) and DMAP
(cat.) in pyridine (5 mL) was added BzC1 (0.20 mL, 1.8 mmol) and
stirred at room temperature overnight. The solution was added Sat.
NaHCO.sub.3 aq., extracted with AcOEt, washed with brine, dried
with MgSO.sub.4, and evaporated to dryness. The residue was
purified by flash column chromatography on silica gel (Hex.: AcOEt
10:1) to give dibenzoylated product (471 mg) as a colorless oil. To
a stirred solution of this compound in dry MeOH (5 mL) was added
TFA (10 mL) dropwise at 0.degree. C. After 2 h, the solution was
evaporated to dryness and co-evaporated with toluene 3 times. The
residue was dissolved in dioxane (15 mL) and Sat. NaHCO.sub.3 aq.
(15 mL). To a stirred solution, Na.sub.2CO.sub.3 (155 mg) and
Boc.sub.2O (320 mg, 1.5 mmol) were added and stirred overnight.
This solution was extracted with AcOEt, washed brine, dried with
MgSO.sub.4 and evaporated to dryness. The residue was purified by
flash column chromatography on silica gel (Hex.: AcOEt 5:1) to give
D (301 mg, 67% over 3 steps) as a colorless oil. .sup.1H NMR (500
MHz, CDCl.sub.3) .delta.: 8.05 (2H, d, J=7.2 Hz), 7.95 (2H, d,
J=7.1 Hz), 7.63 (1H, t, J=7.5 Hz), 7.46-7.54 (3H, m), 7.38 (2H, t,
J=7.5 Hz), 5.50 (1H, d, J=9.6 Hz), 5.40 (1H, dd, J=2.4 Hz, 9.3 Hz),
5.33 (1H, d, J=9.5 Hz), 4.00-4.07 (1H, m), 3.64-3.67 (2H, m),
2.55-2.65 (1H, m), 1.96-2.10 (2H, m), 1.48 (9H, s), 1.20-1.45 (24H,
m), 0.88 (3H, t, J=7.0 Hz). .sup.13C NMR (125 MHz, CDCl.sub.3)
.quadrature..delta.: 167.03, 166.15, 155.54, 133.73, 133.04,
129.95, 129.64, 129.15, 128.75, 128.63, 128.36, 80.00, 73.81,
73.72, 60.40, 51.46, 31.90, 29.67, 29.65, 29.63, 29.59, 29.53,
29.51, 29.34, 28.30, 25.72, 22.67, 14.11. HRMS (ESI-TOF) for
C.sub.37H.sub.55NO.sub.7Na.sup.+ [M+Na].sup.+ calcd 648.3871. found
648.3866.
Synthesis of Compound E
##STR00146##
[0520] To a stirred solution of D (1.5 g, 2.5 mmol), d (1.8 g, 3.0
mmol) and AW300 (2.0 g) in Et.sub.2O-THF (7:1, 34 mL) was cooled to
-40.degree. C. and added BF.sub.3.OEt.sub.2 (0.63 mL, 5 mmol). The
solution was stirred for 4 h at ambient temperature, and warmed to
room temperature. The solution was filtered, added sat. NaHCO.sub.3
aq. and extracted with AcOEt. The organic layer was dried with
MgSO.sub.4 and evaporated to dryness. The residue was purified by
flash column chromatography on silica gel (Hex.: AcOEt 5:1) to give
a coupled product (980 mg, 38%) as a colorless oil.
[0521] To a stirred solution of this compound (980 mg, 0.95 mmol)
in EtOH (30 mL) was added 10% Pd--C (490 mg) and stirred vigorously
under H.sub.2 atmosphere overnight. The solution was filtered and
concentrated to dryness. The residue and DMAP (cat.) were dissolved
with pyridine (10 mL) and Ac.sub.2O (10 mL), stirred at room
temperature overnight. The solution was concentrated, dissolved
with AcOEt, washed with brine and concentrated to dryness. The
residue was purified by flash column chromatography on silica gel
(Hex.: AcOEt 2:1) to give E (790 mg, 87%) as a colorless oil.
.sup.1H NMR (600 MHz, CDCl.sub.3) .delta.: 8.00 (2H, d, J=7.3 Hz),
7.93 (2H, d, J=7.5 Hz), 7.60 (1H, t, J=7.4 Hz), 7.52 (1H, t, J=7.4
Hz), 7.47 (2H, t, J=7.8 Hz), 7.37 (2H, t, J=7.7 Hz), 5.66 (1H, dd,
J=2.3 Hz, 9.6 Hz), 5.41-5.48 (2H, m), 5.29-5.33 (2H, m), 5.16 (1H,
dd, J=3.6 Hz, 10.9 Hz), 4.82 (1H, d, J=3.5 Hz), 4.26 (1H, t, J=9.8
Hz), 4.17 (1H, t, J=6.7 Hz), 4.06 (1H, dd, J=6.1 Hz, 11.3 Hz), 4.00
(1H, dd, J=7.1 Hz, 11.3 Hz), 3.78 (1H, dd, J=2.4 Hz, 10.7 Hz), 3.49
(1H, dd, J=2.4 Hz, 10.7 Hz), 2.10 (3H, s), 2.02 (3H, s), 1.99 (3H,
s), 1.98 (3H, s), 1.90-1.99 (2H, m), 1.52 (9H, s), 1.20-1.37 (24H,
m), 0.88 (3H, t, J=7.0 Hz). .sup.13C NMR (150 MHz, CDCl.sub.3)
.quadrature..quadrature.: 170.58, 170.28, 170.11, 170.01, 166.11,
164.97, 155.11, 133.34, 132.90, 129.92, 129.70, 129.56, 129.37,
129.52, 128.22, 97.35, 80.28, 73.88, 71.81, 67.84, 67.70, 67.60,
66.97, 66.50, 61.67, 49.94, 31.83, 29.60, 29.58, 29.56, 29.51,
29.43, 29.27, 29.20, 28.27, 28.13, 25.63, 22.60, 20.60, 20.58,
20.52, 20.49, 14.05. HRMS (ESI-TOF) for
C.sub.51H.sub.73NO.sub.16Na.sup.+ [M+Na].sup.+ calcd 978.4821.
found 978.4814.
[0522] Alternatively, to a stirred solution of D (1.0 g, 1.6 mmol)
d (1.4 g, 2.4 mmol) and AW300 (2.0 g) in Et2O-THF (2:1, 30 mL) was
cooled to -30.degree. C. and added TMSOTf (18 .mu.L, 0.081 mmol).
The solution was stirred for 1 h at ambient temperature, and d (0.9
g, 1.5 mmol) and TMSOTf (20 .mu.L, 0.090 mmol) was added. The
solution was stirred an additional 1 h and then warmed to
-10.degree. C. The solution was added triethylamine (2 mL), then
filtered, added sat. NaHCO.sub.3 aq. and extracted with AcOEt. The
organic layer was dried with MgSO4 and evaporated to dryness. The
residue was purified by flash column chromatography on silica gel
(Hex.: AcOEt 5:1) to give a coupled product (1.3 g, 77%) as a
colorless oil.
[0523] To a stirred solution of this compound (1.3 g, 1.2 mmol) in
AcOEt (30 mL) and EtOH (30 mL) was added 10% Pd--C (600 mg) and
stirred vigorously under H.sub.2 atmosphere overnight. The solution
was filtered and concentrated to dryness. The residue and DMAP
(cat.) were dissolved in pyridine (10 mL) and Ac.sub.2O (10 mL),
stirred at room temperature overnight. The solution was
concentrated, dissolved with AcOEt, washed with brine and
concentrated to dryness. The residue was purified by flash column
chromatography on silica gel (Hex.: AcOEt 2:1) to give 42 (1.1 g,
93%) as a colorless oil. .sup.1H NMR (600 MHz, CDCl.sub.3) .delta.:
8.00 (2H, d, J=7.3 Hz), 7.93 (2H, d, J=7.5 Hz), 7.60 (1H, t, J=7.4
Hz), 7.52 (1H, t, J=7.4 Hz), 7.47 (2H, t, J=7.8 Hz), 7.37 (2H, t,
J=7.7 Hz), 5.66 (1H, dd, J=2.3 Hz, 9.6 Hz), 5.41-5.48 (2H, m),
5.29-5.33 (2H, m), 5.16 (1H, dd, J=3.6 Hz, 10.9 Hz), 4.82 (1H, d,
J=3.5 Hz), 4.26 (1H, t, J=9.8 Hz), 4.17 (1H, t, J=6.7 Hz), 4.06
(1H, dd, J=6.1 Hz, 11.3 Hz), 4.00 (1H, dd, J=7.1 Hz, 11.3 Hz), 3.78
(1H, dd, J=2.4 Hz, 10.7 Hz), 3.49 (1H, dd, J=2.4 Hz, 10.7 Hz), 2.10
(3H, s), 2.02 (3H, s), 1.99 (3H, s), 1.98 (3H, s), 1.90-1.99 (2H,
m), 1.52 (9H, s), 1.20-1.37 (24H, m), 0.88 (3H, t, J=7.0 Hz).
.sup.13C NMR (150 MHz, CDCl.sub.3) .delta.: 170.58, 170.28, 170.11,
170.01, 166.11, 164.97, 155.11, 133.34, 132.90, 129.92, 129.70,
129.56, 129.37, 129.52, 128.22, 97.35, 80.28, 73.88, 71.81, 67.84,
67.70, 67.60, 66.97, 66.50, 61.67, 49.94, 31.83, 29.60, 29.58,
29.56, 29.51, 29.43, 29.27, 29.20, 28.27, 28.13, 25.63, 22.60,
20.60, 20.58, 20.52, 20.49, 14.05. HRMS (ESI-TOF) for
C.sub.51H.sub.73NO.sub.16Na.sup.+ [M+Na].sup.+ calcd 978.4821.
found 978.4814.
[0524] General procedure of synthesis of fatty acid chain analogues
was as follows:
##STR00147##
[0525] To a stirred solution of E (240 mg, 0.25 mmol) in
CH.sub.2Cl.sub.2 (2.4 mL) was added TFA (2.4 mL) at 0.degree. C.
and stirred for 2 h at ambient temperature. The solution was
evaporated to dryness and co-evaporated with toluene 3 times to
give deprotected amine. This compound was dissolved in
CH.sub.2Cl.sub.2 and used for the next reaction without further
purification.
[0526] To the deprotected amine (0.021 mmol) in CH.sub.2Cl.sub.2
(1.0 mL) was added R--COOH (0.031 mmol), HBTu (12 mg, 0.031 mmol)
and NMM (31 mg, 0.3 mmol) and stirred at room temperature
overnight. The solution was purified by flash column chromatography
on silica gel (Hex.: AcOEt 2:1) to give the coupled product as a
amorphic solids.
[0527] These compounds were dissolved in MeOH (2.0 mL) and 0.5
mol/L NaOMe in MeOH (4 drops) was added. The solution was stirred
overnight at room temperature and evaporated to dryness. The
residues were purified by flash column chromatography on silica gel
(CHCl.sub.3:MeOH 10:1) to give R.sub.1,2.
[0528] Compound R.sub.1,2 were synthesized in a manner similar to
that described above.
##STR00148##
[0529] Intermediate of R.sub.1:
[0530] Yield 28 mg (65%). .sup.1H NMR (600 MHz, CDCl.sub.3)
.quadrature.: 8.00 (2H, dd, J=1.2 Hz, 8.2 Hz), 7.91 (2H, dd, J=1.2
Hz, 8.3 Hz), 7.61 (1H, t, J=7.4 Hz), 7.53 (1H, t, J=7.4 Hz), 7.47
(2H, t, J=7.8 Hz), 7.38 (2H, t, J=7.8 Hz), 7.25-7.28 (2H, m),
7.15-7.20 (3H, m), 6.57 (1H, d, J=9.7 Hz), 5.69 (1H, dd, J=2.4 Hz,
9.9 Hz), 5.43 (1H, d, J=3.3 Hz), 5.33 (1H, dd, J=3.4 Hz, 10.9 Hz),
5.29-5.32 (1H, m), 5.15 (1H, dd, J=3.6 Hz, 10.9 Hz), 4.81 (1H, d,
J=3.6 Hz), 4.62 (1H, tt, J=2.5 Hz, 9.9 Hz), 4.11 (1H, dd, J=6.6 Hz,
13.3 Hz), 4.05 (1H, dd, J=6.0 Hz, 11.0 Hz), 3.96 (1H, dd, J=7.0 Hz,
11.3 Hz), 3.73 (1H, dd, J=2.8 Hz, 10.9 Hz), 3.49 (1H, dd, J=2.4 Hz,
10.9 Hz), 2.64 (2H, t, J=7.8 Hz), 2.35 (2H, t, J=7.7 Hz), 2.10 (3H,
s), 1.994 (3H, s), 1.986 (3H, s), 1.94 (3H, s), 1.89-1.93 (2H, m),
1.66-1.78 (4H, m), 1.42-1.48 (2H, m), 1.18-1.35 (24H, m), 0.87 (3H,
t, J=7.1 Hz). .sup.13C NMR (150 MHz, CDCl.sub.3)
.quadrature..quadrature.: 172.90, 170.59, 170.39, 170.21, 170.15,
166.50, 165.03, 142.53, 133.49, 133.07, 129.87, 129.77, 129.62,
129.30, 128.62, 128.38, 128.32, 128.24, 125.62, 97.29, 74.14,
71.45, 67.90, 67.53, 67.32, 67.10, 66.68, 61.74, 48.18, 36.65,
35.76, 31.90, 31.22, 29.65, 29.63, 29.60, 29.56, 29.53, 29.34,
29.32, 28.99, 27.86, 25.69, 25.57, 22.67, 20.67, 20.62, 20.58,
20.50, 14.11. HRMS (ESI-TOF) for C.sub.36H.sub.64NO.sub.9.sup.+
[M+H].sup.+ calcd 1030.5522. found 1030.5507.
##STR00149##
[0531] R.sub.1:
[0532] Yield 14 mg (79%). .sup.1H NMR (500 MHz, CDCl.sub.3-MeOH
4:1) .quadrature.: 7.25-7.29 (2H, m), 7.15-7.19 (3H, m), 4.90 (1H,
d, J=3.9 Hz), 4.17-4.21 (1H, m), 3.94 (1H, d, J=3.2 Hz), 3.87 (1H,
d, J=4.7 Hz), 3.67-3.81 (6H, m), 3.51-3.56 (2H, m), 2.61 (2H, t,
J=7.8 Hz), 2.20 (2H, t, J=7.6 Hz), 1.44-1.70 (6H, m), 1.21-1.41
(26H, m), 0.88 (3H, t, J=7.0 Hz). .sup.13C NMR (125 MHz,
CDCl.sub.3-MeOH 4:1) .quadrature..quadrature.: 174.08, 142.25,
128.10, 128.01, 125.42, 99.49, 74.64, 71.86, 70.49, 70.04, 69.53,
68.70, 67.27, 61.69, 50.17, 36.14, 35.46, 32.49, 31.67, 30.93,
29.54, 29.49, 29.46, 29.40, 29.11, 29.00, 28.67, 25.60, 25.40,
22.42, 13.76. HRMS (ESI-TOF) for C.sub.36H.sub.64NO.sub.9.sup.+
[M+H].sup.+ calcd 654.4575. found 654.4568.
##STR00150##
[0533] Intermediate of R.sub.2:
[0534] Yield 28 mg (65%). .sup.1H NMR (600 MHz, CDCl.sub.3)
.delta.: 7.99 (2H, d, J=7.7 Hz), 7.91 (2H, d, J=7.9 Hz), 7.61 (1H,
t, J=7.4 Hz), 7.53 (1H, t, J=7.4 Hz), 7.47 (2H, t, J=7.7 Hz), 7.37
(2H, t, J=7.7 Hz), 7.24-7.28 (2H, m), 7.14-7.18 (3H, m), 6.62 (1H,
d, J=9.8 Hz), 5.71 (1H, dd, J=2.3 Hz, 9.9 Hz), 5.43 (1H, d, J=3.2
Hz), 5.35 (1H, dd, J=3.3 Hz, 10.9 Hz), 5.29-5.31 (1H, m), 5.15 (1H,
dd, J=3.6 Hz, 10.9 Hz), 4.81 (1H, d, J=3.6 Hz), 4.59-4.64 (1H, m),
4.09-4.12 (1H, m), 4.06 (1H, dd, J=5.9 Hz, 11.2 Hz), 3.97 (1H, dd,
J=7.0 Hz, 11.3 Hz), 3.73 (1H, dd, J=2.7 Hz, 10.8 Hz), 3.48 (1H, dd,
J=2.2 Hz, 10.9 Hz), 2.60 (2H, t, J=7.8 Hz), 2.35 (2H, t, J=7.7 Hz),
2.10 (3H, s), 2.005 (3H, s), 1.996 (3H, s), 1.94 (3H, s), 1.89-1.93
(2H, m), 1.59-1.76 (4H, m), 1.19-1.43 (30H, m), 0.87 (3H, t, J=7.0
Hz). .sup.13C NMR (150 MHz, CDCl.sub.3) .quadrature..quadrature.:
172.99, 170.60, 170.38, 170.23, 170.16, 166.52, 165.03, 142.80,
133.47, 133.07, 129.87, 129.76, 129.61, 129.30, 128.61, 128.36,
128.31, 128.18, 125.52, 97.24, 74.16, 71.35, 67.93, 67.52, 67.29,
67.12, 66.70, 61.77, 48.15, 36.71, 35.92, 31.89, 31.47, 29.66,
29.63, 29.60, 29.56, 29.53, 29.34, 29.30, 29.26, 29.23, 29.19,
27.80, 25.72, 25.67, 22.66, 20.67, 20.63, 20.58, 20.49, 14.11. HRMS
(ESI-TOF) for C.sub.60H.sub.84NO.sub.15.sup.+ [M+H].sup.+ calcd
1058.5835. found 1058.5819.
##STR00151##
[0535] R.sub.2:
[0536] Yield 14 mg (79%). .sup.1H NMR (500 MHz, CDCl.sub.3-MeOH
4:1) .delta.: 7.25-7.29 (2H, m), 7.15-7.18 (3H, m), 4.91 (1H, d,
J=3.8 Hz), 4.17-4.22 (1H, m), 3.94 (1H, d, J=3.2 Hz), 3.87 (1H, d,
J=4.7 Hz), 3.67-3.81 (6H, m), 3.51-3.56 (2H, m), 2.60 (2H, t, J=7.7
Hz), 2.19 (2H, t, J=7.7 Hz), 1.49-1.70 (6H, m), 1.20-1.41 (30H, m),
0.88 (3H, t, J=7.0 Hz). .sup.13C NMR (125 MHz, CDCl.sub.3-MeOH 4:1)
.quadrature..quadrature.: 174.18, 142.54, 128.11, 127.97, 125.34,
99.49, 74.64, 71.86, 70.48, 70.05, 69.54, 68.71, 67.28, 61.71,
50.17, 36.28, 36.23, 35.67, 32.47, 31.67, 31.24, 29.54, 29.49,
29.47, 29.41, 29.11, 29.04, 29.01, 28.92, 25.60, 25.57, 22.42,
13.76. HRMS (ESI-TOF) for C.sub.38H.sub.68NO.sub.9.sup.+
[M+H].sup.+ calcd 682.4888. found 682.4880.
[0537] In general, the phytosphingosine skeleton was constructed by
modification of a method described by Savage and co-workers [R. D.
Goff, et al. J. Am. Chem. Soc., 2004, 126, 13602-13603] Garner's
aldehyde A was coupled with a Wittig reagent prepared from
phosphonium bromide B according to Berova's method [O. Shirota, et
al., Tetrahedron, 1999, 55, 13643-13658] to give cis olefin B in
66% yield. Treatment of olefin B with osmium tetroxide gave a
corresponding diol C and its undesired isomer. The two hydroxyl
groups of diol C were protected with benzoyl groups, and then the
isopropylidene group was removed by the successive treatment of
TFA, followed by Boc anhydride protection to afford
phytosphingosine acceptor D in 67% yield over 3 steps.
[0538] Glycosylation of phytosphingosine acceptor D and donor d in
the presence of BF.sub.3.OEt.sub.2 gave a predominantly
.alpha.-configured product. Hydrogenation was avoided as the final
deprotection step to ensure accessibility to a more diverse set of
compounds. The galactose protecting groups were removed and then
protected with acetates to furnish the key intermediate E in 33%
over 3 steps.
[0539] Compound E was deprotected with TFA to give the deprotected
amine. A variety of fatty acyl chain analogues were then couples to
the amine to form R after removal of the acetyl groups.
[0540] A synthetic scheme for Phytosphingosine Analogues is
provided in scheme 2 below:
Synthesis of Phytosphingosine Analogues of .alpha.-GalCer
##STR00152##
[0542] wherein R is selected from Table 2 below to provide the
corresponding compound.
TABLE-US-00002 TABLE 2 Compound No. R = 112 ##STR00153## 113
##STR00154##
Synthesis of Phytosphingosine Chain Analogues
[0543] The synthesis of phytosphingosine chain analogues PC6Ph,
PC8Ph, PC11Ph and PC13Ph are summarized in scheme 2. The synthetic
route is essentially similar to the one reported in J. Am. Chem.
Soc. 2006, 128, 9022-9023, supporting information.
##STR00155## ##STR00156##
Characterization Data:
[0544] Compound 112 (lot. MFJ3-017-1):
[0545] .sup.1H NMR (500 MHz, CDCl.sub.3-MeOH 4:1) .delta.: 7.26 (m,
2H), 7.23-7.19 (m, 2H), 7.18-7.14 (m, 1H), 4.90 (d, J=3.9 Hz, 1H),
4.24-4.19 (m, 1H), 3.86 (dd, J=10.8, 5.2 Hz, 1H), 3.82-3.62 (m,
7H), 3.58-3.53 (m, 2H), 2.92-2.84 (m, 1H), 2.67 (ddd, J=13.7, 9.3,
7.5 Hz, 1H), 2.16 (m, 2H), 2.06-1.98 (m, 1H), 1.74-1.65 (m, 1H),
1.62-1.53 (m, 2H), 1.33-1.19 (m, 44H), 0.88 (t, J=7.0 Hz, 3H).
.sup.13C NMR (125 MHz, CDCl.sub.3-MeOH 4:1) .quadrature..delta.:
174.06, 141.93, 128.25, 128.01, 125.43, 99.48, 74.60, 70.75, 70.44,
69.99, 69.52, 68.66, 67.03, 61.69, 50.15, 50.06, 36.27, 34.13,
31.67, 31.59, 29.43, 29.31, 29.15, 29.09, 25.55, 22.41, 17.60,
13.76. HRMS (ESI-TOF) for C.sub.44H.sub.80NO.sub.9.sup.+
[M+H].sup.+ calcd 766.5827. found 766.5813.
Compound 113 (lot. MFJ3-018-1)
[0546] .sup.1H NMR (400 MHz, CDCl.sub.3-MeOH 4:1) .delta.: 7.26 (m,
2H), 7.19-7.13 (m, 3H), 4.91 (d, J=3.8 Hz, 1H), 4.20 (q, J=4.4 Hz,
1H), 3.95-3.85 (m, 2H), 3.83-3.61 (m, 6H), 3.59-3.50 (m, 2H), 2.63
(t, J=7.5 Hz, 2H), 2.20 (t, J=7.5 Hz, 2H), 1.78-1.54 (m, 6H),
1.47-1.17 (m, 46H), 0.89 (t, J=6.9 Hz, 3H). .sup.13C NMR (100 MHz,
CDCl.sub.3-MeOH 4:1) .quadrature..delta.: 174.16, 142.27, 127.91,
127.77, 125.14, 99.33, 74.28, 71.38, 70.42, 69.86, 69.33, 68.51,
66.84, 61.40, 50.02, 36.04, 35.52, 31.93, 31.51, 31.21, 29.26,
29.14, 28.99, 28.94, 25.47, 25.08, 22.25, 13.51. HRMS (ESI-TOF) for
C.sub.46H.sub.84NO.sub.9.sup.+ [M+H].sup.+ calcd 794.6140. found
794.6129.
Example 8
Recognition of Glycolipids by Murine NKT Cell Lines Results in IL-2
Secretion
Materials and Methods
Glycolipids
[0547] The compound KRN 7000 was purchased (Kirin, Japan). The
remaining compounds were synthesized as described hereinabove.
1.2 Hybridoma Assay
[0548] CD1d reactive T cell hybridomas with an invariant
V.alpha.14i T cell antigen receptor a chain were used, as described
(Sidobre, S., et al. (2004) Proc. Natl. Acad. Sci. USA 101,
12254-12259). T cell hybridomas were stimulated with 0.0001-1
.mu.g/ml of the indicated glycolipids added to CD1d transfected A20
B lymphoma cells, as described (Elewaut, D., et al. (2003) J. Exp.
Med. 198, 1133-1146). As a measure of T cell activation, IL-2 and
IFN-.gamma. release into the tissue culture medium was measured
after 16 hours culture by an ELISA assay.
In Vitro Cytokine Secretion Assay Using Human NKT Cell Lines
[0549] IL-2 secretion by the V.alpha. 24i human NKT cell line was
determined by ELISA (BD Pharmingen, San Diego, Calif.) after
culture for 16 hours. For these assays, 1.times.10.sup.5 V.alpha.
24i human NKT cells were co-cultured with 4.times.10.sup.5
irradiated, immature CD14.sup.+ dendritic cells, in the presence of
the glycolipid compounds at 10 .mu.g/ml in a 96-well flat-bottom
plate [Wu et al. PNAS 2005 102: 1351].
Results
[0550] In order to test whether glycolipids with modifications the
lipid moiety of .alpha.-GalCer affected the immunogenicity of the
compound, a series of glycolipids with varied modifications of this
region were synthesized and assayed.
[0551] Mouse V.alpha.14i NKT cells immortalized by cell fusion
provided a convenient method for assaying the ability of the
synthetic glycolipids to activate T cells. As shown in FIG. 12, a
number of the compounds (60, 61, 62, 64, 65, 74, 77) stimulated
significant IL-2 release from the hybridomas when used at 1
.mu.g/ml, however KRN7000 (.alpha.-GalCer) appeared to stimulate
the greatest amount of IL-2 release.
[0552] Another series of compounds were evaluated for their ability
to stimulate IL-2 release, when provided at various concentrations
(FIG. 13). In this case, several of these compounds stimulated
greater IL-2 release, as compared to KRN7000 (.alpha.-GalCer), in
particular, compounds with a terminal phenyl substituent.
[0553] The simplest benzoyl analog 58 showed only slight activity.
Introduction of either electron donating groups (68; 4-OCH3, 69;
4-CH3) or withdrawing groups (70; 4-Cl, 59; 4-CF3) onto the benzene
ring increased their activity. The other benzoyl analogues,
4-Pyridyl 80, 3-pyridyl 71, indole analog 81 and biphenyl analogues
72, 63 and 80 also exhibited similar trends. However, their
activities still remained about half of .alpha.-GalCer.
[0554] Benzyl analogues 60, 61, 69, 73 and 74 showed improved IL-2
production compared with the benzoyl analogues. Of these compounds,
smaller aromatic groups such as 60, 61 and 69 showed better
activity than that of analogues 73, 74 bearing larger aromatic
groups. The activities of thiophene analog 69 and benzene analog 60
were comparable.
[0555] Phenylethylene analogues 62, 77, 87 and 88 demonstrated
comparable or even more potent IL-2 production compared with the
benzyl analogues. The 4-CF3 analog 87 and 4-isobutyl analog 82
possessed slightly better activities compared with the 4-OCH3
analog 62 and 4-F analog 88. Substitution of the phenyl group with
the 3-pyridyl group 86 diminished IL-2 production dramatically,
which contradicted the trends of benzoyl analogues (58 and 71). In
addition, the introduction of a trans-double bond as a spacer group
significantly reduced IL-2 production compared with the saturated
analog (75 and 77). The biphenyl analog 93 also showed a decreased
activity. Introduction of a basic functional group, such as
piperidinylethyl analog 91 demonstrated a significant reduction in
cytokine production. This result may be because of repulsion
between the basic amine moiety and the hydrophobic residue in the
binding pocket. The 4-Fluorophenoxymethyl analog 65 gave a similar
activity as the corresponding carbon analog 88. On the other hand,
the 2,6-dimethyl substituted analog 66 exhibited a reduced
activity, suggesting that the binding pocket was not large enough
to accept bulky substituents. Similar results were observed in
compounds 72, 89 and 90, bearing bulky substituent such as
4-biphenylmethyl, 2,2-diphenylmethyl and 9-fluorenyl,
respectively.
[0556] Further extension of spacer chain length gave best results
under these conditions. The activity of 3-phenylpropyl analog 82
was moderate. However, the 5-phenylpentyl 83, 7-phenylheptyl 84 and
10-phenyloctyl 85 all showed a significant increase of IL-2
production. Compounds 83-85 were much more potent than
.alpha.-GalCer.
Example 9
Recognition of Glycolipids by Human NKT Cell Lines Results in NKT
Cell IFN-.gamma. and IL-4 Secretion
Materials and Methods
Generation of V.alpha.24i Human NKT Cell Line
[0557] Human NKT cell lines, expressing the V.alpha.24i T cell
receptor as well as CD161, were generated as follows: Anti-CD161
monoclonal antibodies, and anti-CD14 monoclonal antibodies, each
coupled to magnetic beads (Miltenyi biotec, Auburn, Calif.), were
used sequentially to isolate CD161.sup.+ cells and CD14+ cells from
leukopaks. Immature dendritic cells were generated from the
CD14.sup.+ cells after a two-day incubation in the presence of 300
U/ml GM-CSF (R&D systems, Minneapolis, Minn.) and 100 U/ml IL-4
(R&D systems, Minneapolis, Minn.). Following irradiation with
2000 rads, the immature dendritic cells were co-cultured with
syngeneic CD161.sup.+ cells in the presence of 100-0.1 ng/ml of
alpha-galactosylceramide and 10 IU/ml of IL-2 (Invitrogen, Carlsbad
Calif.) for 10 to 14 days. After stimulating the CD161.sup.+ cells
a second time with alpha-galactosylceramide-pulsed, irradiated
immature dendritic cells, NKT cell lines were shown by flow
cytometry to express both CD161.sup.+ and V 24i TCR (99%
purity).
[0558] In some cases, Hela cells were transfected with a human CD1d
construct [Xing et al. 2005. Bioorg Med Chem 13: 2907], and were
used to present the glycolipids, via pulsing with the respective
compounds at the indicated concentration, to NKT lines.
[0559] IFN-.gamma. secretion by the V.alpha. 24i human NKT cell
line was determined by ELISA (BD Pharmingen, San Diego, Calif.)
after culture for 16 hours. For these assays, 1.times.10.sup.5
V.alpha. 24i human NKT cells were co-cultured with 4.times.10.sup.5
irradiated, immature CD14.sup.+ dendritic cells, in the presence of
the glycolipid compounds at 10 .mu.g/ml in a 96-well flat-bottom
plate.
Results
[0560] IFN-.gamma. secretion from a V.alpha.24i NKT cell line were
assessed, after stimulation with irradiated, syngeneic CD14.sup.+
immature dendritic cells in the presence of 10, 1 and 0.1 .mu.g/ml
of the respective glycolipids and 10 IU/ml of IL-2 (FIGS. 14, 15
and 16).
[0561] Stimulation of the NKT cell line by many of the glycolipid
compounds resulted in significant IFN-.gamma. secretion, when
compared to the negative control, with some specific compounds
providing greater IFN-.gamma. secretion as compared to
.alpha.-GalCer.
[0562] As illustrated in FIGS. 17 and 18, additional glycolipid
compounds were prepared and evaluated for
interferon-.gamma.secretion by human NKT cells in response to
glycolipid presentation by CD14.sup.+ DCs, as compared to
.alpha.-GalCer, at a concentration of 100-0.1 ng/mL. Compounds 83,
84 and 85 in these figures consistently stimulating greater
IFN-.gamma. secretion, at all doses evaluated, as compared to KRN,
and other compounds.
[0563] Hela cells expressing human CD1d were also effective in
presenting the glycolipids to human NKT cells, with similar
profiles in terms of stimulating NKT cell IFN-.gamma.secretion
(FIG. 19).
[0564] Compounds effective in stimulating IFN-g secretion were also
found to stimulate IL-4 secretion (FIG. 20).
[0565] The longer alkyl chain analogues 83-85 were more potent
toward both IFN-.gamma. and IL-4 production than the shorter alkyl
chain analogues. The 7-phenylheptyl analog 83 exhibited a high
ratio of IFN-.gamma./IL-4 activity and was the best among these
compounds. However, compounds 73 and 77 are more selective for IL-4
production while 82 is specific for IFN-.gamma.production.
Example 10
Possible Structural Basis for Glycolipid Recognition
[0566] Recent glycolipid-CD1d protein crystal structures revealed
the existence of various aromatic residues, Tyr73, Phe114, Phe70
and Tip114, which might be able to interact with the phenyl group
of the present compounds comprising fatty acyl chain analogues.
According to these crystal structures, the benzoyl analogues 8-14,
which have no spacer chain, seemed to be too short to interact with
these aromatic residues. To further investigate the interactions
between the phenyl analogues and human CD1d, Autodock 3.0 [G. M.
Morris, et al., J. Comp. Chem., 1998, 19, 1639-1662] was utilized
to model the binding of these compounds in the hCD1d hydrophobic
groove (FIG. 21). Compounds 40-42 were individually docked and
their results did not vary significantly from the crystal structure
of .alpha.-GalCer bound to hCD1d [M. Koch, et al., Nat. Immunol.,
2005, 6,819-826]. In each case, the phytosphingosine tail extended
into the F' pocket and the A' pocket was occupied by the fatty acyl
chain with the galactose headgroup presented in nearly all the same
configuration. However, introduction of a terminal phenyl group in
the .alpha.-GalCer analogues seemed to promote additional specific
interactions between compounds 40, 41 and the phenol ring of Tyr73
and between 42 and Trp40.
[0567] Biphenyl analogues 16-18 and cinnamoyl analogues 30, 32,
lacked a flexible fatty acyl chain and may not have been able to
extend into the A' pocket deep enough to make any specific
interactions. Extension of the spacer chain length, such as the
benzyl analogues 19-22, the phenylethylene analogues 24-28, and the
4-fluorophenoxymethyl analog 34, allowed for tighter binding via
.pi.-.pi. interaction, possibly with the aromatic side-chain of
Tyr73. Further extension of the spacer chain length, such as
pentamethylene analog 83, heptamethylene analog 84 and
decamethylene analog 85, were more suitable for tighter binding.
These results suggest that the introduction of .pi.-.pi.
interaction potentiates IL-2 production, probably through the
formation of a tighter ligand-CD1d protein complex.
[0568] These fatty acyl chain analogues bearing aromatic groups
seemed to possess more potent activity than the corresponding
simple fatty acyl chain analogues. Compounds 83-85 bearing 5, 7, 10
carbons spacer chain, respectively, demonstrated much more potent
IL-2 production than that of other groups, with .alpha.-GalCer
bearing a C26 fatty acyl chain. These results suggest that
introduction of a terminal aromatic group on the fatty acyl chain
causes an enhancement of the activity through interactions between
the aromatic residues in the hydrophobic pocket of CD1d protein and
the lipid tail.
Example 11
In Vivo Mouse Assay
[0569] Wild-type C57BL/6 (B6) mice were administered intravenously
with 1 .mu.g of each glycolipid or with nothing, and serum samples
were obtained at 2, 6, 12, 24, 48, and 72 hours after injection for
ELISA analyses of IL-4, IFN-.gamma., and IL-12 concentrations. The
serum concentrations of IFN-.gamma. and IL-4 were measured by way
of a sandwich ELISA (e-bioscience, San Diego, Calif.). The serum
concentrations of IL-12p70 were also measured by way of a sandwich
ELISA (PharMingen, San Diego, Calif.). Results are provided in FIG.
22.
[0570] It will be appreciated by a person skilled in the art that
the present invention is not limited by what has been particularly
shown and described hereinabove. Rather, the scope of the invention
is defined by the claims that follow:
* * * * *