U.S. patent application number 11/244327 was filed with the patent office on 2006-04-06 for method for activating human antigen presenting cells, activated human antigen - presenting cells, and use thereof.
This patent application is currently assigned to Kirin Brewery Company, Limited. Invention is credited to Yasuhiko Koezuka, Kazuhiro Motoki, Yasunori Yamaguchi.
Application Number | 20060073122 11/244327 |
Document ID | / |
Family ID | 18410441 |
Filed Date | 2006-04-06 |
United States Patent
Application |
20060073122 |
Kind Code |
A1 |
Koezuka; Yasuhiko ; et
al. |
April 6, 2006 |
Method for activating human antigen presenting cells, activated
human antigen - presenting cells, and use thereof
Abstract
The invention relates to a method for activating human-derived
antigen-presenting cells by in vitro cultivation with at least one
of the glycoside compounds represented by formula (A) or salts
thereof [preferred example:
(2S,3S,4R)-1-(.alpha.-D-galactopyranosyloxy)-2-hexacosanoylamino-3,4-octa-
decanediol], and to human antigen-presenting cells activated by the
method. The invention also relates to a method for treatment of
cancer and infectious diseases including AIDS with the activated
human antigen-presenting cells, and to a use of the activated human
antigen-presenting cells in the preparation of medicines for
treating such diseases. The invention can provide a satisfactory
therapeutic effect on cancer and infectious diseases including AIDS
without the need to pulse the human antigen-presenting cells with
tumor antigens. ##STR1##
Inventors: |
Koezuka; Yasuhiko; (Gunma,
JP) ; Yamaguchi; Yasunori; (Gunma, JP) ;
Motoki; Kazuhiro; (Gunma, JP) |
Correspondence
Address: |
FOLEY AND LARDNER LLP;SUITE 500
3000 K STREET NW
WASHINGTON
DC
20007
US
|
Assignee: |
Kirin Brewery Company,
Limited
|
Family ID: |
18410441 |
Appl. No.: |
11/244327 |
Filed: |
October 6, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09721768 |
Nov 27, 2000 |
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11244327 |
Oct 6, 2005 |
|
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09331789 |
Jun 25, 1999 |
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PCT/JP97/04832 |
Dec 25, 1997 |
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09721768 |
Nov 27, 2000 |
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Current U.S.
Class: |
424/93.7 ;
514/23 |
Current CPC
Class: |
C12N 5/0639 20130101;
C12N 2501/23 20130101; A61K 2039/5154 20130101; A61P 31/12
20180101; A61P 35/00 20180101; C12N 2501/22 20130101; C12N 2501/90
20130101; C07H 15/04 20130101 |
Class at
Publication: |
424/093.7 ;
514/023 |
International
Class: |
A61K 35/14 20060101
A61K035/14; A61K 31/70 20060101 A61K031/70 |
Foreign Application Data
Date |
Code |
Application Number |
Dec 27, 1996 |
JP |
350429/1996 |
Claims
1-23. (canceled)
24. A method for activating a human antigen-presenting cell,
comprising culturing human dendritic cells in vitro with at least
one of the glycoside compounds represented by formula (A) or salts
thereof: ##STR12## wherein R.sub.1 is H or OH; X is an integer of
from 7 to 25; R.sub.2 is a substituent defined by any one of the
following (a) to (e): (a) --CH.sub.2(CH.sub.2).sub.YCH.sub.3; (b)
--CH(OH)(CH.sub.2).sub.YCH.sub.3; (c)
--CH(OH)(CH2).sub.vCH(CH.sub.3).sub.2; (d)
--CH.dbd.CH(CH2).sub.YCH.sub.3; and (e)
--CH(OH)(CH.sub.2).sub.YCH(CH.sub.3)CH.sub.2CH.sub.3 wherein Y is
an integer of from 5 to 17; R.sub.3 is H; R.sub.4 is OH; R.sub.5 is
OH; R.sub.6 is H; one of R.sub.7 and R.sub.8 is H and the other is
OH; and R.sub.9 is H, CH.sub.3 or CH.sub.2OH.
25. The method of claim 24, wherein the human dendritic cells are
obtained by culturing human monocytes in vitro in the presence of
GM-CSF and IL-4,
26. The method of claim 25, wherein the human monocyte is prepared
from human peripheral blood.
27. The method of claim 25, wherein the human monocyte is prepared
from human umbilical cord blood.
28. The method of claim 25, wherein the human monocyte is prepared
from a human bone marrow cell.
29. The method of claim 25, wherein the human monocyte is prepared
from human epidermis.
30. The method of claim 24 or 25, wherein the glycoside compound is
a compound represented by formula (B): ##STR13## wherein: R.sub.1
is H or OH; X is an integer of from 7 to 25; R.sub.2 is a
substituent defined by any one of the following (a) to (e): (a)
--CH.sub.2(CH.sub.2).sub.YCH.sub.3; (b)
--CH(OH)(CH.sub.2).sub.YCH.sub.3; (c)
--CH(OH)(CH.sub.2).sub.vCH(CH.sub.3).sub.2; (d)
--CH.dbd.CH(CH.sub.2).sub.YCH.sub.3; and (e)
--CH(OH)(CH.sub.2).sub.YCH(CH.sub.3)CH.sub.2CH.sub.3 wherein Y is
an integer of from 5 to 17; R.sub.3 to R.sub.9 are substituents
defined by any one of the following (i) to (ii): (i) R.sub.3,
R.sub.6 and R.sub.8 are each H; R.sub.4 is OH; R.sub.5 is OH;
R.sub.7 is OH; and R.sub.9 is H, CH.sub.3, or CH.sub.2OH; (ii)
R.sub.3, R.sub.6 and R.sub.7 are each H; R.sub.4, R.sub.5 and
R.sub.9 are as defined as in (i); and R.sub.6 is OH.
31. The method of claim 24 or 25, wherein the glycoside compound is
a compound represented by formula (B): ##STR14## wherein: R.sub.1
is H or OH; X is an integer of from 7 to 25; R.sub.2 is a
substituent defined by any one of the following (a) to (e): (a)
--CH.sub.2(CH.sub.2).sub.YCH.sub.3; (b)
--CH(OH)(CH.sub.2).sub.YCH.sub.3; (c)
--CH(OH)(CH.sub.2).sub.YCH(CH.sub.3).sub.2; (d)
--CH.dbd.CH(CH.sub.2).sub.YCH.sub.3; and (e)
--CH(OH)(CH.sub.2).sub.YCH(CH.sub.3)CH.sub.2CH.sub.3 wherein Y is
an integer of from 5 to 17; R.sub.3, R.sub.6, and R.sub.8 are each
H; R.sub.4, R.sub.5, and R.sub.7 are each OH; and R.sub.9 is
CH.sub.2OH.
32. The method of claim 24 or 25, wherein the glycoside compound is
a compound represented by formula (B): ##STR15## wherein R.sub.1 is
H or OH; X is an integer of from 7 to 25; R.sub.2 is a substituent
defined by any one of the following: (b)
--CH(OH)(CH.sub.2).sub.YCH.sub.3; (c)
--CH(OH)(CH.sub.2).sub.YCH(CH.sub.3).sub.2; and (d)
--CH(OH)(CH.sub.2).sub.YCH(CH.sub.3)CH.sub.2CH.sub.3; wherein Y is
an integer of from 5 to 17; and R.sub.3, R.sub.6 and R.sub.8 are
each H; R.sub.4, R.sub.5 and R.sub.7 are each OH; and R.sub.9 is
CH.sub.2OH.
33. The method of claim 24 or 25, wherein the glycoside compound is
a compound represented by formula (B): ##STR16## wherein X is an
integer of from 7 to 25; R.sub.1 is H; R.sub.2 is
--CH(OH)(CH.sub.2).sub.YCH.sub.3 where Y is an integer of from 5 to
17; H.sub.3, R.sub.6 and R.sub.8 are each H; R.sub.4, R.sub.5 and
R.sub.7 are each OH; and R.sub.9 is CH.sub.2OH.
34. The method of claim 24 or 25, wherein the glycoside compound is
a compound represented by formula (B): ##STR17## wherein X is an
integer of from 7 to 25; R.sub.1 is H; R.sub.2 is
--CH(OH)(CH.sub.2).sub.YCH.sub.3 where Y is an integer of from 5 to
17 and where the OH group is of R configuration; and R.sub.3,
R.sub.6 and R.sub.8 are each H; R.sub.4, R.sub.5 and R.sub.7 are
each OH; and R.sub.9 is CH.sub.2OH.
35. The method of claim 24 or 25, wherein the glycoside compound is
a compound represented by formula (B): ##STR18## wherein X is from
21 to 25 and Y is an integer of from 11 to 15; R.sub.1 is H;
R.sub.2 is --CH(OH)(CH.sub.2).sub.YCH.sub.3 where Y is an integer
of from 5 to 17 and where the OH group is of R configuration; and
R.sub.3, R.sub.6 and R.sub.8 are each H; R.sub.4, R.sub.5 and
R.sub.7 are each OH; and R.sub.9 is CH2OH.
36. The method of claim 24 or 25, wherein the glycoside compound is
selected from the group consisting of: (2S, 3S,
4R)-1-(.alpha.-D-galactopyranosyloxy)-2-hexacosanoylamino-3,4-octadecaned-
iol; (2S,
3R)-1-(.alpha.-D-galactopyranosyloxy)-2-[(R)-2-hydroxytetracosa-
noylamino]-3-octadecanol; (2S,
3R)-1-(.alpha.-D-galactopyranosyloxy)-2-tetracosanoylamino-3-octadecanol;
(2S,
3R)-1-(6'-deoxy-.alpha.-D-galactopyranosyloxy)-2-tetracosanoylamino-
-3-octadecanediol; (2S, 3S,
4R)-1-(.alpha.-D-galactopyranosyloxy)-2-[(R)-2-hydroxytetracosanoylamino]-
-3,4-octadecanediol; and (2S, 3S,
4R)-1-(.alpha.-D-galactopyranosyloxy)-2-tetracosanoylamino-3,4-octadecane-
diol.
Description
BACKGROUND OF THE INVENTION
[0001] 1. Field of the Invention
[0002] The present invention relates to a novel activated human
antigen-presenting cell, a method for activating a human
antigen-presenting cell in vitro, a method for treating cancer and
infectious diseases including AIDS using the activated human
antigen-presenting cell, and a use of the activated human
antigen-presenting cell in preparing medicines for such
treatment.
[0003] 2. Disclosure of Related Art
[0004] There have been various strategies for the therapeutical
treatment of cancer patients, including surgical extraction of
tumor foci, chemotherapy, radiotherapy and immunotherapy. However,
the cure rates of these strategies are not as high as they are
expected. Therefore, there exists a strong demand for developing
new therapeutic agents and methods which can provide improved cure
rates in the treatment of cancer. Macrophages, B cells and
dendritic cells, which are members of antigen-presenting cell
(APC), have been known to be cells essential for immune response.
Recently, an idea that using APCs, particularly dendritic cells, to
induce cancer immunity may be effective in treating cancer (Grabbe,
S. et al., 1995, Immunol. Today, 16, 117) has attracted much
attention.
[0005] Several processes for preparing dendritic cells from mouse
have been known, such as from the spleen (Clowley, M. et al., 1989,
Cell. Immunol., 118, 108), from the bone marrow (Inaba, K. et al.,
1992, J. Exp. Med., 176, 1693) and from the epidermis (the
epidermis-derived dendritic cells are known as "Langerhans's cells)
(Witmer-Pack, M. et al., 1987, J. Exp. Med., 166, 1487).
Furthermore, it was demonstrated that pulsing the dendritic cells
prepared from the murine bone marrow with tumor antigen and
administrating the pulsed dendritic cells into a subject prior to
and after the implantation of tumor cells into the subject can
elicit tumor immunity (Celluzzi, C. M. et al., 1996, J. Exp. Med.,
183, 283; Paglia, P. et al., 1996, J. Exp. Med., 183, 317).
[0006] The following processes are known for preparing human APCs,
particularly dendritic cells, from peripheral blood or umbilical
cord blood: a process in which FcR.sup.+ cells, T cells, B cells
and NK cells are removed from human peripheral blood using the
antibodies against them from peripheral blood monocytes to give
APCs (Hsu et al., 1996, Nature Med., 2, 52); and a process in which
adherent cells in human peripheral blood monocytes from which
CD19.sup.+ B cells and CD2.sup.+ T cells were removed are cultured
with a granulocyte-macrophage colony stimulating factor (GM-CSF)
and interleukin 4 (IL-4) for about one week (Sallusto, F. et al.,
1994, J. Exp. Med., 179, 1109). It has been reported that such
human peripheral blood-derived APCs have both allogeneic and
autologous MLR (mixed leukocyte reaction) enhancing effects. On the
other hand, for preparing APCs from human umbilical cord blood or
bone marrow cells, a process is known where GM-CSF and tumor
necrosis factor-.alpha. (TNF-.alpha.) are used to prepare APCs from
CD34.sup.+ cells found in umbilical cord blood or bone marrow
(Santiago-Schwartz, F. et al., 1992, J. Leukocyte Biol., 52, 274;
Caux, C. et al., 1992, Nature, 360, 258). However, although the
APCs prepared from human umbilical cord blood or bone marrow cells
by such a process as mentioned above have an allogeneic MLR
enhancing effect, they have no (Santiago-Schwartz, F. et. al.) or,
if any, extremely poor (Caux et al.) antologous MLR enhancing
effect.
[0007] Recently, it has been demonstrated that a therapy with the
APCs that had been pulsed with tumor antigens is effective for
treating B cell lymphoma patients (Hsu, F. J. et al., 1996, Nature
Med., 2, 52). That is, a therapy for B cell lymphoma patients was
successfully achieved by culturing APCs prepared from peripheral
blood of a B cell lymphoma patient together with B cell lymphoma
antigens in vitro and administrating the cultured APCs into the B
cell lymphoma patient.
[0008] As shown in this exemplary therapy, methods in which APCs
are pulsed with tumor antigens may be very effective for treating
cancer if the cancer can be clearly identified by their tumor
antigens, like B cell lymphoma. However, such methods as mentioned
above take enormous time and costs, because the tumor antigens are
generally specific to the individual patients and, therefore, must
be specified for the respective patients, and the specified tumor
antigens must be produced in large quantities to pulse the APCs.
Furthermore, since it is impossible for most of the cancer patients
to identify their tumor antigens, the range of application of the
method in which APCs are pulsed with tumor antigens is limited. In
view of the above, in order to apply the therapy with APCs
(hereinafter referred to as "the APC therapy") recognized to be a
very effective therapy for cancer, to as many patients as possible,
there has been a strong demand for development of a method for
preparing human APCs effective for treating cancer without the aid
of tumor antigens.
[0009] Within a living body, various types of
.beta.-galactosylceramides and .beta.-glucosylceramides are
present, each of which has a sugar linked to a ceramide via a
.beta.-linkage (Svennerholm, L. et al., 1972, Biochem. Biophys.
Acta., 280, 626; Karlsson, K. A. et al., 1973, Biochim. Biophys.
Acta., 316, 317). On the other hand, the inventors have found that
.alpha.-galactosylceramides have remarkable immunostimulatory
properties and anti-tumor properties (Morita, M. et al., 1995, J.
Med. Chem., 38, 2176), and that such properties of
.alpha.-galactosylceramides and .alpha.-glucosylceramides are far
more potent than those of the corresponding .beta.-anomers (Motoki,
K. et al., 1995, Biol. Pharm. Bull., 18, 1487). It was also found
that compounds having an .alpha.-glycosylceramide structure show a
protecting effect against radiation when administered into a living
subject (Motoki, K. et al., 1995, Bioorg. Med. Chem. Lett., 5,
2413) and cause an increase in the number of platelets and white
blood cells (Motoki, K. et al., 1996, Biol. Pharm. Bull., 19, 952).
The inventors also have found that an .alpha.-galactosylceramide,
KRN7000, can enhance the functions of dendritic cell-rich APCs
prepared from the murine spleen, and that the enhanced APCs exhibit
an anti-tumor effect when administered into a subject before tumors
are implanted into the subject (Koezuka, Y. et al., 1995, The
9.sup.th International Congress of Immunology, Abstract, 55;
Yamaguchi, Y. et al., 1996, Oncol. Res. 8, 399).
[0010] However, the effect of glycoside compounds, such as KRN7000,
or salts thereof on APCs rich in dendritic cells from murine bone
marrows and Langerhans's cells from murine epidermis has not been
clarified. In addition, there is no report on anti-tumor effects of
the APCs rich in murine spleen- and bone marrow-derived dendritic
cells, which have been stimulated with KRN7000, upon administration
into a subject after tumors are implanted into the subject. No
effect of glycoside compounds such as KRN7000 or salts thereof on
human APCs has also been known.
[0011] To address the above-mentioned demands, the present
invention provides a novel human APC activating agent which is
useful in preparing activated human APCs that exhibit a sufficient
therapeutic effect on cancer and various infectious diseases
including AIDS without the need to pulse the antigen-presenting
cells with tumor antigens, and also provides a method for
activating human APCs with such an activating agent.
SUMMARY OF THE INVENTION
[0012] The present invention provides a method for activating human
APCs, which comprises culturing human-derived APCs in vitro with at
least one of glycoside compounds represented by formula (A) or
salts thereof: ##STR2## wherein R.sub.1 to R.sub.9 and X are to be
defined later.
[0013] According to one embodiment, the present invention provides
a method for activating human APCs which comprises culturing
human-derived APCs in vitro with a glycoside compound
(2S,3S,4R)-1-(.alpha.-D-galactopyranosyloxy)-2-hexacosanoylamino-3,4-octa-
decanediol or a salt thereof.
[0014] The present invention also provides activated human APCs
which can be prepared by culturing human-derived APCs in vitro with
at least one of the glycoside compounds represented by formula (A)
above or salts thereof.
[0015] The present invention further provides a method for treating
cancer and infectious diseases including AIDS, which comprises
applying an APC therapy with such activated human APCs as defined
above.
[0016] The present invention further provides a use of such
activated human APCs as defined above in the preparation of a
medicine for treating cancer and infectious diseases including AIDS
by application of the APC therapy.
BRIEF DESCRIPTION OF THE DRAWINGS
[0017] FIG. 1 shows the amount of .sup.3H-TdR taken up into the
cells variously treated as described. In FIG. 1, "V-APC" and
"KRN-APC" refer to human peripheral blood-derived APCs pretreated
with a vehicle and KRN7000, respectively.
[0018] FIG. 2 shows a relationship between the number of APCs and
the amount of .sup.3H-TdR taken up into an allogeneic T cells. In
FIG. 2, "V-APC" and "KRN-APC" refer to human umbilical cord
blood-derived APCs (CD1c.sup.+ cells) stimulated with a vehicle and
KRN7000, respectively.
[0019] FIG. 3 shows a relationship between the number of APCs and
the amount of .sup.3H-TdR taken up into autologous T cells. In FIG.
3, "V-APC" and "KRN-APC" refer to the same APCs as those used in
FIG. 2.
[0020] FIG. 4 shows a relationship between the number of APCs and
the amount of .sup.3H-TdR taken up into spleen cells. In FIG. 2,
"V-APC", "KRN-APC", "583-APC", "517-APC", "564-APC", "563-APC" and
"562-APC" refer to murine spleen-derived APCs pretreated with a
vehicle, KRN7000, AGL-583, AGL-517, AGL-564, AGL-563 and AGL-562,
respectively.
[0021] FIG. 5 shows the survival periods of EL-4-bearing mice when
murine spleen-derived APCs pretreated with a vehicle or KRN7000
were injected intravenously to the mice.
[0022] FIG. 6 shows a relationship between the number of APCs and
the amount of .sup.3H-TdR taken up into T cells. In FIG. 6, "V-APC"
and "KRN-APC" refer to murine bone marrow-derived APCs pretreated
with a vehicle and KRN7000, respectively.
[0023] FIG. 7 shows the weight of the liver of a tumor-bearing
mouse when the V-APC or KRN-APC prepared in the same manner as in
FIG. 6 was injected intravenously into the mouse.
[0024] FIG. 8 shows a relationship between the number of APCs and
the amount of 3H-TdR taken up into spleen cells. In FIG. 8, "V-APC"
and "KRN-APC" refer to murine epidermis-derived APCs pretreated
with a vehicle and KRN7000, respectively.
[0025] FIG. 9 shows the volume of a subcutaneous tumor for each of
the tumor-bearing mice when the APCs pretreated with a vehicle, a
vehicle+B16-tumor cell lysate, KRN7000 or KRN7000+B16-tumor cell
lysate (V-APC, V-T-APC, KRN-APC or KRN-T-APC, respectively),
respectively, was injected intravenously to the mice.
[0026] FIG. 10 shows a relationship between the number of APCs
given after culturing for 3 days and the amount of .sup.3H-TdR
taken up into human peripheral blood-derived FcR.sup.- cells. In
FIG. 10, "V-APC", "KRN-APC", "V-APC-G-M" and "KRN-APC-GM" refer to
human peripheral blood-derived APCs pretreated with a vehicle,
KRN7000, a vehicle+GM-CSF+IL4 and KRN+GM-CSF+IL4, respectively.
[0027] FIG. 11 shows a relationship between the number of APCs
given after culturing for 7 days and the amount of .sup.3H-TdR
taken up into human peripheral blood-derived FcR.sup.- cells. In
FIG. 11, "V-APC-GM" and "KRN-APC-GM" refer to the same APCs as
those used in FIG. 10.
[0028] FIG. 12 shows a relationship between the number of APCs
given after culturing for 11 days and the amount of .sup.3H-TdR
taken up into human peripheral blood-derived FcR cells. In FIG. 12,
"V-APC-GM" and "KRN-APC-GM" refer to the same APCs as those used in
FIG. 10, and "MCM.sup.+" and "MCM.sup.-" respectively mean that the
APCs are cultured with and without a monocyte conditioned medium
(MCM).
[0029] FIG. 13 shows a relationship between the number of APCs
given after culturing for 3 days and the amount of .sup.3H-TdR
taken up into human umbilical cord blood-derived FcR.sup.- cells.
In FIG. 13, "V-APC-GM" and "KRN-APC-GM" refer to the human
umbilical cord blood-derived APCs respectively pretreated with a
vehicle+GM-CSF+IL4 and KRN7000+GM-CSF+IL4.
[0030] FIG. 14 shows activation effect of murine spleen-derived
APCs when they are stimulated by Compounds Nos. 1, 2, 5, 10 and 16
(except KRN7000), termed AGL506, AGL514, AGL571, AGL512 and AGL525,
respectively.
DETAILED DESCRIPTION OF THE INVENTION
[0031] As a result of extensive and intensive studies by the
inventors on the above-mentioned problems, it was found that the
glycoside compounds according to the present invention
(representatively, KRN7000) can significantly enhance the functions
of human APCs prepared from human peripheral blood and human
umbilical cord blood. The glycoside compounds can also
significantly enhance the functions of dendritic cell-rich APCs
from a murine bone marrow and Langerhans's cell-rich APCs from
murine auricle, as well as dendritic cell-rich APCs from a murine
spleen. It was also found that a significant anti-tumor effect can
be observed when dendritic cell-rich APCs from a murine spleen or a
murine bone marrow which have been cultured in vitro with the
glycoside compounds of the present invention (representatively,
KRN7000) are injected into a mouse into which tumor cells have been
implanted. Based on these findings, the invention was
completed.
[0032] That is, the present invention relates to an activating
agent (i.e., an antigen-presenting function enhancing agent)
comprising a specific glycoside compound, which is useful for
preparing activated human APCs having a therapeutic effect on
cancer and infectious diseases including AIDS. Specifically, the
present invention relates to an activating agent for APCs prepared
from human peripheral blood, human umbilical cord blood, human bone
marrow cells or human epidermis (i.e., APCs to be activated). More
specifically, the present invention relates to an activating agent
for human monocytes, human dendritic cells or human CD1c.sup.+
cells.
[0033] According to the present invention, there is provided a
method for activating human antigen-presenting cells which
comprises culturing human-derived antigen-presenting cells in vitro
with at least one of the glycoside compounds represented by formula
(A) or salts thereof: ##STR3## wherein:
[0034] R.sub.1 is H or OH;
[0035] X is an integer of from 7 to 25;
[0036] R.sub.2 is a substituent defined by any one of the following
(a) to (e): [0037] (a) --CH.sub.2(CH.sub.2).sub.YCH.sub.3; [0038]
(b) --CH(OH)(CH.sub.2).sub.YCH.sub.3; [0039] (c)
--CH(OH)(CH.sub.2).sub.YCH(CH.sub.3).sub.2; [0040] (d)
--CH.dbd.CH(CH.sub.2).sub.YCH.sub.3; and [0041] (e)
--CH(OH)(CH.sub.2).sub.YCH(CH.sub.3)CH.sub.2CH.sub.3; [0042]
wherein Y is an integer of from 5 to 17;
[0043] R.sub.3 is H; ##STR4##
[0044] one of R.sub.5 and R.sub.6 is H and the other is OH,
##STR5##
[0045] one of R.sub.7 and R.sub.8 is H and the other is OH,
##STR6##
[0046] In a preferred embodiment, the present invention provide a
method for activating human antigen-presenting cells which
comprises using at least one of the glycoside compounds represented
by formula (B) or salts thereof: ##STR7## wherein:
[0047] R.sub.1, X and R.sub.2 are as defined as in the case of
formula (A); and
[0048] R.sub.3 to R.sub.9 are substituents defined by any one of
the following (i) to (iii): [0049] (i) [galactose type] [0050] each
of R.sub.3, R.sub.6 and R.sub.8 is H; ##STR8## [0051] (ii) [glucose
type] OH [0052] each of R.sub.3, R.sub.6 and R.sub.7 is H; [0053]
R.sub.4, R.sub.5 and R.sub.9 are as defined as in (i) and ##STR9##
[0054] (iii) [allose type] [0055] each of R.sub.3, R.sub.5 and
R.sub.7 is H; [0056] each of R.sub.4, R.sub.6 and R.sub.8 is OH;
and [0057] R.sub.9 is H, CH.sub.3 or CH.sub.2OH.
[0058] The glycoside compounds defined by formula (A) or (B) above
are comprised of a sugar moiety and an aglycone moiety, and some of
them are also referred to as .alpha.-cerebrosides,
.alpha.-glycosylceramides, .alpha.-glucosylceramides,
.alpha.-galactocerebrosides or a galactosylceramides. These
compounds are characterized by having the .alpha.-form of anomeric
configuration.
[0059] In the glycoside compound, the sugar moiety is preferably of
[galactose type] as defined in (i), and more preferably of one
wherein each of R.sub.3, R.sub.6 and R.sub.8 is H, each of R.sub.4,
R.sub.5 and R.sub.7 is OH and R.sub.9 is CH.sub.2OH (i.e.,
.alpha.-galactopyranosyl).
[0060] In the glycoside compound, the aglycone moiety preferably
has R.sub.2 being any one of the substituents (b) (c) and (e)
above, and more preferably has R.sub.1 being H (i.e., kerasin type)
and R.sub.2 being the substituent (b). X is preferably an integer
of 21 to 25 and Y is preferably an integer of 11 to 15.
[0061] Preferable examples of the glycoside compound of the present
invention are listed below. In the list, compounds (1)-(9),
(10)-(24), (25)-(31), (32)-(33), and (34) are those compounds in
which R.sub.2 is the substituent (a), (b), (c), (d) or (e) above,
respectively. The alphabet letters A, B, C and D behind the
compounds, name indicate the reference specifications of
WO93/05055, WO94/02168, WO94/09020 and WO94/24142, respectively,
which describe the synthesis methods of the annoted compounds.
Among the glycoside compounds below, compound (14)
(2S,3S,4R)-1-((.alpha.-D-galactopyranosyloxy)-2-hexacosanoylamino-3,4-oct-
adecanediol (referred to as "KRN7000 hereinbelow), is most
preferable. With respect to this compound, an example of the
synthesis process will be illustrated in the Production Example and
Scheme 1 below. TABLE-US-00001 (1)
(2S,3R)-1-(.alpha.-D-galactopyranosyloxy)-2-[(R)-2- A
hydroxytetracosanoylamino]-3-octadecanol (2)
(2S,3R)-1-(.alpha.-D-galactopyranosyloxy)-2- A
tetracosanoylamino]-3-octadecanol (3)
(2S,3R)-1-(.alpha.-D-galactopyranosyloxy)-2- A
tetradecanoylamino-3-octadecanol (4)
(2S,3R)-1-(.alpha.-D-glucopyranosyloxy)-2- C
tetradecanoylamino-3-octadecanol (5)
(2S,3R)-1-(6'-deoxy-.alpha.-D-galactopyranosyloxy)-2- C
tetradecanoylamino-3-octadecanol (6)
(2S,3R)-1-(.beta.-L-arabinopyranosyloxy)-2- C
tetradecanoylamino-3-octadecanol (7)
(2S,3R)-1-(.alpha.-D-galactopyranosyloxy)-2- A
tetradecanoylamino-3-hexadecanol (8)
(2R,3R)-1-(.alpha.-D-galactopyranosyloxy)-2- A
tetradecanoylamino-3-hexadecanol (9)
(2R,3S)-1-(.alpha.-D-galactopyranosyloxy)-2- A
tetradecanoylamino-3-hexadecanol (10)
(2S,3S,4R)-1-(.alpha.-D-galactopyranosyloxy)-2-[(R)-2- A
hydroxytetracosanoylamino]-3,4-octadecanediol (11)
(2S,3S,4R)-1-(.alpha.-D-galactopyranosyloxy)-2-[(R)-2- A
hydroxytetracosanoylamino]-3,4-undecanediol (12)
(2S,3S,4R)-1-(.alpha.-D-galactopyranosyloxy)-2-[(R)-2- A
hydroxyhexacosanoylamino]-3,4-icosanediol (13)
(2S,3S,4R)-1-(.alpha.-D-galactopyranosyloxy)-2-[(S)-2- A
hydroxytetracosanoylamino]-3,4-heptadecanediol (14)
(2S,3S,4R)-1-(.alpha.-D-galactopyranosyloxy)-2- Production
hexacosanoylamino-3,4-octadecanediol Example (15)
(2S,3S,4R)-1-(.alpha.-D-galactopyranosyloxy)-2- B
octacosanoylamino-3,4-heptadecanediol (16)
(2S,3S,4R)-1-(.alpha.-D-galactopyranosyloxy)-2- A
tetracosanoylamino-3,4-octadecanediol (17)
(2S,3S,4R)-1-(.alpha.-D-galactopyranosyloxy)-2- A
tetracosanoylamino-3,4-undecanediol (18)
(2S,3S,4R)-1-(.alpha.-D-galactopyranosyloxy)-2- C
hexacosanoylamino-3,4-octadecanediol (19)
O-.beta.-D-galactofuranosyl-(1.fwdarw.3)-O-.alpha.-D- D
galactopyranosyl-(1.fwdarw.1)-(2S,3S,4R)-2-amino-N-[(R)-2-
hydroxytetracosanoyl]-1,3,4-octadecanetriol (20)
O-.alpha.-D-galactopyranosyl-(1.fwdarw.6)-O-.alpha.-D-glucopyranosyl-
D (1.fwdarw.1)-(2S,3S,4R)-2-amino-N-hexacosanoyl-1,3,4-
octadecanetriol (21)
O-.alpha.-D-galactopyranosyl-(1.fwdarw.6)-O-.alpha.-D- D
galactopyranosyl-(1.fwdarw.1)-(2S,3S,4R)-2-amino-N-
hexacosanoyl-1,3,4-octadecanetriol (22)
O-.alpha.-D-glucopyranosyl-(1.fwdarw.4)-O-.alpha.-D-glucopyranosyl-(1
D .fwdarw.1)-(2S,3S,4R)-2-amino-N-hexacosanoyl-1,3,4-
octadecanetriol (23)
O-(N-acetyl-2-amino-2-deoxy-.alpha.-D-galactopyranosyl-(1 D
.fwdarw.3)-O-[.alpha.-D-glucopyranosyl-(1.fwdarw.2)]-O-.alpha.-D-
galactopyranosyl-(1.fwdarw.1)-(2S,3S,4R)-2-amino-N-[(R)-2-
hydroxyhexacosanoyl-1,3,4-octadecanetriol (24)
O-(N-acetyl-2-amino-2-deoxy-.alpha.-D-galactopyranosyl-(1 D
.fwdarw.3)-O-[.alpha.-D-glucopyranosyl-(1.fwdarw.2)]-O-.alpha.-D-
galactopyranosyl-(1.fwdarw.1)-(2S,3S,4R)-2-amino-N-[(R)-2-
hydroxytetracosanoyl-1,3,4-hexadecanediol (25)
(2S,3S,4R)-1-(.alpha.-D-galactopyranosyloxy)-2-[(R)-2- A
hydroxytricosanoylamino]-16-methyl-3,4-heptadecanediol (26)
(2S,3S,4R)-1-(.alpha.-D-galactopyranosyloxy)-2-[(S)-2- A
hydroxytetracosanoylamino]-16-methyl-3,4-heptadecanediol (27)
(2S,3S,4R)-1-(.alpha.-D-galactopyranosyloxy)-16-methyl-2- A
tetracosanoylamino]-3,4-heptadecanediol (28)
O-.beta.-D-galactofuranosyl-(1.fwdarw.3)-O-.alpha.-D- D
galactopyranosyl-(1.fwdarw.1)-(2S,3S,4R)-2-amino-N-[(R)-2-
hydroxytetracosanoyl]-17-methyl-1,3,4-octadecanetriol (29)
O-.beta.-D-galactofuranosyl-(1.fwdarw.3)-O-.alpha.-D- D
galactopyranosyl-(1.fwdarw.1)-(2S,3S,4R)-2-amino-N-[(R)-2-
hydroxytetracosanoyl]-15-methyl-1,3,4-hexadecanediol (30)
O-(N-acetyl-2-amino-2-deoxy-.alpha.-D-galactopyranosyl-(1 D
.fwdarw.3)-O-[.alpha.-D-glucopyranosyl-(1.fwdarw.2)]-O-.alpha.-D-
galactopyranosyl-(1.fwdarw.1)-(2S,3S,4R)-2-amino-N-[(R)-2-
hydroxyhexacosanoyl-16-methyl-1,3,4-octadecanetriol (31)
O-(N-acetyl-2-amino-2-deoxy-.alpha.-D-galactopyranosyl-(1 D
.fwdarw.3)-O-[.alpha.-D-glucopyranosyl-(1.fwdarw.2)]-O-.alpha.-D-
galactopyranosyl-(1.fwdarw.1)-(2S,3S,4R)-2-amino-N-[(R)-2-
hydroxytetracosanoyl-16-methyl-1,3,4-heptadecanetriol (32)
(2S,3S,4E)-1-(.alpha.-D-galactopyranosyloxy)-2- A
octadecanoylamino-4-octadecene-3-ol (33)
(2S,3S,4E)-1-(.alpha.-D-galactopyranosyloxy)-2- A
tetradecanoylamino-4-octadecene-3-ol (34)
(2S,3S,4R)-1-(.alpha.-D-galactopyranosyloxy)-2-[(R)-2- A
hydroxypentacosanoylamino]-16-methyl-3,4-octadecanediol
[0062] The glycoside compound defined by formula (A) or (B) may
form an acid addition salt with a pharmaceutically acceptable acid.
Examples of the acid to be used for formation of such an acid
addition salt include inorganic acids such as hydrochloric acid,
sulfuric acid, nitric acid and phosphoric acid; and organic acids
such as acetic acid, propionic acid, maleic acid, oleic acid,
palmitic acid, citric acid, succinic acid, tartaric acid, fumaric
acid, glutamic acid, pantothenic acid, lauryl sulfonic acid,
methanesulfonic acid and phthalic acid.
[0063] The human APC-activating agent according to the present
invention can be used to activate the APCs prepared from various
human tissues such as peripheral blood, umbilical cord blood, bone
marrow cells and epidermis. Typically, the activating agent of the
present invention can be added in vitro during or after the
preparation of the human APCs (for example, from human peripheral
blood, umbilical cord blood or bone marrow cells and, if necessary,
with GM-CSF and IL-4) to give the activated human APCs. The
activated human APCs may be prepared more efficiently by adding
substance(s) such as SCF, IL-1, IL-3, TNF-a and Flk2/Flt3 ligand or
a monocyte conditioned medium (MCM) in addition to GM-CSF and IL-4.
Moreover, in the preparation of the activated human APCs, tumor
antigens, viral antigens or antigen-specific peptides thereof can
be added to give the activated human APCs that are more effective
for treating cancer and infectious diseases.
[0064] The human APC-activating agent of the present invention may
be dissolved in an appropriate dissolving solvent such as DMSO and
then diluted to an appropriate concentration with physiological
saline, a culture medium or the like. The final concentration of
the activating agent in a suitable cultivation for activating human
APCs is within the range from 0.1 ng/mL to 10 .mu.g/mL, and
preferably from 0.01 .mu.g/mL to 1 .mu.g/mL. It is preferable that
the human APC-activating agent according to the present invention
be prepared and added immediately before it is used.
[0065] Accordingly, the present invention also provides activated
human APCs which can be prepared by culturing human-derived APCs in
vitro with at least one of the glycoside compounds represented by
formula (A) or (B) defined above or salts thereof.
[0066] In an embodiment of the present invention, the human-derived
APCs are CD1c-positive or CD1c-rich cells. Such cells may be
prepared from, but not limited to, human peripheral blood, human
umbilical cord blood, human bone marrow cells, human epidermis, and
the like.
[0067] The use of the human APCs activated with the activating
agent of the present invention leads to effective APC therapy for
patients suffering from cancer. Furthermore, it is expected that
the cytotoxic T lymphocytes (CTLs) prepared by co-culturing the
activated APCs and peripheral blood from a patient can be used for
an effective therapy for patients suffering from cancer or
infectious diseases including AIDS.
[0068] Accordingly, the present invention also provides a method
for treating cancer and infectious diseases including AIDS with the
activated human APCs.
[0069] The present invention further provides a use of the
activated human APCs in the preparation of medicines for treating
cancer and infectious diseases including AIDS by application of an
APC therapy.
[0070] The following examples illustrate the present invention more
clearly; however, these examples are not be construed to limit the
scope of the invention.
EXAMPLES
Production Examples
[0071] ##STR10## ##STR11##
Synthesis of Compound G1
[0072] To a solution of D-lyxose (200 g, 1.33 mol) in acetone (3.0
L) dried over calcium chloride was added sulfuric acid (0.5 mL).
The mixture was stirred at room temperature for 18 hours, and
Molecular Sieves 4A powder (100 g) was then added thereto. After
neutralizing, the reaction solution was filtered with celite and
the residue was washed with acetone. The filtrate and the washings
were combined and concentrated under reduced pressure, thereby
giving a crude product of Compound G1 in an amount of 240 g (yield
95%). For the subsequent reaction, the crude product was used
without any further purification. For the preparation of a sample
for analyses, a portion of the crude product was subjected to
further purification by chromatography on a silica gel column with
hexane:acetone (9:1) as an eluent.
[0073] mp 76-78.degree. C.; FDMS m/z 191 (M+1).sup.+; .sup.1H-NMR
(500 MHz, CDCl.sub.3) .delta.5.45 (1H, d, J=1.8 Hz), 4.83 (1H, dd,
J=3.7, 5.5 Hz), 4.64 (1H, d, J=6.1 Hz), 4.27-4.30(1H, m),
3.90-3.99(2H, m) 1.48 (3H, s), 1.32 (3H, s).
Synthesis of Compound G2
[0074] To a solution (168 mL) of Compound G1 (239 g, about 1.26
mol) in methylene chloride were added pyridine (10 mL) and trityl
chloride (39.0 g), and the mixture was stirred at 32.degree. C. for
4 hours. Ethanol (8 mL) was then added to the solution dropwise and
stirred at room temperature for 2 hours. The solution was washed
with a saturated aqueous ammonium chloride solution, a saturated
aqueous sodium hydrogencarbonate solution and the brine, and was
concentrated under reduced pressure. The residue was dissolved in
ethyl acetate and then cooled to 0.degree. C. to cause
precipitation, thereby giving a product of Compound G2 in an amount
of 501 g (yield based on the amount of D-lyxose: 87%).
[0075] mp 174-176.degree. C.; FDMS m/z 432 M.sup.+; .sup.1H-NMR
(500 MHz, CDCl.sub.3) .delta.7.21-7.49 (15H, m), 5.38 (1H, d, J=2.4
Hz), 4.75 (1H, dd, J=3.7, 6.1 Hz), 4.59 (1H, d, J=6.1 Hz),
4.31-4.35 (1H, m), 3.43 (1H, dd, J=4.9, 9.8 Hz), 3.39 (1H, dd,
J=6.7, 9.8 Hz), 1.29 (3H, s), 1.28 (3H, s)
Synthesis of Compound G3
[0076] To a solution (1500 mL) of tridecanetriphenylphosphonium
bromide (962 g, 1.16 mol; a product prepared by heating
1-bromotridecane and triphenylphosphine at 140.degree. C. for 4.5
hours) in THF was added a 2.5 M solution of n-butyl lithium in
hexane (462 mL, 1.16 mol) dropwise in an atmosphere of argon at
0.degree. C. After the dropwise addition was completed, the
resultant solution was stirred for 15 minutes. To the solution was
added a solution (450 mL) of Compound G2 (250 g, 579 mmol) in THF
dropwise. The resultant solution was stirred for 18 hours while
gradually elevating the solution temperature to room temperature.
The reaction solution was concentrated under reduced pressure. To
the residue was added a mixed solution of hexane:methanol:water
(10:7:3, 1000 mL) and washed with a saturated aqueous ammonium
chloride solution. The aqueous layer was extracted with hexane (500
mL) several times. All of the organic layers obtained were
combined, dried over anhydrous magnesium sulfate, and then
concentrated under reduced pressure, thereby giving a crude product
of Compound G3 in an amount of 339 g (yield: 98%). For the
subsequent reaction, the crude product was used without any further
purification. For the preparation of a sample for analyses, a
portion of the crude product was subjected to further purification
by chromatography on a silica gel column with hexane:ethyl acetate
(9:1) as an eluent.
[0077] FDMS m/z 598M.sup.+; .sup.1H-NMR (500 MHz, CDCl.sub.3)
.delta. 7.21-7.45 (15H, m), 5.48-5.59(2H, m), 4.91(0.7H, t, J=7.3
Hz), 4.44(0.3H, t, J=7.3 Hz), 4.26(0.3H, dd, J=4.3, 7.3 Hz),
4.21(0.7H, dd, J=4.3, 6.7 Hz), 3.75(0.7H, m), 3.69(0.3H, m),
3.24(0.3H, dd, J=4.9, 9.8 Hz), 3.17(0.7H, dd, J=4.9, 9.8 Hz),
3.09-3.14 (1H, (3.11, dd, J=4.9, 9.2 Hz), H1bE overlapped)
1.75-2.03(2H, m), 1.49(3H, s), 1.39 and 1.38(3H, each s),
1.21-1.34(20H, m), 0.88(3H, t, J=6.7 Hz).
Synthesis of Compound G4
[0078] To a solution (1500 mL) of Compound G3 (338 g, about 565
mmol) in methylene chloride was added pyridine (500 mL).
Subsequently methanesulfonyl chloride (49 mL, 633 mmol) was added
dropwise thereto and then stirred at 31.degree. C. for 24 hours.
Ethanol (40 mL) was added dropwise thereto and stirred at room
temperature for 1 hour. After the reaction solution was
concentrated under reduced pressure, a mixed solution of
hexane:methanol:water (10:7:3, 1000 mL) was added to the residue to
cause separation of the solution into an aqueous layer and an
organic layer. The aqueous layer was extracted with hexane (200 mL)
3 times. All of the organic layers obtained were combined, dried
over anhydrous magnesium sulfate, and then concentrated under
reduced pressure, thereby giving a crude product of Compound G4 in
an amount of 363 g (yield: 95%). For the subsequent reaction, the
crude product was used without any further purification. For the
preparation of a sample for analyses, a portion of the crude
product was subjected to further purification by chromatography on
a silica gel column with hexane:ethyl acetate (9:1) as an
eluent.
[0079] FDMS m/z 676M.sup.+; .sup.1H-NMR (500 MHz, CDCl.sub.3)
.delta. 7.21-7.47 (15H, m), 5.41(0 .7H, ddd, J=5.5, 9.2, 11.0 Hz),
5.32(0.7H, bt, J=11.0 Hz), 5.22 (0.3H, bdd, J=9.2, 15.0 Hz),
5.02(0.3H, dt, J.sub.t=7.3 Hz, J.sub.d=15.0 Hz), 4.8 (0.7H, ddd,
J=3.1, 5.5, 7.9 Hz), 4.73(0.7H, dd, J=5.5, 9.8 Hz), 4.64-4.67
(0.3H, m), 4.61(0.3H, dd, J=5.5, 9.2 Hz), 4.48(0.7, dd, J=5.5, 7.9
Hz), 4.22(0.3H, dd, J=5.5, 9.2 Hz) 3.55(0.3H, dd, J=2.4, 11.6 Hz),
3.45(0.7H, dd, J=3.2, 11.0 Hz), 3.06-3.12(4H, (3.12,S), (3.11,S),
(3.09, dd, J=3.1, 11.0 Hz)), 1.66-1.82 (2H, m), 1.47 and 1.46 (3H,
each s), 1.39 (3H, s), 1.13-1.35 (20H, m), 0.88 (3H, t, J=6.8
Hz).
Synthesis of Compound G5
[0080] To a solution (1500 mL) of Compound G4 (362 g, about 536
mmol) in methylene chloride was added methanol (350 mL).
Concentrated hydrochloric acid (200 mL) was added to the solution
dropwise and then stirred at room temperature for 5 hours. The
reaction solution was neutralized with sodium hydrogencarbonate and
then subjected to filtration. The filtrate was concentrated under
reduced pressure, and ethyl acetate was added to the residue and
then washed with brine. The aqueous layer was extracted with ethyl
acetate several times. All of the organic layers obtained were
combined, dried over anhydrous magnesium sulfate, and then
concentrated under reduced pressure. The resultant product was
crystallized from hexane to give a product of Compound G5 in an
amount of 161 g (yield based on the amount of Compound G2:
70%).
[0081] mp 66-67.degree. C.; FDMS m/z 377(M-H.sub.2O)+;
.sup.1H-NMR(500 MHz, CDCl.sub.3+D.sub.20) .delta. 5.86(0.3H, dt,
J.sub.t=7.3 Hz, J.sub.d=14.7 Hz), 5.77(0.7H, dt, J.sub.t=7.3,
J.sub.d=10.4 Hz), 5.55(0.3H, br.dd, J=7.3, 14.7 Hz), 5.49(0.7H, bt,
J=9.8 Hz), 4.91-4.97(1H, m), 4.51 (0.7H, bt, J=9.8 Hz), 4.11(0.3H,
bt, J=7.3 Hz), 3.94-4.03 (2H, m), 3.67-3.73 (1H, (3.70, dd, J=3.1,
6.7 Hz), (3.69, dd, J=3.1, 7.3 Hz)], 3.20 and 3.19 (3H, each s),
2.05-2.22(2H, m), 1.22-1.43(20H, m), 0.88(3H, t, J=6.7 Hz).
Synthesis of Compound G6
[0082] To a solution (780 mL) of Compound G5 (160 g, 405 mmol) in
THF was added 5% palladium on barium sulfate (16 g). After the
reaction vessel was purged with hydrogen gas, the mixture was
stirred at room temperature for 20 hours. The reaction solution was
filtered with celite and the filter cake was washed with a mixed
solution of chloroform:methanol (1:1). The filtrate and the
washings were combined and concentrated under reduced pressure. The
residue was crystallized from ethyl acetate to give a product of
Compound G6 in an amount of 146 g (yield: 91%).
[0083] [.alpha.].sub.23.sup.D+12.sup.O(c 1, CHCl.sub.3/MeOH=1:1);
mP 124-126.degree. C.; FDMS m/z 397 (M+1).sup.+; .sup.1H-NMR (500
MHz, CDCl.sub.3/CD.sub.3=1:1) .delta. 4.93-4.96(1H, m, H2),
3.91(1H, dd, J=6.7, 12.2 Hz), 3.85 (1H, dd, J=4.9, 12.2 Hz),
3.54-3.60(1H, m), 3.50(1H, dd, J=1.8, 8.5 Hz), 3.19(3H, s),
1.75-1.83(1H, m), 1.53-1.62(1H, m), 1.2-1.45(24H, m), 0.89(3H, t,
J=6.7 Hz).
Synthesis of Compound G7
[0084] To a solution (1000 mL) of Compound G6 (145 g, 365 mmol) in
DMF was added sodium azide (47 g, 730 mmol), and the mixture was
stirred at 95.degree. C. for 4 hours. The reaction solution was
concentrated. Ethyl acetate (450 mL) was added to the residue, and
the resultant solution was washed with water. The aqueous layer was
re-extracted with ethyl acetate several times. All of the organic
layers obtained were combined, washed with brine, dried over
anhydrous magnesium sulfate and then concentrated under reduced
pressure, thereby giving a crude product of Compound G7 in an
amount of 122 g (yield: 97%). For the subsequent reaction, the
crude product was used without any further purification. For the
preparation of a sample for analyses, a portion of the crude
product was subjected to further purification by chromatography on
a silica gel column with hexane:ethyl acetate (9:1) as an
eluent.
[0085] [.alpha.].sub.23.sup.D+16.5.sup.O(c 0.5, CHCl.sub.3/MeOH
=1:1); mP 92-93.degree. C.; FDMS m/z 344(M+1).sup.+; .sup.1H-NMR
(500 MHz, CD.sub.3OD) .delta. 3.91(1H, dd, J=3.7, 11.6 Hz),
3.75(1H, dd, J=7.9, 11.6 Hz), 3.49-3.61 (3H, m), 1.50-1.71(2H, m),
1.22-1.46(24H,m), 0.90(3H, t, J=6.7 Hz)
Synthesis of Compound G8
[0086] To a solution (750 mL) of Compound G7 (121 g, about 352
mmol) in methylene chloride were added pyridine (250 mL) and trityl
chloride (124 g, 445 mL), and the mixture was stirred at room
temperature for 16 hours. Ethanol (30 mL) was added dropwise
thereto and then stirred at room temperature for 30 minutes. After
washing with a saturated sodium hydrogencarbonate solution, a
saturated aqueous ammonium chloride solution and brine, the
reaction solution was dried over anhydrous magnesium sulfate and
then concentrated under reduced pressure. The residue was purified
by chromatography on a silica gel column with hexane:ethyl acetate
(10:1) as an eluent, thereby giving a product of Compound G8 in an
amount of 34.4 g (yield based on the amount of Compound G6:
52%).
[0087] [.alpha.].sub.24.sup.D+11.9.sup.O(c 0.9, CHCl.sub.3), FDMS
m/z 585 M.sup.+; .sup.1H-NMR (500 MHz, CDCl.sub.3+D.sub.20) .delta.
7.24-7.61(15H, m), 3.62-3.66(2H, m), 3.51-3.57(2H, m), 3.42(1H, dd,
J=6.0, 10.4 Hz), 1.23-1.56(26H, m), 0.88(3H, t, J=6.7 Hz).
Synthesis of Compound G9
[0088] To a solution (300 mL) of Compound G8 (33.5 g, 57.3 mmol) in
DMF was added 60% hydrogenated sodium (5.5 g, about 138 mmol in
terms of NaH), and the mixture was stirred for 40 minutes. The
reaction solution was cooled to 0.degree. C., and then benzyl
chloride (15 mL, 120 mmol) was added dropwise thereto. The
resultant solution was stirred for 18 hours while gradually
elevating the solution temperature to room temperature. Ice-cooled
water (100 mL) was added to the reaction solution to stop the
reaction, and the reaction solution was then extracted with ethyl
acetate. The extract was washed with brine 3 times. All of the
organic layers obtained were combined, dried over anhydrous
magnesium sulfate, and then concentrated under reduced pressure,
thereby giving a crude product of Compound G9 in an amount of 42.2
g (yield: 96%). For the subsequent reaction, the crude product was
used without any further purification. For the preparation of a
sample for analyses, a portion of the crude product was subjected
to further purification by chromatography on a silica gel column
with hexane:ethyl acetate (100:1) as an eluent.
[0089] [.alpha.].sub.24.sup.D+9.8.sup.O(c 1.0, CHCl.sub.3), FDMS
m/z 738 (M-N.sub.2)+; .sup.1H-NMR (500 MHz, CDCl.sub.3) .delta.
7.07-7.48(25H, m), 4.57(1H, d, J=11.6 Hz), 4.44(1H, d, J=11.6 Hz),
4.41 (2H, s), 3.73-3.79 (1H, m), 3.46-3.56 (2H, m), 3.37 (1H, dd,
J=8.6, 10.4 Hz), 1.20-1.64 (26H, m), 0.88 (3H, t, J=6.7 Hz).
Synthesis of Compounds G10 and G11
[0090] To a solution (250 mL) of Compound G9 (41.2 g, about 54
mmol) in 1-propanol was added methanol (30 mL), and subsequently 5%
palladium on carbon (4.1 g) and ammonium formate (27.1 g, 4.3 mol)
were added thereto. After the mixture was stirred at room
temperature for 16 hours, it was diluted with ethyl acetate and
then filtered with celite. The filtrate was concentrated under
reduced pressure and then dissolved in ethyl acetate. The resultant
solution was washed 3 times with a saturated aqueous sodium
hydrogencarbonate solution and subsequently with brine. All of the
organic layers obtained were combined, dried over anhydrous
magnesium sulfate, and then concentrated under reduced pressure,
thereby giving a crude product of Compound G10 in an amount of 38.9
g (98%). For the subsequent reaction, the crude product (Compound
G10) was used without any further purification.
[0091] To a solution (300 mL) of Compound G10 in methylene chloride
were added hexacosanic acid (22.4 g, 56.5 mmol) and WSC
hydrochloride (12.6 g, 64.6 mmol), and the mixture was refluxed
with heating for 2 hours. After cooling to room temperature, the
reaction solution was concentrated under reduced pressure. Ethyl
acetate (500 mL) was added to the residue, and the resultant
solution was washed several times with a 0.5 M aqueous solution of
hydrochloric acid, brine, a saturated aqueous sodium
hydrogencarbonate solution and then brine. All of the organic
layers obtained were combined, dried over anhydrous magnesium
sulfate, and then concentrated under reduced pressure, thereby
giving a crude product of Compound G11 in an amount of 53.2 g
(yield: 88%). For the subsequent reaction, the crude product
(Compound G11) was used without any further purification. For the
preparation of a sample for analyses, a portion of the crude
product was subjected to further purification by chromatography on
a silica gel column with hexane:ethyl acetate (100:1) as an
eluent.
[0092] [.alpha.].sub.24.sup.D+5.3.sup.O(c 0.4, CHCl.sub.3); FDMS
m/z 1118 M.sup.+; .sup.1H-NMR (500 MHz, CDCl.sub.3) .delta.
7.20-7.38(25H, m), 5.57(1H, d, J=9.1 Hz), 4.80 (1H, d, J=11.6 Hz),
4.48-4.50 (3H, m), 4.24-4.32(1H, m), 3.83 (1H, dd, J=3.0, 6.7 Hz),
3.43-3.51(2H, m, H1a), 3.29(1H, dd, J=4.3, 9.8 Hz), 1.92(2H, t,
J=7.3 Hz), 1.28-1.60(72H, m), 0.88(6H, t, J=6.7 Hz).
Synthesis of Compound G12
[0093] To a solution (180 mL) of Compound G11 (52.2 g, about 47
mmol) in methylene chloride was added methanol (36 mL), and a 10%
solution of hydrochloric acid in methanol (3.0 mL) was added
dropwise thereto. The mixture was stirred at room temperature for 2
hours. The reaction solution was neutralized with sodium
hydrogencarbonate powder (18 g) and then filtered with celite. The
residue was washed with methylene chloride. The filtrate and the
washings were combined, and then washed with brine. The organic
layer obtained was dried over anhydrous magnesium sulfate, and then
concentrated under reduced pressure. The residue was dissolved in
hot acetone and then cooled to 0.degree. C. to cause precipitation
of a product of Compound G12 in a purified form in an amount of
38.6 g (yield based on the amount of G9: 77%).
[0094] [.alpha.].sub.24.sup.D-29.7.sup.O(c 0.7, CHCl.sub.3); mp
75-76.5.degree. C.; FDMS m/z 876M.sup.+; .sup.1H-NMR (500 MHz,
CDCl.sub.3) .delta. 7.30-7.47(10H, m), 6.03(1H, d, J=7.9 Hz), 4.72
(1H, d, J=11.6 Hz), 4.66(1H, d, J=11.6 Hz), 4.61(1H, d, J=11.6 Hz),
4.45(1H, d, J=11.6 Hz), 4.12-4.17(1H, m), 4.00(1H, dt, J.sub.t=4.3,
J.sub.d=7.3 Hz), 3.67-3.72(2H, m), 3.61(1H, ddd, J=4.3, 8.6, 11.6
Hz), 1.94-2.05(2H, m), 1.15-1.69(72H, m), 0.88(6H, t, J=6.1
Hz).
Synthesis of Compound G13
[0095] (1) 2,3,4,6-Tetra-O-benzyl-D-galactopyranosyl acetate (79.8
g) was dissolved in a mixed solution of toluene (160 mL) and
isopropyl ether (520 mL) and then cooled to -10-0.degree. C. To the
solution was added a solution containing 2.0 equivalent amount of
HBr in isopropyl ether (2.8 mmol/mL, about 100 mL). After the
reaction solution was stirred at -10-0.degree. C. for about 90
minutes, a 5% aqueous solution of sodium hydrogencarbonate was
poured thereto and stirred to neutralize the excess HBr. The whole
of the solution was transferred to a separatory funnel to cause
separation into an aqueous layer and an organic layer. The aqueous
layer obtained was discarded and the organic layer was washed with
a 10% aqueous solution of sodium chloride twice. The organic layer
was concentrated under reduced pressure, thereby giving a syrup
product of 2,3,4,6-tetra-O-benzyl-.alpha.-D-galactopyranosyl
bromide (Gal-Br).
[0096] (2) To a solution (420 mL) containing Compound G12 (60.0 g,
68.6 mmol), tetrahexylammonium bromide (89.4 g, 206 mmol) and
Molecular Sieves 4A powder (60 g) in toluene were added DMF (140
mL) and subsequently a solution (250 mL) of Gal-Br (about 137 mmol)
in toluene. The mixture was stirred at room temperature for 72
hours. Methanol (12 mL) was added to the reaction solution and
stirred for 2 hours. After the solution was filtered with celite,
the filtrate was washed with a saturated aqueous sodium
hydrogencarbonate solution and brine, dried over anhydrous
magnesium sulfate, and then concentrated under reduced pressure.
Acetonitrile was added to the residue, and the resultant solution
was stirred for 2 hours to cause precipitation. The precipitate
obtained was dried under reduced pressure to give a dry powder
product. The powder product was purified by chromatography on a
silica gel column with hexane:ethyl acetate (8:1) as an eluent,
thereby giving Compound G13 in an amount of 70.9 g (yield:
74%).
[0097] [.alpha.].sub.24.sup.D+18.8.sup.O(c 0.9, CHCl.sub.3); mp
74-75.degree. C.; FDMS m/z 1399 M+1).sup.+; .sup.1H-NMR (500 MHz,
CDCl.sub.3) .delta. 7.21-7.37(30H, m), 6.12 (1H, d, J=9.0 Hz),
4.91(1H, d, J=11.6 Hz), 4.84(1H, d, J=3.7 Hz), 4.72-4.80 (4H, m),
4.35-4.65 (7H, m), 4.12-4.18 (1H, m), 3.99-4.05(2H, m), 3.84-3.93
(4H, m), 3.73(1H, dd, J=3.7, 11.0 Hz), 3.47-3.51(2H, m), 3.42(1H,
dd, J=6.1, 9.1 Hz), 1.87-1.99(2H, m), 1.18-1.70(72H, m), 0.88(6H,
t, J=7.4 Hz).
(2S, 3R,
4R)-1-O-(.alpha.-D-Galactopyranosyl)-N-hexacosanoyl-2-amino-1,3,4-
-octadecanetriol (KRN7000)
[0098] Compound G13 (60.0 g, 42.9 mmol) was suspended into ethanol
(960 mL), and a 20% suspension of palladium hydroxide (6.0 g) in
ethanol was added thereto. 4-Methylcyclohexene (120 mL, 93.5 mmol)
was added to the suspension as a donor compound for hydrogen, and
then refluxed under heating for 4 hours. Thereafter, the resultant
solution was filtered to remove the catalyst. The residue was
washed with hot ethanol. The filtrate was allowed to stand at room
temperature to cause precipitation of a white product. The white
precipitate was filtered out and then dried under reduced pressure.
The powder product obtained was suspended into ethanol:water (92:8,
3.5 L), dissolved by heating while stirring, and then allowed to
stand to cause precipitation again. The precipitate solution was
filtered and the filter cake obtained was dried under reduced
pressure, thereby giving the title compound in a white powder form
in an amount of 35.0 g (yield: 95%).
[0099] [.alpha.].sub.24.sup.D+43.6.sup.O(c 1.0, pyridine); mp
189.5-190.5.degree. C.; negative FABMS m/z 857(M-H).sup.-;
IR(cm.sup.-1, KBr) 3300, 2930, 2850, 1640, 1540, 1470, 1070;
.sup.1H-NMR(500 MHz, C.sub.5D.sub.5N) .delta. 8.47(1H, d, J=8.5
Hz), 5.58(1H, d, J=3.7 Hz), 5.27(1H, m), 4.63-4.70 (2H, m),
4.56(1H, m), 4.52(1H, t, J=6.1 Hz), 4.37-4.47 (4H, m), 4.33(2H, m),
2.45(2H, t, J=7.3 Hz), 2.25-2.34 (1H, m), 1.87-1.97(2H, m),
1.78-1.85(2H, m), 1.62-1.72(1H, m), 1.26-1.45(66H,m), 0.88(6H, t,
J=6.7 Hz). .sup.13C-NMR (125 MHz, C.sub.5D.sub.5N) .delta.
173.2(s), 101.5(d), 76.7(d), 73.0(d), 72.5(d), 71.6(d), 71.0(d),
70.3(d), 68.7(t), 62.7(t), 51.4(d), 36.8(t), 34.4(t), 32.1(t),
30.4(t), 30.2(t), 30.03(t), 30.00(t), 29.93(t), 29.87(t), 29.81(t),
29.76 (t), 29.6(t), 26.5(t), 26.4(t), 22.9(t), 14.3(q).
Pharmacological Tests
[Pharmacological Test 1] Effort of KRN 7000 on Human Peripheral
Blood-Derived APC
[0100] This experiment was designed to study an effect of KRN7000
(Compound No.14, a representative glycoside compound of the
invention) on APCs prepared from human peripheral blood. The APCs
(mainly comprised of dendritic cells) derived from human peripheral
blood were prepared essentially according to the method of Hsu et
al. (supra). Briefly, human peripheral blood was layered over
Ficoll-Paque and centrifuged at 2,000 rpm for 35 minutes to give a
mononuclear cell fraction. A portion of the mononuclear cell
fraction was suspended into PBS (Ca.sup.++, Mg.sup.++ free)
supplemented with 5% of inactivated human AB serum. The resultant
solution was layered over 50% Percol, and then centrifuged at 3,000
rpm for 20 minutes to give a high-density fraction. The
high-density fraction was panned using a human
.gamma.-globulin-coated dish to remove FcR.sup.+ cells therefrom
(i.e., a monocyte-removed fraction). The resultant fraction was
reacted with anti-CD3 monoclonal antibodies, anti-CD21 monoclonal
antibodies and anti-CD56 monoclonal antibodies. After washing, the
resultant solution was panned using an anti-mouse IgG
antibody-coated dish to remove cells that were reactive with the
above antibodies (i.e., T cells, B cells and NK cells). The cell
fraction thus obtained (i.e., a fraction from which monocytes, T
cells, B cells and NK cells were removed) was cultured in RPMI 1640
medium supplemented with 5% of inactivated human AB serum in the
presence of a vehicle (DMSO, final concentration: 0.1%) and KRN7000
(final concentration: 100 ng/mL) for 40 hours. After washing, the
resultant cells were provided for use as APCs.
[0101] On the other hand, as responder cells, CD4.sup.+ T cells
were purified from the rest of the above-prepared mononuclear cell
fraction using a human CD4.sup.+ T cell-enrichment column, and were
cultured in RPMI 1640 medium supplemented with 5% of inactivated
human AB serum until they were subjected to use with the APCs.
[0102] The APCs (5.times.10.sup.4 cells/well) and the responder
cells (1.times.10.sup.5 cells/well) both prepared in the above
steps were added to wells of a 96-well flat-bottomed plate, and
then subjected to autologous MLR assay. During the last 12 hours of
the 6-day culture period, tritium-thymidine (.sup.3H-TdR) was added
to each of the wells (0.5 .mu.Ci/well), and then the cells were
harvested. The amount of the 3H-TdR taken up into the cells was
determined using a liquid scintillation counter. The results (mean
value and standard deviation of 3 wells) are shown in FIG. 1. In
FIG. 1, "V-APC" and "KRN-APC" refer to the APCs pretreated with the
vehicle and KRN7000, respectively.
[0103] As shown in FIG. 1, when each of CD4.sup.+ T cell, the
vehicle-pretreated APC (V-APC) and the KRN7000-pretreated APC
(KRN-APC) were cultured singly, in either case, the cells took up
no or a negligible amount of .sup.3H-TdR. In contrast, when the
above-prepared T cell was mixed with V-APC, an obvious .sup.3H-TdR
uptake was observed. When the T cell was mixed with KRN-APC, a
significantly remarkable .sup.3H-TdR uptake was observed. These
results demonstrate that KRN-APC has a more significant autologous
MLR enhancing effect than V-APC. Accordingly, KRN7000 has an
activating effect on the APCs prepared from human peripheral blood
(i.e., an antigen-presenting function enhancing effect).
(Pharmacological Test 2] Effect of KRN7000 on Human Umbilical Cord
Blood-Derived APC
[0104] APCs derived from human umbilical cord blood (CD1c positive
cells; CD1c.sup.+ cells) were prepared in the following manner.
That is, human umbilical cord blood was layered over Lymphoprep and
centrifuged at 1500 rpm for 20 minutes to give a mononuclear cell
fraction. A portion of the mononuclear cell fraction was washed
with PBS supplemented with 2% inactivated FBS and 1 mM EDTA twice,
and FcR was blocked with human .gamma.-globulin. The solution was
reacted with anti-CD1c antibodies, and washed with PBS supplemented
with 2% FBS and 1 mM EDTA twice. The solution was further reacted
with anti-mouse IgG microbeads, washed with PBS supplemented with
2% FBS and 1 mM EDTA, and then suspended into PBS supplemented with
0.5% BSA and 5 mM EDTA. CD1c.sup.+ cells were obtained from the
suspension using a MiniMACS magnetic column previously washed with
PBS supplemented with 0.5% BSA and 5 mM EDTA. The CD1c.sup.+ cells
obtained were suspended into RPMI 1640 medium supplemented with 10%
inactivated human AB blood serum to adjust its density to
1.times.10.sup.6 cells/mL. The cell suspension was added to wells
of a 24-well plate (2 mL/well), and then a vehicle (DMSO, final
concentration: 0.1%) or KRN7000 (final concentration: 100 ng/mL)
was added to each of the wells. After incubating for 3 days, the
plate was washed 3 times and provided for use as APCs.
[0105] CD4.sup.+ T cells were purified from the rest of the
above-prepared mononuclear cell fraction using a human CD4.sup.+ T
cell-enrichment column. The T cells obtained were suspended into
RPMI 1640 medium supplemented with 10% inactivated human AB blood
serum to adjust its density to 1.times.10.sup.6 cells/well. The
suspension was added to wells of a 24-well plate (2 mL/well) and
cultured for 3 days. The T cells thus obtained were used as
responder cells for autologous MLR assay. On the other hand, a
peripheral blood was collected from a normal volunteer, and
CD4.sup.+ T cells were purified therefrom using a human CD4.sup.+ T
cell-enrichment column. The T cells thus obtained was used as
responder cells for allogeneic MLR assay. The responder cells
(1.times.10.sup.5 cells/50 mL/well) and the stimulator cells (0,
1.000, 10000 cells/50 mL/well) were added to wells of a 96-well
flat-bottomed plate, and the plate was incubated. For allogeneic
MLR assay, the plate was incubated for 4 days, .sup.3H-TdR was
added to each of the wells (0.5 .mu.Ci/well). After 20 hours, the
cells were harvested and the amount of the .sup.3H-TdR taken up
into the cells was determined using a liquid scintillation counter.
The results (mean value and standard deviation of 3 wells) are
shown in FIG. 2.
[0106] For autologous MLR assay, the plate was incubated for 7
days, .sup.3H-TdR was added to each of the wells (0.5 .mu.Ci/well).
After 8 hours, the cells were harvested and the amount of the
.sup.3H-TdR taken up into the cells was determined using a liquid
scintillation counter. The results (mean value and standard
deviation of 3 wells) are shown in FIG. 3. In FIGS. 2 and 3,
"V-APC" and "KRN-APC" refer to the APCs (CD1c.sup.+ cells)
pretreated with the vehicle and KRN7000, respectively.
[0107] As shown in FIG. 2., the CD1c.sup.+ cells stimulated with
the vehicle (APCs; V-APC) promoted the uptake of .sup.3H-TdR
(proliferation of allogeneic T cells) in a manner dependent on the
number of the V-APC, whereas the CD1c.sup.+ cells stimulated with
KRN7000 (APCs; KRN-APC) showed a stronger allogeneic T
cell-proliferation-promoting effect than V-APC.
[0108] As shown in FIG. 3, when autologous T cells were used as the
responder cells, the CD1c.sup.+ cells stimulated with the vehicle
(V-APC) hardly stimulated the proliferation of the autologous T
cells even if the APCs were added to the wells in an amount of
10000 cells/well; whereas the CD1c cells treated with KRN7000
(KRN-APC) significantly stimulated the proliferation of the
autologous T cells in a manner dependent on the number of the APCs,
similar to the case for the allogeneic T cells.
[Pharmacological Test 3] Effect of .alpha.- and
.beta.-galactosylceramides and .alpha.- and
.beta.-glucosylceramides on Murine Spleen-Derived APC
[0109] The preparation of dendritic cell-rich APCs from a murine
spleen was performed essentially in accordance with the method of
Clowley, M. et al. (supra). Briefly, the spleen was removed from a
BDF1 mouse. After the treatment with 100 U/mL of collagenase, the
spleen was dissected with tweezers to separate into a cell
suspension and a tissue debris. The tissue debris was suspended
into a 400 U/mL solution of collagenase. After culturing in a
CO.sub.2 incubator for 20 minutes, the suspension was passed
through a stainless steel mesh with an inner syringe to give a cell
suspension. Two of the cell suspensions were combined, and
subjected to centrifugation. The cell suspension was subjected to
density gradient centrifugation with a high-density BSA to give a
low-density cell fraction. The cell fraction was plated onto a 60
mm dish and cultured for 2 hours. The procedure for removing the
floating cells was repeated 3 times. RPMI 1640 medium supplemented
with 10% inactivated FBS was added to the dish and subsequently any
one of KRN7000 (Compound No.14), AGL583 (a .beta.-anomer of
KRN7000), AGL517 (Compound No.3), AGL564 (a .beta.-anomer of
AGL517), AGL563 (Compound No.4), AGL562 (a .beta.-anomer of
AGL-563) and a vehicle (DMSO, final concentration: 0.1%) was also
added thereto to a final concentration of 100 ng/mL. After
culturing overnight, the non-adherent cells were collected, and the
resultant cells were provided for use as APCs.
[0110] On the other hand, BDF1 mouse spleen cells were treated with
a red blood cell-lysing buffer comprised of NH.sub.4Cl and Tris-HCl
to hemolyze red blood cells, and the resultant spleen cells were
suspended into RPMI 1640 medium supplemented with 10% inactivated
FBS. The spleen cells were inoculated onto a 100 mm dish, and
cultured in a CO.sub.2 incubator for 2 hours. Thereafter, the
non-adherent cells were recovered, which were provided for use as
responder cells.
[0111] The above-prepared APCs (1.times.10.sup.3,
3.3.times.10.sup.3 or 1.times.10.sup.4 cells/well) and the
responder cells (2.5.times.10.sup.5 cells/well) were added to wells
of a 96-well plate for syngeneic MLR assay. After culturing for 1
day, .sup.3H-TdR was added to each of the wells (0.5 .mu.Ci/well).
After 16 hours, the cells were harvested, and the amount of
.sup.3H-TdR taken up into the cells was determined using a liquid
scintillation counter. The results (mean value and standard
deviation of 3 wells) are shown in FIG. 4.
[0112] In FIG. 4, "V-APC", "KRN-APC", "583-APC", "517-APC",
"564-APC", "563-APC" and "562-APC" refer to the APCs pretreated
with the vehicle, KRN7000, AGL-583, AGL-517, AGL-564, AGL-563 and
AGL-562, respectively.
[0113] As shown in FIG. 4, the APCs pretreated with
.alpha.-galactosylceramides such as KRN-APC and 517-APV showed a
significant syngeneic MLR enhancing effect, whereas the APCs
pretreated with .beta.-galactosylceramides such as 583-APC and
564-APC did not show any such effect. The APCs pretreated with
.alpha.-glucosylceramides such as 563-APC also showed a remarkable
syngeneic MLR enhancing effect, whereas the APCs pretreated with
.beta.-glucosylceramides such as 562-APC did not show any such
effect.
[Pharmacological Test 4] APC Therapy With Murine Spleen-Derived
APCs Pretreated With KRN7000
[0114] To study an anti-tumor effect of murine spleen-derived APCs
activated with KRN7000 on mice implanted with tumor cells,
experiments were performed with BDF1 mice (6-week-old, female). All
experiments included 6 mice per group. Murine T cell lymphoma EL-4
cells were injected intravenously into each mouse at a level of
1.times.10.sup.5 cells/mouse (injection day: day 0). On day 1, the
vehicle-pretreated APCs (V-APC) and the KRN7000-pretreated (100
ng/mL) APCs (KRN-APC) both prepared in the same manner as in
Pharmacological Test 3 were each injected intravenously into the
mice at a level of 5.times.10.sup.5 cells/mouse. As a positive
control, KRN7000 was injected intravenously into the mice at a dose
of 100 .mu.g/kg on days 1, 5 and 9. Each mouse was checked for a
sign of life daily to determine its survival period. The results
are shown in FIG. 5.
[0115] As shown in FIG. 5, when V-APC was injected, no prolongation
of survival period was observed. However, when KRN-APC was
injected, a remarkable increase in survival period was observed and
50% of the KRN-APC-injected mice were cured completely. When
KRN7000 was injected intravenously at a dose of 100 .mu.g/kg 3
times, a remarkable increase in survival period was observed,
however, it was found that the potency of the effect was weaker
than that when KRN-APC was injected only once.
(Pharmacological Test 5] Antigen Presenting Function Enhancing
Effort of KRN7000 on Murine Bone Marrow-Derived Dendritic Cell
[0116] Dendritic cell-rich APCs derived from murine bone marrow
were prepared according to the method of Inaba, K. et al. (supra)
with some modifications (Yamaguchi Y. et al., Stem Cells,
1977:15:144-153). Briefly, bone marrow cells from BALB/c mice were
prepared. After hemolyzing the red blood cells with a NH.sub.4Cl
solution, FcR.sup.+ cells were removed by panning using a human
.gamma.-globulin-coated dish. The cells thus obtained were
suspended in RPMI medium supplemented with 10% FCS, and cultured in
the presence of 10 ng/mL of mouse rGM-CSF and 10 ng/mL of human
rTGF-.beta. for 6 days at 5.times.10.sup.5 cells/well (1 ml/well, a
24-well plate). Every 2 days, each well was washed briefly using a
Pasteur pipette and then about 75% of the medium was sucked out and
supplemented with 1 ml of a fresh medium containing the above
factors. After culturing for 6 days, non-adherent cells were
recovered, and FcR.sup.+ cells were removed therefrom by panning
using a human .gamma.-globulin-coated dish. The cells thus prepared
were cultured in a culture medium supplemented with 10 ng/mL of
mouse rGM-CSF and 10 ng/mL of human rTNF-.alpha. for additional 2
days. During this culture, either a vehicle (DMSO, final
concentration: 0.1%) or KRN7000 (final concentration: 100 ng/mL)
was added to the culture medium. The cells were collected and
washed 3 times to provide for use as APCs. On the other hand, as
responder cells, T cells were prepared from a BALB/c murine spleen
using a T cell enrichment column (R&D). T cells were added to
wells of a 96-well plate (3.times.10.sup.5 cells/well), and the
above-prepared APCs were further added thereto for syngeneic MLR
assay (3.times.10.sup.4, 1.times.10.sup.4, 3.times.10.sup.3 and
1.times.10.sup.3 cells/well, respectively). After culturing for 2
days. .sup.3H-TdR was added to each of the wells (0.5 .mu.Ci/mL).
After 6 hours, the cells were harvested and determined on the
uptake of .sup.3H-TdR into the cells using a liquid scintillation
counter. The results (mean value and standard deviation of 3 wells)
are shown in FIG. 6. In FIG. 6, "V-APC" and "KRN-APC" refer to the
APCs pretreated with the vehicle and KRN7000, respectively.
[0117] As shown in FIG. 6, the murine bone marrow-derived APCs
which were stimulated with KRN7000 (KRN-APC) showed a remarkable
syngeneic MLR enhancing effect compared to the APCs stimulated with
the vehicle (V-APC).
(Pharmacological Test 6] APC Therapy With Murine Bone Marrow
Derived-APCs Pretreated with KRN7000
[0118] The experiment was performed to study an anti-tumor effect
of murine bone marrow-derived APCs activated with KRN7000 on mice
implanted with tumor cells. All experiments were performed with
CDF1 mice (6-week-old, female), which were divided into groups
consisting of 5 mice. Murine colon adenocarcinoma Colon26 cells
were implanted into each mouse intrasplenically at a level of
2.times.10.sup.6 cells/mouse (inoculation day: day 0). On day 1,
the vehicle-pretreated APCs (V-APC) and the KRN7000-pretreated (100
ng/mL) APCs (KRN-APC) both prepared in the same manner as in
Pharmacological Test 5 were individually injected intravenously to
the mice at a level of 8.times.10.sup.5 cells/mouse. As a positive
control, KRN7000 was injected intravenously to the mice at a dose
of 100 .mu.g/kg on days 1, 5 and 9. On day 14, the liver was
removed from each of the mice and weighed. The results (mean value
and standard deviation of the weights of the liver of 5 mice) are
shown in FIG. 7. Here, the liver from the mouse that had not been
implanted with the tumor cells was about 1 g. Accordingly, the
substantial tumor weight was determined by subtracting 1 g from the
weight of the liver of the tumor-implanted mouse.
[0119] As shown in FIG. 7, when V-APC was injected, a slight
suppressive effect on tumor growth was observed; whereas when
KRN-APC was injected, a remarkable inhibitory effect on tumor
growth was observed and no tumor nodule in the liver was observed
by naked eyes in 3 of the 5 mice. When KRN7000 was injected
intravenously 3 times at a dose of 100 .mu.g/kg, a remarkable
inhibitory effect on tumor growth was observed, which was at the
same level as that when KRN-APC was injected once.
[Pharmacological Test 7] Antigen Presenting function Enhancing
Effect of KRM7000 on Murine Epidermis-Derived APCs
[0120] Langerhans's cell-rich APCs were prepared from murine
auricle essentially in accordance with the method of Witmer-Pack,
M. et al. (supra). Briefly, the epidermis of auricle of a BALB/c
mouse was peeled into front and back epidermal sheets, and each of
the epidermal sheets was soaked into Hanks's solution supplemented
with 1% trypsin at 37.degree. C. for 30 minutes to 1 hour. The
epidermal sheets obtained were placed on a mesh, and shaken up and
down in Hanks's solution supplemented with 10% FCS. The cells
detached from the epidermal sheets were collected, and then
suspended into RPMI solution supplemented with 10% FCS at 10.sup.6
cells/mL. After addition of either a vehicle (DMSO, final
concentration: 0.1%) or KRN7000 (final concentration: 100 ng/mL),
the cells were cultured under 5% CO.sub.2 at 37.degree. C. for 3
days. Thereafter, the cells that were non-adherent to the dish were
collected and layered over Lympholite-M. After centrifugation at
1400 rpm for 10 minutes, the low-density cells were collected,
washed with RPMI twice, which were provided for use as APCs.
[0121] As responder cells, spleen cells were prepared from BALB/c
mice in the same manner as described in Pharmacological Test 3. The
responder cells thus obtained were added to wells of a 96-well
plate (2.5.times.10.sup.5 cells/well), and the APCs were also added
thereto for syngeneic MLR assay (4.times.10.sup.4, 4/3.times.10 and
4/9.times.10.sup.4 cells/well, respectively). After culturing for 2
days, .sup.3H-TdR was added to each well (0.5 .mu.Ci/mL). After 6
hours, the cells were harvested and determined on the amount of
.sup.3H-TdR taken up into the cells using a liquid scintillation
counter. The results (mean value and standard deviation of 3 wells)
are shown in FIG. 8. In FIG. 8, "V-APC" and "KRN-APC" refer to the
APCs pretreated with the vehicle and KRN7000, respectively.
[0122] As shown in FIG. 8, the murine epidermis-derived APCs
stimulated with KRN (KRN-APCs) showed a more remarkable syngeneic
MLR enhancing effect compared to those stimulated with the vehicle
(V-APCs).
[Pharmacological Test 8] APC Therapy With Murine Spleen-Derived
APCs pretreated With KRN7000 and Tumor Antigen
[0123] The experiment was performed with BDF1 mice (6-week-old,
female), which were divided into groups consisting of 6 mice.
Murine melanoma B16 cells were implanted subcutaneously into each
mouse at a level of 1.5.times.10.sup.6 cells/mouse (implantation
day: day 0). Several types of pretreated APCs were prepared in the
same manner as in Pharmacological Test 3, in which APCs were
pretreated with a vehicle (DMSO, final concentration: 0.1%)
(V-APC), with both the vehicle (DMSO, final concentration: 0.1%)
and a B16-tumor cell lysate (V-T-APC), with KRN7000 (final
concentration: 100 ng/mL) (KRN-APC), and with both KRN7000 (final
concentration: 100 ng/mL) and a B16-tumor cell lysate (KRN-T-APC),
respectively. Each of the pretreated APCs was injected
intravenously into the mice at a dose of 5.times.10.sup.5
cells/mouse on day 1. As a positive control, KRN7000 was injected
intravenously into the mice at a dose of 100 .mu.g/kg on days 1, 5
and 9. The volume of the subcutaneous tumor in each mouse was
measured. The results (the average of the tumor volumes of 6 mice)
are shown in FIG. 9.
[0124] As shown in FIG. 9, little or no tumor growth-suppressive
effect was observed when V-APCs and V-T-APCs were injected. On the
contrary, when KRN-APCs were injected once, a tumor
growth-inhibitory effect was observed, which was at the same level
as that when KRN7000 was injected 3 times intravenously at a dose
of 100 .mu.g/kg. It is interesting to note that the one injection
of KRN-T-APC produced a more remarkable tumor growth inhibitory
effect than the one injection of KRN-APC or the three intravenous
injections of KRN7000 at a dose of 100 .mu.g/kg.
[Pharmacological Test 9] Effect of KRN7000 on Human Peripheral
Blood-Derived APCs
[0125] A monocyte fraction was prepared from human peripheral blood
mononuclear cells by Percol density-gradient centrifugation. The
monocyte fraction was cultured in the presence or absence of GM-CSF
(50 ng/mL) and IL-4 (100 ng/mL.), and simultaneously either a
vehicle (v; DMSO, 0.1%) or KRN7000 (KRN; 100 ng/mL) was added
thereto. After culturing for 3 days, the cells were collected and
washed with the medium 3 times to give V-APC, KRN-APC, V-APC-GM and
KRN-APC-GM (where "GM" refers to the APCs cultured in the presence
of GM-CSF and IL-4), which were then provided for use as APCs. As
responder cells, human peripheral blood-derived FcR.sup.- cells
were used to perform allogeneic MLR assay.
[0126] As shown in FIG. 10, all of the APCs enhanced the allogeneic
MLR response in a manner dependent on the number of the APCs,
however, KRN-APC and KRN-APC-GM showed a clearly stronger
stimulatory effect on the proliferation of responder cells compared
to V-APC and V-APC-GM, respectively. Furthermore, it was also found
that the effect of KRN-APC-GM was stronger than KRN-APC. It is
assumed that the suppressed proliferation of the responder cells by
the addition of KRN-APC-GM in an amount of 20000 cells/well is due
to the over growth of the responder cells.
[Pharmacological Test 101 Effort of XRN7000 on Human Peripheral
Blood-derived APC
[0127] To study the effect of long-term culture of APC, the
following experiment was performed. A monocyte fraction was
prepared from human peripheral mononuclear cells by Percol
density-gradient centrifugation. The monocyte fraction was cultured
in the presence of GM-CSF (50 ng/mL) and IL-4 (100 ng/mL), and
simultaneously either a vehicle (V; 0.1% DMSO) or KRN7000 (KRN; 100
ng/mL) was added thereto. After culturing for 7 days, the cells
were recovered and washed with the medium 3 times to give V-APC-GM
and KRN-APC-GM, respectively, which were provided for use as APCs.
As responder cells, human peripheral blood-derived FcR.sup.- cells
were used to perform autologous MLR assay.
[0128] As shown in FIG. 11, V-APC-GM did not stimulate the
proliferation of the responder cells, whereas KRN-APC-GM enhanced
the autologous MLR response in a manner dependent on the number of
the APCs.
(Pharmacological Test 11] Effect of KRN7000 on Human Peripheral
Blood-Derived APC
[0129] To study the effect of long-term culture of APC, the
following experiment was performed. A monocyte fraction was
prepared from human peripheral mononuclear cells by Percol
density-gradient centrifugation. The monocyte fraction was cultured
in the presence of GM-CSF (50 ng/mL) and IL-4 (100 ng/mL) with or
without monocyte conditioned medium (MCM) prepared by the method of
Bender et al (Bender A. et al., 1996, J. Immunol. Method., 196,
121). Simultaneously either a vehicle (V; 0.1% DMSO) or KRN7000
(KRN; 100 ng/mL) was added to the fraction. After culturing for 11
days, the cells were recovered and washed with the medium 3 times
to give V-APC-GM (MCM-), KRN-APC-GM (MCM-), V-APC-GM (MC-M+) and
KRN-APC-GM (MCM+), respectively, which were provided for use as
APCs. As responder cells, human peripheral blood-derived FcR.sup.-
cells were used to perform autologous MLR assay.
[0130] As shown in FIG. 12, V-APC-GM (MCM-) did not stimulate the
proliferation of the responder cells, whereas KRN-APC-GM (MCM-)
enhanced the autologous MLR response in a manner dependent on the
number of the APCs. Both V-APC-GM (MCM+) and KRN-APC-GM (MCM+)
enhanced the autologous MLR response in a manner dependent on the
number of the APCs. The effect of KRN-APC-GM (MCM+) was stronger
than that of V-APC-GM (MCM+).
[0131] From the results of Pharmacological Tests 9, 10 and 11
above, it has been found that KRN7000 can activate the APCs from
human peripheral blood by various methods.
[Pharmacological Test 12] Effort of KRN7000 on Human Umbilical Cord
Blood-Derived APC
[0132] A monocyte fraction was prepared from human umbilical cord
blood mononuclear cells by Percol density-gradient centrifugation.
The monocyte fraction was cultured in the presence of GM-CSF (50
ng/mL) and IL-4 (100 ng/mL), and simultaneously either a vehicle
(V; 0.1% DMSO) or KRN7000 (KRN; 100 ng/mL) was added thereto. After
culturing for 3 days, the cells were recovered and washed with the
medium 3 times to give V-APC-GM and KRN-APC-GM, respectively, which
were provided for use as APCs. As response cells, human umbilical
cord blood-derived FcR.sup.- cells were used for autologous MLR
assay.
[0133] As shown in FIG. 13, V-APC-GM did not stimulate the
proliferation of the responder cells, whereas KRN-APC-GM enhanced
the autologous MLR response in a manner dependent on the number of
the APCs. It is assumed that the suppressed proliferation of the
responder cells by the addition of KRN-APC-GM in an amount of
20,000 cells/well is due to the over growth of the responder
cells.
[0134] This result shows that KRN7000 can activate the APCs derived
from human umbilical cord blood by various methods.
(Pharmacological Test 13] Effect of .alpha.-galactosylceramides on
Murine Spleen-Derived APC
[0135] To demonstrate that the .alpha.-galactosylceramide
derivatives of the present invention other than KRN7000 also have
an APC-activating effect, the following pharmacological test was
performed.
[0136] The dendritic cell-rich APCs were prepared from murine
spleen in the same manner as described in Pharmacological Test 3.
Briefly, the APCs were treated with any one of KRN7000 (compound
No.14 of the invention), AGL512 (Compound No.10 of the invention),
AGL525 (Compound No.16 of the invention), AGL506 (Compound No.1 of
the invention), AGL514 (Compound No.2 of the invention), AGL571
(Compound No.5 of the invention) and a vehicle (DMSO, final
concentration: 0.1%) at the final concentration of 100 ng/mL. Each
of the APCs thus treated (1.times.10.sup.4 cells/well) and the
responder cells (2.5.times.10.sup.5 cells/well) were added to wells
of a 96-well plate and then subjected to syngeneic MLR assay. After
culturing for 2 days, .sup.3H-TdR was added to each well (0.5
.mu.Ci/well). After 8 hours, the cells were harvested, and the
amount of the .sup.3H-TdR taken up into the cells was determined
using a liquid scintillation counter. The results are shown in FIG.
14.
[0137] In FIG. 14, "V-APC", "KRN-APC", "512-APC", "525-APC",
"506-APC", "514-APC" and "571-APV" refer to the APCs pretreated
with the vehicle, KRN7000, AGL-512, AGL-525, AGL-506, AGL-514 and
AGL-571, respectively.
[0138] As shown in FIG. 14, the APCs pretreated with
.alpha.-galactosylceramide derivatives showed a remarkable
syngeneic MLR enhancing effect.
[0139] This result shows that the compounds of the present
invention (.alpha.-galactosylceramides and
.alpha.-glucosylceramides) other than KRN7000 also have an effect
of activating the dendritic cell-rich APCs derived from murine
spleen (i.e., an APC function-enhancing effect).
* * * * *