U.S. patent application number 11/894364 was filed with the patent office on 2008-07-17 for reversible inhibitors of s-adenosyl-l-homocysteine hydrolase and uses thereof.
Invention is credited to Chong-Sheng Yuan.
Application Number | 20080171049 11/894364 |
Document ID | / |
Family ID | 33130865 |
Filed Date | 2008-07-17 |
United States Patent
Application |
20080171049 |
Kind Code |
A1 |
Yuan; Chong-Sheng |
July 17, 2008 |
Reversible inhibitors of S-adenosyl-L-homocysteine hydrolase and
uses thereof
Abstract
The present invention provides compositions and methods for
reversibly inhibiting S-adenosyl-L-homocysteine (SAH) hydrolase.
The compounds of the present invention can be used in combination
with an anti-hemorrhagic viral infection agent, an
immunosuppressant, a homocysteine lowering agent, or an
anti-neoplasm agent. The compositions and methods of the present
invention can be used for the prevention and treatment of
hemorrhagic virus infection, autoimmune diseases, autograft
rejection, neoplasm, hyperhomocysteineuria, cardiovascular disease,
stroke, Alzheimer's disease, or diabetes.
Inventors: |
Yuan; Chong-Sheng; (San
Diego, CA) |
Correspondence
Address: |
MORRISON & FOERSTER LLP
12531 HIGH BLUFF DRIVE, SUITE 100
SAN DIEGO
CA
92130-2040
US
|
Family ID: |
33130865 |
Appl. No.: |
11/894364 |
Filed: |
August 20, 2007 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
11649996 |
Jan 5, 2007 |
|
|
|
11894364 |
|
|
|
|
10410879 |
Apr 9, 2003 |
7196093 |
|
|
11649996 |
|
|
|
|
Current U.S.
Class: |
424/141.1 ;
424/130.1; 424/184.1; 514/263.1; 544/264 |
Current CPC
Class: |
A61K 31/52 20130101;
A61P 37/06 20180101; G16B 15/00 20190201; A61P 3/10 20180101; A61P
31/14 20180101; A61P 25/28 20180101; A61K 31/52 20130101; C07D
473/34 20130101; A61P 31/20 20180101; A61P 37/00 20180101; A61K
2300/00 20130101; G16B 20/00 20190201; A61K 45/06 20130101; A61P
35/00 20180101 |
Class at
Publication: |
424/141.1 ;
544/264; 514/263.1; 424/184.1; 424/130.1 |
International
Class: |
A61K 39/395 20060101
A61K039/395; C07D 473/00 20060101 C07D473/00; A61P 35/00 20060101
A61P035/00; A61P 37/00 20060101 A61P037/00; A61K 31/52 20060101
A61K031/52; A61K 39/38 20060101 A61K039/38 |
Claims
1. A compound or a pharmaceutically acceptable salt thereof, having
the formula (I): ##STR00007## wherein Z is selected from the group
consisting of carbon and nitrogen, R1 and R2 are the same or
different, and are selected from the group consisting of hydrogen,
hydroxy, alkyl, cycloalkyl, alkenyl, alkoxy, amino, aryl,
heteroaryl, and halogen; R3 and R4 are the same or different and
are selected from the group consisting of hydrogen, alkyl, acetyl,
alkenyl, aryl, and heteroaryl; X is selected from the group
consisting of oxygen, nitrogen, and sulfur; and Y is selected from
the group consisting of hydrogen, a C.sub.1-10 alkyl group,
alkenyl, vinyl, aryl, and heteroaryl, provided that the compound is
not (4-adenine-9-yl)-2-hydroxybutanoic acid.
2. The compound of claim 1, wherein R1, R2, R3, and R4 are
hydrogen.
3. The compound of claim 1, wherein X is oxygen.
4. The compound of claim 1, wherein Y is a C.sub.1-10 alkyl
group.
5. The compound of claim 1, wherein R1, R2, R3, and R4 are
hydrogen, X is oxygen, and Y is a C.sub.1-10 alkyl group.
6. The compound of claim 1, wherein the .beta. carbon of said
compound has a configuration selected from the group consisting of
S, R, and a racemic mixture thereof.
7. The compound of claim 1, wherein said compound has a K.sub.i
value less than 100 nM for a mammalian S- adenosyl-L-homocysteine
(SAH) hydrolase in a biological medium.
8. The compound of claim 7, wherein said mammal is human.
9. The compound of claim 1, wherein said compound has a K.sub.i
value between about 1 nM and about 100 nm for a mammalian SAH
hydrolase in a biological medium.
10. The compound of claim 9, wherein said mammal is human.
11. A pharmaceutical composition comprising a compound of claim 1
and a pharmaceutically acceptable excipient.
12. The pharmaceutical composition of claim 11, wherein said
compound has a K.sub.i value less than 100 nM for a mammalian SAH
hydrolase in a biological medium.
13. The pharmaceutical composition of claim 12, wherein said mammal
is human.
14. The pharmaceutical composition of claim 11, wherein said
compound has a K.sub.i value between about 1 nM and about 100 nM
for a mammalian SAH hydrolase in a biological medium.
15. The pharmaceutical composition of claim 14, wherein said mammal
is human.
16. The pharmaceutical composition of claim 11, wherein said
composition is formulated for oral, parenteral, intranasal,
topical, or injectable administration.
17. The pharmaceutical composition of claim 11, wherein said
composition is formulated in a solid or liquid dosage form.
18. The pharmaceutical composition of claim 11, wherein said
composition is formulated for oral administration in a dosage
ranging from about 0.1 to about 20 mg/kg per day.
19. The pharmaceutical composition of claim 11, wherein said
composition is formulated for injectable administration in a dosage
ranging from about 0.1 to about 20 mg/kg per day.
20. The pharmaceutical composition of claim 19, wherein said
injectable administration is selected from the group consisting of
intracavernous injection, subcutaneous injection, intravenous
injection, intramuscular injection, and intradermal injection.
21. A kit, comprising an effective amount of said composition of
claim 11, and an instruction means for administering said
composition.
22. A method for reversibly inhibiting activity of a
S-adenosyl-L-homocysteine (SAH) hydrolase in a mammal, comprising
administering to a mammal to which such reversible inhibition is
needed or desirable, an effective amount of a compound or a
pharmaceutically acceptable salt thereof, having the formula (I):
##STR00008## wherein Z is selected from the group consisting of
carbon and nitrogen, R1 and R2 are the same or different, and are
selected from the group consisting of hydrogen, hydroxy, alkyl,
cycloalkyl, alkenyl, alkoxy, amino, aryl, heteroaryl, and halogen;
R3 and R4 are the same or different and are selected from the group
consisting of hydrogen, alkyl, acetyl, alkenyl, aryl, and
heteroaryl; X is selected from the group consisting of oxygen,
nitrogen, and sulfur; and Y is selected from the group consisting
of hydrogen, a C.sub.1-10 alkyl group, alkenyl, vinyl, aryl, and
heteroaryl, thereby reversibly inhibiting the activity of SAH
hydrolase in said mammal.
23. The method of claim 22, wherein said mammal is human.
24. The method of claim 23, wherein said mammal is suspected of
having a disease selected from the group consisting of hemorrhagic
viral infection, autoimmune disease, autograft rejection, neoplasm,
hyperhomocysteineuria, cardiovascular disease, stroke, Alzheimer
disease, and diabetes.
25. The method of claim 24, wherein said hemorrhagic viral
infection is caused by a virus selected from the group consisting
of a Bunyaviridaea, a Filoviridae, a Flaviviridae, and an
Arenaviridae virus.
26. The method of claim 25, wherein said Filoviridae virus is Ebola
virus.
27. The method of claim 22, wherein said administering step
comprises administering an effective amount of said compound in the
treatment of an autoimmune disease in said mammal.
28. The method of claim 22, wherein said administering step
comprises administering an effective amount of said compound in the
treatment of allograft rejection in said mammal.
29. The method of claim 22, wherein said administering step
comprises administering an effective amount of said compound for
lowering homocysteine in said mammal.
30. The method of claim 22, wherein said administering step
comprises administering an effective amount of said compound in the
treatment of a neoplasm in said mammal.
31. The method of claim 30, wherein said neoplasm is selected from
the group consisting of adrenal gland, anus, auditory nerve, bile
ducts, bladder, bone, brain, breast, bruccal, central nervous
system, cervix, colon, ear, endometrium, esophagus, eye, eyelids,
fallopian tube, gastrointestinal tract, head and neck, heart,
kidney, larynx, liver, lung, mandible, mandibular condyle, maxilla,
mouth, nasopharynx, nose, oral cavity, ovary, pancreas, parotid
gland, penis, pinna, pituitary, prostate gland, rectum, retina,
salivary glands, skin, small intestine, spinal cord, stomach,
testes, thyroid, tonsil, urethra, uterus, vagina, vestibulocochlear
nerve, and vulva neoplasm.
32. The method of claim 31, wherein said neoplasm is selected from
the group consisting of breast, ovary, stomach, prostate, colon and
lung cancer.
33. The method of claim 22, wherein said compound or a
pharmaceutically acceptable salt thereof is not
(4-adenine-9-yl)-2-hydroxybutanoic acid.
34. A combination, comprising: a) an effective amount of a compound
or a pharmaceutically acceptable salt thereof, having the formula
(I): ##STR00009## wherein Z is selected from the group consisting
of carbon and nitrogen, R1 and R2 are the same or different, and
are selected from the group consisting of hydrogen, hydroxy, alkyl,
cycloalkyl, alkenyl, alkoxy, amino, aryl, heteroaryl, and halogen;
R3 and R4 are the same or different and are selected from the group
consisting of hydrogen, alkyl, acetyl, alkenyl, aryl, and
heteroaryl; X is selected from the group consisting of oxygen,
nitrogen, and sulfur; and Y is selected from the group consisting
of hydrogen, a C.sub.1-10 alkyl group, alkenyl, vinyl, aryl, and
heteroaryl; and b) an effective amount of a compound selected from
the group consisting of an anti-hemorrhagic viral infection agent,
an immunosuppressant, a homocysteine lowering agent, and an
anti-neoplasm agent.
35. The combination of claim 34, wherein said anti-hemorrhagic
viral infection agent inhibits interleukin-1 (IL-1), tumor necrosis
factor (TNF), or a combination thereof.
36. The combination of claim 34, wherein said anti-hemorrhagic
viral infection agent is selected from the group consisting of an
anti-viral vaccine, an anti-viral antibody, a viral-activated
immune cell, and a viral-activated immune serum.
37. The combination of claim 34, wherein said immunosuppressant is
selected from the group consisting of cyclosporine, tacrolimus, an
adrenocortical steroid, azathioprine, mycophenolate,
cyclophosphamide, methotrexate, chlorambucil, vincristine,
vinblastine, dactinomycin, an antithymocyte globulin, muromonab-CD3
monoclonal antibody, Rh.sub.0(D) immune globulin, methoxsalen, and
thalidomide.
38. The combination of claim 34, wherein said homocysteine lowering
agent is selected from the group consisting of vitamin B.sub.6,
vitamin B.sub.12 and folate.
39. The combination of claim 34, wherein said anti-neoplasm agent
is selected from the group consisting of an anti-angiogenic agent,
an alkylating agent, an antimetabolite, a natural product, a
platinum coordination complex, an anthracenedione, a substituted
urea, a methylhydrazine derivative, an adrenocortical suppressant,
a hormone, an antagonist, an oncogene inhibitor, a tumor suppressor
gene or protein, an anti-oncogene antibody, and an anti-oncogene
antisense oligonucleotide.
40. The combination of claim 34, further comprising a
pharmaceutically acceptable carrier or excipient.
41. The combination of claim 34, wherein said compound or a
pharmaceutically acceptable salt thereof is not
(4-adenine-9-yl)-2-hydroxybutanoic acid.
42. A method for reversibly inhibiting activity of a SAH hydrolase
in a mammal, comprising administering to a mammal to which such
reversible inhibition is needed or desirable, an effective amount
of a combination, wherein the combination comprises: a) an
effective amount of a compound or a pharmaceutically acceptable
salt thereof, having the formula (I): ##STR00010## wherein Z is
selected from the group consisting of carbon and nitrogen, R1 and
R2 are the same or different, and are selected from the group
consisting of hydrogen, hydroxy, alkyl, cycloalkyl, alkenyl,
alkoxy, amino, aryl, heteroaryl, and halogen; R3 and R4 are the
same or different and are selected from the group consisting of
hydrogen, alkyl, acetyl, alkenyl, aryl, and heteroaryl; X is
selected from the group consisting of oxygen, nitrogen, and sulfur;
and Y is selected from the group consisting of hydrogen, a
C.sub.1-10 alkyl group, alkenyl, vinyl, aryl, and heteroaryl; and
b) an effective amount of a compound selected from the group
consisting of an anti-hemorrhagic viral infection agent, an
immunosuppressant, a homocysteine lowering agent, and an
anti-neoplasm agent, thereby reversibly inhibiting said activity of
SAH hydrolase in said mammal.
43. The method of claim 42, wherein said compound or a
pharmaceutically acceptable salt thereof is not
(4-adenine-9-yl)-2-hydroxybutanoic acid.
44. A kit, comprising an effective amount of a combination of claim
34 and an instruction means for administering said combination.
45. A method for identifying a candidate inhibitor compound capable
of inhibiting S-adenosyl-L-homocysteine hydrolase (SAH) activity,
comprising the steps of: a) constructing a computer model of the
SAH binding pocket; b) screening a plurality of compounds having
the structure ##STR00011## wherein Z is selected from the group
consisting of carbon and nitrogen; and c) identifying a compound
that computationally binds to said binding pocket.
46. The method of claim 45, further comprising the step of assaying
said compound to determine the ability of said compound to inhibit
SAH activity.
47. A method for reversibly inhibiting activity of a SAH hydrolase,
comprising contacting a SAH hydrolase with an effective amount of
said compound of claim 1 to reversibly inhibit the activity of said
SAH hydrolase.
48. A method for reversibly inhibiting activity of a SAH hydrolase,
comprising contacting a SAH hydrolase with an effective amount of
said combination of claim 34 to reversibly inhibit the activity of
said SAH hydrolase.
49. The method of claim 22, which is used to inhibit lymphocyte
proliferation, to inhibit production and/or release of IL-12P40,
IL-12P70 and TNF-.alpha. or to inhibit primary antibody production
in the mammal.
50. The method of claim 23, wherein said mammal is suspected of
having a disease selected from the group consisting of inflammatory
Bowel disease, multiple sclerosis and autoimmune neuritis.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] The present application is a continuation of U.S. patent
application Ser. No. 11/649,996, filed Jan. 5, 2007, now pending, a
continuation of U.S. patent application Ser. No. 10/410,879, filed
Apr. 9, 2003, now U.S. Pat. No. 7,196,093. The contents of the
above patent applications are incorporated by reference herein in
their entirety.
BACKGROUND OF THE INVENTION
[0002] SAH hydrolase has been an attractive target for antiviral
drug design based on the observation that many viruses require
5'-capped, methylated structures on their mRNA for efficient
translation of viral proteins. Yuan et al., Exp. Opin. Ther.
Patents, 9: 1197-1206 (1999); Yuan et al., in Adv. Antiviral Drug
Des. vol 2, pp. 41-88, De Clercq (ed), JAI Press, Inc. London, UK
(1996). Inhibition of SAH hydrolase results in inhibition of
S-adenosyl-L-methionine (SAM)-dependent methylation reactions,
including viral mRNA methylation, thus inhibiting viral replication
(Scheme 1).
##STR00001##
Scheme 1. Mechanism of Methylation Based Inhibition of Viral
Replication
[0003] Numerous inhibitors of SAH hydrolase have been identified
from naturally occurring compounds and synthetic compounds. Most
potent inhibitors are irreversible inhibitors, which irreversibly
inactivate SAH hydrolase in a time-dependent fashion. Studies have
demonstrated that irreversible inhibitors only produce narrow
therapeutic windows due to their severe cytotoxic effects (Wolfe
and Borchardt, Journal of Medicinal Chemistry, 34:1521-1530
(1991)). Since SAH hydrolase is a ubiquitous cellular enzyme with a
very slow turnover rate (t.sub.1/2=24 hours in mouse liver),
irreversible inhibitors can cause prolonged inhibition of the
enzyme activity. For instance, it can take up to seven days for
complete recovery of enzyme activity, which can lead to unwanted
side effects. The severe cytotoxicity associated with irreversible
inhibitors has been the major factor that has impaired the
development of these inhibitors into clinically useful drugs.
Because of the cytotoxicity associated with irreversible
inhibitors, reversible inhibitors are preferred.
[0004] However, at present, there are no known reversible SAH
hydrolase inhibitors that are potent enough to produce substantial
inhibitory activity against the enzyme when tested in vivo. For
example, the reversible inhibitor
(S)-9-(2,3-dihydroxypropyl)adenine ((S)-DHPA), which has a K.sub.i
value of 3.5 .mu.M against SAH hydrolase, lacks inhibitory potency.
(Votruba and Holy, Coll. Czech. Chem. Commun., 45:3039 (1980)).
Though (s)-DHPA was reported to be a reversible inhibitor of
isolated AdoHcy hydrolase (Votruba and Holy, Coll. Czech. Chem.
Commun., 45:3039 (1980)), it was also reported to be a irreversible
inhibitor of intracellular AdoHcy hydrolase (Schanche et al.,
Molecular Pharmacology, 26:553-558 (1984)). Thus, existing
reversible inhibitors are not clinically useful therapeutic agents,
and there remains a need for SAH hydrolase inhibitors that exhibit
potency without the undesired cytotoxic effects.
BRIEF SUMMARY OF THE INVENTION
[0005] The present invention provides novel reversible inhibitors
of SAH hydrolase. The compounds of the present invention are useful
as agents demonstrating biological activities related to their
ability to inhibit SAH hydrolase. In one embodiment, the reversible
inhibitors of SAH hydrolase have the formula (I), and
pharmaceutically acceptable salts thereof:
##STR00002##
[0006] wherein Z is carbon or nitrogen, R1 and R2 are the same or
different, and are hydrogen, hydroxy, alkyl, cycloalkyl, alkenyl,
alkoxy, amino, aryl, heteroaryl, or halogen; R3 and R4 are the same
or different and are hydrogen, alkyl, acetyl, alkenyl, aryl, or
heteroaryl; X is oxygen, nitrogen, or sulfur; and Y is hydrogen, a
C.sub.1-10 alkyl group, alkenyl, vinyl, aryl, or heteroaryl. In a
particular embodiment, the compound is not
(4-adenine-9-yl)-2-hydroxybutanoic acid.
[0007] Compounds of formula I can have an S configuration at the
.beta. carbon, an R configuration at the .beta. carbon, or comprise
a racemic mixture. In one embodiment, the compounds have a K.sub.i
value less than 100 nM for a mammalian SAH hydrolase in a
biological medium, e.g., serum. In other embodiments, the compounds
have a K.sub.i value between about 1 nM and about 100 nM for a
mammalian SAH hydrolase in a biological medium. The compounds
preferably have a K.sub.i value less than 100 nM, or a K.sub.i
value between about 1 nM and about 100 nM for a human SAH hydrolase
in a biological medium.
[0008] Compounds of formula I can have substituents wherein R1, R2,
R3, and R4 are hydrogen. In one aspect of the present invention, X
is oxygen. In another aspect of the present invention, Y is
hydrogen or a C.sub.1-10 alkyl group. In yet another aspect of the
present invention, R1, R2, R3, and R4 are hydrogen, X is oxygen,
and Y is hydrogen or a C.sub.1-10 alkyl group.
[0009] The present invention also relates to a pharmaceutical
composition comprising an effective amount of a compound of formula
I or pharmaceutically acceptable salts thereof, and a
pharmaceutically acceptable carrier or diluent. Pharmaceutical
compositions may be administered by oral, parenteral (e.g.,
intramuscular, intraperitoneal, intravenous, intracisternal
injection or infusion, subcutaneous injection, or implant),
inhalation spray, nasal, vaginal, rectal, sublingual, or topical
routes of administration. The pharmaceutical compositions may be
formulated in suitable dosage unit formulations appropriate for
each route of administration.
[0010] It is not intended that the present invention be limited to
particular formulations or particular modes of administration. In
one embodiment, the composition is formulated for oral, parenteral,
intranasal, topical, or injectable administration. Non-limited
examples of injectable administration are intracavernous injection,
subcutaneous injection, intravenous injection, intramuscular
injection and intradermal injection. The pharmaceutical composition
can be formulated for oral administration in a dosage ranging from
about 0.1 to about 20 mg/kg per day. The pharmaceutical composition
can also be formulated for injectable administration in a dosage
ranging from about 0.1 to about 20 mg/kg per day.
[0011] Pharmaceutical compositions of the present invention can be
formulated in a solid or liquid dosage form. For example, the
pharmaceutical compositions may be formulated as a solid in the
form of tablets, capsules, granules, powders, and similar
compounds. The pharmaceutical compositions may also be formulated
as a liquid in the form of syrups, injection mixtures, and the
like.
[0012] The present invention also provides a kit comprising an
effective amount of the composition of the present invention, and
an instruction means for administering the composition.
[0013] Furthermore, the present invention provides methods for
reversibly inhibiting the activity of a S-adenyl-L-homocysteine
(SAH) hydrolase. In one embodiment, the present invention provides
a method for reversibly inhibiting activity of a
S-adenosyl-L-homocysteine (SAH) hydrolase in a mammal, comprising
administering to a mammal to which such reversible inhibition is
needed or desirable, an effective amount of a compound or a
pharmaceutically acceptable salt thereof, having the formula
(I):
##STR00003##
[0014] wherein Z is selected from the group consisting of carbon
and nitrogen, R1 and R2 are the same or different, and are selected
from the group consisting of hydrogen, hydroxy, alkyl, cycloalkyl,
alkenyl, alkoxy, amino, aryl, heteroaryl, and halogen; R3 and R4
are the same or different and are selected from the group
consisting of hydrogen, alkyl, acetyl, alkenyl, aryl, and
heteroaryl; X is selected from the group consisting of oxygen,
nitrogen, and sulfur; and Y is selected from the group consisting
of hydrogen, a C.sub.1-10 alkyl group, alkenyl, vinyl, aryl, and
heteroaryl, thereby reversibly inhibiting the activity of SAH
hydrolase in said mammal. In a particular embodiment, the
administered compound or a pharmaceutically acceptable derivative
thereof is not (4-adenine-9-yl)-2-hydroxybutanoic acid.
[0015] In preferred embodiments, the mammal is suspected of having
a disease selected from the group consisting of hemorrhagic viral
infection, autoimmune disease, autograft rejection, neoplasm,
hyperhomocysteineuria, cardiovascular disease, stroke, Alzheimer's
disease, diabetes, inflammatory Bowel disease, multiple sclerosis
and autoimmune neuritis. However, it is not intended that the
present invention be limited to the prevention and treatment of
particular diseases.
[0016] It is an object of the present invention to provide methods
for preventing and treating hemorrhagic viral infections. In one
aspect, the method comprises administering an effective amount of
compounds having formula I in the treatment of hemorrhagic viral
infections in a mammal. In particular embodiments, the hemorrhagic
viral infection is caused by a virus selected from the group
consisting of a Bunyaviridaea, a Filoviridae, a Flaviviridae, and
an Arenaviridae virus. In other particular embodiments, the
Filoviridae virus is Ebola virus.
[0017] It is also an object of the present invention to provide
methods for preventing and treating autoimmune diseases. In one
aspect, the method comprises administering an effective amount of
compounds having formula I in the treatment of an autoimmune
disease in a mammal.
[0018] It is also an object of the present invention to provide
methods for preventing and treating allograft rejection. In one
aspect, the method comprises administering an effective amount of
compounds having formula I in the treatment of allograft rejection
in a mammal.
[0019] Furthermore, it is an object of the present invention to
provide methods for preventing or treating hyperhomocysteineuria,
or for lowering plasma homocysteine in a mammal. In one aspect, the
method comprises administering an effective amount of compounds
having formula I for lowering plasma homocysteine in a mammal.
[0020] Further, it is an object of the present invention to provide
methods for preventing or treating neoplasm. In one aspect, the
method comprises administering an effective amount of compounds
having formula I for in the treatment of neoplasm in a mammal.
Non-limiting examples of neoplasm are neoplasm of the adrenal
gland, anus, auditory nerve, bile ducts, bladder, bone, brain,
breast, bruccal, central nervous system, cervix, colon, ear,
endometrium, esophagus, eye, eyelids, fallopian tube,
gastrointestinal tract, head and neck, heart, kidney, larynx,
liver, lung, mandible, mandibular condyle, maxilla, mouth,
nasopharynx, nose, oral cavity, ovary, pancreas, parotid gland,
penis, pinna, pituitary, prostate gland, rectum, retina, salivary
glands, skin, small intestine, spinal cord, stomach, testes,
thyroid, tonsil, urethra, uterus, vagina, vestibulocochlear nerve,
and the vulva.
[0021] The present invention also provides a combination,
comprising an effective amount of a compound having formula I, and
an effective amount of an anti-hemorrhagic viral infection agent,
an immunosuppressant, a plasma homocysteine lowering agent, and an
anti-neoplasm agent. The combination can further comprise a
pharmaceutically acceptable carrier or excipient. In a particular
embodiment, the combination does not include
(4-adenine-9-yl)-2-hydroxybutanoic acid.
[0022] In a particular embodiment, the anti-hemorrhagic viral
infection agent inhibits interleukin-1 (IL-1), tumor necrosis
factor (TNF), or a combination thereof. The anti-hemorrhagic viral
infection agent can be an anti-viral vaccine, an anti-viral
antibody, a viral-activated immune cell, or a viral-activated
immune serum.
[0023] In another embodiment, the immunosuppressant is
cyclosporine, tacrolimus, an adrenocortical steroid, azathioprine,
mycophenolate, cyclophosphamide, methotrexate, chlorambucil,
vincristine, vinblastine, dactinomycin, an antithymocyte globulin,
muromonab-CD3 monoclonal antibody, Rh.sub.0(D) immunoglobulin,
methoxsalen, or thalidomide.
[0024] In other particular embodiments, the homocysteine lowering
agent is vitamin B.sub.6, vitamin B.sub.12, or folate.
[0025] In yet other embodiments, the anti-neoplasm agent is an
anti-angiogenic agent, an alkylating agent, an antimetabolite, a
natural product, a platinum coordination complex, an
anthracenedione, a substituted urea, a methylhydrazine derivative,
an adrenocortical suppressant, a hormone, an antagonist, an
oncogene inhibitor, a tumor suppressor gene or protein, an
anti-oncogene antibody, or an anti-oncogene antisense
oligonucleotide.
[0026] The present invention also provides a kit comprising an
effective amount of the combination of the present invention, and
an instruction means for administering the combination.
[0027] Furthermore, the present invention provides a method for
reversibly inhibiting activity of a SAH hydrolase in a mammal,
comprising administering to a mammal to which such reversible
inhibition is needed or desirable, an effective amount of a
combination, wherein the combination comprises: a) an effective
amount of a compound or a pharmaceutically acceptable salt thereof,
having the formula (I):
##STR00004##
[0028] wherein Z is selected from the group consisting of carbon
and nitrogen, R1 and R2 are the same or different, and are selected
from the group consisting of hydrogen, hydroxy, alkyl, cycloalkyl,
alkenyl, alkoxy, amino, aryl, heteroaryl, and halogen; R3 and R4
are the same or different and are selected from the group
consisting of hydrogen, alkyl, acetyl, alkenyl, aryl, and
heteroaryl; X is selected from the group consisting of oxygen,
nitrogen, and sulfur; and Y is selected from the group consisting
of hydrogen, a C.sub.1-10 alkyl group, alkenyl, vinyl, aryl, and
heteroaryl; and b) an effective amount of a compound selected from
the group consisting of an anti-hemorrhagic viral infection agent,
an immunosuppressant, a homocysteine lowering agent, and an
anti-neoplasm agent, thereby reversibly inhibiting said activity of
SAH hydrolase in said mammal. In a particular embodiment, the
administered combination does not include
(4-adenine-9-yl)-2-hydroxybutanoic acid.
[0029] The combination can be used with any other pharmaceutical
composition to modulate SAH hydrolase activity in a mammal. The
combination can also be used in the prevention and treatment of
diseases such as hemorrhagic viral infection, autoimmune disease,
autograft rejection, neoplasm, and hyperhomocysteineuria,
cardiovascular disease, stroke, Alzheimer's disease, diabetes,
inflammatory Bowel disease, multiple sclerosis or autoimmune
neuritis, as described above. However, it is not intended that the
combination be limited to the prevention and uses of particular
diseases.
BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING(S)
[0030] FIG. 1 illustrates effects of DZ2002 on Quantitative
hemolysis of Sheep Red Blood Cells (QHS) assay. Data were expressed
as means.+-.SD. **: P<0.01 compared with control.
[0031] FIG. 2 illustrates that DZ2002 suppresses T cell
proliferation in mixed lymphocyte reaction. Data were expressed as
means.+-.SD. ***: P<0.001 compared with control.
[0032] FIG. 3 illustrates that DZ2002 have no cytotoxicity in
spleen cell.
[0033] FIG. 4 illustrates effects of DZ2002 on DTH ear swelling in
Balb/c mice.
[0034] FIG. 5 illustrates effects of DZ2002 on TNF-.alpha.
production from TG induced peritoneal cells.
[0035] FIG. 6A illustrates effects of DZ2002 on the expression of
MHC-II on THP-1 cells; FIG. 6B illustrates effects of DZ2002 on the
expression of CD80 on THP-1 cells; and FIG. 6C illustrates effects
of DZ2002 on the expression of CD86 on THP-1 cells.
[0036] FIGS. 7A and 7B illustrate effects of DZ2002 on IL-12P40 and
IL-12P70 production from THP-1 cells.
DETAILED DESCRIPTION OF THE INVENTION
[0037] For clarity of disclosure, and not by way of limitation, the
detailed description of the invention is divided into the
subsections that follow.
A. Definitions
[0038] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as is commonly understood by one
of ordinary skill in the art to which this invention belongs. All
patents, applications, published applications and other
publications referred to herein are incorporated by reference in
their entirety. If a definition set forth in this section is
contrary to or otherwise inconsistent with a definition set forth
in the patents, applications, published applications and other
publications that are herein incorporated by reference, the
definition set forth in this section prevails over the definition
that is incorporated herein by reference.
[0039] As used herein, "a" or "an" means "at least one" or "one or
more."
[0040] As used herein, a "composition" refers to any mixture of two
or more products or compounds. It may be a solution, a suspension,
liquid, powder, a paste, aqueous, non-aqueous, or any combination
thereof.
[0041] As used herein, a "combination" refers to any association
between two or among more items.
[0042] As used herein, "homocysteine" (Hcy) refers to a compound
with the following molecular formula:
HSCH.sub.2CH.sub.2CH(NH.sub.2)COOH. Biologically, Hcy is produced
by demethylation of methionine and is an intermediate in the
biosynthesis of cysteine from methionine. The term "Hcy"
encompasses free Hcy (in the reduced form) and conjugated Hcy (in
the oxidized form). Hcy can conjugate with proteins, peptides,
itself or other thiols through a disulfide bond.
[0043] As used herein, "SAH hydrolase" refers to an enzyme which
catalyzes hydrolysis of SAH to adenosine (Ado) and Hcy. The enzyme
is an ubiquitous eukaryotic enzyme, which is also found in some
prokaryotes. SAH hydrolase also catalyzes the formation of SAH from
Ado and Hcy. The co-enzyme of SAH hydrolase is NAD.sup.+/NADH. SAH
hydrolase may have several catalytic activities. In the hydrolytic
direction, the first step involves oxidation of the 3'-hydroxyl
group of SAH (3'-oxidative activity) by enzyme-bound NAD.sup.+
(E-NAD.sup.+), followed by .beta.-elimination of L-Hcy to give
3'-keto-4',5'-didehydro-5'-deoxy-Ado. Michael addition of water to
the 5'-position to this tightly bound intermediate (5'-hydrolytic
activity) affords 3'-keto-Ado, which is then reduced by
enzyme-bound NADH (E-NADH) to Ado (3'-reduction activity). It is
intended to encompass SAH hydrolase with conservative amino acid
substitutions that do not substantially alter its activity.
[0044] As used herein, the terms "pharmaceutically acceptable
salts" or "pharmaceutically acceptable derivatives" of the
compounds of the present invention encompass any salts, esters or
derivatives that may be readily prepared by those of skill in this
art. Pharmaceutically acceptable salts of the compounds of this
invention include, for example, those derived from pharmaceutically
acceptable inorganic and organic acids and bases. Salts derived
from appropriate bases include, but are not limited to, alkali
metal (e.g., sodium), alkaline earth metal (e.g., magnesium),
ammonium and N(C.sub.1-4 alkyl).sub.4.sup.+ salts. Examples of
suitable acids include, but are not limited to, hydrochloric,
hydrobromic, sulfuric, nitric, perchloric, fumaric, maleic,
phosphoric, glycolic, lactic, salicylic, succinic,
toluene-p-sulfonic, tartaric, acetic, citric, methanesulfonic,
formic, benzoic, malonic, naphthalene-2-sulfonic, and
benzenesulfonic acids. Other acids, such as oxalic, while not in
themselves pharmaceutically acceptable, may be employed in the
preparation of salts useful as intermediates in obtaining the
compounds of the invention and their pharmaceutically acceptable
acid salts.
[0045] As used herein, "biological activity" refers to the in vivo
activities of a compound or physiological responses that result
upon in vivo administration of a compound, composition, or other
mixture. Biological activity, thus, encompasses therapeutic effects
and pharmaceutical activity of such compounds, compositions and
mixtures. Biological activities may be observed in in vitro systems
designed to test or use such activities.
[0046] As used herein, "plasma" refers to the fluid, noncellular
portion of the blood, distinguished from the serum obtained after
coagulation.
[0047] As used herein, "serum" refers to the fluid portion of the
blood obtained after removal of the fibrin clot and blood cells,
distinguished from the plasma in circulating blood.
[0048] As used herein, "fluid" refers to any composition that can
flow. Fluids thus encompass compositions that are in the form of
semi-solids, pastes, solutions, aqueous mixtures, gels, lotions,
creams, and other such compositions.
[0049] As used herein, the abbreviations for any protective groups,
amino acids and other compounds, are in accord with their common
usage, recognized abbreviations, or the IUPAC-IUB Commission on
Biochemical Nomenclature, unless otherwise indicated (see
Biochemistry 11: 1726 (1972)).
[0050] As used herein, "disease or disorder" refers to a
pathological condition in an organism, which is characterizable by
identifiable symptoms.
[0051] As used herein, the term "a therapeutic agent" refers to any
conventional drug or drug therapies which are known to those
skilled in the art, including, but not limited to vaccines.
[0052] As used herein, "vaccine" refers to any compositions
intended for active immunological prophylaxis. A vaccine may be
used therapeutically to treat a disease, to prevent development of
a disease, or to decrease the severity of a disease either
proactively or after infection. Exemplary vaccines include, but are
not limited to, preparations of killed microbes of virulent
strains, living microbes of attenuated (variant or mutant) strains,
or microbial, fungal, plant, protozoa, or metazoa derivatives or
products. The term also encompasses protein/peptide and nucleotide
based vaccines.
[0053] As used herein, the term "therapeutically effective amount"
refers to that amount that is sufficient to ameliorate, or in some
manner reduce the symptoms associated with the disease. Such amount
may be administered as a single dosage or according to a regimen.
Repeated administration may be required to achieve the desired
amelioration of symptoms.
[0054] As used herein, the terms "administration" or
"administering" a compound refers to any suitable method of
providing a compound of the invention or a pro-drug of a compound
of the invention to a subject.
[0055] As used herein, the term "treatment" refers to any manner in
which the symptoms of a condition, disorder or disease are
ameliorated or otherwise beneficially altered. Treatment also
encompasses any pharmaceutical use of the compositions herein.
Amelioration of symptoms of a particular disorder refers to any
lessening of symptoms, whether permanent or temporary, that can be
attributed to or associated with administration of the
composition.
[0056] As used herein, the term "substitute" refers to the
replacement of a hydrogen atom in a compound with a substituent
group.
[0057] As used herein, the term "alkyl" encompasses straight or
branched alkyl groups, including alkyl groups that are optionally
substituted with one or more substituents. For example, the alkyl
group can be optionally substituted with hydroxy, halogen, aryl,
alkoxy, acyl, or other substituents known in the art. One of more
carbon atoms of the alkyl group can also be optionally replaced by
one or more heteroatoms.
[0058] As used herein, the term "K.sub.i" refers to a numerical
measure of the effectiveness of a compound in inhibiting the
activity of a target enzyme such as ICE. Lower values to K.sub.i
reflect higher effectiveness. The K.sub.i value is derived by
fitting experimentally determined rate data to standard enzyme
kinetic equations (Segel, Enzyme Kinetics, Wiley-Interscience,
1975).
[0059] As used herein, "an anti-neoplastic treatment" refers to any
treatment designed to treat the neoplasm, tumor or cancer by
lessening or ameliorating its symptoms. Treatments that prevent the
occurrence or lessen the severity of neoplasm, tumor or cancer are
also contemplated.
[0060] As used herein, "neoplasm (neoplasia)" refers to abnormal
new growth, and thus means the same as tumor, which may be benign
or malignant. Unlike hyperplasia, neoplastic proliferation persists
even in the absence of the original stimulus.
[0061] As used herein, "an anti-neoplasm agent (used
interchangeably with anti-neoplastic agent, anti-tumor or
anti-cancer agent)" refers to any agents used in the anti-neoplasm
treatment. These include any agents, that when used alone or in
combination with other compounds, can alleviate, reduce,
ameliorate, prevent, place or maintain in a state of remission
clinical symptoms or diagnostic markers associated with neoplasm,
tumor or cancer. The anti-neoplasm agent that can be used in the
combinations of the present invention include, but are not limited
to, anti-angiogenic agents, alkylating agents, antimetabolite,
certain natural products, platinum coordination complexes,
anthracenediones, substituted ureas, methylhydrazine derivatives,
adrenocortical suppressants, certain hormones and antagonists,
anti-cancer polysaccharides, and certain herb extracts such as
Chinese herb extracts.
[0062] As used herein, "tumor suppressor gene" (also referred to as
anti-oncogene or cancer susceptibility gene) refers to a gene that
encodes a product which normally negatively regulates the cell
cycle, and which must be mutated or otherwise inactivated before a
cell can proceed to rapid division. Exemplary tumor suppressor
genes include, but are not limited to, p16, p21, p53, RB
(retinoblastoma), WT-1 (Wilm's tumor), DCC (deleted in colonic
carcinoma), NF-1 (neurofibrosarcoma) and APC (adenomatous
polypospis coli).
[0063] As used herein, "oncogene" refers to a mutated and/or
overexpressed version of a normal gene of animal cells (the
proto-oncogene) that in a dominant fashion can release the cell
from normal restraints on growth. Thus, an oncogene alone, or in
concert with other changes, converts a cell into a tumor cell.
Exemplary oncogenes include, but are not limited to, abl, erbA,
erbB, ets, fes (fps), fgr, fms, fos, hst, int1, int2, jun, hit,
lym, mas, met, mil (raf), mos, myb, myc, N-myc, neu (ErbB2), ral
(mil), Ha-ras, Ki-ras, N-ras, rel, ros, sis, src, ski, trk and
yes.
[0064] As used herein, "antisense polynucleotides" refer to
synthetic sequences of nucleotide bases complementary to mRNA or
the sense strand of double stranded DNA. Admixture of sense and
antisense polynucleotides under appropriate conditions leads to the
binding of the two molecules, or hybridization. When these
polynucleotides bind to (hybridize with) mRNA, inhibition of
protein synthesis (translation) occurs. When these polynucleotides
bind to double stranded DNA, inhibition of RNA synthesis
(transcription) occurs. The resulting inhibition of translation
and/or transcription leads to an inhibition of the synthesis of the
protein encoded by the sense strand.
[0065] As used herein, "antibody" includes antibody fragments, such
as Fab fragments, which are composed of a light chain and the
variable region of a heavy chain.
[0066] As used herein, "humanized antibodies" refers to antibodies
that are modified to include "human" sequences of amino acids so
that administration to a human will not provoke an immune response.
Methods for preparing such antibodies are known. For example, the
hybridoma that expresses the monoclonal antibody is altered by
recombinant DNA techniques to express an antibody in which the
amino acid composition of the non-variable regions is based on
human antibodies. Computer programs have been designed to identify
such regions.
[0067] As used herein, "anti-hemorrhagic virus agent" or
"anti-viral hemorrhagic agent" refer to any agent used in the
treatment of hemorrhagic viral infections. These include any
agents, alone or in combination with other compounds, that can
alleviate, reduce, ameliorate, prevent, or maintain in a place of
remission clinical symptoms or diagnostic markers associated with
viral hemorrhagic diseases, or disorders. Non-limiting examples of
antiviral-hemorrhagic agents include interleukin-1 (IL-1)
inhibitors, tumor necrosis factor (TNF) inhibitors, anti-viral
vaccines, anti-viral antibodies, viral-activated immune cells, and
viral-activated immune sera.
[0068] As used herein, "an anti-hemorrhagic virus treatment" refers
to any treatment designed to treat hemorrhagic viral infections by
lessening or ameliorating the symptoms. Treatments that prevent the
infection or lessen its severity are also contemplated.
[0069] As used herein, "IL-1 inhibitor" encompasses any substances
that prevent or decrease production, post-translational
modifications, maturation, or release of IL-1, or any substances
that interfere with or decrease the efficacy of the interaction
between IL-1 and IL-1 receptor. Preferably, the IL-1 inhibitor is
an anti-IL-1 antibody, an anti-IL-1 receptor antibody, an IL-1
receptor antagonist, an IL-1 production inhibitor, an IL-1 receptor
production inhibitor, or an IL-1 releasing inhibitor.
[0070] As used herein, "tumor necrosis factor" ("TNF") refers to a
group of proinflammatory cytokines encoded within the major
histocompatibility complex. The TNF family members include TNF
.alpha. and TNFR (also known as cachectin and lymphotoxin,
respectively). Complementary cDNA clones encoding TNA .alpha. and
TNFR have been isolated. Thus, reference to "TNF" encompasses all
proteins encoded by the TNF gene family, including TNF .alpha. and
TNF, or an equivalent molecule obtained from any other source or
that has been prepared synthetically. It is intended to encompass
TNF with conservative amino acid substitutions that do not
substantially alter its activity.
[0071] As used herein, "TNF inhibitor" encompasses any substances
that prevent or decrease production, post-translational
modifications, maturation, or release of TNF, or any substances
that interfere with or decrease the efficacy of the interaction
between TNF and TNF receptor. Preferably, the TNF inhibitor is an
anti-TNF antibody, an anti-TNF receptor antibody, a TNF receptor
antagonist, a TNF production inhibitor, a TNF receptor production
inhibitor, or a TNF releasing inhibitor.
B. Reversible Inhibitors of S-Adenosyl-L-Homocysteine Hydrolase
[0072] One approach for minimizing mechanism-based cytotoxicity is
to optimize the pharmacokinetic profiles of SAH hydrolase
inhibitors, such that the inhibitors exhibit reversible inhibiting
activity. Pharmacokinetic profiles can be optimized by optimizing
K.sub.Off values. For example, K.sub.Off values are optimized such
that they are small enough to produce desired antiviral effects,
but large enough to allow adequate recovery of the enzyme activity
before the next dose.
Eritadenine Derivatives as Reversible Inhibitors of SAH
Hydrolase
[0073] The present invention relates to novel inhibitors of
S-adenosyl-L-homocysteine compositions that are reversible and
potent. For example, the present invention provides compounds with
a K.sub.i value of less than 100 nM. In one embodiment, the present
invention provides 4(adenine-9-yl)-2-hydroxybutanoic acid, its
derivatives, and pharmaceutically acceptable salts thereof, and
methods for reversibly inhibiting SAH hydrolase using such
compounds.
[0074] The reversible inhibitor, 4(adenine-9-yl)-2-hydroxybutanoic
acid is synthesized from deoxyl modification of eritadenine at the
beta carbon. Eritadenine is a naturally occurring compound and a
potent irreversible inhibitor of SAH hydrolase. Deoxyl modification
of eritadenine at the beta carbon results in a compound that is a
reversible inhibitor, while retaining inhibitory potency.
Derivatives of 4(adenine-9-yl)-2-hydroxybutanoic acid can be
synthesized using conventional synthetic methods known to one of
ordinary skill in the art. (See e.g., Yuan et al., Adv. Antiviral
Drug Des. 2: 41-88 (1996); Holy et al., Coll. Czechoslovak Chem.
Commun. 50: 245-279 (1985)).
[0075] Examples of 4(adenine-9-yl)-2-hydroxybutanoic acids
derivatives include, but are not limited to, base-modified
derivatives, and side-chain substituted derivatives. Base modified
derivatives are derivatives of 4(adenine-9-yl)-2-hydroxybutanoic
acids with modifications at the adenyl ring base. The adenyl ring
can be modified with various modifying groups at the amino group.
The adenyl ring can also be modified with various substituents at
the C2 and C8 positions of the adenyl ring.
[0076] In one embodiment, the reversible inhibitors of SAH
hydrolase have the following formula (I), and pharmaceutically
acceptable salts thereof:
##STR00005##
wherein Z is carbon or nitrogen, R1 and R2 are the same or
different, and are hydrogen, hydroxy, alkyl, cycloalkyl, alkenyl,
alkoxy, amino, aryl, heteroaryl, or halogen; R3 and R4 are the same
or different and are hydrogen, alkyl, acetyl, alkenyl, aryl, or
heteroaryl; X is oxygen, nitrogen, or sulfur; and Y is hydrogen, a
C.sub.1-10 alkyl group, alkenyl, vinyl, aryl, or heteroaryl.
[0077] The different R groups can be optionally substituted with
other substituents. These substituents may be halogen, hydroxy,
alkoxy, nitro, cyano, carboxylic acid, alkyl, alkenyl, cycloalkyl,
thiol, amino, acyl, carboxylate, aryl, carbamate, carboxamide,
sulfonamide, a heterocyclic group, or any appropriate substituent
known in the art. In a particular embodiment, each R group is
hydrogen, or a lower straight chain alkyl such as methyl. In
another embodiment, one or more carbon atoms in the alkyl or alkoxy
groups may be replaced by one or more heteroatoms.
[0078] The amino group may also be substituted once or twice to
form a secondary or tertiary amine. Non-limiting examples of
substituents include alkyls or an optionally substituted alkyl
group; alkene or an optionally substituted alkenyl group;
cycloalkyl or an optionally substituted cycloalkyl group; aryl,
heterocyclic; aralkyl (e.g. phenyl C.sub.1-4 alkyl); heteroalkyl
such as phenyl, pyridine, phenylmethyl, phenethyl, pyridinylmethyl,
pyridinylethyl; and other substituents. The heterocyclic group may
be a 5 or 6 membered ring containing 1-4 heteroatoms.
[0079] The amino group may be substituted with an optionally
substituted C.sub.2-4 alkanoyl (e.g. acetyl, propionyl, butyryl,
isobutyryl etc.); a C.sub.1-4 alkylsulfonyl (e.g. methanesulfonyl,
ethanesulfonyl, etc.); a carbonyl or sulfonyl substituted aromatic
or heterocyclic ring (e.g. benzenesulfonyl, benzoyl,
pyridinesulfonyl, pyridinecarbonyl etc.).
[0080] The CO--X--Y group can be an optionally substituted
carboxylate group. Examples of the optionally substituted
carboxylate group include, but are not limited to, an optionally
substituted alkyl (e.g. C.sub.1-10 alkyl); an optionally
substituted cycloalkyl (e.g. C.sub.3-7 cycloalkyl); an optionally
substituted alkenyl (e.g. C.sub.2-10 alkenyl); an optionally
substituted cycloalkenyl (e.g. C.sub.3-7 cycloalkenyl); an
optionally substituted aryl (e.g. phenyl, naphthyl, C.sub.1-4 aryl
such as benzyl); and other appropriate substituents. Groups such as
methoxymethyl, methoxyethyl, and related groups are also
encompassed.
Structure-Based Drug Design of Novel SAH Hydrolase Inhibitors
[0081] It is also an object of the present invention to provide
structure-based drug design using the compounds of the present
invention as an initial template molecule. Recently, X-ray
structures of SAH hydrolase have become available for both "open"
and "closed" forms of the enzyme. Using structure-based design, one
of ordinary skill in the art can design novel compounds for
screening SAH hydrolase inhibitors. The design or selection of
candidate compounds can begin with the selection of various
moieties which fill binding pockets of the SAH hydrolase. (See
e.g., U.S. Pat. No. 5,756,466; Klebe, J. Mol. Med. 78: 69-281
(2000); and Maignan et al., Curr. Top. Med. Chem. 1: 161-174
(2001)).
[0082] There are a number of ways to select moieties to fill
individual binding pockets. These include visual inspection of a
physical model or computer model of the active site and manual
docking of models of selected moieties into various binding
pockets. Modeling software that is well known and available in the
art can be used. These include, but are not limited to, QUANTA
(Molecular Simulations, Inc., Burlington, Mass., 1992); SYBYL
(Molecular Modeling Software, Tripos Associates, Inc., St. Louis,
Mo., 1992); AMBER (Weiner et al., J. Am. Chem. Soc. 6: 765-784
(1984)); CHARMM (Brooks et al., J. Comp. Chem. 4: 187-217 (1983)).
The modeling step can be followed by energy minimization with
standard molecular mechanics forcefields such as CHARMM and AMBER.
In addition, there are a number of more specialized computer
programs to assist in the process of selecting the binding moieties
of this invention. These include, but are not limited to:
[0083] 1. GRID (Goodford, "A Computational Procedure for
Determining Energetically Favorable Binding Sites on Biologically
Important Macromolecules," J. Med. Chem. 28: 849-857 (1985)). GRID
is available from Oxford University, Oxford, UK.
[0084] 2. MCSS (Miranker et al., "Functionality Maps of Binding
Sites: A Multiple Copy Simultaneous Search Method," in Proteins:
Structure, Function and Genetics" 11: 29-34 (1991)). MCSS is
available from Molecular Simulations, Burlington, Mass.
[0085] 3. AUTODOCK (Goodsell et al., "Automated Docking of
Substrates to Proteins by Simulated Annealing," in PROTEINS:
Structure, Function and Genetics 8: 195-202 (1990)). AUTODOCK is
available from the Scripps Research Institute, La Jolla, Calif.
[0086] 4. DOCK (Kuntz et al., "A Geometric Approach to
Macromolecule-Ligand Interactions," J. Mol. Biol. 161: 269-288
(1982)). DOCK is available from the University of California, San
Francisco, Calif.
[0087] Once suitable binding moieties have been selected, they can
be assembled into a single inhibitor. This assembly may be
accomplished by connecting the various moieties to a central
scaffold. The assembly process may, for example, be done by visual
inspection followed by manual model building, again using software
such as QUANTA or SYBYL. A number of other programs may also be
used to help select ways to connect the various moieties. These
include, but are not limited to:
[0088] 1. CAVEAT (Bartlett et al., "CAVEAT: A Program to Facilitate
the Structure-Derived Design of Biologically Active Molecules," in
Molecular Recognition in Chemical and Biological Problems, Special
Pub., Royal Chem. Soc. 78: 182-196 (1989)). CAVEAT is available
from the University of California, Berkeley, Calif.
[0089] 2. 3D Database systems such as MACCS-3D (MDL Information
Systems, San Leandro, Calif.). This area has been recently reviewed
by Martin (Martin, "3D Database Searching in Drug Design," J. Med.
Chem. 35: 2145-2154 (1992)).
[0090] 3. HOOK (available from Molecular Simulations, Burlington,
Mass.)
[0091] In addition to the above computer assisted modeling of
inhibitor compounds, the inhibitors of this invention may be
constructed de novo using either an empty active site or optionally
including some portions of a known inhibitor. Such methods are well
known in the art. They include, for example:
[0092] 1. LUDI (Bohm, "The Computer Program LUDI: A New Method for
the De Novo Design of Enzyme Inhibitors," J. Comp. Aid. Molec.
Design 6: 61-78 (1992)). LUDI is available from Biosym
Technologies, San Diego, Calif.
[0093] 2. LEGEND (Nishibata et al., Tetrahedron, 47: 8985 (1991)).
LEGEND is available from Molecular Simulations, Burlington,
Mass.
[0094] 3. LeapFrog (available from Tripos associates, St. Louis,
Mo.).
[0095] A number of techniques commonly used for modeling drugs may
be employed (see e.g., Cohen et al., J. Med. Chem. 33: 883-894
(1990)). Likewise a number of examples in the chemical literature
of techniques can be applied to specific drug design projects. (For
a review, see, Navia et al., Curr. Opin. Struc. Biol. 2: 202-210
(1991)). Using the novel combination of steps of the present
invention, the skilled artisan can advantageously avoid time
consuming and expensive experimentation to determine enzymatic
inhibition activity of particular compounds. The method is also
useful in facilitating rational design of SAH hydrolase inhibitors,
and therapeutic and prophylactic agents against SAH
hydrolase-mediated diseases. Accordingly, the present invention
relates to such inhibitors, and methods for identifying or
selecting such inhibitors.
[0096] A variety of conventional techniques may be used to carry
out each of the above evaluations, as well as evaluations necessary
in screening a candidate compound for SAH hydrolase inhibiting
activity. Generally, these techniques involve determining the
location and binding proximity of a given moiety, the occupied
space of a bound inhibitor, the deformation energy of binding of a
given compound and electrostatic interaction energies. Examples of
conventional techniques useful in the above evaluations include,
but are not limited to, quantum mechanics, molecular mechanics,
molecular dynamics, Monte Carlo sampling, systematic searches and
distance geometry methods (Marshall, Ann. Ref. Pharmacol. Toxicol.
27: 193 (1987)). Specific computer software has been developed for
use in carrying out these methods. Examples of programs designed
for such uses include: Gaussian 92 (Gaussian, Inc., Pittsburgh,
Pa.); AMBER; QUANTA/CHARMM; and Insight II/Discover (Biosysm
Technologies Inc., San Diego, Calif.). These programs may be
implemented, for instance, using a Silicon Graphics Indigo2
workstation or IBM RISC/6000 workstation model 550. Other hardware
systems and software packages will be known and be of evident
applicability to those skilled in the art.
[0097] Different classes of active SAH hydrolase inhibitors,
according to this invention, may interact in similar ways with the
various binding pockets of the SAH hydrolase active site. The
spatial arrangement of these important groups is often referred to
as a pharmacophore. The concept of the pharmacophore has been well
described in the literature (See Mayer et al., J. Comp. Aided
Molec. Design 1: 3-16 (1987); Hopfinger et al. in Concepts and
Applications of Molecular Similarity, Johnson and Maggiora (eds.),
Wiley (1990))
[0098] Different classes of SAH hydrolase inhibitors of this
invention may also use different scaffolds or core structures that
allow the necessary moieties to be placed in the active site such
that the specific interactions necessary for binding may be
obtained. These compounds are best defined in terms of their
ability to match the pharmacophore, i.e., their structural identity
relative to the shape and properties of the active site of SAH
hydrolase. Various scaffolds have been described in, for example,
Klebe, G., J. Mol. Med. 78: 269-281 (2000); Maignan et al., Curr.
Top. Med. Chem. 1: 161-174 (2001); and U.S. Pat. No. 5,756,466 to
Bemis et al.).
S-Adenosyl-L-Homocysteine Hydrolase to be Inhibited
[0099] The compounds of the present invention can be used to
reversibly inhibit any SAH hydrolase. It is not intended that the
present invention be limited to reversibly inhibiting any specific
SAH hydrolase.
[0100] In one embodiment, the compounds of the present invention
can be used to reversibly inhibit SAH hydrolase encoded by nucleic
acids containing nucleotide sequences with the following GenBank
accession Nos.: AF129871 (Gossypium hirsutum); AQ003753
(Cryptosporidium parvum); AF105295 (Alexandrium fundyense);
AA955402 (Rattus norvegicus); AA900229 (Rattus norvegicus);
AA874914 (Rattus norvegicus); AA695679 (Drosophila melanogaster
ovary); AA803942 (Drosophila melanogaster ovary; AI187655 (Manduca
sexta male antennae); U40872 (Trichomonas vaginalis); AJ007835
(Xenopus Laevis); AF080546 (Anopheles gambiae); AI069796 (T. cruzi
epimastigote); Z97059 (Arabidopsis thaliana); AF059581 (Arabidopsis
thaliana); U82761 (Homo sapiens); AA754430 (Oryza sativa); D49804
(Nicotiana tabacum); D45204 (Nicotiana tabacum); X95636 (D.
melanogaster); T18277 (endosperm Zea mays); R75259 (Mouse brain);
Z26881 (C. roseus); X12523 (D. discoideum); X64391 (Streptomyces
fradiae); W21772 (Maize Leaf); AH003443 (Rattus norvegicus); U14963
(Rattus norvegicus); U14962 (Rattus norvegicus); U14961 (Rattus
norvegicus); U14960 (Rattus norvegicus); U14959 (Rattus
norvegicus); U14937 (Rattus norvegicus); U14988 (Rattus
norvegicus); U14987 (Rattus norvegicus); U14986 (Rattus
norvegicus); U14985 (Rattus norvegicus); U14984 (Rattus
norvegicus); U14983 (Rattus norvegicus); U14982 (Rattus
norvegicus); U14981 (Rattus norvegicus); U14980 (Rattus
norvegicus); U14979 (Rattus norvegicus); U14978 (Rattus
norvegicus); U14977 (Rattus norvegicus); U14976 (Rattus
norvegicus); U14975 (Rattus norvegicus); L32836 (Mus musculus);
L35559 (Xenopus laevis); Z19779 (Human foetal Adrenals tissue);
L23836 (Rhodobacter capsulatus); M15185 (Rat); L11872 (Triticum
aestivum); M19937 (Slime mold (D. discoideum); M80630 (Rhodobacter
capsulatus).
[0101] In another embodiment, the compounds of the present
invention can be used to reversibly inhibit SAH hydrolase encoded
by nucleic acids containing nucleotide sequences with the GenBank
accession Nos. M61831-61832 (see also Coulter-Karis and Hershfield,
Ann. Hum. Genet., 53(2):169-175 (1989)). The compounds of the
present invention can also be used to reversibly inhibit SAH
hydrolase encoded by nucleic acids containing the nucleotide or
amino acid sequences set forth in U.S. Pat. No. 5,854,023.
C. Use as Therapeutic Agents
[0102] Reversible inhibition of SAH hydrolase using
4(adenine-9-yl)-2-hydroxybutanoic acid, its derivatives, and
pharmaceutically acceptable salts results in significantly reduced
cytotoxicity while retaining its therapeutic effects. With its
potency and reversibility, the compounds of the present invention
can be used as therapeutic agents without the severe toxicity
associated with other irreversible inhibitors. The compounds of the
present invention are useful as agents demonstrating biological
activities related to their ability to inhibit SAH hydrolase. The
inhibitory effect on SAH hydrolase can be evaluated using the ratio
of the initial rates of SAH hydrolysis in the presence or absence
of the inhibitor, or using any methods known to one of ordinary
skill in the art. The present invention provides compositions and
methods for the prevention and treatment of diseases such as
hemorrhagic viral infection, autoimmune disease, autograft
rejection, neoplasm, hyperhomocysteineuria, cardiovascular disease,
stroke, Alzheimer's disease, and diabetes. However, it is not
intended that the present invention be limited to the prevention
and treatment of particular diseases.
[0103] 1. Hemorrhagic Fever Viruses
[0104] The present invention provides compositions and methods for
the treatment of viral hemorrhagic fever. The reversible inhibitors
of the present invention can serve as a broad-spectrum antiviral
agent against all types of viruses causing hemorrhagic fever,
including, but not limited to, togavirus, arenavirus, nairovirus,
and hantavirus. Broad-spectrum antiviral drugs offer many
advantages over narrow-spectrum agents. Because of the difficulty
associated with clinical diagnoses of viral pathogens, diagnostic
results often arrive too late for the choice of a specific
antiviral drug. Immediate action is often necessary to prevent the
condition of the patient from worsening, particularly in acute
infections where viral chemotherapy must start as soon as the
patient presents clinical symptoms.
[0105] Inhibitors of S-adenosyl-L-homocysteine (SAH) hydrolase have
been reported to be effective in the treatment of Ebola viral
infections. The compounds of the present invention can also be used
against other hemorrhagic diseases, such as those described in WO
00/64479. Although the mechanism of inhibition is not necessary in
practicing the methods of the present invention, the mechanism of
action by which the compounds of the present invention inhibit
viral replication may be based on inhibition of viral
methylation.
[0106] 2. Autoimmune Diseases and Diseases Associated With
Immunosuppression
[0107] The present invention contemplates compositions and methods
for preventing and treating autoimmune diseases. If a person has an
autoimmune disease, the immune system mistakenly attacks the cells,
tissues, and organs of a person's own body. As a group, autoimmune
diseases afflict millions of Americans. Most autoimmune diseases
strike women more often than men. Examples of autoimmune diseases
can be found from the National Institute of Health, "Understanding
Autoimmune Disease"
(http://www.niaid.nih.gov/publications/autoimmune/autoimmune.htm.).
[0108] Compounds that modulate SAH hydrolase activity may also be
used for the treatment of diseases that are associated with
immunosuppression. Immunosuppression can be due to chemotherapy,
radiation therapy, enhanced wound healing, enhanced burn treatment,
or other drug therapy such as corticosteroid therapy, or a
combination of drugs used in the treatment of autoimmune diseases
and graft/transplantation rejection. Immunosuppression can also be
due to congenital deficiency in receptor function, infectious
diseases, parasitic diseases, or other causes.
[0109] 3. Neoplasm and Cancer
[0110] The present invention also contemplates compositions and
methods for preventing and treating neoplasms, including, but not
limited to neoplasm associated with the adrenal gland, anus,
auditory nerve, bile ducts, bladder, bone, brain, breast, bruccal,
central nervous system, cervix, colon, ear, endometrium, esophagus,
eye, eyelids, fallopian tube, gastrointestinal tract, head, neck,
heart, kidney, larynx, liver, lung, mandible, mandibular condyle,
maxilla, mouth, nasopharynx, nose, oral cavity, ovary, pancreas,
parotid gland, penis, pinna, pituitary, prostate gland, rectum,
retina, salivary glands, skin, small intestine, spinal cord,
stomach, testes, thyroid, tonsil, urethra, uterus, vagina,
vestibulocochlear nerve, vulva, and neoplasm associated with other
organs. In particular embodiments, the pharmaceutical compositions
of the present invention are useful for the treatment of non-small
cell lung cancer, lung cancer, breast cancer, and prostate cancer.
The present invention further contemplates compositions and methods
for preventing and treating cancers, including, but not limited to
those associated with solid tumors, lymphoma, metastatic tumors,
glioblastoma tumors, and other carcinomas tumors.
[0111] 4. Diseases Associated With Increased Homocysteine
Levels
[0112] Furthermore, it is contemplated that the compounds of the
present invention can be used as a plasma homocysteine lowering
agent for the prevention and treatment of diseases associated with
increased levels of homocysteine. Diseases which have been found to
be linked with increased homocysteine levels (i.e.,
hyperhomocysteinemia) include, but are not limited to
cardiovascular diseases, stroke, Alzheimer's disease and diabetes.
For example, various studies have shown a relation between
hyperhomocysteinemia and coronary heart disease (CHD), peripheral
vascular disease, stroke, and venous thrombosis.
[0113] The increased risk of stroke from high homocysteine levels
also increase the chance of developing Alzheimer's disease. Recent
studies have also shown that people with dementia of the
Alzheimer's type have elevated levels of homocysteine in their
blood. (Selhub et al., "Plasma homocysteine as a risk factor for
dementia and Alzheimer's disease," N. Eng. J. Med. 46: 476-483
(2002)). Elevated homocysteine has also been linked to
complications in diabetes, lupus, and other chronic diseases.
D. Pharmaceutical Compositions
[0114] Pharmaceutical compositions of the present invention
comprise any of the compounds of the present invention and
pharmaceutically acceptable salts thereof, alone or in combination
with any pharmaceutically acceptable carriers, adjuvant or vehicle.
Acceptable compositions and methods for their administration that
can be employed for use in this invention include, but are not
limited to those described in U.S. Pat. Nos. 5,736,154; 6,197,801;
5,741,511; 5,886,039; 5,941,868; 6,258,374 and 5,686,102. Examples
of pharmaceutically acceptable carriers, adjuvants and vehicles
that can be used in the pharmaceutical compositions of this
invention include, but are not limited to, ion exchangers, alumina,
aluminum stearate, lecithin, serum proteins, such as human serum
albumin, buffer substances such as phosphates, glycine, sorbic
acid, potassium sorbate, partial glyceride mixtures of saturated
vegetable fatty acids, water, salts or electrolytes such as
protamine sulfate, disodium hydrogen phosphate, potassium hydrogen
phosphate, sodium chloride, zinc salts, colloidal silica, magnesium
trisilicate, polyvinyl pyrrolidone, cellulose-based substances,
polyethylene glycol, sodium carboxymethylcellulose, polyacrylates,
waxes, polyethylene-polyoxypropylene-block polymers, polyethylene
glycol and wool fat.
[0115] The formulation, dosage and route of administration can be
determined according to methods known in the art (see e.g.,
Remington: The Science and Practice of Pharmacy, Alfonso R. Gennaro
(Editor) Mack Publishing Company, April 1997; Therapeutic Peptides
and Proteins: Formulation, Processing, and Delivery Systems, Banga,
1999; and Pharmaceutical Formulation Development of Peptides and
Proteins, Hovgaard and Frkjr (Ed.), Taylor & Francis, Inc.,
2000; Biopharmaceutical Drug Design and Development, Wu-Pong and
Rojanasakul (Ed.), Humana Press, 1999). In the treatment or
prevention of conditions which require SAH hydrolase modulation, an
appropriate dosage level will generally be about 0.01 to 500 mg per
kg body weight per day. Preferably, the dosage level will be about
0.1 to about 250 mg/kg per day. In more preferred embodiments, the
dosage level will range from about 0.1 to about 20 mg/kg per day.
The appropriate dosage can be administered in single or multiple
dose. It will be understood that the specific dose level and
frequency of dosage for any particular subject may be varied and
will depend upon a variety of factors, including the activity of
the specific compound used, the metabolic stability and length of
action of that compound, the age, body weight, general health, sex,
diet, mode and time of administration, rate of excretion, drug
combination, the severity of the particular condition, and the
patient undergoing therapy.
[0116] The pharmaceutical compositions of this invention can be
administered orally, parenterally, by inhalation spray, topically,
rectally, nasally, buccally, vaginally, via an implanted reservoir,
or any suitable form of administration. The term parenteral as used
herein includes subcutaneous, intracutaneous, intravenous,
intramuscular, intra-articular, intrasynovial, intrasternal,
intrathecal, intralesional and intracranial injection or infusion
techniques. The most suitable route in any given case will depend
on the nature and severity of the condition being treated and on
the nature of SAH hydrolase inhibitor being used.
[0117] The pharmaceutical compositions may be in the form of a
sterile injectable preparation, for example, as a sterile
injectable aqueous or oleaginous suspension. This suspension may be
formulated according to techniques known in the art using suitable
dispersing or wetting agents (e.g., Tween 80), and suspending
agents. The sterile injectable preparation may also be a sterile
injectable solution or suspension in a non-toxic
parenterally-acceptable diluent or solvent. For example, the
pharmaceutical composition may be a solution in 1,3-butanediol.
Other examples of acceptable vehicles and solvents that may be
employed in the compositions of the present invention include, but
are not limited to, mannitol, water, Ringer's solution and isotonic
sodium chloride solution. In addition, sterile, fixed oils are
conventionally employed as a solvent or suspending medium. For this
purpose, any bland fixed oil may be employed including synthetic
mono- or diglycerides. Fatty acids, such as oleic acid and its
glyceride derivatives are useful in the preparation of injectables,
as are natural pharmaceutically-acceptable oils, such as olive oil
or castor oil, especially in their polyoxyethylated versions. These
oil solutions or suspensions may also contain a long-chain alcohol
diluent or dispersant.
[0118] The pharmaceutical compositions of this invention may be
orally administered in any orally acceptable dosage form including,
but not limited to, capsules, tablets, and aqueous suspensions and
solutions. In the case of tablets for oral use, commonly used
carriers include, but are not limited to, lactose and corn starch.
Lubricating agents, such as magnesium stearate, can also be added.
For oral administration in a capsule form, useful diluents include
lactose and dried corn starch. When aqueous suspensions are
administered orally, the active ingredient is combined with
emulsifying and suspending agents. If desired, certain sweetening,
flavoring, and coloring agents may be added.
[0119] The pharmaceutical compositions of this invention may also
be administered in the form of suppositories for rectal
administration. These compositions can be prepared by mixing a
compound of this invention with a suitable non-irritating
excipient. In particular embodiments, the excipient is solid at
room temperature but liquid at the rectal temperature. Thus, the
excipient will melt in the rectum to release the active components.
Such materials include, but are not limited to, cocoa butter,
beeswax and polyethylene glycols.
[0120] The pharmaceutical compositions of this invention may be
administered by nasal aerosol or inhalation. Such compositions are
prepared according to techniques well-known in the art of
pharmaceutical formulation. For example, such composition may be
prepared as solutions in saline, employing benzyl alcohol or other
suitable preservatives, absorption promoters to enhance
bioavailability, fluorocarbons, and/or other solubilizing or
dispersing agents known in the art.
[0121] The pharmaceutical compositions of this invention may also
be administered topically. For topical application to the skin, the
pharmaceutical composition may be formulated with a suitable
ointment containing the active components suspended or dissolved in
a carrier. Carriers for topical administration of the compounds of
this invention include, but are not limited to, mineral oil, liquid
petroleum, white petroleum, propylene glycol, polyoxyethylene
polyoxypropylene compound, emulsifying wax and water.
Alternatively, the pharmaceutical composition can be formulated
with a suitable lotion or cream containing the active compound
suspended or dissolved in a carrier. Suitable carriers include, but
are not limited to, mineral oil, sorbitan monostearate, polysorbate
60, cetyl esters wax, cetaryl alcohol, 2-octyldodecanol, benzyl
alcohol and water. The pharmaceutical compositions of this
invention may also be topically applied to the lower intestinal
tract by rectal suppository formulation or in a suitable enema
formulation. Topically-transdermal patches are also included in
this invention.
[0122] The invention also provides kits for carrying out the
therapeutic regimens of the invention. Such kits comprise
therapeutically effective amounts of an SAH hydrolase inhibitor,
alone or in combination with other agents, in pharmaceutically
acceptable form. Preferred pharmaceutical forms include inhibitors
in combination with sterile saline, dextrose solution, buffered
solution, or other pharmaceutically acceptable sterile fluid.
Alternatively, the composition may be lyophilized or desiccated. In
this instance, the kit may further comprise a pharmaceutically
acceptable solution, preferably sterile, to form a solution for
injection purposes. In another embodiment, the kit may further
comprise a needle or syringe, preferably packaged in sterile form,
for injecting the composition. In other embodiments, the kit
further comprises an instruction means for administering the
composition to a subject. The instruction means can be a written
insert, an audiotape, an audiovisual tape, or any other means of
instructing the administration of the composition to a subject.
E. Combinations for Reversibly Inhibiting SAH Hydrolase
Activity
[0123] The present invention also provides combinations and kits
for reversibly inhibiting SAH hydrolase activity. In one
embodiment, the present invention provides a combination,
comprising an effective amount of a compound having formula I; and
an effective amount of an anti-hemorrhagic viral infection agent,
an immunosuppressant, a plasma homocysteine lowering agent, or an
anti-neoplasm agent. The combination can further comprise a
pharmaceutically acceptable carrier or excipient. In yet another
aspect, the present invention provides a kit, comprising an
effective amount of the combination as described, and an
instruction means for administering the combination to a
subject.
[0124] Any agent that can alleviate or ameliorate clinical symptoms
or diagnostic markers associated with viral hemorrhagic diseases
can be used in the combination of the present invention. Anti-viral
therapeutic agents include, but are not limited to, anti-viral
vaccines, anti-viral antibiotics, viral-activated immune cells and
viral-activated immune sera. WO 00/64479 describes examples of
anti-viral therapeutic agents that can be used in the combination
of the present invention. Preferred embodiments are antiviral
therapeutic agents that exhibit biological activity against viral
hemorrhagic diseases caused by infection of a Bunyaviridaea, a
Filoviridae, a Flaviviridae, or an Arenaviridae virus.
[0125] Any agent that suppresses the ability of the body's immune
system to fight disease can be used in the combination of the
present invention. Non-limiting examples of immunosuppressants are
cyclosporine, prednisilone, azathioprine, tacrolimus, an
adrenocortical steroid, mycophenolate, cyclophosphamide,
methotrexate, chlorambucil, vincristine, vinblastine, dactinomycin,
an antithymocyte globulin, muromonab-CD3 monoclonal antibody,
Rh.sub.0(D) immune globulin, methoxsalen, and thalidomide (See,
Goodman & Gilman's The Pharmacological Basis of Therapeutics,
(9th Ed.) McGraw-Hill 1996, pages 1294-1304). The immunosuppressant
can be taken as a combination of drugs. For example, most people
start on a combination of drugs (e.g., cyclosporin, azathioprine,
and prednisilone combination) after their transplant. Over a period
of time, the doses of each drug and the number of drugs taken may
be reduced as the risks of rejection decline.
[0126] Any agent that lowers homocysteine levels can be used in the
combination of the present invention. Folic acid is known to be an
effective homocysteine-lowering agent. Other homocysteine-lowering
agents include, but are not limited to, betaine, trimethylglycin,
cyanobalamin, and other group vitamins. The combination can also
include any multi-vitamin and mineral supplement for use in
lowering homocysteine. Examples of multivitamin and mineral
supplements that can be used in the combinations of the present
invention, include, but are not limited to, those described in U.S.
Pat. Nos. 6,361,800; 6,353,003; 6,323,188; 6,274,170; 6,210,686;
6,203,818; and 5,668,173.
[0127] Any anti-neoplasm agent can be used in the combination of
the present invention. Examples of anti-neoplasm agents that can be
used in the compositions and methods of the present invention are
described in U.S. Patent Application No. 2002/044919. In one
embodiment, the anti-neoplasm agent used is an anti-angiogenic
agent. The anti-angiogenic agent can be an inhibitor of basement
membrane degradation, an inhibitor of cell migration, an inhibitor
of endothelial cell proliferation, and an inhibitor of
three-dimensional organization and establishment of potency.
Examples of such anti-angiogenic agent are illustrated in Auerbach
and Auerbach, Pharmacol. Ther., 63: 265-311 (1994); O'Reilly,
Investigational New Drugs, 15: 5-13 (1997); J. Nat'l Cancer
Instit., 88: 786-788 (1996); and U.S. Pat. Nos. 5,593,990;
5,629,327 and 5,712,291. In another embodiment, the anti-neoplasm
agent used is an alkylating agent, an antimetabolite, a natural
product, a platinum coordination complex, an anthracenedione, a
substituted urea, a methylhydrazine derivative, an adrenocortical
suppressant, a hormone, and an antagonist.
[0128] Other anti-neoplasm agents include, but are not limited to,
cytidine, arabinosyladenine (araC), daunomycin, doxorubicin,
methotrexate (MTX), fluorinated pyrimidines such as 5-fluorouracil
(5U), hydroxyurea, 6-mercaptopurine, plant alkaloids such as
vincristine (VCR), VP-16 and vinblastine (VLB), alkylating agent,
cisplatin, nitrogen Mustard, trisamine, procarbazine, bleomycin,
mitomycin C, actinomycin D, or an enzyme such as L-Asparaginase.
The anti-neoplasm agent can also be an oncogene inhibitor such as
an anti-oncogene antibody or an anti-oncogene antisense
oligonucleotide. In another embodiment, the anti-neoplastic agent
is a cellular matrix inhibitor such as an anti-cellular-matrix
antibody or an anti-cellular-matrix antisense oligonucleotide. For
example, antibodies and antisense oligonucleotides against
caveolin-1, decorin, cadherins, catenins, integrins, and other
cellular matrix or cellular matrix genes can be used.
[0129] In a specific embodiment, the combination further comprises
a tumor suppressor gene for combined intratumoral therapy and gene
therapy. The gene can be used in the form of naked DNA, complexed
DNA, cDNA, plasmid DNA, RNA or other mixtures thereof as components
of the gene delivery system. In another embodiment, the tumor
suppressor gene is included in a viral vector. Any viral vectors
that are suitable for gene therapy can used in the combination. For
example, an adenovirus vector (U.S. Pat. No. 5,869,305), a simian
virus vector (U.S. Pat. No. 5,962,274), a conditionally replicating
human immunodeficiency viral vector (U.S. Pat. No. 5,888,767),
retrovirus, SV40, Herpes simplex viral amplicon vectors and
vaccinia virus vectors can be used. In addition, the genes can be
delivered in a non-viral vector system such as a liposome wherein
the lipid protects the DNA or other biomaterials from oxidation
during the coagulation.
F. EXAMPLES
Example 1
Synthesis of Reversible Inhibitors of SAH Hydrolase
[0130] .sup.1H (Me.sub.4Si) NMR spectra were determined with
solution in CDCl.sub.3 at 400 MHz, .sup.13C (Me.sub.4Si) at 100.6
MHz unless otherwise noted. Mass spectra (MS) were obtained by
atmospheric pressure chemical ionization (APCI) technique. Reagent
grade chemicals were used. Solvents were dried by reflux over and
distillation from CaH.sub.2 under an argon atmosphere, except THF,
which was distilled from benzophenone and potassium. TLC was
performed on Merck kieselgel 60-F.sub.254 with MeOH/CHCl.sub.3
(1:9) and EtOAc/MeOH (95:5) as developing systems, and products
were detected with 254 nm light. Merck kieselgel 60 (230-400 mesh)
was used for column chromatography.
[0131] Elemental analyses were determined by Galbraith
Laboratories, Knoxville, Tenn. Spectral data for isolated compounds
were consistent with reported data. (Holy, Coll. Czech. Chem.
Commun. 43, 3444-3464 (1978); Holy et al., Coll. Czech. Chem.
Commun. 50: 262-279 (1985); Japanese Patent 69-50781; Chem. Abstr.
1972: 514811). The syntheses are schematically shown in Scheme
2.
9-(3,4-O-Isopropylidene-3,4-dihydroxybutyl)adenine (1)
[0132] .sup.1H NMR .delta. 1.36 (s, 3, CH.sub.3), 1.45 (s, 3,
CH.sub.3), 2.00-2.05 (m, 1, H2'), 2.22-2.24 (m, 1, H2''), 3.57-3.59
(m, 1, H3'), 4.03-4.06 (m, 2, H4',4''), 4.34-4.43 (m, 2, H1',1''),
5.76 (br s, 2, NH.sub.2), 7.86 (s, 1, H8), 8.38 (s, 1, H2); MS
(APCI) m/z 264 (100, MH.sup.+).
9-(3,4-Dihydroxybutyl)adenine (2)
[0133] A solution of 1 (110 mg, 0.18 mmol) in CF.sub.3COOH/H.sub.2O
(9:1) (5 ml) was stirred for 20 min at .about.0.degree. C.
Volatiles were evaporated, coevaporated with toluene (3.times.) and
EtOH (2.times.) to give 2 (73 mg, 78%) after crystallization from
EtOH with spectra data as reported.
9-(4-O-Butyldimethylsilyl-3,4-dihydroxybutyl)adenine (3)
[0134] TBDMS-Cl (186 mg, 1.23 mmol) and imidazole (168 mg, 2.46
mmol) were added to a stirred solution of 2 (250 mg, 1.12 mmol) in
dry DMF (8 mL). The mixture was stirred at ambient temperature for
5 h, then reaction mixture was partitioned between
EtOAc/NH.sub.4Cl/H.sub.2O. The water layer was extracted with next
portion of EtOAc. The combined organic phase was washed (brine),
dried (Na.sub.2SO.sub.4), evaporated and the residue was column
chromatographed (CHCl.sub.3/MeOH; 97:3) to give 3 (234 mg, 62%):
.sup.1H NMR .delta. 0.04 (s, 6, 2.times.CH.sub.3), 0.88 (s, 9,
t-Bu), 1.81-1.89 (m, 1, H2'), 2.03-2.11 (m, 1, H2''), 3.50-3.52 (m,
2, H4',4''), 3.58-3.59 (m, 1, H3'), 4.32-4.45 (m, 2, H1',1''), 6.10
(br s, 2, NH.sub.2), 7.87 (s, 1, H8), 8.36 (s, 1, H2); .sup.13C NMR
.delta. -5.0 & -4.9 (233 CH.sub.3), 18.7 (t-Bu), 26.3 (t-Bu),
33.7 (C2'), 40.9 (C1'), 67.4 (C4'), 68.4 (C3'), 119.9 (C5), 141.4
(C8), 150.5 (C4), 153.1 (C2), 155.8 (C6); MS (APCI) m/z 338 (100,
MH.sup.+). Anal. Calcd for C.sub.15H.sub.27N.sub.5O.sub.2Si
(337.50): C, 53.38; H, 8.06; N, 20.75.
9-[4-O-Butyldimethylsilyl-3-O-(1-ethoxyethyl)-3,4-dihydroxybutyl]adenine
(4)
[0135] Ethyl vinyl ether (214 mg, 0.28 mL, 2.96 mmol) and
pyridinium p-toluenesulfonate (15 mg, mmol) were added to a
solution of 3 (250 mg, 0.74 mmol) in dry CH.sub.2Cl.sub.2 (30 mL),
and the mixture was stirred at ambient temperature under N.sub.2
until no starting material was detected by TLC (usually 5-6 days).
Then reaction mixture was washed with water, dried
(Na.sub.2SO.sub.4), and was evaporated. Column chromatography
(EtOAc/MeOH; 97:3) gave 4 (160 mg, 53%) as .about.1:1 mixture of
diastereoisomers: .sup.1H NMR .delta. 0.04 (s, 6,
2.times.CH.sub.3), 0.88 (s, 9, t-Bu), 1.18-1.34 (complex m, 6,
2.times.CH.sub.3), 2.05-2.21 (m, 2, H2',2''), 3.50-3.63 (complex m,
4, H4',4'', CH.sub.2), 3.70-3.82 (m, 1, H3'), 4.31-4.40 (m, 2,
H1',1''), 4.72-4.78 & 4.88-4.93 (2.times.m, 1, CH), 5.99 &
6.06 (2.times.br s, 2, NH.sub.2), 7.86 & 8.04 (2.times.s, 1,
H8), 8.37 (s, 1, H2); .sup.13C NMR .delta. -5.1 (2.times.CH.sub.3),
15.7 & 15.9 (CH.sub.3), 18.6 (t-Bu), 20.7 & 21.0
(CH.sub.3), 26.2 (t-Bu), 32.5 & 32.7 (C2'), 41.0 & 41.1
(C1'), 61.0 & 62.1 (CH.sub.2), 65.5 & 66.1 (C4'), 73.6
& 74.8 (C3'), 100.2 & 100.5 (CH), 119.9 (C5), 141.1 &
142.2 (C8), 150.5 (C4), 152.1 & 152.5 (C2), 155.3 (C6); MS
(APCI) m/z 410 (100, MH.sup.+). Anal. Calcd for
C.sub.19H.sub.35N.sub.5O.sub.3Si (409.60): C, 55.71; H, 8.61; N,
17.10.
9-[3-O-(1-Ethoxyethyl)-3,4-dihydroxybutyl]adenine (5)--Procedure
A
[0136] TBAF/THF (0.88 mL, 1M) was added to a solution of 4 (180 mg,
0.44 mmol) in dry THF (6 mL) and the mixture was stirred at ambient
temperature for 20 min. Volatiles were evaporated and the residue
was column chromatographed (EtOAc/MeOH; 78:12) to give 5 (120 mg,
92%) as .about.1:1 mixture of diastereoisomers: .sup.1H NMR .delta.
1.21-1.25 (complex m, 3, CH.sub.3), 1.35 & 1.39 (d, J=5.3 Hz,
3, CH.sub.3), 2.05-2.25 (m, 2, H2',2''), 3.53-3.82 (complex m, 5,
H3',4',4'', CH.sub.2), 4.32-4.39 (m, 2, H1',1''), 4.69 &
4.88-4.87 (q, J=5.3 Hz, 1, CH), 5.88 & 5.92 (2.times.br s, 2,
NH.sub.2), 7.82 & 7.94 (2.times.s, 1, H8), 8.36 (s, 1, H2);
.sup.13C NMR .delta. 15.5 & 15.7 (CH.sub.3), 20.4 & 20.6
(CH.sub.3), 32.5 & 32.7 (C2'), 40.0 & 41.1 (C1'), 60.9
& 62.4 (CH.sub.2), 64.9 & 65.9 (C4'), 73.2 & 78.9
(C3'), 99.6 & 101.5 (CH), 119.7 (C5), 140.8 & 141.5 (C8),
150.5 (C4), 152.8 & 153.1 (C2), 155.6 & 155.7 (C6); MS
(APCI) m/z 296 (100, MH.sup.+). Anal. Calcd for
C.sub.13H.sub.21N.sub.5O.sub.3 (295.34): C, 52.87; H, 7.17; N,
23.71.
Methyl 4-(Adenin-9-yl)-2-hydroxybutanoate (6)--Procedure B
[0137] To a suspension of 5 (90 mg, 0.31 mmol) in
CH.sub.3CN/CCl.sub.4/H.sub.2O (1:1:1.5; 1.5 mL), NaHCO.sub.3 (161
mg, 0.88 mmol), NaIO.sub.4 (353 mg, 1.65 mmol) and RuCl.sub.3
(trace) were added. The mixture was stirred at ambient temperature
for 48 h until no starting material was detected on TLC. Then water
(5 mL) and CHCl.sub.3 (4 mL) were added, the two layers were
separated and water phase was washed with CHCl.sub.3 (3 mL). The
aqueous layer was acidified with HCl to pH .about.4 and applied on
a column of Dowex 50W.times.2 (H.sup.+). Column was washed with 200
mL of water then product was eluted with 2.5% NH.sub.4OH/H.sub.2O.
The combined UV-absorbing ammonia eluate was evaporated and
coevaporated with MeOH (2.times.). The residue was dissolved in
MeOH (5 mL) and a solution of CH.sub.2N.sub.2 in diethyl ether was
added until yellow color of diazomethane was maintained during
several minutes. The solution was concentrated and column
chromatographed (CHCl.sub.3/MeOH; 95:5) to give 6 (31 mg, 41%) as a
white solid with data identical as reported. To avoid formation of
by-product 6' it is imported to keep desired amount of NaHCO.sub.3
in reaction mixture.
4-(Adenin-9-yl)-2-hydroxybutanoic acid (7)--Procedure C
[0138] NaOH/H.sub.2O (1 mL. 0.1 M) was added to a solution of 6 (10
mg, 0.04 mmol) in MeOH/H.sub.2O (2.0 mL). The mixture was stirred
at ambient temperature for 6 h until no starting material was
detected on TLC. Then reaction mixture was acidified with HCl to pH
.about.4 and applied on a column of Dowex 50W.times.2 (H.sup.+).
Column was washed with water (100 mL) and then product was eluted
with 2.5% NH.sub.4OH. The combined UV-absorbing ammonia eluate was
evaporated to give 7 as a ammonium salt (7.6 mg, 75%) with data as
reported.
9-(3,4-O-Di-t-Butyldimethylsilyl-3,4-dihydroxybutyl)adenine (8)
[0139] TBDMS-Cl (593 mg, 3.92 mmol) and imidazole (534 mg, 7.85
mmol) were added to a stirred solution of 2 (350 mg, 1.57 mmol) in
dry DMF (8 mL), and the mixture was stirred at ambient temperature
overnight. Then reaction mixture was partitioned between
EtOAc/NH.sub.4Cl/H.sub.2O. The water layer was extracted with
EtOAc. The combined organic phase was washed (brine), dried
(Na.sub.2SO.sub.4), and evaporated. Column chromatography
(CHCl.sub.3.fwdarw.3% MeOH/CHCl.sub.3) gave 8 (610 mg, 86%):
.sup.1H NMR .delta. 0.04 (s, 6, 2.times.CH.sub.3), 0.09 (s, 6,
2.times.CH.sub.3), 0.88 (s, 9, t-Bu), 0.92 (s, 9, t-Bu), 2.02-2.07
(m, 1, H2'), 2.21-2.26 (m, 1, H2''), 3.46 (dd, J=6.8, 10.0 Hz, 1,
H4'), 3.59 (dd, J=5.2, 10.0 Hz, 1, H4''), 3.78-3.81 (m, 1, H3'),
4.32-4.45 (m, 2, H1',1''), 5.96 (br s, 2, NH.sub.2), 7.84 (s 1,
H8), 8.42 (s, 1, H2); MS (APCI) m/z 452 (100, MH.sup.+). Anal.
Calcd for C.sub.21H.sub.41N.sub.5O.sub.2Si.sub.2 (451.76): C,
55.83; H, 9.15; N, 15.50.
9-(3-O-t-Butyldimethylsilyl-3,4-dihydroxybutyl)adenine (9)
[0140] Compound 8 (400 mg, 0.887 mmol) was added to a solution of
CH.sub.3CO.sub.2H/H.sub.2O/THF (13:7:3; 8 mL) and the mixture was
stirred at ambient temperature until more polar spot of compound 2
starting to appear on TLC. Then reaction mixture was partitioned
(EtOAc//NaHCO.sub.3/H.sub.2O) and the aqueous layer was extracted
with next portion of EtOAc. The combined organic phase was washed
(NaHCO.sub.3, brine), dried (Na.sub.2SO.sub.4), evaporated and
column chromatographed (CHCl.sub.3.fwdarw.4% MeOH/CHCl.sub.3) to
give recovered 8 (140 mg, 35%) and 9 (155 mg, 52%): .sup.1H NMR
.delta. 0.04 (s, 6, 2.times.CH.sub.3), 0.88 (s, 9, t-Bu), 2.16-2.19
(m, 2, H2',2''), 3.61 (dd, J=4.5, 11.4 Hz, 1, H4''), 3.65 (dd,
J=5.2, 11.4 Hz, 1, H4''), 3.88 (q, J=5.2 Hz, 1, H3'), 4.26-4.36 (m,
2, H1',1''), 6.17 (br s, 2, NH.sub.2), 7.80 (s, 1, H8), 8.29 (s, 1,
H2); .sup.13C NMR .delta. -4.4 & 4.1 (CH.sub.3), 18.5 (t-Bu),
26.2 (t-Bu), 34.5 (C2'), 40.8 (C1'), 65.5 (C4'), 70.6 (C3'), 119.8
(C5), 140.7 (C8), 150.4 (C4), 153.0 (C2), 155.5 (C6); MS (APCI) m/z
338 (100, MH.sup.+). Anal. Calcd for
C.sub.15H.sub.27N.sub.5O.sub.2Si (337.50): C, 53.38; H, 8.06; N,
20.75.
Methyl 3-(Adenin-9-yl)propionate (6')
[0141] Treatment of 9 (50 mg, 0.148 mmol) by procedure B (column
chromatography: CHCl.sub.3/MeOH 97:3) gave 6' (9 mg, 27%) with data
as reported.
3-(Adenin-9-yl)propionic acid (7')
[0142] Treatment of 6' (10 mg, 0.045 mmol) by procedure C (column
chromatography: CHCl.sub.3/MeOH 97:3) gave 6' (7.3 mg, 78%) with
data as reported.
##STR00006##
Example 2
DZ2002 Mediates Immunosuppressive Effects
Materials and Methods
[0143] Reagents
[0144] AdoHcy hydrolase inhibitors DZ2002 was synthesized at
Diazyme Laboratories. Con A (Concanavalin A), LPS (Escherichia coli
055:B5) and Sac (Staphylococcus aureus Cowan strain 1) were
obtained from Pansorbin.RTM. cells, Biosciences, inc. (La Jolla,
Calif. 92039, USA). RPMI 1640 and fetal bovine serum (FBS) were
obtained from GIBCO. Purified rat anti-mouse IL-10, IL-12p70,
IL-12p40, IFN-.gamma. and biotinylated anti-mouse IL-10, IL-12p70,
IL-12p40, IFN-.gamma., FITC-anti-mouse-CD11b (Mac-1), Phycorythrin
(PE)-anti-mouse I-Ad, PE-anti-human-CD14, PE-anti-human-ABC,
PE-anti-human-DR, PE-anti-human-CD80 and PE-anti-human-CD86 were
Pharmingen products. Thioglycollate (TG) is available from
Sigma-Aldrich.
[0145] Animal
[0146] Inbred BALB/C mice, 6.about.8 weeks of age, were provided by
Shanghai Experimental Animal Center of Chinese Academy of Sciences
with Certificate No. 99-003. The mice were housed in specific
pathogen-free (SPF) conditions with room temperature of
24.+-.2.degree. C., 12 hr light/dark cycle, and provided with
sterile food and water ad libitum.
[0147] Cells
[0148] Spleens from Balb/c mice were aseptically removed, pooled,
and single cell suspensions prepared in PBS. Erythrocytes were
lysed by treatment with Tris-buffered ammonium chloride (0.155 M
NH.sub.4CL, 0.0165 M Tris, PH 7.2). Mononuclear cells were washed
with PBS and resuspended in RPMI-1640 media supplemented with
benzylpenicillin 100000 UL.sup.-1, and streptomycin 100 mgL.sup.-1.
The cell viability and concentration were determined by trypan blue
exclusion.
[0149] Peritoneal exudate cells were induced in BALB/C mice by an
intraperitoneal injection of 0.5 ml of 3% TG. After 4 days, the
peritoneal exudates cells were harvested by sterile lavage.
[0150] THP-1 (American Type Culture Collection, Manassas, Va.) is a
human monocytic leukemia. THP-1 cells were maintained in suspension
culture in RPMI1640 medium supplemented with 10% FBS. Cultures were
maintained at 37.degree. C. in a humidified atmosphere of 5%
CO.sub.2 in air and were subculture at 1/10 dilution every 5-6
days.
[0151] [.sup.3H]-Thymidine Incorporation to the Splenic
Lymphocytes
[0152] Mouse splenic lymphocytes were cultured in vitro in RPMI1640
supplemented with 10% FBS. Cells were incubated in a 96-well plate
at 1.times.10.sup.5 cells/200 .mu.l/well in a humidified CO.sub.2
incubator at 37.degree. C. for 48 hours with 5 .mu.g/ml of Con A or
10 .mu.g/ml LPS in the presence or absence of various
concentrations of DZ2002. After 40 hour incubation, cells were
pulsed with 0.5 .mu.Ci/well of [.sup.3H]-thymidine and cultured for
another 8 hours. The cells were then harvested onto glass fiber
filters and the incorporated radioactivity was counted using a Beta
Scintillator (MicroBeta Trilux, PerkinElmer Life Sciences).
[0153] MTT Assay of the Splenic Lymphocytes
[0154] Cytotoxicity was assessed with MTT assay. Mouse splenic
lymphocytes were incubated in a 96-well plate at 9.times.10.sup.4
cells/180 .mu.l/well in a humidified CO.sub.2 incubator at
37.degree. C. for 48 hours in the presence or absence of various
concentrations of DZ2002. Fifteen (15) .mu.l of 5 mg/ml of MTT was
pulsed 4 h prior to end of the culture (total 190 .mu.l), and then
80 .mu.l solvent (10% SDS, 50% N,N-dimethy formamide, PH 7.2) was
added. Incubate for 7 h and read OD.sub.590 at a microplate reader
(Bio-rad Model 550 Japan).
[0155] Cytokine Production
[0156] Murine splenic mononuclear cells (5.times.10.sup.6) were
cultured in 24-well plates in a volume of 2 ml/well in the presence
of Sac (1:10000), ConA (5 ug/ml) or LPS (10 ug/ml) in the presence
or absence of various concentrations of DZ2002. After 24 h,
cell-free supernatant was collected and frozen at -20.degree. C.
The concentrations of IL-12p40, IL-12p70, IL-10 and TNF-.alpha.
were determined in an ELISA specific for murine cytokines.
[0157] Murine peritoneal exudate cells (6.25.times.10.sup.5) were
cultured in 24-well plates in a volume of 1 ml/well for 2 hours. In
adherent cells were washed by ice cold RPMI 1640 and adherent cells
were culture in a volume of 2 ml/well in the presence of
IFN-.gamma. (2.5 ng/ml) and LPS (1 .mu.g/ml) in the presence or
absence of various concentrations of DZ2002. After 24 h, cell-free
supernatant was collected and frozen at -20.degree. C. The
concentrations of IL-12p40, IL-12p70, IL-10 and TNF-.alpha. were
determined by ELISA.
[0158] THP-1 cells (6.times.10.sup.5) were cultured in 24-well
plates in a volume of 2 ml/well in the presence of 1.2% and in the
presence or absence of various concentrations of DZ2002. After 24
h, IFN-.gamma. (500 U/ml) was added and another 16 h later, LPS (1
.mu.g/ml) was added. Cell-free supernatant was collected after 24 h
and frozen at -20.degree. C. The concentrations of IL-12p40,
IL-12p70, IL-10 and TNF-.alpha. were determined for ELISA.
[0159] Quantitative Hemolysis of Sheep Red Blood Cells (QHS)
Assay
[0160] Female Balb/c mice were immunized by intraperitoneal
injection with 0.2 ml of 16.7% of SRBC on day 4. Vehicle,
Dexamethasone and DZ2002 were administrated on each group (n=6) by
intraperitoneal injection on 7 consecutive days of 1-7. On day 8,
mice were sacrificed and made a mixed suspension of spleen cells of
2.times.10.sup.6 cells/ml. 1 ml of cell suspension was incubated
with 1 ml of 0.5% SRBC and 1 ml of 1:10 dilution of guinea pig
complement for 1 h at 37.degree. C., then centrifuged (3 min, 3000
g) and determined the supernatant hemolysis at 413 nm, according to
Simpson et al, J. Immunol. Methods., 21(1-2):159-65. (1978) with
some modifications. Each group was triplicated.
[0161] Mixed Lymphocyte Reaction (MLR) Proliferation Assay.
[0162] Balb/c mouse spleen cells were prepared in 10.sup.7 cells/ml
suspension, cultured 2 h with 50 .mu.g/ml of mitomycin. Then cells
were washed and cultured together with fresh C57/B6 mice
splenocytes equally in a final concentration of 1.0.times.10.sup.6
cells/ml in the presence or absence of various concentrations of
DZ2002. After 48 hour incubation, cells were pulsed with 0.5
.mu.Ci/well of [.sup.3H]-thymidine and cultured for another 24
hours. The cells were then harvested onto glass fiber filters and
the incorporated radioactivity was counted using a Beta
Scintillator (MicroBeta Trilux, PerkinElmer Life Sciences).
[0163] DNFB-Induced Delayed Type Hypersensitivity (DTH)
Response
[0164] Female Balb/c mice were sensitized with 20 .mu.l of 0.6%
DNFB dissolved in acetone-olive oil (4:1) on each hind foot on day
0 and 1. On day 7 mice were challenged with 10 .mu.l of 0.5% DNFB
on both sides of left ear, methods according to Phanuphak (1974)
with some modifications. Vehicle, CsA, and DZ2002 (1, 3, 10 mg/kg)
were administrated on each group (n=10) by intraperitoneal
injection on 1 hour before and 12 hours, 24 hours after the
challenge. Ear swelling was expressed as difference between the
weight of the left and right ear patches made by a specific 8-mm
punch 30 h after the challenge.
[0165] Flow Cytometry
[0166] Murine peritoneal exudate cells or THP-1 cells were washed
in cold PBS (staining buffer, containing 0.1% NaN.sub.3, 1% FBS, PH
7.2). Cells were resuspended at 2.0.times.10.sup.7/ml in cold
staining buffer. Optimal concentrations of each
fluorochrome-labeled antibody were added to 50 .mu.L cells. Fc
receptors were blocked using 10 .mu.L normal mouse serum. Cells
were incubated in the dark at 4.degree. C. for 30 min, washed twice
with 2.0 mL staining buffer and resupended in 0.5 mL of PBS, PH
7.2. Cells were stored in the dark at 4.degree. C. and analyzed on
a FACScan flow cytometer (Becton Dickinson, San Jose, Calif.). Data
were analyzed by means of CellQuest.TM. Software (Becton Dickinson,
San Jose, Calif.).
[0167] Statistical Analysis
[0168] Results were expressed as x.+-.s, independent two-tailed
t-test was performed and P values less than 0.05 were considered to
be significant. Each experiment was repeated at least three
times.
Results
[0169] Inhibition of [.sup.3H]-Thymidine Incorporation to the
Splenic Lymphocytes by AdoHcy Hydrolase Inhibitors
[0170] After 48 h of culture, DZ2002 (0.1-10 .mu.molL.sup.-1) have
no effects on the lymphocytes proliferation induced by ConA. DZ2002
(10 .mu.molL.sup.-1) inhibited lymphocytes proliferation induced by
LPS.
[0171] Effect of DZ2002 on IL-10, IL-12P40 and IL-12P70 Production
from Sac Stimulated Murine Splenocytes
[0172] Sac stimulation induced marked increasing of IL-10, IL-12P40
and IFN-.gamma. production from murine splenocytes compared with
resting splenocytes. DZ2002 (.mu.molL.sup.-1) dose dependently
inhibited IL-12P40, IL-12P70 and TNF-.alpha. release, but have no
effect on IL-10 production from Sac stimulated splenocytes. (data
not shown).
[0173] Effects of DZ2002 on Quantitative Hemolysis of Sheep Red
Blood Cells (QHS) Assay
[0174] Quantitative hemolysis of SRBC is a model of primary
antibody production in response to antigenic stimulation. As FIG. 3
shows, consecutively 7-day intraperitoneal injection of DZ2002
inhibited 24.5 and 18.4% of QHS at doses of 0.08 and 2 mg/kg
respectively, compared with 38.1% of that of 5 mg/kg Dethamethasone
(p<0.05 for All experiment groups compared with Vehicle control
group) (FIG. 1).
[0175] DZ2002 Suppress T Cell Proliferation in Mixed Lymphocyte
Reaction
[0176] Mitomycin-treated Balb/c (H-2.sup.d) spleen cell were
applied as allogeneic stimulator to C57BL/6(H-2.sup.b) spleen cells
proliferation. DZ2002 had a strong suppression to MLR with 40.2,
36.9 and 42.3% at doses of 0.1, 1 and 10 .mu.mol/L respectively for
3-day culture. (FIG. 2).
[0177] DZ2002 Have No Cytotoxicity in Spleen Cell
[0178] In two days of culture, 0.1-10 umol/L DZ2002 showed no
cytotoxicity to spleen cells. The OD values of cells incubated with
DZ2002 have no difference with that of the control. (FIG. 3).
[0179] DZ2002 Reversed the Suppression of Mouse DTH Response
Induced by Ethanol Consumption
[0180] Nine mice were prepared for each group. Mice were sensitized
with 0.5% DNFB solution (20 ul) in absolute acetone/olive oil (4:1)
on each hind foot on day 0 and 1. Five days after initial
sensitization, mice were challenged with 0.2% DNFB (10 ul) on both
sides of left ear under light Metofane anesthesia. The right ear
was treated with vehicle alone. DZ2002 were orally administered to
the mice 1 h before DNFB challenge. The degree of ear swelling was
measured 24 h after challenge using a ear puncher and an analytic
balance to measure the weight (mg). Results were expressed as the
difference between the weight of the left and the right ear.
Spleens were taken from four mice in each group after the
measurement of the ear swelling and frozen until analysis.
[0181] Effects of DZ2002 on the Expression of MHC-II on Resident
and TG Induced Peritoneal Cells
[0182] MHC-II expression by peritoneal macrophages was assessed
following a 48-h incubation with media alone or with IFN-.gamma. at
100 U/ml. Cells incubated with IFN-.gamma. in the present of 0.1
and 1 .mu.mol/L DZ2002 enhance the levels of MHC-II expression of
resident peritoneal cells and 10 .mu.mol/L DZ2002 reduce the levels
of MHC-II expression. (data not shown) As reflected in the TG
induced peritoneal cells, 1 and 10 .mu.mol/L DZ2002 decrease the
Mac-1.sup.+ percentage when incubate with media alone. And the
level of MHC-II expression of cells incubated with IFN-.gamma. was
dose-dependently decreased in the presence of 0.1-10 .mu.mol/L
DZ2002. (data not shown).
[0183] Effect of DZ2002 on IL-10, IL-12P40 and TNF-.alpha.
Production from TG Induced Peritoneal Cells
[0184] Cytokines produced by peritoneal macrophages were assessed
following a 24-h incubation with IFN-.gamma. at 25 U/ml and LPS at
1 .mu.g/ml. Resident peritoneal cells produce low levers of
cytokines except for some IL-10 with incubated with IFN-.gamma. and
LPS (data not shown). As for TG induced peritoneal macrophages,
DZ2002 inhibited IL-12P40 and TNF-.alpha. release, but have no
effect on IL-10 production in the dose of 0.1-10 .mu.mol/L (FIG.
5).
[0185] DZ2002 Inhibits Expression of MHC-II, CD80 and CD86 on THP-1
Cells
[0186] MHC-II, CD80 and CD86 expression by THP-1 cells was assessed
following a 48-h incubation with media alone or with IFN-.gamma. at
100 U/ml. Cells incubated with IFN-.gamma. in the present of 10
.mu.mol/L DZ2002 modestly reduce the levels of MHC-II expression of
THP-1 cells and 0.1-10 .mu.mol/L DZ2002 reduce the levels of CD80
and CD86 expression by an dose-dependently way. (FIG. 6A).
[0187] Effect of DZ2002 on IL-10, IL-12P40 and TNF-.alpha.
Production from THP-1 Cells
[0188] Cytokines produced by THP-1 cells were assessed following a
24-h incubation with IFN-.gamma. at 500 U/ml and LPS at 1 .mu.g/ml.
As FIG. 7 shows, DZ2002 inhibited IL-12P40 and TNF-.alpha. release,
in the dose of 0.1-10 .mu.mol/L.
[0189] The above examples are included for illustrative purposes
only and are not intended to limit the scope of the invention. Many
variations to those described above are possible. Since
modifications and variations to the examples described above will
be apparent to those of skill in this art, it is intended that this
invention be limited only by the scope of the appended claims.
* * * * *
References