U.S. patent application number 11/336029 was filed with the patent office on 2006-08-24 for methylphenidate derivatives and uses of them.
Invention is credited to David Bar-Or, Nagaraja R.K. Rao.
Application Number | 20060189655 11/336029 |
Document ID | / |
Family ID | 36692913 |
Filed Date | 2006-08-24 |
United States Patent
Application |
20060189655 |
Kind Code |
A1 |
Bar-Or; David ; et
al. |
August 24, 2006 |
Methylphenidate derivatives and uses of them
Abstract
The present invention provides methods of using compounds of
formula I: ##STR1## and salts and prodrugs thereof, wherein n,
R.sup.1 and R.sup.2 are defined herein. The invention also provides
certain novel compounds of formula I and pharmaceutical
compositions comprising them.
Inventors: |
Bar-Or; David; (Englewood,
CO) ; Rao; Nagaraja R.K.; (Cardiff, GB) |
Correspondence
Address: |
FAEGRE & BENSON LLP;PATENT DOCKETING
2200 WELLS FARGO CENTER
90 SOUTH 7TH STREET
MINNEAPOLIS
MN
55402-3901
US
|
Family ID: |
36692913 |
Appl. No.: |
11/336029 |
Filed: |
January 20, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60663006 |
Mar 18, 2005 |
|
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|
60645778 |
Jan 20, 2005 |
|
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Current U.S.
Class: |
514/317 |
Current CPC
Class: |
A61P 9/00 20180101; A61P
35/04 20180101; A61P 17/06 20180101; A61P 3/04 20180101; A61P 17/00
20180101; A61P 19/02 20180101; A61P 7/00 20180101; A61P 15/00
20180101; A61P 27/02 20180101; A61P 35/00 20180101; A61P 37/06
20180101; A61K 31/445 20130101; A61P 25/22 20180101; A61P 27/06
20180101; A61P 9/10 20180101; A61P 3/10 20180101; A61P 35/02
20180101 |
Class at
Publication: |
514/317 |
International
Class: |
A61K 31/445 20060101
A61K031/445 |
Claims
1. A method of inhibiting angiogenesis in an animal comprising
administering to the animal an effective amount of a compound of
formula I: ##STR7## or a salt or a prodrug thereof, where n is an
integer from 1 to 5, each R.sup.1 is independently aryl,
heteroaryl, alkyl, cycloalkyl, alkoxy, aryloxy, acyl, carboxyl,
hydroxyl, halogen, amino, nitro, sulfo or sulfhydryl, wherein each
alkyl is optionally substituted with hydroxyl, amino or sulfhydryl;
and R.sup.2 is hydrogen or lower alkyl.
2. A method of treating an angiogenic disease or condition in an
animal comprising administering to the animal an effective amount
of a compound of formula I: ##STR8## or a salt or a prodrug
thereof, where n is an integer from 1 to 5, each R.sup.1 is
independently aryl, heteroaryl, alkyl, cycloalkyl, alkoxy, aryloxy,
acyl, carboxyl, hydroxyl, halogen, amino, nitro, sulfo or
sulfhydryl, wherein each alkyl is optionally substituted with
hydroxyl, amino or sulfhydryl; and R.sup.2 is hydrogen or lower
alkyl.
3. A method of treating an ocular angiogenic disease or condition
in an animal comprising administering to the animal an effective
amount of a compound of formula I: ##STR9## or a salt or a prodrug
thereof, where n is an integer from 1 to 5, each R.sup.1 is
independently aryl, heteroaryl, alkyl, cycloalkyl, alkoxy, aryloxy,
acyl, carboxyl, hydroxyl, halogen, amino, nitro, sulfo or
sulfhydryl, wherein each alkyl is optionally substituted with
hydroxyl, amino or sulfhydryl; and R.sup.2 is hydrogen or lower
alkyl.
4. The method of claim 3 wherein the ocular angiogenic disease or
condition is diabetic retinopathy.
5. The method of claim 3 wherein the ocular angiogenic disease or
condition is macular degeneration.
6. The method of claim 3 wherein the ocular angiogenic disease or
condition is retinopathy of prematurity, corneal graft rejection,
neovascular glaucoma, retrolental fibroplasias or rubeosis.
7. A method of treating a neoplastic disease in an animal
comprising administering to the animal an effective amount of a
compound of formula I: ##STR10## or a salt or a prodrug thereof,
where n is an integer from 1 to 5, each R.sup.1 is independently
aryl, heteroaryl, alkyl, cycloalkyl, alkoxy, aryloxy, acyl,
carboxyl, hydroxyl, halogen, amino, nitro, sulfo or sulfhydryl,
wherein each alkyl is optionally substituted with hydroxyl, amino
or sulfhydryl; and R.sup.2 is hydrogen or lower alkyl.
8. The method of claim 7 wherein the neoplastic disease is a
tumor.
9. The method of claim 8 wherein the tumor is a malignant
tumor.
10. The method of claim 9 wherein the tumor is a tumor of the
bladder, brain, breast, cervix, colon, rectum, kidney, liver, lung,
ovary, pancreas, prostate, stomach or uterus.
11. The method of claim 10 wherein the tumor is a tumor of the
brain, breast, colon, liver or pancreas.
12. The method of claim 11 wherein the tumor is a tumor of the
brain.
13. The method of claim 12 wherein the brain tumor is a
glioblastoma.
14. The method of claim 7 wherein the neoplastic disease is tumor
metastasis.
15. A method of treating a proliferative disorder in an animal
comprising administering to the animal an effective amount of a
compound of formula I: ##STR11## or a salt or a prodrug thereof,
where n is an integer from 1 to 5, each R.sup.1 is independently
aryl, heteroaryl, alkyl, cycloalkyl, alkoxy, aryloxy, acyl,
carboxyl, hydroxyl, halogen, amino, nitro, sulfo or sulfhydryl,
wherein each alkyl is optionally substituted with hydroxyl, amino
or sulfhydryl; and R.sup.2 is hydrogen or lower alkyl.
16. The method of claim 15 wherein the proliferative disorder is a
cancer.
17. The method of claim 16 wherein the cancer is a carcinoma, a
sarcoma, a lymphoma or a leukemia.
18. The method of claim 15 wherein the proliferative disorder is a
mesangial cell proliferation disorder.
19. The method of claim 15 wherein the proliferative disorder is a
fibrotic disorder.
20. The method of claim 15 wherein the proliferative disorder is a
hyperproliferative skin disorder.
21. The method of claim 20 wherein the the hyperproliferative skin
disorder is skin cancer.
22. The method of any one of claims 1-21 wherein the compound is:
##STR12##
23. A compound of formula IA: ##STR13## where n is an integer from
1 to 5; each R.sup.1 is independently a moiety of the formula
--C(O)--R.sup.8, --OR.sup.7 or --C(O)--O--R.sup.3; R.sup.2 is
hydrogen or lower alkyl; R.sup.3 is hydrogen, alkyl, cycloalkyl or
aryl; R.sup.7 is aryl; and R.sup.8 is cycloalkyl or aryl.
24. The compound of claim 23 wherein R.sup.1 is a moiety of the
formula --OR.sup.7.
25. The compound of claim 24 wherein R.sup.7 is phenyl.
26. The compound of claim 23 wherein n is 1 or 2.
27. A pharmaceutical composition comprising a
pharmaceutically-acceptable carrier and compound of formula IA or a
salt or a prodrug thereof: ##STR14## where n is an integer from 1
to 5; each R.sup.1 is independently a moiety of the formula
--C(O)--R.sup.8, --OR.sup.7 or --C(O)--O--R.sup.3; R.sup.2 is
hydrogen or lower alkyl; R.sup.3 is hydrogen, alkyl, cycloalkyl or
aryl; R.sup.7 is aryl; and R.sup.8 is cycloalkyl or aryl.
28. The compound of claim 27 wherein R.sup.1 is a moiety of the
formula --OR.sup.7.
29. The compound of claim 28 wherein R.sup.7 is phenyl.
30. The compound of claim 27 wherein n is 1 or 2.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims benefit of provisional applications
Nos. 60/645,778, filed Jan. 20, 2005, and 60/663,006, filed Mar.
18, 2005, the complete disclosures of which are incorporated herein
by reference.
FIELD OF THE INVENTION
[0002] The invention relates to uses of methylphenidate
derivatives. The uses include inhibiting angiogenesis and treating
angiogenic diseases and conditions.
BACKGROUND
[0003] Methylphenidate is the treatment of choice for children and
adults diagnosed with attention deficit/hyperactivity disorder
(ADHD), including its inattentive subtype (formerly known as
attention deficit disorder or ADD). Certain derivatives of
methylphenidate have also been proposed for the treatment of ADD
(see U.S. Pat. No. 6,025,502) and for the treatment of other
neurological disorders and conditions (see U.S. Pat. Nos.
5,859,249, 6,025,502 and 6,486,177 and PCT application WO
99/36403).
[0004] Methylphenidate is a mild central nervous stimulant and is
also taught for treating apathy, fatigue, cognitive decline, and
depression in cancer patients, AIDS patients and other seriously
ill patients. See U.S. Pat. Nos. 5,908,850, 6,127,385, 6,395,752
and 6,486,177, Challman and Lipsky, Mayo Clin. Proc., 75:711-721
(2000) and Leonard et al., Hum. Psychopharmacol. Clin. Exp.,
19:151-180 (2004).
[0005] It has been reported that methylphenidate is not
carcinogenic, and that there is a less than expected rate of
cancer, in rats and humans taking methylphenidate. See Dunnick and
Hailey, Toxicology, 103:77-84 (1995), National Toxicology Program,
Natl. Toxicol. Program Tech. Rep. Ser., 439:1-299 (1995), Dunnick
et al., Cancer Lett., 102:77-83 (1996) and Teo et al., Mutat. Res.,
537:67-79 (2003). However, there is some evidence that
methylphenidate is carcinogenic in mice. Dunnick and Hailey,
Toxicology, 103:77-84 (1995) and National Toxicology Program, Natl.
Toxicol. Program Tech. Rep. Ser., 439:1-299 (1995). Further, some
types of tumors have been reported to be decreased, while other
types of tumors have been reported to be increased. See Dunnick and
Hailey, Toxicology, 103:77-84 (1995), National Toxicology Program,
Natl. Toxicol. Program Tech. Rep. Ser., 439:1-299 (1995) and
Dunnick et al., Cancer Lett., 102:77-83 (1996).
SUMMARY OF THE INVENTION
[0006] The invention provides methods of using a compound of
formula I ##STR2## wherein n is an integer from 1 to 5, and each
R.sup.1 is independently aryl, heteroaryl, alkyl, cycloalkyl,
alkoxy, aryloxy, acyl, carboxyl, hydroxyl, halogen, amino, nitro,
sulfo or sulfhydryl. Each alkyl can optionally be substituted with
hydroxyl, amino or sulfhydryl. R.sup.2 is hydrogen or lower
alkyl.
[0007] In a first embodiment, the invention provides a method of
inhibiting angiogenesis in an animal. The method comprises
administering an effective amount of a compound of formula I, or a
pharmaceutically-acceptable salt or a prodrug thereof, to the
animal.
[0008] In a second embodiment, the invention provides a method of
treating an angiogenic disease or condition in an animal. The
method comprises administering a therapeutically effective amount
of a compound of formula I, or a pharmaceutically-acceptable salt
or a prodrug thereof, to the animal.
[0009] In a third embodiment, the invention provides a method of
treating a proliferative disorder in an animal. The method
comprises administering a therapeutically effective amount of a
compound of formula I, or a pharmaceutically-acceptable salt or a
prodrug thereof, to the animal.
[0010] The invention also provides a compound of formula IA:
##STR3## where
[0011] n is an integer from 1 to 5;
[0012] each R.sup.1 is independently a moiety of the formula
--C(O)--R.sup.8, --OR.sup.7 or --C(O)--O--R.sup.3;
[0013] R.sup.2 is hydrogen or lower alkyl;
[0014] R.sup.3 is hydrogen, alkyl, cycloalkyl or aryl;
[0015] R.sup.7 is aryl; and
[0016] R.sup.8 is cycloalkyl or aryl.
[0017] The invention further provides a pharmaceutical composition
comprising a pharmaceutically-acceptable carrier and a compound of
formula IA: ##STR4## or a salt or prodrug thereof, where
[0018] n is an integer from 1 to 5;
[0019] each R.sup.1 is independently a moiety of the formula
--C(O)--R.sup.8, --OR.sup.7 or --C(O)--O--R.sup.3;
[0020] R.sup.2 is hydrogen or lower alkyl;
[0021] R.sup.3 is hydrogen, alkyl, cycloalkyl or aryl;
[0022] R.sup.7 is aryl; and
[0023] R.sup.8 is cycloalkyl or aryl.
BRIEF DESCRIPTION OF THE DRAWINGS
[0024] FIGS. 1A-C are graphs of OD at 530 nm for various additives
to peripheral blood lymphocyte (PBL) cultures stimulated with 2
.mu.g/ml, 5 .mu.g/ml and 20 .mu.g/ml phytohemagglutinin (PHA),
respectively.
[0025] FIG. 2 is a graph of OD at 530 nm for various additives to
PBL cultures stimulated with 2 .mu.g/ml PHA.
[0026] FIG. 3 is a graph of concentration of IL-13 for various
additives to PBL cultures stimulated with 2 .mu.g/ml PHA.
[0027] FIG. 4 is a graph of concentration of IFN.gamma. for various
additives to PBL cultures stimulated with 2 .mu.g/ml PHA.
[0028] FIGS. 5A-B are graphs of OD at 530 nm for various additives
to PBL cultures stimulated with 2 .mu.g/ml and 5 .mu.g/ml PHA,
respectively.
[0029] FIG. 6 is a graph of concentration of IL-13 for various
additives to PBL cultures stimulated with 5 .mu.g/ml PHA.
[0030] FIG. 7 is a graph of concentration of TNF.alpha. for various
additives to PBL cultures stimulated with 2 .mu.g/ml PHA.
[0031] FIG. 8 is a graph of concentration of IL-8 for various
additives to PBL cultures stimulated with 2 .mu.g/ml PHA.
[0032] FIGS. 9A-B are graphs of OD for various additives to THP-1
monocyte cultures stimulated with lipopolysaccharide (LPS). In FIG.
9B, the top dark gray bar in each instance is c-Jun and the bottom
light gray bar is NF.kappa.B.
DETAILED DESCRIPTION OF THE INVENTION
[0033] In one aspect, compounds of formula I are useful in the
practice of the present invention. ##STR5##
[0034] In Formula I, n is an integer from 1 to 5. Preferably n is 1
or 2.
[0035] Each R.sup.1, which may be the same or different, is aryl,
heteroaryl, alkyl, cycloalkyl, alkoxy, aryloxy, acyl, carboxyl,
hydroxyl, halogen, amino, nitro, sulfo or sulfhydryl. Each alkyl
can optionally be substituted with hydroxyl, amino or sulfhydryl.
R.sup.1 is preferably aryl, alkyl, cycloalkyl, alkoxy, aryloxy or
acyl. More preferably R.sup.1 is aryl, alkyl or cycloalkyl, even
more preferably aryl, most preferably phenyl.
[0036] In formula I, R.sup.2 is hydrogen or lower alkyl.
Preferably, R.sup.2 is --CH.sub.3.
[0037] In one specific embodiment, the compound of formula II is
particularly useful in the present invention: ##STR6##
[0038] "Acyl" means a moiety of the formula --C(O)--R.sup.3,
wherein R.sup.3 is H, alkyl, cycloalkyl or aryl.
[0039] "Amino" means a moiety of the formula --NR.sup.4R.sup.5,
wherein each of R.sup.4 and R.sup.5 is independently H or lower
alkyl, preferably lower alkyl.
[0040] "Alkoxy" means a moiety of the formula --OR.sup.6, wherein
R.sup.6 is alkyl. An example of an alkoxy group is methoxy
(--O--CH.sub.3).
[0041] "Alkyl" means a monovalent saturated straight-chain or
branched hydrocarbon containing 1-8 carbon atoms. Each alkyl may,
optionally, be substituted with one or more amino, hydroxyl or
sulfhydryl groups.
[0042] "Aryl" means a monovalent mono-, bi- or tricyclic aromatic
hydrocarbon moiety of 6 to 14 ring carbon atoms. Preferred is
phenyl.
[0043] "Aryloxy" means a moiety of the formula --OR.sup.7, wherein
R.sup.7 is aryl. An example of an alkoxy group is phenoxy.
[0044] "Carboxyl" means a moiety of the formula --C(O)--OR.sup.3,
wherein R.sup.3 is H, alkyl, cycloalkyl or aryl.
[0045] "Cycloalkyl" means a saturated, monovalent mono- or bicyclic
hydrocarbon moiety of three to ten ring carbon atoms. Preferably
the cycloalkyl contains 4-8 ring carbon atoms. The most preferred
cycloalkyl is cyclohexyl.
[0046] "Halogen" means chlorine, fluorine, bromine or iodine.
Preferred is chlorine or bromine.
[0047] "Heteroaryl" means a monovalent monocyclic or bicyclic
aromatic moiety of 5 to 12 ring atoms containing one, two, or three
ring heteroatoms each of which is independently selected from N, O,
and S, the remaining ring atoms being C.
[0048] "Hydroxyl" means --OH.
[0049] "Lower alkyl" means a saturated straight-chain or branched
hydrocarbon containing 1-4 carbon atoms.
[0050] "Nitro" means --NO.sub.2.
[0051] "Sulfhydryl" means --SH.
[0052] "Sulfo" means --SO.sub.3H.
[0053] "Prodrug" means any compound which releases an active parent
drug according to formula I in vivo when such prodrug is
administered to a mammalian subject. Prodrugs of a compound of
formula I are prepared by modifying one or more functional group(s)
present in the compound of formula I in such a way that the
modification(s) may be cleaved in vivo to release the parent
compound. Prodrugs include compounds of formula I wherein a
hydroxy, amino, or sulfhydryl group in a compound of formula I is
bonded to any group that may be cleaved in vivo to generate the
free hydroxyl, amino, or sulfhydryl group, respectively. Examples
of prodrugs include, but are not limited to, esters (e.g., acetate,
formate, and benzoate derivatives), carbamates (e.g.,
N,N-dimethylaminocarbonyl) of hydroxy functional groups in
compounds of formula I, and the like.
[0054] "Inhibit" or "inhibiting" is used herein to mean to reduce
(wholly or partially) or to prevent.
[0055] "Treating" or "treatment" of a disease or condition
includes: (1) preventing the disease or condition, i.e., causing
the clinical symptoms of the disease or condition not to develop in
a mammal that may be exposed to or predisposed to the disease or
condition, but does not yet experience or display symptoms of the
disease or condition; (2) inhibiting the disease or condition,
i.e., arresting or reducing the development of the disease or
condition or its clinical symptoms; or (3) relieving the disease or
condition, i.e., causing regression of the disease or condition or
its clinical symptoms, including curing the disease or
condition.
[0056] An "effective amount" means the amount of a compound that,
when administered to an animal for treating a disease or condition
or for causing an effect is sufficient to do so. The "effective
amount" can and will most likely vary depending on the compound,
the disease or condition and its severity, or the effect sought to
be caused, and the age, weight, etc., of the animal to be
treated.
[0057] Methods of synthesizing the compounds of formula I useful in
the present invention are known in the art. See, e.g., U.S. Pat.
Nos. 5,859,249 and 6,025,502, PCT application WO 99/36403, Pan et
al., Eur. J. Pharmacol., 264, 177-182 (1994), Gatley et al., Life
Sci., 58, 231-239 (1996), Deutsch et al., J. Med. Chem., 39,
1201-1209 (1996), Thai et al., J. Med. Chem., 41, 591-601 (1998),
and Wayment et al., J. Neurochem., 72:1266-1274 (1999), Krim, Lori,
Thesis (Ph.D. in Chemistry) (2001), University Of Pennsylvania,
Chemistry Library Reading Room (Call No. QD001 2001.K92),
University Microfilms Order No. 3031684, ISBN 0-493-44179-4, the
complete disclosures of which along with the references cited
therein are incorporated herein by reference.
[0058] If the compound of the present invention contains one or
more chiral centers, the compound can be synthesized
enantioselectively or a mixture of enantiomers and/or diastereomers
can be prepared and separated. The resolution of the compounds of
the present invention, their starting materials and/or the
intermediates may be carried out by known procedures, e.g., as
described in the four volume compendium Optical Resolution
Procedures for Chemical Compounds: Optical Resolution Information
Center, Manhattan College, Riverdale, N.Y., and in Enantiomers,
Racemates and Resolutions, Jean Jacques, Andre Collet and Samuel H.
Wilen; John Wiley & Sons, Inc., New York, 1981, which are
incorporated herein in their entirety. Basically, the resolution of
the compounds is based on the differences in the physical
properties of diastereomers by attachment, either chemically or
enzymatically, of an enantiomerically pure moiety, resulting in
forms that are separable by fractional crystallization,
distillation or chromatography.
[0059] The pharmaceutically-acceptable salts of the compounds of
formula I may also be used in the practice of the invention.
Pharmaceutically-acceptable salts include conventional non-toxic
salts, such as salts derived from inorganic acids (such as
hydrochloric, hydrobromic, sulfuric, phosphoric, nitric, and the
like), organic acids (such as acetic, propionic, succinic,
glycolic, stearic, lactic, malic, tartaric, citric, glutamic,
aspartic, benzoic, salicylic, oxalic, ascorbic acid, and the like)
or bases (such as the hydroxide, carbonate or bicarbonate of a
pharmaceutically-acceptable metal cation or organic cations derived
from N,N-dibenzylethylenediamine, D-glucosamine, or
ethylenediamine). The salts are prepared in a conventional manner,
e.g., by reacting the free base form of the compound with an
acid.
[0060] It is to be understood that the scope of this invention
encompasses not only the use of the compounds of formula I
themselves, but also the salts and prodrugs thereof. In addition,
the present invention contemplates the use of the isomers of the
compounds of formula I, and of the salts and prodrugs thereof,
including pure isomers and various mixtures of isomers.
[0061] Compounds of formula I, pharmaceutically-acceptable salts
thereof or prodrugs thereof, can be used to inhibit angiogenesis.
Angiogenesis is the process of new blood vessel formation in the
body. Angiogenesis is also used herein to mean the same as, or to
include, neovascularization, vascularization, arterialization and
vasculogenesis.
[0062] Compounds of formula I, pharmaceutically-acceptable salts
thereof or prodrugs thereof, can also be used to treat angiogenic
diseases and conditions. An angiogenic disease or condition is a
disease or condition involving, caused by, exacerbated by, or
dependent on, angiogenesis. Specific angiogenic diseases and
conditions treatable according to the invention include neoplastic
diseases, hypertrophy (e.g., cardiac hypertrophy induced by thyroid
hormone), connective tissue disorders (e.g., rheumatoid arthritis
and atherosclerosis), psoriasis, ocular angiogenic diseases,
cardiovascular diseases, cerebral vascular diseases, endometriosis,
polyposis, obesity, diabetes-associated diseases and hemophiliac
joints. The compounds of formula I, pharmaceutically-acceptable
salts thereof or prodrugs thereof, can also be used to inhibit the
vascularization required for embryo implantation, thereby providing
a method of birth control.
[0063] The compounds of formula I, pharmaceutically-acceptable
salts thereof or prodrugs thereof, will be particularly useful for
the treatment of ocular angiogenic diseases. Ocular angiogenic
diseases include diabetic retinopathy, retinopathy of prematurity,
macular degeneration, corneal graft rejection, neovascular
glaucoma, retrolental fibroplasias, and rubeosis. The compounds of
formula I, pharmaceutically-acceptable salts thereof or prodrugs
thereof, will be especially useful for the treatment of diabetic
retinopathy and macular degeneration.
[0064] The compounds of formula I, pharmaceutically-acceptable
salts thereof or prodrugs thereof, will also be particularly useful
for the treatment of neoplastic diseases. Neoplastic diseases
treatable with the compounds of formula I,
pharmaceutically-acceptable salts thereof or prodrugs thereof,
include malignant tumors (e.g., tumors of the bladder, brain,
breast, cervix, colon, rectum, kidney, liver, lung, ovary,
pancreas, prostate, stomach and uterus), tumor metastasis, and
benign tumors (e.g., hemangiomas, acoustic neuromas, neurofibromas,
trachomas and pyrogenic granulomas)). The compounds of formula I,
pharmaceutically-acceptable salts thereof or prodrugs thereof, will
be especially useful for the treatment of tumors of the brain,
breast, colon, liver and pancreas, most especially tumors of the
brain (e.g., glioblastomas).
[0065] In addition to being able to inhibit angiogenesis, the
compounds of formula I, pharmaceutically-acceptable salts thereof
or prodrugs thereof, have been found to be able to inhibit the
proliferation of cells, reduce the growth of cancer cells, inhibit
the production of cytokines, inhibit Ras and RAP-1, and inhibit the
production of NF.kappa.B and AP-1. Thus, the compounds of formula
I, pharmaceutically-acceptable salts thereof or prodrugs thereof,
will also be particularly useful for the treatment of a variety of
proliferative disorders, including angiogenic diseases and
conditions, especially neoplastic diseases (see above), and other
cancers and other proliferative disorders.
[0066] Cancers treatable with the compounds of formula I,
pharmaceutically-acceptable salts thereof or prodrugs thereof,
include carcinomas, sarcomas, lymphomas, leukemias, solid tumors
and hematologic malignancies. Specific cancers treatable with the
compounds of formula I, pharmaceutically-acceptable salts thereof
or prodrugs thereof, include brain cancers, head and neck cancers,
breast cancers, ovarian cancers, prostate cancers, gastric cancers,
colon cancers, pancreatic cancers, bladder cancers, thyroid
cancers, hepatic cancers, lung cancers, bone cancers and skin
cancers. The compounds of formula I, pharmaceutically-acceptable
salts thereof or prodrugs thereof, will be especially useful for
the treatment of brain cancers, breast cancers, colon cancers,
liver cancers, pancreatic cancers, skin cancers, lymphomas and
leukemias.
[0067] Other proliferative disorders include mesangial cell
proliferation disorders, fibrotic disorders and hyperproliferative
skin disorders. Mesangial cell proliferative disorders refer to
disorders brought about by abnormal proliferation of mesangial
cells. Mesangial cell proliferative disorders include renal
diseases, such as glomerulonephritis, diabetic nephropathy,
malignant nephrosclerosis, thrombotic microangiopathy syndromes and
glomerulopathies. Fibrotic disorders refer to the abnormal
formation of extracellular matrices. Examples of fibrotic disorders
include hepatic cirrhosis, pulmonary fibrosis and atherosclerosis.
Hyperproliferative skin disorders include psoriasis, skin cancer
and epidermal hyperproliferation.
[0068] To treat an animal in need of treatment, a compound of
formula I, pharmaceutically-acceptable salt thereof or prodrug
thereof, is administered to the animal. Preferably, the animal is a
mammal, such as a rabbit, goat, dog, cat, horse or human. Most
preferably, the animal is a human.
[0069] Effective dosage forms, modes of administration and dosage
amounts for the compounds of the invention may be determined
empirically, and making such determinations is within the skill of
the art. It is understood by those skilled in the art that the
dosage amount will vary with the particular compound employed, the
disease or condition to be treated, the severity of the disease or
condition, the route(s) of administration, the rate of excretion of
the compound, the duration of the treatment, the identify of any
other active ingredient(s)s being administered to the animal, the
age, size and species of the animal, and like factors known in the
medical and veterinary arts. In general, a suitable daily dose of a
compound of the present invention will be that amount of the
compound which is the lowest dose effective to produce a
therapeutic effect. However, the daily dosage will be determined by
an attending physician or veterinarian within the scope of sound
medical judgment. If desired, the effective daily dose may be
administered as two, three, four, five, six or more sub-doses,
administered separately at appropriate intervals throughout the
day. Administration of the compound should be continued until an
acceptable response is achieved.
[0070] The compounds useful in the present invention (i.e., the
compounds of formula I and the pharmaceutically-acceptable salts
and prodrugs thereof) may be administered to an animal patient for
therapy by any suitable route of administration, including orally,
nasally, rectally, vaginally, parenterally (e.g., intravenously,
intraspinally, intraperitoneally, subcutaneously, or
intramuscularly), intracisternally, transdermally, intracranially,
intracerebrally, and topically (including buccally and
sublingually). The preferred routes of administration are orally
and topically.
[0071] While it is possible for a compound useful in the present
invention to be administered alone, it is preferable to administer
the compound as a pharmaceutical formulation (composition). The
pharmaceutical compositions useful in the invention comprise one or
more compounds of formula I, or pharmaceutically-acceptable salts
or prodrugs thereof, as active ingredient(s) in admixture with one
or more pharmaceutically-acceptable carriers and, optionally, with
one or more other compounds, active ingredient(s) or other
materials. Each carrier must be "acceptable" in the sense of being
compatible with the other ingredients of the formulation and not
injurious to the animal. Pharmaceutically-acceptable carriers are
well known in the art. Regardless of the route of administration
selected, the compounds of the present invention are formulated
into pharmaceutically-acceptable dosage forms by conventional
methods known to those of skill in the art. See, e.g., Remington's
Pharmaceutical Sciences.
[0072] Formulations of the invention suitable for oral
administration may be in the form of capsules, cachets, pills,
tablets, powders, granules or as a solution or a suspension in an
aqueous or non-aqueous liquid, or an oil-in-water or water-in-oil
liquid emulsions, or as an elixir or syrup, or as pastilles (using
an inert base, such as gelatin and glycerin, or sucrose and
acacia), and the like, each containing a predetermined amount of a
compound or compounds useful in the present invention as an active
ingredient. A compound or compounds useful in the present invention
may also be administered as bolus, electuary or paste.
[0073] In solid dosage forms for oral administration (capsules,
tablets, pills, dragees, powders, granules and the like), the
active ingredient(s) is (are) mixed with one or more
pharmaceutically acceptable carriers, such as sodium citrate or
dicalcium phosphate, and/or any of the following: (1) fillers or
extenders, such as starches, lactose, sucrose, glucose, mannitol,
and/or silicic acid; (2) binders, such as, for example,
carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone,
sucrose and/or acacia; (3) humectants, such as glycerol; (4)
disintegrating agents, such as agar-agar, calcium carbonate, potato
or tapioca starch, alginic acid, certain silicates, and sodium
carbonate; (5) solution retarding agents, such as paraffin; (6)
absorption accelerators, such as quaternary ammonium compounds; (7)
wetting agents, such as, for example, cetyl alcohol and glycerol
monosterate; (8) absorbents, such as kaolin and bentonite clay; (9)
lubricants, such as talc, calcium stearate, magnesium stearate,
solid polyethylene glycols, sodium lauryl sulfate, and mixtures
thereof; and (10) coloring agents. In the case of capsules, tablets
and pills, the pharmaceutical compositions may also comprise
buffering agents. Solid compositions of a similar type may be
employed as fillers in soft and hard-filled gelatin capsules using
such excipients as lactose or milk sugars, as well as high
molecular weight polyethylene glycols and the like.
[0074] A tablet may be made by compression or molding optionally
with one or more accessory ingredients. Compressed tablets may be
prepared using binder (for example, gelatin or hydroxypropylmethyl
cellulose), lubricant, inert diluent, preservative, disintegrant
(for example, sodium starch glycolate or cross-linked sodium
carboxymethyl cellulose), surface-active or dispersing agent.
Molded tablets may be made by molding in a suitable machine a
mixture of the powdered compound moistened with an inert liquid
diluent.
[0075] The tablets, and other solid dosage forms of the
pharmaceutical compositions of the present invention, such as
dragees, capsules, pills and granules, may optionally be scored or
prepared with coatings and shells, such as enteric coatings and
other coatings well known in the pharmaceutical-formulating art.
They may also be formulated so as to provide slow or controlled
release of the active ingredient therein using, for example,
hydroxypropylmethyl cellulose in varying proportions to provide the
desired release profile, other polymer matrices, liposomes and/or
microspheres. They may be sterilized by, for example, filtration
through a bacteria-retaining filter. These compositions may also
optionally contain opacifying agents and may be of a composition
that they release the active ingredient only, or preferentially, in
a certain portion of the gastrointestinal tract, optionally, in a
delayed manner. Examples of embedding compositions which can be
used include polymeric substances and waxes. The active ingredient
can also be in microencapsulated form.
[0076] Liquid dosage forms for oral administration of the compounds
of the invention include pharmaceutically-acceptable emulsions,
microemulsions, solutions, suspensions, syrups and elixirs. In
addition to the active ingredient(s), the liquid dosage forms may
contain inert diluents commonly used in the art, such as, for
example, water or other solvents, solubilizing agents and
emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl
carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate,
propylene glycol, 1,3-butylene glycol, oils (in particular,
cottonseed, groundnut, corn, germ, olive, castor and sesame oils),
glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty
acid esters of sorbitan, and mixtures thereof.
[0077] Besides inert diluents, the oral compositions can also
include adjuvants such as wetting agents, emulsifying and
suspending agents, sweetening, flavoring, coloring, perfuming and
preservative agents.
[0078] Suspensions, in addition to the active compound(s), may
contain suspending agents as, for example, ethoxylated isostearyl
alcohols, polyoxyethylene sorbitol and sorbitan esters,
microcrystalline cellulose, aluminum metahydroxide, bentonite,
agar-agar and tragacanth, and mixtures thereof.
[0079] Pharmaceutical formulations for intraocular injection of a
compound or compounds of the invention into the eyeball include
solutions, emulsions, suspensions, particles, capsules,
microspheres, liposomes, matrices, etc. See, e.g., U.S. Pat. No.
6,060,463, U.S. Patent Application Publication No. 2005/0101582,
and PCT application WO 2004/043480, the complete disclosures of
which are incorporated herein by reference. For instance, a
pharmaceutical formulation for intraocular injection may comprise
one or more compounds of the invention in combination with one or
more pharmaceutically-acceptable sterile isotonic aqueous or
non-aqueous solutions, suspensions or emulsions, which may contain
antioxidants, buffers, suspending agents, thickening agents or
viscosity-enhancing agents (such as a hyaluronic acid polymer).
Examples of suitable aqueous and nonaqueous carriers include water,
saline (preferably 0.9%), dextrose in water (preferably 5%),
buffers, dimethylsulfoxide, alcohols and polyols (such as glycerol,
propylene glycol, polyethylene glycol, and the like). These
compositions may also contain adjuvants such as wetting agents and
emulsifying agents and dispersing agents. In addition, prolonged
absorption of the injectable pharmaceutical form may be brought
about by the inclusion of agents which delay absorption such as
polymers and gelatin. Injectable depot forms can be made by
incorporating the drug into microcapsules or microspheres made of
biodegradable polymers such as polylactide-polyglycolide. Examples
of other biodegradable polymers include poly(orthoesters),
poly(glycolic) acid, poly(lactic) acid, polycaprolactone and
poly(anhydrides). Depot injectable formulations are also prepared
by entrapping the drug in liposomes (composed of the usual
ingredients, such as dipalmitoyl phosphatidylcholine) or
microemulsions which are compatible with eye tissue. Depending on
the ratio of drug to polymer or lipid, the nature of the particular
polymer or lipid components, the type of liposome employed, and
whether the microcapsules or microspheres are coated or uncoated,
the rate of drug release from microcapsules, microspheres and
liposomes can be controlled.
[0080] The compounds of the invention can also be administered
surgically as an ocular implant. For instance, a reservoir
container having a diffusible wall of polyvinyl alcohol or
polyvinyl acetate and containing a compound or compounds of the
invention can be implanted in or on the sclera. As another example,
a compound or compounds of the invention can be incorporated into a
polymeric matrix made of a polymer, such as polycaprolactone,
poly(glycolic) acid, poly(lactic) acid, poly(anhydride), or a
lipid, such as sebacic acid, and may be implanted on the sclera or
in the eye. This is usually accomplished with the animal receiving
a topical or local anesthetic and using a small incision made
behind the cornea. The matrix is then inserted through the incision
and sutured to the sclera.
[0081] A preferred embodiment of the invention is local topical
administration of the compounds of the invention to the eye, and a
particularly preferred embodiment of the invention is a topical
pharmaceutical composition suitable for application to the eye.
Topical pharmaceutical compositions suitable for application to the
eye include solutions, suspensions, dispersions, drops, gels,
hydrogels and ointments. See, e.g., U.S. Pat. No. 5,407,926 and PCT
applications WO 2004/058289, WO 01/30337 and WO 01/68053, the
complete disclosures of all of which are incorporated herein by
reference.
[0082] Topical formulations suitable for application to the eye for
treatment of an angiogenic disease or condition comprise one or
more compounds of the invention in an aqueous or nonaqueous base.
The topical formulations can also include absorption enhancers,
permeation enhancers, thickening agents, viscosity enhancers,
agents for adjusting and/or maintaining the pH, agents to adjust
the osmotic pressure, preservatives, surfactants, buffers, salts
(preferably sodium chloride), suspending agents, dispersing agents,
solubilizing agents, stabilizers and/or tonicity agents. Topical
formulations suitable for application to the eye for treatment of
an angiogenic disease or condition will preferably comprise an
absorption or permeation enhancer to promote absorption or
permeation of the compound or compounds of the invention into the
eye and/or a thickening agent or viscosity enhancer that is capable
of increasing the residence time of a compound or compounds of the
invention in the eye. See PCT applications WO 2004/058289, WO
01/30337 and WO 01/68053. Exemplary absorption/permeation enhancers
include methysulfonylmethane, alone or in combination with
dimethylsulfoxide, carboxylic acids and surfactants. Exemplary
thickening agents and viscosity enhancers include dextrans,
polyethylene glycols, polyvinylpyrrolidone, polysaccharide gels,
Gelrite.RTM., cellulosic polymers (such as hydroxypropyl
methylcellulose), carboxyl-containing polymers (such as polymers or
copolymers of acrylic acid), polyvinyl alcohol and hyaluronic acid
or a salt thereof.
[0083] Liquid dosage forms (e.g., solutions, suspensions,
dispersions and drops) can be prepared, for example, by dissolving,
dispersing, suspending, etc. a compound or compounds of the
invention in a vehicle, such as, for example, water, saline,
aqueous dextrose, glycerol, ethanol and the like, to form a
solution, dispersion or suspension. If desired, the pharmaceutical
formulation may also contain minor amounts of non-toxic auxillary
substances, such as wetting or emulsifying agents, pH buffering
agents and the like, for example sodium acetate, sorbitan
monolaurate, triethanolamine sodium acetate, triethanolamine
oleate, etc.
[0084] Aqueous solutions and suspensions can include, in addition
to a compound or compounds of the invention, preservatives,
surfactants, buffers, salts (preferably sodium chloride), tonicity
agents and water. If suspensions are used, the particle sizes
should be less than 10 .mu.m to minimize eye irritation. If
solutions or suspensions are used, the amount delivered to the eye
should not exceed 50 .mu.l to avoid excessive spillage from the
eye.
[0085] Colloidal suspensions are generally formed from
microparticles (i.e., microspheres, nanospheres, microcapsules or
nanocapsules, where microspheres and nanospheres are generally
monolithic particles of a polymer matrix in which the formulation
is trapped, adsorbed, or otherwise contained, while with
microcapsules and nanocapsules the formulation is actually
encapsulated). The upper limit for the size of these microparticles
is about 5.mu. to about 10.mu..
[0086] Ophthalmic ointments include a compound or compounds of the
invention in an appropriate base, such as mineral oil, liquid
lanolin, white petrolatum, a combination of two or all three of the
foregoing, or polyethylene-mineral oil gel. A preservative may
optionally be included.
[0087] Ophthalmic gels include a compound or compounds of the
invention suspended in a hydrophilic base, such as Carpobol-940 or
a combination of ethanol, water and propylene glycol (e.g., in a
ratio of 40:40:20). A gelling agent, such as
hydroxylethylcellulose, hydroxypropylcellulose,
hydroxypropylmethylcellulose or ammoniated glycyrrhizinate, is
used. A preservative and/or a tonicity agent may optionally be
included.
[0088] Hydrogels are formed by incorporation of a swellable,
gel-forming polymer, such as those listed above as thickening
agents or viscosity enhancers, except that a formulation referred
to in the art as a "hydrogel" typically has a higher viscosity than
a formulation referred to as a "thickened" solution or suspension.
In contrast to such preformed hydrogels, a formulation may also be
prepared so to form a hydrogel in situ following application to the
eye. Such gels are liquid at room temperature but gel at higher
temperatures (and thus are termed "thermoreversible" hydrogels),
such as when placed in contact with body fluids. Biocompatible
polymers that impart this property include acrylic acid polymers
and copolymers, N-isopropylacrylamide derivatives and ABA block
copolymers of ethylene oxide and propylene oxide (conventionally
referred to as "poloxamers" and available under the Pluronic.RTM.
tradename from BASF-Wayndotte).
[0089] Preferred dispersions are liposomal, in which case the
formulation is enclosed within liposomes (microscopic vesicles
composed of alternating aqueous compartments and lipid
bilayers).
[0090] Eye drops can be formulated with an aqueous or nonaqueous
base also comprising one or more dispersing agents, solubilizing
agents or suspending agents. Drops can be delivered by means of a
simple eye dropper-capped bottle or by means of a plastic bottle
adapted to deliver liquid contents dropwise by means of a specially
shaped closure.
[0091] The compounds of the invention can also be applied topically
by means of drug-impregnated solid carrier that is inserted into
the eye. Drug release is generally effected by dissolution or
bioerosion of the polymer, osmosis, or combinations thereof.
Several matrix-type delivery systems can be used. Such systems
include hydrophilic soft contact lenses impregnated or soaked with
the desired compound of the invention, as well as biodegradable or
soluble devices that need not be removed after placement in the
eye. These soluble ocular inserts can be composed of any degradable
substance that can be tolerated by the eye and that is compatible
with the compound of the invention that is to be administered. Such
substances include, but are not limited to, poly(vinyl alcohol),
polymers and copolymers of polyacrylamide, ethylacrylate and
vinylpyrrolidone, as well as cross-linked polypeptides or
polysaccharides, such as chitin.
[0092] Dosage forms for the other types of topical administration
(i.e., not to the eye) or for transdermal administration of
compounds of the invention include powders, sprays, ointments,
pastes, creams, lotions, gels, solutions, patches, drops and
inhalants. The active ingredient may be mixed under sterile
conditions with a pharmaceutically-acceptable carrier, and with any
buffers, or propellants which may be required. The ointments,
pastes, creams and gels may contain, in addition to the active
ingredient, excipients, such as animal and vegetable fats, oils,
waxes, paraffins, starch, tragacanth, cellulose derivatives,
polyethylene glycols, silicones, bentonites, silicic acid, talc and
zinc oxide, or mixtures thereof. Powders and sprays can contain, in
addition to the active ingredient, excipients such as lactose,
talc, silicic acid, aluminum hydroxide, calcium silicates and
polyamide powder or mixtures of these substances. Sprays can
additionally contain customary propellants such as
chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons,
such as butane and propane. Transdermal patches have the added
advantage of providing controlled delivery of compounds of the
invention to the body. Such dosage forms can be made by dissolving,
dispersing or otherwise incorporating one or more compounds of the
invention in a proper medium, such as an elastomeric matrix
material. Absorption enhancers can also be used to increase the
flux of the compound across the skin. The rate of such flux can be
controlled by either providing a rate-controlling membrane or
dispersing the compound in a polymer matrix or gel.
[0093] Formulations of the pharmaceutical compositions for rectal
or vaginal administration may be presented as a suppository, which
may be prepared by mixing one or more compounds of the invention
with one or more suitable nonirritating excipients or carriers
comprising, for example, cocoa butter, polyethylene glycol, a
suppository wax or salicylate, and which is solid at room
temperature, but liquid at body temperature and, therefore, will
melt in the rectum or vaginal cavity and release the active
compound. Formulations of the present invention which are suitable
for vaginal administration also include pessaries, tampons, creams,
gels, pastes, foams or spray formulations containing such carriers
as are known in the art to be appropriate.
[0094] Pharmaceutical formulations include those suitable for
administration by inhalation or insufflation or for nasal or
intraocular administration. For administration to the upper (nasal)
or lower respiratory tract by inhalation, the compounds of the
invention are conveniently delivered from an insufflator, nebulizer
or a pressurized pack or other convenient means of delivering an
aerosol spray. Pressurized packs may comprise a suitable propellant
such as dichlorodifluoromethane, trichlorofluoromethane,
dichlorotetrafluoroethane, carbon dioxide, or other suitable gas.
In the case of a pressurized aerosol, the dosage unit may be
determined by providing a valve to deliver a metered amount.
[0095] Alternatively, for administration by inhalation or
insufflation, the composition may take the form of a dry powder,
for example, a powder mix of one or more compounds of the invention
and a suitable powder base, such as lactose or starch. The powder
composition may be presented in unit dosage form in, for example,
capsules or cartridges, or, e.g., gelatin or blister packs from
which the powder may be administered with the aid of an inhalator,
insufflator or a metered-dose inhaler.
[0096] For intranasal administration, compounds useful in the
invention may be administered by means of nose drops or a liquid
spray, such as by means of a plastic bottle atomizer or
metered-dose inhaler. Liquid sprays are conveniently delivered from
pressurized packs. Typical of atomizers are the Mistometer
(Wintrop) and Medihaler (Riker).
[0097] Drops, such as eye drops or nose drops, may be formulated
with an aqueous or nonaqueous base also comprising one or more
dispersing agents, solubilizing agents or suspending agents. Drops
can be delivered by means of a simple eye dropper-capped bottle or
by means of a plastic bottle adapted to deliver liquid contents
dropwise by means of a specially shaped closure.
[0098] Pharmaceutical compositions suitable for parenteral
administrations comprise one or more compounds useful in the
invention in combination with one or more
pharmaceutically-acceptable sterile isotonic aqueous or non-aqueous
solutions, dispersions, suspensions or emulsions, or sterile
powders which may be reconstituted into sterile injectable
solutions or dispersions just prior to use, which may contain
antioxidants, buffers, solutes which render the formulation
isotonic with the blood of the intended recipient or suspending or
thickening agents.
[0099] Examples of suitable aqueous and nonaqueous carriers which
may be employed in the pharmaceutical compositions of the invention
include water, ethanol, polyols (such as glycerol, propylene
glycol, polyethylene glycol, and the like), and suitable mixtures
thereof, vegetable oils, such as olive oil, and injectable organic
esters, such as ethyl oleate. Proper fluidity can be maintained,
for example, by the use of coating materials, such as lecithin, by
the maintenance of the required particle size in the case of
dispersions, and by the use of surfactants.
[0100] These compositions may also contain adjuvants such as
wetting agents, emulsifying agents and dispersing agents. It may
also be desirable to include isotonic agents, such as sugars,
sodium chloride, and the like in the compositions. In addition,
prolonged absorption of the injectable pharmaceutical form may be
brought about by the inclusion of agents which delay absorption
such as aluminum monosterate and gelatin.
[0101] In some cases, in order to prolong the effect of the active
ingredient(s), it is desirable to slow the absorption of the active
ingredient(s) from subcutaneous or intramuscular injection. This
may be accomplished by the use of a liquid suspension of
crystalline or amorphous material having poor water solubility. The
rate of absorption of the active ingredient(s) then depends upon
its rate of dissolution which, in turn, may depend upon crystal
size and crystalline form. Alternatively, delayed absorption of a
parenterally-administered active ingredient(s) is accomplished by
dissolving or suspending the active ingredient(s) in an oil
vehicle.
[0102] Injectable depot forms are made by forming microencapsule
matrices of the active ingredient(s) in biodegradable polymers such
as polylactide-polyglycolide. Depending on the ratio of active
ingredient(s) to polymer, and the nature of the particular polymer
employed, the rate of release of the active ingredient(s) can be
controlled. Examples of other biodegradable polymers include
poly(orthoesters) and poly(anhydrides). Depot injectable
formulations are also prepared by entrapping the active
ingredient(s) in liposomes or microemulsions which are compatible
with body tissue. The injectable materials can be sterilized for
example, by filtration through a bacterial-retaining filter.
[0103] The formulations may be presented in unit-dose or multi-dose
sealed containers, for example, ampules and vials, and may be
stored in a lyophilized condition requiring only the addition of
the sterile liquid carrier, for example water for injection,
immediately prior to use. Extemporaneous injection solutions and
suspensions may be prepared from sterile powders, granules and
tablets of the type described above.
[0104] Additional objects, advantages, and novel features of this
invention will become apparent to those skilled in the art upon
examination of the following examples thereof, which are not
intended to be limiting.
EXAMPLES
Example 1
[0105] Whole blood was drawn from GR283, a human volunteer with
known allergies, into a glass vacutainer tube containing no
anticoagulant. This blood was allowed to clot, and the serum was
removed by centrifugation and then heat inactivated by placing it
in a water bath at 56.degree. C. for 30 minutes. Whole blood from
GR283 was also drawn into a glass vacutainer tube containing
heparin and used for peripheral blood lymphocytes (PBL) isolation
as follows. Whole blood was layered over room temperature
Histopaque 1077 solution and centrifuged at 2000 rpm for 15 minutes
at room temperature. Cells at the plasma-Histopaque interface were
then removed and washed with culture medium (IMDM medium with 10%
heat-inactivated GR283 serum plus 1% penicillin/streptomycin) at
37.degree. C.
[0106] The compound of formula II (see above) and methylphenidate
(both obtained from Dr. Jeffrey D. Winkler, University of
Pennsylvania, Philadelphia, Pa.) in culture medium were added to
wells of a 96-well plate to give final concentrations of 5
.mu.g/ml, 15 .mu.g/ml and 50 .mu.g/ml of the compound of formula II
and of methylphenidate. Sterile 18 M.OMEGA. water, the solvent for
the compound of formula II, and dexamethasone (obtained from Sigma)
(final concentration of 10 .mu.g/ml in water) were used as
controls. Then, GR283's PBL in culture medium were added to the
wells to give a final concentration of 150,000 cells per well, and
the plates were incubated at 37.degree. C., 5% CO.sub.2 for 24
hours. After this incubation, phytohemagglutinin (PHA) in culture
medium was added to give final concentrations of 2 .mu.g/ml, 5
.mu.g/ml or 20 .mu.g/ml, final total volume of 200 .mu.l/well, and
the cells were incubated for an additional 72 hours at 37.degree.
C., 5% CO.sub.2. All cultures were performed in triplicate.
[0107] At the end of this incubation, cell clumping was examined by
photographing representative wells with a digital camera mounted to
an inverted microscope. The compound of formula II reduced the
amount of cell clumping induced by 5 .mu.g/ml PHA in a
dose-dependent manner. The compound of formula II attenuated cell
clumping, presumably, as a result of decreased expression of
cellular adhesion molecules on the surfaces of the cells.
[0108] Cell proliferation was assayed by adding 20 .mu.l of Promega
cell titer solution to each well and incubating the plate for an
additional 4 hours. Promega cell titer solution is a solution
containing a tetrazolium dye that is reduced by living cells to a
formazan dye, and this reduction is proportional to the number of
living cells present in the well. After the 4-hour incubation, the
optical density (OD) at 530 nm of each well was measured. The OD at
530 nm for blank wells containing no cells was subtracted from the
OD of the experimental wells. The results of the proliferation
assays are presented in FIGS. 1A-C. As can be seen from FIGS. 1A-C,
the compound of formula II (Cpd. II) and dexamethasone (Dex)
significantly inhibited the proliferation of PBL stimulated with
PHA in a dose-dependent manner. Methylphenidate (MP) showed a
significant effect at its highest dose and the lowest PHA dose.
Otherwise, methyphenidate did not significantly reduce the
proliferation of the PHA-stimulated PBL.
Example 2
[0109] Whole blood was drawn from GR467, a human volunteer with
known allergies, and processed as described in Example 1 to give
heat-inactivated serum and PBL. The compound of formula II and
methylphenidate in culture medium (made using heat-inactivated
GR467 serum) were added to wells of a 96-well plate to give final
concentrations of 5 .mu.g/ml, 15 .mu.g/ml and 25 .mu.g/ml of the
compound of formula II and 15 .mu.g/ml methylphendiate. Water and
dexamethasone (final concentration of 10 .mu.M) were used as
controls. Then, GR467's PBL in culture medium were added to the
wells to give a final concentration of 150,000 cells per well, and
the plates were incubated at 37.degree. C., 5% CO.sub.2 for 24
hours. After this incubation, PHA was added to give a final
concentration of 2 .mu.g/ml, final total volume of 200 .mu.l/well,
and the cells were incubated for an additional 72 hours at
37.degree. C., 5% CO.sub.2. All cultures were performed in
triplicate.
[0110] At the end of this incubation, cell proliferation was
determined as described in Example 1. The results are presented in
FIG. 2. As can be seen from FIG. 2, the compound of formula II (Cpd
II) and dexamethasone (Dex) significantly inhibited the
proliferation of PBL, both unstimulated and stimulated with PHA,
whereas methylphenidate did not.
[0111] The release of cytokines by the PBL was also measured by
culturing the PBL in 1 ml tubes, at 1.3.times.10.sup.6 cells per
ml, with 15 .mu.g/ml of the compound of formula II, 15 .mu.g/ml
methylphenidate or 10 .mu.M dexamethasone at 37.degree. C., 5%
CO.sub.2 for 24 hours. After this incubation, PHA was added to give
a final concentration of 2 .mu.g/ml, and the cells were incubated
for an additional 96 hours at 37.degree. C., 5% CO.sub.2. All
cultures were performed in triplicate. Cells were then removed by
centrifugation at 1000 rpm for 10 minutes, and the culture medium
collected.
[0112] IL-13 is made by activated T.sub.H2 cells, and IL-13's
primary targets are B-cells and monocytes. IL-13 stimulates humoral
immune responses, and it has been implicated in the pathogenesis of
asthma. IL-13 is secreted by lymphoma cell lines and may be an
autocrine growth factor. IL-13 is also expressed in pancreatic
cancer. However, IL-13 has also been reported to inhibit the growth
of other types of tumors, such as gliomas and renal cell
carcinomas
[0113] IFN.gamma. is a proinflammatory cytokine made by activated
T-cells and other cells. IFN.gamma. can activate neutrophils,
endothelial cells and macrophages, as well as cause an increase in
MHC molecule expression. IFN.gamma. drives the cell-mediated immune
response. IFN.gamma. plays an important role in the immune-mediated
rejection of established tumors. IFN.gamma. has antiproliferative
effects on some tumors, can have apoptotic effects on others, can
induce the production of angiostatic chemokines and enhances the
immunogenicity of tumor cells.
[0114] Release of IL-13 and interferon gamma (IFN.gamma.) into the
culture medium was measured by ELISA. To perform the ELISA, matched
pairs of antibodies against human IL-13 and IFN.gamma. were
purchased from Pierce Biotechnology and Biosource, respectively.
ELISA strip well plates were coated with 10 .mu.g/ml of antibody
(in phosphate-buffered saline (PBS)) to IL-13 and 4 .mu.g/ml of
antibody to IFN.gamma. (in PBS) overnight at room temperature. The
plates were then blocked using a 4% BSA solution in PBS for one
hour, followed by the addition of 50 .mu.l of experimental culture
medium per well in duplicate. The plates were incubated at room
temperature for one hour and then washed using 50 mM Tris pH 8.0
with 0.1% Tween 20. Then, solutions of 400 ng/ml biotinylated
second antibody to IL-13 and 500 ng/ml biotinylated second antibody
to IFN.gamma. were made in blocking buffer, and 100 .mu.l were
added per well. The plates were incubated for 1 hour and washed
again. A 1:8000 dilution of Strepavidin HRP (Pierce Biotechnology)
conjugate was made in blocking buffer, and 100 .mu.l were added to
the wells and incubation continued for 30 minutes. A final wash
step was performed, after which 100 .mu.l Pierce Biotechnology TMB
substrate were added to each well. Color was developed for 30
minutes and stopped by adding 100 .mu.l 0.18 N H.sub.2SO.sub.4. OD
was determined using microplate reader with a 450 nM filter.
[0115] The results for IL-13 are shown in FIG. 3. As can be seen,
the compound of formula II (Cpd. II) and dexamethasone (Dex)
significantly inhibited IL-13 release induced by PHA.
Methylphenidate (MP) did not inhibit the release of IL-13. Indeed,
methylphenidate increased the release of IL-13 by the
PHA-stimulated cells.
[0116] The results for IFN.gamma. are shown in FIG. 4. As can be
seen, the compound of formula II (Cpd. II) and dexamethasone (Dex)
significantly inhibited IFN.gamma. release in both unstimulated
cells and in cells stimulated with PHA. Methylphenidate (MP) had
some effect on the release of IFN.gamma. by unstimulated cells, but
did not significantly suppress the release of IFN.gamma. from cells
stimulated with PHA. Indeed, methylphenidate increased the release
of IFN.gamma. by the PHA-stimulated cells.
Example 3
[0117] Whole blood was drawn from GR191, a normal human volunteer,
and processed as described in Example 1 to give heat-inactivated
serum and PBL. The compound of formula II and methylphenidate in
culture medium (made using heat-inactivated GR191 serum) were added
to wells of a 96-well plate to give final concentrations of 5
.mu.g/ml, 15 .mu.g/ml, 25 .mu.g/ml and 50 .mu.g/ml of the compound
of formula II and 50 .mu.g/ml methylphendiate. Water, mouse nerve
growth factor (Upstate Biotechnology, Inc) (NGF) (final
concentration of 250 ng/ml) and dexamethasone (final concentration
of 10 .mu.M) were used as controls. Then, GR191's PBL in culture
medium were added to the wells to give a final concentration of
150,000 cells per well, and the plates were incubated at 37.degree.
C., 5% CO.sub.2 for 24 hours. After this incubation, PHA was added
to give final concentrations of 2 .mu.g/ml and 5 .mu.g/ml, final
total volume of 200 .mu.l/well, and the cells were incubated for an
additional 72 hours at 37.degree. C., 5% CO.sub.2. All cultures
were performed in triplicate.
[0118] At the end of this incubation, cell proliferation was
determined as described in Example 1. The results are presented in
FIGS. 5A-B. As can be seen from FIGS. 5A-B, the compound of formula
II (Cpd. II) and dexamethasone (Dex) significantly inhibited the
proliferation of PBL, both unstimulated and stimulated with PHA,
whereas methylphenidate (MP) did not.
[0119] The release of cytokines by the PBL was also measured by
culturing the PBL in 1 ml tubes, at 1.times.10.sup.6 cells per ml,
with 15 .mu.g/ml and 50 .mu.g/ml of the compound of formula II or
10 .mu.M dexamethasone at 37.degree. C., 5% CO.sub.2 for 24 hours.
After this incubation, PHA was added to give a final concentration
of 5 .mu.g/ml, and the cells were incubated for an additional 72
hours at 37.degree. C., 5% CO.sub.2. All cultures were performed in
triplicate. Cells were then removed by centrifugation at 1000 rpm
for 10 minutes.
[0120] The supernatants were collected, and the concentrations of
IL-13 and tumor necrosis factor alpha (TNF.alpha.) in the
supernatants were measured by ELISA. The IL-13 ELISA was performed
as described in Example 2. The results are presented in FIG. 6. As
can be seen in FIG. 6, the compound of formula II (Cpd. II) and
dexamethasone (Dex) significantly inhibited the release of IL-13
from the PHA-stimulated PBL.
[0121] TNF.alpha. is a proinflammatory cytokine made by activated
T-cells and other cells. TNF.alpha. causes endothelial cells to
express adhesion molecules and may play a role in the recruitment
of immune cells to the sites of inflammation. TNF.alpha. has been
detected in multiple solid and hemotologic malignancies. A number
of different intracellular signals are induced by TNF.alpha.,
including signals for both cells survival through NF.kappa.B and
AP-1 and cell death through caspase activation. NF.kappa.B is a key
regulator of cell survival and promoter of carcinogenesis in
multiple tumor types.
[0122] The TNF.alpha. ELISA was performed as described in Example 2
using matched pair antibodies from Pierce Endogen (2 .mu.g/ml for
the coating antibody and 250 ng/ml for the second antibody). The
results are presented in FIG. 7. As can be seen in FIG. 7, the
compound of formula II (Cpd. II) and dexamethasone (Dex)
significantly inhibited the release of TNF.alpha. from
PHA-stimulated PBL.
[0123] The cells were further analyzed by flow cytometry. Annexin
was used to determine populations of dead or dying cells. Anti-CD69
antibody was used to establish the level of cellular activation.
Antibody to T-cell receptor .alpha..beta. (TCR) was also used.
Recombinant Annexin 5 (PE and FITC conjugates) and the antibodies
were all purchase from Caltag (Burlingham, Calif.) and used
following the manufacturer's recommendations. The following results
were observed.
Cell Death:
[0124] Annexin staining of TCR-positive cells increased from 7.3%
(background) to 45% and 23% with 50 .mu.g/ml and 15 .mu.g/ml of the
compound of formula II, respectively, signifying an increase in
cell death in the T-cell population. Stimulation with PHA at 5
.mu.g/ml increased the annexin staining of TCR-positive cells to
67%. This indicates that PHA can also induce cell death in the
T-cell population. Cell death decreased slightly as a result of
treatment with PHA plus 15 .mu.g/ml of the compound of formula II
(62% of the TCR-positive cells stained for annexin with PHA and IMM
0001 versus 67% with PHA alone). PHA plus 50 .mu.g/ml of the
compound of formula II caused 87% cell death in the TCR-positive
subset of cells as seen by annexin staining. These results show
that the higher 50 .mu.g/ml concentration of the compound of
formula II caused significant death of T-cells, whereas the lower
15 .mu.g/ml concentration did not. Dexamethasone rescued the
PHA-induced increase in annexin staining of TCR-positive cells
(decreased from 84% to 48%), demonstrating that the control
compound is working properly.
Activation Of T-Cells:
[0125] CD69+TCR staining (activated T cells) was not detected in
any of the controls (nil, compound of formula II alone and
dexamethasone alone). PHA increased CD69+TCR staining to 84%. Only
PHA caused T-cell activation as detectable by increased CD 69
staining. CD69+TCR staining of PHA-stimulated cells dropped from
84% to 54% with 50 .mu.g/ml of the compound of formula II and to
64% with 15 .mu.g/ml of the compound of formula II. Dexamethasone
was less effective than the compound of formula II at reducing the
CD69+TCR staining of PHA-stimulated cells. Thus, the compound of
formula II is more effective at decreasing T-cell activation than
dexamethasone, a potent anti-inflammatory.
Example 4
[0126] Whole blood was drawn from GR-192, a normal human volunteer,
and processed as described in Example 1 to give heat-inactivated
serum and PBL. Then, GR-192's PBL were cultured in 1 ml tubes, at
1.3.times.10.sup.6 cells per ml, with 15 .mu.g/ml of the compound
of formula II (in culture medium made using 10% heat-inactivated
GR-192 serum) or 10 .mu.M dexamethasone, at 37.degree. C., 5%
CO.sub.2 for 24 hours. After this incubation, PHA was added to give
a final concentration of 2 .mu.g/ml, and the cells were incubated
for an additional 96 hours at 37.degree. C., 5% CO.sub.2. All
cultures were performed in triplicate. Cells were then removed by
centrifugation at 1000 rpm for 10 minutes, and the culture medium
collected.
[0127] Release of IL-8 into the culture medium was measured by
ELISA. IL-8 is a pro-inflammatory cytokine and a potent
chemoattractant and activator of neutrophils. It has also been
reported to be a chemoattractant and activator of T-lymphocytes and
eosinophils. IL-8 is produced by immune cells (including
lymphocytes, neutrophils, monocytes and macrophages), fibroblasts
and epithelial cells. IL-8 has potent angiogenic activity.
[0128] To perform the ELISA, matched pairs of antibodies against
human IL-8 were purchased from Pierce Biotechnology and Biosource,
respectively. ELISA strip well plates were coated with 2 .mu.g/ml
of antibody to IL-8 (in phosphate-buffered saline (PBS)) overnight
at room temperature. The plates were then blocked using a 4% BSA
solution in PBS for one hour, followed by the addition of 50 .mu.l
of experimental culture medium per well in duplicate. The plates
were incubated at room temperature for one hour and then washed
using 50 mM Tris pH 8.0 with 0.1% Tween 20. Then, solutions of 100
ng/ml biotinylated second antibody to IL-8 were made in blocking
buffer, and 100 .mu.l were added per well. The plates were
incubated for 1 hour and washed again. A 1:8000 dilution of
Strepavidin HRP (Pierce Biotechnology) conjugate was made in
blocking buffer, and 100 .mu.l were added to the wells and
incubation continued for 30 minutes. A final wash step was
performed, after which 100 .mu.l Pierce Biotechnology TMB substrate
were added to each well. Color was developed for 30 minutes and
stopped by adding 100 .mu.l 0.18 N H.sub.2SO.sub.4. OD was
determined using microplate reader with a 450 nm filter.
[0129] The results are shown in FIG. 8. As can be seen, the
compound of formula II (Cpd. II) and dexamethasone (Dex)
significantly inhibited IL-8 release induced by PHA.
[0130] A CD4-positive human T-lymphocyte cell line (TRiPS), which
was isolated from an influenza-immunized donor and is specific for
hemagglutinin peptide 307-319, was stimulated for passage using
approximately 4.times.10.sup.5 cells on day 18-20 after a previous
stimulation. Cells were washed once in cold Iscove's Modified
Dulbecco Minimal Essential Medium (IMDM, Sigma) plus 10% fetal
bovine serum (FBS; American Type Culture Collection (ATCC)) and
resuspended in 1.0 ml cold IMDM medium containing a 1:500 dilution
of anti-CD3 monoclonal antibody OKT3 (prepared from mouse ascites
fluid). Cells were incubated with antibody for 30 minutes on ice,
then washed with cold medium without FBS and combined with
approximately 2.times.10.sup.6 4000R-irradiated normal human donor
peripheral blood leukocytes (PBL), as feeder cells, in medium plus
50 U/ml human IL-2 (Xenometrix). Cultures were expanded by the
addition of fresh IMDM medium with FBS plus IL-2 on day 3. Day of
culture is measured from the day of stimulation with OKT3. Cells
can be used for experiments starting on day 7 (at maximum
proliferation), typically on day 14 (most sensitive to
re-stimulation) and up until day 21 (resting cells approaching
senescence).
[0131] Activation experiments were performed by withdrawing an
aliquot of cells and washing twice with warmed (37.degree. C.)
IMDM. For each specific assay, 2.times.10.sup.5 viable cells were
pre-incubated in a total volume of 0.9 ml warmed IMDM medium
containing 15 .mu.g/ml of the compound of formula II or 10 .mu.M
dexamethasone for 15 minutes at 37.degree. C. An aliquot of
2.times.10.sup.5 CD3/CD28 Dynabeads (Dynal), as activating
stimulus, in 0.1 ml warmed IMDM was then added, and the cultures
incubated 24 hours at 37.degree. C. Supernatants of the cell
cultures were harvested after pelleting the cells by
centrifugation.
[0132] Cytokine content was assayed by specific IL-8 ELISA as
described above. It was found that the compound of formula II had
no effect on IL-8 production by the TRiPS cell line.
Example 5
[0133] THP-1 is a monocyte cell line obtained from American Type
Culture Collection (ATCC) (catalog no. TIB-202). THP-1 cells were
placed in medium (RPMI containing 10% FCS and 8 ng/ml
monothioglycerol (obtained from Sigma)) at a concentration of
250,000 cells per ml and incubated with 15 .mu.g/ml of compound of
formula II or 10 .mu.M dexamethasone for one hour at 37.degree. C.
and 5% CO.sub.2. After 1 hour, lipopolysaccharide (LPS) (obtained
from Sigma) was added to the cultures to give a final concentration
of 200 ng/ml, and the cells were then incubated for an additional 4
hours or for an additional 24 hours. After the incubation, the
cells were centrifuged, and the supernatants were collected. The
concentrations of IL-8 and TNF.alpha. in the supernatants were
determined by ELISA.
[0134] The concentrations of IL-8 in the supernatants were
determined by ELISA performed as described in Example 4. The
results are presented in Table 1 below. As can be seen in Table 1,
the compound of formula II (Cpd. II) and dexamethasone (Dex)
significantly inhibited the release of IL-8 from the LPS-stimulated
monocytes.
[0135] The TNF.alpha. ELISA was performed as described in Example
2. The results are presented in Table 2 below. As can be seen in
Table 2, the compound of formula II (Cpd. II) and dexamethasone
(Dex) significantly inhibited the release of TNF.alpha. from the
LPS-stimulated monocytes. TABLE-US-00001 TABLE 1 Time Of Mean IL-8
Sample Incubation Concentration (pg/ml) % Inhibition Control (no
additives) 4 hours 75.96 .+-. 12.73 N/A LPS 4 hours 2844.60 .+-.
180.55 N/A LPS + Cpd II 4 hours 2185.00 .+-. 78.30 23% LPS + Dex 4
hours 2102.18 .+-. 52.20 26% Control (no additives) 24 hours 46.09
.+-. 22.42 N/A LPS 24 hours 6653.20 .+-. 193.18 N/A LPS + Cpd II 24
hours 4490.20 .+-. 264.46 33% LPS + Dex 24 hours 2300.00 .+-.
283.41 66%
[0136] TABLE-US-00002 TABLE 2 Time Of Mean TNF.alpha. Sample
Incubation Concentration (pg/ml) % Inhibition Control (no
additives) 24 hours 1.415 .+-. 1.464 N/A LPS 24 hours 138.655 .+-.
0.601 N/A LPS + Cpd II 24 hours 65.370 .+-. 0.891 53% LPS + Dex 24
hours 94.759 .+-. 8.755 32%
Example 6
[0137] The Jurkat T-lymphocyte leukemia cell line was obtained from
American Type Culture Collection (ATCC), Rockville, Md. (catalog
no. TIB-152). Jurkat cells, at 1.times.10.sup.5 cells/ml, were
cultured at 37.degree. C. and 5% CO.sub.2 in IMDM medium (ATCC)
with 10% FCS for 72 hours with 7.5 .mu.g/ml or 15 .mu.g/ml of the
compound of formula II (Cpd II). Following the incubation, the
cells were washed with Hepes buffered saline, split into three
equal volumes, and then incubated with 5 .mu.M ethidium bromide
dimer-1 (ETH-D1) (obtained from Molecular Probes) and 5 .mu.M
calcein AM solution (obtained from Promega) for one hour at
37.degree. C. and 5% CO.sub.2 in 96-well culture plates to assay
for cell viability. The fluorescence in each well was measured
using a plate reader at excitation/emission 485/530 nm and 530/645
nm. Relative percentage of dead to live cells was calculated by
dividing ETH-D1 fluorescence by calcein AM fluorescence. The
results are shown in Table 3 below. TABLE-US-00003 TABLE 3 Sample
Relative Percentage Dead/Live Control (no additives) 20.85% .+-.
1.42% 7.5 .mu.g/ml Cpd II 16.74% .+-. 2.15% 15 .mu.g/ml Cpd II
40.79% .+-. 1.81%
Example 7
[0138] Passage 4 (i.e., four cell population doublings) human
umbilical vein endothelial cells (HUVECs), human source lot number
9713 (obtained from ATCC) in 1 ml of endothelial growth medium-2
(EGM-2) (obtained from Cambrex) were mixed with 30 .mu.g of the
compound of formula II (Cpd II) in endothelial basal medium-2
(EBM-2) (Cambrex) or 30 .mu.g methylphenidate (MP) in EBM-2. Water
(vehicle for the two test compounds) was used as a control, and the
PI3 kinase inhibitor, LY 294002 (Sigma), at 50 .mu.M, was included
as a positive control. Then, the cells were seeded at 10,000
cells/well into the wells of a plate contained in a tube formation
assay kit obtained from BD Biosciences, Rockville, Md. The wells of
the plate contained an extracellular matrix protein gel. Fetal calf
serum (FCS) (ATCC) was added to a final concentration of 5% to
initiate tube formation. Then, the plates were incubated for 18
hours at 37.degree. C. and 5% CO.sub.2. Following the incubation,
the plates were photographed with a digital camera attached to an
inverted microscope (Olympus IMT-2 set at a phase contrast (PC) of
10).
[0139] When endothelial cells are cultured on extracellular matrix
protein gels in the presence of angiogenic signals, they arrange
themselves into structures loosely resembling capillary blood
vessels. To establish the basal tube formation for this assay,
cells were treated with the same amount of water as present in the
solutions of Cpd II and MP. This treatment produced a lattice of
endothelial cell structures with multiple branch points. Treatment
with Cpd II and LY 294002 reduced the amount of branching and
cellular interaction in the wells, leaving the cells in isolated
clusters. MP had no observable effect on the ability of the
endothelial cells to organize into structures resembling capillary
blood vessels. These data indicate that Cpd II, but not MP,
interferes with this step of angiogenesis.
Example 8
[0140] Passage 4 HUVECs, lot number 9713, in either EGM-2 plus 50
ng/ml vascular endothelial growth factor (VEGF) (obtained from
Sigma) or in EGM-2 complete medium (containing 2% FCS,
hydrocortisone, human fibroblast growth factor B, VEGF, recombinant
insulin-like growth factor-1, ascorbate, human epithelial growth
factor, gentamycin and heparin) (obtained from Cambrex) were put
into the wells of a 96-well tissue culture plate at 5,000
cells/well. The following additives were added to the cells: water
(vehicle control); 5 .mu.g/ml of the compound of formula II (Cpd
II); 15 .mu.g/ml Cpd II; or 30 .mu.g/ml of Cpd II. After 48 hours
of culture at 37.degree. C. and 5% CO.sub.2, cell proliferation was
evaluated by the Promega cell titer assay as described in Example
1, except that the plates were incubated for only 2 hours after
addition of the Promega cell titer reagent.
[0141] The results are shown in Table 4 below. As can be seen from
Table 4, Cpd II reduced the number of cells detected in the wells
in a dose-dependent manner. The reductions seen with 15 .mu.g/ml
Cpd II and 30 .mu.g/ml Cpd II were statistically significant. Since
wells with no growth factors were not included, it is not possible
to determine if the reductions in cell numbers seen with Cpd II are
due to inhibition of proliferation or a cytotoxic effect.
TABLE-US-00004 TABLE 4 p value (versus Sample Medium Mean OD at 530
nm vehicle control) Control (no additives) EGM-2 + VEGF 0.141 .+-.
0.004 N/A Vehicle control (water added) EGM-2 + VEGF 0.224 .+-.
0.011 N/A 5 .mu.g/ml Cpd II EGM-2 + VEGF 0.189 .+-. 0.014 0.0324 15
.mu.g/ml Cpd II EGM-2 + VEGF 0.132 .+-. 0.022 0.0069 30 .mu.g/ml
Cpd II EGM-2 + VEGF 0.046 .+-. 0.012 0.0003 Control (no additives)
EGM-2 + growth factors 0.243 .+-. 0.002 N/A Vehicle control (water
added) EGM-2 + growth factors 0.299 .+-. 0.011 N/A 5 .mu.g/ml Cpd
II EGM-2 + growth factors 0.271 .+-. 0.022 0.1131 15 .mu.g/ml Cpd
II EGM-2 + growth factors 0.239 .+-. 0.019 0.0283 30 .mu.g/ml Cpd
II EGM-2 + growth factors 0.066 .+-. 0.003 0.0001
Example 9
[0142] HepG2 is a human hepatic cancer cell line, which was
obtained from ATCC. HepG2 cells were grown to confluence in 25
cm.sup.2 flasks in IMDM medium containing 10% FCS. Then, the cells
were trypsinized as follows. The medium in each flask was aspirated
and replaced with 5 ml of 0.025% trypsin/EDTA (Cambrex). The cells
were monitored on a microscope until they no longer adhered to the
flasks. Then, 5 ml of trypsin neutralizing solution (TNS) (Cambrex)
were added to each flask to stop the reaction. The cell suspension
was centrifuged at 1000 rpm for 10 minutes, and the supernatants
were aspirated. The cells were reconstituted in fresh medium and
counted. Then, 4 ml of the cell suspension in medium containing at
1.22.times.10.sup.6 cells/ml were mixed with an additional 1 ml of
medium. Next, 0.5 ml/well of the resulting cell suspension was
added to wells in a 24-well culture plate (about 500,000
cells/well). The cells were treated as indicated in Table 5 below
and incubated for 24 hours at 37.degree. C., with or without 5%
CO.sub.2.The supernatants were removed from the wells and
centrifuged to remove debris. Next, the supernatants were analyzed
for erythropoietin (EPO) production. EPO was measured by ELISA
using a kit obtained from R & D Systems, Minneapolis, Minn.
(catalog no. DE900) following the manufacturer's instructions.
[0143] The results are shown in Table 5 below. As can be seen from
Table 5, Cpd II significantly inhibited the release of EPO from the
HepG2 cells. A decrease in EPO would have an inhibitory effect on
angiogenesis. A viability assay was not performed, but the
morphology of the cells appeared normal based on microscopic
analysis. TABLE-US-00005 TABLE 5 p value versus Treatment Mean
Units/ml EPO hypoxia alone Control (no treatment) 74.90 .+-. 2.65
N/A Hypoxia (5% CO.sub.2) 108.39 .+-. 2.81 N/A Hypoxia + 15
.mu.g/ml Cpd II 71.60 .+-. 2.01 0.005 Hypoxia + 25 .mu.M LY 294002
52.99 .+-. 1.04 0.016
Example 10
[0144] Passage 4 HUVECs, lot number 9713, were put into the wells
of a 48-well tissue culture plate at 20,000 cells/well in 500 .mu.l
of EGM-2 complete medium (but without serum or ascorbate)
supplemented with ITSS (insulin, transferrin and sodium selenite)
(obtained from Sigma). Also, passage 4 HUVECs, human source lot
number 7016 (obtained from ATCC), were put into the wells of a
48-well tissue culture plate at 20,000 cells/well in 500 .mu.l of
EGM-2 complete medium (but without serum or ascorbate) supplemented
with ITSS. The following additives were added to the cells: water
(vehicle control) and 15 .mu.g/ml of the compound of formula II
(Cpd II). After incubation for 1 hour at 37.degree. C. and 5%
CO.sub.2, LPS was added to give a final concentration of 200 ng/ml,
and the cells were incubated overnight at 37.degree. C. and 5%
CO.sub.2. After this incubation, the supernatants were collected,
and the amount of IL-8 in the supernatants determined by ELISA as
described in Example 4.
[0145] The results are shown in Table 6 below. As can be seen in
Table 6, Cpd II complete eliminated IL-8 release by the 7016 HUVECs
and decreased IL-8 release by 90% in the 9713 HUVECs.
TABLE-US-00006 TABLE 6 Cells Treatment IL-8 (pg/ml) 7016 HUVECs
Control (LPS only) 53.3 7016 HUVECs LPS + 15 .mu.g/ml Cpd II Below
detection 9713 HUVECs Control (LPS only) 485.0 9713 HUVECs LPS + 15
.mu.g/ml Cpd II 49.8
Example 11
[0146] Passage 4 HUVECs, human source lot number 8710 (obtained
from ATCC), were put into the wells of a 24-well tissue culture
plate at 5,000 cells/well in EGM-2 medium and cultured for 72 hours
at 37.degree. C. and 5% CO.sub.2. Then, the medium was replaced
with fresh medium, and the following additives were added to the
cells: water (vehicle control); 1 .mu.g/ml, 5 .mu.g/ml, 10
.mu.g/ml, 15 .mu.g/ml or 30 .mu.g/ml of the compound of formula II
(Cpd II); 15 .mu.g/ml methylphenidate (MP); 10 .mu.M LY 294002; or
10 .mu.M dexamethasone (Dex). After incubation for 1 hour at
37.degree. C. and 5% CO.sub.2, TNF.alpha. (Pierce) was added to
give a final concentration of 10 ng/ml, and the cells were
incubated for an additional 18 hours at 37.degree. C. and 5%
CO.sub.2. After this incubation, the supernatants were collected,
and the amount of IL-8 in the supernatants determined by ELISA as
described in Example 4.
[0147] The results are shown in Table 7 below. As can be seen in
Table 7, Cpd II decreased IL-8 release stimulated by TNF.alpha. in
a dose-dependent manner, although there did appear to be some cell
death caused by the highest dose (30 .mu.g/ml). Dex and MP slightly
decreased IL-8 release and LY 294002 significantly decreased IL-8
release. TABLE-US-00007 TABLE 7 % Treatment Mean IL-8 (pg/ml) p
value inhibition No additives 207.15 .+-. 66.17 30 .mu.g/ml Cpd II
0 15 .mu.g/ml Cpd II 400.35 10 ng/ml TNF.alpha. 34695 .+-. 301.9 10
ng/ml TNF.alpha. + water 35572 .+-. 967.74 10 ng/ml TNF.alpha. + 30
.mu.g/ml 4829.8 .+-. 214.13 86.93% Cpd II 10 ng/ml TNF.alpha. + 15
.mu.g/ml 20817 .+-. 674.63 0.002 41.72% Cpd II 10 ng/ml TNF.alpha.
+ 10 .mu.g/ml 22050 .+-. 727.27 0.003 38.24% Cpd II 10 ng/ml
TNF.alpha. + 5 .mu.g/ml 34482 .+-. 2127.22 0.124 3.08% Cpd II 10
ng/ml TNF.alpha. + 1 .mu.g/ml 53657 .+-. 3935.18 0.011 (-51.1%) Cpd
II 10 ng/ml TNF.alpha. + 15 .mu.g/ml 30183 .+-. 3448.01 0.051
15.24% MP 10 ng/ml TNF.alpha. + 10 .mu.M 9196.1 .+-. 150.97 74.58%
LY 294002 10 ng/ml TNF.alpha. + 10 .mu.M Dex 35952 .+-. 2197.14
0.072 6.88%
Example 12
[0148] The transcription factor NF.kappa.B (nuclear factor
.kappa.B) is implicated in the regulation of the expression of a
wide variety of genes that code for mediators of the immune, acute
phase and inflammatory responses. NF.kappa.B is a key regulator of
cell survival and promoter of carcinogenesis. There are five
subunits of the NF.kappa.B family in mammals: p50, p65 (RelA),
c-Rel, p52 and RelB. The p50/p65 heterodimers and the p50
homodimers are the most common dimers found the NF.kappa.B
signaling pathway. NF.kappa.B can be activated by a number of
stimuli, including components of bacterial cell walls, such as
lipopolysaccharide, or inflammatory cytokines, such as TNF.alpha.
or IL-1.beta..
[0149] Activator protein-1 (AP-1) is a transcription factor that is
activated during the cell cycle to promote cell survival,
differentiation and adaptive responses. AP-1 proteins play a role
in the expression of many genes involved in proliferation and cell
cycle progression. For instance, cell transformation by oncogenes
that function in the growth factor signal transduction pathway,
such as ras, rasF and mek, results in a high increase in AP-1
component protein expression. Therefore, AP-1 regulated genes
support the invasive process observed during malignancy and
metastasis. AP-1 belongs to a large family of structurally related
transcription factors that includes ATFI-4, c-Fos, c-Jun, c-Myc and
C/EBP. AP-1 is composed of a mixture of heterodimeric complexes of
proteins derived from the Fos and Jun families, including c-Fos,
FosB, Fra-1, Fra-2, c-Jun, JunB and JunD. Primarily, AP-1 dimers
bind to DNA on a TPA-response element (TRE). AP-1 expression is
induced by multiple stimuli such as serum, growth factors, phorbol
esters, oncogenes, cytokines of the TGF-13, TNF and interferon
families, neuronal depolarization and cellular stress.
[0150] Passage 5 HUVECs, human source lot number 8750, were grown
to confluence in 25 cm.sup.2 flasks in EGM-2 medium. The following
additives were added to the flasks (total volume of 5 ml/flask) in
EGM-2 medium containing 2% FCS, GA1000 (gentamycin), heparin and
ascorbic acid (all from Cambrex): 1 .mu.g/ml of the compound of
formula II (Cpd II); 5 .mu.g/ml Cpd II; 15 .mu.g/ml of Cpd II; 15
.mu.g/ml methylphenidate (MP); or 10 .mu.M LY 294002. The flasks
were incubated overnight at 37.degree. C., 5% CO.sub.2 After this
incubation, VEGF was added to give a final concentration 10 ng/ml,
and the flasks were incubated for an additional 30 minutes.
[0151] Then, the amount of NF.kappa.B was determined using a
TransAM.TM. NF.kappa.B p65/NF.kappa.B p50 Transcription Factor
Assay Kit and a Nuclear Extract Kit from Active Motif North
America, Carlsbad, Calif., according to the manufacturer's
instructions. Briefly, a nuclear extract of the cells was prepared
using the Nuclear Extract Kit. Then, the nuclear extract was added
to the wells of the 96-well plate of the TransAM.TM. kit.
Oligonucleotide containing an NF.kappa.B consensus binding site was
immobilized in the wells, and the activated NF.kappa.B contained in
the nuclear extract was bound to the oligonucleotide. Then, an
antibody directed against the NF.kappa.B p65 or p50 subunit was
added, and the NF.kappa.B complex bound to the oligonucleotide was
detected. A secondary antibody conjugated to horseradish peroxidase
(HRP) was next added to provide a colorimetric readout that was
quantified by spectrophotometry (measurement at 450 nm).
[0152] The amount of c-Jun was determined using a TransAM.TM. AP-1
Family Transcription Factor Assay Kit and a Nuclear Extract Kit
from Active Motif North America, Carlsbad, Calif., according to
manufacturer's instructions. Briefly, a nuclear extract of the
cells was prepared using the Nuclear Extract Kit. Then, the nuclear
extract was added to the wells of a 96-well plate in which
oligonucleotide containing a TPA-responsive element (TRE) was
immobilized. Activator protein-1 (AP-1) dimers contained in the
nuclear extract were bound to this oligonucleotide and were
detected using an antibody specific for c-Jun. A secondary antibody
conjugated to horseradish peroxidase (HRP) was next added to
provide a calorimetric readout that was quantified by
spectrophotometry (measurement at 450 nm).
[0153] The results are shown in Tables 8 and 9 below. As can be
seen from Table 8, VEGF treatment of HUVECs caused almost a
doubling of activated NF.kappa.B as detected by the TransAM assay.
Cpd II at 15 .mu.g/ml and 5 .mu.g/ml reduced the amount of
activated NF.kappa.B back to basal levels. As can be seen from
Table 9, VEGF treatment of HUVECs caused an increase of c-Jun. Cpd
II at 15 .mu.g/ml and 5 .mu.g/ml completely eliminated the increase
in the amount of c-Jun. TABLE-US-00008 TABLE 8 Sample Mean OD 450
nm (NF.kappa.B) Control (no additives) 0.070 .+-. 0.002 VEGF only
0.111 .+-. 0.007 VEGF + 15 .mu.g/ml Cpd II 0.060 .+-. 0.008 VEGF +
5 .mu.g/ml Cpd II 0.065 .+-. 0.010 VEGF + 1 .mu.g/ml Cpd II 0.097
.+-. 0.013 VEGF + 15 .mu.g/ml MP 0.093 .+-. 0.011 VEGF + 10 .mu.M
LY 294002 0.138 .+-. 0.008
[0154] TABLE-US-00009 TABLE 9 Sample Mean OD 450 nm (c-Jun) Control
(no additives) 0.204 .+-. 0.016 VEGF only 0.261 .+-. 0.013 VEGF +
15 .mu.g/ml Cpd II 0.204 .+-. 0.010 VEGF + 5 .mu.g/ml Cpd II 0.185
.+-. 0.025 VEGF + 1 .mu.g/ml Cpd II 0.221 .+-. 0.008 VEGF + 15
.mu.g/ml MP 0.230 .+-. 0.016 VEGF + 10 .mu.M LY 294002 0.340 .+-.
0.020
Example 13
[0155] Passage 8 (human iliac artery endothelial cells (HIAECs)
(obtained from ATCC; catalog no. CC-2545) were grown to confluence
in 25 cm.sup.2 flasks in EGM-2 medium. Eighteen hours prior to the
experiment, the medium was replaced with EGM-2 medium containing
0.1% FCS plus heparin, GA 1000 (gentamycin) and bovine pituitary
extract (all from Cambrex) to place the cells in a resting state.
To perform the experiment the medium was aspirated from the flasks,
and the following additives were added to the flasks in fresh
medium (total volume of 5 ml/flask): 15 .mu.g/ml of the compound of
formula II (Cpd II) or 10 .mu.M LY 294002. The flasks were
incubated 2 hours at 37.degree. C., 5% CO.sub.2 After this
incubation, VEGF or TNF.alpha. was added to give a final
concentration 10 ng/ml, and the flasks were incubated for an
additional 30 minutes. Then, the amount of NF.kappa.B was
determined using a TransAM.TM. NF.kappa.B p65/NF.kappa.B p50
Transcription Factor Assay Kit and a Nuclear Extract Kit from
Active Motif North America, Carlsbad, Calif., as described in
Example 12.
[0156] The results are shown in Table 10 below. As can be seen from
Table 10, TNF.alpha. treatment of HUVECs caused an extremely large
increase in the amount of activated NF.kappa.B as detected by the
TransAM assay. Cpd II at 15 .mu.g/ml reduced the amount of
activated NF.kappa.B about 82%. The treatment with VEGF did not
result in as large an increase in activated NF.kappa.B as achieved
with TNF.alpha., but the increased amount was reduced 70% by Cpd.
II. TABLE-US-00010 TABLE 10 Mean OD 450 nm Percent Sample
(NF.kappa.B) Inhibition Control (no additives) 0.174 .+-. 0.004
TNF.alpha. only 0.881 .+-. 0.021 TNF.alpha. + 15 .mu.g/ml Cpd II
0.302 .+-. 0.003 81.89% TNF.alpha. + 10 .mu.M LY 294002 0.810 .+-.
0.007 10.04% VEGF only 0.220 .+-. 0.007 VEGF + 15 .mu.g/ml Cpd II
0.066 .+-. 0.005 70.00%
Example 14
[0157] Day 18 TRiPS cells, 1.times.10.sup.6, were incubated for 30
minutes at 37.degree. C., either with nothing added ("Nil"), with 1
.mu.l CD3/CD28 Dynabeads (Dynal, Oslo, Norway) ("CD3/CD28 beads")
per 100,000 cells, or with CD3/CD28 beads and 15 .mu.g/ml of the
compound of formula II (Cpd II). After the incubation, the cells
were lysed in Cell-Lytic Mammalian Cell Extraction Reagent (Sigma).
After centrifugation to pellet cellular debris, the supernatants
(cell extracts) were obtained.
[0158] The cell extracts (supernatants) were then analyzed using a
Custom AntibodyArray.TM. manufactured by Hypromatrix Inc.,
Worcester, Mass., following the manufacturer's instructions. The
Custom AntibodyArray.TM. is a nylon membrane blotted with
antibodies to the proteins listed below. Briefly, the cell extracts
were incubated with duplicate Custom AntibodyArray.TM.'s for 2
hours at room temperature with slow shaking, followed by three
washes with Tris buffer (150 mM NaCl, 25 mM Tris, 0.05% Tween-20,
pH 7.5). HRP-labeled antibodies specific for
phosphorylated-tyrosine, phosphorylated-serine and
phosphorylated-threonine in Tris buffer were added, and the arrays
incubated for 2 hours. After three more washes with Tris buffer, a
peroxidase-reactive luminescent substrate was added. The arrays
were visualized by exposure to X-ray film. Densitometry of the
X-ray films was measured by scanning and computer analysis. The
results are summarized in Table 11 below. TABLE-US-00011 TABLE 11
Effect of Cpd II on the protein in Protein CD3/CD28 stimulated
TRiPS cells RAP1 Activated RAP2 Activated JAK2 Activated STAT4
Activated STAT5b Activated PI3kinaseP85 Activated MEK1 Decreased
level to below basal levels (Nil control) JNK1 Decreased level back
to basal levels (Nil control) JNK2 Decreased level back to basal
levels (Nil control) JNK3 Decreased level back to basal levels (Nil
control) MEKK1 Decreased level back to basal levels (Nil control)
IkB-.beta. Decreased level back to basal levels (Nil control) IkB-r
Decreased level back to basal levels (Nil control) IL-2 Decreased
level back to basal levels (Nil control) IL-4 Decreased level back
to basal levels (Nil control) IL-7y Decreased level back to basal
levels (Nil control) 14-3-3 Slightly decreased the level STAT6
Slightly decreased the level IkB-.epsilon. Slightly decreased the
level IkB-.alpha. Slightly decreased the level VAV No effect STAT2
No effect
Example 15
[0159] Cells of the MC/9 murine fibroblast cell line (obtained from
ATCC, catalog no. CRL-8305) were placed into the wells of a 96-well
tissue culture plate at 25,000 cells/well. The culture medium was
Delbecco's Modified Eagle's Medium (DMEM) (obtained from Cambrex)
containing 10% FCS. Nil control wells contained no additives. The
remaining wells contained either 25 ng/ml murine nerve growth
factor (NGF) (obtained from Upstate Biotechnology, Lake Placid,
N.Y.) or 25 ng/ml NGF and 5% TSTIM (a culture supplement prepared
from rats and containing concanavalin A which was obtained from BD
Biosciences). In addition, the following additives were added to
the cells: water (vehicle control); 5 .mu.g/ml of the compound of
formula II (Cpd II); 15 .mu.g/ml Cpd II; or 30 .mu.g/ml of Cpd II.
After 72 hours of culture at 37.degree. C. and 5% CO.sub.2, cell
proliferation was evaluated by the Promega cell titer assay as
described in Example 1. The results are shown in Table 12 below.
TABLE-US-00012 TABLE 12 Additive Mean OD 530 nm No additives 0.058
.+-. 0.008 NGF 0.116 .+-. 0.029 NGF + water 0.101 .+-. 0.022 NGF +
1 .mu.g/ml Cpd II 0.117 .+-. 0.015 NGF + 5 .mu.g/ml Cpd II 0.108
.+-. 0.012 NGF + 15 .mu.g/ml Cpd II 0.049 .+-. 0.016 NGF + TSTIM
0.490 .+-. 0.047 NGF + TSTIM + water 0.365 .+-. 0.026 NGF + TSTIM +
1 .mu.g/ml Cpd II 0.428 .+-. 0.027 NGF + TSTIM + 5 .mu.g/ml Cpd II
0.373 .+-. 0.016 NGF + TSTIM + 15 .mu.g/ml Cpd II 0.326 .+-.
0.024
Example 16
[0160] THP-1 cells were placed in medium (RPMI containing 10% FCS
and 8 ng/ml monothioglycerol) at a concentration of 250,000 cells
per ml and incubated with 5 .mu.g/ml of compound of formula II (Cpd
II) or 15 .mu.g/ml of Cpd II for one hour at 37.degree. C. and 5%
CO.sub.2. After 1 hour, lipopolysaccharide (LPS) was added to the
cultures to give a final concentration of 200 ng/ml, and the cells
were then incubated for an additional 24 hours. After the
incubation, the amount of NF.kappa.B and c-Jun were determined as
described in Example 12. Also, the amount of c-Fos was determined
using a TransAM.TM. AP-1 Family Transcription Factor Assay Kit and
a Nuclear Extract Kit from Active Motif North America, Carlsbad,
Calif., according to manufacturer's instructions. Briefly, a
nuclear extract of the cells was prepared using the Nuclear Extract
Kit. Then, the nuclear extract was added to the wells of a 96-well
plate in which oligonucleotide containing a TPA-responsive element
(TRE) was immobilized. Activator protein-1 (AP-1) dimers contained
in the nuclear extract were bound to this oligonucleotide and were
detected using an antibody specific for c-Fos. A secondary antibody
conjugated to horseradish peroxidase (HRP) was next added to
provide a colorimetric readout that was quantified by
spectrophotometry (measurement at 450 nm). The results are shown in
FIGS. 9A-B.
Example 17
[0161] Day 10 TRiPS cells, 1.times.10.sup.6, were incubated with 15
.mu.g/ml of the compound of formula II (Cpd II) for 1 hour at
37.degree. C. Then, the cells were incubated with CD3/CD28 beads (1
.mu.l per 100,000 cells) (obtained from Dynal) for 10 minutes at
37.degree. C. The cells were then lysed with a mild buffer
(supplied with Pierce EZ-Detect activation kit described below) to
produce cell extracts. Protein concentrations of the resulting
extracts were determined by bicinchoninic acid (BCA) assay (Pierce)
and placed on ice for immediate use.
[0162] Pulldown assays were performed using Pierce EZ-Detect
activation kits according to the manufacturer's instructions
utilizing GST-RAF-1-RBD and GST-RalGDS-RBD for Ras and RAP-1
respectively. Briefly, 400 .mu.g total protein from each extract
was combined with recombinant protein and glutathione resin and
incubated at 4.degree. C. for one hour with gentle shaking. The
resin was then washed to remove unbound protein and the activated
Ras and RAP-1 proteins were removed by boiling in the presence of
SDS-PAGE loading dye containing reducing agent. Ras and RAP-1
western blots were performed to visualize the proteins using
antibodies supplied with the kit. Densitometry of the X-ray films
was done by scanning and computer analysis.
[0163] The results are shown in Table 13. As can be seen from Table
13, incubating the TRiPS cells with Cpd II resulted in very strong
inhibition of Ras protein. Stimulation of the cells with CD3/CD28
beads did not increase the amount of RAP-1 protein as expected, but
Cpd II also appeared to inhibit RAP-1. TABLE-US-00013 TABLE 13
Integrated Optical Integrated Optical Density for Density for
Treatment RAS assay RAP-1 assay No treatment 66.83 259.27 CD3/CD28
beads only 245.91 213.66 CD3/CD28 beads + 84.98 87.26 15 .mu.g/ml
Cpd II
[0164] The foregoing discussion of the invention has been presented
for purposes of illustration and description. The foregoing is not
intended to limit the invention to the form or forms disclosed
herein. Although the description of the invention has included
description of one or more embodiments and certain variations and
modifications, other variations and modifications are within the
scope of the invention, e.g., as may be within the skill and
knowledge of those in the art, after understanding the present
disclosure. It is intended to obtain rights which include
alternative embodiments to the extent permitted, including
alternate, interchangeable and/or equivalent structures, functions,
ranges or steps to those claimed, whether or not such alternate,
interchangeable and/or equivalent structures, functions, ranges or
steps are disclosed herein, and without intending to publicly
dedicate any patentable subject matter.
* * * * *