U.S. patent application number 12/454802 was filed with the patent office on 2009-11-26 for drug design for tubulin inhibitors, compositions, and methods of treatment thereof.
This patent application is currently assigned to BiPar Sciences, Inc.. Invention is credited to John Burnier, Valeria Ossovskaya, Barry Sherman, Max Totrov.
Application Number | 20090291924 12/454802 |
Document ID | / |
Family ID | 39158008 |
Filed Date | 2009-11-26 |
United States Patent
Application |
20090291924 |
Kind Code |
A1 |
Ossovskaya; Valeria ; et
al. |
November 26, 2009 |
Drug design for tubulin inhibitors, compositions, and methods of
treatment thereof
Abstract
The present invention relates to a computer-assisted method of a
designing of a tubulin inhibitor comprising: a) determining an
interaction between a tubulin protein and a chemical known to bind
the tubulin protein by evaluating a binding of the tubulin protein
to the chemical known to bind the tubulin protein; b) based on the
interaction, designing a candidate tubulin inhibitor; c)
determining an interaction between the tubulin protein and the
candidate tubulin inhibitor by evaluating a binding of the tubulin
protein to the candidate tubulin inhibitor; and d) concluding that
the candidate tubulin inhibitor inhibits the tubulin protein
wherein the conclusion is based on the interaction of step c). The
invention also provides compositions and methods of treatment of
diseases with the candidate tubulin inhibitors.
Inventors: |
Ossovskaya; Valeria; (San
Francisco, CA) ; Burnier; John; (Pacifica, CA)
; Sherman; Barry; (Hillsborough, CA) ; Totrov;
Max; (San Diego, CA) |
Correspondence
Address: |
BiPar Sciences Inc. c/o MoFo
755 Page Mill Road
Palo Alto
CA
94304
US
|
Assignee: |
BiPar Sciences, Inc.
South San Francisco
CA
|
Family ID: |
39158008 |
Appl. No.: |
12/454802 |
Filed: |
May 21, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11850620 |
Sep 5, 2007 |
7538252 |
|
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12454802 |
|
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|
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60842480 |
Sep 5, 2006 |
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Current U.S.
Class: |
514/125 ;
436/501; 514/252.12; 514/646; 514/683; 564/430; 568/315;
703/12 |
Current CPC
Class: |
A61K 31/12 20130101;
A61K 31/66 20130101; G16C 20/50 20190201; A61K 31/135 20130101;
A61P 13/00 20180101; A61K 31/497 20130101; A61P 35/00 20180101;
A61P 15/00 20180101; A61P 11/00 20180101; G16B 15/00 20190201 |
Class at
Publication: |
514/125 ;
436/501; 703/12; 514/646; 514/683; 514/252.12; 564/430;
568/315 |
International
Class: |
A61K 31/661 20060101
A61K031/661; G01N 33/53 20060101 G01N033/53; G06G 7/58 20060101
G06G007/58; A61K 31/135 20060101 A61K031/135; A61K 31/12 20060101
A61K031/12; A61K 31/495 20060101 A61K031/495; C07C 213/00 20060101
C07C213/00; C07C 45/00 20060101 C07C045/00; A61P 35/00 20060101
A61P035/00; A61P 5/00 20060101 A61P005/00; A61P 11/00 20060101
A61P011/00; A61P 13/00 20060101 A61P013/00; A61P 15/00 20060101
A61P015/00 |
Claims
1. A computer-assisted method of a designing of a tubulin inhibitor
comprising: a. determining an interaction between a tubulin protein
and a chemical known to bind the tubulin protein by evaluating a
binding of the tubulin protein to the chemical known to bind the
tubulin protein; b. based on the interaction, designing a candidate
tubulin inhibitor; c. determining an interaction between the
tubulin protein and the candidate tubulin inhibitor by evaluating a
binding of the tubulin protein to the candidate tubulin inhibitor;
and d. concluding that the candidate tubulin inhibitor inhibits the
tubulin protein wherein the conclusion is based on the interaction
of step c).
2. The method of claim 1, wherein the tubulin protein is a
three-dimensional structure comprising a binding domain of the
tubulin protein.
3. The method of claim 2, wherein the binding domain of the tubulin
protein is selected from the group consisting of colchicine binding
domain, vinblastine binding domain, and paclitaxel binding
domain.
4. The method of claim 3, wherein the binding domain of the tubulin
protein is a colchicine binding domain.
5. The method of claim 1, wherein the chemical known to bind the
tubulin protein is a three-dimensional structure.
6. The method of claim 1, wherein the tubulin protein is derived
from a crystal of the tubulin protein.
7. The method of claim 1, wherein the designing is performed in
conjunction with a computer modeling.
8. The method of claim 2, wherein the binding domain of the tubulin
protein is an intradimer surface.
9. The method of claim 2, wherein the binding domain of the tubulin
protein is an interdimer surface.
10. The method of claim 2, wherein the binding domain of the
tubulin protein is a surface facing inside of a microtubule near an
interprotofilament interface at a distance from any longitudinal
interface.
11. The method of claim 10, wherein the surface is a paclitaxel
binding site.
12. The method of claim 1, wherein the designing involves replacing
a substituent on the chemical known to bind the tubulin protein
with another substituent wherein the other substituent improves the
binding of the candidate tubulin inhibitor with the tubulin
protein.
13. The method of claim 1, wherein the interaction is steric
interaction, van der Waals interaction, electrostatic interaction,
solvation interaction, charge interaction, covalent bonding
interaction, non-covalent bonding interaction, entropically
favorable interaction, enthalpically favorable interaction, or a
combination thereof.
14. The method of claim 1, wherein the candidate tubulin inhibitor
is an analogue of the chemical known to bind the tubulin
protein.
15. The method of claim 1, wherein the chemical known to bind the
tubulin protein is a ketone that is a thyroxine analogue.
16. The method of claim 1, further comprising a step of chemically
synthesizing the candidate tubulin inhibitor.
17. The method of claim 16, further comprising evaluating a tubulin
inhibitory activity of the candidate tubulin inhibitor and
selecting the candidate tubulin inhibitor based on the
evaluation.
18. The method of claim 17, wherein the evaluating the tubulin
inhibiting activity involves an assay technique.
19. The method of claim 1, wherein the candidate tubulin inhibitor
is a compound of formula I, its pharmaceutically acceptable salts,
or prodrugs thereof: ##STR00048## wherein: R.sub.1 and R.sub.5 are
halogens; R.sub.2, R.sub.3, and R.sub.4 are independently selected
from the group consisting of hydrogen, hydroxyl, halogen, ester,
optionally substituted alkoxy, optionally substituted amine,
phosphate, optionally substituted alkyl, and optionally substituted
acetyl; and R.sub.6, R.sub.7, R.sub.8, R.sub.9, and R.sub.10 are
independently selected from the group consisting of hydrogen,
hydroxyl, halogen, optionally substituted alkoxy, optionally
substituted amine, phosphate, optionally substituted alkyl,
optionally substituted alkenyl, optionally substituted alkynyl,
nitroso, carboxyl, optionally substituted cycloalkyl, and
optionally substituted heterocyclic.
20. The method of claim 19, wherein the candidate tubulin inhibitor
is a compound of formula II or its pharmaceutically acceptable
salts or prodrugs: ##STR00049## wherein: R, R', R.sub.2, R.sub.4,
R.sub.6, R.sub.7, R.sub.9, and R.sub.10 are independently selected
from the group consisting of hydrogen, hydroxyl, halogen,
optionally substituted alkoxy, optionally substituted amine,
phosphate, optionally substituted alkyl, and optionally substituted
acetyl.
21. The method of claim 20, wherein the candidate tubulin inhibitor
is a compound of formula III or its pharmaceutically acceptable
salts or prodrugs: ##STR00050## wherein: R, R.sub.7, R.sub.9, and
R.sub.10 are independently selected from the group consisting of
hydrogen, hydroxyl, optionally substituted amine, phosphate, and
optionally substituted alkyl.
22. The method of claim 21, wherein the candidate tubulin inhibitor
is a compound of formula IIIA or its pharmaceutically acceptable
salts or prodrugs: ##STR00051##
23. The method of claim 21, wherein the candidate tubulin inhibitor
is a compound of formula IIIB or its pharmaceutically acceptable
salts or prodrugs: ##STR00052##
24. The method of claim 21, wherein the candidate tubulin inhibitor
is a compound of formula IIIC or its pharmaceutically acceptable
salts or prodrugs: ##STR00053##
25. The method of claim 21, wherein the candidate tubulin inhibitor
is a compound of formula IIID or its pharmaceutically acceptable
salts or prodrugs: ##STR00054##
26. The method of claim 21, wherein the optionally substituted
alkyl is substituted with an optionally substituted
heterocyclic.
27. The method of claim 26, wherein the optionally substituted
heterocyclic is selected from a group consisting of azeridine,
azetidine, pyrrole, dihydropyrrole, pyrrolidene, pyrazole,
pyrazoline, pyrazolidine, imidazole, benzimidazole, triazole,
tetrazole, oxazole, isoxazole, benzoxazole, oxadiazole, oxazoline,
oxazolidine, thiazole, isothiazole, pyridine, dihydropyridine,
tetrahydropyridine, quinazoline, pyrazine, pyrimidine, pyridazine,
quinoline, isoquinoline, triazine, tetrazine, and piperazine.
28. The method of claim 27, wherein the candidate tubulin inhibitor
is a compound of formula IIIE or its pharmaceutically acceptable
salts or prodrugs: ##STR00055##
29. A computer system containing a set of information to perform a
design of a tubulin inhibitor having a user interface comprising a
display unit, the set of information comprising: a. logic for
inputting an information regarding a binding of a tubulin protein
to a chemical known to bind tubulin protein; b. logic for designing
a candidate tubulin inhibitor based on the binding of the tubulin
protein to the chemical known to bind tubulin protein; c. logic for
determining an information regarding a binding of the tubulin
protein to the candidate tubulin inhibitor; and d. logic for making
a conclusion regarding a tubulin inhibitory properties of the
candidate tubulin inhibitor based on the determination of step
c).
30. A computer-readable storage medium containing a set of
information for a general purpose computer having a user interface
comprising, a display unit, the set of information comprising: a.
logic for inputting an information regarding a binding of a tubulin
protein to a chemical known to bind tubulin protein; b. logic for
designing a candidate tubulin inhibitor based on the binding of the
tubulin protein to the chemical known to bind tubulin protein; c.
logic for determining an information regarding a binding of the
tubulin protein to the candidate tubulin inhibitor; and d. logic
for making a conclusion regarding a tubulin inhibitory properties
of the candidate tubulin inhibitor based on the determination of
step c).
31. The system of claim 29, wherein the chemical is a ketone which
is a thyroxine analogue.
32. The computer readable media of claim 30, wherein the chemical
is a ketone which is a thyroxine analogue.
33. An electronic signal or carrier wave that is propagated over
the internet between computers comprising a set of information for
a general purpose computer having a user interface comprising a
display unit, the set of information comprising a computer-readable
storage medium containing a set of information for a general
purpose computer having a user interface comprising a display unit,
the set of information comprising: a. logic for inputting an
information regarding a binding of a tubulin protein to a chemical
known to bind tubulin protein; b. logic for designing a candidate
tubulin inhibitor based on the binding of the tubulin protein to
the chemical known to bind tubulin protein; c. logic for
determining an information regarding a binding of the tubulin
protein to the candidate tubulin inhibitor; and d. logic for making
a conclusion regarding a tubulin inhibitory properties of the
candidate tubulin inhibitor based on the determination of step
c).
34. A method of treating a disease comprising administering to a
patient in need thereof an effective amount of at least one
compound of formula I, its pharmaceutically acceptable salts, or
prodrugs thereof: ##STR00056## wherein: R.sub.1 and R.sub.5 are
halogens; R.sub.2, R.sub.3, and R.sub.4 are independently selected
from the group consisting of hydrogen, hydroxyl, halogen, ester,
optionally substituted alkoxy, optionally substituted amine,
phosphate, optionally substituted alkyl, and optionally substituted
acetyl; and R.sub.6, R.sub.7, R.sub.8, R.sub.9, and R.sub.10 are
independently selected from the group consisting of hydrogen,
hydroxyl, halogen, optionally substituted alkoxy, optionally
substituted amine, phosphate, optionally substituted alkyl,
optionally substituted alkenyl, optionally substituted alkynyl,
nitroso, carboxyl, optionally substituted cycloalkyl, and
optionally substituted heterocyclic.
35. The method of claim 34, wherein the compound is of formula II
or its pharmaceutically acceptable salts or prodrugs: ##STR00057##
wherein: R, R', R.sub.2, R.sub.4, R.sub.6, R.sub.7, R.sub.9, and
R.sub.10 are independently selected from the group consisting of
hydrogen, hydroxyl, halogen, optionally substituted alkoxy,
optionally substituted amine, phosphate, optionally substituted
alkyl, and optionally substituted acetyl.
36. The method of claim 35, wherein the compound is of formula III
or its pharmaceutically acceptable salts or prodrugs: ##STR00058##
wherein: R.sub.6, R.sub.7, R.sub.9, and R.sub.10 are independently
selected from the group consisting of hydrogen, hydroxyl,
optionally substituted amine, phosphate, and optionally substituted
alkyl.
37. The method of claim 35, wherein the compound is of formula IIIA
or its pharmaceutically acceptable salts or prodrugs:
##STR00059##
38. The method of claim 35, wherein the compound is of formula IIIB
or its pharmaceutically acceptable salts or prodrugs:
##STR00060##
39. The method of claim 35, wherein the compound is of formula IIIC
or its pharmaceutically acceptable salts or prodrugs:
##STR00061##
40. The method of claim 35, wherein the compound is of formula IIID
or its pharmaceutically acceptable salts or prodrugs:
##STR00062##
41. The method of claim 36, wherein the optionally substituted
alkyl is substituted with an optionally substituted
heterocyclic.
42. The method of claim 41, wherein the optionally substituted
heterocyclic is selected from a group consisting of azeridine,
azetidine, pyrrole, dihydropyrrole, pyrrolidene, pyrazole,
pyrazoline, pyrazolidine, imidazole, benzimidazole, triazole,
tetrazole, oxazole, isoxazole, benzoxazole, oxadiazole, oxazoline,
oxazolidine, thiazole, isothiazole, pyridine, dihydropyridine,
tetrahydropyridine, quinazoline, pyrazine, pyrimidine, pyridazine,
quinoline, isoquinoline, triazine, tetrazine, and piperazine.
43. The method of claim 42, wherein the candidate tubulin inhibitor
is a compound of formula IIIE or its pharmaceutically acceptable
salts or prodrugs: ##STR00063##
44. The method of claim 34, wherein the treating comprises
inhibiting tubulin protein function.
45. The method of claim 34, wherein the disease is selected from
the group consisting of cancer, inflammation, metabolic disease,
gout, CVS disease, CNS disease, disorder of the hematolymphoid
system, disorder of endocrine and neuroendocrine, disorder of
urinary tract, disorder of respiratory system, disorder of female
genital system, and disorder of male genital system.
46. A process of manufacturing a compound of formula IIIB or its
pharmaceutically acceptable salts or prodrugs: ##STR00064##
comprising: (a) nitrating
1-(3,5-diiodo-4-(4-methoxyphenoxy)phenyl)ethanone to form as an
intermediate
1-(3,5-diiodo-4-(4-methoxy-3-nitrophenoxy)phenyl)ethanone; and (b)
reducing the intermediate of step (a) to form the compound of
formula IIIB.
47. The process of claim 46, wherein the nitrating is carried out
in the presence of nitric acid and concentrated sulfuric acid in an
organic solvent.
48. The process of claim 47, wherein the organic solvent is
methylene chloride.
49. The process of claim 46, wherein the reducing is carried out in
the presence of tin(II) chloride.
50. A process of manufacturing a compound of formula IIIA or its
pharmaceutically acceptable salts or prodrugs: ##STR00065##
comprising: (a) reacting a compound of formula IIIB: ##STR00066##
with a metal nitrate in the presence of a strong acid to form a
diazonium intermediate; and (b) refluxing the diazonium
intermediate in the presence of a strong acid to form the compound
of formula IIIA.
51. The process of claim 50, wherein the metal nitrate is sodium
nitrate.
52. The process of claim 50, wherein the strong acid of step (a) is
sulfuric acid.
53. The process of claim 50, wherein the strong acid of step (b) is
sulfuric acid.
54. The process of claim 50, wherein the strong acid of step (a)
and step (b) is sulfuric acid.
Description
CROSS-REFERENCE
[0001] This application is a divisional application of U.S. patent
application Ser. No. 11/850,620, filed on Sep. 5, 2007, which
claims priority to U.S. Provisional Patent Application No.
60/842,480, filed Sep. 5, 2006, all of which are incorporated
herein by reference in their entirety.
BACKGROUND OF THE INVENTION
[0002] Tubulin proteins are essential building blocks of the
microtubules of cells, which in turn form a major part of the
cellular cytoskeleton and are involved in important cellular
functions including intracellular transport, maintenance of cell
shape, chromosome segregation during mitosis, and cell motility.
Polymers of alpha and beta tubulin subunit heterodimers form hollow
cylindrical filament structures which comprise the microtubules.
The dynamic between microtubular polymerization and
depolymerization is essential to mitosis.
[0003] There exist multiple isoforms of the alpha and beta tubulin
proteins, with distinct patterns of expression amongst different
types of cells such as brain and hematopoietic cells (Drukman et
al. 2002, International Journal of Oncology, 21:621-28.).
Additionally, some forms of tubulin exist ubiquitously.
[0004] Because of the role microtubules play in mitosis, they are
attractive targets for chemotherapeutic intervention for cancer.
Drugs affecting microtubule function have primarily targeted the
alpha and beta tubulin heterodimers, and have been shown to have
various binding sites on the heterodimer (Hadfield et al., 2003,
Progress in Cell Cycle Research, 5:309-25). Such sites include
colchicine, vinca alkaloid, paclitaxel, and other binding
sites.
[0005] Drugs targeting tubulin form two main families:
microtubule-destabilizers and microtubule-stabilizers.
Destabilizing agents include the vinca alkaloids, such as
vincristine, vinblastine, and vinorelbine. Their mechanism of
action is thought to be through tubulin self-association, causing
formation of structures other than the normal hollow cylinders of
normal microtubules. Stabilizing agents include taxanes like
paclitaxel and docetaxel. Their mechanism of action causes the
microtubule to stabilize and prevents it from depolymerizing,
disrupting the polymerization/depolymerization equilibrium
essential for mitosis.
[0006] Despite the existence of tubulin-targeting drugs, cancer
cells also have exhibited an ability to overcome their effects,
becoming resistant to such chemotherapeutic agents. Examples of
resistance mechanisms to tubulin-targeting drugs include increased
expression and activity of the drug resistant P-glycoprotein
("P-gp") pump, altered expression of tubulin subtypes and isoforms,
mutations in tubulin proteins including in drug binding sites, and
post-translational modifications of tubulin proteins such as
acetylation. These resistance mechanisms reveal that there is a
need for novel chemotherapeutic agents that target tubulin, yet are
less susceptible to chemotherapeutic drug resistance.
[0007] The identification of tubulin inhibitors can be attempted
using methods such as screening of large numbers of random
libraries of natural and/or synthetic compounds. However, this
method is inefficient in that it typically results in a small
number of positive "hits" and is constrained by logistical factors
accompanying large screening processes.
[0008] Another method of such identification is structure-based
drug design ("SBDD"). SBDD comprises a number of integrated
components including structural information (eg. spectroscopic data
like X-ray or magnetic resonance information, relating to enzyme
structure and/or conformation, protein-ligand interactions, etc.),
computer modeling, medicinal chemistry, and biological testing
(both in vivo and in vitro). These components, each alone or in
combination, are useful for accelerating the drug discovery
process, for gaining insight into disease and disease processes,
and for providing a more efficient method for identifying drug
candidates.
[0009] Accordingly, the present invention provides compositions and
methods related to a design of candidate tubulin inhibitor s and
methods of treatment thereof.
SUMMARY OF THE INVENTION
[0010] One aspect of the present invention relates to a
computer-assisted method of a designing of a tubulin inhibitor by
a) determining an interaction between a tubulin protein and a
chemical known to bind the tubulin protein by evaluating a binding
of the tubulin protein to the chemical known to bind the tubulin
protein; b) based on the interaction, designing a candidate tubulin
inhibitor; c) determining an interaction between the tubulin
protein and the candidate tubulin inhibitor by evaluating a binding
of the tubulin protein to the candidate tubulin inhibitor; and d)
ascertaining that the candidate tubulin inhibitor inhibits the
tubulin protein wherein the conclusion is based on the interaction
of step c). In some embodiments, the tubulin protein is a
three-dimensional structure comprising a binding domain of the
tubulin protein. In some embodiments, the binding domain of the
tubulin protein is for example, colchicine binding domain,
vinblastine binding domain, and paclitaxel binding domain. In some
embodiments, the tubulin protein is derived from a crystal of the
tubulin protein. In some embodiments, the designing is performed in
conjunction with a computer modeling. In some embodiments, the
binding domain of the tubulin protein is an intradimer surface. In
some other embodiments, the binding domain of the tubulin protein
is an interdimer surface. In still some embodiments, the binding
domain of the tubulin protein is a surface facing inside of a
microtubule near an interprotofilament interface at a distance from
any longitudinal interface. In some embodiments, the surface is a
paclitaxel binding site.
[0011] In some embodiments of the aforementioned aspect of the
present invention, the designing involves replacing a substituent
on the chemical known to bind the tubulin protein with another
substituent wherein the other substituent improves the binding of
the candidate tubulin inhibitor with the tubulin protein. In some
embodiments, the interaction is for example, steric interaction,
van der Waals interaction, electrostatic interaction, solvation
interaction, charge interaction, covalent bonding interaction,
non-covalent bonding interaction, entropically favorable
interaction, enthalpically favorable interaction, or a combination
thereof. In some embodiments, the candidate tubulin inhibitor is an
analogue of the chemical known to bind the tubulin protein. In some
embodiments, the chemical known to bind the tubulin protein is a
ketone that is a thyroxine analogue. In still some embodiments, the
method further comprises of a step of chemically synthesizing the
candidate tubulin inhibitor. In some embodiments, the method
further comprises of evaluating a tubulin inhibitory activity of
the candidate tubulin inhibitor and selecting the candidate tubulin
inhibitor based on the evaluation. In some embodiments, the
evaluation of the tubulin inhibiting activity involves an assay
technique.
[0012] In some embodiments of the aforementioned computer-assisted
method of a designing of a tubulin inhibitor, the candidate tubulin
inhibitor is a compound of formula I, its pharmaceutically
acceptable salts, or prodrugs thereof:
##STR00001##
wherein: R.sub.1 and R.sub.5 are halogens; R.sub.2, R.sub.3, and
R.sub.4 are independently selected from the group consisting of
hydrogen, hydroxyl, halogen, ester, optionally substituted alkoxy,
optionally substituted amine, phosphate, optionally substituted
alkyl, and optionally substituted acetyl; and R.sub.6, R.sub.7,
R.sub.8, R.sub.9, and R.sub.10 are independently selected from the
group consisting of hydrogen, hydroxyl, halogen, optionally
substituted alkoxy, optionally substituted amine, phosphate,
optionally substituted alkyl, optionally substituted alkenyl,
optionally substituted alkynyl, nitroso, carboxyl, optionally
substituted cycloalkyl, and optionally substituted
heterocyclic.
[0013] In some embodiments of the aforementioned aspect, the
candidate tubulin inhibitor is a compound of formula II or its
pharmaceutically acceptable salts or prodrugs:
##STR00002##
wherein: R, R', R.sub.2, R.sub.4, R.sub.6, R.sub.7, R.sub.9, and
R.sub.10 are independently selected from the group consisting of
hydrogen, hydroxyl, halogen, optionally substituted alkoxy,
optionally substituted amine, phosphate, optionally substituted
alkyl, and optionally substituted acetyl.
[0014] In some embodiments, the candidate tubulin inhibitor is a
compound of formula III or its pharmaceutically acceptable salts or
prodrugs:
##STR00003##
wherein: R.sub.6, R.sub.7, R.sub.9, and R.sub.10 are independently
selected from the group consisting of hydrogen, hydroxyl,
optionally substituted amine, phosphate, and optionally substituted
alkyl.
[0015] In some embodiments, the candidate tubulin inhibitor is
1-(4-(3-hydroxy-4-methoxyphenoxy)-3,5-diiodophenyl)ethanone,
represented by formula IIIA or its pharmaceutically acceptable
salts or prodrugs:
##STR00004##
[0016] In some embodiments, the candidate tubulin inhibitor is
1-(4-(3-amino-4-methoxyphenoxy)-3,5-diiodophenyl)ethanone,
represented by formula IIIB or its pharmaceutically acceptable
salts or prodrugs:
##STR00005##
[0017] In some embodiments, the candidate tubulin inhibitor is a
compound of formula IIIC or its pharmaceutically acceptable salts
or prodrugs:
##STR00006##
[0018] In some embodiments, the candidate tubulin inhibitor is mono
(5-methoxy-3-(4-acetyl-2,6-iodophenoxy)phenyl) phosphoric acid
ester, represented by formula IIID or its pharmaceutically
acceptable salts or prodrugs:
##STR00007##
[0019] In some embodiments, the optionally substituted alkyl is
substituted with an optionally substituted heterocyclic. In some
embodiments, the optionally substituted heterocyclic is for
example, azeridine, azetidine, pyrrole, dihydropyrrole,
pyrrolidene, pyrazole, pyrazoline, pyrazolidine, imidazole,
benzimidazole, triazole, tetrazole, oxazole, isoxazole,
benzoxazole, oxadiazole, oxazoline, oxazolidine, thiazole,
isothiazole, pyridine, dihydropyridine, tetrahydropyridine,
quinazoline, pyrazine, pyrimidine, pyridazine, quinoline,
isoquinoline, triazine, tetrazine, and piperazine.
[0020] In some embodiments, the candidate tubulin inhibitor is the
compound
1-(4-(2-(N-piperazinylprop-3-yl))-4-methoxyphenoxy)-3,5-diiodoph-
enyl)ethanone, represented by formula IIIE or its pharmaceutically
acceptable salts or prodrugs:
##STR00008##
[0021] Another aspect of the invention relates to a computer system
containing a set of information to perform a design of a tubulin
inhibitor having a user interface comprising a display unit, the
set of information comprises a) logic for inputting an information
regarding a binding of a tubulin protein to a chemical known to
bind tubulin protein; b) logic for designing a candidate tubulin
inhibitor based on the binding of the tubulin protein to the
chemical known to bind tubulin protein; c) logic for determining an
information regarding a binding of the tubulin protein to the
candidate tubulin inhibitor; and d) logic for making a conclusion
regarding a tubulin inhibitory properties of the candidate tubulin
inhibitor based on the determination of step c).
[0022] Yet another aspect of the invention relates to a
computer-readable storage medium containing a set of information
for a general purpose computer having a user interface comprising a
display unit, the set of information comprises a) logic for
inputting an information regarding a binding of a tubulin protein
to a chemical known to bind tubulin protein; b) logic for designing
a candidate tubulin inhibitor based on the binding of the tubulin
protein to the chemical known to bind tubulin protein; c) logic for
determining an information regarding a binding of the tubulin
protein to the candidate tubulin inhibitor; and d) logic for making
a conclusion regarding a tubulin inhibitory properties of the
candidate tubulin inhibitor based on the determination of step c).
In some embodiments, the chemical is a ketone which is a thyroxine
analogue. In some embodiments, the chemical is a ketone which is a
thyroxine analogue.
[0023] Yet another aspect of the invention relates to an electronic
signal or carrier wave that is propagated over the internet between
computers comprising a set of information for a general purpose
computer having a user interface comprising a display unit, the set
of information comprising a computer-readable storage medium
containing a set of information for a general purpose computer
having a user interface comprising a display unit, the set of
information comprises a) logic for inputting an information
regarding a binding of a tubulin protein to a chemical known to
bind tubulin protein; b) logic for designing a candidate tubulin
inhibitor based on the binding of the tubulin protein to the
chemical known to bind tubulin protein; c) logic for determining an
information regarding a binding of the tubulin protein to the
candidate tubulin inhibitor; and d) logic for making a conclusion
regarding a tubulin inhibitory properties of the candidate tubulin
inhibitor based on the determination of step c).
[0024] Another aspect of the invention relates to a method of
treating a disease by administering to a patient in need thereof an
effective amount of at least one compound of formula I, its
pharmaceutically acceptable salts, or prodrugs thereof:
##STR00009##
wherein: R.sub.1 and R.sub.5 are halogens; R.sub.2, R.sub.3, and
R.sub.4 are independently selected from the group consisting of
hydrogen, hydroxyl, halogen, ester, optionally substituted alkoxy,
optionally substituted amine, phosphate, optionally substituted
alkyl, and optionally substituted acetyl; and R.sub.6, R.sub.7,
R.sub.8, R.sub.9, and R.sub.10 are independently selected from the
group consisting of hydrogen, hydroxyl, halogen, optionally
substituted alkoxy, optionally substituted amine, phosphate,
optionally substituted alkyl, optionally substituted alkenyl,
optionally substituted alkynyl, nitroso, carboxyl, optionally
substituted cycloalkyl, and optionally substituted
heterocyclic.
[0025] In some embodiments of aforementioned method of treating a
disease, the compound is of formula II or its pharmaceutically
acceptable salts or prodrugs:
##STR00010##
wherein: R, R', R.sub.2, R.sub.4, R.sub.6, R.sub.7, R.sub.9, and
R.sub.10 are independently selected from the group consisting of
hydrogen, hydroxyl, halogen, optionally substituted alkoxy,
optionally substituted amine, phosphate, optionally substituted
alkyl, and optionally substituted acetyl. In some embodiments, the
compound is of formula III or its pharmaceutically acceptable salts
or prodrugs:
##STR00011##
wherein: R.sub.6, R.sub.7, R.sub.9, and R.sub.10 are independently
selected from the group consisting of hydrogen, hydroxyl,
optionally substituted amine, phosphate, and optionally substituted
alkyl. In some embodiments, the compound is
1-(4-(3-hydroxy-4-methoxyphenoxy)-3,5-diiodophenyl)ethanone,
represented by formula IIIA or its pharmaceutically acceptable
salts or prodrugs:
##STR00012##
[0026] In some embodiments, the compound is
1-(4-(3-amino-4-methoxyphenoxy)-3,5-diiodophenyl)ethanone,
represented by formula IIIB or its pharmaceutically acceptable
salts or prodrugs:
##STR00013##
[0027] In some embodiments, the compound is
1-(4-(2-amino-4-methoxyphenoxy)-3,5-diiodophenyl)ethanone,
represented by formula IIIC or its pharmaceutically acceptable
salts or prodrugs:
##STR00014##
[0028] In some embodiments, the compound is mono
(5-methoxy-3-(4-acetyl-2,6-iodophenoxy)phenyl) phosphoric acid
ester, represented by formula IIID or its pharmaceutically
acceptable salts or prodrugs:
##STR00015##
[0029] In some embodiments, the optionally substituted alkyl is
substituted with an optionally substituted heterocyclic. In some
embodiments, the optionally substituted heterocyclic is for
example, azeridine, azetidine, pyrrole, dihydropyrrole,
pyrrolidene, pyrazole, pyrazoline, pyrazolidine, imidazole,
benzimidazole, triazole, tetrazole, oxazole, isoxazole,
benzoxazole, oxadiazole, oxazoline, oxazolidine, thiazole,
isothiazole, pyridine, dihydropyridine, tetrahydropyridine,
quinazoline, pyrazine, pyrimidine, pyridazine, quinoline,
isoquinoline, triazine, tetrazine, and piperazine. In some
embodiments, the candidate tubulin inhibitor is
1-(4-(2-(N-piperazinylprop-3-yl))-4-methoxyphenoxy)-3,5-diiodophenyl)etha-
none, represented by formula IIIE or its pharmaceutically
acceptable salts or prodrugs:
##STR00016##
[0030] In some embodiments, the treating comprises inhibiting
tubulin protein function. In some embodiments, the disease is for
example, cancer, inflammation, metabolic disease, gout, CVS
disease, CNS disease, disorder of the hematolymphoid system,
disorder of endocrine and neuroendocrine, disorder of urinary
tract, disorder of respiratory system, disorder of female genital
system, and disorder of male genital system.
[0031] Another aspect of the present invention relates to a
compound of formula I or its pharmaceutically acceptable salts or
prodrugs:
##STR00017##
wherein: R.sub.1 and R.sub.5 are halogens; R.sub.2, R.sub.3, and
R.sub.4 are independently selected from the group consisting of
hydrogen, hydroxyl, halogen, ester, optionally substituted alkoxy,
optionally substituted amine, phosphate, optionally substituted
alkyl, and optionally substituted acetyl; and R.sub.6, R.sub.7,
R.sub.8, R.sub.9, and R.sub.10 are independently selected from the
group consisting of hydrogen, hydroxyl, halogen, optionally
substituted alkoxy, optionally substituted amine, phosphate,
optionally substituted alkyl, optionally substituted alkenyl,
optionally substituted alkynyl, nitroso, carboxyl, optionally
substituted cycloalkyl, and optionally substituted heterocyclic.
Preferably, the compound is a tubulin inhibitor.
[0032] In some embodiments, the compound is of formula II or its
pharmaceutically acceptable salts or prodrugs:
##STR00018##
wherein: R, R', R.sub.2, R.sub.4, R.sub.7, R.sub.9, and R.sub.10
are independently selected from the group consisting of hydrogen,
hydroxyl, halogen, optionally substituted alkoxy, optionally
substituted amine, phosphate, optionally substituted alkyl, and
optionally substituted acetyl. Preferably, the compound is a
tubulin inhibitor.
[0033] In some embodiments, the compound is of formula III or its
pharmaceutically acceptable salts or prodrugs:
##STR00019## [0034] wherein: R.sub.6, R.sub.7, R.sub.9, and
R.sub.10 are independently selected from the group consisting of
hydrogen, hydroxyl, optionally substituted amine, phosphate, and
optionally substituted alkyl Preferably, the compound is a tubulin
inhibitor.
[0035] In some embodiments, the compound is
1-(4-(3-hydroxy-4-methoxyphenoxy)-3,5-diiodophenyl)ethanone,
represented by formula IIIA or its pharmaceutically acceptable
salts or prodrugs:
##STR00020##
[0036] In some embodiments, the compound is
1-(4-(3-amino-4-methoxyphenoxy)-3,5-diiodophenyl)ethanone
1-(4-(3-amino-4-methoxyphenoxy)-3,5-diiodophenyl)ethanone,
represented by formula IIIB or its pharmaceutically acceptable
salts or prodrugs:
##STR00021##
[0037] In some embodiments, the compound is
1-(4-(2-amino-4-methoxyphenoxy)-3,5-diiodophenyl)ethanone,
represented by formula IIIC or its pharmaceutically acceptable
salts or prodrugs:
##STR00022##
[0038] In some embodiments, the compound is mono
(5-methoxy-3-(4-acetyl-2,6-iodophenoxy)phenyl) phosphoric acid
ester, represented by formula IIID or its pharmaceutically
acceptable salts or prodrugs:
##STR00023##
[0039] In some embodiments of the aforementioned aspect of the
invention, the optionally substituted alkyl is substituted with an
optionally substituted heterocyclic. In some embodiments, the
optionally substituted heterocyclic is for example, azeridine,
azetidine, pyrrole, dihydropyrrole, pyrrolidene, pyrazole,
pyrazoline, pyrazolidine, imidazole, benzimidazole, triazole,
tetrazole, oxazole, isoxazole, benzoxazole, oxadiazole, oxazoline,
oxazolidine, thiazole, isothiazole, pyridine, dihydropyridine,
tetrahydropyridine, quinazoline, pyrazine, pyrimidine, pyridazine,
quinoline, isoquinoline, triazine, tetrazine, and piperazine. In
some embodiments, the compound is
1-(4-(2-(N-piperazinylprop-3-yl))-4-methoxyphenoxy)-3,5-diiodophenyl)etha-
none, represented by formula IIIE or its pharmaceutically
acceptable salts or prodrugs:
##STR00024##
[0040] Some embodiments of the aforementioned aspect of the present
invention relate to a pharmaceutical composition comprising an
effective amount of at least one compound of the present invention
and a pharmaceutically acceptable carrier.
[0041] Some embodiments provide a process of manufacturing a
compound of formula IIIB or its pharmaceutically acceptable salts
or prodrugs:
##STR00025##
[0042] The process comprises: (a) nitrating
1-(3,5-diiodo-4-(4-methoxyphenoxy)phenyl)ethanone to form as an
intermediate
1-(3,5-diiodo-4-(4-methoxy-3-nitrophenoxy)phenyl)ethanone; and (b)
reducing the intermediate of step (a) to form the compound of
formula IIIB. In some embodiments, the nitrating is carried out in
the presence of nitric acid and concentrated sulfuric acid in an
organic solvent. In some embodiments, the organic solvent is
methylene chloride. In some embodiments, the reducing is carried
out in the presence of tin(II) chloride.
[0043] Some embodiments of the invention provide a process of
manufacturing a compound of formula IIIA or its pharmaceutically
acceptable salts or prodrugs:
##STR00026##
The process comprises: (a) reacting a compound of formula IIIB or
its pharmaceutically acceptable salts or prodrugs:
##STR00027##
with a metal nitrate in the presence of a strong acid to form a
diazonium intermediate; and (b) refluxing the diazonium
intermediate in the presence of a strong acid to form the compound
of formula IIIA. In some embodiments, the metal nitrate is sodium
nitrate. In some embodiments, the strong acid of step (a) is
sulfuric acid. In some embodiments, the strong acid of step (b) is
sulfuric acid. In some embodiments, the strong acid of step (a) and
step (b) is sulfuric acid. Other aspects, characteristics and
advantages will become clear upon consideration of the instant
disclosure, including the appended drawings and original
claims.
INCORPORATION BY REFERENCE
[0044] All publications and patent applications mentioned in this
specification are herein incorporated by reference to the same
extent as if each individual publication or patent application was
specifically and individually indicated to be incorporated by
reference.
BRIEF DESCRIPTION OF THE DRAWINGS
[0045] The novel features of the invention are set forth with
particularity in the appended claims. A better understanding of the
features and advantages of the present invention will be obtained
by reference to the following detailed description that sets forth
illustrative embodiments, in which the principles of the invention
are utilized, and the accompanying drawings of which:
[0046] FIG. 1 is a flow chart showing the steps of the methods as
disclosed herein.
[0047] FIG. 2 illustrates a computer for implementing selected
operations associated with the methods disclosed herein.
[0048] FIG. 3 illustrates a tubulin X-ray structural data
analysis.
[0049] FIG. 4A illustrates a binding pose of methyl
3,5-diiodo-4-(4'-methoxyphenoxy)-benzoate (as thick sticks) in
tubulin. A bound conformation of colchicine from experimentally
determined structure is superimposed (thin sticks).
[0050] FIG. 4B depicts the methyl
3,5-diiodo-4-(4'-methoxyphenoxy)-benzoate as thick sticks and
colchicine as thin sticks.
[0051] FIG. 5 illustrates a binding of the tubulin inhibitor with
at least two residues that differ between isotypes.
[0052] FIG. 6 illustrates the cell-cycle arrest caused by
1-(3,5-diiodo-4-(3-amino-4-methoxyphenoxy)phenylethanone, a PARP-1
inhibitor disclosed herein as Formula IIIB
(1-(4-(3-amino-4-methoxyphenoxy)-3,5-diiodophenyl)ethanone), as
compared to control, methyl 3,5-diiodo-4-(4-methoxyphenyloxy)benzyl
ketone (1-[3,5-diiodo-4-(4'-methoxyphenoxy)-phenyl]-ethanone
(DIPE)).
[0053] FIG. 7 illustrates the induction of apoptosis in HeLa cells
induced by the compound of Formula IIIB
(1-(4-(3-amino-4-methoxyphenoxy)-3,5-diiodophenyl)ethanone), as
compared to control, methyl 3,5-diiodo-4-(4-methoxyphenyloxy)benzyl
ketone (1-[3,5-diiodo-4-(4'-methoxyphenoxy)-phenyl]-ethanone
(DIPE)).
DETAILED DESCRIPTION OF THE INVENTION
Definitions
[0054] The term, "aryl" refers to optionally substituted mono- or
bicyclic aromatic rings containing only carbon atoms. The term can
also include phenyl group fused to a monocyclic cycloalkyl or
monocyclic cycloheteroalkyl group in which the point of attachment
is on an aromatic portion. Examples of aryl groups include, e.g.,
phenyl, naphthyl, indanyl, indenyl, tetrahydronaphthyl,
2,3-dihydrobenzofuranyl, dihydrobenzopyranyl, 1,4-benzodioxanyl,
and the like.
[0055] The term, "heterocyclic" refers to an optionally substituted
mono- or bicyclic aromatic ring containing at least one heteroatom
(an atom other than carbon), such as N, O and S, with each ring
containing about 5 to about 6 atoms. Examples of heterocyclic
groups include, e.g., pyrrolyl, isoxazolyl, isothiazolyl,
pyrazolyl, pyridyl, oxazolyl, oxadiazolyl, thiadiazolyl, thiazolyl,
imidazolyl, triazolyl, tetrazolyl, furanyl, triazinyl, thienyl,
pyrimidyl, pyridazinyl, pyrazinyl, benzoxazolyl, benzothiazolyl,
benzimidazolyl, benzofuranyl, benzothiophenyl, furo(2,3-b)pyridyl,
quinolyl, indolyl, isoquinolyl, and the like.
[0056] The term, "computer system" as used herein, means the
hardware means, software means and data storage means used to
perform method of the present invention. Preferably, the computer
system is used to analyze atomic coordinate data. The minimum
hardware means of the computer-based systems of the present
invention comprises a central processing unit (CPU), input means,
output means and data storage means. Desirably a monitor is
provided to visualize the structure data. The computer can be a
stand-alone, or connected to a network and/or shared server. The
data storage means can be RAM or means for accessing computer
readable media of the invention.
[0057] The term, "computer readable media" as used herein, means
any media which can be read and accessed by a computer, for
example, the media is suitable for use in the above-mentioned
computer system. The media include, but are not limited to:
magnetic storage media such as floppy discs, hard disc storage
medium and magnetic tape; optical storage media such as optical
discs or CD-ROM; electrical storage media such as RAM and ROM; and
hybrids of these categories such as magnetic/optical storage
media.
[0058] The term "inhibit" or its grammatical equivalent, such as
"inhibitory," is not intended to require complete reduction in
biological activity, preferably, tubulin activity. Such reduction
is preferably by at least about 50%, at least about 75%, at least
about 90%, and more preferably by at least about 95% of the
activity of the molecule in the absence of the inhibitory effect,
e.g., in the absence of a tubulin inhibitor as disclosed in the
invention. Most preferably, the term refers to an observable or
measurable reduction in activity. In treatment scenarios,
preferably the inhibition is sufficient to produce a therapeutic
and/or prophylactic benefit in the condition being treated.
[0059] The term "model" or its grammatical equivalents, such as,
"modeling" as used herein, means the quantitative and qualitative
analysis of molecular structure and/or function based on atomic
structural information and interaction models. The term "modeling"
includes for example, conventional numeric-based molecular dynamic
and energy minimization models, interactive computer graphic
models, modified molecular mechanics models, distance geometry and
other structure-based constraint models.
[0060] The term "pharmaceutically acceptable salt" as used herein,
means those salts which retain the biological effectiveness and
properties of the compounds of the present invention, and which are
not biologically or otherwise undesirable.
[0061] The term "substituted" includes single or multiple degrees
of substitution by a named substituent.
[0062] The term "candidate tubulin inhibitor" as used herein, means
any compound which is potentially capable of associating with any
of the alpha, beta, or gamma tubulin proteins, and/or inhibiting
tubulin protein activity and/or the ability of tubulin protein to
interact with another molecule or to form microtubule polymers. The
candidate compound may be designed or obtained from a library of
compounds which may comprise peptides, as well as other compounds,
such as small organic molecules and particularly new lead
compounds. By way of example, the candidate compound may be a
natural substance, a biological macromolecule, or an extract made
from biological materials such as bacteria, fingi, or animal
(particularly mammalian) cells or tissues, an organic or an
inorganic molecule, a synthetic test compound, a semi-synthetic
test compound, a carbohydrate, a monosaccharide, an oligosaccharide
or polysaccharide, a glycolipid, a glycopeptide, a saponin, a
heterocyclic compound, a structural or functional mimetic, a
peptide, a peptidomimetic, a derivatized test compound, a peptide
cleaved from a whole protein, or a peptides synthesized
synthetically (such as, by way of example, either using a peptide
synthesizer or by recombinant techniques or combinations thereof),
a recombinant test compound, a natural or a non-natural test
compound, a fusion protein or equivalent thereof and mutants,
derivatives or combinations thereof.
[0063] The term "treating" or its grammatical equivalents as used
herein, means achieving a therapeutic benefit and/or a prophylactic
benefit. By therapeutic benefit is meant eradication or
amelioration of the underlying disorder being treated. Also, a
therapeutic benefit is achieved with the eradication or
amelioration of one or more of the physiological symptoms
associated with the underlying disorder such that an improvement is
observed in the patient, notwithstanding that the patient may still
be afflicted with the underlying disorder. For prophylactic
benefit, the compositions may be administered to a patient at risk
of developing a particular disease, or to a patient reporting one
or more of the physiological symptoms of a disease, even though a
diagnosis of this disease may not have been made.
[0064] The term "anti-vascular" refers to an agent or method that
reduces the amount of blood vessels supplying a tumor that exist
before or immediately before the administration of the agent or the
implementation of the method.
[0065] The term "tubulin heterodimer" refers to an alpha tubulin
protein and beta tubulin protein subunit complex, comprising the
building blocs for microtubule polymerization.
[0066] The term "thyroid hormone analogue" or "thyroid
hormone/analogue" include both naturally-occurring thyroid hormones
such as thyroxine, and chemicals similar in structure to the
naturally-occurring thyroid hormones. The chemicals that are
similar in structure to the naturally-occurring thyroid hormones
may have similar or different biological and pharmacological
properties as compared to the naturally-occurring thyroid
hormones.
[0067] The term "tubulin protein" includes single tubulin gene
products, variants, or splice variants thereof, and also includes
tubulin protein heterodimers such as, but not limited to,
heterodimers of the alpha and beta tubulin protein gene
products.
[0068] The term "tubulin inhibitor" includes any agent that
inhibits the biological activity of a tubulin protein, and includes
analogues of thyroxine.
Methods for a Design of a Tubulin Inhibitor
[0069] One aspect of the present invention relates to methods for
design of a tubulin inhibitor. In some preferred embodiments, the
designing comprises using computer modeling techniques. In
particular, the present invention relates to a computer-assisted
method of a design of a tubulin inhibitor comprising: a)
determining an interaction between a tubulin protein and a chemical
known to bind the tubulin protein by evaluating a binding of the
tubulin protein to the chemical known to bind the tubulin protein;
b) based on the interaction, designing a candidate tubulin
inhibitor; c) determining an interaction between the tubulin
protein and the candidate tubulin inhibitor by evaluating a binding
of the tubulin protein to the candidate tubulin inhibitor; and d)
concluding that the candidate tubulin inhibitor inhibits the
tubulin protein wherein the conclusion is based on the interaction
of step c).
[0070] In some preferred embodiments, a three-dimensional structure
comprising a tubulin protein and a three-dimensional structure of
the chemical is used for determining an interaction between the
tubulin protein and the chemical. In some preferred embodiments, a
three dimensional structure of a binding domain of a tubulin
protein is modeled using a crystal of alpha and beta tubulin
protein heterodimers through x-ray crystallographic techniques.
Preferably, the tubulin protein is the alpha-tubulin/beta tubulin
heterodimer. A three dimensional structure of a known tubulin
inhibitor or thyroid hormone analogue is modeled based on
techniques known in the art. The three dimensional structure of a
known tubulin inhibitor or thyroid hormone is allowed to interact
with the three dimensional structure of a binding domain of the
tubulin protein. Various tubulin inhibitors and thyroid hormone
analogues are known in the art and are within the scope of the
present invention. Some of the examples of the known tubulin
inhibitors and thyroid hormone analogues include, but are not
limited to, vinblastine, vincristine, vinorelbine, paclitaxel,
docetaxel, colchicine, and thyroxine. In some preferred embodiments
of the present invention, the known tubulin inhibitor is
colchicine. In another preferred embodiment of the present
invention, the thyroid hormone analogue is thyroxine.
[0071] An interaction between the tubulin protein or tubulin
protein heterodimer and the known tubulin inhibitor or thyroid
hormone analogue is determined based on an evaluation of a three
dimensional structure of domains of a tubulin protein bound to the
known tubulin inhibitor or thyroid hormone analogue. The evaluation
can comprise evaluation of one or more of steric interactions, van
der Waals interactions, electrostatic interactions, solvation
interactions, charge interactions, covalent bonding interactions,
non-covalent bonding interactions, entropically favorable
interactions, enthalpically favorable interactions, or a
combination thereof. The techniques for the evaluation of the
interaction between the protein and the drug are well known in the
art and are well within the scope of the present invention.
[0072] FIG. 3 illustrates a tubulin X-ray structural data analysis.
Small-molecule inhibitor binding pocket in tubulin is shown (green
blob). A colchicine molecule bound to the tubulin is shown in stick
representation. Surrounding amino-acid residues are labeled, dark
blue for beta-tubulin and magenta for alpha-tubulin.
[0073] Based on the evaluation of the binding of a known tubulin
inhibitor with the tubulin protein, a candidate tubulin inhibitor
can be designed. Preferably, the candidate tubulin inhibitor is
designed using computer modeling. In some preferred embodiments,
the candidate tubulin inhibitor is an analogue of the known tubulin
inhibitor. In still further preferred embodiments, the candidate
tubulin inhibitor is an analogue of the colchicine. In still
further preferred embodiments, the candidate tubulin inhibitor is
an analogue of the thyroid hormone thyroxine. A candidate tubulin
inhibitor can be designed in such a way that it fits equally or
more efficiently in the binding domain of the tubulin protein as
compared to the known tubulin inhibitor or thyroid-hormone
analogue.
[0074] FIG. 4A illustrates a binding pose of methyl
3,5-diiodo-4-(4'-methoxyphenoxy)-benzoate (as thick sticks) in
tubulin. A bound conformation of colchicine from experimentally
determined structure is superimposed (thin sticks). FIG. 4B depicts
the methyl 3,5-diiodo-4-(4'-methoxyphenoxy)-benzoate as thick
sticks and colchicine as thin sticks. The compound structures are
converted to 3D and docked into tubulin binding pocket using
Molsoft's ICM-Dock.
[0075] FIG. 5 illustrates at least two residues that differ between
isotypes (in particular isotype III). The two isotypes are in
contact with the inhibitors. Differential affinity of tubulin
inhibitors to isotypes may be important for efficacy. For example,
isotype 3 may be at least partially responsible for taxol
resistance.
[0076] After the designing of the candidate tubulin inhibitor, an
interaction between the tubulin protein or tubulin protein
heterodimer and the candidate tubulin inhibitor can be determined
based on an evaluation of the three dimensional structure of the
domains of the tubulin protein or tubulin protein heterodimer bound
to the candidate tubulin inhibitor. The evaluation can comprise
evaluation of one or more of steric interactions, van der Waals
interactions, electrostatic interactions, solvation interactions,
charge interactions, covalent bonding interactions, non-covalent
bonding interactions, entropically favorable interactions,
enthalpically favorable interactions, or a combination thereof.
Based on the evaluation a conclusion can be made regarding the
candidate tubulin inhibitor that inhibits tubulin protein
function.
[0077] Alternatively, the tubulin protein or tubulin heterodimer
complex can be co-crystallized with a candidate tubulin inhibitor
in order to provide a crystal suitable for determining the
structure of the complex. A crystal of the tubulin protein or
tubulin heterodimer complex can be soaked in a solution containing
the candidate tubulin inhibitor in order to form co-crystals by
diffusion of the candidate tubulin inhibitor into the crystal of
the tubulin protein. In some embodiments, the structure of the
tubulin protein or tubulin heterodimer complex obtained in the
presence and absence of the candidate tubulin inhibitor can be
compared to determine structural information about the tubulin
protein, identification of possible binding regions of the tubulin
protein and/or determine the interaction between the candidate
tubulin inhibitor and the tubulin protein or tubulin heterodimer
complex.
[0078] The present invention further relates to methods for
synthesizing the candidate tubulin inhibitors by conventional
synthetic chemistry techniques. These techniques are known in the
art and are within the scope of the present invention. The present
invention further relates to assessing the bioactivity, such as
tubulin inhibiting activity, of the synthesized tubulin inhibitor
compounds. The assay techniques for assessing the bioactivity of
the candidate tubulin inhibitor are well known in the art and are
within the scope of the present invention. Another aspect of the
present invention relates to providing methods of treatment of a
disease using the tubulin inhibitors. Preferably, the disease is a
condition that is related to tubulin function.
[0079] The steps to some of the preferable methods of the present
invention are depicted in FIG. 1. Without limiting the scope of the
present invention, the steps can be performed independent of each
other or one after the other. One or more steps can be skipped in
the methods of the present invention. A tubulin protein or tubulin
heterodimer is provided at step 101. The tubulin protein or tubulin
heterodimer may be provided as a three dimensional structure of a
binding domain of a tubulin protein or tubulin heterodimer. The
three dimensional structure of the tubulin protein or tubulin
heterodimer may be modeled from a crystal of tubulin protein or
tubulin protein heterodimer using x-ray crystallography. A chemical
known to bind the tubulin protein or tubulin heterodimer is
provided at step 102. A three dimensional structure of the chemical
may be provided. The three dimensional structure of the chemical
known to bind the tubulin protein or tubulin heterodimer may be
provided by a computer modeling technique. An interaction between
the tubulin protein or tubulin heterodimer and the chemical known
to bind the tubulin protein or tubulin heterodimer can be
determined based on the evaluation of the three dimensional
structure of the binding domain of the tubulin protein or tubulin
protein heterodimer bound to the chemical known to bind the tubulin
protein or tubulin heterodimer at step 103. Based on the
evaluation, a candidate tubulin inhibitor can be designed at step
104. The candidate tubulin inhibitor can be designed by computer
modeling. An interaction between the tubulin protein or tubulin
heterodimer and the candidate tubulin inhibitor can be determined
based on the evaluation of the three dimensional structure of the
binding domain of the tubulin protein or tubulin protein
heterodimer bound to the candidate tubulin inhibitor at step 105.
Based on this evaluation, a conclusion can be made regarding a
candidate tubulin inhibitor that inhibits tubulin protein function
at step 106. Further, the candidate tubulin inhibitor that inhibits
tubulin protein can be chemically synthesized at step 107. The
chemically synthesized candidate tubulin inhibitor can be assayed
for its bioactivity, preferably, activity in inhibiting tubulin
function at step 108. The candidate tubulin inhibitor that inhibits
tubulin protein or tubulin protein heterodimer can be used for
treating diseases at step 109. It shall be understood that the
invention includes other methods not explicitly set forth
herein.
[0080] Tubulin and Microtubules
[0081] Tubulin is a protein that comprises cellular microtubules.
Two distinct tubulin proteins, the alpha and beta protein subunits
form heterodimers that form polymers that in turn form the hollow
cylinders that make up the microtubular structure. Both the alpha
and beta protein subunits bind guanosine triphosphate ("GTP"), but
only the beta protein subunit hydrolyzes GTP to guanosine
diphosphate ("GDP"). The molecular weight of the alpha and beta
protein subunits is approximately 55 kilodaltons ("kDa") each.
[0082] Microtubules are comprised of polymers of alpha/beta tubulin
heterodimers, and form one of the components of the cell's
cytoskeleton. They have a hollow cylindrical shape and their
functions include, but are not limited to, cell motility,
maintenance of cell shape, intracellular transport, and chromosomal
segregation during mitosis. Microtubules grow through
polymerization and nucleate in a microtubule organizing center. In
these centers, an additional type of tubulin protein, the
gamma-tubulin, interacts with several other proteins and forms a
ring structure called the "gamma-tubulin ring complex," which helps
to provide scaffolding for polymerization.
[0083] During polymerization, the microtubule grows by adding more
alpha/beta heterodimers onto the "cap" or end of the microtubule.
The cap of the microtubule contains at least one alpha/beta
heterodimer bound to GTP. Because GDP-bound alpha/beta heterodimers
at the cap tend to depolymerize from the microtubule, the GTP-bound
cap provides protection against depolymerization, even though
alpha/beta lower down the polymer are GDP-bound. Eventually, the
GTP in the cap is hydrolyzed, creating depolymerization of the
microtubule and accounting for the equilibrium between microtubule
formation and depolymerization in cells.
[0084] Binding Domains of Tubulin
[0085] There are three drug families that target tubulin's
function, and therefore at least three binding sites have been
described (for a review, see Downing et al., 1999, Cell Structure
and Function, 24:269-71). These include, but are not limited to,
the colchicine binding site, the vinca alkaloid binding site, and
the paclitaxel binding site. At least two binding sites have been
characterized as sites that induce destabilization of microtubular
polymers: the colchicine and vinca alkaloid binding sites. The
paclitaxel binding site has been connected to increased
microtubular polymer stability, and both stability and
depolymerization have been shown to cause cell death.
[0086] The colchicine binding site is approximately in the dimer's
middle, near the interface of the alpha and beta tubulin proteins.
It also appears to be on the side of the heterodimer that faces the
microtubular luminal surface. The vinca alkaloid binding site is
located near the longitudinal inter-dimer surface, and is near the
GTP/GDP nucleotide binding site. The paclitaxel binding site is on
the beta protein subunit of the dimer, and appears to be involved
in dimer interfacing with other polymer strand dimers. Therefore,
it is thought to be involved with polymer strand lateral
interaction, possibly strengthening it, since it has a
polymerization-stabilizing function.
[0087] Crystal Structure of Tubulin
[0088] Alpha and beta heterodimers of tubulin comprise the
structural subunit of microtubules, and this subunit's crystal
structure has been reported, with crystals induced using zinc (see
Nogales et al., 1998, Nature, 391:199-202). One embodiment of the
invention utilizes the techniques described therein to crystallize
tubulin protein or tubulin heterodimers with which to determine the
atomic coordinates. In one embodiment of the invention, the
structures of alpha and beta tubulin proteins are very similar as
each protein has a core of two beta-sheets surrounded by
alpha-helices. In another embodiment, each monomer has at least
three functional domains including, but not limited to, the
amino-terminal domain (which contains the GTP/GDP nucleotide
binding region, the vinca alkaloid binding-domains, and the
colchicine binding domain), the intermediate domain (which contains
the paclitaxel-binding domain), and the carboxy-terminal domain
(which is thought to contain the binding regions for motor
proteins).
[0089] Examples of methods for determining structure information of
tubulin protein, tubulin protein heterodimer, or either bound with
a inhibitor include: 1) mass spectrometry to determine one or more
properties of a protein, including primary sequence, post
translation modification, protein-small molecule interaction, or
protein-protein interaction ability; 2) NMR, including ID NMR,
multidimensional NMR, and multinuclear NMR, such as
.sup.15N/.sup.1H HSQC spectra, to determine one or more properties
of a protein including three dimensional structure, conformational
states, aggregation level, state of protein folding or unfolding,
or the dynamic properties of the protein; and 3) x-ray
crystallography to determine one or more properties of a protein,
including three dimensional structure, diffraction of its crystal
form or its space group. The present invention preferably uses
x-ray crystallography to determine the structural characteristics
of the of tubulin protein or tubulin heterodimer. In particular,
x-ray diffraction of a crystallized form of the of tubulin protein
or tubulin heterodimer can be used to determine the three
dimensional structure of the of tubulin protein or tubulin
heterodimer.
[0090] Crystals of tubulin protein or tubulin heterodimer can be
produced or grown by a number of techniques including batch
crystallization, vapor diffusion (either by sitting drop or hanging
drop), soaking, and by microdialysis. Seeding of the crystals in
some instances can be required to obtain x-ray quality crystals.
Standard micro and/or macro seeding of crystals can be used.
Preferably, the crystal diffracts x-rays for the determination of
the atomic coordinates of the of tubulin protein or tubulin
heterodimer to a resolution greater than 5.0 Angstroms,
alternatively greater than 3.0 Angstroms, or alternatively greater
than 2.0
[0091] Crystals can be grown from a solution containing a purified
tubulin protein or tubulin heterodimer, or a fragment thereof
(e.g., a stable domain), by a variety of conventional processes
(McPherson, 1982 John Wiley, New York; McPherson, 1990, Eur. J.
Biochem. 189: 1-23; Webber. 1991, Adv. Protein Chem. 41:1-36). In
some embodiments, native crystals of the tubulin protein or tubulin
heterodimer can be grown by adding precipitants to the concentrated
solution of the tubulin protein or tubulin heterodimer. The
precipitants can be added at a concentration just below that
necessary to precipitate the tubulin protein or tubulin
heterodimer. Water can be removed by controlled evaporation to
produce precipitating conditions, which are maintained until
crystal growth ceases. The formation of crystals can depend on
various factors including pH, temperature, tubulin protein or
tubulin heterodimer concentration, the nature of the solvent and
precipitant, as well as the presence of added ions or ligands to
the tubulin protein or tubulin heterodimer. In addition, the
sequence of the tubulin protein or tubulin heterodimer being
crystallized can have an effect on the success of obtaining
crystals. Many routine crystallization experiments can be needed to
screen all these factors for the few combinations that might give
crystal suitable for x-ray diffraction analysis. Crystallization
robots can automate and speed up the work of reproducibly setting
up large number of crystallization experiments. Once the conditions
for growing the crystal are optimized, variations of the condition
can be systematically screened in order to find the set of
conditions which allow the growth of sufficiently large, single,
well ordered crystals. In some embodiments, the tubulin protein or
tubulin heterodimer can be co-crystallized with a compound that
stabilizes the tubulin protein or tubulin heterodimer.
[0092] Before the data collection, the tubulin protein or tubulin
heterodimer crystal can be frozen to protect it from radiation
damage. A number of different cryo-protectants can be used to
assist in freezing the crystal, such as methyl pentanediol (MPD),
isopropanol, ethylene glycol, glycerol, formate, citrate, mineral
oil, or a low-molecular-weight polyethylene glycol (PEG). As an
alternative to freezing the crystal, the crystal can also be used
for diffraction experiments performed at temperatures above the
freezing point of the solution. In these instances, the crystal can
be protected from drying out by placing it in a narrow capillary of
a suitable material (generally glass or quartz) with some of the
crystal growth solution included in order to maintain vapor
pressure.
[0093] X-ray diffraction results can be recorded by a number of
ways know to one of skill in the art. Examples of area electronic
detectors include charge coupled device detectors, multi-wire area
detectors and phosphoimager detectors (Amemiya, Y, 1997. Methods in
Enzymology, Vol. 276. Academic Press, San Diego, pp. 233-243;
Westbrook, E. M., Naday; 1. 1997. Methods in Enzymology, Vol. 276.
Academic Press, San Diego, pp. 244-268; 1997. Kahn, R. &
Fourme, R. Methods in Enzymology, Vol. 276. Academic Press, San
Diego, pp. 268-286). Collection of X-ray diffraction patterns are
well known by those skilled in the art and are within the scope of
the present invention.
[0094] Modeling of the three dimensional structure of the tubulin
protein or tubulin heterodimer can be accomplished by either the
crystallographer using a computer graphics program such as TURBO or
O (Jones, T A. et al., Acta Crystallogr. A47, 100-119, 1991) or,
under suitable circumstances, by using a fully automated model
building program, such as WARP (Anastassis et al. Nature Structural
Biology, May 1999 Volume 6 Number 5 pp 458-463) or MAID (Levitt, D.
G., Acta Crystallogr. D 2001 V57: 1013-9). This structure can be
used to calculate model-derived diffraction amplitudes and
phases.
[0095] The three dimensional structure of the crystal of the
tubulin protein or tubulin heterodimer can be modeled using
molecular replacement. The term "molecular replacement" refers to a
method that involves generating a preliminary model of a molecule
or complex whose structure coordinates are unknown, by orienting
and positioning a molecule whose structure coordinates are known
within the unit cell of the unknown crystal, so as best to account
for the observed diffraction pattern of the unknown crystal. Phases
can then be calculated from this model and combined with the
observed amplitudes to give an approximate Fourier synthesis of the
structure whose coordinates are unknown. This, in turn, can be
subject to any of the several forms of refinement to provide a
final, more accurate structure of the unknown crystal.
[0096] Homology modeling (also known as comparative modeling or
knowledge-based modeling) methods can also be used to develop a
three dimensional structure of the tubulin protein or tubulin
heterodimer. The method utilizes a computer model of a known
protein, a computer representation of the amino acid sequence of
the polypeptide (e.g., tubulin protein or tubulin heterodimer) with
an unknown structure, and standard computer representations of the
structures of amino acids. This method is well known to those
skilled in the art (Greer, 1985, Science 228, 1055; Bundell et al
1988, Eur. J. Biochem. 172, 513).
[0097] A three dimensional structure of the tubulin protein or
tubulin heterodimer can be described by the set of atoms that best
predict the observed diffraction data. Files can be created for the
structure that defines each atom by its chemical identity, spatial
coordinates in three dimensions, root mean squared deviation from
the mean observed position and fractional occupancy of the observed
position. Hydrogen bonds and other atomic interactions, both within
the protein and to bound ligands, can be identified. A model can
represent the secondary, tertiary and/or quaternary structure of
the tubulin protein or tubulin heterodimer. The model itself can be
in two or three dimensions.
[0098] It is known in the art that a set of structure coordinates
for a protein, complex or a portion thereof, is a relative set of
points that define a shape in three dimensions. Thus, it is
possible that an entirely different set of coordinates could define
a similar or identical shape. Moreover, slight variations in the
individual coordinates can have little effect on overall shape.
Such variations in coordinates can be generated because
ofmathematical manipulations of the structure coordinates. For
example, structure coordinates could be manipulated by
crystallographic permutations of the structure coordinates,
fractionalization of the structure coordinates, integer additions
or subtractions to sets of the structure coordinates, inversion of
the structure coordinates or any combination of the above.
[0099] The three-dimensional structure of a tubulin protein or
tubulin heterodimer, a known tubulin inhibitor or thyroid
hormone/analogue, a candidate tubulin inhibitor, or a tubulin
protein/tubulin heterodimer bound to a known tubulin inhibitor or
thyroid hormone/analogue or a candidate tubulin inhibitor (tubulin
protein-tubulin inhibitor complex), can be determined by
conventional means as described above or as known in the art. The
structure factors from the three-dimensional structure coordinates
of tubulin protein or tubulin heterodimer can be utilized to aid
the structure determination of the tubulin protein-tubulin
inhibitor complex. Structure factors include mathematical
expressions derived from three-dimensional structure coordinates of
the tubulin protein or tubulin heterodimer. These mathematical
expressions include, for example, amplitude and phase information.
The three-dimensional structure of the tubulin protein or tubulin
heterodimer, a known tubulin inhibitor or thyroid hormone/analogue,
a candidate tubulin inhibitor or a tubulin protein or tubulin
heterodimer-tubulin inhibitor complex can be determined using
molecular replacement analysis. This analysis utilizes a known
three-dimensional structure as a search model to determine the
structure of a closely related tubulin protein or tubulin
heterodimer, a known tubulin inhibitor or thyroid hormone/analogue,
a candidate tubulin inhibitor or a tubulin protein or tubulin
heterodimer-tubulin inhibitor complex.
[0100] In some embodiments, the tubulin protein or tubulin
heterodimer can be soluble, purified and/or isolated tubulin
protein or tubulin heterodimer which can optionally comprise a tag
or label to facilitate expression, purification and/or structural
or functional characterization. In some embodiments, a tubulin
protein or tubulin heterodimer which is used in accordance with the
methods of the invention is labeled with an isotopic label to
facilitate its detection and or structural characterization using
nuclear magnetic resonance or another applicable technique.
Exemplary isotopic labels include radioisotopic labels such as, for
example, potassium-40 (.sup.40K), carbon-14 (.sup.14C), tritium
(.sup.3H), sulfur-35 (.sup.35S), phosphorus-32 (.sup.32P),
technetium-99m (.sup.99mTc), thallium-201 (.sup.201TI), gallium-67
(.sup.67Ga), indium-111 (.sup.11In), iodine-123 (.sup.123I),
iodine-131 (.sup.131I), yttrium-90 (.sup.90Y), samarium-153
(.sup.153Sm), rhenium-186 (.sup.186Re), rhenium-188 (.sup.188Re),
dysprosium-165 (.sup.165Dy) and holmium-166 (.sup.166Ho). The
isotopic label can also be an atom with non zero nuclear spin,
including, for example, hydrogen-1 (.sup.1H), hydrogen-2 (.sup.2H),
hydrogen-3 (.sup.3H), phosphorous-31 (.sup.31P), sodium-23
(.sup.23Na), nitrogen-14 (.sup.14N), nitrogen-15 (.sup.15N),
carbon-13 (.sup.13C) and fluorine-19 (.sup.19F).
[0101] In certain embodiments, the tubulin protein or tubulin
heterodimer is uniformly labeled with an isotopic label, for
example, wherein about 50%, 70%, 80%, 90%, 95%, or 98% of the
possible labels in the tubulin protein or tubulin heterodimer are
labeled, e.g., wherein about 50%, 70%, 80%, 90%, 95%, or 98% of the
nitrogen atoms in the tubulin protein or tubulin heterodimer are
.sup.15N, and/or wherein about 50%, 70%, 80%, 90%, 95%, or 98% of
the carbon atoms in the tubulin protein or tubulin heterodimer are
.sup.13C, and/or wherein about 50%, 70%, 80%, 90%, 95%, or 98% of
the hydrogen atoms in the tubulin protein or tubulin heterodimer
are .sup.2H. In other embodiments, the isotopic label is located in
one or more specific locations within the tubulin protein or
tubulin heterodimer. The invention also encompasses the embodiment
wherein a single tubulin protein or tubulin heterodimer comprises
two or more different isotopic labels, for example, the tubulin
protein or tubulin heterodimer comprises both .sup.15N and .sup.13C
labeling.
[0102] In yet another embodiment, the tubulin protein or tubulin
heterodimer which can be used in accordance with the methods of the
invention is labeled to facilitate structural characterization
using x-ray crystallography or another applicable technique.
Exemplary labels include heavy atom labels such as, for example,
cobalt, selenium, krypton, bromine, strontium, molybdenum,
ruthenium, rhodium, palladium, silver, cadmium, tin, iodine, xenon,
barium, lanthanum, cerium, praseodymium, neodymium, samarium,
europium, gadolinium, terbium, dysprosium, holmium, erbium,
thulium, ytterbium, lutetium, tantalum, tungsten, rhenium, osmium,
iridium, platinum, gold, mercury, thallium, lead, thorium and
uranium.
[0103] Designing a Tubulin Inhibitor
[0104] Designing as disclosed in the present invention involves
designing a chemical substance, particularly a candidate tubulin
inhibitor that interacts in some way with receptor or binding
domains of the tubulin protein or tubulin heterodimer. Typically,
for a drug to effectively interact with the binding domains of the
tubulin protein or tubulin heterodimer, it can be necessary that
the three-dimensional shape ("conformation") of tubulin protein or
tubulin heterodimer assumes a compatible conformation that allows
the drug and the binding domain of the tubulin protein or tubulin
heterodimer to fit and bind together in a way that produces a
desired result. Preferably, the desired result is an efficient
binding of the drug with the tubulin protein or tubulin heterodimer
resulting in an inhibition of the tubulin activity. In such
instance, the complex shape or conformation of the binding domain
of the tubulin protein or tubulin heterodimer can be compared to a
"lock", and the corresponding requisite shape or conformation of
the drug as a "key" that unlocks (i.e., produces the desired result
within) the binding domain of the tubulin protein or tubulin
heterodimer. This "lock-and-key" analogy emphasizes that only a
properly conformed key (drug patterned thereafter) is able to fit
within the lock (the binding domain of the tubulin protein or
tubulin heterodimer) in order to "unlock" it (produce a desired
result). Further, even if the key fits in the lock, it must have
the proper composition in order for it to perform its function.
That is, the drug contains the elements in the spatial arrangement
and position in order to properly bind with the binding domain of
the tubulin protein or tubulin heterodimer. The design as disclosed
herein can include knowing or predicting the conformation of the
binding domain of the tubulin protein or tubulin heterodimer, and
also controlling and/or predicting the conformation of the drug,
i.e., a candidate tubulin inhibitor that is to interact with the
binding domain of the tubulin protein or tubulin heterodimer.
[0105] Determination of the binding domain of the tubulin protein
or tubulin heterodimer, and in particular the recognition of the
role of domains helps in identifying binding of tubulin inhibitors
or thyroid hormone analogues in the binding domains of the tubulin
protein or tubulin heterodimer. A known tubulin inhibitor or
thyroid hormone analogue is used to evaluate its binding with the
binding domain of the tubulin protein or tubulin heterodimer. Based
on this evaluation, computational techniques for drug design are
used to design candidate tubulin inhibitors based on the structure
of a known tubulin inhibitor or thyroid hormone analogue. For
example, automated ligand-receptor docking programs which require
accurate information on the atomic coordinates of target receptors
are used to design candidate tubulin inhibitors. The candidate
tubulin inhibitors can be designed de novo or can be analogs of
known tubulin inhibitors or thyroid hormone analogues. Preferably,
the candidate tubulin inhibitor is designed based on a known
tubulin inhibitor or thyroid hormone analogue. More preferably, the
candidate tubulin inhibitor is an analogue of thyroxine.
Alternatively, the candidate tubulin inhibitors can be synthesized
and formed into a complex with tubulin protein or tubulin protein
heterodimer, and the complex can then be analyzed by x-ray
crystallography to identify the actual position of the bound
tubulin inhibitor. The structure and/or functional groups of the
candidate tubulin inhibitor can then be adjusted, if necessary, in
view of the results of the x-ray analysis, and the synthesis and
analysis sequence repeated until an optimized tubulin inhibitor is
obtained.
[0106] The designing of the candidate tubulin inhibitor can involve
computer-based in silico screening of compound databases (such as
the Cambridge structural database) with the aim of identifying
compounds which interact with the binding cavity or sites of the
target tubulin protein or tubulin heterodimer. Screening selection
criteria can be based on pharmacokinetic properties such as
metabolic stability and toxicity. Determination of the mechanism of
the tubulin inhibition allows the architecture and the chemical
nature of tubulin binding sites to be better defined, which in turn
allows the geometric and functional constraints of a substituent on
the candidate tubulin inhibitor to be derived more accurately. The
substituent can be a type of virtual 3-D pharmacophore, which can
be used as selection criteria or filter for database screening.
[0107] In some preferred embodiments of the present invention, the
candidate tubulin inhibitor is an analogue of thyroxine. Based on
the interaction of the thyroxine with the binding domain of the
tubulin protein or tubulin heterodimer, a candidate tubulin
inhibitor can be designed.
[0108] In some embodiments, the compound is of formula I, its
pharmaceutically acceptable salts or prodrugs thereof:
##STR00028##
wherein: R.sub.1 and R.sub.5 are halogens; R.sub.2, R.sub.3, and
R.sub.4 are independently selected from the group consisting of
hydrogen, hydroxyl, halogen, ester, optionally substituted alkoxy,
optionally substituted amine, phosphate, optionally substituted
alkyl, and optionally substituted acetyl; R.sub.6, R.sub.7,
R.sub.8, R.sub.9, and R.sub.10 are independently selected from the
group consisting of hydrogen, hydroxyl, halogen, optionally
substituted alkoxy, optionally substituted amine, phosphate,
optionally substituted alkyl, optionally substituted alkenyl,
optionally substituted alkynyl, nitroso, carboxyl, optionally
substituted cycloalkyl, and optionally substituted heterocyclic.
The optionally substituted heterocyclic can be for example,
azeridine, azetidine, pyrrole, dihydropyrrole, pyrrolidene,
pyrazole, pyrazoline, pyrazolidine, imidazole, benzimidazole,
triazole, tetrazole, oxazole, isoxazole, benzoxazole, oxadiazole,
oxazoline, oxazolidine, thiazole, isothiazole, pyridine,
dihydropyridine, tetrahydropyridine, quinazoline, pyrazine,
pyrimidine, pyridazine, quinoline, isoquinoline, triazine,
tetrazine, and piperazine. Preferably, the compounds as provided
herein are tubulin inhibitors.
[0109] In some embodiments of the present invention, the compound
is of formula II, its pharmaceutically acceptable salts or prodrugs
thereof:
##STR00029##
wherein: R, R', R.sub.2, R.sub.4, R.sub.6, R.sub.7, R.sub.9, and
R.sub.10 are independently selected from the group consisting of
hydrogen, hydroxyl, halogen, optionally substituted alkoxy,
optionally substituted amine, phosphate, optionally substituted
alkyl, and optionally substituted acetyl. Preferably, the compound
is a tubulin inhibitor.
[0110] In some embodiments of the present invention, the compound
is of formula III, its pharmaceutically acceptable salts or
prodrugs thereof:
##STR00030##
wherein R.sub.6, R.sub.7, R.sub.9, and R.sub.10 are independently
selected from the group consisting of hydrogen, hydroxyl,
optionally substituted amine, phosphate, and optionally substituted
alkyl. Preferably, the compound is a tubulin inhibitor.
[0111] In some preferred embodiments, the compound is
1-(4-(3-hydroxy-4-methoxyphenoxy)-3,5-diiodophenyl)ethanone,
represented by formula IIIA, its pharmaceutically acceptable salts
or prodrugs thereof:
##STR00031##
[0112] In some preferred embodiments, the compound is
1-(4-(3-amino-4-methoxyphenoxy)-3,5-diiodophenyl)ethanone,
represented by formula IIIB, its pharmaceutically acceptable salts
or prodrugs thereof:
##STR00032##
[0113] In some preferred embodiments, the compound is
1-(4-(2-amino-4-methoxyphenoxy)-3,5-diiodophenyl)ethanone,
represented by formula IIIC, its pharmaceutically acceptable salts
or prodrugs thereof:
##STR00033##
[0114] In some preferred embodiments, the compound is mono
(5-methoxy-3-(4-acetyl-2,6-iodophenoxy)phenyl) phosphoric acid
ester, represented by formula IIID, its pharmaceutically acceptable
salts or prodrugs thereof:
##STR00034##
[0115] In some preferred embodiments, the compound is
1-(4-(2-(N-piperazinylprop-3-yl))-4-methoxyphenoxy)-3,5-diiodophenyl)etha-
none, represented by formula IIIE, its pharmaceutically acceptable
salts or prodrugs thereof:
##STR00035##
[0116] Typical salts are those of the inorganic ions, such as, for
example, sodium, potassium, calcium, magnesium ions, and the like.
Such salts include salts with inorganic or organic acids, such as
hydrochloric acid, hydrobromic acid, phosphoric acid, nitric acid,
sulfuric acid, methanesulfonic acid, p-toluenesulfonic acid, acetic
acid, turmeric acid, succinic acid, lactic acid, mandelic acid,
malic acid, citric acid, tartaric acid or maleic acid. In addition,
if the compound(s) contain a carboxy group or other acidic group,
it can be converted into a pharmaceutically acceptable addition
salt with inorganic or organic bases. Examples of suitable bases
include sodium hydroxide, potassium hydroxide, ammonia,
cyclohexylamine, dicyclohexyl-amine, ethanolamine, diethanolamine,
triethanolamine, and the like.
[0117] The candidate tubulin inhibitors described herein can
contain one or more asymmetric centers and thus occur as racemates
and racemic mixtures, single enantiomers, individual diastereomers
and diastereomeric mixtures. All such isomeric forms of these
compounds are expressly included in the present invention. The
tubulin inhibitors described herein can also be represented in
multiple tautomeric forms, all of which are included herein. The
tubulin inhibitors can also occur in cis- or trans- or E- or
Z-double bond isomeric forms. All such isomeric forms of such
inhibitors are expressly included in the present invention. All
crystal forms of the tubulin inhibitors described herein are
expressly included in the present invention. The tubulin inhibitors
can also be present as their pharmaceutically acceptable salts,
derivatives or prodrugs.
[0118] The known or a candidate tubulin inhibitor molecule or
thyroid analogue can be examined through the use of computer
modeling using a docking program such as GRID, DOCK, or AUTODOCK
(see Wolfgang B. Fischer, Anal. Bioanal. Chem. 2003, 375, 23-25).
This procedure can include computer fitting of a three dimensional
structure of a known or a candidate tubulin inhibitor molecule to a
binding domain of the tubulin protein or tubulin heterodimer to
ascertain how well the shape and the chemical structure of the
known or the candidate tubulin inhibitor molecule will complement
the binding domain of the tubulin protein or tubulin heterodimer.
Computer programs can also be employed to estimate the attraction,
repulsion, and steric hindrance of the known or the candidate
tubulin inhibitor to the binding domain of the tubulin protein or
tubulin heterodimer. Typically, the tighter the fit (e.g., the
lower the steric hindrance, and/or the greater the attractive
force) the more potent the tubulin inhibitor will be since these
properties are consistent with a tighter binding constant.
Furthermore, the more specificity in the design of a candidate
tubulin inhibitor the more likely it can be that the candidate
tubulin inhibitor will not interfere with other properties of the
tubulin protein or tubulin heterodimer or other proteins. This can
minimize potential side-effects due to unwanted interactions with
other proteins.
[0119] Numerous computer programs are available and suitable for a
drug design and the processes of computer modeling, model building,
and computationally identifying, selecting and evaluating candidate
tubulin inhibitors in the methods described herein. These include,
for example, GRID (available form Oxford University, UK), MCSS
(available from Molecular Simulations Inc., Burlington, Mass.),
AUTODOCK (available from Oxford Molecular Group), FLEX X (available
from Tripos, St. Louis. Mo.), DOCK (available from University of
California, San Francisco), CAVEAT (available from University of
California, Berkeley), HOOK (available from Molecular Simulations
Inc., Burlington, Mass.), and 3D database systems such as MACCS-3D
(available from MDL Information Systems, San Leandro, Calif.),
UNITY (available from Tripos, St. Louis. Mo.), and CATALYST
(available from Molecular Simulations Inc., Burlington, Mass.). The
computer program that can be used in the present invention is ICM
(available from Molsoft L L C, La Jolla, Calif.).
[0120] Potential tubulin inhibitors can also be computationally
designed "de novo" using such software packages as LUDI (available
from Biosym Technologies, San Diego, Calif.), LEGEND (available
from Molecular Simulations Inc., Burlington, Mass.), and LEAPFROG
(Tripos Associates, St. Louis, Mo.). Compound deformation energy
and electrostatic repulsion, can be evaluated using programs such
as GAUSSIAN 92, AMBER, QUANTA/CHARMM, AND INSIGHT II/DISCOVER.
These computer evaluation and modeling techniques can be performed
on any suitable hardware including for example, workstations
available from Silicon Graphics, Sun Microsystems, and the like.
The computer workstation that can be used in the present invention
is Apple Power Mac G5. The techniques, methods, hardware and
software as disclosed herein are representative and are not
intended to be limiting to the scope of the present invention.
Other modeling techniques known in the art can also be employed in
accordance with this invention.
[0121] Another aspect of the invention relates to a computer system
containing a set of information to perform a design of a tubulin
inhibitor having a user interface comprising a display unit, the
set of information comprising: [0122] a) logic for inputting an
information regarding a binding of a tubulin protein to a chemical
known to bind tubulin protein; [0123] b) logic for designing a
candidate tubulin inhibitor based on the binding of the tubulin
protein to the chemical known to bind tubulin protein; [0124] c)
logic for determining an information regarding a binding of the
tubulin protein to the candidate tubulin inhibitor; and [0125] d)
logic for making a conclusion regarding a tubulin inhibitory
properties of the candidate tubulin inhibitor based on the
determination of step c).
[0126] In some preferred embodiments, the steps of the methods of
the present invention are performed using a computer as depicted in
FIG. 2. FIG. 2 illustrates a computer for implementing selected
operations associated with the methods of the present invention.
The computer 200 includes a central processing unit 201 connected
to a set of input/output devices 202 via a system bus 203. The
input/output devices 202 can include a keyboard, mouse, scanner,
data port, video monitor, liquid crystal display, printer, and the
like. A memory 204 in the form of primary and/or secondary memory
is also connected to the system bus 203. These components of FIG. 2
characterize a standard computer. This standard computer is
programmed in accordance with the invention. In particular, the
computer 200 can be programmed to perform various operations of the
methods of the present invention.
[0127] The memory 204 of the computer 200 can store a
modeling/determining module 205. In other words, the
modeling/determining module 205 can perform the operations
associated with steps of FIG. 1. The modeling/determining module
includes modeling a three dimensional structure of tubulin protein
or tubulin heterodimer from a crystal of the tubulin protein or
tubulin heterodimer, modeling a three dimensional structure of a
binding domain of a tubulin protein or tubulin heterodimer,
modeling a three dimensional structure of a known tubulin inhibitor
or thyroid hormone analogue, modeling and determining a binding of
the three dimensional structure of the binding domain of a tubulin
protein or tubulin heterodimer with the tubulin inhibitor or
thyroid hormone analogue, modeling a three dimensional structure of
a candidate tubulin inhibitor, modeling and determining a binding
of the three dimensional structure of the binding domain of the
tubulin protein or tubulin heterodimer with the candidate tubulin
inhibitor, and evaluating the binding of the known tubulin
inhibitor or thyroid hormone analogue or the candidate tubulin
inhibitor with the tubulin protein or tubulin heterodimer. The
modeling module can also include a conclusion module which includes
a conclusion regarding the candidate tubulin inhibitor that
inhibits tubulin activity.
[0128] The candidate tubulin inhibitor as disclosed herein can be
prepared by employing standard synthetic techniques known in the
art. The candidate tubulin inhibitors can be analyzed for their
bioactivity. Preferably, the bioactivity relates to inhibition of
tubulin activity. The compounds which display tubulin inhibiting
activity can be candidate tubulin inhibitors, while the compounds
which do not display tubulin inhibiting activity help define
portions of the molecule which are particularly involved in
imparting tubulin inhibiting activity to the candidate tubulin
inhibitor. Where analogue compounds are not bioactive, additional
analogue compounds can be designed, subjected to the methods of the
present invention, and then tested for bioactivity. Additional
candidate tubulin inhibitors can be devised by either repeating the
above-described process, or seeking to render other portions of the
target structure chemically modified.
[0129] In some embodiments of the present invention, pertinent
physical and chemical properties (i.e., sites of hydrogen bonding,
surface area, atomic and molecular volume, charge density,
directionality of the charges, etc.) of candidate tubulin
inhibitors can be used to develop a collection of parameters
required for the desired bioactivity. A database of known compounds
(e.g., the Cambridge crystal structure database) can then be
searched for structures which contain the steric parameters
required for the desired bioactivity. Compounds which are found to
contain the desired steric parameters can be retrieved, and further
analyzed to determine which of the retrieved compounds also have
the desired electronic properties, relative to the candidate
tubulin inhibitor. Compounds that are found to contain both the
desired steric and electronic properties can be additional
candidates as tubulin inhibitors.
[0130] Known compounds which also possess the collection of
parameters required for the desired bioactivity can then be tested
to see if they also possess the desired bioactivity. Alternatively,
known compounds which also possess the collection of parameters
required for the desired bioactivity can be modified to remove
excess functionality which is not required for the particular
bioactivity being tested. Such a modified compound can be a simple,
readily prepared tubulin inhibitor.
[0131] The tubulin inhibitors described herein are also useful for
inhibiting the biological activity of any protein comprising
greater than 90%, alternatively greater than 85%, or alternatively
greater than 70% sequence homology with a tubulin protein sequence.
The tubulin inhibitors described herein are also useful for
inhibiting the biological activity of any protein comprising a
subsequence, or variant thereof, of any protein that comprises
greater than 90%, alternatively greater than 85%, or alternatively
greater than 70% sequence homology with a tubulin subsequence. Such
subsequence preferably comprises greater than 90%, alternatively
greater than 85%, or alternatively greater than 70% sequence
homology with the sequence of an active site or subdomain of a
tubulin protein.
[0132] Synthesis Schemes for Candidate Tubulin Inhibitors
[0133] The candidate tubulin inhibitor s as disclosed herein can be
prepared by employing standard synthetic techniques known in the
art and such techniques are within the scope of the present
invention. Without limiting the scope of the present invention some
of the synthesis schemes for the candidate Tubulin inhibitor s are
provided as below.
[0134] An example of a synthesis scheme for candidate tubulin
inhibitor of a compound of formula IIIA from pyrocatechol (CAS
120-80-9) is as provided below (Evans D. A. et al., J. Am. Chem.
Soc. 2001, 123, 12411-12413; Evans D. A. et al., Tetrahedron Lett.
1998, 39, 2933-2936; Saimoto H. et al., Tetrahedron Lett., 1986,
27, 1607; Covello M. et. al., Chem. Abstr. 1962, 56, 5929i).
##STR00036##
[0135] An alternate synthesis of a compound of formula IIIA starts
with a compound of formula IIIB, which is first converted to a
diazonium salt in the presence of a metal nitrate, such as sodium
nitrate. This nitronium ion is then transformed into the hydroxy by
reflux in the presence of a strong acid, such as sulfuric acid.
[0136] First, the compound of formula IIIB is obtained as described
herein and is suspended in 6 N sulfuric acid. The reaction mixture
is cooled to Ti=0-5.degree. C. Sodium nitrate is dissolved in water
and is added dropwise under the surface of the starting material
suspension. As the suspension is very thick, further water is added
and stirring is continued for 2.5 hour at Ti=0-5.degree. C. Further
sodium nitrate is added and stirring is continued for 3 hours. A
few crystals of urea are added to decompose any excess sodium
nitrate. Test with iodine-starch paper.
##STR00037##
[0137] The diazonium suspension is added to pre-heated 6N sulfuric
acid within 1 hour. Heating is continued for 24 hours. The
suspension is cooled to room temperature and filtered off and
washed with water. The solid is dried in vacuo at 40.degree. C. The
crude product may be purified by chromatography on silica gel.
##STR00038##
[0138] An example of a synthesis scheme for candidate tubulin
inhibitor of a compound of formula IIIB from 4-bromoanisole (CAS
104-92-7) is as provided below (Evans D. A. et al., J. Am. Chem.
Soc. 2001, 123, 12411-12413; Evans D. A. et al., Tetrahedron Lett.
1998, 39, 2933-2936; Muathen H. A., Molecules, 2003, 8, 593-598;
Saimoto H. et al., Tetrahedron Lett., 1986, 27, 1607; Hantson A. L.
et al., 5.sup.th International Conference on Isotopes, Brussels,
Belgium, Apr. 25-29, 2005, 279-283; Covello M. et. al., Chem.
Abstr. 1962, 56, 5929i).
##STR00039##
[0139] An alternate synthetic scheme for a compound of formula IIIB
is begins with 1-(3,5-diiodo-4-(4-methoxyphenoxy)phenyl)ethanone
(DIPE) as starting material. First, DIPE is nitrated to form a
3-nitro adduct. The nitration may be carried out in a suitable
reagent, such as nitric acid, e.g. in the presence of a strong acid
such as sulfuric acid. Once the 3-nitro adduct (nitro intermediate)
has been formed, it can then be reduced to form the 3-amino adduct,
which is the compound of formula IIIB.
[0140] The reduction of the nitro intermediate must be carried out
with a reducing reagent that will selectively reduce the aryl nitro
moiety but not the aryl carbonyl moiety. It has been found that
tin(II) chloride (SnCl.sub.2) in 37% HCl is such a reagent; however
other reagent systems that reduce the nitro moiety and spare the
aryl carbonyl moiety may be employed. In some embodiments it may be
critical to choose a reagent such as SnCl.sub.2/HCl that gives a
nearly quantitative (>95% or >97.5%, >99% purity)
conversion of 3-nitro to 3-amino moiety.
[0141] In step 1, DIPE is first dissolved in methylene chloride. A
solution of nitric acid and sulfuric acid is cooled to Ti
-0-5.degree. C. The acid mixture is added to the starting material
solution at Ti<5.degree. C. A solution of the nitro-intermediate
is obtained. The reaction mixture is then diluted with methylene
chloride until solids dissolve completely. The organic phase is
washed with water, 10% aqueous sodium bicarbonate solution and then
water. The organic phase is dried over sodium sulfate and
concentrated in vacuo.
##STR00040##
[0142] In step 2, a solution of ethanol and 37% HCl is cooled to
Ti<10.degree. C., to which is added anhydrous tin(II) chloride.
This produces a clear, colorless solution. The nitro intermediate
is then added immediately. The ice bath is removed and the
suspension is warmed to room temperature within 30 min. The
suspension is then warmed to Ti=30-35.degree. C. This reaction
mixture is then stirred at room temperature overnight. The reaction
mixture is then filtered and the filtrate washed with ethanol and
dried in vacuo at 38.degree. C. The crude product may then be
re-crystallized from TBME.
##STR00041##
[0143] An example of a synthesis scheme for candidate tubulin
inhibitor of a compound of formula IIIC from
1-iodo-4-methoxy-2-nitrobenzene (CAS 50590-07-3) is as provided
below (Beringer et al. JACS, 1959, 81, 343; Organic Syntheses,
1995, Coll. Vol. 3, p. 355; 1942, Vol. 22, p. 52; Hantson A. L. et
al., 5.sup.th International Conference on Isotopes, Brussels,
Belgium, Apr. 25-29, 2005, 279-283; Gowda D. et al., Ind. J. Chem.
Sect. B, 2001, 40, 75-77; Covello M. et. al., Chem. Abstr. 1962,
56, 5929i).
##STR00042##
[0144] An example of a synthesis scheme for candidate tubulin
inhibitor of a compound of formula IIID from pyrocatechol (CAS
120-80-9) is as provided below (Evans D. A. et al., J. Am. Chem.
Soc. 2001, 123, 12411-12413; Evans D. A. et al., Tetrahedron Lett.
1998, 39, 2933-2936; Perich J. W. et al., Synthesis 1988, 142-144;
Covello M. et. al., Chem. Abstr. 1962, 56, 5929i).
##STR00043##
[0145] An example of a synthesis scheme for candidate tubulin
inhibitor of a compound of formula IIIE from
3-(3-methoxyphenyl)proprionic acid (CAS 10516-71-9) is as provided
below (Beringer et al., JACS, 1959, 81, 343; Organic Syntheses,
1995, Coll. Vol. 3, p. 355; 1942, Vol. 22, p. 52; Wing-Wah Sy,
Tetrahedron Lett. 1993, 39, 6223-6224; Hantson A. L. et al.,
5.sup.th International Conference on Isotopes, Brussels, Belgium,
Apr. 25-29, 2005, 279-283; Covello M. et. al., Chem. Abstr. 1962,
56, 5929i; B. W. Yoo et. al., Bull. Korean Chem. Soc. 2004, Vol.
25, No. 11, 1633-1634).
##STR00044##
[0146] Techniques for the Measurement of Tubulin Inhibiting
Activity of Tubulin Inhibitors
[0147] In some embodiments, a tubulin inhibiting activity of the
candidate tubulin inhibitor is evaluated to characterize the
ability of a candidate tubulin inhibitor to bind to a tubulin
protein or tubulin heterodimer, and/or characterize the ability of
the candidate tubulin inhibitor to modify the activity of a tubulin
protein or tubulin heterodimer. Preferably, the technique used for
evaluation is an assay technique. Both in vitro and in vivo assays
can be used in accordance with the methods of the invention
depending on the identity of the tubulin protein or tubulin
heterodimer being investigated. Appropriate activity or functional
assays can be readily determined by the skilled artisan based on
the disclosure herein. The candidate tubulin inhibitors described
herein can be used in assays, including radiolabeled, antibody
detection and fluorometric assays, for the isolation,
identification, or structural or functional characterization of the
tubulin protein or tubulin heterodimer.
[0148] The assay can be an enzyme inhibition assay or tubulin
inhibition assay utilizing a full length or truncated tubulin
protein or tubulin heterodimer. The tubulin protein or tubulin
heterodimer can be contacted with the candidate tubulin inhibitor
and a measurement of the binding affinity of the candidate tubulin
inhibitor against a standard is determined. Such assays are known
to one of ordinary skill in the art and are within the scope of the
present invention. The assay for evaluating tubulin inhibiting
activity of the candidate tubulin inhibitor can be a cell-based
assay. The candidate tubulin inhibitor is contacted with a cell and
a measurement of an inhibition of a standard marker produced in the
cell is determined. Cells can be either isolated from an animal,
including a transformed cultured cell, or can be in a living
animal. Such assays are also known to one of ordinary skill in the
art and are within the scope of the present invention.
[0149] The candidate tubulin inhibitors of the present invention
can be identified using, for example, immunoassays such as enzyme
linked immunoabsorbent assays (ELISA) and radioimmunoassays (RIA)
or binding assays such as Biacore assays. Binding assays can employ
kinetic or thermodynamic methodology using a wide variety of
techniques including, but not limited to, microcalorimetry,
circular dichroism, capillary zone electrophoresis, nuclear
magnetic resonance spectroscopy, fluorescence spectroscopy, and
combinations thereof. Without limiting the scope of the present
invention, some of the examples of the techniques for measurement
of the bioactivity of the tubulin inhibitors, are provided
below.
[0150] Fluorescence Microscopy
[0151] Some embodiments of the invention include fluorescence
microscopy for measuring the tubulin inhibiting activity of the
candidate tubulin inhibitors of the present invention. Fluorescence
microscopy enables the molecular composition of the structures
being observed to be identified through the use of
fluorescently-labeled probes of high chemical specificity such as
antibodies. It can be done by directly conjugating a fluorophore to
a tubulin protein or tubulin heterodimer and introducing this back
into a cell. Fluorescent analogue can behave like the native
protein and can therefore serve to reveal the distribution and
behavior of this tubulin protein or tubulin heterodimer in the
cell. Along with NMR, infrared spectroscopy, circular dichroism and
other techniques, protein intrinsic fluorescence decay and its
associated observation of fluorescence anisotropy, collisional
quenching and resonance energy transfer are techniques for tubulin
detection. The naturally fluorescent proteins can be used as
fluorescent probes. The jellyfish Aequorea victoria produces a
naturally fluorescent protein known as green fluorescent protein
(GFP). The fusion of these fluorescent probes to a target protein
enables visualization by fluorescence microscopy and quantification
by flow cytometry.
[0152] By way of example only, some of the probes are labels such
as, fluorescein and its derivatives, carboxyfluoresceins,
rhodamines and their derivatives, atto labels, fluorescent red and
fluorescent orange: cy3/cy5 alternatives, lanthanide complexes with
long lifetimes, long wavelength labels--up to 800 rn, DY cyanine
labels, and phycobili proteins. By way of example only, some of the
probes are conjugates such as, isothiocyanate conjugates,
streptavidin conjugates, and biotin conjugates. By way of example
only, some of the probes are enzyme substrates such as, fluorogenic
and chromogenic substrates. By way of example only, some of the
probes are fluorochromes such as, FITC (green fluorescence,
excitation/emission=506/529 nm), rhodamine B (orange fluorescence,
excitation/emission=560/584 nm), and Nile blue A (red fluorescence,
excitation/emission=636/686 nm). Fluorescent nanoparticles can be
used for various types of immunoassays. Fluorescent nanoparticles
are based on different materials, such as, polyacrylonitrile, and
polystyrene etc. Fluorescent molecular rotors are sensors of
microenvironment restriction that become fluorescent when their
rotation is constrained. Few examples of molecular constraint
include increased dye (aggregation), binding to antibodies, or
being trapped in the polymerization of actin. IEF (isoelectric
focusing) is an analytical tool for the separation of ampholytes,
mainly proteins. An advantage for IEF-gel electrophoresis with
fluorescent IEF-marker is the possibility to directly observe the
formation of gradient. Fluorescent IEF-marker can also be detected
by UV-absorption at 280 nm (20.degree. C.).
[0153] A peptide library can be synthesized on solid supports and,
by using coloring receptors, subsequent dyed solid supports can be
selected one by one. If receptors cannot indicate any color, their
binding antibodies can be dyed. The method can not only be used on
protein receptors, but also on screening binding ligands of
synthesized artificial receptors and screening new metal binding
ligands as well. Automated methods for HTS and FACS (fluorescence
activated cell sorter) can also be used.
[0154] Immunoassays
[0155] Some embodiments of the invention include immunoassay for
measuring the tubulin inhibiting activity of the candidate tubulin
inhibitors of the present invention. In immunoblotting like the
western blot of electrophoretically-separated proteins a single
protein can be identified by its antibody. An immunoassay can be a
competitive binding immunoassay where analyte competes with a
labeled antigen for a limited pool of antibody molecules (e.g.
radioimmunoassay, EMIT). An immunoassay can be non-competitive
where an antibody is present in excess and is labeled. As analyte
antigen complex is increased, the amount of labeled
antibody-antigen complex can also increase (e.g. ELISA). Antibodies
can be polyclonal if produced by antigen injection into an
experimental animal, or monoclonal if produced by cell fusion and
cell culture techniques. In an immunoassay, the antibody can serve
as a specific reagent for the analyte antigen.
[0156] Without limiting the scope and content of the present
invention, some of the types of immunoassays are, but not limited
to, RIAs (radioimmunoassay), enzyme immunoassays like ELISA
(enzyme-linked immunosorbent assay), EMIT (enzyme multiplied
immunoassay technique), microparticle enzyme immunoassay (MEIA),
LIA (luminescent immunoassay), and FIA (fluorescent immunoassay).
The antibodies--either used as primary or secondary ones--can be
labeled with radioisotopes (e.g. 125I), fluorescent dyes (e.g.
FITC) or enzymes (e.g. HRP or AP) which can catalyze fluorogenic or
luminogenic reactions.
[0157] Biotin, or vitamin H is a co-enzyme which inherits a
specific affinity towards avidin and streptavidin. This interaction
makes biotinylated peptides a useful tool in various biotechnology
assays for quality and quantity testing. To improve
biotin/streptavidin recognition by minimizing steric hindrances, it
can be necessary to enlarge the distance between biotin and the
peptide itself. This can be achieved by coupling a spacer molecule
(e.g., 6-aminohexanoic acid) between biotin and the peptide.
[0158] The biotin quantitation assay for biotinylated proteins
provides a sensitive fluorometric assay for accurately determining
the number of biotin labels on a protein. Biotinylated peptides are
widely used in a variety of biomedical screening systems requiring
immobilization of at least one of the interaction partners onto
streptavidin coated beads, membranes, glass slides or microtiter
plates. The assay is based on the displacement of a ligand tagged
with a quencher dye from the biotin binding sites of a reagent. To
expose any biotin groups in a multiply-labeled protein that are
sterically restricted and inaccessible to the reagent, the protein
can be treated with protease for digesting the protein.
[0159] EMIT is a competitive binding immunoassay that avoids the
usual separation step. A type of immunoassay in which the protein
is labeled with an enzyme, and the enzyme-protein-antibody complex
is enzymatically inactive, allowing quantitation of unlabelled
protein. Some embodiments of the invention include ELISA to analyze
tubulin proteins or tubulin heterodimers. ELISA is based on
selective antibodies attached to solid supports combined with
enzyme reactions to produce systems capable of detecting low levels
of proteins. It is also known as enzyme immunoassay or EIA. The
protein is detected by antibodies that have been made against it,
that is, for which it is the antigen. Monoclonal antibodies are
often used.
[0160] The test can require the antibodies to be fixed to a solid
surface, such as the inner surface of a test tube, and a
preparation of the same antibodies coupled to an enzyme. The enzyme
can be one (e.g., .beta.-galactosidase) that produces a colored
product from a colorless substrate. The test, for example, can be
performed by filling the tube with the antigen solution (e.g.,
protein) to be assayed. Any antigen molecule present can bind to
the immobilized antibody molecules. The antibody-enzyme conjugate
can be added to the reaction mixture. The antibody part of the
conjugate binds to any antigen molecules that were bound
previously, creating an antibody-antigen-antibody "sandwich". After
washing away any unbound conjugate, the substrate solution can be
added. After a set interval, the reaction is stopped (e.g., by
adding 1 N NaOH) and the concentration of colored product formed is
measured in a spectrophotometer. The intensity of color is
proportional to the concentration of bound antigen.
[0161] ELISA can also be adapted to measure the concentration of
antibodies, in which case, the wells are coated with the
appropriate antigen. The solution (e.g., serum) containing antibody
can be added. After it has had time to bind to the immobilized
antigen, an enzyme-conjugated anti-immunoglobulin can be added,
consisting of an antibody specific for the antibodies being tested
for. After washing away unreacted reagent, the substrate can be
added. The intensity of the color produced is proportional to the
amount of enzyme-labeled antibodies bound (and thus to the
concentration of the antibodies being assayed).
[0162] Some embodiments of the invention include radioimmunoassays
for measuring the tubulin inhibiting activity of the candidate
tubulin inhibitors of the present invention. Radioactive isotopes
can be used to study in vivo metabolism, distribution, and binding
of small amount of compounds. Radioactive isotopes of .sup.1H,
.sup.12C, .sup.31P, .sup.32S, and .sup.127I, in body are used such
as .sup.3H, .sup.14C, .sup.32P, .sup.35S, and .sup.125I. In
receptor fixation method in 96 well plates, receptors can be fixed
in each well by using antibody or chemical methods and radioactive
labeled ligands can be added to each well to induce binding.
Unbound ligands can be washed out and then the standard can be
determined by quantitative analysis of radioactivity of bound
ligands or that of washed-out ligands. Then, addition of screening
target compounds can induce competitive binding reaction with
receptors. If the compounds show higher affinity to receptors than
standard radioactive ligands, most of radioactive ligands would not
bind to receptors and can be left in solution. Therefore, by
analyzing quantity of bound radioactive ligands (or washed-out
ligands), testing compounds' affinity to receptors can be
indicated.
[0163] The filter membrane method can be needed when receptors
cannot be fixed to 96 well plates or when ligand binding needs to
be done in solution phase. In other words, after ligand-receptor
binding reaction in solution, if the reaction solution is filtered
through nitrocellulose filter paper, small molecules including
ligands can go through it and only protein receptors can be left on
the paper. Only ligands that strongly bound to receptors can stay
on the filter paper and the relative affinity of added compounds
can be identified by quantitative analysis of the standard
radioactive ligands.
[0164] Some embodiments of the invention include fluorescence
immunoassays for measuring the tubulin inhibiting activity of the
candidate tubulin inhibitors of the present invention. Fluorescence
based immunological methods are based upon the competitive binding
of labeled ligands versus unlabeled ones on highly specific
receptor sites. The fluorescence technique can be used for
immunoassays based on changes in fluorescence lifetime with
changing analyte concentration. This technique can work with short
lifetime dyes like fluorescein isothiocyanate (FITC) (the donor)
whose fluorescence can be quenched by energy transfer to eosin (the
acceptor). A number of photoluminescent compounds can be used, such
as cyanines, oxazines, thiazines, porphyrins, phthalocyanines,
fluorescent infrared-emitting polynuclear aromatic hydrocarbons,
phycobiliproteins, squaraines and organo-metallic complexes,
hydrocarbons and azo dyes.
[0165] Fluorescence-based immunological methods can be, for
example, heterogeneous or homogenous. Heterogeneous immunoassays
comprise physical separation of bound from free labeled analyte.
The analyte or antibody can be attached to a solid surface.
Homogenous immunoassays comprise no physical separation.
Double-antibody fluorophore-labeled antigen participates in an
equilibrium reaction with antibodies directed against both the
antigen and the fluorophore. Labeled and unlabeled antigen can
compete for a limited number of anti-antigen antibodies.
[0166] Some of the fluorescence immunoassay methods include simple
fluorescence labeling method, fluorescence resonance energy
transfer (FRET), time resolved fluorescence (TRF), and scanning
probe microscopy (SPM). The simple fluorescence labeling method can
be used for receptor-ligand binding, enzymatic activity by using
pertinent fluorescence, and as a fluorescent indicator of various
in vivo physiological changes such as pH, ion concentration, and
electric pressure.
Method of Treatment with Tubulin Inhibitors
[0167] The present invention relates to a pharmaceutical
composition, medicament, drug, or other composition of the
candidate tubulin inhibitors comprising compounds of formulae I-III
where III includes IIIA-E, for treatment of diseases. Preferably,
the diseases are tubulin-mediated diseases. The candidate tubulin
inhibitors of the present invention can have therapeutic benefit in
the treatment of various diseases including cancer and metabolic
diseases, and as an adjunct therapy with chemotherapeutic
agents/radiation in therapy for cancer.
[0168] The methods of the present invention also comprise
administering one or more the candidate tubulin inhibitors in
combination with other therapies. The condition being treated will
determine the type of therapy that will be co-administered with the
candidate tubulin inhibitors. For example, for treating cancer, the
compound of some embodiments of the invention can be used in
combination with antibody (polyclonal or monoclonal) therapy,
chemotherapy, radiation therapy, bone marrow transplantation,
side-effect-limiting therapy, nucleotide therapy, gene therapy, or
a combination thereof.
[0169] In addition, the candidate tubulin inhibitors can be used to
treat a variety of diseases including metabolic diseases. Such
diseases, include, but are not limited to, gout.
[0170] In another aspect of the invention, the candidate tubulin
inhibitors can be utilized to treat cancer, and to radiosensitize
and/or chemosensitize cancer cells. The candidate tubulin
inhibitors of the present invention can be "anticancer agents,"
which term also encompasses "anti-tumor cell growth agents,"
"pro-differentiating reagents," "anti-angiogenic agents,"
"pro-angiogenic agents," "anti-mitotic agents," "anti-proliferative
agents," "pro-apoptotic agents," "anti-vascular agents," and
"pro-necrotic agents." Radiosensitizers are known to increase the
sensitivity of cancerous cells to the toxic effects of
electromagnetic radiation of x-rays. Examples of x-ray activated
radiosensitizers include, but are not limited to, the following:
metronidazole, misonidazole, desmethylmisonidazole, pimonidazole,
etanidazole, nimorazole, mitomycin C, RSU 1069, SR 4233, E09, RB
6145, nicotinamide, 5-bromodeoxyuridine (BrDU), 5-iododeoxyuridine
(IUdR), bromodeoxycytidine, fluorodeoxyuridine (FudR), hydroxyurea,
cisplatin, and therapeutically effective analogs and derivatives of
the same.
[0171] Photodynamic therapy (PDT) of cancers employs visible light
as the radiation activator of the sensitizing agent. Examples of
photodynamic radiosensitizers include the following, but are not
limited to: hematoporphyrin derivatives, photofrin, benzoporphyrin
derivatives, NPe6, tin etioporphyrin SnET2, pheoborbide-.alpha.,
bacteriochlorophyll-.alpha., naphthalocyanines, phthalocyanines,
zinc phthalocyanine, and therapeutically effective analogs and
derivatives of the same.
[0172] Chemosensitizers are also known to increase the sensitivity
of cancerous cells to the toxic effects of chemotherapeutic
compounds. Exemplary chemotherapeutic agents that can be used in
conjunction with candidate tubulin inhibitors include, but are not
limited to, adriamycin, camptothecin, dacarbazine, carboplatin,
cisplatin, daunorubicin, docetaxel, doxorubicin, interferon (alpha,
beta, ganma), interleukin 2, innotecan, paclitaxel, streptozotocin,
temozolomide, topotecan, and therapeutically effective analogs and
derivatives of the same. In addition, other therapeutic agents
which can be used in conjunction with candidate tubulin inhibitors
include, but are not limited to, 5-fluorouracil, leucovorin,
5'-amino-5'-deoxythymidine, oxygen, carbogen, red cell
transfusions, perfluorocarbons (e.g., Fluosol-DA), 2,3-DPG, BW12C,
calcium channel blockers, pentoxyfylline, antiangiogenesis
compounds, hydralazine, and L-BSO.
[0173] The methods of treatment as disclosed herein can be via oral
administration, transmucosal administration, buccal administration,
nasal administration, inhalation, parental administration,
intravenous, subcutaneous, intramuscular, sublingual, transdermal
administration, and rectal administration.
[0174] Pharmaceutical compositions of the candidate tubulin
inhibitors of the present invention, include compositions wherein
the active ingredient is contained in a therapeutically or
prophylactically effective amount, i.e., in an amount effective to
achieve therapeutic or prophylactic benefit. The actual amount
effective for a particular application will depend, inter alia, on
the condition being treated and the route of administration.
Determination of an effective amount is well within the
capabilities of those skilled in the art. The pharmaceutical
compositions comprise the candidate tubulin inhibitor, one or more
pharmaceutically acceptable carriers, diluents or excipients, and
optionally additional therapeutic agents. The compositions can be
formulated for sustained or delayed release.
[0175] A preferred therapeutic composition of the present invention
also includes an excipient, an adjuvant and/or carrier. Suitable
excipients include compounds that the subject to be treated can
tolerate. Examples of such excipients include water, saline,
Ringer's solution, dextrose solution, Hank's solution, and other
aqueous physiologically balanced salt solutions. Nonaqueous
vehicles, such as fixed oils, sesame oil, ethyl oleate, or
triglycerides can also be used. Other useful formulations include
suspensions containing viscosity enhancing agents, such as sodium
carboxymethylcellulose, sorbitol, or dextran. Excipients can also
contain minor amounts of additives, such as substances that enhance
isotonicity and chemical stability. Examples of buffers include
phosphate buffer, bicarbonate buffer and Tris buffer, while
examples of preservatives include thimerosal, o-cresol, formalin
and benzyl alcohol. Standard formulations can either be liquid
injectables or solids which can be taken up in a suitable liquid as
a suspension or solution for injection. Thus, in a non-liquid
formulation, the excipient can comprise dextrose, human serum
albumin, preservatives, etc., to which sterile water or saline can
be added prior to administration. In one embodiment of the present
invention, a therapeutic composition can include a carrier.
Carriers include compounds that increase the half-life of a
therapeutic composition in the treated subject. Suitable carriers
include, but are not limited to, polymeric controlled release
vehicles, biodegradable implants, liposomes, bacteria, viruses,
other cells, oils, esters, and glycols.
[0176] The oral form in which the therapeutic agent is administered
can include powder, tablet, capsule, solution, or emulsion. The
effective amount can be administered in a single dose or in a
series of doses separated by appropriate time intervals, such as
hours. Pharmaceutical compositions can be formulated in
conventional manner using one or more physiologically acceptable
carriers comprising excipients and auxiliaries which facilitate
processing of the active compounds into preparations which can be
used pharmaceutically. Proper formulation is dependent upon the
route of administration chosen. Suitable techniques for preparing
pharmaceutical compositions of the therapeutic agents of the
present invention are well known in the art.
[0177] It will be appreciated that appropriate dosages of the
active compounds, and compositions comprising the active compounds,
can vary from patient to patient. Determining the optimal dosage
will generally involve the balancing of the level of therapeutic
benefit against any risk or deleterious side effects of the
treatments of the present invention. The selected dosage level will
depend on a variety of factors including, but not limited to, the
activity of the particular candidate tubulin inhibitor, the route
of administration, the time of administration, the rate of
excretion of the compound, the duration of the treatment, other
drugs, compounds, and/or materials used in combination, and the
age, sex, weight, condition, general health, and prior medical
history of the patient. The amount of compound and route of
administration will ultimately be at the discretion of the
physician, although generally the dosage will be to achieve local
concentrations at the site of action which achieve the desired
effect without causing substantial harmful or deleterious
side-effects.
[0178] Administration in vivo can be effected in one dose,
continuously or intermittently (e.g. in divided doses at
appropriate intervals) throughout the course of treatment. Methods
of determining the most effective means and dosage of
administration are well known to those of skill in the art and will
vary with the formulation used for therapy, the purpose of the
therapy, the target cell being treated, and the subject being
treated. Single or multiple administrations can be carried out with
the dose level and pattern being selected by the treating
physician.
[0179] Some of the examples of diseases treatable by candidate
tubulin inhibitors of the present invention are disclosed herein
but they are not in any way limiting to the scope of the present
invention.
[0180] Examples of Various Diseases
[0181] Various diseases that can be treated by the candidate
tubulin inhibitors of the present invention include, but are not
limited to, cancer types including adrenal cortical cancer, anal
cancer, aplastic anemia, bile duct cancer, bladder cancer, bone
cancer, bone metastasis, adult CNS brain tumors, children CNS brain
tumors, breast cancer, castleman disease, cervical cancer,
childhood Non-Hodgkin's lymphoma, colon and rectum cancer,
endometrial cancer, esophagus cancer, Ewing's family of tumors, eye
cancer, gallbladder cancer, gastrointestianl carcinoid tumors,
gastrointestinal stromal tumors, gestational trophoblastic disease,
Hodgkin's disease, Kaposi'sarcoma, kidney cancer, laryngeal and
hypopharyngeal cancer, acute lymphocytic leukemia, acute myeloid
leukemia, children's leukemia, chronic lymphocytic leukemia,
chronic myeloid leukemia, liver cancer, lung cancer, lung carcinoid
tumors, Non-Hodgkin's lymphoma, male breast cancer, malignant
mesothelioma, multiple myeloma, myelodysplastic syndrome, nasal
cavity and paranasal cancer, nasopharyngeal cancer, neuroblastoma,
oral cavity and oropharyngeal cancer, osteosarcoma, ovarian cancer,
pancreatic cancer, penile cancer, pituitary tumor, prostate cancer,
retinoblastoma, rhabdomyosarcoma, salivary gland cancer, sarcoma
(adult soft tissue cancer), melanoma skin cancer, nonmelanoma skin
cancer, stomach cancer, testicular cancer, thymus cancer, thyroid
cancer, uterine sacrcoma, vaginal cancer, vulvar cancer,
Waldenstrom's macroglobulinemia, chronic lymphocyte leukemia, and
reactive lymphoid hyperplasia.
[0182] The diseases include cancer angiogenesis, new or established
vascularization of tumors, and tumor cell proliferation and
metastasis. Certain of the compounds described herein are also
useful in the promotion of angiogenesis, thus one embodiment of the
present invention is to treat diseases requiring growth of new
blood vessels. Such diseases include, but are not limited to,
myocardial hypertrophy, angina, and high blood pressure. The
invention in an embodiment also provides methods to treat other
diseases, such as, metabolic diseases like gout.
[0183] Examples of Cancer
[0184] The cancer include but are not limited to, adrenal cortical
cancer, anal cancer, aplastic anemia, bile duct cancer, bladder
cancer, bone cancer, bone metastasis, Adult CNS brain tumors,
Children CNS brain tumors, breast cancer, blood cancer, Castleman
Disease, cervical cancer, Childhood Non-Hodgkin's lymphoma, colon
and rectum cancer, endometrial cancer, esophagus cancer, Ewing's
family of tumors, eye cancer, gallbladder cancer, gastrointestianl
carcinoid tumors, gastrointestinal stromal tumors, gestational
trophoblastic disease, Hodgkin's disease, Kaposi'sarcoma, kidney
cancer, laryngeal and hypopharyngeal cancer, acute lymphocytic
leukemia, acute myeloid leukemia; children's leukemia, chronic
lymphocytic leukemia, chronic myeloid leukemia, liver cancer, lung
cancer, lung carcinoid tumors, Non-Hodgkin's lymphoma, male breast
cancer, malignant mesotheliorna, multiple myeloma, myelodysplastic
syndrome, nasal cavity and paranasal cancer, nasopharyngeal cancer,
neuroblastoma, oral cavity and oropharyngeal cancer, osteosarcoma,
ovarian cancer, pancreatic cancer, penile cancer, pituitary tumor,
prostate cancer, retinoblastoma, rhabdomyosarcoma, salivary gland
cancer, sarcoma (adult soft tissue cancer), melanoma skin cancer,
nonmelanoma skin cancer, stomach cancer, testicular cancer, thymus
cancer, thyroid cancer, uterine sacrcoma, vaginal cancer, vulvar
cancer, and Waldenstrom's macroglobulinemia.
[0185] Carcinoma of the thyroid gland is the most common malignancy
of the endocrine system. Carcinoma of the thyroid gland include
differentiated tumors (papillary or follicular) and poorly
differentiated tumors (medullary or anaplastic). Carcinomas of the
vagina include squamous cell carcinoma, adenocarcinoma, melanoma
and sarcoma. Testicular cancer is broadly divided into seminoma and
nonseminoma types.
[0186] Thymomas are epithelial tumors of the thymus, which may or
may not be extensively infiltrated by non-neoplastic lymphocytes.
The term thymoma is customarily used to describe neoplasms that
show no overt atypia of the epithelial component. A thymic
epithelial tumor that exhibits clear-cut cytologic atypia and
histologic features no longer specific to the thymus is known as a
thymic carcinoma (also known as type C thymoma).
[0187] The methods provided by the invention can comprise the
administration of the tubulin inhibitor sin combination with other
therapies. The choice of therapy that can be co-administered with
the compositions of the invention can depend, in part, on the
condition being treated. For example, for treating acute myleoid
leukemia, a tubulin inhibitor can be used in combination with
radiation therapy, monoclonal antibody therapy, chemotherapy, bone
marrow transplantation, gene therapy, immunotherapy, or a
combination thereof.
[0188] Her-2 Related Cancer
[0189] Her-2 disease is a type of breast cancer. Characterized by
aggressive growth and a poor prognosis, it can be caused by the
presence of excessive numbers of a gene called HER2 (human
epidermal growth factor receptor-2) in tumor cells. Therapies that
can used in combination with the tubulin inhibitor s as disclosed
herein include, but are no limited to Her-2 antibodies such as
herceptin, anti-hormones (e.g., selective oestrogen receptor
modulator (SERM) tamoxifen), chemotherapy and radiotherapy,
aromatase inhibitors (e.g. anastrazole, letrozole and exemestane)
and anti-oestrogens (e.g., fulvestrant (Faslodex)).
Blood Cancer
[0190] Lymphoma
[0191] B-Cell Lymphomas
[0192] Non-Hodgkin's Lymphomas caused by malignant (cancerous)
B-Cell lymphocytes represent a large subset (about 85% in the US)
of the known types of lymphoma (the other 2 subsets being T-Cell
lymphomas and lymphomas where the cell type is the Natural Killer
Cell or unknown). Cells undergo many changes in their life cycle
dependent on complex signaling processes between cells and
interaction with foreign substances in the body. Apparently various
types of lymphoma or leukemia can occur in the B-Cell life
cycle.
[0193] Breast Cancer
[0194] A lobular carcinoma in situ and a ductal carcinoma in situ
are breast cancers that develop in the lobules and ducts,
respectively, but may not have spread to the fatty tissue
surrounding the breast or to other areas of the body. An
infiltrating (or invasive) lobular and a ductal carcinoma are
cancers that have developed in the lobules and ducts, respectively,
and have spread to either the breast's fatty tissue and/or other
parts of the body. Other cancers of the breast that can benefit
from treatment provided by the methods of the present invention are
medullary carcinomas, colloid carcinomas, tubular carcinomas, and
inflammatory breast cancer.
[0195] In some embodiments, the invention provides for treatment of
so-called "triple negative" breast cancer. There are several
subclasses of breast cancer identified by classic biomarkers such
as estrogen receptor (ER) and/or progesterone receptor (PR)
positive tumors, HER2-amplified tumors, and ER/PR/HER2-negative
tumors. These three subtypes have been reproducibly identified by
gene expression profiling in multiple breast cancer and exhibit
basal-like subtype expression profiles and poor prognosis. Triple
negative breast cancer is characterized by ER/PR/HER2-negative
tumors.
[0196] Ovarian Cancer
[0197] The ovarian cancer includes but is not limited to,
epithelial ovarian tumors, adenocarcinoma in the ovary and an
adenocarcinoma that has migrated from the ovary into the abdominal
cavity. Treatments for ovarian cancer that can be used in
combination with the tubulin inhibitors of the present invention
include but are not limited to, surgery, immunotherapy,
chemotherapy, hormone therapy, radiation therapy, or a combination
thereof. Some possible surgical procedures include debulking, and a
unilateral or bilateral oophorectomy and/or a unilateral or
bilateral salpigectomy.
[0198] Anti-cancer drugs that can be used in the combination
therapy include cyclophosphamide, etoposide, altretamine, and
ifosfamide. Hormone therapy with the drug tamoxifen can be used to
shrink ovarian tumors. Radiation therapy can be external beam
radiation therapy and/or brachytherapy.
[0199] Cervical Cancer
[0200] The cervical cancer includes, but is not limited to, an
adenocarcinoma in the cervix epithelial. Two main types of this
cancer exist: squamous cell carcinoma and adenocarcinomas. Some
cervical cancers have characteristics of both of these and are
called adenosquamous carcinomas or mixed carcinomas.
[0201] Prostate Cancer
[0202] The prostate cancer includes, but is not limited to, an
adenocarcinoma or an adenocarinoma that has migrated to the bone.
Prostate cancer develops in the prostate organ in men, which
surrounds the first part of the urethra.
[0203] Pancreatic Cancer
[0204] The pancreatic cancer includes, but is not limited to, an
epitheliod carcinoma in the pancreatic duct tissue and an
adenocarcinoma in a pancreatic duct. Treatments that can be used in
combination with the tubulin inhibitor s of the present invention
include but are not limited to, surgery, immunotherapy, radiation
therapy, and chemotherapy. Possible surgical treatment options
include a distal or total pancreatectomy and a
pancreaticoduodenectomy (Whipple procedure). Radiation therapy can
be an option for pancreatic cancer patients, such as external beam
radiation where radiation is focused on the tumor by a machine
outside the body. Another option is intraoperative electron beam
radiation administered during an operation.
[0205] Bladder Cancer
[0206] The bladder cancer includes, but is not limited to, a
transitional cell carcinoma in urinary bladder. Bladder cancers are
urothelial carcinomas (transitional cell carcinomas) or tumors in
the urothelial cells that line the bladder. The remaining cases of
bladder cancer are squamous cell carcinomas, adenocarcinomas, and
small cell cancers. Several subtypes of urothelial carcinomas exist
depending on whether they are noninvasive or invasive and whether
they are papillary, or flat. Noninvasive tumors are in the
urothelium, the innermost layer of the bladder, while invasive
tumors have spread from the urothelium to deeper layers of the
bladder's main muscle wall. Invasive papillary urothelial
carcinomas are slender finger-like projections that branch into the
hollow center of the bladder and also grow outward into the bladder
wall. Non-invasive papillary urothelial tumors grow towards the
center of the bladder. While a non-invasive, flat urothelial tumor
(also called a flat carcinoma in situ) is confined to the layer of
cells closest to the inside hollow part of the bladder, an invasive
flat urothelial carcinoma invades the deeper layer of the bladder,
particularly the muscle layer.
[0207] The therapies that can be used in combination with the
tubulin inhibitors of the present invention for the treatment of
bladder cancer include surgery, radiation therapy, immunotherapy,
chemotherapy, or a combination thereof. Some surgical options are a
transurethral resection, a cystectomy, or a radical cystectomy.
Radiation therapy for bladder cancer can include external beam
radiation and brachytherapy.
[0208] Immunotherapy is another method that can be used to treat a
bladder cancer patient. One method is Bacillus Calmete-Guerin (BCG)
where a bacterium sometimes used in tuberculosis vaccination is
given directly to the bladder through a catheter. The body mounts
an immune response to the bacterium, thereby attacking and killing
the cancer cells. Another method of immunotherapy is the
administration of interferons, glycoproteins that modulate the
immune response. Interferon alpha is often used to treat bladder
cancer.
[0209] Anti-cancer drugs that can be used in combination to treat
bladder cancer include thitepa, methotrexate, vinblastine,
doxorubicin, cyclophosphamide, paclitaxel, carboplatin, cisplatin,
ifosfamide, gemcitabine, or combinations thereof.
[0210] Acute Myeloid Leukemia
[0211] The acute myeloid leukemia (AML) includes acute
promyleocytic leukemia in peripheral blood. AML begins in the bone
marrow but can spread to other parts of the body including the
lymph nodes, liver, spleen, central nervous system, and testes. AML
can be characterized by immature bone marrow cells usually
granulocytes or monocytes, which can continue to reproduce and
accumulate.
[0212] AML can be treated by other therapies in combination with
the tubulin inhibitors of the present invention. Such therapies
include but are not limited to, immunotherapy, radiation therapy,
chemotherapy, bone marrow or peripheral blood stem cell
transplantation, or a combination thereof. Radiation therapy
includes external beam radiation and can have side effects.
Anti-cancer drugs that can be used in chemotherapy to treat AML
include cytarabine, anthracycline, anthracenedione, idarubicin,
daunorubicin, idarubicin, mitoxantrone, thioguanine, vincristine,
prednisone, etoposide, or a combination thereof.
[0213] Monoclonal antibody therapy can be used to treat AML
patients. Small molecules or radioactive chemicals can be attached
to these antibodies before administration to a patient in order to
provide a means of killing leukemia cells in the body. The
monoclonal antibody, gemtuzumab ozogamicin, which binds CD33 on AML
cells, can be used to treat AML patients unable to tolerate prior
chemotherapy regimens. Bone marrow or peripheral blood stem cell
transplantation can be used to treat AML patients. Some possible
transplantation procedures are an allogenic or an autologous
transplant.
[0214] Other types of leukemia's that can be treated by the methods
provided by the invention include but not limited to, Acute
Lymphocytic Leukemia, Chronic Lymphocytic Leukemia, Chronic Myeloid
Leukemia, Hairy Cell Leukemia, Myelodysplasia, and
Myeloproliferative Disorders.
[0215] Lung Cancer
[0216] The common type of lung cancer is non-small cell lung cancer
(NSCLC), which is divided into squamous cell carcinomas,
adenocarcinomas, and large cell undifferentiated carcinomas.
Treatment options for lung cancer in combination with the tubulin
inhibitors of the present invention include surgery, immunotherapy,
radiation therapy, chemotherapy, photodynamic therapy, or a
combination thereof. Some possible surgical options for treatment
of lung cancer are a segmental or wedge resection, a lobectomy, or
a pneumonectomy. Radiation therapy can be external beam radiation
therapy or brachytherapy.
[0217] Some anti-cancer drugs that can be used in chemotherapy to
treat lung cancer include cisplatin, carboplatin, paclitaxel,
docetaxel, gemcitabine, vinorelbine, irinotecan, etoposde,
vinblastine, gefitinib, ifosfamide, methotrexate, or a combination
thereof. Photodynamic therapy (PDT) can be used to treat lung
cancer patients.
[0218] Skin Cancer
[0219] There are several types of cancer that start in the skin.
The most common types are basal cell carcinoma and squamous cell
carcinoma, which are non-melanoma skin cancers. Actinic keratosis
is a skin condition that sometimes develops into squamous cell
carcinoma. Non-melanoma skin cancers rarely spread to other parts
of the body. Melanoma, the rarest form of skin cancer, is more
likely to invade nearby tissues and spread to other parts of the
body.
[0220] Different types of treatments that can be used in
combination with the tubulin inhibitors of the present invention
include but are not limited to, surgery, radiation therapy,
chemotherapy and photodynamic therapy. Some possible surgical
options for treatment of skin cancer are mohs micrographic surgery,
simple excision, electrodesiccation and curettage, cryosurgery,
laser surgery. Radiation therapy can be external beam radiation
therapy or brachytherapy. Other types of treatments include
biologic therapy or immunotherapy, chemoimmunotherapy, topical
chemotherapy with fluorouracil and photodynamic therapy.
[0221] Eye Cancer, Retinoblastoma
[0222] Retinoblastoma is a malignant tumor of the retina. The tumor
can be in one eye only or in both eyes. Treatment options that can
be used in combination with the tubulin inhibitors of the present
invention include enucleation (surgery to remove the eye),
radiation therapy, cryotherapy, photocoagulation, immunotherapy,
thermotherapy and chemotherapy. Radiation therapy can be external
beam radiation therapy or brachytherapy.
[0223] Eye Cancer, Intraocular Melanoma
[0224] Intraocular melanoma is a disease in which cancer cells are
found in the part of the eye called the uvea. The uvea includes the
iris, the ciliary body, and the choroid. Intraocular melanoma
occurs most often in people who are middle aged. Treatments that
can be used in combination with the tubulin inhibitors of the
present invention include surgery, immunotherapy, radiation therapy
and laser therapy. Surgery is the most common treatment of
intraocular melanoma. Some possible surgical options are
iridectomy, iridotrabeculectomy, iridocyclectomy, choroidectomy,
enucleation and orbital exenteration. Radiation therapy can be
external beam radiation therapy or brachytherapy. Laser therapy can
be an intensely powerful beam of light to destroy the tumor,
thermotherapy or photocoagulation.
[0225] Endometrium Cancer
[0226] Endometrial cancer is a cancer that starts in the
endometrium, the inner lining of the uterus. Some of the examples
of the cancer of uterus and endometrium include, but are not
limited to, adenocarcinomas, adenoacanthomas, adenosquamous
carcinomas, papillary serous adenocarcinomas, clear cell
adenocarcinomas, uterine sarcomas, stromal sarcomas, malignant
mixed mesodermal tumors, and leiomyosarcomas.
[0227] Liver Cancer
[0228] Primary liver cancer can occur in both adults and children.
Different types of treatments that can be used in combination with
the tubulin inhibitors of the present invention include surgery,
immunotherapy, radiation therapy, chemotherapy and percutaneous
ethanol injection. The types of surgery that can be used are
cryosurgery, partial hepatectomy, total hepatectomy and
radiofrequency ablation. Radiation therapy can be external beam
radiation therapy, brachytherapy, radiosensitizers or radioiabel
antibodies. Other types of treatment include hyperthermia therapy
and immunotherapy.
[0229] Kidney Cancer
[0230] Kidney cancer (also called renal cell cancer or renal
adenocarcinoma) is a disease in which malignant cells are found in
the lining of tubules in the kidney. Treatments that can be used in
combination with the tubulin inhibitors of the present invention
include surgery, radiation therapy, chemotherapy and immunotherapy.
Some possible surgical options to treat kidney cancer are partial
nephrectomy, simple nephrectomy and radical nephrectomy. Radiation
therapy can be external beam radiation therapy or brachytherapy.
Stem cell transplant can be used to treat kidney cancer.
[0231] Thyroid Cancer
[0232] Thyroid cancer is a disease in which cancer (malignant)
cells are found in the tissues of the thyroid gland. The four main
types of thyroid cancer are papillary, follicular, medullary and
anaplastic. Thyroid cancer can be treated by surgery,
immunotherapy, radiation therapy, hormone therapy and chemotherapy.
Some possible surgical options that can be used in combination with
the tubulin inhibitors of the present invention include but are not
limited to, lobectomy, near-total thyroidectomy, total
thyroidectomy and lymph node dissection. Radiation therapy can be
external radiation therapy or can require intake of a liquid that
contains radioactive iodine. Hormone therapy uses hormones to stop
cancer cells from growing. In treating thyroid cancer, hormones can
be used to stop the body from making other hormones that might make
cancer cells grow.
[0233] AIDS-Related Lymphoma
[0234] AIDS-related lymphoma is a disease in which malignant cells
form in the lymph system of patients who have acquired
immunodeficiency syndrome (AIDS). AIDS is caused by the human
immunodeficiency virus (HIV), which attacks and weakens the body's
immune system. The immune system is then unable to fight infection
and diseases that invade the body. People with HIV disease have an
increased risk of developing infections, lymphoma, and other types
of cancer. Lymphomas are cancers that affect the white blood cells
of the lymph system. Lymphomas are divided into two general types:
Hodgkin's lymphoma and non-Hodgkin's lymphoma. Both Hodgkin's
lymphoma and non-Hodgkin's lymphoma can occur in AIDS patients, but
non-Hodgkin's lymphoma is more common. When a person with AIDS has
non-Hodgkin's lymphoma, it is called an AIDS-related lymphoma.
Non-Hodgkin's lymphomas can be indolent (slow-growing) or
aggressive (fast-growing). AIDS-related lymphoma is usually
aggressive. The three main types of AIDS-related lymphoma are
diffuse large B-cell lymphoma, B-cell immunoblastic lymphoma and
small non-cleaved cell lymphoma.
[0235] Highly-active antiretroviral therapy (HAART) is used to slow
progression of HIV. Medicine to prevent and treat infections, which
can be serious, is also used. AIDS-related lymphomas can be treated
by chemotherapy, immunotherapy, radiation therapy and high-dose
chemotherapy with stem cell transplant. Radiation therapy can be
external beam radiation therapy or brachytherapy. AIDS-related
lymphomas can be treated by monoclonal antibody therapy.
[0236] Kaposi's Sarcoma
[0237] Kaposi's sarcoma is a disease in which cancer cells are
found in the tissues under the skin or mucous membranes that line
the mouth, nose, and anus. Kaposi's sarcoma can occur in people who
are taking immunosuppressants. Kaposi's sarcoma in patients who
have Acquired Immunodeficiency Syndrome (AIDS) is called epidemic
Kaposi's sarcoma. Kaposi's sarcoma can be treated with surgery,
chemotherapy, radiation therapy and immunotherapy. External
radiation therapy is a common treatment of Kaposi's sarcoma.
Treatments that can be used in combination with the tubulin
inhibitors of the present invention include but are not limited to,
local excision, electrodeiccation and curettage, and
cryltherapy.
[0238] Viral-Induced Cancers
[0239] The virus-malignancy systems include hepatitis B virus
(HBV), hepatitis C virus (HCV), and hepatocellular carcinoma; human
lymphotropic virus-type 1 (HTLV-1) and adult T-cell
leukemia/lymphoma; and human papilloma virus (HPV) and cervical
cancer.
[0240] Virus-Induced Hepatocellular Carcinoma
[0241] HBV and HCV and hepatocellular carcinoma or liver cancer can
appear to act via chronic replication in the liver by causing cell
death and subsequent regeneration. Treatments that can be used in
combination with the tubulin inhibitors of the present invention
include but are not limited to, include surgery, immunotherapy,
radiation therapy, chemotherapy and percutaneous ethanol injection.
The types of surgery that can be used are cryosurgery, partial
hepatectomy, total hepatectomy and radiofrequency ablation.
Radiation therapy can be external beam radiation therapy,
brachytherapy, radiosensitizers or radiolabel antibodies. Other
types of treatment include hyperthermia therapy and
immunotherapy.
[0242] Viral-Induced Adult T Cell Leukemia/Lymphoma
[0243] Adult T cell leukemia is a cancer of the blood and bone
marrow. The treatments for adult T cell leukemia/lymphoma that can
be used in combination with the tubulin inhibitors of the present
invention include but are not limited to, radiation therapy,
immunotherapy, and chemotherapy. Radiation therapy can be external
beam radiation therapy or brachytherapy. Other methods of treating
adult T cell leukemia/lymphoma include immunotherapy and high-dose
chemotherapy with stem cell transplantion.
[0244] Viral-Induced Cervical Cancer
[0245] Infection of the cervix with human papillomavirus (HPV) is a
cause of cervical cancer. The treatments for cervical cancers that
can be used in combination with the tubulin inhibitors of the
present invention include but are not limited to, surgery,
immunotherapy, radiation therapy and chemotherapy. The types of
surgery that can be used are conization, total hysterectomy,
bilateral salpingo-oophorectomy, radical hysterectomy, pelvic
exenteration, cryosurgery, laser surgery and loop electrosurgical
excision procedure. Radiation therapy can be external beam
radiation therapy or brachytherapy.
[0246] CNS Cancers
[0247] Brain and spinal cord tumors are abnormal growths of tissue
found inside the skull or the bony spinal column, which are the
primary components of the central nervous system (CNS). Benign
tumors are non-cancerous, and malignant tumors are cancerous.
Tumors that originate in the brain or spinal cord are called
primary tumors. Primary tumors can result from specific genetic
disease (e.g., neurofibromatosis, tuberous sclerosis) or from
exposure to radiation or cancer-causing chemicals.
[0248] The primary brain tumor in adults comes from cells in the
brain called astrocytes that make up the blood-brain barrier and
contribute to the nutrition of the central nervous system. These
tumors are called gliomas (astrocytoma, anaplastic astrocytoma, or
glioblastoma multiforme). Some of the tumors are, but not limited
to, Oligodendroglioma, Ependymoma, Meningioma, Lymphoma,
Schwannoma, and Medulloblastoma.
[0249] Neuroepithelial Tumors of the CNS
[0250] Astrocytic tumors, such as astrocytoma; anaplastic
(malignant) astrocytoma, such as hemispheric, diencephalic, optic,
brain stem, cerebellar; glioblastoma multiforme; pilocytic
astrocytoma, such as hemispheric, diencephalic, optic, brain stem,
cerebellar; subependymal giant cell astrocytoma; and pleomorphic
xanthoastrocytoma. Oligodendroglial tumors, such as
oligodendroglioma; and anaplastic (malignant) oligodendroglioma.
Ependymal cell tumors, such as ependymoma; anaplastic ependymoma;
myxopapillary ependymoma; and subependymoma. Mixed gliomas, such as
mixed oligoastrocytoma; anaplastic (malignant) oligoastrocytoma;
and others (e.g. ependymo-astrocytomas). Neuroepithelial tumors of
uncertain origin, such as polar spongioblastoma; astroblastoma; and
gliomatosis cerebri. Tumors of the choroid plexus, such as choroid
plexus papilloma; and choroid plexus carcinoma (anaplastic choroid
plexus papilloma). Neuronal and mixed neuronal-glial tumors, such
as gangliocytoma; dysplastic gangliocytoma of cerebellum
(Lhermitte-Duclos); ganglioglioma; anaplastic (malignant)
ganglioglioma; desmoplastic infantile ganglioglioma, such as
desmoplastic infantile astrocytoma; central neurocytoma;
dysembryoplastic neuroepithelial tumor; olfactory neuroblastoma
(esthesioneuroblastoma. Pineal Parenchyma Tumors, such as
pineocytoma; pineoblastoma; and mixed pineocytoma/pineoblastoma.
Tumors with neuroblastic or glioblastic elements (embryonal
tumors), such as medulloepithelioma; primitive neuroectodermal
tumors with multipotent differentiation, such as medulloblastoma;
cerebral primitive neuroectodermal tumor; neuroblastoma;
retinoblastoma; and ependymoblastoma.
[0251] Other CNS Neoplasms
[0252] Tumors of the sellar region, such as pituitary adenoma;
pituitary carcinoma; and craniopharyngioma. Hematopoietic tumors,
such as primary malignant lymphomas; plasmacytoma; and granulocytic
sarcoma. Germ Cell Tumors, such as germinoma; embryonal carcinoma;
yolk sac tumor (endodermal sinus tumor); choriocarcinoma; teratoma;
and mixed germ cell tumors. Tumors of the Meninges, such as
meningioma; atypical meningioma; and anaplastic (malignant)
meningioma. Non-menigothelial tumors of the meninges, such as
Benign Mesenchymal; Malignant Mesenchymal; Primary Melanocytic
Lesions; Hemopoietic Neoplasms; and Tumors of Uncertain
Histogenesis, such as hemangioblastoma (capillary
hemangioblastoma). Tumors of Cranial and Spinal Nerves, such as
schwannoma (neurinoma, neurilemoma); neurofibroma; malignant
peripheral nerve sheath tumor (malignant schwannoma), such as
epithelioid, divergent mesenchymal or epithelial differentiation,
and melanotic. Local Extensions from Regional Tumors; such as
paraganglioma (chemodectoma); chordoma; chodroma; chondrosarcoma;
and carcinoma. Metastatic tumours, Unclassified Tumors and Cysts
and Tumor-like Lesions, such as Rathke cleft cyst; Epidermoid;
dermoid; colloid cyst of the third ventricle; enterogenous cyst;
neuroglial cyst; granular cell tumor (choristoma, pituicytoma);
hypothalamic neuronal hamartoma; nasal glial herterotopia; and
plasma cell granuloma.
[0253] Chemotherapeutics available are, but not limited to,
alkylating agents such as, Cyclophosphamide, Ifosphamide,
Melphalan, Chlorambucil, BCNU, CCNU, Decarbazine, Procarbazine,
Busulfan, and Thiotepa; antimetabolites such as, Methotraxate,
5-Fluorouracil, Cytarabine, Gemcitabine (Gemzar.RTM.),
6-mercaptopurine, 6-thioguanine, Fludarabine, and Cladribine;
anthracyclins such as, daunorubicin. Doxorubicin, Idarubicin,
Epirubicin and Mitoxantrone; antibiotics such as, Bleomycin;
camptothecins such as, irinotecan and topotecan; taxanes such as,
paclitaxel and docetaxel; and platinums such as, Cisplatin,
carboplatin, and Oxaliplatin.
[0254] PNS Cancers
[0255] The peripheral nervous system consists of the nerves that
branch out from the brain and spinal cord. These nerves form the
communication network between the CNS and the body parts. The
peripheral nervous system is further subdivided into the somatic
nervous system and the autonomic nervous system. The somatic
nervous system consists of nerves that go to the skin and muscles
and is involved in conscious activities. The autonomic nervous
system consists of nerves that connect the CNS to the visceral
organs such as the heart, stomach, and intestines. It mediates
unconscious activities.
[0256] Acoustic neuromas are benign fibrous growths that arise from
the balance nerve, also called the eighth cranial nerve or
vestibulocochlear nerve. The malignant peripheral nerve sheath
tumor (MPNST) is the malignant counterpart to benign soft tissue
tumors such as neurofibromas and schwannomas. It is most common in
the deep soft tissue, usually in close proximity of a nerve trunk.
The most common sites include the sciatic nerve, brachial plexus,
and sarcal plexus.
[0257] The MPNST can be classified into three major categories with
epithelioid, mesenchymal or glandular characteristics. Some of the
MPNST include but not limited to, subcutaneous malignant
epithelioid schwannoma with cartilaginous differentiation,
glandular malignant schwannoma, malignant peripheral nerve sheath
tumor with perineurial differentiation, cutaneous epithelioid
malignant nerve sheath tumor with rhabdoid features, superficial
epithelioid MPNST, triton Tumor (MPNST with rhabdomyoblastic
differentiation), schwannoma with rhabdomyoblastic differentiation.
Rare MPNST cases contain multiple sarcomatous tissue types,
especially osteosarcoma, chondrosarcoma and angiosarcoma. These
have sometimes been indistinguishable from the malignant
mesenchymoma of soft tissue.
[0258] Other types of PNS cancers include but not limited to,
malignant fibrous cytoma, malignant fibrous histiocytoma, malignant
meningioma, malignant mesothelioma, and malignant mixed Mullerian
tumor.
[0259] Oral Cavity and Oropharyngeal Cancer
[0260] Cancers of the oral cavity include but are not limited to,
hypopharyngeal cancer, laryngeal cancer, nasopharyngeal cancer, and
oropharyngeal cancer.
[0261] Stomach Cancer
[0262] There are three main types of stomach cancers: lymphomas,
gastric stromal tumors, and carcinoid tumors. Lymphomas are cancers
of the immune system tissue that are sometimes found in the wall of
the stomach. Gastric stromal tumors develop from the tissue of the
stomach wall. Carcinoid tumors are tumors of hormone-producing
cells of the stomach.
[0263] Testicular Cancer
[0264] Testicular cancer is cancer that typically develops in one
or both testicles in young men. Cancers of the testicle develop in
certain cells known as germ cells. The two types of germ cell
tumors (GCTs) that occur in men are seminomas (60%) and
non-seminomas (40%). Tumors can also arise in the supportive and
hormone-producing tissues, or stroma, of the testicles. Such tumors
are known as gonadal stromal tumors. The two types are Leydig cell
tumors and Sertoli cell tumors. Secondary testicular tumors are
those that start in another organ and then spread to the testicle.
Lymphoma is a secondary testicular cancer.
[0265] Thymus Cancer
[0266] The thymus is a small organ located in the upper/front
portion of your chest, extending from the base of the throat to the
front of the heart. The thymus contains two main types of cells,
thymic epithelial cells and lymphocytes. Thymic epithelial cells
can give origin to thymomas and thymic carcinomas. Lymphocytes,
whether in the thymus or in the lymph nodes, can become malignant
and develop into cancers called Hodgkin disease and non-Hodgkin
lymphomas. The thymus cancer includes Kulchitsky cells, or
neuroendocrine cells, which normally release certain hormones.
These cells can give rise to cancers, called carcinoids or
carcinoid tumors.
[0267] Treatments that can be used in combination with the tubulin
inhibitors of the present invention include but are not limited to,
surgery, immunotherapy, chemotherapy, radiation therapy,
combination of chemotherapy and radiation therapy or biological
therapy. Anticancer drugs that have been used in the treatment of
thymomas and thymic carcinomas are doxorubicin (Adriamycin),
cisplatin, ifosfamide, and corticosteroids (prednisone).
EXAMPLES
Synthetic Examples
Synthetic Example 1
Synthesis of
1-(4-(3-amino-4-methoxyphenoxy)-3,5-diiodophenyl)ethanone (Formula
IIIB)
[0268] The synthesis of
1-(4-(3-amino-4-methoxyphenoxy)-3,5-diiodophenyl)ethanone,
represented by formula IIIB, was carried out as follows:
##STR00045##
[0269] The compound of formula IIIB was synthesized from
1-(3,5-diiodo-4-(4-methoxyphenoxy)phenyl)ethanone (DIPE) according
to the following procedure: The synthesis of DIPE has been
previously described, e.g. in U.S. Pat. No. 5,922,775, U.S. Pat.
No. 6,303,629, U.S. Pat. No. 5,908,861 and U.S. Pat. No.
6,326,402.
Nitration of DIPE to form
1-(3,5-diiodo-4-(4-methoxy-3-nitrophenoxy)phenyl)ethanone
[0270] Nitration of DIPE was performed with a mixture of 1.4 equiv.
69% nitric acid and 2.2 equiv. 96% sulfuric acid at Ti
<5.degree. C. in methylene chloride. The reaction was complete
after 30 min. According to TLC a single product was formed,
indicating that mono-nitration took place. The product isolated by
extractive workup was obtained in 95% yield with a purity of 99.9
area %. As the nitro-reduced product was eventually identified as
the compound of formula IIIB, it is inferred that the product of
this reaction is
1-(3,5-diiodo-4-(4-methoxy-3-nitrophenoxy)phenyl)ethanone (3-nitro
intermediate).
[0271] Reduction of Nitro Group to Form the Compound of Formula
IIIB
[0272] The reduction of the nitro group was carried out with 5.5
equiv. of anhydrous tin(II) chloride in ethanol, 37% HCl solution
at Ti<10.degree. C.; afterwards the solution was allowed to warm
to room temperature and was stirred at Ti=30-35.degree. C. for 7
hours. The product was isolated as its hydrochloride salt, which
was transformed into the free base upon basification with 1N NaOH
solution. An impurity at RRT=1.08 was present in 1.9 area %. This
impurity was determined to be the nitroso impurity. The crude
product was re-crystallized three times from ethyl acetate. The
nitroso impurity was thereby reduced to 0.4 area % and the product
of formula IIIB was obtained in 99.4 area % purity as a white
solid. The structure of formula IIIB and that of the 3-nitro
intermediate were determined by NMR studies (.sup.1H-, .sup.13C-,
HSQC-, HMBC-, NOESY- and COSY-experiments).
Synthetic Example 2
Synthesis of
1-(4-(3-hydroxy-4-methoxyphenoxy)-3,5-diiodophenyl)ethanone
(Formula IIIA)
[0273] The synthesis of
1-(4-(3-hydroxy-4-methoxyphenoxy)-3,5-diiodophenyl)ethanone,
represented by formula IIIA, was carried out as follows:
##STR00046##
[0274] Diazonium Salt Formation
[0275] A diazonium salt:
##STR00047##
was formed by adding an aqueous solution of NaNO.sub.2 (2.0 equiv.;
c=1.4 g/mL) at Ti=0-5.degree. C. to a suspension of the compound of
formula IIIB, as obtained in synthetic example 1. The reaction was
followed by HPLC. The diazonium salt is eluted at RRT of 0.78.
Diazonium salt formation was complete within 5 hours. The
conversion was 91% and the purity of the diazonium salt was 87.9
area %. Excess nitrous acid was destroyed by addition of urea; and
the reaction mixture was tested with starch iodine paper.
[0276] Conversion of the Diazonium Salt to the Compound of Formula
IIIA
[0277] The diazonium salt was added to 6N H.sub.2SO.sub.4 (30.6
equiv.) and the reaction mixture stirred at Ti=100.degree. C. for
24 hours. The diazonium salt of the compound of formula IIIB was
surprisingly stable. Silica gel chromatography (methylene chloride
as mobile phase) followed by normal and reverse phase
chromatography resulted in 3.9 mg of compound of formula IIIA with
a purity of 74.2 area % to 80.9 area %.
Example 1
Effect on Tubulin Polymerization
[0278] In the following experiments the candidate tubulin
inhibitors, separately or in combination, at 1 to 5 mM
concentrations inhibit the GTP-dependent polymerization of MTP as
determined by an optical test. This inhibition is critically
dependent on the concentration of GTP. The quantitative correlation
between the concentrations of candidate tubulin inhibitors and GTP,
under conditions of a linear rate of MTP polymerization, follows
Michaelis-Menten kinetics and the inhibition portrays a "mixed"
type, where km for GTP and Vmax are altered simultaneously.
Candidate tubulin inhibitors inhibit MTP polymerization parallel to
their anti-tumorigenic action in vivo. The MTP site is one of the
early cellular response sites of the candidate tubulin
inhibitors.
[0279] Exposure of human mammary cancer cells (MDA-MB-231) to 1 mM
of a candidate tubulin inhibitor induces abnormal spindle
structures within 18 hours of drug treatment, thus a putative
candidate tubulin inhibitor-microtubule-protein (MTP) interaction
appears to be a component of early cellular responses to the drug.
Abnormal spindle structures could be the result of candidate
tubulin inhibitor-MTP interaction or reactions of a candidate
tubulin inhibitor with components of the microtubule organizing
center or with as yet undefined systems sequentially or in concert.
Since time-dependent quantitative analysis of the MTP system in
situ is unsuitable for initial velocity measurement we adapted the
in vitro assembly system of neurotubules as a model for a
quantitative analysis of the interaction of a candidate tubulin
inhibitor with MTP. As demonstrated by Gaskin, et al., 1974,
"Turbidimetric studies of the in vitro assembly and disassembly of
porcine neurotubules", J. Mol. Biol. 89:737-758; and Kirschner, et
al., 1974, "Microtubules from mammalian brain: some properties of
their depolymerization products and a proposed mechanism of
assembly and disassembly", Proc. Natl. Acad. Sci. U.S.A.
71:1159-1163; this system is suitable for kinetic assay of MTP
assembly in vitro. The time course of MTP assembly consists of
initiation and propagation and termination steps, Gaskin, et al.,
1974, "Turbidimetric studies of the in vitro assembly and
disassembly of porcine neurotubules", J. Mol. Biol. 89:737-758. The
rate of propagation under defined conditions is sufficiently linear
to permit kinetic analysis that can be evaluated with respect
candidate tubulin inhibitor and GTP concentrations. As we show here
the inhibition of MTP assembly by candidate tubulin inhibitors
occurs in the same range of drug concentration as required to
inhibit tumorigenesis in vivo, or to inhibit cell replication or
induce eventual cell death; Mendeleyev, et al., 1997. "Structural
specificity and tumoricidal action of
methyl-3,5-diiodo-4-(4'-methoxyphenoxy)benzoate (DIME)" Int. J.
Oncol., 10:689-695. Therefore the a candidate tubulin inhibitor-MTP
interaction is most probably a component of the apparently
pleiotropic cellular mechanism of action of DIME.
Isolation of Microtubule Proteins (MTP)
[0280] Preparation of MTP and an optical test for polymerization is
adopted from published methods, Gaskin, et al., 1974,
"Turbidimetric studies of the in vitro assembly and disassembly of
porcine neurotubules", J. Mol. Biol. 89:737-758; Tiwari, et al.,
1993, "A pH and temperature-dependent cycling method that doubles
the yield of microtubule protein", Anal. Biochem. 215:96-103.
Bovine or rabbit brain is homogenized in an equal volume of ice
cold buffer containing 100 mM Pipes/K+ (pH 7.4), 4 mM EGTA. 1 mM
MgCl.sub.2, 0.5 mM DTT and 0.1 mM PMSF, and centrifuged at 39,000 g
for 1 hour at 4.degree. C. To the supernatant, DMSO (8% final
concentration) and GTP (1 mM final concentration) are added,
followed by incubation at 37.degree. C. for 30 minutes.
Microtubules are pelleted at 100,000 g at 37.degree. C. for 30
minutes. The pellets are incubated on ice for 15 minutes, followed
by resuspension in ice cold PEM buffer (100 mM Pipes/K+ (pH 6.9), 1
mM EGTA, 1 mM MgCl.sub.2). This warm polymerization and cold
depolymerization cycle is repeated once more and the cold,
resuspended monomeric MTP (8-10 mg/ml protein) is used for the
optical test for polymerization kinetics. Both rabbit or bovine
brain yields identical MTP preparations.
[0281] The polymerization reaction is started by the addition of
100 ml of MTP solution (equivalent to 0.8-1.0 mg protein) and
initial linear rates of increase in absorbance at 350 nm follows
and is recorded at 37.degree. C. in a Perkin-Elmer 552 double beam
spectrophotometer, equipped with a thermostatically controlled
cuvette holder.
[0282] Inhibition of MTP polymerization can have highly complex
cellular consequences. In cytokinesis, this inhibition can
interfere with traction forces of tubulin and prevent the formation
of a cleavage furrow which is essential for cell division, Burton,
et al., 1997, "Traction forces of cytokinesis measured with
optically modified elastic substrate", Nature 385:450-454. The
inhibition of MTP polymerization by candidate tubulin inhibitors
should be correlated with the biochemical sites of this drug.
[0283] On the basis of these experiments, it can be seen that
thyroxine type analogues, such as the candidate tubulin inhibitors,
are capable of blocking mitosis in cancer cells.
Example 2
Anti-Tumor Efficacy of Antitubulin Inhibitors
[0284] In Vitro Cytotoxicity Screen Against Human Tumor Cell
Lines.
[0285] Human tumor cell lines are exposed continuously to varying
concentrations of test agents (i.e., candidate tubulin inhibitors)
and the viability of the cells are measured at set time points (1,
3, and 7 days) using the alamar Blue.TM. assay. When alamar
Blue.TM. dye is added to culture medium, the dye is reduced by
cellular mitochondrial enzymes yielding a soluble product with
substantially enhanced fluorescence. This fluorescence is measured
with a fluorimeter, whereby the signal is directly proportional to
cell number.
[0286] Various tumor cell lines of human origin representing a wide
diversity of cancer phenotypes and genotypes are exposed in vitro
to the candidate tubulin inhibitors to evaluate the drugs'
effective range. Tumor lines that are screened include MDA-MB-231
(breast), MCF-7 (breast), MDA-MB-468 (breast), Siha (squamous cell
carcinoma), A549 (non-small cell lung), HL-60 (leukemia), and
Ovcar-3 (ovarian). The test agents are prepared in serial dilutions
from 10 mM stock solutions in DMSO yielding a 66-fold dose range of
20 .mu.M, 10 .mu.M, 3 .mu.M, 10 .mu.M and 0.3 .mu.M concentrations.
In each case, cells are maintained at 37.degree. C. in 5 percent
CO.sub.2 in air. All cell lines are incubated in Dulbecco's
modified Eagle medium (DMEM) supplemented with 10% heat-inactivated
fetal bovine serum and graded doses of candidate tubulin
inhibitors. 5-Fluorouracil (5-FU) is included as a positive control
in experiments with some the cell lines. The experiments are
conducted in 96-well tissue culture plates (Falcon) with an initial
seeding density of 1000 cells per well in 250 .mu.L aliquots. The
next day, medium is replaced with 100 .mu.L of drug dilutions in
triplicate.
[0287] After a 7 day exposure to the various agents, the alamar
Blue.TM. dye is diluted to 20% in DMEM, and 100 .mu.L is added to
each well. The cells are incubated for 8 hours until obvious color
changes indicate sufficient amounts of reduced dye for
quantitation. Relative cell number is evaluated by fluorimetry on a
Millipore 2300 CytoFluor fluorescence measurement system.
Measurements are taken directly from 96-well plates after
excitation at 560 nm with concomitant emission at 590 nm. IC50
values are calculated vs. control (non-treated cells).
Example 3
In Vitro Metabolism of Candidate Tubulin Inhibitors in Human
Leukemia Cells
[0288] In humans and laboratory animals, enzyme systems in the
liver are capable of metabolizing a large number of chemicals to
inactive forms, and some chemotherapeutic agents are inactive
unless they are metabolized to active forms. To test for either of
these possibilities, the candidate tubulin inhibitors are incubated
with a microsomal enzyme fraction, referred to as S9.
[0289] Human leukemia (HL60) cells are used to evaluate drug
metabolism. Exposure to the test article is performed in the
presence and absence of S9, prepared from the liver of adult male
rats given a single intraperitoneal injection of Arachlor 1254 (500
mg/kg). The S9 consists of the 9000.times.G supernatant of liver
homogenized in 0.25 M sucrose-100 mM phosphate buffer (pH 7.4)
(Molecular Toxicology, Inc.). Cofactors are 1 mM NADP and 5 mM
sodium isocitrate. On the day of exposure, cultures of cells, with
or without the metabolism mixture, are incubated for 4 hours in
RPMI containing 5% FBS and graded doses of candidate tubulin
inhibitors. The candidate agents are prepared in serial dilutions
from 10 nM stock solutions in DMSO yielding a dose range 100 .mu.M,
50 .mu.M, 25 .mu.M, 10 .mu.M and 5 .mu.M concentrations and a final
DMSO concentration of 1%. In each case, cells are maintained at
37.degree. C. in 5 percent CO.sub.2 in air. The experiments are
conducted in 6-well tissue culture plates (Falcon) with an initial
seeding density of 2.times.10.sup.6 cells per well in 2 ml
aliquots. The test compounds are added immediately to the medium in
duplicate cultures. Treatment solution is removed by a series of
low-speed centrifugations to pellet the cells, followed by removal
of the supernatant and resuspension of cells in fresh RPMI
containing 10% FBS.
Example 4
In Vivo Efficacy of Candidate Tubulin Inhibitors Against Met-1
Mouse Mammary Tumors
[0290] Mammary fat pads of Balb/c immunodeficient (nude) mice are
implanted with 1 mm.sup.3 of mammary tumor tissue. These mammary
tumors originate from the met-1 mouse mammary tumor cell line and
are been propagated by passaging in vivo. When palpable tumors
appear, animals are subdivided into various treatment groups and
treated daily with intraperitoneal injections of control vehicle,
and candidate tubulin inhibitors (50 mg/kg). A group of untreated
control animals are included in the study, because agents used in
the vehicle for these test articles (DMSO/ethanol/Cremophor EL/PEG
400, 1:0.5:0.5:6) are themselves found to have cytotoxic activity.
Twice weekly, the volumes of the tumors in each animal in each
group are measured to gather information on tumor growth (volume)
as a function of type of treatment, dose, and time.
Example 5
Anti-Angiogenic Properties: the Chick Chorioallantoic Membrane
(CAM) Assay
[0291] Candidate tubulin inhibitors are tested in the CAM assay.
The endpoint of the CAM assay is a quantitative determination of
basement membrane biosynthesis by measuring the incorporation of
.sup.14C-proline into Type IV collagenous protein.
[0292] The CAM assay involves the development of live chick embryos
in Petri dishes under special sterile conditions. Therefore, only
limited numbers of embryos can be used for evaluation of compounds
in a single experiment. Candidate tubulin inhibitors are tested in
separate assays. A known angiogenesis inhibitor, 2-methoxyestradiol
(2-ME), is used as the positive control, and human fibroblast
growth factor (hFGF) is used to induce angiogenesis in the CAM.
[0293] Fertilized eggs are supplied by Melody. Ranch, Aptos, Calif.
L-[U-.sup.14C] proline (specific activity, 290 mCi/mmol) is
purchased from New England Nuclear, Boston, Mass. Collagenase and
2-ME were obtained from Sigma Chemical Co., St. Louis, Mo. Silicone
ring cups are obtained by cutting silicone tubing (3 mm diameter)
into small "O" rings 1 mm in thickness. These silicone ring cups
can be reused many times if they are sterilized prior to each
assay. Plastic Petri dishes (20.times.100 mm) are purchase from
Baxter diagnostics, Inc., Hayward, Calif. hFGF-B is obtained from
Clonetics Corporation, San Diego, Calif.
[0294] For testing, a minimum amount of acetone-methanol (1:1) is
added to the test compounds for sterilization. The acetone-methanol
mixture is then evaporated to dryness in a sterile hood. The
compounds are dissolved in dimethyl sulfoxide (DMSO) first and
subsequently diluted with saline containing methylcellulose. The
final concentrations are 2% DMSO and 0.5% methylcellulose. All test
solutions are added to each CAM in 20-ml aliquots.
[0295] The method of Folkman, et al. (Folkman, et al. (1974) Dev.
Biol. 41:391-394) with some modifications, is used to cultivate
chicken embryos as follows:
[0296] Fresh fertile eggs are incubated for three days in a
standard egg incubator. On Day 3, eggs are cracked under sterile
conditions and embryos are placed in 20.times.100 mm plastic Petri
dishes and cultivated at 37.degree. C. in an embryo incubator with
a water reservoir on the bottom shelf. Air is continuously bubbled
into the water reservoir by using a small pump so that the humidity
in the incubator is kept constant. Observations are made daily to
ensure that all embryos are healthy. Dead or unhealthy embryos are
removed from the incubator immediately to avoid contamination. On
Day 9, a sterile silicone ring cup is placed on each CAM and 0.5
mCi of .sup.14C-proline with or without the test compound plus 2.5
ng of hFGF dissolved in saline containing 0.5% methylcellulose is
delivered into each ring cup in a sterile hood. 2-ME id tested in
parallel to serve as a reference compound. After addition of test
materials, the embryos are returned to the incubator and
cultivation continued. On Day 12, all embryos are transferred to a
cold room at 4-10.degree. C. The anti-angiogenic effect of each
test compound is determined by using the collagenase assay to
measure .sup.14C-proline incorporation into collagenous
protein.
Example 6
Collagenase Assay for Measurement of .sup.14C-Proline Incorporation
into Collagenous Protein
[0297] Using the procedure outlined in Maragoudakis, et al., (1989)
J. Pharm Exp. Ther. 251:679-682, the embryos are placed on ice, and
a piece of CAM 10 mm in diameter is cut off under each ring cup and
placed in a separate tube. To each tube is added 1.0 ml of
phosphate-buffered saline (PBS, pH 7.3) containing 0.11 mg
cycloheximide and 0.17 mg dipyridyl. The tubes are placed in a
boiling water bath for 10 min and then cooled to room temperature.
The PBS in each tube is discarded after centrifugation at
3000.times.G for 10 min. The CAM residue is washed once with 3 ml
of 15% TCA and then three times with 3 ml of 5% TCA. Centrifugation
is carried out as described above between each washing. At this
point all non-protein bound radioactivity was removed, and the CAM
containing the newly synthesized .sup.14C-collagenous protein is
suspended in 0.9 ml of 0.1 N NaOH and 1.1 ml of HEPES buffer at pH
7.4. The pH of the sample is neutralized with 0.8 N HCl, using
phenol red as indicator.
[0298] To digest the .sup.14C-collagenous protein, 7.5 units of
collagenase and 500 mmol of calcium chloride in 40 ml of HEPES
buffer is added to the above samples: and mixtures are incubated at
37.degree. C. for 4 h. The reaction is stopped by adding 1.0 ml of
20% TCA containing 5 mg of tannic acid into each tube. After vortex
mixing, the samples are centrifuged at 3000.times.G for 10 min. An
aliquot of the clear supernatant was taken for scintillation
counting to quantitate the radiolabeled tripeptides corresponding
to basement membrane collagen and other collagenous materials
synthesized by the CAM from .sup.14C-proline. The CAM pellets in
each tube are solubilized in 0.5 ml of 1.0 N NaOH by boiling in a
water bath for 5 min. An aliquot of the dissolved CAM is used for
protein determination using the method provided by Pierce Chemical
Co. The radioactivity per milligram of protein from the CAM treated
with a test compound relative to that from the control CAM gives
the percent of angiogenesis inhibition.
Example 7
Assays Used to Screen for Anti-Mitotic Properties: Microtubule
Assembly Inhibition Assay
[0299] A cell-free assay for measuring inhibition of the
microtubule assembly can be performed by first mixing tubulin and
rhodamine-labeled tubulin at a ratio of 4:1 (Hyman, A., et al.
(1990) Meth. Enzymol. 196, 478-485; Belmont, L. D., et al. (1996)
Cell 84, 623-631). This tubulin solution is then added on ice to a
buffer (BRB 80; 80 mM potassium salt of PIPES (pH 7.5), 5 mM
MgCl.sub.2, 1 mM EGTA) containing 1 mM GTP and 1 mM DTr to a 15
.mu.M final concentration. Drugs at different concentrations (0.5
.mu.l) are added to 50 .mu.l samples of the buffered tubulin, and
10 .mu.l of each solution is transferred to microfuge tubes. Each
tube receives 0.4 .mu.l of microtubule seeds (Belmont, L. D.,
supra). Tubes are incubated at 37.degree. C. for 10 min before
adding 100 .mu.l of BRB 80 containing 1% glutaraldehyde. Each
reaction mixture (2.5 .mu.l) is transferred to a microscope slide
for fluorescence microscopy.
[0300] Microtubules in intact cells are visualized by using a mouse
anti-tubulin monoclonal antibody and a fluorescein-labeled donkey
anti-mouse polyclonal antibody. Briefly, HeLa cells are plated in
2-well chamber slides (Nunc, Napierville, Ill.) at
1.5.times.10.sup.4/ml and incubated in 5% CO.sub.2 at 37.degree. C.
for 24 h before treatment with drugs for 1 h. After removing the
medium, cellular microtubules are stabilized by using BRB 80
containing 4 mM EGTA and 0.5% Triton X-100. The cells are fixed for
3 min in methanol chilled at -20.degree. C., washed with TBS buffer
(0.15 M NaCl, 0.02 M Tris-HCl, pH 7.4), and permeabilized with
TBS/0.5% Triton X-100. After several washes with TBS/0.1% Triton
X-100, the cells are blocked with an antibody dilution buffer (TBS,
0.1% Triton X-100, 2% BSA, 0.1% sodium azide) for 10 min. The cells
are stained in the dark with the primary antibody for 1.5 h, and
then the secondary antibody is added in the antibody dilution
buffer containing 1 .mu.g/ml Hoechst 33342 and incubated in the
dark for 45 min. The slides are mounted with n-propyl gallate (2%
w/v in 30% 0.1 M Tris/glycerol, pH 9.0) and sealed under glass
cover slips.
Example 8
Competitive Tubulin-Binding Assay
[0301] The competitive binding of a compound to the colchicine
binding site of tubulin is performed by using a spin column method
(Woods, J. A., et al. (1995) British J. Cancer 71, 705-711).
.sup.14C-labeled candidate tubulin inhibitor (20 .mu.M, 10 nCi) is
mixed with tubulin and colchicine at different concentrations and
incubated at room temperature for 1.5 h in a buffer containing 0.1
M MES (pH 6.8), 1 mM EGTA, 1 mM EDTA, and 1 mM MgCl.sub.2. Each
reaction mixture is loaded onto a column containing 1 ml of
Sephadex G50 equilibrated with a buffer containing 40 mM MES (pH
7.5), 40 mM Tris, and 1 mM MgSO.sub.4. The columns are centrifuged
at 900.times.G for 3 min and the eluents are each mixed with 3 ml
of CytoScint (ICN) for analysis by liquid scintillation
counting.
Example 9
Use of the Candidate Tubulin Inhibitors as Sensitizers for
Radiotherapy: In Vitro Radiosensitization Assay
[0302] Radiosensitization studies are performed on cancer cells
grown in culture. The test compound is added to the cells prior to,
during, or after irradiation. Radiation dosages are typically
measured in units of Gy/min, with one Gy is equal to 100 rads,
while one rad is the quantity of ionizing radiation that results in
the absorption of 100 ergs of energy per gram of irradiated
material. Sensitization enhancement ratios (SER) are determined at
a 10% survival level. The C1.6 value (i.e., a concentration of test
compound yielding an SER of 1.6) is determined by plotting SER
values against test compound concentration.
[0303] HeLa S-3 cells are grown and maintained in modified Eagles's
medium containing 10% fetal bovine serum in a humidity and
CO.sub.2-controlled incubator. Experimental studies are initiated
on exponential cultures growing in 60 mm tissue culture dishes at
treatment densities of 3-7.times.10.sup.5 cells/dish. The doubling
time of HeLa S-3 cultures is typically approximately 18 hours.
[0304] A compound of formula (I) is dissolved in distilled water
just prior to use and diluted 1:100 through addition of the
appropriate volume to cultured cells. For ultraviolet irradiation
studies, compound is added only during the repair period following
irradiation. For X-irradiation studies, drug is added to cultures 1
hour prior to irradiation and remains in contact with cells during
irradiation.
[0305] For ultraviolet (non-ionizing) irradiation, all medium is
removed from cultures and, with the lid removed, dishes are exposed
to 1.4 J/M2 of UV254 nm light emitted from a G.E. germicidal lamp.
Fresh media with or without test compound is then added to cultures
during the subsequent repair period. In some cases, cells are
harvested immediately in order to establish a T0 value for DNA
strand breaks. X-irradiation (ionizing) of cultures is carried out
in a TFI Bigshot X-ray unit at 3 mA, 50 keV, filtered with 1.5 mm
Be and delivering 0.56 Gy/min to the cells (through the lid and 5
mls of media) as determined by a Victoreen ionization chamber
calibrated in the 10 to 50 keV range. Following X-irradiation,
cultures are harvested immediately for colony forming ability
assays. D0 values are calculated from survival curves computer
plotted by linear regression analysis.
[0306] The ability of cells to form colonies after irradiation is
determined by standard methods. Cultures treated with test compound
are irradiated and immediately trypsinized, counted and re-plated
in 5 ml medium containing the appropriate number of cells (500 for
untreated cultures and cultures exposed to 2.8 Gy X-rays; 2,000 for
cultures exposed to 20 J/M2 UV; 5,000 for cultures exposed to 5.6
Gy X-rays and 10,000 for cultures exposed to 8.4 Gy X-rays).
Cultures are grown for 10 days at which time colonies of 1.0-2.0 mm
(50-200 cells) are evaluated by methanol fixation and staining.
Untreated HeLa cells exhibit cloning efficiencies in the range of
34 to 46% using this protocol.
[0307] In Vivo Radiosensitization Assay
[0308] This example describes one method by which one of skill in
the art can assay the effect of the compounds of the invention on
radiotherapy of malignant tumors. The model system used in this
study is well established for determining the effects of radiation
on tumor tissue. See, e.g., Twentyman, et al. (1980) J. Nat'l.
Cancer Inst. 64: 595-603; Brown, et al. (1980) J. Nat'l. Cancer
Inst. 64: 603-611; Bernstein, et al. (1982) Radiation Res. 91:
624-637. The model uses RIF-1 tumor cells, which are well suited to
studies of radiation response, including in vitro cell survival and
in vivo tumor studies, in part because of its rapid growth rate,
with a doubling time of 65 hours and a cell cycle time of 12 hours.
The RIF-1 tumor is minimally immunogenic, and metastasizes only at
a late stage of growth.
[0309] Tumors are produced by the subcutaneous inoculation into the
lower backs of mice. This inoculation consists of a suspension of
2.times.10.sup.5 RIF-1 cells from culture in 0.25 ml of alpha
minimum essential media (MEM, Gibso) supplemented with 10% fetal
bovine serum (Johns Scientific). Male C3H/He mice (Harlan Sprague
Dawley Inc., Indianapolis, Ind.) that are 5 to 7 weeks old at the
time of inoculation are suitable for these experiments. Animals are
anaesthetized for the inoculation.
[0310] The tumors are then allowed to grow to 1 cm in average
diameter. Measurements are made using a caliper, talking the tumor
length and width and calculating the average of these two. Tumor
diameter measurements are taken every 2 to 3 days from the time the
tumor cells were implanted.
[0311] Tumors are allowed to grow to approximately 1 cm average
diameter without any intervention. Upon the tumors reaching this
average diameter, the subject animals are randomized into one of
three groups. One group receives no radiation and no test compound,
a second group receives radiation and no test compound and a third
group receives radiation and test compound.
[0312] For radiation treatment, each animal receives a general
anesthetic. Animals immobilized and placed in the radiotherapy
apparatus for the same period of time. The radiation exposure
consists of a single dose of 3000 cGy of 150 KeV X-irradiation
(mean time 10 minutes, 40 seconds). Preferably, the radiotherapy
equipment (Protea ionization chamber) is calibrated before and
after each session to ensure absolute uniform dosing. The
radiotherapy is administered with a cone over the tumor and lower
back of the animal, which in every case assures a uniform maximal
delivery dose to the tumor while minimizing dose delivery to the
sensitive structures of the abdomen and upper pelvis.
[0313] Those animals randomized to the test compound-treated group
receive an intravenous dose of a candidate tubulin inhibitor. The
day the subject animal is treated is designated "Day Zero". At
frequent intervals, usually every other day, the tumors are
measured in the same fashion as previously described, and an
average of two diameters calculated. These data are plotted as a
function of time. The end-point of the study occurs when the tumor
reaches double the original treatment diameter, or approximately 2
cm. Animals are euthanized in a CO.sub.2 chamber and the tumors
removed surgically post-mortem. Representative tumors are
sectioned, paraffin embedded and slides are stained using H&E
stain and examined to confirm the histological presence of RIP-1
fibrosarcoma. Animals are sacrificed and tumors harvested before
the tumor reaches twice the original diameter in the following
situations: premature death of animal following treatment;
ulceration of the tumor; or infection or inflammation of the
injection site.
[0314] Following the termination of the experiment, growth curves
for each subject tumor are completed and a line of best fit
assigned for purposes of interpolation between data points. The
average diameter (AD) of each tumor is then determined for each day
of the study from the lane of best fit.
Example 10
Use of Candidate Tubulin Inhibitors as Sensitizers for
Chemotherapy
[0315] Assays for determining appropriate dosages of the compounds
of the invention for use as sensitizers for chemotherapy and
immunotherapy are similar to those described for radiotherapy. To
examine the "pre-incubation effect" of the compounds of the
invention, cancer cells are exposed to a fixed concentration of a
candidate tubulin inhibitor test compound for 2 hours, followed by
exposure to varying concentrations of a chemotherapeutic or
immunotherapeutic agent for 1 hour at 37.degree. C., and then
assayed for colony formation. In evaluating the effect of
"pre-incubation time" on chemosensitization, cancer cells are
exposed to fixed concentrations of the test compound for 0 to 4
hours at 37.degree. C., followed by exposure to a chemotherapeutic
agent under aerobic conditions for 1 hour at 37.degree. C., and
then assayed for colony formation. Test compound dose-dependent
potentiation also is examined by exposing cancer cells to various
test compound concentrations for 2 hours at 37.degree. C., and then
to a fixed dose of each chemotherapeutic agent for 1 hour at
37.degree. C. under aerobic conditions. Experiments using a
simultaneous addition of the sensitizer and chemotherapeutic agent
for 1 hour at 37.degree. C. under aerobic conditions can also be
performed.
Example 11
Chemoprevention
[0316] The efficacy of the claimed compounds as a chemopreventive
agent can be demonstrated using in vitro and in vivo models of
5AzadC-induced carcinogenesis. A suitable model system uses
pre-malignant murine fibroblasts (cell lines 4C8 and PR4) that
express a transcriptionally activated c-Ha-ras protooncogene. These
non-tumorigenic cells, which are highly susceptible to malignant
conversion by pharmacological doses of 5AzadC, are subclones of
mouse NIH 3T3 fibroblasts, PR4N and 4C8-A10 (designated here PR4
and 4C8) and have been previously described (see, e.g., Wilson, et
al. (1986) Anal. Biochem., 152: 275-284; Dugaiczyk (1983) Biochem.
22:1605-1613). Both cell lines are phenotypic revertants isolated
from LTR/c-Ha-ras 1-transformed 3T3 cells after long-term treatment
with murine interferon alpha/beta. Cultures are maintained in
Dulbecco's modified Eagle's medium (DM supplemented with 10% heat
inactivated fetal calf serum (Gibco) and antibiotics. The sodium
salts of phenylacetic and phenylbutyric acids (Elan Pharmaceutical
Corporation) are dissolved in distilled water. 5AzadC (Sigma St.
Louis, Mo.) is dissolved in phosphate buffered saline (PBS) and
stored in aliquots at -20.degree. C. until use. Exposure of 5AzadC
to direct light is avoided at all times to prevent drug
hydrolysis.
[0317] For in vitro tests, cells are plated at 1-2.times.10.sup.5
cells in 100 mm dishes and the test compound added to the growth
medium at 20 and 48 hrs later. The cells are subsequently
subcultured and observed for phenotypic alterations. Whereas
untreated 4C8 and PR4 form contact-inhibited monolayers composed of
epithelial-like cells, transient exposure of these cultures to 0.1
.mu.M 5AzadC during logarithmic phase of growth results in rapid
and massive neoplastic transformation. Within one week of 5AzadC
treatment, the great majority of the cell population becomes
refractile and spindly in shape and form multilayered cultures with
increased saturation densities, which is indicative of loss of
contact inhibition of growth. Treatment of the cells with the test
compound reduces or prevents these phenotypic changes. The test
compound can be administered prior to treatment of the cells with
5AzadC, simultaneously with 5 AzadC treatment, or after 5AzadC
treatment.
[0318] For in vivo tests of the ability of the test compounds to
prevent malignancy, 6-9 week-old female athymic nude mice are
inoculated subcutaneously (s.c.) with 0.5.times.10.sup.6 cells.
Twenty four hours later 400 .mu.g of freshly prepared 5AzadC in 200
.mu.l PBS is administered intraperitoneally (i.p.) into each animal
(approximately 20 mg/kg). The test compound is also administered to
the animal. The number, size, and weight of tumors is recorded
after 3-4 weeks. For histological examination, tumors are excised,
fixed in Bouin's solution (picric acid: 37% formaldehyde: glacial
acetic acid, 15:5:1 vol/vol), and stained with H&E. A single
i.p. injection of mice with 5AzadC (20 mg/kg) typically results in
tumor development at the site of 4C8 cell inoculation in controls.
However, animals protected by a compound of the invention either
fail to develop tumors or form slow-growing lesions at the site of
4C8 inoculation.
[0319] An additional test of the ability of the compounds of the
invention to prevent malignant growth involves inhibition of cell
growth on matrigel, which is a reconstituted basement membrane
(Collaborative Research). This assay models the ability of cells to
degrade and cross tissue barriers. Cells are exposed for 48 hrs in
plastic tissue culture dishes with 5AzadC alone or in combination
with the test compound. Treatment with the test compound is
continued for an additional 1-2 weeks, after which cells are
re-plated onto 16 mm dishes that were previously coated with 250
.mu.l of matrigel (10 mg/ml). The test compound is either added to
the dishes or omitted in order to determine whether the effect is
reversible. In the absence of test compound, net-like formation
characteristic of invasive cells typically occurs within 12 hours,
and invasion into the matrigel is evident after 6-9 days.
Example 12
Assay for Measuring the Ability of Candidate Tubulin Compounds to
Reduce the Levels of Tumor Necrosis Factor (TNF-Alpha)
[0320] This example provides an assay that can be used to screen
compounds of Formula I for their ability to reduce the expression
of cytokines (e.g., TNF-alpha) in a mammal.
[0321] Cell Line
[0322] The murine macrophage PU5-1.8 cell line is purchased from
the American Type Culture Collection (ATCC, Rockville, Md.). Cells
are grown in DMEM medium supplemented with 100 mM sodium pyruvate,
0.1 mM nonessential amino acids, 2 mM glutamine and 5% fetal bovine
serum (Life Technologies, Staten Island, N.Y.). Cells are
maintained in a humidified atmosphere of 5% CO.sub.2-95% air at
37.degree. C. Cells are passaged twice weekly by firmly tapping the
side of the flask to dislodge the adherent cells. Both non-adherent
and adherent cells are passaged. Exponentially growing cells are
seeded at 5.times.10.sup.5/mL, 4 mL per 60-mm dish 24 h prior to
the experiment. Test compounds are delivered in 1 mL volumes of the
medium added to each dish at the start of the experiment. All
dishes are incubated at 37.degree. C. in 5% CO.sub.2-95% air for 3
h.
[0323] Reagents.
[0324] The Tumor Necrosis Factor (TNF-alpha) cDNA is obtained from
the ATCC (Rockville, Md.). [alpha-32 P]-dCTP (250 .mu.Ci) and nylon
membranes (Hybond N) are obtained from Amersham (Arlington Heights,
Ill.). Colchicine (used as a control) is purchased from the Sigma
Chemical Company (St. Louis, Mo.). Lipopolysaccharide (LPS) from
Escherichia coli is purchased from DIFCO Laboratories (Detroit,
Mich.). All plastic supplies are purchased from VWR Scientific
products (San Francisco, Calif.).
[0325] Northern Blotting
[0326] Total RNA is isolated by the guanidinium-cesium chloride
method as described before (N. S. Waleh, et al., 1994, Cancer Res.,
54:838-843). Five to 10 .mu.g of total RNA is electrophoresed in 1%
agarose gels containing 6% formaldehyde. Following electrophoresis,
gels are stained with ethidium bromide to visualize the positions
of 28S and 18S RNA. The RNA is then transferred to nylon membranes
(Amersham Hybond N) by capillary blotting and fixed to the filter
by exposure to UV light. The blots are probed with .sup.32P-labeled
cDNA sequences of human TNF-alpha obtained from the American Type
Culture Collection (ATCC). The TNF-alpha cDNA is a 1.1 kb PstI
fragment of plasmid pE4 in E. coli MM294 (ATCC 39894).
Hybridizations is carried out at 42.degree. C. in 50% formamide,
5.times.SSC, 5.times.Denhardt's solution, 0.1% SDS, and 0.3 mg/mL
salmon sperm DNA. Filters are washed by 1.times.SSC, 0.1% SDS,
twice at room temperature for 15 min and once at 55.degree. C. in
0.1.times.SSC, 0.1% SDS for 1 hr. Filters are exposed to X-ray film
at -70.degree. C. using an intensifying screen (Coronex
Hi-Plus).
[0327] Hybridized bands are quantified by analyzing the images
obtained by using a video densitometer (Applied Imaging
Corporation, Santa Clara, Calif.). Film densities are calibrated
using an optical-density wedge.
[0328] Treatment of PU5-1.8 murine macrophages with LPS (100 ng/mL)
for 3 h results in a significant increase (>7 fold) in the level
of TNF-alpha mRNA as determined by Northern blot analysis.
Treatment of cells with colchicine at 10 .mu.M concentration has no
effect on TNF-alpha mRNA expression. However, addition of
colchicine at 10 .mu.M to the LPS treated cultures resulted in
substantial reduction of TNF-alpha mRNA accumulation. The
inhibition levels were 68% for colchicine.
[0329] To establish a concentration-effect relationship,
macrophages are exposed to various concentrations of candidate
tubulin inhibitors in the presence of the stimulus LPS for 3 h. The
amounts of TNF-alpha mRNA declines with increasing concentrations
of the candidate tubulin inhibitors. Thus, the above assay can be
used to show that candidate tubulin inhibitors have the ability to
reduce the level of TNF-alpha produced by a cell.
Example 13
Cell Cycle Analysis with Flow Cytometry
[0330] The bromodeoxyuridine (BrdU) method for the analysis of the
cell cycle can be used for the study of cell kinetics.
Bromodeoxyuridine is an analogue of the DNA base thymidine and can
compete with that base for uptake during the synthesis of DNA. So
those cells that have been actively synthesizing DNA during the
time that BrdU is present can be positive for it. It can be
detected by a monoclonal antibody and by simultaneously staining
for DNA content with propidium iodide. The percentage of cells in
G0/G1, S and G2/M can be determined. By altering the time that BrdU
is present cell cycle times can also be assessed. The protocol used
in this analysis is as follows. Cells are treated with 10 .mu.M
BrdU for an appropriate time (30 mins). After harvesting and
washing cells, the cells are fixed in ice-cold 70% ethanol while
vortexing. Samples can stay in ethanol for up to 7 days. Sample is
spun at 2000 rpm, 5 min. Sample is washed twice in PBS. Cells are
treated with 2M hydrochloric acid for 20 min. at room temperature
with frequent mixing. Acid is spun off (2000 rpm, 5 min.) and
washed twice in PBS and once in PBS-T (PBS+0.5% Tween+0.05% w/v
BSA). A 50 .mu.l anti-BrdU antibody (Sera Lab) is added, and left
for 15 min. at room temperature. It is then washed twice in PBS-T.
A 50 .mu.l FITC-conjugated goat-anti-rabbit immunoglobulins (DAKO)
is added to it, and left for 15 min. at room temperature. It is
then washed once in PBS-T. A 100 .mu.l Ribonuclease, and 400 .mu.l
propidium iodide (Stock is 50 .mu.g/1 ml) is then added. Analyses
is done by flow cytometry (Fabbri F. et al. 2006, BMC Cell Biology,
7:6).
[0331] It is to be understood that the above descriptions are
intended to be illustrative and not restrictive. Many embodiments
will be apparent to those of skill in the art upon reading the
above descriptions. The scope of the invention should, therefore,
be determined not with reference to the above descriptions, but
should instead be determined with reference to the appended claims,
along with the full scope of equivalents to which such claims are
entitled. The disclosures of all articles and references, including
patent applications and publications, are incorporated herein by
reference for all purposes.
Example 14
Cell Cycle Analysis
[0332] HCT116 and Hela cells were obtained from ATCC. For DNA
content analysis, 2.times.10.sup.5 cells were washed twice with PBS
and fixed in 70% ethanol. Cells were treated with 100 units/mL
RNase A for 20 minutes at 37.degree. C., resuspended in cold PBS
containing Alexa Fluor.RTM. 405 fluorescent stain (Invitrogen)
according to the manufacturer's protocol. Cells were analyzed by
flow cytometry.
[0333] For DNA replication analysis, 2.times.10.sup.5 cells were
incubated with 50 .mu.mol/L bromodeoxyuridine (BrdUrd) for 30
minutes. Cells were fixed in 70% ethanol and BrdUrd incorporation
was determined by flow cytometric analysis using an
anti-BrdUrd-FITC antibody (Becton Dickinson, Franklin Lakes, N.J.)
according to the manufacturer's protocol. To assess the degree of
G2/M checkpoint, mitotic cells were detected by flow cytometry
using the mitosis-specific antibody GF-7. Fixed cells were
incubated for 30 minutes with GF7-phycoerythrin (PE) antibody (BD
Biosciences Pharmingen), washed with PBS and analyzed by flow
cytometry. FIG. 6 compares the results obtained with DIPE and
formula IIIB.
Example 15
Cell Cycle Analysis with Flow Cytometry
[0334] Programmed cell death (apoptosis) is an important
homeostatic mechanism in the immune and other systems. The
phenomenon of nuclear disintegration during apoptosis can be used
as a marker to detect cells undergoing this process. While a
variety of methods are available to detect apoptotic cells the
TUNEL assay allows the rapid phenotypic identification of
individual apoptotic cells using flow cytometry (FACS). Terminal
transferase dUTP nick end labeling (TUNEL) is a common method for
detecting DNA fragmentation that results from apoptosis. The assay
based on the presence of nicks in the DNA that can be identified by
terminal transferase, an enzyme that will catalyze the addition of
dUTPs that are secondarily labeled with a marker. It may also label
cells undergoing necrosis or cells that have suffered severe DNA
damage.
[0335] Hela cells were obtained from ATCC. For DNA content
analysis, 2.times.10.sup.5 cells were washed twice with PBS and
fixed in 70% ethanol. Cells were treated with 100 units/mL RNase A
for 20 minutes at 37.degree. C., resuspended in cold PBS containing
Alexa Fluor.RTM. 405 fluorescent stain (Invitrogen) according to
the manufacturer's protocol. Cells were analyzed by flow cytometry
to characterize the cell cycle. To perform the TUNEL assay, cells
were also fixed with 0.25% formaldehyde in the medium before the
ethanol treatment and then stained with the Apo-Direct.RTM. kit
(eBioscience, San Diego, Calif.). The results of this assay for
DIPE (control) and formula IIIB are shown in FIG. 7.
[0336] While preferred embodiments of the present invention have
been shown and described herein, it will be obvious to those
skilled in the art that such embodiments are provided by way of
example only. Numerous variations, changes, and substitutions will
now occur to those skilled in the art without departing from the
invention. It should be understood that various alternatives to the
embodiments of the invention described herein may be employed in
practicing the invention. It is intended that the following claims
define the scope of the invention and that methods and structures
within the scope of these claims and their equivalents be covered
thereby.
* * * * *