U.S. patent application number 15/275050 was filed with the patent office on 2017-08-17 for use of modulators of ccr5 in the treatment of cancer and cancer metastasis.
The applicant listed for this patent is Richard G. Pestell. Invention is credited to Richard G. Pestell.
Application Number | 20170231991 15/275050 |
Document ID | / |
Family ID | 49549081 |
Filed Date | 2017-08-17 |
United States Patent
Application |
20170231991 |
Kind Code |
A1 |
Pestell; Richard G. |
August 17, 2017 |
Use Of Modulators Of CCR5 In The Treatment Of Cancer And Cancer
Metastasis
Abstract
This disclosure is directed, in part, to a method of determining
whether a subject having cancer is at risk for developing
metastasis of the cancer. In one embodiment, the method comprises
(a) obtaining a biological sample from the subject having cancer;
(b) determining CCR5 expression level and/or expression level of at
least one of CCR5 ligands in the biological sample; and (c) if the
expression level of CCR5 and/or of at least one of CCR5 ligands
determined in step (b) is increased compared to CCR5 expression
level and/or expression level of at least one of CCR5 ligands in a
control sample, then the subject is identified as likely at risk
for developing metastasis of the cancer.
Inventors: |
Pestell; Richard G.;
(Philadelphia, PA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Pestell; Richard G. |
Philadelphia |
PA |
US |
|
|
Family ID: |
49549081 |
Appl. No.: |
15/275050 |
Filed: |
September 23, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13893791 |
May 14, 2013 |
9453836 |
|
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15275050 |
|
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61646593 |
May 14, 2012 |
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61646586 |
May 14, 2012 |
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Current U.S.
Class: |
514/210.18 |
Current CPC
Class: |
C12Q 2600/118 20130101;
A61P 35/04 20180101; A61K 31/46 20130101; A61K 31/4375 20130101;
C12Q 2600/158 20130101; A61P 35/00 20180101; A61K 31/506 20130101;
A61P 35/02 20180101; A61K 31/4184 20130101; G01N 2333/705 20130101;
C12Q 1/6886 20130101; G01N 2800/56 20130101; G01N 33/57415
20130101; G01N 2333/7158 20130101; G01N 33/5091 20130101; G01N
33/57484 20130101; G01N 33/57492 20130101 |
International
Class: |
A61K 31/506 20060101
A61K031/506; G01N 33/574 20060101 G01N033/574; A61K 31/4375
20060101 A61K031/4375; C12Q 1/68 20060101 C12Q001/68; A61K 31/46
20060101 A61K031/46; A61K 31/4184 20060101 A61K031/4184 |
Goverment Interests
STATEMENT REGARDING FEDERALLY SPONSORED R&D
[0002] This invention was supported in part by PASPA-UNAM
(M.A.V-V.), NIH grants R01CA070896, R01CA075503, R01CA132115,
R01CA107382, R01CA086072 (R.G.P.), R01CA120876 (M.P.L), the Kimmel
Cancer Center NIH Cancer Center Core grant P30CA056036 (R.G.P.),
generous grants from the Dr. Ralph and Marian C. Falk Medical
Research Trust and the Margaret Q. Landenberger Research
Foundation, and a grant from Pennsylvania Department of Health
(R.G.P.). Accordingly, the United States government has certain
rights in the invention.
Claims
1-14. (canceled)
15. A method of preventing metastasis of breast cancer in a subject
having breast cancer, wherein the subject is at risk of developing
metastasis of the breast cancer, the method comprising:
administering to the subject who is at risk of developing
metastasis of the breast cancer a therapeutically effective amount
of a CCR5 antagonist to prevent the metastasis of the breast
cancer.
16. The method of claim 15, wherein the CCR5 antagonist is selected
from the group consisting of
4,4-difluoro-N-[(1S)-3-[(1R,5S)-3-(3-methyl-5-propan-2-yl-1,2,4-triazol-4-
-yl)-8-azabicyclo[3.2.1]octan-8-yl]-1-phenylpropyl]cyclohexane-1-carboxami-
de or a pharmaceutically acceptable salt thereof and
(4,6-dimethylpyrimidin-5-yl)-[4-[(3
S)-4-[(1R)-2-methoxy-1-[4-(trifluoromethyl)pheny]ethyl]-3-methylpiperazi--
n-1-yl]-4-methylpiperidin-1-yl]methanone or a pharmaceutically
acceptable salt thereof.
17. The method of claim 15, wherein the breast cancer is basal
breast cancer.
18. The method of claim 15, wherein said CCR5 antagonist is
selected from the group consisting of:
4,4-difluoro-N-[(1S)-3-[(1R,5S)-3-(3-methyl-5-propan-2-yl-1,2,4-triazol-4-
-yl)-8-azabicyclo[3.2.1]octan-8-yl]-1-phenylpropyl]cyclohexane-1-carboxami-
de;
N-(1S)-3-3-(3-Isopropyl-5-methyl-4H-1,2,4-triazol-4-yl)-exo-8-azabicyc-
lo[3.2.1]oct-8-yl-1-phenylpropylcyclobutanecarboxamide;
N-(1S)-3-3-(3-Isopropyl-5-methyl-4H-1,2,4-triazol-4-yl)-exo-8-azabicyclo[-
3.2.1]oct-8-yl-1-phenylpropylcyclopentanecarboxamide;
N-(1S)-3-3-(3-Isopropyl-5-methyl-4H-1,2,4-triazol-4-yl)-exo-8-azabicyclo[-
3.2.1]oct-8-yl-1-phenylpropyl-4,4,4-trifluorobutanamide;
N-(1S)-3-3-(3-Isopropyl-5-methyl-4H-1,2,4-triazol-4-yl)-exo-8-azabicyclo[-
3.2.1]oct-8-yl-1-phenylpropyl-4,4-difluorocyclohexanecarboxamide;
N-(1S)-3-3-(3-Isopropyl-5-methyl-4H-1,2,4-triazol-4-yl)-exo-8-azabicyclo[-
3.2.1]oct-8-yl-1-(3-fluorophenyl)propyl-4,4-difluorocyclohexanecarboxamide-
; and pharmaceutically acceptable salts or solvates thereof.
19. The method of claim 15, wherein said CCR5 antagonist is
selected from the group consisting of:
N-{3-[3-exo-(2-Methyl-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-8-yl]--
1-phenylpropyl}cyclobutanecarboxamide;
N-{(1S)-3-[3-exo-(2-Methyl-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-8-
-yl]-1-phenylpropyl}cyclobutanecarboxamide;
N-{(1S)-3-[3-endo-(2-Methyl-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct--
8-yl]-1-phenylpropyl}cyclobutanecarboxamide;
N-{(1S)-3-[3-exo-(2-Methyl-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-8-
-yl]-1-phenylpropyl}tetrahydro-2H-pyran-4-carboxamide;
1-Acetyl-N-{(1S)-3-[3-exo-(2-methyl-1H-benzimidazol-1-yl)-8-azabicyclo[3.-
2.1]oct-8-yl]-1-phenylpropyl}3-azetidine carboxamide;
1-Hydroxy-N-{(1S)-3-[3-exo-(2-methyl-1H-benzimidazol-1-yl)-8-azabicyclo[3-
.2.1]oct-8-yl]-1-phenylpropyl}cyclopentanecarboxamide;
2-Methyl-N-{(1S)-3-[3-exo-(2-methyl-1H-benzimidazol-1-yl)-8-azabicyclo[3.-
2.1]oct-8-yl]-1-phenylpropyl}cyclopropanecarboxamide;
2-Cyclopropyl-N-{1S)-3-[3-exo-(2-methyl-1H-benzimidazol-1-yl)-8-azabicycl-
o [3.2.1]oct-8-yl]-1-phenylpropyl}acetamide;
N-{(1S)-3-[3-exo-(2-methyl-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-8-
-yl]-1-phenylpropyl}tetrahydro-3-furancarboxamide;
3,3,3-Trifluoro-N-{(1S)-3-[3-exo-(2-methyl-1H-benzimidazol-1-yl)-8-azabic-
yclo[3.2.1]oct-8-yl]-1-phenylpropyl}propanamide;
N-{(1S)-3-[3-exo-(2-Methyl-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-8-
-yl]-1-phenylpropyl}tetrahydro-2-furancarboxamide;
1-(Acetylamino)-N-{(1S)-3-[3-exo-(2-methyl-1H-benzimidazol-1-yl)-8-azabic-
yclo[3.2.1]oct-8-yl]-1-phenylpropyl}cyclopentanecarboxamide;
N-{(1S)-3-[3-exo-(2-Methyl-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-8-
-yl]-1-phenylpropyl}acetamide;
1-Methoxy-N-{(1S)-3-[3-exo-(2-methyl-1H-benzimidazol-1-yl)-8-azabicyclo[3-
.2.1]oct-8-yl]-1-phenylpropyl}cyclopentanecarboxamide;
1-Amino-N-{(1S)-3-[3-exo-(2-methyl-1H-benzimidazol-1-yl)-8-azabicyclo[3.2-
.1]oct-8-yl]-1-phenylpropyl}cyclopentanecarboxamide;
1-Methyl-N-{(1S)-3-[3-exo-(2-methyl-1H-benzimidazol-1-yl)-8-azabicyclo[3.-
2.1]oct-8-yl]-1-phenylpropyl}-2-oxo-4-pyrrolidinecarboxamide;
1-Acetyl-N-{(1S)-3-[3-endo-(2-methyl-1H-benzimidazol-1-yl)-8-azabicyclo[3-
.2.1]oct-8-yl]-1-phenylpropyl)3-azetidinecarboxamide;
N-{(1S)-3-[3-endo-(2-Methyl-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct--
8-yl]-1-phenylpropyl}acetamide;
N-{(1S)-3-[6-(2-Methyl-1H-benzimidazol-1-yl)-3-azabicyclo[3.1.0]hex-3-yl]-
-1-phenylpropyl}cyclobutanecarboxamide;
2-Cyclopropyl-N-{(1S)-3-[3-exo-(3-{4-[(methylsulfonyl)amino]benzyl}-1,2,4-
-oxadiazol-5-yl)-8-azabicyclo[3.2.1]oct-8-yl]-1-phenylpropyl}acetamide;
N-{(1S)-3-[7-exo-(2-Methyl-1H-benzimidazol-1-yl)-3-oxa-9-azabicyclo[3.3.1-
]non-9-yl]-1-phenylpropyl}cyclobutanecarboxamide;
2-Cyclopropyl-N-{(1S)-3-[7-exo-(2-methyl-1H-benzimidazol-1-yl)-3-oxa-9-az-
abicyclo[3.3.1]non-9-yl]-1-phenylpropyl}acetamide;
3,3,3-Trifluoro-N-{(1S)-3-[7-exo-(2-methyl-1H-benzimidazol-1-yl)-3-oxa-9--
azabicyclo[3.3.1]non-9-yl]-1-phenylpropyl}propanamide;
N-{(1S)-3-[7-endo-(2-Methyl-1H-benzimidazol-1-yl)-3-oxa-9-azabicyclo[3.3.-
1]non-9-yl]-1-phenylpropyl}cyclobutanecarboxamide;
2-Cyclopropyl-N-{(1S)-3-[7-endo-(2-methyl-1H-benzimidazol-1-yl)-3-oxa-9-a-
zabicyclo[3.3.1]non-9-yl]-1-phenylpropyl}acetamide;
N-{(1S)-3-[7-exo-(2-Methyl-1H-benzimidazol-1-yl)-3-thia-9-azabicyclo[3.3.-
1]non-9-yl]-1-phenylpropyl}cyclobutanecarboxamide;
2-Cyclopropyl-N-[(1S)-3-(3-endo-{[2-(4-fluorophenyl)acetyl]amino}-8-azabi-
cyclo[3.2.1]oct-8-yl)-1-phenylpropyl]acetamide;
N-[(1S)-3-(3-{[3-endo-(4-Fluorophenyl)ppropanoyl]amino}-8-azabicyclo[3.2.-
1]oct-8-yl)-1-phenylpropyl]cyclobutanecarboxamide;
N-[(1S)-3-(3-{[3-exo-(4-Fluorophenyl)prpropanoyl]amino}-8-azabicyclo[3.2.-
1]oct-8-yl)-1-phenylpropyl]cyclobutanecarboxamide;
2-Cyclopropyl-N-[(1S)-3-(3-exo-{[2-(4-fluorophenyl)acetyl]amino}-8-azabic-
yclo[3.2.1]oct-8-yl)-1-phenylpropyl]acetamide;
N-{(1S)-3-[3-exo-(2-Methyl-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-8-
-yl]-1-phenylpropyl)}1-propionyl-3-azetidinecarboxamide;
N-{(1S)-3-[3-endo-(2-Methyl-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct--
8-yl]-1-phenylpropyl}tetrahydro-3-furancarboxamide;
N-{(1S)-3-[3-endo-(2-Methyl-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct--
8-yl]-1-phenylpropyl}tetrahydro-2H-pyran4-carboxamide;
N-{(1S)-3-[3-endo-(2-Methyl-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct--
8-yl]-1-phenylpropyl}tetrahydro-2-furancarboxamide;
1-Acetyl-N-{(1S)-3-[3-endo-(1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct--
8-yl]-1-phenylpropyl}-3-azetidinecarboxamide;
N-{(1S)-3-[3-endo-(1H-Benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-8-yl]-1-p-
henylpropyl}-1-propionyl-3-azetidinecarboxamide; Methyl
3-[({(1S)-3-[3-exo-(2-methyl-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-
-8-yl]-1-phenylpropyl}amino)carbonyl]-1-azetidinecarboxylate;
N-{(1S)-3-[3-[3-endo(2-Methyl-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oc-
t-8-yl]-1-phenylpropyl}-1-propionyl-3-azetidinecarboxamide
1-Acetyl-N-{(1S)-3-endo-(2-methyl-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.-
1]oct-8-yl]-1-phenylpropyl}-2-azetidinecarboxamide;
2-[Acetyl(methyl)amino]-N-{(1S)-3-[3-endo-(2-methyl-1H-benzimidazol-1-yl)-
-8-azabicyclo[3.2.1]oct-8-yl]-1-phenylpropyl}acetamide;
3-[Acetyl(methyl)amino]-N-{(1S)-3-[3-endo-(2-methyl-1H-benzimidazol-1-yl)-
-8-azabicyclo[3.2.1]oct-8-yl]-1-phenylpropyl}propanamide;
2-Methoxy-N-{(1S)-3-[3-endo-(2-methyl-1H-benzimidazol-1-yl)-8-azabicyclo[-
3.2.1]oct-8-yl]-1-phenylpropyl}acetamide;
3-Methoxy-N-{(1S)-3-[3-endo-(2-methyl-1H-benzimidazol-1-yl)-8-azabicyclo[-
3.2.1]oct-8-yl]-1-phenylpropyl}propanamide;
1-Acetyl-N-{(1S)-3-[3-[3-endo-(2-methyl-1H-benzimidazol-1-yl)-8-azabicycl-
o[3.2.1]oct-8-yl]-1]-1-phenylpropyl}-3-pyrrolidinecarboxamide;
1-Methyl-N-{(1S)-3-[3-endo-(2-methyl-1H-benzimidazol-1-yl)-8-azabicyclo[3-
.2.1]oct-8-yl]-1-phenylpropyl}-2-oxo-4-pyrrolidinecarboxamide;
1-Acetyl-N-{(1S)-3-[3-exo-(2-ethyl-1H-benzimidazol-1-yl)-8-azabicyclo[3.2-
.1]oct-8-yl]-1-phenylpropyl}-3-azetidinecarboxamide;
N-{(1S)-3-[3-exo-(2-Ethyl-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-8--
yl]-1-phenylpropyl}-1-propionyl-3-azetidinecarboxamide;
1-Acetyl-N-((1S)-1-phenyl-3-{3-exo[2-(trifluoromethyl)-1H-benzimidazol-1--
yl]-8-azabicyclo[3.2.1]oct-8-yl}propyl)-3-azetidinecarboxamide;
N-((1S)-1-Phenyl-3-{3-exo-[2-(trifluoromethyl)-1H-benzimidazol-1-yl]-8-az-
abicyclo[3.2.1]oct-8-yl}propyl)-1-propionyl-3-azetidinecarboxamide;
N-((1S)-1-Phenyl-3-{3-exo[2-(trifluoromethyl)-1H-benzimidazol-1-yl]-8-aza-
bicyclo[3.2.1]oct-8-yl}propyl)acetamide;
2-[Acetyl(methyl)amino]-N-((1S)-1-phenyl-3-{3-exo-[2-(trifluoromethyl)-1H-
-benzimidazol-1-yl]-8-azabicyclo[3.2.1]oct-8-yl}propyl)acetamide;
1-Acetyl-N-{(1S)-3-[3-exo-(1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-8-
-yl]-1-phenylpropyl}-3-azetidinecarboxamide;
N(1S)-3-[3-exo-(1H-Benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-8-yl]-1-phen-
ylpropyl}-1-propionyl-3-azetidinecarboxamide;
1-acetyl-N(1S)-3-[3-exo-(5-fluoro-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.-
1]oct-8-yl]-1-phenylpropyl)3-azetidinecarboxamide;
N-{(1S)-3-[3-exo-(5-Fluoro-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-8-
-yl]-1-phenylpropyl}-1-propionyl-3-azetidinecarboxamide;
1-Acetyl-N-{(1S)-3-[3-exo-(5-fluoro-2-methyl-1H-benzimidazol-1-yl)-8-azab-
icyclo[3.2.1]oct-8-yl]-1-phenylpropyl}3-azetidinecarboxamide;
N-{(1S)-3-[3-exo-(5-Fluoro-2-methyl-1H-benzimidazol-1-yl)-8-azabicyclo[3.-
2.1]oct-8-yl]-1-phenylpropyl}-1-propionyl-3-azetidinecarboxamide;
N-{(1S)-3-[3-exo-(2-methyl-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-8-
-yl]-1-phenylpropyl}-3-azetidinecarboxamide;
1-methyl-N-{(1S)-3-[3-exo-(2-methyl-1H-benzimidazol-1-yl)-8-azabicyclo[3.-
2.1]oct-8-yl]-1-phenylpropyl}-3-azetidinecarboxamide;
(2S)-1-acetyl-N-{(1S)-3-[3-exo-(2-methyl-1H-benzimidazol-1-yl)-8-azabicyc-
lo[3.2.1]oct-8-yl]-1-phenylpropyl}-2-azetidinecarboxamide;
(2R)-1-acetyl-N-{(1S)-3-[3-exo-(2-methyl-1H-benzimidazol-1-yl)-8-azabicyc-
lo[3.2.1]oct-8-yl]-1-phenylpropyl}-2-azetidinecarboxamide;
2-[acetyl(methyl)amino]-N-{(1S)-3-[3-exo-(2-methyl-1H-benzimidazol-1-yl)--
8-azabicyclo[3.2.1]oct-8-yl]-1-phenylpropyl}acetamide;
3-[acetyl(methyl)amino]-N-{(1S)-3-[3-exo-(2-methyl-1H-benzimidazol-1-yl)--
8-azabicyclo[3.2.1]oct-8-yl]-1-phenylpropyl}propanamide;
1-acetyl-N-{(1S)-3-[3-exo-(2-methyl-1H-benzimidazol-1-yl)-8-azabicyclo[3.-
2.1]oct-8-yl]-1-phenylpropyl}-3-pyrrolidinecarboxamide;
N-{(1S)-3-[3-exo-(2-methyl-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-8-
-yl]-1-phenylpropyl}-1-(trifluoromethyl)cyclopropanecarboxamide;
2-methoxy-N-{(1S)-3-[3-exo-(2-methyl-1H-benzimidazol-1-yl)-8-azabicyclo[3-
.2.1]oct-8-yl]-1-phenylpropyl}acetamide;
3-methoxy-N-{(1S)-3-[3-exo-(2-methyl-1H-benzimidazol-1-yl)-8-azabicyclo[3-
.2.1]oct-8-yl]-1-phenylpropyl}propanamide;
1-Acetyl-N-{(1S)-3[3-exo-(4-fluoro-2-methyl-1H-benzimidazol-1-yl)-8-azabi-
cyclo[3.2.1]oct-8-yl]-1-phenylpropyl}-3-azetidinecarboxamide;
N-{(1S)-3-[3-exo-(4-Fluoro-2-methyl-1H-benzimidazol-1-yl)-8-azabicyclo[3.-
2.1]oct-8-yl]1-1-phenylpropyl}-3-azetidinecarboxamide;
1-Methyl-N-{(1S)-3-[3-exo-(4-fluoro-2-methyl-1H-benzimidazol-1-yl)-8-azab-
icyclo[3.2.1]oct-8-yl]-1-phenylpropyl}-3-azetidinecarboxamide;
N-{(1S)-3-[3-exo-(4-Fluoro-2-methyl-1H-benzimidazol-1-yl)-8-azabicyclo[3.-
2.1]oct-8-yl]-1-phenylpropyl}-1-propionyl-3-azetidinecarboxamide;
2-Methoxy-N-{(1S)-3[3-exo-(4-Fluoro-2-methyl-1H-benzimidazol-1-yl)-8-azab-
icyclo[3.2.1]oct-8-yl]-1-phenylpropyl}acetamide;
N-{(1S)-3-[3-exo-(4-Fluoro-2-Methyl-1H-benzimidazol-1-yl)-8-azabicyclo[3.-
2.1]oct-8-yl]-1-phenylpropyl}acetamide;
3-Methoxy-N-{(1S)-3-[3-exo-(4-fluoro-2-methyl-1H-benzimidazol-1-yl)-8-aza-
bicyclo[3.2.1]oct-8-yl]-1-phenylpropyl}propanamide;
2-[Acetyl(methyl)amino]-N-{(1S)-3-[3-exo-(4-fluoro-2-methyl-1H-benzimidaz-
ol-1-yl)-8-azabicyclo[3.2.1]oct-8-yl]-1-phenylpropyl}acetamide;
3-[Acetyl(methyl)amino]-N-{(1S)-3-[3-exo-(4-fluoro-2-methyl-1H-benzimidaz-
ol-1-yl)-8-azabicyclo[3.2.1]oct-8-yl]-1-phenylpropyl}propanamide;
N-{(1S)-3-[3-exo-(4-fluoro-2-methyl-1H-benzimidazol-1-yl)-8-azabicyclo[3.-
2.1]oct-8-yl]-1-phenylpropyl}-3-methyl-3-oxetanecarboxamide;
3-Ethyl-N-{(1S)-3-[3-exo-(4-fluoro-2-methyl-1H-benzimidazol-1-yl)-8-azabi-
cyclo[3.2.1]oct-8-yl]-1-phenylpropyl}-3-oxetanecarboxamide;
N-{(1S)-3-[3-exo-(4-Fluoro-2-methyl-1H-benzimidazol-1-yl)-8-azabicyclo[3.-
2.1]oct-8-yl]-1-phenylpropyl}-3-oxetanecarboxamide;
3-Ethyl-N-{(1S)-3-[3-exo-(4-fluoro-1H-benzimidazol-1-yl)-8-azabicyclo[3.2-
.1]oct-8-yl]-1-phenylpropyl}-3-oxetanecarboxamide;
N-{(1S)-3-[3-exo-(4-Fluoro-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-8-
-yl]-1-phenylpropyl}-3-methyl-3-oxetanecarboxamide;
N-{(1S)-3-[3-exo-(4-Fluoro-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-8-
-yl]-1-phenylpropyl}-3-oxetanecarboxamide;
N-{(1S)-3[3-exo-(4-Fluoro-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-8--
yl]-1-phenylpropyl}-3-azetidinecarboxamide;
N-{(1S)-3-[3-exo-(4-Fluoro-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-8-
-yl]-1-phenylpropyl}-1-methyl-3-azetidinecarboxamide;
1-Acetyl-N-{(1S)-3-[3-exo-(4-fluoro-1H-benzimidazol-1-yl)-8-azabicyclo[3.-
2.1]oct-8-yl]-1-phenylpropyl}-3-azetidinecarboxamide;
N-{(1S)-3-[3-exo-(4-Fluoro-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-8-
-yl]-1-phenylpropyl}-1-propionyl-3-azetidinecarboxamide;
N-{(1S)-3-[3-exo-(4-Fluoro-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-8-
-yl]-1-phenylpropyl}-2-methoxyacetamide;
N-{(1S)-3-[3-exo-(4-Fluoro-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-8-
-yl]-1-phenylpropyl}acetamide;
N{-1S)-3-[3-exo-(4-fluoro-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-8--
yl]-1-phenylpropyl}-3-methoxypropanamide;
2-[Acetyl(methyl)amino]-N-{(1S)-3-[3-exo-(4-fluoro-1H-benzimidazol-1-yl)--
8-azabicyclo[3.2.1]oct-8-yl]-1-phenylpropyl}acetamide;
3-[Acetyl(methyl)amino]-N-{(1S)-3-[3-exo-(4-fluoro-1H-benzimidazol-1-yl)--
8-azabicyclo[3.2.1]oct-8-yl]-1-phenylpropyl}propanamide; and
pharmaceutically acceptable salts thereof.
20. The method of claim 15, wherein the method prevents the
metastasis of the breast cancer to one or more organs selected from
the group consisting of liver, brain, bladder, lung, adrenal gland,
kidney, bone and combinations thereof
21. The method of claim 15, wherein the CCR5 antagonist is
administered concomitantly or concurrently with an additional
therapeutic.
22. The method of claim 15, further comprising identifying the
subject with breast cancer as being at risk of developing
metastasis of the breast cancer prior to the administration of the
CCR5 antagonist, the identifying comprising: (a) obtaining a
biological sample from the subject having breast cancer; (b)
measuring CCR5 level of expression and/or level of expression of at
least one of CCR5 ligands in the biological sample, wherein if the
measured CCR5 level of expression and/or the level of expression of
at least one of CCR5 ligands determined in step (b) is increased
compared to CCR5 level of expression and/or level of expression of
at least one of CCR5 ligands in a normal control breast tissue
sample, then the subject is identified at risk for developing
metastasis of the breast cancer.
23. The method of claim 22, wherein measuring CCR5 level of
expression and/or level of expression of at least one of CCR5
ligands in the biological sample comprises performing a an
RNA-based assay selected from the group consisting reverse
transcription polymerase chain reaction or a microarray assay.
24. The method of claim 22, wherein measuring CCR5 level of
expression and/or level of expression of at least one of CCR5
ligands in the biological sample comprises performing an
immunoassay selected from the group consisting of
immunohistochemical staining or fluorescence activated cell
sorting.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 13/893,791, filed May 14, 2013, which claims the benefit of
U.S. Provisional Patent Application Nos. 61/646,586, filed May 14,
2012, and entitled "Use Of Modulators Of CCR5 In The Treatment Of
Cancer And Cancer Metastasis"; and 61/646,593, filed May 14, 2012,
and entitled "Use Of Modulators Of CCR5 In The Treatment Of Cancer
And Cancer Metastasis", each of which are incorporated by reference
herein in their entirety.
FIELD OF THE INVENTION
[0003] This disclosure is directed, in part, to a method of
determining whether a subject having cancer is at risk for
developing metastasis of the cancer and a method of blocking cancer
methastasis.
BACKGROUND OF THE INVENTION
[0004] Breast cancer causes the death of 40,000 women in the USA
and 410,000 women in the world annually..sup.1 Despite advances in
the treatment of the disease, 20% to 30% of patients with early
breast cancers will experience relapse with distant metastatic
disease..sup.2 In those patients, metastasis is the main cause of
death. Patients with basal tumors have increased risk of metastasis
and lower survival rate..sup.3' .sup.4 Kennecke et al. studied
3,726 breast cancer patients and reported that the basal tumors
have higher frequencies of metastases and reduced time from
identification of metastases to death compared to that of patients
with luminal A or B tumors..sup.4 The absence of AR, ER, and HER-2
commonly found in basal breast tumors.sup.5 means that they are
unlikely to respond to hormone therapies or HER-2 targeted
therapies. Currently, chemotherapy, radiation, and surgery are the
only choices for patients with basal breast cancers, but all
demonstrate poor outcomes..sup.6 The need for a specific targeted
therapy for basal breast cancer remains urgent.
BRIEF SUMMARY OF THE INVENTION
[0005] Certain aspects of embodiments disclosed herein by way of
example are summarized below. It should be understood that these
aspects are presented merely to provide the reader with a brief
summary of certain forms an invention disclosed and/or claimed
herein might take and that these aspects are not intended to limit
the scope of any invention disclosed and/or claimed herein. Indeed,
any invention disclosed and/or claimed herein may encompass a
variety of aspects that may not be set forth below.
[0006] The present disclosure generally relates to various methods,
including methods of determining whether a subject has cancer or is
at risk for developing cancer and/or is at risk for developing
cancer metastasis. In some embodiments, the methods of the present
invention include methods of treating, preventing, or managing a
neoplasm or a cancer metastasis in a patient. In some embodiments,
the methods of the present invention include in vivo methods for
down regulating CCR5 expression in a set of one or more prostate
cancer cell lines derived from transduction of murine epithelial or
prostate epithelial cells and transformed by at least one oncogene
selected from the group consisting of NeuT, Ha-Ras, and c-Src. In
some exemplary embodiments drugs that target the HIV receptor CCRS,
which the virus uses to enter and infect host cells, are used to
prevent migration and spread of cancer cells from their primary
tissue to secondary sites in the body of the patient. In one
embodiment, a drug that targets the HIV receptor CCR5 may be used
as an adjuvant therapy, or adjuvant care, wherein the drug is
administered to the patient in addition to a primary, main or
initial treatment for cancer. In some embodiments, such adjuvant
therapy may be administered concomitantly or concurrently as other
therapies for cancer. In one embodiment, such adjuvant therapy may
be given concurrently as other adjuvant therapies or following
other adjuvant therapies.
[0007] In some embodiments, when CCR5 receptor antagonists are used
as adjuvant therapy they improve prognosis in cancer patients. In
some embodiments, where a plurality of anticancer drugs are used in
combination for cancer therapy, a CCR5 receptor antagonist may be
included as an adjuvant therapy in order to improve the therapeutic
effect by blocking metastasis of the cancer being treated, thereby
contributing to improving clinical outcomes of the cancer
therapy.
BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS
[0008] The foregoing summary, as well as other features, aspects,
and advantages of the present invention and the following detailed
description of embodiments of the invention, will be better
understood when read in conjunction with the claims and the
appended drawings of an exemplary embodiments, wherein:
[0009] FIG. 1A illustrates a heatmap of the expression of CCL5 and
its receptor CCR5 in samples from patients with breast cancer
divided by molecular subtype (luminal A, luminal B, basal,
normal-like, and Her-2 ) based on their gene expression
pattern;
[0010] FIG. 1B includes plots 1B1 through 1B5, which illustrates
scatter plots and correlation analysis (Student t test) of the
expression of CCL5 and CCR5 among the breast cancer molecular
subtypes of luminal A, luminal B, basal, normal-like, and
Her-2;
[0011] FIG. 1C illustrates quantification of the proportions of the
breast cancer samples overexpressing CCL5 and CCR5, fraction of the
bars representing upper right quadrants of the scatter plots shown
in Figure B;
[0012] FIG. 1D illustrates metastasis-free Kaplan-Meier plots and
log-rank analysis for the different molecular subtypes of breast
cancer in the analyzed database, which is described in Materials
and Methods section of this of this application;
[0013] FIG. 2A illustrates flow cytometric histograms of the CCR5
expression in MDA-MB-231 breast cancer cells identified a
subpopulation of CCR5.sup.+ cells;
[0014] FIG. 2B includes plot 2B1 and illustrates induction of
calcium signaling in cells loaded with Fluo-4-AM before the
sequential addition of CCL5 (60 .mu.g/mL) and FBS (5%);
[0015] FIG. 2C illustrates 3D invasion into collagen gels by breast
cancer cell lines, using CCL5 (15 .mu.g/mL) as chemoattractant;
[0016] FIG. 2D illustrates mean distances of invasion.+-.SEM from 3
independent experiments;
[0017] FIG. 2E 3D invasion assays for MCF-10A cells and
MCF-10A-NeuT, -Ras, and -Src derivatives showing that CCL5 -induced
invasion is activated by oncogenic transformation;
[0018] FIG. 2F illustrates quantification (F, mean.+-.SEM, n=3) 3D
invasion assays for MCF-10A cells and MCF-10A-NeuT, -Ras, and -Src
derivatives showing that CCL5 -induced invasion is activated by
oncogenic transformation shown in FIG. 2E;
[0019] FIG. 2G illustrates CCR5+ and CCR5- subpopulations from
SUM-159 cell line that were isolated by FACS and their respected
invasion into collagen gels evaluated using fetal bovine serum
(FBS) as chemoattractant;
[0020] FIG. 2H illustrates quantification of the invasion of CCR5+
and CCR5- subpopulations into collagen as mean.+-.SEM of the two
independent experiments shown in FIG. 2G;
[0021] FIG. 3A includes image 3A1, image 3A2, graph 3A3 and graph
3A4 and illustrates intensity versus time analysis of Fluo-4
AM-loaded MDA-MB-231 cells treated with the CCR5 antagonists
maraviroc or vicriviroc (100 nmol/L) for 30 minutes before the
addition of CCL5 (60 m/mL);
[0022] FIG. 3B illustrates a comparison of the fraction of cells
with increased fluorescence intensity upon addition of CCL5;
[0023] FIG. 3C includes image 3C1, image 3C2, graph 3C3 and graph
3C4 and shows CCL5-induced calcium signaling blocked by CCR5
antagonists in Hs578T cells;
[0024] FIG. 3D illustrates quantification (mean.+-.SEM) of 3 to 4
independent experiments shown in FIGS. 3A-3C;
[0025] FIG. 4A illustrates 3D reconstruction of FBS-induced
invasion into collagen gels by Hs578T breast cancer cells in
presence of CCR5 antagonists (100 nmol/L);
[0026] FIG. 4B illustrates quantifications (mean.+-.SEM, n=3) and
analysis (Bonferroni t test) of the FBS-induced invasion into
collagen gels by Hs578T breast cancer cells shown in FIG. 4A;
[0027] FIG. 4C illustrates 3D reconstruction of FBS-induced
invasion into collagen gels by SUM-159 breast cancer cells in
presence of CCR5 antagonists (100 nmol/L);
[0028] FIG. 4D illustrates quantifications (mean.+-.SEM, n=3) and
analysis (Bonferroni t test) of the FBS-induced invasion into
collagen gels by SUM-159 breast cancer cells shown in FIG. 4A;
[0029] FIG. 5A shows exemplary in vivo bioluminescent images (BLIs)
of vehicle- or maraviroc-treated (8 mg/kg every 12 hours) of
nonobese diabetic (NOD)/severe combined immunodeficiency (SCID)
mice (hereinafter abbreviated as NOD/SCID mice);
[0030] FIG. 5B shows quantification (mean.+-.SEM, n=6) of in vivo
bioluminescent images (BLIs) in the control (vehicle-treated
NOD/SCID mice, red line/squares) and treated groups
(maraviroc-treated NOD/SCID mice, blue line/triangles);
[0031] FIG. 5C includes images 5C1 and 5C2 and shows the presence
of pulmonary tumors and the differences between treatments
corroborated by ex vivo imaging (left) and India ink staining
(right);
[0032] FIG. 5D showing the fraction of mice with metastatic tumors
being significantly larger in the control group (P<0.0001,
Fisher exact test);
[0033] FIG. 5E showing histological analysis (hematoxylin and eosin
staining, .times.100) of the area covered by metastatic tumors in
lung slides;
[0034] FIG. 5F shows quantification of the area covered by
metastatic tumors in lung slides shown in FIG. 5E;
[0035] FIG. 6A, effect of CCR5 antagonist on breast cancer cell
viability. MDA-MB-231 cells were exposed to increasing
concentrations of maraviroc (inverted triangles) or vicriviroc
(squares) for 48 hours and the cell viability was evaluated by MTT
assay.
[0036] FIG. 6B illustrates flow cytometric analysis showing CCR5
expression in MDA-MB-231 cells stably transfected with
pcDNA3.1.sup.+/Zeo.sup.+(MDA.Vector) or human CCR5 cloned into
pCDNA3.sup.+Zeo.sup.+ (MDA.CCR5).
[0037] FIG. 6C illustrates a comparison of in vitro proliferation
rates of MDA-MB-231 in MDA.Vector and MDA.CCRS;
[0038] FIG. 6D illustrates evaluation of the in vivo effect of
maraviroc on growth of established metastasis in mice, wherein
treatment of mice was initiated 10 days after injection of
MDA.pFULG cells as illustrated, and in vivo bioluminescence imaging
(BLI) of the mice for evaluating the efficacy of the treatment was
carried out in days: 0, 10, 17, 24, 31 and 38;
[0039] FIG. 6E illustrates quantification (mean.+-.SEM, n=5) of in
vivo BLI in the control (red/squares) and treated groups
(blue/triangles) showed no differences in the growth rate;
[0040] FIG. 6F illustrates an schema of the experimental design
used to evaluate CCR5 role in lung colonization by MDA-MB-231
cells;
[0041] FIG. 6G illustrates exemplary confocal images of eGFP.sup.+
cells in lungs of mice 24 hours after injection of the mice with
MDA.pFULG cells, wherein cells expressing eGFP were counted in 3
random fields of 2 different histologic sections (separated 700
.mu.m from each other) per mouse (n=5 mice per group);
[0042] FIG. 6H illustrates quantification (H) of the number of
eGFP.sup.+ cells in lungs of mice 24 hours after injection of the
mice with MDA.pFULG cells, wherein cells expressing eGFP were
counted in 3 random fields of 2 different histologic sections
(separated 700 .mu.m from each other) per mouse (n=5 mice per
group);
[0043] FIG. 7A illustrates GSEA analysis using KEGG and GO of tumor
samples to determine the gene expression signaling pathway
associated with enrichment of CCR5 and CCL5, wherein genes in the
examined breast cancer dataset are ranked by a signal-to-noise
metric representing their differential expression in highest 2.5%
CCL5/CCR5 -expressing samples versus lowest (2.5%) CCL5/CCR5
expressing samples (N=54), depicted with a color gradient, where
red indicates positive correlation with CCL5/CCR5 expression and
blue represents negative correlation with CCL5/CCR5 expression;
[0044] FIG. 7B includes scatter plots 7B1 through 7B5 and
illustrates scatter plots and correlation analysis using students'
t-tests for the expression of CCR1 and CCR5 among the breast cancer
molecular subtypes from a set of 2250 human breast cancer data
shown in FIG. 1A;
[0045] FIG. 7C includes scatter plots 7C1 through 7C5 and shows
scatter plots and correlation analysis for the expression of CCR3
and CCL5 among the breast cancer molecular subtypes from a set of
2250 human breast cancer data shown in FIG. 1A;
[0046] FIG. 7D includes Kaplan Meier curves 7D1 and 7D2 which are
Kaplan Meier curves for patients with breast cancer samples
enriched for the highest level of CCR5 (up of 50%) versus the lower
level CCR5 expression (lower 50%);
[0047] FIG. 8A illustrates heat map of CCL5 expression and its
receptors CCR5, CCR1 and CCR3 in healthy population.
[0048] FIG. 8B illustrates heat map representing cross correlation
of CCL5 expression and its receptors CCR5, CCR1 and CCR3 amongst
healthy population;
[0049] FIG. 8C illustrates heat map representing cross correlation
of CCL5 expression and its receptors CCR5, CCR1 and CCR3 amongst
breast cancer patients derived from a collection of 2550 breast
cancers;
[0050] FIG. 9A illustrates flow cytometry plot of the CCR5
expression in HS578T cells stably transfected with allophycocyanin
(APC)-labeled antibody to CCR5;
[0051] FIG. 9B includes plot 9B1 and illustrates induction of
calcium signaling in HS578T cells loaded with Fluo-4-AM before the
sequential addition of CCL5 (60 .mu.g/mL) and fetal bovine serum
(FBS) (5%), wherein RFI represents relative fluorescence
intensities. The red traces are of cells that did not respond to
the addition of CCL5 ligand--but do respond to FBS (fetal bovine
serum), the green line is the trace for the cells that do respond
to the additionaof CCL5, and also respond to FBS. This data tells
us that the MDA-MB-231 cells are a population of cells in which
some cells have the receptor and respond to CCL5- and some cells
that do not have the CCR5 receptor and do not respond to its ligand
CCL5;
[0052] FIG. 9C illustrates flow cytometry plot of the CCR5
expression in SUM159 cells stably transfected with allophycocyanin
(APC)-labeled antibody to CCR5.
[0053] FIG. 9D includes plot 9D1 and illustrates induction of
calcium signaling in SUM159 cells loaded with Fluo-4-AM before the
sequential addition of CCL5 (60 .mu.g/mL) and fetal bovine serum
(FBS) (5%), wherein RFI represents relative fluorescence
intensities. The red traces are of cells that did not respond to
the addition of CCL5 ligand--but do respond to FBS (fetal bovine
serum), the green line is the trace for the cells that do respond
to the additionaof CCL5 , and also respond to FBS. This data tells
us that the SUM-159 cells are a population of cells in which some
cells have the receptor and respond to CCL5- and some cells that do
not have the CCR5 receptor and do not respond to its ligand
CCL5;
[0054] FIG. 10A includes plots 10A1 and 10A2 and illustrates
fluorescence-activated cell sorting (FACS) analysis for the
abundance of CCR5 receptor in either the SUM159-vector control
cells or SUM159 cells stably overexpressing the CCR5 receptor.
APC-labeled antibody to CCR5 was used to follow the CCR5 positive
cells;
[0055] FIG. 10B illustrates schematic representation of the
timeline for induction of calcium signaling in which CCL5 is added
to the SUM159-vector control cells or the SUM159 cells stably
overexpressing the CCR5 receptor at 60 seconds and FBS is added at
320 seconds;
[0056] FIG. 10C illustrates induction of calcium signaling (the
Ca.sup.-2 response to CCL5 versus FBS) in the SUM159-vector control
cells loaded with Fluo-4-AM before and upon the addition of CCL5 or
FBS;
[0057] FIG. 10D illustrates the average fluorescence for the
population of SUM159-vector control cells;
[0058] FIG. 10E illustrates induction of calcium signaling (the
Ca.sup.-2 response to CCL5 versus FBS) in the SUM159 cells stably
overexpressing the CCR5 receptor at 60 seconds and FBS is added at
320 second;
[0059] FIG. 1OF illustrates the average fluorescence for the
population of SUM159-CCR5 cells;
[0060] FIG. 11A illurates that fact that maraviroc reduces
MDA-MB-231 breast cancer lung metastasis burden as evidenced by the
quantification results of weekly BLI, conducted for 5 weeks on
maraviroc-treated NOD/SCID mice, wherein the lung metastatic tumor
radiance antemortem was used as a surrogate measurement of tumor
burden to the lungs (per FIG. 5B).
[0061] FIG. 11B illustrates the fact that maraviroc reduces
MDA-MB-231 breast cancer lung metastasis burden as evidenced by the
quantification illustrates results of weekly BLI, conducted for 5
weeks on vehicle-treated NOD/SCID mice, wherein the lung metastatic
tumor radiance antemortem was used as a surrogate measurement of
tumor burden.
[0062] FIG. 12 includes plots 12A-12C and 12C1 and illustrates that
fact that pulmonary tumors are enriched in CCR5.sup.+4 cells as
evidenced by the FACS analysis of the proportion of CCR5+ cells
into metastatic tumors caused by tail vein injection of MDA.pFULG
cells. The proportion of CCR5+ cells within eGFP+MDA.pFULG cells in
culture (FIG. 12A) was compared with that of cells isolated from
metastatic tumors (FIG. 12B). Analysis of data (FIG. 12C) showed an
eight-fold increase in CCR5+ fraction in tumors (mean.+-.SEM, n=6;
Student's t test);
[0063] FIG. 13A includes plots 13A1 through 13A5 and illustrates
cell surface expression of CCR1 protein in cell lines MDA-MB-23,
HS578T, SUM159, MCF-7, Jurkat cells by FACS analysis;
[0064] FIG. 13B includes plots 13B1 through 13B5 and illustrates
cell surface expression of CCR3 in cell lines MDA-MB-23, HS578T,
SUM159, MCF-7, Jurkat cells by FACS analysis;
[0065] FIG. 13C includes plots 13C1 through 13C5 and illustrates
cell surface expression of CCR5 protein in cell lines MDA-MB-23,
HS578T, SUM159, MCF-7, Jurkat cells by FACS analysis;
[0066] FIG. 13D shows relative abundance of cell surface expression
of CCR1, CCR3 and CCR5 of cell lines MDA-MB-23, HS578T, SUM159,
MCF-7, Jurkat cells as determined by FACS analysis;
[0067] FIG. 14A illustrates immunohistochemical staining of CCR5 in
breast cancer tissue, showing staining was localized primarily to
the breast cancer epithelial cell compared with normal breast
tissue;
[0068] FIG. 14B illustrates immunohistochemical staining of CCR5 in
normal breast tissue, showing staining of CCR5 in normal breast
tissue is very low, demonstrating lack of CCR5 in normal breast
compared with breast tumor;
[0069] FIG. 14C Immunohistochemical staining of CCR5 in breast
cancer tissue, demonstrating CCR5 immunohistochemical staining
being localized primarily to the breast cancer epithelial cell
compared with normal breast tissue.
[0070] FIG. 14D shows immunohistochemical staining of CCR5 in
normal breast tissue, demonstrating lack of CCR5 in normal breast
tissue (of different patients than shown in FIG. 14B) compared with
breast tumor;
DETAILED DESCRIPTION OF THE INVENTION
[0071] Chemokine (C-C motif) ligand 5 ("CCL5"), also known as
RANTES (an acronym for Regulated on Activation, Normal T cell
Expressed and Secreted) is a protein which in humans is encoded by
the CCL5 gene. Its receptor, C-C chemokine receptor type 5 ("CCR5"
or "CD195") is a protein found on the white blood cells. CCR5 is
the main coreceptor used by macrophage (M)-tropic strains of human
immunodeficiency virus type 1 (HIV-1) and HIV-2, which are
responsible for viral transmission. CCR5 therefore plays an
essential role in HIV pathogenesis. A number of inflammatory
CC-chemokines, including MIP-1 alpha, MIP-1 beta, RANTES, MCP-2,
and HCC-1[9-74] act as CCR5 agonists, while MCP-3 is a natural
antagonist of the receptor. CCR5 is mainly expressed in memory
T-cells, macrophages, and immature dendritic cells, and is
upregulated by proinflammatory cytokines.
[0072] Classes of antiretroviral medications with activity against
HIV include nucleoside analogs, nonnucleoside reverse transcriptase
inhibitors, protease inhibitors, fusion inhibitors, integrase
inhibitors, and CCR5 receptor antagonists. CCR5 antagonists exert
their antiviral activity against HIV by blocking entry of CCR5
-tropic viruses into the CD4 T cell. As a result, CCR5 antagonists
have historically only been associated with expression in
inflammatory cells in the immune system.
[0073] Prior to the present disclosure, the roles of the chemokine
CCL5 and its receptor CCR5 in cancer progression were unclear. As
disclosed herein, patients with cancers expressing CCL5 and its
receptor CCR5, present new drug targets. As disclosed herein,
treating cancers that expressed CCL5 and its receptor CCR5 with
drugs that blocking their activities respecting CCL5 and its
receptor CCR5 selectively affect these cancer cells that express
mutant chemokine CCL5 and its receptor CCR5. In one embodiment, a
subpopulation of human breast cancer cell lines were found to
express CCR5 displayed a functional response to CCL5.
[0074] A microarray analysis conducted on 2,254 human breast cancer
specimens found increased expression of CCL5 and its receptor CCR5,
but not CCR3, in the basal and HER-2 genetic subtypes. The
subpopulation of human breast cancer cell lines found to express
CCR5 displayed a functional response to CCL5. Also, oncogene
transformation induced CCR5 expression, and the subpopulation of
cancer cells that expressed functional CCR5 also displayed
increased invasiveness.
[0075] In one embodiment, the CCR5 antagonists developed initially
to block CCR5 HIV co-receptor function, reduced in vitro invasion
of cancer cells without affecting cell proliferation or viability.
In one embodiment, the CCR5 antagonists include maraviroc and
vicriviroc. In one embodiment, the subpopulation of cancer cells
that expressed functional CCR5 include basal breast cancer cells.
In one embodiment, the CCR5 antagonists include maraviroc and
vicriviroc reduced in vitro invasion of basal cancer cells without
affecting the basal cancer cell proliferation or viability. In one
embodiment, the CCR5 antagonists include maraviroc and vicriviroc
reduced in vivo invasion of basal cancer cells without affecting
the basal cancer cell proliferation or viability.
[0076] However, as disclosed herein, CCL5 and its receptor CCR5
were found to be expressed in cancer cells, including breast cancer
cells, were also found to regulate cancer metastasis, spread of
cancer from its primary site to other sites in the body (e.g.,
brain, liver, lung). Moreover, it was found that blocking the CCR5
receptor with CCR5 antagonists, such as Maraviroc and Vicriviroc,
prevented migration and spread of the cancer from its primary site
to other sites in the body.
[0077] As described herein, CCR5 and CCL5 were found to play a key
role in cancer invasiveness. For example, it was shown that CCR5
antagonists slowed down and/or prevented the invasion of secondary
sites in the body by cancers that express CCL5 and/or its receptor
CCR5, demonstrating usefulness of CCR5 antagonists as viable
adjuvant therapy for reducing the risk of metastasis in cancer
patients, including patients having basal breast cancer molecular
subtype that express CCL5 and/or its receptor CCR5.
[0078] Cell receptor status can determine whether or not a cancer
patient is susceptible to a particular anti-cancer treatment. For
example, patients diagnosed with the basal breast cancer subtype,
where the basal breast cancer subtype does not express the androgen
or estrogen receptors or HER-2, current treatment choices,
including chemotherapy, radiation, or surgery, are not only the
only choices for these patients, but all show poor outcomes for
these patients. Notwithstanding the fact that currently there are
no other effective therapies available to them, the patients with
this variant of basal breast cancer are further disadvantaged by
the fact that this variant of basal breast cancer is also typically
associated with metastasis. New cancer treatments present the only
hope of cancer free survival for these patients. Accordingly, an
urgent need exists for a specific targeted therapy for the basal
breast cancer subtype.
[0079] In one aspect the present invention relates to the use of
CCR5 modulators to treat, prevent, or manage a neoplasm or
metastasis of the neoplasm. In one embodiment the neoplasm is
cancer. Exemplary cancers and related disorders that can be
treated, prevented, or managed in accordance with the exemplary
embodiments of the methods of the present invention include, but
are not limited to, leukemias, such as but not limited to, acute
leukemia, acute lymphocytic leukemia, acute myelocytic leukemias,
such as, myeloblastic, promyelocytic, myelomonocytic, monocytic,
and erythroleukemia leukemias and myelodysplastic syndrome; chronic
leukemias, such as but not limited to, chronic myelocytic
(granulocytic) leukemia, chronic lymphocytic leukemia, hairy cell
leukemia; polycythemia vera; lymphomas such as but not limited to
Hodgkin's disease, non-Hodgkin's disease; multiple myelomas such as
but not limited to smoldering multiple myeloma, nonsecretory
myeloma, osteosclerotic myeloma, plasma cell leukemia, solitary
plasmacytoma and extramedullary plasmacytoma; Waldenstrom's
macroglobulinemia; monoclonal gammopathy of undetermined
significance; benign monoclonal gammopathy; heavy chain disease;
bone and connective tissue sarcomas such as but not limited to bone
sarcoma, osteosarcoma, chondrosarcoma, Ewing's sarcoma, malignant
giant cell tumor, fibrosarcoma of bone, chordoma, periosteal
sarcoma, soft-tissue sarcomas, angiosarcoma (hemangiosarcoma),
fibrosarcoma, Kaposi's sarcoma, leiomyosarcoma, liposarcoma,
lymphangiosarcoma, neurilemmoma, rhabdomyosarcoma, synovial
sarcoma; brain tumors such as but not limited to, glioma,
astrocytoma, brain stem glioma, ependymoma, oligodendroglioma,
nonglial tumor, acoustic neurinoma, craniopharyngioma,
medulloblastoma, meningioma, pineocytoma, pineoblastoma, primary
brain lymphoma; breast cancer including but not limited to ductal
carcinoma, adenocarcinoma, lobular (small cell) carcinoma,
intraductal carcinoma, medullary breast cancer, mucinous breast
cancer, tubular breast cancer, papillary breast cancer, Paget's
disease, and inflammatory breast cancer; adrenal cancer such as but
not limited to pheochromocytom and adrenocortical carcinoma;
thyroid cancer such as but not limited to papillary or follicular
thyroid cancer, medullary thyroid cancer and anaplastic thyroid
cancer; pancreatic cancer such as but not limited to, insulinoma,
gastrinoma, glucagonoma, vipoma, somatostatin-secreting tumor, and
carcinoid or islet cell tumor; pituitary cancers such as but
limited to Cushing's disease, prolactin-secreting tumor,
acromegaly, and diabetes insipius; eye cancers such as but not
limited to ocular melanoma such as iris melanoma, choroidal
melanoma, and cilliary body melanoma, and retinoblastoma; vaginal
cancers such as squamous cell carcinoma, adenocarcinoma, and
melanoma; vulvar cancer such as squamous cell carcinoma, melanoma,
adenocarcinoma, basal cell carcinoma, sarcoma, and Paget's disease;
cervical cancers such as but not limited to, squamous cell
carcinoma, and adenocarcinoma; uterine cancers such as but not
limited to endometrial carcinoma and uterine sarcoma; ovarian
cancers such as but not limited to, ovarian epithelial carcinoma,
borderline tumor, germ cell tumor, and stromal tumor; esophageal
cancers such as but not limited to, squamous cancer,
adenocarcinoma, adenoid cystic carcinoma, mucoepidermoid carcinoma,
adenosquamous carcinoma, sarcoma, melanoma, plasmacytoma, verrucous
carcinoma, and oat cell (small cell) carcinoma; stomach cancers
such as but not limited to, adenocarcinoma, fungating (polypoid),
ulcerating, superficial spreading, diffusely spreading, malignant
lymphoma, liposarcoma, fibrosarcoma, and carcinosarcoma; colon
cancers; rectal cancers; liver cancers such as but not limited to
hepatocellular carcinoma and hepatoblastoma; gallbladder cancers
such as adenocarcinoma; cholangiocarcinomas such as but not limited
to pappillary, nodular, and diffuse; lung cancers such as non-small
cell lung cancer, squamous cell carcinoma (epidermoid carcinoma),
adenocarcinoma, large-cell carcinoma and small-cell lung cancer;
testicular cancers such as but not limited to germinal tumor,
seminoma, anaplastic, classic (typical), spermatocytic,
nonseminoma, embryonal carcinoma, teratoma carcinoma,
choriocarcinoma (yolk-sac tumor), prostate cancers such as but not
limited to, prostatic intraepithelial neoplasia, adenocarcinoma,
leiomyosarcoma, and rhabdomyosarcoma; penal cancers; oral cancers
such as but not limited to squamous cell carcinoma; basal cancers;
salivary gland cancers such as but not limited to adenocarcinoma,
mucoepidermoid carcinoma, and adenoidcystic carcinoma; pharynx
cancers such as but not limited to squamous cell cancer, and
verrucous; skin cancers such as but not limited to, basal cell
carcinoma, squamous cell carcinoma and melanoma, superficial
spreading melanoma, nodular melanoma, lentigo malignant melanoma,
acral lentiginous melanoma; kidney cancers such as but not limited
to renal cell carcinoma, adenocarcinoma, hypemephroma,
fibrosarcoma, transitional cell cancer (renal pelvis and/or
uterer); Wilms' tumor; bladder cancers such as but not limited to
transitional cell carcinoma, squamous cell cancer, adenocarcinoma,
carcinosarcoma. In addition, cancers include myxosarcoma,
osteogenic sarcoma, endotheliosarcoma, lymphangioendotheliosarcoma,
mesothelioma, synovioma, hemangioblastoma, epithelial carcinoma,
cystadenocarcinoma, bronchogenic carcinoma, sweat gland carcinoma,
sebaceous gland carcinoma, papillary carcinoma and papillary
adenocarcinomas.
[0080] In one embodiment, antagonists of CCR5 are used to treat
CCR5-expressing neoplasm or metastasis of the CCR5-expressing
neoplasm. In one embodiment, antagonists of CCR5 are used to
prevent the neoplasm or metastasis of said neoplasm. In one
embodiment, antagonists of CCR5 are used to manage the neoplasm
metastasis of said neoplasm. In one embodiment, antagonists of CCR5
are used slow the progression of the CCR5-expressing neoplasm or
metastasis of the CCR5-expressing neoplasm. In one embodiment,
antagonists of CCR5 are used to delay metastasis of the
CCR5-expressing neoplasm.
[0081] In one embodiment, antagonists of CCR5 that are suitable for
use in accordance with the exemplary methods of the present
invention include, but are not limited to, the chemical compounds
that are described in U.S. Pat. No. 6,667,314 by Perros et al. The
chemical compounds of Perros et al. and all formulations and dosage
forms including them are incorporated by reference into the present
application. Preferred examples of the compounds by Perros et al.
include: [0082]
4,4-difluoro-N-[(1S)-3-[(1R,5S)-3-(3-methyl-5-propan-2-yl-1,2,4-triazol-4-
-yl)-8-azabicyclo[3.2.1]octan-8-yl]-1-phenylpropyl]cyclohexane-1-carboxami-
de ("Maraviroc"); [0083]
N-(1S)-3-3-(3-Isopropyl-5-methyl-4H-1,2,4-triazol-4-yl)-exo-8-azabicyclo[-
3.2.1]oct-8-yl-1-phenylpropylcyclobutanecarboxamide; [0084]
N-(1S)-3-3-(3-Isopropyl-5-methyl-4H-1,2,4-triazol-4-yl)-exo-8-azabicyclo[-
3.2.1]oct-8-yl-1-phenylpropylcyclopentanecarboxamide; [0085]
N-(1S)-3-3-(3-Isopropyl-5-methyl-4H-1,2,4-triazol-4-yl)-exo-8-azabicyclo[-
3.2.1]oct-8-yl-1-phenylpropyl-4,4,4-trifluorobutanamide; [0086]
N-(1S)-3-3-(3-Isopropyl-5-methyl-4H-1,2,4-triazol-4-yl)-exo-8-azabicyclo[-
3.2.1]oct-8-yl-1-phenylpropyl-4,4-difluorocyclohexanecarboxamide;
[0087]
N-(1S)-3-3-(3-Isopropyl-5-methyl-4H-1,2,4-triazol-4-yl)-exo-8-azabicyclo[-
3.2.1]oct-8-yl-1-(3-fluorophenyl)propyl-4,4-difluorocyclohexanecarboxamide-
; and pharmaceutically acceptable salts or solvates thereof.
[0088] In one embodiment, the modulators of CCR5 receptor that are
suitable for treating the neoplasm or metastasis of said neoplasm;
or preventing the neoplasm or metastasis of said neoplasm; or
managing the neoplasm metastasis of said neoplasm; or slowing the
progression of the neoplasm or metastasis of said neoplasm; or
delaying the neoplasm or metastasis of said neoplasm; include, but
are not limited to the chemical compounds that are described in
U.S. Pat. No. 6,586,430 by Armour et al. The chemical compounds of
Armour et al. and all formulations or dosage forms including them
are incorporated by reference into the present application.
Preferred examples of the compounds by Armour et al. include:
[0089]
N-{3-[3-exo-(2-Methyl-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-8-yl]--
1-phenylpropyl}cyclobutanecarboxamide; [0090]
N-{(1S)-3[3-exo-(2-Methyl-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-8--
yl]-1-phenylpropyl}cyclobutanecarboxamide; [0091]
N-{(1S)-3-[3-endo-(2-Methyl-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct--
8-yl]-1-phenylpropyl}cyclobutanecarboxamide; [0092]
N-{(1S)-3-[3-exo-(2-Methyl-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-8-
-yl]-1-phenylpropyl}tetrahydro-2H-pyran-4-carboxamide; [0093]
1-Acetyl-N-{(1S)-3-[3-exo-(2-methyl-1H-benzimidazol-1-yl)-8-azabicyclo[3.-
2.1]oct-8-yl]-1-phenylpropyl}3-azetidine carboxamide; [0094]
1-Hydroxy-N-{(1S)-3-[3-exo-(2-methyl-1H-benzimidazol-1-yl)-8-azabicyclo[3
.2.1]oct-8-yl]-1-phenylpropyl}cyclopentanecarboxamide; [0095]
2-Methyl-N-{(1S)-3-[3-exo-(2-methyl-1H-benzimidazol-1-yl)-8-azabicyclo[3.-
2.1]oct-8-yl]-1-phenylpropyl}cyclopropanecarboxamide; [0096]
2-Cyclopropyl-N-{1S)-3-[3-exo-(2-methyl-1H-benzimidazol-1-yl)-8-azabicycl-
o [3.2.1]oct-8-yl]-1-phenylpropyl}acetamide; [0097]
N-{(1S)-3-[3-exo-(2-methyl-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-8-
-yl]-1-phenylpropyl}tetrahydro-3-furancarboxamide; [0098]
3,3,3-Trifluoro-N-{(1S)-3-[3-exo-(2-methyl-1H-benzimidazol-1-yl)-8-azabic-
yclo[3.2.1]oct-8-yl]-1-phenylpropyl}propanamide; [0099]
N-{(1S)-3-[3-exo-(2-Methyl-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-8-
-yl]-1-phenylpropyl}tetrahydro-2-furancarboxamide; [0100]
1-(Acetylamino)-N-{(1S)-3-[3-exo-(2-methyl-1H-benzimidazol-1-yl)-8-azabic-
yclo[3.2.1]oct-8-yl]-1-phenylpropyl}cyclopentanecarboxamide; [0101]
N-{(1S)-3-[3-exo-(2-Methyl-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-8-
-yl]-1-phenylpropyl}acetamide; [0102]
1-Methoxy-N-{(1S)-3-[3-exo-(2-methyl-1H-benzimidazol-1-yl)-8-azabicyclo[3-
.2.1]oct-8-yl]-1-phenylpropyl}cyclopentanecarboxamide;
[0103]
1-Amino-N-{(1S)-3-[3-exo-(2-methyl-1H-benzimidazol-1-yl)-8-azabicyc-
lo[3.2.1]oct-8-yl]-1-phenylpropyl}cyclopentanecarboxamide; [0104]
1-Methyl-N-{(1S)-3-[3-exo-(2-methyl-1H-benzimidazol-1-yl)-8-azabicyclo[3.-
2.1]oct-8-yl]-1-phenylpropyl}-2-oxo-4-pyrrolidinecarboxamide;
[0105]
1-Acetyl-N-{(1S)-3-[3-endo-(2-methyl-1H-benzimidazol-1-yl)-8-azabicyclo[3-
.2.1]oct-8-yl]-1-phenylpropyl}3-azetidinecarboxamide; [0106]
N-{(1S-3-[3-endo-(2-Methyl-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-8-
-yl]-1-phenylpropyl}acetamide; [0107]
N-{(1S)-3[6-(2-Methyl-1H-benzimidazol-1-yl)-3-azabicyclo[3.1.0]hex-3-yl]--
1-phenylpropyl}cyclobutanecarboxamide; [0108]
2-Cyclopropyl-N-{(1S)-3-[3-exo-(3-{4-[(methylsulfonyl)amino]benzyl}-1,2,4-
-oxadiazol-5-yl)-8-azabicyclo[3.2.1]oct-8-yl]-1-phenylpropyl}acetamide;
[0109]
N-{(1S)-3-[7-exo-(2-Methyl-1H-benzimidazol-1-yl)-3-oxa-9-azabicycl-
o[3.3.1]non-9-yl]-1-phenylpropyl}cyclobutanecarboxamide; [0110]
2-Cyclopropyl-N-{(1S)-3-[7-exo-(2-methyl-1H-benzimidazol-1-yl)-3-oxa-9-az-
abicyclo[3.3.1]non-9-yl]-1-phenylpropyl}acetamide; [0111]
3,3,3-Trifluoro-N-{(1S)-3-[7-exo-(2-methyl-1H-benzimidazol-1-yl)-3-oxa-9--
azabicyclo[3.3.1]non-9-yl]-1-phenylpropyl}propanamide; [0112]
N-{(1S)-3-[7-endo-(2-Methyl-1H-benzimidazol-1-yl)-3-oxa-9-azabicyclo[3.3.-
1]non-9-yl]-1-phenylpropyl}cyclobutanecarboxamide; [0113]
2-Cyclopropyl-N-{(1S)-3-[7-endo-(2-methyl-1H-benzimidazol-1-yl)-3-oxa-9-a-
zabicyclo[3.3.1]non-9-yl]-1-phenylpropyl}acetamide; [0114]
N-{(1S)-3-[7-exo-(2-Methyl-1H-benzimidazol-1-yl)-3-thia-9-azabicyclo[3.3.-
1]non-9-yl]-1-phenylpropyl}cyclobutanecarboxamide; [0115]
2-Cyclopropyl-N-[(1S)-3-(3-endo-{[2-(4-fluorophenyl)acetyl]amino}-8-azabi-
cyclo[3.2.1]oct-8-yl)-1-phenylpropyl]acetamide; [0116]
N-[(1S)-3-(3-{[3-endo-(4-Fluorophenyl)ppropanoyl]amino}-8-azabicyclo[3.2.-
1]oct-8-yl)-1-phenylpropyl]cyclobutanecarboxamide; [0117]
N-[(1S)-3-(3-{[3-exo-(4-Fluorophenyl)prpropanoyl]amino}-8-azabicyclo[3.2.-
1]oct-8-yl)-1-phenylpropyl]cyclobutanecarboxamide; [0118]
2-Cyclopropyl-N-[(1S)-3-(3-exo-{[2-(4-fluorophenyl)acetyl]amino}-8-azabic-
yclo[3.2.1]oct-8-yl)-1-phenylpropyl]acetamide; [0119]
N-{(1S)-3-[3-exo-(2-Methyl-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-8-
-yl]-1-phenylpropyl)}1-propionyl-3-azetidinecarboxamide; [0120]
N-{(1S)-3-[3-endo-(2-Methyl-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct--
8-yl]-1-phenylpropyl}tetrahydro-3-furancarboxamide; [0121]
N-{(1S-3-[3-endo-(2-Methyl-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-8-
-yl]-1-phenylpropyl}tetrahydro-2H-pyran4-carboxamide;
[0122]
N-{(1S)-3-[3-endo-(2-Methyl-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.-
1]oct-8-yl]-1-phenylpropyl}tetrahydro-2-furancarboxamide; [0123]
1-Acetyl-N-{(1S)-3-[3-endo-(1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct--
8-yl]-1-phenylpropyl}-3-azetidinecarboxamide; [0124]
N-{(1S)-3-[3-endo-(1H-Benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-8-yl]-1-p-
henylpropyl}-1-propionyl-3-azetidinecarboxamide; [0125] Methyl
3-[({(1S)-3-[3-exo-(2-methyl-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-
-8-yl]-1-phenylpropyl}amino)carbonyl]-1-azetidinecarboxylate;
[0126]
N-{(1S)-3-[3-endo-(2-Methyl-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct--
8-yl]-1-phenylpropyl }-1-propionyl-3-azetidinecarboxamide
1-Acetyl-N-{(1S)-3-[3-endo-(2-methyl-1H-benzimidazol-1-yl)-8-azabicyclo[3-
.2.1]oct-8-yl]-1-phenylpropyl}-2-azetidinecarboxamide; [0127]
2-[Acetyl(methyl)amino]-N-{(1S)-3-[3-endo-(2-methyl-1H-benzimidazol-1-yl)-
-8-azabicyclo[3.2.1]oct-8-yl]-1-phenylpropyl}acetamide; [0128]
3-[Acetyl(methyl)amino]-N-{(1S)-3-[3-endo-(2-methyl-1H-benzimidazol-1-yl)-
-8-azabicyclo[3.2.1]oct-8-yl]-1-phenylpropyl}propanamide; [0129]
2-Methoxy-N-{(1S)-3-[3-endo-(2-methyl-1H-benzimidazol-1-yl)-8-azabicyclo[-
3.2.1]oct-8-yl]-1-phenylpropyl}acetamide; [0130]
3-Methoxy-N-{(1S)-3-[3-endo-(2-methyl-1H-benzimidazol-1-yl)-8-azabicyclo[-
3.2.1]oct-8-yl]-1-phenylpropyl}propanamide; [0131]
1-Acetyl-N-{(1S)-3-[3-endo-(2-methyl-1H-benzimidazol-1-yl)-8-azabicyclo[3-
.2.1]oct-8-yl]-1-phenylpropyl}-3-pyrrolidinecarboxamide; [0132]
1-Methyl-N-{(1S)-3-[3-endo-(2-methyl-1H-benzimidazol-1-yl)-8-azabicyclo[3-
.2.1]oct-8-yl]-1-phenylpropyl}-2-oxo-4-pyrrolidinecarboxamide;
[0133]
1-Acetyl-N-{(1S)-3-[3-exo-(2-ethyl-1H-benzimidazol-1-yl)-8-azabicyclo[3.2-
.1]oct-8-yl]-1-phenylpropyl}-3-azetidinecarboxamide; [0134]
N-{(1S)-3-[3-exo-(2-Ethyl-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-8--
yl]-1-phenylpropyl}-1-propionyl-3-azetidinecarboxamide; [0135]
1-Acetyl-N-((1S)-1-phenyl-3-{3-exo[2-(trifluoromethyl)-1H-benzimidazol-1--
yl]-8-azabicyclo[3.2.1]oct-8-yl}propyl)-3-azetidinecarboxamide;
[0136]
N-((1S)-1-Phenyl-3-{3-exo-[2-(trifluoromethyl)-1H-benzimidazol-1-yl]-8-az-
abicyclo[3.2.1]oct-8-yl}propyl)-1-propionyl-3-azetidinecarboxamide;
[0137]
N-((1S)-1-Phenyl-3-{3-exo[2-(trifluoromethyl)-1H-benzimidazol-1-yl]-8-aza-
bicyclo[3.2.1]oct-8-yl}propyl)acetamide; [0138]
2-[Acetyl(methyl)amino]-N-((1S)-1-phenyl-3-{3-exo-[2-(trifluoromethyl)-1H-
-benzimidazol-1-yl]-8-azabicyclo[3.2.1]oct-8-yl}propyl)acetamide;
[0139]
1-Acetyl-N-{(1S)-3-[3-exo-(1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-8-
-yl]-1-phenylpropyl}-3-azetidinecarboxamide; [0140]
N(1S)-3-[3-exo-(1H-Benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-8-yl]-1-phen-
ylpropyl}-1-propionyl-3-azetidinecarboxamide; [0141]
1-acetyl-N(1S)-3-[3-exo-(5-fluoro-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.-
1]oct-8-yl]-1-phenylpropyl)3-azetidinecarboxamide; [0142]
N-{(1S)-3-[3-exo-(5-Fluoro-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-8-
-yl]-1-phenylpropyl}-1-propionyl-3-azetidinecarboxamide; [0143]
1-Acetyl-N-{(1S)-[3-exo-(5-fluoro-2-methyl-1H-benzimidazol-1-yl)-8-azabic-
yclo[3.2.1]oct-8-yl]-1-phenylpropyl}3-azetidinecarboxamide; [0144]
N-{(1S)-3-[3-exo-(5-Fluoro-2-methyl-1H-benzimidazol-1-yl)-8-azabicyclo[3.-
2.1]oct-8-yl]-1-phenylpropyl}-1-propionyl-3-azetidinecarboxamide;
[0145]
N-{(1S)-3-[3-exo-(2-methyl-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-8-
-yl]-1-phenylpropyl}-3-azetidinecarboxamide; [0146]
1-methyl-N-{(1S)-3-[3-exo-(2-methyl-1H-benzimidazol-1-yl)-8-azabicyclo[3.-
2.1]oct-8-yl]-1-phenylpropyl}-3-azetidinecarboxamide; [0147]
(2S)-1-acetyl-N-{(1S)-3-[3-exo-(2-methyl-1H-benzimidazol-1-yl)-8-azabicyc-
lo[3.2.1]oct-8-yl]-1-phenylpropyl}-2-azetidinecarboxamide; [0148]
(2R)-1-acetyl-N-{(1S)-3-[3-exo-(2-methyl-1H-benzimidazol-1-yl)-8-azabicyc-
lo[3.2.1]oct-8-yl]-1-phenylpropyl}-2-azetidinecarboxamide; [0149]
2-[acetyl(methyl)amino]-N-{(1S)-3-[3-exo-(2-methyl-1H-benzimidazol-1-yl)--
8-azabicyclo[3.2.1]oct-8-yl]-1-phenylpropyl}acetamide; [0150]
3-[acetyl(methyl)amino]-N-{(1S)-3-[3-exo-(2-methyl-1H-benzimidazol-1-yl)--
8-azabicyclo[3.2.1]oct-8-yl]-1-phenylpropyl}propanamide; [0151]
1-acetyl-N-{(1S)-3-[3-exo-(2-methyl-1H-benzimidazol-1-yl)-8-azabicyclo[3.-
2.1]oct-8-yl]-1-phenylpropyl}-3-pyrrolidinecarboxamide; [0152]
N-{(1S)-3-[3-exo-(2-methyl-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-8-
-yl]-1-phenylpropyl}-1-(trifluoromethyl)cyclopropanecarboxamide;
[0153]
2-methoxy-N-{(1S)-3-[3-exo-(2-methyl-1H-benzimidazol-1-yl)-8-azabicyclo[3-
.2.1]oct-8-yl]-1-phenylpropyl}acetamide; [0154]
3-methoxy-N-{(1S)-3-[3-exo-(2-methyl-1H-benzimidazol-1-yl)-8-azabicyclo[3-
.2.1]oct-8-yl]-1-phenylpropyl}propanamide; [0155]
1-Acetyl-N-{(1S)-3-[3-exo-(4-fluoro-2-methyl-1H-benzimidazol-1-yl)-8-azab-
icyclo[3.2.1]oct-8-yl]-1-phenylpropyl}-3-azetidinecarboxamide;
[0156]
N-{(1S)-3-[3-exo-(4-Fluoro-2-methyl-1H-benzimidazol-1-yl)-8-azabicyclo[3.-
2.1]oct-8-yl]-1-phenylpropyl}-3-azetidinecarboxamide; [0157]
1-Methyl-N-{(1S)-3-[3-exo-(4-fluoro-2-methyl-1H-benzimidazol-1-yl)-8-azab-
icyclo[3.2.1]oct-8-yl]-1-phenylpropyl}-3-azetidinecarboxamide;
[0158]
N-{(1S)-3[3-exo-(4-Fluoro-2-methyl-1H-benzimidazol-1-yl)-8-azabicyclo[3.2-
.1]oct-8-yl]-1-phenylpropyl}-1-propionyl-3-azetidinecarboxamide;
[0159]
2-Methoxy-N-{(1S)-3-[3-exo-(4-Fluoro-2-methyl-1H-benzimidazol-1-yl)-8-aza-
bicyclo[3.2.1]oct-8-yl]-1-phenylpropyl}acetamide; [0160]
N-{(1S)-3[3-exo-(4-Fluoro-2-Methyl-1H-benzimidazol-1-yl)-8-azabicyclo[3.2-
.1]oct-8-yl]-1-phenylpropyl}acetamide;
3-Methoxy-N-{(1S)-3-[3-exo-(4-fluoro-2-methyl-1H-benzimidazol-1-yl)-8-aza-
bicyclo[3.2.1]oct-8-yl]-1-phenylpropyl}propanamide; [0161]
2-[Acetyl(methyl)amino]-N-{(1S)-3-[3-exo-(4-fluoro-2-methyl-1H-benzimidaz-
ol-1-yl)-8-azabicyclo[3.2.1]oct-8-yl]-1-phenylpropyl}acetamide;
[0162]
3-[Acetyl(methyl)amino]-N-{(1S)-3-[3-exo-(4-fluoro-2-methyl-1H-benzimidaz-
ol-1-yl)-8-azabicyclo[3.2.1]oct-8-yl]-1-phenylpropyl}propanamide;
[0163]
N-{(1S)-3[3-exo-(4-fluoro-2-methyl-1H-benzimidazol-1-yl)-8-azabicyclo[3.2-
.1]oct-8-yl]-1-phenylpropyl}-3-methyl-3-oxetanecarboxamide; [0164]
3-Ethyl-N-{(1S)-3[3-exo-(4-fluoro-2-methyl-1H-benzimidazol-1-yl)-8-azabic-
yclo[3.2.1]oct-8-yl]-1-phenylpropyl}-3-oxetanecarboxamide; [0165]
N-{(1S)-3-[3-exo-(4-Fluoro-2-methyl-1H-benzimidazol-1-yl)-8-azabicyclo[3.-
2.1]oct-8-yl]-1-phenylpropyl}-3-oxetanecarboxamide; [0166]
3-Ethyl-N-{(1S)-3[3-exo-(4-fluoro-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.-
1]oct-8-yl]-1-phenylpropyl}-3-oxetanecarboxamide; [0167]
N-{(1S)-3-[3-exo-(4-Fluoro-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-8-
-yl]-1-phenylpropyl}-3-methyl-3-oxetanecarboxamide; [0168]
N-{(1S)-3-[3-exo-(4-Fluoro-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-8-
-yl]-1-phenylpropyl}-3-oxetanecarboxamide; [0169]
N-{(1S)-3-[3-exo-(4-Fluoro-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-8-
-yl]-1-phenylpropyl}-3-azetidinecarboxamide; [0170]
N-{(1S)-3-[3-exo-(4-Fluoro-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-8-
-yl]-1-phenylpropyl}-1-methyl-3-azetidinecarboxamide; [0171]
1-Acetyl-N-{(1S)-3[3-exo-(4-fluoro-1H-benzimidazol-1-yl)-8-azabicyclo[3.2-
.1]oct-8-yl]-1-phenylpropyl}-3-azetidinecarboxamide;
[0172]
N-{(1S)-3-[3-exo-(4-Fluoro-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1-
]oct-8-yl]-1-phenylpropyl}-1-propionyl-3-azetidinecarboxamide;
[0173]
N-{(1S)-3-[3-exo-(4-Fluoro-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-8-
-yl]-1-phenylpropyl}-2-methoxyacetamide; [0174] N-{(1S)-3-[3
-exo-(4-Fluoro-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-8-yl]-1-pheny-
lpropyl}acetamide; [0175]
N{-1S)-3-[3-exo-(4-fluoro-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-8--
yl]-1-phenylpropyl}-3-methoxypropanamide; [0176]
2-[Acetyl(methyl)amino]-N-{(1S)-3-[3-exo-(4-fluoro-1H-benzimidazol-1-yl)--
8-azabicyclo[3.2.1]oct-8-yl]-1-phenylpropyl}acetamide;
3-[Acetyl(methyl)amino]-N-{(1S)-3-[3exo-(4-fluoro-1H-benzimidazol-1-yl)-8-
-azabicyclo[3.2.1]oct-8-yl]-1-phenylpropyl}propanamide; and
pharmaceutically acceptable salts thereof.
[0177] In yet another embodiment, modulators of CCR5 receptor that
are suitable for treating neoplasm or metastasis of the neoplasm;
or preventing the neoplasm or metastasis of the neoplasm; or
managing the neoplasm metastasis of the neoplasm; or slowing the
progression of the neoplasm or metastasis of the neoplasm; or
delaying the neoplasm or metastasis of said neoplasm; include, but
are not limited to the chemical compounds that are described in
U.S. Pat. Nos. 6,689,765 and 7,384,944 both by Baroudy et al. The
chemical compounds of Baroudy et al. and all formulations or dosage
forms including them are incorporated by reference into the present
application. A preferred example of the compounds by Baroudy et al.
includes:
(4,6-dimethylpyrimidin-5-yl)-[4-[(3S)-4-[(1R)-2-methoxy-1-[4-(trifluorome-
thyl)phenyl]ethyl]-3-methylpiperazin-1-yl]-4-methylpiperidin-1-yl]methanon-
e (Vicriviroc, also previously named SCH 417690 and SCH-D), which
has an alternative IUPAC name of
5-({4-[(3S)-4-{2-methoxy-1-[4-(trifluoromethyl)phenyl]ethyl
}-3-methylpiperazin-1-yl]-4-methylpiperidin-1-yl}carbonyl)-4,6-dimethylpy-
rimidine; and pharmaceutically acceptable salts or solvates
thereof.
[0178] In one aspect, the present invention provides a method of
determining whether a subject has cancer or is at risk for
developing cancer and/or is at risk for developing metastasis of In
one embodiment, the method includes obtaining a biological sample
from a subject having or suspected of having cancer and assessing
the level of expression of CCR5 and/or of at least one of CCR5
ligands in the biological sample. In one embodiment, the expression
level of CCR5 and/or of at least one of CCR5 ligands in the
biological sample is compared to an expression level of CCR5 and/or
of at least one of CCR5 ligands in a control sample. In one
embodiment, if the expression level of CCR5 and/or of at least one
of CCR5 ligands in the biological sample is higher than the level
of expression of CCR5 and/or of at least one of CCR5 ligands in the
control sample, then the subject is diagnosed as likely to have
cancer. In one embodiment, if the expression level of CCR5 and/or
of at least one of CCR5 ligands in the biological sample is higher
than the level of expression of CCR5 and/or of at least one of CCR5
ligands in the control sample, then the subject is diagnosed as at
increased risk for developing cancer. In one embodiment, if the
expression level of CCR5 and/or of at least one of CCR5 ligands in
the biological sample is higher than the level of expression of
CCR5 and/or of at least one of CCR5 ligands in the control sample,
then the subject is diagnosed as at increased risk for developing
cancer metastasis.
[0179] The term "subject" as used herein is intended to include
animals. In particular embodiments, the subject is a mammal, a
human or nonhuman primate, a dog, a cat, a horse, a cow or a
rodent.
[0180] In another aspect, the present invention provides a method
for molecular classification of cancer based on a level of
expression of CCR5 and/or of at least one of CCR5 ligands in the
biological sample of the cancer. In one embodiment, the method for
molecular classification of cancer comprises (a) obtaining a
biological sample of cancer from subject; (b) determining level of
expression of CCR5 and/or level of expression of at least one of
CCR5 ligands in the biological sample; and (c) if the level of
expression of CCR5 and/or level of expression of at least one
of
[0181] CCR5 ligands determined in step (b) is higher than the level
of expression of CCR5 and/or of at least one of CCR5 ligands in a
control sample, then the cancer is classified as CCR5 -expressing
cancer. In one embodiment, a cancer subject whose cancer has been
classified as CCR5 -expressing cancer is diagnosed as likely at
risk for developing metastasis of the cancer. In one embodiment,
the cancer is breast cancer. In one embodiment, the cancer is
prostate cancer. In one embodiment, the cancer is selected from the
group consisting of leukemias, such as but not limited to, acute
leukemia, acute lymphocytic leukemia, acute myelocytic leukemias,
such as, myeloblastic, promyelocytic, myelomonocytic, monocytic,
and erythroleukemia leukemias and myelodysplastic syndrome; chronic
leukemias, such as but not limited to, chronic myelocytic
(granulocytic) leukemia, chronic lymphocytic leukemia, hairy cell
leukemia; polycythemia vera; lymphomas such as but not limited to
Hodgkin's disease, non-Hodgkin's disease; multiple myelomas such as
but not limited to smoldering multiple myeloma, nonsecretory
myeloma, osteosclerotic myeloma, plasma cell leukemia, solitary
plasmacytoma and extramedullary plasmacytoma; Waldenstrom's
macroglobulinemia; monoclonal gammopathy of undetermined
significance; benign monoclonal gammopathy; heavy chain disease;
bone and connective tissue sarcomas such as but not limited to bone
sarcoma, osteosarcoma, chondrosarcoma, Ewing's sarcoma, malignant
giant cell tumor, fibrosarcoma of bone, chordoma, periosteal
sarcoma, soft-tissue sarcomas, angiosarcoma (hemangiosarcoma),
fibrosarcoma, Kaposi's sarcoma, leiomyosarcoma, liposarcoma,
lymphangiosarcoma, neurilemmoma, rhabdomyosarcoma, synovial
sarcoma; brain tumors such as but not limited to, glioma,
astrocytoma, brain stem glioma, ependymoma, oligodendroglioma,
nonglial tumor, acoustic neurinoma, craniopharyngioma,
medulloblastoma, meningioma, pineocytoma, pineoblastoma, primary
brain lymphoma; breast cancer including but not limited to ductal
carcinoma, adenocarcinoma, lobular (small cell) carcinoma,
intraductal carcinoma, medullary breast cancer, mucinous breast
cancer, tubular breast cancer, papillary breast cancer, Paget's
disease, and inflammatory breast cancer; adrenal cancer such as but
not limited to pheochromocytom and adrenocortical carcinoma;
thyroid cancer such as but not limited to papillary or follicular
thyroid cancer, medullary thyroid cancer and anaplastic thyroid
cancer; pancreatic cancer such as but not limited to, insulinoma,
gastrinoma, glucagonoma, vipoma, somatostatin-secreting tumor, and
carcinoid or islet cell tumor; pituitary cancers such as but
limited to Cushing's disease, prolactin-secreting tumor,
acromegaly, and diabetes insipius; eye cancers such as but not
limited to ocular melanoma such as iris melanoma, choroidal
melanoma, and cilliary body melanoma, and retinoblastoma; vaginal
cancers such as squamous cell carcinoma, adenocarcinoma, and
melanoma; vulvar cancer such as squamous cell carcinoma, melanoma,
adenocarcinoma, basal cell carcinoma, sarcoma, and Paget's disease;
cervical cancers such as but not limited to, squamous cell
carcinoma, and adenocarcinoma; uterine cancers such as but not
limited to endometrial carcinoma and uterine sarcoma; ovarian
cancers such as but not limited to, ovarian epithelial carcinoma,
borderline tumor, germ cell tumor, and stromal tumor; esophageal
cancers such as but not limited to, squamous cancer,
adenocarcinoma, adenoid cystic carcinoma, mucoepidermoid carcinoma,
adenosquamous carcinoma, sarcoma, melanoma, plasmacytoma, verrucous
carcinoma, and oat cell (small cell) carcinoma; stomach cancers
such as but not limited to, adenocarcinoma, fungating (polypoid),
ulcerating, superficial spreading, diffusely spreading, malignant
lymphoma, liposarcoma, fibrosarcoma, and carcinosarcoma; colon
cancers; rectal cancers; liver cancers such as but not limited to
hepatocellular carcinoma and hepatoblastoma; gallbladder cancers
such as adenocarcinoma; cholangiocarcinomas such as but not limited
to pappillary, nodular, and diffuse; lung cancers such as non-small
cell lung cancer, squamous cell carcinoma (epidermoid carcinoma),
adenocarcinoma, large-cell carcinoma and small-cell lung cancer;
testicular cancers such as but not limited to germinal tumor,
seminoma, anaplastic, classic (typical), spermatocytic,
nonseminoma, embryonal carcinoma, teratoma carcinoma,
choriocarcinoma (yolk-sac tumor), prostate cancers such as but not
limited to, prostatic intraepithelial neoplasia, adenocarcinoma,
leiomyosarcoma, and rhabdomyosarcoma; penal cancers; oral cancers
such as but not limited to squamous cell carcinoma; basal cancers;
salivary gland cancers such as but not limited to adenocarcinoma,
mucoepidermoid carcinoma, and adenoidcystic carcinoma; pharynx
cancers such as but not limited to squamous cell cancer, and
verrucous; skin cancers such as but not limited to, basal cell
carcinoma, squamous cell carcinoma and melanoma, superficial
spreading melanoma, nodular melanoma, lentigo malignant melanoma,
acral lentiginous melanoma; kidney cancers such as but not limited
to renal cell carcinoma, adenocarcinoma, hypemephroma,
fibrosarcoma, transitional cell cancer (renal pelvis and/or
uterer); Wilms' tumor; bladder cancers such as but not limited to
transitional cell carcinoma, squamous cell cancer, adenocarcinoma,
carcinosarcoma. In addition, cancers include myxosarcoma,
osteogenic sarcoma, endotheliosarcoma, lymphangioendotheliosarcoma,
mesothelioma, synovioma, hemangioblastoma, epithelial carcinoma,
cystadenocarcinoma, bronchogenic carcinoma, sweat gland carcinoma,
sebaceous gland carcinoma, papillary carcinoma and papillary
adenocarcinomas.
[0182] In another aspect, the present invention provides for a
method of treating, preventing, or managing a CCR-5 expressing
neoplasm or a metastasis of the CCR5-expressing neoplasm in a
subject. In one embodiment, the a method of treating or managing a
CCR-5 expressing neoplasm or a metastasis of the CCR5-expressing
neoplasm in subject having the CCR5-expressing neoplasm or at risk
for developing metastasis of the CCR5-expressing neoplasm,
comprises administering to the subject a CCR5 modulator. In one
embodiment, the CCR5 modulator comprises a CCR5 antagonist. In one
embodiment, the CCR5 antagonist comprises
4,4-difluoro-[(1S)-3-[(1R,5S)-3-(3-methyl-5-propan-2-yl-1,2,4-triazol-4-y-
l)-8-azabicyclo[3.2.1]octan-8-yl]-1-phenylpropyl]cyclohexane-1-carboxamide
("Maraviroc"). In one embodiment, the CCR5 modulator comprises a
CCR5 antagonist. In one embodiment, the CCR5 antagonist comprises
(4,6-dimethylpyrimidin-5-yl)-[4-[(3S)-4-[(1R)-2-methoxy-1-[4-(trifluorome-
thyl)phenyl]ethyl]-3-methylpiperazin-1-yl]-4-methylpiperidin-1-yl]methanon-
e ("Vicriviroc"). In one embodiment, the CCR5 antagonist antagonist
is selected from the group consisting of
4,4-difluoro-N-[(1S)-3-[(1R,5S)-3-(3-methyl-5-propan-2-yl-1,2,4-triazol-4-
-yl)-8-azabicyclo[3.2.1]octan-8-yl]-1-phenylpropyl]cyclohexane-1-carboxami-
de ("Maraviroc") and
(4,6-dimethylpyrimidin-5-yl)-[4-[(3S)-4-[(1R)-2-methoxy-1-[4-(trifluorome-
thyl)phenyl]ethyl]-3-methylpiperazin-1-yl]-4-methylpiperidin-1-yl]methanon-
e ("Vicriviroc").
[0183] In one embodiment, sutiable CCR5 antagonist is selected from
the group consisting of: 4,4-difluoro-N-[(1S)-3-[(1R,
5S)-3-(3-methyl-5-propan-2-yl-1,2,4-triazol-4-yl)-8-azabicyclo[3.2.1]octa-
n-8-yl]-1-phenylpropyl]cyclohexane-1-carboxamide ("Maraviroc");
[0184]
N-(1S)-3-3-(3-Isopropyl-5-methyl-4H-1,2,4-triazol-4-yl)-exo-8-azabicyclo[-
3.2.1]oct-8-yl-1-phenylpropylcyclobutanecarboxamide;
[0185]
N-(1S)-3-3-(3-Isopropyl-5-methyl-4H-1,2,4-triazol-4-yl)-exo-8-azabi-
cyclo[3.2.1]oct-8-yl-1-phenylpropylcyclopentanecarboxamide;
[0186]
N-(1S)-3-3-(3-Isopropyl-5-methyl-4H-1,2,4-triazol-4-yl)-exo-8-azabi-
cyclo[3.2.1]oct-8-yl-1-phenylpropyl-4,4,4-trifluorobutanamide;
[0187]
N-(1S)-3-3-(3-Isopropyl-5-methyl-4H-1,2,4-triazol-4-yl)-exo-8-azabi-
cyclo[3.2.1]oct-8-yl-1-phenylpropyl-4,4-difluorocyclohexanecarboxamide;
[0188]
N-(1S)-3-3-(3-Isopropyl-5-methyl-4H-1,2,4-triazol-4-yl)-exo-8-azabi-
cyclo[3.2.1]oct-8-yl-1-(3-fluorophenyl)propyl-4,4-difluorocyclohexanecarbo-
xamide; and pharmaceutically acceptable salts or solvates
thereof.
[0189] In one embodiment, suitable CCR5 antagonist is selected from
the group consisting of:
[0190]
N-{3-[3-exo-(2-Methyl-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct--
8-yl]-1-phenylpropyl}cyclobutanecarboxamide;
[0191]
N-{(1S)-3-[3-exo-(2-Methyl-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1-
]oct-8-yl]-1-phenylpropyl}cyclobutanecarboxamide;
[0192]
N-{(1S)-3-[3-endo-(2-Methyl-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.-
1]oct-8-yl]-1-phenylpropyl}cyclobutanecarboxamide;
[0193]
N-{(1S)-3-[3-exo-(2-Methyl-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1-
]oct-8-yl]-1-phenylpropyl}tetrahydro-2H-pyran-4-carboxamide;
[0194]
1-Acetyl-N-{(1S)-3-[3-exo-(2-methyl-1H-benzimidazol-1-yl)-8-azabicy-
clo[3.2.1]oct-8-yl]-1-phenylpropyl}3-azetidine carboxamide;
[0195]
1-Hydroxy-N-{(1S)-3-[3-exo-(2-methyl-1H-benzimidazol-1-yl)-8-azabic-
yclo[3.2.1]oct-8-yl]-1-phenylpropyl}cyclopentanecarboxamide;
[0196]
2-Methyl-N-{(1S)-3-[3-exo-(2-methyl-1H-benzimidazol-1-yl)-8-azabicy-
clo[3.2.1]oct-8-yl]-1-phenylpropyl}cyclopropanecarboxamide;
[0197]
2-Cyclopropyl-N-{1S)-3-[3-exo-(2-methyl-1H-benzimidazol-1-yl)-8-aza-
bicyclo [3.2.1]oct-8-yl]-1-phenylpropyl}acetamide;
[0198]
N-{(1S)-3-[3-exo-(2-methyl-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1-
]oct-8-yl]-1-phenylpropyl}tetrahydro-3-furancarboxamide;
[0199]
3,3,3-Trifluoro-N-{(1S)-3-[3-exo-(2-methyl-1H-benzimidazol-1-yl)-8--
azabicyclo[3.2.1]oct-8-yl]-1-phenylpropyl}propanamide;
[0200]
N-{(1S)-3-[3-exo-(2-Methyl-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1-
]oct-8-yl]-1-phenylpropyl}tetrahydro-2-furancarboxamide;
[0201]
1-(Acetylamino)-N-{(1S)-3-[3-exo-(2-methyl-1H-benzimidazol-1-yl)-8--
azabicyclo[3.2.1]oct-8-yl]-1-phenylpropyl}cyclopentanecarboxamide;
[0202]
N-{(1S)-3-[3-exo-(2-Methyl-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1-
]oct-8-yl]-1-phenylpropyl}acetamide;
[0203]
1-Methoxy-N-{(1S)-3-[3-exo-(2-methyl-1H-benzimidazol-1-yl)-8-azabic-
yclo[3.2.1]oct-8-yl]-1-phenylpropyl}cyclopentanecarboxamide;
[0204]
1-Amino-N-{(1S)-3-[3-exo-(2-methyl-1H-benzimidazol-1-yl)-8-azabicyc-
lo[3.2.1]oct-8-yl]-1-phenylpropyl}cyclopentanecarboxamide;
[0205]
1-Methyl-N-{(1S)-3-[3-exo-(2-methyl-1H-benzimidazol-1-yl)-8-azabicy-
clo[3.2.1]oct-8-yl]-1-phenylpropyl}-2-oxo-4-pyrrolidinecarboxamide;
[0206]
1-Acetyl-N-{(1S)-3-[3-endo-(2-methyl-1H-benzimidazol-1-yl)-8-azabic-
yclo[3.2.1]oct-8-yl]-1-phenylpropyl}3-azetidinecarboxamide;
[0207]
N-{(1S)-3-[3-endo-(2-Methyl-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.-
1]oct-8-yl]-1-phenylpropyl}acetamide;
[0208]
N-{(1S)-3[6-(2-Methyl-1H-benzimidazol-1-yl)-3-azabicyclo[3.1.0]hex--
3-yl]-1-phenylpropyl}cyclobutanecarboxamide;
[0209]
2-Cyclopropyl-N-{(1S)-3-[3-exo-(3-{4-[(methylsulfonyl)amino]benzyl}-
-1,2,4-oxadiazol-5-yl)-8-azabicyclo[3.2.1]oct-8-yl]-1-phenylpropyl}acetami-
de;
[0210]
N-{(1S)-3-[7-exo-(2-Methyl-1H-benzimidazol-1-yl)-3-oxa-9-azabicyclo-
[3.3.1]non-9-yl]-1-phenylpropyl}cyclobutanecarboxamide;
[0211]
2-Cyclopropyl-N-{(1S)-3-[7-exo-(2-methyl-1H-benzimidazol-1-yl)-3-ox-
a-9-azabicyclo[3.3.1]non-9-yl]-1-phenylpropyl}acetamide;
[0212]
3,3,3-Trifluoro-N-{(1S)-3-[7-exo-(2-methyl-1H-benzimidazol-1-yl)-3--
oxa-9-azabicyclo[3.3.1]non-9-yl]-1-phenylpropyl}propanamide;
[0213]
N-{(1S)-3-[7-endo-(2-Methyl-1H-benzimidazol-1-yl)-3-oxa-9-azabicycl-
o[3.3.1]non-9-yl]-1-phenylpropyl}cyclobutanecarboxamide;
[0214]
2-Cyclopropyl-N-{(1S)-3-[7-endo-(2-methyl-1H-benzimidazol-1-yl)-3-o-
xa-9-azabicyclo[3.3.1]non-9-yl]-1-phenylpropyl}acetamide;
[0215]
N-{(1S)-3-[7-exo-(2-Methyl-1H-benzimidazol-1-yl)-3-thia-9-azabicycl-
o[3.3.1]non-9-yl]-1-phenylpropyl}cyclobutanecarboxamide;
[0216]
2-Cyclopropyl-N-[(1S)-3-(3-endo-{[2-(4-fluorophenyl)acetyl]amino}-8-
-azabicyclo[3.2.1]oct-8-yl)-1-phenylpropyl]acetamide;
[0217]
N-[(1S)-3-(3-{[3-endo-(4-Fluorophenyl)ppropanoyl]amino}-8-azabicycl-
o[3.2.1]oct-8-yl)-1-phenylpropyl]cyclobutanecarboxamide;
[0218]
N-[(1S)-3-(3-{[3-exo-(4-Fluorophenyl)prpropanoyl]amino}-8-azabicycl-
o[3.2.1]oct-8-yl)-1-phenylpropyl]cyclobutanecarboxamide;
[0219]
2-Cyclopropyl-N-[(1S)-3-(3-exo-{[2-(4-fluorophenyl)acetyl]amino}-8--
azabicyclo[3.2.1]oct-8-yl)-1-phenylpropyl]acetamide;
[0220]
N-{(1S)-3-[3-exo-(2-Methyl-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1-
]oct-8-yl]-1-phenylpropyl)}1-propionyl-3-azetidinecarboxamide;
[0221]
N-{(1S)-3-[3-endo-(2-Methyl-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.-
1]oct-8-yl]-1-phenylpropyl}tetrahydro-3-furancarboxamide;
[0222]
N-{(1S-3-[3-endo-(2-Methyl-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1-
]oct-8-yl]-1-phenylpropyl}tetrahydro-2H-pyran4-carboxamide;
[0223]
N-{(1S)-3-[3-endo-(2-Methyl-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.-
1]oct-8-yl]-1-phenylpropyl}tetrahydro-2-furancarboxamide;
[0224]
1-Acetyl-N-{(1S)-3-[3-endo-(1H-benzimidazol-1-yl)-8-azabicyclo[3.2.-
1]oct-8-yl]-1-phenylpropyl}-3-azetidinecarboxamide;
[0225]
N-{(1S)-3-[3-endo-(1H-Benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-8-y-
l]-1-phenylpropyl}-1-propionyl-3-azetidinecarboxamide;
[0226] Methyl
3-[({(1S)-3-[3-exo-(2-methyl-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-
-8-yl]-1-phenylpropyl}amino)carbonyl]-1-azetidinecarboxylate;
[0227]
N-{(1S)-3-[3-endo-(2-Methyl-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.-
1]oct-8-yl]-1-phenylpropyl}-1-propionyl-3-azetidinecarboxamide
1-Acetyl-N-{(1S-3-[3-endo-(2-methyl-1H-benzimidazol-1-yl)-8-azabicyclo[3.-
2.1]oct-8-yl]-1-phenylpropyl}-2-azetidinecarboxamide;
[0228]
2-[Acetyl(methyl)amino]-N-{(1S)-3-[3-endo-(2-methyl-1H-benzimidazol-
-1-yl)-8-azabicyclo[3.2.1]oct-8-yl]-1-phenylpropyl}acetamide;
[0229]
3-[Acetyl(methyl)amino]-N-{(1S)-3-[3-endo-(2-methyl-1H-benzimidazol-
-1-yl)-8-azabicyclo[3.2.1]oct-8-yl]-1-phenylpropyl}propanamide;
[0230]
2-Methoxy-N-{(1S)-3-[3-endo-(2-methyl-1H-benzimidazol-1-yl)-8-azabi-
cyclo[3.2.1]oct-8-yl]-1-phenylpropyl}acetamide;
[0231]
3-Methoxy-N-{(1S)-3-[3-endo-(2-methyl-1H-benzimidazol-1-yl)-8-azabi-
cyclo[3.2.1]oct-8-yl]-1-phenylpropyl}propanamide;
[0232]
1-Acetyl-N-{(1S)-3-[3-endo-(2-methyl-1H-benzimidazol-1-yl)-8-azabic-
yclo[3.2.1]oct-8-yl]-1-phenylpropyl}-3-pyrrolidinecarboxamide;
[0233]
1-Methyl-N-{(1S)-3-[3-endo-(2-methyl-1H-benzimidazol-1-yl)-8-azabic-
yclo[3.2.1]oct-8-yl]-1-phenylpropyl}-2-oxo-4-pyrrolidinecarboxamide;
[0234]
1-Acetyl-N-{(1S)-3[3-exo-(2-ethyl-1H-benzimidazol-1-yl)-8-azabicycl-
o[3.2.1]oct-8-yl]-1-phenylpropyl}-3-azetidinecarboxamide;
[0235]
N-{(1S)-3-[3-exo-(2-Ethyl-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]-
oct-8-yl]-1-phenylpropyl}-1-propionyl-3-azetidinecarboxamide;
[0236]
1-Acetyl-N-((1S)-1-phenyl-3-{3-exo[2-(trifluoromethyl)-1H-benzimida-
zol-1-yl]-8-azabicyclo[3.2.1]oct-8-yl}propyl)-3-azetidinecarboxamide;
[0237]
N-((1S)-1-Phenyl-3-{3-exo-[2-(trifluoromethyl)-1H-benzimidazol-1-yl-
]-8-azabicyclo[3.2.1]oct-8-yl}propyl)-1-propionyl-3-azetidinecarboxamide;
[0238]
N-((1S)-1-Phenyl-3-{3-exo[2-(trifluoromethyl)-1H-benzimidazol-1-yl]-
-8-azabicyclo[3.2.1]oct-8-yl}propyl)acetamide;
[0239]
2-[Acetyl(methyl)amino]-N-((1S)-1-phenyl-3-{3-exo-[2-(trifluorometh-
yl)-1H-benzimidazol-1-yl]-8-azabicyclo[3.2.1]oct-8-yl}propyl)acetamide;
[0240]
1-Acetyl-N-{(1S)-3[3-exo-(1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]-
oct-8-yl]-1-phenylpropyl}-3-azetidinecarboxamide;
[0241]
N(1S)-3[3-exo-(1H-Benzimidazol-1-yl)-8-azabicyclo[3.2.1]oct-8-yl]-1-
-propionyl-3-azetidinecarboxamide;
[0242]
1-acetyl-N(1S)-3-[3-exo-(5-fluoro-1H-benzimidazol-1-yl)-8-azabicycl-
o[3.2.1]oct-8-yl]-1-phenylpropyl)3-azetidinecarboxamide;
[0243]
N-{(1S)-3-[3-exo-(5-Fluoro-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1-
]oct-8-yl]-1-phenylpropyl}-1-propionyl-3-azetidinecarboxamide;
[0244]
1-Acetyl-N-{(1S)-3[3-exo-(5-fluoro-2-methyl-1H-benzimidazol-1-yl)-8-
-azabicyclo[3.2.1]oct-8-yl]-1-phenylpropyl}3-azetidinecarboxamide;
[0245]
N-{(1S)-3[3-exo-(5-Fluoro-2-methyl-1H-benzimidazol-1-yl)-8-azabicyc-
lo[3.2.1]oct-8-yl]-1-phenylpropyl}-1-propionyl-3-azetidinecarboxamide;
[0246]
N-{(1S)-3-[3-exo-(2-methyl-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1-
]oct-8-yl]-1-phenylpropyl}-3-azetidinecarboxamide;
[0247]
1-methyl-N-{(1S)-3-[3-exo-(2-methyl-1H-benzimidazol-1-yl)-8-azabicy-
clo[3.2.1]oct-8-yl]-1-phenylpropyl}-3-azetidinecarboxamide;
[0248]
(2S)-1-acetyl-N-{(1S)-3-[3-exo-(2-methyl-1H-benzimidazol-1-yl)-8-az-
abicyclo[3.2.1]oct-8-yl]-1-phenylpropyl}-2-azetidinecarboxamide;
[0249]
(2R)-1-acetyl-N-{(1S)-3-[3-exo-(2-methyl-1H-benzimidazol-1-yl)-8-az-
abicyclo[3.2.1]oct-8-yl]-1-phenylpropyl}-2-azetidinecarboxamide;
[0250]
2-[acetyl(methyl)amino]-N-{(1S)-3-[3-exo-(2-methyl-1H-benzimidazol--
1-yl)-8-azabicyclo[3.2.1]oct-8-yl]-1-phenylpropyl}acetamide;
[0251]
3-[acetyl(methyl)amino]-N-{(1S)-3-[3-exo-(2-methyl-1H-benzimidazol--
1-yl)-8-azabicyclo[3.2.1]oct-8-yl]-1-phenylpropyl}propanamide;
[0252]
1-acetyl-N-{(1S)-3-[3-exo-(2-methyl-1H-benzimidazol-1-yl)-8-azabicy-
clo[3.2.1]oct-8-yl]-1-phenylpropyl}-3-pyrrolidinecarboxamide;
[0253]
N-{(1S)-3-[3-exo-(2-methyl-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1-
]oct-8-yl]-1-phenylpropyl}-1-(trifluoromethyl)cyclopropanecarboxamide;
[0254]
2-methoxy-N-{(1S)-3-[3-exo-(2-methyl-1H-benzimidazol-1-yl)-8-azabic-
yclo[3.2.1]oct-8-yl]-1-phenylpropyl}acetamide;
[0255]
3-methoxy-N-{(1S)-3-[3-exo-(2-methyl-1H-benzimidazol-1-yl)-8-azabic-
yclo[3.2.1]oct-8-yl]-1-phenylpropyl}propanamide;
[0256]
1-Acetyl-N-{(1S)-3[3-exo-(4-fluoro-2-methyl-1H-benzimidazol-1-yl)-8-
-azabicyclo[3.2.1]oct-8-yl]-1-phenylpropyl}-3-azetidinecarboxamide;
[0257]
N-{(1S)-3[3-exo-(4-Fluoro-2-methyl-1H-benzimidazol-1-yl)-8-azabicyc-
lo[3.2.1]oct-8-yl]-1-phenylpropyl}-3-azetidinecarboxamide;
[0258]
1-Methyl-N-{(1S)-3[3-exo-(4-fluoro-2-methyl-1H-benzimidazol-1-yl)-8-
-azabicyclo[3.2.1]oct-8-yl]-1-phenylpropyl}-3-azetidinecarboxamide;
[0259]
N-{(1S)-3[3-exo-(4-Fluoro-2-methyl-1H-benzimidazol-1-yl)-8-azabicyc-
lo[3.2.1]oct-8-yl]-1-phenylpropyl}-1-propionyl-3-azetidinecarboxamide;
[0260]
2-Methoxy-N-{(1S)-3-[3-exo-(4-Fluoro-2-methyl-1H-benzimidazol-1-yl)-
-8-azabicyclo[3.2.1]oct-8-yl]-1-phenylpropyl}acetamide;
[0261]
N-{(1S)-3[3-exo-(4-Fluoro-2-Methyl-1H-benzimidazol-1-yl)-8-azabicyc-
lo[3.2.1]oct-8-yl]-1-phenylpropyl}acetamide;
[0262]
3-Methoxy-N-{(1S)-3-[3-exo-(4-fluoro-2-methyl-1H-benzimidazol-1-yl)-
-8-azabicyclo[3.2.1]oct-8-yl]-1-phenylpropyl}propanamide;
[0263]
2-[Acetyl(methyl)amino]-N-{(1S)-3-[3-exo-(4-fluoro-2-methyl-1H-benz-
imidazol-1-yl)-8-azabicyclo[3.2.1]oct-8-yl]-1-phenylpropyl}acetamide;
[0264]
3-[Acetyl(methyl)amino]-N-{(1S)-3-[3-exo-(4-fluoro-2-methyl-1H-benz-
imidazol-1-yl)-8-azabicyclo[3.2.1]oct-8-yl]-1
-phenylpropyl}propanamide;
[0265]
N-{(1S)-3[3-exo-(4-fluoro-2-methyl-1H-benzimidazol-1-yl)-8-azabicyc-
lo[3.2.1]oct-8-yl]-1-phenylpropyl}-3-methyl-3-oxetanecarboxamide;
[0266]
3-Ethyl-N-{(1S)-3[3-exo-(4-fluoro-2-methyl-1H-benzimidazol-1-yl)-8--
azabicyclo[3.2.1]oct-8-yl]-1-phenylpropyl}-3-oxetanecarboxamide;
[0267]
N-{(1S)-3[3-exo-(4-Fluoro-2-methyl-1H-benzimidazol-1-yl)-8-azabicyc-
lo[3.2.1]oct-8-yl]-1-phenylpropyl}-3-oxetanecarboxamide;
[0268]
3-Ethyl-N-{(1S)-3[3-exo-(4-fluoro-1H-benzimidazol-1-yl)-8-azabicycl-
o[3.2.1]oct-8-yl]-1-phenylpropyl}-3-oxetanecarboxamide;
[0269]
N-{(1S)-3-[3-exo-(4-Fluoro-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1-
]oct-8-yl]-1-phenylpropyl}-3-methyl-3-oxetanecarboxamide;
[0270]
N-{(1S)-3-[3-exo-(4-Fluoro-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1-
]oct-8-yl]-1-phenylpropyl}-3-oxetanecarboxamide;
[0271]
N-{(1S)-3-[3-exo-(4-Fluoro-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1-
]oct-8-yl]-1-phenylpropyl}-3-azetidinecarboxamide;
[0272]
N-{(1S)-3-[3-exo-(4-Fluoro-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1-
]oct-8-yl]-1-phenylpropyl}-1-methyl-3-azetidinecarboxamide;
[0273]
1-Acetyl-N-{(1S)-3[3-exo-(4-fluoro-1H-benzimidazol-1-yl)-8-azabicyc-
lo[3.2.1]oct-8-yl]-1-phenylpropyl}-3-azetidinecarboxamide;
[0274]
N-{(1S)-3-[3-exo-(4-Fluoro-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1-
]oct-8-yl]-1-phenylpropyl}-1-propionyl-3-azetidinecarboxamide;
[0275]
N-{(1S)-3-[3-exo-(4-Fluoro-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1-
]oct-8-yl]-1-phenylpropyl}-2-methoxyacetamide;
[0276]
N-{(1S)-3-[3-exo-(4-Fluoro-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1-
]oct-8-yl]-1-phenylpropyl}acetamide;
[0277]
N{-1S)-3[3-exo-(4-fluoro-1H-benzimidazol-1-yl)-8-azabicyclo[3.2.1]o-
ct-8-yl]-1-phenylpropyl}-3-methoxypropanamide;
[0278]
2-[Acetyl(methyl)amino]-N-{(1S)-3-[3-exo-(4-fluoro-1H-benzimidazol--
1-yl)-8-azabicyclo[3.2.1]oct-8-yl]-1-phenylpropyl}acetamide;
[0279]
3-[Acetyl(methyl)amino]-N-{(1S)-3-[3-exo-(4-fluoro-1H-benzimidazol--
1-yl)-8-azabicyclo[3.2.1]oct-8-yl]-1-phenylpropyl}propanamide; and
pharmaceutically acceptable salts thereof.
[0280] As disclosed herein, one aspect of the present invention
provides for a method of treating, preventing, or managing a CCR-5
expressing neoplasm or a metastasis of the CCR5-expressing neoplasm
in a subject by administering to the subject a CCR5 receptor
antagonist. In one embodiment, CCR5 receptor anatagonists are
administered in the form of pharmaceutical formulations or dosage
forms that include the CCR5 receptor antagonists. In one
embodiment, the CCR5 receptor antagonists are used in the form of
acids, esters, or other suitable chemical derivatives. In one
embodiment, the CCR5 receptor antagonists are in the form of
pharmaceutically acceptable salts derived from various organic and
inorganic acids and bases in accordance with procedures well known
in the art. In one embodiment, the expression "pharmaceutically
acceptable salt" as used herein is intended to mean an active
ingredient comprising a CCR5 receptor antagonist utilized in the
form of a salt thereof, especially where the salt form confers on
the CCR5 receptor antagonist improved pharmacokinetic properties as
compared to the free form of the CCR5 receptor antagonist or other
previously disclosed salt form. In one embodiment, a
pharmaceutically acceptable salt form of the CCR5 receptor
antagonist may also initially confer a desirable pharmacokinetic
property on the CCR5 receptor antagonist which it did not
previously possess, and may even positively affect the
pharmacodynamics of the CCR5 receptor antagonist with respect to
its therapeutic activity in the body. In one embodiment, the
pharmacokinetic properties of the CCR5 receptor antagonist which
may be favorably affected include, e.g., the manner in which the
CCR5 receptor antagonist is transported across cell membranes,
which in turn may directly and positively affect the absorption,
distribution, biotransformation or excretion of the CCR5 receptor
antagonist.
[0281] While the route of administration of the pharmaceutical
composition is important and various anatomical, physiological and
pathological factors can critically affect bioavailability, the
solubility of the CCR5 receptor antagonist is usually dependent
upon the character of the particular salt form thereof which it
utilized. Further, an aqueous solution may provide the most rapid
absorption of an active ingredient into the body of a patient being
treated, while lipid solutions and suspensions, as well as solid
dosage forms, may result in less rapid absorption. Oral ingestion
of the CCR5 receptor antagonist is the most preferred route of
administration for reasons of safety, convenience, and economy, but
absorption of such an oral dosage form can be adversely affected by
physical characteristics such as polarity, emesis caused by
irritation of the gastrointestinal mucosa, destruction by digestive
enzymes and low pH, irregular absorption or propulsion in the
presence of food or other drugs, and metabolism by enzymes of the
mucosa, the intestinal flora, or the liver. Formulation of the CCR5
receptor antagonist into different pharmaceutically acceptable salt
forms may be effective in overcoming or alleviating one or more of
the above-recited problems encountered with absorption of oral
dosage forms. Well-known pharmaceutically acceptable salts include,
but are not limited to acetate, adipate, alginate, aspartate,
benzoate, benzenesulfonate, besylate, bisulfate, butyrate, citrate,
camphorate, camphorsulfonate, cyclopentanepropionate, digluconate,
dodecysulfate, ethanesulfonate, fumarate, glucoheptanoate,
gluconate, glycerophosphate, hemisuccinate, hemisulfate,
heptanoate, hexanoate, hippurate, hydrochloride, hydrobromide,
hydroiodide, 2-hydroxyethanesulfonate, isethionate, lactate,
lactobionate, maleate, mandelate, methanesulfonate,
2-naphthalenesulfonate, nicotinate, nitrate, oxalate, oleate,
pamoate, pectinate, persulfate, 3-phenylpropionate, phosphonate,
picrate, pivalate, propionate, salicylate, sodium phosphate,
stearate, succinate, sulfate, sulfosalicylate, tartrate,
thiocyanate, thiomalate, tosylate, and undecanoate.
[0282] Base salts of the compounds of suitable CCR5 receptor
antagonists include, but are not limited to ammonium salts; alkali
metal salts such as sodium and potassium; alkaline earth metal
salts such as calcium and magnesium; salts with organic bases such
as dicyclohexylamine, meglumine, N-methyl-D-glucamine,
trishydroxymethyl)methylamine (tromethamine), and salts with amino
acids such as arginine, lysine, etc. Compounds of the present
invention which comprise basic nitrogen-containing groups may be
quaternized with such agents as (C.sub.1C.sub.4) alkyl halides,
e.g., methyl, ethyl, iso-propyl and tert-butyl chlorides, bromides
and iodides; di(C.sub.1-C.sub.4) alkyl sulfate, e.g., dimethyl,
diethyl and diamyl sulfates; (C.sub.10-C.sub.18) alkyl halides,
e.g., decyl, dodecyl, lauryl, myristyl and stearyl chlorides,
bromides and iodides; and aryl-(C.sub.1-C.sub.4) alkyl halides,
e.g., benzyl chloride and phenethyl bromide. Such salts permit the
preparation of both water-soluble and oil-soluble compounds of the
present invention.
[0283] Among the above-recited pharmaceutical salts those which are
preferred include, but are not limited to acetate, besylate,
citrate, fumarate, gluconate, hemisuccinate, hippurate,
hydrochloride, hydrobromide, isethionate, mandelate, meglumine,
nitrate, oleate, phosphonate, pivalate, sodium phosphate, stearate,
sulfate, sulfosalicylate, tartrate, thiomalate, tosylate, and
tromethamine.
[0284] Multiple salts forms are included within the scope of the
present invention where a CCR5 receptor antagonist ontains more
than one group capable of forming such pharmaceutically acceptable
salts. Examples of typical multiple salt forms include, but are not
limited to bitartrate, diacetate, difumarate, dimeglumine,
diphosphate, disodium, and trihydrochloride. Suitable CCR5 receptor
antagonists can be administered alone but will generally be
administered in admixture with one or more suitable pharmaceutical
excipients, diluents or carriers selected with regard to the
intended route of administration and standard pharmaceutical
practice.
[0285] For example, a CCR5 receptor antagonist can be administered
orally or sublingually in the form of tablets, capsules, ovules,
elixirs, solutions or suspensions, which may contain flavouring or
colouring agents, for immediate or controlled release applications.
Such tablets may contain excipients such as microcrystalline
cellulose, lactose, sodium citrate, calcium carbonate, dicalcium
phosphate and glycine, disintegrants such as starch (preferably
corn, potato or tapioca starch), alginic acid and certain complex
silicates, and granulation binders such as polyvinylpyrrolidone,
sucrose, gelatin and acacia. Additionally, lubricating agents such
as magnesium stearate, sodium lauryl sulfate and talc may be
included. Solid compositions of a similar type may also be employed
as fillers in gelatin capsules. Preferred excipients in this regard
include lactose or milk sugar as well as high molecular weight
polyethylene glycols. For aqueous suspensions and/or elixirs, the
CCRS receptor antagonist may be combined with various sweetening or
flavouring agents, colouring matter or dyes, with emulsifying
and/or suspending agents and with diluents such as water, ethanol,
propylene glycol and glycerin, and combinations thereof
[0286] Suitable CCR5 receptor antagonists can also be injected
parenterally, for example, intravenously, intraperitoneally,
intrathecally, intraventricularly, intrastemally, intracranially,
intramuscularly or subcutaneously, or they may be administered by
infusion techniques. They are best used in the form of a sterile
aqueous solution which may contain other substances, for example,
enough salts or glucose to make the solution isotonic with blood.
The aqueous solutions should be suitably buffered (preferably to a
pH of from 3 to 9), if necessary. The preparation of suitable
parenteral formulations under sterile conditions is readily
accomplished by standard pharmaceutical techniques well known to
those skilled in the art.
[0287] For oral and parenteral administration to human subjects,
the daily dosage level of the CCR5 receptor antagonist will usually
be from 1 microgram/kg to 25 mg/kg (in single or divided doses).
Thus tablets or capsules of the CCR5 receptor antagonist may
contain from 0.05 mg to 1.0 g of active compound for administration
singly or two or more at a time, as appropriate. The physician in
any event will determine the actual dosage which will be most
suitable for any individual patient and it will vary with the age,
weight and response of the particular patient. The above dosages
are exemplary of the average case. There can, of course, be
individual instances where higher or lower dosage ranges are
merited and such are within the scope of this invention. Suitable
CCR5 receptor antagonists can also be administered intranasally or
by inhalation and are conveniently delivered in the form of a dry
powder inhaler or an aerosol spray presentation from a pressurised
container or a nebuliser with the use of a suitable propellant, eg
dichlorodifluoromethane, trichlorofluoromethane,
dichlorotetrafluoroethane, a hydrofluoroalkane such as
1,1,1,2-tetrafluorethane (HFA 134a), carbon dioxide or other
suitable gas. In the case of a pressurised aerosol, the dosage unit
may be determined by providing a valve to deliver a metered amount.
The pressurized container or nebulizer may contain a solution or
suspension of the active compound, eg using a mixture of ethanol
and the propellant as the solvent, which may additional contain a
lubricant, e.g. sorbitan trioleate. Capsules and cartridges (made,
for example, from gelatin) for use in an inhaler or insufflator may
be formulated to contain a powder mix of a CCR5 receptor antagonist
and a suitable powder base such as lactose or starch.
[0288] Aerosol or dry powder formulations are preferably arranged
so that each metered dose or "puff" contains from 20 .mu.g to 20 mg
of a CCR5 receptor antagonist for delivery to the subject. The
overall daily dose with an aerosol will be in the range of from 20
.mu.g to 20 mg which may be administered in a single dose or, more
usually, in divided doses throughout the day. Alternatively,
Suitable CCR5 receptor antagonists can be administered in the form
of a suppository or pessary, or they may be applied topically in
the form of a lotion, solution, cream, ointment or dusting powder.
The CCR5 receptor antagonist may also be transdermally administered
by the use of a skin patch. They may also be administered by the
ocular route, particularly for treating neurological disorders of
the eye.
[0289] For ophthalmic use, the compounds can be formulated as
micronised suspensions in isotonic, pH adjusted, sterile saline,
or, preferably, as solutions in isotonic, pH adjusted, sterile
saline, optionally in combination with a preservative such as
benzylalkonium chloride. Alternatively, they may be formulated in
an ointment such as petrolatum.
[0290] For application topically to the skin, the compounds of the
formula (I) can be formulated as a suitable ointment containing the
active compound suspended or dissolved in, for example, a mixture
with one or more of the following: mineral oil, liquid petrolatum,
white petrolatum, propylene glycol, polyoxyethylene
polyoxypropylene compound, emulsifying wax and water.
Alternatively, they can be formulated as a suitable lotion or
cream, suspended or dissolved in, for example, a mixture of one or
more of the following: mineral oil, sorbitan monostearate, a
polyethylene glycol, liquid paraffin, polysorbate 60, cetyl esters
wax, cetearyl alcohol, 2-octyldodecanol, benyl alcohol and
water.
[0291] In some embodiments, the compounds described herein can
modulate CCR5 chemokine receptor activity and consequent or
associated pathogenic processes subsequently mediated by the CCR5
receptor and its ligands. The expression "modulate CCR5 chemokine
receptor activity" as used herein is intended to refer to
manipulation of the basic physiological processes and agencies
which involve CCR5 chemokine receptors and their ligands. Included
within the scope of this intended meaning are all types and
subtypes of CCR5 receptors, in whatever tissues of a particular
patient they are found, and in or on whatever components of the
cells comprising those tissues they may be located. Most commonly,
CCR5 receptors are situated on the cell membranes of particular
cell types such as monocytes. CCR5 receptors participate in and
define, along with various endogenous ligands to which they are
naturally bound, signaling pathways which control important
cellular and tissue functions by means of the influence which they
exert on the movement of agents such as the chemokines, into and
out of those cells and tissues.
[0292] The dosage and dose rate of the compounds of Formula (I)
effective for treating or preventing diseases and conditions in a
patient which are mediated by or associated with modulation of CCR5
chemokine receptor activity as described herein, as well as for
favorably affecting the outcome thereof in the patient, in
accordance with the methods of treatment of the present invention
comprising administering to the patient a therapeutically effective
amount of a CCR5 receptor antagonist, will depend on a variety of
factors such as the nature of the CCR5 receptor antagonist, the
size of the patient, the goal of the treatment, the nature of the
pathology being treated, the specific pharmaceutical composition
used, the concurrent treatments that the patient may be subject to,
and the observations and conclusions of the treating physician.
[0293] Generally, however, the effective therapeutic dose of a
suitable CCR5 receptor antagonist which will be administered to a
subject will be between about 10 .mu.g (0.01 mg)/kg and about 60.0
mg/kg of body weight per day, preferably between about 100 .mu.g
(0.1 mg)/kg and about 10 mg/kg of body weight per day, more
preferably between about 1.0 mg/kg and about 6.0 mg/kg of body
weight per day, and most preferably between about 2.0 mg/kg and
about 4.0 mg/kg of body weight per day of the CCR5 receptor
antagonist.
[0294] Included within the scope of the present invention are
embodiments comprising coadministration of, and compositions which
contain, in addition to a CCR5 receptor antagonist as active
ingredient, additional therapeutic agents and active ingredients.
Such multiple drug regimens, often referred to as combination
therapy, may be used in the treatment and prevention of any of the
diseases or conditions mediated by or associated with CCR5
chemokine receptor modulation, particularly cancer metastasis. The
use of such combinations of therapeutic agents is especially
pertinent with respect to the treatment, prevention, or management
of cancer metastasis within a subject in need of treatment cancer,
prevention cancer, or management of risk of cancer metastasis.
[0295] Exemplary CCR5 receptor antagonists may be administered in
accordance with a regimen of 1 to 4 times per day, preferably once
or twice per day. The specific dose level and frequency of dosage
for any particular patient may be varied and will depend upon a
variety of factors including the activity of the specific compound
employed, the metabolic stability and length of action of that
compound, the age, body weight, general health, sex, diet, mode and
time of administration, rate of excretion, drug combination, the
severity of the particular condition, and the host undergoing.
[0296] As disclosed in U.S. Pat. No. 6,667,314 by Perros et al.
CCR5 antagonists can be administered orally, buccally or
sublingually in the form of tablets, capsules, multi-particulates,
gels, films, ovules, elixirs, solutions or suspensions, which may
contain flavouring or colouring agents, for immediate-, delayed-,
modified-, sustained-, pulsed- or controlled-release applications.
The CCR5 receptor antagonists may also be administered as
fast-dispersing or fast-dissolving dosage forms or in the form of a
high energy dispersion or as coated particles. Suitable
formulations of the compounds of the CCR5 receptor antagonist may
be in coated or uncoated form, as desired. Such solid
pharmaceutical compositions, for example, tablets, may contain
excipients such as microcrystalline cellulose, lactose, sodium
citrate, calcium carbonate, dibasic calcium phosphate, glycine and
starch (preferably corn, potato or tapioca starch), disintegrants
such as sodium starch glycollate, croscarmellose sodium and certain
complex silicates, and granulation binders such as
polyvinylpyrrolidone, hydroxypropylmethylcellulose (HPMC),
hydroxypropylcellulose (HPC), sucrose, gelatin and acacia.
Additionally, lubricating agents such as magnesium stearate,
stearic acid, glyceryl behenate and talc may be included.
[0297] "Metastasis", as used herein, is defined as the transfer of
malignant tumor cells, or neoplasm, via the circulatory or
lymphatic systems or via natural body cavities, usually from the
primary site of neoplasia to a distant site in the body, and
subsequent development of secondary tumors or colonies in the new
location. In some exemplary embodiments of the methods of the
present invention, metastasis comprises a tumor metastasis in or
more organs selected from the group consisting of liver, brain,
bladder, lung, adrenal gland, kidney, bone, skin or pancreas or
control kidney and combinations thereof.
[0298] In another aspect, the present invention provides a method
of identifying a compound that reduces or prevents or treats cancer
metastasis. In one embodiment, the method identifies a candidate
compound that selectively interferes with proliferation or
viability of neoplastic cells that over express CCR5 and/or over
express at least one of CCR5 receptor ligand. In one embodiment,
the method identifies a candidate compound that selectively blocks
activity of CCR5 and/or at least one of CCR5 receptor ligand in
neoplastic cells that over express CCR5 and/or over express at
least one of CCR5 receptor ligand. In one exemplary embodiment, the
method for identifying a candidate compound that reduces or
prevents or treats cancer metastasis in neoplastic cells that over
express CCR5 and/or over express at least one of CCR5 receptor
ligand, comprises (a) contacting a one or more neoplastic cells
that over express CCR5 and/or over express at least one of CCR5
receptor ligand with one or more candidate compounds; and (b)
detecting activity of and/or proliferation or viability of the one
or more neoplastic cells that over express CCR5 and/or over express
at least one of CCR5 receptor ligand, wherein decreased activity
and/or decreased proliferation and/or decreased viability of the
one or more neoplastic cells (relative to as compared to a control
sample) identifies the candidate compound as a compound that that
selectively reduces or prevents or treats cancer metastasis in
neoplastic cells that over express CCR5 and/or over express at
least one of CCR5 receptor ligand. In one embodiment, if
proliferation of the one or more neoplastic cells is
decreased/depressed compared to untreated (control) neoplastic
cells that over express CCR5 and/or over express at least one of
CCR5 receptor ligand, the candidate compound is identified as a
compound that selectively reduces or prevents or treats metastasis
of a neoplasm cells that over express CCR5 and/or over express at
least one of CCR5 receptor ligand. In one embodiment, if viability
of the one or more neoplastic cells is decreased/depressed compared
to untreated (control) neoplastic cells that over express CCR5
and/or over express at least one of CCR5 receptor ligand, the
candidate compound is identified as a compound that that
selectively reduces or prevents or treats metastasis of a neoplasm
cells that over express CCR5 and/or over express at least one of
CCR5 receptor ligand. In one embodiment, the one or more CCR5
receptor ligands comprise CCLS. In one embodiment, the one or more
CCR5 receptor ligands comprise CCLS. In one embodiment, the one or
more CCR5 receptor ligands comprise CCL7.
[0299] For the study disclosed herein, CCL5 and CCR5 expression in
human breast cancer cell lines were investigated as well as the
effect of CCR5 antagonists in vitro and in vivo. An interrogation
was conducted using a microarray dataset to evaluate CCR5 and CCL5
expression in the context of 2,254 patient breast cancer samples.
Samples in the dataset were assigned to five breast cancer
subtypes, including luminal A, luminal B, normal-like, basal and
HER-2 overexpressing disease. The analysis revealed an increased
expression of CCL5 and CCR5 in patients with basal and HER-2
subtypes. 58% the cancer samples indicated a positive CCR5 and CCL5
signature. It was found that oncogenes turn on the CCR5 receptor in
normal breast cells as they became transformed into cancer cells.
Metastasis those cells was also found to be regulated by CCR5.
[0300] To evaluate the functional relevance of CCR5 in cellular
migration and invasion in vitro, the drugs were tested in 3-D
invasion assays with two different cell lines. It was found that
both antagonists inhibited breast cancer cell invasiveness.
[0301] To evaluate the functional relevance of CCR5 in cellular
migration and invasion in vivo, mice were injected with the
antagonists and invasiveness of the basal breast cancer cells to
other tissue, i.e. lung, was tracked with bioluminescence imaging.
It was found that mice treated with the drug showed a more than 90%
reduction in both the number and size of pulmonary metastases
compared to untreated mice. This and the other preclinical studies
provide the rational basis for studying the use of CCR5 antagonists
as new treatments to block the dissemination of basal breast
cancers. These findings may also have implications for other
cancers where CCR5 promotes metastasis, such as prostate and
gastric.
[0302] Referring to the drawings in detail, wherein like reference
numerals indicate like elements throughout, there are exemplary
embodiments of the present invention shown in FIGS. 1A-14D.
[0303] Referring to FIG. 1A, there is shown a heatmap of the
expression of CCL5 and its receptor CCR5 in samples from patients
with breast cancer divided by molecular subtypes of breast cancer,
namely luminal A, luminal B, basal, normal-like, and Her-2, based
on their gene expression pattern. The heatmap shows that relative
abundances of CCL5 and CCR5 are increased (over expressed) in
patients with the basal and HER-2 breast cancer subtypes. The
heatmap also shows that CCL5 and CCR5 are over expressed in the
Her-2 breast cancer subtype.
[0304] Referring to FIG. 1B, there is shown fluorescence-activated
cell sorting (FACS) scatter plots and correlation analysis (Student
t test) of the expression of CCL5 and CCR5 among the breast cancer
molecular subtypes whose expression of CCL5 and its receptor CCR5
is shown FIG. 1A. Consistent with the expression of CCL5 and its
receptor CCR5 observed in A, the scatter plots show CCL5 and CCR5
are over expressed in patients with the basal and HER-2 breast
cancer subtypes.
[0305] Referring to FIG. 1C, there is shown quantification of the
proportions of the breast cancer samples overexpressing CCL5 and
CCR5 (fraction of the bars representing upper right quadrants of
the scatter plots shown in Figure B) displayed in FIG. 1B. The
number of samples in each subtype is indicated at the top of the
bar.
[0306] Referring to FIG. 1D, there is shown metastasis-free
Kaplan-Meier plots and log-rank analysis for the different genetic
subtypes in the analyzed database described in Materials and
Methods section of the present disclosure. The metastasis-free
Kaplan-Meier plots show that patients with the basal or HER-2
subtypes of breast cancer display increased probability to develop
metastasis.
[0307] FIGS. 2A-2H, show that human breast cancer cell lines that
express CCR5 respond to CCL5. In FIG. 1A, flow cytometric
histograms of the CCR5 expression in MDA-MB-231 breast cancer cells
identified a subpopulation of CCR5+ cells. In FIG. 2B, induction of
calcium signaling in cells loaded with Fluo-4-AM before the
sequential addition of CCL5 (60 .mu.g/mL) and FBS (5%) is shown. A
fraction of cells responded to CCL5 (closed arrowheads in the
middle of micrographs) whereas the rest did not (open arrowheads).
The average changes in fluorescence on 5 responsive (green line)
and 5 nonresponsive (red line) cells are represented in the far
right graphs. Data shown are representative of 3 to 5 independent
experiments for each cell line (Bar, 100 .mu.m). In FIG. 2C, 3D
invasion into collagen gels by breast cancer cell lines, using CCL5
(15 .mu.g/mL) as chemoattractant is shown. Figure D shows mean
distances of invasion.+-.SEM from 3 independent experiments whose
3D invasion are shown in FIG. 2C. FIG. 2E shows 3D invasion assays,
and their corresponding quantification shown in FIG. 2F
(mean.+-.SEM, n=3), for MCF-10A cells and MCF-10A-NeuT, -Ras, and
-Src derivatives showing that CCL5 -induced invasion is activated
by oncogenic transformation. FIG. 2G shows CCR5+ cells display
increased invasiveness. CCR5+ and CCR5- subpopulations from SUM-159
cell line were isolated by FACS and invasion into collagen gels was
evaluated using FBS as chemoattractant. Quantification of FACS and
invasion into collagen gels experiments of the samples in FIG. 2G
is shown in FIG. 2H as mean.+-.SEM of 2 independent experiments.
Statistical analysis was conducted using the Student t test.
[0308] FIGS. 3A-3D show that CCR5 antagonists block CCL5 -induced
calcium signaling. In FIG. 3A, there is shown intensity versus time
analysis of Fluo-4 AM-loaded MDA-MB-231 cells treated with the CCR5
antagonists maraviroc or vicriviroc (100 nmol/L) for 30 minutes
before the addition of CCL5 (60 .mu.g/mL). Micrographs illustrate
the axis (x-x') of the pseudoline scan plot. Those axes were used
to construct the adjacent intensity versus time plots. In FIG. 3B,
there is shown comparison of the fraction of cells with increased
fluorescence intensity upon addition of
[0309] CCL5. In FIG. 3C, CCL5 -induced calcium signaling was also
blocked by CCR5 antagonists in Hs578T cells. The corresponding
quantification is shown in FIG. 3D. The data in FIGS. 3B and 3D are
mean.+-.SEM of 3 to 4 independent experiments. Statistical analysis
was conducted using the Student t test.
[0310] FIGS. 4A-4D show that CCR5 antagonists block FBS-induced
breast cancer cell invasion. In FIG. 4A, there is shown 3D
reconstruction of FBS-induced invasion into collagen gels by Hs578T
breast cancer cells in presence of CCR5 antagonists (100 nmol/L).
In FIG. 3C there is shown 3D reconstruction of FBS-induced invasion
into collagen gels by SUM-159 breast cancer cells in presence of
CCR5 antagonists (100 nmol/L). The corresponding quantifications
(mean.+-.SEM, n=3) and analysis (Bonferroni t test) are displayed
in FIGS. 4B and 4D.
[0311] Referring to FIGS. 5A-5F, the data presented show that the
CCR5 antagonist maraviroc inhibits lung metastases in vivo. In FIG.
5A, MDA-MB-231 cells transduced with Luc2-eGFP fusion protein were
injected into the tail vein of NOD/SCID mice and the in vivo
bioluminescent signal was quantified weekly. Representative in vivo
images of vehicle- or maraviroc-treated (8 mg/kg every 12 hours)
mice are shown in FIG. 5A. In FIG. 5B, there is shown
quantification (mean.+-.SEM, n=6) of BLI in the control (red line)
and treated groups (blue line. Statistical comparison (*, P =0.048)
was carried out using Student t test with Welch correction for
heterogeneous variances. As shown in FIG. 5C, the presence of
pulmonary tumors and the differences between treatments were
corroborated by ex vivo imaging (left) and India ink staining
(right). As shown in FIG. 5D, the fraction of mice with metastatic
tumors was significantly larger in the control group (P<0.0001,
Fisher exact test). FIG. 5E shows histologic analysis (hematoxylin
and eosin staining, .times.100) and the corresponding
quantification (shown in FIG. 5F) of the area covered by metastatic
tumors in lung slides. Tumor area was quantified with the Nikon
Elements BR 3.0 software analyzing at low magnification (.times.40)
2 random fields of 2 different histologic sections (separated 600
.mu.m from each other) per mouse. Statistical analysis was
conducted using the Student t test with Welch correction for
heterogeneous variances (n=33 and 12 for control and treated
groups, respectively).
[0312] Referring to FIGS. 6A-6H, it is shown that maraviroc reduces
lung colonization but does not modify cell proliferation. In FIG.
6A, there is shown the effect of CCR5 antagonist on breast cancer
cell viability. MDA-MB-231 cells were exposed to increasing
concentrations of maraviroc (inverted triangles) or vicriviroc
(squares) for 48 hours and the cell viability was evaluated by MTT
assay. Graph is from a representative experiment carried out by
sextuplicate. No statistical differences were found (ANOVA) in 3
independent experiments. In FIG. 6B, there is shown CCR5 expression
in MDA-MB-231 cells stably transfected with pcDNA3.1+/Zeo+
(MDA.Vector) or human CCR5 cloned into pCDNA3+Zeo+ (MDA.CCRS). In
FIG. 6C, comparison of in vitro proliferation rates of MDA.Vector
versus MDA.CCR5 showed no differences (ANOVA). Representative
experiment from 2 carried out by sextuplicate. In FIG. 6D, to
evaluate the in vivo effect of maraviroc on growth of established
metastasis, treatment of mice was initiated 10 days after injection
of MDA.pFULG cells as illustrated. In FIG. 6E, quantification (mean
.+-.SEM, n=5) of in vivo BLI in the control (red/squares) and
treated groups (blue/triangles) showed no differences in the growth
rate. There is no effect of maraviroc of the growth rate of the
tumors once established in the lungs. Thus, the reduction in tumor
mass in the lungs is due to the inhibition of "homing" or spread of
tumors to the lung. FIG. 6F shows schema of the experimental design
used to evaluate CCR5 role in lung colonization. FIG. 6G shows
representative confocal images and quantification (FIG. 6H) of the
number of eGFP+ cells in lungs 24 hours after injection of
MDA.pFULG cells. Cells expressing eGFP were counted in 3 random
fields of 2 different histologic sections (separated 700 .mu.m from
each other) per mouse (n=5 mice per group). Statistical analysis
was conducted using Student t test. Bar in micrographs, 100
.mu.m.
[0313] Reference is now made to FIGS. 7A-7D. To determine the gene
expression signaling pathway associated with enrichment of CCR5 and
CCL5, GSEA analysis using KEGG and GO was conducted of the tumor
samples discussed herein. FIG. 7A shows the GSEA analysis. These
studies showed enrichment for gene expression of pathways including
lymphocyte activation, Janus-activated kinase (JAK)-STAT signaling,
and Toll-like receptor activation. The receptors for CCL5 include
CCR1 and CCR3. Increased expression of CCL5 associated with
increased CCR1, but not CCR3, in the basal and HER-2 genetic tumor
type (FIGS. 7B and 7C). In ER-negative patients receiving
chemotherapy, there was an insignificant trend toward reduced
metastasis-free survival and relapse-free survival in the increased
CCR5 population, compared with the population with reduced CCR5
expression (FIG. 7D).
[0314] In FIGS. 8A-8D, a comparison of expression levels for CCL5
versus CCR5, CCR1, and CCR3, comparing normal breast with breast
cancer showed increased correlation between receptor and ligand
expression levels in tumors compared with healthy breast tissue.
FIG. 8A illustrates heatmap of CCL5 expression and its receptors
CCR5, CCR1 and CCR3 in healthy population. The healthy population
was previously described as part of a collection of 2550 breast
cancers (Ertel, A., Dean, J. L., Rui, H., Liu, C., Witkiewicz, A.
K., Knudsen, K. E., and Knudsen, E. S. RB-pathway disruption in
breast cancer: differential association with disease subtypes,
disease-specific prognosis and therapeutic response. Cell Cycle, 9:
4153-4163, 2010.).
[0315] Referring to FIGS. 9A-9D, three human breast cancer cell
lines with a basal phenotype and molecular signature: MDA-MB-231,
Hs578T, and SUM-159 (34-37) were used as models in the studies.
Analysis of CCR5 expression by FACS showed that a small
subpopulation of cells were positive for the receptor in all 3 cell
lines (FIG. 2A for MDA-MB-231 and FIG. 9A and FIG. 9C for Hs578T
and SUM-159). Because CCR5 activation induces calcium flux (38,
39), the activation of calcium signaling was assessed by CCL5.
Addition of CCL5 to the cultures induced immediate calcium fluxes
in a subpopulation of cells (FIG. 2B for MDA-MB-231 and FIGS. 9B
and 9D for Hs578T and SUM-159), providing evidence that CCR5 is
functional in basal breast cancer cells. As a positive control, the
same cultures were exposed to 5% FBS (40). Calcium flux, assessed
by relative fluorescence intensity, increased in more than 95% of
the cells after FBS addition (FIG. 2B and FIGS. 9B and 9D). To
further distinguish CCL5 -dependent signaling, SUM159 cells were
stably transduced with a CCR5 expression vector and the Ca.sup.+2
response to CCL5 versus FBS was conducted (FIGS. 10 C and 10D vs.
FIGS. 10E and 10F). CCR5 induced Ca.sup.+2 signaling in the CCR5
-overexpressing cells, whereas both lines responded similarly to
FBS induced Ca.sup.+2 activation (FIGS. 10A-10F).
[0316] In view of the finding that CCR5 inhibition by CCR5
antagonists reduced calcium signaling and cell invasion, the in
vivo effect of maraviroc on lung metastasis was determined.
MDA-MB-231 cells were transduced within the Luc2-eGFP lentiviral
vector (MDA.pFULG cells) in an experimental metastasis model. The
Luc2 gene is a codon-optimized version of Luc and cells expressing
this reporter were 10 to 100 times brighter than the unmodified Luc
gene (30). After injection of MDA.pFULG cells into the tail vein of
mice, noninvasive BLI enabled the early detection of breast cancer
metastasis (41). Weekly BLI was conducted for 5 weeks and the
radiance antemortem was used as a surrogate measurement of tumor
burden. Mice treated with maraviroc (8 mg/kg twice daily) showed a
significant reduction in both the number and the size of pulmonary
metastases compared with vehicle-treated mice (FIGS. 5A and 5B,
FIGS. 11A and 11B). To avoid the possibility that metastases were
missed because of inappropriate imaging, ex vivo imaging, India ink
staining (FIG. 5C), and histology (FIG. 5E) of the lungs were
conducted. Histologic analysis corroborated that tumor burden
corresponds to bioluminescence, as previously shown (30).
Metastatic tumors were still detectable in 50% of the
maraviroc-treated mice, but their mean size was reduced by 65%
(FIGS. 5D and 5F). Interestingly, analysis of CCR5 expression in
lungs from control mice showed an 8-fold enrichment of the
CCRS+fraction (FIG. 12). Collectively, these results provide
evidence that CCR5 antagonists reduce breast cancer metastasis in
vivo.
[0317] Given the aggressive clinical behavior of basal breast
cancer and the lack of targeted therapies for it, the importance of
the CCLS/CCR5 was evaluated in invasion and metastasis in the human
breast cancer cell lines MDA-MB-231, Hs578T and SUM-159. These cell
lines reflect the clinicopathologic features of the basal subtype
of breast cancer (including the lack of HER-2, ER, and progesterone
receptor), a basal-like molecular signature, the activation of
specific signaling pathways (e.g., hypoxic or EGF receptor
responses) and over expression of epithelial-mesenchymal transition
proteins (FN, VIM, ad matrix metalloproteinase 2; refs. 34-37).
Only a small fraction of cells within the cell lines used in this
study expressed CCR5 as evaluated by FACS analysis. The studies
confirmed the expression of CCR5 in MDA-MB-23 cells by reverse
transcriptase PCR and showed the presence of the CCR5 protein by
FACS analysis (FIGS. 13A-13D), and showed that CCR5
immunohistochemical staining was localized primarily to the breast
cancer epithelial cell, compared with normal breast tissue (FIGS.
14A-14D).
[0318] The results disclosed herein show that CCL5 activates
calcium flux in basal-like human breast cancer cells. By using the
selective CCR5-antagonists maraviroc and vicriviroc (both with IC50
below 30 nmol/L; refs. 44, 45), it was shown that CCLS-activated
signaling is mediated by CCRS. However, the fraction of
CCL5-responsive cells (10% and 12% for MDA-MB-231 and Hs578T cells,
respectively) is higher than the percentage of CCR5-expressing
cells determined by FACS. This may be due to the greater
sensitivity of the Ca2+ activation assays compared with the
sensitivity of analysis by FACS. In addition, CCL5 -induced calcium
redistribution is not completely blocked by CCR5 antagonists. This
may be caused by the expression of other receptors to CCL5, namely
CCR1 and CCR3. CCR5 has been identified as the main CCL5 receptor
in MDA-MB-231 cells (13) and CCR1 and CCR3 transcripts are absent
in both MDA-MB-231 or Hs578T cell lines (8) and breast tumor
samples (11). CCR1 and CCR3 were able to be detected by FACS (FIGS.
13A-13D), suggesting a possible mechanism for the incomplete
response to the CCR5 antagonist.
[0319] In one aspect, the present invention provides an in vivo
method for identifying a candidate compound that down regulate
expression of CCR5 and/or one or more of its ligand in tumor cells
overexpressing endogenous or virally transduced oncogene selected
from the group consisting of NeuT, Ha-Ras, c-Src and combinations
thereof. In one embodiment, the method comprises (a) administering
a candidate compound to an animal model of the tumor cells; (b) and
measuring the level of CCR5 RNA or protein expression in the animal
model, wherein if the level of CCR5 RNA or protein expression in
the animal model is decreased compared to the level of CCR5 RNA or
protein expression in untreated animal model, then the candidate
compound is identified as a compound that down regulates expression
of CCR5 and/or one or more of its ligand in tumor cells
overexpressing endogenous or virally transduced oncogene selected
from the group consisting of NeuT, Ha-Ras, c-Src and combinations
thereof. In one embodiment, the tumor cells comprise mammalian
prostate cancer cells. In one embodiment, tumor cells comprise
mammalian prostate cancer cell line having at least one or more of
a set of primary mammalian epithelial cells which have been
infected with a retroviral vector carrying an oncogene selected
from the group consisting of NeuT, Ha-Ras, c-Src and combinations
thereof.
[0320] An exemplary mammalian prostate cancer cell lines suitable
for use in the present invention include, but are not limited to,
mammalian prostate cancer cell lines disclosed in U.S. Provisional
Patent Application No. 61/646,586, filed May 14, 2012, whose
contents are incorporated by reference herein in their entirety. In
one embodiment, the mammalian prostate cancer cell line comprises
at least one or more of a set of primary mammalian epithelial cells
which have been infected with a retroviral vector carrying an
oncogene. In one embodiment, the oncogene is selected from the
group consisting of c-Myc, Ha-Ras, NeuT, c-Src and combinations
thereof. In one embodiment of the mammalian prostate cancer cell
line, an oncogene selected from the group consisting of c-Myc,
Ha-Ras, NeuT, c-Src and combinations thereof is expressed. The
mammalian prostate cancer cell line can include any suitable
mammalian cell, including primary murine epithelial cells. In some
embodiments, the primary mammalian epithelial cells are derived
from any immune competent mammal. In one embodiment, the primary
mammalian epithelial cells are derived from an immune competent
mammal selected from the group consisting of rodents, including
rats and mice.
[0321] In some embodiments, the suitable animal model of cancer
comprises an immune competent mammal implanted with a cancer cell
line transformed with one or more of a set of oncogenes selected
from the group consisting of c-Myc, Ha-Ras, NeuT, c-Src and
combinations thereof. In one embodiment, the animal model of cancer
is an immunocompetent transgenic mouse created using the mammalian
prostate cancer cell line of the present invention develops a
prostate tumor capable of producing a detectable molecular genetic
signature based on an expression level of one or more of a set of
oncogenes selected from the group consisting of c-Myc, Ha-Ras,
NeuT, c-Src and combinations thereof.
[0322] In some embodiments, the suitable animal model of cancer is
produced by an in vitro method. In one embodiment, in vitro method
for producing the suitable animal model includes production of
immortalized primary mammalian epithelial cells. In one embodiment,
the method for the in vitro production of immortalized primary
mammalian epithelial cells, comprises infecting primary mammalian
epithelial cells with a retroviral vector carrying an oncogene
selected from the group consisting of c-Myc, Ha-Ras, NeuT, c-Src
and combinations thereof to provide infected cells, wherein the
primary mammalian epithelial cells are capable of being infected by
said retroviral vector and under conditions whereby the c-Myc,
Ha-Ras, NeuT, c-Src and combinations thereof are expressed in said
infected cells.
I. EXAMPLES
[0323] A. Materials and Methods
[0324] A.1. Breast Cancer Patients Data Set and Statistical
Analysis
[0325] A microarray data set that was previously compiled (21) from
the public repositories Gene Expression Omnibus (23) and
ArrayExpress (24) was used to evaluate CCR5 and CCL5 expression in
the context of clinical samples. Samples in this data set were
assigned to 5 canonical breast cancer subtypes, including luminal
A, luminal B, normal-like, basal, and HER-2 - overexpressing
disease. The classification of microarray samples among these 5
subtypes was achieved by computing their correlation against an
expression profile centroid representative of each subtype and
assigning samples to the subtype with the highest corresponding
correlation coefficient (25). Samples with a maximum correlation
coefficient below 0.3 were considered unclassified. Analysis of
CCL5 and CCR5 transcript was then conducted specifically among the
luminal A, luminal B, basal, normal-like, and HER-2 subtypes.
Differential expression of the averaged gene signature magnitude
among these sample subsets was evaluated using 2-tailed Student t
test. Kaplan-Meier analysis was used to evaluate survival trends
within the sample subsets. Scatter plots of CCL5 versus CCR5
samples were also generated to observe coregulation patterns
specific to each subtype. For these scatter plots, gene profiles
were median-centered and scaled to unitary SD.
[0326] A.2. Cell Lines and Cell Culture
[0327] MDA-MB-231, MCF-7, and Hs578T cells were maintained in
Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10%
FBS. SUM-159 cells (kindly provided by Dr. Stephen Ethier, Wayne
State University, Detroit, Mich.) were maintained in Ham's F-12
supplemented with 4 .mu.g/mL of insulin, 1 .mu.g/mL of
hydrocortisone, and 5% FBS. Oncogene-transformed derivatives of
MCF-10A cells (MCF10A-NeuT, MCF10A-Src, and MCF10A-Ras; ref 26)
were maintained in DMEM:Ham's F-12 (50 of 50) supplemented with 4
mg/mL of insulin, 10 ng/mL of EGF, and 1 mg/mL of hydrocortisone. A
total of 100 .mu.g/mL of each penicillin and streptomycin were
included in all media. Cells were cultured in 5% CO.sub.2 at
37.degree. C. For in vitro treatments, maraviroc was dissolved in
dimethyl sulfoxide (DMSO) and diluted in culture medium. The final
concentration of DMSO in treated and control cultures was 0.5%.
Vicriviroc was dissolved in culture medium.
[0328] A.3. Fluorescence-Activated Cell-Sorting Analysis
[0329] Cell labeling and fluorescence-activated cell-sorting (FACS)
analysis for CCR5 were based on prior publications (27) with minor
modifications. Before labeling, the cells were blocked with normal
mouse IgG (1 of 100) and purified rat anti-mouse Fc.gamma. III/II
receptor antibody (1 of 100; Pharmingen) for 30 minutes and then
incubated with allophycocyanin (APC)-labeled CCRS antibody (R&D
Systems). All experiments were conducted at 4.degree. C. Sample
analysis was conducted on FACSCalibur flow cytometer (BD
Biosciences). These data were analyzed with FlowJo software (Tree
Star, Inc.).
[0330] A.4. Invasion Assay
[0331] The 3-dimensional invasion assay was conducted as previously
reported (12). Briefly, 100 .mu.L of 1.67 mg/mL Rat Tail collagen
type I (BD Biosciences) was pipetted into the top chamber of a
24-well 8 .mu.m pore Transwell (Corning). The Transwell was
incubated at 37.degree. C. overnight to allow the collagen to
solidify. A total of 30,000 cells were then seeded on the bottom of
the Transwell membrane and allowed to attach. Serum-free growth
medium was placed into the bottom chamber, whereas 15 ng/mL CCL5 or
5% FBS was used as a chemoattractant in the medium of the upper
chamber. The cells were then chemoattracted across the filter
through the collagen above for 3 days. Cells were fixed in 4%
formaldehyde, permeabilized with 0.2% Triton-X in PBS, and then
stained with 40 .mu.g/mL propidium iodide (PI) for 2 hours.
Fluorescence was analyzed by confocal z-sections (one section every
20 .mu.m) at .times.10 magnification from the bottom of the filter
using a Zeiss LSM 510 Meta inverted confocal microscope at the
Kimmel Cancer Center Bioimaging Facility.
[0332] A.5. Intracellular Calcium Assay
[0333] Calcium responses induced either by CCL5 or FBS in human
cancer cell lines were monitored under fluorescence confocal
microscope as previously reported (28). Briefly, breast cancer
cells were seeded in 4-well labtek chambers (Nunc) at 10.sup.4
cells/cm.sup.2 and incubated for 1 day.
[0334] After 12-hour starvation, cells were labeled by incubating
them with 2 mmol/L Fluo-4-AM (Molecular Probes) in HBSS for 30
minutes, washed twice, and incubated for additional 30 minutes
before imaging under the microscope. Time-lapse images were
collected using a Zeiss LSM 510 Meta inverted confocal microscope
with the incubator at 37.degree. C. Relative intracellular
Ca.sup.2+ concentration was determined by the changes in
fluorescent intensity (FI) of Fluo-4-AM upon the addition of CCL5
(60 ng/mL) or FBS (5%) and was calculated as
(FI.sub.t-FI.sub.0)/FI.sub.0.
[0335] A.6. MTT Assay
[0336] The MTT assay is a colorimetric assay for measuring the
activity of cellular enzymes that reduce the tetrazolium dye,
3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, a
yellow tetrazole (MTT), to its insoluble formazan, giving a purple
color. These assay measures cellular metabolic activity via
NAD(P)H-dependent cellular oxidoreductase enzymes and may, under
defined conditions, reflect the number of viable cells (cell
proliferation). Tetrazolium dye assay can also be used to measure
cytotoxicity (loss of viable cells) or cytostatic activity (shift
from proliferative to resting status) of potential medicinal agents
and toxic materials. MTT Assay usually done in the dark because MTT
reagent is sensitive to light.
[0337] The effects of CCR5 antagonists on cell viability and
proliferation rate were estimated using the soluble tetrazolium
salt MTT assay (29). MTT is reduced by the mitochondria of viable
cells, and the amount of reduced formazan is proportional to the
number of viable cells. After 72 hours of exposure to the drugs,
cells were incubated with 1 mg/mL of MTT for 90 minutes. Then, the
reduced (insoluble and colored) formazan was dissolved in DMSO and
measured spectrophotometrically at 570 nm. The effect of CCR5 over
expression in breast cancer cell proliferation was studied in
MDA-MB-231 cells transfected with full-length human CCR5 subcloned
into pcDNA3.1.sup.+/Zeo.sup.+ vector (kindly provided by Dr.
Eleanor Fish, University of Toronto, Toronto, ON, Canada) and
selected with Zeocin (200.mu.g/mL) as previously described (18).
MTT assays were conducted in sextuplicate using 96-well
microplates.
[0338] A.7. Viral Cell Transduction
[0339] A lentiviral vector encoding firefly luciferase 2
(Luc2)-eGFP fusion protein was a generous gift from Dr. Sanjiv S.
Gambhir (School of Medicine, Stanford University, Stanford, Calif.;
ref. 30). Lentivirus propagation was conducted following the
protocol described by Zahler and colleagues (31). Breast cancer
cell lines were transduced at a multiplicity of infection of 20 in
the presence of 8 mg/mL polybrene (Sigma) for 24 hours (30,
31).
[0340] A.B. Experimental Metastasis Assay And Bioluminescence
Imaging
[0341] MB-MDA-231 cells expressing Luc2-eGFP (called MDA.pFLUG for
the rest of the article) were detached with a nonenzymatic cell
dissociation buffer (4 mmol/L EDTA in Ca.sup.2+ and Mg.sup.2--free
PBS), resuspended in Dulbecco's PBS without Ca.sup.2+ and Mg.sup.2+
and immediately injected into the tail vein of 8-week-old, female
nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice
(NCI, Bethesda Md.). Each mouse received 10.sup.6 cells. Mice were
treated by oral gavage with maraviroc (8 mg/kg every 12 hours) or
vehicle (5% DMSO in acidified water; ref. 32). Treatment was
started immediately after injection or 10 days later for the
experiments analyzing the proliferation of established metastasis.
For in vivo bioluminescence imaging (BLI), mice were given an
intraperitoneal (i.p.) injection with 200 .mu.L of d-luciferin (30
mg/mL). Mice were anesthetized with isoflurane (2% in 1 L/min
oxygen), and bioluminescence images were acquired 10 to 15 minutes
after d-luciferin injection using the IVIS XR system (Caliper Life
Sciences). Acquisition times ranged from 10 seconds (for later time
points) to 5 minutes (for early time points). Data are expressed as
total photon flux and were analyzed using Living Image 3.0 software
(Caliper Life Sciences). For ex vivo BLI, d-luciferin was diluted
in PBS to a final concentration of 300 .mu.g/mL and used to soak
freshly isolated lungs for 2 to 3 minutes before imaging. Some
lungs were stained with India ink, as previously reported (33), or
processed and stained with hematoxylin and eosin to corroborate the
presence of pulmonary tumors. For homing assays, mice were
euthanized 24 hours after the intravenous injection of MDA.pFULG
cells. Lungs were perfused with PBS, fixed with freshly prepared
formaldehyde (4% in PBS), and frozen in optimum cutting temperature
(Sakura Finetek). Cryosections (10 .mu.m) were counterstained with
4',6-diamidino-2-phenylindole analyzed by confocal microscopy.
Animal experiments were approved by the Thomas Jefferson
University's Institutional Animal Care and Use Committee.
[0342] A.9. Reagents and Antibodies
[0343] CCL5 (catalog no. 278-RN) and anti-CCR5 APC antibody
(catalog no. FAB1802A) were purchased from R&D Systems. A
rabbit anti-human CCR5 polyclonal antibody (GenScipt; catalog no.
A00979) was used for immunohistochemical staining. Rat tail
collagen type I was purchased from BD Biosciences. Vicriviroc and
maraviroc were obtained from Selleck Chemicals. Luciferin was
obtained from Gold Biotechnology.
[0344] B. Results
[0345] B.1. Active CCL5/CCR5 Signaling In Basal Breast Cancer
[0346] To examine the relative abundance of CCL5 and its receptor
CCR5 by genetic subtype, interrogation was conducted within a
combined microarray database comprising 2,254 human breast cancer
samples from 27 independent studies (21). The relative abundances
of CCL5 and CCRS were significantly increased in the basal and
HER-2 subtypes compared with the normal-like, luminal A and luminal
B subtypes (FIG. 1A). The increased expression of CCL5 and CCRS
correlated positively in individual breast cancer samples and the
correlation was highly significant in the basal and HER-2 subtypes
(FIG. 1B). The proportion of patients with a CCL5/CCR5-positive
signature was more than 58% in the basal and HER-2 subtypes (FIG.
1C). In agreement with previous reports, clinical information of
the cases in this database showed that the probability to develop
metastasis is increased in the basal, luminal B, and HER-2 subtypes
(FIG. 1D).
[0347] To determine the gene expression signaling pathway
associated with enrichment of CCR5 and CCL5, GSEA analysis using
KEGG and GO was conducted of these tumor samples (FIG. 7A). These
studies showed enrichment for gene expression of pathways including
lymphocyte activation, Janus-activated kinase (JAK)-STAT signaling,
and Toll-like receptor activation (FIG. 7A). The receptors for CCL5
include CCR1 and CCR3. Increased expression of CCL5 associated with
increased CCR1, but not CCR3, in the basal and HER-2 genetic tumor
type (FIGS. 7B and 7C). In ER-negative patients receiving
chemotherapy, there was an insignificant trend toward reduced
metastasis-free survival and relapse-free survival in the increased
CCR5 population, compared with the population with reduced CCR5
expression (FIG. 7D). A comparison of expression levels for CCL5
versus CCR5, CCR1, and CCR3, comparing normal breast with breast
cancer showed increased correlation between receptor and ligand
expression levels in tumors compared with healthy breast tissue
(FIG. 8A).
[0348] B.2. Ccl5 Promotes Breast Cancer Ca.sup.2+ Signaling and
Cellular Invasion
[0349] Three human breast cancer cell lines with a basal phenotype
and molecular signature: MDA-MB-231, Hs578T, and SUM-159 (34-37)
were used as models in the studies described herein. Analysis of
CCR5 expression by FACS showed that a small subpopulation of cells
were positive for the receptor in all 3 cell lines (FIG. 2A for
MDA-MB-231 and FIGS. 9A and 9C for Hs578T and SUM-159). Because
CCR5 activation induces calcium flux (38, 39), the activation of
calcium signaling was assessed by CCL5. Addition of CCL5 to the
cultures induced immediate calcium fluxes in a subpopulation of
cells (FIG. 2B for MDA-MB-231 and FIGS. 9B and 9D for Hs578T and
SUM-159), providing evidence that CCR5 is functional in basal
breast cancer cells. As a positive control, the same cultures were
exposed to 5% FBS (40). Calcium flux, assessed by relative
fluorescence intensity, increased in more than 95% of the cells
after FBS addition (FIG. 2B and FIGS. 9B and 9D). To further
distinguish CCL5 -dependent signaling, SUM159 cells were stably
transduced with a CCR5 expression vector and the Ca.sup.+2 response
to CCL5 versus FBS was conducted (FIGS. 10C and 10D vs. 10E and
10F). CCR5 induced Ca.sup.+2 signaling in the CCR5-overexpressing
cells, whereas both lines responded similarly to FBS induced
Ca.sup.+2 activation (FIGS. 10A-10F).
[0350] Next, the effect of CCR5 activation on breast cancer cell
invasion was assessed using 3D migration assays. CCL5 induced
invasion of the basal MDA-MB-231, Hs578T, SUM-159 but not the
luminal MCF-7 cells (FIGS. 2C and D). CCL5 promoted invasion of
MCF-10A cells engineered to express either NeuT, H-Ras, or c-Src
oncogenes, compared with MCF10A vector-transduced cells (FIG. 2E
and F), suggesting that CCL5 responsiveness may be acquired during
transformation and requires specific cooperative oncogenic signals.
The finding that CCL5 induced cellular invasion led us to examine
the migratory capacity of CCR5.sup.+ cells versus that of
CCR5.sup.- cells. Within the same SUM-159 breast cancer cell line,
CCR5.sup.+ cells showed an approximately 40-fold greater cellular
invasiveness (FIG. 2G and H), indicating that the expression of
CCR5 correlates with a proinvasive phenotype.
[0351] B.3. CCR5 Antagonists Block Breast Cancer Calcium Signaling
and Cell Invasion
[0352] The importance of CCR5 in HIV infection led to the
development of different drugs that target this receptor.
Therefore, examination of whether the CCR5 antagonists maraviroc
and vicriviroc were capable of blocking the CCL5/CCR5 signaling in
basal breast cancer cells were conducted. Both CCR5 antagonists
blocked CCL5 -induced calcium mobilization. In MDA-MB-231 cells,
maraviroc and vicriviroc inhibited calcium responses by 65% and
90%, respectively (FIG. 3A and 3B). Similar observations were made
with both drugs in Hs578T cells (FIG. 3C and D), indicating that
CCR5 expressed in different basal breast cancer cells is sensitive
to pharmacologic inhibition.
[0353] To evaluate the functional relevance of CCR5 in cellular
migration and invasion, the effects of maraviroc and vicriviroc
were tested in 3D invasion assays. Using 2 different cell lines, it
was found that both CCR5 antagonists inhibited FBS-induced breast
cancer cell invasion at the clinically relevant concentration of
100 nmol/L (FIGS. 4A-4D). Thus, the proinvasive effect of CCR5 can
be abrogated by using specific antagonists.
[0354] B.4. CCR5 Inhibition Blocks Breast Cancer Metastasis In
Vivo
[0355] In view of the finding that CCR5 inhibition by CCR5
antagonists reduced calcium signaling and cell invasion, the in
vivo effect of maraviroc on lung metastasis was determined.
MDA-MB-231 cells transduced within the Luc2-eGFP lentiviral vector
(MDA.pFULG cells) were used in an experimental metastasis model.
The Luc2 gene is a codon-optimized version of Luc and cells
expressing this reporter were 10 to 100 times brighter than the
unmodified Luc gene (30). After injection of MDA.pFULG cells into
the tail vein of mice, noninvasive BLI enabled the early detection
of breast cancer metastasis (41). Weekly BLI was conducted for 5
weeks and the radiance antemortem was used as a surrogate
measurement of tumor burden. Mice treated with maraviroc (8 mg/kg
twice daily) showed a significant reduction in both the number and
the size of pulmonary metastases compared with vehicle-treated mice
(FIGS. 5A and B, FIGS. 11A and 11B). To avoid the possibility that
metastases were missed because of inappropriate imaging, ex vivo
imaging, India ink staining (FIG. 5C), and histology (FIG. 5E) of
the lungs were conducted. Histologic analysis corroborated that
tumor burden corresponds to bioluminescence, as previously shown
(30). Metastatic tumors were still detectable in 50% of the
maraviroc-treated mice, but their mean size was reduced by 65%
(FIGS. 5D and F). Interestingly, analysis of CCR5 expression in
lungs from control mice showed an 8-fold enrichment of the
CCR5.sup.+fraction (FIG. 12). Collectively, these results provide
evidence that CCR5 antagonists reduce breast cancer metastasis in
vivo.
[0356] B.5. CCR5 Antagonist Impairs Lung Colonization but Not Cell
Proliferation or Tumor Growth
[0357] It was determined whether the reduction in metastatic tumors
by maraviroc involved changes in cellular proliferation and/or
target organ colonization. The effect of CCR5 inhibition on cell
viability and proliferation both in vitro and in vivo was analyzed.
Maraviroc or vicriviroc treatment of MDA-MB-231 cells for 48 hours
did not affect the MTT reduction, which was used as a surrogate
measurement of cancer cell number (FIG. 6A). In agreement, over
expression of CCR5 in MDA-MB-231 cells did not modify their
proliferation rate compared with cells transfected with the empty
vector (FIGS. 6B and 6C). Finally, maraviroc treatment of mice with
established pulmonary metastasis did not modify tumor growth (FIGS.
6D and 6E), indicating that CCR5 activation does not promote the
proliferation of basal breast cancer cells in vitro nor in the
pulmonary microenvironment of immunocompromised mice.
[0358] On a different in vivo experiment, the effect of maraviroc
on breast cancer cell homing to lungs was examined. To reach a
steady-state concentration in plasma and tissues, mice were given
10 administrations of maraviroc (twice a day for 5 days) before the
intravenous injection of MDA.pFULG cells (FIG. 6F). Inoculation of
equal numbers of MDA.pFULG cells in control and treated groups was
corroborated by BLI immediately after injection. Maraviroc reduced
the number of eGFP.sup.- cells in the lungs by 40% (FIGS. 6G and
6H), suggesting that the in vivo antimetastatic effect of maraviroc
is caused by a reduction in the number of cancer cells that
colonize the target organ from the circulation.
[0359] C. Discussion
[0360] The current studies show for the first time that: (i)
enrichment of CCL5/CCR5 expression occurs in patients with basal
and Her2 positive genetic subtypes of breast cancer; (ii) oncogenic
transformation of immortalized human breast cells by distinct
oncogenes induces CCLS responsiveness; and (iii) maraviroc, an
FDA-approved drug for the treatment of CCR5 -trophic HIV infection,
reduce metastatic tumor burden in vivo.
[0361] Previous studies showed that CCL5 levels are elevated in
breast primary and metastatic tumors (9-11), suggesting a role of
CCL5 in the acquisition of malignancy. The present disclosure show
that increased expression of CCL5 and CCR5 are associated and that
CCL5/CCR5 expression levels are different among the different
genetic subtypes of breast cancer. Increased expression of CCL5 and
CCR5 is found in the basal and HER-2 subtypes. In agreement,
increased CCL5 expression has been found predominantly in
ER-negative patients (42). Increased CCL5 also correlated with
increased CCR1 in basal and Her2 genetic subtypes of breast cancer.
A trend toward reduced metastasis-free survival and relapse-free
survival was observed among the CCR5-overexpressing tumors in
patients who received chemotherapy.
[0362] Given the aggressive clinical behavior of basal breast
cancer and the lack of targeted therapies for it, the importance of
the CCL5/CCR5 axis in invasion and metastasis was evaluated in the
human breast cancer cell lines MDA-MB-231, Hs578T and SUM-159.
These cell lines reflect the clinicopathologic features of the
basal subtype of breast cancer (including the lack of HER-2, ER,
and progesterone receptor), a basal-like molecular signature, the
activation of specific signaling pathways (e.g., hypoxic or EGF
receptor responses) and over expression of epithelial-mesenchymal
transition proteins (FN, VIM, and matrix metalloproteinase 2; refs.
34-37). Only a small fraction of cells within the cell lines used
in this study expressed CCR5 as evaluated by FACS analysis. Our
findings are consistent with studies by Muller and colleagues who
showed CCR5 expression in MDA-MB-231 by quantitative real-time PCR
(8). These studies confirmed the expression of CCR5 in MDA-MB-23
cells by reverse transcriptase PCR and showed the presence of the
CCR5 protein by FACS analysis (FIGS. 13A-13D), and showed that CCR5
immunohistochemical staining was localized primarily to the breast
cancer epithelial cell, compared with normal breast tissue (FIGS.
14A-14D).
[0363] The results herein show that CCL5 activates calcium flux in
basal-like human breast cancer cells, as previously described in
cells of the immune system (39, 43) and CCR5 -transfected cells
(27, 44, 45). By using the selective CCR5 -antagonists maraviroc
and vicriviroc (both with IC.sub.50 below 30 nmol/L; refs. 44, 45),
it was shown that CCL5 -activated signaling is mediated by CCR5.
However, the fraction of CCL5 -responsive cells (10% and 12% for
MDA-MB-231 and Hs578T cells, respectively) is higher than the
percentage of CCR5-expressing cells determined by FACS. This may be
due to the greater sensitivity of the Ca.sup.2+ activation assays
compared with the sensitivity of analysis by FACS. In addition,
CCL5-induced calcium redistribution is not completely blocked by
CCR5 antagonists. This may be caused by the expression of other
receptors to CCL5, namely CCR1 and CCR3. CCR5 has been identified
as the main CCL5 receptor in MDA-MB-231 cells (13) and CCR1 and
CCR3 transcripts are absent in both MDA-MB-231 or Hs578T cell lines
(8) and breast tumor samples (11). CCR1 and CCR3 were able to be
detected by FACS (FIGS. 13A-13D), suggesting a possible mechanism
for the incomplete response to the CCR5 antagonist.
[0364] It was observed that the subpopulation of CCR5.sup.+ cells
displayed increased invasiveness, indicating that CCR5 favors cell
migration and invasion in basal-like breast cancer cells. The
failure of luminal-like MCF-7 cells to respond to CCL5 is in
agreement with previous publications (12). These studies also
showed that CCR5 inhibition with either maraviroc or vicriviroc
reduced in vitro FBS-induced breast cancer cellular invasion
without affecting cellular viability. The finding that CCR5
antagonists block FBS-induced invasion is novel and suggested that
CCR5 activation contribute to the production of metastasis in vivo
where different chemotactic and growth signals are present. The
mechanisms involved in CCR5 regulation of FBS-activated
invasiveness are uncharacterized but they may include
heterodimerization and ligand affinity regulation of other GPCRs
(46), or the transactivation of growth factor receptor (47) or
integrin-mediated signaling (48), as described in noncancerous
cells.
[0365] The in vivo antimetastatic effect of maraviroc was shown by
injecting MDA.pFULG cells into the circulation of immunodeficient
mice and treating them with clinically relevant doses of the drug.
In humans, oral doses of 300 mg produce an average C.sub.max of
1,200 nmol/L (49), whereas in mice 16 mg/kg produce an average
C.sub.max of 1,045 nmol/L (32). Because the drug is taken twice a
day in the clinical setting, 16 mg/kg/d divided into 2 doses and
administered during the experiments described herein. Maraviroc
significantly reduced the pulmonary tumor burden. Although it has
been proposed that pharmacologic CCR5 inhibition may be beneficial
for patients with breast cancer, to our knowledge this is the first
study showing that systemic administration of a CCR5 antagonist
reduces metastatic colonization of basal breast cancer cells.
[0366] The antimetastatic effect of maraviroc is not caused by
alterations in growth of established metastasis. CCR5 activation by
CCL5 drives proliferation in CCR5 -transfected MCF-7 breast cancer
cells (18) and prostate cancer cells (50), but this study and
others (13) showed that the CCL5/CCR5 axis does not play a role in
cell proliferation or survival in the basal-like MDA-MB-231 cells.
Furthermore, inhibition of CCR5 surface expression through a
dominant-negative form of CCR5 (CCR5.DELTA.32) in MDA-MB-231 cells
does not change in vivo proliferation or apoptotic response (17).
On the other hand, it was found that maraviroc reduced lung
colonization by MDA.pFULG cancer cells. This result is consistent
with previous studies in which inhibition of CCR5 expression within
breast cancer cells or administration of anti-CCL5 neutralizing
antibody to tumor-bearing mice reduced the enhanced metastatic
capability induced by coinjection of mesenchymal stem cells (MSC;
ref 13). The authors identified cancer cell extravasation as the
crucial metastatic step affected by CCL5/CCR5 inhibition (13).
Together, these data support a role for CCR5 antagonists in
blocking the ability of basal breast cancer cells to reach the
metastatic sites instead of inhibiting their proliferation or
survival after arrival. Blocking the homing of cancer cells to
metastatic sites is a desirable characteristic in a true
antimetastatic drug (51). Therefore, CCR5 antagonists may be useful
as adjuvant therapy for breast basal tumors with CCR5 over
expression or other tumor types where CCR5 promotes metastasis,
such as prostate cancer (50) or gastric cancer (52).
[0367] It will be appreciated by those skilled in the art that
changes could be made to the exemplary embodiments shown and
described above without departing from the broad inventive concept
thereof. It is understood, therefore, that this invention is not
limited to the exemplary embodiments shown and described, but it is
intended to cover modifications within the spirit and scope of the
present invention as defined by the claims. For example, specific
features of the exemplary embodiments may or may not be part of the
claimed invention and features of the disclosed embodiments may be
combined. Unless specifically set forth herein, the terms "a", "an"
and "the" are not limited to one element but instead should be read
as meaning "at least one".
[0368] It is to be understood that at least some of the figures and
descriptions of the invention have been simplified to focus on
elements that are relevant for a clear understanding of the
invention, while eliminating, for purposes of clarity, other
elements that those of ordinary skill in the art will appreciate
may also comprise a portion of the invention. However, because such
elements are well known in the art, and because they do not
necessarily facilitate a better understanding of the invention, a
description of such elements is not provided herein.
[0369] Further, to the extent that the method does not rely on the
particular order of steps set forth herein, the particular order of
the steps should not be construed as limitation on the claims. The
claims directed to the method of the present invention should not
be limited to the performance of their steps in the order written,
and one skilled in the art can readily appreciate that the steps
may be varied and still remain within the spirit and scope of the
present invention.
[0370] All references, including publications, patent applications,
and patents, cited herein are hereby incorporated by reference to
the same extent as if each reference were individually and
specifically indicated to be incorporated by reference and were set
forth in its entirety herein.
II. REFERENCES
[0371] 1. Parkin D M, Fernandez L M. Use of statistics to assess
the global burden of breast cancer. Breast J 2006; 12 Suppl 1:
S70-80. [0372] 2. Group EBCTC. Effects of chemotherapy and hormonal
therapy for early breast cancer on recurrence and 15-year survival:
an overview of the randomised trials. Lancet 2005; 365: 1687-717.
[0373] 3. Meyers M O, Klauber-Demore N, 0llila D W, Amos K D, Moore
D T, Drobish A A, et al. Impact of breast cancer molecular subtypes
on locoregional recurrence in patients treated with neoadjuvant
chemotherapy for locally advanced breast cancer. Ann Surg Oncol
2011; 18: 2851-7. [0374] 4. Kennecke H, Yerushalmi R, Woods R,
Cheang M C, Voduc D, Speers C H, et al. Metastatic behavior of
breast cancer subtypes. J Clin Oncol 2010; 28: 3271-7. [0375] 5.
Perou C M, Sorlie T, Eisen M B, van de Rijn M, Jeffrey S S, Rees C
A, et al. Molecular portraits of human breast tumours. Nature 2000;
406: 747-52. [0376] 6. Reis-Filho J S, Lakhani S R . Breast cancer
special types: why bother? J Pathol 2008; 216: 394-8. [0377]
Kakinuma T, Hwang S T . Chemokines, chemokine receptors, and cancer
metastasis. J Leukoc Biol 2006; 79: 639-51. [0378] 8. Muller A,
Homey B, Soto H, Ge N, Catron D, Buchanan M E, et al. Involvement
of chemokine receptors in breast cancer metastasis. Nature 2001;
410: 50-6. [0379] 9. Luboshits G, Shina S, Kaplan O, Engelberg S,
Nass D, Lifshitz-Mercer B, et al. Elevated expression of the CC
chemokine regulated on activation, normal T cell expressed and
secreted (RANTES) in advanced breast carcinoma. Cancer Res 1999;
59: 4681-7. [0380] 10. Niwa Y, Akamatsu H, Niwa H, Sumi H, Ozaki Y,
Abe A . Correlation of tissue and plasma RANTES levels with disease
course in patients with breast or cervical cancer. Clin Cancer Res
2001; 7: 285-9. [0381] 11. Zhang Y, Yao F, Yao X, Yi C, Tan C, Wei
L, et al. Role of CCL5 in invasion, proliferation and proportion of
CD44+/CD24- phenotype of MCF-7 cells and correlation of CCL5 and
CCR5 expression with breast cancer progression. Oncol Rep 2009; 21:
1113-21. Medline 12. Jiao X, Katiyar S, Willmarth NE, Liu M, Ma X,
Flomenberg N, et al. c-Jun induces mammary epithelial cellular
invasion and breast cancer stem cell expansion. J Biol Chem 2010;
285: 8218-26. [0382] 13. Karnoub A E, Dash A B, Vo A P, Sullivan A,
Brooks M W, Bell G W, et al. Mesenchymal stem cells within tumour
stroma promote breast cancer metastasis. Nature 2007; 449: 557-63.
[0383] 14. Locati M, Deuschle U, Massardi M L, Martinez F O, Sironi
M, Sozzani S, et al. Analysis of the gene expression profile
activated by the CC chemokine ligand 5/RANTES and by
lipopolysaccharide in human monocytes. J Immunol 2002; 168:
3557-62. [0384] 15. Robinson S C, Scott K A, Balkwill F R .
Chemokine stimulation of monocyte matrix metalloproteinase-9
requires endogenous TNF-alpha. Eur J Immunol 2002; 32: 404-12.
[0385] 16. Robinson S C, Scott K A, Wilson J L, Thompson R G,
Proudfoot A E, Balkwill F R . A chemokine receptor antagonist
inhibits experimental breast tumor growth. Cancer Res 2003; 63:
8360-5. [0386] 17. Manes S, Mira E, Colomer R, Montero S, Real L M,
Gomez-Mouton C, et al. CCRS expression influences the progression
of human breast cancer in a p53-dependent manner. J Exp Med 2003;
198: 1381-9.
[0387] 18. Murooka T T, Rahbar R, Fish E N . CCL5 promotes
proliferation of MCF-7 cells through mTOR-dependent mRNA
translation. Biochem Biophys Res Commun 2009; 387: 381-6. [0388]
19. Stormes K A, Lemken C A, Lepre J V, Marinucci M N, Kurt R A .
Inhibition of metastasis by inhibition of tumor-derived CCLS.
Breast Cancer Res Treat 2005; 89: 209-12. [0389] 20. Jayasinghe M
M, Golden J M, Nair P, O'Donnell C M, Werner M T, Kurt R A .
Tumor-derived CCL5 does not contribute to breast cancer
progression. Breast Cancer Res Treat 2008; 111: 511-21. [0390] 21.
Ertel A, Dean J L, Rui H, Liu C, Witkiewicz A K, Knudsen K E, et
al. RB-pathway disruption in breast cancer: differential
association with disease subtypes, disease-specific prognosis and
therapeutic response. Cell Cycle 2010; 9: 4153-63. [0391] 22.
Wilkin T J, Su Z, Krambrink A, Long J, Greaves W, Gross R, et al.
Three-year safety and efficacy of vicriviroc, a CCR5 antagonist, in
HIV-1-infected treatment-experienced patients. J Acquir Immune
Defic Syndr 2010; 54: 470-6. [0392] 23. Barrett T, Troup D B,
Wilhite S E, Ledoux P, Rudnev D, Evangelista C, et al. NCBI
GEO:
[0393] mining tens of millions of expression profiles--database and
tools update. Nucleic Acids Res 2007; 35: D760-5. [0394] 24. Brazma
A, Parkinson H, Sarkans U, Shojatalab M, Vilo J, Abeygunawardena N,
et al. ArrayExpress-a public repository for microarray gene
expression data at the EBI. Nucleic Acids Res 2003; 31: 68-71.
[0395] 25. Hu Z, Fan C, Oh D S, Marron J S, He X, Qaqish B F, et
al. The molecular portraits of breast tumors are conserved across
microarray platforms. BMC Genomics 2006; 7: 96. [0396] 26. Liu M,
Casimiro M C, Wang C, Shirley L A, Jiao X, Katiyar S, et al.
p21CIP1 attenuates Ras- and c-Myc-dependent breast tumor epithelial
mesenchymal transition and cancer stem cell-like gene expression in
vivo . Proc Natl Acad Sci U S A 2009; 106: 19035-9. [0397] 27.
Nguyen D H, Taub D . Cholesterol is essential for macrophage
inflammatory protein 1 beta binding and conformational integrity of
CC chemokine receptor 5. Blood 2002; 99: 4298-306. [0398] 28.
Janowski E, Jiao X, Katiyar S, Lisanti M P, Liu M, Pestell R G, et
al. c-Jun is required for TGF-beta-mediated cellular migration via
nuclear Ca(2) signaling. Int J Biochem Cell Biol 2011; 43: 1104-13.
[0399] 29. Velasco-Velazquez M A, Agramonte-Hevia J, Barrera D,
Jimenez-Orozco A, Garcia-Mondragon M J, Mendoza-Patino N, et al.
4-Hydroxycoumarin disorganizes the actin cytoskeleton in B16-F10
melanoma cells but not in B82 fibroblasts, decreasing their
adhesion to extracellular matrix proteins and motility. Cancer Lett
2003; 198: 179-86. [0400] 30. Liu H, Patel M R, Prescher J A,
Patsialou A, Qian D, Lin J, et al. Cancer stem cells from human
breast tumors are involved in spontaneous metastases in orthotopic
mouse models. Proc Natl Acad Sci U S A 2010; 107: 18115-20. [0401]
31. Zahler M H, Irani A, Malhi H, Reutens A T, Albanese C,
Bouzahzah B, et al. The application of a lentiviral vector for gene
transfer in fetal human hepatocytes. J Gene Med 2000; 2: 186-93.
[0402] 32. Walker D K, Abel S, Comby P, Muirhead G J, Nedderman A
N, Smith D A . Species differences in the disposition of the CCR5
antagonist, UK-427,857, a new potential treatment for HIV. Drug
Metab Dispos 2005; 33: 587-95. [0403] 33. Wu K, Katiyar S, Li A,
Liu M, Ju X, Popov V M, et al. Dachshund inhibits oncogene-induced
breast cancer cellular migration and invasion through suppression
of interleukin-8. [0404] 34. Charafe-Jauffret E, Ginestier C,
Monville F, Finetti P, Adelaide J, Cervera N, et al. Gene
expression profiling of breast cell lines identifies potential new
basal markers. Oncogene 2006; 25: 2273-84. [0405] 35. Riaz M,
Elstrodt F, Hollestelle A, Dehghan A, Klijn J G, Schutte M .
Low-risk susceptibility alleles in 40 human breast cancer cell
lines. BMC Cancer 2009; 9: 236. [0406] 36. Hollestelle A, Nagel J
H, Smid M, Lam S, Elstrodt F, Wasielewski M, et al. Distinct gene
mutation profiles among luminal-type and basal-type breast cancer
cell lines. Breast Cancer Res Treat 2010; 121: 53-64. [0407] 37.
Kao J, Salari K, Bocanegra M, Choi Y L, Girard L, Gandhi J, et al.
Molecular profiling of breast cancer cell lines defines relevant
tumor models and provides a resource for cancer gene discovery.
PLoS ONE 2009; 4: e6146. [0408] 38. Mueller A, Mahmoud N G,
Goedecke M C, McKeating J A, Strange P G. Pharmacological
characterization of the chemokine receptor, CCR5. Br J Pharmacol
2002; 135: 1033-43. [0409] 39. Petkovic V, Moghini C, Paoletti S,
Uguccioni M, Gerber B. I-TAC/CXCL11 is a natural antagonist for
CCR5. J Leukoc Bio 2004; 76: 701-8. [0410] 40. Miyashita M, Smith M
W, Willey J C, Lechner J F, Trump B F, Harris C C . Effects of
serum, transforming growth factor type beta, or
12-O-tetradecanoyl-phorbol-13-acetate on ionized cytosolic calcium
concentration in normal and transformed human bronchial epithelial
cells. Cancer Res 1989; 49: 63-7. [0411] 41. Prescher J A, Contag C
H . Guided by the light: visualizing biomolecular processes in
living animals with bioluminescence. Curr Opin Chem Bio 2010; 14:
80-9. [0412] 42. Yaal-Hahoshen N, Shina S, Leider-Trejo L, Barnea
I, Shabtai EL, Azenshtein E, et al. The chemokine CCL5 as a
potential prognostic factor predicting disease progression in stage
II breast cancer patients. Clin Cancer Res 2006; 12: 4474-80.
[0413] 43. Shideman C R, Hu S, Peterson P K, Thayer S A . CCL5
evokes calcium signals in microglia through a kinase-,
phosphoinositide-, and nucleotide-dependent mechanism. J Neurosci
Res 2006; 83: 1471-84. [0414] 44. Dorr P, Westby M, Dobbs S,
Griffin P, Irvine B, Macartney M, et al. Maraviroc (UK-427,857), a
potent, orally bioavailable, and selective small-molecule inhibitor
of chemokine receptor CCR5 with broad-spectrum anti-human
immunodeficiency virus type 1 activity. Antimicrob Agents Chemother
2005; 49: 4721-32. [0415] 45. Strizki J M, Tremblay C, Xu S, Wojcik
L, Wagner N, Gonsiorek W, et al. Discovery and characterization of
vicriviroc (SCH 417690), a CCR5 antagonist with potent activity
against human immunodeficiency virus type 1. Antimicrob Agents
Chemother 2005; 49: 4911-9. [0416] 46. Isik N, Hereld D, Jin T .
Fluorescence resonance energy transfer imaging reveals that
chemokine-binding modulates heterodimers of CXCR4 and CCR5
receptors. PLoS ONE 2008; 3: e3424. [0417] 47. Mira E, Lacalle R A,
Gonzalez M A, Gomez-Mouton C, Abad J L, Bernad A, et al. A role for
chemokine receptor transactivation in growth factor signaling. EMBO
Rep 2001; 2: 151-6. [0418] 48. Thirkill T L, Lowe K, Vedagiri H,
Blankenship T N, Barakat A I, Douglas G C . Macaque trophoblast
migration is regulated by RANTES. Exp Cell Res 2005; 305: 355-64.
[0419] 49. Abel S, van der Ryst E, Rosario M C, Ridgway C E,
Medhurst C G, Taylor-Worth R J, et al. Assessment of the
pharmacokinetics, safety and tolerability of maraviroc, a novel
CCR5 antagonist, in healthy volunteers. Br J Clin Pharmacol 2008;
65 Suppl 1: 5-18. [0420] 50. Vaday G G, Peehl D M, Kadam P A,
Lawrence D M. Expression of CCL5 (RANTES) and CCR5 in prostate
cancer. Prostate 2006; 66: 124-34. [0421] 51. Perret G Y, Crepin M
. New pharmacological strategies against metastatic spread. Fundam
Clin Pharmacol 2008; 22: 465-92. [0422] 52. Sugasawa H, Ichikura T,
Tsujimoto H, Kinoshita M, Morita D, Ono S, et al. Prognostic
significance of expression of CCLS/RANTES receptors in patients
with gastric cancer. J Surg Oncol 2008; 97: 445-50.
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