U.S. patent application number 15/542480 was filed with the patent office on 2018-10-25 for compositions and methods for enhancing oncolytic virus efficacy.
This patent application is currently assigned to Ottawa Hospital Research Institute. The applicant listed for this patent is Ottawa Hospital Research Institute, University of Ottawa. Invention is credited to John C. Bell, Christopher Noyce Boddy, Jean-Simon Diallo, Mark Dornan, Ramya Krishnan, Fabrice Le Boeuf, Andrew Macklin, Jeffrey Smith.
Application Number | 20180305345 15/542480 |
Document ID | / |
Family ID | 56542075 |
Filed Date | 2018-10-25 |
United States Patent
Application |
20180305345 |
Kind Code |
A1 |
Diallo; Jean-Simon ; et
al. |
October 25, 2018 |
COMPOSITIONS AND METHODS FOR ENHANCING ONCOLYTIC VIRUS EFFICACY
Abstract
Provided are compounds that enhance the efficacy of viruses by
increasing spread of the virus in cells, increasing the titer of
virus in cells, or increasing the cytotoxicity of virus to cells.
Other uses, compositions and methods of using same are also
provided.
Inventors: |
Diallo; Jean-Simon;
(Ontario, CA) ; Boddy; Christopher Noyce; (Ottawa,
CA) ; Dornan; Mark; (Ottawa, CA) ; Krishnan;
Ramya; (Ottawa, CA) ; Le Boeuf; Fabrice;
(Gatineau, CA) ; Bell; John C.; (Ottawa, CA)
; Macklin; Andrew; (Ottawa, CA) ; Smith;
Jeffrey; (Ottawa, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Ottawa Hospital Research Institute
University of Ottawa |
Ottawa
Ottawa |
|
CA
CA |
|
|
Assignee: |
Ottawa Hospital Research
Institute
Ottawa
ON
University of Ottawa
Ottawa
ON
|
Family ID: |
56542075 |
Appl. No.: |
15/542480 |
Filed: |
January 26, 2016 |
PCT Filed: |
January 26, 2016 |
PCT NO: |
PCT/CA2016/050062 |
371 Date: |
July 10, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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62107923 |
Jan 26, 2015 |
|
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62107908 |
Jan 26, 2015 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07D 471/04 20130101;
A61K 45/06 20130101; C07D 405/04 20130101; C12N 2760/16134
20130101; C07D 237/14 20130101; C07D 317/64 20130101; C07D 413/06
20130101; C12N 2760/16151 20130101; C12N 2760/20234 20130101; C07D
307/58 20130101; C12N 2710/16651 20130101; C07D 231/08 20130101;
C12N 2710/10334 20130101; C07D 307/30 20130101; C12N 7/00 20130101;
C07D 209/48 20130101; C07D 307/66 20130101; C12N 2710/10351
20130101; C12N 2750/14151 20130101; C07D 409/06 20130101; A61K
31/341 20130101; C07D 403/06 20130101; C07D 417/06 20130101; C12N
2760/20252 20130101; A61P 35/02 20180101; C12N 2760/20251 20130101;
C07D 207/38 20130101; C07D 401/06 20130101; A61K 2300/00 20130101;
C12N 2710/24151 20130101; C12N 2760/16152 20130101; C12N 2710/10352
20130101; C07D 405/06 20130101; C07D 405/12 20130101; C07D 207/36
20130101; C07D 207/456 20130101; C07D 307/60 20130101 |
International
Class: |
C07D 413/06 20060101
C07D413/06; C07D 207/38 20060101 C07D207/38; C07D 207/456 20060101
C07D207/456; C07D 209/48 20060101 C07D209/48; C07D 231/08 20060101
C07D231/08; C07D 237/14 20060101 C07D237/14; C07D 307/58 20060101
C07D307/58; C07D 307/60 20060101 C07D307/60; C07D 307/66 20060101
C07D307/66; C07D 317/64 20060101 C07D317/64; C07D 401/06 20060101
C07D401/06; C07D 403/06 20060101 C07D403/06; C07D 405/04 20060101
C07D405/04; C07D 405/12 20060101 C07D405/12; C07D 409/06 20060101
C07D409/06; C07D 471/04 20060101 C07D471/04; A61P 35/02 20060101
A61P035/02 |
Claims
1. A method of enhancing or increasing the infection, spread,
titer, or cytotoxicity of an oncolytic virus in cancer or tumor
cells, comprising administering a compound, or a combination of
compounds, defined by formula (I): ##STR00138## an N-oxide,
pharmaceutically acceptable addition salt, quarternary amine or
stereochemically isomeric form thereof, wherein: A is a 5-membered
heterocyclic ring comprising 0 or 1 double bond and 1 heteroatom
selected from O, substituted or unsubstituted N; R.sup.1 and
R.sup.4 are each independently H, oxo, hydroxyl, alkynyloxy,
phenyl, substituted phenyl, benzyl, substituted benzyl, triazolyl,
substituted triazolyl, or indolyl; R.sup.2 and R.sup.3 are each
independently hydrogen, halogen, alkynylamino, isobutylamino, or
benzylamino; to said cells prior to, after, or concurrently with
infection of the cells with the virus.
2. A method of enhancing or increasing the infection, spread,
titer, or cytotoxicity of an oncolytic virus in cancer or tumor
cells, comprising administering a compound, or a combination of
compounds, selected from the group consisting of:
.alpha.,.beta.-dichloro-.gamma.-hydroxy-N-benzyl-crotonic lactam;
3,4-dichloro-5-prop-2-ynyloxy-5H-furan-2-one;
3,4-dibromo-5-prop-2-ynyloxy-5H-furan-2-one;
3-chloro-5-phenyl-4-prop-2-ynylamino-5H-furan-2-one;
3-chloro-4-isobutylamino-5-prop-2-ynyloxy-5H-furan-2-one;
1-benzyl-3,4-dichloro-5-prop-2-ynyloxy-1,5-dihydro-pyrrol-2-one;
3,4-dichloro-5-hydroxy-1-(2-methoxy-benzyl)-1,5-dihydro-pyrrol-2-one;
4-benzylamino-3-chloro-5-prop-2-ynyloxy-5H-furan-2-one;
3,4-dichloro-5-hydroxy-1-prop-2-ynyl-1,5-dihydro-pyrrol-2-one;
3,4-dichloro-5H-furan-2-one; benzo[1,3]dioxole-5,6-dione;
4,5-dichloro-2H-pyridazin-3-one;
4,5-dichloro-2-phenyl-2H-pyridazin-3-one;
3,4-dichloro-1-phenyl-pyrrole-2,5-dione;
3,4-dichloro-5-(1H-indol-3-yl)-5H-furan-2-one, indole-3-crotonic
acid; 3,4-dichloro-5-hydroxy-1,5-dihydro-pyrrol-2-one;
5-phenyl-4,5-dihydro-3aH-pyrrolo[1,2-a]quinolin-1-one;
5-phenyl-4,5-dihydro-3aH-pyrrolo[1,2-a]quinolin-1-one;
3,4-dichloro-5-(3-nitro-phenyl)-5H-furan-2-one;
3,4-dichloro-5-hydroxy-1-methyl-1,5-dihydro-pyrrol-2-one;
3,4-dichloro-1-prop-2-ynyl-5-prop-2-ynyloxy-1,5-dihydro-pyrrol-2-one;
3,4-dichloro-1-(2-chloro-benzyl)-5-hydroxy-1,5-dihydro-pyrrol-2-one;
3,4-dichloro-5-hydroxy-1-propyl-1,5-dihydro-pyrrol-2-one;
1-phenyl-pyrrole-2,5-dione;
3,4-dichloro-1-propyl-pyrrole-2,5-dione;
1-benzyl-3,4-dichloro-pyrrole-2,5-dione;
3,4-dichloro-5-hydroxy-1-phenyl-1,5-dihydro-pyrrol-2-one;
1-(1-benzyl-1H-[1,2,3]triazol-4-ylmethyl)-3,4-dichloro-5-hydroxy-1,5-dihy-
dro-pyrrol-2-one;
[4-(4-chloro-3-isobutylamino-5-oxo-2,5-dihydro-furan-2-yloxymethyl)-[1,2,-
3]triazol-1-yl]-acetic acid;
[4-(3,4-dichloro-2-hydroxy-5-oxo-2,5-dihydro-pyrrol-1-ylmethyl)-[1,2,3]tr-
iazol-1-yl]-acetic acid;
3,4-dichloro-5-hydroxy-1-phenethyl-1H-pyrrol-2(5H)-one;
3,4-dichloro-5-hydroxy-1-(2-morpholinoethyl)-1H-pyrrol-2(5H)-one;
3,4-dichloro-1-cyclopropyl-5-hydroxy-1H-pyrrol-2(5H)-one;
3,4-dichloro-5-hydroxy-1-(2-mercaptoethyl)-1H-pyrrol-2(5H)-one;
2-(3,4-dichloro-2-hydroxy-5-oxo-2,5-dihydro-1H-pyrrol-1-yl)ethanaminium
2,2,2-trifluoroacetate;
3,4-dichloro-5-hydroxy-1-(3-phenylprop-2-ynyl)-1H-pyrrol-2(5H)-one;
3,4-dichloro-5-hydroxy-1-(4-(trifluoromethyl)benzyl)-1H-pyrrol-2(5H)-one;
1-(biphenyl-4-ylmethyl)-3,4-dichloro-5-hydroxy-1H-pyrrol-2(5H)-one;
3,4-dichloro-5-hydroxy-1-(4-nitrobenzyl)-1H-pyrrol-2(5H)-one;
3,4-dichloro-5-hydroxy-1-(2-methoxybenzyl)-1H-pyrrol-2(5H)-one;
3,4-dichloro-1-(2-chlorobenzyl)-5-hydroxy-1H-pyrrol-2(5H)-one;
1-benzhydryl-3,4-dichloro-5-hydroxy-1H-pyrrol-2(5H)-one;
3,4-dichloro-5-hydroxy-1-(naphthalen-1-ylmethyl)-1H-pyrrol-2(5H)-one;
3,4-dichloro-5-hydroxy-1-(1-phenylethyl)-1H-pyrrol-2(5H)-one;
3,4-dichloro-5-hydroxy-1-(pyridin-3-ylmethyl)-1H-pyrrol-2(5H)-one;
3,4-dichloro-5-hydroxy-1-(pyridin-4-ylmethyl)-1H-pyrrol-2(5H)-one;
3,4-dichloro-5-hydroxy-1-(pyridin-2-ylmethyl)-1H-pyrrol-2(5H)-one;
3,4-dichloro-1-(furan-2-ylmethyl)-5-hydroxy-1H-pyrrol-2(5H)-one;
N-(2-(3,4-dichloro-2-hydroxy-5-oxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl)-5-(d-
imethylamino)naphthalene-1-sulfonamide;
(3aS)-2,3-dichloro-5-phenyl-4,5-dihydropyrrolo[1,2-a]quinolin-1(3aH)-one;
3,4-diiodo-2-phenyl-2,5-dihydrofuran; D-Gluconamide, N-octyl;
(S)-11-amino-4,7,10,14-tetraoxo-15-((2R,3R,4R,5R,6R)-3,4,5-trihydroxy-6-(-
hydroxymethyl)tetrahydro-2H-pyran-2-yl)-3,6,9,13-tetraazapentadecan-1-oic
acid; 1-allyl-3,4-dichloro-5-hydroxy-1H-pyrrol-2(5H)-one;
3,4-dichloro-5-hydroxy-1-(2-hydroxybenzyl)-1H-pyrrol-2(5H)-one;
3,4-dichloro-5-hydroxy-1-(thiophen-2-ylmethyl)-1H-pyrrol-2(5H)-one;
3,4-dichloro-5-hydroxy-1-(4-(methylsulfonyl)benzyl)-1H-pyrrol-2(5H)-one;
3,4-dichloro-1-((4,5-dimethyloxazol-2-yl)methyl)-5-hydroxy-1H-pyrrol-2(5H-
)-one;
3,4-dichloro-5-hydroxy-1-(3,4,5-trifluorobenzyl)-1H-pyrrol-2(5H)-on-
e; 3,4-dichloro-5-hydroxy-1-(4-methoxybenzyl)-1H-pyrrol-2(5H)-one;
4,5-dichloro-2-(2,2,2-trifluoroethyl)pyridazin-3(2H)-one;
4,5-dichloro-2-cyclohexylpyridazin-3(2H)-one; methyl
2-(4-((3,4-dichloro-2-hydroxy-5-oxo-2,5-dihydro-1H-pyrrol-1-yl)methyl)-1H-
-1,2,3-triazol-1-yl)acetate;
4,5-dichloro-2-o-tolylpyridazin-3(2H)-one;
4,5-dichloro-2-(2-(dimethylamino)ethyl)pyridazin-3(2H)-one
hydrochloride; and
4,5-dichloro-2-(4-fluorophenyl)pyridazin-3(2H)-one or any compound
or group of compounds defined in Table 1; to said cells prior to,
after, or concurrently with infection of the cells with the
virus.
3. A method of enhancing or increasing the infection, spread,
titer, or cytotoxicity of an oncolytic virus in cancer or tumor
cells, comprising administering a compound, or a combination of
compounds, defined by formula (VIII): ##STR00139## or a
pharmaceutically acceptable salt, or stereochemically isomeric form
thereof, wherein: X.sub.12 is O, NH, or substituted N; X.sub.13 is
halogen such as Cl, or NHX.sub.14, wherein X.sub.14 is a
substituted or unsubstituted linear or branched alkyl, alkenyl, or
alkynyl, or substituted or unsubstituted aryl or heteroaryl; and
X.sub.15 is H, OH, .dbd.O, substituted or unsubstituted mono- or
bi-cycloaryl or -heteroaryl such as substituted or unsubstituted
phenyl, or OX.sub.16, wherein X.sub.16 is H, linear or branched
substituted or unsubstituted alkyl, alkenyl, alkynyl, or acyl, or
X.sub.16 is acetyl, methyl, or --CH.sub.2--C.ident.CH; to said
cells prior to, after, or concurrently with infection of the cells
with the virus.
4. The method according to claim 3, wherein the compound, or at
least one of the compounds of the combination of compounds, is
defined by formula (III): ##STR00140## wherein X.sub.5 is H,
substituted or unsubstituted linear or branched C.sub.1-C.sub.12
alkyl, alkenyl, or alkynyl, substituted or unsubstituted mono- or
bi-cycloaryl or -heteroaryl, substituted or unsubstituted
cycloalkyl or heterocycloalkyl, or wherein X.sub.5 is substituted
or unsubstituted alkynyloxy, phenyl, alkylphenyl, substituted
phenyl, benzyl, substituted benzyl, triazolyl, substituted
triazolyl, naphthalenyl, substituted naphthalenyl, substituted or
unsubstituted pyridinyl, substituted or unsubstituted furanyl or
thiofuranyl, thiophenyl, sulfonobenzyl, methylsulfonobenzyl,
pyrrolyl, substituted or unsubstituted morpholine, cycloalkyl,
alkylthiol, substituted or unsubstituted alkyamine, or substituted
or unsubstituted oxazoline.
5. The method according to claim 3, wherein the compound, or at
least one of the compounds of the combination of compounds, is
defined by formula (IV): ##STR00141## wherein X.sub.6 is H,
substituted or unsubstituted linear or branched alkyl, alkenyl,
alkynyl, or acyl, or wherein X.sub.6 is substituted or
unsubstituted methyl, alkyl triazolyl, acetyl, or
--CH.sub.2--C.ident.CH.
6. The method according to claim 3, wherein the compound, or at
least one of the compounds of the combination of compounds, is
defined by formula (VI): ##STR00142## wherein X.sub.8 is
substituted or unsubstituted linear or branched alkyl, alkenyl, or
alkyny, or substituted or unsubstituted aryl or heteroaryl, or
X.sub.8 is substituted or unsubstituted benzyl.
7. The method according to claim 3, wherein the compound, or at
least one of the compounds of the combination of compounds, is
defined by formula (VII): ##STR00143## wherein X.sub.9 is H, OH,
OX.sub.11, or .dbd.O, wherein X.sub.11 is H, substituted or
unsubstituted linear or branched alkyl, alkenyl, alkynyl, or acyl,
or X.sub.11 is acetyl, methyl, or --CH.sub.2--C.ident.CH.
8-10. (canceled)
11. The method of claim 1, wherein the cells are cancer cells in
vivo, or in vitro.
12. The method of claim 11 wherein the in vivo cancer cells are
from a mammalian subject.
13. The method of claim 8, wherein the mammalian subject is a human
subject.
14. A method of increasing the oncolytic activity of an oncolytic
virus in cancer or tumor cells, comprising administering a
compound, or a combination of compounds, as defined in claim 1 to
said cancer or tumor cells prior to, concurrently with or after the
oncolytic virus.
15. The method of claim 14, wherein the cancer cells are in vivo,
or in vitro.
16. The method of claim 14 wherein the in vivo cancer cells are
from a mammalian subject.
17. The method of claim 16, wherein the mammalian subject is a
human subject.
18-21. (canceled)
22. The method of claim 1, wherein the compound, or at least one of
the compounds of the combination of compounds, exhibits a
degradation half-life greater than 20, 40, 60, 80, 100, 120, 240,
360 minutes or more in phosphate buffer at pH 7.4.
23. The method of claim 1, wherein the compound, or at least one of
the compounds of the combination of compounds, exhibits a half-life
greater than 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70,
75, 80, 85, 90, 95, 100, 200, 300, 400 minutes or more in a
glutathione stability assay.
24. The method of claim 1, wherein the compound, or at least one of
the compounds of the combination of compounds, exhibits a lower
LD50 in the presence of an oncolytic virus than its absence.
25. The method of claim 24, wherein the difference in LD50 is 10,
20, 30, 40, 50, 60, 70, 80, 90, 100 .mu.M or more in the presence
of an oncolytic virus compared to its absence.
26. The method of claim 1, wherein the compound, or at least one of
the compounds of the combination of compounds, has a viral
sensitizer activity on VSV.DELTA.51 in 786-0 cells which is about
0.01 or greater when reported as peak fold change in viral
expression unit normalized to
3,4-dichloro-5-phenyl-2,5-dihydrofuran-2-one.
27. The method of claim 1, wherein the method further comprises
administration of an anticancer agent or cancer therapeutic.
28. A method of enhancing or increasing the infection, spread,
titer, or cytotoxicity of an oncolytic virus in cancer or tumor
cells, comprising administering VSe1 and MD03011, ##STR00144## to
said cells prior to, after, or concurrently with infection of the
cells with the virus.
Description
FIELD OF INVENTION
[0001] The present invention relates to compounds, methods, and
compositions that enhance viral infection, growth, spread or
virus-induced cytotoxicity.
BACKGROUND OF THE INVENTION
[0002] Genetically attenuated viruses form the basis of a growing
number of biotechnology and pharmaceutical platforms. Emerging in
the field of cancer therapeutics, oncolytic virotherapy has shown
significant promise over the last decade. A number of oncolytic
viruses (OV) based on a wide range of viral backbones from small
RNA viruses (eg. rhabdoviruses), to large DNA viruses (eg.
poxviruses, herpesviruses) are currently being evaluated in
clinical trials to treat a range of cancer types. Generating
substantial excitement for this form of cancer therapy, approval of
the first-in-class OV based on herpes-simplex virus-1 (HSV-1) for
treatment of melanoma is expected in the next year.
[0003] Oncolytic viruses (OVs) are self-amplifying biotherapeutic
agents that have been selected or engineered to preferentially
infect and kill cancer cells. When effective, OVs lead to tumor
eradication not only by direct lysis of cancer cells but also
through downstream generation of anti-cancer immune responses,
vascular shutdown, and therapeutic transgene expression. As a basis
for their selectivity, OVs exploit cellular defects that are
inherent to the cancerous phenotype. This includes dysfunctional
anti-viral responses and immune evasion, increased cell
proliferation and metabolism, and leaky tumor vasculature. The
biological environment ensuing from tumorigenesis is well suited to
support the growth of genetically attenuated OVs that are otherwise
harmless to normal cells.
[0004] OVs stand to be an attractive therapeutic modality for
cancer because of their curative potential and their relatively
mild side effects amounting to acute flu-like symptoms. However,
heterogeneity in the clinical response to OVs remains a significant
hurdle to overcome, as demonstrated in several human clinical
trials. This heterogeneity in response may be attributed to factors
that impede effective OV delivery and spread within tumors.
[0005] It is well recognized in the OV field that viral engineering
or combinations of therapeutic modalities are critical areas that
need to be explored to improve efficacy. Recently, a
high-throughput small molecule phenotypic screen was conducted to
identify compounds that sensitize resistant cancer cells to
infection with the rhabdovirus-based OV named VSV.DELTA.51.
VSV.DELTA.51 is an engineered mutant of vesicular stomatitis virus
(VSV) that is highly sensitive to interferon (IFN) and its
antiviral effects. Much like HSV-1 and many other OVs, VSV.DELTA.51
faces a roadblock in tumors that retain effective cellular
antiviral responses.
[0006] VSe1 (3,4-dichloro-5-phenyl-2,5-dihydrofuran-2-one, FIG. 1a,
also referred to as compound 1 herein) emerged as one of the most
active hits from this phenotypic screen and was shown to enhance
VSV.DELTA.51 oncolysis in vitro and in vivo, increasing virus
output by as much as 1,000-fold preferentially in cancer cells.
While the molecular target remains elusive, the compound has been
shown to dampen the activation of antiviral responses--most
prominently the transcriptional response to type I IFN. Given the
potential use of VSe1 for co-administration with OVs in humans, the
electrophilic nature of VSe1 prompted us to investigate the
scaffold to identify active analogues with more favourable
physiochemical properties and explore structure-activity
relationships. In this study we report the development of a new
class of small molecules derived from VSe1 that significantly
enhances OV propagation leading to oncolysis in resistant cancers.
Other beneficial effects are also demonstrated.
[0007] The prior art associated with this subject area includes the
subject matter encompassed by references 1-74 (and citations
therein) provided in the reference section of this document. All of
the citations are herein incorporated by reference in their
entirety.
SUMMARY OF THE INVENTION
[0008] The present invention relates to compounds, compositions and
methods that enhance viral infection, growth, spread or
cytotoxicity.
[0009] In an embodiment, there is provided herein a method of
enhancing or increasing the infection, spread, titer, or
cytotoxicity of an oncolytic virus in cancer or tumor cells,
comprising administering a compound, or a combination of compounds,
defined by formula (I):
##STR00001## [0010] an N-oxide, pharmaceutically acceptable
addition salt, quarternary amine or stereochemically isomeric form
thereof, wherein: [0011] A is a 5-membered heterocyclic ring
comprising 0 or 1 double bond and 1 heteroatom selected from O,
substituted or unsubstituted N; [0012] R.sup.1 and R.sup.4 are each
independently H, oxo, hydroxyl, alkynyloxy, phenyl, substituted
phenyl, benzyl, substituted benzyl, triazolyl, substituted
triazolyl, or indolyl; and [0013] R.sup.2 and R.sup.3 are each
independently hydrogen, halogen, alkynylamino, isobutylamino, or
benzylamino; to said cells prior to, after, or concurrently with
infection of the cells with the virus.
[0014] In another embodiment, there is provided herein a method of
enhancing or increasing the infection, spread, titer, or
cytotoxicity of an oncolytic virus in cancer or tumor cells,
comprising administering a compound, or a combination of compounds,
selected from the group consisting of: [0015]
.alpha.,.beta.-dichloro-.gamma.-hydroxy-N-benzyl-crotonic lactam;
3,4-dichloro-5-prop-2-ynyloxy-5H-furan-2-one;
3,4-dibromo-5-prop-2-ynyloxy-5H-furan-2-one;
3-chloro-5-phenyl-4-prop-2-ynylamino-5H-furan-2-one;
3-chloro-4-isobutylamino-5-prop-2-ynyloxy-5H-furan-2-one;
1-benzyl-3,4-dichloro-5-prop-2-ynyloxy-1,5-dihydro-pyrrol-2-one;
3,4-dichloro-5-hydroxy-1-(2-methoxy-benzyl)-1,5-dihydro-pyrrol-2-one;
4-benzylamino-3-chloro-5-prop-2-ynyloxy-5H-furan-2-one;
3,4-dichloro-5-hydroxy-1-prop-2-ynyl-1,5-dihydro-pyrrol-2-one;
3,4-dichloro-5H-furan-2-one; benzo[1,3]dioxole-5,6-dione;
4,5-dichloro-2H-pyridazin-3-one;
4,5-dichloro-2-phenyl-2H-pyridazin-3-one;
3,4-dichloro-1-phenyl-pyrrole-2,5-dione;
3,4-dichloro-5-(1H-indol-3-yl)-5H-furan-2-one, indole-3-crotonic
acid; 3,4-dichloro-5-hydroxy-1,5-dihydro-pyrrol-2-one;
5-phenyl-4,5-dihydro-3aH-pyrrolo[1,2-a]quinolin-1-one;
5-phenyl-4,5-dihydro-3aH-pyrrolo[1,2-a]quinolin-1-one;
3,4-dichloro-5-(3-nitro-phenyl)-5H-furan-2-one;
3,4-dichloro-5-hydroxy-1-methyl-1,5-dihydro-pyrrol-2-one;
3,4-dichloro-1-prop-2-ynyl-5-prop-2-ynyloxy-1,5-dihydro-pyrrol-2-one;
3,4-dichloro-1-(2-chloro-benzyl)-5-hydroxy-1,5-dihydro-pyrrol-2-one;
3,4-dichloro-5-hydroxy-1-propyl-1,5-dihydro-pyrrol-2-one;
1-phenyl-pyrrole-2,5-dione;
3,4-dichloro-1-propyl-pyrrole-2,5-dione;
1-benzyl-3,4-dichloro-pyrrole-2,5-dione;
3,4-dichloro-5-hydroxy-1-phenyl-1,5-dihydro-pyrrol-2-one;
1-(1-benzyl-1H-[1,2,3]triazol-4-ylmethyl)-3,4-dichloro-5-hydroxy-1,5-dihy-
dro-pyrrol-2-one;
[4-(4-chloro-3-isobutylamino-5-oxo-2,5-dihydro-furan-2-yloxymethyl)-[1,2,-
3]triazol-1-yl]-acetic acid;
[4-(3,4-dichloro-2-hydroxy-5-oxo-2,5-dihydro-pyrrol-1-ylmethyl)-[1,2,3]tr-
iazol-1-yl]-acetic acid;
3,4-dichloro-5-hydroxy-1-phenethyl-1H-pyrrol-2(5H)-one;
3,4-dichloro-5-hydroxy-1-(2-morpholinoethyl)-1H-pyrrol-2(5H)-one;
3,4-dichloro-1-cyclopropyl-5-hydroxy-1H-pyrrol-2(5H)-one;
3,4-dichloro-5-hydroxy-1-(2-mercaptoethyl)-1H-pyrrol-2(5H)-one;
2-(3,4-dichloro-2-hydroxy-5-oxo-2,5-dihydro-1H-pyrrol-1-yl)ethanaminium
2,2,2-trifluoroacetate;
3,4-dichloro-5-hydroxy-1-(3-phenylprop-2-ynyl)-1H-pyrrol-2(5H)-one;
3,4-dichloro-5-hydroxy-1-(4-(trifluoromethyl)benzyl)-1H-pyrrol-2(5H)-one;
1-(biphenyl-4-ylmethyl)-3,4-dichloro-5-hydroxy-1H-pyrrol-2(5H)-one;
3,4-dichloro-5-hydroxy-1-(4-nitrobenzyl)-1H-pyrrol-2(5H)-one;
3,4-dichloro-5-hydroxy-1-(2-methoxybenzyl)-1H-pyrrol-2(5H)-one;
3,4-dichloro-1-(2-chlorobenzyl)-5-hydroxy-1H-pyrrol-2(5H)-one;
1-benzhydryl-3,4-dichloro-5-hydroxy-1H-pyrrol-2(5H)-one;
3,4-dichloro-5-hydroxy-1-(naphthalen-1-ylmethyl)-1H-pyrrol-2(5H)-one;
3,4-dichloro-5-hydroxy-1-(1-phenylethyl)-1H-pyrrol-2(5H)-one;
3,4-dichloro-5-hydroxy-1-(pyridin-3-ylmethyl)-1H-pyrrol-2(5H)-one;
3,4-dichloro-5-hydroxy-1-(pyridin-4-ylmethyl)-1H-pyrrol-2(5H)-one;
3,4-dichloro-5-hydroxy-1-(pyridin-2-ylmethyl)-1H-pyrrol-2(5H)-one;
3,4-dichloro-1-(furan-2-ylmethyl)-5-hydroxy-1H-pyrrol-2(5H)-one;
N-(2-(3,4-dichloro-2-hydroxy-5-oxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl)-5-(d-
imethylamino)naphthalene-1-sulfonamide;
(3aS)-2,3-dichloro-5-phenyl-4,5-dihydropyrrolo[1,2-a]quinolin-1(3
aH)-one; 3,4-diiodo-2-phenyl-2,5-dihydrofuran; D-Gluconamide,
N-octyl;
(S)-11-amino-4,7,10,14-tetraoxo-15-((2R,3R,4R,5R,6R)-3,4,5-trihydroxy-6-(-
hydroxymethyl)tetrahydro-2H-pyran-2-yl)-3,6,9,13-tetraazapentadecan-1-oic
acid; 1-allyl-3,4-dichloro-5-hydroxy-1H-pyrrol-2(5H)-one;
3,4-dichloro-5-hydroxy-1-(2-hydroxybenzyl)-1H-pyrrol-2(5H)-one;
3,4-dichloro-5-hydroxy-1-(thiophen-2-ylmethyl)-1H-pyrrol-2(5H)-one;
3,4-dichloro-5-hydroxy-1-(4-(methylsulfonyl)benzyl)-1H-pyrrol-2(5H)-one;
3,4-dichloro-1-((4,5-dimethyloxazol-2-yl)methyl)-5-hydroxy-1H-pyrrol-2(5H-
)-one;
3,4-dichloro-5-hydroxy-1-(3,4,5-trifluorobenzyl)-1H-pyrrol-2(5H)-on-
e; 3,4-dichloro-5-hydroxy-1-(4-methoxybenzyl)-1H-pyrrol-2(5H)-one;
4,5-dichloro-2-(2,2,2-trifluoroethyl)pyridazin-3 (2H)-one;
4,5-dichloro-2-cyclohexylpyridazin-3(2H)-one; methyl
2-(4-((3,4-dichloro-2-hydroxy-5-oxo-2,5-dihydro-1H-pyrrol-1-yl)methyl)-1H-
-1,2,3-triazol-1-yl)acetate;
4,5-dichloro-2-o-tolylpyridazin-3(2H)-one;
4,5-dichloro-2-(2-(dimethylamino)ethyl)pyridazin-3(2H)-one
hydrochloride; and
4,5-dichloro-2-(4-fluorophenyl)pyridazin-3(2H)-one or any compound
or group of compounds defined in Table 1; to said cells prior to,
after, or concurrently with infection of the cells with the
virus.
[0016] In another embodiment, there is provided herein a method of
enhancing or increasing the infection, spread, titer, or
cytotoxicity of an oncolytic virus in cancer or tumor cells,
comprising administering a compound, or a combination of compounds,
defined by formula (II):
##STR00002##
or [0017] a pharmaceutically acceptable salt, or stereochemically
isomeric form thereof, wherein: [0018] X.sub.1 is a heteroatom such
as O, NH, or substituted N; [0019] X.sub.2 is halogen (such as, for
example, Cl), or NHX.sub.3, wherein X.sub.3 is a substituted or
unsubstituted linear or branched alkyl, alkenyl, or alkynyl, or
substituted or unsubstituted aryl or heteroaryl; [0020] i is 0 when
X.sub.1 is O, or 0 or 1 when X.sub.1 is NH or substituted N; [0021]
represents a double bond which is present when i is 1, and absent
when i is 0 such that X.sub.1 is directly bonded to the
X.sub.4-bearing carbon through a single bond when i is 0; and
[0022] X.sub.4 is H, OH, .dbd.O, substituted or unsubstituted mono-
or bi-cycloaryl or -heteroaryl (such as, for example, substituted
or unsubstituted phenyl), or OX.sub.10, wherein X.sub.10 is H,
linear or branched substituted or unsubstituted alkyl, alkenyl,
alkynyl, or acyl (for example, X.sub.10 may be acetyl, methyl, or
--CH.sub.2--C.ident.CH); to said cells prior to, after, or
concurrently with infection of the cells with the virus.
[0023] In yet another embodiment, there is provided herein a method
of enhancing or increasing the infection, spread, titer, or
cytotoxicity of an oncolytic virus in cancer or tumor cells,
comprising administering a compound, or a combination of compounds,
defined by formula (VIII):
##STR00003## [0024] or [0025] a pharmaceutically acceptable salt,
or stereochemically isomeric form thereof, wherein: [0026] X.sub.12
is O, NH, or substituted N; [0027] X.sub.13 is halogen such as Cl,
or NHX.sub.14, wherein X.sub.14 is a substituted or unsubstituted
linear or branched alkyl, alkenyl, or alkynyl, or substituted or
unsubstituted aryl or heteroaryl; and [0028] X.sub.15 is H, OH,
.dbd.O, substituted or unsubstituted mono- or bi-cycloaryl or
-heteroaryl such as substituted or unsubstituted phenyl, or
OX.sub.16, wherein X.sub.16 is H, linear or branched substituted or
unsubstituted alkyl, alkenyl, alkynyl, or acyl, or X.sub.16 is
acetyl, methyl, or --CH.sub.2--C.ident.CH; to said cells prior to,
after, or concurrently with infection of the cells with the
virus.
[0029] In another embodiment of any one of the method or methods
described herein, the compound, or at least one of the compounds of
the combination of compounds, may be defined by formula (III):
##STR00004##
or [0030] a pharmaceutically acceptable salt, or stereochemically
isomeric form thereof, wherein: [0031] X.sub.5 is H, substituted or
unsubstituted linear or branched C.sub.1-C.sub.12 alkyl, alkenyl,
or alkynyl, substituted or unsubstituted mono- or bi-cycloaryl or
-heteroaryl, substituted or unsubstituted cycloalkyl or
heterocycloalkyl, or wherein X.sub.5 is substituted or
unsubstituted alkynyloxy, phenyl, alkylphenyl, substituted phenyl,
benzyl, substituted benzyl, triazolyl, substituted triazolyl,
naphthalenyl, substituted naphthalenyl, substituted or
unsubstituted pyridinyl, substituted or unsubstituted furanyl or
thiofuranyl, thiophenyl, sulfonobenzyl, methylsulfonobenzyl,
pyrrolyl, substituted or unsubstituted morpholine, cycloalkyl,
alkylthiol, substituted or unsubstituted alkyamine, or substituted
or unsubstituted oxazoline.
[0032] In yet another embodiment of any one of the method or
methods described herein, the compound, or at least one of the
compounds of the combination of compounds, may be defined by
formula (IV):
##STR00005##
or a pharmaceutically acceptable salt, or stereochemically isomeric
form thereof, wherein: [0033] wherein X.sub.6 is H, substituted or
unsubstituted linear or branched alkyl, alkenyl, alkynyl, or acyl,
or wherein X.sub.6 is substituted or unsubstituted methyl, alkyl
triazolyl, acetyl, or --CH.sub.2--C.ident.CH.
[0034] In yet another embodiment of any one of the method or
methods described herein, the compound, or at least one of the
compounds of the combination of compounds, may be defined by
formula (V):
##STR00006##
or [0035] a pharmaceutically acceptable salt, or stereochemically
isomeric form thereof, wherein: [0036] X.sub.7 is H, substituted or
unsubstituted aryl or heteroaryl, substituted or unsubstituted
linear or branched alkyl, alkenyl, or alkynyl, or substituted or
unsubstituted cycloalkyl. For example, X.sub.7 may be substituted
or unsubstituted alkylamine, or substituted or unsubstituted
phenyl.
[0037] In yet another embodiment of any one of the method or
methods described herein, the compound, or at least one of the
compounds of the combination of compounds, may be defined by
formula (VI):
##STR00007##
or [0038] a pharmaceutically acceptable salt, or stereochemically
isomeric form thereof, wherein: [0039] wherein X.sub.8 is
substituted or unsubstituted linear or branched alkyl, alkenyl, or
alkyny, or substituted or unsubstituted aryl or heteroaryl, or
X.sub.8 is substituted or unsubstituted benzyl.
[0040] In yet another embodiment of any one of the method or
methods described herein, the compound, or at least one of the
compounds of the combination of compounds, may be defined by
formula (VII):
##STR00008##
or [0041] a pharmaceutically acceptable salt, or stereochemically
isomeric form thereof, wherein: [0042] wherein X.sub.9 is H, OH,
OX.sub.11, or .dbd.O, wherein X.sub.11 is H, substituted or
unsubstituted linear or branched alkyl, alkenyl, alkynyl, or acyl,
or X.sub.11 is acetyl, methyl, or --CH.sub.2--C.ident.CH.
[0043] In still another embodiment of any one of the method or
methods described herein, the compound or combination of compounds
may be present in a composition comprising the compound(s) and a
carrier, diluent or excipient.
[0044] In another embodiment, there is provided herein a
composition comprising one or more of the compounds having a
structure as defined in any of the method or methods as described
above, and one or more of a) a virus, a genetically modified virus,
an attenuated virus, a cancer vaccine, a cancer gene therapy
vector, or an oncolytic virus, b) one or more cancer cells, c) a
carrier, diluent or excipient, d) a pharmaceutically acceptable
carrier, diluent or excipient, e) non-cancer cells; f) cell culture
media; g) one or more cancer therapeutics; or any combination of
a)-g).
[0045] In yet another embodiment, there is provided herein a kit
comprising one or more of the compounds having a structure as
defined in any of the method or methods as described above, and one
or more of a) a virus, a genetically modified virus, an attenuated
virus, a cancer vaccine, a cancer gene therapy vector, or an
oncolytic virus, b) one or more cancer cells, c) a pharmaceutically
acceptable carrier, diluent or excipient, d) non-cancer cells; e)
cell culture media; f) one or more cancer therapeutics, g) a cell
culture plate or multi-well dish; h) an apparatus to deliver the
compound to a cell, medium or to a subject; i) instructions for
using the compound or any component in the kit, j) a carrier
diluent or excipient, or any combination of a)-j).
[0046] In another embodiment, the cells may be cancer cells in
vivo, or in vitro.
[0047] In a further embodiment, the in vivo cancer cells may be
from a mammalian subject.
[0048] In still a further embodiment, the mammalian subject may be
a human subject.
[0049] In yet another embodiment, there is provided herein a method
of increasing the oncolytic activity of an oncolytic virus in
cancer or tumor cells, comprising administering a compound, or a
combination of compounds, having a structure as defined in any of
the method or methods as described above to said cancer or tumor
cells prior to, concurrently with or after the oncolytic virus.
[0050] In another embodiment, the cancer cells may be in vivo, or
in vitro.
[0051] In still another embodiment, the in vivo cancer cells may be
from a mammalian subject.
[0052] In yet another embodiment, the mammalian subject may be a
human subject.
[0053] In another embodiment, there is provided herein a compound
having a structure as defined in any of the method or methods as
described above. In still another embodiment, the compound may be
for use as a viral sensitizer for sensitizing a cancer or tumor
cell to an oncolytic virus.
[0054] In an embodiment, there is provided herein a compound having
a structure as defined in any of the method or methods as described
above, for use as a viral sensitizer to enhance or increase
oncolytic virus activity in cancer or tumor cells.
[0055] In a further embodiment, the compound may be for enhancing
or increasing oncolytic virus titer in cancer or tumor cells.
[0056] In another embodiment, the compound may be for enhancing or
increasing cytotoxicity of the oncolytic virus in cancer or tumor
cells.
[0057] In another embodiment, there is provided herein a compound
having a structure as defined in any of the method or methods
described above, for use in enhancing or increasing the infection,
spread, titer, or cytotoxicity of an oncolytic virus in cancer or
tumor cells.
[0058] In another embodiment, there is provided herein a use of a
compound having a structure as defined in any of the method or
methods described above for enhancing or increasing the infection,
spread, titer, or cytotoxicity of an oncolytic virus in cancer or
tumor cells.
[0059] In another embodiment, there is provided herein a use of a
compound having a structure as defined in any of the method or
methods described above in the manufacture of a medicament for
enhancing or increasing the infection, spread, titer, or
cytotoxicity of an oncolytic virus in cancer or tumor cells.
[0060] In a further embodiment of any of the compounds,
compositions, method or methods described above, the compound, or
at least one of the compounds of the combination of compounds, may
exhibit a degradation half-life greater than 20, 40, 60, 80, 100,
120, 240, 360 minutes or more in phosphate buffer at pH 7.4.
[0061] In another embodiment of any of the compounds, compositions,
method or methods described above, the compound, or at least one of
the compounds of the combination of compounds, may exhibit a
half-life greater than 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55,
60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 300, 400 minutes or more
in a glutathione stability assay.
[0062] In still another embodiment of any of the compounds,
compositions, method or methods described above, the compound, or
at least one of the compounds of the combination of compounds,
exhibits a lower LD50 in the presence of an oncolytic virus than in
its absence.
[0063] In a further embodiment, the difference in LD50 may be 10,
20, 30, 40, 50, 60, 70, 80, 90, 100 .mu.M or more in the presence
of an oncolytic virus compared to its absence.
[0064] In yet another embodiment of any of the compounds,
compositions, method or methods described above, the compound, or
at least one of the compounds of the combination of compounds, may
exhibit a viral sensitizer activity on VSV.DELTA.51 in 786-0 cells
which is about 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08,
0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, or greater,
or any range bounded at a lower end by any one of these values, any
range bounded at an upper end by any one of these values, or any
range falling between any two of these values, when reported as
peak fold change in viral expression unit normalized to
3,4-dichloro-5-phenyl-2,5-dihydrofuran-2-one. In one embodiment,
for example, the compound may be a compound which exhibits a viral
sensitizer activity on VSV.DELTA.51 in 786-0 cells which is greater
than or equal to (.gtoreq.) about 0.01 when reported as peak fold
change in viral expression unit normalized to
3,4-dichloro-5-phenyl-2,5-dihydrofuran-2-one.
[0065] In still another embodiment, a compound as described above
may be a compound for which greater than about 0.5%, 1%, 5%, 10%,
15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 70%, 75%, 80%,
90%, or greater, or any range bounded at a lower end by any one of
these values, any range bounded at an upper end by any one of these
values, or any range falling between any two of these values, of
the compound remains after 3 hour incubation at 37.degree. C. in
aqueous, protein-rich Balb/c mouse plasma buffered 1:1 with pH 7.4
phosphate buffered saline (PBS). For example, a compound as
described above may be a compound for which greater than or equal
to (.gtoreq.) about 0.5% of the compound remains after 3 hour
incubation at 37.degree. C. in aqueous, protein-rich Balb/c mouse
plasma buffered 1:1 with pH 7.4 phosphate buffered saline
(PBS).
[0066] In yet another embodiment, a compound as described above may
be a compound having an LD.sub.50 in the presence of virus which is
greater than or equal to (.gtoreq.) about 1 .mu.m, 5 .mu.m, 10
.mu.m, 15 .mu.m, 20 .mu.m, 25 .mu.m, 30 .mu.m, 35 .mu.m, 40 .mu.m,
45 .mu.m, 50 .mu.m, 55 .mu.m, 60 .mu.m, 65 .mu.m, 70 .mu.m, 75
.mu.m, 80 .mu.m, 85 .mu.m, 90 .mu.m, 95 .mu.m, 100 .mu.m, or 105
.mu.m, or 110 .mu.m, or any range falling between any two of these
values, less than (<) the LD.sub.50 of the same compound in the
absence of virus as determined in, for example, 786-0 cells where
the virus is VSV.
[0067] In still another embodiment of any of the method or methods
described above, the method may further comprise administration of
an anticancer agent or cancer therapeutic.
[0068] In another embodiment, there is provided herein a method of
enhancing or increasing the infection, spread, titer, or
cytotoxicity of an oncolytic virus in cancer or tumor cells,
comprising administering VSe1 and MD03011,
##STR00009##
to said cells prior to, after, or concurrently with infection of
the cells with the virus.
[0069] In an embodiment of any of the method or methods above, the
oncolytic virus may be any suitable oncolytic virus known in the
art which preferentially infects and lyses cancer or tumor cells as
compared to non-cancer or normal cells. Examples of oncolytic
viruses known in the art which may be employed herein may include,
without limitation, reovirus, newcastle disease virus, adenovirus,
herpes virus, polio virus, mumps virus, measles virus, influenza
virus, vaccinia virus, rhabdoviruses such as vesicular stomatitis
virus and derivatives/variants thereof. In a preferred embodiment,
the virus may be a Vesicular stomatitis virus (VSV), or a related
rhabdovirus variant/derivative thereof (such as, for example, a
maraba virus such as MG1), for example, selected under specific
growth conditions, one that has been subjected to a range of
selection pressures, one that has been genetically modified using
recombinant techniques known within the art, or a combination
thereof. In another preferred embodiment, the virus may be
VSV.DELTA.51 (Stojdl et al., VSV strains with defects in their
ability to shutdown innate immunity are potent systemic anti-cancer
agents., Cancer Cell. 2003 October; 4(4):263-75, herein
incorporated by reference). Other derivatives or variants may be
based on viruses such as Maraba (MG-1, for example), Rabies,
Rotavirus, Influenza, Hepatitis A, Mumps, Measles, Rubella,
Herpesvirus, Reovirus, Parvovirus, Dengue Virus, Cickungunya Virus,
Vaccinia virus, Modified Vaccinia Ankara, Respiratory Syncitial
Virus, Varicella, LCMV, HSV-1, HSV-2, adenovirus, adeno-associated
virus, lentivirus, or replicating retrovirus, for example.
[0070] In another embodiment of any of the method or methods above,
the one or more types of cancer or tumor cells may be cancer or
tumor cells in vitro or in vivo from any cell, cell line, tissue or
organism, for example, but not limited to human, rat, mouse, cat,
dog, pig, primate, horse and the like. In a preferred embodiment,
the one or more cancer or tumor cells comprise human cancer or
tumor cells, for example, but not limited to lymphoblastic
leukemia, myeloid leukemia, adrenocortical carcinoma, AIDS-related
cancers, AIDS-related lymphoma, anal cancer, appendix cancer,
astrocytoma, atypical teratoid/rhabdoid tumor, basal cell
carcinoma, bile duct cancer, bladder cancer, bone cancer,
osteosarcoma, malignant fibrous histiocytoma, brain stem glioma,
brain tumor, cerebellar astrocytoma, cerebral astrocytoma/malignant
glioma, craniopharyngioma, ependymoblastoma, medulloblastoma,
pineal parenchymal tumors of intermediate differentiation,
supratentorial primitive neuroectodermal tumors and pineoblastoma,
visual pathway and hypothalamic glioma, spinal cord tumors, breast
cancer, bronchial tumors, Burkitt lymphoma, carcinoid tumor,
central nervous system lymphoma, cervical cancer, chordoma, chronic
lymphocytic leukemia, chronic myelogenous leukemia, chronic
myeloproliferative disorders, colon cancer, cutaneous T-Cell
lymphoma, embryonal tumors, endometrial cancer, ependymoblastoma,
ependymoma, esophageal cancer, extracranial germ cell tumor,
extragonadal germ cell tumor, extrahepatic bile duct cancer, eye
cancer, intraocular melanoma, retinoblastoma, gallbladder cancer,
gastric (stomach) cancer, gastrointestinal carcinoid tumor,
gastrointestinal stromal tumor (GIST), gastrointestinal stromal
cell tumor, germ cell tumors, extracranial, extragonadal, ovarian,
gestational trophoblastic tumor, glioma, hairy cell leukemia, head
and neck cancer, hepatocellular (Liver) cancer, histiocytosis,
Langerhans cell cancer, Hodgkin lymphoma, hypopharyngeal cancer,
islet cell tumors, Kaposi sarcoma, kidney cancer, laryngeal cancer,
lymphocytic leukemia, hairy cell leukemia, lip and oral cavity
cancer, liver cancer, non-small cell lung cancer, small cell lung
cancer, Hodgkin lymphoma, non-Hodgkin lymphoma, malignant fibrous
histiocytoma of bone and osteosarcoma, medulloblastoma,
medulloepithelioma, melanoma, intraocular melanoma, Merkel cell
carcinoma, mesothelioma, metastatic squamous neck cancer, mouth
cancer, multiple endocrine neoplasia syndrome, multiple
myeloma/plasma cell neoplasm, nasal cavity and paranasal sinus
cancer, nasopharyngeal cancer, neuroblastoma, oral cancer,
oropharyngeal cancer, ovarian cancer, pancreatic cancer,
parathyroid cancer, penile cancer, pharyngeal cancer,
pheochromocytoma, pineal parenchymal tumors, pineoblastoma and
supratentorial primitive neuroectodermal tumors, pituitary tumor,
plasma cell neoplasm/multiple myeloma, pleuropulmonary blastoma,
primary central nervous system lymphoma, prostate cancer, rectal
cancer, renal cell (kidney) cancer, renal pelvis and ureter cancer,
transitional cell cancer, respiratory tract carcinoma,
retinoblastoma, rhabdomyosarcoma, salivary gland cancer, uterine
sarcoma, skin cancer, Merkel cell skin carcinoma, small intestine
cancer, soft tissue sarcoma, squamous cell carcinoma, squamous neck
cancer, stomach (Gastric) cancer, supratentorial primitive
neuroectodermal tumors, T-Cell lymphoma, testicular cancer, throat
cancer, thymoma and thymic carcinoma, thyroid cancer, trophoblastic
tumor, urethral cancer, uterine cancer, endometrial cancer, uterine
sarcoma, vaginal cancer, vulvar cancer, or Wilms tumor. However,
the compounds and compositions described herein possible may be
used to treat other cancers or tumor in vivo or in vitro.
[0071] According to the present invention there is provided a
compound or group of compounds defined by formula (I)
##STR00010##
an N-oxide, pharmaceutically acceptable addition salt, quarternary
amine or stereochemically isomeric form thereof, wherein A is a
5-membered heterocyclic ring comprising 0 or 1 double bond and 1
heteroatom selected from O, substituted or unsubstituted N, R.sub.1
and R.sub.4 are each independently H, oxo, hydroxyl, alkynyloxy,
phenyl, substituted phenyl, benzyl, substituted benzyl, triazolyl,
substituted triazolyl, or indolyl and R.sub.2 and R.sub.3 are each
independently hydrogen, halogen, alkynylamino, isobutylamino, or
benzylamino.
[0072] Also provided is one or more compounds or group of compounds
selected from the group consisting of
.alpha.,.beta.-dichloro-.gamma.-hydroxy-N-benzyl-crotonic lactam;
3,4-dichloro-5-prop-2-ynyloxy-5H-furan-2-one;
3,4-dibromo-5-prop-2-ynyloxy-5H-furan-2-one;
3-chloro-5-phenyl-4-prop-2-ynylamino-5H-furan-2-one;
3-chloro-4-isobutylamino-5-prop-2-ynyloxy-5H-furan-2-one;
1-benzyl-3,4-dichloro-5-prop-2-ynyloxy-1,5-dihydro-pyrrol-2-one;
3,4-dichloro-5-hydroxy-1-(2-methoxy-benzyl)-1,5-dihydro-pyrrol-2-one;
4-benzylamino-3-chloro-5-prop-2-ynyloxy-5H-furan-2-one;
3,4-dichloro-5-hydroxy-1-prop-2-ynyl-1,5-dihydro-pyrrol-2-one;
3,4-dichloro-5H-furan-2-one; benzo[1,3]dioxole-5,6-dione;
4,5-dichloro-2H-pyridazin-3-one;
4,5-dichloro-2-phenyl-2H-pyridazin-3-one;
3,4-dichloro-1-phenyl-pyrrole-2,5-dione;
3,4-dichloro-5-(1H-indol-3-yl)-5H-furan-2-one, indole-3-crotonic
acid; 3,4-dichloro-5-hydroxy-1,5-dihydro-pyrrol-2-one;
5-phenyl-4,5-dihydro-3aH-pyrrolo[1,2-a]quinolin-1-one;
5-phenyl-4,5-dihydro-3aH-pyrrolo[1,2-a]quinolin-1-one;
3,4-dichloro-5-(3-nitro-phenyl)-5H-furan-2-one;
3,4-dichloro-5-hydroxy-1-methyl-1,5-dihydro-pyrrol-2-one;
3,4-dichloro-1-prop-2-ynyl-5-prop-2-ynyloxy-1,5-dihydro-pyrrol-2-one;
3,4-dichloro-1-(2-chloro-benzyl)-5-hydroxy-1,5-dihydro-pyrrol-2-one;
3,4-dichloro-5-hydroxy-1-propyl-1,5-dihydro-pyrrol-2-one;
1-phenyl-pyrrole-2,5-dione;
3,4-dichloro-1-propyl-pyrrole-2,5-dione;
1-benzyl-3,4-dichloro-pyrrole-2,5-dione;
3,4-dichloro-5-hydroxy-1-phenyl-1,5-dihydro-pyrrol-2-one;
1-(1-benzyl-1H-[1,2,3]triazol-4-ylmethyl)-3,4-dichloro-5-hydroxy-1,5-dihy-
dro-pyrrol-2-one;
[4-(4-chloro-3-isobutylamino-5-oxo-2,5-dihydro-furan-2-yloxymethyl)-[1,2,-
3]triazol-1-yl]-acetic acid;
[4-(3,4-dichloro-2-hydroxy-5-oxo-2,5-dihydro-pyrrol-1-ylmethyl)-[1,2,3]tr-
iazol-1-yl]-acetic acid;
3,4-dichloro-5-hydroxy-1-phenethyl-1H-pyrrol-2(5H)-one;
3,4-dichloro-5-hydroxy-1-(2-morpholinoethyl)-1H-pyrrol-2(5H)-one;
3,4-dichloro-1-cyclopropyl-5-hydroxy-1H-pyrrol-2(5H)-one;
3,4-dichloro-5-hydroxy-1-(2-mercaptoethyl)-1H-pyrrol-2(5H)-one;
2-(3,4-dichloro-2-hydroxy-5-oxo-2,5-dihydro-1H-pyrrol-1-yl)ethanaminium
2,2,2-trifluoroacetate;
3,4-dichloro-5-hydroxy-1-(3-phenylprop-2-ynyl)-1H-pyrrol-2(5H)-one;
3,4-dichloro-5-hydroxy-1-(4-(trifluoromethyl)benzyl)-1H-pyrrol-2(5H)-one;
1-(biphenyl-4-ylmethyl)-3,4-dichloro-5-hydroxy-1H-pyrrol-2(5H)-one;
3,4-dichloro-5-hydroxy-1-(4-nitrobenzyl)-1H-pyrrol-2(5H)-one;
3,4-dichloro-5-hydroxy-1-(2-methoxybenzyl)-1H-pyrrol-2(5H)-one;
3,4-dichloro-1-(2-chlorobenzyl)-5-hydroxy-1H-pyrrol-2(5H)-one;
1-benzhydryl-3,4-dichloro-5-hydroxy-1H-pyrrol-2(5H)-one;
3,4-dichloro-5-hydroxy-1-(naphthalen-1-ylmethyl)-1H-pyrrol-2(5H)-one;
3,4-dichloro-5-hydroxy-1-(1-phenylethyl)-1H-pyrrol-2(5H)-one;
3,4-dichloro-5-hydroxy-1-(pyridin-3-ylmethyl)-1H-pyrrol-2(5H)-one;
3,4-dichloro-5-hydroxy-1-(pyridin-4-ylmethyl)-1H-pyrrol-2(5H)-one;
3,4-dichloro-5-hydroxy-1-(pyridin-2-ylmethyl)-1H-pyrrol-2(5H)-one;
3,4-dichloro-1-(furan-2-ylmethyl)-5-hydroxy-1H-pyrrol-2(5H)-one;
N-(2-(3,4-dichloro-2-hydroxy-5-oxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl)-5-(d-
imethylamino)naphthalene-1-sulfonamide;
(3aS)-2,3-dichloro-5-phenyl-4,5-dihydropyrrolo[1,2-a]quinolin-1(3aH)-one;
3,4-diiodo-2-phenyl-2,5-dihydrofuran; D-Gluconamide, N-octyl;
(S)-11-amino-4,7,10,14-tetraoxo-15-((2R,3R,4R,5R,6R)-3,4,5-trihydroxy-6-(-
hydroxymethyl)tetrahydro-2H-pyran-2-yl)-3,6,9,13-tetraazapentadecan-1-oic
acid; 1-allyl-3,4-dichloro-5-hydroxy-1H-pyrrol-2(5H)-one;
3,4-dichloro-5-hydroxy-1-(2-hydroxybenzyl)-1H-pyrrol-2(5H)-one;
3,4-dichloro-5-hydroxy-1-(thiophen-2-ylmethyl)-1H-pyrrol-2(5H)-one;
3,4-dichloro-5-hydroxy-1-(4-(methylsulfonyl)benzyl)-1H-pyrrol-2(5H)-one;
3,4-dichloro-1-((4,5-dimethyloxazol-2-yl)methyl)-5-hydroxy-1H-pyrrol-2(5H-
)-one;
3,4-dichloro-5-hydroxy-1-(3,4,5-trifluorobenzyl)-1H-pyrrol-2(5H)-on-
e; 3,4-dichloro-5-hydroxy-1-(4-methoxybenzyl)-1H-pyrrol-2(5H)-one;
4,5-dichloro-2-(2,2,2-trifluoroethyl)pyridazin-3(2H)-one;
4,5-dichloro-2-cyclohexylpyridazin-3(2H)-one; methyl
2-(4-((3,4-dichloro-2-hydroxy-5-oxo-2,5-dihydro-1H-pyrrol-1-yl)methyl)-1H-
-1,2,3-triazol-1-yl)acetate;
4,5-dichloro-2-o-tolylpyridazin-3(2H)-one;
4,5-dichloro-2-(2-(dimethylamino)ethyl)pyridazin-3(2H)-one
hydrochloride; and
4,5-dichloro-2-(4-fluorophenyl)pyridazin-3(2H)-one. Any combination
of 2, 3, 4, 5 or more compounds from above is also
contemplated.
[0073] The present invention also provides a composition comprising
the compound(s) as described herein, and an acceptable carrier,
diluent or excipient. In a further embodiment, the carrier is a
pharmaceutically acceptable carrier.
[0074] Also provided is a composition comprising the compound(s) as
described herein, and one or more of a) a virus, a genetically
modified virus, an attenuated virus, a cancer vaccine, a cancer
gene therapy vector, or an oncolytic virus, b) one or more type of
cells such as, for example, cancer cells, c) a carrier, diluent or
excipient, d) a pharmaceutically acceptable carrier, diluent or
excipient, e) non-cancer or normal cells; f) cell culture media; g)
one or more cancer therapeutics; or any combination of a)-g).
[0075] The present invention further provides a kit comprising the
compound(s) as described herein, and one or more of a) a virus, a
genetically modified virus, an attenuated virus, a cancer vaccine,
a cancer gene therapy vector, or an oncolytic virus, b) one or more
cancer cells, c) a carrier, diluent or excipient, d) a
pharmaceutically acceptable carrier, diluent or excipient, e)
non-cancer cells, f) cell culture media, g) one or more cancer
therapeutics, h) a cell culture plate or multi-well dish, i) an
apparatus to deliver the compound to a cell, medium or to a
subject; j) instructions for using the compound or any component in
the kit, or any combination of a)-j).
[0076] In one non-limiting embodiment of a composition or kit as
described above, the composition or kit may comprise a cell
infected with a virus such as, for example, an oncolytic virus.
[0077] Also provided is a method of enhancing or increasing the
infection, spread and/or titer, and/or cytotoxicity of a virus in
cells comprising, administering the compound(s) as described herein
to the cells prior to, after or concurrently with the virus, and
culturing the virus and cells to enhance or increase the infection,
spread and/or titer, or cytotoxicity of the virus in said cells.
Preferably, the cells are cancer cells, tumor cells or cells which
have been immortalized. More preferably, the cells are in vivo
cancer cells from a mammalian, still more preferably a human
subject and the method is practiced in vivo.
[0078] Also provided is a method of enhancing or increasing the
oncolytic activity of an oncolytic virus in cancer cells
comprising, administering the compound(s) as described herein to
the cancer cells or subject prior to, concurrently with or after
the oncolytic virus and culturing the oncolytic virus and cancer
cells. In a further embodiment, the cancer cells are in vivo cancer
cells. In a separate embodiment, the cancer cells are in vitro
cancer cells. The cells may be from a mammalian subject, preferably
a human subject.
[0079] The present invention also provides a composition comprising
the compound as described above, and a carrier, diluent or
excipient, more preferably a pharmaceutically acceptable carrier,
diluent or excipient.
[0080] Also, the present invention provides a composition
comprising the compound as described above and one or more of a) a
virus, an attenuated virus, a genetically modified virus, a cancer
vaccine, a cancer gene therapy vector, or an oncolytic virus; b)
one or more cancer cells; c) a carrier, diluent or excipient; d) a
pharmaceutically acceptable carrier, diluent or excipient; e)
non-cancer or normal cells, f) cell culture media, g) one or more
cancer therapeutics, or any combination of a)-g). The present
invention also contemplates embodiments wherein any one or a
combination of a-g) are specifically excluded from the composition
or kit. Any component or group of components may be excluded if
desired.
[0081] In a particular embodiment, which is not meant to be
limiting in any manner, there is provided a compound as described
above and a medium for growing, culturing or infecting cells with a
virus and optionally, one or more cells which are capable of being
infected by the virus. In a further embodiment, the cells are
cancer cells, tumor cells or immortalized cells.
[0082] Also provided is a kit comprising the compound as described
above and a) a virus, preferably an attenuated or genetically
modified virus, a cancer vaccine, a cancer gene therapy vector, or
an oncolytic virus; b) one or more cancer cells; c) a
pharmaceutically acceptable carrier, diluent or excipient; d)
non-cancer cells; e) cell culture media; f) one or more cancer
therapeutics, g) a cell culture plate or multi-well dish; h) an
apparatus to deliver the viral sensitizing compound to a cell,
medium or to a subject; i) instructions for using the viral
sensitizing agent; j) a carrier diluent or excipient, or any
combination of a)-j). The present invention also contemplates kits
wherein any one or a combination thereof of a)-j) are specifically
excluded.
[0083] In a particular embodiment, which is not meant to be
limiting in any manner, there is provided a kit comprising a
compound as described above and a medium for growing, culturing or
infecting cells with a virus and optionally, one or more cells
which are capable of being infected by the virus. The kit may also
comprise instructions for using any component or combination of
components and/or practicing any method as described herein.
[0084] The present invention also provides a method of enhancing or
increasing the infection, spread and/or titer, or cytotoxicity of a
virus in cells comprising, administering the compound as described
herein to the cells prior to, after or concurrently with the virus.
The method is preferably practiced in vivo but in vitro
applications are also contemplated.
[0085] The present invention also provides a method of enhancing or
increasing the infection, spread and/or titer, or cytotoxicity of
an attenuated virus or a genetically modified virus in cells
comprising, administering the compound as described above to the
cells prior to, after or concurrently with the attenuated or
genetically modified virus.
[0086] The present invention also provides a method of enhancing or
increasing the spread of an oncolytic virus in tumor or cancer
cells comprising, administering the compound as described above to
the cancer or tumor cells prior to, after or concurrently with the
oncolytic virus. The cancer or tumor cells may be in vivo, or in
vitro, preferably in vivo from a mammalian subject such as, but not
limited to, a human subject.
[0087] Also provided is a method of enhancing or increasing the
oncolytic activity of an oncolytic virus in cancer or tumor cells
comprising, administering the compound as described above to the
cancer or tumor cells prior to, concurrently with or after the
oncolytic virus. The cancer or tumor cells may be in vivo, or in
vitro, preferably from a mammalian subject such as, but not limited
to a human subject.
[0088] The present invention also contemplates a method of
producing a virus by growing the virus in an appropriate medium in
the presence of the compound as described above.
[0089] The present invention also contemplates a method of
producing an attenuated virus by growing the virus in an
appropriate medium in the presence of the compound as described
above.
[0090] The present invention also contemplates a method of
producing a genetically modified virus by growing the virus in an
appropriate medium in the presence of the compound as described
above.
[0091] The present invention also contemplates a method of
producing an oncolytic virus by growing the virus in an appropriate
medium in the presence of the compound as described above.
[0092] The present invention also contemplates a method of
producing a cancer vaccine by growing the virus in an appropriate
medium in the presence of the compound as described above.
[0093] The present invention also contemplates a method of
producing a cancer gene therapy vector by growing the virus in an
appropriate medium in the presence of the compound as described
above.
[0094] This summary of the invention does not necessarily describe
all features of the invention.
BRIEF DESCRIPTION OF THE DRAWINGS
[0095] These and other features of the invention will become more
apparent from the following description in which reference is made
to the appended drawings wherein:
[0096] FIG. 1 shows the physicochemical properties and activity
profile of parent compound VSe1
(3,4-dichloro-5-phenyl-2,5-dihydrofuran-2-one, FIG. 1a, also
referred to as compound 1 herein). (A) Structure of compound 1
(3,4-dichloro-5-phenyl-5H-furan-2-one). (B) Stability of VSe1 in 20
mM phosphate buffer over time measured by HPLC. (C) VSe1 was
incubated in sterile water for 0 h, 1.5 h, 3 h or 24 h before being
used to treat 786-0 cells at different concentrations. 4 hours
post-treatment, cells were infected with VSV.DELTA.51 expressing
firefly luciferase (VSV.DELTA.51-FLuc at a multiplicity of
infection (MOI) of 0.005. 40 hours later, virus output in viral
expression units (VEUs) per milliliter was measured with a
previously described luciferase reporter assay known in the art.
(D) 786-0 cells were treated with VSe1 at various doses. VSe1 was
removed and replaced with fresh media after 1 h, 1.5 h, 2 h, 2.5 h
and 6 h. VSe1 was not removed in the control condition. 4 hours
post-treatment, cells were infected with VSV.DELTA.51 expressing
firefly luciferase VSV.DELTA.51-FLuc at an MOI of 0.005. For the
condition where VSe1 was replaced with fresh media 6 h after
treatment, infection was performed immediately following media
replacement. 40 hours later, virus output in viral expression units
(VEUs) per milliliter was measured with a previously described
luciferase reporter assay known in the art;
[0097] FIG. 2 shows chemical synthesis routes for VSe1 (DCPDF)
analogs starting from mucochloric acid. (a) AlCl3 (1.5 equiv.),
benzene, rt, 16 h (b) amine (3 equiv.), DMF (c) NaBH4 (1.5 equiv.),
MeOH, 0.degree. C., 30 min. then, H2SO4 (1 equiv.), 20 min., O
.degree. C. to rt. (d) H2SO4 cat., alcohol, rt. (e) NH.sub.4OH,
Na2CO3, H2O, 0.degree. C. to rt, 16 h (f) EtOAc, reflux, 3 hr. (g)
benzylamine (1.1 equiv.), NaBH(OAc)3 (1.5 equiv.), CHCl3, 2 hr.,
rt. (h) SOCl2, DMF, reflux, 16 hr. (i) amine (2.2 equiv.), dioxane,
rt, 16 hr. (j) MeOH, H2SO4, reflux, 16 hr. (k) pyridine (2 equiv.),
Ac2O, rt, 6 hr. (1) CuI (0.1 equiv.), -78.degree. C., 20 min., then
RMgCl (2.5 equiv.) -78.degree. C. to rt, 16 hr. (m) SNAC, DMSO,
40.degree. C., 3 hr;
[0098] FIG. 3 shows the physicochemical and activity profile of
pyrolle-based VSe1 derivatives (see also Table 1 for more
information). (A) Stability of compound 10 in 20 mM phosphate
buffer over time measured by HPLC. (B) 250 .mu.L of a 40 mM DMSO
stock solution of compound 10 was added to L-glutathione (15.4 mg,
5 mol equiv.) suspended in 250 .mu.L of DMSO. The resulting mixture
was placed in a 37.degree. C. shaker. 10 .mu.L aliquots were
removed and quenched in 990 .mu.L of water (containing 0.5% formic
acid) at various time points, including at t=0 minutes and t=60
minutes shown above. Analysis by ESI-LC-MS allowed for the
identification and quantification of compound 10 and the
glutathione adduct. (C) 786-0 cells were treated with compound 10
at various doses. Compound 10 was removed and replaced with fresh
media after 1 h, 1.5 h, 2 h, 2.5 h and 6 h. Compound 10 was not
removed in the control condition. 4 hours post-treatment, cells
were infected with VSV.DELTA.51 expressing firefly luciferase
(VSV.DELTA.51-FLuc at a multiplicity of infection (MOI) of 0.005.
For the condition where compound 10 was replaced with fresh media 6
h after treatment, infection was performed immediately following
media replacement. 40 hours later, virus output in viral expression
units (VEUs) per milliliter was measured with a previously describe
luciferase reporter known in the art assay. (D) & (E) Pyrolle
based VSe1 analogs enhance oncolytic Herpes Simplex Virus type-1
(HSV-1) replication in cancer cells. D) Mouse mammary carcinoma
(4T1) cells were left untreated or, treated with VSe1 analog
compound 10 for 4h at various concentrations: 2.5 .mu.M, 5 .mu.M,
10 .mu.M, 15 .mu.M or 20 .mu.M. ICP0-null HSV-N212eGFP was then
added at MOI 0.005. eGFP fluorescence was detected 48h after HSV
infection (D). In (E), HSV titers were determined 48 h after
infection. Mean.+-.SEM from 3 independent experiments when error
bars are shown;
[0099] FIG. 4 shows that VSe1 (or Compound 1) and its analogues
selectively enhance the replication of oncolytic vesicular
stomatitis virus in ex vivo tumor tissues. (A) CT26 (murine colon
carcinoma) tumors were grown subcutaneously in Balb/c mice for 24
days and subsequently excised and cored, along with normal brain,
lung and spleen tissues. Tissue samples were treated in triplicate
with various concentrations of compounds for 4 hours prior to
infection with 1.times.10.sup.4 plaque-forming units of vesicular
stomatitis virus expressing GFP (VSV.DELTA.51-GFP). Virus
replication was assessed by fluorescence microscopy 24 hours
post-infection. Representative images from each triplicate set for
an effective concentration are shown. (B-G) Infected cores and
corresponding supernatants were collected 36 hours post-infection.
VSV.DELTA.51-GFP infection was quantified by standard plaque assay
(8). Cores were homogenized prior to titering. Graphs show the sum
of infectious titer from core and supernatant for each compound in
each tissue type. Doses shown here are those that are depicted in
panel (A). The horizontal black line on each graph at
1.times.10.sup.4 PFU/mL represents the amount of VSV.DELTA.51-GFP
used to initially infect each core;
[0100] FIG. 5 shows the results of dose escalation studies with
VSe1 (compound 1), compound 10, compound 24 and compound 28 in
mice. Six-week old female Balb/c mice were given (A) compound 1,
(B) compound 10, (C) compound 24, or (D) compound 28 dissolved in
DMSO via intraperitoneal administration. Five mice were assigned to
each dose group for each chemical. The dose was adjusted for
individual mice based on weight. Graphs stop when the first mouse
in the group was euthanized. (A-B) Mice were injected on Day 1 and
weights were recorded over a 10 day period. (C-D) Mice were
injected on Day 1, 3 and 5. Weights were recorded over an 18 day
period. For all groups, weights are reported relative to the
initial weight on Day 1;
[0101] FIG. 6 shows that pyrrole-based VSe1 analogs enhance the
spread of oncolytic virus in resistant tumors in vivo.
VSV.DELTA.51-resistant CT26 cells were subcutaneously engrafted
into female Balb/c mice. After 11 days (tumors were approximately 5
mm.times.5 mm in size), mice were given 30 uL of vehicle (DMSO) (A,
C), 50 mg/kg of compound 28 (B), or 40 mg/kg of compound 10 (D) by
intratumoral injection. 4 hours later, mice were treated with
1.times.10.sup.8 plaque-forming units of VSV.DELTA.51-FLuc. Virus
replication was measured by quantifying luciferase expression (in
mean relative light units) with an in-vivo imaging system (IVIS) 24
hours post-infection for groups A-B and 48 hours post-infection for
groups C-D;
[0102] FIG. 7 shows that VSe1 and its analogues may be combined for
synergistic effects. Human renal carcinoma (786-0) cells were
treated with various concentrations of VSe1 alone, MD03011 alone,
or co-treated with both compounds. After 4 hours, cells were
infected with VSV.DELTA.51 expressing firefly luciferase
(VSV.DELTA.51Fluc) at an MOI of 0.005. 40 hours later, virus output
in viral expression units (VEUs) per milliliter was measured with a
previously described luciferase reporter assay. Data is reported as
fold change in VEU relative to the VSe1 19 uM condition (Garcia et
al J Vis Exp 2014; herein incorporated by reference in its
entirety);
[0103] FIG. 8 shows that VSe1 and its analogues may enhance
infection of cells with oncolytic Maraba virus. Human renal
carcinoma (786-0) cells were left untreated or, treated with
compound at various concentrations. After 4 hours, cells were
infected with MGI-eGFP at MOI 0.005. eGFP fluorescence was detected
24h after MG1 infection and quantified using a Cellomics ArrayScan
high content screening microscope. Compound labels correspond to
Table 1;
[0104] FIG. 9 shows that VSe1 and its analogs enhance infection of
cells with Herpes Simplex Virus-1. Human renal carcinoma (786-0)
cells were left untreated or, treated with compound at various
concentrations. After 4 hours, cells were infected with ICP0-null
HSV-N212eGFP at MOI 0.01. eGFP fluorescence was detected 48 h after
HSV infection and quantified using a Cellomics ArrayScan high
content screening microscope. Compound labels correspond to Table
1; and
[0105] FIG. 10 shows that VSe1 analogue 28 (see Table 1) enhances
therapeutic activity of oncolytic VSV in a human colon cancer
xenograft model. (A) VSV.DELTA.51-resistant HT29 cells (human colon
carcinoma) were subcutaneously engrafted into female Balb/c mice.
After tumors reached a size of at least 5 mm.times.5 mm, mice were
given 25 .mu.L of vehicle (DMSO), or 40 mg/kg of compound 28 by
intratumoral injection. Four hours later, mice were treated with
1.times.10.sup.8 plaque-forming units of VSV.DELTA.51-FLuc. Tumor
volume (width.sup.2.times.length/2) was monitored using electronic
calipers and average tumour volume relative to the treatment day
are shown. Mice were euthanized when tumors reached a size of 1600
mm.sup.2. Tumor growth curves were stopped when the first mouse in
each group was euthanized. (B) Survival was monitored over
time.
DETAILED DESCRIPTION
[0106] The following description is of one or more preferred
embodiments. Several inventions may be described herein with
compounds, compositions, and kits provided with identical, similar
or distinct uses or methods of use.
[0107] In a first aspect, there is provided one or more compounds,
alone or in combination which enhances or increases viral activity,
for example, but not limited to, at least one of infection,
production, titer, spread or cytotoxicity in cells as compared to
when the one or more compounds are not employed. The compounds
described herein may be considered viral sensitizing compounds,
viral sensitizers or viral sensitizing agents.
[0108] In still a further embodiment, which is not meant to be
limiting, there is provided one or more compounds, alone or in
combination which enhances or increases at least one of oncolytic
viral production or cytotoxicity in cells, for example, but not
limited to, by increasing oncolytic viral titers in cells,
increasing oncolytic viral spread in cells, increasing oncolytic
viral cytotoxicity in cells or any combination thereof, as compared
to when the one or more compounds are not employed.
[0109] In still a further embodiment, which is not meant to be
limiting, there is provided one or more compounds, alone or in
combination which enhances or increases at least one of viral
production or cytotoxicity in cancer or tumor cells for example,
but not limited to, by increasing viral titers in cancer or tumor
cells, increasing viral spread in cancer or tumor cells, increasing
viral cytotoxicity in cancer or tumor cells or any combination
thereof, as compared to when the one or more compounds are not
employed. In a further aspect, which is not meant to be limiting,
oncolytic virus production or cytotoxicity is enhanced or increased
in cancer or tumor cells as compared to viral production or
cytotoxicity in normal or non-cancer or tumor cells.
[0110] It will be understood by the person of skill in the art
having regard to the teachings herein that enhancing or increasing
viral activity, production, oncolytic activity, or cytotoxicity may
include enhancing or increasing at least one of viral infection
and/or rate thereof, viral production and/or rate thereof, viral
titer and/or rate at which full titer may be reached, viral spread
and/or rate thereof, cell lysis and/or rate thereof, viral
cytotoxicity and/or rate thereof, or any combination thereof, as
compared to when the one or more compounds are not used.
[0111] In still a further embodiment, which is not meant to be
limiting, there is provided one or more compounds, alone or in
combination which enhances or increases at least one of oncolytic
viral production or cytotoxicity in cancer or tumor cells, for
example, but not limited to by increasing oncolytic viral
production in cancer or tumor cells, increasing oncolytic viral
titers in cancer or tumor cells, increasing oncolytic viral spread
in cancer or tumor cells, increasing oncolytic viral cytotoxicity
in cancer or tumor cells or any combination thereof, as compared to
when the one or more compounds are not employed. In a further
aspect, which is not meant to be limiting, oncolytic viral
production or cytotoxicity is enhanced in cancer or tumor cells as
compared to oncolytic viral production or cytotoxicity in normal or
non-cancer or non-tumor cells.
[0112] Based on results obtained for specific compounds in various
tests and screens as described herein and having regard to the
results obtained from several structure-function analyses, a broad
class of compounds and several subclasses was identified which
exhibit one or more of the properties as described above, or which
may be employed as controls or otherwise in in-vivo or in-vitro
experiments or in additional structure-function analyses to
determine additional compounds with interesting features as
described herein.
[0113] The present invention concerns compounds of formula
##STR00011##
an N-oxide, pharmaceutically acceptable addition salt, quarternary
amine or stereochemically isomeric form thereof, wherein: A is a
5-membered heterocyclic ring comprising 0 or 1 double bond and 1
heteroatom selected from O, and substituted or unsubstituted N;
R.sub.1 and R.sub.4 are each independently H, oxo, hydroxyl,
alkynyloxy, phenyl, substituted phenyl, benzyl, substituted benzyl,
triazolyl, substituted triazolyl, or indolyl; R.sub.2 and R.sub.3
are each independently hydrogen, halogen, alkynylamino,
isobutylamino, or benzylamino.
[0114] In a particular embodiment, R.sub.4 is oxo. In a further
embodiment R.sub.1 is hydroxyl. In still a further embodiment
R.sub.4 is oxo and R.sub.1 is hydroxyl.
[0115] In an embodiment which is not meant to be considered
limiting in any manner, there is provided a viral sensitizing
compound as described above, wherein A is a 5-membered heterocyclic
ring comprising, for example, but not limited to, unsubstituted N
or N substituted with C1-C12 alkyl, alkenyl, alkynyl, alkynyloxy,
phenyl, substituted phenyl, benzyl, substituted benzyl, triazolyl,
substituted triazolyl, naphthalenyl, pyridinyl, furanyl,
thiophenyl, sulfonobenzyl, methylsulfonobenzyl, or pyrrolyl. Other
substituents are also contemplated.
[0116] In a particular embodiment, the N in the 5-membered
heterocyclic ring is unsubstituted. In a further embodiment, the N
in the 5-membered heterocyclic ring is substituted, for example,
but without limitation with phenyl or benzyl.
[0117] In a further embodiment of the present invention, there is
provided a viral sensitizing compound as described above, wherein A
is a 5-membered heterocyclic ring comprising, for example, but not
limited to, N substituted with methyl, ethyl, propyl, cyclopropyl,
phenyl, benzyl, halogen substituted benzyl, methoxybenzyl,
benzyltriazolyl, morpholinoethyl, --CH2-C.ident.CH, --C.ident.CH,
mercaptoethyl, --CH2CH2NH3; CH2-C.ident.C-phenyl,
trifluoromethylbenzyl, or fluoroethyl.
[0118] By the term "viral sensitizing compound", "viral sensitizing
agent", or "viral sensitizer", it is meant a compound that
increases or enhances the infection of a virus, for example, but
without limitation, a genetically modified virus or attenuated
virus, a cancer vaccine, a cancer gene therapy vector or more
preferably an oncolytic virus; increases or enhances the spread of
a virus, preferably a genetically modified virus or attenuated
virus, a cancer vaccine, a cancer gene therapy vector, or an
oncolytic virus in one or more types of cells; increases or
enhances the cytotoxicity/oncolytic activity of an oncolytic virus
against one or more cancer or tumor cells; increases or enhances
the production, yield, titer, or reproductive capacity of a virus,
more preferably a genetically modified virus, an attenuated virus,
a cancer vaccine, a cancer gene therapy vector, or an oncolytic
virus; or any combination of the above. It is also preferred that
the viral sensitizing compound reduces the viability of a cancer or
tumor cell by either killing the cancer or tumor cells or limiting
its growth for a period of time.
[0119] By the term "oncolytic virus" it is meant a virus that
preferentially infects and lyses cancer or tumor cells as compared
to non-cancer or normal cells. In a preferred embodiment, the virus
in the presence of one or more compounds or compositions described
herein preferentially infects and lyses cancer cells or tumor cells
as compared to the virus alone and as compared to normal cells
alone or in the presence of the compound or composition alone.
Cytotoxic/oncolytic activity of the virus may be present, observed
or demonstrated in vitro, in vivo, or both. Preferably, the virus
exhibits cytotoxic/oncolytic activity in vivo. It is to be
understood that the oncolytic virus need not always preferentially
infect all cancer or tumor cells over all normal cells or tissues.
For example, but not wishing to be limiting, FIG. 4G shows an
example of an oncolytic virus which preferentially infects and
enhances replication in tumors in the presence of compound 40 even
though compound 40 also increases infection and replication in
brain tissue in the presence of compound 40.
[0120] Additional examples of oncolytic viruses known in the art
which may be employed herein include, without limitation, reovirus,
newcastle disease virus, adenovirus, herpes virus, polio virus,
mumps virus, measles virus, influenza virus, vaccinia virus,
rhabdoviruses such as vesicular stomatitis virus and
derivatives/variants thereof.
[0121] By a "derivative" or "variant" of a virus, it is meant a
virus obtained by selecting the virus under different growth
conditions, one that has been subjected to a range of selection
pressures, that has been genetically modified using recombinant
techniques known within the art, or one that has been engineered to
be replication defective and/or express transgenes, or any
combination thereof. Examples of such viruses are known in the art,
for example from US patent applications 20040115170, 20040170607,
20020037543, WO 00/62735; U.S. Pat. Nos. 7,052,832, 7,063,835,
7,122,182 (which are hereby incorporated by reference) and others.
Preferably the virus is a Vesicular stomatitis virus (VSV), or a
related rhabdovirus variant/derivative thereof, for example,
selected under specific growth conditions, one that has been
subjected to a range of selection pressures, one that has been
genetically modified using recombinant techniques known within the
art, or a combination thereof. In a preferred embodiment, the virus
is VSV.DELTA.51 (Stojdl et al., VSV strains with defects in their
ability to shutdown innate immunity are potent systemic anti-cancer
agents., Cancer Cell. 2003 October; 4(4):263-75, herein
incorporated by reference). Other derivatives or variants may be
based on viruses such as Maraba MG-1, Rabies, Rotavirus, Influenza,
Hepatitis A, Mumps, Measles, Rubella, Herpesvirus, Reovirus,
Parvovirus, Dengue Virus, Cickungunya Virus, Vaccinia virus,
Modified Vaccinia Ankara, Respiratory Syncitial Virus, Varicella,
LCMV, HSV-1, HSV-2, adenovirus, adeno-associated virus, lentivirus,
or replicating retrovirus.
[0122] The one or more types of cancer or tumor cells may be cancer
or tumor cells in vitro or in vivo from any cell, cell line, tissue
or organism, for example, but not limited to human, rat, mouse,
cat, dog, pig, primate, horse and the like. In a preferred
embodiment, the one or more cancer or tumor cells comprise human
cancer or tumor cells, for example, but not limited to
lymphoblastic leukemia, myeloid leukemia, adrenocortical carcinoma,
AIDS-related cancers, AIDS-related lymphoma, anal cancer, appendix
cancer, astrocytoma, atypical teratoid/rhabdoid tumor, basal cell
carcinoma, bile duct cancer, bladder cancer, bone cancer,
osteosarcoma, malignant fibrous histiocytoma, brain stem glioma,
brain tumor, cerebellar astrocytoma, cerebral astrocytoma/malignant
glioma, craniopharyngioma, ependymoblastoma, medulloblastoma,
pineal parenchymal tumors of intermediate differentiation,
supratentorial primitive neuroectodermal tumors and pineoblastoma,
visual pathway and hypothalamic glioma, spinal cord tumors, breast
cancer, bronchial tumors, Burkitt lymphoma, carcinoid tumor,
central nervous system lymphoma, cervical cancer, chordoma, chronic
lymphocytic leukemia, chronic myelogenous leukemia, chronic
myeloproliferative disorders, colon cancer, cutaneous T-Cell
lymphoma, embryonal tumors, endometrial cancer, ependymoblastoma,
ependymoma, esophageal cancer, extracranial germ cell tumor,
extragonadal germ cell tumor, extrahepatic bile duct cancer, eye
cancer, intraocular melanoma, retinoblastoma, gallbladder cancer,
gastric (stomach) cancer, gastrointestinal carcinoid tumor,
gastrointestinal stromal tumor (GIST), gastrointestinal stromal
cell tumor, germ cell tumors, extracranial, extragonadal, ovarian,
gestational trophoblastic tumor, glioma, hairy cell leukemia, head
and neck cancer, hepatocellular (Liver) cancer, histiocytosis,
Langerhans cell cancer, Hodgkin lymphoma, hypopharyngeal cancer,
islet cell tumors, Kaposi sarcoma, kidney cancer, laryngeal cancer,
lymphocytic leukemia, hairy cell leukemia, lip and oral cavity
cancer, liver cancer, non-small cell lung cancer, small cell lung
cancer, Hodgkin lymphoma, non-Hodgkin lymphoma, malignant fibrous
histiocytoma of bone and osteosarcoma, medulloblastoma,
medulloepithelioma, melanoma, intraocular melanoma, Merkel cell
carcinoma, mesothelioma, metastatic squamous neck cancer, mouth
cancer, multiple endocrine neoplasia syndrome, multiple
myeloma/plasma cell neoplasm, nasal cavity and paranasal sinus
cancer, nasopharyngeal cancer, neuroblastoma, oral cancer,
oropharyngeal cancer, ovarian cancer, pancreatic cancer,
parathyroid cancer, penile cancer, pharyngeal cancer,
pheochromocytoma, pineal parenchymal tumors, pineoblastoma and
supratentorial primitive neuroectodermal tumors, pituitary tumor,
plasma cell neoplasm/multiple myeloma, pleuropulmonary blastoma,
primary central nervous system lymphoma, prostate cancer, rectal
cancer, renal cell (kidney) cancer, renal pelvis and ureter cancer,
transitional cell cancer, respiratory tract carcinoma,
retinoblastoma, rhabdomyosarcoma, salivary gland cancer, uterine
sarcoma, skin cancer, Merkel cell skin carcinoma, small intestine
cancer, soft tissue sarcoma, squamous cell carcinoma, squamous neck
cancer, stomach (Gastric) cancer, supratentorial primitive
neuroectodermal tumors, T-Cell lymphoma, testicular cancer, throat
cancer, thymoma and thymic carcinoma, thyroid cancer, trophoblastic
tumor, urethral cancer, uterine cancer, endometrial cancer, uterine
sarcoma, vaginal cancer, vulvar cancer, or Wilms tumor. However,
the compounds and compositions described herein possible may be
used to treat other cancers or tumor in vivo or in vitro.
[0123] The present invention also provides a composition comprising
a) one or more compounds as described herein and b) one or more
additional components, for example, but not limited to 1) a
carrier, diluent or excipient, 2) a pharmaceutically acceptable
carrier, diluent or excipient, 3) a virus, for example, but not
limited to an attenuated virus, a genetically modified virus or an
oncolytic virus, 4) cancer or tumor cells, 5) non-cancerous or
normal cells, 6) cell culture media, 7) one or more cancer
therapeutics, for example, but not limited to chemotherapeutics. As
an example, but not to be considered limiting in any manner,
cyclophosphamide (CPA) is a common chemotherapy drug used primarily
for the treatment of lymphoma, chronic lymphocytic leukemia and
breast, ovarian and bladder cancers. CPA is converted into its
active metabolites, 4-hydroxycyclophosphamide and aldophosphamide
by liver oxidases. Use of CPA as an immune suppressant to enhance
viral oncolysis has improved virotherapy efficacy in combination
with oncolytic variants of HSV, adenoviruses, measles virus,
reovirus, and vaccinia virus.
[0124] A further cancer therapeutic known in the art is cisplatin.
Cisplatin binds and cross-links cellular DNA leading to apoptosis
when DNA is not repaired. Cisplatin has been investigated in
combination with oncolytic adenoviruses, herpes viruses,
parvovirus, vaccinia virus, and vesicular stomatitis virus.
Enhanced therapeutic activity in vitro and in vivo has been
observed when combining cisplatin with oncolytic variants of
adenovirus, herpesvirus, parvovirus and vaccinia virus whereas
slight inhibition was observed for oncolytic variant of vesicular
stomatitis virus.
[0125] Mitomycin C (MMC) is a DNA cross-linking antibiotic with
antineoplastic properties. MMC exhibited synergistic cytotoxicty
with oncolytic HSV. In vivo, combination of oncolytic herpes virus
and MMC significantly improved therapeutic effects in models of
gastric carcinomatosis and non-small cell lung cancer.
[0126] Doxorubicin is an anthracycline antibiotic that intercalates
into DNA and prevents the action of topoisomerase II. Doxorubicin
was synergistically cytotoxic when combined with oncolytic
adenovirus (ONYX-015) and the combination reduced tumor growth
relative to the monotherapies. ONYX-015 was successfully combined
with MAP (mitomycin C, doxorubicin and cisplatin) chemotherapy in a
phase I-II clinical trial for treatment of advanced sarcomas.
[0127] Gancyclovir (GCV) is a widely used antiviral agent,
originally developed for the treatment of cytomegalovirus
infections. GCV is a guanasine analogue prodrug that upon
phosphorylation by herpes virus thymidine kinase (TK) competes with
cellular dGTP for incorporation into DNA resulting in elongation
termination. Oncolytic viruses encoding the HSV TK gene lead to an
accumulation of toxic GCV metabolites in tumor cells which
interfere with cellular DNA synthesis leading to apoptosis.
Targeted oncolytic HSV viruses in combination with GCV
significantly improved survival in models of human ovarian cancer
and rat gliosarcoma. Adenoviruses, engineered to express the HSV TK
gene, also show enhanced anti-tumor activity when combined with
GCV.
[0128] CD/5-FC enzyme/pro-drug therapy has also proven successful
in combination with oncolytic virotherapy. 5-FU is a pyrimidine
analogue that inhibits the synthesis of thymidine. The anti-tumor
activity of two different oncolytic vaccinia viruses expressing CD
was significantly enhanced when combined with 5-FC therapy in
immune-competent ovarian cancer and immune suppressed colon cancer
models.
[0129] Taxanes are a class of chemotherapy drugs, including
paclitaxel and docetaxel, which cause stabilization of cellular
microtubules thereby preventing function of the cellular
cytoskeleton, a requirement for mitosis. Combination of docetaxel
or paclitaxel with an urothelium- or prostate-targeted oncolytic
adenovirus significantly reduced in vivo tumor volume and resulted
in synergistic in vitro cytotoxicity.
[0130] Rapamycin (sirolimus) is an immunosuppressant commonly used
in transplant patients however it has also been shown to
significantly enhance the oncolytic effects of the oncolytic
variants of poxviruses myxoma and vaccinia virus.
[0131] The prototypical proteosome inhibitor MG-132 enhanced
cellular CAR expression in Lovo colon carcinoma cells, which was
accompanied with enhanced oncolytic adenovirus target gene
expression and oncolysis.
[0132] The efficacy of oncolytic VSV against chronic lymphocytic
leukemia cells was increased by combination therapy with the BCL-2
inhibitor EM20-25.
[0133] One group showed that a single dose of angiostatic cRGD
peptide treatment before oncolytic virus treatment enhanced the
antitumor efficacy of oncolytic HSV.
[0134] The present invention also provides a kit comprising one or
more compound(s) alone or in combination or a composition
comprising same, for example as described herein. The kit may also
comprise one or more of a cell culture dish/plate or multi-well
dish/plate, and/or an apparatus or device(s) to deliver the
compound(s) or a composition comprising the same to a cell, tissue,
cell culture or cell culture medium, or to a subject in vivo. The
kit may also comprise instructions for administering or using the
compound(s), and/or virus(es) as described herein, for example, but
not limited to attenuated viruses, genetically modified viruses,
cancer vaccines, cancer gene therapy vectors, oncolytic viruses,
any combination thereof, or any combination of distinct
viruses.
[0135] For in vivo therapeutic applications, preferably there is
provided a pharmaceutical composition comprising one or more
compounds and a pharmaceutically acceptable carrier, diluent or
excipient, optionally containing other solutes such as dissolved
salts and the like. In a preferred embodiment, the solution
comprises enough saline, glucose or the like to make the solution
isotonic.
[0136] Pharmaceutical compositions and methods of preparing
pharmaceutical compositions are known in the art and are described,
for example, in "Remington: The Science and Practice of Pharmacy"
(formerly "Remingtons Pharmaceutical Sciences"); Gennaro, A.,
Lippincott, Williams & Wilkins, Philadelphia, Pa. (2000),
herein incorporated by reference.
[0137] Administration of such compositions may be via a number of
routes depending upon whether local and/or systemic treatment is
desired and upon the area to be treated. In a first embodiment,
which is not meant to be limiting, the compound is administered
locally to the area to be treated. Administration may be topical
(including ophthalmic and to mucous membranes including vaginal and
rectal delivery), pulmonary (e.g. by inhalation or insufflation of
powders or aerosols, including by nebulizer), intratracheal,
intranasal, epidermal and transdermal, oral or parenteral.
Parenteral administration includes intravenous, intraarterial,
subcutaneous, intraperitoneal or intramuscular injection or
infusion, or intracranial, e.g. intrathecal or intraventricular,
administration. Also contemplated is intra-tumor injection,
perfusion or delivery into the general vicinity of the tumor or
injection into the vasculature supplying a tumor. Alternatively,
the viral sensitizing compounds may be formulated in a tablet or
capsule for oral administration. Alternate dosage forms, including
slow-release, sustained-release, extended release, as would be
known in the art are also contemplated.
[0138] For administration by inhalation or insufflation, the
compounds can be formulated into an aqueous or partially aqueous
solution, which can then be utilized in the form of an aerosol. For
topical use, the modulators can be formulated as dusting powders,
creams or lotions in pharmaceutically acceptable vehicles, which
are applied to affected portions of the skin.
[0139] Without wishing to be liming, the dosage requirements for
the viral sensitizing compounds of the present invention may vary
with the particular compositions employed, the route of
administration and the particular subject being treated. Dosage
requirements can be determined by standard clinical techniques
known to a worker skilled in the art. Typically, treatment will
generally be initiated with small dosages less than the optimum
dose of the compound or compound/virus. Thereafter, the dosage is
increased until the optimum or satisfactory effect under the
circumstances is reached. In general, the viral sensitizing agent
or pharmaceutical compositions comprising the viral sensitizing
agent are administered at a concentration that will generally
afford effective results without causing significant harmful or
deleterious side effects. Administration can be either as a single
unit dose or, if desired, the dosage can be divided into convenient
subunits that are administered at suitable times throughout the
day.
[0140] The viral sensitizing compound may be employed in sequential
administration, for example, before, after or both before and after
administration of a virus, for example, but not limited to an
attenuated virus, a genetically modified virus, a cancer vaccine, a
cancer gene therapy vector or an oncolytic virus. Alternatively,
the viral sensitizing compound may be administered concurrently or
in combination with a virus as described above, preferably in
combination with an oncolytic virus. In addition, the viral
sensitizing agent may be used with an oncolytic virus as described
above and in combination with one or more cancer therapeutics or
cancer therapies as is known to a person of skill in the art, for
example but not limited to interferon therapy, interleukin therapy,
colony stimulating factor therapy, chemotherapeutic drugs, for
example, but not limited to 5-fluorodeoxyuridine amsacrine,
bleomycin, busulfan, capecitabine, carboplatin, carmustine,
chlorambucil, cisplatin, cladribine, clofarabine, crisantaspase,
cyclophosphamide, cytarabine, dacarbazine, dactinomycin,
daunorubicin, docetaxel, doxorubicin, epirubicin, etoposide,
fludarabine, fluorouracil, gemcitabine, gliadel, hydroxycarbamide,
idarubicin, ifosfamide, irinotecan, leucovorin, lomustine,
melphalan, mercaptopurine, mesna, methotrexate, mitomycin,
mitoxantrone, oxaliplatin, paclitaxel, pemetrexed, pentostatin,
procarbazine, raltitrexed, satraplatin, streptozocin,
tegafur-uracil, temozolomide, teniposide, thiotepa, tioguanine,
topotecan, treosulfan, vinblastine, vincristine, vindesine,
vinorelbine or a combination thereof. Further, anti-cancer
biologics may also be employed, for example, but without
limitation, monoclonal antibodies and the like.
[0141] The present invention also contemplates methods and uses of
the compounds as described herein for increasing or enhancing the
spread of a virus, for example, a genetically modified virus, an
attenuated virus, a cancer vaccine, a cancer gene therapy vector,
or an oncolytic virus in one or more cells, for example, but not
limited to one or more types of cancer or tumor cells, increasing
or enhancing the cytotoxicity/oncolytic activity of an oncolytic
virus against one or more cancer or tumor cells, increasing or
enhancing the production, yield or reproductive capacity of a
virus, for example, a genetically modified virus, an attenuated
virus, cancer vaccine, cancer gene therapy vector, an oncolytic
virus, or, any combination of the above. In an embodiment, which is
not meant to be limiting in any manner, the viral sensitizing
compound reduces the viability of a cancer or tumor cell by either
killing the cancer or tumor cell or limiting its growth for a
period of time. The compounds may also be used for the production
of a medicament for accomplishing same.
[0142] In an embodiment of the present invention, which is not
meant to be limiting in any manner, the one or more compounds are
.alpha.,.beta.-dichloro-.gamma.-hydroxy-N-benzyl-crotonic lactam;
3,4-dichloro-5-prop-2-ynyloxy-5H-furan-2-one;
3,4-dibromo-5-prop-2-ynyloxy-5H-furan-2-one;
3-chloro-5-phenyl-4-prop-2-ynylamino-5H-furan-2-one;
3-chloro-4-isobutylamino-5-prop-2-ynyloxy-5H-furan-2-one;
1-benzyl-3,4-dichloro-5-prop-2-ynyloxy-1,5-dihydro-pyrrol-2-one;
3,4-dichloro-5-hydroxy-1-(2-methoxy-benzyl)-1,5-dihydro-pyrrol-2-one;
4-benzylamino-3-chloro-5-prop-2-ynyloxy-5H-furan-2-one;
3,4-dichloro-5-hydroxy-1-prop-2-ynyl-1,5-dihydro-pyrrol-2-one;
3,4-dichloro-5H-furan-2-one; benzo[1,3]dioxole-5,6-dione;
4,5-dichloro-2H-pyridazin-3-one;
4,5-dichloro-2-phenyl-2H-pyridazin-3-one;
3,4-dichloro-1-phenyl-pyrrole-2,5-dione;
3,4-dichloro-5-(1H-indol-3-yl)-5H-furan-2-one, indole-3-crotonic
acid; 3,4-dichloro-5-hydroxy-1,5-dihydro-pyrrol-2-one;
5-phenyl-4,5-dihydro-3aH-pyrrolo[1,2-a]quinolin-1-one;
5-phenyl-4,5-dihydro-3aH-pyrrolo[1,2-a]quinolin-1-one;
3,4-dichloro-5-(3-nitro-phenyl)-5H-furan-2-one;
3,4-dichloro-5-hydroxy-1-methyl-1,5-dihydro-pyrrol-2-one;
3,4-dichloro-1-prop-2-ynyl-5-prop-2-ynyloxy-1,5-dihydro-pyrrol-2-one;
3,4-dichloro-1-(2-chloro-benzyl)-5-hydroxy-1,5-dihydro-pyrrol-2-one;
3,4-dichloro-5-hydroxy-1-propyl-1,5-dihydro-pyrrol-2-one;
1-phenyl-pyrrole-2,5-dione;
3,4-dichloro-1-propyl-pyrrole-2,5-dione;
1-benzyl-3,4-dichloro-pyrrole-2,5-dione;
3,4-dichloro-5-hydroxy-1-phenyl-1,5-dihydro-pyrrol-2-one;
1-(1-benzyl-1H-[1,2,3]triazol-4-ylmethyl)-3,4-dichloro-5-hydroxy-1,5-dihy-
dro-pyrrol-2-one;
[4-(4-chloro-3-isobutylamino-5-oxo-2,5-dihydro-furan-2-yloxymethyl)-[1,2,-
3]triazol-1-yl]-acetic acid;
[4-(3,4-dichloro-2-hydroxy-5-oxo-2,5-dihydro-pyrrol-1-ylmethyl)-[1,2,3]tr-
iazol-1-yl]-acetic acid;
3,4-dichloro-5-hydroxy-1-phenethyl-1H-pyrrol-2(5H)-one;
3,4-dichloro-5-hydroxy-1-(2-morpholinoethyl)-1H-pyrrol-2(5H)-one;
3,4-dichloro-1-cyclopropyl-5-hydroxy-1H-pyrrol-2(5H)-one;
3,4-dichloro-5-hydroxy-1-(2-mercaptoethyl)-1H-pyrrol-2(5H)-one;
2-(3,4-dichloro-2-hydroxy-5-oxo-2,5-dihydro-1H-pyrrol-1-yl)ethanaminium
2,2,2-trifluoroacetate;
3,4-dichloro-5-hydroxy-1-(3-phenylprop-2-ynyl)-1H-pyrrol-2(5H)-one;
3,4-dichloro-5-hydroxy-1-(4-(trifluoromethyl)benzyl)-1H-pyrrol-2(5H)-one;
1-(biphenyl-4-ylmethyl)-3,4-dichloro-5-hydroxy-1H-pyrrol-2(5H)-one;
3,4-dichloro-5-hydroxy-1-(4-nitrobenzyl)-1H-pyrrol-2(5H)-one;
3,4-dichloro-5-hydroxy-1-(2-methoxybenzyl)-1H-pyrrol-2(5H)-one;
3,4-dichloro-1-(2-chlorobenzyl)-5-hydroxy-1H-pyrrol-2(5H)-one;
1-benzhydryl-3,4-dichloro-5-hydroxy-1H-pyrrol-2(5H)-one;
3,4-dichloro-5-hydroxy-1-(naphthalen-1-ylmethyl)-1H-pyrrol-2(5H)-one;
3,4-dichloro-5-hydroxy-1-(1-phenylethyl)-1H-pyrrol-2(5H)-one;
3,4-dichloro-5-hydroxy-1-(pyridin-3-ylmethyl)-1H-pyrrol-2(5H)-one;
3,4-dichloro-5-hydroxy-1-(pyridin-4-ylmethyl)-1H-pyrrol-2(5H)-one;
3,4-dichloro-5-hydroxy-1-(pyridin-2-ylmethyl)-1H-pyrrol-2(5H)-one;
3,4-dichloro-1-(furan-2-ylmethyl)-5-hydroxy-1H-pyrrol-2(5H)-one;
N-(2-(3,4-dichloro-2-hydroxy-5-oxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl)-5-(d-
imethylamino)naphthalene-1-sulfonamide;
(3aS)-2,3-dichloro-5-phenyl-4,5-dihydropyrrolo[1,2-a]quinolin-1
(3aH)-one; 3,4-diiodo-2-phenyl-2,5-dihydrofuran; D-Gluconamide,
N-octyl;
(S)-11-amino-4,7,10,14-tetraoxo-15-((2R,3R,4R,5R,6R)-3,4,5-trihydroxy-6-(-
hydroxymethyl)tetrahydro-2H-pyran-2-yl)-3,6,9,13-tetraazapentadecan-1-oic
acid; 1-allyl-3,4-dichloro-5-hydroxy-1H-pyrrol-2(5H)-one;
3,4-dichloro-5-hydroxy-1-(2-hydroxybenzyl)-1H-pyrrol-2(5H)-one;
3,4-dichloro-5-hydroxy-1-(thiophen-2-ylmethyl)-1H-pyrrol-2(5H)-one;
3,4-dichloro-5-hydroxy-1-(4-(methylsulfonyl)benzyl)-1H-pyrrol-2(5H)-one;
3,4-dichloro-1-((4,5-dimethyloxazol-2-yl)methyl)-5-hydroxy-1H-pyrrol-2(5H-
)-one;
3,4-dichloro-5-hydroxy-1-(3,4,5-trifluorobenzyl)-1H-pyrrol-2(5H)-on-
e; 3,4-dichloro-5-hydroxy-1-(4-methoxybenzyl)-1H-pyrrol-2(5H)-one;
4,5-dichloro-2-(2,2,2-trifluoroethyl)pyridazin-3(2H)-one;
4,5-dichloro-2-cyclohexylpyridazin-3 (2H)-one; methyl
2-(4-((3,4-dichloro-2-hydroxy-5-oxo-2,5-dihydro-1H-pyrrol-1-yl)methyl)-1H-
-1,2,3-triazol-1-yl)acetate;
4,5-dichloro-2-o-tolylpyridazin-3(2H)-one;
4,5-dichloro-2-(2-(dimethylamino)ethyl)pyridazin-3(2H)-one
hydrochloride; and
4,5-dichloro-2-(4-fluorophenyl)pyridazin-3(2H)-one or any compound
or group of compounds as described herein. Table 2 below includes
structures of several interesting viral sensitizing compounds such
as these.
TABLE-US-00001 TABLE 2 Structures, Chemical Names and Reference
Codes for Viral Sensitizing Compounds Name Chemical name Structure
MD01145 .alpha.,.beta.-dichloro-.gamma.-hydroxy-N-benzyl-crotonic
lactam ##STR00012## MD01155, MD02140
3,4-Dichloro-5-prop-2-ynyloxy-5H-furan-2- one, ##STR00013##
CM01013, MD02182 3,4-Dibromo-5-prop-2-ynyloxy-5H-furan-2- one
##STR00014## CM01027 3-Chloro-5-phenyl-4-prop-2-ynylamino-5H-
furan-2-one ##STR00015## MD01165, MD02142
3-Chloro-4-isobutylamino-5-prop-2-ynyloxy- 5H-furan-2-one
##STR00016## MD01171, MD02180
1-Benzyl-3,4-dichloro-5-prop-2-ynyloxy-1,5- dihydro-pyrrol-2-one
##STR00017## CM01025 3,4-Dichloro-5-hydroxy-1-(2-methoxy-
benzyl)-1,5-dihydro-pyrrol-2-one ##STR00018## MD01159
4-Benzylamino-3-chloro-5-prop-2-ynyloxy- 5H-furan-2-one
##STR00019## MD01151 3,4-Dichloro-5-hydroxy-1-prop-2-ynyl-1,5-
dihydro-pyrrol-2-one ##STR00020## MD02068
3,4-Dichloro-5H-furan-2-one ##STR00021## TD193
Benzo[1,3]dioxole-5,6-dione ##STR00022## MD02026
4,5-Dichloro-2H-pyridazin-3-one ##STR00023## MD02054
4,5-Dichloro-2-phenyl-2H-pyridazin-3-one ##STR00024## MD01139
3,4-Dichloro-1-phenyl-pyrrole-2,5-dione ##STR00025## MD01041
3,4-Dichloro-5-(1H-indol-3-yl)-5H-furan-2- one, indole-3-crotonic
acid ##STR00026## MD01033, MD02052
3,4-Dichloro-5-hydroxy-1,5-dihydro-pyrrol- 2-one ##STR00027##
MD01071F1 5-Phenyl-4,5-dihydro-3aH-pyrrolo[1,2- a]quinolin-1-one
##STR00028## MD01071F2 5-Phenyl-4,5-dihydro-3aH-pyrrolo[1,2-
a]quinolin-1-one ##STR00029## MD01085
3,4-Dichloro-5-(3-nitro-phenyl)-5H-furan-2- one ##STR00030##
CM01031, CP01026 3,4-Dichloro-5-hydroxy-1-methyl-1,5-
dihydro-pyrrol-2-one ##STR00031## MD01169
3,4-Dichloro-1-prop-2-ynyl-5-prop-2-
ynyloxy-1,5-dihydro-pyrrol-2-one ##STR00032## MD01179
3,4-Dichloro-1-(2-chloro-benzyl)-5-hydroxy-
1,5-dihydro-pyrrol-2-one ##STR00033## MD02010
3,4-Dichloro-5-hydroxy-1-propyl-1,5- dihydro-pyrrol-2-one
##STR00034## MD01037 1-Phenyl-pyrrole-2,5-dione ##STR00035##
MD01129 3,4-Dichloro-1-propyl-pyrrole-2,5-dione ##STR00036##
MD01133 1-Benzyl-3,4-dichloro-pyrrole-2,5-dione ##STR00037##
MD01147 3,4-Dichloro-5-hydroxy-1-phenyl-1,5- dihydro-pyrrol-2-one
##STR00038## CM01099 1-(1-Benzyl-1H-[1,2,3]triazol-4-ylmethyl)-
3,4-dichloro-5-hydroxy-1,5-dihydro-pyrrol- 2-one ##STR00039##
MD02136 [4-(4-Chloro-3-isobutylamino-5-oxo-2,5-
dihydro-furan-2-yloxymethyl)-[1,2,3]triazol- 1-yl]-acetic acid
##STR00040## MD02124 [4-(3,4-Dichloro-2-hydroxy-5-oxo-2,5-
dihydro-pyrrol-1-ylmethyl)-[1,2,3]triazol-1- yl]-acetic acid
##STR00041## MD03009 3,4-dichloro-5-hydroxy-1-phenethyl-1H-
pyrrol-2(5H)-one ##STR00042## MD03011 3,4-dichloro-5-hydroxy-1-(2-
morpholinoethyl)-1H-pyrrol-2(5H)-one ##STR00043## MD03013
3,4-dichloro-1-cyclopropyl-5-hydroxy-1H- pyrrol-2(5H)-one
##STR00044## MD03007 3,4-dichloro-5-hydroxy-1-(2-
mercaptoethyl)-1H-pyrrol-2(5H)-one ##STR00045## CP01046
2-(3,4-dichloro-2-hydroxy-5-oxo-2,5-
dihydro-1H-pyrrol-1-yl)ethanaminium 2,2,2- trifluoroacetate
##STR00046## MD01183 3,4-dichloro-5-hydroxy-1-(3-phenylprop-2-
ynyl)-1H-pyrrol-2(5H)-one ##STR00047## MD03017
3,4-dichloro-5-hydroxy-1-(4-
(trifluoromethyl)benzyl)-1H-pyrrol-2(5H)- one ##STR00048## CP01042
1-(biphenyl-4-ylmethyl)-3,4-dichloro-5- hydroxy-1H-pyrrol-2(5H)-one
##STR00049## CP01001 3,4-dichloro-5-hydroxy-1-(4-nitrobenzyl)-
1H-pyrrol-2(5H)-one ##STR00050## CP01005
3,4-dichloro-5-hydroxy-1-(2- methoxybenzyl)-1H-pyrrol-2(5H)-one
##STR00051## CP01011 3,4-dichloro-1-(2-chlorobenzyl)-5-hydroxy-
1H-pyrrol-2(5H)-one ##STR00052## CP01035
1-benzhydryl-3,4-dichloro-5-hydroxy-1H- pyrrol-2(5H)-one
##STR00053## CP01039 3,4-dichloro-5-hydroxy-1-(naphthalen-1-
ylmethyl)-1H-pyrrol-2(5H)-one ##STR00054## CP01037
3,4-dichloro-5-hydroxy-1-(1-phenylethyl)- 1H-pyrrol-2(5H)-one
##STR00055## CP01047 3,4-dichloro-5-hydroxy-1-(pyridin-3-
ylmethyl)-1H-pyrrol-2(5H)-one ##STR00056## CP01048
3,4-dichloro-5-hydroxy-1-(pyridin-4- ylmethyl)-1H-pyrrol-2(5H)-one
##STR00057## CP01036 3,4-dichloro-5-hydroxy-1-(pyridin-2-
ylmethyl)-1H-pyrrol-2(5H)-one ##STR00058## PL01010
3,4-dichloro-1-(furan-2-ylmethyl)-5- hydroxy-1H-pyrrol-2(5H)-one
##STR00059## CP01020 N-(2-(3,4-dichloro-2-hydroxy-5-oxo-2,5-
dihydro-1H-pyrrol-1-yl)ethyl)-5-
(dimethylamino)naphthalene-1-sulfonamide ##STR00060## CP01034
(3aS)-2,3-dichloro-5-phenyl-4,5-
dihydropyrrolo[1,2-a]quinolin-1(3aH)-one ##STR00061## CP01012
3,4-diiodo-2-phenyl-2,5-dihydrofuran ##STR00062## PL01013
1-allyl-3,4-dichloro-5-hydroxy-1H-pyrrol- 2(5H)-one ##STR00063##
PL01017 3,4-dichloro-5-hydroxy-1-(2-hydroxybenzyl)-
1H-pyrrol-2(5H)-one ##STR00064## PL01018
3,4-dichloro-5-hydroxy-1-(thiophen-2- ylmethyl)-1H-pyrrol-2(5H)-one
##STR00065## PL01019 3,4-dichloro-5-hydroxy-1-(4-
(methylsulfonyl)benzyl)-1H-pyrrol-2(5H)- one ##STR00066## PL01020
3,4-dichloro-1-((4,5-dimethyloxazol-2-
yl)methyl)-5-hydroxy-1H-pyrrol-2(5H)-one ##STR00067## PL01021
3,4-dichloro-5-hydroxy-1-(3,4,5-
trifluorobenzyl)-1H-pyrrol-2(5H)-one ##STR00068## MD01187
3,4-dichloro-5-hydroxy-1-(4- methoxybenzyl)-1H-pyrrol-2(5H)-one
##STR00069## PL01023 4,5-dichloro-2-(2,2,2-
trifluoroethyl)pyridazin-3(2H)-one ##STR00070## PL01024
4,5-dichloro-2-cyclohexylpyridazin-3(2H)- one ##STR00071## PL01012
methyl 2-(4-((3,4-dichloro-2-hydroxy-5-oxo-
2,5-dihydro-1H-pyrrol-1-yl)methyl)-1H- 1,2,3-triazol-1-yl)acetate
##STR00072## PL01025 4,5-dichloro-2-o-tolylpyridazin-3(2H)-one
##STR00073## PL01026 4,5-dichloro-2-(2-
(dimethylamino)ethyl)pyridazin-3(2H)-one hydrochloride ##STR00074##
PL01027 4,5-dichloro-2-(4-fluorophenyl)pyridazin- 3(2H)-one
##STR00075##
[0143] As will be appreciated by a person of skill in the art, the
general class structures and specific compounds as identified
herein may be employed alone or in combination in any variety of
compositions as required by a person of skill in the art. Without
wishing to be bound by theory, potential uses for the compounds as
described herein may include increasing infection, spread and/or
viral titer in specific cells, for example, in cancer or tumor
cells/tissues or cells derived from cultures that have been
immortalized, increasing cytotoxicity of viruses, including
oncolytic viruses in specific cells, for example, in cancer or
tumor cells/tissues, for the production of viruses which may be
subsequently used in the production of cancer gene therapy vectors
or cancer vaccines. Also, importantly, the compounds as described
herein may also be employed as internal controls or in
structure-function analyses to determine additional classes or
specific molecules which exhibit similar or improved properties to
those currently described herein.
[0144] As Vse1 (compound 1) is known in the art, it is contemplated
that any of the compositions described herein, uses thereof which
employ this compound may be specifically excluded if desired. Any
other compound or use thereof as described herein also may be
specifically excluded if that compound and/or the use of it as
described herein is disclosed in the art.
[0145] In an embodiment, there is provided herein a method of
enhancing or increasing the infection, spread, titer, or
cytotoxicity of an oncolytic virus in cancer or tumor cells,
comprising administering a compound, or a combination of compounds,
defined by formula (II):
##STR00076##
or [0146] a pharmaceutically acceptable salt, or stereochemically
isomeric form thereof, wherein: [0147] X.sub.1 is a heteroatom such
as O, NH, or substituted N; [0148] X.sub.2 is halogen (such as, for
example, Cl), or NHX.sub.3, wherein X.sub.3 is a substituted or
unsubstituted linear or branched alkyl, alkenyl, or alkynyl, or
substituted or unsubstituted aryl or heteroaryl; [0149] i is 0 when
X.sub.1 is O, or 0 or 1 when X.sub.1 is NH or substituted N; [0150]
represents a double bond which is present when i is 1, and absent
when i is 0 such that X.sub.1 is directly bonded to the
X.sub.4-bearing carbon through a single bond when i is 0; and
[0151] X.sub.4 is H, OH, .dbd.O, substituted or unsubstituted mono-
or bi-cycloaryl or -heteroaryl (such as, for example, substituted
or unsubstituted phenyl), or OX.sub.10, wherein X.sub.10 is H,
linear or branched substituted or unsubstituted alkyl, alkenyl,
alkynyl, or acyl (for example, X.sub.10 may be acetyl, methyl, or
--CH.sub.2--C.ident.CH); to said cells prior to, after, or
concurrently with infection of the cells with the virus.
[0152] In certain non-limiting embodiments, substituted N may
include N substituted with H, substituted or unsubstituted linear
or branched C.sub.1-C.sub.12 alkyl, alkenyl, or alkynyl,
substituted or unsubstituted mono- or bi-cycloaryl or -heteroaryl,
substituted or unsubstituted cycloalkyl or heterocycloalkyl, for
example. For example, N may be substituted with substituted or
unsubstituted alkynyloxy, phenyl, alkylphenyl, substituted phenyl,
benzyl, substituted benzyl, triazolyl, substituted triazolyl,
naphthalenyl, substituted naphthalenyl, substituted or
unsubstituted pyridinyl, substituted or unsubstituted furanyl or
thiofuranyl, thiophenyl, sulfonobenzyl, methylsulfonobenzyl,
pyrrolyl, substituted or unsubstituted morpholine, cycloalkyl,
alkylthiol, substituted or unsubstituted alkyamine, or substituted
or unsubstituted oxazoline.
[0153] In certain non-limiting embodiments, substituted or
unsubstituted linear or branched alkyl, alkenyl, or alkynyl may
include any suitable substituted or unsubstituted linear or
branched alkyl, alkenyl, or alkynyl, such as an optionally
substituted linear or branched alkyl, alkenyl, or alkynyl
comprising a C.sub.1-C.sub.12 carbon chain and, in the case of
alkenyl or alkynyl, at least one carbon-carbon double or triple
bond, respectively.
[0154] In certain non-limiting embodiments, substituted or
unsubstituted aryl or heteroaryl may include any suitable mono- or
bi-cyclic aryl or heteroaryl group which may be optionally
substituted. Examples of aryl and heteroaryl groups may include
5-membered, 6-membered, or >6-membered aryl or heteroaryl
groups.
[0155] In certain non-limiting embodiments, acyl may include a
group having the formula R--C(.dbd.O)--, wherein R is substituted
or unsubstituted linear or branched alkyl, alkenyl, or alkynyl, for
example.
[0156] In certain non-limiting embodiments, substituted or
unsubstituted cycloalkyl or heterocycloalkyl may include any
suitable cycloalky or heterocycloalkyl group having a ring size
which is .gtoreq.3, and which may be optionally substituted.
[0157] In certain non-limiting embodiments, substituted or
unsubstituted alkynyloxy may include any suitable group having the
formula --O--R, wherein R comprises a substituted or unsubstituted
linear or branched C.sub.1-C.sub.12 alkynyl group (i.e. a carbon
chain having at least one carbon-carbon triple bond).
[0158] Non-limiting examples of suitable substituents of compounds
of formula (II) may be found in the compound structures shown in
Tables 1 and 2, for example.
[0159] Examples of compounds of formula (II) are described in
detail herein, and may be found in both Tables 1 and 2. Subsets of
compounds of formula (II), which share certain structural and/or
pharmacophore features therewith, may include:
[0160] Compounds defined by formula (VIII):
##STR00077##
or [0161] a pharmaceutically acceptable salt, or stereochemically
isomeric form thereof, wherein: [0162] X.sub.12 is O, NH, or
substituted N; [0163] X.sub.13 is halogen such as Cl, or
NHX.sub.14, wherein X.sub.14 is a substituted or unsubstituted
linear or branched alkyl, alkenyl, or alkynyl, or substituted or
unsubstituted aryl or heteroaryl; and [0164] X.sub.15 is H, OH,
.dbd.O, substituted or unsubstituted mono- or bi-cycloaryl or
-heteroaryl such as substituted or unsubstituted phenyl, or
OX.sub.16, wherein X.sub.16 is H, linear or branched substituted or
unsubstituted alkyl, alkenyl, alkynyl, or acyl, or X.sub.16 is
acetyl, methyl, or --CH.sub.2--C.ident.CH;
[0165] Compounds defined by formula (III):
##STR00078##
or [0166] a pharmaceutically acceptable salt, or stereochemically
isomeric form thereof, wherein: [0167] X.sub.5 is H, substituted or
unsubstituted linear or branched C.sub.1-C.sub.12 alkyl, alkenyl,
or alkynyl, substituted or unsubstituted mono- or bi-cycloaryl or
-heteroaryl, substituted or unsubstituted cycloalkyl or
heterocycloalkyl, or wherein X.sub.5 is substituted or
unsubstituted alkynyloxy, phenyl, alkylphenyl, substituted phenyl,
benzyl, substituted benzyl, triazolyl, substituted triazolyl,
naphthalenyl, substituted naphthalenyl, substituted or
unsubstituted pyridinyl, substituted or unsubstituted furanyl or
thiofuranyl, thiophenyl, sulfonobenzyl, methylsulfonobenzyl,
pyrrolyl, substituted or unsubstituted morpholine, cycloalkyl,
alkylthiol, substituted or unsubstituted alkyamine, or substituted
or unsubstituted oxazoline;
[0168] Compounds defined by formula (IV):
##STR00079##
or a pharmaceutically acceptable salt, or stereochemically isomeric
form thereof, wherein: [0169] wherein X.sub.6 is H, substituted or
unsubstituted linear or branched alkyl, alkenyl, alkynyl, or acyl,
or wherein X.sub.6 is substituted or unsubstituted methyl, alkyl
triazolyl, acetyl, or --CH.sub.2--C.ident.CH;
[0170] Compounds defined by formula (V):
##STR00080##
or [0171] a pharmaceutically acceptable salt, or stereochemically
isomeric form thereof, wherein: [0172] X.sub.7 is H, substituted or
unsubstituted aryl or heteroaryl, substituted or unsubstituted
linear or branched alkyl, alkenyl, or alkynyl, or substituted or
unsubstituted cycloalkyl. For example, X.sub.7 may be substituted
or unsubstituted alkylamine, or substituted or unsubstituted
phenyl;
[0173] Compounds defined by formula (VI):
##STR00081##
or [0174] a pharmaceutically acceptable salt, or stereochemically
isomeric form thereof, wherein: [0175] wherein X.sub.8 is
substituted or unsubstituted linear or branched alkyl, alkenyl, or
alkyny, or substituted or unsubstituted aryl or heteroaryl, or
X.sub.8 is substituted or unsubstituted benzyl; and/or
[0176] Compounds defined by formula (VII):
##STR00082##
or [0177] a pharmaceutically acceptable salt, or stereochemically
isomeric form thereof, wherein: [0178] X.sub.9 is H, OH, OX.sub.11,
or .dbd.O, wherein X.sub.11 is H, substituted or unsubstituted
linear or branched alkyl, alkenyl, alkynyl, or acyl, or X.sub.11 is
acetyl, methyl, or --CH.sub.2--C.ident.CH.
[0179] In an embodiment of any one of the method or methods
described herein, the compound or combination of compounds
described above may be present in a composition comprising the
compound(s) and a carrier, diluent or excipient.
[0180] In another embodiment, there is provided herein a
composition comprising one or more of the compounds having a
structure as defined above, and one or more of a) a virus, a
genetically modified virus, an attenuated virus, a cancer vaccine,
a cancer gene therapy vector, or an oncolytic virus, b) one or more
cancer cells, c) a carrier, diluent or excipient, d) a
pharmaceutically acceptable carrier, diluent or excipient, e)
non-cancer cells; f) cell culture media; g) one or more cancer
therapeutics; or any combination of a)-g).
[0181] It will be understood by the person of skill in the art
having regard to the teachings herein that two or more of the
compounds described herein may be used in combination. For example,
two or more of the compounds described herein may be administered
to a cell simultaneously, sequentially, or in combination. As shown
in FIG. 7 and described in further detail in the examples below,
using VSe1 and MD03011 in combination produced a remarkable
synergistic effect under the conditions tested, demonstrating that
combinations of two or more compounds as described herein may be
desirable in certain applications.
[0182] In yet another embodiment, there is provided herein a kit
comprising one or more of the compounds having a structure as
defined above, and one or more of a) a virus, a genetically
modified virus, an attenuated virus, a cancer vaccine, a cancer
gene therapy vector, or an oncolytic virus, b) one or more cancer
cells, c) a pharmaceutically acceptable carrier, diluent or
excipient, d) non-cancer cells; e) cell culture media; f) one or
more cancer therapeutics, g) a cell culture plate or multi-well
dish; h) an apparatus to deliver the compound to a cell, medium or
to a subject; i) instructions for using the compound or any
component in the kit, j) a carrier diluent or excipient, or any
combination of a)-j).
[0183] In another embodiment, the cells may be cancer cells in
vivo, or in vitro. In a further embodiment, the in vivo cancer
cells may be from a mammalian subject. In still a further
embodiment, the mammalian subject may be a human subject.
[0184] In yet another embodiment, there is provided herein a method
of increasing the oncolytic activity of an oncolytic virus in
cancer or tumor cells, comprising administering a compound, or a
combination of compounds, having a structure as described above to
said cancer or tumor cells prior to, concurrently with or after the
oncolytic virus. In another embodiment, the cancer cells may be in
vivo, or in vitro. In still another embodiment, the in vivo cancer
cells may be from a mammalian subject. In yet another embodiment,
the mammalian subject may be a human subject.
[0185] In another embodiment, there is provided herein a compound
having a structure as described above. In still another embodiment,
the compound may be for use as a viral sensitizer for sensitizing a
cancer or tumor cell to an oncolytic virus.
[0186] In an embodiment, there is provided herein a compound having
a structure as described above, for use as a viral sensitizer to
enhance or increase oncolytic virus activity in cancer or tumor
cells. In a further embodiment, the compound may be for enhancing
or increasing oncolytic virus titer in cancer or tumor cells. In
another embodiment, the compound may be for enhancing or increasing
cytotoxicity of the oncolytic virus in cancer or tumor cells.
[0187] In a further embodiment of any of the compounds,
compositions, method or methods described above, the compound, or
at least one of the compounds of the combination of compounds, may
exhibit a degradation half-life greater than 20, 40, 60, 80, 100,
120, 240, 360 minutes or more in phosphate buffer at pH 7.4.
[0188] In still another embodiment, a compound as described herein
may be a compound for which greater than about 0.5%, 1%, 5%, 10%,
15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 70%, 75%, 80%,
90%, or greater, or any range bounded at a lower end by any one of
these values, any range bounded at an upper end by any one of these
values, or any range falling between any two of these values, of
the compound remains after 3 hour incubation at 37.degree. C. in
aqueous, protein-rich Balb/c mouse plasma buffered 1:1 with pH 7.4
phosphate buffered saline (PBS). For example, a compound as
described above may be a compound for which greater than or equal
to (.gtoreq.) about 0.5% of the compound remains after 3 hour
incubation at 37.degree. C. in aqueous, protein-rich Balb/c mouse
plasma buffered 1:1 with pH 7.4 phosphate buffered saline (PBS).
For example, a compound as described herein may be a compound for
which greater than or equal to (.gtoreq.) about 0.5% of the
compound remains after 3 hour incubation at 37.degree. C. in
aqueous, protein-rich Balb/c mouse plasma buffered 1:1 with pH 7.4
phosphate buffered saline (PBS).
[0189] In another embodiment of any of the compounds, compositions,
method or methods described above, the compound, or at least one of
the compounds of the combination of compounds, may exhibit a
half-life greater than 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55,
60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 300, 400 minutes or more
in a glutathione stability assay.
[0190] In still another embodiment of any of the compounds,
compositions, method or methods described above, the compound, or
at least one of the compounds of the combination of compounds, may
exhibit a lower LD.sub.50 in the presence of an oncolytic virus
than in its absence. In a further embodiment, the difference in
LD.sub.50 may be 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 .mu.M or
more in the presence of an oncolytic virus compared to its
absence.
[0191] In still another embodiment of any of the compounds,
compositions, method or methods described above, the compound, or
at least one of the compounds of the combination of compounds, may
be a compound having an LD.sub.50 in the presence of virus which is
greater than or equal to (.gtoreq.) about 1 .mu.m, 5 .mu.m, 10
.mu.m, 15 .mu.m, 20 .mu.m, 25 .mu.m, 30 .mu.m, 35 .mu.m, 40 .mu.m,
45 .mu.m, 50 .mu.m, 55 .mu.m, 60 .mu.m, 65 .mu.m, 70 .mu.m, 75
.mu.m, 80 .mu.m, 85 .mu.m, 90 .mu.m, 95 .mu.m, 100 .mu.m, or 105
.mu.m, or 110 .mu.m, or any range falling between any two of these
values, less than (<) the LD.sub.50 of the same compound in the
absence of virus as determined in, for example, 786-0 cells where
the virus is VSV.
[0192] In still another embodiment of any of the compounds,
compositions, method or methods described above, the compound, or
at least one of the compounds of the combination of compounds, may
exhibit a viral sensitizer activity on VSV.DELTA.51 in 786-0 cells
which is about 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08,
0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, or greater,
or any range bounded at a lower end by any one of these values, any
range bounded at an upper end by any one of these values, or any
range falling between any two of these values, when reported as
peak fold change in viral expression unit normalized to
3,4-dichloro-5-phenyl-2,5-dihydrofuran-2-one. In one embodiment,
for example, the compound may be a compound which exhibits a viral
sensitizer activity on VSV.DELTA.51 in 786-0 cells which is greater
than or equal to (.gtoreq.) about 0.01 when reported as peak fold
change in viral expression unit normalized to
3,4-dichloro-5-phenyl-2,5-dihydrofuran-2-one. It will be understood
by the person of skill in the art having regard to the teachings
herein that in certain embodiments, PFC between two compounds need
not always be calculated at the same compound concentration
(although this may indeed be done). For example, PFC between two
compounds could be determined at a dose of each compound which is
experimentally determined to be particularly effective or near
optimal, such as the most effective dose determined from a
dose-response curve.
[0193] Thus, in a non-limiting embodiment, a compound as described
above may be a compound which exhibits a viral sensitizer activity
on VSV.DELTA.51 in 786-0 cells which is about 0.01, 0.02, 0.03,
0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6,
0.7, 0.8, 0.9, 1.0, or greater, or any range bounded at a lower end
by any one of these values, any range bounded at an upper end by
any one of these values, or any range falling between any two of
these values, when reported as peak fold change in viral expression
unit normalized to 3,4-dichloro-5-phenyl-2,5-dihydrofuran-2-one
(VSe1) taken with both compounds being used at the same
concentration (for example, a particularly effective or near
optimal VSe1 dose, such as the most effective dose of VSe1
determined from a dose-response curve), or taken with the compound
and VSe1 being used at different concentrations (for example, a
dose of each compound which is experimentally determined to be
particularly effective or near optimal for each compound, such as
the most effective dose for each compound determined from a
dose-response curve).
[0194] In one embodiment, for example, the compound may be a
compound which exhibits a viral sensitizer activity on VSV.DELTA.51
in 786-0 cells which is greater than or equal to (.gtoreq.) about
0.01 when reported as peak fold change in viral expression unit
normalized to 3,4-dichloro-5-phenyl-2,5-dihydrofuran-2-one when
both compounds are being used at the same concentration (for
example, a particularly effective or near optimal VSe1 dose, such
as the most effective dose of VSe1 determined from a dose-response
curve), or when the compound and VSe1 are being used at different
concentrations (for example, a dose of each compound which is
experimentally determined to be particularly effective or near
optimal for each compound, such as the most effective dose for each
compound determined from a dose-response curve).
[0195] In still another embodiment of any of the method or methods
described above, the method may further comprise administration of
an anticancer agent or cancer therapeutic.
[0196] In another embodiment, there is provided herein a method of
enhancing or increasing the infection, spread, titer, or
cytotoxicity of an oncolytic virus in cancer or tumor cells,
comprising administering VSe1 and MD03011,
##STR00083##
to said cells prior to, after, or concurrently with infection of
the cells with the virus.
[0197] The present invention will be further illustrated in the
following examples.
EXAMPLES
Example 1: Chemical and Biological Characterization of Parent
Compound VSe1 (Compound 1)
[0198] To gain a better understanding of the physiochemical nature
of compound 1, we measured the compound's stability in phosphate
buffer (pH=7.4) with an HPLC assay (FIG. 1B). Degradation occurred
quickly (t1/2=28.8 min) leading to a complex mixture of products.
Given the short half-life of compound 1 we considered that a
degradation product in an aqueous media could be responsible for
the impact on virus output from cancer cells, which can be observed
up to at least 48h post-treatment with compound 1. To address this,
we treated VSV.DELTA.51-resistant 786-0 renal carcinoma cells with
compound 1 and pre-incubated for up to 24 h in aqueous media. 40 h
following infection with VSV.DELTA.51 expressing luciferase
(VSV.DELTA.51-FLuc), virus output in viral expression units (VEUs)
per ml was measured with a luciferase-reporter assay. Supporting
compound 1 as an active agent as opposed to a degradation product,
pre-incubation of compound 1 in media for as little as 90 minutes
did not lead to robust enhancement of viral titers otherwise
observed (FIG. 1C). However, removal of compound 1 following
treatment at various times before infection with VSV.DELTA.51-FLuc
revealed that the induced effect of compound 1 on viral growth is
rapid and sustained (FIG. 1D).
[0199] Analogue Synthesis and Evaluation
[0200] The above observations provided a rationale to derive novel
compounds based on compound 1 with increased stability. Taking
advantage of the versatile reactions mucohalic acids can undergo
(FIG. 2), a diverse set of analogues was synthesized to reveal
structure-activity relationships (SAR, see Table 1). Table 1 shows
the structure activity and physico-chemical property relationship
of VSe1 derivatives. Compounds were evaluated for viral sensitizer
activity on VSV in 786-0 cells as described. Viral sensitizer
activity is reported as Peak Fold Change (PFC) in Viral Expression
Unit normalized to VSe1..sup.a Values in parenthesis (uM) indicates
a particularly effective dose tested. LD50 indicates in vitro 50%
lethal dose, which is provided with and without addition of
virus..sup.b GSH half life was determined and is a measure of
compound electrophilicity (low values indicate high
electrophilicity) and stability. Stability in aqueous, protein-rich
mouse plasma was also measured. A high % remaining after 3 hours
indicates high stability. .sup.CNE indicates the compound did not
detectably increase viral output. .sup.DND indicates not
determined..sup.e NR indicates not reactive.
[0201] Analogue compounds were screened for their ability to
augment VSV.DELTA.51-FLuc activity in 786-0 cells as above and
their pro-viral activity was compared to compound 1. Cytotoxicity
in presence and absence of virus was also assessed using an
alamarBlue.RTM. metabolic dye. Analogue stability was measured with
both a glutathione stability assay indicating physiochemical
susceptibility to act as a Michael-acceptor, and a plasma assay
indicating metabolic stability. Substitution of the .beta.-chlorine
with an alkyl amine resulted in compounds with dramatically
increased stability but loss of viral enhancement activity
(compounds 3, 4 (see Table 1)). Removal of the aryl group (compound
5) or replacement with a methoxide group (compound 6) resulted in
active compounds with similar stability issues as compound 1.
Encouragingly, compounds with the
3,4-dichloro-5-hydroxy-1,5-dihydropyrrol-2-one scaffolds (compounds
9, 10) enhanced viral expression and showed remarkably improved
stability with both the GSH and plasma assays. In addition,
compound 10 was found to be stable over time in phosphate buffer
(pH=7.4) (FIG. 3A). The LC/MS trace observed in the glutathione
stability assay also showed that compound 10, contrary to compound
1, cleanly reacted with the nucleophile to form the glutathione
adduct as the major detectable product (FIG. 3B). However,
similarly to compound 1, the impact of compound 10 on viral growth
was found to be rapid and sustained (FIG. 3C). In addition, both
compounds 1 and 10 increased growth of oncolytic HSV-1 expressing
GFP in 786-0 cancer cells as observed by fluorescence microscopy
and standard plaque assay (FIG. 3D-E).
Given this information, we decided to further explore the
1,5-dihydropyrrol-2-one scaffold. A set of analogue compounds was
synthesized to reveal further SAR (Table 1). Analogues with
alterations to the hydroxyl group (compounds 11-15) reduced
activity, as did alterations to the dichloro-moiety (compounds
16-21) when compared to compound 1. We then decided to investigate
3,4-dichloro-5-hydroxy-1,5-dihydropyrrol-2-one scaffold with
various substitutions on the amine (Table 1). Analogues with
saturated and unsaturated alkyl chains (compounds 22-27) were
synthesized and evaluated which lead to the identification of
cyclopropyl derivative compound 25, which displayed retained
activity and remarkably improved toxicity and stability profiles.
Morpholine containing analogue compound 28 also displayed promising
activity with improved physiochemical properties. The
phenylpropylamine derivative compound 30 showed improved activity
over similar analogues with varying alkyl chain lengths (compounds
10, 29, 31). A set of aromatic and hetero-aromatic compounds were
also evaluated (compounds 36-53), leading to the identification of
compound 40, a 4-trifluoromethyl benzylamine derivative which
displayed activity 1.6 fold greater than compound 1 in addition to
improved stability.
[0202] Ex-Vivo Assessment of Analogue Activity
[0203] To facilitate evaluation of a larger number of compounds
prior to in vivo testing, we chose to test a subset of analogues
for their ability to enhance VSV.DELTA.51 oncolysis in ex vivo
tissue samples. Tissue samples from VSV.DELTA.51 resistant CT26
murine colon cancer tumors as well as normal mouse brain, lung and
spleens were cored and put in tissue culture. Viable cores were
selected for subsequent treatment with a compound and VSV.DELTA.51
expressing green fluorescent protein (VSV.DELTA.51-GFP). FIG. 4A
shows representative images of infected cores that were pre-treated
with a select dose of compound. Corresponding viral titers as
determined by plaque assay are shown in FIGS. 4B-G. To varying
extents, compound 1 and analogues enhanced VSV.DELTA.51-GFP titers
in CT26 colon cancer specimens. For compound 28 in particular,
there was robust enhancement of VSV.DELTA.51 output in the tumor
specimens (nearly 100-fold) but little to no impact in normal
tissue specimens, indicating that the specificity of VSV.DELTA.51
towards tumor tissue is maintained following treatment with the
compound.
[0204] In Vivo Evaluation
[0205] We next proceeded to evaluate the in vivo tolerability of a
subset of analogues, selected based on desirable physiochemical
characteristics, in vitro activity and ex vivo activity/selectivity
profiles. Compounds were administered intraperitoneally to Balb/c
mice and body weight was monitored over several days. Mice were
sacrificed when they reached the endpoint of 20% loss of body
weight or showed significant outward signs of toxicity. FIG. 5
shows that compound 1 leads to toxicity starting at a dose of 10
mg/kg. In contrast analog compound 10 was well tolerated up to a
dose of 50 mg/kg and analog compounds 24 and 28 up to 100
mg/kg.
[0206] Because it effectively and specifically enhanced OV output
in tumors ex vivo and was very well tolerated in mice, we proceeded
to evaluate analog compound 28 for its ability to increase the
infection of tumors with VSV.DELTA.51-FLuc in vivo. Balb/c mice
were subcutaneously engrafted with CT26 cells and treated
intra-tumorally with VSV.DELTA.51-FLuc alone or in combination with
analog compound 28. We used an in vivo imaging system (IVIS) to
measure luciferase activity associated to virus replication 48 h
post treatment. FIG. 6 shows that compared to VSV.DELTA.51-FLuc
alone, analog compound 28 (FIG. 6A-B) significantly enhanced
tumor-specific viral replication specifically within the tumor.
Similar results were obtained with analog compound 10 (FIGS.
6C-D).
[0207] In this study, we carried out SAR studies leading to the
identification of new pyrrole derivatives of compound 1 with
substantially improved stability, reduced electrophilicity, and
retained or improved ability to enhance growth of OVs such as HSV-1
and VSV.DELTA.51 in resistant cancer cell lines in vitro. In vivo
evaluation further demonstrated that compounds such as analog 28
are exceptionally well tolerated (>10.times. the dose of parent
compound 1) and effective at enhancing OV replication specifically
within tumors. Ex vivo evaluation of compounds provided a useful
pre-screening tool for selecting active and tumor-selective leads.
In summary, we have derived a novel class of compounds that
sensitize cells to virus infection and that could be useful for a
broad range of applications utilizing viral vectors.
[0208] Experimental Methodology
[0209] Cell Lines
[0210] 786-0 (human renal carcinoma) and Vero (monkey kidney) cells
were obtained from the American Type Culture Collection and
maintained in Dulbecco's Modified Eagle's medium (Corning)
supplemented with 10% fetal bovine serum and buffered with 30 mM
Hepes. All cell lines were incubated at 37.degree. C. with 5%
CO2.
[0211] Viruses
[0212] VSV.DELTA.51 is a recombinant variant of the Indiana
serotype of VSV harbouring a deletion of the 51st methionine in the
M protein. VSV.DELTA.51 expressing green fluorescent protein (GFP)
or firefly luciferase (FLuc) are recombinant derivatives of
VSV.DELTA.51. All virus stocks were propagated in Vero cells,
purified on Optiprep gradient and titered on Vero cells. HSV-1 N212
(an ICPO-deleted oncolytic strain) expressing GFP was obtained from
Dr. Karen Mossman.
[0213] High-Throughput Screening of Analogs
[0214] High-throughput assessment of analog activity and
cytotoxicity was performed as previously described (Garcia V, et
al. J Vis Exp 2014). Briefly, stock preparations of compounds in
dimethylsulfoxide (DMSO) were first diluted in 5% DMSO (in water)
and then in cell culture media to obtain indicated concentration
range. Diluted compounds were applied to confluent 786-0 cells in
96-well plates for indicated times prior to infection with
VSV.DELTA.51 (or mock) at a multiplicity of infection of 0.01.
Vehicle alone (DMSO) was used as a negative control. 48 h later,
infected and mock infected cells were either incubated with Alamar
Blue.RTM. according to manufacturer's instructions (Life
Technologies) to assess viability or supernatants were collected
for virus quantification by luciferase assay on confluent Vero
cells in 96-well plates infected for 5-6 hours. To generate the
standard curve, known amounts of virus (in plaque-forming units, or
pfu) were added to the Vero cells at the same time as transfer of
786-0 supernatant. Upon measurement of bioluminescence, input pfu
was plotted against mean relative light units for the standard
curve, which was fit by four-parameter non-linear regression
analysis. This standard curve was used to interpolate viral
expression units (VEUs) which correlate with virus titer. For these
experiments, compound 1 was used as a control and peak fold change
(PFC) in VEU for each analog was normalized to peak PFC for VSe1 to
generate the data in Table 1. Thus, all the data presented for
analog compounds is in relation to results obtained for compound
1.
[0215] Glutathione Stability Experiment
[0216] 250 .mu.L of a 40 mM DMSO stock solution of each compound
was added to L-glutathione (15.4 mg, 5 mol equiv.) suspended in 250
.mu.L of DMSO. The resulting mixture was placed in a 37.degree. C.
shaker. 10 .mu.L aliquots were removed and quenched in 990 .mu.L of
water (containing 0.5% formic acid) at various time points,
including at t=0 min, for analysis by ESI-LC-MS. All ESI-LC-MS
analyses were collected on an API2000 LC/MS/MS System (Applied
Biosystems) equipped with a turbo-ion spray ESI probe interfaced
with a Prominence UFLC (Shimadzu) equipped with a reverse phase BDS
Hypersil C18 50.times.2.1 mm column, particle size 3 m (Thermo
Scientific). HPLC/LCMS UV absorption was monitored at 254 nm and
210 nm. Both the compound and the glutathione adduct were
identified by MS. Area of the UV peak was recorded for each time
point.
[0217] Plasma Stability Assay
[0218] MRM Development. 10 mM stock solutions of each analogue were
prepared in methanol and diluted with aqueous formic acid (0.1%) to
a final concentration of 1 .mu.M. 5 .mu.L of the diluted solution
was inserted into a Proxeon nanoelectrospray emitter (Thermo
Scientific, Odense, Denmark) and analyzed in positive ion mode via
nanoESI MS using a QStarXL hybrid quadrupole time-of-flight mass
spectrometer (AB Sciex, Framingham, Mass., USA). Full mass and
product ion spectra were collected for each compound using a
nanoESI voltage of 1000 V, a declustering potential of 30 V and a
focusing potential of 120 V. The product ion spectra were used to
determine two multiple reaction monitoring (MRM) transitions for
each compound with optimized collision energies: a "quantitative
transition" to determine the relative quantities of each compound
as well as a "confirmatory transition" to eliminate isobaric
interference in the measurements.
[0219] Plasma Incubation. 1 mM stock solutions of each analogue
were prepared in methanol and mixed with Balb/c mouse plasma
(Innovative Research, Novi, Mich., USA) that was buffered 1:1 with
PBS (pH=7.4). To increase the through-put of the assay, compounds
were multiplexed in groups of three and analyzed in triplicate. The
compounds were added to the plasma to a final concentration of 10
.mu.M in a total volume of 400 .mu.L. Immediately upon mixing, 200
.mu.L of the sample mixture was quenched with 300 .mu.L of aqueous
formic acid (5%) to prevent degradation of the analogues. The
remainder of the sample mixture was incubated at 37.degree. C. for
three hours and quenched in an identical fashion. The quenched
samples were passed through a 3 kDa Amicon molecular weight cut off
filter (Millipore, Billerica, Mass., USA) by centrifugation at
14,000 rpm for 15 mins.
[0220] LC-MRM. 20 .mu.L of the filtrates was subjected to MRM
analysis via a Qtrap 4000 (AB Sciex, Framingham, Mass., USA) hybrid
triple quadrupole linear ion trap mass spectrometer with a Turbo V
ion spray source coupled to a Dionex Ultimate3000 HPLC (Thermo
Fisher Scientific, Waltham, Mass., USA) (see Supporting
Information). Fritted fused silica columns (200 .mu.m ID) (Molex,
Lisle, Ill., USA) were packed with 5 m Magic C18 (MICHROM
Bioresources Inc., Auburn, Calif., USA) reversed phase beads to a
length of 5 cm using an in house high-pressure vessel.
Chromatographic separations were employed using reversed phase
solvents (water and acetonitrile both containing 0.1% formic acid)
over 10 minutes. Spectra were obtained using an ion spray voltage
of 5000 V and a declustering potential of 25 V. Automatic
quantitation was achieved using MultiQuant (AB Sciex, Framingham,
Mass., USA) via integration of the peak areas for the extracted ion
chromatogram of the quantitative MRM transition. The plasma
stability of each compound was calculated as a percentage of the
compound detected after three hours of incubation in plasma
relative to the amount detected after immediate quenching.
[0221] Ex-Vivo Studies
[0222] Balb/c mice were implanted with CT26-WT (murine colon
carcinoma) cells. Mice were sacrificed 24 days later, after tumors
had reached at least 10 mm.times.10 mm in size. Tumor, lung,
spleen, brain, and abdominal muscle tissue were extracted from the
mice, cut into 2 mm thick slices and cored into 2 mm.times.2 mm
pieces via punch biopsy. Each tissue core was incubated in 1 mL of
Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10%
fetal bovine serum, 30 mM HEPES and amphotericin B, in a
37.degree., 5% CO2 humidified incubator. In order to assess the
viability of each core, alamarBlue.RTM. was added to each well for
a 4-hour incubation period. Viable cores were selected and treated
with various concentrations of VSe1 and analogues. The cores were
then infected with VSV.DELTA.51 expressing a GFP transgene
(VSV.DELTA.51-GFP) four hours post treatment. GFP pictures were
taken for each core 24 hours post infection. Cores and supernatants
were collected 30 hours post infection and titered by plaque
assay.
[0223] Further Analogue Evaluation
[0224] Synergistic Effect Studies
[0225] It was hypothesized that combinations of two or more
compounds as described herein may also be used. An assay was
conducted in which human renal carcinoma (786-0) cells were
co-treated with VSe1 and the VSe1 analogue MD03011 (structure shown
in Table 2). Assay results are provided in FIG. 7. As can be seen,
a surprising synergist effect was observed when cells were treated
with both VSe1 and MD03011, with fold change in VEU/mL climbing
substantially. These results suggest that synergistic effects may
be obtained using combinations of two or more compounds as
described herein. Under the conditions tested, VSe1 combined with
MD03011 provided an excellent synergistic effect.
[0226] Infection of Cells with Oncolytic Maraba Virus
[0227] FIG. 8 shows that VSe1 and its analogues may enhance
infection of cells with oncolytic Maraba virus. Human renal
carcinoma (786-0) cells were left untreated or, treated with
compound at various concentrations. After 4 hours, cells were
infected with MG1-eGFP at MOI 0.005. eGFP fluorescence was detected
24 h after MG1 infection and quantified using a Cellomics ArrayScan
high content screening microscope. Compounds tested include
compounds 1, 10, 27, 28, and 29 as shown in Table 1, each of which
showed increased GFP counts in this assay.
[0228] Infection of Cells with Herpes Simplex Virus-1
[0229] FIG. 9 shows that VSe1 and its analogs enhance infection of
cells with Herpes Simplex Virus-1. Human renal carcinoma (786-0)
cells were left untreated or, treated with compound at various
concentrations. After 4 hours, cells were infected with ICPO-null
HSV-N212eGFP at MOI 0.01. eGFP fluorescence was detected 48 h after
HSV infection and quantified using a Cellomics ArrayScan high
content screening microscope. Compounds tested include compounds 1,
10, 27, 28, and 29 as shown in Table 1, each of which showed
increased GFP counts in this assay.
[0230] VSe1 Analogue Effect on Therapeutic Activity of Oncolytic
VSV in a Cancer Xenograft Model
[0231] FIG. 10 shows that VSe1 analogue 28 (see structure shown in
Table 1) enhances therapeutic activity of oncolytic VSV in a human
colon cancer xenograft model. FIG. 10(A) shows results obtained
when VSV.DELTA.51-resistant HT29 cells (human colon carcinoma) were
subcutaneously engrafted into female Balb/c mice, and the xenograft
mice were then treated with compound 28 by intratumoral injection
followed by VSV.DELTA.51-FLuc. Mouse survival was monitored over
time, as shown in FIG. 10(B). These results demonstrate that
compound 28 reduced tumor growth, and increased mouse survival
rates, in these experimental conditions.
[0232] All citations are hereby incorporated by reference.
[0233] The present invention has been described with regard to one
or more embodiments. However, it will be apparent to persons
skilled in the art that a number of variations and modifications
can be made without departing from the scope of the invention as
defined in the claims.
REFERENCES
[0234] 1. Adusumilli P S, Chan M K, Chun Y S, Hezel M, Chou T C,
Rusch V W et al. (2006). Cisplatin-induced GADD34 upregulation
potentiates oncolytic viral therapy in the treatment of malignant
pleural mesothelioma. Cancer Biol Ther; 5: 48-53. [0235] 2. Ahmed,
M., S. D. Cramer, and D. S. Lyles, Sensitivity of prostate tumors
to wild type and M protein mutant vesicular stomatitis viruses.
Virology, 2004. 330(1): p. 34-49. [0236] 3. Bennett J J, Adusumilli
P, Petrowsky H, Burt B M, Roberts G, Delman K A et al. (2004).
Upregulation of GADD34 mediates the synergistic anticancer activity
of mitomycin C and a gamma134.5 deleted oncolytic herpes virus
(G207). FASEB J; 18: 1001-1003. [0237] 4. Brideau, C., B. Gunter,
B. Pikounis, and A. Liaw, Improved statistical methods for hit
selection in high-throughput screening. J Biomol Screen, 2003.
8(6): p. 634-47. [0238] 5. Chalikonda S, Kivlen M H, O'Malley M E,
Eric Dong X D, McCart J A, Gorry M C et al. (2008). Oncolytic
virotherapy for ovarian carcinomatosis using a
replication-selective vaccinia virus armed with a yeast cytosine
deaminase gene. Cancer Gene Ther; 15: 115-125. [0239] 6. Chang, H.
M., M. Paulson, M. Holko, C. M. Rice, B. R. Williams, I. Marie, and
D. E. Levy, Induction of interferon-stimulated gene expression and
antiviral responses require protein deacetylase activity. Proc Natl
Acad Sci USA, 2004. 101(26): p. 9578-83. [0240] 7. Chen G, Zhou J,
Gao Q, Huang X, Li K, Zhuang L et al. (2006). Oncolytic
adenovirus-mediated transfer of the antisense chk2 selectively
inhibits tumor growth in vitro and in vivo. Cancer Gene Ther; 13:
930-939. [0241] 8. Cheong S C, Wang Y, Meng J H, Hill R, Sweeney K,
Kim D et al. (2008). E1A-expressing adenoviral E3B mutants act
synergistically with chemotherapeutics in immunocompetent tumor
models. Cancer Gene Ther; 15: 40-50. [0242] 9. Chou, T. C. and P.
Talaly, A simple generalized equation for the analysis of multiple
inhibitions of Michaelis-Menten kinetic systems. J Biol Chem, 1977.
252(18): p. 6438-42. [0243] 10. Ebert, O., S. Harbaran, K.
Shinozaki, and S. L. Woo, Systemic therapy of experimental breast
cancer metastases by mutant vesicular stomatitis virus in
immune-competent mice. Cancer Gene Ther, 2005. 12(4): p. 350-8.
[0244] 11. Foloppe J, Kintz J, Futin N, Findeli A, Cordier P,
Schlesinger Y et al. (2008). Targeted delivery of a suicide gene to
human colorectal tumors by a conditionally replicating vaccinia
virus. Gene Ther; 15: 1361-1371. [0245] 12. Freytag S O, Barton K
N, Brown S L, Narra V, Zhang Y, Tyson D et al. (2007). Replication
competent adenovirus-mediated suicide gene therapy with radiation
in a preclinical model of pancreatic cancer. Mol Ther; 15:
1600-1606. [0246] 13. Fukuda K, Abei M, Ugai H, Kawashima R, Seo E,
Wakayama M et al. (2009). E1A, E1B double restricted replicative
adenovirus at low dose greatly augments tumor-specific suicide gene
therapy for gallbladder cancer. Cancer Gene Ther; 16: 126-136.
[0247] 14. Galanis E, Okuno S H, Nascimento A G, Lewis B D, Lee R
A, Oliveira A M et al. (2005). Phase I-II trial of ONYX-015 in
combination with MAP chemotherapy in patients with advanced
sarcomas. Gene Ther; 12: 437-445. [0248] 15. Gao Q, Zhou J, Huang
X, Chen G, Ye F, Lu Y et al. (2006). Selective targeting of
checkpoint kinase 1 in tumor cells with a novel potent oncolytic
adenovirus. Mol Ther; 13: 928-937. [0249] 16. Graat H C, Witlox M
A, Schagen F H, Kaspers G J, Helder M N, Bras J et al. (2006).
Different susceptibility of osteosarcoma cell lines and primary
cells to treatment with oncolytic adenovirus and doxorubicin or
cisplatin. Br J Cancer; 94: 1837-1844. [0250] 17. Hsieh J L, Lee C
H, Teo M L, Lin Y J, Huang Y S, Wu C L et al. (2009).
Transthyretin-driven oncolytic adenovirus suppresses tumor growth
in orthotopic and ascites models of hepatocellular carcinoma.
Cancer Sci; 100: 537-545. [0251] 18. Hsu K F, Wu C L, Huang S C,
Hsieh J L, Huang Y S, Chen Y F et al. (2008). Conditionally
replicating E1B-deleted adenovirus driven by the squamous cell
carcinoma antigen 2 promoter for uterine cervical cancer therapy.
Cancer Gene Ther; 15: 526-534. [0252] 19. Ikeda K, Ichikawa T,
Wakimoto H, Silver J S, Deisboeck T S, Finkelstein D et al. (1999).
Oncolytic virus therapy of multiple tumors in the brain requires
suppression of innate and elicited antiviral responses. Nat Med 5:
881-887. [0253] 20. Ikeda K, Wakimoto H, Ichikawa T, Jhung S,
Hochberg F H, Louis D N et al. (2000). Complement depletion
facilitates the infection of multiple brain tumors by an
intravascular, replication conditional herpes simplex virus mutant.
J Virol; 74: 4765-4775. [0254] 21. Kambara H, Saeki Y, Chiocca E A
(2005). Cyclophosphamide allows for in vivo dose reduction of a
potent oncolytic virus. Cancer Res; 65: 11255-11258. [0255] 22.
Kasuya H, Nishiyama Y, Nomoto S, Goshima F, Takeda S, Watanabe I et
al. (2007). Suitability of a US3-inactivated HSV mutant (L1BR1). as
an oncolytic virus for pancreatic cancer therapy. Cancer Gene Ther;
14: 533-542. [0256] 23. Kim E J, Yoo J Y, Choi Y H, Ahn K J, Lee J
D, Yun C O et al. (2008). Imaging of viral thymidine kinase gene
expression by replicating oncolytic adenovirus and prediction of
therapeutic efficacy. Yonsei Med J; 49: 811-818. [0257] 24. Kottke
T, Thompson J, Diaz R M, Pulido J, Willmon C, Coffey M et al.
(2009). Improved Systemic Delivery of Oncolytic Reovirus to
Established Tumors Using Preconditioning with
Cyclophosphamide-Mediated Treg Modulation and Interleukin-2. Clin
Cancer Res; 15: 561-569. [0258] 25. Kramm C M, Rainov N G,
Sena-Esteves M, Barnett F H, Chase M, Herrlinger U et al. (1996).
Longterm survival in a rodent model of disseminated brain tumors by
combined intrathecal delivery of herpes vectors and ganciclovir
treatment. Hum Gene Ther; 7: 1989-1994. [0259] 26. Kurozumi K,
Hardcastle J, Thakur R, Shroll J, Nowicki M, Otsuki A et al.
(2008). Oncolytic HSV-1 infection of tumors induces angiogenesis
and upregulates CYR61. Mol Ther; 16: 1382-1391. [0260] 27. Kurozumi
K, Hardcastle J, Thakur R, Yang M, Christoforidis G, Fulci G et al.
(2007). Effect of tumor microenvironment modulation on the efficacy
of oncolytic virus therapy. J Natl Cancer Inst; 99: 1768-1781.
[0261] 28. Lun X Q, Jang J N, Tang N, Deng H, Bell J C, Stojdl D F
et al. (2009). Efficacy of systemically administered oncolytic
vaccinia virotherapy for malignant gliomas is enhanced by
combination therapy with rapamycin or cyclophosphamide. Clin Cancer
Res; 15: 2777-2788. [0262] 29. Lun X Q, Zhou H, Alain T, Sun B,
Wang L, Barrett J W et al. (2007). Targeting human medulloblastoma:
oncolytic virotherapy with myxoma virus is enhanced by rapamycin.
Cancer Res; 67: 8818-8827. [0263] 30. Lun, X., D. L. Senger, T.
Alain, A. Oprea, K. Parato, D. Stojdl, B. Lichty, A. Power, R. N.
Johnston, M. Hamilton, I. Parney, J. C. Bell, and P. A. Forsyth,
Effects of intravenously administered recombinant vesicular
stomatitis virus (VSV(deltaM51)) on multifocal and invasive
gliomas. J Natl Cancer Inst, 2006. 98(21): p. 1546-57. [0264] 31.
Mace A T, Harrow S J, Ganly I, Brown S M (2007). Cytotoxic effects
of the oncolytic herpes simplex virus HSV 1716 alone and in
combination with cisplatin in head and neck squamous cell
carcinoma. Acta Otolaryngol; 127: 880-887. [0265] 32. Mai, A., S.
Valente, A. Nebbioso, S. Simeoni, R. Ragno, S. Massa, G. Brosch, F.
De Bellis, F. Manzo, and L. Altucci, New pyrrole-based histone
deacetylase inhibitors: binding mode, enzyme- and cell-based
investigations. Int J Biochem Cell Biol, 2009. 41(1): p. 235-47.
[0266] 33. McCart J A, Puhlmann M, Lee J, Hu Y, Libutti S K,
Alexander H R et al. (2000). Complex interactions between the
replicating oncolytic effect and the enzyme/prodrug effect of
vaccinia mediated tumor regression. Gene Ther; 7: 1217-1223. [0267]
34. Monneret, C., Histone deacetylase inhibitors. Eur J Med Chem,
2005. 40(1): p. 1-13. [0268] 35. Mullerad M, Bochner B H,
Adusumilli P S, Bhargava A, Kikuchi E, Hui-Ni C et al. (2005).
Herpes simplex virus based gene therapy enhances the efficacy of
mitomycin C for the treatment of human bladder transitional cell
carcinoma. J Urol; 174: 741-746. [0269] 36. Nawa A, Nozawa N,
Goshima F, Nagasaka T, Kikkawa F, Niwa Y et al. (2003). Oncolytic
viral therapy for human ovarian cancer using a novel
replication-competent herpes simplex virus type I mutant in a mouse
model. Gynecol Oncol; 91: 81-88. [0270] 37. Nguyen, T. L., H.
Abdelbary, M. Arguello, C. Breitbach, S. Leveille, J. S. Diallo, A.
Yasmeen, T. A. Bismar, D. Kim, T. Falls, V. E. Snoulten, B. C.
Vanderhyden, J. Werier, H. Atkins, M. J. Vaha-Koskela, D. F.
Stojdl, J. C. Bell, and J. Hiscott, Chemical targeting of the
innate antiviral response by histone deacetylase inhibitors renders
refractory cancers sensitive to viral oncolysis. Proc Natl Acad Sci
USA, 2008. 105(39): p. 14981-6. [0271] 38. Pan Q, Liu B, Liu J, Cai
R, Wang Y, Qian C (2007). Synergistic induction of tumor cell death
by combining cisplatin with an oncolytic adenovirus carrying TRAIL.
Mol Cell Biochem; 304: 315-323. [0272] 39. Pan Q W, Zhong S Y, Liu
B S, Liu J, Cai R, Wang Y G et al. (2007). Enhanced sensitivity of
hepatocellular carcinoma cells to chemotherapy with a Smac-armed
oncolytic adenovirus. Acta Pharmacol Sin; 28: 1996-2004. [0273] 40.
Parato, K. A., D. Senger, P. A. Forsyth, and J. C. Bell, Recent
progress in the battle between oncolytic viruses and tumors. Nat
Rev Cancer, 2005. 5(12): p. 965-76. [0274] 41. Pawlik T M, Nakamura
H, Mullen J T, Kasuya H, Yoon S S, Chandrasekhar S et al. (2002).
Prodrug bioactivation and oncolysis of diffuse liver metastases by
a herpes simplex virus 1 mutant that expresses the CYP2B1
transgene. Cancer; 95: 1171-1181. [0275] 42. Qiao J, Wang H, Kottke
T, White C, Twigger K, Diaz R M et al. (2008). Cyclophosphamide
Facilitates Antitumor Efficacy against Subcutaneous Tumors
following Intravenous Delivery of Reovirus. Clin Cancer Res; 14:
259-269. [0276] 43. Reddy, P. S., K. D. Burroughs, L. M. Hales, S.
Ganesh, B. H. Jones, N. Idamakanti, C. Hay, S. S. Li, K. L. Skele,
A. J. Vasko, J. Yang, D. N. Watkins, C. M. Rudin, and P. L.
Hallenbeck, Seneca Valley virus, a systemically deliverable
oncolytic picornavirus, and the treatment of neuroendocrine
cancers. J Natl Cancer Inst, 2007. 99(21): p. 1623-33. [0277] 44.
Ryan P C, Jakubczak J L, Stewart D A, Hawkins L K, Cheng C, Clarke
L M et al. (2004). Antitumor efficacy and tumor-selective
replication with a single intravenous injection of OAS403, an
oncolytic adenovirus dependent on two prevalent alterations in
human cancer. Cancer Gene Ther; 11: 555-569. [0278] 45. Sieben M,
Herzer K, Zeidler M, Heinrichs V, Leuchs B, Schuler M et al.
(2008). Killing of p53-deficient hepatoma cells by parvovirus H-1
and chemotherapeutics requires promyelocytic leukemia protein.
World J Gastroenterol; 14: 3819-3828. [0279] 46. Stanford M M,
Barrett J W, Nazarian S H, Werden S, McFadden G (2007). Oncolytic
virotherapy synergism with signaling inhibitors: Rapamycin
increases myxoma virus tropism for human tumor cells. J Virol; 81:
1251-1260. [0280] 47. Stanford M M, Shaban M, Barrett J W, Werden S
J, Gilbert P A, Bondy-Denomy J et al. (2008). Myxoma virus
oncolysis of primary and metastatic B16F10 mouse tumors in vivo.
Mol Ther 16: 52-59. [0281] 48. Stojdl, D. F., B. Lichty, S.
Knowles, R. Marius, H. Atkins, N. Sonenberg, and J. C. Bell,
Exploiting tumor-specific defects in the interferon pathway with a
previously unknown oncolytic virus. Nat Med, 2000. 6(7): p. 821-5.
[0282] 49. Stojdl, D. F., B. D. Lichty, B. R. tenOever, J. M.
Paterson, A. T. Power, S. Knowles, R. Marius, J. Reynard, L.
Poliquin, H. Atkins, E. G. Brown, R. K. Durbin, J. E. Durbin, J.
Hiscott, and J. C. Bell, VSV strains with defects in their ability
to shutdown innate immunity are potent systemic anti-cancer agents.
Cancer Cell, 2003. 4(4): p. 263-75. [0283] 50. Sung C K, Choi B,
Wanna G, Genden E M, Woo S L, Shin E J (2008). Combined VSV
oncolytic virus and chemotherapy for squamous cell carcinoma.
Laryngoscope; 118: 237-242. [0284] 51. Taneja, S., J. MacGregor, S.
Markus, S. Ha, and I. Mohr, Enhanced antitumor efficacy of a herpes
simplex virus mutant isolated by genetic selection in cancer cells.
Proc Natl Acad Sci USA, 2001. 98(15): p. 8804-8. [0285] 52. Thomas
M, Spencer J F, Toth K, Sagartz J E, Phillips N J, Wold W S (2008).
Immunosuppression enhances oncolytic adenovirus replication and
antitumor efficacy in the Syrian hamster model. Mol Ther;
16:1665-1673. [0286] 53. Tomicic M T, Thust R, Kaina B (2002).
Ganciclovir-induced apoptosis in HSV-1 thymidine kinase expressing
cells: critical role of DNA breaks, Bcl-2 decline and caspase-9
activation. Oncogene; 21: 2141-2153. [0287] 54. Toyoizumi T, Mick
R, Abbas A E, Kang E H, Kaiser L R, Molnar-Kimber K L (1999).
Combined therapy with chemotherapeutic agents and herpes simplex
virus type 1 ICP34.5 mutant (HSV-1716). in human non-small cell
lung cancer. Hum Gene Ther; 10: 3013-3029. [0288] 55. Tumilasci V
F, Oliere S, Nguyen T L, Shamy A, Bell J, Hiscott J (2008).
Targeting the apoptotic pathway with BCL-2 inhibitors sensitizes
primary chronic lymphocytic leukemia cells to vesicular stomatitis
virus-induced oncolysis. J Virol; 82: 8487-8489. [0289] 56.
Ungerechts G, Springfeld C, Frenzke M E, Lampe J, Parker W B,
Sorscher E J et al. (2007). An immunocompetent murine model for
oncolysis with an armed and targeted measles virus. Mol Ther; 15:
1991-1997. [0290] 57. Yoon A R, Kim J H, Lee Y S, Kim H, Yoo J Y,
Sohn J H et al. (2006). Markedly enhanced cytolysis by E1B-19
kD-deleted oncolytic adenovirus in combination with cisplatin. Hum
Gene Ther; 17: 379-390. [0291] 58. Yu D C, Chen Y, Dilley J, Li Y,
Embry M, Zhang H et al. (2001). Antitumor synergy of CV787, a
prostate cancer-specific adenovirus, and paclitaxel and docetaxel.
Cancer Res; 61: 517-525. [0292] 59. Yu Y A, Galanis C, Woo Y, Chen
N, Zhang Q, Fong Y et al. (2009). Regression of human pancreatic
tumor xenografts in mice after a single systemic injection of
recombinant vaccinia virus GLV-1h68. Mol Cancer Ther; 8: 141-151.
[0293] 60. Zhang J, Ramesh N, Chen Y, Li Y, Dilley J, Working P et
al. (2002). Identification of human uroplakin II promoter and its
use in the construction of CG8840, a urothelium-specific adenovirus
variant that eliminates established bladder tumors in combination
with docetaxel. Cancer Res; 62: 3743-3750. [0294] 61. Zhang N H,
Song L B, Wu X J, Li R P, Zeng M S, Zhu X F et al. (2008).
Proteasome inhibitor MG-132 modifies coxsackie and adenovirus
receptor expression in colon cancer cell line lovo. Cell Cycle; 7:
925-933. [0295] 62. Zhou J, Gao Q, Chen G, Huang X, Lu Y, Li K et
al. (2005). Novel oncolytic adenovirus selectively targets
tumor-associated polo-like kinase 1 and tumor cell viability. Clin
Cancer Res; 11: 8431-8440. [0296] 63. Garcia V, Krishnan R, Davis
C, Batenchuk C, Le Boeuf F, Abdelbary H, Diallo J S (2014).
High-throughput Titration of Luciferase-expressing Recombinant
Viruses. J Vis Exp. 2014 Sep. 19; (91).
[0297] 64. Ilkow, C. S., Swift, S. L., Bell, J. C. & Diallo,
J.-S. From scourge to cure: tumor-selective viral pathogenesis as a
new strategy against cancer. PLoS Pathog. 10, e1003836 (2014).
[0298] 65. Russell, S. J., Peng, K.-W. & Bell, J. C. NIH Public
Access. Nat. Biotechnol. 30, 1-29 (2014). [0299] 66. Breitbach, C.
J. et al. Intravenous delivery of a multi-mechanistic
cancer-targeted oncolytic poxvirus in humans. Nature 477, 99-102
(2011). [0300] 67. Heo, J. et al. Randomized dose-finding clinical
trial of oncolytic immunotherapeutic vaccinia JX-594 in liver
cancer. Nat. Med. 19, 329-36 (2013). [0301] 68. Stojdl, D. F. et
al. VSV strains with defects in their ability to shutdown innate
immunity are potent systemic anti-cancer agents. 4, 263-275 (2003).
[0302] 69. Ottolino-Perry, K., Diallo, J.-S., Lichty, B. D., Bell,
J. C. & McCart, J. A. Intelligent design: combination therapy
with oncolytic viruses. Mol. Ther. 18, 251-63 (2010). [0303] 70.
Forbes, N. E., Krishnan, R. & Diallo, J.-S. Pharmacological
modulation of anti-tumor immunity induced by oncolytic viruses.
Front. Oncol. 4, 191 (2014). [0304] 71. Diallo, J.-S. et al. A
high-throughput pharmacoviral approach identifies novel oncolytic
virus sensitizers. Mol. Ther. 18, 1123-9 (2010). [0305] 72. Zhang,
J., Sarma, K. & Curran, T. Recent Progress in the Chemistry of
Mucohalic Acids: Versatile Building Blocks in Organic Synthesis.
Synlett 24, 550-569 (2013). [0306] 73. Garcia, V. et al.
High-throughput Titration of Luciferase-expressing Recombinant
Viruses. J. Vis. Exp. 1-8 (2014). doi:10.3791/51890 [0307] 74.
Budke, B. et al. An optimized RAD51 inhibitor that disrupts
homologous recombination without requiring Michael acceptor
reactivity. J. Med. Chem. 56, 254-63 (2013).
TABLE-US-00002 [0307] TABLE 1 Normalized PFC GSH Plasma stab. (PFC
dose LD50 LD50 with half-life % remaning at Compound Structure
(.mu.M)).sup.a (.mu.M) virus (.mu.M) (min).sup.b 3 hrs 1
##STR00084## 1.00 (60 .mu.M) 79 16 <5 0 2 ##STR00085## 0.37 (72
.mu.M) 87 50 <5 0 3 ##STR00086## 0.19 (96 .mu.M) 140 140
NR.sup.e 65.6 .+-. 6.5 4 ##STR00087## 0.18 (80 .mu.M) 90 90 NR 0 5
##STR00088## 0.27 (36 .mu.M) 41 27 <5 0 6 ##STR00089## 0.21 (60
.mu.M) 73 51 <5 0 7 ##STR00090## 0.17 (60 .mu.M) 52 17 <5 0 8
##STR00091## NE.sup.c >180 >180 NR 88.3 .+-. 9.3 9
##STR00092## 0.67 (120 .mu.M) 148 87 117 0 10 ##STR00093## 0.37 (48
.mu.M) 67 51 32 19.8 .+-. 0.4 11 ##STR00094## 0.03 (240 .mu.M) 332
332 64 42.5 .+-. 9.6 12 ##STR00095## 0.28 (180 .mu.M) 206 203 118
47.6 .+-. 1.4 13 ##STR00096## 0.16 (60 .mu.M) 61 45 21 0 14
##STR00097## 0.03 (17.8 .mu.M) 104 98 <5 70.2 .+-. 8.4 15
##STR00098## NE 66 66 340 14.9 .+-. 7.1 16 ##STR00099## NE >360
>360 NR.sup.e 98.2 .+-. 3.7 17 ##STR00100## NE >360 >360
NR 82.0 .+-. 10.2 18 ##STR00101## NE >360 >360 -- ND.sup.d 19
##STR00102## NE 250 245 -- ND 20 ##STR00103## NE >360 >360 NR
102.9 .+-. 1.6 21 ##STR00104## NE >90 >90 NR 102.7 .+-. 10.8
##STR00105## 22 ##STR00106## 0.47 (96 .mu.M) 119 76 68 72.0 .+-.
3.0 23 ##STR00107## 0.06 (72 .mu.M) >90 55 -- ND 24 ##STR00108##
0.48 (120 .mu.M) 174 96 61 91.6 .+-. 5.2 25 ##STR00109## 0.74 (80
.mu.M) 127 51 53 54.8 .+-. 3.6 26 ##STR00110## 0.52 (96 .mu.M) 110
66 46 64.8 .+-. 7.7 27 ##STR00111## 0.04 (80 .mu.M) 100 60 21 9.0
.+-. 1.4 28 ##STR00112## 1.00 (80 .mu.M) 153 55 96 38.9 .+-. 5.2 29
##STR00113## 0.51 (72 .mu.M) 74 27 74 57.6 .+-. 6.6 30 ##STR00114##
0.57 (32 .mu.M) 36 20 50 42.9 .+-. 7.2 31 ##STR00115## 0.26 (40
.mu.M) 40 34 72 40.1 .+-. 9.8 32 ##STR00116## 0.11 (27 .mu.M) 28 5
24 ND 33 ##STR00117## 0.30 (18 .mu.M) 18 12 24 0 34 ##STR00118##
0.33 (72 .mu.M) 74 6 31 48.0 .+-. 16.5 35 ##STR00119## 0.14 (27
.mu.M) 36 23 43 63.8 .+-. 3.2 36 ##STR00120## 0.07 (180 .mu.M)
>180 >180 34 28.2 .+-. 2.6 37 ##STR00121## 0.56 (48 .mu.M) 58
38 41 0.7 .+-. 0.1 38 ##STR00122## 0.35 (216 .mu.M) 215 107 32 25.7
.+-. 2.9 39 ##STR00123## 0.51 (60 .mu.M) >90 25 34 41.4 .+-. 5.4
40 ##STR00124## 1.6 (27 .mu.M) 36 13 32 15.3 .+-. 2.5 41
##STR00125## 0.19 (40 .mu.M) 39 30 35 51.4 .+-. 8.2 42 ##STR00126##
0.03 (40 .mu.M) 55 17 40 49.1 .+-. 12.4 43 ##STR00127## 0.15 (60
.mu.M) >90 45 69 58.3 .+-. 0.6 44 ##STR00128## 0.08 (60 .mu.M)
63 39 31 45.9 .+-. 8.1 45 ##STR00129## 0.06 (48 .mu.M) 43 37 31
54.2 .+-. 4.2 46 ##STR00130## 0.09 (40 .mu.M) 42 36 32 23.1 .+-.
0.8 47 ##STR00131## 0.10 (40 .mu.M) 36 35 35 22.7 .+-. 8.4 48
##STR00132## 0.65 (32 .mu.M) 38 24 14 36.5 .+-. 7.6 49 ##STR00133##
0.31 (96 .mu.M) 131 67 64 44.6 .+-. 1.2 50 ##STR00134## 0.42 (60
.mu.M) 85 29 54 39.6 .+-. 2.6 51 ##STR00135## 0.31 (72 .mu.M) 89 28
53 44.0 .+-. 1.0 52 ##STR00136## 0.25 (60 .mu.M) 67 28 45 54.1 .+-.
5.1 53 ##STR00137## 0.51 (60 .mu.M) 64 27 40 50.1 .+-. 10.5
* * * * *