Patent Application
20120134963
The present invention relates to compounds that enhance viral growth, spread or cytotoxicity. More specifically, the present invention relates to compounds that enhance oncolytic viral efficacy and methods of using same.
Oncolytic viruses (OV) are novel replicating therapeutics selected or designed to preferentially grow in and kill cancer cells. Diverse OV platforms have shown promise for the treatment of several types of cancers (1-5). Due to the self-replicating nature of OVs, the principle challenge in OV therapy is not initial saturation of all the tumors but rather efficient spreading within tumor cells upon infection of a reasonable amount of cancerous tissue. Much like most live vaccines, essentially all OVs have been genetically modified or selected for attenuated growth. While this limits the spread of OVs in normal host tissues, it can also blunt their natural ability to rapidly spread within and between tumors (6).
Vesicular stomatitis virus (VSV) is an OV that has shown outstanding efficacy in several in vivo cancer models (2, 3, 7). VSV is a small, enveloped, negative strand RNA rhabdovirus that is particularly sensitive to type I Interferons (IFN), a key component of normal innate cellular anti-viral immunity. In most cancers, IFN response pathways are defective and VSV can be extremely effective (2, 3). However, several cancers retain robust anti-viral defenses, rendering VSV much less effective when used alone (5, 8).
We have previously shown that histone deacetylase inhibitors (HDI) enhance the ability of VSV to infect and kill resistant tumor cells due in part to HDI-induced dampening of IFN mediated anti-viral response (9) and to enhanced apoptosis (10). Continual administration of HDIs prior to and following VSV administration in mice led to better spread of the virus preferentially within tumors and led to reduced tumor growth in the combination treatment as opposed to either treatment alone. Because continuous administration of HDIs can lead to various toxicities including cardiac toxicity in humans, it is desirable to identify other small molecules that could be used to enhance OV efficacy.
There is a need in the art to identify compounds and compositions that enhance virus growth, spread or cytotoxicity. There is also a need in the art to identify compounds and compositions that enhance oncolytic virus efficacy. Further, there is a need in the art to identify novel methods for treating cancer cells in vitro and in vivo.
The present invention relates to compounds that enhance viral growth, spread or cytotoxicity. More specifically, the present invention relates to compounds that enhance oncolytic viral efficacy and methods of using same.
According to the present invention there is provided a compound of formula
an N-oxide, pharmaceutically acceptable addition salt, quarternary amine or stereochemically isomeric form thereof, wherein:
A is a 5-membered heterocylic ring comprising 1-4 heteroatoms selected from O, N or S and 1 or 2 double bonds;
R1 is H, oxo, alkoxycarbonyl, hydrazinylcarbonylalkyl or amino;
R2 is nothing, alkyl, halogen, carboxyl, heteroarylcarbonylamino or hydroxyl;
R3 is nothing, H, alkyl, halogen or heterocyclylaminosulfonyl, and
R4 is H, alkyl, unsubstituted aryl or aryl substituted with 1-3 halogens.
According to a further embodiment, there is provided a compound as described above wherein A is
R1 is H, oxo, alkoxycarbonyl, hydrazinylcarbonylalkyl or amino;
R2 is nothing, alkyl, halogen, carboxyl, heteroarylcarbonylamino or hydroxyl;
R3 is nothing, H, alkyl, halogen or heterocyclylaminosulfonyl, and
R4 is H, alkyl, unsubstituted aryl or aryl substituted with 1-3 halogens.
The present invention also provides a compound as described above represented by
wherein,
X2, X3 and X4 are independently C or N;
R1 is H, oxo, alkoxycarbonyl, hydrazinylcarbonylalkyl or amino;
R2 is nothing, alkyl, halogen, carboxyl, heteroarylcarbonylamino or hydroxyl;
R3 is nothing, H, alkyl, halogen or heterocyclylaminosulfonyl;
R4 is H, alkyl, unsubstituted aryl or aryl substituted with 1-3 halogens;
wherein the bond between the atoms X2 and X3 is a single or a double bond;
wherein the bond between the atoms X3 and X4 is a single or a double bond;
wherein the bond between the atoms X4 and X5 is a single or a double bond;
wherein the bond between the atoms X5 and X1 is a single or a double bond;
wherein when the bond between the atoms X2 and X3 and the bond between X4 and X5 are each single bonds, the bond between the atoms X3 and X4 is a double bond, the bond between the atoms X5 and X1 is a single bond, X2 is C and R1 is oxo; or
wherein when the bond between the atoms X2 and X3 and the bond between X4 and X5 are each single bonds, the bond between the atoms X3 and X4 is a double bond, the bond between the atoms X5 and X1 is a double bond and X1 is N, or
wherein when the bond between the atoms X2 and X3 and the bond between X4 and X5 are each double bonds, the bond between the atoms X3 and X4 is a single bond and the bond between X5 and X1 is a single bond.
In a further embodiment, there is provided a compound as described above represented by
wherein:
R2 is alkyl, halogen, carboxyl or hydroxyl;
R3 is alkyl or halogen, and
R4 is alkyl, unsubstituted aryl or aryl substituted with 1-3 halogens.
Also provided is a compound as described above selected from the group consisting of 3,4-dichloro-5-phenyl-2,5-dihydrofuran-2-one, 2-phenyl-1H-imidazole-4-carboxylic acid 1.5 hydrate, 3-[5-(2,3-dichlorophenyl)-2H-1,2,3,4-azepanyl)amino]sulfonyl}1H-pyrrole-2-carboxylate, 2-amino-5-phenyl-3-thiophenecarboxylic acid, methyl 3-[(quinolin-6-ylcarbonyl)amino]thiophene-2-carboxylate, 5-(2-chloro-6-fluorophenyl)-3-hydroxy-4-methyl-2,5-dihydrofuran-2-one, and 5-(2,6-dichlorophenyl)-3-hydroxy-4-methyl-2,5-dihydrofuran-2-one.
The present invention also provides a compound having formula
wherein,
R5 is hydroxyalkyl, heteroaryl, unsubstituted aryl, aryl substituted with one or more substituents selected from the group consisting of alkyl and halogen, heterocyclyl or benzodioxylalkyl; and
R6 is amino, aryl or aryl substituted with one or more substituents selected from the group consisting of alkyl and halogen.
Also provided is a compound as described above, wherein the compound is selected from N-(3,4-dimethylphenyl)-N′-(2-pyridyl)thiourea, N1-(2,6-diethylphenyl)hydrazine-1-carbothioamide, N-(2-hydroxyethyl)-N′-(2-methylphenyl)thiourea, N1-(2-chloro-6-methylphenyl)hydrazine-1-carbothioamide, and N-(4-chlorophenyl)-N-(2,3-dihydro-1,4-benzodioxin-2-ylmethyl)urea.
The present invention also provides a compound as described above selected from the group consisting of 3,4-dichloro-5-phenyl-2,5-dihydrofuran-2-one, 2-phenyl-1H-imidazole-4-carboxylic acid 1.5 hydrate, 3-[5-(2,3-dichlorophenyl)-2H-1,2,3,4-tetraazol-2-yl]propanohydrazide, ethyl 3,5-dimethyl-4-{[(2-oxo-3-azepanyl)amino]sulfonyl}1H-pyrrole-2-carboxylate, 2-amino-5-phenyl-3-thiophenecarboxylic acid, methyl 3-[(quinolin-6-ylcarbonyl]amino]thiophene-2-carboxylate, 5-(2-chloro-6-fluorophenyl)-3-hydroxy-4-methyl-2,5-dihydrofuran-2-one, and 5-(2,6-dichlorophenyl)-3-hydroxy-4-methyl-2,5-dihydrofuran-2-one, N-(3,4-dimethylphenyl)-N′-(2-pyridyl)thiourea, N1-(2,6-diethylphenyl)hydrazine-1-carbothioamide, N-(2-hydroxyethyl)-N′-(2-methylphenyl)thiourea, N1-(2-chloro-6-methylphenyl)hydrazine-1-carbothioamide, N-(4-chlorophenyl)-N′-(2,3-dihydro-1,4-benzodioxin-2-ylmethyl)urea, 4-(benzyloxy)-2-methyl-1-nitrobenzene, 1-{4-[(2-methylquinolin-4-yl)amino]phenyl}ethan-1-one, N1-(1,2,3,10-tetramethoxy-9-oxo-5,6,7,9-tetrahydrobenzo[a]heptalen-7-yl)acetamide, methyl N-[4-(dimethylamino)benzylidene]aminomethanehydrazonothioate, methyl N-(4-chlorophenyl)-(dimethylamino)methanimidothioate hydroiodide, 4′,5′-dihydro-4′-(5-methoxyphenyl)spiro[2H-1-benzothiopyran-3(4H)m3′-[3H]pyrazole]-4-one, 1H-benzo[d]imidazole-2-thiol, N-(2-furylmethylidene)-(4-{[(2-furylmethylidene)amino]methyl}cyclohexyl)methanamine; 2-[4-(diethoxymethyl)benzylidene]malononitrile; 2-(cyclopropylcarbonyl)-3-(3-phenoxy-2-thienyl)acrylonitrile; N′-(3,5-dichlorophenyl)-2,4-difluorobenzohydrazide; 10-(hydroxymethylene)phenanthren-9(10H)-one; N1-(2,5-difluorophenyl)-4-({[4-(trifluoromethyl)phenyl]sulfonyl}amino)benzene-1-sulfonamide; N-[4-(4-chlorophenyl)-2,5-dioxopiperazino]-2-(2,3-dihydro-1H-indol-1-yl)acetamide, 4-{[(4-{[(3-carboxyacryloyl)amino]methyl}cyclohexyl)methyl]amino}-4-oxo-2-butenoic acid; 5-oxo-3-phenyl-5-{4-[3-(trifluoromethyl)-1H-pyrazol-1-yl]anilino}pentanoic acid, N1-(4-chlorophenyl)-2-({4-methyl-5-[1-methyl-2-(methylthio)-1H-imidazol-5-yl]-4H-1,2,4-triazol-3-yl}thio)acetamide, 6-[2-(4-methylphenyl)-2-oxoethyl]-3-phenyl-2,5-dihydro-1,2,4-triazin-5-one; N1-[2-(tert-butyl)-7-methyl-5-(trifluoromethyl)pyrazolo[1,5-a]pyrimidin-3-yl]acetamide; 4-(2,3-dihydro-1H-inden-5-yl)-6-(trifluoromethyl)pyrimidin-2-amine; ethyl 1-(2,3-dihydro-1-benzofuran-5-ylsulfonyl)-4-piperidinecarboxylate, 2,3-diphenylcycloprop-2-en-1-one, 1-cyclododecyl-1H-pyrrole-2,5-dione, 1-(4-methylphenyl)-2,5-dihydro-1H-pyrrole-2,5-dione, 2-[(4-phenoxyanilino)methyl]isoindoline-1,3-dione, 2-{[1-(3-chloro-4-methylphenyl)-2,5-dioxotetrahydro-1H-pyrrol-3-yl]thio}benzoic acid, 1-(1,3-benzodioxol-5-ylmethyl)-2,5-dihydro-1H-pyrrole-2,5-dione, 4-chloro-N-[3-chloro-2-(isopropylthio)phenyl]benzamide, and N-({5-[({2-[(2-furylmethyl)thio]ethyl}amino)sulfonyl]-2-thienyl}methyl)benzamide. In a preferred embodiment, the compound is 3,4-dichloro-5-phenyl-2,5-dihydrofuran-2-one (DCPDF).
The present invention also provides a composition comprising the compound as described above, and a pharmaceutically acceptable carrier, diluent or excipient.
Also, the present invention provides a composition comprising the compound as described above and one or more of a) a virus, preferably an attenuated virus, a genetically modified virus 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).
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 immortalized cells, cancer cells or tumor cells. In an alternate embodiment, the cells are MDCK, HEK293, Vero, HeLa or PER.C6cells.
Also provided is a kit comprising the compound as described above and a) a virus, preferably an attenuated or genetically modified virus 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).
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.
The present invention also provides a method of enhancing the spread of a virus in cells comprising, administering the compound as described above to the cells prior to, after or concurrently with the virus. The method is preferably practiced in vitro.
The present invention also provides a method of enhancing the spread 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.
The present invention also provides a method of enhancing 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.
Also provided is a method of 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.
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.
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.
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.
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.
This summary of the invention does not necessarily describe all features of the invention.
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:
a-j) show results of 4T1 cells pre-treated for 2 hours with selected drugs identified from a screen of more than 13500 compounds. Cells were then challenged with RFP-expressing VSVΔ51 at indicated MOI (0.03 to 0.003). 48 hours later, fluorescence pictures were taken and coomassie stains were performed.
The following description is of a preferred embodiment.
In a first aspect, there is provided compounds which increase or enhance viral spread in cancer cells, tumors or immortalized cells, such as, for example, CT-26, 4T1 breast cancer cells, 786-0, U-251, B16 melanoma cells and colon tumors but not normal or non-immortalized cells.
In a further aspect, there is provided compounds which increase or enhance viral titers in cells, for example, CT-26 cells, 786-0, 4T1, colon tumor, vulvar tumor and bone tumor cells but not normal or non-immortalized cells.
In still a further aspect, there is provided compounds which increase cytotoxicity of viruses, particularly oncolytic viruses in cells.
Based on results obtained for specific compounds in various comprehensive screens as described herein and having regard to the results obtained from several structure-functional 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 in in-vivo or in-vitro experiments or in additional structure-function analyses to determine additional compounds with interesting features as described herein.
The present invention concerns viral sensitizing compounds of formula
an N-oxide, pharmaceutically acceptable addition salt, quarternary amine or stereochemically isomeric form thereof, wherein:
A is a 5-membered heterocylic ring comprising 1-4 heteroatoms selected from O, N or S and 1 or 2 double bonds;
R1 is H, oxo, alkoxycarbonyl, hydrazinylcarbonylalkyl or amino;
R2 is nothing, alkyl, halogen, carboxyl, heteroarylcarbonylamino or hydroxyl;
R3 is nothing, H, alkyl, halogen or heterocyclylaminosulfonyl, and
R4 is H, alkyl, unsubstituted aryl or aryl substituted with 1-3 halogens.
An interesting group of compounds are those compounds of formula (I) wherein,
R1 is H, oxo, alkoxycarbonyl, hydrazinylcarbonylalkyl or amino;
R2 is nothing, alkyl, halogen, carboxyl, heteroarylcarbonylamino or hydroxyl;
R3 is nothing, H, alkyl, halogen or heterocyclylaminosulfonyl, and
R4 is H, alkyl, unsubstituted aryl or aryl substituted with 1-3 halogens.
A further interesting group of compounds are those compounds of formula (I), for example, but not limited to formula
wherein,
X2, X3 and X4 are independently C or N;
R1 is H, oxo, alkoxycarbonyl, hydrazinylcarbonylalkyl or amino;
R2 is nothing, alkyl, halogen, carboxyl, heteroarylcarbonylamino or hydroxyl;
R3 is nothing, H, alkyl, halogen or heterocyclylaminosulfonyl;
R4 is H, alkyl, unsubstituted aryl or aryl substituted with 1-3 halogens;
wherein the bond between the atoms X2 and X3 is a single or a double bond;
wherein the bond between the atoms X3 and X4 is a single or a double bond;
wherein the bond between the atoms X4 and X5 is a single or a double bond;
wherein the bond between the atoms X5 and X1 is a single or a double bond;
wherein when the bond between the atoms X2 and X3 and the bond between X4 and X5 are each single bonds, the bond between the atoms X3 and X4 is a double bond, the bond between the atoms X5 and X1 is a single bond, X2 is C and R1 is oxo; or
wherein when the bond between the atoms X2 and X3 and the bond between X4 and X5 are each single bonds, the bond between the atoms X3 and X4 is a double bond, the bond between the atoms X5 and X1 is a double bond and X1 is N, or
wherein when the bond between the atoms X2 and X3 and the bond between X4 and X5 are each double bonds, the bond between the atoms X3 and X4 is a single bond and the bond between X5 and X1 is a single bond.
A further interesting group of compounds are those compounds of formula (I), for example, but not limited to formula (III)
wherein:
R2 is alkyl, halogen, carboxyl or hydroxyl;
R3 is alkyl or halogen, and
R4 is alkyl, unsubstituted aryl or aryl substituted with 1-3 halogens.
A further interesting group of compounds are those of formula (I), for example, but not limited to
The present invention also concerns compounds of formula
wherein,
R5 is hydroxyalkyl, heteroaryl, unsubstituted aryl, aryl substituted with one or more substituents selected from the group consisting of alkyl and halogen, heterocyclyl or benzodioxylalkyl; and
R6 is amino, aryl or aryl substituted with one or more substituents selected from the group consisting of alkyl and halogen.
A further interesting group of compounds are those compounds of formula (IV), for example, but not limited to
Additional interesting compounds are identified in Table 1.
Additional interesting viral sensitizing compounds are described below in Table 2 and may be referred to by their chemical name, code or structure.
By the term “viral sensitizing compound” or “viral sensitizing agent” it is meant a compound that increases or enhances the spread of a virus, preferably a genetically modified virus or attenuated virus, more preferably an oncolytic virus in one or more types of cells, preferably cancer or tumor cells but not normal or non-immortalized 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 or reproductive capacity of a virus, more preferably a genetically modified, attenuated or 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.
By the term “oncolytic virus” it is meant a virus that preferentially infects and lyses cancer or tumor cells as compared to normal cells. 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. Examples of oncolytic viruses known in the art include, without limitation, reovirus, newcastle disease virus, adenovirus, herpes virus, polio virus, mumps virus, measles virus, influenza virus, vaccinia virus, rhabdovirus, vesicular stomatitis virus and derivatives/variants thereof.
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, one that has been genetically modified using recombinant techniques known within the art, 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 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 VSVΔ51 (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).
By the term “alkyl” it is meant a straight or branched chain alkyl having 1 to 20 carbon atoms, more particularly 1 to 8 carbon atoms, or even more particularly 1 to 6 carbon atoms. Examples of alkyl groups include methyl, ethyl, propyl, isopropyl, butyl, isobutyl, secondary butyl, tertiary butyl, pentyl, isopentyl, neopentyl, hexyl, heptyl, octyl, 2-ethylhexyl, 1,1,3,3-tetramethylbutyl, nonyl, decyl, dodecyl, tetradecyl, hexadecyl, octadecyl and eicosyl.
The one or more types of cancer or tumor cells may be cancer 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 may be used to treat any other cancer or tumor in vivo or in vitro.
The present invention also provides a composition comprising a) one or more viral sensitizing compounds as described herein and b) one or more additional components, for example, but not limited to, a carrier, diluent or excipient, a pharmaceutically acceptable carrier, diluent or excipient, a virus, for example, but not limited to an attenuated virus, a genetically modified virus or an oncolytic virus, cancer or tumor cells, non-cancerous cells, cell culture media, 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 HSV (15-18), adenoviruses (19), measles virus (20) reovirus (21, 22) and vaccinia virus (23).
Cisplatin binds and cross-links cellular DNA leading to apoptosis when DNA is not repaired. Cisplatin has been investigated in combination with oncolytic adenoviruses (25-34), herpes viruses (35-37), parvovirus (38), vaccinia virus (39) and vesicular stomatitis virus (40). Enhanced therapeutic activity in vitro and in vivo has been observed when combining cisplatin with adenovirus, herpesvirus, parvovirus and vaccinia virus whereas slight inhibition was observed for vesicular stomatitis virus.
Mitomycin C (MMC) is a DNA cross-linking antibiotic with antineoplastic properties. MMC exhibited synergistic cytotoxicty with HSV (40, 41). In vivo, combination herpes virus and MMC significantly improved therapeutic effects in models of gastric carcinomatosis (43) and non-small cell lung cancer (41).
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 (42, 44) and the combination reduced tumor growth relative to the monotherapies (45). 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 (30).
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 (46). Targeted oncolytic HSV viruses in combination with GCV significantly improved survival in models of human ovarian cancer (47) and rat gliosarcoma (48). Adenoviruses, engineered to express the HSV TK gene, also show enhanced anti-tumor activity when combined with GCV (49-51).
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 vaccinia viruses expressing CD was significantly enhanced when combined with 5-FC therapy in immune-competent ovarian cancer (52) and immune suppressed colon cancer models (53,54).
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 prostatetargeted adenovirus significantly reduced in vivo tumor volume and resulted in synergistic in vitro cytotoxicity (55, 56).
Rapamycin (sirolimus) is an immunosuppressant commonly used in transplant patients however it has also been shown to significantly enhance the oncolytic effects of the poxviruses myxoma and vaccinia virus (23, 57-59).
The prototypical proteosome inhibitor MG-132 enhanced cellular CAR expression in Lovo colon carcinoma cells, which was accompanied with enhanced adenovirus target gene expression and oncolysis (60).
The efficacy of oncolytic VSV against chronic lymphocytic leukemia cells was increased by combination therapy with the BCL-2 inhibitor EM20-25 (61).
One group showed that a single dose of angiostatic cRGD peptide treatment before oncolytic virus treatment enhanced the antitumor efficacy of oncolytic HSV (24, 62).
The present invention also provides a kit comprising one or more viral sensitizing compound(s) or a composition comprising same. The kit may also comprise a cell culture dish/plate or multi-well dish/plate, an apparatus to deliver the viral sensitizing compounds or a composition comprising the same to a cell, cell culture or cell culture medium, or to a subject in vivo. The kit may also comprise instructions for administering or using the viral sensitizing compound, virus, for example, but not limited to attenuated virus, genetically modified virus, oncolytic virus, any combination thereof, or any combination of distinct viruses.
For in vivo therapeutic applications, there is provided a pharmaceutical composition comprising one or more viral sensitizing 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 or glucose to make the solution isotonic. 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, Philidelphia, Pa. (2000), herein incorporated by reference.
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 viral sensitizing 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, as would be known in the art are also contemplated.
For administration by inhalation or insufflation, the viral sensitizing compounds can be formulated into an aqueous or partially aqueous solution, which can then be utilised 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.
The dosage requirements for the viral sensitizing compounds of the present invention 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. Thereafter, the dosage is increased until the optimum 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.
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 or an oncolytic virus. Alternatively, the viral sensitizing compound may be administered 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 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, monoclonal antibodies and the like.
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 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, 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.
In an embodiment of the present invention there is provided a composition comprising a viral sensitizing compound as described herein for example, one or more of 3,4-dichloro-5-phenyl-2,5-dihydrofuran-2-one, 2-phenyl-1H-imidazole-4-carboxylic acid 1.5 hydrate, 3-[5-(2,3-dichlorophenyl)-2H-1,2,3,4-tetraazol-2-yl]propanohydrazide, ethyl 3,5-dimethyl-4-{[(2-oxo-3-azepanyl)amino]sulfonyl}1H-pyrrole-2-carboxylate, 2-amino-5-phenyl-3-thiophenecarboxylic acid, methyl 3-[(quinolin-6-ylcarbonyl)amino]thiophene-2-carboxylate, 5-(2-chloro-6-fluorophenyl)-3-hydroxy-4-methyl-2,5-dihydrofuran-2-one, and 5-(2,6-dichlorophenyl)-3-hydroxy-4-methyl-2,5-dihydrofuran-2-one, N-(3,4-dimethylphenyl)-N′-(2-pyridyl)thiourea, N1-(2,6-diethylphenyl)hydrazine-1-carbothioamide, N-(2-hydroxyethyl)-N′-(2-methylphenyl)thiourea, N1-(2-chloro-6-methylphenyl)hydrazine-1-carbothioamide, N-(4-chlorophenyl)-N′-(2,3-dihydro-1,4-benzodioxin-2-ylmethyl)urea, 4-(benzyloxy)-2-methyl-1-nitrobenzene, 1-{4-[(2-methylquinolin-4-yl)amino]phenyl}ethan-1-one, N1-(1,2,3,10-tetramethoxy-9-oxo-5,6,7,9-tetrahydrobenzo[a]heptalen-7-yl)acetamide, methyl N-[4-(dimethylamino)benzylidene]aminomethanehydrazonothioate, methyl N-(4-chlorophenyl)-(dimethylamino)methanimidothioate hydroiodide, 4′,5′-dihydro-4′-(5-methoxyphenyl)spiro[2H-1-benzothiopyran-3(4H)m3′-[3H]pyrazole]-4-one, 1H-benzo[d]imidazole-2-thiol, N-(2-furylmethylidene)-(4-{[(2-furylmethylidene)amino]methyl}cyclohexyl)methanamine; 2-[4-(diethoxymethyl)benzylidene]malononitrile; 2-(cyclopropylcarbonyl)-3-(3-phenoxy-2-thienyl)acrylonitrile; N′-(3,5-dichlorophenyl)-2,4-difluorobenzohydrazide; 10-(hydroxymethylene)phenanthren-9(10H)-one; N1-(2,5-difluorophenyl)-4-({[4-(trifluoromethyl)phenyl]sulfonyl}amino)benzene-1-sulfonamide; N-[4-(4-chlorophenyl)-2,5-dioxopiperazino]-2-(2,3-dihydro-1H-indol-1-yl)acetamide, 4-{[(4-{[(3-carboxyacryloyl)amino]methyl}cyclohexyl)methyl]amino}-4-oxo-2-butenoic acid; 5-oxo-3-phenyl-5-{4-[3-(trifluoromethyl)-1H-pyrazol-1-yl]anilino}pentanoic acid, N1-(4-chlorophenyl)-2-({4-methyl-5-[1-methyl-2-(methylthio)-1H-imidazol-5-yl]-4H-1,2,4-triazol-3-yl}thio)acetamide, 6-[2-(4-methylphenyl)-2-oxoethyl]-3-phenyl-2,5-dihydro-1,2,4-triazin-5-one; N1-[2-(tert-butyl)-7-methyl-5-(trifluoromethyl)pyrazolo[1,5-a]pyrimidin-3-yl]acetamide; 4-(2,3-dihydro-1H-inden-5-yl)-6-(trifluoromethyl)pyrimidin-2-amine; ethyl 1-(2,3-dihydro-1-benzofuran-5-ylsulfonyl)-4-piperidinecarboxylate, 2,3-diphenylcycloprop-2-en-1-one, 1-cyclododecyl-1H-pyrrole-2,5-dione, 1-(4-methylphenyl)-2,5-dihydro-1H-pyrrole-2,5-dione, 2-[(4-phenoxyanilino)methyl]isoindoline-1,3-dione, 2-{[1-(3-chloro-4-methylphenyl)-2,5-dioxotetrahydro-1H-pyrrol-3-yl]thio}benzoic acid, 1-(1,3-benzodioxol-5-ylmethyl)-2,5-dihydro-1H-pyrrole-2,5-dione, 4-chloro-N-[3-chloro-2-(isopropylthio)phenyl]benzamide, and N-({5-[({2-[(2-furylmethyl)thio]ethyl}amino)sulfonyl]-2-thienyl}methyl)benzamide, parbendazole, methiazole, colchicine, vinorelbine base, ethyl 4-amino-2-anilino-5-nitrothiophene-3-carboxylate, 2-[di(methylthio)methylidene]malononitrile, N-(1H-indol-3-ylmethyl)-N-methyl-2-phenylethanamine oxalate, 3-(2-furyl)-N-(4,5,6,7-tetrahydro-1,3-benzothiazol-2-yl)acrylamide, albendazole, 2-phenyl-4-quinolinamine oxalate, paclitaxel, nocodazole, (2,5-dimethoxyphenyl)[(2-methoxy-1-naphthyl)methyl]amine, DBPDF, BB90, L1EA, R1EA, or LT33 (see
In a further embodiment there is provided a composition comprising a viral sensitizing compound as described above except that the composition does not comprise one or more compounds selected from the group consisting of 3,4-dichloro-5-phenyl-2,5-dihydrofuran-2-one, 2-phenyl-1H-imidazole-4-carboxylic acid 1.5 hydrate, 3-[5-(2,3-dichlorophenyl)-2H-1,2,3,4-tetraazol-2-yl]propanohydrazide, ethyl 3,5-dimethyl-4-{[(2-oxo-3-azepanyl)amino]sulfonyl}1H-pyrrole-2-carboxylate, 2-amino-5-phenyl-3-thiophenecarboxylic acid, methyl 3-[(quinolin-6-ylcarbonyl)amino]thiophene-2-carboxylate, 5-(2-chloro-6-fluorophenyl)-3-hydroxy-4-methyl-2,5-dihydrofuran-2-one, and 5-(2,6-dichlorophenyl)-3-hydroxy-4-methyl-2,5-dihydrofuran-2-one, N-(3,4-dimethylphenyl)-N′-(2-pyridyl)thiourea, N1-(2,6-diethylphenyl)hydrazine-1-carbothioamide, N-(2-hydroxyethyl)-N′-(2-methylphenyl)thiourea, N1-(2-chloro-6-methylphenyl)hydrazine-1-carbothioamide, N-(4-chlorophenyl)-N′-(2,3-dihydro-1,4-benzodioxin-2-ylmethyl)urea, 4-(benzyloxy)-2-methyl-1-nitrobenzene, 1-{4-[(2-methylquinolin-4-yl)amino]phenyl}ethan-1-one, N1-(1,2,3,10-tetramethoxy-9-oxo-5,6,7,9-tetrahydrobenzo[a]heptalen-7-yl)acetamide, methyl N-[4-(dimethylamino)benzylidene]aminomethanehydrazonothioate, methyl N-(4-chlorophenyl)-(dimethylamino)methanimidothioate hydroiodide, 4′,5′-dihydro-4′-(5-methoxyphenyl)spiro[2H-1-benzothiopyran-3(4H)m3′-[3H]pyrazole]-4-one, 1H-benzo[d]imidazole-2-thiol, N-(2-furylmethylidene)-(4-{[(2-furylmethylidene)amino]methyl}cyclohexyl)methanamine; 2-[4-(diethoxymethyl)benzylidene]malononitrile; 2-(cyclopropylcarbonyl)-3-(3-phenoxy-2-thienyl)acrylonitrile; N′-(3,5-dichlorophenyl)-2,4-difluorobenzohydrazide; 10-(hydroxymethylene)phenanthren-9(10H)-one; N1-(2,5-difluorophenyl)-4-({[4-(trifluoromethyl)phenyl]sulfonyl}amino)benzene-1-sulfonamide; N-[4-(4-chlorophenyl)-2,5-dioxopiperazino]-2-(2,3-dihydro-1H-indol-1-yl)acetamide, 4-{[(4-{[(3-carboxyacryloyl)amino]methyl}cyclohexyl)methyl]amino}-4-oxo-2-butenoic acid; 5-oxo-3-phenyl-5-{4-[3-(trifluoromethyl)-1H-pyrazol-1-yl]anilino}pentanoic acid, N1-(4-chlorophenyl)-2-({4-methyl-5-[1-methyl-2-(methylthio)-1H-imidazol-5-yl]-4H-1,2,4-triazol-3-yl}thio)acetamide, 6-[2-(4-methylphenyl)-2-oxoethyl]-3-phenyl-2,5-dihydro-1,2,4-triazin-5-one; N1-[2-(tert-butyl)-7-methyl-5-(trifluoromethyl)pyrazolo[1,5-a]pyrimidin-3-yl]acetamide; 4-(2,3-dihydro-1H-inden-5-yl)-6-(trifluoromethyl)pyrimidin-2-amine; ethyl 1-(2,3-dihydro-1-benzofuran-5-ylsulfonyl)-4-piperidinecarboxylate, 2,3-diphenylcycloprop-2-en-1-one, 1-cyclododecyl-1H-pyrrole-2,5-dione, 1-(4-methylphenyl)-2,5-dihydro-1H-pyrrole-2,5-dione, 2-[(4-phenoxyanilino)methyl]isoindoline-1,3-dione, 2-{[1-(3-chloro-4-methylphenyl)-2,5-dioxotetrahydro-1H-pyrrol-3-yl]thio}benzoic acid, 1-(1,3-benzodioxol-5-ylmethyl)-2,5-dihydro-1H-pyrrole-2,5-dione, 4-chloro-N-[3-chloro-2-(isopropylthio)phenyl]benzamide, and N-({5-[({2-[(2-furylmethyl)thio]ethyl}amino)sulfonyl]-2-thienyl}methyl)benzamide, parbendazole, methiazole, colchicine, vinorelbine base, ethyl 4-amino-2-anilino-5-nitrothiophene-3-carboxylate, 2-[di(methylthio)methylidene]malononitrile, N-(1H-indol-3-ylmethyl)-N-methyl-2-phenylethanamine oxalate, 3-(2-furyl)-N-(4,5,6,7-tetrahydro-1,3-benzothiazol-2-yl)acrylamide, albendazole, 2-phenyl-4-quinolinamine oxalate, paclitaxel, nocodazole, or (2,5-dimethoxyphenyl)[(2-methoxy-1-naphthyl)methyl]amine, DBPDF, BB90, L1EA, R1EA, or LT33.
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 be selected from the group consisting of increasing 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 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.
A High-Throughput Screen for the Identification of Virus Sensitizers
Anti-viral signaling pathways involve several layers of regulation spanning from the cellular plasma membrane (eg TLRs and IFN receptors), through the cytoplasm (eg. IKKs, Jak RIG-I,) into the nucleus (eg. IRFs, STATs, NF-κB) and back out again. Without wishing to be bound by theory or limiting in any manner this suggested that a high throughput, infected cell based screen could potentially identify viral sensitizing compounds that are active at multiple levels to enhance virus replication, etc. To test this idea, an initial screen of 12,280 synthetic drug-like molecules was carried out in combination with VSVΔ51 and a breast cancer cell line (4T1) known to be only partially permissive to this particular virus. We compared and contrasted the cytotoxicity of a given compound alone or in combination with a low dose of VSVΔ51 as described herein. Low concentrations of virus (0.03 plaque forming units per cell) were used so that virus alone caused minimal cell death over the time of the assay, thus favoring the selection of compounds that promote virus replication and spread in cell culture. A number of compounds appeared to increase virus killing of 4T1 cells and these lead candidates (see for example dot plot of
VSe1 disrupts anti-viral signaling
We examined the ability of VSe1 to block interferon activated transcription programs. HEK 293 cells were transfected with a reporter plasmid that contains the luciferase gene under the control of an interferon responsive promoter element (ISRE). When treated with human interferon alpha, the transfected cells expressed luciferase in a dose dependent fashion however interferon dependent transcription could be blunted by the addition of increasing doses of VSe1 to the cultures (see for example
VSe1 Represses Virus-Induced Cellular Gene Expression
Without wishing to be bound by theory or limiting in any manner, the results presented above collectively suggest that one of the key effects of VSe1 and other viral sensitizers could be to reduce transcriptional levels of anti-viral gene products. To test this idea, we used gene expression arrays and compared and contrasted mRNA profiles in cells infected with VSVΔ51 in the presence or absence of VSe1. CT26 colon cancer cells were pre-treated with VSe1 or vehicle and subsequently infected with VSVΔ51 (MOI 0.03) or mock-infected with media. RNA was extracted 24 hours post-infection and mRNA expression was compared. We found that under these conditions VSVΔ51 infection leads to increased transcription of over 80 cellular genes including a variety of known antiviral genes (e.g. OAS, Mx2). SAHA pre-treatment significantly blunted virus induced transcription of many of these genes (79%) but in select cases appeared to further increase the transcription of genes induced by the virus (six genes over 2-fold increase relative to virus alone). Consistent with its ability to enhance the replication and spread of VSVΔ51, VSe1 potently reduced the induction of over 96% of the cellular antiviral transcripts induced by virus infection alone.
VSe1 Augments VSVΔ51 Oncolytic Activity In Vivo and in Primary Human Tumor Samples
As VSe1 enhanced the oncolytic activity of VSVΔ51 in cancer cells but not normal cells in vitro we sought to determine if this level of specificity would be observed in vivo and/or in freshly explanted patient tumor material. Balb/C mice were engrafted with a VSVΔ51-resistant CT26 colon cancer cell line and tumor growth was evaluated following treatment with vehicle control, VSe1, vehicle/VSVΔ51 or VSe1/VSVΔ51. Whereas neither VSe1 nor VSVΔ51 alone had a significant effect on tumor growth, the combination of VSe1 and VSVΔ51 led to a significant delay in tumor progression. Importantly, when animals were treated with VSVΔ51 harboring the GFP gene in the presence or absence of VSe1 there was no detectable virus in any of the normal tissues of treated animals. This same specificity and magnitude of virus enhancement was seen when primary human tumour explants were infected in vitro in the presence of VSe1. An example of these experiments is demonstrated wherein VSVΔ51-GFP was added to a colon cancer sample in the presence or absence of VSe1. While in this patient sample VSVΔ51-GFP replicated poorly on its own, its growth and spread (as visualized by green fluorescence) was significantly enhanced in the presence of VSe1. The titers of virus produced in primary human tumor samples was determined in the presence of increasing amounts of VSe1. As was observed in previous tumor cell line experiments, we found that VSe1 could increase VSVΔ51 from 10 to 100 fold in primary human tumor samples of various origins. In one colorectal cancer case, adjacent normal colon tissue was isolated and VSVΔ51 on its own grew better in tumour versus adjacent normal tissues. Importantly, while treatment of the explants with VSe1 did not increase the replication of VSVΔ51 in normal tissues, it led to over 100-fold growth of VSVΔ51 in the tumour tissue, leading to roughly 1000-fold differential in replication between normal and cancerous tissues.
The present invention will be further illustrated in the following examples.
In order to identify viral sensitizing agents in a high-throughput fashion, we developed an assay using 96-well plates to quantify oncolytic viral activity against cancer cells. Initially, the assay examined VSVΔ51-associated cytotoxicity against 4T1 breast cancer cells. This assay uses HEPES-buffered, phenol red free cell culture media (Gibco®) to minimize background and to minimize problems associated with pH changes that can influence VSV growth. For this assay, 30 000 4T1 cells per well are plated in 96-well plates and the cells are allowed to adhere to the plates overnight in the aforementioned media. The next day cells are pre-treated for 4 hours with a 10 μM concentration of library compounds (dissolved in 5% dimethylsulfoxide (DMSO)), which is added using a Biomek FX liquid handler, and subsequently challenged with VSVΔ51 at a multiplicity of infection (MOI) of 0.03 or a control (added using a Biotek μFill). Duplicate plates are run for each condition. On each plate, internal controls comprising cells pre-treated (at the same time as the library compounds) with either 5% DMSO (negative control) or 5 μM SAHA (positive control) are included. 40 hours later, plates are removed from the incubators and allowed to equilibrate for 2 hours at room temperature and Alamar Blue® reagent is added to the plates and a first fluorescence reading is taken immediately using a fluorescence plate reader (Perkin Elmer EnVision plate reader, 530 nm excitation, 590 emission). Plates are then incubated at room temperature for 2 hours and a second fluorescence reading is taken. The difference between the second and first reading is calculated and employed in further calculations of metabolic activity. While taking the difference between the second and the first reading is known to reduce the interfering effect of auto-fluorescent compounds, we have found that pre-equilibration of plate temperatures for 2 hours and incubation at room temperature following addition of Alamar blue is an effective way to reduce plate position effects.
Identification of Viral Sensitizing Compounds
Preferred compounds were subsequently identified on the basis of normalized metabolic activity values (cytotoxicity), where low cytotoxicity in absence of virus and high cytotoxicity in presence of virus is favored. Notably, the ratio of cell metabolic activity observed between the compound used in combination with VSVΔ51 over that of the compound used alone and the difference between these two values are used as selection criteria. The B-score is also considered for choosing hit compounds. Specifically, compounds exhibiting negative B-scores in presence of virus but exhibiting B-scores near zero without virus are considered to be potential viral sensitizers. B-score normalization uses a two-way median polish, thus taking into account row/column positional effects (Brideau et al., J Biomol Screen. 2003 December; 8(6):634-47; which is herein incorporated by reference) and preventing selection of hits based on position. To this effect, compounds which on their own exhibit near zero B-scores but show highly negative B-scores in presence of virus (suggestive of more cytotoxicity) are favored. Additionally, the difference between B-scores obtained in the presence and in absence of virus is considered as a parameter (ΔB-score) in order to identify compounds exhibiting large differences in activity in infected versus uninfected wells. Finally, duplication of the results is considered in the selection of the hits. For each parameter, stringency thresholds were established based on the mean and the standard deviation and/or based on the hit rate. Potential viral sensitizers are then identified based on four different weighted scores calculated from the different parameters described above and a compiled list of the top quartile scoring compounds was generated.
Validation of Novel Viral Sensitizing Compounds
Using the method described above, we have screened more than 13500 compounds and have identified many viral sensitizing agents. Those compounds that met stringent cut-off criteria were analyzed further for structural similarities as described herein. Compounds were validated when independent tests twice showed enhanced VSVΔ51 spread and oncolytic activity in 4T1 cells by two or more of the following methods: fluorescence microscopy (viral spread), coomassie blue assay (cell detachment, cytotoxicity), alamar blue assay (metabolic activity, cytotoxicity), or plaque assay (viral titer). Initial testing is done within a given dose range near that used for the screen (between 20 μM and 2.5 μM).
One viral sensitizing agent identified by the screening method that exhibited high activity was 3,4-dichloro-5-phenyl-2,5-dihydrofuran-2-one (DCPDF). We confirmed that DCPDF increased the spread of VSVΔ51 in 4T1 breast cancer cells, and found it also enhanced the spread of VSVΔ51 in CT26 colon cancer cells, 786-0 human renal carcinoma cells and U251 human glioblastoma cells (
DCPDF increased the titers of VSVΔ51 in several cell lines including 786-0, CT26 and 4T1 (
Isobologram analyses based on the method of Chou and Talalay (Chou and Talalay (1977) J. Biol. Chem. 252:6438-6442, which is herein incorporated by reference) confirmed that DCPDF led to bone fide synergistic cell killing when used in combination with VSVΔ51 in both 4T1 and CT26 cells (
We also examined whether DCPDF could enhance the spread of other oncolytic viruses. In B16 melanoma cells as well as in 4T1 cells, we observed enhanced spread and viral output of a genetically attenuated strain of vaccinia virus (VVdd, deleted for thymidine kinase and vaccinia growth factor genes;
Other Novel Viral Sensitizing Compounds
Subsequent to validating the highly active viral sensitizing agent DCPDF, we went on to validate other viral sensitizing compounds according to the criteria mentioned previously. The compounds were initially tested as soon as they were identified from the screen; subsequently, priority was given to molecules exhibiting a similar structure to DCPDF, based on the characteristic 5-atom furane ring and possible substitution of the oxygen atom of the furane ring with another electronegative atom such as nitrogen or sulfur. Additional viral sensitizing compounds were identified and validated (see
Materials, Methods and Other Technical Details
Drugs and Chemicals: Compounds used for the high throughput screen were a selected subset from the Maybridge HitFinder, Chembridge DIVERSet, Microsource Spectrum, Prestwick, BIOMOL, and Sigma LOPAC screening collections. Selection of compounds was based on chemical diversity and non-overlap. All compounds were dissolved at 10 mM in DMSO. For further in vitro testing, VSe1 (also known as 3,4-dichloro-5-phenyl-2,5-dihydrofuran-2-one or DCPDF) was obtained from Ryan Scientific (Mt. Pleasant, S.C., USA) and dissolved in DMSO at 10 mM. For in vivo use, VSe1 was dissolved fresh in 30% ethanol, 5% DMSO (in PBS) at 0.4 mg per 50 μl. SAHA was obtained from Exclusive Chemistry (Obninsk, Russia) and also dissolved in DMSO at 10 mM. Both were stored at −20° C. IFNα treatment was performed using Intron A (Shering), stored at 40 C at stock concentration 10×106 IU/ml.
Cell lines: The following cell lines were purchased from the American Type Culture Collection: 4T1 (mouse breast adenocarcinoma), B16 (mouse melanoma), 786-0 (human renal cancer), U251 (human glioma), GM38 (normal human fibroblast), HEK 293T (Human embryonic kidney), U2OS (human osteosarcoma) and Vero (monkey kidney cells). All cell lines, except GM38, were cultured in HyQ High glucose Dulbecco's modified Eagle medium (DMEM) (HyClone) supplemented with 10% fetal calf serum (CanSera, Etobicoke, Canada). GM38 cells were grown in DMEM supplemented with 20% fetal bovine serum (Gibco). All cell lines were incubated at 37 degrees in a 5% CO2 humidified incubator.
Viruses: The Indiana serotype of VSV (mutant or wild type) was used throughout this study and was propagated in Vero cells. VSVΔ51 is a naturally occurring interferon-inducing mutant of the heat-resistant strain of wild-type VSV Indiana, while VSVΔ51 expressing RFP or GFP are recombinant derivatives of VSVΔ51 (49). Virions were purified from cell culture supernatants by passage through a 0.2 μm Steritop filter (Millipore, Billerica, Mass.) and centrifugation at 30,000 g before resuspension in PBS (HyClone, Logan, Utah). For the High throughput screen, 30% sucrose was added to increase virus stability. For in vivo studies, virus was further purified on 5-50% Optiprep® (Sigma) gradient. Doubled deleted vaccinia virus (VVdd) expressing fluorescent Cherry protein was obtained by homologous recombination with VVdd-GFP and was propagated in U20S cells.
High Throughput Screening: 4T1 cells were plated in HEPES-buffered, phenol red free DMEM (Gibco) in 96-well plates and allowed to adhere overnight. The next day, 95% confluent cells were pre-treated for 4 hours with a 10 μM concentration of library compounds added using a Biomek FX liquid handler (Beckman Coulter, Fullerton, Calif., USA), and subsequently challenged with VSVΔ51 at an MOI of 0.0325 or a control added using a μFill liquid handler (Biotek, Winooski, Vt., USA). Duplicate plates were run for each condition. On each plate, internal controls consisting of cells pre-treated (at the same time as the library compounds) with DMSO (negative control) were included. 40 hours later, plates were incubated with Alamar Blue® and fluorescence emission rate was assessed using an EnVision plate reader (Perkin Elmer, Waltham, Mass., USA). Cytotoxicity of each drug was determined in both presence and absence of virus and was defined as follows: Cytotoxicity in presence of VSVΔ51 (VSV)=fluorescence rate in presence of drug+VSVΔ51 divided by average fluorescence rate of the DMSO+VSVΔ51 controls (eight per plate). Cytotoxicity in absence of VSVΔ51 (CTRL)=fluorescence rate in presence of drug (but no virus) divided by average fluorescence rate of the DMSO control (no virus, eight per plate). Cell killing induced by virus alone was assessed by comparing DMSO controls on infected and mock-infected plates and was below 10%. Average Log (VSV/CTRL) values for the duplicates were used as the parameter for selection of hits, where −0.3 was the cutoff value. Reproducibility of the Log (VSV/CTRL) values across the duplicates was also considered in the selection.
Viral titers: Supernatants from each treatment condition were collected at the specified time point. A serial dilution was then performed in serum-free DMEM and 500 μl of each dilution was applied to a confluent monolayer of Vero cells for 45 minutes. Subsequently, the plates were overlayed with 0.5% agarose in DMEM-10% FBS and the plaques were grown for 24 h. Carnoy fixative (Methanol:Acetic Acid is 3:1) was then applied directly on top of the overlay for 5 minutes. The overlay was gently lifted off using a spatula and the fixed monolayer was stained via 0.5% coomassie blue for 30 minutes, after which the plaques were counted. VVDD samples were tittered on U2OS monolayer using 1.5% carboxyl methyl-cellulose in DMEM-10% FBS for 48 h. The overlay was removed and the monolayer stained via 0.5% coomassie blue for 30 minutes, after which the plaques were counted.
Assessment of combination index: 25 000 4T1 or CT26 cells were plated per well in 96 well plates and left to adhere over night. The following day, cells were pre-treated for 4 hours with serial dilutions of Vse1 (200 μM to 1.5 μM, 1:2 dilution steps) then infected with serial dilutions of VSVΔ51 (100000 PFU to 780 PFU) keeping a fixed ratio combination of VSVΔ51 and VSe1 (500 PFU to 1 μM). Cytotoxicity was assessed using Alamar blue reagent after 48 h. Combination indices (CI) were calculated using the Calcusyn Software (Biosoft, Ferguson Mo., USA) according to the method of Chou and Talalay (Chou and Talalay (1977) J. Biol. Chem. 252:6438-6442).
Reporter assays: HEK 293T cells were plated at 1.3×105 cells/well in 24-well dishes. The following day, cells were co-transfected with an ISRE-driven luciferase reporter plasmid as described previously (Lai, F et al. (2008). J Virol Methods 153: 276-279) and a CMV-driven β-galactosidase control plasmid. 6 Hours post-transfection, cells were treated with VSe1 or mock treated with vehicle. Approximately 20 hours after receiving VSe1, cells were then treated with IFN-α with a complete media change. The following day, cells were lysed and measured for luciferase using the BD Monolight kit (Becton Dickinson, Franklin Lakes, N.J., USA). β-galactosidase activity was measured using the Luminescent β-galactosidase kit (Clontech, Mountainview, Calif., USA).
HDAC enzymatic activity assays: Activity of recombinant HDACs 1 through 11 were tested in presence of either VSe1 20 μM or TSA 20 μM by Reaction Biology Corp. (Malvern, Pa., USA) using 50 μM of a fluorogenic peptide from p53 residues 379-382 (RHKKAc for HDAC 1-7 and 9-11 or RHKAcKAc for HDAC8). HDAC activity was compared to control treated with DMSO (vehicle) and expressed as a percentage of HDAC activity in the control. All conditions were tested in duplicate.
Microarrays: CT26 cells were plated at a density of 1.5×106 in 100 mm petris and allowed to adhere overnight. The next day, cells were treated with either DMSO, 20 μM VSe1 or 5 μM SAHA. Four hours later, VSVΔ51 (or control media) was added at an MOI of 0.03. Twenty four hours post-infection, cells were collected using a rubber scraper in a small volume of PBS. Cell pellets were subsequently used for total RNA extraction using Qiagen QiaShredder columns and the Qiagen RNeasy extraction kit (Qiagen, Valencia, Calif., USA). A pooled duplicate sample RNA was used for subsequent hybridization on microarray. RNA quality was confirmed using an Agilent 2100 Bioanalyzer (Santa Clara, Calif., USA) prior to labeling of RNA and hybridization onto Affymetrix mouse gene 1.0 ST arrays according to manufacturer instructions. Low signal genes (<50 in DMSO-treated, mock-infected control) were removed from the data set. Expression of the remaining genes was normalized to average overall signal for each array. Subsequently the fold change in gene expression was calculated for each gene in relation to the mock-infected, DMSO-treated control. A 2-fold change in gene expression relative to the control was used as a cutoff for selection of treatment-perturbed genes. Analysis was done using Microsoft Excel.
Animal tumor model: Syngenic colon carcinoma tumors were established subcutaneously in the hind flanks of 6 week old female Balb/c mice by injecting 3×105 of VSVΔ51-resistant CT26 cells suspended in 100 μl PBS. By day 11 post-implantation, tumors had reached an approximate average size of 220 mm3 and mice were treated with a 0.4 mg dose of VSe1 resuspended in 30% ethanol 5% DMSO, 65% PBS (or vehicle control) administered intraperitoneally. VSVΔ51 (1×108 pfu) was introduced intratumorally 4 h following the first VSe1 dose. Subsequently, VSe1 (or vehicle) was re-administered on day 13 and day 15 post implantation (0.4 mg/injection/mouse). Tumor sizes were measured every 2-3 days using an electronic caliper. Tumor volume was calculated as =(length×width2)/2. Relative tumor size for each mouse at each time point was calculated relative to the initial tumor size measured on day 11. ANOVA was used to assess statistical significance of observed differences at each time point.
Treatment and processing of primary tissue specimens: Primary tissue specimens were obtained from consenting patients who underwent tumor resection.
All tissues were processed within 48 h post surgical excision. 300 μm tissue slices were obtained using a Krumdieck tissue slicer (Alabama research and development, Munford, Ala., USA) and plated in DMEM supplemented with 10% FBS. After the indicated treatment conditions, samples were visualized by fluorescence microscopy. Tissues were subsequently weighed and homogenized in 1 ml of PBS using a homogenizer (Kinematica AG-PCU-11). Serial dilutions of tissue homogenates were prepared in serum free media and viral titers were quantified by standard plaque assay.
Realtime PCR: 2 μg RNA was used to synthesize cDNA using the SuperScript first-strand synthesis system (random hexamer method) according to manufacturers instructions (Invitrogen, ON, Canada). The QuantiTect SYBR Green PCR kit was used as recommended (Qiagen, ON, Canada). Real time PCR reactions were performed on a Rotor-gene RG-300 (Corbett Research, Australia). Optimal threshold and reaction efficiency were determined using the Rotor-gene software. Melt curves for each primer exhibited a single peak, indicating specific amplification, which was also confirmed by agarose gel. Ct values were determined using the Rotor-gene software at the optimal threshold previously determined for each gene. Gene expression relative to GAPDH was calculated. Fold induction was calculated relative to the DMSO treated control for each gene. Primers were designed using Primer 3 v 4.0
All citations are hereby incorporated by reference.
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.
| Number | Date | Country | Kind |
|---|---|---|---|
| 2689707 | Nov 2009 | CA | national |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/CA10/01057 | 7/7/2010 | WO | 00 | 2/2/2012 |
| Number | Date | Country | |
|---|---|---|---|
| 61270345 | Jul 2009 | US |
