Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
  • A61K35/66—Microorganisms or materials therefrom
  • A61K35/76—Viruses; Subviral particles; Bacteriophages
  • A61K35/763—Herpes virus
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
  • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca

Definitions

• the present invention relates to the use of an oncolytic virus and a JAK inhibitor in the treatment of cancer.
• Oncolytic virotherapy concerns the use of lytic viruses which selectively infect and kill cancer cells.
• Some oncolytic viruses are promising therapies as they display extraordinarily selection for replication in cancer cells and their self-limiting propagation within tumors results in fewer toxic side effects.
• Several oncolytic viruses have shown great promise in the clinic (Bell, J., Oncolytic Viruses: An Approved Product on the Horizon? Mol
• JAKs Janus kinases
• pseudokinase domain dual specificity kinase activity has now been identified for this JH2 domain and inactivation of JH2 catalytic activity increased JAK2 basal activity and downstream signalling.
• JAK2-activating mutations including the V617F in myeloproliferative neoplasms and R683G in acute lymphoblastic leukemias are localized to the JH2 domain and function by releasing the autoinhibitory interactions between the JH 1 and JH2 domains. It is the presence of two active kinase domains which provides the rationale for naming them Janus kinases, after the two-faced Roman god.
• the JAK/STAT pathway is the major signalling cascade activated by cytokine, chemokine and growth factor receptors
• the signalling network comprises the JAK family of nonreceptor tyrosine kinases and the STAT (signal transduction and transcription) family of transcription factors ( Figure 1). Cytokine or growth factor receptor binding stimulates JAKs, which phosphorylate and activate STATs, whose dimerization and nuclear translocation results in the transcription of STAT-dependent genes.
• JAK/STAT signalling is normally transient and tightly regulated.
• STATs can be phosphorylated by other kinases including SRC, ABL and EGFR.
• Activated JAK2 can epigenetically regulate expression of STAT-independent genes by a direct phosphorylation of histone H3 leading to expression of oncogenes such as MYC and has been implicated in primary mediastinal B-cell lymphomas (PMBL) and other hematopoietic cancers.
• PMBL primary mediastinal B-cell lymphomas
• JAK inhibitors Jakinibs
• Phases l-lll JAK inhibitors
• Virology 338 (2005) 173-181) reported that HSV-1 infection rapidly induced SOCS3 and that SOCS3 induction was inhibited by the addition of a JAK3 inhibitor WHI-P131 , with treatment of cells with WHI-P131 , or transfection of antisense oligonucleotides specific for SOCS3, dramatically supressing replication of HSV-1 in FL cells. Because of the apparent ability of the JAK3 inhibitor to suppress HSV- 1 infection and propagation JAK3 inhibitors were proposed by Yokota et al as a potential treatment for viral infection.
• the present invention concerns the use of an oncolytic virus to treat cancer, wherein the subject receives the oncolytic virus and a chemotherapeutic agent as part of the programme of treatment.
• the chemotherapeutic agent is preferably a JAK inhibitor.
• the oncolytic virus and chemotherapeutic agent are administered as part of a method of treating cancer in the subject. They may be administered simultaneously, e.g. as a combined preparation or as separate preparations one administered immediately after the other. Alternatively, they may be administered separately and sequentially, where one agent is administered and then the other administered later after a predetermined time interval.
• an oncolytic virus for use in a method of treating cancer, the method comprising simultaneous or sequential administration of an oncolytic virus and a JAK inhibitor.
• the use of an oncolytic virus in the manufacture of a medicament for use in a method of treatment of cancer is provided, wherein the method of treatment comprises administering a JAK inhibitor to the patient in need of treatment.
• a method of treating cancer is provided, the method comprising administration of an oncolytic virus and a JAK inhibitor to a patient in need of treatment, thereby treating the cancer.
• an oncolytic virus for use in a method of treating cancer, wherein the method of treatment comprises administering a JAK inhibitor to the patient in need of treatment.
• a JAK inhibitor for use in a method of treating cancer, wherein the method of treatment comprises administering an oncolytic virus to the patient in need of treatment.
• the use of a JAK inhibitor in the manufacture of a medicament for use in a method of treatment of cancer is provided, wherein the method of treatment comprises administering an oncolytic virus to the patient in need of treatment.
• composition or medicament comprising an oncolytic virus and a JAK inhibitor.
• the JAK inhibitor is Ruxolitinib, Tofacitinib, AZD1480 or MS-1020 .
• the JAK inhibitor may be a different JAK inhibitor, e.g. as described herein.
• the JAK inhibitor is a JAK3 inhibitor, optionally having combined inhibitory action at one or more, or all, of JAK1 , JAK2 or TYK2 or optionally not having combined inhibitory action at one or more, or all, of JAK1 , JAK2 or TYK2.
• the oncolytic virus is an oncolytic herpes simplex virus.
• the oncolytic herpes simplex virus is a type 1 herpes simplex virus (HSV-1) or a type-2 herpes simplex virus (HSV-2) or an intertypic recombinant of HSV-1 and HSV- 2.
• HSV-1 herpes simplex virus
• all copies of the ICP34.5 gene in the genome of the oncolytic herpes simplex virus are modified such that the ICP34.5 gene is incapable of expressing a functional ICP34.5 gene product.
• the oncolytic herpes simplex virus may be an ICP34.5 null mutant.
• one or both of the ICP34.5 genes in the genome of the oncolytic herpes simplex virus are modified such that the ICP34.5 gene is incapable of expressing a functional ICP34.5 gene product.
• the oncolytic herpes simplex virus is a mutant of HSV-1 strain 17.
• the oncolytic herpes simplex virus is HSV1716 (ECACC
• herpes simplex virus is a mutant of HSV-1 strain 17 mutant 1716.
• the oncolytic herpes simplex virus preferably is not part of an artificial chromosome, or does not have genomic DNA that is part of an artificial chromosome, such as a BAC (bacterial artificial chromosome) or YAC (yeast artificial chromosome).
• the oncolytic herpes simplex virus preferably has a genome that functions (e.g. replicates) in an independent manner that corresponds with other wild type herpes simplex viruses.
• the oncolytic herpes simplex virus according to the present invention may be variants of and/or derived from naturally occurring strains of the herpes simplex virus.
• the oncolytic virus is selected from one of: an oncolytic reovirus, an oncolytic vaccinia virus, an oncolytic adenovirus, an oncolytic Coxsackie virus, an oncolytic Newcastle Disease Virus, an oncolytic parvovirus, an oncolytic poxvirus, an oncolytic paramyxovirus.
• kits comprising a predetermined amount of oncolytic virus and a predetermined amount of chemotherapeutic agent is provided, wherein the chemotherapeutic agent is a JAK inhibitor.
• the kit may be provided together with instructions for the administration of the oncolytic virus and a JAK inhibitor sequentially or simultaneously in order to provide a treatment for cancer.
• an oncolytic virus preferably HSV1716
• the products may be pharmaceutically acceptable formulations and may optionally be formulated as a combined preparation for coadministration.
• the oncolytic virus may be any oncolytic virus. Preferably it is a replication-competent virus, being replication-competent at least in the target tumor cells. In some
• the oncolytic virus is selected from one of an oncolytic herpes simplex virus, an oncolytic reovirus, an oncolytic vaccinia virus, an oncolytic adenovirus, an oncolytic Newcastle Disease Virus, an oncolytic Coxsackie virus, an oncolytic measles virus.
• An oncolytic virus is a virus that will lyse cancer cells (oncolysis), preferably in a selective manner. Viruses that selectively replicate in dividing cells over non-dividing cells are often oncolytic. Oncolytic viruses are well known in the art and are reviewed in Molecular Therapy Vol.18 No.2 Feb 2010 pg 233-234.
• the oncolytic virus is a herpes simplex virus.
• the herpes simplex virus (HSV) genome comprises two covalently linked segments, designated long (L) and short (S). Each segment contains a unique sequence flanked by a pair of inverted terminal repeat sequences.
• the long repeat (RL or R L ) and the short repeat (RS or R s ) are distinct.
• the HSV ICP34.5 also called ⁇ 34.5) gene, which has been extensively studied, has been sequenced in HSV-1 strains F and syn17+ and in HSV-2 strain HG52. One copy of the ICP34.5 gene is located within each of the RL repeat regions.
• Mutants inactivating one or both copies of the ICP34.5 gene are known to lack neurovirulence, i.e. be avirulent/ non-neurovirulent (non-neurovirulence is defined by the ability to introduce a high titre of virus (approx 10 6 plaque forming units (pfu)) to an animal or patient without causing a lethal encephalitis such that the LD 50 in animals, e.g. mice, or human patients is in the approximate range of ⁇ 10 6 pfu), and be oncolytic.
• avirulent/ non-neurovirulent is defined by the ability to introduce a high titre of virus (approx 10 6 plaque forming units (pfu)) to an animal or patient without causing a lethal encephalitis such that the LD 50 in animals, e.g. mice, or human patients is in the approximate range of ⁇ 10 6 pfu), and be oncolytic.
• Oncolytic HSV that may be used in the present invention include HSV in which one or both of the ⁇ 34.5 (also called ICP34.5) genes are modified (e.g. by mutation which may be a deletion, insertion, addition or substitution) such that the respective gene is incapable of expressing, e.g. encoding, a functional ICP34.5 protein.
• ⁇ 34.5 also called ICP34.5 genes
• both copies of the ⁇ 34.5 gene are modified such that the modified HSV is not capable of expressing, e.g. producing, a functional ICP34.5 protein.
• the oncolytic herpes simplex virus may be an ICP34.5 null mutant where all copies of the ICP34.5 gene present in the herpes simplex virus genome (two copies are normally present) are disrupted such that the herpes simplex virus is incapable of producing a functional ICP34.5 gene product.
• the oncolytic herpes simplex virus may lack at least one expressible ICP34.5 gene.
• the herpes simplex virus may lack only one expressible ICP34.5 gene.
• the herpes simplex virus may lack both expressible ICP34.5 genes.
• each ICP34.5 gene present in the herpes simplex virus may not be expressible.
• Oncolytic herpes simplex virus may be derived from any HSV including any laboratory strain or clinical isolate (non-laboratory strain) of HSV.
• the HSV is a mutant of HSV-1 or HSV-2.
• the HSV may be an intertypic recombinant of HSV-1 and HSV-2.
• the mutant may be of one of laboratory strains HSV-1 strain 17, HSV-1 strain F or HSV-2 strain HG52.
• the mutant may be of the non- laboratory strain JS-1.
• the mutant is a mutant of HSV-1 strain 17.
• the herpes simplex virus may be one of HSV-1 strain 17 mutant 1716, HSV-1 strain F mutant R3616, HSV-1 strain F mutant G207, HSV-1 mutant NV1020, or a further mutant thereof in which the HSV genome contains additional mutations and/or one or more heterologous nucleotide sequences. Additional mutations may include disabling mutations, which may affect the virulence of the virus or its ability to replicate. For example, mutations may be made in any one or more of ICP6, ICPO, ICP4, ICP27. Preferably, a mutation in one of these genes (optionally in both copies of the gene where appropriate) leads to an inability (or reduction of the ability) of the HSV to express the corresponding functional polypeptide.
• the additional mutation of the HSV genome may be accomplished by addition, deletion, insertion or substitution of nucleotides.
• the oncolytic herpes simplex virus preferably is not part of an artificial chromosome, or does not have genomic DNA that is part of an artificial chromosome, such as a BAC (bacterial artificial chromosome) or YAC (yeast artificial chromosome).
• the oncolytic herpes simplex virus preferably has a genome that functions (e.g. replicates) in an independent manner that corresponds with other wild type herpes simplex viruses.
• the oncolytic herpes simplex virus according to the present invention may be variants of and/or derived from naturally occurring strains of the herpes simplex virus.
• oncolytic herpes simplex viruses are known in the art. Examples include HSV1716, R3616 (e.g. see Chou & Roizman, Proc. Natl. Acad. Sci. Vol.89, pp.3266- 3270, April 1992), G207 (Toda et al, Human Gene Therapy 9:2177-2185, October 10, 1995), NV1020 (Geevarghese et al, Human Gene Therapy 2010 Sep; 21 (9): 11 19-28), RE6 (Thompson et al, Virology 131 , 171-179 (1983)), and OncovexTM (Simpson et al, Cancer Res 2006; 66:(9) 4835-4842 May 1 , 2006; Liu et al, Gene Therapy (2003): 10, 292-303), dlsptk, hrR3,R4009, MGH-1 , MGH-2, G47A, Myb34.5, DF3v34.5, HF10,
• the herpes simplex virus is HSV-1 strain 17 mutant 1716 (HSV1716).
• HSV 1716 is an oncolytic, non-neurovirulent HSV and is described in EP
• HSV 1716 has been deposited on 28 January 1992 at the European Collection of Animal Cell Cultures, Vaccine Research and Production Laboratories, Public Health Laboratory Services, Porton Down, Salisbury, Wiltshire, SP4 0JG, United Kingdom under accession number V92012803 in accordance with the provisions of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure (herein referred to as the 'Budapest Treaty').
• the herpes simplex virus is a mutant of HSV-1 strain 17 modified such that both ICP34.5 genes do not express a functional gene product, e.g. by mutation (e.g. insertion, deletion, addition, substitution) of the ICP34.5 gene, but otherwise resembling or substantially resembling the genome of the wild type parent virus HSV-1 strain 17+. That is, the virus may be a variant of HSV1716, having a genome mutated so as to inactivate both copies of the ICP34.5 gene of HSV-1 strain 17+ but not otherwise altered to insert or delete/modify other protein coding sequences.
• oncolytic virus examples include oncolytic poxvirus (e.g. orthopoxviruses) such as vaccinia virus JX-954 and GLV-1 h68 (Park, BH et al. (2008) Lancet Oncol 9:533-542; Kelly et al. Human Gene Therapy 19:744-782 (March
• an oncolytic virus such as A13, A15, A18, A21 (Au et al, Virology Journal 201 1 , 8:22), oncolytic Newcastle Disease Virus (Mansour et al, J Virol 2011 , Jun; 85(12):6015- 23), and oncolytic parvoviruses such as H-1 PV and MVM (Wennier et al. Expert Rev Mol Med. 13 e18 5 Dec 201 1).
• the genome of an oncolytic virus according to the present invention may be further modified to contain nucleic acid encoding at least one copy of a polypeptide that is heterologous to the virus (i.e.
• the oncolytic virus may also be an expression vector from which the polypeptide may be expressed. Examples of such viruses are described in WO2005/049846 and WO2005/049845.
• nucleic acid encoding the polypeptide is preferably operably linked to a regulatory sequence, e.g. a promoter, capable of effecting transcription of the nucleic acid encoding the polypeptide.
• a regulatory sequence e.g. promoter
• a regulatory sequence that is operably linked to a nucleotide sequence may be located adjacent to that sequence or in close proximity such that the regulatory sequence can effect and/or control expression of a product of the nucleotide sequence.
• the encoded product of the nucleotide sequence may therefore be expressible from that regulatory sequence.
• Oncolytic viruses may be formulated as medicaments and pharmaceutical compositions for clinical use and in such formulations may be combined with a pharmaceutically acceptable carrier, diluent or adjuvant.
• the composition may be formulated for topical, parenteral, systemic, intracavitary, intravenous, intra-arterial, intramuscular, intrathecal, intraocular, intratumoral, subcutaneous, oral or transdermal routes of administration which may include injection.
• Suitable formulations may comprise the virus in a sterile or isotonic medium.
• Medicaments and pharmaceutical compositions may be formulated in fluid (including gel) or solid (e.g. tablet) form. Fluid formulations may be formulated for administration by injection or via catheter to a selected region of the human or animal body.
• Administration is preferably in a "therapeutically effective amount", this being sufficient to show benefit to the individual.
• the actual amount administered, and rate and time-course of administration, will depend on the nature and severity of the disease being treated. Prescription of treatment, e.g. decisions on dosage etc, is within the responsibility of general practitioners and other medical doctors, and typically takes account of the disorder to be treated, the condition of the individual patient, the site of delivery, the method of administration and other factors known to practitioners. Examples of the techniques and protocols mentioned above can be found in Remington's Pharmaceutical Sciences, 20th Edition, 2000, pub. Lippincott, Williams & Wilkins.
• Targeting therapies may be used to deliver the oncolytic virus to certain types of cell, e.g. by the use of targeting systems such as antibody or cell specific ligands. Targeting may be desirable for a variety of reasons; for example if the virus is unacceptably toxic in high dose, or if it would otherwise require too high a dosage, or if it would not otherwise be able to enter the target cells.
• HSV capable of targeting cells and tissues are described in (PCT/GB2003/000603; WO 03/068809), hereby incorporated in its entirety by reference.
• An oncolytic virus may be administered alone or in combination with other treatments, either simultaneously or sequentially dependent upon the condition to be treated.
• Such other treatments may include chemotherapy (including either systemic treatment with a chemotherapeutic agent or targeted therapy using small molecule or biological molecule (e.g. antibody) based agents that target key pathways in tumor development,
• Chemotherapy refers to treatment of a tumor with a drug.
• the drug may be a chemical entity, e.g. small molecule pharmaceutical, protein inhibitor (e.g. enzyme inhibitor, kinase inhibitor), or a biological agent, e.g. antibody, antibody fragment, nucleic acid or peptide aptamer, nucleic acid (e.g. DNA, RNA), peptide, polypeptide, or protein.
• protein inhibitor e.g. enzyme inhibitor, kinase inhibitor
• a biological agent e.g. antibody, antibody fragment, nucleic acid or peptide aptamer, nucleic acid (e.g. DNA, RNA), peptide, polypeptide, or protein.
• the drug may be formulated as a pharmaceutical composition or medicament.
• the formulation may comprise one or more drugs (e.g. one or more active agents) together with one or more pharmaceutically acceptable diluents, excipients or carriers.
• a treatment may involve administration of more than one drug.
• a drug may be administered alone or in combination with other treatments, either simultaneously or sequentially dependent upon the condition to be treated.
• the chemotherapy may be a co-therapy involving administration of two drugs/agents, one or more of which may be intended to treat the tumor.
• an oncolytic virus and chemotherapeutic may be administered simultaneously, separately, or sequentially which may allow the two agents to be present in the tumor requiring treatment at the same time and thereby provide a combined therapeutic effect, which may be additive or synergistic.
• the chemotherapy may be administered by one or more routes of administration, e.g. parenteral, intra-arterial injection or infusion, intravenous injection or infusion,
• Administration is preferably in a "therapeutically effective amount", this being sufficient to show benefit to the individual.
• the actual amount administered, and rate and time-course of administration, will depend on the nature and severity of the disease being treated. Prescription of treatment, e.g. decisions on dosage etc, is within the responsibility of general practitioners and other medical doctors, and typically takes account of the disorder to be treated, the condition of the individual patient, the site of delivery, the method of administration and other factors known to practitioners. Examples of the techniques and protocols mentioned above can be found in Remington's Pharmaceutical Sciences, 20th Edition, 2000, pub. Lippincott, Williams & Wilkins.
• the chemotherapy may be administered according to a treatment regime.
• the treatment regime may be a pre-determined timetable, plan, scheme or schedule of chemotherapy administration which may be prepared by a physician or medical practitioner and may be tailored to suit the patient requiring treatment.
• the treatment regime may indicate one or more of: the type of chemotherapy to administer to the patient; the dose of each drug; the time interval between
• JAK inhibitors are examples of chemotherapeutic agents that may be used together with an oncolytic virus to treat a cancer in accordance with the present invention.
• a JAK inhibitor is an agent capable of inhibiting the activity of one of the JAK kinases (JAK1 , JAK2, JAK3 and TYK2), preferably a mammalian JAK kinase, more preferably a human JAK kinase.
• a JAK inhibitor may be selected that is an inhibitor of one or more of the JAK kinases.
• it may be an inhibitor that is specific for only one of the JAK kinases, or may exhibit inhibition at more than one of the JAK kinases.
• a JAK inhibitor that exhibits inhibition at the JAK3 kinase may be chosen.
• a JAK inhibitor will typically be a chemical entity, e.g. small molecule pharmaceutical, antibiotic, or a biological agent, e.g. antibody, antibody fragment, nucleic acid or peptide aptamer, nucleic acid (e.g. DNA, RNA), peptide, polypeptide, or protein.
• a biological agent e.g. antibody, antibody fragment, nucleic acid or peptide aptamer, nucleic acid (e.g. DNA, RNA), peptide, polypeptide, or protein.
• JAK kinase activity can be tested using routine procedures known to those of ordinary skill in the art, thus allowing one to confirm whether a given agent is a JAK kinase inhibitor. Suitable methods include the use of in vitro kinase assays, such as those described in Changelian et al (The specificity of JAK3 kinase inhibitors. Blood February 15, 2008 vol. 11 1 no. 4 2155-2157), Yin et al (Development of a high- throughput cell-based reporter assay for screening of JAK3 inhibitors. J Biomol Screen. 201 1 Apr; 16(4):443-9.
• Oh et al A receptor-independent, cell-based JAK activation assay for screening for JAK3-specific inhibitors.
• a commercially available kit designed for screening of JAK inhibitors may be selected such as, for JAK1 and JAK2, the Z'-LYTE® assay, LanthaScreenTM assay, and Omnia® kinase assay (all Invitrogen, Inc., USA).
• IC50 for JAK inhibitors may be determined using the Cellular LanthaScreenTM assay, phosphoELISATM assay or CellSensor® pathway reported assay (all Invitrogen, Inc., USA).
• Other commercially available JAK kinase assay kits include Phospho-STAT1 , Phospho-STAT3 & Total STAT3, Phospho-STAT5 (TGR Biosciences), ADP-GloTM JAK3 Kinase Assay (Promega Corporation, Wl, USA, Cat# V9101 , V3701 , V9441).
• JAK inhibitors are known, as discussed below.
• Ruxolitinib (INCB018424) is an orally bioavailable JAK inhibitor with potential
• JAK1/JAK2 inhibitor IC50 values for JAK1 , JAK2 and JAK3 are 3, 5 and 332nM, respectively.
• JAK1480 is a novel potent small JAK2 inhibitor with an IC50 of 0.26 nM. Inhibition of JAK2 activity is associated with abrogation of STAT3 nuclear translocation and tumorigenesis. AZD1480 suppresses the growth of human solid tumor xenografts harboring persistent STAT3 activity.
• AZD1480 inhibited the phosphorylation of STAT5 with an IC50 of 46 nM in TEL-Jak2 cells, whereas little or no inhibition of STAT5 phosphorylation was observed in the TEL-Jak3, TEL-Jak1 , or TEL-Tyk2 cells at or below 1 ⁇ AZD1480 (Cancer Cell 2009 December 8; 16(6): 487-497).
• AZD1480 The structure of AZD1480 is shown in Figure 16.
• AT9283 is a small molecule multi-targeted c-ABL, JAK2, Aurora A and B inhibitor with IC50 of 4, 1.2, 1 , 1 and approximate 3 nM for Bcr-Abl(T3151), Jak2 and Jak3, aurora A and B, respectively. AT9283 inhibited growth and survival of multiple solid tumor cell lines and was efficacious in mouse xenograft models. AT9283-treatment resulted in
• Cell lines harboring mutated JAK2 alleles CHRF-288-11 or Ba/F3-TEL-JAK2 were inhibited more potently than the corresponding pair harboring mutated JAK3 alleles (CMK or Ba/F3-TEL-JAK3), and STAT-5
• AG 490 is a potent epidermal growth factor receptor kinase autophosphorylation inhibitor with an IC50 of 100 nM and 56.8 ⁇ for EGFR and JAK, respectively. It inhibits cytokine- independent cell growth in vitro and tumor cell invasion in vivo. It selectively blocks leukemic cell growth in vitro and in vivo by inducing programmed cell death, with no harmful effect on normal hematopoiesis. It inhibits the constitutive activation of STAT-3 DNA binding and IL-2-induced growth of MF tumor cells.
• LY2784544 is a small molecule selective JAK2 kinase inhibitor with an IC50 of 68 nM (Curr Hematol Malig Rep. 2010 Jan;5(1): 15-21).
• WP1066 markedly inhibits the growth of HEL cells carrying the JAK2 V617F mutant isoform in a dose-dependent manner with IC50 of 2.3 ⁇ .
• WP1066 ( ⁇ 4.0 ⁇ ) inhibits the phosphorylation of JAK2, STAT3, STAT5, and ERK1/2 without affecting the
• WP1066 at concentrations of 2.5 ⁇ enhances the cytotoxic effects of CTX in B16 cells (Int J Cancer 2012; 131 (1), 8-17). WP1066 at concentrations ranging from 0.5-3.0 ⁇ inhibits the proliferation of AML colony-forming cells obtained from patients and that of the AML cell lines OCIM2 and K562 in a dose-dependent manner.
• WP1066 ( ⁇ 4.0 ⁇ ) dose-dependently decreases JAK2 and pJAK2 protein levels as well as downstream phosphorylation levels of STAT3, STAT5, and AKT in OCIM2 and K562 cells.
• WP1066 at concentrations of 5 ⁇ prevents the phosphorylation of STAT3, and at concentrations of 2.5 ⁇ WP1066 significantly inhibits cell survival and proliferation in Caki-1 and 786-0 renal cancer cells.
• WP1066 at concentrations of 5 ⁇ suppresses HIF1 a and HIF2a expression and VEGF production in Caki-1 and 786-0 renal cancer cells.
• TG101348 (SAR302503) is a potent, highly selective and ATP- competitive JAK2 inhibitor with an IC50 of 3 nM for JAK2 and JAK2 V617F (Cancer Cell 2008 Apr;3(4):311-20).
• TG101348 (SAR302503) potently prevents JAK2V617F enzyme activity and in human cells.
• TG 101348 (SAR302503) was selected as a clinical development candidate.
• TG101348 (SAR302503) reveals remarkable kinase specificity as shown by 83X selectivity versus JAK3 and potent suppression of ⁇ 2% of the kinases evaluated in a commercial, phylogenetically diverse panel of 212 kinases.
• TG101209 is a potent and small molecule JAK2-selective kinase inhibitor with IC50 of 6, 25, 17 and 169 nM for JAK2, FLT3, RET and JAK3, respectively.
• TG101209 potently prevents myeloproliferative disorder-related JAK2V617F and MPLW515L/K mutations.
• NVP-BSK805 dihydrochloride is a potent and selective quinoxaline JAK2 inhibitor with IC50 of 0.48, 0.56 and 0.58 nM for JAK2 JH1 , FL JAK V617F and FL JAK2 wt, respectively.
• NVP-BSK805 dihydrochloride displays more than 20-fold selectivity over the other JAK family members and more than 100-fold selectivity over a panel of kinases in vitro.
• NVP- BSK805 dihydrochloride blocks the growth of JAKV617F cells and induces apoptosis.
• AZ960 is a small molecule JAK2 kinase inhibitor with an IC50 and Ki of 3 mM and 0.45 nM in vitro, respectively. AZ960 was also shown to be active against other kinases, including TrkA, Aurora-A, and FAK, with IC50 of around 0.1 ⁇ (The Journal of Biological Chemistry; November 21 , 2008;283:32334-32343; Mol Cancer Ther 2010;9:3386-3395).
• LY3009104 (INCB28050; Baricitinib) is an oral JAK1 and JAK2 inhibitor.
• MS-1020 (ab141465) is a cell-permeable inhibitor of Janus Kinase 3 (JAK3), which selectively inhibits constitutive autophosphorylation of JAK3 and suppression of interleukin-2 induced JAK3/STAT5 signalling but not prolactin induced JAK2/STAT5 signalling (Kim, BH et al, Br J Haematol (2010) 148(1): 132-43).
• JAK3 Janus Kinase 3
• MS-1020 is specific for JAK3.
• the structure of ms-1020 is shown in Figure 37.
• JAK inhibitors include ONX0803 (SB1518; Pacritinib), SB1578, SB1317, AC-430, AEG1174, BMS-91 1543, XL-019 and WHI-P131 (e.g. see Seavey and Dobrzanski, The many faces of Janus kinase, Biochemical Pharmacology 83 (2012) 1 136-11).
• the active compound of a given chemotherapeutic agent may be provided in the form of a corresponding salt, solvate, or prodrug.
• chemotherapeutic agent includes reference to such forms.
• a corresponding salt of the active compound for example, a pharmaceutically-acceptable salt.
• a pharmaceutically-acceptable salt examples of pharmaceutically acceptable salts are discussed in Berge et al., 1977, "Pharmaceutically Acceptable Salts," J. Pharm. Sci., Vol. 66, pp. 1-19.
• a salt may be formed with a suitable cation.
• suitable inorganic cations include, but are not limited to, alkali metal ions such as Na + and K + , alkaline earth cations such as Ca 2+ and Mg 2+ , and other cations such as ⁇ 3 .
• suitable organic cations include, but are not limited to, ammonium ion (i.e., NH 4 + ) and substituted ammonium ions (e.g., NH 3 R + , NH 2 R 2 + , NHR 3 + , NR 4 + ).
• suitable substituted ammonium ions are those derived from:
• ethylamine diethylamine, dicyclohexylamine, triethylamine, butylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine, benzylamine, phenylbenzylamine, choline, meglumine, and tromethamine, as well as amino acids, such as lysine and arginine.
• An example of a common quaternary ammonium ion is N(CH 3 ) 4 + .
• a salt may be formed with a suitable anion.
• suitable inorganic anions include, but are not limited to, those derived from the following inorganic acids: hydrochloric, hydrobromic, hydroiodic, sulfuric, sulfurous, nitric, nitrous,
• suitable organic anions include, but are not limited to, those derived from the following organic acids: 2-acetyoxybenzoic, acetic, ascorbic, aspartic, benzoic, camphorsulfonic, cinnamic, citric, edetic, ethanedisulfonic, ethanesulfonic, fumaric, glucheptonic, gluconic, glutamic, glycolic, hydroxymaleic, hydroxynaphthalene carboxylic, isethionic, lactic, lactobionic, lauric, maleic, malic, methanesulfonic, mucic, oleic, oxalic, palmitic, pamoic, pantothenic, phenylacetic, phenylsulfonic, propionic, pyruvic, salicylic, stearic, succinic, sulfanilic, tartaric, toluenesulfonic, and vale
• solvate is used herein in the conventional sense to refer to a complex of solute (e.g., active compound, salt of active compound) and solvent. If the solvent is water, the solvate may be conveniently referred to as a hydrate, for example, a mono-hydrate, a di-hydrate, a tri-hydrate, etc.
• prodrug refers to a compound which, when metabolised (e.g., in vivo), yields the desired active compound.
• the prodrug is inactive, or less active than the active compound, but may provide advantageous handling, administration, or metabolic properties.
• a reference to a particular compound also include prodrugs thereof.
• prodrugs are activated enzymatically to yield the active compound, or a compound which, upon further chemical reaction, yields the active compound (for example, as in ADEPT, GDEPT, LIDEPT, etc.).
• the prodrug may be a sugar derivative or other glycoside conjugate, or may be an amino acid ester derivative.
• compositions may be administered alone or in combination with other treatments, either simultaneously or sequentially dependent upon the condition to be treated.
• an oncolytic virus and chemotherapeutic agent may be administered simultaneously or sequentially.
• Simultaneous administration refers to administration of the oncolytic virus and
• chemotherapeutic agent together, for example as a pharmaceutical composition containing both agents, or immediately after each other and optionally via the same route of administration, e.g. to the same artery, vein or other blood vessel.
• Sequential administration refers to administration of one of the oncolytic virus or chemotherapeutic agent followed after a given time interval by separate administration of the other agent. It is not required that the two agents are administered by the same route, although this is the case in some embodiments.
• the time interval may be any time interval.
• simultaneous or sequential administration is intended such that both the oncolytic virus and chemotherapeutic agent are delivered to the same tumor tissue to effect treatment it is not essential for both agents to be present in the tumor tissue in active form at the same time.
• the time interval is selected such that the oncolytic virus and chemotherapeutic agent are expected to be present in the tumor tissue in active form at the same time, thereby allowing for a combined, additive or synergistic effect of the two agents in treating the tumor.
• the time interval selected may be any one of 5 minutes or less, 10 minutes or less, 15 minutes or less, 20 minutes or less, 25 minutes or less, 30 minutes or less, 45 minutes or less, 60 minutes or less, 90 minutes or less, 120 minutes or less, 180 minutes or less,
• synergy may be measured using the CompuSyn software (ComboSyn Incorporated, USA) [Chou TC and Martin N. CompuSyn for Drug
• JAK inhibitor The existence of a synergism between a JAK inhibitor and virus is not essential to the present invention, although may be an advantageous characteristic.
• a cancer may be any unwanted cell proliferation (or any disease manifesting itself by unwanted cell proliferation), neoplasm or tumor or increased risk of or predisposition to the unwanted cell proliferation, neoplasm or tumor.
• the cancer may be benign or malignant and may be primary or secondary (metastatic).
• a neoplasm or tumor may be any abnormal growth or proliferation of cells and may be located in any tissue. Examples of tissues include the adrenal gland, adrenal medulla, anus, appendix, bladder, blood, bone, bone marrow, brain, breast, cecum, central nervous system (including or excluding the brain) cerebellum, cervix, colon, duodenum, endometrium, epithelial cells (e.g.
• kidney oesophagus
• glial cells heart, ileum, jejunum, kidney, lacrimal glad, larynx, liver, lung, lymph, lymph node, lymphoblast, maxilla, mediastinum, mesentery, myometrium, nasopharynx, omentume, oral cavity, ovary, pancreas, parotid gland, peripheral nervous system, peritoneum, pleura, prostate, salivary gland, sigmoid colon, skin, small intestine, soft tissues, spleen, stomach, testis, thymus, thyroid gland, tongue, tonsil, trachea, uterus, vulva, white blood cells.
• Tumors to be treated may be nervous or non-nervous system tumors.
• Nervous system tumors may originate either in the central or peripheral nervous system, e.g. glioma, medulloblastoma, meningioma, neurofibroma, ependymoma, Schwannoma,
• Non-nervous system Neurofibrosarcoma, astrocytoma and oligodendroglioma.
• cancers/tumors may originate in any other non-nervous tissue, examples include melanoma, mesothelioma, lymphoma, myeloma, leukemia, Non-Hodgkin's lymphoma (NHL), Hodgkin's lymphoma, chronic myelogenous leukemia (CML), acute myeloid leukemia (AML), myelodysplasia syndrome (MDS), cutaneous T-cell lymphoma (CTCL), chronic lymphocytic leukemia (CLL), hepatoma, epidermoid carcinoma, prostate carcinoma, breast cancer, lung cancer , colon cancer, ovarian cancer, pancreatic cancer, thymic carcinoma, NSCLC, haematologic cancer and sarcoma.
• NHL Non-Hodgkin's lymphoma
• CML chronic myelogenous leukemia
• AML acute myeloid leukemia
• MDS myelodysplasia syndrome
• CTCL
• Cancers (and subjects) to be treated may also be selected by selecting cancers in which oncolytic virus replication is increased when the cells are contacted (e.g. in vitro by obtaining a sample of tumor cells from the cancer) with the JAK inhibitor, virus or both. Accordingly, in some embodiments of the invention treatment of cancers may be for subjects grouped/selected according to this criteria
• the subject to be treated may be any animal or human.
• the subject is preferably mammalian, more preferably human.
• the subject may be a non-human mammal, but is more preferably human.
• the subject may be male or female.
• the subject may be a patient.
• a subject may have been diagnosed with a cancer, or be suspected of having a cancer.
• Cancers to be treated with a combination of an herpes simplex virus and a JAK inhibitor may be those which respond to combined (e.g. simultaneous or sequential) administration of herpes simplex virus and JAK inhibitor by enhancing herpes simplex virus replication in cells of the cancer.
• This property may be used to define cancers that will respond to the treatment, and therefore allows for subjects to be selected on the basis of determining the response of a subject's cancer.
• this may involve determining, preferably in vitro, whether contacting cells of the cancer, or cancer cells of a corresponding type, with the herpes simplex virus and JAK inhibitor enhances replication of the herpes simplex virus in cells of the cancer.
• cancers that are expected to respond to treatment may be identified by determining, preferably in vitro, whether contacting cells of the cancer, or cancer cells of a corresponding type, with the herpes simplex virus and JAK inhibitor enhances replication of the herpes simplex virus in cells of the cancer.
• This property may therefore be used independently to identify cancers that are expected to respond to treatment and/or to select subjects for combined treatment with a herpes simplex virus and JAK inhibitor.
• the combination of a herpes simplex virus and JAK inhibitor may be used to treat subjects having cancer in which:
• the subject has a cancer which responds to combined treatment with a herpes simplex virus and JAK inhibitor by enhancing herpes simplex virus replication in cells of the cancer;
• the cancer responds to combined treatment with a herpes simplex virus and JAK inhibitor by enhancing herpes simplex virus replication in cells of the cancer;
• the cancer has been selected as one that responds to combined treatment with a herpes simplex virus and JAK inhibitor by enhancing herpes simplex virus replication in cells of the cancer;
• the method of treatment comprises determining whether combined (e.g.
• Herpes simplex virus replication and its enhancement may be measured by a number of well-known assay techniques including plaque assay or marker expression, e.g. using an HSV expressing a detectable marker under control of a constitutive promoter.
• an HSV is created that expresses a non-HSV protein such as GFP or luciferase under the control of a constitutive promoter.
• the effectiveness of the HSV variant as a reporter for HSV replication can be established by comparing expression of the marker following infection of known permissive and non-permissive cell types.
• Enhancement of replication may be determined relative to one or more controls, e.g. relative to permissive and non-permissive cell types, known to respectively permit or not permit HSV infection and replication.
• permissive and non-permissive cell types known to respectively permit or not permit HSV infection and replication.
• Vero and BHK cells are permissive and the murine mesothelioma cell line AB12 and murine NIH3T6 cells are non- permissive.
• Enhancement of replication may be an increase in the level of replication normally seen in the cell type concerned for the virus being analysed.
• the enhancement may be a potentiation of virus replication in the presence of the JAK inhibitor.
• Enhancement can normally be assessed by comparing the level of replication of virus in a cell not contacted with JAK inhibitor against the level of replication in the same cell type when contacted with an JAK inhibitor.
• An increase in the level of replication in cells contacted with JAK inhibitor in addition to virus of at least 10% may be considered an enhancement, although the level of increase may be greater, e.g. one of 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or greater.
• cancers to be treated with a combination of an herpes simplex virus and a JAK inhibitor may be further defined by those which exhibit an improved response to combined (e.g. simultaneous or sequential) administration of herpes simplex virus and JAK inhibitor compared to the response seen when the cancer is treated with either agent alone (i.e. not in combination).
• the improved response is at least additive.
• the improved response is synergistic, e.g. a response that is greater than the sum of the individual responses or a cooperative response that creates an enhanced combined effect.
• the existence of a synergistic response may be measured by Chou Talalay analysis, as described herein.
• the improvement of response helps further define cancers that will respond to the treatment, and therefore allows for subjects to be selected on the basis of determining the existence of an improved response of a subject's cancer. In particular, this may involve determining, preferably in vitro, whether contacting cells of the cancer, or cancer cells of a corresponding type, with the herpes simplex virus and JAK inhibitor produces an improved response.
• cancers selected for treatment may be identified by determining, preferably in vitro, whether contacting cells of the cancer, or cancer cells of a corresponding type, with the herpes simplex virus and JAK inhibitor produces an improved response. This property may be used independently to select subjects (or cancers) for combined treatment with a herpes simplex virus and JAK inhibitor.
• Determining the presence of an improved response may be measured by a number of well-known assay techniques.
• the response being measured may be cell death and this may be measured by one of the MTS, LDH or DCP assays described herein.
• an herpes simplex virus for use in a method of treating cancer in a subject, the method comprising simultaneous or sequential administration of a herpes simplex virus and a JAK inhibitor, wherein the cancer is characterised in that in response to simultaneous or sequential administration of the herpes simplex virus and JAK inhibitor herpes simplex virus replication in cells of the cancer is enhanced.
• an herpes simplex virus in the manufacture of a medicament for use in a method of treating cancer in a subject comprising simultaneous or sequential administration of a herpes simplex virus and JAK inhibitor, wherein the cancer is characterised in that in response to simultaneous or sequential administration of the herpes simplex virus and JAK inhibitor herpes simplex virus replication in cells of the cancer is enhanced.
• a method of treating a subject having a cancer comprising:
• a JAK inhibitor for use in a method of treating cancer in a subject, the method comprising simultaneous or sequential administration of a herpes simplex virus and a JAK inhibitor, wherein the cancer is characterised in that in response to simultaneous or sequential administration of the herpes simplex virus and JAK inhibitor herpes simplex virus replication in cells of the cancer is enhanced.
• an herpes simplex virus for use in a method of treating cancer in a subject, the method comprising simultaneous or sequential administration of a herpes simplex virus and a JAK inhibitor, wherein the method comprises determining whether simultaneous or sequential administration of the herpes simplex virus and JAK inhibitor enhances replication of the herpes simplex virus in cells of the cancer.
• an herpes simplex virus in the manufacture of a medicament for use in a method of treating cancer in a subject comprising simultaneous or sequential administration of a herpes simplex virus and a JAK inhibitor, wherein the method comprises determining, preferably in vitro, whether simultaneous or sequential administration of the herpes simplex virus and JAK inhibitor enhances replication of the herpes simplex virus in cells of the cancer.
• a method of treating a subject having a cancer comprising:
• a JAK inhibitor for use in a method of treating cancer in a subject, the method comprising simultaneous or sequential administration of a herpes simplex virus and a JAK inhibitor, wherein the method comprises determining, preferably in vitro, whether simultaneous or sequential administration of the herpes simplex virus and JAK inhibitor enhances replication of the herpes simplex virus in cells of the cancer.
• the use of a JAK inhibitor in the manufacture of a medicament for use in a method of treating cancer in a subject comprising simultaneous or sequential administration of a herpes simplex virus and a JAK inhibitor, wherein the method comprises determining, preferably in vitro, whether simultaneous or sequential administration of the herpes simplex virus and JAK inhibitor enhances replication of the herpes simplex virus in cells of the cancer.
• a method of selecting a subject for combined treatment of a cancer with an oncolytic herpes simplex virus (HSV) and a JAK inhibitor comprising:
• herpes simplex virus and JAK inhibitor enhances replication of the herpes simplex virus in cells of the cancer.
• a method of selecting a subject for combined treatment of a cancer with an oncolytic herpes simplex virus (HSV) and a JAK inhibitor comprising: infecting, in vitro, at least one cell of a sample of the cancer requiring treatment with an oncolytic herpes simplex virus and a JAK inhibitor,
• a method of selecting a JAK inhibitor for combined treatment of a cancer with a herpes simplex virus comprising
• a method of determining the expected response of a cancer to combined treatment with an oncolytic herpes simplex virus (HSV) and a JAK inhibitor comprising:
• a method of selecting a subject for treatment of a cancer with an oncolytic herpes simplex virus (HSV) and a JAK inhibitor comprising:
• determining the level of replication of the herpes simplex virus in the infected cell(s) and based on the level of replication selecting a subject for treatment with an oncolytic herpes simplex virus and JAK inhibitor.
• chemotherapeutic agents In addition to treating a cancer by using an oncolytic virus with a JAK inhibitor, subjects being treated may also receive treatment with other chemotherapeutic agents.
• chemotherapeutic agents may be selected from:
• alkylating agents such as cisplatin, carboplatin, mechlorethamine
• alkaloids and terpenoids such as vinca alkaloids (e.g. vincristine, vinblastine, vinorelbine, vindesine), podophyllotoxin, etoposide, teniposide, taxanes such as paclitaxel (TaxolTM), docetaxel;
• topoisomerase inhibitors such as the type I topoisomerase inhibitors
• camptothecins irinotecan and topotecan or the type II topoisomerase inhibitors amsacrine, etoposide, etoposide phosphate, teniposide;
• antitumor antibiotics e.g. anthracyline antibiotics
• dactinomycin e.g. dactinomycin
• doxorubicin (AdriamycinTM), epirubicin, bleomycin, rapamycin;
• antibody based agents such as anti-VEGF, anti-TNFa, anti-IL-2,
• anti-Gpllb/llla anti-CD-52, anti-CD20, anti-RSV, anti-HER2/neu(erbB2), anti- TNF receptor, anti-EGFR antibodies, monoclonal antibodies or antibody fragments
• examples include: cetuximab, panitumumab, infliximab, basiliximab, bevacizumab (Avastin®), abciximab, daclizumab, gemtuzumab, alemtuzumab, rituximab (Mabthera®), palivizumab, trastuzumab, etanercept, adalimumab, nimotuzumab,
• EGFR inihibitors such as erlotinib, cetuximab and gefitinib
• anti-angiogenic agents such as bevacizumab (Avastin®).
• Viruses, chemotherapeutic agents, medicaments and pharmaceutical compositions may be formulated for administration by a number of routes, including but not limited to, parenteral, intravenous, intra-arterial, intramuscular, intratumoural and oral.
• Viruses, chemotherapeutic agents, medicaments and compositions may be formulated in fluid or solid form. Fluid formulations may be formulated for administration by injection to a selected region of the human or animal body. Dosage regime
• Multiple doses of the oncolytic virus may be provided.
• One or more, or each, of the doses may be accompanied by simultaneous or sequential administration of a chemotherapeutic agent.
• Multiple doses may be separated by a predetermined time interval, which may be selected to be one of 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31 days, or 1 , 2, 3, 4, 5, or 6 months.
• doses may be given once every 7, 14, 21 or 28 days (plus or minus 3, 2, or 1 days).
• the dose of oncolytic virus given at each dosing point may be the same, but this is not essential. For example, it may be appropriate to give a higher priming dose at the first, second and/or third dosing points. Kits
• kits of parts may have at least one container having a predetermined quantity of oncolytic virus, e.g. predetermined viral dose or number/quantity/concentration of viral particles.
• the oncolytic virus may be formulated so as to be suitable for injection or infusion to a tumor or to the blood.
• the kit may further comprise at least one container having a predetermined quantity of chemotherapeutic agent.
• the chemotherapeutic agent may also be formulated so as to be suitable for injection or infusion to the tumor or to the blood, or alternatively may be formulated for oral administration.
• a container having a mixture of a predetermined quantity of oncolytic virus and predetermined quantity of chemotherapeutic agent is provided, which may optionally be formulated so as to be suitable for injection or infusion to the tumor or to the blood.
• the kit may also contain apparatus suitable to administer one or more doses of the oncolytic virus and/or chemotherapeutic agent.
• apparatus may include one or more of a catheter and/or needle and/or syringe, such apparatus preferably being provided in sterile form.
• the kit may further comprise instructions for the administration of a therapeutically effective dose of the oncolytic virus and/or chemotherapeutic agent.
• the invention includes the combination of the aspects and preferred features described except where such a combination is clearly impermissible or expressly avoided.
• FIG. 1 Illustration of Janus kinase (JAK)-signal transducer and activator of transcription (STAT) networks response to growth factors and cytokines to coordinate proliferation, growth, hematopoiesis and the immune response.
• JK Janus kinase
• STAT activator of transcription
• FIG. 1 Diagram of involvement of JAK/STAT signalling in multiple pathways of carcinogenesis and cancer metastasis (from Seavey and Dobrzanski, The many faces of Janus kinase, Biochemical Pharmacology 83 (2012) 1136-1 145). JAK/STAT signalling pathways can play various roles in cancer development and progression including tumour cell survival, metastasis, drug resistance and importantly the response to cytokines from tumour-driven inflammation and inflammatory responses that can lead to tumorigenesis.
• the different boxes highlight an overlap between the JAK-mediated signalling and hallmarks of cancer as defined by Hanahan and Weinberg: invasion metastasis, survival, proliferation, angiogenesis represent the original hallmarks (Cell 2000; 100:57-70);
• FIG. 3 Chart showing combination analysis of HSV1716 with tofacitinib (a) and ruxolitinib (b) in UVW cells. Virus doses were 5000, 2500, 1250, 625 and 312 pfu and drug doses were 50, 25, 5, 2.5, 0.5 and ⁇ .
• Figure 5 Graph showing virus toxicity of ruxolitinib and tofacitinib in Ovcar3 cells.
• Figure 6 Graph showing virus toxicity of ruxolitinib and tofacitinib in one58 cells.
• Figure 7 Comparison of HSV1716 kill on different cancer cell lines by MTS cell survival assay. Graph showing cell survival (as a % of control uninfected cells). Cell survival decreases with increasing virus pfu (312, 625, 1250, 2500 and 5000 pfu/well) HSV1716 on five selected cancer cell lines.
• FIG. 10 Table 1 showing summary of results from LDH leakage assay for tofacitinib and ruxolitinib in combination with HSV1716 in UVW, one58, SKOV3 and HuH7 cells.
• Figure 11. Table 2 showing summary of results for tofacitinib and ruxolitinib in combination with HSV1716 in HepG2, Ovcar3 and CP70 cells.
• Figure 14 Structure of ruxolitinib.
• Figure 15. Structure of tofacitinib.
• Figure 16. Structures of selected JAK inhibitors.
• FIG. 17 Diagram illustrating dead cell protease coupled reaction for measuring cell death.
• the AFF-Glo peptide is a substrate for dead cell protease and cleavage releases aminoluciferin.
• Aminoluciferin is a substrate for a modified recombinant luciferase but not for wild-type luciferase.
• FIG. 1 Chart showing moi-dependent DCP leakage from various cell lines infected with HSV1716.
• Figure 19 Chart showing moi-dependent DCP leakage from UVW cells infected with HSV1716.
• Figure 20 Chart showing effects of Ovcar3 cell density on DCP readings after treatment with HSV1716 at moi 10, 1 or 0.1 for 72 hours.
• Figure 21 Chart showing effects of UVW cell density on DCP readings after treatment with HSV1716 at moi 10, 1 or 0.1 for 72 hours.
• FIG 22 Chart showing GFP fluorescence measured by FITC fluoremetry following infection of SKOV3, HuH7, UVW, one58, HepG2-luc and AB12 cells with HSV1716gfp at moi 0.0001 , 0.001. 0.01 , 0.1 , 0.5 and 1.
• Figure 23 Charts showing toxicity profiles for MS-1020 in Hep3B (a), HuH7 (b), Ovcar3 (c), UVW (d) and SPC111 (e) cells. Cells were incubated with MS-1020 at concentrations 50, 25, 10, 5, 2.5, 1 , 0.5, 0.25 and 0.1 ⁇ and DCP leakage determined after 72 hrs. Each concentration was assayed in triplicate at least and values are graphed as % of control (cells alone with no drug) for MS-1020 IC50 determinations.
• FIG. 25 Charts showing MS-1020 effects on gfp expression during infection with HSV1716gfp.
• MS-1020 was incubated with Hep3B (a), HuH7 (b), Ovcar3 (c), UVW (d) and SPC11 1 (e) cells plus virus at moi 0.5/0.05 at 5, 2.5, 1.25 and 0.25uM for 72 hrs and gfp fluorescence measured.
• Drug-mediated virus toxicities are presented as percentage of control cells (virus alone and no drug) in Hep3B, HuH7, Ovcar3, UVW, and SPC-1 11.
• Figure 26 Table 6 showing summary of MS-1020 effects on HSV1716 replication from Figure 25.
• ++ stimulation >15% of control value
• + stimulation >5% control
• +/- no effect
• - inhibition ⁇ 5% of control
• - inhibition ⁇ 15% control.
• FIG. 27 Charts showing Chou/Talalay plots for HSV1716 in combination with MS- 1020 in Hep3B (a), HuH7 (b), Ovcar3 (c), UVW (d), and SPC-1 11 (e) cells.
• the relevant tables of Fa and CI values for the individual MS-1020 concentrations and HSV1716 moi accompany each Chou/Talalay plot. If the Fa value was negative then the corresponding CI value could not be determined and CI values above 4 are not presented in the graphs.
• Figure 28 Table 7 showing Summary of Chou/Talalay analysis for MS-1020 in combination with HSV1716 at moi 0.5 and 0.05 in Hep3B, HuH7, Ovcar3, UVW and
• FIG. 29 Chou/Talalay plots for HSV1716 in combination with ruxolitinib in Hep3B, U87 and SPC-11 1 cells.
• the relevant tables of Fa and CI values for the individual ruxolitinib concentrations and HSV1716 moi accompany each Chou/Talalay plot. If the Fa value was negative then the corresponding CI value could not be determined and CI values above 4 are not presented in the graphs.
• FIG. 30 Table 8 showing summary of Chou/Talalay analysis of ruxolitinib in combination with HSV1716 at moi 2 and 0.2 in Hep3B, U87 and SPC11 1 cells. Numbers of synergistic combinations at the two moi are indicated and the overall
• FIG. 31 Chou/Talalay plots for HSV1716 in combination with tofacitinib in Hep3B, U87 and SPC-11 1 cells.
• the relevant tables of Fa and CI values for the individual tofacitinib concentrations and HSV1716 moi accompany each Chou/Talalay plot. If the Fa value was negative then the corresponding CI value could not be determined and CI values above 4 are not presented in the graphs.
• Figure 34 Table 10 showing AZD1480 IC50 values as estimated from the curves shown in Figure 33.
• FIG. 35 Charts showing Chou/Talalay plots for HSV1716 in combination with AZD1480 in Hep3B, HepG2-luc, CP70, Ovcar3, SKOV3, U87, UVW and SPC-11 1 cells.
• the relevant tables of Fa and CI values for the individual AZD1480 concentrations and HSV1716 moi accompany each Chou/Talalay plot. If the Fa value was negative then the corresponding CI value could not be determined and CI values above 2 are not presented in the graphs.
• FIG. 37 Structure of MS-1020.
• Results obtained in Examples 7 and 8 allowed us to concluded that JAK inhibitors show synergy in combination with HSV1716 with synergy signals being very strong in most cell types with the JAK3 inhibitor tofacitinib. Some synergies were also detected for ruxolitinib.
• Results obtained in Example 9 show synergistic action of HSV1716 with MS-1020, ruxolitinib, tofacitinib and AZD1480 in causing cell death across a range of tumor cell types.
• JAK inhibitors especially JAK3 inhibitors, stimulate virus replication in tumor cells leading to increased progeny production, which is a necessary precursor to enhanced viral lysis of the tumor cell.
• JAK3 activation by HSV infection acts to reduce subsequent replication efficiency
• JAK inhibition appears to alleviate the repression.
• Example 1 Cell line panel for combinatorial studies
• CP70, SKOV3, A2780 and Ovcar 3 are human ovarian cancer cell lines.
• HuH7 and HepG2-luc are human hepatocellular carcinoma cell lines.
• One58 is a human malignant pleural mesothelioma cell line.
• U87MG-luc and UVW are human glioma cell lines.
• HepG2-luc and U87MG-luc are luciferase expressing cell lines which were stably transfected with the firefly luciferase gene (Caliper Life Sciences, Inc., USA).
• the CP70 cell line is a cisplatin-resistant derivative of A2780 cells and the cells have approximately 13-fold more resistance to cisplatin than the parental A2780 line.
• MTS assays are a straightforward and convenient method to assess the viability of cultured cells and can be easily adapted for a high throughput format.
• the MTT/MTS assay is a colorimetric assay for measuring the activity of cellular enzymes that reduce the tetrazolium dye, MTT, to its insoluble formazan, giving rise to a purple color. These assays measure cellular metabolic activity via NAD(P)H-dependent cellular
• Tetrazolium dye assays can also be used to measure cytotoxicity (loss of viable cells) or cytostatic activity (shift from proliferative to resting status) of potential medicinal agents and toxic materials.
• MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H- tetrazolium), in the presence of phenazine methosulfate (PMS), produces a formazan product that has an absorbance maximum at 490-500 nm in phosphate-buffered saline. Precautions are needed to ensure accuracy when using this assay and there are strong arguments for confirming MTS results using qualitative observations under a microscope. Tetrazolium (formazan) dye reduction relies on NAD(P)H-dependent oxidoreductase enzymes largely in the cytosolic compartment of the cell.
• MTS and other tetrazolium dyes increases with cellular metabolic activity due to elevated NAD(P)H flux and reduces as cells are killed and numbers decrease. Rapidly dividing cells exhibit high rates of MTS reduction whereas resting cells that are viable but metabolically quiet reduce very little MTS. Culture conditions and/or the introduction of active components that alter metabolic activity within cells can result in altered tetrazolium dye reduction and an apparent increase cell numbers although survival has not been affected.
• MTS assays were able to measure cell killing by HSV1716 in different cells lines ( Figure 7) and to a limited extent this reflected the replication competence of the virus in these cell lines with HSV1716 replicating best in Ovcar 3 and HuH7. However the percentage decrease relative to control cells is limited in both one58 and U87MG cells although the virus does replicate well in these cell lines. Note that at the lowest virus dose there is in apparent increase in MTS signal in UVW, one58 and U87MG.
• MTS assay analysis was considered useful with the proviso that it can generate unreliable data when virus and/or drug increases the metabolic rate of cells. Because of this supporting data from a cell death assay has also been obtained in order to confirm MTS results.
• Lactate dehydrogenase is a soluble cytosolic enzyme present in most eukaryotic cells that is released into culture medium upon cell death due to damage of plasma membrane.
• the increase in the LDH activity in culture supernatant is proportional to the number of lysed cells.
• LDH The activity of LDH can be readily measured (e.g. using the Promega LDH CytoTox-ONE assay kit) in tissue culture supernatants with a threshold of approximately 2000 dead cells/well and virus toxicities were clearly observed in different cell types in a cell density dependent assay (data not shown).
• the LDH kits were purchased from Promega (CytoTox-ONE) and used as follows. 40 ⁇ of PBS was added to clean 96 well ELISA plate and 10 ⁇ of cell media was transferred to each of the wells from the corresponding well on the virus/drug combination test plate. Lactate dehydrogenase substrate was then added (40 ⁇ ) and after 30 mins incubation in the dark at room temperature the absorbance at 490nm was determined. Virus infection over 72 hours caused an increase in LDH leakage from the various cell lines which was dependent on the HSV1716 multiplicity of infection (moi). The LDH basal level and the amount of activity leaked into the medium varied with the different cell lines and probably reflected the intrinsic LDH amounts present in each cell line.
• moi multiplicity of infection
• Combination of drugs with HSV1716 was performed in 96-well tissue culture plates. For each drug/virus combination two 96 well plates were set up with combination grids as shown in Figure 8. No outside wells were used giving 6x10 usable wells per plate. The plates were split into columns 3 wells wide and 6 wells in length. Each column was then infected with decreasing concentrations of virus per column. This was then followed by the addition of drug to the plates in decreasing concentrations per row. Initial attempts were made using preliminary IC50 analysis to identify suitable drug concentrations to test but this could be highly cell type dependent or the drugs displayed very limited toxicities.
• virus doses were selected to cover the range of HSV1716 replication competencies in the cell panel. Virus doses were 5000pfu, 2500pfu, 1250pfu, 625pfu and 312 pfu which are approximately equivalent to moi 0.5, 0.25, 0.125, 0.063 and 0.032. The higher mois are required for cell lines which have low HSV1716 replication kinetics such as CP70 and SKOV3.
• Results principally LDH but also MTS as supportive data were derived from each of these plates after 72hours of exposure to drug and/or virus and values were entered into an analysis spreadsheet. Results calculated in the spreadsheet were readily entered into the Compusyn programme (Combosyn, Inc., USA) for calculation of the combinatorial index and combination plots were generated. Compusyn generates Chou-Talalay combination plots as shown in Figure 9.
• the fraction affected (Fa) is a measurement of the number of cells that are effected (i.e. killed) by the drug or virus treatment. If, for example, a drug dose killed 80% of the cells then the Fa value would be 0.8.
• Example 6 Virus health assay to detect drugs that block virus replication- using HSV1716luc to monitor direct toxicity of drugs on virus
• HSV1716 direct toxicity assay using a fixed amount of HSV1716 (1x10 5 pfu total comprising HSV1716 spiked with 20% HSV1716luc [HSV1716luc is a modified HSV1716 that expresses luciferase]) and various drug concentrations was a useful adjunct to drug combination studies.
• the microtitre plate based assay allowed high throughput screening and the assay measures drug effects directly on virus replication as virus and drug are added simultaneously and effects are measured during the first replication cycle of the virus. The results complement data generated by LDH and MTS assay.
• luciferase activity was determined in each well by the addition of 20 ⁇ luciferin substrate.
• Spiked virus was used to prevent carry-over of light into adjacent wells since the assays were performed in clear 96-well tissue culture plates.
• Optimum light output from the spiked HSV1716luc was after 24hrs of infection as demonstrated in the three cell lines tested (Ovcar 3, one58 and UVW) and examples of drug toxicity profiles were determined.
• Ruxolitinib was mostly toxic at high doses except in SKOV3 cells where it had no effect on virus replication. Tofacitinib was mostly non-toxic in one58, Ovcar3, SKOV3 and UVW with some toxicity at high doses in HuH7 cells. There was some suggestion of stimulation of virus replication at high doses of tofacitinib in one58 and SKOV3 cells.
• tofacitinib CP69 alone is non-toxic but enhances virus-mediated cell killing at all doses except for the lowest virus dose in which there is a small reduction in toxicity.
• Ruxolitinb alone is toxic to UVW cells with a dose-dependent increase in LDH leakage. At the higher doses of drug cell killing is impaired by combination with most doses of HSV1716 (except 1250pfu). At lower ruxolitinib doses (0.5 and 2.5 ⁇ ) cell killing is increased by most doses of HSV1716 except 1250pfu suggesting some synergy between drug and HSV1716.
• tofacitinib (CP69) alone displays weak dose-dependent cytotoxicity which is enhanced by HSV1716 especially at low (312, 625 and to some extent 1250pfu) doses of virus. There is little distinction in the toxicity curves for tofacitinib at the two higher virus doses and for 1250pfu at 25 and 50 ⁇ drug.
• Ruxolitinb has dose dependent toxicity in HuH7 cells which is unaffected in combination with HSV1716 at most virus doses except at 1250pfu where cell killing is enhanced.
• Example 9 - DCP Assay Analysis HSV1716 in combination with one of MS-1020, ruxolitinib, tofacitinib or AZD1480 in tumor cells
• the CytoTox Glo cytotoxicity assay uses a light-based luciferase assay to detect leakage of dead cell protease (DCP) from dead cells. This was used in conjunction with HSV1716gfp and a fluorescence detection assay to monitor drug effects on virus replication. Using this assay system, we analysed HSV1716 in combination with the JAK inhibitors MS-1020, ruxolitinib, tofacitinib or AZD1480.
• Dead cell protease was assayed using the CytoTox-Glo Cytotoxicity kit from Promega.
• the kit provides a lumiogenic peptide substrate, AAF-Glo, to measure dead cell protease activity in the medium.
• AAF-Glo lumiogenic peptide substrate
• the peptide substrate cannot cross the intact cell membrane of a live cell and will only be cleaved ( Figure 17) when dead cell protease has been released into the medium as cells die.
• the assay then uses the Ultra-Glo recombinant luciferase which can use the released aminoluciferin as substrate to generate a readily detectable luminescence signal.
• the DCP assay is more sensitive than the LDH assay and can potentially detect as few as 200-500 dead cells/well.
• HSV1716 increased DCP levels above base line (base line ⁇ 0.0001 on log scale of Figs 18 and 19) and DCP levels increased in a dose-dependent manner to reach a maximum at moi 10. In all cell lines the increase at moi 1 was at least >50% of base line.
• Drug toxicity could also be detected using the DCP assay (data not shown).
• the DCP assay was cell density dependent as shown for Ovcar3 and UVW cells incubated for 72 hrs with virus in Figs 20 and 21.
• Ovcar3 and UVW cell numbers varied from 8000 cells/well to 250 cells/well and, 24 hrs after plating out at these densities they were treated either with HSV1716 at moi 10, 1 or 0.1 for 72 hours.
• DCP assays were then performed as described above. DCP readings were highest and maximal differences between virus-treated and controls occurred at the highest density of 8000 cells/well and this was used in all subsequent studies.
• HCG High Grade Glioma
• HCC Hepatocellular carcinoma
• COSMIC Catalogue Of Somatic Mutations In Cancer entry indicates somatic mutations in CDKN2A, CDKN2C, CDKN2a(p14) and PTEN.
• U87 cells are highly permissive for HSV1716 replication in tissue culture. After 72 hrs of replication HSV-1 17+ yields approximately 43100 +/- 13988 pfu progeny/input virus and HSV1716 yields 8806 +/- 2713 pfu progeny/input virus equivalent to a replication competence ratio (HSV1716 yield/HSV-1 17+ yield) of 0.2. Thus HSV1716 replication in U87 cells is impaired 5-fold compared to wild-type HSV-1 17+. U87 cells readily form subcutaneous xenografts in nude mice.
• UVW aka MOG-G-UVW. ECCAC - 86022703
• UVW are permissive for HSV-1 17+ and HSV1716 replication.
• HSV1716 replication in UVW cells is similar to wild-type HSV-1 17+.
• UVW cells readily form subcutaneous xenografts in nude mice
• HuH-7 is a well-differentiated, hepatocyte-derived cellular carcinoma cell line that was originally taken from a liver tumor in a 57-year-old Japanese male.
• HuH-7 are epithelial- like tumorigenic cells which are able to form subcutaneous xenografts in nude mice. According to their entry in COSMIC HuH7 cells have mutated FAM123B and TP53 genes
• HuH7 cells are highly permissive for HSV1716 replication both in tissue culture and in subcutaneous xenografts. After 72 hrs of replication HSV-1 17+ yields approximately 3250 +/- 814 pfu progeny/input virus and HSV1716 yields 28500 +/- 4815 pfu
• HSV1716 replication in HuH7 cells is approximately 9-fold higher compared to wild-type HSV-1 17+.
• HuH7 cells from the Virttu cell bank readily form subcutaneous xenografts in nude mice.
• cells Derived from an 8 year old male, cells contain integrated Hepatitis B virus genome.
• Hep3B cells are fully permissive for HSV-1 17+ and HSV1716 replication. After 72 hrs of replication HSV-1 17+ yields approximately 14430 +/- 3085 pfu progeny/input virus and HSV1716 yields 4820 +/- 2182pfu progeny/input virus equivalent to a replication competence ratio (HSV1716 yield/HSV-1 17+ yield) of 0.33. Thus HSV1716 replication in Hep3B cells is impaired 3-fold compared to wild-type HSV-1 17+.
• HepG2-luc (Caliper HT1080-/uc-?)
• the Hep G2 cell line was isolated from a liver biopsy of a male Caucasian aged 15 years, with a well differentiated hepatocellular carcinoma.
• the cells secrete a variety of major plasma proteins e.g. albumin, alpha2-macroglobulin, alpha 1-antitrypsin, transferrin and plasminogen but Hepatitis B virus surface antigens have not been detected. No entry in COSMIC.
• HepG2-/uc2 is a luciferase expressing cell line stably transfected with the firefly luciferase gene (Iuc2).
• the cell line was established by transducing lentivirus containing luciferase 2 gene under the control of human ubiquitin C promoter.
• HepG2-luc are fully permissive for HSV-1 17+ and HSV1716 replication. After 72 hrs of replication HSV-1 17+ yields approximately 40670 +/- 5690 pfu progeny/input virus and HSV1716 yields 60030 +/- 9870 pfu progeny/input virus equivalent to a replication competence ratio (HSV1716 yield/HSV-1 17+ yield) of 1.5. Thus HSV1716 replication in HepG2-luc cells is slightly better compared to wild-type HSV-1 17+.
• This cell line was derived from the pleural fluid of a patient with malignant mesothelioma. The patient had known exposure to crocidolite asbestos. Cells express cytokeratin and epithelial membrane antigen (EMA) but not mucin. Cells are epithelial-like and spindle- shaped with few vacuoles. No entry in COSMIC.
• EMA epithelial membrane antigen
• HSV-1 17+ and HSV1716 replication are permissive for HSV-1 17+ and HSV1716 replication. After 72 hrs of replication HSV-1 17+ yields approximately 13650 pfu progeny/input virus and HSV1716 yields 12650 pfu progeny/input virus equivalent to a replication competence ratio (HSV1716 yield/HSV-1 17+ yield) of 1. Thus HSV1716 replication in one58 cells is similar to wild- type HSV-1 17+.
• SPC1 11 was derived from the pleural effusion of a 55-year old male patient, prior to treatment, with a known history of exposure to asbestos.
• the cells are
• HSV-1 17+ and HSV1716 replication are permissive for HSV-1 17+ and HSV1716 replication. After 72 hrs of replication HSV-1 17+ yields approximately 11920 +/- 1002 pfu progeny/input virus and HSV1716 yields 2710 +/- 1343 pfu progeny/input virus equivalent to a replication competence ratio (HSV1716 yield/HSV-1 17+ yield) of 0.23 Thus HSV1716 replication in
• SPC1 11 cells is impaired 4-fold compared to wild-type HSV-1 17+.
• the cell line is aneuploid human female, with
• COSMIC entry indicates somatic mutation in TP53.
• Ovcar3 are fully permissive for HSV-1 17+ and HSV1716 replication. After 72 hrs of replication HSV-1 17+ yields approximately 95543 +/- 25174 pfu progeny/input virus and HSV1716 yields 179009 +/- 20662 pfu progeny/input virus equivalent to a replication competence ratio (HSV1716 yield/HSV-1 17+ yield) of 1.9. Thus HSV1716 replication in Ovcar3 cells is slightly better compared to wild-type HSV-1 17+.
• HSV-1 17+ and HSV1716 replication are permissive for HSV-1 17+ and HSV1716 replication. After 72 hrs of replication HSV-1 17+ yields approximately 27849 +/- 7511 pfu progeny/input virus and HSV1716 yields 913 +/- 299 pfu progeny/input virus equivalent to a replication
• HSV1716 replication in SKOV3 cells is severely impaired 30-fold compared to wild-type HSV-1 17+.
• CP70 is a human, ovarian cancer derived cell line.
• the CP70 cell line is a cisplatin- resistant derivative of A2780 cells and the cells have approximately 13-fold more resistance to cisplatin than the parental A2780 line.
• the A2780 human ovarian cancer cell line was established from tumour tissue from an untreated patient. According to their entry in COSMIC they have a mutated PTEN gene.
• HSV-1 17+ yields approximately 223 +/- 59 pfu progeny/input virus and HSV1716 yields 57 +/- 7pfu progeny/input virus equivalent to a replication
• HSV1716 yield/HSV-1 17+ yield competence ratio (HSV1716 yield/HSV-1 17+ yield) of 0.26.
• HSV1716 replication in CP70 cells is impaired 4-fold compared to wild-type HSV-1 17+.
• CP70 cells readily form subcutaneous xenografts in nude mice.
• HSV1716 variant expressing green fluorescent protein was used for combination analysis.
• HSV1716gfp was produced from the parental HSV1716 by insertion of a CMV- gfp expression cassette in the UL-43 gene. The virus stock for use was created on
• HSV1716gfp is indistinguishable from HSV1716 in in vitro replication assays in all cell lines tested (Conner, unpublished data).
• the 5ml of 1x10 7 pfu/ml stock is diluted into 45ml medium to generate a 1x10 6 pfu/ml stock, 6ml aliquots of which are stored for use in assays.
• GFP was measured using a Perkin Elmer 1420 multilabel counter Victor 3 in FITC fluoremetry mode for 1 sec/well. This assay is effective at assessing HSV1716gfp replication as shown in Figure 53. Levels of gfp fluorescence increase with increasing moi of HSV1716gfp in SKOV3, HuH7, UVW, one58 and HepG2-luc but not in the murine mesothelioma cell line AB12 in which, like murine NIH3T6 cells, HSV1716 fails to replicate (Conner, unpublished data). The largest percentage increases in gfp
• MS-1020 is a cell-permeable inhibitor of Janus Kinase 3 (JAK3), which selectively inhibits constitutive autophosphorylation of JAK3 and suppression of interleukin-2 induced JAK3/STAT5 signalling but not prolactin induced JAK2/STAT5 signalling (Kim, BH et al, Br J Haematol (2010) 148(1): 132-43).
• JAK3 Janus Kinase 3
• MS-1020 is specific for JAK3 and has no effect on other JAK isoforms or several other kinases including Src, Akt, EGFR and ERK1/2.
• MS-1020 blocks phosphorylation of downstream signal transduction and activators of transcription (STAT) isoforms, reducing the expression of anti-apoptotic genes, leading to cell death. This suggests MS-1020 may have therapeutic potential in the treatment of cancers harbouring aberrant JAK3 signalling.
• MS-1020 was obtained from Sigma-Aldrich (SML0204) and was > 98% pure. MS-1020 was dissolved in DMSO to give a 50mM stock solution which was stored at -70°C until required. Prior to use, the stock was allowed to thaw and equilibrate to room temperature for 30 mins before dilution in medium for combination analysis. Results
• MS-1020 was analysed in combination with HSV1716 in vitro with Hep3B, HuH7, Ovcar3, UVW and SPC1 11 cells. Toxicity was assessed in each well using DCP cell death assays. IC50 curves for each cell line tested are shown in Figure 23. MS-1020 displayed cytotoxicity in Hep3B, HuH7, Ovcar3, UVW and SPC11 1 cells with IC50s of 0.75uM, 0.1 uM, 0.2uM, 5uM and 0.1 uM respectively Table 5 ( Figure 24).
• MS-1020 had no toxicity with respect to HSV1716 as assessed by gfp expression during infection of the cell lines with HSV1716gfp. MS-1020 was incubated with cells plus virus at 5, 2.5, 1.25 and 0.25 ⁇ for 72 hrs and gfp fluorescence measured. Drug-mediated virus toxicities are presented in the graphs in Figure 25 as percentage of control cells (virus alone and no drug) in Hep3B, HuH7, Ovcar3, UVW, and SPC-1 11. Virus was used at moi 0.5 or moi 0.05. MS-1020 had little effect on gfp expression during HSV1716gfp infection Hep3B, HuH7, Ovcar3, UVW and SPC1 11 cells Table 6 ( Figure 26).
• Combination analysis MS-1020 was tested at 4 different doses 5, 2.5, 1.25 and 0.25 ⁇ in Hep3B, HuH7, Ovcar3, UVW, and SPC1 11 cells in combination with HSV1716 at moi 0.5 and 0.05 and cell death after 72 hours exposure was assessed by DCP assay.
• Data was analysed for synergy/antagonism by calculating combination indices and graphing them against Fa (FaCI/Chou-Talalay plots) using a spreadsheet designed to automatically calculate Fa and CI and graph the corresponding results from the raw DCP readings. The analysis was repeated on three separate plates and representative individual plots along with their respective Fa and CI values are presented in Figure 27 with synergies/antagonisms summarised in Table 7 ( Figure 28).
• the CI values for the 4 drug concentrations at the two HSV1716 moi were determined and if less than 1 then the combination of virus and drug must act synergistically to enhance cell death. If 4 or more out of the 8 available combinations resulted in CI values ⁇ 1 then the drug was identified as combining synergistically with HSV1716 in that cell line. If 1-3 out of the 8 combinations resulted in Cl ⁇ 1 , this suggests some limited synergy with HSV1716. If 0 of the 8 available combinations resulted in CI values >1 then the drug was seen as antagonistic with HSV1716. A negative Fa value occurs when the test DCP value is less than the control (i.e. indicative of improved survival) and is therefore scored as antagonistic.
• MS-1020 in combination with HSV1716 was synergistic at both moi and all MS- 1020 concentrations in Hep3B and at both moi and all concentration of MS-1020 except 0.5 ⁇ in UVW cells.
• MS1020 in combination with HSV1716 in SPC11 1 cells was synergistic only at moi 0.05 with all MS-1020 concentrations except 0.5 ⁇ although the combination was additive at this drug concentration. There were no synergies at moi 0.5 in SPC1 11 cells although the combination at this moi with 0.5uM MS-1020 was additive.
• Tofacitinib (trade name Xeljanz, formerly tasocitinib, CP-690550) is a drug of the Janus Kinase (JAK) inhibitor class, discovered and developed by Pfizer. Tofacitinib is a novel inhibitor of JAK3 with IC50 of 1 nM and has approximately 20- to 100-fold less activity against JAK2 and JAK1.
• Tofacitinib is currently approved for the treatment of rheumatoid arthritis (RA) in the United States and is being studied for treatment of psoriasis, inflammatory bowel disease, and other immunological diseases, as well as for the prevention of organ
• RA rheumatoid arthritis
• Tofacitinib was not approved by the EMA because of concerns over efficacy and safety.
• tofacitinib rapidly improved disease by inhibiting the production of inflammatory mediators and suppressing STAT1 -dependent genes in joint tissue.
• This efficacy in this disease model correlated with the inhibition of both JAK1 and 3 signalling pathways, suggesting that tofacitinib may exert therapeutic benefit via pathways that are not exclusive to inhibition of JAK3 ( Ghoreschi et al (201 1) J. Immunol. 186(7): 4234-4243).
• Tofacitinib generated a significant pro-apoptotic effect on murine FDCP-EpoR cells carrying
• JAK2 V6 7F JAK2 V6 7F
• This activity is coupled with the inhibition of phosphorylation of the key JAK2 V6 7F -dependent downstream signalling effectors STAT3, STAT5, and v-akt murine thymoma viral oncogene homolog (AKT) (Manshouri et al. (2008) Cancer Sci. 99 (6): 1265-73).
• Ruxolitinib (trade names Jakafi and Jakavi, by Incyte Pharmaceuticals and Novartis) is the first potent, selective, JAK1/2 inhibitor to enter the clinic with IC50 of 3.3 nM/2.8 nM respectively and 130-fold selectivity for JAK1/2 versus JAK3. Ruxolitinib potently and selectively inhibits JAK2 V6 7F -mediated signalling and proliferation in Ba/F3 cells and HEL cells and markedly increases apoptosis in a dose dependent manner in Ba/F3 cells (Quintas-Cardama et al (2010) Blood 1 15 (15): 3109-31 17).
• Ruxolitinib is approved for the treatment of intermediate or high-risk myelofibrosis, a type of bone marrow cancer and is also being investigated for the treatment of other types of cancer (such as lymphomas and pancreatic cancer), for polycythemia vera and for plaque psoriasis.
• ruxolitinib was approved by the U.S. Food and Drug Administration (FDA) for the treatment of intermediate or high-risk myelofibrosis based on results of the COMFORT-I and COMFORT-II Trials ( Harrison et al (2012) NEJM 366 (9): 787-789, Verstovsek et al. (2012) NEJM 366 (9): 799-807).
• Ruxolitinib was obtained from Selleckchem UK (S1378) and was > 99.22% pure.
• Ruxolitinib was dissolved in DMSO to give a 50mM stock solution which was stored at - 70°C until required. Prior to use, the stock was allowed to thaw and equilibrate to room temperature for 30 mins before dilution in medium for combination analysis.
• Tofacitinib citrate was obtained from Selleckchem UK (S5001) and was > 99.32% pure. Tofacitinib was dissolved in DMSO to give a 50mM stock solution which was stored at - 70°C until required. Prior to use, the stock was allowed to thaw and equilibrate to room temperature for 30 mins before dilution in medium for combination analysis.
• Ruxolitinib and tofacitinib were analysed in combination with HSV1716 in vitro in Hep3B, U87 and SPC1 11 cells. Toxicity was assessed in each well using DCP cell death assay.
• Tofacitinib and ruxolitinib displayed some toxicity in Hep3B, U87 and SPC1 11 cells and Chou-Talalay analysis was performed with both drugs in combination with HSV1716.
• Ruxolitinib and tofacitinib were tested at 4 different doses 10, 7.5, 5 and 2.5 ⁇ in Hep3B, U87 and SPC1 11 in combination with HSV1716 at moi 2 and 0.2 and cell death after 72 hours exposure was assessed by DCP assay. Data was analysed for
• AZD1480 (AstraZeneca) is an orally bio available, ATP competitive inhibitor of JAK1 and JAK2, which has been shown to inhibit the growth of solid tumours including breast, ovarian and prostate (Hedvat, M et al, Cancer Cell, 2009: 16 487-97).
• the ability of AZD1480 to limit tumour volume is attributed to the inhibition of STAT3 activation.
• the JAK/STAT pathway which is involved in inflammation, proliferation and
• STAT3 has been implicated in multiple cancers due to its unregulated and aberrantly activated in many cancers including breast, colon, prostate and GBM.
• AZD1480 was analysed in combination with HSV1716 in vitro using Hep3B, HepG2-luc, CP70, Ovcar3, SKOV3, U87, UVW and SPC11 1 cells. Toxicity was assessed in each well using DCP cell death assays.
• IC50 curves for each cell line tested are shown in Figure 33 and the estimated IC50s are shown in Table 10 ( Figure 34).
• AZD1480 was toxic in all cell lines with IC50s in the range 0.5-25 uM with limited cell line variability in toxicity apart from HepG2-luc which was the most sensitive cell line to this inhibitor Table 10 ( Figure 34).
• AZD1480 was tested at 4 different doses 12.5, 5, 2.5 and 0.25 ⁇ in Hep3B, HepG2-luc, CP70, Ovcar3, SKOV3, U87, UVW and SPC111 in combination with HSV1716 at moi 0.5 and 0.05 and cell death after 72 hours exposure was assessed by DCP assay. Data was analysed for synergy/antagonism by calculating combination indices and graphing them against Fa (FaCI/Chou-Talalay plots) using a spreadsheet designed to automatically calculate Fa and CI and graph the corresponding results from the raw DCP readings. The analysis was repeated on three separate plates and representative individual plots along with their respective Fa and CI values are presented in Figure 35 with
• ⁇ AZD1480 is toxic in all of the cell lines tested.
• AZD1480 was synergistic in Hep3B, HepG2-luc, CP70, SKOV3 and SPC111 at both moi.

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