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WO2005113013A2 - Compositions de vmn et methodes d'utilisation de ces compositions pour le traitement du cancer - Google Patents

Compositions de vmn et methodes d'utilisation de ces compositions pour le traitement du cancer Download PDF

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Publication number
WO2005113013A2
WO2005113013A2 PCT/IL2005/000518 IL2005000518W WO2005113013A2 WO 2005113013 A2 WO2005113013 A2 WO 2005113013A2 IL 2005000518 W IL2005000518 W IL 2005000518W WO 2005113013 A2 WO2005113013 A2 WO 2005113013A2
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WO
WIPO (PCT)
Prior art keywords
ndv
administration
administering
eid
tumor
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/IL2005/000518
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English (en)
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WO2005113013A3 (fr
Inventor
Zichria Zakay-Rones
Amos Panet
Evgeniya Greenbaum
Eithan Galun
Arnold I. Freeman
Linda Rasooly
Charles S. Irving
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Theravir Management LP
Original Assignee
Theravir Management LP
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Filing date
Publication date
Application filed by Theravir Management LP filed Critical Theravir Management LP
Publication of WO2005113013A2 publication Critical patent/WO2005113013A2/fr
Publication of WO2005113013A3 publication Critical patent/WO2005113013A3/fr
Priority to US11/561,510 priority Critical patent/US7615209B2/en
Priority to IL179420A priority patent/IL179420A0/en
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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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
    • A61K35/768—Oncolytic viruses not provided for in groups A61K35/761 - A61K35/766
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/18011—Paramyxoviridae
    • C12N2760/18111—Avulavirus, e.g. Newcastle disease virus
    • C12N2760/18132—Use of virus as therapeutic agent, other than vaccine, e.g. as cytolytic agent

Definitions

  • the present invention relates to methods for inducing regression of a tumor in a subject comprising administering to the subject a pharmaceutical composition comprising as an active ingredient a lentogenic strain of Newcastle disease virus having oncolytic activity.
  • the present invention provides methods for inducing regression of a tumor in a subject unresponsive to conventional anti-cancer therapies.
  • GBM glioblastoma multiforme
  • Newcastle Disease Virus has a long history as a broad spectrum oncolytic agent that can destroy tumor cells and stimulate the immune system (Bar Eli, N., et al., J. Cancer Res. Clin. Oncol. 122: 409- 415, 1996; Tzado -David, Y., et al., J. Cancer Res. Clin. Oncol. 121: 169-174, 1995; Schirrmacher, V., et al., Int. J. Oncol. 18: 945-952, 2001).
  • NDV is a single stranded RNA virus, whose natural host is poultry.
  • NDV strains have been classified as pathogenic (mesogenic or velogenic) or non- pathogenic (lentogenic) to poultry.
  • the 73T, MTH68 and PV701 (MK107) mesogenic strains of NDV have been the subject of several clinical studies.
  • NDV-PV701 has recently been evaluated in a Phase I study of patients with advanced solid tumors; however, patients with CNS tumors were excluded from these studies (Pecora, A.L., et al., J Clin Oncol 20:2251-66, 2002).
  • the anti-neoplastic responses to MTH68 in malignant glioma have been reported (Csatary, L.K., et al. J. Neurooncol. 67: 83-93, 2004).
  • Lentogenic strains of NDV have also been shown to kill some cancer cell lines (Schirrmacher, V., et al. ibid). Infection of tumor cells by lentogenic NDV has been found to generate several innate danger signals leading to apoptosis.
  • the lentogenic Ulster strain of NDV has been combined with various tumor cells as a tumor vaccine for different cancers including glioblastoma, however the use of a lentogenic NDV strain alone in virotherapy has not been evaluated.
  • WO 00/62735 of Pro-Virus discloses the use of any interferon sensitive strain of virus for killing neoplastic cells that are deficient in the interferon response.
  • the Pro- Virus disclosure supplies a catalog of viral strains including three mesogenic strains of NDV (MK107, NJ Roakin, and Connecticut-70726) shown to be useful for treatment of human tumor xenografts in athymic mice. NDV administration to these mice caused tumor regression, which was attributed to more efficient and selective replication of NDV in tumor cells versus normal cells. The differential sensitivity of tumor cells to killing by NDV was disclosed to be correlated to an inability of the cells to manifest interferon- mediated antiviral response.
  • the above patent application claims methods of infecting neoplasms or tumors and methods of treating neoplasms or tumors by interferon-sensitive, replication competent RNA or DNA viruses. European Patent No.
  • 0696326 discloses a use of NDV in manufacturing of a medicament for treatment of cancer, wherein the NDV is of moderate virulence and is cytolytic.
  • European Patent Application No. 1314431 discloses a composition comprising NDV for use in the treatment of cancer, wherein the NDV is of moderate virulence and is cytolytic.
  • European Patent Application No. 01486211 claims a use of NDV in the manufacture of medicament for treatment of cancer in a mammal having a tumor wherein the medicament is administered systematically in multiple doses to said mammal in an amount sufficient to cause tumor regression.
  • 01486211 refers to various strains of NDV, both cytolytic and non-cytolytic, the applicants of the European application provide in vivo effects of two mesogenic strains of NDV in animal models. No clinical studies nor examples of in vivo effects of lentogenic strains of NDV are disclosed in the European Patent Application No. 01486211.
  • International Patent Application WO 2005/018580 claims a method of treating a mammalian subject having a tumor comprising administering to the subject an amount of a NDV. Though the NDV according to WO 2005/018580 can be of low (lentogenic), moderate (mesogenic) or high (velogenic) virulence, the application relates to a mesogenic strain of NDV.
  • the negative-stranded RNA virus is a replication competent oncolytic virus, particularly a NDV, and more particularly a mesogenic strain of NDV.
  • the present invention provides methods for inducing tumor regression in a subject, the methods comprise administering to the subject a pharmaceutical composition comprising a lentogenic oncolytic strain of NDV.
  • the methods of inducing tumor regression in cancer patients as disclosed herein which comprise administering a pharmaceutical composition comprising a lentogenic strain of NDV to a cancer patient, are efficacious in decreasing tumor size as well as inhibiting tumor growth.
  • the methods of the present invention are particularly efficient in cancer patients unresponsive to conventional therapies.
  • the methods of inducing tumor regression of the present invention do not cause inflammatory responses, which are a major concern for viral therapy in cancer patients.
  • the use of a lentogenic strain of NDV reduces the concerns related to environmental impact of virus shedding.
  • a lentogenic oncolytic strain of NDV is capable of inhibiting the proliferation and killing of a wide range of tumor cell lines.
  • the tumor cell lines affected are rat glioma cells, human glioblastoma cell lines, human prostate cancer cell lines, human bladder cancer cell lines, mouse and human lung cancer cell lines, breast cancer cell lines, and human colon cancer cell lines. While the inhibitory effect of the lentogenic strain of NDV on the proliferation of tumor cell lines was highly pronounced, the virus had minor effect on the proliferation of normal cells such as fibroblasts and Peripheral Blood Mononuclear Cells (PBMC).
  • PBMC Peripheral Blood Mononuclear Cells
  • inducing regression of a tumor in a subject by administering to the subject a pharmaceutical composition comprising a lentogenic oncolytic strain of NDV lead to an extended median survival and an improvement in the clinical condition of said subject for a certain period of time.
  • the methods of the present invention are highly useful for cancer patients unresponsive to conventional therapies.
  • the principles of the present invention are exemplified herein below with glioblastoma patients, who underwent surgical procedures, radiotherapy, chemotherapy or a combination thereof, which did not improve the patient's clinical condition.
  • the results demonstrate that administering a pharmaceutical composition comprising a lentogenic oncolytic strain of NDV to glioblastoma patients resulted in disappearance of the tumor, as evidenced by radiological tests, for a limited period of time. Life expectancy of the patients extended from about 14 months from time of diagnosis to about 27 months, accompanied with minor or mild adverse effects, i.e., grade I and II fever.
  • the methods of the present invention are safe, efficient, and particularly useful in cancer patients unresponsive to conventional therapies.
  • the present invention provides a method for inducing regression of a tumor in a subject comprising administering to the subject a pharmaceutical composition comprising a therapeutically effective amount of a lentogenic oncolytic strain of NDV and a pharmaceutically acceptable carrier.
  • the lentogenic oncolytic strain of NDV is the HUJ strain of NDV disclosed in the International Patent Application WO 2003/022202, which is incorporated by reference as if fully set forth herein.
  • any type of tumor can be treated with the pharmaceutical composition of the invention.
  • tumors examples include, but are not limited to lung carcinoma, breast carcinoma, prostate carcinoma, colon adenocarcinoma, cervical carcinoma, endometrial carcinoma, ovarian carcinoma, bladder carcinoma, Wilm's tumor, fibrosarcoma, osteosarcoma, melanoma, synovial sarcoma, epidermoid carcinoma, pancreas carcinoma, endocrine system carcinoma, astrocytoma, oligodendroglioma, menigioma, neuroblastoma, glioblastoma, ependyoma, Schwannoma, neurofibrosarcoma, neuroblastoma, and medullablastoma.
  • administering the pharmaceutical composition to the subject is selected from the group consisting of parenteral, oral, rectal, vaginal, topical, intranasal, inhalation, buccal, and ophthalmic administration.
  • parenteral administration includes, but is not limited to, intravenous and intraarterial infusion, intravenous, subcutaneous, intraperitoneal, intraarterial, intramuscular, and intralesional injection. The intralesional injection can be directly into the tumor or adjacent to the tumor.
  • administering the pharmaceutical composition of the invention is by intravenous infusion.
  • the therapeutically effective amount of the HUJ strain of NDV is a daily dose from about lxl 0 8 to about 5xl0 n egg infective dose (50%) - EID 50 .
  • the therapeutically effective amount of the NDV HUJ is a daily dose of about l.lxlO 10 EID50.
  • the daily dose can be administered in a single or multiple administrations.
  • the pharmaceutical composition is administered at fixed intervals, e.g., daily, weekly, biweekly, etc.
  • the pharmaceutical composition is administered in a dosage cycle administration wherein an identical dose of the pharmaceutical composition is administered throughout the dosage cycle, which is either continuous or interrupted by intervals without treatment.
  • the dosage cycle administration comprises administering to the subject a daily dose of the pharmaceutical composition for five successive days followed by a halt of administration.
  • the halt of administration according to the principles of the present invention is of at least one day.
  • the halt of administration is of two days.
  • the halt of administration is of nine days.
  • the present invention encompasses a dosage cycle administration wherein administering the daily dose of the pharmaceutical composition is for two successive days, three successive days, four successive days or more successive days followed by a halt of administration.
  • the dose, the duration of administration and the halt of administration in the dosage cycle administration of the pharmaceutical composition of the invention can vary and will be determined by a physician based on radiological and/or clinical evidence of the disease progression.
  • the dosage cycle administration is performed at least once.
  • the dosage cycle is administered at least twice.
  • the amount of the oncolytic lentogenic strain of NDV administered daily in a later dosage cycle is higher than the amount administered in a former dosage cycle.
  • administering the dosage cycle further comprises administering to the subject a maintenance dose at least once a week.
  • the maintenance dose can range from about 5xl0 9 to about 5xl0 u EID 5 o.
  • the maintenance dose of NDV HUJ is 6.3x10 9 E ⁇ D 50 .
  • the maintenance dose is administered twice a week. It will be understood that the maintenance dose preferably will not exceed the highest dose administered to a subject.
  • the maintenance dose and the schedule of administration of the maintenance dose will be determined by a physician based on radiological and/or clinical evidence of the disease progression.
  • the subject administered with the pharmaceutical composition of the invention is unresponsive to at least one anti-cancer therapy.
  • the subject administered with the pharmaceutical composition of the invention is unresponsive to at least one of tumor resection, radiotherapy and/or chemotherapy.
  • the tumor is glioblastoma.
  • the method for inducing regression of glioblastoma comprises administering the pharmaceutical composition by intravenous infusion.
  • the therapeutically effective amount of NDV HUJ for inducing regression of glioblastoma is a daily dose of about lxlO 8 to about 5.5xl0 10 EID 50 .
  • administering the pharmaceutical composition to the subject having glioblastoma comprises a dosage cycle administration.
  • the method of inducing regression of glioblastoma in a subject comprises at least one of the following steps: (i) administering a daily dose of about lxlO 8 EID 50 for five successive days followed by no administration for at least one day; (ii) administering a daily dose of about 5x10 s EID 5 Q for five successive days followed by no administration for at least one day; (iii) administering a daily dose of about lxl 0 9 EID 50 for five successive days followed by no administration for at least one day; (iv) administering a daily dose of about 5xl0 9 EID 5 Q for five successive days followed by no administration for at least one day; (v) administering a daily dose of about lxlO 10 EID 50 for five successive days followed by no administration for at least one day; (vi) administering a daily dose of about 5xl0 10 EID 50 for five successive days followed by no administration for at least one day; and optionally (vii) repeating at least one
  • the method of inducing regression of glioblastoma in a subject comprises at least one of the following steps: (i) administering a daily dose of about lxl 0 8 EID 50 for five successive days followed by no administration for two days; (ii) administering a daily dose of about 5xl0 8 EID 50 for five successive days followed by no administration for two days; (iii) administering a daily dose of about lxl 0 9 EID 50 for five successive days followed by no administration for nine days; (iv) administering a daily dose of about 5xl0 9 EID 5 o for five successive days followed by no administration for nine days; (v) administering a daily dose of about lxlO 10 EID 50 for five successive days followed by no administration for nine days; (vi) administering a daily dose of about 5xl0 10 EID 50 for five successive days followed by no administration for nine days; and optionally (vii) repeating at least one of steps (i) to (vi).
  • the methods of inducing regression of glioblastoma in a subject further comprise as a last step administering a maintenance dose of the pharmaceutical composition at least once a week.
  • the maintenance dose is of about 5xl0 9 EID 5 0 to about 5x10 l EID 50 of NDV HUJ at least once a week.
  • the maintenance dose is of about 6.3x10 9 EID 50 of NDV HUJ at least twice a week.
  • the glioblastoma patient administered with the pharmaceutical composition of the invention is unresponsive to at least one anti-cancer therapy.
  • FIGs. 1A-D show photomicrographs of rat glioblastoma cells (RG2) incubated for three days in the presence of various concentrations of NDV HUJ.
  • A RG2 grown without virus;
  • B RG2 grown in the presence of 100 MOI NDV HUJ;
  • C RG2 grown in the presence of 200 MOI NDV HUJ;
  • D RG2 grown in the presence of 400 MOI
  • FIG. 2 shows the amount of NDV HUJ in the supernatant of rat glioblastoma cells (RG2) incubated with 400 MOI of NDV HUJ.
  • the supernatant of RG2 cells infected with 400 MOI of NDV HUJ was sampled after 1, 24, or 72 hours of incubation and was applied to human fibrosarcoma HT1080 cells (positive control). Viability of HT1080 cells after 3 days of incubation was determined by the XTT assay. The amount of the virus in the supernatant, which affected HT1080 viability, was expressed as MOI.
  • FIG. 3 shows FACS analysis of annexin expression in cells treated with NDV HUJ.
  • FIGs. 4A-B show the effect of increasing MOI doses of NDV HUJ on cell viability of glioblastoma cell lines in vitro as determined by the XTT assay (panel A) and by the Alamar Blue assay (panel B).
  • FIG. 5 shows a dose response relationship between increasing amounts of virus per cell (MOI) and the decrease in the viability of human fibrosarcoma cells (HT1080) incubated with NDV HUJ for 3 days.
  • FIG. 6 shows the effect of increasing MOI doses of NDV HUJ on cell viability of prostate cancer cell lines in vitro as determined by the XTT assay.
  • FIG. 7 shows the effect of increasing MOI doses of NDV HUJ on cell viability of T24P bladder cancer cell line in vitro as determined by the XTT and Alamar Blue assays.
  • FIG. 8 shows the effect of increasing MOI doses of NDV HUJ on cell viability of
  • FIG. 9 shows the effect of increasing MOI doses of NDV HUJ on cell viability of HT29 colon cancer cell line in vitro as determined by the XTT and Alamar Blue assays.
  • FIG. 10 shows the effect of increasing MOI doses of NDV HUJ on cell viability of breast cancer cell lines in vitro as determined by the XTT assay.
  • FIGs. 11A-B show the absence of an effect of NDV HUJ on mouse stroma fibroblasts (MS-5) and on human peripheral blood mononuclear cells (PBMC).
  • FIG. 12 shows the effect of NDV HUJ treatment on tumor size in nude athymic mice injected with prostate cancer cells.
  • FIG. 13 shows a scheme of patient enrollment to the NDV HUJ treatment.
  • FIGs. 14A-F shows the gadolinium enhanced Tl MRI scans obtained from a glioblastoma patient treated with NDV HUJ.
  • Viruses are known to exert oncolytic effects on tumor cells and the use of oncolytic viruses as therapeutic agents has been reported. Some effort has been done to use non- human viruses exhibiting medium to high pathogenicity for their natural hosts in the treatment of cancer patients.
  • the present invention discloses methods for inducing regression of tumors in human subjects, the methods utilize a lentogenic strain of Newcastle disease virus (NDV), which is non-pathogenic to poultry but exhibits oncolytic properties.
  • NDV Newcastle disease virus
  • the disclosed methods provide safe, effective and reliable means to induce regression of a tumor in an individual in need thereof. These methods overcome the drawbacks of using pathogenic strains of viruses for human therapy.
  • the present invention provides a method for inducing regression of a tumor in a subject, the method comprises the step of administering to the subject a pharmaceutical composition comprising a therapeutically effective amount of a lentogenic oncolytic strain of NDV.
  • the lentogenic oncolytic strain of NDV is NDV HUJ.
  • regression of a tumor means decreasing tumor size or arresting tumor growth or tumor progression, which have their commonly understood meaning of suppressing tumor growth.
  • oncolytic virus refers to a virus capable of exerting a cytotoxic or killing effect in vitro and in vivo to tumor cells with little or no effect on normal cells.
  • oncolytic activity refers to cytotoxic or killing activity of a virus to tumor cells.
  • the oncolytic activity exerted by a lentogenic strain of NDV, particularly NDV HUJ is probably primarily due to cell apoptosis and to a lesser extent to plasma membrane lysis, the latter is accompanied by release of viable progeny into the cell's milieu that subsequently infect adjacent cells.
  • the cytotoxic effects under in vitro or in vivo conditions can be detected by various means as known in the art, for example, by inhibiting cell proliferation, by detecting tumor size using gadolinium enhanced MRI scanning, by radiolabeling of a tumor, and the like.
  • clonal virus can be produced according to any method available to the skilled artisan.
  • clonal virus can be produced by limiting dilution or by plaque purification.
  • a cloned lentogenic NDV strain denoted NDV HUJ is disclosed in the International Patent Application WO 2003/022202, the content of which is incorporated by reference as if fully set forth herein.
  • the clonal NDV HUJ strain was prepared by limiting dilution and further purified on a sucrose gradient. All types of tumors can be included in the scope of the present invention.
  • the following solid tumors can be treated: skin (e.g., squamous cell carcinoma, basal cell carcinoma, or melanoma), breast, colorectal, prostate, brain and nervous system, head and neck, testicular, ovarian, pancreatic, lung, liver (e.g., hepatoma), kidney, bladder, gastrointestinal, bone, endocrine system (e.g., thyroid and pituitary tumors), and lymphatic system (e.g., Hodgkin's and non-Hodgkin's lymphomas) tumors.
  • skin e.g., squamous cell carcinoma, basal cell carcinoma, or melanoma
  • breast e.g., colorectal
  • prostate e.g., colorectal
  • brain and nervous system e.g., brain and nervous system
  • head and neck testicular, ovarian
  • pancreatic lung
  • liver e.g., hepatoma
  • kidney e.g.,
  • Tumors of the nervous system include, for example, astrocytoma, oligodendroglioma, menigioma, neuroblastoma, glioblastoma, ependyoma, Schwannoma, neurofibrosarcoma, neuroblastoma, and medullablastoma.
  • Other types of tumors include fibrosarcoma, epidermoid carcinoma, and any other solid tumor.
  • benign and malignant proliferative diseases of the blood for example, leukemia.
  • the methods of the invention can be used to induce regression of primary tumors and tumor metastases.
  • the NDV administered according to the methods of the invention follow the same pathways as metastasizing tumor cells, thus enhancing the likelihood of NDV reaching those areas within the lymphatic system, e.g., lymph nodes that are at greatest risk for harboring metastatic disease.
  • the pharmaceutical compositions of the invention comprise as an active ingredient a lentogenic oncolytic strain of NDV, particularly the NDV HUJ, in a form suitable for administration to a human subject.
  • the pharmaceutical compositions can further comprise one or more pharmaceutically acceptable carriers, one or more additional ingredients, or a combination thereof.
  • pharmaceutically acceptable means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans.
  • the term “pharmaceutically acceptable” means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans.
  • carrier refers to a diluent, adjuvant, excipient, or vehicle with which the therapeutic active agent is administered. Carriers are more or less inert substances when added to a pharmaceutical composition to confer suitable consistency or form to the composition.
  • the formulations of the pharmaceutical compositions described herein can be prepared by any method known in the art of pharmacology and biotechnology. In general, such preparatory methods include the step of bringing the active ingredient into association with a carrier or one or more other accessory ingredients, and then, if necessary or desirable, shaping or packaging the product into a desired single- or multi-dose unit.
  • compositions that are useful in the methods of the invention can be prepared in formulations suitable for parenteral, oral, rectal, vaginal, topical, intranasal, inhalation, buccal, ophthalmic, or any other route of administration, depending on the anticipated site at or to which the composition is to be administered.
  • a pharmaceutical composition of the invention can be prepared in bulk, as a single unit dose or as a plurality of single unit doses.
  • a "unit dose” is a discrete amount of the pharmaceutical composition comprising a predetermined amount of the virus.
  • parenteral administration" of a pharmaceutical composition includes any route of administration characterized by physical breaching of a tissue of a subject and administration of the pharmaceutical composition through the breach in the tissue.
  • Parenteral administration thus includes, but is not limited to, administration of a pharmaceutical composition by injection of the composition, by application of the composition through a surgical incision, by application of the composition through a tissue- penetrating non-surgical wound, and the like.
  • parenteral administration is contemplated to include, but is not limited to, intravenous injection, subcutaneous injection, intraperitoneal injection, intraarterial injection, intramuscular injection, intravenous infusion and intraarterial infusion.
  • the administration of the pharmaceutical composition comprising NDV HUJ is by intravenous infusion or by subcutaneous injection.
  • the volume of the composition administered is from about 10 milliliters to about 500 milliliters. In an exemplary embodiment, the volume of the composition administered intravenously to a patient is of about 15 ml.
  • convection methods which include multiple lines can be placed around the tumor and a pump can be used to bathe the tumor with a solution of the pharmaceutical composition of the invention, which solution can be recycled during a few days.
  • Formulations of the pharmaceutical composition suitable for parenteral administration can comprise the active ingredient combined with a pharmaceutically acceptable carrier, such as sterile water or sterile isotonic saline.
  • Such formulations can be prepared in a form suitable for bolus administration or for continuous administration.
  • Injectable formulations can be prepared in a unit dosage form, such as in ampules, in multi- dose containers containing a preservative, or in single-use devices for auto-injection or injection by a medical practitioner.
  • Formulations for parenteral administration include, but are not limited to, suspensions, solutions, emulsions in oily or aqueous vehicles, pastes, and implantable sustained-release or biodegradable formulations.
  • Such formulations can further comprise one or more additional ingredients including, but not limited to, suspending agents, emulsifying agents, dispersing agents and stabilizing agents.
  • a formulation for parenteral administration can be provided in a dry (i.e., powder or granular) form for reconstitution with a suitable vehicle (e.g., sterile pyrogen-free water) prior to parenteral administration of the reconstituted composition.
  • a suitable vehicle e.g., sterile pyrogen-free water
  • the pharmaceutical compositions can be prepared in the form of a sterile injectable aqueous or oily suspension or solution.
  • an "oily" suspension or solution is one which comprises a carbon-containing liquid molecule and which exhibits a less polar character than water.
  • These suspensions or solutions can be formulated according to the known art, and can comprise, in addition to the active ingredient, additional ingredients such as dispersing agents, wetting agents, suspending agents, and preservatives.
  • Such sterile injectable formulations can be prepared using a non-toxic parenterally-acceptable diluent or solvent, such as water or 1,3-butane diol, for example.
  • a non-toxic parenterally-acceptable diluent or solvent such as water or 1,3-butane diol, for example.
  • Other acceptable diluents and solvents include, but are not limited to, Ringer's solution, isotonic sodium chloride solution, phosphate buffered saline, and oils such as synthetic mono- or di-glycerides.
  • Other parentally-administrable formulations, which are useful include those, which comprise the active ingredient in microcrystalline form, in a liposomal preparation, or as a component of biodegradable polymer systems.
  • compositions for sustained release or implantation can comprise pharmaceutically acceptable polymeric materials such as an ion exchange resin, a sparingly soluble polymer, and the like.
  • Known suspending agents include, but are not limited to, hydrogenated edible fats, sodium alginate, polyvinylpyrrolidone, gum tragacanth, gum acacia, and cellulose derivatives such as sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethyl-cellulose.
  • Known dispersing or wetting agents include, but are not limited to, naturally-occurring phosphatides such as lecithin, condensation products of an alkylene oxide with a fatty acid, with a long chain aliphatic alcohol, with a partial ester derived from a fatty acid and a hexitol, or with a partial ester derived from a fatty acid and a hexitol anhydride (e.g., polyoxyethylene stearate, heptadecaethyleneoxycetanol, polyoxyethylene sorbitol monooleate, and polyoxyethylene sorbitan monooleate, respectively).
  • Known emulsifying agents include, but are not limited to, lecithin and acacia.
  • Known preservatives include, but are not limited to, methyl, ethyl, or n-propyl-para- hydroxybenzoates, ascorbic acid, and sorbic acid.
  • the virus can be used in a composition with an adjuvant such as alum hydroxide.
  • the pharmaceutical composition can be formulated in an emulsions or submicron emulsion.
  • the pharmaceutical composition can further comprise other known additives such as, for example, human serum albumin or sucrose.
  • a formulation of a pharmaceutical composition of the invention suitable for oral administration can be prepared in the form of a discrete solid unit dose including, but not limited to, a tablet, a hard or soft capsule, a cachet, a troche, or a lozenge, each containing a predetermined amount of the active ingredient.
  • Other formulations suitable for oral administration include, but are not limited to, a powdered or granular formulation, an aqueous or oily suspension, an aqueous or oily solution, or an emulsion.
  • Formulations suitable for topical administration include, but are not limited to, liquid or semi-liquid preparations such as liniments, lotions, oil-in-water or water-in-oil emulsions such as creams, ointments or pastes, and solutions or suspensions.
  • Formulations for topical administration can further comprise one or more of the additional ingredients described herein above.
  • the pharmaceutical compositions of the invention can thus be in any form suitable for administration according to principles well known in the art. It is understood that the ordinarily skilled physician will readily determine and prescribe effective amounts of a lentogenic oncolytic strain of NDV to kill tumor cells in a subject. In so proceeding, the physician can, for example, prescribe relatively low doses at first, subsequently increasing the doses until an appropriate response is obtained.
  • the specific dose levels for any particular subject will depend upon a variety of factors including the extent, density, location, and type of tumor cells to be killed, the age, body weight, general health, gender, and diet of the subject, the time of administration, the route of administration, the rate of excretion, and a drug combination. Administration of the pharmaceutical composition should be continued until tumor regresses and health has been restored to the subject.
  • the therapeutically effective amount of a lentogenic oncolytic strain of NDV, particularly NDV HUJ is a daily dose in a range from about lxl 0 8 to about 5.5xlO ⁇ egg-infective dose 50% (EID 50 ).
  • the EID 50 as used herein refers to virus infectious units, which are measured by the dilution of a virus suspension that causes the infection of 50% of the hen's eggs determined by a qualitative hemagglutination test.
  • the daily dose of NDV HUJ is about lxlO 10 EID5 0 . It will be understood that the daily dose can be an escalating dose so that a low daily dose can be administered first, and subsequently higher daily dose can be administered until an appropriate response is achieved. Also, the daily dose of the composition can be administered to the subject in multiple administrations in the course of a single twenty-four hour period in which a portion of the daily dose is administered at each administration.
  • the daily dose is administered in a single administration.
  • a daily dose can be administered during a period of a few minutes to a few hours, depending on the practitioner's decision.
  • administering the pharmaceutical composition comprising a lentogenic oncolytic strain of NDV is performed in dosage cycle administration.
  • dosage cycle administration refers to a schedule of administration wherein the subject is administered continuously with a defined dose of the pharmaceutical composition after which a continuous administration of another dose, preferably higher dose, of the pharmaceutical composition is administered.
  • a dosage cycle refers to administering the pharmaceutical composition in intervals, e.g., a defined dose of the composition is administered for one, two, three, or more days after which the subject is not treated so that a halt in administration of the pharmaceutical composition takes place.
  • a dosage cycle consists of a daily administration of the composition for five successive days after which at least a one- day halt is performed.
  • the present invention also encompasses a dosage cycle, which includes a daily administration for less or for more than five successive days after which a halt of administration takes place.
  • the halt of administration lasts for two days.
  • the halt of administration lasts for nine days.
  • a dosage cycle is repeated at least once. According to an exemplary embodiment, a dosage cycle is repeated at least twice. According to an exemplary embodiment, a dosage cycle is repeated three times. It will be understood that the daily dose of NDV administered in successive dosage cycles can be identical or escalating. However, the duration of a dosage cycle, the duration of administration, the duration of the halt of administration and the dose administered can vary according to the clinical condition of the subject, the type of tumor, and the like. According to some embodiments, a maintenance dose is administered after completion of the dosage cycles.
  • a "maintenance dose" as used herein refers to a dose of a lentogenic oncolytic strain of NDV, which inliibits re-growth or further growth of the tumor.
  • the maintenance daily dose is about 6.3x10 9 EID 50 of NDV HUJ.
  • the maintenance dose is typically administered at least once a week, preferably at least twice a week. It will be understood that the maintenance dose can be determined by a physician based on radiological and/or clinical evidence of disease progression.
  • the methods of the invention can also include the use of an additional anticancer therapy. For example, the methods can be carried out in conjunction with chemotherapy, radiotherapy, biological therapy, gene therapy and/or any other therapy known in the art including, but not limited to, anti-inflammatory treatment with anti-inflammatory agents, for example, corticosteroids.
  • alkylating agents that can be used in the methods of the invention include temozolomide, busulfan, carboplatin, carmustine, chlorambucil, cisplatin, cyclophosphamide (i.e., cytoxan), dacarbazine, ifosfamide, lomustine, mecholarethamine, melphalan, procarbazine, streptozocin, and thiotepa.
  • antineoplastic antibiotics include bleomycin, dactinomycin, daunorubicin, doxorubicin, idarubicin, mitomycin (e.g., mitomycin C), mitoxantrone, pentostatin, and plicamycin.
  • antimetabolites include fluorodeoxyuridine, cladribine, cytarabine, floxuridine, fludarabine, fluorouracil (e.g., 5- fluorouracil (5FU)), gemcitabine, hydroxyurea, mercaptopurine, methotrexate, and thioguanine.
  • natural source derivatives include docetaxel, etoposide, irinotecan, paclitaxel, teniposide, topotecan, vinblastine, vincristine, vinorelbine, taxol, prednisone, tamoxifen, asparaginase, and mitotane.
  • the biological therapy that can be used in conjunction with the methods of the invention can involve administration of an immunomodulatory molecule, such as a molecule selected from the group consisting of tumor antigens, antibodies, cytokines (e.g., interleukins, interferons, tumor necrosis factor (TNF), granulocyte macrophage colony stimulating factor (GM-CSF), macrophage colony stimulating factor (M-CSF), and granulocyte colony stimulating factor (G-CSF)), chemokines, complement components, complement component receptors, immune system accessory molecules, adhesion molecules, and adhesion molecule receptors.
  • cytokines e.g., interleukins, interferons, tumor necrosis factor (TNF), granulocyte macrophage colony stimulating factor (GM-CSF), macrophage colony stimulating factor (M-CSF), and granulocyte colony stimulating factor (G-CSF)
  • chemokines chemokines
  • complement components complement component receptors
  • immune system accessory molecules adh
  • viruses used in the methods of the invention can be augmented, if desired, by including heterologous nucleic acid sequences encoding one or more therapeutic products in the viruses.
  • nucleic acid sequences encoding cytotoxins, immunomodulatory proteins (i.e., proteins that enhance or suppress patient immune responses to antigens), tumor antigens, antisense RNA molecules, siRNAs or ribozymes can be included in the viruses.
  • immunomodulatory proteins examples include, e.g., cytokines (e.g., interleukins, for example, any of interleukins 1-15, ⁇ , ⁇ , or ⁇ -interferons, tumor necrosis factor (TNF), granulocyte macrophage colony stimulating factor (GM-CSF), macrophage colony stimulating factor (M-CSF), and granulocyte colony stimulating factor (G-CSF)), chemokines (e.g., neutrophil activating protein (NAP), macrophage chemoattractant and activating factor (MCAF), and macrophage inflammatory peptides, complement components and their receptors, immune system accessory molecules (e.g., B7.1 and B7.2), adhesion molecules (e.g., ICAM-1, 2, and 3), and adhesion receptor molecules.
  • cytokines e.g., interleukins, for example, any of interleukins 1-15, ⁇ , ⁇ , or ⁇ -interferons
  • TNF tumor nec
  • heterologous nucleic acid sequences for use in the methods of the invention can be readily selected by those of skill in the art.
  • the heterologous nucleic acid sequences can be inserted into the viruses for use in the methods of the invention in a location that renders them under the control of regulatory sequences of the viruses.
  • the heterologous nucleic acid sequences can be inserted as part of an expression cassette that includes regulatory elements, such as promoters and/or enhancers.
  • Appropriate regulatory elements can be selected by those of skill in the art based on, for example, the desired tissue-specificity and level of expression. For example, a cell-type specific or tumor-specific promoter can be used to limit expression of a gene product to a specific cell type.
  • Tumor specific promoters can also be selected for use in the invention, based on the etiology of the cancer. Examples of promoters that function specifically in tumor cells include the stromelysin 3 promoter, which is specific for breast cancer cells (Basset et al., Nature 348: 699,1990); the surfactant protein A promoter, which is specific for non-small cell lung cancer cells (Smith et al., Hum. Gene Ther.
  • the mtsl promoter which is specific for metastatic tumors (Tulchinsky et al., Proc. Natl. Acad. Sci. U. S. A. 89: 9146,1992)
  • the c-erbB-2 promoter which is specific for pancreatic, breast, gastric, ovarian, and non-small cell lung cells (Harris et al., Gene Ther. 1:170,1994)
  • the c- erbB-3 promoter which is specific for breast cancer cells (Quin et al., Histopathology 25: 247,1994), and the like.
  • NDV-HUJ NDV-HUJ is a highly purified isolate originally derived from the naturally attenuated Bl NDV vaccine strain (ATCC, 1971).
  • the HUJ strain was classified as lentogenic and having no pathogenicity for its natural host, poultry, on the basis of its intra-cranial pathogenicity index (ICPI) of 0.0 determined by the Israel Ministry of Agriculture and a 112 G-R-Q-G-R-L 117 cleavage site sequence of its surface fusion protein, as well as having a Mean Death Time (MDT) of > lOOhrs.
  • ICPI intra-cranial pathogenicity index
  • MDT Mean Death Time
  • NDV-HUJ Clinical lots of NDV-HUJ were prepared as follows: The attenuated Bl NDV vaccine strain was passaged four times in hen's eggs to prepare a research stock.
  • the infected allantoic fluid from the fourth passage (E4 stock) was stored at -70°C.
  • the infected allantoic fluid from the E4 stock underwent 50 regular passages in 10-11 day old embryonated eggs.
  • the allantoic fluid was labeled "NDV lento" and was divided into vials stored at -80°C.
  • the "NDV lento" was cloned in 10-11 days old embryonated eggs by limiting dilution.
  • NDV lento (cloned) Allantoic fluid from the egg infected with the highest dilution was labeled "NDV lento (cloned)" and was stored at -70°C.
  • the "NDV lento (cloned)” strain was shown to be oncolytic and was renamed "NDV HUJ”.
  • the NDV HUJ strain was further cloned twice by limiting dilution in 10-11 day old embryonated SPF (specific pathogens free) eggs (obtained from ALPES (Aves Libres de Pat ⁇ genos Especificos S.A. de C.V), Pueblo, Mexico, a subsidiary of SPAFAS Charles River Lab.) to produce a Virus Master Seed Bank consisting of 220 tubes.
  • the tubes were stored at -80°C and contained the harvested allantoic fluid frozen without any further purification.
  • One tube from the Master seed bank was expanded into a Virus Working Seed Bank consisting of 300 tubes following the same procedure as used in the production of the master bank.
  • the working bank tubes were stored at -65°C.
  • the tubes contained the harvested allantoic fluid frozen without any further purification.
  • the starting material for virus production for clinical studies was NDV HUJ Working
  • Virus production for a clinical study used approximately 3000 eggs. The production was divided into several harvests ( ⁇ 500 eggs). For each harvest, a vial of working bank was thawed and the virus suspension was diluted in Gibco PBS (10 5 EID 50 /egg). A small hole was manually punched in the top of the egg and an aliquot of the virus suspension was injected into the amino-allantoic cavity of the egg. The hole in each egg was sealed with sterile acrylic cement and the eggs were incubated for 72 hrs. The eggs were checked for viability. Eggs which appeared upon candling to have died within the last 12- 24 hrs were set aside for harvesting and eggs which had appeared to have died earlier were discarded.
  • the virus was then concentrated by high speed centrifugation and purified in sucrose gradients as follows: Clarified crude bulk virus after having been stored at 4-7°C for between 1-6 weeks was re-clarified by low speed centrifugation (3000 rpm 30 min). Aliquots of re-clarified bulk from each harvest were taken and stored at -80°C for further testing and additional aliquots were taken for in-process sterility testing. The re-clarified bulk was then centrifuged at high speed 12,500 rpm for 1.5 hrs at 4°C and the pelleted virus was re- suspended in Gibco Dulbeco PBS.
  • a total of 12,260ml of reclarified bulk fluids from five harvests were concentrated to a total of 100 ml of resuspended pelleted virus with a 50% average yield based on EID 50 titers, ranging from 29% to 82% yields for individual harvests. Sterility was tested on aliquots from each tube of re-suspended concentrated virus. All samples of the concentrated virus passed sterility testing. The concentrated virus (100 ml) was centrifuged in Sorvall SurespinTM 630/36ml swinging bucket head at 22,000 rpm (89,744g) in a 20/40/60% sucrose gradient for 2.5 hrs at 4°C.
  • Clinical dosages were prepared from combined harvests of purified virus by diluting the viral suspension with sterile saline to achieve a concentration of approximately lxl0 9 EID 5 o /ml.
  • the purified virus was formulated as a suspension in PBS buffer, vialed as 1.1 ml aliquots and stored at -70°C until immediately before use.
  • Clinical lots of NDV-HUJ met the release criteria approved by the Israel Ministry of Health.
  • NOAEL Non-Observed Adverse Effect Level
  • Egg infectious dose (EID 50 ) was determined by inoculation of serial dilutions into the allantoic sac of 10-11 day old embryonated eggs and checking the fluids for hemagglutination 72-96 hrs post inoculation according to routinely used methods (Sever J. L., et al. J. Immunol. 80: 320-329, 1962). The EID 50 value was calculated by the method of Reed and Muench (Reed, L. J., et al. Amer. J. Hyg. 27: 493-497, 1938).
  • EXAMPLE 1 Effect of NDV HUJ on glioblastoma cell lines in vitro Cell lines
  • Cell lines were cultured in DMEM with 10% FBS, and 1% penicilin/streptomicin, 1% L- glutamine (Biological Industries, Kibbutz Bet Haemek, Israel).
  • the cell lines were periodically tested for mycoplasma infection using the EZ-PCR Mycoplasma Test Kit (Biological Industries, Kibbutz Bet Haemek, Israel). All cell lines were originally obtained from ATCC, except for T24P (obtained from Prof. A. Hochberg, Hebrew University, Jerusalem, Israel), MS-5 (obtained from Prof. A. Peled, Hebrew University, Jerusalem, Israel) and Ml 09 (obtained from Prof. E. Galun, Hebrew University, Jerusalem, Israel).
  • NDV HUJ Cell killing Cell killing by NDV HUJ was visualized by incubating RG2 rat glioblastoma cells at a concentration of 3.4xl0 5 cells/ml in a 96 well tissue culture plate with various concentrations of NDV HUJ for 3 days. The cells were visualized and photographed using an inverted microscope.
  • NDV HUJ Multiplicity of Infection
  • XTT and Alamar blue assays Cells were plated at 10 4 cells/well in 96 well tissue culture plates. The cells were immediately incubated with various levels of virus for 3 days in a 37 ° C 5% CO 2 incubator. After the incubation, XTT assay was performed using the Cell Proliferation kit - XTT based colorimetric assay (Biological Industries, Kibbutz Bet Haemek, Israel). This assay also indicates the metabolic state of the cells.
  • Alamar blue assay which determines the energy status of a cell, was performed using the Alamar blue reagent (Serotec Ltd, Israel). Optical density readings for XTT at 450/630nm and Alamar blue at 570/600nm assays were performed following 2 and 3-hour incubations, respectively.
  • RG2 cells were incubated overnight with 50 MOI of NDV HUJ, medium alone or l ⁇ M Staurosporine (Sigma-Aldrich Israel). Following incubation, cells were collected and analyzed for Annexin expression as follows: Annexin staining was performed using the Annexin V FITC (IQ Products, Groningen, The Netherlands) according to the manufacturer's instructions.
  • PI staining was performed by fixation of lxlO 6 cells in 1ml cold ethanol (95%), washing in PBS, digestion of RNA by the addition of RNAse A (Sigma-Aldrich Israel) to a concentration of lO ⁇ g/ml, and staining with 50 ⁇ l PI (Sigma-Aldrich Israel) for 15 minutes prior to FACS analysis. FACS analysis was performed using the Cellquest software on a FACS Calibur.
  • NDV HUJ Replication of NDV HUJ in RG2 tumor cells
  • RG2 tumor cells were incubated with NDV HUJ and the supernatants were collected at various time points of incubation.
  • the original concentration of NDV HUJ remained unchanged 1-hour post application (FIG. 2, circles).
  • the amount of the virus in the supernatant dramatically decreased and after 3 days of incubation barely detectable levels of virus were present in the supernatant (FIG. 2, circles).
  • the levels of the virus in the supernatant were identical in wells containing glioblastoma cells (RG2) and in wells where virus was incubated in medium alone (FIG. 2, squares).
  • Apoptosis of RG2 cells following overnight exposure to NDV HUJ RG2 cells were exposed overnight to either a known apoptosis inducer (Staurosporine) or to 50 MOI of NDV HUJ.
  • Staurosporine a known apoptosis inducer
  • One of the first markers of apoptosis - Annexin staining - was detected in Staurosporine induced cells.
  • Staurosporine FIG. 3B
  • NDV HUJ Treatment of RG2 cells with NDV HUJ increased the proportion of apoptotic cells even more than the known inducer of apoptosis (Staurosporine) and caused a more dramatic shift to the right in the annexin stained cell population (FIG. 3C). These results indicate that NDV HUJ caused a more marked apoptosis in rat glioblastoma cells than the known apoptosis inducer - Staurosporine. The presence of apoptotic RG2 cells following incubation with NDV HUJ can suggest that the mechanism for the cytotoxic effect of NDV HUJ on malignant glioma cells is primarily apoptotic.
  • NDV HUJ Effect of NDV HUJ on the viability of glioblastoma cell lines Viability of glioblastoma cell lines was determined by XTT and Alamar Blue assays as described herein above. As shown in FIGs. 4A and 4B, NDV HUJ significantly affected glioblastoma cell viability in vitro.
  • EXAMPLE 2 Effect of NDV HUJ on fibrosarcoma cell line in vitro Human fibrosarcoma HT1080 cells were incubated in the presence of increasing MOI of NDV HUJ and cell viability was determined by XTT and Alamar Blue assays. As shown in FIG. 5, NDV HUJ exerted a pronounced killing effect on HT1080 cells as determined by both assays.
  • Viability of prostate cancer cell lines was determined by the XTT assay as described in Example 1 herein above. As shown in FIG. 6, NDV HUJ significantly affected prostate cancer cell viability in vitro.
  • Viability of a bladder cancer cell line was determined by the XTT and Alamar Blue assays as described in Example 1 herein above. As shown in FIG. 7, NDV HUJ significantly affected bladder cancer cell viability in vitro.
  • Viability of a mouse lung cancer cell line was determined by the XTT and Alamar Blue assays as described in Example 1 herein above. As shown in FIG. 8, NDV HUJ significantly affected lung cancer cell viability in vitro. The effect of NDV-HUJ on the viability of an additional mouse lung cancer cell line (3LL) was examined. Cell monolayers (3 day old) of mouse 3LL (Table 1) were infected with the virus at the indicated MOI. Total number of cells and percent of dead cells was determined at 24-hour interval by trypan blue exclusion.
  • Viability of a colon cancer cell line was determined by the XTT and Alamar Blue assays as described in Example 1 herein above. As shown in FIG. 9, NDV HUJ significantly affected colon cancer cell viability in vitro.
  • EXAMPLE 7 Effect of NDV HUJ on breast cancer cell lines in vitro Viability of breast cancer cell lines was determined by the XTT assay as described in
  • NDV HUJ affected breast cancer cell viability in vitro.
  • EXAMPLE 8 Absence of effect of NDV HUJ on normal cells in vitro
  • PBMCs peripheral blood mononuclear cells
  • the cells were incubated in the presence of lOng/ml phorbol myristate acetate (PMA) and 200ng/ml ionomycin.
  • PMA phorbol myristate acetate
  • the cells were incubated with increasing MOI of NDV HUJ and cell viability was determined by XTT and Alamar Blue assays. As shown in FIG. 11B, PBMCs viability was unaffected by NDV HUJ.
  • EXAMPLE 9 Effect of NDV HUJ on tumor progression in an animal model Luciferase-transfected PC3 prostate cancer cells expressing high levels of the CxCr4 receptor were obtained from Prof. A. Peled of Hadassah University Hospital, Jerusalem, Israel. Nude athymic, male, 6 week old mice were injected subcutaneously on the rear right flank with 8xl0 6 luciferase-transfected PC3 prostate cancer cells suspended in PBS. Mice were observed until tumors appeared at the site of injection. When the size of some of the tumors reached 5mm in either direction, in vivo imaging of luciferase activity was carried out using a CCCD camera, and the tumor size was calculated on the basis of luciferase activity.
  • mice were assigned to three groups so that each group contained identical number of mice with tumors of similar sizes.
  • One group was treated once per week with an intra-peritoneal injection of lxlO 9 EID50 of NDV-HUJ and another was treated twice per week.
  • a third group received only PBS injections.
  • In vivo imaging of tumor size based on luciferase activity was carried out at biweekly intervals to follow tumor progression.
  • FIG. 12 shows the average size of tumors in the control group (hashed shading), once per week treated group (solid shading) and twice per week treated group (no shading) at 17, 32 and 46 days after the start of treatment.
  • Gd-MRI gadolinium enhanced magnetic resonance imaging
  • Hepatic, renal and bone marrow function requirements were: Hb > 9 g%; WBC > 1000/mm 3 ; platelet count > 30,000 mm 3 ; creatinine ⁇ 2.5 mg%; liver function tests less than twice normal.
  • Patients were not allowed to receive any investigational agents other than NDV-HUJ or any other anti- cancer agent during the study.
  • a history of allergy to egg ovalbumin was a cause for exclusion.
  • Other exclusion criteria included acute severe or life threatening infection, severe depression or psychosis or history of non-compliance to therapy.
  • a written informed consent document approved by the Hadassah Hospital Institutional Review Board had to be signed by the patients and by a spouse, parent or guardian.
  • NDV-HUJ Intravenous administration of NDV-HUJ
  • a thawed and sonicated NDV-HUJ suspension was diluted into 20 ml of saline in a burrette (Voluset, Teva Medical) immediately before use and administered over 15 minutes through a peripheral or central venous line.
  • the first part of the study utilized a modified Simons Type 4B intra-patient, single step, dose accelerated dosage titration scheme (Simon, R., et al., J. Natl. Cancer Inst, 89:
  • NCI CTC Cancer Institute Common Toxicity Criteria
  • NDV-HUJ efficacy was assessed by tumor response measured by gadolinium enhanced MRI. Patients were evaluated for response within two weeks following completion of the study dosing and again 10 weeks later. During maintenance dosing, patients were evaluated at 4 to 8 week intervals. Tumor response was determined from the change in the tumor's cross-section (cm ) and was scored as complete response (CR), partial response (PR), stable disease (SD) or progressive disease (PD) using standard criteria (McDonald, D. R., et al., J. Clin. Oncol. 8: 1277-1280, 1990). Clinical diagnosis of progressive disease was made by clinical/neurological deterioration in conjunction with increase in size of tumor determined by CT or MRI.
  • Plasma samples were collected at various time intervals, stored at -70°C and later evaluated for NDV-HUJ viral recovery. If cerebrospinal fluid (CSF) and biopsy samples were available these were also analyzed. Samples were screened for infectious virus by inoculation into the allantoic and yolk sacs of 10-11 day old embryonated eggs and checking the fluids for qualitative and quantitative hemagglutination (HA) 72-96 hrs post inoculation according to routine methods (Sever J.L., et al. J. Immunol. 80: 320-329, 1962). If a sample was found to be negative in qualitative HA test, samples were further passaged three times in order to enable viral multiplication and further evaluated for qualitative/quantitative HA.
  • CSF cerebrospinal fluid
  • HA hemagglutination
  • Dx diagnosis
  • GBM glioblastoma multiforme
  • I primary GBM
  • II secondary GBM
  • VT viral therapy
  • B biopsy
  • PR partial resection
  • GTR gross total resection
  • Rx treatment
  • RT radiotherapy
  • IAC intra-arterial carboplatin
  • TMZ temozolomide
  • R right; L, left.
  • patients #02, 05 and 06 in Part I completed all three cycles of the 55 BIU maximum dose step #6, while patient #01 was withdrawn in the middle of the third cycle of step #6 due to clinical and radiological disease progression.
  • patients #07, 09 and 11 completed all three cycles of dosage step #5 (11 BIU).
  • patients #07 continued with maintenance dosing until radiological disease progression. Additional treatments that the patients received following withdrawal from maintenance dosing are given in Table 5.
  • Time to progression (clinical and radiological) and overall survival are given in Table 5. Survival ranged from 3 to 66 weeks from the start of virotherapy. Time to progression ranged from 3 to 53 weeks.
  • Tumor Response All patients had obvious measurable disease (>2 cm 2 contrast enhanced tumor cross-section) at treatment initiation (Table 4). As seen in FIG. 14, one patient (#09) had stable disease at first follow-up, a partial remission at second follow-up and complete remission during maintenance dosing, at which time there was a significant improvement in neurological status and corticosteroid therapy was discontinued. The complete response did not prove durable (Table 5) and on routine imaging three months after complete disappearance of the tumor a lesion subsequently shown by histology to be GBM reappeared in the tumor bed. Three patients (#02, 05 and 06) had an increase in enhanced tumor cross-section without a deterioration of their neurological status. As part of their clinical management, their tumors were biopsied.
  • Inflammatory cells were found in the biopsied tumor of patients #02 and #05, but not in that of patient 06; however, it was not possible to determine whether the level of inflammatory cells was greater than those routinely found in GBM.
  • Another patient (#11) did not have a change in neurological status and did not have an increase in enhanced tumor cross-section, but was judged to have radiological disease progression based on increased enhancement within the resection cavity and changes in the FLAIR and rCBV MRI images. As seen in Table 2, the time to clinical progression in patients #05 and 11 occurred considerably later than their radiological progression. All patients in the study eventually developed both clinical and radiological progression (Table 6). Reliable CBV and MRS data were not available on most of the cases for technical reasons.
  • Virology A total of 101 blood, saliva and urine samples from five patients (#01, 02, 03, 05 and 09) were tested for infectious NDV by inoculation into the allantoic sac of embryonated eggs, a highly sensitive biological infectivity assay that indicated their presence or absence. All baseline samples were negative. In all five patients, infectious NDV particles were recovered from blood, urine and saliva samples taken during the first dosage cycle. Infectious NDV particles were recovered from blood, saliva and urine samples taken after the patients developed anti-hemagglutinin NDV antibodies. Infectious NDV was also recovered from 9 out of the 10 blood samples taken 9 days after the last dose of NDV-HUJ from the previous dosage cycle.
  • Infectious NDV particles were recovered from a tumor biopsy sample of patient #02, but not from the biopsy of patient #05. Tumor cystic fluid samples obtained via an implanted Ommaya device from patient #02 also tested positive for infectious NDV. Identity of the isolated virus was determined using anti-NDV antibodies.
  • the non-lytic HUJ strain is lentogenic and has a G-R-Q-G-R-L sequence.
  • lentogenic strains produce defective progeny, are monocyclic and cannot easily spread between tissues. This property significantly decreases and often completely eliminates the pathogenicity of the virus for its natural avian host and allows these strains to be used as poultry vaccines.
  • Non-lytic, lentogenic strains are considered to be safer with respect to their natural hosts than lytic, mesogenic strains, whose use and importation is banned in many countries.
  • the absence of pathogenicity of NDV HUJ for poultry reduces concerns related to the environmental impact of virus shedding.
  • NDV HUJ The dosing schedule of NDV HUJ used for glioblastoma patients was used in the treatment of other types of cancer.
  • the patient received three cycles of 6xl0 9 EID 50 NDV HUJ administered on five consecutive days every 14 days.

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Abstract

La présente invention concerne des méthodes destinées à induire la régression d'une tumeur chez un sujet, qui consistent à administrer audit sujet une composition pharmaceutique comprenant une souche lentogène du virus de la maladie de Newcastle (VMN). Une souche virale de VMN préférée pouvant induire la régression d'une tumeur chez un sujet est une souche clonale de VMN appelée HUJ. Les méthodes de l'invention sont particulièrement utiles pour induire la régression de tumeurs chez des patients insensibles aux traitements classiques contre le cancer.
PCT/IL2005/000518 2001-09-12 2005-05-19 Compositions de vmn et methodes d'utilisation de ces compositions pour le traitement du cancer Ceased WO2005113013A2 (fr)

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Cited By (4)

* Cited by examiner, † Cited by third party
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US7767200B2 (en) 2005-07-14 2010-08-03 Wellstat Biologics Corporation Cancer treatment using viruses, fluoropyrimidines and camptothecins
US8377450B2 (en) 2009-11-30 2013-02-19 United Cancer Research Institute Clone of Newcastle disease virus, its manufacture and its application in the medical treatment of cancer
US20130202558A1 (en) * 2012-02-01 2013-08-08 James C. Phillips Recombinant replication competent oncolytic viruses and methods of use thereof for the treatment of cancer
US9844574B2 (en) 2004-04-27 2017-12-19 Wellstat Biologics Corporation Cancer treatment using viruses and camptothecins

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US20030044384A1 (en) * 1997-10-09 2003-03-06 Pro-Virus, Inc. Treatment of neoplasms with viruses

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9844574B2 (en) 2004-04-27 2017-12-19 Wellstat Biologics Corporation Cancer treatment using viruses and camptothecins
US7767200B2 (en) 2005-07-14 2010-08-03 Wellstat Biologics Corporation Cancer treatment using viruses, fluoropyrimidines and camptothecins
US8377450B2 (en) 2009-11-30 2013-02-19 United Cancer Research Institute Clone of Newcastle disease virus, its manufacture and its application in the medical treatment of cancer
US20130202558A1 (en) * 2012-02-01 2013-08-08 James C. Phillips Recombinant replication competent oncolytic viruses and methods of use thereof for the treatment of cancer

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