WO2001022970A1 - Therapie a base de 2-5a et d'interferon - Google Patents

Therapie a base de 2-5a et d'interferon Download PDF

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WO2001022970A1
WO2001022970A1 PCT/US2000/041038 US0041038W WO0122970A1 WO 2001022970 A1 WO2001022970 A1 WO 2001022970A1 US 0041038 W US0041038 W US 0041038W WO 0122970 A1 WO0122970 A1 WO 0122970A1
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ifn
analog
biostable
subject
cells
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PCT/US2000/041038
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WO2001022970A9 (fr
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Robert H. Silverman
Lorraine Rusch
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The Cleveland Clinic Foundation
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Priority to EP00977293A priority patent/EP1229920A1/fr
Priority to AU14948/01A priority patent/AU1494801A/en
Priority to CA002388025A priority patent/CA2388025A1/fr
Publication of WO2001022970A1 publication Critical patent/WO2001022970A1/fr
Publication of WO2001022970A9 publication Critical patent/WO2001022970A9/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
    • A61K31/7048—Compounds having saccharide radicals and heterocyclic rings having oxygen as a ring hetero atom, e.g. leucoglucosan, hesperidin, erythromycin, nystatin, digitoxin or digoxin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/7125—Nucleic acids or oligonucleotides having modified internucleoside linkage, i.e. other than 3'-5' phosphodiesters
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/21—Interferons [IFN]
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/543—Lipids, e.g. triglycerides; Polyamines, e.g. spermine or spermidine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • IFNs interferons
  • IFNs interferons
  • cytokines that are responsible for providing vertebrates with innate immunity against a wide-range of viruses and other microbial pathogens (Stark, et al., Annual Review of Biochemistry, 67, 227-264, 1998)).
  • IFNs regulate cell proliferation, apoptosis and the immune system, properties that underlie their uses in treatment of cancer. IFNs alter patterns of gene expression in cells by binding to specific cell surface receptors and activating JAK/STAT pathways.
  • Type I IFN consists of the IFN - ⁇ , - ⁇ , and - ⁇ , all of which are structurally related and encoded in a gene cluster on the short arm of human chromosome 9 (Knight E. (1976), IFN: purification and initial characterization from human diploid cells, Pore. Natl. Acad. Sci. USA, 73:520-523).
  • the Type I IFN genes are differentially regulated. All nucleated mammalian cells produce type I IFNs in response to viral infections.
  • Type II IFN consists solely of IFN- ⁇ , which is produced by T-cells and natural killer cells and has no structural similarity to type I IFNs (Young H., Hardy K. (1990), IFN- ⁇ producer cells, activation stimuli and molecular genetic regulation, Pharmacol. Ther., 45:137-151).
  • the human IFN ⁇ gene is located on chromosome 12.
  • IFNs have been used in the clinic against a wide range of human malignancies.
  • IFN- ⁇ 2 as a single agent has been used to treat hairy cell leukemia, Kaposi's sarcoma, chronic myelogenous leukemia (CML) , B-cell and T-cell lymphomas, melanomas, myelomas, and renal cell carcinoma.
  • IFN- ⁇ 2 has been used in combination with doxorubicin for treating follicular lymphoma.
  • IFN- ⁇ 2 has also been used in combination with 1- ⁇ -D arabinofuanosylctyosine to improve the cytogenic response in CML.
  • IFN ⁇ has been used to treat hepatocellular carcinoma. IFNs have also been used to treat various viral diseases including chronic active hepatitis and recurring papillomas. While current IFN therapies have proved beneficial, their efficacy is somewhat limited, in part due to significant side-effects. Therefore, there is a need for additional anti-cancer and antiviral therapies, particularly for therapies which enhance the efficacy of IFNs.
  • the present invention provides new methods of treating malignancies in a subject, particularly a human subject. Such methods comprise administering 2', 5' oligoadenylate (2-5A) or a biostable analog of 2-5 A to the subject.
  • IFN is also administered to the subject.
  • Interferon and 2-5 A or the biostable analog thereof are administered concurrently or sequentially.
  • Interferon and 2-5 A or biostable analog thereof are administered separately in different pharmaceutical compositions or together in a single pharmaceutical composition.
  • Interferon and 2-5A or the biostable analog thereof are administered in therapeutically effective amounts.
  • cancers which may be treated according to the present method include, but are not limited to, hairy cell leukemia, Kaposi's sarcoma, CML, B-cell and T-cell lymphomas, melanomas, myelomas, renal cell carcinoma, ovarian, breast, bronchogenic, bladder, and gastrointestinal carcinomas, acute leukemias, malignant glioma and fibrosarcoma.
  • the present invention also relates to new methods of treating viral diseases in a subject, particularly a human subject.
  • Such methods comprise administering interferon and 2', 5' oligoadenylate (2-5A) or a biostable analog of 2-5A to a subject.
  • Interferon and 2-5A or the biostable analog thereof are administered concurrently or sequentially.
  • Interferon and the 2-5A or the biostable analog thereof are administered in amounts sufficient to reduce the pathological effects of a given virus in the subject.
  • viral diseases which may be treated according to this method include, but are not limited to, hepatitis C, hepatitis B, and viral infections caused by human papilloma virus or a picornavirus.
  • the present invention also relates to a method of selectively inducing programmed cell death (apoptosis) in cancer cells either in vitro or in vivo.
  • the method comprises administering 2-5A or a biostable analog thereof to the cancer cells.
  • IFN is also administered to the cell.
  • FIGURES Figure 1 is a schematic of the IFN-regulated system.
  • Figure 2 shows the structure of the trimeric species of 2-5 A and the structure of the 2-5 A analog, sp(A2'p) 3 5'A2'p3'dC (2-5 AdC).
  • Figure 3 shows the effect of IFN and 2-5 A therapy on levels of apoptosis in ovarian carcinoma cells (HEY IB) and normal ovarian epithelial cells (NOE) as determined by TUNEL assays.
  • HEY IB and NOE cells were plated (2xl0 5 cells/plate) in six-well tissue culture plates and incubated in the absence or presence of IFN (2,000 U/ml) overnight prior to transfection with either A2'p5'A or 2-5A (6 ⁇ M) in serum-free Opti-MEM for 3.5h. Samples were diluted in growth media 24h for TUNEL analysis.
  • Figure 4 shows the kinetics of apoptosis induced in HEY1B cells by the combination of IFN and 2-5A as determined by Annexin V binding assays.
  • HEY IB cells were plated (2xl0 5 cells/plate) in six-well tissue culture plates and incubated in the absence or presence of EFN (2,000 U/ml) overnight prior to transfection with either A2'p5'A or 2-5A (6 ⁇ M) in serum-free Opti-MEM for 3.5h and then placed in growth media. Cells were harvested at various times for Annexin V binding assays by FACS analysis.
  • Figure 5 shows the effect of 2-5A treatment on the death of human malignant glioma
  • U373 cells as determined by trypan blue exclusion assay. U373 cells were transfected with the trimeric species of 2-5 A, ppp5'A2'p5'A2'p5'A, which was complexed with lipofectamine PLUS. Cell death was determined after incubation of the cells for 16 h by trypan blue exclusion assay and counting live and dead cells under a microscope with a hemocytometer.
  • Figure 6 shows the effect of 2-5A and IFN treatment on the viability of ovarian carcinoma cells (HEY IB) as determined using Cell Titer 96 Aqueous Non-Radioactive Cell Proliferation Assay. Data displayed is the mean and standard deviation of triplicate analysis for 24h (A), 48h (B), and 72h (C).
  • Figure 7 shows the effect of treatment with IFN and the 2-5A analog, 2-5 AdC on the viability of ovarian carcinoma cells, as determined by trypan blue dye exclusion assay. HEY1B cells were incubated overnight the absence or presence of 2,000 units per ml of IFN- ⁇ 2a.
  • Figure 8 shows the effect of IFN and 2-5A therapy on the growth of human ovarian carcinoma in nude (athymic) mice.
  • HEY IB human ovarian carcinoma cells 3 x 10 6 /site, were injected into groups of 4 nude mice. When tumors reached volumes of 100 mm 3 , they were directly injected daily with 50 ⁇ l of 5 ⁇ M 2-5 A. On day 6, the 2-5 A treated tumors were injected with 1,000 U of IFN- ⁇ 2a.
  • the present invention provides methods for treating malignancies and viral diseases in a subject, particularly a human subject.
  • the methods comprise administering a small molecule known as 2-5 A to the subject.
  • interferon is also administered to the subject.
  • Such methods are particularly useful for treating ovarian carcinoma, malignant gliomas, and breast cancer.
  • a combination of IFN and 2-5A is highly effective in selectively inducing programmed cell death (apoptosis) of human ovarian cancer cells (HEY IB) without affecting the viability of normal ovarian epithelial cells.
  • a combination of IFN and 2-5A was also highly effective in selectively inducing apoptosis of human malignant glioma U373 cells.
  • the molecular mechanism of cell death induced by IFN and 2-5 A was investigated. Interferon treatment of the HEY IB cells led to enhanced levels of cytochrome C.
  • pan-caspase inhibitor BD-f k and the caspase 3 inhibitor DEVD-fmk partially suppressed the IFN and 2-5 A apoptotic response in human malignant glioma U373 cells.
  • interferon refers to the type I interferons which include IFN- ⁇ , with all of its subtypes, IFN- ⁇ , IFN- ⁇ and IFN- ⁇ , as well as the Type II interferon, IFN- ⁇ .
  • interferon also refers to IFN con, which is a consensus type I interferon produced by Amgen, and chemically-modified interferons such as pegylated interferon.
  • the interferon is a type I interferon, IFN con, or a chemically-modified version thereof.
  • 2-5 A is a series of short, heat-stable oligoadenylates with unusual 2', 5' phosphodiester linkages in contrast to the typical 3'-5' linkage that is found RNA and DNA chains (Kerr I. M., Brown R.E. (1978), pppA2'p5'A2'p5'A: an inhibitor of protein synthesis synthesized with an enzymes fraction from IFN-treated cells, Proc. Natl. Acad. Sci. USA, 75:256-260).
  • 2-5 A is a mixture of oligomeric species. In other embodiments, 2-5A is a single species, e.g., the trimeric, tetrameric, or pentameric form of the oligoadenylate or an analog thereof. (See Figure 2)
  • Interferon and 2-5A from whatever source derived, including without limitation from recombinant and non-recombinant sources, may be administered separately in individual pharmaceutical compositions or together as a single pharmaceutical composition.
  • Such composition or compositions may further comprise a pharmaceutically acceptable carrier.
  • Such pharmaceutical composition may also contain diluents, fillers, salts, buffers, stabilizers, solubilizers, antioxidants and other additives well known in the art.
  • pharmaceutically acceptable means a non-toxic material that does not interfere with the effectiveness of the activity of the interferon or the 2-5 A. The characteristics of the carrier will depend on the route of administration.
  • the pharmaceutical composition may further contain other agents which either enhance the activity of interferon or 2-5 A or complement its activity or use in treating the viral disease or malignancy. Such additional factors and/or agents may be included in the pharmaceutical composition to produce a synergistic effect with the interferon or 2-5A, or to minimize side effects.
  • the pharmaceutical compositions of the invention may be in the form of a liposome in which 2-5A is combined, in addition to other pharmaceutically acceptable carriers, with amphipathic agents such as lipids which exist in aggregated form as micelles, insoluble monolayers, liquid crystals, or lamellar layers in aqueous solution.
  • Suitable lipids for liposomal formulation include, without limitation, monoglycerides, diglycerides, sulfatides, lysolecithin, phospholipids, saponin, bile acids, and the like. Preparation of such liposomal formulations is within the level of skill in the art.
  • the 2-5 A or analog is formulated with a cationic lipid carrier, such as lipofectamine or a cationic liposome.
  • the cationic lipid carrier may further comprise a molecule which targets the resulting complex of carrier and 2-5A to a particular cell type, such as for example a tumor cell. Examples of such targeting molecules are folate, transferrin and antibodies or fragments thereof.
  • Interferon and 2-5 A are administered to a host subject in a therapeutically effective amount.
  • therapeutically effective amount means the total amount of interferon and that is sufficient to show a meaningful benefit, i.e., treatment, healing, or amelioration of the relevant malignancy or virally-induced disease, or an increase in rate of treatment, healing, or amelioration of such malignancy or disease.
  • the amount of interferon or 2-5A in the pharmaceutical composition will depend upon the nature and severity of the condition being treated, and on the nature of prior treatments which the subject has undergone and the type of cancer or virus being targeted. Ultimately, the attending physician will decide the amount of 2-5A and interferon with which to treat each individual subject. Initially, the attending physician will administer low doses of 2-5A and interferon and observe the subject's response. Larger doses of 2-5A and interferon may be administered until the optimal therapeutic effect is obtained for the patient, and at that point the dosage is not increased further.
  • compositions used to practice the method of the present invention should contain from about 0.1 million to about 10 million units per dose of interferon and from about 1 mg to about 1 gm per dose of 2- 5A or the analog thereof. Although a single dose of interferon and 2-5A may be sufficient to ameliorate the pathological effects of the virus or the malignancy, it is expected that multiple doses of interferon and the will be prefened.
  • compositions comprising interferon, or 2-5 A, or both may be carried out in a variety of conventional ways, such as cutaneous, subcutaneous, intramuscular, intraperitoneal, parenteral or intravenous injection.
  • the interferon pharmaceutical composition is administered first and then the 2-5A pharmaceutical composition is administered thereafter.
  • the 2-5A composition is administered first and the interferon containing composition is administered thereafter.
  • the second composition is administered from several hours to about 3 day post treatment with the first composition.
  • both the interferon and the 2-5 A or 2-5A analog are administered simultaneously. Both agents may be administered simultaneously by continuous intravenous infusion.
  • the interferon may be administered by subcutaneous or intramusclar injection while 2-5A or 2-5A analog is administered at the same time by continuous infusion.
  • the duration of therapy using the pharmaceutical composition of the present invention will vary, depending on the severity of the disease being treated and the condition and response of each individual patient. Ultimately the attending physician will decide on the appropriate duration of therapy using the pharmaceutical composition of the present invention.
  • 2-5A was prepared using synthetase isolated from extracts of IFN-treated HeLa cells on an activating affinity matrix of poly(I):polyC-agrarose (AGPoly(I):poly(C)TM-type 6;
  • IFN ⁇ 2a (Roferon, Roche) for 16h. Cells were washed in PBS and harvested by scraping. A post-mitochondrial fraction was prepared by lysing the cells in 7 ml of buffer A (0.5% NP-40; 90 mM KC1; 1 mM Mg acetate, 10 mM Hepes, pH 7.6, lO ⁇ g/ml leupeptin and 2 mM 2-mercaptoethanol), vortexing briefly, and centrifuging at 10,000 g for 10 min. The supernatant containing the synthetase in crude extract was removed.
  • buffer A (0.5% NP-40; 90 mM KC1; 1 mM Mg acetate, 10 mM Hepes, pH 7.6, lO ⁇ g/ml leupeptin and 2 mM 2-mercaptoethanol
  • Poly(I):poly(C)-agarose [1.2 ml suspension containing 6 mg of poly(I):poly(C)] was washed twice by centrifuging at 1,500 g for 5 min, discarding supernatant, and resuspending in 50 ml of buffer B (10 mM Hepes, pH 7.5; 50 mM KC1; 1.5 mM MgAC; 7 mM mercaptoethanol and 20%) v/v glycerol).
  • the poly (I):poly(C)Sepharose was resuspended in the post-mitochondrial fraction of Hela cells and incubated for 1 h at 4°C on a rotating shaker at a low speed.
  • the complex of synthetase bound to the resin was centrifuged at 1,500 g for 5 min and the supernatant discarded. The pellet was washed three times in 15 ml of buffer B by centrifuging at 1,500 g for 5 min, discarding supernatant and resuspending. The final pellet was resuspended in Buffer B supplemented with 4 mM ATP adjusted to pH 7 (Sigma). The first round of synthesis was performed by incubation for 20h at 37°C with gentle, continuous shaking. The resin complex was separated from the crude 2-5 A preparation by centrifuging and removing the supernatant containing the 2-5 A. A second round of synthesis was performed by resuspending in fresh buffer B with 4 mM ATP and incubating an additional 37°C for 72h. The 2-5A was stored at -70°C.
  • 2-5A was analyzed on a Beckman HPLC system using System Gold integration parameters. Crude extract was analyzed on a reverse phase HPLC column (C-18, 60A, 5 ⁇ Princeton Sphere) in a 50% methanol and ammonium phosphate (pH 7.2) mobile phase system ramping from zero to 25% methanol over 60 min. The crude preparation was compared to known standards of ATP (Sigma), dimeric (A2'p5'A, Sigma) and trimer (p 3 (A2'p) 2 A) forms for identification. Over 60%) of the material consisted of the trimeric and tetramer forms of 2-5A while the remaining fraction consisted of ATP, dimeric, pentamer and hexamer. Unless otherwise stated, "2-5 A" as used in this example refers to this mixture of 2-5 A oligomers.
  • Human HEY IB cells were cultured in RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum, 100 units/ml penicillin, 100 ⁇ g/ml streptomycin/penicillin.
  • NOE Normal ovarian epithelial cells
  • the combined mixture was incubated for an additional 15 min and diluted up to the appropriate volume in Opti- MEM.
  • 1 ml of the transfection material was placed on individual wells of a six-well tissue culture plate seeded with 2 xlO 5 cells, some of which had been pre-treated overnight with 2,000 U/ml IFN (Roferon ⁇ 2a).
  • the transfection material was incubated on cells at 37° C for 3.5h and an equal volume of DMEM media supplemented with 10%> FBS and 1% penicillin/streptomycin was added for the noted period of time.
  • Cells were transfected with 6 ⁇ M of a natural mixture of 2-5 A and apoptosis was monitored by FACS TUNEL assays for DNA breakage.
  • Cells were harvested at 24 h for TUNEL analysis by removing the transfection material and media to a 15-ml tube and rinsing the cells once with PBS. Cells were trypsinized and transferred to the same tube. The tissue culture wells were washed again and the wash was also added to the same tube. This material was centrifuged at 2,500 x g for 10 min, washed with PBS and centrifuged again. The cell pellet was resuspended in 1%> paraformaldehyde prepared in PBS and incubated on ice for 1 min. The cells were centrifuged and washed prior to suspension in 70% ethanol. Samples were stored at -20°C.
  • the APO-BRDUTM kit (Pharmingen, Becton Dickinson, San Jose, CA) was used to perform TUNEL analysis.
  • the kit uses a two color staining method, one for labeling DNA breaks and another for labeling total cellular DNA.
  • the enzyme, terminal deoxynucleotidyl transferase (TdT) catalyzes a template independent addition of brominated deoxyuridylate monophosphate (Br-dUMP) to the 3'-hydroxyl (-OH) ends of double- and single-stranded DNA. After Br-dUTP incorporation, sites are identified by an FITC-labeled anti-BrdU monoclonal antibody.
  • Non- clumped cells are gated and separate boxes are drawn around cells that stain positive (upper box) and negative (lower box) with the FITC-BRDU mAb. Double-stained cells in the upper right quadrant determine the percent of apoptotic cells.
  • Annexin V binds to PS present on the cell surface and can be monitored using FITC- labeled annexin V with FACS analysis. Results were consistent with the TU ⁇ EL assays showing induction of annexin V binding by 16 hrs after treatment with 2-5 A. The levels of annexin V positive cells was greatly increased by pretreating the HEYIB cells with IF ⁇ - ⁇ 2 prior to transfections. There were 28.7% annexin V positive, apoptotic cells after 24 hr of 2-5A treatment whereas the combination of IFN and 2-5 A caused 71.2% apoptosis. These results are in close agreement with the TUNEL assays. Therefore, IFN plus 2-5A induced HEYIB cells to undergo apoptosis, apparently in a highly selective manner.
  • cytochrome C is an IFN induced protein using western blots. Cytochrome C levels increased after HEYIB cells were treated with 100 or 1,000 units per ml of IFN- ⁇ 2a. Interestingly, the transfection of into HEYIB cells induced the rapid release of cytochrome C from the mitochondria into the cytoplasm. A sharp decrease in mitochondrial cytochrome C levels was observed within the first hour of 2-5A treatment. The molecular events by which leads to cytochrome C release from mitochondria is unknown. However, we have found RNase L in a mitochondrial fraction (P10).
  • the CellTiter 96® AQueous Non-Radioactive Cell Proliferation Assay is a colorimetric method for determining the number of viable cells in proliferation or chemosensitivity assays. It is composed of solutions of a novel tetrazolium compound (3-(4,5dimethylthiazol-2-yl)-5- ⁇ -carboxymethoxyphenyl)-2-(4- sulfophenyl)-2H-tetrazolium, inner salt; MTS) and an electron coupling reagent (phenazine methosulfate; PMS).
  • a novel tetrazolium compound 3-(4,5dimethylthiazol-2-yl)-5- ⁇ -carboxymethoxyphenyl)-2-(4- sulfophenyl)-2H-tetrazolium, inner salt; MTS
  • an electron coupling reagent phenazine methosulfate
  • MTS (Owen's reagent) is bioreduced by cells into a formazan that is soluble in tissue culture medium.
  • the absorbance of the formazan at 490 nm can be measured directly from 96 well assay plates without additional processing.
  • the conversion of MTS into the aqueous soluble formazan is accomplished by dehydrogenase enzymes found in metabolically active cells.
  • the quantity of formazan product as measured by the amount of 490 nm absorbance is directly proportional to the number of living cells in culture.
  • 50 ⁇ l of the MTS reagent (30% solution in PBS) was added to each well of the 96 well plate and incubated for 2h prior to monitoring the absorbance at 490 nn.
  • IFN- ⁇ treatments also produced a dose-dependent anti-cellular effect. After four days, cell viability was reduced by 27, 54, and 85% with 20, 100 and 1000 units per ml of IFN- ⁇ , respectively. The combined treatments were particularly effective in decreasing cell viability. For instance, after 48 h of treatment with 2-5 A (1 mM), IFN-b (1,000 units per ml), or the combination of 2-5A and IFN- ⁇ , cell viability was reduced by 60%, 78% and 96%, respectively. The enhanced anti-cellular effect of combining 2- 5A and IFN- ⁇ treatments was further explored by monitoring apoptosis.
  • EXAMPLE 3 Treatment of Human Glioblastoma Cells with 2-5 A in the Absence and Presence of IFN.
  • a mixture of 2-5A was prepared as described above in example 1 and used in combination with IFN ⁇ to treat human glioblastoma cells.
  • the glioblastoma cell line, U373, was derived from a patient diagnosed with fatal and invasive brain cancer. These cells can induce tumors when injected in mice.
  • RNase L Prior to treatment, the 2-5A was assayed with purified, recombinant RNase L fusion protein.
  • RNase L One of the most convenient sources of RNase L for functional analysis of 2-5 A involves the E. coli expression and purification of a glutathione-S-transferase (GST) fusion protein.
  • GST glutathione-S-transferase
  • the cDNAs for RNase L in plasmid pGEX4-T-3 is transformed into E.
  • the harvested cell pellets are washed with PBS, resuspended in 5 volumes of PBS-C (PBS with 10% v/v of glycerol, 1 mM EDTA, 0.1 mM ATP, 5mM MgCt ⁇ , 14 mM 2-mercaptoethanol, 1 ⁇ g per ml of leupeptin and 1 mM PMSF) supplemented with 1 ⁇ g per ml lysozyme, and incubated at room temperature for 20 min.
  • PBS-C PBS with 10% v/v of glycerol, 1 mM EDTA, 0.1 mM ATP, 5mM MgCt ⁇ , 14 mM 2-mercaptoethanol, 1 ⁇ g per ml of leupeptin and 1 mM PMSF
  • the suspended cells are lysed by sonicating on ice for 20 sec four times, Triton-X 100 is added to a final concentration of 1% v/v and the cell lysates are incubated at room temperature for 20 min. The supernatants are collected after centrifugation at 16,700 g for 20 min at 4°C. Purification of fusion proteins is performed as described by the manufacturer of the glutathione-sepharose 4B (Pharmacia Biotech) with modifications. Briefly, glutathione-sepharose 4B (200 ⁇ l of a 50%> slurry in PBS-C) is added to extract from 200 ml cultures of bacteria at room temperature for 20 min with shaking.
  • the fusion proteins are eluted with 20 mM of glutathione in 50 mM Tris-HCl, pH 8.0 containing 1 ⁇ g per ml leupeptin, with shaking at room temperature for 20 min.
  • Expression and purity of the protein preparations are determined by SDS/PAGE and coomassie blue staining and by western blots with monoclonal antibody to RNase L.
  • oligouridylic acid U25
  • [5'- 32 P]-pCp 3,000 Ci/mmole) (Du Pont NEN) with T4 RNA ligase
  • the cells are stained simultaneously with fluoroscein isothocynate (FITC)-Annexin V [green fluorescence] and propidium iodide (PI) (red fluorescence) for 15 min at room temperature in the dark as described [Pharmingen-Becton Dickinson, San Diego].
  • FITC fluoroscein isothocynate
  • PI propidium iodide
  • Cells are gently resuspended, bivariate flow cytometry using a FACScan is performed and the data analyzed with CellQuest software (Becton Dickinson, San Jose, CA).
  • the results show that apoptosis increased as a function of time. There was 41 %> and 56% apoptosis after 16 h in the presence of 2-5 A alone or 2-5A plus IFN, respectively. Dimer failed to increase apoptosis over background levels in the presence or absence of IFN. Similarly, IFN by itself did not cause apopto
  • EXAMPLE 4 Treatment of HEYIB cells with a spA2'p5'A2'p5'A2'p3'dC (2-5AdCl and UN.
  • dC SpA2'p5'A2'p5'A2'p3'dC
  • Fig. 7 The compound dC is stablized at both termini against enzymes that can degrade. Specifically, the 5'-terminus is protected against phosphatase activity by a thiophosphate group. The 2',3 '-terminus is protected against 3' to 5' phosphodiesterase activity linking an inverted deoxycytosine in 2' to 3' linkage to the final (third)
  • Tetrahydrofuran (TEAA) was added (1ml) to the preparation and the THF was evaporated on the speedvac.
  • dc was purified by RP-HPLC using a RLP-1 column (Hamilton). Effluent was evaporated to dryness and dissolved in deionized water and desalted on Sep-Pak cartridges
  • HEYIB ovarian cancer cells were incubated overnight in the absence or presence of IFN- ⁇ 2a and then transfected with 2-5 AdC (Fig. 7).
  • 3 and 6 ⁇ M 2-5 AdC caused 12.6+6.1% and 44.6+9.1%) cell death, respectively.
  • 3 and 6 ⁇ M 2-5AdC caused 20.5+7%> and 92.8+0.07% cell death. Therefore, dC has an enhanced cell killing effect on the HEYIB tumor cells.
  • TBAF tetrabutylammonium fluoride
  • THF tetrahydrofuran
  • HHYIB Human ovarian cancer cells
  • Fig. 8 Human ovarian cancer cells
  • rum reached volumes of about 100 mm 3
  • four mice had their tumors directly injected with 50 ⁇ l of 5 ⁇ M 2-5A complexed with lipofectamine (time Day 0, 3 and 5 in the Figure).
  • Control mice were injected with lipofectamine alone at the same times.
  • the treated animals were injected in opposite flanks with 1,000 U of IFN- 2A, continuing with alternate IFN and 2-5A treatments (in the same amounts) on every other day.
  • the human adenocarcinoma cell line MCF-7 was obtained from a pleural effusion from the mammary gland of a female patient. The growth of this cell line is inhibited by tumor necrosis factor alpha. An aggressive epithelial carcinoma, MCF-7 cells showed effective killing by 2-5 A alone, or with 2-5 A and a low concentration of IFN (20U). High concentrations of IFN were effective inhibitors of MCF-7 cell growth , even in the absence of 2-5A. Low concentration of 2-5A decreased viability by 50% (4 ⁇ M in the presence or absence of 20 U/ml IFN). These results demonstrate that the MCF-7 cells were sensitive to the antiproliferative/apoptotic effects of 2-5 A and IFN.
  • EXAMPLE 8 Treatment of Bladder Carcinoma Cells with 2-5 A and IFN.
  • T24 cells are transitional bladder carcinoma cells which responded to 2-5 A treatment at higher IC-50 concentrations of 2-5 A (32 ⁇ M). The cell viability decreased further with IFN pre-treatment and the IC-50 concentrations of 2-5A were decreased to 30, 20 and 18 ⁇ M for 20, 100, and 1,000 U/ml IFN, respectively.
  • RT4 cells are transitional bladder epithelial papillomas, a much less invasive form of cancer. These cells resisted treatment to both and IFN.
  • the IC-50 concentrations remained greater than 36 ⁇ M except for 1,000 U/ml IFN, where the IC-50 concentration of 2-5 A decreased to 12 ⁇ M.
  • the enhanced anti-cellular effect of 2-5 A and JEN in the more aggressive T24 transitional carcinoma cells as compared with RT4 papilloma cells suggests a preferred therapeutic window for treating bladder cancer.
  • Fibroblasts HT 1080 and WI-38 cells were transfected with (0-2 ⁇ M) in the presence or absence of IFN in order to compare the ability of RNase L to decrease cell viability through apoptosis.
  • the HT1080 fibrosarcoma cells were very sensitive to the effects of 2-5 A activation of RNaseL and apoptosis induction.
  • HT 1080 fibrosarcoma cells had an IC-50 concentration of only 0.25 ⁇ M after 72h of treatment of 2-5A alone and was decreased by the addition of IFN pretreatment.
  • the normal lung fibroblast primary cells WI-38 required almost 1 ⁇ M of 2-5 A (72h) in the absence of JEN.

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Abstract

L'invention concerne des procédés relatifs au traitement de tumeurs malignes, qui consistent à administrer du 2', 5' oligoadénylate (2-5A) ou un analogue biostable de 2-5A au patient. Eventuellement, on administre également de l'interféron (IFN). Il est possible de traiter par exemple les cancers suivants (liste non exhaustive) : leucémie à cellules 'chevelues', sarcome de Kaposi, leucémie myéloïde chronique, lymphomes B et T, mélanomes, myélomes, hypernéphromes, cancer des ovaires, du sein, des bronches,de la vessie, carcinomes gastrointestinaux, leucémie aiguë, gliomes malins et fibrosarcomes. L'invention concerne également des procédés relatifs au traitement de maladies virales, qui consistent à administrer à la fois de l'IFN et du 2', 5' oligoadénylate (2-5A) ou un analogue biostable de 2-5A au patient. Il est possible de traiter par exemple les maladies virales suivantes (liste non exhaustive) : hépatite C, hépatite B, et infections virales induites par un papillomavirus ou un picornavirus humain. L'invention concerne en outre un procédé permettant d'induire de manière sélective la mort cellulaire programmée (apoptose) dans les cellules cancéreuses, in vitro ou in vivo, en administrant à ces cellules du 2-5A ou un analogue biostable.
PCT/US2000/041038 1999-09-29 2000-09-29 Therapie a base de 2-5a et d'interferon WO2001022970A1 (fr)

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JP2001526181A JP2003510281A (ja) 1999-09-29 2000-09-29 ２−５ａおよびインターフェロンを用いる治療
EP00977293A EP1229920A1 (fr) 1999-09-29 2000-09-29 Therapie a base de 2-5a et d'interferon
AU14948/01A AU1494801A (en) 1999-09-29 2000-09-29 Therapy with 2-5a and interferon
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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002060455A1 (fr) * 2000-10-24 2002-08-08 Dana-Farber Cancer Institute, Inc. Procedes et compositions utilisant des 2',5'-oligoadenylates pour le traitement de troubles de la proliferation cellulaire
WO2004046161A1 (fr) * 2002-11-19 2004-06-03 Sankyo Company, Limited Nouveaux analogues d'acide 2',5'-oligoadenylique
JP2004182725A (ja) * 2002-11-19 2004-07-02 Sankyo Co Ltd 新規２’，５’−オリゴアデニル酸類縁体
WO2005105136A1 (fr) * 2004-04-27 2005-11-10 University Of South Florida Therapie nanogenique pour troubles causes par la proliferation cellulaire
US7354908B2 (en) 2002-04-30 2008-04-08 University Of South Florida Materials and methods for prevention and treatment of RNA viral diseases
US7595303B1 (en) 2002-09-05 2009-09-29 University Of South Florida Genetic adjuvants for immunotherapy

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20050084967A1 (en) 2002-06-28 2005-04-21 Xcyte Therapies, Inc. Compositions and methods for eliminating undesired subpopulations of T cells in patients with immunological defects related to autoimmunity and organ or hematopoietic stem cell transplantation

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US5550111A (en) * 1984-07-11 1996-08-27 Temple University-Of The Commonwealth System Of Higher Education Dual action 2',5'-oligoadenylate antiviral derivatives and uses thereof
US5908621A (en) * 1995-11-02 1999-06-01 Schering Corporation Polyethylene glycol modified interferon therapy
US5962431A (en) * 1993-09-24 1999-10-05 Budowsky; Edward I. Method of treating papillomavirus using 2',5'-oligoadenylate

Patent Citations (3)

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Publication number Priority date Publication date Assignee Title
US5550111A (en) * 1984-07-11 1996-08-27 Temple University-Of The Commonwealth System Of Higher Education Dual action 2',5'-oligoadenylate antiviral derivatives and uses thereof
US5962431A (en) * 1993-09-24 1999-10-05 Budowsky; Edward I. Method of treating papillomavirus using 2',5'-oligoadenylate
US5908621A (en) * 1995-11-02 1999-06-01 Schering Corporation Polyethylene glycol modified interferon therapy

Cited By (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002060455A1 (fr) * 2000-10-24 2002-08-08 Dana-Farber Cancer Institute, Inc. Procedes et compositions utilisant des 2',5'-oligoadenylates pour le traitement de troubles de la proliferation cellulaire
US7354908B2 (en) 2002-04-30 2008-04-08 University Of South Florida Materials and methods for prevention and treatment of RNA viral diseases
US8293717B2 (en) 2002-04-30 2012-10-23 University Of South Florida Materials and methods for prevention and treatment of RNA viral diseases
US8802647B2 (en) 2002-04-30 2014-08-12 University Of South Florida Materials and methods for prevention and treatment of RNA viral diseases
US7595303B1 (en) 2002-09-05 2009-09-29 University Of South Florida Genetic adjuvants for immunotherapy
US8603458B2 (en) 2002-09-05 2013-12-10 University Of South Florida Genetic adjuvants for immunotherapy
WO2004046161A1 (fr) * 2002-11-19 2004-06-03 Sankyo Company, Limited Nouveaux analogues d'acide 2',5'-oligoadenylique
JP2004182725A (ja) * 2002-11-19 2004-07-02 Sankyo Co Ltd 新規２’，５’−オリゴアデニル酸類縁体
US7651999B2 (en) 2002-11-19 2010-01-26 Sankyo Company, Limited 2′, 5′-oligoadenylate analogs
US7994152B2 (en) 2002-11-19 2011-08-09 Sankyo Company, Limited Method of treating a cancer by administering A 2′,5′-oligoadenylate analog
KR101117998B1 (ko) 2002-11-19 2012-04-12 상꾜 가부시키가이샤 신규 2', 5'-올리고아데닐산 유사체
WO2005105136A1 (fr) * 2004-04-27 2005-11-10 University Of South Florida Therapie nanogenique pour troubles causes par la proliferation cellulaire

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