WO2004070008A2 - Techniques et compositions d'utilisation de suramine, de polysulfate pentosane, d'antisens de telomerase et d'inhibiteurs de la telomerase - Google Patents

Techniques et compositions d'utilisation de suramine, de polysulfate pentosane, d'antisens de telomerase et d'inhibiteurs de la telomerase Download PDF

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WO2004070008A2
WO2004070008A2 PCT/US2004/002609 US2004002609W WO2004070008A2 WO 2004070008 A2 WO2004070008 A2 WO 2004070008A2 US 2004002609 W US2004002609 W US 2004002609W WO 2004070008 A2 WO2004070008 A2 WO 2004070008A2
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telomerase
suramin
tumor
cancer
patient
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PCT/US2004/002609
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WO2004070008A3 (fr
WO2004070008A8 (fr
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Jessie L.-S. Au
M. Guillaume Wientjes
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Au Jessie L-S
Wientjes Guillaume M
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Priority to EP04706977A priority Critical patent/EP1606302A4/fr
Priority to AU2004209428A priority patent/AU2004209428A1/en
Priority to JP2006503176A priority patent/JP2007525414A/ja
Priority to CA002515000A priority patent/CA2515000A1/fr
Publication of WO2004070008A2 publication Critical patent/WO2004070008A2/fr
Publication of WO2004070008A3 publication Critical patent/WO2004070008A3/fr
Priority to US11/193,883 priority patent/US20050282893A1/en
Publication of WO2004070008A8 publication Critical patent/WO2004070008A8/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
    • A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
    • A61K31/737—Sulfated polysaccharides, e.g. chondroitin sulfate, dermatan sulfate
    • 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
    • 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
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1137—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against enzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y207/00—Transferases transferring phosphorus-containing groups (2.7)
    • C12Y207/07—Nucleotidyltransferases (2.7.7)
    • C12Y207/07049—RNA-directed DNA polymerase (2.7.7.49), i.e. telomerase or reverse-transcriptase
    • 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
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/11—Antisense
    • 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
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/11—Antisense
    • C12N2310/111—Antisense spanning the whole gene, or a large part of it

Definitions

  • the present invention describes an antisense molecule that targets the RNA portion of human telomerase (hTR-antisense) and inhibits telomerase activity in human cancer cells.
  • the invention also describes the use of suramin, pentosan polysulfate (PPS), or hTR-antisense to inhibit telomerase activity.
  • PPS pentosan polysulfate
  • the invention further teaches the use of another telomerase inhibitor, 3'-azido-deoxythymidine (AZT), at nontoxic doses producing plasma concentrations in the nanomolar range, to treat tumors.
  • the present invention further teaches a method of treating a tumor by using a telomerase inhibitor concurrently with or after other means of surgical or non-surgical tumor size reduction (cytoreduction).
  • the invention further teaches the use of a telomerase inhibitor prior to non-surgical tumor size reduction treatments, to sensitize tumor cells to the effects of such treatments.
  • the invention further teaches the use of a telomerase inhibitor, e.g., suramin, PPS, or hTR-antisense, to prevent the development of cancer.
  • Telomeres are the structures capping the ends of chromosomes, and are critical to the maintenance of chromosomal integrity and replication potential. Due to the inability to replicate the 3 1 end of chromosomes by DNA polymerases, telomeres are shortened by 50 to 200 bp per cell division. Loss of telomeres to below a critical minimum length results in cell death (Lingner, et al., Science, 269:1533-1534, 1995), or causes cells to enter senescence (e.g., loss of proliferative capacity) (Goldstein, Science, 249:1129-1133, 1990; Martin, et al., Lab.
  • telomere length The enzyme telomerase, consisting of RNA and protein components, is capable of restoring telomere length, and is nearly universally present in tumor cells and usually absent in normal somatic cells (Hiyama, et al., J Natl Cancer Inst, 88:116-122, 1996).
  • telomerase inhibitors are useful therapeutic anticancer agents (e.g. , in Huminiecki, L, Acta Biochimica Polonica, 43:531-538, 1996). This proposed use was based on the hypothesis that active telomerase is needed for initiating and maintaining neoplasia. No evidence of clinical effectiveness of telomerase inhibition has been reported. In fact, several important findings have since been described; all of which indicate that telomerase inhibitors are of limited therapeutic value.
  • telomere lengthening mechanisms that are independent of telomerase (e.g., Gan, et al., FEBS Lett, 527:10-14, 2002).
  • the pre-existing telomeres in tumor cells are usually of sufficient length to support multiple rounds of cell proliferation, even when telomerase is completely inhibited. Accordingly, telomerase inhibitors do not cause cytotoxicity until after a significant lag time. For example, telomerase inhibitors resulted in cytotoxicity in HeLa cells after 23 to 26 cell doublings (Feng, et al., Science, 269:1236-1241 , 1995).
  • telomerase inhibitors impractical and not useful therapeutic agents.
  • no drugs specifically identified as telomerase inhibitors have been tested as anticancer agents in human patients, even though the hypothesis of using telomerase inhibitors to treat cancer first appeared nearly 10 years ago (U.S. Patent No. 5,489,508).
  • telomere damage thereby inducing telomerase activity and leading to resistance to paclitaxel treatment in the cancer
  • co-administration of a telomerase inhibitor e.g., AZT, hTR- antisense
  • a telomerase inhibitor diminishes this resistance and enhances the anti-tumor effect of paclitaxel.
  • the present application extends Applicants' earlier invention and teaches methods to make telomerase inhibition an effective antitumor treatment.
  • the invention teaches using a cytoreductive treatment, administered prior to or concurrent with the administration of a telomerase inhibitor, to reduce the tumor burden, so that the tumor burden would not reach the lethal level before the telomerase inhibitor can erode the telomere length to below the critical level for apoptosis and cell senescence to occur and, thereby, allow telomerase inhibition to become an effective treatment.
  • the invention teaches methods to use telomerase inhibitors to combat cancer, comprising of administering to the cancer patient a cytoreductive treatment (i.e., treatment that reduces the tumor size, e.g., surgery, radiation therapy, chemotherapy, etc.) followed by administering a telomerase inhibitor, wherein the plasma concentrations of the telomerase inhibitor are maintained at telomerase-inhibitory levels for a long duration, e.g., several weeks or several months.
  • the telomerase inhibitor also may be given concurrently with the cytoreductive treatment.
  • This teaching is based on Applicants' discovery that maintenance of suramin in plasma at telomerase-inhibitory, but nontoxic, concentrations during and/or after completion of a cytoreduction treatment, e.g., chemotherapy, enhanced tumor shrinkage, retarded tumor progression and extended the survival time in human cancer patients.
  • a cytoreduction treatment e.g., chemotherapy
  • Another aspect of the current invention teaches the use of a telomerase inhibitor after terminating a cytotoxic treatment, e.g., due to the dose-limiting toxicity of the cytotoxic treatment. Cytotoxic treatments, such as chemotherapy, usually cause toxicity in cancer patients and, therefore, cannot be given indefinitely (i.e., as a maintenance treatment for a maintenance time period).
  • telomerase inhibitors due to the selective expression of telomerase in tumor cells, are not likely to cause toxicity and, therefore, can be given indefinitely or over long period of time. The ability of the telomerase inhibitors to inhibit cell proliferation or induce cell senescence, in turn, will prevent tumor regrowth and lead to survival advantage.
  • paclitaxel 225 mg/m 2
  • the neurotoxicity of paclitaxel is cumulative and becomes dose-limiting usually after 4 to 6 treatments, necessitating the termination of therapy.
  • the tumor typically starts to resume growth within several months, resulting in average survival time of about 8 months (Schiller, et al., 2002).
  • WO 97 38013 A teaches the use of peptide nucleic acid antisense molecules to telomerase (PNA) and antineoplastic agents in combination. Kondo described that the inhibition of telomerase increases the susceptibility of tumors to DNA damaging drugs (Kondo, et al., Oncogene, 16:3323-3330, 1998). Hence, these previous publications teach the generally accepted concept that a combined treatment of a cancer with two effective modalities may be more effective than treatment with each of the separate modalities. However, these publications did not disclose the use of a cytoreductive treatment for the purpose of reducing the tumor size sufficiently to make treatment with a telomerase inhibitor effective.
  • the present invention also teaches that pretreatment with a telomerase inhibitor enhances the antitumor effect of chemotherapy.
  • the chemosensitization effect of suramin was observed when low doses of suramin were administered before or after tumor establishment, or when the tumor burden was not life-threatening. This discovery is novel as no such use or results have been previously described.
  • telomerase inhibitors The current application teaches the use of suramin, PPS, or hTR-antisense to inhibit telomerase. This is based on Applicants' discovery that suramin, PPS, and hTR-antisense are effective telomerase inhibitors and reduce the telomere length in tumors implanted in animals. The prior art teaches several compounds with telomerase-inhibitory properties (e.g. , those described in U.S. Patents Nos. 5,656,638, 5,760,062, 5,767,278, 5,770,613, and 5,863,936).
  • telomerase inhibition possibly due to crosslinking of the telomeric repeat sequences
  • PNA peptide nucleic acid
  • the prior art further teaches the use of peptide nucleic acid (PNA) antisense molecules and phosphorothioate oligonucleotides to inhibit telomerase activity by targeting the RNA component of telomerase (Norton, et; al., Nat Biotechnol, 14:615-619, 1996).
  • PNA peptide nucleic acid
  • telomerase inhibitors as chemopreventives. Chemoprevention using telomerase inhibitors has been proposed. For example, Akama indicated the possibility of administering the telomerase inhibitors of the class of thiazolidinone compounds as a prophylactic (Akama, et al., U.S. Patent No. 6,452,014, 2002). However, Akama does not teach using suramin, PPS, or hTR antisense to inhibit telomerase.
  • Suramin a polysulfonated naphthylurea
  • has multiple pharmacological activities (Ahmann, et al., Proc.Am.Soc.Clin.Oncol., 10:178, 1991 ; Armand, Bonnay, Gandia, Cvitkovic, De Braud , Bertheault, Droz, Carde, Schlumberger, and Fourcault, 1991 ; Dreicer, et al., 1999; Falcone, et al., 1998; Falcone, et al., 1999; Garrett, et al., Proc.Natl.Acad.Sci.USA, 81 :7466-7470, 1984; Grazioli, et al., Int J Immunopharmacol, 14:637-642, 1992; Hawking, Adv.Pharmacol.Chemother., 5:289-322, 1978; Hensey, et al., FASEB, 258:156-
  • the therapeutic plasma concentration of suramin is between 100 to 200 ⁇ M (140-280 ⁇ g/ml) (Funayama, et al., Anticancer Res., 13:1981-1988, 1993), with 70 to 210 ⁇ M (100- 300 ⁇ g/ml) indicated as widest limits (Klohs, et al., US 5,597,830, 1997).
  • suramin shows significant toxicities and only modest activity in patients.
  • Multiple groups of investigators have shown that single agent therapy using suramin has no appreciable antitumor effects in human patients and that a combination of high dose suramin and a cytotoxic agent does not produce beneficial results in human patients as compared to single agents.
  • suramin either a single agent, or as combination of suramin in any dose with a cytotoxic agent, or to use the combination of subtherapeutic and nontoxic doses of suramin with a cytotoxic agent.
  • the only exception would be the use of suramin at subtherapeutic doses, to enhance the action of other treatment modalities.
  • This use discovered by Applicants, and described in detail in U.S. Patent 6,599,912, requires the administration of suramin shortly before, during, and shortly after, treatment of the patient with other modalities, such as chemotherapy or radiation.
  • the current invention supports additional and different uses of suramin, and emphasizes the need for long total treatment duration, e.g., weeks, months, years, or indefinite time, so as to inhibits telomerase and cause sufficient telomere shortening to inhibit cell proliferation or cause cell death.
  • the present invention teaches the requirement of continuous inhibition of telomerase. Hence, a compound with a slow elimination from the body is desired.
  • Suramin fulfills such requirement.
  • the pharmacokinetics of suramin in human patients is characterized by a triphasic plasma concentration decline, with half-lives of 5.5 hours, 4.1 days, and 78 days.
  • the total body clearance is 0.0095 liter/hour/m 2 (Jodrell, et al., J Clin. Oncol., 12:166-175, 1994).
  • the disposition of suramin in dogs is also slow with a terminal half-life of about 13 days (See Example 8).
  • PPS is a semi-synthetic heparinoid and has anti-coagulant effect (Wellstein, et al., Breast Cancer Res. Treat, 38:109-119, 1996). At concentrations that had no anticoagulation effect in patient sera and that were 1 , 000- fold lower than its cytotoxic effects, PPS inhibits the binding of FGF to their receptors and also inhibits angiogenesis in the chicken chorioallantoic membrane assay (Parker, et al., J.Natl. Cancer Inst, 85:1068-1073, 1993; Wellstein, et al., 1996). Anticoagulation is only found at concentrations above 1 ⁇ g/ml (Parker, et al., 1993).
  • PPS inhibits the growth of the rat MAT-LyLu tumor, if treatment is started when the tumor is not palpable, but has little effect against established tumors and cannot inhibit the metastasis of MCF7 tumor in mice (McLeskey, et al., Br.J. Cancer, 73:1053-1062, 1996; Nguyen, et al., Anticancer Res., 13:2143-2148, 1993; Wellstein, et al., J.Natl.Cancer Inst, 83:716-720, 1991).
  • the antitumor activity of PPS in preclinical tumor models has led to its clinical evaluation.
  • the doses of PPS used for antitumor activity evaluation were about 400 mg per meter squared per day, equivalent to about 10 mg/kg per day, and were given orally. Plasma concentrations increased over time of continuous daily administration, reaching 200-460 ng/ml on the fifteenth day of treatment. At this dose level, hematologic toxicities such as diarrhea, gastrointestinal bleeding, or proctitis, usually occurred within 1 to 3 month. Proctitis was the dose limiting toxicity in this trial (Marshall et al., Clin. Cancer Res., 3:2347-2354, 1997). The PPS concentrations required for 50% inhibition of telomerase activity (0.5-0.6 ⁇ g/ml, see
  • Example 3 were substantially higher than the concentrations that cause proctitis toxicity.
  • This consideration together with the discovery that local administration of suramin to the targeted organ resulted in telomerase-inhibitory concentrations in the targeted tissues (e.g., 5 to 100 ⁇ g/g) but very low concentrations of suramin in the plasma (e.g., 0.1 ⁇ g/ml, see Example 13), led to the invention of local administration of PPS to the organs where telomerase inhibition is desired. This new use eliminates the potential problem of systemic host toxicities.
  • AZT is used to treat patients infected with the human immunodeficiency virus. AZT originally was developed as an antitumor agent.
  • AZT antitumor agent
  • AZT has since been tested in Phase I and II clinical trials, either as single agent or in combination with other drugs, in the treatment of gastrointestinal cancers. All of these earlier studies used doses of AZT (i.e., 7 to 10 g/m 2 /day) that would produce plasma concentrations in excess of 10 micromolar, as calculated based on the available data, as follows.
  • a minimal dose of 3 g/m /day is used (U.S. Patent No. 5,116,823), which converts to approximately 85 mg/kg/day.
  • Linear extrapolation of the steady-state concentration from 1.06 ⁇ g/ml for 15 mg/kg/day yields an expected plasma concentration of 6 ⁇ g/ml, or 22 ⁇ M, for a dose of 85 mg/kg/day.
  • AZT is a telomerase inhibitor; the concentrations that produces 50% inhibition ranged from micromolar to millimolar (Strahl, et al., Nucleic Acid Res, 22:893-900, 1994; Strahl, et al., Mol.Cell Biol., 16:53-65, 1996).
  • the present invention teaches the use of low doses of AZT.
  • Applicants discovered that administration of nontoxic doses of AZT that delivered nanomolar plasma concentrations resulted in elimination of well-established tumors in animals. Addition of such nontoxic doses of AZT also enhanced the antitumor activity of paclitaxel in animals.
  • AZT has been evaluated as an antitumor agent and a telomerase inhibitor, no prior art describes the use or the effectiveness of AZT at such low doses.
  • AZT at nanomolar concentrations, enhances the activity of chemotherapy is also surprising in view of the prior art showing that the cytotoxic or telomerase-inhibitory concentrations of AZT are in the micromolar or millimolar range (Strahl, et al., 1994; Strahl, et al., 1996; (Melana, et al., Clin.Cancer Res., 4:693-696, 1998; Table 1 in Example 1).
  • telomere antisense constructs Inhibition of telomerase activity by antisense constructs has been reported, and the possibility of their application in the treatment of cancer has been proposed (Kelland, Lancet Oncol, 2:95-102, 2001). However, the hTR antisense reported here has not been described. Further, combining long-term treatment of telomerase inhibition through the use of telomere antisense constructs, with cytoreductive treatments, have not been proposed or described.
  • telomere inhibition and telomere shortening are effective telomerase inhibitors and reduce the telomere length in tumors implanted in animals.
  • the second discovery is that pretreatment with suramin enhances the activity of chemotherapy against well-established tumors in tumor-bearing animals.
  • the third discovery is that telomerase inhibitors, such as suramin and PPS, enhanced the anticancer activity of cancer chemotherapy and radiation.
  • the fourth discovery is that maintenance of suramin in plasma at telomerase-inhibitory concentrations during and after completion of a cytoreductive treatment promoted tumor shrinkage, delayed tumor growth and extended the survival time in human cancer patients.
  • the fifth discovery is that local administration of suramin to a tissue that was the intended target for treatment resulted in local tissue concentrations that were sufficient to inhibit telomerase and shorten telomere in the tissue but at the same time resulted in very low suramin concentrations in the plasma that would not have been sufficient to inhibit telomerase.
  • the sixth discovery was that administration of nontoxic doses of AZT that delivered nanomolar plasma concentrations resulted in elimination of well- established tumors in animals and enhanced the antitumor activity of paclitaxel in animals.
  • the present invention teaches methods of inhibiting telomerase by contacting telomerase or cells containing telomerase with suramin, PPS, or hTR-antisense.
  • a second and related aspect teaches methods of inhibiting telomerase activity in a patient, preferably a mammal, suffering from a telomerase-mediated condition or disease, comprising administration to the patient of a therapeutically effective amount of suramin, or another telomerase inhibitor.
  • a third aspect teaches methods of improving the treatment outcome of a cancer patient, preferably a mammal, comprising administration to the patient of a telomerase-inhibitory amount of suramin, or another telomerase inhibitor.
  • the telomerase-inhibiting treatment is given in such a way that the tumor is exposed to a telomerase inhibitor for a duration of at least several cycles of proliferation and, more preferably, at least 10 or 20 cycles of proliferation.
  • a fourth aspect teaches methods of enhancing the treatment of a cancer patient, preferably a mammal, comprising administering to the patient a telomerase- inhibitory amount of suramin, or another telomerase inhibitor, during and after completion of a cytoreductive treatment.
  • cytoreductive treatment can be surgical excision of tumors or non-surgical treatments, e.g., radiation therapy, chemotherapy, photodynamic therapy.
  • One or more telomerase inhibitors may be used to obtain better treatment results.
  • the invention teaches a method of treating a cancer in a patient by first identifying a patient about to have a cancer, or harboring a cancer that is too small to be detected, and then administering a telomerase inhibitory agent to the patient such that treatment of the cancer, or prevention of cancer development, is achieved.
  • Treatment of such patients with a telomerase inhibitory agent would be effective, as the small initial size of the tumor would allow many tumor proliferation cycles before the tumor burden would threaten the health and well being of the patient.
  • the invention is a method of treating a patient in remission, after a successful treatment of his or her cancer, but where the patient remains at a substantial risk of developing a new or recurrent cancer. This is especially so for an agent of minimal or no toxicity to the patient, as it provides for a favorable risk-to- benefit ratio.
  • Suramin, PPS, or AZT inter alia, at the low amounts needed to inhibit telomerase activity, are minimally or not toxic to the patient, and provide such favorable risk-to-benefit ratio.
  • a seventh aspect teaches methods of inhibiting telomerase by transfecting cells with the hTR-antisense.
  • the invention teaches the use of nontoxic doses of AZT yielding plasma concentrations below the micromolar range, e.g., in the nanomolar range, to combat cancer.
  • the telomerase inhibitor can be administered locally, near the site of a known tumor, or in an organ where the current or future occurrence of a tumor is suspected.
  • the locally administered telomerase inhibitor will provide effective telomerase-inhibitory concentrations to tumor cells or tissue located in proximity to the administration site.
  • Local administration e.g., injection or implantation, can be in the form of a depot, such as a slow release device.
  • Local administration of the telomerase inhibitor is to provide telomerase-inhibitory concentrations in the tissues that are targets of the treatment, but does not need to result in telomerase-inhibitory concentrations in plasma or other organs that are not the targets of the treatment.
  • Applicants described the discovery that acidic and basic fibroblast growth factors (FGF) cause broad spectrum tumor resistance to chemotherapy and that inhibitors of FGF enhance the antitumor activity of cancer chemotherapy and radiation therapy.
  • FGF acidic and basic fibroblast growth factors
  • Suramin and PPS are FGF inhibitors.
  • the current invention teaches that the same compounds inhibit telomerase activity.
  • the concentrations of suramin and PPS that produce FGF inhibition also produce telomerase inhibition.
  • suramin or PPS can be administered to a patient in combination with a cytotoxic anti-cancer treatment to enhance be effect of the cytotoxic agents, while at the same time inhibiting telomerase to decrease the telomere length and thereby achieve additional, beneficial antitumor effect.
  • suramin or PPS administration can be maintained to achieve a long-term inhibition of telomerase activity.
  • the use of the same agent(s) to target both the FGF-resistance and telomerase-resistance mechanisms represents a convenience to the patient and to the treating physician and improves the outcome of the cytotoxic treatment.
  • telomerase inhibitor requires inhibition of telomerase activity over multiple proliferation cycles of the tumor cells.
  • Such continuous inhibition is most effectively achieved with an agent that has a long terminal half-life in the patient, for example longer than one week, so as to continuously maintain inhibition of telomerase activity with treatment at infrequent intervals.
  • Suramin with an elimination half-life in excess of one week in humans (Jordell et al., 1994) and nonhuman animals (see Example 8), fulfills this requirement.
  • the compounds of the invention have many valuable uses as inhibitors of deleterious telomerase activity, such as, for example, in the treatment of cancer in mammals, such as humans.
  • the pharmaceutical compositions of the invention may be employed in treatment regimens in which cancer cells are killed, in vivo, or can be used to kill cancer cells ex vivo.
  • this invention provides therapeutic compounds and compositions for treating cancer, and methods for treating cancer and other telomerase-mediated conditions or diseases in humans and other mammals (e.g., dogs, cats, cows, horses, and other animals of veterinary interest).
  • the term "aberrant growth” refers to a cell phenotype, which differs from the normal phenotype of the cell, particularly those associated either directly or indirectly with a disease such as cancer.
  • administering refers to the introduction of an agent to a cell, e.g., in vitro, a cell in an animal, i.e., in vivo, or a cell later placed back in the animal (i.e., ex vivo).
  • agent As used herein, the terms “agent”, “drug”, “compound”, “anticancer agent”, “chemotherapeutic”, “antineoplastic”, and “antitumor agent” are used interchangeably and refer to agent/s (unless further qualified) that have the property of inhibiting or reducing aberrant cell growth, e.g., a cancer. The foregoing terms are also intended to include cytotoxic, cytocidal, or cytostatic agents.
  • agent includes small molecules, macromolecules (e.g. , peptides, proteins, antibodies, or antibody fragments), and nucleic acids (e.g., gene therapy constructs, recombinant viruses, nucleic acid fragments (including, e.g., synthetic nucleic acid fragments).
  • apoptosis refers to any non-necrotic, cell-regulated form of cell death, as defined by criteria well established in the art.
  • cancer As used herein, the terms “cancer”, “tumor cell”, “tumor”, “leukemia”, or “leukemic cell” are used interchangeably and refer to any neoplasm ("new growth"), such as a carcinoma (derived from epithelial cells), adenocarcinoma (derived from glandular tissue), sarcoma (derived from connective tissue), lymphoma (derived from lymph tissue), or cancer of the blood (e.g., leukemia or erythroleukemia).
  • carcinoma derived from epithelial cells
  • adenocarcinoma derived from glandular tissue
  • sarcoma derived from connective tissue
  • lymphoma derived from lymph tissue
  • cancer of the blood e.g., leukemia or erythroleukemia
  • cancer of the blood e.g., leukemia or erythroleukemia.
  • cancer e.g., leukemia or erythroleukemia
  • cancer and tumor cell are intended to encompass cancers or cells that may be either benign, premalignant, or malignant.
  • a cancer or tumor cell exhibits various art recognized hallmarks such as, e.g., growth factor independence, lack of cell/cell contact growth inhibition, and/or abnormal karyotype.
  • a normal cell typically can only be passaged in culture for a finite number of passages and/or exhibits various art recognized hallmarks attributed to normal cells (e.g., growth factor dependence, contact inhibition, and/or a normal karyotype).
  • the term "cell” includes any eukaryotic cell, such as somatic or germ line mammalian cells, or cell lines, e.g., HeLa cells (human), NIH3T3 cells (murine), embryonic stem cells, and cell types such as hematopoietic stem cells, myoblasts, hepatocytes, lymphocytes, and epithelial cells and, e.g., the cell lines described herein.
  • eukaryotic cell such as somatic or germ line mammalian cells, or cell lines, e.g., HeLa cells (human), NIH3T3 cells (murine), embryonic stem cells, and cell types such as hematopoietic stem cells, myoblasts, hepatocytes, lymphocytes, and epithelial cells and, e.g., the cell lines described herein.
  • the term "identifying a patient having or about to have” refers to a patient having been determined to have, or to be statistically likely to have, a cancer using various art recognized diagnostic or prognostic techniques including, e.g., the prostate specific antigen (PSA) test, BRCA1 and/or BRCA2 genotyping, genetic profiling, etc.
  • PSA prostate specific antigen
  • BRCA1 and/or BRCA2 genotyping genetic profiling, etc.
  • the term is also intended to include the mere knowing or receipt of any information (e.g., a prognosis, diagnosis) indicating that the patient is having or about to have a cancer.
  • the term "inhibiting or reducing the growth of a cell” refers to the slowing, interrupting, or arresting of its growth and/or metastasis, and can, but does not necessarily require, e.g., a total elimination of the aberrant growth of the cell.
  • the term is also intended to encompass inhibiting or reducing cell growth via cell death (apoptosis) or necrosis.
  • the terms “locally” and “regionally” are used interchangeably, and refer to the administration of a therapy into a tumor mass, into a tumor-bearing organ, or in a general tumor field or area suspected to be seeded with metastases, or premalignant lesions, e.g. an organ specific for a tumor type such as prostate for prostate cancer.
  • tumor burden a term widely recognized in the art, refers to the partial or total mass, volume or size of tumor tissues in a patient.
  • the tumor size is determined by standard clinical means, usually consisting of palpation, or imaging methods (e.g., X-ray, CAT scan, PET scan, ultrasound sonogram).
  • the tumor burden can be estimated from a summation of the tumor sizes.
  • the tumor burden is an art recognized indicator of the clinical course of the disease, where a large tumor burden indicates a bad prognosis, while a small tumor burden is a positive prognostic sign. Tumor burden is often regarded in relationship to the lethality of the tumor for the patient.
  • a metastatic tumor growing in the brain can be lethal at a size of a few centimeters, while much larger tumors in the liver or viscera can be tolerated.
  • a lethal tumor burden for a patient with a tumor metastatic to the brain can be much smaller than a lethal tumor burden for a patient with no such metastases.
  • systemically refers to the administration of a therapy with the intent that the agent will be widely disseminated throughout subject, such as by oral or intravenous administration.
  • systemic concentrations refer to concentrations throughout the body, such as found in circulating plasma.
  • pharmaceutically acceptable carrier includes a pharmaceutically acceptable material, composition or vehicle, suitable for administering compounds of the present invention to mammals.
  • the term "pharmaceutical composition” includes preparations suitable for administration to mammals, e.g., humans.
  • the compounds of the present invention are administered as pharmaceuticals to mammals, e.g., humans, they can be given per se or as a pharmaceutical composition containing, for example, 0.1 to 99.5% (more preferably, 0.5 to 90%) of active ingredient (e.g., a therapeutically-effective amount) in combination with a pharmaceutically acceptable carrier.
  • the term "subject” is intended to include human and non-human animals (e.g., mice, rats, rabbits, cats, dogs, livestock, and primates).
  • Preferred human animals include a human patient having a disorder characterized by aberrant cell growth, e.g., a cancer.
  • telomere refers to the end of a eukaryotic chromosome, which is frequently abnormally extended in a cancer cell.
  • telomerase refers to the cellular enzyme or enzyme activity directed to the nucleotide polymerization or maintenance of chromosome ends known as telomeres.
  • telomerase inhibitory agent and telomerase inhibitor refer to an agent that inhibits (completely or partially) the activity of the enzyme telomerase.
  • inhibition of telomerase refers to a directly measurable inhibition of the telomerase enzyme, for example, as demonstrated by using the modified TRAP assay described by Gan et al., (Gan et al., Pharm. Res., 18:488-493, 2001), or based on the reduction of the average telomere length in all of the cells as demonstrated by using the TALA assay described by Gan et al., (Gan et al., Pharm. Res., 18:1655-1659, 2001) or erosion of individual telomeres in individual cells using the FISH assay described by Gan ef al., (Gan et al., Pharm. Res., 18:1655-1659, 2001).
  • chemosensitizer refers to an agent that increases the antitumor effect of a second agent, e.g., an anticancer chemotherapeutic agent.
  • chemosensitization refers to an increase in antitumor activity of a cancer chemotherapeutic agent by a chemosensitizer when compared to the antitumor activity of the cancer chemotherapeutic agent given without the chemosensitizer.
  • chemoprevention refers to using an agent to prevent the development of a tumor, a cancer.
  • chemopreventive refers to an agent that prevents the development of a tumor, a cancer.
  • telomerase inhibitory agent and/or chemotherapeutic refers to the amount of such an agent which, alone or in combination, is effective, upon single- or multiple-dose administration to the subject, e.g., a human patient, at inhibiting telomerase activity (for a telomerase inhibitory agent), or at inhibiting or reducing aberrant cell growth, e.g., a cancer (for a chemotherapeutic).
  • pentosan polysulfate or "PPS” refers to a semi- synthetic sulfated polyanion composed of beta-D-xylopyranose residues with properties similar to heparin, with molecular weight ranges from 1500-5000.
  • the compound is, for example, described in the Merck index, 10th edition, page 1025, Merck & Co, Inc, 1983.
  • Other names used to describe this compound are, inter alia, xylan hydrogen sulfate; xylan polysulfate; CB 8061 ; Fibrase; Hemoclar.
  • biomarkers is intended as art recognized and refers to molecules or compounds, e.g., protein or gene, whose presence or levels indicates the presence of a disease or the heightened likelihood of developing a disease, e.g., cancer.
  • a disease e.g., cancer.
  • patients that have elevated levels of prostate specific antigen are likely to have or to develop prostate cancer.
  • Patients that have a mutation in BRCA1 or BRCA2 genes are likely to have or to develop breast and ovarian cancers.
  • ex vivo refers to tests performed using living cells in tissue culture.
  • telomere activity has been demonstrated by analysis of telomerase activity (Kim et al., 1994, Science, 266:2011-2014).
  • the invention is a method for inhibiting the ability of a cell to proliferate or replicate.
  • one or more compounds described in the present invention and that are capable of inhibiting telomerase enzyme activity are provided during cell replication.
  • telomeres play a critical role in allowing the end of the linear chromosomal DNA to be replicated completely without the loss of terminal bases at the 5'-end of each strand.
  • telomere activity in hematopoietic stem cells is useful for the treatment of a condition associated with an increased rate of proliferation of a cell, such as in cancer (telomerase-activity in malignant cells), and hematopoiesis (telomerase activity in hematopoietic stem cells), for example.
  • the present invention teaches using suramin, PPS, and hTR-antisense to inhibit telomerase, and thereby preventing or treating many types of malignancies.
  • the compounds of the present invention can provide a highly general method of treating malignancies, as demonstrated by the high percentage of human tumor cell lines and tumors that express telomerase.
  • the compounds described in the present invention can be effective in providing treatments that selectively target malignant cells, thus avoiding many of the deleterious side-effects usually associated with cytotoxic chemotherapeutic agents that kill dividing cells indiscriminately.
  • the compounds of the invention extend to analogues of suramin and PPS that also inhibit telomerase.
  • the present invention provides compounds, pharmaceutical compositions and methods relating to these compounds, or their pharmaceutically acceptable salts, for inhibiting a telomerase enzyme, comprising contacting the telomerase enzyme with a compound, or its pharmaceutically acceptable salt, where the compounds are suramin or PPS.
  • the telomerase to be inhibited is a mammalian telomerase, such as a human telomerase.
  • a second and related aspect of the present invention is the discovery that suramin, when maintained in a subject in amounts that are effective in the inhibition of telomerase activity, can shorten the telomere length in a tumor.
  • this aspect of the present invention teaches methods of inhibiting telomerase activity in a patient, preferably a mammal, suffering from a telomerase-mediated condition or disease, comprising administration to the patient of a therapeutically effective amount of suramin, or another telomerase inhibitor.
  • the compounds described in the present invention inhibit telomerase in cell extracts, cultured cells and in intact animals.
  • the activities of the compounds of the invention can also be demonstrated using the methods described herein.
  • One method used to identify compounds of the invention that inhibit telomerase activity involves placing cells, tissues, or preferably a cellular extract or other preparation containing telomerase, in contact with several known concentrations of a test compound in a buffer compatible with telomerase activity. The level of telomerase activity for each concentration of test compound is measured and the IC 50 (the concentration of the test compound that produced 50% inhibition of the enzyme activity relative to its original or control value) or IC 9 o for the compound is determined using standard techniques. Other methods for determining the inhibitory concentration of a compound of the invention against telomerase can be employed as will be apparent to those of skill in the art based on the disclosure herein.
  • IC 50 values for suramin and PPS were determined and found to be below 10 ⁇ M.
  • telomere-dependent cell lines Treatment of telomerase-dependent cell lines, e.g., human pharynx FaDu cells, with a compound of the invention is also expected to induce a reduction of telomere length in the treated cells.
  • Compounds described in the present invention also are expected to induce telomere reduction during cell division in human tumor cell lines, such as FaDu and human prostate PC3.
  • the observed reduction in telomere length is expected to be no different from cells that are treated only with the vehicle, e.g., physiological saline.
  • Compounds described in the invention also are expected to demonstrate no significant cytotoxic effects in normal cells at the telomerase inhibitory concentrations of their proposed use.
  • telomerase the specificity of the compounds described in the present invention for telomerase can be determined by comparing their activity (IC 50 ) on telomerase to their activity on other enzymes.
  • Enzymes are, as art recognized, molecules that facilitate a biological reaction.
  • enzymes similar nucleic acid binding or modifying activity similar to telomerase in vitro include DNA Polymerase I, HeLa RNA Polymerase II, T3 RNA Polymerase, MMLV Reverse Transcriptase, Topoisomerase I, Topoisomerase II, Terminal Transferase and Single-Stranded DNA Binding Protein (SSB).
  • SSB Single-Stranded DNA Binding Protein
  • telomerase Compounds having lower IC 5 o values for telomerase as compared to the IC 5 o values toward the other enzymes being screened are said to possess specificity for telomerase.
  • compounds having lower IC 90 values for telomerase as compared to the IC 90 values toward the other enzymes being screened are said to possess specificity for telomerase.
  • mice treated with a compound of the invention are expected to have tumor masses that, on average, may decrease, remain unchanged, or even increase for a period following the initial dosing, but will shrink in mass with continuing treatment.
  • control animals treated with physiological saline are expected to have tumor masses that continue to increase.
  • the present invention also provides methods for selecting treatment regimens involving administration of a compound of the invention.
  • TRF terminal restriction fragment
  • DNA from tumor cells is analyzed by digestion with restriction enzymes specific for sequences other than the telomeric (T 2 AG 3 ) n sequence.
  • An example of such analysis is described in a previous patent application (U.S. Patent Application Serial No. 10/464,018).
  • gel electrophoresis is performed to separate the restriction fragments according to size.
  • the separated fragments then are probed with nucleic acid probes specific for telomeric sequences to determine the lengths of the terminal fragments containing the telomere DNA of the cells in the sample.
  • nucleic acid probes specific for telomeric sequences to determine the lengths of the terminal fragments containing the telomere DNA of the cells in the sample.
  • a third aspect of the present invention is the discovery that treatment with suramin prior to the initiation of cancer chemotherapy, in the presence of minimal residual disease and where the suramin is administered in amounts that are effective to inhibit telomerase activity, enhances the efficacy of the cancer chemotherapy.
  • this aspect of the present invention teaches methods of improving the treatment outcome of a cancer patient, preferably a mammal, comprising administration to the patient of a telomerase-inhibitory amount of suramin, or another telomerase inhibitor.
  • the telomerase-inhibiting treatment is given in such a way that the tumor is exposed to a telomerase inhibitor for a duration of at least several cycles of proliferation, and more preferably at least 10 to 20 cycles of proliferation.
  • a preferred method is the administration of the telomerase inhibitor before the administration of a cytotoxic regimen.
  • An alternative preferred method is the administration of the telomerase inhibitor before and during the administration of a cytotoxic regimen.
  • the telomerase inhibitor is suramin, where the suramin is administered in an amount that is effective in the inhibition of telomerase activity but insufficient to produce antitumor activity.
  • a fourth aspect of the present invention is the discovery that maintenance of suramin in plasma at telomerase-inhibitory concentrations during and after completion of a cytoreductive treatment promoted tumor shrinkage, delayed tumor growth, and extended the survival time in human cancer patients.
  • this aspect of the present invention provides methods of enhancing the treatment of a cancer patient, preferably a mammal, comprising administering to the patient a therapeutically effective amount of suramin, or another telomerase inhibitor, during and after completion of a cytoreductive treatment.
  • Such cytoreductive treatment can be surgical excision of tumors or non-surgical treatments, e.g., radiation therapy, chemotherapy, photodynamic therapy.
  • the telomerase-inhibiting treatment is given in such a way that the tumor is exposed to a telomerase inhibitor at a plasma concentration that is known to produce telomerase inhibition in tumor cells, for a duration that is equivalent to at least several cycles of proliferation, and more preferably at least 10 to 20 cycles of proliferation.
  • a preferred method is the administration of the telomerase inhibitor during and after the administration of a cytotoxic regimen, where the cytotoxic regimen will reduce the tumor load in the patient and thereby provide sufficient lead time for telomerase inhibitors to result in shortening of telomere to below the critical length to induce cell death and senescence.
  • reduction of the tumor size is accomplished by surgical means.
  • a telomerase inhibitor is administered after a cytotoxic regimen is terminated, e.g., due to dose-limiting toxicity in the cancer patient, as a means to combat cancer.
  • a telomerase inhibitor is administered after a cytotoxic regimen is terminated., and the administration of a telomerase inhibitor is continued for at least several weeks, months, years, or more preferably, indefinite period.
  • the invention teaches a method of treating a cancer in a patient by identifying a patient about to have a cancer, or harboring a cancer that is too small to be detected by conventional means, and administering a telomerase inhibitory agent to the patient such that treatment of the cancer or prevention of cancer development is achieved.
  • some of the conventional methods to detect tumors are physical methods (e.g., palpation), pathological methods (e.g., blood in urine or stool), or imaging methods (e.g., X-ray, CAT scan, PET scan, ultrasound sonogram). Identification of patients that are likely to have a cancer or harboring an undetectable cancer can also be achieved by monitoring biomarkers or genetic defects.
  • a patient may have a blood level of the prostate specific antigen (PSA) that is above the normal limit of 4 ng/ml, but may not have a tumor palpable by digital rectal exam, or visible by ultrasound imaging.
  • PSA prostate specific antigen
  • the elevated PSA level would indicate a high likelihood of the formation or the presence of a cancer of the prostate, while the absence of physical detection indicates that the tumor is extremely small, or in a precursor state.
  • a female patient not currently having a detectable tumor could have a mutation in the BRCA1 or BRCA2 gene, showing a strong predisposition for the development of a breast or ovarian cancer.
  • Other art recognized methods for assessing the likelihood of tumor occurrence and the tumor burden are also included.
  • the invention is a method of treating a patient in remission, after a successful treatment of his or her cancer, but where the patient remains at a substantial risk of developing a new or recurrent cancer.
  • This patient would benefit from a treatment with a telomerase inhibitor.
  • the telomerase inhibitor would effectively combat the patient's cancer if it reappears.
  • a seventh aspect of the present invention is based on the Applicants' finding that transfection of cells with the hTR-antisense effectively inhibits the telomerase enzyme activity in vitro.
  • the nucleotide sequence defining the hTR-antisense is obvious from Example 4.
  • the present invention teaches methods of inhibiting telomerase by transfecting cells with the hTR-antisense.
  • the invention teaches the use of nontoxic doses of AZT yielding plasma concentrations below the micromolar range, e.g., in the nanomolar range, to combat cancer.
  • AZT is given to a cancer patient, without other accompanying treatment.
  • AZT is given to a cancer patient after a surgical cytoreductive treatment.
  • AZT is given prior to, concurrently with, or after a nonsurgical cytoreductive treatment.
  • telomerase inhibitors Local administration of telomerase inhibitors to a target site or organ
  • the telomerase inhibitor can be administered locally or regionally, near the site of a known tumor, or in an organ where the current or future occurrence of a tumor is suspected.
  • the locally administered telomerase inhibitor will provide effective inhibitory concentrations to tumor cells or tissues located in proximity to the administration site.
  • Local administration e. g., injection or implantation, can be in the form of a depot, such as a slow release device. Implantation may require a surgical procedure, depending on the site of the treatment. For example, a patient successfully treated for a superficial bladder cancer of high grade would have no known remaining tumor, but would be at an elevated statistical risk of tumor recurrence.
  • a device that slowly releases a telomerase inhibitor over a period of weeks or months or years could be placed directly into the urinary bladder by transurethral insertion.
  • the released telomerase inhibitor would provide effective inhibitory concentrations to the mucosal or muscle cell layers of the urinary bladder, and thus inhibit tumor formation and eventually any recurring tumor.
  • PSA prostate specific antigen
  • local administration or implantation of a slow-release preparation in or near the prostate to deliver one or more telomerase inhibitors can be used to reduce the chance of the development of symptomatic tumors.
  • telomerase inhibitor Local administration of the telomerase inhibitor is to provide telomerase- inhibitory concentrations in the tissues that are targets of the treatment, but does not need to result in telomerase-inhibitory concentrations in plasma or other organs that are not the targets of the treatment.
  • This aspect of the present invention is based on the discovery that local administration of suramin to the targeted organ resulted in telomerase-inhibitory concentrations in the targeted tissues (e.g., 5 to 100 ⁇ g/g) but very low concentrations of suramin in the plasma (e.g., 0.1 ⁇ g/ml or less).
  • telomerase inhibitor has certain advantages over systemic routes of administration.
  • One advantage is that it avoids the need of frequent drug administrations, while assuring that telomerase inhibition is continuous and uninterrupted.
  • Another advantage is that the local administration diminishes the possibility of toxicity of a telomerase inhibitor to other tissues that are not the intended targets of the treatment.
  • a telomerase inhibitor e.g. suramin
  • a reduction of exposure of non-tumor-bearing organs will further reduce the chance that a hypersensitivity reaction, or other rare event, occurs.
  • this advantage would be of even greater significance.
  • the invention can be used to treat bladder interstitial cystitis by use of bladder local administration, e.g., injection or implantation, can be in the form of a depot, such as a slow release device.
  • bladder local administration e.g., injection or implantation
  • This method is for enhancing therapeutic outcome of treating patient having bladder interstitial and is implemented by locally administering to the patient an effective amount of one or more of suramin, a pharmaceutically acceptable salt of suramin, pentosan polysulfate (PPS), a pharmaceutically acceptable salt of PPS.
  • PPS pentosan polysulfate
  • the invention features methods for inhibiting or reducing cell growth, e.g. , aberrant growth, e.g. , hyperplastic or hypertrophic cell growth, by contacting the cells with at least one cytoreductive agent and at least one telomerase inhibiting agent.
  • the methods include a step of contacting pathological hyperproliferative cells (e.g., a cancer cell) with an amount of at least one telomerase inhibiting agent which is effective to reduce or inhibit the proliferation of the cell, or induce cell killing.
  • the present methods can be performed on cells in culture, e.g., in vitro or ex vivo, or can be performed on cells present in a subject, e.g., as part of an in vivo therapeutic protocol.
  • the therapeutic regimen can be carried out on a human or on other animal subjects.
  • the enhanced therapeutic effectiveness of the combination therapy of the present invention represents a promising alternative to conventional highly toxic regimens of anticancer agents.
  • telomerase inhibitory agent can be utilized alone, the agent is preferably combined with a cytotoxic agent for a therapeutic effect that is greater than expected for each of the agents alone.
  • these agents may be further combined with other anticancer agents, e.g., antimicrotubule agents, topoisomerase I inhibitors, topoisomerase II inhibitors, antimetabolites, mitotic inhibitors, alkylating agents, intercalating agents, agents capable of interfering with a signal transduction pathway (e.g., a protein kinase C inhibitors, e.g., an antihormone, e.g., an antibody against growth factor receptors), agents that promote apoptosis and/or necrosis, biological response modifiers (e.g. interferons, e.g. interleukins, e.g. tumor necrosis factors), surgery, or radiation.
  • a signal transduction pathway e.g., a protein kinase C inhibitors, e.g., an anti
  • the enhanced, and preferably synergistic, action of the cytotoxic agent when used in combination with a telomerase inhibitory agent improves the efficacy of the anticancer agent/s allowing for the administration of lower doses of one or more of these agents (even, e.g., a subtherapeutic dose of an agent, if only tested or used alone rather than in combination); thus, reducing the induction of side effects in a subject, such as a human cancer patient (e.g., any art recognized side effects associated with the administration of an unmodified dose of a chemotherapeutic, e.g., hair loss, neutropenia, intestinal epithelial cell sloughing, efc.).
  • a chemotherapeutic e.g., hair loss, neutropenia, intestinal epithelial cell sloughing, efc.
  • the methods of the invention can be used in treating malignancies of the various organ systems, such as those affecting lung, breast, lymphoid, gastrointestinal (e.g. , colon, rectum), and the genitourinary tract (e.g., prostate, bladder, testes), pharynx, as well as adenocarcinomas which include malignancies such as colon cancer, rectal cancer, renal cell carcinoma, prostate cancer and/or testicular tumors, non-small cell carcinoma of the lung, cancer of the small intestine, and cancer of the esophagus.
  • gastrointestinal e.g. , colon, rectum
  • genitourinary tract e.g., prostate, bladder, testes
  • pharynx e.g., pharynx
  • adenocarcinomas which include malignancies such as colon cancer, rectal cancer, renal cell carcinoma, prostate cancer and/or testicular tumors, non-small cell carcinoma of the lung, cancer of the small intestine
  • Exemplary solid tumors that can be treated include, e.g. , fibrosarcoma, myosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, a n g i o s a r c o m a , e n d o t h e l i o s a r c o m a , lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, gastric cancer, esophageal cancer, colon carcinoma, rectal cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, uterine cancer, cancer of the head and neck, skin cancer, brain cancer, squamous cell carcinoma, sebaceous gland carcinoma, papillar
  • the methods of the invention also can be used to inhibit or reduce the growth of a cell of hematopoietic origin, e.g., arising from the myeloid, lymphoid, or erythroid lineages, or any precursor cells thereof.
  • a cell of hematopoietic origin e.g., arising from the myeloid, lymphoid, or erythroid lineages, or any precursor cells thereof.
  • the present invention contemplates the treatment of various myeloid disorders including, but not limited to, acute promyeloid leukemia (APML), acute myelogenous leukemia (AML), and chronic myelogenous leukemia (CML) (reviewed in Vaickus, L. (1991) Crit Rev. Oncol. /Hemotol. 11 :267-97).
  • APML acute promyeloid leukemia
  • AML acute myelogenous leukemia
  • CML chronic myelogenous leukemia
  • Lymphoid malignancies which can be treated by the method, include, but are not limited, to acute lymphoblastic leukemia (ALL; which includes B-lineage ALL and T-lineage ALL), chronic lymphocytic leukemia (CLL), prolymphocytic leukemia (PLL), hairy cell leukemia (HLL), and Waldenstrom's macroglobulinemia (WM).
  • ALL acute lymphoblastic leukemia
  • CLL chronic lymphocytic leukemia
  • PLL prolymphocytic leukemia
  • HLL hairy cell leukemia
  • WM Waldenstrom's macroglobulinemia
  • malignant lymphomas contemplated by the treatment method of the present invention include, but are not limited to, non-Hodgkin's lymphoma and variants thereof, e.g., peripheral T-cell lymphomas, adult T-cell leukemia/lymphoma (ATL), cutaneous T-cell lymphoma (CTCL), and large granular lymphocytic leukemia (LGF).
  • non-Hodgkin's lymphoma and variants thereof e.g., peripheral T-cell lymphomas, adult T-cell leukemia/lymphoma (ATL), cutaneous T-cell lymphoma (CTCL), and large granular lymphocytic leukemia (LGF).
  • malignancies which can be treated by the subject methods, include erythroleukemias, lymphomas, Hodgkin's disease, and malignancies of uncertain origin, e.g., which are not easily categorized and may, e.g., exhibit multiple cell types, such as certain embryonic carcinomas or teratomas.
  • the subject can be a patient with non-small cell lung cancer, and is treated with a combination of paclitaxel, carboplatin, and long-term suramin, where the suramin treatment is continued well after the paclitaxel/carboplatin combination needed to be discontinued for reasons of drug-related toxicity or because the patient ceased to respond.
  • a patient with non-small cell lung cancer can be treated with a combination of gemcitabine, cisplatin, and long- term suramin.
  • the subject can be a patient with hormone refractory prostate cancer, who is treated with a combination of estramustine phosphate, taxotere, and long-term suramin, or with a combination of doxorubicin, ketoconazole, and long-term suramin.
  • the subject can be a patient with metastatic breast cancer, who is treated with a combination of cyclophosphamide, doxorubicin, 5- fluorouracil, and long-term suramin, or a combination of doxorubicin, taxotere, and long-term suramin.
  • the subject is a patient with advanced breast cancer that overexpresses the HER2/neu oncogene, who is treated with Herceptin and long-term suramin, with or without paclitaxel or cisplatin.
  • the subject can be a patient with advanced or metastatic colorectal cancer, who is treated with a combination of irinotecan and long-term suramin.
  • the subject is a patient with advanced colon cancer, who is treated with a combination of 5-fluorouracil, leucovorin, and long-term suramin.
  • the telomerase inhibitory agent is administered systemically.
  • the selected agent can be administered parenterally (e.g., subcutaneously, intravenously, intramuscularly, intraperitoneally, intradermally, intrathecally, etc.), orally, nasally, intrapulmonary by inhalation, rectally, and/or transdermally.
  • the telomerase inhibitory agent is administered locally or regionally.
  • the selected agent can be administered intravesically (i.e. , into the urinary bladder), intraprostatically, intratumorally, or topically.
  • the method further includes repeated dosages of the same, or a different agent, and such particulars are further discussed below.
  • a physician or veterinarian having ordinary skill in the art can readily determine and prescribe the effective amount of the agent or agents (e.g., in the form of a pharmaceutical composition) required.
  • the physician or veterinarian typically may start doses of the agents of the invention at levels lower than that required in order to commence the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved.
  • a suitable dose of an agent of the invention will be that amount of the agent which is the lowest dose effective to produce a therapeutic effect; i.e., treat a condition in a subject, e.g., cancer.
  • Such an effective dose will generally depend upon the factors described above.
  • intravenous and subcutaneous doses of the agents of this invention for a patient will range from about 0.0001 to about 100 mg per kilogram of body weight, more preferably from about 0.01 to about 10 mg per kg, and still more preferably from about 0.10 to about 4 mg per kg.
  • the effective daily dose of the active agent may be administered as two, three, four, five, six, or more sub-doses administered separately at appropriate intervals throughout the day, optionally, in unit dosage forms.
  • the telomerase inhibitor is suramin and the suramin is present in a concentration that is sufficient to inhibit telomerase activity, but is not sufficient to produce one or more of: (i) significant inhibition of cell proliferation; (ii) significant cell death in human and/or animal tumor cells, (iii) a measurable antitumor effect in a subject, e.g., a human subject, and/or (iv) cell cycle arrest.
  • the determination of effect on cultured cells can be determined with the system described in Example 14.
  • the telomerase inhibitor is suramin and it is administered at levels such that the plasma concentration of suramin that is present does not result in one or more of: (i) significant cell cycle arrest, (ii) significant cell death, or (iii) significant inhibition of cell growth, e.g., the concentration in plasma is of a level that, if the same concentration of suramin is provided in cultured cells, at least 50%, more preferably at least 80%, and most preferably at least 99% of the treated cultured cells continue to be involved in one or more of: cycling cells continue to progress through the cell cycle, cells remain viable, or cells remain capable of proliferating, following treatment with suramin.
  • suramin is administered in an amount that results in a plasma concentration ranging from about 0.001 to 100 ⁇ g/ml, preferably about 0.1 to 70 ⁇ g/ml, even more preferably, about 0.5 to 30 ⁇ g/ml.
  • the pharmacokinetics of suramin is characterized by a triphasic concentration decline, with half-lives of 5.5 hours, 4.1 days and 78 days. The total body clearance is 0.0095 liter/hour/m 2 (Jodrell et al., J Clin Oncol 12:166-175, 1994).
  • an initial dose of approximately 240 mg/m 2 should be administered to the average patient to achieve plasma concentrations declining from about 90 ⁇ g/ml (63 ⁇ M) to about 14 ⁇ g/ml (10 ⁇ M) over 168 hours.
  • the 168-hour, or 1 week, duration is chosen as an example, as this time interval is frequently used for repeat visits to a treating physician.
  • Similar calculations can be performed to identify the initial suramin dose to deliver the preferred suramin plasma concentrations over other desired treatment durations. Maintenance doses to adjust the plasma concentrations for later treatment cycles can be similarly calculated.
  • the total suramin exposure in the plasma preferably is less than 7,840 ⁇ M-day over 112 days, less than 7,100 ⁇ M-day over 112 days, less than 5,880 ⁇ M-day over 84 days, less than 5,300 ⁇ M-day over 84 days, less than 2,000 ⁇ M-day over 20 days, preferably less than 800 ⁇ M-day over 96 hours, preferably less than 600 ⁇ M-day over 96 hours, preferably less than 500 ⁇ M- day over 96 hours, preferably less than 400 ⁇ M-day over 96 hours, preferably less than 300 ⁇ M-day over 96 hours, preferably less than 252 ⁇ M-day over 96 hours, preferably less than 200 ⁇ M-day over 96 hours, preferably less than 150 ⁇ M-day over 96 hours, preferably less than 100 ⁇ M-day over 96 hours, and most preferably less than 52 ⁇ M-day over 96 hours.
  • the total suramin exposure is a product of the drug plasma concentration in ⁇ M-day (e.g., the average micromolarity over 24 hours) and the treatment duration in days. For example, treatment of a subject with 13 ⁇ M of suramin for four days would result in a total drug exposure of 52 ⁇ M-day over 96 hours.
  • suramin is administered in an amount that results in a plasma concentration of less than 100 ⁇ g/ml, preferably less than 90 ⁇ g/ml, preferably less than 80 ⁇ g/ml, preferably less than 60 ⁇ g/ml, preferably less than 40 ⁇ g/ml, more preferably less than 15 ⁇ g/ml, and most preferably less than 10 ⁇ g/ml.
  • the telomerase inhibitor is suramin and the time period over which the suramin is administered or over which the suramin is maintained at the plasma concentration sufficient to inhibit telomerase activity is more than one month, or preferably more then one year, or even more preferably indefinitely.
  • the telomerase inhibitor is suramin and the time period over which the suramin is administered or over which the suramin is maintained at the plasma concentration sufficient to inhibit telomerase activity is longer than 60 days, preferably longer than 100 days, preferably longer than 150 days, preferably longer than one year, more preferably longer than two years, and most preferably for indefinite time period, beyond the time duration where a cytoreductive treatment is applied.
  • the telomerase inhibitor is suramin and suramin is administered locally to the target organ, and the time period over which the suramin concentration in the tissues intended for suramin treatment is sufficient to inhibit telomerase activity is more than one month, or preferably more than one year, or more preferably indefinite.
  • suramin as a cancer preventative, treatment may require years of use stretching to the rest of the patient's life.
  • the telomerase inhibitor is suramin and suramin is administered locally to the target organ, and the time period over which the suramin concentration in the tissues intended for suramin treatment is sufficient to inhibit telomerase activity is longer than 60 days, preferably longer than 100 days, preferably longer than 150 days, preferably longer than one year, more preferably longer than two years, and most preferably for indefinite time period, beyond the time duration where a cytoreductive treatment is applied.
  • the telomerase inhibitor is suramin and the time period over which the suramin is administered or over which the suramin is maintained at the plasma concentration sufficient to inhibit the telomerase activity or to enhance the efficacy of the cytoreductive treatment begins more than 30 days, preferably more than 60 days, preferably more than 100 days, preferably more than 150 days, preferably more than one year, and most preferably more than two years before the first day on which the cytoreductive treatment is administered.
  • suramin to enhance the antitumor effect of a cytoreductive treatment
  • the suramin dose is selected to deliver a plasma concentration of below 100 ⁇ g/ml, preferably below 80 ⁇ g/ml, preferably below 60 ⁇ g/ml, more preferably below 40 ⁇ g/ml, and most preferably below 15 ⁇ g/ml in a mammal treated with a cytoreductive treatment.
  • the suramin dose is administered before, simultaneously with, or after the administration of at least one anticancer agent or other cytoreductive treatment.
  • mice with two weekly intravenous bolus suramin doses of 10 mg/kg for 6 weeks enhances the antitumor effect of the subsequently administered anticancer drugs (e.g., paclitaxel), but does not result in additional body weight loss.
  • This dose is calculated to result in a plasma suramin concentration of about 10 ⁇ M ( ⁇ 14 ⁇ g/ml) at 72 hours after dose administration.
  • the methods of the art use high dose suramin, either alone or in combination with a cytotoxic agent, where for a human subject, maintenance of plasma suramin concentrations of between 150 to 300 ⁇ g/ml is needed to produce a measurable antitumor effect (Eisenberger et al., (1995) J Clin Oncol 13:2174-2186; Klohs, U.S. Patent Numbers 5,597,830 and 5,767,110).
  • a typical suramin dosing schedule aimed at maintaining suramin plasma concentrations between 150 and 300 ⁇ g/ml consists of an initial administration of 2100 mg/m 2 over the first week with the subsequent doses repeated every 28 days for 6 months or longer; the subsequent doses are adjusted using the Bayesian pharmacokinetic method (Dawson et al., Clin Cancer Res 4:37-44, 1998; Falcone et al., Cancer 86:470-476, 1999).
  • suramin causes the following toxicity in a human patient: adrenal insufficiency, coagulopathy, peripheral neuropathy, and proximal muscle weakness (Dorr and Von Hoff, Cancer Chemotherapy Handbook, 1994, pp 859-866). The incidence and severity of these toxicities are positively related to cumulated dose and are minimized in the methods described herein.
  • the telomerase inhibitor is PPS.
  • the PPS is present in a concentration that is sufficient to inhibit telomerase activity and induce shortening of telomeres in tumor cells, but is not sufficient to produce one or more of: (i) significant anti-coagulation activity; (ii) significant cell death in human and/or animal tumor cells, (iii) a measurable antitumor effect in a subject, e.g., a human subject, and/or (iv) cell cycle arrest.
  • an agent of the present invention to be administered alone or in combination with another agent, it is preferable to administer the agent(s) as a pharmaceutical composition.
  • the telomerase inhibitory agent is suramin.
  • the invention features a pharmaceutical composition, which includes at least one telomerase inhibitory agent and a pharmaceutically acceptable carrier.
  • the agent(s) are present in an amount effective to inhibit the telomerase activity in the tumor of the patient, and to enhancing the killing, of a hyperproliferative cell.
  • compositions are packaged with instructions for use as described herein.
  • the invention also encompasses timed-release formulations, for example, a slow release formulation of a telomerase inhibitory agent, and a pharmaceutically acceptable carrier.
  • the pharmaceutical composition is suitable for intravenous injection.
  • the composition may also be suitable for local, regional, or systemic administration.
  • the pharmaceutical composition may comprise one or more pharmaceutically acceptable carriers.
  • the invention pertains to nanoparticles, which comprise a cross-linked gelatin and a therapeutic agent, e.g., a telomerase inhibitory agent, such as, for example, suramin or PPS.
  • a therapeutic agent e.g., a telomerase inhibitory agent, such as, for example, suramin or PPS.
  • the invention pertains to compositions containing the nanoparticles and a pharmaceutically acceptable carrier.
  • the carrier for example, can be suitable for systemic, regional, or local administration.
  • the nanoparticles are about 500 to about 1 ⁇ m, or about 600 nm to about 800 nm in diameter.
  • the invention also pertains to microparticles comprising a therapeutic agent, e.g. a telomerase inhibitory agent, such as suramin or PPS.
  • a therapeutic agent e.g. a telomerase inhibitory agent, such as suramin or PPS.
  • the microparticle is about 500 nm to about 100 ⁇ m, about 500 nm to about 50 ⁇ m, about 500 nm to about 25 ⁇ m, about 500 nm to about 20 ⁇ m, about 500 nm to about 15 ⁇ m, about 500 nm to about 10 ⁇ m, about 750 nm to about 10 ⁇ m, about 1 ⁇ m to about 10 ⁇ m, about 750 nm to about 7.5 ⁇ m, about 1 ⁇ m to about 7.5 ⁇ m, about 2 ⁇ m to about 7.5 ⁇ m, 3 ⁇ m to about 7 ⁇ m, or about 5 ⁇ m in diameter.
  • the invention pertains to a composition, which comprises the microparticles and a pharmaceutically acceptable carrier.
  • the pharmaceutically acceptable carrier may be, for example, suitable for administration to a patient locally, regionally, or systemically.
  • the invention also pertains to a method for treating a patient, comprising administering to the patient microparticles of the invention and a pharmaceutically acceptable carrier.
  • the invention features a microparticle suitable for administration to a patient locally, regionally, or systemically, comprising paclitaxel, wherein said microparticle has a diameter of about 5 ⁇ m.
  • the invention features microparticles suitable for administration to a patient locally, regionally, or systemically, comprising suramin or PPS, wherein said microparticle has a diameter of about 5 ⁇ m.
  • the invention also pertains to a kit, i.e., an article of manufacture, for the treatment of a cancer.
  • the kit contains a telomerase inhibitory agent in a pharmaceutically acceptable carrier, a container, and directions for using said telomerase inhibitory agent for inhibiting or reducing the growth of a cell, e.g., aberrant growth associated with, e.g., a cancer or a tumor.
  • a kit of the invention may comprise a telomerase inhibitory agent for previous, subsequent, or concurrent administration.
  • the kit may also provide the telomerase inhibitory agent formulated in dosages and carriers appropriately for local, regional, or systemic administration.
  • the kit may also provide for the prognosing, diagnosing, and/or staging of a cancer for, e.g., determining the susceptibility or resistance of the cancer.
  • compositions comprising compounds of the invention may contain wetting agents, emulsifiers and lubricants, such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweetening, flavoring, and perfuming agents, and preservatives.
  • wetting agents such as sodium lauryl sulfate and magnesium stearate
  • coloring agents such as sodium lauryl sulfate and magnesium stearate
  • coloring agents such as sodium lauryl sulfate and magnesium stearate
  • coloring agents such as sodium lauryl sulfate and magnesium stearate
  • coloring agents such as sodium lauryl sulfate and magnesium stearate
  • coloring agents such as sodium lauryl sulfate and magnesium stearate
  • coating agents such as sweetening, flavoring, and perfuming agents, and preservatives.
  • Formulations of the present invention include those suitable for oral, nasal, topical, inhalation, transdermal, buccal, sublingual, rectal, vaginal, and/or parenteral administration. They are given by forms suitable for each administration route. For example, they are administered in tablets or capsule form, by injection, inhalation, eye lotion, ointment, suppository, etc. administration by injection, infusion or inhalation; topical by lotion or ointment; and rectal by suppositories.
  • the formulations conveniently may be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy.
  • the amount of active ingredient that can be combined with a carrier material to produce a single dosage form will generally be that amount of the agent that produces a therapeutic effect. Generally, out of one hundred per cent, this amount will range from about 1 per cent to about ninety-nine percent of active ingredient, preferably from about 5 per cent to about 70 per cent, most preferably from about 10 per cent to about 30 per cent.
  • Formulations of the invention suitable for oral administration may be in the form of capsules, cachets, pills, tablets, lozenges (using a flavored basis, usually sucrose and acacia or tragacanth), powders, granules, or as a solution or a suspension in an aqueous or non-aqueous liquid, or as an oil-in-water or water-in-oil liquid emulsion, or as an elixir or syrup, or as pastilles (using an inert base, such as gelatin and glycerin, or sucrose and acacia) and/or as mouth washes and the like.
  • Transdermal patches have the added advantage of providing controlled delivery of a compound of the present invention to the body.
  • Such dosage forms can be made by dissolving or dispersing the compound in the proper medium.
  • Absorption enhancers can also be used to increase the flux of the compound across the skin. The rate of such flux can be controlled by either providing a rate controlling membrane or dispersing the active compound in a polymer matrix or gel.
  • Ophthalmic formulations are also contemplated as being within the scope of this invention.
  • compositions of this invention suitable for parenteral administration comprise one or more compounds of the invention in combination with one or more pharmaceutically acceptable sterile isotonic aqueous or non-aqueous solutions, dispersions, suspensions, or emulsions, or sterile powders which may be reconstituted into sterile injectable solutions or dispersions just prior to use, which may contain buffers, bacteriostats, solutes which render the formulation isotonic with the blood of the intended recipient or suspending or thickening agents.
  • sterile injectable solutions or dispersions just prior to use
  • buffers bacteriostats, solutes which render the formulation isotonic with the blood of the intended recipient or suspending or thickening agents.
  • the rate of absorption of the drug then depends upon its rate of dissolution, which, in turn, may depend upon crystal size and crystalline form.
  • delayed absorption of a parenterally-administered drug form is accomplished by dissolving or suspending the drug in an oil vehicle.
  • injectable depot forms are made by forming microencapsule matrices of the subject compounds in biodegradable polymers, such as, for example, polylactide- polyglycolide. Depending on the ratio of drug to polymer, and the nature of the particular polymer employed, the rate of drug release can be controlled. Examples of other biodegradable polymers include, for example, poly(orthoesters) and poly(anhydrides). Depot injectable formulations are also prepared by entrapping the drug in liposomes or microemulsions, which are compatible with body tissue.
  • the compounds of the present invention which may be used in a suitable hydrated form, and/or the pharmaceutical compositions of the present invention, are formulated into pharmaceutically acceptable dosage forms by conventional methods known to those of skill in the art.
  • Actual dosage levels of the active ingredients in the pharmaceutical compositions of this invention may be varied so as to obtain an amount of the active ingredient, which is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient.
  • the selected dosage level will depend upon a variety of factors including, inter alia, the activity of the particular compound of the present invention employed, or the ester, salt or amide thereof, the route of administration, the time of administration, the rate of excretion of the particular compound being employed, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular compound employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts.
  • the required materials e.g., drugs, chemicals and reagents, human breast MCF7 cells, pharynx FaDu cells, prostate PC3 cells
  • the FaDu tumor xenograft in immunodeficient mice and 3-dimensional tumor histocultures were obtained, prepared and used described in U.S. Patent Application Serial No. 09/587,662, and Gan et al., FEBS Letters, 527:10-14, 2002.
  • Measurement of drug effect in cultured cells was as described in U.S. Patent Application Serial No. 09/587,662.
  • telomerase activity in cell lysates and intact cells was measured using intracellular TRAP, as follows. Cells (1 x 10 5 ) were washed twice with PBS and centrifuged. The cell pellet was resuspended in 100 ⁇ l of serum-free RPMI 1640 containing 5 u/ml of Streptolysin O, 2 ⁇ M of TS primer, and 50 ⁇ M of dNTP, and incubated at room temperature for 5 min.
  • TRAP Telomeric Repeat Amplification Protocol
  • the enzyme streptolysin O was used to increase the cell membrane permeability to the TS primer.
  • the TS primer was elongated by the intracellular telomerase in situ.
  • the elongated TS primer was then isolated from the cells and used as the template for PCR amplification.
  • 200 ⁇ l of RPMI 1640 medium containing 10% FBS was added to the cells to stop the permeating process.
  • the mixture was incubated at 30° C for 30 min to allow the extension of intracellular TS primer by telomerase.
  • the cells were then lyzed and the cell lysates, which contained the already extended TS primer, was directly analyzed by TRAP.
  • telomere length in cultured cells. Two methods were used to measure telomere length. The first method was a solution hybridization based telomere amount and length assay (TALA) (Gan, et al., Pharm. Res., 18:1655-1659, 2001) that measures the mean length of the terminal restriction fragments (TRF). The second method was fluorescence in situ hybridization (FISH) to detect telomere signal and to estimate the approximate length of individual telomere structures at the end of chromosomes. The FISH method is as described in U.S. Patent Application Serial No. 09/587,662 and Gan, et al., 2001.
  • Senescent cells were identified by 4- galactosidase staining as described (Dimri, et; al., 1995).
  • telomerase Inhibition of telomerase.
  • Suramin an agent with mild reverse transcriptase inhibitory activity but not known to inhibit telomerase, was studied in multiple human cancer cell lines, including human pharynx FaDu, human prostate PC3, and human breast MCF7. Its activity was compared with that of AZT.
  • Treatment Protocol Treatment with suramin or AZT was initiated after cells were allowed to attach to the growth surface in culture flasks. On the day of experiments, the culture medium was removed and replaced with inhibitor-containing medium. Drug concentrations of 0, 0.1 , 1 , 5, 10, 50 ⁇ M suramin, or O, 0.1 , 1 , 10, 100 ⁇ M AZT were employed. Telomerase activity in cell lysates and intact cells, and the telomere length, after 7-15 weeks of growth in medium containing suramin or AZT concentrations ranging from 0 to 50 ⁇ M, were measured. Effect of suramin and AZT on telomerase activity. Telomerase activity was inhibited by suramin and AZT in a concentration-dependent manner in human cancer cells. Concentrations resulting in 50% inhibition are shown in Table 1. The concentrations required for 90% inhibition were less than 50 micromolar.
  • telomerase occurs only in intact cells and not in cell extracts.
  • telomere length Effect of suramin and AZT on telomere length. Prolonged treatment (7-15 weeks) with suramin or AZT resulted in 34-55% telomere shortening in FaDu cells, and about 30% shortening in PC3 cells. Conclusion. Suramin effectively inhibits telomerase at low micromolar concentrations, and behaves similarly to the known telomerase inhibitor AZT.
  • Suramin inhibits telomerase and shortens telomeres in vivo.
  • the in vivo effectiveness of suramin as an inhibitor of telomerase activity was evaluated by measuring the telomere length in tumor cells implanted in immunosuppressed mice.
  • FaDu cells 0.5 ⁇ 1 x 10 6 cells in a volume of 100 ⁇ l
  • mice received, by intravenous injection into the tail vein, repeated doses of 10 mg/kg suramin.
  • the first dose was administered immediately after tumor implantation, with repeat doses given twice a week thereafter.
  • Tumors were collected after 2 to 6 weeks of suramin treatment.
  • Frozen tissue sections were analyzed for telomere length in individual cells using fluorescent in situ hybridization. About 10 microscope fields at 400-fold magnification were randomly chosen for each section, and the percentage of tumor cells with attenuated or lost telomere signals were counted.
  • mice treated with suramin or physiologic saline showed an equal tumor establishment rate of 100%, and undistinguishable rates of bodyweight increases.
  • the FISH result showed a gradual shortening of the telomeres of tumor cells over time.
  • the fraction of cells with a reduced or eliminated telomere signal remained at the control level of approximately 10% for the first two weeks of treatment, increasing to approximately 40% of cells at week 3, 75% at week 4, over 80% at week 5 and 95% at week 6. No change was observed in the tumor cells of saline-treated control animals.
  • Suramin effectively reduces telomere length in tumors grown in vivo.
  • EXAMPLE 3 PPS IS AN EFFECTIVE TELOMERASE INHIBITOR PPS inhibits telomerase. The inhibition of telomerase activity in FaDu cells after exposure to PPS was studied.
  • Treatment Protocol Treatment with PPS was initiated after cells were allowed to attach to the growth surface in culture flasks. On the day of experiments, the culture medium was removed and replaced with inhibitor-containing medium. Drug concentrations of 0, 0.1 , 1 , 10, 100, 1000 ⁇ g/ml of PPS were employed.
  • Telomerase activity was measured by the modified quantitative TRAP assay.
  • telomerase activity was inhibited by PPS in a concentration-dependent manner in FaDu and SKOV-3 cells.
  • the concentrations resulting in 50% inhibition were 0.56 and 0.60 ⁇ g/ml, respectively.
  • the concentrations resulting in 80% inhibition were less than 10 ⁇ g/ml for both cells.
  • the concentrations resulting in 90% inhibition were less than 100 ⁇ g/ml for both cells.
  • PPS is an effective inhibitor of telomerase activity in cells.
  • concentrations at which PPS produces 50% inhibition of telomerase activity are lower than the concentrations required for anticoagulation (above 1 ⁇ g/ml).
  • hTR ANTISENSE INHIBITS HUMAN TELOMERASE hTR antisense inhibits telomerase.
  • the antisense study consisted of the following steps: (a) construction of a sense and antisense to the RNA portion of the human telomerase (hTR), (b) stable transfection of cells with the hTR antisense (or hTR sense control), and (c) determination of the ability of the hTR antisense to inhibit telomerase activity and induce telomere shortening.
  • the methodologies are detailed in the art, for example, by Mo et al., Cancer Res. 2003.
  • Antisense and sense constructs The sense and anti-sense expression plasmids for human RNA portion of telomerase were prepared. The 185 basepair sense and antisense sequences are given below, and were found to agree with the GenBank sequence (accession No. NR_001566). These procedures resulted in 5 clones that contained the hTR fragment. Sequence analysis indicated that one clone was sense, whereas the other 4 clones were antisense. These hTR sense and hTR antisense expression plasmids were then transfected into human pharynx FaDu cancer cells.
  • Transfection procedures Transfection of the antisense construct used an IPTG-inducible mammalian expression system. The resulting clones were used for experiments.
  • Table 2 summarizes the results which show a slower growth rate for the antisense + IPTG cells (i.e., cells that were transfected by hTR antisense and treated with IPTG to induce the expression of hTR) compared to cells that had either not been transfected with the antisense, not transfected but treated with IPTG, transfected with the sense and treated with IPTG, or transfected with the antisense but without the IPTG induction (i.e., control, + IPTG, + sense + IPTG, and antisense).
  • IPTG cells i.e., cells that were transfected by hTR antisense and treated with IPTG to induce the expression of hTR
  • hTR antisense Effect of hTR antisense on the cytotoxic effect of paclitaxel.
  • Two clones of cells that were stably transfected with the hTR antisense were studied.
  • the cytotoxic effect of paclitaxel was quantified using the SRB method, which measures the total cellular proteins.
  • the cells transfected with hTR antisense were treated with IPTG for 44 (clone#1) to 57 (clone #2) days, and then with paclitaxel for 96 hours.
  • Telomere length (referred to as terminal restriction fragment) was measured using the TALA method. Telomerase activity was measured using the improved TRAP method. The results, summarized in Table 2, show that the hTR antisense reduced the telomere length and telomerase activity.
  • Tumor size reduction after low-dose suramin in some animals was determined in immunosuppressed mice implanted with FaDu tumors.
  • mice were injected subcutaneously in the thigh area with 5x10 5 FaDu cells.
  • Suramin (10 mg/kg) was administered by intraperitoneal injection, twice-weekly for 6 weeks.
  • Suramin administration was initiated on the day of tumor cell inoculation.
  • the control animals were treated identically, except the suramin solution for injection was replaced by a physiologic saline solution. Tumor sizes were observed twice-weekly. Effect of low-dose suramin treatment of tumor growth.
  • Six animals received suramin. In five animals, tumor size increased with time. The tumor in one of the remaining animals initially grew, reaching a size of about 4 mm after 2 weeks, but then declined, and completely disappeared by 6 weeks.
  • ACTIVITY OF CHEMOTHERAPY IN TUMOR-BEARING ANIMALS This example describes the enhancement of the antitumor effect of a chemotherapy agent after prolonged pre-treatment with a telomerase inhibitor. Telomerase inhibition treatment was initiated when the tumor burden is low and not yet palpable, comparable to situation of minimal or undetectable tumors.
  • IIIB/IV nonsmall cell lung cancer were treated with paclitaxel, carboplatin, and suramin. Treatment was administered about every 3 weeks.
  • the loading dose of suramin was approximately 240 mg/m 2 and the subsequent doses were calculated based on a mathematical equation Applicants have developed (PCT Application No. PCT/US02/30210). These suramin doses resulted in plasma concentration between about 2 to about 90 micromolar over at least 21 days. As shown in Example 1 , these concentrations are sufficient to inhibit telomerase.
  • a total of 54 patients were treated. The first 6 patients received suramin as a single dose, and the remaining patients received suramin in two split doses given 24 hours apart. No toxicity attributed to the use of suramin was observed.
  • the overall response rate was 40.8% (consisting of 6% complete response which corresponds to no measurable disease and 34.8% partial response which corresponds to at least 50% tumor shrinkage), the time to disease progression (TTP) was longer than 6 months, and the median survival time (MST) was longer than 12 months (Villalona- Calero, et al., Clin. Cancer Res., 9:3303-3311, 2003; Villalona-Calero, et al., IASLC Meeting, Vancouver, August, 2003).
  • Treatment protocol Four beagle dogs, weighing 11.4 ⁇ 0.4 kg, were used. The animals were cannulated in the cephalic veins of both front legs. Suramin (6.75 mg/kg) was infused intravenously over 30 minutes into one vein, while blood samples were obtained from the other vein. Suramin was administered as an aqueous solution of sodium suramin. Blood samples were taken at 5, 30 minutes, 1 , 2, 4, 6, 9, 12, 24, 48, 72 hours, and on day 7, 14, and 21 , placed in heparinized tubes, and plasma prepared by centrifugation.
  • Plasma concentrations of suramin were determined using high performance liquid chromatography, as previously described (Kassack, et al., J Chromatogr.B Biomed.Appl., 686:275-284, 1996). Non- compartmental pharmacokinetic analysis was performed by standard means (Gibaldi, et al., Pharmacokinetics., 1982).
  • This Example teaches that a second telomerase inhibitor, pentosan polysulfate (PPS), enhances the antitumor activity of a cytotoxic agent in cultured tumor cells and primary cultures of patient tumors.
  • PPS pentosan polysulfate
  • PPS occurs at the telomerase-inhibitory concentrations of 10 and 100 ⁇ g/ml, which are >10-fold and >100-fold lower than the PPS concentrations that produce antitumor activity (Wellstein, et al., J. Natl. Cancer Inst, 83:716-720, 1991 ; Switzerlandmaier, et al., Ann. NYAcad. Sciences, 886:243-248, 1999).
  • 5-Fluorouracil was used as the chemotherapeutic agent.
  • Cells were treated with 5-fluorouracil for 96 hours, with and without PPS.
  • the drug effect was measured as inhibition of the incorporation of a DNA precursor (bromodeoxyuridine or BrdU) using ELISA.
  • the results show that PPS had no cytotoxicity at 10 and 100 ⁇ g/ml; the IC 50 of PPS, as a single agent, was ⁇ 1 ,400 ⁇ g/ml.
  • Table 4 shows that the IC 50 values (i.e., drug concentrations required to produce 50% inhibition) of 5- fluorouracil was reduced by the addition of PPS.
  • Results in Table 4 further show that addition of a second telomerase inhibitor, suramin at a telomerase-inhibitory concentration (i.e., 30 ⁇ M), further reduced the IC 50 of 5-fluorouracil, indicating that the chemosensitization effect of telomerase inhibitors are additive.
  • This example describes the enhancement of the antitumor effect of a chemotherapeutic (i. e. , paclitaxel), by the telomerase inhibitor AZT, in immunodeficient mice bearing human head and neck cancer FaDu xenografts.
  • a chemotherapeutic i. e. , paclitaxel
  • AZT telomerase inhibitor
  • paclitaxel The activity of paclitaxel, with or without AZT, was evaluated in immunodeficient mice (male Balb/c nu/nu mice, 6-8 weeks old) bearing the human pharynx FaDu xenografts.
  • Xenografts were formed by subcutaneous injection of 10 6 viable tumor cells in 0.1 ml physiologic saline in the right and left flank areas, and were allowed to grow for about 14 days to reach a size of >15 mm 3 before drug treatment was started.
  • the four treatment groups were: saline control, AZT, paclitaxel, paclitaxel + AZT.
  • the saline control group received injections of 200 ⁇ l/day of physiological saline for five consecutive days.
  • the paclitaxel group received injections of 10 mg/kg/day paclitaxel dissolved in Cremophor and ethanol (i.e., Taxol) in a volume of 200 ⁇ l for five consecutive days.
  • the AZT group received a seven-day infusion of AZT at a rate of 200 ng/hour by a subcutaneously implanted Alzet minipump.
  • the paclitaxel + AZT group received the combined treatment of the paclitaxel group and the AZT group, where the AZT infusion was started one day prior to the start of the paclitaxel injections. Animal weights and tumor sizes were measured on days 1 , 3, 6, 8, and 10 after initiation of the paclitaxel treatment.
  • the antitumor effect of the drug treatments was measured in three ways. The first was the reduction in tumor size. Tumor sizes were determined by first preparing a mold of the extruding tumor using Jeltrate, a rapidly setting molding material, and then preparing and weighing the countermold. Second, the apoptotic effect was measured. The animals were euthanized on day 10, and the tumors were harvested and fixed in formalin. Histologic sections of 5-micron thickness were prepared and stained with hematoxylin and eosin. The tumor sections were evaluated morphologically for tumor cell density, and density of apoptotic cells. Because apoptotic cells disappear over time, the density of non-apoptotic cells is a secondary indicator of apoptosis.
  • Cell densities were determined by counting the number of cells in four randomly selected microscopic fields at 400 x magnification, using image analysis procedures (Song, et al., Proc.Natl.Acad.Sci.USA, 97:8658- 8663, 2000). Third, the ability of drug treatment to prolong the survival time was measured. For this study, the animals were monitored for 100 days, or until moribundity, defined by a tumor length exceeding 1.0 cm, was reached. The concentration of AZT in plasma was determined in a parallel experiment, using three mice, subcutaneously implanted with an Alzet 1002 osmotic minipump. Drug infusion was allowed to take place for four days before the blood of the animals was harvested. This long duration of infusion guaranteed that constant steady-state plasma concentrations had been achieved. AZT concentrations in plasma were determined using a commercially available ELISA assay (Neogen, Lexington, KY).
  • Treatments with single agents produced minimal toxicity, with no toxicity-related death and minimal body weight loss compared to the pretreatment weight ( ⁇ 3%).
  • the addition of AZT to paclitaxel did not enhance the body weight loss, indicating that AZT did not enhance the host toxicity of paclitaxel.
  • the concentration of AZT in plasma was 9.6 nM. This concentration is well below the concentration required for inhibition of telomerase (2 ⁇ M in FaDu cells, see Table 1), or induction of cytotoxicity (20 ⁇ M in FaDu cells; Mo et al., Cancer Res. 63:579-585, 2003). Hence the result of this study is surprising.
  • Tumor size reduction after AZT in animals The effect of repeated administration of single agent AZT on tumor size was determined in immunosuppressed mice implanted with FaDu tumors.
  • AZT Treatment Protocol. Immunosuppressed mice were injected subcutaneously in both flanks with 10 6 FaDu cells per flank. AZT was administered by continuous subcutaneous infusion using an Alzet® osmotic minipump at a rate of 5 ⁇ g/mouse/day. This AZT dose resulted in steady state plasma concentrations of about 10 ng/ml. AZT administration was started 14 days after inoculation, and lasted 14 days. The control animals were treated identically, except the AZT solution for injection was replaced by a physiologic saline solution. Tumor sizes were observed twice-weekly. Effect of AZT treatment of tumor growth. All 16 animals implanted with tumor cells showed measurable tumors prior to AZT treatment.
  • the tumors stopped growing upon AZT administration, and subsequently declined in size.
  • the tumors were not palpable on day 38, and completely disappeared on day 59, as confirmed by necropsy examination.
  • the average initial tumor size was 35 mm 3 (range, 18-80 mm 3 ), which was similar to the tumor sizes in the remainder of the animals.
  • This example describes the expected enhancement of the antitumor effect of a cytoreductive treatment when the cytoreductive treatment is combined with long-term treatment with a telomerase inhibitor.
  • the treatment with the telomerase inhibitor can start prior to, concurrently with, or after completion of the cytoreductive treatment.
  • telomere inhibitors Effect of long-term treatment with a telomerase inhibitor in combination with a cytoreductive treatment
  • the tumor burden in the host must be small so that the tumor burden does not reach the lethal level before the telomerase inhibitor can erode the telomere length to below the critical level for inhibiting proliferation and for inducing apoptosis and cell senescence. This can be accomplished by using cytoreductive treatments.
  • telomere lengths in the tumor cells will decrease over time, and that, eventually, apoptosis or cell senescence will be induced.
  • Treatment protocol Patients, presenting with a cancer for which the best treatment option is a form of cytoreductive therapy, would be treated with the cytoreductive treatment of choice.
  • the cytoreductive treatment selected for an individual patient would depend on the patient's cancer, health status, age, previous treatment history, and other factors usually considered in the selection of a treatment protocol.
  • the patient Upon completion of the cytoreductive treatment regimen, and if the patient is not considered cured, or recurrence of the disease is considered likely, the patient would receive continuing treatment with a telomerase inhibitor, at a dose level that is sufficient to induce inhibition of telomerase.
  • the duration of the continuing treatment would be protracted, lasting at least two weeks, but preferably longer than two months, or more preferably longer than six months, or more preferably until the patient is considered cured.
  • An alternative treatment protocol where the telomerase inhibitor treatment is initiated prior to, or concurrently with the cytoreductive treatment, and continued after completion of the cytoreductive treatment regimen, would be advantageous, as the tumor would be exposed to the telomerase inhibiting effect for a longer period of time.
  • This example describes regional administration of a telomerase inhibitor, to achieve telomerase inhibitory concentrations in the targeted tissue or organ, while presenting low and not effective telomerase inhibitory concentrations in the plasma.
  • the animals were cannulated in a cephalic vein for administration of anesthetics, and in a jugular vein for blood sampling.
  • a urethral catheter was inserted for dose instillation and sampling of the bladder contents.
  • Animals were given intravesical doses of suramin (20 ml containing 6 mg/ml suramin) in water.
  • Concentrations of the bladder contents and systemic plasma were sampled at frequent intervals for 120 minutes, at which time the bladder tissue was harvested and the animal sacrificed.
  • Tissue sections of approximately 2 cm x 2 cm surface area were cut from the bladder wall, and rapidly frozen on a flat stainless steel plate cooled on dry ice. The outer edges of the tissue samples were trimmed to avoid contamination with instillation fluid, and the frozen tissues cut into 40 ⁇ m sections parallel to the urothelial surface.
  • Suramin concentrations in the tissue layers, plasma and bladder contents were determined by HPLC analysis.
  • Cell culture assay experiments can be performed in the human prostate PC3 tumor cells, the human breast MCF7 cells, or the human pharynx FaDu cells. If the requirements of the invention are met in any of the three cell cells, the choice and the dosage of the telomerase inhibitor is suitable for use in the invention. Preferably the PC3 cells are used.
  • Human prostate PC3 tumor cells, breast MCF7 cells, or pharynx FaDu cells can be obtained from the American Type Culture Collection. The doubling time of all three cell lines is approximately 24-hour. All three cell lines should be cultured as monolayers in a humidified environment containing 5% CO 2 and 95% air, at 37° C. PC3 cells should be maintained in RPMI 1640 medium, MCF7 cells in either RPMI 1640 or Minimal Essential Medium (MEM), and FaDu cells in MEM.
  • All culture media should be supplemented with 9% heat-inactivated fetal bovine serum, 2 mM I- glutamine, 0.1% 10 mM non-essential amino acids, 90 ⁇ g/ml gentamicin, and 90 ⁇ g/ml cefotaxime sodium.
  • Cells are harvested from subconfluent cultures using trypsin and resuspended in fresh medium before plating. Cells with > 90% viability, as determined by trypan blue exclusion, are used to evaluate the cytotoxicity of a telomerase inhibitor, e.g., suramin.
  • a telomerase inhibitor e.g., suramin.
  • Cells are plated in 96 well microtiter plates at a density such that confluence would not be achieved at the end of the drug treatment period.
  • Cells are allowed to attach to the plate surface by growing in drug-free medium for 20 to 24 hr. Afterward, cells are incubated with the FGF antagonist (one example used 0.2 ml of suramin)-containing culture medium, at concentrations spanning at least 4 log scales. The drug effect should be measured as inhibition of BrdU incorporation, e.g., according to the Cell Proliferation ELISA BrdU (Boehringer Mannheim).
  • Suramin is a potent inhibitor of vascular endothelial growth factor. A contribution to the molecular basis of its antiangiogenic action., J. Mol.Cell Cardiol., 28, 1523-1529, 1996.

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Abstract

La présente invention concerne des techniques et des compositions permettant d'inhiber l'activité télomérase et un traitement de maladies ou d'états induits par la télomérase. Ces techniques, ces composés et ces compositions peuvent être utilisés seuls ou en combinaison avec d'autres agents pharmaceutiquement actifs, une intervention chirurgicale ou un traitement par rayons pour traiter des états ou des maladies induites par l'activité télomérase, par exemple pour traiter un cancer.
PCT/US2004/002609 2003-01-31 2004-01-30 Techniques et compositions d'utilisation de suramine, de polysulfate pentosane, d'antisens de telomerase et d'inhibiteurs de la telomerase WO2004070008A2 (fr)

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EP04706977A EP1606302A4 (fr) 2003-01-31 2004-01-30 Techniques et compositions d'utilisation de suramine, de polysulfate pentosane, d'antisens de telomerase et d'inhibiteurs de la telomerase
AU2004209428A AU2004209428A1 (en) 2003-01-31 2004-01-30 Methods and compositions for using suramin, pentosan polysulfate, telomerase antisense, and telomerase inhibitors
JP2006503176A JP2007525414A (ja) 2003-01-31 2004-01-30 スラミン、ペントサン・ポリサルフェート、テロメラーゼアンチセンス、及びテロメラーゼ阻害剤を用いるための方法及び成分
CA002515000A CA2515000A1 (fr) 2003-01-31 2004-01-30 Techniques et compositions d'utilisation de suramine, de polysulfate pentosane, d'antisens de telomerase et d'inhibiteurs de la telomerase
US11/193,883 US20050282893A1 (en) 2004-01-30 2005-07-29 Methods and compositions for using suramin, pentosan, polysulfate, telomerase antisense and telomerase inhibitors

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EP3554505A4 (fr) * 2016-12-13 2020-09-16 Beta Therapeutics Pty. Ltd. Méthodes de traitement de troubles oculaires
US11718609B2 (en) 2016-12-13 2023-08-08 Beta Therapeutics Pty Ltd Heparanase inhibitors and use thereof
US11787783B2 (en) 2016-12-13 2023-10-17 Beta Therapeutics Pty Ltd Heparanase inhibitors and use thereof

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NZ708920A (en) * 2012-12-07 2020-08-28 Geron Corp Use of telomerase inhibitors for the treatment of myeloproliferative disorders and myeloproliferative neoplasms
JPWO2022114111A1 (fr) * 2020-11-27 2022-06-02

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US5882893A (en) * 1997-12-04 1999-03-16 Millennium Pharmaceuticals, Inc. Nucleic acids encoding muscarinic receptors and uses therefor

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP3554505A4 (fr) * 2016-12-13 2020-09-16 Beta Therapeutics Pty. Ltd. Méthodes de traitement de troubles oculaires
US11718609B2 (en) 2016-12-13 2023-08-08 Beta Therapeutics Pty Ltd Heparanase inhibitors and use thereof
US11787783B2 (en) 2016-12-13 2023-10-17 Beta Therapeutics Pty Ltd Heparanase inhibitors and use thereof

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AU2004209428A1 (en) 2004-08-19
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EP1606302A2 (fr) 2005-12-21
WO2004070008A8 (fr) 2005-09-29
CN1768075A (zh) 2006-05-03
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