WO2004112730A2 - Method and compositions for treating tumors - Google Patents

Method and compositions for treating tumors Download PDF

Info

Publication number
WO2004112730A2
WO2004112730A2 PCT/US2004/019999 US2004019999W WO2004112730A2 WO 2004112730 A2 WO2004112730 A2 WO 2004112730A2 US 2004019999 W US2004019999 W US 2004019999W WO 2004112730 A2 WO2004112730 A2 WO 2004112730A2
Authority
WO
WIPO (PCT)
Prior art keywords
tumor
nucleoside analog
patient
tezacitabine
prodrug
Prior art date
Application number
PCT/US2004/019999
Other languages
French (fr)
Other versions
WO2004112730A3 (en
Inventor
Sharon Lea Aukermann
Michelle D. Bronson
Wendy Fantl
Barbara Hibner
Original Assignee
Chiron Corporation
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Chiron Corporation filed Critical Chiron Corporation
Publication of WO2004112730A2 publication Critical patent/WO2004112730A2/en
Publication of WO2004112730A3 publication Critical patent/WO2004112730A3/en

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/106—Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers

Definitions

  • the invention relates to treating tumors resistant to nucleoside analogs, particularly gemcitabine-resistant tumors.
  • Anticancer agents of all types can become less efficacious over time. This decreased sensitivity is called resistance. Resistance can be classified as “innate” in which case the tumor is refractory to a certain therapy or resistance may be “acquired” during or after a particular therapy. A variety of mechanisms of resistance have been described in the literature, which include "host factors,” such as poor absorption, increased metabolism, low tolerance, or poor penetration of the drug to the tumor site. Kinsella & Smith, Gen. Pharmacol. 30, 623-26, 1998; Tannock et al., Clin. Cancer Res. 8, 878-84, 2002. Polymorphic genes present in human populations affect each of these parameters.
  • nucleoside analogs [04] Several resistance mechanisms have been described for nucleoside analogs. First, increased levels of cytidine deaminase may lead to low serum levels of prodrug through increased metabolism. Neff & Blau, Cancer Exp. Hematol. 11, 1340-46, 1996. Second, uptake of the analog may be reduced through alterations in expression of the equilabrative nucleotide transporters (ENTs) or concentrative nucleotide transporters (CNTs).
  • ENTs equilabrative nucleotide transporters
  • CNTs concentrative nucleotide transporters
  • gemcitabine is a substrate for four of the nucleoside transporters found in humans, ei, es, cib and cit, and nucleotide transporter activity is required for gemcitabine toxicity in cells. Mackey et al., Cancer Res.
  • Nucleotide transporter deficiency may confer significant gemcitabine resistance. Mackey et al., Clin. Cancer Res. 8, 110-16, 2002. Resistance also can result from other means of altering the balance of drugs within cells, such as the presence or activation of efflux pumps (e.g., MDR), changes in membrane lipids, or increased metabolism of the drug inside the cell. Once inside the cell, there may also be activating or deactivating mutations in the target genes, modulation of expression of target genes such as deoxycytidine kinase and ribonucleotide reductase (Bergman et al., Drug Resist. Update 5, 19, 2002; Zhou et al, Cytogenet. Cell. Genet. 95, 34-42 2001), amplification of target genes, increased DNA repair, or changes in the DNA damage response pathways leading to altered apoptotic responses. Shi et al., Oncology 62, 354-62, 2002.
  • efflux pumps e.g., MDR
  • a method to aid in determining whether a tumor of a patient is likely to respond to gemcitabine comprising the step of: examining a sample of a tumor of a patient to detect a mutation in a ras gene.
  • ras gene is H-ras. 7. The method of embodiment 1 further comprising the step of detecting a level of cytidine deaminase activity or an amount of cytidine deaminase expression in a biological sample of the patient.
  • a kit comprising: an oligonucleotide probe for detecting a mutation in a ras gene; and a package insert that describes the method of embodiment 1.
  • a method to aid in determining whether a tumor of a patient is likely to respond to tezacitabine comprising the step of: examining a sample of the tumor to detect a mutation in a ras gene.
  • the method of embodiment 11 further comprising the step of detecting a level of cytidine deaminase activity or an amount of cytidine deaminase expression in a biological sample of the patient.
  • kits comprising: an oligonucleotide probe for detecting a mutation in a ras gene; and a package insert that describes the method of embodiment 11.
  • a method to aid in determining whether a tumor of a patient is likely to respond to gemcitabine comprising the steps of: detecting a level of cytidine deaminase activity or an amount of cytidine deaminase expression in a biological sample of the patient.
  • a kit comprising: a substrate for cytidine deaminase or a reagent for detecting cytidine deaminase expression; and a package insert that describes the method of embodiment 21.
  • a method to aid in determining whether a tumor of a patient is likely to respond to tezacitabine comprising the steps of: examining a first sample of the tumor to detect a level of cytidine deaminase activity or an amount of cytidine deaminase expression.
  • a kit comprising: a substrate for cytidine deaminase or a reagent for detecting cytidine deaminase expression; and a package insert that describes the method of embodiment 26.
  • a method to aid in determining whether a tumor of a patient is likely to respond to a combination of tezacitabine and (a) a nucleoside analog that inhibits thymidylate synthase or (b) a prodrug of the nucleoside analog comprising the step of: examining a sample of the tumor to detect a level of thymidylate synthase activity or an amount of thymidylate synthase expression.
  • kits comprising: a substrate for thymidylate synthase or a reagent for detecting thymidylate synthase expression; and a package insert that describes the method of embodiment 30.
  • a method of treating a patient having a nucleoside analog-resistant tumor comprising the step of administering to the patient a therapeutically effective amount of tezacitabine.
  • nucleoside analog-resistant tumor is resistant to gemcitabine.
  • nucleoside analog-resistant tumor is a tumor of the breast, prostate, lung, colon, stomach, pancreas, ovary, brain, colon, rectum, esophagus, kidney, bladder, or blood.
  • nucleoside analog-resistant tumor is a gastroesophageal tumor.
  • kits comprising: a pharmaceutical composition comprising tezacitabine; and a package insert that describes the method of embodiment 34.
  • a method of treating a patient having a nucleoside analog-resistant tumor comprising the steps of: administering to the patient a therapeutically effective amount of tezacitabine; and administering to the patient a therapeutically effective amount of a secondary nucleoside analog or a prodrug thereof.
  • the method of embodiment 44 further comprising the step of detecting a level of thymidine phosphorylase activity or expression in a biological sample of the patient before administering the secondary nucleoside analog or the prodrug.
  • nucleoside analog-resistant tumor is a tumor of the breast, prostate, lung, colon, stomach, pancreas, ovary, brain, colon, rectum, esophagus, kidney, bladder, or blood.
  • nucleoside analog-resistant tumor is a gastroesophageal tumor.
  • nucleoside analog-resistant tumor is a colorectal tumor.
  • a kit comprising: a pharmaceutical composition comprising tezacitabine; and a package insert that describes the method of embodiment 44.
  • kit of embodiment 62 further comprising a pharmaceutical composition comprising a secondary nucleoside analog or a prodrug thereof.
  • a method of treating a patient having a tumor, wherein cells of the tumor comprise a mutation in a ras gene comprising the step of administering to the patient a therapeutically effective amount of a primary nucleoside analog selected from the group consisting of tezacitabine and gemcitabine.
  • a primary nucleoside analog selected from the group consisting of tezacitabine and gemcitabine.
  • tumor is a tumor of the breast, prostate, lung, colon, stomach, pancreas, ovary, brain, colon, rectum, esophagus, kidney, bladder, or blood.
  • kits comprising: a pharmaceutical composition comprising tezacitabine or gemcitabine; and a package insert that describes the method of embodiment 64.
  • kit of embodiment 78 wherein the package insert further describes the method of embodiment 71.
  • a method of treating a patient having a tumor wherein cytidme deaminase activity in a first biological sample of the patient is at least about 20 nmol/hr/mg or wherein the amount of cytidine deaminase expression in a second biological sample is at least about 10% higher than a baseline amount of cytidine deaminase expression in a corresponding biological sample of an individual who does not have a tumor, comprising the step of: administering to the patient a therapeutically effective amount of tezacitabine.
  • cells of the tumor comprise a mutation in a ras gene.
  • the tumor is a tumor of the breast, prostate, lung, colon, stomach, pancreas, ovary, brain, colon, rectum, esophagus, kidney, bladder, or blood.
  • a kit comprising: a pharmaceutical composition comprising tezacitabine or gemcitabine; and a package insert that describes the method of embodiment 80.
  • FIG. 1 Correlation of tezacitabine sensitivity with a K-ras mutation.
  • the invention provides various methods to aid in to aid in determining whether a tumor of a patient is likely to respond to gemcitabine or tezacitabine or to a combination of gemcitabine or tezacitabine and a secondary nucleoside analog or prodrug thereof.
  • a tumor "responds" to a particular therapy if the size of the tumor does not substantially change or if its size decreases by at least 5, 10, 15, 20, 25, or 50% or more over a predetermined period of time (e.g., 1 week, 2 weeks, 3 weeks, 1 month, 2 months, etc.).
  • Patients having a variety of solid or liquid tumors including, but not limited to, gastroesophageal tumors, esophageal tumors, and tumors of the breast, prostate, lung, colon, stomach, pancreas, ovary, brain, colon, rectum, esophagus, kidney, bladder, or blood, can benefit from screening methods of the invention.
  • the activation of the Ras oncogene is the most frequent gain-of-function mutation detected in human cancer occurring in approximately 30% of all human tumors.
  • Patients whose tumors contain a ras mutation are likely to respond to treatment with gemcitabine or tezacitabine or to a combination of gemcitabine or tezacitabine and a secondary nucleoside analog or a prodrug thereof. See FIG. 1.
  • a sample of a tumor of a patient is examined to detect a mutation in a ras gene.
  • the ras gene can be K-ras ras, N-ras, or H-ras, including splice variants of ras genes such as K-ras 4A and K-ras 4B.
  • Ras mutations can be detected by any known methods including, but not limited to, direct sequencing or single strand conformational polymorphism of PCR products generated from total tumor RNA derived from patient biopsies (Easwaran et al., Cancer Research 63, 3145, 2003).
  • Secondary nucleoside analogs useful in combination with tezacitabine or gemcitabine are nucleoside analogs other than tezacitabine or gemcitabine; if a tumor is resistant to nucleoside analogs, the secondary nucleoside analog or the prodrug preferably is different that the particular nucleoside analog(s) to which a tumor is resistant.
  • Secondary nucleoside analogs useful in the present invention include, but are not limited to, gemcitabine, cytarabine, fludarabine, cladribine, troxacitabine, thioguanine, triapine, and pentostatin.
  • Capecitabine for example, is a prodrug of 5-fluorouracil.
  • a level of cytidine deaminase activity or an amount of cytidine deaminase expression is determined in a biological sample of the patient.
  • Biological samples include, for example, blood samples and tumor samples.
  • Cytidine deaminase expression can be measured in a variety of ways. In one method, aliquots of purified cytidine deaminase protein or cell lysates are incubated with a substrate, such as 5'-dFCrd. Samples are incubated in a buffered reaction mixture, and the amount of product, 5'-dUrd, is measured in the supernatant using HPLC after centrifugation. See Watanabe & Uchica, Biochem. Biophys. Acta 1312, 99-104, 1996).
  • cytidine deaminase activity can be measured by evaluating the conversion of a radiolabeled substrate, such as tritiated cytidine, into uridine. Labeled cytidine remaining in the mixture is captured on filters and read by scintillation counting. Neff & Blau, Exptl. Hematol. 24, 1340-46, 1996. It is also possible to evaluate the rate of deamination by cytidine deaminase by determining the decrease in absorbance in a spectrophotometer at 286 nm. Bouffard et al., Biochem. Pharmacol. 45, 1857-61, 1993.
  • Levels of cytidine deaminase protein or mRNA expression also can be determined as an indicator of cytidine deaminase activity. For example, Western blot analysis or other immunocytochemically-based methods can be used to detect levels of cytidine deaminase protein. Levels of cytidme deaminase mRNA can be determined, for example, by Northern blot analysis or PCR-based methods.
  • both a ras mutation and a level of cytidine deaminase activity or an amount of cytidme deaminase expression are determined.
  • Patients whose tumors contain a ras mutation and whose cells, particularly tumor cells, contain increased levels of cytidme deaminase activity or expression are particularly suited for treatment with tezacitabine, either alone or in combination with a secondary nucleoside analog or a prodrug thereof.
  • Patients whose tumors contain a ras mutation and whose cells, particularly tumor cells contain a low or baseline amount of cytidine deaminase activity or expression can be treated with gemcitabine or with tezacitabine.
  • the level of cytidine deaminase activity in the biological sample of the patient is preferably at least about 20, 30, 40, 50, 60, 70, 80, 90, or 100 or more nmole/hr/mg or the amount of cytidine deaminase expression in a biological sample of the patient preferably is at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 250, 300, 350, 400, 450, or 500% higher than a baseline amount of cytidine deaminase expression in a corresponding biological sample of an individual who does not have a tumor.
  • the invention also provides methods to aid in determining whether a tumor of a patient is likely to respond to a combination of tezacitabine and (a) a nucleoside analog that inhibits thymidylate synthase or (b) a prodrug of the nucleoside analog.
  • Nucleoside analogs that inhibit thymidylate synthase include, but are not limited to, AG-331, nolatrexed, 5- fluorouracil, raltitrexed, BGC-9331, FO-152, lometrexol, LY-225693, LY-288784, LY- 295248, pemetrexed, DMPDDF, OSI-7904L, CB-30900, 5-fiuorocytosine derivatives, UCN-01, NSC-678515, NB-1011, doxifluridine, AO-90, OGT-719, trimexetrate, pyrmidine deoxynucleoside analogs, galocitabine, edatrexate, MVA-FCU1, sorivudine, ICI-198583, and ZM-246315.
  • the prodrug is capecitabine.
  • Such screening methods can be applied to assess a variety of tumors including, but not limited to, gastroesophageal tumors, esophageal tumors, and tumors of the breast, prostate, lung, colon, stomach, pancreas, ovary, brain, colon, rectum, esophagus, kidney, bladder, or blood.
  • One embodiment of the invention provides a method to aid in determining whether a tumor of a patient is likely to respond to a combination of tezacitabine and (a) a nucleoside analog that inhibits thymidylate synthase or (b) a prodrug of the nucleoside analog, such as capecitabine.
  • a sample of the tumor is examined to detect a level of thymidylate synthase activity or an amount of thymidylate synthase expression.
  • Thymidylate synthase mRNA expression can be measured as described above.
  • thymidylate synthase mRNA expression is in the range of 0.9 - 20.1 x 10 "3 relative to expression of a housekeeping gene (e.g., ⁇ -actin)
  • a tumor likely will respond to a combination of tezacitabine and (a) a nucleoside analog that inhibits thymidylate synthase or (b) a prodrug of the nucleoside analog, such as capecitabine.
  • Thymidylate synthase mRNA expression preferably is 10 - 15 x 10 "3 , 5 - 10 x 10 "3 , or 4 - 6 x 10 "3 relative to expression of a housekeeping gene. See, e.g., Shirota et al., J. Clin. Oncol. 19, 4298-4304, 2001.
  • Corresponding levels of thymidylate synthase protein can be measured as described, for example, in U.S. Patent 6,569,634. Corresponding levels of thymidylate synthase activity can be measured using any methods known in the art. See, e.g., Baba et al., Cancer Chemother Pharmacol. 2003 Sep 11 (e-pub); Higashiyama et al., Lung Cancer. 2001 Dec;34(3):407-16.
  • the invention also provides methods for treating patients who have nucleoside analog- resistant tumors, particularly gemcitabine-resistant tumors.
  • Patients with nucleoside analog-resistant tumors have a cancer that has progressed either while being treated with the nucleoside analog or within six months of completing treatment with the nucleoside analog.
  • Such patients can be treated with (E)-2'-deoxy-2'-(fluoromethylene)cytidine (FMdC, tezacitabine), either alone or in combination with an effective amount of one or more secondary nucleoside analog.
  • Nucleoside analog-resistant tumors that can be treated according to the invention include any tumor that has become resistant to a nucleoside analog, particularly gemcitabine.
  • Such tumors include, but are not limited to, gastroesophageal, colorectal, breast, prostate, lung, colon, stomach, pancreatic, ovarian, brain and hematopoietic cancers, non-small cell lung cancer, colorectal cancer, leukemia, lymphoma, esophageal carcinoma, renal cell carcinoma, bladder cancer, head and neck cancer, and sarcomas such as cholangiosarcoma and esophageal sarcoma.
  • Tezacitabine is an analog of deoxycytidine that possesses anti-tumor activity. Once inside a cell, tezacitabine is phosphorylated to yield the mono-, di- and triphosphate forms. Tezacitabine diphosphate irreversibly inhibits ribonucleotide reductase, resulting in decreased deoxynucleotide pools inside the cell. Tezacitabine triphosphate can be incorporated by DNA polymerase into DNA; this incorporation causes DNA synthesis chain termination and eventual cell death. Together these two activities are self- potentiating; decreased deoxynucleotide pools lead to increased incorporation of tezacitabine triphosphate in DNA in a self-potentiating mechanism. In this way, tezacitabine is a dual-acting nucleoside analog.
  • tezacitabine differs from gemcitabine in a number of characteristics, including its susceptibility to deamination, its potency of ribonucleotide reductase (RNR) inhibition, and its affinities for deoxycytidine kinase and DNA polymerase ⁇ (see Tables 1 and 2). Because tezacitabine is a more potent and irreversible RNR inhibitor, tezacitabine can overcome gemcitabine resistance due to overexpression or activation of ribonucleotide reductase.
  • RNR ribonucleotide reductase
  • tezacitabine can be used to treat tumors that have become resistant to other nucleoside analogs that differ from tezacitabine in one or more aspects, such as having a different mechanism for entering the cell, different NT sensitivities, a different drug metabolism, different effects or utilization of alternative DNA repair mechanisms, and the like.
  • nucleoside analog-resistant tumors Treatment of nucleoside analog-resistant tumors with a combination of tezacitabine or gemcitabine and a secondary nucleoside analog or a prodrug of a secondary nucleoside analog
  • deoxycytidine analogs require phosphorylation by deoxycytidine kinase to form the monophosphate form, and this is typically the rate limiting step.
  • Deoxycytidine kinase is regulated by the levels of the nucleotide pools, which in turn can be controlled by ribonucleotide reductase.
  • Treatment with tezacitabine a potent and irreversible inhibitor of RNR, could lead to greater activity of deoxycytidine kinase, thereby potentiating its own activity and the activity of any other nucleoside analog that has deoxycytidine kinase as a rate limiting step.
  • a decrease in the intracellular pool of deoxynucleotides can increase the activity of chemotherapy agents that induce DNA damage, such as oxaliplatin, cisplatin, topotecan, irinotecan and adriamycin.
  • irreversible inhibition of RNR by tezacitabine may allow better incorporation of other nucleoside analogs even in tumors that were previously resistant to nucleoside analog therapy.
  • Tezacitabine or gemcitabine and a secondary nucleoside analog or prodrug thereof can be administered together in the same formulation or separately. Separate formulations are useful for treatment protocols in which there is a predetermined time between the administration of tezacitabine or gemcitabine and the secondary nucleoside analog or prodrug thereof, with either agent being administered first.
  • a biological sample of a patient can be assessed to detect a level of thymidine phosphorylase activity or expression in a biological sample of the patient before administering the secondary nucleoside analog or the prodrug.
  • Suitable samples include blood and tumor samples.
  • Capecitabine is a preferred prodrug.
  • the secondary nucleoside analog or prodrug preferably is administered when the level of thymidine phosphorylase activity or expression in the patient's sample is at least 10% greater than a baseline thymidine phosphorylase level measured in a corresponding non-patient sample.
  • Thymidine phosphorylase activity can be measured as described, for example, in U.S. Patent 4,219,621, U.S. Patent 6,290,953, or US 2003/0091990.
  • Levels of thymidine phosphorylase protein or mRNA expression also can be determined as an indicator of thymidine phosphorylase activity.
  • Western blot analysis or other immunocytochemically-based methods can be used to detect levels of thymidine phosphorylase protein.
  • Levels of thymidine phosphorylase mRNA can be determined, for example, by Northern blot analysis or PCR-based methods.
  • levels of cytokines such as TNF- ⁇ can be measured in a blood sample to serve as a surrogate marker for thymidine phosphorylase activity or expression.
  • compositions of tezacitabine and/or secondary nucleoside analogs or their prodrugs can include a suitable pharmaceutical carrier or excipient.
  • a suitable pharmaceutical carrier or excipient for example, an active ingredient can be mixed with an excipient, diluted by an excipient, or enclosed within a carrier that can be in the form of a capsule, sachet, paper or other container.
  • the excipient serves as a diluent, it can be a solid, semi-solid, or liquid material, which acts as a vehicle, carrier or medium for the active ingredient.
  • compositions can be in a variety of forms, such as tablets, pills, powders, lozenges, sachets, cachets, elixirs, suspensions, emulsions, solutions, syrups, sterile injectable solutions, and sterile packaged powders.
  • excipients include lactose, dextrose, sucrose, sorbitol, mannitol, starches, gum acacia, calcium phosphate, alginates, tragacanth, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, sterile water, syrup, and methyl cellulose.
  • Formulations can also include lubricating agents such as talc, magnesium stearate, and mineral oil; wetting agents; emulsifying and suspending agents; preserving agents such as methylparaben and propylparaben; sweetening agents; and flavoring agents.
  • the compositions used in this invention can be formulated so as to provide quick, sustained or delayed release of the active ingredient after administration to the patient by employing procedures known in the art.
  • Tablets or pills can be coated or otherwise compounded to provide a dosage form affording the advantage of prolonged action.
  • the tablet or pill can comprise an inner dosage and an outer dosage component, the latter being in the form of an envelope over the former.
  • the two components can separated by enteric layer which serves to resist disintegration in the stomach and permit the inner component to pass intact into the duodenum or to be delayed in release.
  • enteric layers or coatings such materials including a number of polymeric acids and mixtures of polymeric acids with such materials as shellac, cetyl alcohol, and cellulose acetate.
  • the liquid forms in which the drugs used in this invention may be incorporated for administration orally include aqueous solutions, suitably flavored syrups, aqueous or oil suspensions, and flavored emulsions with edible oils such as cottonseed oil, sesame oil, coconut oil, or peanut oil, as well as elixirs and similar pharmaceutical vehicles.
  • the liquid forms in which the drugs used in this invention may be used for parenteral administration include aqueous solutions, liposomal preparations, aqueous microemulsions, lipid solutions or suspensions, and the like.
  • therapeutically effective amounts of either tezacitabine alone or of tezacitabine in combination with one or more secondary nucleoside analogs or their prodrugs are administered to treat cancer patients having nucleoside analog-resistant tumors, particular gemcitabine-resistant tumors.
  • a "therapeutically effective amount” refers to an amount of active compound, such as tezacitabine or a secondary nucleoside analog or prodrug thereof, which, when administered to a cancer patient, is effective to cause a reduction of symptoms of the patient's cancer, e.g., a shrinking of tumor size, tumor cell death, and the like.
  • Patients include both human and veterinary patients.
  • the amount of tezacitabine administered to the patient for treatment ranges from about 2 mg m to about 800 mg/m , depending upon the dose schedule. In some cases, clinical doses of 50-600 mg/m may overcome nucleoside analog resistance. The dose determination is well within the skill of the physician administering the treatment and will generally be determined based upon the body weight, gender, age, health, body surface area and other factors considered by a skilled physician. [35] Combination therapy using tezacitabine and fluoropyrimidines is schedule dependent both in vitro and in vivo.
  • Clonogenic assays with HCT116 cells showed that 24-hour pretreatment with tezacitabine sensitized the cells to 5-FU or its metabolite 5-fluoro-2'- deoxy uridine (FUdR). Efficacy of 5-FU and tezacitabine administered as single bolus injection in an HCT116 xenograft model is not schedule dependent at the doses tested, and the combination is not significantly different from that of tezacitabine alone. HCT116 xenograft studies with tezacitabine in combination with capecitabine, an oral prodrug of 5-FU showed schedule dependent efficacy. Capecitabine administered daily for two weeks in combination with a single bolus injection of tezacitabine showed schedule dependent tumor regression over a variety of doses.
  • Tezacitabine-induced potentiation of FUdR cell killing in HCT116 cells can be explained by a significant increase in Caspase 3-7 activation after 24 hours exposure to FUdR.
  • Tezacitabine doses that are cytotoxically therapeutic or subtherapeutic can be a strong modulator of cell responses to clinically used anticancer agents like fluoropyrimidines and may overcome possible mechanisms of resistance to DNA-interacting drugs.
  • the initial amount of secondary nucleoside analog or prodrug to be administered can be obtained from the package insert for each analog. Because of synergistic effects when administered in conjunction with tezacitabine, however, the dose of any particular secondary nucleoside analog or prodrug may be less than the dose recommended on the package insert. Determination of a lower dose than recommended on the package insert can readily be determined by the skilled physician. Routes of Administration
  • Tezacitabine and secondary nucleoside analogs or their prodrugs can be administered by a variety of routes including oral, transdermal, parenteral, subcutaneous, intravenous, intraarterial, intraperitoneal and intramuscular. Different routes of administration can be used for tezacitabine and for the secondary nucleoside analog or prodrug, as may be appropriate for the particular combination of agents being used and the particular tumor type being treated. Selection of appropriate routes of administration is well within the skill of the treating physician.
  • kits for carrying out various methods of the invention comprise a container that comprises a pharmaceutical preparation comprising tezacitabine and/or a secondary nucleoside analog or prodrug.
  • Suitable containers for the pharmaceutical preparations include, for example, bottles, vials, syringes, and test tubes.
  • the containers can be formed from a variety of materials, including glass or plastic.
  • the container may have a ste ⁇ le access port (for example, the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle).
  • kits also can further comprise a second container comprising a pharmaceutically- acceptable buffer, such as phosphate-buffered saline, Ringer's solution, or dextrose solution. It can also contain other materials useful to the end-user, including other buffers, diluents, filters, needles, and syringes.
  • a kit can also comprise a second or third container with another active agent, for example another secondary nucleoside analog or prodrug or other anti-cancer agent.
  • kits of the invention can comprise a reagent for detecting a mutation in a ras gene or a reagent for detecting cytidine deaminase, thymidine phosphatase, or thymidylate synthase expression (e.g., at least one oligonucleotide probe or antibody), a substrate for cytidine deaminase (e.g., 5'-dFCrd or radiolabeled 5'-dFCrd), a substrate for thymidine phosphatase (such as thymidine or radiolabeled thymidine), or a substrate for thymidylate synthase (e.g., dUMP or radiolabeled dUMP).
  • the kit can also comprise a package insert containing printed instructions for carrying out methods of the invention.
  • the package insert may be an unapproved draft package insert or may be a package insert approved by the Food and Drug Administration (FDA) or
  • the invention also provides methods of identifying therapeutic combinations of tezacitabine and one or more secondary nucleoside analogs. Cytotoxicity of tezacitabine alone and in combination with different anticancer agents can be evaluated in cell lines deficient in a specific DNA repair pathway (see Table 4). Nucleoside analog-resistant tumor cell lines whose particular defect is unknown also can be screened for sensitivity to tezacitabine or other nucleoside analogs. Tumor cell lines can be made resistant to a nucleoside analog by any means that affects one or more of the parameters discussed above. For example, transfecting the cells with a target gene, thereby increasing its expression and rendering the cells resistant to the drug may alter cell line's sensitivity. Typically, however, cells are treated in culture with low levels of the drug over months and are periodically tested for sensitivity. Resistant cells with a stable phenotype can then be used in screening assays.
  • each nucleoside analog-resistant or D ⁇ A repair deficient cell line is compared with the cytotoxicity induced by tezacitabine alone or in combination, in the parental wild type cell line. If the results identify hypersensitivity to tezacitabine alone in one of the deficient cell lines, this suggests an active involvement of that particular D ⁇ A repair pathway in removing the base analogue from D ⁇ A. For the combination studies, if a particular combination and schedule appears synergistic in the wild type cell line but not in one of the mutants, the specific D ⁇ A repair pathway involved is likely to be responsible for the beneficial effects of the combination under study.
  • Human tumor cell lines are tested for sensitivity to tezacitabine or to tezacitabine in combination with one or more secondary nucleoside analogs by determining the inhibitory concentration at which cellular proliferation is decreased by 50% (IC 50 ).
  • the cells are plated in plastic dishes (typically 96 or 384 wells per plate), and 24 hours later the tezacitabine or other nucleoside analog is added to the media in the wells.
  • Drug concentrations are chosen to cover the ICso range for the drug as previously determined.
  • the second drug is added either at the same time or at a later time point.
  • Drug interactions can be evaluated by using Loewe's equation, in which ⁇ is determined as a measure of the combination's synergy, additivity or antagonism. See Chou & Talalay, Adv. Enzyme Regul. 22, 27-55, 1984.
  • Tezacitabine- or other nucleoside analog-induced cytotoxicity can be evaluated, for example, by the clonogenic survival assay.
  • cells are serially diluted (range 2xl0 2 -2xl0 3 cells/ 60 mm dish) and plated in complete growth medium and allowed to adhere for 18 hours.
  • the cells can then be treated with tezacitabine (10-100 nM) for 24 or 48 hours.
  • Tezacitabine-containing medium is then removed, cultures are washed with PBS and incubated for 7-10 days. Colonies (>50 cells) are then stained and counted.
  • the induction and repair of DNA damage by tezacitabine alone or in combination can be studied with the aid of the Comet assay (Single Gel Cell Electrophoresis).
  • the principle of the assay is based upon the ability of denatured, cleaved DNA fragments to migrate out of the cell under the influence of an electric field, whereas undamaged DNA migrates slower and remains within the confines of the nucleoid when an electric current is applied.
  • the assay is performed according to a standard protocol. After drug treatment, the cells are washed, resuspended in ice cold PBS at the concentration of lxlO 5 , and mixed at a ratio of 1 to 10 (v/v) with molten LMAgarose (at 42°C).
  • Electrophoresis is carried out according to standard protocols. Following electrophoresis, slides are dipped in ethanol for 5 minutes and air dried. To score the comets, 50 ⁇ l of diluted SYBR green is placed onto each circle of dried agarose. Stained slides can be viewed by epifluorescence microscopy (fluorescein filter, excitation/emission 494/521 nm). Evaluation of the DNA comet tail shape and migration pattern allows for assessment of DNA damage.
  • BER can repair small chemical changes of the DNA bases, such uracil incorporated instead of thymidine.
  • NER repairs bulky adducts such as cisplatin intrastrand adducts and damage caused by UV-light.
  • HR and NHEJ repair double strand breaks and HR also interstrand crosslinks caused by radiation and DNA damaging agents.
  • CHO DNA repair mechanisms are very similar to the mechanisms active in human cells and therefore represent a good model for understanding the role played by DNA repair in cytotoxicity.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Engineering & Computer Science (AREA)
  • Immunology (AREA)
  • Pathology (AREA)
  • Analytical Chemistry (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Genetics & Genomics (AREA)
  • Hospice & Palliative Care (AREA)
  • Biochemistry (AREA)
  • Microbiology (AREA)
  • Molecular Biology (AREA)
  • Biophysics (AREA)
  • Physics & Mathematics (AREA)
  • Oncology (AREA)
  • Biotechnology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Saccharide Compounds (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

Tezacitabine, either alone or in combination with one or more nucleoside analogs or prodrugs thereof, can be use to treat nucleoside analog-resistant tumor, particularly gemcitabine-resistant tumors. Screening tumor samples for ras mutations or screening tumor or other patient samples for levels of cytidine deaminase activity or expression, for levels of thymidine phosphorylase activity, or for levels of thymidylate synthase activity or expression are useful for assessing whether a tumor of a patient is likely to respond to treatment with various nucleoside analogs, particularly tezacitabine, gemcitabine, and combinations of nucleoside analogs or prodrugs thereof.

Description

METHODS AND COMPOSITIONS FOR TREATING TUMORS
[01] This application incorporates by reference co-pending provisional applications Serial No. 60/482,874 filed June 27, 2003 and Serial No. 60/479,155 filed June 18, 2003.
FIELD OF THE INVENTION
[02] The invention relates to treating tumors resistant to nucleoside analogs, particularly gemcitabine-resistant tumors.
BACKGROUND OF THE INVENTION
[03] Anticancer agents of all types can become less efficacious over time. This decreased sensitivity is called resistance. Resistance can be classified as "innate" in which case the tumor is refractory to a certain therapy or resistance may be "acquired" during or after a particular therapy. A variety of mechanisms of resistance have been described in the literature, which include "host factors," such as poor absorption, increased metabolism, low tolerance, or poor penetration of the drug to the tumor site. Kinsella & Smith, Gen. Pharmacol. 30, 623-26, 1998; Tannock et al., Clin. Cancer Res. 8, 878-84, 2002. Polymorphic genes present in human populations affect each of these parameters. However, most of the focus on tumor resistance to therapy has been on the epigenetic and genetic alterations that occur in the cancer cells within the tumor. Some of these changes, such as the loss of a particular enzyme necessary for the conversion of a prodrug into its active metabolite, may result in resistance to only a select set of drugs. In other cases, changes may occur that lead to multi-drug resistance, in which drugs with a variety of mechanisms of action are rendered inactive.
[04] Several resistance mechanisms have been described for nucleoside analogs. First, increased levels of cytidine deaminase may lead to low serum levels of prodrug through increased metabolism. Neff & Blau, Cancer Exp. Hematol. 11, 1340-46, 1996. Second, uptake of the analog may be reduced through alterations in expression of the equilabrative nucleotide transporters (ENTs) or concentrative nucleotide transporters (CNTs). For example, gemcitabine is a substrate for four of the nucleoside transporters found in humans, ei, es, cib and cit, and nucleotide transporter activity is required for gemcitabine toxicity in cells. Mackey et al., Cancer Res. 58, 4349-57, 1998. Nucleotide transporter deficiency may confer significant gemcitabine resistance. Mackey et al., Clin. Cancer Res. 8, 110-16, 2002. Resistance also can result from other means of altering the balance of drugs within cells, such as the presence or activation of efflux pumps (e.g., MDR), changes in membrane lipids, or increased metabolism of the drug inside the cell. Once inside the cell, there may also be activating or deactivating mutations in the target genes, modulation of expression of target genes such as deoxycytidine kinase and ribonucleotide reductase (Bergman et al., Drug Resist. Update 5, 19, 2002; Zhou et al, Cytogenet. Cell. Genet. 95, 34-42 2001), amplification of target genes, increased DNA repair, or changes in the DNA damage response pathways leading to altered apoptotic responses. Shi et al., Oncology 62, 354-62, 2002.
[05] There is a need in the art to provide anti-cancer therapies useful against tumors that have become resistant to nucleoside analogs, particularly gemcitabine-resistant tumors.
BRIEF SUMMARY OF THE INVENTION
[06] The invention provides at least the following embodiments:
1. A method to aid in determining whether a tumor of a patient is likely to respond to gemcitabine, comprising the step of: examining a sample of a tumor of a patient to detect a mutation in a ras gene.
2. The method of embodiment 1 wherein the ras gene is K-ras.
3. The method of embodiment 2 wherein the K-ras is K-ras 4A.
4. The method of embodiment 2 wherein the K-ras is K-ras 4B .
5. The method of embodiment 1 wherein the ras gene is N-ras.
6. The method of embodiment 1 wherein the ras gene is H-ras. 7. The method of embodiment 1 further comprising the step of detecting a level of cytidine deaminase activity or an amount of cytidine deaminase expression in a biological sample of the patient.
8. The method of embodiment 7 wherein the biological sample is a sample of the tumor.
9. The method of embodiment 1 wherein the tumor is a gastroesophageal tumor.
10. A kit comprising: an oligonucleotide probe for detecting a mutation in a ras gene; and a package insert that describes the method of embodiment 1.
11. A method to aid in determining whether a tumor of a patient is likely to respond to tezacitabine, comprising the step of: examining a sample of the tumor to detect a mutation in a ras gene.
12. The method of embodiment 11 wherein the ras gene is K-ras.
13. The method of embodiment 12 wherein the K-ras is K-ras 4 A.
14. The method of embodiment 12 wherein the K-ras is K-ras 4B.
15. The method of embodiment 11 wherein the ras gene is N-ras.
16. The method of embodiment 11 wherein the ras gene is H-ras.
17. The method of embodiment 11 further comprising the step of detecting a level of cytidine deaminase activity or an amount of cytidine deaminase expression in a biological sample of the patient.
18. The method of embodiment 17 wherein the biological sample is a tumor sample.
19. The method of embodiment 11 wherein the tumor is a gastroesophageal tumor.
20. A kit comprising: an oligonucleotide probe for detecting a mutation in a ras gene; and a package insert that describes the method of embodiment 11.
21. A method to aid in determining whether a tumor of a patient is likely to respond to gemcitabine, comprising the steps of: detecting a level of cytidine deaminase activity or an amount of cytidine deaminase expression in a biological sample of the patient.
22. The method of embodiment 21 wherein the biological sample is a tumor sample.
23. The method of embodiment 21 further comprising the step of examining a second sample of the tumor to detect a mutation in a ras gene.
24. The method of embodiment 21 wherein the tumor is a gastroesophageal tumor.
25. A kit comprising: a substrate for cytidine deaminase or a reagent for detecting cytidine deaminase expression; and a package insert that describes the method of embodiment 21.
26. A method to aid in determining whether a tumor of a patient is likely to respond to tezacitabine, comprising the steps of: examining a first sample of the tumor to detect a level of cytidine deaminase activity or an amount of cytidine deaminase expression.
27. The method of embodiment 26 further comprising the step of examining a second sample of the tumor to detect a mutation in a ras gene.
28. The method of embodiment 26 wherein the tumor is a gastroesophageal tumor.
29. A kit comprising: a substrate for cytidine deaminase or a reagent for detecting cytidine deaminase expression; and a package insert that describes the method of embodiment 26.
30. A method to aid in determining whether a tumor of a patient is likely to respond to a combination of tezacitabine and (a) a nucleoside analog that inhibits thymidylate synthase or (b) a prodrug of the nucleoside analog, comprising the step of: examining a sample of the tumor to detect a level of thymidylate synthase activity or an amount of thymidylate synthase expression.
31. The method of embodiment 30 wherein the prodrug is capecitabine.
32. The method of embodiment 30 wherein the tumor is a gastroesophageal tumor.
33. A kit comprising: a substrate for thymidylate synthase or a reagent for detecting thymidylate synthase expression; and a package insert that describes the method of embodiment 30.
34. A method of treating a patient having a nucleoside analog-resistant tumor, comprising the step of administering to the patient a therapeutically effective amount of tezacitabine.
35. The method of embodiment 62 wherein cells of the nucleoside analog-resistant tumor contain a mutated ras gene.
36. The method of embodiment 62 wherein the nucleoside analog-resistant tumor is resistant to gemcitabine. 37. The method of embodiment 62 wherein the nucleoside analog-resistant tumor is a tumor of the breast, prostate, lung, colon, stomach, pancreas, ovary, brain, colon, rectum, esophagus, kidney, bladder, or blood.
38. The method of embodiment 62 wherein the nucleoside analog-resistant tumor is a gastroesophageal tumor.
39. The method of embodiment 34 wherein cytidine deaminase activity in cells of the patient is at least about 20 nmole/hr/mg.
40. The method of embodiment 39 wherein the cells are tumor cells.
41. The method of embodiment 34 wherein the amount of cytidine deaminase expression in a biological sample of the patient is at least 10% higher than a baseline amount of cytidine deaminase expression in a corresponding biological sample of an individual who does not have a tumor.
42. The method of embodiment 41 wherein the biological sample of the patient is a tumor sample.
43. A kit comprising: a pharmaceutical composition comprising tezacitabine; and a package insert that describes the method of embodiment 34.
44. A method of treating a patient having a nucleoside analog-resistant tumor, comprising the steps of: administering to the patient a therapeutically effective amount of tezacitabine; and administering to the patient a therapeutically effective amount of a secondary nucleoside analog or a prodrug thereof. 45. The method of embodiment 44 further comprising the step of detecting a level of thymidine phosphorylase activity or expression in a biological sample of the patient before administering the secondary nucleoside analog or the prodrug.
46. The method of embodiment 44 wherein the prodrug is capecitabine.
47. The method of embodiment 44 wherein cells of the nucleoside analog-resistant tumor contain a mutated ras gene.
48. The method of embodiment 44 wherein the nucleoside analog-resistant tumor is resistant to gemcitabine.
49. The method of embodiment 44 wherein the nucleoside analog-resistant tumor is resistant to 5-fluorouracil.
50. The method of embodiment 44 wherein the secondary nucleoside analog is cytarabine, fludarabine, cladribine, troxacitabine, thioguanine, triapine, and pentostatin.
51. The method of embodiment 44 wherein the tezacitabine and the secondary nucleoside analog or prodrug thereof are administered in the same pharmaceutical composition.
52. The method of embodiment 44 wherein the tezacitabine and the secondary nucleoside analog or prodrug thereof are administered in different pharmaceutical compositions.
53. The method of embodiment 44 wherein the tezacitabine and the secondary nucleoside analog or prodrug thereof are administered simultaneously.
54. The method of embodiment 44 wherein the tezacitabine and the secondary nucleoside analog or prodrug thereof are administered sequentially.
55. The method of embodiment 44 wherein the nucleoside analog-resistant tumor is a tumor of the breast, prostate, lung, colon, stomach, pancreas, ovary, brain, colon, rectum, esophagus, kidney, bladder, or blood. 56. The method of embodiment 44 wherein the nucleoside analog-resistant tumor is a gastroesophageal tumor.
57. The method of embodiment 44 wherein the nucleoside analog-resistant tumor is a colorectal tumor.
58. The method of embodiment 44 wherein cytidine deaminase activity in cells of the patient is at least about 20 nmole hr/mg.
59. The method of embodiment 58 wherein the cells are tumor cells.
60. The method of embodiment 44 wherein the amount of cytidine deaminase expression in a biological sample of the patient is at least 10% higher than a baseline amount of cytidme deaminase expression in a corresponding biological sample of an individual who does not have a tumor.
61. The method of embodiment 60 wherein the biological sample of the patient is a tumor sample.
62. A kit comprising: a pharmaceutical composition comprising tezacitabine; and a package insert that describes the method of embodiment 44.
63. The kit of embodiment 62 further comprising a pharmaceutical composition comprising a secondary nucleoside analog or a prodrug thereof.
64. A method of treating a patient having a tumor, wherein cells of the tumor comprise a mutation in a ras gene, comprising the step of administering to the patient a therapeutically effective amount of a primary nucleoside analog selected from the group consisting of tezacitabine and gemcitabine. 65. The method of embodiment 64 wherein the primary nucleoside analog is tezacitabine.
66. The method of embodiment 64 wherein the primary nucleoside analog is gemcitabine.
67. The method of embodiment 64 wherein cytidine deaminase activity in cells of the patient is at least about 20 nmol/hr/mg and the primary nucleoside analog is tezacitabine.
68. The method of embodiment 64 wherein cytidine deaminase activity in cells of the tumor is at least about 20 nmol/hr/mg and the primary nucleoside analog is tezacitabine.
69. The method of embodiment 64 wherein the tumor is a tumor of the breast, prostate, lung, colon, stomach, pancreas, ovary, brain, colon, rectum, esophagus, kidney, bladder, or blood.
70. The method of embodiment 64 wherein the tumor is a gastroesophageal tumor.
71. The method of embodiment 64, further comprising the step of: administering to the patient a therapeutically effective amount of a secondary nucleoside analog or a prodrug thereof.
72. The method of embodiment 70 wherein the secondary nucleoside analog is cytarabine, fludarabine, cladribine, troxacitabine, thioguanine, triapine, and pentostatin.
73. The method of embodiment 70 wherein the prodrug is capecitabine.
74. The method of embodiment 70 wherein the primary nucleoside analog and the secondary nucleoside analog or prodrug thereof are administered in the same pharmaceutical composition. 75. The method of embodiment 70 wherein the primary nucleoside analog and the secondary nucleoside analog or prodrug thereof are administered in different pharmaceutical compositions.
76. The method of embodiment 70 wherein the primary nucleoside analog and the secondary nucleoside analog or prodrug thereof are administered simultaneously.
77. The method of embodiment 70 wherein the primary nucleoside analog and the secondary nucleoside analog or prodrug thereof are administered sequentially.
78. A kit comprising: a pharmaceutical composition comprising tezacitabine or gemcitabine; and a package insert that describes the method of embodiment 64.
79. The kit of embodiment 78 wherein the package insert further describes the method of embodiment 71.
80. A method of treating a patient having a tumor, wherein cytidme deaminase activity in a first biological sample of the patient is at least about 20 nmol/hr/mg or wherein the amount of cytidine deaminase expression in a second biological sample is at least about 10% higher than a baseline amount of cytidine deaminase expression in a corresponding biological sample of an individual who does not have a tumor, comprising the step of: administering to the patient a therapeutically effective amount of tezacitabine.
81. The method of embodiment 80 wherein the first biological sample of the patient is a tumor sample.
82. The method of embodiment 80 wherein cells of the tumor comprise a mutation in a ras gene. 83. The method of embodiment 80 wherein the tumor is a tumor of the breast, prostate, lung, colon, stomach, pancreas, ovary, brain, colon, rectum, esophagus, kidney, bladder, or blood.
84. The method of embodiment 80 wherein the tumor is a gastroesophageal tumor.
85. *■ The method of embodiment 80, further comprising the step of: administering to the patient a therapeutically effective amount of a secondary nucleoside analog or a prodrug thereof.
86. The method of embodiment 85 wherein the secondary nucleoside analog is cytarabine, fludarabine, cladribine, troxacitabine, thioguanine, triapine, and pentostatin.
87. The method of embodiment 85 wherein the prodrug is capecitabine.
88. The method of embodiment 80 wherein the primary nucleoside analog and the secondary nucleoside analog or prodrug thereof are administered in the same pharmaceutical composition.
89. The method of embodiment 80 wherein the primary nucleoside analog and the secondary nucleoside analog or prodrug thereof are administered in different pharmaceutical compositions.
90. The method of embodiment 80 wherein the tezacitabine and the secondary nucleoside analog or prodrug thereof are administered simultaneously.
91. The method of embodiment 80 wherein the tezacitabine and the secondary nucleoside analog or prodrug thereof are administered sequentially.
92. A kit comprising: a pharmaceutical composition comprising tezacitabine or gemcitabine; and a package insert that describes the method of embodiment 80. BRIEF DESCRIPTION OF THE FIGURE
[07] FIG. 1. Correlation of tezacitabine sensitivity with a K-ras mutation.
DETAILED DESCRIPTION OF THE INVENTION
[08] The invention provides various methods to aid in to aid in determining whether a tumor of a patient is likely to respond to gemcitabine or tezacitabine or to a combination of gemcitabine or tezacitabine and a secondary nucleoside analog or prodrug thereof.
[09] As used herein, a tumor "responds" to a particular therapy if the size of the tumor does not substantially change or if its size decreases by at least 5, 10, 15, 20, 25, or 50% or more over a predetermined period of time (e.g., 1 week, 2 weeks, 3 weeks, 1 month, 2 months, etc.).
[10] Patients having a variety of solid or liquid tumors including, but not limited to, gastroesophageal tumors, esophageal tumors, and tumors of the breast, prostate, lung, colon, stomach, pancreas, ovary, brain, colon, rectum, esophagus, kidney, bladder, or blood, can benefit from screening methods of the invention.
[11] For example, the activation of the Ras oncogene is the most frequent gain-of-function mutation detected in human cancer occurring in approximately 30% of all human tumors. Patients whose tumors contain a ras mutation are likely to respond to treatment with gemcitabine or tezacitabine or to a combination of gemcitabine or tezacitabine and a secondary nucleoside analog or a prodrug thereof. See FIG. 1. Thus, in one embodiment, a sample of a tumor of a patient is examined to detect a mutation in a ras gene. The ras gene can be K-ras ras, N-ras, or H-ras, including splice variants of ras genes such as K-ras 4A and K-ras 4B. Ras mutations can be detected by any known methods including, but not limited to, direct sequencing or single strand conformational polymorphism of PCR products generated from total tumor RNA derived from patient biopsies (Easwaran et al., Cancer Research 63, 3145, 2003).
[12] "Secondary nucleoside analogs" useful in combination with tezacitabine or gemcitabine are nucleoside analogs other than tezacitabine or gemcitabine; if a tumor is resistant to nucleoside analogs, the secondary nucleoside analog or the prodrug preferably is different that the particular nucleoside analog(s) to which a tumor is resistant. Secondary nucleoside analogs useful in the present invention include, but are not limited to, gemcitabine, cytarabine, fludarabine, cladribine, troxacitabine, thioguanine, triapine, and pentostatin. Capecitabine, for example, is a prodrug of 5-fluorouracil.
[13] In another embodiment, a level of cytidine deaminase activity or an amount of cytidine deaminase expression is determined in a biological sample of the patient. Biological samples include, for example, blood samples and tumor samples.
[14] Cytidine deaminase expression can be measured in a variety of ways. In one method, aliquots of purified cytidine deaminase protein or cell lysates are incubated with a substrate, such as 5'-dFCrd. Samples are incubated in a buffered reaction mixture, and the amount of product, 5'-dUrd, is measured in the supernatant using HPLC after centrifugation. See Watanabe & Uchica, Biochem. Biophys. Acta 1312, 99-104, 1996). Alternatively, cytidine deaminase activity can be measured by evaluating the conversion of a radiolabeled substrate, such as tritiated cytidine, into uridine. Labeled cytidine remaining in the mixture is captured on filters and read by scintillation counting. Neff & Blau, Exptl. Hematol. 24, 1340-46, 1996. It is also possible to evaluate the rate of deamination by cytidine deaminase by determining the decrease in absorbance in a spectrophotometer at 286 nm. Bouffard et al., Biochem. Pharmacol. 45, 1857-61, 1993.
[15] Levels of cytidine deaminase protein or mRNA expression also can be determined as an indicator of cytidine deaminase activity. For example, Western blot analysis or other immunocytochemically-based methods can be used to detect levels of cytidine deaminase protein. Levels of cytidme deaminase mRNA can be determined, for example, by Northern blot analysis or PCR-based methods.
[16] In other embodiments, both a ras mutation and a level of cytidine deaminase activity or an amount of cytidme deaminase expression are determined. Patients whose tumors contain a ras mutation and whose cells, particularly tumor cells, contain increased levels of cytidme deaminase activity or expression are particularly suited for treatment with tezacitabine, either alone or in combination with a secondary nucleoside analog or a prodrug thereof. Patients whose tumors contain a ras mutation and whose cells, particularly tumor cells, contain a low or baseline amount of cytidine deaminase activity or expression can be treated with gemcitabine or with tezacitabine.
[17] For treatment with tezacitabine, the level of cytidine deaminase activity in the biological sample of the patient is preferably at least about 20, 30, 40, 50, 60, 70, 80, 90, or 100 or more nmole/hr/mg or the amount of cytidine deaminase expression in a biological sample of the patient preferably is at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 250, 300, 350, 400, 450, or 500% higher than a baseline amount of cytidine deaminase expression in a corresponding biological sample of an individual who does not have a tumor.
[18] The invention also provides methods to aid in determining whether a tumor of a patient is likely to respond to a combination of tezacitabine and (a) a nucleoside analog that inhibits thymidylate synthase or (b) a prodrug of the nucleoside analog. Nucleoside analogs that inhibit thymidylate synthase include, but are not limited to, AG-331, nolatrexed, 5- fluorouracil, raltitrexed, BGC-9331, FO-152, lometrexol, LY-225693, LY-288784, LY- 295248, pemetrexed, DMPDDF, OSI-7904L, CB-30900, 5-fiuorocytosine derivatives, UCN-01, NSC-678515, NB-1011, doxifluridine, AO-90, OGT-719, trimexetrate, pyrmidine deoxynucleoside analogs, galocitabine, edatrexate, MVA-FCU1, sorivudine, ICI-198583, and ZM-246315. In a preferred embodiment, the prodrug is capecitabine. Such screening methods can be applied to assess a variety of tumors including, but not limited to, gastroesophageal tumors, esophageal tumors, and tumors of the breast, prostate, lung, colon, stomach, pancreas, ovary, brain, colon, rectum, esophagus, kidney, bladder, or blood.
[19] One embodiment of the invention provides a method to aid in determining whether a tumor of a patient is likely to respond to a combination of tezacitabine and (a) a nucleoside analog that inhibits thymidylate synthase or (b) a prodrug of the nucleoside analog, such as capecitabine. In this embodiment, a sample of the tumor is examined to detect a level of thymidylate synthase activity or an amount of thymidylate synthase expression. Thymidylate synthase mRNA expression can be measured as described above. If thymidylate synthase mRNA expression is in the range of 0.9 - 20.1 x 10"3 relative to expression of a housekeeping gene (e.g., β-actin), a tumor likely will respond to a combination of tezacitabine and (a) a nucleoside analog that inhibits thymidylate synthase or (b) a prodrug of the nucleoside analog, such as capecitabine. Thymidylate synthase mRNA expression preferably is 10 - 15 x 10 "3, 5 - 10 x 10"3, or 4 - 6 x 10"3 relative to expression of a housekeeping gene. See, e.g., Shirota et al., J. Clin. Oncol. 19, 4298-4304, 2001.
[20] Corresponding levels of thymidylate synthase protein can be measured as described, for example, in U.S. Patent 6,569,634. Corresponding levels of thymidylate synthase activity can be measured using any methods known in the art. See, e.g., Baba et al., Cancer Chemother Pharmacol. 2003 Sep 11 (e-pub); Higashiyama et al., Lung Cancer. 2001 Dec;34(3):407-16.
[21] The invention also provides methods for treating patients who have nucleoside analog- resistant tumors, particularly gemcitabine-resistant tumors. Patients with nucleoside analog-resistant tumors have a cancer that has progressed either while being treated with the nucleoside analog or within six months of completing treatment with the nucleoside analog. Such patients can be treated with (E)-2'-deoxy-2'-(fluoromethylene)cytidine (FMdC, tezacitabine), either alone or in combination with an effective amount of one or more secondary nucleoside analog. Nucleoside analog-resistant tumors that can be treated according to the invention include any tumor that has become resistant to a nucleoside analog, particularly gemcitabine. Such tumors include, but are not limited to, gastroesophageal, colorectal, breast, prostate, lung, colon, stomach, pancreatic, ovarian, brain and hematopoietic cancers, non-small cell lung cancer, colorectal cancer, leukemia, lymphoma, esophageal carcinoma, renal cell carcinoma, bladder cancer, head and neck cancer, and sarcomas such as cholangiosarcoma and esophageal sarcoma.
Treatment of nucleoside analog-resistant tumors with tezacitabine
[22] Tezacitabine is an analog of deoxycytidine that possesses anti-tumor activity. Once inside a cell, tezacitabine is phosphorylated to yield the mono-, di- and triphosphate forms. Tezacitabine diphosphate irreversibly inhibits ribonucleotide reductase, resulting in decreased deoxynucleotide pools inside the cell. Tezacitabine triphosphate can be incorporated by DNA polymerase into DNA; this incorporation causes DNA synthesis chain termination and eventual cell death. Together these two activities are self- potentiating; decreased deoxynucleotide pools lead to increased incorporation of tezacitabine triphosphate in DNA in a self-potentiating mechanism. In this way, tezacitabine is a dual-acting nucleoside analog.
[23] The mechanisms of cellular resistance to tezacitabine may be substantially different from resistance mechanisms to other nucleoside analogs. For example, tezacitabine differs from gemcitabine in a number of characteristics, including its susceptibility to deamination, its potency of ribonucleotide reductase (RNR) inhibition, and its affinities for deoxycytidine kinase and DNA polymerase α (see Tables 1 and 2). Because tezacitabine is a more potent and irreversible RNR inhibitor, tezacitabine can overcome gemcitabine resistance due to overexpression or activation of ribonucleotide reductase. Thus, tezacitabine can be used to treat tumors that have become resistant to other nucleoside analogs that differ from tezacitabine in one or more aspects, such as having a different mechanism for entering the cell, different NT sensitivities, a different drug metabolism, different effects or utilization of alternative DNA repair mechanisms, and the like.
[24] Some mechanisms of gemcitabine-resistance are listed in Table 3.
PATENT PP20669.003
Table 1. Metabolic Characteristics
Figure imgf000018_0001
PATENT PP20669.003
Table 2. Cellular Pharmacology Characteristics
Figure imgf000019_0001
PATENT PP20669.003
Table 3. Mechanisms of Gemcitabine Resistance
Figure imgf000020_0001
Treatment of nucleoside analog-resistant tumors with a combination of tezacitabine or gemcitabine and a secondary nucleoside analog or a prodrug of a secondary nucleoside analog
[25] Specific drug combinations with tezacitabine can be synergistic for treating nucleoside- resistant tumors. For example, deoxycytidine analogs require phosphorylation by deoxycytidine kinase to form the monophosphate form, and this is typically the rate limiting step. Deoxycytidine kinase is regulated by the levels of the nucleotide pools, which in turn can be controlled by ribonucleotide reductase. Treatment with tezacitabine, a potent and irreversible inhibitor of RNR, could lead to greater activity of deoxycytidine kinase, thereby potentiating its own activity and the activity of any other nucleoside analog that has deoxycytidine kinase as a rate limiting step. A decrease in the intracellular pool of deoxynucleotides can increase the activity of chemotherapy agents that induce DNA damage, such as oxaliplatin, cisplatin, topotecan, irinotecan and adriamycin. In addition, irreversible inhibition of RNR by tezacitabine may allow better incorporation of other nucleoside analogs even in tumors that were previously resistant to nucleoside analog therapy.
[26] Tezacitabine or gemcitabine and a secondary nucleoside analog or prodrug thereof can be administered together in the same formulation or separately. Separate formulations are useful for treatment protocols in which there is a predetermined time between the administration of tezacitabine or gemcitabine and the secondary nucleoside analog or prodrug thereof, with either agent being administered first.
[27] Optionally, a biological sample of a patient can be assessed to detect a level of thymidine phosphorylase activity or expression in a biological sample of the patient before administering the secondary nucleoside analog or the prodrug. Suitable samples include blood and tumor samples. Capecitabine is a preferred prodrug. The secondary nucleoside analog or prodrug preferably is administered when the level of thymidine phosphorylase activity or expression in the patient's sample is at least 10% greater than a baseline thymidine phosphorylase level measured in a corresponding non-patient sample.
[28] Thymidine phosphorylase activity can be measured as described, for example, in U.S. Patent 4,219,621, U.S. Patent 6,290,953, or US 2003/0091990. Levels of thymidine phosphorylase protein or mRNA expression also can be determined as an indicator of thymidine phosphorylase activity. For example, Western blot analysis or other immunocytochemically-based methods can be used to detect levels of thymidine phosphorylase protein. Levels of thymidine phosphorylase mRNA can be determined, for example, by Northern blot analysis or PCR-based methods. Alternatively, levels of cytokines such as TNF-α can be measured in a blood sample to serve as a surrogate marker for thymidine phosphorylase activity or expression.
Pharmaceutical Formulations
[29] Pharmaceutical formulations of tezacitabine and/or secondary nucleoside analogs or their prodrugs can include a suitable pharmaceutical carrier or excipient. For example, an active ingredient can be mixed with an excipient, diluted by an excipient, or enclosed within a carrier that can be in the form of a capsule, sachet, paper or other container. When the excipient serves as a diluent, it can be a solid, semi-solid, or liquid material, which acts as a vehicle, carrier or medium for the active ingredient. Pharmaceutical formulations can be in a variety of forms, such as tablets, pills, powders, lozenges, sachets, cachets, elixirs, suspensions, emulsions, solutions, syrups, sterile injectable solutions, and sterile packaged powders.
[30] Examples of suitable excipients include lactose, dextrose, sucrose, sorbitol, mannitol, starches, gum acacia, calcium phosphate, alginates, tragacanth, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, sterile water, syrup, and methyl cellulose. Formulations can also include lubricating agents such as talc, magnesium stearate, and mineral oil; wetting agents; emulsifying and suspending agents; preserving agents such as methylparaben and propylparaben; sweetening agents; and flavoring agents. The compositions used in this invention can be formulated so as to provide quick, sustained or delayed release of the active ingredient after administration to the patient by employing procedures known in the art.
[31] Tablets or pills can be coated or otherwise compounded to provide a dosage form affording the advantage of prolonged action. For example, the tablet or pill can comprise an inner dosage and an outer dosage component, the latter being in the form of an envelope over the former. The two components can separated by enteric layer which serves to resist disintegration in the stomach and permit the inner component to pass intact into the duodenum or to be delayed in release. A variety of materials can be used for such enteric layers or coatings, such materials including a number of polymeric acids and mixtures of polymeric acids with such materials as shellac, cetyl alcohol, and cellulose acetate.
[32] The liquid forms in which the drugs used in this invention may be incorporated for administration orally include aqueous solutions, suitably flavored syrups, aqueous or oil suspensions, and flavored emulsions with edible oils such as cottonseed oil, sesame oil, coconut oil, or peanut oil, as well as elixirs and similar pharmaceutical vehicles. The liquid forms in which the drugs used in this invention may be used for parenteral administration include aqueous solutions, liposomal preparations, aqueous microemulsions, lipid solutions or suspensions, and the like.
Dosages
[33] According to the invention, therapeutically effective amounts of either tezacitabine alone or of tezacitabine in combination with one or more secondary nucleoside analogs or their prodrugs are administered to treat cancer patients having nucleoside analog-resistant tumors, particular gemcitabine-resistant tumors. A "therapeutically effective amount" refers to an amount of active compound, such as tezacitabine or a secondary nucleoside analog or prodrug thereof, which, when administered to a cancer patient, is effective to cause a reduction of symptoms of the patient's cancer, e.g., a shrinking of tumor size, tumor cell death, and the like. Patients include both human and veterinary patients.
[34] The amount of tezacitabine administered to the patient for treatment ranges from about 2 mg m to about 800 mg/m , depending upon the dose schedule. In some cases, clinical doses of 50-600 mg/m may overcome nucleoside analog resistance. The dose determination is well within the skill of the physician administering the treatment and will generally be determined based upon the body weight, gender, age, health, body surface area and other factors considered by a skilled physician. [35] Combination therapy using tezacitabine and fluoropyrimidines is schedule dependent both in vitro and in vivo. Clonogenic assays with HCT116 cells showed that 24-hour pretreatment with tezacitabine sensitized the cells to 5-FU or its metabolite 5-fluoro-2'- deoxy uridine (FUdR). Efficacy of 5-FU and tezacitabine administered as single bolus injection in an HCT116 xenograft model is not schedule dependent at the doses tested, and the combination is not significantly different from that of tezacitabine alone. HCT116 xenograft studies with tezacitabine in combination with capecitabine, an oral prodrug of 5-FU showed schedule dependent efficacy. Capecitabine administered daily for two weeks in combination with a single bolus injection of tezacitabine showed schedule dependent tumor regression over a variety of doses.
[36] The cellular responses to tezacitabine in HCT116 human colon carcinoma cells using a clonogenic assay as measure of cell survival following drug treatment show a time- dependency of tezacitabine-induced cell killing (IC50 decreased - 3-fold when the exposure time was increased from 24 to 48 hours). Similarly, tezacitabine induced Caspases 3-7 in HCT116 cells in a time-dependent fashion, suggesting that the observed cytotoxicity is the result of a cascade of programmed cell death events. Tezacitabine can therefore be used to modulate the mechanisms involved in sensitivity or resistance to 5- fluoro-2'-deoxyuridine (FUdR) and other fluoropyrimidines. The tezacitabine-induced potentiation of FUdR cell killing in HCT116 cells can be explained by a significant increase in Caspase 3-7 activation after 24 hours exposure to FUdR. Tezacitabine doses that are cytotoxically therapeutic or subtherapeutic can be a strong modulator of cell responses to clinically used anticancer agents like fluoropyrimidines and may overcome possible mechanisms of resistance to DNA-interacting drugs.
[37] The initial amount of secondary nucleoside analog or prodrug to be administered can be obtained from the package insert for each analog. Because of synergistic effects when administered in conjunction with tezacitabine, however, the dose of any particular secondary nucleoside analog or prodrug may be less than the dose recommended on the package insert. Determination of a lower dose than recommended on the package insert can readily be determined by the skilled physician. Routes of Administration
[38] Tezacitabine and secondary nucleoside analogs or their prodrugs can be administered by a variety of routes including oral, transdermal, parenteral, subcutaneous, intravenous, intraarterial, intraperitoneal and intramuscular. Different routes of administration can be used for tezacitabine and for the secondary nucleoside analog or prodrug, as may be appropriate for the particular combination of agents being used and the particular tumor type being treated. Selection of appropriate routes of administration is well within the skill of the treating physician.
Kits
[39] The invention also provides kits for carrying out various methods of the invention. Some kits of the invention comprise a container that comprises a pharmaceutical preparation comprising tezacitabine and/or a secondary nucleoside analog or prodrug. Suitable containers for the pharmaceutical preparations include, for example, bottles, vials, syringes, and test tubes. The containers can be formed from a variety of materials, including glass or plastic. The container may have a steήle access port (for example, the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle).
[40] Such kits also can further comprise a second container comprising a pharmaceutically- acceptable buffer, such as phosphate-buffered saline, Ringer's solution, or dextrose solution. It can also contain other materials useful to the end-user, including other buffers, diluents, filters, needles, and syringes. A kit can also comprise a second or third container with another active agent, for example another secondary nucleoside analog or prodrug or other anti-cancer agent.
[41] Other kits of the invention can comprise a reagent for detecting a mutation in a ras gene or a reagent for detecting cytidine deaminase, thymidine phosphatase, or thymidylate synthase expression (e.g., at least one oligonucleotide probe or antibody), a substrate for cytidine deaminase (e.g., 5'-dFCrd or radiolabeled 5'-dFCrd), a substrate for thymidine phosphatase (such as thymidine or radiolabeled thymidine), or a substrate for thymidylate synthase (e.g., dUMP or radiolabeled dUMP). [42] The kit can also comprise a package insert containing printed instructions for carrying out methods of the invention. The package insert may be an unapproved draft package insert or may be a package insert approved by the Food and Drug Administration (FDA) or other regulatory body.
Methods of identifying therapeutic combinations
[43] The invention also provides methods of identifying therapeutic combinations of tezacitabine and one or more secondary nucleoside analogs. Cytotoxicity of tezacitabine alone and in combination with different anticancer agents can be evaluated in cell lines deficient in a specific DNA repair pathway (see Table 4). Nucleoside analog-resistant tumor cell lines whose particular defect is unknown also can be screened for sensitivity to tezacitabine or other nucleoside analogs. Tumor cell lines can be made resistant to a nucleoside analog by any means that affects one or more of the parameters discussed above. For example, transfecting the cells with a target gene, thereby increasing its expression and rendering the cells resistant to the drug may alter cell line's sensitivity. Typically, however, cells are treated in culture with low levels of the drug over months and are periodically tested for sensitivity. Resistant cells with a stable phenotype can then be used in screening assays.
Table 4. CHO cell lines
Figure imgf000027_0001
[44] The cytotoxicity observed in each nucleoside analog-resistant or DΝA repair deficient cell line is compared with the cytotoxicity induced by tezacitabine alone or in combination, in the parental wild type cell line. If the results identify hypersensitivity to tezacitabine alone in one of the deficient cell lines, this suggests an active involvement of that particular DΝA repair pathway in removing the base analogue from DΝA. For the combination studies, if a particular combination and schedule appears synergistic in the wild type cell line but not in one of the mutants, the specific DΝA repair pathway involved is likely to be responsible for the beneficial effects of the combination under study.
[45] Human tumor cell lines are tested for sensitivity to tezacitabine or to tezacitabine in combination with one or more secondary nucleoside analogs by determining the inhibitory concentration at which cellular proliferation is decreased by 50% (IC50). The cells are plated in plastic dishes (typically 96 or 384 wells per plate), and 24 hours later the tezacitabine or other nucleoside analog is added to the media in the wells. Drug concentrations are chosen to cover the ICso range for the drug as previously determined. The second drug is added either at the same time or at a later time point. Drug interactions can be evaluated by using Loewe's equation, in which αis determined as a measure of the combination's synergy, additivity or antagonism. See Chou & Talalay, Adv. Enzyme Regul. 22, 27-55, 1984.
[46] Any method of assessing tezacitabine- or other nucleoside analog-induced cytotoxicity can be used. Tezacitabine- or other nucleoside analog-induced cytotoxicity can be evaluated, for example, by the clonogenic survival assay. In this assay cells are serially diluted (range 2xl02 -2xl03 cells/ 60 mm dish) and plated in complete growth medium and allowed to adhere for 18 hours. For example, the cells can then be treated with tezacitabine (10-100 nM) for 24 or 48 hours. Tezacitabine-containing medium is then removed, cultures are washed with PBS and incubated for 7-10 days. Colonies (>50 cells) are then stained and counted. For combination studies, cells are exposed for variable period of times sequentially or simultaneously to tezacitabine plus another anticancer agent and the resulting colonies are stained and counted like after treatment with tezacitabine alone. The effect of drug treatment(s) is expressed as decreased cell clonogenicity in treated cells compared to untreated control.
[47] The induction and repair of DNA damage by tezacitabine alone or in combination can be studied with the aid of the Comet assay (Single Gel Cell Electrophoresis). The principle of the assay is based upon the ability of denatured, cleaved DNA fragments to migrate out of the cell under the influence of an electric field, whereas undamaged DNA migrates slower and remains within the confines of the nucleoid when an electric current is applied. The assay is performed according to a standard protocol. After drug treatment, the cells are washed, resuspended in ice cold PBS at the concentration of lxlO5, and mixed at a ratio of 1 to 10 (v/v) with molten LMAgarose (at 42°C). Immediately, 75 μl are loaded onto a CometSlide, and the slide is placed flat at 4°C in the dark for 10 minutes. Following solidification of the agarose, slides are immersed in chilled lysis solution and left at 4°C for 30 to 60 minutes. Slides are next immersed in an alkaline solution (ph>13) for 20 to 60 minutes at room temperature in the dark, then placed into an horizontal electrophoresis apparatus. TBE (for neutral electrophoresis) or alkaline solution (for alkaline electrophoresis) is then poured into the chamber until level just covers samples.
[48] Electrophoresis is carried out according to standard protocols. Following electrophoresis, slides are dipped in ethanol for 5 minutes and air dried. To score the comets, 50 μl of diluted SYBR green is placed onto each circle of dried agarose. Stained slides can be viewed by epifluorescence microscopy (fluorescein filter, excitation/emission 494/521 nm). Evaluation of the DNA comet tail shape and migration pattern allows for assessment of DNA damage.
[49] The drug interactions between tezacitabine and DNA damaging agents can also be studied by first determining the role of repair mechanisms on tezacitabine IC50s, and then using this information to guide the evaluation of various drug combinations. The availability of CHO cellular models characterized by deficiencies in specific DNA repair proteins is a useful tool to clarify the mechanism of action of DNA-interacting compounds like tezacitabine. CHO (Chinese Hamster Ovary) cell lines carrying mutations in BER (Base Excision Repair), NER (Nucleotide Excision Repair), HR (Homologous Recombination) and NHEJ (Non Homologous End Joining) are now available and well genetically characterized (Table 3). BER can repair small chemical changes of the DNA bases, such uracil incorporated instead of thymidine. NER repairs bulky adducts such as cisplatin intrastrand adducts and damage caused by UV-light. HR and NHEJ repair double strand breaks and HR also interstrand crosslinks caused by radiation and DNA damaging agents. CHO DNA repair mechanisms are very similar to the mechanisms active in human cells and therefore represent a good model for understanding the role played by DNA repair in cytotoxicity.
[50] All patents, patent applications, and references cited in this disclosure are expressly incorporated herein by reference. The above disclosure generally describes the present invention. Additional advantages and modifications will readily occur to those skilled in the art. Therefore, the invention in its broader aspects is not limited to the specific details and representative embodiments shown and described herein.

Claims

We claim:
1. A method to aid in determining whether a tumor of a patient is likely to respond to gemcitabine, comprising the step of: examining a sample of a tumor of a patient to detect a mutation in a ras gene.
2. The method of claim 1 wherein the ras gene is K-ras.
3. The method of claim 2 wherein the K-ras is K-ras 4 A.
4. The method of claim 2 wherein the K-ras is K-ras 4B.
5. The method of claim 1 wherein the ras gene is N-ras.
6. The method of claim 1 wherein the ras gene is H-ras.
7. The method of claim 1 further comprising the step of detecting a level of cytidine deaminase activity or an amount of cytidine deaminase expression in a biological sample of the patient.
8. The method of claim 7 wherein the biological sample is a sample of the tumor.
9. The method of claim 1 wherein the tumor is a gastroesophageal tumor.
10. A kit comprising: an oligonucleotide probe for detecting a mutation in a ras gene; and a package insert that describes the method of claim 1.
11. A method to aid in determining whether a tumor of a patient is likely to respond to tezacitabine, comprising the step of: examining a sample of the tumor to detect a mutation in a ras gene.
12. The method of claim 11 wherein the ras gene is K-ras.
13. The method of claim 12 wherein the K-ras is K-ras 4 A.
14. The method of claim 12 wherein the K-ras is K-ras 4B.
15. The method of claim 11 wherein the ras gene is N-ras.
16. The method of claim 11 wherein the ras gene is H-ras.
17. The method of claim 11 further comprising the step of detecting a level of cytidine deaminase activity or an amount of cytidine deaminase expression in a biological sample of the patient.
18. The method of claim 17 wherein the biological sample is a tumor sample.
19. The method of claim 11 wherein the tumor is a gastroesophageal tumor.
20. A kit comprising: an oligonucleotide probe for detecting a mutation in a ras gene; and a package insert that describes the method of claim 11.
21. A method to aid in determimng whether a tumor of a patient is likely to respond to gemcitabine, comprising the steps of: detecting a level of cytidine deaminase activity or an amount of cytidine deaminase expression in a biological sample of the patient.
22. The method of claim 21 wherein the biological sample is a tumor sample.
23. The method of claim 21 further comprising the step of examining a second sample of the tumor to detect a mutation in a ras gene.
24. The method of claim 21 wherein the tumor is a gastroesophageal tumor.
25. A kit comprising: a substrate for cytidine deaminase or a reagent for detecting cytidine deaminase expression; and a package insert that describes the method of claim 21.
26. A method to aid in determining whether a tumor of a patient is likely to respond to tezacitabine, comprising the steps of: examining a first sample of the tumor to detect a level of cytidine deaminase activity or an amount of cytidine deaminase expression.
27. The method of claim 26 further comprising the step of examining a second sample of the tumor to detect a mutation in a ras gene.
28. The method of claim 26 wherein the tumor is a gastroesophageal tumor.
29. A kit comprising: a substrate for cytidine deaminase or a reagent for detecting cytidine deaminase expression; and a package insert that describes the method of claim 26.
30. A method to aid in determining whether a tumor of a patient is likely to respond to a combination of tezacitabine and (a) a nucleoside analog that inhibits thymidylate synthase or (b) a prodrug of the nucleoside analog, comprising the step of: examining a sample of the tumor to detect a level of thymidylate synthase activity or an amount of thymidylate synthase expression.
31. The method of claim 30 wherein the prodrug is capecitabine.
32. The method of claim 30 wherein the tumor is a gastroesophageal tumor.
33. A kit comprising: a substrate for thymidylate synthase or a reagent for detecting thymidylate synthase expression; and a package insert that describes the method of claim 30.
34. A method of treating a patient having a nucleoside analog-resistant tumor, comprising the step of administering to the patient a therapeutically effective amount of tezacitabine.
35. The method of claim 62 wherein cells of the nucleoside analog-resistant tumor contain a mutated ras gene.
36. The method of claim 62 wherein the nucleoside analog-resistant tumor is resistant to gemcitabine.
37. The method of claim 62 wherein the nucleoside analog-resistant tumor is a tumor of the breast, prostate, lung, colon, stomach, pancreas, ovary, brain, colon, rectum, esophagus, kidney, bladder, or blood.
38. The method of claim 62 wherein the nucleoside analog-resistant tumor is a gastroesophageal tumor.
39. The method of claim 34 wherein cytidine deaminase activity in cells of the patient is at least about 20 nmole/hr/mg.
40. The method of claim 39 wherein the cells are tumor cells.
41. The method of claim 34 wherein the amount of cytidine deaminase expression in a biological sample of the patient is at least 10% higher than a baseline amount of cytidine deaminase expression in a corresponding biological sample of an individual who does not have a tumor.
42. The method of claim 41 wherein the biological sample of the patient is a tumor sample.
43. A kit comprising: a pharmaceutical composition comprising tezacitabine; and a package insert that describes the method of claim 34.
44. A method of treating a patient having a nucleoside analog-resistant tumor, comprising the steps of: administering to the patient a therapeutically effective amount of tezacitabine; and administering to the patient a therapeutically effective amount of a secondary nucleoside analog or a prodrug thereof.
45. The method of claim 44 further comprising the step of detecting a level of thymidine phosphorylase activity or expression in a biological sample of the patient before administering the secondary nucleoside analog or the prodrug.
46. The method of claim 44 wherein the prodrug is capecitabine.
47. The method of claim 44 wherein cells of the nucleoside analog-resistant tumor contain a mutated ras gene.
48. The method of claim 44 wherein the nucleoside analog-resistant tumor is resistant to gemcitabine.
49. The method of claim 44 wherein the nucleoside analog-resistant tumor is resistant to 5-fluorouracil.
50. The method of claim 44 wherein the secondary nucleoside analog is cytarabine, fludarabine, cladribine, troxacitabine, thioguanine, triapine, and pentostatin.
51. The method of claim 44 wherein the tezacitabine and the secondary nucleoside analog or prodrug thereof are administered in the same pharmaceutical composition.
52. The method of claim 44 wherein the tezacitabine and the secondary nucleoside analog or prodrug thereof are administered in different pharmaceutical compositions.
53. The method of claim 44 wherein the tezacitabine and the secondary nucleoside analog or prodrug thereof are administered simultaneously.
54. The method of claim 44 wherein the tezacitabine and the secondary nucleoside analog or prodrug thereof are administered sequentially.
55. The method of claim 44 wherein the nucleoside analog-resistant tumor is a tumor of the breast, prostate, lung, colon, stomach, pancreas, ovary, brain, colon, rectum, esophagus, kidney, bladder, or blood.
56. The method of claim 44 wherein the nucleoside analog-resistant tumor is a gastroesophageal tumor.
57. The method of claim 44 wherein the nucleoside analog-resistant tumor is a colorectal tumor.
58. The method of claim 44 wherein cytidine deaminase activity in cells of the patient is at least about 20 nmole/hr/mg.
59. The method of claim 58 wherein the cells are tumor cells.
60. The method of claim 44 wherein the amount of cytidine deaminase expression in a biological sample of the patient is at least 10% higher than a baseline amount of cytidine deaminase expression in a corresponding biological sample of an individual who does not have a tumor.
61. The method of claim 60 wherein the biological sample of the patient is a tumor sample.
62. A kit comprising: a pharmaceutical composition comprising tezacitabine; and a package insert that describes the method of claim 44.
63. The kit of claim 62 further comprising a pharmaceutical composition comprising a secondary nucleoside analog or a prodrug thereof.
64. A method of treating a patient having a tumor, wherein cells of the tumor comprise a mutation in a ras gene, comprising the step of administering to the patient a therapeutically effective amount of a primary nucleoside analog selected from the group consisting of tezacitabine and gemcitabine.
65. The method of claim 64 wherein the primary nucleoside analog is tezacitabine.
66. The method of claim 64 wherein the primary nucleoside analog is gemcitabine.
67. The method of claim 64 wherein cytidine deaminase activity in cells of the patient is at least about 20 nmol/hr/mg and the primary nucleoside analog is tezacitabine.
68. The method of claim 64 wherein cytidine deaminase activity in cells of the tumor is at least about 20 nmol/hr/mg and the primary nucleoside analog is tezacitabine.
69. The method of claim 64 wherein the tumor is a tumor of the breast, prostate, lung, colon, stomach, pancreas, ovary, brain, colon, rectum, esophagus, kidney, bladder, or blood.
70. The method of claim 64 wherein the tumor is a gastroesophageal tumor.
71. The method of claim 64, further comprising the step of: administering to the patient a therapeutically effective amount of a secondary nucleoside analog or a prodrug thereof.
72. The method of claim 70 wherein the secondary nucleoside analog is cytarabine, fludarabine, cladribine, troxacitabine, thioguanine, triapine, and pentostatin.
73. The method of claim 70 wherein the prodrug is capecitabine.
74. The method of claim 70 wherein the primary nucleoside analog and the secondary nucleoside analog or prodrug thereof are administered in the same pharmaceutical composition.
75. The method of claim 70 wherein the primary nucleoside analog and the secondary nucleoside analog or prodrug thereof are administered in different pharmaceutical compositions.
76. The method of claim 70 wherein the primary nucleoside analog and the secondary nucleoside analog or prodrug thereof are administered simultaneously.
77. The method of claim 70 wherein the primary nucleoside analog and the secondary nucleoside analog or prodrug thereof are administered sequentially.
78. A kit comprising: a pharmaceutical composition comprising tezacitabine or gemcitabine; and a package insert that describes the method of claim 64.
79. The kit of claim 78 wherein the package insert further describes the method of claim 71.
80. A method of treating a patient having a tumor, wherein cytidme deaminase activity in a first biological sample of the patient is at least about 20 nmol/hr/mg or wherein the amount of cytidine deaminase expression in a second biological sample is at least about 10% higher than a baseline amount of cytidine deaminase expression in a corresponding biological sample of an individual who does not have a tumor, comprising the step of: administering to the patient a therapeutically effective amount of tezacitabine.
81. The method of claim 80 wherein the first biological sample of the patient is a tumor sample.
82. The method of claim 80 wherein cells of the tumor comprise a mutation in a ras gene.
83. The method of claim 80 wherein the tumor is a tumor of the breast, prostate, lung, colon, stomach, pancreas, ovary, brain, colon, rectum, esophagus, kidney, bladder, or blood.
84. The method of claim 80 wherein the tumor is a gastroesophageal tumor.
85. The method of claim 80, further comprising the step of: administering to the patient a therapeutically effective amount of a secondary nucleoside analog or a prodrug thereof.
86. The method of claim 85 wherein the secondary nucleoside analog is cytarabine, fludarabine, cladribine, troxacitabine, thioguanine, triapine, and pentostatin.
87. The method of claim 85 wherein the prodrug is capecitabine.
88. The method of claim 80 wherein the primary nucleoside analog and the secondary nucleoside analog or prodrug thereof are administered in the same pharmaceutical composition.
89. The method of claim 80 wherein the primary nucleoside analog and the secondary nucleoside analog or prodrug thereof are administered in different pharmaceutical compositions.
90. The method of claim 80 wherein the tezacitabine and the secondary nucleoside analog or prodrug thereof are administered simultaneously.
91. The method of claim 80 wherein the tezacitabine and the secondary nucleoside analog or prodrug thereof are administered sequentially.
92. A kit comprising: a pharmaceutical composition comprising tezacitabine or gemcitabine; and a package insert that describes the method of claim 80.
PCT/US2004/019999 2003-06-18 2004-06-18 Method and compositions for treating tumors WO2004112730A2 (en)

Applications Claiming Priority (6)

Application Number Priority Date Filing Date Title
US47915503P 2003-06-18 2003-06-18
US60/479,155 2003-06-18
US48287403P 2003-06-27 2003-06-27
US60/482,874 2003-06-27
US51767503P 2003-11-06 2003-11-06
US60/517,675 2003-11-06

Publications (2)

Publication Number Publication Date
WO2004112730A2 true WO2004112730A2 (en) 2004-12-29
WO2004112730A3 WO2004112730A3 (en) 2005-03-10

Family

ID=33545335

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2004/019999 WO2004112730A2 (en) 2003-06-18 2004-06-18 Method and compositions for treating tumors

Country Status (1)

Country Link
WO (1) WO2004112730A2 (en)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009021551A1 (en) * 2007-08-13 2009-02-19 Universite De La Mediterranee A method for assessing the risk of toxicity in a chemotherapy
EP2348131A1 (en) * 2010-01-22 2011-07-27 Institut Curie Low levels of cytidine deaminase as a marker for genetic predisposition to develop cancer

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0843019A2 (en) * 1996-11-08 1998-05-20 Kyowa Medex Co., Ltd. Method for determination of specific nucleic acid sequence, and a reagent therefor

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0843019A2 (en) * 1996-11-08 1998-05-20 Kyowa Medex Co., Ltd. Method for determination of specific nucleic acid sequence, and a reagent therefor

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
EDA H. ET AL.: 'The antiproliferative activity of DMDC is modulated by inhibition of cytidine deaminase' CANCER RESEARCH vol. 58, 15 March 1998, pages 1165 - 1169, XP008042567 *
ELLEDGE R.M. ET AL.: 'Evaluation of thymidilate synthase RNA expression by polymerase chain reaction' MOLECULAR AND CELLULAR PROBES vol. 8, 1994, pages 67 - 72, XP002983414 *
RODRIGUEZ G.I.: 'Phase I clinical trials of tezacitabine Ä(E)-2'deoxy-2'-(fluoromethylene)cytidineÜ in patients with refractory solid tumors' CLINICAL CANCER RESEARCH vol. 8, September 2002, pages 2828 - 2834, XP002983415 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009021551A1 (en) * 2007-08-13 2009-02-19 Universite De La Mediterranee A method for assessing the risk of toxicity in a chemotherapy
EP2348131A1 (en) * 2010-01-22 2011-07-27 Institut Curie Low levels of cytidine deaminase as a marker for genetic predisposition to develop cancer
WO2011089197A1 (en) * 2010-01-22 2011-07-28 Institut Curie Low levels of cytidine deaminase as a marker for predisposition to develop cancer

Also Published As

Publication number Publication date
WO2004112730A3 (en) 2005-03-10

Similar Documents

Publication Publication Date Title
Tsai et al. Enhancement of chemosensitivity by tyrphostin AG825 in high-p185neu expressing non-small cell lung cancer cells
CN107849113B (en) Compositions and methods for treating patients with RTK mutant cells
CA2335617C (en) Use of 3'-azido-2',3'-dideoxyuridine in combination with further anti-hiv drugs for the manufacture of a medicament for the treatment of hiv
CN106102745A (en) Treat and prevent the method for graft versus host disease(GVH disease)
TWI697329B (en) Medicament for use in the treatment of a hematological cancer and use thereof
US20110312528A1 (en) Novel synergistic combination of gemcitabine with P276-00 or P1446A in treatment of cancer
KR102595395B1 (en) Method for treating cancer by combined use
US20230122940A1 (en) Combination therapy for treating cancer
KR20220061190A (en) ATP-Based Cell Sorting and Hyperproliferative Cancer Stem Cells
Tsai et al. Cytotoxic effects of gemcitabine-containing regimens against human non-small cell lung cancer cell lines which express different levels of p185neu
EP2755662B1 (en) Combinations of ribavirin and gdc-0449 for the treatment of leukaemia
US20110151020A1 (en) Active substance combination with gemcitabine for the treatment of epithelial cancer
Zhang et al. Flavopiridol (Alvocidib), a cyclin-dependent kinases (CDKs) inhibitor, found synergy effects with niclosamide in cutaneous t-cell lymphoma
EA001988B1 (en) METHOD FOR DEPLETING OF ADENOSINE 5'-MONOPHOSPHATE IN METHYTHIAODENSINEPHOEPHORYLASE (MTAse) DEFICIENT HOST CELLS IN HUMANS
ES2956508T3 (en) Deoxycytidine derivatives for use in cancer therapies
Untch et al. chemosensitivity testing in gynecologic oncology—dream or reality?
WO2004112730A2 (en) Method and compositions for treating tumors
WO2006053252A2 (en) Methods of treating hematological malignancies with nucleoside analog drugs
CN1427719A (en) Use of substituted acryloyl distamycin derivatives in treatment of tumors associated with high levels of glutathione
Mantel et al. P21waf-1-Chk1 pathway monitors G1 phase microtubule integrity and is crucial for restriction transition
EP3340978A1 (en) Compositions and methods for treating ewing family tumors
KR101340949B1 (en) Biomarker and screening method of methotrexate having pulmonary toxicity using thereof
JP2022520802A (en) Combination therapy for use in the treatment of cancer
US20100004193A1 (en) Combination therapy
Bergman et al. Increased sensitivity to gemcitabine of P-gP and MRP overexpressing human non-small cell lung cancer cell lines

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A2

Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BW BY BZ CA CH CN CO CR CU CZ DE DK DM DZ EC EE EG ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX MZ NA NI NO NZ OM PG PH PL PT RO RU SC SD SE SG SK SL SY TJ TM TN TR TT TZ UA UG US UZ VC VN YU ZA ZM ZW

AL Designated countries for regional patents

Kind code of ref document: A2

Designated state(s): GM KE LS MW MZ NA SD SL SZ TZ UG ZM ZW AM AZ BY KG KZ MD RU TJ TM AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HU IE IT LU MC NL PL PT RO SE SI SK TR BF BJ CF CG CI CM GA GN GQ GW ML MR NE SN TD TG

121 Ep: the epo has been informed by wipo that ep was designated in this application
122 Ep: pct application non-entry in european phase