WO1993023014A1 - Combinaison de drogues chimiotherapeutiques - Google Patents

Combinaison de drogues chimiotherapeutiques Download PDF

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WO1993023014A1
WO1993023014A1 PCT/US1993/004775 US9304775W WO9323014A1 WO 1993023014 A1 WO1993023014 A1 WO 1993023014A1 US 9304775 W US9304775 W US 9304775W WO 9323014 A1 WO9323014 A1 WO 9323014A1
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mmpr
inhibitor
tumor
group
pala
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PCT/US1993/004775
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English (en)
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Daniel S. Martin
Robert L. Stolfi
Joseph R. Colofiore
L. D. Nord
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Martin Daniel S
Stolfi Robert L
Colofiore Joseph R
Nord L D
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Application filed by Martin Daniel S, Stolfi Robert L, Colofiore Joseph R, Nord L D filed Critical Martin Daniel S
Priority to AU43834/93A priority Critical patent/AU684709B2/en
Priority to EP93914010A priority patent/EP0641193A4/fr
Priority to JP6503842A priority patent/JPH08506317A/ja
Publication of WO1993023014A1 publication Critical patent/WO1993023014A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/455—Nicotinic acids, e.g. niacin; Derivatives thereof, e.g. esters, amides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
    • A61K31/52—Purines, e.g. adenine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00

Definitions

  • the present invention relates to compositions
  • the invention also relates to combinations of such compounds with compounds which induce apoptosis.
  • the invention also relates to the use of these compositions and combinations in the treatment of anti-neoplastic disease.
  • Adenosine triphosphate is the key energy source in major metabolic processes such as biosynthesis, active transport and DNA repair. Consequently, if ATP production is inhibited, consumption of existing ATP will result in an energy deficiency that would adversely affect the functional and morphologic integrity of the cell.
  • the nicotinamide antagonist 6-aminonicotinamide 6-aminonicotinamide
  • NAD dinucleotide
  • 6-ANAD 6-ANAD
  • 6-ANADP 6-ANADP
  • 6-AN has demonstrated preclinical anti-cancer activity (Martin et al., Cancer Res. 17:600-604 (1957), Hunting et al., Nature Biochem. Pharmacol. 34:3999-4003 (1985)), but at tolerated doses, did not have antitumor efficacy as a single agent in humans (Herter et al., Cancer Research 21:31-37 (1961)).
  • MMPR adenosine analog 6-methylmercaptopurine riboside
  • NAD nicotinamide mononucleotide
  • ATP nicotinamide mononucleotide
  • thio-ITP thio-ITP
  • MMPR and 6-AN are synergistic if the timing of their administration is appropriate, because the lowering of NAD levels by MMPR favors the competition of 6-ANAD with NAD, and thereby enhances the magnitude of ATP depletion that is achieved by either drug alone.
  • the combination of 6-AN and MMPR produces regressions of advanced murine breast tumors which cannot be obtained with either drug alone (Martin, Mt. Yale J. Med. 52:426-434 (1985)).
  • MMPR in high dosage, is reported to decrease pyrimidine ribonucleotide
  • N-(phosphonacetyl)-L-aspartic acid (PALA) in low
  • non-toxic dosage can lower pyrimidine levels in vivo selectively in certain tumors (Martin et al., Cancer Res. 43:2317-2321 (1983)).
  • compositions which deplete cellular energy are provided.
  • the subject invention relates to chemotherapeutic drug combinations and their use in the treatment of antineoplastic disease.
  • drug combinations comprise: a) cellular energy depletion compounds, and b) at least one apoptosis inducing agent.
  • Figure 1 shows changes in PCr/Pi and NTP/Pi after treatment with the triple drug combination PALA, MMPR, and 6-AN.
  • the change in PCr/Pi and NTP/Pi at 10 hours is statistically significant (P less than 0.01, p less than 0.02).
  • the change in NTP/Pi between the 10 and 24 hour measurement is not significant.
  • FIG. 2 shows results obtained in Example 2.
  • CD8F1 mice bearing spontaneous, autochthonous breast tumors received three courses of treatment on a q 10-11 day schedule with Adria alone at 11 mg/kg, or with Adria at 6 mg/kg administered 21 ⁇ 2 hrs after PALA + MMPR + 6-AN (PALA at 100 mg/kg 17 hrs before MMPR at 150 mg/kg plus 6-AN at 10 mg/kg), or with the same regimen of PALA + MMPR + 6-AN without Adria.
  • Tumors averaged 304 mgs at initiation of therapy.
  • the subject invention relates to drug combinations comprising cellular energy depletion compounds and at least one apoptosis inducing agent.
  • the invention also relates to the use of such drug combinations in the treatment of antineoplastic disease.
  • apoptosis inducing agents are used in conjunction with cellular energy depletion compounds.
  • apoptosis is a process by which cells are removed from embryonic and developing somatic tissues, and has been implicated in terminal differentiation of myeloid cells, and in hormone-dependent tissue atrophy. It has also been documented in the cytoxic T-cell killing of tumor cells, and in tumor regression (Wyllie et al., Int. Rev. Cytology 68:251-306 (1980)). Cell death via necrosis is characterized by cell swelling, chromatin flocculation and disruption of cell integrity followed by cell lysis (Wyllie et al., Int. Rev. Cytology 68:251-306 (1980)).
  • apoptosis inducing agents include:
  • the invention provides drug combinations and methods for impairing cellular energy and nucleotide metabolism, and thereby dramatically increasing the antitumor efficacy of a wide variety of apoptosis-inducing antineoplastic agents, without a corresponding increase in host
  • the PALA-MMPR-6-AN combination produces a level of wide biochemical damage in cancer cells that complements, and is complemented by, the apoptosis-inducing biochemical effects of DNA-damaging agents, and thereby result in enhanced kill of tumor cells.
  • 5-fluorouracil 5-fluorouracil
  • adriamycin adriamycin, taxol, radiotherapy, mitomycin C, cis-platinum, cytoxan, phenylalanine mustard, arid
  • etoposide with the PALA-MMPR-6-AN triple combination demonstrate greater anticancer activity than that observed with the individual drugs alone at maximum tolerated dose (MTD) or with the PALA-MMPR-6-AN combination alone.
  • Intracellular ATP has been negelected as a primary target for cancer chemotherapy because ATP has long been considered too important a source of energy for all cells to warrant the expectation that primarily directed
  • anti-ATP chemotherapy could be selective for cancer cells and safe for host tissues.
  • the subject application documents novel compositions and methods for impairing tumor cell energy metabolism, with striking therapeutic activity, a safe therapeutic index, and an associated depletion of ATP levels in the in vivo treated tumors.
  • the energy-depleting compositions of the invention When administered in conjunction with agents which trigger apoptosis (programmed cell death) in tumors, the energy-depleting compositions of the invention produce therapeutic activity against spontaneous tumors in vivo substantially greater than can be obtained by either energy depletion compositions or
  • NADH reduced NAD
  • NAD nicotinamide adenine
  • dinucleotide phosphate are oxidizing agents which function as coenzymes in critical biochemical reactions. Disruptions in NAD synthesis and metabolism has profoundly adverse effects on cellular integrity because of the central role played by these coenzymes in intermediary metabolism including the generation of ATP from ADP. A limitation of adenine or NAD, or both, are keys to ATP depletion. In the subject invention, these metabolites are targets for chemotherapy designed to result in
  • the cellular energy depletion compounds of the invention are typically used in combination and typically include:
  • DNA-damaging drugs is increased when the DNA repair potential of the cell is decreased.
  • DNA-damaging drugs by virtue of its ability to deplete both the deoxyribonucleotides, and the energy source required for the DNA repair processes.
  • Inhibitors of purine nucleotide biosynthesis of the invention include the following: a) Direct Inhibitors
  • MMPR and folate antagonists which are relatively selective inhibitors of the enzyme glycinamide ribonucleotide transformylase, e.g. DDATHF or DACTHF.
  • Nicotinamide antagonists of the invention include the following:
  • Inhibitors of pyrimidine nucleotide biosynthesis of the invention include the following:
  • PALA is an inhibitor of de novo pyrimidine
  • the triple combination is less toxic when the
  • interval between courses of PALA + MMPR + 6-AN treatment is extended from 7 days to 10 or 11 days, and this change in the schedule of administration reduces toxicity
  • an apoptosis inducing agent such as FUra
  • GSH is a major cellular reductant (Meister et al., Pharmac. Ther.
  • biochemical modulation refers to the pharmacologic manipulation of metabolic pathways by an agent (the modulating agent) to produce the selective enhancement of the antitumor effect of a second agent (the “effector” agent) (Martin, Biochemical modulation ⁇
  • the triple combination, PALA + MMPR + 6-AN may be viewed as biochemical modulation employed to establish in tumor cells a wide array of biochemical changes ⁇ i.e., a primary diminution of ATP lowering of all of the
  • DNA-damaging anticancer agents e.g., FUra, cisplatinum, BCNU
  • FUra cisplatinum
  • BCNU DNA-damaging anticancer agents
  • This modulation and complementation results in enhanced cancer cell deaths which reflect in improved tumor regression rates.
  • Intracellular ATP has been neglected as a primary target for cancer chemotherapy because ATP has long been considered too important a source of energy for all cells to warrant the expectation that primarily directed
  • anti-ATP chemotherapy could be selective for cancer cells and safe for host tissues.
  • the subject has
  • cellular energy depletion compounds are administered with 5-fluoroaracil as the apoptosis inducing agent.
  • the subject invention relates to a highly active chemotherapeutic drug
  • fluoropyrimidine-induced "thymineless death” has been shown to be due to "programmed cell death” (apoptosis) activated by DNA strand breakage (Kyprianou et al., Biochem. Biophys. Res. Communications 165:73-81,
  • PALA-MMPR-6-AN drug combination reveal severe ATP loss, inhibition of macromolecular synthesis, inhibition of the pentose phosphate shunt, NAD depletion, reduction of ribonucleoside triphosphates and inhibition of protein synthesis, a pattern of findings which overlap with those reported in "thymineless death” and apoptosis.
  • apoptosis-inducing anticancer agent such as FUra
  • PALA-MMPR-6-AN induces elevation of PRPP levels and reduction of UTP pools.
  • the triple drug combination also increases the therapeutic activity of FUra by increasing the anabolism of FUra to its nucleotides, as well as by favoring the competition of the analog over the natural pyrimidine intermediates whose levels have been reduced by PALA.
  • a combination of PALA plus FUra has proven to be significantly more active than FUra alone in the clinical treatment of colorectal cancer (Ardalan et al., J. Clin. Oncol. 6:1053-1058 (1988), 0'Dwyer et al., J. Clin. Oncol. 8:1497-1503 (1990)).
  • MMPR can result in the elevation of PRPP levels in human tumors such as colon, ovary and breast (Peters et al., Cancer Chemother.
  • cellular energy depletion compounds are administered with adriamycin as the apoptosis inducing agent.
  • This combination yields significantly enhanced anti-cancer activity over that produced by either Adria alone at maximum tolerated dose (MTD), or by the triple drug combination, against large, spontaneous,
  • Adriamycin cannot be given to breast cancer patients for long periods because its cumulative dosage reaches
  • cellular energy depletion compounds are administered with taxol as the apoptosis inducing agent.
  • An advantageous embodiment of the invention comprises;
  • ATP-depleting drug combination administered in conjunction with taxol results in the inhibition of energy-dependent resistance mechanisms to taxol.
  • the new antimicrotubule agent, taxol, an antimitotic agent and the first compound with a taxane ring has demonstrated significant antineoplastic activity in patients with refractory ovarian cancer, refractory breast cancer, non-small cell lung cancer, and other cancers (Rowinsky et al., Pharmac. Ther. 52:35-84 (1991)). Its antitumor activity, novel mechanism of action, and unique structure have generated excitement.
  • Taxol is a plant product (obtained from the bark of the Pacific Yew tree, Taxol brevifolia), and because it is obtained from a limited resource there is a supply problem critical to its widespread clinical use.
  • Taxol is not considered a DNA-damaging agent. Taxol is an antimitotic agent that binds
  • microtubules preferentially to microtubules in the absence of the cofactors tubulin and GTP, a mechanism unlike that of other ant ⁇ microtuble agents in cancer chemotherapeutics (e.g., vincristine, and colchicine). It blocks cells in the mitotic phase of the cell cycle so that these cells are unable to replicate normally, and cell death ensues (Rowinsky et al., Pharmac. Ther. 52:35-84 (1991)).
  • cancer chemotherapeutics e.g., vincristine, and colchicine
  • taxol-induced disruption of the microtubular network of cancer cells induces death of cancer cells by apoptosis (Martin et al., Cell Ti ⁇ sue Kinet. 23:545-559 (1991), and Lennon et al., Cell Prolif. 24:203-214 (1991)), as Adria does (Marks et al., Biochem. Pharmacol. 42:1859-1867 (1990)).
  • Another similarity between taxol and Adria is that cells resistant to both agents usually display the
  • MDR multi-drug resistant
  • MDR cells are cross-resistant to both Adria and Taxol (Rowinsky et al., Pharmac. Ther. 52:35-84 (1991)) and Gerlach et al., Cancer Surveys 5:26-46 (1986)), and, since inhibitors of energy production (e.g., azide) when added to such resistant cells in vitro increase the net accumulation of drug in MDR-cells (Gerlach et al., Cancer Surveys 5:26-46 (1986) and Dano, Biochim. Biophys. Acta. 323:466-483 (1973)), the ATP-depleting effects of the PALA-MMPR-6-AN triple combination reverses Adria-and taxol-resistant cells to chemosensitivity.
  • inhibitors of energy production e.g., azide
  • Taxol is similarly enhanced in therapeutic activity by the prior administration of the PALA-MMPR-6-AN triple combination. A markedly lower dose of taxol is necessary in the quadruple combination. Although taxol had been previously reported to be ineffective against the CD8F1 murine mammary carcinoma (Rowinsky et al., Pharmac. Ther. 52:35-84 (1991)), it was considered that the high degree of chemotherapeutic correlation that had been observed between this model and the human disease
  • Example 3 shows that taxol alone is active against the CD8F1 tumor, that the administration of the
  • PALA-MMPR-6-AN triple drug combination prior to taxol significantly enhanced antitumor activity over that produced by either taxol alone at MTD, or the triple drug combination alone, and that only one-third the dose of taxol (in combination) is required for this greater antitumor activity.
  • cellular energy depletion compounds are administered with radiation as the apoptosis inducing agent.
  • the PALA-MMPR-6-AN combination sensitizes tumors to ionizing radiation therapy (see Example IV).
  • the drug combinations of the subject invention are useful in treating a wide variety of neoplastic diseases.
  • the apoptosis inducing agent of the combination of the invention (which comprises the cellular energy depletion compounds plus an apoptosis inducing agent), is selected on the basis of demonstrated antitumor activity when it is used without the cellular energy depleting combination.
  • depletion compounds e.g. PALA-MMPR-6-AN
  • PALA-MMPR-6-AN apoptosis inducing agents to which the tumors are already susceptible to some degree.
  • fluorouracil (and therefore, the
  • PALA-MMPR-6-AN is useful for treating tumors of the colon, stomach, breast, head-and-neck, and pancreas.
  • Taxol (and therefore the combination of taxol with the cellular energy depletion compounds) is useful for
  • Adriamycin (and therefore the combination of adriamycin with the energy depletion compounds of the subject invention) is useful in a wide variety of tumors: acute leukemias, malignant lymphomas, cancers of the ovary, breast, lung, bladder, thyroid, endometrium, testes, prostate, cervix,
  • Ionizing radiation (and therefore the combination of ionizing radiation with the cellular energy depletion compounds of the invention) is useful for treating a variety of tumor types, including, lymphomas, and cancers of the breast, pelvis, and lung. Furthermore, since the energy depleting combinations of the subject invention render tumors more sensitive to each apoptosis inducing agent, the range of tumors
  • susceptible to treatment with a given apoptosis inducing agent in combination with the cellular energy depleting combinations of the invention is broader than the range of tumor types treatable with the apoptosis inducing agent alone.
  • a combination of two or more apoptosis inducing agents is optionally administered in conjunction with an energy depleting composition of the invention.
  • MDR Multiple drug resistance
  • adriamycin, taxol, and vinca alkaloids from cells or critical regions of cells, such as the nucleus
  • the response to adriamycin decreases over time during the three courses of drug treatment, indicating that the tumors are developing resistance to the drug.
  • the response rate actually improves over time during the three courses of treatment, indicating that the tumors are not developing resistance to adriamycin, and in fact, an unprecedented 100% partial regression rate was observed in these animals ( Figure 2).
  • the compounds of the invention are administered in therapeutically effective amounts.
  • the term is administered in therapeutically effective amounts.
  • terapéuticaally effective amount refers to that amount which provides therapeutic effects for a given condition and administration regimen.
  • the compounds and compositions of the invention are administered orally, by parenteral injection, intravenously, or topically, depending on the condition being treated.
  • an inhibitor of pyrimidine biosynthesis e.g. PALA
  • an inhibitor of purine nucleotide biosynthesis e.g. MMPR
  • a nicotinamide antagonist e.g. 6-AN
  • apoptosis inducing agent(s) is administered after the cellular energy depleting combination, typically about 2 to 3 hours afterwards (although the timing may be modified for particular drugs according to observed clinical benefit).
  • the doses of the particular agents are determined according to clinical response, alterations in biochemical indices of efficacy, and observed signs of toxicity.
  • PALA is typically administered in a dose of 250 mg/square meter; this dose has been found to be suitable in clinical studies in which PALA was administered as a modulator of fluorouracil.
  • a typical single dose of 6-AN is 10 to 50 mg/square meter. The optimum dose range for 6-AN is determined by taking tissue biopsies before and after administration of increasing doses of 6-AN and determining activity of enzymes requiring pyridine courses of treatment.
  • a typical single dose of MMPR is 200 to 400 mg/square meter; a dose of 225 mg/kg has been safely administered to humans in combination with PALA.
  • the compounds and compositions of the invention are formulated in pharmaceutically acceptable carriers.
  • Niacin or niacinamide ameliorate toxicity due to 6-AN (or other nicotinamide antagonists; uridine, prodrugs of uridine, or other pyrimidine nucleotide precursors reverse the biochemical deficits produced by PALA or other inhibitors of pyrimidine nucleotide
  • purine nucleotide poois affected by MMPR or other inhibitors of purine nucleotide biosynthesis
  • appropriate purine nucleotide precursors e.g., inosine, AICAR, adenosine, or hypoxanthine
  • an inhibitor of purine degradation such as allopurinol
  • doses of apoptosis-inducing agents are typically less than or equal to the doses that would be administered in the absence of the
  • pretreatment with PALA-MMPR-6-AN permits substantial reductions in the doses of adriamycin and taxol required to produce optimum benefit. This is very important, since adriamycin produces cumulative cardiotoxicity, limiting the total amount that a patient can safely receive. Supplies of taxol are currently limited (since it is obtained from the bark of the Pacific Yew, a relatively uncommon tree). A substantial reduction in the dose of taxol needed to produce an optimum clinical effect (as is demonstrated in Example III), permits more patients to be effectively treated with taxol (or other scarce but effective apoptosis-inducing agents).
  • a course of treatment (PALA-MMPR-6-AN followed by an apoptosis-inducing agent) is repeated about every 10 days. Patients typically receive three or more
  • the compounds and compositions of the invention are dissolved or suspended in aqueous medium such as sterile physiological saline.
  • aqueous medium such as sterile physiological saline.
  • solubilizing agents like ethanol, propylene glycol, or polyoxyethylated castor oil are used.
  • Example I PALA, MMPR, 6-AN and FUra
  • each treatment group Individual tumors ranged in size from 100 to 500 mg, and the average tumor weight in all groups was 260 mg at the beginning of treatment.
  • Murine Breast Tumor System First Passage CD8F1 Mammary Carcinoma ⁇
  • each individual cancer has a heterogeneous neoplastic cell population.
  • the first generation transplants of CD8F1 breast tumors are obtained from a tumor cell brei made by pooling 3-4 spontaneously-arising tumors.
  • the individual transplants in each experiment develop from a single brei that, although common to all the mice in that experiment, has a neoplastic cell composition that is likely slightly different from that in another
  • the CD8F1 first generation breast tumor is included in the murine tumor testing panel of the National Cancer Drug Screening Program (Goldin et al., Eur. J. Cancer 17:129-142 (1981)).
  • tumor-bearing mice were distributed among experimental groups so that mice carrying tumors of approximately equal weight were represented in each treatment group.
  • the average tumor weight was close to 125 mg at the beginning of treatment.
  • Tumor Measurements Two axes of the tumor (the longest axis, L, and the shortest axis, W) were measured with the aid of a Vernier caliper. Tumor weight was estimated according to the formula: tumor weight (mg) L (mm) ⁇ (W(MM) 2 )/2.
  • Chemotherapeutic Agents ⁇ MMPR, 6-AN and FUra were obtained from Sigma Chemical Co., St. Louis, MO. Each of these agents was dissolved in 0.85% NaCl solution
  • chemotherapy i.e., MMPR + 6-AN
  • MMPR + 6-AN a chemotherapeutic sequence
  • partial tumor regression is defined as a reduction in tumor volume of 50% or greater compared to the tumor volume at the time of initiation of treatment.
  • the partial regression rate obtained from a particular treatment is expressed as a percentage; i.e., number of partial regressions per group/total number of animals per group ⁇ 100.
  • Radiolabeled precursors, 32P and (3H)-L-Leucine were administered i.p.; the labeling period was 2 hrs; At the end of the labeling period, animals were sacrificed by cervical dislocation. Tumor tissues were homogenzied in
  • TNE buffer containing 1% Triton-X 100 TNE: 0.01 M
  • fructose-6-phosphate were measured on perchloric acid extracts by published methods (6-phosphogluconate (Haid, Methods of Enzymatic Analysis (Bergmeyer, H.U., ed). New York: Academic Press pp. 1248-1250 (1974));
  • NTP levels in Tumor ⁇ HPLC analysis in tumor was performed on a Waters 840 HPLC system with a WISP automatic sampler. NTP levels were analyzed by ion-exchange gradient chromatography using a Waters SAX column starting with 3 mM NH 4 H 2 PO 4 , pH 3.5, proceeding in two steps to 70% 0.5 M NH 4 H 2 PO 4 , pH 5.0, plus 30% starting buffer. The run time for each 100 ul of extracted sample was 60 min. Tumor NTP levels are expressed as micrograms nucleotide triphosphate per milligram of protein.
  • 31P NMR (Nuclear Magnetic Reeonance) Spectra ⁇ 31P NMR spectra were obtained using techniques described previously (Koutcher et al., Cancer Res. 50:7252-7256 (1990)). Briefly, spectra were obtained on a General Electric NT-300 wide bore spectrometer operating at 121.5 MHz. Experimental parameters included a spectral width of +/- 12,000 Hz, 60° tip angle, recycle delay of 2 seconds, 512-1024 averaged free induction decays (FID's), and 1024 data points. The spectra are partially saturated using these experimental conditions. Four turn solenoid coils with a Faraday shield (Ng et al., J. Magn. Reson. 49:526 (1982)), positioned between the body of the mouse and the coil were used to detect the NMR signal. Control
  • Spectral peak areas were estimated by fitting the spectra to a series of Lorentzian peaks, using a program (GEMCAP) available on the spectrometer, after fitting the baseline to a third order polynomial (using standard General
  • beta-NTP was used for calculating NTP peak area ratios.
  • Thvmidine kinase and Thymidylate svnthase assays were homogenzied (Potter-Elvehjem homogenizer) as a 20% (wt/vol) solution in Tris-Cl (100 mM, pH 7.6),
  • the assay mixture contained Tris-Cl (100 mM, pH 7.6), ATP (5 mM), MgCl 2 and (5-C 3 H 3 )thymidine (25 uM, 1.0 Ci/mmol) and cytosolic protein. Thymidylate synthase activity was measured by the release of tritium from (5- 3 H)dUMP (10 uM, 1.0 Ci/mmol), CH 2 H 4 PteGlu (100 uM) and cytosolic fraction (25 uL) (Roberts, Biochemistry 5:3546-3548 (1966)). Reactions were terminated by the addition of perchloric acid (10 uL, 0.7M). Protein was determined by the method of Lowry et al., J. Biol. chem. 193:265-275 (1951).
  • FUra into RNA was determined by isolating tumor RNA by the acid-guanidine isothiocyanate procedure (Chomczynski et al., Anal. Biochem. 162:156-159 (1987)), after treatment with (5- 3 H)FUra (2.0 mCi/mmol). Tissues were harvested
  • spontenaous CD8F1 breast tumor transplants were treated with the triple combination of PALA + MMPR + 6-AN.
  • PALA 100 mg/kg
  • MMPR 150 mg/kg
  • 6-AN 10 mg/kg
  • a second group (Group 2) received the same treatment with PALA + MMPR + 6-AN, followed 2% hours later by FUra (75 mg/kg).
  • Partial tumor regressions were observed in 8 of the surviving 48 mice (17%), treated with PALA + MMPR + 6-AN (Group 1). The range of regression rate in individual experiments varied from 0 to 30%. The addition of FUra to the same regimen of PALA + MMPR + 6-AN (Group 2) produced a significant and meaningful increase in therapeutic activity. Thirty-seven of the 50 treated mice (i.e., 74%) experienced partial tumor regressions (with a range in the individual experiments of 60-90%). This level of
  • the PALA + MMPR + 6-AN regimen (group 1) produced a partial tumor regression rate of 38% in mice bearing spontaneous, autochthonous breast tumors (24 partial tumor regressions in 64 surviving mice) with an acceptable mortality rate (4%, 3 deaths in 67 treated mice).
  • the addition of FUra to the three-drug regimen (Group 2) resulted in 41 partial regressions in the 61 surviving mice, or 67% (with a range of 50-90% in the individual experiments) without an increase in the mortality rate (only 7%), and with only a 10% body weight loss.
  • PALA-MMPR-6-AN resulted in significant inhibition of macromolecular synthesis in first passage CD8F1 breast tumors detectable at the earliest time point examined, 2.5 hours after drug administration, and progressing to 80% inhibition of DNA synthesis, 85% inhibition of RNA
  • 6-phosphogluconate (6-PG) dehydrogenase (Herken et al., Biochem. Biophys. Res. Comm. 36:93-100 (1969)).
  • the accumulation of this substrate results in feed-back inhibition of phosphoglucose isomerase and prevents the formation of fructose-6-phosphate from glucose-6-phosphate (G-6-P) (Racker, In: Mechanisms in Bioenergetics.
  • NMR spectra were obtained from first passage CD8F1 breast tumors prior to treatment, and at 2, 10, and 24 hours post treatment with PALA-MMPR-6-AN. Baseline spectra were similar to those obtained in previous studies (Koutcher et al., Magnetic Resonance in Medicine
  • PALA-MMPR-6-AN Group 4
  • ATP levels in first passage CD8F1 breast tumors were significantly depressed, reaching a level of 32% of control in tumors from mice treated with the 3-drug combination at 24 hours post treatment.
  • b ( ) % of saline control (Group 1).
  • RNA synthesis Since the inhibition of RNA synthesis is one of the earliest measurable events after the administration of the triple combination, the effect of the combination upon the incorporation of FUra into RNA is of interest.
  • the amount of tumor (FU)RNA in the group which received PALA + MMPR + 6-AN + FUra was appreciable (355 ⁇ 127 cpm/mg RNA). Thus, although RNA synthesis was significantly inhibited
  • thymidylate synthase activity 24 hours after treatment are shown in Table 5.
  • **avg. control activity 12.71 pmol/min/mg protein
  • Thymidylate synthase activity was decreased by nearly 50% after treatment with PALA-MMPR-6-AN, even without the addition of FUra (Group 2, Table 5). This depression of enzyme activity is likely due to the general inhibition of protein synthesis following treatment with the 3-drug combination described above. However, the addition of FUra 2.5 hours following treatment with the 3-drug
  • This example shows an impressive increase in tumor regression rates when FUra was administered in conjunction with PALA, MMPR and 6-AN in the therapy of either advanced first passage, or spontaneous, murine breast tumors, and shows the results of measurements of biochemical
  • CD8F1 hybrid mice bearing single spontaneous, autochthonous breast tumors arising during the preceeding week were selected from a colony which has been described previously (Stolfi et al., Cancer Chemother. Rep.
  • mice were distributed among experimental groups so that mice carrying tumors of approximately equal weight were represented in each treatment group.
  • each individual cancer has a heterogeneous cell population and therefore, unlike long-transplanted tumor lines, one spontaneous tumor may differ from another of the same histiotype in
  • Chemotherapeutic Agents MMPR and 6-AN were obtained from Sigma Chemical Co., St. Louis, MO. Adria was obtained from Adria
  • partial tumor regression is defined as a reduction in tumor volume of 50% or greater compared to the tumor volume at the time of initiation of treatment.
  • the partial regression rate obtained from a particular treatment is expressed as a percentage; i.e., Number of partial regressions per group/Total number of animals per group ⁇ 100.
  • Complete tumor regression was defined as the inability to detect tumor by palpation at the initial site of tumor appearance.
  • a comparable group of mice was treated with Adria alone at 11 mg/kg which had been determined previously to be the MTD of Adria alone when administered in this 10-11 day treatment schedule. Results were observed 7 days after the third course of treatment.
  • c Adria at 11 mg/kg is the MTD of Adria alone in a 10-11 day treatment schedule.
  • the PALA + MMPR + 6-AN regimen (Group 1) produced a partial tumor regression rate of 76% in mice bearing spontaneous, autochthonous breast tumors (32 partial tumor regressions in 42 surviving mice with a range of 50-92% in the individual experiments) with no toxic deaths in the treated mice.
  • the addition of Adria to the three-drug regimen (Group 2) resulted in 42 partial tumor regressions in the 42 surviving mice, or 100% (i.e., with no range in the individual experiments), without a significant
  • treatment groups is plotted at 7 days after each of the 3 courses of treatment. Note the diminishing therapeutic effect in mice treated with Adria alone at it's MTD of 11 mg/kg every 10-11 days. After the first course, 43% of the tumors had regressed to 50% or less of their initial size. However, 7 days after the second course, only 21% were partially regressed, and 7 days after the third course, only 16% were still in partial regression. In contrast, in mice treated with PALA + MMPR + 6-AN followed by Adria at 6 mg/kg, the regression rate was 66% after the first course, and then it increased to 93% after the second course and to 100% after the third course.
  • CD8F1 hybrid mice bearing single spontaneous, autochthonous breast tumors arising during the preceding week were selected from a colony (Stolfi et al., Cancer Chemother. Rep. 55:239-251 (1971) and Martin et al., Cancer Chemother. Rep., Part 2, 5:89-109 (1975)).
  • a tumor cell brei prepared by pooling 3-4 spontaneously arising CD8F1 breast tumors, was transplanted into syngeneic three-month old mice.
  • the tumor-bearing mice were distributed among experimental groups so that mice carrying tumors of approximately equal weight were represented in each treatment group. Therapy was begun when the tumors were advanced and relatively large; the average tumor weight was close to 130 mg at the beginning of treatment.
  • each individual cancer has a heterogeneous cell population.
  • the first generation transplants of CD8F1 breast tumors are obtained from a tumor cell brei made by pooling 3-4 spontaneously arising tumors.
  • the individual transplants in each experiment develop from a single brei that, although common to all the mice in that experiments, has a neoplastic cell composition that is likely slightly different from that in another
  • the CD8F1 first generation breast tumor is included in the murine tumor testing panel of the National Cancer Drug Screening Program (Goldin et al., Eur. J. Cancer
  • MMPR and 6-AN were obtained from Sigma Chemical Co., St. Louis, MO. Each of these agents was dissolved in 0.85% NaCl solution immediately before use. PALA and taxol were obtained from the Department of Health,
  • Taxol was received already soluabilizxed in polyoxyethylated castor oil and dehydrated alcohol. Because of the known toxicity of this diluent, the Taxol stock was diluted, depending upon the dose to be administered, a minimum of 6-fold in saline before injection. For doses below 10 mg/kg, taxol was administered in 0.1 ml/10 g of bodyweight. For doses above 10 mg/kg, an appropriate additional volume was administered.
  • Taxol was administered in a fractionated schedule as follows: Taxol (4 mg/kg) simultaneously with MMPR + 6-AN then, 1 1/2 hours later, Taxol (4 mg/kg) q 3 hours ⁇ 7.
  • partial tumor regression is defined as a reduction in tumor volume of 50% or greater compared to the tumor volume at the time of initiation of treatment.
  • the partial regression rate obtained from a particular treatment is expressed as a percentage; i.e., Number of partial regressions per
  • Table 7 reports a series of four experiments in CD8F1 mice bearing first passage spontaneous CD8F1 advanced breast tumor transplants. Each individual experiment compared a group of saline-treated controls with a second group that received the maximal tolerated dose (MTD) of taxol alone (80 mg/kg) in a q 10-11 day administration schedule for a total of 3 courses, and observations were recorded 6 days after the last course of treatment.
  • MTD maximal tolerated dose
  • Taxol 80 -5 0/10 ( 0%) 1,749
  • Taxol was added to the triple regimen in the indicated fractionated schedule (Group 2). The weight loss (25%) and absent mortality (0%) in the taxol-containing four drug
  • the average tumor weight (138 mg) of the three-drug regimen + Taxol, Group 2 although much smaller than the average tumor weight (271 mg) of its 3-drug control without taxol, Group 1, nevertheless was not significantly different from that of its control (Group 1) But there were 5/9 partial tumor regressions, or a 55% PR, compared to only 1/9 (11%PR) in its three-drug control. Group 1.
  • Treatment was repeated at 10 or 11 day intervals in all groups for a total of 3 courses, and observations were recorded 6 days after the last course of treatment.
  • Group 4 the taxol-containing four drug combination, had tumors that were significantly inhibited compared to the tumors in the three drug combination without taxol, Group 3, and to taxol alone, Group 2, and this was achieved with little toxicity (13% weight loss; 0% mortality). It should be noted that the superior anti-tumor activity of Group 4 was achieved with a dose of taxol (25 mg/kg) that was less than one-third that of taxol alone (80 mg/kg), Group 2.
  • the PALA-MMPR-6-A ⁇ combination also sensitizes tumors to ionizing radiation therapy. Mice with advanced transplanted CD8F1 breast tumors (initial tumor weight 150mg) were divided into four
  • mice had regressions (tumor size less than 50% of original weight), and 3 of those regressions were complete.
  • tumor w't. 150 mg when treatment initiated.
  • Three courses of the indicated treatment were administered with a 10-11 day interval between courses. Subscripts refer to doses in mg/kg. Radiation was administered at 15 Gy. Observations are recorded at 36 days after the third course of treatment.

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Abstract

L'invention se rapporte à certaines compositions et combinaisons de composés de déplétion de l'ATP et d'agents provoquant l'apoptose. L'invention concerne égalament des procédés de traitement antinéoplasique par l'administration de ces combinaisons.
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EP1171118A2 (fr) * 1999-04-09 2002-01-16 Jessie L.S. Au Methodes et compositions ameliorant la diffusion d'agents therapeutiques a l'interieur de tissus
EP1349555A1 (fr) * 2000-12-04 2003-10-08 Sloan-Kettering Institute For Cancer Research Traitement du cancer par reduction d'energie intracellulaire et a l'aide de pyrimidines
WO2003105862A1 (fr) * 2002-06-13 2003-12-24 Sloan-Kettering Institute For Cancer Research Strategies d'appauvrissement d'energie in vivo permettant de tuer des cellules cancereuses resistant aux medicaments
JP2008133291A (ja) * 1995-01-17 2008-06-12 Aventis Pharma Sa 化増殖症の併用療法
EP2711009A1 (fr) * 2012-09-19 2014-03-26 Institut Univ. de Ciència i Tecnologia, S.A. Composés destinés à être utilisés dans le traitement ou la prévention du cancer primitif et métastatique du sein et de la prostate

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AU2002342808A1 (en) * 2002-10-09 2004-05-04 Unibioscreen S.A. Extract with anti-tumor and anti-poisonous activity

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US5114951A (en) * 1989-04-11 1992-05-19 Burroughs Wellcome Company Agents for combating multiple drug resistance

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US5114951A (en) * 1989-04-11 1992-05-19 Burroughs Wellcome Company Agents for combating multiple drug resistance

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See also references of EP0641193A4 *

Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2008133291A (ja) * 1995-01-17 2008-06-12 Aventis Pharma Sa 化増殖症の併用療法
JP2012036201A (ja) * 1995-01-17 2012-02-23 Aventis Pharma Sa 過増殖症の併用療法
EP1171118A2 (fr) * 1999-04-09 2002-01-16 Jessie L.S. Au Methodes et compositions ameliorant la diffusion d'agents therapeutiques a l'interieur de tissus
EP1349555A1 (fr) * 2000-12-04 2003-10-08 Sloan-Kettering Institute For Cancer Research Traitement du cancer par reduction d'energie intracellulaire et a l'aide de pyrimidines
EP1349555A4 (fr) * 2000-12-04 2007-04-25 Sloan Kettering Inst Cancer Traitement du cancer par reduction d'energie intracellulaire et a l'aide de pyrimidines
US7381713B2 (en) 2000-12-04 2008-06-03 Sioan-Kettering Institute For Cancer Research Treatment of cancer by reduction of intracellular energy and pyrimidines
WO2003105862A1 (fr) * 2002-06-13 2003-12-24 Sloan-Kettering Institute For Cancer Research Strategies d'appauvrissement d'energie in vivo permettant de tuer des cellules cancereuses resistant aux medicaments
US7514413B2 (en) * 2002-06-13 2009-04-07 Sloan-Kettering Institute For Cancer Research In-vivo energy depleting strategies for killing drug-resistant cancer cells
EP2711009A1 (fr) * 2012-09-19 2014-03-26 Institut Univ. de Ciència i Tecnologia, S.A. Composés destinés à être utilisés dans le traitement ou la prévention du cancer primitif et métastatique du sein et de la prostate

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