WO2018234574A1 - Combination therapy for the treatment of cancer - Google Patents

Combination therapy for the treatment of cancer Download PDF

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Publication number
WO2018234574A1
WO2018234574A1 PCT/EP2018/066834 EP2018066834W WO2018234574A1 WO 2018234574 A1 WO2018234574 A1 WO 2018234574A1 EP 2018066834 W EP2018066834 W EP 2018066834W WO 2018234574 A1 WO2018234574 A1 WO 2018234574A1
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polyamine
drug
subject
dfmo
inhibitor
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PCT/EP2018/066834
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French (fr)
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Yann GASTON-MATHE
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Ygm Consult Sas
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Publication of WO2018234574A1 publication Critical patent/WO2018234574A1/en

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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
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/365—Lactones
    • 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
    • A61K31/13—Amines
    • A61K31/155—Amidines (), e.g. guanidine (H2N—C(=NH)—NH2), isourea (N=C(OH)—NH2), isothiourea (—N=C(SH)—NH2)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A61K31/195—Carboxylic acids, e.g. valproic acid having an amino group
    • A61K31/197—Carboxylic acids, e.g. valproic acid having an amino group the amino and the carboxyl groups being attached to the same acyclic carbon chain, e.g. gamma-aminobutyric acid [GABA], beta-alanine, epsilon-aminocaproic acid or pantothenic acid
    • A61K31/198—Alpha-amino acids, e.g. alanine or edetic acid [EDTA]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • the application relates to a combination therapy for the treatment of cancer, more particularly to the combination of a polyamine-vectored cytotoxic agent with polyamine depletion for improved cancer therapy.
  • Polyamines are present in mammalians as a result of endogenous synthesis (e.g., intracellular synthesis) as well as of exogenous import (e.g., via food intake). Tumor growth is generally associated with upregulation of the polyamine level.
  • WO 2005/100363 in the name of PIERRE FABRE MEDICAMENT describes podophyllotoxin derivatives, such as the F14512 drug (2- ⁇ 3-[4-(3- aminopropylamino)butylamino]propylamino ⁇ -N-[9-(4-hydroxy-3,5-dimethoxyphenyl)-8-oxo- 5,5a,6,8,8a,9-hexahydrofuro[3';4';6,7]naphto[2,3-d][l,3]dioxol-5-yl]acetamide).
  • the F14512 drug (2- ⁇ 3-[4-(3- aminopropylamino)butylamino]propylamino ⁇ -N-[9-(4-hydroxy-3,5-dimethoxyphenyl)-8-oxo- 5,5a,6,8,8a,9-hexahydrofuro[3';4';6,7]naphto[2,
  • enzyme inhibitors to inhibit the polyamine cycle.
  • enzyme inhibitors notably comprise the Ornithine Decarboxylase (ODC) inhibitor eflornithine (DFMO) (cf. e.g., Cipolla et al. 2010, Biomedecine & Pharmacotherapy 64: 363-368) and the SAMDC inhibitor mitoguazone (MGBG or METHYL-GAG) (cf. e.g., Murray-Stewart et al. 2016, Biochemical Journal 473: 2937-2953).
  • ODC Ornithine Decarboxylase
  • DFMO eflornithine
  • MGBG or METHYL-GAG SAMDC inhibitor mitoguazone
  • polyamine enzyme inhibitors have led to discrepant anti-tumor responses or to insufficient therapeutic efficacy, notably concerning relapsed or refractory cancers.
  • the application provides means for combination therapy in the treatment of cancer.
  • the means of the application combine a polyamine-vectored cytotoxic agent with polyamine depletion.
  • the means of the application notably provide improved therapeutic efficacy.
  • the application generally relates to the combination of a polyamine-vectored cytotoxic agent with polyamine depletion for improved cancer therapy.
  • the application relates more particularly to a drug comprising a cytotoxic agent linked to a polyamine moiety for use in cancer therapy, wherein said cancer therapy comprises administering said drug to a subject in need thereof and simultaneously, sequentially or separately submitting said subject to polyamine depletion.
  • the application also relates more particularly to a drug comprising a cytotoxic agent linked to a polyamine moiety for use in cancer therapy, wherein said cancer therapy comprises administering said drug to a subject in need thereof and simultaneously and separately submitting said subject to polyamine depletion.
  • a pre-treatment for instance with at least one inhibitor of polyamine synthesis and/or polyamine metabolism like DFMO before a simultaneous treatment with a drug comprising a cytotoxic agent linked to a polyamine moiety and at least one inhibitor of polyamine synthesis and/or polyamine metabolism like DFMO is performed.
  • Said polyamine depletion may advantageously comprise the (at least partial) depletion of endogenous polyamines, i.e., of intracellular polyamines, which are contained in cancer cells of said subject.
  • Depletion of endogenous polyamines may e.g., comprise administering at least one inhibitor of polyamine synthesis and/or at least one inhibitor of polyamine metabolism to said subject.
  • Depletion of endogenous polyamines is intended to increase the activity of the Polyamine Transport System (PTS) of the cancer cells of said subject.
  • PTS Polyamine Transport System
  • Said polyamine depletion may alternatively or complementarily comprise the (at least partial) depletion of exogenous polyamines, i.e., of polyamines, which are contained in the extracellular fluid (e.g. in the circulating blood) of the subject (and which may originate e.g., from food or medicine intake, or from the metabolism of the gut microorganisms).
  • Depleting exogenous polyamines may comprise submitting the subject to a polyamine deficient or reduced diet, and/or administering a gut decontaminant and/or administering at least one polyamine scavenging agent.
  • Depletion of the exogenous polyamines is intended to decrease the competition, which said cytotoxic agent linked to a polyamine moiety faces for entry into the cancer cells of said subject, and to increase the need for polyamines by the cancer cells of said subject, thus inducing a higher activity of the PTS of the cancer cells of said subject.
  • the polyamine depletion starts prior to the administration of a polyamine- vectored drug.
  • the application also relates to a kit (or kit-of-parts) for simultaneous, sequential or separate use of a first drug and of a second drug in cancer therapy, wherein said first drug is the drug, which comprises a cytotoxic agent linked to a polyamine moiety, and wherein said second drug is the at least one inhibitor of polyamine synthesis and/or at least one inhibitor of polyamine metabolism.
  • Figure 2 describes the effect of various concentrations of F14512 or etoposide on the survival of L1210 cells after 72h incubation with or without DFMO ⁇ following 72h pre-incubation with or without DFMO 200 ⁇ + Putrescine 20 ⁇ .
  • Figure 3 describes the effect of various concentrations of F14512, alone or in combination with DFMO 100 or 500 ⁇ , on the cell proliferation of 5 human AML cell lines, after 120h of incubation
  • Figure 4 describes the effect of various concentrations of F14512 or etoposide, alone or in combination with DFMO ⁇ , on the proliferation of HCT116 (top) and A2780 cells, after 96h of incubation following 24h pre-treatment with DFMO or control
  • Figure 5 describes the effect of various concentrations of F14512 or etoposide, alone or associated with a DFMO ⁇ alone or ⁇ 100 ⁇ +/-5 ⁇ 486 ⁇ ⁇ . ⁇ (figure 5A), DFMO ⁇ +/-Verapamil (VE ) 10 ⁇ (figure 5B), or DFMO ⁇ +/-Verapamil (VER) 30 ⁇ (figure 5C) on the proliferation of A2780 cells, after 5 days of incubation following 24h pre-treatment
  • Figure 6 describes the survival of mice implanted with L1210 cells according to the treatment received. DETAILED DESCRIPTION
  • the application relates to a polyamine-vectored cytotoxic agent, more particularly to a drug or kit comprising a polyamine-vectored cytotoxic agent, as well as to the medical applications thereof, notably in the field of cancer treatment.
  • the application relates to a combination therapy, which comprises combining or associating:
  • the polyamine-vectored cytotoxic agent is a cytotoxic agent, which is linked, more particularly covalently linked to a polyamine moiety.
  • the polyamine moiety is intended to act as a tail or tag, which targets the cytotoxic agent to the cells of a subject in need thereof, more particularly to the tumor or cancer cells of the subject.
  • Mammalian cells can import exogenous polyamines through the Polyamine Transport System (PTS).
  • PTS Polyamine Transport System
  • the PTS has been described e.g., in Poulin et al. 2012 (Amino Acids 42:711-723).
  • the activity of the PTS usually is superior in proliferating cells (e.g., in tumor or cancer cells) than in resting cells (e.g., in non-tumor resting cells) (cf. e.g. Palmer and Wallace 2010 (Amino Acids (2010) 38:415-422)).
  • the polyamine moiety which is linked, more particularly covalently linked to the cytotoxic agent, is intended to act as a tail or tag, which targets the cytotoxic agent through the PTS of the cells of a subject in need thereof, more particularly through the PTS of the tumor or cancer cells of the subject.
  • the polyamine-vectored cytotoxic agent is (preferentially or specifically) delivered into the tumor or cancer cells of the subject.
  • tumor is intended in accordance with its broadest meaning in the field, and includes the meaning of malign tumor or cancer.
  • tumor cells is intended in accordance with its broadest meaning in the field, and includes the meaning of malign tumor cells or of cancer cells.
  • the phrase "subject" is intended in accordance with its broadest meaning in the field, and includes the meaning of mammalian subject, non-human mammalian subject, or human subject, more particularly the meaning of (mammalian, non-human mammalian, or human) subject in need of tumor or cancer treatment.
  • the administration of the polyamine-vectored cytotoxic agent to a subject in need thereof is associated or combined with the submission of said subject to polyamine depletion.
  • the administration of the polyamine-vectored cytotoxic agent to a subject in need thereof is simultaneous, sequential or separate from the submission of said subject to polyamine depletion.
  • the application thus relates to a drug comprising a cytotoxic agent covalently linked to a polyamine moiety for use in (tumor or) cancer therapy, wherein said (tumor or) cancer therapy comprises administering said drug to a subject in need thereof and simultaneously, sequentially or separately submitting said subject to polyamine depletion.
  • the application relates to a drug comprising a cytotoxic agent linked to a polyamine moiety for use in treating tumor or cancer in a subject in need thereof, wherein said subject has been, is being or will be (more particularly has been or is being) simultaneously, sequentially or separately submitted to polyamine depletion.
  • said cytotoxic agent is a topoisomerase II inhibitor.
  • said cytotoxic agent is a topoisomerase II inhibitor, which is vectored to, or preferentially delivered to, or specifically delivered to, PTS expressing cells, more particularly to tumor or cancer cells.
  • said cytotoxic agent may comprise or be the epipodophyllotoxin moiety of etoposide.
  • Said epipodophyllotoxin moiety is of formula (I)
  • the number of amino groups of said polyamine moiety can e.g., be of 2, 3 or 4, more particularly of 4.
  • Said polyamine moiety may comprise or be at least one moiety chosen from among spermine, spermidine, homospermidine, putrescine and cadaverine, more particularly (at least one) spermine, which is of formula (II)
  • the cytotoxic agent and the polyamine moiety may be directly or indirectly linked together.
  • the linkage advantageously is a covalent linkage.
  • Said polyamine-vectored cytotoxic agent may be a non-naturally occurring product.
  • said polyamine-vectored cytotoxic agent comprises or is:
  • epipodophyllotoxin moiety of etoposide said epipodophyllotoxin moiety being of formula (I),
  • covalent linkage is a direct linkage or an indirect linkage, more particularly an
  • said polyamine-vectored cytotoxic agent comprises or is the F14512 drug, which is of formula (III):
  • HI i.e., 2 3-[4-(3-aminopropylamino)butylamino]propylamino ⁇ -N-[9-(4-hydroxy-3,5- dimethoxyphenyl)-8-oxo-5,5a,6,8,8a,9-hexahydrofuro[3';4';6,7]naphto[2,3-d][l,3]dioxol-5- yljacetamide.
  • polyamine-vectored cytotoxic agent administered to a subject in need thereof is associated or combined with the application of an (at least partial) depletion of the polyamines of the subject, more particularly an (at least partial) depletion of the free polyamines of the subject.
  • Said (free) polyamines notably comprise:
  • the (free) polyamines that are located intracellular ⁇ are located intracellular ⁇ (more particularly inside the tumor or cancer cells of the subject), and
  • the (free and) extracellular polyamines that are contained in the extracellular fluid circulating blood, interstitial fluid and transcellular fluid including cerebrospinal fluid or urine) of the subject.
  • Polyamine depletion may comprise:
  • endogenous polyamines is intended in accordance with its ordinary meaning in the field.
  • the expression “endogenous polyamines” is generally intended to designate polyamines, which are produced endogenously, i.e., polyamines, which are (endogenously) produced by cells of the subject, more particularly by tumor or cancer cells of the subject (and which are contained in or secreted from said (tumor or cancer) cells). More particularly, the expression “endogenous polyamines” encompasses polyamines, which are intracellular ⁇ produced by (tumor or cancer) cells of the subject, e.g., by intracellular enzymes of said (tumor or cancer) cells. More particularly, the expression “endogenous polyamines” encompasses intracellularly produced polyamines, which are produced by (and contained in or secreted from) tumor or cancer cells of the subject.
  • exogenous polyamines is intended in accordance with its ordinary meaning in the field.
  • the expression “exogenous polyamines” is generally intended to designate polyamines, which are not produced endogenously, i.e., which are exogenously produced and taken up by the subject.
  • the expression “exogenous polyamines” is notably intended to designate polyamines, which can be present in the body of said subject, but which are not produced by the (tumor or cancer) cells of the subject.
  • exogenous polyamines may e.g., encompass:
  • polyamine depletion comprises (at least partial) depletion of endogenous polyamines, and optionally further comprises (at least partial) depletion of exogenous polyamines.
  • Means for depleting endogenous polyamines may e.g., comprise administering to said subject at least one inhibitor of polyamine synthesis and/or of polyamine metabolism.
  • inhibitor of polyamine synthesis is intended in accordance with its ordinary meaning in the field, and generally refers to a compound inhibiting one or several of the enzymes involved in the intra-cellular synthesis of polyamines, e.g. ornithine decarboxylase (ODC), S-Adenosyl Methionine decarboxylase (SAMDC), spermidine synthase, and spermine synthase.
  • ODC ornithine decarboxylase
  • SAMDC S-Adenosyl Methionine decarboxylase
  • spermidine synthase spermine synthase
  • the phrase "inhibitor of polyamine metabolism” is intended in accordance with its ordinary meaning in the field, and generally refers to a compound modulating (decreasing) the metabolism of polyamines in the cell by modulating the expression of the enzymes involved in polyamine synthesis and catabolism, either by down-regulating the expression of genes or gene products involved in polyamine synthesis, or by up-regulating the expression of genes or genes products involved in polyamine catabolism, or by up-regulating the expression of genes or gene products that decrease polyamine synthesis or a combination of several of the above.
  • Means for depleting exogenous polyamines may e.g., comprise submitting said subject to a polyamine reduced (or deficient) diet and/or administering a gut (microbial) decontaminant to said subject and/or administering a polyamine scavenging agent to prevent absorption of exogenous polyamines from the gut into the circulating blood of the subject.
  • Submitting said subject to polyamine depletion may thus comprise one, or at least two (e.g. two or three or four) of the following four polyamine depleting treatments i.-iv.:
  • submitting said subject to polyamine depletion may comprise the polyamine depleting treatment of i. ((at least partial) depletion of endogenous polyamines) and, optionally, at least one (i.e., one or two or three) of the polyamine depleting treatments ii., iii., and iv. ((at least partial) depletion of exogenous polyamines).
  • polyamine depletion comprises the (at least partial) depletion of the endogenous polyamines.
  • At least partial depletion of endogenous polyamines advantageously induces or stimulates the activity of the Polyamine Transport System (PTS) of the (tumor or cancer) cells of said subject, and/or the intracellular uptake of circulating (extracellular) polyamines.
  • PTS Polyamine Transport System
  • the increase in PTS activity can be viewed as a mean, by which the (tumor or cancer) cells compensate for the deficit in endogenous polyamines by allowing for an increase of the intracellular importation or uptake of circulating (extracellular) polyamines (via increased PTS activity).
  • the polyamine moiety which is linked to the cytotoxic agent, acts as a tail or tag, which targets the cytotoxic agent through the PTS of the (tumor or cancer) cells. Therefore, the depletion of endogenous polyamines increases the capacity of the tumor or cancer cells of the subject to intracellular ⁇ import or uptake the polyamine-vectored cytotoxic agent (via their increased PTS activity).
  • the depletion of endogenous polyamines is intended to increase the PTS activity of the tumor or cancer cells of the subject, and to thereby increase the quantity or rate at which the tumor or cancer cells of the subject uptake the polyamine-vectored cytotoxic agent.
  • the depletion of endogenous polyamines is intended to improve the therapeutic effectiveness of the polyamine-vectored cytotoxic agent.
  • polyamine depletion advantageously comprises (the (at least partial) depletion of the endogenous polyamines, wherein said (at least partial) depletion of the endogenous polyamines comprises):
  • Said (at least partial) depletion and/or inhibition may be achieved e.g., by administration of at least one inhibitor of polyamine synthesis and/or of polyamine metabolism.
  • polyamine depletion advantageously comprises (the (at least partial) depletion of the endogenous polyamines, wherein said (at least partial) depletion of the endogenous polyamines comprises) administering at least one inhibitor of polyamine synthesis and/or of polyamine metabolism to said subject.
  • Said at least one inhibitor of polyamine synthesis and/or of polyamine metabolism may comprise or be chosen from among:
  • ODC Ornithine Decarboxylase
  • DFMO eflornithine
  • SAMDC S-Adenosyl-DeCarboxylase
  • SSAT spermidine/spermine-N-acetyltransferase
  • NSAIDs non-steroidal anti- inflammatory drugs
  • ornithine decarboxylase such as curcumin, MYC inhibitors, BET inhibitors, and Retinoic acid and its derivatives, including all-trans retinoic acid (ATRA),
  • Estrogen Receptor (ER) antagonists such as tamoxifen and fulvestrant
  • aromatase inhibitors e.g., anastrazole, letrozole, exemestane
  • AR Androgen Receptor
  • LHRH agonists e.g., triptorelin, leuprolide
  • LHRH antagonists e.g., degarelix
  • cyp-17 antagonists e.g., abiraterone
  • glucocorticoids such as dexamethasone, betamethasone, prednisone,
  • anti-cancer agents which induce a down-regulation of polyamine synthesis (inhibitors of the PI3K / AKT / mTOR pathway, BET or c-myc inhibitors, MAP / ERK pathway inhibitors such as EGFR inhibitors, inhibitors RAF or MEK, MAPK inhibitors) and/or an up-regulation of polyamine catabolism, such as platinum agents (more particularly oxaliplatin and cisplatin); more preferably said anti-cancer agents do not comprise platinum agents;
  • polyamine metabolism pathway regulation such as Nl, Nll-diethylnorspermine (DENSPM).
  • said at least one inhibitor of polyamine synthesis and/or of polyamine metabolism may comprise or be chosen from among:
  • ODC Ornithine Decarboxylase
  • DFMO eflornithine
  • SAMDC S-Adenosyl-DeCarboxylase
  • MGBG mitoguazone
  • METHYL-GAG METHYL-GAG
  • GCP48664 or SAM486A sardomozide
  • NSAIDs non-steroidal anti-inflammatory drugs
  • sulindac such as sulindac, celecoxib, piroxicam, and aspirin
  • glucocorticoids such as dexamethasone, betamethasone, prednisone,
  • - calcium channel antagonist such as verapamil, amlodipine, dicardipine, diltiazem, and - polyamine analogues depleting the intracellular pool of polyamines through the modulation polyamine metabolism pathway regulation, such as Nl, Nll-diethylnorspermine (DENSPM).
  • said at least one inhibitor of polyamine synthesis and/or of polyamine metabolism may comprise or be at least one inhibitor chosen from among:
  • - calcium channel antagonist such as verapamil, amlodipine, dicardipine, diltiazem,
  • NSAIDs non-steroidal anti-inflammatory drugs
  • Sulindac such as Sulindac, celecoxib, piroxicam, and aspirin
  • glucocorticoids such as dexamethasone, betamethasone, prednisone.
  • said at least one inhibitor of polyamine synthesis and/or of polyamine metabolism may comprise or be at least one inhibitor chosen from among:
  • said at least one inhibitor of polyamine synthesis and/or of polyamine metabolism may comprise or be at least one inhibitor chosen from among:
  • Eflornithine (also known, or referred to, as DFMO) is 2,5-diamino-2-(difluoromethyl)pentanoic acid (C6H12F2N202; PUBCHEM ® ID 3009).
  • said at least one inhibitor of polyamine synthesis and/or of polyamine metabolism may comprise or be at least one ODC inhibitor, more particularly at least the ODC inhibitor DFMO.
  • said at least one inhibitor of polyamine synthesis and/or of polyamine metabolism may comprise or be at least one SAMDC inhibitor, more particularly at least the SAMDC inhibitor SAM486A, MGBG, berenil, pentamidine, more particularly SAM486A.
  • said at least one inhibitor of polyamine synthesis and/or of polyamine metabolism may comprise or be at least one calcium channel antagonist, more particularly verapamil.
  • the present invention concerns a drug for the use as indicated above which comprises the following combinations:
  • the (at least partial) depletion of endogenous polyamines may e.g., be to an extent sufficient for significantly increasing the activity of the Polyamine Transport System (PTS) of the (tumor or) cancer cells of said subject.
  • PTS Polyamine Transport System
  • said at least one inhibitor of polyamine synthesis and/or polyamine metabolism may e.g., be administered (to a subject in need thereof) at a dose or at a dosage regimen, which increases the activity of the Polyamine Transport System (PTS) of the (tumor or) cancer cells of said subject, and/or the intracellular uptake of circulating (extracellular) polyamines.
  • PTS Polyamine Transport System
  • said at least one inhibitor of polyamine synthesis and/or of polyamine metabolism is administered to said subject at a dose lower than the dose at which said inhibitor of polyamine synthesis and/or of polyamine metabolism (is or) would be administered (to said subject) as sole active principle for (tumor or) cancer therapy, more particularly for (tumor or) cancer monotherapy and/or at doses intended to stop (tumor or) cancer cell proliferation.
  • administering the said inhibitor of polyamine synthesis and/or of polyamine metabolisms at a dose intended to have a cytotoxic or cytostatic effect and/or to stop or prevent the growth of (tumor) cells would result in a decrease in (tumor or) cancer cell proliferation and a down- regulation of topoisomerase II in these cells, whereas said drug anti-tumor efficacy requires a proliferation of (tumor or) cancer cells, and a significant activity of topoisomerase II, which is the intracellular target of said cytotoxic agent, and would prevent said drug to reach its target and induce (tumor) cell death and exert its cytotoxic effect
  • administering the said inhibitor of polyamine synthesis and/or of polyamine metabolism is intended as a means to increase said drug anti-tumor efficacy, while having no, or very limited impact on patient safety. More particularly, at the dose administered, the toxicity of said inhibitor of polyamine synthesis and/or of polyamine metabolism is expected to be minimal compared to the toxicity of usual chemotherapy agents.
  • DFMO is administered per os chronically for the prevention of colon cancer in high risk patients with very good patient safety.
  • verapamil is administered chronically for the prevention and treatment of cardiovascular disease.
  • said at least one inhibitor of polyamine synthesis and/or of polyamine metabolism is DFMO administered per os, it may be administered at a dose lower than 5 g/m2/day, - more particularly at a dose of 1-4 g/m2/day,
  • said at least one inhibitor of polyamine synthesis and/or of polyamine metabolism is DFMO administered per os, it may be administered at a dose lower than 15 g/day,
  • said at least one inhibitor of polyamine synthesis and/or of polyamine metabolism when administered intravenously, it may be administered at a dose lower than 400 mg/kg/day, more particularly at a dose of 30-300 mg/kg/day, more particularly at a dose of 50-200 mg/kg/day, more particularly at a dose of about 100 mg/kg/day.
  • said at least one inhibitor of polyamine synthesis and/or of polyamine metabolism is SAM486A administered intravenously, it may be administered at a dose lower than 100 mg/m 2 /day, more particularly lower than 50 mg/m 2 /day, more particularly at a dose lower than 30 mg/m 2 /day, more particularly at a dose around 20 mg/m 2 /day.
  • said at least one inhibitor of polyamine synthesis and/or of polyamine metabolism is verapamil administered per os, it may be administered at a dose of 300 mg/day to 1000 mg/day, more particularly at a dose around 500 mg/day.
  • said drug comprising a cytotoxic agent linked to a polyamine moiety
  • said at least one inhibitor of polyamine synthesis and/or of polyamine metabolism are administered to a subject in need thereof at a dosage regimen which is synergistically effective, more particularly at a dosage regimen which is synergistically effective for improved cytotoxicity against the tumor or cancer cells of the subject.
  • dose regimen is understood in accordance with its ordinary meaning in the field. It generally encompasses the schedule of doses of the therapeutic agents per unit of time, which may include one or more of the following features:
  • the time when the dose(s) are to be given e.g., at 8 a.m. and 4 p.m. daily, or after or prior to administration of a certain other product or treatment
  • the amount of a medicine or of active principle(s) e.g., number of capsules or the quantity of active principle(s) to be given at each specific time.
  • drug regimen encompasses the quantity of different active principles to be administered for treating the subject and the relative timing of their respective administrations.
  • dose is intended in accordance with its general meaning in the field. Its meaning notably encompasses the quantity of active principle to be given at a specific time.
  • depletion of exogenous polyamines may be alternative to, or complementary to the depletion of endogenous polyamines.
  • Depletion of exogenous polyamines may notably be intended to decrease the concentration of circulating (extracellular) polyamines in the extracellular fluid (ECF)of said subject.
  • the result may advantageously be that there are less circulating polyamines to compete with the polyamine- vectored cytotoxic agent for intracellular importation (through the PTS) and that the decrease in circulating polyamine will lead to a higher activity of the PTS as a compensatory mechanism.
  • the decrease in circulating polyamines may thereby favor the entry of the polyamine-vectored cytotoxic agent into the tumor or cancer cells (via the PTS).
  • depletion of exogenous polyamines advantageously decreases the competition, which said cytotoxic agent linked to a polyamine moiety faces for entry into the cancer cells of said subject. More particularly, depletion of exogenous polyamines advantageously is conducted or applied at a dose or dose regimen, which decreases the competition, which said cytotoxic agent linked to a polyamine moiety faces for entry into the cancer cells of said subject.
  • the depletion of exogenous polyamines is intended to improve the therapeutic effectiveness of the polyamine-vectored cytotoxic agent (by decreasing the competition for its entry into the tumor or cancer cells of the subject).
  • Submitting said subject to exogenous polyamine depletion may comprise one or several of the following three polyamine depleting treatments a.-c:
  • said diet may e.g., be a diet, which lowers the quantity or concentration of (extracellular) polyamines in extracellular fluids (e.g. the circulating blood or urine) of said subject.
  • said gut decontaminant is administered to said subject at a dose or in accordance with a dose regimen, which lowers the quantity or concentration of (extracellular) polyamines in extracellular fluids (e.g. the circulating blood or urine) of said subject.
  • the polyamine scavenging agent comprises at least one agent that is capable of binding or adsorbing polyamines without being absorbed by the intestine is preferably selected from clayey minerals, or active or activated charcoal.
  • said polyamine scavenging agent comprises at least one agent that is capable of binding or adsorbing polyamines in the intestine without being absorbed by the intestine; said agent is a clayey minerals which is preferably selected from:
  • the substance that is capable of binding or adsorbing polyamines without being absorbed by the intestine is a clayey minerals which is selected from diosmectite, beidellitic montmorillonite, (Activated) Mormoiron Attapulgite, Monmectite and Kaolinite or their mixture thereof, and more preferably diosmectite.
  • said polyamine reduced or deficient diet is a diet, which lowers the quantity or concentration of extracellular polyamines in extracellular fluids (e.g, the circulating blood or urine) of said subject down to an extent sufficient for significantly decreasing the competition for entry into the tumor or cancer cells of the subject, which the polyamine-vectored cytotoxic agent faces with respect to extracellular polyamines.
  • extracellular fluids e.g, the circulating blood or urine
  • the degree and duration of polyamine reduction or deficiency, which is applied by the diet are sufficient for significantly decreasing said competition (to thereby favor the entry of the polyamine-vectored cytotoxic agent into the tumor or cancer cells).
  • polyamine reduced or deficient diet examples include any polyamine reduced or deficient diet, which the person of average skill in the art may find appropriate.
  • polyamine reduced or deficient diet may e.g., comprise a diet, wherein high polyamine-containing foodstuff (e.g., > 201 nmol/g/mL of polyamine) are prohibited.
  • high polyamine-containing foodstuff e.g., > 201 nmol/g/mL of polyamine
  • polyamine reduced or deficient diet may e.g., comprise a diet, wherein median polyamine-containing foodstuff (e.g., 101-200 nmol/g/mL of polyamine) are limited to 3 or 4 times a week.
  • median polyamine-containing foodstuff e.g., 101-200 nmol/g/mL of polyamine
  • polyamine reduced or deficient diet may e.g., comprise a diet, wherein low polyamine-containing foodstuff (e.g., ⁇ 100 nmol/g/mL of polyamine) can be eaten at will.
  • low polyamine-containing foodstuff e.g., ⁇ 100 nmol/g/mL of polyamine
  • polyamine reduced or deficient diet may e.g., comprise a diet, wherein:
  • - high polyamine-containing foodstuff e.g., > 201 nmol/g/mL of polyamine
  • - median polyamine-containing foodstuff e.g., 101-200 nmol/g/mL of polyamine
  • - low polyamine-containing foodstuff e.g., ⁇ 100 nmol/g/mL of polyamine
  • diet duration may e.g., comprise a period of at least 5 days, or of at least one week, or of at least two weeks, or of at least two months or of at least 3 months.
  • depletion of exogenous polyamines may comprise the administration of at least one gut decontaminant.
  • the gut microorganisms more particularly the gut bacteria and/or the gut fungi, produce polyamines, which are released in the circulating blood of the subject.
  • the at least one gut decontaminant is intended to decrease or suppress these microorganisms, more particularly these bacteria or fungi, to thereby decrease or suppress the quantity or concentration of polyamines, which is produced by these microorganisms (and released in the circulating blood of the subject).
  • the at least one gut decontaminant may e.g., be chosen from among the antibiotics, more particularly broad spectrum antibiotics, more particularly at least one antibiotic comprising at least one active principle chosen from among:
  • neomycin e.g., neomycin sulfate
  • colistine e.g., colistine sulfate
  • Neomycin is (2 S,3S,4S,5 )-5-amino-2-(aminomethyl)-6-((2 ,3S,4 ,5S)-5-((l ,2 ,5 ,6 )-3,5- diamino-2-((2R,3S,4R,5S)-3-amino-6-(aminomethyl)-4,5-dihydroxytetrahydro-2H-pyran-2-yloxy)-
  • Nifuroxazide is 4-hydroxy-N-[(5-nitrofuran-2-yl)methylene]benzohydrazide.
  • Colistine is N-(4-amino-l-(l-(4-amino-l-oxo-l-(3,12,23-tris(2-aminoethyl)-20-(l-hydroxyethyl)-
  • Fidaxomicine is 3-(((6-Deoxy-4-0-(3,5-dichloro-2-ethyl-4,6-dihydroxybenzoyl)-2-0-methyl- -D- mannopyranosyl)oxy)-methyl)-12(R)-[(6-deoxy-5-C-methyl-4-0-(2-methyl-l-oxopropyl)- -D-lyxo- hexopyranosyl)oxy]-ll(S)-ethyl-8(S)-hydroxy-18(S)-(l(R)-hydroxyethyl)-9,13,15- trimethyloxacyclooctadeca-3,5,9,13,15-pentaene-2-one.
  • Sulfaguanidine is 4-Amino-N-[amino(imino)methyl]benzenesulfonamide.
  • Tiliquinol is 5-methylquinolin-8-ol.
  • Nifurzide is 5-Nitro-N ' -[(lE,2E)-3-(5-nitro-2-furyl)-2-propen-l-ylidene]-2- thiophenecarbohydrazide.
  • ifaximine is (2S,16Z,18E,20S,21S,22R,23R,24R,25S,26S,27S,28E)-5,6,21,23,25-pentahydroxy-27- methoxy-2,4,ll,16,20,22,24,26-octamethyl-2,7-(epoxypentadeca-[l,ll,13]trienimino)benzofuro [4,5-e]pyrido[l,2-a]-benzimida-zole-l,15(2H)-dione,25-acetate.
  • Tilbroquinol is 7-bromo-5-methylquinolin-8-ol.
  • the at least one gut decontaminant is administered to said subject at a dose or dose regimen, which participates in lowering the quantity or concentration of extracellular polyamines, which are circulating in the blood of the subject, more particularly at a dose or dose regimen, which lowers the quantity or concentration of polyamines produced by the gut microorganisms, down to an extent sufficient for significantly decreasing the competition for entry into the tumor or cancer cells of the subject, which the polyamine-vectored cytotoxic agent faces with respect to extracellular polyamines, which are circulating in the blood of the subject.
  • the dose or dosage regimen of the at least one gut decontaminant is sufficient for significantly contributing to said competition decrease (to thereby favor the entry of the polyamine-vectored cytotoxic agent into the tumor or cancer cells).
  • depletion of exogenous polyamines may comprise the administration of at least one polyamine scavenging agent, more particularly at a dose or in accordance with a dose regimen, which lowers the quantity or concentration of (extracellular) polyamines in the extracellular fluids (e.g. the circulating blood or urine) of said subject.
  • the polyamine depletion comprises the depletion of the endogenous polyamines and further comprises the depletion of exogenous polyamines.
  • the increase in the level of PTS activity by tumor or cancer cells i.e., the increase in polyamine uptake by the tumor or cancer cells
  • the decrease in the level of circulating polyamines i.e., the decrease in the competition for entry of the polyamine-vectored drug into the tumor or cancer cells (lower competition between the polyamine vectored drug and the circulating polyamines).
  • submitting said subject to polyamine depletion advantageously comprises administering at least one inhibitor of polyamine synthesis and/or of polyamine metabolism to said subject, and further comprises one or several of the following three polyamine depleting treatments a.-c:
  • the application of polyamine depletion starts prior to the administration of the polyamine-vectored cytotoxic agent.
  • sequentially or separately submitting said subject to polyamine depletion advantageously comprises submitting said subject to polyamine depletion prior to administering said drug to said subject.
  • sequentially or separately submitting said subject to polyamine depletion may advantageously comprise submitting said subject to polyamine depletion (e.g., to endogenous and optionally exogenous polyamine depletion) prior to administering the drug (i.e., the drug, which comprises the polyamine-vectored cytotoxic agent) to said subject, more particularly prior to any administration of said drug.
  • the drug i.e., the drug, which comprises the polyamine-vectored cytotoxic agent
  • sequentially or separately submitting said subject to polyamine depletion may for example comprise submitting said subject to (e.g., endogenous and optionally exogenous) polyamine depletion:
  • sequentially or separately submitting said subject to polyamine depletion may for example comprise:
  • sequentially or separately submitting said subject to polyamine depletion may for example comprise:
  • DFMO e.g., per os at 7.5 g/day, 3 times a day
  • verapamil e.g., per os, 500 mg/day
  • inhibitor of polyamine synthesis or inhibitor of polyamine metabolism is DFMO administered intravenously, simultaneously, sequentially or separately submitting said subject to polyamine depletion may for example comprise:
  • simultaneously, sequentially or separately submitting said subject to polyamine depletion may then comprise, for each cycle of said drug administration: at day 1: starting the exogenous polyamine depletion (and continuing until the end of the treatment);
  • DFMO e.g., iv at 50-200 mg/kg/day, more particularly at 100 mg/kg/day, by continuous perfusion of 1 to 6 hours, preferably 3 hours
  • 1 to 6 hours, more particularly, 2 to 4 hours, more particularly 3 hours before the administration of said drug
  • o starting the administration of said drug 1 to 6 hours, more particularly 2 to 4 hours, more particularly 3 hours after the start of the administration of DFMO, and administering the drug by continuous perfusion of 1 to 24 hours, more particularly 3 hours, or alternatively, more particularly 24 hours; and o continuing the administration of DFMO simultaneously to the administration of said drug and until the end of the administration of said drug.
  • simultaneously, sequentially or separately submitting said subject to polyamine depletion may then comprise, for each cycle of said drug administration: at day 1: starting the exogenous polyamine depletion (and continuing until the end of the treatment);
  • DFMO e.g., iv at 50-200 mg/kg/day, more particularly at 100 mg/kg/day, by continuous perfusion of 1 to 6 hours, preferably 3 hours
  • SAM486A e.g., iv at 10-50 mg/m 2 /day, more particularly at 20 mg/m 2 /day
  • l to 6 hours more particularly, 2 to 4 hours, more particularly 3 hours, before the administration of said drug
  • DFMO+SAM486A and administering the drug by continuous perfusion of 1 to 24 hours, more particularly 3 hours, or alternatively, more particularly 24 hours; and o continuing the administration of DFMO and SAM486A simultaneously to the administration of said drug and until the end of the administration of said drug.
  • said subject prior to implementation of said polyamine depletion, said subject has:
  • circulating polyamines more particularly of circulating extracellular polyamines more particularly in blood and/or in urine and/or of polyamines that are contained in circulating blood cells, more particularly in circulating erythrocytes, and/or
  • said subject prior to implementation of said polyamine depletion, has a high concentration of circulating extracellular polyamines and/or of polyamines that are contained in circulating cells, more particularly in circulating erythrocytes.
  • the combination therapy of the application is intended for treating a (malignant) tumor or cancer, more particularly:
  • hematological malignancies such as
  • solid tumors such as
  • NSCLC non-small cell lung cancer
  • liver cancer more particularly hepatocarcinoma
  • prostate cancer more particularly a hormone-resistant prostate cancer
  • brain cancer more particularly glioma, more particularly glioblastoma;
  • stomach cancer a colorectal cancer, a pancreas cancer, a biliary tract cancer, or a Gastro-lntestinal Stromal Tumor (GIST).
  • GIST Gastro-lntestinal Stromal Tumor
  • Said (malignant) tumor or cancer may more particularly be:
  • lymphoma more particularly Burkitt's lymphoma
  • NSCLC non-small cell lung cancer
  • hepatocarcinoma head and neck cancer
  • glioma more particularly glioblastoma
  • Said (malignant) tumor or cancer may more particularly be:
  • leukemia more particularly acute myeloid leukemia
  • lymphoma more particularly Burkitt's lymhoma
  • glioma more particularly glioblastoma
  • Said (malignant) tumor or cancer may more particularly be:
  • leukemia more particularly acute myeloid leukemia
  • lymphoma more particularly Burkitt's lymhoma
  • glioma more particularly glioblastoma
  • Said (malignant) tumor or cancer may more particularly be:
  • leukemia more particularly acute myeloid leukemia
  • glioma more particularly glioblastoma
  • the cancer therapy may further comprise the administration of further anti-cancer drugs and/or the application of radiotherapy.
  • said cancer therapy may further comprise submitting said subject to radiotherapy and/or administering to said subject at least one further anti-cancer drug, more particularly at least one further anti-cancer drug, which is chosen from among the following:
  • platinum agents more particularly cisplatin, carboplatin and oxaliplatin
  • - intercalating agents including anthracyclins (more particularly doxorubicin, daunorubicin and mitoxantrone);
  • alkylating agents more particularly nitrosoureas, melphalan, temozolomide and cyclophosphamide
  • nucleotide antimetabolites and nucleotide analogues (more particularly cytarabine and gemcitabine);
  • topoisomerase I inhibitors (more particularly irinotecan);
  • microtubule inhibitors including taxanes (more particularly paclitaxel and docetaxel) and vinca- alkaloids (more particularly vincristine);
  • - demethylating agents more particularly azacytidine and decitabine
  • -EGF inhibitors both antibodies (more particularly cetuximab and panitumumab), and small molecules (more particularly erlotinib, afatinib and gefitinib);
  • PI3K/AKT/mTORCl pathway inhibitors such as PI3K inhibitors (more particularly idelalisib), AKT inhibitors and mTOR inhibitors (more particularly sirolimus, everolimus, temsirolimus);
  • MAPK/ERK pathway inhibitors such as RAF inhibitors (more particularly sorafenib, encorafenib and vemurafenib), MEK inhibitors (more particularly cobimetinib, trametinib), and MAPK inhibitors.
  • said cancer therapy may further comprise administering to said subject at least one further anti-cancer drug which down-regulates or inhibits polyamine metabolism, more particularly at least one further anti-cancer drug, which is chosen from among the following:
  • - EGFR inhibitors both antibodies (more particularly cetuximab and panitumumab), and small molecules (more particularly erlotinib, afatinib, lapatinib and gefitinib);
  • PI3K/AKT/mTORCl pathway inhibitors such as PI3K inhibitors (more particularly idelalisib), AKT inhibitors and mTOR inhibitors (more particularly sirolimus, everolimus, temsirolimus); and
  • said cancer therapy may also further comprise administering to said subject at least one further anti-cancer drug which anti-tumor activity is enhanced by polyamine depletion, more particularly at least one further anti-cancer drug, which is chosen from among the following:
  • intercalating agents including anthracyclins (more particularly doxorubicin, daunorubicin and mitoxantrone),
  • alkylating agents more particularly nitrosoureas, melphalan, temozolomide and cyclophosphamide
  • the application also relates to a pharmaceutical composition
  • a pharmaceutical composition comprising a fixed dose combination of a first active ingredient and a second active ingredient for use in cancer therapy, wherein:
  • said first active ingredient is the drug of the application, i.e., a drug, which comprises a cytotoxic agent linked to a polyamine moiety as herein defined, and wherein
  • said second active ingredient is the at least one inhibitor of polyamine synthesis and/or of polyamine metabolism as herein defined, preferably DFMO, preferably for intravenous administration.
  • the application relates to a drug comprising a cytotoxic agent of formula (I) linked to a polyamine moiety of formula (II), and at least one inhibitor of polyamine synthesis and/or of polyamine metabolism for use in (tumor or) cancer therapy, wherein said (tumor or) cancer therapy comprises administering said drug to a subject in need thereof and simultaneously, submitting said subject to polyamine depletion.
  • a particular embodiment of the present application concerns a drug for said use, which comprises or consists in the F14512 drug, and the at least one inhibitor of polyamine synthesis and/or of polyamine metabolism is DFMO.
  • Said particular combination may be administered by the intravenous or per os route.
  • the application also relates to a kit (or kit-of-parts) for simultaneous, sequential or separate use (or administration) of a first drug and of a second drug in cancer therapy, wherein:
  • said first drug is the drug of the application, i.e., a drug, which comprises a cytotoxic agent linked to a polyamine moiety as herein defined, and wherein
  • the application also relates to a kit (or kit-of-parts) for simultaneous, (or administration) of a first drug and of a second drug in cancer therapy, wherein:
  • said first drug is the pharmaceutical composition as herein defined comprising the drug of the application, i.e., a drug, which comprises a cytotoxic agent linked to a polyamine moiety as herein defined, and the at least one inhibitor of polyamine synthesis and/or of polyamine metabolism as herein defined, preferably DFMO, for intravenous administration, and wherein said second drug is the at least one inhibitor of polyamine synthesis and/or of polyamine metabolism as herein defined.
  • the kit of the application may further comprise:
  • said third drug is or comprises at least one (human) gut decontaminant (cf. above for further features that may define such a decontaminant) or a polyamine scavenging agent.
  • Example 1 Cell viability assays on the L1210 cell lines incubated with F 14512 or etoposide combined with DFMO in various experimental conditions
  • the murine lymphocytic leukemia cell line L1210 was provided by Dr. Claude Boucheix (UM S935, INSERM, Paul Brousse).
  • DFMO was solubilized in water at 0.1M, F14512 and etoposide in DMSO at 10- 2 M then diluted in the complete cell culture medium accordingly. Putrescine was solubilized at 10-2M in the complete medium and diluted accordingly.
  • L1210 cells were cultivated in RPMI-1640 supplemented with 10% foetal calf serum, L-glutamine and Penicillin/Streptomycin. Cells were maintained and propagated at 37°C, 5% C0 2 and saturating humidity.
  • Pre-treatments L1210 cells were seeded in 25 cm 2 cell culture T- flasks at a density of 10 4 cell/ml in complete culture medium, kept one hour in the cell incubator before DFMO and/or Putrescine being added. After 72h incubation cells were counted to assess the potential toxicity of pre- treatment conditions. Each cell suspension was then adjusted to 11 000 cell/ml.
  • Cells were seeded in 96 flat-bottomed cell culture plates at a density of 1000 cells per well in complete culture medium +/- DFMO and +/- putrescine in a volume of 90 ⁇ . Plates were kept at 37°C in the cell culture incubator before 10 ⁇ /well of lOx F14512 or etoposide solution was added. Each concentration was tested in triplicate. After 72h incubation, cell survival was measured using the Premix WST-1 cell proliferation system (Takara) by reading 450 nm absorbances, (Pherastar multimode reader, BMG Labtech).
  • L1210 cells were pre-treated in T-flasks with or without DFMO at 50 ⁇ , ⁇ , 200 ⁇ , 300 ⁇ , with or without addition of putrescine (Put) ⁇ , then plated and treated with various concentrations of F14512 associated or not with DFMO as per below:
  • cells were pre-treated in T-flasks for 72h without DFMO or with DFMO with or without putrescine ⁇ , then plated with F14512 with or without DFMO
  • DFMO at low, non-cytotoxic concentrations (100 ⁇ ), and following 72h pre-incubation in the same conditions or at 200 ⁇ , potentiated the activity of F14512 on L1210 cell lines at 72h. Higher concentrations of DFMO had significant impact on control cell survival, thus preventing interpretation of results.
  • Pre-treatment with DFMO 200 ⁇ also induced an increased sensitivity to F14512 without having any impact on control cell growth, supporting the hypothesis that pre- treatment with DFMO increased the uptake of F14512 in tumor cells.
  • DFMO also increased sensitivity of L1210 cells to etoposide, although at a much lower extent.
  • Table 3 F14512 and Etoposide IC50 for L1210 cell line after incubation alone or in combination with DFMO in various conditions of pre-treatment and treatment 1
  • Example 2 Cell viability assays on human AML cell lines incubated with F14512 in presence or absence of DFMO
  • AML cell lines (HL-60, HEL, IMS-M2, KGla, MOLM-13, MOLM-14, MV4-11, NB4, NOMO-1, OCI-AML3, THP1, U937, Kasumil, ME-1) were obtained from the Stegmaier Lab (Dana Farber Cancer Institute, Boston). Cells were seeded on 384-well plates at 0.015 to 0.075.10 s cells/mL (depending on the growth rate of each cell line) in PMI 1640 medium (ThermoFisher) supplemented with 10% or 20% Fetal Bovine Serum (FBS) and 1% Penicillin/streptomycin.
  • PMI 1640 medium ThermoFisher
  • FBS Fetal Bovine Serum
  • F14512 (Villapharma) was added (concentration range: 10 to 0.001 ⁇ . Plates were incubated at 37°C, 20% 0 2 , 5% C0 2 . After 120h, 15 ⁇ of CellTiter-Glo ® Luminescent Cell Viability Assay (Promega) was added in each well, plates were agitated for 25 minutes before analysis of luminescence by a SpectraMax ® i3x plate reader (Molecular Devices). Each experimental condition was measured in 6-plicate. IC50s were determined using GraphpadPrism 5.0.
  • F14512 was found to be active on all cell lines tested at concentrations below ⁇ . F14512 IC50 concentrations at 5 days ranged between 0.6nM (MOLM-14 cell lines) and 680nM (ME-1) (Table 4).
  • DFMO at ⁇ and 500 ⁇ potentiated the cytotoxicity of F14512 for 5/5 human AML cell lines by a factor ranging from 1.3 to >100. In most cell lines, potentiation was higher with DFMO 500 ⁇ .
  • Example 3 Cell viability assays on solid tumor cell lines HCT116 and A2780 incubated with F14512 in presence or absence of DFMO
  • DMSO molecular biology reagent quality Sterile injectable water (Lonza) Putrescine (Sigma), F14512 (Villapharma) DFMO (Rusan), etoposide (Sigma), verapamil (Sigma), SAM486A (Medkoo Sciences).
  • A2780 cells were cultivated in RPMI 1640 supplemented with 10% fcetal calf serum, L-gutamine and Penicillin/Streptomycin. HCT116 cells in DMEM supplemented with 10% fcetal calf serum, L-gutamine and Penicillin/Streptomycin. Cells were maintained and propagated at 37°C, 5% C0 2 and saturating humidity
  • Pretreatments A2780 and HCT116 cells were seeded in 96 well flat-bottomed 96 well plates at 2000 cells/well and 1000 cells/well respectively under a 90 ⁇ /well volume. Cells were kept overnight in the cell culture incubator to allow cell adherence. Pretreatment agents were added (10 ⁇ /well of lOx solutions). Plates were kept for 24h in the incubator
  • F14512 alone IC50 for HCT116 and A2780 cells was respectively 196nM and 52nM.
  • Addition of DFMO ⁇ to F14512 resulted into an increase of F14512 cytotoxicity as measured by F14512 IC50 in the presence of DFMO ⁇ : 51nM for HCT116 (3.8 fold increase in cytotoxicity vs F14512 alone) and llnM in A2780 (4.7 fold increase in cytotoxicity).
  • the efficacy of etoposide was not increased by the addition of DFMO ⁇ (table 6, figure 4).
  • Table 6 F14512 IC50 for HCT116 and A2780 cell lines after incubation alone or in combination with DFMO ⁇ 1
  • F14512 was supplied from Villapharma, DFMO from Rusan, etoposide from Hopital Louis Herriot.
  • mice were weighted twice per week for 4 weeks and monitored daily for behavior and development of the ascites. They were euthanatized when their health visibly deteriorated. Survival data at day 28 were analyzed using SAS JMP vl4.0 software. Survival median and 95% confidence intervals, as well as statistical differences between groups were determined using a Weibull model.
  • mice that received Special Diet comprising diosmectite in addition to DFMO had a longer survival compared to the group receiving only F14512 + DFMO.
  • mice treated with F14512 In contrast to the benefit provided by DFMO +/- diosmectite observed with in mice treated with F14512, no benefit of polyamine depletion was observed in mice treated with etoposide, as mice receiving etoposide + DFMO + SD had a lower 28-day survival rate than the mice treated with etoposide alone.
  • Cipolla et al. 2010 (Biomedicine & Pharmacotherapy 64: 363-368) Evageliou and Hogarty 2009 (Clin. Cancer Res. 15(19): 5956-5961) Levin et al. 2007. (International Journal of Cancer 121(10): 2279-2283)

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Abstract

The application relates to a combination therapy for the treatment of cancer, more particularly to the combination of a polyamine-vectored cytotoxic drug with polyamine depletion for improved anticancer therapy.

Description

TITLE
COMBINATION THERAPY FOR THE TREATMENT OF CANCER FIELD
The application relates to a combination therapy for the treatment of cancer, more particularly to the combination of a polyamine-vectored cytotoxic agent with polyamine depletion for improved cancer therapy.
BACKGROUND
Polyamines are present in mammalians as a result of endogenous synthesis (e.g., intracellular synthesis) as well as of exogenous import (e.g., via food intake). Tumor growth is generally associated with upregulation of the polyamine level.
A strategy in drug design based on polyamine therefore uses polyamine as a means to preferentially target tumor cell and to improve drug delivery into these cells. This strategy has led to the development of various drug-polyamine conjugates as potential anti-cancer targeted drugs. For example, WO 2005/100363 in the name of PIERRE FABRE MEDICAMENT describes podophyllotoxin derivatives, such as the F14512 drug (2-{3-[4-(3- aminopropylamino)butylamino]propylamino}-N-[9-(4-hydroxy-3,5-dimethoxyphenyl)-8-oxo- 5,5a,6,8,8a,9-hexahydrofuro[3';4';6,7]naphto[2,3-d][l,3]dioxol-5-yl]acetamide). A review of the molecular mechanisms of drug-polyamine conjugates can be found e.g., in Xie et al. 2010 (Expert Opinion on Drug Delivery 7(9): 1049-1061) or Evageliou and Hogarty 2009 (Clin. Cancer Res. 15(19): 5956-5961).
However, the use of a polyamine tail to target the drug to cancer cells has not always provided improved anti-cancer efficacy. The discrepancy in the anti-cancer efficacies is not clearly elucidated.
An alternative strategy involves the use of enzyme inhibitors to inhibit the polyamine cycle. Such enzyme inhibitors notably comprise the Ornithine Decarboxylase (ODC) inhibitor eflornithine (DFMO) (cf. e.g., Cipolla et al. 2010, Biomedecine & Pharmacotherapy 64: 363-368) and the SAMDC inhibitor mitoguazone (MGBG or METHYL-GAG) (cf. e.g., Murray-Stewart et al. 2016, Biochemical Journal 473: 2937-2953). However, polyamine enzyme inhibitors have led to discrepant anti-tumor responses or to insufficient therapeutic efficacy, notably concerning relapsed or refractory cancers.
The application provides means for combination therapy in the treatment of cancer. The means of the application combine a polyamine-vectored cytotoxic agent with polyamine depletion. The means of the application notably provide improved therapeutic efficacy.
SUMMARY
The application generally relates to the combination of a polyamine-vectored cytotoxic agent with polyamine depletion for improved cancer therapy.
The application relates more particularly to a drug comprising a cytotoxic agent linked to a polyamine moiety for use in cancer therapy, wherein said cancer therapy comprises administering said drug to a subject in need thereof and simultaneously, sequentially or separately submitting said subject to polyamine depletion.
The application also relates more particularly to a drug comprising a cytotoxic agent linked to a polyamine moiety for use in cancer therapy, wherein said cancer therapy comprises administering said drug to a subject in need thereof and simultaneously and separately submitting said subject to polyamine depletion.
In certain cases it may be advantageous to perform a pre-treatment, for instance with at least one inhibitor of polyamine synthesis and/or polyamine metabolism like DFMO before a simultaneous treatment with a drug comprising a cytotoxic agent linked to a polyamine moiety and at least one inhibitor of polyamine synthesis and/or polyamine metabolism like DFMO is performed.
Said polyamine depletion may advantageously comprise the (at least partial) depletion of endogenous polyamines, i.e., of intracellular polyamines, which are contained in cancer cells of said subject. Depletion of endogenous polyamines may e.g., comprise administering at least one inhibitor of polyamine synthesis and/or at least one inhibitor of polyamine metabolism to said subject. Depletion of endogenous polyamines is intended to increase the activity of the Polyamine Transport System (PTS) of the cancer cells of said subject.
Said polyamine depletion may alternatively or complementarily comprise the (at least partial) depletion of exogenous polyamines, i.e., of polyamines, which are contained in the extracellular fluid (e.g. in the circulating blood) of the subject (and which may originate e.g., from food or medicine intake, or from the metabolism of the gut microorganisms). Depleting exogenous polyamines may comprise submitting the subject to a polyamine deficient or reduced diet, and/or administering a gut decontaminant and/or administering at least one polyamine scavenging agent. Depletion of the exogenous polyamines is intended to decrease the competition, which said cytotoxic agent linked to a polyamine moiety faces for entry into the cancer cells of said subject, and to increase the need for polyamines by the cancer cells of said subject, thus inducing a higher activity of the PTS of the cancer cells of said subject.
Advantageously, the polyamine depletion starts prior to the administration of a polyamine- vectored drug.
The application also relates to a kit (or kit-of-parts) for simultaneous, sequential or separate use of a first drug and of a second drug in cancer therapy, wherein said first drug is the drug, which comprises a cytotoxic agent linked to a polyamine moiety, and wherein said second drug is the at least one inhibitor of polyamine synthesis and/or at least one inhibitor of polyamine metabolism.
BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 describes the effect of various concentrations of F14512 on the survival of L1210 cells after 72h incubation with or without DFMO following 72h pre-incubation with or without DFMO +/- Putrescine ΙΟμΜ.
Figure 2 describes the effect of various concentrations of F14512 or etoposide on the survival of L1210 cells after 72h incubation with or without DFMO ΙΟΟμΜ following 72h pre-incubation with or without DFMO 200μΜ + Putrescine 20μΜ.
Figure 3 describes the effect of various concentrations of F14512, alone or in combination with DFMO 100 or 500μΜ, on the cell proliferation of 5 human AML cell lines, after 120h of incubation Figure 4 describes the effect of various concentrations of F14512 or etoposide, alone or in combination with DFMO ΙΟΟμΜ, on the proliferation of HCT116 (top) and A2780 cells, after 96h of incubation following 24h pre-treatment with DFMO or control
Figure 5 describes the effect of various concentrations of F14512 or etoposide, alone or associated with a DFMO ΙΟΟμΜ alone or ϋΡΜΟ100μΜ+/-5ΑΜ486Α Ο.ΙμΜ (figure 5A), DFMO ΙΟΟμΜ +/-Verapamil (VE ) 10μΜ (figure 5B), or DFMO ΙΟΟμΜ +/-Verapamil (VER) 30μΜ (figure 5C) on the proliferation of A2780 cells, after 5 days of incubation following 24h pre-treatment Figure 6 describes the survival of mice implanted with L1210 cells according to the treatment received. DETAILED DESCRIPTION
In the application, unless specified otherwise or unless a context dictates otherwise, all the terms have their ordinary meaning in the relevant field(s).
The application relates to a polyamine-vectored cytotoxic agent, more particularly to a drug or kit comprising a polyamine-vectored cytotoxic agent, as well as to the medical applications thereof, notably in the field of cancer treatment.
More particularly, the application relates to a combination therapy, which comprises combining or associating:
the administration of a polyamine-vectored cytotoxic agent, with
the application of a polyamine depleting treatment.
The polyamine-vectored cytotoxic agent is a cytotoxic agent, which is linked, more particularly covalently linked to a polyamine moiety. The polyamine moiety is intended to act as a tail or tag, which targets the cytotoxic agent to the cells of a subject in need thereof, more particularly to the tumor or cancer cells of the subject.
Mammalian cells, more particularly human cells, can import exogenous polyamines through the Polyamine Transport System (PTS). The PTS has been described e.g., in Poulin et al. 2012 (Amino Acids 42:711-723). The activity of the PTS usually is superior in proliferating cells (e.g., in tumor or cancer cells) than in resting cells (e.g., in non-tumor resting cells) (cf. e.g. Palmer and Wallace 2010 (Amino Acids (2010) 38:415-422)). The polyamine moiety, which is linked, more particularly covalently linked to the cytotoxic agent, is intended to act as a tail or tag, which targets the cytotoxic agent through the PTS of the cells of a subject in need thereof, more particularly through the PTS of the tumor or cancer cells of the subject.
Hence, the polyamine-vectored cytotoxic agent is (preferentially or specifically) delivered into the tumor or cancer cells of the subject.
Throughout the application, the phrase "tumor" is intended in accordance with its broadest meaning in the field, and includes the meaning of malign tumor or cancer. Similarly, the expression "tumor cells" is intended in accordance with its broadest meaning in the field, and includes the meaning of malign tumor cells or of cancer cells.
Throughout the application, the phrase "subject" is intended in accordance with its broadest meaning in the field, and includes the meaning of mammalian subject, non-human mammalian subject, or human subject, more particularly the meaning of (mammalian, non-human mammalian, or human) subject in need of tumor or cancer treatment.
In accordance with the application, the administration of the polyamine-vectored cytotoxic agent to a subject in need thereof is associated or combined with the submission of said subject to polyamine depletion. Hence, the administration of the polyamine-vectored cytotoxic agent to a subject in need thereof is simultaneous, sequential or separate from the submission of said subject to polyamine depletion.
The application thus relates to a drug comprising a cytotoxic agent covalently linked to a polyamine moiety for use in (tumor or) cancer therapy, wherein said (tumor or) cancer therapy comprises administering said drug to a subject in need thereof and simultaneously, sequentially or separately submitting said subject to polyamine depletion.
In other words, the application relates to a drug comprising a cytotoxic agent linked to a polyamine moiety for use in treating tumor or cancer in a subject in need thereof, wherein said subject has been, is being or will be (more particularly has been or is being) simultaneously, sequentially or separately submitted to polyamine depletion.
Advantageously, said cytotoxic agent is a topoisomerase II inhibitor. Advantageously, said cytotoxic agent is a topoisomerase II inhibitor, which is vectored to, or preferentially delivered to, or specifically delivered to, PTS expressing cells, more particularly to tumor or cancer cells.
More particularly, said cytotoxic agent may comprise or be the epipodophyllotoxin moiety of etoposide. Said epipodophyllotoxin moiety is of formula (I)
Figure imgf000006_0001
(I). The number of amino groups of said polyamine moiety can e.g., be of 2, 3 or 4, more particularly of 4. Said polyamine moiety may comprise or be at least one moiety chosen from among spermine, spermidine, homospermidine, putrescine and cadaverine, more particularly (at least one) spermine, which is of formula (II)
Figure imgf000007_0001
(II).
The cytotoxic agent and the polyamine moiety may be directly or indirectly linked together. The linkage advantageously is a covalent linkage.
More particularly, the cytotoxic agent and the polyamine moiety may be covalently linked together via a nitrogen-containing moiety, more particularly a moiety comprising at least one amide (functional group), e.g., a moiety comprising at least one (amide functional) group of formula HN-C=0. Said polyamine-vectored cytotoxic agent may be a non-naturally occurring product.
Advantageously, said polyamine-vectored cytotoxic agent comprises or is:
the epipodophyllotoxin moiety of etoposide, said epipodophyllotoxin moiety being of formula (I),
covalently linked to
- the polyamine moiety of formula (II),
wherein said covalent linkage is a direct linkage or an indirect linkage, more particularly an
(indirect) linkage via a nitrogen-containing moiety, more particularly a moiety comprising at least one amide (functional group), e.g., a moiety comprising at least one (amide functional) group of formula HN-C=0.
Advantageously, said polyamine-vectored cytotoxic agent comprises or is the F14512 drug, which is of formula (III):
Figure imgf000008_0001
(HI), i.e., 2 3-[4-(3-aminopropylamino)butylamino]propylamino}-N-[9-(4-hydroxy-3,5- dimethoxyphenyl)-8-oxo-5,5a,6,8,8a,9-hexahydrofuro[3';4';6,7]naphto[2,3-d][l,3]dioxol-5- yljacetamide.
The administration of said polyamine-vectored cytotoxic agent to a subject in need thereof is associated or combined with the application of an (at least partial) depletion of the polyamines of the subject, more particularly an (at least partial) depletion of the free polyamines of the subject. Said (free) polyamines notably comprise:
the (free) polyamines that are located intracellular^ (more particularly inside the tumor or cancer cells of the subject), and
the (free and) extracellular polyamines that are contained in the extracellular fluid (circulating blood, interstitial fluid and transcellular fluid including cerebrospinal fluid or urine) of the subject.
Polyamine depletion may comprise:
- the (at least partial) depletion of endogenous polyamines, and/or
- the (at least partial) depletion of exogenous polyamines.
Throughout the application, the expression "endogenous polyamines" is intended in accordance with its ordinary meaning in the field. The expression "endogenous polyamines" is generally intended to designate polyamines, which are produced endogenously, i.e., polyamines, which are (endogenously) produced by cells of the subject, more particularly by tumor or cancer cells of the subject (and which are contained in or secreted from said (tumor or cancer) cells). More particularly, the expression "endogenous polyamines" encompasses polyamines, which are intracellular^ produced by (tumor or cancer) cells of the subject, e.g., by intracellular enzymes of said (tumor or cancer) cells. More particularly, the expression "endogenous polyamines" encompasses intracellularly produced polyamines, which are produced by (and contained in or secreted from) tumor or cancer cells of the subject.
Throughout the application, the expression "exogenous polyamines" is intended in accordance with its ordinary meaning in the field. The expression "exogenous polyamines" is generally intended to designate polyamines, which are not produced endogenously, i.e., which are exogenously produced and taken up by the subject. The expression "exogenous polyamines" is notably intended to designate polyamines, which can be present in the body of said subject, but which are not produced by the (tumor or cancer) cells of the subject. The expression "exogenous polyamines" may e.g., encompass:
- polyamines, which are delivered in the body of the subject by the food (including beverage) and/or by the pharmaceutical product(s), which is/are taken up by or administered to the subject (e.g., via the oral route or the systemic route), and/or
- polyamines, which are produced in said subject by the microbiota of said subject, more specifically the gut microorganisms of said subject, e.g., by the gut bacteria and/or by the gut fungi of the subject.
Throughout the application, the phrase "depletion" (or depleting or any grammatical derivative thereof) is intended in accordance with its ordinary meaning in the field, and generally refers to a decrease in quantity or concentration. Throughout the application, the phrase "depletion" encompasses the meaning of "partial depletion", of "at least partial depletion", and of "full depletion". More particularly, the phrase "depletion" encompasses the meaning of "at least partial depletion". Advantageously, polyamine depletion comprises (at least partial) depletion of endogenous polyamines, and optionally further comprises (at least partial) depletion of exogenous polyamines.
Means for depleting endogenous polyamines may e.g., comprise administering to said subject at least one inhibitor of polyamine synthesis and/or of polyamine metabolism.
Throughout the application, the phrase "inhibitor of polyamine synthesis" is intended in accordance with its ordinary meaning in the field, and generally refers to a compound inhibiting one or several of the enzymes involved in the intra-cellular synthesis of polyamines, e.g. ornithine decarboxylase (ODC), S-Adenosyl Methionine decarboxylase (SAMDC), spermidine synthase, and spermine synthase.
Throughout the application, the phrase "inhibitor of polyamine metabolism" is intended in accordance with its ordinary meaning in the field, and generally refers to a compound modulating (decreasing) the metabolism of polyamines in the cell by modulating the expression of the enzymes involved in polyamine synthesis and catabolism, either by down-regulating the expression of genes or gene products involved in polyamine synthesis, or by up-regulating the expression of genes or genes products involved in polyamine catabolism, or by up-regulating the expression of genes or gene products that decrease polyamine synthesis or a combination of several of the above.
Means for depleting exogenous polyamines may e.g., comprise submitting said subject to a polyamine reduced (or deficient) diet and/or administering a gut (microbial) decontaminant to said subject and/or administering a polyamine scavenging agent to prevent absorption of exogenous polyamines from the gut into the circulating blood of the subject.
Submitting said subject to polyamine depletion may thus comprise one, or at least two (e.g. two or three or four) of the following four polyamine depleting treatments i.-iv.:
i. administering at least one inhibitor of polyamine synthesis and/or of polyamine metabolism to said subject;
ii. submitting said subject to a polyamine reduced diet or to a polyamine deficient diet;
iii. administering at least one gut decontaminant to said subject; and
iv. administering at least one polyamine scavenging agent to said subject.
For example, submitting said subject to polyamine depletion may comprise the polyamine depleting treatment of i. ((at least partial) depletion of endogenous polyamines) and, optionally, at least one (i.e., one or two or three) of the polyamine depleting treatments ii., iii., and iv. ((at least partial) depletion of exogenous polyamines).
Advantageously, polyamine depletion comprises the (at least partial) depletion of the endogenous polyamines.
(At least partial) depletion of endogenous polyamines advantageously induces or stimulates the activity of the Polyamine Transport System (PTS) of the (tumor or cancer) cells of said subject, and/or the intracellular uptake of circulating (extracellular) polyamines. The increase in PTS activity can be viewed as a mean, by which the (tumor or cancer) cells compensate for the deficit in endogenous polyamines by allowing for an increase of the intracellular importation or uptake of circulating (extracellular) polyamines (via increased PTS activity).
The polyamine moiety, which is linked to the cytotoxic agent, acts as a tail or tag, which targets the cytotoxic agent through the PTS of the (tumor or cancer) cells. Therefore, the depletion of endogenous polyamines increases the capacity of the tumor or cancer cells of the subject to intracellular^ import or uptake the polyamine-vectored cytotoxic agent (via their increased PTS activity).
In other words, the depletion of endogenous polyamines is intended to increase the PTS activity of the tumor or cancer cells of the subject, and to thereby increase the quantity or rate at which the tumor or cancer cells of the subject uptake the polyamine-vectored cytotoxic agent. In other words, the depletion of endogenous polyamines is intended to improve the therapeutic effectiveness of the polyamine-vectored cytotoxic agent.
Hence, polyamine depletion advantageously comprises (the (at least partial) depletion of the endogenous polyamines, wherein said (at least partial) depletion of the endogenous polyamines comprises):
(at least partially) depleting tumor or cancer cells of the subject from the intracellular polyamines which are contained in said cells (or which are produced by these cells); and/or
(at least partially) inhibiting the (endogenous) synthesis of (intracellular) polyamines by tumor or cancer cells of the su bject.
Said (at least partial) depletion and/or inhibition may be achieved e.g., by administration of at least one inhibitor of polyamine synthesis and/or of polyamine metabolism.
Hence, polyamine depletion advantageously comprises (the (at least partial) depletion of the endogenous polyamines, wherein said (at least partial) depletion of the endogenous polyamines comprises) administering at least one inhibitor of polyamine synthesis and/or of polyamine metabolism to said subject.
Said at least one inhibitor of polyamine synthesis and/or of polyamine metabolism may comprise or be chosen from among:
- inhibitors of Ornithine Decarboxylase (ODC), such as, but not limited to, eflornithine (DFMO),
- inhibitors of S-Adenosyl-DeCarboxylase (SAMDC), such as, but not limited to, mitoguazone (MGBG or METHYL-GAG), 4-amidinoindanon-l-[2'amidino]hydrazone (SAM486A or GCP48664 or sardomozide), l,3-tris-[4'-amidinophanyl]triazine (berenil), and ρ,ρ'-
[pentamethylenedioxyjdibenzamidine (pentamidine), - inhibitors of polyamine oxidase
- inhibitors of other polyamine metabolism enzymes, for example Spermidine Synthase inhibitors and/or Spermine Synthase inhibitors,
- inducers of spermidine/spermine-N-acetyltransferase (SSAT) including non-steroidal anti- inflammatory drugs (NSAIDs), such as sulindac, celecoxib, piroxicam, and aspirin,
- down-regulators of ornithine decarboxylase such as curcumin, MYC inhibitors, BET inhibitors, and Retinoic acid and its derivatives, including all-trans retinoic acid (ATRA),
- Estrogen Receptor (ER) antagonists such as tamoxifen and fulvestrant,
- estrogen blockade therapies such as aromatase inhibitors (e.g., anastrazole, letrozole, exemestane),
- Androgen Receptor (AR) antagonists such as enzalutamide, bicalutamide
- androgen blockade therapies such as LHRH agonists (e.g., triptorelin, leuprolide), LHRH antagonists (e.g., degarelix), cyp-17 antagonists (e.g., abiraterone),
- glucocorticoids such as dexamethasone, betamethasone, prednisone,
- calcium channel inhibitors such as verapamil, amlodipine, dicardipine, diltiazem7
- anti-cancer agents which induce a down-regulation of polyamine synthesis (inhibitors of the PI3K / AKT / mTOR pathway, BET or c-myc inhibitors, MAP / ERK pathway inhibitors such as EGFR inhibitors, inhibitors RAF or MEK, MAPK inhibitors) and/or an up-regulation of polyamine catabolism, such as platinum agents (more particularly oxaliplatin and cisplatin); more preferably said anti-cancer agents do not comprise platinum agents;
- polyamine analogues depleting the intracellular pool of polyamines through the modulation of polyamine metabolism pathway regulation, such as Nl, Nll-diethylnorspermine (DENSPM).
More particularly, said at least one inhibitor of polyamine synthesis and/or of polyamine metabolism may comprise or be chosen from among:
- inhibitors of Ornithine Decarboxylase (ODC), such as, but not limited to, eflornithine (DFMO),
- inhibitors of S-Adenosyl-DeCarboxylase (SAMDC), such as, but not limited to, mitoguazone (MGBG or METHYL-GAG) or sardomozide (GCP48664 or SAM486A),
- non-steroidal anti-inflammatory drugs (NSAIDs), such as sulindac, celecoxib, piroxicam, and aspirin,
- down-regulators of ornithine decarboxylase such as curcumin and Retinoic acid and its derivatives,
- glucocorticoids such as dexamethasone, betamethasone, prednisone,
- calcium channel antagonist such as verapamil, amlodipine, dicardipine, diltiazem, and - polyamine analogues depleting the intracellular pool of polyamines through the modulation polyamine metabolism pathway regulation, such as Nl, Nll-diethylnorspermine (DENSPM).
More particularly, said at least one inhibitor of polyamine synthesis and/or of polyamine metabolism may comprise or be at least one inhibitor chosen from among:
- the ODC inhibitor DFMO,
- the SAMDC inhibitor SAM486A,
- calcium channel antagonist such as verapamil, amlodipine, dicardipine, diltiazem,
- non-steroidal anti-inflammatory drugs (NSAIDs) such as Sulindac, celecoxib, piroxicam, and aspirin, and
- glucocorticoids such as dexamethasone, betamethasone, prednisone.
More particularly, said at least one inhibitor of polyamine synthesis and/or of polyamine metabolism may comprise or be at least one inhibitor chosen from among:
- the ODC inhibitor DFMO, and
- the SAMDC inhibitor SAM486A.
Alternatively, said at least one inhibitor of polyamine synthesis and/or of polyamine metabolism may comprise or be at least one inhibitor chosen from among:
- the ODC inhibitor DFMO, and
- the calcium channel antagonist verapamil.
Eflornithine (also known, or referred to, as DFMO) is 2,5-diamino-2-(difluoromethyl)pentanoic acid (C6H12F2N202; PUBCHEM® ID 3009).
For example, said at least one inhibitor of polyamine synthesis and/or of polyamine metabolism may comprise or be at least one ODC inhibitor, more particularly at least the ODC inhibitor DFMO. For example, said at least one inhibitor of polyamine synthesis and/or of polyamine metabolism may comprise or be at least one SAMDC inhibitor, more particularly at least the SAMDC inhibitor SAM486A, MGBG, berenil, pentamidine, more particularly SAM486A.
For example, said at least one inhibitor of polyamine synthesis and/or of polyamine metabolism may comprise or be at least one calcium channel antagonist, more particularly verapamil.
More particularly, the present invention concerns a drug for the use as indicated above which comprises the following combinations:
- the F14512 drug and eflornithine (DFMO)
- the F14512 drug and verapamil
- the F14512 drug and eflornithine (DFMO) and verapamil
- the F14512 drug and eflornithine (DFMO) and SAM486A The (at least partial) depletion of endogenous polyamines may e.g., be to an extent sufficient for significantly increasing the activity of the Polyamine Transport System (PTS) of the (tumor or) cancer cells of said subject.
More particularly, said at least one inhibitor of polyamine synthesis and/or polyamine metabolism may e.g., be administered (to a subject in need thereof) at a dose or at a dosage regimen, which increases the activity of the Polyamine Transport System (PTS) of the (tumor or) cancer cells of said subject, and/or the intracellular uptake of circulating (extracellular) polyamines.
Advantageously, said at least one inhibitor of polyamine synthesis and/or of polyamine metabolism is administered to said subject at a dose lower than the dose at which said inhibitor of polyamine synthesis and/or of polyamine metabolism (is or) would be administered (to said subject) as sole active principle for (tumor or) cancer therapy, more particularly for (tumor or) cancer monotherapy and/or at doses intended to stop (tumor or) cancer cell proliferation.
Indeed, administering the said inhibitor of polyamine synthesis and/or of polyamine metabolisms at a dose intended to have a cytotoxic or cytostatic effect and/or to stop or prevent the growth of (tumor) cells would result in a decrease in (tumor or) cancer cell proliferation and a down- regulation of topoisomerase II in these cells, whereas said drug anti-tumor efficacy requires a proliferation of (tumor or) cancer cells, and a significant activity of topoisomerase II, which is the intracellular target of said cytotoxic agent, and would prevent said drug to reach its target and induce (tumor) cell death and exert its cytotoxic effect
Indeed, administering the said inhibitor of polyamine synthesis and/or of polyamine metabolism is intended as a means to increase said drug anti-tumor efficacy, while having no, or very limited impact on patient safety. More particularly, at the dose administered, the toxicity of said inhibitor of polyamine synthesis and/or of polyamine metabolism is expected to be minimal compared to the toxicity of usual chemotherapy agents. For example, DFMO is administered per os chronically for the prevention of colon cancer in high risk patients with very good patient safety. For example, verapamil is administered chronically for the prevention and treatment of cardiovascular disease.
For example, when said at least one inhibitor of polyamine synthesis and/or of polyamine metabolism is DFMO administered per os, it may be administered at a dose lower than 5 g/m2/day, - more particularly at a dose of 1-4 g/m2/day,
- more particularly at a dose of 1.5-3.5 g/m2/day,
- more particularly at a dose of about 2 g/m2/day,
In another example, when said at least one inhibitor of polyamine synthesis and/or of polyamine metabolism is DFMO administered per os, it may be administered at a dose lower than 15 g/day,
- more particularly at a dose of 3-12 g/day
- more particularly at a dose of 4.5-9 g/day
- more particularly at a dose of about 7.5 g/day.
For example, when said at least one inhibitor of polyamine synthesis and/or of polyamine metabolism is DFMO administered intravenously, it may be administered at a dose lower than 400 mg/kg/day, more particularly at a dose of 30-300 mg/kg/day, more particularly at a dose of 50-200 mg/kg/day, more particularly at a dose of about 100 mg/kg/day.
For example, when said at least one inhibitor of polyamine synthesis and/or of polyamine metabolism is SAM486A administered intravenously, it may be administered at a dose lower than 100 mg/m2/day, more particularly lower than 50 mg/m2/day, more particularly at a dose lower than 30 mg/m2/day, more particularly at a dose around 20 mg/m2/day.
For example, when said at least one inhibitor of polyamine synthesis and/or of polyamine metabolism is verapamil administered per os, it may be administered at a dose of 300 mg/day to 1000 mg/day, more particularly at a dose around 500 mg/day.
Advantageously,
said drug comprising a cytotoxic agent linked to a polyamine moiety, and
said at least one inhibitor of polyamine synthesis and/or of polyamine metabolism are administered to a subject in need thereof at a dosage regimen which is synergistically effective, more particularly at a dosage regimen which is synergistically effective for improved cytotoxicity against the tumor or cancer cells of the subject.
Throughout the application, the expression "dosage regimen" is understood in accordance with its ordinary meaning in the field. It generally encompasses the schedule of doses of the therapeutic agents per unit of time, which may include one or more of the following features:
- the time between doses (e.g., every 6 hours),
- the time when the dose(s) are to be given (e.g., at 8 a.m. and 4 p.m. daily, or after or prior to administration of a certain other product or treatment), and - the amount of a medicine or of active principle(s) (e.g., number of capsules or the quantity of active principle(s)) to be given at each specific time.
More particularly, the expression "dosage regimen" encompasses the quantity of different active principles to be administered for treating the subject and the relative timing of their respective administrations.
Throughout the application, the phrase "dose" is intended in accordance with its general meaning in the field. Its meaning notably encompasses the quantity of active principle to be given at a specific time. The depletion of exogenous polyamines may be alternative to, or complementary to the depletion of endogenous polyamines.
Depletion of exogenous polyamines may notably be intended to decrease the concentration of circulating (extracellular) polyamines in the extracellular fluid (ECF)of said subject. The result may advantageously be that there are less circulating polyamines to compete with the polyamine- vectored cytotoxic agent for intracellular importation (through the PTS) and that the decrease in circulating polyamine will lead to a higher activity of the PTS as a compensatory mechanism. The decrease in circulating polyamines may thereby favor the entry of the polyamine-vectored cytotoxic agent into the tumor or cancer cells (via the PTS).
Hence, depletion of exogenous polyamines advantageously decreases the competition, which said cytotoxic agent linked to a polyamine moiety faces for entry into the cancer cells of said subject. More particularly, depletion of exogenous polyamines advantageously is conducted or applied at a dose or dose regimen, which decreases the competition, which said cytotoxic agent linked to a polyamine moiety faces for entry into the cancer cells of said subject.
In other words, the depletion of exogenous polyamines is intended to improve the therapeutic effectiveness of the polyamine-vectored cytotoxic agent (by decreasing the competition for its entry into the tumor or cancer cells of the subject).
Submitting said subject to exogenous polyamine depletion (i.e. to at least partial depletion of the (extracellular) polyamines which are contained or circulating in the extracellular fluid (blood and interstitial fluid (and optionally in the urine) of the subject) may comprise one or several of the following three polyamine depleting treatments a.-c:
a. submitting said subject to a polyamine reduced diet or to a polyamine deficient diet; and b. administering at least one gut decontaminant to said subject, and c. administering at least polyamine scavenging agent to said subject.
More particularly, said diet may e.g., be a diet, which lowers the quantity or concentration of (extracellular) polyamines in extracellular fluids (e.g. the circulating blood or urine) of said subject. More particularly, said gut decontaminant is administered to said subject at a dose or in accordance with a dose regimen, which lowers the quantity or concentration of (extracellular) polyamines in extracellular fluids (e.g. the circulating blood or urine) of said subject.
In one embodiment, the polyamine scavenging agent comprises at least one agent that is capable of binding or adsorbing polyamines without being absorbed by the intestine is preferably selected from clayey minerals, or active or activated charcoal.
More particularly, said polyamine scavenging agent comprises at least one agent that is capable of binding or adsorbing polyamines in the intestine without being absorbed by the intestine; said agent is a clayey minerals which is preferably selected from:
Kaolinite, Nacrite, Dickite, Halloysite, Antigorite, Chysotile, Lizardite, Cronstedite, Berthierine, Amesite, Pyrophyllite, Smectites including Montmorillonite, Beidellite, Nontronite, Stevensite, Hectorite, Saponite, Bowlingite, Sauconite, Smectite dioctahedrale also called Diosmectite, Vermiculite, Batavite, Muscovite, lllite, Sericite, Damouzite, Paragonite, Glauconite, Celadonite, Talcum powder, Minnesotaite, Phlogopite, lllite, Biotite, Lepidolite, Ledikite, Donbassite, Cookeite, Sudoite, Franklinfurnaceite, Diabantite, Penninite, Chamosite, Brunsvigite, Clinochlore, Thuringite, Ripidolite, Sheridanite, Bentonite, Attapulgite, (Activated) Mormoiron Attapulgite, Monmectite, Akipula, aluminum, aluminium silicate, anhydrous aluminum silicates, askipula, beidellitic montmorillonite, benditos, bioelectrical minerals, cipula, chalk, clay dirt, clay dust, clay lozenges, clay suspension substances, clay tablets, colloidal minerals, colloidal trace minerals, fossil farina, humic shale, Indian healing clay, kaolin, kipula, magnesium silicate, mountain meal, NovaSil, NS, panito del senor, plant-derived liquid minerals, tirra santa, Terra sigillata, white mud, white clay, green clay or their mixture thereof.
More preferably, the substance that is capable of binding or adsorbing polyamines without being absorbed by the intestine is a clayey minerals which is selected from diosmectite, beidellitic montmorillonite, (Activated) Mormoiron Attapulgite, Monmectite and Kaolinite or their mixture thereof, and more preferably diosmectite. More particularly, said polyamine reduced or deficient diet is a diet, which lowers the quantity or concentration of extracellular polyamines in extracellular fluids (e.g, the circulating blood or urine) of said subject down to an extent sufficient for significantly decreasing the competition for entry into the tumor or cancer cells of the subject, which the polyamine-vectored cytotoxic agent faces with respect to extracellular polyamines.
Advantageously, the degree and duration of polyamine reduction or deficiency, which is applied by the diet, are sufficient for significantly decreasing said competition (to thereby favor the entry of the polyamine-vectored cytotoxic agent into the tumor or cancer cells).
Examples of polyamine reduced or deficient diet include any polyamine reduced or deficient diet, which the person of average skill in the art may find appropriate.
Examples of polyamine reduced or deficient diet may e.g., comprise a diet, wherein high polyamine-containing foodstuff (e.g., > 201 nmol/g/mL of polyamine) are prohibited.
Examples of polyamine reduced or deficient diet may e.g., comprise a diet, wherein median polyamine-containing foodstuff (e.g., 101-200 nmol/g/mL of polyamine) are limited to 3 or 4 times a week.
Examples of polyamine reduced or deficient diet may e.g., comprise a diet, wherein low polyamine-containing foodstuff (e.g., < 100 nmol/g/mL of polyamine) can be eaten at will.
Examples of polyamine reduced or deficient diet may e.g., comprise a diet, wherein:
- high polyamine-containing foodstuff (e.g., > 201 nmol/g/mL of polyamine) are prohibited, - median polyamine-containing foodstuff (e.g., 101-200 nmol/g/mL of polyamine) are limited to 3 or 4 times a week, and
- low polyamine-containing foodstuff (e.g., < 100 nmol/g/mL of polyamine) can be eaten at will. Examples of diet duration may e.g., comprise a period of at least 5 days, or of at least one week, or of at least two weeks, or of at least two months or of at least 3 months.
Examples of polyamine reduced or deficient diet have been described in Cipolla et al. 2010 (Biomedicine & Pharmacotherapy 64: 363-368).
Alternatively or complementarily to said polyamine reduced or deficient diet, depletion of exogenous polyamines (more particularly of extracellular polyamines, which are contained in extracellular fluids (e.g. the circulating blood or urine) of the subject) may comprise the administration of at least one gut decontaminant.
The gut microorganisms, more particularly the gut bacteria and/or the gut fungi, produce polyamines, which are released in the circulating blood of the subject. The at least one gut decontaminant is intended to decrease or suppress these microorganisms, more particularly these bacteria or fungi, to thereby decrease or suppress the quantity or concentration of polyamines, which is produced by these microorganisms (and released in the circulating blood of the subject).
The at least one gut decontaminant may e.g., be chosen from among the antibiotics, more particularly broad spectrum antibiotics, more particularly at least one antibiotic comprising at least one active principle chosen from among:
- neomycin (e.g., neomycin sulfate),
- nifuroxazide,
- colistine (e.g., colistine sulfate),
- fidaxomicine,
- sulfaguanidine,
- tiliquinol,
- nifurzide,
- rifaximine, and
- tilbroquinol.
Neomycin is (2 S,3S,4S,5 )-5-amino-2-(aminomethyl)-6-((2 ,3S,4 ,5S)-5-((l ,2 ,5 ,6 )-3,5- diamino-2-((2R,3S,4R,5S)-3-amino-6-(aminomethyl)-4,5-dihydroxytetrahydro-2H-pyran-2-yloxy)-
6-hydroxycyclohexyloxy)-4-hydroxy-2-(hydroxymethyl)tetrahydrofuran-3-yloxy)tetrahydro-2H- pyran-3,4-diol.
Nifuroxazide is 4-hydroxy-N-[(5-nitrofuran-2-yl)methylene]benzohydrazide.
Colistine is N-(4-amino-l-(l-(4-amino-l-oxo-l-(3,12,23-tris(2-aminoethyl)-20-(l-hydroxyethyl)-
6,9-diisobutyl-2,5,8,ll,14,19,22-heptaoxo-l,4,7,10,13,18-hexaazacyclotricosan-15- ylamino)butan-2-ylamino)-3-hydroxybutan-2-ylamino)-l-oxobutan-2-yl)-N,5- dimethylheptanamide.
Fidaxomicine is 3-(((6-Deoxy-4-0-(3,5-dichloro-2-ethyl-4,6-dihydroxybenzoyl)-2-0-methyl- -D- mannopyranosyl)oxy)-methyl)-12(R)-[(6-deoxy-5-C-methyl-4-0-(2-methyl-l-oxopropyl)- -D-lyxo- hexopyranosyl)oxy]-ll(S)-ethyl-8(S)-hydroxy-18(S)-(l(R)-hydroxyethyl)-9,13,15- trimethyloxacyclooctadeca-3,5,9,13,15-pentaene-2-one.
Sulfaguanidine is 4-Amino-N-[amino(imino)methyl]benzenesulfonamide.
Tiliquinol is 5-methylquinolin-8-ol.
Nifurzide is 5-Nitro-N'-[(lE,2E)-3-(5-nitro-2-furyl)-2-propen-l-ylidene]-2- thiophenecarbohydrazide. ifaximine is (2S,16Z,18E,20S,21S,22R,23R,24R,25S,26S,27S,28E)-5,6,21,23,25-pentahydroxy-27- methoxy-2,4,ll,16,20,22,24,26-octamethyl-2,7-(epoxypentadeca-[l,ll,13]trienimino)benzofuro [4,5-e]pyrido[l,2-a]-benzimida-zole-l,15(2H)-dione,25-acetate.
Tilbroquinol is 7-bromo-5-methylquinolin-8-ol.
More particularly, the at least one gut decontaminant is administered to said subject at a dose or dose regimen, which participates in lowering the quantity or concentration of extracellular polyamines, which are circulating in the blood of the subject, more particularly at a dose or dose regimen, which lowers the quantity or concentration of polyamines produced by the gut microorganisms, down to an extent sufficient for significantly decreasing the competition for entry into the tumor or cancer cells of the subject, which the polyamine-vectored cytotoxic agent faces with respect to extracellular polyamines, which are circulating in the blood of the subject. Advantageously, the dose or dosage regimen of the at least one gut decontaminant is sufficient for significantly contributing to said competition decrease (to thereby favor the entry of the polyamine-vectored cytotoxic agent into the tumor or cancer cells).
Alternatively or complementarily to said polyamine reduced or deficient diet and/or to the administration of at least one gut decontaminant, depletion of exogenous polyamines (more particularly of extracellular polyamines, which are contained in the circulating blood (and/or urine) of the subject) may comprise the administration of at least one polyamine scavenging agent, more particularly at a dose or in accordance with a dose regimen, which lowers the quantity or concentration of (extracellular) polyamines in the extracellular fluids (e.g. the circulating blood or urine) of said subject.
Advantageously, the polyamine depletion comprises the depletion of the endogenous polyamines and further comprises the depletion of exogenous polyamines.
Combining the depletion of endogenous polyamines with the depletion of exogenous polyamines is particularly advantageous, because that it combines:
- the increase in the level of PTS activity by tumor or cancer cells, i.e., the increase in polyamine uptake by the tumor or cancer cells, and
- the decrease in the level of circulating polyamines, i.e., the decrease in the competition for entry of the polyamine-vectored drug into the tumor or cancer cells (lower competition between the polyamine vectored drug and the circulating polyamines).
Therefore, submitting said subject to polyamine depletion advantageously comprises administering at least one inhibitor of polyamine synthesis and/or of polyamine metabolism to said subject, and further comprises one or several of the following three polyamine depleting treatments a.-c:
a. submitting said subject to a polyamine reduced diet or to a polyamine deficient diet;
b. administering at least one gut decontaminant to said subject; and
c. administering at least polyamine scavenging agent to said subject.
Advantageously, the application of polyamine depletion starts prior to the administration of the polyamine-vectored cytotoxic agent. In other words, simultaneously, sequentially or separately submitting said subject to polyamine depletion advantageously comprises submitting said subject to polyamine depletion prior to administering said drug to said subject.
Simultaneously, sequentially or separately submitting said subject to polyamine depletion may advantageously comprise submitting said subject to polyamine depletion (e.g., to endogenous and optionally exogenous polyamine depletion) prior to administering the drug (i.e., the drug, which comprises the polyamine-vectored cytotoxic agent) to said subject, more particularly prior to any administration of said drug.
Simultaneously, sequentially or separately submitting said subject to polyamine depletion may for example comprise submitting said subject to (e.g., endogenous and optionally exogenous) polyamine depletion:
- prior to any administration of said drug and after at least one administration of said drug; or
- prior to at least one administration of said drug and after at least one administration of said drug; or
- prior to any administration of said drug, simultaneously with at least one administration of said drug and after at least one administration of said drug; or
- prior to at least one administration of said drug, simultaneously with at least one administration of said drug and after at least one administration of said drug (wherein each of said at least administration are independent from each other).
Simultaneously, sequentially or separately submitting said subject to polyamine depletion may for example comprise:
starting the exogenous polyamine depletion through the separate or combined means of polyamine reduced or deficient diet, and/or gut decontamination, and/or polyamine scavenging agent administration at least 3 days, e.g., 3 to 5 days, before the administration of said drug, and continuing the exogenous polyamine depletion until the end of the administration cycle of said drug, and/or, more particularly and starting the administration of at least one inhibitor of polyamine synthesis and/or of polyamine metabolism prior to, more particularly at least 1 day before, e.g., 1 to 3 days before the administration of said drug and continuing the administration of said at least one inhibitor until the end of the administration cycle of said drug.
Simultaneously, sequentially or separately submitting said subject to polyamine depletion may for example comprise:
at day 1: starting the exogenous polyamine depletion (and continuing until the end of the treatment);
at day 3: optionally administering DFMO (e.g., per os at 7.5 g/day, 3 times a day), and continuing until the end of the treatment;
at day 3: optionally administering verapamil (e.g., per os, 500 mg/day), and continuing until the end of the treatment; and
at day 5: starting the administration of said drug (and continuing as required).
In another embodiment, wherein said inhibitor of polyamine synthesis or inhibitor of polyamine metabolism is DFMO administered intravenously, simultaneously, sequentially or separately submitting said subject to polyamine depletion may for example comprise:
starting the exogenous polyamine depletion through the separate or combined means of polyamine reduced or deficient diet, and/or gut decontamination, and/or polyamine scavenging agent administration at least 3 days, e.g., 3 to 5 days, before the administration of said drug, and continuing the exogenous polyamine depletion until the end of the administration cycle of said drug, and/or, more particularly and
starting the administration of at least one inhibitor of polyamine synthesis and/or of polyamine metabolism prior to, more particularly less than 6 hours before, e.g., 1 to 6 hours before the administration of said drug and continuing the administration of said at least one inhibitor until the beginning of the administration of said drug, or, preferably, until the end of the administration of said drug.
For example, in this embodiment, simultaneously, sequentially or separately submitting said subject to polyamine depletion may then comprise, for each cycle of said drug administration: at day 1: starting the exogenous polyamine depletion (and continuing until the end of the treatment);
from day 5 and until the end of said drug administration cycle:
o starting the administration of DFMO (e.g., iv at 50-200 mg/kg/day, more particularly at 100 mg/kg/day, by continuous perfusion of 1 to 6 hours, preferably 3 hours), 1 to 6 hours, more particularly, 2 to 4 hours, more particularly 3 hours, before the administration of said drug; and o starting the administration of said drug, 1 to 6 hours, more particularly 2 to 4 hours, more particularly 3 hours after the start of the administration of DFMO, and administering the drug by continuous perfusion of 1 to 24 hours, more particularly 3 hours, or alternatively, more particularly 24 hours; and o continuing the administration of DFMO simultaneously to the administration of said drug and until the end of the administration of said drug.
For example, in this embodiment, simultaneously, sequentially or separately submitting said subject to polyamine depletion may then comprise, for each cycle of said drug administration: at day 1: starting the exogenous polyamine depletion (and continuing until the end of the treatment);
from day 5 and until the end of said drug administration cycle:
o starting the combined administration of DFMO (e.g., iv at 50-200 mg/kg/day, more particularly at 100 mg/kg/day, by continuous perfusion of 1 to 6 hours, preferably 3 hours) and SAM486A (e.g., iv at 10-50 mg/m2/day, more particularly at 20 mg/m2/day), l to 6 hours, more particularly, 2 to 4 hours, more particularly 3 hours, before the administration of said drug; and
o starting the administration of said drug, 1 to 6 hours, more particularly 2 to 4 hours, more particularly 3 hours after the start of the administration of
DFMO+SAM486A, and administering the drug by continuous perfusion of 1 to 24 hours, more particularly 3 hours, or alternatively, more particularly 24 hours; and o continuing the administration of DFMO and SAM486A simultaneously to the administration of said drug and until the end of the administration of said drug.
Advantageously, prior to implementation of said polyamine depletion, said subject has:
a high concentration of circulating polyamines, more particularly of circulating extracellular polyamines more particularly in blood and/or in urine and/or of polyamines that are contained in circulating blood cells, more particularly in circulating erythrocytes, and/or
a high concentration of intra-tumor polyamines and/or a high level of expression and/or activity of polyamine synthesis enzyme(s), especially ODC and/or SAMDC, and/or a tumor molecular phenotype that is known to be associated with high levels of expression of intra-tumor polyamine synthesis enzyme(s), such as MYC amplification, MYCN amplification, AS mutated phenotype, BRAF mutated phenotype, EGFR mutated phenotype, ER positive phenotype, AR positive phenotype, etc.
Advantageously, prior to implementation of said polyamine depletion, said subject has a high concentration of circulating extracellular polyamines and/or of polyamines that are contained in circulating cells, more particularly in circulating erythrocytes.
The combination therapy of the application is intended for treating a (malignant) tumor or cancer, more particularly:
hematological malignancies, such as
- leukemia, more particularly acute myeloid leukemia; or
lymphoma; or
myeloma.
or
solid tumors, such as
- small cell lung cancer;
non-small cell lung cancer (NSCLC);
sarcoma;
liver cancer, more particularly hepatocarcinoma;
head and neck cancer;
- prostate cancer, more particularly a hormone-resistant prostate cancer;
ovarian cancer;
breast cancer;
brain cancer, more particularly glioma, more particularly glioblastoma;
renal cell carcinoma;
- neuroblastoma;
testicular cancer; or
digestive tumors, more particularly a stomach cancer, a colorectal cancer, a pancreas cancer, a biliary tract cancer, or a Gastro-lntestinal Stromal Tumor (GIST).
Said (malignant) tumor or cancer may more particularly be:
- leukemia, more particularly acute myeloid leukemia;
lymphoma, more particularly Burkitt's lymphoma;
small cell lung cancer;
non-small cell lung cancer (NSCLC);
hepatocarcinoma; head and neck cancer;
breast cancer;
glioma, more particularly glioblastoma;
renal cell carcinoma;
- neuroblastoma; or
testicular cancer.
Said (malignant) tumor or cancer may more particularly be:
leukemia, more particularly acute myeloid leukemia;
lymphoma, more particularly Burkitt's lymhoma;
- small cell lung cancer;
head and neck cancer;
glioma, more particularly glioblastoma;
renal cell carcinoma,
neuroblastoma; or
- testicular cancer.
Said (malignant) tumor or cancer may more particularly be:
leukemia, more particularly acute myeloid leukemia;
lymphoma, more particularly Burkitt's lymhoma;
small cell lung cancer; or
- glioma, more particularly glioblastoma; or
neuroblastoma.
Said (malignant) tumor or cancer may more particularly be:
leukemia, more particularly acute myeloid leukemia;
small cell lung cancer; or
- glioma, more particularly glioblastoma; or
neuroblastoma.
In addition to the combination therapy of the application, the cancer therapy may further comprise the administration of further anti-cancer drugs and/or the application of radiotherapy. Hence, said cancer therapy may further comprise submitting said subject to radiotherapy and/or administering to said subject at least one further anti-cancer drug, more particularly at least one further anti-cancer drug, which is chosen from among the following:
- platinum agents (more particularly cisplatin, carboplatin and oxaliplatin); - intercalating agents, including anthracyclins (more particularly doxorubicin, daunorubicin and mitoxantrone);
- alkylating agents (more particularly nitrosoureas, melphalan, temozolomide and cyclophosphamide);
- proteasome inhibitors (more particularly bortezomib);
- nucleotide antimetabolites and nucleotide analogues (more particularly cytarabine and gemcitabine);
- antifolates (more particularly methotrexate and pemetrexed);
- topoisomerase I inhibitors (more particularly irinotecan);
- microtubule inhibitors, including taxanes (more particularly paclitaxel and docetaxel) and vinca- alkaloids (more particularly vincristine);
- histone deacetylase inhibitors (more particularly Vorinostat or SAHA);
- demethylating agents (more particularly azacytidine and decitabine);
-EGF inhibitors, both antibodies (more particularly cetuximab and panitumumab), and small molecules (more particularly erlotinib, afatinib and gefitinib);
- BET inhibitors or MYC inhibitors acting to suppress c-Myc or n-Myc activity;
- PI3K/AKT/mTORCl pathway inhibitors such as PI3K inhibitors (more particularly idelalisib), AKT inhibitors and mTOR inhibitors (more particularly sirolimus, everolimus, temsirolimus);
- MAPK/ERK pathway inhibitors such as RAF inhibitors (more particularly sorafenib, encorafenib and vemurafenib), MEK inhibitors (more particularly cobimetinib, trametinib), and MAPK inhibitors.
More particularly, said cancer therapy may further comprise administering to said subject at least one further anti-cancer drug which down-regulates or inhibits polyamine metabolism, more particularly at least one further anti-cancer drug, which is chosen from among the following:
- EGFR inhibitors, both antibodies (more particularly cetuximab and panitumumab), and small molecules (more particularly erlotinib, afatinib, lapatinib and gefitinib);
- BET inhibitors or MYC inhibitors acting to suppress c-Myc or n-Myc activity;
- PI3K/AKT/mTORCl pathway inhibitors such as PI3K inhibitors (more particularly idelalisib), AKT inhibitors and mTOR inhibitors (more particularly sirolimus, everolimus, temsirolimus); and
- MAPK/ERK pathway inhibitors such as RAF inhibitors (more particularly sorafenib, encorafenib and vemurafenib), MEK inhibitors (more particularly cobimetinib, trametinib), and MAPK inhibitors. Alternatively, or additionally, said cancer therapy may also further comprise administering to said subject at least one further anti-cancer drug which anti-tumor activity is enhanced by polyamine depletion, more particularly at least one further anti-cancer drug, which is chosen from among the following:
- intercalating agents, including anthracyclins (more particularly doxorubicin, daunorubicin and mitoxantrone),
- alkylating agents (more particularly nitrosoureas, melphalan, temozolomide and cyclophosphamide), and
- antimetabolites (more particularly 5-FU).
The application also relates to a pharmaceutical composition comprising a fixed dose combination of a first active ingredient and a second active ingredient for use in cancer therapy, wherein:
said first active ingredient is the drug of the application, i.e., a drug, which comprises a cytotoxic agent linked to a polyamine moiety as herein defined, and wherein
- said second active ingredient is the at least one inhibitor of polyamine synthesis and/or of polyamine metabolism as herein defined, preferably DFMO, preferably for intravenous administration.
More particularly, the application relates to a drug comprising a cytotoxic agent of formula (I) linked to a polyamine moiety of formula (II), and at least one inhibitor of polyamine synthesis and/or of polyamine metabolism for use in (tumor or) cancer therapy, wherein said (tumor or) cancer therapy comprises administering said drug to a subject in need thereof and simultaneously, submitting said subject to polyamine depletion.
A particular embodiment of the present application concerns a drug for said use, which comprises or consists in the F14512 drug, and the at least one inhibitor of polyamine synthesis and/or of polyamine metabolism is DFMO.
Said particular combination may be administered by the intravenous or per os route.
The application also relates to a kit (or kit-of-parts) for simultaneous, sequential or separate use (or administration) of a first drug and of a second drug in cancer therapy, wherein:
- said first drug is the drug of the application, i.e., a drug, which comprises a cytotoxic agent linked to a polyamine moiety as herein defined, and wherein
said second drug is the at least one inhibitor of polyamine synthesis and/or of polyamine metabolism as herein defined. The application also relates to a kit (or kit-of-parts) for simultaneous, (or administration) of a first drug and of a second drug in cancer therapy, wherein:
said first drug is the pharmaceutical composition as herein defined comprising the drug of the application, i.e., a drug, which comprises a cytotoxic agent linked to a polyamine moiety as herein defined, and the at least one inhibitor of polyamine synthesis and/or of polyamine metabolism as herein defined, preferably DFMO, for intravenous administration, and wherein said second drug is the at least one inhibitor of polyamine synthesis and/or of polyamine metabolism as herein defined. The kit of the application may further comprise:
- a leaflet containing instructions for a polyamine reduced diet or a polyamine deficient diet (cf. above for further features that may define such a diet), and/or
- a third drug, wherein said third drug is or comprises at least one (human) gut decontaminant (cf. above for further features that may define such a decontaminant) or a polyamine scavenging agent.
Throughout the application, the term "comprising", which is synonymous with "including" or "containing", is open-ended, and does not exclude additional, un-recited element(s), ingredient(s) or method step(s), whereas the term "consisting of" is a closed term, which excludes any additional element, step, or ingredient which is not explicitly recited.
The term "essentially consisting of" is a partially open term, which does not exclude additional, un-recited element(s), step(s), or ingredient(s), as long as these additional element(s), step(s) or ingredient(s) do not materially affect the basic and novel properties of the invention.
The term "comprising" (or "comprise(s)") hence includes the term "consisting of" ("consist(s) of"), as well as the term "essentially consisting of" ("essentially consist(s) of"). Accordingly, the term "comprising" (or "comprise(s)") is, in the present application, meant as more particularly encompassing the term "consisting of" ("consist(s) of"), and the term "essentially consisting of" ("essentially consist(s) of").
In an attempt to help the reader of the present application, the description has been separated in various paragraphs or sections. These separations should not be considered as disconnecting the substance of a paragraph or section from the substance of another paragraph or section. To the contrary, the present description encompasses all the combinations of the various sections, paragraphs and sentences that can be contemplated.
The following examples are offered by way of illustration, and not by way of limitation. EXAMPLES
Example 1: Cell viability assays on the L1210 cell lines incubated with F 14512 or etoposide combined with DFMO in various experimental conditions
Materials and methods
Cell lines: The murine lymphocytic leukemia cell line L1210 was provided by Dr. Claude Boucheix (UM S935, INSERM, Paul Brousse).
Chemicals: RPMI-1640 (PanBiotech), L-gutamine (Gibco), antibiotics (Gibco), and FCS (Origin South America, Heat inactivated, Dominique Dutscher ), DMSO molecular biology reagent quality (Sigma), Sterile injectable water (Lonza), Putrescine (Sigma), F14512 (Villapharma), Etoposide (Sigma) DFMO (Rusan).
DFMO was solubilized in water at 0.1M, F14512 and etoposide in DMSO at 10-2 M then diluted in the complete cell culture medium accordingly. Putrescine was solubilized at 10-2M in the complete medium and diluted accordingly.
Cell culture: L1210 cells were cultivated in RPMI-1640 supplemented with 10% foetal calf serum, L-glutamine and Penicillin/Streptomycin. Cells were maintained and propagated at 37°C, 5% C02 and saturating humidity.
Pre-treatments: L1210 cells were seeded in 25 cm2 cell culture T- flasks at a density of 104 cell/ml in complete culture medium, kept one hour in the cell incubator before DFMO and/or Putrescine being added. After 72h incubation cells were counted to assess the potential toxicity of pre- treatment conditions. Each cell suspension was then adjusted to 11 000 cell/ml.
Treatments: Cells were seeded in 96 flat-bottomed cell culture plates at a density of 1000 cells per well in complete culture medium +/- DFMO and +/- putrescine in a volume of 90 μΙ. Plates were kept at 37°C in the cell culture incubator before 10 μΙ /well of lOx F14512 or etoposide solution was added. Each concentration was tested in triplicate. After 72h incubation, cell survival was measured using the Premix WST-1 cell proliferation system (Takara) by reading 450 nm absorbances, (Pherastar multimode reader, BMG Labtech). Experiment 1: L1210 cells were pre-treated in T-flasks with or without DFMO at 50μΜ, ΙΟΟμΜ, 200μΜ, 300μΜ, with or without addition of putrescine (Put) ΙΟμΜ, then plated and treated with various concentrations of F14512 associated or not with DFMO as per below:
Figure imgf000030_0001
Experiment 2: L1210 cells were pre-treated in T-flasks then plated and treated with various concentrations of F14512 or etoposide associated or not with DFMO as per below:
Figure imgf000030_0002
Calculations: Percentages of cell survival were given by the net 450 nm absorbances ratio to th respective controls. IC50s were determined using GraphpadPrism 5.0.
Results
Experiment 1: At the end of the pre-treatment phase, the number of cells was significantly decreased in the cells treated with DFMO 200μΜ (35% vs control) or DFMO 300μΜ (25%). This was partially reversed by Put ΙΟμΜ (54% vs control in cells treated with DFMO 200μΜ or DFMO 300μΜ + Put ΙΟμΜ). Cells treated with DFMO ΙΟΟμΜ were less affected (72% vs control, 81% for the cells with Put ΙΟμΜ). DFMO 50μΜ had no effect on cell proliferation (table 1). Table 1: Cell survival after 72h incubation with various concentrations of DFMO +/- Putrescine ΙΟμΜ
Figure imgf000031_0001
A the end of the treatment phase, cells treated with F14512+DFMO ΙΟΟμΜ after pre-treatment with DFMO ΙΟΟμΜ or DFMO ΙΟΟμΜ+Put 10μΜ appeared to be more significantly more sensitive to F14512 (IC50=7.5nM and 7.7nM respectively) than the Control cells (IC50=14.9nM) (figure 1A, table 2). Control cell survival was not affected by treatment with DFMO ΙΟΟμΜ. Cells pre-treated with DFMO ΙΟΟμΜ that did not receive DFMO during the treatment phase did not show increased sensitivity to F14512 (data not shown). Conversely, the growth of cells treated with DFMO 200μΜ following pre-incubation with DFMO 200μΜ +/- Put ΙΟμΜ was importantly decreased versus control cells (12% and 18%, respectively), preventing the interpretation of F14512 IC50 results. However, it was observed that cells pre- treated with DFMO 200μΜ or 200μΜ+Ρυΐ ΙΟμΜ had an increased sensitivity to F14512
(potentiation ratio 3.7 and 3.1 respectively), despite the fact that DFMO was not present during the treatment phase (figure 2B, table 2).
Table 2: F14512 IC50 for L1210 cell line after incubation alone or in combination with DFMO in various conditions of pre-treatment and treatment1 F14512 Cell surv3
(% s
Association (pre- IC50 [95% CI] Potentiation
treatment/treatment) (nM) ratio2 none/none) none/none 14.9 [9.4;23.5] NA NA
DFMO ΙΟΟμΜ / DFMO ΙΟΟμΜ 7.5 [5.9;9.6] 2.0 97.2
DFMO ΙΟΟμΜ+Put ΙΟμΜ/DFMO
7.7 [6.0;9.8] 1.9 124.5
ΙΟΟμΜ
DFMO 200μΜ / None 4.8 [3.7;6.3] 3.1 77.8
DFMO 200μΜ + PUT ΙΟμΜ/none 4.0 [3.3;4.8] 3.7 104.0
1 : cells were pre-treated in T-flasks for 72h without DFMO or with DFMO with or without putrescine ΙΟμΜ, then plated with F14512 with or without DFMO
2: ratio between IC50 of F14512 alone and IC50 of F14512 combined with DFMO
3: ratio between the number of cells treated with DFMO alone in absence of F14512 and the number of untreated cells
Experiment 2: After pre-treatment with DFMO 200μΜ +/- Putrescine at 10, 20 or 40μΜ, cell viability was 30% of control for cells exposed to DFMO alone, 45% for cells with Put 10μΜ, 80% for cells with Put 20μΜ and 66% for cells with Put 40μΜ. Based on these results, cells incubated with DFMO + Put 20μΜ were then selected for treatment with F14512 or etoposide +/- DFMO ΙΟΟμΜ, in comparison with control cells. The results on F14512 or etoposide are indicated in table 3 and figure 2. Control cell survival was not affected, and sensitivity to F14512 was increased by a factor 4 for cells pre-treated with DFMO 200μΜ + Put 20μΜ and co-treated with DFMO ΙΟΟμΜ. In the same conditions, the sensitivity to etoposide was increased but only by a factor 2. Potentiation was also observed to a lesser extent in DFMO pre-treated cells that di not receive DFMO during the treatment phase (table 3).
Conclusions
DFMO at low, non-cytotoxic concentrations (100 μΜ), and following 72h pre-incubation in the same conditions or at 200μΜ, potentiated the activity of F14512 on L1210 cell lines at 72h. Higher concentrations of DFMO had significant impact on control cell survival, thus preventing interpretation of results. Pre-treatment with DFMO 200μΜ also induced an increased sensitivity to F14512 without having any impact on control cell growth, supporting the hypothesis that pre- treatment with DFMO increased the uptake of F14512 in tumor cells. DFMO also increased sensitivity of L1210 cells to etoposide, although at a much lower extent. Table 3: F14512 and Etoposide IC50 for L1210 cell line after incubation alone or in combination with DFMO in various conditions of pre-treatment and treatment1
F14512 Etoposide Cell surv3
(% vs
IC50 [95% CI] Potentiation IC50 [95% CI] Potentiation
Association (pre-treatment/treatment) none/none)
(nM) ratio1 (nM) ratio2
none/none 25.5 [6.1;107] NA 53.5 [20.2;142] NA NA
DFMO 200μΜ +PUT 2(^M/DFMO ΙΟΟμΜ 6.3 [4.1;9.8] 4.0 23.1 [14.3;37.3] 2.3 90.4
DFMO 200μΜ + PUT 20μΜ/ηοηθ 10.0 [4.3;23.4] 2.6 29.0 [14.1;59.6] 1.8 130.5
1: cells were pre-treated in T-flasks for 72h without DFMO or with DFMO 200μΜ with or without putrescine 20μΜ, then plated with F14512 or etoposide with or without DFMO ΙΟΟμΜ
2: ratio between IC50 of F14512 or etoposide alone andlC50 of F14512 or etoposide combined with DFMO
3: ratio between the nb of cells treated with DFMO alone in absence of F14512/etoposide the and the nb of cells untreated
Example 2: Cell viability assays on human AML cell lines incubated with F14512 in presence or absence of DFMO
Materials and methods
Experiment 1: AML cell lines (HL-60, HEL, IMS-M2, KGla, MOLM-13, MOLM-14, MV4-11, NB4, NOMO-1, OCI-AML3, THP1, U937, Kasumil, ME-1) were obtained from the Stegmaier Lab (Dana Farber Cancer Institute, Boston). Cells were seeded on 384-well plates at 0.015 to 0.075.10s cells/mL (depending on the growth rate of each cell line) in PMI 1640 medium (ThermoFisher) supplemented with 10% or 20% Fetal Bovine Serum (FBS) and 1% Penicillin/streptomycin. F14512 (Villapharma) was added (concentration range: 10 to 0.001 μΜ. Plates were incubated at 37°C, 20% 02, 5% C02. After 120h, 15μΙ of CellTiter-Glo® Luminescent Cell Viability Assay (Promega) was added in each well, plates were agitated for 25 minutes before analysis of luminescence by a SpectraMax® i3x plate reader (Molecular Devices). Each experimental condition was measured in 6-plicate. IC50s were determined using GraphpadPrism 5.0.
Experiment 2: 5 cell lines with F14512 IC50 > Ο.ΟΙμΜ were selected to be tested with F14512 (same concentration range) in combination or not with DFMO (Sigma) at ΙΟΟμΜ or 500μΜ in the same experimental conditions.
Results
Experiment 1: F14512 was found to be active on all cell lines tested at concentrations below ΙμΜ. F14512 IC50 concentrations at 5 days ranged between 0.6nM (MOLM-14 cell lines) and 680nM (ME-1) (Table 4).
Table 4: F14512 IC50 after 5 days of incubation in 15 AML cell lines
Cell line CI50 (nM) 95% CI
MOLM-14 0.632 2.793 to 6.577
IMS-M2 0.9111 5.353 to 12.40
HL-60 0.9744 1.467 to 4.638
MV4-11 0.9774 5.253 to 17.15
NOMO-1 2.702 2.483 to 2.942
MOLM-13 3.311 2.454 to 4.466
U937 5.393 2.775 to 10.48 OCI-AML3 16.21 10.79 to 24.34
KASUMI-1 26.41 10.77 to 64.73
HEL 40.53 24.06 to 68.26
KGla 43.54 26.71 to 70.96
THP-1 104.9 69.81 to 157.7
ME-1 679.3 298.2 to 1547
Experiment 2: The 5 most resistant cell lines from experiment 1 (KASUMI-1, HEL, KGla, THP-1, ME-1) were tested with F14512 alone or in combination with DFMO 100 or 500μΜ. At these concentrations, DFMO was not toxic for cell lines (data not shown). Impact of DFMO on IC50 cytotoxicity is showed in Figure 3. In all cell lines, a potentiation of F14512 cytotoxicity was observed with gains in IC50 for F14512+DFMO ΙΟΟμΜ vs F14512 alone ranging from a factor 1.3 to 30, and gains for F14512+DFMO 500μΜ vs F14512 alone from a factor 3 to 160 (table 5).
Table 5: F14512 IC50 after 5 days of incubation alone or in combination with DFMO ΙΟΟμΜ or DFMO 500μΜ in 5 AML cell lines selected for their relative resistance to F14512 alone
Association Potentiation % cell surv.
Cell line CI50 (nM) 95% conf. interval
with DFMO by DFMO1 (/ctrl none)2
HEL none 42.56 14.63 to 123.8 NA
DFMO 100 μΜ 27.72 6.402 to 120.0 1.5 116.5
DFMO 500 μΜ 0.2617 0.04339 to 1.579 163 104.5
KGla none 76.47 18.60 to 314.3 NA
DFMO 100 μΜ 16.17 9.726 to 26.87 4.7 105.4
DFMO 500 μΜ 0.594 0.2630 to 1.341 129 123.6
THP1 none 77.26 56.68 to 105.3 NA
DFMO 100 μΜ 41.01 7.746 to 217.1 1.9 91.7
DFMO 500 μΜ 24.48 1.590 to 376.8 3.2 91.1
KASUMI-1 none 7.806 2.888 to 21.10 NA
DFMO 100 μΜ 5.874 1.438 to 24.00 1.3 84.2
DFMO 500 μΜ 0.8069 0.1314 to 4.955 9.7 91.7
ME-1 none 656.4 41.47 to 10391 NA
DFMO 100 μΜ 21.5 6.413 to 72.06 30.5 173.7
DFMO 500 μΜ 55.1 25.38 to 119.6 11.9 111.4 1: ratio between IC50 of F14512 alone andlC50 of F14512 combined with DFMO
2: ratio between the nb of cells with DFMO and the nb of cells untreated
Conclusions
In the conditions tested in this experiment, at sub-toxic concentrations, co-administration of
DFMO at ΙΟΟμΜ and 500μΜ potentiated the cytotoxicity of F14512 for 5/5 human AML cell lines by a factor ranging from 1.3 to >100. In most cell lines, potentiation was higher with DFMO 500μΜ. Example 3: Cell viability assays on solid tumor cell lines HCT116 and A2780 incubated with F14512 in presence or absence of DFMO
Materials and methods
Cell lines: A2780 (human ovarian carcinoma) HCT116 (human colorectal carcinoma)
Cell culture reagents and chemicals: RPMI-1640(PanBiotech), DMEM (PanBiotech) L-gutamine (Gibco) antibiotics (Gibco) FCS (Origin South America, Heat inactivated, Dominique Dutscher ) DMSO molecular biology reagent quality (Sigma) Sterile injectable water (Lonza) Putrescine (Sigma), F14512 (Villapharma) DFMO (Rusan), etoposide (Sigma), verapamil (Sigma), SAM486A (Medkoo Sciences).
Cell culture: A2780 cells were cultivated in RPMI 1640 supplemented with 10% fcetal calf serum, L-gutamine and Penicillin/Streptomycin. HCT116 cells in DMEM supplemented with 10% fcetal calf serum, L-gutamine and Penicillin/Streptomycin. Cells were maintained and propagated at 37°C, 5% C02 and saturating humidity
Pretreatments: A2780 and HCT116 cells were seeded in 96 well flat-bottomed 96 well plates at 2000 cells/well and 1000 cells/well respectively under a 90 μΙ/well volume. Cells were kept overnight in the cell culture incubator to allow cell adherence. Pretreatment agents were added (10μΙ /well of lOx solutions). Plates were kept for 24h in the incubator
Treatments: After 24h, 10 μΙ /well of llx F14512 or etoposide solution were added to the cell culture medium. After 5 days of additional incubation, cell survival was measured using the Premix WST-1 cell proliferation system (Takara) by reading 450 nm absorbances, (Pherastar multimode reader, BMG Labtech). All concentrations were tested in triplicate.
Experiment 1: HCT116 and A2780 cell lines, association of F14512 or etoposide with control or DFMO ΙΟΟμΜ
Experiment 2: A2780 cell line, association of F14512 or etoposide with the following drugs:
None (control)
- DFMO ΙΟΟμΜ - DFMO 100μΜ+5ΑΜ486Α Ο.ΙμΜ
- Verapamil ΙΟμΜ
Verapamil 30μΜ
- DFMO 100μΜ+ Verapamil ΙΟμΜ
- DFMO 100μΜ+ Verapamil 30μΜ
Calculations: Percentages of cell survival were given by the net 450 nm absorbances ratio to the respective controls. IC50s were determined using GraphpadPrism v5.0 software.
Results:
Experiment 1: F14512 alone IC50 for HCT116 and A2780 cells was respectively 196nM and 52nM. Addition of DFMO ΙΟΟμΜ to F14512 resulted into an increase of F14512 cytotoxicity as measured by F14512 IC50 in the presence of DFMO ΙΟΟμΜ: 51nM for HCT116 (3.8 fold increase in cytotoxicity vs F14512 alone) and llnM in A2780 (4.7 fold increase in cytotoxicity). The efficacy of etoposide was not increased by the addition of DFMO ΙΟΟμΜ (table 6, figure 4).
Table 6: F14512 IC50 for HCT116 and A2780 cell lines after incubation alone or in combination with DFMO ΙΟΟμΜ1
Figure imgf000037_0001
1: cells incubated for 24h with DFMO alone, then co-incubated with F14512 or etoposide+DFMO for 5 days
2: ratio between IC50 of F14512 or etoposide alone and IC50 of F14512 or etoposide when combined with DFMO
Experiment 2: Potentiation of F14512 by DFMO ΙΟΟμΜ was confirmed. Addition of SAM486A Ο.ΙμΜ to DFMO ΙΟΟμΜ increased the potentiating effect of DFMO on F14512 anti-tumor activity. At sub-toxic concentrations (10μΜ and 30μΜ), verapamil also increased F14512 activity, 1.8 and 3.1 fold respectively. Combination of DFMO and verapamil produced a synergistic effect on F14512 potentiation (4.8 fold increase in F14512 potency for DFMO ΙΟΟμΜ+verapamil 10μΜ; 9.7 fold increase for DFMO ΙΟΟμΜ+verapamil 30μΜ). Detailed results are shown in table 7 and figure 5. Table 7: F14512 and etoposide IC50 for A2780 cell lines after incubation alone or in combination with various polyamine metabolism inhibitors1
Figure imgf000038_0001
cells incubated for 24h with polyamine metabolism inhibitors, then co-incubated with F14512 etoposide+polyamine metabolism inhibitors for 5 days
2: ratio between IC50 of F14512/etoposide alone and IC50 of F14512/etoposide when combined with polyamine metabolism inhibitors
Conclusions:
Addition of DFMO at sub-toxic concentration (ΙΟΟμΜ) potentiated the cytotoxicity of F14512, but not etoposide, in HCT116 and A2780 cell lines. SAM486A, a SAMDC inhibitor, and verapamil, a calcium channel antagonist known to modulate ODC activity, appeared to increase DFMO potentiating effect on F14512 toxicity in the A2780 cell line. Verapamil at sub-toxic concentration 10μΜ and 30μΜ also potentiated F14512 activity in the A2780 cell line. Example 4: Assessment of the impact of co-treatment with DFMO or DFMO + smectite on the survival of B6D2F1 mice bearing L1210 murine leukaemia cells and treated with F14512 or etoposide
Materials and methods
Eighty (80) B6D2F1 immunocompetent mice were injected intraperitoneally with each 2.10s L1210 syngeneic leukemic cells, at DO.
At Dl, the treatments started as below, with exception of DFMO per os and/or SD which were started at D-2: Group # animals Treatment Dose (mg/kg) Route Treatment schedule
1 10 Control — —
In drinking water, ad
DFMO po1 1% Per os
2 10 libitum
SD2 N/A Per os Special diet, ad libitum
3 10 F14512 0.25 IP Every day (5/7)
F14512 0.25 IP Every day (5/7)
In drinking water, ad
4 10 DFMO po1 1% Per os
libitum
SD2 N/A Per os Special diet, ad libitum
F14512 0.25 IP Every day (5/7)
5 10
DFMO ip 200 IP Twice per day for 5 days
F14512 0.25 IP Every day (5/7)
6 10 DFMO ip 200 IP Twice per day for 5 days
SD2 N/A Per os Special diet, ad libitum
7 10 Etoposide 2 mg/kg IP Every day (5/7)
Etoposide 2 mg/kg IP Every day (5/7)
8 10 DFMO ip 200 IP Twice per day for 5 days
SD2 N/A Per os Special diet, ad libitum
1: DFMO per os was started at D-2
2: Special diet consisting of standard rodent chow (SAFE) supplemented with lOg/kg diosmectite (Smecta, Ipsen) was started at D-2
F14512 was supplied from Villapharma, DFMO from Rusan, etoposide from Hopital Louis Herriot.
The mice were weighted twice per week for 4 weeks and monitored daily for behavior and development of the ascites. They were euthanatized when their health visibly deteriorated. Survival data at day 28 were analyzed using SAS JMP vl4.0 software. Survival median and 95% confidence intervals, as well as statistical differences between groups were determined using a Weibull model.
Results
The detailed 28-day survival data for groups 1 to 6 are shown in figure 6. At 28 days, the survival rates in the different groups were as in table 8. Table 8: 28-day survival rate of B6D2F1 mice implanted with L1210 cells at DO according to treatment received
Figure imgf000040_0001
Survival duration comparisons between groups provided the following results: p-value
Comparison Interpretation
(Weibull)
Control vs DFMO + SD 0.0004 DFMO + SD > Control
Control vs F14512 < 0.0001 F14512 > Control
trend for superiority of F14512 + DFMO ip
F14512 vs F14512 + DFMO ip 0.1703 vs F14512
F14512 vs F14512 + DFMO ip + SD 0.0047 F14512 + DFMO ip + SD > F14512
F14512 vs F14512 + DFMO po + SD 0.0046 F14512 + DFMO po + SD > F14512
trend for superiority of F14512 + DFMO ip
F14512 + DFMO ip vs F14512 + DFMO ip + SD 0.0901 + SD vs F14512 + DFMO ip
Control vs Etoposide < 0.0001 Etoposide > control
Trend for superiority of Etoposide vs
Etoposide vs Etoposide + DFMO po + SD 0.0837 Etoposide + DFMO po + SD
Conclusions
Addition of DFMO ip and DFMO po to F14512 was associated with an extended duration of survival compared to mice treated with F14512 alone. In the groups receiving F14512 + DFMO, mice that received Special Diet comprising diosmectite in addition to DFMO had a longer survival compared to the group receiving only F14512 + DFMO.
In contrast to the benefit provided by DFMO +/- diosmectite observed with in mice treated with F14512, no benefit of polyamine depletion was observed in mice treated with etoposide, as mice receiving etoposide + DFMO + SD had a lower 28-day survival rate than the mice treated with etoposide alone.
BIBLIOGRAPHIC REFERENCES
Bergeron et al. 1997 (Leukemia 11: 31-36)
Cipolla et al. 2010 (Biomedicine & Pharmacotherapy 64: 363-368) Evageliou and Hogarty 2009 (Clin. Cancer Res. 15(19): 5956-5961) Levin et al. 2007. (International Journal of Cancer 121(10): 2279-2283)
Lugghezzani et al. 2010 (European Journal of Cancer 46: 1927 -1935) Murray-Stewart et al. 2016 (Biochemical Journal 473: 2937-2953) Palmer and Wallace 2010 (Amino Acids (2010) 38:415-422)
Poulin et al. 2012 (Amino Acids 42:711-723)
Takahashi et al. 2015 (Br. J. Cancer. 17; 113(10): 1493-1501)
WO 2005/100363 in the name of PIERRE FABRE MEDICAMENT Xie et al. 2010 (Expert Opinion on Drug Delivery 7(9): 1049-106

Claims

1. A drug comprising a cytotoxic agent linked to a polyamine moiety for use in cancer therapy, wherein said cancer therapy comprises administering said drug to a subject in need thereof and simultaneously, sequentially or separately submitting said subject to polyamine depletion, wherein said cytotoxic agent comprises or is the epipodophyllotoxin moiety of etoposide, said epipodophyllotoxin moiety being of formula (I)
Figure imgf000043_0001
and wherein said polyamine moiety comprises or is spermine, spermidine, putrescine or cadaverine, more particularly spermine moiety, which is of formula (II):
Figure imgf000043_0002
2. The drug for the use of claim 1, wherein said polyamine depletion comprises the at least partial depletion of the intracellular polyamines, which are contained in cancer cells of said subject.
3. The drug for the use of claim 2, wherein the at least partial depletion of the intracellular polyamines, which are contained in cancer cells of said subject, comprises administering at least one inhibitor of polyamine synthesis and/or of polyamine metabolism to said subject.
4. The drug for the use of any one of claims 1-3, wherein said polyamine depletion comprises administering at least one inhibitor of polyamine synthesis and/or of polyamine metabolism to said subject, and wherein said at least one inhibitor of polyamine synthesis and/or of polyamine metabolism comprises or is at least one of the following two inhibitors:
- an inhibitor of Ornithine Decarboxylase (ODC) and
- an inhibitor of S-Adenosyl-DeCarboxylase (SAMDC),
more particularly at least one of the following two inhibitors:
- eflornithine (DFMO) and
- MGBG or SAM486A
5. The drug for the use of any one of claims 1-4, wherein said polyamine depletion comprises administering at least one inhibitor of polyamine synthesis and/or of polyamine metabolism to said subject, and wherein said at least one inhibitor of polyamine synthesis and/or of polyamine metabolism comprises or is at least one of the following two inhibitors:
- eflornithine (DFMO) and
-SAM486A
6. The drug for the use of any one of claims 1-3, wherein said at least one inhibitor of polyamine synthesis and/or of polyamine metabolism comprises or is at least one calcium channel antagonist, more particularly verapamil.
7. The drug for the use of any one of claims 1-3, wherein said at least one inhibitor of polyamine synthesis and/or of polyamine metabolism comprises or is at least one of the following two inhibitors:
- an inhibitor of Ornithine Decarboxylase (ODC, more particularly eflornithine (DFMO), and
- a calcium channel antagonist, more particularly verapamil, and
- a Non Steroidal Anti-Inflammatory drug (NSAID), more particularly sulindac, celecoxib, piroxicam, or aspirin, and
- a glucocorticoid, more particularly dexamethasone, betamethasone, or prednisone.
8. The drug for the use of any one of claims 1-3 which comprises the following combinations: - the F14512 drug and eflornithine (DFMO)
- the F14512 drug and verapamil
- the F14512 drug and eflornithine (DFMO) and verapamil
- the F14512 drug and eflornithine (DFMO) and SAM486A
9. The drug for the use of any one of claims 3-8, wherein said at least one inhibitor of polyamine synthesis and/or of polyamine metabolism is administered to said subject at a dose or at a dosage regimen, which increases the activity of the Polyamine Transport System (PTS) of the cancer cells of said subject.
10. The drug for the use of any one of claims 3-9, wherein:
- said drug comprising a cytotoxic agent linked to a polyamine moiety, and
- said at least one inhibitor of polyamine synthesis and/or of polyamine metabolism
are administered to said subject at a dosage regimen which is synergistically effective for improved cytotoxicity against the cancer cells of the subject.
11. The drug for the use of any one of claims 1-10, comprising a fixed dose combination of:
a cytotoxic agent of formula (I) linked to a polyamine moiety of formula (II), and at least one inhibitor of polyamine synthesis and/or of polyamine metabolism.
12. The drug for the use of claim 11, wherein the cytotoxic agent is the F14512 drug, and the at least one inhibitor of polyamine synthesis and/or of polyamine metabolism is DFMO.
13. The drug for the use of any one of claims 1-12, wherein said polyamine depletion comprises the at least partial depletion of the polyamines, which are contained in the extracellular fluids of the subject.
14. The drug for the use of claim 13, wherein the at least partial depletion of the polyamines, which are contained in the circulating blood of the subject, comprises one, two or the three of the following three polyamine depleting treatments a.-c:
a. submitting said subject to a polyamine reduced or deficient diet;
b. administering at least one gut decontaminant to said subject; and
c. administering at least one polyamine scavenging agent to said subject.
15. The drug for the use of claim 13 or 14, wherein the at least partial depletion of the polyamines, which are contained in the extracellular fluid of the subject, decreases the competition, which said cytotoxic agent linked to a polyamine moiety faces for entry into the cancer cells of said subject.
16. The drug for the use of any one of claims 1-15, wherein simultaneously, sequentially or separately submitting said subject to polyamine depletion comprises submitting said subject to polyamine depletion prior to administering said drug to said subject.
17. The drug for the use of any one of claims 1-16, wherein said cancer is leukemia, small cell lung cancer, glioma or neuroblastoma.
18. A kit-of-parts for simultaneous, sequential or separate use of a first drug and of a second drug in cancer therapy, wherein said first drug is the drug, which comprises a cytotoxic agent linked to a polyamine moiety, as defined in any one of claims 1-17, and wherein said second drug is the at least one inhibitor of polyamine synthesis and/or of polyamine metabolism as defined in any one of claims 3-11.
19. The kit-of-parts for the use of claim 18, which further comprises at least one of the following two items:
- a leaflet containing instructions for a polyamine reduced or deficient diet, and
- a third drug, wherein said third drug is or comprises at least one gut decontaminant.
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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005100363A1 (en) 2004-04-16 2005-10-27 Pierre Fabre Medicament (poly) aminoacetamide derivatives of epipodophyllotoxin their process of preparation and their applications in therapeutics as anticancer agents

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005100363A1 (en) 2004-04-16 2005-10-27 Pierre Fabre Medicament (poly) aminoacetamide derivatives of epipodophyllotoxin their process of preparation and their applications in therapeutics as anticancer agents

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Title
BERGERON ET AL., LEUKEMIA, vol. 11, 1997, pages 31 - 36
CIPOLLA ET AL., BIOMEDECINE & PHARMACOTHERAPY, vol. 64, 2010, pages 363 - 368
CIPOLLA ET AL., BIOMEDICINE & PHARMACOTHERAPY, vol. 64, 2010, pages 363 - 368
EVAGELIOU; HOGARTY, CLIN. CANCER RES., vol. 15, no. 19, 2009, pages 5956 - 5961
KRUCZYNSKI ET AL: "F14512, A POLYAMINE VECTORIZED ANTI-CANCER DRUG EXHIBITS A MARKED ANTILEUKEMIC ACTIVITY ALONE AND IN COMBINATION WITH ARA-C", EUROPEAN JOURNAL OF CANCER,, vol. 48, no. SUP5, 1 January 2012 (2012-01-01), pages S223 - S224, XP002775860 *
LEBLOND PIERRE ET AL: "Activity of the polyamine-vectorized anti-cancer drug F14512 against pediatric glioma and neuroblastoma cell lines", INVESTIGATIONAL NEW DRUGS, MARTINUS NIJHOFF PUBLISHERS, BOSTON, US, vol. 32, no. 5, 11 July 2014 (2014-07-11), pages 883 - 892, XP035906289, ISSN: 0167-6997, [retrieved on 20140711], DOI: 10.1007/S10637-014-0132-3 *
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MOUAWAD ET AL: "THE ANTITUMOR DRUG F14512 ENHANCES CISPLATIN AND IONIZING RADIATION EFFECTS IN HEAD AND NECK SQUAMOUS CARCINOMA CELL LINES", ORAL ONCOLOGY,, vol. 50, 1 January 2014 (2014-01-01), pages 113 - 119, XP002775861, DOI: 10.1016/J.ORALONCOLOGY.2013.11.003 *
MURRAY-STEWART ET AL., BIOCHEMICAL JOURNAL, vol. 473, 2016, pages 2937 - 2953
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XIE ET AL., EXPERT OPINION ON DRUG DELIVERY, vol. 7, no. 9, 2010, pages 1049 - 106
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