EP2303255A1 - Compositions pharmaceutiques utiles pour le traitement de cancers, en particulier d'une leucémie myéloïde aiguë et d'une leucémie promyélocytique aiguë - Google Patents

Compositions pharmaceutiques utiles pour le traitement de cancers, en particulier d'une leucémie myéloïde aiguë et d'une leucémie promyélocytique aiguë

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Publication number
EP2303255A1
EP2303255A1 EP09757550A EP09757550A EP2303255A1 EP 2303255 A1 EP2303255 A1 EP 2303255A1 EP 09757550 A EP09757550 A EP 09757550A EP 09757550 A EP09757550 A EP 09757550A EP 2303255 A1 EP2303255 A1 EP 2303255A1
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Prior art keywords
camp
retinoic acid
inn
dichloropyrid
pdei
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EP09757550A
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German (de)
English (en)
Inventor
Marie-Claude Guillemin
Rihab Nasr
Hugues De The
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Universite Paris Diderot Paris 7
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Universite Paris Diderot Paris 7
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Priority to EP09757550A priority Critical patent/EP2303255A1/fr
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Withdrawn legal-status Critical Current

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • 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
    • A61K31/20—Carboxylic acids, e.g. valproic acid having a carboxyl group bound to a chain of seven or more carbon atoms, e.g. stearic, palmitic, arachidic acids
    • A61K31/203—Retinoic acids ; Salts thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
    • A61K31/52—Purines, e.g. adenine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K33/00—Medicinal preparations containing inorganic active ingredients
    • A61K33/24—Heavy metals; Compounds thereof
    • A61K33/36—Arsenic; Compounds thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5011—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing antineoplastic activity
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/575—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57505—Immunoassay; Biospecific binding assay; Materials therefor for cancer of the blood, e.g. leukaemia
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/10—Screening for compounds of potential therapeutic value involving cells

Definitions

  • the present invention relates to new pharmaceutical compositions useful for the treatment of cancers, in particular acute myeloid leukemia (AML) and acute promyelocytic leukemia (APL).
  • AML acute myeloid leukemia
  • APL acute promyelocytic leukemia
  • APL Acute promyelocytic leukemia
  • APL is characterized by a differentiation blockage at the promyelocytic stage and a specific t(15,17) translocation, which encodes a PML/RARA fusion protein.
  • PML/RARA is a potent transcriptional repressor with both gains of function and dominant-negative properties, resulting in transcriptional repression of retinoic acid (RA) or non-RA target genes (de The, H., et al.
  • the PML-RAR alpha fusion mRNA generated by the t(15;17) translocation in acute promyelocytic leukemia encodes a functionally altered RAR.
  • Gene silencing involves enhanced recruitment of nuclear receptor corepressors, the polycomb complex or Daxx, resulting in changes in chromatin organization and DNA- methylation (Minucci, S., et al. Oligomerization of RAR and AMLl transcription factors as a novel mechanism of oncogenic activation. MoI. Cell 5, 811-820 (2000); Lin, R. & Evans, R. Acquisition of oncogenic potential by RAR chimeras in acute promyelocytic leukemia through formation of homodimers. Molecular Cell 5, 821-830. (2000); Di Croce, L., et al. Methyltransferase recruitment and DNA hypermethylation of target promoters by an oncogenic transcription factor. Science 295, 1079-1082.
  • RA binds PML/RARA, turns it into a transcriptional activator and triggers its degradation
  • PML/RAR alpha oncoprotein is a direct molecular target of retinoic acid in acute promyelocytic leukemia cells.
  • Blood 88, 2826-2832 (1996); Nervi, C, et al.
  • Caspases mediate retinoic acid-induced degradation of the acute promyelocytic leukaemia PML/RARalpha fusion protein.
  • Blood 92, 2244-2251 (1998); Zhu, J., et al.
  • Retinoic acid induces proteasome-dependent degradation of retinoic acid receptor alpha (RAR alpha) and oncogenic RAR alpha fusion proteins. Proc. Natl. Acad. Sci. USA 96, 14807-14812 (1999)).
  • arsenic activates kinases targeting PML/RARA or its obligatory RXR partner, modulates PML and PML/RARA sumoylation and triggers their degradation (Zhu, J v et al. A sumoylation site in PML/RARA is essential for leukemic transformation. Cancer Cell 7, 143-153 (2005); Lallemand-Breitenbach, V., et al.
  • Nuclear receptors harbour two distinct transcriptional activation domains, AFl and AF2. The later overlaps the ligand-binding domain (LBD) and is under the direct control of ligand-induced conformational changes.
  • AF-I is inlaid within the variable N- terminal domain and is regulated by phosphorylation, but its contribution to nuclear receptor signaling has never been established in vivo.
  • RARA AFl is phosphorylated by the cdk7/cyclin H sub-complex of TFIIH (Rochette-Egly, C, Adam, S., Rossignol, M., EgIy, J.-M. & Chambon, P.
  • Phosphorylation by PKA potentiates retinoic acid receptor alpha activity by means of increasing interaction with and phosphorylation by cyclin H/cdk7. Proc Natl Acad Sci U S A 103, 9548-9553 (2006)). Both APL differentiation and PML/RARA transactivation are enhanced by cAMP signaling, and some RA-resistant APL cell-lines differentiate upon cAMP exposure (Kamashev, D.E., Vitoux, D. & De The, H. PML/RARA-RXR oligomers mediate retinoid- and rexinoid- /cAMP in APL cell differentiation. J. Exp. Med. 199, 1-13.
  • RA arsenic derivatives or a combination thereof are able to suppress the differentiation blockage at the promyelocytic stage leading thus to a remission of a patient having AML or APL disease but are unable to eradicate the LIC.
  • cyclic AMP adenosine 3 '-5' cyclic monophosphate
  • activation of the cAMP signal transduction pathway differentiates many acute myeloid leukemia cell-lines and strongly synergizes with other differentiating agents (Olsson, LL. , and T. R. Breitman. 1982.
  • compositions comprising at least one compound activating the cAMP signal transduction pathway, in particular theophylline in association with a cell- differentiation factor such as RA and an apoptose inducer such as AS 2 O 3 , useful for the treatment of cancers, have been described (WO 2004/026319).
  • said compositions only acts on the differentiation of cells inducing only remission of the disease, not on the eradication of LIC leading to complete healing of the disease.
  • WO 2004/062671 relates to PDE IV inhibitors such as N-(3,5-dichloropyrid-4-yl)-3- cyclopentyloxy-4-methoxybenzamide (INN: piclamilast) or 3-cyclopropylmethoxy-4- difluoromethoxy-N-(3,5-dichloropyrid-4-yl)-benzamide (INN: roflumilast) alone or in combination with differentiation inducing agents such as RA or As 2 Ch for the treatment of neoplasm of lymphoid cells.
  • PDE IV inhibitors such as N-(3,5-dichloropyrid-4-yl)-3- cyclopentyloxy-4-methoxybenzamide (INN: piclamilast) or 3-cyclopropylmethoxy-4- difluoromethoxy-N-(3,5-dichloropyrid-4-yl)-benzamide (INN: roflumilast) alone or in combination with differentiation
  • the chemical name or INN name of piclamilast will be employed and will refer to the same molecule.
  • the chemical name or INN name of roflumilast will be employed and will refer to the same molecule.
  • An object of the present invention is to provide new pharmaceutical compositions for the treatment of cancers, in particular AML and APL, allowing to prevent the reemergence of the disease from remaining stem cells after cessation of the classical treatments and thus eradicate the disease, or to treat RA resistant patient.
  • Another object of the present invention is to provide a method for screening drugs liable to be used for the manufacture of medicaments intended for the eradication of LIC and in particular for the treatment of pathologies such as AML or APL.
  • the present invention relates to the use of a composition comprising retinoic acid (RA) or a related compound thereof such as a retinoid, in combination with at least one phosphodiesterase inhibitor (PDEI) or with an arsenic derivative, or with at least one phosphodiesterase inhibitor (PDEI) and an arsenic derivative, for the manufacture of a drug intended to suppress the differentiation blockage of cancer cells such as neuroblastoma cells or leukaemia cells, and to eradicate cancer stem cells such as neuroblastoma initiating cells (NIC) or leukaemia initiating cells, for the treatment of pathologies such as cancer, in particular neuroblastoma, acute myelocytic leukaemia (AML) including acute promyelocyte leukaemia (APL).
  • RA retinoic acid
  • PDEI phosphodiesterase inhibitor
  • PDEI phosphodiesterase inhibitor
  • arsenic derivative or with at least one phosphodiesterase inhibitor (PDEI) and an arsen
  • retinoic acid is meant all-trans retinoic acid or cis derivatives of ATRA such as 9-cis retinoic acid or 13-cis retinoic acid, vitamin A (retinol), carotene or rexinoids or pharmacologically acceptable salts thereof.
  • related compound thereof means an analogue of retinoic acid, i.e. compound able to bind to and activate nuclear receptors, for instance retinoids.
  • retinoid receptors There are six known retinoid receptors, the retinoic acid receptors: RAR ⁇ , ⁇ and ⁇ and the retinoid X receptors: RXR ⁇ , ⁇ and ⁇ .
  • RAR ⁇ or RARA are used independently and have the same meaning.
  • retinoid examples of retinoid but without being limited to, can be found in Beard et al. ⁇ Handbook of experimental pharmacology, retinoids the biochemical and molecular of vitamin A and retinoid action; Nau, H., Blaner W. S., Eds.; Springer: Berlin Heidelberg, 1999; Vol. 139, pl85) or in Beard et al (Bioorg. Med. Chem. Lett. 12 (2002), 3145-3148).
  • PDEI are drugs that blocks one or more of the five subtypes of the enzyme phosphodiesterase (PDE), therefore preventing the inactivation of the intracellular second messengers, cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP), by the respective PDE subtype(s).
  • PDE phosphodiesterase
  • -non selective inhibitors such as caffeine, theophylline, aminophylline, isobutylmethyl xantine,
  • - PDEl -selective inhibitors such as vinpocetine, - PDE2-selective inhibitors such as EHNA,
  • PDE3-selective inhibitors such as enoximone and milrinone
  • PDE4-selective inhibitors such as mesembrine, rolipram, ibudilast, N-(3,5- dichloropyrid-4-yl)-3-cyclopentyloxy-4-methoxybenzamide (INN: piclamilast) or 3- cyclopropylmethoxy-4-difluoromethoxy-N-(3,5-dichloropyrid-4-yl)-benzamide (INN: roflumilast), methoxyquinazoline,
  • PDE5-selective inhibitors such as sildenafil, tadalafil and vardenafil, udenafil and avanafil, zaprinast.
  • Arsenic derivative is a common naturally occurring substance that can exist under three inorganic forms: red (arsenic disulfide referred to as realgar, pararealgar or sandacara), yellow (arsenic trisulf ⁇ de referred to as arsenikon, aurum pigmentum or orpiment) and white (arsenic trioxide).
  • the expression "suppress the differentiation blockage of" means that the composition is able to eliminate the blockage of the differentiation of the cells, in particular at their promyelocyte stage for the leukaemia cells and thus trigger the differentiation of the cells (figure 1).
  • FACS analysis allows distinguishing differentiated cells from undifferentiated cells. Indeed, the lin- cells obtained in figure 2A are completely undifferentiated and have no differentiation markers (Gr-I " , CD-I Ib " , lower left zone delimited by 10 1 on the x and y axis of the square fig 3A). After growing on MC, cells acquire the differentiation marker GR-I and become only partially differentiated (Gr-I + , CD-I Ib " , zone (a) of the square (fig 3A)). The left part of the square (zones (a) and (b) corresponds therefore to promyelocytes and cells in figures 3 A are constituted of promyelocytes only.
  • the cells acquire the differentiation marker CD-l ib and are constituted of granulocytes only (Gr-I + , CD-I Ib + , zone (c) of the square and Gr-I " , CD-I Ib + , zone (d), (fig 3C)).
  • the right part of the square corresponds therefore to granulocytes.
  • Neuroblastoma is the most common extracranial solid cancer in childhood and the most common cancer in infancy. In cancers, a small number of cells with stem- like properties have the ability to regenerate new tumors. In AML or APL, these cells with stem-like properties are called leukaemia initiating cells (LIC).
  • LIC leukaemia initiating cells
  • Eradication can be evaluated according to the protocol described in example 6 or 7.
  • Example 5 and 7 shows that RA treatment alone, whatever the treatment length, only relapse but never eradicate APL. Indeed, there is a complete dissociation between efficient RA-induced differentiation, leading to a remission of the disease but not a complete treatment of the disease, and eradication of LIC.
  • one of the advantages of the present invention is to provide a composition comprising RA or a retinoid in combination with at least a PDEI and/or an arsenic derivative, said composition being advantageously synergic and able to both suppress the differentiation blockage and at the same time to eradicate the LIC.
  • composition of the invention can comprise at least two compounds (RA or related compound and at least one PDEI or an arsenic derivative), or at least three compound (RA or related compound and at least one PDEI and an arsenic derivative).
  • the composition defined above comprises retinoic acid
  • composition of the invention can thus comprise at least two compounds (RA or related compound and at least one PDEI, or at least three compounds (RA or related compound and at least one PDEI and an arsenic derivative).
  • the composition defined above comprises retinoic acid (RA) or a related compound thereof such as a retinoid, in combination with an arsenic derivative.
  • RA retinoic acid
  • arsenic derivative an arsenic derivative
  • composition of the invention comprises two compounds (RA or related compound and an arsenic derivative).
  • the invention relates to the use of a composition comprising retinoic acid (RA) or a related compound thereof such as a retinoid, in combination with at least one phosphodiesterase inhibitor (PDEI) or at least one agent enabling to increase the cellular content of cAMP or derivatives thereof with respect to the originally present cellular content of said cAMP and an arsenic derivative, or with at least one phosphodiesterase inhibitor (PDEI) or at least one agent enabling to increase the cellular content of cAMP or derivatives thereof with respect to the originally present cellular content of said cAMP, for the manufacture of a drug intended to suppress the differentiation blockage of cancer cells such as neuroblastoma cells or leukaemia cells, and to eradicate cancer stem cells such as neuroblastoma initiating cells (NIC) or leukaemia initiating cells, for the treatment of pathologies such as cancer, in particular neuroblastoma, acute myelocytic leukaemia (AML) including acute promyelocytic
  • composition comprises:
  • -RA or a retinoid in combination with a PDEI or one agent enabling to increase the cellular content of cAMP and an arsenic derivative, or,
  • cAMP corresponds to the following formula:
  • cAMP derivatives of cAMP are well known to the man skilled in the art, they notably comprise 8-Cl-cAMP, 8-CPT-cAMP, 8-Br-cAMP and dibutyryl-cAMP, or pharmacologically acceptable salts thereof.
  • the "originally present cellular content of cAMP" relates to the cAMP content of cells prior to the addition to said cells of any compound liable to modify the cellular concentration of cAMP.
  • cAMP cyclopentasergic asergic asergic asergic asergic asergic asergic asergic asergic asergic asergic asergic asergic asergic asergic asergic asergic asergic asergic asergic asergic phosphate, or of its derivatives, can be measured according to methods well known to the man skilled in the art.
  • the cAMP content of a cell results from an equilibrium between two opposite reaction types, i.e. reaction concurring to the synthesis of cAMP, such as reactions catalyzed by adenylate cyclases, and reactions concurring to the degradation of cAMP, such as reactions catalyzed by phosphodiesterases (PDE). Consequently, a rise in the cellular content of cAMP can be observed following addition of compounds either activating cAMP synthesis or inhibiting cAMP degradation.
  • reaction concurring to the synthesis of cAMP such as reactions catalyzed by adenylate cyclases
  • PDE phosphodiesterases
  • an "agent enabling to increase the cellular content of cAMP or derivatives thereof can be for instance, cAMP or a derivative thereof in itself, or an agent activating the intracellular synthesis of cAMP, or an agent inhibiting the intracellular degradation of cAMP or derivatives thereof, provided it is added to cells in an amount sufficient to lead to an increase of the cAMP content of said cells.
  • the invention relates to the use of the composition defined above for the manufacture of a drug intended to degrade PML-RARA of leukaemia cells for the treatment of pathologies such as leukaemia, in particular acute promyelocytic leukaemia (APL).
  • APL acute promyelocytic leukaemia
  • degrade PML-RARA means that the PML/RARA fusion protein encoded by the specific t(15,17) translocation characteristic of APL is degraded.
  • the catabolism of PML/RARA is produced by a conjugation to the ubiquitin-like peptide SUMO that triggers the degradation of the fusion protein.
  • Example 8 shows that a proteasome inhibitor (Velcade ® ) delays the APL regression, blocks the restoration of normal haematopoiesis and antagonizes loss of LIC triggered by RA/arsenic association. PML/RARA degradation is thus a critical molecular determinant of LIC eradication.
  • Velcade ® proteasome inhibitor
  • compositions are able to both suppress the differentiation blockage and at the same time to degrade the PML/RARA fusion protein encoded by the specific t(15,17) translocation, by sumoylation and/or by S873 phosphorylation and thus eradicate the LIC.
  • the invention relates to the use of a composition defined above wherein said related compound of RA is a compound able to degrade PML/RARA.
  • the present invention relates to the use of the composition defined above, wherein said composition comprises retinoic acid (RA) or a related compound thereof such as a retinoid, in combination with at least one phosphodiesterase inhibitor (PDEI) said PDEI being selected from the list consisted of N- (3,5-dichloropyrid-4-yl)-3-cyclopentyloxy-4-methoxybenzamide (INN: piclamilast) or 3- cyclopropylmethoxy-4-difluoromethoxy-N-(3,5-dichloropyrid-4-yl)-benzamide (INN: roflumilast) and an arsenic derivative.
  • the composition comprises RA or a retinoid, in combination with piclamilast or roflumilast and an arsenic derivative.
  • the present invention relates to the use of one of the above defined compositions wherein the drug is for administration of a RA dose in the range from 200 mg/m 2 to 20 mg/m 2 , preferably from 175 mg/m 2 to 20 mg/m 2 , preferably from 150 mg/m 2 to 20 mg/m 2 , preferably from 150 mg/m 2 to 20 mg/m 2 , preferably from 100 mg/m 2 to 20 mg/m 2 , preferably from 75 mg/m 2 to 20 mg/m 2 , preferably from 45 mg/m 2 to 20 mg/m 2 , preferably from 40 mg/m 2 to 20 mg/m 2 , more preferably from 30 mg/m 2 to 20 mg/m 2 , in particular 30 mg/m 2 .
  • RA is usually used at a dosage of about 10 mg/m 2 , but when used alone, RA at this dose is able to promote the differentiation but its concentration is too low to complete the PML/RARA catabolism. Much higher RA concentrations are required for full PML/RARA catabolism than for efficient target gene activation (example 11, figure 21).
  • still another advantage of the invention is to provide compositions wherein high RA doses, i.e. from 200 mg/m 2 to 20 mg/m 2 , preferably from 175 mg/m 2 to 20 mg/m 2 , preferably from 150 mg/m 2 to 20 mg/m 2 , preferably from 150 mg/m 2 to 20 mg/m 2 , preferably from 100 mg/m 2 to 20 mg/m 2 , preferably from 75 mg/m 2 to 20 mg/m 2 , preferably from 45 mg/m 2 to 20 mg/m 2 , more preferably from 40 mg/m 2 to 20 mg/m 2 , more preferably from 30 mg/m 2 to 20 mg/m 2 , in particular 30 mg/m 2 , that allow to both suppress the differentiation blockage and at the same time to degrade the fusion protein and thus eradicate the LIC.
  • high RA doses i.e. from 200 mg/m 2 to 20 mg/m 2 , preferably from 175 mg/m 2 to 20 mg/m 2 , preferably from 150 mg
  • the present invention relates to the use of one of the above defined compositions wherein the drug is for administration of a RA dose in the range from 100 mg/m 2 to 46 mg/m 2 , preferably from 90 mg/m 2 to 50 mg/m 2 , more preferably from 80 mg/m 2 to 50 mg/m 2 , more preferably from 70 mg/m 2 to 50 mg/m 2 , more preferably from 60 mg/m 2 to 50 mg/m 2 , in particular 50 mg/m 2 .
  • the present invention relates to the use of one of the above defined compositions wherein the drug is for administration of a RA dose in the range from less than 20 mg/m 2 to 10 mg/m 2 , preferably from 15 mg/m 2 to 10 mg/m 2 , in particular 10 mg/m 2 .
  • RA is usually used at a dosage of about 10 mg/m 2 , but when used alone, RA at this dose is only able to promote the differentiation but not a complete catabolism.
  • examples 10 and 11 show that activation of cAMP signalling by analogs of cAMP or phosphodiesterase inhibitors synergizes with RA to induce degradation.
  • the synergic effect is represented in figure 20 wherein the survival of secondary transplant recipient of primary APL mice treated with cAMP taken alone is about 20 days, and 32 days for the mice treated with RA 10 mg/m 2 taken alone, while it is 68 days for the combination of cAMP and RA.
  • Another advantage of the invention is to provide compositions wherein classic RA doses in the range from less than 20 mg/m 2 to 10 mg/m 2 , preferably from 15 mg/m 2 to 10 mg/m 2 , in particular 10 mg/m 2 are synergized with PDEI and allow to both suppress the differentiation blockage and at the same time to degrade the fusion protein and thus eradicate the LIC.
  • the present invention relates to the use of one of the above defined compositions, wherein said composition comprises retinoic acid (RA) or a related compound thereof such as a retinoid, in combination with at least one phosphodiesterase inhibitor (PDEI) said PDEI is selected from the list consisted of N-(3,5- dichloropyrid-4-yl)-3-cyclopentyloxy-4-methoxybenzamide (INN: piclamilast) or 3- cyclopropylmethoxy-4-difluoromethoxy-N-(3,5-dichloropyrid-4-yl)-benzamide (INN: roflumilast).
  • the composition comprises RA or a retinoid, in combination with piclamilast or roflumilast, without an arsenic derivative.
  • the present invention relates to the use of one of the above defined compositions wherein the drug is for administration of a RA dose in the range from 1 mg/m 2 to less than 10 mg/m 2 , preferably from 1 mg/m 2 to 5 mg/m 2 , in particular 1.5 mg/m 2 .
  • a RA dose in the range from 1 mg/m 2 to less than 10 mg/m 2 , preferably from 1 mg/m 2 to 5 mg/m 2 , in particular 1.5 mg/m 2 .
  • RA is usually used at a dosage of more than 10 mg/m 2 but presents serious side effects over a long term use, such as mucocutaneous toxicity, hypertriglyceridemie and headache (Agnish N. D.; Kochar, D. M., In Retinoid and clinical practice; Korean, G., Ed.;
  • Example 6 shows that a RA dosage reproducing this situation, i.e. a RA dosage of 1.5 mg/m 2 , suppresses the differentiation blockage but not the LIC eradication.
  • Example 9 shows that cyclic AMP or PDEI synergizes with suboptimal RA dosage to induce LIC loss and in opposition to RA taken alone, the combination allows suppressing the differentiation blockage and the LIC eradication. Nevertheless, the synergic effect is only seen for the eradication not for the differentiation confirming the complete uncoupling between differentiation and eradication.
  • 3-cyclopentyloxy-4-methoxybenzamide (INN: piclamilast) taken alone is about 23 days, and 23 days for the mice treated with RA 1.5 mg/m 2 taken alone while it is 39 days for the combination PDEI and RA.
  • the synergy is dramatically raised with a combination AS 2 O3 and RA 1.5 mg/m 2 that results in 165 days of survival.
  • composition of the invention can therefore comprise at least two compounds
  • the present invention relates to the use of one of the above defined compositions wherein the drug is for administration of a RA dose selected from the group consisting of 2 mg/m 2 , 2.5 mg/m 2 , 3 mg/m 2 or 3.5 mg/m 2 .
  • the present invention relates to the use of one of the above defined compositions, wherein RA is selected from the group consisting of all-trans retinoic acid (ATRA) or 9-cis retinoic acid or 13-cis retinoic acid, in particular all-trans retinoic acid.
  • ATRA all-trans retinoic acid
  • 9-cis retinoic acid or 13-cis retinoic acid, in particular all-trans retinoic acid.
  • the present invention relates to the use of one of the above defined compositions, wherein said retinoid is a stable analogue of retinoic acid, in particular a RARA agonist poorly sensitive to CYP26A1 degradation.
  • Retinoid can exhibit different selectivity relative to the RAR ⁇ , RAR RAR ⁇ and ⁇ , for instance an improved RAR ⁇ binding selectivity relative to RAR ⁇ and ⁇ . Distinct RAR subtypes are expressed in different tissues. For example, the most abundant receptor in the skin is RAR ⁇ and studies strongly suggest that the mucocutaneous toxicity caused by retinoids is trough activation of RAR ⁇ (Bioorg. Med. Chem. Lett. 12 (2002), 3145-3148).
  • another advantage of the present invention is to provide a composition having lowered side effects, in particular the mucocutaneous toxicity, while maintaining suppression of the differentiation blockage and at the same time the degradation of the fusion protein and thus eradication of LIC.
  • CYP26A1 is a retinoic acid metabolising enzyme produced by a gene which is normally activated in response to RA in mouse APL. The expression of this gene leads therefore to the degradation of RA.
  • the use of a stable analogue of RA in particular a RARA agonist poorly sensitive to CYP26A1 degradation, i.e. RARA agonists resistant to the degradation or the catabolism caused by CYP26A1, allows treating patients who are resistant to retinoic acid and thus wherein RA treatment to trigger the differentiation is no more possible.
  • the present invention relates to the use of one of the above defined composition, wherein said pathologies such as AML and APL are resistant to conventional leukaemia treatments against leukaemia such as radiotherapy, chemotherapy, or retinoic acid administration. Some patients are resistant to retinoic acid and thus RA treatment taken alone to trigger the differentiation is no more possible.
  • RA is at a high dosage, i.e. from 200 mg/m 2 to 20 mg/m 2 , preferably from 175 mg/m 2 to
  • 20 mg/m 2 preferably from 150 mg/m 2 to 20 mg/m 2 , preferably from 150 mg/m 2 to 20 mg/m 2 , preferably from 150 mg/m 2 to 20 mg/m 2 , preferably from 100 mg/m 2 to 20 mg/m 2 , preferably from 75 mg/m 2 to 20 mg/m 2 , preferably from 45 mg/m 2 to 20 mg/m 2 , preferably from 40 mg/m 2 to 20 mg/m 2 , more preferably from 30 mg/m 2 to 20 mg/m 2 , in particular 30 mg/m 2 , or at a classic RA dosage in the range from less than 20 mg/m 2 to 10 mg/m 2 , preferably from 15 mg/m 2 to 10 mg/m 2 , in particular 10 mg/m 2 , or at a low dosage in the range from 1 mg/m 2 to less than 10 mg/m 2 , preferably from 1 mg/m 2 to 5 mg/m 2 , in particular 1.5 mg/m 2 , able to circumvent the problems of non
  • the present invention relates to the use of the above defined composition comprising retinoic acid (RA) at a RA dose in the range from 1 mg/m 2 to less than 10 mg/m 2 , preferably from 1 mg/m 2 to 5 mg/m 2 , in particular 1.5 mg/m 2 , or at a RA dose, in the range from 200 mg/m 2 to 20 mg/m 2 , preferably from 175 mg/m 2 to 20 mg/m 2 , preferably from 150 mg/m 2 to 20 mg/m 2 , preferably from 150 mg/m 2 to 20 mg/m 2 , preferably from 100 mg/m 2 to 20 mg/m 2 , preferably from 75 mg/m 2 to 20 mg/m 2 , preferably from 45 mg/m 2 to 20 mg/m 2 , preferably from 40 mg/m 2 to 20 mg/m 2 , more preferably from 30 mg/m 2 to 20 mg/m 2 , in particular 30 mg/m 2 , or a related compound thereof such as a
  • the composition comprises a low or high dosage of RA as above defined or a retinoid, in combination with a PDEI or with at least one agent enabling to increase the cellular content of cAMP or derivatives thereof with respect to the originally present cellular content of said cAMP selected from the group comprising cAMP, 8-Cl-cAMP, 8-CPT-cAMP, 8-Br-cAMP, dibutyryl-cAMP or pharmacologically acceptable salts thereof, as above defined and an arsenic derivative or not.
  • the present invention relates to the use of the above defined composition
  • the composition comprises RA at a classic dosage, i.e.
  • the present invention relates to the use of the above defined composition
  • retinoic acid (RA) or a related compound thereof such as a retinoid in combination with an arsenic derivative, or with at least one phosphodiesterase inhibitor (PDEI) and an arsenic derivative, wherein said arsenic derivative is selected from the group consisting of arsenic trioxide (AS 2 O3) or arsenic sulfide (As 4 S 4 ).
  • the composition comprises RA at a high dosage, i.e. from 200 mg/m 2 to 20 mg/m 2 , preferably from 175 mg/m 2 to 20 mg/m 2 , preferably from 150 mg/m 2 to 20 mg/m 2 , preferably from 150 mg/m 2 to 20 mg/m 2 , preferably from 100 mg/m 2 to 20 mg/m 2 , preferably from 75 mg/m 2 to 20 mg/m 2 , preferably from 45 mg/m 2 to 20 mg/m 2 , preferably from 40 mg/m 2 to 20 mg/m 2 , more preferably from 30 mg/m 2 to 20 mg/m 2 , in particular 30 mg/m 2 , or at a classic RA dosage in the range from less than 20 mg/m 2 to 10 mg/m 2 , preferably from 15 mg/m 2 to 10 mg/m 2 , in particular 10 mg/m 2 , or at a low dosage in the range from 1 mg/m 2 to less than 10 mg/m 2 , preferably from
  • the present invention relates to the use of the above defined composition wherein the active substance of the composition consists in retinoic acid (RA) or a related compound thereof such as a retinoid, in combination with at least one phosphodiesterase inhibitor (PDEI) selected from the group consisting of methylxanthines such as caffeine, theophylline, aminophylline, isobutyl-methylxanthine, rolipram, sildenafil, vardenaf ⁇ l, zaprinast, methoxyquinazoline, N-(3,5-dichloropyrid-4-yl)- 3-cyclopentyloxy-4-methoxybenzamide (INN: piclamilast) or 3-cyclopropylmethoxy-4- difluoromethoxy-N-(3,5-dichloropyrid-4-yl)-benzamide (INN: roflumilast) or with at least one agent enabling to increase the cellular content of RA or retinoic
  • the composition comprises RA at a high dosage, i.e. at a high dosage, i.e. from 200 mg/m 2 to 20 mg/m 2 , preferably from 175 mg/m 2 to 20 mg/m 2 , preferably from 150 mg/m 2 to 20 mg/m 2 , preferably from 150 mg/m 2 to 20 mg/m 2 , preferably from 100 mg/m 2 to 20 mg/m 2 , preferably from 75 mg/m 2 to 20 mg/m 2 , preferably from 45 mg/m 2 to 20 mg/m 2 , preferably from 40 mg/m 2 to 20 mg/m 2 , more preferably from 30 mg/m 2 to 20 mg/m 2 , in particular 30 mg/m 2 , or at a low dosage in the range from 1 mg/m 2 to less than 10 mg/m 2 , preferably from 1 mg/m 2 to 5 mg/m 2 , in particular 1.5 mg/m 2 , or a retinoid whatever the dosage, as above defined, in combination with any P
  • the present invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising as active substance, retinoic acid (RA) or a related compound thereof such as a retinoid, in combination with at least one phosphodiesterase inhibitor (PDEI) or with an arsenic derivative, or with at least one phosphodiesterase inhibitor (PDEI) or at least one agent enabling to increase the cellular content of cAMP or derivatives thereof with respect to the originally present cellular content of said cAMP and an arsenic derivative, as defined above, in a pharmacologically acceptable vehicle.
  • the pharmaceutical composition comprises RA at a high dosage, i.e.
  • the pharmaceutical composition comprises RA at a high dosage, i.e. from 200 mg/m 2 to 20 mg/m 2 , preferably from 175 mg/m 2 to 20 mg/m 2 , preferably from 150 mg/m 2 to 20 mg/m 2 , preferably from 150 mg/m 2 to 20 mg/m 2 , preferably from 100 mg/m 2 to 20 mg/m 2 , preferably from 75 mg/m 2 to 20 mg/m 2 , preferably from 45 mg/m 2 to 20 mg/m 2 , preferably from 40 mg/m 2 to 20 mg/m 2 , more preferably from 30 mg/m to 20 mg/m , in particular 30 mg/m , or at a classic RA dosage in the range from less than 20 mg/m to 10 mg/m , preferably from 15 mg/m 2 to 10 mg/m 2 , in particular 10 mg/m 2 , or at a low RA dosage in the range from 1 mg/m 2 to less than 10 mg/m 2 , preferably from 1 mg/m 2 to less
  • the dosage of RA in the above defined compositions is in the range from 100 mg/m 2 to 46 mg/m 2 , preferably from 90 mg/m 2 to 50 mg/m 2 , more preferably from 80 mg/m 2 to 50 mg/m 2 , more preferably from 70 mg/m 2 to 50 mg/m 2 , more preferably from 60 mg/m 2 to 50 mg/m 2 , in particular 50 mg/m 2 .
  • the present invention relates to the composition above defined, comprising as active substance, retinoic acid (RA) or a related compound thereof such as a retinoid, in combination with at least one phosphodiesterase inhibitor (PDEI), and possibly an arsenic derivative, in a pharmacologically acceptable vehicle.
  • RA retinoic acid
  • PDEI phosphodiesterase inhibitor
  • the pharmaceutical composition comprises RA at a 200 mg/m 2 to 20 mg/m 2 , preferably from 175 mg/m 2 to 20 mg/m 2 , preferably from 150 mg/m 2 to 20 mg/m 2 , preferably from 150 mg/m 2 to 20 mg/m 2 , preferably from 100 mg/m 2 to 20 mg/m 2 , preferably from 75 mg/m 2 to 20 mg/m 2 , preferably dosage, i.e.
  • the present invention relates to the composition above defined, comprising as active substance, retinoic acid (RA) or a related compound thereof such as a retinoid, in combination with an arsenic derivative, in a pharmacologically acceptable vehicle.
  • RA retinoic acid
  • an arsenic derivative in a pharmacologically acceptable vehicle.
  • the pharmaceutical composition comprises RA at a high dosage, i.e. from 200 mg/m 2 to 20 mg/m 2 , preferably from 175 mg/m 2 to 20 mg/m 2 , preferably from 150 mg/m 2 to 20 mg/m 2 , preferably from 150 mg/m 2 to 20 mg/m 2 , preferably from 100 mg/m 2 to 20 mg/m 2 , preferably from 75 mg/m 2 to 20 mg/m 2 , preferably from 45 mg/m 2 to 20 mg/m 2 , preferably from 40 mg/m 2 to 20 mg/m 2 , more preferably from 30 mg/m 2 to 20 mg/m 2 , in particular 30 mg/m 2 , or
  • the present invention relates to the pharmaceutical composition above defined, comprising as active substance, RA or a related compound thereof such as a retinoid, in combination with at least one phosphodiesterase inhibitor (PDEI) selected from the list consisted of N-(3,5-dichloropyrid-4-yl)-3-cyclopentyloxy-4- methoxybenzamide (INN: piclamilast) or 3-cyclopropylmethoxy-4-difluoromethoxy-N- (3,5-dichloropyrid-4-yl)-benzamide (INN: roflumilast).
  • PDEI phosphodiesterase inhibitor
  • the pharmaceutical composition comprises RA at a classic dosage in the range from less than 20 mg/m 2 to 10 mg/m 2 , preferably from 15 mg/m 2 to 10 mg/m 2 , in particular 10 mg/m 2 , or a retinoid, different from RA, whatever the dosage as above defined, from 1 mg/m 2 to less than 200 mg/m 2 , and piclamilast or roflumilast, without arsenic derivative.
  • the present invention relates to the composition above defined, wherein the RA dose is in the range comprised from 45 mg/m 2 to 20 mg/m 2 , preferably from 40 mg/m 2 to 20 mg/m 2 , more preferably from 30 mg/m 2 to 20 mg/m 2 , in particular 30 mg/m 2 and possibly an arsenic derivative in combination with at least one
  • the present invention relates to the composition above defined, wherein the RA dose is in the range comprised from less than 20 mg/m 2 to 10 mg/m 2 , in particular 10 mg/m 2 and possibly an arsenic derivative in combination with at least one PDEI, in a pharmacologically acceptable vehicle.
  • the present invention relates to the composition above defined, wherein the RA dose is in the range comprised from 1 mg/m 2 to less than 10 mg/m 2 , preferably 1 to 5 mg/m 2 , in particular 1.5 mg/m 2 and possibly an arsenic derivative in combination with at least one PDEI, in a pharmacologically acceptable vehicle.
  • the pharmaceutical compositions comprises a low dosage of RA or of ATRA or cis derivatives of ATRA, or a retinoid as above defined, in combination with any PDEI above defined and possibly an arsenic derivative.
  • the pharmaceutical compositions above defined are in a form appropriate for the administration of RA at a dose selected from the group consisting of 2 mg/m 2 , 2.5 mg/m 2 , 3 mg/m 2 or 3.5 mg/m 2 .
  • the retinoic acid of one of the above defined pharmaceutical compositions is selected from the group consisting of all-trans retinoic acid (ATRA) or 9-cis retinoic acid or 13-cis retinoic acid, in particular all-trans retinoic acid.
  • ATRA all-trans retinoic acid
  • 9-cis retinoic acid or 13-cis retinoic acid, in particular all-trans retinoic acid.
  • the pharmaceutical compositions comprise ATRA or 9- cis retinoic acid or 13-cis retinoic acid at a 200 mg/m 2 to 20 mg/m 2 , preferably from 175 mg/m 2 to 20 mg/m 2 , preferably from 150 mg/m 2 to 20 mg/m 2 , preferably from 150 mg/m 2 to 20 mg/m 2 , preferably from 100 mg/m 2 to 20 mg/m 2 , preferably from 75 mg/m 2 to 20 mg/m 2 , preferably dosage, i.e. from 45 mg/m 2 to 20 mg/m 2 , preferably from 40 mg/m 2 to 20 mg/m 2 , more preferably from 30 mg/m 2 to 20 mg/m 2 , in particular 30 mg/m 2 , or
  • the present invention relates to the composition above defined, wherein said retinoid is a stable analogue of retinoic acid, in particular a RARA agonist poorly sensitive to CYP26A1 degradation.
  • the pharmaceutical composition comprises an stable analogue of retinoic acid, in particular a RARA agonist poorly sensitive to CYP26A1 degradation, in combination with any PDEI above defined and possibly an arsenic derivative.
  • the present invention relates to the composition above defined, wherein the active substance of the composition consists in said RARA agonist poorly sensitive to CYP26A1 degradation, N-(3,5-dichloropyrid-4-yl)-3-cyclopentyloxy-4- methoxybenzamide (INN: piclamilast) and an arsenic derivative such arsenic trioxide
  • the pharmaceutical compositions above defined are in a form appropriate for the administration of RA at a low dose in the range comprised from 1 mg/m 2 to less than 10 mg/m 2 , preferably 1 to 5 mg/m 2 , in particular 1.5 mg/m 2 , or at a high dose in the range from 200 mg/m 2 to 20 mg/m 2 , preferably from 175 mg/m 2 to 20 mg/m 2 , preferably from 150 mg/m 2 to 20 mg/m 2 , preferably from 150 mg/m 2 to 20 mg/m 2 , preferably from 150 mg/m 2 to 20 mg/m 2 , preferably from 100 mg/m 2 to 20 mg/m 2 , preferably 75 mg/m 2 to 20 mg/m 2 , preferably 45 mg/m 2 to 20 mg/m 2 , preferably from 40 mg/m 2 to 20 mg/m 2 , more preferably from 30 mg/m 2 to 20 mg/m 2 , in particular 30 mg/m 2 , or a retinoid in combination with at least one P
  • the pharmaceutical compositions comprise RA at a low dosage in the range from 1 mg/m 2 to less than 10 mg/m 2 , preferably from 1 mg/m 2 to 5 mg/m 2 , in particular 1.5 mg/m 2 , or at a high dosage in the range from 200 mg/m 2 to 20 mg/m 2 , preferably from 175 mg/m 2 to 20 mg/m 2 , preferably from 150 mg/m 2 to 20 mg/m 2 , preferably from 150 mg/m 2 to 20 mg/m 2 , preferably from 100 mg/m 2 to 20 mg/m 2 , preferably from 75 mg/m 2 to 20 mg/m 2 , preferably from 45 mg/m 2 to 20 mg/m 2 , preferably from 40 mg/m 2 to 20 mg/m 2 , more preferably from 30 mg/m 2 to 20 mg/m 2 , in particular 30 mg/m 2 , or a retinoid whatever the dosage, and a PDEI above defined and possibly an arsenic derivative.
  • the pharmaceutical composition above defined comprises as active substance, RA or a related compound thereof such as a retinoid, in combination with at least one PDEI selected from the group consisting of N-(3,5- dichloropyrid-4-yl)-3-cyclopentyloxy-4-methoxybenzamide (INN: piclamilast) or 3- cyclopropylmethoxy-4-difluoromethoxy-N-(3,5-dichloropyrid-4-yl)-benzamide (INN: roflumilast) or with at least one agent enabling to increase the cellular content of cAMP or derivatives thereof with respect to the originally present cellular content of said cAMP selected from the group comprising cAMP, 8-Cl-cAMP, 8-CPT-cAMP, 8-Br-cAMP, dibutyryl-cAMP or pharmacologically acceptable salts thereof and possibly an arsenic derivative as defined above.
  • PDEI selected from the group consisting of N-(3,5-
  • the pharmaceutical composition comprises RA or ATRA or cis derivatives of ATRA at a high dosage, i.e. from 200 mg/m 2 to 20 mg/m 2 , preferably from 175 mg/m 2 to 20 mg/m 2 , preferably from 150 mg/m 2 to 20 mg/m 2 , preferably from 150 mg/m 2 to 20 mg/m 2 , preferably from 100 mg/m 2 to 20 mg/m 2 , preferably from 75 mg/m 2 to 20 mg/m 2 , preferably from 45 mg/m 2 to 20 mg/m 2 , preferably from 40 mg/m 2 to 20 mg/m 2 , more preferably from 30 mg/m 2 to 20 mg/m 2 , in particular 30 mg/m 2 , or
  • RA or ATRA or cis derivatives of ATRA at a low dosage in the range from 1 mg/m 2 to less than 10 mg/m 2 , preferably from 1 mg/m 2 to 5 mg/m 2 , in particular 1.5 mg/m 2 , or a retinoid whatever the dosage, in combination with N-(3,5-dichloropyrid-4-yl)-3-cyclopentyloxy-4- methoxybenzamide (INN: piclamilast) or 3-cyclopropylmethoxy-4-difluoromethoxy-N- (3,5-dichloropyrid-4-yl)-benzamide (INN: roflumilast).
  • the arsenic derivative of the pharmaceutical compositions above defined is selected from the group consisting of arsenic trioxide (AS 2 O3) or arsenic sulfide (AS 4 S 4 ).
  • the pharmaceutical compositions above defined are in a form appropriate for the administration of about 10 mg/m 2 to 45 mg/m 2 of RA, and of about 0.014 mg/kg/day to about 0.43 mg/kg/day of arsenic trioxide, and N-(3,5-dichloropyrid-4- yl)-3-cyclopentyloxy-4-methoxybenzamide (INN: piclamilast) or 3-cyclopropylmethoxy-4- difiuoromethoxy-N-(3 ,5-dichloropyrid-4-yl)-benzamide (INN: roflumilast).
  • the pharmaceutical composition comprises RA at a high dosage, i.e. from 200 mg/m 2 to 20 mg/m 2 , preferably from 175 mg/m 2 to 20 mg/m 2 , preferably from 150 mg/m 2 to 20 mg/m 2 , preferably from 150 mg/m 2 to 20 mg/m 2 , preferably from 100 mg/m 2 to 20 mg/m 2 , preferably from 75 mg/m 2 to 20 mg/m 2 , preferably from about 20 mg/m 2 to about 45 mg/m 2 , preferably from about 20 mg/m 2 to about 40 mg/m 2 , more preferably from about 20 mg/m 2 to about 30 mg/m 2 , in particular 30 mg/m 2 , or RA at a classic dosage, i.e.
  • the arsenic trioxide dose of the pharmaceutical compositions above defined is comprised from about 0.014 mg/kg/day to about 0.43 mg/kg/day, preferably from about 0.014 mg/kg/day to about 0.25 mg/kg/day, preferably from about 0.014 mg/kg/day to about 0.14 mg/kg/day, more preferably from about 0.05 mg/kg/day to about 0.14 mg/kg/day, in particular about 0.014 mg/kg/day.
  • the pharmaceutical composition above defined is in a form appropriate for the administration of about 1 mg/m 2 to less than 10 mg/m 2 of RA, and of about 0.014 mg/kg/day to about 0.43 mg/kg/day of arsenic trioxide, and N-(3,5- dichloropyrid-4-yl)-3-cyclopentyloxy-4-methoxybenzamide (INN: piclamilast) or 3- cyclopropylmethoxy-4-difluoromethoxy-N-(3,5-dichloropyrid-4-yl)-benzamide (INN: roflumilast).
  • the pharmaceutical composition comprises a low dosage of RA dosage in the range from 1 mg/m 2 to less than 10 mg/m 2 , preferably from 1 mg/m 2 to 5 mg/m 2 , in particular 1.5 mg/m 2 , or a retinoid, in combination with N-(3,5-dichloropyrid- 4-yl)-3-cyclopentyloxy-4-methoxybenzamide (INN: piclamilast) or 3-cyclopropylmethoxy- 4-difluoromethoxy-N-(3,5-dichloropyrid-4-yl)-benzamide (INN: roflumilast) and arsenic trioxide.
  • RA dosage in the range from 1 mg/m 2 to less than 10 mg/m 2 , preferably from 1 mg/m 2 to 5 mg/m 2 , in particular 1.5 mg/m 2 , or a retinoid, in combination with N-(3,5-dichloropyrid- 4-yl)-3-cyclopenty
  • the pharmaceutical composition above defined comprises retinoic acid (RA) or a related compound thereof such as a retinoid, in combination with at least one phosphodiesterase inhibitor (PDEI) selected from the group consisting of methylxanthines such as caffeine, theophylline, aminophylline, isobutyl- methylxanthine, rolipram, sildenafil, vardenafil, zaprinast, methoxyquinazoline, N-(3,5- dichloropyrid-4-yl)-3-cyclopentyloxy-4-methoxybenzamide (INN: piclamilast) or 3- cyclopropylmethoxy-4-difluoromethoxy-N-(3,5-dichloropyrid-4-yl)-benzamide (INN: roflumilast).
  • PDEI phosphodiesterase inhibitor
  • the pharmaceutical composition comprises RA at a high dosage, i.e. from 200 mg/m 2 to 20 mg/m 2 , preferably from 175 mg/m 2 to 20 mg/m 2 , preferably from 150 mg/m 2 to 20 mg/m 2 , preferably from 150 mg/m 2 to 20 mg/m 2 , preferably from 100 mg/m 2 to 20 mg/m 2 , preferably from 75 mg/m 2 to 20 mg/m 2 , preferably from 45 mg/m 2 to 20 mg/m 2 , preferably from 40 mg/m 2 to 20 mg/m 2 , more preferably from 30 mg/m 2 to 20 mg/m 2 , in particular 30 mg/m 2 , or
  • RA at a low dosage in the range from 1 mg/m 2 to less than 10 mg/m 2 , preferably from 1 mg/m 2 to 5 mg/m 2 , in particular 1.5 mg/m 2 , or a retinoid whatever the dosage as above defined, in combination with a PDEI above defined.
  • the present invention relates to a product containing: - a first pharmaceutical composition comprising as active substance: * RA at a dosage in the range from 1 mg/m 2 to less than 10 mg/m 2 , preferably from
  • PDEI a least one PDEI selected from the group consisting of methylxanthines such as caffeine or theophylline, aminophylline, isobutyl-methylxanthine, rolipram, sildenafil, vardenafil, zaprinast, methoxyquinazoline, N-(3,5-dichloropyrid-4-yl)-3-cyclopentyloxy-4- methoxybenzamide (INN: piclamilast) or 3-cyclopropylmethoxy-4-difluoromethoxy-N- (3,5-dichloropyrid-4-yl)-benzamide (INN: roflumilast) or with at least one agent enabling to increase the cellular content of cAMP or derivatives thereof with respect to the originally present cellular content of said cAMP selected from the group comprising cAMP, 8-C1- cAMP, 8-CPT-cAMP, 8-Br-cAMP, dibutyryl-c
  • RA retinoic acid
  • PDEI phosphodiesterase inhibitor
  • methylxanthines such as caffeine, theophylline, aminophylline, isobutyl-methylxanthme, rolipram, sildenafil, vardenaf ⁇ l, zaprinast, methoxyquinazoline, N-(3,5-dichloropyrid-4-yl)- 3-cyclopentyloxy-4-methoxybenzamide (INN: piclamilast) or 3-cyclopropylmethoxy-4- difiuoromethoxy-N-(3 ,5-dichloropyrid-4-yl)-benzamide (INN: roflumilast), as a combined preparation for simultaneous, separate or sequential use in cancer therapy, in particular in APL , AML or lymphoid leukemia.
  • PDEI phosphodiesterase inhibitor
  • the first pharmaceutical composition comprises RA at low dosage in association with an PDEI and an arsenic derivative.
  • the second pharmaceutical composition comprises RA at low dosage in association with piclamilaste or roflumilaste, without an arsenic derivative.
  • the first pharmaceutical composition is intended for an induction treatment or therapy and the second pharmaceutical composition is intended for a maintenance treatment or therapy.
  • the induction treatment can be carried out during three to five months, and the maintenance treatment can be carried out during one to two years. Maintenance treatment follows immediately after the end of the induction treatment.
  • the PDEI of said first pharmaceutical is selected from the list consisted of piclamilaste or roflumilaste.
  • the present invention relates to a product containing: - a first pharmaceutical composition comprising as active substance:
  • *retinoic acid (RA) at a high dosage i.e. from 200 mg/m 2 to 20 mg/m 2 , preferably from 175 mg/m 2 to 20 mg/m 2 , preferably from 150 mg/m 2 to 20 mg/m 2 , preferably from 150 mg/m 2 to 20 mg/m 2 , preferably from 100 mg/m 2 to 20 mg/m 2 , preferably from 75 mg/m 2 to 20 mg/m 2 , preferably from 45 mg/m 2 to
  • PDEI phosphodiesterase inhibitor
  • methylxanthines such as caffeine, theophylline, aminophylline, isobutyl-methylxanthine, rolipram, sildenafil, vardenaf ⁇ l, zaprinast, methoxyquinazoline, N-(3,5-dichloropyrid-4-yl)-3-cyclopentyloxy-4- methoxybenzamide (INN: piclamilast) or 3-cyclopropylmethoxy-4- difluoromethoxy-N-(3,5-dichloropyrid-4-yl)-benzamide (INN: roflumilast), and,
  • PDEI phosphodiesterase inhibitor
  • the first pharmaceutical composition comprises RA at a high dosage in association with piclamilaste or roflumilaste, and an arsenic derivative.
  • the second pharmaceutical composition comprises RA at low dosage in association with piclamilaste or roflumilaste, without an arsenic derivative.
  • the first pharmaceutical composition is intended for an induction treatment or therapy and the second pharmaceutical composition is intended for a maintenance treatment or therapy.
  • the induction treatment can be carried out during three to five months, and the maintenance treatment can be carried out during one to two years. Maintenance treatment follows immediately after the end of the induction treatment.
  • the invention relates to a process of in vitro screening of molecules to test having the capacity to eradicate the leukaemia initiating cells comprising the step of contacting leukaemia initiating cells with a molecule to test in combination with a dose of retinoic acid in the range from 1 mg/m 2 to less than 10 mg/m 2 , preferably from 1 mg/m 2 to 5 mg/m 2 , in particular 1.5 mg/m 2 , and an PDEI.
  • the process above defined comprises the following steps:
  • Culture medium are numerous and commercially available.
  • methyl cellulose but without being limited to, can be used and is available from Stem Cell Technologies, Vancouver.
  • the mammal can be a mouse, a rat ...
  • small colonies refers to small size colonies constituted in small cells.
  • step 2 The absence of small colonies obtained in step 2. means that LIC have been eradicated.
  • the invention relates to a process of in vitro screening of molecules to test having the capacity to eradicate the leukaemia initiating cells comprising the following steps : 1. culturing, in a culture plate containing a culture medium, previously isolated bone marrow transformed primary haematopoietic progenitor cells of a mammal, in particular expressing PML/RARA,
  • step 3 selecting the molecules of step 2. giving rise to said phosphorylation.
  • step 2 The presence of the phosphorylation in step 2. means that PML//RARA has been degraded leading thus to the eradication of LIC. Therefore, a molecule to test will be selected if there is phosphorylation in step 2. and further studied in particular by in vivo screening.
  • the present invention relates to a process of in vivo screening of molecules to test having the capacity to eradicate the leukaemia initiating cells comprising the step of injecting to a second mammal recipient cells isolated from bone marrow of a first mammal recipient treated with a molecule to test in combination with RA at a low dosage in the range from 1 mg/m 2 to less than 10 mg/m 2 , preferably from 1 mg/m 2 to 5 mg/m 2 , in particular 1.5 mg/m 2 ,and AS 2 O3.
  • the process of in vivo screening above defined comprising the following steps: 1. treating a first mammal recipient with a dose of RA of 1.5 mg/m 2 in association with a PDEI and AS 2 O 3 for a week,
  • step 7 8. measuring the time to death in said other second mammal recipient of step 7, 9. selecting the molecule for which the time to death of step 7 is statistically higher than the time to death of step 4.
  • mimal recipient is meant a mouse, a rat ...
  • a control can be obtained by making the same four first steps on another mammal recipient without treatment with RA of 1.5 mg/m 2 in association with a PDEI and As 2 Ch for a week.
  • Each first mammal recipient and second mammal recipient must be different (control, treated with RA of 1.5 mg/m 2 in association with a PDEI and AS 2 O3 for a week, treated with RA of 1.5 mg/m 2 in association with a molecule to test and AS 2 O 3 for a week)
  • the time to death obtained in step 4 when treated with RA of 1.5 mg/m 2 in association with a PDEI and AS 2 O3 for a week is statistically higher than the time to death obtained in control, because the leukaemia stem cells of the control have not been eradicated.
  • an interesting molecule to select in step 9 is a molecule with statistically higher time to death than the time to death of step 4.
  • Figures IA and IB represent the model for APL pathogenesis (Grignani; Cell 1993,
  • Figures 2 to 4 show that the RA trigger APL cell differentiation and loss of clonogenic precursors are uncoupled:
  • Figure 2A, Figure 2B, Figure 2C, Figure 2D and Figure 2E represent the colonies obtained with or without treatment by RA.
  • Figure 2A represents the ex vivo PML/RARA-transformed primary haematopoietic progenitors grown in methyl-cellulose (MC). Briefly, mice are treated with 5-Fluoro-Uracyl and after 5 days, the bone marrow is isolated and treated by method well known by the man skilled in the art to remove lineage + (lin +), for example Gr-I + , TERl 19 + , CD-I Ib + and isolate lin - which are transformed by PML/RARA and grown on methyl cellulose (MC) medium to give colonies (mean of 5 independent experiments)
  • Figures 2B to 2D represent the exposition of colonies obtained in figure 2A to 10 "6 M RA for a week (figure B) and regrown: large colonies in MC in the absence of RA for another week (figure 2C), small colonies and isolated cells in MC in the absence of RA for another week
  • Figures 2E and 2F represent the schematic indication of the nature of the myeloid cells in RA-treated (E) or untreated colonies (F). Despite RA-triggered terminal differentiation, a significant number of clonogenic cells are recovered.
  • Figure 3A, Figure 3B, Figure 3C, Figure 3D, Figure 3E, Figure 3F, Figure 3G, and Figure 3H represent the comparative FACS analysis with two myeloid differentiation markers and May Grunwald Giemsia stain of the recovered cells of the figures 2A to F.
  • Figures 3 A and B represent the FACS and May Grunwald Giemsia stain of the non treated cells (see figure 2A).
  • Figures 3C and D represent the FACS and May Grunwald Giemsia stain of the treated cells (see figure 2B).
  • Figures 3 E and F represent the FACS and May Grunwald Giemsia stain of the non treated cells after regrowing of the large colonies (see figure 2C).
  • Figures 3 G and H represent the FACS and May Grunwald Giemsia stain of the non treated cells after regrowing of the small colonies (see figure 2D).
  • RA treated cells lead to large colonies and small colonies which are regrowing without RA. Large colonies lead to a composition of differentiated and undifferentiated cells (Fig.3 E and 3F) and small colonies lead to undifferentiated cells only (Fig. 3 G and 3H) indicating that significant clonogenic cells are recovered.
  • Figure 4A, Figure 4B, Figure 4C, Figure 4D, Figure 4E, Figure 4F, Figure 4G, Figure 4H, and Figure 41 represent the morphologically bone marrow from RA-treated (RA 10 mg/m (RA 10) for a week) APL mouse containing a large number of remaining leukaemia initiating cells (LIC).
  • RA-treated RA 10 mg/m (RA 10) for a week
  • LIC leukaemia initiating cells
  • Figure 4A represents the spleen weight of untreated or RA-treated APL mice for a week with dose RA pellets (RAlO for a week) at D7 (left histogram: control APL, middle histogram RA 10, right histogram: normal mice).
  • Figures 4B and C represent the bone marrow May Grunwald Giemsa (MGG) stains (B: control, C: RA 10 treatment for a week).
  • Figures 4D and E represent FACS analysis with two myeloid differentiation markers, CD-I Ib (Mac) and GR-I (D: control, E: RA 10 treatment for a week).
  • Figure 4F represents the abundance of the PML/RARA gene determined by quantitative PCR (left histogram: control, right histogram: RA).
  • Figure 4G represents the peripheral white blood cells for the control, treated mice (RA 10 for a week) and normal mice (left histogram: control, middle histogram RA 10, right histogram: normal mice).
  • Figure 4H represents the treatment protocol of mice.
  • Figure 41 represents the mean time to death in 6 independent experiments obtained after secondary transplant 7 days post treatment (upper histogram: RA, lower histogram: control).
  • Figures 5 to 8 show further dissociation of differentiation and loss of clonogenic potential.
  • Figure 5A, Figure 5B, Figure 5C, Figure 5D, Figure 5E, and Figure 5F represent the MGG-stained bone marrows of mice treated with classic (RAlO mg/m 2 ) or lower (RAl .5 mg/m 2 ) retinoic acid concentrations for a length of time of D3 or D7.
  • Figure 5A and D represent the control at D3 and D7 respectively.
  • Figure 5B and E represent the RA 1.5 treatment at D3 and D7 respectively.
  • Figure 5 C and F represent the RA 10 treatment at D3 and D7 respectively.
  • Figure 5G, Figure 5H, Figure 51, Figure 5J, Figure 5K, and Figure 5L represent the FACS analysis of the corresponding bone marrows.
  • Figure 5 G and J represent the control at D3 and D7 respectively.
  • Figure 5H and K represent the RA 1.5 treatment at D3 and D7 respectively.
  • Figure 51 and L represent the RA 10 treatment at D3 and D7 respectively.
  • Figure 6A, Figure 6B, and Figure 6C present the results obtained with the different treatments with RA.
  • Figure 6A represent the spleen weight of the mice (light grey D3 and dark grey D7; left histogram: control; middle histogram: RAl.5 mg/m 2 (RA 1.5), right histogram: RA 10)
  • Figure 6B represents PML/RARA content at day 7 (left histogram: control; middle histogram: RA 1.5, right histogram: RA 10).
  • Figure 6C represents LIC abundance, as assessed by secondary transplantation showing the time to death in secondary transplant 7 days post treatment (upper histogram: RA 10; middle histogram: RA 1.5, lower histogram: control).
  • FIG. 7 A, Figure 7B, Figure 7C, Figure 7D, and Figure 7E show that the PLZF/RARA murine APLs, another fusion protein similar to PML/RARA, terminally differentiate in response to RA, without any restoration of haematopoiesis or significant LIC loss.
  • Treatments with dose RA pellets (RAlO) were for 7 or 12 days.
  • Figures 7A and B represent the bone marrow May Grunwald Giemsa (MGG) stains showing complete terminal granulocytic differentiation at D 12 (A: control, B: RA).
  • Figures 7C and D represent the FACS analysis at D12 (A: control, B: RA).
  • Figure 7E represents the peripheral white blood cells at D7 (left histogram: control, right histogram: RA 10).
  • Figure 8A, Figure 8B, Figure 8C represent present the results obtained with the treatment with RA at D7.
  • Figure 8A represents the spleen weight of untreated or RA-treated PZF/RARA APL mouse (left histogram: control APL, middle histogram RA 10, right histogram: normal mice).
  • Figure 8B represents abundance of the PLZF/RARA gene determined by quantitative PCR (left histogram: control, right histogram: RA 10).
  • Figure 8C represents mean time to death of syngenic animals injected with 10 7 bone marrow cells from RA-treated of untreated leukaemic animals (left histogram: RA 10, right histogram: control).
  • Leukaemia initiating cells are as abundant in the differentiated marrow as in the blastic one.
  • Figure 9 to 12 show that LIC eradication by the RA/arsenic association is dependent on active proteolysis.
  • Figure 9A, Figure 9B, Figure 9C, Figure 9D, Figure 9E, Figure 9F, Figure 9G, and Figure 9H show that the RA 10 mg/m 2 /arsenic association allows differentiation and synergizes for LIC eradication.
  • Figure 9A, Figure 9B, Figure 9C, Figure 9D represents MGG stains of RAlO- or arsenic-treated mice for 3 days (A: control , B: RA, C: AS 2 O3, D: Ra + AS 2 O3).
  • Figure 9E, Figure 9F, Figure 9G, Figure 9H represents the FACS of RAlO- or arsenic-treated mice for 3 days (A: control , B: RA, C: AS2O3, D: Ra + AS2O3).
  • Figure 1OA represents spleen weights at day 7 with the different treatments (from left to right histograms: control, RA, AS2O3, Ra + AS2O3).
  • Figure 1 OB represents the luciferase imaging of APL mouse treated for 3 days (from left to right images: control, RA, AS 2 O3, Ra + AS 2 O3).
  • Figure 1OC represents luciferase activity (arbitrary unit) of secondary transplants from the mice imaged in figure 1OB.
  • Figure 1OD represents survival of secondary transplants after 7 days treatment of the primary mice with RA, arsenic or the combination (mean of 3 independent experiments; from upper to lower histograms: Ra + AS 2 O3, AS 2 O3, RA, control).
  • Figure 1 IG, Figure 1 IH, Figure 1 II, Figure 1 IJ, Figure 1 IK, Figure 1 IL show that the RA/arsenic synergy is dependent on active proteolysis and represent the RA/arsenic association evaluation as above, in the presence or absence of Velcade ® , a clinically used proteasome inhibitor.
  • Figure HA, Figure HB, Figure HC, Figure HD, Figure HE, Figure HF represent
  • MGG stains after 6 days treatment A: control, B: RAlO, C: As 2 O 3 , D: Velcade ® , E: Ra + As 2 O 3 , F: Ra + As 2 O 3 + Velcade ® ).
  • Figure HG, Figure 11H, Figure 111, Figure I U, Figure HK, and Figure 1 IL represent FACS of the bone marrows after 6 days treatment (G: control, H: RAlO, I: As 2 O 3 , J: Velcade ® , K: Ra + As 2 O 3 , L: Ra + As 2 O 3 + Velcade ® ).
  • Figure 12A represents the PML/RARA DNA in the marrows after 3 days treatment of the primary mice (from left to right histograms: control, RA + AS 2 O3, Velcade ® , Ra + As 2 O 3 + Velcade ® ).
  • Figure 12B represents the time to death in secondary transplant 3 days post treatment (from upper to lower histograms: RA + As 2 O 3 + Velcade ® , Velcade ® , RA + As 2 O 3 , control).
  • Velcade ® induces significant differentiation on its own, it hampers leukaemia eradication, restoration of normal haematopoiesis and LIC loss.
  • Figure 13 to 16 show that activation of cAMP signalling dramatically synergizes with low-dose RA to induce LIC loss.
  • Figure 13A represents the spleen weights (A) at day 7 of mice treated with normal or low doses RA, in the presence or absence of cAMP (from left to right histograms: control, RA 10, RA 1.5, As 2 O 3 , cAMP, RA 1.5 + cAMP).
  • Figure 13B represents the light units (B) of mice treated with normal or low doses
  • RA in the presence or absence of cAMP at day -1 (white histogram), 3 (light grey histogram, 6 (dark grey histogram) (from left to right histograms: control, RA 1.5, RA 10, As 2 O 3 , cAMP, RA 1.5 + cAMP).
  • Figure 14A, Figure 14B represent the MGG stains of bone marrows of the treated animals with RA 10 at D3 (A) and D7 (B).
  • Figure 14C, Figure 14D represent the MGG stains of bone marrows of the treated animals with RA 1.5 at D3 (C) and D7 (D).
  • Figure 14E, Figure 14F represent the MGG stains of bone marrows of the treated animals with cAMP at D3 (E) and D7 (F).
  • Figure 14G, Figure 14H represent the MGG stains of bone marrows of the treated animals with RA 1.5 and cAMP at D3 (G) and D7 (H).
  • Figure 15A, Figure 15B, Figure 15C, Figure 15D represent the FACS analysis at day 3 post treatment (A: control, B: RA 1.5, C: RA 10, D: RA1.5 + cAMP). These figures demonstrate a similar induction of differentiation for RAl .5 in the presence or absence of cAMP.
  • Figure 16A represents the survival of secondary transplant recipients of primary APL mice treated at day 7 (from upper to lower histograms: RA 1.5 + As 2 O 3 + Piclamilast, As 2 O 3 + Piclamilast, RA 1.5 + Piclamilast, Piclamilast, RA 1.5 + As 2 O 3 , As 2 O 3 , RA 1.5, control).
  • Figure 16B represents the survival of secondary transplant recipients of primary APL mice treated at day 3 (from upper to lower histograms: RA 1.5 + AS 2 O3 + Piclamilast, As 2 O 3 + Piclamilast, RA 1.5 + Piclamilast, Piclamilast, RA 1.5 + As 2 O 3 , As 2 O 3 , RA 1.5, control).
  • Figure 17 to 20 show that mutation of S873 in PML/RARA yields RA-resistant transgenic-derived APLs.
  • Figure 17 represents the spleen weights at day 7 of mice treated with RA or As 2 O 3 , in the presence or absence of cAMP (from left to right histograms: control, cAMP, RA 10, RA 10 + cAMP, As 2 O 3 , As 2 O 3 + cAMP, RA 10 + As 2 O 3 ).
  • Figure 18A, Figure 18B, Figure 18C, Figure 18D, Figure 18E, Figure 18F represent MGG stains after 6 days treatment (A: control, B: cAMP, C: RAlO, D: RA 10 + cAMP, E: As 2 O 3 , F: As 2 O 3 + cAMP, G: Ra + As 2 O 3 ).
  • Figure 19A, Figure 19B, Figure 19C, Figure 19D, Figure 19E and Figure 19F represent the FACS analysis at day 3 post treatment (A: control, B: cAMP, C: RAlO, D: RA 10 + cAMP, E: As 2 O 3 , F: As 2 O 3 + cAMP). These figures demonstrate a similar induction of differentiation for RAl .5 in the presence or absence of cAMP.
  • Figure 2OA represents the white blood cells content (WBC) obtained at D3 and D6 in presence (right histogram) or absence (left.histogram) of RA 10.
  • Figure 2OB represents the PML/RARA abundance in presence (right histogram) or absence (left histogram) of RA 10.
  • Figure 2OC represents the survival of secondary transplant recipients of primary
  • APL mice treated at day 6 (from upper to lower histograms: control, RA 10, cAMP, As 2 O 3 , As 2 O 3 + cAMP, RA 10 + As 2 O 3 ).
  • Figures 21-23 show that the PKA phosphorylation site in RARA or PML/RARA is dispensable for RA-induced activation of target genes, but desensitizes PML/RARA to RA-induced degradation.
  • Figures 21 A and 21B represent the activation of rarb (A) and cyp26a (B) gene expression in RARA (black) or RARAS369A (grey)-transduced rara,b,g -/- MEFs after 6 hours at different RA doses.
  • Figure 22 represents the activation of tgll, rarb and cyp26a by PML/RARA or PML/RARA-S873A in mouse APLs (Tg), retroviraly transduced progenitors (BM) or rara,b,g -/- MEFs. Mean +/- standard deviation of 3 independent experiments.
  • Figure 23 represents the PML/RARA degradation in mouse APLs triggered by 15 hours exposure to the indicated compounds.
  • the mutant APL is less sensitive to RA - induced degradation than the wild-type one.
  • synergistic effects of RA (10 ⁇ 7 M) and the phospho-diesterase inhibitor (PDEI: piclamilast) in normal, but not mutant APLs. representative experiment from 3 independent ones).
  • Figure 24 represents the white blood cells content (WBC/mm 3 , full line) and the blast content (%, dotted line) obtained after two cycles of a high RA dose treatment (45 mg/ni 2 /day).
  • Retroviruses were obtained following transient transfection of the Plat-E packaging cell line by the different pMSCV vectors. They were used to infect lineage-depleted murine hematopoeitic cells collected from donor mice that were given intraperitoneal injections of 5-Fluorouracil 5 days prior to BM isolation as described previously (Zhu, J., et al. A sumoylation site in PML/RARA is essential for leukemic transformation. Cancer Cell 7, 143-153 (2005)).
  • transduced cells were cultured in methylcellulose medium (Stem Cell Technologies, Vancouver) supplemented with 100 ng/ml stem cell factor (SCF) and 10 ng/ml of each of IL-3, IL-6 and GM-CSF (Abcys) in the presence of G418 selection.
  • SCF stem cell factor
  • neomycin-selected cells were recovered from Methylcellulose and either analyzed or replated at a density of 10,000 cells/well in the presence or absence of Retinoic acid (RA) 1 ⁇ M (Sigma- Aldrich). Serial replating assays were performed in duplicate every 7 days.
  • RA Retinoic acid
  • Immortalized MEFs in which the three RARs were excised (Altucci, L., et al. Rexino id-triggered differentiation and tumours selective apoptosis of AML by protein kinase- A-mediated de-subordination of RXR. Cancer Res 65, 8754-8765. (2005)) were retrovirally transduced with either RARA, PML/RARA or the corresponding mutants followed by G418 selection. Cells were cultured in DMEM containing 10% FBS and treated overnight with RA 1 ⁇ M.
  • bone marrow cells were collected from bilateral femurs and tibiae of control or treated leukemic mice by flushing marrow cavities with cRPMI through a 21 -Gauge needle. Then cells were cultured ex vivo in cRPMI medium supplemented with IL-3, IL-6 and SCF in the presence or absence of RA.
  • Transgenic mice were obtained as previously described in the FVB strain, using the MRP8 expression cassette. All experiments including mice were repeated at least three times. Animal handling was done according to the guidelines of institutional animal treatment committees using approved protocols. The transplantation model of APL was previously described (Lallemand- Boothnbach, V., et al. Retinoic acid and arsenic synergize to eradicate leukemic cells in a mouse model of acute promyelocytic leukemia. J. Exp. Med. 189, 1043-1052 (1999)). Briefly, PML/RARA APLs were transplanted in syngenic female mice, while PLZF/RARA+RARA/PLZF APLs were serially transplanted in Nude mice. For transplantation of secondary recipients, bone marrow cells were isolated from the tibiae and femurs of control and treated mice. 10 7 cells were re-injected intravenously in syngeneic recipient mice and survival was monitored.
  • RA was administered to mice via subcutaneous implantation of a 21 -day-release pellet containing 10 mg or 1,5 mg RA (Innovative Research of America), and cAMP by subcutaneous implantation of Alzet pumps (Guillemin, M. C, et al. In Vivo Activation of cAMP Signaling Induces Growth Arrest and Differentiation in Acute Promyelocytic Leukemia. J Exp Med 196, 1373-1380, (2002)).
  • Arsenic was administered by daily intraperitoneal injections. Morphological differentiation was assessed by May-Grunwald- Giemsa- stained cytospin slides.
  • Tissues and cells from autopsied mice were analyzed as before (Lallemand-Breitenbach, V., et al. Retinoic acid and arsenic synergize to eradicate leukemic cells in a mouse model of acute promyelocytic leukemia. J. Exp. Med. 189, 1043- 1052 (1999)).
  • Leukemias were transduced by retroviral infection (kind gift of S. Kogan) to allow for luciferase expression.
  • In vivo imaging was performed using a Xenogen IVISlOO facility according to the manufacturer's instructions.
  • Protein lysates were prepared from treated and untreated cells. Total cellular proteins were loaded onto 7% acrylamide gels, subjected to electrophoresis and transferred onto nitrocellulose membranes. The blots were blocked for one hour at room temperature in 5% skimmed milk in TBS. The membranes were then probed overnight with primary antibody at 4°C. PML/RARA expression was evaluated using an anti-RARA rabbit serum (RPl 15) kindly provided by P. Chambon. Detection was performed with the chemiluminescent substrate SuperSignal WestPico (Pierce biotechnology). Loading of equal amounts of protein was assessed by reprobing membranes with an anti- ⁇ -actin antibody (Sigma- Aldrich) FACS analysis :
  • cellular Fc receptors were first blocked with normal rat Immunoglobulin G. Immunophenotypic analysis was then performed using fluorochrome-conjugated monoclonal antibodies to c-Kit (Clone 2B8), Mac-1 and Gr-I (clone RB6-8-C5). Staining was performed at 4°C for 20 min. Cells were washed twice and resuspended in 0.5 ⁇ g/ml Propidium Iodide. Analysis was performed using the CellQuest software. Dead cells were gated out by high-Propidium Iodide staining and forward light scatter.
  • APL differentiation does not parallel loss of leukaemia initiating cells ex vivo and in vivo
  • PML/RARA expression in murine primary haematopoietic progenitor cells induces a sharp differentiation arrest and allows indefinite replating in methyl-cellulose cultures, less than 1% of transformed cells yielding colonies (Zhu, J., et al. A sumoylation site in PML/RARA is essential for leukemic transformation. Cancer Cell 7, 143-153 (2005)).
  • RA Retinoic acid and arsenic synergize to eradicate leukemic cells in a mouse model of acute promyelocytic leukemia. J. Exp. Med. 189, 1043-1052 (1999)).
  • RA lead to a rapid differentiation, followed at day 7, by the complete disappearance of APL cells from the marrow, as assessed by morphology and quantitative PCR on the PML/RARA gene ( Figure 4).
  • RA normalized the spleen weights and the blood counts of treated mice. However, the ability of these morphologically normal RA-treated marrows to initiate APL development in secondary recipient mice was only modestly reduced, indicating that many APL LIC remained ( Figure 4).
  • the curative RA/arsenic association triggers an immediate LIC loss that requires active proteolysis
  • the RA/arsenic association eradicates mouse APLs (Lallemand-Breitenbach, V v et al. Retinoic acid and arsenic synergize to eradicate leukemic cells in a mouse model of acute promyelocytic leukemia. J. Exp. Med. 189, 1043-1052 (1999); Rego, E.M., He, L.Z., Warrell, R.P., Jr., Wang, Z.G. & Pandolfi, P.P.
  • RA and arsenic share the ability to induce PML/RARA degradation through non- overlapping pathways, which may therefore cooperate to clear the fusion protein (reviewed in (Zhu, J., Lallemand-Breitenbach, V. & de The, H. Pathways of retinoic acid- or arsenic trioxide-induced PML/RARalpha catabolism, role of oncogene degradation in disease remission. Oncogene 20, 7257-7265. (2001)).
  • mice were treated with the RA/arsenic association in the presence or absence of the proteasome inhibitor Velcade ® . While the inhibitor induced terminal differentiation and APL cell loss on its own, when combined to the RA/arsenic association, it retarded APL regression and blocked the restoration of normal haematopoiesis ( Figure 11, 12).
  • Velcade ® sharply also antagonized loss of LIC triggered by the RA/arsenic association, implying that PML/RARA degradation is an important molecular determinant of LIC eradication by this association.
  • cAMP has been combined with sub-optimal RA doses in the APL mouse. Cyclic AMP alone induced a significant reduction in tumor mass, accompanied by apoptosis induction (Guillemin, M.C., et al. In Vivo Activation of cAMP Signaling Induces Growth Arrest and Differentiation in Acute Promyelocytic Leukemia. J Exp Med 196, 1373-1380. (2002)).
  • target genes activation was analyzed in different cellular backgrounds, including RARA- or PML/RARA-transduced mouse embryo fibroblasts (MEF) derived from rara,b,g -/- embryos (Zhou, J., et al. Dimerization- induced corepressor binding and relaxed DNAbinding specificity are critical for PML/RARA-induced immortalization. Proc Natl Acad Sci USA 103, 9238-9243 (2006)).
  • MEF mouse embryo fibroblasts
  • PML/RARA phosphorylation at S873 regulates the RA-triggered degradation of the fusion, but not the activation of direct transcriptional targets.
  • EXAMPLE 12 Effect of a high RA dose (45 mg/m 2 /day) on a patient diagnosed with t(ll,17) and

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Abstract

La présente invention porte sur de nouvelles compositions pharmaceutiques utiles pour le traitement de cancers, en particulier la leucémie myéloïde aiguë (AML) et la leucémie promyélocytique aiguë (APL). Lesdites compositions contiennent de l'acide rétinoïque ou un composé y afférent associé à au moins un inhibiteur de phosphodiestérase pour augmenter le contenu cellulaire de cAMP. Eventuellement, lesdites compositions contiennent aussi un dérivé d'arsenic.
EP09757550A 2008-06-03 2009-06-03 Compositions pharmaceutiques utiles pour le traitement de cancers, en particulier d'une leucémie myéloïde aiguë et d'une leucémie promyélocytique aiguë Withdrawn EP2303255A1 (fr)

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EP09757550A EP2303255A1 (fr) 2008-06-03 2009-06-03 Compositions pharmaceutiques utiles pour le traitement de cancers, en particulier d'une leucémie myéloïde aiguë et d'une leucémie promyélocytique aiguë

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