Classifications

• • 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
  • A61K31/33—Heterocyclic compounds
  • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
  • A61K31/55—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having seven-membered rings, e.g. azelastine, pentylenetetrazole
  • A61K31/553—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having seven-membered rings, e.g. azelastine, pentylenetetrazole having at least one nitrogen and one oxygen as ring hetero atoms, e.g. loxapine, staurosporine
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
  • A61K31/7088—Compounds having three or more nucleosides or nucleotides
• • 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
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00

Definitions

• the present invention relates to a method of treating myelodysplastic syndromes, lymphomas and leukemias, in particular Systemic mastocytosis (SM), and also acute myeloid leukemia (AML) and solid tumors such as e.g. colorectal cancer (CRC) and non-small cell lung cancer (NSCLC) with a pharmaceutical combination of a FLT-3 kinase inhibitor and an MCL-I inhibitor, such as a mcl-1 -specific nucleic acid construct.
• An mcl-1 -specific nucleic acid construct includes, but is not limited to an antisense mcl-1 oligonucleotide or a mcl-1 -specific RNAi construct.
• An mcl-1 -specific RNAi construct includes but is not limited to a short interfering RNA (siRNA), a micro RNA (miRNA) or a hairpin RNA (shRNA) construct.
• the invention also relates to the use of a pharmaceutical combination of antisense oligonucleotide or a mcl-1 -specific RNAi, and a FLT-3 kinase inhibitor for the treatment of the diseases or malignancies mentioned above and the use of such a pharmaceutical composition for the manufacture of a medicament for the treatment of these diseases or malignancies.
• Mastocytosis is a term collectively used for a group of disorders characterized by abnormal accumulation of mast cells (MC) in one or more organ systems. Cutaneous and systemic variants of the disease have been described. Cutaneous mastocytosis (CM) typically develops in early childhood and shows a benign course with frequent spontaneous regression. Systemic mastocytosis (SM) can develop at any age and is characterized by involvement of one or more visceral organs with or without skin involvement. In contrast to CM, SM is a persistent clonal disorder of MC. In a high proportion of cases, the transforming KIT mutation D816V is detectable. In advanced SM, the mutation may also be detectable in other myeloid lineages or even in B lymphocytes.
• SM is a disease of multilineage hematopoietic progenitors.
• the concept that SM arises from a multilineage progenitor is also supported by the notion that these patients can develop an associated hematologic non mast cell lineage disease (AHNMD).
• HNMD hematologic non mast cell lineage disease
• ISM indolent systemic mastocytosis
• SM-AHNMD mastocytosis with AHNMD
• ASM aggressive SM
• MCL mast cell leukemia
• TK novel tyrosine kinase inhibitors, like such as yV- ⁇ S.lO ⁇ l l ⁇ B ⁇ O.l L ⁇ .lS-hexahydro-lO-methoxy-Q-methyl-l-oxo-Q.lS-epoxy-l// ⁇ //- Case 50270A
• neoplastic MC diindolo[l,2,3-gh:3',2',r-lm]pyrrolo[3,4-j][l,7]benzodiazonin-l l-yl]-jV-methylbenzamide or nilotinib (4-methyl-3-[[4-(3-pyridinyl)-2-pyrimidinyl] amino]-N-[5-(4-methyl-lH-imidazol-l-yl)-3- (trifluoromethyl)phenyl] benzamide ), may also counteract growth of neoplastic MC. Again, however, long lasting responses have yet to be described.
• imatinib inhibits the growth of neoplastic MC in patients with SM.
• the effect of imatinib is only seen in patients in whom neoplastic MC harbour wild type KIT, the KIT mutation F522C, or in patients who have coexistent eosinophilia associated with the FIPlLl/PDGFR ⁇ fusion gene.
• imatinib showed little if any effects on neoplastic MC exhibiting KIT D816V.
• this KIT mutation which is detectable in a majority of patients with SM, confers resistance against imatinib.
• McI-I is a well characterized member of the BcI-2 family that is considered to act anti- apoptotic in various neoplastic cells. See, e.g., Mendelian Inheritance in Man, MIM accno. 159552. Originally, McI-I was described as a survival-enhancing molecule, expressed during TPA-induced differentiation of leukemic ML-I cells. Consecutive studies show that McI-I is constitutively expressed in primary neoplastic cells in chronic myeloid leukemia. However, little is known so far about the expression and role of McI-I in other myeloid neoplasms.
• FLT-3 kinase inhibitors in combination with an antisense oligonucleotide or a mcl-1 -specific RNAi construct possess therapeutic properties.
• These findings render the combination of a FLT-3 kinase inhibitor and a mcl-1 inhibitor as particularly useful for the treatment myelodysplastic syndromes, lymphomas and leukemias, in particular Systemic mastocytosis, and also acute myeloid leukemia (AML) and also solid tumors such as e.g. colorectal cancer (CRC) and non-small cell lung cancer (NSCLC).
• CRC colorectal cancer
• NSCLC non-small cell lung cancer
• FLT-3 kinase inhibitors of particular interest for use in the inventive combination are 4- methyl-3-[[4-(3-pyridinyl)-2-pyrimidinyl]amino]-N-[5-(4-methyl-lH-imidazol-l-yl)-3- (trifluoromethyl)phenyl] benzamide, and imatinib and staurosporine derivatives of formula
• Ri and R 2 are, independently of one another, unsubstituted or substituted alkyl, hydrogen, halogen, hydroxy, etherified or esterified hydroxy, amino, mono- or disubstituted amino, cyano, nitro, Case 50270A
• n and m are, independently of one another, a number from and including 0 to and including 4; n' and m' are, independently of one another, a number from and including 0 to and including 4; R 3 , R 4 , R 8 and R, o are, independently of one another, hydrogen, -O ' , acyl with up to 30 carbon atoms, an aliphatic, carbocyclic, or carbocyclic-aliphatic radical with up to 29 carbon atoms in each case, a heterocyclic or heterocyclic-aliphatic radical with up to 20 carbon atoms in each case, and
• Z stands for hydrogen or lower alkyl; and either the two bonds characterised by wavy lines are absent in ring A and replaced by 4 hydrogen atoms, and the two wavy lines in ring B each, together with the respective parallel bond, signify a double bond; or the two bonds characterised by wavy lines are absent in ring B and replaced by a total of 4 hydrogen atoms, and the two wavy lines in ring A each, together with the respective parallel bond, signify a double bond; or both in ring A and in ring B all of the 4 wavy bonds are absent and are replaced by a total of 8 hydrogen atoms; or a salt thereof, if at least one salt-forming group is present.
• Lower alkyl is especially methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, or tert-butyl, and also pentyl, hexyl, or heptyl.
• Unsubstituted or substituted alkyl is preferably Ci-C 2 oalkyl, especially lower alkyl, typically methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, or tert-butyl, which is unsubstituted or substituted especially by halogen, such as fluorine, chlorine, bromine, or iodine, Ce- Ci 4 aryl, such as phenyl or naphthyl, hydroxy, etherified hydroxy, such as lower alkoxy, phenyl- lower alkoxy or phenyloxy, esterified hydroxy, such as lower alkanoyloxy or benzoyloxy, amino, mono- or disubstituted amino, such as lower alkylamino, lower alkanoylamino, phenyl-lower alkylamino, N,N-di-lower alkylamino,
• Halogen is preferably fluorine, chlorine, bromine, or iodine, especially fluorine or chlorine.
• Etherified hydroxy is especially lower alkoxy, C 6 -C 14 aryloxy, such as phenyloxy, or C 6 - Ci 4 aryl-lower alkoxy, such as benzyloxy.
• Esterified hydroxy is preferably lower alkanoyloxy or C ⁇ -C ⁇ arylcarbonyloxy, such as benzoyloxy.
• Mono- or disubstituted amino is especially amino monosubstituted or disubstituted by lower alkyl, C 6 -Ci 4 aryl, C 6 -Ci 4 aryl-lower alkyl, lower alkanoyl, or
• Substituted mercapto is especially lower alkylthio, C 6 -Ci 4 arylthio, C 6 -Ci 4 aryl-lower alkylthio, lower alkanoylthio, or C 6 -Ci 4 aryl-lower alkanoylthio.
• Esterified carboxy is especially lower alkoxycarbonyl, C 6 -Ci 4 aryl-lower alkoxycarbonyl or C 6 -C, 4 aryloxycarbonyl.
• N-Mono- or N,N-disubstituted carbamoyl is especially carbamoyl N-monosubstituted or N,N-disubstituted by lower alkyl, C ⁇ -C ⁇ aryl or C 6 -Ci 4 aryl-lower alkyl.
• Case 50270A is especially carbamoyl N-monosubstituted or N,N-disubstituted by lower alkyl, C ⁇ -C ⁇ aryl or C 6 -Ci 4 aryl-lower alkyl.
• Substituted sulfonyl is especially C ⁇ -C ⁇ arylsulfonyl, such as toluenesulfonyl, C 6 -Ci 4 aryl-lower alkanesulfonyl or lower alkanesulfonyl.
• N-Mono- or N,N-disubstituted aminosulfonyl is especially aminosulfonyl N- monosubstituted or N,N-disubstituted by lower alkyl, C 6 -Ci 4 aryl or C 6 -Ci 4 aryl-lower alkyl.
• C ⁇ -C ⁇ Aryl is an aryl radical with 6 to 14 carbon atoms in the ring system, such as phenyl, naphthyl, fluorenyl, or indenyl, which is unsubstituted or is substituted especially by halogen, such as fluorine, chlorine, bromine, or iodine, phenyl or naphthyl, hydroxy, lower alkoxy, phenyl-lower alkoxy, phenyloxy, lower alkanoyloxy, benzoyloxy, amino, lower alkylamino, lower alkanoylamino, phenyl-lower alkylamino, N,N-di-lower alkylamino, N,N-di-(phenyl-lower alkyl)amino, cyano, mercapto, lower alkylthio, carboxy, lower alkoxycarbonyl, carbamoyl, N-lower alkylcarbamoyl,
• indices n and m are in each case preferably 1, 2 or especially 0.
• compounds of formula I in which n and m are in each case 0 (zero) are especially preferred.
• An aliphatic carbohydrate radical R 3 , R 4 , Rg or R 10 with up to 29 carbon atoms which is substituted by acyclic substituents and preferably has a maximum of 18, especially a maximum of 12, and as a rule not more than 7 carbon atoms, may be saturated or unsaturated and is especially an unsubstituted or a straight-chain or branched lower alkyl, lower alkenyl, lower alkadienyl, or lower alkinyl radical substituted by acyclic substituents.
• Lower alkyl is, for example, methyl, ethyl, n- propyl, isopropyl, n-butyl, isobutyl, sec-butyl or tert-butyl, and also n-pentyl, isopentyl, n-hexyl, isohexyl and n-heptyl; lower alkenyl is, for example, allyl, propenyl, isopropenyl, 2- or 3-methallyl and 2- or 3-butenyl; lower alkadienyl is, for example, l-penta-2,4-dienyl; lower alkinyl is, for example, propargyl or 2-butinyl.
• the double bond is especially located in a position higher than the ⁇ -position in relation to the free valency.
• Substituents are especially the acyl radicals defined hereinbelow as substituents of R°, preferably free or esterified carboxy, such as carboxy or lower alkoxycarbonyl, cyano or di-lower alkylamino.
• a carbocyclic or carbocyclic-aliphatic radical R 3 , R 4 , R 8 or R 1O with up to 29 carbon atoms in each case is especially an aromatic, a cycloaliphatic, a cycloaliphatic-aliphatic, or an aromatic-aliphatic radical which is either present in unsubstituted form or substituted by radicals referred to hereinbelow as substituents of R 0 .
• aromatic radical (aryl radical) R 3 or R 4 is most especially a phenyl, also a naphthyl, such as 1- or 2-naphthyl, a biphenylyl, such as especially 4- biphenylyl, and also an anthryl, fluorenyl and azulenyl, as well as their aromatic analogues with one or more saturated rings, which is either present in unsubstituted form or substituted by radicals referred to hereinbelow as substituents of R 0 .
• Preferred aromatic-aliphatic radicals are aryl-lower Case 50270A
• aryl-lower alkenyl radicals e.g. phenyl-lower alkyl or phenyl-lower alkenyl with a terminal phenyl radical, such as for example benzyl, phenethyl, 1-, 2-, or 3-phenylpropyl, diphenylmethyl (benzhydryl), trityl, and cinnamyl, and also 1- or 2-naphthylmethyl.
• aryl radicals carrying acyclic radicals such as lower alkyl, special mention is made of o-, m- and p-tolyl and xylyl radicals with variously situated methyl radicals.
• a cycloaliphatic radical R 3 , R 4 , R 8 or R, o with up to 29 carbon atoms is especially a substituted or preferably unsubstituted mono-, bi-, or polycyclic cycloalkyl-, cycloalkenyl-, or cycloalkadienyl radical.
• Preference is for radicals with a maximum of 14, especially 12, ring-carbon atoms and 3- to 8-, preferably 5- to 7-, and most especially 6-member rings which can also carry one or more, for example two, aliphatic hydrocarbon radicals, for example those named above, especially the lower alkyl radicals, or other cycloaliphatic radicals as substituents.
• Preferred substituents are the acyclic substituents named hereinbelow for R 0 .
• O with up to 29 carbon atoms is a radical in which an acyclic radical, especially one with a maximum of 7, preferably a maximum of 4 carbon atoms, such as especially methyl, ethyl, and vinyl, carries one or more cycloaliphatic radicals as defined hereinabove.
• an acyclic radical especially one with a maximum of 7, preferably a maximum of 4 carbon atoms, such as especially methyl, ethyl, and vinyl
• Preferred substituents are the acyclic substituents named herein below
• Heterocyclic radicals R 3 , R 4 , R 8 or R 10 with up to 20 carbon atoms each and up to 9 heteroatoms each are especially monocyclic, but also bi- or polycyclic, aza-, thia-, oxa-, thiaza-, oxaza-, diaza-, triaza-, or tetrazacyclic radicals of an aromatic character, as well as corresponding heterocyclic radicals of this type which are partly or most especially wholly saturated, these radicals - if need be - possibly carrying further acyclic, carbocyclic, or heterocyclic radicals and/or possibly mono-, di-, or polysubstituted by functional groups, preferably those named hereinabove as substituents of aliphatic hydrocarbon radicals.
• pyrryl for example 2-pyrryl or 3-pyrryl
• pyridyl for example 2-, 3-, or 4-pyridyl
• thienyl for example 2- or 3-thienyl
• furyl for example 2-furyl
• analogous bicyclic radicals with an oxygen, sulfur, or nitrogen atom are, for example, indolyl, typically 2- or 3-indolyl, quinolyl, typically 2- or 4-quinolyl, isoquinolyl, typically 3- or 5- isoquinolyl, benzofuranyl, typically 2-benzofuranyl, chromenyl, typically 3-chromenyl, or benzo- thienyl, typically 2- or 3-benzothienyl
• preferred monocyclic and bicyclic radicals with several heteroatoms are, for example, imidazolyl,
• 2-thiazolyl, and benzimidazolyl typically 2-benzimidazolyl, benzoxazolyl, typically 2- benzoxazolyl, or quinazolyl, typically 2-quinazolinyl.
• benzimidazolyl typically 2-benzimidazolyl
• benzoxazolyl typically 2- benzoxazolyl
• quinazolyl typically 2-quinazolinyl.
• Appropriate partially or, especially, completely saturated analogous radicals may also be considered, such as 2-tetrahydrofuryl, 2- or 3- pyrrolidinyl, 2-, 3-, or 4-piperidyl, and also 2-or 3-morpholinyl, 2- or 3-thiomo ⁇ holinyl, 2- piperazinyl and N-mono- or N,N'-bis-lower alkyl-2-piperazinyl radicals.
• These radicals may also carry one or more acyclic, carbocyclic, or heterocyclic radicals, especially those mentioned hereinabove.
• the free valency of the heterocyclic radicals R 3 or R 4 must emanate from one of their carbon atoms.
• Heterocyclyl may be unsubstituted or substituted by one or more, preferably one or two, of the substituents named hereinbelow for R 0 .
• Heterocyclic-aliphatic radicals R 3 , R 4 , Rs or Rio especially lower alkyl radicals, especially with a maximum of 7, preferably a maximum of 4 carbon atoms, for example those named hereinabove, which carry one, two, or more heterocyclic radicals, for example those named in the preceding paragraph, the heterocyclic ring possibly being linked to the aliphatic chain also by one of its nitrogen atoms.
• a preferred heterocyclic-aliphatic radical R is, for example, imidazol-1- ylmethyl, 4-methylpiperazin-l-ylmethyl, piperazin-1-ylmethyl, 2-(morpholin-4-yl)ethyl and also pyrid-3-ylmethyl.
• Heterocyclyl may be unsubstituted or substituted by one or more, preferably one or two, of the substituents named hereinbelow for R°.
• a heteroaliphatic radical R 3 , R 4 , Rg or Ri 0 with up to 20 carbon atoms each and up to 10 heteroatoms each is an aliphatic radical which, instead of one, two, or more carbon atoms, contains identical or different heteroatoms, such as especially oxygen, sulfur, and nitrogen.
• R 3 , R 4 , Rg or Ri 0 apart from acyl, is lower alkyl, particlularly methyl or ethyl; lower alkoxycarbonyl-lower alkyl, especially methoxycarbonylmethyl or 2-(tert- butoxycarbonyl)ethyl; carboxy-lower alkyl, especially carboxymethyl or 2-carboxyethyl; or cyano- lower alkyl, especially 2-cyanoethyl.
• An acyl radical R 3 , R 4 , Re, R 7 , Re, R 9 , or Ri O with up to 30 carbon atoms derives from a carboxylic acid, functionally modified if need be, an organic sulfonic acid, or a phosphoric acid, such as pyro- or orthophosphoric acid, esterified if need be.
• R 0 HN- or R 0 R 0 N- wherein the R 0 radicals may be identical or different from one another.
• the hydrocarbyl (hydrocarbon radical) R° is an acyclic (aliphatic), carbocyclic, or carbocyclic-acyclic hydrocarbon radical, with up to 29 carbon atoms each, especially up to 18, and preferably up to 12 carbon atoms, and is saturated or unsaturated, unsubstituted or substituted.
• it may contain identical or different heteroatoms, such as especially oxygen, sulfur, and nitrogen in the acyclic and/or cyclic part; in the latter case, it is described as a heterocyclic radical (heterocyclyl radical) or a heterocyclic-acyclic radical.
• Unsaturated radicals are those, which contain one or more, especially conjugated and/or isolated, multiple bonds (double or triple bonds).
• cyclic radicals includes also aromatic and non-aromatic radicals with conjugated double bonds, for example those wherein at least one 6- member carbocyclic or a 5- to 8-member heterocyclic ring contains the maximum number of non- cumulative double bonds.
• An acyclic unsubstituted hydrocarbon radical R 0 is especially a straight-chained or branched lower alkyl-, lower alkenyl-, lower alkadienyl-, or lower alkinyl radical.
• Lower alkyl R° is, for example, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl or tert-butyl, and also n- pentyl, isopentyl, n-hexyl, isohexyl and n-heptyl; lower alkenyl is, for example, allyl, propenyl, isopropenyl, 2- or 3-methallyl and 2- or 3-butenyl; lower alkadienyl is, for example, l-penta-2,4- dienyl; lower alkinyl is, for example, propargyl or 2-butinyl.
• the double bond is especially located in a position higher than the ⁇ -position in relation to the free valency.
• a carbocyclic hydrocarbon radical R 0 is especially a mono-, bi-, or polycyclic cycloalkyl-, cycloalkenyl-, or cycloalkadienyl radical, or a corresponding aryl radical. Preference is for radicals with a maximum of 14, especially 12, ring-carbon atoms and 3- to 8-, preferably 5- to 7- , and most especially 6-member rings which can also carry one or more, for example two, acyclic radicals, for example those named above, especially the lower alkyl radicals, or other carbocyclic radicals.
• Carbocyclic-acyclic radicals are those in which an acyclic radical, especially one with a maximum of 7, preferably a maximum of 4 carbon atoms, such as especially methyl, ethyl and vinyl, carries one or more carbocyclic, if need be aromatic radicals of the above definition. Special mention is made of cycloalkyl-lower and aryl-lower alkyl radicals, as well as their analogues which are unsaturated in the ring and/or chain, and which carry the ring at the terminal carbon atom of the chain. Case 50270A
• Cycloalkyl R 0 has most especially from 3 up to and including 10 carbon atoms and is, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl, as well as bicyclo[2,2,2]octyl, 2-bicyclo[2,2,l]heptyl, and adamantyl, which may also be substituted by 1, 2, or more, for example lower, alkyl radicals, especially methyl radicals; cycloalkenyl is for example one of the monocyclic cycloalkyl radicals already named which carries a double bond in the 1-, 2-, or 3 position.
• Cycloalkyl-lower alkyl or -lower alkenyl is for example a -methyl, -1- or -2-ethyl, -1- or -2-vinyl, -1-, -2-, or -3-propyl or -allyl substituted by one of the above-named cycloalkyl radicals, those substituted at the end of the linear chain being preferred.
• An aryl radical R 0 is most especially a phenyl, also a naphthyl, such as 1- or 2-naphthyl, a biphenylyl, such as especially 4-biphenylyI, and also an anthryl, fluorenyl and azulenyl, as well as their aromatic analogues with one or more saturated rings.
• Preferred aryl-lower alkyl and -lower alkenyl radicals are, for example, phenyl-lower alkyl or phenyl-lower alkenyl with a terminal phenyl radical, such as for example benzyl, phenethyl, 1-, 2-, or 3-phenylpropyl, diphenylmethyl (benzhydryl), trityl, and cinnamyl, and also 1- or 2-naphthylmethyl.
• Aryl may be unsubstituted or substituted.
• Heterocyclic radicals including heterocyclic-acyclic radicals, are especially monocyclic, but also bi- or polycyclic, aza-, thia-, oxa-, thiaza-, oxaza-, diaza-, triaza-, or tetrazacyclic radicals of an aromatic character, as well as corresponding heterocyclic radicals of this type which are partly or most especially wholly saturated; if need be, for example as in the case of the above-mentioned carbocyclic or aryl radicals, these radicals may carry further acyclic, carbocyclic, or heterocyclic radicals and/or may be mono-, di-, or polysubstituted by functional groups.
• heterocyclic-acyclic radicals has for example the meaning indicated for the corresponding carbocyclic-acyclic radicals.
• they are unsubstituted or substituted monocyclic radicals with a nitrogen, oxygen, or sulfur atom, such as 2-aziridinyl, and especially aromatic radicals of this type, such as pyrrolyl, for example 2-pyrrolyl or 3-pyrrolyl, pyridyl, for example 2-, 3-, or 4-pyridyl, and also thienyl, for example 2- or 3-thienyl, or furyl, for example 2- furyl; analogous bicyclic radicals with an oxygen, sulfur, or nitrogen atom are, for example, indolyl, typically 2- or 3-indolyl, quinolyl, typically 2- or 4-quinolyl, isoquinolyl, typically 3- or 5- isoquinolyl, benzofuranyl, typically 2-benzofuranyl, chromenyl, typically 3-chromenyl, or benzo-
• radicals may also carry one or more acyclic, carbocyclic, or heterocyclic radicals, especially those mentioned hereinabove.
• Heterocyclic-acyclic radicals are especially derived from acyclic radicals with a maximum of 7, preferably a maximum of 4 carbon atoms, for example those named hereinabove, and may carry one, two, or more heterocyclic radicals, for example those named hereinabove, the ring possibly being linked to the aliphatic chain also by one of its nitrogen atoms.
• a hydrocarbyl may be substituted by one, two, or more identical or different substituents (functional groups); one or more of the following substituents may be considered: lower alkyl; free, etherified and esterified hydroxyl groups; carboxy groups and esterified carboxy groups; mercapto- and lower alkylthio- and, if need be, substituted phenylthio groups; halogen atoms, typically chlorine and fluorine, but also bromine and iodine; halogen-lower alkyl groups; oxo groups which are present in the form of formyl (i.e.
• aldehydo aldehydo
• keto groups also as corresponding acetals or ketals; azido groups; nitro groups; cyano groups; primary, secondary and preferably tertiary amino groups, amino-lower alkyl, mono- or disubstituted amino-lower alkyl, primary or secondary amino groups protected by conventional protecting groups (especially lower alkoxycarbonyl, typically tert-butoxycarbonyl) lower alkylenedioxy, and also free or functionally modified sulfo groups, typically sulfamoyl or sulfo groups present in free form or as salts.
• protecting groups especially lower alkoxycarbonyl, typically tert-butoxycarbonyl
• the hydrocarbyl radical may also carry carbamoyl, ureido, or guanidino groups, which are free or which carry one or two substituents, and cyano groups.
• groups is taken to imply also an individual group.
• Halogen-lower alkyl contains preferably 1 to 3 halogen atoms; preferred is trifiuoromethyl or chloromethyl.
• An etherified hydroxyl group present in the hydrocarbyl as substituent is, for example, a lower alkoxy group, typically the methoxy-, ethoxy-, propoxy-, isopropoxy-, butoxy-, and tert- butoxy group, which may also be substituted, especially by (i) heterocyclyl, whereby heterocyclyl can have preferably 4 to 12 ring atoms, may be unsaturated, or partially or wholly saturated, is mono- or bicyclic, and may contain up to three heteroatoms selected from nitrogen, oxygen, and sulfur, and is most especially pyrrolyl, for example 2-pyrrolyl or 3-pyrrolyl, pyridyl, for example 2-, 3- or 4-pyridyl, and also thienyl, for example 2- or 3-thienyl, or furyl, for example 2-furyl, indolyl, typically 2- or 3-indolyl, quinolyl, typically 2- or 4-quinolyl, isoquinolyl, typically
• Such etherified hydroxyl groups are also unsubstituted or substituted phenoxy radicals and phenyl-lower alkoxy radicals, such as especially benzyloxy, benzhydryloxy, and triphenylmethoxy (trityloxy), as well as heterocyclyloxy radicals, wherein heterocyclyl can have preferably 4 to 12 ring atoms, may be unsaturated, or partially or wholly saturated, is mono- or bicyclic, and may contain up to three heteroatoms selected from nitrogen, oxygen, and sulfur, and is most especially pyrrolyl, for example 2-pyrrolyl or 3-pyrrolyl, pyridyl, for example 2-, 3- or 4-pyridyl, and also thienyl, for example 2- or 3-thienyl, or furyl, for example 2-furyl, indolyl, typically 2- or 3-indolyl, quinolyl, typically 2- or 4-quinolyl, isoquinolyl, typically 3- or 5-isoquinoly
• Etherified hydroxyl groups in this context are taken to include silylated hydroxyl - groups, typically for example tri-lower alkylsilyloxy, typically trimethylsilyloxy and dimethyl-tert- butylsilyloxy, or phenyldi-lower alkylsilyloxy and lower alkyl-diphenylsilyloxy.
• An esterified hydroxyl group present in the hydrocarbyl as a substituent is, for example, lower alkanoyloxy.
• a carboxyl group present in the hydrocarbyl as a substituent is one in which the hydrogen atom is replaced by one of the hydrocarbyl radicals characterised hereinabove, preferably a lower alkyl- or phenyl-lower alkyl radical; an example of an esterified carboxyl group is lower alkoxycarbonyl or phenyl-lower alkoxycarbonyl substituted if need be in the phenyl part, especially the methoxy, ethoxy, tert-butoxy, and benzyloxycarbonyl group, as well as a lactonised carboxyl group.
• a primary amino group -NH 2 as substituent of the hydrocarbyls may also be present in a form protected by a conventional protecting group.
• a secondary amino group carries, instead of one Case 50270A
• a hydrocarbyl radical preferably an unsubstituted one, typically one of the above-named, especially lower alkyl, and may also be present in protected form.
• a tertiary amino group present in the hydrocarbyl as substituent carries 2 different or, preferably, identical hydrocarbyl radicals (including the heterocyclic radicals), such as the unsubstituted hydrocarbyl radicals characterised hereinabove, especially lower alkyl.
• a preferred amino group is one with the formula Rn(Ri 2 )N-, wherein R ⁇ ⁇ and R 12 are independently in each case hydrogen, unsubstituted acyclic C r C 7 -hydrocarbyl (such as especially C r C 4 alkyl or C 2 -C 4 alkenyl) or monocyclic aryl, aralkyl, or aralkenyl, substituted if necessary by C r C-alkyl, Ci-C 4 -alkoxy, halogen, and/or nitro, and having a maximum of 10 carbon atoms, where the carbon-containing radicals may be interlinked through a carbon-carbon bond or an oxygen atom, a sulfur atom, or a nitrogen atom substituted if necessary by hydrocarbyl.
• Rn(Ri 2 )N- wherein R ⁇ ⁇ and R 12 are independently in each case hydrogen, unsubstituted acyclic C r C 7 -hydrocarbyl (such as especially
• di-lower alkylamino typically dimethylamino or diethylamino, pyrrolidino, imidazol-1-yl, piperidino, piperazino, 4-Iower alkylpiperazino, morpholino, thiomorpholino and piperazino or 4-methylpiperazino, as well as diphenylamino and dibenzylamino substituted if need be, especially in the phenyl part, for example by lower-alkyl, lower-alkoxy, halogen, and/or nitro; of the protected groups, especially lower alkoxycarbonylamino, typically tert-butoxycarbonylamino, phenyl-lower alkoxycarbonylamino, typically 4-methoxybenzyloxycarbonylamino, and 9-fluorenyl
• Amino-lower alkyl is most especially substituted in the 1-position of the lower alkyl chain by amino and is especially aminomethyl.
• Mono- or disubstituted amino-lower alkyl is amino-lower alkyl substituted by one or two radicals, wherein amino-lower alkyl is most especially substituted by amino in the 1-position of the lower alkyl chain and is especially aminomethyl; the amino substituents here are preferably (if 2 substituents are present in the respective amino group independently of one another) from the group comprising lower alkyl, such as especially methyl, ethyl or n-propyl, hydroxy-lower alkyl, typically 2-hydroxyethyl, C 3 -C 8 cycloalkyl, especially cyclohexyl, amino-lower alkyl, typically 3- aminopropyl or 4-aminobutyl, N-mono- or N,N-di(lower alkyl)-amino-Iower alkyl, typically 3- (N,N-dimethylamino)propyl, amino, N-mono- or N,N-di-lower alkyl)
• Disubstituted amino-lower alkyl is also a 5 or 6-membered, saturated or unsaturated heterocyclyl bonded to lower alkyl via a nitrogen atom (preferably in the 1-position) and having 0 to 2, especially 0 or 1, other heteroatoms selected from oxygen, nitrogen, and sulfur, which is unsubstituted or substituted, especially by one or two radicals from the group comprising lower alkyl, Case 50270A
• - 14 - typically methyl, and also oxo.
• Preferred here is pyrrolidino (1-pyrrolidinyl), piperidino (1 -pi- peridinyl), piperazino (1-piperazinyl), 4-lower alkylpiperazino, typically 4-methylpiperazino, imi- dazolino (1-imidazolyl), mo ⁇ holino (4-morpholinyl), or also thiomorpholino, S-oxo-thio- morpholino, or S,S-dioxothiomorpholino.
• Lower alkylenedioxy is especially methylenedioxy.
• a carbamoyl group carrying one or two substituents is especially aminocarbonyl (carbamoyl) which is substitiuted by one or two radicals at the nitrogen; the amino substituents here are preferably (if 2 substituents are present in the respective amino group independently of one another) from the group comprising lower alkyl, such as especially methyl, ethyl or n-propyl, hydroxy-lower alkyl, typically 2-hydroxyethyl, C3-Cscycloalkyl, especially cyclohexyl, amino- lower alkyl, typically 3-aminopropyl or 4-aminobutyl, N-mono- or N,N-di(lower alkyl)-amino- lower alkyl, typically 3-(N,N-dimethylamino)propyl, amino, N-mono- or N,N-di-lower alkylamino and N-mono- or N,N-di-(hydroxy-lower al
• Preferred here is pyrrolidino (1-pyrrolidinyl), piperidino (1-pi- peridinyl), piperazino (1-piperazinyl), 4-lower alkylpiperazino, typically 4-methylpiperazino, imi- dazolino (1-imidazolyl), morpholino (4-morpholinyl), or also thiomorpholino, S-oxo-thio- morpholino, or S,S-dioxothiomor ⁇ holino.
• An acyl derived from an organic sulfonic acid which is designated Ac 2
• Ac 2 is especially one with the subformula R°-S ⁇ 2 -, wherein R 0 is a hydrocarbyl as defined above in the general and specific meanings, the latter also being generally preferred here.
• R 0 is a hydrocarbyl as defined above in the general and specific meanings, the latter also being generally preferred here.
• Especially preferred is lower al- kylphenylsulfonyl, especially 4-toluenesulfonyl.
• An acyl derived from a phosphoric acid, esterified if necessary, which is designated Ac 3 , is especially one with the subformula R°O(R°O)P( O)-, wherein the radicals R 0 are, independently of one another, as defined in the general and specific meanings indicated above.
• R 0 has the following preferred meanings: lower alkyl, especially methyl or ethyl, amino-lower alkyl, wherein the amino group is unprotected or is protected by a conventional amino protecting group - especially by lower alkoxycarbonyl, typically tert-lower alkoxycarbonyl, for example tert- butoxycarbonyl - e.g. aminomethyl, R,S-, R- or preferably S-1-aminoethyl, tert- Case 50270A
• Preferred acyl radicals Ac 1 are acyl radicals of a carboxylic acid which are characterised by the subformula R°-CO-, wherein R° has one of the above general and preferred meanings of the hydrocarbyl radical R°.
• Especially preferred radicals R° here are lower alkyl, especially methyl or ethyl, amino-lower alkyl, wherein the amino group is unprotected or protected by a conventional amino protecting group, especially by lower alkoxycarbonyl, typically tert-lower alkoxycarbonyl, for example tert-butoxycarbonyl, e.g.
• a further preferred Acyl Ac' is derived from monoesters of carbonic acid and is characterised by the subformula R°-O-CO-.
• the lower alky! radicals, especially tert-butyl, are especially preferred hydrocarbyl radicals R° in these derivatives.
• a preferred acyl Ac 2 of subformula R°-SO 2 -, wherein R° is a hydrocarbyl as defined in the above general and specific meanings, is lower alkylphenylsulfonyl, typically 4-toluenesulfonyl.
• Z is especially lower alkyl, most especially methyl or hydrogen.
• the compounds of the invention may also be present in the form of pharmaceutically, i.e. physiologically, acceptable salts, provided they contain salt-forming groups.
• pharmaceutically unacceptable salts may also be used.
• therapeutic use only pharmaceutically acceptable salts are used, and these salts are preferred.
• compounds of formula I having free acid groups may exist as a salt, preferably as a physiologically acceptable salt with a salt-forming basic component.
• a salt-forming basic component may be primarily metal or ammonium salts, such as alkali metal or alkaline earth metal salts, for example sodium, potassium, magnesium or calcium salts, or ammonium salts with ammonia or suitable organic amines, especially tertiary monoamines and heterocyclic bases, for example triethylamine, tri-(2-hydroxyethyl)-amine, N-ethylpiperidine or N,N'-dimethylpiperazine.
• Compounds of the invention having a basic character may also exist as addition salts, especially as acid addition salts with inorganic and organic acids, but also as quaternary salts.
• compounds which have a basic group, such as an amino group, as a substituent may form acid addition salts with common acids.
• Suitable acids are, for example, hydrohalic acids, e.g.
• hydrochloric and hydrobromic acid sulfuric acid, phosphoric acid, nitric acid or perchloric acid, or aliphatic, alicyclic, aromatic or heterocyclic carboxylic or sulfonic acids, such as formic, acetic, propionic, succinic, glycolic, lactic, malic, tartaric, citric, fumaric, maleic, hydroxymaleic, oxalic, pyruvic, phenylacetic, benzoic, p-aminobenzoic, anthranilic, p-hydroxybenzoic, salicylic, p- aminosalicylic acid, pamoic acid, methanesulfonic, ethanesulfonic, hydroxyethanesulfonic, ethylenedisulfonic, halobenzenesulfonic, toluenesulfonic, naphthalenesulfonic acids or sulfanilic acid, and also methionine, tryptophan,
• any reference hereinbefore and hereinafter to the free compounds is to be understood as referring also to the corresponding salts, and the solvates thereof, e.g,. hydrates, as appropriate and expedient.
• Ri and R 2 independently of each other are lower alkyl, lower alkyl substituted by halogen, C 6 -Ci 4 aryl, hydroxy, lower alkoxy, phenyl-lower alkoxy, phenyloxy, lower alkanoyloxy, benzoyloxy, amino, lower alkylamino, lower alkanoylamino, phenyl-lower alkylamino, N,N-di- lower alkylamino, N,N-di-(phenyl-lower alkyl)amino, cyano, mercapto, lower alkylthio, carboxy, lower alkoxycarbonyl, carbamoyl, N-lower alkylcarbamoyl, N,N-di-lower alkylcarbamoyl, sulfo, lower alkanesulfonyl, lower alkoxysulfonyl, aminosulfonyl, N-lower alkyl
• n and m are independently of each other 0 or 1 or 2, preferably 0;
• R 3 , R 4 , R 8 , R 10 are independently of each other hydrogen, lower alkyl, lower alkenyl or lower alkadienyl, which are each unsubstituted or monosubstituted or polysubsituted, preferably monosubstituted or disubstituted by a substituent independently selected from lower alkyl; hydroxy; lower alkoxy, which may be unsubstituted or mono-, di-, or trisubstituted by (i) heterocyclyl with 4 to 12 ring atoms, which may be unsaturated, wholly saturated, or partly saturated, is monocyclic or bicyclic and may contain up to three heteroatoms selected from nitrogen, oxygen and sulfur, and is most especially pyrrolyl, for example 2-pyrrolyl or 3-pyrrolyl, pyridyl, for example 2-, 3- or 4- pyridyl, or in a broader sense also thienyl, for example 2- or 3-thienyl, or
• phenyl, naphthyl, phenyl-lower alkyl or phenyl-lower alkenyl with a terminal phenyl radical which is unsubstituted or monosubstituted or disubstituted by the radicals named above as substituents of lower alkyl, lower alkenyl or lower alkadienyl;
• heterocyclyl-lower alkyl wherein heterocyclyl is pyrrolyl, for example 2-pyrrolyl or 3-pyrrolyl, pyridyl, for example 2-, 3- or 4-pyridyl, or in a broader sense also thienyl, for example 2- or 3-thienyl, or furyl, for example 2-furyl, indolyl, typically 2- or 3-indolyl, quinolyl, typically 2- or 4-quinolyl, isoquinolyl, typically 3- or 5-isoquinolyl, benzofuranyl, typically 2-benzofuranyl, chro- menyl, typically 3-chromenyl, benzothienyl, typically 2- or 3-benzothienyl; imidazolyl, typically 1- or 2-imidazolyl, pyrimidinyl, typically 2-or 4-pyrimidinyl, oxazolyl, typically 2-oxazolyl, isoxazolyl, typically 3-isoxazolyl, thi
• R 4 may also be absent for the compound of formula II;
• R 4 is absent for compounds of formula II, hydrogen or CH 3 for compounds of formula I, and
• [0088] is acyl of the subformula R°-SO 2 -
• R 0 in the said radicals has the following meanings: substituted or unsubstituted lower alkyl, especially methyl or ethyl, amino-lower alkyl hydroxy-lower alkyl, wherein the amino group is unprotected or is protected by a conventional amino protecting group - especially by lower alkoxycarbonyl, typically tert-lower alkoxycarbonyl, for example tert-butoxycarbonyl - e.g. aminomethyl, R,S-, R- or preferably S-1-aminoethyl, tert-butoxycarbonylaminomethyl or R 5 S-, R-, Case 50270A
• p is 0 if R 4 is absent, or is 1 if R 3 and R 4 are both present and in each case are one of the aforementioned radicals (for compounds of formula II);
• Rs is hydrogen or lower alkyl, especially hydrogen
• X stands for 2 hydrogen atoms, for O, or for 1 hydrogen atom and hydroxy; or for 1 hydrogen atom and lower alkoxy;
• Z is hydrogen or especially lower alkyl, most especially methyl
• R 3 and R 4 are independently of each other hydrogen, lower alkyl unsubstituted or mono- or disubstituted, especially monosubstituted, by radicals selected independently of one another from carboxy; lower alkoxycarbonyl; and cyano;
• R 4 is hydrogen or -CH 3 .
• R 3 is as defined above or preferably R 3 is, acyl of the subformula R°-CO, wherein R 0 is lower alkyl; amino-lower alkyl, wherein the amino group is present in unprotected form or is protected by lower alkoxycarbonyl; tetrahydropyranyloxy-lower alkyl; phenyl; imidazolyl-lower alkoxyphenyl; carboxyphenyl; lower alkoxycarbonylphenyl; halogen-lower alkylphenyl; imidazol- 1-ylphenyl; pyrrolidino-lower alkylphenyl; piperazino-lower alkylphenyl; (4-lower alkylpiperazinomethyl)phenyl; morpholino-lower alkylphenyl; piperazinocarbonylphenyl; or (4- lower alkylpiperazino)phenyl;
• acyl of the subformula R°HN-C( W)-, wherein W is oxygen and R° has the following meanings: morpholino-lower alkyl, phenyl, lower alkoxyphenyl, carboxyphenyl, or lower alkoxycarbonylphenyl;
• R 3 is lower alkylphenylsulfonyl, typically 4-toluenesulfonyl;
• R 5 is hydrogen or lower alkyl, especially hydrogen
• X stands for 2 hydrogen atoms or for O
• Z is methyl or hydrogen; fOOl 11 ) or a salt thereof, if at least one salt-forming group is present.
• R 3 and R 4 are independently of each other hydrogen, lower alkyl unsubstituted or mono- or disubstituted, especially monosubstituted, by radicals selected independently of one another from carboxy; lower alkoxycarbonyl; and cyano; whereby R 4 may also be absent;
• R 3 is acyl from the subformula R°-CO, wherein R 0 is lower alkyl, especially methyl or ethyl; amino-lower alkyl, wherein the amino group is unprotected or protected by lower alkoxycarbonyl, typically tert-lower alkoxycarbonyl, for example tert-butoxycarbonyl, e.g.
• acyl of the subformula R°HN-C( W)-, wherein W is oxygen and R 0 has the following preferred meanings: morpholino-lower alkyl, typically 2-morpholinoethyl, phenyl, lower alkoxyphenyl, typically 4-methoxyphenyl or 4-ethoxyphenyl, carboxyphenyl, typically 4- carboxyphenyl, or lower alkoxycarbonylphenyl, typically 4-ethoxycarbonylphenyl;
• p is 0 if R 4 is absent, or is 1 if R 3 and R 4 are both present and in each case are one of the aforementioned radicals;
• R 5 is hydrogen or lower alkyl, especially hydrogen, [00123] X stands for 2 hydrogen atoms or for O; [00124
• R 4 is selected from N-[0-(tetrahydropyran-4-yl )- D-lactoyl]; N-[2-(tetrahydro-pyran-4-yloxy)-acetyl)]
• CAS CHEMICAL ABSTRACTS registry number
• the preferred STAUROSPORINE DERIVATIVE according to the invention is N- [(95, 1 OR, 1 IR, 13 ⁇ )-2,3, 10,1 1,12, 13-hexahydro- 10-methoxy-9-methyl- 1 -oxo-9, 13-epoxy-l//,9//- diindolo[l,2,3-gh:3',2',r-lm]pyrrolo[3,4-j][l,7]benzodiazonin-l l-yl]- ⁇ f -methylbenzamide ofthe formula (VII): Case 50270A
• Compound of formula VII is also known as MIDOSTAURIN [International Nonproprietary Name] or PKC412.
• MIDOSTAURIN is a derivative of the naturally occurring alkaloid staurosporine, and has been specifically described in the European patent No. 0 296 110 published on December 21, 1988, as well as in US patent No. 5;093,330 published on March 3, 1992, and Japanese Patent No. 2 708 047.
• Nucleic acid molecules that inhibit apoptosis or programmed cell death include, but are not limited to, antisense oligonucleotides and RNAi constructs specific to the gene encoding the antiapoptotic MCLl protein or to transcripts of this gene. Most preferred for use in the inventive combination is McI-I specific RNAi construct, including especially a mcl-1 specific siRNA. Exemplary mcl-1 RNAi constructs of the invention are described in the examples.
• the protein encoded by myeloid cell leukemia sequence 1 belongs to the Bcl-2 family. Alternative splicing occurs at this gene locus, resulting in two transcript variants encoding distinct isoforms.
• the longer gene product (isoform 1) enhances cell survival by inhibiting apoptosis.
• the alternatively spliced shorter gene product (isoform 2) promotes apoptosis and is death-inducing.
• Aliases of the mcl-1 gene include but are not limited to EAT, MCLlL, MCLlS, MGC 104264, MGC 1839, and TM.
• Alternative designations of the MCLl polypeptide include, but are not limited to: induced myeloid leukemia cell differentiation protein Mcl-1; myeloid cell leukemia sequence 1; and myeloid cell leukemia sequence 1, isoform 1.
• nucleic acid molecule is intended to include DNA molecules (e.g., cDNA or genomic DNA or oligonucleotides) and RNA molecules (e.g., mRNA or RNAi constructs such as siRNA, shRNA) and analogs of the DNA or RNA molecules generated using nucleoside analogs.
• the nucleic acid molecule can be single stranded or double stranded. Where single-stranded, the nucleic acid can be a sense strand or an antisense strand.
• the term includes synthetic (e.g., chemically synthesized) DNA.
• Nucleic acids can be synthesized using nucleoside analogs or derivatives (e.g., inosine or phosphorothioate nucleotides). Such nucleosides can be used, for example, to prepare nucleic acids that have altered base-pairing abilities or increased resistance to nucleases.
• nucleoside analogs or derivatives e.g., inosine or phosphorothioate nucleotides.
• Such nucleosides can be used, for example, to prepare nucleic acids that have altered base-pairing abilities or increased resistance to nucleases.
• the invention encompasses RNAi or antisense nucleic acid molecules specific to a mcll nucleotide sequence, including, e.g., the mcl-1 cDNA.
• the cDNA sequence of the longer mcl-1 isoform 1 (GenBank accno. NM_021960) is provided in SEQ ID NO:1.
• GenBank accno. NM [_021960, 4020 bp linearmRNA , PRI 12-JUN-2006 [Homo sapiens] (SEQ ID NO:1)
• the invention further encompasses nucleic acid molecules that differ from the nucleotide sequence ofSEQ ID NO.l due to degeneracy ofthe genetic code but encode the same mcl-1 protein as that encoded by SEQ ID NO:1.
• the 350 aa MCLl polypeptide (GenPep accno. NP068779) encoded by the nucleotide ofSEQ ID NO:1 is provided as SEQ ID NO:2.
• DNA sequence polymorphisms that lead to changes in the amino acid sequences of mcl-1 may exist within a given population (e.g., a human population). Such genetic polymorphisms may exist within a population due to natural allelic variation. Such natural allelic variations can typically result in 15% variance in the nucleotide sequence of the mcl-1 gene. Any and all such nucleotide variations, whether "silent" or resulting in amino acid polymorphisms that are the result of natural allelic variation and that do not alter the functional activity of mcl-1, are intended to be within the scope of the invention. Thus, Case 50270A
• amino acids in mcl-1 can be replaced by another amino acid, e.g., by conservative substitutions.
• allelic variants of the mcl-1 sequence that may exist in the population, skilled practitioners will appreciate that changes can be introduced by mutating the nucleotide sequence of SEQ ID NO:1, thereby leading to changes in the amino acid sequence of the encoded protein without altering the functional ability of the protein. For example, one can make nucleotide substitutions leading to amino acid substitutions at "non essential” amino acid residues.
• a "non essential” amino acid residue is a residue that can be altered from the wild type sequence of human mcl-1 protein without altering the biological activity of the mcl-1 protein, whereas an "essential" amino acid residue is required for biological activity.
• amino acid residues that are conserved among mcl-1 proteins of various species are predicted to be particularly unamenable to alteration.
• nucleic acid molecules encoding mcl-1 proteins that contain changes in amino acid residues that are not essential for activity are included in the present invention.
• Such mcl-1 proteins differ in amino acid sequence from SEQ ID NO:2, and yet retain at least a portion of the wild-type's biological activity.
• the isolated nucleic acid molecule includes a sequence encoding a protein that includes an amino acid sequence at least about 75% identical, e.g., 80%, 85%, 90%, 95% or 98% identical to the amino acid sequence of SEQ ID NO:2, over the entire length of SEQ ID NO:2.
• An isolated nucleic acid molecule encoding a mcl-1 protein having a sequence differing from SEQ ID NO:2 can be created by introducing one or more nucleotide substitutions, additions or deletions into SEQ ID NO:1, such that one or more amino acid substitutions, additions or deletions are introduced into the encoded protein. Mutations can be introduced by standard techniques, e.g., site directed mutagenesis and PCR mediated mutagenesis. Conservative amino acid substitutions can be made at one or more predicted non essential amino acid residues. Thus, for example, up to 1%, 2%, 3%, 5%, or 10% of the amino acids can be replaced by conservative substitution.
• a “conservative amino acid substitution” is one in which an amino acid residue is replaced with another having a similar side chain.
• Families of amino acid residues having similar side chains are known in the art. They include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).
• mutants can be screened for mcl-1 biological activity to identify mutants that retain activity.
• the protein can be expressed and its activity determined.
• the present invention also encompasses antisense nucleic acids molecules, i.e., molecules that are complementary to a sense strand, e.g., complementary to the coding strand of a double stranded cDNA molecule or complementary to an mRNA sequence.
• An antisense nucleic acid can hydrogen bond to the corresponding sense strand.
• the antisense nucleic acid can be complementary to the entire mcl-1 -coding sequence, or to only a portion thereof.
• An antisense nucleic acid molecule can be antisense to a noncoding region of the sense strand.
• the noncoding regions are the 5' and 3' sequences that flank the coding region of a gene or mRNA and are not translated.
• the antisense nucleic acid molecule can be complementary to the entire coding region of mcl-1 mRNA, or, in some instances, complementary to only a portion of the coding or 3' or 5' noncoding region of mcl-1 mRNA.
• An antisense oligonucleotide can be, for example, about 5, e.g., about 10, 15, 18, 20, 25, 30, 35, 40, 45 or about 50 nucleotides in length.
• antisense nucleic acids For general guidance concerning antisense nucleic acids, see, e.g., Goodchild, "Inhibition of Gene Expression by Oligonucleotides," in Topics in Molecular and Structural Biology, Vol. 12: Oligodeoxynucleotides (Cohen, ed.), MacMillan Press, London, pp. 53-77 (1989).
• exemplary useful antisense oligonucleotides include, but are not limited to, antisense oligonucleotides that include a sequence of at least 12 nucleotides complementary to a region between nucleotides 131 and 1183 of SEQ ID NO:1. See also SEQ ID NOS:7-9 in the Examples.
• An antisense nucleic acid of the invention can be constructed using chemical synthesis, enzymatic ligation reactions and other art-known procedures.
• an antisense nucleic acid e.g., an antisense oligonucleotide
• an antisense nucleic acid can be synthesized using naturally occurring nucleotides or variously modified nucleotides designed to increase the biological stability of the molecules and/or physical stability of the duplex formed between antisense and sense nucleic acids.
• phosphorothioate derivatives and acridine substituted nucleotides can be used.
• modified nucleotides useful for generating antisense nucleic acids include 5 fluorouracil, 5 bromouracil, 5 chlorouracil, 5 iodouracil, hypoxanthine, xanthine, 4 acetylcytosine, 5 (carboxyhydroxylmethyl) uracil, 1 methylguanine, 5 carboxymethylaminomethyl 2 thiouridine, 2,2 dimethylguanine, dihydrouracil, beta D galactosylqueosine, 5 carboxymethylaminomethyluracil, inosine, N6 isopentenyladenine, 1 methylinosine, 2 methyladenine, 2 methylguanine, 4 thiouracil, 3 methylcytosine, 5 methylcytosine, N6 adenine, 7 methylguanine, 5 methoxyuracil, 5 methylaminomethyluracil, 5 methoxyaminomethyl 2 thiouracil, pseudouracil, que
• - 34 - isopentenyladenine, uracil 5 oxyacetic acid (v), wybutoxosine, 2 thiocytosine, 2 thiouracil, 5 methyluracil, uracil 5 oxyacetic acid methylester, 5 methyl 2 thiouracil, 3 (3 amino 3 N 2 carboxypropyl) uracil, (acp3)w, and 2,6 diaminopurine.
• an antisense nucleic acid can be produced using an expression vector, into which a nucleic acid has been subcloned in an antisense orientation (i.e., RNA transcribed from the inserted nucleic acid will be complementary to a target nucleic acid of interest, described further in the following subsection).
• the antisense nucleic acid molecules of the invention can be administered to a subject or generated in situ such that they hybridize with or bind to cellular mRNA and/or genomic DNA encoding a mcl-1 protein.
• the antisense nucleic acids can inhibit expression of the protein, e.g., by inhibiting transcription and/or translation.
• Hybridization can be by conventional nucleotide complementarity to form a stable duplex or, in the case of an antisense nucleic acid molecule that binds to DNA duplexes, through specific interactions in the major groove of the double helix.
• Antisense nucleic acids can be administered by any method known in the art, e.g., by direct injection at a tissue site.
• antisense nucleic acid molecules can be adapted to target select cells, so that they can be administered systemically.
• antisense molecules can be modified such that they bind specifically to receptors or antigens expressed on a selected cell surface, e.g., by linking the antisense nucleic acid molecules to peptides or antibodies that bind to cell surface receptors or antigens.
• Antisense nucleic acid molecules can also be delivered to cells using certain vectors described herein. Vector constructs in which the sequence to be transcribed is placed under the control of a strong pol II or pol III promoter can be used to achieve sufficient intracellular concentrations of antisense transcripts.
• An antisense nucleic acid molecule of the invention can be an ⁇ anomeric nucleic acid molecule.
• An ⁇ anomeric nucleic acid molecule forms specific double stranded hybrids with complementary RNA in which, contrary to the usual ⁇ units, the strands run parallel to each other (Gaultier et al. (1987) Nucleic Acids Res 15:6625 6641).
• the antisense nucleic acid molecule can also include a 2' o methylribonucleotide (Inoue et al. (1987) Nucleic Acids Res 15:6131 6148) or a chimeric RNA DNA analogue (Inoue et al. (1987) FEBS Lett 215:327 330).
• RNAi RNA interference
• dsRNA double-stranded RNA
• the invention also includes nucleic acids capable of suppressing expression of mcl-1 by way of RNA interference (RNAi).
• RNAi is a process whereby double-stranded RNA (dsRNA) induces the sequence-specific degradation of homologous mRNA in animals and plant cells (Hutvagner and Zamore (2002) Cur. Opin Gene. Dev 12:225-232; Sharp (2001) Genes Dev. 15:485- 490.
• RNAi can be triggered by, e.g., 21-nucleotide (nt) duplex RNAs, also called small inhibitory RNAs (siRNAs) (Chiu et al (2001) MoI Cell 10:549-561; Elbashir et al. Case 50270A
• RNA polymerase III promoters Zeng et al. (2002) MoI Cell 9:1327-1333; Paddison et al. (2002) Genes Dev 16:948-958; Lee et al.
• RNAi by expression of siRNAs and shRNAs in mammalian cells has been described by Yu et al. ((2002) Proc Natl Acad Sci USA 99:6047-6052).
• the present invention includes siRNAs, duplexes thereof (i.e., dsRNAs), shRNAs, and miRNAs, which interact with (e.g., bind) a mcl-1 target sequence (e.g., a mcl-1 mRNA (e.g., an mRNA corresponding to human mcl-1 sequences such as those shown herein)).
• a mcl-1 target sequence e.g., a mcl-1 mRNA (e.g., an mRNA corresponding to human mcl-1 sequences such as those shown herein)
• Such nucleic acids can be, e.g., chemically synthesized RNA oligonucleotides (see Elbashir et al., supra).
• Exemplary RNAi sequences encompassed by and useful in the present invention include at least one of the following sequences, or its complementary strand sequence:
• Plasmids can be used to transcribe shRNAs that can function as siRNAs (see Sui et al. (2002) Proc Natl Acad Sci USA 99:5515-5520; Brummelkamp et al. (2002) Science 296:550-553).
• Such plasmids can contain, e.g., an RNA polymerase III promoter, followed by sequence that is transcribed into an RNA containing a sense sequence, a loop structure, and an antisense sequence.
• Vectors e.g., plasmids
• a promoter e.g., an RNA polymerase III promoter
• a sequence that is transcribed into an RNA containing a mcl-1 "sense" sequence, a loop structure, and the corresponding mcl- 1 "antisense” sequence can be made using procedures that are routine in the art.
• vectors of the present invention can include a U6 promoter and sequences that are transcribed into siRNAs with uridine overhangs (e.g., 1-6 uridines at the 3' terminus).
• uridine overhangs e.g., 1-6 uridines at the 3' terminus.
• An exemplary shRNA encompassed by and useful in the present invention is described in the Examples section, which is transcribed from the sequences set forth herein as SEQ ID NOs: 9 and 10, respectively (see Table 1, below).
• miRNAs are excised from an approximately 70-nucleotide precursor RNA stem-loop (the shRNA), possibly by the RNase Ill-type enzyme known as Dicer or a homolog thereof.
• the shRNA RNA stem-loop
• Dicer RNase Ill-type enzyme
• a vector construct that expresses the miRNA can be used to produce siRNAs to initiate RNAi against the target in mammalian cells (Zeng, supra).
• the nucleic acids of the invention can be included in, and expressed by, viral vectors. Accordingly, the invention features viral vectors that induce specific silencing of mcl-1 through expression of siRNA.
• mcl-1 -specific adenoviral vectors can be constructed by generating recombinant adenoviruses expressing siRNA under RNA Pol II promoter transcription control.
• a siRNAs or other nucleic acid-based inhibitor can be administered to animals and humans using any art-known method.
• a "high-pressure" delivery technique can be used, such as a rapid injection (for example, an injection that is complete within a few seconds) of a large volume of an siRNA-containing solution into the animal via an artery or vein (see, e.g., Lewis (2002) Nature Genetics 32:107-108).
• Microparticles, nanoparticles and liposomes can also be used to deliver siRNAs.
• siRNAs that target mcl-1 mRNA and that are associated with a microparticle, nanoparticle or liposome, or that are otherwise formulated for delivery to a biological cell are within the scope of the present invention.
• a dsRNA molecule of the present invention can include about 15 or more (e.g., 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more) nucleotides in each strand.
• the strands of the dsRNA can be substantially complementary to a target region of mcl-1 mRNA.
• one strand can be about 90% (e.g., about 90%, 95%, or 100%) complementary to a target region of mcl-1 mRNA.
• the target region can be any region of the mcl-1 nucleic acid sequence, e.g., a region corresponding to nucleotides 1 to 228 of SEQ ID NO:1, e.g., within the region encoding a polypeptide of amino acids 1 to 76 of SEQ ID NO:2.
• a dsRNA molecule of the invention can be produced by, e.g., chemically synthesizing the molecule, transcribing the molecule in vitro from a DNA template, making the molecule in vivo from, e.g., shRNA (generation of dsRNAs and other inhibitory nucleic acids is discussed further above), or using any other method known in the art.
• each strand of a dsRNA can be chemically synthesized using naturally occurring nucleotides or variously modified nucleosides designed to increase the biological stability of the molecules or to increase the physical stability of the duplex formed between the antisense and sense strands of the dsRNA (e.g., phosphorothioate derivatives and acridine substituted nucleosides can be used).
• naturally occurring nucleotides or variously modified nucleosides designed to increase the biological stability of the molecules or to increase the physical stability of the duplex formed between the antisense and sense strands of the dsRNA (e.g., phosphorothioate derivatives and acridine substituted nucleosides can be used).
• the dsRNA can also be produced using an expression vector into which a nucleic acid including both the sense and antisense sequences in the same strand has been cloned (e.g., an RNA transcribed from the inserted nucleic acid may form a hairpin, the stem of which forms the dsRNA that includes an antisense strand that is complementary to a mcl-1 RNA). Accordingly, one can make a dsRNA that inhibits mcl-1 expression by generating (or designing) a dsRNA that includes a sequence that is the reverse and complement of a given mcl-1 mRNA sequence.
• the dsRNA can be tested in mcl-1 -expressing cells.
• An anti- mcl-1 dsRNA will decrease expression of mcl-1 (i.e., will decrease the levels of mcl-1 protein and mcl-1 mRNA) in the cells, mcl-1 expression can be assayed by methods known in the art.
• levels of mcl-1 protein can be assayed by Western blot analysis, by immunoprecipitation, by ELISA, or by a functional assay.
• Levels of mcl-1 mRNA can be assayed by Northern blot, in situ analysis, or reverse transcription coupled with polymerase chain reaction (RT-PCR).
• Ribozymes are catalytic RNA molecules with ribonuclease activity that are capable of cleaving a single stranded nucleic acid (e.g., mRNA) to which they have a complementary region.
• ribozymes e.g., hammerhead ribozymes (described in Haselhoff and Gerlach (1988) Nature 334:585 591)
• a ribozyme having specificity for a mcl-1 encoding nucleic acid can be designed based upon the nucleotide sequence of a mcl-1 cDNA disclosed herein.
• a derivative of a Tetrahymena L 19 IVS RNA can be constructed in which the nucleotide sequence of the active site is complementary to the nucleotide sequence to be cleaved in a mcl-1 encoding mRNA. See, e.g., Cech et al. U.S. Patent No. 4,987,071; and Cech et al. U.S. Patent No. 5,116,742.
• mcl-1 mRNA can be used to Case 50270A
• RNA molecules having a specific ribonuclease activity from a pool of RNA molecules. See, e.g., Bartel and Szostak (1993) Science 261 :141 1 1418.
• the invention also encompasses nucleic acid molecules that form triple helical structures.
• mcl-1 gene expression can be inhibited by targeting nucleotide sequences complementary to a regulatory region of the mcl-1 gene (e.g., the mcl-1 promoter and/or enhancers) to form triple helical structures that prevent transcription of the mcl-1 gene in target cells.
• a regulatory region of the mcl-1 gene e.g., the mcl-1 promoter and/or enhancers
• the nucleic acid molecules of the invention are modified at the base moiety, sugar moiety or phosphate backbone to improve, e.g., the stability, hybridization or solubility of the molecule.
• the deoxyribose phosphate backbone of the nucleic acids can be modified to generate peptide nucleic acids (see Hyrup et al. (1996) Bioorganic & Medicinal Chemistry 4(1):5 23).
• peptide nucleic acids refer to nucleic acid mimics, e.g., DNA mimics, in which the deoxyribose phosphate backbone is replaced by a pseudopeptide backbone and only the four natural nucleobases are retained.
• the neutral backbone of PNAs has been shown to allow for specific hybridization to DNA and RNA under conditions of low ionic strength.
• the synthesis of PNA oligomers can be performed using standard solid phase peptide synthesis protocols as described in Hyrup et al. (1996) supra; Perry O'Keefe et al. (1996) Proc. Natl. Acad. Sci. USA 93:14670 675.
• mcl-1 PNAs can be used for therapeutic and diagnostic applications.
• PNAs can be used as antisense or antigene agents for sequence specific modulation of gene expression by, e.g., inducing transcription or translation arrest or inhibiting replication of the gene
• mcl-1 PNAs can also be used, e.g., in the analysis of single base pair mutations in a gene by, e.g., PNA directed PCR clamping; as artificial restriction enzymes when used in combination with other enzymes, e.g., Sl nucleases (Hyrup (1996) supra); or as probes or primers for DNA sequence and hybridization (Hyrup (1996) supra; Perry O'Keefe et al. (1996) Proc. Natl. Acad. Sci. USA 93: 14670 675).
• mcl-1 PNAs can be modified, e.g., to enhance their stability or cellular uptake, by attaching lipophilic or other helper groups to PNA, by the formation of PNA DNA chimeras, or by the use of liposomes or other techniques of drug delivery known in the art.
• mcl- 1 PNA DNA chimeras can be generated that may combine the advantageous properties of PNA and DNA.
• Such chimeras allow DNA recognition enzymes, e.g., RNAse H and DNA polymerases, to interact with the DNA portion while the PNA portion provides high binding affinity and specificity.
• PNA DNA chimeras can be linked using linkers of appropriate lengths Case 50270A
• a DNA chain can be synthesized on a solid support using standard phosphoramidite coupling chemistry and modified nucleoside analogs, e.g., 5' (4 methoxytrityl)amino 5' deoxy thymidine phosphoramidite, can be used as a linker between the PNA and the 5' end of DNA (Mag et al. (1989) Nucleic Acid Res.
• PNA monomers are then coupled in a stepwise manner to produce a chimeric molecule with a 5' PNA segment and a 3' DNA segment (Finn et al. (1996) Nucleic Acids Research 24(17):3357 63).
• chimeric molecules can be synthesized with a 5' DNA segment and a 3' PNA segment (Peterser et al. (1975) Bioorganic Med. Chem. Lett. 5: 11 19 11124).
• the oligonucleotide may include other appended groups such as peptides (e.g., for targeting host cell receptors in vivo) or agents facilitating transport across the cell membrane (see, e.g., Letsinger et al. (1989) Proc. Natl. Acad. Sci. USA 86:6553 6556; Lemaitre et al. (1987) Proc. Natl. Acad. Sci. USA 84:648 652; PCT Publication No. WO 88/09810) or the blood brain barrier (see, e.g., PCT Publication No. WO 89/10134).
• peptides e.g., for targeting host cell receptors in vivo
• agents facilitating transport across the cell membrane see, e.g., Letsinger et al. (1989) Proc. Natl. Acad. Sci. USA 86:6553 6556; Lemaitre et al. (1987) Proc. Natl. Acad.
• oligonucleotides can be modified with hybridization triggered cleavage agents (see, e.g., Krol et al. (1988) Bio/Techniques 6:958 976) or intercalating agents (see, e.g., Zon (1988) Pharm. Res. 5:539 549).
• the oligonucleotide may be conjugated to another molecule, e.g., a peptide, hybridization triggered cross linking agent, transport agent or hybridization triggered cleavage agent.
• vectors e.g., expression vectors, containing a mcl-1 nucleic acid (or a portion thereof).
• vector refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.
• a plasmid i.e., a circular double stranded DNA loop into which additional DNA segments can be ligated.
• a viral vector Another type is a viral vector, wherein additional DNA segments can be ligated into the viral genome.
• Certain vectors are capable of autonomous replication in a host cell into which they are introduced. Therefore, also included in the invention are host cells containing the mcl-1 nucleic acid containing vector. The choice of vector and host cell is routine to a skilled practicioner of the art.
• the present invention provides a method of treating myelodysplastic syndromes, lymphomas and leukemias, in particular Systemic mastocytosis, and also acute myeloid leukemia (AML) and also solid tumors such as e.g. colorectal cancer (CRC) and non-small cell lung cancer (NSCLC), comprising administering to a mammalin need of such a treatment a therapeutically Case 50270A
• a FLT-3 kinase inhibitor and mcl-1 inhibitor such as an antisense oligonucleotide or a mcl- 1 -specific RNAi construct
• the instant invention provides a method for treating mammals, especially humans, suffering from myelodysplastic syndromes, lymphomas and leukemias, in particular Systemic mastocytosis, and also acute myeloid leukemia (AML) and also solid tumors such as e.g.
• myelodysplastic syndromes lymphomas and leukemias, in particular Systemic mastocytosis, and also acute myeloid leukemia (AML) and also solid tumors such as e.g.
• lymphomas and leukemias in particular Systemic mastocytosis
• AML acute myeloid leukemia
• solid tumors such as e.g.
• CRC colorectal cancer
• NSCLC non-small cell lung cancer
• administering to a mammal in need of such treatment an therapeutically effective amount of a combination of N- [(95, 10 ⁇ , 11 R, 13 ⁇ )-2,3 , 10, 11 , 12, 13-hexahydro- 10-methoxy-9-methy 1- 1 -oxo-9, 13-epoxy- 1 H, 9H- diindolo[ 1 ,2,3-gh:3',2', 1 '-lm]pyrrolo[3,4-j][l ,7]benzodiazonin- 11 -yl]-7V-methylbenzamide of the formula (VII), or a pharmaceutically acceptable salt thereof and an antisense oligonucleotide or a mcl-1 -specific RNAi construct.
• the instant invention relates to the use of a combination of a FLT-3 kinase inhibitor and antisense oligonucleotide or a mcl-1 -specific RNAi construct for treating myelodysplastic syndromes, lymphomas and leukemias, in particular Systemic mastocytosis, and also acute myeloid leukemia (AML) and also solid tumors such as e.g. colorectal cancer (CRC) and non-small cell lung cancer (NSCLC).
• CRC colorectal cancer
• NSCLC non-small cell lung cancer
• the instant invention relates to the use of a combination of a FLT-3 kinase inhibitor and a antisense oligonucleotide or a mcl-1 -specific RNAi construct for the preparation of a pharmaceutical composition for treating myelodysplastic syndromes, lymphomas and leukemias, in particular Systemic mastocytosis, and also acute myeloid leukemia (AML) and also solid tumors such as e.g. colorectal cancer (CRC) and non-small cell lung cancer (NSCLC).
• CRC colorectal cancer
• NSCLC non-small cell lung cancer
• a combination of N- [(95,10/?, 11 ⁇ ,13 ⁇ )-2,3,1O,1 1,12,13- hexahydro- 10-methoxy-9-methyl- 1 -oxo-9,13-epoxy- l//,9//-diindolo[ 1 ,2,3-gh:3',2', 1 '- lm]pyrrolo[3,4-j][l,7]benzodiazonin-l l-yl]-N-methylbenzamide of the formula (VII), or a pharmaceutically acceptable salt thereof and an antisense oligonucleotide or a mcl-1 -specific RNAi construct are the preferred combinations of a FLT-3 kinase inhibitor and an antisense oligonucleotide or a mcl-1 -specific RNAi construct.
• myelodysplastic syndromes lymphomas and leukemias, in particular Systemic mastocytosis, and also acute myeloid leukemia (AML) and also solid tumors such as e.g. colorectal cancer (CRC) and non-small cell lung cancer (NSCLC)
• CRC colorectal cancer
• NSCLC non-small cell lung cancer
• the present invention also relates to a combination, such as a combined preparation or a pharmaceutical composition, which comprises (a) a FLT-3 inhibitor, especially the FLT-3 inhibitors specifically mentioned hereinbefore, in particular those mentioned as being preferred, and (b) an an antisense oligonucleotide or a mcl-1 -specific RNAi construct, in which the active ingredients (a) and (b) are present in each case in free form or in the form of a pharmaceutically acceptable salt or suitable biopharmaceutical formulation, for simultaneous, concurrent, separate or sequential use.
• suitable biopharmaceutical formulation are known to one skilled in the art, wherein the formulation isoptimized depending upon whether the therapeutical administration is of an RNAi construct or for an antisense oligonucleotide construct.
• a combined preparation defines especially a “kit of parts” in the sense that the combination partners (a) and (b) as defined above can be dosed independently or by use of Case 50270A
• kits of parts can then, e.g., be administered simultaneously or chronologically staggered, that is at different time points and with equal or different time intervals for any part of the kit of parts.
• the ratio of the total amounts of the combination partner (a) to the combination partner (b) to be administered in the combined preparation can be varied, e.g. in order to cope with the needs of a patient sub-population to be treated or the needs of the single patient which different needs can be due to the particular disease, severity of the disease, age, sex, body weight, etc. of the patients.
• the precise dosage of the FLT-3 inhibitor and the antisense oligonucleotide or a mcl-1 -specific RNAi construct be employed for treating the diseases and conditions mentioned hereinbefore depends upon several factors including the host, the nature and the severity of the condition being treated, the mode of administration. However, in general, satisfactory results are achieved when the FLT-3 inhibitor is administered parenterally, e.g., intraperitoneally, intravenously, intramuscularly, subcutaneously, intratumorally, or rectally, or enterally, e.g., orally, preferably intravenously or, preferably orally, intravenously at a daily dosage of 0.1 to 10 mg/kg body weight, preferably 1 to 5 mg/kg body weight.
• a preferred intravenous daily dosage is O.lto 10 mg/kg body weight or, for most larger primates, a daily dosage of 200-300 mg.
• a typical intravenous dosage is 3 to 5 mg/kg, three to five times a week.
• the FLT-3 inhibitors are administered orally, by dosage forms such as microemulsions, soft gels or solid dispersions in dosages up to about 250 mg/day, in particular 225 mg/day, administered once, twice or three times daily.
• a small dose is administered initially and the dosage is gradually increased until the optimal dosage for the host under treatment is determined.
• the upper limit of dosage is that imposed by side effects and can be determined by trial for the host being treated.
• the FLT-3 inhibitors the antisense oligonucleotide or a mcl-1 -specific RNAi construct may be combined with one or more pharmaceutically acceptable carriers and, optionally, one or more other conventional pharmaceutical adjuvants and administered enterally, e.g. orally, in the form of tablets, capsules, caplets, etc. or parenterally, e.g., intraperitoneally or intravenously, in the form of sterile injectable solutions or suspensions.
• enteral and parenteral compositions may be prepared by conventional means.
• the infusion solutions according to the present invention are preferably sterile. This may be readily accomplished, e.g. by filtration through sterile filtration membranes. Aseptic formation of any composition in liquid form, the aseptic filling of vials and/or combining a Case 50270A
• composition of the present invention with a suitable diluent under aseptic conditions are well known to the skilled addressee.
• the FLT-3 inhibitors and an antisense oligonucleotide or a mcl-1 -specific RNAi construct may be formulated into enteral and parenteral pharmaceutical compositions containing an amount of the active substance that is effective for treating the diseases and conditions named hereinbefore, such compositions in unit dosage form and such compositions comprising a pharmaceutically acceptable carrier.
• compositions of FLT-3 inhibitors are described in the European patent No. 0 657 164 published on June 14, 1995.
• the described pharmaceutical compositions comprise a solution or dispersion of compounds of formula I such as MIDOSTAURIN in a saturated polyalkylene glycol glyceride, in which the glycol glyceride is a mixture of glyceryl and polyethylene glycol esters of one or more C8-C18 saturated fatty acids.
• Composition A [00228]
• Gelucire 44/14 (82 parts) is melted by heating to 60° C.
• Powdered MIDOSTAURIN (18 parts) is added to the molten material.
• the resulting mixture is homogenised and the dispersion obtained is introduced into hard gelatin capsules of different size, so that some contain a 25mg dosage and others a 75mg dosage of the MIDOSTAURIN.
• the resulting capsules are suitable for oral administration.
• Composition B [00230]
• Gelucire 44/14 (86 parts) is melted by heating to 60° C.
• Powdered MIDOSTAURIN (14 parts) is added to the molten material.
• the mixture is homogenised and the dispersion obtained is introduced into hard gelatin capsules of different size, so that some contain a 25mg dosage and others a 75mg dosage of the MIDOSTAURIN.
• the resulting capsules are suitable for oral administration.
• Gelucire 44/14 available commercially from Gattefosse is a mixture of esters of C8- Cl 8 saturated fatty acids with glycerol and a polyethylene glycol having a molecular weight of about 1500, the specifications for the composition of the fatty acid component being, by weight, 4- 10% caprylic acid, 3-9% capric acid, 40-50% lauric acid, 14-24% myristic acid, 4-14% palmitic acid and 5-15% stearic acid.
• Case 50270A is a mixture of esters of C8- Cl 8 saturated fatty acids with glycerol and a polyethylene glycol having a molecular weight of about 1500, the specifications for the composition of the fatty acid component being, by weight, 4- 10% caprylic acid, 3-9% capric acid, 40-50% lauric acid, 14-24% myristic acid, 4-14% palmitic acid and 5-15% stearic acid.
• Gelucire formulation consists of:
• MIDOSTAURIN 3.0 g filled into a 60 mL Twist off flask (00234)
• a preferred example of soft gel will contain the following Microemulsion:
• Mcl-1 The expression and functional role of Mcl-1 in neoplastic human MC has is examined. It can be shown that primary neoplastic MC in all variants of SM including ASM and MCL as well as the MCL cell line HMC-I, express Mcl-1 in a constitutive manner.
• FCM flow cytometry
• the preferred FLT-3 inhibitor also inhibits the surface expression of p-FLT-3 but not of FLT-3 (as can be determined by FCM) on MV cells.
• treatment with a preferred HDAI compound attenuates both FLT-3 and p- Case 50270A
• FLT-3 levels in a dose-dependent manner in MV and RS cells as can be determined both by Western and FCM analyses. Exposure to a preferred HDAI compound (20 to 100 nM) also down regulates the levels of p-FLT-3, p-AKT and p-ERKl/2. Significantly, co-treatment with a preferred FLT-3 inhibitor and a preferred HDAI compound hereingly induces apoptosis of MV and RS cells. This is associated with more attenuation of p-FLT-3, p-AKT and p-ERKl/2 in MV cells.
• there is at least one beneficial effect e.g., a mutual enhancing of the effect of the first and second active ingredient, in particular a synergism, e.g. a more than additive effect, additional advantageous effects, less side effects, a combined therapeutical effect in a otherwise noneffective dosage of one or both of the first and second active ingredient, and especially a strong synergism the active ingredients.
• a beneficial effect e.g., a mutual enhancing of the effect of the first and second active ingredient, in particular a synergism, e.g. a more than additive effect, additional advantageous effects, less side effects, a combined therapeutical effect in a otherwise noneffective dosage of one or both of the first and second active ingredient, and especially a strong synergism the active ingredients.
• the molar ratio of FLT-3 inhibitor/ an antisense oligonucleotide or a mcl-1 -specific RNAi construct in the combination is generally from 1/10 to 10/1, preferably from 1/5 to 5/1, e.g. Vi, 1/1, 2/1, or 3/1.
• Imatinib (STI571), PKC41228, and nilotinib (4-methyl-3-[[4-(3-pyridinyl)-2- pyrimidinyl]amino]-N-[5-(4-methyl-lH-imidazol-l-yl)-3-(t ⁇ fluoromethyl)phenyl] benzamide )29 are provided by Novartis Pharma AG (Basel, Switzerland).
• Interleukin-4 (IL-4) is purchased from Peprotech (Rocky Hill, NJ), RPMI 1640 medium and fetal calf serum (FCS) from PAA laboratories (Pasching, Austria), L- glutamine and Iscove's modified Dulbecco's medium (IMDM) from Gibco Life Technologies (Gaithersburg, MD), 3 H-thymidine from Amersham (Aylesbury, UK), and claribine (2- chlorodeoxyadenosine, 2CdA) from Janssen-Cilag (Beerse, Belgium).
• FCS fetal calf serum
• IMDM L- glutamine and Iscove's modified Dulbecco's medium
• 2CdA 2CdA
• Antisense oligonucleotides are obtained from VBC Genomics (Vienna, Austria) or ISIS Pharmaceuticals (Carlsbad, CA), and siRNA from Dharmacon (Lafayette, CO).
• Bone marrow (BM) cells are obtained from the posterior iliac crest and collected in syringes containing preservative-free heparin. In each case, informed consent is obtained before biopsy. Diagnoses are established according to WHO criteria. In two patients with mast cell leukemia (MCL) and one with mast cell sarcoma (MCS), primary neoplastic MC are purified to homogeneity (purity >98%) using the PE-labeled mAb YB5.B8 (Pharmingen, San Diego, CA) and a FACS- Vantage cell sorter (Becton Dickinson, San Jose, CA) as described. All experiments are conducted with approval by the institutional review board and in accordance with the declaration of Helsinki.
• HMC-I human mast cell line generated from leukemic cells in a patient with MCL48 are obtained (Mayo Clinic, Rochester, MN). Two subclones of HMC-I are used, namely HMC- 1.1 harboring the KIT mutation G560V but not D816V, and HMC- 1.2 cells exhibiting both the G560V mutation and D816V mutation of KIT.32 HMC-I cells are grown in IMDM medium supplemented with 10% FCS, L-glutamine, and antibiotics at 37°C and 5% CO 2 . HMC-I cells are re-thawed from an original stock every 4 to 8 weeks and passaged weekly. As control of 'phenotypic stability', HMC-I cells are periodically checked for expression of KIT and the down-modulating effect of IL-4 (100 U/ml, 48 hours).
• HMC-I cells are incubated with various concentrations of the KIT D816V-targeting TK inhibitor PKC412 (100 pM through 10 ⁇ M), various concentrations of 4-methyl-3-[[4-(3-pyridinyl)- 2-pyrimidinyl]amino]-N-[5-(4-methyl-lH-imidazol-l-yl)-3-(trifluoromethyl)phenyl] benzamide (1 nM through 100 ⁇ M), imatinib (3 nM through 300 ⁇ M), 2CdA (0.1 - 10,000 ng/mL), or control medium at 37°C and 5% CO 2 for up to 48 hours.
• PKC412 100 pM through 10 ⁇ M
• HMC-I cells are transfected with mcl-1 -specific ASO before being exposed to inhibitory drugs (see below).
• cells are transfected with various concentrations of Mcl-1 -specific ASO (50 nM - 250 nM) for up to 12 hours or with siRNA (200 nM) for 12 hours without further exposure to targeted drugs.
• Mcl-1 -specific ASO 50 nM - 250 nM
• siRNA 200 nM
• cells are subjected to 3 H-thymidine uptake experiments, analysis of cell viability and apoptosis, or Western blotting.
• RNA is isolated from HMC-I cells using Trizol (Invitrogen, Carlsbad, CA) according to the manufacturers' instructions. RNA preparation and Northern blotting are performed essentially as described. In brief, 15 ⁇ g of total RNA are size-fractionated on 1.0% formaldehyde- agarose gels and transferred to nylon membranes (Hybond N, Amersham, Aylesbury, U.K.) as described. Hybridization is performed in rapid-hyb buffer (Amersham) using 32 P-labeled cDNAs specific for mcl-1 or ⁇ -actin. The mcl-1 probe is generated by excising the full length mcl-1 cDNA Case 50270A
• Bound radioactivity is visualized by exposure to Biomax MS film (Kodak, Rochester, NY) at -80 0 C using intensifying screens (Kodak). mRNA expression levels are quantified by densitometry of autoradiograms using the E. A. S. Y. Win32 software (Herolab, Wiesloch, Germany). RT-PCR reactions are performed using the Protoscript First Strand cDNA Synthesis kit (New England Biolabs, Beverly, MA) using 1 ⁇ g RNA in a 50 ⁇ L reaction volume. PCR conditions are as follows: initial denaturation at 94°C for 60 seconds, annealing at 55°C for 60 seconds, polymerization at 72°C for 60 seconds (35 cycles), and terminal extension at 72°C for 10 minutes.
• PCR products are resolved in 1% agarose gels containing 0.5 ⁇ g/mL ethidium bromide.
• Immunocytochemistry is performed on cytospin preparations of HMC-I cells as described using anti-Mcl-1 antibody (work dilution 1 :200) and biotinylated goat anti-rabbit IgG (Biocarta, San Diego, CA). In select experiments, an McI-I- blocking peptide (Santa Cruz) is applied (work dilution 1 :40). As chromogen, alkaline phosphatase complex (Biocarta) is used. Antibody-reactivity is made visible using Neofuchsin (Nichirei, Tokyo, Japan).
• HMC-1.1 and HMC-1.2 cells are transfected with an mcl-1 -specific 2'-O- methoxyethyl/2'-deoxynucleotide chimeric phosphorothioate ASO (5'-TTG GCT TTG TGT CCT TGG CG-3') (SEQ ID NO:7) or with a scramble control oligonucleotide pool.
• Scramble control represents a mixture of A (adenine), G (guanine), T (thymine), and C (cytosine) bases, the resulting preparation containing an equimolar mixture of oligonucleotides.
• an annealed, purified, and desalted double-stranded mcl-1 siRNA (AAG AAA CGC GGU AAU CGG ACU) (SEQ ID NO:8) and AGU CCG AUU ACC GCG UUU CUU 3' (mcll, SEQ ID NO:9)) and a control siRNA against luciferase (CUU ACG CUG AGU ACU UCG A) (SEQ ID NO: 10), both obtained from Dharmacon (Lafayette, CO), are applied.
• 800,000 cells are seeded in 75 cm 2 culture plates at 37°C for 24 hours.
• Mcl-1-ASO and Mcl-1 siRNA are complexed with Lipofectin-Reagent (Invitrogen) in RPMI 1640 medium essentially as described.
• HMC-1.1 or HMC-1.2 cells are incubated with various concentrations of mcl-1 ASO (50-250 nM) or 200 nM mcl-1 -specific siRNA at 37°C for 4 hours.
• mcl-1 ASO 50-250 nM
• 200 nM mcl-1 -specific siRNA at 37°C for 4 hours.
• cells are washed and cultured in RPMI 1640 medium with 10% FCS in the presence or absence of various concentrations of TK inhibitors for another 12 hours before being analyzed.
• siRNA-treated cells are kept in control medium for 12 hours (without TK inhibitors) before being examined for the percentage of apoptotic cells and Western blotting.
• McI-I ASO or control oligonucleotides cells are cultured in 96-well microtiter plates (5 x 10 4 cells per well) in the absence or presence of various concentrations of PKC412 (100 pM through 10 ⁇ M), 4-methyl-3-[[4-(3-pyridinyl)-2-pyrimidinyl]amino]-N-[5-(4-methyl-lH-imidazol-l-yl)-3- (trifluoromethyl)phenyl] benzamide (1 nM through 100 ⁇ M), imatinib (3 nM through 300 ⁇ M), or cladribine (2CdA; HMC- 1.1 : 0.1 - 10,000 ng/mL; HMC- 1.2: 0.5 - 500 ng/mL), for 24 hours.
• PKC412 100 pM through 10 ⁇ M
• RT-PCR analysis of mcl-1 mRNA expression in K.562 cells, HMC- 1.1 cells, HMC-1.2 cells, as well as purified (purity >98%) neoplastic human mast cells is obtained from one patient with mast cell sarcoma (MCS) (#1) and two patients (#2 and #3) with mast cell leukemia (MCL).
• MCS mast cell sarcoma
• MCL mast cell leukemia
• HMC- 1.2 1.1 and HMC- 1.2 is also found to express the McI-I protein as determined by immunocytochemistry.
• the reactivity of the anti-Mcl-1 antibody with HMC- 1.1 cells after preincubation with control medium or an McI-I -specific blocking peptide is performed, and the same result is obtained with HMC- 1.2 cells.
• mcl-1 mRNA in primary neoplastic MC and HMC-I cells we examine expression of mcl-1 mRNA in primary neoplastic MC and HMC-I cells. As assessed by Northern blotting, both HMC- 1.1 cells and HMC- 1.2 cells are found to express mcl-1 mRNA. Expression of mcl-1 mRNA in HMC-I cells is confirmed by RT-PCR analysis. Moreover, we are able to demonstrate expression of mcl-1 mRNA in highly purified primary neoplastic MC in patients with MCL or MCS, by RT-PCR analysis.
• METHOD Effects of mcl- 1 antisense oligonucleotides on McI-I protein expression and cell viability in HMC- 1.1 and HMC- 1.2 cells are analyzed.
• HMC- 1.1 cells and HMC- 1.2 cells harbouring KIT D816V are transfected with an mcl-1 antisense oligonucleotide at 250 nM, a scramble control, or are left untransfected for 12 hours before being analyzed.
• Western Blot analysis of Mcl-1 expression is performed using an anti-Mcl-1 antibody.
• ⁇ -Actin served as loading control.
• Evaluation of non-viable (trypan blue-positive) cells is expressed as percentage of all nucleated cells. Numbers (%) of apoptotic cells. Results represent the mean ⁇ S.D. from 3 independent experiments.
• ⁇ -Actin serves as loading control.
• Time-dependent effects of mcl-1 antisense oligonucleotides (250 nM) and a scramble Control (250 nM) on expression of the Mcl-1 protein in HMC-1.1 cells and HMC-1.2 cells is observed.
• Mcl-1 expression is determined by Western blotting with ⁇ -Actin serving as a loading control. The time-dependent effects of the mcl-1 antisense oligonucleotides (250 nM) and of the scramble Control (250 nM) on cell viablity, i.e. the Case 50270A
• McI-I As a survival molecule and potential target in SM, McI-I is knocked down in HMC- 1.1 cells as well as in HMC- 1.2 cells by an antisense oligonucleotide (ASO) approach. As determined by Western blotting, transfection of both cell lines with mcl-1 ASO (250 nM) virtually abolished expression of the McI-I protein when comparing to the scramble control or to non-transfected cells.
• ASO antisense oligonucleotide
• HMC-I neoplastic MC
• METHOD [001] Effects of mcl-1 -siRNA are analyzed in neoplastic mast cells on expression of Mcl-1 protein and cell viability in HMC-1.1 cells and HMC-1.2 cells.
• Cells are left untransfected (Control) or are transfected with either an mcl-1 -specific siRNA (mcl-1 -siRNA; 200 nM) or a luciferase-specific (control)-siRNA (luc-siRNA; 200 nM) as described herein.
• Mcl-1 protein expression is determined by Western blotting using a polyclonal anti-Mcl-1 antibody. Equal loading is confirmed by probing for ⁇ -Actin.
• Cell viability is analyzed by recording the percentage (%) of apoptotic cells. Results represent the mean ⁇ S.D. from 3 independent experiments in each set of experiments.
• RESULTS In a next step, we apply an mcl-1 -specific siRNA to further demonstrate the role of mcl-1 as a survival-related molecule and potential target in neoplastic MC.
• the mcl-1 siRNA is found to downregulate expression of the Mcl-1 protein in HMC- 1.1 cells and HMC-1.2 cells.
• the siRNA induced down-regulation of Mcl-1 is found to be associated with an increase in apoptotic cells in HMC- 1.1 cells as well as in HMC-1.2 cells.
• METHOD Effects of PKC412, nilotinib (i.e., 4-methyl-3-[[4-(3-pyridinyl)-2- pyrimidinyl]amino]-N-[5-(4-methyl-lH-imidazol-l-yl)-3-(trifluoromethyl)phenyl] benzamide), imatinib (i.e., GLIVEC or GLEEVEC) or 2CdA on expression of the Mcl-1 protein and on cell viability in HMC-1.1 cells lacking KITOS 16V and in HMC-1.2 cells exhibiting KITOi 16V is Case 50270A
• McI-I protein expression is determined by Western blotting using a polyclonal anti-McI-1 antibody. Equal loading is confirmed by probing for ⁇ -Actin. Cell viability is analyzed by recording the percentage (%) of apoptotic cells. Results documenting drug effects on cell viability represent the mean ⁇ S.D. from 3 independent experiments.
• McI-I ASO cooperate with PKC412, 4-methyl-3-[[4-(3-pyridinyl)-2- pyrimidinyl]amino]-N-[5-(4-methyl-lH-imidazol-l-yl)-3-(trifluoromethyl)phenyl] benzamide (nilotinib), and imatinib in producing growth inhibition in neoplastic MC.
• PKC412 50-250 nM 50-250 nM 0.5-3 ⁇ M 0.5-3 ⁇ M nilotinib** 3-10 nM 1-3 ⁇ M 30-80 nM >10 ⁇ M
• ** nilotinib is 4-methyl-3-[[4-(3-pyridinyl)-2-pyrimidinyl]amino]-N-[5-(4-methyl-lH-imidazol-l- yl)-3-(trifluoromethyl)phenyl] benzamide
• METHOD Effects of various concentrations of PKC412, 4-methyl-3-[[4-(3-pyridinyl)- 2-pyrimidinyl]amino]-N-[5-(4-methyl-lH-imidazol-l-yl)-3-(trifluoromethyl)phenyl] benzamide (nilotinib), STI571 (imatinib), or 2CdA on 3 H-thymidine uptake by HMC- 1.1 cells transfected with a scramble control or with mcl-1 antisense oligonucleotides, 50 nM is performed.
• oligonucleotides 80 nM are analyzed. Results are expressed as percent of control i.e. scramble control without targeted drug and represent the mean ⁇ SD of 3 independent experiments. Effects of mcl-1 antisense oligonucleotides or scramble control (each 100 nM) applied with PKC412, nilotinib, STI571 (imatinib), or 2CdA or without the respective drug on cell viability, that is, percentage (%) of apoptotic cells are analyzed. Results represent the mean ⁇ SD of 3 independent experiments.
• HMC- 1.2 cells synergistic effects are only seen with combinations of ASO and PKC412, and ASO and 2CdA, but not with ASO and 4-methyl-3- t[4-(3-pyridinyl)-2-pyrimidinyl]amino]-N-[5-(4-methyl-lH-imidazol-l-yl)-3-(trifluoromethyl) phenyl] benzamide or ASO and imatinib. All in all, these data show that Mcl-1 can be employed as a new drug target in neoplastic MC and that an mcl-1 -specific ASO is capable of sensitizing HMC-I cells against 2CdA and novel TK inhibitors.
• Mcl-1 is a well characterized member of the Bcl-2 family that has recently been implicated in the pathogenesis of various myeloid neoplasms.
• Systemic mastocytosis SM is a myeloid neoplasm characterized by abnormal growth and accumulation of MC in visceral organs.
• neoplastic MC in patients with SM as well as the human mast cell leukemia cell line HMC-I express Mcl-1 in a constitutive manner.
• Mcl-1 is a critical survival-molecule in neoplastic MC
• targeting of Mcl-1 by ASO or siRNA is associated with decreased survival of neoplastic MC and with an increased response to KIT tyrosine kinase inhibitors, including PKC412 or 4-methyl-3-[[4-(3-pyridinyl)-2- pyrimidinyl]amino]-N-[5-(4-methyl-lH-imidazol-l-yl)-3-(trifluoromethyl)phenyl] benzamide.
• McI-I must be regarded as a crucial survival factor expressed in neoplastic MC.
• PKC412 4-methyl-3-[[4-(3- pyridinyl)-2-pyrimidinyl]amino]-N-[5-(4-methyl-lH-imidazol-l-yl)-3-(trifluoromethyl)phenyl] benzamide, and imatinib are found to downregulate expression of McI-I in HMC- 1.1 (lacking KIT D816V but expressing KIT G560V), whereas in HMC-1.2 cells expressing KIT D816V, only PKC412 and to a lesser degree 4-methyl-3-[[4-(3-pyridinyl)-2-pyrimidinyl]arnino]-N-[5-(4-rnethyl- lH-imidazol-l-yl)-3-(trifluoromethyl)phenyl] benzamide, decreased the expression of the McI-I protein.
• KIT D816V or have KIT D816V-negative sublcones that may be selected during TK-targeted therapy to cause relapse.
• McI-I is a well established survival molecule that counteracts apoptosis in myeloid cells.
• HMC-I cells are transfected with mcl-1 -specific ASO or mcl-1 -specific siRNA.
• the specific ASO- and siRNA-induced knock-down of the Mcl-1 protein is demonstrable by Western blotting and is accompanied by a significant increase in apoptotic cells.
• the proliferation of HMC-I cells decreased after transfection with Mcl-1 ASO compared to cells transfected with a scramble control.
• ASM and MCL are high grade MC malignancies with a grave prognosis and a short survival. In these patients, a number of different factors may contribute to abnormal growth and survival of MC, and it is difficult to identify drugs counteracting growth of neoplastic cells in these patients.
• PKC412 a novel TK inhibitor that counteracts TK activity of mutants of Flt3, wild type KIT as well as KIT D816V.
• PKC412 is found to counteract growth and McI- 1 expression in HMC-1.2 cells expressing KIT D816V in the present study. With regard to growth inhibition, these data confirm previous observations by us and by others. However, even PKC412 as a single drug may not be able to induce durable complete remissions in patients with ASM or MCL.
• a most attractive approach in TK-driven, drug-resistant myeloid neoplasms is to combine targeted drugs with each other or with conventional drugs. In case of ASM or MCL, it may thus be reasonable to consider combinations of drugs employing PKC412, other TK inhibitors, and other targeted drugs.
• 2CdA is a drug that has recently been introduced as a novel effective cyto-reductive agent for the treatment of advanced SM.
• 2CdA inhibits growth and Mcl-1 expression in HMC- 1.2 cells much more potently compared to HMC- 1.1 cells, which may be of great clinical importance, since most patients with SM including those with ASM and MCL, display this KIT mutation.
• 2CdA is the first drug described to act better in MC expressing KIT D816 V as on MC lacking this KIT mutant.
• 2CdA did not produce synergistic anti-proliferative effects with mcl-1 ASO in the two HMC-I subclones analyzed.

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