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WO2009000297A1 - Inhibiteurs de la tpp ii en vue d'une utilisation en combinaison avec une chimiothérapie destinée au traitement du cancer - Google Patents

Inhibiteurs de la tpp ii en vue d'une utilisation en combinaison avec une chimiothérapie destinée au traitement du cancer Download PDF

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Publication number
WO2009000297A1
WO2009000297A1 PCT/EP2007/005621 EP2007005621W WO2009000297A1 WO 2009000297 A1 WO2009000297 A1 WO 2009000297A1 EP 2007005621 W EP2007005621 W EP 2007005621W WO 2009000297 A1 WO2009000297 A1 WO 2009000297A1
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compound
alkyl
phenyl
unbranched
unsaturated
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PCT/EP2007/005621
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English (en)
Inventor
Rickard Glas
Hong Xu
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Oncoreg Ab
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Priority to PCT/EP2007/005621 priority Critical patent/WO2009000297A1/fr
Publication of WO2009000297A1 publication Critical patent/WO2009000297A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/06—Tripeptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • the present invention relates to compounds for use in combination with chemotherapy for the treatment of cancer. 5
  • TPP II tripeptidyl-peptidase Il
  • TPPII regulates transduction of apoptotic signals as well as centrosome homeostasis, by unclear mechanisms (Hong X, Lei L, Glas R. Tumors acquire inhibitor of apoptosis protein (IAP)- mediated apoptosis resistance through altered specificity of cytosolic proteolysis.
  • the present invention provides a compound for use in enhancing the efficacy of cancer chemotherapy or increasing the in vivo cancer chemotherapy susceptibility of tumour cells, wherein said compound is a TPP Il inhibitor.
  • cancer chemotherapy covers the treatment of a cancerous condition by a chemical compound which is known to have some therapeutic effect against cancer.
  • the TPP Il inhibitor may be used in combination with one or more drugs of which at least one is known to possess anti-cancer properties.
  • the therapy herein may also include preventative therapy and the treatment of a precancerous condition.
  • tumor cells includes cancerous or pre-cancerous cells. Such cells may have cancerous or pre-cancerous defects. Thus the cells may have acquired one or several alterations characteristic of malignant progression.
  • the invention not only allows chemotherapy-resistant tumours to be treated, but is also advantageous even with tumours that can be treated with chemotherapy, in allowing lower doses of chemotherapy to be used.
  • the present invention provides a compound for use in enhancing the efficacy of cancer chemotherapy or increasing the in vivo cancer chemotherapy susceptibility of tumour cells, wherein said compound is selected from the following formula (i) or is a pharmaceutically acceptable salt thereof:
  • a 1 , A 2 and A 3 are amino acid residues having the following definitions according to the standard one-letter abbreviations or names:
  • a 1 is G, A, V, L, I, P, 2-a mi no butyric acid, norvaline or tert-butyl glycine,
  • a 2 is G, A, V, L 1 I 1 P, F 1 W, C, S 1 K 1 R 1 2-aminobutyric acid, norvaline, norleucine, tert-butyl alanine, alpha-methyl leucine, 4,5-dehydro-leucine, allo-isoleucine, alpha- methyl valine, tert-butyl glycine, 2-allylglycine, ornithine or alpha, gamma- diaminobutyric acid,
  • a 3 is G, A, V, L, I 1 P, F, W, D, E, Y, 2-aminobutyric acid, norvaline or tert-butyl glycine,
  • R N1 and R N2 are each attached to the N terminus of the peptide, are the same or different, and are each independently
  • linker! may be absent, i.e. a single bond, or CH 2 , CH 2 CH 2 , CH 2 CH 2 CH 2 ,
  • R N3 and R N4 are the same or different and are hydrogen or any of the following optionally substituted groups: saturated or unsaturated, branched or unbranched Ci -6 alkyl; saturated or unsaturated, branched or unbranched C 3 . 12 cycloalkyl; benzyl; phenyl; naphthyl; mono- or bicyclic C MO heteroaryl; or non-aromatic d.-io heterocyclyl;
  • R N3 and/or R N4 which may be: hydroxy-; thio-: amino-; carboxylic acid; saturated or unsaturated, branched or unbranched C 1-6 alkyloxy; saturated or unsaturated, branched or unbranched C 3-12 cycloalkyl;
  • N-, O-, or S- acetyl carboxylic acid saturated or unsaturated, branched or unbranched Ci -6 alkyl ester; carboxylic acid saturated or unsaturated, branched or unbranched C 3-I2 cycloalkyl ester phenyl; mono- or bicyclic Ci -10 heteroaryl; non-aromatic Ci.i 0 heterocyclyl; or halogen;
  • R C1 is attached to the C terminus of the tripeptide, and is: O-R C2 ,
  • R c2 and/or R C3 and/or R C4 which may be one or more of: hydroxy-; thio-: amino-; carboxylic acid; saturated or unsaturated, branched or unbranched Ci -6 alkyloxy; saturated or unsaturated, branched or unbranched C 3 _i 2 cycloalkyl; N-, O-, or S- acetyl; carboxylic acid saturated or unsaturated, branched or unbranched Ci -6 alkyl ester; carboxylic acid saturated or unsaturated, branched or unbranched C 3-I2 cycloalkyl ester phenyl; halogen; mono- or bicyclic Ci_io heteroaryl; or non-aromatic C MO heterocyclyl.
  • N and CO indicated in the general formula for formula (i) are the nitrogen atom of amino acid residue A 1 and the carbonyl group of amino acid residue A 3 respectively.
  • the invention provides a method of enhancing the efficacy of cancer chemotherapy or increasing the in vivo cancer chemotherapy susceptibility of tumour cells comprising administering to a patient in need thereof a therapeutically effective amount of a TPPII inhibitor or a compound selected from formula (i) or a pharmaceutically acceptable salt thereof.
  • the compound may be administered in combination with cancer chemotherapy in order to decrease resistance to said cancer chemotherapy.
  • the administration is preferably repeated until treatment of the tumour is enhanced.
  • the present invention provides the use of a TPPII inhibitor or a compound selected from formula (i) or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for enhancing the efficacy of cancer chemotherapy or increasing the in vivo cancer chemotherapy susceptibility of tumour cells.
  • TPP Il inhibitors are useful in combination with cancer chemotherapy in the treatment of cancer.
  • the present invention provides a pharmaceutical composition comprising a TPP Il inhibitor and a cancer chemotherapy compound.
  • Said pharmaceutical composition may also comprise a pharmaceutically acceptable diluent or carrier.
  • Said pharmaceutical composition may comprise more than one cancer drug.
  • the TPP Il inhibitor and cancer chemotherapy compound may be present in the same composition, so that they can be administered together.
  • the TPP Il inhibitor and the cancer therapy agent may be present in a kit of parts so that they may be administered separately.
  • the present invention provides a combination of a TPP Il inhibitor and a cancer chemotherapy compound for use as in therapy.
  • the combination may be such that the TPP Il inhibitor and cancer chemotherapy compound are administrable simultaneously or sequentially.
  • the invention provides a method for identifying a compound suitable for enhancing the efficacy of cancer chemotherapy or increasing the in vivo cancer chemotherapy susceptibility of tumour cells comprising contacting TPP Il with a compound to be screened, and identifying whether the compound inhibits the activity of TPP II.
  • the cancer chemotherapy referred to herein may for example be cytostatic therapy or angiogenesis inhibition.
  • two classes of cancer chemotherapeutic agents which may be used in combination with TPP Il inhibitors include cytostatic drugs and angiogenesis inhibitors.
  • Cytostatic drugs are substances with an inhibitory effect on cancer cell growth, which may be used for the treatment of cancer.
  • Cytostatic drugs comprise, but are not limited to, DNA damaging drugs (e.g. topoisomerase inhibitors, alkylators, or anti-metabolites), tubulin inhibitors, proteasome inhibitors, Hsp90 inhibitors, Corticosteroids, inhibitors of growth factor signaling (e.g. inhibitors of PI3K or MAPK pathways), inhibitors of the anti-apoptotic signals (e.g. antagonists of Bcl-2 or XIAP) or activators of pro-apoptotic signals (e.g. p53 activators).
  • DNA damaging drugs e.g. topoisomerase inhibitors, alkylators, or anti-metabolites
  • tubulin inhibitors e.g. cystulin inhibitors, proteasome inhibitors, Hsp90 inhibitors, Corticosteroids, inhibitors of growth factor signaling (e.g. inhibitors of PI3K or MAPK
  • the present invention therefore relates, inter alia, to the use of molecules that inhibit or antagonize the function of TPPII in combination with cytostatic drugs.
  • In vivo data presented below support the use of TPP Il inhibitors in combination with numerous types of cytostatic drugs across the spectrum of cytostatic therapy.
  • the TPP Il inhibitor may be used in combination with a drug selected from any one or more of the following classes: DNA damaging drugs (e.g. topoisomerase inhibitors, alkylators, or anti-metabolites), tubulin inhibitors, proteasome inhibitors, Hsp90 inhibitors, Corticosteroids, inhibitors of growth factor signaling (e.g. inhibitors of PI3K or MAPK pathways), inhibitors of the anti-apoptotic signals (e.g. antagonists of Bcl-2 or XIAP) or activators of pro-apoptotic signals (e.g. p53 activators).
  • DNA damaging drugs e.g. topoisomerase inhibitors, alkylators, or anti-metabolites
  • tubulin inhibitors e.g. proteasome inhibitors, Hsp90 inhibitors, Corticosteroids
  • inhibitors of growth factor signaling e.g. inhibitors of PI3K or MAPK pathways
  • inhibitors of the anti-apoptotic signals e.g. antagonist
  • cytostatic drugs for use in combination with TPP Il inhibitors is the class of alkylators, for example cyclophosphamide.
  • Another preferred class of cytostatic drugs for use in combination with TPP Il inhibitors is the class of tubulin inhibitors, for example paclitaxel, taxoter or vinorelbine, preferably paclitaxel.
  • Angiogenesis inhibitors treat cancer by targeting the growth of blood vessels, since these are needed to supply nutrients and oxygen to an expanding tumour mass (Folkman, J. Angiogenesis. Annnu Rev. Med. 2006;57:1-18).
  • This concept has been much studied in pre-clinical studies with inhibitors that block growth factor receptors on endothelial cells (e.g. VEGF-R), which in some cases can cause complete regression of a growing tumour mass in mice.
  • VEGF-R endothelial cells
  • Recent studies in human patients have also shown significant, although less dramatic, effects on tumour growth. This has led to approval of certain inhibitors of angiogenesis for therapy in cancer patients.
  • the present invention therefore relates, inter alia, to the use of molecules that inhibit or antagonize the function of TPPII in combination with angiogenesis inhibitors.
  • TPP Il inhibitors in combination with angiogenesis inhibitors.
  • TNP- 470 One preferred angiogenesis inhibitor for use in combination with TPP Il inhibitors is TNP- 470. Another is Thalidomide.
  • PIKKs Phospho-lnositide-3-OH- Kinase-related kinases
  • PIKKs play a role as signal transducers from sensor molecules in response to stress, and phosphorylate a network of regulatory factors to initiate DNA repair and cell cycle arrest; pathways often constitutively activated in transformed cells (Bartkova J, Horejsi Z, Koed K, Kramer A, Tort F, Zieger K, et. al. Activation of the DNA damage checkpoint and genomic instability in human precancerous lesions. Nature 2005;434:907-13) (Bartkova J, Horejsi Z, Koed K, Kramer A, Tort F, Zieger K, et. al. DNA damage response as a candidate anti-cancer barrier in early human tumorigenesis. Nature 2005;434:864-70).
  • Cancer therapy frequently depends on the induction of DNA damage, e.g. treatment with gamma-irradiation or DNA topoisomerase inhibitors.
  • Nuclear PIKKs i.e. ATM, ATR and DNA-PKcs
  • Incubation of tumour cells with inhibitors of these PIKKs block DNA repair responses, which increases susceptibility to gamma-irradiation- induced apoptosis in vitro (Cowell IG, Durkacz BW, Tilby MJ. Sensitization of breast carcinoma cells to ionizing radiation by small molecule inhibitors of DNA- dependent protein kinase and ataxia telangiectsia mutated.
  • mTOR a cytosolic PIKK-family member
  • Akt kinase activation Akt kinase activation
  • Inhibitors of mTOR sensitize tumors to gamma-irradiation in mice, with the occasional observation of tumor regression, and such inhibitors show promising results in trials against several forms of cancer (Faivre S, Kroemer G, Raymond E. Current development of mTOR inhibitors as anticancer agents.
  • Glioma stem cells promote radioresistance by preferential activation of the DNA damage response. Nature 2006;444:756-60) (Reinhardt HC, Aslanian AS, Lees JA, Yaffe MB. p53-deficient cells rely on ATM- and ATR-mediated checkpoint signaling through the p38MAPK/MK2 pathway for survival after DNA damage. Cancer Cell 2007;11 :175-89). It is yet unclear which PIKKs, or PIKK-dependent pathways, represent targets for efficient cancer therapy.
  • TPPII has a crucial role in gamma- irradiation-induced DNA damage responses in vitro and in resistance to gamma-irradiation-based cancer therapy in vivo.
  • TPPII requires PIKK signaling, and that TPPII is rapidly translocated into the nucleus of gamma-irradiated cells. These events are dependent on mTOR, a cytosolic/ mitochondrial PIKK that is activated by gamma-irradiation. Lymphoma cells with inhibited expression of TPPII fail to efficiently stabilize p53, and have reduced ability to arrest proliferation in response to gamma-irradiation.
  • BRCT BRCA C-terminal
  • TPPII tri-peptide-based inhibitors of TPPII which cause complete in vivo tumour regression in mice, in response to relatively low doses of gamma-irradiation (3-4 Gy/week). We have observed this with established mouse and human tumours of diverse tissue backgrounds, with no tumour re- growth after cancellation of treatment. We have also found that these TPPII inhibitors do not have adverse cellular toxicity. Our data indicate that TPPII connects signaling by cytosolic/mitochondrial and nuclear PIKK-dependent pathways, and that TPPII can be 5 targeted for inhibition of tumor therapy resistance.
  • TPP Il accepts a relatively broad range of substrates. All the compounds falling within formula (i) are peptides or peptide analogues. Compounds of formulae (i) are readily synthesizable by methods known in the art (see for example Ganellin et al., J. Med. Chem. 10 2000, 43, 664-674) or are readily commercially available (for example from Bachem AG). In a preferred aspect the compound may be selected from formulae (i). Such tripeptides and derivatives are particularly effective therapeutic agents.
  • the compound for use in enhancing the efficacy of cancer 15 chemotherapy or increasing the in vivo cancer chemotherapy susceptibility of tumour cells may be a compound which is known to be a TPP Il inhibitor in vivo.
  • the compound may be selected from compounds identified in Winter et al., Journal of Molecular Graphics and Modelling 2005, 23, 409-418 as TPP Il inhibitors.
  • the 20 compounds may be selected from the following formula (ii) because these compounds are particularly suited to the TPP Il pharmacophore:
  • R 1 5 wherein R' is H, CH 3 , CH 2 CH 3 , CH 2 CH 2 CH 3 or CH(CH 3 ) 2 ,
  • R" is H, CH 2 CH 3 , CH 2 CH 2 CH 3 , CH(CH 3 ) 2, CH 2 CH 2 CH 2 CH 3 , CH 2 CH(CH 3 ) 2 , CH(CH 3 )CH 2 CH 3 or C(CH 3 ) 3 , and R 111 Js H 1 CH 31 OCH 31 F 1 Cl Or Br;
  • the compound may be selected from compounds identified in US 6,335,360 of Schwartz et al. as TPP Il inhibitors.
  • Such compounds include those of the following formula (iii).
  • each R 1 may be the same or different, and is selected from the group consisting of halogen, OH; Ci -C 6 alkyl optionally substituted by one or more radicals selected from the group consisting of halogen and OH; (Ci -C 6 ) alkenyl optionally substituted by one or more radicals selected from the group consisting of halogen and OH; (C1 -C 6 ) alkynyl, optionally substituted by one or more radicals selected from the group consisting of halogen and OH, X(Ci -C 6 )alkyl, wherein X is S 1 0 or OCO, and the alkyl is optionally substituted by one or more radicals selected from the group consisting of halogen and OH; SO 2 (Ci -C 6 )alkyl, optionally substituted by at least one halogen, YSO 3 H, YSO 2 (Ci -C 6 )alkyl, wherein Y is O or NH and the al
  • n is from 0 to 4;
  • R 2 is CH 2 R 4 , wherein R 4 is Ci -C 6 alkyl substituted by one or more radicals selected from the group consisting of halogen and OH;
  • (CH 2 ) p Z(CH 2 )qCH 3 wherein Z is O or S, p is from 0 to 5 and q is from 0 to 5, provided that p+q is from 0 to 5;
  • R 2 is (Ci -C 6 )alkyl or O(Ci -C 6 )alkyl, each optionally substituted by at least one halogen;
  • R 3 is H; (Ci -C 6 )alkyl optionally substituted by at least one halogen; (CH 2 ) P ZR 5 wherein p is from 1 to 3, Z is O or S and R 5 is H or (Ci -C 3 )alkyl; benzyl.
  • a 1 is G, A, V, L, I, P, S, T, C, N, Q, 2-aminobutyric acid, norvaline, norleucine, tert- butyl alanine, alpha-methyl leucine, 4,5-dehydro-leucine, allo-isoleucine, alpha- methyl valine, tert-butyl glycine or 2-allylglycine,
  • a 2 is G, A, V, L, I, P, S, T 1 C, N, Q, F, Y, W, K 1 R, histidine, 2-aminobutyric acid, norvaline, norleucine, tert-butyl alanine, alpha-methyl leucine, 4,5-dehydro-leucine, allo-isoleucine, alpha-methyl valine, tert-butyl glycine, 2-allylglycine, ornithine, alpha.gamma-diaminobutyric acid or 4,5-dehydro-lysine, and
  • a 3 is G, A 1 V 1 L, I, P 1 S 1 T, C, N 1 Q, D, E, F, Y 1 W, 2-aminobutyric acid, norvaline, norleucine, tert-butyl alanine, alpha-methyl leucine, 4,5-dehydro-leucine, allo- isoleucine, alpha-methyl valine, tert-butyl glycine or 2-allylglycine.
  • amino acids of natural (L) configuration are preferred, particularly at the A 2 position.
  • R N1 is hydrogen
  • R N2 is:
  • R N3 is hydrogen or any of the following unsubstituted groups: saturated or unsaturated, branched or unbranched Ci -4 alkyl; benzyl; phenyl; or monocyclic heteroaryl.
  • R C1 is: O-R
  • R C1 is OH 1 O-Ci- 6 alkyl, 0-Ci -6 alkyl-phenyl, NH-Ci -6 alkyl, or NH-Ci -6 alkyl-phenyl.
  • a 1 is G, A or 2-aminobutyric acid,
  • a 2 is L, I, norleucine, V, norvaline, tert-butyl alanine, 4,5-dehydro-leucine, allo-isoleucine, 2- allylglycine, P, 2-aminobutyric acid, alpha-methyl leucine, alpha-methyl valine or tert-butyl glycine,
  • a 3 is G, A 1 V, P, 2-aminobutyric acid or norvaline,
  • R N1 is H
  • R C1 is OH, O-Ci. 6 alkyl, 0-Ci -6 alkyl-phenyl, NH-Ci -6 alkyl, or NH-Ci -6 alkyl-phenyl.
  • a 1 is G, A or 2-aminobutyric acid
  • a 2 is L, I 1 norleucine, V, norvaline, tert-butyl alanine, 4,5-dehydro-leucine, allo-isoleucine or
  • a 3 is G, A 1 V, P, 2-aminobutyric acid or norvaline,
  • R N1 is H
  • R C1 is OH, O-Ci. 6 alkyl, O-Ci -6 alkyl-phenyl, NH-C 1-6 alkyl, or NH-Ci -6 alkyl-phenyl.
  • a 2 is L, I 1 or norleucine, A 3 is G or A, R N1 is H,
  • R C2 is hydrogen or any of the following unsubstituted groups: saturated or unsaturated, branched or unbranched C1. 5 alkyl; benzyl; phenyl; or monocyclic Ci.i 0 heteroaryl.
  • R N1 is hydrogen
  • R m is hydrogen
  • R N2 is hydrogen
  • R N1 is hydrogen
  • R N2 is a is benzyloxycarbonyl, benzyl, benzoyl, tert-butyloxycarbonyl, 9- fluorenylmethoxycarbonyl or FA, more preferably benzyloxycarbonyl or FA.
  • R C1 is OH, 0-C 1-6 alkyl, 0-Ci -6 alkyl-phenyl, NH-Ci -6 alkyl, or NH-Ci -6 alkyl-phenyl, more preferably OH.
  • a 1 is G, A, V, L, I 1 P, 2-aminobutyric acid, norvaline or tert-butyl glycine
  • a 2 is G, A, V, L, I, P, F, W, C, S, K, R, 2-aminobutyric acid, norvaline, norleucine, tert-butyl alanine, alpha-methyl leucine, 4,5-dehydro-leucine, allo-isoleucine, alpha-methyl valine, tert-butyl glycine, 2-allylglycine, ornithine or alpha, gamma-diaminobutyric acid
  • a 3 is G, A, V 1 L, I, P, F, W, D, E, Y, 2-aminobutyric acid, norvaline or tert-butyl glycine
  • R N1 is H,
  • a first set of specific preferred compounds are those in which: A 1 is G, A 2 is L,
  • a 3 is G, A, V, L, I, P, F, W, D, E, Y, 2-aminobutyric acid, norvaline or tert-butyl glycine, more preferably G, A, V, P 1 2-aminobutyric acid or norvaline, more preferably G or A, R N1 is hydrogen, R N2 is benzyloxycarbonyl, and R C1 is OH.
  • a second set of specific preferred compounds are those in which: A 1 is G,
  • a 2 is G, A, V, L, I, P, F, W, C, S, 2-aminobutyric acid, norvaline, norleucine, tert-butyl alanine, alpha-methyl leucine, 4,5-dehydro-leucine, allo-isoleucine, alpha-methyl valine, tert-butyl glycine or 2-allylglycine, more preferably L, I, norleucine, V, norvaline, tert-butyl alanine, 4,5-dehydro-leucine, allo-isoleucine, 2-allylglycine, P, 2-aminobutyric acid, alpha- methyl leucine, alpha-methyl valine or tert-butyl glycine, more preferably L, I, norleucine, V, norvaline, tert-butyl alanine, 4,5-dehydro-leucine, allo-isoleucine or 2-allylg
  • R N2 is benzyloxycarbonyl
  • R C1 is OH
  • a third set of specific preferred compounds are those in which: A 1 is G, A, V, L, I, P, 2-aminobutyric acid, norvaline or tert-butyl glycine, more preferably G,
  • a or 2-aminobutyric acid more preferably G or A,
  • a 2 is L
  • a 3 is A
  • R N1 is hydrogen
  • R N2 is benzyloxycarbonyl
  • R C1 is OH
  • sequence A 1 -A 2 -A 3 is GLA 1 GLF 1 GVA 1 GIA, GPA or ALA 1 most preferably GLA 1 and: R N1 is hydrogen,
  • R N2 is benzyloxycarbonyl
  • R C1 is OH
  • alkyl groups are described as saturated or unsaturated, this encompasses alkyl, alkenyl and alkynyl hydrocarbon moieties.
  • Ci -6 alkyl is preferably Ci -4 alkyl, more preferably methyl, ethyl, n-propyl, isopropyl, or butyl (branched or unbranched), most preferably methyl.
  • C 3- i 2 cycloalkyl is preferably C 5-I0 cycloalkyl, more preferably C 5 .7 cycloalkyl.
  • aryl is an aromatic group, preferably phenyl or naphthyl
  • hetero as part of a word means containing one or more heteroatom(s) preferably selected from N, O and S.
  • heteroaryl is preferably pyridyl, pyrrolyl, quinolinyl, furanyl, thienyl, oxadiazolyl, thiadiazolyl, thiazolyl, oxazolyl, pyrazolyl, triazolyl, tetrazolyl, isoxazolyl, isothiazolyl, imidazolyl, pyrimidinyl, indolyl, pyrazinyl, indazolyl, pyrimidinyl, thiophenetyl, pyranyl, carbazolyl, acridinyl, quinolinyl, benzimidazolyl, benzthiazolyl, purinyl, cinnolinyl or pteridinyl.
  • non-aromatic heterocyclyl is preferably pyrrolidinyl, piperidyl, piperazinyl, morpholinyl, tetrahydrofuranyl or monosaccharide.
  • halogen is preferably Cl or F, more preferably Cl. Further preferred compounds of formula (i)
  • a 1 may preferably be selected from G, A or 2-aminobutyric acid; more preferably G or A 1 most preferably G.
  • a 2 may preferably be selected from L, I, norleucine, V, norvaline, tert-butyl alanine, 4,5-dehydro-leucine, allo-isoleucine, 2-allylglycine, P, K, 2-aminobutyric acid, alpha-methyl leucine, alpha-methyl valine or tert-butyl glycine; more preferably L, I, norleucine, V, norvaline, tert-butyl alanine, 4,5-dehydro-leucine, allo-isoleucine, 2- allylglycine, P or K; more preferably L, I, norleucine, P or K; more preferably L or P.
  • a 3 may preferably be selected from G, A, V, P, 2-aminobutyric acid or norvaline; more preferably G or A.
  • G One general preference is that A 3 is G.
  • a 3 is A, particularly when R C1 is OH.
  • R N1 is hydrogen
  • R N2 is preferably:
  • linkeri may be absent, i.e. a single bond, or CH 2 , CH 2 CH 2 , CH 2 CH 2 CH 2 ,
  • R N3 is hydrogen or any of the following unsubstituted groups: saturated or unsaturated, branched or unbranched Ci. 4 alkyl; benzyl; phenyl; or monocyclic heteroaryl.
  • R N2 is more preferably hydrogen, benzyloxycarbonyl, benzyl, benzoyl, tert- butyloxycarbonyl, 9-fluorenylmethoxycarbonyl or FA, more preferably hydrogen, benzyloxycarbonyl or FA.
  • R C1 is: O-R C2 ,
  • R C2 is hydrogen or any of the following unsubstituted groups: saturated or unsaturated, branched or unbranched C 1-5 alkyl; benzyl; phenyl; or monocyclic Ci -10 heteroaryl.
  • R C1 is more preferably OH, 0-Ci -6 alkyl, 0-C 1-6 alkyl-phenyl, NH 2 , NH-Ci -6 alkyl, or NH-C 1-6 alkyl-phenyl, more preferably OH, 0-Ci -6 alkyl, NH 2 , or NH-C 1-6 alkyl, more preferably OH or NH 2 .
  • Compounds of particular interest include those wherein A 2 is P.
  • Compounds of particular interest include those wherein R C1 is NH 2 .
  • a 3 In general the following amino acids are less preferred for A 3 : F, W, D, E and Y. Similarly, in general A 3 may be selected not to be P and/or E due to compounds containing these exhibiting lower activity.
  • R' is CH 2 CH 3 or CH 2 CH 2 CH 3 ,
  • R" is CH 2 CH 2 CH 3 or CH(CH 3 ) 2 .
  • R'" is H or Cl.
  • Preferred compounds of formula (NH a) is H or Cl.
  • Z-GLA-OH i.e. tripeptide GLA which is derivatized at the N-terminus with a Z group and which is not derivatized at the C- terminus.
  • Z denotes benzyloxycarbonyl.
  • R N1 is H
  • R N2 is Z
  • a 1 is G
  • a 2 is L
  • a 3 is A
  • R C1 is OH.
  • This compound is available commercially from Bachem AG and has been found to inhibit the bacterial homologue of the eukaryotic TPP II, Subtilisin.
  • Z-GLA-OH is of low cost and works well in vivo to induce rejection of tumours that are resistant to cancer chemotherapy. Novel treatments of therapy resistant cancers are of substantial interest to public health.
  • any disclosures of any compounds or groups of compounds herein may optionally be subject to the proviso that the sequence A 1 A 2 A 3 is not GLA, or the proviso that the compound is not selected from the group consisting of Z-GLA-OH, Bn-GLA-OH, FA-GLA-OH or H-GLA-OH, or the proviso that the compound is not Z-GLA-OH.
  • Z-GLA- OH or other compounds described herein may be administered to improve such treatment in patients with malignant disease, for example increasing the in vivo response to such treatment in solid tumours.
  • a 1 A 2 A 3 is GPG, such as GPG-NH 2 or Z- GPG-NH 2 .
  • the compounds described herein may be administered in any suitable manner.
  • the administration may be parenteral, such as intravenous or subcutaneous, oral, transdermal, intranasal, by inhalation, or rectal.
  • the compounds are administered by injection.
  • pharmaceutically acceptable addition salts for use in the pharmaceutical compositions of the present invention include those derived from mineral acids, such as hydrochloric, hydrobromic, phosphoric, metaphosphoric, nitric and sulphuric acids, and organic acids, such as tartaric, acetic, citric, malic, lactic, fumaric, benzoic, glycolic, gluconic, succinic, and arylsulphonic acids.
  • the pharmaceutically acceptable excipients described herein for example, vehicles, adjuvants, carriers or diluents, are well-known to those who are skilled in the art and are readily available to the public.
  • the pharmaceutically acceptable carrier may be one that is chemically inert to the active compounds and that has no detrimental side effects or toxicity under the conditions of use. Pharmaceutical formulations are found e.g. in Remington: The Science and Practice of Pharmacy, 19th ed., Mack Printing Company, Easton, Pennsylvania (1995).
  • the composition may be prepared for any route of administration, e.g. oral, intravenous, cutaneous or subcutaneous, nasal, intramuscular, or intraperitoneal.
  • routes of administration e.g. oral, intravenous, cutaneous or subcutaneous, nasal, intramuscular, or intraperitoneal.
  • a parenterally acceptable aqueous solution is employed, which is pyrogen free and has requisite pH, tonicity and stability.
  • Those skilled in the art are well able to prepare suitable solutions and numerous methods are described in the literature. A brief review of methods of drug delivery is also found in e.g. Langer, Science 249:1527-1533 (1990).
  • the dose administered to a mammal, particularly a human, in the context of the present invention should be sufficient to effect a therapeutic response in the mammal over a reasonable time frame.
  • dosage will depend upon a variety of factors including the age, condition and body weight of the patient, as well as the stage/severity of the disease.
  • the dose will also be determined by the route (administration form) timing and frequency of administration.
  • the dosage can vary for example from about 0.01 mg to about 10 g, preferably from about 1 mg to about 8 g, preferably from about 10 mg to about 5 g, more preferably from about 10 mg to about 2 g, more preferably from about 100 mg to about 1 g per day of a compound or the corresponding amount of a pharmaceutically acceptable salt thereof.
  • TPP Il protein may be purified in a first step, and a TPP ll-preferred fluorogenic substrate may be used in a second step. This results in an effective method to measure TPP Il activity.
  • TPP II 100 x 10 6 cells (such as EL-4 cells) were sedimented and ' lysed by vortexing in glass beads and homogenisation buffer (50 mM Tris Base pH 7.5, 250 mM Sucrose, 5 mM MgCI 2 , 1 mM DTT). Cellular lysates were subjected to differential centrifugation; first the cellular homogenate was centrifuged at 14,000 rpm for 15 min, and then the supernatant was transferred to ultra-centrifugation tubes.
  • homogenisation buffer 50 mM Tris Base pH 7.5, 250 mM Sucrose, 5 mM MgCI 2 , 1 mM DTT.
  • the sample was ultra-centrifugated at 100,000 x g for 1 hour, and the supernatant (denoted as cytosol in most biochemical literature) was subjected to 100,000 x g centrifugation for 5 hours, which sedimented high molecular weight cytosolic proteins/protein complexes.
  • the resulting pellet dissolved in 50 mM Tris Base pH 7.5, 30%Glycerol, 5 mM MgCb, and 1 mM DTT, and 1 ug of high molecular weight protein was used as enzyme in peptidase assays.
  • Cleavage activity may be measured for example by emission at 460 nm in a LS50B Luminescence Spectrometer (Perkin Elmer, Boston, MA).
  • the compounds of use in the present invention may be defined as those which result in partial or preferably complete tumour regression compared to control experiments when used in an in vivo model which comprises the steps of: (i) inoculation of tumor cells into mice; (ii) administration of a cancer chemotherapeutic agent to said mice and administration of compound to said mice; and (iii) measuring the tumour size at periodic intervals.
  • the step of administration of a cancer chemotherapeutic agent is omitted in the control experiments. Further details and examples of tumour growth experiments are described below. We found it convenient to inject the compound shortly after application of cancer chemotherapy, but the invention should not be understood as limited to this sequence of administration.
  • the compounds used in the present invention are sufficiently serum-stable, i.e. in vivo they retain their identity long enough to exert the desired therapeutic effect.
  • TPPII activity enzyme cleavage of AAF-AMC, top
  • expression by western blotting with anti-TPPU, bottom
  • E Cell cycle analysis of EL-4.wt (top) versus EL-4.TPPN' cells (bottom), before or 20 hours after exposure to 10 Gy of gamma- irradiation.
  • F Phospho-Ser139-H2AX (gamma-H2AX) expression in EL-4.wt control versus EL-4.TPPII' cells exposed to 2,5 Gy of gamma-irradiation. 5
  • TPPII expression is required for stabilization of p53.
  • A p53 expression in EL-4.wt control versus EL-4.TPPN' cells.
  • B p21 expression in EL-4.wt control versus EL-4. TPPII' cells.
  • C p53 expression in EL-4.pcDNA3control versus EL- 0 4.pcDNA3-TPPII cells.
  • D Western blotting analysis of TPPII using p53- immunoprecipitates from lysates of EL-4.wt versus EL-4.
  • TPPII 1 cells top; or from EL-4.wt cells treated with 1 micro-M wortmannin, versus untreated (bottom). Lanes labeled "+” indicates gamma-irradiated cells, whereas "-" were untreated (incubated for 16 hours at 37 0 C, prior to lysis).
  • E p53 expression in ALC.pcDNA3 versus ALC.pSUPER-TPPII 1 (left), YAC-1 versus YAC-1.pSUPER-TPPII' (middle) and LLCpSUPER control versus LLC.TPPir cells (right), exposed to gamma-irradiation.
  • TPPII controls pathways that respond to PIKK signaling.
  • A Western blotting analysis of Akt kinase expression, total Akt and Ser473-phosphorylated (p-Akt), in EL-4.wt control versus EL-4.TPPII 1 cells (top), or in EL-4.pcDNA3 versus EL-4.pcDNA3- TPPII cells (bottom).
  • B Growth in vitro of EL-4.wt and EL-4.TPPII 1 cells in cell culture medium with either high (5%, left) or low (1%, right) serum content. Both live (empty circles) and dead (filled circles) cells were counted.
  • TPPII controls interactions that mediate p53 stabilization.
  • TPPII is required for in vivo tumor resistance to gamma-irradiation.
  • C Tumor growth of 5 x 10 6 EL-4.ATM 1 cells in syngeneic C57BI/6 mice, left untreated (top) or gamma-irradiated with 4 Gy at time-points indicated with arrows (bottom).
  • D Tumor growth of 5 x 10 6 EL- 4.TPPirVG725E cells in syngeneic C57BI/6 mice, left untreated (top) or gamma-irradiated (bottom).
  • FIG. 6 The Subtilisin inhibitor Z-Gly-Leu-Ala-OH inhibits TPPII and allows efficient radio-sensitization of tumors in vivo.
  • (A) Cleavage of AAF-AMC by partially purified TPPII enzyme, as measured by fluorimetry, in the presence of Z-GLA-OH or butabindide.
  • C Tumor growth of 10 6 EL-4 lymphoma cells in syngeneic C57BI/6 mice, treated with gamma-irradiation doses of 3 Gy, 2 Gy or 1 Gy in combination with Z-GLA-OH injection (left panel); versus gamma-irradiation doses of 4 Gy or Z-GLA-OH alone and untreated (middle panel).
  • A Flow cytometric analysis of DBA/2 spleen cells 13 days post-transplantation of stem cells transduced with pMSCV-Bcl-XL-IRES-E-GFP and pMSCV-c-Myc-IRES-E-YFP.
  • B In vivo tumor growth of DBA/2-c-myc/Bcl-xL cells in the presence or absence of gamma- irradiation treatment and Z-GLA-OH.
  • C-G Flow cytometric detection of vector encoded YFP (c-Myc+) and GFP (Bcl-xL+) from DBA-c-Myc/Bcl-xL cells in tissues derived from tumour-carrying mice from untreated (C-E) versus treated (F, G) mice (gamma-irradiation and Z-GLA-OH), tissues used were from subcutaneous tumor (C) 1 lung (D, F), and spleen (E, G). Gates indicated in top panels correspond to cells analyzed for GFP/YFP- fluorescence in bottom panels.
  • H-J Histological sections of livers from mice inoculated with DBA/2-c-Myc/Bcl-xL cells, receiving no treatment (H), gamma-irradiation (I) or both gamma-irradiation and Z-GLA-OH (J). Arrows indicate sinusoids filled with tumor cells.
  • Tumour size vertical axis, mm 3
  • time horizontal axis, days
  • FIG. 9 Inhibition of TPP Il affects Mre11 foci formation
  • Lewis Lung Carcinoma (LLC, A) 1 ALC (B) and YAC-1 (C) cells were stably transfected with pSUPER- TPPIIi, or with empty pSUPER vector, and were exposed to 5 Gy of gamma-irradiation.
  • Immunocytochemical expression of TPPII and Mre11 was measured, as indicated in figure, and DAPI was used for nuclear control staining.
  • FIGS. 10 and 11 Treatment with Z-GLA-OH in combination with a range of cytostatic drugs enhances therapeutic effects in vivo.
  • Tumour size vertical axis, mm 3
  • time horizontal axis, days
  • Figure 13 shows growth of EL-4 T-lymphoma cells in vivo, in syngeneic mice, treated with Dexamethasone (5 mg/kg) and/or Z-GLA-OH (13.8 mg/kg), or left untreated.
  • EL-4 is a Benzpyrene-induced lymphoma cell line derived from the C57BI/6 mouse strain.
  • EL-4.wt and EL-4.TPPII 1 are EL-4 cells transfected with the pSUPER vector (Brummelkamp, TR, Bernards, R, Agami, R. A system for stable expression of short interfering RNAs in mammalian cells. Science 2002;296:550-3), empty 5 versus containing the siRNA directed against TPPII.
  • HeLa cells are human cervical carcinoma cells.
  • YAC-1 is a Moloney Leukemia Virus-induced lymphoma cell line derived from the A/Sn mouse strain.
  • ALC is a T cell lymphoma induced by radiation leukemia virus D-RadLV, derived from the C57BI/6 mouse strain.
  • D-RadLV radiation leukemia virus
  • PBS Phosphate Buffered Saline
  • NLVS is an inhibitor of the proteasome that preferentially targets the 15 chymotryptic peptidase activity, and efficiently inhibits proteasomal degradation in live cells.
  • Butabindide is described in the literature (Rose, C, Vargas, F, Facchinetti, P, Bourgeat, P, Bambal, RB, Bishop, PB, et. al. Characterization and inhibition of a cholecystokinin- inactivating serine peptidase. Nature 1996;380:403-9).
  • Z-Gly-Leu-Ala-OH is an inhibitor of Subtilisin (Bachem, Weil am Rhein, Germany), a bacterial enzyme with an 20 active site that is homologous to that of TPPII.
  • Wortmannin is an inhibitor of PIKK (PI3- kinase-related) -family kinases (Sigma, St. Louis, MO). All inhibitors were dissolved in DMSO and stored at -2O 0 C until use.
  • 35 Cleavage activity was measured by emission at 460 nm in a LS50B Luminescence Spectrometer (Perkin Elmer, Boston, MA).
  • a DNA topoisomerase Il inhibitor commonly used as an apoptosis-inducing agent, to starvation (50% PBS).
  • Cells were seeded at 10 6 cells/ml in 12-well plates and incubated for the indicated times, usually 18-24 hours.
  • DNA from EL-4 control and adapted cells was purified by standard chloroform extraction, and 2.5 micro-g of DNA was loaded on 1.8% agarose gel for detection of DNA from apoptotic cells.
  • TPPII siRNA-expressing pSUPER (Brummelkamp, TR, Bernards, R, Agami, R. A system for stable expression of short interfering RNAs in mammalian cells. Science 2002;296:550-3.) plasmids were constructed as follows. Non- phosphorylated DNA oligomers (Thermo Hybaid, UIm, Germany) were resuspended to a concentration of 3 micro-g/micro-l.
  • annealing buffer 100 mM KAc; 30 mM HEPES-KOH pH 7.4; 2 mM MgAc
  • 2 micro-l of annealed oligomers were mixed with 100 ng of pSUPER plasmid (digested with BgIII and Hindlll), ligated, transformed, and plated on Amp/LP plates, as previously described (Brummelkamp, TR, Bernards, R, Agami, R. A system for stable expression of short interfering RNAs in mammalian cells. Science 2002;296:550-3.). Colonies were screened for the presence of inserts by EcoRI-Hindlll digestion and DNA sequencing. Annealed oligomer pairs were as follows, for pSUPER-TPPN', forward primer:
  • Akt by rabbit anti-Akt serum (Cell Signaling Technology, Beverly, MA); Phospho- Akt (Ser 473) by 193H2 rabbit anti-phospho-Akt serum (Cell Signaling Technology, Beverly, MA); gamma-H2AX by rabbit anti-gamma-H2AX (Cell Signalling Technology, Beverly, MA); Mre11 by polyclonal rabbit anti-human Mre1 1 (Cell Signalling Technology, Beverly, MA); p21 by SX118 (R & D Systems, Minneapolis, MN); p53 (R & D Systems, Minneapolis, MN); Rae-1 by monoclonal Rat anti-mouse Rae-1 , 199215 (R &D Systems, Minneapolis, MN); XIAP by monoclonal mouse anti-human XIAP, 117320 (R&D Systems, Minneapolis, MN).
  • Tumor Growth Experiments. Tumor cells were washed in PBS and resuspended in a volume of 200 micro-l per inoculate. The cells were then inoculated into the right flank at 10 6 per mouse and growth of the tumor was monitored by measurement two times per week. The initiation of anti-tumor treatment of the mice was to some extent individualized according to when tumor growth started in each mouse. The mice were irradiated with 4 Gy prior to tumor inoculation in order to inhibit anti-tumor immune responses. The tumor volume was calculated as the mean volume in mice with tumors growth, according to (ai x a 2 x a 3 )/2 (the numbers a, denote tumor diameter, width and depth).
  • c-Myc was amplified from human cDNA (brain) by PCR using the following primers: 5 ⁇ CGTGAATTCCACCATGCCCCTCAACGTTAGCTTC and 3TACGTCTCGAGCTTACGCACAAGAGTTCCGTAG and inserted in the EcoRI site of the retroviral expression vector pMSCV-IRES-EYFP.
  • hBcl-x L was excised from the pLXIN-hBcl- x L (Djerbi, M., Darreh-Shori, T., Zhivotovsky, B. & Grandien, A.
  • retroviral vectors were transiently transfected into Phoenix-Eco packaging cells using the LipofectAMINE 2000 Reagent (Invitrogen, Life Technologies Inc., Paisley, UK) and viral supernatants containing viral particles were harvested and used to transduce lineage negative cells obtained from bone marrow of 5-fluorouracil treated mice. These cells were thereafter injected into lethally irradiated recipient mice. Between 7 and 14 days after transplantation, the mice developed an acute myeloid leukaemia-like disease. Cells from spleen of such mice could be grown in vitro in regular RPMI medium supplemented with, glutamin and fetal calf serum.
  • Detection of GFP and YFP expression was performed using a CyanTM ADP cytometer (Dako, Glostrup, Denmark) where after excitation at 488 nm, a 525-nm long-pass dichroic mirror was used to initially separate the signals followed by a 510/21-nm bandpass filter for detection of EGFP and a 550/30-nm band pass filter for EYFP. Data were analyzed using FlowJo software (Tree Star, Inc., San Carlos, CA).
  • ATM Ataxia Telangiectasia Mutated
  • BRCT BRCA C-terminal repeat
  • NLVS ⁇ hydroxy- ⁇ -iodo-S-nitrophenylacetyl-Leu-Leu-Leu-vinyl sulphone
  • Pl Propidium Iodide
  • PIKKs Phosphoinositide-3-OH-kinase-related kinases
  • TPPII Tripeptidyl-peptidase Il
  • FA 3-(2-furyl)acryloyl
  • YFP Yellow Fluorescent Protein
  • GFP Green Fluorescent Protein
  • the invention also makes use of several unnatural alpha-amino acids.
  • Gamma-irradiation-induced cell cycle arrest depends on TPPII expression.
  • TPPII expression is increased by several types of stress we tested whether this was controlled by PIKKs.
  • Western blotting analysis of the T cell lymphoma line EL-4 with TPPII anti-serum we found that TPPII expression was increased by gamma-irradiation. Further, this increase was not present in gamma-irradiated EL-4 cells treated with 1 micro- M wortmannin, a PIKK inhibitor, which instead reduced TPPII expression (Fig. 1A).
  • EL-4.TPPII 1 cells had both inhibited expression and activity of TPPII, in comparison to EL-4.wt cells (transfected with empty pSUPER vector, Fig. 1 B).
  • gamma- irradiation 5 Gy.
  • TPPII was previously reported as a soluble cytosolic peptidase (Reits, E, Neijssen, J, Herberts, C, Benckhuijsen, W 1 Janssen, L 1 Drijfhout, JW, et. al. A major role for TPPII in trimming proteasomal degradation products for MHC class I antigen presentation.
  • PIKKs Activation of PIKKs is required to halt DNA synthesis in response to DNA damage (Bakkenist, CJ 1 Kastan MB. Initiating cellular stress responses. Cell 2004;118:9-17)
  • TPPII' cells arrested almost uniformly in G2/M after exposure to gamma-irradiation, whereas EL-4.wt control cells showed both G1 and G2/M arrest, suggesting an absence of a G1/S checkpoint in EL-4.TPPN' cells (Fig. 1 E).
  • TPPII 1 cells as measured by western blotting of gamma-H2AX (Ser139-phosphorylated H2AX, Fig. 1 F).
  • H2AX is phosphorylated in response to ATM activation, which triggers the formation of DNA repair foci (Bakkenist, CJ, Kastan MB. Initiating cellular stress responses.
  • TPPII is rapidly translocated into the nucleus following gamma- irradiation-exposure, and required to efficiently halt DNA synthesis in EL-4 cells, but not for phosphorylation of H2AX.
  • the transcription factor p53 initiates cell cycle arrest in response to many types of stress, and its expression is controlled by direct phosphorylation by PIKKs.
  • PIKKs direct phosphorylation by PIKKs.
  • p21 a transcriptional target of p53
  • EL-4.TPPH' cells following exposure to gamma-irradiation, compared to EL-4.wt control cells (Fig. 2B).
  • EL-4.pcDNA- TPPII cells that stably over-express TPPII, showed increased levels of p53 following exposure to gamma-irradiation in comparison to EL-4.pcDNA3 cells (Wang, EW, Kessler, BM, Borodovsky, A, Cravatt, BF, Bogyo, M, Ploegh, HL, et. al.
  • TPPII controls activation of several pathways that depend on PIKK signaling.
  • TPPII expression was a requirement for stabilization of p53 we tested also other stress-induced pathways that depend on PIKK signaling (Gasser, S, Orsulic, S 1 Brown, EJ, Raulet, DH.
  • the DNA damage pathway regulates innate immune system ligands of the NKG2D receptor. Nature 2005;436: 1186-90) (Viniegra, JG, Martinez, N, Modirassari, P, Losa, JH, Parada Cobo, C, Lobo, VJ, et. al. Full activation of PKB/Akt in response to insulin or ionizing radiation is mediated through ATM. J Biol Chem.
  • Akt kinase is important for transduction of cell survival signals, and is over- activated in many tumors.
  • EL-4. TPPII 1 cells showed an increased rate of proliferation, compared to EL-4.wt, but also an increased accumulation of dead cells (Fig. 3C). Further, by lowering serum concentrations to 1% this accumulation was accelerated, compared to EL-4.wt cells, suggesting that cell survival mechanisms
  • Akt kinase a direct substrate of Akt kinase (Dan, HC, Sun, M, Kaneko, S, Feldman, Rl, Nicosia, SV, Wang, HG, et. al. Akt
  • XIAP X-linked inhibitor of apoptosis protein
  • BRCA C-terminal repeat (BRCT)-domains are often contained within proteins controlling DNA damage signaling pathway, where they control interactions with ATM substrates (Bork, P, Hofmann, K, Bucher, P, Neuwald, AF, Altschul, SF, Koonin, EV.
  • ATM substrates Bork, P, Hofmann, K, Bucher, P, Neuwald, AF, Altschul, SF, Koonin, EV.
  • TPPir7G725E 25 mutation in position 725 (denoted TPPir7G725E).
  • TPPIf 1 as well as TPPII wt /G725E mutant molecules were stably expressed in EL-4 cells co-transfected with pSUPER-TPPII' (Fig. 4B).
  • the expression of p53 was analyzed in EL-4.TPPir and EL-4.TPPirVG725E transfectant cells exposed to gamma-irradiation.
  • EL-4.TPPII wt /G725E cells showed much reduced expression of p53, compared to EL-
  • p53 fails to be recruited to such sites.
  • NLVS-treated EL-4.TPPII 1 cells also failed to show ATM, 53BP1 and Mre1 1 in p53-immunoprecipitates (Fig. 4E-G).
  • p53 and ATM are found in proximity to DNA repair foci components is in line with that certain p53 isoforms accumulate at these foci, where they may interact with ATM kinase (Al Rashid, ST, Dellaire, G, Cuddihy, A, JaIaIi, F, Vaid, M, Coackley, C, et. al. Evidence for the direct binding of phosphorylated p53 to sites of DNA breaks in vivo. Cancer Res. 2005;65: 10810- 21 ).
  • Our data support that a physical link between p53 and ATM, as well as DNA repair foci components 53BP1 and Mre11 requires TPPII.
  • TPPII expression controls gamma-irradiation resistance of EL-4 tumors in vivo.
  • PIKKs are possible target molecules for the development of novel cancer therapies (Choudhury, A, Cuddihy, A, Bristow, RG. Radiation and new molecular agents part I: targeting ATM-ATR checkpoints, DNA repair, and the proteasome. Semin Radiat Oncol 2006; 16:51 -8).
  • TPPII-mediated growth regulation was important for in vivo tumor growth.
  • mice carrying either tumors of EL-4.wt or EL-4.TPPII' cells with 2- 4 doses of 4 Gy (400 Rad's) gamma-irradiation. We found that this had minor effects on tumor size after inoculation with 10 6 EL-4.wt cells that continued to grow despite gamma- irradiation (Fig. 5 A, gamma-irradiation indicated with arrow).
  • mice carrying tumors of EL-4. TPPII 1 cells responded to gamma-irradiation treatment with complete regression of established tumors (Fig. 5B). These data resembled those obtained with tumors of EL-4.ATM 1 or EL-4.TPPII wt /G725E cells, since these also failed to resist gamma- irradiation in vivo (Fig. 5C, D).
  • the data support TPPII as a target to increase in vivo gamma-irradiation susceptibility of tumor cells.
  • Tri-peptide-based TPPII inhibitors radio-sensitize tumors in vivo.
  • TPPII is a Subtilisin-type Serine peptidase, with a catalytic domain that is homologous to bacterial Subtilisins (Tomkinson, B, Wemstedt, C, Hellman, U, Zetterqvist, O. Active site of tripeptidyl peptidase Il from human erythrocytes is of the subtilisin type. Proc Natl Acad Sci U S A. 1987;84:7508-12).
  • Z-GLA-OH tri-peptide Subtilisin inhibitor Z-G Iy-Le u-Ala- OH
  • LLC tumors were virtually insensitive to repeated gamma-irradiation doses of 4 Gy, and Z-GLA- OH only (in the absence of gamma-irradiation) gave no effect (Fig. 6D).
  • Z-GLA-OH in the absence of gamma-irradiation
  • TPPII is an evolutionary conserved enzyme with an identity of 96% at the amino acid level between human and mouse, and we observed strong tumor regression also of human HeLa cervical carcinoma cells in Z-GLA-OH-treated SCID mice in response to gamma- irradiation (Fig. 6E).
  • a reduced dose of gamma-irradiation (1 ,5 Gy/dose) was used, since SCID mice have substantially reduced radio-resistance.
  • mice inoculated with DBA/2-c-Myc/Bcl-x L cells we found tumor dissemination into the liver, as observed by histological analysis of fixed organs (Fig. 7 H). These malignant cells were also detected by flow cytometry showing YFP + /GFP + cells in the spleen, lung and liver, using the cells from the primary tumor as control (Fig. 7 C-G).
  • gamma-irradiation 4 Gy/dose, 1 dose/week
  • Fig. 7 F, G, J we failed to find tumor cells in either lung, spleen or liver in these Z-GLA-OH-treated mice (Fig. 7 F, G, J).
  • Gamma-irradiation was required for this treatment response, since no reduction of tumor size was observed in mice receiving Z- GLA-OH only (Fig. 7 B).
  • Table 1 contains in vitro data, in fluorometric units which are arbitrary but relative, for the inhibition of cleavage of AAF-AMC (H-Ala-Ala-7-amido-4-methylcoumarin) by compounds at several concentrations. Some beneficial effect is seen for most of the compounds tested.
  • TPP Il protein was enriched, and then a TPP ll-preferred fluorogenic substrate AAF-AMC was used.
  • 100 x 10 6 cells were sedimented and lysed by vortexing in glass beads and homogenisation buffer (50 mM Tris Base pH 7.5, 250 mM Sucrose, 5 mM MgCI 2 , 1 mM DTT).
  • Cellular lysates were subjected to differential centrifugation; first the cellular homogenate was centrifuged at 14,000 rpm for 15 min, and then the supernatant was transferred to ultra-centrifugation tubes.
  • the sample was ultra-centrifugated at 100,000 x g for 1 hour, and the supernatant (denoted as cytosol in most biochemical literature) was subjected to 100,000 x g centrifugation for 5 hours, which sedimented high molecular weight cytosolic proteins/protein complexes.
  • the resulting pellet dissolved in 50 mM Tris Base pH 7.5, 30%Glycerol, 5 mM MgCI 2 , and 1 mM DTT, and 1 ug of high molecular weight protein was used as enzyme in peptidase assays.
  • TPP Il To test the activity of TPP Il we used the substrate and AAF-AMC (Sigma, St. Louis, MO), at 100 uM concentration in 100 ul of test buffer composed of 50 mM Tri Base pH 7.5, 5 mM MgCI 2 and 1 mM DTT. To stop reactions we used dilution with 900 ul 1% SDS solution. Cleavage activity was measured by emission at 460 nm in a LS50B Luminescence Spectrometer (Perkin Elmer, Boston, MA).
  • FA 3-(2-furyl)acryloyl
  • PBS phosphate-buffered saline.
  • the text (Z, FA, H, etc.) at the start of each compound name is the substituent at the N-terminus; H indicates that the N- terminus is free NH 2 .
  • the text (OH, NBu, etc.) at the end of each compound name is the substituent at the C-terminus; OH indicates that the C-terminus is free CO 2 H.
  • Table 2 contains in vivo data, showing tumor volume in mm 3 , in groups of 4 mice with LLC (Lewis Lung Carcinoma). Mice were sacrificed if the tumor volume exceeded 1000 mm 3 . Some mice were administered with the compounds alone; others were additionally administered with irradiation. Mice were given the compounds, and in some cases also gamma irradition (400 Rad), at days 7, 10, 14, 18 and 21. In combination with irradiation some compounds showed excellent results.
  • the fact that the dipeptide derivative Z-GL- OH performs poorly in vitro as well as in vivo supports the theory that the in vitro results can be extrapolated to in vivo effects.
  • 6,0 144,0 600,0 600 mean 6,00 168,00 495,00 800,00
  • FA-GLA-OH 4 0 100,0 90,0 48 18,0 irradiated 0,0 90,0 120,0 48 48,0
  • 18,0 320,0 864,0 1372 mean 7,00 207,00 672,00 1076,50 1654,00 Table 2 days after tumor inoculation
  • Table 3 contains further in vivo data, showing tumor volume in mm 3 , in groups of 7-8 mice, according to the EL-4 tumor model described above. 1.000.000 EL-4 lymphoma cells were inoculated subcutaneously at day 0. No palpable tumors were observed until day 22. At each treatment (twice weekly) mice with palpable tumors were given 400 Rads irradiation alone, or in combination with 14 micro-l 5OmM solution of Z-GLA-OH. Mice with no palpable tumors were not treated, i.e. in mice with rejected tumors, treatment was terminated and the mice were kept under observation. Table 3 shows excellent results, namely complete rejection of established tumors, not just arrest of tumor growth, decreased volume, or a delay of tumor growth.
  • the compound was inoculated intraperitoneally, whereas tumors were always inoculated subcutaneously.
  • GPG-NH 2 and Z-GPG-NH 2 were tested in the same manner as Z-GLA-OH. These were injected twice weekly at 13.8 mg/kg in tumor bearing mice, and compared to Z-GLA-OH for their ability to mediate sensitization to gamma-irradiation in vivo. We found that both GPG- NH 2 and Z-GPG-NH 2 mediated complete regression of established EL-4 tumors following gamma-irradiation.
  • TPPII is rapidly translocated into the nucleus of gamma-irradiated cells.
  • the results of further immunocytochemical experiments are shown in Figure 9.
  • TPPII does not appear to form foci, which would have instead shown a dotted appearance (Fig. 9, shown for cells with inhibited TPPII expression, LLC, ALC and YAC-1 ).
  • This failure of cells with inhibited TPP Il expression to assemble Mre1 1 foci upon gamma-irradiation exposure provides further support for the use of TPP Il inhibitors in the present invention.
  • TPPII inhibitors allow increased efficiency of in vivo chemotherapy
  • TPPII inhibition complemented cytostatic drugs, of eight different classes, in experimental cancer therapy in mice.
  • Z-GLA-OH could increase the efficiency of common cytostatic drugs clinically used, including compounds that belong to the groups of DNA Topoisomerase inhibitors, DNA inter-calators, Alkylators, Anti-metabolites, Tubulin inhibitors, Proteasome inhibitors and Hsp90 inhibitors (Table 4).
  • cytostatic drugs were injected twice weekly in mice with established tumors at doses previously used in therapeutic cancer models in mice, and we observed a reduced tumor growth in most treated mice, causing a substantial delay in tumor growth (Fig. 10).
  • DNA Topoisomerase inhibitors Etoposide, 12,5 mg/kg; Doxorubicin, 16 mg/kg or Irinotecan, 60 mg/kg
  • a DNA inter-calating drug Ciplatinum, 12mg/kg
  • DNA damaging drugs 5-Fluoro-Uracil (anti-metabolite, 20 mg/kg) and Cyclophosphamide (alkylator, 220 mg/kg) also showed therapeutic effect on tumor growth in combination with Z-GLA-OH (Fig. 11 ).
  • Cyclophosphamide was very effective in combination with Z-GLA-OH, with complete regression of established EL-4 tumors in most tested mice.
  • Many of the tested cytostatic drugs caused occasional complete regressions, e.g. using Velcade or Geldanamycin, although these treatment responses were mostly partial (Fig. 1 , exp. 2).
  • Paclitaxel 22 mg/kg
  • Taxoter 22 mg/kg
  • Vinorelbine Tubulin inhibitors
  • TPPII inhibition allows greatly improved therapy with several common cytostatic drugs in clinical use.
  • Tri-peptide TPPII inhibitors allow increased efficiency of treatment with angiogenesis inhibitors in vivo.
  • TNP-470 and Thalidomide were treated three times per week from the time point of tumor detection (i.e. at least 1 mm 3 size) until the tumors reached 1000 mm 3 or regressed.
  • both TNP-470 or Thalidomide treatments had partial effects on the growth of EL-4.wt control tumors, which reached the size of 1000 mm 3 with a delay of 1-2 weeks, compared to untreated mice ( Figure 12).
  • Z-GLA-OH to these injections with angiogenesis inhibitors caused a substantial improvement of the anti-tumour effects, with frequent complete regressions occurring; in about 50% of the mice ( Figure 12).
  • Dexamethasone-treatment is a standard method to treat patients with auto-immune, inflammatory as well as transplantation rejection diseases. It is however clear that disease symptoms, as well as immune activation and proliferation, are sometimes not controlled by Dexamethasone, or other Cortisone derivatives. Certain cytostatic drugs, e.g. Sendoxan or Cyclophosphamide, are treatment options when others have failed.

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  • Proteomics, Peptides & Aminoacids (AREA)
  • Immunology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Engineering & Computer Science (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Organic Chemistry (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

Les inhibiteurs de la TPP II (tripeptidyl-peptidase II) sont utiles pour amplifier l'efficacité d'une chimiothérapie du cancer ou augmenter la sensibilité in vivo à la chimiothérapie du cancer des cellules tumorales. Les composés appropriés comprennent des composés de tripeptides de formule générale RN1RN2-A1-A2-A3-CO-RC1 dans laquelle RN1, RN2, A1, A2, A3 et RC1 sont tels que définis dans ce document et qui comprennent, par exemple, les séquences tripeptidiques GLA et GPG. Des expériences in vivo montrent que les inhibiteurs de la TPPII sont efficaces en combinaison avec chacun de tout un éventail d'agents chimiothérapeutiques de divers types.
PCT/EP2007/005621 2007-06-25 2007-06-25 Inhibiteurs de la tpp ii en vue d'une utilisation en combinaison avec une chimiothérapie destinée au traitement du cancer WO2009000297A1 (fr)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8362068B2 (en) 2009-12-18 2013-01-29 Idenix Pharmaceuticals, Inc. 5,5-fused arylene or heteroarylene hepatitis C virus inhibitors
WO2015040235A1 (fr) 2013-09-23 2015-03-26 Dr. August Wolff Gmbh & Co. Kg Arzneimittel Tripeptides anti-inflammatoires
US12036286B2 (en) 2021-03-18 2024-07-16 Seagen Inc. Selective drug release from internalized conjugates of biologically active compounds

Citations (2)

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WO2003105835A1 (fr) * 2002-06-14 2003-12-24 President And Fellows Of Harvard College Methodes d'utilisation d'inhibiteurs de tripeptidyl peptidase ii comme agents anticancereux
WO2007080194A2 (fr) * 2006-01-13 2007-07-19 Oncoreg Ab Utilisation de composés en combinaison avec l'irradiation gamma pour le traitement du cancer

Patent Citations (2)

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WO2003105835A1 (fr) * 2002-06-14 2003-12-24 President And Fellows Of Harvard College Methodes d'utilisation d'inhibiteurs de tripeptidyl peptidase ii comme agents anticancereux
WO2007080194A2 (fr) * 2006-01-13 2007-07-19 Oncoreg Ab Utilisation de composés en combinaison avec l'irradiation gamma pour le traitement du cancer

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GANELLIN C R ET AL: "Inhibitors of tripeptidyl peptidase II. 2. Generation of the first novel lead inhibitor of cholecystokinin-8-inactivating peptidase: A strategy for the design of peptidase inhibitors", JOURNAL OF MEDICINAL CHEMISTRY, AMERICAN CHEMICAL SOCIETY. WASHINGTON, US, vol. 43, no. 4, 2000, pages 664 - 674, XP002389454, ISSN: 0022-2623 *
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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8362068B2 (en) 2009-12-18 2013-01-29 Idenix Pharmaceuticals, Inc. 5,5-fused arylene or heteroarylene hepatitis C virus inhibitors
US9187496B2 (en) 2009-12-18 2015-11-17 Idenix Pharmaceuticals Llc 5,5-fused arylene or heteroarylene hepatitis C virus inhibitors
WO2015040235A1 (fr) 2013-09-23 2015-03-26 Dr. August Wolff Gmbh & Co. Kg Arzneimittel Tripeptides anti-inflammatoires
US12036286B2 (en) 2021-03-18 2024-07-16 Seagen Inc. Selective drug release from internalized conjugates of biologically active compounds

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