[go: up one dir, main page]

WO2018185516A1 - Methods and pharmaceutical compositions for treating cardiovascular toxicity induced by anti-cancer therapy - Google Patents

Methods and pharmaceutical compositions for treating cardiovascular toxicity induced by anti-cancer therapy Download PDF

Info

Publication number
WO2018185516A1
WO2018185516A1 PCT/IB2017/000566 IB2017000566W WO2018185516A1 WO 2018185516 A1 WO2018185516 A1 WO 2018185516A1 IB 2017000566 W IB2017000566 W IB 2017000566W WO 2018185516 A1 WO2018185516 A1 WO 2018185516A1
Authority
WO
WIPO (PCT)
Prior art keywords
sunitinib
cancer
compound
endothelin
treatment
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/IB2017/000566
Other languages
French (fr)
Inventor
Bertrand Tavitian
Neeraj DHAUN
Joevin SOURDON
Pierre-Louis Tharaux
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Assistance Publique Hopitaux de Paris APHP
Institut National de la Sante et de la Recherche Medicale INSERM
Universite Paris Descartes
University of Edinburgh
Original Assignee
Assistance Publique Hopitaux de Paris APHP
Institut National de la Sante et de la Recherche Medicale INSERM
Universite Paris Descartes
University of Edinburgh
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Assistance Publique Hopitaux de Paris APHP, Institut National de la Sante et de la Recherche Medicale INSERM, Universite Paris Descartes, University of Edinburgh filed Critical Assistance Publique Hopitaux de Paris APHP
Priority to PCT/IB2017/000566 priority Critical patent/WO2018185516A1/en
Publication of WO2018185516A1 publication Critical patent/WO2018185516A1/en
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Classifications

    • 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
    • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
    • A61K31/403—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil condensed with carbocyclic rings, e.g. carbazole
    • A61K31/404—Indoles, e.g. pindolol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/506—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim not condensed and containing further heterocyclic rings
    • 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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system

Definitions

  • the present invention relates to methods and pharmaceutical compositions for treating cardiovascular toxicity induced by an anti-cancer and an anti-angiogenic compound.
  • anticancer drugs The major factor limiting therapeutic administration of anticancer drugs is their toxic side effect on off-target organs. It is well known that classical anticancer drugs, e.g. anthracyclines, antimetabolites, alkylating agents, taxanes, induce serious cardiovascular toxicity (1,2). Newer anticancer agents such as interferon and tyrosine kinase receptor (TKR) inhibitors also have cardiovascular side-effects (3) that, although often less severe than those observed with anthracyclines, are frequent and may be life-threatening (4). The clinical importance of cardiotoxicity associated with cancer therapy has led to the emergence of cardio- oncology, an interdisciplinary field that aims to better understand and limit the cardiotoxicity of cancer therapy (5).
  • classical anticancer drugs e.g. anthracyclines, antimetabolites, alkylating agents, taxanes
  • TTR tyrosine kinase receptor
  • VEGFRs Vascular Endothelial Growth Factor Receptors
  • Sunitinib (Sutent; Pfizer, USA) is an anti-angiogenic TKR inhibitor of VEGFRs, platelet-derived growth factor receptors (PDGF-Rs), and c-kit (13), approved in 2006 by the FDA for the treatment of renal cell carcinoma (14), imatinib-resistant gastrointestinal stromal tumor (15) and neuroendocrine tumors (16).
  • PDGF-Rs platelet-derived growth factor receptors
  • c-kit 13
  • sunitinib leads to mitochondrial dysfunction; in mice, to increased apoptosis of cardiomyocytes (8).
  • a poor coronary flow response to bradykinin was reported in sunitinib-treated hearts, supporting microvascular dysfunction as a direct cardiac side effect of the drug (21).
  • sunitinib inhibits AMPK (22) and induces mitochondrial damage (8) opens up the possibility that some or all of its side effects could result from a direct deregulation of cardiac metabolism.
  • ET receptor blockers can reduce blood pressure (BP) and renal injury in animals and patients treated with sunitinib (21,23-25).
  • BP blood pressure
  • ET receptor antagonism in treating or preventing the cardiotoxic effects of sunitinib have not been studied.
  • the inventors explored cardiac metabolism after sunitinib treatment in mice using PET-FDG.
  • the inventors aimed to (i) better clarify the cardiac metabolic pathways deregulated during the early stages of sunitinib treatment, (ii) determine if the cardiac side effects are mediated by the endothelin pathway (iii) test the blockade of the endothelin system to prevent the cardiac side effects of sunitinib, and (iv) confirm that PET- FDG can be useful to monitor cardiac metabolic remodeling.
  • the present invention relates to methods and pharmaceutical compositions for treating cardiovascular toxicity induced by an anti-cancer and an anti-angiogenic compound.
  • the growing field of cardio-oncology addresses the side effects of cancer treatment on the cardiovascular system.
  • sunitinib explored the cardiotoxicity induced by the antiangiogenic therapy, sunitinib, in the mouse heart from a diagnostic and therapeutic perspective.
  • the inventors showed that sunitinib induces an anaerobic switch of cellular metabolism within the myocardium which is associated with the development of myocardial fibrosis as demonstrated by echocardiography.
  • the capacity of positron emission tomography with [ 18 F]fluorodeoxyglucose to detect the changes in cardiac metabolism caused by sunitinib was dependent on fasting status and duration of treatment.
  • Pan proteomic analysis in the myocardium showed that sunitinib induced (i) an early metabolic switch with enhanced glycolysis and reduced oxidative phosphorylation, and (ii) a metabolic failure to use glucose as energy substrate, similar to the insulin resistance found in type 2 diabetes.
  • Co-administration of macitentan, the endothelin receptor antagonist, to sunitinib-treated animals prevented both metabolic defects, restored glucose uptake and cardiac function, and prevented myocardial fibrosis.
  • These results support the endothelin system in mediating the cardiotoxic effects of sunitinib and endothelin receptor antagonism as a potential therapeutic approach to prevent cardiotoxicity.
  • metabolic and functional imaging can monitor the cardiotoxic effects and the benefits of endothelin antagonism in a theranostics approach.
  • the present invention relates to a compound selected from the group consisting of endothelin receptor antagonist and inhibitor of endothelin receptor expression for use in the treatment of cardiovascular toxicity induced by anti-cancer compound in a subject in need thereof.
  • the present invention relates to a compound selected from the group consisting of endothelin receptor antagonist and inhibitor of endothelin receptor expression for use in the treatment of cardiovascular toxicity induced by anti-angiogenic compound in a subject in need thereof.
  • the term "subject” denotes a mammal. Typically, a subject according to the invention refers to any subject (preferably human) receiving anti-cancer therapy. Typically, a subject according to the invention refers to any subject (preferably human) receiving anti- angiogenic therapy. In a particular embodiment, the term “subject” refers to a subject afflicted or at risk to be afflicted with cardiovascular toxicity induced by anti-cancer therapy or anti- angiogenic therapy. In a particular embodiment, the term “subject” refers to a subject afflicted with cancer. In a particular embodiment, the term “subject” refers to a subject afflicted with angiogenesis-related diseases.
  • the term “subject” refers to a subject afflicted with cancer or angiogenesis-related diseases receiving anti-cancer therapy or anti- angiogenic therapy.
  • the term “subject” refers to a subject afflicted with cancer or angiogenesis-related diseases, and afflicted or at risk to be afflicted with cardiovascular toxicity induced by anti-cancer therapy or anti-angiogenic therapy.
  • treatment refers to both prophylactic or preventive treatment as well as curative or disease modifying treatment, including treatment of subjects at risk of contracting the disease or suspected to have contracted the disease as well as subjects who are ill or have been diagnosed as suffering from a disease or medical condition, and includes suppression of clinical relapse.
  • the treatment may be administered to a subject having a medical disorder or who ultimately may acquire the disorder, in order to prevent, cure, delay the onset of, reduce the severity of, or ameliorate one or more symptoms of a disorder or recurring disorder, or in order to prolong the survival of a subject beyond that expected in the absence of such treatment.
  • therapeutic regimen is meant the pattern of treatment of an illness, e.g., the pattern of dosing used during therapy.
  • a therapeutic regimen may include an induction regimen and a maintenance regimen.
  • the phrase “induction regimen” or “induction period” refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the initial treatment of a disease.
  • the general goal of an induction regimen is to provide a high level of drug to a subject during the initial period of a treatment regimen.
  • An induction regimen may employ (in part or in whole) a "loading regimen", which may include administering a greater dose of the drug than a physician would employ during a maintenance regimen, administering a drug more frequently than a physician would administer the drug during a maintenance regimen, or both.
  • maintenance regimen refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the maintenance of a subject during treatment of an illness, e.g., to keep the subject in remission for long periods of time (months or years).
  • a maintenance regimen may employ continuous therapy (e.g., administering a drug at a regular intervals, e.g., weekly, monthly, yearly, etc.) or intermittent therapy (e.g., interrupted treatment, intermittent treatment, treatment at relapse, or treatment upon achievement of a particular predetermined criteria [e.g., disease manifestation, etc.]).
  • cardiovascular toxicity has its general meaning in the art and refers to cardiotoxicity induced by anti-cancer therapy.
  • cardiac toxicity also refers to cardiotoxicity induced by anti-angiogenic therapy.
  • cardiovascular toxicity also refers to cardiovascular side-effects of anti-cancer therapy and anti-angiogenic therapy.
  • cardiac toxicity also refers to metabolic defects, increase myocardial fibrosis, reduction of myocardial glucose uptake, cardiac dysfunction, cardiac ischemia and switch toward anaerobic metabolism.
  • cardiovascular toxicity also refers to anaerobic switch of cellular metabolism within the myocardium which is associated with the development of myocardial fibrosis, early metabolic switch with enhanced glycolysis and reduced oxidative phosphorylation, and a metabolic failure to use glucose as energy substrate, similar to the insulin resistance found in type 2 diabetes.
  • the compound of the invention is used in the treatment of vascular toxicity induced by anti-cancer therapy and anti-angiogenic therapy.
  • vascular toxicity refers to microvascular dysfunction and damage in the heart, kidney, retina, brain and other target organs such as renal, retinal and cerebrovascular circulation dysfunction induced by anti-cancer therapy and anti-angiogenic therapy.
  • cancer has its general meaning in the art and includes, but is not limited to, solid tumors and blood borne tumors.
  • the term cancer includes diseases of the skin, tissues, organs, bone, cartilage, blood and vessels.
  • the term “cancer” further encompasses both primary and metastatic cancers. Examples of cancers include, but are not limited to, cancer cells from the adrenal, bladder, blood, bone, bone marrow, brain, breast, colon, esophagus, gastrointestine, gum, head, kidney, liver, lung, nasopharynx, neck, ovary, prostate, skin, stomach, sympathic and parasympathic ganglia, testis, tongue, or uterus.
  • the cancer may specifically be of the following histological type, though it is not limited to these: neoplasm, malignant; carcinoma; carcinoma, undifferentiated; giant and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; pilomatrix carcinoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; gastrinoma, malignant; cholangiocarcinoma; hepatocellular carcinoma; combined hepatocellular carcinoma and cholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma in adenomatous polyp; adenocarcinoma, familial polyposis coli; solid carcinoma; carcinoid tumor, malignant; branchiolo-alveolar adenocarcinoma; papillary adenocarcinoma; chromophobe carcinoma; acid
  • the subject suffers from a cancer selected from the group consisting of breast cancer, triple negative breast cancer, colon cancer, lung cancer, prostate cancer, testicular cancer, brain cancer, skin cancer, rectal cancer, gastric cancer, esophageal cancer, sarcomas, adrenal cancer, sympathic and parasympathic ganglia cancer, tracheal cancer, head and neck cancer, pancreatic cancer, liver cancer, ovarian cancer, lymphoid cancer, cervical cancer, vulvar cancer, melanoma, mesothelioma, renal cancer, bladder cancer, thyroid cancer, bone cancers, carcinomas, sarcomas, and soft tissue cancers.
  • a cancer selected from the group consisting of breast cancer, triple negative breast cancer, colon cancer, lung cancer, prostate cancer, testicular cancer, brain cancer, skin cancer, rectal cancer, gastric cancer, esophageal cancer, sarcomas, adrenal cancer, sympathic and parasympathic ganglia cancer, tracheal cancer
  • angiogenesis-related diseases has its general meaning in the art and refers to diseases associated with or supported by pathological angiogenesis (i.e., inappropriate, excessive or undesired formation of blood vessels), which may be induced by various angiogenic factors.
  • pathological angiogenesis i.e., inappropriate, excessive or undesired formation of blood vessels
  • angiogenesis-related diseases also relates to angiogenic diseases associated with abnormal neovascularisation.
  • Angiogenesis-related diseases include but are not limited to cancer, tumor angiogenesis, primary and metastatic solid tumors, including carcinomas of breast, colon, rectum, lung, oropharynx, hypopharynx, esophagus, stomach, pancreas, liver, gallbladder and bile ducts, small intestine, kidney, bladder, urothelium, female genital tract, (including cervix, uterus, and ovaries as well as choriocarcinoma and gestational trophoblastic disease), male genital tract (including prostate, seminal vesicles, testes and germ cell tumors), endocrine glands (including the thyroid, adrenal, and pituitary glands), and skin, as well as hemangiomas, melanomas, sarcomas (including those arising from bone and soft tissues as well as Kaposi's sarcoma) and tumors of the brain, nerves, eyes, such as astrocytomas,
  • Angiogenesis-related diseases also relate to tumors arising from hematopoietic malignancies such as leukemias as well both Hodgkin's and non-Hodgkin's lymphomas.
  • Angiogenesis-related diseases also relate to various ocular diseases such as diabetic retinopathy, retinopathy of prematurity, corneal graft rejection, retrolental fibroplasia, neovascular glaucoma, rubeosis, retinal neovascularization due to macular degeneration, hypoxia, angiogenesis in the eye associated with infection or surgical intervention, and other abnormal neovascularization conditions of the eye.
  • Angiogenesis- related diseases also relate to rheumatoid, immune and degenerative arthritis.
  • Angiogenesis-related diseases also relate to skin diseases such as psoriasis; blood vessel diseases such as hemagiomas, and capillary proliferation within atherosclerotic plaques; Osier-Webber Syndrome; myocardial angiogenesis; plaque neovascularization; telangiectasia; haemophiliac joints; angiofibroma; and wound granulation.
  • Angiogenesis-related diseases also relate to diseases characterized by excessive or abnormal stimulation of endothelial cells, including but not limited to intestinal adhesions, Crohn's disease, atherosclerosis, scleroderma, and hypertrophic scars, i.e. keloids., diseases that have angiogenesis as a pathologic consequence such as cat scratch disease (Rochele ninalia quintosa) and ulcers (Helicobacter pylori).
  • anti-cancer compound has its general meaning in the art and refers to compounds used in anti-cancer therapy such as anti-angiogenic compound, tyrosine kinase inhibitors, tyrosine kinase receptor (TKR) inhibitors, Vascular Endothelial Growth Factor Receptors (VEGFRs) pathway inhibitors, interferon therapy, anti-HER2 compounds, anti- EGFR compounds, alkylating agents, anti-metabolites, immunotherapeutic agents, Interferons (IFNs), Interleukins, and chemotherapeutic agents such as described below.
  • anti-angiogenic compound has its general meaning in the art and refers to compounds used in anti-angiogenic therapy such as tyrosine kinase inhibitors, anti-angiogenic tyrosine kinase receptor (TKR) inhibitors, anti-angiogenics targeting the Vascular Endothelial Growth Factor Receptors (VEGFRs) pathway, interferon therapy and anti-HER2 compounds such as Trastuzumab (herceptin) and pertuzumab.
  • anti- angiogenic compound refers to Sunitinib (Sutent), an anti-angiogenic TKR inhibitor of VEGFRs, platelet-derived growth factor receptors (PDGF-Rs), and c-kit.
  • tyrosine kinase inhibitor refers to any of a variety of therapeutic agents or drugs that act as selective or non-selective inhibitors of receptor and/or non-receptor tyrosine kinases. Tyrosine kinase inhibitors and related compounds are well known in the art and described in U.S Patent Publication 2007/0254295, which is incorporated by reference herein in its entirety.
  • a compound related to a tyrosine kinase inhibitor will recapitulate the effect of the tyrosine kinase inhibitor, e.g., the related compound will act on a different member of the tyrosine kinase signaling pathway to produce the same effect as would a tyrosine kinase inhibitor of that tyrosine kinase.
  • tyrosine kinase inhibitors and related compounds suitable for use in methods of embodiments of the present invention include, but are not limited to, sunitinib (Sutent; SU11248), dasatinib (BMS-354825), PP2, BEZ235, saracatinib, gefitinib (Iressa), erlotinib (Tarceva; OSI-1774), lapatinib (GW572016; GW2016), canertinib (CI 1033), semaxinib (SU5416), vatalanib (PTK787/ZK222584), sorafenib (BAY 43-9006), imatinib (Gleevec; STI571), leflunomide (SU101), vandetanib (Zactima; ZD6474), MK-2206 (8-[4-aminocyclobutyl)phenyl]-9-phenyl- l,2,4-triazolo[3,4
  • the tyrosine kinase inhibitor is a small molecule kinase inhibitor that has been orally administered and that has been the subject of at least one Phase I clinical trial, more preferably at least one Phase II clinical, even more preferably at least one Phase III clinical trial, and most preferably approved by the FDA for at least one hematological or oncological indication.
  • inhibitors include, but are not limited to, Gefitinib, Erlotinib, Lapatinib, Canertinib, BMS-599626 (AC-480), Neratinib, KR -633, CEP-11981, Imatinib, Nilotinib, Dasatinib, AZM-475271, CP-724714, TAK-165, Sunitinib, Vatalanib, CP- 547632, Vandetanib, Bosutinib, Lestaurtinib, Tandutinib, Midostaurin, Enzastaurin, AEE-788, Pazopanib, Axitinib, Motasenib, OSI-930, Cediranib, KR -951, Dovitinib, Seliciclib, SNS- 032, PD-0332991, MKC-I (Ro-317453; R-440), Sorafenib, ABT
  • anti-angiogenic compound refers to compounds targeting the vascular endothelial growth factor (VEGF) pathway such anti-VEGF antibody bevacizumab (Avastin) and VEGF receptor tyrosine kinase inhibitor (TKI) compounds such as sunitinib (Sutent), vandetanib (Zactima), pazopanib (Votrient), sorafenib (Nexavar) and cediranib.
  • VEGF vascular endothelial growth factor
  • TKI VEGF receptor tyrosine kinase inhibitor
  • endothelin has its general meaning in the art and refers to the vaso constricting peptide endothelin- 1 (ET-1) produced primarily in the endothelium and acting on its both receptors ETA and ETB .
  • the endothelin (ET- 1 ) can be from any source, but typically is a mammalian (e.g., human and non-human primate) endothelin, particularly a human endothelin.
  • An exemplary native endothelin- 1 amino acid sequence is provided in UniProt database under accession number P05305 and an exemplary native nucleotide sequence encoding for endothelin- 1 is provided in GenBank database under accession number NM_001955.
  • endothelin receptor has its general meaning in the art and refers to G-protein- coupled receptors ETA and ETB.
  • ETA has its general meaning in the art and refers to the endothelin receptor type A.
  • ETA can be from any source, but typically is a mammalian (e.g., human and non-human primate) ETA, particularly a human ETA.
  • An exemplary native ETA amino acid sequence is provided in UniProt database under accession number P25101 and an exemplary native nucleotide sequence encoding for ETA is provided in GenBank database under accession number NM_001957.
  • ETB has its general meaning in the art and refers to the endothelin receptor type B.
  • ETB can be from any source, but typically is a mammalian (e.g., human and non-human primate) ETB, particularly a human ETB.
  • An exemplary native ETB amino acid sequence is provided in UniProt database under accession number P24530 and an exemplary native nucleotide sequence encoding for ETB is provided in GenBank database under accession number NM 000115.
  • endothelin receptor antagonist refers to a compound that selectively blocks or inactivates endothelin receptor.
  • selectively blocks or inactivates refers to a compound that preferentially binds to and blocks or inactivates endothelin receptor with a greater affinity and potency, respectively, than its interaction with the other sub-types or isoforms of G protein-coupled receptors family.
  • Compounds that prefer endothelin receptor, but that may also block or inactivate other G protein-coupled receptors sub-types, as partial or full antagonists, are contemplated.
  • endothelin receptor antagonist refers to any compound that can directly or indirectly block the signal transduction cascade related to the endothelin receptor.
  • endothelin receptor antagonist should be understood broadly and encompasses compounds acting directly (by binding) on endothelin, ETA or ETB proteins and able to prevent the interaction or binding between endothelin and its receptor(s).
  • endothelin receptor antagonist also refers to dual ETA and ETB receptor antagonist (ETA/ETB antagonist), selective ETA receptor antagonist or selective ETB receptor antagonist.
  • the "endothelin receptor antagonist” may also consist in compounds that inhibit the binding of the endothelin to endothelin receptor.
  • endothelin receptor antagonist also refers to inhibition of endothelin downstream signalling, phospholipase C, protein kinase C and MAPK1;ERK2.
  • endothelin receptor antagonist also refers to endothelin inhibitor such as compound able to prevent the action of endothelin (ET-1) on its receptors ETA and ETB, inhibitor of endothelin formation and inhibitor of endothelin expression.
  • an endothelin receptor antagonist is a small organic molecule, an oligonucleotide, a polypeptide, an aptamer, an antibody or an intra-antibody.
  • Tests and assays for determining whether a compound is an endothelin receptor antagonist are well known by the skilled person in the art such as described in Aubert and Juillerat-Jeanneret, 2017; Okada M. et al, 2002; US5,292,740 and US5,883,254.
  • the endothelin receptor antagonists are well-known in the art as illustrated by Aubert and Juillerat-Jeanneret, 2017; Okada M. et al, 2002; US5,292,740 and US5,883,254.
  • the endothelin receptor antagonist is selected from the group consisting of macitentan, bosentan, darusentan, sitaxsentan, tezosentan, ambrisentan, atrasentan, avosentan, clazosentan, zibotentan, edonentan, enrasentan, danusentan, A- 182086, A- 192621, ABT-627, BMS193884, BQ-123, BQ-788, CI 1020, FR-139317, S-0139, CPU0213, J- 104132, SB-209670, TA-0201, TAK-044, TBC3711, YM-598, ZD-1611, ZD-4054 and compounds described in Aubert
  • the endothelin receptor antagonist is an ETA/ETB antagonist such as macitentan, bosentan (US5,292,740 and US5, 883,254), A-182086, CPU0213, J-104132 and SB209670.
  • ETA/ETB antagonist such as macitentan, bosentan (US5,292,740 and US5, 883,254), A-182086, CPU0213, J-104132 and SB209670.
  • the endothelin receptor antagonist is a selective ETB receptor antagonist such as BQ-788 (Okada M. et al, 2002) and A- 192621.
  • a selective ETB receptor antagonist exhibits an affinity (as expressed by dissociation constant Ki) for ETB less than about ⁇ and a selectivity for ETB over ETA (as expressed by the ratio KiETB/KiETA) is at least about 50.
  • KiETB is less than 5 nM, more particularly less than 2 nM and even more particularly less than 1 Nm.
  • the ratio KiETB/KiETA is at least about 100, more particularly about 500 and even more particularly about 1000.
  • the endothelin receptor antagonist is an endothelin inhibitor such as a molecule that decreases or prevents endothelin formation.
  • ECE inhibitor Endothelin-Converting Enzyme inhibitor
  • FR901533 also called WS79089B, Tsurumi Y, et al, 1994 and Tsurumi Y, et al, 1995
  • CGS 26303 De Lombaert S et al, 1994
  • EAE Endothelin-Converting Enzyme
  • the endothelin receptor antagonist of the invention is an aptamer.
  • Aptamers are a class of molecule that represents an alternative to antibodies in term of molecular recognition.
  • Aptamers are oligonucleotide sequences with the capacity to recognize virtually any class of target molecules with high affinity and specificity.
  • Such ligands may be isolated through Systematic Evolution of Ligands by Exponential enrichment (SELEX) of a random sequence library, as described in Tuerk C. and Gold L., 1990.
  • the random sequence library is obtainable by combinatorial chemical synthesis of DNA. In this library, each member is a linear oligomer, eventually chemically modified, of a unique sequence.
  • Peptide aptamers consists of a conformationally constrained antibody variable region displayed by a platform protein, such as E. coli Thioredoxin A that are selected from combinatorial libraries by two hybrid methods (Colas et al, 1996). Then after raising aptamers directed against endothelin receptor of the invention as above described, the skilled man in the art can easily select those inhibiting endothelin receptor.
  • the endothelin receptor antagonist of the invention is an antibody (the term including "antibody portion") directed against endothelin receptors ETA and/or ETB or endothelin.
  • the antibody is a monoclonal antibody. In one embodiment of the antibodies or portions thereof described herein, the antibody is a polyclonal antibody. In one embodiment of the antibodies or portions thereof described herein, the antibody is a humanized antibody. In one embodiment of the antibodies or portions thereof described herein, the antibody is a chimeric antibody. In one embodiment of the antibodies or portions thereof described herein, the portion of the antibody comprises a light chain of the antibody. In one embodiment of the antibodies or portions thereof described herein, the portion of the antibody comprises a heavy chain of the antibody. In one embodiment of the antibodies or portions thereof described herein, the portion of the antibody comprises a Fab portion of the antibody.
  • the portion of the antibody comprises a F(ab')2 portion of the antibody. In one embodiment of the antibodies or portions thereof described herein, the portion of the antibody comprises a Fc portion of the antibody. In one embodiment of the antibodies or portions thereof described herein, the portion of the antibody comprises a Fv portion of the antibody. In one embodiment of the antibodies or portions thereof described herein, the portion of the antibody comprises a variable domain of the antibody. In one embodiment of the antibodies or portions thereof described herein, the portion of the antibody comprises one or more CDR domains of the antibody.
  • antibody includes both naturally occurring and non-naturally occurring antibodies. Specifically, “antibody” includes polyclonal and monoclonal antibodies, and monovalent and divalent fragments thereof. Furthermore, “antibody” includes chimeric antibodies, wholly synthetic antibodies, single chain antibodies, and fragments thereof. The antibody may be a human or nonhuman antibody. A nonhuman antibody may be humanized by recombinant methods to reduce its immunogenicity in man.
  • Antibodies are prepared according to conventional methodology. Monoclonal antibodies may be generated using the method of Kohler and Milstein (Nature, 256:495, 1975). To prepare monoclonal antibodies useful in the invention, a mouse or other appropriate host animal is immunized at suitable intervals (e.g., twice-weekly, weekly, twice-monthly or monthly) with antigenic forms of endothelin receptors ETA and/or ETB or endothelin. The animal may be administered a final "boost" of antigen within one week of sacrifice. It is often desirable to use an immunologic adjuvant during immunization.
  • Suitable immunologic adjuvants include Freund's complete adjuvant, Freund's incomplete adjuvant, alum, Ribi adjuvant, Hunter's Titermax, saponin adjuvants such as QS21 or Quil A, or CpG-containing immunostimulatory oligonucleotides.
  • Other suitable adjuvants are well-known in the field.
  • the animals may be immunized by subcutaneous, intraperitoneal, intramuscular, intravenous, intranasal or other routes. A given animal may be immunized with multiple forms of the antigen by multiple routes.
  • the antigen may be provided as synthetic peptides corresponding to antigenic regions of interest in endothelin receptors ETA and/or ETB or endothelin.
  • lymphocytes are isolated from the spleen, lymph node or other organ of the animal and fused with a suitable myeloma cell line using an agent such as polyethylene glycol to form a hydridoma.
  • cells are placed in media permissive for growth of hybridomas but not the fusion partners using standard methods, as described (Coding, Monoclonal Antibodies: Principles and Practice: Production and Application of Monoclonal Antibodies in Cell Biology, Biochemistry and Immunology, 3rd edition, Academic Press, New York, 1996).
  • cell supernatants are analyzed for the presence of antibodies of the desired specificity, i.e., that selectively bind the antigen.
  • Suitable analytical techniques include ELISA, flow cytometry, immunoprecipitation, and western blotting. Other screening techniques are well-known in the field. Preferred techniques are those that confirm binding of antibodies to conformationally intact, natively folded antigen, such as non-denaturing ELISA, flow cytometry, and immunoprecipitation.
  • an antibody from which the pFc' region has been enzymatically cleaved, or which has been produced without the pFc' region designated an F(ab')2 fragment, retains both of the antigen binding sites of an intact antibody.
  • an antibody from which the Fc region has been enzymatically cleaved, or which has been produced without the Fc region designated an Fab fragment, retains one of the antigen binding sites of an intact antibody molecule.
  • Fab fragments consist of a covalently bound antibody light chain and a portion of the antibody heavy chain denoted Fd.
  • the Fd fragments are the major determinant of antibody specificity (a single Fd fragment may be associated with up to ten different light chains without altering antibody specificity) and Fd fragments retain epitope-binding ability in isolation.
  • CDRs complementarity determining regions
  • FRs framework regions
  • CDR1 through CDRS complementarity determining regions
  • compositions and methods that include humanized forms of antibodies.
  • humanized describes antibodies wherein some, most or all of the amino acids outside the CDR regions are replaced with corresponding amino acids derived from human immunoglobulin molecules.
  • Methods of humanization include, but are not limited to, those described in U.S. Pat. Nos. 4,816,567, 5,225,539, 5,585,089, 5,693,761, 5,693,762 and 5,859,205, which are hereby incorporated by reference.
  • the above U.S. Pat. Nos. 5,585,089 and 5,693,761, and WO 90/07861 also propose four possible criteria which may used in designing the humanized antibodies.
  • the first proposal was that for an acceptor, use a framework from a particular human immunoglobulin that is unusually homologous to the donor immunoglobulin to be humanized, or use a consensus framework from many human antibodies.
  • the second proposal was that if an amino acid in the framework of the human immunoglobulin is unusual and the donor amino acid at that position is typical for human sequences, then the donor amino acid rather than the acceptor may be selected.
  • the third proposal was that in the positions immediately adjacent to the 3 CDRs in the humanized immunoglobulin chain, the donor amino acid rather than the acceptor amino acid may be selected.
  • the fourth proposal was to use the donor amino acid reside at the framework positions at which the amino acid is predicted to have a side chain atom within 3 A of the CDRs in a three dimensional model of the antibody and is predicted to be capable of interacting with the CDRs.
  • the above methods are merely illustrative of some of the methods that one skilled in the art could employ to make humanized antibodies.
  • One of ordinary skill in the art will be familiar with other methods for antibody humanization.
  • humanized forms of the antibodies some, most or all of the amino acids outside the CDR regions have been replaced with amino acids from human immunoglobulin molecules but where some, most or all amino acids within one or more CDR regions are unchanged. Small additions, deletions, insertions, substitutions or modifications of amino acids are permissible as long as they would not abrogate the ability of the antibody to bind a given antigen.
  • Suitable human immunoglobulin molecules would include IgGl, IgG2, IgG3, IgG4, IgA and IgM molecules.
  • a "humanized" antibody retains a similar antigenic specificity as the original antibody.
  • the affinity and/or specificity of binding of the antibody may be increased using methods of "directed evolution", as described by Wu et al, /. Mol. Biol. 294: 151, 1999, the contents of which are incorporated herein by reference.
  • Fully human monoclonal antibodies also can be prepared by immunizing mice transgenic for large portions of human immunoglobulin heavy and light chain loci. See, e.g., U.S. Pat. Nos. 5,591,669, 5,598,369, 5,545,806, 5,545,807, 6,150,584, and references cited therein, the contents of which are incorporated herein by reference. These animals have been genetically modified such that there is a functional deletion in the production of endogenous (e.g., murine) antibodies. The animals are further modified to contain all or a portion of the human germ-line immunoglobulin gene locus such that immunization of these animals will result in the production of fully human antibodies to the antigen of interest.
  • monoclonal antibodies can be prepared according to standard hybridoma technology. These monoclonal antibodies will have human immunoglobulin amino acid sequences and therefore will not provoke human anti-mouse antibody (KAMA) responses when administered to humans.
  • KAMA human anti-mouse antibody
  • the present invention also provides for F(ab') 2 Fab, Fv and Fd fragments; chimeric antibodies in which the Fc and/or FR and/or CDR1 and/or CDR2 and/or light chain CDR3 regions have been replaced by homologous human or non-human sequences; chimeric F(ab')2 fragment antibodies in which the FR and/or CDR1 and/or CDR2 and/or light chain CDR3 regions have been replaced by homologous human or non-human sequences; chimeric Fab fragment antibodies in which the FR and/or CDR1 and/or CDR2 and/or light chain CDR3 regions have been replaced by homologous human or non-human sequences; and chimeric Fd fragment antibodies in which the FR and/or CDR1 and/or CDR2 regions have been replaced by homologous human or non-human sequences.
  • the present invention also includes so-called single chain antibodies.
  • the various antibody molecules and fragments may derive from any of the commonly known immunoglobulin classes, including but not limited to IgA, secretory IgA, IgE, IgG and IgM.
  • IgG subclasses are also well known to those in the art and include but are not limited to human IgGl, IgG2, IgG3 and IgG4.
  • the endothelin receptor antagonist of the invention is a Human IgG4.
  • the antibody according to the invention is a single domain antibody.
  • the term “single domain antibody” (sdAb) or “VHH” refers to the single heavy chain variable domain of antibodies of the type that can be found in Camelid mammals which are naturally devoid of light chains. Such VHH are also called “nanobody®”. According to the invention, sdAb can particularly be llama sdAb.
  • VHH refers to the single heavy chain having 3 complementarity determining regions (CDRs): CDR1, CDR2 and CDR3.
  • CDRs complementarity determining region
  • CDR complementarity determining region
  • VHH according to the invention can readily be prepared by an ordinarily skilled artisan using routine experimentation.
  • VHH variants and modified form thereof may be produced under any known technique in the art such as in- vitro maturation.
  • VHHs or sdAbs are usually generated by PCR cloning of the V-domain repertoire from blood, lymph node, or spleen cDNA obtained from immunized animals into a phage display vector, such as pHEN2.
  • Antigen- specific VHHs are commonly selected by panning phage libraries on immobilized antigen, e.g., antigen coated onto the plastic surface of a test tube, biotinylated antigens immobilized on streptavidin beads, or membrane proteins expressed on the surface of cells.
  • immobilized antigen e.g., antigen coated onto the plastic surface of a test tube, biotinylated antigens immobilized on streptavidin beads, or membrane proteins expressed on the surface of cells.
  • VHHs often show lower affinities for their antigen than VHHs derived from animals that have received several immunizations.
  • VHHs from immune libraries are attributed to the natural selection of variant VHHs during clonal expansion of B-cells in the lymphoid organs of immunized animals.
  • the affinity of VHHs from non-immune libraries can often be improved by mimicking this strategy in vitro, i.e., by site directed mutagenesis of the CDR regions and further rounds of panning on immobilized antigen under conditions of increased stringency (higher temperature, high or low salt concentration, high or low pH, and low antigen concentrations).
  • VHHs derived from camelid are readily expressed in and purified from the E. coli periplasm at much higher levels than the corresponding domains of conventional antibodies.
  • VHHs generally display high solubility and stability and can also be readily produced in yeast, plant, and mammalian cells.
  • the "Hamers patents” describe methods and techniques for generating VHH against any desired target (see for example US 5,800,988; US 5,874, 541 and US 6,015,695).
  • the "Hamers patents” more particularly describe production of VHHs in bacterial hosts such as E. coli (see for example US 6,765,087) and in lower eukaryotic hosts such as moulds (for example Aspergillus or Trichoderma) or in yeast (for example Saccharomyces, Kluyveromyces, Hansenula or Pichia) (see for example US 6,838,254).
  • the compound of the invention is an inhibitor of endothelin receptor expression or inhibitor of endothelin expression.
  • a gene product can be the direct transcriptional product of a gene (e.g., mRNA, tRNA, rRNA, antisense RNA, ribozyme, structural RNA or any other type of RNA) or a protein produced by translation of a mRNA.
  • Gene products also include messenger RNAs, which are modified, by processes such as capping, polyadenylation, methylation, and editing, and proteins (e.g., endothelin receptor ETA, endothelin receptor ETB and endothelin) modified by, for example, methylation, acetylation, phosphorylation, ubiquitination, SUMOylation, ADP-ribosylation, myristilation, and glycosylation.
  • proteins e.g., endothelin receptor ETA, endothelin receptor ETB and endothelin
  • an “inhibitor of expression” refers to a natural or synthetic compound that has a biological effect to inhibit the expression of a gene.
  • Inhibitors of expression for use in the present invention may be based on antisense oligonucleotide constructs.
  • Anti-sense oligonucleotides including anti-sense RNA molecules and anti-sense DNA molecules, would act to directly block the translation of mRNA by binding thereto and thus preventing protein translation or increasing mRNA degradation, thus decreasing the level of proteins, and thus activity, in a cell.
  • antisense oligonucleotides of at least about 15 bases and complementary to unique regions of the mRNA transcript sequence encoding endothelin receptor ETA, endothelin receptor ETB and endothelin can be synthesized, e.g., by conventional phosphodiester techniques and administered by e.g., intravenous injection or infusion.
  • Methods for using antisense techniques for specifically alleviating gene expression of genes whose sequence is known are well known in the art (e.g. see U.S. Pat. Nos. 6,566,135; 6,566,131; 6,365,354; 6,410,323; 6,107,091; 6,046,321; and 5,981,732).
  • Small inhibitory RNAs can also function as inhibitors of expression for use in the present invention.
  • Gene expression can be reduced by contacting the subject or cell with a small double stranded RNA (dsRNA), or a vector or construct causing the production of a small double stranded RNA, such that endothelin receptor ETA, endothelin receptor ETB and endothelin expression is specifically inhibited (i.e. RNA interference or RNAi).
  • dsRNA small double stranded RNA
  • RNAi RNA interference
  • Methods for selecting an appropriate dsRNA or dsRNA-encoding vector are well known in the art for genes whose sequence is known (e.g. see Tuschl, T. et al. (1999); Elbashir, S. M. et al.
  • Ribozymes can also function as inhibitors of expression for use in the present invention.
  • Ribozymes are enzymatic RNA molecules capable of catalysing the specific cleavage of RNA.
  • the mechanism of ribozyme action involves sequence specific hybridization of the ribozyme molecule to complementary target RNA, followed by endonucleo lytic cleavage.
  • Engineered hairpin or hammerhead motif ribozyme molecules that specifically and efficiently catalyze endonucleo lytic cleavage of mRNA sequences are thereby useful within the scope of the present invention.
  • ribozyme cleavage sites within any potential RNA target are initially identified by scanning the target molecule for ribozyme cleavage sites, which typically include the following sequences, GUA, GUU, and GUC. Once identified, short RNA sequences of between about 15 and 20 ribonucleotides corresponding to the region of the target gene containing the cleavage site can be evaluated for predicted structural features, such as secondary structure, that can render the oligonucleotide sequence unsuitable. The suitability of candidate targets can also be evaluated by testing their accessibility to hybridization with complementary oligonucleotides, using, e.g., ribonuclease protection assays.
  • antisense oligonucleotides and ribozymes useful as inhibitors of expression can be prepared by known methods. These include techniques for chemical synthesis such as, e.g., by solid phase phosphoramadite chemical synthesis. Alternatively, anti-sense RNA molecules can be generated by in vitro or in vivo transcription of DNA sequences encoding the RNA molecule. Such DNA sequences can be incorporated into a wide variety of vectors that incorporate suitable R A polymerase promoters such as the T7 or SP6 polymerase promoters. Various modifications to the oligonucleotides of the invention can be introduced as a means of increasing intracellular stability and half-life.
  • Possible modifications include but are not limited to the addition of flanking sequences of ribonucleotides or deoxyribonucleotides to the 5' and/or 3' ends of the molecule, or the use of phosphorothioate or 2'-0-methyl rather than phosphodiesterase linkages within the oligonucleotide backbone.
  • Antisense oligonucleotides siRNAs and ribozymes of the invention may be delivered in vivo alone or in association with a vector.
  • a "vector" is any vehicle capable of facilitating the transfer of the antisense oligonucleotide siRNA or ribozyme nucleic acid to the cells and preferably cells expressing endothelin receptor ETA, endothelin receptor ETB and endothelin.
  • the vector transports the nucleic acid to cells with reduced degradation relative to the extent of degradation that would result in the absence of the vector.
  • the vectors useful in the invention include, but are not limited to, plasmids, phagemids, viruses, other vehicles derived from viral or bacterial sources that have been manipulated by the insertion or incorporation of the antisense oligonucleotide siRNA or ribozyme nucleic acid sequences.
  • Viral vectors are a preferred type of vector and include, but are not limited to nucleic acid sequences from the following viruses: retrovirus, such as moloney murine leukemia virus, harvey murine sarcoma virus, murine mammary tumor virus, and rouse sarcoma virus; adenovirus, adeno-associated virus; Simian Virus (SV)40-type viruses; polyomaviruses; Herpes viruses; papilloma viruses; vaccinia virus; poliovirus; and RNA virus such as a retrovirus.
  • retrovirus such as moloney murine leukemia virus, harvey murine sarcoma virus, murine mammary tumor virus, and rouse sarcoma virus
  • adenovirus adeno-associated virus
  • Simian Virus (SV)40-type viruses Simian Virus (SV)40-type viruses
  • polyomaviruses Herpes viruses
  • Non-cytopathic viruses include retroviruses (e.g., lentivirus), the life cycle of which involves reverse transcription of genomic viral RNA into DNA with subsequent pro-viral integration into host cellular DNA. Retroviruses have been approved for human gene therapy trials. Most useful are those retroviruses that are replication-deficient (i.e., capable of directing synthesis of the desired proteins, but incapable of manufacturing an infectious particle). Such genetically altered retroviral expression vectors have general utility for the high-efficiency transduction of genes in vivo.
  • viruses for certain applications are the adenoviruses and Adeno-Associated Viruses (AAV), which are double-stranded DNA viruses that have already been approved for human use in gene therapy.
  • AAV can be engineered to be replication deficient and is capable of infecting a wide range of cell types and species. It further has advantages such as, heat and lipid solvent stability; high transduction frequencies in cells of diverse lineages, including hematopoietic cells; and lack of super-infection inhibition thus allowing multiple series of transductions.
  • AAV can integrate into human cellular DNA in a site-specific manner, thereby minimizing the possibility of insertional mutagenesis and variability of inserted gene expression characteristic of retroviral infection.
  • wild-type AAV infections have been followed in tissue culture for greater than 100 passages in the absence of selective pressure, implying that AAV genomic integration is a relatively stable event.
  • AAV can also function in an extra-chromosomal fashion.
  • Plasmid vectors have been extensively described in the art and are well known to those of skill in the art. See e.g., SANBROOK et al, "Molecular Cloning: A Laboratory Manual," Second Edition, Cold Spring Harbor Laboratory Press, 1989. In the last few years, plasmid vectors have been used as DNA vaccines for delivering antigen- encoding genes to cells in vivo. They are particularly advantageous for this because they do not have the same safety concerns as with many of the viral vectors. These plasmids, however, having a promoter compatible with the host cell, can express a peptide from a gene operatively encoded within the plasmid.
  • Plasmids may be delivered by a variety of parenteral, mucosal and topical routes.
  • the DNA plasmid can be injected either by intramuscular, intradermal, subcutaneous, or other routes. It may also be administered by intranasal sprays or drops, rectal suppository and orally. It may also be administered into the epidermis or a mucosal surface using a gene-gun. Plasmids may be given in an aqueous solution, dried onto gold particles or in association with another DNA delivery system including but not limited to liposomes, dendrimers, cochleate and microencapsulation.
  • the compounds according to the invention as described above are administered to the subject in a therapeutically effective amount.
  • a “therapeutically effective amount” of the compound of the present invention as above described is meant a sufficient amount of the compound for treating cardiovascular toxicity induced by anti-angiogenic compound at a reasonable benefit/risk ratio applicable to any medical treatment. It will be understood, however, that the total daily usage of the compounds and compositions of the present invention will be decided by the attending physician within the scope of sound medical judgment.
  • the specific therapeutically effective dose level for any particular subject will depend upon a variety of factors including the disorder being treated and the severity of the disorder; activity of the specific compound employed; the specific composition employed, the age, body weight, general health, sex and diet of the subject; the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidental with the specific compound employed; and like factors well known in the medical arts.
  • the daily dosage of the products may be varied over a wide range from 0.01 to 1,000 mg per adult per day.
  • the compositions contain 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 25.0, 50.0, 100, 250 and 500 mg of the compound of the present invention for the symptomatic adjustment of the dosage to the subject to be treated.
  • a medicament typically contains from about 0.01 mg to about 500 mg of the compound of the present invention, preferably from 1 mg to about 100 mg of the compound of the present invention.
  • An effective amount of the drug is ordinarily supplied at a dosage level from 0.0002 mg/kg to about 20 mg/kg of body weight per day, especially from about 0.001 mg/kg to 7 mg/kg of body weight per day.
  • the compound according to the invention may be used in a concentration between 0.01 ⁇ and 20 ⁇ , particularly, the compound of the invention may be used in a concentration of 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 20.0 ⁇ .
  • the present invention relates to the compound according to the invention in combination with one or more anti-cancer compound for use in the treatment of cardiovascular toxicity induced by anti-cancer compound in a subject in need thereof.
  • the present invention relates to the compound according to the invention in combination with one or more anti-angiogenic compound for use in the treatment of cardiovascular toxicity induced by anti-angiogenic compound in a subject in need thereof.
  • the compound of the invention is administered sequentially or concomitantly with one or more anti-cancer compound and anti-angiogenic compound.
  • the compound of the present invention is administered to the subject in the form of a pharmaceutical composition.
  • the compound of the present invention may be combined with pharmaceutically acceptable excipients, and optionally sustained-release matrices, such as biodegradable polymers, to form therapeutic compositions.
  • pharmaceutically acceptable excipients or “pharmaceutically acceptable” refer to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to a mammal, especially a human, as appropriate.
  • a pharmaceutically acceptable carrier or excipient refers to a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type.
  • the active principle in the pharmaceutical compositions of the present invention for oral, sublingual, subcutaneous, intramuscular, intravenous, transdermal, local or rectal administration, can be administered in a unit administration form, as a mixture with conventional pharmaceutical supports, to animals and human beings.
  • Suitable unit administration forms comprise oral-route forms such as tablets, gel capsules, powders, granules and oral suspensions or solutions, sublingual and buccal administration forms, aerosols, implants, subcutaneous, transdermal, topical, intraperitoneal, intramuscular, intravenous, subdermal, transdermal, intrathecal and intranasal administration forms and rectal administration forms.
  • the pharmaceutical compositions contain vehicles which are pharmaceutically acceptable for a formulation capable of being injected.
  • vehicles which are pharmaceutically acceptable for a formulation capable of being injected.
  • These may be in particular isotonic, sterile, saline solutions (monosodium or disodium phosphate, sodium, potassium, calcium or magnesium chloride and the like or mixtures of such salts), or dry, especially freeze-dried compositions which upon addition, depending on the case, of sterilized water or physiological saline, permit the constitution of injectable solutions.
  • the pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions; formulations including sesame oil, peanut oil or aqueous propylene glycol; and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions.
  • the form In all cases, the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi.
  • Solutions comprising compounds of the invention as free base or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.
  • the compound of the present invention can be formulated into a composition in a neutral or salt form.
  • Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like.
  • inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like.
  • Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine,
  • the carrier can also be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetables oils.
  • the proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • the prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like.
  • isotonic agents for example, sugars or sodium chloride.
  • Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminium monostearate and gelatin.
  • Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with several of the other ingredients enumerated above, as required, followed by filtered sterilization.
  • dispersions are prepared by incorporating the various sterilized agent of the present inventions into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above.
  • sterile powders for the preparation of sterile injectable solutions the typical methods of preparation are vacuum-drying and freeze- drying techniques which yield a powder of the compound of the present invention plus any additional desired ingredient from a previously sterile- filtered solution thereof.
  • the preparation of more, or highly concentrated solutions for direct injection is also contemplated, where the use of DMSO as solvent is envisioned to result in extremely rapid penetration, delivering high concentrations of the active agents to a small tumor area.
  • solutions Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective.
  • the formulations are easily administered in a variety of dosage forms, such as the type of injectable solutions described above, but drug release capsules and the like can also be employed.
  • aqueous solutions For parenteral administration in an aqueous solution, for example, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose.
  • aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration.
  • sterile aqueous media which can be employed will be known to those of skill in the art in light of the present disclosure. Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject.
  • the compound of the present invention is administered sequentially or concomitantly with one or more therapeutic active agent such as chemotherapeutic or radiotherapeutic.
  • the compound of the present invention is administered with a chemotherapeutic agent.
  • chemotherapeutic agent refers to chemical compounds that are effective in inhibiting tumor growth.
  • examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, trietylenephosphoramide, triethylenethiophosphaorarnide and trimethylolomelamine; acetogenins (especially bullatacin and bullatacinone); a carnptothecin (including the synthetic analogue topotecan); bryostatin; callystatin; CC-1065 (including its adozeles).
  • calicheamicin especially calicheamicin (11 and calicheamicin 211, see, e.g., Agnew Chem Intl. Ed. Engl. 33: 183-186 (1994); dynemicin, including dynemicin A; an esperamicin; as well as neocarzinostatin chromophore and related chromoprotein enediyne antiobiotic chromomophores), aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, carabicin, canninomycin, carzinophilin, chromomycins, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (including morpholino- doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrol
  • paclitaxel (TAXOL®, Bristol-Myers Squibb Oncology, Princeton, N.].) and doxetaxel (TAXOTERE®, Rhone-Poulenc Rorer, Antony, France); chlorambucil; gemcitabine; 6- thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin and carboplatin; vinblastine; platinum; etoposide (VP- 16); ifosfamide; mitomycin C; mitoxantrone; vincristine; vinorelbine; navelbine; novantrone; teniposide; daunomycin; aminopterin; xeloda; ibandronate; CPT-1 1 ; topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoic acid; capecitabine; and pharmaceutically acceptable salts, acids or derivatives of any of the above.
  • anti-estrogens including for example tamoxifen, raloxifene, aromatase inhibiting 4(5)-imidazoles, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, and toremifene (Fareston); and anti-androgens such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; and phannaceutically acceptable salts, acids or derivatives of any of the above.
  • the compound of the present invention is administered with a targeted cancer therapy.
  • Targeted cancer therapies are drugs or other substances that block the growth and spread of cancer by interfering with specific molecules ("molecular targets") that are involved in the growth, progression, and spread of cancer.
  • Targeted cancer therapies are sometimes called “molecularly targeted drugs", “molecularly targeted therapies”, “precision medicines”, or similar names.
  • the targeted therapy consists of administering the subject with a tyrosine kinase inhibitor as defined above.
  • compound of the present invention is administered with an immunotherapeutic agent.
  • immunotherapeutic agent refers to a compound, composition or treatment that indirectly or directly enhances, stimulates or increases the body's immune response against cancer cells and/or that decreases the side effects of other anticancer therapies. Immunotherapy is thus a therapy that directly or indirectly stimulates or enhances the immune system's responses to cancer cells and/or lessens the side effects that may have been caused by other anti-cancer agents. Immunotherapy is also referred to in the art as immunologic therapy, biological therapy biological response modifier therapy and biotherapy.
  • immunotherapeutic agents examples include, but are not limited to, cytokines, cancer vaccines, monoclonal antibodies and non-cytokine adjuvants.
  • the immunotherapeutic treatment may consist of administering the subject with an amount of immune cells (T cells, NK, cells, dendritic cells, B cells).
  • Immunotherapeutic agents can be non-specific, i.e. boost the immune system generally so that the human body becomes more effective in fighting the growth and/or spread of cancer cells, or they can be specific, i.e. targeted to the cancer cells themselves immunotherapy regimens may combine the use of non-specific and specific immunotherapeutic agents.
  • Non-specific immunotherapeutic agents are substances that stimulate or indirectly improve the immune system.
  • Non-specific immunotherapeutic agents have been used alone as a main therapy for the treatment of cancer, as well as in addition to a main therapy, in which case the non-specific immunotherapeutic agent functions as an adjuvant to enhance the effectiveness of other therapies (e.g. cancer vaccines).
  • Non-specific immunotherapeutic agents can also function in this latter context to reduce the side effects of other therapies, for example, bone marrow suppression induced by certain chemotherapeutic agents.
  • Non-specific immunotherapeutic agents can act on key immune system cells and cause secondary responses, such as increased production of cytokines and immunoglobulins. Alternatively, the agents can themselves comprise cytokines.
  • Nonspecific immunotherapeutic agents are generally classified as cytokines or non-cytokine adjuvants.
  • cytokines have found application in the treatment of cancer either as general non-specific immunotherapies designed to boost the immune system, or as adjuvants provided with other therapies.
  • Suitable cytokines include, but are not limited to, interferons, interleukins and colony- stimulating factors.
  • Interferons (IFNs) contemplated by the present invention include the common types of IFNs, IFN-alpha (IFN-a), IFN-beta (IFN- ⁇ ) and IFN- gamma (IFN- ⁇ ).
  • IFNs can act directly on cancer cells, for example, by slowing their growth, promoting their development into cells with more normal behaviour and/or increasing their production of antigens thus making the cancer cells easier for the immune system to recognise and destroy.
  • IFNs can also act indirectly on cancer cells, for example, by slowing down angiogenesis, boosting the immune system and/or stimulating natural killer (NK) cells, T cells and macrophages.
  • Recombinant IFN-alpha is available commercially as Roferon (Roche Pharmaceuticals) and Intron A (Schering Corporation).
  • Interleukins contemplated by the present invention include IL-2, IL-4, IL-11 and IL-12. Examples of commercially available recombinant interleukins include Proleukin® (IL-2; Chiron Corporation) and Neumega® (IL- 12; Wyeth Pharmaceuticals). Zymogenetics, Inc.
  • Colony-stimulating factors contemplated by the present invention include granulocyte colony stimulating factor (G-CSF or filgrastim), granulocyte-macrophage colony stimulating factor (GM-CSF or sargramostim) and erythropoietin (epoetin alfa, darbepoietin). Treatment with one or more growth factors can help to stimulate the generation of new blood cells in subjects undergoing traditional chemotherapy.
  • CSFs can be helpful in decreasing the side effects associated with chemotherapy and can allow for higher doses of chemo therapeutic agents to be used.
  • Various-recombinant colony stimulating factors are available commercially, for example, Neupogen® (G-CSF; Amgen), Neulasta (pelfilgrastim; Amgen), Leukine (GM-CSF; Berlex), Procrit (erythropoietin; Ortho Biotech), Epogen (erythropoietin; Amgen), Arnesp (erytropoietin).
  • immunotherapeutic agents can be active, i.e. stimulate the body's own immune response, or they can be passive, i.e.
  • Passive specific immunotherapy typically involves the use of one or more monoclonal antibodies that are specific for a particular antigen found on the surface of a cancer cell or that are specific for a particular cell growth factor.
  • Monoclonal antibodies may be used in the treatment of cancer in a number of ways, for example, to enhance a subject's immune response to a specific type of cancer, to interfere with the growth of cancer cells by targeting specific cell growth factors, such as those involved in angiogenesis, or by enhancing the delivery of other anticancer agents to cancer cells when linked or conjugated to agents such as chemotherapeutic agents, radioactive particles or toxins.
  • Monoclonal antibodies currently used as cancer immunotherapeutic agents that are suitable for inclusion in the combinations of the present invention include, but are not limited to, rituximab (Rituxan®), trastuzumab (Herceptin®), ibritumomab tiuxetan (Zevalin®), tositumomab (Bexxar®), cetuximab (C-225, Erbitux®), bevacizumab (Avastin®), gemtuzumab ozogamicin (Mylotarg®), alemtuzumab (Campath®), and BL22.
  • Other examples include anti-CTLA4 antibodies (e.g.
  • antibodies include B cell depleting antibodies.
  • Typical B cell depleting antibodies include but are not limited to anti-CD20 monoclonal antibodies [e.g.
  • the immunotherapeutic treatment may consist of allografting, in particular, allograft with hematopoietic stem cell HSC.
  • the immunotherapeutic treatment may also consist in an adoptive immunotherapy as described by Nicholas P. Restifo, Mark E.
  • NK cells circulating lymphocytes
  • the activated lymphocytes or NK cells are most preferably be the subject's own cells that were earlier isolated from a blood or tumor sample and activated (or "expanded") in vitro.
  • the compound of the present invention is administered with a radio therapeutic agent.
  • radiotherapeutic agent as used herein, is intended to refer to any radiotherapeutic agent known to one of skill in the art to be effective to treat or ameliorate cancer, without limitation.
  • the radiotherapeutic agent can be an agent such as those administered in brachytherapy or radionuclide therapy.
  • Such methods can optionally further comprise the administration of one or more additional cancer therapies, such as, but not limited to, chemotherapies, and/or another radiotherapy.
  • said additional active compounds may be contained in the same composition or administrated separately.
  • the pharmaceutical composition of the invention relates to combined preparation for simultaneous, separate or sequential use in the treatment of cardiovascular toxicity induced by anti-cancer compound and anti-angiogenic compound in a subject in need thereof.
  • the present invention also relates to a method for treating cardiovascular toxicity induced by anti-cancer compound in a subject in need thereof, comprising the step of administering to said subject the compound of the invention.
  • the present invention also relates to a method for treating cardiovascular toxicity induced by anti-angiogenic compound in a subject in need thereof, comprising the step of administering to said subject the compound of the invention.
  • kits comprising the compound of the invention. Kits containing the compound of the invention find use in therapeutic methods.
  • the present invention relates to a method of screening a candidate compound for use as a drug for the treatment of cardiovascular toxicity induced by anti-cancer compound and anti-angiogenic compound in a subject in need thereof, wherein the method comprises the steps of:
  • a candidate compound such as small organic molecule, an oligonucleotide, a polypeptide, an aptamer, antibody or an intra-antibody, measuring the cardiovascular toxicity,
  • cardiovascular toxicity is measured such as described in the example.
  • measuring the cardiovascular toxicity involves determining a Ki on the endothelin receptor cloned and transfected in a stable manner into a CHO cell line, measuring the endothelin receptor downstream signalling phospho lipase C, protein kinase C and MAPK1;ERK2, measuring aerobic and anaerobic metabolism, measuring myocardial metabolism, measuring glucose uptake, measuring cardiac fibrosis, measuring diastolic dysfunction and myocardial flux dysfunction and performing nuclear imaging of the heart using Sicintigraphy, Single photon emiccion tomograpgy (SPECT) or PET with various radiopharmaceuticals and with or without kinetics analysis of dynamic scans of the heart for monitoring cardiac metabolic remodeling.
  • SPECT Single photon emiccion tomograpgy
  • the present invention relates to a method of monitoring cardiovascular toxicity induced by an anti-cancer compound and an anti-angiogenic compound by performing PET-FDG scan such as described in the example.
  • the present invention relates to a method of monitoring the efficacy of the compound of the invention in the treatment of cardiovascular toxicity induced by an anticancer compound and anti-angiogenic compound by performing PET-FDG scan such as described in the example.
  • FIGURES are a diagrammatic representation of FIGURES.
  • FIG. 1 represents study design: (A) represents investigation for short-term cardiotoxic effects on immunodeficient tumor-bearing mice (nude). Sunitinib-treated mice were studied at baseline and week 1 using a cancer PET protocol compared to vehicle. (B) represents investigation for short-term cardiotoxic effects on immunocompetent mice (C57BL/6). Sunitinib-treated mice were studied at baseline and week 1 using a cancer PET protocol and echocardiography compared to vehicle. (C) represents study design for long-term treatment on immunocompetent mice (C57BL/6). Sunitinib-treated mice were followed at baseline, week 1, week 2 and week 3 using a cardiac PET protocol and echocardiography compared to vehicle and sunitinib+macitentan groups.
  • FDG 2'-deoxy-2'-[18F]fluoro-D-glucose.
  • FIG. 3 Sunitinib-induced increased FDG uptake in fasted mice is associated with increased fibrosis:
  • A Quantification of microvascular density normalized by cell number in nude and C57B1/6 mice treated with vehicle (open circles) or sunitinib (black circles).
  • B Quantification of fibrosis (normalized by tissue area) in nude and C57B1/6 mice treated with vehicle (open circles) or sunitinib (filled circles).
  • C Representative quantification for GLUT1, HK2 and PGCl (normalized by cyclophilin B) mice treated with vehicle (open circles) or sunitinib (filled circles). Data expressed as mean ⁇ SEM; $$p ⁇ 0.01 $$$p ⁇ 0.001 compared to vehicle.
  • HK2 Hexokinase 2
  • GLUT1 glucose transporter 1
  • PGCl Peroxisome proliferator- activated receptor gamma coactivator 1-alpha
  • S sunitinib
  • V vehicle.
  • FIG. 4 Macitentan prevents sunitinib-induced diastolic dysfunction.
  • C Difference treatment - baseline of left ventricular internal diameter at diastole for the three groups.
  • D Difference treatment - baseline of aortic velocity time integral for both groups.
  • AoVTI Aortic velocity tracking integral
  • CO cardiac output
  • LVID left ventricular internal diameter
  • SUV standard uptake value
  • MRGlu metabolic rate of glucose.
  • FIG. 7 Schematic representation of the mechanism of sunitinib-induced cardiac side effects:
  • Sunitinib upregulates glycolysis and downregulates oxidative metabolism in cardiac mitochondria.
  • Sunitinib induces resistance to insulin stimulation of cardiac glucose uptake.
  • the metabolic switch is an immediate early response to sunitinib while insulin resistance either appears later or is masked by the metabolic switch during the early stages of sunitinib treatment. Both mechanisms depend on signaling by the endothelin pathway, and lead to myocardial fibrosis and impaired cardiac function, and are reversed by the endothelin receptors antagonist macitentan. Red indicates upregulated proteins and pathways, blue indicates downregulated protein and pathways.
  • ATP Adenosine triphosphate
  • ET-1 endothelin 1
  • ETA endothelin receptor type A
  • FA fatty acid
  • GLUT glucose transporter protein
  • 02 oxygen
  • OXPHOS oxidative phosphorylation
  • TCA tricarboxylic acid.
  • mice from groups A and B were fasted overnight. Mice from panel C were not fasted and had free access to food and water. Mice were anesthetized (2 ⁇ 0.5% isoflurane in air), weighted and glycemia was measured in blood drawn from the caudal ventral artery using an Accu-Chek® Aviva Nano A (Accu-Chek, France). A catheter home-made from a 26G needle (Fischer Scientific, France) connected to a 5cm polyethylene tubing (Tygon Microbore Tubing, 0.010" x 0.030"OD; Fisher Scientific, France) was inserted in the caudal vein for radiotracer injection. Mice were then installed into the PET-CT dedicated bed and respiration and body temperature were registered.
  • CT was acquired in a PET- CT scanner (nanoScan PET-CT; Mediso, Hungary) using the following acquisition parameters: semi-circular mode, 39kV tension, 720 projections full scan, 300ms per projection, binning 1 :4. Then, PET acquisition was started and, 30 seconds later, lOMBq of 2'-deoxy-2'-[18F]fluoro-D- glucose (FDG; Advanced Applied Applications, France) in 0.2mL saline was injected via the catheter. The first scan was a dynamic acquisition of 30.5min and was followed by a gated cardiac scan of 30min duration.
  • FDG 2'-deoxy-2'-[18F]fluoro-D- glucose
  • PET data were collected in list mode and binned using a 5ns time window, with a 400-600keV energy window and a 1 :5 coincidence mode.
  • Data were reconstructed using the Tera-Tomo reconstruction engine (3D-OSEM based manufactured customized algorithm) with expectation maximization iterations, scatter and attenuation correction.
  • the first scan was reconstructed starting 10s before FDG injection with the following time sequence: 26 x 5s; 6 x 30s; 5 x 120s; 3 x 300s and 3 x 600s.
  • the second scan reconstructed in a single time frame from 45 to 60min post-injection.
  • FDG accumulation was quantified as mean Standard Uptake Value (SUV, ratio of the radioactivity concentration in myocardium on the whole body concentration of the injected radioactivity) between 45 and 60min post-injection in 3D volumes-of- interest (VOI) delineated semi-automatically by iso-contours at 45% threshold of maximal value in the myocardium on PET/CT fusion slices using the PMOD software package (PMOD Technologies Ltd, Zurich, Switzerland).
  • Metabolic flux was quantified using compartmental modeling tool of PMOD software using the same VOI as above and a VOI semi-automatically delineated on the vena cava for the arterial input function as previously described (65). Metabolic rate of glucose were calculated by the multiplication of the metabolic flux by [plasma glucose (mmol/l)/lumped constant (fixed at 0.69)].
  • LVID Left Ventricular Internal Diameter
  • LVPW Left Ventricular Posterior Wall thickness
  • CO Cardiac Output
  • FS fractional shortering
  • Ascending aorta diameter (Ao) and Left Atrium diameter (LA) were measured using M-mode in parasternal long axis.
  • Aortic velocity tracking integral (AoVTI) was measured using PW Doppler, in suprasternal view allowing measurement of mean aortic velocity and peak aortic velocity.
  • Cardiac microvessels were stained using Isolectine B4 Griffonia Simplicifolia-FITC (Sigma Aldrich). Nuclei were counter stained with DAPI. Microvessels and nuclei were counted in 4 fields at a magnification of x200 in 2 independent sections from each heart using Matlab® based software. Microvessel density was normalized to the number of nuclei.
  • Chemiluminescence detection was performed using the ECL kit (Clarity Western ECl substrate; BioRad). Quantitation of immunoblots was done on digitalized images using ImageJ software. The intensity of immunoreactive bands was normalized by the loading control (Cyclophilin B, 1 : 1000, abl6045, Abeam).
  • Tissue sample preparation Frozen mouse hearts were individually ground under liquid nitrogen to yield a fine powder using a pestle and mortar. The tissue powder was weighted and solubilized in lysis buffer (4% SDS, lOOmM Tris-HCl, pH 8.0). Protein extracts were clarified by centrifugation at 21,000 X G, 1 hour, 4°C. Protein concentration of the supernatant was determined using bicinchoninic acid assay (BCA, Pierce). Peptides were prepared by Filter Aided Separation method (FASP) essentially as described (66).
  • FASP Filter Aided Separation method
  • solubilization buffer 50 mM Tris/HCl, pH8.5, SDS 2%, 20mM TCEP, 50 mM chloroacetamide
  • extracts were diluted with 300 ⁇ Tris Urea buffer (Urea 8M, Tris/HCl 50mM (pH 8.5) and transferred onto 30kDa centrifuged filters and prepared for digestion as described (66). Proteins were digested during 14h at 37°C with ⁇ g trypsin (Promega) and peptides were desalted on C18 StageTips (67).
  • peptides were solubilized in 2% trifiuoroacetic acid (TFA) and fractionated by strong cationic exchange (SCX) StageTips, mainly as described (Kulak et al, 2014) except that fractions 1 and 2 were pooled in most experiments.
  • Mass spectrometry analysis Mass spectrometry analyses were performed on a Dionex U3000 RSLC nano-LC- system coupled to either a Q-Exactive or a LTQ Orbitrap- Velos mass spectrometer, all from Thermo Fisher Scientific. After drying, peptides from SCX StageTip fractions were solubilized in 10 ⁇ of 0.1% TFA containing 2% acetonitrile (ACN).
  • ACN acetonitrile
  • the mass spectrometer acquired data throughout the elution process and operated in a data-dependent scheme with full MS scans acquired with the Orbitrap, followed by up to 10 MS/MS HCD fragmentations in the Q-Exactive (Thermo Fisher) on the most abundant ions detected. Settings were essentially as in (68) with slight modifications: the recurrent loop of the 10 most intense nLC-eluting peptides were HCD- fragmented between each full scan (data dependent mode). Resolution was set to 70,000 for full scans at AGC target 1,10e6 within 60ms MIIT. The MS scans spanned from 350 to 1500m/z.
  • Precursor selection window was set at 2Th, and MS/MS scan resolution was set at 17,500 with AGC target 1,10e5 within 60ms MIIT. HCD Normalized Collision Energy (NCE) was set at 27%. Dynamic exclusion was set to 30s duration. Spectra were recorded in profile mode. The mass spectrometry data were analyzed using Maxquant version 1,5,2,8 (69). The database used was a concatenation of human sequences from the Uniprot-Swissprot database (Uniprot, release 2015-02) and a list of contaminant sequences from Maxquant. The enzyme specificity was trypsin. The precursor mass tolerance was set to 4.5ppm and the fragment mass tolerance to 20ppm for Q-Exactive data.
  • Protein copy numbers per cell were then calculated using the "Protein ruler” plugin of Perseus by standardization on total histone MS signal as described (70).
  • RNA extraction and qPCR R A from hearts and aorta was isolated using TRI Reagent Solution (Invitrogen). Any DNA present was degraded using RQ1 RNAse-Free DNase (Promega) according to the manufacturer's instructions.
  • cDNA was synthesized using a High Capacity cDNA Reverse Transcription kit with RNase inhibitor (Invitrogen).
  • qRT-PCR was performed using Fast SYBR Green Master Mix (Applied Biosystems) on the ABI7900 System (Applied Biosystems). Primers for 18s, prepro-ET-1, ETA and ETB receptors were generated. The amplification reaction mixture was heated at 95°C for 20s, then subject to 40 cycles of 95°C for Is then 60°C for 20s. Values obtained for experimental gene measurements were normalized against expression of 18s.
  • Sunitinib downregulates oxidative energy metabolism pathways
  • Ingenuity® analysis highlighted that sunitinib treatment induced mitochondrial dysfunction and a clear switch towards anaerobic glycolytic metabolism, similar to the one seen during cardiac hypertrophy (31).
  • proteins of the fatty acid degradation pathway such as acyl-CoA dehydrogenase (Acad8), phospholipases, and fatty acid transferases, those controlling glycogen breakdown (Pgaml, Me3) and the synthesis of the cofactor flavine adenine dinucleotide (FAD) were reduced in sunitinib-treated hearts.
  • Endothelin receptor antagonism prevents sunitinib-induced diastolic dysfunction and myocardial flux dysfunction
  • Macitentan reduces cardiac fibrosis and downregulates myocardial ETA receptors
  • Macitentan reverses the sunitinib-induced aerobic to anaerobic switch
  • Protein clusters involved in myocardial infarction, endothelial cell dysfunction and apoptosis, atherosclerosis, thrombosis, inflammation and hypertension were targeted by sunitinib. Comparing protein expression in the sunitinib-treated group with that in the vehicle and sunitinib plus macitentan groups highlighted once again the protective effects of macitentan, notably on the following clusters: myocardial infarction, endothelial cell dysfunction and apoptosis (data not shown), glycogen metabolism, TCA cycle, acetyl CoA biosynthesis.
  • the level of expression of the pyruvate dehydrogenase (PDH) components were maintained at control levels by macitentan in the oxidative phosphorylation deficient myocardium induced by sunitinib (data not shown).
  • the levels of expression of some proteins involved in glycolysis were augmented by 3 weeks of sunitinib treatment (e.g. glucose-6-phosphate isomerase and phosphoglycerate mutase), while those of others were reduced, e.g. muscle-type phosphofructokinase and glyceraldehyde-3 -phosphate dehydrogenase.
  • Sunitinib-treated hearts showed higher lactate dehydrogenase (LDH) that is released during tissue hypoxia and damage.
  • LDH lactate dehydrogenase
  • sunitinib induced a dramatic (8-fold) increase in the expression of GLUT4 (SLC2a4), the insulin-regulated transporter of glucose at the plasma membrane.
  • GLUT4 SLC2a4
  • RablO a small ras-family GTPase required for translocation of GLUT4
  • Macitentan only partially reverted the effects of sunitinib on the level of GLUT4 and RablO, but significantly increased the level of expression of the pleiotropic regulatory protein sirtuin 2, in line with the role of this protein in energetic metabolism preservation.
  • Sunitinib treatment is limited by its cardiovascular side effects.
  • a recent study reported that sunitinib induces an early switch of cardiac metabolism to anaerobic glycolysis and impairs heart function (30).
  • myocardial remodeling by sunitinib also induces a reduced glucose uptake resembling the one found during insulin resistance, and show that sunitinib cardiotoxicity is a combination of several complex mechanisms occurring over a sequential time course.
  • sunitinib-induced cardiac injury and dysfunction are prevented through inhibition of endothelin signaling, strongly supporting a role for this pathway in sunitinib 's cardio toxic effects (Figure 7).
  • FDG uptake is higher in non-fasting than in fasting conditions (37) and in oncology studies FDG PET imaging is acquired under fasting conditions in order to minimize muscular and myocardial uptake and improve tumor detection.
  • cardiac PET-FDG often utilizes an euglycemic clamp (glucose load with additional insulin administration after overnight fasting) in order to maximize heart uptake (37,38).
  • ischemic territories appear as hot spots with higher FDG uptake than the intact myocardium after fasting (39) while they may not differentiate from intact tissue in non-fasted conditions after a glucose load (40-42).
  • sunitinib Systemic administration of sunitinib rapidly induces a metabolic switch towards glycolysis with reduced expressions of key enzymes of the TCA cycle and key proteins for mitochondrial oxidative phosphorylation (OXPHOS) and for the beta-oxidation of fatty acids.
  • OXPHOS mitochondrial oxidative phosphorylation
  • OXPHOS mitochondrial oxidative phosphorylation
  • sunitinib-treated hearts presented anaerobic metabolism and it is known that PDH inhibition leads to a slow recovery of glucose uptake and uncoupling of glycolysis (49), and that lactate accumulation decreases glucose uptake (43).
  • patterns of protein expression in the 3-week sunitinib-treated heart resembled the one seen in diabetic patients in which the FDG metabolic flux is reduced (50), as well as the pattern in diabetic rats where myocardial glucose uptake is reduced under ischemic conditions (51).
  • ischitinib-treated hearts we observed a dramatic increase of GLUT4 activated in high glycaemia-high insulinemia conditions (36).
  • Macitentan is a mixed ETA/ETB antagonist approved for the treatment of pulmonary arterial hypertension and has no known direct or indirect interaction with sunitinib of other TKI (FDA). Furthermore, non-selective ET receptors antagonism prevents hypertension and renal injury (24) induced by sunitinib. In the future, it will be interesting to test if the use of a selective ETA receptor antagonism produces comparable cardioprotection since ET-1 is known to exert a pro-fibrotic action (62) and hypertrophy (34) via the ETA receptor.
  • TCA cycle Isobutyryl-CoA dehydrogenase, mitochondrial Acad8 ⁇ -1.30
  • TCA cycle Isocitrate dehydrogenase [NAD] subunit Idh3g 0.03 -1.24 gamma 1, mitochondrial
  • Mitochondria Mitochondrial import receptor subunit TOM70 Tomm70 0.06 -1.27 a
  • AMPK 5' AMP-activated protein kinase
  • cdp cytidine diphospho
  • ERR estrogen-related receptor
  • NAD nicotinamide adenine dinucleotide
  • NADP nicotinamide adenine dinucleotide phosphate
  • PGC-1 peroxisome proliferator- activated receptor gamma coactivator 1 -alpha
  • TCA tricarboxylic acid. sunitinib sunitinib sunitinib + versus vehicle versus macitentan sunitinib + versus macitentan vehicle
  • Glucogenolysis RAB10, member RAS Ra 0,071 -1,30 0,405 1,07 0,030
  • Glucogenolysis Phosphoglycerate mutase 1 Pga 0,011 1,48 0,141 1,18 0,057 1,26 ml
  • TCA cycle Isobutyryl-CoA Aca 0,026 -1,21 0,002 -1,20 0,932
  • Mitochondria 3-hydroxyisobutyryl-CoA Hib 0,403 -1,06 0,018 -1,18 0,020 1,12 FA hydrolase, mitochondrial ch Mitochondria 3-hydroxyisobutyrate Hib 0,738 1,04 0,036 -1,26 0,005 1,30 dehydrogenase, adh
  • Mitochondria Mitochondrial-processing Pm 0,049 -1,17 0,011 -1,36 0,142 1,16 peptidase subunit beta pcb
  • Mitochondria Mitochondrial import To 0,299 1,16 0,006 -1,46 0,001 1,70 receptor subunit TOM22 mm
  • Mitochondria Mitochondrial Rho Rh 0,008 3,11 0,862 1,01 0,010 3,08
  • Mitochondria Mitochondrial import Ti 0,474 1,08 0,418 -1,08 0,010 1,17 membrane translocase mm
  • Mitochondria Mitochondrial import To 0,024 1,23 0,923 1,01 0,022 1,22 receptor subunit TOM40 mm
  • Mitochondria Mitochondrial Mte 0,045 -2,39 0,005 -2,27 0,821
  • Mitochondria A-kinase anchor protein 1, Ak 0,185 -1,19 0,057 -1,31 0,471 1,10 mitochondrial apl
  • Atherosclerosis Apolipoprotein A-IV Ap 0,013 1,62 0,212 1,24 0,106 1,31 oa4
  • ATP Adenosine triphosphate
  • CD36 cluster of differentiation 36
  • FA fatty acid
  • GTP Guanosine- 5 '-triphosphate
  • IR insulin resitance
  • K+ potassium
  • Na+ sodium
  • NAD nicotinamide adenine dinucleotide
  • RAB Ras-related protein
  • RNA Ribonucleic acid
  • TCA tricarboxylic acid.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Health & Medical Sciences (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Epidemiology (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Cardiology (AREA)
  • Heart & Thoracic Surgery (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

The invention relates to methods for treating cardiovascular toxicity induced by anti-cancer and anti-angiogenic compound. The inventors explored the cardiotoxicity induced by the antiangiogenic therapy, sunitinib, in the mouse heart. The inventors showed that sunitinib induces an anaerobic switch of cellular metabolism within the myocardium which is associated with the development of myocardial fibrosis as demonstrated by echocardiography. The capacity of positron emission tomography to detect the changes in cardiac metabolism caused by sunitinib was dependent on fasting status and duration of treatment. Pan proteomic analysis in the myocardium showed that sunitinib induced (i) an early metabolic switch with enhanced glycolysis and reduced oxidative phosphorylation, and (ii) a metabolic failure to use glucose as energy substrate, similar to the insulin resistance found in type 2 diabetes. Co-administration of macitentan, the endothelin receptor antagonist, to sunitinib-treated animals prevented both metabolic defects, restored glucose uptake and cardiac function, and prevented myocardial fibrosis. Thus, the invention relates to a compound selected from the group consisting of endothelin receptor antagonist and inhibitor of endothelin receptor expression for use in the treatment of cardiovascular toxicity induced by anti-cancer and anti-angiogenic compound.

Description

METHODS AND PHARMACEUTICAL COMPOSITIONS FOR TREATING CARDIOVASCULAR TOXICITY INDUCED BY ANTI-CANCER THERAPY
FIELD OF THE INVENTION:
The present invention relates to methods and pharmaceutical compositions for treating cardiovascular toxicity induced by an anti-cancer and an anti-angiogenic compound.
BACKGROUND OF THE INVENTION:
The major factor limiting therapeutic administration of anticancer drugs is their toxic side effect on off-target organs. It is well known that classical anticancer drugs, e.g. anthracyclines, antimetabolites, alkylating agents, taxanes, induce serious cardiovascular toxicity (1,2). Newer anticancer agents such as interferon and tyrosine kinase receptor (TKR) inhibitors also have cardiovascular side-effects (3) that, although often less severe than those observed with anthracyclines, are frequent and may be life-threatening (4). The clinical importance of cardiotoxicity associated with cancer therapy has led to the emergence of cardio- oncology, an interdisciplinary field that aims to better understand and limit the cardiotoxicity of cancer therapy (5).
Anti-angiogenics targeting the Vascular Endothelial Growth Factor Receptors (VEGFRs) pathway are considered as an essential asset for cancer treatment given the importance of the neovascularization process in tumor development (6,7). However, they are associated with a spectrum of cardiovascular side effects (8). The major treatment- limiting side effects of antiangiogenic TKR inhibitors include nausea, fatigue, hypertension, myocardial infarction, left ventricular cardiac and renal dysfunction, QT prolongation, neutropenia, proteinuria, thyroid impairment, thrombosis and hemorrhage (9-12).
Sunitinib (Sutent; Pfizer, USA) is an anti-angiogenic TKR inhibitor of VEGFRs, platelet-derived growth factor receptors (PDGF-Rs), and c-kit (13), approved in 2006 by the FDA for the treatment of renal cell carcinoma (14), imatinib-resistant gastrointestinal stromal tumor (15) and neuroendocrine tumors (16). In 2007, a study reported hypertension in -50% of sunitinib-treated patients (8), decreased left ventricular ejection fraction (LVEF) in -30% of patients, and congestive heart failure in -10% of imatinib-resistant patients treated with sunitinib (8). Regarding sunitinib-induced hypertension, Lankhorst et al. listed as plausible mechanisms: deregulation of the nitric oxide signaling pathway, microvascular rarefaction, activation of endothelin (ET) system, salt sensitivity and oxidative stress (17). However, there are contradictory reports on the potential of sunitinib to up- or downregulate the nitric oxide pathway (17) and to cause microvascular rarefaction (18). Inhibition of myocardial VEGFRs reduces response to stress, activates the pro-apoptotic pathway and maintains a high level of HIF-Ι that is known to induce cardiomyopathy (20). Inhibition of PDGFRs by sunitinib induces a loss of coronary microvascular pericytes, suggesting a possible direct effect of sunitinib on myocardial blood supply (19). In rats, sunitinib leads to mitochondrial dysfunction; in mice, to increased apoptosis of cardiomyocytes (8). Ex vivo, a poor coronary flow response to bradykinin was reported in sunitinib-treated hearts, supporting microvascular dysfunction as a direct cardiac side effect of the drug (21). Finally, the fact that sunitinib inhibits AMPK (22) and induces mitochondrial damage (8) opens up the possibility that some or all of its side effects could result from a direct deregulation of cardiac metabolism.
An activated ET system likely contributes to vasoconstriction, hypertension, renal injury and proteinuria (23). Previous studies have shown that ET receptor blockers can reduce blood pressure (BP) and renal injury in animals and patients treated with sunitinib (21,23-25). However, the potential benefits of ET receptor antagonism in treating or preventing the cardiotoxic effects of sunitinib have not been studied.
Clinical imaging of the heart is a method of choice to explore the cardiac side effects of anticancer therapy (26). Echocardiography and magnetic resonance imaging (MRI) show reduced left ventricular ejection fraction (LVEF) in advanced stages of cardio toxicity (27). In fact, most imaging studies have focused on the late stages of cardiotoxicity, either because imaging was prescribed after the clinical signs became evident, or because imaging failed to detect earlier signs. Nevertheless, a few studies have shown the capacity of nuclear imaging techniques to assess early cardiotoxicity associated with anthracyclines and trastuzumab (28). Borde et al. described a higher uptake of 2'-deoxy-2'-[18F]fluoro-D-glucose (FDG) in the myocardium of patients treated with adriamycin, highlighting the capacity of positron emission tomography (PET) to detect a deregulation of myocardial metabolism induced by a cancer treatment (29). Recently, O'Farrell et al described an early increase of the metabolic rate of glucose in sunitinib-treated rodents (30). This is particularly interesting from a clinical perspective as PET can simultaneously stage cancer and explore cardiac metabolism.
In the present invention, the inventors explored cardiac metabolism after sunitinib treatment in mice using PET-FDG. The inventors aimed to (i) better clarify the cardiac metabolic pathways deregulated during the early stages of sunitinib treatment, (ii) determine if the cardiac side effects are mediated by the endothelin pathway (iii) test the blockade of the endothelin system to prevent the cardiac side effects of sunitinib, and (iv) confirm that PET- FDG can be useful to monitor cardiac metabolic remodeling. SUMMARY OF THE INVENTION:
The present invention relates to methods and pharmaceutical compositions for treating cardiovascular toxicity induced by an anti-cancer and an anti-angiogenic compound.
DETAILED DESCRIPTION OF THE INVENTION:
The growing field of cardio-oncology addresses the side effects of cancer treatment on the cardiovascular system. Here, the inventors explored the cardiotoxicity induced by the antiangiogenic therapy, sunitinib, in the mouse heart from a diagnostic and therapeutic perspective. The inventors showed that sunitinib induces an anaerobic switch of cellular metabolism within the myocardium which is associated with the development of myocardial fibrosis as demonstrated by echocardiography. The capacity of positron emission tomography with [18F]fluorodeoxyglucose to detect the changes in cardiac metabolism caused by sunitinib was dependent on fasting status and duration of treatment. Pan proteomic analysis in the myocardium showed that sunitinib induced (i) an early metabolic switch with enhanced glycolysis and reduced oxidative phosphorylation, and (ii) a metabolic failure to use glucose as energy substrate, similar to the insulin resistance found in type 2 diabetes. Co-administration of macitentan, the endothelin receptor antagonist, to sunitinib-treated animals prevented both metabolic defects, restored glucose uptake and cardiac function, and prevented myocardial fibrosis. These results support the endothelin system in mediating the cardiotoxic effects of sunitinib and endothelin receptor antagonism as a potential therapeutic approach to prevent cardiotoxicity. Furthermore, metabolic and functional imaging can monitor the cardiotoxic effects and the benefits of endothelin antagonism in a theranostics approach.
Accordingly, the present invention relates to a compound selected from the group consisting of endothelin receptor antagonist and inhibitor of endothelin receptor expression for use in the treatment of cardiovascular toxicity induced by anti-cancer compound in a subject in need thereof.
In a further aspect, the present invention relates to a compound selected from the group consisting of endothelin receptor antagonist and inhibitor of endothelin receptor expression for use in the treatment of cardiovascular toxicity induced by anti-angiogenic compound in a subject in need thereof.
As used herein, the term "subject" denotes a mammal. Typically, a subject according to the invention refers to any subject (preferably human) receiving anti-cancer therapy. Typically, a subject according to the invention refers to any subject (preferably human) receiving anti- angiogenic therapy. In a particular embodiment, the term "subject" refers to a subject afflicted or at risk to be afflicted with cardiovascular toxicity induced by anti-cancer therapy or anti- angiogenic therapy. In a particular embodiment, the term "subject" refers to a subject afflicted with cancer. In a particular embodiment, the term "subject" refers to a subject afflicted with angiogenesis-related diseases. In a particular embodiment, the term "subject" refers to a subject afflicted with cancer or angiogenesis-related diseases receiving anti-cancer therapy or anti- angiogenic therapy. In a particular embodiment, the term "subject" refers to a subject afflicted with cancer or angiogenesis-related diseases, and afflicted or at risk to be afflicted with cardiovascular toxicity induced by anti-cancer therapy or anti-angiogenic therapy.
As used herein, the term "treatment" or "treat" refer to both prophylactic or preventive treatment as well as curative or disease modifying treatment, including treatment of subjects at risk of contracting the disease or suspected to have contracted the disease as well as subjects who are ill or have been diagnosed as suffering from a disease or medical condition, and includes suppression of clinical relapse. The treatment may be administered to a subject having a medical disorder or who ultimately may acquire the disorder, in order to prevent, cure, delay the onset of, reduce the severity of, or ameliorate one or more symptoms of a disorder or recurring disorder, or in order to prolong the survival of a subject beyond that expected in the absence of such treatment. By "therapeutic regimen" is meant the pattern of treatment of an illness, e.g., the pattern of dosing used during therapy. A therapeutic regimen may include an induction regimen and a maintenance regimen. The phrase "induction regimen" or "induction period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the initial treatment of a disease. The general goal of an induction regimen is to provide a high level of drug to a subject during the initial period of a treatment regimen. An induction regimen may employ (in part or in whole) a "loading regimen", which may include administering a greater dose of the drug than a physician would employ during a maintenance regimen, administering a drug more frequently than a physician would administer the drug during a maintenance regimen, or both. The phrase "maintenance regimen" or "maintenance period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the maintenance of a subject during treatment of an illness, e.g., to keep the subject in remission for long periods of time (months or years). A maintenance regimen may employ continuous therapy (e.g., administering a drug at a regular intervals, e.g., weekly, monthly, yearly, etc.) or intermittent therapy (e.g., interrupted treatment, intermittent treatment, treatment at relapse, or treatment upon achievement of a particular predetermined criteria [e.g., disease manifestation, etc.]).
The term "cardiovascular toxicity" has its general meaning in the art and refers to cardiotoxicity induced by anti-cancer therapy. The term "cardiovascular toxicity" also refers to cardiotoxicity induced by anti-angiogenic therapy. The term "cardiovascular toxicity" also refers to cardiovascular side-effects of anti-cancer therapy and anti-angiogenic therapy. The term "cardiovascular toxicity" also refers to metabolic defects, increase myocardial fibrosis, reduction of myocardial glucose uptake, cardiac dysfunction, cardiac ischemia and switch toward anaerobic metabolism. The term "cardiovascular toxicity" also refers to anaerobic switch of cellular metabolism within the myocardium which is associated with the development of myocardial fibrosis, early metabolic switch with enhanced glycolysis and reduced oxidative phosphorylation, and a metabolic failure to use glucose as energy substrate, similar to the insulin resistance found in type 2 diabetes.
In some embodiment, the compound of the invention is used in the treatment of vascular toxicity induced by anti-cancer therapy and anti-angiogenic therapy. The term "vascular toxicity" refers to microvascular dysfunction and damage in the heart, kidney, retina, brain and other target organs such as renal, retinal and cerebrovascular circulation dysfunction induced by anti-cancer therapy and anti-angiogenic therapy.
As used herein, the term "cancer" has its general meaning in the art and includes, but is not limited to, solid tumors and blood borne tumors. The term cancer includes diseases of the skin, tissues, organs, bone, cartilage, blood and vessels. The term "cancer" further encompasses both primary and metastatic cancers. Examples of cancers include, but are not limited to, cancer cells from the adrenal, bladder, blood, bone, bone marrow, brain, breast, colon, esophagus, gastrointestine, gum, head, kidney, liver, lung, nasopharynx, neck, ovary, prostate, skin, stomach, sympathic and parasympathic ganglia, testis, tongue, or uterus. In addition, the cancer may specifically be of the following histological type, though it is not limited to these: neoplasm, malignant; carcinoma; carcinoma, undifferentiated; giant and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; pilomatrix carcinoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; gastrinoma, malignant; cholangiocarcinoma; hepatocellular carcinoma; combined hepatocellular carcinoma and cholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma in adenomatous polyp; adenocarcinoma, familial polyposis coli; solid carcinoma; carcinoid tumor, malignant; branchiolo-alveolar adenocarcinoma; papillary adenocarcinoma; chromophobe carcinoma; acidophil carcinoma; oxyphilic adenocarcinoma; basophil carcinoma; clear cell adenocarcinoma; granular cell carcinoma; follicular adenocarcinoma; papillary and follicular adenocarcinoma; nonencapsulating sclerosing carcinoma; adrenal cortical carcinoma; endometroid carcinoma; skin appendage carcinoma; apocrine adenocarcinoma; sebaceous adenocarcinoma; ceruminous; adenocarcinoma; mucoepidermoid carcinoma; cystadenocarcinoma; papillary cystadenocarcinoma; papillary serous cystadenocarcinoma; mucinous cystadenocarcinoma; mucinous adenocarcinoma; signet ring cell carcinoma; infiltrating duct carcinoma; medullary carcinoma; lobular carcinoma; inflammatory carcinoma; paget's disease, mammary; acinar cell carcinoma; adenosquamous carcinoma; adenocarcinoma w/squamous metaplasia; thymoma, malignant; ovarian stromal tumor, malignant; thecoma, malignant; granulosa cell tumor, malignant; and roblastoma, malignant; Sertoli cell carcinoma; leydig cell tumor, malignant; lipid cell tumor, malignant; paraganglioma, malignant; extra- mammary paraganglioma, malignant; pheochromocytoma; glomangio sarcoma; malignant melanoma; amelanotic melanoma; superficial spreading melanoma; malign melanoma in giant pigmented nevus; epithelioid cell melanoma; blue nevus, malignant; sarcoma; fibrosarcoma; fibrous histiocytoma, malignant; myxosarcoma; liposarcoma; leiomyosarcoma; rhabdomyosarcoma; embryonal rhabdomyosarcoma; alveolar rhabdomyosarcoma; stromal sarcoma; mixed tumor, malignant; mullerian mixed tumor; nephroblastoma; hepatoblastoma; carcinosarcoma; mesenchymoma, malignant; brenner tumor, malignant; phyllodes tumor, malignant; synovial sarcoma; mesothelioma, malignant; dysgerminoma; embryonal carcinoma; teratoma, malignant; struma ovarii, malignant; choriocarcinoma; mesonephroma, malignant; hemangio sarcoma; hemangioendothelioma, malignant; kaposi's sarcoma; hemangiopericytoma, malignant; lymphangiosarcoma; osteosarcoma; juxtacortical osteosarcoma; chondrosarcoma; chondroblastoma, malignant; mesenchymal chondrosarcoma; giant cell tumor of bone; ewing's sarcoma; odontogenic tumor, malignant; ameloblastic odontosarcoma; ameloblastoma, malignant; ameloblastic fibrosarcoma; pinealoma, malignant; chordoma; glioma, malignant; ependymoma; astrocytoma; protoplasmic astrocytoma; fibrillary astrocytoma; astroblastoma; glioblastoma; oligodendroglioma; oligodendroblastoma; primitive neuroectodermal; cerebellar sarcoma; ganglioneuroblastoma; neuroendocrine carcinoma; neuroblastoma; retinoblastoma; olfactory neurogenic tumor; meningioma, malignant; neurofibrosarcoma; neurilemmoma, malignant; granular cell tumor, malignant; malignant lymphoma; Hodgkin's disease; Hodgkin's lymphoma; paragranuloma; malignant lymphoma, small lymphocytic; malignant lymphoma, large cell, diffuse; malignant lymphoma, follicular; mycosis fungoides; other specified non-Hodgkin's lymphomas; malignant histiocytosis; multiple myeloma; mast cell sarcoma; immunoproliferative small intestinal disease; leukemia; lymphoid leukemia; plasma cell leukemia; erythro leukemia; lymphosarcoma cell leukemia; myeloid leukemia; basophilic leukemia; eosinophilic leukemia; monocytic leukemia; mast cell leukemia; megakaryoblastic leukemia; myeloid sarcoma; and hairy cell leukemia. In some embodiments, the subject suffers from a cancer selected from the group consisting of breast cancer, triple negative breast cancer, colon cancer, lung cancer, prostate cancer, testicular cancer, brain cancer, skin cancer, rectal cancer, gastric cancer, esophageal cancer, sarcomas, adrenal cancer, sympathic and parasympathic ganglia cancer, tracheal cancer, head and neck cancer, pancreatic cancer, liver cancer, ovarian cancer, lymphoid cancer, cervical cancer, vulvar cancer, melanoma, mesothelioma, renal cancer, bladder cancer, thyroid cancer, bone cancers, carcinomas, sarcomas, and soft tissue cancers.
As used herein, the term "angiogenesis-related diseases" has its general meaning in the art and refers to diseases associated with or supported by pathological angiogenesis (i.e., inappropriate, excessive or undesired formation of blood vessels), which may be induced by various angiogenic factors. The term "angiogenesis-related diseases" also relates to angiogenic diseases associated with abnormal neovascularisation. Angiogenesis-related diseases include but are not limited to cancer, tumor angiogenesis, primary and metastatic solid tumors, including carcinomas of breast, colon, rectum, lung, oropharynx, hypopharynx, esophagus, stomach, pancreas, liver, gallbladder and bile ducts, small intestine, kidney, bladder, urothelium, female genital tract, (including cervix, uterus, and ovaries as well as choriocarcinoma and gestational trophoblastic disease), male genital tract (including prostate, seminal vesicles, testes and germ cell tumors), endocrine glands (including the thyroid, adrenal, and pituitary glands), and skin, as well as hemangiomas, melanomas, sarcomas (including those arising from bone and soft tissues as well as Kaposi's sarcoma) and tumors of the brain, nerves, eyes, such as astrocytomas, gliomas, glioblastomas, retinoblastomas, neuromas, neuroblastomas, Schwannomas, and meningiomas. Angiogenesis-related diseases also relate to tumors arising from hematopoietic malignancies such as leukemias as well both Hodgkin's and non-Hodgkin's lymphomas. Angiogenesis-related diseases also relate to various ocular diseases such as diabetic retinopathy, retinopathy of prematurity, corneal graft rejection, retrolental fibroplasia, neovascular glaucoma, rubeosis, retinal neovascularization due to macular degeneration, hypoxia, angiogenesis in the eye associated with infection or surgical intervention, and other abnormal neovascularization conditions of the eye. Angiogenesis- related diseases also relate to rheumatoid, immune and degenerative arthritis. Angiogenesis- related diseases also relate to skin diseases such as psoriasis; blood vessel diseases such as hemagiomas, and capillary proliferation within atherosclerotic plaques; Osier-Webber Syndrome; myocardial angiogenesis; plaque neovascularization; telangiectasia; haemophiliac joints; angiofibroma; and wound granulation. Angiogenesis-related diseases also relate to diseases characterized by excessive or abnormal stimulation of endothelial cells, including but not limited to intestinal adhesions, Crohn's disease, atherosclerosis, scleroderma, and hypertrophic scars, i.e. keloids., diseases that have angiogenesis as a pathologic consequence such as cat scratch disease (Rochele ninalia quintosa) and ulcers (Helicobacter pylori).
The term "anti-cancer compound" has its general meaning in the art and refers to compounds used in anti-cancer therapy such as anti-angiogenic compound, tyrosine kinase inhibitors, tyrosine kinase receptor (TKR) inhibitors, Vascular Endothelial Growth Factor Receptors (VEGFRs) pathway inhibitors, interferon therapy, anti-HER2 compounds, anti- EGFR compounds, alkylating agents, anti-metabolites, immunotherapeutic agents, Interferons (IFNs), Interleukins, and chemotherapeutic agents such as described below.
The term "anti-angiogenic compound" has its general meaning in the art and refers to compounds used in anti-angiogenic therapy such as tyrosine kinase inhibitors, anti-angiogenic tyrosine kinase receptor (TKR) inhibitors, anti-angiogenics targeting the Vascular Endothelial Growth Factor Receptors (VEGFRs) pathway, interferon therapy and anti-HER2 compounds such as Trastuzumab (herceptin) and pertuzumab. In one embodiment, the term "anti- angiogenic compound" refers to Sunitinib (Sutent), an anti-angiogenic TKR inhibitor of VEGFRs, platelet-derived growth factor receptors (PDGF-Rs), and c-kit.
The term "tyrosine kinase inhibitor" refers to any of a variety of therapeutic agents or drugs that act as selective or non-selective inhibitors of receptor and/or non-receptor tyrosine kinases. Tyrosine kinase inhibitors and related compounds are well known in the art and described in U.S Patent Publication 2007/0254295, which is incorporated by reference herein in its entirety. It will be appreciated by one of skill in the art that a compound related to a tyrosine kinase inhibitor will recapitulate the effect of the tyrosine kinase inhibitor, e.g., the related compound will act on a different member of the tyrosine kinase signaling pathway to produce the same effect as would a tyrosine kinase inhibitor of that tyrosine kinase. Examples of tyrosine kinase inhibitors and related compounds suitable for use in methods of embodiments of the present invention include, but are not limited to, sunitinib (Sutent; SU11248), dasatinib (BMS-354825), PP2, BEZ235, saracatinib, gefitinib (Iressa), erlotinib (Tarceva; OSI-1774), lapatinib (GW572016; GW2016), canertinib (CI 1033), semaxinib (SU5416), vatalanib (PTK787/ZK222584), sorafenib (BAY 43-9006), imatinib (Gleevec; STI571), leflunomide (SU101), vandetanib (Zactima; ZD6474), MK-2206 (8-[4-aminocyclobutyl)phenyl]-9-phenyl- l,2,4-triazolo[3,4-f][l,6]naphthyridin-3(2H)-one hydrochloride) derivatives thereof, analogs thereof, and combinations thereof. Additional tyrosine kinase inhibitors and related compounds suitable for use in the present invention are described in, for example, U.S Patent Publication 2007/0254295, U.S. Pat. Nos. 5,618,829, 5,639,757, 5,728,868, 5,804,396, 6,100,254, 6,127,374, 6,245,759, 6,306,874, 6,313,138, 6,316,444, 6,329,380, 6,344,459, 6,420,382, 6,479,512, 6,498,165, 6,544,988, 6,562,818, 6,586,423, 6,586,424, 6,740,665, 6,794,393, 6,875,767, 6,927,293, and 6,958,340, all of which are incorporated by reference herein in their entirety. In certain embodiments, the tyrosine kinase inhibitor is a small molecule kinase inhibitor that has been orally administered and that has been the subject of at least one Phase I clinical trial, more preferably at least one Phase II clinical, even more preferably at least one Phase III clinical trial, and most preferably approved by the FDA for at least one hematological or oncological indication. Examples of such inhibitors include, but are not limited to, Gefitinib, Erlotinib, Lapatinib, Canertinib, BMS-599626 (AC-480), Neratinib, KR -633, CEP-11981, Imatinib, Nilotinib, Dasatinib, AZM-475271, CP-724714, TAK-165, Sunitinib, Vatalanib, CP- 547632, Vandetanib, Bosutinib, Lestaurtinib, Tandutinib, Midostaurin, Enzastaurin, AEE-788, Pazopanib, Axitinib, Motasenib, OSI-930, Cediranib, KR -951, Dovitinib, Seliciclib, SNS- 032, PD-0332991, MKC-I (Ro-317453; R-440), Sorafenib, ABT-869, Brivanib (BMS- 582664), SU-14813, Telatinib, SU-6668, (TSU-68), L-21649, MLN-8054, AEW-541, and PD- 0325901.
In one embodiment, the term "anti-angiogenic compound" refers to compounds targeting the vascular endothelial growth factor (VEGF) pathway such anti-VEGF antibody bevacizumab (Avastin) and VEGF receptor tyrosine kinase inhibitor (TKI) compounds such as sunitinib (Sutent), vandetanib (Zactima), pazopanib (Votrient), sorafenib (Nexavar) and cediranib.
The term "endothelin" has its general meaning in the art and refers to the vaso constricting peptide endothelin- 1 (ET-1) produced primarily in the endothelium and acting on its both receptors ETA and ETB . The endothelin (ET- 1 ) can be from any source, but typically is a mammalian (e.g., human and non-human primate) endothelin, particularly a human endothelin. An exemplary native endothelin- 1 amino acid sequence is provided in UniProt database under accession number P05305 and an exemplary native nucleotide sequence encoding for endothelin- 1 is provided in GenBank database under accession number NM_001955.
The term "endothelin receptor" has its general meaning in the art and refers to G-protein- coupled receptors ETA and ETB.
The term "ETA" has its general meaning in the art and refers to the endothelin receptor type A. ETA can be from any source, but typically is a mammalian (e.g., human and non-human primate) ETA, particularly a human ETA. An exemplary native ETA amino acid sequence is provided in UniProt database under accession number P25101 and an exemplary native nucleotide sequence encoding for ETA is provided in GenBank database under accession number NM_001957.
The term "ETB" has its general meaning in the art and refers to the endothelin receptor type B. ETB can be from any source, but typically is a mammalian (e.g., human and non-human primate) ETB, particularly a human ETB. An exemplary native ETB amino acid sequence is provided in UniProt database under accession number P24530 and an exemplary native nucleotide sequence encoding for ETB is provided in GenBank database under accession number NM 000115.
The term "endothelin receptor antagonist" refers to a compound that selectively blocks or inactivates endothelin receptor. As used herein, the term "selectively blocks or inactivates" refers to a compound that preferentially binds to and blocks or inactivates endothelin receptor with a greater affinity and potency, respectively, than its interaction with the other sub-types or isoforms of G protein-coupled receptors family. Compounds that prefer endothelin receptor, but that may also block or inactivate other G protein-coupled receptors sub-types, as partial or full antagonists, are contemplated. The term "endothelin receptor antagonist" refers to any compound that can directly or indirectly block the signal transduction cascade related to the endothelin receptor. The term "endothelin receptor antagonist" should be understood broadly and encompasses compounds acting directly (by binding) on endothelin, ETA or ETB proteins and able to prevent the interaction or binding between endothelin and its receptor(s). The term "endothelin receptor antagonist" also refers to dual ETA and ETB receptor antagonist (ETA/ETB antagonist), selective ETA receptor antagonist or selective ETB receptor antagonist. The "endothelin receptor antagonist" may also consist in compounds that inhibit the binding of the endothelin to endothelin receptor. The term "endothelin receptor antagonist" also refers to inhibition of endothelin downstream signalling, phospholipase C, protein kinase C and MAPK1;ERK2. The term "endothelin receptor antagonist" also refers to endothelin inhibitor such as compound able to prevent the action of endothelin (ET-1) on its receptors ETA and ETB, inhibitor of endothelin formation and inhibitor of endothelin expression. Typically, an endothelin receptor antagonist is a small organic molecule, an oligonucleotide, a polypeptide, an aptamer, an antibody or an intra-antibody.
Tests and assays for determining whether a compound is an endothelin receptor antagonist are well known by the skilled person in the art such as described in Aubert and Juillerat-Jeanneret, 2017; Okada M. et al, 2002; US5,292,740 and US5,883,254.
The endothelin receptor antagonists are well-known in the art as illustrated by Aubert and Juillerat-Jeanneret, 2017; Okada M. et al, 2002; US5,292,740 and US5,883,254. In one embodiment of the invention, the endothelin receptor antagonist is selected from the group consisting of macitentan, bosentan, darusentan, sitaxsentan, tezosentan, ambrisentan, atrasentan, avosentan, clazosentan, zibotentan, edonentan, enrasentan, danusentan, A- 182086, A- 192621, ABT-627, BMS193884, BQ-123, BQ-788, CI 1020, FR-139317, S-0139, CPU0213, J- 104132, SB-209670, TA-0201, TAK-044, TBC3711, YM-598, ZD-1611, ZD-4054 and compounds described in Aubert and Juillerat-Jeanneret, 2017; Okada M. et al, 2002; US5,292,740 and US5,883,254.
In one embodiment of the invention, the endothelin receptor antagonist is an ETA/ETB antagonist such as macitentan, bosentan (US5,292,740 and US5, 883,254), A-182086, CPU0213, J-104132 and SB209670.
In one embodiment of the invention, the endothelin receptor antagonist is a selective ETB receptor antagonist such as BQ-788 (Okada M. et al, 2002) and A- 192621. A selective ETB receptor antagonist exhibits an affinity (as expressed by dissociation constant Ki) for ETB less than about ΙΟηΜ and a selectivity for ETB over ETA (as expressed by the ratio KiETB/KiETA) is at least about 50. In a particular embodiment, KiETB is less than 5 nM, more particularly less than 2 nM and even more particularly less than 1 Nm. In another particular embodiment, the ratio KiETB/KiETA is at least about 100, more particularly about 500 and even more particularly about 1000.
In one embodiment of the invention, the endothelin receptor antagonist is an endothelin inhibitor such as a molecule that decreases or prevents endothelin formation.
Such molecules are particularly Endothelin-Converting Enzyme inhibitor (ECE inhibitor), such as FR901533 (also called WS79089B, Tsurumi Y, et al, 1994 and Tsurumi Y, et al, 1995) or CGS 26303 (De Lombaert S et al, 1994).
In another embodiment, the use of an inhibitor of Endothelin-Converting Enzyme (ECE) gene expression can also be envisaged.
In another embodiment, the endothelin receptor antagonist of the invention is an aptamer. Aptamers are a class of molecule that represents an alternative to antibodies in term of molecular recognition. Aptamers are oligonucleotide sequences with the capacity to recognize virtually any class of target molecules with high affinity and specificity. Such ligands may be isolated through Systematic Evolution of Ligands by Exponential enrichment (SELEX) of a random sequence library, as described in Tuerk C. and Gold L., 1990. The random sequence library is obtainable by combinatorial chemical synthesis of DNA. In this library, each member is a linear oligomer, eventually chemically modified, of a unique sequence. Possible modifications, uses and advantages of this class of molecules have been reviewed in Jayasena S.D., 1999. Peptide aptamers consists of a conformationally constrained antibody variable region displayed by a platform protein, such as E. coli Thioredoxin A that are selected from combinatorial libraries by two hybrid methods (Colas et al, 1996). Then after raising aptamers directed against endothelin receptor of the invention as above described, the skilled man in the art can easily select those inhibiting endothelin receptor.
In another embodiment, the endothelin receptor antagonist of the invention is an antibody (the term including "antibody portion") directed against endothelin receptors ETA and/or ETB or endothelin.
In one embodiment of the antibodies or portions thereof described herein, the antibody is a monoclonal antibody. In one embodiment of the antibodies or portions thereof described herein, the antibody is a polyclonal antibody. In one embodiment of the antibodies or portions thereof described herein, the antibody is a humanized antibody. In one embodiment of the antibodies or portions thereof described herein, the antibody is a chimeric antibody. In one embodiment of the antibodies or portions thereof described herein, the portion of the antibody comprises a light chain of the antibody. In one embodiment of the antibodies or portions thereof described herein, the portion of the antibody comprises a heavy chain of the antibody. In one embodiment of the antibodies or portions thereof described herein, the portion of the antibody comprises a Fab portion of the antibody. In one embodiment of the antibodies or portions thereof described herein, the portion of the antibody comprises a F(ab')2 portion of the antibody. In one embodiment of the antibodies or portions thereof described herein, the portion of the antibody comprises a Fc portion of the antibody. In one embodiment of the antibodies or portions thereof described herein, the portion of the antibody comprises a Fv portion of the antibody. In one embodiment of the antibodies or portions thereof described herein, the portion of the antibody comprises a variable domain of the antibody. In one embodiment of the antibodies or portions thereof described herein, the portion of the antibody comprises one or more CDR domains of the antibody.
As used herein, "antibody" includes both naturally occurring and non-naturally occurring antibodies. Specifically, "antibody" includes polyclonal and monoclonal antibodies, and monovalent and divalent fragments thereof. Furthermore, "antibody" includes chimeric antibodies, wholly synthetic antibodies, single chain antibodies, and fragments thereof. The antibody may be a human or nonhuman antibody. A nonhuman antibody may be humanized by recombinant methods to reduce its immunogenicity in man.
Antibodies are prepared according to conventional methodology. Monoclonal antibodies may be generated using the method of Kohler and Milstein (Nature, 256:495, 1975). To prepare monoclonal antibodies useful in the invention, a mouse or other appropriate host animal is immunized at suitable intervals (e.g., twice-weekly, weekly, twice-monthly or monthly) with antigenic forms of endothelin receptors ETA and/or ETB or endothelin. The animal may be administered a final "boost" of antigen within one week of sacrifice. It is often desirable to use an immunologic adjuvant during immunization. Suitable immunologic adjuvants include Freund's complete adjuvant, Freund's incomplete adjuvant, alum, Ribi adjuvant, Hunter's Titermax, saponin adjuvants such as QS21 or Quil A, or CpG-containing immunostimulatory oligonucleotides. Other suitable adjuvants are well-known in the field. The animals may be immunized by subcutaneous, intraperitoneal, intramuscular, intravenous, intranasal or other routes. A given animal may be immunized with multiple forms of the antigen by multiple routes.
Briefly, the antigen may be provided as synthetic peptides corresponding to antigenic regions of interest in endothelin receptors ETA and/or ETB or endothelin. Following the immunization regimen, lymphocytes are isolated from the spleen, lymph node or other organ of the animal and fused with a suitable myeloma cell line using an agent such as polyethylene glycol to form a hydridoma. Following fusion, cells are placed in media permissive for growth of hybridomas but not the fusion partners using standard methods, as described (Coding, Monoclonal Antibodies: Principles and Practice: Production and Application of Monoclonal Antibodies in Cell Biology, Biochemistry and Immunology, 3rd edition, Academic Press, New York, 1996). Following culture of the hybridomas, cell supernatants are analyzed for the presence of antibodies of the desired specificity, i.e., that selectively bind the antigen. Suitable analytical techniques include ELISA, flow cytometry, immunoprecipitation, and western blotting. Other screening techniques are well-known in the field. Preferred techniques are those that confirm binding of antibodies to conformationally intact, natively folded antigen, such as non-denaturing ELISA, flow cytometry, and immunoprecipitation.
Significantly, as is well-known in the art, only a small portion of an antibody molecule, the paratope, is involved in the binding of the antibody to its epitope (see, in general, Clark, W. R. (1986) The Experimental Foundations of Modern Immunology Wiley & Sons, Inc., New York; Roitt, I. (1991) Essential Immunology, 7th Ed., Blackwell Scientific Publications, Oxford). The Fc' and Fc regions, for example, are effectors of the complement cascade but are not involved in antigen binding. An antibody from which the pFc' region has been enzymatically cleaved, or which has been produced without the pFc' region, designated an F(ab')2 fragment, retains both of the antigen binding sites of an intact antibody. Similarly, an antibody from which the Fc region has been enzymatically cleaved, or which has been produced without the Fc region, designated an Fab fragment, retains one of the antigen binding sites of an intact antibody molecule. Proceeding further, Fab fragments consist of a covalently bound antibody light chain and a portion of the antibody heavy chain denoted Fd. The Fd fragments are the major determinant of antibody specificity (a single Fd fragment may be associated with up to ten different light chains without altering antibody specificity) and Fd fragments retain epitope-binding ability in isolation.
Within the antigen-binding portion of an antibody, as is well-known in the art, there are complementarity determining regions (CDRs), which directly interact with the epitope of the antigen, and framework regions (FRs), which maintain the tertiary structure of the paratope (see, in general, Clark, 1986; Roitt, 1991). In both the heavy chain Fd fragment and the light chain of IgG immunoglobulins, there are four framework regions (FR1 through FR4) separated respectively by three complementarity determining regions (CDR1 through CDRS). The CDRs, and in particular the CDRS regions, and more particularly the heavy chain CDRS, are largely responsible for antibody specificity.
It is now well-established in the art that the non CDR regions of a mammalian antibody may be replaced with similar regions of conspecific or heterospecific antibodies while retaining the epitopic specificity of the original antibody. This is most clearly manifested in the development and use of "humanized" antibodies in which non-human CDRs are covalently joined to human FR and/or Fc/pFc' regions to produce a functional antibody.
This invention provides in certain embodiments compositions and methods that include humanized forms of antibodies. As used herein, "humanized" describes antibodies wherein some, most or all of the amino acids outside the CDR regions are replaced with corresponding amino acids derived from human immunoglobulin molecules. Methods of humanization include, but are not limited to, those described in U.S. Pat. Nos. 4,816,567, 5,225,539, 5,585,089, 5,693,761, 5,693,762 and 5,859,205, which are hereby incorporated by reference. The above U.S. Pat. Nos. 5,585,089 and 5,693,761, and WO 90/07861 also propose four possible criteria which may used in designing the humanized antibodies. The first proposal was that for an acceptor, use a framework from a particular human immunoglobulin that is unusually homologous to the donor immunoglobulin to be humanized, or use a consensus framework from many human antibodies. The second proposal was that if an amino acid in the framework of the human immunoglobulin is unusual and the donor amino acid at that position is typical for human sequences, then the donor amino acid rather than the acceptor may be selected. The third proposal was that in the positions immediately adjacent to the 3 CDRs in the humanized immunoglobulin chain, the donor amino acid rather than the acceptor amino acid may be selected. The fourth proposal was to use the donor amino acid reside at the framework positions at which the amino acid is predicted to have a side chain atom within 3 A of the CDRs in a three dimensional model of the antibody and is predicted to be capable of interacting with the CDRs. The above methods are merely illustrative of some of the methods that one skilled in the art could employ to make humanized antibodies. One of ordinary skill in the art will be familiar with other methods for antibody humanization.
In one embodiment of the humanized forms of the antibodies, some, most or all of the amino acids outside the CDR regions have been replaced with amino acids from human immunoglobulin molecules but where some, most or all amino acids within one or more CDR regions are unchanged. Small additions, deletions, insertions, substitutions or modifications of amino acids are permissible as long as they would not abrogate the ability of the antibody to bind a given antigen. Suitable human immunoglobulin molecules would include IgGl, IgG2, IgG3, IgG4, IgA and IgM molecules. A "humanized" antibody retains a similar antigenic specificity as the original antibody. However, using certain methods of humanization, the affinity and/or specificity of binding of the antibody may be increased using methods of "directed evolution", as described by Wu et al, /. Mol. Biol. 294: 151, 1999, the contents of which are incorporated herein by reference.
Fully human monoclonal antibodies also can be prepared by immunizing mice transgenic for large portions of human immunoglobulin heavy and light chain loci. See, e.g., U.S. Pat. Nos. 5,591,669, 5,598,369, 5,545,806, 5,545,807, 6,150,584, and references cited therein, the contents of which are incorporated herein by reference. These animals have been genetically modified such that there is a functional deletion in the production of endogenous (e.g., murine) antibodies. The animals are further modified to contain all or a portion of the human germ-line immunoglobulin gene locus such that immunization of these animals will result in the production of fully human antibodies to the antigen of interest. Following immunization of these mice (e.g., XenoMouse (Abgenix), HuMAb mice (Medarex/GenPharm)), monoclonal antibodies can be prepared according to standard hybridoma technology. These monoclonal antibodies will have human immunoglobulin amino acid sequences and therefore will not provoke human anti-mouse antibody (KAMA) responses when administered to humans.
In vitro methods also exist for producing human antibodies. These include phage display technology (U.S. Pat. Nos. 5,565,332 and 5,573,905) and in vitro stimulation of human B cells (U.S. Pat. Nos. 5,229,275 and 5,567,610). The contents of these patents are incorporated herein by reference. Thus, as will be apparent to one of ordinary skill in the art, the present invention also provides for F(ab') 2 Fab, Fv and Fd fragments; chimeric antibodies in which the Fc and/or FR and/or CDR1 and/or CDR2 and/or light chain CDR3 regions have been replaced by homologous human or non-human sequences; chimeric F(ab')2 fragment antibodies in which the FR and/or CDR1 and/or CDR2 and/or light chain CDR3 regions have been replaced by homologous human or non-human sequences; chimeric Fab fragment antibodies in which the FR and/or CDR1 and/or CDR2 and/or light chain CDR3 regions have been replaced by homologous human or non-human sequences; and chimeric Fd fragment antibodies in which the FR and/or CDR1 and/or CDR2 regions have been replaced by homologous human or non- human sequences. The present invention also includes so-called single chain antibodies.
The various antibody molecules and fragments may derive from any of the commonly known immunoglobulin classes, including but not limited to IgA, secretory IgA, IgE, IgG and IgM. IgG subclasses are also well known to those in the art and include but are not limited to human IgGl, IgG2, IgG3 and IgG4. In a preferred embodiment, the endothelin receptor antagonist of the invention is a Human IgG4.
In another embodiment, the antibody according to the invention is a single domain antibody. The term "single domain antibody" (sdAb) or "VHH" refers to the single heavy chain variable domain of antibodies of the type that can be found in Camelid mammals which are naturally devoid of light chains. Such VHH are also called "nanobody®". According to the invention, sdAb can particularly be llama sdAb. The term "VHH" refers to the single heavy chain having 3 complementarity determining regions (CDRs): CDR1, CDR2 and CDR3. The term "complementarity determining region" or "CDR" refers to the hypervariable amino acid sequences which define the binding affinity and specificity of the VHH.
The VHH according to the invention can readily be prepared by an ordinarily skilled artisan using routine experimentation. The VHH variants and modified form thereof may be produced under any known technique in the art such as in- vitro maturation.
VHHs or sdAbs are usually generated by PCR cloning of the V-domain repertoire from blood, lymph node, or spleen cDNA obtained from immunized animals into a phage display vector, such as pHEN2. Antigen- specific VHHs are commonly selected by panning phage libraries on immobilized antigen, e.g., antigen coated onto the plastic surface of a test tube, biotinylated antigens immobilized on streptavidin beads, or membrane proteins expressed on the surface of cells. However, such VHHs often show lower affinities for their antigen than VHHs derived from animals that have received several immunizations. The high affinity of VHHs from immune libraries is attributed to the natural selection of variant VHHs during clonal expansion of B-cells in the lymphoid organs of immunized animals. The affinity of VHHs from non-immune libraries can often be improved by mimicking this strategy in vitro, i.e., by site directed mutagenesis of the CDR regions and further rounds of panning on immobilized antigen under conditions of increased stringency (higher temperature, high or low salt concentration, high or low pH, and low antigen concentrations). VHHs derived from camelid are readily expressed in and purified from the E. coli periplasm at much higher levels than the corresponding domains of conventional antibodies. VHHs generally display high solubility and stability and can also be readily produced in yeast, plant, and mammalian cells. For example, the "Hamers patents" describe methods and techniques for generating VHH against any desired target (see for example US 5,800,988; US 5,874, 541 and US 6,015,695). The "Hamers patents" more particularly describe production of VHHs in bacterial hosts such as E. coli (see for example US 6,765,087) and in lower eukaryotic hosts such as moulds (for example Aspergillus or Trichoderma) or in yeast (for example Saccharomyces, Kluyveromyces, Hansenula or Pichia) (see for example US 6,838,254).
In one embodiment, the compound of the invention is an inhibitor of endothelin receptor expression or inhibitor of endothelin expression.
The term "expression" when used in the context of expression of a gene or nucleic acid refers to the conversion of the information, contained in a gene, into a gene product. A gene product can be the direct transcriptional product of a gene (e.g., mRNA, tRNA, rRNA, antisense RNA, ribozyme, structural RNA or any other type of RNA) or a protein produced by translation of a mRNA. Gene products also include messenger RNAs, which are modified, by processes such as capping, polyadenylation, methylation, and editing, and proteins (e.g., endothelin receptor ETA, endothelin receptor ETB and endothelin) modified by, for example, methylation, acetylation, phosphorylation, ubiquitination, SUMOylation, ADP-ribosylation, myristilation, and glycosylation.
An "inhibitor of expression" refers to a natural or synthetic compound that has a biological effect to inhibit the expression of a gene.
Inhibitors of expression for use in the present invention may be based on antisense oligonucleotide constructs. Anti-sense oligonucleotides, including anti-sense RNA molecules and anti-sense DNA molecules, would act to directly block the translation of mRNA by binding thereto and thus preventing protein translation or increasing mRNA degradation, thus decreasing the level of proteins, and thus activity, in a cell. For example, antisense oligonucleotides of at least about 15 bases and complementary to unique regions of the mRNA transcript sequence encoding endothelin receptor ETA, endothelin receptor ETB and endothelin can be synthesized, e.g., by conventional phosphodiester techniques and administered by e.g., intravenous injection or infusion. Methods for using antisense techniques for specifically alleviating gene expression of genes whose sequence is known are well known in the art (e.g. see U.S. Pat. Nos. 6,566,135; 6,566,131; 6,365,354; 6,410,323; 6,107,091; 6,046,321; and 5,981,732).
Small inhibitory RNAs (siRNAs) can also function as inhibitors of expression for use in the present invention. Gene expression can be reduced by contacting the subject or cell with a small double stranded RNA (dsRNA), or a vector or construct causing the production of a small double stranded RNA, such that endothelin receptor ETA, endothelin receptor ETB and endothelin expression is specifically inhibited (i.e. RNA interference or RNAi). Methods for selecting an appropriate dsRNA or dsRNA-encoding vector are well known in the art for genes whose sequence is known (e.g. see Tuschl, T. et al. (1999); Elbashir, S. M. et al. (2001); Hannon, GJ. (2002); McManus, MT. et al. (2002); Brummelkamp, TR. et al. (2002); U.S. Pat. Nos. 6,573,099 and 6,506,559; and International Patent Publication Nos. WO 01/36646, WO 99/32619, and WO 01/68836).
Ribozymes can also function as inhibitors of expression for use in the present invention. Ribozymes are enzymatic RNA molecules capable of catalysing the specific cleavage of RNA. The mechanism of ribozyme action involves sequence specific hybridization of the ribozyme molecule to complementary target RNA, followed by endonucleo lytic cleavage. Engineered hairpin or hammerhead motif ribozyme molecules that specifically and efficiently catalyze endonucleo lytic cleavage of mRNA sequences are thereby useful within the scope of the present invention. Specific ribozyme cleavage sites within any potential RNA target are initially identified by scanning the target molecule for ribozyme cleavage sites, which typically include the following sequences, GUA, GUU, and GUC. Once identified, short RNA sequences of between about 15 and 20 ribonucleotides corresponding to the region of the target gene containing the cleavage site can be evaluated for predicted structural features, such as secondary structure, that can render the oligonucleotide sequence unsuitable. The suitability of candidate targets can also be evaluated by testing their accessibility to hybridization with complementary oligonucleotides, using, e.g., ribonuclease protection assays.
Both antisense oligonucleotides and ribozymes useful as inhibitors of expression can be prepared by known methods. These include techniques for chemical synthesis such as, e.g., by solid phase phosphoramadite chemical synthesis. Alternatively, anti-sense RNA molecules can be generated by in vitro or in vivo transcription of DNA sequences encoding the RNA molecule. Such DNA sequences can be incorporated into a wide variety of vectors that incorporate suitable R A polymerase promoters such as the T7 or SP6 polymerase promoters. Various modifications to the oligonucleotides of the invention can be introduced as a means of increasing intracellular stability and half-life. Possible modifications include but are not limited to the addition of flanking sequences of ribonucleotides or deoxyribonucleotides to the 5' and/or 3' ends of the molecule, or the use of phosphorothioate or 2'-0-methyl rather than phosphodiesterase linkages within the oligonucleotide backbone.
Antisense oligonucleotides siRNAs and ribozymes of the invention may be delivered in vivo alone or in association with a vector. In its broadest sense, a "vector" is any vehicle capable of facilitating the transfer of the antisense oligonucleotide siRNA or ribozyme nucleic acid to the cells and preferably cells expressing endothelin receptor ETA, endothelin receptor ETB and endothelin. Preferably, the vector transports the nucleic acid to cells with reduced degradation relative to the extent of degradation that would result in the absence of the vector. In general, the vectors useful in the invention include, but are not limited to, plasmids, phagemids, viruses, other vehicles derived from viral or bacterial sources that have been manipulated by the insertion or incorporation of the antisense oligonucleotide siRNA or ribozyme nucleic acid sequences. Viral vectors are a preferred type of vector and include, but are not limited to nucleic acid sequences from the following viruses: retrovirus, such as moloney murine leukemia virus, harvey murine sarcoma virus, murine mammary tumor virus, and rouse sarcoma virus; adenovirus, adeno-associated virus; Simian Virus (SV)40-type viruses; polyomaviruses; Herpes viruses; papilloma viruses; vaccinia virus; poliovirus; and RNA virus such as a retrovirus. One can readily employ other vectors not named but known to the art.
Preferred viral vectors are based on non-cytopathic eukaryotic viruses in which nonessential genes have been replaced with the gene of interest. Non-cytopathic viruses include retroviruses (e.g., lentivirus), the life cycle of which involves reverse transcription of genomic viral RNA into DNA with subsequent pro-viral integration into host cellular DNA. Retroviruses have been approved for human gene therapy trials. Most useful are those retroviruses that are replication-deficient (i.e., capable of directing synthesis of the desired proteins, but incapable of manufacturing an infectious particle). Such genetically altered retroviral expression vectors have general utility for the high-efficiency transduction of genes in vivo. Standard protocols for producing replication-deficient retroviruses (including the steps of incorporation of exogenous genetic material into a plasmid, transfection of a packaging cell lined with plasmid, production of recombinant retroviruses by the packaging cell line, collection of viral particles from tissue culture media, and infection of the target cells with viral particles) are provided in KRIEGLER (A Laboratory Manual," W.H. Freeman CO., New York, 1990) and in MUR Y ("Methods in Molecular Biology," vol.7, Humana Press, Inc., Cliffton, N.J., 1991).
Preferred viruses for certain applications are the adenoviruses and Adeno-Associated Viruses (AAV), which are double-stranded DNA viruses that have already been approved for human use in gene therapy. AAV can be engineered to be replication deficient and is capable of infecting a wide range of cell types and species. It further has advantages such as, heat and lipid solvent stability; high transduction frequencies in cells of diverse lineages, including hematopoietic cells; and lack of super-infection inhibition thus allowing multiple series of transductions. Reportedly, AAV can integrate into human cellular DNA in a site-specific manner, thereby minimizing the possibility of insertional mutagenesis and variability of inserted gene expression characteristic of retroviral infection. In addition, wild-type AAV infections have been followed in tissue culture for greater than 100 passages in the absence of selective pressure, implying that AAV genomic integration is a relatively stable event. AAV can also function in an extra-chromosomal fashion.
Other vectors include plasmid vectors. Plasmid vectors have been extensively described in the art and are well known to those of skill in the art. See e.g., SANBROOK et al, "Molecular Cloning: A Laboratory Manual," Second Edition, Cold Spring Harbor Laboratory Press, 1989. In the last few years, plasmid vectors have been used as DNA vaccines for delivering antigen- encoding genes to cells in vivo. They are particularly advantageous for this because they do not have the same safety concerns as with many of the viral vectors. These plasmids, however, having a promoter compatible with the host cell, can express a peptide from a gene operatively encoded within the plasmid. Some commonly used plasmids include pUC18, pUC19, pRC/CMV, and pBlueScript. Other plasmids are well known to those of ordinary skill in the art. Additionally, plasmids may be custom designed using restriction enzymes and ligation reactions to remove and add specific fragments of DNA. Plasmids may be delivered by a variety of parenteral, mucosal and topical routes. For example, the DNA plasmid can be injected either by intramuscular, intradermal, subcutaneous, or other routes. It may also be administered by intranasal sprays or drops, rectal suppository and orally. It may also be administered into the epidermis or a mucosal surface using a gene-gun. Plasmids may be given in an aqueous solution, dried onto gold particles or in association with another DNA delivery system including but not limited to liposomes, dendrimers, cochleate and microencapsulation.
Typically the compounds according to the invention as described above are administered to the subject in a therapeutically effective amount. By a "therapeutically effective amount" of the compound of the present invention as above described is meant a sufficient amount of the compound for treating cardiovascular toxicity induced by anti-angiogenic compound at a reasonable benefit/risk ratio applicable to any medical treatment. It will be understood, however, that the total daily usage of the compounds and compositions of the present invention will be decided by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular subject will depend upon a variety of factors including the disorder being treated and the severity of the disorder; activity of the specific compound employed; the specific composition employed, the age, body weight, general health, sex and diet of the subject; the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidental with the specific compound employed; and like factors well known in the medical arts. For example, it is well within the skill of the art to start doses of the compound at levels lower than those required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved. However, the daily dosage of the products may be varied over a wide range from 0.01 to 1,000 mg per adult per day. Typically, the compositions contain 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 25.0, 50.0, 100, 250 and 500 mg of the compound of the present invention for the symptomatic adjustment of the dosage to the subject to be treated. A medicament typically contains from about 0.01 mg to about 500 mg of the compound of the present invention, preferably from 1 mg to about 100 mg of the compound of the present invention. An effective amount of the drug is ordinarily supplied at a dosage level from 0.0002 mg/kg to about 20 mg/kg of body weight per day, especially from about 0.001 mg/kg to 7 mg/kg of body weight per day.
In a particular embodiment, the compound according to the invention may be used in a concentration between 0.01 μΜ and 20 μΜ, particularly, the compound of the invention may be used in a concentration of 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 20.0 μΜ.
In a further aspect, the present invention relates to the compound according to the invention in combination with one or more anti-cancer compound for use in the treatment of cardiovascular toxicity induced by anti-cancer compound in a subject in need thereof.
In a further aspect, the present invention relates to the compound according to the invention in combination with one or more anti-angiogenic compound for use in the treatment of cardiovascular toxicity induced by anti-angiogenic compound in a subject in need thereof. According to the present invention, the compound of the invention is administered sequentially or concomitantly with one or more anti-cancer compound and anti-angiogenic compound.
According to the invention, the compound of the present invention is administered to the subject in the form of a pharmaceutical composition. Typically, the compound of the present invention may be combined with pharmaceutically acceptable excipients, and optionally sustained-release matrices, such as biodegradable polymers, to form therapeutic compositions. "Pharmaceutically" or "pharmaceutically acceptable" refer to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to a mammal, especially a human, as appropriate. A pharmaceutically acceptable carrier or excipient refers to a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type.
In the pharmaceutical compositions of the present invention for oral, sublingual, subcutaneous, intramuscular, intravenous, transdermal, local or rectal administration, the active principle, alone or in combination with another active principle, can be administered in a unit administration form, as a mixture with conventional pharmaceutical supports, to animals and human beings. Suitable unit administration forms comprise oral-route forms such as tablets, gel capsules, powders, granules and oral suspensions or solutions, sublingual and buccal administration forms, aerosols, implants, subcutaneous, transdermal, topical, intraperitoneal, intramuscular, intravenous, subdermal, transdermal, intrathecal and intranasal administration forms and rectal administration forms.
Typically, the pharmaceutical compositions contain vehicles which are pharmaceutically acceptable for a formulation capable of being injected. These may be in particular isotonic, sterile, saline solutions (monosodium or disodium phosphate, sodium, potassium, calcium or magnesium chloride and the like or mixtures of such salts), or dry, especially freeze-dried compositions which upon addition, depending on the case, of sterilized water or physiological saline, permit the constitution of injectable solutions. The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions; formulations including sesame oil, peanut oil or aqueous propylene glycol; and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. Solutions comprising compounds of the invention as free base or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms. The compound of the present invention can be formulated into a composition in a neutral or salt form. Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like. The carrier can also be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetables oils. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminium monostearate and gelatin. Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with several of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized agent of the present inventions into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the typical methods of preparation are vacuum-drying and freeze- drying techniques which yield a powder of the compound of the present invention plus any additional desired ingredient from a previously sterile- filtered solution thereof. The preparation of more, or highly concentrated solutions for direct injection is also contemplated, where the use of DMSO as solvent is envisioned to result in extremely rapid penetration, delivering high concentrations of the active agents to a small tumor area. Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective. The formulations are easily administered in a variety of dosage forms, such as the type of injectable solutions described above, but drug release capsules and the like can also be employed. For parenteral administration in an aqueous solution, for example, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration. In this connection, sterile aqueous media which can be employed will be known to those of skill in the art in light of the present disclosure. Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject.
In some embodiments, the compound of the present invention is administered sequentially or concomitantly with one or more therapeutic active agent such as chemotherapeutic or radiotherapeutic.
In some embodiments, the compound of the present invention is administered with a chemotherapeutic agent. The term "chemotherapeutic agent" refers to chemical compounds that are effective in inhibiting tumor growth. Examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, trietylenephosphoramide, triethylenethiophosphaorarnide and trimethylolomelamine; acetogenins (especially bullatacin and bullatacinone); a carnptothecin (including the synthetic analogue topotecan); bryostatin; callystatin; CC-1065 (including its adozelesin, carzelesin and bizelesin synthetic analogues); cryptophycins (particularly cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including the synthetic analogues, KW-2189 and CBI-TMI); eleutherobin; pancratistatin; a sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil, chlornaphazine, cholophosphamide, estrarnustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, ranimustine; antibiotics such as the enediyne antibiotics (e.g. calicheamicin, especially calicheamicin (11 and calicheamicin 211, see, e.g., Agnew Chem Intl. Ed. Engl. 33: 183-186 (1994); dynemicin, including dynemicin A; an esperamicin; as well as neocarzinostatin chromophore and related chromoprotein enediyne antiobiotic chromomophores), aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, carabicin, canninomycin, carzinophilin, chromomycins, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (including morpholino- doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin, idanrbicin, marcellomycin, mitomycins, mycophenolic acid, nogalarnycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptomgrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogues such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine, 5-FU; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; anti- adrenals such as aminoglutethimide, mitotane, trilostane; folic acid replenisher such as frolinic acid; aceglatone; aldophospharnide glycoside; aminolevulinic acid; amsacrine; bestrabucil; bisantrene; edatraxate; defo famine; demecolcine; diaziquone; elfornithine; elliptinium acetate; an epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidamine; maytansinoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidamol; nitracrine; pentostatin; phenamet; pirarubicin; podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK®; razoxane; rhizoxin; sizofiran; spirogennanium; tenuazonic acid; triaziquone; 2,2',2"- trichlorotriethylarnine; trichothecenes (especially T-2 toxin, verracurin A, roridinA and anguidine); urethan; vindesine; dacarbazine; mannomustine; mitobromtol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa; taxoids, e.g. paclitaxel (TAXOL®, Bristol-Myers Squibb Oncology, Princeton, N.].) and doxetaxel (TAXOTERE®, Rhone-Poulenc Rorer, Antony, France); chlorambucil; gemcitabine; 6- thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin and carboplatin; vinblastine; platinum; etoposide (VP- 16); ifosfamide; mitomycin C; mitoxantrone; vincristine; vinorelbine; navelbine; novantrone; teniposide; daunomycin; aminopterin; xeloda; ibandronate; CPT-1 1 ; topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoic acid; capecitabine; and pharmaceutically acceptable salts, acids or derivatives of any of the above. Also included in this definition are antihormonal agents that act to regulate or inhibit hormone action on tumors such as anti-estrogens including for example tamoxifen, raloxifene, aromatase inhibiting 4(5)-imidazoles, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, and toremifene (Fareston); and anti-androgens such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; and phannaceutically acceptable salts, acids or derivatives of any of the above.
In some embodiments, the compound of the present invention is administered with a targeted cancer therapy. Targeted cancer therapies are drugs or other substances that block the growth and spread of cancer by interfering with specific molecules ("molecular targets") that are involved in the growth, progression, and spread of cancer. Targeted cancer therapies are sometimes called "molecularly targeted drugs", "molecularly targeted therapies", "precision medicines", or similar names. In some embodiments, the targeted therapy consists of administering the subject with a tyrosine kinase inhibitor as defined above.
In some embodiments, compound of the present invention is administered with an immunotherapeutic agent. The term "immunotherapeutic agent," as used herein, refers to a compound, composition or treatment that indirectly or directly enhances, stimulates or increases the body's immune response against cancer cells and/or that decreases the side effects of other anticancer therapies. Immunotherapy is thus a therapy that directly or indirectly stimulates or enhances the immune system's responses to cancer cells and/or lessens the side effects that may have been caused by other anti-cancer agents. Immunotherapy is also referred to in the art as immunologic therapy, biological therapy biological response modifier therapy and biotherapy. Examples of common immunotherapeutic agents known in the art include, but are not limited to, cytokines, cancer vaccines, monoclonal antibodies and non-cytokine adjuvants. Alternatively the immunotherapeutic treatment may consist of administering the subject with an amount of immune cells (T cells, NK, cells, dendritic cells, B cells...). Immunotherapeutic agents can be non-specific, i.e. boost the immune system generally so that the human body becomes more effective in fighting the growth and/or spread of cancer cells, or they can be specific, i.e. targeted to the cancer cells themselves immunotherapy regimens may combine the use of non-specific and specific immunotherapeutic agents. Non-specific immunotherapeutic agents are substances that stimulate or indirectly improve the immune system. Non-specific immunotherapeutic agents have been used alone as a main therapy for the treatment of cancer, as well as in addition to a main therapy, in which case the non-specific immunotherapeutic agent functions as an adjuvant to enhance the effectiveness of other therapies (e.g. cancer vaccines). Non-specific immunotherapeutic agents can also function in this latter context to reduce the side effects of other therapies, for example, bone marrow suppression induced by certain chemotherapeutic agents. Non-specific immunotherapeutic agents can act on key immune system cells and cause secondary responses, such as increased production of cytokines and immunoglobulins. Alternatively, the agents can themselves comprise cytokines. Nonspecific immunotherapeutic agents are generally classified as cytokines or non-cytokine adjuvants. A number of cytokines have found application in the treatment of cancer either as general non-specific immunotherapies designed to boost the immune system, or as adjuvants provided with other therapies. Suitable cytokines include, but are not limited to, interferons, interleukins and colony- stimulating factors. Interferons (IFNs) contemplated by the present invention include the common types of IFNs, IFN-alpha (IFN-a), IFN-beta (IFN-β) and IFN- gamma (IFN-γ). IFNs can act directly on cancer cells, for example, by slowing their growth, promoting their development into cells with more normal behaviour and/or increasing their production of antigens thus making the cancer cells easier for the immune system to recognise and destroy. IFNs can also act indirectly on cancer cells, for example, by slowing down angiogenesis, boosting the immune system and/or stimulating natural killer (NK) cells, T cells and macrophages. Recombinant IFN-alpha is available commercially as Roferon (Roche Pharmaceuticals) and Intron A (Schering Corporation). Interleukins contemplated by the present invention include IL-2, IL-4, IL-11 and IL-12. Examples of commercially available recombinant interleukins include Proleukin® (IL-2; Chiron Corporation) and Neumega® (IL- 12; Wyeth Pharmaceuticals). Zymogenetics, Inc. (Seattle, Wash.) is currently testing a recombinant form of IL-21, which is also contemplated for use in the combinations of the present invention. Colony-stimulating factors (CSFs) contemplated by the present invention include granulocyte colony stimulating factor (G-CSF or filgrastim), granulocyte-macrophage colony stimulating factor (GM-CSF or sargramostim) and erythropoietin (epoetin alfa, darbepoietin). Treatment with one or more growth factors can help to stimulate the generation of new blood cells in subjects undergoing traditional chemotherapy. Accordingly, treatment with CSFs can be helpful in decreasing the side effects associated with chemotherapy and can allow for higher doses of chemo therapeutic agents to be used. Various-recombinant colony stimulating factors are available commercially, for example, Neupogen® (G-CSF; Amgen), Neulasta (pelfilgrastim; Amgen), Leukine (GM-CSF; Berlex), Procrit (erythropoietin; Ortho Biotech), Epogen (erythropoietin; Amgen), Arnesp (erytropoietin). In addition to having specific or non-specific targets, immunotherapeutic agents can be active, i.e. stimulate the body's own immune response, or they can be passive, i.e. comprise immune system components that were generated external to the body. Passive specific immunotherapy typically involves the use of one or more monoclonal antibodies that are specific for a particular antigen found on the surface of a cancer cell or that are specific for a particular cell growth factor. Monoclonal antibodies may be used in the treatment of cancer in a number of ways, for example, to enhance a subject's immune response to a specific type of cancer, to interfere with the growth of cancer cells by targeting specific cell growth factors, such as those involved in angiogenesis, or by enhancing the delivery of other anticancer agents to cancer cells when linked or conjugated to agents such as chemotherapeutic agents, radioactive particles or toxins. Monoclonal antibodies currently used as cancer immunotherapeutic agents that are suitable for inclusion in the combinations of the present invention include, but are not limited to, rituximab (Rituxan®), trastuzumab (Herceptin®), ibritumomab tiuxetan (Zevalin®), tositumomab (Bexxar®), cetuximab (C-225, Erbitux®), bevacizumab (Avastin®), gemtuzumab ozogamicin (Mylotarg®), alemtuzumab (Campath®), and BL22. Other examples include anti-CTLA4 antibodies (e.g. Ipilimumab), anti-PDl antibodies, anti-PDLl antibodies, anti-TIMP3 antibodies, anti-LAG3 antibodies, anti-B7H3 antibodies, anti-B7H4 antibodies or anti-B7H6 antibodies. In some embodiments, antibodies include B cell depleting antibodies. Typical B cell depleting antibodies include but are not limited to anti-CD20 monoclonal antibodies [e.g. Rituximab (Roche), Ibritumomab tiuxetan (Bayer Schering), Tositumomab (GlaxoSmithKline), AME-133v (Applied Molecular Evolution), Ocrelizumab (Roche), Ofatumumab (HuMax-CD20, Gemnab), TRU-015 (Trubion) and IMMU-106 (Immuno medics)], an anti-CD22 antibody [e.g. Epratuzumab, Leonard et al, Clinical Cancer Research (Z004) 10: 53Z7-5334], anti-CD79a antibodies, anti-CD27 antibodies, or anti-CD19 antibodies (e.g. U.S. Pat. No. 7,109,304), anti-BAFF-R antibodies (e.g. Belimumab, GlaxoSmithKline), anti- APRIL antibodies (e.g. anti- human APRIL antibody, ProSci inc.), and anti-IL-6 antibodies [e.g. previously described by De Benedetti et al, J Immunol (2001) 166: 4334-4340 and by Suzuki et al, Europ J of Immunol (1992) 22 (8) 1989-1993, fully incorporated herein by reference]. The immunotherapeutic treatment may consist of allografting, in particular, allograft with hematopoietic stem cell HSC. The immunotherapeutic treatment may also consist in an adoptive immunotherapy as described by Nicholas P. Restifo, Mark E. Dudley and Steven A. Rosenberg "Adoptive immunotherapy for cancer: harnessing the T cell response, Nature Reviews Immunology, Volume 12, April 2012). In adoptive immunotherapy, the subject's circulating lymphocytes, NK cells, are isolated amplified in vitro and readministered to the subject. The activated lymphocytes or NK cells are most preferably be the subject's own cells that were earlier isolated from a blood or tumor sample and activated (or "expanded") in vitro.
In some embodiments, the compound of the present invention is administered with a radio therapeutic agent. The term "radiotherapeutic agent" as used herein, is intended to refer to any radiotherapeutic agent known to one of skill in the art to be effective to treat or ameliorate cancer, without limitation. For instance, the radiotherapeutic agent can be an agent such as those administered in brachytherapy or radionuclide therapy. Such methods can optionally further comprise the administration of one or more additional cancer therapies, such as, but not limited to, chemotherapies, and/or another radiotherapy. In one embodiment, said additional active compounds may be contained in the same composition or administrated separately.
In another embodiment, the pharmaceutical composition of the invention relates to combined preparation for simultaneous, separate or sequential use in the treatment of cardiovascular toxicity induced by anti-cancer compound and anti-angiogenic compound in a subject in need thereof.
In a further aspect, the present invention also relates to a method for treating cardiovascular toxicity induced by anti-cancer compound in a subject in need thereof, comprising the step of administering to said subject the compound of the invention.
In a further aspect, the present invention also relates to a method for treating cardiovascular toxicity induced by anti-angiogenic compound in a subject in need thereof, comprising the step of administering to said subject the compound of the invention.
The invention also provides kits comprising the compound of the invention. Kits containing the compound of the invention find use in therapeutic methods.
In a further aspect, the present invention relates to a method of screening a candidate compound for use as a drug for the treatment of cardiovascular toxicity induced by anti-cancer compound and anti-angiogenic compound in a subject in need thereof, wherein the method comprises the steps of:
- providing an endothelin and endothelin receptors, providing a cell, tissue sample or organism expressing an endothelin and endothelin receptors,
providing anti-cancer compound and anti-angiogenic compound,
- providing a candidate compound such as small organic molecule, an oligonucleotide, a polypeptide, an aptamer, antibody or an intra-antibody, measuring the cardiovascular toxicity,
- selecting positively candidate compounds that inhibit cardiovascular toxicity induced by the anti-cancer compound and anti-angiogenic compound. Methods for measuring cardiovascular toxicity are well known in the art. For example, the cardiovascular toxicity is measured such as described in the example. For example, measuring the cardiovascular toxicity involves determining a Ki on the endothelin receptor cloned and transfected in a stable manner into a CHO cell line, measuring the endothelin receptor downstream signalling phospho lipase C, protein kinase C and MAPK1;ERK2, measuring aerobic and anaerobic metabolism, measuring myocardial metabolism, measuring glucose uptake, measuring cardiac fibrosis, measuring diastolic dysfunction and myocardial flux dysfunction and performing nuclear imaging of the heart using Sicintigraphy, Single photon emiccion tomograpgy (SPECT) or PET with various radiopharmaceuticals and with or without kinetics analysis of dynamic scans of the heart for monitoring cardiac metabolic remodeling.
In a further aspect, the present invention relates to a method of monitoring cardiovascular toxicity induced by an anti-cancer compound and an anti-angiogenic compound by performing PET-FDG scan such as described in the example.
In a further aspect, the present invention relates to a method of monitoring the efficacy of the compound of the invention in the treatment of cardiovascular toxicity induced by an anticancer compound and anti-angiogenic compound by performing PET-FDG scan such as described in the example.
The invention will be further illustrated by the following figures and examples. However, these examples and figures should not be interpreted in any way as limiting the scope of the present invention.
FIGURES:
Figure 1. Study design: (A) represents investigation for short-term cardiotoxic effects on immunodeficient tumor-bearing mice (nude). Sunitinib-treated mice were studied at baseline and week 1 using a cancer PET protocol compared to vehicle. (B) represents investigation for short-term cardiotoxic effects on immunocompetent mice (C57BL/6). Sunitinib-treated mice were studied at baseline and week 1 using a cancer PET protocol and echocardiography compared to vehicle. (C) represents study design for long-term treatment on immunocompetent mice (C57BL/6). Sunitinib-treated mice were followed at baseline, week 1, week 2 and week 3 using a cardiac PET protocol and echocardiography compared to vehicle and sunitinib+macitentan groups. FDG: 2'-deoxy-2'-[18F]fluoro-D-glucose.
Figure 2. Sunitinib increases myocardial FDG uptake in fasted mice: (A) Difference post-treatment - baseline of myocardial FDG-SUV for sunitinib and vehicle groups in nude mice (n=6 for vehicle, n=10 for sunitinib) and C57BL/6 (n=6 for each). (B) Difference post- treatment - baseline of myocardial metabolic flux for sunitinib and vehicle groups in nude mice (n=5 for vehicle, n=10 for sunitinib) and C57BL/6 (n=2 for each). (C) Difference in cardiac output (CO, Heart Rate times stroke volume) for sunitinib and vehicle in C57BL/6 mice (n=8 for each groups). Data expressed as mean±SEM; *p <0.05 compared to baseline, **p <0.01 and ***p <0.001 compared to baseline, $p <0.05 compared to vehicle. CO: cardiac output; SUV: standard uptake values
Figure 3. Sunitinib-induced increased FDG uptake in fasted mice is associated with increased fibrosis: (A) Quantification of microvascular density normalized by cell number in nude and C57B1/6 mice treated with vehicle (open circles) or sunitinib (black circles). (B) Quantification of fibrosis (normalized by tissue area) in nude and C57B1/6 mice treated with vehicle (open circles) or sunitinib (filled circles). (C) Representative quantification for GLUT1, HK2 and PGCl (normalized by cyclophilin B) mice treated with vehicle (open circles) or sunitinib (filled circles). Data expressed as mean ± SEM; $$p <0.01 $$$p <0.001 compared to vehicle. HK2: Hexokinase 2; GLUT1 : glucose transporter 1; PGCl ; Peroxisome proliferator- activated receptor gamma coactivator 1-alpha; S: sunitinib; V: vehicle.
Figure 4: Macitentan prevents sunitinib-induced diastolic dysfunction. (A) Difference treatment - baseline of myocardial FDG-SUV for vehicle (n=9), sunitinib (n=10) and sunitinib+macitentan treated mice (n=8) at week 1 and week 3. (B) Difference treatment - baseline values of CO is represented for vehicle (n=7), sunitinib (n=10) and sunitinib+macitentan (n=8) groups at week 1 and 3. (C) Difference treatment - baseline of left ventricular internal diameter at diastole for the three groups. (D) Difference treatment - baseline of aortic velocity time integral for both groups. Data are expressed as mean ± SEM, *p <0.05 compared to baseline, ***p <0.001 compared to baseline, $p <0.05 compared to other at week 3. AoVTI: Aortic velocity tracking integral; CO: cardiac output; LVID: left ventricular internal diameter; SUV: standard uptake value.
Figure 5. Macitentan prevents myocardial flux dysfunction induced by sunitinib.
(A) Difference treatment - baseline of myocardial metabolic flux and metabolic rate of glucose (MRglu) for vehicle (n=5), sunitinib (n=5) and sunitinib+macitentan (n=6) groups. Data are expressed as mean ± SEM, *p <0.05 compared to baseline, $p <0.05 compared to other at week 3. #p <0.05 compared to sunitinib group at week 3. (B) Quantification of vessels area reported on number of cells for vehicle (n=9), sunitinib (n=9) and sunitinib+macitentan (n=7) groups at D22 in non-fasted C57B1/6 mice. MRGlu: metabolic rate of glucose.
Figure 6. Protective effects of macitentan involve ET receptors: (A) ng of ET-1,
ETA and ETB receptor mRNA reported on ng of 18s RNA in the myocardium (n=6 for each).
(B) Quantification of fibrosis for vehicle (n=9), sunitinib (n=9) and sunitinib+ macitentan (n=7) groups at D22 in non-fasted C57B1/6 mice. Data are expressed as mean ± SEM, $p <0.05; $$$p <0.001 compared to other. EDN1 : Preproendothelin-1; EDNRA: endothelin receptor type A; EDNRB: endothelin receptor type B.
Figure 7: Schematic representation of the mechanism of sunitinib-induced cardiac side effects: (A) Sunitinib upregulates glycolysis and downregulates oxidative metabolism in cardiac mitochondria. (B) Sunitinib induces resistance to insulin stimulation of cardiac glucose uptake. The metabolic switch is an immediate early response to sunitinib while insulin resistance either appears later or is masked by the metabolic switch during the early stages of sunitinib treatment. Both mechanisms depend on signaling by the endothelin pathway, and lead to myocardial fibrosis and impaired cardiac function, and are reversed by the endothelin receptors antagonist macitentan. Red indicates upregulated proteins and pathways, blue indicates downregulated protein and pathways. ATP: Adenosine triphosphate; ET-1 : endothelin 1; ETA: endothelin receptor type A; FA: fatty acid; GLUT: glucose transporter protein; 02: oxygen; OXPHOS: oxidative phosphorylation; TCA: tricarboxylic acid.
EXAMPLE:
Material & Methods
Experimental design
Animal experiments were authorized by the French Ethical committee for animal experimentation under No. 15-045 and performed by certified personal following the French law of animal welfare on animal experimentation n°2013-118. Female nude (nu/nu) and C57BL/6 mice (Janvier Labs, France) 15 weeks aged were maintained in controlled temperature (24°C) and relative humidity (50%) on a 12/12-light/dark cycle and were fed ad libitum. Sunitinib malate (Sutent®, Pfizer, USA) was dissolved at lOmg/mL in DMSO/PBS (1 :4). 40 mg/kg/ml is the minimum dosage that permit to demonstrate anti-tumor effect and produce comparable plasma concentrations to those found in patients (8,63,64). However, preliminary results in our institution demonstrated the variability in the results with this dosage. Based on that, we administered sunitinib daily by oral gavage in a dose of 50 mg/kg body weight to improve homogeneity and reduce number of animals. In the sunitnib+macitentan group, macitentan (20mg/mL) was added to the gavage solution, with the volume maintained at 150μί.
This study followed a standard protocol for monitoring of short-term response to therapy in fasted tumor-bearing nude mice. Mice were injected bilaterally in the left and right flanks subcutaneously with 1 x 107 Cricetulus griseus (CCL-39) tumor cells suspended in 300μ1 of culture medium. The PET scan was performed before and 2 days after a 5 -day course of 50mg/kg sunitinib malate (treatment group) or vehicle (vehicle group) (Figure 1 A). To check whether the increased SUV was independent of the presence of a tumor in immunodeficient animals, the same protocol was reproduced in C57BL/6 immunocompetent mice fasted before the PET examination. Chow diet was removed the day before the experiment for a 12 hours fasting. Cardiac echography was also performed to measure heart function (Figure IB). Finally, to explore the pathophysiological mechanisms of sunitinib cardiotoxicity, PET-FDG and echocardiography were performed in non-fasted mice at baseline and 1 and 3 weeks of a three- week course of sunitinib or vehicle. A third group with co-administration of sunitinib and the endothelin receptor antagonist macitentan was added to test the hypothesis that the cardiac effects of sunitinib were, in part, mediated by the endothelin pathway (Figure 1C). For all the panels, animals of both groups were randomly assessed, experiment and analysis were realized blinded and animals from both groups were image the same day.
Cardiac Positron Emission Tomography (PET)
All mice from groups A and B were fasted overnight. Mice from panel C were not fasted and had free access to food and water. Mice were anesthetized (2±0.5% isoflurane in air), weighted and glycemia was measured in blood drawn from the caudal ventral artery using an Accu-Chek® Aviva Nano A (Accu-Chek, France). A catheter home-made from a 26G needle (Fischer Scientific, France) connected to a 5cm polyethylene tubing (Tygon Microbore Tubing, 0.010" x 0.030"OD; Fisher Scientific, France) was inserted in the caudal vein for radiotracer injection. Mice were then installed into the PET-CT dedicated bed and respiration and body temperature were registered. Body temperature was maintained at 37°C and anesthesia was controlled on the breathing rate throughout the entire examination. CT was acquired in a PET- CT scanner (nanoScan PET-CT; Mediso, Hungary) using the following acquisition parameters: semi-circular mode, 39kV tension, 720 projections full scan, 300ms per projection, binning 1 :4. Then, PET acquisition was started and, 30 seconds later, lOMBq of 2'-deoxy-2'-[18F]fluoro-D- glucose (FDG; Advanced Applied Applications, France) in 0.2mL saline was injected via the catheter. The first scan was a dynamic acquisition of 30.5min and was followed by a gated cardiac scan of 30min duration. PET data were collected in list mode and binned using a 5ns time window, with a 400-600keV energy window and a 1 :5 coincidence mode. Data were reconstructed using the Tera-Tomo reconstruction engine (3D-OSEM based manufactured customized algorithm) with expectation maximization iterations, scatter and attenuation correction. The first scan was reconstructed starting 10s before FDG injection with the following time sequence: 26 x 5s; 6 x 30s; 5 x 120s; 3 x 300s and 3 x 600s. The second scan reconstructed in a single time frame from 45 to 60min post-injection.
FDG accumulation was quantified as mean Standard Uptake Value (SUV, ratio of the radioactivity concentration in myocardium on the whole body concentration of the injected radioactivity) between 45 and 60min post-injection in 3D volumes-of- interest (VOI) delineated semi-automatically by iso-contours at 45% threshold of maximal value in the myocardium on PET/CT fusion slices using the PMOD software package (PMOD Technologies Ltd, Zurich, Switzerland). Metabolic flux was quantified using compartmental modeling tool of PMOD software using the same VOI as above and a VOI semi-automatically delineated on the vena cava for the arterial input function as previously described (65). Metabolic rate of glucose were calculated by the multiplication of the metabolic flux by [plasma glucose (mmol/l)/lumped constant (fixed at 0.69)].
Echocardiography
Conventional echocardiography was performed in anesthetized mice using a Vevo 2100 high resolution ultrasound device (Visualsonics, Toronto, Canada) with a 40MHz probe (MS- 550). Mice were anesthetized with 3% isoflurane in air for induction and maintained with 1.5%. Mice were depilated in the thoracic region and then placed in the supine position on a dedicated heating platform, allowing monitoring of ECG, temperature and respiratory frequency. All acquisitions were performed within body temperature limits 36-37.5°C. Parasternal long axis views were recorded and 3 consecutive measurements in M-mode were drawn to determine Left Ventricular Internal Diameter (LVID) at diastole (d) and systole (s) and Left Ventricular Posterior Wall thickness (LVPW) in both telediastole (d) and telesystole (s). Cardiac Output (CO) and the percentage of fractional shortering (FS) were then calculated using the VevoLab Software (Visualsonics). Mitral flow deceleration time was calculated from mitral flow velocities using Pulsed Wave Doppler (PW Doppler). Ascending aorta diameter (Ao) and Left Atrium diameter (LA) were measured using M-mode in parasternal long axis. Aortic velocity tracking integral (AoVTI) was measured using PW Doppler, in suprasternal view allowing measurement of mean aortic velocity and peak aortic velocity.
Assessment of microvascular density
Cardiac microvessels were stained using Isolectine B4 Griffonia Simplicifolia-FITC (Sigma Aldrich). Nuclei were counter stained with DAPI. Microvessels and nuclei were counted in 4 fields at a magnification of x200 in 2 independent sections from each heart using Matlab® based software. Microvessel density was normalized to the number of nuclei.
Assessment of cardiac fibrosis
Frozen sections were incubated with Picrosirius red (VWR) 0.1% in picric acid (Sigma) in a Leica ST5020 automatic stainer during 30min and dehydrated in ethanol and xylene. Whole sections were observed at a magnification of x20 using a Nanozoomer HT 2.0 (Hamamatsu) and fibrosis was quantified using Matlab® based software.
Western blotting
Twenty micrograms of each heart lysate were loaded onto a 10% SDS-PAGE gels (mini- protean TGX gels, BioRad) and transferred to nitrocellulose membranes. The membranes were blocked and immunoblotted with the following primary antibodies: GLUT1 (1 : 1000, ab652, Abeam), PGCla (1 : 1000, ab54481, Abeam) and hexokinase II (1 :500, bs-3993R, Interchim). Membranes were then incubated with horseradish peroxidase (HRP)-conjugated anti-rabbit secondary antibodies (1 : 10000, 474-1506, KPL). Chemiluminescence detection was performed using the ECL kit (Clarity Western ECl substrate; BioRad). Quantitation of immunoblots was done on digitalized images using ImageJ software. The intensity of immunoreactive bands was normalized by the loading control (Cyclophilin B, 1 : 1000, abl6045, Abeam).
Proteomics
Tissue sample preparation: Frozen mouse hearts were individually ground under liquid nitrogen to yield a fine powder using a pestle and mortar. The tissue powder was weighted and solubilized in lysis buffer (4% SDS, lOOmM Tris-HCl, pH 8.0). Protein extracts were clarified by centrifugation at 21,000XG, 1 hour, 4°C. Protein concentration of the supernatant was determined using bicinchoninic acid assay (BCA, Pierce). Peptides were prepared by Filter Aided Separation method (FASP) essentially as described (66). Briefly, 50μg of proteins from whole lysates were diluted to ΙΟΟμί in solubilization buffer (50 mM Tris/HCl, pH8.5, SDS 2%, 20mM TCEP, 50 mM chloroacetamide) and heated for 5 min at 95°C. After cooling to room temperature, extracts were diluted with 300μί Tris Urea buffer (Urea 8M, Tris/HCl 50mM (pH 8.5) and transferred onto 30kDa centrifuged filters and prepared for digestion as described (66). Proteins were digested during 14h at 37°C with μg trypsin (Promega) and peptides were desalted on C18 StageTips (67). After drying, peptides were solubilized in 2% trifiuoroacetic acid (TFA) and fractionated by strong cationic exchange (SCX) StageTips, mainly as described (Kulak et al, 2014) except that fractions 1 and 2 were pooled in most experiments. Mass spectrometry analysis: Mass spectrometry analyses were performed on a Dionex U3000 RSLC nano-LC- system coupled to either a Q-Exactive or a LTQ Orbitrap- Velos mass spectrometer, all from Thermo Fisher Scientific. After drying, peptides from SCX StageTip fractions were solubilized in 10 μί of 0.1% TFA containing 2% acetonitrile (ACN). One μΐ, was loaded, concentrated and washed for 3min on a C 18 reverse phase precolumn (3 μιη particle size, 100 A pore size, 75 μιη inner diameter, 2 cm length, Dionex, Thermo Fisher Scientific). Peptides were separated on a CI 8 reverse phase resin (2 μιη particle size, ΙΟθΑ pore size, 75 μιη inner diameter, 25 cm length from Dionex) with a 3 -hour gradient starting from 99% of solvent A containing 0.1% formic acid in H20 and ending in 40% of solvent B containing 80% acetonitrile, 0.085% formic acid in H20. The mass spectrometer acquired data throughout the elution process and operated in a data-dependent scheme with full MS scans acquired with the Orbitrap, followed by up to 10 MS/MS HCD fragmentations in the Q-Exactive (Thermo Fisher) on the most abundant ions detected. Settings were essentially as in (68) with slight modifications: the recurrent loop of the 10 most intense nLC-eluting peptides were HCD- fragmented between each full scan (data dependent mode). Resolution was set to 70,000 for full scans at AGC target 1,10e6 within 60ms MIIT. The MS scans spanned from 350 to 1500m/z. Precursor selection window was set at 2Th, and MS/MS scan resolution was set at 17,500 with AGC target 1,10e5 within 60ms MIIT. HCD Normalized Collision Energy (NCE) was set at 27%. Dynamic exclusion was set to 30s duration. Spectra were recorded in profile mode. The mass spectrometry data were analyzed using Maxquant version 1,5,2,8 (69). The database used was a concatenation of human sequences from the Uniprot-Swissprot database (Uniprot, release 2015-02) and a list of contaminant sequences from Maxquant. The enzyme specificity was trypsin. The precursor mass tolerance was set to 4.5ppm and the fragment mass tolerance to 20ppm for Q-Exactive data. Carbamidomethylation of cysteins was set as constant modification and acetylation of protein N-terminus and oxidation of methionines were set as variable modification. Second peptide search was allowed and minimal length of peptides was set at 7 amino acids. False discovery rate (FDR) was kept below 1% on both peptides and proteins. Label-free protein quantification (LFQ) was done using both unique and razor peptides. At least 2 such peptides were required for LFQ. The "match between runs" (MBR) option was allowed with a match time 0.7 min window and an alignment time window of 20min. For analysis, LFQ results from MaxQuant were imported into the Perseus software (version 1.5.1.6). Reverse and contaminant proteins were excluded from analysis. Contaminating proteins from culture medium, essentially coming from added serum, such as immunoglobulins or transferrin were also removed from the protein list. Protein copy numbers per cell were then calculated using the "Protein ruler" plugin of Perseus by standardization on total histone MS signal as described (70).
Modeling with Ingenuity® and Pathway Studio®
We used ingenuity pathway analysis (Ingenuity Systems, Redwood City, CA) and Pathway Studio (https://www.pathwaystudio.com/) to study pathways deregulated by sunitinib as already described (71). For both, data from proteomic were classified according to the fold change and p-value compared to reference group (i.e: vehicle) using a paired t.test and only significant deregulated proteins were enter in the software. Briefly, these software create hypothetical networks (including several proteins) to highlight networks that are significantly (p <0.05) different compared to a reference group (i.e: vehicle group or sunitnib + macitentan group). Those networks were assigned to a biological function(s), pathway(s) and/or disease(s) using the Ingenuity Pathways Knowledge Base, Pathway Studio database and the scientific literature.
RNA extraction and qPCR R A from hearts and aorta was isolated using TRI Reagent Solution (Invitrogen). Any DNA present was degraded using RQ1 RNAse-Free DNase (Promega) according to the manufacturer's instructions. cDNA was synthesized using a High Capacity cDNA Reverse Transcription kit with RNase inhibitor (Invitrogen). qRT-PCR was performed using Fast SYBR Green Master Mix (Applied Biosystems) on the ABI7900 System (Applied Biosystems). Primers for 18s, prepro-ET-1, ETA and ETB receptors were generated. The amplification reaction mixture was heated at 95°C for 20s, then subject to 40 cycles of 95°C for Is then 60°C for 20s. Values obtained for experimental gene measurements were normalized against expression of 18s.
Statistical analysis
Statistical analysis and data comparison for proteomics data were done using either Perseus or Excel software to classify the data using the two tailed Student t.test. In modeling software, p-value cutoff was 0.05 calculated with right-tailed Fisher Exact Test (Ingenuity®) and with Mann-Whitney U-test (Pathway Studio®). Data are expressed as mean ± SEM. Analyses were performed with Graphpad Prism (7.00). Unpaired and paired t tests were used to compare two data sets, one-way ANOVA was used to compare three data sets, and two-way ANOVA was used to compare three groups followed across time.
Results
The study design followed a standard protocol of mouse oncology studies for monitoring of short-term response to therapy with PET in fasted, tumor-bearing, nude mice (Figure 1A). The same protocol was repeated in C57BL/6 immunocompetent mice with addition of echocardiography (Figure IB). In an other group of C57BL/6 mice, treatment duration was extended to three weeks (Figure 1C). Histology and proteome analysis were performed in all groups after treatment completion.
Sunitinib increases myocardial FDG uptake
After 5 days of sunitinib treatment, a significantly higher accumulation of FDG in the heart of fasted tumor-bearing nude mice was observed than in the vehicle group (data not shown). Compared to baseline, the mean standard uptake value (SUV) increased in the sunitinib group whereas it remained unchanged in the vehicle group (p <0.001, Figure 2A). Similar results were obtained in non-tumorized immunocompetent C57BL/6 mice using the same protocol: 5 days of sunitinib significantly increased FDG uptake compared to baseline (p <0.05; Figure 2A). Dynamic PET scans obtained under fasting conditions confirmed that sunitinib increased cardiac FDG metabolic flux in nude and C57BL/6 mice (Figure 2B). These findings support a sunitinib-induced shift towards glycolysis. Accordingly, hearts from sunitinib-treated mice showed a trend for higher expression of the glucose transporter protein GLUTl and of Hexokinase II, the glucose phosphorylating enzyme, together with reduced expression of PGCl , a key regulator of energy metabolism (Figure 3C). Moreover, 5 days of sunitinib treatment in C57BL/6 mice raised cardiac FDG uptake and lowered cardiac output (p <0.05, sunitinib vs. vehicle) (Figure 2C).
Sunitinib-induced increased myocardial FDG uptake is associated with increased cardiac fibrosis
We then explored whether the sunitinib-induced increase in FDG uptake was associated with a direct effect on the cardiac micro vasculature, but found no difference in vessel density after sunitinib treatment (Figure 3 A). This is in keeping with previous studies that have shown that microvascular rarefaction is not responsible for sunitinib-induced coronary dysfunction (19). By contrast, sunitinib treatment significantly increased myocardial fibrosis compared to hearts from vehicle-treated mice (p<0.001 and p<0.01 for nude and C57BL/6 mice, respectively) (Figure 3B).
Sunitinib downregulates oxidative energy metabolism pathways
Label-free pan-analysis of protein expression in mouse hearts revealed that 5 days of sunitinib treatment led to a significant downregulation of major oxygen-dependent metabolic pathways, including isocitrate dehydrogenase and succinate dehydrogenase (both enzymes of the tricarboxylic acid (TCA) cycle), phosphoenolpyruvate carboxykinase (first enzyme of gluconeogenesis), phosphoglycerate mutase and phosphorylase B kinase (key enzymes of carbohydrate metabolism) and carnitine O-palmitoyltransferase (mitochondrial transporter of fatty acids). All the proteins for which we found different levels of expression in the sunitinib and vehicle groups are listed in Table 1. Ingenuity® analysis highlighted that sunitinib treatment induced mitochondrial dysfunction and a clear switch towards anaerobic glycolytic metabolism, similar to the one seen during cardiac hypertrophy (31). In particular, proteins of the fatty acid degradation pathway such as acyl-CoA dehydrogenase (Acad8), phospholipases, and fatty acid transferases, those controlling glycogen breakdown (Pgaml, Me3) and the synthesis of the cofactor flavine adenine dinucleotide (FAD) were reduced in sunitinib-treated hearts. Taken together, these results are in agreement with a switch towards the utilization of glucose as major energy source and with the activation of glycolysis in the myocardium of sunitinib-treated mice evidenced by increased FDG uptake.
The effect of sunitinib on cardiac FDG uptake depends on glucose metabolism of the heart Cardiac FDG uptake varies widely with plasmatic concentrations of glucose and insulinemia, hence with the post-prandial time. Oncology PET scans are performed in fasted patients and animals in order to reduce the cardiac uptake of FDG in oncology studies and better delineate the tumor uptake. Conversely, imaging of glucose metabolism in the heart is often performed under an euglycemic clamp (glucose plus insulin administration) in order to increase metabolism of glucose and FDG uptake in the heart. Since sunitinib has been shown to induce hypoglycemia in patient and animal studies (32), we tested whether the effect of the drug on cardiac FDG uptake would differ in fasted versus non- fasted mice. Interestingly, in contrast to fasted mice, the FDG SUV were similar in sunitinib and vehicle-treated non-fasted animals after 1 and 3 weeks of treatment (Figure 4A). However, the effects of sunitinib on heart function were maintained in non-fasted animals, i.e. cardiac output was reduced after 1 and 3 weeks of sunitinib with respect to pre-sunitinib baseline values (Figure 4B), with significant reductions in diastolic (Figure 4C) and stroke volumes. Sunitinib also significantly reduced blood flow as shown by the fall in aortic flow (aortic velocity tracking integral) (Figure 4D).
Endothelin receptor antagonism prevents sunitinib-induced diastolic dysfunction and myocardial flux dysfunction
It has been demonstrated that the endothelin (ET) pathway mediates the hypertension and renal complications of sunitinib (24). Macitentan, a clinically-available mixed ETA and ETB receptor antagonist, was given concomitantly with sunitinib to mice during 3 weeks. A comparison of protein expression in the sunitinib-treated group with that in the vehicle group and sunitinib plus macitentan group evidenced the protective effects of macitentan on diastolic dysfunction. This was confirmed by expression levels of several actin and myosin protein iso forms and associated fibrillar protein, for which macitentan countered the effect of sunitinib on their level of expression (Table 2). It shoul be noted that macitentan treatment normalized both the diastolic volume and the cardiac output, allowing the maintenance of aortic flow (Figure 4B - 4D).
Kinetics analysis of dynamic FDG-PET scans of the heart showed a trend towards a higher metabolic flux and a higher metabolic rate of glucose (MRGlu) after 1 and 2 weeks of sunitinib treatment that did not reach statistical significance (Figure 5A). In contrast, both parameters were significantly reduced after 3 weeks of sunitinib, showing that the early promotion of cardiac glycolysis by sunitinib was followed by a secondary drop in glucose utilization. Macitentan plus sunitinib co-administration maintained metabolic flux and MRGlu to their pre-treatment baseline levels after 3 weeks of treatment (Figure 5A). In line with our previous observations at 1 week of treatment, 3 weeks of sunitinib was not associated with any change in myocardial vessel density (Figure 5B). A proteomics analysis of the macitentan and sunitinib treated hearts and comparison with the sunitinib-only or vehicle-treated groups confirmed the protective effects of endothelin antagonism by macitentan on endothelial cell dysfunction (Table 2).
Macitentan reduces cardiac fibrosis and downregulates myocardial ETA receptors
We studied the effect of sunitinib, with or without the addition of macitentan, on cardiac fibrosis. Sunitinib treatment was associated with the development of cardiac fibrosis that was prevented by co-administration of macitentan (data not shown). Since the ETA receptor is known to mediate fibrosis (33) and cardiac hypertrophy (34), it can be assumed that sunitinib- induced cardiac injuries are mediated through the ETA receptor. We further explored the effects of macitentan on the ET pathway in order to define which receptor is involved in sunitinib- induced cardiac injuries. Macitentan did not affect ET-1 or ETB receptor expression, but significantly downregulated myocardial ETA receptor expression (Figure 6A).
Moreover, both ETA and ETB receptors of the aorta were increased in mice treated with sunitinib and sunitinib+macitentan (data not shown). Since elevated ET-1 is found in patients and rodents treated with sunitinib (21) and elevated ET receptors characterizes hypertension (35), we speculated that the ET pathway was activated by sunitinib. Accordingly, the ET downstream signaling, phospholipase C, protein kinase C and MAPK1;ERK2, was high in all samples (n=6) of sunitinib-treated hearts but was undetectable in the vehicle group. In addition, these effectors were detected only in 3 samples out of 6 (50%) in the sunitinib+macitentan group and were expressed at lower level than the sunitinib-group.
Macitentan reverses the sunitinib-induced aerobic to anaerobic switch
Label-free pan-analysis of protein expression in mouse hearts collected from the three groups (vehicle, sunitinib and sunitinib+macitentan) is shown in Table 2. Data analysis using Ingenuity® and Pathway Studio® softwares showed that sunitinib induced an impairment in carbohydrate and fatty acid oxidative metabolisms and in the TCA cycle. Mitochondrial homeostasis was also deregulated, as shown by reduced expression in both the inner and outer mitochondrial membrane complexes, in mitochondrial transporters and in mitochondrial translation factors. In line with the effect of sunitinib on myocardial flux dysfunction, several patterns of protein clusters were drastically different in the sunitinib and vehicle groups (data not shown). Protein clusters involved in myocardial infarction, endothelial cell dysfunction and apoptosis, atherosclerosis, thrombosis, inflammation and hypertension, were targeted by sunitinib. Comparing protein expression in the sunitinib-treated group with that in the vehicle and sunitinib plus macitentan groups highlighted once again the protective effects of macitentan, notably on the following clusters: myocardial infarction, endothelial cell dysfunction and apoptosis (data not shown), glycogen metabolism, TCA cycle, acetyl CoA biosynthesis. The level of expression of the pyruvate dehydrogenase (PDH) components (alpha and beta El components of the complex, PDH phosphatase) were maintained at control levels by macitentan in the oxidative phosphorylation deficient myocardium induced by sunitinib (data not shown). Interestingly, the levels of expression of some proteins involved in glycolysis were augmented by 3 weeks of sunitinib treatment (e.g. glucose-6-phosphate isomerase and phosphoglycerate mutase), while those of others were reduced, e.g. muscle-type phosphofructokinase and glyceraldehyde-3 -phosphate dehydrogenase. Sunitinib-treated hearts showed higher lactate dehydrogenase (LDH) that is released during tissue hypoxia and damage. Surprisingly, sunitinib induced a dramatic (8-fold) increase in the expression of GLUT4 (SLC2a4), the insulin-regulated transporter of glucose at the plasma membrane. However, the level of RablO, a small ras-family GTPase required for translocation of GLUT4, was significantly decreased. Together with a trend for elevated blood sugar in the sunitinib treated group (190±38 mg/dL), these results were coherent with an insulin-resistant type of diabetic metabolic profile of the sunitinib treated hearts. Addition of macitentan to sunitinib suppressed the diabetic-like clusters (data not shown), returned glycaemia to pre-treatment levels (171±17 mg/dL and 173±23 mg/dL in the vehicle and sunitinib+macitentan treated groups, respectively), and activated clusters for glycolysis. Macitentan only partially reverted the effects of sunitinib on the level of GLUT4 and RablO, but significantly increased the level of expression of the pleiotropic regulatory protein sirtuin 2, in line with the role of this protein in energetic metabolism preservation.
Discussion
Sunitinib treatment is limited by its cardiovascular side effects. A recent study reported that sunitinib induces an early switch of cardiac metabolism to anaerobic glycolysis and impairs heart function (30). Here, we show that in addition to this metabolic switch, myocardial remodeling by sunitinib also induces a reduced glucose uptake resembling the one found during insulin resistance, and show that sunitinib cardiotoxicity is a combination of several complex mechanisms occurring over a sequential time course. Moreover, we show that sunitinib-induced cardiac injury and dysfunction are prevented through inhibition of endothelin signaling, strongly supporting a role for this pathway in sunitinib 's cardio toxic effects (Figure 7).
Our results confirm that FDG-PET is able to quantify the metabolic changes induced by sunitinib administration. It remains unclear why this has not been noted previously given the widespread use of FDG-PET in the staging and follow-up of cancer patients. One explanation might be that myocardial glucose metabolism is a complex, tightly regulated mechanism that varies according to nutrient and oxygen levels. Under physiological conditions, the heart preferentially uses oxidization of fatty acids as fuel and switches to glycolysis in high glucose and insulin or low oxygen conditions by activating synthesis and translocation to the plasma membrane of the glucose transporter GLUT4 (36). Therefore, myocardial FDG uptake varies according to fasting status, diet, diabetes and ischemia (37). In mouse and man, FDG uptake is higher in non-fasting than in fasting conditions (37) and in oncology studies FDG PET imaging is acquired under fasting conditions in order to minimize muscular and myocardial uptake and improve tumor detection. In contrast, cardiac PET-FDG often utilizes an euglycemic clamp (glucose load with additional insulin administration after overnight fasting) in order to maximize heart uptake (37,38). Interestingly, in the case of vascular dysfunction, PET images of the heart look different under fasting and non-fasting conditions: ischemic territories appear as hot spots with higher FDG uptake than the intact myocardium after fasting (39) while they may not differentiate from intact tissue in non-fasted conditions after a glucose load (40-42). In the absence of sunitinib treatment, FDG-SUV and metabolic flux were lower in fasted than in non-fasted states. We show here that the increase in FDG uptake after one week of sunitinib was more pronounced in fasted than in non- fasted mice. Therefore, dietary status also influences the FDG cardiac uptake under sunitinib treatment.
Systemic administration of sunitinib rapidly induces a metabolic switch towards glycolysis with reduced expressions of key enzymes of the TCA cycle and key proteins for mitochondrial oxidative phosphorylation (OXPHOS) and for the beta-oxidation of fatty acids. We observed this switch regardless of whether the animals were fasted or not (although with a different intensity in both conditions) at one and 3 weeks after beginning of the anti-angiogenic treatment. O'Farrel et al. also showed increased FDG uptake as early 2-3 days after introduction of sunitinib in mice and 5 days in rats (30). Overall, the sunitinib-treated heart tends to switch towards an anaerobic glycolysis metabolism with accumulation of lactate, similar to the one found in hypoxic muscles and during heart failure (43). At the proteomic level, the changes in the expression of proteins involved in metabolic pathways in the hearts of non-fasted animals treated during one and three weeks were largely consistent. For instance, 3 weeks of treatment decreased the PDH complex and increased levels of LDH, confirming the glycolytic switch combining inhibition of the pathways leading to acetyl Co A biosynthesis and diversion toward anaerobic conversion of pyruvate to lactate. Sunitinib-treated hearts were also unlikely to use glycogen as energy source since they expressed very low phosphorylase B kinase, in agreement with the elevated glycogen level described by Rees et al (32). Interestingly, the mechanism of sunitinib's cardiac toxicity is not due to a reduction in blood vessel density in the myocardium. This is different from the action on tumor vessels, whose number is drastically reduced after a short course of sunitinib (44).
Here, macitentan treatment prevented the deficiency in acetyl CoA biosynthesis and impairment of the TCA cycle and suppressed the protein patterns typical of endothelial cell apoptosis and myocardial infarction. Others have shown that macitentan also prevented the hypertension associated with sunitinib (25). Our results suggest that the cardiotoxic effects of sunitinib are mediated by the ETA receptor. The current explanation for the cardiac toxicity of sunitinib is a direct toxicity on the myocardium with mitochondrial dysfunction, that is exacerbated by hypertension since cardiomyocytes use glucose as main energy source when subjected to pressure overload (45). Reduced availability of oxygen in the myocardium will also lead to rapid metabolic changes with increased FDG uptake. This is the case in the hearts of fasted mice at one day after transverse aortic constriction surgery (46). Moreover, during fasting the ischemic territories of the heart show a higher FDG uptake than healthy myocardium that prefers fatty acids as energy substrate (47). The present data support a role for the endothelin system in mediating switch towards anaerobic metabolism.
We observed a decreased metabolic flux in the hearts of mice treated with sunitinib for 3 weeks. Although they did not elaborate on this result, O'Farrell et al observed the same trend and showed a similar metabolic rate of glucose at 3 weeks of treatment in sham and sunitinib treated animals (30). Interestingly, in their study, as in ours, the apparent reversal of the early metabolic switch induced by sunitinib was not accompanied by an improvement in the left ventricular ejection fraction. This delayed effect of sunitinib may be in line with a case report describing decreased myocardial FDG uptake in patients treated with imatinib plus sorafenib who later developed a cardiac event (48). Therefore, we explored the possible mechanisms underlying this observation. On the one hand, sunitinib-treated hearts presented anaerobic metabolism and it is known that PDH inhibition leads to a slow recovery of glucose uptake and uncoupling of glycolysis (49), and that lactate accumulation decreases glucose uptake (43). On the other hand, patterns of protein expression in the 3-week sunitinib-treated heart resembled the one seen in diabetic patients in which the FDG metabolic flux is reduced (50), as well as the pattern in diabetic rats where myocardial glucose uptake is reduced under ischemic conditions (51). Despite the lower metabolic rate of glucose in sunitinib-treated hearts, we observed a dramatic increase of GLUT4 activated in high glycaemia-high insulinemia conditions (36). However, the expression of RablO, that controls the plasma membrane insertion of GLUT4 and whose activation is under control of the AS 160 GTPase, a substrate of AMPK which is known to be inhibited by sunitinib (22), was reduced. Therefore, it is likely that although overexpressed in 3 -week sunitinib treated hearts, GLUT4 was not inserted in the plasma membrane and not actively transporting glucose or FDG inside the cardiomyocytes.
Overall our results indicate that, in mice, a 3 -week sunitinib course (the regimen is usually 4 weeks in patients) induces a form of cardiac insulin resistance. This is not totally surprising as sunitinib and TKI are known to affect glucose levels to the point that they have been considered as potential drugs for type 2 diabetes (52). In our study, the addition of macitentan largely improved the metabolic rate of glucose and suppressed the sunitinib-induced insulin resistance and diabetic patterns. Moreover, ETA activation is associated with impaired glucose uptake via the inhibition of the AMPK/Akt signaling pathway of the translocation of GLUT4 in skeletal muscle (53). Here, we demonstrate that the sunitinib-induced reduction in glucose uptake after prolonged treatment is mediated by the ETA receptor through the inhibition of insulin- stimulated AMPK. Other studies have shown that disruption of the endothelin pathway is associated with impaired glucose uptake in skeletal muscle. Shemyakin et al. showed decreased insulin-stimulated Akt phosphorylation by ET- 1 , and infusion of ET- 1 reduced insulin sensitivity in humans and in animals (54,55). In addition, ET-1 blockade by ETA/ETB receptors inhibition increases glucose uptake in patients with insulin resistance (54,55).
Our results suggest that two mechanisms are at work in the cardiotoxic effects of sunitinib: (i) in the first days after initiation of treatment, a metabolic switch towards glycolysis with increased FDG uptake and, (ii) evidenced after at least 3 weeks of treatment, a type of insulin resistance that leads to subnormal FDG uptake. Our present data show that the cardiotoxicity of sunitinib is for a good part related to a deregulation of cardiac metabolism, which in some respect is similar to the one observed in other situations. It has been demonstrated that during ischemia, the heart is not able to use its metabolic reserve (56) and is associated with lactate acidosis leading to contractile dysfunction (57). Similarly, hypertrophic hearts are not able to increase glucose uptake enough to sustain the energy demand (58), and by-products from glycolysis and glycogen utilization are required for proper myocardium relaxation (59). This metabolic dead end is concomitant with major deleterious consequences on three key components of cardiac physiology: (i) mitochondrial homeostasis; (ii); fibrosis and (iii) contractile function. Fibrosis is responsible for impaired myocardial relaxation and we show here that impaired cardiac output was due to a decreased left ventricular volume during the diastole (LVID,d), responsible for diastolic dysfunction (60,61). This has functional consequences with decreased flow velocities, leading to cardiac dysfunction. The therapeutic perspective is to control and, whenever possible, to prevent the car dio toxicity of anticancer drugs. Regarding sunitinib, our results show that the administration of macitentan reverts most, if not all, the effects of sunitinib on cardiac metabolism and prevents impairment of left ventricular function and fibrosis. Macitentan is a mixed ETA/ETB antagonist approved for the treatment of pulmonary arterial hypertension and has no known direct or indirect interaction with sunitinib of other TKI (FDA). Furthermore, non-selective ET receptors antagonism prevents hypertension and renal injury (24) induced by sunitinib. In the future, it will be interesting to test if the use of a selective ETA receptor antagonism produces comparable cardioprotection since ET-1 is known to exert a pro-fibrotic action (62) and hypertrophy (34) via the ETA receptor.
We have shown here that co-administration of macitentan prevents deregulation of myocardial metabolism and cardiac fibrosis and restores the diastolic function impaired by sunitinib. Taken together, these results support the administration of a mixed ETA/ETB inhibitor such as macitentan to protect the myocardium during sunitinib treatment. Our preclinical results call for clinical imaging studies aiming to identify the risk of developing side effects at an early stage of sunitinib treatment, and consequently to administer preventive therapy (22). Because of its clinical importance, further exploration of the capacity of macitentan therapy to protect the heart from sunitinib-induced cardiac dysfunction is worth exploring in a theranostics approach.
sunitinib versus vehicle
Categories Protein name Gene ID p- FC value
Glucogenolysis Phosphoglycerate mutase 1 Pgaml 0.01 -1.28
Glucogenolysis NADP-dependent malic enzyme 3, Me3 0.01 -1.17 mitochondrial
TCA cycle Isobutyryl-CoA dehydrogenase, mitochondrial Acad8 < -1.30
0.001
TCA cycle Pyruvate dehydrogenase (acetyl-transferring) Pdk2 0.08 -1.21 kinase isozyme 2, mitochondrial
TCA cycle Isocitrate dehydrogenase [NAD] subunit Idh3g 0.03 -1.24 gamma 1, mitochondrial
TCA cycle Isocitrate dehydrogenase [NAD] subunit alpha, Idh3a 0.05 -1.14 mitochondrial
TCA cycle Succinate dehydrogenase complex flavoprotein Sdha 0.02 -1.12 subunit A, mitochondrial
AMPK Phosphoenolpyruvate carboxykinase 2, Pck2 <
mitochondrial 0.000
1
AMPK Carnitine O-palmitoyltransferase 1, muscle Cptlb 0.21 -1.19 isoform
Hypertrophy Carnitine O-palmitoyltransferase 1, liver Cptla 0.04 -1.31 isoform
Hypertrophy Phospholipase C beta 3 Plcb3 0.01 -
Hypertrophy Transforming protein hoA RhoA 0.02 -1.59
Hypertrophy Caveolin-1 Cavl 0.04 -1.30
Hypertrophy Telethonin Tcap < 0.01 -1.70
Hypertrophy A-kinase anchor protein 1, mitochondrial Akapl 0.02 -1.45
Mitochondria Heat shock protein beta-1 Hspbl 0.02 -1.23
Mitochondria Heat shock 70 kDa protein 4L Hspa4l 0.01 -1.44
Mitochondria Mitochondrial import receptor subunit TOM70 Tomm70 0.06 -1.27 a
Mitochondria Electron transfer flavoprotein dehydrogenase Etfdh 0,04 -1.34
Mitochondria Sulfite oxidase, mitochondrial Suox 0.02 1.22 Energy NAD-dependent protein deacetylase sirtuin-4 Sirt4
metabolism
Energy PGC-1 and E -induced regulator in muscle Perml 0.09 -1.35 metabolism protein 1
Hypoxia ubiquitin specific peptidase 19 Uspl9 <
0.000
1
Hypoxia Hypoxia up-regulated protein 1 Hyoul 0.06 1.26
Nitric oxide Guanylate cyclase soluble subunit beta-1 Gucylb3 0.03 -1.55
Oxidative stress Glutathione reductase, mitochondrial Gsr 0.02 1.41
Glycometabolism l-acylglycerol-3-phosphate O-acyltransferase 2 Agpat2 < -2.13
0.001
Glycometabolism Dolichyl-diphosphooligosaccharide-non- Ddost 0.02 1.15 catalytic subunit
Glycometabolism CDP-diacylglycerol-inositol 3- Cdipt 0.02 1.63 phosphatidyltransferase
Sodium-dependent phosphate transporter 2 Slc20a2 0.03 -1.28
Sodium/potassium-transporting ATPase Atplbl 0.03 -1.31 subunit beta-1
Flavin adenine dinucleotide synthetase 1 Fladl 0.03 -1.29
Pirin Pir 0.04 -
Table 1. Major changes in protein expression levels of the myocardium after one week of treatment: Results from Label- free protein quantification showing the ratios of protein expression levels in sunitinib versus vehicle. Numbers indicate the fold change (FC) between two groups and the p-value of the two tailed Student t.test. The sign "-" indicates undetected protein in the denominator, group. n=3 for each. AMPK: 5' AMP-activated protein kinase; cdp: cytidine diphospho; ERR: estrogen-related receptor; NAD: nicotinamide adenine dinucleotide; NADP: nicotinamide adenine dinucleotide phosphate; PGC-1 : peroxisome proliferator- activated receptor gamma coactivator 1 -alpha; TCA: tricarboxylic acid. sunitinib sunitinib sunitinib + versus vehicle versus macitentan sunitinib + versus macitentan vehicle
Categories Protein ID p- FC Rvalue FC p- FC value value
Glucogenolysis facilitated glucose Sic 0,009 7,78 0,219 1,57 0,024 4,97 transporter member 4 2a4
Glucogenolysis RAB10, member RAS Ra 0,071 -1,30 0,405 1,07 0,030
oncogene family blO 1,39
Glycogen, IR 1,4-alpha-glucan- Gb 0,038 -1,22 0,010 -1,23 0,908 1,01 branching enzyme 1 el
Glucogenolysis Acyl-CoA dehydrogenase Aca 0,010 -1,18 0,114 -1,11 0,172
family member 9 d9 1,06
Glucogenolysis phosphorylase, glycogen, Pyg 0,046 -1,14 0,213 -1,09 0,424
muscle m 1,05
Glucogenolysis Glucose-6-phosphate Gpi 0,003 1,15 0,276 -1,07 0,003 1,23 isomerase
Glucogenolysis Glucose-6-phosphate 1- G6 0,855 -1,09 0,360 1,98 0,015
dehydrogenase pdx 2,17
Glucogenolysis Phosphofructokinase, Pfk 0,071 -1,20 0,959 1,00 0,071
muscle m 1,20
Glucogenolysis phosphofructokinase, Pfk 0,024 1,26 0,013 -1,42 0,001 1,79 platelet P
Glucogenolysis Phosphoglycerate mutase 1 Pga 0,011 1,48 0,141 1,18 0,057 1,26 ml
Glucogenolysis Glyceraldehyde-3- Ga 0,074 -1,12 0,043 -1,24 0,265 1,10 phosphate dehydrogenase pdh
FA metabolism, CD36 molecule Cd3 0,012 1,36 0,999 1,00 0,014 1,36 IR 6
FA metabolism Acyl-CoA synthetase family Acs 0,038 1,28 0,022 -1,37 0,518 1,08 member 2 £2
TCA cycle, FA Malonyl-CoA Mly 0,024 1,23 0,208 -1,15 0,477 metabolism, IR decarboxylase, cd 1,08 mitochondrial
TCA cycle Isobutyryl-CoA Aca 0,026 -1,21 0,002 -1,20 0,932
dehydrogenase, d8 1,01 mitochondrial
TCA cycle Pyruvate dehydrogenase (El Pdh 0,004 -1,20 0,003 -1,36 0,144 1,13 component) beta b
TCA cycle Pyruvate dehydrogenase (El Pdh 0,041 -1,17 0,009 -1,22 0,491 1,04 component) alpha al
TCA cycle Pyruvate dehydrogenase Pdp 0,008 -1,24 0,047 -1,13 0,106
hosphatase regulatory subunit r 1,10
TCA cycle Dihydrolipoamide S- Dlat 0,003 -1,30 0,012 -1,28 0,814
acetyltransferase 1,01
TCA cycle Succinate dehydrogenase ; Sdh 0,022 -1,18 0,089 -1,14 0,649
flavoprotein subunit a 1,03
TCA cycle Isocitrate dehydrogenase Idh 0,017 -1,42 0,001 -1,31 0,496
[NAD] subunit 3g 1,08
TCA cycle 2-oxoglutarate Ogd 0,074 -1,17 0,041 -1,21 0,446 1,04 dehydrogenase, h
mitochondrial
L-lactate dehydrogenase A Ldh 0,001 1,60 0,958 -1,01 <0,001 1,61 a
Mitochondria, 3-hydroxyisobutyryl-CoA Hib 0,403 -1,06 0,018 -1,18 0,020 1,12 FA hydrolase, mitochondrial ch Mitochondria 3-hydroxyisobutyrate Hib 0,738 1,04 0,036 -1,26 0,005 1,30 dehydrogenase, adh
mitochondrial
Mitochondria Mitochondrial-processing Pm 0,049 -1,17 0,011 -1,36 0,142 1,16 peptidase subunit beta pcb
Mitochondria Mitochondrial import To 0,299 1,16 0,006 -1,46 0,001 1,70 receptor subunit TOM22 mm
22
Mitochondria Translocase of inner Ti 0,015 1,80 0,613 1,09 0,004 1,65 mitochondrial membrane 8 mm
8al
Mitochondria Mitochondrial Rho Rh 0,008 3,11 0,862 1,01 0,010 3,08
GTPase 1 otl
Mitochondria Translocase of outer To 0,001 -1,41 0,118 -1,13 0,010
mitochondrial membrane mm 1,25 70 70a
Mitochondria Mitochondrial import Ti 0,474 1,08 0,418 -1,08 0,010 1,17 membrane translocase mm
subunit 50
Mitochondria Mitochondrial import To 0,024 1,23 0,923 1,01 0,022 1,22 receptor subunit TOM40 mm
homolog 40
Mitochondria Translation factor Gufl, Guf 0,026 -4,87 0,022 -5,32 0,791 1,09 mitochondrial 1
Mitochondria Elongation factor G, Gf 0,011 -1,24 0,021 -1,20 0,422
mitochondrial ml 1,03
Mitochondria Dynamin-like protein, Op 0,099 -1,16 0,069 -1,16 0,994 1,00 mitochondrial al
Mitochondria, Cytochrome c oxidase Cox 0,021 3,65 0,369 1,42 0,113 2,57 IR subunit 6A2 6a2
Mitochondria Cytochrome c oxidase Coa 0,007 - 0,934 1,03 0,002 - assembly factor 3 3
Mitochondria Mitochondrial Mte 0,045 -2,39 0,005 -2,27 0,821
transcription termination rf2 1,05 factor 2
Mitochondria ATP synthase Fl complex Atp 0,249 1,33 0,106 -1,21 0,040 1,62 assembly factor 2 af2
Mitochondria ATP synthase subunit Atp 0,145 1,22 0,277 -1,15 0,006 1,41 beta, mitochondrial 5b
Mitochondria BolA-like protein 3 Bol 0,010 -2,77 0,025 -2,26 0,164
a3 1,22
Mitochondria A-kinase anchor protein 1, Ak 0,185 -1,19 0,057 -1,31 0,471 1,10 mitochondrial apl
Mitochondria Aldehyde dehydrogenase, Aid 0,013 -1,20 0,467 1,05 0,001
mitochondrial h2 1,26
Mitochondria 3-oxoacyl-[acyl-carrier- Oxs 0,890 1,01 0,013 -1,20 0,037 1,22 protein] synthase, m
mitochondrial
Mitochondria Elongation factor 1-delta Eef 0,011 1,22 0,065 1,17 0,529 1,04
Id
Mitochondria NADH dehydrogenase 1 Nd 0,221 1,33 0,840 -1,04 0,020 1,38 repirator beta subcomplex subunit 3 ufb
chain 3
Mitochondria NADH dehydrogenase 1 Nd 0,288 1,22 0,611 -1,08 0,027 1,32 repiratory alpha subcomplex subunit ufa
chain 13 13
Mitochondria NADH dehydrogenase 1 Nd 0,171 1,25 0,915 -1,01 0,006 1,27 repiratory beta subcomplex subunit ufb
chain 10 10 Mitochondria NADH dehydrogenase Nd 0,335 1,13 0,325 -1,12 0,008 1,27 repiratory flavoprotein 2, ufv
chain mitochondrial 2
Mitochondria NADH dehydrogenase 1 Nd 0,010 1,99 0,704 1,05 0,009 1,90 repiratory alpha subcomplex subunit ufa
chain 11 11
Mitochondria NADH dehydrogenase 1 Nd 0,160 1,28 0,876 1,02 0,038 1,25 repiratory alpha subcomplex subunit ufa
chain 9 9
Mitochondria NADH dehydrogenase Nd 0,391 -1,09 0,040 -1,26 0,031 1,16 repiratory iron-sulfur protein 2 ufs
chain 2
Energy Creatine kinase M-type Ck 0,066 -1,24 0,032 -1,25 0,948 1,00 metabolism m
Energy Nucleoside diphosphate Ndk -1,38 0,032 -2,65 0,030 1,92 metabolism kinase 3 3 0,640
Energy NAD-dependent protein Sirt 0,297 -1,16 0,044 -1,47 0,196 1,27 metabolism deacetylase sirtuin-3 3
Energy NAD-dependent protein Sirt 0,142 -1,17 0,033 -1,18 0,867 1,01 metabolism deacylase sirtuin-5 5
Energy NAD-dependent protein Sirt 0,039 -1,63 0,033 -1,72 0,715 1,06 metabolism, deacetylase sirtuin-2 2
Inflammation Macrophage migration Mif 0,797 1,13 <0,001 -1,93 0,040 2,19 inhibitory factor
Inflammation Interferon-inducible Irg 0,038 -3,04 0,275 -4,79 0,617 1,57
GTPase 1 a6
Inflammation Serpin family F member 2 Ser 0,025 1,58 0,915 1,02 0,011 1,55 pinf
2
Inflammation Fibrinogen beta chain Fgb 0,032 1,48 0,361 -1,27 0,058 1,87
Inflammation Mitogen-activated protein Ma 0,011 -1,32 0,291 -1,14 0,107
kinase kinase 1 p2k 1,16
1
Inflammation, Plasma kallikrein Bl Klk 0,033 2,10 0,311 1,30 0,073 1,62 hypertension bl
Inflammation, Kininogenl Kn 0,002 1,72 0,335 -1,13 0,0002 1,94 hypertension gi
Inflammation, Plasminogen Pig 0,008 1,60 0,521 1,09 0,010 1,47 atherosclerosis
Inflammation, Ras homolog family rho 0,010 1,58 0,117 1,23 0,086 1,29 atherosclerosis member A A
Inflammation, Fibronectin Fnl 0,007 1,55 0,252 1,16 0,038 1,33 atherosclerosis
Inflammation, Signal transducer and Stat 0,031 -2,94 0,223 -3,00 0,969 1,02 atherosclerosis, activator of transcription 1 1
IR
Atherosclerosis Intercellular adhesion lea 0,037 2,07 0,273 1,33 0,270 1,56 molecule 1 ml
Atherosclerosis Apolipoprotein A-IV Ap 0,013 1,62 0,212 1,24 0,106 1,31 oa4
Atherosclerosis Glutathione S-transferase Gst 0,026 1,25 1,152 1,15 0,243 1,08 , infarction Mu 1 ml
Thrombosis, Heparin cofactor 2 Ser 0,033 2,68 0,621 1,11 0,028 2,42 pin
dl
Thrombosis, Antithrombin-III Ser 0,014 1,56 0,167 1,21 0,052 1,29 endothelial cell pin
apoptosis cl
Figure imgf000052_0001
Cellular Myosin-XVIIIa My 0,014 -1,26 <0,001 -1,22 0,620 morphology ol8 1,03 a
Cellular Titin Ttn 0,014 -1,37 0,016 -1,15 0,099 morphology 1,20
Cellular Myocardial zonula adherens My 0,001 -1,33 0,004 -1,26 0,286 morphology protein zap 1,05
Cellular Myomesin-2 My 0,019 -1,17 0,045 -1,17 0,981 morphology om 1,00
2
Cellular Actin-related protein 2/3 Arp 0,752 1,04 0,037 1,35 0,003 morphology complex subunit 2 c2 1,30
Cellular Valine-tRNA ligase Var 0,151 -1,13 0,108 1,16 <0,001 morphology s 1,32
Cellular GTP-binding nuclear protein Ran 0,010 1,41 0,009 1,42 0,925 morphology Ran 1,01
Eiidotliclin Phospholipase C Pic 0,009 2,08
downstream b4
signaling
Eiidotliclin Mapkl; Erk2 0,343 1,25 0,103 1,53 0,330 1,22 downstream Afamin Af <0,00 5,15 0,064 1,40 0,001 3,67 signaling m 1
Flavin adenine dinucleotide Fla 0,010 -1,62 0,005 -1,38 0,249 synthetase 1 dl 1,17
Musashi RNA binding Msi 0,022 -1,29 0,011 -1,49 0,298 1,15 protein 2 2
Pre-mRNA-processing Prpf 0,046 -1,35 0,298 -1,22 0,480 factor 19 19 1,10
Adenylosuccinate lyase Ads 0,031 -1,31 0,017 -1,41 0,584 1,08
1
Vinculin Vcl 0,005 1,15 0,002 1,13 0,600 1,02
Table 2. Major changes in protein expression levels of the myocardium after three weeks of treatments: Results from Label-free protein quantification showing the ratios of protein expression levels in sunitinib versus vehicle, sunitinib versus sunitinib+macitentan and sunitinib+macitentan versus vehicle. Numbers indicate the fold change (FC) between two groups and the p-value of the two tailed Student t.test Data are expressed as Fold change (FC). The sign "-" indicates undetected protein in the denominator group. n=6 for each. ATP: Adenosine triphosphate; CD36: cluster of differentiation 36; FA: fatty acid; GTP: Guanosine- 5 '-triphosphate; IR: insulin resitance; K+: potassium; Na+: sodium; NAD: nicotinamide adenine dinucleotide; RAB: Ras-related protein; RNA: Ribonucleic acid; TCA: tricarboxylic acid.
REFERENCES:
Throughout this application, various references describe the state of the art to which this invention pertains. The disclosures of these references are hereby incorporated by reference into the present disclosure. 1. Tolba KA, Deliargyris EN. Cardiotoxicity of cancer therapy. Cancer Invest. 1999;17(6):408-22.
2. Monsuez J- J, Charniot J-C, Vignat N, Artigou J-Y. Cardiac side-effects of cancer chemotherapy. Int J Cardiol. 2010 Sep 24;144(1):3-15.
3. Herrmann J, Yang EH, Iliescu CA, Cilingiroglu M, Charitakis K, Hakeem A, et al. Vascular Toxicities of Cancer Therapies: The Old and the New—An Evolving Avenue. Circulation. 2016 Mar 29;133(13): 1272-89.
4. Gottlieb S. Cancer drug may cause heart failure. BMJ. 2000 Jul 29;321(7256):259.
5. Kounis NG, Koniari I, Hahalis G. Cardio-oncology, Immuno -oncology, Onco- cardiology and Onco-immunology. Int J Cardiol. 2016 Nov 15;223:254-7.
6. Verheul HMW, Pinedo HM. Possible molecular mechanisms involved in the toxicity of angiogenesis inhibition. Nat Rev Cancer. 2007 Jun;7(6):475-85.
7. Kerbel R, Folkman J. Clinical translation of angiogenesis inhibitors. Nat Rev Cancer. 2002 Oct;2(10):727-39.
8. Chu TF, Rupnick MA, Kerkela R, Dallabrida SM, Zurakowski D, Nguyen L, et al. Cardiotoxicity associated with tyrosine kinase inhibitor sunitinib. Lancet Lond Engl. 2007 Dec 15;370(9604):2011-9.
9. Kappers MH, van Esch JHM, Sleijfer S, Danser AHJ, van den Meiracker AH. Cardiovascular and renal toxicity during angiogenesis inhibition: clinical and mechanistic aspects. J Hypertens. 2009 Dec;27(12):2297-309.
10. Kappers MHW, van Esch JHM, Smedts FMM, de Krijger RR, Eechoute K, Mathijssen RHJ, et al. Sunitinib-induced hypothyroidism is due to induction of type 3 deiodinase activity and thyroidal capillary regression. J Clin Endocrinol Metab. 201 1 Oct;96(10):3087-94.
11. Kamba T, McDonald DM. Mechanisms of adverse effects of anti-VEGF therapy for cancer. Br J Cancer. 2007 Jun 18;96(12): 1788-95.
12. Chrisoulidou A, Mandanas S, Margaritidou E, Mathiopoulou L, Boudina M, Georgopoulos K, et al. Treatment compliance and severe adverse events limit the use of tyrosine kinase inhibitors in refractory thyroid cancer. OncoTargets Ther. 2015 Sep 3;8:2435-42.
13. Hartmann JT, Kanz L. Sunitinib and periodic hair depigmentation due to temporary c-KIT inhibition. Arch Dermatol. 2008 Nov;144(l l): 1525-6. 14. Motzer RJ, Hutson TE, Tomczak P, Michaelson MD, Bukowski RM, Rixe O, et al. Sunitinib versus interferon alfa in metastatic renal-cell carcinoma. N Engl J Med. 2007 Jan 11;356(2): 115-24.
15. Demetri GD, van Oosterom AT, Garrett CR, Blackstein ME, Shah MH, Verweij J, et al. Efficacy and safety of sunitinib in patients with advanced gastrointestinal stromal tumour after failure of imatinib: a randomised controlled trial. Lancet Lond Engl. 2006 Oct 14;368(9544): 1329-38.
16. Raymond E, Dahan L, Raoul J-L, Bang Y-J, Borbath I, Lombard-Bohas C, et al. Sunitinib malate for the treatment of pancreatic neuroendocrine tumors. N Engl J Med. 201 1 Feb 10;364(6):501-13.
17. Lankhorst S, Kappers MHW, van Esch JHM, Danser AHJ, van den Meiracker AH. Mechanism of hypertension and proteinuria during angiogenesis inhibition: evolving role of endothelin-1. J Hypertens. 2013 Mar;31(3):444-454.
18. Meiracker AH van den, Danser AHJ, Sleijfer S, Kappers MHW. Re: Hypertension as a Biomarker of Efficacy in Patients With Metastatic Renal Cell Carcinoma
Treated With Sunitinib . J Natl Cancer Inst [Internet] . 2011 Aug 23 [cited 2016 Oct 4] ; Available from: http://jnci.oxfordjournals.org/content/early/2011/08/23/jnci.djr328
19. Chintalgattu V, Rees ML, Culver JC, Goel A, Jiffar T, Zhang J, et al. Coronary microvascular pericytes are the cellular target of sunitinib malate-induced cardiotoxicity. Sci Transl Med. 2013 May 29;5(187): 187ra69.
20. Moslehi J, Minamishima YA, Shi J, Neuberg D, Charytan DM, Padera RF, et al. Loss of hypoxia-inducible factor prolyl hydroxylase activity in cardiomyocytes phenocopies ischemic cardiomyopathy. Circulation. 2010 Sep 7; 122(10): 1004— 16.
21. Kappers MHW, Esch JHM van, Sluiter W, Sleijfer S, Danser AHJ, Meiracker AH van den. Hypertension Induced by the Tyrosine Kinase Inhibitor Sunitinib Is Associated
With Increased Circulating Endothelin-1 Levels. Hypertension. 2010 Oct 1;56(4):675-81.
22. Yang Y, Bu P. Progress on the cardiotoxicity of sunitinib: Prognostic significance, mechanism and protective therapies. Chem Biol Interact. 2016 Sep 25;257: 125- 31.
23. Dhaun N, Webb DJ. Receptor tyrosine kinase inhibition, hypertension, and proteinuria: is endothelin the smoking gun? Hypertens Dallas Tex 1979. 2010 Oct;56(4):575- 7. 24. Lankhorst S, Kappers MHW, van Esch JHM, Smedts FMM, Sleijfer S, Mathijssen RHJ, et al. Treatment of hypertension and renal injury induced by the angiogenesis inhibitor sunitinib: preclinical study. Hypertens Dallas Tex 1979. 2014 Dec;64(6): 1282-9.
25. Dhaun N, Maclntyre IM, Kerr D, Melville V, Johnston NR, Haughie S, et al. Selective endothelin-A receptor antagonism reduces proteinuria, blood pressure, and arterial stiffness in chronic proteinuric kidney disease. Hypertens Dallas Tex 1979. 2011 Apr;57(4):772-9.
26. Kongbundansuk S, Hundley WG. Noninvasive imaging of cardiovascular injury related to the treatment of cancer. JACC Cardiovasc Imaging. 2014 Aug;7(8):824-38.
27. Pizzino F, Vizzari G, Qamar R, Bomzer C, Carerj S, Zito C, et al. Multimodality
Imaging in Cardiooncology. J Oncol. 2015;2015:263950.
28. D'Amore C, Gargiulo P, Paolillo S, Pellegrino AM, Formisano T, Mariniello A, et al. Nuclear imaging in detection and monitoring of cardiotoxicity. World J Radiol. 2014 Jul 28;6(7):486-92.
29. Borde C, Kand P, Basu S. Enhanced myocardial fluorodeoxyglucose uptake following Adriamycin-based therapy: Evidence of early chemotherapeutic cardiotoxicity? World J Radiol. 2012 May 28;4(5):220-3.
30. O'Farrell AC, Evans R, Silvola JMU, Miller IS, Conroy E, Hector S, et al. A Novel Positron Emission Tomography (PET) Approach to Monitor Cardiac Metabolic Pathway Remodeling in Response to Sunitinib Malate. PloS One. 2017;12(l):e0169964.
31. Kolwicz SC, Tian R. Glucose metabolism and cardiac hypertrophy. Cardiovasc Res. 201 1 May 1;90(2): 194-201.
32. Rees ML, Subramaniam J, Li Y, Hamilton DJ, Frazier OH, Taegtmeyer H. A PKM2 signature in the failing heart. Biochem Biophys Res Commun. 2015 Apr 10;459(3):430- 6.
33. Hafizi S, Wharton J, Chester AH, Yacoub MH. Profibrotic effects of endothelin- 1 via the ETA receptor in cultured human cardiac fibroblasts. Cell Physiol Biochem Int J Exp Cell Physiol Biochem Pharmacol. 2004;14(4-6):285-92.
34. Ehmke H, Faulhaber J, Miinter K, Kirchengast M, Wiesner RJ. Chronic ETA receptor blockade attenuates cardiac hypertrophy independently of blood pressure effects in renovascular hypertensive rats. Hypertens Dallas Tex 1979. 1999 Apr;33(4):954-60.
35. Nilsson D, Wackenfors A, Gustafsson L, Edvinsson L, Paulsson P, Ingemansson R, et al. Increased ETA and ETB receptor contraction in the left internal mammary artery from patients with hypertension. J Hum Hypertens. 2007 Dec 13;22(3):226-9. 36. Hue L, Beauloye C, Marsin A-S, Bertrand L, Horman S, Rider MH. Insulin and Ischemia Stimulate Glycolysis by Acting on the Same Targets Through Different and Opposing Signaling Pathways. J Mol Cell Cardiol. 2002 Sep;34(9): 1091-7.
37. Kreissl MC, Stout DB, Wong K-P, Wu H-M, Caglayan E, Ladno W, et al. Influence of dietary state and insulin on myocardial, skeletal muscle and brain [ 18 F]- fluorodeoxyglucose kinetics in mice. EXNMMI Res. 2011 Jul 6;1(1):8.
38. Vitale GD, deKemp RA, Ruddy TD, Williams K, Beanlands RS. Myocardial glucose utilization and optimization of (18)F-FDG PET imaging in patients with non- insulin- dependent diabetes mellitus, coronary artery disease, and left ventricular dysfunction. J Nucl Med Off Publ Soc Nucl Med. 2001 Dec;42(12): 1730-6.
39. Dilsizian V. 18F-FDG Uptake as a Surrogate Marker for Antecedent Ischemia. J Nucl Med. 2008 Dec 1 ;49(12): 1909-11.
40. Haas F, Jennen L, Heinzmann U, Augustin N, Wottke M, Schwaiger M, et al. Ischemically compromised myocardium displays different time-courses of functional recovery: Correlation with morphological alterations? ResearchGate. 2001 Sep l;20(2):290-8.
41. Schinkel AFL, Poldermans D, Elhendy A, Bax JJ. Assessment of Myocardial Viability in Patients with Heart Failure. J Nucl Med. 2007 Jul 1;48(7): 1135-46.
42. Rahimtoola SH, Dilsizian V, Kramer CM, Marwick TH, Vanoverschelde J-LJ. Chronic Ischemic Left Ventricular Dysfunction. JACC Cardiovasc Imaging. 2008 Jul l;l(4):536-55.
43. Piper HM. Pathophysiology of Severe Ischemic Myocardial Injury. Kluwer Academic Publishers; 1990. 448 p.
44. Kumar RMR, Arlt MJ, Kuzmanov A, Born W, Fuchs B. Sunitinib malate (SU- 11248) reduces tumour burden and lung metastasis in an intratibial human xenograft osteosarcoma mouse model. Am J Cancer Res. 2015 Jun 15;5(7):2156-68.
45. Goodwin GW, Taylor CS, Taegtmeyer H. Regulation of energy metabolism of the heart during acute increase in heart work. J Biol Chem. 1998 Nov 6;273(45):29530-9.
46. Zhong M, Alonso CE, Taegtmeyer H, Kundu BK. Quantitative PET imaging detects early metabolic remodeling in a mouse model of pressure-overload left ventricular hypertrophy in vivo. J Nucl Med Off Publ Soc Nucl Med. 2013 Apr;54(4):609-15.
47. Gropler RJ, Peterson LR. Imaging of Myocardial Metabolism. ResearchGate. 2010 Dec 31;641-56. 48. Toubert M-E, Vercellino L, Faugeron I, Lussato D, Hindie E, Bousquet G. Fatal heart failure after a 26-month combination of tyrosine kinase inhibitors in a papillary thyroid cancer. Thyroid Off J Am Thyroid Assoc. 2011 Apr;21(4):451-4.
49. Kobayashi K, Neely JR. Effects of ischemia and reperfusion on pyruvate dehydrogenase activity in isolated rat hearts. J Mol Cell Cardiol. 1983 Jun;15(6):359-67.
50. Voipio-Pulkki LM, Nuutila P, Knuuti MJ, Ruotsalainen U, Haaparanta M, Teras M, et al. Heart and skeletal muscle glucose disposal in type 2 diabetic patients as determined by positron emission tomography. ResearchGate. 1994 Jan l;34(12):2064-7.
51. Stanley WC, Hall JL, Hacker TA, Hernandez LA, Whitesell LF. Decreased myocardial glucose uptake during ischemia in diabetic swine. Metabolism. 1997
Feb;46(2): 168-72.
52. Malek R, Davis SN. Tyrosine kinase inhibitors under investigation for the treatment of type II diabetes. Expert Opin Investig Drugs. 2016;25(3):287-96.
53. Horinouchi T, Hoshi A, Harada T, Higa T, Karki S, Terada K, et al. Endothelin- 1 suppresses insulin- stimulated Akt phosphorylation and glucose uptake via GPCR kinase 2 in skeletal muscle cells. Br J Pharmacol. 2016 Mar;173(6): 1018-32.
54. Shemyakin A, Salehzadeh F, Bohm F, Al-Khalili L, Gonon A, Wagner H, et al. Regulation of Glucose Uptake by Endothelin-1 in Human Skeletal Muscle in Vivo and in Vitro. J Clin Endocrinol Metab. 2010 May l;95(5):2359-66.
55. Shemyakin A, Salehzadeh F, Duque-Guimaraes DE, Bohm F, Rullman E,
Gustafsson T, et al. Endothelin-1 Reduces Glucose Uptake in Human Skeletal Muscle In Vivo and In Vitro. Diabetes. 2011 Aug l;60(8):2061-7.
56. Nagata D, Hirata Y. The role of AMP-activated protein kinase in the cardiovascular system. Hypertens Res. 2009 Nov 13;33(l):22-8.
57. Orchard CH. The role of the sarcoplasmic reticulum in the response of ferret and rat heart muscle to acidosis. J Physiol. 1987 Mar;384:431-49.
58. Kassiotis C, Rajabi M, Taegtmeyer H. Metabolic Reserve of the Heart: The
Forgotten Link Between Contraction and Coronary Flow. Prog Cardiovasc Dis. 2008;51(1):74-
88.
59. Kusuoka H, Marban E. Mechanism of the diastolic dysfunction induced by glycolytic inhibition. Does adenosine triphosphate derived from glycolysis play a favored role in cellular Ca2+ homeostasis in ferret myocardium? J Clin Invest. 1994 Mar;93(3): 1216-23.
60. Burlew BS, Weber KT. Cardiac fibrosis as a cause of diastolic dysfunction. Herz. 2002 Mar;27(2):92-8. 61. Mandinov L, Eberli FR, Seiler C, Hess OM. Diastolic heart failure. Cardiovasc Res. 2000 Mar 1;45(4):813-25.
62. Clozel M, Salloukh H. Role of endothelin in fibrosis and anti-fibrotic potential of bosentan. Ann Med. 2005;37(1):2-12.
63. Chien M-H, Lee L-M, Hsiao M, Wei L-H, Chen C-H, Lai T-C, et al. Inhibition of Metastatic Potential in Breast Carcinoma In Vivo and In Vitro through Targeting VEGFRs and FGFRs. Evid-Based Complement Altern Med ECAM [Internet]. 2013 [cited 2017 Mar 10];2013. Available from: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3686112/
64. Mendel DB, Laird AD, Xin X, Louie SG, Christensen JG, Li G, et al. In vivo antitumor activity of SU11248, a novel tyrosine kinase inhibitor targeting vascular endothelial growth factor and platelet-derived growth factor receptors: determination of a pharmacokinetic/pharmacodynamic relationship. Clin Cancer Res Off J Am Assoc Cancer Res. 2003 Jan;9(l):327-37.
65. Thackeray JT, Bankstahl JP, Bengel FM. Impact of Image-Derived Input Function and Fit Time Intervals on Patlak Quantification of Myocardial Glucose Uptake in
Mice. J Nucl Med Off Publ Soc Nucl Med. 2015 Oct;56(10): 1615-21.
66. Wisniewski JR, Zougman A, Nagaraj N, Mann M. Universal sample preparation method for proteome analysis. Nat Methods. 2009 May;6(5):359-62.
67. Rappsilber J, Mann M, Ishihama Y. Protocol for micro-purification, enrichment, pre-fractionation and storage of peptides for proteomics using StageTips. Nat Protoc.
2007;2(8): 1896-906.
68. Wisniewski JR, Dus K, Mann M. Proteomic workflow for analysis of archival formalin- fixed and paraffin-embedded clinical samples to a depth of 10 000 proteins. Proteomics Clin Appl. 2013 Apr;7(3-4):225-33.
69. Cox J, Matic I, Hilger M, Nagaraj N, Selbach M, Olsen JV, et al. A practical guide to the MaxQuant computational platform for SILAC-based quantitative proteomics. Nat Protoc. 2009;4(5):698-705.
70. Wisniewski JR, Hein MY, Cox J, Mann M. A "proteomic ruler" for protein copy number and concentration estimation without spike-in standards. Mol Cell Proteomics MCP. 2014 Dec;13(12):3497-506.
71. Previlon M, Gall ML, Chafey P, Federeci C, Pezet M, Clary G, et al. Comparative differential proteomic profiles of nonfailing and failing hearts after in vivo thoracic aortic constriction in mice overexpressing FKBP12.6. Physiol Rep [Internet]. 2013 Aug [cited 2017 Jan 20]; 1(3).

Claims

CLAIMS:
1. A compound selected from the group consisting of endothelin receptor antagonist and inhibitor of endothelin receptor expression for use in the treatment of cardiovascular toxicity induced by anti-cancer compound in a subject in need thereof.
2. The compound for use according to claim 1 , wherein said anti-cancer compound is anti- angiogenic compound.
3. The compound for use according to claim 2, wherein said anti-angiogenic compound is a tyrosine kinase receptor (TK ) inhibitor such as sunitinib.
4. The compound for use according to any of claims 1 to 3, wherein said endothelin receptor antagonist is selected from the group consisting of a small organic molecule, an oligonucleotide, a polypeptide, an aptamer, an antibody or an intra-antibody.
5. The compound for use according to any of claims 1 to 3, wherein said endothelin receptor antagonist is selected from the group consisting of dual ETA and ETB receptor antagonist (ETA/ETB antagonist), selective ETA receptor antagonist or selective ETB receptor antagonist.
6. The compound for use according to any of claims 1 to 3, wherein said endothelin receptor antagonist is selected from the group consisting of macitentan, bosentan, darusentan, sitaxsentan, tezosentan, ambrisentan, atrasentan, avosentan, clazosentan, zibotentan, edonentan, A-182086, A-192621, ABT-627, BMS193884, BQ123, BQ-788, CI1020, FR-139317, S-0139, CPU0213, J-104132, SB-209670, TA-0201, TAK-44, TBC3711, YM-598, ZD-1611, ZD4054.
7. A method for treating cardiovascular toxicity induced by anti-cancer compound in a subject in need thereof, comprising the step of administering to said subject a compound selected from the group consisting of endothelin receptor antagonist and inhibitor of endothelin receptor expression.
PCT/IB2017/000566 2017-04-05 2017-04-05 Methods and pharmaceutical compositions for treating cardiovascular toxicity induced by anti-cancer therapy Ceased WO2018185516A1 (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
PCT/IB2017/000566 WO2018185516A1 (en) 2017-04-05 2017-04-05 Methods and pharmaceutical compositions for treating cardiovascular toxicity induced by anti-cancer therapy

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/IB2017/000566 WO2018185516A1 (en) 2017-04-05 2017-04-05 Methods and pharmaceutical compositions for treating cardiovascular toxicity induced by anti-cancer therapy

Publications (1)

Publication Number Publication Date
WO2018185516A1 true WO2018185516A1 (en) 2018-10-11

Family

ID=58745279

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/IB2017/000566 Ceased WO2018185516A1 (en) 2017-04-05 2017-04-05 Methods and pharmaceutical compositions for treating cardiovascular toxicity induced by anti-cancer therapy

Country Status (1)

Country Link
WO (1) WO2018185516A1 (en)

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113209096A (en) * 2021-05-17 2021-08-06 武汉大学 Application of pexidinib in preparation of medicines for preventing, relieving and/or treating myocardial infarction and related diseases thereof
WO2021158663A1 (en) * 2020-02-06 2021-08-12 Perfuse Therapeutics, Inc. Compositions for treatment of ocular diseases
WO2022232586A1 (en) * 2021-04-30 2022-11-03 Perfuse Therapeutics, Inc. Treatment of ocular diseases using endothelin receptor antagonists
US11738007B2 (en) 2019-10-30 2023-08-29 Perfuse Therapeutics, Inc. Treatment of glaucoma using endothelin receptor antagonists
US11786510B2 (en) 2021-04-30 2023-10-17 Perfuse Therapeutics, Inc. Pharmaceutical compositions and intravitreal drug delivery systems for the treatment of ocular diseases

Citations (53)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4816567A (en) 1983-04-08 1989-03-28 Genentech, Inc. Recombinant immunoglobin preparations
WO1990007861A1 (en) 1988-12-28 1990-07-26 Protein Design Labs, Inc. CHIMERIC IMMUNOGLOBULINS SPECIFIC FOR p55 TAC PROTEIN OF THE IL-2 RECEPTOR
US5225539A (en) 1986-03-27 1993-07-06 Medical Research Council Recombinant altered antibodies and methods of making altered antibodies
US5229275A (en) 1990-04-26 1993-07-20 Akzo N.V. In-vitro method for producing antigen-specific human monoclonal antibodies
US5292740A (en) 1991-06-13 1994-03-08 Hoffmann-La Roche Inc. Sulfonamides
US5545806A (en) 1990-08-29 1996-08-13 Genpharm International, Inc. Ransgenic non-human animals for producing heterologous antibodies
US5545807A (en) 1988-10-12 1996-08-13 The Babraham Institute Production of antibodies from transgenic animals
US5565332A (en) 1991-09-23 1996-10-15 Medical Research Council Production of chimeric antibodies - a combinatorial approach
US5567610A (en) 1986-09-04 1996-10-22 Bioinvent International Ab Method of producing human monoclonal antibodies and kit therefor
US5573905A (en) 1992-03-30 1996-11-12 The Scripps Research Institute Encoded combinatorial chemical libraries
US5585089A (en) 1988-12-28 1996-12-17 Protein Design Labs, Inc. Humanized immunoglobulins
US5591669A (en) 1988-12-05 1997-01-07 Genpharm International, Inc. Transgenic mice depleted in a mature lymphocytic cell-type
US5598369A (en) 1994-06-28 1997-01-28 Advanced Micro Devices, Inc. Flash EEPROM array with floating substrate erase operation
US5618829A (en) 1993-01-28 1997-04-08 Mitsubishi Chemical Corporation Tyrosine kinase inhibitors and benzoylacrylamide derivatives
US5639757A (en) 1995-05-23 1997-06-17 Pfizer Inc. 4-aminopyrrolo[2,3-d]pyrimidines as tyrosine kinase inhibitors
US5728868A (en) 1993-07-15 1998-03-17 Cancer Research Campaign Technology Limited Prodrugs of protein tyrosine kinase inhibitors
US5800988A (en) 1992-08-21 1998-09-01 Vrije Universiteit Brussel Immunoglobulins devoid of light chains
US5804396A (en) 1994-10-12 1998-09-08 Sugen, Inc. Assay for agents active in proliferative disorders
US5859205A (en) 1989-12-21 1999-01-12 Celltech Limited Humanised antibodies
US5883254A (en) 1996-11-08 1999-03-16 Hoffmann-La Roche Inc. Process for making pyrimidine derivatives
WO1999032619A1 (en) 1997-12-23 1999-07-01 The Carnegie Institution Of Washington Genetic inhibition by double-stranded rna
US5981732A (en) 1998-12-04 1999-11-09 Isis Pharmaceuticals Inc. Antisense modulation of G-alpha-13 expression
US6046321A (en) 1999-04-09 2000-04-04 Isis Pharmaceuticals Inc. Antisense modulation of G-alpha-i1 expression
US6100254A (en) 1997-10-10 2000-08-08 Board Of Regents, The University Of Texas System Inhibitors of protein tyrosine kinases
US6107091A (en) 1998-12-03 2000-08-22 Isis Pharmaceuticals Inc. Antisense inhibition of G-alpha-16 expression
US6127374A (en) 1997-07-29 2000-10-03 Warner-Lambert Company Irreversible inhibitors of tyrosine kinases
US6150584A (en) 1990-01-12 2000-11-21 Abgenix, Inc. Human antibodies derived from immunized xenomice
WO2001036646A1 (en) 1999-11-19 2001-05-25 Cancer Research Ventures Limited Inhibiting gene expression with dsrna
US6245759B1 (en) 1999-03-11 2001-06-12 Merck & Co., Inc. Tyrosine kinase inhibitors
WO2001068836A2 (en) 2000-03-16 2001-09-20 Genetica, Inc. Methods and compositions for rna interference
US6306874B1 (en) 1999-10-19 2001-10-23 Merck & Co., Inc. Tyrosine kinase inhibitors
US6313138B1 (en) 2000-02-25 2001-11-06 Merck & Co., Inc. Tyrosine kinase inhibitors
US6316444B1 (en) 1999-06-30 2001-11-13 Merck & Co., Inc. SRC kinase inhibitor compounds
US6329380B1 (en) 1999-06-30 2001-12-11 Merck & Co., Inc. SRC kinase inhibitor compounds
US6344459B1 (en) 1996-04-12 2002-02-05 Warner-Lambert Company Irreversible inhibitors of tyrosine kinases
US6365354B1 (en) 2000-07-31 2002-04-02 Isis Pharmaceuticals, Inc. Antisense modulation of lysophospholipase I expression
US6410323B1 (en) 1999-08-31 2002-06-25 Isis Pharmaceuticals, Inc. Antisense modulation of human Rho family gene expression
US6420382B2 (en) 2000-02-25 2002-07-16 Merck & Co., Inc. Tyrosine kinase inhibitors
US6479512B1 (en) 1999-10-19 2002-11-12 Merck & Co., Inc. Tyrosine kinase inhibitors
US6498165B1 (en) 1999-06-30 2002-12-24 Merck & Co., Inc. Src kinase inhibitor compounds
US6566131B1 (en) 2000-10-04 2003-05-20 Isis Pharmaceuticals, Inc. Antisense modulation of Smad6 expression
US6566135B1 (en) 2000-10-04 2003-05-20 Isis Pharmaceuticals, Inc. Antisense modulation of caspase 6 expression
US6573099B2 (en) 1998-03-20 2003-06-03 Benitec Australia, Ltd. Genetic constructs for delaying or repressing the expression of a target gene
US6586424B2 (en) 1999-09-10 2003-07-01 Merck & Co., Inc. Tyrosine kinase inhibitors
US6740665B1 (en) 1999-02-10 2004-05-25 Ramachandran Murali Tyrosine kinase inhibitors and methods of using the same
US6765087B1 (en) 1992-08-21 2004-07-20 Vrije Universiteit Brussel Immunoglobulins devoid of light chains
US6794393B1 (en) 1999-10-19 2004-09-21 Merck & Co., Inc. Tyrosine kinase inhibitors
US6838254B1 (en) 1993-04-29 2005-01-04 Conopco, Inc. Production of antibodies or (functionalized) fragments thereof derived from heavy chain immunoglobulins of camelidae
US6875767B2 (en) 2001-06-22 2005-04-05 Merck & Co., Inc. (5-cyano-2-thiazolyl)amino-4-pyridine tyrosine kinase inhibitors
US6927293B2 (en) 2001-08-30 2005-08-09 Merck & Co., Inc. Tyrosine kinase inhibitors
US6958340B2 (en) 2001-08-01 2005-10-25 Merck & Co., Inc. Tyrosine kinase inhibitors
US7109304B2 (en) 2003-07-31 2006-09-19 Immunomedics, Inc. Humanized anti-CD19 antibodies
US20070254295A1 (en) 2006-03-17 2007-11-01 Prometheus Laboratories Inc. Methods of predicting and monitoring tyrosine kinase inhibitor therapy

Patent Citations (61)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4816567A (en) 1983-04-08 1989-03-28 Genentech, Inc. Recombinant immunoglobin preparations
US5225539A (en) 1986-03-27 1993-07-06 Medical Research Council Recombinant altered antibodies and methods of making altered antibodies
US5567610A (en) 1986-09-04 1996-10-22 Bioinvent International Ab Method of producing human monoclonal antibodies and kit therefor
US5545807A (en) 1988-10-12 1996-08-13 The Babraham Institute Production of antibodies from transgenic animals
US5591669A (en) 1988-12-05 1997-01-07 Genpharm International, Inc. Transgenic mice depleted in a mature lymphocytic cell-type
US5585089A (en) 1988-12-28 1996-12-17 Protein Design Labs, Inc. Humanized immunoglobulins
WO1990007861A1 (en) 1988-12-28 1990-07-26 Protein Design Labs, Inc. CHIMERIC IMMUNOGLOBULINS SPECIFIC FOR p55 TAC PROTEIN OF THE IL-2 RECEPTOR
US5693761A (en) 1988-12-28 1997-12-02 Protein Design Labs, Inc. Polynucleotides encoding improved humanized immunoglobulins
US5693762A (en) 1988-12-28 1997-12-02 Protein Design Labs, Inc. Humanized immunoglobulins
US5859205A (en) 1989-12-21 1999-01-12 Celltech Limited Humanised antibodies
US6150584A (en) 1990-01-12 2000-11-21 Abgenix, Inc. Human antibodies derived from immunized xenomice
US5229275A (en) 1990-04-26 1993-07-20 Akzo N.V. In-vitro method for producing antigen-specific human monoclonal antibodies
US5545806A (en) 1990-08-29 1996-08-13 Genpharm International, Inc. Ransgenic non-human animals for producing heterologous antibodies
US5292740A (en) 1991-06-13 1994-03-08 Hoffmann-La Roche Inc. Sulfonamides
US5565332A (en) 1991-09-23 1996-10-15 Medical Research Council Production of chimeric antibodies - a combinatorial approach
US5573905A (en) 1992-03-30 1996-11-12 The Scripps Research Institute Encoded combinatorial chemical libraries
US5874541A (en) 1992-08-21 1999-02-23 Vrije Universiteit Immunoglobulins devoid of light chains
US5800988A (en) 1992-08-21 1998-09-01 Vrije Universiteit Brussel Immunoglobulins devoid of light chains
US6765087B1 (en) 1992-08-21 2004-07-20 Vrije Universiteit Brussel Immunoglobulins devoid of light chains
US6015695A (en) 1992-08-21 2000-01-18 Vrije Universiteit Brussel Immunoglobulins devoid of light chains
US5618829A (en) 1993-01-28 1997-04-08 Mitsubishi Chemical Corporation Tyrosine kinase inhibitors and benzoylacrylamide derivatives
US6838254B1 (en) 1993-04-29 2005-01-04 Conopco, Inc. Production of antibodies or (functionalized) fragments thereof derived from heavy chain immunoglobulins of camelidae
US5728868A (en) 1993-07-15 1998-03-17 Cancer Research Campaign Technology Limited Prodrugs of protein tyrosine kinase inhibitors
US5598369A (en) 1994-06-28 1997-01-28 Advanced Micro Devices, Inc. Flash EEPROM array with floating substrate erase operation
US5804396A (en) 1994-10-12 1998-09-08 Sugen, Inc. Assay for agents active in proliferative disorders
US5639757A (en) 1995-05-23 1997-06-17 Pfizer Inc. 4-aminopyrrolo[2,3-d]pyrimidines as tyrosine kinase inhibitors
US6344459B1 (en) 1996-04-12 2002-02-05 Warner-Lambert Company Irreversible inhibitors of tyrosine kinases
US5883254A (en) 1996-11-08 1999-03-16 Hoffmann-La Roche Inc. Process for making pyrimidine derivatives
US6127374A (en) 1997-07-29 2000-10-03 Warner-Lambert Company Irreversible inhibitors of tyrosine kinases
US6562818B1 (en) 1997-07-29 2003-05-13 Warner-Lambert Company Irreversible inhibitors of tyrosine kinases
US6100254A (en) 1997-10-10 2000-08-08 Board Of Regents, The University Of Texas System Inhibitors of protein tyrosine kinases
US6506559B1 (en) 1997-12-23 2003-01-14 Carnegie Institute Of Washington Genetic inhibition by double-stranded RNA
WO1999032619A1 (en) 1997-12-23 1999-07-01 The Carnegie Institution Of Washington Genetic inhibition by double-stranded rna
US6573099B2 (en) 1998-03-20 2003-06-03 Benitec Australia, Ltd. Genetic constructs for delaying or repressing the expression of a target gene
US6107091A (en) 1998-12-03 2000-08-22 Isis Pharmaceuticals Inc. Antisense inhibition of G-alpha-16 expression
US5981732A (en) 1998-12-04 1999-11-09 Isis Pharmaceuticals Inc. Antisense modulation of G-alpha-13 expression
US6740665B1 (en) 1999-02-10 2004-05-25 Ramachandran Murali Tyrosine kinase inhibitors and methods of using the same
US6544988B1 (en) 1999-03-11 2003-04-08 Merck & Co., Inc. Tyrosine kinase inhibitors
US6245759B1 (en) 1999-03-11 2001-06-12 Merck & Co., Inc. Tyrosine kinase inhibitors
US6046321A (en) 1999-04-09 2000-04-04 Isis Pharmaceuticals Inc. Antisense modulation of G-alpha-i1 expression
US6498165B1 (en) 1999-06-30 2002-12-24 Merck & Co., Inc. Src kinase inhibitor compounds
US6329380B1 (en) 1999-06-30 2001-12-11 Merck & Co., Inc. SRC kinase inhibitor compounds
US6316444B1 (en) 1999-06-30 2001-11-13 Merck & Co., Inc. SRC kinase inhibitor compounds
US6410323B1 (en) 1999-08-31 2002-06-25 Isis Pharmaceuticals, Inc. Antisense modulation of human Rho family gene expression
US6586424B2 (en) 1999-09-10 2003-07-01 Merck & Co., Inc. Tyrosine kinase inhibitors
US6586423B2 (en) 1999-09-10 2003-07-01 Merck & Co., Inc. Tyrosine kinase inhibitors
US6479512B1 (en) 1999-10-19 2002-11-12 Merck & Co., Inc. Tyrosine kinase inhibitors
US6794393B1 (en) 1999-10-19 2004-09-21 Merck & Co., Inc. Tyrosine kinase inhibitors
US6306874B1 (en) 1999-10-19 2001-10-23 Merck & Co., Inc. Tyrosine kinase inhibitors
WO2001036646A1 (en) 1999-11-19 2001-05-25 Cancer Research Ventures Limited Inhibiting gene expression with dsrna
US6313138B1 (en) 2000-02-25 2001-11-06 Merck & Co., Inc. Tyrosine kinase inhibitors
US6420382B2 (en) 2000-02-25 2002-07-16 Merck & Co., Inc. Tyrosine kinase inhibitors
WO2001068836A2 (en) 2000-03-16 2001-09-20 Genetica, Inc. Methods and compositions for rna interference
US6365354B1 (en) 2000-07-31 2002-04-02 Isis Pharmaceuticals, Inc. Antisense modulation of lysophospholipase I expression
US6566135B1 (en) 2000-10-04 2003-05-20 Isis Pharmaceuticals, Inc. Antisense modulation of caspase 6 expression
US6566131B1 (en) 2000-10-04 2003-05-20 Isis Pharmaceuticals, Inc. Antisense modulation of Smad6 expression
US6875767B2 (en) 2001-06-22 2005-04-05 Merck & Co., Inc. (5-cyano-2-thiazolyl)amino-4-pyridine tyrosine kinase inhibitors
US6958340B2 (en) 2001-08-01 2005-10-25 Merck & Co., Inc. Tyrosine kinase inhibitors
US6927293B2 (en) 2001-08-30 2005-08-09 Merck & Co., Inc. Tyrosine kinase inhibitors
US7109304B2 (en) 2003-07-31 2006-09-19 Immunomedics, Inc. Humanized anti-CD19 antibodies
US20070254295A1 (en) 2006-03-17 2007-11-01 Prometheus Laboratories Inc. Methods of predicting and monitoring tyrosine kinase inhibitor therapy

Non-Patent Citations (88)

* Cited by examiner, † Cited by third party
Title
"A Laboratory Manual", 1990, W.H. FREEMAN C.O.
AGNEW CHEM INTL. ED. ENGL., vol. 33, 1994, pages 183 - 186
BORDE C; KAND P; BASU S: "Enhanced myocardial fluorodeoxyglucose uptake following Adriamycin-based therapy: Evidence of early chemotherapeutic cardiotoxicity?", WORLD J RADIOL., vol. 4, no. 5, 28 May 2012 (2012-05-28), pages 220 - 3
BURLEW BS; WEBER KT: "Cardiac fibrosis as a cause of diastolic dysfunction", HERZ, vol. 27, no. 2, March 2002 (2002-03-01), pages 92 - 8
CHIEN M-H; LEE L-M; HSIAO M; WEI L-H; CHEN C-H; LAI T-C ET AL.: "Inhibition of Metastatic Potential in Breast Carcinoma In Vivo and In Vitro through Targeting VEGFRs and FGFRs", EVID-BASED COMPLEMENT ALTERN MED ECAM [INTERNET, 2013, Retrieved from the Internet <URL:http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3686112>
CHINTALGATTU V; REES ML; CULVER JC; GOEL A; JIFFAR T; ZHANG J ET AL.: "Coronary microvascular pericytes are the cellular target of sunitinib malate-induced cardiotoxicity", SCI TRANSL MED., vol. 5, no. 187, 29 May 2013 (2013-05-29), pages 187RA69
CHRISOULIDOU A; MANDANAS S; MARGARITIDOU E; MATHIOPOULOU L; BOUDINA M; GEORGOPOULOS K ET AL.: "Treatment compliance and severe adverse events limit the use of tyrosine kinase inhibitors in refractory thyroid cancer", ONCOTARGETS THER, vol. 8, 3 September 2015 (2015-09-03), pages 2435 - 42
CHU TF; RUPNICK MA; KERKELA R; DALLABRIDA SM; ZURAKOWSKI D; NGUYEN L ET AL.: "Cardiotoxicity associated with tyrosine kinase inhibitor sunitinib", LANCET LOND ENGL, vol. 370, no. 9604, 15 December 2007 (2007-12-15), pages 2011 - 9, XP022388886, DOI: doi:10.1016/S0140-6736(07)61865-0
CLARK, W. R.: "The Experimental Foundations of Modern Immunology", 1986, WILEY & SONS, INC.
CLOZEL M; SALLOUKH H: "Role of endothelin in fibrosis and anti-fibrotic potential of bosentan", ANN MED., vol. 37, no. 1, 2005, pages 2 - 12, XP008083012
CODING: "Biochemistry and Immunology", 1996, ACADEMIC PRESS, article "Monoclonal Antibodies: Principles and Practice: Production and Application of Monoclonal Antibodies in Cell Biology"
COX J; MATIC I; HILGER M; NAGARAJ N; SELBACH M; OLSEN JV ET AL.: "A practical guide to the MaxQuant computational platform for SILAC-based quantitative proteomics", NAT PROTOC., vol. 4, no. 5, 2009, pages 698 - 705
D'AMORE C; GARGIULO P; PAOLILLO S; PELLEGRINO AM; FORMISANO T; MARINIELLO A ET AL.: "Nuclear imaging in detection and monitoring of cardiotoxicity", WORLD J RADIOL., vol. 6, no. 7, 28 July 2014 (2014-07-28), pages 486 - 92
DE BENEDETTI ET AL., J IMMUNOL, vol. 166, 2001, pages 4334 - 4340
DEMETRI GD; VAN OOSTEROM AT; GARRETT CR; BLACKSTEIN ME; SHAH MH; VERWEIJ J ET AL.: "Efficacy and safety of sunitinib in patients with advanced gastrointestinal stromal tumour after failure of imatinib: a randomised controlled trial", LANCET LOND ENGL., vol. 368, no. 9544, 14 October 2006 (2006-10-14), pages 1329 - 38, XP025093067, DOI: doi:10.1016/S0140-6736(06)69446-4
DHAUN N; MACLNTYRE IM; KERR D; MELVILLE V; JOHNSTON NR; HAUGHIE S ET AL.: "Selective endothelin-A receptor antagonism reduces proteinuria, blood pressure, and arterial stiffness in chronic proteinuric kidney disease", HYPERTENS DALLAS TEX 1979, vol. 57, no. 4, April 2011 (2011-04-01), pages 772 - 9
DHAUN N; WEBB DJ: "Receptor tyrosine kinase inhibition, hypertension, and proteinuria: is endothelin the smoking gun?", HYPERTENS DALLAS TEX 1979, vol. 56, no. 4, October 2010 (2010-10-01), pages 575 - 7
DILSIZIAN V.: "18F-FDG Uptake as a Surrogate Marker for Antecedent Ischemia", J NUCL MED., vol. 49, no. 12, 1 December 2008 (2008-12-01), pages 1909 - 11
EHMKE H; FAULHABER J; MUNTER K; KIRCHENGAST M; WIESNER RJ: "Chronic ETA receptor blockade attenuates cardiac hypertrophy independently of blood pressure effects in renovascular hypertensive rats", HYPERTENS DALLAS TEX 1979, vol. 33, no. 4, April 1999 (1999-04-01), pages 954 - 60
GOODWIN GW; TAYLOR CS; TAEGTMEYER H: "Regulation of energy metabolism of the heart during acute increase in heart work", J BIOL CHEM., vol. 273, no. 45, 6 November 1998 (1998-11-06), pages 29530 - 9
GOTTLIEB S: "Cancer drug may cause heart failure", BMJ, vol. 321, no. 7256, 29 July 2000 (2000-07-29), pages 259
GROPLER RJ; PETERSON LR: "Imaging of Myocardial Metabolism", RESEARCHGATE, 31 December 2010 (2010-12-31), pages 641 - 56
HAAS F; JENNEN L; HEINZMANN U; AUGUSTIN N; WOTTKE M; SCHWAIGER M ET AL.: "Ischemically compromised myocardium displays different time-courses of functional recovery: Correlation with morphological alterations?", RESEARCHGATE, vol. 20, no. 2, 1 September 2001 (2001-09-01), pages 290 - 8
HAFIZI S; WHARTON J; CHESTER AH; YACOUB MH: "Profibrotic effects of endothelin-1 via the ETA receptor in cultured human cardiac fibroblasts", CELL PHYSIOL BIOCHEM INT J EXP CELL PHYSIOL BIOCHEM PHARMACOL., vol. 14, no. 4-6, 2004, pages 285 - 92
HARTMANN JT; KANZ L: "Sunitinib and periodic hair depigmentation due to temporary c-KIT inhibition", ARCH DERMATOL, vol. 144, no. 11, November 2008 (2008-11-01), pages 1525 - 6
HERRMANN J; YANG EH; ILIESCU CA; CILINGIROGLU M; CHARITAKIS K; HAKEEM A ET AL.: "Vascular Toxicities of Cancer Therapies: The Old and the New--An Evolving Avenue", CIRCULATION, vol. 133, no. 13, 29 March 2016 (2016-03-29), pages 1272 - 89
HORINOUCHI T; HOSHI A; HARADA T; HIGA T; KARKI S; TERADA K ET AL.: "Endothelin-1 suppresses insulin-stimulated Akt phosphorylation and glucose uptake via GPCR kinase 2 in skeletal muscle cells", BR J PHARMACOL, vol. 173, no. 6, March 2016 (2016-03-01), pages 1018 - 32
HUE L; BEAULOYE C; MARSIN A-S; BERTRAND L; HORMAN S; RIDER MH: "Insulin and Ischemia Stimulate Glycolysis by Acting on the Same Targets Through Different and Opposing Signaling Pathways", J MOL CELL CARDIOL., vol. 34, no. 9, September 2002 (2002-09-01), pages 1091 - 7
KAMBA T; MCDONALD DM: "Mechanisms of adverse effects of anti-VEGF therapy for cancer", BR J CANCER, vol. 96, no. 12, 18 June 2007 (2007-06-18), pages 1788 - 95
KAPPERS MH; VAN ESCH JHM; SLEIJFER S; DANSER AHJ; VAN DEN MEIRACKER AH: "Cardiovascular and renal toxicity during angiogenesis inhibition: clinical and mechanistic aspects", J HYPERTENS, vol. 27, no. 12, December 2009 (2009-12-01), pages 2297 - 309
KAPPERS MHW; ESCH JHM VAN; SLUITER W; SLEIJFER S; DANSER AHJ; MEIRACKER AH VAN DEN: "Hypertension Induced by the Tyrosine Kinase Inhibitor Sunitinib Is Associated With Increased Circulating Endothelin-1 Levels", HYPERTENSION, vol. 56, no. 4, 1 October 2010 (2010-10-01), pages 675 - 81
KAPPERS MHW; VAN ESCH JHM; SMEDTS FMM; DE KRIJGER RR; EECHOUTE K; MATHIJSSEN RHJ ET AL.: "Sunitinib-induced hypothyroidism is due to induction of type 3 deiodinase activity and thyroidal capillary regression", J CLIN ENDOCRINOL METAB, vol. 96, no. 10, October 2011 (2011-10-01), pages 3087 - 94
KASSIOTIS C; RAJABI M; TAEGTMEYER H: "Metabolic Reserve of the Heart: The Forgotten Link Between Contraction and Coronary Flow", PROG CARDIOVASC DIS., vol. 51, no. 1, 2008, pages 74 - 88, XP026039171, DOI: doi:10.1016/j.pcad.2007.11.005
KERBEL R; FOLKMAN J: "Clinical translation of angiogenesis inhibitors", NAT REV CANCER, vol. 2, no. 10, October 2002 (2002-10-01), pages 727 - 39, XP008031663, DOI: doi:10.1038/nrc905
KIBRIA GOLAM ET AL: "Anti-tumor effect via passive anti-angiogenesis of PEGylated liposomes encapsulating doxorubicin in drug resistant tumors", INTERNATIONAL JOURNAL OF PHARMACEUTICS, ELSEVIER, AMSTERDAM, NL, vol. 509, no. 1, 24 May 2016 (2016-05-24), pages 178 - 187, XP029623682, ISSN: 0378-5173, DOI: 10.1016/J.IJPHARM.2016.05.047 *
KOBAYASHI K; NEELY JR: "Effects of ischemia and reperfusion on pyruvate dehydrogenase activity in isolated rat hearts", J MOL CELL CARDIOL., vol. 15, no. 6, June 1983 (1983-06-01), pages 359 - 67, XP023001461, DOI: doi:10.1016/0022-2828(83)90320-6
KOHLER; MILSTEIN, NATURE, vol. 256, 1975, pages 495
KOLWICZ SC; TIAN R: "Glucose metabolism and cardiac hypertrophy", CARDIOVASC RES., vol. 90, no. 2, 1 May 2011 (2011-05-01), pages 194 - 201
KONGBUNDANSUK S; HUNDLEY WG: "Noninvasive imaging of cardiovascular injury related to the treatment of cancer", JACC CARDIOVASC IMAGING, vol. 7, no. 8, August 2014 (2014-08-01), pages 824 - 38
KOUNIS NG; KONIARI I; HAHALIS G: "Cardio-oncology, Immuno-oncology, Onco-cardiology and Onco-immunology", INT J CARDIOL, vol. 223, 15 November 2016 (2016-11-15), pages 254 - 7, XP029763808, DOI: doi:10.1016/j.ijcard.2016.08.219
KREISSL MC; STOUT DB; WONG K-P; WU H-M; CAGLAYAN E; LADNO W ET AL.: "Influence of dietary state and insulin on myocardial, skeletal muscle and brain [ 18 F]-fluorodeoxyglucose kinetics in mice", EJNMMI RES, vol. 1, no. 1, 6 July 2011 (2011-07-06), pages 8
KUMAR RMR; ARLT MJ; KUZMANOV A; BORN W; FUCHS B: "Sunitinib malate (SU-11248) reduces tumour burden and lung metastasis in an intratibial human xenograft osteosarcoma mouse model", AM J CANCER RES., vol. 5, no. 7, 15 June 2015 (2015-06-15), pages 2156 - 68
KUSUOKA H; MARBAN E.: "Mechanism of the diastolic dysfunction induced by glycolytic inhibition. Does adenosine triphosphate derived from glycolysis play a favored role in cellular Ca2+ homeostasis in ferret myocardium?", J CLIN INVEST, vol. 93, no. 3, March 1994 (1994-03-01), pages 1216 - 23
LANKHORST S; KAPPERS MHW; VAN ESCH JHM; DANSER AHJ; VAN DEN MEIRACKER AH: "Mechanism of hypertension and proteinuria during angiogenesis inhibition: evolving role of endothelin-1", J HYPERTENS, vol. 31, no. 3, March 2013 (2013-03-01), pages 444 - 454
LANKHORST S; KAPPERS MHW; VAN ESCH JHM; SMEDTS FMM; SLEIJFER S; MATHIJSSEN RHJ ET AL.: "Treatment of hypertension and renal injury induced by the angiogenesis inhibitor sunitinib: preclinical study", HYPERTENS DALLAS TEX 1979, vol. 64, no. 6, December 2014 (2014-12-01), pages 1282 - 9
LEONARD ET AL., CLINICAL CANCER RESEARCH, vol. 10, 2004, pages 53Z7 - 5334
MALEK R; DAVIS SN: "Tyrosine kinase inhibitors under investigation for the treatment of type II diabetes", EXPERT OPIN INVESTIG DRUGS, vol. 25, no. 3, 2016, pages 287 - 96
MANDINOV L; EBERLI FR; SEILER C; HESS OM: "Diastolic heart failure", CARDIOVASC RES., vol. 45, no. 4, 1 March 2000 (2000-03-01), pages 813 - 25, XP002903841, DOI: doi:10.1016/S0008-6363(99)00399-5
MATTHIAS SCHWEBE ET AL: "Protective effects of endothelin receptor A and B inhibitors against doxorubicin-induced cardiomyopathy", BIOCHEMICAL PHARMACOLOGY, vol. 94, no. 2, 1 March 2015 (2015-03-01), US, pages 109 - 129, XP055428077, ISSN: 0006-2952, DOI: 10.1016/j.bcp.2015.01.014 *
MEIRACKER AH VAN DEN; DANSER AHJ; SLEIJFER S; KAPPERS MHW: "Re: Hypertension as a Biomarker of Efficacy in Patients With Metastatic Renal Cell Carcinoma Treated With Sunitinib", J NATL CANCER INST [INTERNET, 23 August 2011 (2011-08-23), Retrieved from the Internet <URL:http://jnci.oxfordjournals.org/content/early/2011/08/23/jnci.djr328>
MENDEL DB; LAIRD AD; XIN X; LOUIE SG; CHRISTENSEN JG; LI G ET AL.: "In vivo antitumor activity ofSU1 1248, a novel tyrosine kinase inhibitor targeting vascular endothelial growth factor and platelet-derived growth factor receptors: determination of a pharmacokinetic/pharmacodynamic relationship", CLIN CANCER RES OFF J AM ASSOC CANCER RES., vol. 9, no. 1, January 2003 (2003-01-01), pages 327 - 37
MONSUEZ J-J; CHARNIOT J-C; VIGNAT N; ARTIGOU J-Y: "Cardiac side-effects of cancer chemotherapy", INT J CARDIOL, vol. 144, no. 1, 24 September 2010 (2010-09-24), pages 3 - 15, XP027402170
MOSLEHI J; MINAMISHIMA YA; SHI J; NEUBERG D; CHARYTAN DM; PADERA RF ET AL.: "Loss of hypoxia-inducible factor prolyl hydroxylase activity in cardiomyocytes phenocopies ischemic cardiomyopathy", CIRCULATION, vol. 122, no. 10, 7 September 2010 (2010-09-07), pages 1004 - 16
MOTZER RJ; HUTSON TE; TOMCZAK P; MICHAELSON MD; BUKOWSKI RM; RIXE O ET AL.: "Sunitinib versus interferon alfa in metastatic renal-cell carcinoma", N ENGL J MED., vol. 356, no. 2, 11 January 2007 (2007-01-11), pages 115 - 24
MURRY: "Methods in Molecular Biology", vol. 7, 1991, HUMANA PRESS, INC.
NAGATA D; HIRATA Y: "The role of AMP-activated protein kinase in the cardiovascular system", HYPERTENS RES., vol. 33, no. L, 13 November 2009 (2009-11-13), pages 22 - 8
NICHOLAS P. RESTIFO; MARK E. DUDLEY; STEVEN A. ROSENBERG: "Adoptive immunotherapy for cancer: harnessing the T cell response", NATURE REVIEWS IMMUNOLOGY, vol. 12, April 2012 (2012-04-01), XP055034896, DOI: doi:10.1038/nri3191
NILSSON D; WACKENFORS A; GUSTAFSSON L; EDVINSSON L; PAULSSON P; INGEMANSSON R ET AL.: "Increased ETA and ETB receptor contraction in the left internal mammary artery from patients with hypertension", J HUM HYPERTENS, vol. 22, no. 3, 13 December 2007 (2007-12-13), pages 226 - 9
O'FARRELL AC; EVANS R; SILVOLA JMU; MILLER IS; CONROY E; HECTOR S ET AL.: "A Novel Positron Emission Tomography (PET) Approach to Monitor Cardiac Metabolic Pathway Remodeling in Response to Sunitinib Malate", PLOS ONE, vol. 12, no. L, 2017, pages E0169964
ORCHARD CH: "The role of the sarcoplasmic reticulum in the response of ferret and rat heart muscle to acidosis", J PHYSIOL, vol. 384, March 1987 (1987-03-01), pages 431 - 49
PIPER HM: "Pathophysiology of Severe Ischemic Myocardial Injury", 1990, KLUWER ACADEMIC PUBLISHERS, pages: 448
PIZZINO F; VIZZARI G; QAMAR R; BOMZER C; CAREIJ S; ZITO C ET AL.: "Multimodality Imaging in Cardiooncology", J ONCOL. 2015, 2015, pages 263950
PREVILON M; GALL ML; CHAFEY P; FEDERECI C; PEZET M; CLARY G ET AL.: "Comparative differential proteomic profiles of nonfailing and failing hearts after in vivo thoracic aortic constriction in mice overexpressing FKBP12.6", PHYSIOL REP [INTERNET, vol. 1, no. 3, August 2013 (2013-08-01)
RAHIMTOOLA SH; DILSIZIAN V; KRAMER CM; MARWICK TH; VANOVERSCHELDE J-LJ: "Chronic Ischemic Left Ventricular Dysfunction", JACC CARDIOVASC IMAGING, vol. 1, no. 4, 1 July 2008 (2008-07-01), pages 5 3 6,5 5, XP023182552, DOI: doi:10.1016/j.jcmg.2008.05.009
RAPPSILBER J; MANN M; ISHIHAMA Y: "Protocol for micro-purification, enrichment, pre-fractionation and storage of peptides for proteomics using StageTips", NAT PROTOC, vol. 2, no. 8, 2007, pages 1896 - 906
RAYMOND E; DAHAN L; RAOUL J-L; BANG Y-J; BORBATH I; LOMBARD-BOHAS C ET AL.: "Sunitinib malate for the treatment of pancreatic neuroendocrine tumors", N ENGL J MED., vol. 364, no. 6, 10 February 2011 (2011-02-10), pages 501 - 13
REES ML; SUBRAMANIAM J; LI Y; HAMILTON DJ; FRAZIER OH; TAEGTMEYER H.: "A PKM2 signature in the failing heart", BIOCHEM BIOPHYS RES COMMUN, vol. 459, no. 3, 10 April 2015 (2015-04-10), pages 430 - 6, XP029211256, DOI: doi:10.1016/j.bbrc.2015.02.122
ROITT, I.: "Essential Immunology", 1991, BLACKWELL SCIENTIFIC PUBLICATIONS
S. BIEN ET AL: "The Endothelin Receptor Blocker Bosentan Inhibits Doxorubicin-Induced Cardiomyopathy", CANCER RESEARCH, vol. 67, no. 21, 1 November 2007 (2007-11-01), US, pages 10428 - 10435, XP055428026, ISSN: 0008-5472, DOI: 10.1158/0008-5472.CAN-07-1344 *
SANBROOK ET AL.: "Molecular Cloning: A Laboratory Manual", 1989, COLD SPRING HARBOR LABORATORY PRESS
SCHINKEL AFL; POLDERMANS D; ELHENDY A; BAX JJ: "Assessment of Myocardial Viability in Patients with Heart Failure", J NUCL MED, vol. 48, no. 7, 1 July 2007 (2007-07-01), pages 1135 - 46
SHEMYAKIN A; SALEHZADEH F; BOHM F; AL-KHALILI L; GONON A; WAGNER H ET AL.: "Regulation of Glucose Uptake by Endothelin-1 in Human Skeletal Muscle in Vivo and in Vitro", J CLIN ENDOCRINOL METAB., vol. 95, no. 5, 1 May 2010 (2010-05-01), pages 2359 - 66
SHEMYAKIN A; SALEHZADEH F; DUQUE-GUIMARAES DE; BOHM F; RULLMAN E; GUSTAFSSON T ET AL.: "Endothelin-1 Reduces Glucose Uptake in Human Skeletal Muscle In Vivo and In Vitro", DIABETES, vol. 60, no. 8, 1 August 2011 (2011-08-01), pages 2061 - 7
STANLEY WC; HALL JL; HACKER TA; HERNANDEZ LA; WHITESELL LF: "Decreased myocardial glucose uptake during ischemia in diabetic swine", METABOLISM, vol. 46, no. 2, February 1997 (1997-02-01), pages 168 - 72, XP004538972, DOI: doi:10.1016/S0026-0495(97)90297-3
SUZUKI ET AL., EUROP J OF IMMUNOL, vol. 22, no. 8, 1992, pages 1989 - 1993
THACKERAY JT; BANKSTAHL JP; BENGEL FM: "Impact of Image-Derived Input Function and Fit Time Intervals on Patlak Quantification of Myocardial Glucose Uptake in Mice", J NUCL MED OFF PUBL SOC NUCL MED., vol. 56, no. 10, October 2015 (2015-10-01), pages 1615 - 21
TOLBA KA; DELIARGYRIS EN: "Cardiotoxicity of cancer therapy", CANCER INVEST., vol. 17, no. 6, 1999, pages 408 - 22
TOUBERT M-E; VERCELLINO L; FAUGERON I; LUSSATO D; HINDIE E; BOUSQUET G: "Fatal heart failure after a 26-month combination of tyrosine kinase inhibitors in a papillary thyroid cancer", THYROID OFF J AM THYROID ASSOC, vol. 21, no. 4, April 2011 (2011-04-01), pages 451 - 4
VERHEUL HMW; PINEDO HM: "Possible molecular mechanisms involved in the toxicity of angiogenesis inhibition", NAT REV CANCER, vol. 7, no. 6, June 2007 (2007-06-01), pages 475 - 85
VITALE GD; DEKEMP RA; RUDDY TD; WILLIAMS K; BEANLANDS RS: "Myocardial glucose utilization and optimization of (18)F-FDG PET imaging in patients with non-insulin-dependent diabetes mellitus, coronary artery disease, and left ventricular dysfunction", J NUCL MED OFF PUBL SOC NUCL MED., vol. 42, no. 12, December 2001 (2001-12-01), pages 1730 - 6
VOIPIO-PULKKI LM; NUUTILA P; KNUUTI MJ; RUOTSALAINEN U; HAAPARANTA M; TERAS M ET AL.: "Heart and skeletal muscle glucose disposal in type 2 diabetic patients as determined by positron emission tomography", RESEARCHGATE, vol. 34, no. 12, 1 January 1994 (1994-01-01), pages 2064 - 7
WISNIEWSKI JR; DUS K; MANN M: "Proteomic workflow for analysis of archival formalin-fixed and paraffin-embedded clinical samples to a depth of 10 000 proteins", PROTEOMICS CLIN APPL, vol. 7, no. 3-4, April 2013 (2013-04-01), pages 225 - 33
WISNIEWSKI JR; HEIN MY; COX J; MANN M. A: "proteomic ruler'' for protein copy number and concentration estimation without spike-in standards", MOL CELL PROTEOMICS MCP, vol. 13, no. 12, December 2014 (2014-12-01), pages 3497 - 506
WISNIEWSKI JR; ZOUGMAN A; NAGARAJ N; MANN M: "Universal sample preparation method for proteome analysis", NAT METHODS, vol. 6, no. 5, May 2009 (2009-05-01), pages 359 - 62
WU ET AL., MOL. BIOL., vol. 294, 1999, pages 151
YANG Y; BU P: "Progress on the cardiotoxicity of sunitinib: Prognostic significance, mechanism and protective therapies", CHEM BIOL INTERACT, vol. 257, 25 September 2016 (2016-09-25), pages 125 - 31, XP029708061, DOI: doi:10.1016/j.cbi.2016.08.006
YANG YI ET AL: "Progress on the cardiotoxicity of sunitinib: Prognostic significance, mechanism and protective therapies", CHEMICO-BIOLOGICAL INTERACTIONS, ELSEVIER SCIENCE IRLAND, IR, vol. 257, 13 August 2016 (2016-08-13), pages 125 - 131, XP029708061, ISSN: 0009-2797, DOI: 10.1016/J.CBI.2016.08.006 *
ZHONG M; ALONSO CE; TAEGTMEYER H; KUNDU BK: "Quantitative PET imaging detects early metabolic remodeling in a mouse model of pressure-overload left ventricular hypertrophy in vivo", J NUCL MED OFF PUBL SOC NUCL MED., vol. 54, no. 4, April 2013 (2013-04-01), pages 609 - 15

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11738007B2 (en) 2019-10-30 2023-08-29 Perfuse Therapeutics, Inc. Treatment of glaucoma using endothelin receptor antagonists
WO2021158663A1 (en) * 2020-02-06 2021-08-12 Perfuse Therapeutics, Inc. Compositions for treatment of ocular diseases
US11873279B2 (en) 2020-02-06 2024-01-16 Perfuse Therapeutics, Inc. Compositions for treatment of ocular diseases
WO2022232586A1 (en) * 2021-04-30 2022-11-03 Perfuse Therapeutics, Inc. Treatment of ocular diseases using endothelin receptor antagonists
US11786510B2 (en) 2021-04-30 2023-10-17 Perfuse Therapeutics, Inc. Pharmaceutical compositions and intravitreal drug delivery systems for the treatment of ocular diseases
CN113209096A (en) * 2021-05-17 2021-08-06 武汉大学 Application of pexidinib in preparation of medicines for preventing, relieving and/or treating myocardial infarction and related diseases thereof
CN113209096B (en) * 2021-05-17 2022-06-14 武汉大学 Application of pecidatinib in the preparation of drugs for preventing, relieving and/or treating myocardial infarction and related diseases

Similar Documents

Publication Publication Date Title
Hu et al. IFNα potentiates anti–PD-1 efficacy by remodeling glucose metabolism in the hepatocellular carcinoma microenvironment
TWI577695B (en) Vegf-specific antagonists for adjuvant and neoadjuvant therapy and the treatment of early stage tumors
WO2018185516A1 (en) Methods and pharmaceutical compositions for treating cardiovascular toxicity induced by anti-cancer therapy
EP3108255B1 (en) Methods and pharmaceutical compositions for the treatment of diseases mediated by the nrp-1/obr complex signaling pathway
Qin et al. Pirfenidone facilitates immune infiltration and enhances the antitumor efficacy of PD-L1 blockade in mice
WO2019106126A1 (en) Mdm2 modulators for the diagnosis and treatment of liposarcoma
US20210239702A1 (en) Methods for predicting responsiveness of lung cancer patients to her2-targeting therapies
WO2017158396A1 (en) Cytidine deaminase inhibitors for the treatment of pancreatic cancer
WO2019158689A1 (en) Methods and compositions for treating liver diseases
US20200164034A1 (en) Methods for improving sex-dimorphic responses to targeted therapy in melanoma
WO2018189403A1 (en) Methods and pharmaceutical compositions for the treatment of cancer
WO2012074842A2 (en) Compositions and methods for treating neoplasia
WO2019211370A1 (en) Methods and pharmaceutical compositions for treating cancer
EP4037714A1 (en) Methods and compositions for modulating macrophages polarization
WO2016123679A1 (en) A method of treatment
US10647752B2 (en) Polypeptides capable of inhibiting the binding between leptin and Neuropilin-1
EP3067369A1 (en) Methods and compositions for the treatment of anti-angiogenic resistant cancer
US20180064789A1 (en) Methods and pharmaceutical compositions for the treatment of pancreatic cancer
EP4424711A2 (en) Methods for the diagnosis and treatment of cytokine release syndrome
US20210047696A1 (en) Methods and pharmaceutical compositions for treating cancer
WO2019211369A1 (en) Methods and pharmaceutical compositions for treating cancer
US20230416346A1 (en) C-terminal sparc fragments for treating cancer
WO2024218697A1 (en) Methods and systems for improving anti-cancer therapies
US20240316061A1 (en) Bet inhibitors for treating pab1 deficient cancer
Principe Transforming Growth Factor β Signaling in the Pancreatic Tumor Microenvironment

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 17724895

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 17724895

Country of ref document: EP

Kind code of ref document: A1