WO2016118014A2 - Anti-senescence compounds and uses thereof - Google Patents
Anti-senescence compounds and uses thereof Download PDFInfo
- Publication number
- WO2016118014A2 WO2016118014A2 PCT/NL2016/050057 NL2016050057W WO2016118014A2 WO 2016118014 A2 WO2016118014 A2 WO 2016118014A2 NL 2016050057 W NL2016050057 W NL 2016050057W WO 2016118014 A2 WO2016118014 A2 WO 2016118014A2
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- cancer
- peptide
- inhibitor
- loss
- dri
- Prior art date
Links
- 0 CCOC(C(C(N[C@@]1*C*C)=O)=C(CC(C)=*CC=CC#C2)C1=C(*)C2=O)=O Chemical compound CCOC(C(C(N[C@@]1*C*C)=O)=C(CC(C)=*CC=CC#C2)C1=C(*)C2=O)=O 0.000 description 1
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
- A61K38/10—Peptides having 12 to 20 amino acids
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
- A61P1/14—Prodigestives, e.g. acids, enzymes, appetite stimulants, antidyspeptics, tonics, antiflatulents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
- A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P11/00—Drugs for disorders of the respiratory system
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P11/00—Drugs for disorders of the respiratory system
- A61P11/06—Antiasthmatics
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P11/00—Drugs for disorders of the respiratory system
- A61P11/14—Antitussive agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P13/00—Drugs for disorders of the urinary system
- A61P13/12—Drugs for disorders of the urinary system of the kidneys
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P17/00—Drugs for dermatological disorders
- A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P17/00—Drugs for dermatological disorders
- A61P17/14—Drugs for dermatological disorders for baldness or alopecia
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P19/00—Drugs for skeletal disorders
- A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P19/00—Drugs for skeletal disorders
- A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P19/00—Drugs for skeletal disorders
- A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
- A61P19/10—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease for osteoporosis
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P21/00—Drugs for disorders of the muscular or neuromuscular system
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
- A61P25/04—Centrally acting analgesics, e.g. opioids
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
- A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
- A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
- A61P25/16—Anti-Parkinson drugs
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
- A61P25/20—Hypnotics; Sedatives
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
- A61P25/22—Anxiolytics
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
- A61P25/24—Antidepressants
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
- A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P27/00—Drugs for disorders of the senses
- A61P27/02—Ophthalmic agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P27/00—Drugs for disorders of the senses
- A61P27/02—Ophthalmic agents
- A61P27/12—Ophthalmic agents for cataracts
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P27/00—Drugs for disorders of the senses
- A61P27/16—Otologicals
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
- A61P29/02—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID] without antiinflammatory effect
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P3/00—Drugs for disorders of the metabolism
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P3/00—Drugs for disorders of the metabolism
- A61P3/02—Nutrients, e.g. vitamins, minerals
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P3/00—Drugs for disorders of the metabolism
- A61P3/04—Anorexiants; Antiobesity agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P3/00—Drugs for disorders of the metabolism
- A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
- A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
- A61P35/02—Antineoplastic agents specific for leukemia
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
- A61P35/04—Antineoplastic agents specific for metastasis
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
- A61P37/02—Immunomodulators
- A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P7/00—Drugs for disorders of the blood or the extracellular fluid
- A61P7/06—Antianaemics
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P7/00—Drugs for disorders of the blood or the extracellular fluid
- A61P7/12—Antidiuretics, e.g. drugs for diabetes insipidus
-
- 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
-
- 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
- A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
-
- 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
- A61P9/12—Antihypertensives
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
- C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
- C07K14/4702—Regulators; Modulating activity
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
- C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
- C07K14/4747—Apoptosis related proteins
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
- C07K2319/10—Fusion polypeptide containing a localisation/targetting motif containing a tag for extracellular membrane crossing, e.g. TAT or VP22
Definitions
- the invention is in the field of medicine. More specifically, it is in the field of the treatment of diseases or conditions wherein the removal of senescent cells is beneficial.
- the invention relates to
- the invention also relates to methods of treating an individual suffering, or suspected of suffering, from a disease or condition wherein the removal of senescent cells is beneficial.
- Apoptosis (programmed cell death) and cellular senescence
- telomere shortening may be induced by telomere shortening, oxidative stress, DNA damage, chromatin remodeling, tumor suppressor loss and oncogene induction.
- Apoptosis and cellular senescence play major roles in human aging, age-related diseases and in keeping the development of cancer within bounds.
- Apoptosis provides for the killing of damaged cells.
- Dying cells that undergo the final stages of apoptosis display phagocytic molecules, marking these cells for phagocytosis by cells possessing the appropriate receptors, such as macrophages.
- Non-dividing senescent cells can also be cleared by immune cells, but this process is to large extent inefficient. Consequently, whereas apoptosis kills cells, senescent cells are not cleared from tissues and remain metabolically active.
- senescent cells accumulate with age, and at sites of age-related pathology. Further, senescent cells can acquire mutations that allow them to re-enter a proliferative state. Benign senescent lesions thus retain the capacity to become malignant.
- senescent cells whether in response to inter alia telomere malfunction, DNA damage, oxidative damage, chromatin remodeling, or oncogenic alterations— exhibit great changes in their transcriptomes.
- senescent cells affect and alter the tissue microenvironment and potentially the systemic milieu by secreting proinflammatory cytokines or chemokines, growth factors and/or matrix degrading proteases.
- This phenotype is termed the senescence-associated secretory phenotype (SASP).
- SASP senescence-associated secretory phenotype
- senescent fibroblast have been implicated in decreased milk -production in breast
- senescent pulmonary artery smooth muscle cells are implicated in pulmonary hypertension
- senescent skin cells are related to epidermal thinning and reduced collagen content
- senescent astrocytes and the SASP are implicated in Alzheimers and Parkinson's disease
- senescent chondrocytes and SASP have been brought in connection with osteoarthritis.
- the pathologies that have been brought into connection with cellular senescence and the SASP are even longer and further comprise: Atherosclerosis, lung emphysema, diabetic ulcers, renal disease, kyphosis, osteoporosis, macular degeneration, COPD and insulin resistance, diabetes, obesity, laminopaties such as Hutchinson Gilford's progeria, hernia, sarcopenia and cachexia, arthritis, scoliosis and cancer.
- the inventors solved this problem by their unexpected finding that a peptide according to the invention, and the inhibitors NQDI and R406 for use according to the invention, selectively induce apoptosis in senescent cells and, as a consequence, are applicable in the treatment of diseases associated with the presence of senescent cells.
- the inventors showed inter alia that compounds according to the invention are effective in the (i) removal of senescent cells in vitro, ex vivo and in vivo (ii) countering symptoms of aging and age-related diseases in a fast aging mouse model, (iii) protection of organ function in an in vivo model for chemotherapy- induced toxicity and (iv) sensitization of a resistant cancer to a
- the peptide further comprises a cell-penetrating peptide sequence, preferably said cell- penetrating peptide sequence has the amino acid sequence
- amino acids in said cell- penetrating peptide sequence are D-amino acid residues.
- a peptide of the invention further comprising a cell-penetrating peptide sequence, said cell-penetrating peptide is fused to the C-terminal part of said peptide.
- the invention provides a pharmaceutical
- composition comprising a peptide according to the invention.
- the pharmaceutical composition further comprises a
- the invention provides a retro-inverso peptide of the Forkhead box protein O4, preferably wherein the amino acid sequence of said Forkhead box protein 04 is indicated in Figs 14 or 15, or a homologue or a fragment thereof, wherein said homologue has an amino acid sequence identity of at least 70% to the retro-inverso amino acid sequence of the Forkhead box protein 04, wherein said homologue exhibits apoptosis- inducing activity in senescent cells, and wherein said fragment has apoptosis-inducing activity in senescent cells.
- the invention provides a nucleic acid encoding a peptide according to the invention, optionally comprised in a vector.
- the invention provides a host cell comprising a nucleic acid or vector according to the invention.
- the invention provides a peptide, pharmaceutical composition, or nucleic acid according to the invention for use as a
- pSerl5-p53 active p53 signaling
- said disorder is selected from the group of age-related disorders consisting of atherosclerosis; chronic inflammatory diseases such as arthritis or arthrosis; cancer; osteoarthritis; glomerulosclerosis, diabetes including diabetes type I and II; diabetic ulcers; kyphosis; scoliosis; hepatic insufficiency; cirrhosis; Hutchinson- Gilford progeria syndrome (HGPS); laminopaties; osteoporosis; dementia; (cardio)vascular diseases; obesity; metabolic syndrome; acute myocardial infarction; emphysema; insulin sensitivity; boutonneuse fever; sarcopenia; neurodegenerative diseases such as Alzheimer's, Huntington's or Parkinson's disease; cataracts; anemia; hypertension;
- the invention provides a peptide, pharmaceutical composition, or nucleic acid according to the invention for use as a medicament, or for use in the treatment of a disorder wherein the removal of senescent cells is beneficial, preferably wherein said disorder is selected from the group of age-related disorders consisting of atherosclerosis; chronic inflammatory diseases such as arthritis or arthrosis; cancer; osteoarthritis; glomerulosclerosis, diabetes including diabetes type II; diabetic ulcers; kyphosis; scoliosis; hepatic insufficiency; cirrhosis; Hutchinson- Gilford progeria syndrome (HGPS); laminopaties; osteoporosis; dementia;
- atherosclerosis chronic inflammatory diseases such as arthritis or arthrosis
- cancer osteoarthritis
- glomerulosclerosis diabetes including diabetes type II; diabetic ulcers; kyphosis; scoliosis; hepatic insufficiency; cirrhosis; Hutchinson- Gilford prog
- Parkinson's disease cataracts; anemia; hypertension; fibrosis; age-related macular degeneration; COPD; asthma; renal insufficiency; reducing or preventing graft failure after organ or tissue transplantation; incontinence; hearing loss such as deafness; vision loss such as blindness; sleeping disturbances; pain such as joint pain or leg pain; imbalance; fear;
- the disorder is cancer, and wherein the use is for administration to a mammalian subject, preferably a human, before, during and/or after subjecting said subject to radiation therapy, and/or before, during or after administering to said subject at least one chemotherapeutic agent.
- the cancer is a cancer resistant to therapy.
- a peptide, pharmaceutical composition, or nucleic acid for use according to the invention wherein the disorder is cancer and wherein the cancer is a cancer resistant to therapy
- said therapy-resistant cancer is metastatic melanoma, breast cancer or glioblastoma, preferably metastatic melanoma
- said therapy to which said cancer is resistant is radiation therapy and/or chemotherapy involving a RAF, MEK or ERK inhibitor or a composition comprising 5' FluoroUracil, Doxorubicin and Cyclofosfamide (FAC) as chemotherapeutic agent, preferably wherein said RAF, MEK or ERK inhibitor is RAF265, trametinib, dabrafenib, selumetinib, vemurafenib, cobemitinib and/or trametinib, more preferably vemurafenib and/or trametinib, and
- the invention provides for a peptide
- compositions, or nucleic acid according to the invention for use in removing senescent cells in a human subject suffering from, or expected to suffer from, atherosclerosis; chronic inflammatory diseases such as arthritis or arthrosis; cancer; osteoarthritis; glomerulosclerosis, diabetes including type II diabetes; diabetic ulcers; kyphosis; scoliosis; hepatic insufficiency; cirrhosis; Hutchinson- Gilford progeria syndrome (HGPS); laminopaties; osteoporosis; dementia; (cardio)vascular diseases; obesity; metabolic syndrome; acute myocardial infarction; emphysema; insulin sensitivity; boutonneuse fever; sarcopenia; neurodegenerative diseases such as Alzheimer's, Huntington's or Parkinson's disease; cataracts; anemia; hypertension; fibrosis; age-related macular degeneration; COPD; asthma; renal insufficiency; reducing or preventing graft failure after organ or tissue transplantation,
- the invention provides for a peptide
- composition or nucleic acid according to the invention for use in countering p21 ci P 1 expression and/or removing cells that express p21 ci P 1 , in a human subject suffering from, or expected to suffer from, atherosclerosis, chronic inflammatory diseases such as arthritis or athrosis, cancer, osteoarthritis, glomerulosclerosis, diabetes including type II diabetes, diabetic ulcers, kyphosis, scoleosis, hepatic insufficiency, cirrhosis, Hutchinson- Gilford progeria syndrome (HGPS), laminopaties, osteoporosis, dementia, (cardio)vascular diseases, obesity, metabolic syndrome, acute myocardial infarction, emphysema, insulin sensitivity, boutonneuse fever, sarcopenia, neurodegenerative diseases such as Alzheimer's , Huntington's or Parkinson's disease cataracts, anemia, hypertension, fibrosis, age-related macular degeneration, COPD, asthma, renal in
- the invention provides for a peptide
- composition or nucleic acid according to the invention for use in countering p i 6INK4a expression and/or removing cells that express pl6INK4a in a subject suffering from, or expected to suffer from,
- Atherosclerosis chronic inflammatory diseases such as arthritis or athrosis, cancer, osteoarthritis, glomerulosclerosis, diabetes including type II diabetes, diabetic ulcers, kyphosis, scoleosis, hepatic insufficiency, cirrhosis, Hutchinson- Gilford progeria syndrome (HGPS), laminopaties, osteoporosis, dementia, (cardio)vascular diseases, obesity, metabolic syndrome, acute myocardial infarction, emphysema, insulin sensitivity, boutonneuse fever, sarcopenia, neurodegenerative diseases such as Alzheimer's , Huntington's or Parkinson's disease cataracts, anemia, hypertension, fibrosis, age-related macular degeneration, COPD, asthma, renal insufficiency, reducing or preventing graft failure after organ or tissue transplantation, incontinence, hearing loss such as deafness, vision loss such as blindness, sleeping disturbances, pain such as joint pain or leg pain, imbalance,
- the invention provides for a peptide
- composition or nucleic acid according to the invention for use in countering, or reducing the number of, nuclear serine- 15- phosphorylated p53 foci in a subject suffering, or expected to suffer, from atherosclerosis, chronic inflammatory diseases such as arthritis or athrosis, cancer, osteoarthritis, glomerulosclerosis, diabetes including type II diabetes, diabetic ulcers, kyphosis, scoleosis, hepatic insufficiency, cirrhosis, Hutchinson- Gilford progeria syndrome (HGPS), laminopaties, osteoporosis, dementia, (cardio)vascular diseases, obesity, metabolic syndrome, acute myocardial infarction, emphysema, insulin sensitivity, boutonneuse fever, sarcopenia, neurodegenerative diseases such as Alzheimer's , Huntington's or Parkinson's disease cataracts, anemia, hypertension, fibrosis, age-related macular degeneration, COPD, asthma, renal insufficiency,
- the invention provides a kit comprising a first container containing a peptide or nucleic acid according to the invention and a second container containing a chemotherapeutic agent.
- the invention provides an inhibitor of ASK 1 for use in the treatment of a disorder wherein the removal of senescent cells is beneficial.
- the inhibitor of ASK 1 is NQDI.
- the disorder is selected from the group of age-related disorders consisting of atherosclerosis; chronic inflammatory diseases such as arthritis or arthrosis; cancer; osteoarthritis; glomerulosclerosis, diabetes including diabetes type II; diabetic ulcers; kyphosis; scoliosis; hepatic insufficiency; cirrhosis; Hutchinson- Gilford progeria syndrome (HGPS); laminopaties; osteoporosis; dementia; (cardio)vascular diseases; obesity; metabolic syndrome; acute myocardial infarction; emphysema; insulin sensitivity; boutonneuse fever; sarcopenia; neurodegenerative diseases such as Alzheimer's, Huntington's or Parkinson's disease; cataracts; anemia; hypertension; fibrosis; age-related macular degeneration; CO
- the disorder is cancer
- a mammalian subject preferably a human
- a mammalian subject before, during and/or after subjecting said subject to radiation therapy, and/or before, during or after administering to said subject at least one chemotherapeutic agent.
- an inhibitor of ASK1 for use according to the invention wherein the disorder is cancer, said cancer is a cancer resistant to therapy.
- an inhibitor of ASK1 for use according to the invention wherein the disorder is cancer and wherein the cancer is a cancer resistant to therapy, said cancer resistant to therapy is metastatic melanoma, breast cancer or glioblastoma, preferably metastatic melanoma, and wherein said therapy to which said cancer is resistant is chemotherapy involving a RAF, MEK or ERK inhibitor as chemotherapeutic agent, preferably wherein said RAF, MEK or ERK inhibitor is RAF265, trametinib, dabrafenib, selumetinib, vemurafenib and/or trametinib, more preferably vemurafenib and/or trametinib.
- the invention provides a pharmaceutical
- combination comprising an inhibitor of ASK1, preferably NQDI, and a chemotherapeutic agent.
- the invention provides a kit for use in medicine, preferably for use in the treatment of cancer, the kit comprising a first container containing an inhibitor of ASK1, preferably NQDI, and a second container containing a chemotherapeutic agent.
- the invention provides an inhibitor of SYK for use in the treatment of a disorder wherein the removal of senescent cells is beneficial, wherein said disorder is not asthma, immune thrombocytopenia, hemolytic anemia, myeloid leukemia and/or lymphoma, preferably wherein said disorders are those listed in claim 15.
- the inhibitor of SYK is R406.
- said disorder is cancer
- the use is for administration to a mammalian subject, preferably a human, before, during and/or after subjecting said subject to radiation therapy and/or before, during or after administering at least one chemotherapeutic agent to said subject, with the proviso that said cancer is not a lymphoma or leukemia, preferably not lymphoma or myeloid leukemia.
- the cancer is a cancer that is resistant to therapy.
- said cancer resistant to therapy is metastatic melanoma, breast cancer or glioblastoma, preferably metastatic melanoma
- said therapy to which said cancer is resistant is chemotherapy involving a RAF, MEK or ERK inhibitor as chemotherapeutic agent, preferably wherein said RAF, MEK or ERK inhibitor is RAF265, trametinib, dabrafenib, selumetinib, vemurafenib and/or trametinib, more preferably vemurafenib and/or trametinib.
- the invention provides a pharmaceutical
- combination comprising an inhibitor of SYK, preferably R406, and a chemotherapeutic agent.
- the invention provides a kit for use in medicine, the kit comprising a first container containing an inhibitor of SYK, preferably R406, and a second container containing a chemotherapeutic agent.
- the invention provides a kit for use in the treatment of cancer, wherein the cancer is not a lymphoma or leukemia, preferably not lymphoma or myeloid leukemia, and wherein the kit comprises a first container containing an inhibitor of SYK, preferably R406, and a second container containing a chemotherapeutic agent.
- Figure 1 shows that the overall fitness of the bubrl H/H ink attac - 3 fast aging mouse model is significantly improved when senescent cells were genetically removed (mice above: senescent cells removed, mice below:
- mice showed reduced signs of aging measured by kyphosis (excessive bone curvature), muscle strength, fat deposition and cataracts (Baker et al., 2011. Nature 479(7372):232-6).
- Senescence is thus causally linked to aging.
- Figure 2 shows in panels A and B the sequence and 3D structure of FOXO4 used for the design of FOXO4 DRI which has the sequence: H- ltlrkepaseiaqsileaysqngwanrrsggkrppprrrqrrkkrg-OH (MW: 5358.2), in which all amino acid residues are in the D-isoform, i.e., the FOX04 DRI peptide is the D-Retro-Inverso isoform of
- Senescent and control IMR90 were incubated with increasing doses of FOX04 DRI and cell viability was determined 6 days later using the AQueousOne Solution Cell Proliferation Assay (Promega) according to the manufacturer's protocol.
- the selectivity index (S.I.) for the EC50 values was determined after non- regression analysis (GraphPad Prism) of the curves.
- Panel D) shows FOX04 DRI selectively reduces senescent cell density in time starting at 36 hours after administration. Real-time cell density was measured using an xCELLigence detection system (ACEA Biosciences). Prior to the start of the measurement 50 ⁇ ⁇ DMEM 10% FCS was added to each well of an E-plate view 16 (Roche) to determine background signal.
- Non-senescent (2000 cells per well) and senescent (5000 cells/well) IMR90 fibroblasts were then plated in 150 ⁇ medium. 16 hours later the E-plate was placed in the xCELLigence reader and the cell density was recorded every 30 minutes. The cells were treated with 25uM FOX04 peptide 8 hours after starting the
- Panel E shows that FOX04 DRI, but not a peptide of identical amino acid sequence in L-isoform, nor an unrelated FOXMl DRI peptide (Kruiswijk et al, Oncogene, 2015), selectively eliminates senescent cells in a dose- dependent manner (6.25, 12.5 and 25 ⁇ , respectively). Experiment as in Fig. 2C, but with the indicated peptides.
- Panel F shows that FOXO4 DRI (25 ⁇ ) selectively induces Caspase-3/7 activation in time in senescent, but not control, IMR90 cells.
- Senescent IMR90 cells were incubated with the pan-caspase inhibitors QVD-OPH or ZVAD-FMK (20 ⁇ ) and exposed to FOX04 DRI. After 6 days cell viability was determined as in Fig. 2C). Both QVD-OPH and ZVAD-FMK potently blocked the effect of FOXO4 DRI to reduce senescent cell viability, indicating FOXO4 DRI causes cell death of senescent IMR90 through Caspase-3/7 mediated apoptosis. Panel H) shows that FOXO4 DRI selectively reduces viability of senescent, but not control, normal human cell types: IMR90, BJ and Wi-38. Experiment as in Fig.
- Panel J shows that FOXO4 DRI selectively kills cells expressing high levels of SASP proteins.
- Senescent IMR90 cells which show elevated foci of active, Sl5-phosphorylated p53, were plated on coverslips and either mock treated or incubated with 25 ⁇ FOX04 DRI in the presence of the pan-caspase inhibitor QVD-OPH to prevent dying cells from dethatching from the coverslips. After 3 days the media was replaced with media containing fresh QVD-OPH, but no additional FOXO4 DRI. At day 5 after FOXO4 DRI exposure the cells were fixed and stained for Ser-15-phosphorylated p53 and DAPI to indicate cell nuclei. A significant reduction of Serl5-p53 foci in the nucleus was apparent, which was caused by translocation to the Cytosol by FOXO4 DRI.
- Panel L shows that FOXO4 DRI reduces expression of the senescence- associated p53 target p2 lCip l.
- p53 signaling causes upregulation of the cell-cycle arrest and pro-survival protein p2 lCip l.
- FOX04 DRI not only lowers expression of pro -survival factors as p2 lCip l, but also causes an elevation in pro-apoptotic signaling by p53.
- Panel N shows that FOX04 DRI (25 ⁇ ) reduces senescent cell viability in a p53-dependent manner.
- Fig. 2c but with control and senescent IMR90 stable transduced with shRNAs against p53 or a control shRNA (shGFP).
- shGFP shRNA
- Loss of p53 decreased the potency of FOX04 DRI for reducing senescent cell viability.
- FOX04 DRI selectively kills senescent cells through p53- mediated apoptosis.
- Figure 3 shows in panel A) a schematic representation of the construct used to detect p l6INK4A-positive senescent cells in vivo.
- the promoter of the prominent senescence gene p l6INK4A drives expression of the bioluminescent enzyme Renilla Luciferase (RLUC).
- RLUC Renilla Luciferase
- XpdTM'TM mice (de Boer et al, 1998 and 2002) crossed with the P16-3MR model mouse, which contains RLUC (hereinafter also referred to as
- P16::3MR senescence-detection model show elevated levels of p l6ink4a- positive senescence in vivo, allowing longitudinal analysis of senescence.
- Panel C) shows that fast aging ⁇ ⁇ / ⁇ mice show elevated expression of FOX04, similar to normally aged (130w) old wildtype mice. Wildtype and 3 ⁇ 4?d TTD/TTD mice were sacrificed at the indicated ages and the kidneys were stained for FOX04. The number of FOX04 positive tubular cells (T) were quantified.
- FOX04 is elevated in 3 ⁇ 4?d TTD/TTD mice, which also show elevated levels of senescence. This allow for testing of FOX04 DRI to see if senescence is influenced.
- Panel D) shows that FOX04 DRI strongly reduces pl6 INK4A - positive senescence in 26w old ⁇ ⁇ mice.
- Panel E shows an anecdotal example showing that FOXO4 DRI can improve kyphosis (bone curvature) and muscle mass oiXpd TTD/TTD mice.
- CT images of the spine and tibia of a mouse treated as in Fig. 3D were taken before and after treatment with FOXO4 DRI.
- FOXO4 DRI lowered the curvature of the spine (Top) and increased the muscle mass (Bottom:
- Panel F shows old wildtype mice and 26w young Xpd TTD/TTD show reduced renal filtering capacity evident from elevated presence of plasma [Urea].
- Blood samples of 4-5 wildtype or Xpd TTD/TTD mice were collected at the indicated time-points and analyzed for the presence of Urea in the plasma fraction, as a marker for reduced renal filtering capacity.
- [Urea] was determined using a kit according to the manufacturer's protocol (Gentaur). * p ⁇ 0.05, ** pO.01. One-way ANOVA with Bonferroni post-test correction (GraphPad Prism). Note that in time the plasma [Urea] in wildtype animals increased.
- 26w young Xpd TTD/TTD mice show a similar loss of renal filtering capacity, validating Xpd TTD/TTD mice of this age as a model for loss of renal function.
- Panel G shows Xpd TTD/TTD mice having reduced fitness, indicated by impaired running wheel behavior and loss of hair.
- Six wildtype and Xpd TTD/TTD mice were placed in running wheel cages and the turns/day were continuously measured for four days.
- Panel H shows a treatment plan for addressing organ function, running wheel behavior and fur score of 26w old wildtype and Xpd TTD/TTD mice following Mock or FOX04 DRI treatment. 4-5 wildtype or Xpd TTD/TTD mice were placed in runningwheel cages and after allowing sufficient time for adjustment a baseline peripheral (cheek) blood sample was taken.
- mice After 5 days of recovery the mice were treated on day 0, 2 and 4 with 5mg/kg FOX04 DRI through intravenous injection. At 28d after the first injection another blood sample was taken and the mice were sacrificed right after. Panel I) shows FOX04 DRI reduces spontaneously occurring renal glomerulosclerosis and improved renal filtering capacity in fast aging Xpd TTD/TTD mice. 26w old wildtype and Xpd TTD/TTD mice were treated as in H). Kidney tissues were fixed in formalin right after sacrifice and embedded in paraffin. 5 m thick sections were stained in Periodic acid- Schiff (PAS) solution and scored for glomerulosclerosis as described in Adamcszak et al, 2003, JASN.
- PAS Periodic acid- Schiff
- Panel J shows a representative example of FOX04 DRI inducing an improvement in running wheel behavior of an Xpd TTD/TTD , but not wildtype mouse. Note that since
- Xpd TTD/TTD ice show strongly reduced running wheel capacity (Fig. 3G) the data are normalized to 100% for the respective running wheel behavior at the start of the experiment for each animal.
- Panel K) shows a quantification of the average changes in running wheel behavior in wildtype and
- Panels M) and N) show that FOX04 DRI counteracts the weight loss seen in fast aging Xpd TTD/TTD mice . More specifically, panel M) shows that fast aging Xpd TTD/TTD mice show reduced body weight compared to wildtype littermates (26w of age). Panel N) shows that FOX04 DRI improves the body weight specifically in the fast aging Xpd TTD/TTD mice.
- mice treated as in A)-L) were analyzed for changes in body weight over the time course of the experiment and the % change plotted.
- Panels 0)-R) show that FOX04 DRI does not induce toxicity to the hematopoietic system.
- Wildtype and fast aging Xpd TTD/TTD mice were treated as in Fig. 3H)-L) and the indicated blood parameters were determined at the end of the
- Figure 4 shows in panel A) that Doxorubicin induces SA-6-GAL expression in IMR90 cells in vitro. IMR90 cells were exposed twice to 0.5 ⁇ Doxorubicin with one day in between. SA-6-GAL activity was determined 7 days later.
- Panel B) shows that Doxorubicin induces expression of p l6 ink4a and FOX04 in IMR90 in vitro.
- Panel C) shows that FOX04 DRI selectively reduces viability of Doxorubicin-induced senescent cells in vitro.
- Panel D) shows that Doxorubicin induces IL-6, indicative of elevated SASP. This figure complements Fig.
- Panel E shows that doxorubicin induces senescence in vivo.
- mice were i.p. injected with lOmg/kg Doxorubicin and analyzed for RLUC on the indicated time-points. Changes in RLUC intensity for 5 animals are plotted for the indicated timepoints after injection.
- Panel G shows that FOX04 DRI lowers Doxorubicin-induced p l6 ink4a -driven RLUC expression indicative of senescence in vivo.
- Mice treated as indicated in Fig. 4F were analyzed for Bioluminescence.
- FOX04 DRI -treated animals showed markedly less Doxorubicin-induced p 16 ink4a -positive senescence compared to PBS-treated animals. This shows that, similar to data in Fig. 2 for spontaneous senescence, FOX04 DRI potently counteracts induced senescence by chemotherapy.
- H To improve the window for measuring healthspan changes, the treatment regime from Fig. 4F) was extended with one additional dose of Doxorubicin and milder FOX04 DRI treatment.
- Doxorubicin exposure in vivo.
- Figure 5 shows in panel A) a viability assay showing different sensitivity in three distinct metastatic melanoma cell lines to the
- Panels C-E) shows that Vemurafenib-treatment of resistant LOX-IMVI cells causes an upregulation in FOXO4 on mRNA (B), and protein levels (C+D). This identifies LOX-IMVI as a potential target for studying the effects of FOXO4 DRI as Vemurafenib itself only shows marginal effects in this line, but it does upregulate FOXO4.
- Panel F) shows that FOXO4 DRI shows synthetic lethality with RAF/MEK inhibitors in therapy resistant LOX-IMVI.
- the cells were co-treated with the pan-caspase inhibitor QVD-OPH to block caspase- dependent cell death. After 3 days the media was refreshed with new QVD- OPH containing media, but in the absence of FOXO4 DRI or Vemurafenib. At day 5 after initial treatment the cells were fixed and stained for
- Cytochrome C is present in the mitochondria in viable cells and is released into the cytosol when apoptosis is activated. Plotted are the number of cells showing release of Cytochrome C. This shows that while Vemurafenib fails to induce apoptosis in resistant cells FOXO4 overcomes this resistance and strongly synergizes with Vemurafenib to induce apoptosis. Panel G), right, shows that the reduction in viability by the combination of Vemurafenib and FOXO4 DRI leads to a loss in cell density.
- Panel H shows that Vemurafenib treatment promotes its own resistance.
- Panel I shows a similar experiment, but with the MEK inhibitor Trametinib as the second drug.
- Panel K shows that A375 which acquired resistance to Vemurafenib and LOX-IMVI which are intrinsically resistant to
- Vemurafenib can be resensitized to that drug by FOX04 DRI.
- Parental A375, Vemurafenib resistant A375 (survivors of 3wks chronic Vemurafenib exposure) and intrinsically resistant LOX-IMVI were treated with
- Vemurafenib or FOXO4 DRI alone or in combination While regular A375 were sensitive to both drugs individually, the Vemurafenib-resistant A375 and LOX-IMVI showed resistance to Vemurafenib.
- FOXO4 DRI strongly resensitized these resistant lines to Vemurafenib. Altogether these results show that FOXO4 is a potent anti-melanoma drug in many cases, but in case it fails to directly kills them it at least shows a synthetic lethal function with the most common chemotherapeutic anti-melanoma agent Vemurafenib and other RAF/MEK inhibitors.
- Panel L shows that the potency of FOXO4 DRI is enhanced by reactive chemical species such as ROS.
- LOX-IMVI were assessed for viabihty in the presence of FOXO4 DRI, the oxidative stress inhibitor N-Acetyl Cysteine (NAC) or the oxidative stressor H2O2. While FOXO4 DRI and H2O2 individually only marginally influenced LOX-IMVI viability, together they showed synthetic lethality. This result indicates that FOX04 DRI is effective against situations and diseases with excessive ROS. This result shows that FOX04 DRI is of use against any situation where there is excessive ROS, such as cancer or Parkinson's disease. Panels M+N) show that FOXO4 DRI potently
- melanoma organoid cultures were generated as described (Kruiswijk et al, Oncogene, 2015) and treated as in Fig. 5F for viability (M) or stained for Apoptosis using TUNEL (N) according to the manufacturer's protocol (Roche). These results show that not only on cell lines, but also on fresh human melanoma tissue FOXO4 DRI has a potent effect to enhance therapy sensitivity by Vemurafenib.
- Figure 6 shows in Panels A+B) that FOXO4 DRI is not only potent against melanoma, but also against breast cancer and Glioblastoma.
- GL261 cells were irradiated (8Gy), incubated with FOXO4 DRI of exposed to combined treatment.
- Panel C) shows that FOXO4 DRI potently and selectively induces apoptosis in FAC-surviving MCF7 cells. MCF7cells were treated with FAC.
- FAC stands for the clinically used breast cancer cocktail comprising 5'FluoroUracil, Doxorubicin (Adriamycin) and
- Cyclofosfamide It is generally noted in this context that it is found that FOXO4 expression is (i) increased in invasive breast carcinoma as compared to normal breast tissue and (ii) is increased in metastasized breast cancer as compared to primary site breast cancer. In addition, in the same context recurrence of breast cancer within five years is linked to an increase in FOX04 expression.
- Figure 7 shows that FOX04 DRI is safer to non-senescent cells than ABT-737 (an alleged senescent cell clearing compound) and shows higher potential for clearing senescent cells.
- A) Control IMR90 and IMR90 induced to senesce through lOGy IR were plated in triplicate for each condition in a 96-well plate. The next day the cells were incubated with the indicated doses of ABT-737 or FOX04 DRI. After 6 days viability was determined through AqeousOne celltiter assay (Promega) according to the
- Figure 8 is the same as Figure 7), except the compounds were added 3x at 1/3, with 3 days in between. (See also Figure 10 below for schedule, except there Doxorubicin was used). Also here ABT-737 proved to be toxic to non-senescent cells and only showed limited effectiveness on killing senescent cells, whereas FOX04 DRI proved to be safe to non-senescent cells and efficient in clearing senescent cells.
- Figure 9 is the same as Figures 7+8, but using IMR90 cells that were induced to senesce through chemotherapy (2x 0.5uM Doxorubicin) instead of IR. The same conclusions apply to Figures 7+8.
- Figure 10 shows the result of a similar experiment as in Figure 9, except the cells were treated with 3x 1/3 of the indicated doses with 2d apart. Also for this experiment it is clear that FOX04 DRI is safe to non- senescent IMR90, whereas ABT-737 lowers their viability already at lower doses. Moreover, FOX04 DRI shows a high selectivity at EC70 and EC50 for clearing senescent, but not non-senescent cells.
- Figure 11 shows that the reported compounds Quesrcetin and Dasatinib are inefficent towards clearing senescent IMR90. Experiments as in Figure 7, except the cells were incubated with Quesrcetin alone (Top) for the indicated concentrations, or a combination of O. luM Quesrcetin in addition to the indicated doses of Dasatinib (Bottom). There was no apparent difference in viability of these compounds for killing senescent vs. non-senescent cells.
- Figure 12 shows that an ASK1 inhibitor selectively induces apoptosis in senescent cells.
- the ASK1 inhibitor NQDI clearly reduces cell viability in senescent, but not non- senescent IMR90 fibroblasts. Senescent and non-senescent IMR90 fibroblasts were incubated with the indicated concentrations of NQDI and after 6 days cell viability was determined by using the CellTiter 96®
- AQueous One Solution Cell Proliferation Assay B) Sequential rounds of NQDI enhance its effectivity towards senescent cell clearance. Senescent and non-senescent IMR90 fibroblasts were plated for longitudinal cell density measurement using the xCELLigence system (ACEA Biosciences, Inc., San Diego, CA). Once baseline measurements were established the cells were incubated on three consecutive days with 2 ⁇ NQDI (24, 48 and 72h, respectively). Cell density after time of plating was followed as indicated. The plot shows cell density data normalized to the density of that condition at the beginning of the experiment. C) The histogram shows a quantification of the change in cell density at the end of the measurement (96h) over baseline.
- Figure 13 shows that a SYK inhibitor selectively induces apoptosis in senescent cells in vitro.
- the graph shows % viability over Mock treated senescent and non-senescent IMR90 cells.
- the SYK inhibitor R406 selectively reduces senescent cell viability.
- peptide preferably relates to peptides having less than 200 amino acid residues, more preferably less than 100 amino acid residues and most preferably less than 50 or 40 amino acid residues.
- a peptide according to the invention may consist of less than 30, 29, 28, 27, 26, 25, 24, 23, 22, 21 or 20 amino acid residues.
- a peptide of the invention contains at least 4, 5, 6, 7, 8, 9 , 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 , 20, 25, 30, 35, 40 or more amino acid residues.
- a peptide according to the invention may further comprise an amino acid sequence that facilitates entry of the peptide into a cell, such as the amino acid sequence
- D-Retro- Inverso Isoform refers to the D-Retro- Inverso Isoform, in which the amino acid sequence is reversed and placed in the D- instead of the L-isoform, in particular with reference to the FOXO4 protein, or a peptide fragment thereof.
- D-isoform refers to an amino acid sequence in which at least part of the amino acid residues have the molecular spatial configuration referred to as "D" (Latin dexter; right).
- a peptide of the invention preferably contains at least one, more preferably at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, or more D- amino acid residues.
- a peptide of the invention preferably contains at least 50%, more preferably at least 75%, even more preferably at least 90, 95, 98 or 99%, most preferably 100% D-amino acid residues.
- a DRI peptide according to the invention may contain a combination of L-amino acid residues and D-amino acid residues, or may consist entirely of D-amino acid residues.
- % sequence identity is defined herein as the percentage of nucleotides in a nucleic acid sequence, or amino acids in an amino acid sequence, that is identical with the nucleotides, resp. amino acids, in a nucleic acid or amino acid sequence of interest, after aligning the sequences and optionally introducing gaps, if necessary, to achieve the maximum percent sequence identity. Methods and computer programs for alignments are well known in the art. Sequence identity is calculated over substantially the whole length, preferably the whole (full) length, of an amino acid sequence of interest. The skilled person understands that consecutive amino acid residues in one amino acid sequence are compared to consecutive amino acid residues in another amino acid sequence. Preferably, the configuration of an amino acid residue, for example D or L, is not relevant for determining amino acid sequence identity. For example, a D-Val exhibits sequence identity to a L-Val in the context of the invention.
- apoptosis refers to a mechanism of cell death affecting single cells, marked by shrinkage of the cell, condensation of chromatin, and fragmentation of the cell into membrane-bound bodies that are eliminated by phagocytosis.
- apoptosis is often used
- apoptosis-inducing activity refers to the intrinsic property of a compound to selectively invoke apoptosis in a (i) particular cell type and/or (ii) cell in a particular stage of development or differentiation, due to internal or external stimuli.
- the skilled person is aware of the existence of in vitro standard assays for determining the extent of apoptosis in a cell culture, for example tests that assess levels of cytoplasmic Cytochrome C (marker for apoptosis) and levels of TUNEL (marker for apoptosis). Using these standard assays, the skilled person can easily assess and compare the apoptosis-inducing activity of different compounds with regard to different cell type or cells in a different
- apoptosis assay is an Annexin V assay and a cleaved caspase-3 staining. To detect cell viability, which is essentially the opposite of apoptosis, an Annexin V assay and a cleaved caspase-3 staining. To detect cell viability, which is essentially the opposite of apoptosis, an Annexin V assay and a cleaved caspase-3 staining. To detect cell viability, which is essentially the opposite of apoptosis, an Annexin V assay and a cleaved caspase-3 staining. To detect cell viability, which is essentially the opposite of apoptosis, an Annexin V assay and a cleaved caspase-3 staining. To detect cell viability, which is essentially the opposite of apoptosis, an Annexin V assay and a cleaved caspase-3 staining. To detect cell viability, which
- MTT assay colorimetric assay for assessing cell viability
- an ATP-detection assay a real-time cell density (for instance xCELLigence) assay or a colony formation assay
- a real-time cell density for instance xCELLigence
- a colony formation assay can be used.
- senescent cells should be interpreted in the context of cellular senescence.
- senescent cells includes cells that are characterized by having an essentially permanent growth arrest. Senescent cells are essentially irresponsive to proliferation-cues. For recognition or detection of senescent cells, molecular markers may be used. Several markers for senescent cells have been developed. The term
- SASP senescence-associated secretory phenotype
- SASP markers such as IL1, IL6 and/or IL8
- IL1 IL1
- IL6 IL6
- IL8 senescence-associated secretory phenotype
- the skilled person knows when a cell is considered to be in essentially permanent growth arrest, for example by assessing EdU incorporation and/or Ki67 positivity.
- the phenomenon of senescence can occur at the end of the proliferative lifespan of normal cells or in normal or tumor cells in response to, for example, chemotherapeutic agents, radiation, DNA damage or other cellular insults.
- senescent cells from inter alia terminally differentiated cells, which in general do not have the characteristics of senescent cells as described hereinabove.
- a senescent cell is characterized by having an essentially permanent or permanent growth arrest, preferably indicated by a lack of proliferation markers in the presence of growth factors.
- a senescent cell is characterized by (i) having an essentially permanent or permanent growth arrest, preferably indicated by a lack of proliferation markers in the presence of growth factors, (ii) p l6 INK4a activation and/or expression, and/or p21 ci P 1 , preferably p53/p21 ci P 1 , activation and/or expression, and/or (iii) FOX04 activation and/or
- a senescent cell is characterized by (i) having an essentially permanent or permanent growth arrest, (ii) p l6 INK4a activation and/or expression and/or p21 ci P 1 , preferably p53/p21 ci P 1 , activation and/or expression, (iii) expression of SA-B-Gal and/or (iv) FOX04 activation and/or expression.
- expression refers to an increase in gene expression products (RNA) or an increase in protein products as compared to non-senescent cells.
- the increase in expression as compared to non-senescent cells is preferably with a factor of at least 1.05, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 3.0, 4.0, 5.0, 6.0 or higher.
- SA-B-Gal can be measured by applying an enzymatic assay using an endogenous enzyme in the lysosomes of senescent cells. Such an assay is normally performed under acidic conditions on fresh material (Dimri et al, PNAS, vol. 92, p. 9363-9367 ((1995). Further
- non-senescent cell refers to a "normal” cell, i.e. a cell which in response to growth factors is able to divide, but does not do so in absence of growth factors. The latter would include a cancer cell. Senescent cells are essentially irresponsive to proliferation-cues.
- a non- senescent cell can be a young human IMR90 lung fibroblast cell ((ATCC® CCL-186TM, preferably less than 50 population doublings)) or a normal or healthy cell from a subject that is to be treated.
- the control can be from a different subject or a pool of such subjects, for instance a subject or group of subjects having an age between 18 and 30 years.
- the control is preferably from the same tissue and/or organ.
- the term "increased” refers to an increase in gene expression products (RNA) or an increase in protein products as compared to a control.
- the increase in expression as compared to a control is preferably with a factor of at least 1.05, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 3.0, 4.0, 5.0, 6.0 or higher.
- the expression of p53 is the expression of active pSerl5-p53, which can inter alia be detected and quantified by immunofluorescence and/or
- the control is preferably a normal or healthy cell responding to growth factors and able to divide, but does not do so in absence of growth factors.
- a control cell can be a human IMR90 lung fibroblast cell ((ATCC® CCL-186TM) or a cell from a subject that is to be treated.
- the control can be from a different subject than the one to be treated or a pool of such subjects, for instance a subject or pool of subjects having an age between 18 and 30.
- the control is preferably from the same tissue and/or organ.
- a target cell as described in this context is further characterized by having oxidative stress, detectable by markers activated upon oxidative stress, such as superoxide dismutases SOD 1 (Anti- Superoxide Dismutase 1 antibody, ab20926, Abeam, UK) and SOD2 (Anti- SOD2 antibody, Stressgen Biotech, Victoria, BC), catalase (Anti-Catalase antibody, ab 16731, Abeam, UK) and/or phosphorylated JNK
- a control as described hereinabove can be used.
- a target cell as described in this paragraph may additionally have one or more of the senescence markers as described herein.
- composition refers to a composition that is made under conditions such that it is suitable for administration to mammals, preferably humans, e.g., it is made under GMP conditions.
- a pharmaceutical composition according to the invention may comprise pharmaceutically acceptable excipients, e.g., without limitation, stabilizers, bulking agents, buffers, carriers, diluents, vehicles, solubilizers, and binders.
- pharmaceutically acceptable excipients e.g., without limitation, stabilizers, bulking agents, buffers, carriers, diluents, vehicles, solubilizers, and binders.
- composition according to the invention is preferably adapted for parenteral administration.
- chemotherapeutic agent refers to a compound that inhibits or prevents the viability and/or function of cells, and/or causes destruction of cells (cell death), and/or exerts anti-tumor/anti- proliferative effects, for example, prevents directly or indirectly the development, maturation or spread of tumor cells.
- the term also includes agents that cause a cytostatic effect only and not a mere cytotoxic effect.
- chemotherapeutic agent includes alkylating agents such as platinum drugs (e.g.
- cisplatin, carboplatin, and oxalaplatin antimetabolites such as 5-fluorouracil (5-FU), 6-mercaptopurine (6-MP), capecitabine (Xeloda), cladribine, clofarabine, cytarabine (Ara-C), floxuridine, fludarabine, gemcitabine (Gemzar), hydroxyurea and methotrexate, anti-tumor antibiotics, preferably Doxorubicin, topoisomerase inhibitors, mitotic inhibitors, corticosteroids, anti-angiogenic agents, tyrosine kinase
- inhibitors protein kinase A inhibitors, members of the cytokine family, radioactive isotopes, inhibitors of the RAF, MEK or ERK families of kinases including, but not limited to vemurafenib, dabrafenib, Raf265, selumetinib and trametinib, and agents commonly used in treatment of melanoma, preferably metastatic melanoma, such as vemurafenib, dabrafenib, Raf265, selumetinib and trametinib.
- Vemurafenib, Dabrafenib and RAF265 are RAF inhibitors; Trametinib and Selumetinib are MEK inhibitors. Also included as chemotherapeutic agents in the context of this invention are Taxanes such as Paclitaxel or Doxetaxel.
- diseases or conditions wherein the removal of senescent cells is beneficial refers to any disease or condition in a mammalian, preferably human, subject wherein removal or clearance or reduced viability of senescent cells is beneficial to the subject suffering from said disease or condition.
- the term encompasses the situation wherein senescent cells are one, or the only, cause of a disease.
- the term further relates to the situation wherein senescent cells might become, in the future, the cause of a disease or condition in said subject.
- the treatment of a disease or condition wherein the removal of senescent cells is beneficial is a disease or condition prevented or preventable by removing senescent cells.
- chemotherapeutic agents and radiation therapy induce cellular senescence. It is advantageous to remove these senescent cells in order to prevent the onset of diseases or conditions associated with cellular senescence.
- the term further encompasses diseases or conditions wherein removal of senescent cells alleviates or reduces symptoms of a disease or condition. It is clear to the skilled person that said removal of senescent cells is beneficial if inter alia the disease or condition can be healed, prevented or if the symptoms of the disease or condition or reduced or alleviated.
- a peptide and inhibitor according to the invention have apoptosis-inducing activity in senescent cells.
- a peptide or inhibitor according to the invention removes, kills, clears or reduces the viability of at least 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 40, 50, 60, 70 or 80% of the senescent cells in a mammalian, preferably human, subject, preferably of the senescent cells in an organ or tissue of a mammal, preferably human, subject.
- a mammalian, preferably human, subject preferably of the senescent cells in an organ or tissue of a mammal, preferably human, subject.
- numerous diseases are linked to senescent cells and treatment for such diseases benefits from the removal of senescent cells.
- the disease or condition wherein the removal of senescent cells is beneficial is a disease or condition that is associated with increased levels of reactive oxygen species or reactive nitrogen species, preferably reactive oxygen species such as H2O2, in a subject, as compared to a subject not having such a disease or disorder.
- a disease or disorder is preferably selected from the list formed by cancer, preferably melanoma, more preferably metastatic melanoma, chronic inflammatory diseases or neurological diseases such as Alzheimer's disease or Parkinson's disease. It was found that the apoptosis- inducing activity of a peptide, pharmaceutical composition or nucleic acid according to the invention was enhanced by the aforementioned reactive species (vide Figure 5L).
- the increase in the level of reactive oxygen species and/or reactive nitrogen species is preferably with a factor of at least 1.05, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 3.0, 4.0, 5.0, 6.0 or higher in a sample representing a disease phenotype, such as a cancer sample, of a subject.
- a sample representing a disease phenotype such as a cancer sample
- the disease or condition wherein the removal of senescent cells is beneficial is selected from the group formed by atherosclerosis, chronic inflammatory diseases such as arthritis or athrosis, cancer, osteoarthritis, diabetes, diabetic ulcers, kyphosis, scoleosis, hepatic insufficiency, cirrhosis, Hutchinson- Gilford progeria syndrome (HGPS), laminopaties, osteoporosis, dementia, (cardio)vascular diseases, obesity, metabolic syndrome, acute myocardial infarction, emphysema, insulin sensitivity, boutonneuse fever, sarcopenia, neurodegenerative diseases such as Alzheimer's , Huntington's or Parkinson's disease, cataracts, anemia, hypertension, fibrosis, age-related macular degeneration, COPD, asthma, renal insufficiency, incontinence, hearing loss such as deafness, vision loss such as blindness, sleeping disturbances, pain such as joint pain or leg pain,
- inflammation preferably chronic inflammation, in a mammalian, preferably human, subject, wherein said inflammation is provided or mediated by senescent cells.
- said senescent cells providing or mediating said inflammation at least partially co-localize in the same organ, more
- the organ preferably tissue, affected by said disease or condition.
- diseases or conditions associated with the presence of senescent cells refers to any disease or condition in a mammalian, preferably human, subject wherein the presence of senescent cells, or presence of cellular senescence, in a mammalian, preferably human, subject is linked to said disease or condition in said subject.
- association with or “linked to”, as used herein, refer to a connection between the presence of senescent cells, or presence of cellular senescence, and said disease or condition.
- association with or “linked to” can inter alia refer to the senescent cells or cellular senescence (i) as the at least partial cause of a disease or condition, (ii) or as a symptom of a disease or disorder.
- the disease or condition associated with the presence of senescent cells is selected from the group formed by atherosclerosis, chronic inflammatory diseases such as arthritis or athrosis, cancer, osteoarthritis, diabetes, diabetic ulcers, kyphosis, scoleosis, hepatic insufficiency, cirrhosis, Hutchinson-Gilford progeria syndrome (HGPS), laminopaties, osteoporosis, dementia, (cardio)vascular diseases, obesity, metabolic syndrome, acute myocardial infarction, emphysema, insulin sensitivity, boutonneuse fever, sarcopenia,
- chronic inflammatory diseases such as arthritis or athrosis, cancer, osteoarthritis, diabetes, diabetic ulcers, kyphosis, scoleosis, hepatic insufficiency, cirrhosis, Hutchinson-Gilford progeria syndrome (HGPS), laminopaties, osteoporosis, dementia, (cardio)
- neurodegenerative diseases such as Alzheimer's , Huntington's or
- Parkinson's disease cataracts anemia, hypertension, fibrosis, age-related macular degeneration, COPD, asthma, renal insufficiency, incontinence, hearing loss such as deafness, vision loss such as blindness, sleeping disturbances, pain such as joint pain or leg pain, imbalance, fear,
- a particularly preferred disease or condition wherein the removal of senescent cells is beneficial is a disease or condition associated with or linked to inflammation, preferably chronic inflammation, in a mammalian, preferably human, subject, wherein said inflammation is provided or mediated by senescent cells.
- said senescent cells providing or mediating said inflammation at least partially co-localize in the same organ, more preferably in the same tissue, as the organ, preferably tissue, affected by said disease or condition.
- condition refers inter alia to sleeping disturbances, pain such as joint pain or leg pain, imbalance, fear, depression, breathlessness, weight loss, hair loss, muscle loss, loss of bone density, frailty and reduced fitness.
- degenerative disease relates to diseases or conditions in which the function and/or structure of a tissue or organ will increasingly deteriorate over time - normally with increasing age -, resulting in a so-called "loss-of-function". The skilled person understands that a degenerative disease may occur in younger people, for-example teens and young adults, as a result of a genetic disorder or unhealthy life-style.
- degenerative disease also includes conditions associated with aging such as sleeping disturbances, pain such as joint pain or leg pain, imbalance, fear, depression, breathlessness, weight loss, hair loss, muscle loss, loss of bone density, frailty and reduced fitness.
- administering refers to introducing a peptide, inhibitor, preferably NQDI or R406, or
- compositions in a mammalian subject, preferably a human, in a pharmaceutically acceptable form and in a pharmaceutically effective amount by a route of administration such as (i) oral administration, preferably in the form of tablets, capsules, syrups, suspensions, and the like; (ii) administration by injection, preferably in the form of a liquid such as water and administered parenterally, such as intravenous, intramuscular, intraperitoneal, subcutaneous, intra- arterial, intracerebral and the like, (hi) transdermal, preferably via topical administration for example in the form of a cream, jelly, powder, or patch; (iv) inhalation, for example in the form of inhalation powders, sprays, suspensions, and the like, and (v) rectal.
- a route of administration such as (i) oral administration, preferably in the form of tablets, capsules, syrups, suspensions, and the like; (ii) administration by injection, preferably in the form of a liquid such as water and administered parenterally,
- compositions or kit are preferably administered parentally, e.g. in a liquid or fluid such as water.
- adjuvant refers to a pharmacologically active compound, preferably a peptide or inhibitor, both according to the invention, that is co-administered together with another pharmacologically active compound, preferably a chemotherapeutic agent.
- An adjuvant may be administered to counter, alleviate or reduce side-effects resulting from a pharmacologically active compound providing the main treatment.
- a peptide or inhibitor according to the invention together with a chemotherapeutic agent as part of a single dosage form (such as a pharmaceutical composition according to the invention) or as multiple dosage forms, e.g. multiple unit dose wherein the distinct products are packaged together, but formulated separately, e.g. in separate containers.
- the peptide or inhibitor according to the invention may be administered prior to or before, consecutively with or during, or following or after the administration of a chemotherapeutic agent.
- both a peptide or inhibitor according to the invention and a chemotherapeutic agent are administered via any of the routes as described above and are preferably administered in such a way that the whole provides a synergistic and/or desired effect.
- single dosage form refers to a dosage form wherein all compounds are present in a single formulation
- multiple dosage form refers to at least two different - and separate - dosage forms.
- a particularly preferred multiple dosage form e.g. in the context of a kit according to the invention, is a multiple unit dose wherein the distinct compounds are packaged together, but formulated separately, e.g. in separate containers.
- subject refers to an animal, preferably a mammal such as a human or dog, most preferably a human, who is in need of treatment of a disease or condition wherein the removal of senescent cells is beneficial or who is suffering or suspected to suffer from a disease or condition wherein the removal of senescent cells is beneficial.
- a subject is at least 30 or at least 40 years old. More preferably, the subject is at least 50 years old.
- a therapeutically or therapeutically effective amount refers to an amount, which has a therapeutic effect or is the amount required to produce a therapeutic effect in a subject.
- a therapeutically or pharmaceutically effective amount of a peptide or inhibitor according to the invention or a pharmaceutical composition of the invention is the amount required to produce a desired therapeutic effect as may be judged by clinical trial results, model animal studies, and/or in vitro studies.
- the pharmaceutically effective amount depends on several factors, including but not limited to, characteristics of the subject (for example height, weight, sex, age and medical history), the specific disease and the particular type of compound that is used.
- radiation therapy refers to an exposure to ionizing radiation, preferably therapeutic, and may include, for example, external beam radiotherapy, photon radiotherapy including X-rays, electron radiotherapy, proton radiotherapy, carbon ion radiotherapy, lithium ion radiotherapy, silicon ion radiotherapy, helium ion radiotherapy,
- radioisotope therapy injectable isotopes, e.g., isotopes adhered to or within or admixed with a matrix of any sort.
- the radiation therapy is preferably photon radiotherapy such as X-rays.
- cancer includes cancers such as adrenocortical carcinoma, anal cancer, appendix cancer, astrocytomas, basal cell carcinoma, bile duct cancer, bladder cancer, bone cancer, brain stem glioma, brain tumor, breast cancer, bronchial tumors, carcinoid tumor, cardiac (heart) tumors, central nervous system tumor, cervical cancer, chordoma, colon cancer, colorectal cancer, craniopharyngioma, ductal carcinoma, embryonal tumors, endometrial cancer, ependymoma,
- cancers such as adrenocortical carcinoma, anal cancer, appendix cancer, astrocytomas, basal cell carcinoma, bile duct cancer, bladder cancer, bone cancer, brain stem glioma, brain tumor, breast cancer, bronchial tumors, carcinoid tumor, cardiac (heart) tumors, central nervous system tumor, cervical cancer, chordoma, colon cancer, colorectal cancer, craniopharyngio
- esophageal cancer esthesioneuroblastoma, ewing sarcoma, extracranial germ cell tumor, extragonadal germ cell tumor, extrahepatic bile duct cancer, eye cancer, fallopian tube cancer, gallbladder cancer, gastric
- stomach gastrointestinal carcinoid tumor, germ cell tumor, head and neck cancer, hepatocellular (liver) cancer, hypopharyngeal cancer, kidney cancer, lung cancer, lip and oral cavity cancer, male breast cancer, metastatic squamous neck cancer, mouth cancer, nasal cavity and paranasal sinus cancer, ovarian cancer, pancreatic cancer, parathyroid cancer, penile cancer, prostate cancer, rectal cancer, salivary gland cancer, skin cancer, small intestine cancer, stomach (gastric) cancer, thyroid cancer, lymphomas, urethral cancer, vaginal cancer, and/or vulvar cancer.
- a particularly preferred cancer is a cancer having a high FOX04 expression level.
- the FOX04 expression is at least a factor 1.05, preferably a factor 1.1, more preferably a factor 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 3.0, 4.0, 5.0, or a factor 6.0 higher than FOX04 expression in non-cancerous cells, preferably non-cancerous or healthy or normal cells, more preferably non-cancerous, or healthy or normal cells in the direct vicinity of said cancer.
- resistant cancer is used interchangeable with the term “cancer resistant to therapy” and refers to both (i) a cancer that is resistant to at least one chemotherapeutic agent, wherein the resistance is acquired after treatment with said at least one chemotherapeutic agent, i.e. a resistance-acquired cancer and (ii) a cancer that is resistant to at least one chemotherapeutic agent wherein the resistance is de novo, i.e. a de novo resistant cancer wherein the resistance is present prior to treatment with said at least one chemotherapeutic agent.
- resistant cancer refers to cancer cells that are able to survive in the presence of at least one chemotherapeutic agent whereas a normal, non-resistant cancer cell would either show signs of cell toxicity, cell death or cellular senescence.
- the skilled person can easily assess whether a cancer is a resistant cancer, namely by assessing cell viability or apoptosis-inducing activity after bringing a suitable chemotherapeutic agent in contact with a cancer originating from a subject.
- the resistant cancer may be a peptide or inhibitor of ASK1 according to the invention.
- the resistant cancer is preferably not a lymphoma or myeloid leukemia. More preferably, the resistant cancer is a resistant adrenocortical carcinoma, anal cancer, appendix cancer, astrocytomas, basal cell carcinoma, bile duct cancer, bladder cancer, bone cancer, brain stem glioma, brain tumor, breast cancer, bronchial tumors, carcinoid tumor, cardiac (heart) tumors, central nervous system tumor, cervical cancer, chordoma, colon cancer, colorectal cancer, craniopharyngioma, ductal carcinoma, embryonal tumors, endometrial cancer, ependymoma, esophageal cancer, esthesioneuroblastoma, ewing sarcoma, extracranial germ cell tumor, extragonadal germ cell tumor, extrahepatic bile duct cancer, glioblastoma, eye cancer, fallopian tube cancer, gall
- said resistant cancer is preferably metastatic melanoma, breast cancer or glioblastoma, more preferably metastatic melanoma, and is resistant to treatment with at least one chemother apeutic agent, preferably a RAF, MEK or ERK inhibitor including RAF265, trametinib, dabrafenib, selumetinib, vemurafenib and/or trametinib, more preferably vemurafenib and/or trametinib.
- RAF RAF
- MEK or ERK inhibitor including RAF265, trametinib, dabrafenib, selumetinib, vemurafenib and/or trametinib, more preferably vemurafenib and/or trametinib.
- the chemotherapeutic agent to which said cancer is resistant is preferably a chemotherapeutic composition comprising 5'FluoroUracil, Doxorubicin (Adriamycin) and Cyclofosfamide (FAC). Every possible combination of a resistant cancer and at least one chemotherapeutic agent, preferably a RAF, MEK or ERK inhibitor, is envisaged herein.
- a particularly preferred resistant cancer is a cancer with an increased FOX04 expression level.
- a resistant cancer has an increased FOX04 expression level if its FOX04 expression level is at least a factor 1.05, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 3.0, 4.0, 5.0, 6.0 or higher than the FOX04 expression level of a non-resistant cancer.
- expression preferably refers to gene expression products (RNA) or protein products. The skilled person is well aware of methods for measuring expression product levels.
- counteracting refers to the effect of opposing, counteracting, inhibiting, blocking, delaying, or reducing the symptom or phenomenon in a subject.
- counteracting refers to counteracting.
- sensitizing refers to the process of making resistant cancer cells susceptible to the effect of a chemotherapeutic agent. Said term refers to the process wherein a cancer cell moves from a state in which it is able to survive in the presence of at least one
- chemotherapeutic agent to a state wherein it shows signs of cell toxicity, cell death and/or cellular senescence as a result of the effect of said at least one chemotherapeutic agent.
- sensitizing encompasses and preferably relates to "re-sensitizing", the latter of which is applicable in the context of sensitizing a resistance-acquired cancer.
- the term "sensitizing” refers to increasing the susceptibility of a resistant cancer in a subject towards at least one chemotherapeutic agent said cancer is resistant to, such that, upon administration of said at least one chemotherapeutic agent, cell viability of said resistant cancer is reduced by at least 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45 ,50, 60, 70, 75, 80 or 90% as compared to the cell viability of a non-sensitized resistant cancer equivalent to said resistant cancer treated with said at least one chemotherapeutic agent.
- Cell viability is measurable by a MTT assay, an ATP-detection assay, a cell density assay or a colony formation assay.
- the term "sensitizing” may refer to increasing the susceptibility of a resistant cancer in a subject towards at least one chemotherapeutic agent said cancer is resistant to, such that, upon administration of said at least one chemotherapeutic agent, the level of apoptosis is increased by at least 25, 30, 35, 40, 45, 50, 55, 60, 70, 80, 90, 100, 200 or 300% as compared to the level of apoptosis in a non-sensitized resistant cancer equivalent to said resistant cancer treated with said at least one chemotherapeutic agent.
- compound refers to any kind of molecule, preferably a pharmacologically active molecule such as a peptide or inhibitor according to the invention or a chemotherapeutic agent.
- tainer refers to a receptacle adapted for holding or storing a compound, preferably a compound (peptide or inhibitor according to the invention or a chemotherapeutic agent)
- formulation is a formulation adapted for parenteral administration.
- the receptacle may suitable be a vial, bottle, jar, or flexible packaging.
- kits should not be explained in such a way that the container only contains, or consists of, a peptide or inhibitor according to the invention or a chemotherapeutic agent.
- the container may suitably contain other compounds or substances than a peptide or inhibitor according to the invention or a chemotherapeutic agent.
- inhibitor refers to any compound, natural or synthetic, which can reduce the activity of a gene product.
- an inhibitor may inhibit the activity of a protein that is encoded by a gene either directly or indirectly.
- Direct inhibition can be obtained, for instance, by binding to a protein and thereby preventing the protein from binding a target (such as a binding partner) or preventing protein activity (such as enzymatic activity).
- Indirect inhibition can be obtained, for instance, by binding to a protein's intended target, such as a binding partner, thereby blocking or reducing activity of the protein.
- the inhibitor is an ASK1 inhibitor or SYK inhibitor. More preferably, said ASK1 inhibitor and SYK inhibitor are kinase inhibitors, i.e. inhibit kinase activity of ASK1 and SYK. It is particularly preferred that said ASK1 inhibitor and SYK inhibitor are kinase inhibitors that also exhibit apoptosis-inducing active in senescent cells. Most preferred are ASK1 inhibitor NQDI and SYK inhibitor R406. The skilled person is well- aware of standard inhibition assays wherein screening for target
- the ASK1 inhibitor or SYK inhibitor is an interfering RNA molecule, preferably siRNA, inhibiting protein expression of ASK1 or SYK.
- the inhibitor of ASK1 or SYK is a morpholino oligomer.
- interfering RNA molecule refers to all RNA or RNA-like molecules that can interact with RISC and participate in RISC-mediated changes in gene expression.
- interfering RNA molecules that can interact with RISC include short hairpin RNAs
- siRNAs single-stranded siRNAs
- miRNAs microRNAs
- morpholino oligomer refers to a polymer of the genetic nitrogeneous bases, adenine, guanine, cytosine, and thymine, in which the nitrogenous bases are linked to a 6-membered morpholine ring; as opposed to ribose or deoxyribose as in RNA or DNA.
- forkhead box 04 (FOXO4) gene refers to the protein- coding gene having Gene ID 4303 (Genbank 7-Dec-2014), and NCBI
- NC_000023.11 and consisting of 7386 nucleotides, also known as AFX; AFXl; or MLLT7.
- FOXO4 peptide and “FOXO4 protein”, which terms are interchangeably herein, refer to a protein translated from a transcript of the forkhead box protein O4 gene, referred to as the Forkhead box protein 04. Isoform 1 ( Figure 14) of this protein is encoded by transcript variant 1 of the FOXO4 gene ( Figure 16). Isoform 2 ( Figure 15) is encoded by transcript variant 2.
- the term “FOXO4 DRI Peptide” refers to the reverse-inverso peptide or protein of the "FOXO4 peptide", preferably, but not necessarily, in an all-D amino acid configuration, and preferably in the form of the isoform 1 protein.
- ASKl refers to mammalian, preferably human apoptosis signal-regulating kinase 1 (ASKl), also known as mitogen- activated protein kinase kinase kinase 5 (MAP3K5).
- ASKl is a MAP kinase and it is suggested that it activates c-Jun N-terminal kinase (JNK).
- JNK mitogen- activated protein kinase kinase kinase 5
- JNK c-Jun N-terminal kinase
- the amino-acid sequences of human and mouse ASKl are known in the art. For example, human ASKl is accessible in Genbank under Acc. No. NP_005914 (03-MAY-2014).
- SYK refers to mammalian, preferably human Spleen Tyrosine Kinase.
- SYK is a member of the Syk family of tyrosine kinases.
- the amino-acid sequences of human and mouse ASKl are known in the art. For example, the amino-acid sequence of human SYK is accessible in Genbank under Acc. No. P43405 (26 Nov 2014).
- NQDI refers to a molecule also
- NQDI 1 having the chemical name 2,7-Dihydro- 2,7-dioxo-3H-naphtho[l,2,3-de]quinoline-l-carboxyhc acid ethyl ester and having the structural formula:
- NQDI is freely available on the market (e.g. Tocris, NQDI 1, Cat. No. 4429). NQDI is preferably formulated in a pharmaceutically acceptable form, more preferably a pharmaceutically accepted form adapted for parental
- R406 refers to a SYK inhibitor.
- R406 is also referred to as 6-(5-fluoro-2-(3,4,5-trimethoxyphenylamino)pyrimidin-4- ylamino)-2,2-dimethyl-2H-pyrido[3,2-b] [l,4]oxazin-3(4H)-one
- R406 is available on the market (e.g. via Selleckchem, US). R406 is preferably formulated in a pharmaceutically acceptable form, more preferably a pharmaceutically accepted form adapted for parental
- R788 also referred to as
- Fostamatinib is a prodrug of R406.
- R406 is a liver degradation product of R788.
- R788 can be administered to a mammalian, preferably human, subject in order to provide R406 to said subject. From a function point of view, the skilled person understands that the term “R406” can be interchanged with "R788” or any other prodrug that releases R406 upon interaction with a
- R788 is available on the market (e.g. Santa Cruz Biotechnology, CAS 901119-35-5) and is known under the chemical name 6- (5 -fluoro-2 -(3 ,4,5 -trimethoxyphenylamino)pyrimidin-4-ylamino) -2 , 2 - dimethyl-3-oxo-2,3-dihydropyrido[3,2-b] [l,4]oxazin-4-yl)methyl dihydrogen phosphate.
- 6- 5 -fluoro-2 -(3 ,4,5 -trimethoxyphenylamino)pyrimidin-4-ylamino) -2 , 2 - dimethyl-3-oxo-2,3-dihydropyrido[3,2-b] [l,4]oxazin-4-yl)methyl dihydrogen phosphate.
- 6- 5 -fluoro-2 -(3 ,4,5 -trimethoxyphenylamino)pyrimidin-4-ylamino
- the SASP may affect and alter the tissue microenvironment and potentially the systemic milieu by secreting pro-inflammatory cytokines or chemokines, growth factors and/or matrix degrading proteases.
- the SASP is generally formed by factors such as IL-6 and IL-8; a variety of monocyte chemo attractant proteins and macrophage inflammatory proteins; and
- inflammation regulating proteins such as granulocyte/macrophage colony- stimulating factor.
- the most highly conserved feature between the SASP of different senescent cells are the pro-inflammatory cytokines. It is the secretion of such factors that cause chronic inflammation, at least locally and possibly systemically. It is commonly accepted that chronic
- Peptides of the invention relate inter alia to a peptide comprising or consisting of an amino acid sequence having at least 50% sequence identity to a peptide having the amino acid sequence SEIAQSILEAYSQNGW or LTLRKEPASEIAQSILEAYSQNGWANRRSGGKRP; or a fragment of said peptide comprising or consisting of the amino acid sequence
- SEIAQSILEAYSQNGW wherein said peptide, preferably said amino acid sequence, and said fragment contain at least one D-amino acid residue, preferably all-D; and wherein said peptide, preferably said amino acid sequence, and said fragment exhibit apoptosis-inducing activity in senescent cells.
- the invention further envisages peptidomimetics of the peptides of the invention.
- the present invention relates to a peptide comprising or consisting of an amino acid sequence having at least 50% sequence identity to a peptide having the amino acid sequence
- LTLRKEPASEIAQSILEAYSQNGWANRRSGGKRPPPRRRQRRKKRG or a fragment of said peptide comprising or consisting of the amino acid sequence SEIAQSILEAYSQNGW; wherein said peptide, preferably said amino acid sequence, and said fragment contain at least one D-amino acid residue, preferably all-D; and wherein said peptide, preferably said amino acid sequence, and said fragment exhibit apoptosis-inducing activity in senescent cells.
- a peptide according to the invention may further comprise an amino acid sequence that is not derived from the FOX04 peptide (or from the FOX04 DRI peptide, meaning that the sequence does not occur therein), such as the sequence "PPRRRQRRKKRG", which facilitates entry of the peptide sequence having the apoptosis-inducing activity into a cell.
- this amino acid sequence can be replaced by any other amino acid sequence that facilitates entry of the peptide into a cell. In other words, said amino acid sequence is not suggested to have any apoptosis-inducing activity.
- Said amino acid sequence preferably contains at least one D-amino acid residue, more preferably all amino acid residues in said amino acid sequence are D-amino acid residues.
- a peptide according to the invention selectively exhibits apoptosis-inducing activity in senescent cells, i.e. not in non-senescent cells.
- a peptide according to the invention favors apoptosis in senescent cells over apoptosis in non-senescent cells by at least a factor 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.5, 3, 4, 5 or higher.
- a peptide according to the invention is preferably isolated.
- a peptide according to the invention exhibits apoptosis-inducing activity in senescent cells.
- a cell culture of senescent cells e.g. obtained by subjecting said cell culture to ionizing radiation or a chemotherapeutic agent and non-senescent cells.
- the presence of senescence cells can be established by testing for SA-B-GAL.
- the second step is to administer to both cell cultures a peptide according to the invention and measure one or more markers of apoptosis, such as (i) staining for cytoplasmic cytochrome C or (ii) staining for TUNEL.
- cytochrome C data can be quantified by counting the number of cells (DAPI can be used to indicate a cell) in which Cytochrome C has been released from the mitochondria to the cytosol or (at later stages) the number of cells that have disappeared completely.
- This assay can be done in presence of a caspase-inhibitor so the cells that are about to undergo apoptosis (indicated by release of
- Cytochrome C into the cytosol are not allowed to actually die as caspases are required for that.
- the benefit of this assay is that it is possible to get a cumulative count on the amount of senescence over several days (for example 5 days).
- the percentage of nuclei (DAPI -positive) which stain positive for TUNEL are counted. This can easily be performed by eye, but it is also possible to use a software tool called Cellprofiler (freeware) to do it objectively.
- a peptide according to the invention is considered to exhibit apoptosis-inducing activity in senescent cells if it kills, clears or removes senescent cells.
- a peptide according to the invention is
- senescent cells considered to exhibit apoptosis-inducing activity in senescent cells if it kills, clears, removes or reduces the viability of at least 5, 10, 15, 20, 25, 30, 40, 50, 60, 70 or 80% of the cells in a senescent cell culture.
- a peptide according to the invention preferably comprises or consists of an amino acid sequence that shows at least 51, 52, 53, 54, 55, 56, 57, 58, 59,60 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence identity to the peptide having the amino acid sequence SEIAQSILEAYSQNGW,LTLRKEPASEIAQSILEAYSQNGWANRRSGGKRP or LTLRKEPASEIAQSILEAYSQNGWANRRSGGKRPPPRRRQRRKKRG; and wherein said peptide contains at least one D-amino acid residue; and wherein said peptide exhibits
- a peptide or fragment of said peptide according to the invention contains at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 27, 27, 28, 29, 30, 31, 32, 33, 34 , 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59,60 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 D-amino acid
- a peptide according to the invention is a DRI -peptide, i.e. a retro- inverso or D-retro-enantiomer of the FOX04 peptide, consisting of D-amino acid derivatives and having the reversed sequence of the FOX04 peptide, preferably with reference to the natural, human FOX04 protein, or a peptide fragment thereof.
- D-amino acid-derivative refers to a FOX04 derivative as defined herein (including the above defined C- and N-terminally truncated peptide fragments having the reverse amino acid sequence of the FOX04 peptide) that include at least one amino acid in the D configuration, preferably more than 25% D, more preferably more than 50% D, even more preferably more than 75% D, even more preferably more than 85% D, and still more preferably more than 95% D amino acid residues.
- a special category of these D-amino acid derivatives are the peptides that are composed of only D amino acids (i.e. in which no L amino acid is present). This special category is herein defined as D-only derivatives.
- a peptide according to the invention can induce apoptosis in senescent cells in vitro, while leaving non-senescent cells intact.
- the inventors even validated their findings in a mouse model which develops cellular senescence at an accelerated rate and exhibits a fast aging phenotype (3 ⁇ 4?d TTD/TTD mouse model, mouse model is described inter alia in WO 2013/152038, e.g. in paragraphs [0135], [0161] and [0244]).
- age-associated phenotypes such as reduced fitness, hair loss and spine curvature can be countered by
- a peptide according to the invention exhibits its function by favoring apoptosis instead of cellular senescence by inhibiting the senescence-favoring interaction between FOX04 and p53 tumor suppressor protein.
- the invention further relates to a pharmaceutical composition comprising a peptide according to the invention.
- a pharmaceutical composition of the invention may comprise a chemotherapeutic agent such as Doxorubicin. It is advantageous to combine a peptide according to the invention and a chemotherapeutic agent in a pharmaceutical composition, i.e. in a single dosage form. It is established that chemotherapeutic agents induce cellular senescence and peptides according to the invention are capable to selectively clear, kill or reduce the viability of senescent cells. In such a way, off-target effects of chemotherapeutic agents are prevented or counteracted (see Figure 4).
- a peptide according to the invention can sensitize a resistant cancer to a chemotherapeutic agent.
- a pharmaceutical composition according to the invention is formulated in a therapeutically effective amount.
- a pharmaceutical composition or peptide according to the invention is in a pharmaceutically acceptable form adapted for administration via a specific route of administration.
- a pharmaceutical composition or peptide according to the invention is most preferably parenterally administered.
- the invention relates to a combination preparation comprising a peptide according to the invention and a chemotherapeutic agent for simultaneous or sequential administration.
- the invention further provides a nucleic acid comprising a DNA sequence that encodes a peptide of the invention.
- a nucleic acid comprising a DNA sequence that encodes a peptide of the invention.
- the person skilled in the art will understand how to generate a DNA sequence that encodes an amino acid sequence of a peptide of the present invention and how to manufacture and isolate a nucleic acid molecule with said DNA sequence using generally known recombinant DNA techniques.
- the present invention also provides an expression vector, preferably a viral expression vector comprising a nucleic acid molecule of the invention. It is envisaged herein that the viral expression vector is suitable for use in gene therapy.
- a peptide according to the invention or a pharmaceutical composition according to the invention are for use as a medicament. More preferably, a peptide according to the invention or a pharmaceutical composition according to the invention are for use in the treatment of a disease or condition wherein the removal of senescent cells is beneficial. Alternatively, a peptide according to the invention or a
- compositions according to the invention are for use in clearing, removing or killing senescent cells. It is envisioned herein that any disease associated with senescent cells having the "senescence associated secretory phenotype (SASP)" in humans, and preferably those having the wildtype p53 tumorsuppressor gene, are sensitive or responsive to
- the invention provides the use of a peptide
- composition or nucleic acid according to the invention for the manufacture of a medicament, preferably a medicament for treating a disease or condition wherein the removal of senescent cells is beneficial.
- said disease or condition is selected from the group formed by atherosclerosis, chronic inflammatory diseases such as arthritis or athrosis, cancer, osteoarthritis, glomerulosclerosis, diabetes including diabetes type II, diabetic ulcers, kyphosis, scoleosis, hepatic insufficiency, cirrhosis, Hutchinson-Gilford progeria syndrome (HGPS), laminopaties, osteoporosis, dementia, (cardio)vascular diseases, obesity, metabolic syndrome, acute myocardial infarction, emphysema, insulin sensitivity, boutonneuse fever, sarcopenia, neurodegenerative diseases such as
- Alzheimer's Huntington's or Parkinson's disease cataracts, anemia, hypertension, fibrosis, age-related macular degeneration, COPD, asthma, renal insufficiency, reducing or preventing graft failure after organ or tissue transplantation, incontinence, hearing loss such as deafness, vision loss such as blindness, sleeping disturbances, pain such as joint pain or leg pain, imbalance, fear, depression, breathlessness, weight loss, hair loss, muscle loss, loss of bone density, frailty and/or reduced fitness.
- graft failure after organ or tissue transplantation is reduced or prevented by administering a peptide, composition or nucleic acid of the invention to a donor subject before transplantation of said organ or tissue; or it is reduced or prevented by subjecting a harvested organ or tissue as such to said peptide, composition or nucleic acid of the invention; or it is reduced or prevented by administering a peptide, composition or nucleic acid of the invention to the recipient subject after transplantation of said organ or tissue.
- a peptide or pharmaceutical composition according to the invention are for use in clearing, removing or killing senescent cells in a human subject suffering from, or expected to suffer from, atherosclerosis, chronic inflammatory diseases such as arthritis or athrosis, cancer, osteoarthritis, diabetes, diabetic ulcers, kyphosis, scoleosis, hepatic insufficiency, cirrhosis, Hutchinson- Gilford progeria syndrome (HGPS), laminopaties, osteoporosis, dementia, (cardio)vascular diseases, obesity, metabolic syndrome, acute myocardial infarction, emphysema, insulin sensitivity, boutonneuse fever, sarcopenia, neurodegenerative diseases such as Alzheimer's, Huntington's or Parkinson's disease cataracts, anemia, hypertension, fibrosis, age-related macular degeneration, COPD, asthma, renal insufficiency, incontinence, hearing loss such as deafness, vision loss such as blind
- a peptide or pharmaceutical composition according to the invention are for use in countering p21 ci P 1 expression in a human subject suffering from, or expected to suffer from, atherosclerosis, chronic
- inflammatory diseases such as arthritis or athrosis, cancer, osteoarthritis, diabetes, diabetic ulcers, kyphosis, scoleosis, hepatic insufficiency, cirrhosis, Hutchinson- Gilford progeria syndrome (HGPS), laminopaties, osteoporosis, dementia, (cardio)vascular diseases, obesity, metabolic syndrome, acute myocardial infarction, emphysema, insulin sensitivity, boutonneuse fever, sarcopenia, neurodegenerative diseases such as Alzheimer's , Huntington's or Parkinson's disease cataracts, anemia, hypertension, fibrosis, age-related macular degeneration, COPD, asthma, renal insufficiency, incontinence, hearing loss such as deafness, vision loss such as blindness, sleeping disturbances, pain such as joint pain or leg pain, imbalance, fear,
- the p21 ci P 1 expression is countered in senescent cells. It is shown in Figure 2 that a peptide according to the invention counters the expression of p21 ci P 1 .
- the human gene p21 ci P 1 is also known as cyclin-dependent kinase inhibitor 1A (CDKNIA), or P21, CIPl, SDI1, WAF1, CAP20, CDKN1 or MDA-6 and is indicated with NCBI gene ID no. 1026.
- a peptide or pharmaceutical composition according to the invention are for use in removing, killing or clearing cells that express pl6 INK4a in a subject suffering from, or expected to suffer from,
- Atherosclerosis chronic inflammatory diseases such as arthritis or athrosis, cancer, osteoarthritis, diabetes, diabetic ulcers, kyphosis, scoleosis, hepatic insufficiency, cirrhosis, Hutchinson- Gilford progeria syndrome (HGPS), laminopaties, osteoporosis, dementia, (cardio)vascular diseases, obesity, metabolic syndrome, acute myocardial infarction, emphysema, insulin sensitivity, boutonneuse fever, sarcopenia, neurodegenerative diseases such as Alzheimer's , Huntington's or Parkinson's disease cataracts, anemia, hypertension, fibrosis, age-related macular degeneration, COPD, asthma, renal insufficiency, incontinence, hearing loss such as deafness, vision loss such as blindness, sleeping disturbances, pain such as joint pain or leg pain, imbalance, fear, depression, breathlessness, weight loss, hair loss, muscle loss, loss of bone density, frailty and/
- a peptide according to the invention removes, kills or clears cells that express p l6 INK4a .
- the human pl6 INK4a gene is also referred to as cyclin- dependent kinase inhibitor 2A and is indicated with NCBI gene ID no. 1029.
- a peptide or pharmaceutical composition according to the invention are for use in countering, or reducing the number of, nuclear serine- 15-phosphorylated p53 foci in a subject suffering, or expected to suffer, from atherosclerosis, chronic inflammatory diseases such as arthritis or athrosis, cancer, osteoarthritis, diabetes, diabetic ulcers, kyphosis, scoleosis, hepatic insufficiency, cirrhosis, Hutchinson-Gilford progeria syndrome (HGPS), laminopaties, osteoporosis, dementia, (cardio)vascular diseases, obesity, metabolic syndrome, acute myocardial infarction, emphysema, insulin sensitivity, boutonneuse fever, sarcopenia,
- chronic inflammatory diseases such as arthritis or athrosis, cancer, osteoarthritis, diabetes, diabetic ulcers, kyphosis, scoleosis, hepatic insufficiency, cirrhosis, Hutchinson
- neurodegenerative diseases such as Alzheimer's , Huntington's or
- Parkinson's disease cataracts anemia, hypertension, fibrosis, age-related macular degeneration, COPD, asthma, renal insufficiency, incontinence, hearing loss such as deafness, vision loss such as blindness, sleeping disturbances, pain such as joint pain or leg pain, imbalance, fear,
- a peptide or pharmaceutical composition according to the invention is preferably administered consecutively in a single dosage form. More preferably, it is administered daily (once in 12h or 24h), for one or more days, preferable consecutive days, such as at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 consecutive days, more preferably 3 consecutive days.
- said peptide or pharmaceutical composition is administered once weekly.
- Such a dosage regimen is also applicable to other compounds mentioned in this application, such as an inhibitor for use or a
- composition - comprising an inhibitor - according to the invention.
- composition for use is for administration, preferably as adjuvant, to a mammalian, preferably human, subject before, during and/or after subjecting said subject to radiation therapy and/or before, during or after administering to said subject a chemotherapeutic agent.
- administration of the peptide or pharmaceutical composition -comprising a peptide according to the invention - for use is adapted for clearing, killing or reducing the viability of cells that have become senescent as a result of treatment by radiation therapy or with a chemotherapeutic agent.
- a peptide or pharmaceutical composition - comprising a peptide according to the invention - could reduce the off-target effects of current chemotherapeutic agents leading to improved organ function by reducing organ toxicity.
- a peptide of the invention has the potential to kill, remove or clear a cancer.
- a peptide according to the invention is in one embodiment suitable for use in the treatment of a cancer, preferably a melanoma, breast cancer, prostate cancer or glioblastoma.
- the resistant cancer is metastatic melanoma, breast cancer or glioblastoma, preferably metastatic melanoma or glioblastoma
- the chemotherapeutic agent is a RAF, MEK or ERK inhibitor such as RAF265, trametinib, dabrafenib, selumetinib, vemurafenib and /or trametinib, more preferably vemurafenib and/or trametinib, or agents as disclosed elsewhere herein, alone or in combination; or radation therapy.
- RAF265 trametinib
- dabrafenib dabrafenib
- selumetinib vemurafenib and /or trametinib
- more preferably vemurafenib and/or trametinib or agents as disclosed elsewhere herein, alone or in combination; or radation therapy.
- a peptide according to the invention
- a peptide or pharmaceutical composition according to the invention are for use in treating a subject suffering from, or expected to suffer from, kyphosis, scoliosis, sarcopenia, cachexia, sclerosis or renal insufficiency.
- the peptide or pharmaceutical composition according to the invention are for use in ameliorating or treating at least one symptom of trichothiodystrophy in a subject suffering from, or expected to suffer from trichothiodystrophy.
- the at least one symptom is selected from the group formed by or consisting of kyphosis, hair loss, fitness decline, scoliosis, sarcopenia, cachexia, sclerosis and renal
- a peptide or pharmaceutical composition according to the invention are preferably for use in countering or treating chemotherapy- induced weight loss in a subject.
- the weight loss is induced by administration of doxorubicin.
- the invention provides a peptide according to the invention for use in sensitizing a resistant cancer in a mammalian, preferably human, subject towards at least one chemotherapeutic agent, preferably at least one chemotherapeutic agent said resistant cancer was or is resistant to.
- said resistant cancer is metastatic melanoma, breast cancer or glioblastoma and said at least one chemotherapeutic agent is a RAF, MEK or ERK inhibitor such as RAF265, trametinib, dabrafenib, selumetinib, vemurafenib and/or trametinib, more preferably vemurafenib and /or trametinib.
- the invention provides for a peptide according to the invention for use in the treatment of a resistant cancer in a mammalian, preferably human, subject, wherein the peptide is for coadministration with a chemotherapeutic agent, preferably a
- chemotherapeutic agent said resistant cancer is resistant to.
- the invention further relates to a kit comprising a first container containing a peptide according to the invention and a second container containing a chemotherapeutic agent.
- the kit may suitably contain instructions regarding the administration in a mammalian, preferably human, subject.
- the human subject is preferably suffering, or suspected to suffer, from cancer, for example a resistant cancer.
- a kit according to the invention - comprising a first container containing a peptide according to the invention - is preferably for use in the treatment of cancer in a mammalian, preferably human, subject.
- a peptide of the invention and a chemotherapeutic agent can be formulated in a single dosage form, e.g. a pharmaceutical composition, it is preferably formulated in a multiple dosage form, wherein a peptide of the invention is in one container and a chemother apeutic agent is in another container.
- a peptide of the invention and a chemother apeutic agent are preferably co-administered.
- said peptide is for administration after or following administration of said chemother apeutic agent.
- a peptide of the invention is
- a peptide according to the invention can reduce the off-target effects of current
- kits according to the invention preferably comprises instructions on dosage regimens obtaining an optimal combined effect of a peptide according to the invention and a chemother apeutic agent.
- the invention provides a method of treating a subject suffering, or suspected to suffer, from a disease or condition wherein the removal of senescent cells is beneficial, comprising a) administering a peptide or a pharmaceutical composition according to the invention to a subject suffering, or suspected to suffer, from a disease or condition wherein the removal of senescent cells is beneficial.
- said disease or condition is cancer; further comprises the step of b) administering a chemother apeutic agent to said subject and/or subjecting said subject to radiation therapy.
- the peptide or the pharmaceutical composition of the invention is administered, preferably as adjuvant, before, during or after administering a chemother apeutic agent to said subject and/or before, during or after subjecting said subject to radiation therapy.
- the cancer is a resistant cancer. In an even more preferred embodiment of any of the methods of the invention described above, wherein said disease or condition is cancer, the cancer is a resistant cancer. In still an even more preferred embodiment of the method described hereinabove, wherein said disease or condition is cancer, the cancer is melanoma, preferably metastatic melanoma, and the
- chemotherapeutic agent is a RAF, MEK or ERK inhibitor such as RAF265, trametinib, dabrafenib, selumetinib, vemurafenib and/or trametinib, more preferably vemurafenib and/or trametinib.
- the invention provides a method of sensitizing a resistant cancer in a mammalian, preferably human, subject to a
- chemotherapeutic agent to which said cancer is resistant comprising a) administering a peptide or a pharmaceutical composition of the invention to said subject.
- said resistant cancer is metastatic melanoma, breast cancer or glioblastoma, preferably metastatic melanoma
- the chemotherapeutic agent is a RAF,MEK or ERK inhibitor such as RAF265, trametinib, dabrafenib, selumetinib, vemurafenib and/or trametinib, more preferably vemurafenib and/or trametinib.
- the invention provides a method of treating a resistant cancer in a mammalian, preferably human, subject, comprising a) administering a peptide or a pharmaceutical composition according to the invention to a mammalian, preferably human, subject suffering, or suspected to suffer, from a resistant cancer; and b) administering a chemotherapeutic agent, preferably a chemotherapeutic agent said resistant cancer was previously resistant to, to said subject.
- a chemotherapeutic agent preferably a chemotherapeutic agent said resistant cancer was previously resistant to, to said subject.
- composition according to the invention is preferably coadministered with said chemotherapeutic agent.
- the resistant cancer is metastatic melanoma, breast cancer or glioblastoma, preferably metastatic melanoma
- the chemotherapeutic agent is a RAF, MEK or ERK inhibitor such as RAF265, trametinib, dabrafenib, selumetinib, vemurafenib and/or
- trametinib more preferably vemurafenib and/or trametinib.
- the invention provides a peptide or composition according to the invention for use in countering chemotherapy-induced toxicity, preferably cytotoxicity, in a subject.
- the toxicity is liver toxicity, preferably as determined by assessing ASAT blood plasma levels, or pancreas toxicity, preferably as determined by assessing amylase blood plasma levels.
- the skilled person is well aware of methods and means for assess liver and pancreas toxicity.
- the invention further relates to an inhibitor of ASK 1 for use in the treatment of a disease or condition wherein the removal of senescent cells is beneficial.
- an inhibitor of ASK1 inhibits MAP kinase activity of ASK1 in senescent cells. It was unexpectedly found that, when selectively inhibiting the MAP kinase function of ASK1 in senescent cells, apoptosis was induced.
- An ASK1 inhibitor may thus suitable be used in selectively clearing, kilhng or reducing the viability of senescent cells (see Figure 12). Particularly preferred is the ASK1 inhibitor NQDI.
- ASK1 MAP kinase signaling is important in maintaining cellular senescence and preventing the induction of apoptosis.
- the invention relates to an inhibitor of ASK 1 for use in clearing, removing or killing senescent cells.
- the disease or condition wherein the removal of senescent cells is beneficial is selected from the group formed by
- Atherosclerosis chronic inflammatory diseases such as arthritis or athrosis, cancer, osteoarthritis, diabetes, diabetic ulcers, kyphosis, scoleosis, hepatic insufficiency, cirrhosis, Hutchinson- Gilford progeria syndrome (HGPS), laminopaties, osteoporosis, dementia, (cardio)vascular diseases, obesity, metabolic syndrome, sarcopenia, acute myocardial infarction, emphysema, insulin sensitivity, boutonneuse fever, neurodegenerative diseases such as alzheimer's disease or parkinson's disease, Huntington's disease, cataracts, anemia, hypertension, fibrosis, age-related macular degeneration, COPD, asthma, renal insufficiency, incontinence, hearing loss such as deafness, vision loss such as blindness, sleeping disturbances, pain such as joint pain or leg pain, imbalance, fear, depression, breathlessness, weight loss, hair loss, muscle loss, loss of bone density
- said inhibitor of ASKl may be administered in a single dosage form once weekly.
- the inhibitor is in a pharmaceutically acceptable form adapted for administration via a specific route of
- An inhibitor of ASKl for use according to the invention is most preferably parenterally administered.
- an inhibitor of ASKl for use according to the invention is cancer; and the inhibitor of ASKl is administered or for administration, preferably as adjuvant, to a mammalian, preferably human, subject before, during and/or after subjecting said subject to radiation therapy and/or before, during or after administering to said subject a chemother apeutic agent.
- administration of the inhibitor of ASKl for use according to the invention is adapted for clearing, killing or reducing the viability of cells that have become senescent as a result of treatment by radiation therapy or with a chemother apeutic agent. It was unexpectedly found that an ASKl inhibitor according to the invention can reduce the off- target effects of current chemotherapeutic agents.
- the cancer is a resistant cancer having resistance to a chemotherapeutic agent. More preferably, said resistant cancer is
- metastatic melanoma breast cancer or glioblastoma, preferably metastatic melanoma
- said chemotherapeutic agent is a RAF, MEK or ERK inhibitor such as RAF265, trametinib, dabrafenib, selumetinib, vemurafenib and/or trametinib, more preferably vemurafenib and/or trametinib.
- the invention provides an inhibitor of ASKl, a pharmaceutical composition comprising an inhibitor of ASKl, or a kit for use in medicine - the kit comprising a first container containing an inhibitor of ASKl -, all according to the invention, for use in sensitizing a resistant cancer in a mammalian, preferably human, subject towards at least one chemotherapeutic agent, preferably at least one chemotherapeutic agent said resistant cancer is resistant to.
- said resistant cancer is metastatic melanoma, breast cancer or glioblastoma and said at least one chemotherapeutic agent is a RAF, MEK or ERK inhibitor such as RAF265, trametinib, dabrafenib, selumetinib, vemurafenib and/or trametinib, more preferably vemurafenib and /or trametinib.
- RAF RAF
- MEK or ERK inhibitor such as RAF265, trametinib, dabrafenib, selumetinib, vemurafenib and/or trametinib, more preferably vemurafenib and /or trametinib.
- the invention provides an inhibitor of ASKl, a pharmaceutical composition comprising an inhibitor of ASKl, or a kit for use in medicine - the kit comprising a first container containing an inhibitor of ASKl -, all according to the invention, for use in the treatment of a resistant cancer in a mammalian, preferably human, subject, wherein said inhibitor of ASKl, pharmaceutical composition comprising an inhibitor of ASKl, or kit for use in medicine - the kit comprising a first container containing an inhibitor of ASKl -, is for co-administration with a
- chemotherapeutic agent preferably a chemotherapeutic agent said resistant cancer is resistant to.
- the invention further relates to a pharmaceutical composition comprising an inhibitor of ASK1, preferably NQDI, and a chemotherapeutic agent.
- a pharmaceutical composition according to the invention is
- a therapeutically effective amount preferably, a pharmaceutically acceptable amount.
- a pharmaceutically effective amount preferably, a pharmaceutically acceptable amount.
- composition according to the invention is in a
- a pharmaceutical composition according to the invention is most preferably parenterally administered. It is advantageous to combine an inhibitor of ASK1 and a chemotherapeutic agent in a single dosage form, since the inhibitor of ASK1 alleviates the off-target effects of a chemotherapeutic agent.
- a pharmaceutical composition according to the invention is preferably for use in the treatment of a cancer, preferably a resistant cancer.
- a pharmaceutical composition according to the invention is preferably administered consecutively in a single dosage form. More preferably, it is administered daily, once in 24h, for one or more days, preferable consecutive days, such as at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 consecutive days, preferably at least three consecutive days. It was found that administration of sequential rounds of NQDI enhances its effectivity towards senescent cell clearance.
- said inhibitor of ASK1 may be administered in a single dosage form once weekly.
- the invention relates to a combination preparation comprising an ASK1 inhibitor and a chemotherapeutic agent for simultaneous or sequential administration.
- the invention also relates to a kit for use in medicine, the kit comprising a first container containing an inhibitor of ASK1, preferably
- kits may suitably contain instructions regarding the administration in a mammalian, preferably human, subject.
- the human subject is preferably suffering, or suspected to suffer, from cancer.
- said kit is for use in the treatment of cancer, preferably a resistant cancer.
- chemother apeutic agent are preferably formulated in a multiple dosage form, wherein an inhibitor of ASK1 is in one container and a
- kits for use according to the invention an inhibitor of ASK1 and a chemotherapeutic agent are preferably co-administered.
- an inhibitor of ASK1 according to the invention is administered as adjuvant in such a way that in can clear, kill or reduce the viabihty of cells that have become senescent as a result of treatment with a chemotherapeutic agent.
- a kit according to the invention preferably comprises instructions on dosage regimens obtaining an optimal combined effect of an inhibitor of ASK1 and a chemotherapeutic agent.
- the invention in another aspect, relates to a method of treating a subject suffering, or suspected to suffer, from a disease or condition wherein the removal of senescent cells is beneficial, comprising a) administering an inhibitor of ASK1, preferable NQDI, to a subject suffering, or suspected to suffer, from a disease or condition wherein the removal of senescent cells is beneficial.
- said disease or condition is cancer, and further comprises the step of b) administering a
- the inhibitor of ASK1 is administered, preferably as adjuvant, before, during or after administering a chemotherapeutic agent to said subject and/or before, during or after subjecting said subject to radiation therapy.
- the cancer is a resistant cancer having resistance to said chemotherapeutic agent.
- said disease or condition is a resistant cancer
- the resistant cancer is metastatic melanoma, breast cancer or glioblastoma, preferably metastatic melanoma
- the chemother apeutic agent is a RAF, MEK or ERK inhibitor such as RAF265, trametinib, dabrafenib,
- selumetinib vemurafenib and/or trametinib, more preferably vemurafenib and/or trametinib.
- the invention provides a method of sensitizing a resistant cancer in a mammalian, preferably human, subject to a
- the resistant cancer is metastatic melanoma, breast cancer or glioblastoma, preferably metastatic melanoma
- the chemotherapeutic agent is a RAF, MEK or ERK inhibitor such as RAF265, trametinib, dabrafenib, selumetinib, vemurafenib and/or trametinib, more preferably vemurafenib and/or trametinib.
- the invention provides a method of treating a resistant cancer in a mammalian, preferably human, subject, comprising a) administering an inhibitor of ASK1 to a mammalian, preferably human, subject suffering, or suspected to suffer, from a resistant cancer; and b) administering a chemotherapeutic agent, preferably a chemotherapeutic agent said resistant cancer is resistant to, to said subject.
- Said inhibitor of ASK1 is preferably co-administered with said chemotherapeutic agent.
- the resistant cancer is metastatic melanoma, breast cancer or glioblastoma, preferably metastatic melanoma
- the chemotherapeutic agent is a RAF, MEK or ERK inhibitor such as RAF265, trametinib, dabrafenib,
- selumetinib vemurafenib and/or trametinib, more preferably vemurafenib and/or trametinib.
- the invention further relates to an inhibitor of SYK for use in the treatment of a disease or condition wherein the removal of senescent cells is beneficial; and wherein said disease or condition is not asthma, immune thrombocytopenia, hemolytic anemia, myeloid leukemia and/or lymphoma, preferably wherein said disease or condition is not asthma, immune thrombocytopenia, anemia, leukemia and/or lymphoma, more preferably wherein said disease or condition is not a lung disease, a bleeding disorder, leukemia and/or lymphoma.
- the invention further relates to an inhibitor of SYK for use in removing, clearing or killing senescent cells.
- an inhibitor of SYK inhibits tyrosine kinase activity of SYK in senescent cells. It was unexpectedly found that, when selectively inhibiting the tyrosine kinase function of SYK in senescent cells, apoptosis was induced.
- a SYK inhibitor may thus suitable be used in selectively clearing, kilhng or reducing the viability of senescent cells (see Figure 13).
- SYK inhibitor R406 is particularly preferred.
- SYK tyrosine kinase signaling is important in maintaining cellular senescence and preventing the induction of apoptosis.
- the invention relates to an inhibitor of SYK for use in the treatment of atherosclerosis, chronic inflammatory diseases such as arthritisor athrosis, diabetes, diabetic ulcers, kyphosis, scoleosis, hepatic insufficiency, cirrhosis, Hutchinson- Gilford progeria syndrome (HGPS), laminopaties, osteoporosis, osteoarthritis, dementia, (cardio)vascular diseases, obesity, metabolic syndrome, acute myocardial infarction, emphysema, insulin sensitivity, boutonneuse fever, neurodegenerative diseases such as alzheimer's disease or parkinson's disease, Huntington's disease, cataracts, sarcopenia, hypertension, fibrosis, age-related macular degeneration, COPD, renal insufficiency, incontinence, hearing loss such as deafness, vision loss such as blindness, sleeping disturbances, pain such as joint pain or leg pain, imbalance, fear, depression, breathlessness,
- esthesioneuroblastoma ewing sarcoma, extracranial germ cell tumor, extragonadal germ cell tumor, extrahepatic bile duct cancer, eye cancer, fallopian tube cancer, gallbladder cancer, gastric (stomach) cancer, gastrointestinal carcinoid tumor, germ cell tumor, head and neck cancer, hepatocellular (liver) cancer, hypopharyngeal cancer, kidney cancer, lung cancer, lip and oral cavity cancer, male breast cancer, metastatic squamous neck cancer, mouth cancer, nasal cavity and paranasal sinus cancer, ovarian cancer, pancreatic cancer, parathyroid cancer, penile cancer, prostate cancer, rectal cancer, salivary gland cancer, skin cancer, small intestine cancer, stomach (gastric) cancer, thyroid cancer, urethral cancer, vaginal cancer, and/or vulvar cancer.
- diseases lymphomas and leukemias are explicitly excluded.
- An inhibitor of SYK for use according to the invention is formulated in a pharmaceutically acceptable form and in a therapeutically effective amount.
- the inhibitor is in a pharmaceutically acceptable form adapted for administration via a specific route of administration.
- An inhibitor of SYK for use according to the invention is most preferably parenterally administered.
- An inhibitor of SYK is preferably administered daily, once in 12h or 24h, for one or more days, preferable consecutive days, such as at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 consecutive days, preferably at least three consecutive days.
- said inhibitor of SYK may be administered in a single dosage form once weekly.
- the disease or condition wherein the removal of senescent cells is beneficial is cancer; and the inhibitor of SYK is administered or for administration, preferably as adjuvant, to a mammalian, preferably human, subject before, during and/or after subjecting said subject to radiation therapy and/or before, during or after administering to said subject a chemother apeutic agent; and wherein the cancer is not a lymphoma or myloid leukemia, preferably leukemia; or wherein the cancer is selected from the group formed by adrenocortical carcinoma, anal cancer, appendix cancer, astrocytomas, basal cell carcinoma, bile duct cancer, bladder cancer, bone cancer, brain stem glioma, brain tumor, breast cancer, bronchial tumors, carcinoid tumor, cardiac (heart) tumors, central nervous system tumor, cervical cancer, chordoma, colon cancer, colorectal cancer,
- craniopharyngioma ductal carcinoma, embryonal tumors, endometrial cancer, ependymoma, esophageal cancer, esthesioneuroblastoma, ewing sarcoma, extracranial germ cell tumor, extragonadal germ cell tumor, extrahepatic bile duct cancer, eye cancer, fallopian tube cancer, gallbladder cancer, gastric (stomach) cancer, gastrointestinal carcinoid tumor, germ cell tumor, head and neck cancer, hepatocellular (liver) cancer, hypopharyngeal cancer, kidney cancer, lung cancer, hp and oral cavity cancer, male breast cancer, metastatic squamous neck cancer, mouth cancer, nasal cavity and paranasal sinus cancer, ovarian cancer, pancreatic cancer, parathyroid cancer, penile cancer, prostate cancer, rectal cancer, salivary gland cancer, skin cancer, small intestine cancer, stomach (gastric) cancer, thyroid cancer, urethral cancer, vaginal cancer, and/or
- the cancer is a resistant cancer having resistance to a chemother apeutic agent.
- the resistant cancer is metastatic melanoma, breast cancer or glioblastoma, preferably metastatic melanoma
- the chemotherapeutic agent is a RAF, MEK or ERK inhibitor such as RAF265, trametinib, dabrafenib, selumetinib, vemurafenib and/or trametinib, more preferably vemurafenib and/or trametinib.
- administration of the inhibitor of SYK for use according to the invention is adapted for clearing, killing or reducing the viability of cells that have become senescent as a result of treatment by radiation therapy or with a chemotherapeutic agent. It was unexpectedly found that a SYK inhibitor according to the invention can reduce the off-target effects of current chemotherapeutic agents.
- the invention provides an inhibitor of SYK, a pharmaceutical composition comprising an inhibitor of SYK, or a kit for use in medicine - the kit comprising a first container containing an inhibitor of SYK -, all according to the invention, for use in sensitizing a resistant cancer in a mammalian, preferably human, subject towards at least one chemotherapeutic agent, preferably at least one chemotherapeutic agent said resistant cancer was or is resistant to.
- said resistant cancer is metastatic melanoma, breast cancer or glioblastoma and said at least one chemotherapeutic agent is a RAF, MEK or ERK inhibitor such as RAF265, trametinib, dabrafenib, selumetinib, vemurafenib and/or trametinib, more preferably vemurafenib and /or trametinib.
- RAF RAF
- MEK or ERK inhibitor such as RAF265, trametinib, dabrafenib, selumetinib, vemurafenib and/or trametinib, more preferably vemurafenib and /or trametinib.
- the invention provides an inhibitor of SYK, a pharmaceutical composition comprising an inhibitor of SYK, or a kit for use in medicine - the kit comprising a first container containing an inhibitor of SYK -, all according to the invention, for use in the treatment of a resistant cancer in a mammalian, preferably human, subject, wherein said inhibitor of SYK, pharmaceutical composition comprising an inhibitor of SYK, or kit for use in medicine - the kit comprising a first container containing an inhibitor of SYK -, is for co-administration with a chemotherapeutic agent, preferably a chemotherapeutic agent said resistant cancer is or was resistant to.
- a chemotherapeutic agent preferably a chemotherapeutic agent said resistant cancer is or was resistant to.
- the invention further relates to a pharmaceutical composition
- a pharmaceutical composition comprising an inhibitor of SYK, preferably R406, and a chemotherapeutic agent.
- a pharmaceutical composition according to the invention is
- a therapeutically effective amount preferably, a pharmaceutically acceptable amount.
- a pharmaceutically effective amount preferably, a pharmaceutically acceptable amount.
- composition according to the invention is in a
- a pharmaceutical composition according to the invention is most preferably parenterally administered. It is advantageous to combine an inhibitor of SYK and a chemotherapeutic agent in a single dosage form, since the inhibitor of SYK alleviates the off-target effects of a chemotherapeutic agent.
- a pharmaceutical composition according to the invention is preferably for use in the treatment of cancer.
- a pharmaceutical composition according to the invention is preferably administered
- consecutively in a single dosage form is administered daily, once in 12h or 24h, for one or more days, preferable consecutive days, such as at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 consecutive days, preferably at least four consecutive days.
- said inhibitor of SYK may be administered in a single dosage form once weekly.
- the invention relates to a combination preparation comprising an inhibitor of SYK and a chemotherapeutic agent for
- the invention also relates to a kit for use in medicine, the kit comprising a first container containing an inhibitor of SYK, preferably
- kits may suitably contain instructions regarding the administration in a mammalian, preferably human, subject.
- the human subject is preferably suffering, or suspected to suffer, from cancer.
- the kit is not for use in the treatment of myeloid leukemia and/or lymphoma.
- a kit according to the invention is preferably for use in the treatment of cancer, more preferably for use in the treatment of a cancer that is not a lymphoma or leukemia.
- chemotherapeutic agent are preferably formulated in a multiple dosage form, wherein an inhibitor of SYK is in one container and a
- chemotherapeutic agent is in another container.
- an inhibitor of SYK and a chemotherapeutic agent are preferably co-administered.
- an inhibitor of SYK according to the invention is administered as adjuvant in such a way that in can clear, kill or reduce the viability of cells that have become senescent as a result of treatment with a chemotherapeutic agent.
- a kit according to the invention preferably comprises instructions on dosage regimens obtaining an optimal combined effect of an inhibitor of SYK and a chemotherapeutic agent.
- the invention provides a method of treating a subject suffering, or suspected to suffer, from a disease or condition wherein the removal of senescent cells is beneficial, comprising a) administering an inhibitor of SYK, preferable R406, to a subject suffering, or suspected to suffer, from a disease or condition wherein the removal of senescent cells is beneficial; and wherein said disease or condition is not asthma, immune thrombocytopenia, hemolytic anemia, myeloid leukemia and/or lymphoma, preferably wherein said disease or condition is not asthma, immune thrombocytopenia, anemia, leukemia and/or lymphoma, more preferably wherein said disease or condition is not a lung disease, a bleeding disorder, leukemia and/or lymphoma.
- the invention provides a method of treating a subject suffering, or suspected to suffer, from a disease or condition wherein the removal of senescent cells is beneficial, comprising a) administering an inhibitor of SYK, preferable R406, to a subject suffering, or suspected to suffer, from atherosclerosis, chronic inflammatory diseases such as arthritis or athrosis, diabetes, diabetic ulcers, kyphosis, scoleosis, hepatic insufficiency, cirrhosis, Hutchinson-Gilford progeria syndrome (HGPS), laminopaties, osteoarthritis, osteoporosis, dementia,
- an inhibitor of SYK preferable R406
- atherosclerosis chronic inflammatory diseases such as arthritis or athrosis, diabetes, diabetic ulcers, kyphosis, scoleosis, hepatic insufficiency, cirrhosis, Hutchinson-Gilford progeria syndrome (HGPS), laminopaties, osteoarthriti
- neurodegenerative diseases such as alzheimer's disease or parkinson's disease, Huntington's disease, cataracts, sarcopenia, hypertension, fibrosis, age-related macular degeneration, COPD, renal insufficiency, incontinence, hearing loss such as deafness, vision loss such as blindness, sleeping disturbances, pain such as joint pain or leg pain, imbalance, fear,
- adrenocortical carcinoma selected from the group formed by adrenocortical carcinoma, anal cancer, appendix cancer, astrocytomas, basal cell carcinoma, bile duct cancer, bladder cancer, bone cancer, brain stem glioma, brain tumor, breast cancer, bronchial tumors, carcinoid tumor, cardiac (heart) tumors, central nervous system tumor, cervical cancer, chordoma, colon cancer, colorectal cancer,
- craniopharyngioma ductal carcinoma, embryonal tumors, endometrial cancer, ependymoma, esophageal cancer, esthesioneuroblastoma, ewing sarcoma, extracranial germ cell tumor, extragonadal germ cell tumor, extrahepatic bile duct cancer, eye cancer, fallopian tube cancer, gallbladder cancer, gastric (stomach) cancer, gastrointestinal carcinoid tumor, germ cell tumor, head and neck cancer, hepatocellular (liver) cancer, hypopharyngeal cancer, kidney cancer, lung cancer, hp and oral cavity cancer, male breast cancer, metastatic squamous neck cancer, mouth cancer, nasal cavity and paranasal sinus cancer, ovarian cancer, pancreatic cancer, parathyroid cancer, penile cancer, prostate cancer, rectal cancer, salivary gland cancer skin cancer, small intestine cancer, stomach (gastric) cancer, thyroid cancer urethral cancer, vaginal cancer, and/or vul
- said disease or condition is cancer
- said method further comprising the step of b) administering a chemotherapeutic agent to said subject and/or subjecting said subject to radiation therapy.
- the inhibitor of SYK is administered or for
- the cancer is a resistant cancer having resistance to said chemother apeutic agent.
- a method of the invention wherein the disease or condition is cancer, said resistant cancer is metastatic melanoma, breast cancer or glioblastoma and said at least one chemotherapeutic agent is a RAF, MEK or ERK inhibitor such as RAF265, trametinib, dabrafenib, selumetinib, vemurafenib and/or trametinib, more preferably vemurafenib and /or trametinib.
- RAF RAF
- MEK or ERK inhibitor such as RAF265, trametinib, dabrafenib, selumetinib, vemurafenib and/or trametinib, more preferably vemurafenib and /or trametinib.
- the invention provides a method of sensitizing a resistant cancer in a mammalian, preferably human, subject to a
- chemotherapeutic agent to which said cancer was or is resistant, comprising administering an inhibitor of SYK, preferably R406, to said subject.
- resistant cancer is metastatic melanoma, breast cancer or glioblastoma and said at least one chemotherapeutic agent is a RAF, MEK or ERK inhibitor such as RAF265, trametinib, dabrafenib, selumetinib, vemurafenib and/or trametinib, more preferably vemurafenib and /or trametinib.
- the invention provides a method of treating a resistant cancer in a mammalian, preferably human, subject, comprising a) administering an inhibitor of SYK to a mammalian, preferably human, subject suffering, or suspected to suffer, from a resistant cancer; and b) administering a chemotherapeutic agent, preferably a chemotherapeutic agent said resistant cancer was or is resistant to, to said subject.
- Said inhibitor of SYK is preferably co-administered with said chemotherapeutic agent.
- the resistant cancer is metastatic melanoma, breast cancer or glioblastoma, preferably metastatic melanoma
- the chemotherapeutic agent is a RAF, MEK or ERK inhibitor such as RAF265, trametinib, dabrafenib,
- selumetinib vemurafenib and/or trametinib, more preferably vemurafenib and/or trametinib.
- Senescent and non-senescent IMR90 fibroblasts were plated for longitudinal cell density measurement using the xCELLigence system (ACE A Biosciences, Inc., San Diego, CA).
- the cells were mock treated (PBS) or exposed to the FOXO4 DRI peptide (50 ⁇ ) and cell density after this treatment was followed in time as indicated. Note that after 36h the FOXO4 DRI peptide selectively and gradually reduced cell density of senescent cells.
- the FOXO4 DRI peptide was ordered from
- the FOXO4 DRI peptide of which the amino acid sequence is in a D-Retro-Inverso Isoform, selectively reduces cell viability of senescent, but not non-senescent IMR90 fibroblast cells in time (.
- Example 2 FOX04 DRI peptide is effective in the treatment of diseases. Trichothiodystrophy is a human progeria syndrome leading to hair loss, neurological defects, bone abnormalities and fitness decline.
- Our department generated a mouse model based on a mutation in the Xpd gene, the Xpd TTD/TTD mouse model, which largely encompasses these features. We observed this mouse model to develop senescence at an accelerated pace, in conjunction with the fast aging phenotype.
- the Xpd TTD/TTD mouse is publicly available and is inter alia described in the literature (De Boer et al.,
- Wildtype (c57bl/6) or Xpd TTD/TTD mice were treated on days 3, 4, 5 and 6 with the FOXO4 DRI peptide by intravenous injection of a lOmg/kg dose. Days 1 and 2 were baseline (control, no treatment) measurements. Ct scans were used to establish the curvature of the spine of the mice as a measure of Kyphosis. Muscle mass was measured by Ct scan followed by 3D volume rendering.
- Example 3 ASKl-inhibitor NQDI clears senescent cells 5000 senescent (obtained as described above) and 2000 non- senescent IMR90 fibroblasts were plated in 96-well plates incubated with concentrations of NQDI between 0-20 ⁇ and after 6 days cell viability was determined by AqueousOne CellTiter (MTT) assay according to the manufacturer's instructions.
- MTT AqueousOne CellTiter
- Senescent and non-senescent IMR90 fibroblasts were plated for longitudinal cell density measurement using the xCELLigence system as described in Example 1 and incubated on three consecutive days with 2 ⁇ NQDI (24, 48 and 72h, respectively). Cell density after time of plating was followed. It was found that the ASK1 inhibitor NQDI reduces cell viability in senescent, but not non-senescent IMR90 fibroblasts. Sequential rounds of NQDI enhance its effectivity towards senescent cell clearance ( Figure 12). NQDI is apparently rather safe even at high doses as as non-senescent cells are not affected by 20uM NQDI.
- Normal human IMR90 fibroblasts were exposed to 10 Gy Ionizing Radiation or treated 3x (bidaily) with 0.5 ⁇ Doxorubicin. Seven days later they were processed for SA-6-Gal staining to visualize senescent cells. Cells exposed to 1x10 Gy Ionizing Radiation or treated 3x (bidaily) with 0.5 ⁇ Doxorubicin were processed for immunofluorescence to detect FOXO4 (Red) and p l6 ink4a (Green). DAPI was used as a counterstain to visualize nuclei. Cells are treated as described before and exposed to a mock treatment or the FOX04 DRI peptide. After 6 days the cell viability was determined using an AqueousOne Celltiter (MTT) assay according to the manufacturer's instructions. The histograms show % viability for the indicated conditions (.
- MTT AqueousOne Celltiter
- the FOX04 DRI peptide selectively targets viability of cells induced to senesce by radiotherapy (IR) or chemotherapy (Doxorubicin).
- p l6::3MR trimodahty reporter
- the 3MR reporter comprises a chimeric protein consisting of Renilla Luciferase (RLUC), Red Fluorescent Protein (RFP) and Thymidine Kinase (TK) from Herpes Simplex Virus.
- RLUC and RFP allow visualization of senescent cells in real-time through bioluminescence and fluorescence, respectively.
- Thymidine kinase allows apoptosis in an induced fashion through the compound Ganciclovir (GCV).
- Ganciclovir Ganciclovir
- mice On day 0, p l6::3MR mice (Demaria et al., Developmental Cell, 31:6 p. 722-733 (2014)) including the Supplemental Experimental procedures) were i.p. injected with lOmg/kg Doxorubicin and RLUC activity as measure of senescence was determined on the indicated timepoints.
- Doxorubicin induces p l6 ink4a -mediated senescence in vivo.
- the FOXO4 DRI peptide and R406 repress Doxorubicin-induced senescence in vivo, shown by reduced bioluminescence and reduced SA-beta- GAL deposits in kidneys.
- mice treated with doxorubicin reduced values of ASAT and amylase, indicators of respectively hver toxicity and pancreas toxicity, were observed in plasma of mice treated with FOX04 DRI peptide, whereas PBS treated mice showed increases toxicity levels as a result of Doxorubicin treatment.
- FOXO 4 DRI peptide can kill cancer cells to some extent as a single entity, i.e. without chemotherapy.
- Example 7 The FOX04 DRI peptide sensitizes metastatic melanoma to Vemurafenib/Trametinib.
- LOX-IMVI cells were plated for (i) cell viability assay as in example 1 by AqueousOne MTT assay, (ii) apoptosis by Cytochrome C release, (iii) longitudinal cell density assay, and incubated with the FOXO4 DRI peptide, Vemurafenib/Trametinib, or their combination, respectively.
- apoptosis by Cytochrome C release experiment 20.000 LOX-IMVI cells were plated on glass coverslips in 24-well plate wells.
- the longitudinal cell density assay is an xCELLigence experiment as mentioned above for the senescence and non-senescent cells. Only in this case 5000 LOX-IMVI cells were used and lOuM of A Rock inhibitor (Y- 27632) was used to prevent anoikis, thus ensuring better attachment of the cells to the electrode.
- the level of FOXO4 is induced by Vemurafenib as determined by
- the FOXO4 DRI peptide strongly enhances the ability of Vemurafenib and Trametinib to kill therapy-resistant LOX-IMVI melanoma cells.
- the first patient group receives the FOXO 4 DRI peptide by intravenous administration using a single-dose injection of the FOXO 4 DRI peptide at a therapeutically effective dose (i.e. a pharmaceutically effective amount), while the second patient group is non-treated or receives a placebo.
- a therapeutically effective dose i.e. a pharmaceutically effective amount
- b is the outcome for non-treated control patients, wherein when a is different from b, it is demonstrated that the FOXO4 DRI peptide is effective in the treatment of atherosclerosis.
- Atherosclerosis disease parameters are inter alia measured by blood tests including measurements of cholesterol, glucose, electrocardiogram, angiography, computerised tomography scan and/or ophthalmoscopy.
- the first patient group receives the FOXO 4 DRI peptide by intravenous administration using a single-dose injection of the FOXO 4 DRI peptide at a therapeutically effective dose (i.e. a pharmaceutically effective amount), while the second patient group is non-treated or receives a placebo.
- a therapeutically effective dose i.e. a pharmaceutically effective amount
- arthritis disease parameters are measured in both patient groups, wherein a is the outcome for treated patients, b is the outcome for non-treated control patients, wherein when a is different from b, it is demonstrated that the FOXO4 DRI peptide is effective in the treatment of arthritis.
- Arthritis disease parameters that are to be measured are inter alia joint symptoms including swelling, pain, stiffness and decreased range of motion.
- the first patient group receives the FOXO 4 DRI peptide by intravenous administration using a single-dose injection of the FOXO 4 DRI peptide at a therapeutically effective dose (i.e. a pharmaceutically effective amount), while the second patient group is non-treated or receives a placebo.
- a therapeutically effective dose i.e. a pharmaceutically effective amount
- metastatic melanoma disease parameters are measured in both patient groups, wherein a is the outcome for treated patients, b is the outcome for non-treated control patients, wherein when a is different from b, it is demonstrated that the FOX04 DRI peptide is effective in the treatment of metastatic melanoma.
- Metastatic melanoma disease parameters that are to be measured are inter alia reduction in tumor size and/or metastazation.
- Example 11 Treatment of resistant breast cancer patients with the FOX04 DRI peptide
- the first patient group receives the FOXO 4 DRI peptide by intravenous administration using a single-dose injection of the FOXO 4 DRI peptide at a therapeutically effective dose (i.e. a
- resistant breast cancer disease parameters are measured in both patient groups, wherein a is the outcome for treated patients, b is the outcome for non-treated control patients, wherein when a is different from b, it is demonstrated that the FOXO4 DRI peptide is effective in the treatment of resistant breast cancer.
- Resistant breast cancer disease parameters that are to be measured are inter alia reduction in tumor size and/or metastazation.
- Example 12 Treatment of radiotherapy resistant glioblastoma patients with the FOX04 DRI peptide
- the first patient group receives the FOXO 4 DRI peptide by intravenous administration using a single-dose injection of the FOXO 4 DRI peptide at a therapeutically effective dose (i.e. a pharmaceutically effective amount) and subjected to radiotherapy, while the second patient group is subjected to radiotherapy but does not receive the FOXO 4 DRI peptide (i.e. is non-treated or receives a placebo.
- a therapeutically effective dose i.e. a pharmaceutically effective amount
- resistant glioblastoma disease parameters are measured in both patient groups, wherein a is the outcome for treated patients, b is the outcome for non-treated control patients, wherein when a is different from b, it is demonstrated that the FOXO4 DRI peptide is effective in the treatment of resistant glioblastoma.
- Resistant glioblastoma disease parameters that are to be measured are inter alia reduction in tumor size and/or metastazation.
- Example 13 Treatment of osteoarthritis patients with the FOX04 DRI peptide
- the first patient group receives the FOXO 4 DRI peptide by intravenous administration using a single-dose injection of the FOXO 4 DRI peptide at a therapeutically effective dose (i.e. a pharmaceutically effective amount), while the second patient group is non-treated or receives a placebo.
- a therapeutically effective dose i.e. a pharmaceutically effective amount
- osteoarthritis disease parameters are measured in both patient groups, wherein a is the outcome for treated patients, b is the outcome for non-treated control patients, wherein when a is different from b, it is demonstrated that the FOXO4 DRI peptide is effective in the treatment of osteoarthritis.
- Osteoarthritis disease parameters that are to be measured are inter alia joint pain, redness, stiffness and/or swelling and joint motion range, X-RAY and/or MRI for bone spurs, blood tests and joint fluid analyses to rule out other causes.
- the first patient group receives the FOXO 4 DRI peptide by intravenous administration using a single-dose injection of the FOXO 4 DRI peptide at a therapeutically effective dose (i.e. a pharmaceutically effective amount), while the second patient group is non-treated or receives a placebo.
- a therapeutically effective dose i.e. a pharmaceutically effective amount
- glomerulosclerosis disease parameters are measured in both patient groups, wherein a is the outcome for treated patients, b is the outcome for non-treated control patients, wherein when a is different from b, it is demonstrated that the FOXO4 DRI peptide is effective in the treatment of glomerulosclerosis.
- Glomerulosclerosis disease parameters that are to be measured are inter alia swellings in limbs, weight gains, changes in urine due to proteinuria, distortion or compression of the small capillaries in the glomerulus that filter blood in a biopsy and plasma [Urea] or [protein], blood pressure, glomerular filtration rate, and/or kidney ultrasound.
- Example 15 Treatment of diabetes type II patients with the FOX04 DRI peptide
- the first patient group receives the FOXO 4 DRI peptide by intravenous administration using a single-dose injection of the FOXO 4 DRI peptide at a therapeutically effective dose (i.e. a pharmaceutically effective amount), while the second patient group is non-treated or receives a placebo.
- a therapeutically effective dose i.e. a pharmaceutically effective amount
- diabetes type II disease parameters are measured in both patient groups, wherein a is the outcome for treated patients, b is the outcome for non-treated control patients, wherein when a is different from b, it is demonstrated that the FOXO4 DRI peptide is effective in the treatment of diabetes type II.
- Diabetes type II disease parameters that are to be measured are inter alia basal blood glucose levels, average blood glucose levels over a period of time (2-3 months; AlC test), fasting plasma glucose, oral glucose tolerance test, plasma glucose test N.B.
- basal blood glucose levels average blood glucose levels over a period of time (2-3 months; AlC test
- fasting plasma glucose oral glucose tolerance test
- plasma glucose test N.B plasma glucose test
- hyperglycemia glucose and ketones in urine
- oral glucose tolerance test AlC test
- the first patient group receives the FOXO 4 DRI peptide by intravenous administration using a single-dose injection of the FOXO 4 DRI peptide at a therapeutically effective dose (i.e. a pharmaceutically effective amount), while the second patient group is non-treated or receives a placebo.
- a therapeutically effective dose i.e. a pharmaceutically effective amount
- b is the outcome for non-treated control patients, wherein when a is different from b, it is demonstrated that the FOXO4 DRI peptide is effective in the treatment of kyphosis.
- Kyphosis disease parameters that are to be
- measured are inter alia measurement of spine curvature by X-RAY, CT and/or MRI.
- the first patient group receives the FOXO 4 DRI peptide by intravenous
- scoliosis disease parameters are measured in both patient groups, wherein a is the outcome for treated patients, b is the outcome for non-treated control patients, wherein when a is different from b, it is demonstrated that the FOXO4 DRI peptide is effective in the treatment of scoliosis.
- Scoliosis disease parameters that are to be measured are inter alia physical examination of spine, ribs, hips and shoulders and/or X-RAY, CT and/or MRI to determine bone curvature.
- the first patient group receives the FOXO 4 DRI peptide by intravenous administration using a single-dose injection of the FOXO 4 DRI peptide at a therapeutically effective dose (i.e. a pharmaceutically effective amount), while the second patient group is non-treated or receives a placebo.
- a therapeutically effective dose i.e. a pharmaceutically effective amount
- Hepatic insufficiency disease parameters are measured in both patient groups, wherein a is the outcome for treated patients, b is the outcome for non-treated control patients, wherein when a is different from b, it is demonstrated that the FOXO4 DRI peptide is effective in the treatment of hepatic insufficiency.
- Hepatic insufficiency disease parameters that are to be measured are inter alia blood AST and ALT values.
- the first patient group receives the FOXO 4 DRI peptide by intravenous administration using a single-dose injection of the FOXO 4 DRI peptide at a therapeutically effective dose (i.e. a pharmaceutically effective amount), while the second patient group is non-treated or receives a placebo.
- a therapeutically effective dose i.e. a pharmaceutically effective amount
- cirrhosis disease parameters are measured in both patient groups, wherein a is the outcome for treated patients, b is the outcome for non-treated control patients, wherein when a is different from b, it is demonstrated that the FOXO4 DRI peptide is effective in the treatment of cirrhosis.
- Cirrhosis disease parameters that are to be measured are inter alia measurements of blood-clotting factors and
- liver biopsy analysis for liver damage.
- the first patient group receives the FOXO 4 DRI peptide by intravenous
- HGPS disease parameters are measured in both patient groups, wherein a is the outcome for treated patients, b is the outcome for non-treated control patients, wherein when a is different from b, it is demonstrated that the FOXO4 DRI peptide is effective in the treatment of HGPS.
- HGPS disease parameters that are to be measured are inter alia features of accelerated aging, hair loss (alopecia), aged-looking skin, joint abnormalities, and a loss of fat under the skin.
- the first patient group receives the FOXO 4 DRI peptide by intravenous administration using a single-dose injection of the FOXO 4 DRI peptide at a therapeutically effective dose (i.e. a pharmaceutically effective amount), while the second patient group is non-treated or receives a placebo.
- a therapeutically effective dose i.e. a pharmaceutically effective amount
- (cardio)vascular disease parameters are measured in both atient groups, wherein a is the outcome for treated patients, b is the outcome for non-treated control patients, wherein when a is different from b, it is demonstrated that the FOXO4 DRI peptide is effective in the treatment of (cardio)vascular disease, (cardio)vascular disease parameters that are to be measured are inter alia cardiac ejection fraction, blood vessel stiffness and blood pressure.
- the first patient group receives the FOXO 4 DRI peptide by intravenous
- Obesity parameters that are to be measured are inter alia body weight, Body-Mass-Index (BMI), waist circumference, waist-to-hip ratio, skinfold thicknesses, and bioelectrical impedance, magnetic resonance imaging anr/or dual energy X-ray absorptiometry
- the first patient group receives the FOXO 4 DRI peptide by intravenous administration using a single-dose injection of the FOXO 4 DRI peptide at a therapeutically effective dose (i.e. a pharmaceutically effective amount), while the second patient group is non-treated or receives a placebo.
- a therapeutically effective dose i.e. a pharmaceutically effective amount
- lung emphysema disease parameters are measured in both patient groups, wherein a is the outcome for treated patients, b is the outcome for non-treated control patients, wherein when a is different from b, it is demonstrated that the FOXO4 DRI peptide is effective in the treatment of lung emphysema.
- Lung emphysema disease parameters that are to be measured are inter alia breathlessness, chest size, decreased breath sounds through the stethoscope, fingertip shape, style of breathing,
- hypoxemia hypercaria, cyanosis, malnutrition.
- the first patient group receives the FOXO 4 DRI peptide by intravenous administration using a single-dose injection of the FOXO 4 DRI peptide at a therapeutically effective dose (i.e. a pharmaceutically effective amount), while the second patient group is non-treated or receives a placebo.
- a therapeutically effective dose i.e. a pharmaceutically effective amount
- b is the outcome for non-treated control patients, wherein when a is different from b, it is demonstrated that the FOXO4 DRI peptide is effective in the treatment of boutonneuse fever.
- Boutonneuse fever disease parameters that are to be measured are inter alia Weil-Felix test
- Example 25 Treatment of Alzheimer's disease patients with the FOX04 DRI peptide
- the first patient group receives the FOXO 4 DRI peptide by intravenous administration using a single-dose injection of the FOXO 4 DRI peptide at a therapeutically effective dose (i.e. a pharmaceutically effective amount), while the second patient group is non-treated or receives a placebo.
- a is the outcome for treated patients
- b is the outcome for non-treated control patients, wherein when a is different from b, it is demonstrated that the FOXO4 DRI peptide is effective in the treatment of Alzheimer's disease.
- Alzheimer's disease parameters that are to be measured are inter alia changes in ability to carry out daily activities, and changes in behavior and personality, tests of memory, problem solving, attention, counting, and language, blood and urine tests, brain scans, such as computed tomography (CT), magnetic resonance imaging (MRI), or positron emission tomography (PET) and /or biomarker analysis.
- CT computed tomography
- MRI magnetic resonance imaging
- PET positron emission tomography
- the first patient group receives the FOXO 4 DRI peptide by intravenous administration using a single-dose injection of the FOXO 4 DRI peptide at a therapeutically effective dose (i.e. a pharmaceutically effective amount), while the second patient group is non-treated or receives a placebo.
- a therapeutically effective dose i.e. a pharmaceutically effective amount
- Parkinson's disease parameters are measured in both patient groups, wherein a is the outcome for treated patients, b is the outcome for non-treated control patients, wherein when a is different from b, it is demonstrated that the FOXO4 DRI peptide is effective in the treatment of Parkinson's disease.
- Parkinson's disease parameters that are to be measured are inter alia analysis for tremors, limb or neck stiffness, general fitness and balance and/or locomotor function.
- the first patient group receives the FOXO 4 DRI peptide by intravenous
- COPD disease parameters that are to be measured are inter alia spirometry and lung functional tests as described for lung emphysema, including breathlessness, chest size, decreased breath sounds through the stethoscope, fingertip shape, style of breathing, hypoxemia, hypercaria, cyanosis, malnutrition. Lung volume, lung ejection capacity, dead volume in the lungs, airflow changes after bronchodilator medication, chest X-RAY and CT scan of the chest and red blood cell counts.
- the first patient group receives the FOXO 4 DRI peptide by intravenous administration using a single-dose injection of the FOXO 4 DRI peptide at a therapeutically effective dose (i.e. a pharmaceutically effective amount), while the second patient group is non-treated or receives a placebo.
- a therapeutically effective dose i.e. a pharmaceutically effective amount
- renal insufficiency disease parameters are measured in both atient groups, wherein a is the outcome for treated patients, b is the outcome for non-treated control patients, wherein when a is different from b, it is demonstrated that the FOX04 DRI peptide is effective in the treatment of renal insufficiency.
- Renal insufficiency disease parameters that are to be measured are inter alia blood pressure, heart/lung sound analysis nervous system exam, urinalaysis for protein content, analysis for creatinine clearance and level of Blood Urea Nitrogen, CT, MRI and/or ultrasound of abdomen and kidneys, kidney biopsy for damage analysis.
- the first patient group receives the FOXO 4 DRI peptide by intravenous administration using a single-dose injection of the FOXO 4 DRI peptide at a therapeutically effective dose (i.e. a pharmaceutically effective amount), while the second patient group is non-treated or receives a placebo.
- a therapeutically effective dose i.e. a pharmaceutically effective amount
- depression parameters are measured in both patient groups, wherein a is the outcome for treated patients, b is the outcome for non-treated control patients, wherein when a is different from b, it is demonstrated that the FOXO4 DRI peptide is effective in the treatment of depression, depression parameters that are to be measured are inter alia physical examination, sadness or depressed mood most of the day, major changes in weight, insomnia or excessive sleep, fatigue or loss of energy most of the day, feelings of hopelessness or worthlessness or excessive guilt, problems with concentration or decision making, recurring thoughts of death or suicide.
- Example 30 Treatment of metabolic syndrome patients with the FOX04 DRI peptide
- the first patient group receives the FOXO 4 DRI peptide by intravenous administration using a single-dose injection of the FOXO 4 DRI peptide at a therapeutically effective dose (i.e. a pharmaceutically effective amount), while the second patient group is non-treated or receives a placebo.
- a is the outcome for treated patients
- b is the outcome for non-treated control patients, wherein when a is different from b, it is demonstrated that the FOXO4 DRI peptide is effective in the treatment of metabolic syndrome.
- Metabolic syndrome disease parameters that are to be measured are inter alia measurements for obesity (see above, e.g. waist circumfence), blood levels of triglicerides, HDL cholesterol, blood pressure, fasting glucose.
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Organic Chemistry (AREA)
- Veterinary Medicine (AREA)
- Pharmacology & Pharmacy (AREA)
- Public Health (AREA)
- Animal Behavior & Ethology (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Neurology (AREA)
- Gastroenterology & Hepatology (AREA)
- Neurosurgery (AREA)
- Diabetes (AREA)
- Biomedical Technology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Immunology (AREA)
- Hematology (AREA)
- Physical Education & Sports Medicine (AREA)
- Zoology (AREA)
- Rheumatology (AREA)
- Orthopedic Medicine & Surgery (AREA)
- Epidemiology (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- Toxicology (AREA)
- Genetics & Genomics (AREA)
- Molecular Biology (AREA)
- Pain & Pain Management (AREA)
- Obesity (AREA)
- Pulmonology (AREA)
- Ophthalmology & Optometry (AREA)
- Heart & Thoracic Surgery (AREA)
- Cardiology (AREA)
- Oncology (AREA)
Abstract
Description
Claims
Priority Applications (11)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
RU2017128116A RU2017128116A (en) | 2015-01-23 | 2016-01-25 | COMPOUNDS PREVENTING AGING, AND WAYS OF THEIR APPLICATION |
SG11201706029VA SG11201706029VA (en) | 2015-01-23 | 2016-01-25 | Anti-senescence compounds and uses thereof |
MX2017009595A MX2017009595A (en) | 2015-01-23 | 2016-01-25 | Anti-senescence compounds and uses thereof. |
CN201680018230.8A CN107531768A (en) | 2015-01-23 | 2016-01-25 | Anti-senescence compounds and application thereof |
EP21189202.1A EP3936141A3 (en) | 2015-01-23 | 2016-01-25 | Anti-senescence compounds and uses thereof |
AU2016209737A AU2016209737A1 (en) | 2015-01-23 | 2016-01-25 | Anti-senescence compounds and uses thereof |
CA2974623A CA2974623A1 (en) | 2015-01-23 | 2016-01-25 | Anti-senescence compounds and uses thereof |
EP16713125.9A EP3247375B1 (en) | 2015-01-23 | 2016-01-25 | Anti-senescence compounds and uses thereof |
JP2017538970A JP6707549B2 (en) | 2015-01-23 | 2016-01-25 | Anti-aging compounds and their use |
US15/545,794 US11723947B2 (en) | 2015-01-23 | 2016-01-25 | Anti-senescence compounds and uses thereof |
BR112017015833-7A BR112017015833A2 (en) | 2015-01-23 | 2016-01-25 | anti-senescence compounds and their uses |
Applications Claiming Priority (4)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
NL2014183 | 2015-01-23 | ||
NL2014183 | 2015-01-23 | ||
EP15193041.9 | 2015-11-04 | ||
EP15193041 | 2015-11-04 |
Publications (2)
Publication Number | Publication Date |
---|---|
WO2016118014A2 true WO2016118014A2 (en) | 2016-07-28 |
WO2016118014A3 WO2016118014A3 (en) | 2016-11-17 |
Family
ID=55642804
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/NL2016/050057 WO2016118014A2 (en) | 2015-01-23 | 2016-01-25 | Anti-senescence compounds and uses thereof |
Country Status (13)
Country | Link |
---|---|
US (1) | US11723947B2 (en) |
EP (2) | EP3936141A3 (en) |
JP (1) | JP6707549B2 (en) |
CN (1) | CN107531768A (en) |
AU (1) | AU2016209737A1 (en) |
BR (1) | BR112017015833A2 (en) |
CA (1) | CA2974623A1 (en) |
CL (1) | CL2017001883A1 (en) |
MX (1) | MX2017009595A (en) |
PE (1) | PE20180128A1 (en) |
RU (1) | RU2017128116A (en) |
SG (1) | SG11201706029VA (en) |
WO (1) | WO2016118014A2 (en) |
Cited By (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2019069070A1 (en) * | 2017-10-02 | 2019-04-11 | Eternans Ltd | Novel senolytic peptides |
JP2020511951A (en) * | 2017-01-03 | 2020-04-23 | バイオアトラ、エルエルシー | Protein therapy for the treatment of senescent cells |
US11040027B2 (en) | 2017-01-17 | 2021-06-22 | Heparegenix Gmbh | Protein kinase inhibitors for promoting liver regeneration or reducing or preventing hepatocyte death |
WO2021165538A1 (en) | 2020-02-21 | 2021-08-26 | Cleara Biotech B.v. | Improved anti-senescence compounds and uses thereof |
US11433073B2 (en) | 2019-12-12 | 2022-09-06 | Ting Therapeutics Llc | Compositions and methods for the prevention and treatment of hearing loss |
EP4249912A1 (en) | 2022-03-24 | 2023-09-27 | Cleara Biotech B.V. | Phosphorylation of p53 as a prognostic or diagnostic marker for the treatment of senescent cells in a mammal |
Families Citing this family (7)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP3705109A1 (en) | 2019-03-05 | 2020-09-09 | Greenaltech, S.L. | Carotenoids in the treatment of senescence-related diseases |
WO2020201444A1 (en) * | 2019-04-02 | 2020-10-08 | Eternans Ltd | Repurposing small molecules for senescence-related diseases and disorders |
CN111346101B (en) * | 2019-04-16 | 2023-01-24 | 中山大学 | Application of TAMATINIB in preparation of medicine for treating glioma |
GB2603645B (en) * | 2019-07-22 | 2023-03-01 | Uniao Brasileira De Educacao Catolica | Polypeptides having anti-senescent effects and uses thereof |
CN113144192A (en) * | 2020-01-22 | 2021-07-23 | 中国科学院上海营养与健康研究所 | Application of MAPK/ERK pathway inhibitor in antagonizing skin aging and radiation-induced skin premature aging |
EP4281034A1 (en) | 2021-01-24 | 2023-11-29 | Forrest, Michael, David | Inhibitors of atp synthase - cosmetic and therapeutic uses |
CN115466321B (en) * | 2022-09-23 | 2024-03-22 | 南方医科大学珠江医院 | FOXO3a-DRI peptide fragment, pharmaceutical composition and application thereof |
Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2013152038A1 (en) | 2012-04-02 | 2013-10-10 | Buck Institute For Research On Aging | Targeting senescent cells and cancer cells by interference with jnk and/or foxo4 |
Family Cites Families (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US8227455B2 (en) * | 2005-04-18 | 2012-07-24 | Rigel Pharmaceuticals, Inc. | Methods of treating cell proliferative disorders |
WO2010065567A2 (en) * | 2008-12-01 | 2010-06-10 | Lifespan Extension Llc | Methods and compositions for altering health, wellbeing, and lifespan |
CN102459313A (en) * | 2009-05-08 | 2012-05-16 | 上海泰飞尔生化技术有限公司 | High penetration prodrug compositions of peptides and peptide-related compounds |
US20130288980A1 (en) * | 2012-04-02 | 2013-10-31 | Buck Institute For Research On Aging | Targeting senescent and cancer cells for selective killing by interference with foxo4 |
US10624948B2 (en) * | 2013-06-26 | 2020-04-21 | Xigen Inflammation Ltd. | Use of cell-permeable peptide inhibitors of the JNK signal transduction pathway for the treatment of various diseases |
CA3048660A1 (en) | 2017-01-03 | 2018-07-12 | Bioatla, Llc | Protein therapeutics for treatment of senescent cells |
-
2016
- 2016-01-25 RU RU2017128116A patent/RU2017128116A/en not_active Application Discontinuation
- 2016-01-25 WO PCT/NL2016/050057 patent/WO2016118014A2/en active Application Filing
- 2016-01-25 CN CN201680018230.8A patent/CN107531768A/en active Pending
- 2016-01-25 CA CA2974623A patent/CA2974623A1/en active Pending
- 2016-01-25 AU AU2016209737A patent/AU2016209737A1/en not_active Abandoned
- 2016-01-25 EP EP21189202.1A patent/EP3936141A3/en not_active Withdrawn
- 2016-01-25 MX MX2017009595A patent/MX2017009595A/en unknown
- 2016-01-25 PE PE2017001261A patent/PE20180128A1/en not_active Application Discontinuation
- 2016-01-25 SG SG11201706029VA patent/SG11201706029VA/en unknown
- 2016-01-25 BR BR112017015833-7A patent/BR112017015833A2/en not_active Application Discontinuation
- 2016-01-25 US US15/545,794 patent/US11723947B2/en active Active
- 2016-01-25 EP EP16713125.9A patent/EP3247375B1/en active Active
- 2016-01-25 JP JP2017538970A patent/JP6707549B2/en active Active
-
2017
- 2017-07-21 CL CL2017001883A patent/CL2017001883A1/en unknown
Patent Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2013152038A1 (en) | 2012-04-02 | 2013-10-10 | Buck Institute For Research On Aging | Targeting senescent cells and cancer cells by interference with jnk and/or foxo4 |
Non-Patent Citations (14)
Title |
---|
ADAMCSZAK ET AL., JASN, 2003 |
BAKER ET AL., NATURE, vol. 479, no. 7372, 2011, pages 232 - 236 |
BERNARD'S LAB NATURE, 2014 |
DE BOER ET AL., CANCER RES., vol. 59, no. 14, 1999, pages 3489 - 3494 |
DE BOER ET AL., MOLECULAR CELL, vol. 1, no. 7, 1998, pages 981 - 990 |
DE BOER ET AL., SCIENCE, vol. 296, no. 5571, 2002, pages 1276 - 1279 |
DEMARIA ET AL., DEVELOPMENTAL CELL, vol. 31, no. 6, 2014, pages 722 - 733 |
DIMRI ET AL., PNAS, vol. 92, 1995, pages 9363 - 9367 |
F. CHOPLIN: "Quantitative Drug Design", 1992, PERGAMON PRESS |
GREER, E. L ET AL.: "FOXO transcription factors at the interface between longevity and tumor suppression", ONCOGENE, vol. 24, 2005, pages 7410 - 7425, XP055125348 |
KRUISWIJK ET AL., ONCOGENE, 2015 |
MIRANDA ET AL., BLOOD, 2003 |
RODIER ET AL., J CELL BIOL, vol. 192, 2011, pages 547 - 556 |
SANTA CRUZ, BIOTECHNOLOGY |
Cited By (8)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP2020511951A (en) * | 2017-01-03 | 2020-04-23 | バイオアトラ、エルエルシー | Protein therapy for the treatment of senescent cells |
US11040027B2 (en) | 2017-01-17 | 2021-06-22 | Heparegenix Gmbh | Protein kinase inhibitors for promoting liver regeneration or reducing or preventing hepatocyte death |
WO2019069070A1 (en) * | 2017-10-02 | 2019-04-11 | Eternans Ltd | Novel senolytic peptides |
US11433073B2 (en) | 2019-12-12 | 2022-09-06 | Ting Therapeutics Llc | Compositions and methods for the prevention and treatment of hearing loss |
WO2021165538A1 (en) | 2020-02-21 | 2021-08-26 | Cleara Biotech B.v. | Improved anti-senescence compounds and uses thereof |
EP4327818A2 (en) | 2020-02-21 | 2024-02-28 | Cleara Biotech B.V. | Improved anti-senescence compounds and uses for the treatment of cancer |
EP4249912A1 (en) | 2022-03-24 | 2023-09-27 | Cleara Biotech B.V. | Phosphorylation of p53 as a prognostic or diagnostic marker for the treatment of senescent cells in a mammal |
WO2023180570A1 (en) | 2022-03-24 | 2023-09-28 | Cleara Biotech B.v. | Phosphorylation of p53 as a prognostic or diagnostic marker for the treatment of senescent cells in a mammal |
Also Published As
Publication number | Publication date |
---|---|
JP6707549B2 (en) | 2020-06-10 |
PE20180128A1 (en) | 2018-01-18 |
RU2017128116A3 (en) | 2019-08-21 |
WO2016118014A3 (en) | 2016-11-17 |
US11723947B2 (en) | 2023-08-15 |
RU2017128116A (en) | 2019-02-26 |
EP3247375B1 (en) | 2021-08-04 |
SG11201706029VA (en) | 2017-08-30 |
US20180015137A1 (en) | 2018-01-18 |
JP2018508196A (en) | 2018-03-29 |
MX2017009595A (en) | 2018-04-20 |
EP3936141A2 (en) | 2022-01-12 |
CL2017001883A1 (en) | 2018-02-02 |
BR112017015833A2 (en) | 2018-03-27 |
EP3936141A3 (en) | 2022-03-23 |
EP3247375A2 (en) | 2017-11-29 |
CN107531768A (en) | 2018-01-02 |
AU2016209737A1 (en) | 2017-08-17 |
CA2974623A1 (en) | 2016-07-28 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
EP3247375B1 (en) | Anti-senescence compounds and uses thereof | |
EP3900774A1 (en) | Using alternating electric fields to increase cell membrane permeability | |
ES2791539T3 (en) | Compounds for the treatment of diseases related to the expression of DUX4 | |
TW201615196A (en) | The new cancer therapy indication of the cellcept | |
KR101877840B1 (en) | Process for the identification of compounds for treating cancer | |
CN107106580B (en) | Composition for treating cancer stem cells | |
Chung et al. | Metronomic oral doxorubicin in combination of Chk1 inhibitor MK-8776 for p53-deficient breast cancer treatment | |
JP7039470B2 (en) | Monocarboxylic Acid Transporter 4 (MCT4) Antisense Oligonucleotide (ASO) Inhibitor for Use as a Therapeutic Agent in the Treatment of Cancer | |
Wang et al. | Theabrownins produced via chemical oxidation of tea polyphenols inhibit human lung cancer cells in vivo and in vitro by suppressing the PI3K/AKT/mTOR pathway activation and promoting autophagy | |
Sun et al. | Arsenic trioxide inhibits angiogenesis in vitro and in vivo by upregulating FoxO3a | |
US9901594B2 (en) | Pharmaceutical composition and uses thereof | |
KR101858128B1 (en) | Pharmaceutical composition for treating cancer having radioresistant phenotype comprising SULF2 inhibitor | |
AU2020255063B2 (en) | Combined use of A-nor-5α androstane compound drug and anticancer drug | |
Zhao et al. | H19 Overexpression Improved Efficacy of Mesenchymal Stem Cells in Ulcerative Colitis by Modulating the miR‐141/ICAM‐1 and miR‐139/CXCR4 Axes | |
EP3969027A1 (en) | Polypeptides for treatment of cancer | |
Chung et al. | Benzamide is an organic compound with the chemical formula of C7H7NO. Menu | |
Zhang et al. | CAFs-Promoted LncRNA DNM3OS Conferred Radioresistance by Regulating DNA Damage Response in a TNFSF4-Dependent Manner in Esophageal Squamous Cell Carcinoma | |
KR20180016357A (en) | METHOD AND APPARATUS FOR DETERMINING INTERFERON HAVING A DIRECT INHIBITING EFFECT ON TUMORS | |
Qu et al. | Induction of apoptosis in human hepatocellular carcinoma cells by synthetic antineoplaston A10 | |
Chen et al. | AKT2 deficiency alleviates doxorubicin-induced cardiac injury via alleviating oxidative stress in cardiomyocytes | |
Wu et al. | OSW-1 Induces Apoptosis and Cyto-Protective Autophagy, and Synergizes with Doxorubicin on Spontaneous Metastasis of Triple Negative Breast Cancer | |
Panchal | Investigation of PARP1 as a Therapeutic Target for Glioblastoma | |
張婷婷 | Silibinin efficacy in a rat model of pulmonary arterial hypertension using monocrotaline and chronic hypoxia | |
Zeng et al. | Ropivacaine Induce G0/G1 Phase Cell Cycle Arrest and Apoptosis of PC12 Cells Via Inhibiting Mitochondrial STAT3 Translocation | |
CN106999526B (en) | Application of polyacetylene glycoside in inhibiting granulosa marrow-derived suppressor cell activity and tumor metastasis |
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: 16713125 Country of ref document: EP Kind code of ref document: A2 |
|
ENP | Entry into the national phase |
Ref document number: 2974623 Country of ref document: CA |
|
ENP | Entry into the national phase |
Ref document number: 2017538970 Country of ref document: JP Kind code of ref document: A |
|
WWE | Wipo information: entry into national phase |
Ref document number: 001261-2017 Country of ref document: PE Ref document number: 11201706029V Country of ref document: SG Ref document number: MX/A/2017/009595 Country of ref document: MX Ref document number: 15545794 Country of ref document: US |
|
NENP | Non-entry into the national phase |
Ref country code: DE |
|
REG | Reference to national code |
Ref country code: BR Ref legal event code: B01A Ref document number: 112017015833 Country of ref document: BR |
|
REEP | Request for entry into the european phase |
Ref document number: 2016713125 Country of ref document: EP |
|
ENP | Entry into the national phase |
Ref document number: 2016209737 Country of ref document: AU Date of ref document: 20160125 Kind code of ref document: A |
|
ENP | Entry into the national phase |
Ref document number: 2017128116 Country of ref document: RU Kind code of ref document: A |
|
ENP | Entry into the national phase |
Ref document number: 112017015833 Country of ref document: BR Kind code of ref document: A2 Effective date: 20170724 |