EP1971356A2 - Hemmung von cxcr4 und/oder der zellmotilität durch phenylalanin, cystein oder diese aminosäuren enthaltende peptide - Google Patents

Hemmung von cxcr4 und/oder der zellmotilität durch phenylalanin, cystein oder diese aminosäuren enthaltende peptide

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Publication number
EP1971356A2
EP1971356A2 EP06821674A EP06821674A EP1971356A2 EP 1971356 A2 EP1971356 A2 EP 1971356A2 EP 06821674 A EP06821674 A EP 06821674A EP 06821674 A EP06821674 A EP 06821674A EP 1971356 A2 EP1971356 A2 EP 1971356A2
Authority
EP
European Patent Office
Prior art keywords
seq
amino acid
peptide
derivative
cysteine
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP06821674A
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English (en)
French (fr)
Inventor
Tsvee Lapidot
Menachem Rubinstein
Asaf Spiegel
Mati Fridkin
Alexander Kalinkovich
Shoham Shivtiel
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Yeda Research and Development Co Ltd
Original Assignee
Yeda Research and Development Co Ltd
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Filing date
Publication date
Application filed by Yeda Research and Development Co Ltd filed Critical Yeda Research and Development Co Ltd
Publication of EP1971356A2 publication Critical patent/EP1971356A2/de
Withdrawn legal-status Critical Current

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    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/06—Tripeptides
    • A61K38/063—Glutathione
    • 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
    • A61K38/08—Peptides having 5 to 11 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A61K31/195—Carboxylic acids, e.g. valproic acid having an amino group
    • A61K31/197—Carboxylic acids, e.g. valproic acid having an amino group the amino and the carboxyl groups being attached to the same acyclic carbon chain, e.g. gamma-aminobutyric acid [GABA], beta-alanine, epsilon-aminocaproic acid or pantothenic acid
    • A61K31/198—Alpha-amino acids, e.g. alanine or edetic acid [EDTA]
    • 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
    • A61K38/05—Dipeptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/06—Tripeptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • 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
    • A61K38/07—Tetrapeptides
    • 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
    • A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • 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]
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    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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    • A61P35/00—Antineoplastic agents
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    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/04—Antineoplastic agents specific for metastasis
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    • 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
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    • 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
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    • C07K5/06—Dipeptides
    • C07K5/06008—Dipeptides with the first amino acid being neutral
    • C07K5/06078—Dipeptides with the first amino acid being neutral and aromatic or cycloaliphatic
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    • C07K5/06—Dipeptides
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    • C07K5/06—Dipeptides
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    • C07K5/06095—Arg-amino acid
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    • C07K5/08—Tripeptides
    • C07K5/0802—Tripeptides with the first amino acid being neutral
    • C07K5/0804—Tripeptides with the first amino acid being neutral and aliphatic
    • C07K5/0808—Tripeptides with the first amino acid being neutral and aliphatic the side chain containing 2 to 4 carbon atoms, e.g. Val, Ile, Leu
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
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    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/08—Tripeptides
    • C07K5/0802—Tripeptides with the first amino acid being neutral
    • C07K5/0804—Tripeptides with the first amino acid being neutral and aliphatic
    • C07K5/081—Tripeptides with the first amino acid being neutral and aliphatic the side chain containing O or S as heteroatoms, e.g. Cys, Ser
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/08—Tripeptides
    • C07K5/0802—Tripeptides with the first amino acid being neutral
    • C07K5/0812—Tripeptides with the first amino acid being neutral and aromatic or cycloaliphatic
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/10—Tetrapeptides
    • C07K5/1002—Tetrapeptides with the first amino acid being neutral
    • C07K5/1005—Tetrapeptides with the first amino acid being neutral and aliphatic
    • C07K5/1013—Tetrapeptides with the first amino acid being neutral and aliphatic the side chain containing O or S as heteroatoms, e.g. Cys, Ser
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/10—Tetrapeptides
    • C07K5/1019—Tetrapeptides with the first amino acid being basic
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/10—Tetrapeptides
    • C07K5/1024—Tetrapeptides with the first amino acid being heterocyclic
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids

Definitions

  • the invention relates to phenylalanine, cysteine, derivatives of said amino acids, peptides comprising them, and to their use in diseases disorders or conditions whose pathology is caused by or associated with CXCR4 activity and/or cell motility.
  • Hematopoietic stem cells are a rare population of cells within the bone marrow microenvironment. Hematopoietic stem cells actively maintain the continuous production of all mature blood cell lineages, which include major components of the immune system such as T and B Lymphocytes throughout life while maintaining a small pool of undifferentiated stem and progenitor cells (Mayani, 2003).
  • stem cells migrate through the blood circulation and home into the bone marrow (BM), repopulating it with immature and maturing myeloid and lymphoid blood cells, which in turn are released into the circulation.
  • BM bone marrow
  • SDF-I chemokine stromal derived factor one
  • SDF-I is highly preserved throughout evolution. Human and mouse SDF-I are cross-reactive and differ in one amino acid. SDF-I (also named CXCL 12) serves as a survival factor for stem and progenitor cells, and is involved in immature B cell and megakaryocyte development (McGrath et al., 1999; Nagasawa et al, 1996). Homing of human stem cells and their subsequent proliferation and differentiation in transplanted immune deficient mice was found to be dependent on interactions between SDF-I, which is expressed by the host bone marrow, and its receptor CXCR4, which is expressed on the donor homing cells.
  • CXCR4 upregulation blocked the low levels of human CD34 + CXCR4 " cell engraftment.
  • the phenotype of repopulating human stem cells was defined as CD34+CD38 "/l0W CXCR4+ cells (Kollet et al., 2002).
  • cytokines such as G-CSF are used to recruit human stem cells from the circulation.
  • Proteolytic enzymes such as neutrophil elastase were found to degrade SDF- 1 in the bone marrow during G-CSF administration.
  • the levels of CXCR4 expression on hematopoietic cells within the bone marrow were found to increase prior to their mobilization.
  • Neutralizing antibody for CXCR4 or SDF-I reduced human and mouse stem cell mobilization, demonstrating SDF-1/CXCR4 signaling in cell egress (Petit et al., 2002).
  • stem cell homing and release/mobilization utilize similar mechanisms, and in both processes SDF-1/CXCR4 interactions play a major role.
  • SDF-I also plays an important role in the migration of leukemic cells. While normal and leukemic cells share similar mechanisms of migration, different homing patterns as well as SDF-I signaling pathways were found when comparing malignant human Pre-B ALL cells (B-cell precursor acute lymphoblastic leukemia) to normal immature CD34+ cells (Spiegel et al., 2004). In another malignant disease, acute myelogenous leukemia (AML), high levels of intracellular CXCR4 and SDF-I have been found in all leukemic cells, including cells that do not express surface CXCR4. CXCR4 is essential for the homing of these cells to the BM of immune deficient mice, demonstrating dynamic regulation of CXCR4 in these cells (Tavor et al., 2005).
  • AML acute myelogenous leukemia
  • CXCR4 is essential for the homing of these cells to the BM of immune deficient mice, demonstrating dynamic regulation of CXCR4 in these cells (Tavor
  • SDF-I activated the major adhesion molecules such as CD44, LFA-I, VLA-4 and VLA-5 on migrating human stem and progenitor cells as part of the multistep process of homing and transendothelial migration (Peled et al., 2000).
  • SDF-I is also involved in proliferation and survival of various cells including normal human CD34+ cells and leukemic cells (Lee et al., 2002; Nishii et al. 1999, and Tavor et al., 2005).
  • the invention relates to the use of the amino acid phenylalanine or a derivative thereof; the amino acid cysteine or a derivative thereof; a combination of amino acids comprising phenylalanine or a derivative thereof and cysteine or a derivative thereof; a peptide comprising up to 22 amino acid residues comprising at least one phenylalanine or a derivative thereof, at least one cysteine or a derivative thereof, or at least one phenylalanine or a derivative thereof and at least one cysteine or a derivative thereof in the manufacture of a medicament for treating or preventing a disease, disorder or condition responsive to inhibition of CXCR4 activity and/or inhibition of cell motility.
  • the amino acid phenylalanine or a derivative thereof is used in the manufacture of a medicament for treating or preventing a disease, disorder or condition responsive to inhibition of CXCR4 activity and/or inhibition of cell motility.
  • the phenylalanine derivative is a solubility-improved derivative of phenylalanine.
  • the phenylalanine derivative is phenylalaninol (Phe-ol) or succinyl phenylalanine (Suc-Phe).
  • the phenylalanine derivative is ⁇ -phenylethylamine.
  • the amino acid cysteine or a derivative thereof is used in the manufacture of a medicament for treating or preventing a disease, disorder or condition responsive to inhibition of CXCR4 activity and/or inhibition of cell motility.
  • cysteine derivative is a solubility-improved derivative of cysteine.
  • cysteine derivative is selected from cysteine ethyl ester, cysteine sulfuric acid, and cysteinamine.
  • the combination of amino acids comprising phenylalanine or a derivative thereof and cysteine or a derivative thereof is used in the manufacture of a medicament for treating or preventing a disease, disorder or condition responsive to inhibition of CXCR4 activity and/or inhibition of cell motility.
  • a peptide of up to 22 amino acid residues comprising at least one phenylalanine or a derivative thereof is used in the manufacture of a medicament for treating or preventing a disease, disorder or condition responsive to inhibition of CXCR4 activity and/or inhibition of cell motility.
  • the peptide consists of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 ,19, 20, or 21 amino acid residues comprising at least one phenylalanine .
  • the peptide comprises about 2, 3, 4, 5, or 6 consecutive amino acid residues including the at least one phenylalanine and at least one non-phenylalanine residue that is a charged amino acid.
  • the at least one non- phenylalanine residue is a positively charged amino acid or a negatively charged amino acid residue such as lysine or arginine.
  • the peptide comprises the amino acid sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or a derivative thereof linked to aminocaproic acid , SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 8.
  • the negatively charged amino acid residue is glutamic acid.
  • the peptide comprises the amino acid sequence of SEQ ID NO: 6 or SEQ ID NO: 7.
  • a peptide of up to 22 amino acid residues comprising at least one cysteine or a derivative thereof is used in the manufacture of a medicament for treating or preventing a disease, disorder or condition responsive to inhibition of CXCR4 activity and/or inhibition of cell motility.
  • the peptide consists of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 ,19, 20, or 21 amino acid residues comprising at least one cysteine.
  • the peptide comprises about 2, 3, 4, 5, or 6 consecutive amino acid residues including the at least one cysteine and at least one non-cysteine residue selected from proline, arginine and glutamic acid.
  • the peptide consists of glutathione.
  • a peptide of up to 22 amino acid residues comprising at least one phenylalanine or a derivative thereof and at least one cysteine or a derivative thereof, except for a peptide from the amino terminus of SDF-I which includes the LSY motif (SEQ ID NO: 19) is used in the manufacture of a medicament for treating or preventing a disease, disorder or condition responsive to inhibition of CXCR4 activity and/or inhibition of cell motility.
  • the peptide consists of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 ,19, 20, and 21 amino acid residues comprising at least one phenylalanine or a derivative thereof and at least one cysteine or a derivative thereof.
  • the peptide comprises about
  • the peptide comprises about 3, 4, 5, 6 or 7 consecutive amino acid residues comprising one cysteine or a derivative thereof and one phenylalanine or a derivative thereof separated by at least one non cysteine or non phenylalanine amino acid residue.
  • the peptide comprises 3 consecutive amino acid residues of SEQ ID NO: 12. In another further embodiment of the invention the peptide comprises 4 consecutive amino acid residues comprising one cysteine or a derivative thereof and one phenylalanine or a derivative thereof separated by at least one non cysteine or non phenylalanine amino acid residue. In another further embodiment of the invention the sequence of the 4 consecutive amino acid residues consists of SEQ ID NO: 11.
  • the first residue of the 4 consecutive amino acid residues is a proline such as in the sequence of the consecutive amino acid residues consisting of SEQ ID NO: 13.
  • the peptide comprises 5 consecutive amino acid residues comprising one cysteine or a derivative thereof and one phenylalanine or a derivative thereof separated by at least one non cysteine or non phenylalanine amino acid residue.
  • the first residue of the 5 consecutive amino acid residues is a proline such as in SEQ ID NO: 16, or a derivative of SEQ ID NO: 16 having instead of a Cysteine a dCys (Trt) residue.
  • the fifth residue of the 5 consecutive amino acid residues is a glutamic acid such as in SEQ ID NO: 10.
  • the peptide comprises 6 consecutive amino acid residues comprising one cysteine or a derivative thereof and one phenylalanine or a derivative thereof separated by at least one non cysteine or non phenylalanine amino acid residue.
  • the sixth residue of the 6 consecutive amino acid residues is glutamic acid and/or the first residue of the consecutive amino acid residues is proline such as in SEQ ID NO: 9.
  • the invention provides the use of a peptide of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,
  • SEQ ID NO: 14 SEQ ID NO: 15, SEQ ID NO: 16 , a derivative of SEQ ID NO: 16 comprising instead of the cysteine a dCys(Trt) residue, or SEQ ID NO: 17 in the manufacture of a medicament for treating a disease, disorder or condition in which CXCR4 activity and/or cell motility is involved in the pathology or course of the disease, disorder or condition.
  • the invention also provides the use of a peptide of 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or 21 amino acid residues comprising the amino acid sequence C-X-X -F (SEQ ID NO: 18) wherein X is an arginine or an alanine in the manufacture of a medicament for treating a disease, disorder or condition in which CXCR4 activity and/or cell motility is involved in the pathology or course of the disease, disorder or condition.
  • the amino acid sequence consists of
  • the peptide is a fusion peptide and/or salt thereof.
  • the use of said amino acid (s) and/or peptide(s) is for the manufacture of a medicament for treating a disease, disorder or condition such as cancer expressing CXCR4, acquired immunodeficiency syndrome (AIDS) and/or inflammation.
  • a disease, disorder or condition such as cancer expressing CXCR4, acquired immunodeficiency syndrome (AIDS) and/or inflammation.
  • AIDS acquired immunodeficiency syndrome
  • the disease is cancer expressing CXCR4, such as leukemia, intraocular lymphoma, non-Hodgkin lymphoma, follicular center lymphoma, multiple myeloma, pancreatic cancer, kidney, prostate, breast, ovary, thyroid, colorectal cancer, oral squamous carcinoma, cervical cancer, neuroblastoma, glioma, astrocytoma, rhabdomyosarcoma, small cell lung cancer and melanoma.
  • CXCR4 such as leukemia, intraocular lymphoma, non-Hodgkin lymphoma, follicular center lymphoma, multiple myeloma, pancreatic cancer, kidney, prostate, breast, ovary, thyroid, colorectal cancer, oral squamous carcinoma, cervical cancer, neuroblastoma, glioma, astrocytoma, rhabdomyosarcoma, small cell lung cancer and melanoma.
  • the cancer is leukaemia, such as acute lymphoblastic leukemia.
  • the use is for the manufacture of a medicament for treating or preventing metastasis.
  • the use is for the manufacture of a medicament for treating AIDS.
  • the use is for treating or preventing an inflammatory disease, disorder or condition such as rheumatoid arthritis, septic arthritis, osteoarthritis, periodontitis, gingivitis, asthma, autoimmune disease, psoriasis, psoriatic arthritis, atopic dermatitis; interstitial nephritis, ocular inflammation, liver cirrhosis, neuroinflammatory disorders, graft versus host disease and inflammatory gastric conditions.
  • the invention provides a peptide of 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22 amino acid residues comprising the amino acid sequence C-X-X-F (SEQ ID NO: 18) wherein X is an arginine or an alanine.
  • the peptide comprises the amino acid sequence of SEQ ID NO: 14 or SEQ ID NO: 15,
  • the invention also provides a peptide of peptide of 3, 4, 5, 6, 7, 8, 9, 10, 11,
  • amino acid residues comprising 3, 4, 5, 6, 7, or 8 consecutive amino acid residues comprising one cysteine or a derivative thereof and one phenylalanine residue or a derivative thereof separated by at least one alanine.
  • the cysteine or derivative thereof and the phenylalanine or derivative thereof are separated by one or two alanine residues.
  • the peptide comprises 4 consecutive amino acid residues comprising one cysteine or a derivative thereof and one phenylalanine or a derivative thereof separated by at least one alanine.
  • the first and/or the last amino acid residue of the 4 consecutive amino acid residues is cysteine or a derivative thereof and phenylalanine or a derivative thereof, respectively.
  • the invention also provides a peptide of up to 22 amino acid residues comprising 3, 4, 5, 6, or 7 consecutive amino acid residues comprising one cysteine or a derivative thereof one phenylalanine residue or a derivative thereof separated by one arginine, except for a peptide from the amino terminus of SDF-I which includes the LSY motif (SEQ ID NO: 19).
  • the peptide of up to 22 amino acid residues comprises 3 consecutive amino acid residues consisting of SEQ ID NO: 12.
  • the peptide of up to 22 amino acid residues comprises 4 consecutive amino acid residues comprising one cysteine or a derivative thereof and one phenylalanine or a derivative thereof separated by one arginine.
  • the first and the last amino acid residue of the consecutive amino acid residues is cysteine or a derivative thereof and phenylalanine or a derivative thereof, respectively such as in the sequence of the 4 consecutive amino acid residues consisting of SEQ ID NO: 11.
  • the first amino acid residue of the 4 consecutive amino acid residues is a proline such as in the sequence of SEQ ID NO: 13.
  • the peptide of up to 22 amino acid residues comprises 5 consecutive amino acid residues comprising one cysteine or a derivative thereof and one phenylalanine or a derivative thereof separated by one arginine.
  • the first amino acid residue of the 5 consecutive amino acid residues is a proline such as in the sequence of SEQ ID NO: 16, or a derivative of SEQ ID NO: 16 having instead of a Cysteine a dCys (Trt) residue.
  • the fifth residue of the 5 consecutive amino acid residues is a glutamic acid such as in the sequence of SEQ ID NO: 10.
  • the peptide of up to 22 amino acid residues comprises 6 consecutive amino acid residues comprising one cysteine or a derivative thereof and one phenylalanine or a derivative thereof separated by one arginine.
  • the sixth residue of the 6 consecutive amino acid residues is glutamic acid.
  • the first residue of the 6 consecutive amino acid residues is proline such as in the sequence of SEQ ID NO: 9.
  • the invention also provides a peptide of up to 22 amino acid residues comprising 4, 5, 6, 7, or 8 consecutive amino acid residues comprising one cysteine or a derivative thereof one phenylalanine residue or a derivative thereof separated by two arginines such as in SEQ ID NO: 15.
  • the invention provides a peptide of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,
  • the invention provides a peptide of 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22 amino acids comprising 3 consecutive amino acid residues consisting of SEQ ID NO: 1 linked to aminocaproic acid or of SEQ ID NO: 8.
  • the invention provides a peptide of about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22 amino acids comprising 4 consecutive amino acid residues consisting of SEQ ID NO: 5.
  • the invention provides a peptide consisting of amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 1 linked to aminocaproic acid , SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, P SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, a derivative of SEQ ID NO: 16 comprising instead of the cysteine a dCys(Trt) residue or SEQ ID NO: 17.
  • the invention provides a peptide of about 3, 4, 5, 6, 7, 8, or 9 amino acid residues comprising the amino acid sequence C-R-F, except for a peptide from the amino terminus of SDF-I including the LSY motif or consisting of 6 to 9 amino acid residues lacking the LSY motif in which the last amino acid residue is serine.
  • Peptides provided by the invention include circularly permuted peptides and/or fusion peptides.
  • a fusion peptide is a peptide fused to a protein such as an immunoglobulin.
  • a fusion peptide is a peptide fused to a high molecular weight polymer such as polyethylene glycol (PEG).
  • PEG polyethylene glycol
  • the invention embraces a salt of a peptide provided by the invention.
  • the invention relates to an isolated DNA encoding a peptide provided by the invention, an expression vector comprising said DNA , a host cell harboring said DNA and/or expression vector, and to a method for producing said peptide comprising growing the host cell and isolating the protein produced.
  • the host cell is a eukaryotic cell, such as a mammalian cell e.g. HeLa, 293 T HEK and CHO, insect cell, or yeast cell.
  • a mammalian cell e.g. HeLa, 293 T HEK and CHO
  • insect cell or yeast cell.
  • the host cell is a prokaryotic cell.
  • the invention relates to the use of a peptide provided by the invention or a salt thereof in the manufacture of a medicament for treating or preventing a disease, disorder or condition selected from cancer expressing CXCR4, acquired immunodeficiency syndrome (AIDS), inflammation and metastasis.
  • a disease, disorder or condition selected from cancer expressing CXCR4, acquired immunodeficiency syndrome (AIDS), inflammation and metastasis.
  • AIDS acquired immunodeficiency syndrome
  • the invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a peptide provided by the invention or a salt thereof.
  • the invention relates to a pharmaceutically acceptable carrier and a peptide of 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22 amino acid residues comprising about 3, 4, 5, 6, or 7 consecutive amino acid residues comprising one cysteine or a derivative thereof one phenylalanine residue or a derivative thereof separated by one arginine, except for a peptide from the amino terminus of SDF-I including the LSY motif.
  • the invention in another further aspect, relates to a pharmaceutical composition
  • a pharmaceutical composition comprising the DNA of the invention and a pharmaceutically acceptable carrier.
  • the invention in another further aspect, relates to a pharmaceutical composition
  • a pharmaceutical composition comprising a vector of the invention and a pharmaceutically acceptable carrier.
  • the invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a mixture of amino acids consisting of phenylalanine or a derivative thereof and cysteine or a derivative thereof.
  • the invention relates to a method of treating or preventing a disease, disorder or condition responsive to inhibition of CXCR4 activity and/or inhibition of cell motility in a subject in need, comprising administering to the subject a therapeutically effective amount of the amino acid phenylalanine or a derivative thereof; the amino acid cysteine or a derivative thereof; a combination of amino acids comprising phenylalanine or a derivative thereof and cysteine or a derivative thereof; a peptide comprising up to 22 amino acid residues comprising at least one phenylalanine or a derivative thereof, at least one cysteine or a derivative thereof, or at least one phenylalanine or a derivative thereof and at least one cysteine or a derivative thereof, except for a peptide from the amino terminus of SDF-I including the LSY motif.
  • the invention relates to a method of treating or preventing a disease, disorder or condition in which the activity of CXCR4 is involved in the pathology or course of the disease, disorder or condition comprising administering to a subject in need a therapeutically effective amount of a peptide comprising a sequence set forth in SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, a derivative of SEQ ID NO: 16 comprising instead of the cysteine a dCys(Trt) residue, SEQ ID NO: 17 or a combination thereof.
  • the invention relates to a method of treating or preventing a disease, disorder or condition in which the activity of CXCR4 is involved in the pathology or course of the disease, disorder or condition comprising administering to a subject in need a therapeutically effective amount of a peptide comprising a sequence set forth in SEQ ID NO: 1, SEQ ID NO: 1 linked to aminocaproic acid , SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 or a combination thereof.
  • the invention relates to a method of treating or preventing a disease, disorder or condition selected from cancer expressing CXCR4, acquired immunodeficiency syndrome (AIDS), inflammation and metastasis comprising administering to a subject in need a therapeutically effective amount of a peptide provided by the invention.
  • a disease, disorder or condition selected from cancer expressing CXCR4, acquired immunodeficiency syndrome (AIDS), inflammation and metastasis
  • Figs. 1A-1D show that a factor found in the conditioned medium of leukemic Bl cells inhibits migration of hematopoietic cells.
  • Bl a line prepared from precursor-B cells from a patient with acute lymphoblastic leukemia B cells [B- ALL]
  • B 1-2 a sub-clone derived from the Bl cells by means of limiting dilution cells grown in 24 well plates at high or low cell densities for 16 hours, were tested for their migration capability towards SDF-I (125ng/ ml) in a transwell migration assay. It was found that cells taken from the high density cultures migrated significantly less towards SDF-I than cells taken from low density cultures *p ⁇ 0.05.
  • CB CD34 + cells comprising hematopoietic precursors
  • leukemic G2 cells another precursor-B cell line prepared from a different precursor-B-ALL patient
  • CB CD34+ or G2 cells were pre-treated for 1 hour with conditioned medium taken from the high density B 1 culture or remained untreated, and their SDF- 1 dependent migration was tested in a transwell migration assay.
  • the tested cells CB CD34+ or G2
  • SDF-I 125 ng/ml
  • C. Shows inhibition of SDF-I mediated ERK phosphorylation in G2 cells preincubated with the conditioned medium of B 1.
  • D. Shows reduced levels of PKC- ⁇ mRNA (detected by PCR) in Bl cells taken from a high-density culture (2x10 6 cells/ml) compared to the levels of PKC- ⁇ mRNA in B l from a low density cultures (1x10 5 cells/ml) or G2 cells taken from high-density cultures (2x10 cells/ml).
  • Bl cell line taken from a high-density culture exhibit reduced expression of PKC- ⁇ .
  • Figs. 2A-2B show the separation of the migration inhibitory factor from the conditioned medium of B l.
  • A. Gel filtration separation was carried out using a superdex peptide column. Low ( ⁇ 3kDa) MW fractions of conditioned medium or appropriate control (consisting of Iscove's Modified Dulbecco's Medium or "IMDM") were loaded and fractionated on the column, and the migration inhibitory capability of the different fractions was tested in transwell assays using G2 cells. The arrow indicates the inhibitory fraction, which is absent in the IMDM.
  • B. Shows that fractions 33-34 inhibit SDF-I mediated migration of G2 cells by 40%.
  • Figs. 3A-3F show the effect of different amino acids, peptides and ⁇ - phenylethylamine on spontaneous and SDF-I mediated migration of cells.
  • A. Upper panel: the indicated concentrations of each of the amino acids Phe, GIu, Met, VaI, Pro, and Cys (upper panels) were added to the lower chamber of the transwell together with medium or with medium supplemented with SDF-I in order to check effect on spontaneous (left) or SDF-I dependent (right) G2 migration, respectively.
  • Phe and Cys were found to be potent inhibitors of both, SDF-I dependent and spontaneous G2 migration.
  • Figs. 4A-4C show the inhibitory effect of Phe-ol on cell adhesion, CXCR4 expression, and SDF-I -mediated calcium flux.
  • A. Shows assessment of adherence of untreated CB CD34+ cells or CD34+ cells pretreated with Phe-ol (at indicated concentration) to stromal cells MS-5 (known to express SDF-I). Adherence of CD34+ cells to MS-5 cells was found to be inhibited by Phe-ol in a dose response manner B.
  • FIGs. 5A-5B show inhibition of homing of transplanted human leukemic G2 cells to the bone marrow (BM) 5 spleen, liver and lung of NOD/SCID by preincubation of the cells with Phe-ol prior to transplantation.
  • NOD/SCID mice were transplanted with untreated or Phe-ol (5 or 10 mM) preincubated G2 cells (10 7 cells/mouse).
  • BM (A), spleen, liver and lungs (B) were harvested and assayed for the levels of human cell homing to such tissues.
  • Data represent the number of CD45 human cells per 10 6 acquired total cells.
  • Phe-ol inhibited homing of leukemic G2 cells to the bone marrow, spleen, liver and lung of NOD/SCID mice in a dose dependent manner.
  • Fig. 6 shows G2 spontaneous and SDF-I -induced (50 ng/ml) cell migration in the presence of L-cysteine, glutathione and cysteine derivates (all at 10 mM).
  • Culture medium alone or medium containing L-cysteine or its derivates were loaded into the bottom of transwell chambers.
  • G2 cells (10 1 VmI) were loaded into the upper chambers and allowed to migrate for 4 hrs at 37 0 C. Cells were collected from the bottom chambers and counted using a fluorescence-activated cell sorter (FACS Calibur).
  • Percentage of migrated cells is shown in: culture with medium alone (control) (1); supplemented with L-cysteine (2); L-cysteine ethyl ester hydrochloride (3); L-cysteine sulfmic acid (4); cysteinamine hydrochloride (5); or glutathione (6).
  • Fig. 7A-7B shows the effect of cysteine and phenylalanine-containing peptides on migration of primary T cells in vitro.
  • the effect of ImM (A) or 100 ⁇ M (B) cysteine and phenylalanine-containing peptides (1) P-C-R-F-F-E (SEQ ID NO: 9); (2) C-R-F-F-E (SEQ ID NO: 10); (3) C-R-F-F (SEQ ID NO: 11); (5) C-R-F (SEQ ID NO: 12); (6) P-C-R-F (SEQ ID NO: 13 ); (7) C-A-A-F (SEQ ID NO: 14); (8) C-R-R-F(SEQ ID NO: 15); and (13) the derivative of P-C-R-F-F (SEQ ID NO: 17), P-dCys(Trt)-R-F-F on SDF-I induced migration of human primary T-cells was explored in vitro.
  • the cells (I x 10 6 /mL) were incubated with the peptides for 2 hrs at 37 0 C, 95% humidity, then added unwashed into the upper chambers of Costar 24-well transwell plates with 5 ⁇ m pore filters (Corning Inc. Corning, NY) and allowed to migrate for 2hrs at 37 0 C, 95% humidity, 5% CO2 towards SDF-I (20 ng/mL).
  • Migrated cells were collected from the lower chambers and counted using a FACSCalibur flow cytometer (Becton Dickinson, San Jose, California). The cells were gated in forward and side scatters set at linear gain. Data are presented as a percentage of migrated cells that were pretreated with peptides compared to the percentage of migrated untreated cells taken as 100%.
  • the present invention relates to the use of the amino acid phenylalanine or a derivative thereof; the amino acid cysteine or a derivative thereof; a combination of amino acids comprising phenylalanine or a derivative thereof and cysteine or a derivative thereof; a peptide comprising up to 22 amino acid residues comprising at least one phenylalanine or a derivative thereof, at least one cysteine or a derivative thereof, or at least one phenylalanine or a derivative thereof and at least one cysteine or a derivative thereof, in the manufacture of a medicament for treating or preventing a disease, disorder or condition responsive to inhibition of CXCR4 activity and/or inhibition of cell motility.
  • agents such as the amino acids cysteine or a derivative thereof, phenylalanine or a derivative thereof, e.g. ⁇ - phenylethylamine which in nature is synthesized from the amino acid phenylalanine by enzymatic decarboxylation, solubility-improved derivatives of phenylalanine such as phenylalaninol, (Phe-ol), succinyl phenylalanine (Suc-Phe), cysteine ethyl ester, cysteine sulfinic acid, cysteinamine; and short peptides including cysteine, phenylalanine or the combination of both amino acids, can inhibit migration of cells and/or CXCR4 expression of cells or both, paves the way to new therapies for diseases, disorder or conditions responsive to inhibition of cell motility and/or CXCR4 activity or whose pathology can be ameliorated, alleviated or prevented by inhibition of cell motility and/or inhibition of CX
  • agents of the invention can be used to decrease the CXCR4 activity or response, without being bound by the mechanism, for example by decreasing expression level of CXCR4 in cells, and/or can be used to inhibit cell motility. Therefore these agents can be used for treating or preventing a disease, disorder, or condition whose pathology is caused by, or associated with the activity or signaling trough CXCR4 in cells and/or motility of cells.
  • a disease, disorder, or condition whose pathology is caused by or associated with the level of CXCR4 in cells and/or motility of cells is a disease, disorder, or condition responsive to inhibition of CXCR4 activity and/or to inhibition of cell motility and which can be prevented or ameliorated by inhibition of CXCR4 activity and/or motility.
  • inhibition of CXCR4 activity refers, for example, to inhibition of activity or inhibition of level of expression of CXCR4.
  • agents of the invention such as cysteine or derivatives thereof; phenylalanine or derivatives thereof such as ⁇ -phenylethylamine; and peptides including phenylalanine; inhibited the spontaneous, as well as the SDF-I dependent motility of Precursor-B ALL (Pre-BLL) cells, as measured in vitro by the transwell assay.
  • Pre-BLL is the most common childhood malignancy and the second most common adult acute leukemia.
  • the migration inhibitory effect of D and L phenylalanine was found to be similar and therefore the migration inhibitory effect is independent of the stereoisomeric form of the amino acid.
  • the findings according to the invention demonstrated that Phe-ol inhibited Pre-BLL cell migration in vivo (or homing) from the circulation to the bone marrow, spleen, lung, and liver.
  • the inhibitory effect of agents according to the invention was detected also in normal non-neoplastic cells.
  • motility of normal cells like hematopoietic cells CD34+ cells, was inhibited by Phe-ol and that motility of normal primary T was inhibited by short peptides including phenylalanine or by short peptides including phenylalanine and cysteine.
  • Findings according to the present invention show that agents of the invention can inhibit activities that are mediated by SDF-I and its receptor CXCR4.
  • the amino acid cysteine or phenylalanine; and derivatives of said amino acid such as phenylalaninol, (Phe- ol), succinyl phenylalanine (Suc-Phe), cysteine ethyl ester, cysteine sulfinic acid, cysteinamine; ⁇ -phenylethylamine; and short peptides which include amino acids cysteine and/or phenylalanine, are able to inhibit SDF-I dependent motility of cells.
  • Phe-ol is capable to mediate; inhibition of homing of Pre-BLL cells to bone marrow, a process known to be dependent on SDF-I activity; inhibition of SDF-I dependent Ca+ influx; and inhibition of adhesion of CD34+ cells to SDF-I producing cells.
  • the levels of expression of CXCR4 in cells treated with Phe-ol was tested according to the invention, and it was found that Phe-ol inhibited CXCR4 expression in neoplastic cells such as Pre BLL cells and in normal cells such as primary T cells and CD34+ cells.
  • the present invention provides the use of the amino acid phenylalanine or a derivative thereof like a solubility-improved derivative, for example, phenylalaninol (Phe-ol) or succinyl phenylalanine (Suc-Phe) or derivatives synthesized from the amino acid phenylalanine like phenylethylamide; the amino acid cysteine or derivatives thereof like cysteine ethyl ester, cysteine sulfmic acid, and cysteinamine; or a mixture of amino acids comprising phenylalanine or a derivative, and cysteine or a derivative thereof; in the manufacture of a medicament for treating or preventing a disease, disorder or condition responsive to inhibition of CXCR4 activity and/or inhibition of cell motility.
  • a solubility-improved derivative for example, phenylalaninol (Phe-ol) or succinyl phenylalanine (Suc-Phe) or derivatives synthe
  • amino acid refers either to a single amino acid or to a derivative thereof, or to a mixture of two ore more amino acids, unless otherwise specified, and includes all the natural occurring amino acids found in proteins and non-natural amino acids.
  • non-natural amino acids include, but are not limited to, 4-hydroxyproline, 5-hydroxylisine, and N-methyl amino acids such as N-methyllysine, ⁇ -carboxyglutamate, desmosine, selenocysteine, citrulline and ornithine.
  • Derivatives of amino acids may be an amino acid residue containing additional chemical moieties not normally part of the amino acid and is encompassed by the invention as long as it retains at least a portion of the function of the amino acid which permits its utility as inhibitor of CXCR4 expression or activity and/or as an inhibitor of spontaneous and/or SDF-I mediated cell migration.
  • ⁇ -phenylethylamine is a derivative of phenylalanine. In nature, it is synthesized from the amino acid phenylalanine by enzymatic decarboxylation.
  • An amino acids may exist in one or more stereoisomeric form, because of the presence of asymmetric atoms or rotational restrictions can exist as a number of stereoisomers with R or S stereochemistry at each chiral centre or as atropisomeres with R or S stereochemistry at each chiral axis.
  • the invention includes use of all such enantiomers and diastereoisomers and mixtures thereof.
  • Phenylalanine and/or cysteine amino acids and/or a derivative thereof like phenylalaninol, succinyl phenylalanine, ⁇ -phenylethylamine, cysteine ethyl ester, cysteine sulfide acid, and cysteinamine can be administered or used according to the invention together with additional amino acids such as, methionine, proline, valine, glutamic acid and/or a combination thereof.
  • the amino acid comprises the combination or mixture of phenylalanine, phenylalaninol, succinyl phenylalanine or phenylethylamine and cysteine, cysteine ethyl ester, cysteine sulfinic acid, or cysteinamine.
  • F-K, K-F, F-R, R-F and F-E were able to inhibit by about 50% of cell migration and F-L-K and F-K- ⁇ -aminocaproic acid were able to inhibit by about 30% of cell migration.
  • the invention provides the use of a short peptide of about 22 amino acid residues comprising at least one phenylalanine or a derivative thereof, or comprising a short peptide of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, and 21 amino acid residues comprising at least one phenylalanine or a derivative thereof.
  • the short peptide may comprise 2, 3, 4, 5 or 6 consecutive amino acid residues which include the at least one phenylalanine and at least one non- penylalanine residue that is a charged amino acid.
  • the charged amino acid can be a positively charged amino acid such as lysine or arginine, or a negatively charged amino acid such as glutamic acid.
  • Non limiting examples of such peptides are short peptides of up to 22 amino acid residues comprising one of the following amino acid sequences F-K, K-F, F-R, R-F, K-F-K-F, F-L-K, F-K- ⁇ -aminocaproic acid F-E, and E-F or a salt thereof. Additional amino acids can be included on either or both of the N- or C- termini of these sequences, of course, these additional amino acid residues should not significantly interfere with the functional activity of the peptides.
  • the invention also relates to the use of a short peptide of up to 22 amino acid comprising at least one cysteine or a derivative thereof, for example to a peptide of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 ,19, 20 or 21 amino acid residues comprising at least one cysteine, for example to a peptide of 2, 3, 4, 5, or 6 consecutive amino acids including the at least one cysteine and at least one non- cysteine residue selected from proline, arginine and glutamic acid.
  • such peptide is glutathione.
  • short peptides containing phenylalanine and cysteine residues such as, for example, P-C-R-F-F-E (SEQ ID NO: 9); C-R-F-F-E (SEQ ID NO: 10); C-R-F-F (SEQ ID NO: 11); C-R-F (SEQ ID NO: 12); P-C-R-F (SEQ ID NO: 13); C-A-A-F (SEQ ID NO: 14); C-R-R-F (SEQ ID NO: 15) and a derivative of P-C-R-F-F (SEQ ID NO: 16), P-dCys(Trt)-R-F-F were very potent inhibitors of SDF-I -mediated T cell migration, as measured by the in vitro transwell assay.
  • Peptide of SEQ ID NO: 9, P-C-R-F- F-E is a hexapeptide from the N-terminus sequence of SDF-I containing one proline residue at position 1 a cysteine residue at position 2 and two phenylalanine at positions 4 and 5, the cysteine and phenylalanine are separated by an arginine and the last residue is glutamic acid.
  • Peptide of SEQ ID NO: 10, C-R-F-F-E is similar to peptide of SEQ ID NO: 9, except that the proline is missing resulting in a pentapetide in which cysteine is at position 1.
  • Peptide of SEQ ID NO: 11, C-R-F-F is similar to peptide of SEQ ID NO: 10, except that the glutamic acid is missing resulting in a tetrapeptide in which cysteine is also at position 1, the last residue is phenylalanine and has in total two phenylalanine residues.
  • Peptide of SEQ ID NO: 12, C-R-F is similar to peptide of SEQ ID NO: 11, except that the last amino acid phenylalanine is missing resulting in a tetrapeptide in which cysteine is at position 1 and has only one phenylalanine residue at position 3 and in which the cysteine and phenylalanine are separated by one arginine.
  • Peptide of SEQ ID NO: 13, P-C-R-F is similar to peptide of SEQ ID NO: 9, except that one phenylalanine residue and one glutamic acid residue at positions 5 and 6 respectively are missing, resulting in a tetrapetide which has a proline at position 1 and the cysteine at position 2 and has only one phenylalanine.
  • Peptide of SEQ ID NO: 14, C-A-A-F is a tertrapeptide like peptide SEQ ID NO: 11 in which the arginine at position 2 and the phenylalanine at position 3 have been substituted by alanines, resulting in a peptide having a cysteine at position 1 and only one phenylalanine at position 4.
  • Peptide of SEQ ID NO: 15, C-R-R-F is a tertrapeptide like peptide of SEQ ID NO: 11 in which the phenylalanine at position 3 has been substituted by arginine resulting in a peptide having a cysteine at position 1 and only one phenylalanine at position 4.
  • Peptide P-dCys (Trt)-R-F-F is a derivative of peptide P-C-R-F-F (SEQ ID NO: 16) in which the cysteine residue which was substituted to the cysteine derivative dCys (TrT).
  • Trt is trity, a protecting group for the SH functional moiety of cysteine.
  • the Fmoc-CYS (Trt) was used as a building block peptide chain assembly. It was found according to the present invention that these peptides used at concentration that were non toxic to the cells, were very potent inhibitors (in range 50-90% inhibition) of T cell migration mediated by SDF-I . These peptides were able to inhibit the spontaneous migration as well, but to a lesser extent.
  • Peptides from the amino terminus of SDF-I of 10 amino acid or larger including the LSY motif were disclosed by Heveker et al. (1998, 2001), Loetscher et al. (1998) and Luo et al. (1999). Of note, all of the active disclosed peptides included the LSY motif. As disclosed by Heveker et al. (2001) the peptides displayed chemotactic effects on polymorphonuclear leucocytes (PMN) but none of them inhibited chemotaxis induced by SDF-I in these cells. The same author has indicated in an earlier publication that the LSY motif (SEQ ID NO: 19) was essential for HIV inhibition (Heveker 1998).
  • the peptides containing cysteine and phenylalanine which according to the invention are from the amino terminus of SDF-I, lack the LSY motif and are capable of inhibiting chemotaxis induced by SDF-I . Since the replication of the HIV, as the chemotactic activity of SDF-I, involves CXCR4 it is likely that these peptides of the invention will also be capable to inhibit HIV virus.
  • the present invention relates to the use of a peptide of up to 22 amino acid residues comprising at least one phenylalanine or a derivative thereof and at least one cysteine or a derivative thereof , except for a peptide from the amino terminus of SDF-I which includes the LSY motif, in the manufacture of a medicament for treating or preventing a disease, disorder or condition responsive to inhibition of CXCR4 activity and/or inhibition of cell motility.
  • the peptide can consist of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 ,19, 20, and 21 amino acid residues comprising at least one phenylalanine or a derivative thereof and at least one cysteine or a derivative thereof, such as a peptide of up to 22 amino acid residues comprising 2, 3, 4, 5, 6 or 7 consecutive amino acid residues including the at least one cysteine residue or a derivative thereof and at the least one phenylalanine residue or a derivative thereof.
  • the peptide consists of 3, 4, 5, 6, or 7 consecutive amino acid residues including one cysteine residue or a derivative thereof and one phenylalanine residue or a derivative thereof separated by one or more arginines such as in the peptides P- C-R-F-F-E (SEQ ID NO: 9), C-R-F-F-E (SEQ ID NO: 10), C-R-F-F (SEQ ID NO: 11), C-R-F (SEQ ID NO: 12), P-C-R-F (SEQ ID NO: 13), C-R-R-F (SEQ ID NO: 15), P-dCys(Trt)-R-F-F (SEQ ID NO: 16).
  • the peptide comprises 3, 4, 5, 6 or 7 consecutive amino acids including one cysteine residue or a derivative thereof and one phenylalanine residue or a derivative thereof separated by one or more alanines like in peptide C-A-A-F.
  • the peptide comprises 4 consecutive amino acid residues wherein the first residue of the consecutive amino acid residues is cysteine or a derivative thereof and the fourth residue is phenylalanine or a derivative thereof like in the peptides C-A-A-F (SEQ ID NO: 14), C-R-R-F (SEQ ID NO: 15) and C-R-F-F (SEQ ID NO: 11).
  • the peptide comprises 4 or 5 consecutive amino acid residues including the at least one cysteine residue or a derivative thereof and at the least one phenylalanine residue or a derivative thereof wherein the first residue of the consecutive amino acid residues is a proline like for example in P-C-R-F (SEQ ID NO: 13) and P-dCys (Trt)-R-F-F (SEQ ID NO: 16), respectively.
  • the peptide comprises 5, 6 or 7 consecutive amino acid residues including the at least one cysteine residue or a derivative thereof and at the least one phenylalanine residue or a derivative thereof wherein the last residue of the consecutive amino cid residues is a glutamic acid such as in C-R-F-F-E (SEQ ID NO: 10), P-C-R-F-F-E (SEQ ID NO: 9) and C-P-C- R-F-F-E (SEQ ID NO: 17), respectively.
  • Exemplary peptides that can be used according to the invention include, but are not limited to, F-K, K-F, F-R, R-F, K-F-K-F, F-E, E-F, F-L-K, F-L- ⁇ - aminocaproic acid and glutathione, and peptides including amino acids of both types phenylalanine and cysteine such as C-P-C-R-F-F-E, P-C-R-F-F-E; C-R-F-F- E; C-R-F-F; C-R-F; P-C-R-F; C-A-A-F; C-R-R-F and P-dCys(Trt)-R-F-F, their salts, functional derivatives, as well as its active mutants, i.e.
  • peptides comprising phenylalanine, and cysteine wherein one or more amino acids of the structure are eliminated or substituted by other amino acids or one or more amino acids are added to that sequence in order to obtain peptides of up to 22 amino acid residues having the same activity such as inhibiting motility and/or CXCR4 activity of cells.
  • the peptides may comprise half-life extending moieties such as a protein or a high molecular weight polymer resulting in "fusion peptides" with extended half- life in body fluids.
  • peptides according to the invention can be fused to a protein such as, for example, an immunoglobulin or to a high molecular weight polymer, such as polyethylene glycol (PEG), or the like.
  • the invention provides peptides, for example it provides a peptide of 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or 21 amino acid residues comprising the amino acid sequence C-X-X-F (SEQ ID NO: 18) wherein X is an arginine or an alanine.
  • the peptide comprises the amino acid sequence of SEQ ID NO: 14 or SEQ ID NO: 15.
  • the invention provides a peptide of 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,
  • amino acid residues comprising 3, 4, 5, 6, 7, or 8 consecutive amino acid residues comprising one cysteine or a derivative thereof one phenylalanine residue or a derivative thereof separated by at least one alanine.
  • the peptide comprises one cysteine or derivative thereof and one phenylalanine or derivative thereof separated by one or two alanine residues.
  • the invention relates also to a peptide of 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22 amino acid residues comprises 4 consecutive amino acid residues wherein the first and the last amino acid residue of the consecutive amino acid residues is cysteine or a derivative thereof and phenylalanine or a derivative thereof, respectively.
  • the invention provides also a peptide of up to 22 amino acid residues comprising 3, 4, 5, 6, or 7 consecutive amino acid residues comprising one cysteine or a derivative thereof one phenylalanine residue or a derivative thereof separated by one arginine, except for a peptide from the amino terminus of SDF-I including the LSY motif and except for a peptide consisting of 6, 7, 8, 9, 10 or 11 amino acid residues which excludes the LSY motif having a serine as the last residue.
  • the peptide comprises 3 consecutive amino acid of SEQ ID NO: 12.
  • the peptide of up to 22 amino acid residues comprises 4 consecutive amino acid residues wherein the first and the last amino acid residue of the consecutive amino acid residues is cysteine or a derivative thereof and phenylalanine or a derivative thereof, respectively like in SEQ ID NO: 1 1.
  • the peptide of up to 22 amino acid residues comprises 4 consecutive amino acid residues wherein the first amino acid residue of the consecutive amino acid residues is a proline like in SEQ ID NO: 13.
  • the peptide of up to 22 amino acid residues comprises 5 consecutive amino acid residues wherein the first amino acid residue of the consecutive amino acid residues is a proline like in SEQ ID NO: 16, or a derivative of SEQ ID NO: 16 having instead of a Cysteine a dCys(Trt) residue.
  • the peptide of up to 22 amino acid residues comprises 5 consecutive amino acid residues wherein the fifth residue of the consecutive amino acid residues is a glutamic acid like in SEQ ID NO: 10.
  • the peptide of up to 22 amino acid residues comprises 6 consecutive amino acid residues wherein the sixth residue of the consecutive amino acid residue is glutamic acid and/or wherein the first residue of the consecutive amino acid residues is proline like in SEQ ID NO: 9.
  • the peptide of up to 22 amino acid residues comprises 7 consecutive amino acid residues wherein the seventh residue of the consecutive amino acid residue is glutamic acid and/or wherein the " first residue of the consecutive amino acid residues is a cysteine like in C-P-C-R-F-F-E (SEQ ID NO: 17).
  • the invention provides a peptide of up to 22 amino acid residues comprising 3, 4, 5, 6, 7, or 8 consecutive amino acid residues comprising one cysteine or a derivative thereof one phenylalanine residue or a derivative thereof separated by two arginines, like in SEQ ID NO: 15.
  • a peptide of 3, 4, 5, 6, 7, 8, or 9 amino acid residues comprising the amino acid sequence C-R-F (SEQ ID NO: 12), except for a peptide from the amino terminus of SDF-I including the LSY motif (SEQ ID NO: 19).
  • the invention also provides a peptide of about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 ,19, 20, 21 or 22 amino acids comprising: 2 consecutive amino acid consisting of F-K, K-F, F-R, R-F, F-E or E-F; 3 consecutive amino acid consisting of F-L-K or F-K- ⁇ -aminocaproic acid; or 4 consecutive amino acid consisting of K-F-K-F.
  • the invention provides a peptide of up to 12 amino acids comprising the amino acid sequence of F-K, K-F, F-R, R-F, K-F-K-F, F-L-K, F-K- ⁇ -aminocaproic acid, F-E, E-F, P-C-R-F-F-E, C-R-F-F-E, C-R-F-F, C-R-F, P-C-R- F, C-A-A-F, C-R-R-F, or P-dCys(Trt)-R-F-F.
  • the peptide consists of F-K, K-F, F-R, R-F, K-F-K-F, F-L-K, F-K- ⁇ -aminocaproic acid, F-E, E-F, P-C-R-F-F-E, C-R-F-F- E, C-R-F-F, C-R-F, P-C-R-F, C-A-A-F, C-R-R-F, P-C-R-F-F or P-dCys(Trt)-R-F- F.
  • Additional amino acids can be included on either or both of the N- or C- termini these sequences, of course, these additional amino acid residues should not significantly interfere with the functional activity of the peptides.
  • the invention provides a peptide consisting of amino acid sequence of SEQ ID NO: I 5 SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 8, SEQ ID NO: 1 linked to aminocaproic acid, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11 5 SEQ ID NO: 12, P SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, a derivative of SEQ ID NO: 16 comprising instead of the cysteine a dCys(Trt) residue or SEQ ID NO: 17 (C-P-C-R-F-F-E.), a derivative thereof, or a combination thereof.
  • K-F-K-F is a dual peptide comprising two K-F peptides which also has inhibitory activity therefore in another embodiment; the present invention provides a multiple peptide comprising a number of the same or different peptides of the present invention.
  • the peptides of the invention can be "protected" at the terminal amino group for example by coupling with any of various amino-terminal protecting groups traditionally employed in peptide synthesis.
  • suitable groups include acyl protecting groups, like formyl, acetyl, and benzoyl; aromatic urethane protecting groups, for example, benzyloxycarbonyl; and aliphatic urethane protecting groups, for example, tert-butoxycarbonyl.
  • the peptides of the invention can be "protected" at the terminal carboxyl group, for example by coupling with any of various carboxy-terminal protecting groups.
  • suitable protecting groups include tert-butyl or benzyl, which are linked to the terminal caroxyl group through an ester or ether bond.
  • the term peptide relates to chains of amino acids and/or amino acid derivatives.
  • a derivative of a peptide according to the invention may contain additional chemical moieties as a part of the peptide. Examples of such chemical moieties are amides of carboxyl groups at the C-terminal end of the peptides and amides of free carboxyl groups of aspartic or glutamic acid residues.
  • any such derivatives which are embraced according to the invention must have substantially similar activity to the peptide of the invention.
  • the peptides may comprise D and/or L amino acids.
  • the advantage of using peptides comprising one or more than one D amino acid residues is that these peptides may be more resistant to proteolytic degradation and therefore may have increased half life in biological fluids such as plasma.
  • the peptide is a short peptide and consists of up to 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22 amino acid residues, up to 10 amino acid residues, or of 2, 3, 4, 5, 6, 7, 8, or 9 amino acid residues.
  • the invention embraces also peptides of the invention that are circularly permuted peptides.
  • the term "circularly permuted” as used herein refers to a linear peptide in which the termini have been joined together, either directly or through a linker, to produce a circular peptide, and then the circular peptide is opened at another location to produce a new linear peptide with termini different from the termini in the original peptide.
  • Circular permutations include those peptides whose structure is equivalent to a peptide that has been circularized and then opened.
  • a circularly permuted peptide may be synthesized de novo as a linear peptide and never go through a circularization and opening step.
  • Circularly permuted peptides are single-chain peptides, which have their normal termini fused, often with a linker, and contain new termini at another position. See Goldenberg, et al. J. MoI. Biol., 165: 407-413 (1983) and Pan et al. Gene 125: 111-114 (1993), both incorporated by reference herein.
  • Circular permutation is functionally equivalent to taking a straight-chain peptide, fusing the ends to form a circular peptide, and then cutting the circular peptide at a different location to form a new straight chain peptide with different termini. Circular permutation thus has the effect of essentially preserving the sequence and identity of the amino acids of a peptide while generating new termini at different locations.
  • the peptides according to the invention can be linear or cyclic peptides. Additional peptides according to the invention comprising different amino acid combinations which include phenylalanine and/or cysteine and exhibit motility and/or CXCR4 inhibitory potency can be produced, for example, by chemical synthesis or by recombinant technology. These methods are known in the art.
  • chemical synthesis is the solid method using a multiple peptide synthesizer (AMS 422. Abimed Analyzer Tech).
  • an Fmoc (N-9 Fluorentylmethoxycarbonyl) strategy was employed following commercial protocols of the company. This approach of peptide synthesis is widely used (Fridkin et al., 2006).
  • peptides that show motility and/or CXCR4 inhibitory potency can be selected by using the transwell in vitro assay as described in the Example section and/or as described below.
  • Expression of a peptide or fusion peptides of the invention in a mammalian cell may be approached by inserting the DNA coding for the peptide or for the fusion peptide into a vector comprising a promoter, optionally an intron sequence and splicing donor/acceptor signals, and further optionally comprising a termination sequence.
  • the above promoter, intron, and termination sequences are operable in mammalian cells.
  • the promoter is preferably a strong promoter such as the above- noted RSV, CMV, or MPSV promoter.
  • the promoter may also be the SV40 early promoter (Everett, et al.
  • a cellular promoter such as the beta-actin promoter or the ELF-I promoter (Tokushige, et al., 1997).
  • a hybrid promoter may be used, such as the hybrid between the lac operator and the human ELF-I alpha promoter as described by Edamatsu et al. 1997, the CMV-beta actin hybrid promoter described by Akagi et al (1997), or the hybrid between the operator sequences and the CMV promoter (Furth et al., 1994, and references therein).
  • Intron sequences which may be inserted as complete sequences, i.e., including the splice donor and acceptor sites, may be inserted into the coding sequence of the polypeptide, which it is desired to express. Insertion if such intron sequences may enhance RNA stability and thus enhance production of the desired polypeptide. While in principle, suitable intron sequences may be selected from any gene containing introns, exemplary intron sequences are the beta-actin intron, the SV 40 intron, and the p55 TNF receptor intron.
  • the intron sequence may contain enhancer elements, which may enhance transcription from the above-noted promoters.
  • intron sequences also contain transcriptional or translational control sequences that confer tissue specific expression. Therefore, when it is desired to express a peptide or fusion peptides of the invention in a tissue-specific manner, such intron sequences may be advantageously employed.
  • An example of an intron containing tissue-specific enhancer elements is the erythroid-specific enhancer located in intron 8 of the human 5-aminolevulinate synthase 2 gene (Surinya et al. 1998), and a discussion of the principle of enhancing protein production using intron sequences, together with example intron sequences, is provided in Huang et al. 1990.
  • Transcriptional termination sequences and polyadenylation signals may be added at the 3 ' end of the DNA coding for the polypeptide that it is desired to express. Such sequences may be found in many or even most genes.
  • the SV 40 polyadenylation signal can be used (Schek et al., 1992, and references therein).
  • Vectors for expression of peptides of invention or fusion peptides in a mammalian cell could be used for example the pcDNA3.1 vector (Invitrogen), which contains the CMV promoter for driving expression of the gene encoding the desired polypeptide and pMPSVEH vectors with the MPSV promoters.
  • pcDNA3.1 vector Invitrogen
  • pMPSVEH vectors with the MPSV promoters.
  • Recombinant polypeptides can be produced either in bacterial or eukaryotic (e.g. CHO) cultured host cells transfected with vectors encoding such polypeptides or in transgenic animals. When using transgenic animals it is particularly advantageous to produce heterologous polypeptides in their milk. Dairy animals such as cattle, sheep and goats are thus exemplary hosts. See, for example, patent specifications WO 88/00239, WO 90/05188, WO 91/02318, and WO 92/11757; and U.S. Pat. Nos. 4,873,191 ; 4,873,316; and 5,304,489, which are incorporated herein by reference in their entirety.
  • the present invention also provides expression vectors comprising the DNA sequence encoding a peptide or fusion peptide of the invention and methods for their production by introducing said vector in prokaryotic or eukaryotic host cells, such as insect cells, yeast cells, or mammalian cell such as HeLa, 293 T HEK and CHO cells, growing the cells and isolating the protein produced.
  • prokaryotic or eukaryotic host cells such as insect cells, yeast cells, or mammalian cell such as HeLa, 293 T HEK and CHO cells, growing the cells and isolating the protein produced.
  • the invention provides a viral vector encoding a peptide, or its fusion peptide.
  • additional peptides according to the invention can be isolated, for example by; chemical synthesis or recombinant expression of cysteine and/or phenylalanine-containing peptides of up to 22 amino acid residues, evaluation of their motility inhibitory effect and/or on SDF-1/CXCR4 inhibitory activity, and selection of peptides exhibiting similar motility inhibitory effect and/or on SDF- 1/CXCR4 inhibitory activity like peptides of SEQ ID NO: 1 to SEQ ID NO: 16.
  • cysteine and/or phenylalanine-containing peptides or fusion peptides can be synthesized and can be tested at various concentrations on SDF-I and/or spontaneous induced migration in vitro or in vivo employing human primary T-cells, CD34+ stem cells or Pre-BLL cells.
  • cells e.g. 1 x 10 6 /mL
  • peptides for example for 2 hrs at 37 0 C, 95% humidity, then added unwashed into the upper chambers of Costar 24-well transwell plates with 5 ⁇ m pore filters (Corning Inc.
  • the peptide may be added to the lower chamber of the transwell assay.
  • Migrated cells can be collected from the lower chambers and counted using a FACSCalibur flow cytometer (Becton Dickinson, San Jose, California). The cells can be gated in forward and side scatters set at linear gain. Data can be presented as a percentage of migrated cells that were pretreated with peptides compared to the percentage of migrated untreated cells taken as 100%.
  • the concentration of the peptide in the lower well and/or in contact with the tested cells can be in the range of 5 nM to 100 nM or 0.1 mM to 10 mM or up to 50 mM and 100 mM; up to 5 or 10 mM; and about 0.1 mM, 1 mM, 5 mM or 10 mM.
  • the invention pertains to a peptide according to the invention as defined above, or to a salt thereof and/or derivative thereof and/or a fusion peptide thereof.
  • salts herein refers to both salts of carboxyl groups and to acid addition salts of amino groups of the peptides of the invention.
  • Salts of a carboxyl group may be formed by means known in the art and include inorganic salts, for example, sodium, calcium, ammonium, ferric or zinc salts, and the like, and salts with organic bases as those formed, for example, with amines, such as triethanolamine, arginine or lysine, piperidine, procaine and the like.
  • Acid addition salts include, for example, salts with mineral acids such as, for example, hydrochloric acid or sulfuric acid, and salts with organic acids such as, for example, acetic acid or oxalic acid. Of course, any such salts must have substantially similar activity to the peptide of the invention.
  • the present invention relates also to the DNA sequence encoding peptides or fusion peptides according to the invention. Moreover, the present invention further concerns the DNA sequences encoding a biologically active peptide, fragment or fusion peptides of a peptide according to the invention.
  • the invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising an agent according to the invention such as phenylalanine or a derivative thereof, cysteine or a derivative thereof, a mixture or combination of phenylalanine or derivative thereof and cysteine or derivative thereof, peptides of up to 22 amino acid residues comprising phenylalanine or derivative thereof and/or cysteine or derivative thereof, a DNA encoding said peptides or a vector harboring said DNA and a pharmaceutically acceptable carrier.
  • an agent according to the invention such as phenylalanine or a derivative thereof, cysteine or a derivative thereof, a mixture or combination of phenylalanine or derivative thereof and cysteine or derivative thereof, peptides of up to 22 amino acid residues comprising phenylalanine or derivative thereof and/or cysteine or derivative thereof, a DNA encoding said peptides or a vector harboring said DNA and a pharmaceutically acceptable carrier.
  • a pharmaceutical or veterinary composition for treating a disease or condition caused by, or associated with CXCR4, or a ' disease or condition responsive to inhibition of CXCR4, comprising an agent according to the invention together with a pharmaceutically or veterinary acceptable excipient or carrier.
  • the present invention provides the use of agents according to the invention in subjects in need such as human patients or in veterinary medicine in the management (namely, treatment or prophylaxis) of a disease, disorder or condition responsive to inhibition of CXCR4 activity or inhibition of motility or disease or condition in which pathogenesis of the disease, disorder or condition is caused by, or associated with, the level or activity of the chemokine receptor CXCR4 and/or with the level of cell motility.
  • the agent according to the invention can be administered in vivo or ex-vivo.
  • a disease, disorder or condition responsive to inhibition of CXCR4, inhibition of cell motility, or inhibition of both CXCR4 or cell motility is a disease, disorder or condition that can be ameliorated or prevented by inhibition of CXCR4 expression or activity in cells involved or responsible for the pathogenesis or course of said disease, disorder or condition, inhibition of cell motility in cells involved or responsible for the pathogenesis or course of said disease, disorder or condition, or inhibition of both CXCR4 expression or activity and inhibition of motility in cells involved or responsible for the pathogenesis or course of said disease, disorder or condition.
  • disorders, diseases or conditions caused by, or associated with CXCR4 activity or responsive to inhibition of CXCR4 activity include, but are not limited to, primary tumor growth, invasion by secondary metastases; inflammatory diseases such as rheumatoid arthritis, septic arthritis, osteoarthritis, periodontitis, gingivitis, asthma, autoimmune disease, psoriasis, psoriatic arthritis, atopic dermatitis; interstitial nephritis, ocular inflammation, liver cirrhosis, an inflammatory condition of the nervous system or neuroinfiammatory disorders, e. g. multiple sclerosis, transplant rejection (e. g. graft versus host disease), inflammatory gastric conditions, e. g.
  • inflammatory diseases such as rheumatoid arthritis, septic arthritis, osteoarthritis, periodontitis, gingivitis, asthma, autoimmune disease, psoriasis, psoriatic arthritis, atopic dermatitis
  • CXCR4 Crohn's disease, inflammatory bowel disease, and ulcerative colitis, and acquired immunodeficiency syndrome (AIDS).
  • CXCR4 is known to be involved in neuronal cell migration and therefore the invention encompasses the use of an agent according to the invention in neurological diseases such as Parkinson's disease and schizophrenia.
  • CXCR4 seems to be commonly expressed on cancer cells and to play a role in migration, invasion, proliferation, survival and other malignant processes. Malignant cells from at least 23 different types of cancer express the chemokine receptor CXCR4 and respond to its ligand SDF-I .
  • CXCR4 receptor was suggested by Balkwill on 2004 to be involved in directed migration of cancer cells to sites of metastasis, increase survival of cancer cells and establishment of a tumor promoting cytokine/chemokine network. Therefore, CXCR4 antagonists are important in cancer therapy. While not wishing to be bound by theory, the inventors believe that an antagonist of the CXCR4 provides effective treatment of cancers that express CXCR4 by decreasing the rate of proliferation or by causing cessation of proliferation or by causing death of cancer cells.
  • the invention relates to the use of an agent according to the invention for treatment of a cancer that expresses CXCR4.
  • CXCR4/SDF-1 is important for blood vessel formation and endothelial cell migration. Therefore, CXCR4 inhibition appears to be advantageous for treatment of a disease in which preventing blood vessel formation is desirable, such as solid cancer.
  • cancers that express CXCR4 include, but are not limited to, B- CLL, AML, B-lineage ALL, intraocular lymphoma Non-Hodgkin lymphoma, follicular center lymphoma, CML, Multiple myeloma, pancreatic cancer, prostate (localized and metastatic cancer), kidney, breast, ovary, thyroid, kidney, colorectal cancer, oral squamous carcinoma, cervical cancer, neuroblastoma, glioma, astrocytoma, rhabdomyosarcoma, small cell lung cancer and melanoma.
  • cancer cells that over express CXCR4 include, but are not limited to, B-CLL, B- lineage ALL, follicular center lymphoma, and astrocytoma (Balkwill, 2004).
  • Precursor-B ALL is the most common childhood malignancy and the second most common adult acute leukemia.
  • Leukemic cells have the ability to infiltrate the liver, spleen, lymph nodes, and central nervous system.
  • High expression of CXCR4 by leukemic cells is strongly predictive for organ invasiveness, including infiltration to the central nervous system in patients with childhood ALL.
  • an agent according to the invention prevented homing of B-cell acute lymphoblastic leukemic cells to the bone marrow, spleen, liver and lung.
  • said agent may be aimed at preventing organ invasiveness of leukemic cells.
  • the invention provides the use of an agent according to the invention for treating or preventing leukemia such as B cell acute lymphoblastic leukemia.
  • leukemia such as B cell acute lymphoblastic leukemia.
  • CXCR4/SDF-1 interaction has been linked to metastasis in some cancer types including prostate cancer, renal cancer, neuroblastoma, ovarian cancer, breast cancer, head and neck and melanoma to organs that are an abundant source of SDF-I, including lymph node, bone marrow, and skin.
  • the invention relates to the use of an agent according to the invention for treating or preventing metastasis.
  • CXCR4 and/or cell motility are known to play a role in inflammation.
  • SDF-I levels are increased in inflammatory liver disease, in synoviocytes of the hyperplastic lining layer of rheumatoid joint.
  • exogenous SDF-I injected in periathric tissues, elicited an inflammatory response (reviewed by Balkwill 2004).
  • Treating mice with CXCR4 antagonist AMD3100 before appearance of first symptoms was found to be beneficial.
  • SDF-I is involved in the destruction of cartilage in osteoarthritis and rheumatoid arthritis (Kanbe et al., 2004).
  • CXCR4 and SDF-I have been implicated inflammation induced inflammation/infection.
  • CXCR4 antagonists and/or inhibitor of cell migration are beneficial for preventing or treating inflammation.
  • the invention provides the use of an agent according to the invention for treating or preventing inflammation.
  • CXCR4 is known in the art to serve as co-receptor for the human immunodeficiency virus (HIV). CXCR4 interact with HIV and with the cellular CD4 receptor to facilitate viral entry into cells. Therapeutic approaches based on antagonist of these receptors have been developed, some of which are currently in clinical trials (Murakami and Yamamoto, 2000). Therefore, CXCR4 antagonists are beneficial in preventing or treating acquired immunodeficiency syndrome (AIDS).
  • HIV human immunodeficiency virus
  • the invention provides the use of an agent according to the invention for treating or preventing AIDS.
  • agents according to the invention may be used to prevent antigen-activated T cell to migrate from the blood to sites of inflammation.
  • agents of the invention, particularly peptides of the invention, that block T cell migration may be used as therapeutic agents for decreasing or inhibiting the pathology of T cell-infiltrative autoimmune diseases.
  • the invention provides a method for treating and/or preventing a disease, disorder or condition whose pathogenesis is caused by, or associated with, the activity of the chemokine receptor CXCR4, comprising administering to a subject in need thereof a therapeutically effective amount of an agent according to the invention or a salt thereof, optionally together with a pharmaceutically acceptable carrier.
  • the invention relates to a method of management (namely, treatment or prophylaxis) of a disease or condition responsive to inhibition of CXCR4 in a subject in need, in particular in a human patient, which method comprises administering to the subject an effective amount of an agent according to the invention defined above, or a pharmaceutical acceptable salt thereof.
  • phenylalanine, cysteine, a derivative of said amino acids and peptides comprising them can be administered as inhibitors of CXCR4 activity and/or cell motility, for example, for the treatment or prevention of cancer expressing CXCR4, acquired immunodeficiency syndrome (AIDS), inflammation and metastasis.
  • Single or multiple administrations of the composition may be administered depending on the dosage and frequency as required and tolerated by the subject.
  • the concentration of composition in these formulations will be so designed as to deliver in the body an amount of molecules sufficient, for obtaining a therapeutic effect.
  • composition will be designed such as to deliver an amount of compound or agent that is sufficient to affect the course and severity of disease or condition mentioned herein, leading to reduction or remission of the disease.
  • the effective amount will depend on the route of administration, the disease to be treated and the condition of the subject.
  • Whether a cancer patient would benefit from treatment with the agent according to the invention can be determined, if desired, by obtaining a tumor sample from the patient and determining whether CXCR4 is expressed in the sample (e.g., by measuring CXCR4 as exemplified below and/or by mRNA levels or any other method well known in the art) or by determining in said sample the ability of SDF-I to modulate proliferation, adhesion, motility, homing, calcium release and other activities signaled by SDF-I .
  • Preventive administration is especially useful in patients having high-risk to be ill or suffer from a disease or condition mentioned herein.
  • agent according to the invention may also be used in a combination therapy in conjunction with other therapeutic agents such as anti-viral agents (e.g., nucleoside or non-nucleoside HIV reverse transcriptase inhibitors, HIV protease inhibitors, and HIV integrase inhibitors), anti-neoplastic agents such as chemotherapy or with other anti-inflammatory agents.
  • anti-viral agents e.g., nucleoside or non-nucleoside HIV reverse transcriptase inhibitors, HIV protease inhibitors, and HIV integrase inhibitors
  • anti-neoplastic agents such as chemotherapy or with other anti-inflammatory agents.
  • pharmaceutically acceptable is meant to encompass any carrier, which does not interfere with effectiveness of the biological activity of the active ingredient and that is not toxic to the host to which it is administered.
  • pharmaceutically acceptable for parenteral administration, the substance according to the invention may be formulated in a unit dosage form for injection in vehicles such as saline, dextrose solution, serum albumin and Ringer's solution.
  • the specific dose level for any particular patient will depend upon a variety of factors including the activity of the specific compound employed, the age, body weight, general health, sex, diet, time of administration, route of administration, rate of excretion, drug combination and the severity of the particular disease undergoing therapy. Optimum dose levels and frequency of dosing will be determined by clinical trial.
  • the compounds with which the invention is concerned may be prepared for administration by any route consistent with their pharmacokinetic properties.
  • compositions may be in the form of tablets, capsules, powders, granules, lozenges, and liquid or gel preparations, such as oral, topical, or sterile parenteral solutions or suspensions.
  • Tablets and capsules for oral administration may be in unit dose presentation form, and may contain conventional excipients such as binding agents, for example syrup, acacia, gelatin, sorbitol, tragacanth, or polyvinyl-pyrrolidone; fillers for example lactose, sugar, maize- starch, calcium phosphate, sorbitol or glycine; tabletting lubricant, for example magnesium stearate, talc, polyethylene glycol or silica; disintegrants for example potato starch, or acceptable wetting agents such as sodium lauryl sulphate.
  • binding agents for example syrup, acacia, gelatin, sorbitol, tragacanth, or polyvinyl-pyrrolidone
  • fillers for example lactose
  • Oral liquid preparations may be in the form of, for example, aqueous or oily suspensions, solutions, emulsions, syrups or elixirs, or may be presented as a dry product for reconstitution with water or other suitable vehicle before use.
  • Such liquid preparations may contain conventional additives such as suspending agents, for example sorbitol, syrup, methyl cellulose, glucose syrup, gelatin hydrogenated edible fats; emulsifying agents, for example lecithin, sorbitan monooleate, or acacia;
  • Non-aqueous vehicles which may include edible oils), for example almond oil, fractionated coconut oil, oily esters such as glycerine, propylene glycol, or ethyl alcohol; preservatives, for example methyl or propyl p-hydroxybenzoate or sorbic acid, and if desired conventional flavouring or colouring agents.
  • the drug may be made up into a cream, lotion or ointment.
  • Cream or ointment formulations which may be used for the drug are conventional formulations well known in the art, for example as described in standard textbooks of pharmaceutics such as the British Pharmacopoeia.
  • the drug may be made up into a solution or suspension in a suitable sterile aqueous or non-aqueous vehicle.
  • Additives for instance buffers such as sodium metabisulphite or disodium edeate; preservatives including bactericidal and fungicidal agents such as phenyl mercuric acetate or nitrate,benzalkonium chloride or chlorhexidine, and thickening agents such ashypromellose may also be included.
  • the active ingredient may also be administered parenterally in a sterile medium.
  • the drug can either be suspended or dissolved in the vehicle.
  • adjuvants such as local anaesthetic, preservative and buffering agents can be dissolved in the vehicle.
  • MS-5 and CD34+ cells were cultured in RPMI 1640 medium supplemented with 10% heat inactivated FCS, antibiotics and glutamine.
  • Murine MS-5 stromal cell line (Millennium Pharmaceuticals, Cambridge, Massachusetts, USA) was previously described (Bleul et al., 1996).
  • pre-B ALL cell lines G2 and B 1 were previously described (Freedman et al., 1993). Bl and G2 cells were cultured in IMDM supplemented with 10% heat inactivated FCS, antibiotics and glutamine
  • Transwell assay (H) Transwell assay. Transwell assays were performed using Costar transwells (6.5 mm/diameter, 5 ⁇ m/pore) as previously described (Aiuti et al., 1997).
  • human G2 cells (10 7 /mouse, for testing homing) were injected into tail vein of 8-week old NOD-SCID mice. 16 hours after injection (for evaluating homing), mice were sacrificed. BM cells were flushed from femurs, tibias, and pelvic bones. Percentages of human cells were determined by flow cytometry as described below.
  • Bl cells a line prepared from B cells from a patient with acute lymphoblastic leukemia B cells [B-ALL]
  • B-ALL acute lymphoblastic leukemia B cells
  • leukemic Bl cells B 1-2 cells (a sub-clone derived from the Bl cells), leukemic G2 cells (another precursor-B cell line prepared from a different precursor-B-ALL patient) or normal cord blood (CB) CD34+ cells pre-treated with medium of B 1 cells from a high density cell culture and were assayed for their ability to migrate towards a gradient of SDF-I in the transwell assay.
  • the upper well of the transwell was loaded with 1-2 xlO 5 cells and the lower well with Bl conditioned medium from a high-density cell culture or with control IMDM.
  • the results summarized in Fig. 1 show inhibition of SDF-I dependent migration of leukemic B l cells, G2 cells and normal CB CD34+ cells by the conditioned medium of Bl (35% and 25% inhibition of G2 and CD34+ cells, respectively).
  • the results obtained show that a cell-migration inhibitory factor is found in the conditioned medium of Bl cells, which were grown to high cell density.
  • PKC- ⁇ is another kinase involved in SDF-I signaling.
  • PKC- ⁇ expression was found significantly downregulated in B l cells growing at the high concentration conditions (Fig. ID). Such down regulation of PKC- ⁇ expression was not recorded in another leukemic G2 cell grown at similar high cell concentration but lacking such a cell-migration inhibitory factor.
  • Example 2 Isolation of the cell-migration inhibitory factor present in the conditioned medium of Bl cells.
  • the conditioned medium of Bl containing the factor was filtered through amicon filters with various pore sizes, and the capability to inhibit cell migration was tested in both filtrate and retentate fractions. Inhibition of G2 migration was observed with filtrate fractions of molecular weight ⁇ 3 IcDa but not with fractions of molecular weight > 3kDa.
  • the amicon-filtered low MW (OkDa) fraction was separated by gel filtration on a 24 ml superdex peptide HR 10/30 column. 0.5 ml fractions were loaded on the column and eluted using triple distilled water. The migration inhibitory capability of the different fractions was measured in the transwell motility assay with G2 cells (0.5 ml of Iscove's Modified Dulbecco's Medium, IMDM was separated in the column as control).
  • the results summarized in Fig. 2A show that fractions 33-34, corresponding to MW of ⁇ 150 Dalton, inhibited migration of G2 cells by 40% (Fig. 2A, B).
  • the low molecular weight cell-migration inhibitory factor was found to be resistant to boiling and low pH conditions (6N HCL).
  • the characteristics of the cell-migration inhibitory factor indicated that it is phenylalanine.
  • Example 3 The amino acids phenylalanine and cysteine are potent cell- migration inhibitory factors.
  • phenylalanine derivatives which were more soluble than phenylalanine, was also tested (Fig. 3 A, lower panel).
  • the inhibitory effect of phenylalanine derivatives such as L-phenylalaninol (Phe-ol) and N-succinyl-L- phenylalanine (suc-Phe) (provided by prof. Mati Fridkin, Weizmann Institute) was evaluated on spontaneous or SDF-I dependent cell migration. Phe-ol and Suc-Phe were very potent inhibitors of spontaneous and SDF-I dependent migration. Either L- or D-phenylalanine added to the lower well of the transwell was found to inhibit migration of G2 cells (Fig 3B).
  • Peptides Phe-lys, Lys-Phe, Phe-Arg, Arg-Phe and Phe-Glu were able to inhibit by about 50% of cell migration and Phe-Leu-Lys and Phe-Lys- ⁇ - aminocaproic acid were able to inhibit by about 30% of cell migration.
  • Example 4 Phe-ol induces CXCR4 down regulation, reduced adhesion and altered SDF-I signaling in G2 cells.
  • phenylalanine and its derivatives have an inhibitory effect on SDF-I activity related to cell migration.
  • SDF-I regulates additional activities in cells.
  • the following experiments were carried out to assess whether the inhibitory effect of the phenylalanine derivatives involves other activities of SDF-I.
  • One such activity is regulation of stem cells adhesion (Peled et al., 1999; Peled et al., 2000).
  • Example 5 Homing of acute lymphoblastic leukemia B cells (Pre-B ALL) to bone marrow, spleen, liver and lung of transplanted NOD-SCID mice is inhibited by Phe-ol.
  • Pre-B ALL acute lymphoblastic leukemia B cells
  • Pre-B ALL is the most common childhood malignancy and the second most common adult acute leukemia.
  • the leukemic cells have the ability to infiltrate the liver, spleen, lymph nodes, and central nervous system.
  • high expression of CXCR4 in the leukemic cells is strongly predictive for organ invasiveness, including infiltration to the central nervous system.
  • the amino acid phenylalanine and its derivatives were capable of inhibiting SDF-I signaling. Since homing of leukemic cell to bone marrow involves SDF-1/CXCR4 interaction, the potential of the phenylalanine derivative Phe-ol to inhibit homing of G2 cells to the bone marrow, spleen, liver and lung of transplanted NOD-SCID mice was tested.
  • G2 cells (10 7 cells/mouse) preincubated for 1 hour with 5 or 10 mM Phe-ol or with medium (as the control) were injected into the tail vein of eight week old NOD/SCID mice, 16 hours after the injection mice were sacrificed, bone marrow (flushed from femurs, tibias, and pelvic bones), spleen, liver and lung were harvested and assayed for the presence of the human G2 cells (as determined by flow cytometry).
  • Example 6 Inhibition of normal and leukemic human cell migration and proliferation by the amino acid cysteine and its derivatives.
  • Chemokines are classified as CXC or CC members based on their composition of the amino acid Cysteine. Our preliminary results reveal that cysteine can inhibit migration of leukemic human progenitors (see Example 3), normal progenitors and of mature cells (not shown). The effect of L-cysteine and its derivates (all at 10 mM) and glutathione (6) on G2 spontaneous and SDF-I -induced (50 ng/ml) migration was tested by the transwell assay.
  • Example 7 inhibition of T cell migration by cysteine and phenylalanine containing peptides.
  • Small peptides were synthesized based on the N-terminus sequence of the SDF-I molecule, which has cysteine and phenylalanine residues (amino acids 9-15 of SDF-I), C-P-C-R-F-F-E (SEQ ID NO: 17).
  • Peptide 1 P-C-R-F-F-E (SEQ ID NO: 9) is a hexapeptide from the N- terminus sequence of SDF-I containing one cysteine residue at position 2 and two phenylalanines at positions 4 and 5.
  • Peptide 2 C-R-F-F-E (SEQ ID NO: 10) is similar to peptide 1, except for the first residue proline resulting in a pentapetide in which cysteine is at position 1.
  • Peptide 3 C-R-F-F (SEQ ID NO: 11) is similar to peptide 2, except that it lacks the last amino acid glutamic acid resulting in a tetrapeptide in which cysteine is also at position 1 and has two phenylalanine residues.
  • Peptide 4 C-R-F (SEQ ID NO: 12) is similar to peptide 3, except that it lacks the last amino acid phenylalanine resulting in a tetrapeptide in which cysteine is also at position 1 and has only one phenylalanine residue.
  • Peptide 5 C-R-F (SEQ ID NO: 12) is another batch of a peptide similar to peptide 4.
  • Peptide 6 P-C-R-F (SEQ ID NO: 13) is similar to peptide 1, except that it lacks the phenylalanine residue and glutamic acid residue at positions 5 and 6 respectively, resulting in a tetrapetide which has the cysteine at position 2 and has one phenylalanine.
  • Peptide 7 C-A-A-F (SEQ ID NO: 14) is a tertrapeptide like peptide 3 in which the arginine at position 2 and the phenylalanine at position 3 have been substituted by alanine, resulting in a peptide having a cysteine at position 1 and only one phenylalanine at position 4.
  • Peptide 8 C-R-R-F (SEQ ID NO: 15) is a tertrapeptide like peptide 3 in which the phenylalanine at position 3 have been substituted by arginine resulting in a peptide having a cysteine at position 1 and only one phenylalanine at position 4.
  • Peptide 12 P-C-R-F-F-E (SEQ ID NO: 9), another batch of peptide #1.
  • Peptide 13 P-dCys (Trt)-R-F-F (a derivative of P-C-R-F-F in SEQ ID NO: 16) is similar to peptide 1 , except for the cysteine residue which was substituted to dCys (TrT) and the glutamic acid residue is missing. Trt is trity a protecting group for the SH functional moiety of cysteine.
  • the Fmoc-CYS (Trt) was used as a building block peptide chain assembly.
  • Migrated cells were collected from the lower chambers and counted using a FACSCalibur flow cytometer (Becton Dickinson, San Jose, California). The cells were gated in forward and side scatters set at linear gain. Data are presented as a percentage of migrated cells that were pretreated with peptides compared to the percentage of migrated untreated cells taken as 100%.
  • Fig. 7A The peptides containing cysteine and phenylalanine at concentration of ImM (Fig. 7A) were found to be very potent inhibitors of T cell migration mediated by SDF-I (in range 50-90% inhibition). The same trend of activity was observed upon testing these peptides at concentration of 100 ⁇ M (Fig.7B). These peptides inhibited spontaneous migration of T cells as well, but the inhibition of spontaneous migration by these peptides was less potent than the inhibition of SDF-I mediated migration (not shown).
  • the chemokine SDF-I is a chemoattractant for human CD34+ hematopoietic progenitor cells and provides a new mechanism to explain the mobilization of CD34+ progenitors to the peripheral blood. J. Exp. Med. 185:111-120 (1997).
  • Fridkin G. Rahimipour S, Ben-Aroya N, Kapitkovsky A, Di-Segni S, Rosenberg M, Kustanovich I 5 Koch Y, Gilon C, Fridkin M. Novel cyclic azo- bridged analogs of gonadotropin-releasing hormone. J Pept Sci Feb; 12(2):106-15. (2006). Freedman, M.H. et al. Autocrine and paracrine growth control by granulocyte-monocyte colony-stimulating factor of acute lymphoblastic leukemia cells. Blood. 81 :3068-3075 (1993).
  • the chemokine SDF-I activates the integrins LFA-I, VLA-4, and VLA-5 on immature human CD34(+) cells: role in transendothelial/stromal migration and engraftment of NOD/SCID mice. Blood. 95(11):3289-96 (2000).

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EP06821674A 2005-12-29 2006-12-27 Hemmung von cxcr4 und/oder der zellmotilität durch phenylalanin, cystein oder diese aminosäuren enthaltende peptide Withdrawn EP1971356A2 (de)

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