US20010016568A1 - Novel metal complexes - Google Patents

Novel metal complexes Download PDF

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US20010016568A1
US20010016568A1 US09/183,974 US18397498A US2001016568A1 US 20010016568 A1 US20010016568 A1 US 20010016568A1 US 18397498 A US18397498 A US 18397498A US 2001016568 A1 US2001016568 A1 US 2001016568A1
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Juan I. Luengo
John G. Gleason
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SmithKline Beecham Corp
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D487/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00
    • C07D487/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00 in which the condensed system contains two hetero rings
    • C07D487/04—Ortho-condensed systems
    • 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
    • A61K31/28—Compounds containing heavy metals
    • A61K31/315—Zinc compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/555—Heterocyclic compounds containing heavy metals, e.g. hemin, hematin, melarsoprol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P21/00—Drugs for disorders of the muscular or neuromuscular system
    • A61P21/02—Muscle relaxants, e.g. for tetanus or cramps
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/04—Anorexiants; Antiobesity agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/10—Antimycotics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/04—Antihaemorrhagics; Procoagulants; Haemostatic agents; Antifibrinolytic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F3/00—Compounds containing elements of Groups 2 or 12 of the Periodic Table
    • C07F3/003—Compounds containing elements of Groups 2 or 12 of the Periodic Table without C-Metal linkages
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/566—Immunoassay; Biospecific binding assay; Materials therefor using specific carrier or receptor proteins as ligand binding reagents where possible specific carrier or receptor proteins are classified with their target compounds
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants

Definitions

  • This invention relates to metal complexed receptor ligands, methods for making and identifying them and their use as agonist of dimeric receptors. More specifically, the invention describes a method to promote the oligomerization of dimeric receptors.
  • Some receptors such as those for erythropoietin (EPO), thrombopoietin (TPO), and granulocyte-colony stimulating factor (G-CSF), contain the ligand-binding and signal-transduction domains within the same polypeptide subunit.
  • Others such as receptors for interleukin-2 (IL-2), IL-3 and IL-6 have separate components for ligand-binding and signal transduction.
  • IL-2 interleukin-2
  • IL-3 interleukin-6
  • This aggregation event can be in the form of homodimerization, in the case of receptors with a single subunit, or heterodimerization, in the case of receptors with different subunits. It has become clear that receptor aggregation is part of the biological signal by which the target cell responds to the presence of specific hormones and growth factors (Young, P. R. “Protein hormones and their receptors”, Curr. Opin. Biotech. 1992, 3, 408-421; Heldin, C. H., “Dimerization of cell surface receptors in signal transduction”). Typical examples of such receptors are growth factor receptors with tyrosine kinase activity as well as cytokine receptors.
  • Monoclonal antibodies have been discovered which have agonist activity to the dimeric receptors such as those from epidermal growth factor (EGF, Fernandez-Pol, J. J. Biol. Chem. 1985, 260, 5003-11; Serrero, G. U.S. Pat. No. 5,723,115), G-CSF (Takahashi, T. et al. J. Biol. Chem. 1996, 271, 17555-17560), tumor necrosis factor (TNF, Fine, S. M. et al. J. Biol. Chem. 1996, 27126, 15303-15306.), growth hormone receptor (Rowlinson, S. W. et al. J. Biol. Chem.
  • one aspect of the present invention is a method for agonizing dimeric cell-surface receptors which comprises contacting the receptor with a zinc chelated receptor ligand.
  • Another aspect of the invention is a method for identifying agonists of dimeric cell-surface receptors.
  • a third aspect of the invention relates to zinc chelated dimeric cell-surface receptor ligands.
  • a fourth aspect of the invention relates to an isolated receptor binding moiety of a zinc chelated dimeric cell-surface receptor ligand.
  • a fifth aspect of the invention is a method for making zinc chelated dimeric cell-surface receptor ligands.
  • FIG. 1 shows the activity of two different samples of compound 1a (from Example 1) on the murine myeloid cell line NFS60 that contained a G-CSF-responsive element linked to a minimal promoter and the gene for luciferase. Activity of compound 1a is below the threshold of 150% over background.
  • the study was performed as a Luciferase assay configured on the G-CSF-responsive NFS60 cell line as described in Tian et al., Science 281, 257-259 (1998).
  • the experiments shown in FIGS. 2 - 8 used the same NFS60 cell line.
  • FIG. 2 shows the same type of experiment in NFS60 cells, but run in the presence of 1 ⁇ M zinc (II).
  • the activity of compound 1a is about 350% over control, which indicates that zinc(II) potentiates the activity of compound 1a.
  • FIG. 3 is an analysis of the effect of ethylenediaminetetraacetic acid (as used herein—EDTA) on the activity of Compound 1a (from Example 1) in NFS60 cells. Shown are luciferase response curves of Compound 1a at the indicated concentrations and in the presence of various concentrations of EDTA. EDTA at 1.2 millimolar concentration antagonized the activity of compound 1a. The media in this assay contained a small amount (1-5 ⁇ M) of zinc(II).
  • EDTA ethylenediaminetetraacetic acid
  • FIG. 4 is an analysis of the effect of EDTA on the activity of recombinant G-CSF on NFS60 cells. Shown are luciferase response curves of recombinant G-CSF at the indicated concentrations and in the presents of various concentrations of EDTA. EDTA at both 1.2 and 5 millimolar has little effect on the activity of recombinant G-CSF in the assay.
  • FIGS. 5 and 6 depict an analysis of the activity of metal chlorides alone on the basal luciferase level of NFS60 cells. Shown are luciferase response curves of the indicated metal chlorides at various concentrations. None of the metals have a meaningful effect on the basal luciferase levels at concentrations equal or less than 10 micromolar.
  • FIGS. 7 and 8 show an analysis of the effect of metal chlorides on the EDTA depleted activity of Compound 1a on NFS60 cells. Shown are luciferase response curves as effected by the indicated metal chlorides. Only zinc (II) at concentrations 0.5-10 micromolar can overcome the inhibition in luciferase activity caused by 50 micromolar concentration of the metal chelator EDTA. None of the other metals tested could overcome the inhibitory effect of EDTA.
  • heteroatom(s) nitrogen, oxygen or sulfur, preferably nitrogen.
  • treating and derivatives thereof as used herein, is meant prophylatic or therapeutic therapy.
  • organic molecule and derivatives thereof as used herein, is meant the standard usage in the art to the ordinary organic chemist and as such excludes inorganic molecules and peptide molecules.
  • the zinc chelated receptor ligands of this invention that have agonist properties towards dimeric cell-surface receptors are compounds that consist of one or more receptor binding moieties, preferably 1 to 4 moieties, most preferably 1 or 2 moieties, wherein each receptor binding moiety forms at least two coordinate bonds to each of one or more zinc ions, preferably each moiety will form two or three coordinate bonds to each of one or two zinc ions.
  • receptor binding moiety means a small organic molecule having a molecular weight from about 100 to about 850, preferably having a molecular weight from about 200 to about 750, most preferably having a molecular weight from about 300 to about 650 and having from 1 to 4 zinc binding motifs, preferably having one or two zinc binding motifs.
  • metal chelation forms a symmetrical multimer, such as a dimer, of the receptor binding moiety.
  • zinc binding motif means a continuation of atoms within a receptor binding moiety that have the following characteristics:
  • each continuation consist of 3 to 10 atoms, preferably 4 to 8 atoms, most preferably 4 or 5 atoms,
  • each continuation further consisting of two or more heteroatoms, preferably from 2 to 4 heteroatoms, most preferably 2 to 3 heteroatoms, preferably at least one of the heteroatoms is nitrogen, wherein the heteroatoms are separated from each other by one to four additional atoms selected from the group consisting of carbon, nitrogen, sulfur and oxygen, preferably carbon or nitrogen, preferably by 2 to 4 additional atoms, most preferably by 2 or 3 additional atoms, and
  • the configuration of heteroatoms within the zinc binding motif allows for chelate coordination to a zinc (II) ion by providing for the formation of at least two coordinate bonds, preferably two or three coordinate bonds, simultaneously to a zinc ion.
  • Examples of zinc binding motifs for use in the present invention include but are not limited to the following: —N—C—C—N—, —N—C ⁇ C—N—, —N—C—C ⁇ N—, —N ⁇ C—C ⁇ N—, —O—C—C—N—, — O—C ⁇ C—N—, —O—C—C ⁇ N—, —O ⁇ C—C ⁇ N—, —S—C—C—N—, —S—C ⁇ C—N—, —S—C—C ⁇ N—, —S ⁇ C—C ⁇ N—, —S— C—C—S—, —N ⁇ C—N—N—, —N—C—N—N—, —O ⁇ C—N—N—, —S ⁇ C—N—N—, —O—C—N—N—, —O—N—C ⁇ O—, —O—N—C ⁇ O—, —N ⁇ C— N— N—C ⁇ N
  • Preferred receptor binding moieties of the present invention comprise one or more of the following functional groups, preferably one or two of the following functional groups: 2-guanidinobenzimidazoles, 2-guanidinobenzoxazoles, 2-guanidionbenzothiazole, 2-mercaptomethylpyridines, acylacetones, acylhydrazines, 2-aminoethanethiols, 2-(imidazol-4-yl)ethylamines, 2-(imidazol-2-yl)ethylamines, 2-(imidazol-4-yl)ethylimines, 2-(imidazol-2-yl)ethylimines, 2-picolylamine, 8-hydroxyquinolines, 8-aminoquinolines, 8-mercaptoquinolines, ethylenediamines, pyridine-2-carboxaldimines, 2,2′-bipyridyls, 2-thiobenzaldimines, 2-hydroxybenzaldimines and 2,5-
  • the above functional groups will generally form part of a larger molecule and may be further substituted in the formation of a receptor binding moiety.
  • Preferred substituents for optional use on the above functional groups consist of one or more groups selected from the following: alkyl, aryl, hydroxy, alkoxy, acyloxy, carbamoyl, amino, N-acylamino, ketone, halogen, cyano, thio, carboxy and carboxamido.
  • an 8 atom zinc binding motif (specifically the —N ⁇ C—N—C ⁇ N—C—C ⁇ N—) is essentially an overlap of a 5 atom zinc binding motif (that is —N ⁇ C—N—C ⁇ N—) and a 4 atom zinc binding motif (that is —N—C—C ⁇ N—) in a continuation.
  • preferred zinc binding motifs of the instant invention consist of a continuation of 4 or 5 atoms either individually or as part of a combination. Further, each atom of a zinc binding motif of the present invention may be further substituted, may be saturated or contain various degrees of unsaturation or may form part of a larger linear system or an aromatic or nonaromatic ring system.
  • the zinc chelated receptor ligands of this invention are included in the pharmaceutical compositions of the invention and used in the methods of the invention.
  • the receptor binding moieties of this invention are included in the pharmaceutical compositions of the invention and used in the methods of the invention.
  • co-administering and derivatives thereof as used herein is meant either simultaneous administration or any manner of separate sequential administration of a zinc chelated receptor ligand, as described herein, and a further active ingredient or ingredients.
  • a zinc chelated receptor ligand as described herein
  • a further active ingredient or ingredients for example, antibacterial agents or antifungal agents.
  • the agents are administered in a close time proximity to each other.
  • the agents are administered in the same dosage form, e.g. one agent may be administered subcutaneously and another agent may be administered orally.
  • the zinc chelated receptor ligands of this invention are prepared by reacting one or more receptor binding moieties and a zinc ion source, such as Zn(NO 3 ) 2 , in a solvent, followed by optional isolation of the zinc chelated receptor ligand.
  • a zinc ion source such as Zn(NO 3 ) 2
  • the order in which the indicated ingredients are utilized in the presently invented process is not critical. All orders of addition of the indicated ingredients are within the scope of the invention.
  • the zinc chelated receptor ligands of this invention can be prepared in vivo by the administration of a receptor binding moiety to a subject and utilization of naturally occurring zinc ions in the body of the subject.
  • the pharmaceutically active compounds of the present invention are active as agonist of dimeric cell-surface receptors they exhibit therapeutic utility in treating disease states associated with compromised function of such dimeric cell-surface receptors.
  • a zinc chelated G-CSF receptor agonist would exhibit efficacy in treating bacterial infections, fungal infections, neutropenia, including chemotherapy-induced neutropenia and bone marrow transplantation and in mobilizing peripheral blood stem cells and other conditions with depressed leukocyte production.
  • Compounds of the present invention are tested for potency as agonist of a dimeric cell-surface receptor in a Luciferase reporter gene assay such as described in Tian et al., Science 281, 257-259 (1998).
  • a Luciferase reporter gene assay such as described in Tian et al., Science 281, 257-259 (1998).
  • G-CSF NFS60 cells Holmes, et al., Proc. Natl. Acad. Sci. USA 82: 6687-6691 (1985)
  • STAT signal transducers and activators of transcription
  • EDTA is a strong metal chelator and had as its only effect, the removal of zinc from the ligand-receptor interaction.
  • Compounds of this invention are also tested for activity in the following assays: CFU-G assay (an example of which is described in King A G, Talmadge J., Badger A M, Pelus L M. Regulation of colony stimulating activity production from bone marrow stromal cells by the hematoregulatory peptide, HP-5. Exp. Hematol. 20:223-228, 1992) and in vivo evaluation of peripheral blood neutrophil and monocyte count in the mouse (an example of which is described in Pelus, L. M.; King, A. G.; Broxmeyer, H. E.; DeMarsh, P. L.; Petteway, S. R.; Bhatnagar, P.
  • Zinc-mediated affinity of compounds from this invention for dimeric cell-surface receptors was measured by isothermal titration microcalorimetry experiments. Titration microcalorimetry detects binding as heat originating from the intrinsic bond forming enthalpy change. In this assay, the compounds were titrated first against zinc(II) alone. In a separate experiment, the compounds were then assayed in the presence of zinc and a dimeric cell-surface receptor/Fc fusion protein (a G-CSF/Fc fusion protein), which contained the extracellular domain of the receptor presented in a dimeric form due to the Fc component.
  • a G-CSF/Fc fusion protein a dimeric cell-surface receptor/Fc fusion protein
  • Compounds 1a and 3a bind to the fusion protein construct with high, submicromolar affinity only in the presence of zinc.
  • Compounds 1, 1a, 2a, and 3a showed activation above 150% of control between the concentration range of 1 to 100 micromolar in the luciferase assay. Further, compound 1a and 3a showed activation above 150% of control between the concentration range of 1 to 100 micromolar in the murine assay.
  • Compounds 1a and 3a showed elevation of peripheral blood neutrophil and monocyte count in the mouse.
  • results depicted in FIG. 4 indicate that the agonist activity of the natural ligand (i.e. G-CSF or recombinant G-CSF as demonstrated herein) is not mediated by metal ions.
  • the natural ligand i.e. G-CSF or recombinant G-CSF as demonstrated herein
  • the pharmaceutically active compounds within the scope of this invention are useful as dimeric cell-surface receptor agonist in mammals, including humans, in need thereof.
  • the present invention therefor provides a method of treating disease states associated with compromised function of dimeric cell-surface receptors, which comprises administering a zinc chelated receptor ligand in a quantity effective to enhance receptor activation.
  • a zinc chelated G-CSF receptor agonist would exhibit efficacy in treating bacterial infections, fungal infections, neutropenia, including chemotherapy-induced neutropenia and bone marrow transplantation and in mobilizing peripheral blood stem cells and other conditions with depressed leukocyte production, through the administration of a zinc chelated G-CSF receptor ligand in a quantity effective to enhance leukocyte production.
  • the zinc chelated receptor ligands of the present invention also provide for a method of treating the above indicated disease states because of their demonstrated ability to act as agonist of dimeric cell-surface receptors.
  • the drug may be administered to a patient in need thereof by any conventional route of administration, including, but not limited to, intravenous, intramuscular, oral, subcutaneous, intradermal, and parenteral.
  • the drug may be formed in vivo by the administration of a dimeric cell-surface receptor binding moiety (or receptor binding moiety as used herein) by the same methods of administration described herein and in about the same amounts as described herein for zinc chelated receptor ligands.
  • a receptor binding moiety of the present invention may be administered with a zinc source so as to facilitate the in vivo formation of a zinc chelated receptor ligand.
  • the pharmaceutically active zinc chelated receptor ligands of the present invention or, when desired and appropriate, the receptor binding moieties of the present invention are incorporated into convenient dosage forms such as capsules, tablets, or injectable preparations.
  • Solid or liquid pharmaceutical carriers are employed.
  • Solid carriers include, starch, lactose, calcium sulfate dihydrate, terra alba, sucrose, talc, gelatin, agar, pectin, acacia, magnesium stearate, and stearic acid.
  • Liquid carriers include syrup, peanut oil, olive oil, saline, and water.
  • the carrier or diluent may include any prolonged release material, such as glyceryl monostearate or glyceryl distearate, alone or with a wax.
  • the amount of solid carrier varies widely but, preferably, will be from about 25 mg to about 1 g per dosage unit.
  • the preparation will be in the form of a syrup, elixir, emulsion, soft gelatin capsule, sterile injectable liquid such as an ampoule, or an aqueous or nonaqueous liquid suspension.
  • the pharmaceutical preparations are made following conventional techniques of a pharmaceutical chemist involving mixing, granulating, and compressing, when necessary, for tablet forms, or mixing, filling and dissolving the ingreidents, as appropriate, to give the desired oral or parenteral products.
  • Doses of the presently invented pharmaceutically active zinc chelated receptor ligands of the present invention or, when desired and appropriate, the receptor binding moieties of the present invention, in a pharmaceutical dosage unit as described above will be an efficacious, nontoxic quantity preferably selected from the range of 0.001 - 125 mg/kg of active compound, preferably 0.001 - 60 mg/kg.
  • the selected dose is administered preferably from 1-6 times daily, orally or parenterally.
  • Preferred forms of parenteral administration include topically, rectally, transdermally, by injection and continuously by infusion.
  • Oral dosage units for human administration preferably contain from 0.05 to 3500 mg of active compound. Oral administration, which uses lower dosages is preferred. Parenteral administration, at high dosages, however, also can be used when safe and convenient for the patient.
  • Optimal dosages to be administered may be readily determined by those skilled in the art, and will vary with the particular zinc chelated receptor ligand or receptor binding moiety in use, the strength of the preparation, the mode of administration, and the advancement of the disease condition. Additional factors depending on the particular patient being treated will result in a need to adjust dosages, including patient age, weight, diet, and time of administration.
  • Another aspect of the present invention is a method for identifying agonists of dimeric cell-surface receptors and receptor ligands identified thereby.
  • the dimeric cell-surface receptor is contacted with receptor ligand candidates in the presence of a micromolar concentration of zinc(II).
  • Ligand candidates which bind to the dimeric cell-surface receptor are selected by receptor binding assays well known to those skilled in the art, such as competitive and non-competitive binding measurements ( Immobilized Affinity Ligand Techniques , G. T. Hermanson, A. K. Mallia, P. K. Smith Eds., Academic Press Inc. San Diego, Calif.
  • the dimeric cell-surface receptor in isolated, immobilized or cell-bound form is contacted with a plurality of zinc chelated receptor ligand candidates and those candidates which bind to and interact with the receptor are selected.
  • the isolated, immobilized or cell-bound receptor is contacted with a variety of metal-chelating receptor ligand candidates in the presence of zinc(II). Binding interaction can be measured directly by using radioactively labeled ligand candidates or indirectly, by using cells expressing the dimeric cell-surface receptor and measuring the occurrence of an event mediated by the formation of a dimeric cell-surface receptor-ligand complex.
  • the ligand candidates can be subjected to competitive binding assays in which the known receptor ligand, labeled preferably with an analytically detectable reagent, most preferably radioactivity, is included with the ligand candidates and a candidate's ability to inhibit the binding of the labeled ligand is measured.
  • Positive receptor ligand candidates are screened for biological function by any one of the receptor function assays well known to those skilled in the art. It is expected that a positive ligand binding candidate will exhibit agonist activity in receptor function assays.
  • G-CSF receptor An example of an appropriate competitive binding assay for the G-CSF receptor involves the immobilization of the G-CSF receptor and incubation with compounds of interest with I 125 radiolabeled G-CSF following the general procedure already described for other cytokine receptors (C. L. Martens et al. J. Biol. Chem. 1995, 270, 21129, E. Whitehorn et al. Biotechnology 1995, 13, 1215, S. D. Yanofsky et al. Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 7381, N. C. Wrighton et al. Science, 1996, 273, 458, S. E. Cwirla, Science, 1997, 276, 1696).
  • the method of this invention of inducing agonist activity at a dimeric cell-surface receptor in mammals, including humans comprises administering to a subject in need of such activity an effective amount of a pharmaceutically active zinc chelated receptor ligand of the present invention or, when desired and appropriate, a receptor binding moiety of the present invention.
  • the invention also provides for the use of a presently invented zinc chelated receptor ligand or a presently invented receptor binding moiety in the manufacture of a medicament for use as an agonist of a dimeric cell-surface receptor.
  • the invention also provides for the use of a zinc chelated receptor ligand or a receptor binding moiety in the manufacture of a medicament for use in therapy.
  • the invention also provides for the use of a zinc chelated receptor ligand or a receptor binding moiety in the manufacture of a medicament for use in enhancing the activity of a dimeric cell-surface receptor.
  • the invention also provides for the use of a zinc chelated receptor ligand or a receptor binding moiety in the manufacture of a medicament for use in treating disease states associated with compromised dimeric cell-surface receptor activity. For example, bacterial and fungal infections.
  • the invention also provides for a pharmaceutical composition for use as an agonist of a dimeric cell-surface receptor which comprises a zinc chelated receptor ligand or a receptor binding moiety and a pharmaceutically acceptable carrier.
  • the invention also provides for a pharmaceutical composition for use in treating bacterial infections which comprises a zinc chelated receptor ligand or a receptor binding moiety and a pharmaceutically acceptable carrier.
  • the invention also provides for a pharmaceutical composition for use in treating fungal infections which comprises a zinc chelated receptor ligand or a receptor binding moiety and a pharmaceutically acceptable carrier.
  • the invention also provides for a process for preparing a pharmaceutical composition containing a pharmaceutically acceptable carrier or diluent and a zinc chelated receptor ligand or a receptor binding moiety which comprises bringing the zinc chelated receptor ligand or the receptor binding moiety into association with the pharmaceutically acceptable carrier or diluent.
  • the pharmaceutically active compounds of the present invention can be co-administered with further active ingredients, such as other compounds known to treat disease states associated with compromised dimeric cell-surface receptor activity.
  • additional active ingredients such as other compounds known to treat disease states associated with compromised dimeric cell-surface receptor activity.
  • compounds to treat bacterial infections and fungal infections are examples of compounds known to treat bacterial infections and fungal infections.
  • the zinc chelated compounds of the invention are utilized as agonists of cell-surface receptors whose signal transduction mechanism involves receptor dimerization or oligomerization. These receptors are divided in five superfamilies (reviewed by Heldin, C. H.
  • PDGFR- ⁇ protein-tyrosine kinase receptors
  • SCFR protein-tyrosine kinase receptors
  • CSF-R protein-tyrosine kinase receptors
  • Flk-2 protein-tyrosine kinase receptors
  • EGFR EGFR
  • Erb2,Erb3, Erb4 FGFR-1, FGFR-2, FGFR-3, FGFR-4, insuline R, IGF-1R, HGFR, MSPR, Flt-1, Flk-1, Trk, TrkB, TrkC, Eph, Elk, Eck, Cck5, Sek, Eck, Erk
  • cytokine receptors GHR, TPOR, EPOR, PRLR, G-CSFR, leptin R, IL-3R, GM-CSFR, IL-5R, IL-6R, LIFR, CNTRFR, IL- 11R, IL-2R, IL-4R, IL-7R, IFN- ⁇ , IFN-
  • dimeric cell-surface receptor(s) refers to the receptors of the five superfamilies as listed above, with the exception of the G-CSF receptor.
  • the zinc chelated compounds of the invention are utilized as agonist of the erythropoietin (EPO) receptor. Additionally, a therapeutically effective amount of a receptor binding moiety of the invention is administered to a subject in need of treatment for anemia.
  • EPO erythropoietin
  • the zinc chelated compounds of the invention are utilized as agonist of the macrophage-colony-stimulating factor (M-CSF) receptor. Additionally, a therapeutically effective amount of a receptor binding moiety of the invention is administered to a subject in need of treatment for neutropenia.
  • M-CSF macrophage-colony-stimulating factor
  • the zinc chelated compounds of the invention are utilized as agonist of the growth hormone (GRH) receptor. Additionally, a therapeutically effective amount of a receptor binding moiety of the invention is administered to a subject in need of treatment for growth hormone deficiency.
  • GSH growth hormone
  • the zinc chelated compounds of the invention are utilized as agonist of the thrombopoietin (TPO) receptor. Additionally, a therapeutically effective amount of a receptor binding moiety of the invention is administered to a subject in need of treatment for thrombocytopenia.
  • TPO thrombopoietin
  • the zinc chelated compounds of the invention are utilized as agonist of the leptin receptor. Additionally, a therapeutically effective amount of a receptor binding moiety of the invention is administered to a subject in need of treatment for obesity.
  • the zinc chelated compounds of the invention are utilized as agonist of the interferon (IFN) alpha receptor. Additionally, a therapeutically effective amount of a receptor binding moiety of the invention is administered to a subject in need of treatment for hepatitis C.
  • IFN interferon
  • the zinc chelated compounds of the invention are utilized as agonist of the interferon (IFN) beta receptor. Additionally, a therapeutically effective amount of a receptor binding moiety of the invention is administered to a subject in need of treatment for multiple sclerosis.
  • IFN interferon
  • the zinc chelated compounds of the invention are utilized as agonist of the insulin receptor. Additionally, a therapeutically effective amount of a receptor binding moiety of the invention is administered to a subject in need of treatment for diabetes.
  • the zinc chelated compounds of the invention are utilized as agonist of the tyrosine kinase (TRK) receptors. Additionally, a therapeutically effective amount of a receptor binding moiety of the invention is administered to a subject in need of treatment for CNS diseases.
  • TRK tyrosine kinase
  • the zinc chelated compounds of the invention are utilized as dimeric cell-surface receptor agonist.
  • the crystalline material was filtered and purified by reversed phase preparative HPLC (Rainin Dynamax, 5 ⁇ M C18 column: 21.4 mm ⁇ 25 cm, elution with gradient acetonitrile-water containing 0.1 % trifluoroacetic acid) to yield the title compound as a white powder (88 mg, 18%).
  • An oral dosage form for administering a presently invented agonist of the G-CSF receptor is produced by filing a standard two piece hard gelatin capsule with the ingredients in the proportions shown in Table I, below.
  • Table I TABLE I INGREDIENTS
  • AMOUNTS Bis ⁇ 2,5-bis[2-benzimidazolylimino]-3a,6a-bis(2-pyridyl)- 25 mg 1,2,3,3a,4,5,6,6a-octahydroimidazo[4,5-d]imidazole-N,N′ ⁇ - zinc(II) (Compound 1) Lactose 55 mg Talc 16 mg Magnesium Stearate 4 mg
  • An injectable form for administering a presently invented agonist of the G-CSF receptor is produced by stirring 1.5% by weight of 2,5-Bis[2-benzimidazolylimino]-3a,6a-diphenyl-1,2,3,3a,4,5,6,6a-octahydroimidazo[4,5-d]imidazole (Compound 2a) in 10% by volume propylene glycol in water.
  • sucrose, calcium sulfate dihydrate and a presently invented agonist of the G-CSF receptor are mixed and granulated in the proportions shown with a 10% gelatin solution.
  • the wet granules are screened, dried, mixed with the starch, talc and stearic acid, screened and compressed into a tablet.

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US20090048318A1 (en) * 2003-04-29 2009-02-19 Connie Erickson-Miller Methods for treating degenerative diseases/injuries
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