+

WO1994027594A2 - Complexes stables de cuivre(i) et leur utilisation comme substances therapeutiques actives - Google Patents

Complexes stables de cuivre(i) et leur utilisation comme substances therapeutiques actives Download PDF

Info

Publication number
WO1994027594A2
WO1994027594A2 PCT/US1994/006247 US9406247W WO9427594A2 WO 1994027594 A2 WO1994027594 A2 WO 1994027594A2 US 9406247 W US9406247 W US 9406247W WO 9427594 A2 WO9427594 A2 WO 9427594A2
Authority
WO
WIPO (PCT)
Prior art keywords
copper
complex
complexes
bcds
stable
Prior art date
Application number
PCT/US1994/006247
Other languages
English (en)
Other versions
WO1994027594A3 (fr
Inventor
Alexander J. Pallenberg
Andrew Branca
Thomas M. Marschner
Leonard M. Patt
Original Assignee
Procyte Corporation
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Procyte Corporation filed Critical Procyte Corporation
Priority to EP94919342A priority Critical patent/EP0701439A1/fr
Priority to JP7501073A priority patent/JPH08511006A/ja
Priority to AU70517/94A priority patent/AU7051794A/en
Priority to ZA949336A priority patent/ZA949336B/xx
Publication of WO1994027594A2 publication Critical patent/WO1994027594A2/fr
Publication of WO1994027594A3 publication Critical patent/WO1994027594A3/fr

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/555—Heterocyclic compounds containing heavy metals, e.g. hemin, hematin, melarsoprol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/47—Quinolines; Isoquinolines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/02—Nutrients, e.g. vitamins, minerals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/06—Antihyperlipidemics
    • 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
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/20—Antivirals for DNA viruses
    • A61P31/22—Antivirals for DNA viruses for herpes viruses
    • 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

Definitions

  • This invention is generally directed to a copper(I) complex and methods relating to the use thereof and, more specifically, to copper(I) complexed by a multi-dentate ligand such that the +1 oxidation state for copper is favored in the resulting complex.
  • Copper is found in both plants and animals, and a number of copper-containing proteins, including enzymes, have been isolated. Copper may exist in a variety of oxidation states, including the 0, +1, +2 and +3 oxidation states (i.e., copper(0), copper(I), copper(II) and copper(III), respectively), with copper(I) and copper(II) the most common.
  • the relative stabilities of copper(I) and copper(II) in aqueous solution depend on the nature of the anions or other ligands present in the solution. Moreover, only low equilibrium concentrations of copper(I) in aqueous solutions (i.e., ⁇ 10 -2 M) can exist.
  • GHK-Cu(II) Various derivatives of GHK-Cu(II) possess similar activity (see U.S. Patent Nos. 4,665,054 and 4,877,770). GHK-Cu(II) and other peptide-copper(II) complexes have also been shown to be effective for stimulating hair growth
  • This invention is generally directed to stable copper(I) complexes and methods relating thereto. More specifically, the stable copper(I) complexes of the present invention comprise copper(I) complexed by a multi-dentate ligand such that the +1 oxidation state for copper is favored.
  • the stable copper(I) complexes have utility for enhancing wound healing in warm-blooded animals, for enhancing or restoring the resistance of warm-blooded animals to oxidative or inflammatory damage associated with reactive oxygen species and/or lipid mediators, for stimulating the growth of hair in warm-blooded animals, for modulating lipid metabolism, for modulating signal transduction in cells by inhibiting protein kinases, and for inhibiting viral activity, including (but not limited to) HIV replication in an HIV-infected animal.
  • Methods of the present invention comprise administering an effective amount of a stable copper(I) complex to the animal.
  • FIG 1 illustrates the activity of a representative copper(I) complex of this invention (i.e., bathocuproine disulfonic acid (“BCDS”) copper(I)) to accelerate wound healing.
  • BCDS bathocuproine disulfonic acid
  • Figure 2 illustrates the ability of a representative copper(I) complex of the present invention, BCDS copper(I), to inhibit viral (i.e., HIV) replication.
  • Figure 3 illustrates synthesis pathways for prostaglandins and leukotrienes, as well as certain key enzymes associated therewith.
  • Figure 4 illustrates a synthesis pathway for cholesterol formation, including the intermediates acetyl CoA and HMG-CoA and the enzymes acetyl CoA synthetase and HMG-CoA reductase.
  • FIG. 5 illustrates the action of Protein Kinase C (PKC) and protein tyrosine kinase in signal transduction
  • PI phosphatidyl inositol
  • IP 3 inositol triphosphate
  • PG phosphatyl glycerol
  • P-Protein phosphorylated protein
  • CDR PK calmoduln-regulated protein kinase
  • PKA Protein Kinase A
  • Protein Kinase Protein Tyrosine Kinase (cytoplasmic)
  • EGF-R Protein Kinase Epidermal growth factor receptor protein tyrosine kinase).
  • This invention is generally directed to copper(I) complexes and methods relating to the use thereof, and more specifically, to copper(I) complexed by a multi-dentate ligand to form a stable copper(I) complex.
  • a "stable copper(I) complex” is copper(I) chelated by at least one multi-dentate ligand such that the resulting complex favors the +1 oxidation state of copper.
  • the most common states of copper(I) are associated with four coordination sites, and are generally of a tetrahedral configuration.
  • chelating agents are coordination compounds in which a single ligand occupies more than one coordination position of a metal ion.
  • a "multi-dentate ligand” is a bi-, tri- or tetra-dentate ligand which occupies two, three or four coordination sites, respectively, of copper (I).
  • the stable copper(I) complexes of this invention include all complexes of copper(I) chelated by at least one multi-dentate ligand which structurally favors the +1 oxidation state of copper.
  • Copper(I) complexes may be formed by reacting a multi-dentate ligand with a source of copper(I) (such as CuCl, Cu 2 O or CuCN) in aqueous solution.
  • the resulting copper(I) complex may then be observed by suitable analytical techniques, such as ESR, NMR and/or UV-VIS, to determine the oxidation state of the copper in the complex (see Munakata et al., Copper Coordination Chemistry: Biochemical and Inorganic Perspectives, Karlin and Zubieta editors, Adenine Press, Guilderland, N.Y., pp. 473-495, 1983).
  • suitable analytical techniques such as ESR, NMR and/or UV-VIS
  • a "stable" copper(I) complex has a half-life of at least 5 minutes, preferably of at least one hour, and more preferably of 24 hours or more (i.e., half of the copper(I) complex remains in the +1 oxidation state) upon exposure to air, at room temperature (23°C) and atmospheric pressure.
  • stable copper(I) complexes of this invention resist oxidation, while non-stable copper(I) complexes are readily oxidized to yield copper(II) complexes upon exposure to air.
  • any multi-dentate ligand which chelates copper(I) to yield a stable copper(I) complex is suitable in the practice of this invention.
  • the multi-dentate ligands of this invention are selected from the following general structures I through VII:
  • a and B represent heteroatoms which may occupy coordination sites of copper(I), and are preferably selected from nitrogen, oxygen, sulfur and phosphorous.
  • the rings of structures I through VII may be aromatic, non-aromatic or a mixture of both aromatic and non-aromatic rings.
  • the following structures are representative of such combinations:
  • Table 1 identifies the structure of the representative multi-dentate ligand, lists the corresponding chemical name, identifies the Chemical Abstracts Registration Number ("CA Reg. No.”), and provides a corresponding reference (if available) describing the synthesis and/or chemistry of the identified multi-dentate ligand.
  • CA Reg. No. Chemical Abstracts Registration Number
  • heteroatoms are selected from nitrogen, oxygen, sulfur, and phosphorus.
  • the compounds listed in Table 2 illustrate further representative multi-dentate ligands of the present invention having additional ring substitutions.
  • Table 2 identifies the structure of the representative multi-dentate ligands, lists the corresponding chemical name, identifies the CA Reg. No., and provides a corresponding reference (if available) describing the synthesis and/or chemistry of the identified multi-dentate ligand.
  • R 1 through R 8 are the same or different, and are selected from the following chemical moieties: -H, -OH, -X, -OX, -COOH, -COOX, -CHO, -CXO, -F, -Cl, -Br, -I, -CN, -NH 2 , -NHX, -NX 2 , -PX 2 , -SO 3 H, -SO 3 Na, -SO 3 K, -SO3X, -PO 3 H, -OPO 3 H, -PO 3 X, -OPO 3 X and -NO 2 .
  • "X" represents and an alkyl moiety or an aryl moiety.
  • alkyl moiety is a straight chain or branched, cyclic or noncyclic, saturated or unsaturated, substituted or unsubstituted carbon chain containing from 1-20 carbon atoms; and an "aryl moiety” is a straight chain or branched, cyclic or noncyclic, saturated or unsaturated, substituted or unsubstituted carbon chain containing at least one substituted or unsubstituted aromatic moiety and containing from 6-20 carbon atoms.
  • Such chemical moieties may also be covalently attached to the ring fusion atoms. Representative examples of the chemical moieties of this invention include, but are not limited to, the moieties identified in Table 3 below.
  • Table 4 identifies the structure of the representative multi-dentate ligands, lists the corresponding chemical name, identifies the CA Reg. No., and provides a corresponding reference (if available) describing the synthesis and/or chemistry of the multi-dentate ligand.
  • the chemical moieties covalently attached to the structural backbone may be joined to yield an aromatic or nonaromatic cyclic chemical moiety.
  • Representative examples of such cyclic chemical moieties are set forth in Table 5, which identifies the structure of the representative multi-dentate ligands, lists the corresponding chemical name, identifies the CA Reg. No., and provides a corresponding reference (if available) describing the synthesis and/or chemistry of the multi- dentate ligand.
  • the multi- dentate ligands are selected from the following structures:
  • R 1 through R 8 are the same or different, and are selected from hydrogen, an alkyl moiety and an aryl moiety.
  • the multi-dentate ligand is 6,6'-dimethyl-2,2'-dipyridine having structure Id:
  • the multi-dentate ligand is neocuproine (2,9-dimethyl-1,10-phenanthroline) having structure lid, or is bathocuproine disulfonic acid ("BCDS") having one of the isomeric structures lie, lie' or IIe'':
  • BCDS refers to a physical mixture of the above isomers (i.e., IIe, IIe' and lie''). Typically, the ratio of the various isomers (i.e., lie: lie' :lie'') vary depending upon the commercial source of BCDS as follows: Aldrich Chemical Co., Inc. (Milwaukee,
  • stable copper(I) complexes of this invention may be made by contacting a multi-dentate ligand with a copper(I) source.
  • the multi-dentate ligands may be obtained from commercial sources, or may be synthesized by known organic synthesis techniques from commercially available reagents.
  • water soluble multi-dentate ligands are complexed with the copper(I) in aqueous solution, employing CuCl, Cu 2 O or CuCN as the copper(I) source.
  • the resulting copper(I) complex may then be recovered by evaporation of solvent to yield the copper(I) complex.
  • copper(I) complexes may be formed by the above procedure employing a suitable non-aqueous (e.g., organic) solvent.
  • the ratio of the multi-dentate ligand to copper(I) may be any ratio which results in a stable copper(I) complex.
  • the ligand to copper ratio is at least 1:1.
  • the ligand to copper ratio ranges from 1:1 to 3:1 (including 2:1).
  • Such copper(I) complexes may be made by the procedures identified in the preceding paragraph by reacting the appropriate molar ratios of the multi-dentate ligand and the copper(I) ion source.
  • copper(I) has enhanced biological activity over copper(II) in certain biological events.
  • copper(I) may be an important intermediate for copper metabolism, including copper uptake and/or transfer, as well as cellular delivery.
  • the reduction of copper(II) to copper(I) is bypassed by direct delivery of copper(I).
  • the stable copper(I) complexes of this invention are suitable for systemic delivery to warm blooded animals, and may provide a sustained release of copper to the animal.
  • the stable copper(I) complexes of this invention possess utility as therapeutic substances, including utility as anti-oxidative and anti-inflammatory agents generally and, more specifically, as wound healing agents.
  • the copper(I) complexes of this invention also possess activity as hair growth agents, lipid modulation agents, signal transduction modulating agents, and anti-viral agents.
  • the various biological activities of the stable copper(I) complexes of this invention are addressed individually below.
  • Highly reactive oxygen species such as the superoxide anion (O 2 ⁇ _ ), hydrogen peroxide (H 2 O 2 ), hydroxyl radical (HO ⁇ ), and lipid peroxides (LOOH) are involved in a number of human diseases.
  • oxygen species have been implicated in autoimmune diseases, arthritis, tissue damage caused by environmental pollutants, cigarette smoke and drugs, tissue injury during, for example, surgery and transplantation, as well as a variety of other conditions (see, e.g., Halliwell, B., Fed, Amer. Soc, Exp, Biol. 1:358-364, 1987).
  • Reactive oxygen species are also generated during the response to injury by phagocytic cells.
  • One of the early events in the wound healing response is the cleansing and sterilization of the wound by neutrophils and macrophages. A mechanism for this sterilization is the generation of the superoxide anion and hydrogen peroxide, and generally results in an inflammatory response.
  • hydroxyl radical is a potent oxidant which initiates the free radical oxidation of fatty acids, as well as the oxidative degradation of other biomolecules.
  • reactive oxygen species cause tissue damage is in post-injury damage to the brain and spinal chord, and in reperfusion injury to ischemic tissue following surgery and transplantation (such as heart surgery and/or transplantation).
  • a sudden inrush of oxygenated blood and activated phagocytic cells leads to superoxide anion and hydrogen peroxide formation.
  • These species do direct damage to tissue, and also react with iron (as discussed above) to generated the very reactive hydroxyl radical.
  • the stable copper(I) complexes of this invention generally serve as anti-oxidative agents which prevent or limit the oxidative damage caused by reactive oxygen species, and further serve as anti-inflammatory agents by reducing the inflammatory response associated with such reactive oxygen species. More specifically, the copper(I) complexes of the present invention are useful in the enhancement and/or restoration of the defense of warm-blooded animals to oxidative or inflammatory damage caused by the highly reactive oxygen species, and may be used in pharmaceutical preparations to inhibit oxidative and inflammatory processes which lead to tissue damage. Moreover, the stable copper(I) complexes of this invention accelerate the wound healing process by "detoxifying" tissue damage by the highly reactive oxygen species.
  • lipid mediators of inflammation e.g., leukotrienes and prostaglandins.
  • IBD inflammatory bowel disease
  • Prostaglandins enhance vasodilation and edema formation
  • leukotrienes are potent chemoattractive agents for leukocytes, especially neutrophils, and stimulate degranulation and the release of damaging lysosomal enzymes and superoxide production.
  • the stable copper(I) complexes of this invention inhibit the formation of prostaglandins and/or leukotrienes by inhibiting the enzymes involved in their formation.
  • the stable copper(I) complexes are effective inhibitors of both cyclooxygenase-1 and cyclooxygenase-2, thereby inhibiting the formation of prostaglandins.
  • the .stable copper(I) complexes are effective inhibitors of 5-lipoxygenase and leukotriene C 4 (LCT 4 ) synthetase, thereby inhibiting the formation of leukotrienes.
  • elastase a neutrophil-released serine protease
  • proteolysis of various cellular targets by elastase has been implicated in a number of pathologic conditions, including emphysema, rheumatoid arthritis, and psoriasis.
  • inhibitors of elastase may be used to treat, prevent or limit the breakdown of normal tissue at the site of inflammation, and the stable copper(I) complexes of this invention are effective inhibitors of elastase.
  • the stable copper(I) complexes of this invention may also be used in the regulation and/or modulation of lipid metabolism in general.
  • hypercholesterolemia and hyperlipidemia are common and serious health problems which are treatable with the stable copper(I) complexes of this invention.
  • Hypercholesterolemia has been observed in marginal and severely copper-deficient rats, as well as other animals, including humans (Lei, "Plasma Cholesterol Response in Copper Deficiency,” Role of Copper in Lipid Metabolism, ed. Lei, CRC Press, pages 1-24, 1990). Elevation in serum cholesterol level has been linked to increases in the activity of hepatic 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG CoA reductase, E.C.I.1.1.34) and glutathione levels (Bunce, "Hypercholesterolemia of Copper Deficiency is Linked to Glutathione Metabolism and Regulation of HMG CoA Reductase," Nutr. Rev. 51: 305-307, 1993; Kim et al., "Inhibition of Elevated Hepatic Glutathione Abolishes Copper Deficiency Cholesterolemia," FASEB J. 6: 467-2471,
  • Acetyl CoA synthetase catalyzes the formation of acetyl CoA from acetate. As illustrated in Figure 4, acetyl CoA can be further metabolized along many different pathways leading primarily to the formation of cholesterol and fatty acids or energy production. Agents which inhibit this enzyme influence the biosynthesis of various lipids.
  • HMG-CoA reductase (3-hydroxy-3-methylglutaryl coenzyme A reductase) is located biochemically later in the lipid synthesis scheme and converts HMG-CoA to mevalonic acid, and is the rate limiting reaction in cholesterol biosynthesis (see Figure 4).
  • Stable copper(I) compounds of this invention inhibit certain key enzymes involved in the formation of lipids, and thus serve as lipid modulating or regulating agents. (The ability of stable copper(I) complexes to inhibit enzymes in the formation of lipids is disclosed in further detail in Examples 12-13.)
  • the stable copper(I) complexes of this invention may also serve as modulating agents of signal transduction in cells.
  • Most intracellular signaling processes are regulated by reversible phosphorylation of specific proteins by kinases. Breakdown of phosphatidylinositol leads to the formation of diacylglycerol and inositol triphosphate, the former acting synergistically with calcium to activate Protein Kinase C (PKC), resulting in translocation of the enzyme from cytosol to the membrane.
  • PKC Protein Kinase C
  • Phosphorylation of proteins by PKC has been implicated as a pivotal regulatory element in signal transduction, cellular regulation and tumor promotion. Inhibitors of PKC, as well as other protein kinases, have the potential to block proliferative signaling in tumor induction, atherosclerosis and immune modulation.
  • factors which stimulate the G-protein linked phospholipase C breakdown of phosphatidylinositol include angiotensin II, bradykinin, endothelin, f-Met-Leu-Phe, and vasopressin. These protein kinase C enzymes are also directly activated by tumor promoters such as phorbol esters.
  • Receptor linked tyrosine kinases include Epidermal Growth Factor, Nerve Growth Factor, and Platelet Derived Growth Factor.
  • cytoplasmic tyrosine kinase activators include cytokines such as Interleukin 2, Interleukin 3, and Interleukin 5. These factors bind to specific lymphocyte receptors which activate the cytoplasmic tyrosine kinase.
  • the action of PKC and protein tyrosine kinase action is illustrated in Figure 5 .
  • the stable copper ( I ) complexes of this invention serve as signal transduction modulating agents by inhibiting one or more enzymes involved in intracellular signal transduction, including PKC and protein tyrosine kinases.
  • the stable copper(I) complexes When administered to an animal to treat the conditions discussed above, the stable copper(I) complexes may first be combined with one or more suitable carriers or diluents to yield a pharmaceutical preparation suitable for topical, oral or parenteral application. Such diluents or carriers, however, should not interact with the stable copper(I) complex to significantly reduce the effectiveness thereof, or oxidize copper(I). Effective administration will preferably deliver a dosage of approximately 0.01 to 100 mg of the stable copper(I) complex per kg of body weight.
  • Suitable carriers for parenteral application include sterile water, physiological saline, bacteriostatic saline (saline containing 0.9 mg/ml benzyl alcohol) and phosphate-buffered saline.
  • the stable copper(I) complexes may be topically applied in the form of liquids, containing pharmaceutically acceptable diluents (such as saline and sterile water) or may be applied as lotions, creams or. gels, containing additional ingredients to impart the desired texture, consistency, viscosity and appearance.
  • pharmaceutically acceptable diluents such as saline and sterile water
  • lotions, creams or. gels containing additional ingredients to impart the desired texture, consistency, viscosity and appearance.
  • Such additional ingredients are familiar to those skilled in the art and include emulsifying agents such as non-ionic ethoxylated and nonethoxylated surfactants, fatty alcohols, fatty acids, organic or inorganic bases, preserving agents, wax esters, steroid alcohols, triglyceride esters, phospholipids such as lecithin and cephalin, polyhydric alcohol esters, fatty alcohol esters, hydrophilic lanolin derivatives, hydrophilic beeswax derivatives, hydrocarbon oils such as palm oil, coconut oil, mineral oil, cocoa butter waxes, silicon oils, pH balancers and cellulose derivatives.
  • emulsifying agents such as non-ionic ethoxylated and nonethoxylated surfactants, fatty alcohols, fatty acids, organic or inorganic bases, preserving agents, wax esters, steroid alcohols, triglyceride esters, phospholipids such as lecithin and cephalin, polyhydric alcohol esters,
  • Topical administration may by accomplished by applying an amount of the preparation directly to the desired area, such as a wound or an inflamed area.
  • the required dosage will vary according to the particular condition to be treated, the severity of the condition, and the duration of the treatment.
  • the preparation may contain about 1% to about 20% of a penetration enhancing agent.
  • penetration enhancing agents include dimethylsulfoxide (DMSO), urea and eucalyptol.
  • the concentration of penetration enhancing agent such as DMSO
  • the stable copper(I) complexes of this invention also possess utility as hair growth agents.
  • Hair loss is a common affliction of humans, the most common being “alopecia” where males lose scalp hair as they get older (also called “male pattern baldness”).
  • Other hair loss afflications include alopecia areata (AA), female pattern baldness and secondary alopecia (e.g., hair loss associated with chemotherapy and/or radiation treatment).
  • the stable copper (I) complexes of this invention are particularly useful in stimulating hair growth associated with any hair loss afflication, including the specific afflications identified above.
  • Terminal hair is coarse, pigmented hair which arises from follicles which are developed deep within the dermis.
  • Vellus hairs are typically thin, non-pigmented hairs which grow from hair follicles which are smaller and located superficially in the dermis.
  • alopecia progresses, there is a change from terminal to vellus type hair.
  • Other changes that contribute to alopecia are alterations in the growth cycle of hair. Hair typically progresses through three cycles, anagen (active hair growth), catagen (transition phase), and telogen (resting phase during which the hair shaft is shed prior to new growth).
  • baldness progresses, there is a shift in the percentages of hair follicles in each phase, with the majority shifting from anagen to telogen.
  • the size of hair follicles is also known to decrease while the total number remains relatively constant.
  • the stable copper(I) complexes of this invention have utility as stimulating agents for the growth of hair in warm-blooded animals.
  • the copper(I) complex may be administered intradermally in the area to be treated, along with a suitable vehicle, at a concentration of approximately 100-500 micrograms of copper(I) complex per 0.1 ml of vehicle.
  • suitable vehicles in this regard include saline, sterile water, and the like.
  • the stable copper(I) complex may be topically applied in the form of a liquid, lotion, cream or gel by applying an effective amount of the topical preparation directly to the scalp. Any quantity sufficient to stimulate the rate of hair growth is effective, and treatment may be repeated as often as the progress of hair growth indicates.
  • suitable topical hair growth preparations contain from about 0.1% to about 20% by weight of the stable copper(I) complex (based on the total weight of the preparation).
  • Topical hair growth preparations of the present invention may contain about 0.5% to about 10% of an emulsifying or surface active agent.
  • Non-ionic surface active agents and ionic surface active agents may be used for the purposes of the present invention.
  • suitable non-ionic surface active agents are nonylphenoxypolyethoxy ethanol (Nonoxynol-9), polyoxyethylene oleyl ether (Brij-97), various polyoxyethylene ethers (Tritons), and block copolymers of ethylene oxide and propylene oxide of various molecular weights (Pluronic 68, for example).
  • Acceptable preparations may also contain about 1% to about 10% of certain ionic surface active agents.
  • These ionic surface active agents may be used in addition to or in place of, the non-ionic surface active agents.
  • Examples of ionic surface active agents are sodium lauryl sulfate and similar compounds.
  • topical hair growth preparations of this invention may contain about 1% to about 20% of a penetration enhancing agent.
  • penetrating enhancing agents are DMSO and Urea.
  • the concentration of a penetrating enhancing agent, such as DMSO may comprise about 30% to about 80% of the topical preparation.
  • the balance of the topical hair growth preparation may comprise an inert, physiologically acceptable carrier.
  • Suitable carriers include, but are not limited to, water, physiological saline, bacteriostatic saline (saline containing 0.9 mg/ml benzyl alcohol), petrolatum based creams (e.g., USP hydrophilic ointments and similar creams, Unibase, Parke-Davis), various types of pharmaceutically acceptable gels, and short chain alcohols and glycols (e.g., ethyl alcohol and propylene glycol).
  • bacteriostatic saline saline containing 0.9 mg/ml benzyl alcohol
  • petrolatum based creams e.g., USP hydrophilic ointments and similar creams, Unibase, Parke-Davis
  • short chain alcohols and glycols e.g., ethyl alcohol and propylene glycol
  • the copper(I) complexes of the present invention also posses utility as anti-viral agents, and are particularly effective in the inhibition of the AIDS virus.
  • Human acquired immunodeficiency syndrome or "AIDS" is a fatal disease for which there is presently no cure.
  • the disease is believed to be caused by a virus known as the human immunodeficiency virus, commonly referred to as "HIV.”
  • HIV The virus is transmitted by HIV-infected individuals through the exchange of bodily fluids. HIV infection results most commonly from sexual contact with an infected partner and the sharing among intravenous drug users of hypodermic syringes previously used by an infected individual.
  • a pregnant HIV-infected mother may infect her unborn child by trans-placental transmission, and HIV-contaminated blood is a possible source of infection for individuals subject to blood transfusion.
  • HIV infection causes a suppression of the immune system.
  • the immune suppression renders the infected individual vulnerable to a variety of opportunistic infections and conditions that are otherwise kept in balance by a healthy immune system.
  • Fatalities result from HIV infection due to the inability of AIDS patients to respond to treatment of the opportunistic infections and conditions as a consequence of their compromised immune systems. Because the virus may often remain dormant, the manifestation of AIDS from HIV infection may take as long as ten years.
  • HIV chronically infects specific immune cells known as T-helper cells, which are required for normal immune response.
  • T-helper cells serve as hosts to the virus and facilitate the reproduction of the virus (the process of viral reproduction is commonly referred to as "replication").
  • the infected host cell eventually dies, the replicated HIV virus is released, and the infection spreads to additional cells. This cycle continues unabated, depleting the population of T-helper cells and, in time, weakens the immune system to the onset of AIDS symptoms.
  • T-helper cells are continuously produced by the body, the population of these cells may be reestablished in the absence of further HIV infection. Therefore, the progression of HIV infection (and the subsequent onset of AIDS) may be arrested by the prevention or inhibition of viral replication, and antiviral agents capable of inhibiting or preventing the replication of HIV should be effective in the treatment of AIDS.
  • HIV replication requires the insertion of viral deoxyribonucleic acid ("DNA”) into the genome of the host cell.
  • the genome of the host cell consists of the cell's own DNA, and is responsible for the synthesis of materials essential to the cell's own function and proliferation.
  • the inserted viral DNA is an enzymatic product derived from viral ribonucleic acid (“RNA”) and the action of an enzyme known as HIV reverse transcriptase. Inhibition of HIV reverse transcriptase precludes the formation of viral DNA required for insertion into the genome of the host. Viral replication is prevented by the absence of viral DNA in the host cell genome. Antiviral agents which inhibit HIV reverse transcriptase are thus potential therapeutic drugs for treatment of AIDS.
  • antiviral agents for inhibiting HIV replication, as well as methods relating to the administration thereof to an HIV-infected patient.
  • the antiviral agents of this invention are the stable copper(I) complexes discloses above, and the methods include administration of a therapeutically effective amount of a composition which includes a stable copper(I) complex in combination with a pharmaceutically acceptable carrier or diluent.
  • the copper(I) complexes of this invention enhance transport of copper(I) into HIV infected cells which, in turn, inhibits or inactivates HIV protease and thus inhibits the replication of HIV.
  • HIV includes the various strains of the virus such as HIV-1 and HIV-2.
  • Administration of the stable copper(I) complexes of the present invention may be accomplished in any manner which will result in a systemic dose of a therapeutically effective amount of the copper(I) complex to an HIV-infected animal or patient (including human patients).
  • administration may be by injection (intramuscular, intravenous, subcutaneous or intradermal), oral, nasal, or suppository applications.
  • preparations of the present invention include stable copper(I) complexes in solution for various forms Of injection, or in preparations which are formulated for the sustained release of the stable copper(I) complexes for oral, nasal, or suppository dosage application and generally include one or more inert, physiological acceptable carriers.
  • the term "effective amount" means an amount of the stable copper(I) complex which inhibits HIV replication in the patient. Suitable dosages may range from approximately 0.01 to 100 mg of stable copper(I) complex per kg body weight.
  • the stable copper(I) complexes of this invention may be screened for their ability to inhibit HIV replication using known techniques.
  • HIV virus replication may be monitored using the Cytopathic Effect (CPE) assay disclosed by Bergeron et al. (J. Virol. 66:5777-5787, 1992).
  • CPE Cytopathic Effect
  • the degree of infection is monitored by the appearance of fused cellular membranes ("syncitium").
  • assays directed to activity of HIV protease may be employed.
  • the assays and techniques disclosed in the following references may be employed: Ashorn et al., Proc. Natl, Acad. Sci. U.S.A. 87:7472-7476, 1990; Schramm et al.,
  • the stable copper (I) complexes of this invention in addition to inhibiting HIV replication, may also inhibit replication of other viruses, including human T-cell leukemia (HTLV) I and/or II, human herpes virus, cytomegalo virus (CMV), encephalomyocarditis virus (EMCV), Epstein Barr virus (EBV), human hepatitis virus, Varicella Zoster virus, Rhinovirus, and rubella virus.
  • HTLV human T-cell leukemia
  • CMV cytomegalo virus
  • EMCV encephalomyocarditis virus
  • EBV Epstein Barr virus
  • human hepatitis virus Varicella Zoster virus
  • Rhinovirus Rhinovirus
  • rubella virus hepatitis virus
  • Example 15 illustrates the inhibitory affect of stable copper(I) complexes of this invention on both encephalomyocarditis virus (EMCV) and cytomegalo virus (CMV).
  • the multi-dentate ligands of this invention also possess biological activity when administered alone as the "free" multi-dentate ligand (i.e., without copper(I)).
  • biological activity includes the activities identified above, including anti-viral activity, as well as a preventative agent against gastric tissue damage.
  • the multi-dentate ligands of this invention when administered as the free ligand, it is believed that they function, at least in part, by scavenging copper(I) to yield the stable copper(I) complex in vivo.
  • Example 1 illustrates the synthesis of neocuproine copper (I) at a molar ratio of 1:1 and 2:1
  • Example 2 illustrates the superoxide dismutase (SOD)-mimetic activity of representative copper(I) complexes of this invention (employing a copper(II)-peptide complex as a positive control)
  • Example. 3 illustrates the wound healing activity of a representative copper(I) complex of this invention
  • Example 4 illustrates hair growth activity of a representative copper(I) complex of this invention
  • Example 5 illustrates inhibition of HIV replication by a representative copper(I) complex of this invention
  • Example 6 illustrates the activity of a representative "free" multi-dentate ligand of this invention for both wound healing and protection against ethanol-induced gastric mucosal damage
  • Examples 7 and 8 illustrates the inhibition of cyclooxygenase-1 and cyclooxygenase-2, respectively, by representative stable copper(I) complexes
  • Example 9 illustrates the inhibition of 5-lipoxygenase by representative stable copper(I) complexes
  • Example 10 illustrates the inhibition of leukotriene C 4 synthetase by representative stable copper(I) complexes
  • Example 11 illustrates the inhibition of elastase by a representative stable copper(I) complex
  • Example 12 illustrates the inhibition of acetyl coenzyme A synthetase by representative stable copper (I) complexes
  • Example 13 illustrates the inhibition of HMG-CoA reductase by representative stable copper(I) complexes
  • Cuprous chloride (1.98g, 20.0mmol) was added to a stirred, vacuum-degassed solution of neocuproine hydrate (4.53g, 20.0mmol) in acetonitrile (150mL). This solution was stirred for 2 hours. The resulting suspension was warmed to boiling and filtered. The filtrate was boiled to a volume of about 10OmL. This solution was allowed to cool slowly to give dark red needles: mp280-284°C (decomp., lit. 310-320°C) (Healy et al., J. Chem. Soc. Dalton Trans.
  • SOD mimetics compounds which possess activity in a superoxide dismutase (SOD) assay are termed "SOD mimetics.”
  • SOD mimetics compounds which possess activity in a superoxide dismutase (SOD) assay.
  • representative copper(I) complexes of this invention were evaluated for SOD mimetic activity as measured by the Xanthine Oxidase/NBT method (see Oberly and Spitz, Handbook of Methods for Oxygen
  • the reactions contained the following: 100 uM Xanthine, 56 uM NBT (Nitro Blue Tetrazolium), 1 unit of Catalase, 50 mM Potassium Phosphate Buffer, pH 7.8.
  • the reaction was initiated by the addition of Xanthine Oxidase in sufficient quantity to obtain an increase in absorbance at 560 nm of approximately 0.025/min. in a total volume of 1.7 ml.
  • the Xanthine Oxidase was prepared fresh daily and stored on ice until used. All the components of the reaction are added except the Xanthine Oxidase and the spectrophotometer was adjusted to zero at 560 nm. The reaction was initiated by the addition of the Xanthine Oxidase. All reagents were obtained from Sigma Chemical Co.
  • BCDS:Cu(I) neocuproine copper(I)
  • NC:Cu(I) neocuproine copper(I)
  • the subcutaneous implantation of stainless steel wound chambers in rats provides a model for the healing of open cavity wounds. Implantation of these chambers triggers a series of responses which reflect the series of phases involved in wound healing - fibrin clot formation, infiltration of white cells, collagen synthesis, and new blood vessel formation.
  • This assay involves the implantation of a stainless steel chamber (1 X 2.5 cm cylindrical 312 SS, 20 mesh, with Teflon end caps) on the dorsal mid-line of rats. After one week to allow for encapsulation of the chamber, the chamber on each rat was injected with a 0.2 ml saline solution containing 2.7 ⁇ mol of the copper(I) complex (i.e., BCDS copper(I) 1:1 or 2:1), or with the same volume of saline (0.2 ml) without the copper(I) complex (i.e., control). Injections were made on days 5, 7, 9, 12, 14, 16 and 19. The chambers were then removed on day 21.
  • a stainless steel chamber (1 X 2.5 cm cylindrical 312 SS, 20 mesh, with Teflon end caps
  • the chambers were lyophilized and the interior contents removed for biochemical analysis.
  • the biochemical parameters examined included the total dry weight, protein content, collagen content (i.e., hydroxyproline content after acid hydrolysis) and glycosaminoglycan content or "GAG" (i.e., uronic acid content after acid hydrolysis).
  • the protein was determined by the method of Lowry et al. (J. Biol. Chem. 193: 265-275, 1951) using Bovine
  • Serum Albumin (BSA) as a standard.
  • the collagen content was determined by acid hydrolysis and a colorimetric assay for hydroxyproline (Bergman et al., Clin. Chim. Acta 27:347-349, 1970), an amino acid specific for collagen.
  • Glycosaminoglycan content was determined by quantitation of the amount of uronic acid (UA). Aliquots of the homogenate were dissolved in 0.5M NaOH, precipitated and washed with ethanol, and uronic acid was determined by a colorimetric assay using 2-phenylphenol as a reagent
  • Glycosaminoglycan content was expressed as ⁇ g of uronic acid per chamber.
  • the following example illustrates the stimulation of hair growth in warm-blooded animals after intradermal injection of a copper(I) complex of this invention.
  • C3H mice 60 days old, telogen hair growth phase
  • a sterile saline solution containing the indicated copper complex was then injected intradermally (i.e., infiltrated under the skin) at two locations within the clipped areas of the mice. Injection at two locations provided two test locations within the clipped area of each mouse.
  • Each injection (0.1 ml) contained the indicated amount of the copper(I) complex (i.e., BCDS copper(I) (1:1) complex at 0.14 ⁇ mol and 1.4 ⁇ mol) within a sterile saline solution.
  • a group of saline injected mice (0.1 ml) served as controls.
  • PBMC peripheral blood mononuclear cells
  • Treated PBMC were pelleted by centrifugation and resuspended to 0.75 ⁇ 10 6 /mL in basal medium with appropriate dilutions of the copper (I) complex or with no copper(I) complex added (i.e., control).
  • To each 0.5 mL aliquot of cells 0.5 mL of appropriate HIV dilution was added. The virus-cell mixture was incubated for 2 hours at 37°C in a 5% CO 2 humidified atmosphere. Following the incubation period, the PBMC were washed twice in phosphate-buffered saline. Cells were resuspended in 5 mL to 7 ⁇ 10 4 cells/mL in basal medium with (or without) the copper (I) complex. Each cell aliquot was dispensed into four replicate wells of a 48 well tissue culture plate. Cells were fed twice a week with appropriate medium.
  • PBMC peripheral blood monoclonal anti-p24 antibody coupled to an enzyme.
  • the amount of p24 was quantified spectrophotometrically.
  • Cyclooxygenase is involved in the formation of prostaglandins and thromboxanes by the oxidative metabolism of arachidonic acid (see Figure 3).
  • cyclooxygenase-1 from ram seminal vesicles was incubated with arachidonic acid (100 uM) for 2 minutes at 37° C in the presence or absence of neocuproine copper(I) (2:1) or BCDS copper(I) (2:1) at increasing concentrations of neocuproine copper (I) or BCDS copper(I) from 0.3 to 300 ⁇ M.
  • cyclooxygenase-1 activity was determined by reading the absorbance at 530 nm (Evans et al., "Actions of Cannabis Constituents on Enzymes of Arachidonate Metabolism:Anti-inflammatory Potential,” Biochem. Pharmacol. 36: 2035- 2037, 1987; Boopathy and Balasubramanian, "Purification and Characterization of Sheep Platelet cyclooxygenase,” Biochem J. 239: 371-377, 1988).
  • TCA trichloroacetic acid
  • Neocuproine copper(I) (2:1) was found to inhibit cyclooxygenase-1 with an IC50 of 23 ⁇ M ( see Table 12).
  • BCDS copper(I) (2:1) complex produced approximately 44% inhibition at a concentration of 300 ⁇ M.
  • Cyclooxygenase-2 also known as prostaglandin H synthase-2, catalyzes the oxygenation of unesterified precursors to form cyclic endoperoxide derivatives, including prostaglandin H (see Figure 3).
  • cyclooxygenase-2 from sheep placenta, 80 units/tube was pre-incubated with 1 mM glutathione (GSH), 1 mM hydroquinone, 2.5 ⁇ M hemoglobin, and either neocuproine copper(I) (2:1) or BCDS copper(I)
  • Neocuproine copper(I) (2:1) was found to inhibit cyclooxygenase-2 at an estimated IC50 of 25 ⁇ M ( see Table 13), which is similar to the results of Example 7 with cyclooxygenase-1.
  • BCDS copper(I) (2:1) produced approximately 34% inhibition at the screening concentration of 300 ⁇ M.
  • the 5-lipoxygenase is the principal lipoxygenase in basophils, polymorphonuclear (PMN) leukocytes, macrophages, mast cells, and any organ undergoing an inflammatory response. As illustrated in Figure 3, the action of 5-lipoxygenase leads to the formation of 5-HPETE and 5-HETE, which are precursors to the leuokotriene LTB4 and LTC 4 .
  • 5-lipoxygenase assays were run using a crude enzyme preparation prepared from rat basophilic leukemia cells (RBL-1).
  • Neocuproine copper(I) (2:1) or BCDS copper(I) (2:1) at increasing concentrations from 0.3 to 300 ⁇ M were pre-incubated with the 5-lipoxygenase for 5 minutes at room temperature, and the reaction was initiated by addition of arachidonic acid substrate. After incubation at room temperature for 8 minutes, the reaction was terminated by the addition of citric acid.
  • 5-HETE RIA Shiuzu et al., "Enzyme with Dual Lipoxygenase Activities Catalyzes Leukotriene A4 Synthesis from Arachidonic Acid," Proc. Natl, Acad, Sci. U.S.A.
  • BCDS copper(I) (2:1) and neocuproine copper (I) (2:1) were found to be inhibitors of 5-lipoxygenase with estimated IC50's of less than 10 ⁇ M (see Table 14). These results show that stable copper(I) complexes of this invention are potent inhibitors of neutrophil 5-lipoxygenase, thus preventing the accumulation of inflammatory lipid mediators at the sites of inflammation.
  • Leukotriene C 4 (LTC 4 ) Synthetase is involved in the formation of LTC 4 from LTA 4 , as illustrated in Figure 3, by the addition of a reduced glutathione at the C6 site.
  • LTC 4 Synthetase was prepared as a crude fraction from rat basophilic leukemia cells (RBL-1). The crude enzyme fraction was incubated with test compounds, LTA 4 methyl ester, albumin (to stabilize the product), and serine borate (to prevent conversion of LTC 4 to LTD 4 ) for 15 minutes at 37° C. The reaction was terminated by the addition of ice cold methanol, and LTC 4 concentration was determined by a specific RIA (Bach et al., "Inhibition by Sulfasalazine of LTC 4 Synthetase and of Rat Liver Glutathione S-Transferases," Biochem. Pharmacol. 34:2695-2704, 1985; Fitzpatrick et al., "Albumin Stabilizes Leukotriene A4," J. Biol. Chem, 257:4680-4683, 1982).
  • Proteolysis of various cellular targets by elastase has been implicated in a number of pathologic conditions, including emphysema, rheumatoid arthritis, and psoriasis.
  • human neutrophil was the source of the elastase.
  • human neutrophil elastase was prepared in crude form from fresh blood following dextran sedimentation, leukocyte isolation, cell lysis and homogenization of sub-cellular granules containing the elastase.
  • BCDS copper (I) (2:1) was incubated with the enzyme and substrate (methoxysuccinly-alanyl-alanyl-propyl-valine-4-nitroanalide) for 8 minutes at 25°C. The reaction is terminated by immersing the test tubes in boiling water for 5 minutes. Spectrophotometric analysis of the proteolytic product is measured at 410 nm (Baugh and Travis, "Human Leukocyte Granule Elastase, Rapid Isolation and Characterization," Biochemistry 15: 836-841, 1976).
  • BCDS copper(I) (2:1) was found to inhibit human neutrophil elastase with an estimated IC50 of 12 ⁇ M (see Table 16). These results show that stable copper(I) complexes of this invention are potent inhibitors of neutrophil elastase, thus preventing or limiting the breakdown of normal tissue at the sites of inflammation. Table 16
  • Neocuproine and BCDS Copper(I) (2:1) In this experiment, the ability of two stable copper(I) complexes, neocuproine copper(I) (2:1) and BCDS copper (I) (2:1), to inhibit certain key enzymes involved in the formation of lipids is demonstrated.
  • CoA synthetase (yeast) activity was monitored by utilization of a labeled substrate, sodium [3H] acetate
  • reaction buffer including 0.1 M glycine-NaOH (pH 9.0), ATP, and the substrate was pre- incubated for 5 minutes at 27°C, followed by addition of 2 nM coenzyme A for an additional 5 minute incubation at 27° C. The reaction was terminated by addition of HCl, and the remaining substrate determined by scintillation counting.
  • stable copper(I) complexes tested were found to inhibit acetyl CoA synthetase with estimated IC 50 's of 30-50 ⁇ M. These results indicate that the stable copper (I) complexes of this invention may serve as lipid modulating (e.g., lipid lowering) agents.
  • HMG-CoA reductase was isolated from rat liver and incubated with [ 14 C]HMG-CoA and either neocuproine copper(I) (2:1) or BCDS copper(I) (2:1) for 15 minutes at 37°C.
  • the reaction is terminated by addition of HCl, and [ 14 C] MVA is separated from the intact substrate by column filtration (Kubo and Strott, "Differential Activity of 3-hydroxy-3-methylglutaryl Coenzyme A Reductase in Zones of the Adrenal Cortex," Endocrinology 120: 214-221, 1987; Heller and Gould, "Solubilization and Partial Purification of Hepatic 3-hydroxy-3-methylglutaryl
  • BCDS Copper(I) (2;1) Isomers The experiments presented in this example demonstrate the effect on anti-HIV activity of different isomers of BCDS copper(I) (2:1). Two experiments utilized p24 antigen capture as a marker for viral replication, while two further experiments utilized reverse transcriptase activity to monitor the course of infection. The infection in all three experiments was performed in cultures of human peripheral blood mononuclear cells (PBMC) treated with HIV-1.
  • PBMC peripheral blood mononuclear cells
  • Structure IIe is referred to herein as the para-para (“PP”) BCDS isomer since both disulfonic acid/sodium salt moieties are located in the para position.
  • structure IIe' and IIe'' are referred to herein as the meta-para (“MP”) and meta-meta (“MM”) BCDS isomers, respectively.
  • MP meta-para
  • MM meta-meta
  • a mixture of the PP, MP and MM BCDS isomers was also tested (referred to herein simply as "BCDS”), having a ratio of PP:MP:MM of approximately 5:39:56.
  • Control (infected cells) 30910.00 3770.00 - - BCDS Copper(I) (10 ⁇ M) 1959.00 317.16 93.66 BCDS Copper(I) (25 ⁇ M) 0.25 0.25 99.99 MP-BCDS Copper(I) (10 ⁇ M) 404.50 124.66 98.69 MP-BCDS Copper(I) (25 ⁇ M) 0.50 0.50 99.99 MM-BCDS Copper(I) (10 ⁇ M) 346.50 106.27 98.88 MM-BCDS Copper(I) (25 ⁇ M) 0.00 0.00 100.00 Week 2
  • the anti-HIV activity of BCDS, PP-BCDS, MP-BCDS and MM-BCDS copper(I) (2:1) was determined by monitoring the same type of culture (i.e., HIV-1, PBMC) by measuring the reverse transcriptase activity as an infection marker.
  • the PBMC culture conditions for this experiment are described above in Example 5.
  • the activity of HIV-1 reverse transcriptase in cellular extracts was determined as a marker for the replication of the virus in culture.
  • HIV-1 reverse transcriptase in PBMC cultures may be performed by known techniques (Chattopadhyay et al., "Purification and Characterization of Heterodimeric Human Immunodeficiency Virus Type 1 (HIV-1) Reverse Transcriptase Produced by an In Vitro Processing of p66 with Recombinant HIV-1 Protease," J. Biol. Chem. 267: 14227-14232, 1992). The results of this experiment are presented in Table 21.
  • A549 cells human lung were infected with EMCV for 24-48 hours in the presence of either BCDS copper (I) or BCDS alone.
  • the cells were cultured in DMEM (10% FBS) for 3-4 days prior to use. The medium was then removed, and the cells incubated with sufficient EMCV in serum free DMEM to kill between 30-90% of the cells in the culture. After 2-3 hours of incubation of the cells with EMCV in their presence (or absence) of the test compounds, complete medium (DMEM + 10% FBS) was added and the cells allowed to incubate for 1-2 days in the presence or absence of the test compounds at concentrations ranging from 0.0001-0.0005 M.
  • CMV Cytomegalo virus
  • cellular viability i.e., mitochondrial function
  • CPE cytopathic effect
  • V t viability of the test culture
  • V v the viability of culture with virus alone
  • V u the viability of uninfected cells
  • This example illustrates the ability of stable copper(I) complexes of this invention to inhibit HIV-1 and HIV-2 proteases.
  • SPA beads Scintillation Proximity Assay
  • the substrate was a 12 residue peptide with the following sequence:
  • the peptide was monoiodinated on the terminal tyrosine residue, biotinylated through the ⁇ -amino group on the terminal lysine, and linked to the SPA bead via a streptavidin link.
  • HIV-1 protease cleaves the peptide substrate at the Phe-Phe bond, releasing the 125 I-fragment from the bead. Once the peptide is cleaved, it can no longer stimulate the scintillant in the SPA bead and the signal is reduced. The rate of reduction is proportional to the activity of the HIV-1 protease. Recombinant HIV-1 protease, affinity purified for kinetic and assay studies, was used in this experiment.
  • the results of this experiment are presented in Table 25.
  • the data presented is the mean ⁇ SD of the percent inhibition relative to a no enzyme control reaction.
  • the IC 50 was estimated from the point at which the dose inhibition line crossed the 50% inhibition line.
  • the estimated IC 50 with this HIV-1 protease assay was 11 ⁇ M.
  • SPA beads were coupled with a peptide substrate to assay for HIV-2 protease.
  • the substrate was the 12 residue peptide identified above and monoiodinated on the terminal tyrosine residue, biotinylated through the ⁇ -amino group on the terminal lysine, and linked to the SPA bead via a streptavidin link.
  • HIV-2 protease cleaves the peptide substrate at the Phe-Phe bond, releasing the 125 I-fragment from the bead. Once the peptide is cleaved, it can no longer stimulate the scintillant in the SPA bead and the signal is reduced. The rate of reduction is proportional to the activity of the HIV-2 protease. Recombinant HIV-2 protease, affinity purified for kinetic and assay studies, was used in this experiment. HIV-2 protease has about 50% sequence homology with HIV-1 protease, and is similar to simian immunodeficiency virus (SIV) protease.
  • SIV simian immunodeficiency virus
  • the results of this experiment are presented in Table 26.
  • the data presented is the mean ⁇ SD of the percent inhibition relative to a no enzyme control reaction.
  • the IC 50 was estimated from the point at which the dose inhibition line crossed the 50% inhibition line.
  • the estimated IC 50 with this HIV-2 protease assay was 10 ⁇ M.
  • This example illustrates the ability of a stable copper(I) complex of this invention, BCDS copper(I) (2:1), to inhibit HIV reverse transcriptase activity.
  • SPA Scintillation Proximity Assay
  • the results of this experiment are presented in Table 27.
  • the data show the mean ⁇ SD of the percent inhibition relative to a no test compound control reaction.
  • the IC 50 is estimated from the point at which the dose inhibition like crosses the 50% inhibition line.
  • the estimated IC 50 was 11 ⁇ M.
  • neocuproine copper(I) (2:1) and neocuproine copper(I) (2:1), to inhibit enzymes involved in intracellular signal transduction.
  • the enzymes tested in this experiment were various protein kinase C isozymes [and protein tyrosine kinases specific for growth factors and cytokines].
  • Protein Kinase C (non-selective) Assay
  • the reaction mixture included 20 mM Tris-HCl, pH 7.4, [32P]-ATP, phosphatidylserine, partially purified PKC from rat brain, and one of the test compounds (Hunnun, et al. "Activation of Protein Kinase C by Triton X-100 Mixed Micelles Containing Diacylglycerol and Phosphatidylserine," J. Biol. Chem.
  • Protein Kinase C ⁇ is one of the major protein kinase
  • Protein kinase C is a family of serine/threonine protein kinases that mediate the actions of a wide variety of growth factor, hormone, and neurotransmitter action.
  • protein Kinase C ⁇ was purified to homogeneity from rat brain using a modification of a the published procedure (3).
  • the purity of the isolated PKC ⁇ was confirmed by SDS/polyacrylamide gel electrophoresis and isoform-specific antibodies.
  • the enzyme was pre-incubated with the test compounds, and its activity is measured by the ability of the enzyme to phosphorylate histone H1 in the absence and presence of calcium, phosphatidylserine, diolein and [32P]ATP. Following a 5 minute incubation, the reaction was terminated by the addition of acetic acid, 50 ul aliquots are removed, spotted on phosphocellulose paper, washed three times in water, dried, and counted to determine phosphorylated product.
  • Table 29 show that the addition of the stable copper(I) complexes inhibit the activity of Protein Kinase C ⁇ .
  • Neocuproine Copper(I) (2:1) 100 87.5 ⁇ 1.8
  • Protein Kinase C ⁇ is another major protein kinase C isoforms. Protein kinase C is a family of serine/threonine protein kinases that mediate the actions of a wide variety of growth factor, hormone, and neurotransmitter action.
  • Protein Kinase C ⁇ (which includes ⁇ I and ⁇ II forms) was purified to homogeneity from rat brain using a modification of a published protocol
  • the purity of the isolated PKC ⁇ was confirmed by SDS/polyacrylamide gel electrophoresis and isoform-specific antibodies.
  • the enzyme was pre-incubated with test compounds, and its activity is measured by the ability of the enzyme to phosphorylate histone H1 in the absence and presence of calcium, phosphatidylserine, diolein and [32P]ATP. Following a 5 minute incubation, the reaction was terminated by the addition of acetic acid, 50 ul aliquots are removed, spotted on phosphocellulose paper, washed three times in water, dried, and counted to determine phosphorylated product.
  • Neocuproine Copper(I) (2:1) 100 84.5 ⁇ 1.9
  • Protein Kinase C ⁇ is another major protein kinase C isoform. Protein kinase C is a family of serine/threonine protein kinases that mediate the actions of a wide variety of growth factor, hormone, and neurotransmitter action.
  • Protein Kinase C ⁇ was purified from insect cells expressing a baculovirus recombinant rabbit brain protein kinase C ⁇ isoform.
  • the enzyme was pre-incubated with the test compounds, and its activity was measured by the ability of the enzyme to phosphorylate histone HI in the absence and presence of calcium, phosphatidylserine, diolein and [32P]ATP.
  • the reaction was terminated by the addition of acetic acid, 50 ul aliquots were removed, spotted on phosphoeellulose paper, washed three times in water, dried, and counted to determine phosphorylated product.
  • Neocuproine Copper(I) (2:1) 100 97
  • Stable Copper(I) Complexes This example illustrates the ability of the representative stable copper(I) complexes, BCDS copper(I) (2:1) and neocuproine copper(I) (2:1), to inhibit enzymes involved in intracellular signal transduction.
  • the enzymes tested in this experiment were protein tyrosine kinases specific for growth factors and cytokines.
  • EGF Epidermal Growth Factor
  • EGF Endothelial growth Factor ⁇
  • TGF- ⁇ Transforming Growth Factor ⁇
  • TGF- ⁇ Transforming Growth Factor ⁇
  • This kinase phosphorylates several cytosolic proteins which lead to induction of intracellular signaling pathways eventually leading to cell mitogenesis and in some cases cellular transformation. Inhibition of the EGF tyrosine kinase is useful for chemotherapy for malignant cells.
  • the kinase assay measures the activity of the 69kD kinase domain by employing an immobilized synthetic polypeptide as a substrate. Following a 10 minute reaction, phosphorylated tyrosine residues were detected by incubation with a monoclonal anti-phosphotyrosine antibody. Bound anti-phosphotyrosine antibody was quantitated by incubation with a biotin-linked anti-mouse IgG, followed by streptavidin linked ⁇ -galactosidase enzyme. Fluorescence resulting from conversion of fluoroscein-di- ⁇ -galactoside to fluorescein was measured. The results of this experiment are presented in Table 33.
  • the lck tyrosine kinase is a member of the sre family of cytoplasmic tyrosine kinases. It is expressed only in T-lymphocytes and NK cells.
  • the p56 lck Tyrosine Kinase is a 56 kD protein that is found associated with the cytoplasmic side of the plasma membrane of these cells. It is responsible of transmission of the IL-2 signal leading to T-lymphocyte activation. The binding of IL-2 to specific IL-2 receptors leads to activation of the p56 tyrosine kinase.
  • the p56 lck Tyrosine Kinase has been found to function in signal transduction for antigen activated CD4 and CD8 T-cell receptors.
  • the p56 l ck Tyrosine Kinase was purified from bovine thymus.
  • the kinase assay measures the activity of the 69kD kinase domain by employing an immobilized synthetic polypeptide as a substrate.
  • the test compounds were pre-incubated with the enzyme of 15 minutes. Following a 10 minute reaction with 100 uM ATP, phosphorylated tyrosine residues are detected by incubation with a monoclonal anti-phosphotyrosine antibody. Bound anti-phosphotyrosine antibody was quantitated by incubation with a biotin-linked anti-mouse IgG, followed by streptavidin linked ⁇ -galactosidase enzyme.
  • Fluorescence resulting from conversion of fluoroscein-di- ⁇ -galactoside to fluorescein was measured (Hatekeyama et al., "Interaction of the IL-2 Receptor with the src-Family Kinase p56 lck : Identification of Novel Intermolecular Association," Science 252:1523-1528, 1991; Caron et al., "Structural Requirements for Enhancement of T-cell Responsiveness by the Lymphocyte Specific Tyrosine Protein Kinase p56 lck ,” Mol. Cell Biol.
  • the fyn tyrosine kinase is also a member of the src family of non-receptor linked cytoplasmic tyrosine kinases.
  • the p59 fyn Tyrosine Kinase is responsible for mediating signal transduction through the T-cell receptor
  • TCR TCR
  • This receptor is responsible for a signal cascade leading to lymphokine secretion and cell proliferation.
  • the p59 fyn Tyrosine Kinase is also one of several kinases associated with the B-cell receptor.
  • the p59 fyn Tyrosine Kinase was purified from bovine thymus.
  • the kinase assay measures the activity of the 69kD kinase domain by employing an immobilized synthetic polypeptide as a substrate.
  • the test compounds are preincubated with the enzyme of 15 minutes.
  • phosphorylated tyrosine residues are detected by incubation with a monoclonal anti-phosphotyrosine antibody.
  • Bound anti-phosphotyrosine antibody is quantitated by incubation with a biotin-linked anti-mouse IgG, followed by streptavidin linked ⁇ -galactosidase enzyme.
  • Fluorescence resulting from conversion of fluoroscein-di- ⁇ -galactoside to fluorescein is measured (Cooke et al., "Regulation of T-cell Receptor Signaling by a src Family Protein Tyrosine Kinase p59 fyn ,” Cell 65:281-291, 1991; Grassman et al, "Protein Tyrosine Kinase p59 fyn is Associated with the T-cell Receptor CD3 Complex in Functional Human Lymphocytes," Eur. J. Immunol.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Veterinary Medicine (AREA)
  • Chemical & Material Sciences (AREA)
  • Public Health (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Animal Behavior & Ethology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Organic Chemistry (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • General Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Virology (AREA)
  • Hematology (AREA)
  • Oncology (AREA)
  • Diabetes (AREA)
  • Epidemiology (AREA)
  • Obesity (AREA)
  • Communicable Diseases (AREA)
  • Nutrition Science (AREA)
  • Pain & Pain Management (AREA)
  • Rheumatology (AREA)
  • Molecular Biology (AREA)
  • Dermatology (AREA)
  • Biotechnology (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Plural Heterocyclic Compounds (AREA)
  • Nitrogen Condensed Heterocyclic Rings (AREA)
  • Heterocyclic Carbon Compounds Containing A Hetero Ring Having Oxygen Or Sulfur (AREA)
  • Nitrogen And Oxygen Or Sulfur-Condensed Heterocyclic Ring Systems (AREA)
  • Acyclic And Carbocyclic Compounds In Medicinal Compositions (AREA)

Abstract

L'invention concerne des composés stables de cuivre(I) et des procédés affins. Les composés stables de cuivre (I) comprennent un ion de cuivre(I) associé à un ligand polydenté qui favorise l'état de d'oxydation +1 du cuivre. Les procédés décrits comprennent l'utilisation des composés stables de cuivre(I) comme agents de guérison de blessures, agents antioxydants, agents anti-inflammatoires, modulateurs de lipides, modulateurs de la transduction de signaux, comme agents stimulant la pousse des cheveux et comme agents antiviraux. Des exemples de composés stables de cuivre(I) comprennent la néocupréine de cuivre(I) et l'acide disulfonique de bathocuproïne de cuivre(I).
PCT/US1994/006247 1993-06-02 1994-06-02 Complexes stables de cuivre(i) et leur utilisation comme substances therapeutiques actives WO1994027594A2 (fr)

Priority Applications (4)

Application Number Priority Date Filing Date Title
EP94919342A EP0701439A1 (fr) 1993-06-02 1994-06-02 Complexes stables de cuivre(i) et leur utilisation comme substances therapeutiques actives
JP7501073A JPH08511006A (ja) 1993-06-02 1994-06-02 安定な銅（▲ｉ▼）錯体及びそれに関する方法
AU70517/94A AU7051794A (en) 1993-06-02 1994-06-02 Stable copper(1) complexes and methods related thereto
ZA949336A ZA949336B (en) 1994-06-02 1994-11-24 Stable copper (I) complexes and methods related thereto.

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US7144093A 1993-06-02 1993-06-02
US08/071,440 1993-06-02

Publications (2)

Publication Number Publication Date
WO1994027594A2 true WO1994027594A2 (fr) 1994-12-08
WO1994027594A3 WO1994027594A3 (fr) 1995-04-27

Family

ID=22101339

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US1994/006247 WO1994027594A2 (fr) 1993-06-02 1994-06-02 Complexes stables de cuivre(i) et leur utilisation comme substances therapeutiques actives

Country Status (8)

Country Link
EP (1) EP0701439A1 (fr)
JP (1) JPH08511006A (fr)
AU (1) AU7051794A (fr)
CA (1) CA2163640A1 (fr)
OA (1) OA10198A (fr)
TW (1) TW239077B (fr)
WO (1) WO1994027594A2 (fr)
ZA (1) ZA943857B (fr)

Cited By (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996039144A1 (fr) * 1995-06-06 1996-12-12 Procyte Corporation Complexes cuivreux stables utilises comme substances a activite therapeutique
WO1997001559A1 (fr) * 1995-06-29 1997-01-16 Procyte Corporation Complexes de zinc(ii) et procedes relatifs a leur utilisation
US6150379A (en) * 1997-11-26 2000-11-21 Axys Pharmaceuticals, Inc. Compounds and compositions as anticoagulants
US6562854B2 (en) 1994-12-14 2003-05-13 Axys Pharmaceuticals, Inc. Compositions comprising a substituted benzimidazole useful for treating immunomediated inflammatory disorders
US6638711B1 (en) 1999-04-29 2003-10-28 The General Hospital Corporation Methods for identifying an agent that inhibits oxygen-dependent hydrogen peroxide formation activity but does not inhibit superoxide-dependent hydrogen peroxide formation
US7045531B1 (en) 1997-03-11 2006-05-16 The General Hospital Corporation Composition comprising a metal chelator and a method of treating amyloidosis by administering the metal chelator
US20140364406A1 (en) * 2004-05-24 2014-12-11 Geoffrey C. GURTNER Method Of Treating Or Preventing Pathologic Effects Of Acute Increases In Hyperglycemia And/Or Acute Increases Of Free Fatty Acid Flux
US9107811B2 (en) 2005-06-20 2015-08-18 Sci-Chem International Pty. Ltd. Composition for treating skin lesions
US10751304B2 (en) 2008-10-10 2020-08-25 The Board Of Trustees Of The Leland Stanford Junior University Topical and transdermal delivery of HIF-1 modulators to prevent and treat chronic wounds
US11331288B2 (en) 2017-09-14 2022-05-17 The Board Of Trustees Of The Leland Stanford Junior University Conditioning irradiated tissue for increasing vascularity
US12303475B2 (en) 2022-04-14 2025-05-20 The Board Of Trustees Of The Leland Stanford Junior University Conditioning irradiated tissue for increasing vascularity

Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CA2933788C (fr) * 2008-09-03 2017-11-28 Nbc Meshtec, Inc. Agent antiviral
TWI580787B (zh) * 2012-12-28 2017-05-01 簡宏堅 重組蛋白質、含有該重組蛋白質之醫藥組成物及該重組蛋白質之製備方法
WO2019236596A1 (fr) * 2018-06-04 2019-12-12 Chemistryrx. Compositions topiques pour stimuler la pousse des cheveux

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4760051A (en) * 1985-01-24 1988-07-26 Pickart Loren R Use of GHL-Cu as a wound-healing and anti-inflammatory agent
JPS6259213A (ja) * 1985-09-10 1987-03-14 Eisai Co Ltd ス−パ−オキサイド除去剤

Cited By (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6562854B2 (en) 1994-12-14 2003-05-13 Axys Pharmaceuticals, Inc. Compositions comprising a substituted benzimidazole useful for treating immunomediated inflammatory disorders
WO1996039144A1 (fr) * 1995-06-06 1996-12-12 Procyte Corporation Complexes cuivreux stables utilises comme substances a activite therapeutique
WO1997001559A1 (fr) * 1995-06-29 1997-01-16 Procyte Corporation Complexes de zinc(ii) et procedes relatifs a leur utilisation
US7045531B1 (en) 1997-03-11 2006-05-16 The General Hospital Corporation Composition comprising a metal chelator and a method of treating amyloidosis by administering the metal chelator
US6150379A (en) * 1997-11-26 2000-11-21 Axys Pharmaceuticals, Inc. Compounds and compositions as anticoagulants
US6638711B1 (en) 1999-04-29 2003-10-28 The General Hospital Corporation Methods for identifying an agent that inhibits oxygen-dependent hydrogen peroxide formation activity but does not inhibit superoxide-dependent hydrogen peroxide formation
US20140364406A1 (en) * 2004-05-24 2014-12-11 Geoffrey C. GURTNER Method Of Treating Or Preventing Pathologic Effects Of Acute Increases In Hyperglycemia And/Or Acute Increases Of Free Fatty Acid Flux
US9107811B2 (en) 2005-06-20 2015-08-18 Sci-Chem International Pty. Ltd. Composition for treating skin lesions
US10751304B2 (en) 2008-10-10 2020-08-25 The Board Of Trustees Of The Leland Stanford Junior University Topical and transdermal delivery of HIF-1 modulators to prevent and treat chronic wounds
US11160775B2 (en) 2008-10-10 2021-11-02 The Board Of Trustees Of The Leland Stanford Junior University Topical and transdermal delivery of HIF-1 modulators to prevent and treat chronic wounds
US11331288B2 (en) 2017-09-14 2022-05-17 The Board Of Trustees Of The Leland Stanford Junior University Conditioning irradiated tissue for increasing vascularity
US12303475B2 (en) 2022-04-14 2025-05-20 The Board Of Trustees Of The Leland Stanford Junior University Conditioning irradiated tissue for increasing vascularity

Also Published As

Publication number Publication date
TW239077B (fr) 1995-01-21
CA2163640A1 (fr) 1994-12-08
JPH08511006A (ja) 1996-11-19
ZA943857B (en) 1995-02-01
EP0701439A1 (fr) 1996-03-20
AU7051794A (en) 1994-12-20
OA10198A (en) 1996-12-18
WO1994027594A3 (fr) 1995-04-27

Similar Documents

Publication Publication Date Title
Kimura et al. Increases in cyclic GMP levels in brain and liver with sodium azide an activator of guanylate cyclase
EP1088098B2 (fr) Analyse de potentiel biochimique et methodes pour réduire la résistance de vih dans la therapie
Lugnier et al. Selective inhibition of cyclic nucleotide phosphodiesterases of human, bovine and rat aorta
WO1994027594A2 (fr) Complexes stables de cuivre(i) et leur utilisation comme substances therapeutiques actives
Rosser et al. Calpain activity increases in hepatocytes following addition of ATP. Demonstration by a novel fluorescent approach.
JP2009242438A (ja) ノルジヒドログアヤレティク酸誘導体を使用して腫瘍を処置するための方法
CA2222224A1 (fr) Analogues de nucleotides utiles pour le traitement topique de maladies proliferatives de la peau
Hall et al. Anti-inflammatory agents III: Structure–activity relationships of brusatol and related quassinoids
WO1996039144A1 (fr) Complexes cuivreux stables utilises comme substances a activite therapeutique
CA2042569A1 (fr) Composition renfermant un facteur de croissance fibroblaste stabilise
Al-Jafari et al. Kinetics of human erythrocyte acetylcholinesterase inhibition by a novel derivative of physostigmine: Phenserine
EP1231916A2 (fr) Utilisation de derives d'indirubine pour la fabrication de medicaments
US5637311A (en) Zinc(II) complexes and methods related thereto
Diebold et al. Influences of lovastatin administration on the respiratory burst of leukocytes and the phosphorylation potential of mitochondria in guinea pigs
US6008188A (en) Cytokine potentiator and pharmaceutical formulation for cytokine administration
Van Wijk et al. Synthesis and antiviral activity of 3′-azido-3′-deoxythymidine triphosphate distearoylglycerol: a novel phospholipid conjugate of the anti-HIV agent AZT
Van Gelder et al. Inhibition of intestinal metabolism of the antiviral ester prodrug bis (POC)-PMPA by nature-identical fruit extracts as a strategy to enhance its oral absorption: an in vitro study
Baker et al. Synthesis and evaluation of a series of 2'-O-acyl derivatives of 9-. beta.-D-arabinofuranosyladenine as antiherpes agents
Knowles et al. Isolation and characterization of plasma membranes from transplantable human astrocytoma, oat cell carcinoma, and melanomas
EP0316117A1 (fr) Composés antiviraux, compositions contenant de tels composés et méthodes de traitement
Miyagi et al. Multiple forms of mammalian sialidase: altered expression in carcinogenesis
Eames et al. Orientation, accessibility, and mobility of equilenin bound to the active site of steroid isomerase
Satoh et al. Inhibition of Na+, K+-ATPase by the extract of Stephania cephararantha Hayata and bisbenzylisoquinoline alkaloid cycleanine, a major constituent
WicK et al. Antitumor effects of L-glutamic acid dihydroxyanilides against experimental melanoma
ADACHI et al. The effects of 1, 2, 3, 4, 6-penta-O-galloyl-β-D-glucose on rat liver mitochondrial respiration

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A2

Designated state(s): AU BB BG BR BY CA CN CZ FI GE HU JP KG KP KR KZ LK LV MD MG MN MW NO NZ PL RO RU SD SI SK TJ TT UA UZ VN

AL Designated countries for regional patents

Kind code of ref document: A2

Designated state(s): AT BE CH DE DK ES FR GB GR IE IT LU MC NL PT SE BF BJ CF CG CI CM GA GN ML MR NE SN TD TG

DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
121 Ep: the epo has been informed by wipo that ep was designated in this application
AK Designated states

Kind code of ref document: A3

Designated state(s): AU BB BG BR BY CA CN CZ FI GE HU JP KG KP KR KZ LK LV MD MG MN MW NO NZ PL RO RU SD SI SK TJ TT UA UZ VN

AL Designated countries for regional patents

Kind code of ref document: A3

Designated state(s): AT BE CH DE DK ES FR GB GR IE IT LU MC NL PT SE BF BJ CF CG CI CM GA GN ML MR NE SN TD TG

WWE Wipo information: entry into national phase

Ref document number: 2163640

Country of ref document: CA

WWE Wipo information: entry into national phase

Ref document number: 267860

Country of ref document: NZ

WWE Wipo information: entry into national phase

Ref document number: 1994919342

Country of ref document: EP

WWP Wipo information: published in national office

Ref document number: 1994919342

Country of ref document: EP

WWW Wipo information: withdrawn in national office

Ref document number: 1994919342

Country of ref document: EP

点击 这是indexloc提供的php浏览器服务，不要输入任何密码和下载