WO1997021431A1 - Cobalt schiff base compounds - Google Patents
Cobalt schiff base compounds Download PDFInfo
- Publication number
- WO1997021431A1 WO1997021431A1 PCT/US1996/019900 US9619900W WO9721431A1 WO 1997021431 A1 WO1997021431 A1 WO 1997021431A1 US 9619900 W US9619900 W US 9619900W WO 9721431 A1 WO9721431 A1 WO 9721431A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- alkyl
- alcohol
- amine
- cobalt
- compound
- Prior art date
Links
- 229910017052 cobalt Inorganic materials 0.000 title abstract description 28
- 239000010941 cobalt Substances 0.000 title abstract description 28
- -1 Cobalt schiff base compounds Chemical class 0.000 title description 30
- 239000002262 Schiff base Substances 0.000 title description 2
- 150000001869 cobalt compounds Chemical class 0.000 claims abstract description 123
- 150000001875 compounds Chemical class 0.000 claims abstract description 77
- 102000004190 Enzymes Human genes 0.000 claims abstract description 74
- 108090000790 Enzymes Proteins 0.000 claims abstract description 74
- 108090000765 processed proteins & peptides Proteins 0.000 claims abstract description 65
- 230000008685 targeting Effects 0.000 claims abstract description 54
- 102000004196 processed proteins & peptides Human genes 0.000 claims abstract description 42
- 150000007523 nucleic acids Chemical class 0.000 claims abstract description 40
- 108020004707 nucleic acids Proteins 0.000 claims abstract description 39
- 102000039446 nucleic acids Human genes 0.000 claims abstract description 39
- 229920001184 polypeptide Polymers 0.000 claims abstract description 35
- 101710185494 Zinc finger protein Proteins 0.000 claims abstract description 17
- 102100023597 Zinc finger protein 816 Human genes 0.000 claims abstract description 17
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 14
- 108090000623 proteins and genes Proteins 0.000 claims description 91
- 102000004169 proteins and genes Human genes 0.000 claims description 90
- 239000003446 ligand Substances 0.000 claims description 79
- XLJKHNWPARRRJB-UHFFFAOYSA-N cobalt(2+) Chemical compound [Co+2] XLJKHNWPARRRJB-UHFFFAOYSA-N 0.000 claims description 47
- 125000000217 alkyl group Chemical group 0.000 claims description 45
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 36
- 239000001257 hydrogen Substances 0.000 claims description 32
- 229910052739 hydrogen Inorganic materials 0.000 claims description 32
- 150000002431 hydrogen Chemical class 0.000 claims description 29
- 150000001412 amines Chemical class 0.000 claims description 26
- 238000000034 method Methods 0.000 claims description 24
- 125000005233 alkylalcohol group Chemical group 0.000 claims description 23
- 230000002209 hydrophobic effect Effects 0.000 claims description 22
- 125000003118 aryl group Chemical group 0.000 claims description 20
- 150000007524 organic acids Chemical class 0.000 claims description 15
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 claims description 11
- 239000000203 mixture Substances 0.000 claims description 11
- 239000007864 aqueous solution Substances 0.000 claims description 9
- 230000002401 inhibitory effect Effects 0.000 claims description 8
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 claims description 3
- 150000003973 alkyl amines Chemical class 0.000 claims 16
- 229940088598 enzyme Drugs 0.000 abstract description 72
- GUTLYIVDDKVIGB-UHFFFAOYSA-N cobalt atom Chemical compound [Co] GUTLYIVDDKVIGB-UHFFFAOYSA-N 0.000 abstract description 28
- 229960004072 thrombin Drugs 0.000 abstract description 27
- 108090000190 Thrombin Proteins 0.000 abstract description 26
- 125000004429 atom Chemical group 0.000 abstract description 8
- 125000004433 nitrogen atom Chemical group N* 0.000 abstract description 7
- 125000004430 oxygen atom Chemical group O* 0.000 abstract description 2
- 235000018102 proteins Nutrition 0.000 description 70
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 42
- 239000003112 inhibitor Substances 0.000 description 41
- 235000014304 histidine Nutrition 0.000 description 39
- 230000027455 binding Effects 0.000 description 38
- 239000000243 solution Substances 0.000 description 38
- 230000005764 inhibitory process Effects 0.000 description 35
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- 229960002885 histidine Drugs 0.000 description 32
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 31
- 235000013495 cobalt Nutrition 0.000 description 27
- RAXXELZNTBOGNW-UHFFFAOYSA-N imidazole Natural products C1=CNC=N1 RAXXELZNTBOGNW-UHFFFAOYSA-N 0.000 description 27
- 229910000069 nitrogen hydride Inorganic materials 0.000 description 25
- ZMANZCXQSJIPKH-UHFFFAOYSA-N Triethylamine Chemical compound CCN(CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-N 0.000 description 23
- HNDVDQJCIGZPNO-YFKPBYRVSA-N L-histidine Chemical compound OC(=O)[C@@H](N)CC1=CN=CN1 HNDVDQJCIGZPNO-YFKPBYRVSA-N 0.000 description 21
- 230000000694 effects Effects 0.000 description 20
- 125000003729 nucleotide group Chemical group 0.000 description 20
- HCHKCACWOHOZIP-UHFFFAOYSA-N Zinc Chemical compound [Zn] HCHKCACWOHOZIP-UHFFFAOYSA-N 0.000 description 19
- 150000001413 amino acids Chemical class 0.000 description 19
- 239000000460 chlorine Substances 0.000 description 19
- 229910052725 zinc Inorganic materials 0.000 description 19
- 239000011701 zinc Substances 0.000 description 19
- 239000002773 nucleotide Substances 0.000 description 17
- 239000002904 solvent Substances 0.000 description 17
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 17
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 16
- 125000003277 amino group Chemical group 0.000 description 16
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- 125000000487 histidyl group Chemical group [H]N([H])C(C(=O)O*)C([H])([H])C1=C([H])N([H])C([H])=N1 0.000 description 15
- 108090001109 Thermolysin Proteins 0.000 description 14
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- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 13
- 238000011534 incubation Methods 0.000 description 13
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 12
- 230000015572 biosynthetic process Effects 0.000 description 12
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- 239000000047 product Substances 0.000 description 12
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- 238000006243 chemical reaction Methods 0.000 description 11
- 239000002184 metal Substances 0.000 description 11
- 229910052751 metal Inorganic materials 0.000 description 11
- 229910052757 nitrogen Inorganic materials 0.000 description 11
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- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 10
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- 150000004700 cobalt complex Chemical class 0.000 description 10
- 238000005859 coupling reaction Methods 0.000 description 10
- JHJLBTNAGRQEKS-UHFFFAOYSA-M sodium bromide Chemical compound [Na+].[Br-] JHJLBTNAGRQEKS-UHFFFAOYSA-M 0.000 description 10
- 239000004475 Arginine Substances 0.000 description 9
- ODKSFYDXXFIFQN-BYPYZUCNSA-P L-argininium(2+) Chemical compound NC(=[NH2+])NCCC[C@H]([NH3+])C(O)=O ODKSFYDXXFIFQN-BYPYZUCNSA-P 0.000 description 9
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- 125000005647 linker group Chemical group 0.000 description 8
- 230000007246 mechanism Effects 0.000 description 8
- 125000001997 phenyl group Chemical group [H]C1=C([H])C([H])=C(*)C([H])=C1[H] 0.000 description 8
- 239000011780 sodium chloride Substances 0.000 description 8
- 102000035195 Peptidases Human genes 0.000 description 7
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- YRKCREAYFQTBPV-UHFFFAOYSA-N acetylacetone Chemical class CC(=O)CC(C)=O YRKCREAYFQTBPV-UHFFFAOYSA-N 0.000 description 7
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- 235000018417 cysteine Nutrition 0.000 description 7
- 230000002255 enzymatic effect Effects 0.000 description 7
- 150000002411 histidines Chemical class 0.000 description 7
- 230000007062 hydrolysis Effects 0.000 description 7
- 238000006460 hydrolysis reaction Methods 0.000 description 7
- 230000002779 inactivation Effects 0.000 description 7
- 238000000746 purification Methods 0.000 description 7
- 230000009467 reduction Effects 0.000 description 7
- JKMHFZQWWAIEOD-UHFFFAOYSA-N 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid Chemical compound OCC[NH+]1CCN(CCS([O-])(=O)=O)CC1 JKMHFZQWWAIEOD-UHFFFAOYSA-N 0.000 description 6
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 6
- 206010010904 Convulsion Diseases 0.000 description 6
- PIICEJLVQHRZGT-UHFFFAOYSA-N Ethylenediamine Chemical compound NCCN PIICEJLVQHRZGT-UHFFFAOYSA-N 0.000 description 6
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- 238000002835 absorbance Methods 0.000 description 6
- CUJRVFIICFDLGR-UHFFFAOYSA-N acetylacetonate Chemical compound CC(=O)[CH-]C(C)=O CUJRVFIICFDLGR-UHFFFAOYSA-N 0.000 description 6
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- 150000001868 cobalt Chemical class 0.000 description 6
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
-
- 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
- A61P31/12—Antivirals
-
- 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
- C07F15/00—Compounds containing elements of Groups 8, 9, 10 or 18 of the Periodic Table
- C07F15/06—Cobalt compounds
- C07F15/065—Cobalt compounds without a metal-carbon linkage
Definitions
- the invention relates to cobalt compounds, and methods of using such compounds to reduce the biological activity of proteins.
- compositions comprising water soluble tetradentate Schiffs base complexes of Co(II). Further provided are compounds having the structure comprising Formula
- Co is either Co(II) or Co(III), and each of R,. R 2 , R 3 , R 4 , R 5 . R ⁇ ,, R 7 and R 8 is a hydrogen, an alkyl group, an aryl group, a hydrophobic organic acid, an alkyl alcohol, an alcohol, an alkyl amine group, an amine group, or a targeting moiety.
- R, R ; , R 3 and R 4 are each hydrogen, alkyl, or aryl.
- protein-cobalt compound complexes comprising a protein attached to the cobalt compound of Formula 1.
- Figure 1 depicts the inhibition of tlirombin.
- 3.07 X IO "9 M thrombin at 25 C was assayed using Spectrozyme TH (American Diagnostics), and the reaction followed at 406 nm using a Hewlett Packard HP8452A diode array spectrophotometer with temperature control. All assays were performed in 10 mM Tris. 10 mM HEPES, 0.1% polyethylene glycol, 0.5 M NaCl, pH 7.8.
- Co(III) carboxypropyl(NH 3 ) 2 (labeled as Co(carboxypropyl)) was coupled to the active site directed peptide NH 3 -GGGdFPR-CO-NH 2 (labeled as peptide dFPR).
- the observed inhibition greatly exceeded Co(carboxypropyl)(NH 3 ) 2 , the peptide.
- Co(III)acacen(NH 3 ) 2 Cl (labeled as Co(acacen)).
- Figure 2 depicts the structure of the Co(III)(acacen-GGGFPR)(NH 3 ) 2 .
- Figures 3 A and 3B shows the temperature dependence of the enzyme inhibition rate correlates to the ligand exchange rate.
- A temperature dependence (see Example 3 )
- B ligand exchange rate (see Example 3).
- Figure 4 shows the inhibition of thrombin by [ColII(acacen)(NH 3 ) 2 ]Cl.
- Thrombin was incubated for 12 hours at room temperature with (A) 0 M Co(III) and B) 2.5 mM Co(III).
- Spectra were taken every 30 seconds for 30 minutes to monitor the release of p-nitroaniline by enzymatic hydrolysis of a commercial substrate.
- Figure 5 shows that inhibition of tlirombin is dependent on the concentration of the inhibitor, the length of incubation, and the temperature of incubation.
- Figures 6A, 6B, 6C, 6D, 6E, 6F, 6G, 6H, and 61 depict the structures of cobalt compounds of the invention that have been made. Unless noted, all of the compounds are Co(III).
- the present invention is directed to cobalt compounds that can exchange or bind functional moieties such as histidine on a protein's surface resulting in the inactivation of a biological activity of the protein due to the complexing of the functional moiety to the cobalt compound.
- Co(II) also depicted herein as Co+2
- Co(III) also depicted herein as Co+3
- Co(II) compounds have up to four coordination atoms, and may contain a first axial ligand, although it is possible that water molecules may be weakly associated in one or both axial ligand positions.
- Co(III) compounds have up to six coordination atoms, of which two are defined herein as axial ligand positions.
- axial ligand herein is meant a ligand L, or L2 located at either the fifth or sixth coordination sites, generally depicted in the structure below:
- the cobalt compounds of the invention derive their biological activity by the substitution or addition of ligands in the axial positions.
- the biological activity of the cobalt compounds of the invention results from the binding of a new axial ligand, most preferably the nitrogen atom of imidazole of the side chain of histidine.
- the amino acid serving as a new axial ligand of the cobalt compound is required by the target protein for its biological activity.
- proteins such as enzymes that utilize a histidine in the active site, or proteins that use histidine, for example, to bind essential metal ions, can be inactivated by the binding of the histidine in an axial ligand position of the cobalt compound, thus preventing the histidine from participating in its normal biological function.
- the Co(III) complex is synthesized or formulated with two particular axial ligands, and then when the complex is added to a protein, for example, the original axial ligand or ligands are replaced by one or more ligands from a protein. This will occur either when the affinity of the protein axial ligand is higher for the cobalt compound as compared to the original axial ligand, or when the new axial ligand is present in elevated concentrations such that the equilibrium of axial ligand binding favors the binding of the new axial ligand from the protein.
- Co(III) complexes are made with axial ligands that can be substituted with other ligands.
- Co(II) compounds of the invention are preferably synthesized with no axial ligands.
- certain moieties such as the nitrogen atom of the imidazole of the side chain of histidine, within the protein can become an axial ligand.
- a tightly-bound protein-cobalt compound complex This occurs when the Co(II) compound, with its four coordinating atoms from the Schiff s base, binds an imidazole moiety, for example, and is oxidized to a Co(III) compound.
- this may be considered a redox reaction, since the Co(II) compound is oxidized to a Co(III) compound upon binding to the protein.
- the imidazole axial ligand serves as a fifth coordinating atom, and is tightly bound.
- the nitrogen atom of an imidazole side chain of the amino acid residue histidine, contained within a target protein, is the new axial ligand. While the examples and disclosure herein particularly describe this hisitidine embodiment, any "cobalt-reactive amino acid” may serve as the new axial ligand.
- a "cobalt-reactive amino acid” is one which is capable of binding to the cobalt compounds of the invention as an axial ligand.
- nitrogent of the imidazole side chain of histidine is particularly preferred
- alternative embodiments utilize the nitrogen atom of the aromatic indole side chain of tryptophan, the sulfur atoms of the side chains of cysteine and methionine, the amino groups of arginine, lysine. asparagine or glutamine as the moieties which may become axial ligands as outlined above.
- the availability of these moieties may depend on the pH of the solution containing the protein or enzyme, since in the protonated state many of these moieties are not good electron donors suitable as axial ligands.
- the present invention provides cobalt compounds that may be complexed with a protein containing a suitable new axial ligand.
- the present invention provides water-soluble tetradentate
- the Schiffs base compound which is a ligand for the Co(II)
- the Schiffs base compound has four coordinating atoms. In a preferred embodiment, there are two nitrogen atoms and two oxygen atoms which serve as the coordinating atoms.
- the term “Schif s base” herein is meant a substituted imine. The substituent groups are outlined below. Schiffs bases are generally the condensation products of amines and aliphatic aldehydes forming azomethines substituted on the nitrogen atom.
- cobalt compound herein is meant a tetradentate Schiffs base with a bound cobalt atom.
- the Schiffs base may be substituted or unsubstituted, and the cobalt may be Co(II) or Co(III).
- the cobalt compounds have the structure depicted in Formula 1 :
- Co is either Co(II) or Co(III).
- R,, R 2 , R 3 , R , R R 6 , R 7 and R s is a hydrogen, an alkyl group, an aryl group, a hydrophobic organic acid, an alkyl alcohol, an alcohol, an alkyl amine group, an amine group, or a targeting moiety.
- Co is Co(II)
- at least one of R, through R 8 is hydrophilic such that the compound is soluble in aqueous solution.
- Co is Co(III).
- at least one of R, through R g is a targeting moiety.
- alkyl or “alkyl group” or grammatical equivalents herein is meant a straight or branched chain alkyl group, with straight chain alkyl groups being preferred. If branched, it may be branched at one or more positions, and unless specified, at any position. Also included within the definition of an l group are cycloalkyl groups such as C5 and C6 rings. In some cases, two R groups may be part of a ring structure, that is, they may be linked to form a cyclic structure.
- the alkyl group may range from about 1 to 20 carbon atoms (Cl - C20). with a preferred embodiment utilizing from about 1 to about 10 carbon atoms (C 1 -
- the alkyl group may be larger, particularly if it is a straight chain alkyl. Particularly preferred is methyl.
- aryl or "aryl group” herein is meant aromatic rings including phenyl, benzyl, and naphthyl, heterocyclic aromatic rings such as pyridine, furan, thiophene, pyrrole, indole and purine, and heterocyclic rings with nitrogen, oxygen, sulfur or phosphorus.
- the alkyl and aryl groups may be substituted, for example, a phenyl group may be a substituted phenyl group.
- Suitable substitution groups include, but arc not limited to. alkyl and aryl groups, halogens such as chlorine, bromine and fluorine, amines. carboxylic acids, and nitro groups.
- hydrophilic organic acid or grammatical equivalents herein is meant an alkyl group containing one or more carboxyl groups, -COOH. i.e. a carboxylic acid.
- the alkyl group may be substituted or unsubstituted.
- Cl - C20 alkyl groups may be used with at least one carboxyl group attached to any one of the alkyl carbons, with C l - C5 being preferred.
- the carboxyl group is attached to the terminal carbon of the alkyl group.
- Other preferred hydrophilic organic acids include phosphonates and sulfonates.
- a preferred hydrophilic organic acid is propionic acid.
- R groups are a hydrophobic organic acid, since, in the case of Co(III), this may result in a compound that is neutrally charged, and thus may cross the blood-brain barrier.
- Particularly preferred is the structure depicted in Formula 2:
- the length of the alkyl group shown in Formula 2 may be altered, either to encourage or prevent the carboxylic acid from "swinging around" to become an axial ligand.
- amine herein is meant an -NRR' group.
- R and R' may be the same or different, and may be hydrogen, alkyl or aryl.
- a preferred -NRR'group is -NH 2 .
- alkyl amine group herein is meant an alkyl group, as defined above, with a -NRR' group, as defined above. As defined above, the alkyl group may be substituted or unsubstituted.
- Preferred alkyl amine groups are n- propylamine and n-butylamine.
- alkyl alcohol herein is meant an alkyl group w r ith an -OH group. As defined above, the alkyl group may be substituted or unsubstituted.
- the alkyl alcohol may be primary, secondary or tertiary, depending on the alkyl group.
- the alkyl alcohol is a straight chain primary alkyl alcohol, generally containing at least 3 carbon atoms.
- Preferred alkyl alcohols include, but are not limited to, n-propyl alcohol, n-butyl alcohol, n- pentyl alcohol, n-heptyl alcohol, or n-octyl alcohol.
- alcohol herein is meant an -OH group.
- At least one of R,-R a is a targeting moiety. It is preferred that only one of the R groups be a targeting moiety. In an alternative embodiment, more than one of the R groups may be a targeting moiety.
- the Co of Formula 1 is Co(III)
- at least one of R,, R 2 , R 3 , R 4 , R 5 , R 6 , R 7 and R 8 is a targeting moiety.
- targeting moiety herein is meant a functional group that will specifically interact with the target protein, and thus is used to target the cobalt compound to a particular target protein. That is, the cobalt compound is covalently linked to a targeting moiety that will specifically bind or associate with a target protein.
- the cobalt compounds of the invention may include a polypeptide inhibitor that is known to inhibit a protease, thus effectively increasing the local concentration of the cobalt compound at a functional site on the target protein.
- Suitable targeting moieties include, but are not limited to, polypeptides, nucleic acids, carbohydrates, lipids, hormones including proteinaceous and steroid hormones, growth factors, receptor ligands, antigens and antibodies, and the like.
- the cobalt compound containing a targeting moiety as one of the R groups inhibits a protein, which may or may not be an enzyme.
- a protein which may or may not be an enzyme.
- inhibition of a protein herein is meant that a biological activity of the protein is decreased or eliminated upon binding of the inhibitor. In the case of enzymes, inhibition results in a decrease or loss of enzymatic activity.
- polypeptides comprising protease substrates or inhibitors are used as an R group on the cobalt compounds, to form cobalt compounds that will selectively inhibit the protease.
- a cobalt compound containing an R group comprising a nucleic acid that specifically binds to a particular nucleic acid binding protein such as a transcription factor is used to selectively inhibit the transcription factor.
- These targeted cobalt compounds preferentially bind to the target site on the protein, favoring that site over non-specific binding to other sites or other proteins. This makes the resulting compound more effective at lower concentrations since fewer molecules interact at other sites and minimizes the side-effects due to inhibition of other proteins. Secondary interactions also increase the time spent at the target, giving more opportunity for ligand exchange.
- cobalt compound for a particular protein, it is to be understood that the high affinity of the cobalt compound for an imidazole moiety, or the other possible reactive axial ligand moieties, is such that the cobalt compound need not be a perfect fit in the active site. Rather, what is important is that the cobalt compound be able to approach the target axial ligand moiety.
- the cobalt compounds should generally not be larger than typical enzyme substrates or inhibitors. The gross structure and surface properties of the cobalt compound reagent will determine its outer sphere interaction with the desired biological active site.
- the target protein When the target protein is known to have a histidine or other cobalt reactive amino acid that is functionally important, either Co(II) or Co(III) may be used, with Co(III) being preferred.
- Co(II) When the functional mechanism of the target protein is unknown, either Co(II) or Co(III) may be used, with Co(II) being preferred.
- the cobalt reactive amino acid is also close to the binding pocket or site of the targeting moiety.
- polypeptide herein is meant a compound ranging from about 2 to about 15 amino acid residues covalently linked by peptide bonds.
- Preferred embodiments utilize polypeptides from about 2 to about 8 amino acids, with about 4 to about 6 being the most preferred.
- the amino acids are naturally occurring amino acids in the L-configuration, although amino acid analogs are also useful, as outlined below.
- the polypeptide may be only a single amino acid residue.
- the polypeptide may be larger, and may even be a protein, although this is not preferred.
- the polypeptide is glycosylated.
- polypeptide Also included within the definition of polypeptide are peptidomimetic structures or amino acid analogs.
- non-naturally occurring side chains or linkages may be used, for example to prevent or retard in vivo degradations.
- the amino acid side chains may be in the (R) or D- configuration.
- the amino acids, normally linked via a peptide bond or linkage i.e. a peptidic carbamoyl group, i.e. -CONH-, may be linked via peptidomimetic bonds. These peptidomimetic bonds include CI L-NH-.
- nucleic acid may refer to either DNA or RNA, or molecules which contain both deoxy- and ribonucleotides. Generally, the nucleic acid is an oligonucleotide, ranging from about 3 nucleotides to about 50 nucleotides, with from about 12 to about 36 being particularly preferred, and at least 21 nucleotides being especially preferred. When the nucleic acid is used solely to confer solubility, the nucleic acid may be smaller, and in some embodiments may be a single nucleotide.
- the nucleotides may be naturally occurring nucleotides, or synthetic nucleotides, and may be any combination of natural and synthetic nucleotides, although uracil, adenine, thymine, cytosine, guanine, and inosine are preferred.
- the nucleic acids include genomic DNA, cDNA and oligonucleotides including sense and anti-sense nucleic acids.
- the nucleic acid may be double stranded, single stranded, or contain portions of both double stranded or single stranded sequence. In a preferred embodiment, for example when the nucleic acid is used to target a zinc finger transcription factor, the nucleic acid is double stranded, as zinc fingers bind to the major groove of double stranded nucleic acids.
- a nucleic acid will generally contain phosphodiester bonds, although in some cases, as outlined below, a nucleic acid may have an analogous backbone, comprising, for example, phosphoramide (Beaucage et al.. Tetrahedron
- Monosaccharides, disaccharides, and oligo- or polysaccharides are all included within the definition and comprise polymers of various sugar molecules linked via glycosidic linkages.
- Particularly preferred carbohydrates are those that comprise all or part of the carbohydrate component of glycosylated proteins, including monomers and oligomers of galactose, mannose, fucose, galactosamine, (particularly N-acetylglucosamine), glucosamine, glucose and sialic acid, and in particular the glycosylation component that allows binding to certain receptors such as cell surface receptors.
- Other carbohydrates comprise monomers and polymers of glucose, ribose, lactose, raffinose, fructose, and other biologically significant carbohydrates.
- Lipid as used herein includes fats, fatty oils, waxes, phospholipids, glycolipids, terpenes, fatty acids, and glycerides. particularly the triglycerides. Also included within the definition of lipids are the eicosanoids. steroids and sterols, some of which are also hormones, such as prostaglandins, opiates, and cholesterol. Hormones include both steroid hormones and proteinaceous hormones, including, but not limited to, epinephrine, thyroxine, oxytocin, insulin, thyroid-stimulating hormone, calcitonin.
- Receptor ligands include ligands that bind to receptors such as cell surface receptors, which include hormones, lipids, proteins, glycoproteins, signal transducers, growth factors, cytokines, and others.
- the targeting moiety is chosen just to confer solubility on the Co(II) or cobalt compound.
- the actual sequence of amino acid residues or nucleotides is not critical.
- the targeting moiety is chosen to target a particular protein or enzyme, and thus, when the targeting moiety is a polypeptide for example, the actual sequence of amino acids is important.
- At least one of R,-R g of Formula 1 is a polypeptide.
- polypeptide is chosen on the basis of the target protein or enzyme to be inhibited.
- the polypeptide when the target enzyme is a protease, the polypeptide will mimic or comprise an enzyme substrate or the reactive site of an inhibitor.
- the inhibitor may be either a reversible or irreversible inhibitor.
- the sequence of the polypeptide is chosen to allow the binding of the polypeptide to the active site of the protease.
- polypeptide and the site of attachment of the polypeptide to the cobalt compound will be chosen to maximize the interaction of the cobalt with the active site histidine. That is, as is explained below, the polypeptide may be attached to the cobalt compound at the N-tcrminal end, the C-terminal end, or internally, via one or more amino acid side chains.
- the active site histidine of many enzymes is close to the Sl -S l ' position of the enzyme's substrate (or inhibitor) binding site.
- examples include the serine and cysteine proteases.
- the polypeptide is chosen to allow optimum interaction of the cobalt compound with the active site histidine.
- the polypeptide may comprise roughly the P4 through Pl residues of a substrate or inhibitor (which occupy the S4 to S 1 positions of the enzyme's binding site), and be attached at the C-terminal end (Pl) to the cobalt compound, to maximize the steric interaction of the cobalt compound with the active site of the enzyme, and particularly the active site histidine.
- the polypeptide may comprise the PL tlirough P4' residues (corresponding to the SI ' to S4' positions), with attachment at the N-terminus (P 1 ').
- the polypeptide spans the Pl -Pl' site, but has an internal attachment at or near the Pl or PL residues, to similarly maximize the interaction of the cobalt compound with the active site histidine.
- the present invention allows a known enzymatic substrate to be used as an inhibitor, as well as increasing the efficiency of known inhibitors, for example via decreasing the K,.
- a wide variety of enzyme substrates and inhibitors for a variety of proteases containing either an active site histidine or an essential metal ion coordinated by a histidine are known in the art.
- the morphological properties of enzymes for which the crystal structures are known are used to design appropriate cobalt compounds.
- Alternative embodiments utilize known characteristics about surface charge and hydrophobicity, and substrate and inhibitor specificity.
- the K, of the polypeptide inhibitor is decreased as a result of attachment to the cobalt compound. That is, the inhibitor becomes a better inhibitor as a result of the attachment of the cobalt compound.
- the cobalt compound is effective at lower concentrations since fewer molecules are wasted at other sites.
- At least one of the R groups is a nucleic acid used to target the cobalt compound to a particular protein or enzyme.
- the target protein can be a nucleic acid binding protein that has at least one histidine that is important in biological activity, such as a zinc finger protein.
- nucleic acid binding protein As is known for zinc finger proteins that bind nucleic acids, it appears that each zinc finger interacts or binds to three base pairs of nucleic acid (see Berg, supra). Thus, the actual sequence of the nucleic acid used to target a nucleic acid binding protein will vary depending on the target protein. Nucleic acid sequences and their target binding proteins are known in the art.
- the cobalt compound can be attached to the nucleic acid in a variety of ways in a variety of positions: the actual methods are described below.
- the attachment site is chosen to maximize the interaction of a cobalt-reactive amino acid such as histidine that is essential for metal ion binding (or an active site histidine) with the cobalt compound.
- the backbone of the nucleic acid is modified to contain a functional group that can be used for attachment to the cobalt compound. This functional group may be added to either the 5' or 3' end of the nucleic acid, or to an internal nucleotide.
- the nucleic acid may be synthesized to contain amino-modified nucleotides using techniques well known in the art (see for example Imazawa et al., J. Org. Chem. 44:2039-2041 (1979); Miller et al., Nucleosides. Nucleotides 12:785-792 (1993): and W095/15971 , and references cited therein).
- amine groups are added to the ribophosphate backbone at the 2' or 3' position, thus allowing the attaclmient of the nucleic acid to the cobalt at either the 5' or 3' end, or to an internal nucleotide.
- nucleic acid is then used to couple the nucleic acid to the cobalt compound.
- nucleotide dimers containing phosphoramide, phosphorothioate, phosphorodithioate, or O- methylphosphoroamidite linkages may be made, and added to the nucleic acid at any position during synthesis, and the nitrogen or sulfur atom used for attachment using well known techniques, as outlined below.
- the phosphorus atom of the backbone may be used, or linkers, as is known in the art (see for example Thuong et al., Angew. Chem. Intl. Ed. Engl. 32:666-690 ( 1993); and Mergny et al.. Nucleic Acid Res. 22:920-928 (1994)).
- R When Co is Co(II) in Formula 1. at least one of R,, R 2 , R 3 , R 4 , R 5 , R 6 , R 7 and R 8 is hydrophilic such that the Co(II) compound is soluble in aqueous solution.
- R is hydrophilic. for example, n-propyl alcohol.
- two. three, four, five, six, seven or eight R groups are hydrophilic.
- the hydrophilic group is a targeting moiety, and preferably either a polypeptide or a nucleic acid.
- solubility is meant that the Co(II) compound has appreciable solubility in aqueous solution and other physiological buffers and solutions. Solubility may be measured in a variety of ways. In one embodiment, solubility is measured using the United States Pharmacopeia solubility classifications, with the Co(II) compound being either very soluble (requiring less than one part of solvent for 1 part of solute), freely soluble
- cobalt containing compounds are generally colored, the appearance of a color upon addition to a colorless aqueous solution indicates an acceptable level of solubility. For example, many Co(II) Schiffs base complexes have a yellow or orange color.
- the cobalt compounds depicted in Formula 1 have a regiospecific hydrophilicity. That is, R,, R 2 , R 3 , and R 4 , are either hydrogen, alkyl or aryl, and are therefore hydrophobic. and at least one of R 5 , R 6 , R 7 and R 8 is hydrophilic.
- R, R 2 , R 3 , and R 4 are either hydrogen, alkyl or aryl, and are therefore hydrophobic. and at least one of R 5 , R 6 , R 7 and R 8 is hydrophilic.
- R 5 , R 6 , R 7 and R 8 is hydrophilic.
- R 5 , R 6 , R 7 and R 8 is hydrophilic.
- this regiospecific hydrophilicity allows better positioning of the cobalt compound in or near the active site or on the surface of a protein or enzyme, as is discussed below. Without being bound b ⁇ theory. it appears that this regiospecific hydrophilicity/hydrophobicity allows the cobalt compound
- Particularly preferred embodiments of the present invention include the structure depicted in Formula 4, wherein R, is n-propyl alcohol.
- R 2 is hydrogen, R- is methyl, R ⁇ , is methyl.
- R 7 is hydrogen, R 8 is methyl, and R and
- R s are hydrogen: -ormula l 4
- the Co can be either Co(II) or Co(III).
- the cobalt complexes may have groups that alter the redox potential, oxidation stability, or ability of the compound to exchange an axial ligand.
- many of the Co(II) complexes of the invention are sensitive to air oxidation. That is, in the presence of atmospheric oxygen, they may be oxidized.
- the Co(Il) complexes are synthesized and utilized in the absence of air.
- the Co complexes are additionally modified to make them air stable compounds.
- replacement of a methyl R group in a complex of the invention with a trifluoromethyl group results in a positive shift of the metal oxidation potential, stabilizing the metal complex with respect to air oxidation.
- 1.1.1 -trifluoro-2,4-pcntanedione is commercially available, and may be used to synthesize trifluoromethyl derivatives of the Co(II) complexes disclosed herein.
- Further well known modifications such as chlorination of the Schiffs base macrocycle also greatly enhance the stability of these complexes with respect to air oxidation.
- the use of trifluoromethyl groups alone or in conjunction with chlorination of the macrocycle will result in a soluble air stable Co(II) macrocycle complex.
- the addition of electron donating or electron withdrawing groups may effect the activity of the cobalt compound with respect to the ability to exchange an axial ligand.
- the addition of trifluoromethyl R groups at the R, and R s positions basically eliminates the reactivity of the Co(III ) compound towards new axial ligands.
- Electron withdrawing or donating groups are preferably added at the R, and/or R 8 positions, as this is easiest for synthesis, as well as the preferred position for electronic coupling.
- the R 2 and/or R 7 positions are also preferred.
- Suitable electron withdrawing groups include, but are not limited to, halides (F, Cl, Br, I, in decreasing order of electron withdrawing strength), phenyl and substituted phenyl groups such as nitro-phenyl, amines and quaternary amines, thiols, nitro groups, carboxy groups, nitrile, alkynes and alkanes, sulfonyls, and others known in the art.
- Suitable electron donating groups include, but are not limited to, -OCH 3 , methyl, carboxylate, and ether.
- cobalt compounds of the invention are synthesized as generically disclosed below in Scheme I, using the general methods of Costa et al.. L Organometal. Chem.. 6: 181 (1966), which describes the preparation of derivatives of the components used to make the ligands used in the invention, such as acetylacetone ethylenediamine (acacen).
- the axial ligands are usually added in the last step.
- the amino-derivative of the core cobalt compound is synthesized as follows:
- the NCS group may then be used for coupling, as is known in the art.
- the cobalt compounds are generally constructed in three phases.
- the core cobalt compound is synthesized with a functional moiety that can be used to couple the targeting moiety.
- the core cobalt compound is made with an amine, a carboxy or a sulfhydryl group.
- the R group, comprising a targeting moiety is made, which also contains a functional moiety that can be used for attachment. In some instances, other reactive groups of the targeting moiety are protected to prevent them from reacting with the functional group of the core cobalt compound.
- amino acid side chains containing amino groups may need to be protected to prevent the side chain from reacting, although in some embodiments the attachment is done via a functional moiety of an amino acid side chain.
- Protecting groups and techniques are well known in the art.
- the core cobalt compound and the R group are made, they can be attached by reacting the functional groups.
- the linkage is direct; for example a cobalt compound containing a carboxy R group may be directly linked to an amino terminus of a polypeptide, as is depicted in the Examples.
- C-terminal attachment may be done using a cobalt compound with a amino functional moiety.
- this direct linkage may be done in organic solvents or alternatively using coupling reagents such as l-(3-dimethylaminopropyl)-3-ethylcarboiimide (EDC) (see generally, March, Advanced Organic Chemistry, 3rd Ed. Kiley & Sons, Inc. (1985) ; see also the 1994 Pierce Chemical Company catalog, technical section on cross-linkers, pages 155-200. inco ⁇ orated herein by reference).
- EDC l-(3-dimethylaminopropyl)-3-ethylcarboiimide
- the linkage between the two functional moieties may utilize a linker, also well known in the art.
- a linker also well known in the art.
- two amino groups may be linked via a stable bifunctional groups as are well known in the art, including homobi functional and heterobifunctional linkers (see Pierce Catalog and Handbook, pages 155-200).
- carboxy groups (either from the polymer or from the cell targeting moiety) may be derivatized using well known linkers (see the Pierce catalog).
- carbodiimides activate carboxy groups for attack by good nucleophiles such as amines (see Torchilin et al., Critical Rev. Therapeutic Drug Carrier Systems, 7(4):275-308 (1991 ), expressly incorporated herein).
- Sulfhydryl groups may be added to amines or carboxy groups with heterobifunctional linkers (see the Pierce catalog).
- attachment may be done in a variety of ways, including those listed above. What is important is that manner of attachment does not significantly alter the functionality of the targeting moiety; that is, they are still able to bind to the target protein. As will be appreciated by those in the art, this is easily verified.
- a number of functional groups of the targeting moiety may be used for covalent coupling, such as alcohols, amino groups, and carboxy groups.
- the targeting moiety may be derivatized to contain a functional moiety, such as through the addition of a linker containing a functional moiety.
- a linker containing a functional moiety When a polypeptide is to be used as an R group, a preferred embodiment utilizes an amino group of the polypeptide.
- the N- terminal amino group may be used, or alternatively, an amino group of an amino acid side chain, such as the amine groups of arginine, asparagine, glutamine, lysine, histidine and tryptophan.
- the linkage may be accomplished using the sulfur atoms of the side chains of methionine or cysteine.
- the carboxy groups of the side chains of glutamic acid and aspartic acid may also be used.
- the R group is a nucleic acid
- a variety of positions may be used as the site of covalent attachment to the cobalt compound.
- the ribophosphate backbone of the nucleic acid is modified to contain a functional moiety (see for example Meade et al., Angewandte Chemie, English Edition, 34(3):352-354 (1995), and references cited therein; Imazawa et al, supra. Miller et al., supra).
- an amino group is added at the 2' or 3' position of the sugar using techniques well known in the art.
- this is done by adding additional nucleotides that have an added amino group to the nucleic acid; that is, as shown in the Examples, one or more "extra" nucleotides is added to the targeting nucleic acid.
- the phosphodiester linkage between two nucleotides may be altered to form phosphoramide, phosphorothioate, phosphorodithioate, or O-methylphosphoroamidite linkages, as is known in the art.
- the nitrogen or sulfur atoms are then used as functional moieties.
- the nucleotide dimer, containing the altered linkage may be added to the nucleotide at any position.
- nucleotide bases themselves may also be used, such as the amino groups on adenosine and cytosine, or modified bases such as is known for thymine (see for example Telser et al. , J. Amer. Chem. Soc. 111 :7221-7226 (1991); Unglisch et al. , ⁇ ngew. Chem 103:629-646 (1991); Angew. Chem. Int. Ed. Engl. 30:613- 629 (1991); Goodchild, Bioconjugate Chem. 1 : 165-187 (1990); and Brun et al. , J. Amer. Chem. Soc. 113 :8153-8159 (1991)). Then the nucleic acid containing the functional group may be added to the cobalt compound either directly or via a linker, as is outlined above for polypeptides.
- targeting moieties such as carbohydrate, lipid, and hormone targeting moieties may be altered to contain functional groups for linkages, as will be appreciated in the art, or derivatized with linkers containing functional groups.
- the functional group for coupling should not prevent the binding of the targeting moiety to the target protein, and preferably does not affect the binding.
- these targeting moieties containing suitable functional groups are made using well known techniques. Once synthesized, the cobalt compounds of the invention find use in a number of applications. At the broadest level, the Co(II) compounds are useful as reducing agents in aqueous solution.
- the cobalt compounds of the invention are useful as general bacteriostatic or bactericidal agents, antimicrobial agents and/or antiviral agents, for both topical and other therapeutic applications.
- topical antimicrobial agents may be useful in cleaning and disinfectant compositions, as will be appreciated in the art.
- Therapeutic uses of antimicrobial and antiviral agents are also well known.
- the compounds are assayed for antiviral, antimicrobial and antibacterial activity using techniques well known in the art; for example, bactericidal activity may be measured using the techniques outlined in example VI of U.S. Patent 5,049,557. Both in vitro and in vivo antiviral activity may be measured using the techniques outlined in U.S. Patent No. 5.210,096.
- the cobalt compounds of the invention can also be used to label proteins.
- the cobalt compounds of the invention can also be used to label proteins.
- Co(II) compounds of the invention are preferably made with no axial ligands, and the Co(III) compounds are generally made with two axial ligands.
- certain moieties on the protein will become axial ligands, resulting in a tightly bound protein-cobalt compound complex.
- cobalt-containing compounds may be detected spcctrophotometrically, the result is a labeled protein.
- the preferred axial ligand from a protein is the imidazole side chain of histidine.
- a protein with one or more histidine residues either at the surface of the protein or otherwise accessible to the solvent can be labeled using the cobalt compounds of the invention.
- the cobalt compounds of the invention are added or contacted with the target protein.
- the excess cobalt compound may be separated, and the labeled protein, with the attached Co(III) compound, is detected spectrophotometrically.
- the Co(III) compounds are generally detected at 280, 338, and 451 nm, although a broad range from 280 to 500 nm may be useful.
- the stoichiometry of the bound cobalt compound to protein will vary depending on the number of potential axial ligands in or at the active site or on the surface of the protein, and may be determined spectrophotomctrically. as is understood in the art. Thus, for example, a protein which has four accessible histidines will generally bind four cobalt compounds, etc.
- cobalt compounds of the present invention are also useful in probing the surface characteristics of a protein.
- the cobalt compounds When used to bind or label proteins, the cobalt compounds can be coupled, using standard technology, to affinity chromatography columns. These columns may then be used to separate proteins from a sample. For example, depending on the specificity of the cobalt complex, proteins may be removed from a sample, or specific proteins, such as those containing histidines at or near the active site may be separated from other components of the sample.
- the cobalt compounds are useful as enzyme inhibitors.
- the mechanism of inactivation is similar to the mechanism of protein labeling.
- an enzyme has one or more moieties capable of binding in an axial position in the cobalt compounds of the invention.
- moieties are also functionally important for enzymatic activity, and are inactivated upon contact with the cobalt compounds of the invention.
- enzymes which have histidine as an active site catalytic residue or have histidines which are functionally important for enzymatic activity are particularly preferred.
- Enzymes such as the serine proteases (trypsin, subtilisin. chymotrypsin, elastase, thrombin, factor Xa, lysozyme, and others known in the art), cysteine proteases such as the cathepsins and interleukin converting enzyme; RNAse H, thermolysin and lactate dehydrogenase all have active site histidines and thus may be inhibited with the compounds of the present invention.
- a cobalt compound is contacted with the target enzyme.
- the imidazole side chain of an active site histidine binds to the cobalt compound as an axial ligand.
- the binding (and oxidation, in the case of the Co(II) compound) results in the inhibition of the enzyme.
- the exact mechanism of the inactivation is unknown; however, several possibilities exist.
- the bound cobalt compound, which after binding and oxidation is a Co(III) compound, may sterically interfere with catalytic activity, i.e. it may be bound in or near the catalytic active site.
- the bound cobalt compound may interfere with the catalytic mechanism, i.e. by binding to a catalytic histidine.
- Co(II) it is also possible that a functionally important moiety at the active site is reduced by the Co(II) compound, and thus the enzyme is inactivated.
- the inactivation of the enzyme by the cobalt compound inhibitor is effectively irreversible.
- the reactive axial ligand from the enzyme is the indole side chain of tryptophan or the side chains of cysteine, methionine, arginine, lysine, asparagine, glutamine, aspartate or glutamate.
- the availability of these moieties may depend on the pH of the solution containing the protein or enzyme, since in the protonated state these moieties arc not good electron donors suitable as axial ligands.
- enzymes with these groups within the active site, or enzymes which have functionally important tryptophans, cysteines, or methionines may be inactivated by the cobalt compounds of the present invention, as outlined above.
- metalloproteins are inactivated with the cobalt compounds of the present invention.
- the metals of metalloproteins have ligands such as histidine, cysteine and methionine. If one or more of these residues are inactivated using these cobalt compounds, the binding of the metal atom may be decreased or eliminated, thus reducing or eliminating biological activity.
- Particular metalloproteins include, but are not limited to, nucleic acid binding proteins such as "zinc finger” proteins and hemerythrin. Zinc finger proteins utilize histidine and cysteine to bind zinc ions (see Berg, Ann. Rev. Biophys. Biophys. Chem 19:405-421 (1990), Berg. Science 232:485
- Zinc finger proteins have been shown to bind nucleic acids and thus play a role in a variety of gene regulator ' processes. Zinc finger proteins include transcription factors and other nucleic acid-binding and gene-regulatory proteins (see Berg, Science, supra), and are found in eukaryotes, prokaryotes, and viruses. Other zinc finger proteins suitable for inactivation by the compounds of the present invention include the nucleic acid binding domain of steroid and thyroid hormone receptors and the human oncogene product GLI (see Pavletch et al., Science 261 :1701 (1993); Kinzler et al..
- one or more of the zinc finger domains utilizes at least one histidine to bind zinc, with the proteins that utilize two histidines being preferred. In some cases the metal is bound exclusively by cysteines.
- metalloprotein When the metalloprotein is a metalloenzyme, displacement of the active site metal by the cobalt complex may modulate enzyme activity.
- metalloenzymes include, but are not limited to, the carboxypeptidases, carbonic anhydrase, thermolysin, collagenase, histidinol dehydrogenase, leukotriene A4 hydrolase. adenosine deaminase, superoxidc dismutasc, alcohol dehydrogenase, lactate dehydrogenase, stromalycin. aminoacyclase, tryptophanyl-tRNA synthetase, and others known in the art.
- serine and cysteine proteases are inhibited.
- the enzyme to be inhibited is carbonic anhydrase.
- Carbonic anhydrase has been implicated in diabetes, ocular disease such as glaucoma, and seizures and convulsions. Accordingly, inhibitors of carbonic anhydrase, such as the Co(II) complexes of the present invention, are useful in the treatment of these conditions.
- the Co(II) complexes are useful in the treatment of elevated intraocular pressure and glaucoma.
- Carbonic anhydrase has been implicated in elevated intraocular pressure, and carbonic anhydrase inhibitors have been shown to be efficacious in decreasing this pressure in animals and humans (see Sharir et al., Experimental Eve Res. 58( 1): 107-1 16 (1994); Rassam et al., Eye 7(Pt 5):697-702 (1993); Gunning et al., Graefes Archive for Clinical and Experimental Ophthalmology 231 (7 :384 1993)V
- the Co(II) compounds are useful in the treatment of seizures and convulsions.
- Carbonic anhydrase II deficient mice have been shown to have increased resistance to chemically induced seizures, and pretreatment with carbonic anhydrase inhibitors has been shown to increase the resistance of normal mice to chemically induced seizures. See Velisek et al., Epilepsy Res. 14(2): 1 15-121 (1993).
- the Co(II) compounds are useful in the treatment of diabetes and abnormal renal function. Elevated levels of carbonic anhydrase have been associated with metabolic diseases like diabetes mellitus and hypertension, and carbonic anhydrase inhibitors have been suggested for treatment. See Parui et al., Biochem. International 26(5): 809-820 (1992); Parui et al, Biochem. International 23(4):779-89 (1991); Dodgson et al.. Arch. Biochem. Biophys. 277(2):410-4 (1990); Hanncdouchc et al.. Clinical Sci.
- the cobalt compounds find use in the inhibition of proteins and enzymes of tumor cells.
- Co(III) "acacen” compounds can exchange an axial ligand for a different one by a dissociative mechanism with the slow loss of one axial ligand to form a five coordinate intermediate, followed by binding to another suitable ligand.
- ligand exchange is a slow process because there is a large loss of ligand field stabilization energy when a ligand is removed from an octahedral d 6 complex (see Huheey et al., Inorganic Chemistry: Principles of Structure and Reactivity, 4th Ed. Ha ⁇ erCollins, N.Y., chapter 13).
- the exchange is slow; for example, [Co(III)(acacen)(NH 3 ) 2 ]Cl in water with excess imidazole exchanges ammonia for imidazole with a half-life under an hour at 25 °C, with the rate of exchange increasing with temperature.
- reduction to cobalt(II) puts an electron into the antibonding d z2 orbital, labilizing the axial ligands.
- Typical one-electron reduction potentials with irreversible loss of an axial ligand are around -360 mV vs NHE (Darbieu et al.,
- Raising the reduction potential of a cobalt acacen compound with substituents such as halides may place it high enough for reduction to occur readily in tumor cells, but not in healthy cells.
- Ware and coworkers use a similar approach to attempt selective release of cobalt-bound cytotoxins in cancer cells (see Ware et al., supra).
- testing the efficacy of the cobalt compounds as inhibitors is routine, as will be appreciated in the art.
- the target protein is an enzyme
- testing is similar to testing any enzyme inhibitor, as is known in the art.
- the enzyme is assayed in the presence and absence of the putative inhibitor, and kinetic parameters are calculated as is known in the art.
- the amount of cobalt compound inhibitor needed to inhibit a given enzyme will vary depending on the number of other reactive axial ligands on the surface of the enzyme, as is outlined above for protein labeling. For example, an enzyme with an active site histidine and two other "surface" histidines will generally require at least a 3: 1 ratio of cobalt compound inhibitor:enzyme.
- the total amount bound to the enzyme may be determined spectrophotometrically, as outlined above.
- the Co(II) compound inhibitors are generated in situ by reducing the corresponding Co(III) compound.
- corresponding Co(III) compound herein is meant a Co(III) compound which has the identical
- R groups as the Co(II) compound.
- the Co(III) compounds are synthesized with axial ligands, such as, but not limited to, amines. 2-methyl imidazole, and water.
- the Co(III) compound is synthesized, and then added to the enzyme under conditions which can result in the reduction of the Co(III) to
- the in situ environment of the enzyme may be a reducing environment for the Co(III) compound, such that the Co(III) is reduced to Co(II).
- the Co(III) compound may contain an electron acceptor group as one of the R groups, such that in a given in situ environment, the electron acceptor group will pick up an electron and donate the electron to the Co(III), thus reducing it to the Co(II) form.
- Suitable electron acceptor groups include, but are not limited to, cations such as methyl violgen (N-N-dimethyl 4,4' bipyridine), or ethyl or propyl violgen, as is understood in the art.
- the reduction potential of the compound may be tailored such that introducing the compound into a particular environment causes reduction; for example, by glutathione in physiological systems.
- the resulting Co(II) compound then reacts with the reactive axial ligands of the enzyme to inhibit the enzyme as outlined above.
- the Co(II) compound is generated in situ; that is, a Co(III) compound is added to an enzyme, is reduced to the Co(II) form, which in turn inhibits the enzyme.
- the Co(III ) compounds may be inert with respect to a selected enzymatic target in a given oxidation state, yet inactivate the enzyme target in a second oxidation state. This mechanism allows the in situ addition of a cobalt compound, whether in vitro or in vivo, in an inactive form, with activation to the Co(II) compound form in a particular reducing environment.
- the compounds of the present invention may be formulated into pharmaceutical compositions, and administered in therapeutically effective dosages.
- therapeutically effective dose herein is meant a dose that produces the effects for which it is administered. The exact dose will depend on the disorder to be treated and the protein to be inhibited, and will be ascertainable by one skilled in the art using known techniques.
- the pharmaceutical compositions of the invention are in a water soluble form, and contain a pharmaceutically acceptable carrier in addition to the cobalt compound.
- the pharmaceutical compositions may be administered in a variety of ways, including, but not limited to, orally, subcutaneously, intravenously, intraperitoneally, or topically.
- the target protein is contacted or exposed to a cobalt compound.
- the cobalt compound has the structure depicted in Formula 1.
- the cobalt compound can be targetted to a particular protein by the addition of a targeting moiety, such as a polypeptide or a nucleic acid.
- Also provided are methods for inhibiting a zinc finger protein comprising contacting a zinc finger protein with a cobalt compound.
- inhibiting a zinc finger protein herein is meant that the biological activity of the zinc finger protein is decreased or eliminated upon exposure to the cobalt compound.
- R ⁇ and R F are the same or different and each is an alkyl group, a phenyl group or a substituted derivative of a phenyl group.
- R n and R,. are the same or different and each is hydrogen, an unbranched alkyl group, a halide or a group having the structure: G — C
- R Q is hydrogen, an alkoxide group, an alkyl group, or OH.
- R t - and R D are the same or different and each is hydrogen or an alkyl group.
- Tris(hydroxymethyl)aminomethane Tris, Trizma Base
- polyethylene glycol PEG 8000
- EDC l-(3-dimethylaminopropyl)-3-ethylcarbodiimide
- HPES N-(2-hydroxyethyl)piperazine-N'-2-ethanesulfonic acid
- Cobaltous acetate tetrahydrate was obtained from
- Human a-thrombin and the assay agent Spectrozyme TH H-D-hexahydrotyrosyl-L -alanyl-L-arginine-p-nitroanilide diacetate
- Antithrombotic peptides were manufactured as amides by the Beckman Institute Biopolymer Synthesis group at Caltech using solid phase methods.
- the compound was further purified using flash silica gel chromatography using 95:5:0.5 (v:v:v) CH 2 Cl 2 :MeOH:Et 3 N as the eluant.
- the resulting monoacacen was characterized by NMR.
- Monoacacen (0.5 g. 3.5 X 10 "3 mol) was dissolved in 5 mL of ethanol and 7- hydroxy-2,4-heptanedione (0.51 g. 3.5 X 10 3 mol) was added.
- the dione was synthesized as described previously (Detty, M.R. J. Org. Chem., 44:2073-2077 (1979)). The reaction was allowed to proceed for 4 hours and the solvent was removed in vacuo.
- the sample was purified using flash silica gel chromatography using 93:7 (v:v) CH 2 Cl 2 :MeOH as the eluant.
- the resulting hydroxypropyl acacen was characterized by NMR.
- Bzacacacen (the Formula 1 compound with Rl , R3 and R6 as methyl. R2 and R7 as hydrogen, and R8 as phenyl) : 1 equiv. benzoylacetone in CH 2 C1 2 was added to a solution of monoacacen in CH 2 C1 2 . Removal of solvent gave a white powder containing some acacen impurity. Purification was accomplished by flash chromatography on silica using 97 CH 2 C1 2 / 3 MeOH/ 0.5 TEA.
- Aciden (compound 1 1 in Scheme III): A solution of 1 equiv. 4.6-dioxoheptanoic acid in CH 2 C1 2 was added to 1 equiv. ethylenediamine in CH 2 C1 2 and the insoluble 1 : 1 condensation product immediately precipitated. The product was collected over a frit and dried in vacuo. The melting point was 140°C, with decomposition. A direct reaction of 4,6-dioxoheptanoic acid with monoacacen did not work, despite repeated attempts. Evidently, the acid group was effecting decomposition, even under anhydrous conditions. Nor did using excess triethylamine to neutralize the diketoacid give satisfactory results.
- N- r/-butyloxycarbonyl (Boc) amino acid derivatives for Merrifield solid-phase synthesis on an ABI Model 430A peptide synthesizer The terminal Boc protecting group was removed with trifluoroacetic acid (TFA). Side chain protecting groups and the peptide-resin bond were cleaved under HF conditions (90% HF. 5%/7-cresol, 5% -thiocresol). After removal of HF under vacuum, the peptide/resin mixture was washed on a fritted funnel with ether. The peptide was then dissolved in 10% aqueous acetic acid and filtered through, leaving the resin behind.
- TFA trifluoroacetic acid
- the crude peptide solution was subjected to gel filtration on anion exchange resin AG 1-X2 to remove the scavengers.
- the peptide can be further purified by reversed-phase HPLC on a Vydac C8 column using a 30-min. linear gradient of 6-26% acetonitrile/water/0.1% TFA with a 2.0 mL/min. flow rate.
- Coupled product [Co(III)(acacen-GGFPR)(NH 3 ) 2 ; shown in Figure 2]:
- One potential difficulty in coupling the cobalt complex to this peptide is that the peptide' s arginine side chain is more reactive than its N-terminus if the arginine is not protected or protonated (Bodzansky, Peptide Chemistry. A Practical Textbook. Springer-Verlag, Berlin, 1988). Since arginine has a pK a around 12.0, it is easily protonated, but this renders the hydrophilic peptide insoluble in the organic solvents, such as dioxane, used for most coupling reactions.
- EDC water-soluble coupling reagent l -(3-dimethylamino-propyl) -3-ethylcarbodiimide
- HPLC purification of a basic, hydrophilic peptide usually calls for a small amount of an organic acid in the eluting solvent to aid retention. Since such an acid would attack the imine bonds of the free ligand, purification was attempted without it, but neither reverse phase C8 and Cl 8 nor normal phase cyano columns were effective in resolving the mixture. Later, some progress was made using basic ammonium acetate buffer and acetonitrile on reversed phase, but there still was some decomposition of the product due to hydrolysis of the imine bonds. In order to prevent this, the imines were protected by inserting the metal into the ligand before attaching the peptide.
- Co(III)hydroxypropyl acacen (30 mg, 7.6 X 10 "5 mol) dissolved in 0.5 mL of H 2 0. This solution was incubated for 48 hours. The excess cobalt complex was separated from the protein using a PD-10 gel filtration column equilibrated with Tris buffer (pH 8, 0.05 M). This enzyme, which was incubated with the Co(III) complex, retained 100% of its activity.
- bovine carbonic anhydrase (30 mg, 1 X I O '6 mol) were dissolved in 3 mL of degassed Tris buffer (pH 8. 0.05 M). To one of the samples was added Co(II)hydroxypropyl acacen (30 mg, 9 X I O "5 mol). The other sample of bovine carbonic anhydrase served as the control. The solutions were incubated under inert atmosphere (glove box) and a 1 mL aliquot was removed from each sample after 48 and 96 hours of incubation.
- This solution was further purified using gel filtration chromatography on an FPLC using a Superdex 75 column (Pharmacia) equilibrated with 0.1 M Tris, 0.1 M NaBr, 0.01 CaCl 2 , pH 7.2. This stock solution was stored at 4C.
- thermolysin substrate was obtained from Sigma as the thermolysin substrate.
- a stock solution of FAGLA (4.0 mM) was prepared by dissolving the substrate in dimethylformadie (DMF) and diluting it with buffer to a final concentration of 0.1 M Tris, 0.1 NaBr and 10 mM CaCL, pH 7.0 (final concentration of DMF was 2.5%; see Feder et al., Biochem. 9:2784-2791 (1970)).
- concentration of enzyme and substrate was 50 nM and 2.0 mM respectively.
- the peptidase activity of thermolysin was determined by following the decrease in abso ⁇ tion at 346 due to the enzymatic hydrolysis of FAGLA. Initial velocities were determined for ⁇ 10% of the reaction.
- thermolysin 2 X 10 "5 mol
- concentration of thermolysin was 5 X 10" 8 M
- concentration of the cobalt inhibitor was 1.25 X IO" 5 M.
- thermolysin Stock solution of thermolysin was mixed with the cobalt compound dissolved in 0.1 M Tris, 0.1 M NaBr, 0.01 CaCL, pH 7.2 (run buffer) to yield a final enzyme concentraiton of 10 mM and a cobalt concentration of 2.5 mM. These solutions were incubated at 25C or 37C for several hours along with a control lacking cobalt compound. Periodically 5 ml aliquots of these solutions were assayed for residual enzyme activity by their addition to a cuvette contain 495 mL of run buffer and 500 ml of FAGLA stock solution, and following the abso ⁇ tion decrease at 346 nm as described above. All enzyme assays were performed at 25C. The results are shown in Figure 3.
- Phosphoramidon binds to thermolysin at the active site, and this enzyme-inhibitor complex has by crystallographically characterized (Weaver et al.. J. Mol. Bio. 1 14:1 19-132 (1977)). After incubation with the cobalt compound overnight, the inhibitor was separated from the enzyme using gel filtration chromatography on an FPLC using a Superdex 75 column (Pharmacia) equilibrated with 0.1 M Tris, 5 mM CaCl 2 , pH 9. The resulting solution was transferred into the Tris running buffer using a PD-10 column (Pharmacia), and was characterized. There was no detectable loss of enzyme activity due to irreversible inactivation by the cobalt complex after removal of the inhibitor.
- thermolysin completely inactivated by the cobalt complex
- the inhibition of the enzyme is a consequence of the binding of one cobalt compound at the enzyme active site.
- Thrombin was chosen as the first target enzyme. Several crystal structures of thrombin are available from the Protein Data Bank (e.g. file 1PPB; see Bode et al, EMBO J. 8:3467 (1989)). Thrombin is a 34 kD serine protease with a well defined mechanism of action involving a histidine residue. It is vital to the coagulation cascade, but an unwanted clot is a severe, life-threatening condition. Thrombin was chosen for this investigation because its structure and mechanism are well understood, there is a simple activity assay, and antithrombotic drugs are useful in the treatment of strokes.
- Thrombin inhibition assay As with any purified blood product, proper care was taken to avoid the transmission of blood-borne pathogens. Thrombin was taken as received (about 1 mL at 30 ⁇ M in 0.75 M sodium chloride storage solution) and divided into 100 ⁇ L aliquots. Each aliquot was diluted to 10 mL using clean, filtered (2 ⁇ ) aqueous 0.75 M NaCl and divided into 1 mL samples. Each sample was stored frozen at -80°C until ready for use. The protein should not be stored at -20 °C as this is too close to the eutectic point for the thrombin-salt mixture and freeze-thaw cycling may damage the protein.
- Thrombin was preincubated with inhibitor in the kinetics buffer (total volume of tlirombin, buffer, and inhibitor of 992mL) for the times specified in Figure 1.
- the substrate, spectrozyme TH was added (8mL of 5mM spectrozyme), and the thrombin-catalyzed hydrolysis rates were monitored at 406 nm.
- the final concentration of thrombin in the experiments was 3 nM. and the concentration of substrate was 40 mM.
- the inhibitor concentrations are outlined in Figure 1.
- the rates of hydrolysis were determined from the linear portion of the saturation-kinetics plots. The percent activity is determined by dividing the rate of spectrozyme hydrolysis with inhibitor by the rate of hydrolysis without inhibitor and multiplying by 100.
- a vial of thrombin prepared as above was thawed in warm water. 100 ⁇ L aliquots were added to 100 ⁇ L samples of the cobalt acacen solutions. One 100 ⁇ L aliquot of thrombin solution was diluted with 100 ⁇ L 0.75M NaCl to be used as a control. Samples were incubated as needed before assay. 980 ⁇ L of the assay buffer was placed in a 1.0 mL, 1 cm quartz cuvette and allowed to equilibrate to 25 °C (Hewett-Packard Peltier constant temperature cell holder). 10 ⁇ L of a sample was mixed thoroughly into the cell's contents. The spectrophotometer was set for a 30 second delay during which 10 ⁇ L
- Spectrozyme TH solution was added and mixed into the cell's contents by inverting the capped cell a few times before replacing it in the spectrophotometer. Scans from 250 to 500 nm were taken every 30 seconds for 10 minutes, although a single-wavelength scan at 406 nm would suffice. After the runs, the control was allowed to hydrolyze to completion before determining the end point.
- thrombin from the same source was incubated without inhibitor for the same length of time. A portion of each incubated solution was assayed at 25°C with an excess of a commercial substrate, Spectrozyme TH, whose proteolysis releases a chromophore, p-nitroaniline. The pseudo-first order production of p-nitroaniline was monitored spectrophotometrically and the rate constant extracted for each run. The activity of the control sample was normal, but the cobalt-containing sample was completely inactive (Figure XX). The cobalt-free ligand had no effect.
- the Co(III) compound was a more effective inhibitor (0% activity) than the Co(II) compound (42% activity relative to its control). Perhaps the overall positive charge on the Co(III) compound assists in attracting the inhibitor to the active site as is seen in a different system (Bagger. J. Inorg. Biochem. 52: 165 ( 1993)).
- a crude attempt to determine the number of cobalt complexes bound to the enzyme was made by first passing the inhibited protein down a size exclusion column to remove most of the unbound cobalt complex and then determining the absorbances at two wavelengths for which the extinction coefficients of both pure thrombin and cobalt compound were known; this information gives the concentrations of each based on Beer's law.
- the initial estimate for two separate samples is 5 - 8 cobalts per enzyme. There are only five histidines in thrombin, but binding to other residues and electrostatic binding cannot be discounted.
- the targeting approach requires attaching a recognition element to the cobalt complex for binding specifically to the active site of thrombin.
- the tripeptide sequence dPhe-Pro-Arg is a known inhibitor of tlirombin. the arginine binding tightly to the Pl aspartate.
- the peptides GGdFPR. GGGdFPR, GGFPR and GGGFPR were obtained as amides and assayed against thrombin. As expected from the crystal structures and inhibition data for similar peptides, (Bajusz et al.. Int. J. Peptide Protein Res.
- the dPhe-containing peptides were found to be better inhibitors since they can access a hydrophobic binding pocket more efficiently than the natural isomers.
- the K; for (Gly) 3 dPheProArg is about 209 ⁇ M.
- Human Spl transcription factor which contains three CCHFI zinc fingers and a synthetic peptide representing the first zinc finger region of retroviral nucleocapsid protein.
- Filter Binding Assay The above samples were applied to nitrocellulose (0.45 um filters, Schleicher and Schuell) and washed twice with washing buffer (100 mM HEPES, pH 7.5, 1 mM EDTA). Membranes were incubated for 15 minutes at room temperature with Filtron-X (National Diagnostics) and bound counts were detected by liquid scintillation (Beckman Instruments). Increasing amounts of cobalt compound resulted in decreased counts bound to the filter, indicating a loss of binding between Spl and oligonucleotide.
- HIV nucleocapsid protein was synthesized and examined in a series of structural studies.
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EP96944811A EP1021176A4 (en) | 1995-12-12 | 1996-12-12 | Cobalt schiff base compounds |
JP52223997A JP2001503376A (en) | 1995-12-12 | 1996-12-12 | Cobalt-Schiff base compounds |
IL12484496A IL124844A0 (en) | 1995-12-12 | 1996-12-12 | Cobalt schiff base compounds |
AU13336/97A AU720841B2 (en) | 1995-12-12 | 1996-12-12 | Cobalt schiff base compounds |
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Cited By (17)
Publication number | Priority date | Publication date | Assignee | Title |
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EP0794782A1 (en) * | 1994-12-15 | 1997-09-17 | California Institute Of Technology | Cobalt schiff base compounds |
WO1999021592A1 (en) * | 1997-10-27 | 1999-05-06 | California Institute Of Technology | Magnetic resonance imaging agents for the delivery of therapeutic agents |
WO1999025389A2 (en) * | 1997-11-17 | 1999-05-27 | Research Corporation Technologies, Inc. | Magnetic resonance imaging agents for the detection of physiological agents |
EP1232159A1 (en) * | 1999-07-23 | 2002-08-21 | Biomolecular Research Institute Ltd. | Beta-amyloid peptide inhibitors |
US6521209B1 (en) | 1996-07-31 | 2003-02-18 | California Institute Of Technology | Bifunctional detection agents |
US6656450B2 (en) | 2000-07-17 | 2003-12-02 | California Institute Of Technology, Inc. | Macrocyclic magnetic resonance imaging contrast agents |
US6673333B1 (en) | 2000-05-04 | 2004-01-06 | Research Corporation Technologies, Inc. | Functional MRI agents for cancer imaging |
US6713045B1 (en) | 1995-06-02 | 2004-03-30 | Research Corporation Technologies, Inc. | Targeted magnetic resonance imaging agents for the detection of physiological processes |
US6713046B1 (en) | 1997-10-27 | 2004-03-30 | Research Corporation Technologies | Magnetic resonance imaging agents for the delivery of therapeutic agents |
US6770261B2 (en) | 1995-06-02 | 2004-08-03 | Research Corporation Technologies | Magnetic resonance imaging agents for the detection of physiological agents |
US7029655B2 (en) | 2000-10-04 | 2006-04-18 | California Institute Of Technology | Magnetic resonance imaging agents for in vivo labeling and detection of amyloid deposits |
WO2011146143A2 (en) | 2010-05-21 | 2011-11-24 | Ohmx Corporation | Detection of cancer by assaying psa enzymatic activity |
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US7118916B2 (en) * | 2002-10-21 | 2006-10-10 | Lifescan, Inc. | Method of reducing analysis time of endpoint-type reaction profiles |
JP7184282B2 (en) * | 2018-12-13 | 2022-12-06 | 株式会社Ihi | Drug delivery system containing metal acene complexes |
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1996
- 1996-12-12 AU AU13336/97A patent/AU720841B2/en not_active Ceased
- 1996-12-12 JP JP52223997A patent/JP2001503376A/en active Pending
- 1996-12-12 IL IL12484496A patent/IL124844A0/en unknown
- 1996-12-12 CA CA002240183A patent/CA2240183A1/en not_active Abandoned
- 1996-12-12 WO PCT/US1996/019900 patent/WO1997021431A1/en not_active Application Discontinuation
- 1996-12-12 EP EP96944811A patent/EP1021176A4/en not_active Withdrawn
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Publication number | Publication date |
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EP1021176A1 (en) | 2000-07-26 |
JP2001503376A (en) | 2001-03-13 |
EP1021176A4 (en) | 2001-04-11 |
CA2240183A1 (en) | 1997-06-19 |
AU1333697A (en) | 1997-07-03 |
AU720841B2 (en) | 2000-06-15 |
IL124844A0 (en) | 1999-01-26 |
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