WO1992015329A1 - Methods and pharmaceutical compositions for inhibiting protease from human immunodeficiency virus - Google Patents

Methods and pharmaceutical compositions for inhibiting protease from human immunodeficiency virus Download PDF

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Publication number
WO1992015329A1
WO1992015329A1 PCT/US1992/001731 US9201731W WO9215329A1 WO 1992015329 A1 WO1992015329 A1 WO 1992015329A1 US 9201731 W US9201731 W US 9201731W WO 9215329 A1 WO9215329 A1 WO 9215329A1
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Prior art keywords
copper
protease
delivery agent
copper ion
ion delivery
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PCT/US1992/001731
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French (fr)
Inventor
Rodney L. Levine
Anders R. Karlstrom
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The United States Of America, Represented By The Secretary, United States Department Of Commerce
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Publication of WO1992015329A1 publication Critical patent/WO1992015329A1/en

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    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00Medicinal preparations containing organic active ingredients
    • A61K31/70Carbohydrates; Sugars; Derivatives thereof
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00Medicinal preparations containing organic active ingredients
    • A61K31/095Sulfur, selenium, or tellurium compounds, e.g. thiols
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00Medicinal preparations containing organic active ingredients
    • A61K31/185Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19Carboxylic acids, e.g. valproic acid
    • A61K31/195Carboxylic acids, e.g. valproic acid having an amino group
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K33/00Medicinal preparations containing inorganic active ingredients
    • A61K33/24Heavy metals; Compounds thereof
    • A61K33/34Copper; Compounds thereof
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00Medicinal preparations containing peptides
    • A61K38/04Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/06Tripeptides
    • A61K38/063Glutathione
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca

Definitions

  • the present invention relates to methods for inhibiting human immunodeficiency viruses and pharmaceuti ⁇ cal compositions useful therein. More specifically, the present invention relates to methods for inhibiting HIV protease with copper ion, as well as pharmaceutical compositions employing a copper ion delivery agent for use in these methods. Description of Related Art
  • the aspartyl protease encoded by the human immuno ⁇ deficiency virus type 1 is essential for the pro- cessing of the viral polyproteins encoded by the gag and pol genes into mature viral proteins. Mutation or dele ⁇ tion of the protease gene blocks replication of the virus, making the protease an attractive target for antiviral therapy of the acquired immunodeficiency syndrome (AIDS) .
  • the inhibitors reported thus far are peptides or peptide analogues, some of which were originally studied as inhibitors of other structurally related aspartyl proteases such as pepsin or renin.
  • Fe/ascorbate/0 2 is a well-studied non-enzymic system (Levine, R.L. (1983) J. Biol . Chem . 258, 11828-1833). For the latter, it is believed that the iron binds to the enzyme at a specific cation binding site. Oxidation of the reduced form of the metal generates a very reactive oxidizing species, such as the hydroxyl radical. The radical reacts with an a ino acid residue very close to its site of generation, gener ⁇ ally inactivating the enzyme. In the case of gluta ine synthetase, the site specificity has been studied in detail. Specificity results from the binding of the redox-capable cation to the two binding sites on the enzyme which would normally bind magnesium.
  • the aspartyl proteases especially pepsin, have been studied in great detail.
  • the active site of this class of proteases always contains two aspartyl residues, and these could conceivably bind a cation.
  • X-ray crystal- lographic studies of several aspartyl proteases have employed heavy-metal derivatives ( lodawer, A. et al. , (1989) Science 245, 616-620; Miller, M. et al., (1989) Nature (London) 337, 576-579; and Navia, M. et al., (1989) Nature (London) 337, 615-620) thus establishing the existence of cation binding sites in these proteins.
  • a pharmaceutical composition for treating viral infections of a host in need thereof wherein the composi- tion contains a copper ion delivery agent and a pharmaceu ⁇ tically acceptable excipient.
  • Figure 1 illustrates the effect of cations on recombinant wild-type protease activity
  • Figure 2 is a time course for inactivation of the recombinant wild-type protease by 25 ⁇ m CuCl 2 ;
  • Figure 3 illustrates the concentration dependence of copper and mercury mediated inactivation of the recom ⁇ binant wild-type protease;
  • Figure 4 illustrates the concentration dependence of copper mediated inactivation of the synthetic mutant protease, its dependence on added thiol, and its lack of dependence on oxygen.
  • the present invention is generally based on the discovery by the inventors that copper potently inhibits the protease from the human immunodeficiency virus-1
  • HIV-l protease is inhibited by micromolar concentrations of Cu 2+ ions.
  • the protease at 2.5 ⁇ M was 50% inhibited by exposure to 5 ⁇ M copper ion for 5 minutes while exposure to 25 ⁇ M caused complete inhibition.
  • Inactivation by Cu + was rapid and not reversed by subsequent exposure to EDTA nor dithiothreitol.
  • Direct inhibition by Cu 2+ required the presence of cysteine residue(s) in the protease.
  • a synthetic mutant protease lacking cysteine residues was not inhibited by exposure to copper ion.
  • a copper ion delivery agent is employed for delivery of copper ions to the target protease thereby causing inhibition thereof.
  • appropriate copper ion delivery agents include copper salts, metal chelators, and peptides and proteins which bind copper. More preferred examples of these agents include copper chloride; EDTA, o-phenanthroline, histidine, and desferrioxamine; histidine-containing peptides, ceruloplasmin, and albumin.
  • the copper ion delivery agent should possess molecular characteristics which allow it to specifically deliver the copper ion to the protease and which in itself would be inhibitory; thus the delivery agent and the copper may act synergistically.
  • Examples of this preferred class of targeted delivery agents are peptide inhibitors of the protease, including pepstatin-type inhibitors.
  • the copper ion delivery agent may also be combined with or administered in the presence of other components, such as thiols, for example, dithiothreitol, cysteine, glutathione, and their esters and other derivatives.
  • the pharmaceutical composition may contain other pharma ⁇ ceuticals in conjunction with the copper ion delivery agent, wherein the pharmaceuticals are used to therapeuti- cally treat acquired immunodeficiency syndrome (AIDS) .
  • AIDS acquired immunodeficiency syndrome
  • Representative examples of these additional pharmaceuti ⁇ cals include antiviral compounds, immunomodulators and immunostimulants, and antibiotics.
  • antiviral compounds include AZT, ddl, ddC, gancyclovir, fluorinated dideoxynucleotides, etc.
  • immunomodulators and immunostimulants include various interleukins, CD4, cytokines, antibody preparations, blood transfusions, cell transfusions, etc.
  • Exemplary antibiotics include antifun- gal agents, antibacterial agents, anti-Pneumcysitis carnii agents, etc.
  • the copper ion delivery agent of the present invention is capable of inhibiting and inactivating viruses, it may also be employed as an active ingredient in a viral disinfectant along with a suitable carrier.
  • the viral disinfection composition should contain an adequate concentration of the copper ion delivery agent necessary for disinfecting a desired target from viruses, such as HIV type viruses.
  • the method and composition of the present inven ⁇ tion may be employed in the treatment of a variety of viruses including, for example, members of the HTLV family, especially human immunodeficiency viruses HIV-l and HIV-2, as well as other protease-dependent retro- viruses such as HTLV-l and animal leukemia viruses.
  • viruses including, for example, members of the HTLV family, especially human immunodeficiency viruses HIV-l and HIV-2, as well as other protease-dependent retro- viruses such as HTLV-l and animal leukemia viruses.
  • Fig 1. shows the effect of cations on recombinant wild-type protease activity.
  • Enzyme 2.5 ⁇ M was incubated with 25 ⁇ M cation in 150 M sodium acetate, pH 5.5, con ⁇ taining 10% (v/v) glycerol for 5 minutes at 37°C.
  • the assay for activity was then begun by the addition of substrate in 150 mM sodium actetate/6 mM EDTA, yielding a final concentration of 1 mM EDTA.
  • the incubation was stopped after 20 minutes additional incubation.
  • Al and Cr were trivalent; Ca, Co, Mg, Mn, Ni, Pb, Zn, Hg, and Cu were divalent; K was monovalent.
  • protease Both cations have high affinity for amino acids and might be inhibiting proteolysis by binding either to the protease or to the nine-residue peptide substrate. Since protease is present at micromolar concentration and peptide at millimolar, the protease was the more likely target. However, a metal-peptide complex could still be the inhibitory species. The protease was shown to be the actual target of copper ion inhibition by incubating either the pept * _e or the protease with 25 ⁇ M copper for 5 min. Then EDTA was added to 1 mM, followed by protease or peptide to provide a complete assayable system.
  • Fig. 2 illustrates the time course for inactivation of the recombinant wild-type protease by 25 ⁇ M CuCl 2 .
  • Protease 2.5 ⁇ M was incubated with '( ⁇ ) or without (g) copper in 150 mM sodium acetate/10% (v/v) glycerol, pH 5.5 at 37°C.
  • the activity assay was initiated by transferring 10 ⁇ l of the incubation solution to a separate tube containing 2 ⁇ l substrate in 150 mM sodium acetate with 6 mM EDTA.
  • Fig. 3 shows concentration dependence of copper ( ⁇ ) and mercury (g) mediated inactivation of the recombinant wild-type protease.
  • Protease was incubated with the cation for 5 minutes and then assayed as described with regard to Fig. 1.
  • the affinity of binding has not yet been determined so one cannot deduce the stoichiometry of binding from the concentration dependence.
  • binding is very tight (stoichio etric)
  • the minimal requirement for inhibition is the binding of about l mercury cation per protease subunit or about 2 copper cations. Further, the binding of the first copper does not appear to affect activity.
  • a thiol may be co-administered with the copper ion delivery agent or the latter administered in the presence of a thiol to achieve desired protease inhibi ⁇ tion. Also, normal plasma and cell content of thiols may be adequate to support copper inhibition properties.
  • the protease was the wild-type.
  • One ⁇ l additions were made to 19 ⁇ l enzyme solution, followed by 2.5 min incuba ⁇ tion at 37°C.
  • Stock solutions used for the additions were 500 ⁇ M CuCl 2 , 500 ⁇ M HgCl 2 , 200 mM dithiothreitol, and 20 mM EDTA.
  • One ⁇ l water was substituted when the addition was "none”.
  • Activity was assayed for 20 minutes following the final incubation. The assay was initiated by adding substrate in 4 ⁇ l 150 mM sodium acetate, pH 5.5, contain ⁇ ing 6 mM EDTA.
  • the protease concentration was 2.0 ⁇ M in the first preparation and 1.64 ⁇ M in the second.
  • the role of cysteine residues in mediating copper inhibition might be probed with protease in which the cysteines were alkylated.
  • the carboxyamidomethylated enzyme was catalytically inactive, so that the effect of copper could not be evaluated.
  • the essential role of cysteine was demonstrated by investigation of a variant protease which lacked cysteine residues.
  • This variant enzyme was produced by solid-phase synthesis, with the two cysteine residues replaced by ⁇ -amino butyric acid (Schneider, J. et al., (1988) Cell 54, 363-368). Copper and mercury did not inhibit this variant protease as shown below in Table 4. Table 4. Effect of oxygen and dithiothreitol on protease activity
  • the concentration of dithiothreitol was 10 mM.
  • Figure 4 illustrates the concentration dependence of copper mediated inactivation of the synthetic mutant protease its dependence on added thiol, and its lack of dependence on oxygen.
  • Enzyme (4.3 ⁇ M) was incubated with the cation for 5 minutes, after which either dithiothreitol or water was added. Following an additional 5 minutes incubation, protease activity was assayed as described in Fig. 1. Incubations were aerobic (squares) or anaerobic (circles) . Open symbols represent incubations without dithiothreitol and closed symbols with 10 mM dithiothreitol.
  • Protease was refolded as follows. First, the enzyme was dialyzed against 6 M guanidine HC1, 50 mM Tris (pH 7.8), 1 mM EDTA, 5 mM dithiothreitol at ambient temperature for 2 hr. The enzyme solution was dialyzed next at 4°C against 3 M guanidine HCl, 50 mM Tris (pH 7.8), 1 mM EDTA, 1 mM dithiothreitol for 2 hr, followed by an additional 2 hr dialysis against 1 M guanidine HCl, 50 mM Tris (pH 7.8), 1 mM EDTA, 1 mM dithiothreitol.
  • the final dialysis was into 20 mM HCl, with an additional change of the HCl solution before overnight dialysis.
  • the dialysis tubing (Spectrum Medical Industries, Los Angeles, CA) had a nominal molecular weight cutoff of 6,000-8,000.
  • the ratio of protease volume to dialysate was 1:100 for guanidine solutions and 1:2000 for the HCl.
  • the absorbance of the 2-nitro-5-thiobenzoate was measured at 412 nm against a reagent blank in a Hewlett Packard model 8450 spectrophotometer.
  • the concentration of the sulfhydryl groups was calculated using a molar absorption coefficient of 13,700 for the dianion (Riddles, P. W. et al., (1979) Anal. Biochem . 94, 75-81.).
  • the carboxyamidomethyl-cysteine derivative of the protease was prepared by treatment with iodoacetamide (Means, G. E. et al. , (1971) Chemical Modification of Proteins (Holden-Day, Inc., San Francisco, CA) , pp. 105- 138) .
  • iodoacetamide Means, G. E. et al. , (1971) Chemical Modification of Proteins (Holden-Day, Inc., San Francisco, CA) , pp. 105- 138
  • one volume of enzyme in 20 mM HCl was mixed with three volumes of 8 M guanidine HCl, 133 mM Tris, 13.3 mM EDTA giving a final pH of 8.0, and then incubated with 5 mM dithio ⁇ threitol for 15 minutes at 37°C.
  • the solution was then made 20 mM in iodoacetamide, incubated at room temperature for 2 hr in the dark, and quenched with excess dithio ⁇ threitol (10 mM) .
  • the sample was then dialyzed into 20 mM HCl as described for enzyme refolding.
  • Oxidized and reduced dithiothreitol were quantitated by high pressure liquid chromatography with monitoring at 210 nm. These compounds are well-separated from the products and substrate of the protease assay and could therefore be quantified using the same analytical system as for the protease assay (Boutelje, J. et al., supra.).
  • the copper ion delivery agents employed in the present invention may be made into pharmaceutical composi ⁇ tions by combination with appropriate pharmaceutically acceptable carriers or diluents, and may be formulated into preparations in solid, semi-solid, liquid or gaseous forms such as tablets, capsules, powders, granules oint- ments, solutions, suppositories, injections, inhalants, and aerosols in the usual ways for their respective route of administration.
  • the following methods and excipients are merely exemplary and are in no way limiting.
  • the copper ion delivery agents employed in the present invention may be used in the form of their pharmaceutically acceptable salts and other compounds, and also may be used alone or in appropriate association, as well as in combination with other pharmaceutically active compounds.
  • the copper ion delivery agents may be used alone or in combination with appropriate additives to make tablets, powders, granules or capsules, e.g.
  • the copper ion delivery agents employed in the present invention may be made into suppos ⁇ itories by mixing with a variety of bases such as emulsi ⁇ fying bases or water-soluble bases.
  • the copper ion delivery agents employed in the present invention may be formulated into preparations for injections by dissolving, suspending or emulsifying them in an aqueous or non-aqueous solvent, such as vegetable oil, synthetic aliphatic acid glycerides, esters of higher aliphatic acids or propylene glycol; and if desired, with conventional additives such as solubilizers, isotonic agents, suspending agents, emulsifying agents, stabilizers and preservatives.
  • an aqueous or non-aqueous solvent such as vegetable oil, synthetic aliphatic acid glycerides, esters of higher aliphatic acids or propylene glycol
  • solubilizers isotonic agents
  • suspending agents emulsifying agents, stabilizers and preservatives.
  • the copper ion delivery agents employed in the invention in the form of a liquid or minute powder may be filled up in an aerosol container with gas or liquid spraying agents, and if desired, together with conven ⁇ tional adjuvants such as humidifying agents. They may also be formulated as pharmaceuticals for non-pressured preparations such as in a nebulizer or an atomizer.
  • a suitable dosage is that which will result in concentration of the copper ion delivery agents in blood and/or tissues harboring the virus which are believed to inhibit the virus.
  • the preferred dosage is that amount sufficient to render a host free of the particular viral infection.
  • the dose may vary when the compounds are used prophylactically.
  • the dose may be adjusted to provide the concentration to bind two copper ions to the protease in order to assure complete inhibition.
  • Unit dosage forms for oral administration such as syrups, elixirs, and suspensions wherein each dosage unit, e.g., teaspoonful, tablespoonful, contains a predetermined amount of the copper ion delivery agents employed in the present invention can be by a pharmaceutically acceptable carrier, such as Sterile Water for Injection, USP, or by normal saline.
  • a pharmaceutically acceptable carrier such as Sterile Water for Injection, USP, or by normal saline.
  • the copper ion delivery agents employed in the present invention can be administered rectally via a suppository.
  • the suppository can include vehicles such as cocoa butter, carbowaxes and polyethylene glycols, which melt at body temperature, yet are solidified at room temperature.
  • the copper ion delivery agents employed in the present invention may also be administered transdermally when combined with appropriate carriers, such as for example dimethylsulfoxide.
  • the copper ion delivery agents employed in the present invention can be utilized in aerosol formulation to be administered via inhalation.
  • the copper ion delivery agents employed in the present invention can be formulated into pressurized acceptable propellants such as dichlorodifluoromethane, propane, nitrogen and the like.
  • unit dosage form refers to physically discrete units suitable as unitary dosages for human and animal subjects, each unit containing a predetermined quantity of the copper ion delivery agents calculated in an amount sufficient to produce the desired effect in association with a pharmaceutically acceptable, diluent, carrier or vehicle.
  • the specifications for the novel unit dosage forms of the present invention depend on the particular compound employed and the effect to be achieved, and the pharmacodynamics associated with each compound in the host.
  • the pharmaceutically acceptable excipients for example, vehicles, adjuvants, carriers or diluents are readily available to the public.

Abstract

A method for inhibiting protease from human immunodeficiency virus and for treating viral infections of a host in need thereof which employs a composition containing a copper ion delivery agent. Treatment of the protease of HIV-1 with micromolar concentrations of copper results in complete inhibition of the protease.

Description

METHODS AND PHARMACEUTICAL COMPOSITIONS FOR INHIBITING PROTEASE FROM HUMAN IMMUNODEFICIENCY VIRUS
BACKGROUND OF THE INVENTION Field of the Invention The present invention relates to methods for inhibiting human immunodeficiency viruses and pharmaceuti¬ cal compositions useful therein. More specifically, the present invention relates to methods for inhibiting HIV protease with copper ion, as well as pharmaceutical compositions employing a copper ion delivery agent for use in these methods. Description of Related Art
The aspartyl protease encoded by the human immuno¬ deficiency virus type 1 (HIV) is essential for the pro- cessing of the viral polyproteins encoded by the gag and pol genes into mature viral proteins. Mutation or dele¬ tion of the protease gene blocks replication of the virus, making the protease an attractive target for antiviral therapy of the acquired immunodeficiency syndrome (AIDS) . The inhibitors reported thus far are peptides or peptide analogues, some of which were originally studied as inhibitors of other structurally related aspartyl proteases such as pepsin or renin.
Another approach to the inhibition of the protease was suggested by studies of metal-catalyzed oxidation of proteins. Both enzymic and non-enzymic metal-catalyzed oxidation systems are capable of oxidatively inactivating many enzymes. Such systems consist of a redox-cycling metal cation such as copper or iron, a reducing agent, and molecular oxygen. Cytochrome P450/NADPH/O2 is an example of an enzymic system (Fucci, L. et al., (1983) Proc . Natl . Acad . Sci . USA 80, 1521-1525) while Fe/ascorbate/02 is a well-studied non-enzymic system (Levine, R.L. (1983) J. Biol . Chem . 258, 11828-1833). For the latter, it is believed that the iron binds to the enzyme at a specific cation binding site. Oxidation of the reduced form of the metal generates a very reactive oxidizing species, such as the hydroxyl radical. The radical reacts with an a ino acid residue very close to its site of generation, gener¬ ally inactivating the enzyme. In the case of gluta ine synthetase, the site specificity has been studied in detail. Specificity results from the binding of the redox-capable cation to the two binding sites on the enzyme which would normally bind magnesium.
The aspartyl proteases, especially pepsin, have been studied in great detail. The active site of this class of proteases always contains two aspartyl residues, and these could conceivably bind a cation. X-ray crystal- lographic studies of several aspartyl proteases have employed heavy-metal derivatives ( lodawer, A. et al. , (1989) Science 245, 616-620; Miller, M. et al., (1989) Nature (London) 337, 576-579; and Navia, M. et al., (1989) Nature (London) 337, 615-620) thus establishing the existence of cation binding sites in these proteins. The effect of metal ions on pepsin has been examined by many groups (Lundblad, R. L. et al. , (1969) J. Biol . Chem . 244, 154-160; Rajagopalan, T. G. et al. , (1966) J. Biol . Chem. 241, 4295-4297; Husain, S. S. et al. , (1971) Proc. Natl . Acad. Sci . USA 68, 2765-2768; and Kirchgessner, M. et al., (1976) Br. J. Nutr. 36, 15-22) . In general, addition of copper to a pepsin/substrate mixture increases the rate of proteolysis, possibly through an effect on the substrate rather than the enzyme. One report suggested that inhibi¬ tion of pepsin might be observable under certain condi¬ tions (Kirchgessner, M. et al., supra.). However, Lundblad and Stein (Lundblad, R. L. et al., supra.) specifically examined the effect of copper on pepsin and reported that there was no change in the catalytic activity. Copper does accelerate the inactivation of pepsin by diazo compounds but this effect was shown to result from the action of copper on the inhibitor (Lundblad, R. L. et al., supra; Husain, S. S. et al., supra.) .
Given this sizeable literature on the aspartyl proteases, direct and specific inhibition of the HIV protease by copper is unexpected. SUMMARY OF THE INVENTION Therefore, it is an object of the present inven¬ tion to provide a method for inhibiting the protease from human immunodeficiency virus. It is another object of the present invention to provide a method of treating viral infections of a host in need thereof by administrating an antiviral effective amount of a pharmaceutical composition containing a copper ion delivery agent. It is a further object of the present invention to provide the basis for a pharmaceutical composition for treating viral infections, such as those caused by human immunodeficiency virus.
It is yet another object of the present invention to provide a viral disinfectant composition and viral disinfecting method wherein a disinfectant composition containing a copper ion delivery agent is employed.
The foregoing objects and others are accomplished in accordance with the present invention by providing a method for inhibiting the protease from human immunodefi¬ ciency virus which includes administering thereto an antiviral effective amount of a copper ion delivery agent. Another method encompassed by the present invention includes treating viral infections of a host in need thereof wherein an antiviral effective amount of a copper ion delivery agent is administered to the host.
In another embodiment of the present invention, a pharmaceutical composition is provided for treating viral infections of a host in need thereof wherein the composi- tion contains a copper ion delivery agent and a pharmaceu¬ tically acceptable excipient.
Further scope of the applicability of the present invention will become apparent from the detailed descrip¬ tion and drawings provided below. However, it should be understood that the detailed description and specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description. BRIEF DESCRIPTION OF THE DRAWINGS The invention is further described in the accom- panying drawings wherein:
Figure 1 illustrates the effect of cations on recombinant wild-type protease activity;
Figure 2 is a time course for inactivation of the recombinant wild-type protease by 25 μm CuCl2; Figure 3 illustrates the concentration dependence of copper and mercury mediated inactivation of the recom¬ binant wild-type protease; and
Figure 4 illustrates the concentration dependence of copper mediated inactivation of the synthetic mutant protease, its dependence on added thiol, and its lack of dependence on oxygen.
DETAILED DESCRIPTION OF THE INVENTION
The present invention is generally based on the discovery by the inventors that copper potently inhibits the protease from the human immunodeficiency virus-1
(HIV-l) . Further, copper inhibition is not oxygen dependent. As evidenced by the results discussed in more detail below, HIV-l protease is inhibited by micromolar concentrations of Cu2+ ions. For example, the protease at 2.5 μM was 50% inhibited by exposure to 5 μM copper ion for 5 minutes while exposure to 25μM caused complete inhibition. Inactivation by Cu + was rapid and not reversed by subsequent exposure to EDTA nor dithiothreitol. Direct inhibition by Cu2+ required the presence of cysteine residue(s) in the protease. Thus, a synthetic mutant protease lacking cysteine residues was not inhibited by exposure to copper ion. However, addition of dithiothreitol as an exogenous thiol rendered even the synthetic mutant protease susceptible to inactivation by copper ion. Oxygen was not required for inactivation of either the recombinant wild-type nor synthetic mutant protease.
In the method and pharmaceutical composition of the present invention, a copper ion delivery agent is employed for delivery of copper ions to the target protease thereby causing inhibition thereof. Generally, examples of appropriate copper ion delivery agents include copper salts, metal chelators, and peptides and proteins which bind copper. More preferred examples of these agents include copper chloride; EDTA, o-phenanthroline, histidine, and desferrioxamine; histidine-containing peptides, ceruloplasmin, and albumin. The copper ion delivery agent should possess molecular characteristics which allow it to specifically deliver the copper ion to the protease and which in itself would be inhibitory; thus the delivery agent and the copper may act synergistically. Examples of this preferred class of targeted delivery agents are peptide inhibitors of the protease, including pepstatin-type inhibitors. The copper ion delivery agent may also be combined with or administered in the presence of other components, such as thiols, for example, dithiothreitol, cysteine, glutathione, and their esters and other derivatives.
In another embodiment of the present invention, the pharmaceutical composition may contain other pharma¬ ceuticals in conjunction with the copper ion delivery agent, wherein the pharmaceuticals are used to therapeuti- cally treat acquired immunodeficiency syndrome (AIDS) .
Representative examples of these additional pharmaceuti¬ cals include antiviral compounds, immunomodulators and immunostimulants, and antibiotics. Exemplary antiviral compounds include AZT, ddl, ddC, gancyclovir, fluorinated dideoxynucleotides, etc. Exemplary immunomodulators and immunostimulants include various interleukins, CD4, cytokines, antibody preparations, blood transfusions, cell transfusions, etc. Exemplary antibiotics include antifun- gal agents, antibacterial agents, anti-Pneumcysitis carnii agents, etc.
Since the copper ion delivery agent of the present invention is capable of inhibiting and inactivating viruses, it may also be employed as an active ingredient in a viral disinfectant along with a suitable carrier. The viral disinfection composition should contain an adequate concentration of the copper ion delivery agent necessary for disinfecting a desired target from viruses, such as HIV type viruses.
The method and composition of the present inven¬ tion may be employed in the treatment of a variety of viruses including, for example, members of the HTLV family, especially human immunodeficiency viruses HIV-l and HIV-2, as well as other protease-dependent retro- viruses such as HTLV-l and animal leukemia viruses.
Inhibition by Copper and Mercury. Pepsin and other aspartyl proteases are generally not inhibited by divalent cations, including copper (Lundblad, R. L. et al., supra.) . However, our studies of the susceptibility of the HIV protease to metal-catalyzed oxidation led to the discovery that micromolar concentrations of copper or mercury caused marked inhibition of the enzyme.
Fig 1. shows the effect of cations on recombinant wild-type protease activity. Enzyme (2.5μM) was incubated with 25μM cation in 150 M sodium acetate, pH 5.5, con¬ taining 10% (v/v) glycerol for 5 minutes at 37°C. The assay for activity was then begun by the addition of substrate in 150 mM sodium actetate/6 mM EDTA, yielding a final concentration of 1 mM EDTA. The incubation was stopped after 20 minutes additional incubation. Al and Cr were trivalent; Ca, Co, Mg, Mn, Ni, Pb, Zn, Hg, and Cu were divalent; K was monovalent.
Addition of the chelator EDTA immediately after adding the metal could not prevent inhibition. When EDTA was added just before the metal, inhibition by copper was blocked but not inhibition by mercury.
Both cations have high affinity for amino acids and might be inhibiting proteolysis by binding either to the protease or to the nine-residue peptide substrate. Since protease is present at micromolar concentration and peptide at millimolar, the protease was the more likely target. However, a metal-peptide complex could still be the inhibitory species. The protease was shown to be the actual target of copper ion inhibition by incubating either the pept* _e or the protease with 25 μM copper for 5 min. Then EDTA was added to 1 mM, followed by protease or peptide to provide a complete assayable system. Preincu- bation of the peptide caused no inhibition of activity while preincubation of the protease led to virtually complete loss of enzymatic activity as shown below in Table 1. Table 1. Effect of order of addition upon inhibition of the recombinant wild-type HIV protease.
Figure imgf000009_0001
Mixtures were incubated 5 min at 37°C between additions. The final concentration of CuCl2 was 25 μM and that of EDTA was 1 mM.
Inhibition is rapid, as shown in Fig. 2 which illustrates the time course for inactivation of the recombinant wild-type protease by 25μM CuCl2. Protease (2.5 μM) was incubated with '(§) or without (g) copper in 150 mM sodium acetate/10% (v/v) glycerol, pH 5.5 at 37°C. The activity assay was initiated by transferring 10 μl of the incubation solution to a separate tube containing 2 μl substrate in 150 mM sodium acetate with 6 mM EDTA.
The concentration dependence of inhibition is plotted in Fig. 3 which shows concentration dependence of copper (§) and mercury (g) mediated inactivation of the recombinant wild-type protease. Protease was incubated with the cation for 5 minutes and then assayed as described with regard to Fig. 1. The affinity of binding has not yet been determined so one cannot deduce the stoichiometry of binding from the concentration dependence. However, if one assumes that binding is very tight (stoichio etric) , then the minimal requirement for inhibition is the binding of about l mercury cation per protease subunit or about 2 copper cations. Further, the binding of the first copper does not appear to affect activity.
Involvement of Thiols. Copper and especially mercury tend to bind to the sulfhydryl group of cysteine residues. The HIV protease has two Cys residues. Cys67 lies on the surface of the enzyme while Cys95 participates in forming the dimer interface of the active protease. Treatment with dithiothreitol restored at least 80% of the activity of the mercury-inhibited enzyme as shown below in Table 2. Although some forms of the viral protease may lack cysteine residues, such as for example HIV-2, inacti¬ vation still occurs if the thiol is supplied exogenously. Therefore, a thiol may be co-administered with the copper ion delivery agent or the latter administered in the presence of a thiol to achieve desired protease inhibi¬ tion. Also, normal plasma and cell content of thiols may be adequate to support copper inhibition properties.
Table 2. Restoration of protease activity
Figure imgf000010_0001
The protease was the wild-type. One μl additions were made to 19 μl enzyme solution, followed by 2.5 min incuba¬ tion at 37°C. Stock solutions used for the additions were 500 μM CuCl2, 500 μM HgCl2, 200 mM dithiothreitol, and 20 mM EDTA. One μl water was substituted when the addition was "none". Activity was assayed for 20 minutes following the final incubation. The assay was initiated by adding substrate in 4 μl 150 mM sodium acetate, pH 5.5, contain¬ ing 6 mM EDTA. Treatment of the copper-inhibited enzyme with dithiothreitol gave a variable, but low recovery of 10-30%. Nevertheless, the copper-treated enzyme had not been irreversibly inhibited. Refolding of the enzyme from 6 M guanidine restored activity to the same level as that of a control not .treated with copper.
The ability of one metal to displace the other from the enzyme was then examined by taking advantage of the observation that the mercury-inhibited enzyme was reactivated by treatment with dithiothrejtol while the copper-inhibited enzyme was not. Neither metal appeared able to displace the other during the 2.5 minutes incuba¬ tion (Table 2) . This result suggests that the off-rate of bound metal is slow, consistent with the observed high affinity of binding. Protease was also titrated with the sulfhydryl reagent 5,5'-dithiobis(2-nitrobenzoic acid) (Ellman's reagent) , although the accuracy of the determination was limited by the amount of protein available. As shown in Table 3 below, untreated protease had about 2 titratable sulfhydryls per subunit under denaturing conditions. Copper-inhibited protease showed a small decrease in sulfhydryl groups while mercury-inhibited protease lost most of its titratable sulfhydryls. Table 3. Titratable sulf ydryl groups in the recombinant wild-type protease.
Protease Treatment
Experiment 1: None
100 μM Cu2+
100 μM Hg2+
Experiment 2:
None 100 μM Cu2+
100 μM Hg2+
Figure imgf000012_0001
The protease concentration was 2.0 μM in the first preparation and 1.64 μM in the second. The role of cysteine residues in mediating copper inhibition might be probed with protease in which the cysteines were alkylated. However, as reported by Meek and colleagues (Meek, T. D. et al., (1989) Proc. Natl. Acad. Sci. USA 86, 1841-1845) , the carboxyamidomethylated enzyme was catalytically inactive, so that the effect of copper could not be evaluated. However, the essential role of cysteine was demonstrated by investigation of a variant protease which lacked cysteine residues. This variant enzyme was produced by solid-phase synthesis, with the two cysteine residues replaced by α-amino butyric acid (Schneider, J. et al., (1988) Cell 54, 363-368). Copper and mercury did not inhibit this variant protease as shown below in Table 4. Table 4. Effect of oxygen and dithiothreitol on protease activity
Protease Metal Dithiothreitol Oxygen Activity,
% of Control
Wild-type Cu2+ , 25 μM - - 0
+ 0
+ + 98
- 98
98
93
Figure imgf000013_0001
When added, the concentration of dithiothreitol was 10 mM.
Effect of Oxygen. The discrepancy of recovery of activity upon dithiothreitol treatment was curious. It seemed likely that both copper and mercury inhibited the protease by direct binding. Addition of dithiothreitol to the inhibited enzyme should reverse the inhibition, if the reactions were relatively fast. However, it is known that iron or copper/ thiol/ oxygen form a potent metal-cata¬ lyzed oxidizing system capable of inactivating many enzymes. Thus, addition of dithiothreitol to the copper- treated enzyme might also cause oxidative modification of the protease. This possibility was considered in experiments which examined the effects of oxygen and dithiothreitol on the inhibition of the recombinant wild-type protease and on the variant protease which lacked cysteine residues. Either anaerobic or aerobic incubation of the recombinant wild-type protease with copper caused loss of enzymatic activity (Table 4) . Incubation of the synthetic protease with copper alone had no effect on proteolytic activity.
However, the addition of dithiothreitol revealed a copper-dependent inactivation which did not require either oxygen or cysteine (Table 4) . That is, the synthetic protease was inactivated when incubated with copper and dithiothreitol, even in the absence of oxygen. No signif¬ icant inactivation occurred when the synthetic protease was exposed to reduced dithiothreitol alone, nor to oxidized dithiothreitol with or without copper.
The extent of inactivation by copper and dithio- threitol varied with the concentration of copper. Figure 4 illustrates the concentration dependence of copper mediated inactivation of the synthetic mutant protease its dependence on added thiol, and its lack of dependence on oxygen. Enzyme (4.3 μM) was incubated with the cation for 5 minutes, after which either dithiothreitol or water was added. Following an additional 5 minutes incubation, protease activity was assayed as described in Fig. 1. Incubations were aerobic (squares) or anaerobic (circles) . Open symbols represent incubations without dithiothreitol and closed symbols with 10 mM dithiothreitol. This observation was consistent with several possible mecha¬ nisms of inactivation, the simplest of which would be direct inhibition by Cu1+, produced through reduction of Cu2+ by dithiothreitol. However, aerobic exposure of the protease to 100 μM CuCl for 10 minutes had no effect on proteolytic activity. Thus, neither the cuprous nor cupric form of copper alone were capable of inhibiting the synthetic protease. However, addition of 10 mM dithio¬ threitol during the last 5 minutes incubation with CuCl caused complete loss of activity, paralleling the results with CuCl2. EXAMPLES MATERIALS AND METHODS HIV-l protease. Production, purification, and assay of the recombinant wild-type protease in Eεcherichia coli were as described (Boutelje, J. et al. , (1990) Arch . Biochem . Biophys . 283, 141-149) . The chemically synthe¬ sized protease (Schneider, J. et al., supra. ) , a generous gift from Dr. Steven Kent, was obtained as a lyophilized powder. Before use, this synthetic protease was dissolved in 6 M guanidine HC1, 50 mM Tris (pH 7.8), 5 mM DTT, and lmM EDTA and refolded as described below. Both the recom¬ binant and the synthetic protease were stored at -70°C in 20 mM HC1 at protein concentrations of 100-200 μg/ml.
Anaerobic experiments were performed in the Anaerobic Laboratory of the National Institutes of Health (Poston, J. M. et al., (1971) Methods Enzymol . 22, 49-54). The atmosphere in this laboratory was constantly monitored and never exceeded an oxygen content of 5 pp . After entry into the anaerobic room, protease and peptide solutions were pump purged ten times and buffers were sparged with purified argon for at least 10 minutes.
Protease was refolded as follows. First, the enzyme was dialyzed against 6 M guanidine HC1, 50 mM Tris (pH 7.8), 1 mM EDTA, 5 mM dithiothreitol at ambient temperature for 2 hr. The enzyme solution was dialyzed next at 4°C against 3 M guanidine HCl, 50 mM Tris (pH 7.8), 1 mM EDTA, 1 mM dithiothreitol for 2 hr, followed by an additional 2 hr dialysis against 1 M guanidine HCl, 50 mM Tris (pH 7.8), 1 mM EDTA, 1 mM dithiothreitol. The final dialysis was into 20 mM HCl, with an additional change of the HCl solution before overnight dialysis. The dialysis tubing (Spectrum Medical Industries, Los Angeles, CA) had a nominal molecular weight cutoff of 6,000-8,000. The ratio of protease volume to dialysate was 1:100 for guanidine solutions and 1:2000 for the HCl.
Cation Inhibition. Stock solutions of l M cations were made by dissolving the salts in water with acidifica¬ tion by HCl to pH 3 - 5. Typically, protease (2.5 μM) was incubated with 25-100 μM cation for 5 minutes at 37° in 10 μl 150 mM sodium acetate, pH 5.5, containing 10% (v/v) glycerol. The assay was started by adding substrate in 2 μl of 150 mM sodium acetate (pH 5.5), 6 mM EDTA, yielding 1 mM EDTA in the assay solution. After 20 minutes at 37°C, products were quantitated by high pressure liquid chromatography as previously described (Boutelje, J. et al., supra. ) .
Sulfhydryl Titration. Ellman's reagent, 5,5'- dithiobis(2-nitrobenzoic acid) was used to quantitate sulfhydryl groups in protein denatured in 6M guanidine HCl. To one volume of protease in 150 mM sodium acetate, pH 5.5, was mixed three volumes of 1.3 mM 5,5'- dithiobis(2-nitrobenzoic acid) in 8 M guanidine HCl, 133 mM Tris, and 13.3 mM EDTA giving a final pH of 7.4. After mixing, the solution was incubated at 37°C for 5 minutes. The absorbance of the 2-nitro-5-thiobenzoate was measured at 412 nm against a reagent blank in a Hewlett Packard model 8450 spectrophotometer. The concentration of the sulfhydryl groups was calculated using a molar absorption coefficient of 13,700 for the dianion (Riddles, P. W. et al., (1979) Anal. Biochem . 94, 75-81.).
The carboxyamidomethyl-cysteine derivative of the protease was prepared by treatment with iodoacetamide (Means, G. E. et al. , (1971) Chemical Modification of Proteins (Holden-Day, Inc., San Francisco, CA) , pp. 105- 138) . To assure reduction of the cysteine residues, one volume of enzyme in 20 mM HCl was mixed with three volumes of 8 M guanidine HCl, 133 mM Tris, 13.3 mM EDTA giving a final pH of 8.0, and then incubated with 5 mM dithio¬ threitol for 15 minutes at 37°C. The solution was then made 20 mM in iodoacetamide, incubated at room temperature for 2 hr in the dark, and quenched with excess dithio¬ threitol (10 mM) . The sample was then dialyzed into 20 mM HCl as described for enzyme refolding.
Analytical Methods. The protease concentration was calculated from the absorbance at 280 nm, corrected for light scatter (Levine, R. L. et al., (1982) Biochemistry 21, 2600-2606) , using molar absorptivities calculated (Mihalyi, E. (1968) J. Chem. En . Data 13, 179- 182) from the sequence of the protease (e=12,300). The accuracy of this method was confirmed by amino acid analysis after acid hydrolysis of the protease. Oxidized dithiothreitol was prepared by stirring a solution of reduced dithiothreitol in room air overnight. Oxidized and reduced dithiothreitol were quantitated by high pressure liquid chromatography with monitoring at 210 nm. These compounds are well-separated from the products and substrate of the protease assay and could therefore be quantified using the same analytical system as for the protease assay (Boutelje, J. et al., supra.).
The copper ion delivery agents employed in the present invention may be made into pharmaceutical composi¬ tions by combination with appropriate pharmaceutically acceptable carriers or diluents, and may be formulated into preparations in solid, semi-solid, liquid or gaseous forms such as tablets, capsules, powders, granules oint- ments, solutions, suppositories, injections, inhalants, and aerosols in the usual ways for their respective route of administration. The following methods and excipients are merely exemplary and are in no way limiting.
In pharmaceutical dosage forms, the copper ion delivery agents employed in the present invention may be used in the form of their pharmaceutically acceptable salts and other compounds, and also may be used alone or in appropriate association, as well as in combination with other pharmaceutically active compounds. In the case of oral preparations, the copper ion delivery agents may be used alone or in combination with appropriate additives to make tablets, powders, granules or capsules, e.g. with conventional additives such as lactose, mannitol, corn starch or potato starch; with binders such as crystalline cellulose, cellulose derivatives, acacia, corn starch or gelatins; with disintegrators such as corn starch, potato starch or sodium carboxymethylcellulose; with lubricants such as talc or magnesium stearate; and if desired, with diluents, buffering agents, moistening agents, preservatives and flavoring agents.
Furthermore, the copper ion delivery agents employed in the present invention may be made into suppos¬ itories by mixing with a variety of bases such as emulsi¬ fying bases or water-soluble bases.
The copper ion delivery agents employed in the present invention may be formulated into preparations for injections by dissolving, suspending or emulsifying them in an aqueous or non-aqueous solvent, such as vegetable oil, synthetic aliphatic acid glycerides, esters of higher aliphatic acids or propylene glycol; and if desired, with conventional additives such as solubilizers, isotonic agents, suspending agents, emulsifying agents, stabilizers and preservatives.
In the cases of inhalations or aerosol prepara¬ tions, the copper ion delivery agents employed in the invention in the form of a liquid or minute powder may be filled up in an aerosol container with gas or liquid spraying agents, and if desired, together with conven¬ tional adjuvants such as humidifying agents. They may also be formulated as pharmaceuticals for non-pressured preparations such as in a nebulizer or an atomizer. A suitable dosage is that which will result in concentration of the copper ion delivery agents in blood and/or tissues harboring the virus which are believed to inhibit the virus. The preferred dosage is that amount sufficient to render a host free of the particular viral infection. The dose may vary when the compounds are used prophylactically. The dose may be adjusted to provide the concentration to bind two copper ions to the protease in order to assure complete inhibition.
Unit dosage forms for oral administration such as syrups, elixirs, and suspensions wherein each dosage unit, e.g., teaspoonful, tablespoonful, contains a predetermined amount of the copper ion delivery agents employed in the present invention can be by a pharmaceutically acceptable carrier, such as Sterile Water for Injection, USP, or by normal saline.
The copper ion delivery agents employed in the present invention can be administered rectally via a suppository. The suppository can include vehicles such as cocoa butter, carbowaxes and polyethylene glycols, which melt at body temperature, yet are solidified at room temperature.
The copper ion delivery agents employed in the present invention may also be administered transdermally when combined with appropriate carriers, such as for example dimethylsulfoxide.
The copper ion delivery agents employed in the present invention can be utilized in aerosol formulation to be administered via inhalation. The copper ion delivery agents employed in the present invention can be formulated into pressurized acceptable propellants such as dichlorodifluoromethane, propane, nitrogen and the like.
The term "unit dosage form" as used herein refers to physically discrete units suitable as unitary dosages for human and animal subjects, each unit containing a predetermined quantity of the copper ion delivery agents calculated in an amount sufficient to produce the desired effect in association with a pharmaceutically acceptable, diluent, carrier or vehicle. The specifications for the novel unit dosage forms of the present invention depend on the particular compound employed and the effect to be achieved, and the pharmacodynamics associated with each compound in the host. The pharmaceutically acceptable excipients, for example, vehicles, adjuvants, carriers or diluents are readily available to the public.
Any necessary adjustments in dose can be readily made to meet the severity of the infection and adjusted accordingly by the skilled practitioner.
The invention 'being thus described, it will be obvious that the same may be varied in many ways. Such variations are not to be regarded as a departure from the spirit and scope of the invention, and all such modifica¬ tions as would be obvious to one skilled in the art are intended to be included within the scope of the following claims.

Claims

WHAT IS CLAIMED IS:
1. A method for treating viral infections of a host needing treatment which comprises administering to the host an antiviral effective amount of a composition comprising a copper ion delivery agent and a pharmaceuti¬ cally acceptable excipient.
2. The method of claim 1, wherein said viral infection is caused by human immunodeficiency virus.
3. The method of claim 1, wherein said copper ion delivery agent is a copper salt.
4. A method for inhibiting protease from a virus by administering thereto an effective amount of a composi¬ tion containing a copper ion delivery agent and a pharma¬ ceutically acceptable excipient.
5. The method of claim 4, wherein said copper ion delivery agent is a copper salt.
6. The method of claim 4, wherein the amount of composition administered is sufficient to effect inhibi¬ tion of human immunodeficiency viruses.
7. A pharmaceutical composition for treating viral infections caused by human immunodeficiency virus which comprises a copper ion delivery agent and a pharma¬ ceutically acceptable excipient.
8. The method of claim 7, wherein said copper ion delivery agent is a copper salt.
9. The method of claim 1, wherein said copper ion delivery agent is selected from the group consisting of copper salts, metal chelators, peptides and proteins which bind copper.
10. The method of claim 4, wherein said copper ion delivery agent is selected from the group consisting of copper salts, metal chelators, peptides and proteins which bind copper.
11. The composition of claim 7, wherein said copper ion delivery agent is selected from the group consisting of copper salts, metal chelators, peptides and proteins which bind copper.
12. The method of claim 1, wherein said composition further comprises a thiol component.
13. The method of claim 4, wherein said composi¬ tion further comprises a thiol component.
14. The composition of claim 7, wherein said composition further comprises a thiol component.
15. The composition of claim 7, wherein the copper ion delivery agent is present in an amount suffi¬ cient to inhibit human immunodeficiency viruses.
16. A viral disinfectant composition for disin- fecting a desired target from viruses which comprises a copper ion delivery agent and a suitable carrier.
17. A method for disinfecting a virally infected target which comprises administering to the target an effective amount of a viral disinfectant composition containing a copper ion delivery agent and a suitable carrier.
18. The composition of claim 7, further compris¬ ing a drug selected from the group consisting of antiviral compounds, immunomodulators, immunostimulants and antibi- otics.
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WO1994018230A1 (en) * 1993-02-02 1994-08-18 Procyte Corporation Inhibition of hiv replication by peptide-copper complexes
WO1996006639A2 (en) * 1994-09-01 1996-03-07 Medico Pharma Vertriebs Gmbh Novel drugs containing chelate-forming agents
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WO2001012168A2 (en) * 1999-08-13 2001-02-22 Faculteit Geneeskunde Universiteit Utrecht Antiviral pharmaceutical compositions containing iron chelators
US6989263B1 (en) * 1994-09-23 2006-01-24 The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services Method for identifying and using compounds that inactivate HIV-1 and other retroviruses by attacking highly conserved zinc fingers in the viral nucleocapsid protein
EP3003045A4 (en) * 2013-03-15 2017-01-25 CDA Research Group, Inc. Topical copper ion treatments in the genital-rectal areas of the body
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WO1994018230A1 (en) * 1993-02-02 1994-08-18 Procyte Corporation Inhibition of hiv replication by peptide-copper complexes
EP0865217A2 (en) 1993-06-15 1998-09-16 Celltrace Communications Limited Telecommunications system
WO1996006639A2 (en) * 1994-09-01 1996-03-07 Medico Pharma Vertriebs Gmbh Novel drugs containing chelate-forming agents
WO1996006639A3 (en) * 1994-09-01 1996-07-25 Medico Pharma Vertriebs Gmbh Novel drugs containing chelate-forming agents
US6989263B1 (en) * 1994-09-23 2006-01-24 The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services Method for identifying and using compounds that inactivate HIV-1 and other retroviruses by attacking highly conserved zinc fingers in the viral nucleocapsid protein
WO1996039144A1 (en) * 1995-06-06 1996-12-12 Procyte Corporation Stable copper(i) complexes as active therapeutic substances
WO2000021369A1 (en) * 1998-10-13 2000-04-20 World Health Research Institute Treating hiv with edta
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