EP1143990A2 - Pharmaceutical compostions comprising peg-asparaginase for the treatment of hiv infections - Google Patents

Pharmaceutical compostions comprising peg-asparaginase for the treatment of hiv infections

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Publication number
EP1143990A2
EP1143990A2 EP00957306A EP00957306A EP1143990A2 EP 1143990 A2 EP1143990 A2 EP 1143990A2 EP 00957306 A EP00957306 A EP 00957306A EP 00957306 A EP00957306 A EP 00957306A EP 1143990 A2 EP1143990 A2 EP 1143990A2
Authority
EP
European Patent Office
Prior art keywords
compound
hiv
peg
compounds
asnase
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP00957306A
Other languages
German (de)
French (fr)
Other versions
EP1143990A3 (en
Inventor
Vassilios L. Avramis
Lewis Cohen
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Enzon Pharmaceuticals Inc
Original Assignee
Aventis Pharmaceuticals Inc
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Filing date
Publication date
Priority claimed from US09/370,390 external-priority patent/US6689762B1/en
Application filed by Aventis Pharmaceuticals Inc filed Critical Aventis Pharmaceuticals Inc
Publication of EP1143990A2 publication Critical patent/EP1143990A2/en
Publication of EP1143990A3 publication Critical patent/EP1143990A3/en
Withdrawn legal-status Critical Current

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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/46—Hydrolases (3)
    • A61K38/50—Hydrolases (3) acting on carbon-nitrogen bonds, other than peptide bonds (3.5), e.g. asparaginase
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/18—Antivirals for RNA viruses for HIV

Definitions

  • the invention is directed to a pharmaceutical composition comp ⁇ smg a PEG- ASNase compound or a pharmaceutically acceptable salt thereof, and optionally at least one compound selected from the group consisting of protease inhibitor compounds, ⁇ bonucleotide reductase inhibitor compounds and HIV reverse transcnptase inhibitor compounds, and a pharmaceutically acceptable carrier.
  • the invention is also directed to a method of inhibiting or treating Human Immunodeficiency Virus (HIV) infection, comprising administering to a patient in need thereof a therapeutically effective amount of a PEG- ASNase compound or a pharmaceutically acceptable salt thereof, and optionally at least one compound selected from the group consisting of protease inhibitor compounds, ⁇ bonucleotide reductase inhibitor compounds and HIV reverse transcnptase inhibitor compounds, or a pharmaceutically acceptable salt thereof.
  • HIV Human Immunodeficiency Virus
  • the human immunodeficiency virus is a retrovirus and is the agent of the complex disease that includes progressive destruction of the immune system (acquired immune deficiency syndrome; AIDS) and degeneration of the central and peripheral nervous system.
  • This retrovirus is previously known as LAV, HTLV-III, or ARV.
  • Protease inhibitor compounds in combination with reverse transc ⁇ ptase (RT) inhibitor compounds have shown success both in vitro and in vivo in patients infected with the virus. Protease inhibitor compounds interfere with the production of new infectious virus.
  • RT reverse transc ⁇ ptase
  • a common feature of the HTV retrovirus replication is extensive post-translational processing of precursor poly-proteins by a virally encoded protease to generate mature viral proteins required for virus assembly and function. Inhibition of this processing prevents the production of new infectious virus.
  • HIV protease inhibitors may prevent proviral integration of infected T-lymphocytes during the early phase of the HIV-1 life cycle, as well as inhibit viral proteolytic processing during its late stage.
  • HTV protease inhibitors have been extensively reviewed (A. Tomasselh et al., Chimica Oggi, 6-27 20 (1991) and T. Meek, J. Enzyme Inhibition 6: 65-98 (19.92).
  • Retroviral replication routinely features post- translational processing of polyproteins. This processing is accomplished by virally encoded HIV protease enzymes. This post-translational process yields mature polypeptides that will subsequently aid in the formation and function of infectious viruses. If this molecular processing is inhibited, then the normal production of HIV is terminated Therefore, it has been discovered that inhibitors of HIV protease may function as anti-HIV viral agents.
  • Retroviruses are widely distributed in vertebrates and are known to cause a variety of diseases in man and animals including HTV, leukemias and lymphomas.
  • the entire retrovirus family is characterized by the presence of a unique enzyme, reverse transcnptase (RT), which transcribes the viral genomic RNA into a double-stranded DNA copy Therefore, considerable efforts are being directed toward the control of HTV by means of inhibition of HIV -reverse transcnptase, required for replication of the virus.
  • RT reverse transcnptase
  • AZT AZT
  • U S. Pat. No. 4,724,232 AZT
  • many of these compounds suffer from toxicity problems, lack of bioavailabihty or are short lived in vivo, viral resistance, or combinations thereof.
  • HTV-reverse transcnptase HTV-reverse transcnptase
  • nucleoside analogue drug combinations It is also known that the inhibition of HTV-reverse transcnptase (HIV-RT) by nucleoside analogue drug combinations indicate that they cannot alone inhibit the RT function completely, but instead can lead to the emergence of drug resistant viral strain. These strains of escape mutants repopulate and render nucleoside analogue therapy ineffective.
  • protease inhibitor compounds to known nucleoside analogue combination therapies has helped to reduce the viral burden for a prolonged period of time.
  • Ribonucleotide reductase is an alloste ⁇ cally regulated enzyme that converts the nucleoside diphosphates to their corresponding deoxynucleoside diphosphates through a complex regulatory mechanism involving one or several electron transfer pathways.
  • HISID 2-hydroxy-lH- ⁇ so ⁇ ndole-l,3-d ⁇ one
  • PEG-asparaginase the polyethylene glycosylated form of E.coh-ASP
  • E.coh-ASP polyethylene glycosylated form of E.coh-ASP
  • PEG-asparaginase has been shown to be useful as a chemotherapeutic agent.
  • PEG-asparaginase has been found to be an alternative preparation with a longer circulating half-life than E.coh L- asparagmase and has been useful m multiagent chemotherapy for childhood acute lymphoblastic leukemia.
  • PEG-ASNase may increase the anti-leukemic effect in isolated CNS relapse.
  • PEG-ASNase is a conjugate of asparaginase with polyethylene glycol. This conjugation occurs through pegylation, a process in which polypeptides, such as enzymes and hormones, are coupled to polyethylene glycol so as to produce a physiologically active non- lmmunogenic water-soluble polypeptide composition.
  • the polyethylene glycol protects the polypeptide from loss of activity and the composition can be injected into the mammalian circulatory system with substantially no lmmunogenic response.
  • the process of pegylation is described in detail in U.S. Patent No. 4,179,337, entitled “Non-Immunogenic Polypeptide", filed July 28, 1977 and issued December 18, 1979, which is incorporated by reference in its entirety herein.
  • Covalent attachment of the polymer to the peptide is affected often by reacting PEG-succinimide derivatives with amino groups on the exterior of protein molecules.
  • Other methods are also disclosed in U S. Patent No. 4,179,337, in Pollack et al., JACS, 98, 289 (1976), U.S. Patent No. 4,847,325 and elsewhere in the art.
  • PEG-asparaginase effectively works alone and synergistically works in combination with one or more of the following: protease inhibitor compounds, HIV reverse transc ⁇ ptase inhibitor compounds, or ribonucleotide reductase inhibitor compounds, to treat infection by HIV.
  • this invention is directed to a pharmaceutical composition comp ⁇ sing a PEG-ASNase compound and optionally at least one compound selected from the group consisting of protease inhibitor compounds, ribonucleotide reductase inhibitor compounds and HTV reverse transcnptase inhibitor compounds, and a pharmaceutically acceptable carrier
  • the invention is also directed to a method of inhibiting or treating Human Immunodeficiency Virus (HTV) infection, comprising administering to a patient in need thereof a therapeutically effective amount of a PEG-ASNase compound or a pharmaceutically acceptable salt thereof, and optionally at least one compound selected from the group consisting of protease inhibitor compounds, ribonucleotide reductase inhibitor compounds and HIV reverse transcnptase inhibitor compounds, or a pharmaceutically acceptable salt thereof
  • HTV Human Immunodeficiency Virus
  • Figure 1 represents the inhibition of HIV-RT in CEM/0 T-cells ( ⁇ PHA) treated with IC50 concentrations of PEG-ASNase or Saquinavir (SAQ) alone or in their combination
  • Figure 2 represents T-Cell (CEM/0) cytotoxicity of Saquinavir, after 72 hours, for different drug concentrations
  • Figure 3 represents T-Cell cytotoxicity of PEG-ASNase and Saquinavir alone, and in sequential combination (PEG-ASNase followed by Saquinavir), for different drug concentrations
  • Figure 4 represents T-Cell cytotoxicity of PEG-ASNase and Saquinavir alone, and in sequential combination (Saquinavir followed by PEG-ASNase), for different drug concentrations
  • Figure 5 represents T-Cell cytotoxicity of PEG-ASNase and Saquinavir alone, and in concurrent combination, for different drug concentrations
  • Figure 6 represents T-Cell synergism of PEG-ASNase and Saquinavir in concurrent combination for different drug concentrations
  • Figure 7 represents the combination index (CI) in CEM/0 of sequential combination of Saquinavir followed by PEG-ASNase, and sequential combination of PEG-ASNase followed by Saquinavir, and concurrent combination of PEG-ASNase and Saquinavir
  • Figure 8 represents the depletion of Asparagme, Glutamine and Aspartic acid concentrations in CEM/0 T-cells after exposure to different concentrations of PEG-ASNase for 24 hours
  • Figure 8a represents a calibration curve of the optical density (OD) of different concentrations of HIV-1 RT
  • Figure 9 represents the number of HTV RNA copies per cell pellets after exposure of the cells to PEG-ASNase and Saquinavir alone and in combination.
  • Figure 9a represents Log ⁇ 0 of the number of HJN R ⁇ A copies per cell pellets after exposure of the cells to PEG-AS ⁇ ase and Saquinavir alone and in combination.
  • Figure 10 represents the calibration curves for the HIV-RT Ehsa assay.
  • Figure 1 1 represents the HIV-1 quantitative R ⁇ A assay of CEM-T-cells treated with single regimens of PEG-AS ⁇ ase, Saquinavir, AZT and MISID, and combination regimens of Saquinavir and PEG-AS ⁇ ase, AZT, Saquinavir and PEG-AS ⁇ ase, and MISID, AZT, Saquinavir and PEG-AS ⁇ ase.
  • acyl means an H-CO- or alkyl-CO- group wherein the alkyl group is as herein described. Preferred acyls contain a lower alkyl. Exemplary acyl groups include formyl, acetyl, propanoyl, 2-methylpropanoyl, butanoyl and palmitoyl.
  • “Acylamino” is an acyl- ⁇ H- group wherein acyl is as defined herein.
  • Alkenyl means an aliphatic hydrocarbon group containing a carbon-carbon double bond and which may be straight or branched having about 2 to about 15 carbon atoms in the chain. Preferred alkenyl groups have 2 to about 12 carbon atoms in the chain; and more preferably about 2 to about 4 carbon atoms in the chain. Branched means that one or more lower alkyl groups such as methyl, ethyl or propyl are attached to a linear alkenyl chain.
  • “Lower alkenyl” means about 2 to about 4 carbon atoms in the chain which may be straight or branched.
  • the alkenyl group may be substituted by one or more halo or cycloalkyl group.
  • exemplary alkenyl groups include ethenyl, propenyl, «-butenyl, r ' -butenyl, 3-methylbut-2- enyl, «-pentenyl, heptenyl, octenyl, cyclohexylbutenyl and decenyl.
  • Alkoxy means an alkyl-O- group wherein the alkyl group is as herein described.
  • Exemplary alkoxy groups include methoxy, ethoxy, n-propoxy, /-propoxy, w-butoxy and heptoxy.
  • Alkoxycarbonyl means an alkyl-O-CO- group, wherein the alkyl group is as herein defined.
  • exemplary alkoxycarbonyl groups include methoxycarbonyl, ethoxycarbonyl, or t- butyloxycarbony 1.
  • Alkyl means an aliphatic hydrocarbon group which may be straight or branched having about 1 to about 20 carbon atoms in the chain. Preferred alkyl groups have 1 to about 12 carbon atoms in the chain. Most preferred alkyl groups have 1 to about 3 carbon atoms in the chain.
  • Branched means that one or more lower alkyl groups such as methyl, ethyl or propyl are attached to a linear alkyl chain
  • “Lower alkyl” means about 1 to about 3 carbon atoms in the chain which may be straight or branched
  • the alkyl may be substituted with one or more "alkyl group substituents" which may be the same or different, and include halo, cycloalkyl, hydroxy, alkoxy, amino, acylamino, aroylamino, carboxy, alkoxycarbonyl,
  • Y and Y are independently hydrogen, optionally substituted alkyl, optionally substituted aryl, optionally
  • alkyl groups include methyl, t ⁇ fluoromethyl, cyclopropylmethyl, cyclopentylmethyl, ethyl, n- propyl, ;-propyl, «-butyl, t-butyl, n-pentyl, 3-pentyl, methoxyethyl, carboxymethyl, formyl, methoxycarbonylethyl, benzyloxycarbonylmethyl, py ⁇ dylmethyloxycarbonylmethyl
  • alkyl substituents are halo, hydroxy, alkoxy, amino, acylamino, aroylamino, carboxy, alkoxycarbonyl, aralkoxycarbonyl, heteroaralkoxycarbonyl, sulfonyl, sulfinyl, acyl, alkanoyl, or Y 1 Y 2 NCO-
  • Alkylthio means an alkyl-S- group wherein the alkyl group is as herein described
  • alkylthio groups include methylthio, ethylthio, i-propylthio and heptylthio
  • Alkylsulfinyl means an alkyl-SO- group wherein the alkyl group is as defined above Preferred groups are those wherein the alkyl group is lower alkyl
  • Alkylsulfonyl means an alkyl-S0 2 -group wherein the alkyl group is as defined above Preferred groups are those wherein the alkyl group is lower alkyl
  • Alkynyl means an aliphatic hydrocarbon group containing a carbon-carbon triple bond and which may be straight or branched having about 2 to about 15 carbon atoms in the chain Preferred alkynyl groups have 2 to about 12 carbon atoms in the chain, and more preferably about 2 to about 4 carbon atoms in the chain Branched means that one or more lower alkyl groups such as methyl, ethyl or propyl are attached to a linear alkynyl chain
  • “Lower alkynyl” means about 2 to about 4 carbon atoms in the chain which may be straight or branched
  • the alkynyl group may be substituted by one or more halo
  • Exemplary alkynyl groups include ethynyl, propynyl, n-butynyl, 2-butynyl, 3-methylbutynyl, ⁇ -pentynyl, heptynyl, octynyl and decynyl
  • “Analogue” means a compound which comprises a chemically modified form of a specific compound or class thereof, and which maintains the pharmaceutical and/or pharmacological activities characteristic of said compound or class
  • Alkoxy means an aralkyl-O- group wherein the aralkyl groups is as herein described
  • Exemplary aralkoxy groups include benzyloxy and 1 - or 2-naphthalenemethoxy
  • Aralkoxycarbonyl means an aralkyl-O-CO- group wherein the aralkyl groups is as herein described.
  • An exemplary aralkoxycarbonyl group is benzyloxycarbonyl.
  • Aralkyl means an aryl-alkyl- group wherein the aryl and alkyl are as herein described. Preferred aralkyls contain a lower alkyl moiety. Exemplary aralkyl groups include benzyl, 2-phenethyl and naphthlenemethyl
  • Alkylthio means an aralkyl-S- group wherein the aralkyl group is as herein described.
  • An exemplary aralkylthio group is benzylthio.
  • Aryloxycarbonyl means an aryl-O-CO- group wherein the aryl group is as defined herein Exemplary aryloxycarbonyl groups include phenoxycarbonyl and naphthoxycarbonyl.
  • Aroylamino is an aroyl-NH- group wherein aroyl is as defined herein
  • Aroyl means an aryl-CO- group wherein the aryl group is as herein described Exemplary groups include benzoyl and 1- and 2-naphthoyl.
  • Aryl means an aromatic monocychc or multicychc ring system of about 6 to about 14 carbon atoms, preferably of about 6 to about 10 carbon atoms.
  • the aryl is optionally substituted with one or more "ring system substituents" which may be the same or different, and are as defined herein.
  • Exemplary aryl groups include phenyl or naphthyl, or phenyl substituted or naphthyl substituted.
  • Aryldiazo means an aryl -diazo- group wherein the aryl and diazo groups are as defined herein.
  • Aryloxy means an aryl-O- group wherein the aryl group is as defined herein.
  • exemplary groups include phenoxy and 2-naphthyloxy.
  • Arylsulfonyl means an aryl-S0 2 - group wherein the aryl group is as defined herein.
  • Arylsulfinyl means an aryl-SO- group wherein the aryl group is as defined herein
  • Arylthio means an aryl-S- group wherein the aryl group is as herein described.
  • Exemplary arylthio groups include phenylthio and naphthylthio.
  • Carboxy means a HO(0)C- (carboxylic acid) group.
  • Cycloalkenyl means a non-aromatic mono- or multicychc ring system of about 3 to about 10 carbon atoms, preferably of about 5 to about 10 carbon atoms, and which contains at least one carbon-carbon double bond. Preferred ring sizes of rings of the ring system include about 5 to about 6 ring atoms.
  • the cycloalkenyl is optionally substituted with one or more "ring system substituents" which may be the same or different, and are as defined herein.
  • Exemplary monocychc cycloalkenyl include cyclopentenyl, cyclohexenyl, cycloheptenyl, and the like.
  • An exemplary multicychc cycloalkenyl is norbornylenyl.
  • Cycloalkyl means a non-aromatic mono- or multicychc ring system of about 3 to about 10 carbon atoms, preferably of about 5 to about 10 carbon atoms. Preferred ring sizes of rings of the ring system include about 5 to about 6 ring atoms.
  • the cycloalkyl is optionally substituted with one or more "ring system substituents" which may be the same or different, and are as defined herein.
  • Exemplary monocychc cycloalkyl include cyclopentyl, cyclohexyl, cycloheptyl, and the like.
  • Exemplary multicychc cycloalkyl include 1-decal ⁇ n, norbornyl, adamant-(l- or 2-)yl, and the like.
  • “Derivative” means a chemically modified compound wherein the modification is considered routine by the ordinary skilled chemist, such as an ester or an amide of an acid, protecting groups, such as a benzyl group for an alcohol or thiol, and tert-butoxycarbonyl group for an amine
  • Effective amount means an amount of a compound/composition according to the present invention effective in producing the desired therapeutic effect
  • Electrode withdrawing group refers to a group that will draw electrons to itself more than a hydrogen atom would if it occupied the same position in the molecule See, J. March, Advanced Organic Chemistry, 3rd Ed., John Wiley & Sons P. 17 (1985). They include such groups as nitro, monohaloalkyl, dihaloalkyl, tnhaloalkyl (e.g., CF 3 ), halogen, formyl, alkylsulfonyl, alkylsulfinyl, and the like. Preferably halogen.
  • Forms suitable for nasal or inhalational administration means formulations which are in a form suitable to be administered nasally or by inhalation to a patient.
  • the formulation may contain a carrier, in a powder form, having a particle size for example in the range 1 to 500 microns (including particle sizes in a range between 20 and 500 microns in increments of 5 microns such as 30 microns, 35 microns, etc.)
  • Suitable formulations wherein the carrier is a liquid, for administration as for example a nasal spray or as nasal drops include aqueous or oily solutions of the active ingredient.
  • Formulations suitable for aerosol administration may be prepared according to conventional methods and may be delivered with other therapeutic agents. Inhalational therapy is readily administered by metered dose inhalers.
  • Forms suitable for oral administration means formulations which are in a form suitable to be administered orally to a patient.
  • the formulations may be presented as discrete units such as capsules, cachets or tablets each containing a predetermined amount of the active ingredient; as a powder or granules; as solution or a suspension in an aqueous liquid or a non-aqueous liquid; or as an oil-in-water liquid emulsion or a water-in-oil liquid emulsion.
  • the active ingredient may also be presented as a bolus, electuary or paste.
  • Forms suitable for parenteral administration means formulations which are in a form suitable to be administered parenterally to a patient.
  • the formulations are sterile and include emulsions, suspensions, aqueous and non-aqueous injection solutions, which may contain suspending agents and thickening agents and anti-oxidants, buffers, bactenostats and solutes which render the formulation lsotonic, and have a suitably adjusted pH, with the blood of the intended recipient
  • Forms suitable for rectal administrations means formulations which are in a form suitable to be administered rectally to a patient
  • the formulation is preferably in the form of suppositories which can be prepared by mixing the compounds useful according to this invention with suitable non-irntating excipients or carriers such as cocoa butter, polyethylene glycol or a suppository wax, which are solid at ordinary temperatures but liquid at body temperature and therefore, melt in the rectum or vaginal cavity and release the active component.
  • suitable for systemic administration means formulations which are in a form suitable to be administered systemically to a patient The formulation is preferably administered by injection, including transmuscular, intravenous, intrapentoneal, and subcutaneous.
  • the compounds useful according to the invention are formulated Swift in liquid solutions, preferably in physiologically compatible buffers such as Hank's solution or Ringer's solution.
  • the compounds may be formulated in solid form and redissolved or suspended immediately prior to use. Lyophilized forms are also included.
  • Systematic administration also can be by transmucosal or transdermal means, or the compounds can be administered orally.
  • penetrants appropriate to the barrier to be permeated are used in the formulation.
  • penetrants are generally known in the art, and include, for example, bile salts and fusidic acid derivatives for transmucosal administration
  • detergents may be used to facilitate permeation.
  • Transmucosal administration may be through use of nasal sprays, for example, or suppositories.
  • the compounds are formulated into conventional oral administration forms such as capsules, tablets, and tonics.
  • "Formulations suitable for topical administration” means formulations which are in a form suitable to be administered topically to a patient.
  • the formulation may be presented as a topical ointment, salves, powders, sprays and inhalants, gels (water or alcohol based), creams, as is generally known in the art, or incorporated into a matrix base for application in a patch, which would allow a controlled release of compound through the transdermal barrier.
  • the active ingredients When formulated in an ointment, the active ingredients may be employed with either a paraffimc or a water-miscible ointment base. Alternatively, the active ingredients may be formulated in a cream with an oil-in-water cream base.
  • Formulations suitable for topical administration in the eye include eye drops wherein the active ingredient is dissolved or suspended in a suitable carrier, especially an aqueous solvent for the active ingredient
  • Formulations suitable for topical administration in the mouth include lozenges comprising the active ingredient in a flavored basis, usually sucrose and acacia or tragacanth, pastilles comprising the active ingredient in an inert basis such as gelatin and glycerin, or sucrose and acacia, and mouthwashes comprising the active ingredient in a suitable liquid carrier
  • Forms suitable for vaginal administration means formulations which are in a form suitable to be administered vagmally to a patient
  • the formulation may be presented as pessaries, tampons, creams, gels, pastes, foams or spray formulations containing in addition to the active ingredient such carriers as are known in the art to be appropriate
  • Heteroaralkoxycarbonyl means an heteroaralkyl-O-CO- group wherein the heteroaralkyl groups is as herein descnbed
  • An exemplary heteroaralkoxycarbonyl group is thienylmethylcarbonyl
  • Heteroaralkyl means a heteroaryl-alkyl- group wherein the heteroaryl and alkyl are as herein described.
  • Preferred heteroaralkyls contain a lower alkyl moiety.
  • Exemplary heteroaralkyl groups may contain thienylmethyl, py ⁇ dylmethyl, lmidazolylmethyl and pyrazmylmethyl
  • Heteroaralkylthio means an heteroaralkyl-S- group wherein the heteroaralkyl group is as herein described.
  • An exemplary heteroaralkylthio group is py ⁇ dylmethylthio
  • Heteroaryl means an aromatic monocychc or multicychc ring system of about 5 to about 14 carbon atoms, preferably about 5 to about 10 carbon atoms, in which one or more of the carbon atoms m the ring system is/are hetero element(s) other than carbon, for example nitrogen, oxygen or sulfur. Preferred ring sizes of rings of the ring system include about 5 to about 6 ring atoms.
  • the “heteroaryl” may also be substituted by one or more "ring system substituents" which may be the same or different, and are as defined herein.
  • heteroaryl The designation of the aza, oxa or thia as a prefix before heteroaryl define that at least a nitrogen, oxygen or sulfur atom is present respectively as a ring atom
  • a nitrogen atom of an heteroaryl may be a basic nitrogen atom and may also be optionally oxidized to the corresponding N-oxide.
  • heteroaryl and substituted heteroaryl groups include pyrazmyl, thienyl, isothiazolyl, oxazolyl, pyrazolyl, furazanyl, pyrrolyl, 1,2,4-th ⁇ ad ⁇ azolyl, py ⁇ dazinyl, quinoxahnyl, phthalazinyl, ⁇ m ⁇ dazo[l ,2-a]py ⁇ d ⁇ ne, ⁇ m ⁇ dazo[2,l-b]th ⁇ azolyl, benzofurazanyl, azaindolyl, benzimidazolyl, benzothienyl, thienopyndyl, thienopynmidyl, pyrrolopy ⁇ dyl, lmidazopy ⁇ dyl, benzoazamdole, 1,2,4-tnazmyl, benzthiazolyl, furanyl, lmidazolyl, indolyl, lnd
  • Heteroaryldiazo means an heteroaryl -diazo- group wherein the heteroaryl and diazo groups are as defined herein.
  • Heteroarylsulfonyl means an aryl-S0 2 - group wherein the heteroaryl group is as defined herein.
  • Heteroarylsulfinyl means an aryl-SO- group wherein the heteroaryl group is as defined herein
  • Heteroarylthio means an aryl-S- group wherein the heteroaryl group is as herein described.
  • exemplary heteroarylthio groups include pyndylthio and py ⁇ midmylthio.
  • Heterocyclenyl means a non-aromatic monocychc or multicychc hydrocarbon ring system of about 3 to about 10 carbon atoms, preferably about 5 to about 10 carbon atoms, in which one or more of the carbon atoms in the ring system is/are hetero element(s) other than carbon, for example nitrogen, oxygen or sulfur atoms, and which contains at least one carbon-carbon double bond or carbon-nitrogen double bond
  • Preferred ring sizes of rings of the ring system include about 5 to about 6 nng atoms.
  • the designation of the aza, oxa or thia as a prefix before heterocyclenyl define that at least a nitrogen, oxygen or sulfur atom is present respectively as a ring atom.
  • the heterocyclenyl may be optionally substituted by one or more ring system substituent, wherein the "ring system substituent" is as defined herein
  • the nitrogen atom of an heterocyclenyl may be a basic nitrogen atom.
  • the nitrogen or sulphur atom of the heterocyclenyl may also be optionally oxidized to the corresponding N-oxide, S-oxide or S,S-d ⁇ ox ⁇ de.
  • Exemplary monocychc azaheterocyclenyl groups include 1,2,3,4- tetrahydrohydropyndine, 1,2-d ⁇ hydropyndyl, 1,4-d ⁇ hydropyndyl, 1,2,3,6-tetrahydropy ⁇ dme, 1,4,5,6-tetrahydropy ⁇ m ⁇ d ⁇ ne, 2-pyrrohnyl, 3-pyrrohnyl, 2- imidazohnyl, 2-pyrazohnyl, and the like.
  • Exemplary oxaheterocyclenyl groups include 3,4- d ⁇ hydro-2H-pyran, dihydrofuranyl, and fluorodihydrofuranyl. Preferred is dihydrofuranyl.
  • An exemplary multicychc oxaheterocyclenyl group is 7-oxab ⁇ cyclo[2 2.1]heptenyl.
  • Preferred monocychc thiaheterocycleny rings include dihydrothiophenyl and dihydrothiopyranyl; more preferred is dihydrothiophenyl.
  • Preferred ring system substituents include amidmo, halogen, hydroxy, alkoxycarbonylalkyl, carboxyalkyl or Y'Y 2 N- as defined herein.
  • Heterocyclyl means a non-aromatic saturated monocychc or multicychc ring system of about 3 to about 10 carbon atoms, preferably about 5 to about 10 carbon atoms, in which one or more of the carbon atoms in the ring system is/are hetero element(s) other than carbon, for example nitrogen, oxygen or sulfur Preferred ring sizes of rings of the ring system include about 5 to about 6 ring atoms
  • the designation of the aza, oxa or thia as a prefix before heterocyclyl define that at least a nitrogen, oxygen or sulfur atom is present respectively as a ring atom
  • the heterocyclyl may be optionally substituted by one or more "ring system substituents" which may be the same or different, and are as defined herein
  • the nitrogen atom of an heterocyclyl may be a basic nitrogen atom
  • the nitrogen or sulphur atom of the heterocyclyl may also be optionally oxidized to the corresponding N-oxide, S-oxide
  • “Hydrate” means a solvate wherein the solvent molecule(s) is/are H 2 0
  • "Hydroxyalkyl” means a HO-alkyl- group wherein alkyl is as herein defined Preferred hydroxyalkyls contain lower alkyl Exemplary hydroxyalkyl groups include hydroxymethyl and 2-hydroxyethyl.
  • “Hygroscopicity” means sorption, implying an acquired amount or state of water sufficient to affect the physical or chemical properties of the substance (Eds. J. Swarb ⁇ ck and J C Boylan, Encyclopedia of Pharmaceutical Technology, Vol 10, p. 33).
  • liquid dosage form means the dose of the active compound to be administered to the patient is in liquid form, for example, pharmaceutically acceptable emulsions, solutions, suspensions, syrups and elixirs
  • the liquid dosage forms may contain inert diluents commonly used in the art, such as water or other solvents, solubihzing agents and emulsifiers, as for example, ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils, in particular, cottonseed oil, groundnut oil, corn germ oil, olive oil, castor oil and sesame oil, glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan or mixtures of these substances, and the like.
  • inert diluents commonly used in the art, such as
  • MISID PL-7
  • Rl is a methyl group
  • R2 is an isopropyl group
  • Modulate means the ability of a compound to either directly (by binding to the receptor as a hgand) or indirectly (as a precursor for a hgand or an mducer which promotes production of a hgand from a precursor) induce expression of gene(s) maintained under hormone control, or to repress expression of gene (s) maintained under such control.
  • "Patient” includes both human and other mammals.
  • PEG-ASNase means the protein synthesis inhibitor compound, asparaginase, conjugated with polyethylene glycol (PEG).
  • the polyethylene glycol preferably has an average molecular weight between about 1000 and 100,000 daltons, more preferably between 4000 and 40,000 daltons, depending, for example, on the molecular weight of the particular protein synthesis inhibitor compound employed. Since the object of the modification is to obtain a conjugated protein with retained biological activity, with enhanced in vivo half-life over the unconjugated protein synthesis inhibitor compound, and with reduced lmmunogenicity, the molecular weight of the polymer will be chosen to optimize these conditions.
  • the PEG homopolymer is substituted at one end with an alkyl group, but it may also be unsubstituted.
  • the alkyl group is a Ci -C 4 alkyl group, and most preferably a methyl group.
  • the polymer is a monomethyl-substituted PEG homopolymer and has a molecular weight of about 4000 to 40,000 daltons.
  • PEG-ASNase is the compound sold under the name ONCASPAR by Rh ⁇ ne-Poulenc Rorer "Pharmaceutical composition” means a composition comp ⁇ sing a compound of the invention and at least one component selected from the group comprising pharmaceutically acceptable carriers, diluents, adjuvants, excipients, or vehicles, such as preserving agents, fillers, disintegrating agents, wetting agents, emulsifying agents, suspending agents, sweetening agents, flavoring agents, perfuming agents, antibacterial agents, antifungal agents, lubricating agents and dispensing agents, depending on the nature of the mode of administration and dosage forms
  • suspending agents include ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalhne cellulose, aluminum metahydroxide, bentomte, agar-agar and tragacanth, or mixtures of these substances.
  • microorganisms Prevention of the action of microorganisms can be ensured by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, and the like. It may also be desirable to include lsotonic agents, for example sugars, sodium chloride and the like. Prolonged absorption of the injectable pharmaceutical form can be brought about by the use of agents delaying absorption, for example, aluminum monosterate and gelatin.
  • suitable carriers, diluents, solvents or vehicles include water, ethanol, polyols, suitable mixtures thereof, vegetable oils (such as olive oil) and injectable organic esters such as ethyl oleate.
  • excipients include lactose, milk sugar, sodium citrate, calcium carbonate, dicalcium phosphate phosphate.
  • disintegrating agents include starch, alginic acids and certain complex silicates.
  • lubricants include magnesium stearate, sodium lauryl sulphate, talc, as well as high molecular weight polyethylene glycols.
  • “Pharmaceutically acceptable” means it is, within the scope of sound medical judgement, suitable for use in contact with the cells of humans and lower animals without undue toxicity, irritation, allergic response and the like, and are commensurate with a reasonable benefit/risk ratio.
  • “Pharmaceutically acceptable dosage forms” means dosage forms of the compound of the invention, and includes, for example, tablets, dragees, powders, elixirs, syrups, liquid preparations, including suspensions, sprays, inhalants tablets, lozenges, emulsions, solutions, granules, capsules and suppositories, as well as liquid preparations for injections, including hposome preparations Techniques and formulations generally may be found in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, PA, latest edition
  • “Pharmaceutically acceptable prodrugs” as used herein means those prodrugs of the compounds useful according to the present invention which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and lower animals with undue toxicity, irritation, allergic response, and the like, commensurate with a reasonable benefit/risk ratio, and effective for their intended use, as well as the zwitte ⁇ onic forms, where possible, of the compounds of the invention
  • prodrug means compounds that are rapidly transformed in vivo to yield the parent compound of the invention, for example by hydrolysis in blood.
  • Functional groups which may be rapidly transformed, by metabolic cleavage, in vivo form a class of groups reactive with the carboxyl group of the compounds of this invention They include, but are not limited to such groups as alkanoyl (such as acetyl, propionyl, butyryl, and the like), unsubstituted and substituted aroyl (such as benzoyl and substituted benzoyl), alkoxycarbonyl (such as ethoxycarbonyl), tnalkylsilyl (such as tnmethyl- and tnethysilyl), monoesters formed with dicarboxylic acids (such as succinyl), and the like Because of the ease with which the metabolically cleavable groups of the compounds useful according to this invention are cleaved in vivo, the compounds bearing such groups act as pro-drugs.
  • alkanoyl such as acetyl, propionyl, butyryl, and the like
  • the compounds bearing the metabolically cleavable groups have the advantage that they may exhibit improved bioavailabihty as a result of enhanced solubility and/or rate of absorption conferred upon the parent compound by virtue of the presence of the metabolically cleavable group.
  • prodrugs A thorough discussion of prodrugs is provided in the following: Design of Prodrugs, H. Bundgaard, ed., Elsevier, 1985; Methods in Enzymology, K. Widder et al, Ed., Academic Press, 42, p.309-396, 1985; A Textbook of Drug Design and Development, Krogsgaard-Larsen and H.
  • Bundgaard ed., Chapter 5; "Design and Applications of Prodrugs” p.113-191, 1991; Advanced Drug Delivery Reviews, H Bundgard, 8, p.1-38, 1992; Journal of Pharmaceutical Sciences, 77, p. 285, 1988; Chem. Pharm. Bull., N. Nakeya et al, 32, p. 692, 1984; Pro-drugs as Novel Delivery Systems, T. Higuchi and V. Stella, Vol 14 of the A.C.S. Symposium Series, and Bioreversible Carriers m Drug Design, Edward B. Roche, ed , American Pharmaceutical Association and Pergamon Press, 1987, which are incorporated herein by reference.
  • “Pharmaceutically acceptable salts” means the relatively non-toxic, inorganic and organic acid addition salts, and base addition salts, of compounds of the present invention These salts can be prepared in situ during the final isolation and purification of the compounds.
  • acid addition salts can be prepared by separately reacting the purified compound in its free base form with a suitable organic or inorganic acid and isolating the salt thus formed
  • Exemplary acid addition salts include the hydrobromide, hydrochlo ⁇ de, sulfate, bisulfate, phosphate, nitrate, acetate, oxalate, valerate, oleate, palmitate, stearate, laurate, borate, benzoate, lactate, phosphate, tosylate, citrate, maleate, fumarate, succinate, tartrate, naphthylate, mesylate, glucoheptonate, lactiobionate, sulphamates, malonates, sahcylates, propionate
  • Base addition salts can also be prepared by separately reacting the purified compound in its acid form with a suitable organic or inorganic base and isolating the salt thus formed.
  • Base addition salts include pharmaceutically acceptable metal and amine salts. Suitable metal salts include the sodium, potassium, calcium, barium, zinc, magnesium, and aluminum salts. The sodium and potassium salts are preferred.
  • Suitable inorganic base addition salts are prepared from metal bases which include sodium hydride, sodium hydroxide, potassium hydroxide, calcium hydroxide, aluminium hydroxide, lithium hydroxide, magnesium hydroxide, zinc hydroxide Suitable amine base addition salts are prepared from amines which have sufficient basicity to form a stable salt, and preferably include those amines which are frequently used in medicinal chemistry because of their low toxicity and acceptability for medical use.
  • ammonia ethylenediamine, N-methyl-glucamine, lysine, argimne, ornithine, cholme, N,N'-d ⁇ benzylethylened ⁇ am ⁇ ne, chloroprocame, diethanolamine, procaine, N-benzylphenethylamme, diethylamine, piperazine, t ⁇ s(hydroxymethyl)-ammomethane, tetramethylammonium hydroxide, t ⁇ ethylamine, dibenzylamme, ephenamme, dehydroabietylamme, N-ethylpipe ⁇ dine, benzylamine, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, t ⁇ methylamine, ethylamme, basic amino acids, e.g., lysine and argmme, and dicyclohexylamine, and the like.
  • Ring system substituents mean substituents attached to aromatic or non-aromatic ring systems inclusive of hydrogen, alkylaryl, heteroaryl, aralkyl, heteroaralkyl, hydroxy, hydroxyalkyl, alkoxy, aryloxy, aralkoxy, acyl, aroyl, halo, nitro, cyano, carboxy, alkoxycarbonyl, aryloxycarbonyl, aralkoxycarbonyl, alkylsulfonyl, arylsulfonyl, heteroarylsulfonyl, alkylsulfinyl, arylsulfinyl, heteroarylsulfinyl, alkylthio, arylthio, heteroarylthio, aralkylthio, heteroaralkylthio, cycloalkyl, cycloalkenyl, heterocyclyl, heterocyclenyl, aryldiazo, heteroaryldiazo,
  • Solid dosage form means the dosage form of the compound useful according to the invention is solid form, for example capsules, tablets, pills, powders, dragees or granules.
  • the compound useful according to the invention is admixed with at least one inert customary excipient (or carrier) such as sodium citrate or dicalcium phosphate or (a) fillers or extenders, as for example, starches, lactose, sucrose, glucose, manmtol and silicic acid, (b) binders, as for example, carboxymethylcellulose, ahgnates, gelatin, polyvinylpyrrohdone, sucrose and acacia, (c) humectants, as for example, glycerol, (d) disintegrating agents, as for example, agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain complex silicates and sodium carbonate, (e) solution retarders, as for example paraffin, (f) inert customary
  • Solvate means a physical association of a compound of this invention with one or more solvent molecules. This physical association includes hydrogen bonding. In certain instances the solvate will be capable of isolation, for example when one or more solvent molecules are incorporated in the crystal lattice of the crystalline solid. "Solvate” encompasses both solution-phase and isolable solvates. Exemplary solvates include hydrates, ethanolates, methanolates, and the like. In a specific embodiment, the term “about” or “approximately” means within 20%, preferably within 10%, and more preferably within 5% of a given value or range.
  • a preferred embodiment according to the invention is a method of inhibiting or treating Human Immunodeficiency Virus (HTV) infection, comprising administering to a patient in need thereof an effective amount of a pharmaceutically acceptable composition comp ⁇ sing a PEG-ASNase compound
  • Another preferred embodiment according to the invention is a method of inhibiting or treating Human Immunodeficiency Virus (HIV) infection, comprising administering to a patient in need thereof an effective amount of a pharmaceutically acceptable composition comprising a PEG-ASNase compound and at least one protease inhibitor compound.
  • HIV Human Immunodeficiency Virus
  • Another preferred embodiment according to the invention is a method of inhibiting or treating Human Immunodeficiency Virus (HIV) infection, comprising administering to a patient in need thereof an effective amount of a pharmaceutically acceptable composition comp ⁇ sing a PEG-ASNase compound and at least one HTV reverse transcnptase inhibitor compound
  • Another preferred embodiment according to the invention is a method of inhibiting or treating Human Immunodeficiency Virus (HTV) infection, comprising administering to a patient in need thereof an effective amount of a pharmaceutically acceptable composition comprising a PEG-ASNase compound and at least one ribonucleotide reductase inhibitor compound.
  • HTV Human Immunodeficiency Virus
  • Another preferred embodiment according to the invention is a method of inhibiting or treating Human Immunodeficiency Virus (HIV) infection, comp ⁇ sing administering to a patient in need thereof an effective amount of a pharmaceutically acceptable composition comprising a PEG-ASNase compound, at least one a protease inhibitor compound and at least one ribonucleotide reductase inhibitor compound.
  • HIV Human Immunodeficiency Virus
  • Another preferred embodiment according to the invention is a method of inhibiting or treating Human Immunodeficiency Virus (HTV) infection, comprising administering to a patient in need thereof an effective amount of a pharmaceutically acceptable composition comprising a PEG-ASNase compound, at least one protease inhibitor compound and at least one HIV reverse transcnptase inhibitor compound.
  • HTV Human Immunodeficiency Virus
  • Another preferred embodiment according to the invention is a method of inhibiting or treating Human Immunodeficiency Virus (HTV) infection, comprising administering to a patient in need thereof an effective amount of a pharmaceutically acceptable composition comprising a PEG-ASNase compound, at least one ribonucleotide reductase inhibitor compound and at least one HTV reverse transcnptase inhibitor compound.
  • HTV Human Immunodeficiency Virus
  • Another preferred embodiment according to the invention is a method of inhibiting or treating Human Immunodeficiency Virus (HTV) infection, comprising administering to a patient in need thereof an effective amount of a pharmaceutically acceptable composition comprising a PEG-ASNase compound, at least one protease inhibitor compound, at least one ribonucleotide reductase inhibitor compound and at least one HIV reverse transcnptase inhibitor compound.
  • HTV Human Immunodeficiency Virus
  • Another preferred embodiment according to the invention is a method of inhibiting or treating Human Immunodeficiency Virus (HIV) infection, comp ⁇ sing administering to a patient in need thereof a therapeutically effective amount of a PEG-ASNase compound and at least one compound selected from the group consisting of protease inhibitor compounds, ribonucleotide reductase inhibitor compounds and HTV reverse transcnptase inhibitor compounds.
  • a preferred embodiment according to the invention is a method of inhibiting or treating Human Immunodeficiency Virus (HTV) infection, comprising administering to a patient in need thereof an effective amount of a pharmaceutically acceptable composition comprising asparaginase.
  • HTV Human Immunodeficiency Virus
  • Another preferred embodiment according to the invention is a method of inhibiting or treating Human Immunodeficiency Virus (HTV) infection, comprising administering to a patient in need thereof an effective amount of a pharmaceutically acceptable composition comprising asparaginase and at least one compound selected from the group consisting of protease inhibitor compounds, ribonucleotide reductase inhibitor compounds and HIV reverse transcnptase inhibitor compounds.
  • HTV Human Immunodeficiency Virus
  • Another preferred embodiment according to the invention is a method of inhibiting or treating Human Immunodeficiency Virus (HTV) infection, comp ⁇ sing administering to a patient in need thereof an effective amount of a pharmaceutically acceptable composition comprising at least one ribonucleotide reductase inhibitor compound of formula I
  • Rl is alkyl, alkenyl, alkynyl or an electron withdrawing group
  • R2 is alkyl, alkenyl, alkynyl; or a pharmaceutically acceptable salt thereof, an N-oxide thereof, a solvate thereof, an acid bioisostere thereof, or prodrug thereof
  • Another preferred embodiment according to the invention is a method of inhibiting the production, or limiting the spread, of HTV comprising exposing a cell population infected with HTV to an effective amount of a PEG-ASNase compound or asparaginase, and optionally at least one compound selected from the group consisting of protease inhibitor compounds, ribonucleotide reductase inhibitor compounds and HIV reverse transcnptase inhibitor compounds.
  • HIV reverse transcnptase activity comprising contacting HIV reverse transc ⁇ ptase with a composition comprising a PEG-ASNase compound or asparaginase, and optionally at least one compound selected from the group consisting of protease inhibitor compounds, ribonucleotide reductase inhibitor compounds and HIV reverse transcnptase inhibitor compounds
  • HIV reverse transc ⁇ ptase activity comprising contacting HIV reverse transcnptase with a composition comp ⁇ sing a compound of formula I.
  • Another preferred embodiment according to the invention is a method of inhibiting
  • HIV reverse transcnptase activity comprising contacting HIV reverse transcnptase with a composition comprising MISID having the formula
  • HIV reverse transcnptase activity comprising contacting HIV reverse transcnptase with a composition comprising a PEG-ASNase compound.
  • Another preferred embodiment according to the invention is method of selectively inhibiting HIV -RNA production comp ⁇ sing exposing a cell population infected with HIV to a pharmaceutically acceptable composition comp ⁇ sing a PEG-ASNase compound and at least one protease inhibitor compound.
  • Another preferred embodiment according to the invention is method of selectively inhibiting HFV-RNA production comprising exposing a cell population infected with HIV to a pharmaceutically acceptable composition comprising a PEG-ASNase compound and Saquinavir
  • Another preferred embodiment according to the invention is a method of inhibiting
  • HIV-RNA production comprising contacting a cell population infected with HIV with a composition comp ⁇ sing a PEG-ASNase compound or asparaginase, and optionally at least one compound selected from the group consisting of protease inhibitor compounds, ribonucleotide reductase inhibitor compounds and HIV reverse transcnptase inhibitor compounds
  • Another preferred embodiment according to the invention is a method of inhibiting HIV-RNA production, comp ⁇ sing contacting a cell population infected with HIV with a composition comprising a compound of formula I
  • Another preferred embodiment according to the invention is a method of inhibiting HIV-RNA production, comp ⁇ sing contacting a cell population infected with HTV with a composition comprising MISID
  • Another preferred embodiment according to the invention is a method of inhibiting HIV-RNA production, comp ⁇ sing contacting a cell population infected with HIV with a composition comprising a PEG-ASNase compound
  • the protease inhibitor compounds are selected from Saqumovir, Nelfmavir, Endmovere, Indinavir, Ritonavir, C ⁇ xivan, Viracept, Norvir, and VX-478
  • the protease inhibitor compound is Saqumovir
  • the HTV reverse transcnptase inhibitor compounds are selected from AZT (Retrovir, zidovudine) ddl (Videx, didanosine) ddC (Hivid, zalcitabine), d4T (Ze ⁇ t, stavudine) and 3TC (Epivir, lamivudine)
  • the HTV reverse transcnptase inhibitor compound is AZT
  • the ribonucleotide reductase inhibitor compounds are selected from Hydroxyurea (HU), BW- 348U87, 3- ammopy ⁇ d ⁇ ne-2-carboxaldehyde thiosemicarbazone (3-AP) Amidox (VF 236, NSC-343341, N,3,4-t ⁇ hydroxybenzenecarbox ⁇ m ⁇ dam ⁇ de), BUD 1257 (2-benzyl-3-phenylprop ⁇ onyl-L-(N- methyl)valyl-L-3-(methyl)valyl-L-(N4,N4-tetramethylene)asparag ⁇ nyl-L-(3,3- tetramethylene)aspartyl-L-(4-methyl)leucme), BILD 1357 (2-benzyl-3-phenylprop ⁇ onyl-L- (HU), BW- 348U87, 3- ammop
  • the ribonucleotide reductase inhibitor compound is the compound of formula I.
  • this invention is directed to a method of inhibiting or treating Human Immunodeficiency Virus (HTV) infection, comprising administering to a patient in need of such treatment a pharmaceutically effective amount of compound of formula I wherein Rl is lower alkyl, lower alkenyl, lower alkynyl, or an electron withdrawing group; and
  • HTV Human Immunodeficiency Virus
  • R2 is lower alkyl, lower alkenyl, lower alkynyl; or a pharmaceutically acceptable salt thereof, an N-oxide thereof, a solvate thereof, an acid bioisostere thereof, or prodrug thereof.
  • this invention is directed to a method of inhibiting or treating Human Immunodeficiency Virus (HIV) infection, comprising administering to a patient in need of such treatment a pharmaceutically effective amount of compound of formula I wherein Rl is lower alkyl, lower alkenyl, lower alkynyl, or a halogen group; and R2 is lower alkyl, lower alkenyl, lower alkynyl; or a pharmaceutically acceptable salt thereof, an N-oxide thereof, a solvate thereof, an acid bioisostere thereof, or prodrug thereof.
  • HAV Human Immunodeficiency Virus
  • this invention is directed to a method of inhibiting or treating Human Immunodeficiency Virus (HTV) infection, comprising administering to a patient m need of such treatment a pharmaceutically effective amount of compound of formula I wherein Rl is lower alkyl or a halogen group; and
  • HTV Human Immunodeficiency Virus
  • R2 is lower alkyl, or a pharmaceutically acceptable salt thereof, an N-oxide thereof, a solvate thereof, an acid bioisostere thereof, or prodrug thereof
  • this invention is directed to a method of inhibiting or treating Human Immunodeficiency Virus (HIV) infection, comprising administering to a patient in need of such treatment a pharmaceutically effective amount of compound of formula I wherein Rl is a halogen group; and
  • HIV Human Immunodeficiency Virus
  • R2 is lower alkyl; or a pharmaceutically acceptable salt thereof, an N-oxide thereof, a solvate thereof, an acid bioisostere thereof, or prodrug thereof
  • this invention is directed to a method of inhibiting or treating Human Immunodeficiency Virus (HTV) infection, comp ⁇ sing administering to a patient in need of such treatment a pharmaceutically effective amount of compound of formula I wherein Rl is lower alkyl; and
  • HTV Human Immunodeficiency Virus
  • R2 is lower alkyl, or a pharmaceutically acceptable salt thereof, an N-oxide thereof, a solvate thereof, an acid bioisostere thereof, or prodrug thereof.
  • this invention is directed to a method of inhibiting or treating Human Immunodeficiency Virus (HIV) infection, comprising administering to a patient in need of such treatment a pharmaceutically effective amount of compound of formula I wherein Rl is a bromine or chlorine atom; and
  • R2 is a methyl group or an isopropyl group; or a pharmaceutically acceptable salt thereof, an N-oxide thereof, a solvate thereof, an acid bioisostere thereof, or prodrug thereof.
  • this invention is directed to a method of inhibiting or treating Human Immunodeficiency Virus (HTV) infection, comp ⁇ sing administering to a patient in need of such treatment a pharmaceutically effective amount of compound of formula I wherein Rl is a methyl group; and R2 is lower alkyl; or a pharmaceutically acceptable salt thereof, an N-oxide thereof, a solvate thereof, an acid bioisostere thereof, or prodrug thereof.
  • HTV Human Immunodeficiency Virus
  • this invention is directed to a method of inhibiting or treating Human Immunodeficiency Virus (HTV) infection, comprising administering to a patient m need of such treatment a pharmaceutically effective amount of compound of formula I wherein Rl is a methyl group; and R2 is an isopropyl group; or a pharmaceutically acceptable salt thereof, an N-oxide thereof, a solvate thereof, an acid bioisostere thereof, or prodrug thereof
  • the protein synthesis inhibitor compound is PEG-ASNase.
  • the protein synthesis inhibitor compound is asparaginase
  • the protease inhibitor compound is Saqumovir or Endinovere.
  • the protease inhibitor compound is Saqumovir.
  • the HTV reverse transcnptase inhibitor compounds are selected from AZT and 3-TC
  • the ribonucleotide reductase inhibitor compound is MISID (PL-7).
  • the compounds of use according to the invention are administered in concurrent combination.
  • the compounds of use according to the invention are administered sequentially.
  • the compounds of use according to the invention are administered sequentially, preferably by administering a protease inhibitor followed by the PEG-ASNase compound or asparaginase.
  • the compounds of use according to the invention are administered sequentially, preferably by administering Saquinavir followed by the PEG-ASNase compound or asparaginase.
  • the compounds of use according to the invention are administered sequentially, preferably administering in the order Saquinavir, followed by the PEG-ASNase compound or asparaginase, followed by one or more compounds selected from the group consisting of HTV reverse transcnptase inhibitor compounds and ribonucleotide reductase inhibitor compounds.
  • HIV Human Immunodeficiency Virus
  • HTV Human Immunodeficiency Virus
  • HIV Human Immunodeficiency Virus
  • a method of inhibiting or treating Human Immunodeficiency Virus (HIV) infection comprising administering to a patient in need thereof a synergistic combination of a PEG-ASNase compound and at least one ribonucleotide reductase inhibitor compound
  • a method of inhibiting or treating Human Immunodeficiency Virus (HIV) infection comprising administering to a patient in need thereof a synergistic combination of a PEG-ASNase compound and at least one HTV reverse transcnptase inhibitor compound
  • HTV Human Immunodeficiency Virus
  • HIV Human Immunodeficiency Virus
  • HTV Human Immunodeficiency Virus
  • kits having a plurality of active ingredients (with or without carrier) which, together, may be effectively utilized for carrying out the novel combination therapies of the invention
  • T-cells are the main cells in the mammalian body that are infected with HIV virus T-cells are considered to be the viral factories for HIV infection
  • the HIV virus In order to re-infect new T-cells, the HIV virus must enter the T-cell and be able to replicate
  • the viral replication and encapsulation process requires the participation of mtracellular enzymes PEG-ASNase, a specific protein synthesis inhibitor compound to thymic-hneage cells, is shown to very useful in inhibiting synthesis of enzymes required for competent replication and assembly of the HTV virus and in providing other antiprohferative effects related to the HIV virus
  • PEG-ASNase a specific protein synthesis inhibitor compound to thymic-hneage cells
  • kit for treating or preventing a physiological condition associated with HIV, said kit comprising a plurality of separate containers, wherein at least one of said containers contains a PEG-ASNase compound or asparaginase, and at least another of said containers contains one or more compounds selected from the group consisting of protease inhibitor compounds, HIV reverse transcnptase inhibitor compounds and ribonucleotide reductase inhibitor compounds, and said containers optionally contain a pharmaceutical carrier, which kit may be effectively utilized for carrying out combination therapies according to the invention
  • kits for treating or preventing a physiological condition associated with HIV comprising a plurality of separate containers, wherein at least one of said containers contains a compound of formula I and at least another of said containers contains one or more compounds selected from the group consisting of PEG-ASNase compounds, protease inhibitor compounds, HTV reverse transcnptase inhibitor compounds and ribonucleotide reductase inhibitor compounds, and said containers optionally contain a pharmaceutical carrier, which kit may be effectively utilized for carrying out combination therapies according to the invention
  • a further embodiment for a kit would be wherein at least one of said containers should contain a PEG-ASNase compound without the presence of a protease inhibitor compound, a HIV reverse transc ⁇ ptase inhibitor compound or a ribonucleotide reductase inhibitor compound, and at least another of said containers should contain one or more compounds selected from the group consisting of protease inhibitor compounds, HTV reverse transcnpta
  • a further embodiment for a kit would be wherein at least one of said containers should contain a compound of formula I without the presence of a PEG-ASNase compound, a protease inhibitor compound, a HTV reverse transcnptase inhibitor compound or another ribonucleotide reductase inhibitor compound, and at least another of said containers should contain one or more compounds selected from the group consisting of protease inhibitor compounds, HIV reverse transc ⁇ ptase inhibitor compounds and another ribonucleotide reductase inhibitor compound, without the presence the same compound of formula I.
  • a further embodiment for a kit would be wherein of said containers at least one of said containers should contain MISID (PL-7) without the presence of a PEG-ASNase compound, a protease inhibitor compound, a HIV reverse transcnptase inhibitor compound or another ribonucleotide reductase inhibitor compound, and at least another of said containers should contain one or more compounds selected from the group consisting of protease inhibitor compounds, HIV reverse transcnptase inhibitor compounds and ribonucleotide reductase inhibitor compounds, without the presence of MISID (PL-7).
  • MISID PL-7
  • the compounds useful according to the invention optionally are supplied as salts Those salts which are pharmaceutically acceptable are of particular interest since they are useful in administering the foregoing compounds for medical purposes. Salts which are not pharmaceutically acceptable are useful in manufacturing processes, for isolation and purification purposes, and in some instances, for use in separating stereoisomenc forms of the compounds of this invention. The latter is particularly true of amine salts prepared from optically active amines.
  • acid addition salts may be formed and are simply a more convenient form for use; and in practice, use of the salt form inherently amounts to use of the free base form
  • compositions may also be mixed another therapeutic compound to form pharmaceutical compositions (with or without diluent or carrier) which, when administered, provide simultaneous administration of a combination of active ingredients resulting in the combination therapy of the invention While it is possible for the compounds useful according to the invention to be administered alone it is preferably to present them as pharmaceutical compositions
  • the pharmaceutical compositions, both for veterinary and for human use, useful according to the present invention comprise at lease one compound of the invention, as above defined, together with one or more acceptable carriers therefor and optionally other therapeutic ingredients.
  • active ingredients necessary in combination therapy may be combined in a single pharmaceutical composition for simultaneous administration.
  • the choice of vehicle and the content of active substance in the vehicle are generally determined in accordance with the solubility and chemical properties of the active compound, the particular mode of administration and the provisions to be observed in pharmaceutical practice.
  • excipients such as lactose, sodium citrate, calcium carbonate, dicalcium phosphate and disintegrating agents such as starch, algmic acids and certain complex silicates combined with lubricants such as magnesium stearate, sodium lauryl sulphate and talc may be used for preparing tablets
  • lactose and high molecular weight polyethylene glycols When aqueous suspensions are used they can contain emulsifying agents or agents which facilitate suspension.
  • the oily phase of the emulsions of this invention may be constituted from known ingredients in a known manner. While the oily phase may comprise merely an emulsifier (otherwise known as an emulgent), it desirably comprises a mixture of at least one emulsifier with a fat or an oil or with both a fat and an oil. Preferably, a hydrophihc emulsifier is included together with a hpophihc emulsifier which acts as a stabilizer. It is also preferred to include both an oil and a fat.
  • the emuls ⁇ fier(s) with or without stab ⁇ hzer(s) make up the emulsifying wax, and the way together with the oil and fat make up the emulsifying ointment base which forms the oily dispersed phase of a cream formulation.
  • Emulgents and emulsion stabilizers suitable for use in the formulation of the present invention include Tween® 60, Span® 80, cetostearyl alcohol, benzyl alcohol, mynstyl alcohol, glyceryl mono- stearate and sodium lauryl sulfate
  • the aqueous phase of the cream base may include, for example, a least
  • the topical formulations may desirably include a compound which enhances absorption or penetration of the active ingredient through the skm or other affected areas.
  • dermal penetration enhancers include dimethyl sulphoxide and related analogue.
  • the choice of suitable oils or fats for the formulation is based on achieving the desired cosmetic properties.
  • the cream should preferably be a non-greasy, non-staining and washable product with suitable consistency to avoid leakage from tubes or other containers.
  • Straight or branched chain, mono- or dibasic alkyl esters such as di-isopropyl my ⁇ state, decyl oleate, isopropyl palmitate, butyl stearate, 2-ethylhexyl palmitate or a blend of branched chain esters known as Crodamol CAP may be used, the last three being preferred esters These may be used alone or in combination depending on the properties required.
  • high melting point hpids such as white soft paraffin and/or liquid paraffin or other mineral oils can be used.
  • Solid compositions of may also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polyethylene glycols, and the like.
  • compositions can be administered in a suitable formulation to humans and animals by topical or systemic administration, including oral, inhalational, rectal, nasal, buccal, subhngual, vaginal, parenteral (including subcutaneous, intramuscular, intravenous, intradermal, intrathecal and epidural), intracisternal and intrapentoneal. It will be appreciated that the preferred route may vary with for example the condition of the recipient.
  • the formulations can be prepared in unit dosage form by any of the methods well known in the art of pharmacy Such methods include the step of bringing into association the active ingredient with the carrier which constitutes one or more accessory ingredients.
  • the formulations are prepared by uniformly and intimately bringing into association the active ingredient with liquid carriers or finely divided solid earners or both, and then, if necessary, shaping the product.
  • a tablet may be made by compression or moulding, optionally with one or more accessory ingredients
  • Compressed tables may be prepared by compressing in a suitable machine the active ingredient in a free-flowing form such as a powder or granules, optionally mixed with a binder, lubricant, inert diluent, preservative, surface active or dispersing agent.
  • Moulded tablets may be made by moulding in a suitable machine a mixture of the powdered compounds moistened with an inert liquid diluent.
  • the tablets may optionally be coated or scored and may be formulated so as to provide slow or controlled release of the active ingredient therein
  • Solid compositions for rectal administration include suppositories formulated in accordance with known methods and containing at least one compound of the invention.
  • the compounds can be microencapsulated in, or attached to, a slow release or targeted delivery systems such as a biocompatible, biodegradable polymer matrices (e.g poly(d,l-lact ⁇ de co-glycohde)), hposomes, and microspheres and subcutaneously or intramuscularly injected by a technique called subcutaneous or intramuscular depot to provide continuous slow release of the compound(s) for a period of 2 weeks or longer.
  • the compounds may be sterilized, for example, by filtration through a bacteria retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved in sterile water, or some other sterile injectable medium immediately before use.
  • compositions of the invention may be varied so as to obtain an amount of active ingredient that is effective to obtain a desired therapeutic response for a particular composition and method of administration
  • the selected dosage level therefore depends upon the desired therapeutic effect, on the route of administration, on the desired duration of treatment and other factors.
  • Total daily dose of the compounds useful according to this invention administered to a host in single or divided doses may be in amounts, for example, of from about 0.001 to about 100 g/kg body weight daily and preferably 0.01 to 10 mg/kg/day.
  • Dosage unit compositions may contain such amounts of such submultiples thereof as may be used to make up the daily dose. It will be understood, however, that the specific dose level for any particular patient will depend upon a variety of factors including the body weight, general health, sex, diet, time and route of administration, rates of absorption and excretion, combination with other drugs and the severity of the particular disease being treated.
  • the amount of each component administered is determined by the attending clinicians taking into consideration the etiology and severity of the disease, the patient's condition and age, the potency of each component and other factors.
  • the formulations may be presented in unit-dose or multi-dose containers, for example sealed ampoules and vials with elastomenc stoppers, and may be stored m a freeze- d ⁇ ed (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use.
  • sterile liquid carrier for example water for injections
  • Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the kind previously described
  • the compounds of the invention, their methods or preparation and their biological activity will appear more clearly from the examination of the following examples which are presented as an illustration only and are not to be considered as limiting the invention m its scope
  • Samples are supernatants and pellets obtained from the viral ⁇ drug flasks (incubation for seven days) They are not heat inactivated. — Centrifuge the samples at 2000 g for 30 minutes at 4° C.
  • Strips have 8 wells each, therefore need strips
  • washing solution is a lOx solution, therefore it must be diluted using autoclaved water.
  • **Make 1 bottle wash solution by adding 225 ml autoclaved water to the bottle provided. Mix well. Keep on ice during the assay.
  • the cell line used for these studies is CCRF/CEM/O, a human T-leukemic cell line.
  • PEG-ASNase ONCASPAR
  • Rh ⁇ ne-Poulenc Rorer Rh ⁇ ne-Poulenc Rorer.
  • Saquinavir is commercially available.
  • RPMI-1640 media Irvine Scientific, Irvme, CA
  • Fetal Calf Serum Gemmi Biosource, Calabasas, CA
  • IM Hepes Buffer solution 5% Non-essential ammo acids
  • 2 x 10 6 cells/ml are stimulated with PHA+ media for 48 hours at 37°C with 5% C0 .
  • the same number of cells are incubated in PHA(phytohemagglutm ⁇ n) free media for 48 hours to serve as the negative confrol.
  • the cells are inoculated with the HIV-1 virus as per standard protocol.
  • PEG-ASNase and/or Saquinavir are added to the cells in the appropriate concenfrations (see above).
  • the confrol cells are resuspended in drug-free media for the duration of the exposure which lasted seven days. At day five, two 1 ml aliquots of media are removed from the flasks and stored under liquid nitrogen. At day seven, two more 1 ml aliquots of media are removed from the flasks and stored under liquid nitrogen. In addition, the remaining cells are pelleted and stored at -80°C.
  • the samples produced from this experiment are itemized and then assayed for HTV- RT using the Reverse Transcnptase Assay, non-radioactive (Boehrmger Mannheim).
  • the standard curve is determined and the HIV-RT levels for the experimental samples are calculated.
  • T-cell pellets themselves are then examined for mtracellular HIV-RT.
  • PEG-AS ⁇ ase and Saquinavir are not only synergistic against T-cells, but also are selectively synergistic against HIV-1. These drugs are sufficiently suppressive in releasing new HIV-1 particles to the media (equivalent serum or plasma of patients). The fact that the lower concentrations of the combination are more suppressive of HIV-RT than the most active of the two drugs Saquinavir at a higher concentration, strongly suggests that the combination is selectively inhibiting HIV at the pro-virus level.
  • these drugs and their combination suppress/inhibit HIV-RT intracellularly, it suggests that they inhibit HIV-1 at the provirus level.
  • the integrated HIV provirus is producing mRNA, which is not translated into viral proteins and hence, the inhibition of the production of RT or complete virus particles to be shed in the media.
  • no further HIV-1 infection could be achieved of unmfected T-cells.
  • CEM/0 A human leukemic T-cell line, hereafter referred to as CEM/0 is used for this experiment
  • PEG-ASNase is obtained from Rhone-Poulenc Rorer Pharmaceuticals Inc under the tradename Oncaspar®.
  • Saquinavir is obtained from Roche Laboratories under the tradename InviraseTM.
  • RPMI- 1640 media obtained from Irvine Scientific, Irvine CA is enriched with 10% Fetal Calf Serum obtained from Gemini Biosource, Calabasas, CA, 5% IM Hepes buffer solution and 5% non-essential amino acids obtained from Irvme Scientific, Irvme CA
  • PEG-ASNase 1.0, 0.75, 0 5, 0 4 , 0.3, 0.2, 0 1, 0 03 IU/ml
  • the PEG-ASNase concentration that produces a cytostatic condition in CEM/0 cells in vitro is approximately 0.5 IU/ml PEG-ASNase concenfrations of 1 and 0 75 IU/ml produced significant cell kill and are cytotoxic to CEM/0 cells by 72 hours.
  • the concentrations of 0.03, 0.1, 0.2, 0.3 and 0 4 IU/ml are marginally effective in preventing cell growth as compared to the control (untreated cells) growth rate
  • the cells treated with 0.5 IU/ml PEG-ASNase however, showed a relatively flat cell growth line Thus, a cytostatic effect is produced with this concentration over 72 hours. Therefore, a range of PEG-ASNase concentrations including 0.5 IU/ml are used in the combination regimen investigations. A drug, concentration and time dependent cytotoxic effect of PEG-ASNase in this T-cell line is shown.
  • the IC 50 of Saquinavir in CEM/0 cells is determined to be 26 ⁇ M after a 72 hour incubation period. The results are illustrated in Figure 2. Multiple independent experiments with Saquinavir showed an IC 50 from 21-28 ⁇ M. Concenfrations from 0 001 to 1 ⁇ M Saquinavir produced no cell kill. Concentrations of lO ⁇ M produced only an 8.76% kill compared to untreated confrol samples The highest concentration tested, 100 ⁇ M, killed 99.50% of the cells compared to control cells. Thus, a range of concentrations of 1 to 40 ⁇ M Saquinavir is used in subsequent experiments to investigate the combination Saquinavir/ PEG-ASNase therapy in CEM/0 cells.
  • PEG-ASNase 1.020, 0.765, 0.510, 0.255 and 0.0255 IU/ml
  • the negative control cells are incubated in a drug-free media for the same duration and under the same conditions as the experimental samples
  • Cell density is measured via cell counting using a Coulter Counter coupled with a Coulter Channelyzer for each of the experimental flasks at 24, 48 and 72 hours after incubation. Additionally, Trypan Blue Exclusion tests are performed for each of these experimental conditions. Cell numbers are corrected for the viability determined by theTrypan Blue test and presented as a percentage of the untreated control.
  • Samples of 50 ⁇ l media and 10 ⁇ l of lmM ammoadopic acid are added to 450 ⁇ l of cold methanol m 1.5 ml microfuge tubes.
  • the mixtures are vortexed and centrifuged at 8700 g for two minutes.
  • the supernatants are transferred to borosihcate glass test tubes (13 x 100 mm) and lyophilized.
  • the specimens are stored at -20° C until they are analyzed by HPLC. Prior to HPLC analysis, the samples are dissolved in a buffer containing 95% 7 nM disodium hydrogen phosphate and 5% acetonit ⁇ le.
  • Experiment 5 illustrates the results of the exposure of HIV-RNA in T-cell pellets to a combination of PEG-ASNase and Saquinavir.
  • the experimental procedure did not remove the HIV-1 particle from the supernatant to simulate the continuous exposure of the T- cells to HIV-1 virus.
  • HIV-1 virus there is always HIV-1 virus in the supernatant of the T-cell cultures.
  • the cell line used for this experiment is CEM/0, a human T- cell leukemic cell line.
  • PEG-ASNase OCASPAR
  • Saquinavir is commercially available.
  • AZT is purchased from Sigma.
  • MISH a ribonucleotide reductase inhibitor, is synthesized described in Nandy P, Lien EJ, Avramis VI, Med Chem Res. 1995, 5:664-679.
  • RPMI- 1640 media (Irvme Scientific, Irvine, CA), is enriched with 10% Fetal Calf Serum (Gemini Biosource, Calabasas, CA), 5% IM Hepes Buffer solution and 5% Non-essential amino acids (Irvme Scientific, Irvine, CA)
  • the drug concentrations used are as follows:
  • the same number of cells are incubated in PHA free media for 48 hours to serve as the negative control. At this point the cells are inoculated with the HIN-1 virus as per standard protocol.
  • the HIV containing supernatant from the PHA-stimulated healthy human peripheral mononuclear cells (PBMC) is not removed from the PHA-stimulated CEM/0 cell culture.
  • PBMC peripheral mononuclear cells
  • the HIV-1 virus is always present in the supernatant, an experimental condition that simulates the in vivo clinical condition of newly produced and unmfected T-cells, which are always under constant exposure to HIV-1 particles. These virus particles are released by already infected T-cells and/or lymph nodes of patients.
  • the experimental drugs are added to the cells in the appropriate concentrations at the same time as viral inoculation or 90 minutes after the viral incubation, a time sufficient for the T-cells to be infected and start producing new HIV-1 virus particles.
  • the control cells are resuspended in drug-free media for the duration of the exposure which lasted seven days. Aliquots of media from the confrol flasks only are obtained on day 5 post Rx. At day seven, three 1 ml each, aliquots of media are removed from the flasks and stored under liquid nitrogen. In addition, the remaining cells are split into three and then pelleted and stored at - 80°C.
  • PEG-ASNase or Saquinavir used as a monotherapy at IC 50 concentrations, inhibited HIV-RT by 54% and 83%, respectively These values are similar to those determined in earlier experiments.
  • MISID PL-7
  • RR ribonucleotide reductase
  • Combinations of PEG + SAQ resulted in complete inhibition of HIV-RT in this expenment.
  • Combinations of three drugs, AZT + PEG + SAQ resulted in complete inhibition of HIV-RT in two of the three wells, and the third well's value is inhibited by 95.3% of confrol.
  • the biochemical rationale of this drug combination is that AZT will inhibit further infection by HIV-RT and that this inhibition will be potentiated by the already very efficacious anti-HTV-RT effect of PEG + SAQ regimen. Since the numbers are nea ⁇ ng 100% inhibition of HJN-RT it is extremely difficult to demonstrate an improved inhibition by the three drug over the two drug regimen m this T-cell model system infected with a wild- type HTV virus. In experiments with HTV partially resistant to AZT, as they appear m patients, the regimen may demonstrate the validity of the above biochemical syllogism
  • the cell line used for this experiment is CEM/0, a human T- cell leukemic cell line.
  • PEG-AS ⁇ ase (ONCASPAR) is provided by Rhone-Poulenc Rorer.
  • Saquinavm (SQ) is commercially available AZT is purchased from Sigma.
  • MISID a ribonucleotide reductase inhibitor, is as described m Nandy P, Lien EJ, Avramis VI, Med. Chem Res. 1995, 5.664-679.
  • RPMI-1640 media (Irvme Scientific, Irvine, CA), is enriched with 10% Fetal Calf Serum (Gemini Biosource, Calabasas, CA), 5% IM Hepes Buffer solution and 5% Non-essential ammo acids (Irvme Scientific, Irvme, CA).
  • the drug concentrations used are as follows: PEG-ASNase IC50 alone: 0.40 IU/ml
  • the experimental drugs are added to the cells in the appropriate concentrations at the same time as viral inoculation or 90 minutes after the viral incubation, a time sufficient for the T-cells to be infected and start producing new HIV-1 virus particles.
  • the control cells are resuspended in drug-free media for the duration of the exposure which lasted seven days. Aliquots of media from the control flasks only are obtained on day 5 post Rx. At day seven, three 1 ml each, aliquots of media are removed from the flasks and stored under liquid nitrogen. In addition, the remaining cells are split into three and then pelleted and stored at - 80°C.
  • the samples produced from this experiment are itemized.
  • the cell pellets of the cellular cultures (See Experiment 7) from the 90 minute viral incubation flasks are assayed for HIV-RNA quantitative assay using a kit for the assay, non-radioactive.
  • the standard curve is determined and the HIV-RNA levels for the experimental samples are calculated as described previously.
  • MISID a new ribonucleotide reductase (RR) inhibitor, used alone at the IC 50 concentration (0.685 ⁇ M), also demonstrated a significant inhibition of HTV RT as a single agent and in combination with the three other drugs. This is the result in the cell pellets as well (see Experiment 7) and is the first evidence that a member of this class of RR inhibitors has demonstrated anti-HTV activity in addition to its anti-leukemic activity both intracellularly and in the supernatant specimens of these T-cell cultures.
  • RR ribonucleotide reductase
  • Combinations of PEG + SAQ resulted in complete inhibition of HTV-RT in this and in the previously reported experiment in the cell pellets. In the previous experiments there is a 96% inhibition of HIV-RT (see Experiment 7).
  • Combinations of three drugs, AZT + PEG + SAQ resulted in complete inhibition of HIV-RT in two of the three wells, and the third well's value is inhibited by 95.3% of control, whereas in the supernatants an identical pattern is seen Specimens #20-22.
  • Combinations of four drugs, MISID + AZT + PEG + SAQ resulted in complete inhibition of HIV-RT in all three of the supernatant specimens, which values correspond with the values determined in the cell pellets from the same expe ⁇ ment. These values are supe ⁇ mposable to the three drug regimen, due to maximum inhibition of HIN-RT, as we determined earlier.
  • MISID the RR inhibitor
  • AZTTP AZT-tnphosphate
  • the cell line used for these studies is CEM/0, a human T-cell leukemic cell line.
  • PEG-AS ⁇ ase (ONCASPAR) is provided by Rhone-Poulenc Rorer. Saquinavin is commercially available.
  • AZT is purchased from Sigma.
  • MISID a ribonucleotide reductase inhibitor, is synthesized as indicated in Nandy P, Lien EJ, Avramis VI, Med. Chem. Res 1995, 5.664-679.
  • RPMI-1640 media (Irvine Scientific, Irvine, CA), is enriched with 10% Fetal Calf Serum (Gemini Biosource, Calabasas, CA), 5% IM Hepes Buffer solution and 5% Non-essential amino acids (Irvine Scientific, Irvme, CA).
  • the drug concentrations used are as follows:
  • the experimental drugs are added to the cells in the appropriate concentrations at the same time as viral inoculation or 90 minutes after the viral incubation, a time sufficient for the T-cells to be infected and start producing new HFV-l virus particles
  • the control cells are resuspended in drug-free media for the duration of the exposure which lasted seven days Aliquots of media from the control flasks only are obtained on day 5 post Rx At day seven, three 1 ml each, aliquots of media are removed from the flasks and stored under liquid nitrogen In addition, the remaining cells are split into three and then pelleted and stored at - 80°C
  • the samples produced from this experiment are itemized
  • the cell pellets of the cellular cultures (see Experiment 7) from the 90 minute viral incubation flasks are assayed for HIV-R ⁇ A quantitative assay using a kit for the assay, non-radioactive
  • the standard curve is determined and the HTV-R ⁇ A levels for the experimental samples are calculated and reported previously
  • Inhibition of HIV R ⁇ A is quantitative terms cause by AZT alone (Specimens #11- 13) are greater (sensitive HIV-1 virus to AZT), with SAQ and MISFD as single agents following Greater inhibition of HIV R ⁇ A is seen by the combination of SAQ + PEG- AS ⁇ ase, 38% of control, and from the three drug combination of PEG-AS ⁇ ase +SAQ + AZT, 30%) of control. All three specimens treated with the four drug combination regimen of PEG-ASNase + SAQ + MISID and AZT, #23, 24 & 25, has the greatest inhibition of HIV- RNA from this experiment, 20% of control, clearly showing the significant contribution of the ribonucleotide reductase inhibitor, MISID.
  • MISID a new ribonucleotide reductase (RR) inhibitor, used alone at the IC 50 concentration (0.685 ⁇ M), also demonstrated a significant inhibition of HTV RNA as a single agent and most importantly, in combination with the three other drugs. This is the result in the cell pellets as well (Experiment 6) and is the repeat evidence that a member of this class of RR inhibitors has demonstrated anti-HIV activity in addition to its anti-leukemic activity both intracellularly and in the supernatant specimens of these T-cell cultures
  • MISFD the RR inhibitor
  • AZTTP AZT-tnphosphate
  • the cell line used for these studies is CEM/0, a human T- cell leukemic cell line PEG-ASNase (ONCASPAR) is provided by Rhone-Poulenc Rorer Saquinavin is commercially available.
  • AZT is purchased from Sigma.
  • MISFD a ribonucleotide reductase inhibitor, is as described in Nandy P, Lien EJ, Avramis VI, Med. Chem. Res. 1995, 5.664-679.
  • RPMI-1640 media (Irvine Scientific, Irvme, CA), is enriched with 10% Fetal Calf Serum (Gemini Biosource, Calabasas, CA), 5% IM Hepes Buffer solution and 5% Non-essential amino acids (Irv e Scientific, Irvine, CA).
  • the drug concentrations used are as follows-
  • the experimental drugs are added to the cells in the appropriate concentrations at the same time as viral inoculation or 90 minutes after the viral incubation, a time sufficient for the T-cells to be infected and start producing new HIV-1 virus particles.
  • the control cells are resuspended in drug-free media for the duration of the exposure which lasted seven days. Aliquots of media from the control flasks only are obtained on day 5 post Rx. At day seven, three 1 ml each, aliquots of media are removed from the flasks and stored under liquid nitrogen. In addition, the remaining cells are split into three and then pelleted and stored at - 80°C. Supernatant specimens are obtained form these T-cell cultures and are frozen at -80°C.
  • the samples produced from this experiment are itemized.
  • the cell pellets of the cellular cultures (see Experiment 9) from the 90 minute viral incubation flasks are assayed for HIV- RNA quantitative assay using a kit for the assay, non-radioactive.
  • the standard curve is determined and the HIV-RNA levels for the experimental samples are calculated and reported previously.
  • the data suggest that: a) these combination regimens must be given continuously under these conditions, i.e , in patients with high HIV-RNA copy number and/or viremia and b) the potentiation of the AZT + SAQ is required by either a third RT inhibitor, such as 3TC or and an RR inhibitor, such as, MISFD or hydroxyurea, to potentiate the activity of AZT t ⁇ phosphate (AZTTP) against HIV-RT.
  • a third RT inhibitor such as 3TC or and an RR inhibitor, such as, MISFD or hydroxyurea
  • MISID a new ribonucleotide reductase (RR) inhibitor, used alone at the IC 50 concentration (0.685 ⁇ M), also demonstrated a significant inhibition of HIV-RNA as a single agent which is approximately equal to the inhibition of either SAQ or AZT.
  • MISFD showed it significant usefulness in combination with the three other drugs both against HFV-RT in T-cell pellets and supernatants (see Experiments 7 and 8), respectively, and in suppressing HFV-RNA left in the supernatant (Table 8).
  • Experiment 1 1 Determination of the Synergistic effect of PEG-ASNase, Saquinavir, AZT and 3TC on HIV-RNA in CEM/0 Cell Supernatants
  • N is the number of virus genomic copies/mL of supernatant or copies cell pellet. Dynamic range of the HIV-I Monitor Assay is 400 to 750.000 copies ml. Inquire with laboratory if quanti tion below 400 copies/mL is needed. Table 2
  • N is the Dumber of virus ge ⁇ omic copies/mL of supernatant or copies/cell pellet. Dynamic range of the HIV- 1 Monitor Assay is 400 to 750,000 copies mL Inquire with laboratory if quantita on below 400 copies mL is needed. Table 5

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Abstract

Method of inhibiting or treating Human Immunodeficiency Virus (HIV) infection, comprising administering to a patient in need thereof an effective amount of a pharmaceutically acceptable composition comprising a PEG-ASNase compound or asparaginase, and optionally at least one compound selected from the group consisting of protease inhibitor compounds, ribonucleotide reductase inhibitor compounds and HIV reverse transcriptase inhibitor compounds.

Description

COMPOSITION AND METHODS FOR TREATMENT OF HIV INFECTION This application is a continuation of US Patent Application No. 09/370,390 filed August 6, 1999 which is a continuation-in-part of International Patent Application No. PCT US99/02480, filed on February 9, 1999, which is, in turn, a continuation-in-part of U.S. Provisional Patent Application No. 60/074,066, filed February 9, 1998, now abandoned.
Field of the Invention
The invention is directed to a pharmaceutical composition compπsmg a PEG- ASNase compound or a pharmaceutically acceptable salt thereof, and optionally at least one compound selected from the group consisting of protease inhibitor compounds, πbonucleotide reductase inhibitor compounds and HIV reverse transcnptase inhibitor compounds, and a pharmaceutically acceptable carrier. The invention is also directed to a method of inhibiting or treating Human Immunodeficiency Virus (HIV) infection, comprising administering to a patient in need thereof a therapeutically effective amount of a PEG- ASNase compound or a pharmaceutically acceptable salt thereof, and optionally at least one compound selected from the group consisting of protease inhibitor compounds, πbonucleotide reductase inhibitor compounds and HIV reverse transcnptase inhibitor compounds, or a pharmaceutically acceptable salt thereof.
The human immunodeficiency virus (HIV) is a retrovirus and is the agent of the complex disease that includes progressive destruction of the immune system (acquired immune deficiency syndrome; AIDS) and degeneration of the central and peripheral nervous system. This retrovirus is previously known as LAV, HTLV-III, or ARV. There have been various therapies to treat HIV infection, including therapies with combination drug regimens. Protease inhibitor compounds in combination with reverse transcπptase (RT) inhibitor compounds have shown success both in vitro and in vivo in patients infected with the virus. Protease inhibitor compounds interfere with the production of new infectious virus. A common feature of the HTV retrovirus replication is extensive post-translational processing of precursor poly-proteins by a virally encoded protease to generate mature viral proteins required for virus assembly and function. Inhibition of this processing prevents the production of new infectious virus.
Inhibition of the HIV protease by protease inhibitors may prevent proviral integration of infected T-lymphocytes during the early phase of the HIV-1 life cycle, as well as inhibit viral proteolytic processing during its late stage. HTV protease inhibitors have been extensively reviewed (A. Tomasselh et al., Chimica Oggi, 6-27 20 (1991) and T. Meek, J. Enzyme Inhibition 6: 65-98 (19.92). Retroviral replication routinely features post- translational processing of polyproteins. This processing is accomplished by virally encoded HIV protease enzymes. This post-translational process yields mature polypeptides that will subsequently aid in the formation and function of infectious viruses. If this molecular processing is inhibited, then the normal production of HIV is terminated Therefore, it has been discovered that inhibitors of HIV protease may function as anti-HIV viral agents.
Retroviruses are widely distributed in vertebrates and are known to cause a variety of diseases in man and animals including HTV, leukemias and lymphomas. The entire retrovirus family is characterized by the presence of a unique enzyme, reverse transcnptase (RT), which transcribes the viral genomic RNA into a double-stranded DNA copy Therefore, considerable efforts are being directed toward the control of HTV by means of inhibition of HIV -reverse transcnptase, required for replication of the virus. (V. Merluzzi et al , "Inhibition of the HIV-1 Replication by a Nonnucleoside Reverse Transcnptase Inhibitor", Science, 25, 1411 (1990)). For example, a currently used therapeutic compound, AZT, is an inhibitor of the viral reverse transcnptase (U S. Pat. No. 4,724,232). Unfortunately, many of these compounds suffer from toxicity problems, lack of bioavailabihty or are short lived in vivo, viral resistance, or combinations thereof.
It is also known that the inhibition of HTV-reverse transcnptase (HIV-RT) by nucleoside analogue drug combinations indicate that they cannot alone inhibit the RT function completely, but instead can lead to the emergence of drug resistant viral strain. These strains of escape mutants repopulate and render nucleoside analogue therapy ineffective. The addition of protease inhibitor compounds to known nucleoside analogue combination therapies has helped to reduce the viral burden for a prolonged period of time. Ribonucleotide reductase is an allosteπcally regulated enzyme that converts the nucleoside diphosphates to their corresponding deoxynucleoside diphosphates through a complex regulatory mechanism involving one or several electron transfer pathways. (Holmgren A. Hydrogen donor system for E. Coh Ribonucleoside diphosphate reductase dependent upon glutathione, Proc. Natl Acad. Sci. USA, 1976, 73, 2275-9; Therlander L., Reductase of Ribonucleotides, Ann. Rev. Biochem. 1979, 46, 133-58; Ashley GW, Stubbe J., Current ideas on the chemical mechanism of ribonucleotide reductase, Pharmac. Ther. 1985, 30, 301-29; Stubbe J., Ribonucleotide Reductase. Amazing and confusing, J Biol. Chem. 1990, 265, 5329-32.) Reduction of the πbonucleotide by ribonucleotide reductase enables the DNA ploymerases to utilize the deoxynbonucleotides (dNTPs) during the process of DNA replication. Ribonucleotide reductase activity is well coordinated to the process of cellular proliferation and is markedly increased in the late Gl and the early S-Phase when the bulk of DNA synthesis occurs. (Corey J.G., Whitford Jr. T.W., Ribonucleotide reductase and DNA synthesis in Ehrhch ascites tumor cancer cells, Cancer Res., 1972, 32, 1301-6; Hammerstan E, Reichard P., Saluste E., Pyπmidme nucleosides as precursors of pyπmidines in polynucleotides, J. Biol. Chem , 1950, 183, 105-109.) The important role of ribonucleotide reductase in the synthesis of DNA makes it a target for chemotherapeutic agents. There has recently been found a class of 2-hydroxy-lH-ιsoιndole-l,3-dιone (HISID) which have been shown to have ribonucleotide reductase inhibitor activity (Nandy P, Lien EJ, Avramis VI, Acta Oncologica, 33, 8, 953-61 , 1994, Nandy P, Lien EJ, Avramis VI, Rec Adv Chemoth 1 -995-996, 1994; Nandy P, Lien EJ, Avramis VI, Med. Chem. Res. 1995, 5 664-679.) PEG-asparaginase (the polyethylene glycosylated form of E.coh-ASP) has been shown to be useful as a chemotherapeutic agent. In particular, PEG-asparaginase has been found to be an alternative preparation with a longer circulating half-life than E.coh L- asparagmase and has been useful m multiagent chemotherapy for childhood acute lymphoblastic leukemia. (Ettinger LJ, Ettmger AG, Avramis VI, Gaynon PS, BioDrugs, 7, 1, 30-39, 1997). Also, PEG-ASNase may increase the anti-leukemic effect in isolated CNS relapse. (Malgolowkm M, Ortega S, Carcich DA, Steele D, Tischer D, Franklin J, Nandy P, Peπclou A, Cohen LJ, Avramis VI, Proceedings of ASCO, 17, 1998.)
PEG-ASNase is a conjugate of asparaginase with polyethylene glycol. This conjugation occurs through pegylation, a process in which polypeptides, such as enzymes and hormones, are coupled to polyethylene glycol so as to produce a physiologically active non- lmmunogenic water-soluble polypeptide composition. The polyethylene glycol protects the polypeptide from loss of activity and the composition can be injected into the mammalian circulatory system with substantially no lmmunogenic response. The process of pegylation is described in detail in U.S. Patent No. 4,179,337, entitled "Non-Immunogenic Polypeptide", filed July 28, 1977 and issued December 18, 1979, which is incorporated by reference in its entirety herein. Covalent attachment of the polymer to the peptide is affected often by reacting PEG-succinimide derivatives with amino groups on the exterior of protein molecules. Other methods are also disclosed in U S. Patent No. 4,179,337, in Pollack et al., JACS, 98, 289 (1976), U.S. Patent No. 4,847,325 and elsewhere in the art.
SUMMARY OF THE INVENTION
Applicants have discovered that PEG-asparaginase (PEG-ASNase) effectively works alone and synergistically works in combination with one or more of the following: protease inhibitor compounds, HIV reverse transcπptase inhibitor compounds, or ribonucleotide reductase inhibitor compounds, to treat infection by HIV. Accordingly, in its principle aspect, this invention is directed to a pharmaceutical composition compπsing a PEG-ASNase compound and optionally at least one compound selected from the group consisting of protease inhibitor compounds, ribonucleotide reductase inhibitor compounds and HTV reverse transcnptase inhibitor compounds, and a pharmaceutically acceptable carrier The invention is also directed to a method of inhibiting or treating Human Immunodeficiency Virus (HTV) infection, comprising administering to a patient in need thereof a therapeutically effective amount of a PEG-ASNase compound or a pharmaceutically acceptable salt thereof, and optionally at least one compound selected from the group consisting of protease inhibitor compounds, ribonucleotide reductase inhibitor compounds and HIV reverse transcnptase inhibitor compounds, or a pharmaceutically acceptable salt thereof
Brief Description of the Figures
Figure 1 represents the inhibition of HIV-RT in CEM/0 T-cells (± PHA) treated with IC50 concentrations of PEG-ASNase or Saquinavir (SAQ) alone or in their combination Figure 2 represents T-Cell (CEM/0) cytotoxicity of Saquinavir, after 72 hours, for different drug concentrations
Figure 3 represents T-Cell cytotoxicity of PEG-ASNase and Saquinavir alone, and in sequential combination (PEG-ASNase followed by Saquinavir), for different drug concentrations
Figure 4 represents T-Cell cytotoxicity of PEG-ASNase and Saquinavir alone, and in sequential combination (Saquinavir followed by PEG-ASNase), for different drug concentrations
Figure 5 represents T-Cell cytotoxicity of PEG-ASNase and Saquinavir alone, and in concurrent combination, for different drug concentrations
Figure 6 represents T-Cell synergism of PEG-ASNase and Saquinavir in concurrent combination for different drug concentrations
Figure 7 represents the combination index (CI) in CEM/0 of sequential combination of Saquinavir followed by PEG-ASNase, and sequential combination of PEG-ASNase followed by Saquinavir, and concurrent combination of PEG-ASNase and Saquinavir Figure 8 represents the depletion of Asparagme, Glutamine and Aspartic acid concentrations in CEM/0 T-cells after exposure to different concentrations of PEG-ASNase for 24 hours
Figure 8a represents a calibration curve of the optical density (OD) of different concentrations of HIV-1 RT Figure 9 represents the number of HTV RNA copies per cell pellets after exposure of the cells to PEG-ASNase and Saquinavir alone and in combination.
Figure 9a represents Logι0of the number of HJN RΝA copies per cell pellets after exposure of the cells to PEG-ASΝase and Saquinavir alone and in combination. Figure 10 represents the calibration curves for the HIV-RT Ehsa assay.
Figure 1 1 represents the HIV-1 quantitative RΝA assay of CEM-T-cells treated with single regimens of PEG-ASΝase, Saquinavir, AZT and MISID, and combination regimens of Saquinavir and PEG-ASΝase, AZT, Saquinavir and PEG-ASΝase, and MISID, AZT, Saquinavir and PEG-ASΝase.
Detailed Description of the Invention
As used above, and throughout the description of the invention, the following terms, unless otherwise indicated, shall be understood to have the following meanings -
"Acyl" means an H-CO- or alkyl-CO- group wherein the alkyl group is as herein described. Preferred acyls contain a lower alkyl. Exemplary acyl groups include formyl, acetyl, propanoyl, 2-methylpropanoyl, butanoyl and palmitoyl.
"Acylamino" is an acyl-ΝH- group wherein acyl is as defined herein. "Alkenyl" means an aliphatic hydrocarbon group containing a carbon-carbon double bond and which may be straight or branched having about 2 to about 15 carbon atoms in the chain. Preferred alkenyl groups have 2 to about 12 carbon atoms in the chain; and more preferably about 2 to about 4 carbon atoms in the chain. Branched means that one or more lower alkyl groups such as methyl, ethyl or propyl are attached to a linear alkenyl chain. "Lower alkenyl" means about 2 to about 4 carbon atoms in the chain which may be straight or branched. The alkenyl group may be substituted by one or more halo or cycloalkyl group. Exemplary alkenyl groups include ethenyl, propenyl, «-butenyl, r'-butenyl, 3-methylbut-2- enyl, «-pentenyl, heptenyl, octenyl, cyclohexylbutenyl and decenyl.
"Alkoxy" means an alkyl-O- group wherein the alkyl group is as herein described. Exemplary alkoxy groups include methoxy, ethoxy, n-propoxy, /-propoxy, w-butoxy and heptoxy.
"Alkoxycarbonyl" means an alkyl-O-CO- group, wherein the alkyl group is as herein defined. Exemplary alkoxycarbonyl groups include methoxycarbonyl, ethoxycarbonyl, or t- butyloxycarbony 1.
"Alkyl" means an aliphatic hydrocarbon group which may be straight or branched having about 1 to about 20 carbon atoms in the chain. Preferred alkyl groups have 1 to about 12 carbon atoms in the chain. Most preferred alkyl groups have 1 to about 3 carbon atoms in the chain. Branched means that one or more lower alkyl groups such as methyl, ethyl or propyl are attached to a linear alkyl chain "Lower alkyl" means about 1 to about 3 carbon atoms in the chain which may be straight or branched The alkyl may be substituted with one or more "alkyl group substituents" which may be the same or different, and include halo, cycloalkyl, hydroxy, alkoxy, amino, acylamino, aroylamino, carboxy, alkoxycarbonyl,
1 2 1 2 aralkoxycarbonyl, heteroaralkoxycarbonyl or Y Y NCO-, wherein Y and Y are independently hydrogen, optionally substituted alkyl, optionally substituted aryl, optionally
1 2 substituted aralkyl or optionally substituted heteroaralkyl, or Y and Y taken together with
1 2 the N through which Y and Y are linked form a 4 to 7 membered heterocyclyl Exemplary alkyl groups include methyl, tπfluoromethyl, cyclopropylmethyl, cyclopentylmethyl, ethyl, n- propyl, ;-propyl, «-butyl, t-butyl, n-pentyl, 3-pentyl, methoxyethyl, carboxymethyl, formyl, methoxycarbonylethyl, benzyloxycarbonylmethyl, pyπdylmethyloxycarbonylmethyl
Preferred alkyl substituents are halo, hydroxy, alkoxy, amino, acylamino, aroylamino, carboxy, alkoxycarbonyl, aralkoxycarbonyl, heteroaralkoxycarbonyl, sulfonyl, sulfinyl, acyl, alkanoyl, or Y1Y2NCO- "Alkylthio" means an alkyl-S- group wherein the alkyl group is as herein described
Exemplary alkylthio groups include methylthio, ethylthio, i-propylthio and heptylthio
"Alkylsulfinyl" means an alkyl-SO- group wherein the alkyl group is as defined above Preferred groups are those wherein the alkyl group is lower alkyl
"Alkylsulfonyl" means an alkyl-S02-group wherein the alkyl group is as defined above Preferred groups are those wherein the alkyl group is lower alkyl
"Alkynyl" means an aliphatic hydrocarbon group containing a carbon-carbon triple bond and which may be straight or branched having about 2 to about 15 carbon atoms in the chain Preferred alkynyl groups have 2 to about 12 carbon atoms in the chain, and more preferably about 2 to about 4 carbon atoms in the chain Branched means that one or more lower alkyl groups such as methyl, ethyl or propyl are attached to a linear alkynyl chain
"Lower alkynyl" means about 2 to about 4 carbon atoms in the chain which may be straight or branched The alkynyl group may be substituted by one or more halo Exemplary alkynyl groups include ethynyl, propynyl, n-butynyl, 2-butynyl, 3-methylbutynyl, π-pentynyl, heptynyl, octynyl and decynyl "Analogue" means a compound which comprises a chemically modified form of a specific compound or class thereof, and which maintains the pharmaceutical and/or pharmacological activities characteristic of said compound or class
"Aralkoxy" means an aralkyl-O- group wherein the aralkyl groups is as herein described Exemplary aralkoxy groups include benzyloxy and 1 - or 2-naphthalenemethoxy "Aralkoxycarbonyl" means an aralkyl-O-CO- group wherein the aralkyl groups is as herein described. An exemplary aralkoxycarbonyl group is benzyloxycarbonyl.
"Aralkyl" means an aryl-alkyl- group wherein the aryl and alkyl are as herein described. Preferred aralkyls contain a lower alkyl moiety. Exemplary aralkyl groups include benzyl, 2-phenethyl and naphthlenemethyl
"Aralkylthio" means an aralkyl-S- group wherein the aralkyl group is as herein described. An exemplary aralkylthio group is benzylthio.
"Aryloxycarbonyl" means an aryl-O-CO- group wherein the aryl group is as defined herein Exemplary aryloxycarbonyl groups include phenoxycarbonyl and naphthoxycarbonyl.
"Aroylamino" is an aroyl-NH- group wherein aroyl is as defined herein
"Aroyl" means an aryl-CO- group wherein the aryl group is as herein described Exemplary groups include benzoyl and 1- and 2-naphthoyl.
"Aryl" means an aromatic monocychc or multicychc ring system of about 6 to about 14 carbon atoms, preferably of about 6 to about 10 carbon atoms. The aryl is optionally substituted with one or more "ring system substituents" which may be the same or different, and are as defined herein. Exemplary aryl groups include phenyl or naphthyl, or phenyl substituted or naphthyl substituted.
"Aryldiazo" means an aryl -diazo- group wherein the aryl and diazo groups are as defined herein.
"Aryloxy" means an aryl-O- group wherein the aryl group is as defined herein. Exemplary groups include phenoxy and 2-naphthyloxy.
"Arylsulfonyl" means an aryl-S02- group wherein the aryl group is as defined herein.
"Arylsulfinyl" means an aryl-SO- group wherein the aryl group is as defined herein "Arylthio" means an aryl-S- group wherein the aryl group is as herein described.
Exemplary arylthio groups include phenylthio and naphthylthio.
"Carboxy" means a HO(0)C- (carboxylic acid) group.
"Compounds of the invention", and equivalent expressions, are meant to embrace compounds of the invention as hereinbefore described, which expression includes the prodrugs, the pharmaceutically acceptable salts, and the solvates, e g. hydrates, where the context so permits. Similarly, reference to intermediates, whether or not they themselves are claimed, is meant to embrace their salts, and solvates, where the context so permits For the sake of clarity, particular instances when the context so permits are sometimes indicated in the text, but these instances are purely illustrative and it is not intended to exclude other instances when the context so permits. "Cycloalkenyl" means a non-aromatic mono- or multicychc ring system of about 3 to about 10 carbon atoms, preferably of about 5 to about 10 carbon atoms, and which contains at least one carbon-carbon double bond. Preferred ring sizes of rings of the ring system include about 5 to about 6 ring atoms. The cycloalkenyl is optionally substituted with one or more "ring system substituents" which may be the same or different, and are as defined herein.
Exemplary monocychc cycloalkenyl include cyclopentenyl, cyclohexenyl, cycloheptenyl, and the like. An exemplary multicychc cycloalkenyl is norbornylenyl.
"Cycloalkyl" means a non-aromatic mono- or multicychc ring system of about 3 to about 10 carbon atoms, preferably of about 5 to about 10 carbon atoms. Preferred ring sizes of rings of the ring system include about 5 to about 6 ring atoms The cycloalkyl is optionally substituted with one or more "ring system substituents" which may be the same or different, and are as defined herein. Exemplary monocychc cycloalkyl include cyclopentyl, cyclohexyl, cycloheptyl, and the like. Exemplary multicychc cycloalkyl include 1-decalιn, norbornyl, adamant-(l- or 2-)yl, and the like. "Derivative" means a chemically modified compound wherein the modification is considered routine by the ordinary skilled chemist, such as an ester or an amide of an acid, protecting groups, such as a benzyl group for an alcohol or thiol, and tert-butoxycarbonyl group for an amine
"Diazo" means a bivalent -N=N- radical. "Effective amount" means an amount of a compound/composition according to the present invention effective in producing the desired therapeutic effect
"Electron withdrawing group" as defined herein refers to a group that will draw electrons to itself more than a hydrogen atom would if it occupied the same position in the molecule See, J. March, Advanced Organic Chemistry, 3rd Ed., John Wiley & Sons P. 17 (1985). They include such groups as nitro, monohaloalkyl, dihaloalkyl, tnhaloalkyl (e.g., CF3), halogen, formyl, alkylsulfonyl, alkylsulfinyl, and the like. Preferably halogen.
"Formulations suitable for nasal or inhalational administration" means formulations which are in a form suitable to be administered nasally or by inhalation to a patient. The formulation may contain a carrier, in a powder form, having a particle size for example in the range 1 to 500 microns (including particle sizes in a range between 20 and 500 microns in increments of 5 microns such as 30 microns, 35 microns, etc.) Suitable formulations wherein the carrier is a liquid, for administration as for example a nasal spray or as nasal drops, include aqueous or oily solutions of the active ingredient. Formulations suitable for aerosol administration may be prepared according to conventional methods and may be delivered with other therapeutic agents. Inhalational therapy is readily administered by metered dose inhalers. "Formulations suitable for oral administration" means formulations which are in a form suitable to be administered orally to a patient. The formulations may be presented as discrete units such as capsules, cachets or tablets each containing a predetermined amount of the active ingredient; as a powder or granules; as solution or a suspension in an aqueous liquid or a non-aqueous liquid; or as an oil-in-water liquid emulsion or a water-in-oil liquid emulsion. The active ingredient may also be presented as a bolus, electuary or paste.
"Formulations suitable for parenteral administration" means formulations which are in a form suitable to be administered parenterally to a patient. The formulations are sterile and include emulsions, suspensions, aqueous and non-aqueous injection solutions, which may contain suspending agents and thickening agents and anti-oxidants, buffers, bactenostats and solutes which render the formulation lsotonic, and have a suitably adjusted pH, with the blood of the intended recipient
"Formulations suitable for rectal administrations" means formulations which are in a form suitable to be administered rectally to a patient The formulation is preferably in the form of suppositories which can be prepared by mixing the compounds useful according to this invention with suitable non-irntating excipients or carriers such as cocoa butter, polyethylene glycol or a suppository wax, which are solid at ordinary temperatures but liquid at body temperature and therefore, melt in the rectum or vaginal cavity and release the active component. "Formulations suitable for systemic administration" means formulations which are in a form suitable to be administered systemically to a patient The formulation is preferably administered by injection, including transmuscular, intravenous, intrapentoneal, and subcutaneous. For injection, the compounds useful according to the invention are formulated „ in liquid solutions, preferably in physiologically compatible buffers such as Hank's solution or Ringer's solution. In addition, the compounds may be formulated in solid form and redissolved or suspended immediately prior to use. Lyophilized forms are also included. Systematic administration also can be by transmucosal or transdermal means, or the compounds can be administered orally. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, bile salts and fusidic acid derivatives for transmucosal administration In addition, detergents may be used to facilitate permeation. Transmucosal administration may be through use of nasal sprays, for example, or suppositories. For oral administration, the compounds are formulated into conventional oral administration forms such as capsules, tablets, and tonics. "Formulations suitable for topical administration" means formulations which are in a form suitable to be administered topically to a patient. The formulation may be presented as a topical ointment, salves, powders, sprays and inhalants, gels (water or alcohol based), creams, as is generally known in the art, or incorporated into a matrix base for application in a patch, which would allow a controlled release of compound through the transdermal barrier. When formulated in an ointment, the active ingredients may be employed with either a paraffimc or a water-miscible ointment base. Alternatively, the active ingredients may be formulated in a cream with an oil-in-water cream base Formulations suitable for topical administration in the eye include eye drops wherein the active ingredient is dissolved or suspended in a suitable carrier, especially an aqueous solvent for the active ingredient Formulations suitable for topical administration in the mouth include lozenges comprising the active ingredient in a flavored basis, usually sucrose and acacia or tragacanth, pastilles comprising the active ingredient in an inert basis such as gelatin and glycerin, or sucrose and acacia, and mouthwashes comprising the active ingredient in a suitable liquid carrier
"Formulations suitable for vaginal administration" means formulations which are in a form suitable to be administered vagmally to a patient The formulation may be presented as pessaries, tampons, creams, gels, pastes, foams or spray formulations containing in addition to the active ingredient such carriers as are known in the art to be appropriate
"Heteroaralkoxycarbonyl" means an heteroaralkyl-O-CO- group wherein the heteroaralkyl groups is as herein descnbed An exemplary heteroaralkoxycarbonyl group is thienylmethylcarbonyl "Heteroaralkyl" means a heteroaryl-alkyl- group wherein the heteroaryl and alkyl are as herein described. Preferred heteroaralkyls contain a lower alkyl moiety. Exemplary heteroaralkyl groups may contain thienylmethyl, pyπdylmethyl, lmidazolylmethyl and pyrazmylmethyl
"Heteroaralkylthio" means an heteroaralkyl-S- group wherein the heteroaralkyl group is as herein described. An exemplary heteroaralkylthio group is pyπdylmethylthio
"Heteroaryl" means an aromatic monocychc or multicychc ring system of about 5 to about 14 carbon atoms, preferably about 5 to about 10 carbon atoms, in which one or more of the carbon atoms m the ring system is/are hetero element(s) other than carbon, for example nitrogen, oxygen or sulfur. Preferred ring sizes of rings of the ring system include about 5 to about 6 ring atoms. The "heteroaryl" may also be substituted by one or more "ring system substituents" which may be the same or different, and are as defined herein. The designation of the aza, oxa or thia as a prefix before heteroaryl define that at least a nitrogen, oxygen or sulfur atom is present respectively as a ring atom A nitrogen atom of an heteroaryl may be a basic nitrogen atom and may also be optionally oxidized to the corresponding N-oxide. Exemplary heteroaryl and substituted heteroaryl groups include pyrazmyl, thienyl, isothiazolyl, oxazolyl, pyrazolyl, furazanyl, pyrrolyl, 1,2,4-thιadιazolyl, pyπdazinyl, quinoxahnyl, phthalazinyl, ιmιdazo[l ,2-a]pyπdιne, ιmιdazo[2,l-b]thιazolyl, benzofurazanyl, azaindolyl, benzimidazolyl, benzothienyl, thienopyndyl, thienopynmidyl, pyrrolopyπdyl, lmidazopyπdyl, benzoazamdole, 1,2,4-tnazmyl, benzthiazolyl, furanyl, lmidazolyl, indolyl, lndohzmyl, isoxazolyl, isoquinolinyl, isothiazolyl, oxadiazolyl, pyrazmyl, pyπdazinyl, pyrazolyl, pyndyl, pynmidinyl, pyrrolyl, qumazohnyl, quinohnyl, 1,3,4-thιadιazolyl, thiazolyl, thienyl and tπazolyl. Preferred heteroaryl groups include pyrazmyl, thienyl, pyndyl, pynmidinyl, isoxazolyl and isothiazolyl.
"Heteroaryldiazo" means an heteroaryl -diazo- group wherein the heteroaryl and diazo groups are as defined herein. "Heteroarylsulfonyl" means an aryl-S02- group wherein the heteroaryl group is as defined herein.
"Heteroarylsulfinyl" means an aryl-SO- group wherein the heteroaryl group is as defined herein
"Heteroarylthio" means an aryl-S- group wherein the heteroaryl group is as herein described. Exemplary heteroarylthio groups include pyndylthio and pyπmidmylthio.
"Heterocyclenyl" means a non-aromatic monocychc or multicychc hydrocarbon ring system of about 3 to about 10 carbon atoms, preferably about 5 to about 10 carbon atoms, in which one or more of the carbon atoms in the ring system is/are hetero element(s) other than carbon, for example nitrogen, oxygen or sulfur atoms, and which contains at least one carbon-carbon double bond or carbon-nitrogen double bond Preferred ring sizes of rings of the ring system include about 5 to about 6 nng atoms. The designation of the aza, oxa or thia as a prefix before heterocyclenyl define that at least a nitrogen, oxygen or sulfur atom is present respectively as a ring atom. The heterocyclenyl may be optionally substituted by one or more ring system substituent, wherein the "ring system substituent" is as defined herein The nitrogen atom of an heterocyclenyl may be a basic nitrogen atom. The nitrogen or sulphur atom of the heterocyclenyl may also be optionally oxidized to the corresponding N-oxide, S-oxide or S,S-dιoxιde. Exemplary monocychc azaheterocyclenyl groups include 1,2,3,4- tetrahydrohydropyndine, 1,2-dιhydropyndyl, 1,4-dιhydropyndyl, 1,2,3,6-tetrahydropyπdme, 1,4,5,6-tetrahydropyπmιdιne, 2-pyrrohnyl, 3-pyrrohnyl, 2- imidazohnyl, 2-pyrazohnyl, and the like. Exemplary oxaheterocyclenyl groups include 3,4- dιhydro-2H-pyran, dihydrofuranyl, and fluorodihydrofuranyl. Preferred is dihydrofuranyl. An exemplary multicychc oxaheterocyclenyl group is 7-oxabιcyclo[2 2.1]heptenyl. Preferred monocychc thiaheterocycleny rings include dihydrothiophenyl and dihydrothiopyranyl; more preferred is dihydrothiophenyl. Preferred ring system substituents include amidmo, halogen, hydroxy, alkoxycarbonylalkyl, carboxyalkyl or Y'Y2N- as defined herein. "Heterocyclyl" means a non-aromatic saturated monocychc or multicychc ring system of about 3 to about 10 carbon atoms, preferably about 5 to about 10 carbon atoms, in which one or more of the carbon atoms in the ring system is/are hetero element(s) other than carbon, for example nitrogen, oxygen or sulfur Preferred ring sizes of rings of the ring system include about 5 to about 6 ring atoms The designation of the aza, oxa or thia as a prefix before heterocyclyl define that at least a nitrogen, oxygen or sulfur atom is present respectively as a ring atom The heterocyclyl may be optionally substituted by one or more "ring system substituents" which may be the same or different, and are as defined herein The nitrogen atom of an heterocyclyl may be a basic nitrogen atom The nitrogen or sulphur atom of the heterocyclyl may also be optionally oxidized to the corresponding N-oxide, S-oxide or S,S-dιoxιde Exemplary monocychc heterocyclyl rings include pipeπdyl, pyrrohdinyl, piperazinyl, morpholmyl, thiomorphohnyl, thiazohdinyl, 1,3-dιoxolanyl, 1,4-dιoxanyl, tetrahydrofuranyl, tetrahydrothiophenyl, tetrahydrothiopyranyl, and the like Preferred heterocyclyl group substituents include amidino, halogen, hydroxy, alkoxycarbonylalkyl, carboxyalkyl or Y1 Y2N- as defined herein
"Hydrate" means a solvate wherein the solvent molecule(s) is/are H20 "Hydroxyalkyl" means a HO-alkyl- group wherein alkyl is as herein defined Preferred hydroxyalkyls contain lower alkyl Exemplary hydroxyalkyl groups include hydroxymethyl and 2-hydroxyethyl. "Hygroscopicity" means sorption, implying an acquired amount or state of water sufficient to affect the physical or chemical properties of the substance (Eds. J. Swarbπck and J C Boylan, Encyclopedia of Pharmaceutical Technology, Vol 10, p. 33).
"Liquid dosage form" means the dose of the active compound to be administered to the patient is in liquid form, for example, pharmaceutically acceptable emulsions, solutions, suspensions, syrups and elixirs In addition to the active compounds, the liquid dosage forms may contain inert diluents commonly used in the art, such as water or other solvents, solubihzing agents and emulsifiers, as for example, ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils, in particular, cottonseed oil, groundnut oil, corn germ oil, olive oil, castor oil and sesame oil, glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan or mixtures of these substances, and the like.
"MISID (PL-7)" means a compound of formula I, wherein Rl is a methyl group; and R2 is an isopropyl group.
"Modulate" means the ability of a compound to either directly (by binding to the receptor as a hgand) or indirectly (as a precursor for a hgand or an mducer which promotes production of a hgand from a precursor) induce expression of gene(s) maintained under hormone control, or to repress expression of gene (s) maintained under such control. "Patient" includes both human and other mammals.
"PEG-ASNase" means the protein synthesis inhibitor compound, asparaginase, conjugated with polyethylene glycol (PEG). The polyethylene glycol preferably has an average molecular weight between about 1000 and 100,000 daltons, more preferably between 4000 and 40,000 daltons, depending, for example, on the molecular weight of the particular protein synthesis inhibitor compound employed. Since the object of the modification is to obtain a conjugated protein with retained biological activity, with enhanced in vivo half-life over the unconjugated protein synthesis inhibitor compound, and with reduced lmmunogenicity, the molecular weight of the polymer will be chosen to optimize these conditions. Preferably the PEG homopolymer is substituted at one end with an alkyl group, but it may also be unsubstituted. Preferably the alkyl group is a Ci -C4 alkyl group, and most preferably a methyl group. Preferably, the polymer is a monomethyl-substituted PEG homopolymer and has a molecular weight of about 4000 to 40,000 daltons. Most preferably, PEG-ASNase is the compound sold under the name ONCASPAR by Rhόne-Poulenc Rorer "Pharmaceutical composition" means a composition compπsing a compound of the invention and at least one component selected from the group comprising pharmaceutically acceptable carriers, diluents, adjuvants, excipients, or vehicles, such as preserving agents, fillers, disintegrating agents, wetting agents, emulsifying agents, suspending agents, sweetening agents, flavoring agents, perfuming agents, antibacterial agents, antifungal agents, lubricating agents and dispensing agents, depending on the nature of the mode of administration and dosage forms Examples of suspending agents include ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalhne cellulose, aluminum metahydroxide, bentomte, agar-agar and tragacanth, or mixtures of these substances. Prevention of the action of microorganisms can be ensured by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, and the like. It may also be desirable to include lsotonic agents, for example sugars, sodium chloride and the like. Prolonged absorption of the injectable pharmaceutical form can be brought about by the use of agents delaying absorption, for example, aluminum monosterate and gelatin. Examples of suitable carriers, diluents, solvents or vehicles include water, ethanol, polyols, suitable mixtures thereof, vegetable oils (such as olive oil) and injectable organic esters such as ethyl oleate. Examples of excipients include lactose, milk sugar, sodium citrate, calcium carbonate, dicalcium phosphate phosphate. Examples of disintegrating agents include starch, alginic acids and certain complex silicates. Examples of lubricants include magnesium stearate, sodium lauryl sulphate, talc, as well as high molecular weight polyethylene glycols.
"Pharmaceutically acceptable" means it is, within the scope of sound medical judgement, suitable for use in contact with the cells of humans and lower animals without undue toxicity, irritation, allergic response and the like, and are commensurate with a reasonable benefit/risk ratio.
"Pharmaceutically acceptable dosage forms" means dosage forms of the compound of the invention, and includes, for example, tablets, dragees, powders, elixirs, syrups, liquid preparations, including suspensions, sprays, inhalants tablets, lozenges, emulsions, solutions, granules, capsules and suppositories, as well as liquid preparations for injections, including hposome preparations Techniques and formulations generally may be found in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, PA, latest edition
"Pharmaceutically acceptable prodrugs" as used herein means those prodrugs of the compounds useful according to the present invention which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and lower animals with undue toxicity, irritation, allergic response, and the like, commensurate with a reasonable benefit/risk ratio, and effective for their intended use, as well as the zwitteπonic forms, where possible, of the compounds of the invention The term "prodrug" means compounds that are rapidly transformed in vivo to yield the parent compound of the invention, for example by hydrolysis in blood. Functional groups which may be rapidly transformed, by metabolic cleavage, in vivo form a class of groups reactive with the carboxyl group of the compounds of this invention They include, but are not limited to such groups as alkanoyl (such as acetyl, propionyl, butyryl, and the like), unsubstituted and substituted aroyl (such as benzoyl and substituted benzoyl), alkoxycarbonyl (such as ethoxycarbonyl), tnalkylsilyl (such as tnmethyl- and tnethysilyl), monoesters formed with dicarboxylic acids (such as succinyl), and the like Because of the ease with which the metabolically cleavable groups of the compounds useful according to this invention are cleaved in vivo, the compounds bearing such groups act as pro-drugs. The compounds bearing the metabolically cleavable groups have the advantage that they may exhibit improved bioavailabihty as a result of enhanced solubility and/or rate of absorption conferred upon the parent compound by virtue of the presence of the metabolically cleavable group. A thorough discussion of prodrugs is provided in the following: Design of Prodrugs, H. Bundgaard, ed., Elsevier, 1985; Methods in Enzymology, K. Widder et al, Ed., Academic Press, 42, p.309-396, 1985; A Textbook of Drug Design and Development, Krogsgaard-Larsen and H. Bundgaard, ed., Chapter 5; "Design and Applications of Prodrugs" p.113-191, 1991; Advanced Drug Delivery Reviews, H Bundgard, 8, p.1-38, 1992; Journal of Pharmaceutical Sciences, 77, p. 285, 1988; Chem. Pharm. Bull., N. Nakeya et al, 32, p. 692, 1984; Pro-drugs as Novel Delivery Systems, T. Higuchi and V. Stella, Vol 14 of the A.C.S. Symposium Series, and Bioreversible Carriers m Drug Design, Edward B. Roche, ed , American Pharmaceutical Association and Pergamon Press, 1987, which are incorporated herein by reference. "Pharmaceutically acceptable salts" means the relatively non-toxic, inorganic and organic acid addition salts, and base addition salts, of compounds of the present invention These salts can be prepared in situ during the final isolation and purification of the compounds. In particular, acid addition salts can be prepared by separately reacting the purified compound in its free base form with a suitable organic or inorganic acid and isolating the salt thus formed Exemplary acid addition salts include the hydrobromide, hydrochloπde, sulfate, bisulfate, phosphate, nitrate, acetate, oxalate, valerate, oleate, palmitate, stearate, laurate, borate, benzoate, lactate, phosphate, tosylate, citrate, maleate, fumarate, succinate, tartrate, naphthylate, mesylate, glucoheptonate, lactiobionate, sulphamates, malonates, sahcylates, propionates, methylene-bis-b-hydroxynaphthoates, gentisates, lsethionates, di-p-toluoyltartrates, methane-sulphonates, ethanesulphonates, benzenesulphonates, p-toluenesulphonates, cyclohexylsulphamates and quinateslaurylsulphonate salts, and the like (See, for example S M. Berge, et al., "Pharmaceutical Salts," J Pharm. Sci., 66' p 1 -19 (1977) which is incorporated herein by reference ) Base addition salts can also be prepared by separately reacting the purified compound in its acid form with a suitable organic or inorganic base and isolating the salt thus formed. Base addition salts include pharmaceutically acceptable metal and amine salts. Suitable metal salts include the sodium, potassium, calcium, barium, zinc, magnesium, and aluminum salts. The sodium and potassium salts are preferred. Suitable inorganic base addition salts are prepared from metal bases which include sodium hydride, sodium hydroxide, potassium hydroxide, calcium hydroxide, aluminium hydroxide, lithium hydroxide, magnesium hydroxide, zinc hydroxide Suitable amine base addition salts are prepared from amines which have sufficient basicity to form a stable salt, and preferably include those amines which are frequently used in medicinal chemistry because of their low toxicity and acceptability for medical use. ammonia, ethylenediamine, N-methyl-glucamine, lysine, argimne, ornithine, cholme, N,N'-dιbenzylethylenedιamιne, chloroprocame, diethanolamine, procaine, N-benzylphenethylamme, diethylamine, piperazine, tπs(hydroxymethyl)-ammomethane, tetramethylammonium hydroxide, tπethylamine, dibenzylamme, ephenamme, dehydroabietylamme, N-ethylpipeπdine, benzylamine, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, tπmethylamine, ethylamme, basic amino acids, e.g., lysine and argmme, and dicyclohexylamine, and the like. "Ring system substituents" mean substituents attached to aromatic or non-aromatic ring systems inclusive of hydrogen, alkylaryl, heteroaryl, aralkyl, heteroaralkyl, hydroxy, hydroxyalkyl, alkoxy, aryloxy, aralkoxy, acyl, aroyl, halo, nitro, cyano, carboxy, alkoxycarbonyl, aryloxycarbonyl, aralkoxycarbonyl, alkylsulfonyl, arylsulfonyl, heteroarylsulfonyl, alkylsulfinyl, arylsulfinyl, heteroarylsulfinyl, alkylthio, arylthio, heteroarylthio, aralkylthio, heteroaralkylthio, cycloalkyl, cycloalkenyl, heterocyclyl, heterocyclenyl, aryldiazo, heteroaryldiazo, Y'Y2N-, Y'Y2N-alkyl-, Y'Y2NCO- or Y'Y2NSθ2- , wherein Y1 and Y2 are independently hydrogen, optionally substituted alkyl, optionally substituted aryl, optionally substituted aralkyl or optionally substituted heteroaralkyl, or for where the substituent is Y'Y2N-, then one of Y1 and Y2 may be acyl or aroyl as defined herein and the other of Y1 and Y2 is as defined previously, or for where the substituent is Y'Y2NCO- or Y'Y2NS02, Y1 and Y2 may also be taken together with the N atom through which Y1 and Y2 are linked to form a 4 to 7 membered heterocyclyl or heterocyclenyl When a ring system is saturated or partially saturated, the "ring system substituents" further include , methylene (H2C=), oxo (0=), thioxo (S=)
"Solid dosage form" means the dosage form of the compound useful according to the invention is solid form, for example capsules, tablets, pills, powders, dragees or granules. In such solid dosage forms, the compound useful according to the invention is admixed with at least one inert customary excipient (or carrier) such as sodium citrate or dicalcium phosphate or (a) fillers or extenders, as for example, starches, lactose, sucrose, glucose, manmtol and silicic acid, (b) binders, as for example, carboxymethylcellulose, ahgnates, gelatin, polyvinylpyrrohdone, sucrose and acacia, (c) humectants, as for example, glycerol, (d) disintegrating agents, as for example, agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain complex silicates and sodium carbonate, (e) solution retarders, as for example paraffin, (f) absorption accelerators, as for example, quaternary ammonium compounds, (g) wetting agents, as for example, cetyl alcohol and glycerol monostearate, (h) adsorbents, as for example, kaolin and bentomte, (I) lubricants, as for example, talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, (j) opacifymg agents, (k) buffering agents , and agents which release the compound(s) useful according to the invention in a certain part of the intestinal tract in a delayed manner.
"Solvate" means a physical association of a compound of this invention with one or more solvent molecules. This physical association includes hydrogen bonding. In certain instances the solvate will be capable of isolation, for example when one or more solvent molecules are incorporated in the crystal lattice of the crystalline solid. "Solvate" encompasses both solution-phase and isolable solvates. Exemplary solvates include hydrates, ethanolates, methanolates, and the like. In a specific embodiment, the term "about" or "approximately" means within 20%, preferably within 10%, and more preferably within 5% of a given value or range.
Preferred Embodiments A preferred embodiment according to the invention is a method of inhibiting or treating Human Immunodeficiency Virus (HTV) infection, comprising administering to a patient in need thereof an effective amount of a pharmaceutically acceptable composition compπsing a PEG-ASNase compound
Another preferred embodiment according to the invention is a method of inhibiting or treating Human Immunodeficiency Virus (HIV) infection, comprising administering to a patient in need thereof an effective amount of a pharmaceutically acceptable composition comprising a PEG-ASNase compound and at least one protease inhibitor compound.
Another preferred embodiment according to the invention is a method of inhibiting or treating Human Immunodeficiency Virus (HIV) infection, comprising administering to a patient in need thereof an effective amount of a pharmaceutically acceptable composition compπsing a PEG-ASNase compound and at least one HTV reverse transcnptase inhibitor compound
Another preferred embodiment according to the invention is a method of inhibiting or treating Human Immunodeficiency Virus (HTV) infection, comprising administering to a patient in need thereof an effective amount of a pharmaceutically acceptable composition comprising a PEG-ASNase compound and at least one ribonucleotide reductase inhibitor compound.
Another preferred embodiment according to the invention is a method of inhibiting or treating Human Immunodeficiency Virus (HIV) infection, compπsing administering to a patient in need thereof an effective amount of a pharmaceutically acceptable composition comprising a PEG-ASNase compound, at least one a protease inhibitor compound and at least one ribonucleotide reductase inhibitor compound.
Another preferred embodiment according to the invention is a method of inhibiting or treating Human Immunodeficiency Virus (HTV) infection, comprising administering to a patient in need thereof an effective amount of a pharmaceutically acceptable composition comprising a PEG-ASNase compound, at least one protease inhibitor compound and at least one HIV reverse transcnptase inhibitor compound.
Another preferred embodiment according to the invention is a method of inhibiting or treating Human Immunodeficiency Virus (HTV) infection, comprising administering to a patient in need thereof an effective amount of a pharmaceutically acceptable composition comprising a PEG-ASNase compound, at least one ribonucleotide reductase inhibitor compound and at least one HTV reverse transcnptase inhibitor compound.
Another preferred embodiment according to the invention is a method of inhibiting or treating Human Immunodeficiency Virus (HTV) infection, comprising administering to a patient in need thereof an effective amount of a pharmaceutically acceptable composition comprising a PEG-ASNase compound, at least one protease inhibitor compound, at least one ribonucleotide reductase inhibitor compound and at least one HIV reverse transcnptase inhibitor compound.
Another preferred embodiment according to the invention is a method of inhibiting or treating Human Immunodeficiency Virus (HIV) infection, compπsing administering to a patient in need thereof a therapeutically effective amount of a PEG-ASNase compound and at least one compound selected from the group consisting of protease inhibitor compounds, ribonucleotide reductase inhibitor compounds and HTV reverse transcnptase inhibitor compounds. A preferred embodiment according to the invention is a method of inhibiting or treating Human Immunodeficiency Virus (HTV) infection, comprising administering to a patient in need thereof an effective amount of a pharmaceutically acceptable composition comprising asparaginase.
Another preferred embodiment according to the invention is a method of inhibiting or treating Human Immunodeficiency Virus (HTV) infection, comprising administering to a patient in need thereof an effective amount of a pharmaceutically acceptable composition comprising asparaginase and at least one compound selected from the group consisting of protease inhibitor compounds, ribonucleotide reductase inhibitor compounds and HIV reverse transcnptase inhibitor compounds. Another preferred embodiment according to the invention is a method of inhibiting or treating Human Immunodeficiency Virus (HTV) infection, compπsing administering to a patient in need thereof an effective amount of a pharmaceutically acceptable composition comprising at least one ribonucleotide reductase inhibitor compound of formula I
wherein Rl is alkyl, alkenyl, alkynyl or an electron withdrawing group; and
R2 is alkyl, alkenyl, alkynyl; or a pharmaceutically acceptable salt thereof, an N-oxide thereof, a solvate thereof, an acid bioisostere thereof, or prodrug thereof Another preferred embodiment according to the invention is a method of inhibiting the production, or limiting the spread, of HTV comprising exposing a cell population infected with HTV to an effective amount of a PEG-ASNase compound or asparaginase, and optionally at least one compound selected from the group consisting of protease inhibitor compounds, ribonucleotide reductase inhibitor compounds and HIV reverse transcnptase inhibitor compounds.
Another preferred embodiment according to the invention is a method of inhibiting
HIV reverse transcnptase activity, comprising contacting HIV reverse transcπptase with a composition comprising a PEG-ASNase compound or asparaginase, and optionally at least one compound selected from the group consisting of protease inhibitor compounds, ribonucleotide reductase inhibitor compounds and HIV reverse transcnptase inhibitor compounds
Another preferred embodiment according to the invention is a method of inhibiting
HIV reverse transcπptase activity, comprising contacting HIV reverse transcnptase with a composition compπsing a compound of formula I. Another preferred embodiment according to the invention is a method of inhibiting
HIV reverse transcnptase activity, comprising contacting HIV reverse transcnptase with a composition comprising MISID having the formula
Another preferred embodiment according to the invention is a method of inhibiting
HIV reverse transcnptase activity, comprising contacting HIV reverse transcnptase with a composition comprising a PEG-ASNase compound.
Another preferred embodiment according to the invention is method of selectively inhibiting HIV -RNA production compπsing exposing a cell population infected with HIV to a pharmaceutically acceptable composition compπsing a PEG-ASNase compound and at least one protease inhibitor compound. Another preferred embodiment according to the invention is method of selectively inhibiting HFV-RNA production comprising exposing a cell population infected with HIV to a pharmaceutically acceptable composition comprising a PEG-ASNase compound and Saquinavir Another preferred embodiment according to the invention is a method of inhibiting
HIV-RNA production, comprising contacting a cell population infected with HIV with a composition compπsing a PEG-ASNase compound or asparaginase, and optionally at least one compound selected from the group consisting of protease inhibitor compounds, ribonucleotide reductase inhibitor compounds and HIV reverse transcnptase inhibitor compounds
Another preferred embodiment according to the invention is a method of inhibiting HIV-RNA production, compπsing contacting a cell population infected with HIV with a composition comprising a compound of formula I
Another preferred embodiment according to the invention is a method of inhibiting HIV-RNA production, compπsing contacting a cell population infected with HTV with a composition comprising MISID
Another preferred embodiment according to the invention is a method of inhibiting HIV-RNA production, compπsing contacting a cell population infected with HIV with a composition comprising a PEG-ASNase compound According to another preferred embodiment of the invention, the protease inhibitor compounds are selected from Saqumovir, Nelfmavir, Endmovere, Indinavir, Ritonavir, Cπxivan, Viracept, Norvir, and VX-478
According to a more preferred embodiment of the invention, the protease inhibitor compound is Saqumovir According to another preferred embodiment of the invention, the HTV reverse transcnptase inhibitor compounds are selected from AZT (Retrovir, zidovudine) ddl (Videx, didanosine) ddC (Hivid, zalcitabine), d4T (Zeπt, stavudine) and 3TC (Epivir, lamivudine)
According to another preferred embodiment of the invention, the HTV reverse transcnptase inhibitor compound is AZT According to another preferred embodiment of the invention, the ribonucleotide reductase inhibitor compounds are selected from Hydroxyurea (HU), BW- 348U87, 3- ammopyπdιne-2-carboxaldehyde thiosemicarbazone (3-AP) Amidox (VF 236, NSC-343341, N,3,4-tπhydroxybenzenecarboxιmιdamιde), BUD 1257 (2-benzyl-3-phenylpropιonyl-L-(N- methyl)valyl-L-3-(methyl)valyl-L-(N4,N4-tetramethylene)asparagιnyl-L-(3,3- tetramethylene)aspartyl-L-(4-methyl)leucme), BILD 1357 (2-benzyl-3-phenylpropιonyl-L- (N-methyl)valyl-L-3-(methyl)valyl-L-(N4,N4-tetramethylene)asparagιnyl-L-(3,3- tetramethylene)aspartιc acid l-[l(R)-ethyl-2,2-dιmethylpropylamιde]), BILD 1633, BILD 733 (3-phenylpropιonyl-L-(N-methyl)valyl-L-[3-methyl)valyl-L-[3-(pyrrohdιn-l- ylcarbonyl)]alanyl-L-(l-carboxycyclopentyl)glycyl-L-(4-methyl)leucιnol), BILD 1263 (2- benzyl-3-phenylpropιonyl-L-(N-methyl)valyl-L-3-(methyl)valyl-L-(N4,N4- tetramethylene)asparagιnyl-L-(3,3-tetramethylene)aspartyl-L-(4-methyl)leucinol), BILDl 351 (l-[l(S)-[5(S)-[3-[(all-cιs)-2,6-dιmethylcyclohexyl]ureιdo]-2(S)-(3,3-dιmethyl-2-oxobutyl)- 6,6-dιmethyl-4-oxoheptanoylamιno]-l-[l(R)-ethyl-2,2- dιmethylpropylcarbamol]methyl]cyclopentanecarboxyhc acid]), CI-F-araA (2-chloro-9-(2- deoxy-2-fluoro-beta-D-arabιnofuranosyl)adenme, DAH (D-aspartic-beta-hydroxamate), DDFC (2'-deoxy-2',2'-dιfluorocytιdιne),Dιdox (VF 147; NSC 324360; N,3,4- tπhydroxybenzamide), Eurd (3'-Ethynyluπdιne),GTI 2040, GTI 2501,IMHAG (1- lsoquinolylmethane-N-hydroxy-N'-aminoguanine), LY 207702 (2',2'-dιfluoro-2'- deoxyπbofuranosyl-2,6-dιamιnopuπne), LY 295501 (N-[[3,4- dιchlorophenyl)amιno]carbonyl]2,3-dιhydro-5-benzofuransulfonamιde), MDL 101731 (FMdC; KW 2331 ; (2E)-2'-deoxy-2'-(fluoromethylene)cytιdme), Parabactin, Sulofenur (LY 186641 ; N-[[(4-chlorophenyl)amιno]carbonyl]-2,3-dιhydro-lH-ιndene-5-sulfonamιde), TAS 106 (Ecyd, 3'-ethynylcytιdιne),Tπapιne (OCX 191; OCX 0191), Tπmidox (VF 233; N,3- 4,5-tetrahydroxybenzene carboximidamide), and a compound of formula I.
According to another preferred embodiment of the invention, the ribonucleotide reductase inhibitor compound is the compound of formula I.
According to another preferred embodiment of the invention, this invention is directed to a method of inhibiting or treating Human Immunodeficiency Virus (HTV) infection, comprising administering to a patient in need of such treatment a pharmaceutically effective amount of compound of formula I wherein Rl is lower alkyl, lower alkenyl, lower alkynyl, or an electron withdrawing group; and
R2 is lower alkyl, lower alkenyl, lower alkynyl; or a pharmaceutically acceptable salt thereof, an N-oxide thereof, a solvate thereof, an acid bioisostere thereof, or prodrug thereof. According to another preferred embodiment of the invention, this invention is directed to a method of inhibiting or treating Human Immunodeficiency Virus (HIV) infection, comprising administering to a patient in need of such treatment a pharmaceutically effective amount of compound of formula I wherein Rl is lower alkyl, lower alkenyl, lower alkynyl, or a halogen group; and R2 is lower alkyl, lower alkenyl, lower alkynyl; or a pharmaceutically acceptable salt thereof, an N-oxide thereof, a solvate thereof, an acid bioisostere thereof, or prodrug thereof.
According to another preferred embodiment of the invention, this invention is directed to a method of inhibiting or treating Human Immunodeficiency Virus (HTV) infection, comprising administering to a patient m need of such treatment a pharmaceutically effective amount of compound of formula I wherein Rl is lower alkyl or a halogen group; and
R2 is lower alkyl, or a pharmaceutically acceptable salt thereof, an N-oxide thereof, a solvate thereof, an acid bioisostere thereof, or prodrug thereof
According to another preferred embodiment of the invention, this invention is directed to a method of inhibiting or treating Human Immunodeficiency Virus (HIV) infection, comprising administering to a patient in need of such treatment a pharmaceutically effective amount of compound of formula I wherein Rl is a halogen group; and
R2 is lower alkyl; or a pharmaceutically acceptable salt thereof, an N-oxide thereof, a solvate thereof, an acid bioisostere thereof, or prodrug thereof
According to another preferred embodiment of the invention, this invention is directed to a method of inhibiting or treating Human Immunodeficiency Virus (HTV) infection, compπsing administering to a patient in need of such treatment a pharmaceutically effective amount of compound of formula I wherein Rl is lower alkyl; and
R2 is lower alkyl, or a pharmaceutically acceptable salt thereof, an N-oxide thereof, a solvate thereof, an acid bioisostere thereof, or prodrug thereof.
According to another preferred embodiment of the invention, this invention is directed to a method of inhibiting or treating Human Immunodeficiency Virus (HIV) infection, comprising administering to a patient in need of such treatment a pharmaceutically effective amount of compound of formula I wherein Rl is a bromine or chlorine atom; and
R2 is a methyl group or an isopropyl group; or a pharmaceutically acceptable salt thereof, an N-oxide thereof, a solvate thereof, an acid bioisostere thereof, or prodrug thereof. According to another preferred embodiment of the invention, this invention is directed to a method of inhibiting or treating Human Immunodeficiency Virus (HTV) infection, compπsing administering to a patient in need of such treatment a pharmaceutically effective amount of compound of formula I wherein Rl is a methyl group; and R2 is lower alkyl; or a pharmaceutically acceptable salt thereof, an N-oxide thereof, a solvate thereof, an acid bioisostere thereof, or prodrug thereof.
According to another preferred embodiment of the invention, this invention is directed to a method of inhibiting or treating Human Immunodeficiency Virus (HTV) infection, comprising administering to a patient m need of such treatment a pharmaceutically effective amount of compound of formula I wherein Rl is a methyl group; and R2 is an isopropyl group; or a pharmaceutically acceptable salt thereof, an N-oxide thereof, a solvate thereof, an acid bioisostere thereof, or prodrug thereof According to another preferred embodiment of the invention, the protein synthesis inhibitor compound is PEG-ASNase.
According to another preferred embodiment of the invention, the protein synthesis inhibitor compound is asparaginase
According to another preferred embodiment of the invention, the protease inhibitor compound is Saqumovir or Endinovere.
According to another preferred embodiment of the invention, the protease inhibitor compound is Saqumovir.
According to another preferred embodiment of the invention, the HTV reverse transcnptase inhibitor compounds are selected from AZT and 3-TC According to another preferred embodiment of the invention, the ribonucleotide reductase inhibitor compound is MISID (PL-7).
According to another preferred embodiment of the invention the compounds of use according to the invention are administered in concurrent combination.
According to another preferred embodiment of the invention the compounds of use according to the invention are administered sequentially.
According to another preferred embodiment of the invention the compounds of use according to the invention are administered sequentially, preferably by administering a protease inhibitor followed by the PEG-ASNase compound or asparaginase.
According to another preferred embodiment of the invention the compounds of use according to the invention are administered sequentially, preferably by administering Saquinavir followed by the PEG-ASNase compound or asparaginase. According to another preferred embodiment of the invention the compounds of use according to the invention are administered sequentially, preferably administering in the order Saquinavir, followed by the PEG-ASNase compound or asparaginase, followed by one or more compounds selected from the group consisting of HTV reverse transcnptase inhibitor compounds and ribonucleotide reductase inhibitor compounds.
According to another preferred embodiment of the invention is a method of inhibiting or treating Human Immunodeficiency Virus (HIV) infection, comprising administering to a patient in need thereof a synergistic combination of a PEG-ASNase compound or asparaginase, and at least one compound selected from the group consisting of protease inhibitor compounds, ribonucleotide reductase inhibitor compounds and HIV reverse transcπptase inhibitor compounds
According to another preferred embodiment of the invention is a method of inhibiting or treating Human Immunodeficiency Virus (HTV) infection, comprising administering to a patient in need thereof a synergistic combination of a PEG-ASNase compound and at least one protease inhibitor compound.
According to another preferred embodiment of the invention is a method of inhibiting or treating Human Immunodeficiency Virus (HIV) infection, comprising administering to a patient in need thereof a synergistic combination of a PEG-ASNase compound and at least one ribonucleotide reductase inhibitor compound According to another preferred embodiment of the invention is a method of inhibiting or treating Human Immunodeficiency Virus (HIV) infection, comprising administering to a patient in need thereof a synergistic combination of a PEG-ASNase compound and at least one HTV reverse transcnptase inhibitor compound
According to another preferred embodiment of the invention is a method of inhibiting or treating Human Immunodeficiency Virus (HTV) infection, comprising administering to a patient in need thereof a synergistic combination of a PEG-ASNase compound and at least one protease inhibitor compound and at least one ribonucleotide reductase inhibitor compound
According to another preferred embodiment of the invention is a method of inhibiting or treating Human Immunodeficiency Virus (HIV) infection, comprising administering to a patient in need thereof a synergistic combination of a PEG-ASNase compound and at least one protease inhibitor compound and at least one HTV reverse transcnptase inhibitor compound.
According to another preferred embodiment of the invention is a method of inhibiting or treating Human Immunodeficiency Virus (HTV) infection, compnsing administering to a patient in need thereof a synergistic combination of a PEG-ASNase compound and at least ribonucleotide reductase inhibitor compound and at least HTV reverse transcπptase inhibitor compound
It is a further object of the invention to provide kits having a plurality of active ingredients (with or without carrier) which, together, may be effectively utilized for carrying out the novel combination therapies of the invention
It is another object of the invention to provide a novel pharmaceutical composition which is effective, m and of itself, for utilization in a beneficial combination therapy because it includes a plurality of active ingredients which may be utilized in accordance with the invention Without being limited by theory, it is believed that the invention operates with the following mechanism T-cells are the main cells in the mammalian body that are infected with HIV virus T-cells are considered to be the viral factories for HIV infection In order to re-infect new T-cells, the HIV virus must enter the T-cell and be able to replicate The viral replication and encapsulation process requires the participation of mtracellular enzymes PEG-ASNase, a specific protein synthesis inhibitor compound to thymic-hneage cells, is shown to very useful in inhibiting synthesis of enzymes required for competent replication and assembly of the HTV virus and in providing other antiprohferative effects related to the HIV virus Furthermore, a combination of PEG-ASNase or asparaginase, and one or more compounds selected from the group consisting of a protease inhibitor compound, a HIV-RT inhibitor compound, and a ribonucleotide reductase inhibitor compound, has a synergistic effect to reduce the viral burden for prolonged periods of time The mtracellular mechanism by which PEG-ASNase is believed to operate is to prevent a T-cell infected with HIV virus from synthesizing mtracellular and viral proteins by adversely affecting the supply of the amino acid asparagme (Asn) Thus, by treating infected T-cells with an asparaginase such as PEG-ASNase, synthesis of cellular and viral proteins is inhibited These proteins are necessary for the transcription and translation of the virally coded genes from the provirus, integrated viral origin DNA into mammalian DNA Once viral-oπgm RNA is transcribed by RNA polymerases from the provirus, there are two pathways that may be followed This RNA may be used by ribonucleotide s to produce viral-oπgm proteins, such as HIV-RT Later, the same RNA may be processed by rev-protem into genomic HIV-1 RNA This RNA will be attached to an already synthesized HIV-RT and when two such molecules are present together they constitute the genomic material of a new HTV virus is constituted HIV proteases are involved in processing the viral origin proteins into the final viral packaging The new HTV-1 virus may then bud off the T-cell as a new, complete virus Thus, a protease inhibitor compound in combination with an asparaginase such as PEG-ASNase, can inhibit the processes required for HIV-1 viral replication in a synergistic manner The compositions and methods of therapy of the present invention are useful in the inhibition of HIV protease, the prevention or treatment of infection by HIV and the treatment of consequent pathological conditions such as AIDS Treating AIDS or preventing or treating infection by HIV is defined by including but not limited to treating a wide range of states of HTV infection, ATDS, ARC (AIDS) related complex, both symptomatic and asymptomatic and actual or potential exposure to HIV For example, the compounds of this invention are useful in treating infection by HTV after suspected past exposure to HTV by, for example, blood transfusion, exchange of body fluids, bites, accidental needle sticks, and exposure to patient blood during surgery In the treatment or prevention method according to the invention the PEG-ASNase compound or asparaginase, and optionally a compound selected from the group consisting of a protease inhibitor compound, a HIV reverse transcπptase inhibitor compound and a ribonucleotide reductase inhibitor compound, may be administered in different ways, such as in combination therapies optionally employing medical procedures For example a PEG- ASNase compound and optionally one or more compounds selected from the group consisting of protease inhibitor compounds, HIV reverse transcπptase inhibitor compounds and ribonucleotide reductase inhibitor compounds, may be administered to a patient concomitantly or at different times provided that they are administered such that at some period of time there are pharmaceutically effective amounts of both compounds present in the patient such that a therapeutic effect according to the invention results
Thus, it is a further object of the invention to provide a kit for treating or preventing a physiological condition associated with HIV, said kit comprising a plurality of separate containers, wherein at least one of said containers contains a PEG-ASNase compound or asparaginase, and at least another of said containers contains one or more compounds selected from the group consisting of protease inhibitor compounds, HIV reverse transcnptase inhibitor compounds and ribonucleotide reductase inhibitor compounds, and said containers optionally contain a pharmaceutical carrier, which kit may be effectively utilized for carrying out combination therapies according to the invention
Thus, it is a further object of the invention to provide a pharmaceutical kit for treating or preventing a physiological condition associated with HIV, said kit comprising a plurality of separate containers, wherein at least one of said containers contains a compound of formula I and at least another of said containers contains one or more compounds selected from the group consisting of PEG-ASNase compounds, protease inhibitor compounds, HTV reverse transcnptase inhibitor compounds and ribonucleotide reductase inhibitor compounds, and said containers optionally contain a pharmaceutical carrier, which kit may be effectively utilized for carrying out combination therapies according to the invention A further embodiment for a kit would be wherein at least one of said containers should contain a PEG-ASNase compound without the presence of a protease inhibitor compound, a HIV reverse transcπptase inhibitor compound or a ribonucleotide reductase inhibitor compound, and at least another of said containers should contain one or more compounds selected from the group consisting of protease inhibitor compounds, HTV reverse transcnptase inhibitor compounds and ribonucleotide reductase inhibitor compounds, without the presence of a PEG-ASNase compound
A further embodiment for a kit would be wherein at least one of said containers should contain a compound of formula I without the presence of a PEG-ASNase compound, a protease inhibitor compound, a HTV reverse transcnptase inhibitor compound or another ribonucleotide reductase inhibitor compound, and at least another of said containers should contain one or more compounds selected from the group consisting of protease inhibitor compounds, HIV reverse transcπptase inhibitor compounds and another ribonucleotide reductase inhibitor compound, without the presence the same compound of formula I. A further embodiment for a kit would be wherein of said containers at least one of said containers should contain MISID (PL-7) without the presence of a PEG-ASNase compound, a protease inhibitor compound, a HIV reverse transcnptase inhibitor compound or another ribonucleotide reductase inhibitor compound, and at least another of said containers should contain one or more compounds selected from the group consisting of protease inhibitor compounds, HIV reverse transcnptase inhibitor compounds and ribonucleotide reductase inhibitor compounds, without the presence of MISID (PL-7).
It is to be understood that this invention covers all appropriate combinations of the particular and preferred groupings referred to herein
Compounds according to the invention, for example, starting materials, intermediates or products, are prepared as described herein or by the application or adaptation of known methods, by which is meant methods used heretofore or described in the literature
Compounds of Formula I may be prepared by the application or adaptation of known methods, by which is meant methods used heretofore or described m the literature. In particular, the known method of making derivatives of the formula I as described in Nandy P, Lien EJ, Avramis VI, Med. Chem. Res. 1995, 5:664-679.
The compounds useful according to the invention optionally are supplied as salts Those salts which are pharmaceutically acceptable are of particular interest since they are useful in administering the foregoing compounds for medical purposes. Salts which are not pharmaceutically acceptable are useful in manufacturing processes, for isolation and purification purposes, and in some instances, for use in separating stereoisomenc forms of the compounds of this invention. The latter is particularly true of amine salts prepared from optically active amines.
Where the compound useful according to the invention contains a carboxy group, or a sufficiently acidic bioisostere, base addition salts may be formed and are simply a more convenient form for use; and in practice, use of the salt form inherently amounts to use of the free acid form
Also, where the compound useful according to the invention contains a basic group, or a sufficiently basic bioisostere, acid addition salts may be formed and are simply a more convenient form for use; and in practice, use of the salt form inherently amounts to use of the free base form
The foregoing compounds useful according to the invention may also be mixed another therapeutic compound to form pharmaceutical compositions (with or without diluent or carrier) which, when administered, provide simultaneous administration of a combination of active ingredients resulting in the combination therapy of the invention While it is possible for the compounds useful according to the invention to be administered alone it is preferably to present them as pharmaceutical compositions The pharmaceutical compositions, both for veterinary and for human use, useful according to the present invention comprise at lease one compound of the invention, as above defined, together with one or more acceptable carriers therefor and optionally other therapeutic ingredients.
In certain preferred embodiments, active ingredients necessary in combination therapy may be combined in a single pharmaceutical composition for simultaneous administration.
The choice of vehicle and the content of active substance in the vehicle are generally determined in accordance with the solubility and chemical properties of the active compound, the particular mode of administration and the provisions to be observed in pharmaceutical practice. For example, excipients such as lactose, sodium citrate, calcium carbonate, dicalcium phosphate and disintegrating agents such as starch, algmic acids and certain complex silicates combined with lubricants such as magnesium stearate, sodium lauryl sulphate and talc may be used for preparing tablets To prepare a capsule, it is advantageous to use lactose and high molecular weight polyethylene glycols. When aqueous suspensions are used they can contain emulsifying agents or agents which facilitate suspension. Diluents such as sucrose, ethanol, polyethylene glycol, propylene glycol, glycerol and chloroform or mixtures thereof may also be used. The oily phase of the emulsions of this invention may be constituted from known ingredients in a known manner. While the oily phase may comprise merely an emulsifier (otherwise known as an emulgent), it desirably comprises a mixture of at least one emulsifier with a fat or an oil or with both a fat and an oil. Preferably, a hydrophihc emulsifier is included together with a hpophihc emulsifier which acts as a stabilizer. It is also preferred to include both an oil and a fat. Together, the emulsιfier(s) with or without stabιhzer(s) make up the emulsifying wax, and the way together with the oil and fat make up the emulsifying ointment base which forms the oily dispersed phase of a cream formulation. Emulgents and emulsion stabilizers suitable for use in the formulation of the present invention include Tween® 60, Span® 80, cetostearyl alcohol, benzyl alcohol, mynstyl alcohol, glyceryl mono- stearate and sodium lauryl sulfate If desired, the aqueous phase of the cream base may include, for example, a least
30% w/w of a polyhydπc alcohol, 1 e an alcohol having two or more hydroxyl groups such as propylene glycol, butane 1,3-dιol, manmtol, sorbitol, glycerol and polyethylene glycol (including PEG 400) and mixtures thereof. The topical formulations may desirably include a compound which enhances absorption or penetration of the active ingredient through the skm or other affected areas. Examples of such dermal penetration enhancers include dimethyl sulphoxide and related analogue.
The choice of suitable oils or fats for the formulation is based on achieving the desired cosmetic properties. Thus the cream should preferably be a non-greasy, non-staining and washable product with suitable consistency to avoid leakage from tubes or other containers. Straight or branched chain, mono- or dibasic alkyl esters such as di-isopropyl myπstate, decyl oleate, isopropyl palmitate, butyl stearate, 2-ethylhexyl palmitate or a blend of branched chain esters known as Crodamol CAP may be used, the last three being preferred esters These may be used alone or in combination depending on the properties required. Alternatively, high melting point hpids such as white soft paraffin and/or liquid paraffin or other mineral oils can be used.
Solid compositions of may also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polyethylene glycols, and the like.
The pharmaceutical compositions can be administered in a suitable formulation to humans and animals by topical or systemic administration, including oral, inhalational, rectal, nasal, buccal, subhngual, vaginal, parenteral (including subcutaneous, intramuscular, intravenous, intradermal, intrathecal and epidural), intracisternal and intrapentoneal. It will be appreciated that the preferred route may vary with for example the condition of the recipient. The formulations can be prepared in unit dosage form by any of the methods well known in the art of pharmacy Such methods include the step of bringing into association the active ingredient with the carrier which constitutes one or more accessory ingredients. In general the formulations are prepared by uniformly and intimately bringing into association the active ingredient with liquid carriers or finely divided solid earners or both, and then, if necessary, shaping the product. A tablet may be made by compression or moulding, optionally with one or more accessory ingredients Compressed tables may be prepared by compressing in a suitable machine the active ingredient in a free-flowing form such as a powder or granules, optionally mixed with a binder, lubricant, inert diluent, preservative, surface active or dispersing agent. Moulded tablets may be made by moulding in a suitable machine a mixture of the powdered compounds moistened with an inert liquid diluent. The tablets may optionally be coated or scored and may be formulated so as to provide slow or controlled release of the active ingredient therein
Solid compositions for rectal administration include suppositories formulated in accordance with known methods and containing at least one compound of the invention. If desired, and for more effective distribution, the compounds can be microencapsulated in, or attached to, a slow release or targeted delivery systems such as a biocompatible, biodegradable polymer matrices (e.g poly(d,l-lactιde co-glycohde)), hposomes, and microspheres and subcutaneously or intramuscularly injected by a technique called subcutaneous or intramuscular depot to provide continuous slow release of the compound(s) for a period of 2 weeks or longer. The compounds may be sterilized, for example, by filtration through a bacteria retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved in sterile water, or some other sterile injectable medium immediately before use.
Actual dosage levels of active ingredient in the compositions of the invention may be varied so as to obtain an amount of active ingredient that is effective to obtain a desired therapeutic response for a particular composition and method of administration The selected dosage level therefore depends upon the desired therapeutic effect, on the route of administration, on the desired duration of treatment and other factors.
Total daily dose of the compounds useful according to this invention administered to a host in single or divided doses may be in amounts, for example, of from about 0.001 to about 100 g/kg body weight daily and preferably 0.01 to 10 mg/kg/day. Dosage unit compositions may contain such amounts of such submultiples thereof as may be used to make up the daily dose. It will be understood, however, that the specific dose level for any particular patient will depend upon a variety of factors including the body weight, general health, sex, diet, time and route of administration, rates of absorption and excretion, combination with other drugs and the severity of the particular disease being treated. The amount of each component administered is determined by the attending clinicians taking into consideration the etiology and severity of the disease, the patient's condition and age, the potency of each component and other factors.
The formulations may be presented in unit-dose or multi-dose containers, for example sealed ampoules and vials with elastomenc stoppers, and may be stored m a freeze- dπed (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the kind previously described The compounds of the invention, their methods or preparation and their biological activity will appear more clearly from the examination of the following examples which are presented as an illustration only and are not to be considered as limiting the invention m its scope
Procedures for evaluating the biological activity of compounds or compositions according to the invention are carried out as described herein or by the application or adaptation of known procedures, by which is meant procedures used heretofore or as described in the literature.
Experimental
General Methodology for the HTV-Reverse Transcnptase Assay, non radioactive (Boehrmger Mannheim)
The following is a general procedure for the HIV-reverse transcnptase assay
Day One
Samples are supernatants and pellets obtained from the viral ± drug flasks (incubation for seven days) They are not heat inactivated. — Centrifuge the samples at 2000 g for 30 minutes at 4° C.
Use 2500 rpm to achieve 2000 g.
Transfer the supernatant to a sterile labeled tube. ~ Add 0.5 ml peg solution
Use 1 2 m nacl as the diluent for peg. Peg solution: 30% w/v, 30 g in 100 ml.
Mix thoroughly
Incubate o/n at 0° C (on ice in the refrigerator) Day Two
Centrifuge 500 μl of the samples at 8000 g for 10 minutes at 4° C Use 8000 rpm to achieve 8000 g
Discard the supernatant. Be careful to remove all drops of peg from the samples
Add 40 μl lysis buffer solution
Re-suspend pellet completely.
Transfer the suspension to a fresh reaction tube
Incubate at rt (25°C) for 30 minutes
Make the standard dilutions
Transfer 40 μl of the standards to reaction tubes (n=7 x 2).
Make reaction buffer solution:
—reconstitute the template (vial 4) in 430 μl autoclaved water
—add 1 ml incubation buffer per vial of nucleotides (vial 3).
—add 100 μl of the reconstituted template (vial 4) to the nucleotide solution vial (vial
3).
Add 20 μl reaction buffer to all tubes, unknowns and standards.
Incubate for up to 15 hours at 37° C m a rack in the incubator.
Day Three
Create a template for the Ehsa assay using wordperfect. Open the foil packets and construct a mtp (microtiter plate) module using the frame and the strips provided in the kit.
Unknown n= therefore, total specimens n= Standard n=
Strips have 8 wells each, therefore need strips
Note: you have to round up to the closest multiple of 8. — Transfer 60 μl from the reaction tubes to the corresponding wells of the mtp module as per template.
Cover the mtp with the cover strip provided. ~ Incubate at 37° C in the incubator for 1 hour.
If necessary, make the washing solution: Note: solution provided is a lOx solution, therefore it must be diluted using autoclaved water.
**Make 1 bottle wash solution by adding 225 ml autoclaved water to the bottle provided. Mix well. Keep on ice during the assay.
Remove the solution completely by decanting.
Wash the plate 5x using 250 μl per rinse with a 30 second soak time before decanting.
Make the anti-dig-pod working solution
**Make the anti-dig-pod solution
--Add 500 μl autoclaved water to the anti-dig-pod vial (vial #6) store at 4° C, do not freeze
** Make the anti-dig-pod working solution
Calculate the necessary volume: wells x 200 μl = ml
Use 50 μl anti-dig-pod solution (vial #6) for each 4.95 ml conjugate dilution buffer (solution #8). Add ml anti-dig-pod solution (vial #6)
To ml conjugate dilution buffer (solution #8)
Add 200 μl anti-dig-pod working solution per well of the mtp. — Cover the mtp with the cover strip provided.
Incubate at 37° C in the incubator for 1 hour. Remove the solution completely by decanting.
Wash the plate 5x using 250 μl per rinse with a 30 second soak time before decanting.
Make the abts substrate solution with enhancer
**Make the abts substrate solution
-Dissolve the abts powder mixture (vial #10) in the bottle of substrate buffer (bottle #9)
Calculate the necessary volume: wells x 200 μl = ml ** Add the appropriate amount of enhancer to the solution Use 1 mg substrate enhancer (vial #11) for each 1 ml abts substrate solution (bottle #9)
Add mg substrate enhancer (vial #11)
To ml abts substrate solution (bottle #9) - Add 200 μl abts substrate solution with enhancer per well of the mtp.
Read the plate at 405 nm (reference wavelength 490 nm) at 10, 20, and 30 minutes.
Example 1 - Combination regimen of PEG-ASNase compound and Saquinavir
Materials and Methods:
The cell line used for these studies is CCRF/CEM/O, a human T-leukemic cell line. PEG-ASNase (ONCASPAR) is provided by Rhόne-Poulenc Rorer. Saquinavir is commercially available. RPMI-1640 media (Irvine Scientific, Irvme, CA) is enriched with 10% Fetal Calf Serum (Gemmi Biosource, Calabasas, CA), 5% IM Hepes Buffer solution and 5% Non-essential ammo acids (Irvine Scientific, Irvine, CA). The drug concentrations used are as follows.
PEG-ASNase IC50 alone: 0.4 IU/ml
Saquinavir IC50 alone: 25 μM PEG/SAQ combo IC50: 0.233 IU/ml + 15.52 μM
Briefly, 2 x 106 cells/ml are stimulated with PHA+ media for 48 hours at 37°C with 5% C0 . Also, the same number of cells are incubated in PHA(phytohemagglutmιn) free media for 48 hours to serve as the negative confrol. At this point the cells are inoculated with the HIV-1 virus as per standard protocol. PEG-ASNase and/or Saquinavir are added to the cells in the appropriate concenfrations (see above). The confrol cells are resuspended in drug-free media for the duration of the exposure which lasted seven days. At day five, two 1 ml aliquots of media are removed from the flasks and stored under liquid nitrogen. At day seven, two more 1 ml aliquots of media are removed from the flasks and stored under liquid nitrogen. In addition, the remaining cells are pelleted and stored at -80°C.
The samples produced from this experiment are itemized and then assayed for HTV- RT using the Reverse Transcnptase Assay, non-radioactive (Boehrmger Mannheim). The standard curve is determined and the HIV-RT levels for the experimental samples are calculated.
Results:
The primary observation from the HIV-RT assays in these specimens from T-cells is that there is no HTV-RT/virus in the supernatant of the CEM/0 T-cells post treatment. The results of this experiment are illustrated in Figure 1.
The T-cell pellets themselves are then examined for mtracellular HIV-RT. PEG- ASNase at
0.4 IU/ml (approximate IC30 concentration) demonstrated about 30% inhibition of HIV-RT. Saquinavir, the HIN protease inhibitor compound, alone at 25 μM (approximate IC50 concentration) depleted HIV-RT activity by about 70%> as compared to untreated control cell cultures HIV-RT. Lastly, we have shown that the concurrent combination of PEG-ASΝase and Saquinavir are synergistic, thus the IC50 concentrations of these drugs in combination are 0.233 rU/ml and 14.5 μM, respectively. These concentrations are much lower than their respective IC50 values in CEM/0 T-cells. The combination regimen of PEG-ASΝase and Saquinavir inhibited HIV-RT intracellularly by about 82.3% as compared to untreated control values.
Discussion:
Since the T-cells did not shed HIV-1 in the supernatant after these drug treatments, it appears that PEG-ASΝase and Saquinavir are not only synergistic against T-cells, but also are selectively synergistic against HIV-1. These drugs are sufficiently suppressive in releasing new HIV-1 particles to the media (equivalent serum or plasma of patients). The fact that the lower concentrations of the combination are more suppressive of HIV-RT than the most active of the two drugs Saquinavir at a higher concentration, strongly suggests that the combination is selectively inhibiting HIV at the pro-virus level.
Because these drugs and their combination suppress/inhibit HIV-RT intracellularly, it suggests that they inhibit HIV-1 at the provirus level. In other words, the integrated HIV provirus is producing mRNA, which is not translated into viral proteins and hence, the inhibition of the production of RT or complete virus particles to be shed in the media. Thus, on a theoretical basis, no further HIV-1 infection could be achieved of unmfected T-cells.
Experiment 2: Determination of Cytotoxicity
Materials and Methods-
A human leukemic T-cell line, hereafter referred to as CEM/0 is used for this experiment PEG-ASNase is obtained from Rhone-Poulenc Rorer Pharmaceuticals Inc under the tradename Oncaspar®. Saquinavir is obtained from Roche Laboratories under the tradename Invirase™. RPMI- 1640 media obtained from Irvine Scientific, Irvine CA is enriched with 10% Fetal Calf Serum obtained from Gemini Biosource, Calabasas, CA, 5% IM Hepes buffer solution and 5% non-essential amino acids obtained from Irvme Scientific, Irvme CA
An experiment is carried out to determine the cytotoxicity of Saquinavir and PEG- ASNase For determining the cytotoxicity of either compound alone, 2 x 105 cells/ml are incubated in 24-well plates with the following drug concentrations.
PEG-ASNase: 1.0, 0.75, 0 5, 0 4 , 0.3, 0.2, 0 1, 0 03 IU/ml
Saquinavir. 10"4, 10"5, 10 6, 10 7, and 10"8 M
Results
The PEG-ASNase concentration that produces a cytostatic condition in CEM/0 cells in vitro is approximately 0.5 IU/ml PEG-ASNase concenfrations of 1 and 0 75 IU/ml produced significant cell kill and are cytotoxic to CEM/0 cells by 72 hours. The concentrations of 0.03, 0.1, 0.2, 0.3 and 0 4 IU/ml are marginally effective in preventing cell growth as compared to the control (untreated cells) growth rate The cells treated with 0.5 IU/ml PEG-ASNase, however, showed a relatively flat cell growth line Thus, a cytostatic effect is produced with this concentration over 72 hours. Therefore, a range of PEG-ASNase concentrations including 0.5 IU/ml are used in the combination regimen investigations. A drug, concentration and time dependent cytotoxic effect of PEG-ASNase in this T-cell line is shown.
The IC50 of Saquinavir in CEM/0 cells is determined to be 26μM after a 72 hour incubation period. The results are illustrated in Figure 2. Multiple independent experiments with Saquinavir showed an IC50 from 21-28 μM. Concenfrations from 0 001 to 1 μM Saquinavir produced no cell kill. Concentrations of lOμM produced only an 8.76% kill compared to untreated confrol samples The highest concentration tested, 100 μM, killed 99.50% of the cells compared to control cells. Thus, a range of concentrations of 1 to 40 μM Saquinavir is used in subsequent experiments to investigate the combination Saquinavir/ PEG-ASNase therapy in CEM/0 cells.
Experiment 3: Initial Sequential Combination studies of Saquinavir and PEG-ASNase
Materials and Methods
In this experiment, the described range of concentrations is used to investigate the combined regimen of Saquinavir and PEG-ASNase. For the sequential combination studies of PEG-ASNase followed by Saquinavir, cells are incubated with the concentrations provided below of PEG-ASNase for 24 hours. Then Saquinavir, in the concentrations provided below, is added to the appropriate cells for an additional twenty-four (24) hours, bringing the total exposure time to forty-eight (48) hours. The exposure involved 2 X 105 cells/ml being incubated in a tissue culture flask with concentrations of the drugs investigated as provided below. The results are illustrated in Figure 3. The drug concentrations used are as follows:
PEG-ASNase: 1.020, 0.765, 0.510, 0.255 and 0.0255 IU/ml
Saquinavir: 40, 30, 20, 10 and 1 μM
In all in vitro studies, the negative control cells are incubated in a drug-free media for the same duration and under the same conditions as the experimental samples Cell density is measured via cell counting using a Coulter Counter coupled with a Coulter Channelyzer for each of the experimental flasks at 24, 48 and 72 hours after incubation. Additionally, Trypan Blue Exclusion tests are performed for each of these experimental conditions. Cell numbers are corrected for the viability determined by theTrypan Blue test and presented as a percentage of the untreated control.
The reverse sequence is also tested. The results are illustrated in Figure 4. Saquinavir is administered and the cells incubated for twenty-four (24) hours followed by addition of PEG-ASNase and an additional incubation period of twenty-four (24) hours , bringing the total exposure time to forty-eight (48) hours. Other than the drug sequence, the methodology is the same as that mentioned immediately above. The concurrent combination regimen is also tested using the same methodology. The results are illustrated in Figures 5, 6, and 7. The concurrent combined methodology experiment had the samples exposed to forty-eight (48) hours of concurrent combined exposure.
Results Each regimen tested showed synergistic effects at certain ranges tested. The sequential combined regimen of PEG-ASNase followed by Saquinavir showed a 1.72-fold synergy after forty-eight hour exposure. This is similar to the synergies shown from the other sequential regiment and the concurrent regimen. The invention shows the very desirable result of producing optimal synergism at a level that allows for some cell survival.
Experiment 4. Determination of Ammo Acid Levels
Materials and Methods
Experiments to determine the amino acid level in cell suspensions and cell media are performed to determine the effect of PEG-ASNase on the amino acid levels, particularly the levels of asparagine, glutamine, and aspartic acid.
Samples of 50μl media and 10 μl of lmM ammoadopic acid are added to 450 μl of cold methanol m 1.5 ml microfuge tubes. The mixtures are vortexed and centrifuged at 8700 g for two minutes. The supernatants are transferred to borosihcate glass test tubes (13 x 100 mm) and lyophilized. The specimens are stored at -20° C until they are analyzed by HPLC. Prior to HPLC analysis, the samples are dissolved in a buffer containing 95% 7 nM disodium hydrogen phosphate and 5% acetonitπle.
Results
After twenty-four hours exposure of CEM/0 cells to various concentrations of PEG- ASNase, a significant depletion of asparagine (Asn) is observed. The asparagine level is less than 3.0% of the untreated control. Also, a dose dependent depletion of glutamine (gin) to levels that are less than 3.0% of the untreated control are observed in the experiment using the highest PEG-ASNase concentration. The aspartic acid (Asp) levels are elevated in comparison to the untreated control to levels representing a 200 to 300% increase. The results are illustrated in Figure 8. The calibration curve used to calculate the amount of HIN- RT is illustrated in Figure 8a.
Even at the lowest levels of PEG-ASΝase tested, a depletion of Asn after twenty-four hours is seen. This is consistent with PEG-ASΝase being able to kill illicit T-cells by depletion of vital ammo acids, particularly asparagine. PEG-ASΝase also depletes Gin levels that may be important in the mechanism of destroying T-cells. Experiment 5 : Determination of the effect of the exposure of HIV-RNA in T-cell pellets to a combination of PEG-ASNase and Saquinavir.
This experiment is carried out using a similar procedure, and the same concentrations of materials as in Experiment 1, however the exposure of HIV-RNA in T-cell pellets to a combination of PEG-ASNase and Saquinavir is measured. The results of this experiment are shown in Tables 1 and 2 and in graphs 9 and 9a. It should be noted that "PEG" in Table 2 represents PEG-ASNase.
These results show that PEG-ASNase had no apparent effect on RNA production of HIV-1 , whereas it had a moderate effect in inhibiting HIV-RT in the same cell culture by day 7 (see experiment 5a) Thus PEG-ASNase is inhibiting protein biosynthesis even at the HIV- RT level (see experiment 5a). Saquinavir alone has an inhibitory effect on HIN-1 RΝA production, inhibiting approximately 36% in comparison with the control (figures 9 and 9a). The combination of PEG-ASΝase and Saquinavir, at synergistically reduced concentrations, resulted in inhibiting approximately 12% of HIV-RT (see experiment 5a). Yet, in the same cultures, the combination of PEG-ASΝase and Saquinavir, at the reduced concentrations, yielded no detectable HTV-1 RΝA up to the lower limits of the assay of 400 copies of RΝA per pellet. This data demonstrates that the protein inhibitor (PEG-ASΝase) plus the HIV-1 protease inhibitor (Saquinavir) act not only synergistically but selectively against HIV-RT and more importantly selectively against HIV-1 RΝA production.
Experiment 5a: Determination of the effect of PEG-ASΝase + Saquinavir on the HIV RT Levels of HTV-1 Infected Cell Pellets
This experiment is carried out using a similar procedure, and the same concentrations of materials as in Experiment 7, however the exposure of the HIV virus in T-cell pellets to PEG-ASΝase and Saquinavir alone, and in combination is measured. The results of this experiment are shown in Table 2a. It should be noted that "PEG" in Table 2a represents PEG- ASΝase.
These results show that PEG-ASΝase had a moderate effect in inhibiting HIV-RT . Thus PEG-ASΝase is inhibiting protein biosynthesis even at the HIV-RT level. Saquinavir alone also has an inhibitory effect on HTV-1 RT, inhibiting approximately 15% in comparison with the. The combination of PEG-ASΝase and Saquinavir, at synergistically reduced concentrations, resulted m inhibiting approximately 12% of HIV-RT . Experiment 6: Determination of the Inhibition of HIV-RNA in Supernatant of CEM/0 T-Cells by a Combination Regimen of PEG-ASNase and Saquinavir
Experiment 5 illustrates the results of the exposure of HIV-RNA in T-cell pellets to a combination of PEG-ASNase and Saquinavir. However, the experimental procedure did not remove the HIV-1 particle from the supernatant to simulate the continuous exposure of the T- cells to HIV-1 virus. Thus, there is always HIV-1 virus in the supernatant of the T-cell cultures.
It is discovered that the combination of PEG-ASNase and Saquinavir inhibited the HIV-RNA in the cells pellets to a significant degree, and, in some wells, the HIV-RNA could not be quantitated after treatments with the PEG + SAQ drug combination, thus, we had achieved complete inhibition of HIV-RNA.
The supernatants of the T-cells from experiment 5 are analyzed for HTV -RNA and the results are shown in Tables 3 and 4. It should be noted that "PEG" in Table 3 represents PEG-ASNase. PEG alone inhibited HIV-RNA in the supernatants by approximately 60% and SAQ approximately by 68%. The combination of PEG + SAQ reduced the HIV-RNA by approximately 75% in the supernatant in comparison to untreated control. This RNA inhibition pattern fits nicely with the ones indicated in the earlier experiments reporting inhibiting HTV-RT and HIV-RNA from the same experiment.
Since these drugs do not "kill" the HIV virus in the supernatant, the reduction of the HIV-RNA can only be achieved by the "loss" due to infection and non-regeneration, via replication, due to the inhibition of the virus replication cycle by these drugs, specifically no HIV-RNA is produced intracellularly to be exported into the media as such or as complete HTV -virus particles. Experiment 7: Determination of the effect of PEG-ASNase ± Saquinavir ± AZT ± MISID (PL-7 on the HTV RT Levels of HIV-1 Infected Cell Pellets
Materials and Methods: The cell line used for this experiment is CEM/0, a human T- cell leukemic cell line. PEG-ASNase (ONCASPAR) is provided by Rhone-Poulenc Rorer. Saquinavir is commercially available. AZT is purchased from Sigma. MISH), a ribonucleotide reductase inhibitor, is synthesized described in Nandy P, Lien EJ, Avramis VI, Med Chem Res. 1995, 5:664-679. RPMI- 1640 media (Irvme Scientific, Irvine, CA), is enriched with 10% Fetal Calf Serum (Gemini Biosource, Calabasas, CA), 5% IM Hepes Buffer solution and 5% Non-essential amino acids (Irvme Scientific, Irvine, CA) The drug concentrations used are as follows:
PEG-ASNase IC50 alone: 0.40 TU/ml
Saquinavir IC50 alone: 25 μM AZT lμM
MISID (PL-7) 0.685 μM
PEG IC50 combination: 0.23 IU/ml
SAQ IC50 combination: 14 52 μM
Briefly, 3 x 106 cells/ml are stimulated with PHA+ media for 48 hours at 37°C with
5% C02. Also, the same number of cells are incubated in PHA free media for 48 hours to serve as the negative control. At this point the cells are inoculated with the HIN-1 virus as per standard protocol. Note that in this experiment, the HIV containing supernatant from the PHA-stimulated healthy human peripheral mononuclear cells (PBMC) is not removed from the PHA-stimulated CEM/0 cell culture. Hence, the HIV-1 virus is always present in the supernatant, an experimental condition that simulates the in vivo clinical condition of newly produced and unmfected T-cells, which are always under constant exposure to HIV-1 particles. These virus particles are released by already infected T-cells and/or lymph nodes of patients.
The experimental drugs are added to the cells in the appropriate concentrations at the same time as viral inoculation or 90 minutes after the viral incubation, a time sufficient for the T-cells to be infected and start producing new HIV-1 virus particles. The control cells are resuspended in drug-free media for the duration of the exposure which lasted seven days. Aliquots of media from the confrol flasks only are obtained on day 5 post Rx. At day seven, three 1 ml each, aliquots of media are removed from the flasks and stored under liquid nitrogen. In addition, the remaining cells are split into three and then pelleted and stored at - 80°C.
The samples produced from this experiment are itemized. The cellular pellets from the 90 minute viral incubation flasks are assayed for HIV-RT using an ELISA kit for the Reverse Transcnptase Assay, non-radioactive (Boehrmger Mannheim). The standard curve is determined (See Figure 10) and the HIV-RT levels for the experimental samples are calculated. The results are shown in Table 5. It should be noted that "PEG" in Table 5 represents PEG-ASNase
Results and Discussion- The first observation from the HIV-RT ELISA assays in these specimens from the cellular pellets of the CEM/0 T-cells is that there is diminished HIN-RT activity due to drug treatment, as compared to the untreated control cells The most dramatic inhibition of HIV-RT is caused by AZT, a nucleoside analog reverse transcnptase inhibitor Our initial experimental design and search for a wild-type HIV virus particle, with no mutations on HIV-RT conferring resistance to AZT, is actually shown with these results that AZT alone is very active against this viral strain.
PEG-ASNase or Saquinavir, used as a monotherapy at IC50 concentrations, inhibited HIV-RT by 54% and 83%, respectively These values are similar to those determined in earlier experiments.
MISID (PL-7), a new ribonucleotide reductase (RR) inhibitor, used alone at the IC50 concentration (0.685 μM), also demonstrated a 73% inhibition of HIV-RT. This is the first evidence that a member of this class of RR inhibitors has demonstrated anti-HIV activity in addition to its anti-leukemic activity. The biochemical rationale for this class of compounds in inhibiting HTV is by depleting dNTP pools intracellularly. Lack of or reduced dNTP pools will inhibit the function of HIV-RT, in converting HIV-RNA into proviral DNA prior to integration into the mammalian genomic DNA.
Combinations of PEG + SAQ resulted in complete inhibition of HIV-RT in this expenment. Combinations of three drugs, AZT + PEG + SAQ, resulted in complete inhibition of HIV-RT in two of the three wells, and the third well's value is inhibited by 95.3% of confrol. The biochemical rationale of this drug combination is that AZT will inhibit further infection by HIV-RT and that this inhibition will be potentiated by the already very efficacious anti-HTV-RT effect of PEG + SAQ regimen. Since the numbers are neaπng 100% inhibition of HJN-RT it is extremely difficult to demonstrate an improved inhibition by the three drug over the two drug regimen m this T-cell model system infected with a wild- type HTV virus. In experiments with HTV partially resistant to AZT, as they appear m patients, the regimen may demonstrate the validity of the above biochemical syllogism
Combinations of four drugs, MISID + AZT + PEG + SAQ, resulted in complete inhibition of HIV-RT in two of the three wells, and the third well's value is inhibited by 95% of control. These values are supeπmposable to the three drug regimen, due to maximum inhibition of HIV-RT The biochemical rational for this combination is that MISID, the RR inhibitor, will deplete dΝTP pools and this will potentiate the activity of AZT-tnphosphate (AZTTP), against HIV-RT This augmented inhibitory effect will be either additive or synergistic to the already selectively synergistic effect of a protein plus protease inhibitors against this virus. Since MISID alone appears to have considerable anti-HTV-RT activity we believe that this syllogism will be shown to be correct in experiments with HIV particles resistant to one more of these classes of drugs or in patients who are infected with multi- resistant HIV variants
Therefore, these results show that the following 3 or 4 drug regimen, of AZT + PEG + SAQ or MISID + AZT + PEG + SAQ, act synergistically against HIV-RT.
Experiment 8. Determination of the Synergistic effect of PEG-ASΝase, Saquinavir, AZT and MISID (PL-7) in CEM/0 Cell Supernatant
Materials and Methods. The cell line used for this experiment is CEM/0, a human T- cell leukemic cell line. PEG-ASΝase (ONCASPAR) is provided by Rhone-Poulenc Rorer. Saquinavm (SQ) is commercially available AZT is purchased from Sigma. MISID, a ribonucleotide reductase inhibitor, is as described m Nandy P, Lien EJ, Avramis VI, Med. Chem Res. 1995, 5.664-679. RPMI-1640 media (Irvme Scientific, Irvine, CA), is enriched with 10% Fetal Calf Serum (Gemini Biosource, Calabasas, CA), 5% IM Hepes Buffer solution and 5% Non-essential ammo acids (Irvme Scientific, Irvme, CA). The drug concentrations used are as follows: PEG-ASNase IC50 alone: 0.40 IU/ml
Saquinavir IC50 alone: 25 μM
AZT lμM
MISΓD 0.685 μM
PEG IC50 combination: 0.23 IU/ml
SAQ IC50 combination: 14.52 μM
Briefly, 3 x 106 cells/ml are stimulated with PHA+ media for 48 hours at 37°C with 5% C02. Also, the same number of cells are incubated m PHA free media for 48 hours to serve as the negative control. At this point the cells are inoculated with the HIN-1 virus as per standard protocol Note that in this experiment, the HIV containing supernatant from the PHA-stimulated healthy human peripheral mononuclear cells (PBMC) is not removed from the PHA-stimulated CEM/0 cell culture. Hence, the HIV-1 virus is always present in the supernatant, an experimental condition that simulates the in vivo clinical condition of newly produced and unmfected T-cells, which are always under constant exposure to HIV- 1 particles. These virus particles are released by already infected T-cells and/or lymph nodes of patients.
The experimental drugs are added to the cells in the appropriate concentrations at the same time as viral inoculation or 90 minutes after the viral incubation, a time sufficient for the T-cells to be infected and start producing new HIV-1 virus particles. The control cells are resuspended in drug-free media for the duration of the exposure which lasted seven days. Aliquots of media from the control flasks only are obtained on day 5 post Rx. At day seven, three 1 ml each, aliquots of media are removed from the flasks and stored under liquid nitrogen. In addition, the remaining cells are split into three and then pelleted and stored at - 80°C.
The samples produced from this experiment are itemized. The cell pellets of the cellular cultures (See Experiment 7) from the 90 minute viral incubation flasks are assayed for HIV-RNA quantitative assay using a kit for the assay, non-radioactive. The standard curve is determined and the HIV-RNA levels for the experimental samples are calculated as described previously.
These supernatant specimens are the ones from the cultures of the T-cell pellets of
Experiment 7. These results are disclosed in Experiment 7. The first observation from the HIV-RT ELISA assays in these specimens from the supernatants of the CEM/0 T-cells is that there is diminished HIV-RT activity on day 7 due to drug treatment, as compared to the untreated control cells on the same day. Of importance is that the untreated controls on day 5 (one value) had a higher HIV-RT O.D than on day 7. We examined O.D. values all of which are less than the minimum quantitated concenfrations based on the linearity of the calibration curve. Only one of the specimen supernatent (#7) treated with PEG-ASNase alone had an O.D. value from the assay higher than the negative control, and still lower than the average of the three untreated control culture supernatants.
Great inhibition of HIV-RT in quantitative terms (less than 66% of the Negative control value of 0.075 O.D or 0.05 O.D ) is caused by SAQ (Specimens #9 & 10), AZT (Specimens #1 1 & 13) and MISID (Specimens #15 & 16) as single agents (See Table 6. It should be noted that "PEG" in Table 6 represents PEG-ASNase ) Even greater inhibition of HIV-RT IS seen by the combination of SAQ + PEG-ASNase, three specimens #17, 18 & 19, one specimen from the three drug combination of PEG-ASNase + SAQ + AZT, specimen
#22, and all three specimens treated with the four drug combination regimen of PEG-ASNase +SAQ + MISID and AZT, #23, 24, & 25
Our initial experimental design and search for a wild-type HIV virus particle, with no mutations on HIV-RT conferring resistance to AZT, is actually shown with these results that AZT alone is very active against this viral strain.
MISID, a new ribonucleotide reductase (RR) inhibitor, used alone at the IC50 concentration (0.685μM), also demonstrated a significant inhibition of HTV RT as a single agent and in combination with the three other drugs. This is the result in the cell pellets as well (see Experiment 7) and is the first evidence that a member of this class of RR inhibitors has demonstrated anti-HTV activity in addition to its anti-leukemic activity both intracellularly and in the supernatant specimens of these T-cell cultures.
Combinations of PEG + SAQ resulted in complete inhibition of HTV-RT in this and in the previously reported experiment in the cell pellets. In the previous experiments there is a 96% inhibition of HIV-RT (see Experiment 7). Combinations of three drugs, AZT + PEG + SAQ, resulted in complete inhibition of HIV-RT in two of the three wells, and the third well's value is inhibited by 95.3% of control, whereas in the supernatants an identical pattern is seen Specimens #20-22. Combinations of four drugs, MISID + AZT + PEG + SAQ, resulted in complete inhibition of HIV-RT in all three of the supernatant specimens, which values correspond with the values determined in the cell pellets from the same expeπment. These values are supeπmposable to the three drug regimen, due to maximum inhibition of HIN-RT, as we determined earlier.
The biochemical rationale for this combination is that MISID, the RR inhibitor, will deplete dΝTP pools and this will potentiate the activity of AZT-tnphosphate (AZTTP), against HIV-RT. This augmented inhibitory effect will be either additive or synergistic to the already selectively synergistic effect of a protein plus protease inhibitors against this virus. Since MISID alone appears to have considerable anti-HIV-RT activity we believe that this syllogism will be shown to be correct in experiments with FUN particles resistant to one or more of these classes of drugs or in patients who are infected with multi-resistant HIV variants.
Experiment 9: Determination of the Synergistic effect of PEG-ASΝase, Saquinavir, AZT and MISED (PL-7) in CEM/0 Cell Pellets
The cell line used for these studies is CEM/0, a human T-cell leukemic cell line. PEG-ASΝase (ONCASPAR) is provided by Rhone-Poulenc Rorer. Saquinavin is commercially available. AZT is purchased from Sigma. MISID, a ribonucleotide reductase inhibitor, is synthesized as indicated in Nandy P, Lien EJ, Avramis VI, Med. Chem. Res 1995, 5.664-679. RPMI-1640 media (Irvine Scientific, Irvine, CA), is enriched with 10% Fetal Calf Serum (Gemini Biosource, Calabasas, CA), 5% IM Hepes Buffer solution and 5% Non-essential amino acids (Irvine Scientific, Irvme, CA). The drug concentrations used are as follows:
PEG-ASNase IC50 alone: 0.40 IU/ml
Saquinavir IC50 alone: 25 μM
AZT lμM
MISID 0.685 μM
PEG IC50 combination- 0.23 IU/ml
SAQ IC50 combination- 14.52 μM
Briefly, 3 x 106 cells/ml are stimulated with PHA+ media for 48 hours at 37°C with
5% C02. Also, the same number of cells are incubated in PHA free media for 48 hours to serve as the negative control. At this point the cells are inoculated with the HIV-1 virus as per standard protocol. Note that in this experiment, the HTV containing supernatant from the PHA-stimulated healthy human peripheral mononuclear cells (PBMC) is not removed from the PHA-stimulated CEM/0 cell culture Hence, the HIV-1 virus is always present in the supernatant, an experimental condition that simulates the in vivo clinical condition of newly produced and unmfected T-cells, which are always under constant exposure to HIN- Iparticles These virus particles are released by already infected T-cells and/or lymph nodes of patients
The experimental drugs are added to the cells in the appropriate concentrations at the same time as viral inoculation or 90 minutes after the viral incubation, a time sufficient for the T-cells to be infected and start producing new HFV-l virus particles The control cells are resuspended in drug-free media for the duration of the exposure which lasted seven days Aliquots of media from the control flasks only are obtained on day 5 post Rx At day seven, three 1 ml each, aliquots of media are removed from the flasks and stored under liquid nitrogen In addition, the remaining cells are split into three and then pelleted and stored at - 80°C
The samples produced from this experiment are itemized The cell pellets of the cellular cultures (see Experiment 7) from the 90 minute viral incubation flasks are assayed for HIV-RΝA quantitative assay using a kit for the assay, non-radioactive The standard curve is determined and the HTV-RΝA levels for the experimental samples are calculated and reported previously
The specimens are from the cultures of the T-cell pellets and are from the same experiment as those we reported for the HIV-RT results These results are discussed in Experiment 7 (cell pellets) & Experiment 8 (supernatant)
The first observation from the HIV RΝA quantitative assays in these specimens of CEM 0 T-cells is that there is diminished HIV RΝA activity on day 7 due to drug treatment, as compared to the untreated control cells on the same day The quantitative results are shown m Table 7 and Figure 11 It should be noted that "PEG" in Table 7 represents PEG- ASΝase
Inhibition of HIV RΝA is quantitative terms cause by AZT alone (Specimens #11- 13) are greater (sensitive HIV-1 virus to AZT), with SAQ and MISFD as single agents following Greater inhibition of HIV RΝA is seen by the combination of SAQ + PEG- ASΝase, 38% of control, and from the three drug combination of PEG-ASΝase +SAQ + AZT, 30%) of control. All three specimens treated with the four drug combination regimen of PEG-ASNase + SAQ + MISID and AZT, #23, 24 & 25, has the greatest inhibition of HIV- RNA from this experiment, 20% of control, clearly showing the significant contribution of the ribonucleotide reductase inhibitor, MISID.
MISID, a new ribonucleotide reductase (RR) inhibitor, used alone at the IC50 concentration (0.685 μM), also demonstrated a significant inhibition of HTV RNA as a single agent and most importantly, in combination with the three other drugs. This is the result in the cell pellets as well (Experiment 6) and is the repeat evidence that a member of this class of RR inhibitors has demonstrated anti-HIV activity in addition to its anti-leukemic activity both intracellularly and in the supernatant specimens of these T-cell cultures
The biochemical rationale for this combination is that MISFD, the RR inhibitor, will deplete dNTP pools and this will potentiate the activity of AZT-tnphosphate (AZTTP), against HIV integration and replication, thus reduced HIV RNA. This augmented inhibitory effect will be either additive or synergistic to the already selectively synergistic effect of a protein plus protease inhibitors against this virus Since MISFD alone appears to have considerable anti-HIV RNA inhibitory activity, we believe that this syllogism will be shown to be correct in experiments with HFV particles resistant to one or more of these classes of drugs or in patients who are infected with multi-resistant HIV variants.
Experiment 10: : Determination of the Synergistic effect of PEG-ASNase, Saquinavir, AZT and MISID (PL-7) m the Suppression of HIV-RT
Materials and Methods: The cell line used for these studies is CEM/0, a human T- cell leukemic cell line PEG-ASNase (ONCASPAR) is provided by Rhone-Poulenc Rorer Saquinavin is commercially available. AZT is purchased from Sigma. MISFD, a ribonucleotide reductase inhibitor, is as described in Nandy P, Lien EJ, Avramis VI, Med. Chem. Res. 1995, 5.664-679. RPMI-1640 media (Irvine Scientific, Irvme, CA), is enriched with 10% Fetal Calf Serum (Gemini Biosource, Calabasas, CA), 5% IM Hepes Buffer solution and 5% Non-essential amino acids (Irv e Scientific, Irvine, CA). The drug concentrations used are as follows-
PEG-ASNase IC50 alone: 0.40 IU/ml
Saquinavir IC50 alone: 25 μM
AZT lμM
MISFD 0.685 μM PEG IC50 combination: 0.23 IU/ml
SAQ IC50 combination: 14.52 μM
Briefly, 3 x 106 cells/ml are stimulated with PHA+ media for 48 hours at 37°C with 5% C0 Also, the same number of cells are incubated in PHA free media for 48 hours to serve as the negative control. At this point the cells are inoculated with the HIV-1 virus as per standard protocol Note that m this experiment, the HIV containing supernatant from the PFIA-stimulated healthy human peripheral mononuclear cells (PBMC) is not removed from the PHA-stimulated CEM/0 cell culture. Hence, the HIV-1 virus is always present in the supernatant, an experimental condition that simulates the in vivo clinical condition of newly produced and unmfected T-cells, which are always under constant exposure to HIV- lparticles. In this experiment we have seen much higher HIV-1 titers as per our control HIV- RNA in the T-cells (see Experiment 9) These virus particles are continuously released by already infected T-cells and/or lymph nodes of patients.
The experimental drugs are added to the cells in the appropriate concentrations at the same time as viral inoculation or 90 minutes after the viral incubation, a time sufficient for the T-cells to be infected and start producing new HIV-1 virus particles. The control cells are resuspended in drug-free media for the duration of the exposure which lasted seven days. Aliquots of media from the control flasks only are obtained on day 5 post Rx. At day seven, three 1 ml each, aliquots of media are removed from the flasks and stored under liquid nitrogen. In addition, the remaining cells are split into three and then pelleted and stored at - 80°C. Supernatant specimens are obtained form these T-cell cultures and are frozen at -80°C. The samples produced from this experiment are itemized. The cell pellets of the cellular cultures (see Experiment 9) from the 90 minute viral incubation flasks are assayed for HIV- RNA quantitative assay using a kit for the assay, non-radioactive. We disclose here the quantitative HIV-RNA results from the supernatants of these T-cells. The standard curve is determined and the HIV-RNA levels for the experimental samples are calculated and reported previously.
Results and Discussion: These specimens are from the cultures of the T-cell pellets from the same experiment as those from which we reported the HIV-RT results (see Experiment 8) and the quantitative HIV-RNA in T-cells (see Experiment 9).
The first observation from the HIV-RNA quantitative assays in the supernatant specimens of CEM/0 T-cells is there is diminished HIV-RNA activity on day 7 due to drug treatment, as compared to the untreated control cells on the same day. The quantitative results are shown in the attached table. (Table 8). It should be noted that "PEG" in Table 8 represents PEG-ASNase. The quantitative control HFV-RNA levels (virus genomic copies / ml) are higher that from previous experiments and similar to the untreated control levels (214,445 in T-cell pellets vs. 195,483 in the supernatants).
Inhibition of HFV-RNA in quantitative terms caused by SAQ, AZT or MISFD alone (Specimens #8-16), are non-statistically significant among themselves (sensitive HIV-1 virus to AZT) Similar inhibition percentages of HIV-RNA in the supernatants is seen by either the combination of SAQ + PEG-ASNase, or the three drug combination of PEG-ASNase + SAQ + AZT, in comparison to untreated control. However, all three specimens treated with the four drug combination regimen of MISFD + AZT + PEG-ASNase + SAQ #23, 24 & 25, has the greatest inhibition of HIV-RNA from this experiment, 50% of control, clearly showing the significant contribution of the ribonucleotide reductase inhibitor, MISID. This latter set of data confirms the earlier observation of HIV-RNA inhibition that is shown in the T-cell pellets of 20% of control (Experiment 9). Data from the previous expeπmentals and this evidence indicate that the higher the HIV titer left in the supernatant, the lesser the inhibition of the virus both in the T-cells and in the supernatant would be. In other words, the data suggest that: a) these combination regimens must be given continuously under these conditions, i.e , in patients with high HIV-RNA copy number and/or viremia and b) the potentiation of the AZT + SAQ is required by either a third RT inhibitor, such as 3TC or and an RR inhibitor, such as, MISFD or hydroxyurea, to potentiate the activity of AZT tπphosphate (AZTTP) against HIV-RT.
MISID, a new ribonucleotide reductase (RR) inhibitor, used alone at the IC50 concentration (0.685μM), also demonstrated a significant inhibition of HIV-RNA as a single agent which is approximately equal to the inhibition of either SAQ or AZT. Most importantly, MISFD showed it significant usefulness in combination with the three other drugs both against HFV-RT in T-cell pellets and supernatants (see Experiments 7 and 8), respectively, and in suppressing HFV-RNA left in the supernatant (Table 8). This is also the result in the T-cell pellets (see Experiments 6 and 9) and is the repeat evidence that a member of this class of RR inhibitors has demonstrated anti-HFV activity in addition to is anti- leukemic activity both lnfrocellularly and in the supernatant specimens of these T-cell cultures. The biochemical rationale for this combination is that MISFD, the RR inhibitor, will deplete dNTP pools and this will potentiate the activity of AZTTP, against HTV-1 reverse transcription, integration and replication, thus reduced HIV-RNA. This augmented inhibitory effect will be either additive or synergistic to the already selectively synergistic effect of a protein plus protease inhibitors against this virus. Since MISID alone appears to have considerable anti-HIV -RNA inhibitory activity we believe that this syllogism will be shown to be correct in experiments with HIV particles resistant to one or more of these classes of drugs or in patients who are infected with multi-resistant HFV variants.
Experiment 10: Determination of the Synergistic effect of PEG-ASNase, Saquinavir, AZT and 3TC on HIV-RT in CEM/0 Cell Pellets
This experiment is carried out using a similar procedure, and the same concentrations of materials as in Experiment 9, however 3TC is used instead of MISID. In this experiment the exposure of HIV-RT in T-cell pellets to a combination of PEG-ASNase, Saquinavir, AZT and 3TC is measured The results of this experiment are shown in Table 9. It should be noted that "PEG" in Table 9 represents PEG-ASNase. It is shown in Table 9 that there is some inhibition of HIV-RT by Saquinavir and PEG-ASNase alone. However, the combination of PEG-ASNase, Saquinavir, AZT and 3TC results in the complete inhibition of HIV-RT.
Experiment 1 1 : Determination of the Synergistic effect of PEG-ASNase, Saquinavir, AZT and 3TC on HIV-RNA in CEM/0 Cell Supernatants
This experiment is carried out using a similar procedure, and the same concentrations of materials as in Experiment 10, however the exposure of HIV-RNA in T-cell supernatants to a combination of PEG-ASNase, Saquinavir, AZT and 3TC is measured. The results of this experiment are shown in Table 10. It should be noted that "PEG" in Table 10 represents PEG-ASNase. It is shown in Table 10 that there is some inhibition of HIV-RNA by Saquinavir and PEG-ASNase alone (approximately 55% and 73% of control, respectively). The combination of PEG-ASNase, Saquinavir, and AZT results in the greater inhibition of HIV-RNA (approximately 21% of control). However, the combination of PEG-ASNase, Saquinavir, AZT and 3TC results in the complete inhibition of HIV-RNA (0% of control)(see Table 10).
The present invention may be embodied in other specific forms without departing from the spirit or essential attributes thereof. Table 1
• N is the number of virus genomic copies/mL of supernatant or copies cell pellet. Dynamic range of the HIV-I Monitor Assay is 400 to 750.000 copies ml. Inquire with laboratory if quanti tion below 400 copies/mL is needed. Table 2
Table 2a
Table 3
Table 4
* N is the Dumber of virus geαomic copies/mL of supernatant or copies/cell pellet. Dynamic range of the HIV- 1 Monitor Assay is 400 to 750,000 copies mL Inquire with laboratory if quantita on below 400 copies mL is needed. Table 5
Table 6
Table 7
Table 8
Table 9
to
Table 10

Claims

Claims
1 A method of inhibiting or treating Human Immunodeficiency Virus (HIV) infection, compπsing administering to a patient, in need thereof, a therapeutically effective amount of a composition comprising a PEG-ASNase compound or a pharmaceutically acceptable salt thereof, and optionally at least one compound selected from the group consistmg of protease inhibitor compounds, ribonucleotide reductase inhibitor compounds and HIV reverse transcnptase inhibitor compounds or a pharmaceutically acceptable salt thereof
2 A method according to claim 1 wherein at least one compound is a protease inhibitor compound.
3. A method according to claim 1 wherein at least one compound is a ribonucleotide reductase inhibitor compound.
4. A method according to claim 1 wherein at least one compound is an HIV reverse transcπptase inhibitor compound.
5. A method according to claim 1 wherein the protease inhibitor compound is selected from Saqumovir, Nelfmavir, Indinavir, Endinovere, Ritonavir, Crixivan, Viracept, Norvir, and VX-478.
6. A method according to claim 3 wherein the ribonucleotide reductase inhibitor compound is selected from Hydroxyurea (HU), BW- 348U87, 3-ammopyπdme-2- carboxaldehyde thiosemicarbazone (3-AP) Amidox (VF 236; NSC-343341 , N,3,4- tπhydroxybenzenecarboximidamide), BILD 1257 (2-benzyl-3-phenylpropιonyl-L-(N- methyl)valyl-L-3-(methyl)valyl-L-(N4,N4-tetramethylene)asparagιnyl-L-(3,3- tetramethylene)aspartyl-L-(4-methyl)leucme), BILD 1357 (2-benzyl-3-phenylpropιonyl-L- (N-methyl)valyl-L-3-(methyl)valyl-L-(N4,N4-tetramethylene)asparagιnyl-L-(3,3- tetramethylene)aspartιc acid l-[l(R)-ethyl-2,2-dιmethylpropylamιde]), BILD 1633, BILD 733 (3-phenylpropιonyl-L-(N-methyl)valyl-L-[3-methyl)valyl-L-[3-(pyrrohdm-l- ylcarbonyl)]alanyl-L-(l-carboxycyclopentyl)glycyl-L-(4-methyl)leucmol), BILD 1263 (2- benzyl-3-phenylpropιonyl-L-(N-methyl)valyl-L-3-(methyl)valyl-L-(N4,N4- tetramethylene)asparagmyl-L-(3,3-tetramethylene)aspartyl-L-(4-methyl)leucmol), BILDl 351 (l-[l(S)-[5(S)-[3-[(all-cιs)-2,6-dιmethylcyclohexyl]ureιdo]-2(S)-(3,3-dιmethyl-2-oxobutyl)- 6,6-dιmethyl-4-oxoheptanoylamιno]-l -[ 1 (R)-ethyl-2,2- dιmethylpropylcarbamol]methyl]cyclopentanecarboxyhc acid]), CI-F-araA (2-chloro-9-(2- deoxy-2-fluoro-beta-D-arabιnofuranosyl)adenιne, DAH (D-aspartic-beta-hydroxamate), DDFC (2'-deoxy-2',2'-dιfluorocytιdιne),Dιdox (VF 147; NSC 324360; N,3,4- tπhydroxybenzamide), Eurd (3'-Ethynyluπdιne),GTI 2040, GTI 2501 MHAG (1- lsoquinolylmethane-N-hydroxy-N'-aminoguanme), LY 207702 (2',2'-dιfluoro-2'- deoxyπbofuranosyl-2,6-dιamιnopuπne), LY 295501 (N-[[3,4- dιchlorophenyl)amιno]carbonyl]2,3-dιhydro-5-benzofuransulfonamιde), MDL 101731 (FMdC; KW 2331; (2E)-2'-deoxy-2'-(fluoromethylene)cytιdιne), Parabactin Sulofenur (LY 186641 , N-[[(4-chlorophenyl)amιno]carbonyl]-2,3-dιhydro-lH-ιndene-5- sulfonamide), TAS 106 (Ecyd; 3'-ethynylcytιdιne),Tπapιne (OCX 191 , OCX 0191), Tπmidox (VF 233; N,3-4,5-tetrahydroxybenzene carboximidamide) and compounds of formula I
wherein
Rl is alkyl, alkenyl, alkynyl or an electron withdrawing group; and
R2 is alkyl, alkenyl, alkynyl; or a pharmaceutically acceptable salt thereof, an N-oxide thereof, a solvate thereof, an acid bioisostere thereof, or prodrug thereof.
7. A method according to claim 6 wherein the ribonucleotide reductase inhibitor compound is a compound of formula I.
8. A method according to claim 7 wherein the ribonucleotide reductase compound is a compound of formula I wherein Rl is lower alkyl or a halogen group; and
R2 is lower alkyl; or a pharmaceutically acceptable salt thereof, an N-oxide thereof, a solvate thereof, an acid bioisostere thereof, or prodrug thereof.
9. A method according to claim 6 wherein the ribonucleotide reductase inhibitor compound of formula I is MISID.
10. A method according to claim 4 wherein the HIV reverse transcnptase inhibitor compound is selected from AZT (Retrovir, zidovudme) ddl (Videx, didanosine) ddC (Hivid, zalcitabine), d4T (Zent, stavudine) and 3TC (Epivir, lamivudine).
1 1 A method according to claim 1 wherein said the compounds are administered concurrently.
12 A method according to claim 1 wherein said compounds are administered sequentially.
13. A method of inhibiting the production, or limiting the spread, of HIV comprising the step of exposing a cell population infected with HIV to an effective amount of PEG-ASNase compound and optionally at least one compound selected from the group consisting of protease inhibitor compounds, ribonucleotide reductase inhibitor compounds and HIV reverse transcnptase inhibitor compounds.
14. A method according to claim 1 wherein the compounds administered comprise a synergistically effective combination of a PEG-ASNase compound and at least one compound selected from the group consisting of protease inhibitor compounds, ribonucleotide reductase inhibitor compounds and HIV reverse transcnptase inhibitor compounds.
15. A pharmaceutical composition comprising a PEG-ASNase compound, and one or more compounds selected from the group consisting of a protease inhibitor compound, a HIV reverse transcnptase inhibitor compound, or a ribonucleotide reductase inhibitor compound, and a pharmaceutically acceptable carrier.
16. A pharmaceutical kit for treating or inhibiting a physiological condition associated with HIV, said kit compnsing a plurality of separate containers, wherein at least one of said containers contains a PEG-ASNase compound and at least another of said containers contains one or more compounds selected from the group consistmg of a protease inhibitor compound, a HIV reverse transcnptase inhibitor compound and a nbonucleotide reductase inhibitor compound, and said containers optionally contain a pharmaceutical earner.
17. A method of inhibiting or treating Human Immunodeficiency Virus (HIV) infection, compnsing administering to a patient, in need thereof, a therapeutically effective amount of asparaginase or a pharmaceutically acceptable salt thereof, and optionally at least one compound selected from the group consisting of protease inhibitor compounds, ribonucleotide reductase inhibitor compounds and HIV reverse transcnptase inhibitor compounds or a pharmaceutically acceptable salt thereof
EP00957306A 1999-08-06 2000-08-07 Pharmaceutical compostions comprising peg-asparaginase for the treatment of hiv infections Withdrawn EP1143990A3 (en)

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