WO1996002268A1 - Inhibition d'un virus par l'oxyde nitrique - Google Patents

Inhibition d'un virus par l'oxyde nitrique Download PDF

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Publication number
WO1996002268A1
WO1996002268A1 PCT/US1995/008763 US9508763W WO9602268A1 WO 1996002268 A1 WO1996002268 A1 WO 1996002268A1 US 9508763 W US9508763 W US 9508763W WO 9602268 A1 WO9602268 A1 WO 9602268A1
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Prior art keywords
nitric oxide
virus
nitroso
group
arginine
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PCT/US1995/008763
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English (en)
Inventor
Jonathan Stamler
Joan Mannick
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Brigham And Women's Hospital
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Priority to AU29705/95A priority Critical patent/AU2970595A/en
Publication of WO1996002268A1 publication Critical patent/WO1996002268A1/fr

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    • 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
    • A61K38/55—Protease inhibitors
    • A61K38/556—Angiotensin converting enzyme inhibitors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A61K31/195—Carboxylic acids, e.g. valproic acid having an amino group
    • A61K31/197—Carboxylic acids, e.g. valproic acid having an amino group the amino and the carboxyl groups being attached to the same acyclic carbon chain, e.g. gamma-aminobutyric acid [GABA], beta-alanine, epsilon-aminocaproic acid, pantothenic acid
    • A61K31/198—Alpha-aminoacids, e.g. alanine, edetic acids [EDTA]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/21—Esters, e.g. nitroglycerine, selenocyanates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
    • A61K31/401—Proline; Derivatives thereof, e.g. captopril
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/425—Thiazoles
    • A61K31/426—1,3-Thiazoles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/05—Dipeptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/06—Tripeptides
    • A61K38/063—Glutathione
    • 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
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans

Definitions

  • nitric oxide in host defense. In addition to is production by several cells central to the immune response, nitric oxide also has direct static and cidal effects on bacteria, fungi, and parasites. However, there are no data s. owing an inhibitory effect of nitric oxide on viral replication. Therapeutic options for these diseases are limited. Acyclovir (and related drugs) , an inhibitor of viral DNA polymerase, is the only current approach available for the treatment of herpes viruses. However, it is clearly often suboptimal therapy and resistance may develop.
  • viruses can both replicate (lytic phase) and also incorporate themselves into the genome (latency) .
  • infectious disease e.g., genital Herpes (HSVII) or ononucleosis (EBV)
  • HSVII genital Herpes
  • EBV ononucleosis
  • certain viruses tend to establish latency more readily than others.
  • the virus may serve as a protooncogene, thereby promoting cancer; and it is also resistant to anti-viral therapy.
  • the establishment of latency prevents elimination of virus with anti-viral therapy.
  • nitric oxide and related donors inhibi viral replication and provides a method for inhibiting th replication of a virus which comprises the step of exposin the virus to nitric oxide or a nitric oxide-releasin substance.
  • the virus can be, for example, a Herpes virus.
  • tte metho comprises administering a virus replication inhibitory amoun of nitric oxide or a nitric oxide-releasing, donating, o transferring compound to an individual having a viru infection.
  • the administration can be topical, by inhalation, oral, or parenteral.
  • the treated infection can be localize or systemic.
  • One aspect of the present invention provides a proces for inhibiting replication of a virus by treatment of a animal (preferably a mammal and more preferably a human) wit nitric oxide.
  • a animal preferably a mammal and more preferably a human
  • the virus is Herpes virus.
  • the treatment with nitric oxide encompasses the use o gaseous nitric oxide and/or the use of a compound which i capable of delivering nitric oxide.
  • nitric oxide generally refers to the reactiv forms of nitric oxide, in particular (1) uncharged nitri oxide (NO) (Gaseous nitric oxide is an unchanged form o nitric oxide) ; (2) negatively charged nitric oxide or NO (nitroxyl) and positively charged nitric oxide, or (3) NO (nitrosonium) .
  • NO uncharged nitri oxide
  • the present invention contemplates th use of gaseous nitric oxide as well as compounds capable o donating or releasing nitric oxide in one of its reactiv forms.
  • the reactive form of nitric oxide is provided by gaseous nitric oxide.
  • the reactive form of nitric oxide is provided by a compound which delivers nitric oxide.
  • Compounds which deliver nitric oxide include, but are not limtied to, S-nitrosothiols, S-nitroso amino acids, S- nitroso-polypeptides, and nitrosoamines.
  • nitric oxide, or a nitric oxide releasing, donating, or transferring substance may be used prophylactically to prevent reactivation of latent virus; for example, in an it ⁇ munocompromised host in which reactivation at an inopportune time (i.e. when defenses are low due to chemotherapy) would cause significant morbidity.
  • nitric oxide and compounds that release nitric oxide or otherwise directly or indirectly deliver or transfer nitric oxide to a site of its activity, such as on a cell membrane, in vivo.
  • nitric oxide encompasses uncharged nitric oxide(NO*) and charged nitric oxide species, particularly including nitrosonium ion(NO * ) and nitroxyl ion(NO”) .
  • nitric oxide releasing, delivering or transferring compounds having the structure X-NO wherein X is a nitric oxide releasing, delivering or transferring moiety, include any and all such compounds which provide nitric oxide to its intended site of action in a form active for their intended purpose.
  • NO adducts encompasses any of such nitric oxide, releasing, delivering or transferring compounds.
  • S-nitrosothiols which are compounds that include at least one -S-NO group.
  • Such compounds include ⁇ -nitroso-polypeptides (the ter "polypeptide” includes proteins and also polyamino acids tha do not possess an ascertained biological function, an derivatives thereof); S-nitrosylated amino acids (includin natural and synthetic amino acids and their stereoisomers an racemic mixtures and derivatives thereof) ; S-nitrosate sugars, S-nitrosated-modified and unmodified oligonucleotide (preferably of at least 5, and more particularly 5-200, nucleotid.es) ; and an S-nitrosated hydrocarbon where th hydrocarbon can be a branched or unbranched, and saturated o unsaturated aliphatic hydrocarbon, or an aromati hydrocarbon; S-nitroso hydrocarbons having one or mor substituent groups in addition to the S-nitroso group; an heterocyclic compounds
  • S-nitroso amino acids where the nitroso group i linked to a sulfur group of a sulfur-containing amino acid o derivative thereof.
  • such compounds include th following: S-nitroso-N-acetylcysteine, S-nitroso-captopril S-nitroso-homocysteine. S-nitroso-cysteine and S-nitroso glutathione.
  • Suitable S-nitrosylated proteins include thiol containing proteins(where the NO group is attached to one o more sulfur group on an amino acid or amino acid derivativ thereof) from various functional classes including enzymes, such as tissue-type plasminogen activator(TPA) and cathepsi B; transport proteins, such as lipoproteins, he e protein
  • E 91 such as hemoglobin and serum albumin; and biologically protective proteins, such as the immunoglobulins and the cytokines.
  • biologically protective proteins such as the immunoglobulins and the cytokines.
  • S-nitrosothiols include those having the structures:
  • x equals 2 to 20 and Y is selected from the group consisting of fluoro, C,-C 6 alkoxy, cyano, carboxamido, C 3 -C 6 cycloalkyl, aralkoxy, C 2 -C 6 alkylsulfinyl, arylthio, C,-C 6 alkylamino, C 2 -C,j dialkylamino, hydroxy, carbamoyl, C,-C 6 N- alkylcarbamoyl, Cj-C.j N,N-dialkylcarbamoyl, amino, hydroxyl, carboxyl, hydrogen, nitro and aryl; wherein aryl includes benzyl, naphthyl, and anthracenyl groups.
  • S-nitrosothiols that are S-nitroso- angiotensin converting enzyme inhibitors (hereinafter referred to as S-nitroso-ACE inhibitors) are described in oscalzo, U.S. Patent No. 5,002,964 (1991) and oscalzo et al . , U.S. Patent No. 5,025,001 (1991) both of which are incorporated in their entirety by reference.
  • S-nitroso-ACE inhibitors include compounds having structure (1) :
  • R is hydroxy, NH 2 , NHR 4 , NR 4 R 5 , or C,-C 7 alkoxy, wherein R 4 and R 5 are C,-C 4 alkyl, or phenyl, or C ⁇ C 4 alkyl substituted by phenyl;
  • R 1 is hydrogen, C,-C 7 alkyl, or C,-C 7 alkyl substituted by phenyl, amino, guanidino, NHR 6 , NR 6 R 7 , wherein R 6 and R 7 are methyl or C,-C 4 alkanoyl;
  • R 2 is hydrogen, hydroxy, C,-C 4 alkoxy, phenoxy, or
  • R 3 is hydrogen, C,-C 4 or C,-C 7 alkyl substituted by phenyl; m is 1 to 3; and n is 0 to 2.
  • S-nitroso-ACE inhibitors include N- acetyl-S-nitroso-D-cysteinyl-L-proline, N-acetyl -S-nitroso- D, L-cysteinyl-L-proline, 1- (4-amino-2-S- nitroso)mercaptomethylbutanoyl) - -proline, 1- [2-hexanoyl] -L- proline, 1- [5-guanidino-2- (S-nitroso) mercaptomethyl- pentanoyl] -L-proline, 1- [5-amino-2- (S-nitroso) mercaptomethyl-pentanoyl] -4-hydroxy-L-proline, l- [5- guanidino-2- (S-nitroso)mercaptomethyl-pentanoyl] -4-hydroxy-L- proline, 1- [2-aminomethyl-3 (S-nitroso) -mercapto
  • X is oxygen or sulfur
  • R 3 0 II 3 II A is ON-S-CH 2 -CH-C;
  • R is selected from hydrogen, lower ( -C 4 ) alkyl, benzyl, benzhydryl, -and salt forming ion;
  • R, and R 2 are independently selected from hydrogen, halogen, lower alkyl, lower alkoxy, halo substituted lower alkyl, nitro, and S ⁇ 2 NH 2 ;
  • Z is -C- or -S-
  • R 3 is hydrogen, lower alkyl, halo substituted lower alkyl, phenyl, benzyl, phenethyl, or cycloalkyl;
  • the S-nitroso-ACE inhibitors can be prepared by various methods of synthesis.
  • Acids which may be used for this purpose include aqueous sulfuric, acetic and hydrochloric acids.
  • Thiol precursors are prepared as described in the following: U.S. Pat. NOS. 4,046,889 (1977); 4,052,511; 4,053,651; 4,113,751, 4,154,840, 4129,571 (1978), and 4,154,960 (1979) to Ondetti et al .
  • Such compounds include O- nitroso-polypeptides (the term "polypeptide” includep proteins and also polyamino acids that do not possess an ascertained biological function, and derivatives thereof) ; O-nitrosylated amino acids (including natural and synthetic amino acids and their stereoisomers and racemic mixtures and derivatives thereof) ; O-nitrosated sugars; O-nitrosated-modified and unmodified oligonucleotides (preferably of at least 5, and more particularly 5-200, nucleotides); and an O-nitrosated hydrocarbon where the hydrocarbon can be a branched or unbranched, saturated or unsaturated aliphatic hydrocarbon, or an aromatic hydrocarbon; O-nitroso hydrocarbons having one
  • NO adducts Another group of such NO adducts is the nitrites whic have an -O-NO group wherein R is a protein, polypeptide amino acid, branched or unbranched and saturated o unsaturated alkyl, aryl or a heterocyclic.
  • R is a protein, polypeptide amino acid, branched or unbranched and saturated o unsaturated alkyl, aryl or a heterocyclic.
  • a preferre example is the nitosylated form of isosorbide.
  • Compounds i this group form S-nitrosothiol intermediates in vivo in th recipient human or other animal to be treated and ca therefore include any structurally analogous precursor R-O-N of the S-nitrosothiols described above.
  • N-nitrosoamine ⁇ which are compounds that include at least one -N-NO group
  • Such compounds include N-nitroso-polypeptides (the ter “polypeptide” includes proteins and also polya ino acids tha do not possess an ascertained biological function, an derivatives thereof) ; N-nitrosylated amino acids (includin natural and synthetic amino acids and their stereoisomers an racemic mixtures) ; N-nitrosated sugars; N-nitrosated-modifie and unmodified oligonucleotides (preferably of at least 5 and more particularly 5-200, nucleotides); and an N nitrosated hydrocarbon where the hydrocarbon can be branched or unbranched, and saturated or unsaturate aliphatic hydrocarbon, or an aromatic hydrocarbon,- N-nitros hydrocarbons having one or more substituent groups i addition to the N-nitroso group; and heterocyclic compounds
  • C-nitros compounds that include at least one -C-NO group.
  • Suc compounds include C-nitroso-polypeptides (the ter "polypeptide” includes proteins and also polyamino acids tha do not possess an ascertained biological function, an
  • C-nit.rosylated amino acids including natural and synthetic amino acids and their stereoisomers and racemic mixtures
  • C-nitrosated sugars including natural and synthetic amino acids and their stereoisomers and racemic mixtures
  • C-nitrosated-modified and unmodified oligonucleotides preferably of at least 5, and more particularly 5-200, nucleotides
  • a C-ritrosated hydrocarbon where the hydrocarbon can be a branched or unbranched, and saturated or unsaturated aliphatic hydrocarbon, or an aromatic hydrocarbon
  • C-nitroso hydrocarbons having one or more substituent groups in addition to the C-nitroso group,- and heterocyclic compounds.
  • polypeptides include proteins and also polyamino acids that do not possess an ascertained biological function, and derivatives thereof
  • polypeptide includes proteins and also polyamino acids that do not possess an ascertained biological function, and derivatives thereof
  • - amino acids including natural and synthetic amino acids and their stereoisomers and racemic mixtures and derivatives thereof
  • sugars modified and unmodified oligonucleotides (preferably of at least 5, and more particularly 5-200, nucleotides) ,- and a hydrocarbon where the hydrocarbon can be a branched or unbranched, and saturated or unsaturated aliphatic hydrocarbon, or an aromatic hydrocarbon; hydrocarbons having one or more substituent groups,- and heterocyclic compounds.
  • a preferred example is nitroglycerin.
  • R includes polypeptides(the term "polypeptide” includes proteins and also polyamino acids that do not possess an ascertained biological function, and derivatives thereof) ,- amino acids (including natural and synthetic amino acids and their stereoisomers and racemic mixtures and derivatives thereof) ,- sugars; modified and unmodified oligonucleotides (preferably
  • x-x 9i of at least 5, and more particularly 5-200, nucleotides
  • hydrocarbon can be a branched or unbranched, and saturated or unsaturated aliphatic hydrocarbon, or an aromatic hydrocarbon
  • hydrocarbons having one or more substituent groups in addition to the A-nitroso group,- and heterocyclic compounds.
  • A is S, 0, or N
  • n and x are each integers independently selected from 1, 2 and 3
  • M is a metal, preferably a transition metal.
  • Preferred metals include iron, copper, manganese, cobalt, selenium and luthidium.
  • N-nitrosylated metal centers such as nitroprusside.
  • R includes polypeptides (the term "polypeptide” includes proteins and also polyamino acids that do not possess an ascertained biological function, and derivatives thereof); amino acids (including natural and synthetic amino acids and their stereoisomers and racemic mixtures and derivatives thereof) ,- sugars,- modified and unmodified oligonucleotides (preferably of at least 5, and more particularly 5-200, nucleotides) ,- and a hydrocarbon where the hydrocarbon can be a branched or unbranched, and saturated or unsaturated aliphatic hydrocarbon, or an aromatic hydrocarbon; hydrocarbons having one or more substituent groups,- and heterocyclic compounds.
  • R is preferably a nucleophilic (basic) moiety.
  • M+ is a metal cation, such as, for example, a Group I metal
  • thionitrates which have the structure R-(S) x -NO wherein x is an integer of at least 2.
  • R is as described above for the S-nitrosothiols.
  • dithiols wherein x is 2.
  • Particularly preferred are those compounds where R is a polypeptide or hydrocarbon and a par or pairs of thiols are sufficiently
  • agents which stimulate endogenous NO synthesis will be suitable.
  • Viruses whose replication may be inhibited or prevented by the compounds described hereinabove include, but are not limited to, Epstein-Barr Virus, Herpes Simplex Virus, cytomegalovirus (CMV) , varicella, and other viruses associated with sexually transmitted diseases, or skin, eye, mouth, lymphoid, or other organ infections in immune- competent and immune-compromised hosts.
  • CMV cytomegalovirus
  • the compounds also may be employed in inhibiting or preventing the replication of viruses which have been implicated in the development of several malignancies, including Burkitt's lymphoma, and nasopharyngeal carcinoma.
  • the hereinabove mentioned compounds may be employed in various therapeutic treatments, including: l) the incorporation of NO donors into creams, salves or lotions for the treatment of topical infections; 2) the use of inhaled nitric oxide and other NO donors for the treatment of lung infections,- 3) the use of topical drops and creams for the treatment of mucocutaneous membrane infections, such as infections of the eye, mouth and genitourinary tract; and 4) oral or parenteral administration for systemic or localized infections.
  • a method of inhibiting or preventing cellular apoptosis which comprises administering to cells an effective amount of nitric oxide.
  • the nitric oxide may be administered to cells in vitro or in vivo.
  • the nitric oxide which is administered may be uncharged nitric oxide (NO*) , which may be in the form of gaseious nitric oxide, nitroxyl ion (NO " ) , or nitrosonium ion (NO*) , as hereinabove described.
  • NO* uncharged nitric oxide
  • NO " nitroxyl ion
  • NO* nitrosonium ion
  • such administration of nitric oxide may be achieved by administering a compound capable of donating or releasing nitric oxide in one of its reactive forms, also as hereinabove described.
  • this aspect is applicable to the inhibition of apoptosis of lymphocytes.
  • the nitric oxide When administered in vivo, the nitric oxide may be administered in combination with pharmaceutical carriers and in dosages described herein.
  • Another aspect of the invention is based on the discovery that nitric oxide synthase inhibitors activate latent virus. This is the first demonstration of a mechanism for eradicating the latent state, i.e., once activated, the virus can then be eliminated with anti-viral therapy. Thus, such aspect of the present invention is directed to the use of nitric oxide synthase inhibitors as a means to eradicate latent virus.
  • the invention provides a method for the treatment of a latent virus infection in an animal (preferably a mammal, and more preferably a human) in need thereof, which comprises administering (i) a virus latency preventing or reversing amount of a nitric oxide synthase inhibitor to render the virus replicative and (ii) a virus replication inhibitory amount of nitric oxide or a nitric oxide-releasing, donating, or transferring substance.
  • Latent virus infections which may be treated with such method, include, but are not limited to, infections associated with Epstein-Barr Virus.
  • latent viruses such as, for example, Epstein-Barr Virus
  • such viruses may serve as protooncogenes leading to cancer.
  • Epstein-Barr Virus latent infection is associated with lymphoma (Burkitt's, Hodgkin's, and non- Hodgkin's), and nasopharyngeal carcinoma.
  • lymphoma lymphoma
  • Hodgkin's Hodgkin's
  • non- Hodgkin's non- Hodgkin's
  • nasopharyngeal carcinoma nasopharyngeal carcinoma.
  • the invention provides a composition for the treatment of a latent virus infection in an individual in need thereof, which composition comprises (i) a virus latency preventing or reversing amo'.int of a nitric oxide synthase inhibitor to render the virus replicative and (ii) a virus replication inhibitory amount of nitric oxide or a nitric oxide-releasing, donating, or transferring substance, in a pharmaceutically acceptable carrier.
  • Suitable nitric oxide synthase inhibitors include arginine derivatives such as N°-monomethyl-L-arginine (NMA) , nitro-arginine, diphenylene iodonium and related iodonium derivatives, N-nitro-L-arginine methyl ester, N-methyl-L- arginine, N-amino-L-arginine, ornithine, N-imino-ethyl-L- ornithine and ornithine derivatives, N-nitro-L-arginine, methylene blue, heme binders, trifluoropiperazine, and calcinarin and other calmodulin binders, such as methotrexate.
  • NMA N°-monomethyl-L-arginine
  • nitro-arginine diphenylene iodonium and related iodonium derivatives
  • N-nitro-L-arginine methyl ester N-methyl-L-
  • Treatment of localized topical Herpes infection with th nitric oxide or nitric oxide releasing agent comprise contacting the surface so as to cause the surface to b coated with the nitric oxide or nitric oxide releasing agent.
  • Coating may be accomplished using standard methods well know to those of ordinary skill in the art. For example, coatin a surface with nitric oxide adducts can be achieved b bathing the surface in a solution containing the nitric oxid adduct.
  • synthetic nitric oxide adducts may be coated onto an artificial surface such as a bandage o covering by a variety of chemical techniques which are well known in the art.
  • Major therapeutic uses include: topical to treat skin infections; topical to treat genital and oral lesions,- topical to treat eye infections (cytomegalovirus) ,- topical NO/or SNAC to treat respiratory infections; and intravenous to treat systemic infection.
  • Modes of administration include but are not limited to transdermal, intramuscular, intraperitoneal, intravenous, vaginal, subcutaneous, intranasal, topical and oral routes.
  • the compounds may be administered by any convenient route, for example by infusion or bolus injection by absorption through epithelial or mucocutaneou ⁇ linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.) and may be administered together with other biologically active agents.
  • the compounds of this invention can be employed in combination with conventional excipients, i. e., pharmaceutically acceptable organic or inorganic carrier substances suitable for parenteral application which do not deleteriously react with the active compounds.
  • suitable pharmaceutically acceptable carriers include, but are not limited to, water, salt solutions, alcohol, vegetable oils, polyethylene glycols, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, perfume oil, fatty acid monoglycerides and diglycerides, petroethral fatty acid esters, hydroxymethy1cellulose, polyvinylpyrrolidone, etc.
  • the pharmaceutical preparations can be sterilized and if desired, mixed with auxiliary agents, e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, colorings, flavoring and/or aromatic substances and the like which do not deleteriously react with the active compounds.
  • auxiliary agents e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, colorings, flavoring and/or aromatic substances and the like which do not deleteriously react with the active compounds.
  • auxiliary agents e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, colorings, flavoring and/or aromatic substances and the like which do not deleteriously react with the active compounds.
  • particularly suitable vehicles consist of solutions
  • the composition can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents.
  • the composition can be a liquid solution, suspension, emulsion, tablet, pill, capsule, sustained release formulation, or powder.
  • the composition can be formulated as a suppository, with traditional binders and carriers such as triglycerides.
  • Oral formulations can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc.
  • Various delivery systems are known and can be used to administer a therapeutic compound or composition of the invention, e.g., encapsulation in liposomes, microparticles, microcapsules and the like.
  • the composition is formulated in accordance with routine procedures as a pharmaceutical composition adapted for intravenous administration to human beings.
  • compositions for intravenous administration are solutions in sterile isotonic aqueous buffer.
  • the composition may also include a solubilizing agent and a local anesthetic to ameliorate any pain at the site of the injection.
  • the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a drylyophilized powder or water free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent.
  • the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline.
  • an ampoule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration.
  • the therapeutics of the invention can be formulated as neutral or salt forms.
  • Pharmaceutically acceptable salts include those formed with free amino groups such as those derived from hydrochloric, phosphoric, sulfuric, acetic, oxalic, tartaric acids, etc., and those formed with free carboxyl groups such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol, histidine, procaine, etc.
  • terapéuticaally effective amount refers to the amount of the nitric oxide adduct which is effective to achieve its intended purpose. While individual needs vary, determination of optimal ranges for effective amounts of each nitric oxide adduct is within the skill of the art. Generally, the dosage required to provide an effective amount of the composition, and which can be adjusted by one of ordinary skill in the art will vary, depending on the age, health, physical condition, sex, weight, extent of disease of the recipient, frequency of treatment and the nature and scope of the disorder.
  • the amount of the therapeutic of the invention which will be effective in the treatment of a particular disorder or condition will depend on the nature of the disorder or condition, and can be determined by standard clinical techniques. The precise dose to be employed in the formulation will also depend on the route of administration, and the seriousness of the disease or disorder, and should be decided according to the judgment of the practitioner and each patient's circumstances.
  • suitable dosage ranges for intravenous administration are generally about 20- 500 micrograms of active compound per kilogram body weight.
  • Suitable dosage ranges for intranasal administration are generally about 0.01 pg/kg body weight to 1 mg/kg body weight.
  • Effective doses may be extrapolated from dose- response curves derived from in vitro or animal model test systems and are in the same ranges or less than as described for the commercially available compounds in the Physician's Desk Reference, Medical Economics Company, Inc., Oradell, N.J. 1990.
  • the invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the pharmaceutical compositions of the invention.
  • a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the pharmaceutical compositions of the invention.
  • Associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration.
  • FIG. 1 EBV-infected B cell lines and EBV-negative Burkitt lymphoma cell lines express iNOS messenger RNA consitutively.
  • cDNA was made from whole cell RNA from the Akata (AK) , BJAB (BJ) and B-958 (B9) cell lines. (+) indicates samples to which reverse transcriptase was added, and (-) indicates control samples to which no reverse transcriptase was added. Equal amounts of
  • ⁇ 20- each cDNA sample was PCR amplified using primers specific for human iNOS.
  • the markers on the left indicate the base pair size of OX DNA fragments generated from a Hae III digest.
  • Figure 2 Reactivation of latent EBV by the NOS inhibitor L-NMA and inhibition of EBV replication by the NO donor SNOP.
  • A Immunoblot analysis of Akata cells grown in fully enriched media containing 0.25 mM L- arginine in the presence or absence (control) of LNMA blotted with a human polyclonal antiserum recognizing EBV early lytic antigens.
  • P135 is a DNA binding protein and EAs are the BMRF- 1 and BMLF-1 antigens expressed during EBV early lytic infection.
  • FIG. 3 The effect of the NOS inhibitor L-NMA and acyclovir on the generation of EBV-transformed lymphoblastoid cell lines.
  • EBV-negative lymphocytes were infected with EBV and grown in complete media containing 0. 1 mM arginine (control) , or the same media containing 1 mM L-NMA (NMA) , 5 ug/ l acycloguanisine (Acyclovir) or 1 mM L-NMA and 5 ug/mi acycloguanisine (N+A) .
  • Akata cells were grown for 5- 7 days in media containing 0.25 mM arginine and 0, 0.5 or 5 M L-NMA added as a single dose on day 1. The percentage of apoptotic cells was determined by acridine orange staining. The data are expressed as the mean of 7 separate experiments +/- SEM.
  • BJAB Melezes et al . , Biomedicine. Vol. 22, pgs. 276-284 (1975), BL-30, and BL-41 are EBV-negative Burkitt lymphoma cell lines.
  • BL-41 /B-958 is a clone stably infected with EBV after in vitro infection (Calender et al . , Proc. Nat. Acad. Sci.. Vol. 84, pgs. 8060-8064 (1987)).
  • LCL 1.7 is a lymphoblastoid cell line latently infected with a recombinant EBV. The cell lines were maintained in complete medium consisting of RPMI 1640 (Sigma) supplemented with 10% fetal bovine serum, 2 mM glutamine and 10 ⁇ g/ml gentamycin.
  • the calcium ionophore A23187 (Sigma) was diluted in ethanol to create a 5mM stock solution.
  • Penicillamine (Sigma) was diluted in 0.5 N HCl to create a 200 mM stock solution.
  • One hundred mM stock solutions of SNOP were synthesized immediately prior to adding them to cell cultures by combining equal volumes of 200mM NaN0 2 diluted in H 2 0 with 200 mM penicillamine diluted in 0.5 N HCl.
  • SNOP (Sigma) was diluted in H 2 0 to create a 100 mM stock solution. All of the above stock solutions were diluted at least 1:10 in phosphate buffered saline prior to being added to cell cultures and pH changes were carefully excluded.
  • L-NMA (Calbiochem) was diluted in H20 to create a 100 mM stock solution which was added directly to cell cultures.
  • Acycloguanisine (Sigma) was diluted in 25 mM NAOH to create a 5 mg/ml stock solution.
  • RNAzolTM method CINNA/BIOTECX
  • First strand cDNA was made from l ⁇ g of RNA using Superscript 11 reverse transcriptase (GIBCO/BRL) and random hexamers (25 pmoles) as primers (Perkin Elmer Cetus) , following the 5' RACE protocol (GIBCO/BRL) .
  • Three ⁇ l (out of a total of 20 ⁇ l) of the resulting cDNA was PCR amplified in 25 ⁇ l reactions using primers specific for human iNOS corresponding to nucleotides 212-231 (CTGTCCTTGGAAATTTCTGTT) and 702-681 (TGGCCAGATGTTCCTCTATT) of the human hepatic iNOS cDNA.
  • the PCR conditions were 15 seconds denaturation at 95°C, 30 seconds annealing at 57°C, and 75 seconds extension at 72°C for 35 cycles using a GeneAmp 9600 PCR system (Perkin Elmer Cetus) .
  • the amplified products were analyzed on a 2% Nusieve agarose GTG (FMC) , 1 % Sea Kem ME agarose (FMC) gel containing 0.5 ⁇ g/ml ethidium bromide.
  • NOS activity was defined as the L-NMA (1 00 ⁇ M, Calbiochem, San Diego, CA) inhibitable rate of conversion of L-arginine to L-citrulline by cell lysates, as previously described (Bredt et al . , 1991).
  • harvested cells were sonicated in 50 mM Tris HCl buffer (pH 7.7) containing 1 mM EDTA, 10 ⁇ g/ml leupeptin, lOug/ml antipain, 10 ⁇ g/ml pepstatin, 10 ⁇ g/ml chymostatin, 10 ⁇ g/ml chymotrypsin- trypsin inhibitor, and lOOug/ml phenylmethylsulfonyl fluoride.
  • the reaction was stopped by the addition of 9 volumes of a solution containing 20 mM Na acetate, 1 mM citrulline, 2 mM EDTA, and 2 mM EGTA.
  • the mixture was applied to an AG 50W-X8 resin column (Bio-Rad) and L- [2,3,4,53H] citrulline was eluted with H 2 0. NOS activity was normalized to the protein concentration, determined by Bradford's method (Bradford, Anal. Biochem.. Vol. 72, pgs. 248-254 (1976)).
  • EBV-negative peripheral blood or umbilical cord blood mononuclear cells were infected with stocks of wild- type recombinant EBV generated from the P3HR-1 cell line as described previously (Mannick et al . , J. Virol.. Vol. 65, pgs. 6826-6837 (1991)).
  • One ml of a given EBV stock in complete medium was used to infect 3Oxl0 6 mononuclear cells for 1-2 hour at 37°C.
  • the cells were washed and resuspended in 15 mis of complete medium containing 0.1 mM L-arginine with or without 1 mM L-NMA and/or 5 ⁇ g/ml acycloguanisine (acyclovir) , plated at a concentration of 2x10* cells/ml in 96 well plates and fed twice weekly for 3 weeks with the same medium in which they were originally plated. After 3 weeks, the cells were fed once per week with complete medium without added drug. Four to six weeks after infection, the number of transformed clones obtained was determined by counting the number of wells in each 96 well plate containing macroscopic clumps of cells.
  • EBV replication was analyzed by immunofluorescent staining and Western blotting as described below and apoptosis was analyzed by acridine orange staining (5 ⁇ g/ml) using a Zeiss Axioskop equipped with epifluorescence.
  • Akata cells were grown to approximately 500,000 cells/ml in complete medium and were plated in 24 well plates containing 1 ml of cells/well. Penicillamine, S-nitroso- penicillamine (SNOP) or SNP at a final concentration of 0.1- 1.0 mM was added to the appropriate wells. Immediately after adding these reagents, EBV reactivation was induced by cross linking cell slg with 100 ⁇ g/mi F(ab') 2 goat-antihuman Ig (Jackson Immunological) (Takada, 1984; Daibata et al . , 1990) . After 24 hours, EBV replication was assessed by immunofluorescent staining and Western blotting as described below.
  • Penicillamine, SNOP or SNP was added at a final concentration ranging from 0. 1 - 1 mM to 1 ml cultures of Akata cells growing logarithmically in complete media in 24 well plates. Immediately after adding these reagents, A23187 was added at a final concentration of 2.5 ⁇ M. After 24 hours, the percentage of apoptotic cells was determined by staining the cells with acridine orange as described above.
  • BL-41 and BL-30 cells growing logarithmically in complete media containing l mM L-arginine were resuspended in complete media containing 0.1 mM L-arginine and plated in 24 well plates with 2 ml of cells per well.
  • Penicillamine, SNOP and SNP were added to the appropriate wells atconcentrationsranging from 0.1-1 .0 mM every 24 hours for 4 days starting at time 0.
  • the percentage of apoptotic cells was determined by acridine orange staining as described above.
  • Human B cell lines were analyzed for expression of iNOS by reverse transcription of RNA followed by DNA polymerase chain reaction (RT PCR) .
  • RT PCR DNA polymerase chain reaction
  • the expected 491 bp PCR product was detected on ethidium stained agarose gels using human iNOS-specific primers ( Figure 1) .
  • the PCR product from one of the cell lines (BJAB) was sequenced and found to be identical to the human hepatic iNOS sequence (data not shown) (Geller et al . , Proc. Nat. Acad. Sci., Vol. 90, pgs. 3491-3495 (1993)) .
  • NOS activity in a variety of B cell lines was assayed by monitoring the conversion of [3H] -arginine to [3H] -citruiline (Bredt et al . , Nature, Vol. 351, pgs. 714-718 (1991)). The results of such assay are shown in Table l below.
  • NOS activity was determined by monitoring the conversion of [ 3 H] -arginine to [ 3 H] -citrulline and is expressed s pmoles of L-NMMA inhibitable [ 3 H] -citrulline produced per mg of cellular protein per minute. ND indicates no detectable activity
  • B958 cells had the highest NOS activity.
  • Low NOS activity was detected in the EBV negative human Burkitt lymphoma cell line BJAB and in the EBV- converted Burkitt lymphoma cell line BL 41 / B-958. NOS activity was not demonstrable in the other B cell lines, consistent with the lower levels of RT PCR products from these cells.
  • endogenous NO Production Inhibits Reactivation of Latent EBV Infection Since endogenous NO is produced from L-arginine, and L- arginine deficient media can increase EBV replication in EBV- positive Burkitt lymphoma cell lines (Henle and Henle, J. Virol.. Vol. 2, pgs. 182-191 (1968)) , we investigated whether the effect of L-arginine depletion on the stimulation of EBV replication could be mediated by a reduction in NOS activity.
  • Endogenous NO levels were pharmacologically lowered using the NOS inhibitor N monomethyl-L-arginine (L- ⁇ MA) and the effect on lytic infection was analyzed by indirect immunofluorescent staining using monoclonal antibodies to the EBV xmmediate early lytic infection protein, BZLF-l (Young et al . , . Virol.. Vol. 65, pgs. 2868-2874 (1991)) or the late lytic infection EBV protein BALF3 (gp 110) (Kishishita et al . , Virology, Vol. 133, pgs. 363-375 (1984) .
  • L- ⁇ MA administered as a one-time dose of either 0.5 or 5 mM to arginine (0.25 mM) containing medium, caused a significant increase in virus replication as assessed 5 or 7 days later.
  • latent infection associated proteins may inhibit the effect of L-NMA on iNOS or the effect of NO on blocking virus replication.
  • L-NMA is a specific NOS inhibitor (Gross et al . , Biochem. Biophys. Res. Commun.. Vol. 170, pgs. 96-103 (1990)
  • L-NMA or iNOS could theoretically affect EBV replication through an NO-independent mechanism. If the effect of L-NMA is mediated by inhibition of NO synthesis, exogenous NO would be expected to decrease EBV replication.
  • Virus replication was induced in the Akata cell line by crosslinking slg (Takada, 1984,- Daibata et al . , J . Immunol. , Vol. 144, pgs.
  • SNOP NO donor S-nitroso-penicillamine
  • penicillamine the control thiol compound penicillamine.
  • virus replication was determined by indirect immunofluorescence or by immunoblotting for BZLF-l or early lytic infection proteins as described above.
  • SNOP but not the control thiol, inhibited EBV BZLF1 and early lytic protein expression in a dose dependent manner over the range of 100 / ⁇ M to l M.
  • SNOP showed no significant toxicity to cells when compared with thiol controls as measured by trypan blue exclusion.
  • the NO donor sodium nitroprusside (SNP) had similar inhibitory effects on EBV early lytic protein expression in Akata cells after crosslinking slg (data not shown) .
  • L-NMA might increase EBV replication, and thereby increase the number of transformed cells.
  • Adult peripheral blood or umbilical cord blood mononuclear cells were infected with EBV and then grown in the presence or absence of L-NMA.
  • Pathways which can trigger EBV replication such as slg crosslinking, ionophore-induced calcium fluxes, or serum starvation, also induce apoptosis in B cell lines (Gregory et al . , Nature, Vol. 349, pgs. 612-614 (1991)).
  • B cell apoptosis was affected by NO.
  • Apoptosis was induced in the Akata cell line using the calcium ionophore A23187 at a concentration of 2.5 ⁇ M.
  • the NO donors SNOP and SNP were added to cultures concurrently with A23187 and the percentage of cells undergoing apoptosis was determined by acridine orange staining after 18-24 hours.
  • L-NMA Endogenous NO Production By Human B Cells Inhibits Apoptosis
  • the effect of penicillamine on apoptosis may be due to an antioxidant effect of the thiol (Sandstrom et al . , 1994) .
  • the additive effects of both the thiol and NO components of SNOP on apoptosis may have physiological relevance as S- nitrosothiols are endogenously formed (Stamler et al . , Science, Vol. 258, pgs.
  • Akata cells were induced to replicate virus by ⁇ lg crosslinking or were treated with 2.5 uM A23187 to induce apoptosis in the presence of the indicated concentrations of 8-bromo-cGMP or SNOP.
  • the percentage of cells expressing the BZLF-l protein was determined by immunofluorescence staining using an anti- BZLF-1 monoclonal antibody and the percentage of apoptotic cell ⁇ wa ⁇ determined by acridine orange staining. The numbers represent the means of two separate experiments +/- SEM.

Abstract

L'invention se rapporte à un procédé d'inhibition de la réplication d'un virus consistant à soumettre le virus à l'oxyde nitrique ou à une substance libérant, délivrant ou transférant de l'oxyde nitrique, et notamment à administrer une quantité, inhibant la réplication du virus, d'oxyde nitrique ou d'une substance libérant de l'oxyde nitrique à un patient infecté par un virus. L'invention se rapporte également à un procédé inhibant ou inversant la latence d'un virus en soumettant le virus latent à un inhibiteur de synthase de l'oxyde nitrique, et notamment à un procédé de traitement d'une infection due au virus latent consistant à administrer à un patient (i) une quantité suffisante, inhibant ou inversant la latence du virus, d'inhibiteur de synthase de l'oxyde nitrique pour que le virus se réplique, puis (ii) une quantité, inhibant la réplication du virus, d'oxyde nitrique ou d'une substance libérant de l'oxyde nitrique, et une composition de (i) et (ii) appliquée dans ce traitement, et (iii) une quantité prophylactique de NO(ii) pour empêcher le virus latent de se répliquer.
PCT/US1995/008763 1994-07-15 1995-07-13 Inhibition d'un virus par l'oxyde nitrique WO1996002268A1 (fr)

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Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6232434B1 (en) 1996-08-02 2001-05-15 Duke University Medical Center Polymers for delivering nitric oxide in vivo
WO2003018102A2 (fr) 2001-08-29 2003-03-06 Vectura Limited Dispositif d'administration topique
US6709681B2 (en) 1995-02-17 2004-03-23 Aberdeen University Acidified nitrite as an antimicrobial agent
DE10303196A1 (de) * 2003-01-28 2004-08-12 Märtz, Hans Helmut Verwendung von Erzeugnissen, Stoffen oder Stoffgemischen, aus welchen im menschlichen oder tierischen Körper Stickoxid (Formel: NO) entsteht, zur Behandlung oder Prophylaxe von Infektionskrankheiten des menschlichen oder tierischen Körpers
WO2004087212A2 (fr) * 2003-04-03 2004-10-14 Aga Ab Utilisation d'oxyde nitrique dans le traitement de l'inflammation
WO2006127482A1 (fr) * 2005-05-20 2006-11-30 Bioenvision, Inc. Therapie au bleu de methylene de maladie virale
WO2010017633A1 (fr) * 2008-08-13 2010-02-18 Chris Miller Utilisation de monoxyde d'azote
US20100331404A1 (en) * 2005-08-17 2010-12-30 Rush University Medical Center Vector Delivery-Based Microbicides

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
J. CLIN. INVEST., Volume 91, No. 6, issued June 1993, KENNETH D. CROEN, "Evidence for an Antiviral Effect of Nitric Oxide", pages 2446-2452. *
SCIENCE, Volume 261, issued 10 September 1993, G. KARUPIAH et al., "Inhibition of Viral Replication by Interferon-gamma-Induced Nitric Oxide Synthase", pages 1445-1448. *

Cited By (16)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6709681B2 (en) 1995-02-17 2004-03-23 Aberdeen University Acidified nitrite as an antimicrobial agent
US6875840B2 (en) 1996-08-02 2005-04-05 Duke University Polymers for delivering nitric oxide in vivo
US7417109B2 (en) 1996-08-02 2008-08-26 Duke University Polymers for delivering nitric oxide in vivo
US6673891B2 (en) 1996-08-02 2004-01-06 Duke University Polymers for delivering nitric oxide in vivo
US6403759B2 (en) 1996-08-02 2002-06-11 Duke University Polymers for delivering nitric oxide in vivo
US6232434B1 (en) 1996-08-02 2001-05-15 Duke University Medical Center Polymers for delivering nitric oxide in vivo
US7087709B2 (en) 1996-08-02 2006-08-08 Duke University Polymers for delivering nitric oxide in vivo
WO2003018102A2 (fr) 2001-08-29 2003-03-06 Vectura Limited Dispositif d'administration topique
DE10303196A1 (de) * 2003-01-28 2004-08-12 Märtz, Hans Helmut Verwendung von Erzeugnissen, Stoffen oder Stoffgemischen, aus welchen im menschlichen oder tierischen Körper Stickoxid (Formel: NO) entsteht, zur Behandlung oder Prophylaxe von Infektionskrankheiten des menschlichen oder tierischen Körpers
WO2004087212A2 (fr) * 2003-04-03 2004-10-14 Aga Ab Utilisation d'oxyde nitrique dans le traitement de l'inflammation
WO2004087212A3 (fr) * 2003-04-03 2004-12-16 Aga Ab Utilisation d'oxyde nitrique dans le traitement de l'inflammation
WO2006127482A1 (fr) * 2005-05-20 2006-11-30 Bioenvision, Inc. Therapie au bleu de methylene de maladie virale
US20100331404A1 (en) * 2005-08-17 2010-12-30 Rush University Medical Center Vector Delivery-Based Microbicides
US9393233B2 (en) * 2005-08-17 2016-07-19 Rush University Medical Center Vector delivery-based microbicides
US9603841B2 (en) 2005-08-17 2017-03-28 Rush University Medical Center Vector delivery-based microbicides
WO2010017633A1 (fr) * 2008-08-13 2010-02-18 Chris Miller Utilisation de monoxyde d'azote

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