WO1996002268A1 - Inhibition of virus by nitric oxide - Google Patents

Abstract

A method for inhibiting the replication of a virus by exposing the virus to nitric oxide or a nitric oxide releasing, delivering or transferring substance, particularly administering a virus replication inhibitory amount of nitric oxide or a nitric oxide releasing substance to an individual having a virus infection. A method for preventing of reversing latency in a virus by exposing the latent virus to a nitric oxide synthase inhibitor, particularly a method for the treatment of a latent virus infection in an individual by administering (i) a virus latency preventing or reversing amount of a nitric oxide synthase inhibitor sufficient to render the virus replicative and then (ii) a virus replication inhibitory amount of nitric oxide or a nitric oxide-releasing substance and a composition of (i) and (ii) for such treatment, (iii) a prophylactic amount of NO(ii) to prevent latent virus from becoming replicative.

Description

INHIBITION OF VIRUS BY NITRIC OXIDE

There is extensive data on the importance of 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.

Certain viruses can both replicate (lytic phase) and also incorporate themselves into the genome (latency) . One associates infectious disease (e.g., genital Herpes (HSVII) or ononucleosis (EBV) ) with the replicative cycle of the virus; however, significant morbidity may be associated with the latent phase as well. Further, certain viruses tend to establish latency more readily than others. When latent, the virus may serve as a protooncogene, thereby promoting cancer; and it is also resistant to anti-viral therapy. Thus, the establishment of latency prevents elimination of virus with anti-viral therapy.

RECTi .) ^'1 .. ?1. An aspect of the invention is based on the discovery b the inventors that 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. In a preferred embodiment of this aspect, 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. In a preferred embodiment, 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.

The term 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) . Thus, 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.

•2-

RECT!.!.- -1. In one embodiment, the reactive form of nitric oxide is provided by gaseous nitric oxide.

In another embodiment, 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.

It is also contemplated within the scope of the present invention that 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 itπmunocompromised host in which reactivation at an inopportune time (i.e. when defenses are low due to chemotherapy) would cause significant morbidity.

Compounds contemplated for use in the invention are 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. As used here, the term "nitric oxide" encompasses uncharged nitric oxide(NO*) and charged nitric oxide species, particularly including nitrosonium ion(NO^*) and nitroxyl ion(NO") . The 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. As used here, the term "NO adducts" encompasses any of such nitric oxide, releasing, delivering or transferring compounds.

One group of such NO adducts is the S-nitrosothiols, which are compounds that include at least one -S-NO group.

-3- fiEC-TirJl- T - "- ^{~ ~} 'p' - r <^■■•^• 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-nitrosothiols and the methods fo preparing them are described in U.S. Patent Application No. 07/943,834, filed September 14, 1992, Oae et al . , Org. Prep. Proc. Int., 15.(3) :165-198 (1983); Loscalzo et al. , J Pharmacol. Exp. Ther., 249(3) :726729 (1989) and Kowaluk e al . , J. Pharmacol. Exp. Ther., 251:1256-1264 (1990), all o which are incorporated in their entirety by reference.

One particularly preferred embodiment of this aspec relates to S-nitroso amino acids where the nitroso group i linked to a sulfur group of a sulfur-containing amino acid o derivative thereof. For example, 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

-4-

E 91 such as hemoglobin and serum albumin; and biologically protective proteins, such as the immunoglobulins and the cytokines. Such nitrosylated proteins are described in PCT Publ. Applic. No. WO 93/09806, published may 27, 1993.

Further examples of suitable S-nitrosothiols include those having the structures:

(i) CH₃(CH₂)-SNO wherein x equals 2 to 20;

(ii ) HS (CH₂) _xSN0 wherein x equals 2 to 20 ; and

( iii ) 0NS (CH₂) .Y wherein x equals 2 to 20 and Y is selected from the group consisting of fluoro, C,-C₆ alkoxy, cyano, carboxamido, C₃-C₆ cycloalkyl, aralkoxy, C₂-C₆ alkylsulfinyl, arylthio, C,-C₆ alkylamino, C₂-C,j dialkylamino, hydroxy, carbamoyl, C,-C₆ N- alkylcarbamoyl, Cj-C.j N,N-dialkylcarbamoyl, amino, hydroxyl, carboxyl, hydrogen, nitro and aryl; wherein aryl includes benzyl, naphthyl, and anthracenyl groups.

Other suitable 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. Examples of such S-nitroso-ACE inhibitors include compounds having structure (1) :

(1)

wherein

R is hydroxy, NH₂, NHR⁴, NR⁴R⁵, or C,-C₇ alkoxy, wherein R⁴ and R⁵ are C,-C₄ alkyl, or phenyl, or Cι~C₄ alkyl substituted by phenyl;

R¹ is hydrogen, C,-C₇ alkyl, or C,-C₇ alkyl substituted by phenyl, amino, guanidino, NHR⁶, NR⁶R⁷, wherein R⁶ and R⁷ are methyl or C,-C₄ alkanoyl;

R² is hydrogen, hydroxy, C,-C₄ alkoxy, phenoxy, or

C,-C₇ alkyl;

R³ is hydrogen, C,-C₄ or C,-C₇ alkyl substituted by phenyl; m is 1 to 3; and n is 0 to 2.

Other suitable 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) -mercaptomethyl- pentanoyl -L-proline, and S-nitroso-L-cysteinyl-L-proline. Additional suitable S-nitroso-ACE inhibitors include those having structures (2-3) :

wherein

X is oxygen or sulfur,-

-A,,-A₂- is CH-NH or -C=N-;

R₃ 0 II³ II A is ON-S-CH₂-CH-C;

R is selected from hydrogen, lower ( -C₄) alkyl, benzyl, benzhydryl, -and salt forming ion;

R, and R₂ are independently selected from hydrogen, halogen, lower alkyl, lower alkoxy, halo substituted lower alkyl, nitro, and Sθ₂NH₂;

0 0 .0

Z is -C- or -S-

R₃ is hydrogen, lower alkyl, halo substituted lower alkyl, phenyl, benzyl, phenethyl, or cycloalkyl; and

* is hydrogen, lower alkyl, halo substituted lower alkyl, hydroxy substituted lower alkyl, -(CH₂)„-N (lower alkyl)₂ or -(CH₂)_q-NH₂ and q is one, two, three or four. Additional suitable compounds include those having structures (4-11) :

The S-nitroso-ACE inhibitors can be prepared by various methods of synthesis. In general, the thiol precursor is prepared first, then converted to the S-nitrosothiol derivative by nitrosation of the thiol group with NaN0₂ under acidic conditions (pH = 1 to 5) which yields the S-nitroso derivative. 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 . ; U.S. Pat. No. 4,626,545 (1986) to Taub; and U.S. Pat. Nos. 4,692,458 (1987) and 4,692,459 (1987) to Ryan et al . , Quadro, U.S. Pat. No. 4,447,419 (1984); Haugwitz et al . ; U.S. Pat. No. 4,681,886 (1987), Bush et al. , U.S. Pat. No. 4,568,675 (1986), Bennion et al. , U.S. Pat. No. 4,748,160 (1988), Portlock, U.S. Pat. No. 4,461,896 (1984), Hoefle et al . , European Patent Application Publication No. 0 088 341 (1983), Huange et al . , U.S. Pat. No. 4,585,758 (1986), European Patent application Publication No. 0 237 239, European Patent application Publication No. 0 174 162, published in 1986, European Patent application Publication No. 0 257 485, published in 1988, all of which are incorporated by reference herein.

Another group of such NO adducts are compounds that include at least one -O-NO group. 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

^■9- or more substituent groups in addition to the O-nitros group; and heterocyclic compounds.

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. 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.

Another group of such NO adducts is the N-nitrosoamineε 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

Another group of such NO adducts is the 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

-10- nrr-r - , ~ _r c '_!' " ^* ' ^■ <: . " derivatives thereof) ; C-nit.rosylated amino acids (including natural and synthetic amino acids and their stereoisomers and racemic mixtures) ,- C-nitrosated sugars,- C-nitrosated-modified and unmodified oligonucleotides (preferably of at least 5, and more particularly 5-200, nucleotides) ; and 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.

Another group of such NO adducts is the nitrates which have at least one -0-N0₂ group. Such compounds include 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. A preferred example is nitroglycerin.

Another group of such NO adducts is the nitroso-metal compounds which have the structure (R)_n-A-M- (NO)_x. 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

-11- ^c~x- -'X SH.::^': x-x 9i) 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 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, and M is a metal, preferably a transition metal. Preferred metals include iron, copper, manganese, cobalt, selenium and luthidium. Also contemplated are N-nitrosylated metal centers such as nitroprusside.

Another group of such NO adducts is the N-oxo-N- nitrosoamines which have an R-N(0^"M^*)-N0 group or an R-NO-NO- group. 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 cation.

Another group of such NO adducts is the 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. Preferred are the 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

-12- ^c,i-cτι-,.-;. :ιi[^τ -F.J.E 9i) structurally proximate, i.e. vicinal, that the pair of thiols will be reduced to a disulfide. Those compounds which form disulfide species release nitroxyl ion(NO^") and uncharged nitric oxide(NO») . Those compounds where the thiol groups are not sufficientl close to form disulfide bridges generally only provide nitric oxide as the NO^" form but not as the uncharged NO* form.

Also, 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. 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. Thus, 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.

The above compounds also have been found to be effective in preventing cellular apoptosis. Thus, in accordance with another aspect of the present invention, there is provided 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. In addition, 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.

In particular, this aspect is applicable to the inhibition of apoptosis of lymphocytes.

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.

Thus, in another aspect, 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. When latent viruses (such as, for example, Epstein-Barr Virus) infect an individual, such viruses may serve as protooncogenes leading to cancer. For example, Epstein-Barr Virus latent infection is associated with lymphoma (Burkitt's, Hodgkin's, and non- Hodgkin's), and nasopharyngeal carcinoma. Thus, the above method is applicable particularly to the prevention and treatment of cancerous growths.

In yet another aspect, 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.

-15- 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. In addition, 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 mucocutaneouε 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. For parenteral application, particularly suitable vehicles consist of solutions, preferably oily or aqueous solutions, as well as suspensions, emulsions, or implants. Aqueous suspensions may contain substances which increase the viscosity of the suspension and include, for example, sodium carboxymethyl cellulose, sorbitol, and/or dextran. Optionally, the suspension may also contain stabilizers.

The composition, if desired, 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.

In a preferred embodiment, the composition is formulated in accordance with routine procedures as a pharmaceutical composition adapted for intravenous administration to human beings. Typically, compositions for intravenous administration are solutions in sterile isotonic aqueous buffer. Where necessary, the composition may also include a solubilizing agent and a local anesthetic to ameliorate any pain at the site of the injection. Generally, 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. Where the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. Where the composition is administered by injection, 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.

The term "therapeutically effective amount," for the purposes of the invention, 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. However, 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. 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.

The invention will now be described with respect to the drawings, described hereinbelow.

Figure l. 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. (B) Western blot of whole cell lysates of Akata cells grown in the presence of complete media containing 1 mM L-arginine (control) or induced to replicate virus by crosslinking surface Ig (Ig) , crosslinking surface Ig in the presence of ImM penicillamine (Ig + ImM P) or ImM S-nitrosopenicillamine (Ig + 1 mM SNOP) , and blotted with the antiserum described above. (C) Western blot of whole cell lysates of Akata cells blotted with the anti-BZLF-l monoclonal antibody BZ.l. Ponceau S staining of the blots confirmed that equivalent amounts of protein were loaded in each lane. Molecular weight markers in kilodaltons are indicated on the left.

Figure 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) . The data are expressed as the mean number of EBV transformed clones arising 4-6 weeks after infection from 4 separate experiments +/- SEM. *, p =0.04 vs control and P = 0.0 1 vs + A.

Figure 4. NO donors inhibit apoptosis, and L-NMA or L- arginine deficiency induces apoptosis in the Akata cell line

(A) . A23187, with or without penicillamine, SNOP, or SNP was added to Akata cells and after 18 hours the percentage of cells undergoing apoptosis was determined by acridine orange staining. The data are expressed as the mean of 3-7 separate experiments for each drug +/ -SEM. *,- P = 0.03 vs control (B) . 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. *, P<0.05 vs control; t, P = 0.003 vs control (C) . BL- 30 and BL-41 cells were resuspended in media containing 0.1 mM arginine. Cells were treated every 24 hours with penicillamine, SNOP or no drug and the percentage of cells undergoing apoptosis was determined after 4 days for BL-30 and after 5 days for BL-41. The data are expressed as the mean of 4 separate experiments (2 for each cell line) +/-

SEM *, P=0.03 vs control; t. P = 0.03 vs pen.

In the following example, the following materials were employed and experimental procedures were followed:

Cell Lines

Akata (Takada, 1984) and P3HR-1 clone 16 (Hestrn et al . , Nature, Vol. 295, pgε. 160-163 (1982)); Rabson et al . , J. Virol. , Vol. 44, pgs. 834-844 (1982) are EBV-positive Burkitt lymphoma cell lines. BJAB (Menezes 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 μg/ml gentamycin.

Reagents

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 NaN0₂ diluted in H₂0 with 200 mM penicillamine diluted in 0.5 N HCl. SNOP (Sigma) was diluted in H₂0 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.

RT-PCR Analysis

Whole cell RNA was prepared from a variety of B cell lines using the RNAzol™ method (CINNA/BIOTECX) . First strand cDNA was made from l μg 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 μl (out of a total of 20 μl) of the resulting cDNA was PCR amplified in 25 μl 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 μg/ml ethidium bromide.

Citruiline Assay

NOS activity was defined as the L-NMA (1 00 μM, Calbiochem, San Diego, CA) inhibitable rate of conversion of L-arginine to L-citrulline by cell lysates, as previously described (Bredt et al . , 1991). Briefly, harvested cells were sonicated in 50 mM Tris HCl buffer (pH 7.7) containing 1 mM EDTA, 10 μg/ml leupeptin, lOug/ml antipain, 10 μg/ml pepstatin, 10 μg/ml chymostatin, 10 μg/ml chymotrypsin- trypsin inhibitor, and lOOug/ml phenylmethylsulfonyl fluoride. After centrifugation at 14,000 g for 15 min at 4°C, the supernatant was incubated at 37°C for 45 min in the presence of substrates and cofactors (0.5 mM NADPH, 1 μM FAD, 1 μM FMN, 1 μM tetrahydrobiopterin (obtained from Dr.B.Schircks), 1.25 mM CaCl₂, and 10 μM of L-arginine with [2,3,4,5-³H]L-arginine, (69 Ci/mmol; Amersham) . 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 H₂0. NOS activity was normalized to the protein concentration, determined by Bradford's method (Bradford, Anal. Biochem.. Vol. 72, pgs. 248-254 (1976)).

EBV Infection and Transformation

Adult 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 3Oxl0⁶ mononuclear cells for 1-2 hour at 37°C. After virus infection, 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 μg/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.

Induction of EBV replication or apoptosis by L-NMA

Cells were grown to saturation (approximately 10⁶ cells/ml) in complete medium containing 1 mM L-arginine, diluted 1:4 in medium devoid of L-arginine for a final concentration of L-arginine of 0.25 mM, and then plated in 24 well plates containing 2 ml of cells/well. L-NMA at a final

^■25- concentration of 0.5-5 mM was added as a single dose on day 1. After a 5-7 day incubation, EBV replication was analyzed by immunofluorescent staining and Western blotting as described below and apoptosis was analyzed by acridine orange staining (5 μg/ml) using a Zeiss Axioskop equipped with epifluorescence.

Reactivation of latent EBV infection by crosslinking slg

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 μg/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.

Induction of Apoptosis by A23187

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 μM. After 24 hours, the percentage of apoptotic cells was determined by staining the cells with acridine orange as described above.

Induction of Apoptosis by L-Arginine Deprivation.

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. On day 4-5, the percentage of apoptotic cells was determined by acridine orange staining as described above.

Western Blot Analysis

Whole cell lysates were prepared as described (Alfieri et al . , 1991) , separated on SDS-polyacrylamide gels (8%) , and transferred onto nitrocellulose. The blots were incubated for 1-2 hours with an anti-BZLF-1 monoclonal antibody (BZ.l) or a human antiserum recognizing the early EBV lytic antigens, and developed using an anti-human or anti-mouse alkaline-phosphatase-conjugated antibody (Promega) .

Immunofluorescence

Cells were fixed in acetone for 15 minutes at -20°C and stained as described previously (Alfieri et al . , 1991) using monocional antibodies to the immediate early transactivator BZLF- l or the late viral protein gp 110. Fluorescein isothiocyanate (FITC) -conjugated goat-antimouse (Jackson Immunological) was used as a secondary antibody. Stained cells were visualized with a Zeiss Axioskop equipped with epiflorescence.

From the above experimental procedures, the following results were obtained.

B cells synthesize iNOS

Human B cell lines were analyzed for expression of iNOS by reverse transcription of RNA followed by DNA polymerase chain reaction (RT PCR) . In 10 out of 10 B cell lines tested, 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)) . No PCR product was detected in control reactions with RNA samples to which reverse transcriptase was not added (Figure 1) . These results demonstrate the constitutive presence of iNOS RNA in transformed human B cell lines including lymphoblastoid cell lines transformed by EBV or Burkitt lymphoma derived cell lines, some of which are not EBV- infected. The iNOS DNA fragment amplified from B-958 cDNA was much more abundant than the fragments amplified from an equivalent amount of cDNA from the other B cell lines (Figure 1) , consistent with the possibility that this marmoset cell line transformed by EBV expresses more iNOS RNA.

To determine whether the iNOS message detected by RT PCR was translated into an active enzyme, 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.

Table 1. NOS Activity In B Cell Lines

Cell Line NOS activity (pmole/mg/min)

B-958 1.74 +/- 0.56 (n=2)

BJAB 0.11 +/- 0.05 (n=6)

BL-41 ND (n=2)

BL-41/B-958 0.11 +/- 0.11 (n=2)

P3HR-1 ND (n=l)

Akata ND (n=l)

NOS activity was determined by monitoring the conversion of [³H] -arginine to [³H] -citrulline and is expressed s pmoles of L-NMMA inhibitable [³H] -citrulline produced per mg of cellular protein per minute. ND indicates no detectable activity

As shown in Table 1, 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-ΝMA) 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) . In 2 out of 7 cell lines tested (Akata and P3HR-1) , L-ΝMA, 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. After 5 or 7 days with 5 mM L-ΝMA, the percentage of lytically infected Akata cells increased 4.5 +/- 1.2 fold (mean +/- SEM) as compared to cells incubated in the absence of L-ΝMA (p .= 0.001, n = 12) . The increase in virus replication seen in the presence of L-ΝMA was confirmed by Western blot using a polyclonal human antiserum which reacts with the early lytic infection EBV proteins (Figure 2A) or using the BZLF-l monoclonal antibody (Figure 2C) . A smaller increase in virus replication was also induced by a single dose of 500 uM LΝMA (Figure 2C) . Similarly, in P3HR-1, a 5 or 7 day incubation with a single dose of 5mM L-ΝMA increased BZLF1 expression 7.7 +/- 1.75 fold (p =0.04, n =4) . These results support the hypothesis that reactivation of latent EBV infection in at least two Burkitt lymphoma cell lines is inhibited by endogenously synthesized NO. The other 5 cell lines tested in which L-NMA had no effect on EBV replication were EBV infected LCLs which express a full set of EBV latent gene products. Of interest, the two B cell lines in which there was demonstrated an effect of NOS inhibition on virus replication were the only two cell lines tested which did not express the EBV nuclear antigen EBNA^" 2 (EBNA2) , a full length EBNA-leader protein (EBNA-LP) , or EBV latent membrane proteins 1 or 2 (LMP1 or LMP2) (Rabson et al . , J. Virol.. Vol. 44, pgs. 834-844 (1982); Takada and Ono, J. Virol.. Vol. 63, pgs 445-449 (1989)). These latent infection associated proteins may inhibit the effect of L-NMA on iNOS or the effect of NO on blocking virus replication.

NO Donors Inhibit EBV Replication

Although 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. 4788-4193 (1990)) and the cells were incubated in the NO donor S-nitroso-penicillamine (SNOP) or the control thiol compound penicillamine. After 24 hours, 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 /μM to l M. SNOP (1 mM) decreased virus replication in Akata cells to 34 + /- 5.9 % of the levels seen in the absence of drug (p = 0.05, n = 5) (Figures 2B and C) . At these levels, 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) .

NO Produced By Human Mononuclear Cells Inhibits EBV Replication.

Since NO inhibits EBV replication in EBV-infected Burkitt lymphoma cell lines, NO might also inhibit EBV replication in primary B cells. One might also reason that if endogenous NO has an inhibitory effect on EBV replication, 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. L-NMA (1 mM) caused a small (140 + /- 25 %) (mean + /-SEM) but statistically significant increase in the number of EBV- transformed clones as compared to untreated cultures (p = 0.04, n = 4) (Figure 3) . To assess whether the effect of L- NMA was due to increased EBV replication, acyclovir, an inhibitor of replication, was added to the cultures. As predicted, when acyclovir was added along with L-NMA, the number of transformed clones decreased to control levels (p = 0.01, n = 4) (Figure 3) . In contrast, acyclovir alone had no statistically significant effect on the number of transformed clones when compared to control cultures (p = 0.2, t test, n = 4) (Figure 3). These results suggest that endogenous NO produced by human mononuclear cells inhibits EBV replication in primary B cells.

NO Donors Inhibit B Cell Apoptosis

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)). We therefore determined whether 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 μM. 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. Both NO donors inhibited A23187-induced apoptosis in a dose- dependent manner (Figure 4A) . SNOP (1 mM) decreased apoptosis to 47+/-14% (mean + /- SEM) of levels seen with ionophore alone (p = 0.03, n = 5 ). Likewise, 1 mM SNP decreased apoptosis to 29 +/- 3% (mean +/- SEM) of control levels (p=0.03, n=3) . The control thiol, penicillamine, had no statistically significant effect on apoptosis (Figure 4A) . SNOP and SNP also inhibited apoptosis induced in Akata cells by slg crosslinking (data not shown) .

Endogenous NO Production By Human B Cells Inhibits Apoptosis Studies with L-NMA further support a role for NO in inhibiting B lymphoblast apoptosis. Growing Akata cells in the presence of L-NMA to inhibit iNOS increased apoptosis in a dose-dependent manner. Maximal effects were seen when cells were grown for 5 days in L-arginine enriched media with an initial concentration of 5 mM L-NMA (i.e. added on day 1) (Figure 4B) . Under these conditions, L-NMA increased apoptosis 5.7 + /- 1.5 fold, as compared to controls without L-NMA (p = 0.003, n = 7) . Also consistent with an anti- apoptotic effect of NO was the finding that Akata cells underwent spontaneous apoptosis when grown in media containing low (0. 1 mM) concentrations of L-arginine (6-34 fold increase in apoptosis, n = 2) . Thus, inhibition of endogenous NO production with L-NMA or by substrate (L- arginine) depletion triggers apoptosis. The effect of arginine deficiency on apoptosis and its potential reversal by NO donors was also evaluated in two EBV-negative Burkitt lymphoma cell lines, BL-30 and BL-41. Growth in L-arginine- deficient media caused high levels of apoptosis (12-41 %) after 4 days in the BL-30 cell line and 5 days in the BL-41

^■33- cell line (Figure 4C) . Longer incubations in L-arginine- deficient media caused close to 100% apoptosis in both cell lines. The addition of the S-nitrosothiol, SNOP inhibited arginine deficiency-induced apoptosis in both the BL-30 and BL-41 cell lines in a dose dependent-manner wit': maximal effects seen at a concentration of 1 mM (Figure 4C) . In both cell lines, 1 mM SNOP decreased apoptosis almost completely (2 +/- 0.3 % of control levels, mean + /- SEM) (p = 0.03, n = 4) . The control thiol penicillamine had a significantly less effect on inhibiting apoptosis (1 6 + /- 5.0 % of control levels) when compared to SNOP (p = 0.03 , n = 4) . 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. 1898-1902 (1992) ; Stamler, et al., Proc. Nat. Acad. Sci., Vol. 89, pgs. 444-448 (1992)) . It is concluded that endogenous production of NO inhibits apoptosis in several Burkitt lymphoma cell lines.

The Mechanism By Which NO Inhibits EBV Replication And Apoptosis Is cGMP Independent

Much of the inhibitory effect of NO on platelets and smooth muscle is mediated by increases in cGMP levels due to activation of guanylate cyclase (Ignarro, 1990; Moncada et al . , 1991) . However, the inhibition of EBV replication and apoptosis by NO appears to be cGMP independent . The cGMP analog 8-bromo-cGMP (100 μM or 1 mM) did not inhibit virus replication in Akata cells after slg crosslinking and did not prevent apoptosis in these cells after A23187 treatment as shown in Table 2 below. Table 2. Effects of Cyclic GMP and SNOP on EBV Replication and Apoptosis in the Akata Cell Line

8-bromo-cGMP ( M) BZLF-l (%) Apoptosis (%)

0 2.4 +/- 0.2 46 +/- 3.0

0.1 2.7 +/- 1.7 51 +/- 1.0

1.0 2.5 +/- O.β 52 +/- 6.1

SNOP (mM)

1.0 0.Θ +/- 0.4 20.5 +/- 2.5

Akata cells were induced to replicate virus by βlg 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 cellβ waβ determined by acridine orange staining. The numbers represent the means of two separate experiments +/- SEM.

It is to be understood, however, that the scope of the present invention is not to be limited to the specific embodiments described above. The invention may be practiced other than as particularly described and still be within the scope of the accompanying claims.

Claims

What Is Claimed Is:

1. A method of inhibiting the replication of a virus comprising administering to an animal an effective virus replication inhibiting amount of nitric oxide.

2. The method of claim 1 wherein the treating is effected by administering gaseous nitric oxide or a compound which delivers nitric oxide.

3. The method of claim 2 wherein the compound is selected from the group consisting of S-nitrosothiols, compounds that include at least one -O-NO group, N- nitrosoamines, C-nitroso compounds including at least one -C- NO group, nitrates having at least one -0-N0₂ group, nitroso- metal compounds, N-oxo-N-nitrosoamines, and thionitrates.

4. The method of claim 3 wherein the compound is an S- nitrosothiol.

5. The method of Claim 4 wherein said S-nitrosothiol is an S-nitroso-amino acid.

6. The method of Claim 4 wherein said S-nitrosothiol is an S-nitrosylated protein.

7. The method of Claim 4 wherein said S-nitrosothiol is an S-nitroso-polypeptide.

8. The method of claim 4 wherein the S-nitrosothiol is selected from the group consisting of those having the structures:

(i) CH₃(CH₂)_xSNO wherein x equals 2 to 20; {ii) HS(CH₂)_xSNO wherein x equals 2 to 20; and {iii) ONS(CH₂)_Y wherein x equals 2 to 20 and Y is selected from the group consisting of fluoro, C,-C₆ alkoxy, cyano, carboxamido, C₃-C₆ cycloalkyl, aralkoxy, C,-C₆ alkylsulfinyl, arylthio, C,-C₆ alkylamino, C₂-C_1$ dialkylamino, hydroxy, carbamoyl, C,-C₆ N- alkylcarbamoyl, C₂-C₁₅ N,N-dialkylcarbamoyl, amino, hydroxyl, carboxyl, hydrogen, nitro and aryl; wherein aryl includes benzyl, naphthyl, and anthracenyl groups.

9. The method of claim 4 wherein the S-nitrosothiol is an S-nitroso-ACE inhibitor selected from the group consisting of compounds having the following structure (1) :

(1)

wherein

R is hydroxy, NH₂, NHR⁴, NR⁴R⁵, or C_ι-C₇ alkoxy, wherein R⁴ and R⁵ are C,-C₄ alkyl, or phenyl, or C,-C₄ alkyl substituted by phenyl,-

R¹ is hydrogen, C,-C₇ alkyl, or C_ι-C₇ alkyl substituted by phenyl, amino, guanidino, NHR⁶, NR^⁷, wherein R⁶ and R⁷ are methyl or C,-C₄ alkanoyl;

R² is hydrogen, hydroxy, C,-C₄ alkoxy, phenoxy, or

C,-C₇ alkyl;

R³ is hydrogen, C,-C₄ or C,-C₇ alkyl substituted by phenyl; m is 1 to 3; and n is 0 to 2.

10. The method of claim 4 wherein the S-nitrosothiol is an S-nitroso-ACE inhibitor selected from the group consisting of N-acetyl-S-nitroso-D-cysteinyl-L-proline, N-acetyl-S- nitroso-D,L-cysteinyl -L-proline, l- (4-amino-2-S- nitroso)mercaptomethylbutanoyl) -L-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 , 1- [5- guanidino-2- (S-nitroso)mercaptomethyl-pentanoyl] -4-hydroxy-L- proline, 1- [2-aminomethyl-3 (S-nitroso) -mercaptomethyl- pentanoyl -L-proline, and S-nitroso-L-cysteinyl-L-proline.

11. The method of claim 4 wherein the S-nitrosothiol is an S-nitroso-ACE inhibitor selected from the group consisting of compounds having structures (2-3) :

wherein

X is oxygen or sulfur,- -A,,-A₂- is CH-NH or -C=N-;

A is 0N-S-CH₂-CH-C;

R is selected from hydrogen, lower ( -C,) alkyl, benzyl, benzhydryl, and salt forming ion;

Ri and R₂ are independently selected from hydrogen, halogen, lower alkyl, lower alkoxy, halo substituted lower alkyl, nitro, and SOTNH_J,-

0 0 0

II w //

Z is -C- or -s- R₃ is hydrogen, lower alkyl, halo substituted lower alkyl, phenyl, benzyl, phenethyl, or cycloalkyl; and

R₄ is hydrogen, lower alkyl, halo substituted lower alkyl, hydroxy substituted lower alkyl, -(CH₂)_q-N (lower alkyl)₂ or -(CH₂)_q-NH₂ and q is one, two, three or four.

-39-

SHE E ^~ (RULE 91)

12. The method of claim 4 wherein the S-nitrosothiol is an S nitroso-ACE inhibitor selected from the group consisting of compounds having structures (4-11) :

13. The method of Claim 1 wherein the virus is an Epstein-Barr virus, cytomegalovirus, Herpes Simplex virus, or varicella virus.

14. The method of claim 1 wherein said administration is topical.

15. The method of claim 14 wherein said administration is by topical administration to a virus infected ucocutaneous membrane.

16. The method of claim 14 wherein said administration is by inhalation.

17. The method of claim 1 wherein said administration is parenteral.

18. A method for preventing or reversing latency in a virus which comprises administering to an animal a nitric oxide synthase inhibitor.

19. The method of claim 18 wherein the nitric oxide synthase inhibitor is N^G-monomethyl-L-arginine.

20. The method of claim 18 wherein the nitric oxide synthase inhibitor is nitro-arginine.

21. The method of claim 18 wherein the nitric oxide synthase inhibitor is selected from the group consisting of 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, trifluoropiperazine, calcinarin, calmadulin binders, and methotrexate.

22. A method for the treatment of a latent virus infection in an individual in need thereof, which comprises administering (i) a virus latency preventing or reversing amount of a nitric oxide synthase inhibitor to sufficient render the virus replicative and (ii) a virus replication inhibitory amount of nitric oxide or a nitric oxide-releasing substance.

23. 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 amount of a nitric oxide synthase inhibitor sufficient to render the virus replicative and (ii) a virus replication inhibitory amount of nitric oxide or a nitric oxide-releasing substance, in a pharmaceutically acceptable carrier.

24. A method of preventing cellular apoptosis comprising administering to cells an effective apoptosis inhibiting amount of nitric oxide.

25. The method of claim 24 wherein said cells are lymphocytes.