CA2244078C - Use of leukotriene b4 or its analogues as antiviral and antineoplastic agents - Google Patents

Abstract

The present invention relates to the use of leukotriene B4 (LTB4), variants and derivatives thereof as a therapeutic agent in the treatment or prophylaxis of viral infections caused by human and animal viruses. The present invention also relates to the use of LTB4, variants and derivatives thereof as an anti-neoplastic agent in the prophylaxis and treatment of cancers induced by tumor viruses and in other neoplastic diseases. The human and animal viruses are DNA viruses, such as parvoviridae, papovaviridae, adenoviridae, herpesviridae, poxviridae and hepadnaviridae; RNA viruses, such as picomaviridae, togaviridae, orthomyxoviridae, paramyxoviridae, coronaviridae, reoviridae, oncomaviridae and filoviridae in general, and Retroviridae such as HIV-1 and HIV-2.

Description

BACKGROUND OF THE INVENTION
(a) Field of the Invention The invention relates to the antiviral activity of leukotriene B4 (LTB4) and to the use of leukotriene B4 (LTB4) as a therapeutic agent for treating viral infections caused by human and animal viruses.
(b) Description of Prior Art Many important infectious diseases afflicting mankind are caused by viruses_ Some are important because they are frequently fatal; among such are rabies, smallpox, poliomyelitis, hepatitis, yellow fever, immune deficiencies and various encephalitic diseases. Others are also important because they are very contagious and create acute discomfort such as influenza, measles, mumps and chickenpox, as well as respiratory-gastrointestinal disorders. Others such as rubella and cytomegalovirus can cause congenital abnormalities. Finally, there are viruses, known as oncoviruses, that can cause tumors and cancer in humans and animals.
Among viruses, the family of Herpesviridae is of great interest. Herpes viruses are highly disseminated in nature and highly pathogenic for men.
For example, Epstein-Barr virus (EBV) is known to cause infectious mononucleosis in late childhood, adolescence or in young adults. The hallmarks of acute infectious mononucleosis are sore throat, fever, headache, lymphadenopathy, enlarged tonsils and atypical dividing lymphocytes in the peripheral blood.
Other manifestations frequently include mild hepatitis, splenomegaly and cerebritis (for review see Miller G., In: Virology, B.N. Fields & D.M. Knipe ed., Raven Press, 1990, pp. 1921-1958). EBV is also associated with two forms of cancer: Burkitt's lymphoma (BL) and the nasopharyngeal carcinoma (NPC)_ In endemic areas of equatorial Africa, BL is the most common childhood malignancy, accounting for approximately 80% of cancers in children. While moderately observed in North American Caucasians, NPC
.is one of the most common cancers a.n Southern China with age incidence of 26 to 55. EBV, like the cytomegalovirus, is also associated with post-transplant lymphoproliferative disease, which is a potentially fatal complication of chronic immunosuppression following solid organ or bone marrow transplantation.
Another Herpes virus, namely Herpes Simplex type 1 (HSV-1) is identified as the etiologic agent of gingivostomatitis_ Manifestations are fever, sore throat, and ulcerative and vesicular lesions in the mouth. The most severe clinical state caused by HSV
is the primary genital herpetic infection. While HSV-1 can cause genital herpetic infection, HSV-2 is the main virus associated with this disease. This HSV
infection is accompanied by vesicles, pustules and ulcers causing lesions on genital parts. A urinary retention syndrome may also be encountered_ More than 80% of people are seropositive to HSV-1 or HSV-2 and which have been studied, have indicated a frequency of recurrence or viral reactivation as high as 60%.
Other diseases are also associated with HSV such as skin and eye infections including chorioretinitis and kerato-conjunctivitis_ Approximately 300,000 cases of HSV infections of the eye are diagnosed yearly in the United States of America.
Human Herpes virus-6 (HHV-6) has a marked tropism for cells of the immune system and therefore, HHV-6 infection may result in alteration of the immune response. It is now clear that HHV-6 is the cause of _ 3 _ exanthem subitum as a primary infection in children.
Recent studies indicate that a significant proportion of organ transplant recipients who are seropositive = before transplantation, decnonstrate serologic evidence of reactivation subsequent to immunosuppression.
Heterophil-negative mononucleosis-like illness and non-A, non-B hepatitis also have been associated with active HHV-6 infection. HHV-6 has often been isolated from patients with human irnmunodeficiency virus (HIV) infections. The fact that HIV and HHV-6 can reside in the same target cell has led to speculation that HHV-6 infection may act as a cofactor in the progression of HIV-seropositive patients to symptomatic AIDS_ Recent studies also suggest that a human herpes virus is closely associated with F3IV diseases_ In fact, Kaposi sarcoma (KS), a neoplasm occurring mainly in HIV-infected person, was found to have an infectious etiology. While the virus has been named KS-associated herpes virus, its formal classification is likely to be HHV-8_ Since in the early 1980's, a new disease has been identified and named Acquired ImmunoDeficiency Syndrome (AIDS). The huznan immunodeficiency virus (HIV), which belongs to the Retroviridae family, is known to be the etiologic agent of AIDS. HIV
infection in humans can lead to a variety of disease states such as mononucleosis like syndrome, prolonged asymptomatic infection and AIDS_ The AIDS' associated diseases include Kaposi's sarcoma, pneumonia, chronic diarrhea, meningitis, toxoplasmosis, encephalopathies, anal-rectal carcinomas and B-lymphocytic lymphomas.
The distinctive symptoms of acute infection include lymphadenopathy, fever, myaLgia, arthralgia, headache, fatigue, diarrhea, sore throat and neurologic manifestations.

It is now accepted that HIV is transmitted by three main routes: a) sexual contact, b) contaminated blood, and c) from the mother to the fetus. A wide variety of organs and tissues in humans can be invaded by HIV, including bone marrow, lymph node, blood, brain and skin, via the interactions of the viral envelope protein gpl20 and the cell surface receptor CD4.
At the end of 1993, an estimated 14 million individuals have been infected with HIV and by the year 2000, this number could be as high as 24 million_ Today, medical treatment is limited to the use of antiviral drugs (in particular 3'-azido-3'-deoxythimidine, AZT) and also to the treatment of the many opportunistic infections. However, those treatments are still not fully efficient .in the control of HIV infection. Thus, the elaboration of new molecules for the treatment of HIV infection must be given major emphasis.
In all infectious diseases, the efficacy of therapy often depends on the host immune response.
This is particularly true for herpes viruses. Indeed, the ability of all herpes viruses to establish latent infections results in an extremely high incidence of reactivated infection in immunocompromised patients_ In renal transplant recipients, 40% to 70% reactivate latent HSV infections, and 80% to 100% reactivate CMV
infections. Such viral reactivations have also been observed in HIV-positive patients (AIDS).
Today, the number of therapeutic agents used for the treatment of viral infections remain relatively limited. The major compounds used in the treatment of herpes virus infections are idoxuridine, vidarabine, acyclovir and ganciclovir and, more recently Ãamciclovir which is converted in the body into penciclovir. Their efficacy is limited and they cause many side effects. Allergic effects have been reported in 35% of patients treated with idoxuridine which is used only to treat HSV infection of the eye _ The most common side effects of vidarabine are gastrointestinal disturbances (15% of patients). The = major side effect of acyclovir is the alteration of renal function. Since acyclovir is a nucleoside analog that can be incorporated in both viral and host cell DNA, normal division of host cell can be affected. The most important side effects of gangciclovir are neutropenia and thrombocytopenia that occur in about 40% of AIDS patients.
Thus, there is an urgent need for the development of more efficacious therapeutic agents for the treatment of viral infections with fewer side effects.
Leukotriene B4 (LTB4) [5S,12R-6,8,10,14 (Z,E,E,Z)-eicosatetraenoic acid] is a known natural molecule. LTB4 is a metabolite of arachidonic acid which is derived from the 5-lipoxygenase pathway.
LTB4 has many reported biological properties. In particular, LTB4 is considered as a potent pro-inflammatory compound; its most important biological activity is its chemotactic and chemokinetic effects on leukocytes. Indeed, LTB4 has been shown to be a potent chemoattractant for human polymorphonuclear leukocytes, monocytes and macrophages, both in vitro and in vi vo. LTB4 also activates other leukocyte functions such as degranulation and superoxide anion synthesis. Because of these pro-inflammatory effects, LTB4 is considered as a putative component in defense mechanisms. Moreover, LTB4 is synthesized by inflammatory cells such as polymorphonuclear leukocytes, monocytes and macrophages and is also synthetized by B lymphocytes.

LTB4 has also been shown to exert immunomodulatory activities. Indeed, LTB4 was found to induce suppressor cell activity in human peripheral blood mononuclear leukocyte cultures; the induced suppressor cell activity inhibited the proliferative response of human lymphocytes to mitogens (Rola-Pleszczynski M_ et.al_, BioChem. Biophys. Res.
Comm., 1982, 108:1531)_ It was also shown that LTB4 increases human natural cytotoxic cell activity against K562 erythroleukemia cells and against the human prostatic adenoma MA-160 cells either non-infected or persistently infected with Herpes simplex virus type 1(HSV-1)(RoZa-Pleszczynski M. et al., BioChem. Biophys. Res. Comm., 1983, 113:531; Gagnon L_, et al., Cell Xmmunol., 1987, 110:243). Other studies have indicated that in addition to LTB4, LTA4, LTD4, 5-hydroperoxy- eicosatetraenoic acid and 15-hydroperoxy- eicosatetraenoic acid also enhance human natural killer cell cytotoxicity (Rola-Pleszczynski, M_ et al, Prostaglandins Leukotr-ienes Med., 1984, 13:113; Bray, R.A_ et al_ J.
Immunol, 1986, 136:1783).
A family of molecules collectively called the prostaglandins (prostaglandins A, B, D, J, E and I) which are structurally related to LTB4, have been repeatedly demonstrated to exert antiviral and anti-cancer activity both in in vitro and in vivo systems. The prostaglandins are derived from arachidonic acid, as for LTB4, but originate from a different bi.osynthetic pathway, the cyclooxygenase pathway.
United States Patent No. 4,689,426 issued on August 25, 1987 in the name of Sugiura et al_ describes cyclopentenone derivatives related to prostaglandin A or D which possess anti-tumor and antiviral activities.

_ 7 _ Although, some prostaglandins have been shown to have antiviral activities, they caused undesirable side effects, and exhibited relatively low activity.
It would be highly desirable to be provided with an antiviral agent with greater efficacy and which would not present the undesirable side effects of the known antiviral agents.

SUMMARY OF THE INVENTION
One aim of the present invention is to provide an antiviral agent and use thereof which would be more efficacious for the prophylaxis and treatment of viral infections and would not present the undesirable side effects of the known antiviral agents.
Another aim of the present invention is to provide an antiviral agent for the prophylaxis or treatment of cancers induced by oncoviruses such as retroviruses, papillomaviruses, adenoviruses and herpesviruses.
Another aim of the present invention is to provide an antiviral agent for the prophylaxis or treatment of viral infections in immunosuppressed patients and animals.
Another aim of the present invention is to provide an anti-neoplastic agent for the treatment of cancer.
in accordance with one aspect of the invention there is provided a use of a pharmacologically acceptable therapeutically effective amount of a leukotriene B4 (LTB4) agent for the prophylaxis or treatment of a viral infection in a human or animal.
In accordance with another aspect of the invention there is provided an antiviral pharmaceutical composition comprising a pharmacologically acceptable, therapeutically effective amount of a LTB4 agent and a pharmaceutically acceptable carrier.
Thus in accordance with the present invention there is provided the use of a leukotriene B4 (LTB4) agent as an antiviral agent, for example, against herpes viruses selected from the group consisting of EBV, HSV-l, HSV-2, CMV, VZV, HHV-6, HHV-7 and HHV-8.

In accordance with the present invention there is provided the use of a LTB4 agent as an antiviral agent against HIV-l and HIV-2 and against other human and animal viruses, including, but not limited to, porcine enteroviruses belonging to the picornaviridae or bovine diarrhea virus belonging to the togaviridae family, or bovine respiratory syncytial virus belonging to the paramyxoviridae.
In accordance with the present invention there is provided the use of a LTB4 agent as an antiviral agent in the treatment of viral infections in humans and animals in association with other antiviral agents, including but not limited to interferon-a, -13, -y, tumor necrosis factor a, ganciclovir, acyclovir, vidarabine, idoxuridine, famciclovi.r 3TC, crixivan, nevarepine and prostaglandins or prostaglandin analogs.
In accordance with the present invention, there is provided the use of a LTB4 agent as an antiviral agent for the prophylaxis and treatment of cancers induced by oncoviruses such as retroviruses, papillomaviruses, adenoviruses and herpesviruses.
In accordance with the present invention, there is provided the use of a LTB4 agent as an antiviral agent against cancers induced by oncoviruses in association with other anti-neoplastic agents including but not limited to adriamycine, cyclophosphamide and methotrexate.

In accordance with the present invention, there is provided the use of a LTB4 agent as an antiviral agent for the prophylaxis and treatment of viral = infections in immunosuppressed patients and animals.
Immunosuppressed patients include patients who underwent organ or tissue transplantation and are treated with immunosuppressive agents including but not limited to azathioprine, corticosteroids, adriamycine, cyclophosphamide and methotrexate.
Immunosuppressed patients also include patients with any form of cancer or neoplasic diseases treated or not with anti-neoplastic chemotherapeutic agents including but not limited to adriamycine, cyclophosphamide and methotrexate. Immunosuppressed patients also include patients with inflammatory diseases treated with anti-inflammatory agents including but not limited to corticosteroids, methotrexate, azathioprine and cyclophosphamide_ Immunosuppressed patients also include patients with shock or severe trauma including but not limited to burn .injury, or patients undergoing chronic hemodialysis.
In accordance with the present invention, there is provided the use of a LTB4 agent as an antiviral agent against viral infections in immunosuppressed patients and animals in association with other antiviral agents.
In accordance with the present invention, there is provided the use of a LTB4 agent as an anti-neoplastic agent for the treatment of cancers.
in accordance with the present invention, there is provided the use of a LTB4 agent as an anti-neoplastic agent for the treatment of cancers in association with other anti-neoplastic agents including but not limited to adriamycine, cyclophosphamide and methotrexate.

In accordance with the present invention, there is provided the use of a LTB4 agent as a therapeutic agent against bacterial Gram + and - infections or fungal infections, alone or in association with other antibacterial or antifungal agents_ In accordance with the present invention, there is also provided the use of a LTB4 agent as an antiviral agent for the prophylaxis and treatment of viral infections in humans and animals in association with other agents including but not limited to granulocyte-macrophage colony-stimulating factor (GM-CSF), granulocyte colony stimulating factor (G-CSF), macrophage colony stimulating factor (M-CSF), interferons, tumor necrosis factor a, interleukin-3 and interleukin-5, which have been shown to prime leukocytes for the synthesis of LTB4 or other arachidonic acid metabolites (including several LTB4 agents) and may potentiate the antiviral activity of the LTB4 agent_ In accordance with the present invention, there is provided the use of a LTB4 agent as an antiviral agent in the prophylaxis or treatment of viral infections in humans and animals in association with retinoids including but not limited to 9-cis-retinoic acid and analogs (such as l3-cis-retinoic acid or all trans-retinoic acid), which are ligands of retinoid receptors, and may potentiate the antiviral activity of the LTB4 agent.
In accordance with the present invention, there is provided the use of a LTB4 agent as an antiviral agent for the prophylaxis and treatment of viral infections in animals and humans in association with non-steroidal anti-inflammatory drugs including but not limited to N-acetyl salicylic acid, indomethacin, ibuprofen, flurbiprofen and naproxen, which are inhibitors of the type I (constitutive) and II

(inducible) cyclo-oxygenases, and might be useful in limiting potential side effects of the administration of LTB4 agents in-humans and animals_ BRIEF DESCRIPTION OF THE DRAWINGS
Fig. 1 illustrates the effects of LTB4 on clump formation induced by EBV;
Fig. 2 illustrates the effects of LTB4 on EBV-induced synthesis of Epstein-Barr Nuclear Antigen (EBNA) protein;
Fig. 3 illustrates the effects of LTB4 on the production of EBV particles;
Fig_ 4 illustrates the effects of LTB4 on HSV-1-induced synthesis of cytoplasmic antigens;
Fig_ 5 illustrates the effects of LTB4 on the production of HSV-1 particles;
Fig. 6 illustrates the effects of LTB4 on reverse transcriptase activity in HIV-1 infected peripheral blood mononuclear cells;
Fig. 7 illustrates the effects of LTB4 on cell viability; and Figs. 8A and 8B illustrate the effects of LTB4 and acyclovir on EBV-i.nduced synthesis of EBNA protein (A), and also on HSV-1-induced synthesis of viral proteins (B).

DETAILED DESCRIPTION OF THE INVENTION
i) LTB4 The leukotriene B4 (LTB4) agent of the present invention is either LTB4 or certain structurally related polyunsaturated fatty acids, or substances structurally unrelated to fatty acids, which stimulate the synthesis of LTB4 or other LTB4 agents by cells, or mimic their biological activi.ty. They are either natural substances or analogs of such natural substances. All of the LTB4 agents can be obtained by =-chemical synthesis by methods described in the literature and most are commercially available.
Examples of LTB4 agents are found in "CRC
Handbook of Eicosanoids: Prostaglandins and Related Lipids", Ed. Anthony L. Willis (CRC Press Inc., Boca Raton, Florida, USA).
As used herein, the term "LTB4 agent" means one or more of the following polyunsaturated fatty acids, which in addition to LTB4 itself, are analogs of LTB4, or precursors or metabolites of LTB4 or LTB4 analogs:
LTB4, 14,15-dihydro-LTB4, 17,18-dehydro-LTB4, 19-hy-droxy-LTB4, 20-hydroxy-LTB4 and their 5(R)hydroxy, 5-keto, 5(S)hydroperoxy, 5(R)-hydroperoxy and 5-deoxy analogs; LTA4; 14,15-dihydro-LTA4, 17,18-dehydro-LTA4;
5(S)-hydroxy-6,8,11,14(E,Z,Z,Z)-eicosatetraenoic acid ("5-HETE"), 14,15-dihydro-S-HETE, 17,18-dehydro-5-HETE, and their 5(R)-hydroxy, 5-keto, 5(S)-hydro-peroxy, 5(R)-hydroperoxy analogs; 12(R)-hydroxy-5,8,10,14(Z,Z,E,Z)-eicosatetraenoic acid ("12-HETE"), 5,6-dihydro-l2-HETE, 14,15-dihydro-l2-HETE, 17,18-de-hydro-l2-HETE and their 12(S)-hydroxy, 12-keto, 12(S)-hydroperoxy and 12(R)-hydroperoxy analogs and 12-oxo-5,8,10(Z,Z,E)-dodecatrienoic acid, 15(S)-hydroxy-5,8,11,13(Z,Z,Z,E)-eicosatetraenoic acid ("15-HETE"), 5,6- dihydro-15-HETE, 17,18-dehydro-l5-HETE and their 15(R)-hydsoxy, 15-keto, 15(S)-hydroperoxy, and 15(R)-hydroperoxy analogs.
The term LTB4 agent also includes other derivatives of polyunsaturated fatty acids; some are derived from the cyclooxygenase pathways, the lipoxygenase pathways (5-, 12- and 15-lipoxygenases) or the cytochrome P450 pathways; others are isomers, analogs or derivatives of naturally formed compounds:
12(S)-hydroxy-5,8,10(Z,E,E)-heptadecatrienoic acid;
leukotrienes C4, D4 and E4 and their 14,15-dihydro or 17,18-dehydro analogs; N-acyl or N-alkyl derivatives ~ir'IVU~
A' 'UD SHEET

~ . - 13 -of leukotrienes C4, D4 and E4, and their 14,15-dihydro or 17,18-dehydro analogs; all isomeric 5,12-dihydroxy-6,8,10,14-eicosatetraenoic acids and their 14,15-dihydro or 17,18-dehydro analogs; all isomeric 5,6-dihydroxy-7,9,11,14-eicosatetraenoic acids and their 14,15-dihydro or 17,18-dehydro analogs; all isomeric 5,15-dihydroxy-6,8,11,13-eicosatetraenoic acids (including 5(S),15(S)-dihydroxy-6,8,11,13(E,Z,Z,E)-eicosatetraenoic acid) and their 17,18-dehydro analogs; all isomeric 8-hydroxy-11(12)-epoxy-5,9,14-eicosatrienoic acids (including hepoxilin A3) and their 5,6-dihydro or 14,15-dihydro or 17,18-dehydro analogs; all isomeric 10-hydroxy-11(12)-epoxy-5,8,14-eicosatrienoic acids (including hepoxilin B3) and their 5,6-dihydro or 14,15-dihydro or 17,18-dehydro analogs; all isomeric 8,11,12-trihydroxy-5,9,14-eicosatrienoic acids (including trioxilin A3) and their 5,6-dihydro or 14,15-dihydro or 17,18-dehydro analogs; all isomeric 10,11,12-trihydroxy-5,8,14-eicosatrienoic acids (including trioxilin B3) and their 5,6-dihydro or 14,15-dihydro or 17,18-dehydro analogs; all isomeric 11(12)-epoxy-5,7,9,14-eicosatetraenoic acids and their 14,15-dihydro or 17,18-dehydro analogs; all isomeric 11,12-dihydroxy-5,7,9,14-eicosatetraenoic acids and their 14,15-dihydro or 17,18-dehydro analogs; all isomeric 8(9)-epoxy-5,10,12,14-eicosatetraenoic acids and their 5,6-dihydro or 17,18-dehydro analogs; all isomeric 8,9-dihydroxy-5,10,12,14-eicosatetraenoic acids and their 5,6-dihydro or 17,18-dehydro analogs; all isomeric 8,15-dihydroxy-5,9,11,13-eicosatetraenoic acids and their 5,6-dihydro or 17,18-dehydro analogs; all isomeric 14 (15) -epoxy- 5, 8, 10, 12-eicosatetraenoic acids and their 5,6-dihydro or 17,18-dehydro analogs; all isomeric 14,15-dihydroxy-5,8,10,12-eicosatetraenoic acids and their 5,6-dihydro or 17,18-dehydro analogs;
C~-{L-ET
~.il~

- 13a -all isomeric 5-hydroxy-14(15)-epoxy-6,8,10,12-eicosatetraenoic acids and their 17,18-dehydro analogs; all isomeric 5,14,15-trihydroxy-6,8,10,12-eicosatetraenoic acids (including lipoxin B4) and ~~.

their 17,18-dehydro analogs; all isomeric 5,6,15-trihydroxy-7,9,11,13-eicosatetraenoic acids (including lipoxin A4) and their 17,18-dehydro analogs; all isomeric 5(6)-epoxy-l5-hydroxy-7,9,11,13-eicosatetraenoic acids and their 17,18-dehydro analogs; all isomeric 5-hydroxy-6,8,11,14-eicosatetraenoic acids and their 14,15-dihydro or 17,18-dehydro analogs; all isomeric 8-hydroxy-5,9,11,14-eicosatetraenoic acids and their 5,6-dihydro or 14,15-dihydro or 17,18-dehydro analogs; all isomeric 9-hydroxy-5,7,11,14-eicosatetraenoic acids and their 14,15-dihydro or 17,18-dehydro analogs; all isomeric 11-hydroxy-5,8,12,14-eicosatetraenoic acids and their 5,6-dihydro or 17,18-dehydro analogs; all isomeric 12-hydroxy-5,8,10,14-eicosatetraenoic acids and their 5,6-dihydro or 14,15-dihydro or 17,18-dehydro analogs; all isomeric 15-hydroxy-5,8,11,13-eicosatetraenoic acid and their 5,6-dihydro or 17,18-dehydro analogs; all isomeric 9-hydroxy-10,12-octadecadienoic acids; all isomeric 13-hydroxy-9,11-octadecadienoic acids; 12(R)-hydroxy-5,8,14(Z,Z,Z)-eicosatrienoic acid; all isomeric 5(6)oxido- or 5,6-dihydroxy-8,11,14-eicosatrienoic acids and their 14,15-dihydro or 17,18-dehydro analogs; all isomeric 8(9)-oxido- or 8,9-di.hydroxy-5,11,14-eicosatrienoic acids and their 5,6-dihydro or 14,15-dihydro or 17,18-dehydro analogs; all isomeric 11(12)-oxido- or 11,12-dihydroxy-5,8,14-eicosatrienoic acids and their 5,6-dihydro or 14,15-dihydro or 17,18-dehydro analogs; all isomeric 14(15)-oxido- or 14,15-dihydroxy-5,8,11-eicosatrienoic acids and their 5,6-dihydro or 17,18-dehydro analogs.
The term LTB4 also includes variants which are non-covalently modified fatty acids such as the sodium or the potassium salts of the LTB4 agents.

The term LTB4 agent also includes variants where a modification is introduced into the molecule by reacting targeted functional groups of the fatty acid with an organic derivatizing agent that is capable of reacting with the selected functional group (yielding for example, ester and ether derivatives of LTB4 agent) or to cause intramolecular rearrangement (such as the formation of lactones with hydroxylated fatty acids). The resulting compounds may have altered biological activity and/or bioavailability.
Thus, the covalently modified fatty acid can be a pro-drug with reduced biological activity which upon in vivo administration is slowly transformed into a more active molecule (underivatized LTB4 agent).
Variants may also be metabolically stable and biologically active analogs of LTB4 agents altered in a way that will result in retarded disposition of the compound (decreased metabolism and/or elimination).
Variants with modifications at the omega end (such as 20,20,20-trifluoromethyl-LTB4) show increased resistance to omega-oxidation (a catabolic process of unsaturated fatty acids); other variants with modification at the omega end at the level of carbons 13 to 20 (such as 19-methyl-LTB4 or 19,19-dimethyl-LTB4 or 19-fluoro-LTB4 or 19,19-difluoro-LTB4 or 18,20-d '.uro-LTB4 or 20-fluoro-LTB4) may show increased resistance to omega-oxidation and variants with modifications at the carboxylic end, at the level of carbon 1, 2, 3 or 4 (for example, 3-thio-LTB4, 3-hydroxy-LTB4, 3-methyl-LTB4 or 3,3-dimethyl-LTB4 or 3-fluoro-LTB4 or 3,3-difluoro-LTB4 or 2,3-difluoro-LTB4, LTB4 methylsulfonylamide, LTB4 methylamide, 1-tetrazole LTB4), may show increased metabolic resistance to beta-oxidation and/or to elimination (such as uptake by probenecide-sensitive organic acid transporter). Other variants with modification(s) at ~1~1(+-i t~~ r.r v :4..-=

carbon 12, such as 12(R)-methyl-LTB4, may show increased resistance to reduction of the 11,12 double bond (a metabolic pathway of LTB4). Other variants are analogs of LTB4 agents with structural changes, such as changes in chain length (chain length increased or decreased by up to 4 carbons), addition of double bond(s), saturation of double bond(s), changes in double bond(s) geometry (cis to trans or vice versa), change of double bond(s) for triple bond(s), change in the configuration of one or several functional group ( s) (R to S or S to R), or where one or several functional group(s) or substituent(s) are either removed, added or changed for other functional groups or substituents (including but not limited to hydroperoxyl, carbonyl, sulfhydryl, sulfoxide, sulfone, cystei.nyl, glutathionyl, cysteinyl-glycine, methyl, isopropyl, benzyl, chloro, fluoro), or where the positions of one or several functional groups and/or one or several double bonds has been moved by one, two or three carbons relative to the omega end.
The LTB4 agent may be a variant carrying one or several of the above mentioned structural modif ications _ The LTB4 agents and variants of LTB4 agents are structurally related to LTB4 and bind or may bind with different affinities to either the cell surface binding sites of LTB4 (or other related eicosanoids,.
including but not limited to 5-HETE, LTD4, lipoxin A4) present on various leukocytes (and other cell types), or to the nuclear binding site of LTB4, the transcription factor PPAR . (peroxisome proliferator-activated receptor alpha) (Devchanct P.R., et al., Nature 384:39, 1996), or to other unknown binding sites of LTB4, resulting in the expression of the biological activities of LTB4 and LTB4 agents. The LTB4 agents and variants show or may show biological activities qualitatively similar to that of LTB4 (but may be more or less active than LTB4 itself ) and thus can be expected to exert an antiviral activity similar to that of LTB4. The LTB4 agents and variants thereof are included within the scope of this invention.
The term LTB4 agent also includes agents not structurally related to LTB4 including but not limited to the chemotactic peptide formyl-met-leu-phe (fMLP) (and analogs such as N-formyl-nle-leu-phe, N-formyl-met-leu-phe-benzylamide, N-formyl-met-leu-phe-methyl-ester and N-formyl-Nle-leu-phe-nle-tyr-lys), the complement fragment C5a and analogs, and the biologically active phospholipid platelet-activating factor, 1-0-hexadecyl-2-0-acetyl-sn-glycero-3-phospho-choline (and analogs such as 1-0-octadecyl-2-0-sn-glycero-3-phosphocholi.ne and 1-0-hexadecyl-2-N-methyl-carbamyl-sn-glycero-3-phosphocholine) that stimulate or may stimulate the release of unsaturated fatty acids in cells (mainly arachidonic acid) and consequently the formation of one or several LTB4 agents, and may therefore exhibit an antiviral activity similar to that of LTB4. The above-mentioned LTB4 agents not structurally related to LTB4 are thus included within the scope of this invention.
The term LTB4 agent also includes formulations of compounds which might contain a mixture of two or several LTB4 agents or an LTB4 agent and one or several equally or less active isomer(s) of the LTB4 agent (positional, geometrical or optical isomers).
The term LTB4 agent also includes antibodies to the LTB4 receptor, or anti-idiotypic antibodies to antibodies raised against LTB4 or one of the above-mentioned analogs or variants of LTB4, which can be expected to elicit an LTB4-like biological response, such as an antiviral effect.

ii) Viral Infections The viral infections which may be treated with the LTB4 agent, in accordance with the invention, are infections caused by human and/or animal viruses.
The expression "human and/or animal viruses" is intended to include, without limitation, DNA and RNA
viruses in general and Retroviridae. DNA viruses include parvoviridae, papovaviridae, adenoviridae, herpesviridae, poxviridae and hepadnaviridae. RNA
viruses include picornaviridae, togaviridae, orthomyxoviridae, paramyxoviridae, coronaviridae, reoviridae, oncornaviridae and filoviridae.
The antiviral activity of LTB4 against two herpes viruses, EBV and HSV-1 and against HIV have been studied. Human peripheral blood mononuclear cells were cultured in the presence or absence of LTB4 at different concentrations_ After ten to twelve days of culture, two parameters were evaluated: the formation of clumps or rosettes, which morphologically characterizes the EBV-infected cells, and the presence of Epstein-Barr Nuclear Antigen (EBNA), a viral antigen synthesized in EBV-infected cells_ The results obtained show that LTB4 markedly affected the formation of clumps. Similarly, the percentage of EBNA-positive cells was strongly decreased by more than 56% with 30 nM LTB4 and by more than 70% with a concentration of 100 nM. Similar results were also obtained with HVS-1 and HIV-1. In fact, the presence of LTB4 (100 nM) in the cellular cultures strongly inhibited the synthesis of specific HSV-1 antigen by more than 60%, and suppressed the reverse transcriptase activity by more than 70% in HIV-1 infected PBMC. Interestingly, in all cellular cultures, the cell viability was comparable to that of the unstimulated cells (controls) cultured during the same period of time, indicating that LTB4 exerts no direct cytotoxic effect on the cells. Furthermore, LTB4 was found to inhibit (>80%) the formation of new EBV particles produced in B95-8 cells. This effect was also observed with HSV-1 (75% inhibition) and HIV-1 (>55% inhibition) using Vero cells and Jl.l cells, respectively_ In an in vi vo experimental model (hairless mice), skin lesions generated by HSV-1 inoculation were smaller and disappeared more rapidly in animals treated with LTB4 (10 g intraperitoneally).
These results clearly show that the LTB4 exerts a very potent antiviral effect against the two herpes viruses and HIV-1 and also exerts a protective effect against HSV-1 in vivo without cytotoxic effect on the uninfected cells.
Some LTB4 agents (other than LTB4 itself) were tested and found to exert an antiviral effect on HSV-1 or EBV in vitro. When added to cell culture media (four consecutive additions of 100 pmol/ml throughout the 7-10 days incubation period), the LTB4 agents 20-hydroxy-LTB4, 12(R)-HETE, 14,15-dihydro-LTA4 methyl ester, and N-formyl-met-leu-phe, inhibited the infection of peripheral blood mononuclear leukocytes by HSV-1 (as assessed by the presence of HSV-1 antigens) or the production of HSV-1 particles in Vero cells, by 40% or more. In the same experiments, the antiviral agent acyclovir used at a 10 times greater concentration had similar effects. The LTB4 agents 5(S),15(S)-dihydroxy-6,8,11,13-(E,Z,Z,E)-eicosatetrae-noic acid (5,15-diHETE), 14,15-dihydro-LTA4 methyl ester, LTB4 methyl ester and N-formyl-met-leu-phe, inhibited the infection of peripheral blood mononuclear leukocytes by EBV (as assessed by the presence of the EBV antigen EBNA) or the production of EBV particles in B95-8 cells by 40% or more. While the mechanism(s) of the antiviral effect of LTB4 agents is unknown, the antiviral activity observed for some LTB4 agents, in particular the 5,15-diHETE, which is a weak agonist of the cell membrane receptors of LTB4, suggests that the site of action of the LTB4 agents may not be the cell surface receptors of LTB4, but rather the intracellular (nuclear) binding site of LTB4, the transcription factor peroxisome proliferator-activated re.ceptor (PPAR(x). Indeed, PPARa is known to be responsive to a wide variety of lipophilic molecules, including unsaturated fatty acids, hypolipidemic drugs (fibrates), glucocorticoids, the prostacyclin (PGI2) stable analog Iloprost and xenobiotics, which indicates a relatively low selectivity of the PPARa binding site-ligand interaction. It thus seems likely that a lipophilic compound such as 5,15-diHETE, which is a close structural analog of LTB4 (both compounds are dihydroxylated derivatives of arachidonic acid) could also be a ligand of PPARa. In fact, it is likely that a wide range of unsaturated fatty acids structurally related to LTB4 could bind to PPARa. PPARS constitute a family of transcription factors that control the expression of a number of enzymes involved in lipid metabolism (including fatty acid degradation) and thus control lipid homeostasis. Because viral replication implies the formation of lipid-containing structures (capsid, envelope), activation of PPARa by LTB4 agents may exert antiviral effects by interfering in viral assembly processes. It is interesting that Steinhart W.L., et al (Virology 70:241, 1976) and Mehl J_K., et al. (Antimicrob. Agents Ch. 18: 269, 1980) have previously reported an antiviral activity of clofibrate and procetophene (two hypolipidemic drugs known to activate PPARa) on HSV-1 in vitro. However, it remains distinctly possible that the binding to and activation of PPARa by LTB4 agents may trigger yet unknown cellular events, resulting in an antiviral activity; furthermore, it is not excluded that LTB4 agents could bind and activate other transcription factor(s).
It is noteworthy that ganciclovir was shown to have IC50 values of 1 and 2.4 M in in vitro assays of viral replication (EBV and HSV, respectively)(Matthews and Boehms, Rev Inf. Dis., 1988, 10, suppl. 3:490).
Preliminary comparative studies show that, in vitro LTB4 exerts potent antiviral activity as compared to acyclovir- It is possible that the structure of LTB4 could be modified in order to provide a molecule with even higher antiviral activity or alternatively, a molecule with increased bioavailability (for example, decreased lipophilicity or enhanced resistance to metabolism in vivo).
Thus, the results indicate that LTB4 is useful in the treatment of viral infections in humans and animals. Because these data show that LTB4 exerts antiviral activity against three types of viruses, and thus is not specific to a single type of virus, it is expected to be useful for the treatment of viral infections caused by any type of viruses_ Prostaglandins (A, B, D, J, E and I) are found to be less active, on a molar basis, than LTB4 when tested in vitro.
iii) Dose Ranges The therapeutically effective amount of the LTB4 agent to be administered will vary with the particular LTB4 agent used, the type or mode of administration, the concurrent use of other active compounds, host age and size, type, severity and spread of infection, response of individual patients, and the like_ In the case of LTB4, it will be administered 3.n sufficient doses to obtain an effective peak or steady-state concentration of about 1 nM to 1000 nM, usually about 200 nM in plasma as suggested by the concentrations of LTB4 found to be effective (in terms of antiviral activity) in in vitro experiments (see Examples I to IV). An effective dose amount of the LTB4 agent is thus be determined by the clinician after a consideration of all the above-mentioned criteria. In the case of LTB4 agents other than LTB4 which have a different biological activity, the effective peak or steady-state concentration required may be different, for instance up to 10 M_ The dosage amount of agent necessary to obtain the desired concentrations in blood can be determined by pharmacokinetic studies, as described in Marleau et al_, J. Immunol. 150: 206, 1993, and Marleau et al, Br. J. Pharmacol. 112: 654, 1994.
iv) Pharmaceutical Compositions Any suitable type or mode of administration may be employed for providing a mammal, especially a human with an effective dosage of a LTB4 agent of the present invention. For example, oral, parenteral and topical may be employed_ Dosage forms include tablets, capsules, powders, solutions, dispersions, suspensions, creams, ointments and aerosols.
The pharmaceutical compositions of the present invention comprise a LTB4 agent as an active ingredient, and a pharmaceutically acceptable carrier and optionally other therapeutic ingredients.
It should be recognized that the LTB4 agent can be used in a variety of ways in vivo_ It can be formulated into pharmaceutical compositions according to any known methods of preparing pharmaceutically useful compositions. In this manner, the fatty acid is combined in admixture with a pharmaceutically acceptable carrier vehicle. Suitable vehicles and their formulation, including human proteins, e.g., human serum albumin, are described for instance in Remington's Pharncaceutical Sciences (16th ed. Osol, A., ed., Mack, Easton, PA [19801). In order to form a pharmaceutically acceptable composition suitable for effective administration, such compositions will contain a therapeutically effective amount of the LTB4 agent or amount resulting in antiviral activity, together with a suitable amount of carrier vehicle.
The amounts required for antiviral effects can be determined by in vivo pharmacological studies.
The LTB4 agent can be formulated as a sterile pharmaceutical composition for therapeutic use which is suitable for intravenous or intraarterial administration. The product may be in a solvent-free form and ready to be reconstituted for use by the addition of a suitable carrier or diluent, or alternatively, it may be in the form of solution which may be aqueous or organic.
For reconstitution of a solvent-free product in accordance with the present invention, one may employ a sterile diluent, which may contain materials generally recognized for approximating physiological conditions. In this manner, the sterile diluent may contain salts and/or buffering agents to achieve a physiologically acceptable tonicity and pH, such as sodium chloride, phosphate and/or other substances which are physiologically acceptable and/or safe for use.
When used as an aqueous solution, the pharmaceutical composition will for the most part contain many of the same substances described above for the reconstitution of a solvent-free product.
When used in solution in an organic solvent, a small volume of the solution containing the fatty acid will be diluted with an aqueous solution that will contain many of the same substances described above for the reconstitution of a solvent-free product. The pharmaceutical composition, for the most part, will thus contain many of the same substances described above for the reconstitution of a solvent-free product.
The LTB4 agent useful in the methods of the present invention may be employed in such forms as, for example, sterile solutions for injection or encapsulated (for instance in liposomes) or embedded (for example in suppositories) for slower long-lasting release.
The LTB4 agent may be used in combination with other agents including, but not limited to, anti-viral agents, anti-cancer agents, immunosuppressive agents, anti-inflammatory agents, cytokines, retinoids and compounds that may reduce uptake, elimination or metabolism of the LTB4 agent such as probenecide or clofibrate.
Where the subject LTB4 agent is to be administered to a host as an anti-viral agent, the agent may be administered, for example, intraarterially, intravenously, intraperitoneally, subcutaneously, intramuscularly, by injection, by suppository, or the like. Because of the high cost of most LTB4 agents and their chemical stability, injection of LTB4 may represent the most advantageous form of administration of the composition of the present invention to a patient in order to achieve a better control of the dosage. The mode of administration by injection includes continuous infusion as well as single or multiple boluses_ Given the short half-life of some LTB4 agents in the circulation (Marleau et al., Br. J. Pharmacol. 112:
654, 1994), their administration as single or multiple boluses may imply the simultaneous use of agents to retard elimination of LTB4 agent and/or to inhibit its metabolism, or alternatively, the use of analogs of LTB4 agents with prolonged half-life in the circulation. Useful administration type or mode also includes the use of implantable internal pumps for continuous infusion into a blood vessel or at different sites such as the peritoneal cavity or.
subcutaneously. Such techniques are disclosed in Cecil's Text Book of Medicine (Chapter 164, 19th Edition, 1992) for the treatment of hepatic cancers.
Transdermal administration by means of a patch containing the LTB4 agent may also be a useful administration mode.
Additional pharmaceutical methods may be employed to control the duration of action. For example, controlled release preparations may be achieved through the use of macromolecules to complex or absorb the agent. The controlled delivery may be achieved by selecting appropriate macromolecules (for example, polyesters, polyamino acids, polyvinyl pyrrolidone, ethylene-vinyl acetate, methyl cellulose, carboxymethyl cellulose, protamine sulfate or serum albumin, the appropriate concentration of macromolecules, as well as the methods of incorporation. In this manner, release of the agent can be controlled.
Another possible method useful in controlling the duration of action by controlled release preparations is the incorporation of the agent into particles of a polymeric material such as polyesters, polyamino acids, hydrogels, poly(lactic acid), or ethylene-vinyl acetate copolymers.
Instead of incorporating the subject fatty acids into polymeric particles, it is also possible to , entrap these materials in microcapsules prepared, for instance, by coacervation techniques or by interfacial polymerization (for example, hydroxymethyl cellulose or gelatin microcapsules and polymethyl methacrylate microcapsules, respectively), in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or in macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences (16th ed_ Osol, A_, ed_, Mack, Easton, PA [19807).
The compositions include compositions suitable for oral or parenteral administration_ Conveniently they are presented in unit dosage form and prepared by any of the methods well-known in the art of pharmacy.
In practical use, the LTB4 agent can be combined as the active ingredient in intimate admixture with a pharmaceutical carrier according to conventional pharmaceutical compounding techniques.
The carrier may take a wide variety of forms depending on the form of preparation desired for administration.
In preparing the compositions for oral dosage form, any of the usual pharmaceutical media may be employed, such as, for example, water, glycols, oils, alcohols, flavoring agents, preservatives, coloring agents and the like in the case of oral liquid preparations, such as, for example, suspensions; elixirs and solutions;
or carriers such as starches, sugars, microcrystalline cellulose, diluents, granulating agents, lubricants, binders, disintegrating agents and the like in the case of oral solid preparations such as, for example, powders, capsules and tablets_ If desired, tablets may be coated by standard aqueous or nonaqueous techniques_ Pharmaceutical compositions of the present invention suitable for oral administration may be presented as discrete units such as capsules, cachets or tablets each containing a predetermined amount of the LTB4 agent, as a powder or granules or as a solution or suspension in an aqueous liquid, a non-aqueous liquid, an oil-in-water emulsion or a water-in-oil emulsion. Such compositions may be prepared by any of the methods of pharmacy such methods including the step of bringing the LTB4 agent into association with the carrier which includes one or more necessary ingredients. In general, the compositions are prepared by uniformly and intimately admixing the LTB4 agent with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product into the desired presentation. For example, a tablet may be prepared by compression of molding, optionally with one or more accessory ingredients.
Compressed tablets may be prepared by compressing in a suitable machine, the active ingredient in a free-flowing form such as powder or granules, optionally mixed with a binder, lubricant, inert diluent, surface active or dispersing agent. Molded tablets may be made by molding in a suitable machine, a mixture of the powdered compound moistened with an inert liquid diluent.
It will be understood that the LTB4 agent is to be administered in pharmacologically or physiologically acceptable amounts, by which is to be understood amounts not harmful to the patient, or amounts where any harmful side effects in individual patients are outweighed by the benefits. Similarly, the LTB4 agent is to administered in a therapeutically effective amount, which is to be understood is an amount meeting the intended therapeutic objectives, and providing the benefits available from administration of LTB4 agent.
The present invention will be more readily understood by referring to the following examples which are given to illustrate the invention rather than to limit its scope.

Assay for EBV-induced clump formation and EBNA
synthesis in peripheral blood mononuclear cells Clump formation Peripheral blood mononuclear cells (PBMC) were obtained from healthy donors after dextran sedimentation and centrifugation on Ficoll-PaqueTM
gradients as previously described by Boyum A_ (Scand.
J. Tmmunol., 1976, 5(5):9). Cells were resuspended in RPMI-1640 medium supplemented with 10% heat inactivated fetal calf serum (FCS) in the presence of infectious EBV, strain B95-8, at a viral titer of 107 transforming units (TFU)/ml. When indicated, EBV-infected PBMC were simultaneously treated (single addition) with different concentrations of LTB4, i.e.
0_3, 3.0 and 30 nM, respectively. Cells were cultured in 96-well microplates (106 cells/ml at 200 l/well) during twelve days, and clump formation, which characterizes the EBV-infected cells was evaluated with an inverted microscope (100 X)(Fig. 1).
Cells were cultured in microplates and the clumps were counted in each well. Results show the mean number of clumps per well + S.D_ in one experiment representative of two (2) other. NS:
nonstimulated cells.
Detection of Epstein-Barr Nuclear Antigen (EBNA) In similar experiments, PBMC were infected with EBV and cultured in the presence or absence of LTB4 (Cascade Biochem Ltd_, Berkshire, U.K.) (LTB4 was added to the concentrations indicated in Fig. 2 at days 0, 3, 6 and 9 of c~_:lture ). After ten days of culture, cells were harvesr-ed for determination of the presence of Epstein-Barr Nuclear Antigen (EBNA), a consequence of EBV infection. Preparation of cell smears, fixation and detection of EBNA by the anti-complement immunofluorescence (ACIF) test were carried out as described by Reedman B.M. and Klein G. (Int. J.
Cancer, 1973, 11:499)_ Smears were prepared by spreading 50 l of a concentrated suspension of washed cells (2 x 106/ml) on clean slides, air dried and fixed in cold acetone (-20 C) during 10 minutes.
Human serum (50 V1) from EBV seropositive donor was used as a source of complement_ Slides were incubated at room temperature in a humid chamber during 45 minutes. Slides were then washed three times in phosphate buffer saline (PBS) and stained with 50 l of fluorescein-5-isothiocyanate (FITC "Isomer I")-conjugated goat IgG fraction anti-human complement C3 (Cappel Research Products, Durham, NC) during 60 minutes at room temperature in a humid chamber.
Slides were washed in PBS (3 times), mounted in PBS:glycerol 1:1 and examined_ Raji and U937 cells were used as positive and negative controls, respectively. The percentage of EBNA-positive cells was decreased by more than 55 % with 30 nM LTB4, and by more than 70% with 100 nM LTB4 (Fig. 2)_ The r=esults illustrated in Fig_ 2 are representative of six (6) other experiments. Cells not exposed to EBV
showed no detectable EBNA antigen.

Synthesis of EBV particles In order to evaluate the effects of LTB4 on the production of newly synthesized viral particles, B95-8 cells, in which EBV replicates, was cultured in the' presence or absence of LTB4 (to the concentrations indicated 3.n Fig. 3 at days 0, 5 and 10) during 14 days. The cells were grown in RPMI-1640 medium supplemented with 10% heat inactivated fetal bovine serum (FBS). When the viability of the cells was <20%, cell-free supernatants were harvested and filtered through a 0.45 m pore size filter, and the viral particles were further concentrated by ultracentrifugation (38,000 x g, 160 min., 4 C).
Viral titers were measured by ACIF test on PBMC and expressed in transforming units per ml (TFU/ml). PBMC
were then infected with these different EBV
preparations and the presence of EBNA was assessed by immunofluorescence (ACIF test). The production of EBV
particles was strongly inhibited by 30 nM and 100 nM
LTB4, as shown by the decrease of EBNA antigen positive cells (70% and 85%, respectively)(Fig. 3).
The results illustrated in Fig. 3 are representative of three (3) other experiments.

ZXAMPLIT II
Assay for HSV-l infection of peripheral blood mononuclear cells Detection of specific HSV-1 antigen PBMC were infected with HSV-1 (strain McIntyre) at a TCID50 of 107/ml and treated or not with different concentrations of LTB4 (LTB4 was added to the concentrations indicated in Fig. 4 at days 0, 2 and 4) as described in Example I. After five (5) days in culture, the presence of a specific HSV-1 related antigen synthetized in the cytoplasma of infected cells was evaluated by immunofluorescence, using a monoclonal antibody (H62)(ImmunoCorp, Montreal, Canada)_ Synthesis of the viral antigen was inhibited by 60% in the presence of 100 nM LTB4 (Fig. 4). The results illustrated in Fig. 4 are representative of five (5) other experiments. Similar results (75%
inhibition) were obtained by using a specific antiserum (from a chronically infected donor) in immunofluorescence assay_ Synthesis of HSV-1 particles In order to evaluate the effect of LTB4 on the synthesis of HSV-1 particles, experiments were performed using Vero cells (obtained from the ATCC).
The cells were grown in M-199 medium (Gibco) WO 97/29751 1'CT/CA97/00099 supplemented with 10% heat-inactivated FBS. When the cells were 80% confluent, supernatants were discarded and adherent cells were infected with HSV-1 (TCID50 107/ml) in M-199 medium supplemented with 2% heat-inactivated FBS, and treated or not with LTB4 (LTB4 was added to the concentrations indicated in Fig_ 5 at days 0, 1 and 3). After five days of culture, cell-free supernatants were harvested and filtered through a 0.45 m pore size filter, and the viral particles were further concentrated by ultra-centrifugation (38,000 x g, 160 min_, 4 C). Concentrated viral preparations were suspended in M-199 medium. Freshly cultured Vero cells were then infected with these different HSV-1 preparations and the percentage of infected cells was evaluated by immunofluorescence using a specific antiserum or the H62 monoclonal antibody_ The synthesis of HSV-1 particles was strongly inhibited in the presence of 30 nM and 100 nM
LTB4 in the cultures as shown by the decrease of HSV-1 antigen positive Vero cells (60% and 70%, respectively) (Fig_ 5)_ The results illustrated in Fig. 5 are representative of four (4) other experiments. Similar results were obtained by infecting PBMC.
Assay for HSV-1 infection in vivo The antiviral effect of LTB4 was also evaluated in an in vivo experimental model-Hairless mice (SKHI
strain, from Charles Rivers, 5-6 week old females) were used in these studies. Stock solution of LTB4 (obtained from Cascade Biochem Ltd. Berkshire, U.K.) in ethanol was filtered through a 0.22 m pore size f ilter. LTB4 dilutions were prepared at a concentration of 10 g/100 l in NaCl 0.9% + glucose 5% (50:50, V/V) containing 0.01% BSA.
For virus inoculation, the mice were immobilized and a small area on the back of the mice was scratched six times with a 27 gauge needle in a crossed-hatched pattern_ Forty (40) l of the virus suspension (HSV-1 strain E-377, 107 TCID50/ml) were applied onto the scratched skin area and the virus suspension was rubbed for 20 seconds on the skin using a plastic tip. The infection induced by virus inoculation generated skin lesions, which appeared at the site of inoculation as early as the third day after inoculation and progressed in the form of a 4-5 mm wide band first towards the sides and then towards the abdomen of the mice. Lesions generally were fully developed 5-6 days after inoculation and formed a continuous band extending from the spinal area to the middle of the abdomen_ HSV-1-infected mice may also develop symptoms such as posterior limb inflammation, skinniness and showed decreased activity level (lethargy). In this model, animals may die from encephalitis after HSV-1 inoculation.
LTB4 was injected intraperitoneally (100 l/mice using 1 ml syringe and 23 gauge needle) immediately before virus inoculation (at day 0) and on days 1, 3, 5, 7 and 9 post-inoculation. The mice were housed in groups of five_ The groups (5 animals/group) consisted of 1) non-inoculated mice (a small area on the back of these animals was scratched and rubbed with 40 l of MEM medium), 2) mice inoculated with HSV-1 receiving intraperitoneal injections of NaCl:glucose + 0.01% BSA, and 3) mice inoculated with HSV-1 receiving intraperitoneal injections of LTB4 dissolved in NaCl:glucose + 0.01% BSA. Twice a day, mice were observed for mE ::urement of skin lesions, assessment of other sympto~ and mortality.
The results obtainea indicate that LTB4 exerts a protective effect against HSV-1 infection in vivo.
As indicated in table 1, uninfected animals (group 1) behaved normally and survived throughout the 14-day period; lesions caused by skin scratching disappeared within 3 or 4 days. HSV-1-infected animals (group 2) developed lesions (as described above), which were maximal (length) between days 4 to 8; during this same period, mice of this group showed posterior limb inflammation, skinniness and were much less active and almost inert when handled. In HSV-1-infected and LTB4-treated animals (group 3), lesions also developed from days 0 to 4, but were much smaller (by -80%) than those observed on animals of group 2, and regressed from day 8_ Furthermore, throughout the experiment, posterior limb inf lammation and skinniness were not observed and no deterioration in the general status of the animals was noted, animals remaining active in cages and when manipulated, as for animals of group 1.
All animals survived throughout the experiment; all surviving animals were sacrificed at day 14.
Table 1 Effect of LTB4 on herpes simplex type 1 infection in vivo GROUP Size of infected skin lesions Survival (cm)/ Infection-associated at day symptoms1 14 Day 4 Day 6 Day 8 Day 10 Non infected 0/0 0/0 0/0 0/0 100%
HSV-1-infected 1-3/1 3-5/2 2-5/3 1-2/2 80%
HSV-1-infected + LTB4 treatment <0.5/0 1-3/0 1-3/0 <0.2/0 100%
1 Observed symptoms on HSV-1-infected mice: a) inflammation (swelling) of posterior limbs); b) skinniness (visual observation); c) reduced activity (lethargy).
Score: 1: symptom a; 2: symptoms a + b; 3: symptoms a + b + c.
FaEAMPL8 I I I

Assay for HIV-l-infection of peripheral blood mononuclear cells The antiviral properties of LTB4 on HIV-1-infection were also evaluated.
Reverse transcriptase activity i.n HIV-1-.infected cells PBMC were resuspended at a density of 106 cells/mi in culture medium (RPMI-1640 supplemented with 10% FBS), and cultured in the presence of 3pg/ml PHA-P (Sigma, St.Louis, MO) and 30 U/ml of recombinant human IL-2 for 2 to 3 days at 37 C under a 5% C02 atmosphere. PHA-stimulated PBMC were resuspended at 1 X 106 cells/ml and were infected with HIV-l=I=$
(various multiplicity of infection: number of infectious virus particles/target cell) in the absence or the presence of .i.ncreasing concentrations of LTB4 (0, 30, 100, and 200 nM). The culture media were changed twice a week and appropriate amounts of LTB4 were added at every medium change. Cell-free culture supernatants were frozen at specific time periods until assayed. Virus replication was monitored either by reverse transcriptase or p24 assays.
Virus stocks were prepared from acutely infected cells. In brief, Molt 4 clone 8 were infected with HIV-l==IB. At the maximal virus production and before extensive cytopathic effects were seen, cells were centrifuged at 300 x g for 5 minutes and the virus-containing supernatant was clarified at 2000 X g for 30 minutes and was filtered through a 0.45 m cellulose acetate membrane to remove cellular debris. Thereafter, the virus-containing supernatants were stored at -80 C in aliquots.

Titration of infectivity was performed by terminal dilution micro assay using the highly susceptible MT-4 cell line.
Reverse transcriptase assay Enzymatic activity was measured with 50 l of cell-free supernatant to which 10 l of a solution A
(5 mM dithiothreitol, 50 mM KC1, 0.05% Triton X-100) and 40 l of a solution B (5 mM MgC12, 0.5 M EGTA, 0.04 mg of poly(rA)-oligo(dT)12-1$= 3 mCi [3H]TTP (40 to 70 Ci/mmol) had been added. After incubation for 1 hour at 37 C, samples were precipitated with one volume of a solution containing 0.15% pyrophosphate and 1.66% trichloroacetic acid prior filtration onto glass fiber filters by using a cell harvester system.
The filters were dried and radioactivity was measured in a liquid scintillation counter (1205/1204 BS Beta-plate; Wallac Oy, Turku, Finland)_ The assays were performed in triplicate_ Lnzymatic p24 assay Quantitative determination of the main viral core p24 protein was achieved with the use of a commercial enzyme-linked immunosorbent assay (Organon Teknika, Durham, NC)_ When LTB4 was present in the culture media, the viral activity of HIV-1 in PBMC evaluated after two weeks of culture was reduced by more than 70% (see Fig. 6). The results illustrated in Fig. 6 are representative of three (3) other experiments.
Similar results were obtained by monitoring the p24 release in supernatants.
Synthes3.s of HIV-1 part,icles This set of experiments was carried out with the Jl_1 cell line, a latently injected cell line derived from the parental cell line Jurkat E6.1. J1.1 cells were resuspended at a density of 106 cells/ml in culture medium (RPMI-1640 supplemented with 10% FBS) and were stimulated with the phorbol ester PMA (20 ng/ml) in the absence or the presence of increasing concentrations of LTB4 (0,30, 100 nM)_ LTB4 was again added (to the same concentrations) 24 hours after the initiation of the cultures. After 48 hours of culture, cell-free supernatants were harvested and the presence of infectious HIV-1 particles was quantitated by end-point titration assay_ End-point titration assay (TCID50) End-point titration was carried out in flat-bottom microtiter wells using four parallel series of ten-fold dilutions of cell-free supernatants. After 5 to 7 days of incubation with MT-4 cells, cell-free supernatants were harvested and tested for the major viral core p24 protein by a commercially available enzymatic assay. The TCID50 was calculated by the method of Reed and Muench.
Our results clearly demonstrate that 100 nM
LTB4 inhibited the synthesis of HIV-1 particles in J1.1 cells by 55% to 79% (Table 2).

Table 2 Inhibition of HIV-1 particles synthesis in J1.1 cells by LTB4 Experiment Non treated LTB4-treated LTB4-treated nM 100 nM
1 10001 511 (49%) 447 (55%) 2 1143 575 (50%) 448 (61%) 3 4630 2053 (56%) 981 (79%) J1.1 cells were treated or not with LTB4_ 1 Number of viral particles eval;_ated by end-point titration assay (TCID50) as described in Example III.
Numbers in parenthesis indicate the percentage of 30 inhibition induced by LTB4.

EXAMPLE IV
Assay for LTB4 cytotoxicity In cell cultures described in Examples I and II, the cytotoxic effect of LTB4 was assessed by the trypan blue dye exclusion test at concentrations up to 30 nM. LTB4 was found to exert no cytotoxic effect (Fig. 7). Cell viability was assessed by the trypan blue exclusion test; values (from 1 experiment representative of 3) represent the mean cell viability in cell cultures (n = 24).
$XAMPLE V
Antiviral effects of LTB4 and acyclovir on EBV
infection PBMC (106 cells/ml) were cultured in microplates (96 wells) at 2 x 105 cells/well and infected with EBV (107 TFU/ml) or HSV-1 (107 TCID50/ml) as described in Examples I and II, respectively. At one hour post-infection, cell cultures were treated with LTB4 (100 nM) or with Acyclovir (acycloguanosine)(1000 nM). Drugs were added every 48 hours of culture. EBV and HSV-1 infections were evaluated at days 7 and 6, respectively, by evaluating the synthesis of viral antigens (Figs. 8a and 8b) as described in Examples I
and II_ The results illustrated in Figs. 8A and 8B
are from 1 experiment, representative of four (4) others. Acyclovir was tested at 1 M only.
Detection of viral antigens were performed by immunofluorescence on 36 cultures.
While the invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains and as may be applied to the essential features hereinbefore set forth, and as follows in the scope of the appended claims.

Claims (17)

WHAT IS CLAIMED IS:

1. Use of a pharmacologically acceptable therapeutically effective amount of a leukotriene B4 (LTB4) agent as an antiviral agent for the prophylaxis or treatment of a viral infection in a human or animal.

2. The use according to claim 1, wherein said viral infection is of a human or animal virus selected from the group consisting of DNA viruses, RNA viruses and Retroviridae.

3. The use according to claim 2, wherein said viruses are DNA viruses selected from the group consisting of parvoviridae, papovaviridae, adenoviridae, herpesviridae, poxviridae and hepadnaviridae.

4. The use according to claim 2, wherein said viruses are herpesviridae selected from the group consisting of EBV, HSV-1, HSV-2, CMV, VZV, HHV-6, HHV-7 and HHV-8.

5. The use according to claim 2, wherein said viruses are RNA viruses selected from the group consisting of picornaviridae, togaviridae, orthomyxoviridae, paramyxoviridae, coronaviridae, reoviridae, oncornaviridae and filoviridae.

6. The use according to claim 2, wherein said virus is a bovine diarrhea virus, a porcine enterovirus, a paramyxoviridae or a bovine respiratory syncytial virus.

7. The use according to claim 1, wherein said LTB4 agent is leukotriene B4[5S,12R-6,8,10,14-(Z,E,E,Z)-eicosatetraenoic acid].

8. The use according to claim 2, wherein said human virus is a Retroviridae selected from HIV-1 or HIV-2.

9. Use of a pharmacologically acceptable therapeutically effective amount of a leukotriene B4 (LTB4) agent as an antiviral agent with one or several different antiviral agents for the prophylaxis or treatment of a viral infection in a human or animal.

10. The use according to claim 9, wherein said different antiviral agents are selected from the group consisting of interferon-.alpha., -.beta., -.gamma., tumor necrosis factor .alpha., ganciclovir, acyclovir, vidarabine, idoxuridine, famciclovir, 3TC, crixivan, nevarepine prostaglandins, prostaglandin analogs and AZT.

11. The use according to claim 1, wherein said human or animal is an immunosuppressed patient or animal, or a patient treated with a drug known to enhance the occurrence of viral infections.

12. The use according to claim 11, wherein said drug is selected from the group consisting of azathioprine, corticosteroids, adriamycine, cyclophosphamide and methotrexate.

13. Use of a pharmacologically acceptable therapeutically effective amount of a leukotriene B4 (LTB4) agent as an antiviral agent for the prophylaxis or treatment of cancer induced by oncogene viruses.

14. The use according to claim 1, wherein said viral infection is of EBV.

15. The use according to claim 1, wherein said viral infection is of HSV-1.

16. The use according to claim 1, wherein said viral infection is of HIV-1.

17. The use according to claim 1, wherein said LTB4 agent is selected from the group consisting of:
LTB4, 14,15-dihydro-LTB4, 17,18-dehydro-LTB4, 19-hydroxy-LTB4, 20-hydroxy-LTB4 and 5(R)-hydroxy, 5-keto, 5(S)hydroperoxy, 5(R)-hydroperoxy and 5-deoxy analogs thereof; leukotriene A4 ("LTA4");
14,15-dihydro-LTA4, 17,18-dehydro-LTA4; 5(S)-hydroxy-6,8,11,14(E,Z,Z,Z)-eicosatetraenoic acid ("5-HETE"), 14,15-dihydro-5-HETE, 17,18-dehydro-5-HETE, and 5(R)-hydroxy, 5-keto, 5(S)-hydroperoxy, 5(R)-hydroperoxy analogs thereof; 12(R)-hydroxy-5,8,10,14(Z,Z,E,Z)-eicosatetraenoic acid ("12-HETE"), 5,6-dihydro-12-HETE, 14,15-dihydro-12-HETE, 17,18-dehydro-12-HETE and 12(S)-hydroxy, 12-keto, 12(S)-hydroperoxy and 12(R)-hydroperoxy analogs thereof; and 12-oxo-5,8,10(Z,Z,E)-dodecatrienoic acid, 15(S)-hydroxy-5,8,11,13(Z,Z,Z,E)-eicosatetraenoic acid ("15-HETE"), 5,6- dihydro-15-HETE, 17,18-dehydro-l5-HETE' and 15(R)-hydroxy, 15-keto, 15(S)-hydroperoxy, and 15(R)-hydroperoxy analogs thereof; 12(S)-hydroxy-5,8,10(Z,E,E)-heptadecatrienoic acid; leukotrienes C4, D4 and E4 and 14,15-dihydro or 17,18-dehydro analogs thereof; N-acyl or N-alkyl derivatives of leukotrienes C4, D4 and E4, and 14,15-dihydro or 17,18-dehydro analogs thereof; 5,12-dihydroxy-6,8,10,14-eicosatetraenoic acid, isomers thereof and 14,15-dihydro or 17,18-dehydro analogs thereof; 5,6-dihydroxy-7,9,11,14-eicosatetraenoic acid, isomers thereof and 14,15-dihydro or 17,18-dehydro analogs thereof; 5,15-dihydroxy-6,8,11,13-eicosatetraenoic acid, 5(S),15(S)-dihydroxy-6,8,11,13(E,Z,Z,E)-eicosatetraenoic acid, isomers thereof and 17,18-dehydro analogs thereof; 8-hydroxy-11(12)-epoxy-5,9,14-eicosatrienoic acid, hepoxilin A3, isomers thereof and 5,6-dihydro or 14,15-dihydro or 17,18-dehydro analogs thereof; 10-hydroxy-11(12)-epoxy-5,8,14-eicosatrienoic acid, hepoxilin B3, isomers thereof and 5,6-dihydro or 14,15-dihydro or 17,18-dehydro analogs thereof; 8,11,12-trihydroxy-5,9,14-eicosatrienoic acid, trioxilin A3, isomers thereof and 5,6-dihydro or 14,15-dihydro or 17,18-dehydro analogs thereof; 10,11,12-trihydroxy-5,8,14-eicosatrienoic acid, trioxilin B3, isomers thereof and 5,6-dihydro or 14,15-dihydro or 17,18-dehydro analogs thereof;
11(12)-epoxy-5,7,9,14-eicosatetraenoic acid, isomers thereof and 14,15-dihydro or 17,18-dehydro analogs thereof; 11,12-dihydroxy-5,7,9,14-eicosatetraenoic acid, isomers thereof and 14,15-dihydro or 17,18-dehydro analogs thereof; 8(9)-epoxy-5,10,12,14-eicosatetraenoic: acid, isomers thereof and 5,6-dihydro or 17,18-dehydro analogs thereof; 8,9-dihydroxy-5,10,12,14-eicosatetraenoic acid, isomers thereof and 5,6-dihydro or 17,18-dehydro analogs thereof; 8,15-dihydroxy-5,9,11.,13-eicosatetraenoic acid, isomers thereof and 5,6-dihydro or 17,18-dehydro analogs thereof; 14(15)-epoxy-5,8,10,12-eicosatetraenoic acid, isomers thereof and 5,6-dihydro or 17,18-dehydro analogs thereof; 14,15-dihydroxy-5,8,10,12-eicosatetraenoic acid, isomers thereof and 5,6-dihydro or 17,18-dehydro analogs thereof; 5-hydroxy-14(15)-epoxy-6,8,10,12-eicosatetraenoic acid, isomers thereof and 17,18-dehydro analogs thereof; 5,14,15-trihydroxy-6,8,10,12-eicosatetraenoic acid, lipoxin B4, isomers thereof and 17,18-dehydro analogs thereof; 5,6,15-trihydroxy-7,9,11,13-eicosatetraenoic acid, lipoxin A4, isomers thereof and 17,18-dehydro analogs thereof;
5(6)-epoxy-15-hydroxy-7,9,11,13-eicosatetraenoic acid, isomers thereof and 17,18-dehydro analogs thereof; 5-hydroxy-6,8,11,14-eicosatetraenoic acid, isomers thereof and 14,15-dihydro or 17,18-dehydro analogs thereof; 8-hydroxy-5,9,11,14-eicosatetraenoic acid, isomers thereof and 5,6-dihydro or 14,15-dihydro or 17,18-dehydro analogs thereof; 9-hydroxy-5,7,11,14-eicosatetraenoic acid, isomers thereof and 14,15-dihydro or 17,18-dehydro analogs thereof; 11-hydroxy-5,8,12,14-eicosatetraenoic acid, isomers thereof and 5,6-dihydro or 17,18-dehydro analogs thereof; 12-hydroxy-5,8,10,14-eicosatetraenoic acid, isomers thereof and 5,6-dihydro or 14,15-dihydro or 17,18-dehydro analogs thereof; 15-hydroxy-5,8,11,13-eicosatetraenoic acid, isomers thereof and 5,6-dihydro or 17,18-dehydro analogs thereof; 9-hydroxy-10,12-octadecadienoic acid and isomers thereof; 13-hydroxy-9,11-octadecadienoic acid and isomers thereof; 12(R)-hydroxy-5,8,14(Z,Z,Z)-eicosatrienoic acid and isomers thereof; 5(6)oxido- or 5,6-dihydroxy-8,11,14-eicosatrienoic acid, isomers thereof and 14,15-dihydro or 17,18-dehydro analogs thereof; 8(9)-oxido- or 8,9-dihydroxy-5,11,14-eicosatrienoic acid, isomers thereof and 5,6-dihydro or 14,15-dihydro or 17,18-dehydro analogs thereof; 11(12)-oxido- or 11,12-dihydroxy-5,8,14-eicosatrienoic acid, isomers thereof and 5,6-dihydro or 14,15-dihydro or 17,18-dehydro analogs thereof; 14(15)-oxido- or 14,15-dihydroxy-5,8,11-eicosatrienoic acid, isomers thereof and 5,6-dihydro or 17,18-dehydro analogs thereof; 20,20,20-trifluoromethyl-LTB4; 19-methyl-LTB4, 19,19-dimethyl-LTB4, 19-fluoro-LTB4, 19,19-difluoro-LTB4, 18,20-difluro-LTB4, 20-fluoro-LTB4; 3-thio-LTB4, 3-hydroxy-LTB4, 3-methyl-LTB4, 3,3-dimethyl-LTB4, 3-fluoro-LTB4, 3,3-difluoro-LTB4, 2,3-difluoro-LTB4, LTB4 methyl-sulfonylamide, LTB4 methylamide, 1-tetrazole LTB4;
sodium or potassium salts of said LTB4 agent; and ester or ether derivatives of said LTB4 agent.