Note: Descriptions are shown in the official language in which they were submitted.
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USE OF LEUKOTRIENE B4 OR ITS ANALOGUES AS ANTIVIRAL AND ANTINEOPLASTIC AGENTS
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
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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
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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.
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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
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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.
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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.
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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
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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.
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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.
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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
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(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
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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
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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~
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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
~~.
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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.
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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..-=
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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
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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.
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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
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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
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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
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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
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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,
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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
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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
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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
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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
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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.
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BXA14PLE I
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
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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
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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)
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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
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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
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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)
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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.
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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)
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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.
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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
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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.
