U.S. patent application number 09/761909 was filed with the patent office on 2002-06-06 for biflavanoids and derivatives thereof as antiviral agents.
This patent application is currently assigned to Advanced Life Sciences, Inc.. Invention is credited to Flavin, Michael T., Lin, Yuh-Meei, Schure, Ralph, Zembower, David E., Zhao, Geng-Xian.
Application Number | 20020068757 09/761909 |
Document ID | / |
Family ID | 26740411 |
Filed Date | 2002-06-06 |
United States Patent
Application |
20020068757 |
Kind Code |
A1 |
Lin, Yuh-Meei ; et
al. |
June 6, 2002 |
Biflavanoids and derivatives thereof as antiviral agents
Abstract
Substantially purified antiviral biflavanoids robustaflavone,
hinokiflavone, amentoflavone, agathisflavone, volkensiflavone,
morelloflavone, rhusflavanone, succedaneaflavanone, GB-1a, and
GB-2a are provided. Antiviral biflavanoid derivatives and salt
forms thereof, e.g., robustaflavone tetrasulfate potassium salt,
and methods for preparing the same are also disclosed.
Pharmaceutical compositions which include the antiviral
biflavanoids, derivatives or salts thereof are also provided alone
or in combination with at least one antiviral agent such as 3TC.
Also disclosed is an improved method for obtaining substantially
pure robustaflavone from plant material. The biflavanoid compounds,
derivatives or salts thereof of the invention may be used in a
method for treating and/or preventing viral infections caused by
viral agents such as influenza, e.g., influenza A and B; hepatitis,
e.g., hepatitis B; human immunodeficiency virus, e.g., HIV-1;
Herpes viruses (HSV-1 and HSV-2); Varicella Zoster virus (VZV); and
measles. For instance, semi-synthetic hexa-O-acetate and
hexa-O-methyl ether derivatives of robustaflavone have been found
to be effective in a method for treating or preventing hepatitis B
viral infections. Compositions which include these robustaflavone
derivatives along with methods for preparing and using the same are
also provided. These compositions may be used alone or in
combination with at least one antiviral agent such as 3TC.
Inventors: |
Lin, Yuh-Meei; (Naperville,
IL) ; Zembower, David E.; (La Grange Park, IL)
; Flavin, Michael T.; (Darien, IL) ; Schure,
Ralph; (Darien, IL) ; Zhao, Geng-Xian;
(Woodridge, IL) |
Correspondence
Address: |
McDonnell Boehnen Hulbert & Berghoff
300 South Wacker Drive, Suite 3200
Chicago
IL
60606
US
|
Assignee: |
Advanced Life Sciences,
Inc.
|
Family ID: |
26740411 |
Appl. No.: |
09/761909 |
Filed: |
January 17, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09761909 |
Jan 17, 2001 |
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09060839 |
Apr 15, 1998 |
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09060839 |
Apr 15, 1998 |
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08842625 |
Apr 15, 1997 |
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08842625 |
Apr 15, 1997 |
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08668284 |
Jun 21, 1996 |
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5773462 |
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60000465 |
Jun 23, 1995 |
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Current U.S.
Class: |
514/340 ; 514/27;
514/456 |
Current CPC
Class: |
C07D 311/30 20130101;
A61K 31/70 20130101; A61K 31/47 20130101; A61K 31/7048 20130101;
A61K 45/06 20130101; A61K 38/212 20130101; A61K 31/55 20130101;
A61K 38/212 20130101; A61K 38/212 20130101; A61K 31/47 20130101;
A61K 38/19 20130101; A61K 38/20 20130101; A61K 31/35 20130101; A61K
2300/00 20130101; A61K 31/35 20130101; A61K 2300/00 20130101; A61K
31/35 20130101; A61K 31/35 20130101; A61K 2300/00 20130101; A61K
31/35 20130101; A61K 2300/00 20130101; A61K 38/19 20130101; A61K
38/19 20130101; A61K 31/47 20130101; A61K 31/352 20130101; A61K
31/55 20130101; A61K 31/55 20130101; C07D 311/32 20130101; A61K
31/35 20130101 |
Class at
Publication: |
514/340 ; 514/27;
514/456 |
International
Class: |
A61K 031/7048; A61K
031/44; A61K 031/352 |
Goverment Interests
[0002] This invention was supported in part by NIAID N01-AI45195
and NIH grant No. 1R43AI40745-01. The U.S. government has certain
rights to this invention.
Claims
1. A method for treating or preventing a viral infection which
comprises administering a composition comprising an antivirally
effective amount of at least one biflavanoid and at least one
antiviral agent.
2. The method according to claim 1, wherein said biflavanoid is
selected from the group consisting of robustaflavone,
hinokiflavone, amentoflavone, agathisflavone, volkensiflavone,
rhusflavanone, succedaneaflavanone, morelloflavone, GB-1a, GB-2a,
derivatives or salts thereof.
3. The method according to claim 2, wherein said biflavanoid is
robustaflavone.
4. The method according to claim 2, wherein said derivatives or
salts thereof comprise alkyl ethers, esters, acid adducts, amines
and sulfates.
5. The method according to claim 4, wherein said robustaflavone
salt is robustaflavone tetrasulfate potassium salt.
6. The method according to claim 4, wherein said robustaflavone
salt is robustaflavone hexa-O-methyl ether.
7. The method according to claim 4, wherein said robustaflavone
salt is robustaflavone hexa-O-acetate.
8. The method according to claim 4, wherein said robustaflavone
salt is robustaflavone tetrasodium salt.
9. The method according to claim 1, wherein said antiviral agent
comprises AZT, ddC, ddI, D4T, lamivudine (3TC), alyclovir,
gancyclovir, fluorinated nucleosides, TIBO derivatives, nevirapine,
saquinavir, .alpha.-interferon and recombinant CD4),
imnmunostimulants (e.g., various interleukins and cytokines),
immunomodulators and antibiotics (e.g., antibacterial, antifungal,
anti-pneumocysitis agents).
10. The method according to claim 4, wherein said antiviral agent
is 3TC.
11. The method according to claim 1, wherein said biflavanoid is
robustaflavone and said antiviral agent is 3TC.
12. The method according to claim 1, wherein said viral infection
is by an influenza virus.
13. The method according to claim 12, wherein said influenza virus
is influenza A or influenza B virus.
14. The method according to claim 1, wherein said viral infection
is by a hepatitis virus.
15. The method according to claim 14, wherein said hepatitis virus
is hepatitis B virus.
16. The method according to claim 1, wherein said viral infection
is by a Herpes virus.
17. The method according to claim 16, wherein said Herpes virus is
HSV-1 or HSV-2 virus.
18. The method according to claim 1, wherein said viral infection
is by a Varicella Zoster Virus (VZV).
19. The method according to claim 18, wherein said viral infection
is by a respiratory virus.
20. The method according to claim 19, wherein said respiratory
virus is a measles virus.
21. The method according to claim 1, wherein the viral infection is
by a retrovirus.
22. The method according to claim 21, wherein the retrovirus is a
human immunodeficiency virus (HIV).
23. The method according to claim 22, wherein human
immunodeficiency virus (HIV) is HIV- 1.
24. A pharmaceutical composition for treating and/or preventing
viral infections which comprises an antivirally effective amount of
at least one substantially purified biflavanoid, at least one
antiviral agent and a pharmaceutically acceptable carrier.
25. The composition according to claim 24 wherein said biflavanoid
comprises robustaflavone, hinokiflavone, amentoflavone,
agathisflavone, volkensiflavone, rhusflavanone,
succedaneaflavanone, morelloflavone, GB-1a, GB-2a, derivatives or
salts thereof.
26. The composition according to claim 25, wherein said biflavanoid
is robustaflavone.
27. The composition according to claim 25, wherein said derivatives
or salts thereof comprise alkyl ethers, esters, acid adducts,
amines and sulfates.
28. The composition according to claim 27, wherein said derivatives
or salts thereof is a robustaflavone salt.
29. The composition according to claim 25, wherein said
robustaflavone salt is robustaflavone tetrasulfate potassium
salt.
30. The composition according to claim 25, wherein said
robustaflavone salt is robustaflavone hexa-O-methyl ether.
31. The composition according to claim 25, wherein said
robustaflavone is robustaflavone hexa-O-acetate.
32. The composition according to claim 25, wherein said
robustaflavone is robustaflavone tetrasodium salt.
33. The composition according to claim 24, wherein said antiviral
agent comprises AZT, ddC, ddI, D4T, lamivudine (3TC), alyclovir,
gancyclovir, fluorinated nucleosides, TIBO derivatives, nevirapine,
saquinavir, .alpha.-interferon and recombinant CD4),
immunostimulants (e.g., various interleukins and cytokines),
immunomodulators and antibiotics (e.g., antibacterial, antifungal,
anti-pneumocysitis agents).
34. The composition according to claim 33, wherein said antiviral
agent is 3TC.
35. The composition according to claim 24, wherein said biflavanoid
is robustaflavone and said antiviral agent is 3TC.
36. A pharmaceutical composition for treating and/or preventing
viral infections which comprises an antivirally effective amount of
a biflavanoid derivative.
37. The composition of claim 36 wherein said biflavanoid derivative
comprises robustaflavone hexa-O-methyl ether, robustaflavone
hexa-O-acetate, and robustaflavone tetrasodium salt and a
pharmaceutically acceptable carrier.
38. A method for treating or preventing a viral infection which
comprises administering an antiviral effective amount of a
biflavanoid derivative.
39. The method according to claim 38 wherein said biflavonoid
derivative comprises robustaflavone hexa-O-methyl ether.
40. The method according to claim 38 wherein said biflavonoid
derivative comprises robustaflavone hexa-O-acetate.
41. The method according to claim 38 wherein said biflavonoid
derivative comprises robustaflavone tetrasodium salt.
42. A method for treating or preventing a hepatitis B viral
infection which comprises administering a composition comprising an
antivirally effective amount of robustaflavone hexa-O-acetate.
43. A method for treating or preventing a hepatitis B viral
infection which comprises administering a composition comprising an
antivirally effective amount of robustaflavone hexa-O-methyl
ether.
44. A method for treating or preventing a hepatitis B viral
infection which comprises administering a composition comprising an
antivirally effective amount of robustaflavone tetrasodium
salt.
45. The method according to claims 42, 43, or 44, further
comprising administering at least one other antiviral agent.
46. The method according to claim 45, wherein said antiviral agent
comprises AZT, ddC, ddI, D4T, lamivudine (3TC), alyclovir,
gancyclovir, fluorinated nucleosides, TIBO derivatives, nevirapine,
saquinavir, .alpha.-interferon and recombinant CD4),
immunostimulants (e.g., various interleukins and cytokines),
immunomodulators and antibiotics (e.g., antibacterial, antifungal,
anti-pneumocysitis agent).
47. The method according to claim 45, wherein said antiviral agent
comprises AZT, ddC, ddI, D4T, lamivudine (3TC), alyclovir,
gancyclovir, fluorinated nucleosides, TIBO derivatives, nevirapine,
saquinavir, .alpha.-interferon and recombinant CD4),
immunostimulants (e.g., various interleukins and cytokines),
immunomodulators and antibiotics (e.g., antibacterial, antifungal,
anti-pneumocysitis agents).
48. The method according to claims 45, wherein said antiviral agent
is 3TC.
49. A pharmaceutical composition for treating and/or preventing a
hepatitis B viral infection which comprises an antivirally
effective amount of robustaflavone hexa-O-acetate and a
pharmaceutically acceptable carrier.
50. A pharmaceutical composition for treating and/or preventing a
hepatitis B viral infection which comprises an antivirallv
effective amount of robustaflavone hexa-O-methyl ether and a
pharmaceutically acceptable carrier.
51. A pharmaceutical composition for treating and/or preventing a
hepatitis B viral infection which comprises an antivirally
effective amount of robustaflavone tetradsodium salt and a
pharmaceutically acceptable carrier.
52. The pharmaceutical composition according to claims 49-51
further comprising at least one other anti-viral agent comprising
AZT, ddC, ddI, D4T, lamivudine (3TC), alyclovir, gancyclovir,
fluorinated nucleosides, TIBO derivatives, nevirapine, saquinavir,
.alpha.-interferon and recombinant CD4), immunostimulants (e.g.,
various interleukins and cytokines), immunomodulators and
antibiotics (e.g., antibacterial, antifungal, anti-pneumocysitis
agents).
Description
CROSS-REFERENCE
[0001] This application is a continuation-in-part of U.S. Ser. No.
08/842,625, filed Apr. 15, 1997 which is continuation-in-part of
U.S. Ser. No. 08/668,284, filed Jun. 21, 1996, which in turn is a
continuation-in-part of provisional application No. 60/000465,
filed Jun. 23, 1995.
FIELD OF THE INVENTION
[0003] The present invention relates to substantially pure
antiviral biflavanoids, e.g., robustaflavone, biflavanoid
derivatives and salts thereof such as esters, ethers, amines,
sulfates, ethylene oxide adducts, and acid salts, and
pharmaceutical compositions containing the same. Representative
examples include hexa-O-acetate and hexa-O-methyl ether derivatives
of robustaflavone and robustaflavone tetrasodium salt. The present
invention also relates to methods for extracting substantially pure
robustaflavone from plant material. The present invention also
relates to a method for preventing and/or treating viral infections
such as hepatitis B, influenza A and B, and HIV which employ
robustaflavone or derivatives thereof alone or in combination with
at least one antiviral agent such as 3TC.
BACKGROUND OF THE INVENTION
[0004] Viruses, an important etiologic agent in infectious disease
in humans and other mammals, are a diverse group of infectious
agents that differ greatly in size, shape, chemical composition,
host range, and effects on hosts. After several decades of study,
only a limited number of antiviral agents are available for the
treatment and/or prevention of diseases caused by viruses such as
hepatitis B, influenza A and B and HIV. Because of their toxic
effects on a host, many antiviral agents are limited to topical
applications. Accordingly, there is a need for safe and effective
antiviral agents with a wide-spectrum of anti-viral activity with
reduced toxicity to the host.
[0005] Since the identification of the human immunodeficiency virus
(HIV) as the causative agent of AIDS,.sup.36,46 the search for safe
and effective treatments for HIV infection has become a major focus
for drug discovery groups around the world. Investigations into the
molecular processes of HIV have identified a number of
macromolecular targets for drug design, such as HIV-1 reverse
transcriptase (HIV-RT), protease and integrase enzymes, and
regulatory proteins (e.g., TAT and REV). Other targets are enzymes
which aid in virus attachment and fusion. HIV-RT is an essential
enzyme in the life cycle of HIV, which catalyzes the transcription
of HIV-encoded single-stranded RNA into double-stranded DNA.
Furthermore, the RNA-dependent DNA polymerase function of HIV-RT
does not have an analogous process in mammalian metabolism, and
thus is a suitable target for a chemotherapeutic agent.
[0006] The hepatitis B virus (HBV) infects people of all ages. It
is one of the fastest-spreading sexually transmitted diseases, and
also can be transmitted by sharing needles or by behavior in which
a person's mucus membranes are exposed to an infected person's
blood, semen, vaginal secretions, or saliva. While the initial
sickness is rarely fatal, ten percent of the people who contract
hepatitis are infected for life and run a high risk of developing
serious, long-term liver diseases, such as cirrhosis of the liver
and liver cancer, which can cause serious complications or
death..sup.1 The World Health Organization lists HBV as the ninth
leading cause of death. It is estimated that about 300 million
persons are chronically infected with HBV worldwide, with over 1
million of those in the United States. The Center for Disease
Control estimates that over 300,000 new cases of acute HBV
infection occurs in the United States each year, resulting in 4,000
deaths due to cirrhosis and 1,000 due to hepatocellular
carcinoma..sup.2 The highest rates of HBV infections occur in
Southeast Asia, South Pacific Islands, Sub-Saharan Africa, Alaska,
Amazon, Bahai, Haiti, and the Dominican Republic, where
approximately 20% of the population is chronically
infected..sup.3
[0007] Hepatitis B virus (HBV) infection is currently the most
important chronic virus infection, but no safe and effective
therapy is available at present. The major therapeutic option for
carriers of HBV is alpha interferon, which can control active virus
replication. However, even in the most successful studies, the
response rate in carefully selected patient groups has rarely
exceeded 40%..sup.5,6 One of the reasons cited for interferon
failure is the persistence of viral supercoiled DNA in the
liver..sup.7
[0008] Recently, lamivudine (3TC) has provided encouraging results
against both HBV and HIV in human clinical trials. Lamivudine is
approved for treatment of HIV infection, and is currently being
evaluated as a treatment for HBV. A recent study of 40
HIV-HBV-coinfected patients showed dramatic decreases in the level
of HBV replication following treatment with 3TC over a 12 month
period, with no observable adverse effects (84). Another agent,
famciclovir, an orally active derivative of the acyclic guanine
derivative penciclovir (85), is approved for treatment of herpes
zoster and acute recurrent genital herpes, and has also shown
promising results against HBV infection in clinical trials, again
with little observable toxicity (86). It is hoped that these agents
will eventually provide promising treatment options for HBV
infection.
[0009] Unfortunately, monotherapy of viral infections often results
in selection for mutant viral strains having resistance to the
antiviral drug being used. Indeed, clinical isolates of mutant HBV
strains have been identified having resistance to 3TC following
treatment with that agent (87,88). It is plausible that combination
therapy of HBV would provide an enhanced antiviral response, while
reducing the danger of resistance selection. Combination of agents
has been shown to be superior in treatment of HIV relative to
monotherapy (89). Thus, the identification of compounds which
inhibit HBV, particularly compounds having structures other than
that of the nuceloside analogues, is of critical importance in the
search for effective anti-HBV regimens.
[0010] Influenza is a viral infection marked by fever, chills, and
a generalized feeling of weakness and pain in the muscle, together
with varying signs of soreness in the respiratory tract, head, and
abdomen. Influenza is caused by several types of myxoviruses,
categorized as groups A, B, and C.sub.4. These influenza viruses
generally lead to similar symptoms but are completely unrelated
antigenically, so that infection with one type confers no immunity
against the other. Influenza tends to occur in wavelike epidemics
throughout the world; influenza A tends to appear in cycles of two
to three years and influenza B in cycles of four to five years.
Influenza is one of the few common infectious diseases that are
poorly controlled by modem medicine. Its annual epidemics are
occasionally punctuated by devastating pandemics. For example, the
influenza pandemic of 1918, which killed over 20 million people and
affected perhaps 100 times that number, was the most lethal plague
ever recorded. Since that time, there have been two other pandemics
of lesser severity, the so-called Asian flu of 1957 and the Hong
Kong flu of 1968. All of these pandemics were characterized by the
appearance of a new strain of influenza virus to which the human
population had little resistance and against which previously
existing influenza virus vaccines were ineffective. Moreover,
between pandemics, influenza virus undergoes a gradual antigenic
variation that degrades the level of immunological resistance
against renewed infection..sup.4
[0011] Anti-influenza vaccines, containing killed strains of types
A and B virus currently in circulation, are available, but have
only a 60 to 70% success rate in preventing infection. The standard
influenza vaccine has to be redesigned each year to counter new
variants of the virus. In addition, any immunity provided is
short-lived. The only drugs currently effective in the prevention
and treatment of influenza are amantadine hydrochloride and
rimantadine hydrochloride..sup.11-13 While the clinical use of
amantadine has been limited by the excess rate of CNS side effects,
rimantadine is more active against influenza A both in animals and
human beings, with fewer side effects..sup.14,15 It is the drug of
choice for the chemoprophylaxis of influenza A..sup.13,16,17
However, the clinical usefulness of both drugs is limited by their
effectiveness against only influenza A viruses, by the uncertain
therapeutic efficacy in severe influenza, and by the recent
findings of recovery of drug-resistant strains in some treated
patients..sup.18-22 Ribavirin has been reported to be
therapeutically active, but it remains in the investigational stage
of development..sup.23,24
[0012] In influenza, amantadine and rimantadine have been shown to
be moderately effective against only influenza A viruses; with
amantadine having excessive side effects. Recently, strains of
influenza A resistant to amantadine and rimantadine have been
isolated. Accordingly, there is a need for new types of therapeutic
antiviral agents against both influenza A and influenza B, as well
as against HBV and HIV.
SUMMARY OF THE INVENTION
[0013] The present invention relates to substantially purified
antiviral biflavanoids, derivatives and salts thereof and
pharmaceutical compositions containing the same; improved methods
for extracting substantially pure robustaflavone from plant
material; methods for preparing derivatives and salts from
antiviral biflavanoids; and methods for treating and/or preventing
viral infections using the antiviral biflavanoids, derivatives and
salts thereof.
[0014] The present invention provides substantially purified
biflavanoids comprising robustaflavone, hinokiflavone,
amentoflavone, agathisflavone, morelloflavone, volkensiflavone,
rhusflavanone, succedaneaflavanone, GB-1a, and GB-2a and
pharmaceutical compositions containing the same. Scheme I
illustrates the chemical structures of these biflavanoids. The
biflavanoids of the invention, extractable from plant materials
derived from a variety of natural sources such as Rhus succedanea
and Garcinia multiflora, were found to be effective in inhibiting
viral activity and may be used in a method for treating and/or
preventing a broad range of viral infections such as Influenza A
and B, hepatitis B and HIV-1, HSV-1, HSV-2, VZV, and measles. It
has been discovered that robustaflavone effectively inhibits
activity of influenza A and B viruses, hepatitis B, HIV-1, HSV-1
and HSV-2. Hinokiflavone and morelloflavone exhibited similar
activity against various strains of HIV-1.
[0015] Anti-viral biflavanoid derivatives and salts and
pharmaceutical compositions containing the same are also
contemplated by the invention. Representative derivatives include
ethers, e.g., methyl ethers, esters, amines, ethylene oxide
adducts, and polymers such as trimers and tetramers of apigenin.
Representative salts include sulfates and acid salts. Methods for
preparing these derivatives and salts are also provided. It has
been discovered for instance that salts of robustaflavone, e.g.,
robustaflavone tetrasulfate potassium salt and robustaflavone
tetrasodium salt, effectively inhibit hepatitis B activity.
Furthermore, robustaflavone hexa-O-acetate and hexa-O-methyl ether
derivatives of robustaflavone were found to be not only potent
inhibitors of HBV replication but with greatly reduced
cytotoxicity. Scheme I illustrates several examples of biflavanoid
derivatives.
[0016] Improved methods for extracting robustaflavone from plant
material are also provided. According to one method, a
substantially pure robustaflavone in greater yields can be obtained
through the use of a particular solvent mixture comprising
toluene/ethanol/pyridine. The improved extraction method eliminates
the use of benzene and requires smaller volumes of pyridine from
the prior reported methods.
[0017] A second improved method for purification of robustaflavone
is also provided which involves acetylation of the extracted
pigment to produce acetates of the pigments. Robustaflavone acetate
is then purified by recrystallization and converted to
robustaflavone by hydrolysis. This method eliminates the use of
pyridine in column chromatography and is ideal for large scale
extraction of robustaflavone.
[0018] Finally, a method for treating and/or preventing viral
infections using antiviral biflavanoids alone or in combination
with one or more other antiviral agents is described.
Representative viral infections include influenza A and B viruses,
hepatitis B and human immunodeficiency virus (HIV-1), HSV-1, HSV-2,
VZV, and measles.
[0019] Accordingly, it is an object of the invention to provide
substantially purified antiviral biflavanoids robustaflavone,
hinokiflavone, amentoflavone, agathisflavone, morelloflavone,
rhusflavanone, succedaneaflavanone, GB-1a, and GB-2a.
[0020] It is another object of the invention to provide antiviral
derivatives and salt forms of biflavanoids robustaflavone,
hinokiflavone, amentoflavone, agathisflavone, morelloflavone,
volkensiflavone, rhusflavanone, succedaneaflavanone, GB-1a, and
GB-2a as well as method of preparation thereof. A representative
example of an antiviral biflavanoid derivatives include
robustaflavone tetrasulfate potassium salt, robustaflavone
tetrasodium salt, robustaflavone hexa-O-acetate, and robustaflavone
hexa-O-methyl ether.
[0021] It is yet another object of the invention to provide
pharmaceutical compositions which include at least one antiviral
biflavanoids such as robustaflavone, hinokiflavone, amentoflavone,
agathisflavone, morelloflavone, volkensiflavone, rhusflavanone,
succedaneaflavanone, GB-1a, GB-2a, derivatives or salts thereof.
Pharmaceutical compositions including an effective amount of at
least one antiviral biflavanoid in combination with at least one
other antiviral agent, e.g. robustaflavone with penciclovar or
lamivudine (3TC), for use in combination antiviral therapy are also
contemplated.
[0022] It is a further object of the invention to provide an
improved method for obtaining substantially pure robustaflavone and
in greater yields than prior procedures.
[0023] It is yet a further object of the invention to provide a
method for treating and/or preventing viral infections which
comprises administering an antiviral effective amount of a
biflavanoid. Representative viral infections are caused by viral
agents such as influenza, e.g., influenza A and B; hepatitis, e.g.,
hepatitis B; human immunodeficiency virus, e.g., HIV-1; HSV-1,
HSV-2, VZV, and measles.
[0024] These and other objects of the invention will become
apparent in light of the detailed description below. 1
DESCRIPTION OF THE DRAWINGS
[0025] FIG. 1 illustrates the effect of treatment with
robustaflavone in DMSO on mean arterial oxygen saturation (mean
SaO.sub.2 (%)) in Influenza A virus-infected mice as described in
Example 11.
[0026] FIG. 2 illustrates the effect of treatment with
robustaflavone in DMSO on mean lung scores in Influenza A
virus-infected mice as described in Example 11.
[0027] FIG. 3 illustrates the effect of treatment with
robustaflavone in DMSO on mean lung weights in Influenza A
virus-infected mice as described in Example 11.
[0028] FIG. 4 illustrates the effect of treatment with
robustaflavone in DMSO on mean virus titers in Influenza A
virus-infected mice as described in Example 11.
[0029] FIG. 5 illustrates the effect of treatment with
robustaflavone in CMC on mean arterial oxygen saturation (mean
SaO.sub.2 (%)) in Influenza A virus-infected mice as described in
Example 11.
[0030] FIG. 6 illustrates the effect of treatment with
robustaflavone in CMC on mean lung scores in Influenza A
virus-infected mice as described in Example 11.
[0031] FIG. 7 illustrates the effect of treatment with
robustaflavone in CMC on mean lung weights in Influenza A
virus-infected mice as described in Example 11.
[0032] FIG. 8 illustrates the effect of treatment with
robustaflavone in CMC on mean virus titers in Influenza A
virus-infected mice as described in Example 11.
DETAILED DESCRIPTION OF THE INVENTION
[0033] All references and patents cited herein are hereby
incorporated by reference in their entirety.
[0034] In one embodiment of the invention, substantially pure
biflavanoids robustaflavone, hinokiflavone, amentoflavone,
agathisflavone, morelloflavone, volkensiflavone, rhusflavanone,
succedaneaflavanone, GB-1a, and GB-2a, derivatives and salts of the
biflavanoids, and pharmaceutical compositions containing the same
are disclosed. Methods for extracting and isolating the
biflavanoids were previously reported..sup.28,37,39,40,53-55
Moreover, methods for preparing derivatives such as the
acetate.sup.37,38 and methyl ethers.sup.39,40 for several of these
biflavanoids are also reported. Representative methods for
preparing biflavanoid derivatives are illustrated in the examples
below. Applicants have determined that these biflavanoids,
especially robustaflavone, were surprisingly effective in
inhibiting one or more activities of viruses such as Influenza A
and B, hepatitis B and HIV-1, HSV-1, HSV-2, VZV, and measles.
[0035] Approximately 100 biflavanoids have been isolated to date,
since the first biflavanoid, a biflavone, was isolated in 1929 by
Furukawa from ginkgo biloba L. as a yellow pigment..sup.44,45,61
Biological activities of several biflavanoids, such as ginkgetin,
have been reported. For instance, peripheral vasodilatation,
anti-bradykinin, and anti-spasmogenic activities have been
observed..sup.48,62 Garcinikolin stimulates RNA synthesis in rat
hepatocyte suspensions..sup.57 Also, agathisflavone, kolaviron,
GB-1 and GB-2 have hepatoprotective activity..sup.33,49
Hinokiflavone, kayaflavone, bilobetin, lophirone A, lophiraic acid,
and sotetusflavone demonstrate inhibitory action on the genome
expression of the Epstein-Barr virus (EBV)..sup.51,52,60 GB-1
exhibits molluscicidal activity,.sup.65 while daphnodorin A,
daphnodorin B, and daphnodorin D possess antimicrobial
activity..sup.34 Hinokiflavone exhibits cytotoxicity against tissue
cultured cells of human mouth epidermoid carcinoma (KB)..sup.56
Amentoflavone and morelloflavone exhibit an inhibitory effect on
lipid peroxidation,.sup.41,59,66 and kolaviron produced
hypoglycemic effects..sup.50 None of these references, however,
disclose or suggest that robustaflavone, hinokiflavone,
morelloflavone, amentoflavone, agathisflavone, volkensiflavone,
rhusflavanone, succedaneaflavanone, GB-1a and GB-2a, especially
robustaflavone and its tetrasulfate potassium salt, have an
inhibitory effect against at least one of influenza, e.g.,
influenza A and B; hepatitis, e.g., hepatitis B; human
immunodeficiency virus, e.g., HIV-1; HSV-1, HSV-2, VZV, and
measles.
[0036] In another embodiment of the invention, an improved method
for extracting substantially pure robustaflavone from natural
sources is also provided. Robustaflavone, 1, a naturally occurring
biflavanoid, was previously isolated, purified, and identified from
the seed-kemels of Rhus succedanea..sup.25 Other sources of
robustaflavone include: seed kernel of Rhus succedanea L.;.sup.25
leaves of Selaginella lepidophylla;.sup.27 leaves of Anacardium
occidentale;.sup.28 leaves and branches of Podocarpus neriifolius
D. Doa;.sup.29 Selaginella denticulata;.sup.30 and Selaginella
willdenowii..sup.31
[0037] The drupes of wax-tree, Rhus succedanea L (Anacardiaceae),
are of great economic importance in that they yield Japan wax.
Earlier work on this species has shown the presence of fustin and
fisetin in the wood, rhoifolin in leaves, japanic acid in the wax,
and ellagic acid, fatty acids, and flavanoids in the seed kernels.
Further studies of the pigment in the seed kernels of wax-tree led
to the isolation of eight biflavanoids, four of which were new.
Concentration of the ethanol extract of the seed kernels yielded,
successively, fractions of ellagic acid, pigment A (hinokiflavone
and robustaflavone) and pigment B (amentoflavone). Further
concentrations gave a crude yellow pigment C which, when subjected
to silica gel column chromatography, afforded fractions C.sub.I
(rhusflavanone, succedaneaflavanone and neorhusfiavanone), C.sub.II
(rhusflavone), and C.sub.III (agathisflavone).
[0038] A prior method for extracting and isolating substantially
pure robustaflavone from plant material was reported..sup.55 This
method, however, used large quantities of benzene and pyridine
which is undesirable for use in large scale extractions and
produced mediocre yields of robustaflavone. The Applicants
discovered improved extraction methods which eliminates benzene,
greatly reduces or eliminates the amount of pyridine used and
produces at least double the quantities of substantially pure
robustaflavone compared to the prior method. In one method of the
invention, solvent mixture comprising toluene/ethanol/formic acid
at a volume ratio ranging about 10-30:2-10:1, preferably about
20:5:1, was found to be useful as a developing solvent mixture for
the dry column procedure. This particular solvent mixture was found
to be especially useful in large scale extractions.
[0039] In a second method for purification of robustaflavone,
extracted pigment is acetylated followed by purification and
hydrolysis of the robustaflavone acetate to produce pure
robustaflavone. This method eliminates the use of pyridine in
column chromatography and is ideal for large scale isolation of
purified robustaflavone. According to the second method, pigment A
was converted to the acetate with an acylating agent in solution.
Suitable, but non-limiting, acylating agents include acetic
anhydride and acetyl chloride. The preferred acylating agent is
acetic anhydride. In general, excess amounts of acylating agent
relative to pigment A are used. Suitable, but non-limiting,
solvents for use in this second method include any suitable solvent
generally used for acetylation reactions including pyridine and
triethylamine. In practicing this invention, pyridine is the
preferred solvent.
[0040] The acetylation reaction is generally performed at room
temperature until completion is reached as ascertained by the usual
organic chemical methods such as thin layer chromatography or Gas
liquid chromatography. Thereafter, the crude acetate is
precipitated out in water and collected by filtration. The crude
precipitate is then washed with water and recrystallized out from a
suitable solvent or solvent mixture. In practicing this invention,
robustaflavone acetate is preferrably recrystallized from a 9:1
solvent mixture of methylene chloride and ethyl acetate. If
desired, additional acetate may be recovered from the mother liquor
by column chromatography. The purified robustaflavone acetate is
then hydrolyzed with excess aqueous alkaline solution such as 2M
aqueous sodium hydroxide solution. In practicing this invention,
robustaflavone acetate hydrolysis mixture is preferably to a
temperature ranging between about 30 to about 100.degree. C.,
preferably around 60.degree. C., for a time sufficient to hydrolyze
off the acetate groups. The alkaline reaction mixture is then
cooled in an ice bath and acidified to about pH 3.0 with excess
amounts of aqueous acid solution, e.g., 3N hydrochloric acid, to
precipitate out robustaflavone as a yellow solid.
[0041] Examples of extraction via the improved extraction methods
of the invention are illustrated in the examples below.
[0042] In yet another embodiment of the invention, a method is
provided for treating and/or preventing viral infections in mammals
comprising administering an antivirally effective amount of a
biflavanoid such robustaflavone, hinokiflavone, amentoflavone,
agathisflavone, morelloflavone, volkensiflavone, rhusflavanone,
succedaneaflavanone, GB-1a, and GB-2a. In practicing this
invention, administration of robustaflavone or derivatives thereof
is preferred. Examples of mammals include humans, primates,
bovines, ovines, porcines, felines, canines, etc. Examples of
viruses may include, but not be limited to, HIV-1, HIV-2, herpes
simplex virus (type 1 and 2) (HSV-1 and 2), varicella zoster virus
(VZV), cytomegalovirus (CMV), papilloma virus, HTLV-1, HTLV-2,
feline leukemia virus (FLV), avian sarcoma viruses such as rous
sarcoma virus (RSV), hepatitis types A-E, equine infections,
influenza virus, arboviruses, measles, mumps and rubella viruses.
More preferably the compounds of the present invention will be used
to treat a human infected with hepatitis and/or influenza virus.
Preferably the compounds of the present invention will also be used
to treat a human exposed or infected (i.e., in need of such
treatment) with the human irnmunodeficiency virus, either
prophylactically or therapeutically.
[0043] Antiviral biflavanoids and derivatives thereof may be
formulated as a solution of lyophilized powders for parenteral
administration. Powders may be reconstituted by addition of a
suitable diluent or other pharmaceutically acceptable carrier prior
to use. The liquid formulation is generally a buffered, isotonic,
aqueous solution. Examples of suitable diluents are normal isotonic
saline solution, standard 5% dextrose in water or in buffered
sodium or ammonium acetate solution. Such formulation is especially
suitable for parenteral administration, but may also be used for
oral administration. It may be desirable to add excipients such as
polyvinylpyrrolidone, gelatin, hydroxy cellulose, acacia,
polyethylene glycol, mannitol, sodium choride or sodium
citrate.
[0044] Alternatively, the compounds of the present invention may be
encapsulated, tableted or prepared in an emulsion (oil-in-water or
water-in-oil) syrup for oral administration. Pharmaceutically
acceptable solids or liquid carriers, which are generally known in
the pharmaceutical formulary arts, may be added to enhance or
stabilize the composition, or to facilitate preparation of the
composition. Solid carriers include starch (corn or potato),
lactose, calcium sulfate dihydrate, terra alba, croscarmellose
sodium, magnesium stearate or stearic acid. talc, pectin, acacia,
agar, gelatin, maltodextrins and microcrystalline cellulose, or
collodial silicon dioxide. Liquid carriers include syrup, peanut
oil, olive oil, corn oil, sesame oil, saline and water. The carrier
may also include a sustained release material such as glyceryl
monostearate or glyceryl distearate, alone or with a wax. The
amount of solid carrier varies but, preferably, will be between
about 10 mg to about 1 g per dosage unit.
[0045] The dosage ranges for administration of biflavanoids or
derivatives thereof are those which produce the desired affect
whereby symptoms of infection are ameliorated. For example, as used
herein, a pharmaceutically effective amount for influenza or
hepatitis infection refers to the amount administered so as to
maintain an amount which suppresses or inhibits circulating virus
throughout the period during which infection is evidenced such as
by presence of anti-viral antibodies, presence of culturable virus
and presence of viral antigen in patient sera. The presence of
anti-viral antibodies can be determined through use of standard
ELISA or Western blot assays for example. The dosage will generally
vary with age, extent of the infection, the body weight and
counterindications, if any, for example, immune tolerance. The
dosage will also be determined by the existence of any adverse side
effects that may accompany the compounds. It is always desirable,
whenever possible, to keep adverse side effects to a minimum.
[0046] One skilled in the art can easily determine the appropriate
dosage, schedule, and method of administration for the exact
formulation of the composition being used in order to achieve the
desired effective concentration in the individual patient. However,
the dosage can vary from between about 0.001 mg/kg/day to about 150
mg/kg/day, but preferably between about 1 to about 50
mg/kg/day.
[0047] The pharmaceutical composition may contain other
pharmaceuticals in conjunction with biflavanoids and derivatives
thereof to treat (therapeutically or prophylactically) antiviral
infections. For example, other pharmaceuticals may include, but are
not limited to, other antiviral compounds (e.g., AZT, ddC, ddI,
D4T, 3TC, acyclovir, gancyclovir, fluorinated nucleosides and
nonnucleoside analog compounds such as TIBO derivatives,
nevirapine, saquinavir, a-interfon and recombinant CD4),
immunostimulants (e.g., various interleukins and cytokines),
immunomodulators and antibiotics (e.g., antibacterial, antifungal,
anti-pneumocysitis agents).
[0048] The following examples are illustrative and do not serve to
limit the scope of the invention as claimed. In these examples,
eleven biflavanoids, amentoflavone (1), agathisflavone (2),
robustaflavone (3), hinokiflavone (4), volkensiflavone (5),
morelloflavone (7), rhusflavanone (9), succedaneaflavanone (11),
GB-1a (13), GB-1a 7"-O-b-glucoside (15), and GB-2a (16), isolated
from Rhus succedanea and Garcinia multiflora, and their methyl
ethers, acetate and sulfate potassium salt, volkensiflavone
hexamethyl ether (6), morelloflavone heptamethyl ether (8),
rhusflavanone hexaacetate (10) succedaneaflavanone hexaacetate
(12), GB-1a hexamethyl ether (14) and robustaflavone tetrasulfate
potassium salt were evaluated for their antiviral activities. The
inhibitory activities against HIV-1 RT and various viruses
including herpes viruses (HSV-1, HSV-2, HCMV, and VZV), and
respiratory viruses (influenza A, influenza B, RSV, parainfluenza
3, adenovirus 5, and measles) were investigated.
EXAMPLE 1
Extraction and Isolation of Biflavanoids
Isolation of Compounds
[0049] Compounds tested were isolated from the seed kernels of Rhus
succedanea obtained from Fukkuoka, Japan, and also from the
heartwood of Garcinia multiflora collected in Taiwan.
[0050] Amentoflavone (1),.sup.53 agathisflavone (2),.sup.54
robustaflavone (.sub.3),.sup.55 hinokiflavone (4),.sup.55
rhusflavanone (9),.sup.37 and succedaneaflavanone (11).sup.28 were
isolated from Rhus succedanea. Rhusflavanone hexaacetate (10) and
succedaneaflavanone hexaacetate (12) were prepared directly from
compounds (9) and (11), respectively..sup.37,38 Volkensiflavone
(5), morelloflavone (7), GB-1a (13), GB-1a glucoside (15), and
GB-2a (16) were isolated from Garcinia multiflora..sup.39,40
Volkensiflavone hexamethylether (6), morelloflavone hexamethylether
(8), and GB-1a hexamethylether (14) were prepared from compounds
(5), (7), and (13), respectively..sup.39,40 Robustaflavone
tetrasulfate potassium salt (17) was prepared from robustaflavone
(3).
[0051] In this example, two procedures for isolating robustaflavone
are described. In the first procedure, robustaflavone was isolated
by a dry-column method using benzene/pyridine/formic acid (20:5:1)
as developing solvent, following an earlier reported
procedure..sup.25 In order to eliminate the use of benzene and
large quantities of pyridine, an improved procedure was developed
wherein benzene and pyridine are replaced with other solvents. The
solvent mixture of toluene/ethanol/formic acid in the ratio of
20:5:1 was used as the developing solvent in the dry-column
procedure. Hinokiflavone was eluted completely from the dry-column
and robustaflavone retained in the column. A mixture of ethanol and
pyridine in the ratio of 4:1 was then used to elute robustaflavone
from the column.
[0052] Extraction of Biflavanoids from Rhus succedanea. The seeds
(16 kg) of Rhus succedanea obtained from Fukuoka, Japan were
coarsely powdered and defatted with benzene. The defatted seeds
were exhaustively extracted with boiling 95% EtOH (150 L). The
combined EtOH extracts were concentrated in vacuo. The yellow
pigments obtained during the concentration were filtered to yield
crude pigment A (yield 0.2%) and pigment B (yield 0.2%).
successively. Further concentration yielded yellow pigment C (ca.
2%).
[0053] Isolation of Robustaflavone from Pigment A. One gram of
pigment A dissolved in 10 mL of pyridine was mixed with 5 g of
silica gel (Kiselgel nach Stahl Type 60 Merck) and evaporated in
vacuo to remove pyridine. The dried yellow powder obtained was
packed on the top of a silica gel column (SiO.sub.2100 g,
4.times.20 cm). The solvent mixture (400 mL) of
benzene/pyridine/formic acid (40:10:2) was passed through the
column. The column was sliced into seven bands (bands 1.about.7
from top to bottom). Extraction of the yellow band 4 with EtOAc and
subsequent concentration of the extract yielded yellow crystals
(200 mg), robustaflavone, which were recrystallized from
pyridine-water, m.p. 350-352.degree. C. (dec.). Mg-HCl test (orange
red color), FeCl.sub.3/EtOH test (brown color). IR cm.sup.-1 (KBr):
3300 (OH), 1655, 1645 (CO), 1610, 1570, 1510, 1505, 1485 (aromatic
ring), UVl.sub.max (MeOH) nm (log .epsilon.): 255 (4.71), 275
(4.44), 300 (4.42), 347 (4.49), l.sub.max (NaOAc-MeOH) nm (log
.epsilon.): 257 (4.66), 277 (4.48), 313 (sh, 4.41), 378 (4.38),
l.sub.max (AlCl.sub.3-MeOH) nm (log .epsilon.): 254 (4.80), 278
(4.45), 300 (4.45), 352 (4.50), 388 (4.43); NMR (DMSO-d.sub.6) (60
MHz) d.sub.ppm: 7.87 (1H, d, J=2 Hz, H-2'), 7.94 (1H, dd, J=2 Hz, 9
Hz, H-6'), 7.09 (1H, d, J=9 Hz, H-5'), 7.97 (2H, d, J=9 Hz, H-2"',
6"'), 7.03 (2H, d. J=9 Hz, H-3"', 5"'), 6.23 (1H, d, J=2 Hz, H-6),
6.52 (1H, d, J=2 Hz, H-8), 6.68 (1H, s, H=8"), 6.80 (1H, s, H-3 or
H3"), 6.83 (1H, s, H-3, or 3"), 13.53 (1H, s, HO-5), 13.28 (1H, s,
HO-5"), 11.23.about.8.63 (4H, br., 4.times.OH), Anal, Calcd. for
C.sub.30H.sub.18O.sub.10.H.sub.2O: C, 64.75; H, 3.62, Found: C,
64.5 1; H, 3.83.
Improved Procedure for Isolating Robustaflavone (Method No. 1)
[0054] Pigment A (10 g) was dissolved in 50 mL of pyridine. The
solution was added to 25 g of silica gel and thoroughly mixed. The
pyridine was removed under reduced pressure using a rotary
evaporator and the dry mixture ground to a fine particle size. To a
600 mL fritted filter funnel, incorporating a coarse porosity
sinter with a disc of filter paper placed over the sinter, was
added 250 g of silica gel. The adsorbed Pigment A was then
carefully placed and spread on the top of the silica gel in the
funnel. The solvent system of toluene/ethanol/formic acid (40:10:2)
(2.5 L) was passed through the funnel to remove the hinokiflavone.
The eluent was collected and concentrated to provide 2.01 g of a
yellow solid which was identified as hinokiflavone and a trace of
robustaflavone.
[0055] The silica gel in the fritted funnel was allowed to dry out
overnight. The top layer containing the adsorbed Pigment A was then
scraped off the remaining silica gel and placed into a fritted
filter fuinel of coarse porosity containing a disc of filter paper.
The silica gel containing the adsorbed pigment A was then eluted
using a mixture of toluene/ethanol/formic acid (40:10:2) (2.5 L),
and then ethanol/pyridine (4:1) (4.5 L). The first eluting solution
was concentrated to afford 1.1 g of a yellow solid which was
identified as a mixture of robustaflavone and hinokiflavone, the
major component being robustaflavone. The second eluting solution
(ethanol/pyridine 4:1) was concentrated to afford robustaflavone
(5.65 g). TLC, NMR, MS, and elemental analysis support these
findings. NMR (H-NMR, .sup.13C-NMR, COSY and HETCOR NMR: see Table
1).
Improved Procedure for Isolating Robustaflavone (Method No. 2)
[0056] Pigment A, a mixture of robustaflavone and hinokiflavone,
was converted to the hexaacetate and pentacetate, respectively,
with acetic anhydride in pyridine. The resulting robustaflavone
hexaacetate was purified by recrystallization from the solvent
mixture of dichloromethane and ethyl acetate to obtain
robustaflavone hexaacetate in the yield of 55%. The mother liquor
could be further purified by column chromatography to offer
additional amounts of robustaflavone hexacetate.
[0057] Robustaflavone hexaacetate was hydrolyzed with 2M aqueous
sodium hydroxide at 60.degree. C. for 30 min. The resulting yellow
solution was further stirred at room temperature for additional 2.5
and then acidified with hydrochloric acid (.about.3N) to pH 3 at
0.degree. C. to precipitate robustaflavone as a yellow solid in a
yield of 89.6%.
Robustaflavone Hexaacetate
[0058] To Pigment A (8.84 g) were added 50 mL of pyridine and 50 mL
acetic anhydride. The resulting solution was allowed to stand at
room temperature for 2 days. The reaction mixture was then poured
into ice water (800 mL) with stirring. The precipitate was
collected by filtration and rinsed with ice water to remove
pyridine and acetic anhydride to afford an ivory solid, 12 g (yield
92.5%), which was recrystallized from the solvent mixture of
dichloromethane and ethyl acetate in the ratio of 9:1 to yield
robustaflavone hexaacetate as a white powder, 6.32 g (55% yield),
m.p. 197-198.degree. C., APCI-MS m/z 791.1 [M+H]; H-NMR
.delta..sub.CDC13 7.93 (d, J=8.6 Hz, 1H, H-6"'), 7.92 (dd, J-8.7
HZ, 2.4 HZ, 2H, H-2',6'), 7.85 (d,=2.1 HZ, 1H, H-2"'), 7.43 (d,
J=2.4 Hz, 1H, H-5"'), 7.31 (d, J=2.1 Hz, 1H, H=6"), 7.28 (d, J=2.1
Hz, 1H, H-8"), 6.86 (d, J=2.1 Hz, 1H, H-6"), 6.68 & 6.66 (each
s, 1H, H-3,3"), 2.44, 2.36, 2.34, 2.21, 2.13 & 2.06 (each, s,
3H, 6x OAc); HPLC Rt 13.31 (column: Zorbax 4.6 mm.times.250 mm,
mobile phase: hexane/ethyl acetate 1.2).
Robustaflavone--Hydrolysis of Robustaflavone Hexaacetate
[0059] To robustaflavone hexaacetate (1 g) was added 10 mL of 2M
aqueous NaOH and the mixture was heated to 60.degree. C. for 30
min. The resulting yellow solution was further stirred at room
temperature for an additional 2.5 and then acidified with 3 N of
hydrochloric acid to pH 3 at 0.degree. C. to precipitate
robustaflavone. The yellow precipitate was collected by filtration
and washed with ice water to obtain robustaflavone as yellow solid,
610 mg (yield 89.6%), m.p. 197-198.degree. C.; APCI-MS m/z 539.3
[M+H].sup.+; H--NMR and .sup.13C--NMR identical to the previous
data; HPLC Rt 42.2 (column: C.sub.18, 4.6 mm.times.15 cm, mobile
phase: 20% MeOH/80% of 1% TFA in water to 100% MeOH, gradient).
Characterization of Robustaflavone
[0060] Robustaflavone was recrystallized from pyridine/water, mp.
350-352.degree. C. (dec.). The compound gave an orange-red color in
the Mg-HCl test and a brown color with alcoholic FeCl.sub.3. The IR
spectrum showed a broad hydroxyl absorption at 3250 cm.sup.-1 and a
conjugated carbonyl absorption at 1650 cm.sup.-1. The UV spectrum
in MeOH exhibited four maxima in the region of 347 (log
.epsilon.4.38), 300 (4.42), 275 (4.44) and 255 (4.71) nm, and
underwent a bathochromic shift on addition of NaOAc or AlC.sub.3.
The UV spectrum in AlCl.sub.3-MEOH was similar to that of in
AlCl.sub.3-MEOH upon addition of HCl, indicating the presence of OH
groups at the 5,7 and 4'positions, and the absence of an
o-dihydroxyl group.
[0061] The NMR spectrum (60 MHz) of robustaflavone exhibited six OH
groups at .delta.13.53 (s, 1H), 13.28 (s, 1H) and 11.23-8.63 (br,
4H); the four protons in the 1,4-disubstituted benzene ring
appeared at .delta.7.97 (d, J=9 Hz, 2H) and 7.03 (d, J=9 Hz, 2H);
the three protons in the 1,3,4-trisubstituted benzene ring appeared
at .delta.7.87 (d, J=9 Hz, 1H), 7.94 (dd, J=2 Hz, 9 Hz, 1H) and
7.09 (d, J=9 Hz, 1H); two aromatic protons appeared as meta-coupled
doublets (J=2 Hz) at 6.23 (1H) and 6.52 (1H); three isolated
protons appeared at d 6.83(s), 6.80(s) and 6.68(s) respectively.
The above evidence suggested that the structure of the compound was
composed of two apigenin units joined by an interflavonyl linkage
of C3"'-C6, i.e. robustaflavone, an isomer of amentoflavone. This
was further supported by examination of its acetate and methyl
ether. Acetylation with pyridine/Ac.sub.2O yielded robustaflavone
hexaacetate (3a) as colorless needles, m.p. 199-200.degree. C.
Methylation with Me.sub.2SO.sub.4/K.sub.2CO.sub.3 in dry acetone
afforded a colorless compound, robustaflavone hexamethylether (3b),
m.p. 300-305.degree. C., C.sub.36H.sub.30O.sub.10, M.sup.30 m/z
622. (The induced change in the chemical shifts (ppm) owing to the
addition of Eu(fod).sub.3 on compound (3b) is represented by an
S-value..sup.35) The S-values of MeO-II-5 and MeO-I-5 were 10.85
ppm (largest) and 2.17 ppm respectively, whereas H-I-8 was 0.34
ppm, indicating the presence of a linkage of CII-3"'-CI-6 as
structure (3b) which was characterized as
hexa-O-methylrobustaflavone by comparison with an authentic sample
(TLC, IR, NMR and MS)..sup.35
1TABLE I Assignment of .sup.13C-.sup.1H HETCOR NMR
.sup.13C-.delta..sub.ppm H-.delta..sub.ppm I-2 164.11.sup.a
>C.dbd. II-2 163.86.sup.a >C.dbd. I-3 102.86 .dbd.CH 6.81(s)
II-3 116.10 .dbd.CH 6.84(s) I-4 181.74.sup.b >CO II-4
181.83.sup.b >CO I-5 161.20.sup.c .dbd.C--OH 13.02(s) II-5
159.61.sup.c .dbd.C--OH 13.23(s) I-6 108.89 .dbd.C< II-6 98.82
.dbd.CH 6.20(d,J=2.0 Hz) I-7 162.06.sup.d .dbd.C--OH
10.82-10.00(br) II-7 163.65.sup.d .dbd.C--OH 10.82-10.00(br) I-8
93.44 >CH 6.65(s,1H) II-8 94.05 >CH 6.49(d,J=2.0 Hz) I-9
161.46 .dbd.C--O-- II-9 157.5 .dbd.C--O-- I-10 103.57 .dbd.C<
II-10 103.72 .dbd.C< I-1' 121.22 .dbd.C< II-1' 120.89
.dbd.C< I-2' 128.55 .dbd.CH 7.99(d,J=8.8 Hz) II-2' 130.87
.dbd.CH 7.79(d,J=2.2 Hz) I-3' 116.01 .dbd.CH 6.96(d,J=8.8 Hz) II-3'
120.86 .dbd.C< I-4' 156.35 .dbd.C--OH 10.40(br) II-4' 159.07
.dbd.C--OH 10.20(br) I-5' 116.01 .dbd.CH 6.96(d,J=8.8 Hz) II-5'
102.86 .dbd.CH 7.05(d,J=8.7 Hz) I-6' 128.55 .dbd.CH 7.99(d,J=8.88
Hz) II-6' 127.57 .dbd.CH 7.93(dd,J=8.7 & 2.2Hz) Assignments
bearing the same alphabetical superscript in the spectrum may be
reversed.
[0062] The high resolution CI mass spectrum provided an M+H ion,
m/z 539.096993, C.sub.30H.sub.19O.sub.10, which requires
539.097821572. The infrared spectrum exhibited a broad hydroxyl
absorption at 3250 cm.sup.-1 and a conjugated carbonyl absorption
at 1650 cm.sup.-1. The UV spectrum in MeOH contained four maxima in
the region of 345 (log .epsilon.4.49), 300 (4.42), 275 (4.44) and
255 (4.71 nm, and underwent a bathochromic shift on addition of
NaOAc or AlCl.sub.3. The UV spectrum in AlCl.sub.3-MeOH was similar
to that obtained in AlCl.sub.3-MeOH on addition of HCl, indicating
the presence of OH groups in the 5,7 and 4' positions, and the
absence of an o-dihydroxy group..sup.26 [l.sup.NaOAc-MeOH (log
.epsilon.) 378 (4.38), 313 (sh 4.41), 277 (4.48), 257(4.66) nm;
l.sup.AlC3-MeOH (log .epsilon.) 388 (4.43), 352 (4.50), 300 (4.45),
278 (4.45), 254 (4.80 nm).
[0063] The NMR (300 MHz) spectrum of robustaflavone contained six
OH groups at .delta.13.25 (1H, s), 13.02 (1H, s), 10.83 (1H, s),
10.40 (1H, s), 10.4.about.10.9 (2H, br.); the four protons in the
1,4-disubstituted benzene ring at .delta.7.98 (2H, d, J=8.88 Hz,
H-2"', 6"'and 6.96 (2H, d, J=8.88 Hz, H-3"', 5"'); the three
protons in the 1,3,4-trisubstituted benzene ring at .delta.7.93
(1H, dd., J=8.7 Hz and 2.2 Hz, H-6'), 7.79 (1H, d. J=2.2 Hz, H-2')
and 7.05 (1H, d, J=8.7 Hz, H-5'); the five aromatic protons at
.delta.6.84 (1H, s, H-3'), 6.8 (1H, s, H-3"), 6.65 (1H, s, H-8"),
6.49 (1H, d, J=2.0 Hz, H-8) and 6.20 (1H, d, J=2 Hz, H-6).
EXAMPLE 2
General Procedure for Synthesizing O-Acyl Biflavanoids
[0064] Procedure 1: To a solution of biflavanoid in anhydrous
dichloromethane containing 20% dry pyridine is added an appropriate
acyl chloride or anhydride at 0.degree. C. or at room temperature.
The mixture is allowed to stand overnight, and the volatiles are
evaporated in vacuo. Alternatively, the mixture is poured into
water and extracted with chloroform. The organic layer is washed
with water and brine, dried over anhydrous sodium sulfate, and
concentrated in vacuo. The residue is chromatographed on
preparative TLC or a silica gel column to afford the
product..sup.78
[0065] Procedure 2: Preparation of acetate: Biflavanoid is reacted
with acetic anhydride in pyridine at room temperature overnight.
The reaction mixture is poured into ice water. The precipitate is
filtered and washed with cold 1% hydrochloric acid and then with
water to give biflavanoid acetate..sup.37
[0066] Rhusflavanone hexaacetate: Acetylation of rhusflavanone (200
mg) with Ac.sub.2O/Pyridine at room temperature for 20 h gave
hexaacetate (110 mg) as micro needles, m.p. 130-131.degree. C.,
EIMS M.sup.- m/z 794; IR cm.sup.-1 (KBr) 1770 (acetoxy CO), 1688
(flavanone CO), 1603, 1560, 1510 and 1490 (arom.); H--NMR .delta.
(CDCl.sub.3): 2.02 (3H, s, AcO-7"), 2.10 (3H, s, AcO-7), 2.15 (3H,
s, AcO-5), 2.28 (3H, s, AcO-4"'), 2.32 (3H, s, AcO-4'), 2.40 (3H,
s, AcO-5"), 2.85-3.06 (4H, m, H-3.3"), 5.45-5.35 (2H, m. H-2, 2"),
6.71 (1H, s, H-6"), 6.91 (1H, s, H-8), 7.14 (2H, d, J=9 Hz, H-3"',
5"'), 7.17 (2H, d, J=9 Hz, H-3', 5'), 7.44 (2H, d, J=9 Hz, H-2"',
6"'), 7.55 (2H, d, J=9 Hz, H-2', 6').
[0067] Succedaneaflavanone hexaacetate: Acetylation of
succedaneaflavanone by Procedure No. 2 produced succedaneaflavanone
hexaacetate as white needles. m.p. 252-255.degree. C. (from
CHCl.sub.3-MeOH), IR cm.sup.-1 (KBr): 1770 (OAc), 1688 (flavanone
CO), 1613, 1560, 1510 (arom.); H--NMR .delta. (CDCl.sub.3): 2.10
(6H, s, AcO-7, 7"), 2.17 (6H, s, AcO-5,5"), 2.33 (6H, s, AcO-4',
4"'), 2.83-3.27, 4H, m, H-3, 3"), 5.63 (2H, dd, J=12 Hz, 4 Hz, H-2,
2"), 6.97 (2H, s, H-8, 8"), 7.25 (4H, d, J=8 Hz, H-3', 5', 3", 5"),
7.58 (4H, d, J=8 Hz, H-2', 6', 2"', 6"').
[0068] Robustaflavone hexaacetate: A solution of robustaflavone
(100 mg, 0.186 mmol) in a mixture of pyridine and acetic anhydride
(1 mL each) was allowed to stand at room temperature for 72 h. The
solution was poured into ice water, and the resulting white
precipitate was collected on a fritted glass funnel and rinsed with
water (123 mg, 84.4%). Following recrystallization from EtOAc/MeOH
(1:1, 1.5 mL), 88.5 mg of off-white microcrystalline material was
obtained. Physical and spectral properties were identical to those
previously reported in the literature..sup.55
EXAMPLE 3
General Procedure for Synthesizing Biflavanoid Ethers
[0069] Preparation of biflavanoid alkyl ethers: To a mixture of
biflavanoid and Ag.sub.2O (catalytic amount) in DMF is added a
corresponding alkyl halide at 10-12.degree. C. After stirring for
2.5-4 h, the reaction mixture is kept in a refrigerator overnight.
The catalyst is filtered, and the filtrate is washed with water and
brine and then concentrated in vacuo. The residue is purified by
column chromatography on silica gel to yield the
product..sup.78
[0070] Preparation of biflavanoid methyl ethers: Biflavanoid is
dissolved in anhydrous acetone and potassium carbonate and dimethyl
sulfate are added. The solution is refluxed for 4 h. The
precipitate (potassium carbonate) is filtered and the filtrate is
concentrated under vacuum. The residue is dissolved in chloroform
and washed with brine, dried with magnesium sulfate and
concentrated under vacuum. The resulting crude product is purified
by silica gel column chromatography or preparative thin layer
chromatography and then recrystallized with ethyl acetate, ethanol,
or chloroform to afford biflavanoid methyl ethers..sup.37
[0071] Volkensiflavone hexamethyl ether (6)
[0072] Volkensiflavanone (200 mg) was dissolved in 30 mL of
anhydrous acetone, and 4 g of potassium carbonate and 3 mL of
dimethyl sulfate were added. The solution was refluxed for 4 h. The
precipitate (potassium carbonate) was filtered and the filtrate was
concentrated under vacuum. The reddish brown oily residue was
dissolved in 15 mL of chloroform and the chloroform solution was
washed with brine twice and then water. The chloroform layer was
dried with magnesium sulfate and concentrated under vacuum. The
residue was purified by silica gel column chromatography and eluted
with the mixture of toluene and ethyl acetate in the ratio of 1:1.
The eluent was concentrated under vacuum and the residue was
recrystallized with methanol/chloroform to obtain 135 mg of white
crystals, m.p. 258-260.degree. C., EIMS M.sup.+ m/z 624; IR
cm.sup.-1 (KBr): 2900, 2950, 2850 (OMe), 1680 (flavanone CO), 1645
(flavone CO), 1600, 1580, 1510 and 1490 (arom.) H--NMR d
(CDCl.sub.3): 3.93 (3H, s, OMe), 3.87 (3H, s, OMe); 3.83 (6H, s,
OMe), 3.77 (3H, s, OMe), 3.67 (3H, s, OMe), 4.90 (1H, d, J=12 Hz,
H-3), 5.8 (1H, d, J=12 Hz, H-2), 6.22 (1H, d, J=2 Hz, H-6), 6.23
(1H, s, H-6"'), 6.32 (1H, d, J=2 Hz, H-8), 6.50 (1H, s, H-3"), 6.63
(1H, s, J=9 Hz, H-3', 5'), 6.87 (2H, d, J=9 Hz, H-3"', 5"'), 7.13
(2H, d, J=9 Hz, H-2', 6'), 7.70 (2H, d, J=9 Hz, H-2"', 6").
[0073] GB-1a hexamethyl ether (14)
[0074] GB-1a (200 mg) was methylated by the method described above.
The resulting crude methyl ether was purified by preparative thin
layer chromatography using ethyl acetate as developing solvent. The
band at Rf 0.35 was scraped off and extracted with ethyl acetate.
The ethyl acetate extract was concentrated under vacuum and the
residue was recrystallized from the solvent mixture of acetone and
hexane (1:1) to afford a white solid, 118 mg, m.p. 132-134.degree.
C., EIMS M.sup.+ m/z 626, IR cm.sup.-1 (KBr), 2990, 2930, 2900,
2830 (OMe); 1675 (flavanone CO), 1600, 1570 and 1515 cm.sup.-1
(arom.); H--NMR .delta.(CDCl.sub.3): 2.72 (2H, m, H-3"), 3.90 (6H,
s, 233 OMe), 3.83 (6H, s, 2.times.OMe), 3.90 (6H, s, 2.times.OMe),
4.70 (1H, d, J=12 Hz, 3-H), 5.28 (1H, m, H-2"), 5.73 (1H, d, J=12
Hz, H-2), 6.08 (1H, d J=2 Hz, H-6), 6.15 (1H, s, H-6"), 6.17 (1H,
d, J=2 Hz, H-8), 6.82 (2H, d, J=8 Hz, H-3', 5'), 6.90 (2H, d, J=8
Hz, H-3"', 5"'), 7.28 (2H, d, J=8 Hz, H-2', 6'), 7.32 (2H, d, J=8
Hz, H-2"', 6"').
[0075] Robustaflavone hexamethyl ether. A solution of
robustaflavone (200 mg, 0.272 mnol) in acetone (20 niL) and
dimethylsulfate (2 mL) containing 3.8 g potassium carbonate was
heated at reflux for 72 h with magnetic stirring. Filtration
followed by evaporation of the filtrate afforded a black, oily
semi-solid. Purification via column chromatography (silica gel,
CHCl.sub.3/MeOH, 98:2) provided the desired product as an off-white
solid (140 mg, 60.6%). Physical and spectral properties were
identical to those previously reported in the
literature..sup.55
EXAMPLE 4
General Procedure for Preparation of Biflavanoid Sulfates
[0076] The dicyclohexylcarbodiimide (DDC)-mediated esterification
of flavones and flavonols with tetrabutylammonium hydrogen sulfate
(TBSHS) resulted in the formation of mono-, di-, and trisulfated
products by controlling the reaction temperature and amount of
reagents. Sulfation occurred mainly at positions 7,4' and 3 of the
flavonoid skeleton and followed the order 7>4'>3..sup.80
Biflavanoid partial sulfate esters are prepared by treating the
biflavanoid with TBAHS (tetrabutylammonium hydrogen sulfate) and
DDC (dicyclohexylcarbodiimide) in pyridine using controlled amounts
of reagents and temperature. The reaction product, sulfate ester
TBA-salt, is separated from minor by-products by gel filtration.
The sulfate ester TBA-salt is converted to the potassium salt by
treatment with saturated methanolic potassium carbonate. The
resulting potassium salt is purified by repeated chromatography on
Sephadex G-10 column using a 0-50% gradient of aqueous
methanol..sup..lambda.
Robustaflavone Tetrasulfate K-salt
[0077] A solution of robustaflavone (46.1 mg, 0.086 mM, 1.0
equivalent) in pyridine (5 mL) was treated with
1,3-dicyclohexylcarbodiimide (DCC) (500 mg, 2.423 mM, 28.17
equivalent) and tetrabutylammonium hydrogen sulfate (TBAHS) (97.5
mg, 0.287 mM, 3.34 equivalent) at 4.degree. C. (in refrigerator)
for 86 hours. The reaction solution was diluted with MEOH and the
dicyclohexylurea precipitate was removed by filtration. The
supernatant was chromatographed on Sephadex LH-20 (3 g, in MEOH)
and eluted with MEOH and a MeOH-acetone (1:1) mixture. The yellow
fractions containing robustaflavone tetrasulfate were concentrated
to 5 mL and then treated with 15 mL of saturated K.sub.2CO.sub.3 in
MEOH. The precipitate of robustaflavone tetrasulfate K-salt was
collected by filtration and washed with MEOH (3ml.times.9) and
water 3 mL.times.5), successively. The MEOH and water washes were
collected separately. The water solution was lyophilized to obtain
72 mg robustaflavone-7,4',7",4"'-tetrasulfate K-salt as a yellow
powder, .sup.1H--NMR (DMSO, 300 MHz) .delta.6.56 (1H, bs, H-6),
7.19 (1H, bs, H-8), 6.78 (1H, s, H-3), 7.75 (1H, dd, J=9.0, 2.0 Hz,
H-6'), 7.87 (1H, d, J=9.0 Hz, H-5'), 8.31 (1H, d, J=2.0 Hz, H-2'),
6.85 (1H, s, H-8), 6.75 (1H, s, H-3"), 7.33 (2H, d, J=9.0 Hz,
H-3"', 5"'), 7.94 (2H, d, J=9.0 Hz, H-2"', 6"').
EXAMPLE 5
General Procedure for Preparation of Biflavanoid Acid Salt
[0078] The dried mixture of biflavanoid, appropriate acid
anhydride, and appropriate catalyst, such as
4-dimethylaminopyridine are dissolved in dry pyridine. The solution
is worked-up by standard methods to yield biflavanoid acid adduct.
The biflavanoid acid can be converted to the potassium salt by
treatment with saturated methanolic potassium carbonate..sup.79
Robustaflavone Tetrasodium Salt
[0079] Robustaflavone (53.8 mg, 0.10 mmol) was dissolved in 0.400
mL of 1.0 M NaOH, and the resulting dark yellow solution was freeze
dried. Following drying, 62 mg (99% of theoretical yield) of a
brick-orange glass was obtained. The product is believed a mixture
of salt forms which may include the tetrasodium salt form.
Robustaflavone tetrasodium salt was assayed for activity and was
approximately 10-fold less active than robustaflavone in the vitro
assay against HBV (see Example 6). It was also moderately active
against adenovirus type 1:EC.sub.50 =32 uM; IC.sub.50>160
uM.
EXAMPLE 6
Antiviral HBV Activity of Biflavanoids
[0080] In this example, robustaflavone and related biflavanoids
were screened for hepatitis B (HBV) antiviral and cytotoxicity
activity.
[0081] Antiviral HBV Assay. The inhibition of HBV replication in
cultures of 2.2.15 cells was assayed using chronically
HBV-producing human liver cells which were seeded into 24-well
tissue culture plates and grown to confluence. Test compounds were
added daily for a nine continuous day period; the culture medium
was collected and stored for analysis of extracellular (virion) HBV
DNA after 0, 3, 6, and 9 days of treatment. The treated cells were
lysed 24 hours following day 9 of treatment for the analysis of
intracellular HBV genomic forms. The overall levels of HBV DNA
(both extracellular and intracellular DNA) and the relative rate of
HBV replication (intracellular DNA) were analyzed quantitatively.
The analysis was performed using blot hybridization techniques and
[.sup.32P]-labeled HBV-specific probes. The HBV DNA levels were
measured by comparison to known amounts of HBV DNA standards
applied to every nitrocellulose membrane (gel or slot blot). An
AMBIS beta scanner, which measures the radioactive decay of the
hybridized probes directly from the nitrocellulose membranes, was
used for the quantitative analysis. Standard curves, generated by
multiple analyses, were used to correlate CPM measurements made by
the beta scanner with relative levels of target HBV DNA. The levels
of HBV virion DNA released into the culture medium were analyzed by
a slot blot hybridization procedure. HBV DNA levels were then
compared to those at day 0 to determine the effect of the test
compound. A known positive drug was evaluated in parallel with test
compounds in each test. This drug was 2',3'-dideoxycytosine
(2',3'-ddC) or lamivdine (3TC). The data were expressed as 50%
effective (virus-inhibitory) concentrations (EC.sub.50). The 90%
effective concentration (EC.sub.90), which is that test drug
concentration that inhibits virus yield by 1 log.sub.10, was
determined from these data. Each test compound's antiviral activity
was expressed as a selectivity index (SI), which is the CC.sub.50
or CC.sub.90, the concentration of compound which killed 50% or 90%
of the treated cells, divided by the EC.sub.50. Generally an SI of
10 or greater is indicative of positive antiviral activity,
although other factors, such as a low SI for the positive control,
are also taken into consideration.
[0082] HBV Cytotoxicity Assays. The toxicity of the test compounds
in cultures of 2.2.15 cells, grown to confluence in 96-well
flat-bottomed tissue culture plates and treated with compounds as
described above, were assayed at four concentrations each in
triplicate cultures, in 3 to 10-fold steps. Untreated control
cultures were maintained on each plate. On each plate, wells
containing no cells were used to correct for light scattering. The
toxicity was determined by the inhibition of the uptake of neutral
red dye, determined by absorbance at 510 nm relative to untreated
cells, 24 hours following day 9 of treatment.
[0083] Analysis of HBV Nucleic Acids and Proteins. HBV viron DNA in
culture medium, and intracellular HBV RI and HBV RNA levels were
determined by quantitative blot hydridization analyses (dot,
Southern, and Northern blot, respectively)..sup.81,82 Nucleic acids
were prepared by previously described procedures. Integrated HBV
DNA, which remains at a stable level per cell during the treatment
periods was used to quantitate the amount of cellular DNA
transferred in each Southern gel lane..sup.81,82 For the HBV RNA
analyses, the levels of b-actin RNA were used to quantitate the
amount of cellular RNA transferred in each Northern gel lane.
Previous examinations of b-actin-specific RNA in confluent cultures
of 2.2.15 cells demonstrated a steady state level of approximately
1.0 pg b-actin RNA/mg unfractionated cellular RNA..sup.81 EC.sub.90
values (10-fold depression of HBV DNA levels relative to untreated
control cultures) were determined by linear regression. .sup.82
EC.sub.90 values were used for comparison since, in this culture
system, DNA levels within 3-fold of control values are not
generally statistically significant..sup.83
[0084] Values of HBV proteins were determined by semi-quantitative
EIA performed as previously described..sup.83 For the EIA analyses,
test samples were diluted (2- to 10-fold) so that the assay values
produced were within the linear dynamic range of the EIA assays.
Standard curves using serial dilutions of positive assay controls
were included in each set of EIA analyses. HBV surface antigen
(HBcAg), preSI protein, and HBc antigen (HBcAg) are released as
extracellular products and were therefore analyzed in culture
medium obtained 24 h following the last treatment dose of
oligonucleotides or 2', 3'-ddc. HBV core antigen (HBcAg) is an
intracellular viral protein and was assayed in cell extracts
produced by Triton-X-100 lysis..sup.83
[0085] Cultures for HBV RNA were maintained on 6-well plates,
cultures for HBV virion DNA analyses were maintained on either 96-
or 24-well plates, and cultures for all other HBV parameters were
maintained on 24-well plates.
[0086] The concentrations of antiviral agents used in these studies
approximates the EC.sub.50 values of the individual agents against
intracellular HBV DNA replication intermediates (HBV RI). Cultures
were treated with the indicated agents for 9 days using standard
procedures. Values reported are the levels of the indicated HBV
markers at the end of the treatment period ("DAY 9") expressed as a
percentage (.+-.standard deviation (S.D.) of the average levels in
the control cultures at the beginning of the treatment period ("DAY
0"). The method of expression permits an analysis of the variation
of the HBV markers in the untreated (control) cultures over the
course of the treatment period. HBV nucleic acid levels were
measured by standard blot hydridization (dot, Southern, or
Northern). HBV protein levels were measured by standard
semi-quantitative ELA methods. Cultures for HBV RNA were maintained
in 6-well culture plates. The levels of each of two major classes
of HBV RNA transcripts are listed separately. Cultures for all
other HBV markers were maintained in 24-well culture plates. For
each treatment, a total of 4 separate cultures were used for the
analysis of each HBV marker at both DAY 0 and DAY 9.
[0087] Results. Tables 2 and 3 present evidence that robustaflavone
is an extremely effective anti-HBV agent against the hepatitis B
virus in comparison to the control drug, 2',3'-ddC. It was observed
from the results that robustaflavone exhibited an impressive in
vitro activity against extracellular (virion) HBV DNA, with an
effective average concentration (EC.sub.50) of 0.25 uM and an
average selectivity index (CC.sub.50/EC.sub.90) of 153; compared to
an effective average EC.sub.50 of 1.4 uM and average SI of 31 for
2',3'-ddC. Furthermore, measurement of the relative rate of HBV
replication intermediates (RI) (intracellular DNA) again indicates
the effectiveness of robustaflavone over the control drug,
2',3'-ddC. Robustaflavone exhibits an effective EC.sub.50 of 0.6 uM
and SI of 80; compared to an EC.sub.50 of 2.4 uM and SI of 24 for
2',3'-ddC. Volkensiflavone hexamethyl ether (6), rhusflavanone
acetate (10) and succendaneaflavanone hexaacetate (12) exhibited
moderate anti-HBV activity while amentoflavone (1),
agathistflavone, hinokiflavone (4), volkensiflavone (5),
rhusflavanone (9) and succendaneaflavanone possessed little or no
anti-HBV activity.
[0088] In summary, measurement of the overall levels of HBV DNA
(both extracellular and intracellular DNA) and the relative rate of
HBV replication intermediate (RI) (intracellular DNA) clearly
demonstrates the effectiveness of robustaflavone against HBV.
2TABLE 2 Anti- HBV activity of biflavanoids, biflavanones, and
related semi-synthetic derivatives Hepatitis B Virus (HBV) HBV
Virion EC.sub.50.sup.1 EC.sub.90.sup.2 SI.sup.3 Sample .mu.M .mu.M
(CC.sub.50/EC.sub.90) 2',3'-ddC* 1.8 9.4 28 Lamivudine (3TC) 0.038
0.16 11200 Penciclovir 0.19 0.92 471 Robustaflavone 0.25 2.2(5.6)
153(60) (0.60) Robustaflavone 2.9 28 >36 hexamethyl ether
Robustaflavone 0.73 2.8(11) >360(163) Hexaacetate (3.9)
Amentoflavone (1) >100 >100 ND Agathisflavone (2) >100
>100 ND Robustaflavone (3) 0.25 2.4 153 Hinokiflavone (4)
>100 >100 ND Volkensiflavone (5) >100 >100 ND
Volkensiflavone 11 108 13 Hexamethylether (6) Rhusflavanone (9)
>100 >100 ND Rhusflavanone 7.1 6.2 2.8 hexaacetate (10)
Succedaneaflavanone (11) >100 >100 ND Succedaneaflavanone 3.5
128 1.9 hexaacetate (12) Robustaflavone sodium 3.1 12 88 salt
Robustaflavone 0.4 3.6 110 tetrasulfate (17) *Positive drug
control; *.sup.150% effective dose; *.sup.290% effective dose
(EC.sub.90); *.sup.3selective index: CC.sub.50/EC.sub.90
[0089]
3TABLE 3 Effect of Antiviral Agents on HBV Proteins and Nucleic
Acids in 2.2.15 Cells Relative Levels of HBV Proteins.sup.a and
Nucleic Acids (Day 9, % of Day 0 Control .+-. SD) Viral DNA Viral
mRNA Extra- Intra- 3.6 kb and 3.6 kb and cellular cellular 2.1 kb
2.1 kb Viral Antigens Treatment DNA DNA mRNA mRNA HBsAg HBeAg HbcAG
Untreated 127 .+-. 8 103 .+-. 11 90 .+-. 12 101 .+-. 10 117 .+-. 11
108 .+-. 5 86 .+-. 10 cells 2',3'-ddC 1 + 1 6 + 1 94 + 7 87 + 9 90
- 12 88 + 10 91 + 9 Robusta- 1 .+-. 1 5 .+-. 1 93 .+-. 10 106 .+-.
11 97 .+-. 6 86 .+-. 6 138 .+-. 8 flavone @ 10 .mu.M .sup.aNucleic
acid and protein levels were determined following nine days of
continuous exposure of confluent cultures of 2.2.15 cells to
robustaflavone, ddC, and drug-free media, and are relative to the
levels on day zero, before addition of drug.
EXAMPLE 7
Antivirial HBV Activity of Combinations of Robustaflavone, 3TC and
PCV
[0090] Lamivudine and penciclovir were purchased from Moravek
Biochemical (La Brea, Calif., U.S.A.). Robustaflavone was isolated
from the seeds of Rhus succedanea, as described above.
[0091] Assay of anti-HBV activity and toxicity in 2.2.15 cells.
Determination of therapeutic concentrations (EC.sub.50 and
EC.sub.90) and toxicity concentrations (IC.sub.50) were determined
as previously described (90). Stock solutions of text drugs were
prepared in DMSO. Culture medium was changed daily and analyzed for
extracellular HBV DNA after nine days of continuous drug exposure.
Extracellular HBV DNA was extracted from the culture media and
analyzed by a slot blot hydridization technique, using a
.sup.32P-labeled Eco RI HBV DNA fragment, as previously described
(91), and quantitated via comparison to HBV standards on a
nitrocellulose filter, using an AMBIS beta scanner. Toxicity was
determined by measuring inhibition of neutral red dye uptake.
Effective concentration (EC.sub.50 and EC.sub.90) and toxicity
(IC.sub.50) values were calculated via comparison with drug-free
controls. Effective concentration values for individual agents are
the mean of six cultures per concentration point. Toxicity values
are the mean of three cultures per concentration point.
[0092] Combination Studies of Robustaflavone, 3TC and PCV in 2.2.15
cells. Cultures were treated with combination of agents as
previously described (92). Briefly, antiviral agents were mixed at
approximately equipotent molar concentrations based on the
EC.sub.50 values. Serial dilutions of these mixtures were then used
to treat cultures as described above along with the appropriate
monotherapies. For these studies, eight cultures were used for each
of six 3-fold dilutions.
[0093] Determination of Mechanism of Action. Measurements of
intracellular and extracellular HBV DNA, 3.6 kb and 2.1 kb mRNA
fragments, and HBsAg, HBeAg and HBcAg protein markers were
conducted as previously described (93).
[0094] Results and Discussion. Robustaflavone exhibited activity
against HBV replication in a chronically infected (90) human
hepatoblastoma cell line (2.2.15), inhibiting the replication of
HBV by 50% relative to drug-free controls at a concentration of
0.25 .mu.M (EC.sub.50, mean of two trials), with an in vitro
therapeutic index (TI, IC50/EC90) of 153 (Table 2). None of the
other compounds tested from the series of biflavanoids showed
significant activity against HBV with an acceptable selectivity
index (Table 2); thus, robustaflavone was the only derivative
selected for further evaluation. In a comparison with several
nucleoside antiviral agents, the anti-HBV activity of
robustaflavone was superior to ddC (EC.sub.50=1.4 .mu.M, TI=30) and
similar to penciclovir (EC.sub.50=0.19 .mu.M, TI=471). Lamivudine
was clearly the most active of the agents evaluated
(EC.sub.50=0.038 .mu.M, TI=11200).
[0095] Combinations of antiviral agents are now accepted to be
superior to monotherapy in certain instances, particularly in
treatment of HIV infection, because of the ability of drug
combinations to overcome resistant mutants which accumulate
following exposure to a single agent (89). Clinical isolates of
3TC-resistant mutants of HBV have recently been reported following
monotherapy with that agent (87,88).
[0096] Robustaflavone was evaluated in combination with two leading
anti-HBV candidates, 3TC and penciclovir (the bioactive form of
famciclovir), in an effort to determine if combinations of
robustaflavone with either of these drugs could potentially offer
synergistic benefits if used as part of an anti-HBV regimen. As
shown in Table 4, combinations of robustaflavone (RF) with 3TC at
differing ratios showed varying degrees of syngergism and
antagonism. It is interesting to note that the most effective ratio
of RF to 3TC was 10:1, which exhibited an EC.sub.50 of 0.054 .mu.M
with respect to RF (0.0054 .mu.M with respect to 3TC). The ratio of
10:1 correlates well with the EC.sub.50 ratio for RF and 3TC
(RF/3TC=6.6). The concentration of 3TC in the 10:1 combination
(0.038 .mu.M) necessary to achieve 90% reduction of HBV replication
was lower than that required by 3TC alone (0.16 .mu.M),
illustrating the potential benefits of the combination. Ratios of
RF:30:1 and 3:1 were less effective than the 10:1 conbination.
Combostat.RTM. analysis.sup.96 of the RF/3Tccombination data
indicated that, at a molar ratio of 10:1, the combination exhibited
syngergism at all dilutions. The RC:3TC ratio of 30:1 exhibited
antagonism at lower drug concentrations, and additive or mildly
syngergistic effects at higher concentrations. At a RF:3TC ratio of
3:1, the combination exhibited antagonism at all drug
concentrations.
[0097] The combinations of RF with penciclovir (PCV) also showed a
clear syngergistic effect. Because RF and PCV have similar
effective concentrations (EC.sub.50 values of 0.25 and 0.19 .mu.M,
respectively) and cellular toxicity concentrations (IC.sub.50
values of 337 and 433 .mu.M, respectively), the synergistic effects
of the agents together was much more obvious. At a RF:PCV ratio of
10:1, the selectiveity index was inferior to either drug alone. At
a RF:PCV ratio of 3:1, the selectivity index improved to 570,
higher than either drug alone, while a RF:PCV ratio of 1:1
exhibited an even higher selectivity index of 684. At the 1:1
combination ratio, the concentration of PCV necessary to achieve
90% reduction of HBV replication was 0.51 .mu.M, nearly two-fold
less than the concentration required of PCV alone (0.92 .mu.M).
Combostat.RTM. analysis of the combination data indicated that a
RF:PCV ratio of 1:1 exhibited synergism at all concentrations. At a
RF:PCV ratio of 3:1, the combination still showed a mild degree of
syngergism at all concentrations except for the very lowest, while
a 10:1 ratio exhibited antagonism at most concentrations. Again,
the ratio exhibiting the greatest syngergistic effect was that
which mirrored the EC.sub.50 ratio for the two agents
(RF/PCV=1.3).
[0098] To determine the likely mechanism of action for
robustaflavone, levels of extracellular and intracellular HBV DNA,
mRNA and protein antigen markers were measured in 2.2.15 cells on
day nine following continuous treatment (93). As presented in Table
3, the levels of extracellular and intracellular HBV DNA were
dramatically decreased relative to drug-free controls for both
robustaflavone and ddC, which was used as a positive control.
Neither the levels of viral mRNA (3.6 and 2.1 kb) nor the three
major viral antigens (HBsAg, HBeAg and HBcAg) were significantly
affected by exposure of the cells to robustaflavone. These results
strongly suggest that robustaflavone acts via inhibition of the
viral DNA polymerase, supported by the fact that results for
robustaflavone were essentially identical to those for ddC, an
established inhibitor of viral polymerases, used as a positive
control.
[0099] In conclusion, robustaflavone represents a novel
non-nucleoside natural product possessing impressive activity
against hepatitis B virus replication, determined in a chronically
replicating transfected human cell line. The paucity of agents
available for the treatment of HBV infection underscore the need
for development of new lead compounds, especially non-nucleoside
structures, which would complement the drugs currently in
development.
4TABLE 4 Antiviral and cytotoxicity effects of robustaflavone (RF),
lamivudine (3TC), penciclovir (PCV) and drug combinations in 2.2.15
cells.sup.a Drug EC.sub.50 EC.sub.90 IC50 TI 3TC or (or
combination) (.mu.M).sup.b (.mu.M).sup.b (.mu.M)
(IC.sub.50/EC.sub.90) PCV (.mu.M).sup.c 3TC 0.038 0.16 1790 11200
-- PCV 0.19 0.92 433 471 -- RF 0.25 2.2 337 153 -- RF + 3TC (30:1)
0.33 1.7 325 191 0.057 RP + 3TC (10:1) 0.054 0.38 340 894 0.038 RF
+ 3TC (3:1) 0.16 0.73 363 497 0.24 RF + PCV (10:1) 0.99 2.9 334 115
0.29 RF + PCV (3:1) 0.22 0.63 360 570 0.21 RF + PCV (1:1) 0.11 0.51
349 684 0.51 .sup.aSingle agents studies were conducted as
described in reference 90. Combination studies were conducted as
described in reference 92. .sup.bEffective concentration necessary
to decreased HBV DNA levels by 50% (EC.sub.50) or 90% (EC.sub.90)
relative to drug-free controls. .sup.cConcentration of 3TC or PCV
in combination with RF required to achieve inhibition of HBV
replication by 90% (EC.sub.90)
EXAMPLE 8
Anti-Respiratory Viral Activity of Biflavanoids
[0100] In this example, robustaflavone and related biflavanoids
were screened for respiratory (influenza A and B, RSV,
parainfluenza 3, adenovirus 5, and measles) antiviral and cytotoxic
activities.
[0101] Anti-Respiratory Viral Assay. The viruses used in the
primary screen for antiviral activity against respiratory viruses
consisted of: (1) Influenza A and B--Virus strains: A/Texas/36/91
(H1N1) (Source: Center for Disease Control (CDC), A/Beijing/2/92
(H3N2) (Source: CDC), B/Panama/45/90 (Source: CDC), A/NWS/33 (H1N1)
(Source: American Type Culture Collection [ATCC]). (All but
A/NWS/33 are tested in the presence of trypsin.); cell lines: Madin
Darby canine kidney (MDCK) cells; (2) Respiratory syncytial
virus--Virus strain: Utah 89 (Source: Utah State Diagnostic
Laboratory, cell line: African green monkey kidney (MA-104) cells;
(3) Parainfluenza type 3 virus--Virus stain: C243 (Source: ATCC);
cell line: African green monkey kidney (MA-104) cells; (4) Measles
virus--Virus strain: CC (Source: Pennsylvania State University;
cell line: African green monkey kidney (BSC-1) cells; and (5)
Adenovirus type 5--Virus strain: Adenoid 75 (Source: ATCC); cell
line: Human lung carcinoma (A549) cells.
[0102] Test compounds were assayed for continual activity and
cytotoxicity. Three methods were used for assay of antiviral
activity: (1) inhibition of the viral cytopathic effect (CPE); (2)
increase in the neutral red (NR) dye uptake; and (3) decrease in
the virus yield. Methods for ascertaining cytotoxicity were visual
observation, neutral red uptake, and viable cell count..sup.32
[0103] Inhibition of the Viral Cytopathic Effect (CPE). The test
for CPE was run in 96-well flat-bottomed microplates and was used
for the initial antiviral evaluation of all new test compounds. In
this CPE inhibition test, seven one-half log.sub.10 dilutions of
each test compound were added to 4 cups containing the cell
monolayer; within 5 min, the virus was then added and the plate
sealed, incubated at 37.degree. C. and CPE read microscopically
when untreated infected controls develop a 3 to 4+ CPE
(approximately 72 h). A known positive drug was evaluated in
parallel with test drugs in each test. This drug was ribavirin for
influenza, measles, respiratory syncytial, and parainfluenza
viruses, and (S)-1-(3-hydroxy-2-phosophonylmethoxypropyl)adenine
(HPMPA) for adenovirus. The data were expressed as 50% effective
(virus-inhibitory) concentrations (EC.sub.50).
[0104] Increase in the Neutral Red (NR) Dye Uptake. The test for
increase in the NR dye uptake was run to validate the CPE
inhibition seen in the initial test, and utilizes the same 96-well
microplates after the CPE has been read. Neutral red dye was added
to the medium; cells not damaged by virus take up a greater amount
of dye, which was read on a computerized microplate autoreader. An
EC.sub.50 value was determined from this dye uptake.
[0105] Decrease in virus yield. Compounds considered active by CPE
inhibition and NR uptake were retested using both CPE inhibition,
and, using the same plate, the effect on reduction of virus yield
was determined by assaying frozen and thawed eluates from each cup
for virus titer by serial dilution onto monolayers of susceptible
cells. Development of CPE in these cells was an indication of
presence of infectious virus. As in the initial tests, a known
active drug (ribavirin) was run in parallel as a positive control.
The 90% effective concentration (EC.sub.90), which was that test
drug concentration that inhibits virus yield by 1 log.sub.10, was
determined from these data.
[0106] Cytotoxicity Assays. These assays consist of visual
observation, neutral red dye uptake, and viable cell count.
[0107] Visual Observation--In the CPE inhibition tests, two wells
of uninfected cells treated with each concentration of test
compound were run in parallel with the infected, treated wells. At
the same time CPE was determined microscopically, the toxicity
control cells were examined microscopically for any changes in cell
appearance compared to normal control cells run in the same plate.
These changes were given a designation conforming to the degree of
cytotoxicity seen (e.g., enlargement, granularity, cells with
ragged edges, a cloudy appearance, rounding, detachment from the
surface of the well, or other changes. These changes were given a
designation of T (100% toxic), Pvh (partially toxic-very heavy
80%), Ph (partially toxic-heavy 60%), P (partially toxic-40%), Psi
partially toxic-slight-20%), or 0 (no toxicity -0%), conforming to
the degree of cytotoxicity seen. A 50% cell inhibitory (cytotoxic)
concentration (IC.sub.50) was determined by regression analysis of
the data.
[0108] Neutral Red Dye Uptake--In the neutral red dye uptake phase
of the antiviral test described above, the two toxicity control
wells also receive neutral red dye and the degree of color
intensity was determined spectrophotometrically. A neutral red
IC.sub.50 was subsequently deternmined.
[0109] Viable Cell Count--Compounds considered to have significant
antiviral activity in the initial CPE and NR tests were retested
for their effects on cell growth. In this test, 12-well tissue
culture plates were seeded with cells (sufficient to be
approximately 20% confluent in the well) and exposed to varying
concentrations of the test drug while the cells were dividing
rapidly. The plates were then incubated in a CO.sub.2 incubator at
37.degree. C. for 72 h, at which time the media-drug solution was
removed and the cells washed. Trypsin was added to remove the
cells, which were then counted using a Coulter cell counter. An
IC.sub.50 was then determined using the average of three separate
counts at each drug dilution.
[0110] Each test compound's antiviral activity was expressed as a
selectivity index (SI), which was the IC.sub.50 or IC.sub.90
divided by EC.sub.50. Generally an SI of 10 or greater was
indicative of positive antiviral activity, although other factors,
such as a low SI for the positive control, were also taken into
consideration.
Anti-Influenza A and Anti-Influenza B Activity
[0111] Compounds 1-6 and 9-12 have been screened for inhibitory
activity against influenza A (strains H1N1 and H3N2) and influenza
B viruses. For these compounds both cytopathic effect inhibition
(CPE) and neutral red uptake test methods were investigated. The
results are displayed on Tables 5-7. For the results shown in
Tables 5-7 the selective index (SI) is calculated as IC.sub.50 (50%
cell inhibitor (cytotoxic) concentration) over the EC.sub.50 (50%
effective concentration).
[0112] Influenza A. Tables 5 and 6 provide data that robustaflavone
(3) had significant antiviral activity towards two influenza A
strains, when compared to the control drug, ribavirin. The
effective concentrations (EC.sub.50) of robustaflavone (3) were 1.9
.mu.g/mL for both influenza A H1N1(Table 5) and H3N2 (Table 6)
strains, as compared to 1.9 and 4.1 ug/mL for the control drug,
ribavirin. The IC.sub.50 values for robustaflavone were 18 and 32
ug/mL, respectively for H1N1 and H3N2 in the CPE assay. However,
the selectivity indexes (SI) for ribavirin were 296 and 137 against
influenza A strains H1N1 and H3N2, respectively, as compared to 9.5
and 17 for robustaflavone (3). The effective neutral red
concentrations (EC.sub.50) of robustaflavone (3) were 2.0 and 1.8
ug/mL for influenza A strains H1N1 and H3N2, respectively and the
IC.sub.50 values were .about.32 and .about.100 ug/miL. This
compared favorably with ribavirin, which had effective neutral red
concentrations of 1.4 and 5.7 ug/mL, respectively for these
strains. The SI's for neutral red uptake for ribavirin were 132 and
70, respectively, toward influenza A strains H1N1 and H3N2, whereas
those for robustaflavone (3) were 16 and 56.
[0113] Amentoflavone (1) also demonstrated significant antiviral
activity against both strains of influenza A. The EC.sub.50 values
of amentoflavone (1) were 3.1 and 4.3 .mu.g/mL, respectively in CPE
inhibition tests. The IC.sub.50 values were 22 and >100
.mu.g/mL, therefore it had SI values of 7.1 and >23 for
influenza A strains H1N1 and H3N2. The other biflavanoids assayed
were either inactive or toxic, except for agathisflavone which
produced an SI value of >18 for the neutral red assay, but only
1 for the CPE assay. The acetylation of rhusflavanone (9), to
rhusflavanone hexaacetate (10), slightly increased both the
activity and toxicity against both influenza A strains in both
assays. The acetylation of succedaneaflavanone (11) did not change
the activity or toxicity considerably, and methylation of
volkensiflavone (5) to volkensiflavone hexamethyl ether (6)
resulted in a decrease in both the activity and the toxicity, in
both the CPE inhibition and the neutral red assays. As shown in
Tables 5 and 6, the modifications to these three compounds did
result in changes in activity and toxicity, but none produced
significant changes in the SI value.
5 TABLE 5 Influenza A (H1N1) Virus: Texas/36/91 CPE Inhibition
Neutral Red EC.sub.50*.sup.1 IC.sub.50*.sup.2 EC.sub.50*.sup.1
IC.sub.50*.sup.2 Sample ug/mL ug/mL SI*.sup.3 ug/mL ug/mL SI*.sup.3
Ribavirin* 1.9 562 296 1.4 185 132 Amentoflavone (1) 3.1 22 7.1 5.3
>100 19 Agathisflavone (2) 6.6 6.5 1.0 5.6 >100 18
Robustaflavone (3) 1.9 18 9.5 2.0 32 16 Hinokiflavone (4) >1.0
1.4 <1.4 1.8 2.0 1.1 Volkensiflavone (5) >32 13 0 15 14 1.0
Volkensiflavone .about.100 <24 0 .about.100 .about.100 0
hexamethyl ether (6) Rhusflavanone (9) >10 8.2 0 24 26 1.1
Rhusflavanone >10 7.2 0 5.6 5.7 1.0 hexaacetate (10) Succedanea-
>3.2 4.9 <1.5 5.2 5.0 1.0 flavanone (11) Succedanea- 5.6 8.2
1.5 7.4 7.4 1.0 flavanone hexaacetate (12) *Positive control drug;
*.sup.150% effective dose; *.sup.250% cell inhibitory (cytotoxic)
concentration; *.sup.3selective index: IC.sub.50/EC.sub.50
[0114]
6 TABLE 6 Influenza A (H3N2) Virus: Beijing/32/92 CPE Inhibition
Neutral Red EC.sub.50*.sup.1 IC.sub.50*.sup.2 EC.sub.50*.sup.1
IC.sub.50*.sup.2 Sample ug/mL ug/mL SI*.sup.3 ug/mL ug/mL SI*.sup.3
Ribavirin* 4.1 562 137 5.7 397 70 Amentoflavone (1) 4.3 >100
>23 6.5 >100 >15 Agathisflavone (2) 24 18 0.8 13 19 1.5
Robustaflavone (3) 1.9 .about.32 17 1.8 .about.100 56 Hinokiflavone
(4) >3.2 1.3 0 1.9 2.2 1.2 Volkensiflavone (5) 56 42 0.8 38 37
1.0 Volkensiflavone .about.100 .about.100 0 .about.100 .about.100 0
hexamethyl ether (6) Rhusflavanone (9) >32 24 0 31 31 1.0
Rhusflavanone >10 5.6 0 5.4 5.3 1.0 hexaacetate (10) Succedanea-
>10 12 <1.2 12 12 1.0 flavone (11) Succedanea- 8.8 12 1.4 5.6
5.6 1.0 flavone (12) *Positive control drug; *.sup.150% effective
dose; *.sup.250% cell inhibitory (cytotoxic) concentration;
*.sup.3selective index: IC.sub.50/EC.sub.50
[0115] Influenza B. Table 7 indicates that robustaflavone had
significant antiviral activity towards influenza B, when compared
to the control drug, ribavirin. The effective concentration
(EC.sub.50) of robustaflavone was an impressive 0.23 ug/mL,
compared to 1.5 for ribavirin. The selectivity index (SI) for
ribavirin was >667 against influenza B; as compared to <435
for robustaflavone. The effective neutral red concentration
(EC.sub.50) of robustaflavone was 0.22 ug/mL, compared to the
control drug, ribavirin, 0.48 ug/mL. The SI for neutral red uptake
for ribavirin was 208, compared to 454 for robustaflavone.
7 TABLE 7 Influenza B Virus: Panama/45/90 CPE Inhibition Neutral
Red EC.sub.50*.sup.1 IC.sub.50*.sup.2 EC.sub.50*.sup.1
IC.sub.50*.sup.2 Sample ug/mL ug/mL SI*.sup.3 ug/mL ug/mL SI*.sup.3
Ribavirin* 1.5 >1000 >667 0.48 100 208 Amentoflavone 0.56 100
178 -- -- -- (1) Agathisflavone 3.2 18 5.6 -- -- -- (2)
Robustaflavone 0.23 .about.100 .about.435 0.22 .about.100 454 (3)
Hinokiflavone (4) >1.0 1.2 <1.2 1.9 2.0 1.0 Volkensiflavone
1.1 38 34 4.5 20 4.4 (5) Volkensiflavone 2.6 .about.100 .about.38
<20 .about.100 5.0 hexamethyl ether (6) Rhusflavanone >4.1 38
9.3 -- -- -- (9) Rhusflavanone >10 4.2 0 -- -- -- hexaacetate
(10) Succedanea- 0.97 15 15 2.2 7.0 3.2 flavanone (11) Succedanea-
5.4 12 2.2 5.9 5.9 1.0 flavanone hexaacetate (12) *Positive control
drug; *.sup.150% effective dose; *.sup.250% cell inhibitory
(cytotoxic) concentration; *.sup.3selective index:
IC.sub.50/EC.sub.50
[0116] Amentoflavone (1) (I-3'-II-8 biapigenin), volkensiflavone
(5) (naringenin I-3-II-8 apigenin), volkensiflavone hexamethyl
ether and succedaneaflavanone (11) (I-6-II-6 binaringenin) also
exhibited favorable antiviral activity against influenza B, having
SI values of 178, 34, 38, and 15, respectively in the CPE assay.
Agathisflavone (2)(I-6-11-8 biapigenin) and rhusflavanone (9)
(I-6-II-8 binaringenin) demonstrated activity against influenza B
virus, with SI values of 5.6 and 9.3, for the CPE assay. However in
neutral red uptake tests, these biflavanoids showed no significant
activity. None of the other biflavinoids assayed contributed
significant activity. Methylation of volkensiflavone (5), to
volkensiflavone hexamethyl ether (6) led to lower activity and
decreased cytotoxicity.
[0117] All of these biflavanoids were relatively inactive toward
parainfluenza type 3, respiratory syncytial, measles, and
adenovirus type 5 viruses, as shown in Table 8 and Table 9, except
amentoflavone (1) and rhusflavanone (9) which exhibited some slight
activity against respiratory syncytial virus and measles virus,
respectively.
8 TABLE 8 Measles Virus Adenovirus Type 5 CPI Inhibition Neutral
Red CPE Inhibition Neutral Red EC.sub.50*.sup.1 IC.sub.50*.sup.2
EC.sub.50*.sup.1 IC.sub.50*.sup.2 EC.sub.50*.sup.1 IC.sub.50*.sup.2
EC.sub.50*.sup.1 IC.sub.50*.sup.2 Sample ug/ml ug/ml SI*.sup.3
ug/ml ug/ml SI*.sup.3 ug/ml ug/ml SI*.sup.3 ug/ml ug/ml SI*.sup.3
Ribavirin* 3 150 50 1 150 150 -- -- -- -- -- -- HPMPA* -- -- -- --
-- -- 30 80 3 8 40 5 Amentoflavone (1) <40 <10 0 <20
<60 3 >100 18 0 >100 74 0 Agathisflavone (2) <60 <10
0 <10 <30 3 15 18 1 22 37 1 Robustaflavone (3) <14 <14
1 -30 .about.100 1 >100 56 0 >100 102 0 Hinokiflavone (4)
<3 <4 1 <5 <11 2 >10 22 0 19 27 1 Volkensiflavone
(5) <12 <10 1 <6 <10 1 56 47 1 >32 30 0
Volkensiflavone <70 <60 1 <7 <13 1 >100 47 0 >100
50 0 hexamethyl ether (6) Rhusflavanone (9) 14 21 2 5 40 8 56 47 1
33 15 0 Rhusflavanone .about.3 <4 0 <10 <10 0 >10 22 0
19 26 1 hexaacetate (10) Succedaneaflavone (11) .about.32 <23 0
<12 <20 1 10 22 0 19 27 1 Succedaneaflavone .about.3.2 <4
0 <2 <200 <1 20 19 1 6 6 1 hexaacetate (12) *Positive
control drug; *.sup.150% effective dose; *.sup.250% cell inhibitory
(cytotoxic) concentration, *.sup.3selective index:
IC.sub.50/EC.sub.50
[0118]
9 TABLE 9 Parainfluenza Type 3 Virus Respiratory Syncytial Virus
CPI Inhibition Neutral Red CPE Inhibition Neutral Red
EC.sub.50*.sup.1 IC.sub.50*.sup.2 EC.sub.50*.sup.1 IC.sub.50*.sup.2
EC.sub.50*.sup.1 IC.sub.50*.sup.2 EC.sub.50*.sup.1 IC.sub.50*.sup.2
Sample ug/ml ug/ml SI*.sup.3 ug/ml ug/ml SI*.sup.3 ug/ml ug/ml
SI*.sup.3 ug/ml ug/ml SI*.sup.3 Ribavirin* 25 245 10 17 331 19 12
120 10 6 60 10 Amentoflavone (1) >100 .about.56 0 .about.32
.about.56 2 .about.10 .about.56 6 .about.21 .about.35 2
Agathisflavone (2) >100 .about.33 0 >100 .about.34 0 >10
.about.8 0 .about.24 .about.15 0 Robustaflavone (3) <100 56 0 39
79 2 >100 .about.56 0 .about.50 .about.100 2 Hinokiflavone (4)
<1 1 0 5 7 1 >3.2 2.5 0 >3.2 6 0 Volkensiflavone (5) 47 50
1 15 44 3 32 56 2 28 13 0 Volkensiflavone <32 .about.15 0 >32
.about.33 0 .about.18 .about.30 2 >32 .about.59 >1 hexamethyl
ether (6) Rhusflavanone (9) >100 47 0 85 30 0 >10 18 0 27 20
0 Rhusflavanone >10 22 0 32 19 0 >10 13 0 19 32 2 hexaacetate
(10) Succedaneaflavone (11) >10 .about.22 0 23 .about.29 0
>10 .about.13 0 .about.16 .about.10 0 Succedaneaflavone >10
.about.14 0 >32 .about.13 0 >3 4 0 6 6 1 hexaacetate (12)
*Positive control drug; *.sup.150% effective dose; *.sup.250% cell
inhibitory (cytotoxic) concentration; *.sup.3selective index:
IC.sub.50/EC.sub.50
EXAMPLE 9
Anti-HIV Viral Activity of Biflavanoids
[0119] We have investigated the anti-HIV-1 RT activity of
biflavanoids isolated from Rhus succedanea, amentoflavone (1),
agathisflavone (2), robustaflavone (3), hinokiflavone (4),
rhusflavanone (9), succedaneaflavanone (11), and from Garcinia
multiflora, volkensiflavone (5), morelloflavone (7), GB-1a (13),
GB-1a 7"-O-b-glucoside (15), GB-2a (16), and their sulfate
potassium salt, methyl ether, and acetyl derivative,
volkensiflavone hexaacetate (6), morelloflavone heptamethyl ether
(8), rhusflavanone hexaacetate (10), succedaneaflavanone
hexaacetate (12), GB-1a hexamethyl ether (14), and robustaflavone
tetrasulfate potassium salt (17).
[0120] Anti-HIV-1 RT Assay. The HIV-1 RT is a 66-kDa recombinant
enzyme obtained from an Escherichia coli expression system using a
genetically engineered plasmid; the enzyme was purified to near
homogeneity. Synthetic DNA segments were used to introduce
initiation and termination codons into the HIV-1 RT coding
sequence, which permits expression of large quantities of HIV-1 RT
in E. coli. The enzyme was shown to be active in RT assays and was
inhibited by several known antiretroviral agents (e.g. AZT and
suramin). The purified recombinant enzyme was sufficiently similar
to the viral enzyme that it can be substituted for the latter in
drug screening assays. The recombinant HIV-1 RT preparation used in
all experiments had a protein concentration of 0.11 ug/mL and an
activity of 238 nmol TTP incorporated per 10 min per mg of protein
at 37.degree. C. Prior to performing an experiment, the enzyme was
diluted tenfold with buffer analogous to that used in the
assay.
[0121] The assay mixture (final volume 100 uL) contained the
following: 50 mM Tris-HCl buffer (pH 8.0), 150 mM KCl, 5 mM MgCl,
0.5 mM ethylene glycol-bis-(b-aminoethylether)-N,N'-tetraacetic
acid (EGTA), 5 mM dithiothreitol 0.3 mM glutathione, 2.5 mg/mL
bovine serum albumin, 41 mM poly A [S260 (mM)=7.8], 9.5 mM oligo
(dT)12-18 [S265(mM)=5.6], 0.05% Triton X-100, 20 mM TTP, and 0.5
mCi of [.sup.3H]TTP. The reaction was started by the addition of 10
uL of HIV-1 RT, and the mixture was permitted to incubate at
37.degree. C. for 1 h. Reactions were terminated by the addition of
25 uL of 0.1 M EGTA followed by chilling on ice. Aliquots of each
reaction mixture (100 uL) were then spotted uniformly onto circular
2.5 cm DE-81 (Whatman) filters, kept at ambient temperature for 15
minutes, and washed four times with 5% aqueous
Na.sub.2HPO.sub.47H.sub.2O. This was followed by two more washings
with doubly distilled H.sub.2O. Finally, the filters were
thoroughly dried and subjected to scintillation counting in a
nonaqueous scintillation fluid.
[0122] For testing enzyme inhibition, five serial dilutions of
samples in DMSO (10 uL) were added to the reaction mixtures prior
to the addition of enzyme (10 uL). The final DMSO concentration
used was 10%. The highest concentration of pure natural products
and plant extracts tested was 200 .mu.g/mL. Control assays are
performed without the compounds or extracts, but an equivalent
volume of DMSO was added. Fargaronine chloride was used as the
positive control substance. This compound was isolated from Fagara
xanthoxyloides Lam. Other positive control substances used were
suramin (IC.sub.50 18 .mu.g/mL) and daunomycin (IC.sub.50 125
.mu.g/mL). The assay procedure and the concentration of all
components were the same as that described above..sup.47
[0123] Anti-HIV-1 RT Assay in Primary Human Lymphocytes
[0124] Cell Culture. Human PBM cells from healthy HIV-1
seronegative and hepatitis B virus seronegative donors were
isolated by Ficoll-Hypaque discontinuous gradient centrifugation at
1,000 x g for 30 min, washed twice with phosphate-buffered saline
(pH 7.2, PBS), and pelleted by centrifugation at 300 x g for 10
min. Before infection, the cells were stimulated by
phytohemagglutinin (PHA) at a concentration of 6 .mu.g/mL for 2-3
days in RPMI 1640 medium, supplemented with 15% heat-inactivated
fetal calf serum, 1.5 mM L-glutamine, penicillin (100 U/mL),
streptomycin (100 .mu.g/mL), and 4 mM sodium bicarbonate
buffer.
[0125] Viruses. HIV-1 (strain LAV-1) was obtained from Dr. P.
Feorino (Emory University, Atlanta, Ga.). The virus was propagated
in human PBM cells using RPMI 1640 medium, as described
previously.sup.58 without PHA or fungizone and supplemented with 26
units/mL of recombinant interleukin-2 (Cetus Corporation,
Emeryville, Calif.) and 7 .mu.g/mL DEAE-dextran (Pharmacia,
Uppsala, Sweden). Virus was obtained from cell-free culture
supernatant and was titrated and stored in aliquots at -70.degree.
C. until use.
[0126] Inhibition of Virus Replication in Human PBM Cells.
Uninfected PHA-stimulated human PBM cells were infected in bulk
with a suitable dilution of virus. The mean reverse transcriptase
(RT) activity of the inocula was about 60,000 dpm RT activity/106
cells/10 mL. This represents, by a limiting dilution method in PBM
cells, a multiplicity of infection of about 0.01. After 1 h, the
cells were uniformly distributed among 25 cm.sup.2 flasks to give a
5 mL suspension containing about 2.times.10.sup.6 cells/mL each.
The samples at twice their final concentration in 5 mL of RPMI 1640
medium, supplemented as described above, were added to the
cultures. The cultures were maintained in a humidified 5%
CO.sub.2-95% air incubator at 37.degree. C. for six days after
injection, at which point all cultures were sampled for supematant
RT activity. Previous studies had indicated that maximum RT levels
were obtained at that time.
[0127] RT Activity Assay. A volume of supernatant (1 mL) from each
culture was clarified of cells at 300 x g for 10 min. Virus
particles were pelleted at 12,000 rpm for 2 h using a Jouan
refrigerated microcentrifuge (Model MR 1822) and suspended in 100
.mu.L of virus disrupting buffer (50 mM Tris-HCl, pH 7.8, 800 mM
NaCl, 20% glycerol, 0.5 mM phenylmethyl sulfonyl fluoride, and 0.5%
Triton X-100).
[0128] The RT assay was performed in 96-well microtiter plates, as
described by Spira..sup.69 The reaction mixture, which contained 50
mM Tris-HCl, pH 7.8, 9 mM MgCl.sub.2, 5 mM dithiothreitol, 4.7
.mu.g/mL (rA)n(dT)12-18, 140 .mu.M dAPT, and 0.22 .mu.M
[.sup.3H]TTP (specific activity 78.0 Ci/mmol, equivalent to 17,300
cpm/pmol; NEN Research Products, Boston, Mass.), was added to each
well. The sample (20 .mu.L) was added to the reaction mixture,
which was then incubated at 37.degree. C. for 2 h. The reaction was
terminated by the addition of 100 .mu.L of 10% trichloroacetic acid
(TCA) containing 0.45 mM sodium pyrophosphate. The acid-insoluble
nucleic acids which precipitated were collected on glass filters
using a Skatron semi-automatic harvester (setting 9). The filters
were washed with 5% TCA and 70% ethanol, dried and placed in
scintillation vials. Scintillation fluid (Ecolite, ICN, Irvine,
Calif.) (4 mL) was added and the amount of radioactivity in each
sample was determined using a Beckman liquid scintillation analyzer
(Model LS 3801). The results were expressed in dpm/mL of original
clarified supernatant. The procedures for the anti-HIV assays in
PBM cells described above have been published..sup.67,69
[0129] Cytotoxicity Studies in PBM Cells. The compounds were
evaluated for their potential toxic effects on uninfected
PHA-stimulated human PBM cells. The cells were cultured with and
without drug for 24 h, at which time radiolabeled thymidine was
added. The assay was performed as described previously..sup.35
Alternately, cells are counted on day 6 using a hemacytometer
and/or Coulter counter as described previously..sup.68
[0130] Median-Effect Method. EC.sub.50 and IC.sub.50 values were
obtained by analysis of the data using the median-effect
equation..sup.42 These values were derived from the
computer-generated median effect plot of the dose-effect data using
a commercially available program..sup.43
[0131] The results shown in Table 10 indicate that both
hinokiflavone (4) and robustaflavone (3) demonstrated similar
activity against HIV-1 RT at an IC.sub.50 (50% inhibition dose) of
35.2 .mu.g/mL and 33.7 .mu.g/mL, respectively. The water soluble
form of robustaflavone, robustaflavone tetrasulfate K-salt (17)
exhibited 95.5% inhibition at a concentration of 200 ug/mL, with an
IC.sub.50 value of 144.4 ug/nL. Amentoflavone (1), agathisflavone
(2), morelloflavone (7), GB-1a (13), and GB-2a (16) were moderately
active against HIV-1 RT with IC.sub.50 values of 64.0 .mu.g/mL,
53.8 .mu.g/mL, 64.7 .mu.g/mL, 127.8 .mu.g/mL, and 94.6 .mu.g/mL,
respectively. The other biflavanoids were either slightly active or
inactive against HIV-1 RT.
[0132] The results of both studies are presented in Table 10. The
results of the inhibitory activity tests using HIV-1 RT enzyme
(p66/p51 heterodimer) indicated that the biflavones, two apigenin
units linked either with C--C or C--O--C bonds, exhibited
significant activity. Robustaflavone (3) (two apigenins linked
through an I-6-II-3' linkage) and hinokiflavone (4) (I-6-O-II-4'
linkage) demonstrated similar activity, with 50% inhibition
(IC.sub.50) at doses of 35.2 .mu.g/mL and 33.7 .mu.g/mL,
respectively. The IC.sub.50 values of amentoflavone (1) (I-8-
II-3'linkage) and agathisflavone (2) (I-6-II-8 linkage) were 64.0
.mu.g/mL and 53.8 .mu.g/mL, respectively.
10TABLE 10 Anti-HIV-1 RT Activity of Biflavanoids Anti-HIV-1 RT
Ant-HIV-1 Cytotoxicity % Inhibition in PBM in PBM Selective at 200
IC.sub.50 cells cells Index Compounds ug/ml (uM) EC.sub.50 (uM)
IC.sub.50 (uM) (SI) Apigenin 72 (443) Naringenin 34.9 Weakly Active
Amentoflavone (1) 97.3 (119) >10.94 35 Agathisflavone (2) 99.8
(100) 7.3, 6.0 25 <1 Robustaflavone (3) 91.4 (65) >100 77
<1 Hinokiflavone (4) 89.0 (62) 4.1 9.1 <1 Volkensiflavone (5)
45.3 Weakly Active 2.2 Volkensiflavone 0.00 Inactive Me.sub.2 (6)
Morelloflavone (7) 99.2 (116) 5.7, 8.0 82 12 Rhusflavanone (9) 14.1
Inactive Rhusflavanone Ac.sub.6 (10) 0.00 Inactive Succedanea- 22.1
Inactive flavanone (11) Succedanea- 0.00 Inactive flavanone
Ac.sub.6 (12) GB-1a (13) 86.0 (236) 38.0 88 2.3 GB-1a Me.sub.6 (14)
0.00 Inactive GB-1a glucoside (15) 1.46 Inactive GB-2a (16) 96
(170) Robustaflavone 95.5 144.4 tetrasulfate K-salt
[0133] Biflavanoids constructed of flavanone-flavone units through
I-3-II-8 linkages were moderately to weakly active, i.e.
morelloflavone (7) (naringenin I-3-II-8 quercetin) demonstrated
moderate activity, with an IC.sub.50 value of 64.7 .mu.g/mL, while
volkensiflavone (5)(narnigenin I-3-II-8 apigenin) was weakly
active. Biflavanones consisting of two naringenin units or
naringenin-eriodictol through I-3-II-8 linkages exhibited moderate
activity, such as GB-1a (13) (IC.sub.50 127.8 .mu.g/ML) and GB-2a
(16) (IC.sub.50 94.6 .mu.g/mL). Biflavanones such as rhusflavanone
(9) and succedaneaflavanone (11), comprised of two naringenin units
linked through either I-6-II-8 or I-6-II-6 linkages, were
completely inactive.
[0134] Other structural characteristics were related to activity in
our study. Methylation of the hydroxyl groups of the biflavanoids
resulted in diminished activity. For instance, morelloflavone
heptamethyl ether (8), volkensiflavone hexamethyl ether (6), and
GB-1a hexamethyl ether (14), were inactive; all had exhibited
moderate activity before alkylation. The fact that
GB-1a-7"-O-glucoside (15), demonstrated no activity indicated that
the 7"-hydroxyl group was especially important for anti-HIV-1 RT
activity.
[0135] Six biflavanoids that were determined to be active in the
HIV-1 RT enzyme assay were tested in human PBM cells infected with
HIV-1 (strain LAV). These results are presented in Table 10. It has
been observed that, although robustaflavone (3) exhibited
significant inhibitory activity in the HIV-1 RT enzyme assay, it
was found to be inactive in the assay for the PBM cells infected
with HIV-1. However morelloflavone (7), in the whole cell assay,
exhibited potent inhibitory activity with an EC.sub.50 (50%
effective dose) value of 5.7 (8.0) .mu.g/mL. Morelloflavone only
possessed moderate activity in the anti-HIV-1 RT assay (IC.sub.50
64.7 .mu.g/mL; 116.3 .mu.M). This may suggest that the activity of
these biflavanoids may be dependent upon different cellular
mechanisms.
[0136] Other active compounds were hinokiflavone (4) and GB-1a
(13), which exhibited good activity inhibiting viral replication in
human PBM cells, but also high toxicity against uninfected
PHA-stimulated human PBM cells. The other compounds (amentoflavone
(1) and agathisflavone (2)) assayed in PBM cells appeared to either
lack antiviral potency or display poor selectivity. From these
results, it was concluded that biflavanoids comprised of flavanone
(naringenin) and flavone (luteolin) via a I-3-II-8 bond demonstrate
the most promising anti-HIV-1 activity.
[0137] In the past, some monoflavonoids have been reported to
demonstrate anti-HIV activity. Baicalein (5,6,7-trihydroxyflavone),
tiliroside (kaempferol 3-beta-D (6"-p-coumaroyl)glucoside),
quercetin (3,3',4',5,7-pentahydroxyflavone), kaempferol
(3,4',5,7-tetrahydroxyflavo- ne), and quercetagetin
(3,3',4',5,6,7-hexahydroxyflavone) exhibited inhibitory activity
against HIV-1 reverse transcriptase, whereas luteolin
(3',4',5,7-tetrahydroxyflavone) and apigenin
(4',5,7-trihydroxyflavone) showed moderate to slight inhibition,
and naringenin (4',5,7-trihydroxyflavanone) was completely
inactive..sup.63,64,70 This revealed that the presence of both the
unsaturated double bond between positions 2 and 3 of the flavonoid
pyrone ring (e.g. flavone), and either the 3 hydroxyl groups
introduced at the 5, 6, and 7 positions (bicalein) or the 3, 3',
and 4' positions (quercetin) were a prerequisite for inhibition of
RT activity.
[0138] In our study, apigenin exhibited moderate activity and
naringenin demonstrated slight inhibition. Biflavanoids which
consisted of two apigenin units (amentoflavone (1), agathisflavone
(2), robustaflavone (3), and hinokiflavone (4)) demonstrated
significant activity. Biflavanoids constructed of flavanone and
flavone units (morelloflavone (7)) and biflavanone, linked through
I-3-II-8 (GB-1a (13) and GB-2a (16)) were moderately active, and
biflavanones linked through ring A of two naringenin units
(rhusflavanone (9) and succedaneaflavanone (11)) were inactive.
This structure-activity comparison again demonstrates that hydroxyl
groups and at least one flavone unit in the biflavanoids are
required for activity. A I-3-II-8 linkage is also necessary for
biflavanones to exhibit activity. A firther conclusion is that
previously active compounds become inactive when hydroxy groups are
methylated.
EXAMPLE 10
Anti-Herpes Viral Activity of Biflavanoids
[0139] Anti-Herpes Viral Assay: The viruses used in the primary
screen for anti-viral activity against herpes viruses consisted of:
Herpes Virus 1 (HSV-1 E-377 strain), Herpes Virus 2 (HSV-2 MS
strain), Cytomegalovirus (HCMV AD 169 strain), Varicella Zoster
Virus (VZV Ellen Strain), and Epstein-Barr Virus (EBV),
superinfection of Raji or Daudi cells with P3HR-1.
[0140] The assay for the inhibition of the cytopathic effect (CPE)
for HSV, HCMV and VZV was as follows: Low passage human foreskin
fibroblast cells were seeded in 96-well tissue culture plates 24 h
prior to use, at a cell concentration of 2.5.times.10.sup.4
cells/mL in 0.1 L of minimal essential medium (MEM) supplemented
with 10% fetal bovine serum (FBS). The cells were then incubated
for 24 h at 37.degree. C. in a CO.sub.2 incubator. After
incubation, the medium was removed and 100 mL of MEM containing 2%
FBS was added to all but the first row. In the first row, 125 mL of
the test compound was added in triplicate wells. Medium alone was
added to both cell and virus control wells. The test compound in
the first row was diluted serially 1:5 throughout the remaining
wells by transferring 25 mL using a Cetus Liquid Handling Machine.
After dilution of the compound, 100 mL of the appropriate virus
concentration was added to each well, excluding cell control wells
which received 100 mL of MEM. For HSV-1 and HSV-2 assays, the virus
concentration utilized was 1000 PFUs per well. For CMV and VZV
assays, the virus concentration added was 2500 PFUs per well. The
plates were then incubated at 37.degree. C. in a CO.sub.2 incubator
for three days for HSV-1 and HSV-2, 10 days for VZV, or 14 days for
CMV. After the incubation period, the media was aspirated and the
cells stained with a 0.1% crystal violet solution for 30 min. The
stain was then removed and the plates rinsed using tap water until
all the excess stain was removed. The plates were allowed to dry
for 24 h and then read on a Skatron Plate reader at 620 nm.
[0141] VZV Plaque Reduction Assay. Two days prior to use, HFF cells
were plated into six-well plates and incubated at 37.degree. C.,
with 5% CO.sub.2 atmosphere and 90% humidity. On the date of assay,
the test compound was made up at twice the desired concentration in
2X MEM using six concentrations of the compound. The initial
starting concentrations were usually from 200 ug/mL to 0.06 ug/mL.
The VZV was diluted in 2X MEM containing 10% FBS to a desired
concentration which would give 20-30 plaques per well. The media
was then aspirated from the wells and 0.2 mL of the virus was added
to each well in duplicate, with 0.2 mL of media being added to the
drug toxicity wells. The plates were then incubated for 1 h with
shaking every 15 min. After the incubation period, mean equal
amount of 1% agarose was added to an equal volume of each test
compound dilution. This provided final test compound concentrations
beginning with 100 ug/mL and ending with 0.03 ug/mL, and a final
agarose overlay concentration of 0.5%. The test compound agarose
mixture was applied to each well in 2 mL volumes. The plates were
then incubated, the stain aspirated, and plaques counted using a
stereomicroscope at 10x magnification for ten days, after which the
cells were stained with a 1.5% solution of neutral red dye. On days
three and six an additional 1 mL overlay with equal amounts of 2X
MEM and 1% agarose were added. At the end of the 4-6 h incubation
period, the stain was aspirated and plaques counted using a
stereomicroscope at 10x magnification.
Herpes viruses (HSV-1, HSV-2, HCMV, VZV, and EBV)
[0142] The results of the anti-herpes viruses activity assays of
these biflavanoids are presented in Table 11. Among the compounds
studied, only robustaflavone (3) exhibited significant inhibitory
activities against HSV-1 and HSV-2 viruses. Activity values are
measured by effective concentration (EC.sub.50) and cytotoxicity
concentration (CC.sub.50) at which 50% of cells are free from
pathogens or 50% of cells die. The values for robustaflavone (3)
are an EC.sub.50 of 8.6 .mu.g/mL and CC.sub.50>100 .mu.g/mL,
which results in a selectivity index of >11.6. The anti-viral
activity of robustaflavone (3) against HSV-2 produced an EC.sub.50
value of 8.5 .mu.g/mL, a CC.sub.50 of >100 .mu.g/mL, and a SI of
>11.8. Other results include amentoflavone (1) which
demonstrated only slight activity against HSV-1. Volkensiflavone
(5) exhibited weak inhibitory activity against both HCMV and VZV.
Methylation of volkensiflavone (5) into volkensiflavone hexamethyl
ether (6), resulted in the loss of activity, and a decrease in
toxicity against HCMV, but an increase in activity and toxicity
against VZV. Acetylation of rhusflavanone (9) to rhusflavanone
hexaacetate (10) increased the activity and toxicity against HSV-1
and HSV-2. Acetylation of succedaneaflavanone (11) into
succedaneaflavanone hexaacetate (12) led to a slight decrease of
both activity and toxicity, and resulted in almost equal SI values.
When assayed for activity against VZV, the acetylation product (12)
resulted in an SI value which increased from <3 to 9.6.
11 TABLE 11 HSV-1 (HFF Cells) HSV-2 (HFF Cells) HCMV (HFF Cells)
VZV (HFF Cells) CPE Inhibition CPE Inhibition CPE Inhibition Plaque
Reduction EC.sub.50*.sup.1 CC.sub.50*.sup.2 EC.sub.50*.sup.1
CC.sub.50*.sup.2 EC.sub.50*.sup.1 CC.sub.50*.sup.2 EC.sub.50*.sup.1
CC.sub.50*.sup.2 Sample ug/ml ug/ml SI*.sup.3 ug/ml ug/ml SI*.sup.3
ug/ml ug/ml SI*.sup.3 ug/ml ug/ml SI*.sup.3 ACT* 1.5 -- 0.9 -- --
-- -- -- 0.5 -- -- GVC* -- >100 -- -- -- 0.4 -- -- -- -- --
Amentoflavone (1) 17.9 >100 >5.6 48.0 >100 >2.1 50.8
>100 >1.9 >4.0 9.3 >2.3 Agathisflavone (2) >100
>100 0 >100 >100 0 94.8 >100 >1.0 >4 12.0 <3.0
Robustaflavone (3) 8.6 >100 >11.6 8.5 >100 >11.8 54.8
>100 >1.8 -- -- -- Hinokiflavone (4) >20 77.7 <3.9
>20 77.5 <3/9 >0.8 2.6 <3.2 >4.0 16.8 <4.2
Volkensiflavone (5) >100 >100 0 87.9 >100 >1.1 >4.0
16.8 <4.2 >20 80.0 <4.0 Volkensiflavone >100 >100 0
>100 >100 0 >100 >100 0 3.3 11.1 3.4 hexamethyl ether
(6) Rhusflavanone (9) >100 >100 0 15.7 >100 >6.4
>4.0 13.7 <3.4 >4 16.0 <4 Rhusflavanone >4.0 18.5
<4.6 >4.0 18.5 <4.6 >4.0 13.4 <3.3 11.3 46.7 4.1
hexaacetate (10) Succedaneaflavone (11) >20 60.7 <3.0 >20
60.7 <3.0 >20 55.5 <2.7 >20 60.0 <3.0
Succedaneaflavone >4 17.7 <4.4 >4.0 17.7 <4.4 >4.0
14.4 <3.5 7.1 68.0 9.6 hexaacetate (12) *Positive control drug;
*.sup.150% effective dose; *.sup.250% cell inhibitory (cytotoxic)
concentration; *.sup.3selective index: CC.sub.50/EC.sub.50
EXAMPLE 11
In Vivo Evaluation of Robustaflavone in a Murine Influenza
Model
[0143] In this Example, a series of in vivo experiments were run to
determine if robustaflavone is efficacious against an
experimentally induced influenza virus infection in mice. Prior to
beginning this study, a series of preliminary experiments were run
to determine the maximum tolerated dose of this compound in mice.
Since the compound is not soluble in aqueous medium, it was
suspended in 0.4% carboxymethylcellulose (CMC), a vehicle commonly
used for water-insoluble compounds. When it was found that the
compound was well tolerated at high dosages in this suspension, the
question arose as to whether it was being adequately absorbed by
the animal. Some studies were thus conducted using other vehicles
in which the compound was more soluble. These vehicles included
dimethylsulfoxide (DMSO), dimethyl formamide (DMF), and
polyethylene glycol (PEG).
Materials and Methods
[0144] Animals: Female 13-15 g specific pathogen-free BALB/c mice
were obtained form Simonsen Laboratories (Gilroy, Calif.). They
were quarantined 24 h prior to use, and maintained on Wayne Lab
Blox and tap water. After being infected, their drinking water
contained 0.006% oxytetracycline (Pfizer, New York, N.Y.) to
control possible secondary bacterial infections.
[0145] Virus: A/NWS/33 (H1N1) was obtained from K. W. Cochran,
Univ. of Michigan (Ann Arbor, Mich.). A virus pool was prepared in
MDCK cells; this was titrated in mice, ampuled, and stored at
-80.degree. C. until used.
[0146] Compounds: Robustaflavone was stored at room temperature
until used. Ribavirin, used as a positive control, was obtained
from ICN Pharmaceuticals (Costa Mesa, Calif.). Vehicles considered
included DMSO (Sigma Chemical Co., St. Louis, Mo.), DMF (Sigma),
PEG M.W. 200 (Aldrich Chemical Co. Milwaukee, Wis.), 0.4% CMC
(Sigma) and 1-methyl-2-pyrrolidinone (MPD, Aldrich).
[0147] Arterial Oxygen Saturation (SaO.sub.2) Determinations:
SaO.sub.2 was determined using the Ohmeda Blox 3740 pulse oximeter
(Ohmeda, Louisville, Ohio)). The ear probe attachment was used, the
probe placed on the thigh of the animal, with the slow instrument
mode selected. Readings were made after a 30 second stabilization
time on each animal. Use of this device for measuring effects of
influenza virus on arterial oxygen saturation have been
described..sup.72
[0148] Lung Virus Determinations: Each mouse lung was homogenized
and varying dilutions assayed in triplicate for infectious virus in
MDCK cells as described previously..sup.73
Experiment Design
[0149] 1. Toxicity Determination of robustaflavone in CMC Vehicle:
The compound was suspended in 0.4% CMC at a concentration of 37.5
mg/mL to make a dosage of 500 mg/kg/day. It was injected i.p. into
2 mice daily for 5 days. The mice were weighed and deaths noted
daily.
[0150] 2. Toxicity Determination of robustaflavone in 100% DMSO:
The compound was dissolved in DMSO at a concentration of 25 mg/mL
and in a later experiment in a concentration of 11.25 mg/mL to make
dosages of 250 and 75 mg/kg/day, respectively. The higher dosage
was injected i.p. into mice twice daily for 5 days in a volume of
0.1 mL/injection daily for 5 days in a volume of 0.05 mL/injection.
As controls, mice were treated by the same treatment schedule with
DMSO only in volumes of 0.1 or 0.05 mL/injection. Weight gain and
mortality was determined in these animals.
[0151] 3. 3. Toxicity Determination of DMF and PEG only: DMF and
PEG 200 in a concentration of 100% were injected i.p. into separate
groups of mice daily for 5 days using a volume of 0.05
mL/injection. Again, effects on host weight and deaths of mice were
monitored.
[0152] 4. Effect of robustaflavone in CMC or in DMSO on influenza
virus infection in mice. In the study with CMC, robustaflavone was
used in dosages of 200 and 100 mg/kg/day; using DMSO vehicle, the
dosages were 75 and 37.5 mg/kg/day, with the compound administered
i.p. twice daily for 5 days beginning 4 h pre-virus exposure. The
mice were used in each dose to monitor effects on SaO.sub.2 and
death; from an additional group of similarly infected and treated
mice, 3 animals were killed on days 3, 5, 7 and 9 to assay for lung
score (0=normal, 4=maximal consolidation), weight, and virus titer.
Three to four mice were used as toxicity controls, which were
weighed prior to treatment and again 18 h after treatment
termination, and deaths noted daily. Ribavirin, dissolved in
saline, was used in a dose of 75 mg/kg/day with the same treatment
schedule. Three sets of virus controls were used:
Infected-untreated, infected-treated with CMC only, and
infected-treated with DMSO only. Twenty animals were used in each
of these control groups to monitor SaO.sub.2 and death, with 3
additional mice taken in parallel with treated animals to determine
effects on lung consolidation and virus titer. Two sets of normal
controls were used; one group of three mice was weighed and held in
parallel with the toxicity controls. From the second group three
mice were killed on days 3 and 9 for comparison of lung score and
weight.
[0153] Statistical Evaluation: Increase in survivor number was
evaluated using chi square analysis with Yates' correction. Mean
survival time increases, virus titer and SaO.sub.2 value
differences were analyzed by t-test. Lung consolidation scores were
evaluated by ranked sum analysis.
Results and Discussion
[0154] Toxicological Effects on Various Vehicles: The results of
the various experiments with the vehicles considered are summarized
in Table 12. CMC was the most well tolerated, followed by DMSO. DMF
and PEG 200 were lethally toxic to the mice. One mouse died
immediately following the day 4 i.p. treatment with DMSO; since
this animal died instantly it is probable the death was due to
penetration of an organ by the needle as it was administered into
the peritoneal cavity. Using the 0.05 mL volume of DMSO, the
animals appeared to tolerate this vehicle better than at 0.1 mL.
DMF was highly lethal, killing both animals after two injections,
and PEG 200 was only slightly better, with all mice dying after 3
injections.
[0155] Based on the above data, both CMC and DMSO were used as
solvents for robustaflavone, the latter used in injection volumes
of 0.05 mL.
[0156] Dose Range-Finding Studies with Robustaflavone in Mice:
Using CMC as vehicle, robustaflavone appeared to be quite
insoluble, with dense yellow particulate material seen in the
formulation. When injected i.p. twice daily for 5 days, a dose of
200 mg/kg/day appeared reasonably well tolerated, the treated
animals surviving therapy but losing 0.1 g of weight in the 5-day
treatment period. The material was very soluble in DMSO, forming a
clear solution. A 250 mg/kg/day dose injected i.p. twice daily for
5 days was lethally toxic to the mice, all animals dying by day 5
of treatment and a 6 g weight loss seen. The injection volume in
this experiment was 0.1 mL, when the experiment was repeated using
0.05 mL injection volume, the dosage was lowered to 75 mg/kg/day.
At this dose, all mice survived, although they lost 2 g of weight
during the 5-day treatment period.
[0157] The data using CMC as vehicle suggests the compound was not
being well absorbed in the animal, so for the antiviral experiment
it was decided to use doses of 200 and 100 mg/kg/day. The DMSO
studies indicated 75 mg/kg/day may be approaching the maximum
tolerated dose, so that dose and 37.5 mg/kg/day were chosen for the
in vivo antiviral experiment.
[0158] Effect of robustaflavone in DMSO on Influenza A Virus
Infections in Mice: The results of this experiment are summarized
in Table 14 and FIG. 1 through 4. It was found that the 75
mg/kg/day dose in this antiviral experiment was lethally toxic to
the mice; the 37.5 mg/kg/day dose killed 2 of 3 toxicity control
mice as well. Due to this apparent toxicity, the effects on
survivors and SaO.sub.2 values were inconclusive. This excess
toxicity did not correlate with the earlier-run range-finding
study, although in the latter study marked weight loss was seen
suggesting the compound was approaching a lethally toxic dose.
[0159] A review of FIG. 2 and 3, showing effects of treatment on
lung scores and lung weights, indicates a significant effect of
this compound on lowering lung scores and weights. This effect was
dose-responsive, and suggests robustaflavone may have a significant
influenza-inhibitory effect which may also be seen at a dose more
well tolerated to the mice.
[0160] DMSO used alone was not lethal to the mice, but infected
animals treated with DMSO only died approximately 2 days sooner
than untreated infected controls (Table 12). This suggests the DMSO
injection may result in an enhancement of the infection.
[0161] Ribavirin, run in parallel as a positive control, was highly
active in inhibiting the infection using all evaluation
parameters.
[0162] Effect of robustaflavone in CMC on influenza A virus
infections in mice: The results of this study are seen in Table 14
and in FIGS. 5 through 8. Robustaflavone appeared to be well
tolerated in this experiment, with all toxicity controls surviving
and host weight gain approaching that seen with normal controls run
in parallel observed. The therapy did not prevent death, but did
increase mean survival times in a dose-responsive manner. SaO.sub.2
levels remained high in these treated animals as well (Table 14,
FIG. 5).
[0163] Treatment with this compound also inhibited lung
consolidation in a dose-responsive fashion as seen in FIGS. 6 and
7.
[0164] These data indicate that: 1) Robustaflavone can be
inhibitory to the in vivo influenza infection and 2) there is
apparently at least a partial absorption of the compound since the
dose-responsive effects were seen.
[0165] It may be pertinent to note that two flavones have
previously been reported to have influenza virus-inhibitory
effects. 5,7,8,4'-Tetrahydroxyflavone was reported in 1992.sup.74
to prevent viral proliferation in lungs of infected mice when the
compound was administered either by the intranasal or oral routes.
The related 8-methylether compound,
5,7,4'-trihydroxy-8-methoxyflavone, was similarly effective when
administered intranasally or by the i.p. routes..sup.74-77 Research
by these investigators indicated the compounds reduce viral
replication by inhibiting fusion of the virus with endosome/lysome
membrane which occurs at an early stage of the virus infection
cycle and may also inhibit budding of the progeny virus from the
cells surface..sup.77,78 The vehicle for these flavones was
Na.sub.2CO.sub.3/saline.
Conclusion
[0166] Robustaflavone was evaluated against influenza A/NWS/33
(H1N1) virus infections in mice using two vehicles, 0.4%
carboxymethylcellulose (CMC) and 100% dimethylsulfoxide (DMSO)
Treatment was i.p. twice daily for 5 days beginning 4 h pre-virus
exposure. The compound in DMSO was toxic to the mice at the two
dosages employed, 75 and 37.5 mg/kg/day; despite this toxicity,
significant reduction in lung consolidation was seen. When used in
CMC, the doses of 200 and 100 mg/kg/day used were well tolerated
and both inhibited lung consolidation and slowed the mean day to
death of the animals.
12TABLE 12 Toxicological Effects of CMC, DMSO, DMSF, and PEG 200 in
BALB/c Mice.sup.1 Volume/ Mean Host Injection Surv/ Mean Day Wt.
Vehicle (ml) Total To Death Change (g).sup.2 0.4% 0.1 3 3 >21
1.7 Carboxymethyl- cellulose (CMC) 100% DMSO 0.1 2 2 >21 -.26
100% DMSO 0.05 1 2 4.0 -0.3 100 MF 0.05 0 2 1.0 7 100% PEC 200 0.08
1 2 5.0 -2.2 100% PEC 200 0.1 0 2 2.0 -2.8 .sup.1Treatment i.p. bif
.times. 5. .sup.2Maximum difference between initial weight and
weight after treatment.
[0167]
13TABLE 13 Effect of i.p. Treatment with Robustaflavone in DMSO
Vehicle on influenza A (H1N1) Virus Infections in Mice Animals:
13-15 g female BALB/c Mice Treatment Schedule: bid .times. 5 beg -4
Virus: Influenza A (A/NWS/33 (H1N1), h pre-virus exposure i.n.
Treatment route: i.p. Drug Diluent: Robustaflavone 0.4% Experiment
Duration: 21 days DMSO; Ribavirin Saline Toxicity Controls
Infected, Treated Dosage Surv/ Mean Weight Surv/ MST.sup.b Mean
Compound (mg/kg/day) Total Change (g).sup.a Total (days)
SaO.sub.2.sup.c (%) Robustaflavone 75 0/3 -1.7 0/9 3.2 70.6 37.5
1/3 -0.8 0/10 8.0 73.8 Ribavirin 75 3/3 -0.5 10/10** >21.0**
87.1** DMSO -- -- -- 0/20 9.6 82.6 Untreated -- -- -- 0/20 11.4
84.2 Normals -- 3/3 2.0 -- -- 87.9 .sup.aDifference between initial
weight at start of treatment and weight 18 h following final
treatment of toxicity controls. .sup.bMean survival time of mice
dying on or before day 21. .sup.cMean of days 3-10. **P < 0.01
compared to DMSO-treated controls.
[0168]
14TABLE 14 Effect of i.p. Treatment with Robustaflavone in CMC
Vehicle on influenza A (H1N1) Virus Infections in Mice Animals:
13-15 g female BALB/c Mice Treatment Schedule: bid .times. 5 beg
Virus: Influenza A (A/NWS/33 (H1N1), -4 h pre-virus exposure i.n.
Treatment route: i.p. Drug Diluent: Robustaflavone 0.4% Experiment
Duration: 21 days CMC; Ribavirin Saline Toxicity Infected, Controls
Treated Mean Weight Com- Dosage Surv/ Change Surv MST.sup.b Mean
pound (mg/kg/day) Total (g).sup.a Total (days) SaO.sub.2.sup.c (%)
Robusta- 200 3/3 1.5 0/9 11.1 84.6** flavone 1005 4/4 1.9 0/10 9.8
85.1** Ribavirin 75 3/3 -0.5 10/10** >21.0** 87.1** CMC -- -- --
0/16 9.3 80.4 Untreated -- -- -- 0/20 11.4 84.2 Normals -- 3/3 2.0
-- -- 87.9 .sup.aDifference between initial weight at start of
treatment and weight 18 h following final treatment of toxicity
controls. .sup.bMean survival time of mice dying on or before day
21. .sup.cMean of days 3-10. **P < 0.01 compared to CMC-treated
controls.
CONCLUSION
[0169] Robustaflavone, a naturally occurring biflavanoid isolated
from the seed kernel extract of Rhus succedanea, was found to be a
potent in vitro inhibitor of hepatitis B virus (HBV) replication in
chronically infected human hepatoblastoma 2.2.15 cells, with an
effective concentration (EC.sub.50) of 0.25 .mu.M, and a
therapeutic index (IC.sub.50C.sub.50) of 153. These values were
compared with penciclovir and lamivudine (3TC), which exhibited
EC.sub.50 vlaues of 0.19 and 0.038 .mu.M, respectively, and
therapeutic indexes of 471 and 11200. Combinations of
robustaflavone with penciclovir and lamivudine displayed
synergistic anti-HBV activity, having the most pronounced effects
when the combination ratios were similar to the ratio of EC.sub.50
potencies. Thus, a 1:1 combination of robustaflavone and
penciclovir exhibited an EC.sub.50of 0.11 .mu.M and a therapeutic
index of 684, while a 10:1 combination of robustaflavone and
limivudine exhibited an EC.sub.50 of 0.054 .mu.M and a therapeutic
index of 894. Measurement of extracellular and intracellular HBV
DNA, HBV RNA and viral protein levels following exposure of 2.2.15
cells to robustaflavone indicated that only HBV DNA levels were
affected, suggesting that inhibition of HBV DNA polymerase is the
mechanism of action.
[0170] The results indicated that robustaflavone and robustaflavone
tetrasulfate potassium salt were extremely effective anti-HBV
agents. Robustaflavone also exhibited strong inhibitory effects
against influenza A and influenza B viruses. Both hinokiflavone and
robustaflavone demonstrated similar activity against HIV-1 RT,
producing IC.sub.50 values of 35.2 .mu.g/mL and 33.7 .mu.g/mL,
respectively. Amentoflavone, agathisflavone, morelloflavone, GB-1a
and GB-2a were moderately active against HIV-1 RT, with IC.sub.50
values of 64.0 .mu.g/mL, 53.8 .mu.g/mL, 64.7 .mu.g/mL, 127.8
.mu.g/mL, and 94.6 .mu.g/mL, respectively. Morelloflavone also
demonstrated significant antiviral activity against HIV-1 (strain
LAV in phytohemagglutinin (PHA)-stimulated human peripheral blood
mononuclear (PBM) cells) at an EC50 value of 5.7 .mu.M and an SI
value (selectivity index) of approximately 10. The other
biflavanoids were either slightly active or inactive against these
viruses and HIV-1 RT.
[0171] Amentoflavanone (1), agathisflavone (2), volkensiflavanone
(5), volkensiflavone hexamethyl ether (6), rhusflavanone (9), and
succedaneaflavone (11) exhibited inhibitory activity against
influenza B virus with the selective index (SI) of 178, 5.6, 34,
.about.38, 9.3 and 15, respectively. Amentoflavone (1), and
agathisflavone (2) also demonstrated anti-influenza A activity.
[0172] Robustaflavone (3) produced moderate inhibitory activity
against both HSV-1 and HSV-2. Rhusflavanone (9) was active against
HSV-2, while succedaneaflavanone hexaacetate (12) was moderately
active against VZV.
[0173] Comparison of robustaflavone with a series of other
naturally occurring biflavanoids and biflavanones, as well as
several semi-synthetic derivatives, indicated that robustaflavone
prossesses unique structural features that impart the observed
antiviral activity. The sermi-synthetic derivatives robustaflavone
hexa-O-acetate and robustaflavone hexa-O-methyl ether were
approximately three-fold and 10-fold less potent with regard to
anti-HBV activity, respectively, but neither of these derivatives
exhibited cytotoxicity up to a concentration of 1000 uM.
Volkensiflavone hexa-O-methyl ether, rhusflavanone hexa-O-acetate
and succedaneaflavanone hexa-O-acetate were the only other
non-robustaflavone analogues to inhibit HBV replication, but all
possessed unacceptable selectivity indexes (1.3, 2.8 and 1.9,
respectively). Interestingly, the parent compounds of the latter
three hexa-O-acetate derivatives (volkensiflavone, rhusflavanone
and succedaneaflavanone) were all inactive against HBV replication,
in contrast to the relationship of robustaflavone with its
hexaacetate.
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