U.S. patent application number 14/633320 was filed with the patent office on 2015-08-20 for compositions comprising siraitia grosvenori extracts and methods for the treatment of infection.
The applicant listed for this patent is Cascade Estates Limited. Invention is credited to Patrick T. Prendergast.
Application Number | 20150231190 14/633320 |
Document ID | / |
Family ID | 38670296 |
Filed Date | 2015-08-20 |
United States Patent
Application |
20150231190 |
Kind Code |
A1 |
Prendergast; Patrick T. |
August 20, 2015 |
COMPOSITIONS COMPRISING SIRAITIA GROSVENORI EXTRACTS AND METHODS
FOR THE TREATMENT OF INFECTION
Abstract
A method for the treatment and/or prophylaxis of a viral
infection in a subject is provided wherein the method comprises the
steps of providing a therapeutically effective amount of a
composition comprising an extract of the fruit of Siraitia
grosvenori Swingle and administering the composition to the
subject. The fruit from which the extract is derived is Luo Han
Guo. The extract comprises at least one thterpene glycoside, which
may be in the form of a mogroside compound. The extract has been
shown to be effective in the treatment of viral infections such as
hepatitis C and HIV. Also provided are pharmaceutical compositions
comprising at least one triterpene glycoside, or an analogue,
metabolite, precursor, derivative, pharmaceutically active salt or
pro-drug thereof.
Inventors: |
Prendergast; Patrick T.;
(Byrock, AU) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Cascade Estates Limited |
Quatre Bornes |
|
MU |
|
|
Family ID: |
38670296 |
Appl. No.: |
14/633320 |
Filed: |
February 27, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12679402 |
Apr 23, 2010 |
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PCT/EP2008/062645 |
Sep 22, 2008 |
|
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14633320 |
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60994824 |
Sep 21, 2007 |
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Current U.S.
Class: |
424/758 |
Current CPC
Class: |
A61P 11/00 20180101;
A61P 35/00 20180101; A61K 36/42 20130101; A61P 1/16 20180101; Y02A
50/463 20180101; Y02A 50/30 20180101; A61K 45/06 20130101; A61P
31/16 20180101; A61P 31/20 20180101; A61P 31/12 20180101; A61K
31/405 20130101; A61K 31/704 20130101; A61P 31/18 20180101; A61P
31/14 20180101 |
International
Class: |
A61K 36/42 20060101
A61K036/42; A61K 45/06 20060101 A61K045/06; A61K 31/405 20060101
A61K031/405; A61K 31/704 20060101 A61K031/704 |
Foreign Application Data
Date |
Code |
Application Number |
Sep 21, 2007 |
GB |
0718446.8 |
Nov 12, 2007 |
CN |
2007/10188155.2 |
Claims
1-39. (canceled)
40. An extract of the fruit of Siraitia grosvenori Swingle for use
in the treatment and/or prophylaxis of a viral infection.
41. The extract of claim 40, wherein the fruit is Luo Han Guo.
42. The extract of claim 40, wherein the extract comprises at least
one triterpene glycoside or an analogue, metabolite, precursor,
derivative, pharmaceutically active salt or pro-drug thereof
43. The extract of claim 42, wherein the at least one triterpene
glycoside or analogue thereof is a mogroside.
44. The extract of claim 40, wherein the viral infection is caused
by a virus selected from the group consisting of flavivirus,
retrovirus, influenza virus, hepatitis A, hepatitis B, hepatitis D,
hepatitis E, hepatitis F, hepatitis G, hepatitis H, autoimmune
hepatitis, BVDV and Respiratory Syncytical Virus (RSV).
45. The extract of claim 44, wherein the flavivirus is hepatitis C
virus.
46. (canceled)
47. The extract of claim 44, wherein the retrovirus is HIV.
48. (canceled)
49. The extract of claim 42, wherein the at least one triterpene
glycoside is selected from the group consisting of neogroside,
20-hydroxy-11-oxomogroside IA.sub.1, 11-oxomogroside IIE,
11-oxomogroside IA.sub.1, mogroside IIE, mogroside III, mogroside
IV, mogroside V, siamenoside I, triterpenoid glycoside V,
neogroside, Kaempferol 7-.alpha.--rhamnopyranoside, Kaempferol
3,7-.alpha.--dirhamnopyranoside, 11-oxomogroside III,
11-Dehydroxymogroside III, 11-oxomogroside IV, mogroside II,
mogroside VI, 11-oxo-mogroside and siamcroside-I.
50-68. (canceled)
69. The extract of claim 42, wherein the at least one triterpene
glycoside is halogenated.
70. The extract of claim 40, wherein the extract comprises a
halogen salt.
71. The extract of claim 40, wherein the extract includes a
secondary antiviral compound.
72. The extract of claim 40, wherein the extract includes a statin
or an analogue, metabolite, derivative, pharmaceutically active
salt, precursor or pro-drug thereof.
73. The extract as claimed in of claim 72, wherein the statin is
selected from the group consisting of mevastatin, lovastatin,
pravastatin, simvastatin, cehvastatin, fluindostatin, velbstatin,
fluvastatin, dalvastatin, dihydrocompactin, compactin,
atorvastatin, bervastatin and NK-104, ZD-4522.
74. The extract as claimed in claim 73, wherein the statin is
fluvastatin.
75. The extract as claimed in claim 74, wherein the fluvastatin
comprises a sodium salt.
76-144. (canceled)
145. An extract of the fruit of Siraitia grosvenori Swingle for use
in the treatment and/or prophylaxis of hepatitis C.
146. (canceled)
Description
FIELD OF THE INVENTION
[0001] The present invention relates to methods for the prophylaxis
and/or treatment of viral infections, such as hepatitis C and HIV.
The invention further relates to compositions for use in the
treatment and/or prophylaxis of viral infections.
BACKGROUND TO THE INVENTION
[0002] Luo Han Guo (also known as Luo Han Kuo) is a fruit of the
Siraitia grosvenori Swingle (formerly Momoridca grosvenori Swingle)
belonging to the Cucurbitaceae species. These fruits are cultivated
in parts of China and extract of the Luo Han Guo plant is currently
manufactured and marketed as a natural low-calorie sweetener, for
example, PURELO.TM.. U.S. Pat. No. 5,411,755 describes a process
for preparing a sweet juice from Luo Han Guo fruit. The sweetness
of the extract from Luo Han Guo is due to the presence of highly
stable triterpene glycosides, known as mogrosides, which are about
250 to 300 times sweeter than sucrose. These compounds possess a
triterpene backbone with two to six glucose units attached.
Examples of cucurbitane glycosides purified from Siraitia
grosvenori include 20-hydroxy-11-oxomogroside IA.sub.1,
11-oxomogroside IIE, 11-oxomogroside IA.sub.1, mogroside IIE,
mogroside III, mogroside IV, mogroside V, siamenoside I,
triterpenoid glycoside V, neogroside, Kaempferol
7-.alpha.--rhamnopyranoside, Kaempferol
3,7-.alpha.--dirhamnopyranoside, 11-oxomogroside III,
11-Dehydroxymogroside III, 11-oxomogroside IV, mogroside II,
mogroside VI, 11-oxo-mogroside and siamcroside-I. Mogrosides have
been shown to have beneficial effects in the treatment of diabetes,
cancer and inflammation.
[0003] Statins are compounds that are known to have a lowering
effect on levels of LDL-cholesterol in the human blood. Statins
inhibit hydroxymethylglutaryl coenzyme A (HMG-CoA) reductase, the
rate-determining enzymatic step in cholesterol biosynthesis.
Currently available statins include lovastatin, simvastatin,
pravastatin, fluvastatin (LESCOL.RTM., CANEF.RTM.), cerivastatin
and atorvastatin. Fluvastatin (disclosed in U.S. Pat. No.
4,739,073) is administered as a sodium salt and is an entirely
synthetic compound that is in part structurally distinct from the
fungal derivatives of this class, which contain a
hexahydronaphthalene ring.
[0004] Hepatitis C (HCV), formerly referred to as blood-borne
non-A, non-B hepatitis virus (NANBV), is a transmissible disease
which was first identified in individuals who had received blood
transfusions. Chronic hepatitis C virus is an insidious and
slow-progressing disease having a significant negative impact on
the quality of life. It can eventually result in cirrhosis of the
liver, decompensated liver disease and/or hepatocellular
carcinoma.
[0005] Alpha interferon mono-therapy is commonly used to treat
infection with chronic hepatitis C. However, this treatment is not
always effective and sometimes results in intolerable side effects
depending on the dosage and the duration of therapy.
[0006] The general anti-viral therapeutic Ribavirin has been
proposed as a mono-therapy treatment for chronic hepatitis C
infection. However, this mono-therapy treatment has been found to
be relatively ineffective and, furthermore, presents its own
undesirable side effects.
[0007] An alternative therapeutic approach combining alpha
interferon and Ribavirin has also been proposed. Preliminary
results suggest that such a combination therapy may be more
effective than either alpha interferon or Ribavirin mono-therapy.
However, the combined use of alpha interferon and Ribavirin does
not eradicate HCV-RNA in a long-term, effective manner.
[0008] There is therefore a need to provide a therapy which has
efficacy in the treatment of chronic hepatitis C infection.
Desirably, such a therapeutic composition or treatment regimen will
eradicate HCV-RNA in a long-term, effective manner.
[0009] Furthermore, in addition to the treatment of subjects
infected with HCV, there is a pressing need to provide means of
treating HCV-contaminated blood or blood products. Post-transfusion
hepatitis (PTH) occurs in approximately 10% of transfused patients,
with HCV accounting for up to 90% of these cases. The disease
frequently progresses to chronic liver damage in 25 to 55% of
cases.
[0010] Acquired immune deficiency syndrome (AIDS) is a disease
characterized by failure of the immune system. It is attributable
to a virus called human immunodeficiency virus (HIV), which is a
member of the retrovirus family of lipid envelope viruses, which is
very prevalent in the animal kingdom. HIV infects and takes over
certain cells of the immune system, using these cells to replicate.
This causes the infected cells to function improperly and die
prematurely, thus weakening the immune system. HIV infection
results in disturbances of the entire immune defense mechanism. In
particular, T4 or helper cells are prevented from carrying out
their role in the regulation of the immune response. This
HIV-induced T4 reduction results in the development of frequent and
eventually fatal opportunistic infections caused by pathogenic
organisms such as viruses, bacteria, protozoa or fungi.
[0011] Anti-viral agents that inhibit replication of viruses have
been known since the mid 1980's. The overall goal of anti-HIV
therapy is to slow or stop the replication process, and thereby
slow or stop the progression of HIV disease and the destruction of
the immune system. Many drugs are now available for inhibiting the
replication of the HIV virus. However, their side effects are often
so severe that treatment must be halted allowing HIV resistant
strains to quickly develop. Current anti-HIV therapies can be
categorized into groups based on which step in the virus's life
cycle they target or how they do it. Examples of anti-HIV therapies
include nucleoside analogue reverse-transcriptase inhibitors,
non-nucleoside analogue reverse-transcriptase inhibitors and
protease inhibitors. The first two groups work by mimicking one of
the building blocks of DNA and thus interfering with reverse
transcription, a process essential for HIV replication. The third
group, protease inhibitors, works at a later stage in the viral
life cycle after the virus has successfully infected the cell and
is attempting to replicate. These drugs ultimately slow down the
replication of viral DNA. However, they do not rid the body of the
virus but merely act to slow down and reduce the severity of the
development of the infection.
[0012] The number of patients infected with HIV has significantly
increased in recent years. There is therefore a need for the
development of a more efficacious and less toxic treatment for
HIV.
[0013] Many of the anti-viral therapies which are currently
available are directed towards targeting viral components and are
therefore prone to compensatory viral evasion mechanisms.
Treatments aimed at interfering with, or preventing, viral
replication, either by enhancing antiviral responses or by
inhibiting proviral activities within the host cell have greater
potential for controlling the virus without selective pressure on
the virus itself to mutate in a compensatory manner.
SUMMARY OF THE INVENTION
[0014] According to a first aspect of the present invention, there
is provided a method for the treatment and/or prophylaxis of a
viral infection in a subject, the method comprising the steps of:
[0015] providing a therapeutically effective amount of a
composition comprising at least one triterpene glycoside or an
analogue, metabolite, precursor, derivative, pharmaceutically
active salt or pro-drug thereof; and [0016] administering the
composition to the subject.
[0017] According to a second aspect of the invention there is
provided at least one triterpene glycoside or an analogue,
metabolite, precursor, derivative, pharmaceutically active salt or
pro-drug thereof for use in the treatment and/or prophylaxis of a
viral infection.
[0018] A third aspect of the present invention provides for use of
at least one triterpene glycoside or an analogue, metabolite,
precursor, derivative, pharmaceutically active salt or pro-drug
thereof in the preparation of a medicament for the treatment and/or
prevention of a viral infection.
[0019] According to a further aspect of the invention, there is
provided a composition comprising at least one triterpene glycoside
or an analogue, metabolite, precursor, derivative, pharmaceutically
active salt or pro-drug thereof.
[0020] According to a further aspect of the invention, there is
provided a pharmaceutical composition comprising at least one
triterpene glycoside or an analogue, metabolite, precursor,
derivative, pharmaceutically active salt or pro-drug thereof and at
least one pharmaceutically acceptable diluent, carrier or
excipient.
[0021] According to a further aspect of the invention, there is
provided a composition comprising at least one triterpene glycoside
or an analogue, metabolite, precursor, derivative, pharmaceutically
active salt or pro-drug thereof for use as a medicament.
[0022] According to a further aspect of the present invention,
there is provided a method for the treatment and/or prophylaxis of
a viral infection in a subject, the method comprising the steps of:
[0023] providing a therapeutically effective amount of a
composition comprising an extract of the fruit of Siraitia
grosvenori Swingle; and [0024] administering the composition to the
subject.
[0025] According to a further aspect of the invention there is
provided an extract of the fruit of Siraitia grosvenori Swingle for
use in the treatment and/or prophylaxis of a viral infection.
[0026] A further aspect of the present invention provides for use
of an extract of the fruit of Siraitia grosvenori Swingle in the
preparation of a medicament for the treatment and/or prevention of
a viral infection.
[0027] According to a further aspect of the invention, there is
provided a composition comprising an extract of the fruit of
Siraitia grosvenori Swingle.
[0028] According to a further aspect of the invention, there is
provided a pharmaceutical composition comprising an extract of the
fruit of Siraitia grosvenori Swingle and at least one
pharmaceutically acceptable diluent, carrier or excipient.
[0029] According to a further aspect of the invention, there is
provided a composition comprising an extract of the fruit of
Siraitia grosvenori Swingle for use as a medicament.
DESCRIPTION OF THE FIGURES
[0030] The present invention will now be described with reference
to the following examples which are provided for the purpose of
illustration and are not intended to be construed as being limiting
on the present invention, and further with reference to the figures
as described briefly below.
[0031] FIG. 1 shows the anti-viral activity of an extract of Luo
Han Guo fruit against Bovine Viral Diarrhoea Virus (BVDV);
[0032] FIG. 2 shows the anti-viral activity of mogroside V against
HIV-1 strain IIIB; and
[0033] FIG. 3 shows the anti-viral activity of an extract of Luo
Han Guo fruit against BVDV when the extract is combined with
fluvastatin.
DETAILED DESCRIPTION OF THE INVENTION
[0034] The present invention is directed to the use of at least one
triterpene glycoside and/or an extract of the fruit of Siraitia
grosvenori Swingle as an antiviral.
[0035] In certain embodiments, the at least one triterpene
glycoside or extract is derived from the Cucurbitaceae plant, in
particular from the fruit of the Cucurbitaceae plant. In certain
embodiments, the plant is of the variety Siraitia grosvenori
Swingle and typically the fruit is a fruit of Siraitia grosvenori
Swingle. In certain embodiments, the fruit is Luo Han Guo, also
known as Luo Han Kuo.
[0036] In certain embodiments, the extract is an extract of Luo Han
Guo. In certain embodiments, the extract is derived from the Luo
Han Guo fruit using the process described in U.S. Pat. No.
5,411,755, the entire contents of which are herein incorporated by
reference.
[0037] In certain embodiments, the invention relates to use of an
extract of a Cucurbitaceae plant in the preparation of a medicament
for the treatment and/or prevention of a viral infection.
[0038] In certain embodiments, the extract comprises at least one
triterpene glycoside.
[0039] In certain embodiments, the at least one triterpene
glycoside is a mogroside or an analogue, metabolite, precursor,
derivative, pharmaceutically active salt or pro-drug thereof.
Typically the mogroside is derived from a plant of the
Cucurbitaceae species, in particular the fruit of the Cucurbitaceae
species. In certain embodiments, the plant is Siraitia grosvenori
Swingle (formerly Momoridca grosvenori Swingle). Typically the
fruit is Luo Han Guo.
[0040] In certain embodiments, the at least one triterpene
glycoside is selected from the group consisting of, but not limited
to, 20-hydroxy-11-oxomogroside IA.sub.1, 11-oxomogroside IIE,
11-oxomogroside IA.sub.1, mogroside IIE, mogroside III, mogroside
IV, mogroside V, siamenoside I, triterpenoid glycoside V,
neogroside, Kaempferol 7-.alpha.--rhamnopyranoside, Kaempferol
3,7-.alpha.--dirhamnopyranoside, 11-oxomogroside III,
11-Dehydroxymogroside III, 11-oxomogroside IV, mogroside II,
mogroside VI, 11-oxo-mogroside and siamcroside-I, and analogues,
metabolites, precursors, derivatives, pharmaceutically active salts
or pro-drugs thereof.
[0041] In certain embodiments, the at least one triterpene
glycoside is mogroside V or an analogue, metabolite, precursor,
derivative, pharmaceutically active salt or pro-drug thereof.
[0042] In certain embodiments, the at least one triterpene
glycoside is 20-hydroxy-11-oxomogroside or an analogue, metabolite,
precursor, derivative, pharmaceutically active salt or pro-drug
thereof.
[0043] In certain embodiments, the at least one triterpene
glycoside is 20-hydroxy-11-oxomogroside IA, or an analogue,
metabolite, precursor, derivative, pharmaceutically active salt or
pro-drug thereof.
[0044] In certain embodiments, the at least one triterpene
glycoside is 11-oxomogroside IIE or an analogue, metabolite,
precursor, derivative, pharmaceutically active salt or pro-drug
thereof.
[0045] In certain embodiments, at least one triterpene glycoside is
11-oxomogroside IA.sub.1 or an analogue, metabolite, precursor,
derivative, pharmaceutically active salt or pro-drug thereof.
[0046] In certain embodiments, the at least one triterpene
glycoside is mogroside IIE or an analogue, metabolite, precursor,
derivative, pharmaceutically active salt or pro-drug thereof.
[0047] In certain embodiments, the at least one triterpene
glycoside is mogroside III or an analogue, metabolite, precursor,
derivative, pharmaceutically active salt or pro-drug thereof.
[0048] In certain embodiments, the at least one triterpene
glycoside is mogroside IV or an analogue, metabolite, precursor,
derivative, pharmaceutically active salt or pro-drug thereof.
[0049] In certain embodiments, the at least one triterpene
glycoside is siamenoside I or an analogue, metabolite, precursor,
derivative, pharmaceutically active salt or pro-drug thereof.
[0050] In certain embodiments, the at least one triterpene
glycoside is neogroside or an analogue, metabolite, precursor,
derivative, pharmaceutically active salt or pro-drug thereof.
[0051] In certain embodiments, the at least one triterpene
glycoside is triterpenoid glycoside V or an analogue, metabolite,
precursor, derivative, pharmaceutically active salt or pro-drug
thereof.
[0052] In certain embodiments, the at least one triterpene
glycoside is Kaempferol 7-.alpha.--rhamnopyranoside or an analogue,
metabolite, precursor, derivative, pharmaceutically active salt or
pro-drug thereof.
[0053] In certain embodiments, the at least one triterpene
glycoside is Kaempferol 3,7-.alpha.--dirhamnopyranoside or an
analogue, metabolite, precursor, derivative, pharmaceutically
active salt or pro-drug thereof.
[0054] In certain embodiments, the at least one triterpene
glycoside is 11-oxomogroside III or an analogue, metabolite,
precursor, derivative, pharmaceutically active salt or pro-drug
thereof.
[0055] In certain embodiments, the at least one triterpene
glycoside is 11-Dehydroxymogroside III or an analogue, metabolite,
precursor, derivative, pharmaceutically active salt or pro-drug
thereof.
[0056] In certain embodiments, the at least one triterpene
glycoside is 11-oxomogroside IV or an analogue, metabolite,
precursor, derivative, pharmaceutically active salt or pro-drug
thereof.
[0057] In certain embodiments, the at least one triterpene
glycoside is mogroside II or an analogue, metabolite, precursor,
derivative, pharmaceutically active salt or pro-drug thereof.
[0058] In certain embodiments, the at least one triterpene
glycoside is mogroside VI or an analogue, metabolite, precursor,
derivative, pharmaceutically active salt or pro-drug thereof.
[0059] In certain embodiments, the at least one triterpene
glycoside is 11-oxo-mogroside or an analogue, metabolite,
precursor, derivative, pharmaceutically active salt or pro-drug
thereof.
[0060] In certain embodiments, the at least one triterpene
glycoside is siamcroside-I or an analogue, metabolite, precursor,
derivative, pharmaceutically active salt or pro-drug thereof.
[0061] In certain embodiments, the composition or extract comprises
a mixture of triterpene glycosides, for example mogrosides such as
mogroside IV or mogroside V. Typically, mogroside V is the major
constituent of the mixture. In certain embodiments, the composition
or extract comprises more than 50% mogrosides, typically more than
70% and preferably more than 80%.
[0062] In certain embodiments, the composition or extract comprises
at least one mogroside in an isolated form wherein the at least one
mogroside has been isolated and purified from Luo Han Guo. In
certain embodiments, the composition or extract comprises the at
least one mogroside in the form of a crude extract of Luo Han Guo.
In certain embodiments, the at least one mogroside or extract is
provided in the form of a natural sweetener or juice, for example,
using the process described in U.S. Pat. No. 5,411,755. In certain
embodiments, the juice is dried and the extract provided in a
powder form. In certain embodiments, the at least one mogroside or
extract comprises a bacterially-transformed mogroside. In certain
embodiments, the at least one mogroside or extract comprises a
mogroside in a synthetic form.
[0063] In certain embodiments, the mogroside is selected from the
group of compounds having the chemical formula shown in formula 1
below:
TABLE-US-00001 Formula 1 ##STR00001## R.sub.1 R.sub.2 R.sub.3
R.sub.4 20-hydroxy-11-oxomogroside I A.sub.1(1) --H --Glc .dbd.O
--OH 11-oxomogroside II E --Glc --Glc .dbd.O --H 11-oxomogroside I
A.sub.1 (3) --H --Glc .dbd.O --H mogroside II E (4) --Glc --Glc
##STR00002## --H mogroside III (5) --Glc ##STR00003## ##STR00004##
--H mogroside IV (6) ##STR00005## ##STR00006## ##STR00007## --H
mogroside V (7) ##STR00008## ##STR00009## ##STR00010## --H
[0064] In certain embodiments, the at least one triterpene
glycoside is an analogue, metabolite, precursor, derivative,
pharmaceutically active salt or pro-drug of one of the compounds
shown in formula 1.
[0065] Without wishing to be bound by theory, it is hypothesized
that a composition comprising an extract of the Luo Han Guo fruit,
such as at least one triterpene glycoside or an analogue,
metabolite, precursor, derivative, pharmaceutically active salt or
pro-drug thereof, inhibits or suppresses replication of a viral
infection by interfering with the replicative or other essential
functions of the viral infection or the host cell. Specifically, it
is postulated that triterpene glycoside interacts with the
cytoplasmic environment of the host cell but does not directly
interface with the viral infection. This is advantageous as the
risk of the virus developing resistance to the treatment is
reduced. Accordingly, an effective anti-viral therapy is provided
having minimal risk of conferring resistance to the virus.
[0066] In certain embodiments, the at least one triterpene
glycoside, or an analogue, metabolite, precursor, derivative,
pharmaceutically active salt or pro-drug thereof, is halogenated or
is administered in combination with a halogen salt or with a
halogenated salt. In further embodiments, the halogen or
halogenated salt may be administered before or after the
composition has been administered. Without wishing to be bound by
theory, the inventor predicts that the production and/or activity
of neutrophils and/or monocytes is enhanced when a halogen salt or
a halogenated salt is administered along with the triterpene
glycoside or when the triterpene glycoside is halogenated.
[0067] The invention further relates to a combined medicament
comprising a statin and the at least one triterpene glycoside or an
analogue, metabolite, precursor, derivative, pharmaceutically
active salt or pro-drug thereof. The components of the combined
medicament work in a synergistic manner such that the dose of each
therapeutic compound required in order to achieve a therapeutic
and/or prophylactic effect is reduced when both components are
administered to an individual. Specifically, statins such as
fluvastatin may be used to extend the lower range of the
effectiveness of triterpene glycosides such as mogroside V or
mogroside IV such that lower doses of the triterpene glycoside may
be used. The invention further relates to a combined medicament
comprising a statin and the extract.
[0068] Accordingly, in certain embodiments, the method aspects of
the present invention further comprise the step of: [0069]
administering a therapeutically effective amount of a secondary
composition comprising a statin or an analogue, metabolite,
derivative, pharmaceutically active salt, precursor or pro-drug
thereof.
[0070] In certain embodiments, the statin is selected from the
group consisting of mevastatin, lovastatin, pravastatin,
simvastatin, cerivastatin, fluindostatin, velbstatin, fluvastatin,
dalvastatin, dihydrocompactin, compactin, atorvastatin, bervastatin
and NK-104, ZD-4522.
[0071] In certain embodiments, the statin is fluvastatin.
[0072] In certain embodiments, the statin composition may be
administered to the subject simultaneously with the triterpene
glycoside or extract composition.
[0073] In certain embodiments, the statin composition is
administered separately to the triterpene glycoside or extract
composition.
[0074] In certain embodiments, the statin composition may be
administered to the subject sequentially with the triterpene
glycoside or extract composition. Where the compounds are
administered sequentially, in certain embodiments the statin
composition is administered prior to the triterpene glycoside or
extract composition. In certain further embodiments, the triterpene
glycoside or extract composition is administered prior to the
statin composition.
[0075] In certain embodiments, the statin composition and the
triterpene glycoside or extract composition are co-administered.
Co-administration means that these components may be administered
together as a composition or as part of the same unitary dose or
the same therapeutic regimen. Where the compositions are
administered separately, the "co-administration" of the
compositions does not impose a restriction on the timing,
frequency, dosage or order of when the compounds can be
administered.
[0076] In certain embodiments, the statin and triterpene glycoside
or extract compositions are provided by different routes of
administration. Further, said compositions may be in the same or
different forms, for example a solid and a liquid.
[0077] In certain embodiments, the statin composition and the
triterpene glycoside or extract are provided in a combined
medicament, for example as a single composition.
[0078] In certain embodiments, the statin composition comprises
fluvastatin as a sodium salt.
[0079] The chemical IUPAC name for fluvastatin is
7-[3-(4-fluorophenyl)-1-(1-methylethyl)-1H-indol-2-yl]-3,5-dihydroxy-hept-
-6-enoic acid.
[0080] In one embodiment, fluvastatin is defined by chemical
formula 2 as shown below.
##STR00011##
[0081] The inventor has also identified that the therapeutic
compositions of the present invention comprising at least one
triterpene glycoside or an analogue, metabolite, precursor,
derivative, pharmaceutically active salt or pro-drug thereof, or an
extract of the Luo Han Guo fruit have utility when administered to
a subject along with a further antiviral compound. Such a
combination therapy would, in particular, have utility in relation
to viral infections having a high pathogenicity and, furthermore,
would be effective at reducing the development of viral drug
resistance.
[0082] Accordingly, in certain embodiments, the methods of the
present invention include the step of: [0083] administering a
therapeutically effective amount of a further antiviral
compound.
[0084] In certain embodiments, said further anti-viral compound is
administered in addition to said secondary composition, which
comprises statin or a related compound.
[0085] In certain embodiments, the further antiviral compound may
be administered to the subject simultaneously with the triterpene
glycoside or extract composition.
[0086] In certain embodiments the further antiviral compound is
provided separately to the triterpene glycoside or extract
composition.
[0087] In certain embodiments, the further antiviral compound may
be administered to the subject sequentially with the triterpene
glycoside or extract composition. Where the compounds are
administered sequentially, in certain embodiments the further
antiviral compound is administered prior to the triterpene
glycoside or extract composition. In certain further embodiments,
the triterpene glycoside or extract composition is administered
prior to the further antiviral compound.
[0088] In certain embodiments, the further antiviral compound and
the triterpene glycoside or extract composition are
co-administered.
[0089] In certain embodiments, the further antiviral compound and
the triterpene glycoside or extract composition are provided by
different routes of administration. Further, said further antiviral
compound and composition may be in the same or different forms, for
example a solid and a liquid.
[0090] In certain embodiments, the further antiviral compound and
the triterpene glycoside or extract composition are provided in a
combined medicament, for example as a single composition.
[0091] The antiviral compound may be selected from the group
consisting of nucleoside analogues (AZT; ddC; ddI; d4T; 3TC; BW
1592; PMEA/bis-POM PMEA; dOTC; DAPD); non-nucleoside reverse
transcriptase inhibitors (delavirdine; DMP 266; HBY097; loviride;
nevirapine, emivirine; AG1549; PNU 142721; Calanolide A; DPC961);
protease inhibitors (ABT-378; ritonavir; nelfinavir; BW 141;
KNI-272; indinavir; saquinavir; L-756,423; DMP-450; BMS-232630);
ALX40-4C; hydroxyurea; lobucavir; pentafuside; T-1249; PRO 542;
FP-21399; AMD 3100; HE-2000; peptide T; Abacavir; Acemannan;
Acyclovir; Acyclovir Sodium; Adefovir; Alovudine; Alvircept
Sudotox; Amantadine Hydrochloride; Aranotin; Arildone; Atevirdine
Mesylate; Avridine; Cidofovir; Cipamfylline; Coviracil; Cytarabine
Hydrochloride; Delavirdine Mesylate; Desciclovir; Didanosine;
Disoxaril; Edoxudine; Emivirine; Emtricitabine; Enviradene;
Enviroxime; Epivir; Famciclovir; Famotine Hydrochloride;
Fiacitabine; Fialuridine; Fosarilate; Foscamet Sodium; Fosfonet
Sodium; Ganciclovir; Ganciclovir Sodium; Idoxuridine; Indinavir;
Kethoxal; Lamivudine; Lobucavir; Lodenosine; Lopinavir, Memotine
Hydrochloride; Methisazone; Nelfinavir; Nevirapine; Penciciovir;
Pirodavir; Ribavirin; Rimantadine Hydrochloride; Saquinavir
Mesylate; Ritonavir; Somantadine Hydrochloride; Sorivudine;
Statolon; Stavudine; Tenofovir; Tilorone Hydrochloride;
Trifluridine; Valacyclovir Hydrochloride; Vidarabine; Vidarabine
Phosphate; Vidarabine Sodium Phosphate; Tipranavir, Viroxime;
Zalcitabine; Zidovudine; Zinviroxime and Interferons alpha, beta
and gamma.
[0092] In certain embodiments wherein the virus is HIV, the further
antiviral compound is a drug commonly used in HAART (highly active
antiretroviral therapy). It has been shown that HIV can quickly
mutate to avoid the effect of one drug alone and that it is
therefore advantageous to use drugs effective against HIV in a
combination "cocktail" to fight the virus.
[0093] Some of the compounds (e.g. the at least one triterpene
glycoside, statin and further antiviral compound) for use in the
methods of the present invention may exist as stereoisomers and/or
geometric isomers, e.g. they may possess one or more asymmetric
and/or geometric centres and so may exist in two or more
stereoisomeric and/or geometric forms. The present invention
contemplates the use of all of the individual stereoisomers and
geometric isomers of those compounds and mixtures thereof. The
terms used in the claims encompass these forms, provided said forms
retain the appropriate functional activity (though not necessarily
to the same degree).
[0094] The present invention also includes the use of solvate forms
of these compounds in the methods of the present invention. The
terms used in the claims encompass these forms, provided said forms
retain the appropriate functional activity (though not necessarily
to the same degree).
[0095] The present invention also includes the use of pro-drug
forms of these compounds in the methods of the present invention.
The terms used in the claims encompass these forms. Examples of
pro-drugs include entities that have certain protected group(s) and
which may not possess pharmacological activity as such, but may, in
certain instances, be administered (such as orally or parenterally)
and thereafter metabolised in the body to form pharmacologically
active compounds.
[0096] As defined herein, the term "metabolite" means any substance
which results from, or is produced by, the metabolism or digestion
by a subject of a compound administered to said subject. In
pharmaceutical terms, the phrase relates to the product which
remains after digestion or metabolism, particularly by the liver,
has occurred.
[0097] In certain embodiments of the present invention, the
compounds for use in the methods and compositions of the present
invention, such as triterpene glycoside and/or statin, may be a
mimetic. As used herein, the term "mimetic" relates to any
compound, which includes, but is not limited to, a peptide,
polypeptide, antibody or other organic chemical, which has the same
qualitative activity or effect as the triterpene glycoside or
statin compounds of the invention. The terms used in the claims
encompass mimetics, provided said mimetics retain the appropriate
functional activity (though not necessarily to the same
degree).
[0098] In certain embodiments of the present invention, the
compounds for use in the methods and compositions of the present
invention may be a derivative of a triterpene glycoside compound or
a statin. The term "derivative" as used herein includes chemical
modification of the triterpene glycoside compound and/or statin,
said derivatives retaining the desired chemical activity of the
compounds.
[0099] In certain embodiments of the present invention, the
compounds for use in the methods and compositions of the present
invention may be an analogue of a triterpene glycoside compound or
statin. The term "analogue" as used herein is intended to refer to
compounds having the same functional activity, though not
necessarily to the same degree.
[0100] In certain embodiments of the present invention, the
triterpene glycoside compound may be chemically modified, for
example by halogenation. The chemical modification of the compound
may either enhance or reduce hydrogen bonding interaction, charge
interaction, hydrophobic interaction, van der Waals interaction or
dipole interaction between the compound and the target.
[0101] As herein defined, the expression "pharmaceutically
acceptable salts" extends to both pharmaceutically acceptable acid
addition salts and pharmaceutically acceptable cationic salts. A
"pharmaceutically acceptable cationic salt" includes, but is not
limited to, cationic salts such as the alkali metal salts, for
example, sodium or potassium, as well as alkaline earth metal
salts, for example magnesium, ammonium salts and the like. A
"pharmaceutically acceptable acid addition salts" includes, but is
not limited to salts such as hydrochloride, hydrobromide, sulfate,
hydrogen sulfate, phosphate, hydrogen phosphate,
dihydrogenphosphate, acetate, succinate, citrate, methanesulfonate
(mesylate) and p-toluenesulfonate (tosylate) salts.
[0102] Pharmaceutically-acceptable salts are well known to those
skilled in the art, and for example include those mentioned by
Berge et al, in J. Pharm. Sci. 66,1-19 (1977). Suitable acid
addition salts are formed from acids which form non-toxic salts and
include the hydrochloride, hydrobromide, hydroiodide, nitrate,
sulphate, bisulphate, phosphate, hydrogenphosphate, acetate,
trifluoroacetate, gluconate, lactate, salicylate, citrate,
tartrate, ascorbate, succinate, maleate, fumarate, gluconate,
formate, benzoate, methanesulphonate, ethanesulphonate,
benzenesulphonate and p-toluenesulphonate salts.
[0103] The compounds for use in the methods of the present
invention may be prepared by chemical synthesis techniques. It will
be apparent to those skilled in the art that sensitive functional
groups may need to be protected and deprotected during synthesis of
a compound of the invention. This may be achieved by conventional
techniques, for example as described in "Protective Groups in
Organic Synthesis" by T W Greene and P G M Wuts, John Wiley and
Sons Inc. (1991), and by P. J. Kocienski, in "Protecting Groups",
Georg Thieme Verlag (1994).
[0104] It is possible during some of the reactions that any
stereocentres present could, under certain conditions, be
epimerised, for example if a base is used in a reaction with a
substrate having an optical centre comprising a base-sensitive
group. It should be possible to circumvent potential problems such
as this by choice of reaction sequence, conditions, reagents,
protection/deprotection regimes and the like as these are well
known in the art.
[0105] When reference is made herein to a "triterpene glycoside"
such as mogroside, this term is intended to embrace the triterpene
glycoside in its free base form in addition to pharmaceutically
acceptable salts of such triterpene glycosides. Furthermore, said
triterpene glycoside may extend to an enantiomer or
diasterioisomers of said triterpene glycoside compound, wherein the
desired functional activity of said triterpene glycoside compound
is maintained in that form.
[0106] The triterpene glycoside compounds or related salts for use
in the invention may be separated and purified by conventional
methods.
[0107] In certain embodiments, the statin provided in the statin
composition is at least one statin compound or a stain-like
compound. The terms "statin compound" and "statin-like compound"
embrace analogues, metabolites, derivatives, pharmaceutically
active salts, such as hydroxy acid salts, precursors, pro-drugs,
enantiomers, and stereoisomers of statins or statin-like
compounds.
[0108] In certain embodiments, the statin salt is a hydroxy acid
salt. In certain embodiments, said hydroxy acid salt is a
metabolite of a statin pro-drug which is administered to, and
metabolised by, the subject in need of treatment.
[0109] In certain embodiments, the statin metabolite results from
the metabolism of a compound following administration to a
subject.
[0110] In certain embodiments, the statin pro-drug is a composition
which, following administration to a subject, is metabolised into a
molecular or structural form which is active in terms of providing
the effects desired by a statin compound.
[0111] In certain embodiments, the composition is a combined
medicament comprising at least one statin compound, pro-drug or
metabolite thereof along with triterpene glycoside or a derivative,
analogue or metabolite thereof.
[0112] In certain further embodiments, the statin compound or
statin-like compound may be an optical or geometric isomer, a
nontoxic pharmacologically acceptable acid addition salt, an
N-oxide, an ester, a quaternary ammonium salt, or a prodrug of any
of the above-listed statin compounds.
[0113] In certain embodiments, amorphous forms of the statins may
be administered to a subject.
[0114] In certain embodiments, the pharmaceutically acceptable
diluent, excipient or carrier may be chosen based on the intended
route of administration of the resulting pharmaceutical
composition. In certain embodiments, the composition is formulated
in beta-hydroxycyclodextrin. In certain embodiments, the
pharmaceutically acceptable carrier is selected from the group
consisting of cyclodextrin, alpha-cyclodextrin, beta-cyclodextrin,
(beta-hydroxypropylcyclodextrin) gamma-cyclodextrin and vitamin E
oil.
[0115] In certain embodiments, the composition is formulated in an
enteric coating made of a polymer. Typically, the enteric coating
comprises a polymer or copolymer selected from the group consisting
of poly(lactic-glycolic acid) polyester, cellulose acetate
phthalate, hydroxypropyl-methyl cellulose phthalate poly(butyl
methacrylate), (2-dimethyl aminoethyl) methacrylate and methyl
methacrylate.
[0116] The terms "a combined preparation" or "combined medicament"
or "combined composition" as used herein define a "kit of parts" in
the sense that two or more of the combination partners, i.e. (a)
the triterpene glycoside or extract composition, (b) the statin
composition and (c) the further anti-viral compound, as defined
above, can be dosed independently or by use of different fixed
combinations with distinguished amounts of the combination partners
(a), (b) and (c), i.e., simultaneously or at different time points.
The parts of the kit of parts can then be administered
simultaneously or administered sequentially or separately, for
example chronologically staggered at different time points, and
with equal or different time intervals for any part of the kit of
parts.
[0117] Typically the time intervals of the administration of the
dosage forms are chosen such that the effect on the treated disease
resulting from the combined use of the parts is larger than the
effect which would be obtained by use of only any one of the
combination partners (a), (b) and (c).
[0118] The ratio of the total amounts of the combination partners
to be administered in the combined preparation can be varied, for
example, in order to cope with the needs of a subject to be
treated.
[0119] Typically the amount of the statin composition and the
triterpene glycoside or extract composition is selected such that a
synergism results from the administration of the compositions,
synergism being an effect which is more than an additive effect
resulting from the combination of the statin composition and the
triterpene or extract composition. Further advantages may be the
reduction in side effects or reduction in effective dosage of one
or both compositions.
[0120] In various further aspects, the present invention extends to
a pharmaceutical pack comprising one or more compartments wherein
at least one compartment comprises one or more of the compounds as
defined herein, or a composition or combined medicament as defined
herein or a medicament as defined herein.
[0121] In certain embodiments, the viral infection is by a virus of
the Flaviviridae family, such as a pestivirus, a flavivirus or a
hepacivirus.
[0122] In certain embodiments, the viral infection is by a
flavivirus. In certain embodiments, the flavivirus is hepatitis C.
The compositions and methods of the invention further have utility
in preventing or inhibiting the onset and development of infection
or other conditions associated with hepatitis C, such as cirrhosis
of the liver, decompensated liver disease, chronic liver damage
and/or hepatocellular carcinoma. The compositions and methods of
the invention further have utility in the treatment of hepatitis
contaminated blood products, in particular blood products
contaminated with hepatitis C.
[0123] In certain embodiments, the viral infection is by a
retrovirus. In certain embodiments, the retrovirus is HIV, such as
HIV1 or HIV2. In certain embodiments, the retrovirus is HIV1.
[0124] In certain embodiments, the viral infection may be by any
one of the viruses selected from the group consisting of, but not
limited to, influenza virus, hepatitis A, hepatitis B, hepatitis C,
hepatitis D, hepatitis E, hepatitis F, hepatitis G, hepatitis H,
autoimmune hepatitis and Respiratory Syncytical Virus (RSV). The
influenza virus may be a type A influenza virus or a type B
Influenza virus. In certain embodiments, the virus is a
hepatitis-causing virus selected from the group consisting of, but
not limited to, the Epstein-Barr virus (EBV), cytomegalo virus
(CMV), yellow fever, Bovine Viral Diarrhoea Virus (BVDV), hog
cholera virus and sheep border disease virus.
[0125] In certain embodiments, the viral infection is by any one of
the viruses selected from one or more flavivirus or togavirus
infections including, but not limited to, California encephalitis
virus, St. Louis encephalitis virus, western equine encephalitis
virus, eastern equine encephalitis virus, Colorado tick fever
virus, LaCrosse encephalitis virus, Japanese encephalitis virus,
yellow fever virus, Venezuelan equine encephalitis virus, Murray
valley fever virus, tick-borne encephalitis viruses, GB virus A, GB
virus B, GB virus C, Dengue virus 1, Dengue virus 2, Dengue virus
3, Dengue virus 4, Semliki Forest virus and Sindbis virus.
[0126] In certain further embodiments, the viral infection may be
derived from a rubivirus, such as human rubella virus,
pestiviruses, such as mucosal disease viruses, for example, bovine
virus diarrhoea virus, hog cholera virus and sheep border disease
virus, reteroviruses, such as a human immunodeficiency virus, for
example HIV1 or HIV2, simian immunodeficiency viruses, a
recombinant human simian immunodeficiency virus, a feline
immunodeficiency virus, a feline or murine leukaemia virus, a
feline or murine sarcoma virus, Rote viral infections in children
and marburg viruses.
[0127] In certain embodiments, the viral infection results in an
AIDS related syndrome.
[0128] In certain embodiments, viral replication is inhibited or
suppressed.
[0129] In certain embodiments, the viral infection is a retroviral
infection and the method of the present invention encompasses
treatment and/or prophylaxis of complications or consequences of
retroviral infection.
[0130] In certain embodiments, the viral infection is caused by a
lipid envelope virus.
[0131] As used herein, the term "subject" refers to an animal,
preferably a mammal and in particular a human. A "subject" in the
context of the present invention therefore includes and encompasses
mammals, such as humans, primates and livestock animals (e.g.
sheep, pigs, cattle, horses, donkeys); laboratory test animals,
such as mice, rabbits, rats and guinea pigs; and companion animals,
such as dogs and cats. It is preferred for the purposes of the
present invention that the mammal is a human. The term "subject" is
interchangeable with the term "patient" as used herein.
[0132] In certain embodiments, the subject may be an
immuno-suppressed animal or human. In certain embodiments, the
subject is an immunocompromised AIDS patient or is infected with a
retrovirus such as the HIV virus showing the AIDS related complex
(ARC).
[0133] In certain embodiments, the subject is a neonate.
Administration may be carried out prior to delivery of the neonate
and/or during delivery of the neonate.
[0134] The triterpene glycoside or extract composition may be
administered to a subject via any suitable route. Typically, a
delivery system is selected which will enhance solubility and
provide maximal bioavailability.
[0135] In certain embodiments wherein the viral infection to be
treated is hepatitis, the composition containing the extract or the
at least one triterpene glycoside, such as mogroside, may be
formulated and designed so that upon administration, it has maximum
bioavailability to the liver and hepatitis-infected cells of the
body.
[0136] In certain embodiments, the extract or the triterpene
glycoside, such as mogroside, is delivered to virally infected
cells by incorporating the extract or the triterpene glycoside
composition into a liposome or carbohydrate vehicle. The liposome
or carbohydrate vehicle can be specifically targeted to infected
viral cells by placing antibodies directed to viral antigens on the
surface of the liposome or vehicle. This is advantageous in that it
allows the liposome or carbohydrate vehicle to selectively target
virus-infected cells. In certain embodiments, liposomes are
provided carrying high concentrations of at least one mogroside to
infected cells.
[0137] In certain embodiments, routes of administration may
include, but are not limited to, parenterally (including
subcutaneous, intramuscular and intravenous, by means of, for
example a drip patch), oral, rectal (suppositories), nasal,
gastric, topical (including buccal and sublingual), infusion,
vaginal, intradermal, intraperitoneally, intracranially,
intrathecal and epidural administration. In certain embodiments,
the composition is administered via oral or nasal inhalation. For
administration via the oral or nasal inhalation routes, typically
the active ingredient will be in a suitable pharmaceutical
formulation and may be delivered using a mechanical form including,
but not restricted to an inhaler, nebuliser device or a nasal
spray. Further, where the oral or nasal inhalation route is used,
administration by a SPAG (small particulate aerosol generator) may
be used.
[0138] In certain embodiments, the route of administration is oral
or parenteral, for example, intravenously (that is by injection,
infusion or continuous drip), intramuscularly or rectally by
suppository.
[0139] For intravenous injection, the active ingredient will be in
the form of a parenterally acceptable aqueous solution which is
pyrogen-free and has suitable pH, isotonicity and stability.
Methods of preparing suitable solutions using, for example,
isotonic vehicles such as sodium chloride injection, Ringer's
injection, dextrose solution and Lactated Ringer's injection will
be known to persons skilled in the art. Appropriate non-aqueous
carriers may also be used and examples include cyclodextrin,
preferably hydroxypropyl beta cyclodextrin, mixed oils (vitamin E
oil), polyethylene glycol and ethyl oleate. A preferred carrier is
cyclodextrin in water. Preservatives, stabilisers, buffers,
antioxidants and/or other additives may be included as required to
enhance isotonicity and chemical stability.
[0140] Pharmaceutical compositions for oral administration may be
in tablet, solid, capsule, powder or liquid form. A tablet may
comprise a solid carrier such as gelatin or an adjuvant. Liquid
pharmaceutical compositions generally comprise a liquid carrier,
such as water, petroleum, animal or vegetable oils, mineral oil or
synthetic oil. Physiological saline solution, dextrose or other
saccharide solutions or glycols such as ethylene glycol, propylene
glycol or polyethylene glycol may be included. Suitable
formulations for oral administration further include hard or soft
gelatin capsules, dragees, pills, tablets, including soft-coated
tablets, troches, lozenges, melts, powders, micronized particles,
non-micronized particles, solutions, emulsions, elixirs,
suspensions, syrups or inhalations and controlled release forms
thereof.
[0141] Further forms of oral administration include addition of the
composition to animal feed and/or drinking water for administration
to animals in the treatment and/or prophylaxis of viral infection
in animals.
[0142] In certain embodiments, administration is topical. Suitable
formulations for topical administration include creams, gels,
jellies, mucliages, pastes and ointments. The compounds may be
formulated for transdermal administration, for example in the form
of transdermal patches so as to achieve systemic
administration.
[0143] The triterpene glycoside or extract composition may also be
administered via microspheres, liposomes, other microparticulate
delivery systems or sustained release formulations placed in
certain tissues including blood.
[0144] In certain embodiments, the triterpene glycoside or extract
composition may be implanted into a subject or injected using a
drug delivery system.
[0145] In certain embodiments, the triterpene glycoside or extract
composition may be administered in the form of an infusion solution
or as a nasal inhalation or spray.
[0146] The pharmaceutical composition according to the present
invention may be administered locally or systemically. Systemic
administration is understood to refer to any mode or route of
administration that results in effective amounts of active
triterpene glycoside or extract appearing in the blood or at a site
remote from the site of administration.
[0147] In certain embodiments, the pharmaceutical composition
according to the present invention may be administered
intermittently. This allows the subject to suspend therapy for
periods without the worry of inactivity of the drug resulting from
the development of a viral resistant strain.
[0148] In certain embodiments, the extract or triterpene glycoside,
such as mogroside, is micronized. The term "micronized" is intended
to mean that the compound has been micronized in accordance with
any process for micronizing, a number of which are known in the
art. The micronized particles preferably include a percentage of
particles having a diameter of about 10 microns, or less,
preferably 5 microns or less. For example, in a certain aspect of
the invention, at least 80% of the particles in a formulation of
micronized particles have a diameter of less than 5 microns. An
alternative to micronizing a compound is to solubilize the compound
and put it into liposomes of appropriate size. The manufacture of
liposomes and the insertion of active ingredients into such
liposomes are well known in the art.
[0149] Examples of the techniques and protocols mentioned above and
other techniques and protocols which may be used in accordance with
the invention can be found in Remington's Pharmaceutical Sciences,
18th edition, Gennaro, A. R., Lippincott Williams & Wilkins;
20th edition (Dec. 15, 2000) ISBN 0-912734-04-3 and Pharmaceutical
Dosage Forms and Drug Delivery Systems; Ansel, H. C. et al. 7th
Edition ISBN 0-683305-72-7, the entire disclosures of which is
herein incorporated by reference.
[0150] The triterpene glycoside or extract is preferably
administered to a subject in a "therapeutically effective amount",
this being an amount sufficient to show benefit to the subject. In
particular, the benefit may be the treatment, partial treatment or
amelioration of at least one symptom associated with the viral
infection, or the prevention or partial inhibition of the onset of
at least one symptom associated with the viral infection. The
severity and/or time of onset of the at least one symptom may be
reduced. Where the context demands, a "therapeutically effective
amount" is an amount which induces, promotes, stimulates or
enhances the development of an antiviral response by the
subject.
[0151] The actual amount administered, and the rate and time-course
of administration, will depend on the nature and severity of the
viral infection to be treated. Prescription of treatment, e.g.
decisions on dosage etc., is ultimately within the responsibility
and at the discretion of general practitioners and other medical
doctors, and typically takes account of the viral infection to be
treated, the condition of the individual patient, the site of
delivery, the method of administration and other factors known to
practitioners. The precise dose will depend upon a number of
factors, including the precise nature of the form of the triterpene
glycoside compound to be administered.
[0152] Preferably, a dose is administered such as to produce a
circulating concentration of the triterpene glycoside and/or its
metabolites sufficient to reduce viral loads as monitored by, e.
g., viral titer methods or by PCR.
[0153] In certain embodiments, the composition is in the form of a
unit dose that comprises 5-500 mgs of one or more mogrosides.
[0154] In certain embodiments, the composition is administered at a
concentration of 6 .mu.g/ml. This concentration has been shown to
be particularly effective in the treatment of HIV.
[0155] In certain embodiments, the composition is administered at a
concentration of greater than 40 .mu.g/mL, more preferably greater
than 100 .mu.g/mL. This concentration has been shown to be
particularly effective in the treatment of BVDV.
[0156] In certain embodiments, the composition is administered
daily to a subject.
[0157] In certain embodiments, the composition may be used in ex
vivo applications to kill viruses. The virus may be any of the
viruses listed above. For example, the composition may be used for
disinfecting equipment, walls and floors. The composition may also
be used in the disinfection of blood products, in particular, blood
products contaminated with viruses such as hepatitis C.
[0158] As used herein, the term "treatment" and associated terms
such as "treat" and "treating" mean the prevention or reduction of
the viral infection or the prevention or reduction of the
progression, severity and/or duration of any symptom associated
with the viral infection, wherein said reduction results from the
administration of a composition of the invention. The term
"treatment" refers to any regimen that can benefit a subject. The
treatment may be in respect of an existing condition or may be a
prophylactic (preventative) treatment. Treatment may include
curative, alleviative or prophylactic effects. References herein to
"therapeutic" and "prophylactic" treatments are to be considered in
their broadest context. The term "therapeutic" does not necessarily
imply that a subject is treated until total recovery. Similarly,
"prophylactic" does not necessarily mean that the subject will not
eventually contract a disease condition. Accordingly, therapeutic
and prophylactic treatment includes amelioration of the symptoms of
a particular condition or preventing or otherwise reducing the risk
of developing a particular condition. The term "prophylactic" may
be considered as including reducing the severity or the onset of a
particular condition.
[0159] Unless otherwise defined, all technical and scientific terms
used herein have the meaning commonly understood by a person who is
skilled in the art in the field of the present invention.
[0160] Throughout the specification, unless the context demands
otherwise, the terms "comprise" or "include", or variations such as
"comprises" or "comprising", "includes" or "including" will be
understood to imply the inclusion of a stated integer or group of
integers, but not the exclusion of any other integer or group of
integers.
[0161] As used herein, terms such as "a", "an" and "the" include
singular and plural referents unless the context clearly demands
otherwise. Thus, for example, reference to "an active agent" or "a
pharmacologically active agent" includes a single active agent as
well as two or more different active agents in combination, while
references to "a carrier" includes mixtures of two or more carriers
as well as a single carrier, and the like.
Examples
Example 1
Anti-Viral Activity of Luo Han Guo Extract Against Bovine Viral
Diarrhoea Virus
[0162] Materials and Methods
[0163] An extract of Luo Han Guo was obtained using the process
described in U.S. Pat. No. 5,411,755 and tested for antiviral
activity against Bovine Viral Diarrhoea Virus (BVDV), strain NADL.
BVDV is a flavivirus in the same family as the human hepatitis C
virus. Hepatitis C cannot be grown in tissue culture so BVDV is
used as a model virus due to the structural similarities between
these two viruses.
[0164] The Luo Han Guo extract can be stored at room temperature
and is water soluble. The Luo Han Guo extract was tested
independently at a wide range of concentrations. The extract was
tested in two assays, one at 100, 50, 25, 12.5 and 6.25 .mu.g/mL
and the other at 3600, 1200, 400, 133.3 and 44.4 .mu.g/mL.
[0165] Bos taurus (BT) turbinate cells were used as host cells for
the virus. After the cells were plated and allowed to grow
overnight, The extract was added and then the virus was inoculated
into the cultures. The cultures were allowed to grow for 7 days and
then a fluorometric cytoproliferation assay was run. The data from
all replicate cultures was averaged and compared to the growth of
the negative controls (no virus or extract). The extract was
present during absorption and in the media for the duration of the
assays. A titration was run on the virus under the assay conditions
but without the extract to determine the optimal viral inoculum for
the antiviral assay. A dilution of 10.sup.-4 of the stock virus was
determined to be the correct inoculum for the antiviral assays. The
growth medium used was Dulbecco's Minimum Eagle's Medium and 10%
filtered horse serum.
[0166] Cultures with virus and no extract were used as positive
controls. Cultures of uninfected cells only provided negative
controls. Cytotoxicity controls were provided using the extract at
all concentrations used, but without viral inoculum.
[0167] Results
[0168] Tables 1 and 2 show the cytotoxicity and antiviral
percentage of negative control and the inhibition index for various
concentrations of the extract.
[0169] The results for ranges 2 to 100 .mu.g/mL of extract for the
antiviral percentage of negative control shown in Table 2 are
graphed in FIG. 1.
[0170] The inhibition index was calculated as one minus the ratio
of the average fluorescent units of the positive control to the
average fluorescent units of infected cultures with a particular
concentration of extract (1-(average positive control+average with
extract)). 0 represents no inhibition. 0.9 to 1 represents the best
inhibition. The inhibition index helps define the dosages at which
a drug is most effective.
[0171] In this type of cytoproliferation assay, the outcome is a
race between the virus and the experimental compound. If the virus
is slow to exhibit killing of the cell, the experimental compounds
can do a more thorough job, but if the virus is very aggressive,
like BVDV, and kills quickly, the experimental compounds cannot
catch up. The percent of the negative control does not take into
account how aggressive the virus is. It merely compares the growth
of the cells, in particular cultures, to the growth of the negative
control.
[0172] The inhibition index on the other hand is based on the
positive control. It is an index of how much better the cells grow
with the compound as compared to the positive control (infected
cells without compound) where the closer the value is to 1, the
better the action.
TABLE-US-00002 TABLE 1 Percent of negative control and inhibition
index for high and low range concentrations of Luo Han Guo extract
Cytotoxicity Antiviral (No Virus) % % of Neg. Inhibition Sample of
Neg. Control Control Index Efficacy Luo Han Guo extract - low range
Negative control 100% NA NA NA Positive control NA 8.5% NA NA 100
.mu.g/mL 83.7% 4.1% 0 None 50 .mu.g/mL 101.4% 15.6% 0.46 Partial 25
.mu.g/mL 103.1% 1.5% 0 None 12.5 .mu.g/mL 114.3% 2.8% 0 None 6.25
.mu.g/mL 139.3% 4.6% 0 None Luo Han Guo extract - high range
Negative control 100% NA NA NA Positive control NA 0.8% NA NA 3600
.mu.g/mL 86.9% 28.6% 0.97 Partial 1200 .mu.g/mL 111.5% 18.6% 0.96
Partial 400 .mu.g/mL 88.8% 22.6% 0.96 Partial 133.3 .mu.g/mL 114.2%
10.4% 0.92 Partial 44.4 .mu.g/mL 99.3% 6.0% 0.86 None
TABLE-US-00003 TABLE 2 Percent of negative control and inhibition
index for Luo Han Guo extract Luo Han Guo extract Cytotoxicity (No
Virus) Antiviral % of Neg. % of Neg. Inhibition Sample Control
Control Index Efficacy Negative control (no 100% NA NA NA drug, no
virus) 0 .mu.g/mL Positive control (no drug, NA 8.2% NA NA with
virus) 0 .mu.g/mL 100 .mu.g/mL 135.9% 49.8% 0.84 Partial 36
.mu.g/mL 120.9% 33.2% 0.75 Partial 12 .mu.g/mL 135.6% 9.3% 0.12 No
6 .mu.g/mL 123.0% 10.7% 0.23 No 2 .mu.g/mL 123.1% 12.6% 0.35 No
[0173] The Luo Han Guo extract showed no cytotoxicity over the
entire range and was most effective against BVDV at concentrations
of greater than 100 .mu.g/mL. At concentrations of less than 100
.mu.g/mL, its effectiveness fell and it had little efficacy at
concentrations of 25 .mu.g/mL or less. As noted above, the closer
the value of the inhibition index is to 1, the better the action.
The high ranges of the extract had values close to 1 (0.86 to
0.97).
[0174] The extract was shown not to be able to completely inhibit
BVDV but it had an effective range (40 to 3600 .mu.g/mL) where it
was able to partially inhibit BVDV proliferation. Although it was
unable to completely inhibit BVDV under the conditions of this
study, in the inventor's experience no drug has ever completely
inhibited BVDV in this cell model nor has any drug in this in vitro
system ever been shown to act in any way superior to the extract.
BVDV, which is the most accurate model for hepatitis C available,
is a particularly aggressive virus. Therefore, percentages of the
negative control in the range of 50% for BVDV are considered an
excellent result. Optimum results were obtained for concentrations
of 100 .mu.g/mL where 49.8% negative control was observed.
Example 2
Anti-Viral Activity of the Triterpene Glycoside Mogroside V Against
HIV-1
[0175] Materials and Methods
[0176] Mogroside V was tested for efficacy against HIV-1 strain
IIIB using H9 cells as the viral host.
[0177] Mogroside V can be stored at room temperature in powder form
and is water-soluble. Aqueous stock solution was stored at
4.degree. C. Final reagent dilutions were made in growth media from
the aqueous stock solution.
[0178] Five dilutions of Mogroside V were used: 100, 36, 12, 6 and
2 .mu.g/mL.
[0179] H9 cells were used as host cells for the virus. H9 is a
human T cell clone selected for high yield permissive growth of
HIV-1, cloned from Hut 78, a human T cell lymphoma cell line with
an inducer/helper phenotype derived from the peripheral blood of a
patient with Sezary Syndrome.
[0180] The virus used was HIV-1, Strain IIIB, Catalog No
10-124-000, Lot No. 6M0008-DP. HIV-1 IIIB infects H9 cells but does
not consistently cause cytopathic effect (CPE) and therefore viral
growth is measured by HIV-1 p24 antigen capture ELISA.
[0181] A titration was run on the virus under the assay conditions
without the experimental compounds to determine the viral inoculum
for the antiviral assay, that is the lowest multiplicity of
infection (MOI) that will infect all positive control cultures. The
inoculum was approximately seven times the TCID.sub.50/mL of the
stock virus.
[0182] Cultures with virus and no Mogroside V provided a positive
control. Cultures of uninfected cells only (no Mogroside V)
provided a negative control.
[0183] Cytotoxicity controls were provided using Mogroside V at all
concentrations used, but without viral inoculum. Cytotoxicity was
judged from the microscopic inspection of the cells. If the cells
with Mogroside V grew more slowly than the negative control, had an
altered morphology or appeared necrotic, Mogroside V was judged to
be cytotoxic. If the cytoxicity controls had the same appearance as
the negative controls, Mogroside V was considered non-toxic at the
concentration in question.
[0184] Assay Parameters
[0185] 1 mL cultures were plated with approximately
2.5.times.10.sup.5 cells per culture. Four replicates were run for
each assay condition.
[0186] The virus was absorbed onto the host cells in the presence
of a reduced volume of culture medium for 2 hours. The inoculum was
then washed off the cells with phosphate buffered saline. Cultures
were then fed with appropriate medium containing Mogroside V to
their final volume. The growth medium used was RPMI 1640 with 10%
fetal bovine serum and 50 .mu.g/mL gentamicin. Mogroside V was left
in the growth medium for the duration of the experiment.
[0187] The assay was run for 7 days and re-fed media with
appropriate compound concentrations at 4 days.
[0188] The amount of virus proliferation was measured using an
HIV-1 p24 antigen capture ELISA. At 7 days, supernatants were
harvested and frozen. Supernatants were thawed and diluted as
necessary, and the HIV-1 p24 ELISA assay was run. Excess
supernatant was refrozen and held for 3 months. The HIV-1 p24
.mu.g/mL of replicate cultures were averaged and presented as a
percentage of the positive control.
[0189] Inhibition Index
[0190] The inhibition of Mogroside V at each concentration was
calculated by comparing the average amount of virus produced by the
experimental cultures (with Mogroside V) to the average of the
positive controls (virus but no drug). The inhibition index for
each Mogroside V concentration was computed as 1-(average HIV-1 p24
with Mogroside V+average HIV-1 p24 for the positive control). An
inhibition index of zero indicated no efficacy. The closer to 1 the
inhibition index was, the greater the efficacy.
[0191] Mogroside V was judged to be efficacious against the virus
if cultures with Mogroside V had an inhibition index of 0.90 or
greater, partially efficacious if cultures had an inhibition index
between 0.20 and 0.89 and as having no efficacy if cultures had an
inhibition index of less than 0.20.
[0192] If Mogroside V had a cytotoxic effect on the host cells as
determined by the cytotoxicity control, any decrease in viral
growth was judged to be an effect of the cytotoxicity and not an
antiviral effect.
[0193] Results Positive controls were positive for HIV-1 p24. The
negative control appeared as a healthy growing culture by
microscopic inspection and had no HIV-1 p24 signal.
[0194] Mogroside V was run over a dosage range of 2 to 100 .mu.g/mL
and results are provided in Table 3 below. FIG. 2 shows the
antiviral effect of these ranges. Mogroside V showed no
cytotoxicity, as determined by microscopic inspection, over the
entire range of concentrations used and was most effective against
HIV-1 at a concentration of 6 .mu.g/mL.
TABLE-US-00004 TABLE 3 Cytotoxicity and Inhibition Index for
Mogroside V Sample Cytotoxicity (Mogroside V (Appearance % Positive
Inhibition .mu.g/mL) of cells) Control Index Efficacy Negative
Normal 0 NA NA Control Positive Control NA 100 0 NA 100 .mu.g/mL
Normal 88 0.12 None 36 .mu.g/mL Normal 59 0.41 Partial 12 .mu.g/mL
Normal 34 0.66 Partial 6 .mu.g/mL Normal 21 0.79 Partial 2 .mu.g/mL
Normal 48 0.52 Partial
[0195] Mogroside V was shown not to be able to completely inhibit
HIV-1 under the conditions of this study. However, all
concentrations used (with the exception of the highest
concentration of 100 .mu.g/mL) were able to significantly suppress
viral production. The greatest effect was seen at 6 .mu.g/mL
wherein Mogroside V was nearly 80% effective at suppressing HIV-1
replication, with efficacy falling off as the Mogroside V
concentration was increased or decreased. Without wishing to be
bound by theory, it is hypothesized that the reduction in efficacy
at higher concentrations may be due to chemical interactions in
solution that inhibit absorption by the cells, for example the
formation of inhibiting constructs by Mogroside V.
Example 3
Anti-Viral Activity of Luo Han Guo Extract Against Bovine Viral
Diarrhoea Virus (BVDV) when Combined with Fluvastatin
Materials and Methods
[0196] Materials and methods were similar to those outlined in
Example 1.
[0197] Fluvastatin must be stored between 0.degree. and 5.degree.
C. and is water soluble. The extract and fluvastatin were tested in
combination with two concentrations of the extract, an effective
dose (100 .mu.g/mL) and a dose below its effective range for BVDV
(6 .mu.g/mL), and five doses of fluvastatin in its effective range
(0.313, 0.625, 1.25, 2.5, and 5.0 .mu.g/mL).
[0198] Results
[0199] Table 4 shows the percent of negative control and inhibition
index for the various concentrations of the extract and
fluvastatin. Fluvastatin was shown to extend the lower range of the
effectiveness of the extract. In particular, with 6 .mu.g/mL of
extract, the inhibition index for 5 and 0.3125 .mu.g/mL fluvastatin
was much improved over fluvastatin alone (FIG. 3) or extract alone
(FIG. 1).
TABLE-US-00005 TABLE 4 Percent of negative control and inhibition
index for Luo Han Guo extract and fluvastatin combination.
Cytotoxicity Antiviral (No Virus) % of % of Neg. Inhibition
Fluvastatin Neg. Control Control Index Efficacy Negative control
100% NA NA NA Positive control NA 8.2% NA NA Luo Han Guo Extract
(100 .mu.g/mL)/Fluvastatin 5 .mu.g/mL 93.6% 25.2% 0.67 Partial 2.5
.mu.g/mL 113.5% 51.2% 0.84 Partial 1.25 .mu.g/mL 111.5% 45.3% 0.82
Partial 0.625 .mu.g/mL 108.4% 33.9% 0.76 Partial 0.3125 .mu.g/mL
115.0% 17.0% 0.52 No Luo Han Guo Extract (6 .mu.g/mL)/Fluvastatin 5
.mu.g/mL 88.7% 50.3% 0.84 Partial 2.5 .mu.g/mL 103.3% 39.4% 0.79
Partial 1.25 .mu.g/mL 103.4% 34.2% 0.76 Partial 0.625 .mu.g/mL
113.8% 25.5% 0.68 Partial 0.3125 .mu.g/mL 119.4% 38.0% 0.78
Partial
[0200] Fluvastatin series with 6 .mu.g/mL Luo Han Guo Extract Luo
Han Guo extract at 6 .mu.g/mL in the absence of fluvastatin had no
antiviral activity against BVDV (Table 2), but when coupled with
fluvastatin at any of the concentrations of fluvastatin used, the
combinations all had more antiviral activity than the extract at 6
.mu.g/mL alone (Table 4). At 5 .mu.g/mL, fluvastatin alone had some
cytotoxicity and at 0.3125 .mu.g/mL fluvastatin alone had no
antiviral activity, but when either of these two concentrations of
fluvastatin is combined with the extract at 6 .mu.g/mL, the
antiviral effect is greater than that for either fluvastatin or
extract alone. With 5 .mu.g/mL fluvastatin and 6 .mu.g/mL extract,
the inhibition index was 0.84, and with 0.3125 .mu.g/mL fluvastatin
and 6 .mu.g/mL extract, the inhibition index was 0.78 (Table 4),
both of which are much improved over fluvastatin alone (data not
shown). At the intermediate concentrations of fluvastatin where
fluvastatin alone is most efficacious, fluvastatin alone had more
antiviral activity than the combination. At 2.5 .mu.g/mL there was
not much difference between fluvastatin alone and the combination.
The combination with 1.25 .mu.g/mL fluvastatin was about 10% less
effective than fluvastatin alone, and at 0.625 .mu.g/mL, the
combination was 20% less effective than fluvastatin alone.
[0201] Fluvastatin Series with 100 .mu.g/mL Luo Han Guo Extract
[0202] Luo Han Guo extract showed good inhibition of BVDV by itself
at 100 .mu.g/mL (Table 2). No combination with fluvastatin
increased this effectiveness. In fact most combinations with
fluvastatin showed less activity than extract alone, except for 2.5
.mu.g/mL fluvastatin where the activity with the extract is the
same as if the extract were run alone and the inhibition index was
0.84. Likewise the efficacy of the combination does not improve
over that of fluvastatin alone, except for 2.5 .mu.g/mL where there
is about a 10% increase in efficacy over fluvastatin alone.
[0203] In the ranges where fluvastatin and extract are most
effective individually, there was therefore no advantage in
combining the two drugs. However, fluvastatin was shown to extend
the lower range of the effectiveness of the extract. This would be
advantageous where the extract is not tolerated well by a subject
or is prohibitively expensive.
[0204] All documents referred to in this specification are herein
incorporated by reference. Various modifications and variations to
the described embodiments of the inventions will be apparent to
those skilled in the art without departing from the scope of the
invention. Although the invention has been described in connection
with specific preferred embodiments, it should be understood that
the invention as claimed should not be unduly limited to such
specific embodiments. Indeed, various modifications of the
described modes of carrying out the invention which are obvious to
those skilled in the art are intended to be covered by the present
invention.
* * * * *