NZ625051B2 - Combination therapy with interferon and andrographolides for multiple sclerosis - Google Patents
Combination therapy with interferon and andrographolides for multiple sclerosis Download PDFInfo
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- NZ625051B2 NZ625051B2 NZ625051A NZ62505112A NZ625051B2 NZ 625051 B2 NZ625051 B2 NZ 625051B2 NZ 625051 A NZ625051 A NZ 625051A NZ 62505112 A NZ62505112 A NZ 62505112A NZ 625051 B2 NZ625051 B2 NZ 625051B2
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- A61K31/33—Heterocyclic compounds
- A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
- A61K31/34—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having five-membered rings with one oxygen as the only ring hetero atom, e.g. isosorbide
- A61K31/341—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having five-membered rings with one oxygen as the only ring hetero atom, e.g. isosorbide not condensed with another ring, e.g. ranitidine, furosemide, bufetolol, muscarine
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- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/19—Cytokines; Lymphokines; Interferons
- A61K38/21—Interferons [IFN]
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- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/19—Cytokines; Lymphokines; Interferons
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Abstract
Disclosed is the combination therapy of interferon and a composition of Formula 1, such as andrographolide, for the treatment of demyelinating diseases and fatigue.
Description
COMBINATION THERAPY WITH INTERFERON AND
ANDROGRAPHOLIDES FOR MULTIPLE SCLEROSIS
FIELD OF THE INVENTION
The present invention relates to pharmaceutical compositions for treating Multiple
Sclerosis (MS) and/or other demyelinating diseases, comprising an interferon (IFN),
compound of Formula I, and, optionally, one or more pharmaceutically acceptable
excipients and/or carriers. Another object of the present invention is to provide a
method for treating a subject suffering from MS and/or another demyelinating
disease, and a method for reducing fatigue in a subject in need thereof.
BACKGROUND OF THE INVENTION
Multiple Sclerosis (MS) is a chronic, inflammatory, demyelinating disease of the
Central Nervous System (CNS). It starts typically between 20 and 40 years of age,
and prevails over women. MS is clinically definite diagnosed after at least two
neurologic events, showing demyelination in different areas of the CNS and in
different times (Jacobs et al. 2000, The New England Journal of Medicine. 343(13):
898-904).
The cause of MS is still unknown, but several lines of evidence, derived from the
experimental autoimmune encephalomyelitis, support the autoimmune origin of the
disease (Inglese et al. 2010, NMR Biomed. 23(7): 865-872). MS is characterized by
areas of demyelinated plaques or islands disseminated throughout the CNS with a
predilection for optic nerves, spinal cord, periventricular white matter (WM), corpus
callosum, and cortical and sub-cortical gray matter (GM). (Inglese et al. 2010, NMR
Biomed. 23(7): 865-872). Lesions in MS are very heterogeneous, respect to the
presence and extend of inflammation, demyelination, axonal injury, gliosis and
remyelination (Inglese et al. 2010, NMR Biomed. 23(7): 865-872).
Functional Systems Scores (FSS) and Expanded Disability Status Scale (EDSS)
constitute one of the oldest and most widely utilized assessment instruments in MS
(Kurtzke J.F. 1983, Neurology, 33:1444-1452). Based on a standard neurological
examination, the 7 functional systems are rated. These ratings are then used in
conjunction with observations and information concerning gait and use of assistive
devices to rate the EDSS. Each of the FSS is an ordinal clinical rating scale from 0 to
or 6. The EDSS is an ordinal clinical rating
scale ranging from 0 (normal neurologic examination) to 10 (death due to MS) in half-point
increments.
Magnetic resonance imaging (MRI) of the brain, can add certainty to the diagnosis, by
identifying lesions consistent with the occurrence of demyelination.
Incorporation of IFNs to treatment of MS has opened a new pharmaceutical pathway respect
to traditional immunosuppressive drug (Zaragoza et al. 2002, Farmacia Hospitalaria. 26(5):
294-301).
Interferons are cytokines with antiviral, antiproliferative, and antitumor activity, although they
have different immunomodulatory characteristics. Therefore, these molecules have a great
therapeutic potential in neoplastic and viral diseases. There are different types of IFNs:
interferon-alpha (IFN-a), produced by leucocytes, interferon-beta (IFN-b ), produced by
fibroblasts and interferon-gamma (ING-g ), produced by lymphocytes-T (Zaragoza et al. 2002,
Farmacia Hospitalaria. 26(5):294-301).
Particularly, several data has shown the efficiency of IFN-b in the treatment of MS. Different
randomized, double-blind, placebo-controlled clinical trials has demonstrated a beneficial
effect in a variety of parameters of the disease, reducing disability, frequency of relapsing,
frequency of appearance of new lesions, and improving cerebral atrophy (Zaragoza et al.
2002, Farmacia Hospitalaria. 26(5): 294-301).
Despite teaching the use of IFN-b for MS, however, the art also recognizes that such
treatment is only moderately effective; IFN-b at best merely slows the progression of MS, it
does not cure MS. Further, the relatively high cost of IFN-b renders it financially unavailable
to many patients who need it. The art thus has a long-felt need for a more effective treatment
for MS.
SUMMARY
Our results show that the treatment of MS and other demyelinating diseases using an IFN is
surprisingly improved if the IFN is administered together with a compound of Formula I :
wherein
R is selected from the group consisting of hydrogen, alkyl or hydroxyl,
R is selected from the group consisting of hydroxyalkyl or alkyl-O-Lj, wherein Li is a
carbohydrate moiety,
R is selected from the group consisting of hydrogen or hydroxyl,
X is selected from the roup consisting of C(=CH ), CH(OH), or a spirooxirane-2 moiety
Z is selected from the group consisting of C¾, CH(OH) or C(=0), and
R is selected from the group consisting of an optionally substituted L -alkyl orL2-alkenyl,
wherein L is an optionally substituted 3-furanyl or 3-furenyl moiety. The Formula I
compound may be provided as a pharmaceutically acceptable salt, ester, ether or pro-drug
thereof, and optionally may be formulated into a finished oral dosage form using one or more
pharmaceutically acceptable excipients and/or carriers.
We have found this combination both favors remyelination and reduces inflammation and
fatigue, thus achieving a synergistic effect. We have demonstrated that the administration of
compound of Formula I in combination with IFN-beta reduces significantly the clinical signs
of MS; a synergistic effect of the two active ingredients.
There are examples of combination of interferon and other substances for preparation of
compositions for treating different diseases. Document US2009/0280087, for example,
discloses the combination of Interferon alpha and C-Phycocianin for a pharmaceutical
preparation for autoimmune disease, allergy and cancer treatments. Document US6869600
discloses the use of growth hormone (GH) together with an interferon (IFN) to produce a
pharmaceutical composition for treating multiple sclerosis and/or other demyelinating
diseases. The prior art, however, fails to suggest combining interferon with any compound of
Formula I.
Therefore, a main object of the present invention is to provide pharmaceutical compositions
for treating Multiple Sclerosis (MS) and/or other demyelinating diseases, comprising an
interferon (IFN), compound of Formula I, and, optionally, one or more pharmaceutically
acceptable excipients and/or carriers.
Another object of the present invention is, therefore, to provide a method for treating a
subject suffering from MS and/or another demyelinating disease, the method consisting of
administering the pharmaceutical compositions of the invention to the subject in an effective
amount and for a time sufficient to produce remyelination and to reduce inflammation.
Also another object of the invention is to provide a method to reduce fatigue in a subject in
need thereof, the method consisting of administering the pharmaceutical compositions of the
invention to the subject in an effective amount and for a sufficient time.
An objection of the invention is to provide the use of a compound of Formula I:
wherein
R is selected from the group consisting of hydrogen, alkyl or hydroxyl,
R is selected from the group consisting of hydroxyalkyl or alkyl-O-L , wherein L is
2 1 1
a carbohydrate moiety,
R is selected from the group consisting of hydrogen or hydroxyl,
X is selected from the group consisting of C(=CH ), CH(OH), or a spirooxirane-2
moiety,
Z is selected from the group consisting of CH , CH(OH) or C(=0), and
R is selected from the group consisting of an optionally substituted L -alkyl orL -
4 2 2
alkenyl, wherein L is an optionally substituted 3-furanyl or 3-furenyl moiety,
or a pharmaceutically acceptable salt, ester, or ether thereof,
in the preparation of a medicament to treat a demyelinating disease in a human patient
wherein said patient is provided with an interferon.
Another object of the present invention is the use of an effective amount of interferon and
an effective amount of a compound of Formula I:
wherein
R is selected from the group consisting of hydrogen, alkyl or hydroxyl,
R2 is selected from the group consisting of hydroxyalkyl or alkyl-O-L , wherein L is
a carbohydrate moiety,
R is selected from the group consisting of hydrogen or hydroxyl,
X is selected from the group consisting of C(=CH ), CH(OH), or a spirooxirane-2
moiety.
Z is selected from the group consisting of CH , CH(OH) or C(=0), and
R is selected from the group consisting of an optionally substituted L -alkyl orL -
4 2 2
alkenyl, wherein L is an optionally substituted 3-furanyl or 3-furenyl moiety,
or a pharmaceutically acceptable salt, ester, ether or thereof,
in the preparation of a medicament to reduce fatigue in a human subject in need thereof.
Also provided is the use of at least one compound of Formula I
to manufacture a medicament to treat a human patient diagnosed as having a demyelinating
disease, said medicament to be used in combination with interferon, said medicament having
an amount of said at least one compound of Formula I effective to treat said demyelinating
disease.
DETAILED DESCRIPTION
The present invention provides pharmaceutical compositions and methods for treating
Multiple Sclerosis (MS) and/or other demyelinating diseases, comprising combination
therapy with an interferon (IFN) and at least one compound of the Formula (I):
wherein
R is selected from the group consisting of hydrogen, alkyl or hydroxyl,
[Text continued on page 5]
R is selected from the group consisting of hydroxyalkyl or alkyl-O-Lj, wherein L is
a carbohydrate moiety,
R is selected from the group consisting of hydrogen or hydroxyl,
X is selected from the group consisting of C(=CH ), CH(OH), or a spirooxirane-2
moiety.
Z is selected from the group consisting of C , CH(OH) or C(=0), and
R is selected from the group consisting of an optionally substituted L -alkyl orL -
alkenyl, wherein L is an optionally substituted 3-furanyl or 3-furenyl moiety,
or a pharmaceutically acceptable salt, ester, ether or prodrug thereof, and, optionally, one or
more pharmaceutically acceptable excipients and/or carriers. By "spirooxirane-2 moiety" we
mean an epoxidated C(=CH ) moiety:
In one embodiment, i is methyl.
In another embodiment, R is hydroxymethyl or C Glc, wherein Glc is a glycoside-
forming glucose moiety.
In another embodiment, is an optionally substituted 3-(3-furanyl)-propyl, 3-(3-furanyl)-
prop-l-enyl,3-(3-furanyl)-propenyl, 3-(3-furenyl)-propyl or 3-(3-furenyl)-prop-l-
enyl wherein the 3-furanyl or the 3-furenyl moieties are further optionally substituted.
In one embodiment, Rj, R , R , X and Z are those described above, and R 4 is selected from the
group consisting of:
wherein:
R is selected from the group consisting of hydrogen or hydroxyl,
R 5 and R are independently selected from the group consisting of hydrogen, hydroxyl, or
alkyloxy, or R and R are simultaneously replaced by a single direct bond between the
carbon atoms denoted by *, thus forming a dimer of two monomer molecules of formula (I),
and R and R are independently selected from the group consisting of hydrogen, hydroxyl or
alkyloxy.
In one embodiment, R5, R , R or can be independently methoxy.
In preferred embodiments, the compounds of Formula (I) are selected from the group
consisting of andrographolide, neoandrographolide, 14-deoxyandrographolide 14-deoxy-
11,12-didehydroandrographolide, andrographiside, andrograpanin, 14-deoxy- 11-oxo-
andrographolide, 14-deoxy-l 1-liydroxy-andrographolide, 14-deoxy- 12-hydroxy-
andrographolide, 3,14-dideoxyandrographolide, 3-oxodeoxyandrographolide, 8,17-epoxy-
14-deoxyandrographolide, 14-deoxy- 17-beta-hydroxy andrographolide, 12-
hydroxyandrographolide, bisandrographolide A, 3-oxodeoxy-ll,12-
didehydroandrographolide, 7-hydroxy- 14-deoxyandrographolide, 15-methoxy-3 ,1 -
dihydroxy-8(l 7)1 1,13 -ent-labda-trien-1 6,1 5-olide, andropanolide, 14-deoxy- 12-methoxy-
andrographolide, 14-epi-andrographolide, 19-hidroxi-ent-labda-8(l 7), 13-dien- 15,1 6-olide,
3,13,14, 19-tetrahydroxy-ent-labda-8( 17), 11-dien- 16, 15-olide, 3,19-dihydroxy- 15-methoxy-
ent-labda-8(l 7), 11.13 -trien- 16,15-olide, and 3,19-dihydroxy-ent-labda-8(l 7), 12-dien- 16,15-
olide.
In a most preferred embodiment, the compound of formula I comprises andrographolide.
Andrographolide, CAS Registry No. 55087, systematic name 3-(2-(Decahydro
hydroxy(hydroxymethyl)-5,8a-dimethylmethylenenaphthyl)ethylidene)dihydro
hydroxyfuran-2(3H)-one, is a compound of Formula I, wherein Rl is alkyl, R2 is
hydroxyalkyl, R3 is H, X is C=CH2, Z is CH(OH) and R4 is an (E)hydroxy
propylidenedihydrofuran-2(3H)-one moiety:
Andrographolide is a bitter principle, colorless, neutral crystalline substance, is a diterpene
containing a g -lactone ring. The crystal structure of andrographolide was determined by
Smith et al (1982) and Fujita et al (1984). Andrographolide can be isolated from the aerial
part of Andrographis paniculata by extraction with alcohol or with alkaline solutions.
Hydrolysis of andrographolide under cleavage of the lactone ring yields salts of
andrographolic acid which can be reconverted into andrographolide by acidification (Tang
and Eisenbrand 1992).
In another most preferred embodiment, the compound of formula I comprises
neoandrographolide. Neoandrographolide, CAS Registry No. 272151, systematic name
3-(2-(5-((beta-D-glucopyranosyloxy)methyl)decahydro-5,8a-dimethylmethylene-l-
naphthalenyl)ethyl)-(lR-(lalpha,4abeta,5alpha,8aalpha))-2(5H)-furanone, was first described
by Kleipool (Kleipool, 1952).
The structure of neoandrographolide was described as a diterpene glucoside (Chan et al.,
1971).
In another most preferred embodiment, the compound of Formula I comprises deoxy-
didehydroandrographolide, deoxy-oxoandrographolide or deoxyandrographolide, each
structurally closely related to andrographolide. See Bailmain and Connolly (1973).
In another most preferred embodiment, the compound of Formula I comprises
dideoxyandrographolide (also referred to as andrograpanin, see Fujita et al, (1984)),
andrographiside or a 14-deoxy derivative thereof (e.g., 14-deoxyandrographiside).
Dideoxyandrographolide is the aglicone of neoandrographolide.
Jantan and Waterman (1994) isolated from the aerial part of a Malaysian specimen other
diterpene as the principle constituents, indicating that A . paniculata presents great variation in
chemical compositions depending on the source of origin (Jantan et ah, 1994).
In another most preferred embodiment, the compound of Formula I comprises 3-O -b - -
glucopyranosyl- 14,1 9-dideoxyandrographolide, 14-deoxy- 17-hydroxyandrographolide, 19
[P-D-apiofuranosyl(lf2)-P-D-glucopyranoyl]-3,14-dideoxyandrographolide, 3 b - -
glucopyranosylandrographolide, 12S-hydroxyandrographolide and/or andrographatoside. The
structures of each are taught by Shen et ah 2006.
In another most preferred embodiment, the compound of Formula I comprises andropanolide
or isoandrographolide. The structures of each are taught by Pramanick et ah, 2006.
The prior art teaches that andrographolide reduces interferon gamma production. See Burgos
et al, 2005, United States Patent 8084495 (andrographolide reduces production of interferon
g in T-cells stimulated with concanavalin A). Because the prior art taught that
andrographolide reduces interferon production, the skilled artisan would have predicted that
andrographolide (or similar compounds of Formula I) would have been detrimental if
administered to a patient suffering from a condition (such as multiple sclerosis) which is
known to be treated by administering exogenous interferon. Rather, the artisan would have
predicted that compounds of Formula I would be at best ineffective, and at worst actively
reduce the efficacy of exogenous interferon.
In testing this hypothesis in laboratory mice, however, w e surprisingly found the direct
opposite of what one would have expected: w e found that concomitant administration of a
compound of Formula I increases the effectiveness of interferon, producing an unexpected
and synergistic improvement in health status.
Without intending to limit the legal scope of the legal claims of the instant patent, w e believe
that this synergistic effect may be explained by the following mechanism: w e (and others)
have described that andrographolide can interfere with the Nuclear factor kappaB (NF-KB)
binding to DNA (Hidalgo et ah, 2005; Xia et ah, 2004b) (United States Patent 8084495). NF-
B is a transcription factor found in a great variety of immune cells, participating in the
regulation of genes involved in cellular and physiological processes, such as growth and
apoptosis, and it has an important role in inflammatory and immune responses, by inducing
transcription of pro inflammatory genes (Baeuerle et al, 1996). For instance, the pro
inflammatory mediators such as intercellular adhesion molecule-1, IFNy, iNOS, COX-2 and
IL-8 are proteins regulated by NF-k B . Since andrographolide is able to down-modulate both,
humoral and cellular adaptive immune responses, we suspect this evidence may support an
immune-suppressant effect.
It has been demonstrated in vitro, that this molecule is able to interfere with T cell
proliferation and cytokine release in response to allogenic stimulation. The T cell activation
by dendritic cells (DCs) was completely abolished by exposing DCs to andrographolide
during antigen pulse flruretagoyena et al, 2005). Andrographolide is able to interfere with
maturation of DCs and with their ability to present antigens to T cells.
We also found that andrographolide can interfere with cytokine production in Jurkat cells.
This effect can be mediated by a reduction of IL-2 production by a reduction of signal
transduction pathways and/or interference with transcription factors activation. We proposed
that the cytokine inhibition by andrographolide, can be produced by an interference with
ERK1/2, a MAPK involved in IL-2 and IFN production in T-cells (Burgos et al, 2005).
Other authors showed that andrographolide in dose-dependent manner inhibited macrophages
migration toward C5a. The chemotaxis inhibition is explained because andrographolide
significantly attenuated C5a-stimulated phosphorylation of ERK1/2, and of its upstream
activator, MAP kinase-ERK kinase (MEK1/2) and Akt phosphorylation, a downstream target
protein for PI3K (Tsai et al, 2004). The interference of ERK1/2 phosphorylation also
explains the inhibitoiy effect of andrographolide on TNF-alpha, IL-12a and IL-12b at mRNA
level, and production of TNF-alpha and IL-12p70 proteins in a concentration-dependent
manner in murine macrophages (Qin et al, 2006). An interference of signal transduction
pathways in T-cells has been recently observed. Using anti-CD3 or PMA/Iono we
demonstrated that andrographolide can reduce phosphorylation of ERK1/2 and ERK5. These
pathways are responsible for cytokine production (Dumont et al., 1998; Garaude et al, 2005),
however, a key step is the activation of the Nuclear factor in activated T cells (NAFT), which
is one of the major transcription factors binding to IL-2 gene promoters.
We demonstrated using NFAT-luc that andrographolide can interfere with NFAT activation,
probably by a reduction of translocation to the nucleus, thus explaining the decrease of IL-2
production.
Furthermore, in vivo immune responses such as antibody response to a thymus-dependent
antigen and delayed-type hypersensitivity is drastically diminished in mice by
andrographolide treatment. The andrographolide inhibition of T cells, was applied to interfere
with the onset of experimental autoimmune encephalomyelitis (EAE), an inflammatory
demyelinating disease of the central nervous system that is primarily mediated by CD4 (+) T
cells, which serves as an animal model for human Multiple Sclerosis. Treatment with
andrographolide was able to significantly reduce EAE symptoms in mice by inhibiting T cell
and antibody responses directed to myelin antigens (Iruretagoyena etal, 2005).
Using a MOG-induced (myelin-oligodendrocyte-glycoprotein induced) EAE mice model, we
found that daily treatment with 2 mg/kg s.c. of andrographolide produced an important
improvement in clinical scores of animals treated with andrographolide compared with
animals treated with saline. Similarly, in another model of autoimmune disease, the
administration of andrographolide reduced the susceptibility, prevented the symptoms and
reduced anti-nuclear antibodies and kidney damage of systemic lupus erythematous .
A synergistic effect of andrographolide treatment and IFN-b on the mice model of EAE was
shown. Mean clinical scores of mice injected with IFN-b showed a mild decrease in mean
clinical scores compared with saline injected controls. However, when combined with
andrographolide, the IFN-b treatment showed a more significant reduction in the mean
clinical scores. Our results show a clear beneficial effect of andrographolide on IFN-b
treatment, when administered during the active phase of the disease (day 16-31), which
reduces clinical signs of chronic EAE in mice after immunization with MOG.
Andrographolides potentiate the effect of interferon beta and therefore is a therapeutic tool
from MS and demyelinating diseases stronger than interferon beta alone.
Because andrographolide can modulate several transcription factors or signaling pathways,
involving inflammatory processes and activation of T-cells, andrographolide together with
IFN formulations could be a useful therapy for MS.
Definitions
An "effective amount" refers to an amount of the active ingredients that is sufficient to affect
the course and the severity of the disease, leading to the reduction or remission of such
pathology. The effective amount can be readily determined by a person skilled in the art and
will depend on the route of administration, the weight, the age and the condition of the
patient, and the goal of the administration (therapeutic, prophylactic or diagnostic goal).
The term "interferon", as used in the present patent application, is intended to include any
molecule defined as such in the literature, comprising for example any kinds of IFNs
mentioned in the above section "Background of the Invention". In particular, any kinds of
IFN-alpha, IFN-beta and IFN-gamma are included in the above definition. IFN-beta is the
preferred IFN according to the present invention.
The pharmaceutical compositions of the present invention can comprise naturally occurring,
native, mutant, synthetic or recombinant IFNs.
The term "interferon-beta (IFN-beta)", as used herein, includes human fibroblast interferon, as
obtained by isolation from biological samples or as obtained by recombinant DNA techniques
from prokaryotic or eukaryotic host cells as well as its salts, functional derivatives, variants,
analogs and fragments.
"Derivatives" as used herein covers derivatives which may be any purified compound,
comprising preferably from 90 to 100% of the pure compound, and any compound of
Formula I . Standardized compositions obtained from A . paniculata, containing high
concentrations of compound of Formula I can also be considered as compound(s) of Formula
I as used in the present invention.
The compounds of Formula I are preferably prepared as purified compounds, comprising
preferably at least about 90% of the pure compound, more preferably at least about 98% of
the pure compound. An adequate diluent can also be used, allowing the reconstitution of the
product to the desired concentration prior to administration of the dose. The compounds of
Formula I can be prepared as pharmaceutical finished dosage forms any known manner, such
as a pre-filled syringe, etc.
The pharmaceutical compositions of the invention can comprise purified compounds of
Formula I and/or a mixture of compounds of Formula I. The aforementioned mixture of
compound(s) of Formula I can be synthetically blended or naturally occurring.
"Pharmaceutically acceptable diluents, stabilizers, buffers, preservatives, solubilizers,
emulsifiers, adjuvants and/or carriers" as use herein, is meant that those compounds do not
interfere with the biological activity of the active ingredients and are not toxic to the host to
which is administrated.
In a preferred embodiment a pharmaceutical composition of the invention comprise between 6
and 12 MUI of IFN-beta, and between 50 and 500 mg of compound of Formula I. In a more
preferred embodiment, a pharmaceutical composition of the invention comprise between 6
and 12 MUI of IFN-beta, and between 100 and 200 mg of compound of Formula I.
The administration of the pharmaceutical compositions of the present invention may be by
intravenous, intramuscular, subcutaneous, or oral route, or any other route which may
establish the desired blood levels of the active compounds. For example, for parenteral
administration, the pharmaceutical compositions of the present invention may be formulated
as unit dosage form for injection in vehicles such as sterile water, saline, dextrose solution,
serum albumin and/or Ringer's solution.
The pharmaceutical compositions of the invention are suitable for treating MS and/or other
demyelinating diseases.
The pharmaceutical compositions of the invention are designed for the simultaneous, separate
or sequential use of its active ingredients for the above specified therapy.
The present invention provides a method for treating a subject suffering from MS and/or
another demyelinating disease, the method consisting of administering a pharmaceutical
composition of the invention to the subject in an effective amount, intravenously,
intramuscularly, subcutaneously, or orally every day or every other day and for time
sufficient to produce remyelination and to reduce inflammation. In a preferred embodiment,
the effective amount of the pharmaceutical composition comprises between 6 and 12 MUI of
IFN-beta and between 1 and 5 mg/Kg body weight of compound of Formula I . In a more
preferred embodiment, the effective amount of the pharmaceutical composition comprises
between 6 and 12 MUI of IFN-beta and 2 mg/Kg body weight of compound of Formula I. In a
preferred embodiment, the administration of the pharmaceutical composition is for an
unlimited time.
The present invention also provides a method to reduce fatigue in a subject in need thereof,
the method consisting of administering the pharmaceutical compositions of the invention to
the subject in an effective amount every day or every other day. In a preferred embodiment,
the effective amount of the pharmaceutical composition comprises between 6 and 12 MUI of
IFN-beta and between 1 and 5 mg/Kg body weight of compound of Formula I . In a more
preferred embodiment, the effective amount of the pharmaceutical composition comprises
between 6 and 12 MUI of IFN-beta and 2 g/ g body weight of compound of Formula I. In a
preferred embodiment, the administration of the pharmaceutical composition is for an
unlimited time. The present invention has been described with reference to the preferred
embodiments, but the content of the description comprises all modifications and substitutions
which can be brought by a person skilled in the art without extending beyond the meaning and
purpose of the claims.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1: Effect of IFN-b on disease severity in EAE mice. Mice were treated daily with IFN-
b ( 1 m g of IFN-b in 200 PBS, ip), at the beginning of chronic phase (day 15 post-
immunization) until day 30 post-immunization. As controls MOG-immunized C57BL/6J mice
were injected with PBS (Vehicle).
Figure 2 : Effect of combined therapy on disease severity in EAE mice. This figure shows two
groups of MOG-immunized C57BL/6J mice (150 ug MOG-peptide; 500 g MT; 200 ng PT)
treated daily, or every second day with the combined therapy (4 mg/kg andrographolide and
1 ug IFN-b at the beginning of chronic phase (day 15 post-immunization) until day 30 post-
immunization. As controls MOG-immunized C57BL/6J mice were injected with PBS
(Vehicle).
Figure 3 : Inflammatory infiltrate is reduced in spinal cord with combined therapy in EAE
treated mice. Non-immunized mice (NI) (left panel), MOG-immunized mice treated with
either PBS (middle panel) or combined therapy (CT) EAE treated mice (right panel) were
perfused and 4 % p-formaldehyde fixed. Spinal cords were dissected and analyzed for
inflammatory infiltrate by hematoxylin-eosin staining. Insets show higher magnification
(10X). Mononuclear area fraction was quantified in thoracic spinal cord using 4 different
sections separated by 250 m . Results are shown as mean ± SEM.
Figure 4 : Inflammatory infiltrate and demyelination is reduced in spinal cord with combined
therapy in EAE treated mice. Non-immunized mice (NI) (left panel), MOG-immunized mice
treated with either PBS (middle panel) or combined therapy (CT) EAE treated mice (right
panel) were perfused and 4 % p-formaldehyde fixed. Spinal cords were dissected and
analyzed for inflammatory infiltrate by hematoxylin-eosin staining (H&E) and demyelination
was evaluated by luxol fast blue (LFB) staining. Insets show higher magnification (10X). A :
representative thoracic spinal cord sections. B : magnifications of infiltrating cells and
demyelination.
Figure 5 : Microglial cells from combined therapy (CT) EAE treated mice shows a resting
phenotype. A : spinal cords from non-immunized mice (NI) (left panel), MOG-immunized
mice treated with either PBS (middle panel) or combined therapy (CT) EAE treated mice
(right panel) that were dissected and analyzed for macrophages/microglia by
immunofluorescence using an anti-CDllb antibody followed by incubation with Alexa-fluor
488-conjugated secondary antibody (green). B : insets with higher magnification (20x).
Figure 6:
Figure 7 : The results of treatment with andrographolide mono-therapy. The protocol was
exactly the same as for EAE model of MS as described for interferon mono-therapy (Figure 1)
and for andrographolide + interferon combination therapy (Figures 2 to 5). Andrographolide
4mg/day (without accompanying interferon) was injected (intraperitoneally) daily for days
16-31.
EXAMPLES
The following examples illustrate the invention in detail, but they are not intended to limit the
scope of the invention.
Example 1: Mice with induced Experimental Autoimmune Encephalomyelitis (EAE)
administered with combined therapy of andrographolide and interferon.
Animals
C57BL/6 mice were purchased from Jax® mice laboratories and housed at Pontificia
Universidad Catolicas animal facility. Animal care and use was performed in accordance with
approved animal use protocols and guidelines of Institutional Animal Care and Use
Committee.
Experimental Autoimmune Encephalomyelitis (EAE) induction and treatments
EAE was induced by immunization with myelin oligodendrocyte glycoprotein (MOG)35-55
peptide (MEVGWYRSPFSRVVHLYR) or proteolipid protein (PLP)139-151
(HSLGKWLGHPDKF; CPC Scientific, Sunnyvale, CA, USA) emulsified at 1.5 mg/ml in
PBS with an equal amount of incomplete Freund's adjuvant (IFA; DIFCO, MI, USA)
supplemented with 2.5 mg/ml Mycobacterium tuberculosis, strain H37Ra (Difco, Detroit,
MI). Mice were immunized subcutaneously with 200 ml emulsion. Pertussis toxin (LIST
BIOLOGICAL LABS, CA, USA), 200 ng in 200 m ΐ PBS, was injected intraperitoneally at day
0 and 2 after initial immunization.
Animals were scored for clinical symptoms as follows: 0 = no signs of disease; 1 =lost of tail
tone; 2= flaccid tail; 3= partial hind limb paralysis; 4 = complete hind limb paralysis; 5=
moribund required to sacrifice the animal; 6= death.
For IFN-b assays, C57BL/6, were administered intraperitoneally with 1 g of IFN-b (IFN-b
Rebif 88 m /ml MERCK SORONO) in 200 PBS. Control mice were injected with 200 L
PBS.
IFN-b was administrated from day 15 to 30 post-immunization, daily.
For combined therapy (CT) assays, C57BL/6, were administered intraperitoneally with 4
mg/kg of andrographolide and IFN-b 1 ug in 200 PBS. Control mice were injected with
200 PBS.
Combined therapy (CT) was administrated from day 15 to 30 post-immunization, either daily
or every second day. At day 36 all animals were sacrificed. Clinical score was registered, and
spinal cords were collected for histological analysis.
Histological preparation
Mice were deeply anesthetized with isoflurane and perfused transcardially with ice-cold 1
PBS (25 ml), followed by 4% p-formaldehyde. Spinal cords were dissected and postfixed in
4% PFA overnight at 4°C. Tissue was cryoprotected in 30% (w/v) sucrose at 4°C overnight,
embedded in OCT compound, frozen and sectioning with a cryostat at 20 mhi thick.
Spinal cord inflammatory infiltrate
To evaluate inflammatory infiltrate, hematoxylin eosin staining (H&E) was performed.
Toraxic spinal cord sections were stained with hematoxylin solution modified to Gill
(SIGMA, USA), and with eosin Y (SIGMA, USA). Mononuclear cell infiltration was
determined as the area occupied by positive nuclei in the spinal cord periphery of 4 different
sections.
Spinal cord myelin staining
To evaluate demyelination in EAE spinal cord, luxol fast blue (LFB) staining was performed.
Toraxic spinal cord sections were stained with LFB (SIGMA, USA), and neuron nuclei were
staining with cresyl violet. Demyelination was evaluated as LFB staining free area in spinal
cord white matter.
Immunohistochemistry
Sections of the spinal cord (20 mm) were treated with a permeabilizatioiVblocking solution
containing 10% FCS, 1% glycine, and 0.05% Triton X-100 (Sigma-Aldrich). Primary
antibody rat anti CDllb (1:200; BD, USA) in blocking solution was applied O.N. in a
humidified chamber at 4°C. Secondary antibody goat anti rat conjugated with fluorescein
(Millipore, USA) was applied for 1 h at room temperature.
Disease severity in IFN-b (Figure 1) and combined therapy (CT) EAE mice (Figure 2)
Three groups of MOG-immunized C57BL/6J mice (150 ug MOG-peptide; 500 ug MT; 200
ng PT) were treated daily with IFN-b ( 1 m g of IFN-b (IFN-b ebif 88 m /ml MERCK
SORONO) in 200 PBS, intraperitoneally), daily or every second day with the combined
therapy (CT) (4 mg kg andrographolide plus IFN-b 1 ug) at the beginning of chronic phase
(day 15 post-immunization) until day 30 post-immunization. As controls MOG-immunized
C57BL/6J mice were injected with PBS (Vehicle). At day 36 p.i. all mice were sacrificed
(scores: PBS=2.4; CT daily=0.17; CT every second day= 0.75) and processed for histological
analysis.
Combined therapy decreases significantly clinical symptoms in Chronic EAE mouse model.
Comparing Figures 1 and 2, it is demonstrated, that the addition of compound of Formula I
causes a synergic effect with interferon.
Inflammatory infiltrate is reduced in spinal cord with combined therapy in EAE treated
mice (Figure 3)
Non-immunized mice (NI) (left panel), MOG-immunized mice treated with either PBS
(middle panel) or combined therapy (CT) (right panel) were perfused and 4 % p-
formaldehyde fixed. Spinal cords were dissected and analyzed for inflammatory infiltrate by
hematoxylin-eosin staining. Insets show higher magnification (10X). Mononuclear area
fraction was quantified in thoracic spinal cord using 4 different sections separated by 250 m .
Results are shown as mean ± SEM.
Result: Combined therapy decreases spinal cord cell infiltration in chronic EAE mouse
model.
Inflammatory infiltrate and demyelination is reduced in spinal cord with combined
therapy in EAE treated mice (Figure 4).
Non-immunized mice (NI) (left panel), MOG-immunized mice treated with either PBS
(middle panel) combined therapy (CT) (right panel) were perfused and 4 % p-formaldehyde
fixed. Spinal cords were dissected and analyzed for inflammatory infiltrate by hematoxylin-
eosin staining (H&E) and demyelination was evaluated by luxol fast blue (LFB) staining.
Insets show higher magnification (10X). A shows representative thoracic spinal cord sections.
B shows magnifications of infiltrating cells and demyelination.
Result: Combined therapy decreases spinal cord cell infiltration and demyelination in
Chronic EAE mouse model.
Microglial cells from combined therapy (CT) mice exhibited a resting phenotype (Figure
). Panel A shows spinal cords from non-immunized mice (NI) (left panel), MOG-
immunized mice treated with either PBS (middle panel) or combined therapy (CT) (right
panel) that were dissected and analyzed for macrophages/microglia by immunofluorescence
using an anti-CDllb antibody followed by incubation with Alexa-fluor 488-conjugated
secondary antibody (green). Panel B shows insets with higher magnification ( Ox).
Result: CD1 lb+ cells showed long processes compared to control microglial cells that exhibit
short and ramified processes. Combined therapy might be preventing macrophage infiltration,
microglia activation or both.
Example 2 : Human Multiple sclerosis (MS) patients
MS Patients receiving Andrographolide ONLY
Materials & Methods:
Subjects- Patients
Eight MS patients were not receiving any previous regular pharmacological treatment at all,
some due to clinical reasons, or economical restrictions (e.g., lack of financial access to
commercially-available interferon drug products) and others or both.
Andrographolide product
Andrographolide was obtained as a standardized extract of Andrographispaniculata, purified,
standardized to purity and dried, commercially available from HP Ingredients, Inc., Bradeton,
Florida USA, conforming to an identity specification of >35.0% (w/w) of andrographolide.
The material used in the testing described here had an actual identity assay of an HPLC
content of 47.2% (w/w) of andrographolide. 2.1% (w/w) of neoandrografolide and 3.0%
(w/w) of 14-deoxyandrografolide. (All mass measures refer to dry weight without solvent.)
Treatment regimen
All eight patients were placed on a non-blinded, 42-month treatment regimen of daily intake
(per os) at a dosage of 2mg andrographolide per kilogram of body wieght, 0.1 5mg of
neoandrographolide per kilogram of body weight, and 0.2 mg of deoxyandrographolide per
kilogram of body weight.
Results:
Safety & Tolerability
After 42 months of treatment, no intolerance, adverse interactions or reactions to the test
product have been reported or observed by any of the patients nor the attending physicians. In
contrast to what is normally expected during treatment with interferon beta, none of the
patients treated with andrographolide reported any flu-like side effects.
No patient showed a relapse of multiple sclerosis during the 42 months of treatment, as
measured by clinical and neuroimaging controls.
All patients showed some partial functional recovery of sensitiveness and neuromotricity. In
several patients, the magnitude of this recovery was clinically significant.
Clinical results
1. All patients report some degree of symptomatic control of pain, fatigue, spasticity and
improvement of mood, already noticeable at four months of therapy, but the effect was
significantly less than the group receiving the Combined therapy (andrographolide combined
with interferon)
2 . Two patients with previously very long active disease (21 and 7 years from onset
respectively) presented one early episode of relapse (within the first sixty weeks after initiated
treatment), but symptoms have been very brief and mild, not requiring additional
immunosuppressive treatment as they had before.
3. One patient in full clinical remission, free of symptoms or new neuroimaging lesions after
more than three years, continuous only with monotherapy.
4 . Partial improved focal functionality in 2 of eight patients, such as speech impairment
(dysarthria) and vision at month 6; deglutition (neurologic dysphagia) and fine motricity
(recovering writing, self eating and hygiene) between months 12 -14.
. One of eight patients improved scattered functionality such as fatigue, leg strength,
coordination and walking equilibrium at month six, with sustained progress until now.
6 . Two patients, who previous to treatment were not able to stand up or climb upstairs, have
started antigravity displacement at month 30, beginning perception of initial recovery from
this impairment between four and six months of monotherapy.
7 . No change in the total number and size of demyelinating lesions in the brain as measured
by Magnetic Resonance Imagining (MRI)
8. Some degree of reduction of inflammatory activity of demyelinating lesions comparing
time 0 as measured by MRI Gadolinium contrast medium uptake and 42 months still
pending).
MS Patients receiving Interferon beta ONLY
Materials & Methods:
Subjects- Patients
Ten MS patients diagnosed with Relapsing Remitting type of the disease.
Results:
Safety & Tolerability
1.- Complete safety and tolerability in all patients after 12 months (ongoing) of of an
interferon beta in monotherapy (either Interferon beta-l a (IFNB-la) Avonex(r) 30 g/weekly
(im), Interferon beta-la (IFNB-la) Rebif(r) 22 m g o 44 g/3 times a week (sc)
Interferon beta-lb (IFNB-lb) Betaferon(r)/Betaseron(r) following the instructions of the
physician Some degree of adverse reactions to the test product have been reported or
observed by these patients or physicians.
2 . No relapses during the 12 months of treatment, as measured by clinical and neuroimaging
controls.
3. Appearance of flu-like symptoms such as aches and pains, fever, chills, sweating or
headache in 6 of 8 patients some of which required the use of aspirin or ibuprofen.
4 .- Two patients reported mild depression at the 4th month during treatment
Clinical results
1. All patients reported no degree of symptomatic control of fatigue and improvement of
mood, as seen in the group with ANG or receiving the Combined Theraphy (ANF+INF).
2 . No patients presented episodes of relapse (within the first year of treatment).
3 . No patients showed clinical signs of subjective wellness, despite the fact of appearance of
new neuroimaging lesions.
4 . No improvement on speech impairment (dysarthria) and vision nor deglutition (neurologic
dysphagia) and fine motricity.
. No effect on antigravity displacement.
6 . No change in the total number and size of demyelinating lesions in the brain as measured
by Magnetic Resonance Imagining (MRI)
7 . Some degree of reduction of inflammatory activity of demyelinating lesions comparing
time 0 as measured by MRI Gadolinium contrast medium uptake and 1 months still
pending).
Combined therapy (Andrographolide and Interferon).
Materials & Methods:
Patients already receiving first-line therapy using interferon beta (IFN-b ) were recruited to
receive additional combination therapy of oral tablets containing 55 mg of andrographolide,
twice a day for 60 months. Of recruited patients, three have to date completed the 60 month
treatment period.
Results:
1. Complete safety and tolerability in all patients after 60 months of daily oral intake of
2mg/kg of Andrographolides, associated to Interferon in combined therapy
2 . No relapses during the 60 months, as observed by clinical and neuroimaging follow up.
3 . Early symptomatic synergistic effect of the CT on fatigue, strength and equilibrium
when andrographolide tablets are administered along with Interferon, as observed
between 2 - 3 months of compound of Formula I administration.
4 . Partial, but in some cases significant functional recovery of sensitiveness and
neuromotricity, observed between 24 and 30 months, with the Combined therapy (CT)
. Significant regression of neurological lesions as measured by neuroimaging control
between 14 and 24 months with the Combined therapy (CT)
6 . All patients report total symptomatic control of pain, fatigue, spasticity and
improvement of mood, noticeable already at four months of CT therapy.
7. Patients, who received andrographolide tablets plus Interferon (combined therapy),
responded with an earlier and greater effect on improvement of fatigue and motricity
8. Improvement on focal functionality, such as speech impairment (dysarthria) and vision,
deglutition (severe neurologic dysphagia) and fine motricity (recovering writing, self eating
and hygiene) between months 12 - 14 with the CT.
9. Improvement in scattered functionality such as fatigue, leg strength, coordination and
walking equilibrium with the CT.
. Antigravity displacement at month 30, beginning perception of initial recovery from this
impairment between four and six months with CT .
11. Significant reduction of the size and number of demyelinating lesions in the brain white
matter with the CT.
12. Reduction in inflammatory activity of demyelinating lesions as measured by MRI
Gadolinium contrast medium uptake with the CT.
Given our disclosure here, the artisan can readily derive variations thereof For example, one
may increase the dose of andrographolide, or substitute one compound of Formula I for
another, to achieve a similar effect. We thus intend that the legal coverage of our patent be
defined not by our specific examples taught here, but by our appended legal Claims and
legally permissible equivalents thereof.
Throughout the specification and claims, unless the context requires otherwise, the word “comprise”
or variations such as “comprises” or “comprising”, 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.
Claims (22)
1. Use of a compound of Formula I: wherein R is selected from the group consisting of hydrogen, alkyl or hydroxyl, R is selected from the group consisting of hydroxyalkyl or alkyl-O-L , wherein L is 2 1 1 a carbohydrate moiety, R is selected from the group consisting of hydrogen or hydroxyl, X is selected from the group consisting of C(=CH ), CH(OH), or a spirooxirane-2 moiety, Z is selected from the group consisting of CH , CH(OH) or C(=0), and R is selected from the group consisting of an optionally substituted L -alkyl orL - 4 2 2 alkenyl, wherein L is an optionally substituted 3-furanyl or 3-furenyl moiety, or a pharmaceutically acceptable salt, ester, or ether thereof, in the preparation of a medicament to treat a demyelinating disease in a human patient wherein said patient is provided with an interferon.
2. The use according to claim 1, wherein said demyelinating disease comprises multiple sclerosis.
3. The use according to claim 1, wherein the IFN is selected from the group consisting of: purified naturally-occurring IFN, synthetic IFN and recombinant IFN.
4. The use according to claim 3, wherein the IFN is IFN beta.
5. The use according to claim 1, wherein R is methyl.
6. The use of claim 1, wherein R is selected from the group consisting of: hydroxymethyl and CH2-O-GIC; wherein Glc is a glycoside-forming glucose moiety.
7. The use according to claim 1, wherein R is selected from the group consisting of: 3-(3-furanyl)-propyl, 3-(3-furanyl)-prop-l-enyl, 3-(3-furanyl)-propenyl, 3-(3-fur enyl)-propyl or 3-(3-furenyl)-prop-l-enyl; said 3-furanyl or 3-furenyl moiety further optionally substituted.
8. The use according to claim 1, wherein R is selected from the group consisting wherein, R is selected from the group consisting of: hydrogen and hydroxyl; R and R are independently selected from the group consisting of: hydrogen, hydroxyl and alkyloxy; or R and R are simultaneously replaced by a single direct bond between the carbon atoms denoted by *, thus forming a dimer of two monomer molecules of formula (I), and R and R are independently selected from the group consisting of: hydrogen, hydroxyl and alkyloxy.
9. The use according to claim 8, wherein R , R , R or R can be independently 6 7 8 9 methoxy.
10. The use according to claim 1, wherein the compound of Formula (I) is selected from the group consisting of: andrographolide, neoandrographolide, 14- deoxyandrographolide 14-deoxy-l l,12-didehydroandrographolide, andrographiside, andrograpanin, 14-deoxy- 11-oxo-andrographolide, 14-deoxy-l 1-hydroxy-andrographolide, 14-deoxy- 12-hydroxy-andrographolide, 3,14-dideoxyandrographolide, 3-oxo deoxyandrographolide, 8,17-epoxydeoxyandrographolide, 14-deoxy- 17-beta-hydroxy andrographolide, 12-hydroxyandrographolide, bisandrographolide A, 3-oxodeoxy-l l, 12- didehydroandrographolide, 7-hydroxy- 14-deoxyandrographolide, 15-methoxy-3 , 19- dihydroxy-8(l 7)11,13 -ent-labda-trien-16,15-olide, andropanolide, 14-deoxy- 12-methoxy- andrographolide, 14-epi-andrographolide, 19-hidroxi-ent-labda-8(l 7), 13 -dien- 15,16-olide, 3,13,14, 19-tetrahydroxy-ent-labda-8( 17), 11 -dien- 16, 15 -olide, 3 , 19-dihydroxy methoxy-ent-labda-8(l 7), 11,13 -trien- 16,15-olide, and 3,19-dihydroxy-ent-labda-8(17), 12- dien-16, 15-olide.
11. The use according to claim 1, wherein the interferon is provided in an amount of at least about 6 MUI per month and wherein the compound of Formula I is provided in an amount of at least about 50 mg per day.
12. The use according to claim 11, wherein the interferon is provided in an amount of from about 6 MUI to about 12 MUI per month and wherein the compound of formula I is provided in an amount of from about 50 mg to about 500 mg per day.
13. The use according to claim 1, wherein the interferon and the compound of Formula I are provided in an amount and for a time sufficient to produce remyelination and to reduce inflammation in said human patient.
14. The use according to claim 11, wherein the compound of Formula I is provided in an amount of between about 1 mg and about 5 mg per kilogram of body weight of said human patient.
15. Use of an effective amount of interferon and an effective amount of a compound of Formula I: wherein R is selected from the group consisting of hydrogen, alkyl or hydroxyl, R2 is selected from the group consisting of hydroxyalkyl or alkyl-O-L , wherein L is a carbohydrate moiety, R is selected from the group consisting of hydrogen or hydroxyl, X is selected from the group consisting of C(=CH ), CH(OH), or a spirooxirane-2 moiety. Z is selected from the group consisting of CH , CH(OH) or C(=0), and R is selected from the group consisting of an optionally substituted L -alkyl orL - 4 2 2 alkenyl, wherein L is an optionally substituted 3-furanyl or 3-furenyl moiety, or a pharmaceutically acceptable salt, ester, ether or thereof, in the preparation of a medicament to reduce fatigue in a human subject in need thereof.
16. The use according to claim 15, wherein the effective amount of interferon is from about 6 MUI to about 12 MUI of IFN-beta, and wherein the effective amount of compound of Formula I is between about 1 mg and about 5 mg per kilogram of body weight of said human subject.
17. The use of at least one compound of Formula I to manufacture a medicament to treat a human patient diagnosed as having a demyelinating disease, said medicament to be used in combination with interferon, said medicament having an amount of said at least one compound of Formula I effective to treat said demyelinating disease.
18. The use according to claim 17, wherein said at least one compound of Formula I comprises andrographolide.
19. The use according to claim 17, wherein said medicament is in unit dosage form, and wherein said amount effective to treat said demyelinating disease is not less than about 50 mg per day.
20. The use according to claim 19, wherein said amount effective to treat said demyelinating disease is from about 50 mg to about 500 mg per day.
21. The use according to claim 18, wherein said amount effective to treat said demyelinating disease is from about 50 mg to about 500 mg per day.
22. The use according to claim 18, wherein said demyelinating disease comprises multiple sclerosis.
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US201161578650P | 2011-12-21 | 2011-12-21 | |
US61/578,650 | 2011-12-21 | ||
PCT/US2012/070568 WO2013096423A1 (en) | 2011-12-21 | 2012-12-19 | Combination therapy with interferon and andrographolides for multiple sclerosis |
Publications (2)
Publication Number | Publication Date |
---|---|
NZ625051A NZ625051A (en) | 2016-07-29 |
NZ625051B2 true NZ625051B2 (en) | 2016-11-01 |
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