Note: Descriptions are shown in the official language in which they were submitted.
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INFLAMMATORY DISEASE
The present invention relates to the treatment of inflammatory diseases, and
especially
Th-1 mediated inflammatory diseases. In particular, the invention relates to
the
treatment of Th-1 mediated inflammatory diseases using a range of
compositions, and
to the use of these compositions in methods of treatment. The invention
extends to
adjuvants, and in particular to adjuvants for use in treating a wide variety
of medical
conditions. The invention also provides pharmaceutical compositions and
medicaments
comprising the adjuvant, and to uses of the adjuvant in methods of treatment
and for
eliciting an immune response.
The defence against disease is critical for the survival of all animals, and
the mechanism
employed for this purpose is the animal immune system. The immune system is
complex, and involves two main divisions, (i) innate immunity, and (ii)
adaptive
immunity. The innate immune system includes the cells and mechanisms that
defend
the host from infection by invading organisms, in a non-specific manner.
Leukocytes,
which are involved with the innate system, include inter alia phagocytic
cells, such as
macrophages, neutrophils and dendritic cells. The innate system is fully
functional
before a pathogen enters the host.
In contrast, the adaptive system is only initiated after the pathogen has
entered the host,
at which point it develops a defence specific to that pathogen. The cells of
the adaptive
immune system are called lymphocytes, the two main categories of which are B
cells
and T Cells. B cells are involved in the creation of neutralising antibodies
that circulate
in blood plasma and lymph and form part of the humoral immune response. T
cells
play a role in both the humoral immune response and in cell-mediated immunity.
There are several subsets of activator or effector T cells, including
cytotoxic T cells
(CD8+) and "helper" T cells (CD4+), of which there are two main types known as
Type 1 helper T cells (Th1) and Type 2 helper T cell (Th2).
Th1 cells promote a cell-mediated adaptive immune response, which involves the
activation of macrophages and stimulates the release of various cytokines,
such as
IFNy, TNF-a and IL-12, in response to an antigen. These cytokines influence
the
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function of other cells in the adaptive and innate immune responses, and
result in the
destruction of micro-organisms. Generally, Thl responses are more effective
against
intracellular pathogens, such as viruses and bacteria present inside host
cells. A Th2
response, however, is characterised by the release of IL-4, which results in
the
activation of B cells to make neutralising antibodies, which lead the humoral
immunity.
Th2 responses are more effective against extracellular pathogens, such as
parasites and
toxins located outside host cells. Accordingly, the humoral and cell-mediated
responses
provide quite different mechanisms against an invading pathogen.
Interleukin-10 (IL-10), also known as human cytokine synthesis inhibitory
factor
(CSIF), is an anti-inflammatory cytokine. It is produced primarily by
monocytes and, to
a lesser extent, by lymphocytes, and has pleiotropic effects in
immunoregulation and
inflammation. IL-10 down-regulates the expression of Thl cytokines (such as
IFNy,
TNF-cx and IL-12), MHC class II antigens, and co-stimulatory molecules on
macrophages. It also enhances B cell survival, proliferation, and antibody
production.
Furthermore, IL-10 can block NF-xB activity, and is involved in the regulation
of the
JAK-STAT signaling pathway. Knockout studies in mice have suggested the
function
of IL-10 as an essential immunoregulator in the intestinal tract, and patients
with
Crohn's disease react favorably towards treatment with bacteria producing
recombinant
IL-10, showing the importance of this cytokine for counteracting excessive
immunity in
the human body.
IL-10 is a validated target in inflammatory disease, and has been shown to be
essential
for immunotolerance and the control of Thl immunity (O'Garra et al., 2008,
Immunol.
Rev., 223:114-31). Systemic administration of exogenous IL-10 in early
clinical trials
showed some initial promise in the treatment of various diseases, including
rheumatoid
arthritis, Crohn's disease, psoriasis and cystic fibrosis. However, problems
associated
with these early trials were that systemically administered exogenous IL-10
did not
dampen exaggerated Thl responses as predicted, possibly because tissue levels
of IL-10
were too low (the half-life of IL-10 is short), or because IL-10 was not
presented in the
correct manner. Hence, concentrations of IFNy, TNF-cx and IL-12 actually
increased to
deleterious levels. It will be appreciated that these cytokines are pro-
inflammatory.
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Furthermore, a significant side effect of administering too much exogenous IL-
10 is
that it actually suppresses the immune system, rather than positively
modulating it, and
so results in serious immune-related problems. It will be appreciated that IL-
10, IL-4
and TGF-p are anti-inflammatory.
In view of these problems, there is, therefore, clearly a need in the art for
improved
medicaments for use in the treatment of Thl-mediated diseases, and especially
Thl -
mediated inflammatory disorders, which do not suffer the immune-related
problems
observed in previous IL-10 trials. The inventors investigated, using in vivo
mouse
studies, the effects of non-steroidal anti-inflammatory drugs (NSAID), such as
ibuprofen, on mice that had been previously challenged with influenza virus.
The
inventors formulated ibuprofen in combination with a highly lipophilic
pharmaceutically acceptable vehicle, or adjuvant, which was then orally
administered to
challenged test mice. They observed that ibuprofen administered orally in an
oily
formulation (i.e. the adjuvant) resulted in surprisingly positive effects on
the percentage
survival rate compared to the control mice which had been administered with
just the
oily vehicle or just ibuprofen (not in oil). The inventors also determined
with in vivo
assays that the concentration of IL-10 in the lungs of the surviving animals
dramatically
increased in mice administered with the oily ibuprofen composition.
Hence, in a first aspect, there is provided a pharmaceutical composition
comprising a
therapeutically effective amount of a compound which is capable of increasing
interleukin-10 (IL-10) production, and a pharmaceutically acceptable vehicle
comprising
a lipid and an alcohol, for use in treating a Thl-mediated disease, wherein
the IL-10 is
endogenously produced by Th2 cells, dendritic cells and/or macrophages.
In a second aspect of the invention, there is provided a method of preventing,
treating
and/or ameliorating a Thl-mediated disease, the method comprising
administering, to a
subject in need of such treatment, a pharmaceutical composition comprising a
therapeutically effective amount of a compound which is capable of increasing
interleukin-10 (IL-10) production, and a pharmaceutically acceptable vehicle
comprising
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a lipid and an alcohol, wherein the IL-10 is endogenously produced by Th2
cells,
dendritic cells and/or macrophages.
In a third aspect, there is provided use of a pharmaceutical composition for
treating a
Th1-mediated disease, the composition comprising a therapeutically effective
amount
of a compound which is capable of inducing endogenous production of
interleukin-10
(IL-10) by Th2 cells, dendritic cells and/or macrophages and a
pharmaceutically
acceptable vehicle comprising a lipid and an alcohol.
Surprisingly, as shown in Figure 1, when ibuprofen is administered orally in a
lipophilic
formulation (e.g. at least 30% w/w lipid), or the adjuvant of the invention,
it is shown
to be very effective in the treatment of influenza-induced respiratory
collapse in mice.
Indeed, application of a single dose of the pharmaceutical composition of the
invention
converted the 80% mortality model to an astounding 80% survival outcome, and
this
was totally unexpected. Although the inventors do not wish to be bound by any
theory,
they believe that an explanation for this surprising observation may be due to
the
lipophilicity of NSAIDs, such as ibuprofen (i.e. log P 3.5), which, when
delivered in an
oily formulation having a high lipid content (e.g. at least 30% (w/w) lipid),
results in
them being rapidly absorbed into the systemic circulation via the lymphatic
system.
When a drug/lipid formulation is swallowed, the lipids are mixed with bile in
the
stomach, containing bile salts, and form complexes called micelles or
chylomicrons,
which are large lipoprotein particles that consist of triglycerides,
phospholipids,
cholesterol and proteins, and the NSAID.
The resultant oil/drug/bile salt complex (i.e. micelles or chylomicron) may
then be
absorbed by the proximal gut into the enteric lymphatic system. These
micelles/chylomicrons, carrying the compound which stimulates IL-10
production, are
believed to be transported via the gut lymphatic system to the central venous
vasculature, and then rapidly to the heart, which pumps the active compound-
rich
venous blood to the lung. As a result, the drug is delivered in high
concentrations in
oxygenated blood directly to the lung increasing its bioavailability.
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The inventors believe that lymphatic absorption of the active compound (e.g.
ibuprofen) may be acting as a passive system of distribution of the drug
directly to the
lung, exposing the lung to high concentrations of the compound. The inventors
believe
that this delivery mechanism does not occur when using standard oral
formulations,
which contain no, or only low levels of lipid, which are instead absorbed via
the hepatic
portal vein, with liver-regulated venous absorption, which releases the drug
into
systemic circulation relatively slowly. Accordingly, the inventors believe
that the high
concentration of lipids in the pharmaceutical vehicle used in the composition
of the
first aspect may be the reason for the efficacy of the orally-administered
ibuprofen in
the influenza-induced respiratory collapse assay in mice, as described in the
Examples.
As shown in Figure 2, administration of the pharmaceutical composition of the
invention results in a dramatic increase in the production of endogenous IL-10
in the
lungs of influenza-challenged mice, and this was totally unexpected. Clearly,
when
compared to the concentrations of IL-10 that were produced in the control mice
(i.e.
animals that had been administered with just the oily vehicle or just
ibuprofen and no
oil), the IL-10 concentrations produced in animals administered with the
active agent in
lipid were much more than additive, suggesting that a synergistic effect has
occurred.
As described in Example 4, and as shown in Figures 5-7, the inventors have
demonstrated that NSAIDs (such as ibuprofen), when they are formulated in an
oil and
alcohol (e.g. ethanol) adjuvant, activate endogenous IL-10 production from
macrophages. Thus, it is preferred that the compositions of the invention are
adapted,
in use, to stimulate endogenous production of IL-10 from dendritic cells
and/or
macrophages. One advantage of the compositions of the invention is that they
allow
certain active compounds (i.e. drugs) to be formulated in lipid and taken
orally such
that they are preferentially loaded into macrophages and dendritic cells.
Numerous
disease conditions are believed to be associated with low levels of IL-10
(i.e. hypo-IL-
10 disorders), or in situations where an increase in IL-10 concentration is
either too
slow or insufficient, and any of these conditions may be treated in accordance
with the
invention. Example 5 describes the effects of the compositions of the
invention in an
anti-collagen antibody induced arthritis (ACAIA) murine model. Thus, the Th1-
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mediated disease, which may be treated, may be a Th1-mediated inflammatory
disease,
and preferably systemic inflammatory disease. The composition of the invention
therefore may be an anti-inflammatory pharmaceutical composition.
The disease to be treated may be selected from a group of Th1-mediated
diseases
consisting of rheumatoid arthritis (RA); psoriatic arthritis; psoriasis;
inflammatory
bowel syndrome (IBD); Crohn's disease; ulcerative colitis; multiple sclerosis
(MS); flu,
including pandemic flu; respiratory disorders, for example those caused by
viruses, such
as respiratory syncytial virus (RSV); cystic fibrosis (CF); herpes, including
genital
herpes; asthma and allergies; sepsis and septic shock; bacterial pneumonia;
bacterial
meningitis; dengue hemorrhagic fever; diabetes Type I; endometriosis;
prostatitis;
uveitis; uterine ripening; alopecia areata; ankylosing spondylitis; coeliac
disease;
dermatomyositis; diabetes mellitus Type 1; Goodpasture's syndrome; Graves'
disease;
Guillain-Barre syndrome; juvenile idiopathic arthritis; Hashimoto's
thyroiditis;
idiopathic thrombocytopenic purpura; Lupus erythematosus; mixed connective
tissue
disease; myasthenia gravis; narcolepsy; osteoarthritis; pemphigus vulgaris;
pernicious
anaemia; polymyositis; primary biliary cirrhosis; relapsing polychondritis;
Sjogren's
syndrome; temporal arteritis; vasculitis; Wegener's granulumatosis; and age-
related
macular degeneration.
The Th1-mediated disease may be virally, bacterially or chemically (e.g.
environmentally) induced. For example, a virus causing the Th1-mediated
disease may
cause a chronic or acute infection, which may cause a respiratory disorder.
Suitably, the
virus causing the Th1-mediated disease may be Influenza.
Preferably, the Th1-mediated disease, which may be treated, is systemic
inflammatory
disease, for example inflammatory bowel syndrome (IBD), rheumatoid arthritis
(RA) or
cystic fibrosis (CF). The Th1-mediated disease, which may be treated, is
preferably
IBD.
The inventors carried out investigations on a number of different compounds
that can
induce IL-10 production, as they were aware that IL-10 production is a
characteristic
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that indicates a switch from a Thl to a Th2 response, and that such compounds
could
be used to treat Thl-mediated diseases. They have demonstrated that inducing
the
switch from a Thl to a Th2 response by up-regulating IL-10 production can be
used to
help treat Thl-mediated hyper-inflammation. Indeed, Figure 3 clearly shows
that
administration of the composition of the invention not only increases IL-10
production, it also results in increased IL-4 production, proving that the
switch from a
Thl to a Th2 response has been induced. Advantageously, by stimulating a Th2
response, by inducing the endogenous release of IL-10 of Th2 cells,
macrophages
and/or dendritic cells, B cells are activated, which produce neutralising
antibodies,
which lead the humoral immunity. It will be appreciated that Th2 responses are
much
more effective against extracellular pathogens, such as parasites and toxins
located
outside host cells, than Thl responses.
By way of example, Thl-mediated hyper-inflammation occurs during viral
infections
(e.g. influenza, as demonstrated in the Examples), and so the inventors
believe that
endogenously producing IL-10 by the Th2 cells, macrophages and/or dendritic
cells
upon administration of the composition to the subject could be used to treat a
respiratory collapse caused by a viral infection. It is known that some
viruses, such as
Herpes Simplex Virus (HSV), maintain their presence in the body by stimulating
Thl
immunity, and that therefore, triggering a switch to Th2 immunity would help
treat
herpes infections by allowing the body to attack the virus through natural
killer cell
immunity.
The inventors are of the view that IL-10 is a paracrine cytokine, and that,
therefore, the
compound used in the first, second and third aspects of the invention can be
effectively
used to increase endogenous IL-10 production in a paracrine manner. It will be
appreciated that paracrine signalling can be a form of cell signalling in
which the target
cell is near or local to the signal-releasing cell. Thus, IL-10 can control
the immune
response at sites of inflammation in tandem with cell-cell interactions.
The compound, which is capable of increasing endogenous IL-10 production, may
be
recognised by Th2 cells, dendritic cells and/or macrophages. Preferably,
however, the
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compound, which is capable of increasing endogenous IL-10 production, is
recognised
by dendritic cells and/or macrophages. Th2 cells, dendritic cells and/or
macrophages
may phagocytise the compound (such as a lipid/drug chylomicron), react with
pathways
in each of these cell types, and thereby endogenously produce IL-10.
Preferably,
therefore, the IL-10 is produced by Th2 cells, dendritic cells and/or
macrophages,
which are immuno-competent. Advantageously, in contrast to producing IL-10 by
non-
immune cells, which causes an imbalance in the immune system leading to
infections
and, in some cases cancer, endogenous production of IL-10 by immuno-competent
Th2 cells, dendritic cells and/or macrophages, ensures that the immune system
remains
balanced, thereby avoiding infection.
Hence, the immune system is capable of regulating itself naturally, thereby
reducing the
risk of infections and cancer. As shown in Figures 4-6, the levels of IL-10
generated
due to the regulatory process and feedback loops may modulate (e.g. decrease)
Th1
cytokine concentration, such as IFNy, TNF-a and IL-12, but not suppress their
production completely. This may be important because these Th1 cytokines are
needed
to protect the body from infection. Thus, by stimulating production of IL-10
endogenously, it is possible to overcome the significant immune-related
problems that
are observed when the cytokine is added exogenously, resulting in over-
production of
IFNy, TNF-a and IL-12.
The compound which is capable of inducing endogenous production of interleukin-
10
(IL-10) by Th2 cells, dendritic cells and/or macrophages may be a non-
steroidal anti-
inflammatory drug (NSAID). The NSAID may be a propionic acid derivative, an
acetic
acid derivative, an enolic acid derivative, a fenamic acid derivative, or a
selective- or
non-selective cyclo-oxygenase (COX) inhibitor. The NSAID may be a profen.
Examples of suitable propionic acid NSAID derivatives may include Ibuprofen;
Naproxen; Fenoprofen; Ketoprofen; Flurbiprofen; or Oxaprozin. Examples of
suitable
acetic acid NSAID derivatives may include Aceclofenac; Acemetacin; Actarit;
Alcofenac; Amfenac; Clometacin; Diclofenac; Etodolac; Felbinac; Fenclofenac;
Indometacin; Ketorolac; Metiazinic acid; Mofezolac; Naproxen; Oxametacin;
Suilindac;
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or Zomepirac. Examples of suitable enolic acid NSAID derivatives may include
Piroxicam; Meloxicam; Tenoxicam; Droxicam; Lornoxicam; or Isoxicam. Examples
of
Fenamic acid NSAID derivatives may include Mefenamic acid; Meclofenamic acid;
Flufenamic acid; or Tolfenamic acid.
In embodiments where the NSAID is a cyclooxygenase (COX) inhibitor, it may be
either a cyclooxygenase 1 (COX 1) inhibitor or a cyclooxygenase 2 (COX 2)
inhibitor.
Examples of suitable COX inhibitors may include Ibuprofen; Celecoxib;
Etoricoxib;
Lumiracoxib; Meloxicam; Rofecoxib; or Valdecoxib.
The NSAID may be selected from a group consisting of: Alminoprofen;
Benoxaprofen;
Dexketoprofen; Flurbiprofen; Ibuprofen; Indoprofen; Ketoprofen; Loxoprofen;
Pranoprofen; Protizinic acid; Suprofen; Aceclofenac; Acemetacin; Actarit;
Alcofenac;
Amfenac; Clometacin; Diclofenac; Etodolac; Felbinac; Fenclofenac; Indometacin;
Ketorolac; Metiazinic acid; Mofezolac; Naproxen; Oxametacin; Sulindac;
Zomepirac;
Celecoxib; Etoricoxib; Lumiracoxib; Meloxicam; Rofecoxib; Valdecoxib;
Aloxipirin;
Aminophenazone; Antraphenine; Aspirin; Azapropazone; Benorilate; Benzydamine;
Butibufen; Chlorthenoxacin; Choline Salicylate; Diflunisal; Emorfazone;
Epirizole;
Feclobuzone; Fenbufen; Glafenine; Hydroxylethyl salicylate; Lactyl phenetidin;
Mefenamic acid; Metamizole; Mofebutazone; Nabumetone; Nifenazone; Niflumic
acid;
Phenacetin; Pipebuzone; Propyphenazone; Proquazone; Salicylamide; Salsalate;
Tiaramide; Tinoridine; and Tolfenamic acid.
A preferred NSAID may be Alminoprofen, Benoxaprofen, Dexketoprofen,
Flurbiprofen, Ibuprofen, Indoprofen, Ketoprofen, Loxoprofen, Pranoprofen
protizininic acid, or Suprofen. Preferably, the NSAID is Ibuprofen.
The NSAID may be used in the form of a pharmaceutically acceptable salt,
solvate, or
solvate of a salt, e.g. the hydrochloride.
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NSAIDs described herein may be provided as racemates, or as individual
enantiomers,
including the R- or S-enantiomer. Thus, the NSAID may comprise R-ibuprofen or
S-
ibuprofen, or a combination thereof.
In one embodiment, S-ibuprofen may be used for the treatment of immune-
mediated
diseases which involve pain that is responsive to COX inhibitors, such as
those
described herein (e.g. Celecoxib; Etoricoxib; Lumiracoxib; Meloxicam;
Rofecoxib; or
Valdecoxib). Examples of such diseases may include arthritis, rheumatoid
arthritis,
osteoarthritis, psoriatic arthritis and endometriosis.
In another embodiment, R-ibuprofen may be used for the treatment of immune-
mediated diseases which do not involve pain that is responsive to COX
inhibitors.
Examples of such diseases ma include psoriasis, inflammatory bowel disease,
multiple
sclerosis, pandemic flu, respiratory syncytial virus, cystic fibrosis, genital
herpes, asthma,
bacterial pneumonia, bacterial meningitis, dengue hemorrhagic fever, type I
diabetes,
prostatitis and pre-term labour.
The pharmaceutical vehicle may comprise at least about 10%, 20%, 30%, 35%,
40%,
45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or at least about 99%
(w/w) lipid. The vehicle may comprise between about 35% and 99% (w/w) lipid,
or
between about 45% and 99% (w/w) lipid, or between about 50% and 99% (w/w)
lipid,
or between about 60% and 98% (w/w) lipid, or between about 70% and 97% (w/w)
lipid, or between about 80% and 96% (w/w) lipid, or between about 85% and 95%
(w/w) lipid, or between about 85% and 95% (w/w) lipid, or between about 88%
and
94% (w/w) lipid, or between about 89% and 93% (w/w) lipid.
The pharmaceutical vehicle may comprise a lipid component selected from a
group
consisting of: an oil or oil-based liquid; a fat; a fatty acid (e.g. oleic
acid, stearic acid or
palmitic acid etc.), a fatty acid ester, a fatty alcohol, a glyceride (mono-,
di- or tri-
glyceride); a phospholipid; a glycol ester; a sucrose ester; a wax; a glycerol
oleate
derivative; a medium chain triglyceride; or a mixture thereof. A triglyceride
is an ester
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derived from glycerol and three fatty acids, and is the main constituent of
vegetable oil
and animal fats.
The term "oil" can refer to a fat that is liquid at normal room temperature,
and can be
used for any substance that does not mix with water, and which has a greasy
feel. The
term "fat" can refer to a fat that is solid at normal room temperature. The
term "lipid"
can therefore refer to a liquid or solid fat, as well as to other related
substances.
A suitable oil, which may be used as the lipid component in the pharmaceutical
vehicle,
may be a natural oil or a vegetable oil. Examples of suitable natural oils may
be selected
from a group consisting of linseed oil; soyabean oil; fractionated coconut
oil; mineral
oil; triacetin; ethyl oleate; a hydrogenated natural oil; or a mixture
thereof. Examples of
suitable vegetable oils may be selected from a group consisting of rapeseed
oil; olive oil;
peanut oil; soybean oil; corn oil; safflower oil; arachis oil; sunflower oil;
canola oil;
walnut oil; almond oil; avocado oil; castor oil; coconut oil; corn oil;
cottonseed oil; rice
bran oil; sesame oil; and refined palm oil; or a mixture thereof. Each of
these oils is
commercially available from a number of sources well recognized by those
skilled in the
art.
The lipid component of the pharmaceutical vehicle may comprise a fatty acid
comprising between 8 and 24 carbon atoms, between 10 and 22 carbon atoms,
between
14 and 20 atoms, or between 16 and 20 atoms. The lipid may be saturated or
unsaturated, for example with one, two, three or more double bonds. The lipid
may
comprise a fatty acid selected from a group consisting of myristic acid (C
14:0); palmitic
acid (C 16:0); palmitoleic acid (C 16:1); stearic acid (C 18:0); oleic acid (C
18:1); linoleic
acid (C 18:2); linolenic acid (C 18:3) and arachidic acid (C 20:0); or a
mixture thereof. It
will be appreciated that the first number provided in the brackets corresponds
to the
number of carbon atoms in the fatty acid, and that the second number
corresponds to
the number of double bonds (i.e. unsaturation).
The melting point of the oil is largely determined by the degree of
saturation/unsaturation. The melting points of oleic acid
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(CF13(CH2)7CH=CH(CH2)7COOH), linoleic acid
(CH3(CH2)4(CH=CHCH2)2(CH2)6COOH), and of linolenic acid
(CH3CH2(CH=CHCH2)3(CH2)6COOH), are about 16 C, -5 C and -11 C, respectively.
Thus, the melting point of the lipid may be between about -20 C and 20 C, or
between
about -15 C and 16 C.
The lipid may comprise rapeseed oil. Rapeseed oil is derived from Brassica nap
us, and
contains both omega-6 and omega-3 fatty acids in a ratio of about 2:1.
However, in the
Examples, the inventors found that linseed oil was particularly effective, and
so linseed
oil may be preferred. Linseed oil, also known as flax seed oil, is a clear to
yellowish oil
obtained from the dried ripe seeds of the flax plant (Liman usitatissitimm,
Linaceae). The
oil is obtained by cold pressing, sometimes followed by solvent extraction.
Linseed oil
is a mixture of various triglycerides that differ in terms of their fatty acid
constituents.
For linseed oil, the constituent fatty acids are of the following types: (i)
the saturated
acids palmitic acid (about 7%) and stearic acid (3.4-4.6%); (ii) the
monounsaturated
oleic acid (18.5-22.6%); (iii) the doubly unsaturated linoleic acid (14.2-
17%); and (iii) the
triply unsaturated omega-3 fatty acid cx-linolenic acid (51.9-55.2%). Linseed
oil is also
rich in omega-6 fatty acid. The structure of a representative triglyceride
found in linseed
oil may be represented by formula I:
0
-
0
Thus, the lipid component of the pharmaceutical vehicle may comprise omega 3
and/or omega 6 fatty acid. Omega-3 fatty acids are a family of unsaturated
fatty acids
that have in common a final carbon¨carbon double bond in the n-3 position,
i.e. the
third bond from the methyl end of the fatty acid, and can be represented by
formula II.
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0
9
-10f INVN/N\-/N\F/P-9 N-S12 N\'''<5 ............... N/1;
Omega-6 fatty acids, on the other hand, are a family of unsaturated fatty
acids that have
in common a final carbon¨carbon double bond in the n-6 position, i.e. the
sixth bond,
counting from the end opposite the carboxyl group, and can be represented by
formula
0
He 1; III
Omega-3 and omega-6 fatty acids are derivatives of linolenic acid, the main
difference
being the number and exact position of the double bonds. Accordingly, omega-3
and
The vehicle may comprise less than about 90%, 80%, 70%, 65%, 60%, 55%, 50%,
45%,
40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, or less than about 1% (w/w) alcohol.
The
vehicle may comprise between about 1% and 90% alcohol (w/w), or between about
1%
The alcohol may be a C120 alcohol, a C115 alcohol, a C110 alcohol, a C15
alcohol, or a C2
4 alcohol. The alcohol may be menthol, or a sugar alcohol, such as glycerol,
sorbitol,
erythritol, xylitol, mannitol, isomalt or maltitol.
alcohol is ethanol.
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In one embodiment, the vehicle may comprise between approximately 60% and 95%
(w/w) lipid and between about 5% and 40% (w/w) alcohol. In another embodiment,
the vehicle may comprise between approximately 80% and 95% (w/w) lipid and
between about 5% and 20% (w/w) alcohol.
In one embodiment, the vehicle may comprise between approximately 60% and 95%
(w/w) oil and between about 5% and 40% (w/w) alcohol. In another embodiment,
the
vehicle may comprise between approximately 80% and 95% (w/w) lipid and between
about 5% and 20% (w/w) alcohol. For example, the vehicle may comprise between
approximately 80% and 95% (w/w) olive oil, rapeseed oil or linseed oil, and
between
approximately 5% and 20% (w/w) ethanol. In another embodiment, the vehicle may
comprise between approximately 88% and 92% (w/w) lipid, and between
approximately 8% and 12% (w/w) alcohol. For example, the vehicle may comprise
between approximately 88% and 92% (w/w) olive oil, rapeseed oil or linseed
oil, and
between approximately 8% and 12% (w/w) ethanol. In another embodiment, the
vehicle may comprise approximately 90% (w/w) lipid, and approximately 10%
(w/w)
alcohol. For example, the vehicle may comprise approximately 90% (w/w) olive
oil,
rapeseed oil or linseed oil, and approximately 10% (w/w) ethanol, and
optionally water.
The inventors believe that water has a tendency to increase the instability of
NSAIDs.
Thus, in a preferred embodiment, the vehicle is substantially anhydrous.
Advantageously, the absence of water in embodiments of the vehicle mean that
the
stability of the NSAID in the composition is not compromised, thereby
providing an
improved product.
However, in some embodiments, the vehicle may optionally comprise water. The
vehicle may comprise less than about 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%,
30%, 25%, 20%, 15%, 10%, 5%, or less than about 1% (w/w) water. The vehicle
may
comprise between about 1% and 70% (w/w) water, or between about 1% and 60%
(w/w) water, or between about 1% and 50% (w/w) water, or between about 2% and
40% (w/w) water, or between about 4% and 30% (w/w) water, or between about 6%
and 20% (w/w) water, or between about 8% and 15% (w/w) water.
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It will be appreciated that the composition may be used to treat a Th1-
mediated disease
in a monotherapy (i.e. use of the composition alone). Alternatively, the
compositions
may be used as an adjunct to, or in combination with, known therapies used in
treating
Th1-mediated diseases.
The composition may have a number of different forms depending, in particular,
on
the manner in which the composition is to be used. Thus, for example, the
composition may be in the form of a powder, tablet, capsule, liquid, ointment,
cream,
gel, hydrogel, aerosol, spray, micellar solution, transdermal patch, liposome
suspension
or any other suitable form that may be administered to a person or animal in
need of
treatment. It will be appreciated that the vehicle for medicaments according
to the
invention should be one which is well-tolerated by the subject to whom it is
given, and
preferably enables delivery of the agents across the blood-brain barrier, or
the lungs.
Compositions comprising the active compound and the lipid and alcohol vehicle
(i.e.
the adjuvant) may be used in a number of ways. For instance, oral
administration may
be required in which case the compound may be contained within a composition
that
may, for example be ingested orally in the form of a tablet, capsule or
liquid.
Alternatively, the composition may be administered by injection into the blood
stream.
Injections may be intravenous (bolus or infusion) or subcutaneous (bolus or
infusion).
Alternatively, the composition comprising the active compound may be
administered
by inhalation (e.g. intranasally, or by mouth).
Compositions may also be formulated for topical use. For instance, ointments
may be
applied to the skin. Topical application to the skin is particularly useful
for treating
infections of the skin or as a means of transdermal delivery to other tissues.
Preferably, the composition is orally administrable, i.e. for oral
administration, as
opposed to administration by injection or inhalation etc.
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It will be appreciated that the amount of the active compound that is required
is
determined by its biological activity and bioavailability, which in turn
depends on the
mode of administration, the physicochemical properties of the compound and
whether
the compound is being used as a monotherapy, or in a combined therapy. The
frequency of administration will also be influenced by the above-mentioned
factors and
particularly the half-life of the active compound within the subject being
treated.
Optimal dosages to be administered may be determined by those skilled in the
art, and
will vary with the particular active compound in use, the strength of the
preparation,
the mode of administration, and the advancement of the disease condition.
Additional
factors depending on the particular subject being treated will result in a
need to adjust
dosages, including subject age, weight, gender, diet, and time of
administration.
It will be appreciated that a skilled person will be able to calculate
required doses, and
optimal concentrations of the active compound at a target tissue, based upon
the
pharmacokinetics of the active. Known procedures, such as those conventionally
employed by the pharmaceutical industry (e.g. in vivo experimentation,
clinical trials,
etc.), may be used to establish specific formulations of the active compound
and precise
therapeutic regimes (such as daily doses of the compounds and the frequency of
administration).
Generally, a daily dose of between 0.001 g/kg of body weight and 60mg/kg of
body
weight of the active compound may be used for treating Thl-mediated diseases
depending upon which compound is used. Suitably, the daily dose is between
0.01Kg/kg of body weight and 40mg/kg of body weight, more suitably between
0.01Kg/kg of body weight and 30mg/kg of body weight or between 0.1Kg/kg and 20
mg/kg body weight, and most suitably between approximately 0.111g/kg and 15
mg/kg
body weight.
Daily doses of the active compound may be given as a single administration
(e.g. a
single daily oral dosage form). A suitable daily dose may be between 0.071.tg
and
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1600mg, or
between 10mg and 800mg.
It is envisaged that the composition may be administered more than once to the
subject
in need of treatment. The composition may require administration twice or more
times
during a day. As an example, the composition may be administered as two (or
more
depending upon the severity of the Th1-mediated disease being treated) daily
doses of
between 0.071.tg and 3200mg (i.e. assuming a body weight of 70kg). A patient
receiving
treatment may take a first dose upon waking and then a second dose in the
evening (if
on a two dose regime) or at 3- or 4-hourly intervals thereafter, and so on. It
is
envisaged that the composition may be administered every day (more than once
if
necessary) after the trigger for the Th1-mediated inflammation.
Alternatively, a slow release device may be used to provide optimal doses of
compounds according to the invention to a patient without the need to
administer
repeated doses.
The inventors also believe that compositions of the invention may be
immobilised on
or in a support substrate or matrix forming a lipid-rich formulation, which
may be used
as a delivery device to treat Th1-mediated disorders.
Hence, in a fourth aspect of the invention, there is provided a drug delivery
device
comprising:-
(i) a pharmaceutical composition comprising a therapeutically
effective
amount of a compound which is capable of increasing endogenous interleukin-
10 (IL-10) production by Th2 cells, dendritic cells and/or macrophages, and a
pharmaceutically acceptable vehicle comprising a lipid and an alcohol; and
a support matrix.
The delivery device may be a pessary or a vaginal ring or the like, which may
be worn
by a subject requiring treatment of a Th1-mediated condition. For example the
device
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may be used to treat any disease characterised by a drop in IL-10
concentrations. It is
known that the concentration of IL-10 decreases in pregnant women, as a result
of
foetus tolerance, and this initiates labour during child birth. Thus, the
delivery device
may be used by pregnant women to prevent or delay premature labour.
The support matrix may be made of a substrate material which is suitable for
supporting the composition therein or thereon. The composition may be
immobilised
on the matrix. In one embodiment, the matrix may comprise any material capable
of
melting at, or around, body temperature, such that, over time, the matrix
dissolves
thereby releasing the composition, which is absorbed by the subject.
The support matrix may a suitable gel or wax. For example, conventional
materials for
vaginal administration may be used, such as glycerol, gelatin, glyco-gelatin,
macrogols
(polyethylene glycols), natural, synthetic or semi-synthetic hard fats, and
fractionated
palm kernel oil.
Based on their findings that the compositions described herein may be used to
increase
the levels of endogenous production of IL-10 by Th2 cells, dendritic cells
and/or
macrophages, and thereby reduce the levels of Th1 cytokines, such as TNF-a and
IL-
12, to trigger the Th1 to Th2 switch, the inventors believe that these effects
of the
compounds may be harnessed and used in the manufacture of clinically useful
compositions. As shown in Figures 4-6, the inventors hypothesise that NSAIDs,
when
formulated in oil and alcohol, activate IL-10 production from macrophages.
A "therapeutically effective amount" of the active compound is any amount
which,
when administered to a subject, results in increased levels of IL-10 and IFN-
y, and
preferably decreased concentrations of TNF-a and IL-12, and thereby provides
treatment of a Th1-mediated disease.
For example, the therapeutically effective amount of the active compound used
may be
from about 0.07 lig to about 3200 mg, and preferably from about 0.7 lig to
about 1600
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mg. The amount of the active compound is from about 7 lig to about 1200mg, or
from about 7 lig to about 800 mg.
A "subject" may be a vertebrate, mammal, or domestic animal, and is preferably
a
human being. Hence, medicaments according to the invention may be used to
treat any
mammal, for example human, livestock, pets, or may be used in other veterinary
applications.
A "pharmaceutically acceptable vehicle" as referred to herein can be any
combination
of compounds known to those skilled in the art to be useful in formulating
pharmaceutical compositions, but which comprises a lipid (e.g. at least 30%
(w/w)
lipid) and an alcohol.
In one embodiment, the pharmaceutically acceptable vehicle may be a solid, and
the
composition may be in the form of a powder or tablet. In addition to the lipid
component and alcohol, a solid pharmaceutically acceptable vehicle may
comprise one
or more substances which may also act as flavouring agents, lubricants,
solubilisers,
suspending agents, dyes, fillers, glidants, compression aids, inert binders,
sweeteners,
preservatives, dyes, coatings, or tablet-disintegrating agents. The vehicle
may also be an
encapsulating material. In powders, the vehicle may be a finely divided solid
that is in
admixture with the finely divided active agent (i.e. the compound which
stimulates
endogenous IL-10 production). In tablets, the active agent may be mixed with a
vehicle
having the necessary compression properties in suitable proportions and
compacted in
the shape and size desired. Suitable solid vehicles may comprise, for example
calcium
phosphate, magnesium stearate, talc, sugars, lactose, dextrin, starch,
gelatin, cellulose,
polyvinylpyrrolidine, low melting waxes and ion exchange resins.
In yet another embodiment, the pharmaceutical vehicle may be a liquid, and the
pharmaceutical composition may be in the form of a solution. Liquid vehicles
are used
in preparing solutions, suspensions, emulsions, syrups, elixirs and
pressurized
compositions. The active compound may be dissolved or suspended in a
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pharmaceutically acceptable liquid vehicle such as water, an organic solvent,
a mixture
of both, or pharmaceutically acceptable oils or fats. In addition to the lipid
component,
the liquid vehicle may also comprise other suitable pharmaceutical additives
such as
solubilisers, emulsifiers, buffers, preservatives, sweeteners, flavouring
agents,
suspending agents, thickening agents, colours, viscosity regulators,
stabilizers or osmo-
regulators. Suitable examples of liquid vehicles for oral administration may
include
water (partially containing additives as above, e.g. cellulose derivatives,
preferably
sodium carboxymethyl cellulose solution), alcohols (including monohydric
alcohols and
polyhydric alcohols, e.g. glycols) and their derivatives, and oils (e.g.
fractionated
coconut oil and arachis oil). The vehicle can also be an oily ester, such as
ethyl oleate
or isopropyl myristate.
The composition is preferably administered orally in the form of a sterile
solution or
suspension containing other solutes or suspending agents (for example, enough
saline
or glucose to make the solution isotonic), bile salts, acacia, gelatin,
sorbitan monoleate,
polysorbate 80 (oleate esters of sorbitol and its anhydrides copolymerized
with ethylene
oxide), and the like.
However, the composition may or may not comprise a surfactant. Preferably, the
composition is not emulsified. Examples of surfactants which may or not be
included
in the composition include a phospholipid, such as phosphatidylcholine
(lecithin) and
phosphatidyl ethanolamine; soaps and detergents, including fatty alkali metal,
ammonium, and triethanolamine salts, and detergents, including (a) cationic
detergents
such as, dimethyl dialkyl ammonium halides, and alkyl pyridinium halides; (b)
anionic
detergents such as alkyl, aryl, and olefin sulfonates, alkyl, olefin, ether,
and
monoglyceride sulfates, and sulfosuccinates; (c) non-ionic detergents such as
fatty
amine oxides, fatty acid alkanolamides, and polyoxyethylenepolypropylene
copolymers;
and (d) amphoteric detergents such as alkyl-b-aminopropionates, and 2-alkyl-
imida2oline quaternary ammonium salts. Another example of a detergent may
include
sodium dodecyl sulphate dimethyl sulphoxide. Preferably, the vehicle or
adjuvant of the
invention does not comprise any of these surfactants.
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Many surfactants have certain safety implications, i.e. many are not GRAS
(generally
regarded as safe). Therefore, avoiding the use of a surfactant circumvents
such safety
concerns for the compositions of the invention. In addition, although not
wishing to be
bound by theory, the inventors hypothesise that exclusion of a surfactant
results in
improved uptake and bioavailability of the active agent in the subject being
treated.
The inventors believe that the pharmaceutically acceptable vehicle may
preferably
comprise at least 30% (w/w) lipid, possibly in the absence of ethanol.
Thus, in a fifth aspect, there is provided a pharmaceutical composition
comprising a
therapeutically effective amount of a compound which is capable of increasing
interleukin-10 (IL-10) production, and a pharmaceutically acceptable vehicle
comprising
at least 30% (w/w) lipid, for use in treating a Th1-mediated disease, wherein
the IL-10
is endogenously produced by Th2 cells, dendritic cells and/or macrophages.
In a sixth aspect, there is provided a drug delivery device comprising:-
(i) a pharmaceutical composition comprising a therapeutically
effective
amount of a compound which is capable of increasing endogenous
interleukin-10 (IL-10) production by Th2 cells, dendritic cells and/or
macrophages, and a pharmaceutically acceptable vehicle comprising at
least 30% (w/w) lipid; and
a support matrix.
In a seventh aspect, there is provided a method of preventing, treating and/or
ameliorating a Th1-mediated disease, the method comprising administering, to a
subject
in need of such treatment, a pharmaceutical composition comprising a
therapeutically
effective amount of a compound which is capable of increasing interleukin-10
(IL-10)
production, and a pharmaceutically acceptable vehicle comprising at least 30%
(w/w)
lipid, wherein the IL-10 is endogenously produced by Th2 cells, dendritic
cells and/or
macrophages.
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In eighth aspect, there is provided use of a pharmaceutical composition for
treating a
Th1-mediated disease, the composition comprising a therapeutically effective
amount
of a compound which is capable of inducing endogenous production of
interleukin-10
(IL-10) by Th2 cells, dendritic cells and/or macrophages and a
pharmaceutically
Based on the results demonstrating the surprising immunomodulatory effects of
the
lipid/alcohol vehicle described herein (see Figures 2-6), the inventors
believe that they
have effectively developed a novel adjuvant, which can be used to enhance the
Thus, according to a ninth aspect of the invention, there is provided an oral
adjuvant,
for use in a pharmaceutical composition comprising an immunogen, wherein the
adjuvant comprises a lipid and an alcohol, and stimulates uptake of the
immunogen by
which modifies the effect of other active agents, such as a drug or a vaccine,
while
having few, if any, direct effects when administered by itself. Adjuvants are
frequently
included in vaccines to enhance the recipient's immune response to an
administered
antigen or immunogen, while keeping the administered foreign material to a
minimum.
immunogen, which counteracts the typical characteristic of rapid clearance and
degradation of free antigen. Consideration of which adjuvant to use must take
into
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account the concomitant negative side-effects of adjuvants, such as
undesirable
inflammatory outcomes. For example, many patients suffer "flu-like" symptoms
or
swelling and tenderness at an injection site occurring after a vaccination.
Reducing such
systemic and local unwanted side-effects, while still providing maximal
enhancement of
an immune response, is especially important and continues to drive much of the
research and development of new, improved adjuvants.
The adjuvant of the ninth aspect may be immunostimulatory and/or
immunoinhibiting.
Advantageously, the adjuvant may be capable of enhancing the immunomodulatory
activity of a subject administered with the adjuvant, resulting in the
stimulation of the
immune system, for treating hypo-immune conditions, such as cancer and immuno-
suppression, as well as inhibiting the immune system, for treating hyper-
immune
conditions. Loading immunostimulating drugs (i.e. the immunogen) in
macrophages
and dendritic cells will promote their phenotype to enhance their capacity to
clear
cancer cells, bacteria and virus.
Advantageously, as shown in the Figures, the oily adjuvant of the ninth aspect
displays
not only greater efficacy for promoting the immunological activity of an
antigen or
immunogen (e.g. ibuprofen), but also exhibits improved stability-conferring
characteristics on the antigen or immunogen, and does not induce negative side-
effects
in subjects administered with the adjuvant.
The term "oral adjuvant" can mean that it is orally administrable, i.e. for
oral
administration, as opposed to administration by injection or inhalation etc.
According to a tenth aspect of the invention, there is provided a
pharmaceutical
composition comprising an immunogen and the adjuvant according to the ninth
aspect.
According to an eleventh aspect, there is provided a pharmaceutical
composition
according to the tenth aspect, for use in therapy.
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According to a twelfth aspect, there is provided a pharmaceutical composition
according to the tenth aspect, for use in treating Th1-mediated disease,
cancer, or a
bacterial or viral infection.
According to a thirteenth aspect, there is provided a method of eliciting, in
a subject, an
effective immune response, the method comprising administering, to a subject,
an
effective amount of the pharmaceutical composition of the tenth aspect.
The lipid and alcohol components of the adjuvant may be selected from any of
the
lipids (oil or fat) or alcohols described herein, in any of the described
amounts. For
example, the adjuvant may comprise at least about 10%, 20%, 30%, 35%, 40%,
45%,
50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or at least about 99% (w/w)
lipid. The adjuvant may comprise between about 35% and 99% (w/w) lipid, or
between
about 45% and 99% (w/w) lipid, or between about 50% and 99% (w/w) lipid, or
between about 60% and 98% (w/w) lipid, or between about 70% and 97% (w/w)
lipid,
or between about 80% and 96% (w/w) lipid, or between about 85% and 95% (w/w)
lipid, or between about 85% and 95% (w/w) lipid, or between about 88% and 94%
(w/w) lipid, or between about 89% and 93% (w/w) lipid.
A suitable oil, which may be used as the lipid component in the adjuvant, may
be a
natural oil or a vegetable oil. Examples of suitable natural oils may be
selected from a
group consisting of linseed oil; soyabean oil; fractionated coconut oil;
mineral oil;
triacetin; ethyl oleate; a hydrogenated natural oil; or a mixture thereof.
Examples of
suitable vegetable oils may be selected from a group consisting of rapeseed
oil; olive oil;
peanut oil; soybean oil; corn oil; safflower oil; arachis oil; sunflower oil;
canola oil;
walnut oil; almond oil; avocado oil; castor oil; coconut oil; corn oil;
cottonseed oil; rice
bran oil; sesame oil; and refined palm oil; or a mixture thereof. In one
preferred
embodiment, the lipid may comprise linseed oil.
The adjuvant may comprise less than about 90%, 80%, 70%, 65%, 60%, 55%, 50%,
45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, or less than about 1% (w/w)
alcohol.
The adjuvant may comprise between about 1% and 90% alcohol (w/w), or between
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about 1% and 70% (w/w) alcohol, or between about 1% and 60% (w/w) alcohol, or
between about 1% and 50% (w/w) alcohol, or between about 2% and 40% (w/w)
alcohol, or between about 4% and 30% (w/w) alcohol, or between about 6% and
20%
(w/w) alcohol, or between about 8% and 15% (w/w) alcohol.
The alcohol may be an aliphatic alcohol. The alcohol may be a C120 alcohol, a
C115
alcohol, a C110 alcohol, a C15 alcohol, or a C24 alcohol. The alcohol may be
menthol, or
a sugar alcohol, such as glycerol, sorbitol, erythritol, xylitol, mannitol,
isomalt or
maltitol. The alcohol may be ethanol, propanol or butanol. In one preferred
embodiment, the alcohol is ethanol.
The adjuvant may or may not comprise water. Preferably, the adjuvant is
anhydrous.
Preferably, the adjuvant does not comprise a hydrophilic surfactant, or any of
the
surfactants described herein.
The adjuvant is capable, in use, of stimulating or inducing the dendritic
cells and/or
macrophages in a subject treated with the pharmaceutical composition, to take
up the
immunogen, such that the concentration of immunomodulatory cytokines is
modulated. The immunomodulatory cytokine which is modulated may be selected
from
a group of cytokines including IL-10; IL4; TNF-a; and IFN-y.
As illustrated in Figures 2, 3, 4 and 5, in one embodiment, the adjuvant may
stimulate
the dendritic cells and/or macrophages in the subject to produce or increase
production of an immunomodulatory cytokine. For example, the cytokine which is
produced or increased may be IL-10 and/or TNF- a and/or IL-4.
As illustrated in Figure 6, in another embodiment, the adjuvant may stimulate
the
dendritic cells and/or macrophages in the subject to stop or decrease
production of an
immunomodulatory cytokine. For example, the cytokine for which production is
decreased or prevented may be IFN-y.
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The immunogen present in the composition may be a pharmaceutically active
agent
having a log P value of greater than 2.0 and/or a polar surface area of
between about 25
A2 and 70 A2. The immunogen may preferably be a small molecule having a
molecular
weight of less than 1000 Da.
The term "log P value" will be known to the skilled person, and can refer to
the ratio of
concentrations of a compound (i.e. the immunogen) in the two phases of a
mixture of
two immiscible solvents at equilibrium. The immunogen may have a log P value
which
is greater than 3.0, 4.0, 5.0 or 6Ø The immunogen may have a log P value
which is less
than 7.0, 6.0, 5.0, 4.0 or 3Ø In one embodiment, the immunogen may have a
log P
value of between 2.0 and 7.0, or between 3.0 and 6.0, or between 3.0 and 5Ø
The
immunogen may have a log P value of between 2.0 and 4.0, or between 2.1 and
4.0, or
between 2.2 and 4.0, or between 2.3 and 4Ø In another embodiment, the
immunogen
may have a log P value of between 2.5 and 4.0, or between 3.0 and 4.0, or
between 3.1
and 4Ø In another embodiment, the immunogen may have a log P value of
between
3.3 and 4.0, or between 3.5 and 4Ø In yet another embodiment, the immunogen
may
have a log P value of between 2.0 and 3.0, or between 2.0 and 2.7, or between
2.0 and
2.5. In a further embodiment, the immunogen may have a log P value of between
2.2
and 2.5.
The term "polar surface area" will also be known to the skilled person, and
can refer to
the surface sum over all of the polar atoms in its structure (usually oxygen
and
nitrogen), also including attached hydrogens. In one embodiment, the immunogen
may
have a polar surface area of between 35 A2 and 65 A2, or between 40 A2 and 60
A2, or
between 45 A2 and 55 A2. In another embodiment, the immunogen may have a polar
surface area of between 30 A2 and 50 A2, or between 35 A2 and 45 A2, or
between 40 A2
and 60 A2.
In one embodiment, the immunogen may comprise a peroxidase proliferator-
activator
receptor gamma (PPAR-y) agonist.
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The term "PPAR-y agonist" can mean any molecule that is capable of binding to,
and
triggering a response from, the peroxidase proliferator-activator receptor
gamma
(PPAR-y). This receptor is also known as the glitazone receptor, or nuclear
receptor
subfamily 1, group C, member 3 (NR1C3). PPAR-y is a type II nuclear receptor
that, in
humans, is encoded by the PPAR-y gene. Two isoforms of PPAR-y are detected in
humans, i.e. PPAR-y1, which is found in nearly all tissues except in muscle,
and PPAR-
y2, which is mostly found in adipose tissue and the intestine. The PPAR-y
agonist
present in the composition may be capable of binding to either PPAR-y1 or PPAR-
y2.
The table below provides a list of preferred PPAR-y agonists, which may be
used in the
compositions of the invention. Thus, the PPAR-y agonist may be a compound
selected
from the table.
Table 1 - PPAR-y agonists
Drug C Log P
Gemfibrozil 3.8
Bezafibrate 3.8
Ciprofibrate 3.4
Clofibrate 3.3
Fenofibrate 5.2
Ibuprofen 3.5
Diclofenac 4.4
Indomethacin 4.3
Monascin 3.2
Irbesartan 4.1
Telmisartan 6.9
Mycophenolic acid 3.2
Resveratrol 3.1
Delta(9)- 7.0
tetrahydrocannabinol
Cannabidiol 6.5
Curcumin 3.2
Cilostazol 3.1
Benzbromarone 5.7
6-shogaol 3.7
Glycyrrhetinic acid 6.4
In one preferred embodiment, the PPAR-y agonist may be a fibrate. Ogata et
al., 2009,
Atherosclerosis 205(2): 413-419, describes the PPAR-y agonist properties of
the
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fibrates, gemfibrozil and bezafibrate. The fibrate may be selected from the
group of
fibrates consisting of bezafibrate; ciprofibrate; clofibrate; gemfibrozil; and
fenofibrate.
In yet another embodiment, the PPAR-y agonist may be selected from the group
of
agonists including Monascin; Irbesartan; Telmisartan; Mycophenolic acid;
Resveratrol;
Delta(9)-tetrahydrocannabinol; Cannabidiol; Curcumin; Cilostazol;
Benzbromarone; 6-
shogaol; and Glycyrrhetinic acid.
In another embodiment, the immunogen may be a non-steroidal anti-inflammatory
drug (NSAID). The immunogen may be any of the NSAIDs described herein, for
example ibuprofen.
All of the features described herein (including any accompanying claims,
abstract and
drawings), and/or all of the steps of any method or process so disclosed, may
be
combined with any of the above aspects in any combination, except combinations
where at least some of such features and/or steps are mutually exclusive.
Embodiments of the invention will now be further described, by way of example
only,
with reference to the following Examples, and to the accompanying diagrammatic
drawings, in which:-
Figure 1 shows the effect of one embodiment of the composition of the first
aspect (i.e.
ibuprofen in lipid, which is denoted herein as BC1054), on survival against
Influenza
A/PR/8/34 lethal challenge. 335 g of ibuprofen in a lipid adjuvant (BC1054
adjuvant
oral) was administered to the challenged mice. Two controls were used, i.e.
335 g of
ibuprofen in the absence of the lipid adjuvant (BC1054 oral) and lipid
adjuvant only in
the absence of ibuprofen (control oral);
Figure 2 shows the effect of the formulation comprising 335 g ibuprofen in
lipid
vehicle (BC1054 lipid oral) in vivo on the IL-10 levels in the lungs of
surviving mice.
Two controls were used, i.e. 335 g of ibuprofen in the absence of the lipid
adjuvant
(BC1054 oral) and lipid vehicle only (vehicle oral);
Figure 3 shows the effect of the formulation comprising 335 g ibuprofen in
lipid
vehicle (BC1054 lipid oral) in vivo on the IL-4 levels in the lungs of
surviving mice. Two
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controls were used, i.e. 335 g of ibuprofen in the absence of the lipid
adjuvant
(BC1054 oral) and lipid vehicle only (vehicle oral);
Figure 4 is a graph showing the effect of the BC1054 formulation on IL-10
levels in the
lungs of surviving mice;
Figure 5 is a graph showing the effect of the BC1054 formulation on TNF-alpha
levels
in the lungs of surviving mice;
Figure 6 is a graph showing the effect of the BC1054 formulation on IFN-gamma
levels
in the lungs of surviving mice; and
Figure 7 is a graph showing the effects of the BC1054 formulation in an anti-
collagen
antibody induced arthritis (ACAIA) murine model.
Examples
The inventors carried out a range of in vivo mouse experiments in order to
determine
the effects of ibuprofen on influenza-challenged mice. The inventors have
convincingly demonstrated in the results described below that ibuprofen, when
administered orally in an oil-based formulation (90% linseed oil; 10%
ethanol), results
in a surprising increase in the concentration of endogenous IL-10 and IL-4.
They
observed a concomitant reduction in the viral symptoms (i.e. increase in
survival rate),
and believe that this is because of the elevated IL-10 concentration.
Example 1 - In vivo mouse survival studies
Protocol:
Five groups (n=10) of C57BLK/6 female mice (6-7 weeks old), were divided into
five
experimental groups containing ten animals each. On day 1, animals received an
intranasal lethal dose (50 (11 total, 25 (11 nostril) of Influenza A/PR/8/34
under
halothane-induced anaesthesia.
On day 3, post-challenge with the virus, the animals received the following
treatments:
= Group A received an oral gavage of ibuprofen at a dose of 335.6 g/animal
(equivalent to 20mg/kg/day ; i.e. 1200 mg per person day as maximum
standard dose) dissolved in 100[11 of 10% Ethanol, and 90% linseed oil;
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= Group B was the first control in which mice received vehicle only (gavage
of
10% Ethanol and 90% linseed oil); and
= Group C was the second control in which mice were orally administered
with a
dose of 335.6 g/animal in 10[11 DMSO (no lipid).
The animals were weighed, and monitored for signs of infection daily up to day
6 when
all animals were culled. Figure 1 represents the average animal survival, and
plotted in
Figure 1.
Figure 1 clearly shows that the first control (i.e. oral administration of the
lipid vehicle
only) had an 80% mortality rate, and that the second control (i.e. oral
administration of
ibuprofen only) exhibited a mortality rate of 60%. However the inventors were
surprised to observe that a single dose of BC1054 (i.e. ibuprofen in oil)
converted the
80% mortality rate of the first negative control to an 80% survival rate, and
this was
totally unexpected.
Example 2 - Determination of IL-10 and IL-4 concentrations
Lungs collected at the end of the in vivo phase of the experiment described in
Example
1 were homogenized at 4 C, and the supernatant was collected and stored at -70
C. 60
[IL of capture antibody diluted in coating buffer was added per well. The
plate was
sealed and incubated overnight at 4 C. Wells were then aspirated and washed
with
300 L/of well wash buffer. After the last wash, the plates were inverted and
blotted
on absorbent paper to remove any residual buffer. Plates were washed with
180 L/well of assay diluents and incubated at room temperature for 1 hour.
Samples
were vortexed for 30secs immediately before adding to the plate. Serial
dilutions were
performed within the plate with both the sample and the standards by pipetting
60 L
of assay diluent into each well. The plate was sealed and incubated for 2
hours at room
temperature. IL-4 and IL-10 assays were then carried out. For IL-4, 60 L of
working
detector was added (Detection Antibody + SAv-HRP reagent) to each well. The
plate
was sealed and incubated for 1 hour at room temperature. For IL-10, 60 L of
detection
antibody was diluted in assay diluent to each well.
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Plates were washed and 60 L of SAv-HRP enzyme was diluted in assay diluent and
added to the plate. The plate was sealed and incubated for 20 minutes at room
temperature. Plates were then washed ten times. 60[11 of substrate solution
were added
to each well and the plate was incubated for 30minutes at room temperature in
the
dark. 60 1 of stop solution was added to each well and absorbance was read at
450nm.
IL-10 and IL-4 concentrations were expressed as pg/mg of lung tissue, and then
plotted in Figures 2 and 3.
As can be seen in Figure 2, the two controls (i.e. oral administration of
ibuprofen only,
and oral administration of the lipid vehicle only) resulted in only 2600pg/mg
and
2000pg/mg IL-10, respectively. However, the inventors were astounded to see
that the
test compound, BC1054, resulted in an IL-10 concentration of 6000pg/mg.
Clearly, this
value of IL-10 is much higher than would have been expected if the activity of
the lipid
vehicle and ibuprofen was merely additive (the value would have been only
4600pg/mg). The fact that the IL-10 concentration in BC1054-administered
animals
was 6000pg/mg suggests that the effects are synergistic (i.e. 24% higher than
expected).
The inventors postulate that this increase in endogenous IL-10 production has
at least
contributed to the significant increase in survival shown in Figure 1.
Referring to Figure 3, there is shown the respective concentrations of IL-4 in
the
surviving animals from the assay of Figure 1. IL-4 is a Th2 cytokine, and it
can be seen
that mice administered with BC1054 had much higher concentrations of IL-4 than
either of the two control groups. Accordingly, the inventors have demonstrated
that the
switch from a Th1 to a Th2 response has been induced. Inducing the switch from
a
Th1 to a Th2 response by up-regulating IL-10 production (and IL-4) can be used
to
help treat Th1-mediated hyper-inflammation.
Example 3 - Pessary
The inventors have prepared a delivery device made of a support matrix onto
which the
lipid-rich BC1054 composition is immobilised, and which can be easily used for
treating
any Th1-mediated disorder. The delivery device is formed in the shape of a
pessary,
with a waxy support matrix. Conventional materials for vaginal administration
that have
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been used include glycerol/gelatin, glyco-gelatin, macrogols (polyethylene
glycols),
natural, synthetic or semi-synthetic hard fats, and fractionated palm kernel
oil, each
having BC1054 immobilised thereon. The support matrix melts at body
temperature, so
that, over time, the composition is released and absorbed by the subject.
The inventors are aware that premature labour can be caused in pregnant
mothers due
to a drop in IL-10 concentrations. Therefore, the pessary can be worn by a
pregnant
woman, such that the BC1054 formulation is released, resulting in an increase
in
endogenous IL-10 production, thereby preventing premature labour.
Example 4 ¨ IL10, TNF-alpha and IFN-gamma
The inventors carried out further experiments to show which cytokines are
stimulated
upon administration of the BC1054 formulation.
Lungs were taken from fatally H1N1 infected mice, and IL-10, TNFa and IFNy
levels
of mice subsequently treated with BC1054 (formulation of the invention) and
ibuprofen (not in oil/ethanol vehicle) were measured. The data are shown in
Figures 4-
6. The levels are related to the effect of each treatment on lethality (i.e. a
surrogate for
the anti-inflammatory activity of IL-10).
As shown in Figure 4, administration to the mice of either ibuprofen on its
own, or the
BC1054 formulation, resulted in increased IL-10 levels. However, the inventors
observed that these increases in IL-10 were associated with very different
pharmacodynamic effects. The pattern of pro-inflammatory cytokine reduction
highlights the source of the IL-10 and its relevance to the effect on the
viral survival
model. Importantly, as shown in Figure 6, the levels of IFNy (which is a
lymphocyte-
derived cytokine) were markedly lowered in the ibuprofen-treated mice compared
to
BC1054-treated mice, which was associated with a poor outcome.
Moreover, as shown in Figure 5, TNFa (which is macrophage-related cytokine)
was not
as markedly inhibited. However, in BC1054-treated mice, TNFa levels were
markedly
lowered in the BC1054-treated mice whilst IFNy levels were largely unaffected.
This
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demonstrates that the BC1054 is having its protective effect on the lethality
of H1N1
through macrophage-derived IL-10 levels rather than lymphocyte-derived IL10
("Immunobiology, 5th edition - The Immune System in Health and Disease",
Charles
A Janeway (Jr), Paul Travers, Mark Walport and Mark J Shlomchik. New York:
Garland
Example 5 ¨ Anti-collagen antibody induced arthritis (ACAIA) murine model
The inventors investigated the effects of the BC1054 formulation in an ACAIA
mouse
Materials and Methods
BALB/c mice, on day 0, were intravenously injected with a single inoculation
with and-
collagen II monoclonal antibody (2 mg in 200 (11), followed by an
intraperitoneal
The results of this experiment are shown in Figure 7. As can be seen, as
expected, the
BC1054) displayed the next best results, with 30 mg/kg BC1054 producing a paw
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thickness of only 1.7mm, and_20 mg/kg BC1054 producing a paw thickness of
about
1.73mm. The negative controls (i.e. PBS, 40mg/kg ibuprofen alone, vehicle
alone, and
vehicle methoxycellulose) performed significantly worse than BC1054 with paw
thicknesses of 1.9mm and over. Therefore, these data clearly demonstrate that
the
formulation of the invention can be used to treat systemic inflammation, such
as
arthritis.
Summary
In summary, the inventors were surprised to observe that ibuprofen, when
administered orally in a lipophilic excipient (i.e. linseed oil),
significantly increased
endogenous IL-10 production, and thus surprisingly improved survival in
influenza-
challenged mice. The encouraging results of the in vivo mouse studies clearly
demonstrate that mice infected with a H1N1 virus can be effectively treated by
administration of a single oral dose of ibuprofen present in an oily
formulation. Hence,
any compound which is capable of increasing IL-10 production, when formulated
in a
carrier having a high concentration of lipid, and orally administered will
result in a
much higher bioavailability in the lung. Achieving a high concentration of an
NSAID
for example, such as ibuprofen, in the lung will be advantageous, when
treating Thl-
mediated disorders, for example a respiratory disorder caused by viral
infections.
However, only in the BC1054-treated animals, is the IL-10 signal accompanied
by an
improvement in survival. In the same lungs, there is a more pronounced down-
regulation of IFN-gamma (i.e. a T-cell-derived cytokine) for ibuprofen-treated
mice,
demonstrating that IL-10 in the ibuprofen mice is likely to come from T-
cells. However, in BC1054-treated mice, there is a more pronounced down-
regulation
of pulmonary TNF-alpha (i.e. a macrophage-derived cytokine), demonstrating
that IL-
10 in BC1054-treated mice is more likely to come from macrophages, which
results in
the profound anti-inflammatory effects that were observed.
In summary, therefore, this is evidence that ibuprofen present in the oral
lipid adjuvant
of the invention is more preferentially taken up by macrophages, resulting in
the new
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and surprising anti-inflammatory pharmacology as demonstrated by IBD and RA
model data.
