U.S. patent application number 13/295014 was filed with the patent office on 2012-03-08 for anti-inflammatory modalities.
This patent application is currently assigned to MARSHALL EDWARDS, INC.. Invention is credited to Alan James HUSBAND, Michael John JAMES, Catherine WALKER.
Application Number | 20120059176 13/295014 |
Document ID | / |
Family ID | 37023310 |
Filed Date | 2012-03-08 |
United States Patent
Application |
20120059176 |
Kind Code |
A1 |
WALKER; Catherine ; et
al. |
March 8, 2012 |
ANTI-INFLAMMATORY MODALITIES
Abstract
Anti-inflammatory modalities are described with reference to
select isoflavonoid compounds, compositions containing same and the
use of said compounds and/or compositions in treatment,
particularly for the treatment of inflammatory diseases and related
conditions.
Inventors: |
WALKER; Catherine; (Balmain,
AU) ; HUSBAND; Alan James; (Pyrmont, AU) ;
JAMES; Michael John; (Dulwich, AU) |
Assignee: |
MARSHALL EDWARDS, INC.
San Diego
CA
|
Family ID: |
37023310 |
Appl. No.: |
13/295014 |
Filed: |
November 11, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11909581 |
Jun 23, 2008 |
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PCT/AU2006/000407 |
Mar 24, 2006 |
|
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13295014 |
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60668609 |
Apr 6, 2005 |
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Current U.S.
Class: |
549/403 ;
549/406 |
Current CPC
Class: |
A61P 19/08 20180101;
A61P 43/00 20180101; A61P 3/06 20180101; A61P 9/10 20180101; A61P
29/00 20180101; A61P 1/00 20180101; A61P 11/00 20180101; A61P 11/06
20180101; A61P 5/30 20180101; A61P 9/12 20180101; A61P 9/00
20180101; A61P 19/02 20180101; A61P 35/02 20180101; C07D 493/04
20130101; A61P 1/04 20180101; A61P 37/00 20180101; A61P 37/06
20180101; A61P 35/00 20180101 |
Class at
Publication: |
549/403 ;
549/406 |
International
Class: |
C07D 311/38 20060101
C07D311/38; A61P 29/00 20060101 A61P029/00; A61P 9/12 20060101
A61P009/12; A61P 19/02 20060101 A61P019/02; A61P 11/06 20060101
A61P011/06; A61P 9/10 20060101 A61P009/10; C07D 311/58 20060101
C07D311/58; A61P 19/08 20060101 A61P019/08 |
Foreign Application Data
Date |
Code |
Application Number |
Mar 24, 2005 |
AU |
2005901475 |
Oct 28, 2005 |
AU |
2005905985 |
Claims
1. Use of a compound of formula (I) in the manufacture of a
medicament for the treatment of an inflammatory disease, wherein
the compound of formula (I) is: ##STR00013## and salts thereof
wherein: R.sub.1 and R.sub.2 are independently hydroxy, alkoxy or
acyloxy, R.sub.3 is hydroxy, alkoxy, alkyl or halo, and X is either
.dbd.O or hydrogen and hydroxy.
2. Use according to claim 1, wherein R.sub.1 and R.sub.2 are
hydroxy.
3. Use according to claim 1 or 2, wherein R.sub.3 is methyl, bromo,
chloro or hydroxy.
4. Use according to any one of claims 1 to 3, wherein X is .dbd.O
and the compounds are isoflavan-4-ones of the formula (Ia):
##STR00014## and wherein R.sub.3 is 5-alkyl, 6-halo or 8-halo,
5. Use according to claim 4, wherein R.sub.3 is 5-methyl, 6-chloro
or 8-bromo.
6. Use according to any one of claims 1 to 3, wherin X is hydrogen
and hydroxy and the compounds are isoflavan-4-ols of the formula
(Ib): ##STR00015## and wherein R3 is 8-alkyl or 8-hydroxy.
7. Use according to claim 6, wherein R.sub.3 is 8-methyl or
8-hydroxy.
8. Use according to claim 1, wherein the compound is an
8-substituted isoflavonoid compound of the formula (Ic):
##STR00016## and salts thereof wherein R.sub.1 and R.sub.2 are
independently hydroxy, alkoxy or acyloxy, R.sub.3 is hydroxy, alkyl
or halo, and X is either .dbd.O or hydrogen and hydroxy.
9. Use according to claim 8, wherein R.sub.1 and R.sub.2 are
hydroxy, and R.sub.3 is hydroxy, methyl or bromo.
10. Use according to claim 1 wherein the compound of formula (I) is
selected from compounds 1 to 5: ##STR00017## and pharmaceutically
acceptable salts thereof
11. Use according to any one of claims 1 to 10, wherein the
inflammatory disease or disorder is selected from osteoarthritis,
inflammatory bowel disease (ulcerative colitis and Crohn's
disease), ulcerative proctitis, distal colitis, autoimmune
disorders (SLE, rheumatoid arthritis, glomerulonephritis), asthma
and diseases involving pulmonary inflammation, cardiovascular
disorders including atherosclerosis, hypertension and lipid
dyscrasias and disorders associated with estrogen receptor
activation.
12. Use according to claim 11, wherein the treatment is for pain,
oedema and/or erythema associated with inflammation.
13. Use according to any one of claims 1 to 12, wherein the
treatment of the inflammatory disorder is absent cardiovascular
side effects, is cardioprotective and/or is gut protective.
14. Use of a thromboxane synthase inhibitor for the manufacture of
a medicament for the treatment of inflammation, including pain
associated with inflammation.
15. Use according to claim 14, wherein the treatment is
cardioprotective and/or gut protective.
16. A pharmaceutical agent comprising a compound of formula (I)
according to any one of claims 1 to 10 for the treatment of
inflammatory diseases and disorders or as a thromboxane synthase
inhibitor.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to certain isoflavonoid
compounds, compositions containing same and the use of said
compounds and/or compositions in treatment, particularly for the
treatment of inflammatory diseases and conditions. Inflammatory
conditions include irritable bowel disease (IBD), for example:
ulcerative colitis (UC), ulcerative proctitis, distal colitis;
and/or Crohn's disease (CD), as well as other hepatointestinal
syndromes including primary sclerosing cholangitis (PSC), primary
biliary cirrhosis (PBC), autoimmune hepatitis (AIH) and irritable
bowel syndrome (IBS).
BACKGROUND OF THE INVENTION
[0002] UC causes inflammation of the inner lining of the large
bowel (colon and rectum). CD causes inflammation of the full
thickness of the bowel wall and may involve any part of the
digestive tract from the mouth to the anus. CD can cause recurrent
bowel obstruction, fistulae, abscess formation and sepsis as well
as extra-intestinal manifestations such as arthritis. IBD often
develops between the ages of 15-30, and about 13,000 Australians
have UC and 10, 000 have CD. The Crohn's and Colitis Foundation of
America estimates as many as 1,000,000 Americans have IBD costing
directly and indirectly around $US552 billion annually.
Furthermore, there is research to suggest that persons with IBD are
more likely to develop colon cancer.
[0003] The cause of IBD is unknown. However, both syndromes would
appear to be immunologically-mediated and the inflammatory process
is influenced by environmental and genetic factors. Medical therapy
aims to control the inflammatory process, and is administered for
active and chronic disease, as well as remission maintenance. No
management strategy is totally effective, but current therapy is
targeted at reducing inflammation and/or the immune response using
anti-inflammatories (corticosteroids, aminosalicylates),
immunosuppressives, immunotherapies or surgery. The goals of
treatment are to induce and then maintain remission of disease,
minimising the side-effects which accompany the therapies. Up to
80% of patients with UC and 35% in those with CD relapse within a
year following the induction of remission (Podolsky, D. K. (2002)
"The current future understanding of inflammatory bowel disease."
Best Practice & Research in Clinical Gastroenterology 16(6):
933-43). Additionally, none of the existing therapies are without
significant side effects. The `holy grail` in IBD drug development
is therefore a non-toxic agent which will maintain remission of
disease (Feagan 2003 "Maintenance therapy for inflammatory bowel
disease" The American Journal of Gastroenterology 98 (12,
Supplement 1): S6-S17).
[0004] Primary biliary cirrhosis (PBC), autoimmune hepatitis (AIH)
and primary sclerosing cholangitis (PSC) are chronic liver diseases
that are likely to have an autoimmune basis to their pathogenesis.
PSC appears to be associated with UC. In PSC and PBC, the bile
ducts become inflammed, scarred and eventually blocked, causing
cholestasis, hepatocellular injury and in many cases liver
failure.
[0005] Irritable Bowel Syndrome (IBS) is part of a spectrum of
diseases known as Functional Gastrointestinal Disorders which
include diseases such as non-cardiac chest pain, non-ulcer
dyspepsia, and chronic constipation or diarrhoea. These diseases
are all characterised by chronic or recurrent gastrointestinal
symptoms for which no structural or biochemical cause can be found.
IBS affects between 25 and 55 million people in the USA.
[0006] The prevalence of IBS in the general population of Western
countries varies from 6 to 22%.
[0007] IBS affects 14-24% of women and 5-19% of men. The prevalence
is similar in Caucasians and African Americans, but appears to be
lower in Hispanics. Although several studies have reported a lower
prevalence of IBS among older people, the present studies do not
allow to definitely conclude whether or not an age disparity exists
in IBS. In non-Western countries such as Japan, China, India, and
Africa IBS also appears to be very common.
[0008] Accordingly there is a need for new therapies in the
treatment of inflammation and related diseases and conditions and
new and improved agents and compounds useful for same.
SUMMARY OF THE INVENTION
[0009] Surprisingly the inventors have found that compounds of
formula (I)
##STR00001##
and salts thereof wherein:
[0010] R.sub.1 and R.sub.2 are independently hydroxy, alkoxy or
acyloxy,
[0011] R.sub.3 is hydroxy, alkoxy, alkyl or halo, and
[0012] X is either .dbd.O or hydrogen and hydroxy are particularly
useful in the treatment of inflammatory diseases and cardiovascular
diseases.
[0013] Preferably R.sub.1 and R.sub.2 in compounds of formula (I)
represent hydroxy.
[0014] Preferably R.sub.3 in compounds of formula (I) represents
methyl, bromo, chloro or hydroxy.
[0015] In a preferred embodiment X is .dbd.O and the compounds of
the invention are isoflavan-4-ones of the formula (Ia):
##STR00002##
wherein
[0016] R3 is 5-alkyl, 6-halo or 8-halo,
[0017] more preferably
[0018] R3 is 5-methyl, 6-chloro or 8-bromo.
[0019] In another preferred embodiment X is hydrogen and hydroxy
and the compounds of the invention are isoflavan-4-ols of the
formula (Ib):
##STR00003##
wherein
[0020] R3 is 8-alkyl or 8-hydroxy,
[0021] more preferably
[0022] R3 is 8-methyl, or 8-hydroxy.
[0023] In still another preferred embodiment the compounds are
8-substituted isoflavonoid compounds of the formula (Ic):
##STR00004##
and salts thereof wherein:
[0024] R.sub.1 and R.sub.2 are independently hydroxy, alkoxy or
acyloxy,
[0025] R.sub.3 is hydroxy, alkyl or halo, and
[0026] X is either .dbd.O or hydrogen and hydroxy,
[0027] more preferably
[0028] R.sub.1 and R.sub.2 are hydroxy, and
[0029] R.sub.3 is hydroxy, methyl or bromo.
[0030] Particularly preferred isoflavonoid compounds of formula (I)
and pharmaceutically acceptable salts thereof are selected from
compounds 1 to 5:
##STR00005##
[0031] According to another aspect of the present invention there
is provided the use of one or more compounds of formula (I) as an
anti-inflammatory agent or cardiovascular agent.
[0032] The inflammatory disorders include pain, oedema and erythema
associated with inflammation in general, inflammatory disorders
including osteoarthritis, inflammatory bowel disease (ulcerative
colitis and Crohn's disease), ulcerative proctitis, distal colitis,
autoimmune disorders (SLE, rheumatoid arthritis,
glomerulonephritis), asthma and diseases involving to pulmonary
inflammation, cardiovascular disorders including atherosclerosis,
hypertension and lipid dyscrasias and disorders associated with
estrogen receptor activation. That is the compounds are useful for
the treatment of pain associated with inflammation. In a preferred
embodiment, the treatment of the inflammatory disorders is absent
cardiovascular side effects, cardioprotective and/or is gut
protective.
[0033] According to another aspect of the present invention there
is provided the use of a thromboxane synthase inhibitor for the
manufacture of a medicament for the treatment of inflammation,
including pain associated with inflammation. Preferably the
treatment is cardioprotective and/or gut protective.
[0034] According to another aspect of the invention there is
provided a pharmaceutical agent comprising a compound of formula
(I) for the treatment of inflammatory diseases and disorders or as
a thromboxane synthase inhibitor.
[0035] Throughout this specification and the claims which follow,
unless the context requires otherwise, the word "comprise", and
variations such as "comprises" or "comprising", will be understood
to imply the inclusion of a stated integer or step or group of
integers or steps but not the exclusion of any other integer or
step or group of integers or steps.
BRIEF DESCRIPTION OF THE FIGURES
[0036] FIG. 1 shows the effect of 10 .mu.M test compound on
PGE.sub.2 synthesis by human monocytes stimulated with LPS.
[0037] FIG. 2 shows the effect of 10 .mu.M test compound on
TXB.sub.2 synthesis by human monocytes stimulated with LPS.
[0038] FIG. 3 shows the effect of test compounds on TXB.sub.2
synthesis following the addition of exogenous PGH.sub.2 to U937
cells.
[0039] FIG. 4 shows the percent inhibition of NF.kappa.B promoter
activity in TNF.alpha.-stimulated THP-1 monocyte/macrophages.
[0040] FIG. 5 shows the effect on colonic inflammation of Compounds
1 and 4 dosed orally at 1 mg/kg (mean.+-.SEM).
[0041] FIG. 6 shows the effect on colonic length of Compounds 1 and
4 dosed orally at 1 mg/kg (mean.+-.SEM).
[0042] FIG. 7 shows the effect on clinical score of Compounds 1 and
4 dosed orally at 1 mg/kg. FIG. 8 shows the effect on body weight
of Compounds 1 and 4 dosed orally at 1 mg/kg. FIG. 9 shows the
effect on colonic inflammation of Compounds 1 and 2 pre-dosed
orally at 1 mg/kg (mean.+-.SEM).
[0043] FIG. 10 shows the effect on the length of the colon of
Compounds 1 and 2 pre-dosed orally at 1 mg/kg (mean.+-.SEM).
[0044] FIG. 11 shows the effect on the clinical score of Compounds
1 and 2 pre-dosed orally at 1 mg/kg.
[0045] FIG. 12 shows the effect on the body weight from the
induction of colitis of pre-dosing Compounds 1 and 2 orally at 1
mg/kg for 10 days. FIG. 13 shows the effect on colonic inflammation
of Compound 3a and 3b dosed orally at 1.25 mg/kg (mean.+-.SEM).
[0046] FIG. 14 shows the effect on colonic length of Compound 3a
and 3b dosed orally at 1.25 mg/kg (mean.+-.SEM).
[0047] FIG. 15 shows the effect on clinical score of Compound 3a
and 3b dosed orally at 1.25 mg/kg.
[0048] FIG. 16 shows the effect on body weight of Compound 3a and
3b dosed orally at 1.25 mg/kg.
[0049] FIG. 17 shows the production of PGE.sub.2 and TXB.sub.2 by
colons cultured in the absence of mitogenic stimulation.
[0050] FIG. 18 shows the production of PGE2 and TXB2 by colons
cultured in the presence of ConA.
[0051] FIG. 19 shows the percentage inhibition of the contractile
repsonse of noradrenaline.
[0052] FIG. 20 shows the body weight change in rats given high
doses of Compound 1 orally for 11 days.
[0053] FIG. 21 shows the body weight change following oral
administration of Compound 2 at 5 mg/kg/day or vehicle.
[0054] FIG. 22 shows the body weight change following oral
administration of a mixture of
[0055] Compounds 3a and 3b at 20 mg/kg/day (data sets from
individual mice and mean of group outlined in bold).
[0056] FIG. 23 shows the body weight change following oral
administration of Compound 4 at 5 mg/kg/day or vehicle.
DETAILED DESCRIPTION
[0057] The term alkyl is taken to include straight chain and
branched chain saturated alkyl groups of 1 to 6 carbon atoms, such
as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl,
tertiary butyl, pentyl and the like. The alkyl group more
preferably contains from 1 to 4 carbon atoms, especially methyl,
ethyl, propyl or isopropyl.
[0058] The compounds of the invention include all salts, such as
acid addition salts, anionic salts and zwitterionic salts, and in
particular include pharmaceutically acceptable salts as would be
known to those skilled in the art. The term "pharmaceutically
acceptable salt" refers to an organic or inorganic moiety that
carries a charge and that can be administered in association with a
pharmaceutical agent, for example, as a counter-cation or
counter-anion in a salt. Pharmaceutically acceptable cations are
known to those of skilled in the art, and include but are not
limited to sodium, potassium, calcium, zinc and quaternary amine.
Pharmaceutically acceptable anions are known to those of skill in
the art, and include but are not limited to chloride, acetate,
tosylate, citrate, bicarbonate and carbonate.
[0059] Pharmaceutically acceptable salts include those formed from:
acetic, ascorbic, aspartic, benzoic, benzenesulphonic, citric,
cinnamic, ethanesulphonic, fumaric, glutamic, glutaric, gluconic,
hydrochloric, hydrobromic, lactic, maleic, malic, methanesuiphonic,
naphthoic, hydroxynaphthoic, naphthalenesulphonic,
naphthalenedisulphonic, naphthaleneacrylic, oleic, oxalic,
oxaloacetic, phosphoric, pyruvic, para-toluenesulphonic, tartaric,
trifluoroacetic, triphenylacetic, tricarballylic, salicylic,
sulphuric, sulphamic, sulphanilic and succinic acid.
[0060] The term "pharmaceutically acceptable derivative" or
"prodrug" refers to a derivative of the active compound that upon
administration to the recipient is capable of providing directly or
indirectly, the parent compound or metabolite, or that exhibits
activity itself and includes for example phosphate derivatives and
sulphonate derivatives. Thus, derivatives include solvates,
pharmaceutically active esters, prodrugs or the like.
[0061] The preferred compounds of the present invention also
include all derivatives with physiologically cleavable leaving
groups that can be cleaved in vivo to provide the compounds of the
invention or their active moiety. The leaving groups may include
acyl, phosphate, sulfate, sulfonate, and preferably are mono-, di-
and per-acyl oxy-substituted compounds, where one or more of the
pendant hydroxy groups are protected by an acyl group, preferably
an acetyl group. Typically acyloxy substituted compounds of the
invention are readily cleavable to the corresponding hydroxy
substituted compounds.
[0062] The invention also provides a pharmaceutical composition
comprising a compound of formula (I) and at least one
pharmaceutically acceptable excipient, especially for use in
treatment or in the manufacture of a medicament, for example, for
the treatment of inflammatory bowel disease (IBD), for example:
ulcerative colitis (UC), ulcerative proctitis, distal colitis,
and/or Crohn's disease (CD), as well as other intestinal syndromes
including primary sclerosing cholangitis (PSC), primary biliary
cirrhosis (PBC), autoimmune hepatitis (AIH) and irritable bowel
syndrome (IBS).
[0063] In the context of this application substantially pure is
intended to mean 90% purity or greater such as 95% purity,
particularly 98% purity, especially 99% purity, for example as
assessed by HPLC analysis.
[0064] The invention also extends to employing at least two
compounds of formula (I) in the various aspects of the invention
described herein.
[0065] Pharmaceutical formulations include those suitable for oral,
parenteral (including subcutaneous, intradermal, intramuscular,
intravenous and intraarticular), inhalation (including use of
metered dose pressurised aerosols, nebulisers or insufflators),
rectal and topical (including dermal, buccal, sublingual and
intraocular) administration. The most suitable route may depend
upon for example the condition and disorder of the recipient. The
formulations may conveniently he presented in unit dosage form and
may be prepared by any of the methods well known in the art of
pharmacy. All methods include the step of bringing the active
ingredient into association with the carrier which constitutes one
or more accessory ingredients. In general the formulations are
prepared by uniformly and intimately bringing into association the
active ingredient with liquid carrier or finely divided solid
carriers or both and then, if necessary, shaping the product into
the desired formulation.
[0066] Formulations of the present invention suitable for oral
administration may be presented as discrete units such as capsules
such as gelatine or HPMC capsules, cachets or tablets each
containing a predetermined amount of the active ingredient; as a
powder or granules; as a solution or a suspension in an aqueous
liquid or a non-aqueous liquid; or as an oil-in-water liquid
emulsion or a water-in-oil liquid emulsion. The active ingredient
may also be presented as a paste.
[0067] When compounds of formula (I) are formulated as capsules
preferably the compound is formulated with one or more
pharmaceutically acceptable carrier such as starch, lactose,
microcrystalline cellulose, silicon dioxide and/or a cyclic
oligosaccaride such as cyclodextrin.
[0068] Additional ingredients may include lubricants such as
magnesium stearate and/or calcium stearate.
[0069] Suitable cyclodextrins include a-cyclodextrin,
.beta.-cyclodextrin, .gamma.-cyclodextrin,
demethyl-.beta.-cyclodextrin, 2-hydroxyethyl-.beta.-cyclodextrin,
2-hyroxypropyl-cyclodextrin, 3-hydroxypropyl-.beta.-cyclodestrin
and tri-methyl-(3-cyclodextrin. More preferably the cyclodextrin is
hydroxypropyl-.beta.-cyclodextrin.
[0070] Tablets may be prepared by compression or moulding,
optionally with one or more accessory ingredients. Compressed
tablets may be prepared by compressing in a suitable machine the
active ingredient in a free-flowing form such as a powder or
granules, optionally mixed with a binder, lubricant such as
magnesium stearate or calcium stearate, inert diluent or a surface
active/dispersing agent. Moulded tablets may be made by moulding a
mixture of the powdered compound moistened with an inert liquid
diluent, in a suitable machine. The tablets may optionally be
coated, for example, with an enteric coating and may be formulated
so as to provide slow or controlled release of the active
ingredient therein.
[0071] Formulations for parenteral administration include aqueous
and non-aqueous sterile injection solutions which may contain
anti-oxidants, buffers, bacteriostats and solutes which render the
formulation isotonic with the blood of the intended recipient and
which may include suspending agents and thickening agents.
Preferably a parenteral formulation will comprise a cyclic
oligosaccaride such as hydroxypropyl-.beta.-cyclodextrin. The
formulations may be presented in unit-dose or multi-dose
containers, for example sealed ampoules and vials, and may be
stored in a freeze-dried (lyophilised) condition requiring only the
addition of the sterile liquid carrier, for example saline or
water-for-injection, immediately prior to use. Extemporaneous
injection solutions and suspensions may be prepared from sterile
powders, granules and tablets of the kind previously described.
[0072] Dry powder compositions for topical delivery to the lung by
inhalation may, for example, be presented in capsules and
cartridges of for example gelatine, or blisters of for example
laminated aluminium foil, for use in an inhaler or insufflator.
Formulations generally contain a powder mix for inhalation of the
one or more compounds of the invention and a suitable powder base
(carrier substance) such as lactose or starch. Use of lactose is
preferred. Each capsule or cartridge may generally contain between
20 .mu.g-10 mg of the compound formula (I) optionally in
combination with another therapeutically active ingredient.
Alternatively, the compound or compounds of the invention may be
presented without excipients. Packaging of the formulation may be
for unit dose or multi-dose delivery.
[0073] Spray compositions for topical delivery to the lung by
inhalation may, for example be formulated as aqueous solutions or
suspensions or as aerosols suspensions or solutions delivered from
pressurised packs, such as a metered dose inhaler, with the use of
a suitable liquefied propellant. Suitable propellants include a
fluorocarbon or a hydrogen-containing chlorofluorocarbon or
mixtures thereof, particularly hydrofluoroalkanes, e.g.
dichlorodifluoromethane, trichlorofluoromethane,
dichlorotetra-fluoroethane, especially 1,1,1,2-tetrafluoroethane,
1,1,1,2,3,3,3-heptafluoro-n-propane or a mixture thereof. Carbon
dioxide or other suitable gas may also be used as propellant. The
aerosol composition may be excipient free or may optionally contain
additional formulation excipients well known in the art such as
surfactants eg oleic acid or lecithin and cosolvents eg ethanol.
Pressurised formulations will generally be retained in a canister
(eg an aluminium canister) closed with a valve (eg a metering
valve) and fitted into an actuator provided with a mouthpiece.
[0074] Medicaments for administration by inhalation desirably have
a controlled particle size. The optimum particle size for
inhalation into the bronchial system is usually 1-10 .mu.m,
preferably 2-5 .mu.m. Particles having a size above 20 .mu.m are
generally too large when inhaled to reach the small airways. When
the excipient is lactose it will typically be present as milled
lactose, wherein not more than 85% of lactose particles will have a
MMD of 60-90 .mu.m and not less than 15% will have a MMD of less
than 15 .mu.m.
[0075] Formulations for rectal administration may be presented as a
suppository with carriers such as cocoa butter or polyethylene
glycol, or as an enema wherein the carrier is an isotonic liquid
such as saline. Additional components of the formulation may
include a cyclic oligosaccaride, for example, a cyclodextrin, as
described above, such as hydroxypropyl-.beta.-cyclodextrin, one or
more surfactants, buffer salts or acid or alkali to adjust the pH,
isotonicity adjusting agents and/or anti-oxidants.
[0076] It should be understood that in addition to the ingredients
particularly mentioned above, the formulations of this invention
may include other agents conventional in the art having regard to
the type of formulation in question, for example those suitable for
oral administration may include flavouring agents.
[0077] The compounds and pharmaceutical formulations according to
the invention may be used in combination with or include one or
more other therapeutic agents, for example anti-inflammatory
agents, anticholinergic agents (particularly an M.sub.1, M.sub.2,
M.sub.1/M.sub.2 or M.sub.3 receptor antagonist),
.beta..sub.2-adrenoreceptor agonists, antiinfective agents (e.g.
antibiotics, antivirals), or antihistamines. Preferred are
combinations comprising a compound or compounds of formula (I) or a
pharmaceutically acceptable salt, solvate or physiologically
functional derivative thereof together with a corticosteroid,
and/or an anticholinergic, and/or a PDE-4 inhibitor.
[0078] Suitable anti-inflammatory agents include corticosteroids
and NSAIDs. Suitable corticosteroids, which may be used in
combination with the compounds of the invention are those oral and
inhaled corticosteroids and their pro-drugs which have
anti-inflammatory activity. Examples include methyl prednisolone,
prednisolone, dexamethasone, fluticasone propionate,
6.alpha.,9.alpha.-difluoro-17.alpha.-[(2-furanylcarbonyl)oxy]-11.beta.-hy-
droxy-16a-methyl-3-oxo-androsta-1,4-diene-17.beta.-carbothioic acid
S-fluoromethyl ester,
6.alpha.,9.alpha.-difluoro-11.beta.-hydroxy-16.alpha.-methyl-3-oxo
-17.alpha.-propionyloxy-androsta-1,4-diene-17.beta.-carbothioic
acid S-(2-oxo-tetrahydro-furan-3S-yl) ester, beclomethasone esters
(e.g. the 17-propionate ester or the 17,21-dipropionate ester),
budesonide, flunisolide, mometasone esters (e.g. the furoate
ester), triamcinolone acetonide, rofleponide, ciclesonide and
butixocort propionate. Preferred corticosteroids include
fluticasone propionate, and
6.alpha.,9.alpha.-difluoro-17.alpha.-[(2-furanylcarbonyl)oxy]-11.beta.-hy-
droxy-16.alpha.-methyl-3-oxo-androsta-1,4-diene-17.beta.-carbothioic
acid S-fluoromethyl ester, more preferably
6.alpha.,9.alpha.-difluoro-17.alpha.-[(2-furanylcarbonyl)oxy]-11.beta.-hy-
droxy-16.alpha.-methyl-3-oxo-androsta-1,4-diene-17.beta.-carbothioic
acid S-fluoromethyl ester.
[0079] Suitable NSAIDs include sodium cromoglycate, nedocromil
sodium, phosphodiesterase (PDE) inhibitors (e.g. theophylline, PDE4
inhibitors or mixed PDE3/PDE4 inhibitors), leukotriene antagonists,
inhibitors of leukotriene synthesis, iNOS inhibitors, tryptase and
elastase inhibitors, beta-2 integrin antogonists and adenosine
receptor agonists or antagonists (e.g. adenosine 2a agonists),
cytokine antagonists (e.g. chemokine antagonists) or inhibitors or
cytokine synthesis.
[0080] The co-administration of active ingredients may be
simultaneous or sequential. Simultaneous administration may be
effected by the compounds being in the same unit dose, or in
individual and discrete unit doses administered at the same or
similar time. Sequential administration may be in any order as
required and typically will require an ongoing physiological effect
of the first or initial active agent to be current when the second
or later active agent is administered, especially where a
cumulative or synergistic effect is desired.
[0081] Preferably the formulation is an oral formulation, more
preferably a capsule formulation.
[0082] Preferably the capsule formulation will comprise consist
essentially of or consist of a compound of formula (I) and silicon
dioxide.
[0083] Preferably the capsule will be a HPMC capsule.
[0084] In an alternatively embodiment the formulation is a
suppository or enema, which can be used to direct the active
ingredient more closely to the disease affected area of the
body.
[0085] Preferably compositions according to the present invention,
oral or otherwise, will comprise 5 mg per kg or less, for example,
0.01 to 4 mg per kg such as 0.5 to 3 mg per kg of the body weight
of the patient of compounds of formula (I).
[0086] For example, formulations may comprise 50 to 500 mg of
compounds of formula (I) per unit dose, more preferably 200 to 400
mg such as 250 mg.
[0087] Thus the invention provides one or more compounds of formula
(I) and/or compositions comprising the same for use in treatment or
the manufacture of a medicament.
[0088] The invention also provides a methods of treatment
comprising administering to a patient in need thereof a
therapeutically effective amount of one or more compounds of
formula (I) or a composition comprising same. These methods are
particularly effective for the diseases listed above.
[0089] Preferably the compounds and compositions according to the
invention will be employed in the treatment of IBD, for example,
colitis such as ulcerative colitis, ulcerative proctitis, distal
colitis; Crohn's disease and/or the prevention of colon cancer.
[0090] In another preferred aspect the compounds and compositions
of the present invention are useful in the maintenance of remission
of said diseases, especially IBD.
[0091] Ultimately, the dose prescribed and intervals at which the
prescribed dose is taken will be at the discretion of the physician
with responsibility for the patient.
[0092] Compounds of formula (I) may be administered, for example,
at doses in the range of 50 to 500 mg, for example, 1 to 4 times
daily such as once or twice daily.
[0093] The compounds and compositions comprising the same have
surprisingly low toxicity. Furthermore, they are advantageous in
that they are more selective, less toxic and/or more efficacious,
for example, in reducing clinical symptoms such as weight loss,
gastrointestinal ulceration and the like, than compounds of a
similar structure.
[0094] Whilst not wishing to be bound by theory it is thought that
the compounds of the present invention are selective thromboxane
synthase inhibitors. Thromboxanes (TX) may play a major pathogenic
role in IBD. TXs are produced in excess not only in inflamed gut
mucosa but also by isolated intestinal and peripheral blood
mononuclear cells and uninflamed bowel in Crohn's disease.
[0095] These isoflavan-4-ones and isoflavan-4-ols and derivatives
thereof are readily obtainable by standard procedures known in the
art. Published International patent applications WO 98/08503,
WO00/49009 and WO 01/17986 (all to Novogen Research Pty Ltd), and
references cited therein, which disclosures are incorporated herein
by reference, also provide useful synthetic methods for the
production of isoflavones, isoflavanones, isoflavan-4-ols and
related isoflavonoid compounds as the starting monomers. A
representative general synthesis is set out in Scheme 1 below.
##STR00006##
[0096] Access to various substitution patterns around both the
benzopyran ring and the pendant phenyl ring of the isflavan-4-ols
is made possible by selecting correspondingly substituted
R5-R8-phenols and R2-R4-phenyl acetic acid starting materials. The
ring cyclisation reactions may conveniently be preformed with
R1-substituted methanesulfonyl chloride reactants as would be known
to those skilled in the art. The reduction reaction is successfully
performed with Pd-C or Pd-alumina in an alcoholic solvent in the
presence of hydrogen. The conditions can dictate the end product of
reduction. The person skilled in the art will be aware that other
standard methods of alkylation, cyclisation, hydrogenation and/or
reduction can be employed as appropriate.
[0097] The protection of functional groups on the compounds and
derivatives of the present invention can be carried out by well
established methods in the art, for example as described in T. W.
Greene, Protective Groups in Organic Synthesis, John Wiley &
Sons, New York, 1981.
[0098] Hydroxyl protecting groups include but are not limited to
carboxylic acid esters, eg acetate esters, aryl esters such as
benzoate, acetals/ketals such as acetonide and benzylidene, ethers
such as ortho-benzyl and para-methoxy benzyl ether,
tetrahydropyranyl ether and silyl ethers such as tert-butyldimethyl
silyl ether.
[0099] Protecting groups can be removed by, for example, acid or
base catalysed hydrolysis or reduction, for example, hydrogenation.
Silyl ethers may require hydrogen fluoride or tetrabutylammonium
fluoride to be cleaved.
[0100] Specifically, compound of formula (I) can be prepared, for
example, by the following methods. Herein the substitution pattern
is of a preferred embodiment, being the 8-substituted compounds,
however it will be understood that by varying the starting
materials different substitution patterns may be readily
obtained.
[0101] Isoflavanones and isoflavan-4-ols are readily obtainable by
the selective reduction of isoflavones (see Novogen WO00/49009).
Reducing agents are well known to persons skilled in the art and
can include hydride sources like borohydrides and alkali metal
borohydrides, but would include hydrogen in catalytic hydrogenation
where a suitable catalyst such as palladium on carbon or platinum
oxide is employed. Other suitable hydride sources include sodium
triacetoxyborohydride and sodium cyanoborohydride.
[0102] Preferably the double bond is reduced by hydrogenation.
Preferably the catalyst employed is palladium on carbon or platinum
oxide.
[0103] Resorcinol is the key starting material in Scheme 1 above.
Substituted resorcinols are readily available or can be synthesised
as required. For example, resorcinol may be methylated or
halogenated by treating resorcinol with a halogenating agent, for
example in the presence of a suitable solvent. Suitable
halogenating agents include iodine, bromine, chlorine, or fluorine.
It may be necessary to perform the halogenation step at a reduced
temperature, for example 0.degree. C.
[0104] The compounds of formuala (I) and all intermediate compounds
also form aspects of the invention, as do processes for the
preparation of all the compounds described herein.
[0105] The compound 3,4-epoxy-4',7-dihydroxyisoflavan also shows
anti inflammatory activity. It is obtained by dehydration
(P.sub.2O.sub.5) of 4',7-dihydroxyisoflavan-4-ol to
4',7-dihydroxyisoflav-3-ene. Epoxidation (m-CPBA) of the double
bond affords the epoxide.
[0106] The inventors have found that inhibition of the eicosanoid
thromboxane (TX), by either selectively or non-selectively
inhibiting thromboxane synthase (TXS), has the pleiotropic
functions of:
[0107] 1. anti-inflammatory activity, manifest by a reduction in
the classical clinical signs of pain, erythema and swelling, as
well as
[0108] 2. cardioprotective activity, either by reducing
hypertension, modifying serum lipids and/or by having
anti-atherosclerotic activity, or by eliminating or reducing the
prothrombotic effects of COX-2 inhibition, which contribute to
increased cardiovascular risk.
[0109] 3. gut protective effects via either increased production or
reduced inhibition of prostaglandins, thus avoiding the side
effects of NSAIDS
[0110] Anti-Inflammatory Activity
[0111] Prostaglandins eg PGE.sub.2 and PGI.sub.2 and thromboxanes
(TXs) eg TXA.sub.2 are members of a family of fatty acid
derivatives known as eicosanoids (Penglis et al. 2000). They are
involved in both normal physiology and inflammatory responses, but
have opposing effects on eg cytokine release and platelet
aggregation. Release of arachidonic acid (AA) from membrane
phospholipids provides the primary substrate for eicosanoid
synthesis. Action of the cyclooxygenase (COX) enzymes, regardless
of isotype, causes synthesis of the intermediate prostaglandin
PGH.sub.2, the common precursor for PGE.sub.2, PGI.sub.2 and
TXA.sub.2.
[0112] The inventors have found that inhibition of thromboxane
itself is an anti-inflammatory strategy.
[0113] 1. Prostanoids play an important modulatory role in the
immune response through complex interactions with leucocytes and
parenchymal cells in the inflamed organ. They can produce both pro-
and anti-inflammatory actions depending upon the inflammatory
stimulus, the predominant prostanoid produced, and the profile of
prostanoid receptor expression (Tilley et al. 2001).
[0114] 2. The thromboxane TXA.sub.2 is a prostanoid with actions
that appear to be primarily pro-inflammatory (Thomas et al. 2003).
Enhanced production of TX has been implicated in the pathogenesis
of various immune-mediated diseases eg lupus nephritis.
[0115] 3. The inventors have demonstrated that the isoflavonoid
compounds of the invention and which are TXS inhibitors have marked
anti-inflammatory activity according to an novel and selective mode
of action in a variety of animal models.
[0116] 4. Thromboxanes play a major pathogenic role in inflammatory
bowel disease (IBD). TXs are produced in excess not only in
inflamed mucosa but also in Crohn's disease by uninflamed bowel and
by isolated intestinal and peripheral blood mononuclear cells.
Their cellular source is likely to include platelets, neutrophils,
endothelial and epithelial cells as well as mononuclear cells
(Hawkey et al. 1985; Rampton and Collins 1993; McCartney et al.
1999; Carty et al. 2000; Carty et al. 2002). The pro-inflammatory
effects of TXs are both direct (diapedesis and activation of
neutrophils, mucosal ulceration, reduction of suppressor T-cell
activity) and indirect (vasoconstriction, platelet activation). PGs
are thought to be protective to gastrointestinal mucosa (Carty et
al. 2000). Sulfasalazine, a compound frequently administered in the
treatment of chronic IBD, as well as one of its main metabolites,
sulfapyridine, have been demonstrated to inhibit synthesis of
TXB.sub.2 while enhancing synthesis of PGF.sub.2a or PGE.sub.2,
respectively (Hawkey et al. 1985). In other words, they would
appear to have some level of TX synthase inhibition
[0117] 5. Inhibition of TX synthase leads to reduced formation of
TXs, and because there is an increased availability of the
substrate PGH.sub.2 for PG synthase, an increase in synthesis of
PGs, (Carty et al. 2000; Penglis et al. 2000). An increase in
PGE.sub.2 can exert anti-inflammatory effects. For example:
[0118] a. PGE.sub.2 has been reported to attenuate some acute
inflammatory responses, in particular those initiated by mast cell
degranulation (Raud et al. 1988).
[0119] b. PGE.sub.2 suppresses, whereas TXA.sub.2 increases
TNF.sub..alpha. and IL-1.beta. (Caughey et al. 1997). Inhibition of
TXA.sub.2 is a potential way of inhibiting inflammatory cytokine
production, particularly that of TNF. Currently, biological
therapies which suppress TNF levels (with antibodies or soluble TNF
receptor shave been successful in treating rheumatoid arthritis
which is refractory to, or no longer responsive to other therapies.
A chemical agent which suppressed TNF production and which could be
taken orally would be a great advance. Inhibition of TXA.sub.2
formation may be a means of suppressing production of TNF, a
cytokine which is involved in the signs and symptoms of joint
inflammation and in the longer term degradative phase of joint
inflammation manifest in cartilage degradation, diminution of joint
space and ultimately, joint failure.
[0120] c. PGE.sub.2 inhibits a wide range of T and B cell functions
including inhibition of T lymphocyte activation and proliferation
and Ig production (Tilley et al. 2001). Conversely, TXA.sub.2 may
promote T cell activation and proliferation and facilitate the
development of effector cytolytic T cells (CTLs). Altering this
balance in favour of PG production may facilitate `quenching` of an
inappropriate immune response as occurs in autoimmune disease.
[0121] d. In asthma, PGE.sub.2 promotes vasodilation and increases
vascular permeability (Tilley et al. 2001). As inflammation
progresses, PGE.sub.2 synthesis by macrophages is enhanced due to
increased expression of COX-2 and PGE-synthase. PGE.sub.2 inhibits
leukocyte activation and promotes bronchodilation. TXA.sub.2
synthase inhibitors and thromboxane prostanoid (TP) receptor
antagonists have been developed as anti-asthma drugs (Shi et al.
1998).
[0122] e. In glomerulonephritis there is co-activation of the AA
COX pathway toward synthesis of PGs and TX and of lipoxygenase
pathways toward synthesis of leukotrienes. TXA.sub.2 is the most
abundant eicosanoid synthesized in nephritic glomenili, and
TXA.sub.2 synthase inhibitors (eg Dazmegrel) are now available for
the treatment of glomerulonephritis. In a rat model of nephritis,
Dazmegrel increased PGE.sub.2 synthesis which is useful as
PGE.sub.2 preserves kidney function in glomerulonephritis (Lianos
and Bresnahan 1999).
[0123] f. COX-2-derived prostaglandins are associated with
resolution of inflammation. The profile of PGs being produced in
the inflammatory lesion changes from pro-inflammatory,
PGE.sub.2-dominated during the development of inflammation to
anti-inflammatory, cyclopentenone (cyPG)-dominated during the
resolution of inflammation (Gilroy et al. 1999).
[0124] Cardioprotective Activity
[0125] An inhibition of thromboxane itself is a cardioprotective
strategy, in contrast to other anti-inflammatory agents which
utilise COX inhibition.
[0126] 1. The ratio of PG to TX is dependent on which isoform of
COX (COX-1 or COX-2) is present (Caughey et al. 2001). TXA.sub.2 is
the major COX-1-derived product, but the induction of COX-2 results
in a preferential increase in PGE.sub.2 and PGI.sub.2 synthesis.
Conversely, when COX-2 is inhibited, there is an increase in TX
synthesis.
[0127] 2. TXA.sub.2 is a potent inducer of platelet aggregation.
The increased cardiovascular risk in the rofecoxib (Vioxx) case was
due to the pro-thrombotic consequence of an increase in TX
synthesis caused by selective COX-2 inhibition. Non-selective COX
inhibition also causes some cardiovascular risk, because the
substrate for both TX and PGs, PGH.sub.2 is inhibited by either or
both COX-1 or -2.
[0128] 3. Therefore, if the inhibition of COX is removed from the
anti-inflammatory `equation` entirely, as would happen with
specific TXS inhibition, an inflammatory mediator ie TXA.sub.2 is
inhibited yet there is no increase in cardiovascular risk.
[0129] 4. TXA.sub.2 is also vasoconstrictive, so its inhibition is
likely to contribute to these compounds being anti-hypertensive.
There is a link between angiotensin II and TXA.sub.2 in
cardiovascular function (Dogne et al. 2004). For example, TXA.sub.2
and PGH.sub.2 are involved in angiotensin II-dependent hypertension
by stimulating the contraction of vascular smooth muscle. Moreover,
some commonly used angiotensin II-inhibitors are also thromboxane
receptor (TP) antagonists which inhibit platelet aggregation.
[0130] Gastrointestinal Protective Activity
[0131] Because inhibition of TXS causes an increased availability
of the PG substrate, PGH.sub.2, it is possible that the synthesis
of PGE.sub.2 would be increased, or if not increased, at least not
be inhibited to the extent that occurs with the inhibition of COX.
The use of NSAIDs, which inhibit COX, is associated with
gastrointestinal ulceration, perforation and bleeding, due to the
inhibition of PGE.sub.2.
[0132] Whilst PGs are included in the list of eicosanoids which
mediate the hallmarks of inflammation--pain, swelling and
erythema--they also have a beneficial role in protecting gastric
mucosa from the hydrochloric acid it produces (Vane and Botting
2003). This occurs in a variety of ways:
[0133] 1. Exogenously administered PGs can prevent disruption of
the gastric mucosal barrier, enhance gastric mucosal blood flow and
stimulate mucus and bicarbonate secretion (Miller 1983).
[0134] 2. Inhibition of PGE.sub.2 reduces proliferation of gastric
mucosal cells in vivo (Levi et al. 1990). Consequently, it would
follow that PGE.sub.2 is important in the maintenance of mucosal
integrity.
[0135] 3. Endogenous PGs play a central role in adaptive
cytoprotection induced in the stomach by mild irritants (Robert et
al. 1979). Pre-treatment of cultured gastric mucous cells with
PGE.sub.2 reduced ethanol-caused injury of the cells (Sakamoto et
al. 1993). Furthermore, pre-treatment of gastric mucous cells with
the non-selective COX inhibitor indomethacin enhanced
ethanol-caused injury, suggesting that endogenous PGE.sub.2 is
involved in the cell protection. The receptors activated by
PGE.sub.2 are pharmacologically subdivided into at least four
subtypes (EP.sub.1, EP.sub.2, EP.sub.3, and EP.sub.4) and in vivo,
adaptive gastric cytoprotection is mediated by activation of
EP.sub.1-receptors (Takeuchi et al. 2001).
[0136] 4. The protective role of PGE.sub.2 also involves modulation
of the gastric mucosal microcirculation. Disruption of the balance
between the local release of vasodilator eg PGE.sub.2 and
vasoconstrictor mediators eg endothelium-derived peptide
endothelin-1 (ET-1) is thought to be involved in the pathogenesis
of mucosal injury (Whittle and Lopez-Belmonte 1993).
[0137] PGE.sub.2 reduces radiation-induced apoptosis in the mouse
small intestine and endogenous PGE.sub.2 in the intestine increases
epithelial crypt survival after radiation (Cohn et al. 1997). This
effect is mediated by signalling through EP.sub.2, the
phosphorylation of AKT, and the inhibition of bax translocation
(Tessner et al. 2004).
[0138] It is also surprising the compounds of the present invention
are thought to be chemo-sentising agents, that is they increase the
cytotoxicity of the one or more anticancer drugs co-administered
therewith, and/or radio-sensitising agents by which it is meant
that these compounds either lower the amount of gamma-irradiation
that is required to kill cancerous cells, or they convert cancer
cells from a state of radio-resistance to radio-sensitivity.
[0139] In addition, the compounds of the present invention are
useful in the treatment and prevention of a range of important
human disease including cancers, inflammatory disorders, autoimmune
disorders, cardiovascular disorders, and disorders associated with
estrogen receptor activation.
[0140] The invention is further illustrated by the following
non-limiting Examples and accompanying drawings.
EXAMPLES
Synthetic Methods for Substituted Isoflavanones and
Isoflavan-4-ols
[0141] Condensation of 2-bromoresorcinol with 4-hydroxyphenylacetic
acid--Synthesis of
1-(3-bronio-2,4-dihydroxyphenyl)-2-(4-hydroxyphenyl)-ethanone
##STR00007##
[0142] Borontrifluoride diethyl etherate (30 ml ) was added to a
mixture of 2-bromoresorcinol (5.0 g, 26 mmoles) and
4-hydroxyphenylacetic acid (3.0 g, 26 mmoles) in a round bottom
flask fitted with a condenser and a magnetic stirrer. The mixture
was heated at 100-110.degree. C. for one and a half hour with
stirring. The mixture was then cooled to room temperature and kept
in a freezer overnight. The precipitated solid was filtered and
recrystallised from ethanol to give deoxybenzoin as off-white
crystals. The crystals were then collected and dried in a
desiccator. .sup.1H NMR (CDCl.sub.3): .delta. 4.16 (s, 2H,
CH.sub.2), 6.61 (d, 1H, J 9.0 Hz, ArH), 6.80 (d, 2H, J 8.6 Hz,
ArH), 7.12 (d, 1H, J 8.6 Hz, ArH), 7.75 (d, 1H, J 9.0 Hz, ArH).
Yield: 5.2g, 69%
[0143] Cyclisation of
1-(3-brorno-2,4-dihydroxyphenyl)-2-(4-hydroxyphenyl)-ethanone
--Synthesis of
3-(4-hydroxyphenyl)-7-hydroxy-8-bromo-chromen-4-one
##STR00008##
[0144] Boron trifluoride diethyl etherate (6 ml) was added dropwise
to a solution of 1-(3-bromo-2,4-dihydroxyphenyl)
-2-(4-hydroxyphenyl)-ethanone (2.5 g) in dry dimethyformamide (20
ml) maintained at 50.degree. C. Methanesulfonyl chloride (3.3 ml)
was then added to the solution and the mixture heated at
110.degree. C. for 1 hour. After cooling to room temperature,
hydrochloric acid (2M, 120 ml) was slowly added to the reaction
mixture. The resulting yellow precipitate was filtered, washed
thoroughly with water and dried, .sup.1H NMR (d.sub.6-DMSO):
.delta. 6.79 (d, 2H, J 8.7 Hz, ArH),7.08 (d, 1H, J 8.7 Hz, H6),
7.36 (d, 2H, J 8.7 Hz, ArH), 7.94 (d, 1H, J 8.7 Hz, H5), 8.39 (s,
1H, H2). Yield 2.35 g
[0145] The crude product was used in the next step without any
further purification.
[0146] Acetylation of
3-(4-hydroxyphenyl)-7-hydroxy-8-bromo-chromen-4-one--Synthesis of
3-(4-acetoxyphenyl)-7-acetoxy-8-bromo-chromen-4-one
##STR00009##
[0147] Acetic anhydride (15 ml) and pyridine (2.5 ml) were added to
3-(4-hydroxyphenyl)-7-hydroxy -8-bromo-chromen-4-one (2.35 g) in a
round bottom flask fitted with a condenser and magnetic stirrer.
The mixture was heated to 110.degree. C. for 1 hour. It was then
cooled and left in a refrigerator to crystallise. The white
crystalline solid was filtered and washed several times with water.
Recrystallisation of the crude product from ethanol afforded
3-(4-acetoxyphenyl) -7-acetoxy-8-bromo-chromen-4-one as white
crystalline needles. .sup.1H NMR (CDC13): .delta. 2.32, 2.42 (each
s, 3H, OCOCH.sub.3), 7.17-7.22 (m, 3H, ArH),7.58 (d, 2H, J 8.7 Hz,
ArH), 8.1 (s, 1H, H2), 8.29 (d, 1H, J 9.0 Hz, H5). Yield: 1.5 g
[0148] Hydrogenation of
3-(4-acetoxyphenyl)-7-acetoxy-8-bromo-chromen-4-one--Synthesis of
3-(4-acetoxyphenyl)-7-acetoxy-4-hydroxy-8-bromo-chroman-4-one and
3-(4-acetoxyphenyl) -7-acetoxy-4-hydroxy-8 -bromo-chroman-4-ol
##STR00010##
[0149] A 250 ml round-bottom flask containing a suspension of
3-(4-acetoxyphenyl)-7-acetoxy-8-bromo-chromen-4-one (0.3 g) in
ethyl acetate (80 ml) was evacuated and filled with argon to
provide an inert atmosphere. Platinum oxide (67 mg) was rapidly
added the mixture and the mixture was gently swirled to ensure that
the catalyst is completely covered with ethyl acetate. The mixture
was hydrogenated under standard conditions at room temperature for
3 days. The solution was filtered through a pad of Celite to remove
the catalyst and the solution was evaporated in vacuo to yield a
colourless oil. A tic analysis of the oil indicated it to be
mixture of chroman-4-one and chroman-4-ol. The crude product was
chromatographed on a silica column using dichloromethane/ethyl
acetate (98:2) as the eluent.
[0150] 3-(4-acetoxyphenyl)-7-acetoxy-4-hydroxy-8-bromo-chroman-4-ol
Yield 100 mg
[0151] .sup.1H NMR (CDCl.sub.3): .quadrature. 2.30, 2.35 (each s,
3H, OCOCH.sub.3), 3.32 (dt, 1H, J 3.4 Hz, J 11.7 Hz, H3), 4.48 (m,
1H, H2); 4.65 (dd, 1H, J 10.5 Hz, 11.7 Hz, H2), 4.78 (bs, 1H, H4),
6.75 (d, 1H, J 8.3 Hz, H6), 7.10 (d, 2H, J 8.3 Hz, ArH), 7.25 (d,
1H, J 8.3 Hz, H5), 7.30 (d, 2H, J 8.3 Hz, ArH).
[0152] 3-(4-Acetoxyphenyl)-7-acetoxy-8-bromo-chroman-4-one Yield
170 mg
[0153] .sup.1H NMR (CDCl.sub.3): 2.29, 2.38 (each s, 3H,
OCOCH.sub.3), 4.02 (dd, 1H, J 5.7 and 8.7 Hz, H3), 4.60 (m, 2H,
H2), 6.88 (d, 1H, J 8.6 Hz, H6), 7.09 (d, 2H, J 8.7 Hz, ArH), 7.29
(d, 2H, J 8.7 Hz, H2), 7.96 (d, 1H, J 8.6 Hz, H5).
[0154] 3-(4-hydroxyphenyl)-7-hydroxy-8-bromo-chroman-4-one
##STR00011##
[0155] Imidazole (0.50 g) was added to a suspension of
3-(4-acetoxyphenyl)-7-acetoxy-8-bromo-chroman-4-one (0.17) in
absolute ethanol (7.0 ml) and the mixture was refluxed for 1 h
under argon. The solution was concentrated under reduced pressure
and distilled water (5 ml) and hydrochloric acid (1M, 5 ml)was
added to the residue. The mixture was left overnight in the fridge
and the resulting creamy white solid was filtered, washed
thoroughly with water and dried in a vacuum desiccator.
[0156] Yield 130 mg
[0157] .sup.1H NMR (d.sub.6-acetone): .quadrature. 3.93 (t, 1H, J
6.8 Hz, H3), 4.60 (d, 2H, J 7.2 Hz, H2), 6.75-6.81 (m, 3H, ArH),
7.13 (d, 2H, J 8.7 Hz, ArH), 7.72 (d, 1H, J 8.6 Hz, H5), 8.44 (bs,
1H, OH), 10.2 (bs, 1H, OH).
[0158] A similar deprotection of the acetoxy isoflavan-4-one leads
to the corresponding hydroxy compound. By varying the substitution
pattern of the starting resorcinol, the other compounds of the
invention were synthesised. All compounds synthesised showed
spectral data consistent with the assigned structures.
[0159] The synthesis of Compound 2 started with the condensation of
pyrogallol with 4-hydroxyphenylacetic acid. Further reaction as
above afforded compound 2. Compounds 3, 4 and 5 begin with
2-methylresorcinol, 4-chlororesorcinol and 5-methylresorcinol
respectively.
ANTI-INFLAMMATORY ACTIVITY
[0160] INHIBITION OF EICOSANOIDS INDUCED IN HUMAN MONOCYTES
[0161] Human peripheral blood monocytes (from three separate
individuals) were isolated from buffy coats by lymphoprep gradient
separation of mononuclear cells followed by counter-current
centrifugal elutriation (Demasi et al. 2000). Test compounds were
dissolved in DMSO and added to fresh monocytes to achieve
concentrations of 0, 10 and 100 .mu.M. After 30 min,
lipopolysaccharide (LPS) was added to achieve a final concentration
of 200 ng/mL. After incubation for 18 hrs at 37.degree. C. in 5%
CO.sub.2, supernatants were removed and PGE.sub.2 and TXB.sub.2
(the stable hydrolysis product of TXA.sub.2) production were
measured by radioimmunoassay (RIA). Except for Compound 4, which
was only tested once, each compound was examined in three different
assays. ANOVA followed by Newman-Keuls multiple comparisons test
was used to examine differences between doses and the control
values. A statistical significance level of 0.05 was used and
differences from control values.
[0162] The results obtained at a concentration of 10 .mu.M are
presented in FIG. 1 (PGE.sub.2) and FIG. 2 TXB.sub.2). Because
PGE.sub.2 was either induced in a dose responsive manner by the
test compounds or the effect on PGE.sub.2 was minimal, and
generally TXB.sub.2 was inhibited, these results suggested that the
compounds were thromboxane (TX) synthase inhibitors.
[0163] To examine effects of test compounds directly on TX synthase
(TXS), a different assay system was used. Whereas arachidonic acid
(AA) is the substrate for COX, PGH.sub.2 is the substrate for TX
synthase. To examine directly the effect of these compounds on
TXS,
##STR00012##
[0164] Test compounds at 0 .mu.M, 1 .mu.M, 10 .mu.M and 100 .mu.M
(in DMSO) were incubated with undifferentiated U937 cells
(6.times.10.sup.6/ml) for 30 min at 37.degree. C., after which
PGH.sub.2 (5 .mu.M) was added. After incubation for 10 min, the
reaction was terminated by centrifugation and the supernatants were
collected and TXB.sub.2 production was measured in triplicate by
RIA. ANOVA followed by Newman-Keuls multiple comparisons test was
used to examine differences between doses and the control values. A
statistical significance level of 0.05 was used and differences
from control values are indicated by an asterisk (*). The results
are shown in FIG. 3.
[0165] Because none of these compounds inhibited PGE.sub.2 in the
initial assays it can be inferred that the inhibition demonstrated
in these assays is specific to TXS. These results indicate that
Compounds 1, 2, 4 and 5 are selective thromboxane synthase
inhibitors, whereas the thromoboxane synthase activity displayed by
Compound 3 was less specific.
[0166] INHIBITION OF NF.kappa.B
[0167] Nuclear factor KB (NF-.kappa.B) is a nuclear transcription
factor that regulates expression of a large number of genes
(including COX-2) that are critical for the regulation of
inflammation and various autoimmune diseases, as well as apoptosis,
viral replication and tumorigenesis. NF-.kappa.B is activated by a
variety of stimuli that include growth factors, cytokines,
lymphokines, UV radiation, pharmacological agents and stress.
Inhibition of NF.kappa.B by a test compound suggests that the
compound will have anti-inflammatory activity in vivo.
[0168] Human macrophage THP-1 cells, modified to include the
.beta.-lactamase reporter gene, were seeded into wells of a 96-well
plate (50.times.10.sup.3 cells/well) in the presence of RPMI 1640
medium (70 .mu.l ). TNF.alpha. (7.5 ng/ml) and human sera (final
concentration of 20%) were added to each well. Plates were
incubated for 5 h at 37.degree. C. to allow for NFkB-stimulated
.beta.-lactamase production. LiveBLAzer.TM. FRET B/G Substrate
(CCF4-AM) was then added to assay for .beta.-lactamase activity.
Once CCFA-AM enters a cell, it is converted to negatively charged
CCF4 by endogenous esterases. Excitation of this substrate at 409nm
leads to efficient FRET between the coumarin and fluorescein
moieties, resulting in a green fluorescence detectable at 530 nm.
The presence of .beta.-lactamase leads to cleavage of CCF4 and
results in a loss of FRET, resulting in a robust blue fluorescent
signal detectable at 460 nm. Thus, activity of beta-lactamase (a
marker of NF.kappa.B-promoter activity) is measured as a product to
substrate ratio (blue/green fluorescence ratio: 460 nm/530 nm).
[0169] Compounds 2, 3 and 5 significantly (p<0.001) inhibited
NFKB when compared to vehicle control as shown in FIG. 4.
[0170] INHIBITION OF NITRIC OXIDE
[0171] Nitric oxide (NO) plays an important regulatory/modulatory
role in a variety of inflammatory conditions (Blantz and Munger
2002). Large amounts of NO are produced at sites of inflammation
through the action of inducible nitric oxide synthase (iNOS)
present in both infiltrating leucocytes and activated, resident
tissue cells (Evans 1995). It can be vasodilatory and interfere
with adhesion molecules to prevent neutrophil adhesion. NO release
may also lead to the formation of highly reactive species such as
peroxynitrite and stable nitrosothiols and may cause mitochondrial
damage and nitration of protein tyrosine residues. Excessive NO is
produced during the course of a variety of inflammatory and
autoimmune diseases, including systemic lupus erythematosus (SLE),
Sjogren's syndrome (SS), vasculitis, rheumatoid arthritis, and
osteoarthritis (Clancy et al. 1998). Consequently, its inhibition
is considered to be an anti-inflammatory strategy.
[0172] Exposure of various cells (e.g. macrophages and smooth
muscle cells) to bacterial LPS and proinflammatory cytokines
induces iNOS which generates NO. NO production can be indirectly
quantified by measurement of nitrite (NO2-), a stable breakdown
product of NO. The mouse macrophage cell line RAW 264.7 was
cultured in DMEM supplemented with FCS, 2 mM glutamine and 50 U/ml
penicillin/streptomycin. Cells were treated with either test
compound at 10 .mu.M (in 0.125% DMSO) or vehicle alone, together
with 10 ng/ml LPS. After incubation for 24hrs, culture media was
collected and analysed immediately for NO concentration using the
Griess reaction (Eigler et al. 1995).
[0173] Compounds 1, 3a and 3b inhibited NO synthesis at 10 .mu.M
whereas Compounds 2 and 4 induced it. Compound 5 was not
examined.
TABLE-US-00001 TABLE 1 Effect of test compounds on NO synthesis
difference between Compound effect compared with test group and (10
.mu.M) vehicle control vehicle Compound 1 .dwnarw.by 9% n.s.
Compound 2 .uparw.by 5% p = 0.023 Compound 3a .dwnarw.by 7% n.s.
Compound 3b .dwnarw.by 34% p = 0.0001 Compound 4 .uparw.by 31% p
< 0.0001
[0174] These results indicate yet another mechanism by which the
compounds are anti-inflammatory.
[0175] ANTI-INFLAMMATORY ACTIVITY IN MURINE EAR INFLAMMATION
[0176] Compounds were examined for their ability to inhibit ear
swelling in mice induced by the topical application of arachidonic
acid (AA). The inflammatory response due AA, the immediate
precursor of the eicosanoids, is due to formation of AA metabolites
via both the cyclooxygenase (COX) and lipoxygenase (LOX) pathways
(Young et al. 1984). AA induces an increase in both PGE.sub.2 and
LTC.sub.4 synthesis which precedes the increase in ear thickness
(Opas et al. 1985; Chang et al. 1986).
[0177] Female BALB/c mice (ARC, WA, Australia), weighing 15-21g
were injected with Compound 2 delivered in polyethylene glycol
(PEG) 400:phosphate buffered saline (PBS) 1:1 intraperitoneally
(i/p) at a dose of 25mg/kg either 30min prior to or immediately
before the application of AA to the ears. Mice were anaesthetised
using isoflurane and baseline thickness of both ears was measured
using a spring micrometer. Each mouse received a total of 20 .mu.L
of AA in ethanol (50 mg/ml ) applied to the inner and outer
surfaces of each pinna (i.e.
[0178] 0.5 mg AA per ear). Mice were anaesthetised again to
remeasure the ears at 1 hr post-AA application.
[0179] The difference in ear swelling pre- and post-application of
AA for each ear was calculated, and the average for the two ears of
each mouse calculated. The difference in mean swelling of each test
group compared to the group given vehicle alone was calculated
using a two-tailed unpaired t test (Prism 4, GraphPad
Software).
[0180] All compounds resduced AA-induced ear inflammation.
Treatment with Compounds 2, 3a and 3b resulted in a significant
reduction in ear thickness compared with treatment with vehicle
alone.
TABLE-US-00002 TABLE 2 Change in ear thickness in response to the
application of AA difference between Increase in ear thickness test
group and Compound (mean .+-. SD, .times. 0.01 mm) vehicle Vehicle
14.5 .+-. 4.7 -- Compound 1 9.5 .+-. 2.3 p = 0.3221 Compound 2 6.5
.+-. 2.9 p = 0.0440 Compound 3a 6.7 .+-. 2.1 p = 0.0362 Compound 3b
7.1 .+-. 3.0 p = 0.0268 Compound 4 9.2 .+-. 3.6 p = 0.3381
[0181] These data demonstrate the anti-inflammatory activity of the
test compounds in vivo.
[0182] ANTI-INFLAMMATORY ACTIVITY IN MURINE MODEL OF THE UV
IRRADIATION-INDUCED SKIN OEDEMA
[0183] Acute exposure of mammalian skin to UV irradiation causes an
inflammatory reaction manifested by erythema and oedema. This
reaction is mediated in part by pro-inflammatory prostaglandins
(PGD.sub.2, PGE.sub.2, PGF.sub.2.alpha., and possibly PGI.sub.2)
and leucotrienes, as well as the generation of reactive free
radicals and reactive oxygen species (Sondergaard et al. 1985;
Gonzalez and Pathak 1996; Widyarini et al. 2001).
[0184] Groups of 4-5 female Skh:hr-1 albino mice were irradiated
with 1.times.3 MEdD (minimal edematous dose) of solar simulated UV
radiation (SSUV). Solar-simulated ultraviolet radiation (SSUV) was
provided by a planar bank of 6 UVA tube (Hitachi 40W F4OT 10/BL,
black light) and one UVB tube (Philips TL 40W/12RS) with radiation
filtered through a sheet of 0.125 mm cellulose acetate (Eastman
Chemical Products, Kingport, Tenn., USA) to give
2.96.times.10-4W/cm2 UVA and 1.59.times.10-5W/cm2 UVB. During
irradiation, the cages were rotated below the lights to reduce the
variation in radiation intensity in different positions.
[0185] Either test compound (0.2 ml of a 20 .mu.M solution) or
vehicle (propylene glycol/ethanol/water 1:2:1) was applied to the
irradiated dorsal skin at 30 min, 2 h and 4 h post-irradiation.
Dorsal skin fold measurements were made with a spring micrometer
prior to and at 24 hr and 48 h post-UV exposure. The difference in
skin thickness pre- and post-exposure to UVR was calculated for
each mouse, and the differences examined between test compound and
vehicle control were analysed using an unpaired two-tailed t
test.
[0186] Skin fold thickening was evident at 24 hrs post-UV
irradiation and peaked at 48 hrs, the last time point measured.
Even though test compounds were applied only three times post-UV
irradiation, and dosing was completed 20 hrs prior to the first
skin fold measurement, most of the compounds were active in
reducing UV-induced inflammation, as highlighted in the tables and
graphs below.
[0187] Topical administration of all compounds demonstrated a
tendency to reduce UV-induced inflammation. Compound 3 a
significantly inhibited skin oedema at both time points. Compounds
1 and 2 significantly inhibited at 48 hrs.
TABLE-US-00003 TABLE 3 The change in dorsal skin fold thickness at
24 hrs post-irradiation difference between Increase in skin
thickness test group and Compound (mean .+-. SD, .times. 0.01 mm)
vehicle Vehicle 78 .+-. 23 -- Compound 1 54 .+-. 20 p = 0.0579
Compound 2 68 .+-. 24 p = 0.4275 Compound 3a 32 .+-. 18 p = 0.0023
Compound 3b 56 .+-. 14 p = 0.0671 Compound 4 63 .+-. 22 p =
0.2407
TABLE-US-00004 TABLE 4 The change in dorsal skin fold thickness at
48 hrs post-irradiation difference between Increase in skin
thickness test group and Compound (mean .+-. SD, .times. 0.01 mm)
vehicle Vehicle 147 .+-. 37 -- Compound 1 91 .+-. 7.6 p = 0.0041
Compound 2 105 .+-. 22 p = 0.0299 Compound 3a 69 .+-. 24 p = 0.0011
Compound 3b 113 .+-. 25 p = 0.0746 Compound 4 145 .+-. 11 p =
0.9055
[0188] These results clearly demonstrate the anti-inflammatory
activity of Compounds 1, 2 and 3a. Even though they were
administered topically and within a short interval after the
induction of inflammation, their effects were still evident up to
48 hrs later.
[0189] ANTI-INFLAMMATORY ACTIVITY IN THE RAT AIR POUCH ASSAY
[0190] An alternative assay used to measure anti-inflammatory
efficacy is the air pouch model which involves the repeated
subcutaneous injection of air into the dorsum of rats followed 24 h
later by the intrapouch injection of an inflammatory stimulus
(Gilroy et al. 1998).
[0191] Air pouches were raised on the dorsum of female Dark Agouti
rats, approximately 7 weeks of age. To promote the formation of a
cellular membrane lining the inside of each pouch, the pouches were
maintained by re-inflating on days 2 and 5 after the initial
injection of air. On re-inflation, the pouch was first deflated to
ensure the needle was positioned correctly, before being
re-inflated with 2 mL of sterile air. Using this protocol, the
pouches remained inflated until use on day 7, when they were
injected with 0.5 ml of either test compound at 100 .mu.M or
vehicle control. After 15 min, air pouches were injected with
serum-treated zymozan (STZ -500 .mu.g). Lavage of the air pouch
(4.times.2 ml lavages) was performed at 4h and leucocytes counted,
after which the rats were killed, the air pouch excised and
processed in formalin for histology. The sections were blinded to
the person counting. Using a graticule with 100 squares and the 40x
objective, the number of polymorphs were counted in the pouch
lining at 10 different and non-adjacent sites. Group sizes were 5-6
rats. Data were analysed for statistical significance within each
experiment and using an unpaired t test.
[0192] In general, there was concordance between the two separate
measures of the extent of the inflammatory infiltrate (the numbers
of leucocytes in lavage fluid and polymorphs in tissue sections).
There was also concordance between the two measures for the effects
of the test compounds, which strengthens the validity of each data
set.
[0193] Most compounds tested demonstrated anti-inflammatory
activity. At 1mM application, all compounds except Compounds 1 and
3a significantly suppressed the number of leucocytes in the lavage
fluid. Compounds 2, 3a and 4 suppressed the number of polymorphs in
the tissue sections.
TABLE-US-00005 TABLE 5 Effect of instilling test compounds into
inflammed rat air pouches Polymorphs in Leucocytes in lavage fluid
tissue sections (.times.10.sup.-7) (per 100 squares) Compound 1
0.89 .+-. 0.42 31.0 .+-. 7.25 Vehicle 0.87 .+-. 0.28 31.1 .+-. 10.9
p = 0.9181 p = 0.9921 Compound 2 0.68 .+-. 0.36 14.3 .+-. 5.7
Vehicle.sup.a 1.41 .+-. 0.25 28.9 .+-. 6.6 p = 0.002.sup.b p =
0.002 Compound 3a 0.47 .+-. 0.4 17.9 .+-. 14.2 Vehicle 1.72 .+-.
1.3 27.0 .+-. 17.9 p = 0.1124 p = 0.2196 Compound 3b 0.66 .+-. 0.23
ND Vehicle 1.22 .+-. 0.41 p = 0.0156 Compound 4 1.00 .+-. 0.38 23.4
.+-. 8.8 Vehicle 2.23 .+-. 1.08 34.9 .+-. 14.6 p = 0.025 p = 0.13;
p = 0.065 (1-tailed) .sup.aControl was DMSO/PBS, the vehicle for
the test compounds. The ratio of DMSO/PBS was 1:100. .sup.bunpaired
t-test; 2-tailed unless otherwise indicated
[0194] These results clearly demonstrate the anti-inflammatory
activity of Compounds 2, 3a, 3b and 4 in vivo.
[0195] ANTI-INFLAMMATORY ACTIVITY IN THE MURINE MODEL OF
DSS-INDUCED COLITIS
[0196] Methods
[0197] Female BALB/c mice, 6-7 weeks old, were housed for a minimum
of one week before any experimental work began. To induce colitis,
dextran sulphate sodium (DSS-molecular weight 40 kD, TdB
Consultancy AB, Uppsala, Sweden (Dieleman et al. 1994), was
administered fresh daily in the drinking water at a concentration
of 4.5-5% for 5 days (Okayasu et al. 1990), after which water alone
was given. This regime induced mild colitis, evidenced by diarrhoea
and rectal bleeding, the severity of which was scored, as well as a
reduction in the length of the colon. Histologically, colonic
inflammation was evidenced by ulceration and an infiltration of
neutrophils into the mucosa, which was also scored. The systemic
effect of the colitis causes weight loss, which was also
monitored.
[0198] Results
[0199] In a series of experiments, oral administration of either
Compound 1, Compound 2, Compound 3a, Compound 3b or Compound 4
reduced colonic inflammation. Importantly, the dose rate at which
these compounds were active was relatively low--1.0-1.25mg/kg,
given as a single oral dose per day.
[0200] Experiment I
[0201] Either vehicle (PEG 400:PBS 1:1) Compounds 1 or 4 were
administered at a dose rate of lmg/kg throughout the experiment
until the mice were killed at day 17. Both compounds decreased
colitis. In this assay, Compound 1 offered the better
anti-inflammatory activity (based on a reduction in colonic
ulceration, a delay in the onset of clinical signs, a reduction in
the clinical score, a reduction in the occurrence and severity of
clinical signs, reduced body weight loss and reduced time to
recovery) than Compound 2.
[0202] The mean histology scores are shown FIG. 5, suggesting less
colitis in those mice treated with Compounds 1 and 4. There was
statistically significant (p=0.0135) less ulceration of the colonic
epithelium from those mice given Compound 1 compared with those
given vehicle alone.
[0203] The colonic inflammation induced by ingestion of DSS causes
shortening of the colon, and thus protection conferred by test
agents might be evidenced by a reduction in this shortening. There
was a trend towards the colons from the mice given Compound 1
(p=0.1922) or Compound 4 (p=0.0542) to be longer than those from
the mice given vehicle alone. See FIG. 6.
[0204] The clinical signs appeared in several mice in the groups
given vehicle at day 5, but were not evident until day 6 in the
groups given either Compounds 1 and 4, suggesting that those
compounds may have delayed the onset of colitis. By day 11, the
mean clinical score for the groups given either Compounds 1 and 4
was significantly lower (p=0.017 and p=0.043 respectively) than
that of the group given vehicle alone. These results suggest that
both Compounds 1 and 4 were anti-inflammatory. See FIG. 7.
[0205] The maximum mean weight loss was only 1% for Compound 4 and
2% for Compound 1 but 5% for the group given vehicle alone. The
switch from mean body weight loss to mean body weight gain for a
particular group suggests in part, that resolution of colitis for
that group has occurred. This occurred at day 10 for the group
given Compound 1, at day 12 for Compound 4, but not until day 15
for the vehicle. See FIG. 8.
[0206] Experiment 2
[0207] Either Compounds 1 and 2, at a dose of lmg/kg or vehicle
were administered for 10 days prior to DSS being added to the
drinking water to induce colitis, after which test compound was
stopped. DSS was administered for 5 days and the mice killed 8 days
later (day 23). Inflammation was reduced by both compounds, even
though they were administered only prior to but not during its
induction. See FIG. 9.
[0208] The amount and severity of mucosal ulceration was greater in
the vehicle-treated group, but not significantly so.
[0209] Whilst there was a trend, there were no significant
differences in colon length between either of the test groups and
vehicle. There was a significant difference between the colon
lengths of the mice which were not given DSS and vehicle group
(p=0.0414). However, the colons of the Compound 1-treated groups
were not statistically different in length from those of the
control group, suggesting that Compound I protected against the
inflammatory effects of DSS ingestion to some extent. See FIG.
10.
[0210] One mouse in the group treated with vehicle died at day 18
whereas no mice died in either of the treatment groups. Although
not statistically significant when compared with the
vehicle-treated group, there was solid evidence of a reduction in
the clinical signs of colitis in both treatment groups. See FIG.
11.
[0211] Administration of Compound 2, and to a lesser extent
Compound 1 reduced the body weight loss associated with DSS-induced
colonic inflammation. By day 22, the mice receiving Compound 2
weighed significantly more than those receiving vehicle (p=0.0291).
See FIG. 12.
[0212] Experiment 3
[0213] Either the cis or the trans isomers of Compound 3 (Compound
3a and 3b respectively) at 1.25 mg/kg or vehicle were administered
throughout the experiment until the mice were killed at day 17.
Based on the clinical indicators, colonic length and histological
changes, dosing with Compound 3a and to a lesser extent Compound 3b
reduced colonic inflammation and hastened its resolution.
[0214] Ulceration of the posterior colon was not observed in the
group given Compound 3a and only in 1/8 mice given Compound 3b.
However, ulceration was observed in 3/8 mice given vehicle alone.
The degree of mucosal damage for those mice given Compound 3a was
significantly lower (p=0.018) than that experienced by the
vehicle-treated group. See FIG. 13.
[0215] The colons from the group given Compound 3a were
significantly longer (p=0.039) than those from the group given
vehicle alone. See FIG. 14.
[0216] The mean score of the group receiving Compound 3a was
significantly lower than that of the vehicle group throughout days
8 (p=0.0078), 9 (p=0.0326), 10 (p=0.0386), and 11 (p=0.0252).
However, as the colitis began to resolve in the vehicle-treated
mice, that difference was less evident, and lost statistical
significance. However, by the final days of the experiment, the
mean clinical score was again significantly lower for the group
receiving Compound 3a on both days 15 (p=0.0033) and 16 (p=0.0016).
This suggested Compound 3a exerted an anti-inflammatory effect and
hastened the resolution of inflammation.
[0217] Similarly, the mean score of the group receiving Compound 3b
was significantly lower than that of the vehicle group throughout
days 8 (p=0.0055), 9 (p=0.0057) and 11 (p=0.0252). The mean
clinical score was again significantly lower for the group
receiving Compound 3b on 17 (p=0.0177). This suggested that oral
dosing with Compound 3b also had an anti-inflammatory effect and
hastened the resolution of inflammation. See FIG. 15.
[0218] Whilst the trends were clear, there were no statistically
significant differences in body weight change between the groups
either isomer of Compound 3 and the group given vehicle. The
maximum mean weight loss was less than 2% for the groups given
Compound 3a, whereas it was nearly 5% for the group given vehicle
alone. The switch from mean body weight loss to mean body weight
gain occurred relatively quickly at day 4 for Compound 3a but not
in a substantial way at all for the vehicle group. See FIG. 16.
[0219] Experiment 4
[0220] Groups of mice (n =5) were dosed once daily with Compound 1
1 mg/kg or vehicle throughout the experiment, and killed either on
day 6, 9 or 13. Colons were removed, opened longitudinally and
washed in PBS with pen/strep (100 U/ml/100 .mu.g/ml, Invitrogen)
with added fungizone (2.5 ug/ml, Invitrogen). They were then cut
into small portions and approximately 100 mg colon was placed into
one well of a 24-well plate either in the presence or absence of
ConA (10 m/ml, Sigma) containing 1 ml modified RPMIc (pen/strep and
fungizone as above) for 18-24 hours. Supernatant was collected,
centrifuged and stored at -80.degree. C. PGE.sub.2 and TXB.sub.2 in
the culture supernatants were measured in triplicate by ELISA
(Cayman Chemical).
[0221] By culturing the colons in the absence of mitogenic
stimulation, an assessment of the inflammatory mediators being
produced currently can be made. The inflamed colons from mice
exposed to DSS tend to produce more inflammatory mediators than
healthy, uninflamed colons (Dieleman et al. 1998; Tomoyose et al.
1998; Morteau et al. 2000; Micallef et al. 2002). In this
experiment, where DSS had been ingested, the colons produced
significantly more PGE, at day 9 (p<0.05) and more TXB.sub.2 at
day 6 (p<0.05), when compared with the colons from healthy mice
without colitis. There were, however, trends towards the colons of
mice given DSS to produce more PGE.sub.2 and TXB.sub.2 throughout
the entire experiment, particularly in the early phase. The effect
of Compound 1 at ameliorating these inflammatory responses was
assessed by comparing results from mice given DSS and treated with
Compound 1 with those given DSS and treated with vehicle alone. The
colons from the Compound I -treated mice tended to produced less
PGE.sub.2 at day 9 and day 13, and less TXB.sub.2 at day 6 and day
9 (p<0.05) see FIG. 17.
[0222] When colons were cultured in the presence of a mitogen,
their ability to respond to inflammatory/mitogenic stimuli was
being evaluated. Where DSS had been ingested, the colons produced
significantly more PGE.sub.2 at day 9 (p<0.05) at day 6 in
response to ConA, when compared with the colons from healthy mice
without colitis. Again, there were trends towards the stimulated
colons from mice given DSS to produce more PGE.sub.2 and TXB.sub.2
throughout the entire experiment, particularly in the early phase.
By day 13, there were no differences between healthy and inflamed
colons, suggesting that the acute inflammation caused by DSS had
begun to resolve. There was a trend for treatment with Compound 1
to reduce these responses, although not significantly so. See FIG.
18.
[0223] These results demonstrate that the anti-inflammatory effect
of oral dosing with Compound 1 in the colitis modelwas probably due
to its effect at attenuating eicosanoid synthesis by the inflamed
colon.
[0224] CARDIOPROTECTIVE ACTIVITY VASODILATORY ACTIVITY IN THE RAT
AORTIC RING ASSAY
[0225] The vasodilatory capacity of test compounds was examined ex
vivo using the rat aortic ring assay. The addition of noradrenaline
to the test bath causes the rings to contract, and if that
vasoconstriction is inhibited by a test agent ie it antagonises the
effect of noradrenaline, it suggests that that agent may have
vasodilatory anti-hypertensive activity.
[0226] Methods
[0227] Male Sprague-Dawley rats (250.+-.50 g) were euthanased with
80% CO.sub.2 and 20% O.sub.2. The thoracic aorta was excised and
quickly mounted in organ-baths as described (Chin-Dusting et al.
2001). Full concentration-contractile curves were obtained to
noradrenaline (0.1 nM-10 mM) with and without test compounds
delivered at a concentration of 1 .mu.g/ml. Experiments were
repeated in n=5 different rings from 5 different animals. Only one
compound at any one concentration was tested on any one ring from
any one animal. Sigmoidal dose response curves were fitted for the
data and the logEC.sub.50 calculated (Prism 4, GraphPad Software).
The difference in these values between the presence and absence of
test compound was calculated using a two-tailed paired t test. The
effects of .beta.-oestrodiol and vehicle alone were examined as a
positive and negative control respectively.
[0228] Results
[0229] Compounds 1, 2,4, 5 and the isomeric mixture of Compound 3
were examined in this assay. Compounds 1 and 3 significantly
inhibited the contractile response (logEC.sub.50) of the aortic
ring to noradrenaline compared with vehicle alone. These data
indicate that these compounds, in addition to possessing
anti-inflammatory activity, may have cardiovascular activity as
well. See FIG. 19.
[0230] ABSENCE OF GASTROINTESTINAL TOXICITY
[0231] Compound 1
[0232] Oral toxicity of Compound 1 when administered daily for 11
days was investigated. Compound 1 in CMC 0.1% as administered at
the very high doses of 300 mg/kg and the limit dose of 1000 mg/kg
to groups of four Sprague Dawley rats (two of each gender). Vehicle
was administered to a third group. See FIG. 20.
[0233] The formulation of Compound 1 in CMC 0.1% was stable over a
seven day period at both 4.degree. C., 25.degree. C. and 40.degree.
C. Fresh batches of Compound 1 were supplied after day seven.
[0234] Bodyweights were measured immediately before administration,
then at day eight and again at culling on day 12. All rats were
observed daily for signs of toxicity throughout the experimental
period. At necropsy, gross examination of the external surface of
the body, all orifices and the thoracic and abdominal cavities and
their contents was carried out. Liver, kidney, adrenals, spleen and
gonads were weighed. Terminal haematology, serum biochemistry and
histology of the liver, spleen and bone marrow was done.
[0235] There was no evidence of toxicity of any kind, in particular
gastrointestinal disturbance, weight loss, coagulopathy, anaemia or
other blood dyscrasia which would suggest gastrointestinal
bleeding.
[0236] Compound 2
[0237] Compound 2 at 5 mg/kg (n=3) or vehicle (PEG 400:PBS 1:1;
n=2) was administered via gavage to female BALB/c mice for 10 days.
There were no adverse clinical signs in either group, and body
weight changes were similar. There were no gross pathological
changes evident at necropsy. Histology of caecum and colon were
normal. See FIG. 21.
[0238] Compound 3
[0239] The diastereoisomeric mixture of Compounds 3a and 3b in PEG
400:PBS 1:1 was administered to 7 week old female BALB/c mice (n=8)
by gavage at 20 mg/kg daily for 10 days. There was no vehicle
control group for comparison. However, all mice maintained body
weight and there was a mean increase in the group over the 10 days
of 5% of the starting weight, which was typical of mice of that
age. See FIG. 22.
[0240] Compound 4
[0241] Compound 4 at 5 mg/kg (n=3) or vehicle (n=2, PEG:PBS 1:1)
was administered via gavage to female BALB/c mice for 10 days.
There were no adverse clinical signs in either group, and body
weight changes were similar. There were no gross pathological
changes evident at necropsy. Histology of caecum and colon were
normal.
[0242] These data confirm that the compounds do not cause
gastrointestinal toxicity as evidenced by normal weight gain,
absence of clinical signs and absence of histopathological changes
in those animals receiving them over prolonged periods and often at
extremely high doses.
[0243] To the extent that the word comprising in used in this
specification it is to be understood that the invention in the
alternative also extends to a combination consisting of or
consisting essentially of said elements or integers.
[0244] The reference to any prior art in this specification is not,
and should not be taken as, an acknowledgment or any form of
suggestion that that prior art forms part of the common general
knowledge in the field of endeavour.
[0245] Those skilled in the art will appreciate that the invention
described herein is susceptible to variations and modifications
other than those specifically described. It is to be understood
that the invention includes all such variations and modifications.
The invention also includes all of the steps, features,
compositions and compounds referred to or indicated in this
specification individually or collectively, and any and all
combinations of any two or more of said steps or features.
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