U.S. patent application number 10/146919 was filed with the patent office on 2003-01-23 for compounds which interact with the thyroid hormone receptor for the treatment of fibrotic disease.
Invention is credited to Billingham, Michael Edward John, Fernihough, Janet Katherine.
Application Number | 20030018077 10/146919 |
Document ID | / |
Family ID | 26313780 |
Filed Date | 2003-01-23 |
United States Patent
Application |
20030018077 |
Kind Code |
A1 |
Billingham, Michael Edward John ;
et al. |
January 23, 2003 |
Compounds which interact with the thyroid hormone receptor for the
treatment of fibrotic disease
Abstract
A method of alleviating psoriasis in a warm blooded animal,
which comprises administering an effective amount of at least one
compound having the formula (I) 1 in which X stands for the oxygen
or sulphur atom or for the imino (--NH--) or sulphonyl
(--SO.sub.2--) radical, Y stands for a direct linkage, or for the
oxygen or sulphur atom or for the sulphonyl (--SO.sub.2--) radical
or for the radical of the formula --CR.sup.1R.sup.2--, wherein
R.sup.1 and R.sup.2 which may be the same or different are
hydrogen, alkyl or aryl radicals or R.sup.1 and R.sup.2 may be
joined together to form a cycloalkyl ring, n is an integer having
the value 0 or 1, provided that when n is 0, Y stands for the
oxygen or sulphur atom or for the sulphonyl (--SO.sub.2--) radical,
or an ester, amide or salt thereof.
Inventors: |
Billingham, Michael Edward
John; (Alresford, GB) ; Fernihough, Janet
Katherine; (Oldfield Park, GB) |
Correspondence
Address: |
Martin A. Hay
13 Queen Victoria Street
Macclesfield Cheshire UK
SK11 6LP
GB
|
Family ID: |
26313780 |
Appl. No.: |
10/146919 |
Filed: |
May 17, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10146919 |
May 17, 2002 |
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09986820 |
Nov 13, 2001 |
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6414026 |
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09986820 |
Nov 13, 2001 |
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09674512 |
Nov 16, 2000 |
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6348497 |
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09674512 |
Nov 16, 2000 |
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PCT/GB99/01684 |
May 27, 1999 |
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Current U.S.
Class: |
514/571 ;
514/567; 514/570 |
Current CPC
Class: |
A61P 9/00 20180101; A61P
19/02 20180101; A61P 3/10 20180101; A61P 11/00 20180101; A61K
31/195 20130101; A61P 29/00 20180101; A61P 1/16 20180101; A61P
21/00 20180101; A61P 43/00 20180101; A61P 9/10 20180101; A61K 31/19
20130101 |
Class at
Publication: |
514/571 ;
514/567; 514/570 |
International
Class: |
A61K 031/195; A61K
031/192 |
Foreign Application Data
Date |
Code |
Application Number |
Jun 2, 1998 |
GB |
9811784.9 |
Dec 17, 1998 |
GB |
9827834.4 |
Claims
1. A method of alleviating fibrotic disease by regulating tissue
destructive proteolytic enzyme production in the presence of
thyroid receptor binding but in the substantial absence of
substantive corticosteroid and androgen receptor binding:
2. A method as claimed in claim 1 in which the fibrotic disease is
regulated by administration of an effective amount of at least one
compound having the formula (I) 6in which X stands for the oxygen
or sulphur atom or for the imino (--NH--) or sulphonyl
(--SO.sub.2--) radical, Y stands for a direct linkage, or for the
oxygen or sulphur atom or for the sulphonyl (--SO.sub.2--) radical
or for the radical of the formula --CR.sup.1R.sup.2--, wherein
R.sup.1 and R.sup.2 which may be the same or different are
hydrogen, alkyl or aryl radicals as hereinafter described, ring B
may be optionally substituted by one or more substituents selected
from halogen atoms and alkyl and aryl radicals, n is an integer
having the value 0 or 1, and esters, amides and salts thereof.
3. A method as claimed in claim 1 in which the fibrotic disease is
regulated by administration of an effective amount of at least one
compound having the formula (II) 7
4. A method as claimed in claim 1 in which the fibrotic disease is
regulated by administration of an effective amount of at least one
compound having the formula (III) 8
5 A method of regulating MMP gene activation in the substantial
absence of occupation of either corticosteroid receptor or androgen
receptor.
6 A method of regulating MMP gene activation in by the occupation
of thyroid receptor
7. A method of regulating MMP gene activation by the occupation of
thyroid receptor but in the substantial absence of occupation of
either corticosteroid receptor or androgen receptor
8. A method of regulating MMP gene activation by the occupation of
thyroid receptor but in the substantial absence of occupation of
either corticosteroid receptor or androgen receptor by
administration of an effective amount of at least one compound
having the formula (I)
9. The use of a compound having the structure (I) in the
preparation of a medicament for the structural modification of
fibrotic tissue in a warm blooded animal by regulating tissue
destructive proteolytic enzyme production in the presence of
thyroid receptor binding but in the substantial absence of
substantive corticosteroid and androgen receptor binding 9in which
X stands for the oxygen or sulphur atom or for the imino (--NH--)
or sulphonyl (--SO.sub.2--) radical, Y stands for a direct linkage,
or for the oxygen or sulphur atom or for the sulphonyl
(--SO.sub.2--) radical or for the radical of the formula
--CR.sup.1R.sup.2--, wherein R.sup.1 and R.sup.2 which may be the
same or different are hydrogen, alkyl or aryl radicals as
hereinafter described, ring B may be optionally substituted by one
or more substituents selected from halogen atoms and alkyl and aryl
radicals, n is an integer having the value 0 or 1, and esters,
amides and salts thereof.
10. The use as claimed in claim 4 in which the compound has the
formula (II) 10
11. The use as claimed in claim 4 in which the compound has the
formula (III) 11
12. The use of a compound having the formula (I) in the preparation
of a medicament for the regulation of MMP gene activation in the
substantial absence of occupation of either corticosteroid receptor
or androgen receptor.
13. The use of a compound having the formula (I) in the preparation
of a medicament for the regulation of MMP gene activation in by the
occupation of thyroid receptor.
14 The use of a compound having the formula (I) in the preparation
of a medicament for the regulation of MMP gene activation by the
occupation of thyroid receptor but in the substantial absence of
occupation of either corticosteroid receptor or androgen receptor.
Description
[0001] This invention relates to fibrotic diseases.
[0002] There are many fibrotic diseases in the human. These are
sometimes referred to as chronic connective tissue diseases, and
include degradative and proliferative conditions They include, for
example, deterioration of the joints in arthritis, deformation of
vessel walls in artherosclerosis, accelerated cardiovascular
problems associated with diabetes, lung fibrosis and cirrhosis of
the liver. These fibrotic diseases are generally considered and
studied as separate and different problems. However, it is possible
that common pathways exist in all these fibrotic diseases Indeed,
corticosteriods are used in the treatment of many of these diseases
to relieve symptoms and prevent associated tissue destruction and
scarring; however, the use of corticosteroids is associated with
dose and treatment duration side-effects which limit the usefulness
of this type of compound.
[0003] Some fibrotic diseases are particularly distressing, such
as, for example, those involving deterioration of cartilage and
bone in the joint. Amelioration of such diseases is a particularly
difficult target for the pharmaceutical industry. These conditions
are often accompanied by distressing levels of pain and gradual
impairment of structural body function and movement An added
difficulty in attempting to find a satisfactory cure or improvement
is that joint disease appears to be largely species specific.
Therefore, research into joint disease in man is made more
difficult in that animal models may not be appropriate.
[0004] In mankind, joint disease is generally progressive and
affects all ages, but is particularly prevalent in middle and old
age. Quality of life of the patient is severely reduced as the
level of pain and reduction in mobility increase. Treatments
hitherto have, therefore, been directed at manifestations of the
disease or malfunction which are comparatively less difficult to
treat. In rheumatoid arthritis, these include symptom-modifying
anti-rheumatic drugs (so-called SMARD) for the relief of pain and
decrease ill inflammation at the joint or in the synovial membrane,
such as, for example non-sleroidal anti-inflammatory drugs
(so-called NSAID) Some drugs are said to be disease modifying
(so-called DMARD), such as, for example, non-cytotoxics such as
anti-malarials, gold, sulphasalazine, D-penicillamine, and
cytotoxics such as cyclophosphamide and chlorambucil However, such
drug treatments are often associated with unpleasant or undesirable
side effects, which limit their application and utility A new
category of anti-rheumatic drugs, Disease Controlling
Anti-Rheumatic Drugs, (so-called DCARD) has been proposed to
describe drugs that effectively control the destructive processes
but as yet no marketed anti-rheumatic drugs merit this
classification although corticosteroids have some inhibitory effect
on the articular damage at the low doses that can be tolerated for
long term treatment.
[0005] However, consideration of the above fibrotic diseases as a
class having a common pathway may prove valuable in attempting to
invent new treatments.
[0006] It is known that corticosteriods can regulate gene
transcription by binding to their specific receptor which is one
member of the steroid receptor super-family (Physiology of the
steroid-thyroid hormone nuclear receptor superfamily Williams G R,
Franklyn J A Bailliere's Clinical Endocrinology and Metabolism Vol
8 No 2 pp241-266 1994) This super-family includes the
glucocorticoid, oestrogen, progesterone, androgen, thyroid hormone,
Vitamin D and retinoic acid receptors Such receptors are
responsible for the regulation of many genes including those
encoding tissue destructive proteolytic enzymes, for example,
metalloproteinases (sometimes referred to as MMPs), One such MMP is
collagenase 1. Corticosteroids interact specifically with the
corticoid receptor which then binds to the promoters of the genes
This liganded corticosteroid receptor represses activation of the
collagenase gene by modulating the activity of transcription factor
AP-1 (activating protein 1, heterodimer of c-fos and c-jun
proteins; reviewed by Cato and Wade BioEssays Vol 18 No 5 pp371-378
1996). The liganded steroid receptor thus modulates production of
protein destructive enzymes, thereby reducing degradation of joint
tissues such as cartilage and bone.
[0007] Many of the members of the superfamily of steroid receptors
can modulate tissue destructive proteolytic enzyme production when
liganded with the natural hormone However, use of the natural
hormone at pharmacological levels is associated with severe side
effects due to their biological activity in many physiological
systems. Corticosteroids effectively inhibit proteolytic enzyme
production at the transcriptional level, through modulation of AP1,
but the problem with corticosteroids is that they have additional
transactivation effects on genes that lead to side effects, such as
bone osteoporosis, diminishment of the immune response and water
retention. Some of these side effects can be life threatening.
Therefore, such inhibition or modulation in the substantial absence
of the above hitherto undesirable associated side effects would
represent a major therapeutic advance.
[0008] These undesirable additional transactivation effects are
surprisingly substantially avoided in accordance with the present
invention. It has been found that the use of the compounds
described below have substantially no transactivation properties
and do not transrepress body defence mechanisms signalled by immune
hormones such as the cytokines, IL1 and TNF and the early response
transcription factor NF.kappa.B (Mukaida N et al, J Biol Chem, 269,
13289-13295, 1994). The compounds below, and their associated
pharmaceutical compositions, are not likely, therefore, to
compromise the immune system, which is one of the one of the major
problems associated with the use of corticosteroids. This is a
surprising finding and clearly separates this class of molecule
from the conventional steroid hormones.
[0009] It has been found that compounds (I) hereinafter described
are able to regulate MMP gene activation, but surprisingly not
through occupation of either the corticosteroid receptor or the
androgen receptor. Therefore, surprisingly compounds (I) appear to
regulate MMP gene activation through a different receptor and hence
compounds (I) can thereby substantially prevent transcription of
MMP), including collagenase 1. Indeed, it has been further found
that compounds (I) acts through occupation of the thyroid hormone
receptor. Herein lies the basis of the present invention.
[0010] Thus according to the present invention, a method is
provided of alleviating fibrotic disease by regulating tissue
destructive proteolytic enzyme production in the presence of
thyroid receptor binding, but in the substantial absence of
substantive corticosteroid and androgen receptor binding. This is
effected by administration of an effective amount of at least one
compound having the formula (I) 2
[0011] in which X stands for the oxygen or sulphur atom or for the
imino (--NH--) or sulphonyl (--SO.sub.2--) radical, Y stands for a
direct linkage, or for the oxygen or sulphur atom or for the
sulphonyl (--SO.sub.2--) radical or for the radical of the formula
--CR.sup.1R.sup.2--, wherein R.sup.1 and R.sup.2 which may be the
same or different are hydrogen, alkyl or aryl radicals as
hereinafter described, ring B may be optionally substituted by one
or more substituents selected from halogen atoms and alkyl and aryl
radicals, n is an integer having the value 0 or 1, and esters,
amides and salts thereof.
[0012] In the compounds having the formula (I), R.sup.1 and R.sup.2
may be the same or different and preferably R.sup.1 is selected
from hydrogen or 1-4C alkyl, and R.sup.2 from hydrogen, 1-4C alkyl
or phenyl (which may be optionally substituted with at least one
halogen atom, for example, chlorine, bromine), and R.sup.1 and
R.sup.2 may be joined together to form a cycloalkyl ring (for
example, cyclohexyl); the ring B may be optionally contain one or
more substituents selected from halogen atoms and 1-4C alkyl. Most
preferably, the 1-4C alkyl radicals are methyl or ethyl, and the
halogen atom is chlorine.
[0013] Esters of compound (I) may be useful in the present
invention. Such esters are preferably derived from alcohols having
the formula R.sup.3--OH, where R.sup.3 is preferably I-4C alkyl,
most preferably methyl or ethyl. Salts of compound (I) include
alkali metal and alkaline earth salts, and include magnesium,
aluminium, bismuth, ammonium, and preferably sodium, potassium and
calcium. Where the compound (I) contains a strongly basic
substituent, acid addition salts thereof, such as the
hydrochloride, are comprehended.
[0014] Compounds having the formula (I), in which n is 1, and
R.sup.1 and R.sup.2 which may be the same or different are hydrogen
or alkyl radicals, are described in United Kingdom patent
specification 1140748, the disclosure of which is incorporated
herein. Such compounds are considered to be useful in the treatment
or prophylaxis in humans and animals of such diseases as coronary
artery disease and atherosclerosis. This is because they are said
to reduce the concentration of cholesterol and/or triglycerides in
the blood serum and the level of fibrinogen in blood plasma of
rats. They are also said to possess anti-inflammatory activity in
rats, and are, therefore, considered to be useful in the treatment
of inflammatory signs and symptoms such as rheumatoid arthritis in
man; further work has shown that such compounds have substantially
no anti-inflammatory properties (Billingham M E J and Rushton A,
Anti-inflammatory and Anti-arthritic Drugs, Vol III, Edited by. K D
Rainsford, 31-63, 1985, CRC Press)).
[0015] A particularly preferred compound,
[0016] 1-[4-(4-chlorophenyl)benzyloxy]-1-methylpropionic acid, has
the formula (II) 3
[0017] European patent specification 0 037 698, the disclosure of
which is incorporated herein, describes processes for the
production of compounds having the formula (I) in which n takes the
value 1, Y is a direct link and R.sup.1 and R.sup.2 which may be
the same or different are hydrogen or (1-4C) alkyl. A preferred
compound has the formula (III) 4
[0018] United Kingdom patent specification 860303, the disclosure
of which is incorporated herein, describes compounds having the
formula (I) in which n takes the value 0. A preferred compound has
the formula (IV) and is known as clofibrate. 5
[0019] According to the present invention a method of modifying
fibrotic disease in warm blooded animals is provided which
comprises administering the animal an effective amount of at least
one compound having the formula (I). In a further embodiment of the
present invention a pharmaceutical composition is provided
containing at least one compound having the formula (I) for the
structural modification of fibrotic tissue in a warm blooded
animal.
[0020] In accordance with the present invention, many fibrotic and
proliferative conditions are considered to gain benefit from
treatment with the pharmaceutical composition These include, for
example, rheumatoid arthritis, psoriatic arthritis and psoriasis
itself, the loosening of prosthetic joints, atherosclerosis of
cardiac and coronary vessels and large arteries, the complications
of diabetes, lung fibrosis, liver cirrhosis, systemic sclerosis,
muscular dystrophy.
[0021] In order to be useful in the treatment of the above
conditions in accordance with the present invention, the above
compounds may be administered as a pharmaceutical composition by
any suitable route, but preferably orally, as, for example,
tablets, capsules, suspension, emulsions, powders, syrups, elixirs.
They may be administered as suppositories. The pharmaceutical
compositions may be formulated to include any pharmaceutically
acceptable excipient and may be prepared by any suitable method
known on the art, such as those described in any of the above
patent specifications. The compositions should preferably be
administered to ensure that the patient receives between 0.01 g and
0.5 g of active ingredient per day; the composition containing
suitably between 0.01 g and 0.5 g of active ingredient.
[0022] The invention is illustrated with reference to the following
experimental information.
[0023] Two plasmid constructs were introduced by standard calcium
phosphate precipitation of DNA followed by glycerol shock (Ausubel
F M, Brent R, Kingston D D, Moore J G et al Current protocols in
molecular biology 1994 Greene Publishing
Associates/Wiley-Interscience, New York, N.Y.) using a transformed
African Green Monkey kidney cell line (CV1) (Schneikert J,
Peterziel H, Defossez P- A, Klocker H, de Launoit Y and Cato A C B
Androgen receptor-Ets protein interaction is a novel mechanism for
steroid hormone-mediated down-modulation of matrix
metalloproteinase expression. Journal of Biological Chemistry. Vol
271(39) (pp 23907-23913), 1996).
[0024] Experiment I
[0025] This experiment was intended to show reduction in MMP
promoter activity.
[0026] Accordingly, the following two plasmids were included into
CV1 cells described above
[0027] (a) Either of GR1 or pSG5ARF containing glucocorticoid or
androgen receptor (AR) respectively, both at 2 ug per
5.times.10.sup.5 cells, driven by the Rous sarcoma virus promoter,
and
[0028] (b) a plasmid containing the -73/+63 portion of the
collagenase I promoter linked to luciferase gene at 8 ug per
5.times.105 cells.
[0029] These cells were stimulated using TPA
(12-O-tetradecanoylphorbol-13- -acetate) at 75 ng/ml, which
enhanced the activity of the collagenase promoter. The stimulation
increased luciferase production, which was measured by luminescence
after addition of the luciferase substrate (luciferin) and
co-factors.
[0030] The blank represented the inclusion of these two plasmids
alone with little endogenous activation of collagenase. Addition of
TPA stimulated the activity of this promoter region, resulting in a
high level of luciferase production which is eventually seen as an
enhancement in fluorescence (Y-axis). Both dihydroxytestosterone
(DHT) at 10.sup.-7M and dexamethazone (Dex) at 10.sup.-7M were able
to down regulate this activation through their respective
receptors. CPII was compound II above, clobuzarit. "Empty"
indicated the results when neither GR1 or AR receptors were
present. The results are presented in Table 1.
1 TABLE 1 Empty GR1 AR Raw counts % control Raw counts % control
Raw counts % control Blank 2399 100 12842 100 2668 100 CPII 1882 78
6151 48 1374 51 TPA 5407 225 57519 448 11639 436 TPA + CPII 2747
115 39968 311 5986 224 TPA + DHT TPA + Dex 10981 86
[0031] The above results are presented graphically in FIGS. 1a and
1b. From the results in Table 1, it is shown that Compound II was
also, in addition to DHT or Dex, able to down regulate collagenase
I activation, and also to inhibit partially the background level of
collagenase promoter activity. The level of this reduction which is
achieved by the use of Compound II in both stimulated and
unstimulated conditions is similar, when expressed as ratio of
luciferase counts seen without inclusion of this molecule, over
observable counts when Compound II is included. This suggests that
Compound II does not require the presence of either receptor in
order to exert its action This is further supported by the
observation that Compound II is able to reduce the level of both
TPA stimulated and unstimulated collagenase I (MMP) promoter
activity in the absence of either receptor (Empty).
[0032] Experiment 2
[0033] This experiment was intended to investigate the activation
of androgen and corticosteroid receptors by compound I.
[0034] The CV1 cells described above were transfected with the
following two plasmids
[0035] (a) PGL3MMTV plasmid, containing a transcription site which
is activated by the androgen and glucocorticoid receptor, linked to
the luciferase gene at 9 ug per 5.times.10.sup.5 cells, and
[0036] (b) either one of the plasmids GR1 or pSG5ARFin, containing
glucocorticoid or androgen receptor (AR) respectively, under the
control of constitutively active promoters, both at 2 ug per
5.times.10.sup.5 cells.
[0037] The blank represented the inclusion of these two plasmids
alone, resulting in the absence of receptor activity. Inclusion of
dihydroxytestosterone (DHT) at 10.sup.-7M or dexamethazone (Dex) at
10.sup.-7M to the culture medium enabled the receptor to stimulate
the transcription of the promoter region linked to luciferase which
was eventually seen as an enhancement in fluorescence (Y-axis).
CPII was compound II above, clobuzarit, which was used at
10.sup.-7M.
2 TABLE 2 Raw counts % control AR Blank 4637 100 DHT 1731765 37347
CPII 33130 714 DHT + CPII 1232241 26574 GRI Blank 3569 100 Dex
619085 17346 CPII 3272 92 Dex + CPII 748249 20965
[0038] The above results are presented graphically in FIGS. 2a and
2b, From the results in Table 2, it is shown that Compound II shows
negligible activity to activate the same mechanism as that produced
by the addition of the two steroids DHT and Dex.
[0039] Experiment 3
[0040] This experiment was intended to show the effect of compounds
I on a mediator of the immune response, nuclear factor-.kappa.B
(NF-.kappa.B).
[0041] The following two plasmids were included into CV1 cells
described above
[0042] (a) GR1 as described above at 2 ug per 5.times.10.sup.5
cells, and
[0043] (b) a plasmid designated 3-EnhTK-Luc (at 0.5 ug per
5.times.10.sup.5 cells) which contains a transcription site that is
activated by NF.kappa.B. The cells themselves are capable of
synthesising this protein and hence it is not necessary to
transfect this as well.
[0044] The blank represented the inclusion of these two plasmids
alone; the relatively high value was caused by (i) the high level
of serum in the culture, and (ii) the associated stress that the
cells underwent during the transcription process--both of which
upregulate NF.kappa.B. There was also a related relatively low
activation of TNF (at 4 ng/ml) above a serum containing blank in
the experiment. TNF was tumour necrosis factor alpha.
3 TABLE 3 Raw counts % control Blank 59538 100 CPII 75812 127 TNF
82579 139 TNF + CPII 135886 228 TNF + Dex 18525 31
[0045] The above results are presented in FIG. 3. The results show
that Compound II shows little variation from the background value,
indicating that it is not able to upregulate transcriptional
activity by NF.kappa.B. The lack of inhibitory effect of Compound
II on NF.kappa.B transcription is in contrast to the marked effect
of Dex, showing that Compound II does not act as an
anti-inflammatory agent, so supporting the earlier clinical data
Also, from the lack of substantive stimulation, it can also be
inferred that Compound II, in contrast to DEX, does not interfere
with immune response signalling.
[0046] Experiment 4
[0047] These experiments were intended to identify the receptor
through which compounds I might be acting to block collagenase
promoter activity.
[0048] Transfection procedure hereinbefore described with CV1 cells
was repeated using MCF-7 mammary breast cancer cells derived from
pleural effusion from a breast cancer patient Additionally, each
experiment included transfection of a plasmid (at 8 ug per
5.times.10.sup.5 cells) containing the -517/+63 section of the
collagenase (MMP-1) promoter linked to the luciferase gene. TPA was
used as above to stimulate activity of the collagenase promoter
resulting in luciferase production. Compound II or
3,3,5-triiodo-L-thyronine (T3) was included (both at 10.sup.-8M),
the latter being a known ligand for the thyroid hormone
receptor.
[0049] A series of three experiments was then carried out.
[0050] Experiment 4.1. As MCF-7 cells express endogenous thyroid
hormone receptors, only the collagenase promoter plasmid (at 8 ug
per 5.times.10.sup.5 cells) was transfected into the MCF-7
cells.
[0051] The results are shown in Table 4.1
4 TABLE 4.1 Raw Counts % Control Blank 12575 100 CPII 6029 47.9 TPA
147665 1174.3 TPA + T3 428062 3404.1 TPA + CPII 352376 2802.2
[0052] The results in Table 4 1, presented graphically in FIG. 4.1,
show that in MCF-7 cells, both thyroid hormone and Compound II are
able further to enhance TPA stimulation of luciferase
production.
[0053] Experiment 4.2. The procedure of Experiment 4.1 was repeated
except that a second plasmid, containing 662 bp anti sense
construct for the thyroid receptor (at 8 ug per 5.times.10.sup.5
cells) was additionally transfected.
[0054] The results are shown in Table 4.2.
5 TABLE 4.2 Raw Counts % Control Blank 1458 100 CPII 2379 163.2 TPA
4975 341.2 TPA + T3 19911 1365.6 TPA + CPII 8716 597.8
[0055] The results in Table 4.2, presented graphically in FIG. 4 2,
show that when the thyroid hormone receptor synthesis is inhibited,
luciferase production via the action of T3 and compound II is
substantially prevented.
[0056] Experiment 4.3 A second plasmid was introduced containing
pSP71 vector (Promega) (at 2 ug per 5.times.10.sup.5 cells)
containing the coding sequence for the chick thyroid hormone
receptor which shows high homology to the human thyroid hormone
receptor, driven by the Rous sarcoma virus promoter to ensure
constitutive production
[0057] The results are shown in Table 4.3
6 TABLE 4.3 Raw Counts % Control Blank 2183 100 CPII 1572 72 TPA
11528 528 TPA + T3 1604 73 TPA + CPII 4414 202
[0058] The results in Table 4 3, presented graphically in FIG. 4.3,
show that Compound II, as well as thyroid hormone, is able to
reduce stimulated collagenase transcription.
[0059] Taken together, the results of Experiments 4 1, 4.2 and 4.3
show that compound II mimics the action of T3 which is a known
ligand for the thyroid hormone receptor.
[0060] Experiment 5
[0061] This experiment was designed to demonstrate down-regulation
of pro-MMP-1 and phosphorylation at serine 73 of c-jun, the latter
of which is one half of the protein dimer AP-1, a key regulator of
metalloprotease expression.
[0062] Methods
[0063] Rheumatoid fibroblasts were cultured from synovium obtained
from the knee joint of a patient with rheumatoid arthritis. Cells
were cultured in monolayer with DMEM tissue culture medium (Gibco)
with the inclusion of 5% v/v charcoal stripped serum in 75 cm.sup.2
flasks. The cells were treated for 36 hours with the addition of 10
ng/ml TPA (phorbol ester) plus 5.times.10.sup.-7M thyroid hormone,
Compound II or Compound III. TPA and drugs were added at time zero
and at 30 hours, the TPA addition ensured phosphorylation of
c-jun.
[0064] After 36 hours, cells were lysed in phosphate buffered
saline (PBS) containing protease inhibitors and 0.1% Triton-X-100,
and then boiled for 5 minutes in non-reducing gel loading buffer.
Samples of cell lysate corresponding to equal cell number were
loaded onto a 12.5% SDS polyacrylamide gel, and electrophoresis
using a Bio-Rad.TM., (Bio-Rad Laboratories, Calif. USA) Mini
Protean II cell was performed at approximately 100V for one
hour.
[0065] Transfer of proteins to nitrocellulose membrane was
performed using a Bio-Rad Mini Trans-Blot transfer cell.
[0066] Immunoblotting was performed using polyclonal rabbit
anti-human phosphorylation specific (ser 73) c-jun (cat. No 06659,
Upstate Biotechnology), or MMP-1 (cat. No. RDI-MMP1Habr, Research
Diagnostics) antibodies diluted 1:1000 in Tris buffered saline+3%
fat free milk powder. Swine anti-rabbit horseradish peroxidase
conjugated antibody, (cat. No. p0217, Dako) also at 1:1000 dilution
was used as the secondary antibody. Chemiluminescent detection was
employed using Amersham reagents according to manufacturer's
instructions. Visualisation was achieved by exposing the membrane
to X-ray film (Kodak) for one hour.
[0067] Results are shown in FIG. 5. Left side column of the figure
shows bands corresponding to phosphorylated c-jun (at serine 73),
complexed with DNA, at the top of the figure. Right hand side
column demonstrates a strong regulated band corresponding to
pro-MMP-1 and a faint band at a lower molecular weight
corresponding to active-MMP-1 Lanes 1 and 5 cells treated with TPA
alone; Lanes 2 and 6 cells treated with TPA plus thyroid hormone;
Lanes 3 and 7 cells treated with TPA plus Compound II, Lanes 4 and
8 cells treated with TPA plus Compound III.
[0068] Lanes 2, 3 and 4 show a lower intensity and therefore,
amount of phosphorylation of c-jun, when compared to the TPA alone
treated control in lane 1 Similarly, lanes 6, 7 and 8 show a lower
intensity and therefore, a lower amount of pro-MMP-1, when compared
to the TPA alone treated control in lane 5 Since the lower
molecular weight band across lanes 5, 6, 7 and 8 corresponding to
active MMP-1 is very faint, the regulation by thyroid hormone, and
Compounds II and III appears to be occurring at the transcriptional
level to produce a similar effect to thryoid hormone one the
expression of pro-MMP-1.
[0069] Summary
[0070] Compound II, typical of compounds I, demonstrated steroid
like properties in its ability to reduce collagenase promoter
activity, although this was evident in the substantive absence of
glucocorticoid or androgen receptors. Additionally, compound II did
not activate a construct designed to show responsiveness to the
glucocorticoid receptor It did not enhance or block the activity of
an immune system mediator, demonstrating that it does not possess
anti-inflammatory properties, neither does it compromise immune
response signalling. In a further experiment, the activity of
compound II was identical to T3 in the absence and presence of
endogenous or transfected thyroid hormone receptor. This
demonstrated a common mechanism for these two molecules. The
liganded thyroid hormone receptor is known to be able to down
regulate collagenase promoter activity (A novel mechanism of action
for v-ErbA: abrogation of the inactivation of transcription factor
AP-1 by retinoic acid and thyroid hormone receptors, Desbois-C;
Aubert-D; Legrand-C; Pain-B; Samarut-J Cell. Nov. 15, 1991; 67(4):
731-40), although the mechanism by which this occurs has not yet
been elucidated.
* * * * *