U.S. patent application number 12/977151 was filed with the patent office on 2011-06-09 for therapeutic composition for autoimmune conditions.
This patent application is currently assigned to McEwen Laboratories, Ltd.. Invention is credited to Simon McEwen.
Application Number | 20110135624 12/977151 |
Document ID | / |
Family ID | 9956332 |
Filed Date | 2011-06-09 |
United States Patent
Application |
20110135624 |
Kind Code |
A1 |
McEwen; Simon |
June 9, 2011 |
THERAPEUTIC COMPOSITION FOR AUTOIMMUNE CONDITIONS
Abstract
A therapeutic composition for the treatment, alleviation or
prophylaxis of autoimmune conditions is described. The composition
comprises an enzyme and an immunogen appropriate to the condition
to be treated. The composition may be given in conventional fashion
but is preferably given by intradermal injection.
Inventors: |
McEwen; Simon; (Berkshire,
GB) |
Assignee: |
McEwen Laboratories, Ltd.
Berkshire
GB
|
Family ID: |
9956332 |
Appl. No.: |
12/977151 |
Filed: |
December 23, 2010 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
10820099 |
Apr 7, 2004 |
7901676 |
|
|
12977151 |
|
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|
Current U.S.
Class: |
424/94.61 |
Current CPC
Class: |
A61P 37/06 20180101;
A61K 38/47 20130101; A61P 37/02 20180101; A61P 37/00 20180101; A61P
19/02 20180101; A61P 43/00 20180101; A61K 39/0008 20130101; A61P
29/00 20180101; A61P 19/00 20180101 |
Class at
Publication: |
424/94.61 |
International
Class: |
A61K 38/47 20060101
A61K038/47; A61P 37/00 20060101 A61P037/00; A61P 19/00 20060101
A61P019/00 |
Foreign Application Data
Date |
Code |
Application Number |
Apr 7, 2003 |
GB |
0307989.4 |
Claims
1. A therapeutic composition for the treatment of autoimmune
conditions, wherein the composition comprises purified
.beta.-glucuronidase and purified collagen, wherein the composition
is at a dose that provides a beneficial effect to an individual in
need of treatment of an autoimmune condition.
2. (canceled)
3. (canceled)
4. The composition of claim 1, wherein the .beta.-glucuronidase is
.beta.-D-glucuronoside glucuronosohydrolase (EC 3.2.1.31).
5. The composition of claim 1, wherein the .beta.-glucuronidase is
present at a concentration of between 200 and 10,000 Fishman
units/ml.
6. The composition of claim 1, wherein the .beta.-glucuronidase is
present at a concentration of between 0.5 and 2.5 mg/ml.
7. The composition of claim 1, wherein the composition further
comprises a stabiliser and/or activator.
8. The composition of claim 7, wherein the stabiliser and/or
activator is an inert proteinaceous moiety.
9. The composition of claim 7, wherein the stabiliser and/or
activator is selected from the group consisting of protamine
sulphate and 1,10 diamino decane.
10. The composition of claim 7, wherein the stabiliser and/or
activator is present at a concentration of up to 20 mg/l.
11. A therapeutic composition for the treatment of autoimmune
conditions, the composition comprising .beta.-glucuronidase,
purified collagen and a stabiliser and/or activator, wherein the
composition is at a dose that provides a beneficial effect to an
individual in need of treatment of an autoimmune condition, wherein
the composition further comprises hydroxyl moieties.
12. The composition of claim 11, wherein the hydroxyl moieties are
provided by sugars or diols.
13. The composition of claim 11, wherein the hydroxyl moieties are
provided by 1,3 cyclohexane diol.
14. The composition of claim 11, wherein the hydroxyl moieties are
present at a concentration of up to 20 .mu.g/l.
15. The composition of claim 1 or claim 11, wherein the composition
is buffered to an acid or neutral pH.
16. The composition of claim 15, wherein the composition is
buffered to a pH of between 5 and 6.
17. (canceled)
18. The composition of claim 11, wherein the collagen is present at
a concentration of between 10 and 1.times.10.sup.15
molecules/ml.
19. The composition of claim 1, wherein the composition further
comprises a glycosaminoglycan.
20. The composition according to claim 19, wherein the
glycosaminoglycan is selected from the group consisting of
hyaluronate (D glucuronic acid N acetyl D glucosamine), chondroitin
sulphate (D glucuronic acid N acetyl D galactosamine 4 or 6
sulphate), dermatan sulphate (D glucuronic acid or L iduronic acid
N acetyl D galactosamine), keratan sulphate (D galactose N acetyl D
glucosamine sulphate), and heparan sulphate (D glucuronic acid or L
iduronic acid N acetyl D glucosamine).
21. The composition of claim 19, wherein the glycosaminoglycan is
chondroitin-6-sulphate.
22. The composition of claim 19, wherein the glycosaminoglycan is
present at a concentration of between 0.1 and 1.0 mg/ml.
23. The composition of claim 1, wherein the composition is in a
formulation suitable for transdermal infusion or intradermal
injection.
24. A kit for preparing the composition of claim 1, wherein the kit
comprises an enzyme solution and an immunogen solution, and the two
solutions are introduced to one another and allowed to admix prior
to administration to an individual in need of treatment.
25. A method of treating autoimmune conditions, the method
comprising administering a therapeutically effective amount of the
composition of claim 1 to an individual in need of treatment of an
autoimmune condition.
26. (canceled)
27. (canceled)
28. (canceled)
29. The composition of claim 1 comprising 1,000 to 5,000 Fishman
units/ml of purified .beta.-glucuronidase, 6 .mu.g/ml protamine
sulphate, 1 .mu.g/ml 1,3 cyclohexane diol, and 0.5 mg/ml
chondroitin sulphate, buffered to pH 5.9 and a concentration of
purified collagen selected from the group consisting of
2.5.times.10.sup.12, 2.5.times.10.sup.10 and 2.5.times.10.sup.4
molecules/ml for use in the treatment of multiple sclerosis.
30. The composition of claim 1, wherein the autoimmune condition is
selected from the group consisting of rheumatoid arthritis and
multiple sclerosis.
31. The method of claim 25, wherein the autoimmune condition is
selected from the group consisting of rheumatoid arthritis and
multiple sclerosis.
Description
[0001] This invention relates to a therapeutic composition. More
particularly, the present invention relates to a therapeutic
composition for the treatment, alleviation or prophylaxis of
autoimmune conditions.
[0002] Autoimmunity is present to some extent in everyone and is
usually harmless. However, autoimmunity can cause a broad range of
human illnesses, known collectively as autoimmune diseases,
disorders or conditions. Autoimmune conditions occur when there is
progression from benign autoimmunity to pathogenic autoimmunity
when a misdirected immune response occurs in an individual in which
the immune system attacks the body itself rather than a foreign
body or a xenobiotic or other foreign compound or moiety. This
progression is determined by genetic influences as well as by
environmental triggers.
[0003] Immune reactions are nearly always characterised by
inflammation, which indicates an underlying repair process.
However, in the case of autoimmune conditions or diseases the
inflammation may be chronic, causing tissue damage. For example, in
rheumatoid arthritis chronic inflammation causes characteristic
damage to joints and to cartilage. The precise origin and
pathophysiological processes of these diseases are not fully
known.
[0004] Current treatments for autoimmune conditions are generally
concerned with pain management, the administration of
anti-inflammatory drugs, replacing lost substance (for example, the
provision of insulin in diabetes mellitus) or the administration of
one or more immunosuppressants. These treatments are generally
systemic rather than local and as such may cause adverse effects
elsewhere in the body.
[0005] While these approaches may temporarily alleviate the
conditions, or reduce their progress, they often act to directly
counteract the actual physical state or effect rather than to
remove, reduce or alter the underlying cause or aetiology of the
condition. For example, it is known that many autoimmune reactions
involve a T-cell mediated response, it would therefore be
beneficial to provide a treatment for autoimmune conditions which
acts on this response.
[0006] Autoimmunity is evidenced by the presence of autoantibodies
(antibodies directed against the body) and T-cells which are
reactive with host antigens. Autoimmune conditions may be systemic,
for example systemic lupus erythematosus, or organ specific, for
example thyroiditis. Other examples of autoimmune conditions
include Sjogren's syndrome, Hashimoto's thyroiditis, Myasthenia
gravis, rheumatoid arthritis, juvenile (type 1) diabetes,
polymyositis, scleroderma, Addison's disease, vitiligo, pernicious
anaemia, glomerulonephritis, and pulmonary fibrosis.
[0007] It is an object of the present invention to provide a
therapeutic composition which mediates an effect on an autoimmune
condition by acting on the underlying cause or causes of the
condition. However, the present invention may additionally have an
effect on the physical state or effect of the condition.
[0008] Accordingly, the present invention provides a therapeutic
composition for autoimmune conditions, the composition comprising
an enzyme and an immunogen at a dose which provides a beneficial
effect in an individual in need of treatment.
[0009] Advantageously, the composition of the present invention
mediates a response which acts on or affects the underlying cause
of the autoimmune condition. For example, it may act to
downregulate T-cell mediated reactions. Without wishing to be bound
by theory, the present inventor believes that the immunogens of the
present invention indirectly reduce T-cell activity by means of an
action mediated via the Langerhans or the dendritic cells or by the
thymus, which action redirects antigen sensitive lymphocytes
towards regulatory function (e.g. IL-10 production) or redirects
cell activity away from the target site of the immunogen.
[0010] The term "immunogen" as used herein is intended to define
any substance capable of inducing an immune response. It is not
intended that any of the properties of the immunogen, such as
molecular weight, are to be restricted by this term.
[0011] In the description which follows, the present invention will
be described with particular reference to the treatment of
rheumatoid arthritis. However, the invention finds equal utility in
the treatment of other disorders by the selection of an appropriate
immunogen. For example, multiple sclerosis may be treated by the
use of myelin basic protein as the immunogen, thyroiditis or
Hashimoto's disease may be treated using thyroid proteins as the
immunogen, and diabetes mellitus may be treated using insulin or
.beta.-cell proteins as the immunogen. Additionally, mixtures or
combinations of immunogens may be used, especially where a
condition implicates or is associated with one or more
immunogens.
[0012] Preferably, the enzyme used in the composition is a liver
enzyme or a mucopolysaccharidase. More preferably, the enzyme is a
glucuronidase and most preferably is a .beta.-glucuronidase.
Ideally, the p-glucuronidase is .beta.-D-glucuronoside
glucuronosohydrolase (Registry number EC 3.2.1.31). The source of
the enzyme has been found to make no difference to the activity of
the composition, provided that the enzyme is free from
preservatives or sorbitol. Hence, it may be necessary to purify the
enzyme to enable its use in the composition of the invention. Any
method of purification may be used but it has been found to be
convenient to use gel filtration chromatography or tangential flow
filtration.
[0013] It is preferred that the enzyme is purified to a
concentration of at least 20,000 Fishman units/mg and is present in
the composition at a concentration of between 200 and 10,000
units/ml and ideally between 1,000 and 5,000 units/ml.
[0014] It has been found that contamination by other proteins, even
at very low levels can affect the activity of the enzyme. It is
therefore preferred that a stabiliser and/or activator is present
in the composition. The stabiliser and/or activator is preferably
an inert proteinaceous moiety, for example protamine sulphate or
1,10 diamino decane. Preferably, the stabiliser and/or activator is
present at a concentration of up to 20 mg/l. Where the stabiliser
and/or activator is protamine sulphate, it is preferably present at
a concentration of between 1 and 10 mg/l, more preferably at a
concentration of between 3 and 9 mg/l and ideally at about 6 mg/l
(equivalent to 6 .mu.g/ml).
[0015] The composition may further comprise hydroxyl moieties.
Preferably, the hydroxyl moieties are provided by polyols which
contain at least two hydroxyl moieties and more preferably by
sugars or dials which contain at least two hydroxyl moieties. The
preferred source of the hydroxyl moieties is 1,3 cyclohexane diol.
Preferably, the 1,3 cyclohexane diol is present at a concentration
of up to 20 .mu.g/l, more preferably the 1,3 cyclohexane dot is
present at a concentration of between 0.1 and 10 .mu.g/l and
ideally at a concentration of 1 .mu.g/l. The stereochemistry of the
1,3 cyclohexane diol has been found not to adversely affect the
present invention and hence either the cis, or trans forms or a
racemic mixture may be used.
[0016] The composition is preferably buffered to neutral or an acid
pH. More preferably, the composition is buffered to a pH of between
5 and 6.5 and ideally the composition is buffered to pH 5.9.
[0017] In the preferred embodiment of the invention where the
composition is used in the treatment of rheumatoid arthritis the
preferred immunogen is collagen or fragments, derivatives,
conjugates, mimetics or other products thereof or which have a
collagen-type structure or activity whether natural, synthetic or
modified, regardless of source. The term "collagen" as used
hereafter is intended to include such collagen products as above
described. The collagen is preferably present in a solution. The
collagen may be from any source but it is preferred that the
collagen be free from preservatives or sorbitol or other additives.
Hence, it may be necessary to purify the collagen to enable its use
in the composition of the invention. Any method of purification may
be used but it has been found to be convenient to use gel
filtration chromatography or tangential flow filtration.
[0018] The concentration of collagen present in the composition may
be of between 10 and 1.times.10.sup.15 molecules/ml. More
preferably, the collagen present in the composition may be at a
concentration of between 1.times.10.sup.4 and 1.times.10.sup.13
molecules/ml. Generally, the concentration of the collagen present
in the composition will vary according to the dose required, it is
therefore contemplated that three ranges of collagen dosed
compositions will be made available, these will be vary in strength
from high to low. Compositions in the high strength range will
contain collagen at a concentration of the order of
1.times.10.sup.10 to 1.times.10.sup.15 molecules/ml, and more
preferably will contain about 1.times.10.sup.12 to
1.times.10.sup.13 molecules/ml. Ideally the high strength
composition will contain 2.5.times.10.sup.13 molecules/ml. For
compositions in the mid-strength range, collagen will preferably be
present at a concentration of the order of 1.times.10.sup.9 to
1.times.10.sup.13 molecules/ml, and more preferably will contain
about 1.times.10.sup.10 to 1.times.10.sup.12 molecules/ml. Ideally
the mid-strength composition will contain 2.5.times.10.sup.11
molecules/ml. For compositions in the low strength range, collagen
will preferably be present at a concentration of the order of
1.times.10.sup.9 to 1.times.10.sup.13 molecules/ml, and more
preferably will contain about 1.times.10.sup.10 to
1.times.10.sup.12 molecules/ml. Ideally the mid-strength
composition will contain 2.5.times.10.sup.11 molecules/ml.
[0019] Preferably, the composition further comprises a
glycosaminoglycan or mixtures or combinations thereof. Any
glycosaminoglycan can be used but it is preferred that the
glycosaminoglycan be selected from the group comprising hyaluronate
(D glucuronic acid N acetyl D glucosamine), chondroitin sulphate (D
glucuronic acid N acetyl D galactosamine 1, 3, 4 or 6 sulphate),
dermatan sulphate (D giucuronic acid or L iduronic acid N acetyl
galactosamine), keratan sulphate (D galactose N acetyl D
glucosamine sulphate), and heparan sulphate (D glucuronic acid or L
iduronic acid N acetyl D glucosamine). The most preferred
glycosaminoglycan is chondroitin-6-sulphate.
[0020] The glycosaminoglycan is preferably present in the
composition at a concentration of between 0.1 and 1.0 mg/ml, most
preferably 0.5 mg/ml. Ideally the glycosaminoglycan is free from
preservatives or sugars and to ensure this it may be necessary to
purify the glycosaminoglycan before use. Convenient methods of
purification include gel filtration chromatography or tangential
flow filtration.
[0021] The composition of the invention may be administered in any
conventional manner either systemically or locally, for example by
oral-, parenteral-, intra-dermal-, topical-, rectal-, nasal-routes,
by local injection or by transdermal infusion. At present it is
preferred that the composition is administered by sub-cutaneous
injection, preferably by intradermal injection, or as any form of
trans-dermal infusion. It is not necessary for the composition to
be administered locally to the region of autoimmunity, especially
in rheumatoid arthritis, but it may be preferable to do so in other
conditions in order to minimise any contra-indications or to
expedite an effect at a particular location.
[0022] In a preferred embodiment, the composition is prepared a
short time before administration or even immediately prior to
administration. In this embodiment the composition may be provided
as two preparations, an enzyme preparation and a collagen
preparation, which are introduced to one another and mixed prior to
administration. In this embodiment, the enzyme solution contains
the stabilised enzyme, the hydroxyl moiety and the enzyme in a
buffered solution; all of which are present as described above. The
collagen solution contains the collagen and the glycosaminoglycan,
buffered as described above. Preferably, the composition as
administered contains more collagen solution than enzyme solution,
more preferably at least twice the amount of collagen solution (by
volume) and ideally about 4 parts collagen solution to each part
enzyme solution, by volume.
[0023] Accordingly, the present invention also provides a kit for
preparing the composition of the invention, the kit comprising an
enzyme solution and an immunogen solution, the two solutions being
introduced to one another and allowed to admix prior to
administration to an individual in need of treatment. The kit may
be presented in the form of a multi-chambered or multi-barrelled
dispenser such as a syringe.
[0024] The composition of the present invention may preserved
between formation and use. For example, the composition may be
frozen, dried, freeze-dried, lyophilized, encapsulated or further
preserved with a suitable chosen preservative which has little or
no adverse effect on the in vivo activity of the composition or by
any other preserving technique commonly used or known for
pharmaceuticals. Where the composition is dried or freeze-dried or
otherwise rendered solid, the composition may be formed into a
tablet, capsule, lozenge or other solid dosage form for oral
administration or reconstituted for use in a solution or liquid
form, for example for injection. Where the composition is frozen,
it may be convenient to freeze the composition or its components in
dose unit amounts, optionally in a syringe, to facilitate use by
the individual or medical practitioner.
[0025] Any pharmaceutically acceptable solvent may be used to
produce the liquid form of the composition. Similarly, the usual
binders, excipients, vehicles, and other standard dosage additives
may be used in the composition of the invention.
[0026] The present invention also provides a method of treating or
preventing autoimmune conditions, the method comprising the
administration of a therapeutically effective amount of a
composition comprising an enzyme and an immunogen to an individual
in need of treatment.
[0027] The present invention further provides a method of treating,
alleviating or preventing rheumatoid arthritis, the method
comprising the administration of a therapeutically effective amount
of a composition comprising .beta.-glucuronidase and collagen to an
individual in need of treatment.
[0028] The present invention also provides the use of a
therapeutically effective amount of an enzyme and an immunogen in
the preparation of a medicament for the treatment or prevention of
autoimmune conditions.
[0029] In a further aspect the present invention also provides the
use of a .beta.-glucuronidase and collagen in the preparation of a
medicament for the treatment of rheumatoid arthritis.
[0030] In a final embodiment, the present invention provides a
composition comprising 0.5-2.5 mg/ml .beta.-glucuronidase, 6
.mu.g/ml protamine sulphate, 1 .mu.g/ml 1,3 cyclohexane diol, and
0.5 mg/ml chondroitin sulphate, buffered to pH 5.9 and further
comprising either 2.5.times.10.sup.13, 2.5.times.10.sup.11 or
2.5.times.10.sup.5 molecules/ml of collagen for use in the
treatment of rheumatoid arthritis.
[0031] Embodiments of the invention will now be described, by way
of example only, with reference to the following accompanying
drawings, of which:--
[0032] FIG. 1 is a graph showing the course of arthritis in the
control group (Group B).
[0033] FIG. 2 is a graph showing a comparison of the course of
disease between Group A and Group B over time.
[0034] FIG. 3 shows severity of arthritis in each of the
experimental groups A, B and C on day 29.
[0035] FIG. 4 is a graph showing a comparison of the course of
disease between Group B and Group C over time.
[0036] FIG. 5 shows severity of arthritis in each of the
experimental groups A, B and C on day 52 and indicates that high
dose treatment significantly reduced peak arthritis score.
EXAMPLE 1
[0037] .beta.-glucuronidase (EC 3.2.1.31) (obtained from the marine
mollusc Haliotis midae (South African abalone) was purified by gel
filtration chromatography to remove any preservatives or sorbitol
present.
[0038] The purified .beta.-glucuronidase was added to a buffered
solution at pH 5.9 to give a final concentration of 1.5 mg/ml. 1,3
cyclohexane diol (Sigma, Poole, Dorset, UK) was added to a final
concentration of 1 .mu.g/ml. Protamine sulphate BP was added, with
stirring to prevent precipitation, to a final concentration of 6
.mu.g/ml.
[0039] Separately, natural collagen-type II was purified by gel
filtration chromatography to remove any preservatives or sorbitol
present.
[0040] The purified collagen was dissolved in a solution and
buffered to pH 5.9 to give a final collagen concentration of
2.5.times.10.sup.13. To this solution, chondroitin sulphate was
added to give a final concentration of 0.5 mg/ml.
[0041] 0.01 ml of enzyme solution was introduced to 0.04 ml of
collagen solution and allowed to mix. The 0.05 ml bolus of
composition was used as an intradermal injection in an arthritis
model in mouse. Paw weights and volumes were measured against
control mice receiving vehicle only, collagen only or
.beta.-glucuronidase only.
EXAMPLE 2
Effects of p-Glucuronidase/Type II Collagen on Collagen-Induced
Arthritis in the DBA/1 Mouse
Location
[0042] The test samples were prepared by McEwen Laboratories Ltd.
Pangbourne, Berkshire, England. The test facility was at the
Department of Pathology and Microbiology, School of Medical
Sciences, University of Bristol, England.
Study Schedule
[0043] The study schedule was as follows:
[0044] Study initiation date 2 Feb. 2004
[0045] Assay completion date 24 Mar. 2004
Objective of Study:
[0046] The study was designed to determine the effects of two test
doses of type II collagen in combination with .beta.-glucuronidase
on the incidence and severity of collagen-induced arthritis. Male
DBA/1 mice were chosen for the study and arthritis was initiated
using type II collagen (chicken) in complete Freunds adjuvant as
the initiating stimulus. Treatments were provided as separate
samples of type II collagen and .beta.-glucuronidase. Samples were
kept at 4.degree. C. and were mixed immediately prior to injection.
Treatment was given as a single dose injected subcutaneously into
the scruff of the neck on day 10 post-Induction. Animals were
scored for clinical arthritis from day 17 to day 52 twice weekly by
observation of joint redness and swelling. The study centre was
blinded.
Materials and Methods
Mice
[0047] Thirty male DBA/1 mice were obtained from Harlan Olac at 6
weeks of age. The animals were maintained in the animal house of
The School of Medical Sciences, University of Bristol, until they
had reached 12 weeks of age before the study was initiated. On day
0 all mice were given 100 .mu.g chicken type II collagen emulsified
into complete Freund's adjuvant (CII/CFA) by injection at the base
of the tail. Treatments were given by subcutaneous injection to the
scruff of the neck on day 10 and then clinical joint swelling was
scored twice weekly from day 17 to 52.
Treatment
[0048] .beta.-glucuronidase (E.G. 3.2.1.31) (obtained from the
marine mollusc Haliotis midae) was provided as a freeze-dried
powder. This was further purified (to remove any sorbitol and salts
used in the freeze drying process or as stabilisers) by size
exclusion (gel filtration) chromatography and diluted to a
concentration of 2 mg/ml in a buffer pH5.9. To this solution was
added 1.times.10.sup.-8 mg/ml of 1,3-cyclohexane dial and
6.times.10.sup.-5 mg/ml of protamine sulphate. This solution was
then aseptically filled into vials and stored between 2.degree. C.
and 8.degree. C. until use.
[0049] Type II collagen (obtained from chicken cartilage) was
provided as a freeze-dried powder and was further purified as above
by gel filtration chromatography. This was then diluted in a buffer
solution pH5.9 containing 0.5 mg/ml chondroitin sulphate (purified
from sharks' cartilage). Two dilutions were used representing
approximately 50 ng/ml and 50 fg/ml and these were separately
dispensed aseptically into vials and stored at 2.degree. C. to
8.degree. C. until use.
[0050] Treatments were provided by McEwen Laboratories Ltd and
labelled as follows.
TABLE-US-00001 1. Enzyme A .beta.-glucuronidase solution as
described above 2. CII A Collagen 50 fg/ml as described above 3.
Enzyme B Buffer control 4. CII B Buffer control 5. Enzyme C
.beta.-glucuronidase solution as described above 6. CII C Collagen
50 ng/ml as described above
[0051] The identity of the samples was recorded by McEwen
Laboratories but not disclosed to the University of Bristol in
advance of the end of the in life phase. A, B and C samples were
mixed in a 1 ml syringe no longer than 10 minutes prior to
injection of 200 .mu.l into each animal. Samples were stored at
4.degree. C. until use, and kept on ice following removal from the
refrigerator and up to the point of injection.
Experimental Groups (n=10/group)
[0052] Group A: day 0 CII/CFA, day 10 treatment with mixture A
[0053] Group B: day 0 CII/CFA, day 10 treatment with mixture B
[0054] Group C: day 0 CII/CFA, day 10 treatment with mixture C
Endpoints
[0055] Clinical score of joint swelling. Animals were inspected
twice weekly from day 17 to day 52. On each occasion, each of the
four limbs was given a score according to (0=normal; 1=slight
swelling of whole joint or individual digit inflammation;
2=intermediate swelling of whole joint with redness and/or
inflammation in more than one digit; 3=moderate joint inflammation
and redness spreading to multiple digits, some signs of bone
remodelling; 4=severe joint inflammation and redness spreading to
multiple digits, overt signs of bone remodelling.
Results
[0056] Sample decoding. The identity of the treatment samples was
revealed to the study centre following necropsy.
[0057] Disease was present in the control group (B) with the
expected incidence and severity for this model. Disease was present
at very low level on the first day of inspection, day 17, but the
incidence and severity increased from that day until the end of the
in-life phase (FIG. 1). This progression is in keeping with the
expected course of arthritis in this model. The overall disease
levels in the control group were severe compared to many similar
experiments carried out in the test facility.
[0058] Disease in the low dose treatment group was delayed in onset
compared to the controls (FIG. 2). This meant that a single point T
test revealed a significant difference between severity of
arthritis between Group A and Group B as tested on day 29 (FIG. 3).
Thereafter, disease severity increased in Group A. Although it
remained reduced when compared to Group B for the duration of the
experiment, the overall curve indicated no significant effect of
treatment beyond day 29.
[0059] Disease in the high dose treatment group was lower than that
in Group B from day 25 to the end of the experiment (FIG. 4).
Disease in Group C did not follow the normal course for arthritis
in this model, disease affected a lower than normal number of
animals for the majority of the experiment, and the severity of the
group as a whole was very much lower than expected. With the
exception of two animals, disease severity in Group C remained
extremely low. The lack of protection in these two animals meant
that the variance in the Group C data set was relatively high.
However, the severity of arthritis was significantly lower when
comparing Group C to Group B on days 42, 46 and 52 (Mann-Whitney U
test). The data for day 52 (as the day on which highest disease was
scored) are shown in FIG. 5. In addition, Annova analysis with
Kruskal Wallis post-test reveals an overall significant difference
in the level of disease in Group C compared to Group B
(p=0.0196).
Conclusions
[0060] Both treatment doses altered the course of arthritis in the
experiment. The much less marked reduction, which appeared as a
delay on progression, with low dose treatment was significant
within the experiment. The alteration to the course of disease
observed following high dose treatment is suggestive of a potent
anti-arthritic effect. The levels of disease reduction in Group C
are within the range seen when established anti-arthritic drugs are
given during the course of similar experiments. The fact that this
level of protection was observed following a single treatment is
highly encouraging. Improvement of treatment levels with the low
dose may be achieved by given further doses as disease
progresses.
* * * * *