Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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ANALGESIC AND ANTI-INFLAMMATORY COMPOSITION
FIELD OF THE INVENTION
The present invention relates to an agent having analgesic
and anti-inflammatory activity. In particular the present
invention relates to a composition comprising an effective
amount of an analgesically and anti-inflammatory active
fraction separated from a mixture of plasma and/or serum
and at least one metal, metal ion or metal salt thereof,
wherein said mixture has been denatured.
BACKGROUND OF THE INVENTION
Pain can be defined as an unpleasant sensation ranging
from mild discomfort to agonizing distress, associated
with real or potential tissue damage, or a disorder of the
nervous system. Pain is a response to impulses from the
peripheral nerves in damaged tissue, which pass to nerves
in the spinal cord. All animals experience some degree of
pain during life, whether through injury or disease. As
such, one of the major areas of drug research is the
development of analgesics to be used in pain management.
One area in which pain is more frequently experienced than
in others is inflammation. Pain associated with
inflammation can be caused by pathologic processes in
somatic structures or viscera or by prolonged dysfunction
of parts the peripheral nervous system. Pain associated
with inflammation may be the result of recurrent injuries,
trauma, headache, arthritis including osteoarthritis,
chronic obstructive pulmonary disease, psoriasis, or other
pathologies. Pain associated with inflammation may be
acute or chronic depending on the duration, level and
extent of the inflammation.
Irrespective of the type or cause of pain it is important
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that early treatment is obtained as unrelieved pain can
have profound psychological effects on the patient and
acute pain which is poorly managed initially can
degenerate into chronic pain which may prove more
difficult to treat. However, the difficulty is that pain
perception is a complex psychophysical process that can be
modified by attitude, attention and suggestion. No other
sensation depends as much on cognition and information
processing as does pain. See, for example, Kling, J. W.
and Riggs, L. A., Editors, Woodworth & Schlosberg's
Experimental Psychology, 3rd. edition, Holt, Rinehart and
Winston, Inc., New York, N.Y. (1971).
Therapeutic management of pain includes four steps:
1). peripheral level pain is treated with ice packs,
heat pads, massage or non-steroidal anti-inflammatory
drugs (NSAIDs) like aspirin or ibuprofen to inhibit local
responses to trauma and prevent stimulation of
nociceptors;
2). mild pain is treated with non-opioid analgesics
such as paracetamol;
3). moderate or persisting pain is treated with a
weak opioid like dihydrocodein plus non-opioid analgesics;
and
4). severe pain that persists or increases is treated
with a potent opioid eg nalbuphine plus non-opioid
analgesic.
Despite all of the recent advances in the treatment of
pain and/or inflammation, the majority of the agents used
have side effects or limitations. For example, aspirin can
cause irreversible inhibition of platelet function and
cause gastric irritation. It can precipitate
hypersensitivity reactions including asthma, and there may
be cross sensitivity with other NSAIDs. It also interacts
with a number of other drugs and is especially hazardous
with warfarin.
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Paracetamol does not have the haematological or GI adverse
effects associated with aspirin and side effects are rare;
however, overdosage is particularly dangerous as it may
cause severe or sometimes fatal hepatic damage.
Mild NSAIDs such as ibuprofen have weaker anti-
inflammatory properties than aspirin, but a much lower
risk of GI side effects than aspirin and other NSAIDs.
Dihydrocodeine is a weak opioid which is effective for the
relief of moderate pain of visceral origin. However, it is
known to cause nausea, vomiting and constipation.
Co-dydramol and co-codamol are compound analgesic
preparations which combine paracetamol with a low dose of
an opioid analgesic eg. dihydrocodeine or codeine.
NSAIDs used regularly in full dosage have a lasting
analgesic and anti-inflammatory effect which makes them
particularly useful for treatment of continuous regular
pain associated with inflammation, musculoskeletal and
soft tissue disorders.
Diclofenac combines good efficacy with relatively low
incidence of side effects. It is stronger than ibuprofen
but has more side effects than ibuprofen. It is associated
with intermediate risk of serious upper gastro-intestinal
side effects.
As can be seen, many of currently used analgesics have
associated side effects include dyspepsia, gastric or
small bowel bleeding, ulceration, renal insufficiency,
confusion, rash, headache, hepatic toxicity. NSAIDs also
reversibly inhibit platelet aggregation and prolong
bleeding time. Therefore, the use of analgesic
compositions must be considered within the treatment
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context. At the same time, the treatment context is a
factor that must be taken into account when considering
the pharmacology and physiology of analgesic ingredients.
There is thus a continued need for new analgesics that can
provide fast and reliable analgesia and preferably also
anti-inflammatory benefits.
SUMMARY OF THE INVENTION
In a first aspect, the present invention provides a
composition comprising an effective amount of an
analgesically and/or anti-inflammatory active fraction
separated from a mixture of plasma and/or serum and at
least one metal, metal ion or metal salt thereof, wherein
said mixture has been denatured.
The plasma or serum may be obtained from any animal
source. Preferably, the plasma or serum is isolated from
an animal selected from the group consisting of human,
equine, bovine, ovine, murine, caprine and canine.
In one embodiment, the plasma and/or serum is dried and
lyophilised before use.
Once the plasma and/or serum has been obtained it is mixed
with at least one metal, metal ion or metal salt thereof.
The metal, metal ion or metal salt thereof can be any
metal. In one embodiment, the metal is selected from the
group consisting of nickel, sodium, copper, zinc, cobalt,
iron, magnesium, manganese, potassium, silver and mercury,
ions or salts thereof and mixtures thereof.
Once the metal, metal ion or metal salt thereof has been
mixed with the plasma and/or serum, it is preferably
heated to at least 50 C. Preferably, the mixture is heated
to about 65 C.
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In one embodiment, a protease such as trypsin is
preferably added before heating or after heating. At which
point the resultant mixture is again heated then allowed
to cool to produce an analgesic and/or anti-inflammatory
mixture.
The second heating step is preferably carried out between
about 80 C and about 150 C, more preferably between about
90 C and about 130 C and most preferably, about 120 C to
produce said analgesic and/or anti-inflammatory mixture.
The analgesic and/or anti-inflammatory mixture can be used
directly or further separated to produce an analgesically
and/or anti-inflammatory fraction.
Preferably, the composition of present invention comprises
at least a fraction of an analgesically and/or anti-
inflammatory mixture as described above. More preferably,
the composition of present invention is optionally admixed
with a pharmaceutical carrier. Any pharmaceutical carrier
known in the art may be used.
Accordingly, in a second aspect the present invention
provides an analgesic and/or anti-inflammatory composition
obtained by:
(a) heat denaturing a mixture of plasma and/or
serum and at least one metal, metal ion or metal salt
thereof; and
(b) separating an analgesically active and/or
anti-inflammatory fraction from said denatured mixture.
Preferably, the step of separating the analgesically
active and/or anti-inflammatory fraction is by
chromatography such as affinity chromatography, column
chromatography, partition chromatography, gel-filtration
chromatography with a suitable solvent or solvent mixture.
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In one embodiment, the method further comprises the steps
of incubating said mixture in the presence of a protease
to produce a digested mixture; and heating said digested
mixture. These steps can be undertaken before or after
addition of the at least one metal, metal ion or metal
salt.
Accordingly, in a third aspect the present invention
provides an analgesic and/or anti-inflammatory composition
obtained by:
(a) heat denaturing a mixture of plasma and/or
serum and at least one metal, metal ion or metal salt
thereof;
(b) incubating said mixture in the presence of
a protease to produce a digested mixture;
(c) heating said digested mixture; and
(d) separating an analgesically active and/or
anti-inflammatory fraction from said denatured mixture.
Preferably, the step of separating the analgesically
active and/or anti-inflammatory fraction is by
chromatography such as affinity chromatography, column
chromatography, partition chromatography, gel-filtration
chromatography with a suitable solvent or solvent mixture.
In one embodiment, steps (b) and (c) are performed before
the addition of at least one metal, metal ion or metal
salt thereof. In a further embodiment step (a) further
comprises the addition of NaHCO3.
The step of denaturing the mixture by heat is preferably
carried out at a temperature greater than 65 C.
The fractionation step (d) is preferably performed by
chromatography on a polyamide column; however, any other
method of fractionation may be used.
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In a fourth aspect, the present invention provides a
method for providing analgesia and reduction of
inflammation to a subject, said method comprising
administering to the subject an effective amount of a
composition comprising an effective amount of an
analgesically and/or anti-inflammatory active fraction
separated from a mixture of plasma and/or serum and at
least one metal, metal ion or metal salt thereof, wherein
said mixture has been denatured.
The method of administration may be any method known in
the art. Preferably, the composition is administered
topically, systemically, intramuscularly, subcutaneously,
intraperitoneally, intrapleurally, intraarticularly,
intrathecally, rectally, vaginally, or by inhalation. Most
preferably, the composition is administered topically.
In a fifth aspect, the present invention provides a
composition for reducing inflammation and/or pain in a
subject comprising a pharmaceutically acceptable carrier
and an effective amount of an anti-inflammatory or
analgesically active fraction separated from a mixture of
plasma and/or serum and at least one metal, metal ion or
metal salt thereof, wherein said mixture has been
denatured.
In a sixth aspect, the present invention provides a
physiologically active substance which is extracted from a
mixture of plasma and/or serum and at least one metal,
metal ion or metal salt thereof, wherein said mixture has
been denatured.
Preferably, the physiologically active substance is
further admixed with a pharmaceutically acceptable
carrier. Preferably, the carrier is at least one member
selected from the group consisting of distilled water,
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physiologically saline solution, Ringer's solution, plant
oil, synthetic fatty acid glycerides, higher fatty acid
esters, propylene glycol, lactose, mannitol, corn starch,
crystalline cellulose, gum arabicum, gelatin, potato
starch, carmerose, carmerose calcium, talc, and magnesium
stearate.
In a seventh aspect, the present invention provides a
method for treating a subject afflicted with inflammation
and/or pain comprising administering an effective amount
of an active fraction separated from a mixture of plasma
and/or serum and at least one metal, metal ion or salt
thereof, wherein said mixture has been denatured and
wherein said fraction is admixed with a pharmaceutically
acceptable carrier.
BRIEF DESCRIPTION OF THE FIGURES
Figure 1 shows the effect of one form of the composition
of the present invention (TL-B) comprising zinc chloride,
glycine and trypsinised protein on the TNF-a production by
LPS-stimulated human monocytes.
Figure 2 shows the effect of the composition of the
present invention, containing copper as the metal-
containing solution, on the TNF-a production by LPS-
stimulated human monocytes.
Figure 3 shows the effect of reduced concentrations of one
form of the composition of the present invention (TL-B)
comprising zinc chloride, glycine and trypsinised protein
on the TNF-a production by LPS-stimulated human monocytes.
Figure 4 shows the titration of the effect of different
concentrations of the composition of the present
invention. The purpose was to demonstrate that TL-B does
not compete with the FCS which is being used in the
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culture medium.
Figure 5 shows the effect of the composition on the
metabolism of cells in vitro, with or without LPS
challenge, on a non-radioactive proliferation assay
(CellTiter 960 AQõeou5 Assay) . The purpose was to
demonstrate that the test composition does not reduce the
metabolism of the cells.
DETAILED DESCRIPTION OF THE INVENTION
Before describing the present invention in detail, it is
to be understood that this invention is not limited to
particularly exemplified methods and may, of course, vary.
It is also to be understood that the terminology used
herein is for the purpose of describing particular
embodiments of the invention only, and is not intended to
be limiting which will be limited only by the appended
claims.
All publications, patents and patent applications cited
herein, whether supra or infra, are hereby incorporated by
reference in their entirety. However, publications
mentioned herein are cited for the purpose of describing
and disclosing the protocols and reagents which are
reported in the publications and which might be used in
connection with the invention. Nothing herein is to be
construed as an admission that the invention is not
entitled to antedate such disclosure by virtue of prior
invention.
Furthermore, the practice of the present invention
employs, unless otherwise indicated, conventional
chemistry and pharmacology within the skill of the art.
Such techniques are well known to the skilled worker, and
are explained fully in the literature. See, eg., Coligan,
Dunn, Ploegh, Speicher and Wingfield "Current protocols in
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Protein Science" (1999) Volume I and II (John Wiley & Sons
Inc.); The Merck Index, 12th Edition (1996), Therapeutic
Category and Biological Activity Index; and Remington's
Pharmaceutical Sciences, 17th Edition, Mack Publishing
Company, Easton, Pennsylvania, USA.
It must be noted that as used herein and in the appended
claims, the singular forms "a," "an," and "the" include
plural reference unless the context clearly dictates
otherwise. Thus, for example, a reference to "a metal"
includes a plurality of such metals, and a reference to
"an isolated protein" is a reference to one or more
proteins, and so forth. Unless defined otherwise, all
technical and scientific terms used herein have the same
meanings as commonly understood by one of ordinary skill
in the art to which this invention belongs. Although any
materials and methods similar or equivalent to those
described herein can be used to practice or test the
present invention, the preferred materials and methods are
now described.
In its broadest aspect, the present invention provides a
composition useful as an analgesic and/or anti-
inflammatory agent.
It will be appreciated that the term "anti-inflammatory"
is intended to include an inflammatory response modifier,
including all inflammatory responses such as production of
stress proteins, white blood cell infiltration, fever,
pain, swelling and so forth. Furthermore, the terms
"analgesic," "analgesia," and "analgesically" as used
herein interchangeably are intended to include a pain
reliever that is capable of reducing pain sensation or
nociception, whether the pain incurred is a result of
disease, inflammation, trauma or psychosomatic reaction.
The composition of the present invention will therefore be
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administered as an effective amount to a subject in need
of analgesia or anti-inflammatory treatment. The phrase
"in need of analgesia" as applied to a subject herein
embraces a subject suffering mild to intense pain at the
time of administration of the composition of the present
invention, as well as a subject that can reasonably be
expected to have an imminent onset of mild to intense
pain, eg., within about 1 to about 2 hours and especially
within about 30 minutes, if no analgesic is administered.
The term "effective amount" refers to that amount which is
sufficient to induce or maintain an analgesic effect or
analgesia when administered to a subject; i.e., an
analgesic-producing amount. Equally, the term "effective
amount" when used with reference to the compositions anti-
inflammatory activity means the amount sufficient to
induce or maintain an anti-inflammatory effect. What
constitutes an effective pain-relieving or inflammatory
amount, or dose, of the composition of the present
invention depends, among other factors, on the body weight
of the subject and the intensity of the pain and/or
inflammation being treated. Normally an effective dose
will be found in the range of about 1 to about 6 mg/kg
body weight. For an average 75 kg subject, this range
equates to a dose of about 75 to about 450 mg.
Proportionately smaller or larger doses can be appropriate
for subjects having lesser or greater body weight. Such a
dose can be administered as needed, but typically
administration 1 to about 4 times per day, in most cases 1
or 2 times a day, provides adequate continuing relief of
pain.
An "effective pain-relieving concentration" or "effective
pain-relieving plasma concentration" as used herein is
intended to mean a plasma level in a subject which when
tested in a standardized test involving subject scoring of
the severity of pain, achieves a mean score indicating
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pain relief. In one such test as described herein below,
patients score pain on a scale of from 0 (no reduction in
severity of pain) to 4 (complete relief of pain) and a
mean score equal to or greater than a given value is
deemed to constitute effective pain-relief. A mean score
of 0.5 or greater and, more preferably, 1.0 or greater in
such a test, as exemplified herein, is deemed to
constitute effective pain relief. The skilled artisan will
appreciate, however, that other approaches can be used to
assess the severity of pain and relief from such pain.
Thus, one aspect of the present invention involves a
therapeutic method for analgesia in which a composition
comprising the composition of the present invention is
administered to a subject, in a formulation which provides
detectable pain relief. By "detectable pain relief", it is
meant that the formulation produces effective pain relief
which is measurable by a standard method such as described
above. For example, a formulation, which achieves a mean
score of 0.5 or greater and, more preferably, 1.0 or
greater on a scale of from 0 to 4 in a testing system as
described above, is deemed to provide detectable pain
relief. The invention is not limited to use of any
particular type of formulation, so long as it exhibits the
pharmacokinetic profile defined herein. Examples of
suitable formulation types are described below.
The composition of the present invention essentially
comprises a mixture of plasma and/or serum and at least
one metal, metal ion or metal salt.
The terms "plasma" and "serum" are used herein
interchangeably; however, the term "plasma" typically
refers to the straw-coloured fluid in which the blood
cells are suspended. It consists of various inorganic
salts of sodium, potassium, calcium etc. with a high
concentration of protein (approximately 70g/1) and a
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variety of trace elements. The term "serum" refers to the
fluid that separates from clotted blood or blood plasma
that is allowed to stand. Serum is essentially similar in
composition to plasma, but generally lacks fibrinogen and
others substances that are used in the coagulation
process.
The plasma or serum used in the present invention may be
obtained from any animal source. Preferably, the plasma
and/or serum is isolated from blood taken from an animal
selected from the group consisting of human, equine,
bovine, ovine, murine, caprine and canine:
In one embodiment, the animal source for the plasma or
serum is bovine.
The plasma or serum may be freshly isolated or
alternatively lyophilised. in one embodiment, blood is
isolated from cattle and the haemoglobin is removed by
standard procedures. The plasma is then preferably mixed
with sodium bicarbonate (approx. 20g per litre) and heated
to about 80 C. The coagulated plasma protein is then
removed and lyophilised by standard procedures for further
use.
In one embodiment the lyophilised plasma or serum is
resuspended in water (approximately 50g per litre) and
mixed with at least one metal.
Various metals and/or metal ions are useful in the
composition of the present invention and as such the
present invention embraces all such metals or metal ions.
In one embodiment, the metals are selected from the group
consisting of nickel, sodium, copper, zinc, cobalt, iron,
magnesium, manganese, potassium, silver and mercury.
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In cases where the metals are sufficiently basic or acidic
to form stable non-toxic acid or base salts, the use of
the metals as salts can be appropriate. Examples of
acceptable metal salts include acetate, ascorbate,
benzoate, bicarbonate, chloride, citrate, carbonate, a-
glycerophosphate, a-ketoglutarate, malonate,
methanesulfonate, nitrate, succinate, sulfate, tartarate
and tosylate salts.
Metal salts can be obtained using standard procedures well
known in the art, for example by reacting a sufficiently
basic compound such as an amine with a suitable acid
affording a physiologically acceptable anion. Alkali metal
(for example, sodium, potassium or lithium) or alkaline
earth metal (for example calcium) salts can be made.
In one embodiment for example, the metal is silver (I),
wherein the nitrate salt provides adequate free silver (I)
ion to provide the necessary metal requirement. The
chloride salt on the other hand provides less silver,
being less soluble and with a low dissociation constant
and therefore is less useful in the present invention. The
skilled artisan will be able to readily determine the
suitable salt form of the metal ion that provides the
necessary properties for the present invention.
Furthermore, the skilled artisan will be aware of the
compatibility of the salt forms of the metal(s) and other
components of the composition to maintain adequate levels
of the metal ion(s).
In one embodiment, the metals used in the composition
comprise a mixture of a number of metals. For example, the
mixture of metals could consist essentially of NiSO4.7H20,
NH4V03i NaF, CuSO455H2O, ZnC12, (NH4) 6M07024,4H20, COC12. 6H20,
FeSO4. 7H20, MgSO4. 7H2O, H3BO3, MnCl2. 4H20 and K2CrO4 .
Once the metal, metal ion or metal salt thereof has been
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mixed with the plasma and/or serum, it is preferably
heated to at least 50 C. Preferably, the mixture is heated
to about 65 C.
In one embodiment, a protease selected from the group
consisting of trypsin, chymotrypsin, factor Xa, venom-
protease, thrombin, plasmin and a serine-protease of the
subtilisin family is preferably added before heating or
after heating. Preferably, the protease is trypsin.
The protease can indeed be added before the metal, metal
ion or metal salt is added. Whichever, once the protease
has been added the resulting mixture of plasma/serum and
protease, with or without metal, metal ion or metal salt
is incubated between about 30 C and 45 C for at least 30
minutes. The mixture is then heated again. The second
heating step is preferably carried out between about 80 C
and about 150 C, more preferably between about 90 C and
about 130 C and most preferably, about 120 C to produce
said analgesic and/or anti-inflammatory mixture.
Once the analgesic and/or anti-inflammatory mixture has
been obtained it can be either used directly or
fractionated to obtain an analgesically and/or anti-
inflammatory active fraction. Techniques for fractionating
protein-containing mixtures are well known in the art.
See, for example, "Plasma Protein Fractionation" Heide K,
Haupt H & Schwick H; in The Plasma Proteins, 2nd Edition
Vol 3 (1977) Putnam F. (Ed); US Pat. No. 4,351,710 and US
Pat. No. 4,322,275 both entitled "Fractionation of protein
mixtures"; US Pat. No. 5,138,034 entitled "Method of
fractionating plasma proteins" all incorporated herein by
reference.
As described above, in one embodiment, the present
invention provides a method of relieving pain and/or
inflammation in a subject, the method comprising
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administering to the subject an effective pain- and/or
inflammation-relieving amount of a composition of the
present invention.
The method of the invention can be used to relieve mild to
severe, acute or chronic pain. The method of the invention
is useful for treatment of non-human mammalian subjects or
patients, including domestic, farm and exotic animals,
such as for example dogs horses, zoo animals and the like,
but is primarily useful for treatment of human subjects or
patients.
Generally, the terms "treating," "treatment" and the like
are used herein to mean affecting an individual or
subject, their tissue or cells to obtain a desired
pharmacological and/or physiological effect. The effect
may be prophylactic in terms of completely or partially
preventing the pain or inflammation or sign or symptom
thereof, and/or may be therapeutic in terms of a partial
or complete cure of the pain or inflammation. "Treating"
as used herein covers any treatment of, or prevention of
pain or inflammation in a vertebrate, a mammal,
particularly a human, and includes: (a) preventing the
pain or inflammation from occurring in a subject that may
be predisposed to the pain or inflammation, but has not
yet occurred; (b) inhibiting the pain or inflammation,
i.e., arresting its development; or (c) relieving or
ameliorating the symptoms of the pain or inflammation,
i.e., cause regression of the symptoms of the pain or
inflammation.
While the methods of the present invention are primarily
directed towards pain relief the compositions of the
present invention are also useful in the treatment and/or
prevention of a wide range of conditions and disorders
mediated by COX-2, including but not restricted to
disorders characterized by inflammation, pain and/or
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fever. Such compositions are especially useful as anti-
inflammatory agents, such as in treatment of arthritis,
with the additional benefit of having significantly less
harmful side effects than compositions of conventional
non-steroidal anti-inflammatory drugs (NSAIDs) that lack
selectivity for COX-2 over COX-1. In particular, such
compositions have reduced potential for gastrointestinal
toxicity and gastrointestinal irritation including upper
gastrointestinal ulceration and bleeding, reduced
potential for renal side effects such as reduction in
renal function leading to fluid retention and exacerbation
of hypertension, reduced effect on bleeding times
including inhibition of platelet function, and possibly a
lessened ability to induce asthma attacks in aspirin-
sensitive asthmatic subjects, by comparison with
compositions of conventional NSAIDs. Thus compositions
useful in methods of the invention are particularly useful
as an alternative to conventional NSAIDs where such NSAIDs
are contraindicated, for example in patients with peptic
ulcers, gastritis, regional enteritis, ulcerative colitis,
diverticulitis or with a recurrent history of
gastrointestinal lesions; gastrointestinal bleeding,
coagulation disorders including anemia such as
hypoprothrombinemia, hemophilia or other bleeding
problems; kidney disease; or in patients prior to surgery
or patients taking anticoagulants.
Such compositions are useful to treat a variety of
arthritic disorders, including but not limited to
rheumatoid arthritis, spondyloarthropathies, gouty
arthritis, osteoarthritis, systemic lupus erythematosus
and juvenile arthritis.
Such compositions are useful in treating inflammation in
such diseases as migraine headaches, periarteritis nodosa,
thyroiditis, aplastic anemia, Hodgkin's disease,
sclerodoma, rheumatic fever, type I diabetes,
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neuromuscular junction disease including myasthenia
gravis, white matter disease including multiple sclerosis,
sarcoidosis, nephrotic syndrome, Behcet's syndrome,
polymyositis, gingivitis, nephritis, hypersensitivity,
swelling occurring after injury including brain edema,
myocardial ischemia, and the like.
Such compositions are useful in treatment of pain,
including but not limited to postoperative pain, dental
pain, muscular pain, and pain re=sulting from cancer. For
example, such compositions are useful for relief of pain,
fever and inflammation in a variety of conditions
including rheumatic fever, influenza and other viral
infections including common cold, low back and neck pain,
dysmenorrhea, headache, toothache, sprains and strains,
myositis, neuralgia, synovitis, arthritis, including
rheumatoid arthritis, degenerative joint diseases
(osteoarthritis), gout and ankylosing spondylitis,
bursitis, burns, and trauma following surgical and dental
procedures.
Compositions of the present invention can also be used in
combination therapies with opioids and other analgesics,
including narcotic analgesics, Mu receptor antagonists,
Kappa receptor antagonists, non-narcotic (i.e., non-
addictive) analgesics, monoamine uptake inhibitors,
adenosine regulating agents, cannabinoid derivatives,
Substance P antagonists, neurokinin-1 receptor antagonists
and sodium channel blockers, among others. Preferred
combination therapies comprise a composition useful in
methods of the invention with one or more compounds
selected from aceclofenac, acemetacin, a-acetamidocaproic
acid, acetaminophen, acetaminosalol, acetanilide,
acetylsalicylic acid (aspirin), S-adenosylmethionine,
alclofenac, alfentanil, allylprodine, alminoprofen,
aloxiprin, alphaprodine, aluminum bis(acetylsalicylate),
amfenac, aminochlorthenoxazin, 3-amino-4-hydroxybutyric
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acid, 2-amino-4-picoline, aminopropylon, aminopyrine,
amixetrine, ammoriium salicylate, ampiroxicam, amtolmetin
guacil, anileridine, antipyrine, antipyrine salicylate,
antrafenine, apazone, bendazac, benorylate, benoxaprofen,
benzpiperylon, benzydamine, benzylmorphine, bermoprofen,
bezitramide, a-bisabolol, bromfenac, p-bromoacetanilide,
5-bromosalicylic acid acetate, bromosaligenin, bucetin,
bucloxic acid, bucolome, bufexamac, bumadizon,
buprenorphine, butacetin, butibufen, butophanol, calcium
acetylsalicylate, carbamazepine, carbiphene, carprofen,
carsalam, chlorobutanol, chlorthenoxazin, choline
salicylate, cinchophen, cinmetacin, ciramadol, clidanac,
clometacin, clonitazene, clonixin, clopirac, clove,
codeine, codeine methyl bromide, codeine phosphate,
codeine sulfate, cropropamide, crotethamide, desomorphine,
dexoxadrol, dextromoramide, dezocine, diampromide,
diclofenac sodium, difenamizole, difenpiramide,
diflunisal, dihydrocodeine, dihydrocodeinone enol acetate,
dihydromorphine, dihydroxyaluminum acetylsalicylate,
dimenoxadol, dimepheptanol, dimethylthiambutene,
dioxaphetyl butyrate, dipipanone, diprocetyl, dipyrone,
ditazol, droxicam, emorfazone, enfenamic acid, epirizole,
eptazocine, etersalate, ethenzamide, ethoheptazine,
ethoxazene, ethylmethylthiambutene, ethylmorphine,
etodolac, etofenamate, etonitazene, eugenol, felbinac,
fenbufen, fenclozic acid, fendosal, fenoprofen, fentanyl,
fentiazac, fepradinol, feprazone, floctafenine, flufenamic
acid, flunoxaprofen, fluoresone, flupirtine,
fluproquazone, flurbiprofen, fosfosal, gentisic acid,
glafenine, glucametacin, glycol salicylate, guaiazulene,
hydrocodone, hydromorphone, hydroxypethidine, ibufenac,
ibuprofen, ibuproxam, imidazole salicylate, indomethacin,
indoprofen, isofezolac, isoladol, isomethadone, isonixin,
isoxepac, isoxicam, ketobemidone, ketoprofen, ketorolac,
p-lactophenetide, lefetamine, levorphanol, lofentanil,
lonazolac, lomoxicam, loxoprofen, lysine acetylsalicylate,
magnesium acetylsalicylate, meclofenamic acid, mefenamic
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acid, meperidine, meptazinol, mesalamine, metazocine,
methadone hydrochloride, methotrimeprazine, metiazinic
acid, metofoline, metopon, mofebutazone, mofezolac,
morazone, morphine, morphine hydrochloride, morphine
sulfate, morpholine salicylate, myrophine, nabumetone,
nalbuphine, 1-naphthyl salicylate, naproxen, narceine,
nefopam, nicomorphine, nifenazone, niflumic acid,
nimesulide, 5'-nitro-2'-propoxyacetanilide,
norlevorphanol, normethadone, normorphine, norpipanone,
olsalazine, opium, oxaceprol, oxametacine, oxaprozin,
oxycodone, oxymorphone, oxyphenbutazone, papaveretum,
paranyline, parsalmide, pentazocine, perisoxal,
phenacetin, phenadoxone, phenazocine, phenazopyridine
hydrochloride, phenocoll, phenoperidine, phenopyrazone,
phenyl acetylsalicylate, phenylbutazone, phenyl
salicylate, phenyramidol, piketoprofen, piminodine,
pipebuzone, piperylone, piprofen, pirazolac, piritramide,
piroxicam, pranoprofen, proglumetacin, proheptazine,
promedol, propacetamol, propiram, propoxyphene,
propyphenazone, proquazone, protizinic acid, ramifenazone,
remifentanil, rimazolium metilsulfate, salacetamide,
salicin, salicylamide, salicylamide o-acetic acid,
salicylsulfuric acid, salsalte, salverine, simetride,
sodium salicylate, sufentanil, sulfasalazine, sulindac,
superoxide dismutase, suprofen, suxibuzone, talniflumate,
tenidap, tenoxicam, terofenamate, tetrandrine,
thiazolinobutazone, tiaprofenic acid, tiaramide, tilidine,
tinoridine, tolfenamic acid, tolmetin, tramadol, tropesin,
viminol, xenbucin, ximoprofen, zaltoprofen and zomepirac
(see The Merck Index, 12th Edition (1996), Therapeutic
Category and Biological Activity index, lists therein
headed "Analgesic", "Anti-inflammatory" and
"An.tipyretic" ) .
Still other suitable formulations for use in the present
invention can be found in Remington's Pharmaceutical
Sciences, Mace Publishing Company, Philadelphia, Pa. 17th
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ed. (1985)
The terms "administration," administering," and
"administered" are used herein interchangeably. The
analgesic and/or anti-inflammatory composition of the
present invention may be administered orally including
sublingual, topically, or parenterally in dosage unit
formulations containing conventional non-toxic
pharmaceutically acceptable carriers, adjuvants, and
vehicles. The term "parenteral" as used herein includes
subcutaneous injections, aerosol, intravenous,
intramuscular, intrathecal, intracranial, injection or
infusion techniques or rectal or vaginally. Preferably,
the analgesic and/or anti-inflammatory composition of the
present invention is administered together with a
pharmaceutically acceptable carrier or diluent compatible
with the composition. In preparing such composition, any
conventional pharmaceutically acceptable carrier can be
utilised.
The carrier material can be organic or inorganic inert
carrier material suitable for oral administration.
Suitable carriers include water, gelatin, gum arabic,
lactose, starch, magnesium stearate, talc, vegetable oils,
polyalkylene-glycols, petroleum jelly and the like.
Furthermore, the pharmaceutically active preparations may
contain other pharmaceutically active agents.
Additionally, additives such as flavouring agents,
preservatives, stabilisers, emulsifying agents, buffers
and the like may be added in accordance with accepted
practices of pharmaceutical compounding.
When the analgesic and/or anti-inflammatory composition of
the present invention is administered orally, it is
generally administered at regular intervals, conveniently
at meal times or once daily. The analgesic and/or anti-
inflammatory composition of the present invention can be
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made up in any conventional form including: (a) solid form
for oral, rectal or vaginal administration such as
tablets, capsules (eg. hard or soft gelatine capsules),
pills, sachets, powders, granules, and the like; and (b)
preparations for topical administrations such as
solutions, suspensions, ointments, creams, gels,
micronised powders, sprays, aerosols and the like; (c)
liquid formulations for intravenous administrated may also
be prepared. Pharmaceutical preparations may be sterilised
and/or may contain preservatives, stabilisers, wetting
agents, emulsifiers, salts for varying the osmotic
pressure and/or buffers.
For topical administration to the skin or mucous membrane
the aforementioned analgesic and/or anti-inflammatory
composition of the present invention is preferably
prepared as an ointment, tincture, cream, gel, solution,
lotion, spray; aerosol and dry powder for inhalation,
suspension and the like. In fact, any conventional methods
of preparing topical compositions can be utilised in this
invention. Among the preferred methods of applying the
analgesic and/or anti-inflammatory composition of the
present invention is in the form of an ointment, gel,
cream, lotion, spray; aerosol or dry powder for
inhalation. A pharmaceutical preparation for topical
administration to the skin can be prepared by mixing the
analgesic and/or anti-inflammatory composition of the
present invention with non-toxic, therapeutically inert,
solid or liquid carriers customarily used in such
preparation. These preparations generally contain 0.01 to
5.0 percent by weight, preferably 0.1 to 1.0 percent by
weight, of the analgesic and/or anti-inflammatory
composition of the present invention, based on the total
weight of the peptide preparation.
In preparing the topical preparations described above,
additives such as preservatives, thickeners, perfumes and
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the like conventional in the art of pharmaceutical
compounding of topical preparation can be used. In
addition, conventional antioxidants or mixtures of
conventional antioxidants can be incorporated into the
topical preparations containing the afore-mentioned active
agent. Among the conventional antioxidants which can be
utilised in these preparations are included N-methyl-0-
tocopherolamine, tocopherols, butylated hydroxyanisole,
butylated hydroxytoluene, ethoxyquin and the like. Cream-
base pharmaceutical formulations containing the antigen
preparation, used in accordance with this invention, are
composed of aqueous emulsions containing a fatty acid
alcohol, semi-solid petroleum hydrocarbon, ethylene glycol
and an emulsifying agent.
Ointment formulations containing the analgesic and/or
anti-inflammatory composition of the present invention
comprise admixtures of a semi-solid petroleum hydrocarbon
with a solvent dispersion of the analgesic and/or anti-
inflammatory composition. Cream compositions containing
the analgesic and/or anti-inflammatory composition of this
invention preferably comprise emulsions formed from a
water phase of a humectant, a viscosity stabiliser and
water, an oil phase of a fatty acid alcohol, a semi-solid
petroleum hydrocarbon and an emulsifying agent and a phase
containing analgesic and/or anti-inflammatory composition
dispersed in an aqueous stabiliser-buffer solution.
Stabilisers may be added to the topical preparation. Any
conventional stabiliser can be utilised in accordance with
this invention. In the oil phase, fatty acid alcohol
components function as a stabiliser. These fatty acid
alcohol components function as a stabiliser. These fatty
acid alcohol components are derived from the reduction of
a long-chain saturated fatty acid containing at least 14
carbon atoms.
Formulations for aerosols are described in Drugs and
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Pharmaceutical Sciences, Marcel Dekker, New York, 72: 547-
574 (1996). Furthermore, the analgesic and/or anti-
inflammatory composition of the present invention can be
delivered by dry powder inhalation. Such formulations and
devices are described in Pharmaceutical Technology, June
1997, pp.117-125.
Depending upon the mode or type of administration and the
severity of the pain or inflammation, the treatment regime
will vary. However, typically an individual is monitored
hourly or daily, depending on the above factors, and the
status of pain/inflammation is determined. Administration
of the analgesic and/or anti-inflammatory composition of
the present invention continue until the pain and/or
inflammation is reduced or alleviated.
Protocols for conducting human pharmacokinetic studies are
well known in the art and any standard protocol can be
used to determine whether a particular composition of the
present invention satisfies the pharmacokinetic criteria
set out herein. An example of a suitable protocol is
described below.
In one embodiment, the compositions of the present
invention, upon administration, reduce the amount of TNF-a
present in an individual's tissue as compared to untreated
tissue. Accordingly, the present invention encompasses a
method of reducing the amount of TNF-a in an individual's
tissue comprising the step of administering an effective
amount of a composition comprising an effective amount of
an analgesically and/or anti-inflammatory active fraction
separated from a mixture of plasma and/or serum and at
least one metal, metal ion or metal salt thereof, wherein
said mixture has been denatured, wherein the composition
reduces the amount of TNF-a in the individual's tissue
compared to untreated tissue.
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By "comprising" is meant including, but not limited to,
whatever follows the word comprising". Thus, use of the
term "comprising" indicates that the listed elements are
required or mandatory, but that other elements are
optional and may or may not be present. By "consisting of"
is meant including, and limited to, whatever follows the
phrase "consisting of". Thus, the phrase "consisting of"
indicates that the listed elements are required or
mandatory, and that no other elements may be present. By
"consisting essentially of" is meant including any
elements listed after the phrase, and limited to other
elements that do not interfere with or contribute to the
activity or action specified in the disclosure for the
listed elements. Thus, the phrase "consisting essentially
of" indicates that the listed elements are required or
mandatory, but that other elements are optional and may or
may not be present depending upon whether or not they
affect the activity or action of the listed elements.
The invention will now be further described by way of
reference only to the following non-limiting examples. It
should be understood, however, that the examples following
are illustrative only, and should not be taken in any way
as a restriction on the generality of the invention
described above. In particular, while the invention is
described in detail in relation to the use of specific
animal plasma and metals, it will be clearly understood
that the findings herein are not limited to these
ingredients.
EXAMPLE 1 PREPARATION OF ANALGESIC AND ANTI-
INFLAMMATORY COMPOSITION
200 litres of sterile cattle blood was centrifuged at 1000
- 1300 x g for 10 minutes and the haemoglobin was removed
from the plasma. After centrifugation approximately 100
litres of plasma was gained, and transferred into a dish,
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suitable for heating and continuous mixing. To the plasma
liquid 2kg Sodium Bicarbonate (NaHCO3) was added and mixed
until the NaHCO3 dissolved, then the solution was heated to
80 C. Denatured plasma protein was then recovered and
placed on filter paper to dry. The solid sediment was
then pressed to produce a 60kg solid plasma-protein
"block" which was then lyophilised by standard procedures.
After this process the plasma-protein weighed
approximately 8kg and was=used in the preparation of the
analgesic/antiinflammatory preparation as described below.
A solution was then prepared comprising 152 litres of
water, 8kg dried plasma-protein as prepared above and
200ml of a metal-containing solution. The constituents of
the metal-containing solution are shown in Table 1.
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TABLE 1
METAL-CONTAINING SOLUTION
Ni SO4 7 h2O 10.4g/1
NH4VO3 1. 2g/ 1
Na F 24.Og/1
Cu SO4 5H20 20.Og/1
ZN C12 47.Og/l
(NH4) 6 MO7024 4H20 7. Og/1
CO Cl2 6H2O 20.Og/1
Fe SO4 7H20 100.Og/1
MgSO4 7H2O 80.Og/l
H3BO3 23 . 0g/1
Glucose 50.Og/1
Mn C12 4H20 36.4g/1
K2CrO4 1. Og/1
Glycine 75.0g/1
Citric Acid 20.Og/l
Made up in a 200ml solution with water, which was then
stirred for at least 20 minutes.
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The mixture was then heated up to 120 C and maintained for
two hours with constant mixing. During this time the
plasma-protein dissolved and was sterilized. The
resulting material was then held at a temperature of about
35 C and 0.125g/1 of trypsin was added. The material was
then allowed to incubate for approximately 2 hours. The
digested material was then autoclaved and cooled to
produce the analgesic/anti-inflammatory composition of the
present invention.
EXAMPLE 2 MANUFACTURE OF A TOPICAL ANALGESIC/ANTI-
INFLAMMATORY COMPOSITION
A composition comprising the ingredients shown in Table 2
were mixed at 75-80 C in a 250 litre vacuum homogenizer
equipped with anchor and turbo mixers. Then the
ingredients shown in Table 3 were added and the mixing was
continued at 80-83 C for 10 minutes with the aid of the
turbo mixer.
A slow cooling process was then carried out using the
anchor mixer. When the material reached 60 C, the vacuum
was switched on until the end of the cooling.
At 40-45 C the ingredients shown in Table 4 were added and
mixed for 10 minutes. Mixing with the anchor mixer was
continued until the mixture reached 25 C.
After a standing period of approximately 24 hours, the
topical analgesic was ready for use.
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TABLE 2
Item No. Amount Per Kg Ingredients
1 20g Liposorb S20 (Tween 60)
2 20g Cremaphor A6
3 10g Hydromyristenol
4 40g Cetyl alcohol
70g Corn Oil (Cold Pressed)
6 30g Wheat Germ Oil
7 0.24g Carrot Oil
8 50g Isopropyl Myristate
9 0.2g Butylated Hydroxytoluene B.P.
3g Phenonip
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TABLE 3
11 400g Plasma protein from Example 1
12 15g Propylene Glycol B.P.
13 15g Hygroplex HHG
14 2g Allantoin
15 208g Purified Water B.P.
16 10g Germaben II
17 4g Veegum
18 100g Purified Water B.P.
19 0.04m1 Potassium Bromide 50g/1
20 30.7mg Sodium Sulphide
21 0.04m1 Potassium Iodide 25g/1
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TABLE 4
22 1.4g Chammomile Fragrence
Methodology
1). Add items 1 to 10 in a 250 litre steam pan and heat
75 C;
2). Boil items 15 and 18 in the 150 litre pan and transfer
13 litres to the 50 litre pan and add Veegum and mix until
homogeneous;
3). Add item 14 to the remainder of the Purified Water
B.P. in the 150 litre steam pan at above 90 C and mix.
When dissolved add the items 12, 13 and 16 and maintain
temperature at 75 C with continual mixing;
4). Add the water phase (step 5) to the oil phase (step 3)
and mix using a short shaft air mixer. Then add step 4 to
this using a plastic sieve to ensure that no lumps are
incorporated;
5). Add plasma protein from Example 1 and emulsify for 20
minutes, then continue stirring whilst water cooling to
40 C;
6). Add items 19 to 21 allowing a few minutes in between
each addition whilst mixing. Cool to below 30 C.
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EXAMPLE 3 CLINICAL TRIAL ON TOPICAL ANALGESIC AND
ANTI-INFLAMMATORY COMPOSITION
Twenty-three (23) randomly selected patients in a general
practice setting were supplied with a preparation produced
according to Example 2 above. The patients we advised to
apply the preparation topically three times daily.
Patients were reviewed at regular intervals and divided
into two groups:
Group A - work or injury induced conditions either
acute or sub-acute eg repetitive strain injury (RSI),
tennis elbow, joint and musculo-tendinous injury;
Group B - Arthritic and aging conditions - sub-acute
and chronic eg osteoarthritis.
Table 5 shows the effect of using the topical composition
over a three (3) month period.
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TABLE 5
Patient Details Com lp aint Result Comments
Male - Aged 52 Lateral epicondylitis Improved Full recovery
Male - Aged 28 Musculo-tendinous Full recovery Acute soft tissue injury
Female - Aged 33 Cervical soft tissue Improved Recovery after 1 week
injury
Female - Aged 38 RSI Partial Rapid improvement, but
improvement relapse after cessation of
treatment
Female - Aged 41 RSI No benefit Poor patient selection - no
treatment has worked
Female - Aged 64 Reputed disc No benefit Pathology not treatable
using analgesics/anti-
inflammatory agents
Male - Aged 30 Soft tissue injury Improved Soft tissue injury
Female - Aged 36 RSI No benefit Too greater area to be
treated with a topical agent
Female - Aged 50 Lateral epicondylitis Improved Non-repetitive injury -
Unsuccessfully treated with
physiotherapy/anti-
inflammatory agent
Female - Aged 68 Arthritis Improved Pain relief but movement
still restricted
Male - Aged 47 Soft tissue injury Improved Rapid improvement, but
relapse after cessation of
treatment
Female - Aged 48 Cervical soft tissue Partial Poor compliance
injury improvement
Male - Aged 60 Arthritis Improved Rapid improvement, but
relapse after cessation of
treatment. Intra-articular
cortisone not effective
Female - Aged 70 Arthritis Improved Rapid pain relief obtained
Male - Aged 68 Arthritis Improved Rapid improvement, but
relapse after cessation of
treatment.
Female - Aged 56 Arthritis Improved Pain relief obtained
Female - Aged 49 Gout Improved Pain relief obtained
Female - Aged 80 Calcaneal spur No benefit Condition not treatable with
topical agent
Female - Aged 79 Arthritis Pain relief Pain relief obtained
Female - Aged 69 Arthritis No benefit Non-specific arthritis
Male - Aged 73 Arthritis No benefit Poor patient selection
Male -Aged 67 Inflammation No benefit Topical application not
effective for this rheumatoid
arthritis-like condition
Female - Aged 63 Arthritis No benefit Multiple pathologies
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While these data are more qualitative than quantitative,
it is readily apparent that use of the topical analgesic
composition produced effect.
It was observed that in the more chronic situation,
elderly, local arthritis (in particular osteo-arthritis),
results were more predictable. Clinically, it appears
that local application of the topical analgesic agent to a
pathological joint produces some effect. This may in some
way be related to the "massage" effect, and focusing
attention of the positive aspects of treatment.
One of the most important aspects of the trial was
selection of patient. Inventors believe that the nature
of condition to be treated has an affect on the ability of
a topical agent to work effectively. Of the small numbers
used in this trial, the best results were obtained with
patients having single joint pain or relatively localized
non-joint pain. Patients whose general health was
reasonable was more important than the age of patient.
Finally, non-weight bearing joints responded more quickly
than weight bearing joints.
Of the patients in Group A (acute and sub-acute), the best
results were obtained where local rather than vague
general pain was evident. Repetitive strain injury was
not helped by the use of the analgesic unless the
condition was of the very local category - eg. Tennis-
elbow. Conversely, with the "arthritic" Group B pain from
osteo-arthritis was definitely reduced whilst the
analgesic was being used. However, following cessation of
treatment in may instance, the pain gradually returned.
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EXAMPLE 4 TREATMENT OF OSTEOARTHRITIS IN RPANDOMIZED
DOUBLE BLIND STUDY
Without wishing to be bound by any particular hypothesis
or theory, the inventors believe that the active agents
within the compositions of the present invention are
metallo-peptide complexes. When used topically this
preparation has been shown to be as effective as orally
administered indocid (Indomethacin), a NSAID which reduces
pain, swelling, and inflammation or phenylbutazone, a
NSAID used in the treatment of pain, lameness, laminitis
and osteoarthritis, in an animal model of inflammatory
arthritis. The animal model was inflammation caused by an
injection of mycobacteria into the foot pads of Long-Evans
rats (data not shown).
In has also been demonstrated that the composition
described in Example 2 possessed inhibitory activity
against the serine proteinases-trypsin and human
20. granulocyte elastase (HGE). Since HGE has been implicated
in the destruction of cartilage in inflammatory arthritis
the inhibitory properties of the composition in Example 2
against this or similar enzymes may contribute to its
overall biological activity. Apart from these direct
effects it is postulated that the compositions of the
present invention might also work indirectly by acting as
an agent for specific transdermal transport essential
metals into the affected joints.
In order to test some of these theories patients aged 18
or over were treated three times daily with either the
composition described in Example 2 or a placebo. Neither
the patient nor the physician was aware of which agent
they received. All patients were assessed to have mild to
moderate non-advanced osteoarthritis of hand joints or
knee joints will be entered. The patients were assessed
before using the composition and two weeks after
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commencement of treatment. Joint pain was assessed both
via palpation, movement and scored by the following scale:
0 - Not Tender; 1- Tender; 2 - Tender & Winced; 3 -
Tender, Winced & Withdrew.
Pain, morning stiffness and function were also
subjectively assessed by the patient using the 10 cm
visual analogue scale.
At the end of the two weeks the patients were asked to
assess the composition as to its efficacy, scoring a
percentage between 0 - 1000.
Twenty-two patients completed the trial - three males and
nineteen females. The average age was 60, and the
distribution of placebo and composition was approximately
equal throughout the age range. The degree of severity
and joints involved were also similar for the placebo and
composition groups. Thirteen patients used the
composition from Example 2 and nine used the placebo. The
results are shown in Table 6.
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TABLE 6
Treatment Group
Before Treatment After Treatment
(Mean SD) (Mean SD)
Palpation 2.53 (0.96) 0.38 (0.86)*
Movement 1.70 (0.85) 0.30 (0.85)*
Visual Analogue
Pain 56.5 (16.8) 23.4 (22.0)*
Morning Stiffness
Mins 23.46 (25.85) 11.69 (22.12) NS
Degree 47.30 (30.83) 10.76 (27.98) #
Function 55.38 (20.79) 25.38 (16.98) *
Patient Overall Assessment 75%
Placebo Group
Before Treatment After Treatment
(Mean SD) (Mean SD)
Palpation 2.66 (0.70) 1.88 (0.95) NS
Movement 2.22 (0.83) 1.66 (0.95) NS
Visual Analogue
Pain 54.44 (13.09) 38.33 (27.27) NS
Morning Stiffness
Mins 44.44 (57.46) 17.0 (40.92) NS
Degree 29.44 (30.82) 19.4 (25.98) NS
Function 66.6 (22.51) 50.5 (33.35) NS
Patient Overall Assessment 450
Statistical Significance
*= P<.01 #= P<.1 NS Not Significant
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This double-blind trial was carried out on patients with
clinically well defined osteoarthritis, both placebo and
drug treated groups having a similar degree of severity,
average age and similar spread of joint involvement.
Although the number of patients was not large there was a
clear difference between the drug treated and the placebo
groups (see Table 6). These differences were shown to be
statistically significant to the P < 0.01.
The pain score both on palpation and movement was
significantly reduced after two weeks of treatment with
composition. All measures via the visual analogue scale
were also significantly reduced. In contrast none of the
parameters measured with the placebo group showed a
significant reduction.
When asked to assess the efficacy of the treatment
composition, the treated group scored a 75o approval for
product, while the placebo group only had a 4501 approval
rate.
As the only difference between the two compositions was
the plasma protein from Example 1, it must be assumed that
this product was responsible for the therapeutic effects
observed.
EXAMPLE 5 TOPICAL TREATMENT IN NON-HUMAN ANIMALS
Arthritis is a very common problem in certain dog breeds
such as Rottweilers with clinical signs usually becoming
evident at about 4-6 months of age.
This trial was therefore conducted on a "double blind"
basis to ascertain whether or not the composition
described in Example 2 was capable of reliving symptoms.
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Twenty-seven dogs were admitted into the trial, twenty-six
Rottweilers (including one crossbred Rottweiler) and a
Labrador. There was a fairly even distribution of
immature and mature dogs and both acute and chronic
conditions being treated. A brief summary of the
individual results is given in Table 7. The placebo was
designated A, while the active agent was designated B.
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TABLE 7
No BREED TREATMENT RESULTS
1 Rottweiler B only No response and withdrew
2 Rottweiler B only Possible skin reaction to cream and withdrew
3 Rottweiler B then A Some improvement with B, no change with A
4 Rottweiler A then B Improved with A, then worse with B
5 Rottweiler A then B No response to either
6 Rottweiler A then B No response to either
7 Rottweiler B only Lost to follow up
8 Rottweiler A then B No response to either
9 Rottweiler B then A Improved on B, then worse with A
10 Labrador B then A Marked improvement on B, sustained while on A.
(NB: older dog with chronic arthritis and both elbows treated)
11 Rottweiler A only No change on A, (both legs) then lost to follow up
12 Rottweiler B then A Improved on B then further on A
13 Rottweiler A only Euthanized after 1 week due to severe hip dysplasia.
(Both legs treated)
14 Rottweiler B then A Minimal improvement on B, then further improved on A
(Both legs treated)
15 Rottweiler A then B No change with A, slight improvement with but dog was
rested
16 Rottweiler B then A Improvement on B, then slipped back again on A. (older
dog
with chronic arthritis, post surgery)
17 Rottweiler B only Improved in 2-3 days (old post surgery case) Not given A
18 Rottweiler B then A Improved to soundness in 2 days on B. Sesamoid problem
developed while on A which did not respond to B treatment.
19 Rottweiler B then A Intermittent lameness improved to soundness on B, and
this
sustained while on A and for at least 3 months
20 Rottweiler A then B No response, worse on B than A
21 Rottweiler B only Chronic problem in elbows, coniplicated by sesamoid
fragmentation during treatment. B used on L elbow and R
sesamoid and dog became sound and has remained so.
22 Rottweiler B then A No response. Very severe case, both legs treated. Dog
was
destroyed.
23 Rottweiler A then B No response.
24 Rottweiler A then B Slight improvement on A, but then lameness shifted to
other
leg. No further change on B.
25 Rottweiler X B then A No response. (Chronic post surgery case which was
sound until
slipped on stairs 4 weeks before entering trial)
26 Rottweiler A then B No change on A - results with B to come
27 Rottweiler A then B Results to come
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The most simple way to examine these results is to assess
them on the basis of the "first used" treatment. When
this is done, the results are as follows:
TUBE A TUBE B
No. of dogs treated 11 15
No of dropouts 2 2
No. completing trial 9 13
No. responding (less lame) 2 10
% RESPONDING 22.2 76.9
These results are at first glance better than we expected
when we surveyed the combined results of the double-blind
study. However, the 77% response rate must be interpreted
in the light of a 22% response rate to the placebo (Tube
A). Both these results may have been influenced by the
weather patterns brought about by running this trial in
the Spring. A number of the dog owners suggested that
warmer weather at the time of treatment may have been at
least in part responsible for the improvement in their
dog's lameness. This comment is particularly applicable
to Dog 14, which was recorded as a positive response but
improved further on Tube A after finishing 2 weeks on Tube
B.
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EXAMPLE 6 TEST OF COMPOSITION ON TNF-a PRODUCTION BY
LPS-STIMULATED HUMAN MONOCYTES
The compositions of the present invention are anti-
inflammatory. TNF-a is a cytokine known to be released as
a result of early inflammatory responses. In the present
experiment the aim was to demonstrate that the
compositions of the invention were capable of regulating
or affecting the presence of TNF-a. It was hypothesised
that if the TNF-a levels were reduced in the assay then
this demonstrated that the compositions of the invention
were anti=inflammatory.
Monocytes were isolated by elutriation by centrifugation
standard procedure (Brahmi et al., 1983, Ann Immunol
(Paris) 134D(2): 191-206) from human blood and cultured
overnight in RPMI supplemented with 10% Fetal Calf Serum
(FCS) and 25ng/mL Macrophage Colony Stimulating Factor (M-
CSF)] in the presence of 95o air plus 5o CO2 at 37 C.
Next day monocytes were counted and 5 x 105 cells per well
aliquots placed into wells of a 96 well tissue culture
plate. The volume was made up to 500ja.L per well and then
the cells were stimulated with 500ng/mL lipopolysaccharide
(LPS) in the presence-of 1% Fetal Calf Serum (FCS), with
varying concentrations of test composition for 24 hours.
In the present experiment, the test composition was that
described in Example 1, except that the metal-containing
solution was a simplified version of the metal-containing
solution described in Table 1 in that it only contained
zinc chloride and glycine.
TNF-a levels in the culture supernatants were measured by
ELISA Opti EIA, BD Bioscience following the manufacturer's
instructions.
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The concentrations of test composition used were 40o
(200pL) ; 20% (100lZL) ; 10 0(50pL) ; and 0 0. The control was
LPS (500ng/mL) and there were 3 repeats.
Table 8 together with Figure 1 shows the results.
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TABLE 8
with
Av pg/mL w/o LPS LPS SEM w/o LPS with LPS
Test 400 -117.47 -93.49 40o 3.28 1.25
Test 20% -100.25 -8.14 200 2.17 72.18
Test 100 -55.53 -10.10 100 11.01 12.88
Test Oo -113.79 2700.23 0% 3.72 861.07
SEM: Standard Error of the Mean
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The conclusions that can be drawn from the results are
that the test sample decreases LPS-induced TNF-alpha
secretion in human monocytes, indicating efficacy in
inhibiting inflammatory responses.
EXAMPLE 7 SECOND TEST OF COMPOSITION ON TNF-a
PRODUCTION BY LPS-STIMULATED HUMAN
MONOCYTES
This experiment was essentially a repeat of the experiment
described in Example 6, with the only difference being the
metal-containing solution. In the present experiment, the
test composition was that described in Example 1, except
that the metal-containing solution contained only copper
sulphate.
Table 9 and Figure 2 show the results.
The conclusions that can be drawn from these results are
that the test sample inhibits the inflammatory response of
human monocytes to an LPS challenge.
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TABLE 9
Av pg/mL Ctrl LPS SEM Ctrl LPS
Test 3310.25 5508.58 Test 138.24 1321.58
Ctrl 612.87 26873.00 Ctrl 6.25 932.93
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EXAMPLE 8 TEST OF LOWER CONCENTRATION OF COMPOSITION
ON TNF-a PRODUCTION BY LPS-STIMULATED HUMAN
MONOCYTES
Test of the composition used in Example 6 on TNF-a
production by LPS-stimulated human monocytes was
undertaken, but at lower concentrations.
All other experimental procedures were identical to those
used in Example 6.
Table 10 and Figure 3 show the results.
The conclusions that can be drawn from these results are
that the anti-inflammatory effect of the test sample is
depending on the dosage, further supporting the outcomes
of Example 1, i.e. that LPS-induced TNF-alpha secretion is
inhibited by the test composition.
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TABLE 10
Groups Average (pg/mL) SEM
Test 1001 6627.10 363.07
Test 7.50 7953.37 579.38
Test 5.Oo 9138.62 945.71
Test 2.50 12211.49 412.64
Ctrl 30723.52 1140.03
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EXAMPLE 9 TITRATION OF THE EFFECT OF DIFFERENT
CONCENTRATIONS OF COMPOSITION
Elutriated monocytes were incubated for 24h with a
checker-board pattern of test composition (100, 5%, 2.50 &
Oo) as used in Example 6 with various concentrations of
FCS (100, 50, 1% and 0%). TNF-a was measured by ELISA in
the culture supernatants as described above in Example 6.
Results are shown in Table 11 and Figure 4.
The conclusions that can be drawn from these results are
that the test sample does not compete in inhibiting TNF-
alpha secretion with the FCS.
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TABLE 11.
Av pg/mL Control LPS SEM Control LPS
Test 10%/FCS-10% 823.17 1919.78 Test 10%/FCS-10o 205.20 300.87
Test 10%/FCS-5% 1417.08 1484.48 Test 10a/FCS-5a 184.84 178.36
Test 10o/FCS-19; 1647.46 1273.60 Test 10o/FCS-1% 125.46 20.42
Test 10-o./FCS-0o 5667.25 3059.38 Test 10%/FCS-0% 3320.00 719.32
Test 5%/FCS-10o 402.75 3961.06 Test 5%/FCS-10% 40.86 1191.91
Test 5o/FCS-5% 1123.04 5544.84 Test 5a/FCS-5o 134.77 1394.17
Test 5%/FCS-1% 4037.54 4020.51 Test 5a/FCS-1% 535.61 271.60
Test 5e/FCS-0o 8899.82 7748.21 Test 5%/FCS-0% 1411.18 774.46
Test 2.5o/FCS-109 172.99 18144.74 Test 2.5o/FCS-10% 12.95 5740.39
289.20 9552.10 Test 2.5%/FCS-5o 5.41 1102.56
Test 2.5%/FCS-1o 2139.26 6752.15 Test 2.5e/FCS-1o 117.41 1254.98
Test 2.5o/FCS-0o 11=552.74 17645.83 Test 2.5%/FCS-0% 328.99 504.27
Test 0o/FCS-10% 93.50 11675.28 Test OojFCS-10o 5.63 4217.03
Test 0o/FCS-5% 99.80 8879.63 Test 0o/FCS-5o 7.12 989.86
Test 0o/FCS-1% 101.16 8374.13 Test 0o/FCS-1o 1.85 779.93
Test 0a/FCS-0o 104.32 4422.27 Test 0o/FCS-0% 2.36 251.71
PCT/AU2006/000185
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EXAMPLE 10 AQUEOUS NON-RADIOACTIVE PROLIFERATION ASSAY
In order to show that the compositions of the present
invention do not disturb the metabolism of cells in vitro
and, thus, the TNF-alpha suppressive effect is not due to
a metabolism problem of the cells a non-radioactive
proliferation assay was conducted.
The specific assay used was the CellTiter 960 AQueOlls Non-
Radioactive Cell Proliferation Assay from Promega. This
method is a non-radioactive alternative to the [3H]
thymidine incorporation cell proliferation assay.
Essentially, the manufacturer's instructions were
followed, but briefly, lOOpL of 5 x 106 K562 (human chronic
myelogenous leukaemia) cells in RPMI supplemented with 50
fetal bovine serum (FBS) were added to the wells of a 96-
well plate. Cells were then incubated for 20 hours at 37 C
in a humidified, 596 COz atmosphere. The medium was then
exchanged and allowed to equilibrate for 1 hour, then 20uL
of a solution comprising (3-(4,5-dimethylthiazol-2=-yl)-5-
(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium,
inner salt; (MTS) and phenazine methosulfate (PMS) was
added to each well. A Ohr absorbance reading at 490nm was
taken immediately and then absorbance was measured every
hour thereafter. Readings at 21 and 45 hours after the
addition of the MTS/PMS solution were also taken.
It can be seen from Figure 5 that these cells do not
proliferate. The dye wears off with a higher metabolism,
which is reflected in higher absorbance (y-axis). The data
from TL-treated + LPS challenged cells shows that the test
samples were slightly less metabolically active than the
controls, but at the same time TNF-alpha secretion was
suppressed. These data are not totally unexpected as the
need for a higher metabolism when compared with the
untreated + LPS-challenged cells would be less for these
cells. Non-LPS-challenged cells do not differ in
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metabolism, whether treated with the test compositions or
not.
From these data it can be concluded that the inhibition of
TNF-alpha secretion seen in Examples 6, 7, and 8, was not
due to a reduction in metabolic functioning of the cells.
It should be noted that in all experiments supra the
viability of cells, both test and control, were assessed
visually. In all instances the cells exposed to test
composition were viable as indicated by typical cell
spreading over the culture vessel. The cell spreading
noted was the same as the cell spreading noted fro the
non-challenged/non-treated cells.
