Note: Descriptions are shown in the official language in which they were submitted.
CA 02106695 1999-10-18 L~-~ Z
. ~ tl Barrister ~?
Hu he C~ U ~ ~ Solicitor
Ivor 1VI S'~ Ivor M. Hughes
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Patent f~ Trade Mark Agents O ~ Marcelo k. Sarkis,
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Our Ref.: PT-1205 ' '..::~---.-~-_
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VIA COURIER ;~>~~,~J4:-,-~ ~,.,
October 15, 1999
The Commissioner of Patents
Canadian Intellectual Property Office
INDUSTRY CANADA DEPOT POUR REC. GEN.
50 Victoria St., 12th Ploor DU CANADA 033-1365°t
Place du Portage 1 GSA # 28 4881-10 09-87913
Hull, PQ t;123 300.00
K1A OC9 5291 B# 004 0001-005 11:21A~1 10/20199
Dear Sir:
Re: Canadian Application Serial No. 2,106,695
of Hyal Pharmaceutical Corporation
for HYALURONIC ACID AND FORMS TO PREVENT
ARTERIAL RESTENOSIS
Classification: A61K-31/715
Allowed: April 19, 1999
Due Date: October 19 1999
Pursuant to the Notice of Allowance dated April 19, 1999, a copy of which is
enclosed herewith for your reference, enclosed please find a cheque in the
amount of
$300.00 in payment of the final fee for a large entity.
Also enclosed Applicant submits the required Page 15 in duplicate as requested
in
the Notice of Allowance.
In addition, please find enclosed a stamped, self-addressed verification card
which
we request be acknowledged and returned to this office at the earliest
opportunity.
We thank the Commissioner for his cooperation in this regard.
Respectfully submitted,
9
l ~. ~ ~/~, , ~. ,
';~, , ;,. ~, ~c.,~
Maria Giovannelli
Patent Administrator
MG/mse
Enclosures
175 Commerce Valley Dr. W., Suite 200, Thornhill, Ontario, Canada L3T 7P6
Phone: 905 771-6414 Fax: 905 771-6420
website: www.ivormhughes.com email: mailQivormhughes.com
CA 02106695 1999-10-18
OPIC CIPO
OFFICE DE LA PROPRIETE CANADIAN INTELLECTUAL
INTELLECTUELLE DU CANADA PROPERTY OFFICE
http:llopic.gc.ca Ottawa-Hull K1A OC9 http:llcipo.gc.ca
Ivor M. Hughes 1999/04) 19
200 -175 COIIMIerCe Valley Drive WASt N° de demande/Application No.
THORNHILL Ontario 2,106,695 A61K-31115
L3T 7P6
Votre rtfG~renca / Your rafo~:
1PT-1205
Titre/Title . HYALURONIC ACID AND FORMS TO PREVENT ARTERIAL
RESTENOSIS
Depose Is/Filing date . September 22, 1993
Revendications/Claims . 34 w'
Proprietaire/Owner . Hyal Pharmaceutical Corporation
Pages requises/Pages required: Page 15
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Canada Canada OPIC~CIPO19I
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- 2~.U~~~~
- TITLE OF INVENTION
USE OF HYALURONIC ACID AND FORMS TO PREVENT ARTERIAL
- RESTENOSIS.
FIELD OF INVENTION
This invention relates to the prevention of the narrowing (stenosis)
of tubular walls of an animal after the tubular walls have been traumatized.
In
on.e embodiment, this invention relates to the prevention of arterial
restenosis
after balloon angioplasty.
r
BACKGROUND OF THE INVENTION
Balloon angioplasty is a widely accepted method of opening
blockages in the coronary arteries. The balloon catheter was introduced
experimentally in the early 1960's and was first applied clinically in the
late
1970's. It has since assumed a major therapeutic role in the treatment of
single
and multiple vessel coronary artery disease (Baumgartner, H.R., 1963, Z. Ges.
Exp.
Med., 137:227). However in some patients after successful treatment by balloon
angioplasty, arterial restenosis occurs. This time however the narrowing of
the
inner diameter (ID) of the artery is caused by growth (proliferation) of
endothelial cells in the areas of irritation caused by the balloon
angioplasty.
Thus reblockage occurs not by cholesterol build-up but by build up of
endothelial
cells on the inner wall of the artery reducing the inner diameter (ID) of the
artery
leading to an infarct. In man, the restenotic lesion consists almost entirely,
though not exclusively of vascular smooth muscle cells (Glazier, J.J.,
Williams,
M.G., Madden, S. and Rickards, A.F., 1990, J. Roy. Coll. Phys. Lond., 24:292).
Their
' accumulation within the artery lumen is a result of cell migration and
proliferation. These two events are almost certainly due to the coordinated
interaction of a number of different cytokines likely released by early
accumulation of macrophages at the site of original tissue injury. This
narrowing of the inner diameter (ID) of tubular walls or proliferation of
cells is
not however restricted or limited to the coronary arteries. It can also occur
post
operatively causing restenosis in for example peripheral vascular systems.
-~~.~~~~la
A number of proposals have been made in the prior art to prevent
restenosis.
' - U.S. Patent 5,087,244 (Wolinsky et al.) purports to teach the use of a
catheter having an inelastic balloon at one end thereof, where the balloon has
minute perforations and contains a concentrated heparin solution which will be
released through the perforations contacting an area of the artery after
angioplasty to prevent restenosis.
U.S. Patent 5,116,864 (Hathaway et al.) purports to teach the
r
preventioh of restenosis in peripheral or cardiac vascular systems after
vascular
recanalisation by systemic administration of photo activatable psoralen to
give
serum psoralen levels which inhibit smooth muscle cell growth.
U.S. Patent 5,092,841 (Spears, J.R.) purports to teach the treatment of
an arterial wall injured during angioplasty by delivering bio-protective
material
between the wall and the angioplasty catheter so that the bio-protective
material
is entrapped and permeates into the tissues and vessels of the arterial wall
during opposition of the angioplasty catheter.
EP 356275-A (Petitou et al.) purports to teach the use of new o-
acylated glycosamino-glycan derivatives in the inhibition of post-operative
restenosis.
Berk., B.C. et al in the J. Am. Coll. Cardiol. dated 1991 Vol. 17 #6
Supplement B, pp 111B-117B purports to discuss the pharmacologic roles of
heparin and glucocorticoids to prevent restenosis after coronary angioplasty.
1N0 9209561 (Itoh et al.) purports to teach the use of new ACAT
inhibiting amide derivatives in treatment of restenosis after percutaneous
transluminal coronary angioplasty.
WO 9208472 (Scarborough et al.) purports to teach the use of platelet
antiadhesive peptides) obtained from snake venom for the prevention of
restenosis following angioplasty.
WO 9207852 (Bovy et al.) purports to teach the use of certain
biphenylalkyl xanthine derivatives to prevent post-angioplasty restenosis.
r
-3- 23.0~~~~::~
WO 9205782 (Pill, J.) purports to teach the use of thromboxane-A2-
receptor antagonists (I) in the preparation of medicaments for inhibition of
- proliferative developments in obstructive vascular disorders ie. arterial
restenosis.
WO 9118639 (GAj et al.) purports to teach the inhibition of stenosis
after balloon angioplasty, by the administration of fibronectin by continuous
or
bolus infusion, or by direct infusion into the stenotic region via the
angioplasty
catheter.
CA 2,042,159 laid open application (Ondetti, et al.) purports to teach
the use of ACE inhibitor (via the oral or parenteral route) for preventing or
reducing the risk of restenosis following angioplasty.
U.S. 4,929,602 (Harker, et al.) purports to teach a method of
inhibiting arterial restenosis by administration of D-phenyl alanyl-prolyl-
arginyl-
balomethyl ketone peptide derivative or a hydrolalin acid addition thereof.
U.S. 4,820,732 (Shell, et al.) purports to teach a composition
containing a prostaglandin compound for the reduction of restenosis and abrupt
stenosis.
Applicant is also aware of a company Glycomed developing a
fragment of Heparin that prevents arterial restenosis after balloon
angioplasty.
In the basic research efforts in the latter '70s and the early 80's, there
existed considerable confusion as to what role immunotherapy should take in
cancer. Activation or "hyping" of macrophages was thought to be important.
However, in an examination by Romans and Falk of peritoneal macrophages
obtained from patients with neoplastic disease, there was definite evidence
that
these macrophages were already activated yet were co-existing with cancer
cells
and not causing their destruction.
It has been shown by several independent investigators that the
malfunction of macrophages or the putitive block is due to excessive
prostaglandin and that this can be altered in tissue culture by
corticosteroids,
ASA, and the non-steroidal anti-inflammatory drugs, i.e. indomethacin, and
_,.._
P
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-4- 2~.~~~~
naproxen (NaprosynTM). Again, in animal tumors it was repeatedly
demonstrated that these substances could alter the response to neoplastic
cells
and that various combinations of these substances employed with immune
enhancing agents could produce very credible success in eliminating
experimental tumors. Lala and co-workers combined Indomethacin therapy
with Interleukin 2 and showed that this could effect a cure with experiment
neoplasm.
There were continued problems with the use of any of these agents
P
in the actual human in vivo experience. All of the non-steroidal anti
inflammatory agents . (NSAID) produced major toxicity in terms of gastro
intestinal, neurological, and other areas. Thus, the basis of the present
approach
is that under general circumstances the use of these agents in human disease,
in
sufficient amounts, the drug will penetrate to any pathological tissue to
alter
therapeutically local prostaglandin production. While intravenous preparations
exist of Indomethacin and now of other agents, the data is overwhelming, that
using these drugs alone produces prohibitive side effects in human subjects.
Therefore only insufficient amounts can be brought into the body to effect
more
than occasional responses in neoplasm.
However the majority of the evidence is present to indicate and
therefore it can be postulated that the basis for neoplastic development and
how
the initial cell "sneaks by" the immune surveillance mechanism relates to its
production of prostaglandin. One need postulate only one mutation to alter the
amount of prostaglandin synthesis produced by cells when they become
"malignant" to establish a mechanism of blocking out the initial cell in any
immune reaction, i.e. the macrophage. It therefore became essential to develop
a
combination of NSAIDS for clinical use to produce a major improvement in
response in neoplastic disease and other conditions where excessive
prostaglandin synthesis represents the basis of the pathogenesis of this
disease
state, i.e. arthritis, and various others of the so-called connective tissue
inflammatory disorders and/or auto-aggressive diseases.
r..,; ..:,. ; ,. - :~~. -:: :;-.'.~1
See also:
1. Modulation of Immunity in Cancer Patients by Prostaglandin
Antagonists, Immunit;r to Cancer II Alan R. Liss, Inc.; and
2. Goodwin, J.S. (1981) Prostaglandin E and Cancer Growth
Potential for Immunotherapy with Prostaglandin Synthesis Inhibitors,
Augmentive Agents in Cancer TherapX, Raven Press, New York.
It is therefore an object of this invention to provide a method of
treatment and formulations and pharmaceutical compositions for preventing
arterial restenosis after for example balloon angioplasty when endothelial
cell
proliferation occurs on the inner arterial wall caused by irritation to the
cells by
balloon angioplasty.
It is a further object of the invention to provide such treatment
using hyaluronic acid which is safe and essentially non-toxic.
It is a further object of the invention to provide methods of
treatment and formulations and pharmaceutical compositions generally for
preventing restenosis and inhibiting restenosis for example post operatively
in
peripheral vascular systems.
Further and other objects of the invention will be realized by
persons skilled in the art from the following summary of the invention and
discussion with respect thereto.
SUMMARY OF THE INVENTION
Applicants believe that forms of hyaluronan or hyaluronic acid
(especially hyaluronic acid and salts thereof) will prevent stenosis of the
inner
diameter (ID) of irritated tubular walls and particularly prevent restenosis
of the
arterial walls by for example the proliferation of endothelial cells as a
result of
irritation arising from balloon angioplasty or other treatment. The forms of
hyaluronic acid (for example hyaluronic acid and salts of hyaluronic acid) can
be
administered intravenously or by injection (in the case of direct injection of
small amounts) in effective amounts of about l0mg/70kg person to in excess of
3000mg/70kg person prior to, during and/or after injury.
2~.~~~~~
Hyaluronan or hyaluronic acid is a glycosaminoglycan that is
evolutionarily conserved and composed of repeating dissacharide units of N-
acetyl-glucosamine and glucuronic acid (Laurent and Fraser, 1991, Faseb J.,
6:2397). Hyaluronan exerts effects on cell adhesion, motility, growth and
differentiation and many of these effects are mediated by the expression of
hyaluronan receptors by responding tissues. Thus, hyaluronan was shown to be
able to aggregate white cells as a result of its interaction with receptors
present on
these cells (review, Turley, E.A., 1992, Can, Met. Rev., 11:21). Hyaluronan
accumulates almost exclusively at sites of increased receptor expression or in
the
presence of extracellular hyaluronan binding proteins. Two cell surface
associated receptors have been molecularly characterized and include CD44 and
RHAMM (Receptor for (Hyaluronan) HA - Mediate Motility]. RHAMM is
present in elevated amounts on cells, particularly macrophages and smooth
muscle cells responding to injury.
Therefore according to one aspect of the invention, there is
provided a process for the prevention of the narrowing of the tubular walls of
an
animal after the tubular walls have been traumatized (for example wherein the
tubular walls are arteries which have been subjected to balloon angioplasty)
the
process comprising the administration of a therapeutically effective non-toxic
amount of hyaluronic acid and/or salts thereof and/or homologues, analogues,
derivatives, complexes, esters, fragments and subunits of hyaluronic acid to
the
animal to prevent narrowing of the tubular walls. (The. hyaluronic acid may be
administered before, during and/or after the injury). Preferably the form of
hyaluronic acid is hyaluronic acid and salts thereof. The amount of the form
of
hyaluronic acid administered is preferably between about l0mg/70kg person and
about 3000mg/70kg person.
Thus according to another aspect of the invention, a process is
provided for the prevention of arterial restenosis after balloon angioplasty
in a
human, the process comprising the administration of a therapeutically
effective
non-toxic amount of hyaluronic acid and/or salts thereof and/or homologues,
-~- 21~~~~~5
analogues, derivatives, complexes, esters, fragments, and subunits of
hyaluronic
acid to the human to prevent arterial restenosis. Once again preferably the
form
of hyaluronan or hyaluronic acid is hyaluronic acid and salts thereof and
preferably the amount of the form of hyaluronic acid administered is between
about'l0mg/70kg person and about 3000mg/70kg person. The hyaluronic acid can
be administered before the procedure, for example, balloon angioplasty or
during
or after the procedure (immediately following).
The compositions are preferably administered intravenously in a
m
liquid form and include suitable diluents or other adjuvants as .required for
administration. With respect to the amounts to be administered, they may also
be administered by injection preferably at or proximate the site to be
treated.
A therapeutically effective amount of stenosis inhibiting drug may
be combined with the form of hyaluronic acid for administration. Such drugs
may comprise any of those previously mentioned, and those understood by
persons skilled in the art. One such drug is heparin. Another is a fragment of
heparin.
A therapeutically effective amount of a non-steroidal anti-
inflammatory drug (NSAID) for enhancing the effect of the form of hyaluronic
acid administered in the prevention of the narrowing of the tubular walls may
be administered with the form of the hyaluronic acid. The addition of the non-
steroidal anti-inflammatory agent will enhance the activity of the hyaluronic
acid in preventing the narrowing of the tubular walls for example enhancing
the
arterial restenosis prevention effect of the administered hyaluronic acid
and/or
salts thereof for example by reducing inflammation. The NSAID may be an
NSAID suitable for the purposes and may comprise Diclofenac, Indomethacin
(solubilized in for example N-Methyl Glucamine), Piroxicam, the (~)
tromethamine salt of Ketorolac, acetylsalicylic acid, Naproxen and the like.
The
amounts of NSAID may be appropriate accepted doses preferably administered to
patients. In some cases dose amounts up to l0mg of the NSAID/kg of body
weight (for example 1-2mg of NSAID/kg of body weight) are suitable. With
2~.~~~~1
Uiclofenac much larger amounts are appropriate. Where greater than normal
amounts of NSAIDS are used, in order to reduce side effects caused by excess
NSAID administration, greater than about 200mg of the form of Hyaluronan or
Hyaluronic Acid (HA) per 70kg person may be administered to reduce and
eliminate the side effects such as gastro-intestinal distress, neurological
abnormalities, depression, etc., of administration of the NSAID.
A therapeutically effective amount of a free radical scavenger and
anti-oxidant such as Vitamin C may also be added to the composition to enhance
the effect' of the Hyaluronic Acid and Hyaluronan administered. Such amount
may be up to 50 grams - 100 grams in a dosage as Vitamin C is soluble and is
excreted by the kidneys although much lower amounts are normally used. Other
anti-oxidants and free radical scavengers may also be used. In one embodiment
the composition comprises a form of hyaluronic acid, specifically preferred
hyaluronic acid and/or salts thereof, an NSAID, a stenosis inhibiting drug
and/or Vitamin C for administration for the prevention of the narrowing of the
tubular walls (for example the prevention of arterial restenosis after balloon
angioplasty). The composition may comprise a pluality of dosage amounts from
which one dosage amount may be withdrawn and used, each dosage amount
containing an effective amount of each of the constituents.
Thus according to another aspect of the invention, the use of a
pharmaceutical composition for the prevention of the narrowing of the tubular
walls of an animal or human after the tubular walls have been traumatized is
provided, the use being of a pharmaceutical composition comprising a
therapeutically effective non-toxic amount of hyaluronic acid and/or salts
thereof and/or homologues, analogues, derivatives, complexes, esters,
fragments, and subunits of hyaluronic acid in association with a suitable
diluent
or pharmaceutically acceptable carrier or other adjuvants to prevent narrowing
of the tubular walls - in one embodiment being administered just before the
trauma and in another, immediately after the trauma. Preferably the form of
hyaluronic acid or hyaluronan is hyaluronic acid and salts thereof, for
example
sodium hyaluronate.
According to another aspect of the invention, the use of a
pharmaceutical composition for the prevention of arterial restenosis after
balloon angioplasty in a human is provided, the use being of a pharmaceutical
composition comprising a therapeutically effective non-toxic amount of
hyaluronic acid and/or salts and/or homologues, analogues, derivatives,
complexes, esters, fragments, and subunits of hyaluronic acid in association
with
a suitable diluent, pharmaceutically acceptable carrier or other adjuvants to
prevent arterial restenosis preferably the form of hyaluronic acid is selected
from
hyaluronic acid and salts thereof and the amount of the form of hyaluronic
acid
is between about l0mg/70kg person and about 3000mg/70kg person.
In one embodiment the form of the pharmaceutical composition is .
for intravenous administration and is administered immediately before the
trauma (for example before balloon angioplasty). In another, the composition
is
administered immediately after the trauma.
According to still another aspect of the invention, the
pharmaceutical composition comprises a therapeutically effective amount of
non-steroidal anti-inflammatory drug (NSAID) far example Diclofenac,
Indomethacin (solubilized in N-Methyl Glucamine), Piroxicam, the (~)
tromethamine salt of Ketorolac, acetylsalicylic acid and the like for
enhancing the
effect of the form of hyaluronic acid in the prevention of the narrowing of
the
tubular walls.
Thus according to another aspect of the invention, the use of a
pharmaceutical composition for the prevention of arterial restenosis after
balloon angioplasty is provided, the use being of a pharmaceutical composition
comprising a therapeutically effective non-toxic amount of hyaluronic acid
and/or salts thereof in association with a suitable diluent or
pharmaceutically
acceptable carrier or other adjuvants to prevent arterial restenosis (by
administration for example intravenously of the composition). In some ~
embodiments the amount of the hyaluronic acid and/or salts thereof is between
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about l0mg/70kg person and about 3000mg /70kg person. The composition may .
further comprise a therapeutically effective amount of a non-steroidal anti-
inflammatory drug (NSAID) for enhancing the arterial restenosis prevention
effect of the administered hyaluronic acid and/or salts thereof administered.
The
NSAID may be at accepted appropriate doses depending on the NSAID for
example up to about l0mg/70kg of body weight (for example 1-2mg of NSAID/kg
of body weight). The appropriate dose for Diclofenac is much greater. Where it
is desired to use a dose excess of NSAID, the amount of hyaluronic acid and
salts
thereof preferably exceeds about 200mg/70kg person.
The composition may further comprise a therapeutically effective
amount of Vitamin C or other free radical scavanger or anit-oxidant for
enhancing the effects of the form of hyaluronic acid to prevent narrowing of
the
tubular walls. The Vitamin C may be used in large amounts (for example even
50 - 100 grams) although much smaller amounts are suitable.
The composition may also comprise an effective amount of a
stenosis inhibiting drug.
The composition may comprise hyaluronan or hyaluronic acid and
at least of one of an NSAID, Vitamin C, free radical scavenger, anti-oxidant
and
stenosis inhibiting drug.
According to another aspect of the inventian the use of:
an effective non-toxic amount of hyaluronic acid and/or salts
thereof and/or homologues, analogues, derivatives, complexes, esters,
fragments, and subunits of hyaluronic acid,
in the manufacture of a pharmaceutical composition is provided for
preventing the narrowing of the tubular walls of an animal after the tubular
walls have been traumatized, the use being characterized by a therapeutically
effective non-toxic amount of hyaluronic acid and/or salts thereof and/or
homologues, analogues, derivatives, complexes, esters, fragments, and subunits
of hyaluronic acid being incorporated into the pharmaceutical composition and
being sufficient and effective to prevent the narrowing of the tubular walls
-11-
which were traumatized as for example the arteries being damaged after balloon
angioplasty. Preferably the form of hyaluronic acid is hyaluronic acid and/or
salts thereof and the composition is in a liquid form. Preferably, the form of
hyaluronic acid is utilized at a dose between about l0mg to about 3000mg/70kg
person and more preferably the form of hyaluronic acid is utilized at a dose
greater than 200mg/70kg person. The composition may comprise a plurality of
dosage amounts.
In one embodiment the pharmaceutical composition is for
r
prevention of arterial restenosis after balloon angioplasty in humans. In
embodiments the pharmaceutical composition is given before the balloon
angio~lastv and immediately after the trauma.
According to another aspect of the invention, the use of;
(1) hyaluronic acid and/or salts thereof and/or homologues,
analogues, derivatives, complexes, esters, fragments, and subunits of
hyaluronic
acid, and
(2) an agent selected from a non-steroidal anti-inflammatory
drug (NSAID), a stenosis inhibiting drug, and Vitamin C, free radical
scavenger
and anti-oxidant and combinations thereof is provided
in the manufacture of a pharmaceutical composition (including
diluents, adjuvants and other carriers) for preventing the narrowing of the
tubular walls of an animal after the tubular walls have been traumatized
wherein a therapeutically effective amount of the hyaluronic acid and/or salts
thereof and/or homologues, analogues, derivatives, complexes, esters,
fragments, and subunits of hyaluronic acid is administered to humans together
with a therapeutically effective amount of the agent (2), the use being
characterized in that the amount of component (1) is an effective amount to
prevent the narrowing of the tubular walls of the animal and component (2)
enhances the effect of component (1) in the prevention of the narrowing of the
tubular walls. Once again the pharmaceutical composition may comprise a
plurality of dosage forms from which one dosage amount may be taken.
..:. .,.;:. ..:,.: , "..
C. . . ...:., . ...",.. .... . ..... .. . . . . . . . . .
-12-
Preferably component (1) is hyaluronic acid and/or salts thereof and
the composition is in a liquid form (for example for intravenous use or
injection). Preferably component (1) is utilized at a dose between about l0mg
to
about 3000mg/70kg person. In one use, component (1) is utilized at a dose
greater than 200mg/70kg person.
Component 2 is utilized at amounts effective to enhance the effect
of Component 1. Vitamin C may be utilized in amounts up to 50 - 100 grams per
dose although much smaller amounts are more desirable. The NSAID can be
r
administered in normally acceptable dose amounts depending on the NSAID.
With some NSAIDS the amounts are 1-2mg of NSAID per Kg of body weight, in
others up to about l0mg per kg bodyweight and in others such as Diclofenac,
much larger amounts. Where the NSAID is used in dose excesses (greater
amounts than the normally acceptable dose amounts, the amount of the form of
hyaluronic acid preferably exceeds about 200mg per 70kg person. Suitable
NSAIDS include Diclofenac, Piroxicam, Indomethacin (solubilized in N-methyl
glucamine), acetylsalicylic acid, ~ tromethamine salt of Ketorolac, naproxen
and
the like.
According to another aspect of the invention a pharmaceutical
composition is provided comprising (together with diluents as required) an
effective non-toxic amount of hyaluronic acid and/or salts thereof and/or
homologues, analogues, derivatives, complexes, esters, fragments, and subunits
of hyaluronic acid, for preventing the narrowing of the tubular walls of a
human
after the tubular walls have been traumatized, the composition being
characterized by an effective non-toxic amount of hyaluronic acid and/or salts
thereof and/or homologues, analogues, derivatives, complexes, esters,
fragments, and subunits of hyaluronic acid being incorporated into the
pharmaceutical composition to prevent the narrowing of the tubular walls.
Preferably the form of hyaluronic acid is hyaluronic acid and/or salts thereof
and
preferably the composition is in a liquid form (such as an intraveneous (LV.)
form in an LV. bag with diluents and pharmaceutically acceptable carriers and
t :~. ' .
-13-
adjuvants). The form of hyaluronic acid may b~ ~'u~l~ edv' t doses between
about
l0mg to about 3000mg/70kg person or more and in one embodiment the form of
- hyaluronic acid is utilized at a dose greater than 200mg/70kg person
(especially
where dosage excesses of NSAIDS are employed). In one embodiment the
pharmaceutical composition is for prevention of arterial restenosis after
balloon
angioplasty in humans. The composition may be administered before the
balloon angioplasty and/or after. The pharmaceutical composition may
comprise, a plurality of dosage amounts from which each dosage amount may be
taken.
According .to another aspect of the invention, a pharmaceutical
composition is provided comprising (together with diluents, adjuvants and
other pharmaceutically acceptable carriers as and if desired);
(1) hyaluronic acid and/or salts thereof and/or homologues,
analogues, derivatives, complexes, esters, fragments, and subunits of
hyaluronic
acid, and
(2) an agent selected from a non-steroidal anti-inflammatory
drug, a stenosis inhibiting drug, Vitamin C, an anti-oxidant and free radical
scavenger and combinations thereof ..
for preventing the narrowing of the tubular walls of an animal after
the tubular walls have been traumatized, the composition being characterized
by
an effective non-toxic amount of hyaluronic acid and/or salts thereof and/or
homologues, analogues, derivatives, complexes, esters, fragments, and subunits
of hyaluronic acid being incorporated into the composition together with a
therapeutically effective amount of the agent (2), to prevent the tubular
walls
from narrowing, the composition being characterized that the amount of
component (1) is an effective amount to prevent the narrowing of the tubular
walls of the animal and the amount of component (2) enhances the effect of
component (1) in the prevention of the narrowing of the tubular walls.
Preferably component (1) is hyaluronic acid and/or salts thereof most
preferably
sodium hyaluronate and preferably in a liquid dosage form such as an
-14-
intravenous form (LV. Bag). The composition may be made in bulk and
subsequently put into individual dosage amounts. The composition may be
packaged such that a plurality of dosage amounts are carried in a container
(storage container or reservoir) from which each dosage amount may be
withdrawn when needed for use. In some embodiments component (1) may be
utilized at a dose between about l0mg to about 1000mg/70kg person. In others
the dose amounts may be up to 3000mg/70kg person or more. Preferably
component (1) is utilized at a dose greater than 200mg/70kg person where dose
excesses of the NSAID of component (2) are utilized. In one embodiment the
pharmaceutical composition is for prevention of arterial restenosis after
balloon
angioplasty in humans and may be administered before, during and/or after the
treatment.
Component 2 is utilized at amounts effective to enhance the effect
of Component 1. Vitamin C may be utilized in amounts up to 50 - 100 grams per
dose. The NSAID can be administered in appropriate dose amounts depending
on the NSAID and if given in excess amounts the amount of the form of
hyaluronic acid preferably exceeds about 200mg per 70kg person. Suitable
1.;
NSAIDS are Diclofenac, Piroxicam, Indomethacin (solubilized in N-methyl
glucamine), acetylsalicylic acid, ~ tromethamine salt of Ketorolac, naproxen
and
the like.
When the composition comprises an agent selected from NSAID,
stenosis inhibiting drug, Vitamin C, free radical scavenger and anti-oxidant
and
combinations thereof, Applicants postulate that the hyaluronic acid and/or
salts
thereof and/or the homologues, analogues, derivatives, complexes, esters,
fragments, and/or sub units of hyaluronic acid also facilitates the transport
of the
agent to the site of irritation to enable the agent to penetrate the cells (in
the
artery, endothelial cells) which together will help prevent for example
arterial
restenosis.
By way of example and to illustrate the facilitation of the delivery or
transport of a chemical to a site in a mammal, when ethyl alcohol is injected
-15-
directly into a cancer tumor, and sonographic (ultrasound) assessment is made,
it
is not dispersed throughout the tumor. When the ethyl alcohol to be
administered into a tumor is carried by hyaluronic acid and/or salts thereof,
sonographic assessment of the tumor, demonstrates the dispersion of the ethyl
alcohol throughout the tumor.
While Applicants postulate that the hyaluronic acid facilitates the
transpork and delivery, Applicants' invention may be used as described
irrespective of the actual method of operation of the hyaluronic acid and/or
salts
thereof and/or the homologues, analogues, derivatives, complexes, esters,
fragments and sub units of hyaluronic acid vv~ith the NSAID, stenosis
inhibiting
drug, Vitamin C, free radical scavenger, and /or anti-oxidant.
The combination of hyaluronic acid and salts thereof and other
forms with different chemicals and drugs (for example Vitamin C, NSAIDS,
stenosis inhibiting drug, etc.) alters their distribution and performance in
the
human body and produces an unusual targeting for underperfused tissue and/or
pathological tissue. In this regard the use of ascorbic acid (Vitamin C) as a
free
radical scavenger (50 gm daily - 1000 times the daily dose in therapeutic
purposes
as a Vitamin) administered intravenously with 300 - 500rng of hyahurorvic acid
(sodium hyaluronate) reduces inflammation. The hyaluronic acid enhances the
effect of the ascorbic acid. It is thought that this enhanced activity
eliminates the
free radicals by acting as a free radical sca~renger.
A similar situation occurs with the NSAIDS. As a major amount
of soluble indomethacin is required, the chemical product was solubilized
using
n-methyl glucamine at a dilution of 5mg/ml of n-methyl glucamine (NMG).
This substance is then passed through a 22 micron MiliporeTM filter to produce
sterility. This material is non-toxic at 16 fold the therapeutic dose in
animals and
for this reason was considered appropriate to be used in human conditions.
Thus, IndocidT"' solubilized in NMG is administered to human patients
intravenously or intravascularly at a varying dose up to 10 mg/kg where each
dose of indoxnethacin is combined with for example 200 ~ 1000mg of hyaluronic
CA 02106695 1999-10-18
:;. . ;:.
'1 16 ~ ~. ~ c31~~ e~
acid (for example "LifeCoreTM" hyaluronic acid [sodium hyaluronate]) diluted
in
the original solution of indomethacin and NMG with for example the
"LifeCoreT"'" hyaluronic acid. This produces an appropriate mixture and can be
administered safely by any of the routes. [Similar clinical studies have been
done
with hyaluronic acid prepared by other methods, i.e. extraction. The extracted
material is satisfactory to use for intravenous.]
Thus when an NSAID for example indomethacin (dissolved in n-
methyl glucamine) or other NSAID is administered with greater than 200mg
P
hyaluronic acid for 1 - 2 mg/kg body weight of the NSAID (in one instance
indomethacin and NMG), no major toxic side effects occur such as gastro-
intestinal distress, neurological abnormalities, depression, etc., even at
elevated
amounts of indomethacin (if necessary). If the amount of hyaluronic acid is
decreased below about that amount, the usual side effects may begin to
reoccur.
In addition, the responses that have been observed are superior when the
NSAID (for example IndocidTM) is combined with hyaluronic acid demonstrating '-
~
clearly that the combination is now "targeting" to the tissue when
administered
by the systemic intravenous route. Thus, it has been observed that patients
when receiving in addition to other chemicals (for example ascorbic acid
[Vitamin C], 50 - 200 mg NSAID - hyaluronic acid (sodium hyaluronate) (for
example indomethacin and hyaluronic acid) experience dramatic relief of pain
immediately. Thus Applicants believe that the addition of the NSAID for
example with hyaluronic acid (sodium hyaluronate) prevents enzymatic
production of prostaglandin synthetase which blocks macrophage functioning.
Thus the hyaluronic acid (and salt and other forms) not only enhance the
activity of the NSAID but also reduce any side effects and toxicity that is
associated with the use of the prostaglandin synthesis inhibitors.
The hyaluronic acid and salts thereof may be utilized at varying
doses - 10 to 1000 mg/70 kg person. As there is no toxicity, the hyaluronic
acid
can obviously be administered in a dose excess (for example 3000 mg/70 kg
individual) without any adverse effects.
' ;':::.... .,::.:~. . ~',,.,~ . , ,.. ~ ;. ,. ~ ,:- ~.. . .. ' ,.. '
17
One form of hyaluronic acid and/or salts thereof (far example
sodium salt) and homologues, analogues, derivatives, complexes, esters,
fragments, and sub units of hyaluronic acid, preferably hyaluronic acid and
salts
and thereof suitable for use with Applicant's invention is a fraction supplied
by
Hyal Pharmaceutical Corporation. One such fraction is a 15 ml vial of Sodium
hyaluronate 20mg/ml (300mg/vial - Lot 2F3). The sodium hyaluronate fraction
is a 2% solution with a mean average molecular weight of about 225,000. The
fraction also contains water q.s. which is triple distilled and sterile in
accordance
n
with the ~J.S.P. for injection formulations. The vials of hyaluronic acid
and/or
salts thereof may be carried in a Type 1 borosilicate glass vial closed by a
butyl
stopper which does not react with the contents of the vial.
The fraction of hyaluronic acid and/or salts thereof (for example
sodium salt) and homologues, analogues, derivatives, complexes, esters,
fragments, and/or sub units of hyaluronic acid, preferably hyaluronic acid and
salts thereof may comprise hyaluronic acid and/or salts thereof having the
following characteristics:
a purified, substantially pyrogen-free fraction of hyaluronic acid .
obtained from a natural source having at least one characteristic selected
from
the group consisting of the following:
i) a molecular weight within the range of 150,000-225,000;
ii) less than about 1.25% sulphated mucopoly-saccharides on a
total weight
basis;
iii) less than about 0.6% protein on a total weight basis;
iv) less than about 150 ppm iron on a total weight basis;
v) less than about 15 ppm lead on a total weight basis;
vi) less than 0.0025% glucosamine;
vii) less than 0.025% glucuronic acid;
viii) less than 0.025% N-acetylglucosamine;
ix) less than 0.0025% amino acids;
x) a UV extinction coefficient at 257 nm of less than about
0.275;
~SS.;, ~~:~~~. ":'. ..', .,.., ,',..' ,.,.. ,.., ~ ~.. ,~ . ~.. .~~',,,...
,::'.. ~;~.,.. ":. .
-182~~~~~~
xi) a UV extinction coefficient at 280 nm of less than about 0.25;
and
xii) a pH within the range of 7.3-7.9. Preferably the hyaluronic
acid is mixed with water and the fraction of hyaluronic acid fraction has a
mean
average molecular weight within the range of 150,000-225,000. More preferably
the fraction of hyaluronic acid comprises at least one characteristic selected
from
the group consisting of the following characteristics:
i) less than about 1% sulphated mucopolysaccharides on a total
r
r
weight basis;
ii) less than about 0.4% protein on a total weight basis;
iii) less than about 100 ppm iron on a total weight basis;
iv) less than about 10 ppm lead on a total weight basis;
v ) less than 0.00166% glucosamine;
vi) less than 0.0166% glucuronic acid;
vii) less than 0.0166% N-acetylglucosamine;
viii) less than 0.00166% amino acids;
x) a UV extinction coefficient at 257 nm of less than about
0.23;
xi) a UV extinction coefficient at 280 nm of less than 0.19;
and
xii) a pH within the range of 7.5-7.7
Other forms
of hyaluronic
acid and/or
its salts, and
homologues,
derivatives,
complexes, esters,
fragments and
sub units of
hyaluronic acid
may be
chosen from othersuppliers. Applicants propose the use of sodium hyaluronate
produced and
supplied by
LifeCoreTM Biomedical,
Inc. having
the following
specifications
nSrr:::....
19
Characteristics Specification
Appearance White to cream
colored particles
Odor No perceptible odor
Viscosity Average < 750,000 Daltons
Molecular Weight
UV/Vis Scan, 190-820nm Matches reference scan
r
OD, 260nm < 0.25 OD units
Hyaluronidase Sensitivity Positive response
IR Scan Matches reference
pH, l0mg/g solution 6.2 -.7.8
Water 8% maximum
Protein < 0.3 mcg/mg NaHy
Acetate < 10.0 mcg/mg NaHy
Heavy Metals, maximum ppm
As Cd Cr Co Cu Fe Pb Hg Ni
2.0 5.0 5.0 10.0 10.0 25.0 10.0 10.0 5.0
Microbial Bioburden None observed
Endotoxin < 0.07EU/mg NaHy
Biological Safety Testing Passes Rabbit Ocular
Toxicity Test
Applicants also propose the use of forms of hyaluronic acid
described in the prior art.
The following references
teach hyaluronic acid, sources
thereof and
. ~
... .
: . ..~ ~. , . , . : ' : '... ~
... . ,:. .. .,, . , .. . ~ ~ . ~. . . . .
:1". : , . ,. ,., . .. . , . .. .m. . . . ... . '
-2°-~~ ~~~'~
processes of the manufacture and recovery thereof.
United States Patent 4,141,973 teaches hyaluronic acid fractions
(including sodium salts) having:
"(a) an average molecular weight greater than about 750,000,
preferably greater than about 1,200,000 - that is, a limiting
viscosity number greater than about 1400 cm3/g., and preferably
greater than about 2000 cm3/g.;
' (b) a protein content of less than 0.5% by weight;
(c) ultraviolet light absorbance of a 1% solution of sodium
hyaluronate of less than 3.0 at 257 manometers wavelength and
less than 2.0 at 280 manometers wavelength;
(d) a kinematic viscosity of a 1% solution of sodium
hyaluronate in physiological buffer greater than about 1000
centistokes, preferably greater than 10,000 centistokes;
(e) a molar optical rotation of a 0.1 - 0.2% sodium
hyaluronate solution in physiological buffer of less than -11 X
103 degree - cm2/mole (of disaccharide) measured at 220
manometers;
(f) no significant cellular infiltration of the vitreous and
anterior chamber, no flare in the aqueous humor, no haze or
flare in the vitreous and no pathological changes to the cornea,
lens, iris, retina, and choroid of the owl monkey eye when one
milliliter of a 1% solution of sodium hyaluronate dissolved in
physiological buffer is implanted in the vitreous replacing
approximately one-half the existing liquid vitreous, said HUA
being
(g) sterile and pyrogen free and
(h) non-antigenic."
Canadian Letters Patent 1,205,031 (which refers to United States
..
':
.f.
-21- ~~~lJ i~~
Patent 4,141,973 as prior art) refers to hyaluronic acid fractions having
average
molecular weights of from 50,000 to 100,000; 250,000 to 350,000; and 500,000
to
730,000 and discusses processes of their manufacture.
Where high molecular weight hyaluronic acid (or salts or other
forms thereof) is used, it must be diluted to permit administration and ensure
no
coagulation or interference with body function.
One formulation of Ascorbic Acid (Vitamin C) injection USP is
manufactured by Steris Laboratories, Inc., Phoenix, Arizona, 85043 U.S.A. and
comprises 22 mg/ml (equivalent to sodium ascorbate 250 mg/ml) in 30m1, 50m1,
or 100m1 individual containers, 30m1 size being preferred.
BRIEF DESCRIPTION OF THE DRAWINGS
Surgical procedures were undertaken to illustrate an embodiment
of the invention and analysis of results are illustrated in the enclosed
Figures in
which;
Figure 1 contains photographs of injured and sham operated
arteries.
Figure 2 illustrates in graph form RHAMM (Receptor for H A
(Hyaluronan) l~Iediated Motility) expression by the carotid arteries.
Figure 3 contains photographs of RHAMM and hyaluronan
expression in smooth muscle cells of the carotid artery 4 days after their
injury.
Figure 4 illustrates in a bar graph the effect of RHAMM, HA binding
[Hyaluronan-(Hyaluronic Acid)- binding] peptides (401-411) and anti-Rhamm
antibody on the Chemotaxis of Macrophage cell lines to complement (C5a).
Figure 5 illustrates in bar graph form the effect of RHAMM HA -
Binding peptides (401-411) of the chemotaxis of the neutrophiles to IL-8.
Figure 6 illustrates in bar graph form the effect of RHAMM HA -
binding peptide 401-411 on smooth muscle cell migration 5 hours after
wounding.
-22-
BRIEF DESCRIPTIGN OF THE EMBODIMENT
The following experiments were conducted.
rabbits were anesthetized and balloon angioplasty was performed
on them. The rabbits were perfused with hyaluronan (5 mg/ml) or buffer alone
5 and allowed their recover. Rabbits were sacrificed at 2, 24, 48 hours after
injury
and carotid arteries were processed for histology and serial 5-10 ~m sections
were
taken for processing. Sections were stained with hematoxylin or with anti
RHAMM antibodies. 10 sections of each treatment were analyzed.
The results of the analysis is described below with respect to the
10 Figures.
Figure 1: Injured carotid arteries show denudating of the
endothelia cell layer and adherence of white cells (Figure 1A). White cells
stained positively for RHAMM relative to IgG control background (Figure 1B).
Carotid arteries that were exposed to hyaluronan (Figure 1C) or sham operated
arteries (Figure 1D) show intact endothelial cell layer and do not exhibit
accumulations of white cells.
Figure 2: Western transblot analysis of RHAMM expression by
carotid arteries. Carotid arteries were homogenized, the released proteins
were
1V
electrophoresed on SDS-PAGE and the presence of RHAMM was detected with a
mono-specific antibody. The presence of the antibody was visualized with
chemiluminescence and the relative amounts of bound antibody were quantified
with optical densitometry. Operated animals displayed an acute, large rapid
increase in the presence of RHAMM. Levels of RHAMM had dropped by 5-6
days after tissue injury. Sham operated animals showed no increase in RHAMM
expression.
Figure 3: RHAMM (Figure 3A) and hyaluronan (Figure 3B)
expression in smooth muscle cells of the carotid artery 4 days after their
injury.
RHAMM expression on white cells elevated immediately (Fig. 1) while RHAMM
expression on smooth muscle cells was increased later and concommittent with
the initiation of their locomotion. Smooth muscle cells of sham operated
-23-
2~~~~q
animals did not show a similar increase in the expression of RHAMM (Figure
3C).
Figure 4: The effect of RHAMM peptides on chemotaxis of
neutrophiles in response to IL-8. RHAMM peptides that mimic the hyaluronan
binding domain of RHAMM inhibit the chemotaxis of neutrophiles in a Boyden
chamber assay.
Figure 5: RHAMM peptides and antibodies inhibit chemotaxis
of macrophage cells lines (S1, WEHI-3) in response to complement.
Complement but not heat-inactivated complement (56°C) stimulated
chemotaxis
of macrophage cell lines. RHAMM peptides that mimic hyaluronan binding
domain of RHAMM and anti-RHAMM antibodies inhibit chemotaxis.
Figure 6: RHAMM peptides inhibit the locomotion of smooth
muscle cells responding to injury. The RHAMM peptide that mimics the
hyaluronan binding domain of RHAMM inhibits the locomotion of injury
smooth muscle cells. The scrambled peptide had no effect indicating the
specificity to the "sense" peptide.
RHAMM expression (determined by a method of detection named
Western transblot analysis using mono-specific antibodies to RHAMM (Turley,
e.A., Austin, L., Vandeligt, K. and Clary, C., 1991, J. Cell Biol., 112:1041),
revealed
an acute increase in expression of this receptor that was detectable by two
hours
(Fig. 2), a time frame during which white cells were observed in control
animals
to adhere to the endothelium (Fig. 2). [For a further discussion of RHAMM see
the article "Identification of Two Hyaluronan-binding Domains in the
Hyaluronan Receptor RHAMM", Baihua Yang, Liying Zhang, and Eva Ann
Turley, The Journal of Biological Chemistry, Vol. 268, No. 12, Issue of April
25,
pp. 8617-8623, 1993.] RHAMM was also increased in the contralateral artery
suggesting the release of a soluble factor that regulates RHAMM expression
from
the injured tissue. However, sham operated animals showed little increase in
the expression of RHAMM (Fig. 2). In experimental animals, expression of
RHAMM was maintained for several days, then levels dropped. Examination of
-24-
fixed tissue revealed that the major cells expressing RHAMM were activated
white cells and smooth muscle cells (Fig. 1, 3). The involvement of RHAMM in
white cell and smooth muscle cell locomotion was assessed in vitro using image
analysis to measure random locomotion and Boyden chambers to measure
chemotaxis. Peptides (100 ng/plate) that mimic regions (in particular the
hyaluronan binding domains) of RHAMM, inhibit macrophage (Fig. 4)
neutrophiles (Fig. 5) and smooth muscle cell (Fig. 6) migration to a highly
significant degree (p > 0.0001, Student's "T" test). Collectively, these
results
indicate that RHAMM, and in particular its Hyaluronan binding capability, is
essential for locomotion of white cells and smooth muscle cells and that its
expression is elevated at the site of tissue injury following experimental
ballon
catherization in rabbits.
Hyaluronan treatment of rabbits just prior to their injury abolished
adherence of white cells to endothelium resulting in tissue that appeared
intact
as detected by histological criteria (Fig. 1). Several days after injury,
carotid
arteries of hyaluronan treated rabbits appeared similar to controls displaying
an
intact endothelium.
The rationale for these results is that hyaluronan bound to cells
expressing high levels of its receptor, RHAMM and prevented subsequent
interactions of these cells with the endothelium. It is expected, that
expression of ::
the other hyaluronan receptor, CD44, is also elevated.
For a discussion and illustration of terms and expressions in this
application, reference should be had to the enclosed unpublished article
attached
as "Schedule A" entitled "Neointimal Formation after Balloon Catheter Injury:
A Role of Hyaluronan and the Hyaluronan Receptor RHAMM", the portions
thereof which discuss and illustrate terms and expressions referred to herein
are
incorporated herein by reference.
As many changes can be made to the invention without departing
from the scope of the invention, it is intended that all material contained
herein
be interpreted as illustrative of the invention and not in a limiting sense.
