Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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TITLE OF INVENTION
Modulation of Cellular Activity
FIELD OF INVENTION
This invention relates to the modulation of cellular activity and
particularly to the modulation of cellular activity in a human employing forms of
hyaluronic acid, including specific molecular amounts and fractions of, for example,
sodium hyaluronate and molecules which mimic forms of hyaluronic acid.
BACKGROUND OF INVENTION
Cell adhesion molecules, which were originally thought to be mere
glue that hold cells together in tissues, are now also thought to have a wider
contribution to the function of the human body. In this regard, see BIOTECH GETSA GRIP ON CELL ADHESION, SCIENCE, Volume 260 (May 14, 1993).
Novel therapies have been proposed based on these cell adhesion
molecules. One class of endothelial adhesion molecules referred to includes the
endothelial adhesion molecule ICAM-1. This molecule (ICAM-1) was discovered to
carry the human rhino viruses (which cause about 50% of colds) into the body's
cells. The rhino viruses enter the body's cells by latching on to the adhesion
molecule ICAM-1.
By using soluble ICAM-1 as a decoy, it was proposed that the
infection of the cells can be blocked. However, soluble ICAM just was not very
sticky and therefore, the proposal has not reached the market.
Genetic engineering techniques have been used to fuse antibody
fragments to rhino virus - binding portions of ICAM-1 in an attempt to discover a
prevention for the common cold.
Another adhesion molecule, CD44, is found normally on Iymphocytes.
The CD44 adhesion molecule also studs the surface of pancreatic tumour cells that
are metastatic. The CD44 molecules may have inadvertently disguised the cancer
cells and allows them to circulate freely in the bloodstream.
It has now been discovered that adhesion molecule ICAM-1 is a cell-
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surface receptor for hyaluronic acid (hyaluronan). ICAM-1 is expressed (producedand put on the cell surface) in the liver endothelial cells and corneal epithelium
cells. ICAM-1 is overexpressed in inflammation, cancer and infection.
HARLEC (Hyaluronic Acid [Hyaluronan] Receptors Liver Endothelial
5 Cells) is also a cell-surface receptor for Hyaluronan and is also expressed
(produced and put on the cell surface) in liver endothelial cells and corneal
epithelial cells. (This Inventor believes the two to be one and the same adhesion
molecule or, at the very least, so intimately related to be one another as to be close
to being one and the same.)
The molecules CD44 and RHAMM (Receptor for HA-Mediated
Motility) are also cell-surface receptors for hyaluronic acid (hyaluronan). RHAMM
is a Regulatory molecule. CD44 is an adhesion molecule.
It is therefore an object of the invention to provide new methods of
treatment of disease and conditions, new uses for forms of hyaluronic acid
15 (hyaluronan) (HA), new dosage amounts of pharmaceutical compositions and new
pharmaceutical compositions suitable for use in such treatments and uses of forms
of HA.
It is a further object of this invention to provide new methods of
treatment of disease and conditions which employ molecules that mimic hyaluronic20 acid and pharmaceutically acceptable salts thereof (for example sodium
hyaluronate) in respect of their ability to bind to the same receptors as the forms of
hyaluronic acid (for example, high affinity cell-surface receptors for hyaluronic
acid), new uses for these hyaluronic acid mimicking molecules, new dosage
amounts of pharmaceutical compositions which comprise such molecules and new
25 pharmaceutical compositions suitable for use in such treatments and uses of the
mimicking molecules.
Further and other objects of the invention will be realized to those
skilled in the art from the following summary of the invention and examples.
3 ~1~6013
SUMMARY OF INVENTION
According to one aspect of the invention, a method for the modulation
of cellular activity of tissue and cells expressing a high affinity cell-surface receptor
for hyaluronic acid such as an adhesion molecule (for example, ICAM-1, HARLEC
5 and CD44) and a regulatory molecule (for example, RHAMM) of a human is
provided the method comprising the administration of a non-toxic effective amount
of a form of hyaluronic acid (for example, hyaluronic acid, a pharmaceutically
acceptable salt thereof, [for example, sodium hyaluronate having an average
molecular weight less than 750,000 daltons [for example, less than 50,000 daltons,
between about 100,000 to 150,000 daltons and 225,000 daltons] and from Hyal
Pharmaceutical Corporation within the range of 150,000-225,000 daltons and
those disclosed in U.S. Patent Application 08/143983, and molecular weight
fractions of a form of sodium hyaluronate (for example, fractions disclosed in
Canadian Letters Patent 1205031 (to Fidia) such as those from 50,000-100,000
15 daltons, 250,000-350,000 daltons and 500,000-730,000 daltons, or other fractions
such as less than 50,000 daltons and those between 100,000-150,000 daltons, or
homologues, analogues, derivatives, complexes, esters, fragments and/or sub
units of hyaluronic acid and/or combinations thereof) preferably hyaluronic acid, a
pharmaceutically acceptable salt thereof (for example sodium hyaluronate) and
20 combinations thereof, having a molecular weight less than 750,000 daltons, and
molecules which mimic the forms of hyaluronic acid aforesaid in respect of theirability to bind to the same receptors as the form of hyaluronic acid, to a human to
modulate cellular activity of tissues and/or cells expressing a molecule such as a
high affinity cell-surface receptor for hyaluronic acid (for example, an adhesion or
25 regulatory molecule) in the human body, in a pharmaceutical carrier or excipient
tolerable by the human (for example, sterile water).
Thus, for example, where an inflammatory response (reaction) may
be set up in the area of damage or trauma, the response may include the migration
4 215631~
of inflammatory cells (for example, neutrophils, macrophages, and other white
blood cells) to the area. The damage caused by setting up the inflammatory
response may be greater than the actual damage or trauma caused. The same will
be the case for fibrosis. The administration of the form of hyaluronic acid will bind
to the HA receptor (the high affinity cell-surface receptor) of the molecule, for
example adhesion molecule or regulatory molecule, thus modulating the body's
response and thus, subsequent and consequent damage. In the same way, the
administration of the molecules which mimic the form of hyaluronic acid in respect
of their ability to bind to the same receptors (as the form of hyaluronic acid) will bind
to the HA receptor. Examples of molecules which mimic the form of hyaluronic acid
are for example, monoclonal antibodies which bind to the active site of the
receptor, peptides which fit into the site of the receptor and synthetic chemicals
which fit into the receptor site.
Drugs for the treatment of the disease and for condition may also
accompany the form of hyaluronic acid and/or molecules which mimic the form of
hyaluronic acid in which event the drug enhances the modulation of the form of the
HA and molecule mimicking the HA activity. The form of HA further targets the
drug to the adhesion molecule (for example, ICAM-1, HARLEC, and CD44) and
RHAMM, a Regulatory molecule.
Suitable drugs may comprise for example, a free radical scavenger
(for example ascorbic acid (Vitamin C)), an anti-cancer agent, chemotherapeutic
agent, anti-viral agents for example a nonionic surfactant, e.g. nonoxynol-9
[nonylphenoxy polyethoxy ethanol] found in DelfenTM contraceptive cream, and
anionic surfactants (e.g. cetyl pyridinium chloride) and cationic surfactants (e.g.
benzalkonium chloride), non-steroidal anti-inflammatory drugs (NSAID) for
example indomethacin, naproxen, diclofenac and (+/-) tromethamine salt of
ketorolac (sold under the trademark ToadolTM) and steroidal anti-inflammatory
drugs, anti-fungal agent, detoxifying agents (for example for administration rectally
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in an enema), analgesic, bronchodilator, anti-bacterial agent, antibiotics, drugs for
the treatment of vascular ischemia (for example diabetes and Berger's disease),
anti-body monoclonal agent, minoxidil for topical application for hair growth,
diuretics (for example furosemide (sold under the trademark LasixT M ),
5 immunosuppressants (for example cyclosporins), Iymphokynes (such as interleukin
- 2 and the like), alpha-and-B-interferon, and the like.
According to another aspect of the invention, use of a form of
hyaluronic acid, for example, hyaluronic acid, a salt thereof (for example, sodium
hyaluronate having a molecular weight less than 750,000 daltons, for example,
10 225,000 daltons), molecular fractions thereof [for example, those disclosed from
Hyal Pharmaceutical Corporation in the range of 150,000-225,000 daltons, those
in Canadian Letters Patent 1205031 (to FIDIA) such as those from 50,000-100,000
daltons, 250,000-350,000 daltons and 500,000-730,000, daltons or other fractions],
homologues, analogues, derivatives, complexes, esters, fragments and/or subunits15 of hyaluronic acid and/or combinations thereof and a molecule which mimics the
form of hyaluronic acid in respect of binding to the same receptors as the form of
hyaluronic acid is provided to modulate cellular activity of tissues and/or cells
expressing a high affinity cell-surface receptor in the human body. The form of
hyaluronic acid may be used with a pharmaceutically tolerable excipient or carrier
20 (for example, sterile water). Once again, tissue or cell modulation is enhanced in a
person when suffering a disease and/or condition. The HA and/or HA mimicking
molecule binds to the HA receptors of the molecule such as the high affinity cell-
surface receptor for a form of hyaluronic acid, for example an adhesion or
regulatory molecule. Once again, the form of HA and HA mimicking molecule may
25 be used with a suitable drug (as described previously). Both the form of HA, HA
mimicking molecule and drug are in effective non-toxic amounts.
The form of HA and HA mimicking molecule may also be used to
prevent a disease or condition (such as a cold) by preventing the human rhino
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virus from entering the body's cells by the form of HA and/or HA mimicking
molecule binding with the ICAM-1 adhesion molecules (instead of the Rhino virus
binding or latching on).
Thus, according to another aspect of the invention, Applicant provides
a novel method of preventing a disease and/or condition, the method comprising
administering an effective non-toxic amount of a form of hyaluronic acid (for
example, hyaluronic acid, a pharmaceutically acceptable salt thereof, [for example,
sodium hyaluronate having a molecular weight less than 750,000 daltons for
example, 225,000 daltons, from Hyal Pharmaceutical Corporation in the range of
n 150,000-225,000 daltons and those disclosed in U.S. Patent Application
08/143983 and those molecular weight fractions of a form of sodium hyaluronate
[for example, fractions disclosed in Canadian Letters Patent 125031 (to Fidia) such
as those from 50,000-100,000 daltons, 250,000-350,000 daltons and 500,000-
730,000 daltons or other fractions], homologues, analogues, derivatives,
complexes, esters, fragments and/or subunits of hyaluronic acid and/or
combinations thereof and/or molecules which mimic the forms of hyaluronic acid in
respect of their ability to bind to the same receptors as the form of hyaluronic acid,
to a human to modulate cellular activity of tissues and/or cells expressing a
molecule such as a high affinity cell-surface receptor for a form of hyaluronic acid,
for example, an adhesion or regulatory molecule in the human body to thereby
prevent a disease or condition. The administration of the form of HA or mimicking
molecule is preferably in a pharmaceutically tolerable excipient carrier (for
example, sterile water). Thus, for example, the common cold may be prevented.
Drugs which may assist to prevent a disease or condition may be administered with
the form of hyaluronic acid (HA) and/or HA mimicking molecule. Thus, for example,
acetylsalicylic acid may be administered with a form of HA and/or mimicking
molecule for the prevention of stroke.
The administration of the form of hyaluronic acid (HA) and HA
7 ~15~
.
mimicking molecule thus for example inhibits cellular adhesion and/or modulates
cellular activity in a human. The HA and mimicking molecule bind to the HA
receptors of the molecule at the high affinity cell-surface receptor for a form of the
hyaluronic acid, for example, adhesion molecule (for example ICAM-1, HARLEC
and CD44) and/or regulatory molecule (Rhamm, for example). Thus, the
administration of the form of hyaluronic acid and mimicking molecule will bind to
the HA receptor of the molecule, thus preventing the disease or condition (for
example, preventing the binding of the rhino virus to the adhesion molecule ICAM-
1 thereby preventing the rhino virus entering the body's cells by latching onto the
adhesion molecule ICAM-1).
Where drugs are taken for the prevention of a disease and/or
condition may, as discussed above, accompany the form of hyaluronic acid used toprevent the disease or condition. For example, for the prevention of a stroke,
people today take acetylsalicylic acid (aspirin) in an effective amount. The drug
may be accompanied by a suitable form of hyaluronic acid in an effective non-toxic
amount (for example sodium hyaluronate having a molecular weight less than
750,000 daltons) and/or by a mimicking molecule in an effective amount. Thus, the
taking of the two together will, it is believed, reduce the risk of stroke even more
than the individual taking aspirin alone. The form of HA further targets the drug to
the areas in need of treatment while the form of HA binds with the high affinity cell-
surface receptor for a form of hyaluronic acid as expressed by cells and/or tissue
such as an adhesion molecule (for example, ICAM-1, HARLEC and CD44) and a
Regulatory molecule (for example RHAMM) modulating their activity.
The administration may be given, among other methods,
intravenously, intra-arterially, intraperitoneally, intrapleurally, percutaneously.
(intra-cutaneously - into the skin (for example, targeting the epidermis), by topical
application of effective amounts and by application to the mucosa by topical
application, for example, intranasally, rectally (for example, in the form of an
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enema or by topical application of specific areas in the rectum), and by direct
injection.
Where the form of hyaluronic acid is to be administered, varying
effective doses may be employed - for example, 10 to 1000mg/70kg. person with
5 optimal doses tending to range between 50 and 500mg/70kg. person. As there is
no toxicity in humans, the hyaluronic acid can obviously be administered in a dose
excess (for example 3000mg/70kg. individual) without any adverse effects.
Where the administration is topical (for example, applied to the skin or
mucosa), in excess of 5 mg per cm2 (square centimetre) of skin or exposed tissue10 (including mucosa) of the form of hyaluronic acid, should be applied.
With respect to the treatment of pain, preferably a minimum of 10mg
per cm2 (10mg/cm2) of the form of HA should be applied topically.
The HA mimicking molecules can be given in effective dosage
amounts as persons skilled in the art may use.
Thus, effective dosage amounts can contain at least about 10mg of
the form of hyaluronic acid per 70kg person to in excess of 1000mg per 70kg
person where the administration is non-topical. When an NSAID for example,
indomethacin (dissolved in n-methyl glucamine) or other NSAID is administered
with greater than 200mg hyaluronic acid for example, sodium hyaluronate per 70kg20 person with 1 - 2 mg/kg body weight of the person, 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 belowthat amount, the usual side effects may begin to reoccur. Where administration is
25 topical, the dosage amounts contain at least about 5mg of the form of hyaluronic
acid / cm2 of the skin, mucosa or tissue to which the dosage amount is to be
applied.
The Drugs accompanying the form of hyaluronic acid and/or HA
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mimicking molecule are adjusted with the form of treatment to be an effective non-
toxic amount thereof.
One form of hyaluronic acid and/or pharmaceutically acceptable salts
thereof (for 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
supplied by Hyal Pharmaceutical Corporation. One such form is a 15 ml vial of
Sodium hyaluronate 20mg/ml (300mg/vial - Lot 2F3). The sodium hyaluronate is a
2% solution with a mean average molecular weight of about 225,000 daltons. The
vial also contains water q.s. which is triple distilled and sterile in accordance with
the U.S.P. for injection formulations. The vials of hyaluronic acid and/or saltsthereof 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/amount of hyaluronic acid and/or pharmaceutically
acceptable salts thereof (for example sodium salt) and homologues, analogues,
derivatives, complexes, esters, fragments, and sub units of hyaluronic acid,
preferably hyaluronic acid and pharmaceutically acceptable 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 mucopolysaccharides
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;
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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;
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 of150,000-225,000. More preferably the fraction of hyaluronic acid comprises at
least one characteristic selected from the group consisting of the following
characteristic.
i) less than about 1% sulphated mucopolysaccharides on
a total 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;
ix) a UV extinction coefficient at 257 nm of less than about
0.23;
x) a UV extinction coefficient at 280 nm of less than 0.19;
and
xi) a pH within the range of 7.5-7.7
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In addition, Applicant proposes successful employment of sodium
hyaluronate produced and supplied by LifeCoreTM Biodmedical, Inc. having the
following specifications:
Characteristics Specification
Appearance White to cream colored particles
Odor No perceptible odor
Viscosity Average Molecular Weight < 750,000 Daltons
UV/Vis Scan, 190-820nm Matches reference scan
OD, 260 nm < 0.25 OD units
Hyaluronidase Sensitivity Positive response
IR Scan Matches reference
pH, 10mg/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
Another form of sodium hyaluronate is sold under the name
Hyaluronan HA-M5070 by Skymart Enterprises, Inc. having the following
specifications:
Specification's Test
Results
Lot No. HG1004
pH 6.12
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Condroitin Sulfate not detected
Protein 0.05%
Heavy Metals Not more than 20 ppm
Arsenic Not more than 2 ppm
Loss on Drying 2.07%
Residue on Ignition 16.69%
Intrinsic Viscosity 12.75 dl/s (XW: 679,000)
Nitrogen 3.14%
Assay 104.1 %
Microbiological Counts 80/g
E. coli Negative
Mold and Yeast Not more than 50/g
Other forms of hyaluronic acid and/or its salts, and homologues,
derivatives, complexes, esters, fragments and sub units of hyaluronic acid may be
15 chosen from other suppliers, for example those described in the prior art. The
following references teach hyaluronic acid, sources thereof and processes of themanufacture 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 1,200,00 - 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 nanometers wavelength
and less than 2.0 at 280 nanometers wavelength;
(d) a kinematic viscosity of a 1% solution of sodium hyaluronate in
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'_
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 nanometers;
(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
Patent 4,141,937 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
20 thereof) is used, it must be diluted to permit administration and ensure no
intramuscular coagulation.
According to one aspect of the invention, a dosage amount of a
pharmaceutical composition is provided for the modulation of cellular activity of
tissue and cells expressing a molecule having a high affinity cell-surface receptor
25 for a form of hyaluronic acid, for example, an adhesion molecule (for example,
ICAM-1, HARLEC and CD44) and a Regulatory molecule (for example, RHAMM) of
a human, the dosage amount comprising a non-toxic effective amount of a form of
hyaluronic acid (for example, hyaluronic acid, a pharmaceutically acceptable salt
2156~1~
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thereof, [for example, sodium hyaluronate having a molecular weight less than
750,000 daltons for example, 225,000 daltons], molecular weight fractions of a
form of sodium hyaluronate (for example, fractions from Hyal Pharmaceutical
Corporation in the range of 150,000-225,000 daltons, and those disclosed in U.S.5 Application 08/143983 and those disclosed in Canadian Letters Patent 1205031
(to Fidia) such as those from 50,000-100,000 daltons, 250,000-350,000 daltons
and 500,000-730,000 daltons, or other fractions such as less than 50,000 daltonsand those between about 100,000-150,000 daltons, homologues, analogues,
derivatives, complexes, esters, fragments and/or sub units of hyaluronic acid and/or
combinations thereof) and HA mimicking molecules in respect of their ability to bind
to the same receptors, to a human to modulate cellular activity of tissues and/or
cells expressing a molecule having a high affinity cell-surface receptor for a form of
hyaluronic acid, for example an adhesion molecule and/or a Regulatory molecule
in the human body, in a pharmaceutical carrier excipient tolerable by the human
15 (for example, sterile water).
The result of the treatment by the dosage amount is that tissue and/or
cell modulation is enhanced in the person suffering the disease or condition. The
result of the administration of the form of hyaluronic acid (HA) or HA mimickingmolecule is, for example, the inhibition of cellular adhesion and/or modulation
20 and/or regulation of cellular activity. The HA and HA mimicking molecule eachbinds to the HA receptors of the molecule (for example, adhesion molecule,
Regulatory molecule and the like). Thus prevention of a disease or condition maybe accomplished. The unexpected high affinity of the HA and HA mimicking
molecule for the cell-surface HA receptors enable the modulation of disease or
25 condition for example, the modulation of the inflammatory process, fibrosis, and, for
oncogene control (to prevent cancer and metastases).
The dosage amount of the pharmaceutical composition may also
comprise an effective non-toxic amount of a medicine (drug) or therapeutic agent
~15~13
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for the treatment of the disease and/or condition accompanying the form of
hyaluronic acid or form of hyaluronic acid mimicking molecule for the prevention of
the disease or condition. The drug therefore can enhance the modulation of the
form of the HA and HA mimicking molecule activity. The form of HA further targets
the drug to the cell-surface receptor of the molecule, such as for example, ICAM-1,
HARLEC, and CD44 (examples of adhesion molecules) and RHAMM (a Regulatory
molecule).
Thus, according to another aspect of the invention, a dosage amount
of a pharmaceutical composition may comprise:
i) a medicinal and/or therapeutic agent in a non-toxic
therapeutically effective amount to treat a disease or
condition;
ii) a non-toxic therapeutically effective amount of a form of
hyaluronic acid (for example, hyaluronic acid, a
pharmaceutically acceptable salt thereof, [for example,
sodium hyaluronate having a molecular weight less than
750,000 daltons, for example, 225,000 daltons],
molecular weights of a form of sodium hyaluronate (for
example, from Hyal Pharmaceutical Corporation within
the range of 150,000-225,000 daltons and those
disclosed in U.S. Patent Application 08/143983 and
those disclosed in Canadian Letters Patent 1205031 (to
Fidia) such as those from 50,000-100,000 daltons,
250,000-350,000 daltons, and 500,000-730,000
daltons, or other fractions, such as less than 50,000
daltons and between 100,000-150,000 daltons,
homologues, analogues, derivatives, complexes, esters,
fragments and/or sub units of hyaluronic acid and/or
215S013
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combinations thereof) and HA mimicking molecules
which mimic the form of hyaluronic acid in respect of
their ability to bind to the same receptors, to a human to
modulate cellular activity of tissues and/or cells
expressing a molecule having a high affinity cell-surface
receptor for a form of hyaluronic acid, for example, an
adhesion molecule or regulatory molecule in the human
body, and
iii) a pharmaceutically tolerable excipient (for example, sterile water);
wherein component (ii) is in such form that when the dosage amount
of the pharmaceutical composition is administered/applied, component (ii) is
available to modulate the cellular activity of tissue and cells expressing, for
example, an adhesion molecule (for example, ICAM-1, HARLEC and CD44) and a
Regulatory molecule (RHAMM) of a human by for example, binding with the
receptor for the form of hyaluronic acid and/or HA mimicking molecules, and
component (ii) is immediately available to transport component (i) in the body or
into the skin as the case may be if component (ii) is a form of hyaluronic acid.Suitable medicines (drugs) and therapeutic agents may comprise for
example, a free radical scavenger (for example ascorbic acid (Vitamin C)), an anti-
cancer agent, chemotherapeutic agent, anti-viral agents for example a nonionic
surfactant, e.g. nonoxynol-9 [nonylphenoxy polyethoxy ethanol] found in DelfenTMcontraceptive cream, and anionic surfactants (e.g. cetyl pyridinium chloride) and
cationic surfactants (e.g. benzalkonium chloride), non-steroidal anti-inflammatory
drugs (NSAID) for example indomethacin, naproxen and (+/-) tromethamine salt of
ketorolac (sold under the trademark ToadolTM) and steroidal anti-inflammatory
drugs, anti-fungal agent, detoxifying agents (for example for administration rectally
in an enema), analgesic, bronchodilator, anti-bacterial agent, antibiotics, drugs for
2156~1~
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the treatment of vascular ischemia (for example diabetes and Berger's disease),
anti-body monoclonal agent, minoxidil for topical application for hair growth,
diuretics (for example furosemide (sold under the trademark LasixT M ),
immunosuppressants (for example cyclosporins), Iymphokynes (such as interleukin
5 - 2 and the like), alpha-and-B-interferon, and the like.
Dosage amounts of the pharmaceutical compositions may be
accumulated in containers to supply multiple dosage amounts from which an
individual dosage amount may be taken.
The invention is demonstrated with respect to the following examples:
n Example 1
The polysaccharide hyaluronan (hyaluronic acid; HA or HYA) is
rapidly cleared from the circulation, primarily by endothelial cells of the liver via
receptor-mediated endocytosis. The receptor for HYA on these cells has been
characterized and purified from rat liver endothelial cells (LEC) and polyclonal15 antibodies produced. Other cell-surface "receptors" for HYA have been described
and include the Iymphocyte homing receptor CD44 and a receptor for HYA-
mediated motility of fibroblasts (RHAMM). Many tumours have been reported to be
enriched in HYA and HYA binding sites on cells derived from some tumours have
been described.. Previous findings have shown that transformed tissues such as in
20 mouse mastocytomas and human mammary carcinomas stain positively for the HA-
receptor originally found on liver endothelial cells.
The present work was initiated in order to determine if accessible
HYA binding sites are present in tumour tissue in vivo, and the relation of these
possible sites to previously described HYA-binding proteins. A recently developed
25 1 251-labelling method, that does not significantly alter the Mw of the polysaccharide
nor its receptor-binding properties, was used.
~15601 ~
- 18 -
MATERIALS AND METHODS
Polysaccharides
HA used for labelling and uptake and turnover-studies was extracted
from avian tissue and supplied by Hyal Pharmaceutical Corporation, Toronto,
Canada. The molecular weight distribution of the HA was determined by
chromatography on a calibrated column of Sephacryl HR with porosities noted as
400, 1000 and 2000 (Pharmacia, Uppsala, Sweden) in 0.25M NaCI, 0.05%
chlorbutanol. The HA content in each fraction was monitored by determination of
the absorbance at 214 nm. Radioactivity was measured by gamma-counting on a
Packard auto-gamma gamma-counter. (The mean average molecular weight is in
the order of 450,000 daltons and (made up from powder whose mean average
molecular weight is 500,000-800,000 daltons).
Labelling of HA
The HA was labelled with DL-tyrosine (Sigma chemical company St
Louis, U.S.A.) as previously described after CNBr-activation of the polysaccharide
by the method of Glabe et al [Glabe CG, Harty P.K. and Rosen S.D., Preparation
and properties of fluorescent polysaccharides. Anal. Biochem. 130:287-294 (1983).
Briefly, 15 mg HA was activated at pH 11 by 8 mg CNBr for 5 min. The activated
polysaccharide was separated from the reaction mixture on a small column of
Sephadex G25 (PD 10, Pharmacia, Uppsala, Sweden) equilibrated with 0.2 M
borate buffer pH 8Ø The activated HA was incubated over night with 1 mg tyrosine
(T). The T bound to HYA (T-HA) was separated from unbound T on a PD 10
column equilibrated with phosphate buffered saline (pH 7,5)(PBS), containing
NaCI (8g/l), KCI (0,2 g/l), KH2PO4 (0,2g/l) and Na2HPO4 (1,15 g/l).
A part of the T-HA was iodinated with 1251 by placing 100~ug of T-HA
ïls~l3
._ - 19 -
together with 0.5 mCi 1 25l in a small glass tube covered with a film of 1 Ollg 1,3,4,6-
tetrachloro-3a,6a-diphenylglycouril (Sigma chemical company, St. Louis, U.S.A.).Unincorporated 125l was removed on a PD 10 column equilibrated with PBS and
the iodinated T-HA (1251-T-HA) stored at 5~C. The specific radioactivity was
usually 1500-5000 dpm/ng.
The 1251-T-HA kept a higher molecular weight-profile upon gel
filtration chromatography and was found to be cleared from the circulation with the
kinetics and organ distribution reported for biosynthetically labeled HA of high Mw.
The 1 251-labelled polysaccharide was also taken up by isolated rat liver
endothelial cells both in vivo and in vitro, indicating that the labelling does not
interfere with the binding to specific cell-surface receptors found on these cells
(1 ,2).
15 Immunostaining
All staining was made with the ABC-elite method (VectastainR Elite
ABC). Frozen sections of 6~m were prepared and mounted on glass slides, coated
with gelatin-kromalun, half a gram of kromalun (KCr(SO4)2 x 12 H2O) in 750 ml
aq.dest, and 5g gelatin in 250 ml aq.dest. were, after heating to 56~C, mixed. To
20 remove unspecific binding to serum proteins, the antiserum as well as the
preimmune serum was adsorbed on a SepharoseR 4B gel (Pharmacia LKB
Technology, Uppsala) coupled with rat serum proteins (6 mg protein/ml gel).
Coupling was performed as described by the manufacturer (Affinity
Chromatography; principles & methods, by Pharmacia LKB Biotechnology). The
25 serum and the gel were mixed in equal volumes and incubated for 6h at 4~C. The
frozen sections were fixed in cold methanol for 10 minutes, dried for 10 minutesbefore washing in phosphate buffered saline (PBS). Peroxidase was blocked in
0 3% H2~2 in methanol, and the sections were once more washed in PBS. To
215~013
- 20 -
block endogenous biotin and biotin-binding activity, an avidin/biotin blocking kit
from Vector laboratories was used. The sections were then incubated for 30 min in
PBS containing 4% goat serum (VectastainR Elite ABC). The HARLEC antiserum,
was diluted 1:300 in 4% goat serum in PBS, and incubated for one hour. After
washing in PBS, the sections were incubated with the second goat anti-rabbit
antibody (VectastainR Elite ABC) diluted 1 :200 in PBS for 30 min. After incubation
with the second antibody the sections were washed and incubated with the ABC-
Elite-complex (VectastainR Elite ABC). To develop the colour, 10mg 3-amino-9-
ethyl-carbazol in 6ml dimethylsulphoxide (DMSO) was mixed with 45ml 15mM
acetate buffer pH 5 and
4 1ll Perhydrol (Merck), and the sections were incubated in the mixture
for 7.5 minutes. After washing, the sections were counterstained in Mayers
hematoxylin for 1.5 minutes. The glass slides were mounted in Kelsers glycerol-
gelatin (Merck).
Hyaluronidase treatment of sections
After methanol fixation and washing in PBS the sections were
incubated with 5 U/ml of streptomyces hyaluronidase (Amano Pharmaceutical Co.,
Ltd Japan.), 1mg/ml pepstatin (Sigma), 0.1 M N-Ethylmaleinimid( ), 0.1M EDTA
(Merck) and 5 KlE/ml Trasylol (Bayer) for two hours at 37~C. The regular staining
protocol was then followed.
Uptake studies in vivo
Four female nude (Nu/Nu) rats, weighing 200-250g, were inoculated
subcutaneously with approximately 3X106 cells (in 0.5ml RPMI medium) of the
human celline CCL 218 (American Tissue Type Collection) in one hind leg under
ether anasthesia, 14-30d before the experiments. The animals were anesthetized
with pentobarbital (45 mg/kg body weight) and received an injection in the tail vein
-21- 215~
-
or directly in the approximately 1-2 cm2 large tumour of 3.4-1500~1g 1251-T-HYA (5-
15X106 cpm). To reduce the specific activity and to achieve a higher chemical
amount of HA, sometimes unlabelled HA in 0.05-1.0 ml 0.15 M NaCI, 10mM
NaH2PO4, pH 7.4 was added.
After the study the animals were killed and tumour and organs
assayed for radioactivity. The data were processed using a Macintosh SE/30(~) or a
Macintosh llsi(g) computer (Apple computer Inc. Cupertino, CA, U.S.A.) the graphs
were constructed using the Cricket Graph(g) program (version 1.3, Cricket software,
10 Malvern, PA, U.S.A.) and Canvas (version 3Ø2., Deneba Systems Inc, Miami, Fl,
U.S.A.).
Scintigraphic studies
The rats were anesthetized and injected as described above. In
dynamic studies the injections were made with the rats placed on the standard
medium resolution collimator of the gamma-camera. Images were collected in a
64x64 pixel matrix in word mode with a 34 keV 80% window setting in a Gamma 11
system (Philips). The dynamic sequence was preset at one image per minute for
15 min. After 15 min, static images were collected at different times after injection.
20 Images were then transferred to the Hermes(g) system (Nuclear Diagnostics,
Hagersten, Sweden and London, U.K.) and regions of interest (RO1:6) were drawn.
It was in some instances necessary to draw a ROI covering an area larger than the
tumour or liver itself due to scattering.
25 RESULTS
By immunohistochemistry it was found that polyclonal antibodies
against HARLEC recognize structures in tumours of the human coloncancer-celline
CCL 218 in nude rats. The staining was mainly localized to vessel endothelium
215~-313
- 22 -
and over tumour cells, where it seemed to increase towards the margin of the
tumour (Fig 1a). Preimmune antibodies showed virtually no staining (Fig 1b),
indicating that the HARLEC-staining is specific in this system.
The invention will now be illustrated with reference to the drawings in
5 which:
Figure 1 a) shows immunohistochemical staining of CCI 218 tumour
using polyclonal antibodies to HA receptor HARLEC, b) shows the control using
preimmune antibodies. See Materials and Methods for details.
When tracer doses of the 1251-T-HA was injected intravenously the
radioactivity disappeared rapidly from the blood to the liver and little radioactivity
was left in the blood after a few minutes (Fig 2).
Figure 2 relates to disappearance of radioactivity from blood after
intravenous injection in rats of 5 llg 1251-T-HA. Inset: The recovery of radioactivity
in different organs of the rat 10 min after an intravenous injection of 5 llg 1251-T-
HA. KC denotes Kupffer cells, PC denotes parenchymal cells and LEC denotes
liver endothelial cells.
As the binding-capacity of the liver is so great, not much of the tracer
was left in the blood after passage of the liver and only minor amounts could reach
other tissues. Therefore, amounts exceeding the binding-capacity of the liver were
20 injected and at a dose of 1.5mg HA there was still material in the general
circulation at 24h and an accumulation of radioactivity in the tumours could be
seen by scintigraphy (Fig 3). The major part of radioactivity was seen over the liver,
but significant activity above background was observed over the tumour (Fig 3 a).
The activity over the tumour was severalfold higher than what could be expected
25 from merely increased size of the tissue, and shielding of the liver with lead
produced scintigraphic images where the 50% maximum level corresponded well
with the size of the tumour (Fig 3 b).
Figure 3 depicts scintigraphic images of a nude rat 3 weeks after
2156~13
- 23 -
inoculation with the human coloncancer CCL 218 in one leg, and 24h after
injection of 1.5 mg HA containing 2.5 MBq 1251-T-HA. In 3b the liver is shielded by
lead. Arrow points at tumor.
Immunohistochemical analysis of treated animals showed that
5 hyaluronidase treatment of the sections caused a specific increase in HARLEC
immunoreactivity (Figs. 4 a and b) without increasing the preimmune response
(Figs. 4 c and d). The receptor-staining to a large extent colocalizes with the
staining for HA itself (Figs. 4 e and f). The staining for HA was most prominent in
the marginal zone and in and around vessels, but also other receptor-stained
10 structures were positive for HA.
Figure 4 relates to:
a) Immunohistochemical staining of a CCI 218 tumour, after intravenous
injection of 1251-T-HA, using polyclonal antibodies to the HA receptor HARLEC.
b) Immunohistochemical staining as in 4 a after hyaluronidase treatment.
C) and d) As 4 a and b but with preimmune (control) antibodies.
e) and f) HA-staining of the same tumour as in 4 a-d using biotinylated HA
binding protein.
As a means of reducing uptake in the liver and trying to achieve a
therapeutic response, two animals received single intratumoural injections of 125l-
T-HA. One rat received 3.4 ~lg labelled HA of high specific activity (0.25MBq/~lg)
that to more than 90% stayed in the tumour. Small amounts reached the general
circulation and was taken up by the liver (Fig 5 a). Still 15 d after injection the ratio
of radioactivity over tumour and liver was similar to that observed directly after
injection (Fig 5 b). The animal responded with regression of the tumour from
approximately 2cm2 to 1 cm2 over 7d. There was a gradual decrease and
softening of the tumour until it at d14 not could be sensed by palpation.
Histochemical analysis after sacrifice at d15 showed that the tumour was still
present with a volume of approximately 0.5 cm2. One rat receiving only 0.025MBq
2156~13
- 24 -
but 250 ~,lg HA intratumourally did not respond and had at sacrifice a tumour ofapproximately 5cm2 (the rats were killed at the same time).
Figure 5 depicts a scintigraphic image of a nude rat carrying a CCI
218 derived tumour that 1 d (a) and 15d(b) prior to the image received 3.5~19 125l-
5 T-HA (0.75MBq) intratumourally.
Figure 6 relates to the inhibition of the cell association of 1 251-T-HA
to LEC in culture at 37~C. Results are mean+SD, n=3 (control medium) and n=6
(1A29 medium). See Materials and Methods for details.
Figure 7 depicts the recovery of radioactivity in tumour tissue and
n control muscle tissue 18-20h after an intravenous injection of 2 mg 1251-T-HA. Results are mean+SD, n=3. See Materials and Methods fordetails.
Figure 8 shows frozen sections of tumour tissue stained for HA and
HARLEC/ICAM-1 18-20 h after an intravenous injection of 2 mg 1251-T-HA. See
Materials and Methods for details.
a) Staining for HA
b) Staining for HARLEC/ICAM-1
c) Staining for HARLEC/ICAM-1 after hyaluronidase treatment.
DISCUSSION
The fact that the immunohistochemical staining of HARLEC was
20 evident in the tumours without hyaluronidase treatment (Fig 1) indicated that the
binding sites are to a certain degree unoccupied, and pointed to a possible chance
for uptake of circulating HA. This is in agreement with what is found for the liver
receptors.
25 Second Example
The major site for elimination of HA (Hyaluronic acid and
pharmaceutically acceptable salts thereof) from the bloodstream is via receptor
mediated endocytosis by liver endothelial cells (LEC). The HA receptor (HAR)
215601~
- 25 -
on LEC has been characterized by HA affinity chromatography of surface 125l
labelled rat LEC and purified to homogeneity from rat LEC membranes. This
receptor (termed HARLEC) has a mean molecular weight of approximately 90kD
with a range of 85 to 100 kD and a pl of around 6.7. A monospecific polyclonal
5 antibody against the receptor, which could inhibit binding of HA to LEC and LEC
membranes, (termed anti-HARLEC) was raised. Protein species of 85-90 kD
have also been detected by this antibody in kidney, spleen, thymus and Iymph
noes with immunoblotting. The same tissues, together with the liver, stain
positively with anti-HARLEC, the immunohistochemical staining being mainly
restricted to vascular regions such as the sinusoids in the liver, spleen, and
Iymph nodes and the capillaries in the small intestine, but also to specific
structures such as thymic reticular cells. Corneal characteristics as HARLEC
and are specifically stained by anti-HARLEC. The CEC staining is inhibited if the
corneas are treated with HA prior to staining, while the staining intensity is
15 restored by hyaluronidase treatment.
HARLEC was purified from while rat liver using a series of affinity
chromatographic steps. In the final step, virtually all HA bound to HA-Sepharoseand was specifically eluted with HA-oligosaccharides. Tryptic digestion of
20 purified HARLEC yielded several peptides that were separated by reverse
phase chromatography. Four peptides were sequenced and found to be
identical to intercellular adhesion molecule-1 (ICAM-1). ICAM-1 is a
glycosylated single chain protein of 80 to 114 kD with a core polypeptide of 55
kD. It is normally expressed at low levels, but has been found on normal liver
25 endothelium at the sinusoids and on the endothelium of Iymph nodes, spleen
and some capillaries of the kidney, as well as on corneal endothelial cells. Theimmunohistochemical localization of ICAM-1 corresponds well with the staining
for HARLEC that has been reported. The localization also corresponds to
- 26 ~15~01~
tissues where HA binding and uptake have been found.
HARLEC/ICAM-1 is expressed on tumour endothelium in mouse
mastocytomas and can bind to/with intravenously administered radiolabelled
5 HA. A significant increase of radioactivity in the tumour tissue was found
(approximately 5-fold relative to controls).
By immunohistochemistry it was found that the HA is localized in
areas that also stain positively for ICAM-1, i.e.. mainly vessels. ICAM-1
immunoreactivity is dramatically increased after hyaluronidase treatment of the
sections.
Herein, presented is further evidence that HARLEC/ICAM-1 is a
receptor for HA, that HA targets also human tumours in nude rats and that the
15 targeting is mainly via binding to HARLEC/ICAM-1 on tumour endothelium.
Materials and Methods
Polysaccharides: The HA used for labelling and uptake- and
turnover studies was extracted from avian tissue and supplied by Hyal
20 Pharmaceutical Corporation (HPC), Toronto, Canada. The mean molecular
weight was approximately 450 000 Daltons.
Labelling of HA: The HA was labelled with DL-tyrosine (Sigma
Chemical company St Louis, U.S.A.) and 125l.
Monoclonal antibodies: The monoclonal antibody against rat
25 ICAM-1, clone 1A29, was a kind gift of Professor M Miyasaka, Osaka University,
Japan.
Cultivation of endothelial cells: Rat LEC were isolated after
collagenase perfusion of rat liver and cultured without serum in RPMI medium.
21560~3
- 27 -
Antibody inhibition experiments: 100 000-200 000 LEC
cultured for 4-5h were incubated with 0.5 ~lg/ml 1251-T-HA, and in competition
experiments unlabelled HA or anti-lCAM-1 supernatant medium (diluted 1:2). As
the anti ICAM-1 contained 115 ng/ml HA a control medium containing 115 ng/ml
5 HA was made and used (diluted 1:2) as a control of inhibitory binding activitydue to the HA in the antibody supernatant. The competitors were added prior to
the addition of radiolabelled HA. Total incubation time was 35-40 min. After thetermination of the incubations, the medium was removed, cells washed and
analyzed for radioactivity as previously described(10).
Immunostaining: All staining was made with the ABC-elite
method (Vectastain(g) Elite ABC). Frozen sections of 6~1m were prepared and
mounted on glass slides that were coated with gelatin-kromalun. To remove
unspecific binding to serum proteins, the HARLEC antiserum was adsorbed on a
Sepharose~3) 4B gel (Pharmacia LKB Technology, Uppsala) coupled with rat
serum proteins (6 mg protein/ml gel).
The frozen sections were fixed in cold methanol for 10 minutes,
dried for 10 minutes before being washed with phosphate buffered saline (PBS).
Endogenous peroxidase was blocked in 0.3% H2~2 in methanol, and the
sections were only more washed in PBS. To block endogenous biotin and
biotin-binding activity, an avidin/biotin blocking kit from Vector laboratories was
used. The sections were then incubated for 30 min in PBS containing 4% goat
serum (Vectastain(~ Elite ABC). The HARLEC antiserum, was diluted 1:300 in
4% goat serum in PBS, and incubated for one hour. After washing in PBS, the
sections were incubated with the secondary HRP-conjugated goat anti-rabbit
antibody (Vectastain(g) Elite ABC) diluted 1:200 in PBS for 30 min.
The staining for HA was performed using biotinylated hyaluronan
binding proteins from cartilage (b-HABP), but without CPC treatment, on frozen
sections treated with methanol and blocked for endogenous biotin binding
~lS601~
- 28 -
activity with avidin/biotin blocking kit (Vector laboratories).
After incubation with the second antibody, or b-HABP, the sections
were washed and incubated with the ABC-Elite-complex (Vectastain(~) Elite
ABC). To develop the colour, peroxidase substrate kits (Vector laboratories)
5 containing 3,3 diaminobenzidine or 3-amino-9-ethylcarbazole were used, and
the sections were incubated in the mixture for 5-10 minutes. After washing, the
sections were counterstained in Mayers hematoxylin for 1.5 minutes. The glass
slides were mounted in Kaisers glycerol-gelatin (Merck).
Hyaluronidase treatment of sections: After methanol fixation
10 and washing in PBS the sections were incubated with 5 U/ml of streptomyces
hyaluronidase (Amano Pharmaceutical Co., Ltd Japan.), 1.8 ,ug/ml pepstatin
(Sigma), 1.8 mM EDTA (Merck), 1.8 llg/ml soybean trypsin inhibitor (Sigma), 2.0
mM iodo acetic acid (Sigma), 0.18 mM e-amino-n-caproic acid (Sigma) and 9.0
mM benzamide (Sigma) for two hours at 37~C. The regular staining protocol
was then followed.
Uptake studies in vivo: Female nude (Rowette Nu/Nu) rats,
weighing approximately 200g, were inoculated subcutaneously with
approximately 3X106 cells (in ~11 s-MEM medium (Gibco)) of the human cell line
CCL 218 (American Tissue Type Collection) in one hind leg, while under ether
20 anasthesia, 1 4-30d before the experiments. The animals were anesthetized andreceived an intravenous injection of 2 mg 1251-T-HYA (2-3x106 cpm). To reduce
the specific activity and to achieve a higher concentration of HA, unlabelled HAin 0.15 M NaCI, 10mM NaH2PO4, pH 7.4 (0.05-1.0 ml) was added.
After 18-20h the animals were killed and tumour and organs
25 assayed for radioactivity as previously described (9).
Results:
To further characterize the binding of HA to HARLEC/ICAM-1
~1560~
. - 29 -
experiments were performed with isolated rat LEC, radiolabelled HA and a well
characterized monoclonal antibody towards rat ICAM-1 (lalenti A and Di Rosa
M.; Hyaluronic acid modulates acute and chronic inflammation, Agents Actions
43: 44-47). The cell association of 1251-T-HA to LEC, studied at 37~C, could be
5 inhibited by about 60% (Fig 6). However, approximately 20% of the inhibition
was found to be due to the presence of HA in the antibody tissue culture
medium, as indicated by the inhibition seen in control experiments using
medium containing the same amount of HA but no antibodies (Fig 6).
In order to see if HARLEC/ICAM-1, expressed on endothelial cells
10 in other tissues of the rat, has the same capacity to bind HA as LEC, turnover
and tissue distribution studies were performed in rats carrying a colon carcinoma
of human origin in one hind leg. It was found that 18-20h after an intravenous
injection of 1 251-T-HA (2 mg), most of the radioactivity was, as expected, in the
liver, while very little was in the blood (not shown). The recovery of radioactivity
15 in the tumour was more than three times the amount found in the control muscle
tissue (wet weight adjusted), and this increase was statistically highly significant
(p=0.001, n=3)(Fig 7).
When the tumour tissue was analyzed by histochemical
techniques, it was found that the HA in the tumour was mainly localized to the
20 stroma and vessels of the tumour (Fig 8a), and ICAM-1 in the same areas,
predominantly in vessels (Fig 8b). The ICAM-1 staining, although clearly visible,
was surprisingly weak (Fig 8b). However, after hyaluronidase treatment the
staining was dramatically increased (Fig 8c).
25 Discussion
The finding that monoclonal antibodies to rat ICAM-1 can inhibit the
cell association of labeled HA to rat LEC in culture (Fig 6), provides further
evidence that ICAM-1 is an important cell-surface receptor for HA. The inhibition
2i~GOl~
- 30 -
is only in the order of 50%, but as cell association was studied at 37~C the
inhibition will not be complete due to the continuous appearance of unbound
receptors from the interior of the cells. The affinity of high molecular weight HA
for the receptors on LEC is very high and the labelled HA will probably compete
5 well for binding to the free receptor sites. The possible existence of other HA
binding sites on ICAM-1, not affected by 1A29 binding, also needs to be
considered.
The monoclonal antibodies used in this study have previously
been shown to cause inhibition of leukocyte adherence to endothelial cells. As
10 the antibodies cause inhibition of HA binding to LEC, the HA binds to a site close
to the leukocyte binding site on ICAM-1 and that binding of HA to endothelial
cells causes inhibition of leukocyte adherence to the same cells. Systemic
administration of HA can then have a beneficial effect on inflammatory
conditions. Such effects, similar to the reduced inflammation seen with systemic15 treatments with antibodies to ICAM-1, have been described in animal models of acute and chronic inflammation.
The increased uptake in tumour tissue of the intravenously
administered radiolabelled HA (Fig 7), indicates that binding structures for HA
are present in the tumour. The staining for HA, using a highly specific
20 biotinylated HA binding protein, indicated that the HA was localized in and
around the vessels (Fig 8a). As expected, the staining for HARLEC/ICAM-1 was
also found in vessels, but was rather weak (Fig 8b). Administration of HA to
corneal EC causes a reduced immunohistochemical staining of CEC receptors
which can be restored by hyaluronidase treatment, we also tried such
25 hyaluronidase treatment of the tumour sections. The effect was a dramatic
increase in HARLEC/ICAM-1 staining (Fig 8c), indicating that HA, bound to
HARLEC/ICAM-1, caused an inhibition of the binding of the specific antibody.
Intravenously administered HA is predominantly bound to
21560:~3
- 31 -
endothelial cells via ICAM-1. This points to using HA as a carrier of cytotoxic
drugs to tumours expressing this type of HA-receptor.
As many changes can be made to examples of the invention without
5 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.
