Note: Descriptions are shown in the official language in which they were submitted.
S
2164260
.ITLE OF INVENTION
Targeting of Dosages of Medicine and Therapeutic Agents
FIELD OF THE INVENTION
This invention relates to the targeting of medicines and therapeutic agents
to sites in the body of a mammal in neE~d of treatment and, in one
application,
finds use in the treatment of malignant tumours in humans.
1 0 BACKGROUND OF THE INVENTION
W091 /04058, owned by Hyal Pharmaceutical Corporation, teaches the use
of dosages of at least 10 mg. of forms of hyaluronic acid to transport
effective
amounts of medicines and/or therapeutiic agents to sites in need of treatment
in
1 5 the human body, to penetrate the tissue at the sites in need of treatment,
including scar tissue, through all membranes into the cells to be treated.
At page 25, line 17, the PCT Application teaches the additional benefit of
using at least about 200 mg. of forms crf hyaluronic acid (for example, sodium
2 0 hyaluronate) in a dosage together with the medicine and/or therapeutic
agent for
reducing the side effects of the medicine and/or therapeutic agent when
administered (such as gastro-intestinal distress, neurological abnormalities,
depression, etc. normally associated with the medicine and/or therapeutic
agent)
even at elevated amounts greater than the usual accepted dosage amounts of the
2 5 medicine and/or therapeutic agent when administered alone for example, an
NSAID (non-steroidal anti-inflammatory agent).
The document continues at page 2.5, line 26:
A
21fi4260
"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" the pathological tissue even when administered by
the systemic intravenous route. Thus, it has been observed that
patients with neoplastic diseases when receiving in addition to
other chemicals (for example, ascorbic acid [Vitamin C], phloretin
and anti-cancer drugs), 50 - 200 mg NSAID - hyaluronic acid
(sodium hyaluronate) (for example indomethacin and hyaluronic
acid) experience dramatic relief of pain immediately. This is
followed within a short period of time by a resolution and
resorbtion of neoplastic lesions with an improvement of
pulmonary, and liver function if there is tumor present in these
organs. Thus, the dead tumor material and the debris and tumor
1 5 toxins appear to be better eliminated by the body through the action
of the macrophages whose activiity is enhanced by the addition of
the NSAID (or a steroidal anti-inflammatory drug) administered
with hyaluronic acid (or salt or other form thereof). Thus
Applicants believe that the addition of the NSAID for example with
2 0 hyaluronic acid (sodium hyaluron.ate) deblocks the macrophages by
preventing 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
2 5 with the use of the prostaglandin synthesis inhibitors."
Therefore, if hyaluronic acid is used as a vehicle for a medicine or
therapeutic agent to transport the medicine to a site in the body in need of
treatment, unless the combination is administered directly to the site in need
of
n
2164260
.eatment as by injection into a tumour, much of the combination ends up at the
liver with lesser amounts at the site in need of treatment, unless and until
the
liver has reached its binding capacity for hyaluronan.
In an article entitled "Binding of hyaluronate and chondroitin sulphate to
liver endothelial cells" by Toward C. Laurent, et al., Biochem J. (1986) 234,
653-658,
the authors discussed the fact that "C:irculating sodium hyaluronate (HA) is
efficiently taken up and metabolized by the endothelial cells in the liver
sinusoids", and that "Chondroitin sulphate (CSA) is also taken up and
1 0 metabolized by liver endothelial cells". 'lChe authors also state:
"The partial inhibition of HA binding by CSA (Smedared et al., 1984)
and the inhibition of CSA binding by HA (Smedared et al., 1985)
indicates that the two polysaccharides are recognized by the same
1 5 receptor. We have now confirmed this hypothesis by the use of
oligosaccharides of identical degree of polymerization prepared
from the two polymers."
I have now discovered that the liver endothelial cells carry at least two
2 0 different binding proteins for HA (hyalluronan) including a scavenger
receptor
that binds to both chondroitin sulphate and hyaluronan, (and other
glycosaminoglycan (GAGS)) and, the majority of these different binding
proteins
(the scavenger receptors on the liver) are inhibited from their take-up of
GAGS
(Glycosaminoglycans) including chondroitin sulphate (CS) by their being
2 5 previously bound to, for example, chon~droitin sulphate. I have also
discovered
that the sites in need of treatment for e:Kample, metastatic tumours do not
carry
the same scavenger receptors that bind to chondroitin sulphate. These sites
carry
receptors that do bind to hyaluronan. Thus, by first administering for
example,
chondroitin sulphate for take up by the scavenger receptors of the liver,
2164260
absequently administered hyaluronan for example, will not be taken by the
liver.
It is therefore an object of this invention to provide a novel method of
treatment of a disease or condition in a human which method permits
substantially more of the medicine and/or therapeutic agent to be brought to
the
site in the body in need of treatment.
It is a further object of the invention to target sites in the body in need of
1 0 treatment so that any medicine or therapeutic agent reaches the site in
need of
treatment rather than being taken up elsewhere in the body (for example, the
liver).
It is still a further object of the invention to provide a novel dosage kit or
1 5 combination of materials or chemicals which when used one after the other
will
target the sites in the body in need of treatment for delivery of a medicine
and/or
therapeutic agent to the site in need of treatment.
It is still a further object of the invention to reduce the amount of the
2 0 medicine and/or therapeutic agent that would otherwise be normally
expected
to be used for treating a disease or condii:ion. In this regard, if a
transport agent is
used to transport the medicine and/or therapeutic agent to the site of the
disease
and/or condition, it is a further object of the invention to use less of the
usual
amount of the transport agent (for example, hyaluronan and a pharmaceutically
2 5 acceptable salt thereof) for transporting ithe medicine and/or therapeutic
agent to
transport the medicine and/or therapeutic agent to the site of the treatment.
Thus, for example, less cytotoxic medicine (for example, methotrexate,
cisplatin
and the like) will be needed as an effective dosage amount of the medicine to
achieve successful treatment using less than the usual effective dosage amount
2'~642~A
the hyaluronan for example, as the transport agent.
Further and other objects of the invention will be realized by those skilled
in the art from the following Summary of Invention and Detailed Description of
Examples thereof.
SUMMARY OF INVENTION
I have now discovered that while chondroitin sulphate and hyaluronan
bind to liver cells and particularly, the scavenger receptors on the liver,
chondroitin sulphate does not bind with the receptors on for example, tumours
(for example, metastatic tumours) and particularly, the cell surface receptors
for
hyaluronan comprising, the Regulatory molecule RHAMM (Receptor for H A
Mediated Motility), and adhesion molecules ICAM-1, HARLEC and CD44. This
1 5 has led me to develop my new methods of treatment of disease and
conditions
including metastatic tumours. By "down regulating" the scavenger receptors on
the liver cells by binding them to administered chondroitin sulphate (or other
GAGS [Glycosaminoglycan] such as dextran sulphate, other than a form of
hyaluronan) and subsequently administering the form of hyaluronic acid
2 0 (hyaluronan) with the medicine and/or therapeutic agent, the subsequently
administered amounts of hyaluronan (which transport the medicine and/or
therapeutic agent) are picked up, not by i:he liver whose binding capacity has
been
substantially fulfilled but, by other sates capable of binding with forms of
hyaluronan having excess unfilled hyaluronan receptors (such as on metastatic
2 5 tumours). At the same time, the hyaluronan transports any medicine (or
therapeutic agent) to treat the sites in need of treatment (for example, by an
effective amount of a cytotoxic agent to treat a tumour). As a result, I have,
by
the pre-treatment with for example, chcmdroitin sulphate, found that less of
the
form of the transport agent for example, hyaluronan that is to be subsequently
2164260
aministered is required than previously used. I have also found that less of
the
medicine and/or therapeutic agent is required than was used previously to
provide a successful treatment of the disease and /or condition.
The amount of the chondroitin sulphate that will "down regulate" the
liver cells is preferably in the order of at least about 3-5 mg. of
chondroitin
sulphate per kilogram of body weight of the patient. However, preferably
greater
amounts (mg/kg) are administered to "turn off" the liver. Because the liver
processes the administered and "taken up" chondroitin sulphate rapidly, less
1 0 chondroitin sulphate is not as good as more, as after several hours the
liver has
processed all the chondroitin sulphate. Thus, prolonged "blockage"/"down
regulating" or "immobilization" of the liver cells is preferred. Where
chondroitin sulphate (equivalent dose 1-~2 grams/70 kg person) is
administered,
the take-up of even small amounts of hyaluronan by the liver (0.5-1 mg/70
1 5 kg/person) can be inhibited for an extended time by the administration of
the
chondroitin sulphate. Thus, the hyaluronan is available to transport the
medicine and/or therapeutic agent to the site in need of treatment (for
example,
methotrexate or cisplatin to a tumour or furosemide to a kidney or other use
proposed by the teachings of WO 91 /04058.
The amount thereafter required. of the transport agent for example,
hyaluronan or a pharmaceutically acceptable salt thereof for example, sodium
hyaluronate having for example, a molecular weight less than 750,000 daltons
may be reduced substantially (for example, to an amount of substantially less
2 5 than 10 mg/70 kg person such as 0.1 mg/70 kg person) and the amount of
medicine and/or therapeutic agent likewise substantially reduced to a mere
fraction of what is normally used previously or proposed to be used
previously.
It may be that with the liver shut down, only micrograms (~g)/kg of the body
weight, of the transport agent for example, 20 ~g/kg and micrograms (~,g) of
the
2164260
medicine for example, depending on the medicine 10 ~,g/kg of body weight may
be only required in the dosage.
Other suitable compounds may be substituted for chondroitin sulphate
such as dextran sulphate including other GAGS. Some may be used i n
substantially the same amounts as with chondroitin sulphate; others may be
used in higher or lower amounts. Other GAGS may include Dermatan sulphate,
Heparin sulphate, Heparin or their I'roteoglycan forms, Keratan sulphate.
Keratan sulphate and the like, while not technically a
glycosaminoglycuronoglycan, will be considered to be included as a GAG herein.
Other scavenger receptor ligands may also be used such as acetylated low
density
lipoproteins (LDL), acids such as poly-inosinic acid and the like.
Subsequent to administering the chondroitin sulphate (for example, after 3
to 4 minutes) (for example, by intravenous administration) the combination of
for example, hyaluronan (for example, the amounts, forms and molecular
weights taught in PCT Application WO91/04058, together with the medicine
and/or therapeutic agent (whether excess amounts over and above the normally
administered amounts when at least :?00 mg/70 kg person of the form of
hyaluronic acid is used or the normally used effective amounts of the medicine
or therapeutic agents are used or much llesser amounts (~cg)/kg) is
administered
by any suitable means. Because lesser amounts of medicine and/or therapeutic
agents and for example, hyaluronan may now be used, the amounts specified
above and in the documents above may be substantially reduced. Because the
amount of medicine and/or therapeutic agent is reduced substantially, side
effects are substantially reduced. Thus, even the 200 mg. amount per 70 kg.
person of the form of hyaluronic acid will be reduced. In fact, with the liver
being "blocked", I have found that 5 ~,gi 250 mg rat (20 ~cg/1 kg) of
hyaluronan
targets the site in need of treatment. I have also found that the
B
21s~2so
~ame order of magnitude (~g/250 gm rat) medicine and/or therapeutic agent in
the dosage would be useful for treatment: of the site in need of treatment.
That is
because the liver has now been "blocked". Therefore, the teachings in WO
91 /04058 with respect to dosage amounts may now be modified in light of the
above for use with this invention.
As stated in Application W091 /04058:
(i) at page 17, line 3 to page 18, line 16:
"Applicants have now discovered that combinations and
formulations (for example an injectable formulation) can be
provided for administration to a mammal for the treatment of a
disease or condition, which combinations or formulations employ
1 5 or incorporate as the case may be a therapeutically effective non-
toxic amount of a medicinal and /or therapeutic agent to treat the
disease or condition (for example a free radical scavenger (for
example ascorbic acid (Vitamin C;)), Vitamin C (for the treatment of
mononucleosis), an anti-cancer agent, chemotherapeutic agent, anti-
2 0 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 and (+/-) tromethamine salt of ketorolac
(sold under the trademark ToradolTM) 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 the
r
2164260
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 LasixTM)), innmunosuppressants (for example
cyclosporins), lymphokynes (such as interleukin - 2 and the like),
alpha-and-f3-interferon and the like) administered with, or carried
in, an amount of hyaluronic acid and/or salts thereof (for example
the sodium salt) and/or homologues, analogues, derivatives,
complexes, esters, fragments, and /or sub units of hyaluronic acid
1 0 (preferably hyaluronic acid and salts thereof) sufficient to facilitate
the agent's penetration through the tissue (including scar tissue), at
the site to be treated through. the cell membranes into the
individual cells to be treated. When such combinations and
formulations are administered to patients suffering from the disease
1 5 or condition, the disease or condition is unexpectedly improved.
The formulation can be administered among other methods,
intravenously, intra arterially, intraperitoneally, intrapleurally,
transdermally, on the skin (topically), rectally, orally or by direct
2 0 injection (for example into a tumor, into an abscess or similar
disease focus) or put on a patch to be secured to the skin of the
patient. The hyaluronic acid and/or salts thereof and the agent can
be administered separately but are administered in sufficient
amounts and in an immediate tirr~e sequence or interval (preferably
2 5 concurrently and more preferably simultaneously), preferably at the
identical site (e.g. one given intravenously and the other "piggy
backed"), to treat the disease or condition."
(ii) at page 25, line 18 to page 26, line 7 4:
2164260
"Thus and according to another aspect of the invention when
an NSAID for example indomethacin (dissolved in n-methyl
glucamine) or other NSAID is administered with greater than
200mg 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 di;>tress, neurological abnormalities,
depression, etc., even at elevated amounts of indomethacin (if
necessary). If the amount of hyaluronic acid is decreased below that
1 0 amount, the usual side effects may begin to reoccur. In addition, the
responses that have been observed are superior when the NSAID
(for example IndocidT"') is combined with hyaluronic acid
demonstrating clearly that the combination is now "targeting" to
the pathological tissue even when administered by the systemic
1 5 intravenous route. Thus, it has been observed that patients with
neoplastic diseases when receiving in addition to other chemicals
(for example ascorbic acid [Vitamin C], phloretin and anti-cancer
drugs), 50 - 200 mg NSAID - hyaluronic acid (sodium hyaluronate)
(for example indomethacin and hyaluronic acid) experience
2 0 dramatic relief of pain immediately. This is followed within a short
period of time by a resolution and resorbtion of neoplastic lesions
with an improvement of pulmonary, and liver function if there is
tumor present in these organs. 'Thus the dead tumor material and
the debris and tumor toxins appear to be better eliminated by the
2 5 body through the action of the macrophages whose activity is
enhanced by the addition of the NSAID (or a steroidal anti-
inflammatory drug) administered with hyaluronic acid (or salt or
other form thereof). Thus Applicants believe that the addition of
the NSAID for example with hyaluronic acid (sodium hyaluronate)
il 21 fi42 6 0
~...
deblocks the macrophages by preventing 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 NSAII) but also reduce any side effects
and toxicity that is associated with the use of the prostaglandin
synthesis inhibitors.
Examples of agents suitable for use as chemotherapeutic
agents are NovantroneT"'' (Mitoxantrone), Methotrexate, 5-FU (5-
1 0 Fluouracil), carboplatinum, methyl CCNU administered orally and
Mitomycin C."
(iii) at page 26, lines 32 to 37:
1 5 "The hyaluronic acid and salts thereof may be utilized at
varying doses - 10 to 1000 mg/70 kg person with the optimal doses
tending to range between 50 and ~~50 mg/70 kg individual. As there
is no toxicity, the hyaluronic acid c=an obviously be administered in a
dose excess (for example 3000 mg/70 kg individual) without any
2 0 adverse effects."
(iv) at page 29, line 27 to page 33, line 3.1:
"One form of hyaluronic acid and/or salts thereof (for
2 5 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 Sterivet Laboratories
Limited. One such fraction is a 15 ml vial of Sodium hyaluronate
'2 21 642 6 0
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 with they U.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
I 0 example sodium salt) and homologues, analogues, derivatives,
complexes, esters, fragments, and sub units of hyaluronic acid,
preferably hyaluronic acid and salts thereof may comprise
hyaluronic acid and/or salts thereof having the following
characteristics:
1 5 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;
2 0 ii) less than about 1.25% sulphated mucopoly-
saccharides on a total weight basis;
iii) less than about O.ti% 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;
2 5 vi) less than 0.0025°/j 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
13
2164260
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 of 150,000-225,000. More preferably
the fraction of hyaluroni.c acid comprises at least one
characteristic selected from the group consisting of the
1 0 following characteristics:
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
1 S basis;
iv) less than about 10 ppm lead on a total weight
basis;
v) less than 0.00166% glucosamine;
vi) less than 0.016n% glucuronic acid;
2 0 vii) less than 0.0166% N-acetylglucosamine;
viii) less than 0.001~66% amino acids;
x) a UV extincticm coefficient at 257 nm of less
than about 0.23;
xi) a UV extinction coefficient at 280 nm of less
2 5 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 other suppliers, for
14 21642fi0
example those described in the prior art documents previously
referred to. In addition Applicants have successfully employed
sodium hyaluronate produced and supplied by LifeCoreTM
Biomedical, Inc. having the following specifications
Characteristics Specification
Appearance White to cream
colored particles
1 0 Odor No perceptible odor
Viscosity Average < 750,000 Daltons
Molecular Weight
1 5 UV/Vis Scan, 190-820nm Matches reference scan
OD, 260nm < 0.25 OD units
Hyaluronidase Sensitivity Positive response
IR Scan Matches reference
pH, l0mg/g solution 6.2 - 7.8
2 5 Water 8% maximum
Protein < 0.3 mcg/mg NaHy
Acetate < 10.0 mcg/mg NaHy
21 642 6 0
Heavy Metals, maximum ppm
As Cd Cr Co Cu Fe Pb Hg N i
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
1 0 Biological Safety Testing Passes Rabbit Ocular
Toxicity Test
The following references teach hyaluronic acid, sources
thereof and 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
2 0 viscosity number greater than about 1400 cm3/g., and preferably
greater than about 2000 cm3 / g.;
(b) a protein content of lass 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
2 5 less than 2.0 at 280 nanometers 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
,~ 21646 Q
hyaluronate solution in physiological buffer of less than -11 X
103 degree - cm2/mole (of disaccharide) measured at 220
nanometers;
(f) no significant cellul;~r 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 o~f sodium hyaluronate dissolved in
physiological buffer is implanted in the vitreous replacing
1 0 approximately one-half the existing liquid vitreous, said HUA
being
(g) sterile and pyrogen free and
(h) non-antigenic.'
1 5 Canadian Letters Patent 1,205,031 (which refers to United
States Patent 4,141,973 as prior art;l 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 730,00() 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 intramuscular coagulation."
2 5 (v) and, at page 33, line 37 to page 35, line 30:
"Thus Applicant has combined hyaluronic acid (and sodium
hyaluronate and/or other forms) with medicinal and/or therapeutic
agents for the treatment of conditions and diseases with totally
1' 2164260
unexpected results:
For Example
Condition/Disease Chemicals & Drugs
1. Cancer, increasing activityfree radical scavenger,
of macrophages superoxide dismutase,
ascorbic acid(Vitamin
C)
anti-cancer drugs, NSAID,
Chemotherapeutic Agents,
1 0 detoxifying Agents (e.g.
cholestyramine)
1A. Reduction of swelling
in brain of Dimethyl Sulfoxide
(DMSO)person suffering
brain trauma
1 5 2. Hair growth minoxidil - combination
-
grow more hair when applied
topically
3. Herpes, canker sore, nonionic surfactants,
e.g.,
shingles nonoxynol-9 and
2 0 anionic, (e.g. cetyl
pyridinium chloride)
and
cationic (e.g.
benzalkonium chloride),
surfactants
2 5 4. Renal failure, cardiac diuretics - furosemide
insufficiency, hypertension,,
edema
5. Infection, acne, antibiotics, antibacterials,
mononucleosis antimicrobials, etc.,
3 0 ascorbic
acid and hyaluronic acid
6. Transplants cyclosporins
7. Inflammation, elimination non-steroidal anti-inflamma-
of
tumor break down material tories, NSAID e.g.
3 5 (toxins and debris),
diclofenac,
decreasing side effects, indomethacin, piroxicam,
relief of pain (e.g. ibuprofen, tromethamine
salt
ig 2164260
back pain) of Ketorolac, naproxen,
8. Detoxification enema, detoxifying agent,
peritoneal dialysis
9. Bronchodilation bronchodilators, e.g. beclo-
methasone diproprionate
(sodium cromoglycate although
not specifically a broncho-
dialator), theophylline
10. Vascular ischemia treat limbs in respect of
1 0 diabetes, Berger's disease,
etc. with
suitable medicine e.g. Trental
11. HIV (AIDS) DMSO, Vitamin C, NSAID (e.g.
indomethacin, naproxen,
ketorolac tromethamine),
1 5 interferon, VibramycinTM,
(doxcycline), tetracycline
12. Diabetes insulin
13. Post-menopause estrogens replacement
14. Prevention of topical antimetabolites (e.g. infection
2 0 sulfonamides)
15. Reduction of swelling DMSO
16. Hypertension, cardiac Calcium channel blockers e.g.
insufficiency- Nifedipine f3-
Blockers e.g. atenolol, propranolol
2 5 1~. Prostaglandin acetylsalicylic acid
Synthesis inhibition
18. Enhance oxygenation perfusate
of
tissue by perfusion
fluid
bathing the tissue (forplantation
trans
3 0 purposes"
The above description and proposals will apply herein only modified to
reflect the benefits of this invention. 'Thus, the administration of the form
of
hyaluronic acid and, medicine and/or therapeutic agent described above and
3 5 identified in the Applications may now be preceded by an effective amount
(for
example, exceeding in the order of about 3 - 5 mg/kg of body weight) of
chondroitin sulphate (for example, 200 - 400 mg/70 kg person). As a result,
less
,y 21 fi426 0
_nedicine and/or therapeutic agent for example, a cytotoxic agent, and
transport
agent for example, hyaluronan, than would be normally or usually used for the
treatment, may now be required because the hyaluronan together with the
medicine and/or therapeutic agent novv goes to the site in need of treatment
(tumour, for example) and is not taken up by the liver which has now been
"down regulated". Thus, the liver would not be as damaged by the for example,
cytotoxic agent as in the past.
The chondroitin sulphate preferably may have a molecular weight
1 0 exceeding 20,000 daltons for example, in the order of about between 20,000
and
40,000 daltons although there is a benefit irrespective of the molecular
weight of
chondroitin sulphate administered. Preferably, higher molecular weight
chondroitin sulphate is used so long as it is in a dosage form that can be
administered effectively (for example, in sufficient sterile water for
intravenous
purposes). The dextran sulphate or other agents (such as other
glycosaminoglycans) may have a molecular weight for example, in the range
between about 20,000 and 500,000 daltons or higher provided the dosages can be
effectively administered.
2 0 Thus, according to one aspect o:F the invention, a method of treating a
disease or condition in a human treatable by a medicine and/or therapeutic
agent
which may be transported by an agent (for example, a form of hyaluronic acid
such as sodium hyaluronate) to the site in need of treatment in the body and
which agent may also transport the medicine and/or therapeutic agent to the
2 5 liver (by for example, the transport agent binding to receptors on the
liver) is
provided comprising:
(a) administering an effective amount of a first agent such as
chondroitin sulphate which does not bind to receptors at the
2164260
site in need of treatment but which binds with receptors of
the liver thereby "down regulating" the liver;
and,
(b) thereafter (for example, 3 to 4 minutes after the
administration under sub-:paragraph (a) of this paragraph)
administering an effective non-toxic amount of a medicine
and/or therapeutic agent and an effective amount of a second
agent which is a transport: agent [for the medicine and/or
therapeutic agent] and is a different agent from the first agent
1 0 (for example, a form of hyaluronic acid) and which second
agent binds to the site in need of treatment and would be
capable of binding to the sites of the liver if the liver had not
been "down regulated" so that the liver's binding capacity for
the second agent has been substantially reduced (preferably
1 5 substantially eliminated or blocked) by the up-take by the
liver of the first agent (for example, chondroitin sulphate
Molecular Weight 20,000) administered under sub-paragraph
(a) by for example, binding with the scavenger receptors of the
liver.
Because the liver has been "down regulated", the amounts of medicine
and/or therapeutic agent that may be effective to treat the site in need of
treatment is substantially reduced. As well, the amount of the for example,
form
of hyaluronic acid (transport agent) is substantially reduced so that the
effective
2 5 amount is substantially less than 10 mg/70 kg person (for example, 20
pg/kg of
body weight of the patient being treated).
According to another aspect of the invention, a method of protecting the
liver from taking up medicines and/or i:herapeutic agents toxic to the liver
when
2' 21 642 6 0
..dministering the medicine and/or therapeutic agent to a site in need of
treatment, is provided comprising:
(a) administering an effective amount of a first agent such as
chondroitin sulphate which does not bind to receptors at the
site in need of treatment but which binds with receptors of
the liver thereby "down regulating" the liver;
and,
(b) thereafter (for example, 3 to 4 minutes after the
1 0 administration under sub-paragraph (a) of this paragraph)
administering an effective non-toxic amount of a medicine
and/or therapeutic agent arid an effective amount of a second
agent which is a transport agent and is a different agent from
the first agent, (for example, a form of hyaluronic acid) and
1 5 which second agent binds to the site in need of treatment and
would be capable of binding; to the sites of the liver if the liver
had not been "down regulated" so that its binding capacity for
the second agent has been substantially reduced (preferably
substantially eliminated or blocked) by the up-take by the
2 0 liver of the first agent (for example, chondroitin sulphate,
Molecular Weight 20,000) administered under sub-paragraph
(a) by for example, binding with the scavenger receptors of the
liver.
2 5 The amount of the first agent administered under (a) for example, in the
order of at least about 3 - 5mg/kg of body weight (for example, 200 - 400
mg/70 kg
person) having preferably a molecular weight in the range of 20,000 to 40,000
daltons, may be administered by any suitable manner such as systemically for
example, orally, intravenously, subcutaneously or by direct injection
proximate,
.~~~:
22 2164260
..djacent, or into, the liver (by direct administration into the hepatic
artery).
Thereafter, (for example, after 3 to 4 minutes) the amount of the second agent
for
transport in sub-paragraph (b) for example, sodium hyaluronate having a
molecular weight less than 750,000 d~altons is administered in an effective
amount now found to be substantially lE~ss than l0mg/70kg person discussed in
WO 91/04058 together with the medicine and/or therapeutic agent. The at least
200 mg/70kg person of for example, the sodium hyaluronate provided in
Application W091/04058 to reduce the side effects of the medicine and/or
therapeutic agent may now be substantially reduced because the amount of the
1 0 medicine and/or therapeutic agent that i.s now effective is substantially
less than
previously provided. Thus, less of for e;~cample, the form of hyaluronic acid
may
now be administered in a dosage together with a lesser amount of what is now
an effective amount of medicine and/or therapeutic agent to reduce the side
effects of the medicine and/or therapE~utic agent. Thus, the amounts of the
1 5 medicine and/or therapeutic agent and hyaluronan transport agent may now
be
(~,g) microgram amounts per kilogram of body weight to be effective. .
The first agent may be chondroitin sulphate (preferably) or other suitable
agent (such as dextran sulphate or other GAGS [Glycosaminoglycans] and/or
2 0 their proteoglycan forms which are not a form of hyaluronic acid). Other
scavenger receptor ligands which are effective may also be used as the first
agent.
The second agent is preferably a form o~f hyaluronic acid such as hyaluronan
or
sodium hyaluronate.
2 5 Therefore, according to another aspect of the invention, I have provided a
dosage kit for maximizing the amount of medicine and/or therapeutic agent to
be delivered to a site in the body in need of treatment and/or for protecting
the
liver from taking up medicine and/or therapeutic agent (particularly cytotoxic
agents) when medicines and/or therapeutic agents must be delivered to treat
2 3 2 .~ 6 4 2 6 0
.,...
rtes other than the liver, comprising:
(a) an effective dosage amount of a first agent such as
chondroitin sulphate which does not bind to receptors at the
site in need of treatment but which binds with receptors of
the liver to thereby "down regulate" the liver;
and, separately from the dosage amount of (a), and,
(b) an effective dosage amount comprising an effective non-toxic
dosage amount of a mediciine and/or therapeutic agent and
1 0 an effective amount of a second agent which is a transport
agent and is a different agent from the first agent (for
example, a form of hyaluronic acid) and which second agent
will bind to the site in need of treatment and would be
capable of binding to the sites of the liver if the liver is not
1 5 down regulated so that the liver's binding capacity for the
second agent has been substantially reduced (preferably
eliminated or blocked) by t:he up-take by the liver of the first
agent (for example, chondroitin sulphate Molecular Weight
20,000) when administered after for example, at least about 3-4
2 0 minutes after administration of the dosage amount of the
first agent under sub-paragraph (a) by for example, binding
with the scavenger receptors of the liver.
The dosage amounts for sub-paragraph (b) may be microgram
2 5 (fig) per kilogram of body weight for example, 20 ~g/kg.
Further, according to another aspect of the invention a method is
provided, comprising:
24 21 6 4 2 6 0
(a) administering an effective amount of a first agent which does
not bind to receptors at the site in need of treatment but
which binds with receptors of the liver thereby "down
regulating" the liver;
and,
(b) thereafter administering an effective amount of a medicine
and/or therapeutic agent (for example, an NSAID [non-
steroidal anti-inflammatory agent] or cytotoxic agent (for
example, methotrexate and cisplatin and combinations
1 0 thereof) and an effective amount of a second agent which is a
transport agent and is a different agent from the first agent
and which second agent is a transport agent which binds to
the site in need of treatment and transports to the interstitial
fluid, lymph and lymph nodes, and would be capable of
1 5 binding to the sites of the liver if the liver had not been
"down regulated" so that it binding capacity for the second
agent has been substantiallly reduced by the up-take by the
liver of the first agent administered under sub-paragraph (a)
by binding with the scavenf;er receptors of the liver.
The first agent may be chondroitin sulphate and the second agent may be a
form of hyaluronic acid. The amounts of each may be as previously discussed.
For example, the amount of chondroitin sulphate may exceed at least about 3-
5mg/kg and the effective amount of the form of hyaluronic acid may exceed 0.1
2 5 mg/70kg person and may have a molecular weight less than 750,000 daltons.
The invention will now be illustrated by reference to the following Figures
and examples and discussion with respect thereto:
25 2164260
Figure 1 illustrates the Biodistribution of Labeled Hyaluronan, 18-
20h after Intravenous Injection of 1 mg c~hondroitin Sulphate Followed by 1 mg
Labeled Hyaluronan;
Figure 2 illustrates the Uptake of 1 mg Labeled Hyaluronan (HA)
With or Without Preinjection of 1 mg Cl.~ondroitin Sulphate (CS);
Figure 2b illustrates the Uptake of 1 mg Labeled Hyaluronan;
Figure 3 is made up of two drawings, the top drawing comparing
MCPM/rat v. Time (min.), and the lower drawing comparing the MCPM/organ
when 1 mg of chondroitin sulphate was administered followed by iZSI_HA (125I-
Hyaluronan).
Figure 4 illustrates the inhibition of labeled hyaluronan (HA)
binding to NGW cells at 37°C. (The chondroitin sulphate does not
interfere
whereas the labeled hyaluronan does.)
Figure 5 shows in vivo imal;es of the reduced liver uptake of labeled
1 S hyaluronan (HA) after pre-treatment of rats by chondroitin sulphate (CS).
Figure 6 illustrates the targeting of tumours by trace amount of
labeled HA (hyaluronan) after administration of chondroitin sulphate (CS) in
an
effective amount of 200 - 400 mg/70 kg person of CS.
2 0 A summary of the data I have developed on tumour targeting using
radiolabelled hyaluronan (HA) administered after chondroitin sulphate (CS)
pretreatment is set out in the Figures and is discussed below. I used female
Wistar FU rats (weight in the order of x!50 gm per rat on average) inoculated
in
one hind leg with a rat colon carcinoma called NGW to develop the data. When
2 5 the tumours appeared, the rats received an intravenous injection of 1 mg
chondroitin sulphate (200 - 400 mg/70 k:g person) followed 30 seconds later by
1
mg HA (hyaluronan) of low specific radioactivity. 18 to 20 hours later the
animals were sacrificed and the radioactivity in the organs measured (see Fig.
1).
21 fi426 0
I have with the same model used :L mg HA (hyaluronan) of similar specific
activity alone and found a tumour to nom-tumour ratio of 7.79+5.00.
Using chondroitin sulphate (CS) pretreatment the ratio is increased to
16.23+2.48. The increase is mainly due to a lower uptake in the non-tumour
tissue (muscle of the healthy leg) but there is also a 27% increase in total
amount
bound (see Fig. 2).
The individual values show that 4 out of 5 "controls" have a tumour to
1 0 non-tumour ratio of about 4 (the relativE~ly high ratio of 7.79 is due to
one single
experiment with a high ratio). In earlier published studies with labeled HA
alone, I have seen tumour to non-tumour ratios of about 4 using similar, but
not
identical tumour systems. See "Accessible hyaluronan receptors identical to
ICAM-1 in mouse mast cells", Stefan C~ustafson, et al., Glycoconjugate Journal
1 5 (1995) 12:350-355. Therefore, I have now developed a real improvement
using
chondroitin sulphate (CS) to the delivery of medicine and/or therapeutic
agents.
The administration of chondroitin sulphate (CS) effectively blocks liver
uptake
of labeled HA (hyaluronan) at 10-15 minutes by about 80% (see Fig. 3) (without
chondroitin sulphate (CS) the liver would have absorbed 95% of the
radioactivity
2 0 at this time).
In vitro data also developed by me establishes the NGW tumour cells
have HA (hyaluronan) take-up receptors that are not inhibited
(blocked/immobilized for a period of time from taking up the second agent (for
2 5 example, hyaluronan)) by chondroitin sulphate (CS).
Figure 4 illustrates three determinations which clearly show that the
uptake of radiolabelled hyaluronan is not interfered with by the chondroitin
sulphate but is interfered with by the unlabeled hyaluronan.
21 6426 0
Composite Figure 5 shows in viva images of the reduced liver uptake of
labeled HA after CS pre-treatment of rats. This figure shows that also uptake
of
trace amounts of HA (equivalent to 0.5-1 mg/70 kg person) can be effectively
inhibited for an extended time by CS (equivalent dose 1-2 gm/70 kg person).
Thus, one may use low amounts of n.ot only active drug (medicine and/or
therapeutic agent) (~g/kg) but also of H.A (~g/kg of body weight of the
patient).
Thus, low amounts of HA (much less than 10 mg/70 kg person) can be used as
the transport agent. Thus, I have developed a treatment whereby a low dose of
1 0 HA and low dose of active drug (medicine and/or therapeutic agent) that
really
targets the site of treatment (for examples diseased tissues) which is best
has been
provided and which also reduces side effects.
I have also performed experiments on tumour rats using 1 mg CS (200 -
1 5 400 mg /70 kg person) followed by a trace dose of labeled HA (equivalent
to 0.5-1
mg/70kg person) and seen good targeting to the tumour (see Figure 6). The
experimental conditions are identical to those previously described for NGW
tumour rats, except for the low dose of HA. The relatively high radioactivity
in
the control muscle is most likely due to circulating degradation products at
this
2 0 time point (about 20 h after injection). lEven so, the tumour to non-
tumour ratio
is 8.8 (p<0.001, n=4), showing that the targeting works also at lower HA
doses.
Some preliminary size exclusion chromatographic studies were conducted
on the Mw (molecular weight) distribution of radiolabeled HA (hyaluronan) in
2 5 serum and urine of rats receiving a prei:reatment of 5mg CS and then 5 ~g
125I-
HA (hyaluronan) with a molecular weight of about 400 kDa (400,000 daltons).
Blood was collected at 2-4, 10-12, 22-24 and 70 minutes after the completion
of the
administration. Urine was collected from the bladder at 70 minutes when the
animals were killed and organs collE~cted. The results show that the Mw
2g 21 642 fi 0
,.....
,molecular weight) of hyaluronan goes down (reduces in the body) by time and
at
70 minutes, most of the hyaluronan that is left in the circulation has a
molecular
weight less than 39 kDa (39,000 daltons). At this time, about 10% of the
injected
radioactivity was found in the urine and had a mean Mw (molecular weight) of
about 20 kDa (20,000 daltons). The radioactivity in the liver was only 6-7%
while
kidneys contained about 9%, I believe, due to material being filtered out into
urine. Blood contained around 35% and as other organs contained only
negligible amounts, about 40% has been filtered out into peripheral tissues.
The
chondroitin sulphate (CS) blocking is therefore an ideal way of getting some
1 0 hyaluronan out into the tissues. This is an additional factor in the
increased
binding of intravenously administered hyaluronan (HA) to tumour tissue that I
found using chondroitin sulphate pretreatment. This provides a further method
of delivering a form of hyaluronan (HA) together with a medicine and/or
therapeutic agent into interstitial fluid, lymph and lymph nodes for the
1 5 treatment of disease for example, cancer and metastases. This treatment
may also
be used to prevent metastases.
As many changes can be made to the embodiments without departing
from the scope of the invention, it is intended that all material contained
herein
2 0 be interpreted as illustrative of the invention and not in a limiting
sense.
