Note: Descriptions are shown in the official language in which they were submitted.
WO91/03'26
2 0 6 ~ 7 5 1'CT/~'S90/~4580
PHARMACEUTICAL COMPOSITIONS CONTAINING AROMATIC
POLYMERS AND THERAPEUTIC METHODS USIN~ T~E SAME
Field of Invention
The present invention relates to methods of therapy
involving the use of said pharmaceutical compositions
containing polyaromatic compounds having properties
which mimic pharmacological activities of
glycosaminoglycans and effect the distribution in
tissue of biologically active peptides and proteins
normally bound to glycosaminoglycans,
Glycosaminoglycans (GAG) are linear polysaccharides
formed by characteristic repeating disaccharide units
usually composed of a uronic acid and a hexosamin,e.
The term ~acid mucopolysaccharides~ was used originally
to designate hexosamine-rich acid polysaccharides
extracted from connective tissue. In recent years, the
term ~glycosaminoglycans~ has gained greater acceptance
and is now used in place of mucopolysaccharides. The
hexosamine can be glycocyamine or galactosamine, and
the uronic acid can be glucuronic or iduronic acid.
Sulphate groups are found on all glycosaminoglycans
apart from hyaluronic acid, and all of the sulphated
~091~03226 ~'C~/-S90/04~80
glycosaminoglycans are covalently linked to protein
forming different classes of proteoglycans. However,
i~ would be an oversi~plification to consider
glycosaminoglycans to be si~ple repeat-unit
polysaccharides, since considerable chemical and
configurational variability can be superimposed upon
the component sugars.
Among other functions it has been shown that the
qlycosaminoglycans serve also as a support which binds
various bioactive peptides. This association is based
on a non-coyalent interaction since the bound protein
can be readily released upon the addition of free
glycosaminoglycans. Well known examples of such bound
proteins include enzymes such as lipoprotein lipase
(LPL) or growth-regulating peptides such as fibroblast
growth factor (FGF). Another example of GAG-protein
interaction is that of the enzyme heparinase which
participates in cell-invasion processes. It has been
demonstrated also that the commercially available
glycosaminoglycan, heparin, inhibits the growth of
vascular smooth muscle cells and the proliferation of
kidney mesangial cells. The former cell type is
involved in atherosclerosis while the latter plays a
role in glomerulosclerosis.
Heparin is known also to be involved in the release
of lipoprotein lipase, the inhibition of heparinase and
the release of fibroblast growth factor. The most
common application of heparin is as an anticoagulant
where heparin interacts with proteins which play a ~ey
role in hemostasis.
WO 9 1 /03226
I'C T/1 :S90/04~80
2064375
Glycosaminoglycans such as heparin are a major
constituent participating in the composition of various
biological structures such as hasement membranes,
connective tissues, cartilage and cell-surface
glycocalyx. Connective tissues are responsible for
providing and maintaining form in the body.
Functioning in a mechanical role, they provide a matrix
that serves to connect and bind the cells and organs
and ultimately give support to the body. Unlike the
other tissue types (epithelium, muscle and nerve)
formed mainly by cells, the major constituent of
connective tissue is its extracellular matrix, composed
of protein fibers, an amorphous ground substance, and
tissue fluid, the latter consisting primarily of bound
water of solvation. Embedded within the extracellular
matrix are the connective tissue cells.
In terms of structural composition, connective
tissue can be subdivided into three classes of
components: cells, fibers and ground substance. The
wide variety of connective tissue types in the body
represents modulations in the degree of expression of
these three components.
The amorphous intercellular ground substance fills
the space between cells and fibers of the connective
tissue; it is viscous and acts as a lubricant and also
as a barrier to the penetration of the tissues by
foreign particles. Glycosaminoglycans and structural
glycoproteins are the two principal classes of
components comprising the ground substance.
The present invention is based on the discovery of
a class of compounds exhibiting properties which mimic
w091/032~6
-- PCT/ l !S90/04!i;80
?~64~
the action of glycosaminoglycans and which are capable
of modulating biological systems containing complexes
between bioactive peptideS and/or proteins and
glycosaminoglycans by competing with the binding
interactions of glycosaminoglycans.
SuD ary of the Invention
The present invention relates to a pharmaceutical
composition comprising, in admixture with a
pharmaceutically acceptable carrier, a therapeutically
effective amount of a polyaromatic ring-containing
polymeric compound, substantially free of monomer, and
having properties which mimic the pharmacological
activity of glycosaminoglycans and which are capable of
competing with the binding thereof to bioactive
peptides and/or proteins.
The present invention relates more specifically to
a pharmaceutical composition including as an active
ingredient a therapeutically effective amount of a
polyaromatic ring-containing polymeric compo~nd having
a molecular weight between about 2,000 and about 20,000
Daltons wherein each monomeric unit of said polymer
includes from 1 to about 10 aromatic rings, which may
be substituted by electronegative substituents and/or
negatively charged residues.
This invention relates also to therapeutic methods
comprising the administration of an effective amount of
the above-mentioned pharmaceutical composition to human
or other animal patients in need of cardiovascular
therapy such as anticoagulant and/or antithrombotic
~'0 91/03226 PC~ S9~/()4~80
20~ 75
therapy and/or bone metabolic therapy and/or therapy
for the treatment of neuronal disorders.
Detailed Descrimtion
As employed above and throughout the disclosure,
the following terms, unless otherwise indicated, shall
be understood to have the following meanings:
~ Distribution in tissue~ or ~tissue distribution~
is used herein to mean either the compartmentalization
of a molecular constituent within a given tissue or the
distribution pattern between different tissues.
~ Tissue redistribution~ means a change in the
compartmentalization within a given tissue or a change
in the distribution pattern between tissues. For
example, a peptide may be released from a basement
membrane which can result subsequently in its
consumption by surrounding cells or its transfer into
the bloodstream, or a soluble compound could be bound
to a protein in a competitive binding situation thereby
preventing the association of said compound with
certain tissue-fixed residues.
~ Alkyl~ means a saturated aliphatic hydrocarbon
which may be either straight or branched-chained
containing from about 1 to about 12 carbon atoms.
~ Lower alkyl~ means an alkyl group as above, having
1 to about 4 carbon atoms.
~ Substituted phenyl~ means a phenyl group
substituted with one or more substituents which may be
alkyl, alkoxy, amino, nitro, carboxy, carboalkoxy,
cyano, alkyl amino, halo, hydroxy, hydroxyalkyl,
mercaptyl, alkyl mercaptyl, carboalkyl or
carbamoyl.
WO91/03226 1'Cr/~590/04580
4~ 6
Certain of the polymeric compounds included in the
compositions of the present invention may exist in
enolic or tautomeric forms, and all of these forms are
considered to be included within the scope of this
invention.
The polymeric compounds included in the
compositions of this invention may be useful in the
form of the free base, in the form of salts, esters and
as hydrates. All forms are within the scope of the
invention. Acid addition salts may be formed and are
simply a more convenient form for use; and in practice,
use of the~salt form inherently amounts to use of the
base form. The acids which can be used to prepare the
acid addition salts include preferably those which
produce, when combined with the free base,
pharmaceutically acceptable salts, that is, salts whose
anions are non-toxic to the animal organism in phar-
maceutical doses of the salts, so that the beneficial
pharmacological properties inherent in the free base
are not vitiated by side effects ascribable to the
anions. Although pharmaceutically acceptable salts of
said basic compound are preferred, all acid addition
salts are useful as sources of the free base form even
if the particular salt per se is desired only as an
intermediate product as, for example, when the salt is
formed only for purposes of purification and
identification, or when it is used as an intermediate
in preparing a pharmaceutically acceptable salt by ion
exchange procedures. Pharmaceu~ically acceptable salts
of the compounds useful in the practice of this
invention are those derived from the following acids:
mineral acids such as hydrochloric acid, sulfuric acid,
phosphoric acid and sulfamic acid; and organic acids
such as acetic acid, citric acid, lactic acid, tartaric
w091/03226 PCT/US90/04580
20~7~
acid, malonic acid, methanesulfonic acid,
ethanesulfonic acid, benzenesulfonic acid,
p-toluenesulfonic acid, cyclohexylsulfamic acid, quinic
acid, and the like. The corresponding acid addition
salts comprise the following: hydrochloride, sulfate,
phosphate, sulfamate, acetate, citrate, lactate,
tartarate, methanesulfonate, ethanesulfcnate,
benzenesulfonate, p-toluenesulfonate, cyclohexylsul-
famate and quinate, respectively.
The acid addition salts of the compounds contained
in the compositions of the present invention are
prepared either by dissolving the free base in agueous
or aqueous-alcohol solution or other suitable solvents
containing the appropriate acid and isolating the salt
by evaporating the solution, or by reacting the free
base and acid in an organic solvent, in which case the
salt separates directly or can be obtained by
concentration of the solution.
Preferred pharmaceutical compositions include
polymeric compounds having monomeric units containing
about three to about ten aromatic rings and having, on
at least two of the aromatic rings, one or more
substituent selected from the group consisting of NRRl,
-N~R, -OR, ~O, -NO2, -COOR, halogen, -SO20R, -SO2NHR,
-OSO2OR and R, wherein R is Cl-C12 alkyl or hydrogen
and Rl is lower alkyl, hydrogen, phenyl or substituted
phenyl.
The more preferred number of aromatic rings in each
monomeric unit is about three to about six.
W091~03226 PC~US90/04~X0
~ 6~ 8
Polymers of many known synthetic dyes comprising
monomers having the polyaromatic characteristics
identified above are within the scope of the present
invention and are useful in pharmaceutical
compositions. Such dyes are known to be useful as
color additives, diagnostic aids administered by i.v.
injection, antiseptic agents and/or to treat infectious
disease. Nevertheless, none of these dyes has
previously been described as useful as or used as a
therapeutic agent as a replacement for
glycosaminoglycans.
Exemplary synthetic dyes including polymeric
materials in accordance with this invention include
Aluminon, Halogenated Aluminon, Sulfonated Aluminon,
Sulfonated-Halogenated Aluminon, Anazolene Sodium,
Eosine I Bluish, Eosine Yellowish, Erythrosine, Evan's
Blue, Fast Green FCF, Fucshin(e) Acid, Iodophthalein
Sodium, Rose Bengal, Sulfobromophthalein Sodium,
Suramin Sodium, Trypan Blue, Trypan Red, Rosaniline
Chloride, Crystal Violet, Methyl Blue, Methyl Green,
Coomassie Blue, Basic Fuchsin, Malachite Green,
Brilliant Green, Aniline Blue, Brilliant Cresyl Blue,
Safranin 0, Ethyl Violet, Pararosaniline Acetate and
Methyl Violet.
WO91~03226 PC~ S90/04;80
206437~
More preferred is a polymeric compound of the
structure:
L~(C~2,T
or a salt or ester thereof, wherein a, b and c are
independently 0 or 1 and m is about 5 to about 20, and
dashed lines represent single or double bonds, each
aromatic ring is substituted with at least one
substituent (x, y, z) selected from -NRRl, -N=R, -OR,
=O, -NO2, -COOR, halogen, -SO2OR, SO2NHR, -OSO2OR and R,
wherein R i5 lower alkyl or hydrogen and Rl is lower
alkyl, hydrogen or phenyl and substituted phenyl.
WO9l/03~26 ~CT/US90/04~#0
~,~$ ~ 10
Most preferred is a polymeric compound of the
structure:
_ _ (CH2)a~/COOH
_ ~ ~ _
OOH COOH m
wherein a, b and c are independently 0 or 1 and a+b+c >
2 and m is about 5 to about 20.
The preferred molecular weight of the polymeric
compounds is about 2,000 and about 20,000, and the most
preferred molecular weight is about 2,000 to about
4,000 Daltons, as measured by gel permeation
chromatography.
The following gel per~eation chromatography
conditions has been used to measure the molecular
weight of the polymeric compounds contained in the
product identified commercially, and in the Nerck
Index, as aurintricarboxylic acid (aluminon or ATA~.
Column: Two I-125 (Waters, TN) protein
analysis column in series
Mobil Phase: Water (70$)-propanol (30%)
containing 0.1% (V/V)
trifluoroacetic acid
Detection: W at 220m
w09l/03~26 ~ ,S90/04~80
206~75
Injection: Sample was dissolved in the mobile
phase and injected
Pharmaceutical compositions containing the
pharmacologically active polymeric compounds are
believed to function according to one or more of the
following mechanisms.
1. Removal of cationic proteins from the
glomerular basement membrane or connective-tissues,
thereby preventing local damage (i.e., via ~recharging~
of negatively charge residues in the glomerular
basement membrane).
2. Modulation of LPL, a key enzyme in lipid
distribution among various tissues which could be
implicated in cardiovascular diseases.
3. Release of growth-promoting molecules, such as
fibroblast growth factor (FGF), from basement membranes
in order to enhance the process of angiogenesis and
wound-healing. In addition, FGF can prevent death of
lesioned neurons (see Anderson R.J. et al. Nature
332:360-1 (1988). By the release of endogenously-
stored FGF, the compositions within the scope of the
present invention may be useful in the treatment of
neuronal disorders such as Alzheimer's disease and
other dementia.
4. Blocking the activity of heparinase, an enzyme
which participates in inflammatory processes and
metastases formation.
5. Modulation of bone metabolism.
6. Control of the proliferation of certain cell
types such as smooth muscle cells or mesangial cells.
Preferred embodiments of the present invention are
described in the following non-limiting examples which
~09l/03Z26 ~'C~/l,'S90/~4~X~)
~,~5~ 12
include a description of pharmacological test
procedures believed to correlate to therapeutic
activity in humans and other animals.
EXAMPLE 1
It has been determined that the pharmaceutical
activity of the mixture of compounds commercially
available as ~Aluminon~ (aurintricarboxylic acid or
ATA) is produced by the fraction of the mix~ure
containing polymers having a molecular weight of about
2,000 to about 20,000 Daltons. These polymeric
materials are believed to comprise monomeric units of
ATA.
The following comparative work shows the relative
pharmacological activities of various fractions of the
commercial ATA product. On the basis of the following
worX compositions within the scope of the present
invention exhibit useful anticoagulant and
antithrombotic activity.
Experimental Procedures
ComDarative ComDound A - Commercially available
aurintricarboxylic acid (ATA) from Aldrich Chemical
Co., Wisconsin.
Compound 8 - Polymer containing ATA monomer and having
molecular weight of about 2,000 to about 4,000 Daltons.
This material is prepared using commercially available
aurintricarboxylic acid which is fractionated on Biogel
P-6 by elution with 50% aqueous ethanol with 50mM Tris
at pH 7.5 and then 50% aqueous ethanol with 50mM
glycine at pH 11. The fraction containing polymeric
w09l/03226 PCT/~S90/04580
20~4~75
material having a molecular weight of about 2000 to
about 4000 Daltons is separated.
Comparative Compound C - ATA not containing polymers.
Following the procedure described in Oraanic Svnthesis,
Coll. Vol. I, page 54, the following reaction was
performed to produce what is believed to comprise non-
polymerized ATA. To 17 ml of concentrated sulfuric
acid at 0-5 C and mechanically stirred was added
portionwise sodium nitrite (2.5 gm, 36.2 mmol). The
mixture was stirred until a solution was obtained.
Salicylic acid (5.0 gm, 36.5 mmol) was added
portionwise over 10-15 minutes and the reaction warmed
to room temperature, stirred until a homogeneous
mixture was obtained and cooled to 0-5-C. A solution
of formaldehyde (formalin, 35-40%, 1.25 mL, 16.3 mmol)
was added dropwise over 20 minutes. The reaction was
quenched with the slow addition of 25 gm crushed ice
and then 125 mL of ice water. The resulting solid was
filtered, washed with water and dissolved in dilute
aqueous ammonium hydroxide. Removal of the volatiles
at 90-C and then in vacuo provided the red-brown solid.
Comparative ComDound D - Polymer believed to contain
ATA monomeric units is prepared by using a large excess
of formalin in the above procedure along with the
following reagents.
concentrated sulfuric acid: 17mL
sodium nitrite: 2.5 gm, 36.2 mmol
salicylic acid: 5.0 gm, 36.5 mmol
formalin: 35-40%, 6.0 mL, 81.5 mmol
~9l/03226 ~'Cr/-S9n/04580
~6~ 14
The pharmacological activity of Compounds A through
D is measured according to the following test
procedure.
Activated Partial T~romboplastin Time (APTT~
To an assay cuvette is added 100 ul of normal
pooled plasma (George King Biomedical Inc., Kansas) and
100 ul of a solution containing one of the above
compounds in aqueous 50 mM Tris hydrochloride at p~ 7.5
(0.2 mg of sample in one mL buffer). The sample is
placed in a MLA coagulation timer which automatically
maintains ~the sample at 37 C for 2.5 minutes, 100 ul of
actin activated cephaloplastin reagent is injected,
kept 5 minutes, lO0 ul of 35 mM CaCl is injected, and
clot formation is determined photometrically and the
time recorded. Results are presented in TABLE I below.
TABLE I
ComDound Concentration tma/mL) APTT (sec.)
Normal pooled plasma ~ - 25 - 28
A - Commercial ATA 0.2 40 - 60
C - Non-Polymerized ATA 0.2 28
D - Polymer containing
ATA monomer 0.2 72
B - Polymer having molecular
weight of 2,000 to 4,000
containing ATA monomer 0.2 llO
~ he above comparative test results demonstrate that
a polymer having a molecular weight of 2000 to 4000
Daltons and believed to contain monomers of ATA exhibit
anticoagulant actiYity.
All of the examples presented below involve the use
of commercially available materials. Based on the
~09l/0~226 I'Cr/US90/n4580
2064~ 7~
above example, the polymeric components of the
commercially available products are believed to be the
source of the APTT activity discovered in the
commercial products.
EXAMPLE 2
The polymeric components of the following dyes, at
the concentration listed, significantly prolong the
activated partial thromboplastin time when compared to
a standard buffer solution.
ComDoundMinimum Final Concentration (uq/mll
Rosaniline Chloride 12.5
Crystal Violet8.3
Methyl Blue100.0
Methyl Green50.0
Coomassie Blue150.0
*Basic FuchsinS0.0
Malachite Green150.0
Brilliant Green150.0
Aniline Blue150.0
Brilliant Cresyl Blue 150.0
Safranin O 150.0
Ethyl Violet 50.0
Pararosaniline Acetate 150.0
Methyl Violet 75.0
A~pLE 3
The effect on enzyme levels and enzyme
compartmentalization of compositions within the scope
of the present invention are measured according to the
test procedure described below.
W09l/03226 ~6 ~ PCT/US90/04580
Modulation of Lipoprotein Lipase (LPL) Levels and
Compartmentalization
The enzyme LPL participate in the process of lipid
transfer from the bloodstream to the tissues, LPL is
bound to the external surface of cells via its
association with cell-membrane glycosaminoglycans.
Cultured heart cells is a powerful system in the study
of the enzyme turn-over and the exploration of ways to
manipulate its level since these cells are active in
the biosynthesis of LPL, in addition to the surface
binding of the enzyme.
The following are the materials and methods used:
Fl heart cell cultures are prepared from newborn
rate hearts as previously described (Chajek, T. et al.,
Biochem. Biophys. Acta 528, 456-474 (1978)). They
consist mainly of non-beating mesenchymal cells and are
used 8-12 days after their isolation.
Enzyme activity is determined using aliquots of
medium and of homogenates of cells which are released
from a petri dish with a rubber policeman in 1 ml of
0.025 M NH3/NHjCl buffer (pH 8.1) containing 1 unit/ml
of heparin. The assay system consists of 0.1 ml of
medium or 0.1 ml of cell homogenate ~S0-70 g protein)
and 0.1 ml of substrate containing labeled triolein,
prepared according to the method of Nilsson-Ehle and
Schotz, (J. Lipid Res. 17, 536-541 (1976)).
Incubations are carried out at 37-C for 45 mins. The
reaction is stopped by the addition of
methanol/chloroform/heptane (1.4:1.25:1.v/v) and the
extraction of fatty acids is performed according to the
method of ~3elfrage and Vaughan (J. Lipid Res. 10, 341-
344 (1969), as modified by Nilsson-Ehle and Schotz.
WO 91/03226 PCr/l~S90/04580
2064~ 75
Enzyme activity is calculated according to the formula
of Nilsson-Ehle and Schotz.
Table II below demonstrates that commercially
available Evan's Blue modulates or shifts-up enzyme
levels and influences enzyme compartmentalization.
TABLE II
LPL activity following 24 hrs. incubation of
heart cells with commercially available Evan's Blue
LPL activity: m mole FFA/h/dish
(mean+SEM of triplicates)
Evan's Blue
Concentration % increase
(M) Cells Medium Total in t~e total
0 2420_93 817+14 3237 -(0)
10-5 2757+114 1531+182 4288 +32.5
10-' 2447+48 2783+194 5230 +61.6
10-~ 1698_184 6051+435 7749 +139.4
Similar results are obtained with commercially
available Methyl Blue, Trypan Blue and Aluminon. The
foregoing results indicate that compositions within the
scope of this invention are useful in the treatment of
cardiovascular diseases such as arteriosclerosis.
~XAMPLE 4
The active ingredients in the composition of the
present invention are effective inhibitors of
heparinase activity. The inhibitory activity is
measured by the following procedure.
WO~ 3226 PCT/~S90/04~80
'' `l~
18
Inhibition of HeDarinase Activity
Heparinase is an endoglucuronidase capable of
degrading heparin sulfate (HS) at specific intrachain
sites. Studies on degradation of sulfated
proteoglycans in the subendothelial extracellular
matrix (ECM) demonstrated a correlation between
heparinase activity and the metastatic potential of
various tumor cells. Heparinase activity was also
suggested to play a role in the mobilization of normal
circulating cells of the immune system during
inflammatory processes.
The ability of compositions of the present
invention to inhibit lymphoma-cell derived heparinase
is tested in the assay system described by Vlodavsky et
al. (Cancer Research. 43:2704-2711 (1983)). 35S
labeled ECM is incubated for 24 hours with ESb mouse
lymphoma heparinase in the presence of 10 ugr ml of the
indicated compounds. Degradation of the sulfated
glycosaminoglycan is followed by gel filtration of the
supernatants. Heparinase activity is expressed as the
total amount of labeled low-molecular-weight fragments
released from the EMC substrate.
Exemplary compositions are tested for inhibitory
activity are the results of this test work is
summarized in Table III below.
WO91/03226 PCT/~'S90/04580
TABLE III
Inhibition of heparinase activity by various
commercially available compounds
Total low-mol-wt. HS
Compound fragments released by
rlO uar/ml) hemarinase (c~m) ~ Inhibition
Control 17,756 --
Methyl Blue 13,676 23
Fast Green12,139 32
Evan's Blue 6,986 60
Aluminon 3,822 78
Anticoagulant properties are evaluated in
accordance with the following method.
EXAMPLE 5
The anticoagulant effect of commercially available
Aluminon is demonstrated by measuring the partial
thromboplastin time (PTT) in blood samples from a
normal donor. 0.25 ml of the indicated concentration
of the drug (xlO, diluted in saline) is added to 2.25
ml of fresh human blood, collected in the presence of
citrate. PTT is measured following the addition of
Ca~.
The results are summarized in Table IV below.
U'O 91~0~'26 ~
Co4~ ~cr/~s9n/04~80
TABLE IV
PTT of human blood in the presence of various
concentrations of commercially available Aluminon
~luminon final conc. (ma/ml~ PTT tsec.)
1 >300
0.1 190
0.01 30-5
0 (control)30.5
Commercially available aluminon is an effective
anticoagulant. Studies in laboratory animals, such as
laboratory rats, demonstrate that aluminon can be
absorbed through the gastrointestinal tract. Oral
administration of commercially available aluminon to
rats (1 ml of 10-20% solution) prolongs rats' PTT
considerably (from around 20 sec. at time 0 to >200
sec. after 2-4 hours); concomitantly, plasma levels of
LPL were elevated by about an order of magnitude.
Consequently, the compositions of the present invention
are capable of being administered orally to produce an
anticoagulant effect. In contrast, heparin can only be
administered by injection.
EXAMPLE 6
The active ingredients of the compositions within
the scope of this invention are useful inhibitors of
the proliferation of vascular smooth muscle cells. The
proliferation inhibition of the active ingredients of
the present compositions may be evaluated according to
the following procedure.
wO9l/03226
PCT/US90/04580
2064~75
Inhibition of Vascular-Smooth Muscle Cell Proliferation
Bovine aortal smooth-muscle cells are grown in
tissue culture plates in the presence of glucose-rich
medium (H-21). The cultures (around 35Xl03 cells/well
in 24 wells' plates) are initially supplemented with
10% fetal calf serum (FCS) for 2-3 days. The cell
nutrition is then replaced by a serum-deprived medium
(H-21 plus 0.2% FCS) in order to test the effect of
defined growth factors on the proliferation of smooth-
muscle cells and their possible inhibition by the
compounds with heparin-like activity.
In the~following representative experiment, the
cells transferred to the serum deprived medium are
grown in the presence of thrombin (10-6M) or FGF
(fibroblast growth factor, partially purified from
bovine brain, 250 ngr/ml). Various concentrations of
Aluminon were added to the thrombin or FGF-treated
cells and to non-treated control cultures. 3H
thymidine incorporated into DNA was measured. The
results are presented in Table V below.
TABLE V
Growth inhibition of resting, FGF or thrombin-
stimulated smooth muscle cells by commercially available aluminon
Aluminon H3 thymidine incorporation per well
Concentration (cpm. mean of duplicates)
(u~r/ml) Contxol +FGF +Thrombin
0 9,823 22,574 S4,000
0.2 8,425 22,256 46,857
2 5,120 13,089 14,064
4,105 6,846 6,877
4,343 6,745 5,705
WO 9 1 /03226 PC I / ~. .'i90/04~;8~
~ 4~
The results of Table V show that Aluminon, at a
concentration of 2 ~gr/ml (-4xlO-~), is effective in
inhibiting labeled thymidine incorporation by more than
40% and is capable of mimicking the action of heparin
in this system as a potent inhibitor of the
proliferation of vascular smooth muscle cells.
Compositions of the present invention are useful in
the treatment of cardiovascular diseases, including
arteriosclerosis, bone metabolism and neuronal
disorders.
The compositions of this invention can be normally
administered orally or parenterally, in the treatment
of cardiovascular disorders, bone metabolic disorders
and neuronal disorders in humans or other mammals.
The compositions of this invention may be
formulated for administration in any convenient way,
and the invention includes within its scope
pharmaceutical compositions containing at least one
polymeric compound as described hereinabove adapted for
use in human or veterinary medicine. Such compositions
may be formulated in a conventional manner using one or
more pharmaceutically acceptable carriers or
excipients. Suitable carriers include diluents or
fillers, sterile aqueous media and various non-toxic
organic solvents. The compositions may be formulated
in the form of tablets, capsules, lozenges, troches,
hard candies, powders, aqueous suspensions, or
solutions, injectable solutions, elixirs, syrups and
the like and may contain one or more agents selected
from the group including sweetening agents, flavoring
~0 91/U3226 f'Cr/~590/0$5~0
2064375
23
agents, coloring agents and preserving agents, in order
to provide a pharmaceutically acceptable preparation.
The particular carrier and the ratio of
therapeutically effective compound to carrier are
determined by the solubility and chemical properties of
the compounds, the particular mode of administration
and standard pharmaceutical practice. For example,
excipients such as lactose, sodium citrate, calcium
carbonate and dicalcium phosphate and various disin-
tegratants such as starch, alginic acid and certain
complex silicates, together with lubricating agents
such as magnesium stearate, sodium lauryl; sodium
lauryl sulphate and talc, can be used in producing
tablets. For a c~psule form, lactose and high
molecular weight polyethylene glycols are among the
preferred pharmaceutically acceptable carriers. Where
aqueous suspensions for oral use are formulated, the
carrier can be emulsifying or suspending agents.
Diluents such as ethanol, propylene glycol, glycerin
and chloroform and their combinations can be employed
as well as other materials.
For parenteral administration such as intramuscular
and subcutaneous injection, solutions or suspensions of
the polymeric compounds in sesame or peanut oil or
aqueous propylene glycol solutions, as well as sterile
aqueous solutions can be employed. The aqueous
~olutions using pure distilled water are also useful
for intravenous injection purpoQes, provided that their
pH is properly adjusted, suitably buffered, made
isotonic with sufficient saline or glucose and
sterilized by heating or by microfiltration.
U'O 91/(~3226
l~cr/~sso/w~xo
~,9Co 4~ ~
24
The dosage regimen in carrying out the methods of
this invention is that which insures maximum
therapeutic response
until improvement is obtained and thereafter the
minimum effective level which gives relief. Thus, in
general, the dosages are those that are therapeutically
effective in increasing the clotting time of blood,
decreasing the chances of thrombosis, reducing the
buildup of arterial plaque or in the treatment of bone
metobolic or neuronal disorders such as Alzheimer's
disease. In general, the oral dose may be between
about 1 mg/kg and about 500 mg/kg (preferably in the
range of 1 to 10 mg/kg), and the i.v. dose about 0.1
mg/kg to about 10 mg/kg (preferably in the range of
about 0.1 to about 3 mg/kg), bearing in mind, of
course, that in selecting the appropriate dosage in any
specific case, consideration must be given to the
patient's weight, general health, age and other factors
which may influence response to the drug. The drug may
be administered as frequently as is necessary to
achieve and sustain the desired therapeutic response.
Some patients may respond quickly to a relatively large
or small dose and require little or no maintenance
dosage. On the other hand, other patients may require
sustained dosing from about 1 to about 4 times a day
depending on the physiological needs of the particular
patient. Usually the drug may be administered orally 1
to 4 times per day. It is anticipated that many
patients will require no more than about one to about
two doses daily.
It is also anticipated that the present invention
would be useful as an injectable dosage form which may
be administered in an emergency to a patient suffering
WO91/03226 1~-r/US90/04~80
206~ 7S
from stroke or heart attack. Such treatment may be
followed by intravenous infusion of the active
polymeric compound and the amount of compound infused
into such a patient should be effective to achieve and
maintain the desired therapeutic response.
It will be evident to those skilled in the art that
the invention is not limited to the details of the
foregoing illustrative examples and that the present
invention may be embodied in other specific forms
without departing from the essential attributes
thereof, and it is therefore desired that the present
embodiments and examples be considered in all respects
as illustrative and not restrictive, reference being
made to the appended claims, rather than to the
foregoing description, and all changes which come
within the meaning and range of equivalency of the
claims are therefore intended to be embraced therein.
