Note: Descriptions are shown in the official language in which they were submitted.
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- HOECHSTAKTIENGESELLSCHAFT HOE 96/F 115 Dr. MS/we
Description
5 Antiadhesive properties of polyacrylic acids and polymethacrylic acids
The present invention relates to polyacrylic acids and polymethacrylic acids
of various molar masses for use as pharmaceuticals and, in particular, for
use in the therapy and prophylaxis of diseases associated with excessive
10 cell adhesion, mediated by selectin receptors, in the tissue affected by the
disease.
The circulation of blood cells, such as, for example, leukocytes,
neutrophils, granulocytes and monocytes is a very complex multistage
15 process at the molecular level, only some of the steps in which are known
(Review: T.A.Springer, Cell 76, 301-314, 1994).
The most recent research results have shown that the recirculation of
Iymphocytes which is crucial in immune surveillance, and the localization of
20 neutrophils and monocytes at foci of innan,mation obey very similar
molecular mechanisms. Thus, in acute and chronic inflammatory
processes there is adhesion of the leukocytes to endothelial cells and
migration out into the focus of inflammation and into the secondary
Iymphatic organs.
Numerous specific signal molecules are involved in this process, such as,
for example, interleukins, leukotrienes and tumor necrosis factor (TNF),
their G protein-coupled receptors and, in particular, tissue-specific cell
adhesion molecules, which ensure accurately controlled recognition of
30 immune and endothelial cells. The principal adhesion molecules involved
in this, which are to be referred to as receptors hereinafter, include the
selectins (E, P and L selectins), integrins and the members of the
immunoglobulin superfamily.
35 The three selectin receptors determine the initial phase of leukocyte
CA 0220~121 1997-0~-12
adhesion. E selectin is expressed on endothelial cells a few hours after
stimulation by, for example, interleukin -1 (IL-1~) or tumor necrosis factor
(TNF-a) while P selectin is stored in blood platelets and endothelial cells
and is presented after stimulation by thrombin, peroxide radicals or
substance P, inter alia on cell surfaces. L selectin is permanently
expressed on leukocytes but is rapidly eliminated again from the
leukocytes during the course of the inflammation.
The adhesion of leukocytes to endothelial cells which is mediated by
selectin receptors in the initial phase of inflammatory processes is a natural
and necessary immune response to various inflammatory stimuli and
injuries to vascular tissue. However, the course of a number of acute and
chronic disorders is unfavorably influenced by excessive adhesion of
leukocytes and their infiltration into the affected tissue, and by damage to
healthy tissue in the sense of an autoimmune reaction. These include, for
example, rheumatism, reperfusion injuries such as myocardial
ischaemia/infarct (Ml), acute inflammation of the lungs after a surgical
operation, traumatic shock and stroke, psoriasis, dermatitis, ARDS (adult
respiratory distress syndrome) and the restenosis occurring after surgical
operations (example angioplasty and by-pass surgery).
The natural ligand has already been used successfully in animal
experiments for P selectin-dependent lung injuries (M.S.Mulligan et.al.,
Nature 1993, 364, 149) and for myocardial reperfusion injuries (M.Buerke
et.al., J .Clin. Invest. 1994, 93, 1 140).
In screening for biologically active specific antagonists, it has been found,
surprisingly, that polyacrylic acids and polymethacrylic acids (commercially
available, for example from Fluka) inhibit, depending on their average
30 molar mass, cell adhesion mediated by P selectin.
Accordingly, the present invention relates to a polymer of acrylic acid or of
methacrylic acid or their pharmacologically suitable salts for use as
pharmaceuticals.
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The polymer is preferably polyacrylic acid, preferably its sodium salt.
Particularly suitable for use as pharmaceuticals are polymers of said acids
with molar masses of from 900 to 63,000 g/mol.
Said polymers are particularly suitable for producing a pharmaceutical for
the therapy or prophylaxis of a disease associated with excessive cell
adhesion, mediated by selectin receptor~, in the tissue affected by the
disease, preferably a cardiovascular disorder or a rheumatic disorder.
The pharmaceuticals according to the invention are generally administered
intravenously, orally or parenterally or as implants, but rectal administration
is also possible in principle. Suitable solid or liquid pharmaceutical
formulations are, for example, granules, powders, tablets, coated tablets,
15 (micro)capsules, suppositories, syrups, emulsions, suspensions, aerosols,
drops or injectable solutions in ampule form, and products with protracted
release of active substance, for the production of which normally vehicles
and additives and/or auxiliaries such as disintegrants, binders, coating
agents, swelling agents, glidants or lubricants, flavorings, sweeteners or
20 solubilizers are used. Examples of vehicles or ancillary substances which
are frequently used are magnesium carbonate, titanium dioxide, lactose,
mannitol and other sugars, talc, lactalbumin, gelatin, starch, vitamins,
cellulose and its derivatives, animal and vegetable oils, polyethylene
glycols and solvents such as, for example, sterile water, alcohols, glycerol
25 and polyhydric alcohols.
The pharmaceutical products are preferably produced and administered in
dosage units. Solid dosage units are tablets, capsules and suppositories.
30 The daily doses necessary for treating a patient differ depending on the
activity of the compound, the mode of administration, the nature and
severity of the disorder, and the age and body weight of the patient.
However, higher or lower daily doses may also be appropriate in some
circumstances. The daily dose can be administered either by a single
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administration in the form of a single dosage unit or else several small
dosage units, or by multiple administration of divided doses at particular
intervals. The daily dose to be administered may also depend on the
number of receptors expressed during the course of the disease. It is
5 conceivable that only a few receptors are expressed on the cell surface in
the initial stage of the disease and, accordingly, the daily dose to be
administered is smaller than for patients whose disease is severe.
The pharmaceuticals according to the invention are produced by
10 converting said polymers with conventional vehicles and, where
appropriate, additives and/or auxiliaries into the or a suitable dosage form.
The high molecular weight polyacrylic acids and polymethacrylic acids are
very potent and specific P selectin antagonists. This can be demonstrated
15 by means of the cell adhesion assay described below.
Example 1:
Primary assays for investigating the effect on cell adhesion by
20 recombinant soluble selectin fusion proteins.
In order to test the activity of the polymers on the interaction between the E
and P selectins (old terminology ELAM-1 and GMP-140 respectively) with
their ligands, an assay which is specific in each case for only one of these
25 interactions is used. The ligands are offered in their natural form as surface
structures on promyelocytic HL60 cells. Since HL60 cells have ligands and
adhesion molecules which vary greatly in the specificity, the required
specificity of the assay can be provided only by the binding partner. The
binding partners used were genetically engineered soluble fusion proteins
30 from, in each case, the extracytoplasmic domain of E or P selectin and the
constant region of a human immunoglobulin of subclass IgG1.
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Preparation of L selectin-lgG1
Soluble L selectin-lgG1 fusion protein was prepared by using the genetic
construct "ELAM-Rg" published by Walz et al.,1990.
For expression, the plasmid DNA was transfected into COS-7 cells (ATCC)
using DEAE-dextran (molecular biological methods: see Ausubel, F.M.,
Brent, R., Kingston, R.E., Moore, D.D., Seidman, J.G., Struhl, K. and
Smith, J.A. 1990. Current Protocols in Molecular Biology, John Wiley, New
York). Seven days after the transfection, the culture supernatant was
obtained, centrifuged to remove cells and cell fragments and adjusted to
25 mM Hepes pH 7.0, 0.3 mM PMSF, 0.02% sodium azide and kept at
+4~C. (Walz, G., Aruffo, A., Kolanus, W., Bevilacqua, M. and Seed, B.
1990. Recognition bei ELAM-1 of the sialyl-Lex determinant on myeloid
and tumor cells. Science 250,1132-1135.)
Preparation of P selectin-lgG1
The soluble P selectin-lgG1 fusion protein was prepared by using the
genetic construct "CD62Rg" published by Aruffo et al.,1991. The
subsequent procedure corresponds to the preparation of L selectin-lgG1
described under A1.
Aruffo, A., Kolanus, W., Walz, G., Fredman, P. and Seed, B.1991. CD62/
-P Selectin recognition of myeloid and tumor cell sulfatides. Cell 67, 35-44.
Preparation of CD4-lgG1
30 The soluble CD4-lgG1 fusion protein is prepared by using the genetic
construct "CD4:1gG1 hinge" published by Zettlemeissl et al.,1990. The
subsequent procedure corresponds to the preparation of L selectin-lgG1
described under A1. (Zettelmeissl, G., Gregersen, J.-P., Duport, J. M.
Mehdi, S., Reiner, G. and Seed, B.1990. Expression and characterization
CA 02205121 1997-0~-12
of human CD4: Immunoglobulin Fusion Proteins. DNA and Cell Biology 9,
347-353 )
Procedure for the HL60 cell adhesion assay for recombinant soluble
5 adhesion molecules
1. 96-well microtiter assay plates (Nunc Maxisorb) are incubated with
100 ,ul of a goat anti-human IgG antibody (Sigma), diluted (1 + 100)
in 50 mM Tris pH 9.5, at room temperature for 2 h. Removal of the
antibody solution is followed by one wash with PBS.
2. 150 ,ul of the blocking buffer are left in the wells at room temperature
for 1 h. The composition of the blocking buffer is: 0.1 % gelatin, 1 %
BSA, 5% calf serum, 0.2 mM PMSF, 0.02% sodium azide. Removal
of the blocking buffer is followed by one wash with PBS.
3. 100 ~ul of cell culture supernatant from appropriately transfected and
expressed COS cells are pipetted into each of the wells. Incubation
takes place at room temperature for 2 h. Removal of the ceil culture
supernatant is followed by one wash with PBS.
4. 20 ,ul of binding buffer are placed in the wells. The binding buffer
has the composition: 50 mM Hepes, pH 7,5;
100 mM NaCI; 1 mg/ml BSA;
2 mM MgCI2; 1mMCaCI2, 3mM MnCI2; 0.02% sodium azide; 0.2 mM
PMSF. 5 ,ul of the test substance are pipetted into this column,
mixed by swirling the plate and incubated at room temperature for
10 min.
5. 50 ml of an HL60 cell culture with 200,000 cells/ml are centrifuged
at 350 9 for 4 min. The pellet is resuspended in 10 ml of RPMI 1640
and the cells are again centrifuged. To label the cells, 50 ,ug of
BCECF-AM (Molecular Probes) are dissolved in 5 ,ul of anhydrous
DMSO; then 1.5 ml of RPMI 1640 are added to the BCECF-
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. 7
AM/DMSO solution. The cells are resuspended in this solution and
incubated at 37~C for 30 min. After centrifugation at 350 g for two
minutes, the labeled cell pellet is resuspended in 11 ml of binding
buffer, and the resuspended cells are distributed in 100 ,ul aliquots in
the wells of the microtiter plate. The plate is left to stand at room
temperature for 10 min in order to allow the cells to sediment to the
bottom of the assay plate. During this, the cells have the opportunity
to adhere to the coated plastic.
10 6. To stop the assay, the microtiter plate is completely immersed at anangle of 45~ in the stop buffer (25 mM Tris, pH 7.5; 125 mM NaCI;
0.1% BSA; 2 mM MgCI2; 1 mM CaCI2; 3 mM MnCI2; 0.02% sodium
azide). The stop buffer is removed from the wells by inversion, and
the procedure is repeated twice more.
7. Measurement of the cells adhering in the wells and labeled with
BCECF-AM takes place in a cytofluorimeter (Millipore), with a
sensitivity setting of 4, an excitation wavelength of 485/22 nm and
an emission wavelength of 530/25 nm.
Results:
IC 50 values for P selectin and for E selectin:
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IC50(,uM, IC50(,uM,
P selectin) E selectin)
Polyacrylic acid
165.00 > 2000
995 Na salt
Polyacrylic acid
125.00 ~ 2000
2100 Na salt
Polyacrylic acid
21.00 ~ 2000
4100 Na salt
Polyacrylic acid0.009
62,900 Na salt
(Specifications according to the information in the Fluka 1996 catalog).
Example 2:
Leukocyte adhesion - test of the activity of the compounds according to the
invention in vivo (intravital microscopy on rats):
Tissue destruction by leukocytes which migrate in or block the
microcirculation plays a crucial part in inflammatory processes and other
cytokine-activating conditions. The first phase, which is crucial for the
subsequent disease process, is activation of leukocytes within the blood
stream, in particular in the pre- and postcapillary region. This results, after
the leukocytes have left the axial flow of blood, in an initial adhesion of the
leukocytes to the inner wall of the vessel, i.e. in the vascular endothelium.
All the following leukocyte effects, i.e. active migration through the vessel
wall and subsequent oriented migration in the tissue, are subsequent
reactions (Harlan, J.M., Leukocyte-endothelial interaction, Blood 65, 513-
525, 1985).
This receptor-mediated interaction of leukocytes and endothelial cells is
regarded as an initial sign of the inflammatory process. Besides the
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-
adhesion molecules which are already physiologically expressed, on
exposure to mediators of inflammation (leukotrienes, PAF) and cytokines
(TNF-alpha, interleukins) there is sequential massive expression of
adhesion molecules on the cells. They are at present divided into three
5 groups: 1. Immunoglobulin gene superfamily, 2. Integrins and 3. Selectins~
Whereas adhesion between molecules of the lg gene superfamily takes
place via protein-protein linkages, in the cooperation between selectins it is
lectin-carbohydrate linkages which predominate (Springer, T.A., Adhesion
receptors of the immune system. Nature 346, 425434, 1990; Huges, G.,
10 Cell adhesion molecules - the key to an universal panacea, Scrips
Magazine 6, 30-33,1993; Springer, T.A., Tramc signals for Iymphocyte
recirculation and leukocyte emigration; The multistep paradigm. Cell 76,
301 -314,1994).
15 Method:
The induced adhesion of leukocytes is quantified by a technique of
intravital microscopic investigation on the rat mesenterium (Atherton A. and
Born G.V.R., Quantitative investigations of the adhesiveness of circulation
polymorphonuclear leukocytes to blood vessel walls. J. Physiol. 222, 447-
474,1972; Seiffge, D. Methoden zur Untersuchung der Rezeptor-
vermittelten Interaktion zwischen Leukozyten und Endothelzellen im
Entzundungsgeschehen [Methods for investigating the receptor-mediated
interaction between leukocytes and endothelial cells during inflammation],
in: Ersatz- und Erganzungsmethoden zu Tierversuchen in der
25 biomedizinischen Forschung [Methods for replacing and supplementing
animal experiments in biomedical research], Schofffl, H. et al., (Editors)
Springer, 1995 (in the press)). Under ether-inhalation anesthesia,
prolonged anesthesia is induced by intramuscular injection of urethane
(1.25 mg/kg body weight). After vessels have been exposed by dissection
30 (femoral vein for injection of substances and carotid arteries for
measurement of blood pressure), catheters are tied into these. The
appropriate transparent tissue (mesenterium) is then exposed and
transposed to the microscope stage and covered with liquid paramn at
37~C by standard methods known from the literature (Menger, M.D. and
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Lehr, H., A. Scope and perspectives of intravital microscopy-bridge over
from in vitro to in vivo, Immunology Today 14, 519-522,1993). The test
substance is administered to the animal i.v. (10 mg/kg). The experimental
increase in blood cell adhesion is induced by cytokine activation by
5 systemic administration of lipopolysaccharide (LPS,15 mg/kg) 15 minutes
after administration of the test substance (Foster S.J., McCormick L.M.,
Ntolosi B.A. and Campbell D., Production of TNF-alpha by LPS-stimulated
murine, rat and human blood and its pharmacological modulation, Agents
and Actions 38, C77-C79,1993,18.01.1995). The increased adhesion,
10 caused thereby, of leukocytes to the endothelium is quantified by direct
vital microscopy or with the aid of fluorescent dyes. All the measurement
steps are recorded by video camera and stored in a video recorder. The
number of rolling leukocytes (i.e. all the visibly rolling leukocytes which are
slower than the flowing erythrocytes) and the number of leukocytes
15 adhering to the endothelium (residence time longer than 5 seconds) are
measured every 10 minutes for a period of 60 minutes. After completion of
the experiment, the anesthetized animals are sacrihced painlessly and
without excitation by systemic injection of T61. For the evaluation, the
results in each case from 8 treated and 8 untreated animals (control group)
20 are compared (results stated in percent).
Intravital microskopy on rats:
a) Polyacrylic acid (2100 Na salt): dose: 3 mg/kg; administration: i.v.;
25 species: SPRD (m); weight in 9:
284+/- 9,6; number of vessels: 15; vessel diameter in l~m 28 +/- 2.2;
leukocytes in 103/mm3: 8.2 +/- 2; fibrinogen in mg/100 ml:
96 +/- 10.1; inhibition: -3%
b) Polyacrylic acid (62900 Na salt): dose: 3 mg/kg; administration: i.v.;
species: SPRD (m); weight in g:
276 +/- -13.6; number of vessels: 18; vessel diameter in ~um 27 +/- 5.2;
leukocytes in 103/mm3: 12.5 +/- 3.5; fibrinogen in mg/100 ml:
101 +/- 12.3; inhibition: 64%.
