CA2205121A1 - Antiadhesive properties of polyacrylic acids and polymethacrylic acids - Google Patents

Abstract

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 cell adhesion, mediated by selectin receptors, in the tissue affected by the disease, for example cardiovascular disorders and rheumatism.

Description

CA 0220~121 1997-0~-12 - 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.

CA 0220~121 1997-0~-12 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 CA 0220~121 1997-0~-12 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.

CA 0220~121 1997-0~-12 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-CA 0220~121 1997-0~-12 . 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:

CA 0220~121 1997-0~-12 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 CA 0220~121 1997-0~-12 -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 CA 0220~121 1997-0~-12 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%.

Claims (8)

1. A polymer of acrylic acid or methacrylic acid or its pharmacologically acceptable salts for use as pharmaceutical.

2. A polymer as claimed in claim 1, wherein the polymer is a polyacrylic acid.

3. A polymer as claimed in claim 2, wherein the polymer is the sodium salt of a polyacrylic acid.

4. A polymer as claimed in any of claims 1 to 3, wherein the average molar mass of the polymer is from 900 to 63,000 g/mol.

5. The use of a polymer as claimed in any of claims 1 to 4 for producing a pharmaceutical for the therapy or prophylaxis of a disease associated with excessive cell adhesion, mediated by selectin receptors, in the tissue affected by the disease.

6. The use as claimed in claim 5, wherein the disease is a cardiovascular disorder.

7. The use as claimed in claim 5, wherein the disease is a rheumatic disorder.

8. A pharmaceutical comprising at least one polymer as claimed in any of claims 1 to 4 and pharmaceutical ancillary substances.