EP1725221A2

EP1725221A2 - Use of mrp4-inhibitors for the treatment and/or prophylaxis of cardiovascular diseases

Info

Publication number: EP1725221A2
Application number: EP04797661A
Authority: EP
Inventors: Andreas Greinacher; Heyo K. Kroener; Gabriele Jedlitschky
Original assignee: Ernst Moritz Arndt Universitaet Greifswald
Current assignee: Universitaet Greifswald
Priority date: 2003-11-07
Filing date: 2004-11-05
Publication date: 2006-11-29
Also published as: WO2005044244A2; WO2005044244A3; DE10352511A1; US20070212366A1

Abstract

The invention relates to the use of inhibitors of the multidrug resistance protein 4 (MRP4) in thrombocytes for the treatment and/or prophylaxis of cardiovascular diseases.

Description

Use of MRP4 inhibitors for the treatment and / or prophylaxis of cardiovascular diseases

The present invention 'relates to the use of inhibitors of ^de' s multidrug resistance protein 4 (MRP4) in platelets for the treatment and / or prophylaxis of cardiovascular diseases.

Cardiovascular diseases such as heart attacks, strokes and other arterial vascular occlusions as well as the primary and secondary prophylaxis of arterial thromboembolic complications are carried out in practice according to different approaches. For example, in the treatment of acute coronary syndrome with GPIIb / IIIa blockers. The Long-term therapy with oral platelet aggregation inhibitors has one. essential in the treatment of patients with arteriosclerosis both in the context of the secondary ais and primary prevention. Therapy with the cyclooxygenase inhibitor acetylsalicylic acid (ASA) deserves special mention. Therapy with so-called ADP receptor antagonists has found increasing use in medical practice. Influencing ADP-mediated platelet activation has become of great clinical importance because ADP is one of the most important enhancer substances for platelet activation. The platelet activator ADP is released, for example, from activated platelets and red blood cells. It induces the further activation and aggregation of platelets. Non-steroidal anti-inflammatory drugs (NSAD), such as diclofenac or ibuprofen, inhibit the ADP release that usually starts after platelet activation. ADP receptor antagonists, such as clopidogrel, inhibit the binding of ADP to its receptor on platelets and thus ADP-induced platelet aggregation. Adverse effects such as gastrointestinal side effects (e.g. diarrhea, nausea and vomiting)> severe gastrointestinal bleeding, neutropenia or thrombocytopenia, which are observed with some ADP receptor antagonists, require alternative treatment strategies. Another major disadvantage of the substances used hitherto is that no antidote is available for any of the substances. This leads to major problems with the need for an invasive procedure such as surgery and bleeding.

The object of the present invention is therefore to provide alternative medicaments for the primary and secondary prophylaxis of thromboembolic complications, in particular of Heart attack, stroke and other arterial occlusions, or for the general treatment of cardiovascular diseases.

The object is achieved according to the invention by using inhibitors of the multidrug resistance protein 4 (MRP4) expressed in platelets.

In the context of the present invention it was surprisingly found that MRP4 (also referred to as ABC pump or ABC transporter ABCC4) is expressed in human platelets and is located both on the extracellular membrane and intracellularly in granules (transmitter storage granules). Transport proteins that mediate the accumulation of ADP in the granules are not yet known. MRP4 has now been identified as a transporter for ADP, i.e. Via MRP 4, ADP is only stored in the concentrations in the dense granules of the platelets that are necessary for the activation of further platelets when they are released during platelet activation.

Since stored in delta granules ADP plays a central role in the self-amplification of platelet activation, provides inhibition of the transporter protein MRP4 is a new approach for the ^'treatment of the aforementioned diseases.

The inhibition of MRP 4 reduces the total amount of ADP in the platelets. This inhibits one of the most important mechanisms of self-reinforcement of platelet activation and thus also of excessive platelet activation. This therapeutic principle, which has not yet been used, inhibits the formation of large clots in the arteries and thus the formation Arterial occlusions that cause acute coronary syndrome, stroke and occlusions in peripheral arterial disease.

Studies of patients with genetic defects in the dense granules (synonyms: dense granules, delta granules) have shown that their platelets do not store ADP and therefore cannot release them when platelets are activated. These patients show little tendency to bleed. This is probably because ADP released from other cells can still bind to platelets. The phenotype of these genetically determined diseases is achieved with medication via the therapeutic principle of inhibition of MRP4-mediated storage of ADP mediated in the platelets described here for the first time. This therapeutic approach proposed in the context of the present invention is completely new. None of the drugs available so far to inhibit platelet function aims to reduce the amount of ADP stored in platelets. This therapeutic approach is also safer than previously available therapeutic approaches for inhibiting platelet function. It is known from the therapy of patients with a congenital deficiency of ADP stored in platelets that the vasopressin analogue DDAVP normalizes the tendency to bleed within minutes. It also follows from the principle of action that the ADP content of transfused platelets is not reduced by an MRP4 inhibitor and thus, in contrast to GPIIb / IIIa inhibitors and in contrast to ADP receptor antagonists, platelets transfused can be used effectively to treat severe bleeding. The present invention therefore relates to the use of one or more inhibitors of multidrug resistance protein 4 (MRP4) in platelets for the production of a pharmaceutical preparation for the treatment of disorders of platelet function and for the treatment and / or prophylaxis of cardiovascular diseases. This includes the therapy, primary prophylaxis and / or secondary prophylaxis of acute coronary syndrome, angina pectoris, heart attack, stroke or peripheral arterial occlusive disease, as well as before, during and after stent implantation in vessels.

According to the invention, MRP4 inhibitors are used e.g. Peptides, peptide analogs, peptidomimetics and cyclooxygenase inhibitors, especially non-steroidal, anti-inflammatory active substances, are proposed, which are either applied directly as active substances or as so-called “prodrugs”, from which the active substance arises through the body's own metabolism. Another The active substance in question is, for example, dipyridamole.

According to one embodiment of the invention, amphiphilic organic, neutral or anionic compounds with a molecular weight of approximately 200 to 1000 daltons (Da) are used which inhibit the MRP4-mediated transport of nucleotides (release from platelets). These include: dipyridamole, indomethacin, ibuprofen, inhibitors of organic anion transporters such as sample oath and sulfinpyrazone, pphosphodiesterase inhibitors, in particular structural analogues of cyclic nucleotides such as sildenafil, treguensin, zaprinast, and the leukotriene receptor antagonist MK571 (3 (7-chloro-2-guinolinyl) ethenyl) phenyl) - ((3-dimethylamino-3-oxopropyl) thio) methyl) thio) propanoiae aeid; see. Jones et al. (1989) Can. J. Physiol. Pharmacol. 67, 17 28, Lalloo et at. BMC Med. 2 (2004) 16 and the publications cited in these articles). The knowledge that MRP4 acts as an ADP transporter protein in granules of human platelets can now be used to search for active substances that inhibit this function of the protein MRP4.

The invention therefore also relates to a method for identifying a substance which inhibits the ADP transporter protein MRP4 in platelets - i.e. an active ingredient for the treatment of the aforementioned diseases - in which one

a) the substance to be examined is brought into contact with platelets in vivo or in vitro, a platelet activator is added and the change in the concentration of an activation marker compared to activated platelets which are not brought into contact with the substance to be measured is measured (in vivo or in vitro), and man

b) in MRP4-containing membrane vesicles or granules, which are also brought into contact with the substance to be investigated, the change in labeled (eg with radioactivity), ingested cAMP or cGMP or labeled ADP in comparison to membrane vesicles or granules is measured (eg by measuring the radioactivity of ingested [ ³ H] cAMP or [ ³ H] cGMP) that are not brought into contact with the substance to be examined,

wherein the substance inhibits the ADP transporter protein MRP4 in platelets if the substance in a) and / or b) in each case leads to a reduction in the determined measured value.

According to a particular embodiment, the aforementioned method further comprises a further step in which c) the substance to be examined is brought into contact with platelets in vivo or in vitro and the ADP concentration in the platelets is determined before and after,

the substance inhibiting the ADP transporter protein MRP4 in platelets if the substance in a) and / or b) and / or c) in each case leads to a reduction in the determined measured value.

The aforementioned provisions under a) and b) or a) and b) and c) can of course be carried out in any order. For the initial screening, the determination mentioned under b) can initially be carried out alone.

Alternatively, the method can also be carried out by performing step a) on platelet granule membranes (cf. Example 5).

The substance can also be used to further investigate its effectiveness in one step

d) are administered to a test animal or a test subject, the ADP content of the platelets of the test animal or test subject being determined before and after administration of the substance.

An ADP content that decreases after or as a result of the treatment indicates an MRP4-inhibiting effect of the investigated substance.

A substance which inhibits the ADP transporter protein in platelets is used in the context of the present invention understood a substance that is suitable for reducing the ADP accumulation in granules, ie to block them in whole or in part. These substances are not limited to substances with a specific mechanism of action. The substances can be organic chemical compounds as well as oligopeptides or antibodies.

The term “multidrug resistance protein 4 (MRP4)” is understood in the prior art to mean the human MRP4 gene product and its various isoforms, as well as analogs, homologues and orthologs in other species. These should also be included according to the invention. which encodes MRP4 was initially partially cloned and can be found on chromosome 13 (GenBank accession number U83660, Kool et al., Cancer Res. 57 (1997) 3537-3547).

To carry out the method for identifying MRP4 inhibitors, platelets from non-human sources, such as e.g. Mice, rats, rabbits, primates, transgenic animals, knock-out animals can be used. The active ingredients identified in this way should then preferably be tested for their action on human platelets.

The present invention further provides a method of manufacturing a pharmaceutical composition for the treatment and / or prophylaxis of cardiovascular diseases, including therapy, primary prophylaxis and / or secondary prophylaxis of acute coronary syndrome, angina pectoris, heart attack, stroke or peripheral arterial occlusive disease, as well as during and after stenting I implantation into vessels, provided one The aforementioned method for identifying substances which inhibit the ADP transporter protein MRP4 in platelets is carried out and the substances thus identified are formulated with pharmaceutically acceptable auxiliaries and / or carriers.

The invention thus relates to the use of a substance which inhibits the ADP transporter protein MRP4 in platelets by the abovementioned method for producing a pharmaceutical preparation for the treatment and / or prophylaxis of the abovementioned diseases.

With the knowledge obtained according to the invention that MRP4 acts as an ADP transporter in granules of human platelets, alternative protocols for the therapy, primary and / or secondary prophylaxis of the aforementioned diseases are provided. Individual therapy control is possible by titration of the inhibition of ADP uptake. By determining the platelet ADP content, an individual measurement of the effect of the drug is possible. So far this has been the case with the previous substances, e.g. ADP receptor antagonists, not possible. If there is a need for invasive intervention, the vasopressin analog DDAVP and transfusion of platelets can antagonize the drug effect. This is not possible with GPIIb / IIIa antagonists in the acute phase, nor with the current ADP receptor antagonists because the transfused platelets are immediately inhibited by the drug.

The present invention is explained below with the aid of examples, but without being restricted thereto. EXAMPLES

example 1

The expression and intracellular localization of MRP4 in human platelets was demonstrated by immunoblotting and immunofluorescence microscopy and a functional assay. In immunoblots, the MRP4-specific antibody (rabbit) showed a strong signal at the expected molecular mass of approximately 170 kDa in homogenates of isolated platelets and indicated an MRP4 enrichment in membrane fractions which were separated via a sucrose density gradient.

The MRP4 function, the ³ H-labeled cyclic GMP (cGMP), a known MRP4 substrate was detected was in ^fractions' low density, which contain most plasma membrane proteins, can also be detected such as higher in fractions of density, where intracellular granules.

Immunofluorescence microscopy of platelets with the same

Antibodies showed an accumulation of MRP4 in intracellular granules and a weak signal on the plasma membrane.

In double staining experiments, MRP4-positive granules differed from granules which were positively stained compared to P-selectin, which is predominantly expressed on alpha granules. However, the MRP4 positive granules showed an accumulation of the fluorescent dye mepacrin, which is known to be transported in delta granules. To detect the expression in platelets, washed platelets are incubated on glass plates coated with collagen and adhere to them. Alternatively, any other substances that bind to glass or plastic surfaces and bind to the platelets can be added to collagen, or the platelets are bound directly to a carrier surface. The platelets are fixed, e.g. with 1% paraformaldehyde solution, and the cell membrane is opened by chemicals, e.g. saponin, in such a way that substances that cannot cross the membrane (antibodies,

Receptor binding partner) to intracellular structures. The platelets are then incubated either with a directly fluorescence-labeled antibody against MRP4, or with an ^' unlabeled antibody and in a second step with a labeled secondary antibody which recognizes the primary antibody, for example goat anti rabbit FITC. The localization of the antibody binding is made possible by the coincubation with a monoclonal or polyclonal antibody with known specificity for marker proteins of the platelet granules. The coloring is evaluated with fluorescence microscopy.

Alternatively, the platelets are fixed and incubated with directly or indirectly gold-labeled antibodies against MRP4 and the binding of the antibodies to structures of the dense granules is detected.

To further secure the localization, we examined the binding of anti-MRP4 antibodies to platelets, which were obtained from a patient with Hermansky Pudlak syndrome. In this patient, the dense platelet granules are not present due to a genetic disease, however the ■ other platelet organelles (alpha granules, lysosomes) are preserved. Using the methods described above, it was found that the platelets, which specifically lack the dense granules, do not express MRP4 intracellularly, but do express MRP4 on the thrombocyte surface. The location is thus clearly secured.

For the immunoblot, platelets are washed in physiological saline, solubilized using standard methods and the lysate separated on a gel according to standard methods according to the molecular weight. The proteins in the gel are transferred to a nitrocellulose membrane by applying a voltage gradient. The membrane is then incubated with the primary or secondary labeled antibodies against MRP4 and the binding of the antibodies made visible by: fluorescence, chemical reaction (horseradish-peroxidase reaction) or by radioactive labeling.

Example 2

Measurement of MRP4 function in platelet membranes

The preparation of the membrane vesicles and the transport measurements are based on the method described in detail for MRPl and culture cells by Keppler et al. (Methods Enzy ol. 292 (1998) ^' 607-616).

Wash platelets

About 30 ml of liquid are transferred from the platelet concentrates into a Falcon tube and centrifuged at a centrifugation speed of 1300 * g for 10 min at 4 ° C. After decanting the supernatant the pellet was resuspended in 1 ml of solution B ^(is' 9.3 ml NaCl solution (0.9%), 0.5 ml of solution A (5g EDTA, to 100 ml NaCl solution (0.9%)), 0.2 ml bovine albumin solution (22%)) and made up to a volume of 30 ml with solution B. A further centrifugation is carried out at 1300 ^χ g for 10 min at 4 ° C. The pellet obtained is resuspended in 1 ml of solution C (NaCl solution (0.9%) with lxPBS pH 7.2 to pH 6.5) and made up to a volume of 30 ml with solution C. This step is repeated three times, the platelets only being resuspended on the last wash. The storage then takes place in aliquots of 100 μl at -20 ° C. To minimize the erythrocyte concentration of the platelet concentrate, the loose, white pellet of platelets is removed after the first centrifugation with a ^'Pasteur pipette and thus separated from the more strongly sedimenting erythrocytes.

cell disruption

Ultrasonic

This disruption method is used for washed platelets. The stock solutions of the protease inhibitors leupeptin, aprotinin and phenylmethylsulfonyl fluoride are each added in a ratio of 1: 1000 to the cell suspension with a volume of around 500 μl. The disruption takes place with the Sonopuls device in a cycle of 5 ^χ 10s with a force of 50% with cooling breaks of 2 min between the individual cycles. This is followed by centrifugation at 100,000 ^χ g for 30 min at 4 ° C. The pellet obtained is resuspended in 50 μl of 5 mM Tris solution, pH 7.4 and the protein content is determined Freezing / thawing

The washed platelets are in a cell suspension with a total volume of around 500μl. After adding the stock solutions of the protease inhibitors aprotinin, leupeptin and PMSF in a ratio of 1: 1000, the digestion is carried out by freezing in liquid nitrogen and thawing in a water bath at 37 ° C. This cycle is carried out five times in total. This is followed by centrifugation at 100,000 ^χ g for 30 min at 4 ° C. The pellet obtained is resuspended in 50 μl of 5 mM Tris solution, pH 7.4, and the protein content is determined.

membrane preparation

platelets

The suspension obtained by cell disruption with the help of freezing and thawing is free of cytosolic proteins and thus contains the plasma membranes and other cell organelles, such as mitochondria and granules. It is processed into branch vesicles as follows.

The membrane suspension is transferred to the dounce potter (tight) and diluted to approximately twice the volume with Tris sucrose buffer. The protease inhibitors leupeptin and aprotinin are then added in a volume ratio of the stock solution to the suspension of 1: 1000. These protease inhibitors are competitive inhibitors, whereas PMSF, which has already been added during the digestion, irreversibly inhibits.

30 Potter strokes are then carried out, so that approximately 2 strokes per minute are carried out. Membrane preparation without a sucrose gradient

After the ^' homogenization process, two thirds of the suspension are centrifuged at 100,000 ^χ g at 4 ° C for 30 min. The pellet is resuspended in a volume of Tris sucrose buffer 4 times the pellet volume, transferred again to the dounce potter (tight) and homogenized with 20 potter strokes. Finally, the suspension is drawn up 20 times through a 27G needle in order to produce membrane vesicles that are directed in particular outwards. The storage takes place in liquid nitrogen as 100 μl aliquots.

Membrane preparation with a sucrose gradient

After the homogenization process, a third of the suspension is placed on a sucrose gradient. The 4 ml tubes belonging to the swing-out rotor SW55 of the Beckman centrifuge are loaded as follows. First, 1.5 ml of a 60% sucrose solution in 5mM HEPES, pH 7.4 (m / m) _. placed therein. Then 1.5 ml of a 30% sucrose solution in 5 mM HEPES, pH 7.4 (m / m) are added. 0.5 ml of the membrane suspension is layered over these two sucrose solutions. The subsequent centrifugation with the swing out rotor is carried out at 200,000 ^χ g for 60 min at 4 ° C.

transport measurement

The basis of the method used are membrane vesicles, which are created by shear forces when opening with a needle that is as thin as possible. The method of manufacturing membrane vesicles always produces a certain proportion (around 30%) of inside-out membrane vesicles. The special thing about the inside-out membrane vesicles is that the membrane is oriented the other way round to the plasma membrane. An outward transporter of the plasma membrane would be inside an inside-out vesicle transport. Since the MRP4 is still present in the plasma membrane, inside-out vesicles offer the possibility of a functional test. In addition, there are vesicles in the cytoplasm in platelets and other cells, which result from constrictions in the plasma membrane and are also aligned inside-out and could therefore absorb substances through transport processes.

A tritium-labeled substrate is added to the inside-out vesicles, which can be transported depending on the ATP. By measuring in the presence of AMP and ATP (ATP-containing creatine kinase), the ATP-dependent inward transport becomes measurable, from which the AMP value, which reflects diffusion processes, is subtracted.

The substances in the inside-out vesicles are placed on a filter with a diameter of 0.22μm, so that excess radioactivity is removed by washing. The result is detected by counting the radioactive decays on the filters (see FIG. 1).

The transport experiments were carried out with [ ³ H] -cGMP (tritium-labeled cGMP).

For each batch, 100 μg protein of the membrane vesicle suspension are diluted to 55 μl with Tris-sucrose buffer. In a second approach, 7.5 μl creatine kinase, 6.8 μl ATP or AMP and 7.5 μl of a [ ³ H] -cGMP stock dilution are used for the [ ³ H] -cGMP (1 μCi / μl) in a ratio of 2: 5 is diluted with Tris sucrose buffer. The "final concentration of the [ ³ H] -cGMP corresponds to 4 μM with a radioactivity of 2.7 μCi / 75 μl. The 55 μl of the membrane suspension and the radioactive batch are simultaneously warmed for one minute at 37 ° C. in a thermal shaker before pipetting 20 μl of the radioactive batch into the membrane suspension and the transport experiment is started. During the incubation in a thermal shaker at 37 ° C, 20 μl samples are taken after 1, 10 and 20 min. The samples taken are pipetted into 1 ml of ice-cooled tris-sucrose solution, the total volume being immediately placed on a nitrocellulose filter. The free radioactivity is removed by rinsing with 5 ml of cold Tris sucrose solution with a vacuum. The individual filters are transferred to vials and 10 ml of scintillation fluid (eg from Roth, Zinsser) are added. This liquid converts the existing radioactivity into an evaluable light signal, which can be measured with the Wallac 1409 Liquid Scintillation counter and a special tritium counting program as DPM (decays per minute = decays per minute).

calculation

[ ³ H] -cGMP: 8.8 Ci / mmol = 8.8 μCi / nmol 1 μCi = 2.2 x 10 ⁶ DPM 1 nmol ≡ 8.8 x 2.2 10 ⁶ DPM 0.05 pmol ≡ 1000 DPM

Protein: 100μg / 75μl 26.7 μg / 20μl (sample volume) based on 1 mg 1000 DPM ≡ 1.87 pmol / mg protein: Example 3

Inhibition of transportation:

When testing potential inhibitors, these are added in various concentrations to the incubation together with the labeled substrate. Identification as an inhibitor is given if the transport rate of the labeled substrate is statistically significantly lower in multiple determinations (e.g. Students' T-Test) compared to the control incubation without inhibitor.

Example 4

Identification of MRP4-inhibiting substances by measuring platelet function

A. Born platelet aggregation

The platelet function after inhibition of the MRP4 transporter can be measured with platelet aggregation according to Born (Platelets. In: Human Blood Coagulation, Hae ostasis and Thrombosis, 2nd Ed. Biggs, Editor Blackwell Scientific Publications, London 1976, pp. 168-201) , Platelets are added to a platelet activator and the change in the concentration of an activation marker is measured. The experiment is repeated under identical conditions, but the platelets are first brought into contact with a substance to be investigated before the platelet activator is added. Alternatively, a test animal or a human subject is treated with the MRP4 inhibiting substance and the platelets obtained from this experimental animal or human subject are examined with the same method and either with platelets from untreated experimental animals or untreated subjects or with the platelets from the experimental animal or the human Subjects compared before treatment. The measured values obtained in both tests are compared, the substance indicating an MRP4 inhibitor if the addition of the test substance leads to a reduction in the determined measured value compared to the platelet aggregation test without test substance.

The principle of platelet function analysis using Bornometry is described below.

Measurement method: turbidimetry

Device: 4-channel aggregometer "APACT 4 ^λ (from Laborgeräte + Analysensysteme Vertriebsgesellschaft mbH, Ahrensburg, Germany)

Inducers: ADP, collagen, ristocetin, adrenaline

Execution:

1. Inductors:

The inductors are available as lyophilisates and are prepared for use according to the manufacturer's instructions and can be stored in aliquots at below -20 ° C until use.

The following inducer concentrations (final concentrations) are used: ADP (2.5 μmol / 1, 5 mmol / 1, 10 mmol / 1, 20 mmol / 1) collagen (1 μg / ml, 4 μg / ml) ristocetin (0, 5 mg / ml; 1.5 mg / ml) adrenaline (5 μmol / 1, 10 μmol / 1)

The inductors are thawed shortly before use, mixed vigorously (vortexer) and during use at 4 ° C on ice stored (adrenaline stored at room temperature during the performance).

2. Obtaining Platelet-Rich Plasma (PRP) and Platelet-Low Plasma (PPP):

PRP is first obtained from a healthy blood donor by differential centrifugation of a citrate-anticoagulated, freshly drawn whole blood sample (20 min, 120 x g). The donor is said not to have taken any platelet-reducing medication 10 days before blood collection.

The PRP is transferred to a clean poly tube and adjusted to a platelet count of 300,000 / μl with PPP from the same donor, which is obtained from PRP by high-speed centrifugation (5 min, 860 x g). The set PRP is stored at 37 ° C until use, the tube is closed with parafilm.

3. Device settings and measurement: a) Settings on the APACT 600 sec. Measuring time, stirring speed 1000 rpm, temperature of the heating block 37 ° C. Warm-up time of the device approx. 10 to 15 min.

b) Calibration of the device with PPP 180 μl PPP are mixed with 20 μl 0.9% NaCl solution pH 7.2 in the cuvette provided with a stirring magnet, placed in the measuring channel and measured (corresponds to 100% light transmission).

c) Verification of the device with PRP 180 μl PRP are mixed with 20 μl 0.9% NaCl solution pH 7.2 in the cuvette equipped with a stirring magnet, placed in the measuring channel and measured (corresponds to 0% light transmission).

d) Measurement A clean cuvette provided with a stirring magnet is filled again with 180 μl PRP without bubbles. The measuring channel is started, the recording of the measurement of the light transmittance can be followed on the PC. After approx. 1 min. 20 μl of one of the above are placed in the PRP-filled cell. Pipette inductors (bubble-free, do not remove the cuvette from the measuring channel; make sure that the inductor does not get caught on the edge of the cuvette!). The course of the curve is recorded continuously. The light transmission (%) of the PRP increases with the increase in the aggregated platelets over time.

The cuvette is removed from the measuring channel at the end of the measuring time. The measurements are then repeated as described under b) to d) for each additional inductor. To measure the spontaneous aggregation of the platelets, 20 μl of physiological NaCl solution are pipetted into the PRP instead of the inductor and the measurement is carried out.

4. Quality control:

The measurement of the platelet function of a healthy normal subject serves as a control of the nature of the inductors and the functionality of the aggregometer. The platelet function of, for example, subjects with Va thrombocytopathy is then measured. As mentioned above, the potential MRP4 inhibitors to be investigated are added to the cuvettes before adding the respective inductors.

The vesicle transport studies described in Example 3 are carried out to investigate whether the substance to be examined acts specifically on the MRP4 transporter protein.

B. Expression of Activation Markers in Flow Cytometry

Platelet function after inhibition of the MRP4 transporter can also be determined by expression of activation markers (e.g. PAC-1, CD 63, P-Selectin) in the flow cytometry.

Flow cytometry is a method for characterizing cells based on their size and granularity. It is also used for the quantitative determination of surface molecules and intracellular proteins with the help of fluorescence-labeled antibodies.

The marked cells, which are in suspension, are individually guided past an argon laser (cf. FIG. 2). The resulting fluorescent and scattered light is detected by various photo detectors. The light scattered by the cell in the direction of the laser beam is picked up by the FSC detector (Forward Scatter, FSC) and provides information about the size of the measured cell. The sideward scatter (SSC) correlates with the granularity of the cell. Additional properties of the cells, such as the expression of one or more surface molecules, are registered via various other detectors. The intensity and color of the fluorescence is recorded by a computer for each individual cell. The signals from the photodetectors are transferred via Photo multiplier reinforced. The corresponding data is analyzed using the WinMDI 2.8 computer program.

Activated and resting platelets differ morphologically. Activated platelets are characterized by the formation of microparticles and large platelet aggregates, which leads to a shift in platelets in the FSC / SSC (Matzdorff et al. 1998, J. Lab. Clin. Med. 131 (6): 507-17).

Resting platelets, on the other hand, have a uniform size and therefore form a defined point cloud when considering size and granularity in the flow cytometer. In addition to changing the shape of the platelets, there is surface exposure of P-selectin.

100 μl platelet suspension in 1 ml formaldehyde solution are pipetted for fixation. After two hours to a maximum of four days, the platelets are washed twice with FACS-Flow (1700 x g, 5 min) for staining. The pellet is resuspended in 500 μl FACS-BSA mixture and 25 μl of the sample is incubated with 10 μl antibody (P-selectin, mouse, unlabelled) for 30 min at room temperature in the dark. The mixture is then washed once with FACS-Flow. After the supernatant has been suctioned off, 10 μl of secondary antibodies (anti-mouse, FITC-labeled, diluted 1:30) are added to the platelets. The samples are incubated at room temperature in the dark for 30 min. For the measurement, the platelets are washed once and then taken up in 1 ml of FACS flow.

The measurements are carried out analogously to the investigation of the platelet aggregation according to Born (see under A) by platelet activation in each case in comparison with and without the substance to be investigated, the substance indicates an MRP4 inhibitor if the addition of the test substance leads to a lower platelet activation and thus to a shift of the FSC / SCC ratio towards FSC compared to the measurement without test substance.

C. lumiaggregometry

Platelet function after inhibition of the MRP4 transporter can also be determined by lumia aggregometry. The release of ADP / ATP is measured in lumiaggregometry using the luciferin-luciferase test (cf. Mondoro et al., Blood 96 (2000) 2487-2495 and White et al., Thromb. Haemost. 67 (1992) 572).

The measurements are carried out analogously to the examination of the platelet aggregation according to Born (see under A) or the flow cytometry (see under B) by platelet activation in each case in comparison with and without the substance to be investigated, or of test animals treated with the substance or human test subjects in comparison to test animals or human subjects not treated with the substance, the substance indicating an MRP4 inhibitor if the addition of the test substance leads to a lower platelet activation and thus to a lower luminescence value compared to the measurement without test substance.

The experiment is repeated under identical conditions, but the platelets are first brought into contact with a substance to be investigated. The measured values obtained in both experiments are compared, the substance indicating an MRP4 inhibitor if the addition of the test substance leads to a reduction in the release of ADP / ATP compared to the lumiaggregometry without test substance. In addition to lumie aggregometry, other methods of quantifying ADP in platelets can also be used, such as extraction of ADP and detection using the luciferin-luciferase method, or quantification using chromatographic methods such as HPLC.

Example 5

Involvement of MRP4 in ADP transport in platelet delta granules.

[ ³ H] ADP transport in vesicular fractions from platelets.

To determine whether ADP can be a substrate for MRP4-mediated, ATP-dependent transport, the uptake of [ ³ H] ADP (1 μM) in platelet membrane vesicles (raw membranes) in the presence of 0.4 mmol / 1 ATP measured over a period of 2 minutes. As shown in Figure 3, a time-dependent increase in vesicle-associated [ ³ H] ADP was observed at a rate of 6.74 + 1.9 pmol x mg x protein ^-1 x min ^-1 (mean + standard deviation of three different experiments Triple determination) observed. If you use ATP through 5 '-AMP or the non-hydrolyzable ATP analog AMPPNP, which was commonly used in control incubations to calculate the ATP-dependent component of the transport, was observed to have a three to four times higher background binding of the [ ³ H] ADP to the membranes, which only increased slightly over time (not shown). This lower, non-specific binding of [ ³ H] ADP in the presence of ATP compared to 5 '-AMP is probably due to the presence of unlabeled ADP, which competes with and dilutes ³ H-labeled ADP. The source of this unlabeled ADP could be ADP contamination in commercially available ATP and the formation of ADP by ATP hydrolysis. In order to show the ATP dependence and at the same time to ensure the same initial ADP concentrations, the transport was measured in the presence of ATP with or without 1 mM orthovanadate, an inhibitor of ATP hydrolysis. It was observed a time-dependent increase in the vesicle-associated radioactivity in the presence of ATP ^■, despite the fact that ^[3 H] ADP was diluted by simultaneous formation of unlabeled ADP. This shows that there is active storage that counteracts the thinning effect. The vesicle-associated radioactivity was significantly reduced in the presence of ATP plus orthovanadate, despite the fact that the inhibition of ADP formation should increase the binding of the labeled compound, thus showing that the decrease observed represents the inhibition of the active transport process. The dilution effect on [H] ADP by the formation of unlabeled ADP was also the reason to reduce the ATP concentration from the standard concentration (4 mM) to 0.4 mM.

To determine whether the observed difference in [ ³ H] ADP uptake by the vesicles in the presence and absence of orthovanadate is rather a transmembrane movement than one Reflected binding to the membrane surface, the influence of high osmolarity was investigated. At a concentration of 1 mol / 1 sucrose outside the vesicles, the rate of [ ³ H] ADP transport was significantly reduced in the absence of orthovanadate, indicating active transport (Figure 3B). In the presence of orthovanadate, 1 mol / 1 sucrose only slightly influenced the [ ³ H] ADP association with the vesicles. This shows that the amount of radioactivity measured represents the ratio of [ ³ H] ADP binding to the vesicle membrane, regardless of the transmembrane transport. Furthermore, the vanadate-sensitive ADP accumulation could be detected in the dense granular fraction of the sucrose gradient and was inhibited by dipyridamole, MK571 and cGMP (Table 1).

methods:

Transport of [ ³ H] ADP. An ATP-dependent transport of [ ³ H] ADP (1 μmol / 1) into the membrane vesicles was measured by rapid filtration, as described for [ ³ H] cGMP, with the exception that the vesicles were in with 0.4 mmol / 1 ATP, 10 mmol / 1 MgCl ₂ supplemented incubation buffer in the presence or absence of sodium orthovanadate (1 mmol / 1). To investigate the effect of the osmolarity of the extravesicular medium, the vesicles were preincubated for 45 minutes at 4 ° C. in a buffer containing 1 mol / 1 sucrose or in standard incubation buffer containing 250 mmol / 1 sucrose. Table 1. Inhibition of [ ³ H] ADP transport in dense platelet granules

Dense platelet vesicles (100 μg of protein) were treated with [H] ADP (1 μmol / l) ^' in the presence of 0.4 mmol / 1 ATP or 0.4 mmol / 1 ATP + 1 mmol / 1 orthovanadate for 2 minutes incubated, and the difference in vesicle-associated radioactivity was calculated. ^'The compounds listed were added at the indicated concentrations to the incubations with and without orthovanadate. The difference is given as a percentage of the control (mean + standard deviation of 3 determinations). The control value of the vanadate-sensitive [ ³ H] ADP transport was 3.0 ± 0.4 pmol / mg protein ^-1 after 2 minutes in these experiments.

Description of the figures:

Figure 1:

Principle of transport measurement with inside-out membrane vesicles.

Figure 2:

Structure of a flow cytometer (simplified schematic representation)

Figure 3:

Transport of ADP in platelet membranes.

AD: Raw membrane vesicles from platelets (100 μg of protein) were analyzed with [ ³ H] ADP (1 μmol / 1) in the presence of 0.4 mmol / 1 ATP (squares) or 0.4 mmol / 1 ATP plus ( 1 mmol / L orthovanadate diamonds), and the vesicle-associated radioactivity was determined as described in the section "Methods" (mean ± standard deviation, n ≤ 3; 1 pmol x mg protein ^-1 = 1724 DPM). A, B: Vesicles were preincubated for 45 minutes at 4 ° C in standard incubation buffer containing 250 mmol / l sucrose (A) or in 1 mol / 1 sucrose containing buffer (B). C, D: ADP transport in the absence (C) or presence (D) of 100 μmol / 1 dipyridamole.

Claims

claims:

1. Use of an inhibitor of multidrug resistance protein 4 (MRP4) in platelets for the treatment and / or prophylaxis of cardiovascular diseases.

2. Use according to claim 1, characterized in that the treatment and / or prophylaxis of cardiovascular diseases is the therapy, primary prophylaxis and / or secondary prophylaxis of acute coronary syndrome, angina pectoris, heart attack, stroke or peripheral arterial occlusive disease before, during and after stent implantation in vessels ,

3. Use according to claim 1 and 2, characterized in that the active ingredient is an amphiphilic organic, neutral or anionic compound with a molecular weight of approximately 200 to 1000 daltons (Da), which inhibits the MRP4-mediated transport of ^' nucleotides.

4. Use according to claim 3, characterized in that the active ingredient dipyridamole, indomethacin, ibuprofen, inhibitors of organic anion transporters such as probe egg and sulfinpyrazone, structural analogs of cyclic nucleotides such as sildenafil, trequensin, zaprinast (phosphodiesterase inhibitors) and the leukotriene receptor antagonist MK571.

5. A method for identifying a substance that inhibits the ADP transporter protein MRP4 in platelets, characterized in that a) the substance to be examined is brought into contact with platelets in vivo or in vitro, a platelet activator is added and the change in the concentration of an activation marker compared to activated platelets which are not brought into contact with the substance to be measured is measured (in vivo or in vitro), and b) in MRP4-containing membrane vesicles or granules, which are also brought into contact with the substance to be investigated, the change in labeled, ingested cAMP or cGMP compared to membrane vesicles or granules is measured which are not brought into contact with the substance to be investigated, where ^* the substance inhibits the ADP transporter protein MRP4 in platelets if the substance in a) and / or b) leads to a reduction in the determined measured value.

6. The method according to claim 5, characterized in that c) the substance to be investigated is brought into contact with platelets in vivo or in vitro and the ADP concentration in the platelets is determined before and after, the substance being the ADP transporter protein MRP4 inhibits in platelets if the substance in a) and / or b) and / or c) leads to a reduction in the determined measured value.

7. The method claim 5 or 6, characterized in that one carries out a) and b) or a) and b) and c) in any order.

8. A method for producing a pharmaceutical composition for the treatment and / or prophylaxis of cardiovascular diseases, characterized in that one carries out a method according to claims 4 to 7 and the identified substances are formulated with pharmaceutically acceptable auxiliaries and / or carriers.

9. The method according to claim 8, characterized in that the treatment and / or prophylaxis of cardiovascular diseases is the therapy, primary prophylaxis and / or secondary prophylaxis of acute coronary syndrome, angina pectoris, heart attack, stroke or peripheral arterial occlusive disease before, during and after stent implantation in vessels ,

10. Use of a substance identified by a method of claims 4 to 7 for the treatment and / or prophylaxis of cardiovascular diseases.

11. Use according to claim 10, characterized in that the treatment and / or prophylaxis of cardiovascular diseases is the therapy, primary prophylaxis and / or secondary prophylaxis of acute coronary syndrome, angina pectoris, heart attack, stroke or peripheral arterial occlusive disease before, during and after stent implantation in vessels ,