DE102012025005A1 - Use of resveratrol and/or their analogues e.g. for treating platelet aggregation and cancellation of resistance to acetylsalicylic acid or nonsteroidal anti-inflammatory drugs and/or repealing paradoxical reaction to acetylsalicylic acid - Google Patents

Abstract

Use of resveratrol and/or their analogues for cancellation of resistance to acetylsalicylic acid (ASA) or other nonsteroidal anti-inflammatory drugs (NSAIDs) and/or repealing the paradoxical reaction to acetylsalicylic acid (PRASA) or other NSAIDs, is claimed. An independent claim is also included for a combination comprising at resveratrol and/or their analogs and nonsteroidal anti-inflammatory drugs for preventing or treating platelet aggregation. ACTIVITY : Anticoagulant; Thrombolytic; Hemostatic; Antiinflammatory; Gastrointestinal-Gen; Cardiant; Vasotropic; Antidiabetic; Antilipemic. Test details are described but no results given. MECHANISM OF ACTION : None given.

Description

The invention relates to a method and combinations for improving the platelet aggregation-inhibiting action of non-steroidal anti-inflammatory drugs, as well as corresponding pharmaceutical or nutraceutical compositions, as well as other subject matters of the invention and as specified below.

Platelets play a central role in the development of atherosclerosis and arterial thrombosis.

It is known that certain drugs can act as inhibitors of platelet aggregation by reversibly inhibiting the cyclooxygenase COX-1 catalyzed conversion of arachidonic acid into prostaglandin endoperoxides or irreversibly in the case of acetylsalicylic acid (ASA). The prostaglandin endoperoxides are precursors of prostaglandins, prostacyclin and thromboxane A2.

In the case of ASA, the action takes place by acetylation of the erine residue 530 of COX-1, resulting in blockage of the active site. In addition to the COX-1, there is another cyclooxygenase (isoenzyme), with ASA causing a 170-fold greater binding of COX-1 to COX-2.

Among other things, COX-1 catalyzes the synthesis of prostacyclin in the endothelial cells and thromboxane A2 in the platelets. COX-2 is involved in the synthesis of prostaglandins involved in pain and inflammation and is also detectable in platelets.

The consequence of the inhibition of platelet cyclooxygenase-1 is the inhibition of thromboxane A2 and thus the thromboxane-dependent amplification mechanisms of platelet aggregation. Since platelets have no protein biosynthetic capabilities, the irreversible inhibition of platelet function by ASA inhibits the entire lifetime of a platelet, but by rebuilding thrombocytes per day COX-1 activity is restored by about 10%. To ensure efficacy against platelet aggregation, daily ASA must be taken.

The main indication of ASA is the prevention of thrombotic occlusions of arterial vessels in both acute and long-term therapy of coagulation disorders. However, in a significant proportion of patients treated with ASA, arterial thrombosis occurs and platelet function studies reveal a high variability of the platelet response to ASA ex vivo.

It was shown that a significant proportion of patients treated with ASA still had an arterial thrombosis (ASD resistance) and a high variability of the platelet response to ASA ex vivo could be demonstrated. One also speaks of inadequate response to ASA ("low / non-response"). Only new highly specific methods for the direct measurement of the platelet response to ASA (PADA-RASS, Jenaffin GmbH, Jena) have unequivocally shown that not only an ASD resistance is present in a part of the examined, but also often paradoxical answers to ASA are seen. This, until recently, non-quantifiable, completely new "platelet response" is of high clinical interest because the affected patients are more likely to show enhanced platelet activation / aggregation, thereby promoting acute and chronic coagulation disorders. The pathophysiology of platelet function characterized by the abbreviation "PRASA" (paradoxical reaction to ASA) after first extensive examinations on hundreds of healthy and chronically ill patients, affects more than one third of the tested persons, including those with chronic heart disease and chronic dialysis even higher incidence rates are affected.

Of particular interest are data from those patients who had autoimmune diseases of the gastrointestinal system. These patients have a very high percentage of PRASA reactions. It was thus possible to show for the first time that upregulation of the inducible COX 2 takes place in acute but much more frequent subchronic or chronic inflammatory reactions in the platelets (as well as in other body cells), located safely at the synthe- sizing site of the platelets, in the megakaryocytes of the bone marrow. Depending on the status and duration of the inflammatory mechanisms either an "ASD resistance" - in the platelets are equipotent amounts of COX 1 and COX 2 contain - or a "PRASA" -The platelets have COX 2 mostly "on board". In the interaction of ASA with COX 2, atypical AA metabolites are formed in spite of serine 530 displacement of the AA receptor, including thromboxanes, which may then lead to platelet activation and ultimately to coagulation activation.

The previous consequence of these findings was the strict avoidance of ASA or Coxibe preparations (anti-inflammatory drugs that specifically inhibit the inflammation-regulated COX 2), but also other non-steroidal anti-inflammatory drugs should not be administered. For chronic use is an increased vascular event density has been seen (e.g., in the anti-rheumatic VIOXX).

Patients with coronary heart disease, acute coronary syndrome, heart failure, diabetes mellitus, hyperlipidemia, as well as percutaneous coronary interventions are more likely to have PRASA.

• Kaden T. 2010. Häufigkeit und Profil der ASS-Resistenz bei Patienten mit kardiologischen Erkrankungen im Vergleich zu gesunden Probanden (Dissertation). Jena: Friedrich-Schiller-Universität .
• Hentrich J. 2011. Häufigkeit und Profil der ASS-Resistenz bei chronischen Hämodialyse-Patienten im Vergleich zu gesunden Probanden (Dissertation). Jena: Friedrich-Schiller-Universität .
• Kaden T. Hentrich J, Nowak G. 2009. Prevalence and profilof aspirin resistance in patients with cardiovascular diseases in comparison to healthy volunteers. J Thromb Haemost: 7 (Suppl 2)
• Hentrich J, Kaden T, Nowak G. 2009. Prevalence and profil aspirin resistance in hemodialysis patients in comparison to healthy volunteers. Haemostaseologie, 29: A63 (abstract .

To the previous statements, sources and information can be found in the following dissertations and publications (which are incorporated herein by reference):

• Kaden T. 2010. Frequency and profile of ASD resistance in patients with cardiac disease compared to healthy subjects (dissertation). Jena: Friedrich Schiller University ,
• Hentrich J. 2011. Frequency and profile of ASD-resistance in chronic hemodialysis patients compared to healthy subjects (dissertation). Jena: Friedrich Schiller University ,
• Kaden T. Hentrich J, Nowak G. 2009. Prevalence and profile of aspirin resistance in patients with cardiovascular diseases in comparison to healthy volunteers. J Thromb Haemost: 7 (Suppl 2)
• Hentrich J, Kaden T, Nowak G. 2009. Prevalence and profile of aspirin resistance in hemodialysis patients in comparison to healthy volunteers. Haemostaseology, 29: A63 (abstract ,

It is an object of the present invention to find new approaches to overcoming ASA resistance and PRASA and to provide safer new therapies generally for thrombosis aggregation inhibition.

Resveratrol (the trans-3,5,4'-trihydroxystilbene, predominantly in the more stable trans form, has the formula (in the trans form)

It is found in a number of plants, such as grapes, raspberries, mulberries, plums or peanuts, and the knotweed, Polygonum cuspidatum.

Known effects are those against parasites and fungal infections (it is a phytoalexin, so a protective substance of plants), against cancer cells and positive effects in atherosclerosis, heart disease, Alzheimer's disease, arthritis and some autoimmune diseases and life-prolonging act (see. http://www.resveratrol.de ).

Resveratrol is as a dietary supplement in the form of suitable preparations (eg., As grape extract) z. In the USA and Germany. For example, the substance itself is available from Sigma-Aldrich, CALBIOCHEM, Pharmascience (Montreal), InterHealth (Canada), Pharmascience (Canada), TCI America of Portland, OR; LKT Laboratories of St. Paul, MN; and Samlong Chemical Co., Ltd. from China, or other companies available (see eg. http://www.sigmaaldrich.com/catalog/product/sigma/r5010?lang=de&region=DE ). A commercial method of extracting resveratrol involves the use of alcohol and water to produce Resveratrol from Polygonum cuspidatum.

It has now been found that resveratrol has effects on platelet function: resveratrol inhibits platelet aggregation triggered by aggregation agonists (thrombin, ADP, epinephrine, collagen, AA, PAF); platelet adhesion to collagen or fibrinogen after platelet stimulation is also inhibited by ADP, endotoxin or thrombin greatly reduced. Resveratrol also inhibits calcium-mediated intracellular signal transduction and reduces rapid calcium influx into the platelets upon activation signals. The higher the extracellular calcium concentration, the stronger the resveratrol effect. In contrast to ASA, Resveratrol not only acts on the COX, but as effectively on the peroxidase of the COX. Both enzyme components of the COX are therefore substrates of the resveratrol, the mechanism of action is described with a "hit and run" trait. Resveratrol initially inhibits the peroxidase of COX, but the autocatalytic COX reaction can not be started because the formation of a tyrosyl radical by the peroxidase is omitted. The peroxidases of COX 1 and COX 2 can oxidize resveratrol very quickly, but only COX 1 is also inactivated. COX 2 peroxidase can rapidly oxidize all resveratrol in the microenvironment while remaining active. Thus, the tyrosyl radical mechanism for the COX 2 is started and the proinflammatory or aggregation-triggering mechanisms could become active. The main effect, the COX 2 inhibition, is partially guaranteed even in the oxidized form.

Resveratrol has different effects on COX-1 and COX-2. With respect to COX-1, ASA and resveratrol may behave synergistically, sub-effective concentrations of ASA plus Resveratrol has inhibitory additive effects on platelet function.

In COX 2, no additive effects of the two substances are observed, but resveratrol has retained its platelet function inhibitory effects and thus has significant indications in PRASA and ASD resistance.

In detail, therefore, the invention relates to resveratrol and / or analogs for use in a method for abolishing resistance to ASA or other NSAIDs and / or for reversing the paradoxes response to ASA or other NSAIDs (Paradox Reaction to ASA = PRASA); the use of resveratrol and / or analogs for the preparation of a nutraceutical or in particular a pharmaceutical composition for the abolition of resistance to ASA or other NSAIDs and / or for the abrogation of PRASA; the use of resveratrol and / or analogues to relieve resistance to ASA or other NSAIDs and / or to discontinue PRASA; a corresponding method comprising the administration of resveratrol and / or analogues to relieve resistance to ASA or other NSAIDs and / or to discontinue PRASA; a method or use comprising administration of resveratrol and / or analogs in combination with ASA or other NSAIDs and / or repealing PRASA; a combination comprising resveratol and / or analogs and one or more NSAIDs for prophylactic or therapeutic treatment for platelet aggregation, in particular for the abolition of resistance to ASA or other NSAIDs; and pharmaceutical or nutraceutical formulations or products containing resveratrol and / or analogs and, in the case of the products, one or more NSAIDs for prophylactic or therapeutic treatment for platelet aggregation, in particular for the abolition of resistance to ASA or other NSAIDs and / or for the abrogation of PRASA , and at least one pharmaceutically or nutraceutically suitable carrier material; as well as this new application containing further subject invention.

The more general terms above and below each preferably have the meanings given in the following definitions, wherein in the various subject matters each one or more or all of the more general features can be replaced by more specific definitions, resulting in specific advantageous embodiments of the invention.

Resveratrol and / or analogues in particular resveratrol, especially the trans-form (also referred to as trans-resveratrol, but the term resveratrol here includes both forms or mixtures thereof or in particular trans-resveratrol), or one or more other 3, 5-Dihydroxyphenole, such as corresponding flavones, such as Pentahydroxyflavone, z. B. quercetin (2- (3,4-dihydroxyphenyl) -3,5-7-trihydroxy-4H-1-benzopyran-4-one (obtainable, for example, from green or black tea (Camellia sinensis), red grapes ( Vitis vinifera) or commercially available, for example from Sigma Aldrich, see http://www.sigmaaldrich.com/catalog/product/sigma/g4951?lang=de&region=DE ) or antocyanins such as catechol ((2R, 35) -2- (3,4-dihydroxy) phenyl) -3,4-dihydro-2H-chromen-3,5,7-triol or epicatechin (the (2R, 3R) analogues of the latter compound) (both obtainable, for example, from black or green tea, gerber acacia or blackcurrant or commercially, for example from Sigma-Aldrich, see http://www.sigmaaldrich.com/catalog/product/sigma/g4951?lang=de&region=DE ) to understand. Resveratrol (especially in the trans form) is particularly preferred. Also, extracts which contain resveratrol, in particular a proportion of 0.1 to 99.9 wt .-% z. B. from 0.5 to 4.9 or from 5 to 99.5 wt .-% are possible.

Under ASA or other NSAIDs are acetylsalicylic acid (ASA itself, abbreviated to ASA) or other non-steroidal anti-inflammatory drugs (NSAIDs or English NSAIDs), which may also at least partially lead to a reversible inhibition of platelet aggregation, such as other acetylsalicylic acid derivatives except ASA, acetylpropionic acid derivatives such as flurbiprofen, naproxen ( especially the S-enantiomer), ketoprofen or in particular ibuprofen or tiaprofenic acid, arylacetic acid derivatives such as diclofenac, indoleacetic acid derivatives such as indomethacin, anthranilic acid derivatives such as flufenamine or mefamic acid, or oxicams such as piroxicam, tenoxicam or meloxicam. ASS is particularly preferred.

The methods and uses mentioned above and below relate to use in warm-blooded animals, in particular humans, in particular those (for example due to a risk of thrombosis or a risk of infarction or stroke), in particular for prophylaxis, but also for therapy, for example in the case of a myocardial infarction or another risk of thrombosis) require a (prophylactically and / or therapeutically) antiplatelet treatment, wherein preferably effective amounts of said combination partners are administered. Particular preference is z. B. Patients with inflammatory moderated or inflammatory diseases of the coagulation and the gastrointestinal system.

Under a prophylactic or therapeutic treatment for platelet aggregation, aggregation of the platelets is partially or substantially up to at least 80%. or completely preventing dose of the combination partners, which can be prophylactic or therapeutic, as described or exemplified in the last section. This can be done by all individuals. Test system is the "PADA" described below.

Preferably, it is a treatment that relieves resistance to ASA or other NSAIDs, or the paradox reaction to ASA or other NSAIDs (Paradox Reaction to ASA = PRASA) in patients who are resistant to NSAIDs, particularly ASA, or a PRASE was found.

In particular, resistance to ASA or other NSAIDs means that ASA or other NSAIDs have virtually no detectable antiplatelet effect.

Resistance to ASA or other NSAIDs is particularly evident in the following experimental systems:

1. examination materials

1.1 Devices (Designation Manufacturer Function / Comment)

• KX-21 Sysmex Europe GmbH, 19-parameter fully automatic Hamburg hematology analyzer;
• DADE PFA- ^100® , Dade International Inc., Miami, primary hemostasis disorder analyzer;
• calibrated mini shaker IKA Works, modified JenAffin GmbH, Jena;
• Vortex mixer ZX3 VELP ^®, Scientifica Europe, Usmate (Milano);
• Finnpipette ^® Labsystems, Frankfurt / Main 0.5-10 ul and 40-200 ul;
• Transferpette ^® S Brand GmbH and Co KG, Fix 200 ul and 10 ul Fix, Wertheim
• Gilson Pipetman Abimed, Langenfeld 100-1000 μl

1.2 test kits and ready-to-use components (designation manufacturer function / remark)

• HaemoSys ^® PADA Platelet JenAffin GmbH, Jena Contains control and sample tubes;
• Adhesion assay calibrated particle suspension with HaemoSys ^® PADA-HIT JenAffin GmbH, Jena, stabilizing additive, control solution (0.9% NaCl) with stabilizing additive;
• Stromatolyser ^® -WH, SWH Sysmex Germany GmbH;
• WBC and HGB Lysis Reagent 200 A Norderstedt;
• Cellclean ™ CL-50 Sysmex Europe GmbH, Detergents, Hamburg;
• Cell-Pack PK-301 Sysmex Europe GmbH, Diluent (Thinner), Hamburg;
• Eight Check ^® 3WP H Sysmex Corporation Kobe, Hematology Control, Japan;
• Dade ^® PFA measuring cell collagen / Dade Behring Marburg GmbH 2 μg collagen type I, 50 μg adenosine 5'ADP diphosphate;
• Dade ^® PFA measuring cell collagen / Dade Behring Marburg GmbH 2 μg collagen type I, 10 μg epinephrine epinephrine bitartrate;
• ^Dade® PFA Priming Cartridges Dade Behring Marburg GmbH;
• ^Dade® PFA-100 Cleaning Pads Dade Behring Marburg GmbH;
• Dade ^® PFA Start Solution Dade Behring Marburg GmbH isotonic saline solution;
• ^Dade® PFA-100 O-Ring Dade Behring Marburg GmbH, Service Tool;
• ^Dade® PFA Vacuum Cups Dade Behring Marburg GmbH;
• Dade ^® PFA-100 Spülcartridge Dade Behring Marburg GmbH.

2. Methodology

2.1 Consumables (Designation Manufacturer Function / Comment)

• S-bet Mono ^® LH, 2.6 to 7.5 ml Sarstedt AG & Co, Nümbrecht blood collection system, 16 IU of heparin / ml of blood;
• S-Monowette ^® 5 ml or 2.6 ml Sarstedt AG & Co, Nümbrecht Blood Collection System, trisodium citrate 9NC solution 0.106 mol / l, 0.5 ml or 0.3 ml citrate solution;
• Multi-adapter long Sarstedt AG & Co, Nümbrecht;
• S-bet Mono ^® cannula Sarstedt AG & Co, Nümbrecht 20 G x 1½ ''cannula;
• Micro-Flo DKS Loversan Industria Butterfly Cannula, 21 GA 0.8 × 20 mm, Biomedica S. p. A., Gemonio;
• Plastibrand ^® Brand GmbH and Co KG, pipette tips Nano Cap and 300 ul, Wertheim;
• Ultratip pipette tips Greiner Bio-One, Frickenhausen Pipette tips;
• Parafilm ^® M Brand GmbH and Co KG, sealing film, Wertheim; Reagent tubes Nerbe Plus, Winsen / Luhe

3. Chemicals and Reagents (Designation Manufacturer Function / Comment)

• Aspisol ^® Bayer Vital Leverkusen 0.5 g ASA with 5 ml bidistilled water, in addition, 0.5 μl aggregation buffer was added;
• ^Aspirin® iv Bayer Schering Pharma

4. Blood Collection:

The volunteers took blood samples under a short stowage. After accurate puncture of a forearm or elbow vein (cephalic vein or basilica vein), 5.6 ml and 12.5 ml, respectively, of blood were collected taken. Here either an S-monovette ^® cannula or a butterfly needle was used. The removal took place in citrate and heparin monotypes. Care was taken to ensure that the mono batches were filled exactly to the marking and that foaming during sampling and additional activation of the platelets during transport of the samples were avoided. The blood was mixed by gently swirling the monewinet with the respective anticoagulant. Immediately after collection, the platelet function measurements were made so that a 60-minute window of time between sample acceptance and completion of the study was not exceeded. The blood samples were invariably processed at room temperature. In the run-up to the measurements, a blood count was taken using the KX-21 blood cell machine in both heparin and citrated blood. Quality control in this regard was performed and documented daily (Eightcheck ^® 3WP-H). Regular device-specific maintenance measures (Stromatolyser ^® -WH, CellcleanTM) were also carried out.

4. Principle of the PADA:

The Platelet Adhesion Assay (PADA) is a platelet function test for the quantitative measurement of platelet adhesion in blood and for the study of the effects of GPIIb / IIIa and ADP receptor antagonists on platelet function ( Nowak et al. 2005, Semin. Thromb. Hemost. 31 (4), 470-475 ; Schumann et al. 2005, Semin. Thromb. Hemost. 31 (4), 476-481 ). The test is carried out in citrate-anticoagulated whole blood (3.8%). The blood sample is added a defined number of specially designed polymer particles. These consist of polyphenyl methacrylate (cf. US 7,485,472 ) and have a diameter of 5.5 to 6.5 microns. Due to the special morphological properties of these polymer particles, their surface is immediately and specifically coated with fibrinogen molecules after being added to the blood sample to be examined.

The PADA works with a defined shear stress and without the addition of platelet activating substances. The shaking frequency used here is based on the physiology of platelet activation and corresponds to the shear forces acting on the circulating platelets in the precapillary resistance vessels (arterioles with a diameter of 20-30 μm) ( Nowak et al. 2005 ). During the short incubation phase of five minutes, these platelet-acting shear forces activate part of the platelet population to activate the GPIIb / IIIa and GPIa / IX receptors. This finally results in the adhesion of activated platelets to the fibrinogen-polymer particle complexes.

In the circulation are platelets whose membrane potential has already been modified, for example by mechanical influences or ineffective receptor interactions. These react increasingly and with increasing adhesiveness to the defined shear stress. It is of great importance to limit the shear forces acting in the test in such a way that under no circumstances is a primary aggregation of the platelets induced ( Nowak et al. 2005 ). The measurements include a control sample with no added particles as a reference.

With the help of a blood cell machine, the free platelets, which are not adhered to the polymer particles, are counted both in the particle and in the control sample. On this basis, finally, the calculation of the so-called adhesion index '(AI, according to the formula: AI = 100 × [(PLT control sample - PLT particle sample) / PLT control sample] (PLT = number of free platelets, mean)

The AI thus reflects the adhesiveness of the platelet population as a measure of the current functional state of the platelets in the organism. In previous studies it was independent of the platelet count of the sample (100-350 k / μl), the fibrinogen level (1-5 g / l), hematocrit (25-44%) and sex, with a minimal, non-significant age Had influence ( Nowak et al. 2005 ).

5. Test procedure of the PADA

The PADA and PADA HIT test kit was used to carry out the tests and was used according to the manufacturer's instructions. Prior to use, the appropriate reagents were stored for at least 20 minutes at room temperature. The implementation of the PADA was carried out in compliance with the specified time window of 40-60 minutes after blood collection. After homogenization of the blood sample by gentle swirling, 200 μl of citrated anticoagulated whole blood were pipetted into a sample and a control tube. This air bubble formation was strictly avoided. 10 μl of particle suspension were then added to the sample tube. This was homogenized by vortexing to avoid deviations in the number of particles immediately before addition. The control sample was replaced by the addition of 10 μl of control solution instead of the particle suspension. Subsequently, the two samples were exposed to a shaking incubation phase of exactly five minutes. Here, only a method-specific calibrated shaker was used. This was to avoid a deviation of the shaking frequency in the Warm-up phase at least 20 minutes before use turned on. Immediately after the end of the shake incubation, double measurements of the number of platelets in control and particle samples were made for the calculation of the adhesion index.

6. Evaluation of the PADA

From the duplicate determinations of the number of free platelets (PLT) in the particle and control samples, the mean values (MW) were first calculated and, based on this, the adhesion index (AI) was determined according to the above-mentioned formula.

The standard value of AI in citrated blood is 50 ± 15 (MW ± 2 standard deviation) according to the manufacturer. A PADA AI greater than 65 was considered pathologic in terms of increased platelet adhesiveness.

Structural or qualitative variations of the von Willebrand factor (vWF) lead to reduced adhesiveness ( Nowak et al. 2005 ) and, accordingly, a reduced PADA AI (AI <30). Thus, it is also possible to detect a so-called acquired von Willebrand syndrome.

7. Operating principle of the PADA-RASS

The PADA-RASS (RASS = Resistance to ASA) represents a new method for the investigation of ASA resistance. It is also a modification of the PADA. The PADA-RASS can be used under both current ASA treatment and prior ASA use. The test is carried out exclusively in heparinantikoagulierten whole blood. To the heparin blood sample is added a pharmacodynamically effective dose of ASA.

The underlying value is a sample which contains 0.9% sodium chloride solution instead of ASA.

The PADA-RASS works in the same way as the PADA with the same test procedure (see point 5).

• – ASS-Nonresponder, bei welchen die Blutplättchen keine Reaktion auf ASS zeigen, und
• – die sogenannte PRASA (= paradoxical reaction an ASA). Bei der PRASA-Resistenz kommt es paradoxerweise anstelle einer Inhibition zu einer Aktivierung der Blutplättchen durch ASS, der AI im ASS-haltigen Heparinblut ist größer als der in der Kontrolle ohne ASS.

In the control sample, the heparin molecules bind to glycosaminoglycan receptors. This receptor interaction results in strong platelet activation as a result of the shear stress applied and thus binding of the activated platelets to the fibrinogen-polymer particle complexes. Acetylsalicylic acid in the verum sample, on the other hand, is normally capable of inhibiting this heparin effect in a majority of platelets. The number of activated, bound to the particles platelets is thus lower than in the sample without ASS, resulting in an increased number of free platelets and finally a decreased adhesion index. This is calculated analogously to the PADA in both samples. With the help of the juxtaposition of the adhesion indices, a direct statement can be made about the responsiveness of the platelets to ASA in the presented test situation. In addition to the so-called ASA responders, two ASD resistance groups can be recorded here:

• - ASA nonresponders, in which the platelets show no reaction to ASA, and
• - the so-called PRASA (= paradoxical reaction to ASA). Paradoxically, in the case of PRASA resistance, activation of the platelets by ASA takes place instead of inhibition; the AI in the heparin blood containing ASS is greater than that in the control without ASA.

8. Test procedure of the PADA-RASS

Similar to the implementation of the PADA, attention is paid to a standardized implementation of the individual measuring steps. The materials and reagents used correspond to those from items 1 to 3 above, the added dose of ASA (Aspisol ^® or Aspirin ^® iv) was 1.25 mg ASA per 10 ml.

9. Evaluation of the PADA RASS

From the double measurements of the number of free platelets in the particle and control sample, the respective mean values were calculated first and, based on this, the adhesion index (AI) was determined for both measurement approaches according to a known formula.

Interpretation of the PADA-RASS measurement results was made according to the evaluation scheme incorporated by reference above, which distinguishes between ASA responders, ASA nonresponders and patients with PRASA.

The pharmaceutical or (further) nutraceutical formulations are combination preparations containing both active ingredients (NSAIDs and resveratrol and / or resveratrol analogues) in a unit dosage form ("solid combination") or (combination) products for co-therapeutic and therapeutic use / or prophylactically effective simultaneous and / or sequential administration to patients containing the NSAIDs and resveratrol and / or resveratrol analogues in different dosage unit forms (such as, in particular, kits which may further include instructions for the manner of administration).

Together therapeutically and / or prophylactically effective means in particular that the corresponding products and dosage unit forms are intended for administration in such a way that they partially or completely inhibit platelet aggregation, in particular also in patients in whom resistance to the NSAIDs, in particular ASS, or a PRASE has been found, especially in the above-mentioned experiments.

The solid combinations or products for co-therapeutically effective administration both or in the second case contain both or both of the combination partners NSAIDs and resveratrol and / or resveratrol analogues ("and / or" especially because, whenever using plant extracts, mixtures of resveratrol or its analogue or both), the resveratrol and / or resveratrol analogues (which wherever mentioned also includes a single REsveratrol analog) and, depending on the dosage unit form, at least one pharmaceutically or (further) nutraceutically acceptable carrier material.

Dosage unit forms are understood to be conventional parenterally or in particular enterally administrable administration forms, such as suppositories or nasal sprays or nasal ointments or, in particular, orally administrable administration forms, such as tablets, capsules, pellets, granules, powders (for example in sachets), lozenges, syrups, liquid concentrates , undiluted solutions or other liquid dosage forms. Particularly preferred are tablets and / or capsules, for example conventional soft or hard capsules, such as soft or hard gelatin capsules.

The active ingredient content (NSAR or resveratrol and / or resveratrolanologues) is in each case per dosage unit form, for example, in the range from 0.2 to 99.9% by weight, for example from 2 to 49.9 or 50 to 99% by weight.

The amount per dosage unit form can be z. B. at 10 to 500 mg, z. B. 25 to 200 mg for the NSAID, at z. B. 10 to 3000 mg, z. 25 to 2000 mg for resveratrol and / or resveratrol analogues. Dosage is dependent on weight, age, and other patient-specific criteria, as known to those skilled in the art, and may, for example, be 10 to 500, especially 25 to 200, mg per day in the case of the NSAID for a 70 kg patient. for resveratrol and / or resveratrol analogues at 10 to 3000 mg, in particular at 25 to 2000 mg per day.

Among nutraceutical formulations are, for example, supplements for food, such as concentrates or dosed forms, eg. B. with drug formulations in powder or liquid form, such as capsules, lozenges, tablets, pills, effervescent tablets, powder bags, liquid ampoules, bottles with drip inserts, bottles with food, snacks or the like to understand.

• – Sprengmittel, z. B. mikrokristalline Cellulose (zugleich als Verdünnungsmittel geeignet), oder Carboxymethylcellulose in Salzform, wie Cellulose-carboxymethylether in Form des Alkalimetall, wie Natrium-Salzes, Polyvinylpyrrolidon (1-Vinyl-2-pyrrolidinon-Polymer) (zugleich auch als Binde- und/oder Suspendierungsmittel geeignet) und/oder hochdisperses Siliciumdioxid (zugleich auch als Gleit-, Suspendierungs- und/oder Adsorptionsmittel geeignet);
• – Verdünnungsmittel, z. B. mikrokristalline Cellulose (zugleich als Sprengmittel geeignet), Stärke (zugleich als Binde- und/oder Gleitmittel geeignet), wie Maisstärke, und/oder Talkum (zugleich auch als Gleitmittel geeignet);
• – Suspendierungsmittel, z. B. Polyvinylpyrrolidon (zugleich auch als Binde- und/oder Sprengmittel geeignet) und/oder hochdisperses Siliciumdioxid (zugleich auch als Gleit-, Spreng- und/oder Adsorptionsmittel geeignet);
• – Adsorptionsmittel, wie hochdisperses Siliciumdioxid (zugleich auch als Gleit-, Spreng- und/oder Suspendierungsmittel geeignet);
• – Bindemittel, wie Stärke, z. B. Maisstärke (zugleich als Verdünnungsmittel und/oder Gleitmittel geeignet), und/oder Polyvinylpyrrolidon (zugleich auch als Spreng- und/oder Suspendierungsmittel geeignet);
• – Gleitmittel, wie Stärke (zugleich als Verdünnungs- und/oder Bindemittel geeignet), Talkum (zugleich auch als Verdünnungsmittel geeignet), hochdisperses Siliciumdioxid (zugleich auch als Spreng-, Suspendierungs- und/oder Adsorptionsmittel geeignet) und/oder Magnesiumstearat (vorzugsweise pflanzlichen Ursprungs);

und ein oder mehrere Gemische von zwei oder mehr davon.As pharmaceutically or nutraceutically acceptable carrier materials, materials known to the person skilled in the art are to be used, for example selected from the group which includes the following additives:

• - explosives, z. B. microcrystalline cellulose (also suitable as a diluent), or carboxymethylcellulose in salt form, such as cellulose-carboxymethylether in the form of the alkali metal, such as sodium salt, polyvinylpyrrolidone (1-vinyl-2-pyrrolidinone polymer) (at the same time as binding and / or suspending agent suitable) and / or fumed silica (also also suitable as a slip, suspension and / or adsorbent);
• - Diluent, z. B. microcrystalline cellulose (at the same time suitable as a disintegrant), starch (at the same time suitable as a binder and / or lubricant), such as corn starch, and / or talc (at the same time also suitable as a lubricant);
• - Suspending agent, z. As polyvinylpyrrolidone (also suitable as a binder and / or disintegrants) and / or fumed silica (at the same time also suitable as lubricants, explosives and / or adsorbents);
• Adsorbents, such as fumed silica (also suitable as slip, disintegrants and / or suspending agents);
• - Binders, such as starch, z. B. corn starch (at the same time suitable as a diluent and / or lubricant), and / or polyvinylpyrrolidone (also suitable as blasting and / or suspending agent);
• - Lubricants, such as starch (at the same time suitable as a diluent and / or binder), talc (at the same time suitable as a diluent), fumed silica (also suitable as blasting, suspending and / or adsorbent) and / or magnesium stearate (preferably vegetable origin);

and one or more mixtures of two or more thereof.

Further suitable film-forming agents or binders (especially those with the ability to gel) for controlled, in particular delayed, drug release, such as, for example, etherified or esterified or otherwise modified polypeptides, for example, as an embedding agent for delayed release of active ingredient in the digestive tract, ie preferably in the intestine. Gelatin, resins, e.g. As (meth) acrylic acid esters, waxes, natural resins, eg. Shellac, or in particular polysaccharides, e.g. As dextranes, cellulose acetate, ethylcellulose or especially methylcellulose and / or methylcellulose derivatives which simultaneously contain ethyl, hydroxyethyl, hydroxypropyl or carboxymethyl ether groups. These agents for controlled, in particular delayed drug release can be used as film formers for a core or granules of a pharmaceutical or nutraceutical according to the invention Formulation in the form of films or coatings of the granules or particles used for capsule or tablet production, or preferably as an embedding compound of the delayed-release component (s). The polypeptides and polysaccharides are preferably, in order to allow easy use even with dietary supplements without special authorization and / or labeling obligation to those that are not derived from genetically modified organisms (GMO).

Each of these carrier materials can, based on the finished dosage unit form, in a proportion of 0.5 to 99.8, z. From 5 to 60% by weight.

Further, other additives such as plasticizers suitable for pharmaceutical or in the food supplement industry usual preparations, colorants (such as dyes or pigments), flavorings or the like may be added, preferably in a proportion of 0 to 2 wt .-%, based on the total mass of the product ,

Conventional production methods are used - for example, mixing and tableting (by pressing), granulation (wet or dry) or pelleting (eg by extrusion / extrusion or liquid bed granulation), filling in capsule casings or the like.

Where nutraceutical formulations or nutraceutically acceptable carrier materials are mentioned, this may refer to dietary supplements (for example to the form of capsules, solutions or tablets) or to finished foods or to carrier materials which can be used therein.

Nutraceuticals ("nutraceuticals", "functional foods" or the like) are those products (including drinks) that are suitable for human consumption.

Other nourishing substances may be added, such as vitamins, minerals, unsaturated fatty acids or oils or fats comprising them, flavorings, fragrances, sugars, fruits, dietary fibers, protein, amino acids or the like.

Other common additives are possible, such as stabilizers, thickeners, gelling agents, preservatives, natural or artificial sweeteners other than sugar, acidulants, bitterness-blocking agents, emulsifiers, humectants, and the like. These are also possible with pharmaceutical formulations.

Example:

The following example illustrates the invention without limiting its scope:
In a first clinical study, 17 volunteers between the ages of 23 and 30 were included. Citrate, heparin and hirudin anticoagulated whole blood were analyzed in PADA-RASS and CD62P analyzes (not described here, see above references incorporated by reference). All three anticoagulant blood assays were studied using at least the PADA-RASS technique. Striking was the strong response to PRASA in citrate anticoagulated blood. Here, only one responder (responding patient), but 10 PRASA reactants were found in the subjects. Repeated 60 min after oral intake of 375 mg PADA-RASS trans-resveratrol, 6 subjects returned to responder status and only 5 subjects remained PRASA trait carriers. In principle, the same results after resveratrol administration were to be seen in the other anticoagulant samples.

In the multiplate trial (not described here), these resistance reversal effects of resveratrol could not be seen. The Multiplate test is too rigid and unsuitable for detecting a differentiated ASD resistance.

In principle, in the flow cytometry analyzes for the release of CD62P in unstimulated platelets a reduction of P-selectin and thus a release inhibition of CD 62P to resveratrol could be measured. Parallels to the PADA-RASS antagonism of resveratrol can be seen here.

Assessment of resveratrol effects in the presence of ASA: Resveratrol can largely prevent the PRASA response elicited in COX2-bearing platelets in acute or chronic intestinal and / or vascular inflammatory processes. Even after a single dose of a moderate dose of resveratrol, cardiovascular disease can be delayed or prevented. It is safe to assume that with chronic use of resveratrol the "normal" ASA response rate increases significantly.

The literature and the "scientific community" are increasingly commenting on the problem of ASD resistance. Regarding PRASA data, resveratrol modification of ASD resistance is expected to be of high clinical interest, notably through the additional beneficial effects of resveratrol in inflammation-mediated coagulation and gastrointestinal disorders.

QUOTES INCLUDE IN THE DESCRIPTION

This list of the documents listed by the applicant has been generated automatically and is included solely for the better information of the reader. The list is not part of the German patent or utility model application. The DPMA assumes no liability for any errors or omissions.

Cited patent literature

• US 7485472 [0031]

Cited non-patent literature

• Kaden T. 2010. Frequency and profile of ASD resistance in patients with cardiac disease compared to healthy subjects (dissertation). Jena: Friedrich Schiller University [0012]
• Hentrich J. 2011. Frequency and profile of ASD-resistance in chronic hemodialysis patients compared to healthy subjects (dissertation). Jena: Friedrich Schiller University [0012]
• Kaden T. Hentrich J, Nowak G. 2009. Prevalence and profile of aspirin resistance in patients with cardiovascular diseases in comparison to healthy volunteers. J Thromb Haemost: 7 (Suppl 2) [0012]
• Hentrich J, Kaden T, Nowak G. 2009. Prevalence and profile of aspirin resistance in hemodialysis patients in comparison to healthy volunteers. Haemostaseology, 29: A63 (abstract [0012]
• http://www.resveratrol.de [0016]
• http://www.sigmaaldrich.com/catalog/product/sigma/r5010?lang=en&region=EN [0017]
• http://www.sigmaaldrich.com/catalog/product/sigma/g4951?lang=en&region=EN [0023]
• http://www.sigmaaldrich.com/catalog/product/sigma/g4951?lang=en&region=EN [0023]
• Nowak et al. 2005, Semin. Thromb. Hemost. 31 (4), 470-475 [0031]
• Schumann et al. 2005, Semin. Thromb. Hemost. 31 (4), 476-481 [0031]
• Nowak et al. 2005 [0032]
• Nowak et al. 2005 [0033]
• Nowak et al. 2005 [0035]
• Nowak et al. 2005 [0039]

Claims (19)

Resveratrol and / or analogues for use in a method of reversing resistance to aspirin (ASA) or other non-steroidal anti-inflammatory drugs (NSAIDs) and / or reversing the paradoxes Reaction to ASA (PRASA) or other NSAIDs. Resveratrol for use according to claim 1. Resveratrol for use according to one of claims 1 or 2 for the abolition of resistance to ASA and / or for the removal of the paradoxes Reaction to ASA (PRASA). Resveratrol for use according to one of claims 1 to 3 in patients with inflammation-moderated diseases of the coagulation and / or the gastrointestinal system, with a coronary heart disease, acute coronary syndrome, heart failure, diabetes mellitus, hyperlipidemia or after a percutaneous coronary intervention. Use of resveratrol and / or analogues for the preparation of a nutraceutical or pharmaceutical composition for the purpose of abolishing resistance to ASA or other NSAIDs and / or repealing the PRASA. Use of resveratrol according to claim 5. Use according to one of claims 5 or 6 of resveratrol for the abolition of resistance to ASA and / or for the removal of the paradoxes Reaction to ASA (PRASA). Use of resveratrol according to one of claims 5 to 7 in patients with inflammation-moderated diseases of the coagulation and / or the gastrointestinal system, with coronary heart disease, acute coronary syndrome, cardiac insufficiency, diabetes mellitus, hyperlipidemia or after a percutaneous coronary intervention. Use of resveratrol and / or analogues to relieve resistance to ASA or other NSAIDs and / or to discontinue PRASA. A combination comprising resveratol and / or analogs thereof and one or more NSAIDs for prophylactic or therapeutic treatment for platelet aggregation. A combination according to claim 10 comprising resveratrol and / or analogues thereof and one or more NSAIDs for the abolition of resistance to ASA or other NSAIDs and / or for the abrogation of PRASA. Combination according to either of claims 10 or 11, comprising resveratrol and ASA. A combination according to any one of claims 10 to 12 for treating patients with inflammatory moderated disorders of the coagulation and / or gastrointestinal system, coronary artery disease, acute coronary syndrome, heart failure, diabetes mellitus, hyperlipidemia or after percutaneous coronary intervention. Pharmaceutical or nutraceutical formulation or product comprising resveratrol and / or analogues for prophylactic or therapeutic treatment against platelet aggregation and at least one pharmaceutically or nutraceutically suitable carrier material. A pharmaceutical composition or product according to claim 14 for the abolition of resistance to ASA or other NSAIDs and / or for the abrogation of PRASA. A pharmaceutical product according to any one of claims 14 or 15, including resveratrol and / or analogues thereof in combination with one or more NSAIDs. The pharmaceutical product of claim 16, including resveratrol and ASA. Pharmaceutical product according to claim 16 or 17 in the form of a kit. A pharmaceutical or nutraceutical formulation according to claim 14 or 15 or a pharmaceutical product according to any one of claims 16 to 18 for treatment in patients with inflammatory moderated disorders of coagulation and / or gastrointestinal system, coronary heart disease, acute coronary syndrome, heart failure, diabetes mellitus, hyperlipidemia or after a percutaneous coronary intervention.