AND USE OF XANTHOHUMOL

TECHNICAL FIELD

The object of the present invention is a preparation comprising xanthohumol and/or its pharmaceutically acceptable salt and/or xanthohumol complex with metal as an active substance for use in treatment and/or prevention of cardiologic complications of anticancer therapy (chemotherapy) and use of xanthohumol and/or its pharmaceutically acceptable salt and/or xanthohumol complex with metal in treatment and/or prevention of cardiologic complications of anticancer therapy (chemotherapy).

BACKGROUND OF THE INVENTION

The term chemotherapy used in the present specification is understood as systematic cancer therapy not only with the aid of cytostatic medicines, but also as a therapy, where alkylating medicines, antimetabolites (e.g. methotrexate, 5-fluorouracil), monoclonal antibodies (e.g. alemtuzumab), tyrosine kinase inhibitors (e.g. erlotynib, wemurafenib), hormone medicines (e.g. tamoxifen), antineoplastic antibiotic (e.g. doxorubicin, bleomycin), podophyllotoxin derivatives (e.g. etoposide), spindle poisons (e.g. vincristine, vinblastine), enzymes (e.g. asparaginase), proteasome inhibitors (e.g. bortezomib), and PARP inhibitors (e.g. olaparib) are administered.

Cardiotoxicity induced by chemotherapy used in oncology, is a temporary or permanent disruption of the mechanical or electrical function of the heart muscle. Heart damage can be asymptomatic, in the form of acute or chronic heart failure, myositis, pericarditis, myocardial infarction, development of cardiomyopathy, arrhythmia and sudden myocardial infarction. Acute or subacute cardiotoxicity occurs during chemotherapy (after a single dose or after the whole course of treatment). While chronic cardiotoxicity occurs during 12 months after the last dose of the medicine. There is also a delayed chronic cardiotoxicity occurring from one to five years after the treatment. The mechanism of cardiotoxicity induced by chemotherapy is not fully known. Since myocardial damage or arrhythmias are serious complications, it is very important to protect this important organ during cancer therapy. Publication R. Patel, B. M. Shinde, R. Syed, V. Singh, H. S. Shin pt. Therapeutic potential of quercetin as a cardiovascular agent Eur J Med Chem. 2018 Jul 15;155:889-904 discloses effect of quercetin and the like in improving heart function and suggests its possible use in cardiovascular diseases treatment. Publication M. Bartekova, J. Radosinska, D. Pancza, M. Barancik, T. Ravingerova, Cardioprotective effects of quercetin against ischemia-reperfusion injury are age- dependent, Physiol Res. 2016 Sep 19 discloses use of quercetin in cardiovascular diseases treatment in mammals.

European patent application EP3411017 discloses a pharmaceutical composition for reducing or eliminating cardiotoxicity, particularly cardiotoxicity induced by a cancer treatment or other therapy. In some cases, the methods and compositions prevent or reduce cardiotoxicity caused by anthracy cline treatment. The methods provided herein often comprise administering a protective agent such as myricetin, tricetin, robinetin, ficetin, vitexin, quercetin, dihydrorobinetin, kaempferol, 7,3',4',5'-tetrahydroxyflavone, and myricitrin in conjunction with another protective agent and/or chemotherapeutic . European patent application EP2877167 discloses a method of use of a composition comrpsing a roasted extract and xanthohumol for use in treatment or prevention of cardiovascular diseases.

Patent application US20170304220 discloses use of xanthohumol in treatment of cardiac arrhythmia.

DISCLOSURE OF THE INVENTION

The subject of the present invention is a preparation comprising xanthohumol and/or its pharmaceutically acceptable salt and/or xanthohumol complex with metal as an active substance for use in treatment and/or prevention of cardiologic complications of chemotherapy of a mammal.

During the chemotherapy treatment a chemotherapeutic or chemotherapeutics from the group of anthracyclines are administered.

The preparation comprises at least one pharmaceutically acceptable excipient.

The preparation comprises additionally one and/or more pharmaceutically active substances.

The preparation is administered to a mammal in the amount providing from 0.001 preferably from 0.3 to 10 mg of xanthohumol and/or its pharmaceutically acceptable salt and/or xanthohumol complex with metal based on kg of weight of the mammal, i.e. the preparation comprises such amount of xanthohumol and/or its pharmaceutically acceptable salt and/or xanthohumol complex with metal as to provide a single or multiple dose of from 0.001 preferably from 0.3 to 10 mg of this substance based on kg of weight of the mammal daily.

The preparation according to any of the preceding claims, characterized in that it is administered to a mammal in the amount providing from 1 to 1000 mg of xanthohumol and/or its pharmaceutically acceptable salt and/or xanthohumol complex with metal daily, i.e. the preparation comprises such amount of xanthohumol and/or its pharmaceutically acceptable salt and/or xanthohumol complex with metal at to provide a single or multiple dose from 1 to 1000 mg of this substance to a mammal daily.

The mammal is a human.

The preparation is in the form of a capsule or a tablet or syrup or oral solution or suspension. The preparation is a food product, such as food supplement or foodstuffs intended for particular nutritional uses.

The preparation is in the transdermal form, such as ointment or gel.

The preparation is a medicine.

The preparation is in the form of injection.

The subject of the present invention is also use of xanthohumol and/or its pharmaceutically acceptable salt and/or xanthohumol complex with metal in treatment and/or prevention of cardiologic complications of chemotherapy of a mammal.

During the chemotherapy treatment a chemotherapeutic or chemotherapeutics from the group of anthracyclines are administered, such as doxorubicin, epirubicin or mitoxantron.

Xanthohumol and/or its pharmaceutically acceptable salt and/or xanthohumol complex with metal is administered to a mammal in the amount providing a single or multiple dose of from 0.3 to 10 mg of xanthohumol and/or its pharmaceutically acceptable salt and/or xanthohumol complex with metal based on kg of weight of the mammal. The mammal, to whom xanthohumol and/or its pharmaceutically acceptable salt and/or xanthohumol complex with metal is administered, is a human.

Xanthohumol and/or its pharmaceutically acceptable salt and/or xanthohumol complex with metal is administered to a mammal in such amount as to provide a single or multiple dose from 1 to 1000 mg of xanthohumol and/or its pharmaceutically acceptable salt and/or xanthohumol complex with metal to a mammal daily.

The term“preparation” shall be understood as also a preparation comprising solely raw xanthohumol and/or its pharmaceutically acceptable salt and/or xanthohumol complex with metal without any excipients.

The term "pharmaceutically acceptable salts” shall be understood as salts, which are acceptable from pharmaceutical point of view, such as salts with non-organic bases, such as lithium, sodium, potassium, calcium salts and organic amines and amino acids salts. Xanthohumol complexes with metals, which may be used in the invention, are complexes with transition metals like copper or zinc. In case, when the preparation according to the invention comprises excipients, such excipients are chosen from the group comprising known carriers, preservatives, fragrances, thickeners, sweetening agents, flavoring agents, coating agents, glidants, substances improving absorption (e.g. lipids) and/or improving stability (e.g. cyclodextrin) e.t.c.. The excipient must be pharmaceutically acceptable, useful for preparation of the intended pharmaceutical composition, and shall not negatively impact active substances in the preparation, in particular xanthohumol and/or its pharmaceutically acceptable salt and/or xanthohumol complex with metal.

The preparation comprises defined amount of active substance - the amount therapeutically effective, such as is necessary to obtain, directly or indirectly, therapeutic effect. The preparation is manufactured by combining the active substance which is xanthohumol and/or its pharmaceutically acceptable salt and/or xanthohumol complex with metal with excipients. Excipients are chosen depending on the intended form of the preparation. The preparation may also comprise additional active substances, including it is possible to compose a preparation, in which xanthohumol and/or its pharmaceutically acceptable salt and/or xanthohumol complex is combined with a chemotherapeutic. In this case the protective substance which is xanthohumol and/or its pharmaceutically acceptable salt and/or xanthohumol complex with metal, is administered in one preparation with the chemotherapeutic.

The term„amount therapeutically effective” refers to such amount of xanthohumol and/or its pharmaceutically acceptable salt and/or xanthohumol complex, as to act therapeutically or protectively in cardiologic complications in chemotherapy.

Xanthohumol and/or its pharmaceutically acceptable salt and/or xanthohumol complex is administered to a mammal, in particular a human.

The biological activity of xanthohumol was determined on rat cardiomyocyte lines using the MTT test. Quercetin was used as a comparative substance. Assuming high bioavailability of xanthohumol, the lowest therapeutically effective amount was determined, the determined concentration was converted into milligrams of active substance per kilogram of patient weight. When determining the final therapeutically effective dose, pharmacodynamic and pharmacokinetic aspects as well as other aspects that may affect the bioavailability of the active substance and thus its efficacy should be taken into account. The daily dose of xanthohumol administered orally, up to three times a day, will vary depending on the age, weight and sex of the patient. In general, a daily dose of xanthohumol administered to a human in one or more portions should be from 0.001 to 10 mg/kg body weight, i.e. between 1 and 1000 mg per day.

The state of the art suggests use of isoxanthohumol as one of the ingredients of the preparation used to reduce or eliminate cardiotoxicity, in particular cardiotoxicity induced by cancer therapy or other types of therapy. However, this is not obvious direction for use of xanthohumol for the treatment or prevention of cardiotoxicity in chemotherapy. Xanthohumol and isoxanthohumol are two different molecules. In the stomach, xanthohumol is converted into isoxanthohumol, among others, but isoxanthohumol is not the dominant metabolite. Therefore, the biological effect should be attributed more to xanthohumol, because xanthohumol concentrations in human are significantly higher than xanthohumol concentrations.

The invention is illustrated by the below examples. EXAMPLES

Example 1

Culture of H9c2 rat cardiomy oblast cells line. H9c2 car diomy oblast cells coming from American Tissue Culture Collection were grown in Eagle'a Dulbecco (DMEM) medium with addition of 10% fetal bovine serum (FBS) and mixture of antibiotics (100 U/mL penicillin, and 100 U/mL streptomycin) in a 25-cm ² tissue culture flasks at temperature of 37°C, and atmosphere of 5% CO ₂ . The medium was changed every 2 days, and cells were cultured until reaching 70%-80% confluency.

Cells were seeded in 96-well plate at a density of 1 × 10 ⁴ cells per well. They were cultured for next 24 hours (confluency 80%).

Cells were treated with xanthohumol X (5mM) for 5 hours. After pre-incubation with xanthohumol, the medium was changed to a fresh one comprising damaging substances (H ₂ O ₂ and doxorubicin -- DOX). Incubation with damaging substances was carried out for 90 minutes. Cells viability was measured by the MTT assay (Diagram 1).

The MTT assay was used to determine the cytotoxic effects of the analyzed compounds. After cell incubation in the presence of compounds, 10 ml of MTT tetrazolium reagent was added to experimental plates. After 3 hours of incubation with reagent (37°C, 5% CO ₂ ), the medium was aspirated and the formazan produced in the cells appeared in the form of dark crystals in the bottom of the wells. Next, dimethyl sulfoxide (DMSO) was added to each well in order to dilute emerged formazan crystals. Then the absorbance of the emerged solution was determined at 570 nm wavelength on a plate reader (Spectra Max iD3, Molecular Devices). The number of metabolically active - living, cells was directly proportional to the absorbance of the solution. The results are presented at the diagram as the percentage of control condition ± standard deviation SD.

Diagram 1.

Effect of application of xanthohumol (X) used in combination with doxorubicin (DOX) (low-5 mM/ high- 10 mM) and H ₂ O ₂ (low-0.125 mM/high-0,250mM) on viability of H92c rat cardiomyocytes. MTT assay was used to determined cells viability. Diagram represents the % of viable cells in comparison to the control cells which were not treated with xanthohumol The conducted research proved that xanthohumol in concentration of 5 ml did not cause any cytotoxic effect in H9c2 cells, while xanthohumol co-treatment with doxorubicin prevented doxorubicin induced and H ₂ O ₂ -induced cytotoxicity. Thus the protective action of xanthohumol was demonstrated.

Example 2

Culture of H9c2 rat cardiomyoblast cells was grown in the way described in example 1.

Culture of H9c2 rat cardiomyoblast cells were treated with xanthohumol (X) (5mM) diluted in Eagle'a Dulbecco (DMEM) medium comprising 0.5% fetal bovine serum (FBS) for 5 hours, then doxorubicin (25mM) was added, and incubation was continued for 90 minutes to induce cells injury. Cell viability was measured by the MTT assay (Diagram 2) in the way described in example 1.

Diagram 2.

Viability of H92c rat cardiomyocytes pre-incubated with xanthohumol (X), then damaged by doxorubicin (DOX). Cells were treated with xanthohumol for 5 hours, then DOX (25mM) was added for 90 minutes. MTT assay was used to determined cell viability. Diagram represent the % of viable cells in comparison to the control condition (cells non-treated with xanthohumol). Statistical significance was determined using Mann-Whitney test p<0.05.

Example 3

Culture of H9c2 rat cardiomyoblast cells was grown in the way described in example 1.

Culture of H9c2 rat cardiomyoblast cells were co-treated with xanthohumol (X) (1 or 5mM) and doxorubicin (2.5 or 5mM) for 24 hours. Cell viability was measured by the MTT assay (Diagram 3) in the way described in example 1.

Diagram 3.

Cardiomyocytes were co-treated with xanthohumol ( X) and doxorubicin (DOX) for 24 hours. MTT assay was used to determined cell viability. Diagram represents the % of viable cells in comparison to the control condition (cells non-treated with xanthohumol). Statistical significance was determined using Mann-Whitney test p<0.05.

Xanthohumol protected cardiomiocytes against damage caused by doxorubicin. During pre-incubation of cardiomyocytes xanthohumol (X) protected cells against damaging activity of doxorubicin what resulted in increased viability of cells. Xanthohumol co-treated with doxorubicin increased viability of cardiomyocytes while better effects were noticed in case of cells incubated with higher doses of the agent (5 mM DOX) (Diagram 3). Example 4

Example 4 is a comparative example, where instead of xanthohumol querceting was administered. The tests were carried our in a way similar to the one indicated in Examples 1-3.

Diagram 4

Effect of quercetin (QUE) used in combination with doxorubicin (DOX) (low -5 mM/ high- 10 mM) and H ₂ O ₂ (low-0.125 mM/high-0.250mM) on viability of H92c rat cardiomyocytes. MTT assay was used to determine cell viability. Diagram represents the % of viable cells in compare to the control condition (cells non- treated with quercetin).

Diagram 5

Viability of H92c rat cardiomyocytes pre-incubated with quercetin (QUE), and the, damaged by doxorubicin (DOX). Cardiomyocytes were treated with quercetin for 5 hours, then doxorubicin (25 mM) was added, and incubation was continued for 90 minutes. MET assay was used to determined cell viability. Diagram represents the % of viable cells in comparison to the control condition (cells not treated with quercetin). Statistical significance was determined using Mann-Whitney test p<0.05.

Diagram 6 Cardiomyocytes were co-treatedwith quercetin (QUE) and doxorubicin (DOX) for 24 hours. MET assay was used to determined cell viability. Diagram represents the % of viable cells in comparison to the control condition (cells not treated with quercetin). Statistical significance was determined using Mann-Whitney test p<0.05.

Protective effect of quercetin (QUE) on cardiomyocytes was observed in case of cells damage induced by H ₂ O ₂ in concentration of 0.125 mM (Diagram 4).

During cardiomyocytes pre-incubation quercetin does not protect cells against damaging activity of doxorubicin, what in result does not affect observed viability of cells (Diagram 5). Additionally quercetin applied in twenty- or four- times higher concentration (20 mM), than xanthohumol (accordingly 1 mM and 5 mM), did not increase viability of cardiomyocytes in 24 hours model with use of doxorubicin (given in concentration 5 mM) as the damaging factor (Diagram 6).