Patent Description:
Heart failure is a chronic cardiovascular disease that occurs when the heart muscle is unable to pump enough blood to meet the body's oxygen needs. Heart failure is one of the leading causes of death worldwide. The current classification of heart failure is based on the measurement of the percentage of total blood that is pumped out of the left ventricle during a single cardiac cycle and. Correspondingly, a distinction is made between heart failure with reduced ejection fraction (HFrEF) and heart failure with preserved ejection fraction (HFpEF). Although there are several treatment regimens for HFrEF that increase survival and reduce hospitalisation rates, there is no effective targeted therapy for HFpEF. Patients with HFpEF symptoms do not benefit from the classic treatment regimens used to treat HFrEF, and a search for new therapeutic targets in HFpEF is needed.

Heart failure, both HFrEF and HFpEF, is a systemic disease and therefore the pathological mechanisms and therapeutic efficacy of new compounds cannot be studied in isolated organs or cell cultures, as such, they should be investigated in animal models of HFrEF and HFpEF. Indeed, research into the pathogenesis, new ways of diagnosing and therapy towards heart failure has been successfully conducted in animal models, in particular, in murine models. Several mouse models of heart failure have been developed that can resemble the key clinical manifestations of heart failure, but these are mainly limited to animal models of HFrEF. Currently, the animal models of heart failure are predominantly based on surgical interventions, the administration of pharmacological agents, or the use of an appropriate feeding regimen, which in turn lead to the development of heart failure, as well as combinations of the above methods.

Unlike HFrEF models, HFpEF models are difficult to obtain because they require the application of multiple deleterious factors simultaneously, such as advanced animal age, high-fat diet, pharmacological agents, among others. Therefore, current murine models of HFpEF are almost exclusively based on multiple pathological factors, and when a single deleterious factor is applied, it has to be applied over an extended period of time reaching tens of weeks. Such models, although they exist, have many limitations to their use in testing the efficacy of new therapies.

International patent application <CIT> discloses a method of inducing heart failure with preserved ejection fraction (HFpEF) in a laboratory animal. The disclosed method comprises administering a composition as defined in this application to a laboratory animal. The document also discloses a method of inducing heart failure by additionally feeding the animals a diet containing about <NUM>% - <NUM>% (wt/wt) of a standard diet and a non-standard diet, the standard diet containing about <NUM>% carbohydrate, about <NUM>% fat, about <NUM>% protein, about <NUM>% salt and no cholesterol; the non-standard diet containing about <NUM>% carbohydrate, about <NUM>-<NUM>% fat, about <NUM>% protein, about <NUM>% cholesterol, about <NUM>-<NUM>% sodium chloride. In the execution example, the administration of the diet lasts from <NUM> days to <NUM> days to induce and maintain high blood pressure. In the application example, elevated exercise intolerance and/or diastolic dysfunction (elevated E/A ratio in echocardiographic measurements) were observed in addition to elevated blood pressure.

International patent application <CIT> discloses a method of inducing heart failure with preserved ejection fraction (HFpEF) in a laboratory animal. The disclosed method comprises administering a composition as defined in this application to a laboratory animal. The document also discloses a method of inducing heart failure by additionally administering to the animals a diet containing <NUM>% kcal derived from fat compared to a standard diet, and a combination of administration of the diet and <NUM>-<NUM>% methyl-Nw-nitro-L-arginine ester chloride. In the application example, the administration of the diet lasts from <NUM> days to <NUM> days to induce and maintain reduced contractility and left ventricular hypertrophy. In the application example, left ventricular hypertrophy, increased exercise intolerance and/or diastolic dysfunction (increased E/A ratio in echocardiographic measurements, decreased left ventricular global maximum longitudinal strain as determined by echocardiography) were observed.

Analogously to the above method of inducing heart failure is revealed in an article by <NPL>), which describes the use of a combination of dietary HFD or administration of angiotensin II (instead of L-NAME) in elderly mice, i.e. aged <NUM>-<NUM> months. In contrast, International Application No. <CIT> presents the administration of Angiotensin II as a means of inducing diastolic heart failure in rats in alternative to the rat model with sodium chloride administration (deoxycorticosterone acetate-salt rats, DOCA, rats with increased susceptibility to hypertension induced by chronic sodium chloride administration in drinking water).

On the other hand, Chinese patent application <CIT> discloses a method of inducing heart failure with preserved ejection fraction (HFpEF) in mice aged <NUM>-<NUM> months administered a high-fat diet. The disclosed method comprises the administration of a high-fat diet and deoxycorticosterone pivalate to the mice. The diet disclosed in the application contains <NUM>-<NUM>% fatty acids, <NUM>-<NUM>% carbohydrates, <NUM>-<NUM>% proteins. Deoxycorticosterone pivalate at a dose of <NUM>-<NUM>/kg is administered <NUM>-<NUM> months after the high-fat diet started.

In addition, epidemiological data suggest that there is an increasing number of patients who develop heart failure as a result of prior cancer treatment. In these patients, heart failure may develop independently of other classical risk factors used in stratifying cardiovascular risk in the general population.

Tyrosine kinase inhibitors (TKIs) are a class of therapeutic agents that inhibit tyrosine kinases. A number of TKIs targeting various tyrosine kinases have been shown to be effective anti-cancer and anti-leukemic agents. TKIs carries a significant risk of adverse cardiovascular effects, including, but not limited to, the development of heart failure.

Risto Kerkelä et al. in the article Cardiotoxicity of the cancer therapeutic agent imatinib mesylate (<NPL>) discloses a mouse that is administered with the tyrosine kinase BCR-ABL inhibitor, imatinib. The mice receive between <NUM> and <NUM>/kg/day for <NUM> or <NUM> weeks and have cardiac dysfunction. This document discloses murine model of Heart Failure with Reduced Ejection Fraction (HFrEF) as demonstrated in Tabel <NUM> in D1 however it lacks the information on the age of mice.

The aim of the invention was to develop a method of obtaining a model demonstrating a pathological state of diastolic heart failure with preserved ejection fraction, which was different from known state-of-the-art methods and would produce reproducible results with a reduced intervention time from tens to weeks and eliminate the need for multiple hits to induce HFpEF pathology simultaneously, thereby increasing the controllability and reproducibility of the mouse model of HFpEF. An additional goal was to eliminate the use of animals of advanced age that spontaneously develop HFpEF themselves, reducing the costs associated with housing the animals, thereby reducing the cost of the experiments as well as the time to complete them.

When conducting experiments on laboratory mice administered TKIs, a pathological state of diastolic heart failure with preserved ejection fraction was unexpectedly observed.

Thus, the object of the invention is a method of obtaining a mouse model exhibiting a pathological state of diastolic heart failure with preserved ejection fraction (HFpEF), the method comprising administering to mice a tyrosine kinase inhibitor compound at a dose of at least <NUM>/kg for a period for <NUM> to <NUM> weeks, preferably for <NUM> weeks, wherein said mice are no more than <NUM> weeks old at the start of the administration of TKI compound, preferably from <NUM> to <NUM> weeks old, wherein tyrosine kinase inhibitor is a tyrosine kinase inhibitor having the oncological therapeutic target of BCR-ABL tyrosine kinase, and belonging to the 2nd or 3rd generation of tyrosine kinase inhibitors for the treatment of Chronic Myeloid Leukemia (CML), when in the method of the invention the administered compound is a tyrosine kinase inhibitor selected from the following group:.

The invention also includes a mouse model of diastolic heart failure with preserved ejection fraction obtained by the above method.

The object of the invention is illustrated in embodiments not limiting the scope of the invention, that is defined by the appended claims, and in a drawing, where the <FIG> compares the values of the ratio of the left ventricular filling rate in the early (E) phase of diastole to the left ventricular filling rate in the atrial (A) phase of diastole as a measure of impairment of cardiac diastolic function, for the used TKIs inhibitors.

In the embodiment, a series of experiments were performed in which the following tyrosine kinase inhibitor compounds were administered to mice (male wild-type strain C57/BL6j): Ponatinib at <NUM>, <NUM><NUM>/kg, Asciminib at <NUM>, <NUM> and <NUM>/kg, Nilotinib at <NUM>/kg and Imatinib at <NUM>, <NUM> and <NUM>/kg. These tyrosine kinase inhibitors were administered to mice aged <NUM>-<NUM> weeks for a period of four weeks. This was followed by a cardiac function study using magnetic resonance tomography imaging. During the study, the animals were kept under inhalation anaesthesia using isoflurane at a dose of <NUM>-<NUM>% in a <NUM>:<NUM> mixture of oxygen and air. During the measurement, the animals' condition was monitored using a respiratory sensor, temperature sensor, and ECG system. To assess left ventricular function, a retrospectively gated gradient echo sequence (FLASH) was used. A series of <NUM>-<NUM> imaging slices oriented along the short axis of the heart was obtained to cover the entire left ventricular volume. Based on the obtained imaging data, the cardiac cycle curve, i.e. the change in left ventricular area over time, was calculated according to the protocol described by <NPL>). The following parameters determining left ventricular function were determined from the left ventricular area over time curve:.

Systolic kinetics was assessed as the slope in the initial phase of systole (as the slope of the curve tangent to successive points marking the change in the left ventricular area during the first approximately <NUM>% of the cycle), and filling kinetics was assessed as the slope in the initial filling phase of the left ventricle (in the first approximately <NUM>% of the ventricular inflow phase) referred to as ER (Ejection Rate) and FR (Filling Rate), respectively. The corresponding left ventricular kinetic times: ejection time (ET), filling time (FT), isovolumic_relaxation time (IVRT), and isovolumic contraction time (IVCT) were obtained from the curve by manually marking consecutive parts of the cardiac cycle. The early filling rate (E) and atrial phase (A) were calculated from the derivative of the cardiac cycle curve. Deformability (strain) analysis was performed using MR tagging imaging, peak radial strain and peak circumferential strain were calculated using the SPAMM algorithm.

Parameters describing cardiac systolic function are ejection fraction (EF), stroke volume (SV), cardiac output (CO), end-systolic volume (ESV), ejection rate (ER), and ejection time (ET). On the other hand, parameters describing the diastolic function of the heart are end-diastolic volume (EDV), filling rate (FR), filling time (FT), early phase filling index (E), atrial phase filling index (A) and E/A ratio.

Systolic failure was defined as a significantly reduced ejection fraction (EF) and impairment in at least one of the following additional parameters: stroke volume, cardiac output, end-systolic volume, and ejection rate. Diastolic failure, on the other hand, was defined as a preserved normal ejection fraction (EF) and impaired in at least one of the following parameters: filling rate, early phase filling rate (E), atrial phase filling rate (A), E/A ratio.

The results obtained for the different doses are shown in Table <NUM>. The mean heart rate increased significantly in the Nilotinib <NUM>/kg group of mice and decreased in the Imatinib <NUM> and <NUM>/kg, Asciminib <NUM>, <NUM> and <NUM>/kg, Ponatinib <NUM> and <NUM>/kg groups. Total left ventricular mass increased significantly only in the Ponatinib <NUM>/kg group. End-systolic volume was significantly increased in the Asciminib <NUM>/kg and Ponatinib <NUM>/kg groups, in the other groups it did not differ from the mean as compared to the control group. Importantly, a change in end-systolic volume alone is not sufficient to conclude systolic heart failure (HFrEF). End-diastolic volume was significantly increased in the Asciminib <NUM>/kg group and did not differ in other groups as compared to the control group. The ejection fraction was not significantly altered in any group. In classic systolic failure with reduced ejection fraction, ejection fraction falls to <NUM>-<NUM>% compared to controls. Cardiac output was significantly reduced only in the Imatinib <NUM>/kg group, and in the other groups it did not change as compared to the control group. The cardiac index remained unchanged in all experimental groups. The ejection rate remained unchanged in all experimental groups. The filling rate was significantly reduced in the Nilotinib <NUM>/kg, Imatinib <NUM> and <NUM>/kg, Ponatinib <NUM>, <NUM> and <NUM>/kg groups. Ejection time and filling time remained unchanged in all experimental groups. The isovolumic relaxation time was significantly reduced in the Asciminib <NUM>/kg group. The isovolumetric contraction time was significantly reduced in the Ponatinib <NUM>, <NUM> and <NUM>/kg groups. The early phase filling index (E) was significantly reduced in the Nilotinib <NUM>/kg, Imatinib <NUM>/kg, Asciminib <NUM>/kg, Ponatinib <NUM>, <NUM> and <NUM>/kg groups. The atrial phase filling index (A) was significantly increased in the Asciminib <NUM>, <NUM>/kg, Ponatinib <NUM> and <NUM>/kg groups. The E/A ratio was significantly reduced in all experimental groups (as shown in <FIG>). The peak circumferential strain was significantly reduced in the Asciminib <NUM> and <NUM>/kg and Ponatinib <NUM>/kg groups. The peak radial strain was significantly reduced in the Ponatinib <NUM>/kg group.

A particularly significant impairment of diastolic cardiac function (defined by a reduced E/A parameter with preserved systolic function) was observed in mice in all experimental groups, i.e. when the following compounds constituting TKIs were administered:.

In addition, a comparative study was also conducted using older mice, i.e. aged <NUM>-<NUM> weeks. In this experiment, mice (male wild-type strain C57/BL6j) were administered Nilotinib at a dose of <NUM>/kg for <NUM> weeks. Cardiac function was assessed using magnetic resonance imaging according to the methodology outlined in the application example above.

The results obtained for the different doses are shown in Table <NUM>. A statistically significant reduction in ejection fraction to <NUM>% ± <NUM> as compared to <NUM>% ± <NUM> in the control group was observed, as well as an impairment in ejection volume, cardiac output, and end-systolic volume compared to the control group. Thus, systolic heart failure was observed in older mice. To obtain a mouse model of diastolic heart failure in mice, it is, therefore, necessary to use young mice up to <NUM> weeks of age, and the best results are obtained for mice aged <NUM> weeks.

Claim 1:
A method of obtaining a mouse model of diastolic heart failure with preserved ejection fraction, the method comprising administering to mice a compound which is a tyrosine kinase inhibitor at an effective dose of at least <NUM>/kg for a period for <NUM> to <NUM> weeks, preferably for <NUM> weeks, wherein the age of mice at the start of the administration of compound being not more than <NUM> weeks, preferably from <NUM> to <NUM> weeks, wherein tyrosine kinase inhibitor is a tyrosine kinase inhibitor having the oncological therapeutic target of BCR-ABL tyrosine kinase, and belonging to the 2nd or 3rd generation of tyrosine kinase inhibitors for the treatment of chronic myeloid leukaemia,
wherein the administered compound is a tyrosine kinase inhibitor selected from the following group consisting of:
(a)
<CHM>
wherein Ponatinib is administered at a dose of <NUM> to <NUM>/kg, preferably at a dose of <NUM> to <NUM>/kg, more preferably at a dose of <NUM>/kg,
(b)
<CHM>
wherein Asciminib is administered at a dose of <NUM> to <NUM>/kg, preferably at a dose of <NUM> to <NUM>/kg, more preferably at a dose of <NUM>/kg; and
(c)
<CHM>
wherein Nilotinib is administered at a dose of <NUM> to <NUM>/kg, preferably at a dose of <NUM>/kg.