Treatment of heart failure in human subjects

Abstract

To provide an agent for preventing or treating a cardiac disorder in a human subject.SOLUTION: The present invention provides a therapeutic agent comprising an oligonucleotide that is an effective inhibitor of microRNA miR-132, and its use in medicine.SELECTED DRAWING: None

Description

[Technical Field]

[0001] statement The present invention relates to oligonucleotides that are effective inhibitors of the microRNA miR-132, and their use in medicine, particularly in the prevention or treatment of cardiac and / or fibrotic disorders in human subjects.

[0002] Heart failure (HF) is one of the leading causes of morbidity and mortality worldwide. Myocardial infarction (MI) is the most important cause of HF, as MI subsequently induces adverse cardiac remodeling and leads to HF with poor prognosis. Current therapeutic pharmacological options for HF after MI include angiotensin-modulating drugs, beta-blockers, diuretics, aldosterone antagonists, combinations of angiotensin II receptor blockers and neprilysin inhibitors, vasodilators, inotropes, or SGLT-2 inhibitors. Although several clinical trials have shown that all of these drugs significantly reduce HF mortality, the 5-year mortality rate remains unacceptably high at approximately 50%. Therefore, there is a strong need to develop novel and more efficient therapeutic approaches for HF.

[0003] Pathological hypertrophic growth of cardiomyocytes can lead to cardiac remodeling, heart failure, and sudden cardiac death. Hypertrophic growth of cardiomyocytes occurs as a result of cardiac volume and / or pressure overload. Cardiac hypertrophy is a response to increased cardiac wall stress caused by vasoconstriction. Initially, cardiac hypertrophy is a compensatory mechanism aimed at reducing wall stress and increasing cardiac output. However, prolonged cardiac hypertrophy can progress to systolic dysfunction, cardiac decompensation, and ultimately heart failure. (Hill and Olson, 2008; Barry and Townsend, 2010). The transition from physiological to pathological hypertrophy Degeneration can be caused by many factors, including cardiomyocyte loss through apoptosis and necrosis, altered autophagy, impaired contractile responses, calcium dyshomeostasis, adrenergic receptor desensitization, and myocardial fibrosis (Hill and Olson, 2008; Barry and Townsend, 2010). Hypertrophic signals are primarily mediated by the insulin signaling pathway (Debosch and Muslin, 2008; Barry and Townsend, 2010). Both insulin and insulin-like growth factor-1 (IGF-1) activate prohypertrophic pathways in cardiomyocytes via the IGF-1 receptor, which activates phosphoinositin 3-kinase (PI3K) (McMullen et al., 2004). PI3K activity leads to the phosphorylation and activation of the serine-threonine kinase Akt, which then phosphorylates the antihypertrophic FoxO transcription factors, destabilizing them and preventing their nuclear localization (Datta et al., 1999; Skurk et al., 2005; Ronnebaum and Patterson, 2010). On the other hand, Sirtuin-1 (Sirt-1) Acetylation of FoxO factors by ATP leads to their stabilization and nuclear translocation (Frescas et al., 2005). Stabilized FoxO transcription factors are localized in the nucleus to regulate the expression of antihypertrophic genes. The antihypertrophic function of FoxO proteins is primarily mediated by the suppression of the prohypertrophic calcineurin signaling pathway through the expression of antihypertrophic gene targets of FoxO factors, such as atrogin-1 (Ni et al., 2006; Ronnebaum and Patterson, 2010; Glas, 2010). Furthermore, FoxO transcription factors induce apoptosis and regulate autophagy in cardiomyocytes (Ronnebaum and Patterson, 2010).

[0004] MicroRNAs have been shown to play an important role in adverse cardiac remodeling. WO 2013 / 034653 describes that miR-132 and / or miR-212 induce cardiac hypertrophy and are therefore useful for the treatment of heart failure. It has been described that these proteins may constitute potential therapeutic targets for the treatment of cancer.

[0005] WO2016 / 042561 discloses a polynucleotide that is substantially complementary to the nucleotide sequence of human miR-132. Methods for treating lipid-associated disorders by administering to a subject a therapeutically effective amount of a leutide agent are described.

[0006] The present inventors have identified a novel oligonucleotide that is an effective inhibitor of miR-132 expression in cardiomyocytes. This oligonucleotide analog (hereinafter referred to as CDR132L) ) is a mixmer consisting of DNA and LNA building blocks with phosphorothioate internucleoside linkages.

[0007] Preclinical pharmacology studies of CDR132L were conducted in mice and pigs. In a mouse model of cardiac hypertrophy, CDR132L induced cardiac reverse remodeling associated with decreased expression of miR 132. In a mouse model of post-MI heart failure, CDR132L treatment reduced post-MI heart failure. CDR132L administration was associated with improved cardiac function and reduced miR-132 expression, cardiac stress signaling, and post-MI hypertrophy.

[0008] In a porcine model of post-MI heart failure, CDR132L treatment prevented maladaptive remodeling and reduced left heart Furthermore, CDR132L was found to improve ventricular function. CDR132L normalizes tissue expression of CAR and shifts myosin heavy isoforms (MYH7 / 6 ratio). In a porcine model of post-MI heart failure, CDR132L reverses subacute and chronic heart failure. I found that.

[0009] CDR132L also showed significant toxicity in human hepatocyte cell lines and isolated neonatal rat cardiomyocytes. In vivo toxicity studies in rats and minipigs have shown that the active agent Higher doses of 20 mg / kg and 40 mg / kg, respectively, were found to be well tolerated.

[0010] Pharmacokinetic studies of CDR132L administered intravenously or subcutaneously to healthy rats and pigs confirmed a dose-dependent tissue concentration of CDR132L.

[0011] Furthermore, CDR132L was shown to exhibit superior efficacy compared to other oligonucleotide analogues with the same nucleotide sequence but with different distributions of LNA building blocks.

[0012] In further studies, we investigated the role of fibrosis in an in vivo mouse model of myocardial infarction and liver fibrosis. Antibody activity of the oligonucleotide analog CDR132L in in vitro models of lung disease and pulmonary fibrosis The therapeutic effect on fibrosis was confirmed.

[0013] Furthermore, we found a significant correlation between the amount of miR-132 in circulating body fluids and the therapeutic efficacy of CDR132L. Therefore, the amount of miR-132 in body fluids is relevant for monitoring therapy. It is a biomarker.

[0014] Based on these results, we are conducting a Phase Ib clinical trial to administer CDR132L to human patients with chronic heart failure. A clinical trial protocol has been developed, and this Phase Ib clinical trial has been successfully completed.

[0015] Thus, the oligonucleotide CDR132L is useful in medicine, in particular for treating cardiac disorders and / or fibrosis. These compounds are useful as active agents in the prevention or treatment of sexual disorders.

[0016] Thus, a first aspect of the present invention provides a compound of formula I: 5'-ATGGCTGTAGACTGTT-3' wherein A, T, G, and C are deoxyribonucleotide building blocks, and at least one G or T building block is a bridging nucleotide building block. The oligonucleotide is useful in the prevention and / or treatment of disorders, more particularly cardiac disorders, in human subjects. It is particularly suitable for

[0017] In certain embodiments, the oligonucleotide has Formula II: 5'-A+TG+GC+TG+TA+GACTG+T+T-3' wherein A, T, G and C are deoxyribonucleotide building blocks, +G and +T are bridged nucleotide building blocks and / or morpholino nucleotide building blocks, in particular +G and +T are LNA building blocks. This oligonucleotide is particularly suitable for the prevention or treatment of disorders, more particularly cardiac disorders or fibrotic disorders, in a human subject.

[0018] The oligonucleotide analog of Formula I or Formula II may contain at least one modified internucleoside bond, such as an internucleoside bond that is stabilized against nuclease digestion, such as a phosphorothioate or phosphorodiamidate bond. In a specific embodiment, all internucleoside bonds are modified bonds, particularly phosphorothioate bonds.

[0019] In a more specific embodiment, the invention relates to the oligonucleotide CDR132L described herein.

[0020] The oligonucleotide CDR132L has the formula III: 5'-dA* +T * dG * +G * dC * +T * dG * +T * dA * +G * dA * dC * dT * dG * +T * +T-3' wherein dA is 2' deoxyadenosine, dG is 2' deoxyguanosine, dC is 2' deoxycytidine, and T is thymidine; +T is an LNA-T building block and +G is an LNA-G building block; * Is host The oligonucleotide has the sequence: ##STR00002## where the amino acid sequence is a holothioate bond. This oligonucleotide is particularly suitable for the prevention or treatment of disorders, more particularly cardiac disorders or fibrotic disorders, in human subjects.

[0021] A further aspect of the invention relates to a pharmaceutical composition for use in the prevention or treatment of disorders, more particularly cardiac or fibrotic disorders, particularly in a human subject, comprising as an active agent an oligonucleotide analogue comprising the sequence of Formula I, II or III and a pharmaceutically acceptable carrier.

[0022] As noted above, oligonucleotide analogs comprising the sequences of Formula I, II or III are suitable for medical use.

[0023] In certain embodiments, the medical applications relate to, are associated with, and involve the pathological expression of miR-132. In certain embodiments, the medical application relates to the treatment or prevention of cardiac disorders, particularly disorders associated with cardiac hypertrophy. In certain embodiments, the medical application relates to the treatment of fibrotic disorders, e.g., disorders associated with, accompanied by, and / or caused by pathological fibrosis, particularly cardiac fibrotic disorders, pulmonary fibrotic disorders, or hepatic fibrotic disorders.

[0024] The oligonucleotides of formula I, II or III are deoxyribonucleotide DNA building blocks. The ribonucleotide may be composed of a ribonucleotide building block and a bridged nucleotide building block. A "bridged nucleotide" refers to a modified ribonucleotide containing a two- or three-atom bridge connecting the 2'- and 4'-carbon atoms of the ribose moiety. For example, the bridge may have the structure 2'-O-CH2-4' , 2'-O-CH2-CH2-4', 2'-O-CH(CH3)-4' or the corresponding structures in which O is replaced by S or NH. In certain embodiments, at least one bridged nucleotide building The block is a locked nucleic acid (LNA) building block with a 2'-O-CH2-4'-bridge. It is a block.

[0025] The oligonucleotide of Formula I, II, or III has a length of at least 16 building blocks, e.g., a length of 16 to 20 building blocks. In certain embodiments, the oligonucleotide of Formula I, II, or III has a length of 16 building blocks.

[0026] In some embodiments, the oligonucleotide of Formula I, II, or III has 5 to 10 amino acids, e.g., For example, it contains 6 to 8, especially 7, bridged nucleotide building blocks, such as LNA building blocks.

[0027] In some embodiments, the oligonucleotide of Formula I, II, or III is a naked oligonucleotide. In some embodiments, the oligonucleotide comprises at least one Conjugates to one heterologous site, e.g., a site that does not contribute to the binding of the oligonucleotide to miR-132. The heterologous moiety may be conjugated to the 5'- and / or 3'-end of the oligonucleotide analog by a covalent bond or a spacer. The heterologous moiety may be a moiety that improves targeting and / or cellular uptake, such as a lipid moiety, such as cholesterol or a fatty acid, a sugar or amino sugar moiety, such as an N-galactosamine-containing moiety, a peptide or polypeptide moiety, or a nucleoside or nucleotide moiety, such as an aptamer. The heterologous moiety may be conjugated to the 5'- and / or 3'-end of the oligonucleotide analog by a covalent bond or a spacer. It may be gated.

[0028] The oligonucleotides of the invention are suitable for use in medicine, including human and veterinary medicine. In certain embodiments, the compounds are useful in treating pathological expression, e.g., overexpression of miR-132. The compounds are useful for the prevention or treatment of disorders related to, accompanied by, and / or caused by miR-132. Administration of the compounds has been found to significantly reduce the expression of miR-132 in vitro and in vivo.

[0029] In some embodiments, the compound is administered to patients exhibiting overexpression of miR-132 compared to healthy individuals. In some embodiments, the compound may be administered to a subject in which the level of miR-132 is increased relative to a healthy subject. It is administered to patients who do not show overexpression but still require a reduction in miR-132 levels. That's fine.

[0030] The term "prevention" in the context of the present invention relates to the administration of the compounds to patients known to be at high risk of developing a particular disorder. The term "treatment" in the context of the present invention relates to the administration of the compounds to patients who already develop signs and / or symptoms of a particular disorder. The term "patient" refers to a subject in the human or veterinary medicine field who requires the administration of a compound of the present invention. In certain embodiments, the patient is a human patient.

[0031] In certain embodiments, the compounds of the present invention are useful for preventing or treating cardiac disorders, particularly cardiac hypertrophy-related disorders.For example, the compounds of the present invention are useful for preventing or treating systolic dysfunction, cardiac weakness, heart failure, or for preventing or treating cardiac remodeling after myocardial infarction, myocarditis, valvular heart disease such as aortic stenosis or mitral regurgitation, hereditary cardiac disorders with cardiac hypertrophy, such as hypertrophic non-obstructive cardiomyopathy and obstructive cardiomyopathy, or Fabry disease.In addition, the compounds are useful for preventing or treating cardiac fibrosis.

[0032] The compound is: (i) Patients at high risk of developing heart failure; (ii) Patients suffering from (congestive) heart failure, e.g., those at increased risk of developing heart failure Patients (iii) post-myocardial infarction patients, and / or (iv) aortic and / or pulmonary vein stenosis, atrial or ventricular septal defect, etc., accompanied by cardiac hypertrophy Patients with congenital heart disease It is useful for administration to a patient selected from:

[0033] In certain embodiments, the compounds, particularly CDR132L, are administered to human patients suffering from acute heart failure. The present invention is useful for administration to a human subject suffering from chronic heart failure, a human patient suffering from subacute heart failure, or a human patient suffering from chronic heart failure and / or worsening chronic heart failure.

[0034] In certain embodiments, the compounds, particularly CDR132L, are useful in treating stable heart failure, e.g., non-ischemic and The present invention is useful for administration to human patients suffering from stable heart failure of ischemic and / or ischemic origin.

[0035] In certain embodiments, the compounds, particularly CDR132L, are of non-ischemic and / or ischemic origin. It is useful for administration to human patients suffering from heart failure.

[0036] In certain embodiments, the compounds, particularly CDR132L, are useful in treating less advanced stages of heart failure. It is useful for administration to human patients with complete or advanced stages of heart failure.

[0037] In certain embodiments, the compounds, particularly CDR132L, are selected from the New York Heart Association (NYHA) classification The compositions are useful for administration to human patients suffering from heart failure stage I, II, III and / or IV due to NYHA, e.g., patients suffering from NYHA stage I and / or II heart failure, patients suffering from NYHA stage I, II and / or III heart failure, or patients suffering from NYHA stage III and / or IV heart failure.

[0038] In certain embodiments, the compounds, particularly CDR132L, are administered to patients suffering from heart failure and using an implanted pump, e.g. It is useful for administration to human patients with left ventricular assist devices (LVADs).

[0039] In certain embodiments, the compounds are useful for preventing and / or treating left-sided heart failure, such as systolic heart failure, diastolic heart failure, and conditions associated with systolic and / or diastolic heart failure.

[0040] Systolic heart failure is a type of heart failure associated with a reduced ejection fraction, particularly an ejection fraction of 40% or less, in which the left ventricle loses the ability to contract normally.Administering the compound of the present invention to treat systolic heart failure can lead to the stabilization, increase and / or normalization of ejection fraction.The compound of the present invention is suitable for administration to patients at risk of developing systolic dysfunction, such as patients suffering from hypertension or coronary artery blockage.

[0041] Diastolic heart failure is a type of heart failure associated with impaired left ventricular relaxation, with or without increased filling pressure.In many cases, diastolic heart failure is associated with preserved ejection fraction.Administering the compound of the present invention to treat diastolic heart failure can lead to stabilization, improvement and / or normalization of left ventricular relaxation.The compound of the present invention is suitable for administration to patients at risk of developing diastolic dysfunction, such as patients with hypertension, hyperlipidemia, diabetes, obstructive sleep apnea syndrome, cardiac storage disease, and hereditary heart failure (for example, mutations in titin or other structural genes).

[0042] In a further particular embodiment, the compounds are useful for preventing and / or treating right-sided heart failure, particularly right-sided heart failure resulting from left-sided heart failure.

[0043] In certain embodiments, the compounds of the invention are useful in the prevention or treatment of fibrotic disorders, particularly disorders associated with, accompanied by, and / or resulting from pathological fibrosis.

[0044] Pathological fibrosis is the formation of excess fibrous connective tissue in organs and tissues, particularly those associated with, accompanied by, and / or resulting from pathological conditions. Pathological fibrosis can occur in many different organs and tissues in the body, typically as a result of inflammation or injury.

[0045] In certain embodiments, the fibrosis is cardiac fibrosis, e.g., a condition involving pathological fibrosis of the heart. Exemplary types of cardiac fibrosis include atrial fibrosis, endocardial fibrosis, or fibrosis resulting from a previous myocardial infarction.

[0046] In a further embodiment, the fibrosis is pulmonary fibrosis, e.g., a condition involving pathological fibrosis of the lungs. Exemplary types of pulmonary fibrosis include fibrotic disorders caused by occupational or environmental factors, such as exposure to toxins and pollutants, such as silica dust, asbestos fibers, metal dust, coal dust, grain dust, bird and animal droppings. Other types of pulmonary fibrotic disorders are caused by radiation therapy and / or treatment with pharmaceuticals, such as chemotherapy drugs, cardiac drugs, antibiotics, or anti-inflammatory drugs. Still other types of pulmonary fibrotic disorders are caused by disorders such as idiopathic pulmonary fibrosis, dermatitis, polymyositis, mixed connective tissue disease, rheumatoid arthritis, scleroderma, autoimmune diseases such as Sjögren's syndrome or systemic lupus erythematosus, sarcoidosis, pneumonia, viral infections, or gastroesophageal reflux disease (GERD).

[0047] In a further embodiment, the fibrosis may be liver fibrosis, e.g., a condition involving pathological fibrosis of the liver. Exemplary types of liver fibrosis include those caused by viral infections, e.g., Hepatitis B and / or Caused by hepatitis C virus, inherited metabolic disorders, autoimmune hepatitis, biliary obstruction, and iron overload , caused by non-alcoholic fatty liver diseases such as non-alcoholic fatty liver disease (NAFL) and non-alcoholic steatohepatitis (NASH), as well as alcoholic liver disease.

[0048] In still further embodiments, the fibrosis may also be vascular fibrosis, e.g., arteriosclerosis, skin fibrosis, e.g., keloid formation or nephrogenic systemic fibrosis, arthrofibrosis, some forms of adhesive capsulitis, soft tissue fibrosis such as mediastinal fibrosis or retroperitoneal fibrosis, or fibrosis of the bone marrow, such as myelofibrosis.

[0049] In certain embodiments, the present invention encompasses determining the amount and / or activity of certain physiological parameters in the treated subject before, during, and / or after administration of the compound of the present invention.This accompanying diagnostic procedure can provide support for the above-mentioned medical applications.For example, this diagnostic procedure can provide support in risk assessment, patient stratification, treatment progress monitoring, and / or post-treatment control.

[0050] In certain embodiments, the present invention provides methods for determining the amount and / or activity of miR-132 in a treated subject before, during, and / or after administration of a compound of the present invention. In a further embodiment, the present invention provides a method for determining the amount and / or activity of at least one marker, particularly selected from cardiac markers and / or fibrotic markers. In certain embodiments, the present invention relates to a method for detecting BNP (e.g., NT-proBNP), ANP, or In certain embodiments, the present invention includes determining the amount and / or activity of cardiac markers such as myosin heavy-change isoforms (e.g., MYH7 / 6 ratio), and / or levels of FoxO3 and / or SERCA2. In certain embodiments, the present invention includes determining the amount of NT-proBNP. In yet further embodiments, the present invention provides a method for detecting collagen (e.g., collagen deposition and / or fibrosis marker genes (e.g., collagen 1A1, collagen 1A2, collagen 3A1, determining the amount and / or activity of fibrotic markers, such as expression of procollagen type I C-terminal propeptide (PICP), and / or galectin 3 (Gal-3), and / or matrix metalloproteinases (e.g., matrix metalloproteinase 1 (MMP-1) and / or matrix metalloproteinase 2 (MMP-2)) Contains.

[0051] Determination of the above parameters can be performed in body fluid samples such as blood, plasma, serum or tissue samples at the nucleic acid level and / or protein level according to known methods and may provide useful diagnostic information, for example, about the course of a disease and / or the course and / or success of a therapy.

[0052] Furthermore, the inventors demonstrated that the amount of miR132 in cardiac tissue was significantly higher than that in circulating miR132 by PCR-based detection methods. It has been found that the amount of miR132 in body fluids, such as whole blood, plasma, or serum, is positively correlated with the amount of miR132 in body fluid samples, such as whole blood, plasma, or serum samples. Therefore, measuring the amount of miR132 in body fluid samples, such as whole blood, plasma, or serum samples, provides an indication of the amount of miR132 in target tissues, particularly cardiac tissue.

[0053] In certain embodiments, the invention encompasses determining the amount and / or activity of miR-132 in a treated subject, e.g., a human subject, during the course of therapy. The term "course of therapy" herein is to be understood as the administration of a compound of the invention, in particular the administration of CDR132L, to a subject in need thereof, in particular a human subject, over a period of time, for example at least one day, at least one week, at least two weeks or at least one month. This determination can be performed once or several times during the course of therapy. The amount of miR-132 can be measured in a sample from a body fluid, for example, a circulating sample such as a blood, plasma, or serum sample. The miR-132 is quantitatively determined in a sample from a blood sample. In particular, the sample is a plasma sample. The amount and / or activity is inversely or negatively related to the concentration of the compound in the intended target organ, particularly the heart, but also to its activity and / or therapeutic efficacy, and thus this determination allows for adjustment of the dose administered and / or the interval between individual administrations.

[0054] Furthermore, this assessment allows stratification of patients regarding treatment response, for example, distinguishing between responders and non-responders.In particular, assessment is carried out several times during the course of therapy in patients with chronic diseases, such as patients with chronic heart disease.For example, assessment can be carried out at intervals of weekly, biweekly and / or monthly.

[0055] In certain embodiments, the present invention provides a method for determining changes in ECG parameters over the course of therapy. Relevant ECG parameters for patients with heart failure include, but are not limited to, QRS, T waves, , left bundle branch block (LBBB) and / or right bundle branch block (RBBB); and / or R progression Of particular relevance is the measurement of the QRS.

[0056] According to a further aspect of the invention, the oligonucleotide can be administered in a demand-based dosing regimen, e.g., by adjusting the dose and / or the time interval between individual doses depending on the measured amount of miR132 in the body fluid sample, e.g., if the amount of miR132 in the body fluid sample is found to exceed a predetermined value, a new dose of oligonucleotide is administered.

[0057] Thus, the present invention relates to an oligonucleotide as described above for use in the prevention or treatment of cardiac disorders in a human subject, wherein the oligonucleotide is administered by a demand-based dosing regimen, in particular comprising the steps of: (i) measuring the amount of miR132 in a body fluid sample, such as a whole blood, serum, or plasma sample, of a subject treated with an oligonucleotide; (ii) administering the oligonucleotide at doses and / or time intervals between individual doses determined according to the measured amount of miR132 in step (i), in particular administering a new dose of oligonucleotide if the amount of miR132 in the body fluid sample is found to exceed a predetermined value, e.g., a reference value.

[0058] The compounds of the present invention may be administered as a pharmaceutical composition containing a pharmacologically acceptable carrier. Administration can be carried out by known methods, in which the compound is introduced into the desired target cells or organs of the subject to be treated.

[0059] The compounds may be administered neat or as conjugates with heterologous moieties as described above.

[0060] For pharmaceutical applications, the compositions may be in the form of liquid solutions, eg, injectable solutions, emulsions, suspensions, and the like.

[0061] The composition can be administered by any suitable method, for example, parenterally, in particular by injection such as subcutaneous, intramuscular, intravenous or intraarterial injection or infusion, or by oral or inhalation ingestion, and / or by skin application, or by local application to target organ, for example, by intracoronary perfusion.Carrier can be any suitable pharmaceutical carrier.Preferably, carrier can be used that can increase the efficacy of oligonucleotide molecule entering target cell.Suitable examples of such carrier are liposome, for example cationic liposome, or pre-designed exosome.

[0062] In certain embodiments, the compounds, particularly CDR132L, are administered by intravenous injection or by subcutaneous injection. It is administered by

[0063] The compounds are administered in pharmaceutically effective doses depending on the route of administration and the type or severity of the disease.

[0064] In certain embodiments, the compound is administered to a human subject, for example, by parenteral administration, particularly injection or infusion, e.g., intravenous or subcutaneous injection, at a dose of about 0.1 to 100 mg / kg body weight per dose, e.g., about 0.2 to 50 mg / kg body weight per dose, about 0.8 to 20 mg / kg body weight per dose, or about 3 to 10 mg / kg body weight per dose.

[0065] In pharmacokinetics test, compound has a long half-life of about 3 weeks in cardiac tissue, and has a short biphasic half-life in plasma.These results demonstrate that compound is suitable for various different treatment regimens, for example, treatment regimens that include application at intervals of less than 1 week and treatment regimens that include application at intervals of more than 1 week.

[0066] In certain embodiments, the oligonucleotide is administered to the human subject on a dosing regimen selected from daily administration, every two days, every three days, and every four days, and in particular the oligonucleotide is administered parenterally, more particularly by intravenous or subcutaneous injection, e.g., subcutaneous self-injection by the patient.

[0067] In these embodiments, oligonucleotide can be administered in weight-dependent dose and / or fixed dose.For example, oligonucleotide can be administered in a dose of 0.01mg / kg body weight to 50mg / kg body weight, in a dose of 0.02mg / kg body weight to 10mg / kg body weight, or in a dose of 0.05mg / kg body weight to 5mg / kg body weight per application.Alternatively, oligonucleotide can be administered in a fixed dose of 1mg to 5000mg, in a fixed dose of 2mg to 1000mg, or in a fixed dose of 5mg to 500mg per application.

[0068] In still further embodiments, the oligonucleotide is administered weekly, every 2 weeks, every 3 weeks, every 4 weeks, or monthly, every 6 weeks, every 2 months, every 3 months, every 4 months, or every 6 months. The oligonucleotide is administered to the human subject on a dosing regimen selected from monthly dosing, monthly dosing, and annual dosing, and in particular the oligonucleotide is administered parenterally, more particularly by intravenous or subcutaneous injection, e.g., subcutaneous self-injection by the patient.

[0069] In these embodiments, oligonucleotide can be administered in a weight-dependent dose or a fixed dose.For example, oligonucleotide can be administered in a dose of 0.01mg / kg to 50mg / kg body weight, in a dose of 0.05mg / kg to 20mg / kg body weight, or in a dose of 0.1mg / kg to 10mg / kg body weight per application.Alternatively, oligonucleotide can be administered in a fixed dose of 1mg to 5000mg, in a fixed dose of 5mg to 2000mg, or in a fixed dose of 10mg to 1000mg per application.

[0070] In yet further embodiments, the compound may be administered to a human patient at an initial dose, e.g., one or two initial doses, followed by at least one maintenance dose different from the initial dose. For example, the initial dose may be higher than the maintenance dose, e.g., about 1.5 to 3 times, e.g., about 2 times, the maintenance dose. The initial and / or maintenance doses may be administered as weight-dependent doses or as fixed doses. In a specific embodiment, the initial dose is about 3 to 10 mg / kg, and the maintenance dose is about 1 to 7.5 mg / kg. Furthermore, the maintenance dose may be adjusted, e.g., by titration, based on the amount of miR132 in a body fluid, such as blood, plasma, or serum.

[0071] In a human Phase 1b clinical trial, this compound was confirmed to be well-tolerated and safe in human heart failure patients at single and multiple dose escalation in addition to standard treatment. Furthermore, the pharmacokinetic profile showed high dose linearity with no signs of accumulation. Its unique mechanism of action for heart failure was confirmed by relevant pharmacodynamic parameters and target engagement. No serious adverse events or injection site reactions were observed, and no patients discontinued the study due to adverse events. The compound was also well-tolerated, showing no signs of toxicity at doses up to 10 mg / kg.

[0072] The compounds may be administered alone or in combination with further medicaments, particularly medicaments suitable for the prevention or treatment of cardiac or fibrotic disorders as described above.

[0073] Examples of further medicaments suitable for the prevention or treatment of cardiac disorders are angiotensin modulators, beta-blockers, diuretics, aldosterone antagonists, vasodilators, iontophoretics, statins, neprilysin inhibitors or SGLT-2 inhibitors or combinations thereof (e.g. combinations of neprilysin inhibitors, e.g. combinations of sacubitril and angiotensin II receptor blockers (e.g. valsartan)).

[0074] In certain embodiments, the compound is selected from the group consisting of (i) at least one diuretic, (ii) at least one angiotensin converting enzyme inhibitor, (iii) at least one beta blocker, optionally (iv) an angiotensin II receptor blocker and / or (v) optionally an If channel inhibitor such as ivabradine and optionally (vi) an angiotensin receptor neprilysin inhibitor, and optionally (vii) a glucose co-transporter 2 inhibitor such as empagliflozin and dapagliflozin, and / or optionally (viii) a stem cell therapeutic and / or optionally (ix) an anti-miRNA targeting a different pathway, and / or optionally (x) The compound and inhibitor according to (i) to (x) can be independently selected and combined in any suitable manner.

[0075] Further examples of medicaments suitable for the prevention of the treatment of fibrotic disorders are medicaments for the prevention or treatment of cardiac fibrosis (ACE inhibitors (e.g. lisinopril), angiotensin II receptor blockers (e.g., candesartan, losartan, or olmesartan), aldosterone antagonists (e.g., spironolactone), and / or TGFβ inhibitors (e.g., pirfenidone or tranilast), medicines for the prevention or treatment of pulmonary fibrosis (antifibrotic agents (e.g., nintedanib or pirfenidone), anti-inflammatory agents (e.g., corticosteroids, azathioprine, cyclophosphamide, and mycophenolate mofetil), antireflux agents (e.g., protein pump inhibitors or H2 blockers), and / or anti-tussives), and medicines for the prevention and / or treatment of liver fibrosis (e.g., ACE inhibitors (e.g., benazepril, lixivivir), Nopril or Ramipril), antiviral agents or PPARα-agonists).

[0076] Furthermore, the present invention relates to the use of a compound of the present invention as described hereinabove for the manufacture of a medicament for the prevention or treatment of cardiac disorders.

[0077] Furthermore, the present invention relates to the use of a compound of the invention as described hereinabove for the manufacture of a medicament for the prevention or treatment of a fibrotic disorder.

[0078] Additionally, the present invention provides a method for administering to a subject in need thereof at least one compound described herein. The present invention relates to a method for preventing or treating cardiac disorders comprising administering a therapeutically effective amount of

[0079] Additionally, the present invention provides a method for administering a therapeutically effective amount of at least one compound described herein to a patient in need thereof. The present invention relates to a method for preventing or treating a fibrotic disorder, comprising administering to a subject in need thereof

[0080] Furthermore, the present invention provides a method for determining the amount and / or quality of miR-132 in a subject to which an oligonucleotide has been administered. The present invention relates to a kit for monitoring a therapy using the above-mentioned oligonucleotide, which comprises determining the activity or activity of the oligonucleotide. The kit comprises a primer that binds to DNA encoding miR-132 or a DNA complementary thereto, preferably a primer has-miR-132 that binds to DNA encoding miR-39 or a DNA complementary thereto, preferably a primer cel-miR-39, and optionally a positive control hsa-miR-132 and optionally a positive control cel-miR-39. According to a preferred embodiment, the primers hsa-miR-132 and miR-39 are used together. Additionally, the kit may contain additional compounds, such as a qPCR master mix and nuclease-free water. In a preferred embodiment, the kit contains the primer hsa-miR-132, the primer cel-miR-39, the positive control cited above, and a qPCR master mix and nuclease-free water. Suitable primers are available from the It can be selected by the vendor.

[0081] The present invention will be further explained in more detail by the following figures and examples. [Brief explanation of the drawings]

[0082] [Figure 1] No significant toxicity of CDR132L in HepG2 cells and NRCM was observed in the therapeutic dose range (see Figures 1A and 1B). [Figure 2] Treatment: CDR132L or placebo 20 mg / kg ip weekly (see Figure 2A). miR-132 expression was detected by qPCR. Statistical test: unpaired t-test (Figure 2B). [Figure 3A-B] Representative echocardiographic image of the heart (Figure 3A). CDR132L reversed hypertrophy as measured by myocardial mass and diastolic volume (LVEDV) (Figure 3B). [Figure 3C]CDR132L improves regional contractile function in most segments of the left ventricle (AB, anterior base; AM, anterior mid-segment; AA, anterior apex; PA, posterior apex; PM, mid-segment; and PB, posterior base). [Figure 4] Myocardial infarction (MI) model mice: The coronary artery (LAD) of C57BL / 6N mice was permanently ligated. [Figure 5] CDR132L treatment improved left ventricular dysfunction after MI (Figure 5A), as well as load-independent parameters of systolic function (Figure 5B) (*p<0.05). [Figure 6] CDR132L administration also improves longitudinal strain rate (LSR), thereby reversing post-MI contractile dysfunction in individual myocardial segments in remote parts of the heart (*p<0.05). [Figure 7] CDR123L treatment effectively silences miR-132 expression in cardiac tissue, for example, in the remote (non-infarct) region and peri-infarct zone (Figure 7A). At the histological level, CDR132L reduces cardiomyocyte size in the remote region of the heart after MI (Figure 7B). At the tissue level, CDR132L reduces the expression of the cardiac stress signal ANP in the heart after MI (Figure 7C). [Figure 8] A porcine myocardial infarction model induced by 90 minutes of ischemia (LAD occlusion) followed by reperfusion. [Figure 9] CDR132L treatment prevented maladaptive remodeling and improved function, as determined by measurements of end-diastolic volume, end-systolic volume, ejection fraction, and left ventricular function (Figures 9A-D). CDR132L treatment improved segmental contractility in segments corresponding to viable / remote myocardium; cardiac MRI at endpoint: n = 6 / group, red: p < 0.05 (Figure 9E). [Figure 10] CDR132L treatment avoids maladaptive remodeling and improves LV survival in distal regions as determined by NOGA, an end-point electroanatomical mapping: n=6 / group, placebo vs. CDR132L: p<0.05. [Figure 11]CDR132L normalizes tissue expression of pathological heart failure markers in ANP and BNP and provides a shift in myosin heavy-chain isoforms, i.e., the MYH7 / 6 ratio. [Figure 12] At the histological level, CDR132L treatment effectively reduces cardiomyocyte hypertrophy in representative photographic images (WGA / DAPI staining 20x) (Figure 12A) and graphic depictions (Figure 12B) of the remote LV region, n=6 / group, placebo vs. CDR132L: p<0.05. [Figure 13] A single administration of CDR132L silences cardiac miR-132 levels in a dose-dependent manner. [Figure 14] In vivo treatment with CDR132L does not cause organ toxicity in pigs. [Figure 15] Cardiac FoxO3 and Serca2 mRNA levels were measured four times weekly in control and miR-132TG mice treated with intraperitoneal injections of either a control scrambled oligonucleotide or CDR132L. All values ​​represent mean ± SEM. *P < 0.05 (Figures 15A and 15B). [Figure 16] The experimental design and results are shown in Figure 16. Treatment of NRCM with CDR132L and CDR301 significantly reduced miR-132-3p levels by 96%, while CDR2u1 reduced miRNA expression by 30% (Figures 16A-B). Furthermore, treatment with CDR132L resulted in significant suppression of the miR-132-3p target gene Foxo3, which could not be achieved with CDR2u1 and CDR301 (Figure 16C). [Figure 17] The experimental design is described in FIG. [Figure 18] In vivo evaluation of the antifibrotic effect of CDR132L in post-MI heart failure is shown in Figure 18. Histological results showed that fibrosis was attenuated after CDR132L treatment (Figure 18A), which was also confirmed at the molecular level by reduced gene expression of fibrosis markers such as collagen type III alpha 1 chain (Col3a1) (Figure 18B). [Figure 19]Figure 19 shows an in vitro model of liver fibrosis. Stimulation of HPLFs with transforming growth factor beta (TGF-β) (Figure 19A) resulted in a slight induction of miR-132-3p, which was significantly reduced by CDR132L treatment (Figure 19B). Furthermore, this compound reduced fibroblast proliferation (Figure 19C) and the expression of fibrotic genes such as COL1A1, COL1A2, and MMP2 (Figure 19D). [Figure 20] Figure 20 shows an in vitro lung fibrosis model. In NHLF cells, after fibrosis stimulation with high FBS (5% compared to normal growth conditions of 2% FBS) (Figure 20A), no increase in miR-132-3p was observed (Figure 20B). Nevertheless, treatment with CDR132L resulted in a significant decrease in target microRNAs (Figure 20B) and fibroblast proliferation (Figure 20C). [Figure 21] Circulating miR-132-3p was measured in plasma samples from a placebo-controlled porcine model of myocardial infarction (MI)-induced heart failure (HF). [Figure 22] FIG. 22 shows the correlation analysis between CDR132L concentrations in cardiac tissue (LV MI remote region) of all included animals (IVIV and ICIV) and circulating levels of miR-132-3p normalized to cel-miR-39. [Figure 23] Figure 23 shows the correlation analysis between CDR132L concentrations in cardiac tissue (LV MI remote area) of all animals included (IVIV and ICIV) and plasma levels of NT-proBNP (N-terminal pro-b-type natriuretic peptide). [Figure 24] Figure 24 shows the correlation analysis between functional miR-132-3p levels in cardiac tissue (LV MI remote region) of all included animals (IVIV and ICIV) and circulating levels of this microRNA normalized to cel-miR-39. [Figure 25] Figure 25 shows the correlation analysis between delta EF (EF improvement from day 3 to month 2) of all animals included (IVIV and ICIV) and the circulating levels of this microRNA normalized to cel-miR-39. [Figure 26] Figure 26 shows the correlation analysis between plasma levels of NT-proBNP and delta EF (improvement in EF from day 3 to month 2) for all animals included (IVIV and ICIV). [Figure 27] FIG. 27 shows the correlation analysis between plasma levels of NT-proBNP and circulating levels of miR-132-3p normalized to cel-miR-39 in all animals included (IVIV and ICIV). [Figure 28] Post-myocardial infarction HF model (135 animals, 56 days follow-up). [Figure 29] Significant changes in EF (delta EF) from day 3 to day 56 post-MI were observed in the medium- and high-dose IV / IV and IC / IV groups, indicating functional improvement. [Figure 30] In the mid- and high-dose groups, significant changes in the progression of fibrosis were observed, contributing to functional improvement. [Figure 31] Responder analysis revealed a dose-dependent response in EF improvement: 85.7% of the high-dose IVIV group demonstrated a delta EF greater than 7%, whereas only 4.6% of all placebo animals demonstrated a recovery greater than 7%. [Figure 32] Validation of the efficacy of CDR132L in a porcine model of chronic heart failure (HF) after MI. A chronic model of post-MI HF with a 6-month follow-up. [Figure 33] Significant changes in EF of >7% from 1 to 6 months post-MI were observed in all treatment groups compared with placebo. A strong correlation was observed between treatment dose and EF improvement (delta EF M6-M1). [Figure 34] Cardiac tissue distribution of CDR132L (remote to LV MI) was observed in a dose-dependent manner, with tissue concentrations of CDR132L corresponding to low functional levels of miR-132-3p. [Figure 35] CDR132L strongly improves cardiac function in a porcine model of chronic heart failure. [Figure 36]We found dose-dependent tissue exposure in cardiac tissue samples corresponding to both IV and IC administration of CDR132L (Figure 36a,b). CDR132L activity was confirmed by a reciprocal dose-dependent decrease in target miR-132 levels compared to untreated control animals. There was a strong inverse correlation between cardiac anti-miR-132 concentrations and functional miR-132 levels, independent of the route of administration (Figure 36c). The compound's half-life in myocardial tissue was calculated to be approximately 3 weeks (Figure 36d), and plasma elimination was biphasic, with a rapid alpha phase and a prolonged beta phase (Figure 36e). [Example]

[0083] Example 1 - Silencing of miR-132 expression in cardiomyocytes A quantitative in vitro assay of the miRNA inhibitory activity of a number of structurally similar compounds obtained from the anti-miR-132 library was performed. The silencing of miRNA expression was measured using TaqMan® assay. The results were quantified by quantitative real-time PCR.

[0084] This study investigated the effects of phenylephrine / isoproterenol (10 μM) on hypertrophy stimulation. The study was performed in isolated rat cardiomyocytes. Cells were incubated in standard cell culture medium for 48 hours. Test compounds were administered individually at a concentration of 100 nM. The study was performed in triplicate.

[0085] Compound CDR132L is an LNA-DNA mixmer with a phosphorothioate backbone. It was identified as the most potent compound from the anti-miR-132 structural analogue library.

[0086] The structure of CDR132L is as follows: 5'-dA * +T * dG * +G * dC * +T * dG *+T * dA * +G * dA * dC * dT * dG * +T * +T-3' (wherein dA is 2' deoxyadenosine, dG is 2' deoxyguanosine, dC is 2' deoxycytidine and T is thymidine; +T is the LNA-T building block and +G is the LNA-G building block; and * is a phosphorothioate bond).

[0087] Example 2 - Toxicity Profiling 2.1 Research purpose: Toxicity profiling of CDR132L

[0088] 2.2 Test Overview In vitro cytotoxicity in human hepatocytes (HepG2) and isolated neonatal cardiomyocytes (NRCM) A commercially available colorimetric MTT assay was used to assess cytotoxicity. After adding the compound, the cells were incubated in DMEM medium for 48 hours. Results were compared to a scrambled LNA oligonucleotide (blue) used as a control over the dose range of 0.01–100 μM. The therapeutic dose range is shown in gray.

[0089] 2.3 Results: No significant toxicity of CDR132L in HepG2 cells and NRCM was observed in the therapeutic dose range (see Figures 1A and 1B).

[0090] Example 3 - Left ventricular reverse remodeling in failing hearts by CDR132L administration in a transgenic mouse model 3.1 Test Objective: Testing the efficacy of CDR132L in reversing heart failure in a mouse model of heart failure.

[0091] 3.2 Test Overview Model of cardiac hypertrophy: transgenic (TG) mice overexpressing cardiac miR-132 (Ucar et al. 2012). Treatment: CDR132L or placebo 20 mg / kg ip weekly (see Figure 2A) Groups: wild-type (WT) littermates + placebo, WT + CDR132L, TG + placebo, TG + CDR132L. n=6 / group The expression level of miR-132 was detected by qPCR. Statistical test: unpaired t-test (Figure 2B). **p<0.01, n=6 / group.

[0092] 3.3 Results Representative echocardiographic image of the heart (Figure 3A). CDR132L reversed hypertrophy as measured by myocardial mass and diastolic volume (LVEDV) (Fig. 3B). CDR132L was expressed in most segments of the left ventricle (AB, anterior basal; AM, anterior mid; AA, anterior Improve regional contractile function in the following areas: lateral apex; PA, posterior apex; PM, midbrain; and PB, posterior base (Figure 3C).

[0093] Example 4 - Administration of CDR132L in a mouse model of post-MI heart failure 4.1 Test Objective: Testing the efficacy of CDR132L in a mouse model of post-MI heart failure

[0094] 4.2 Test Overview Myocardial infarction (MI) model mice: The coronary artery (LAD) of C57BL / 6N mice was permanently ligated (Kolk et al. , 2009). Groups: MI or sham, treated with CDR132L or placebo. Treatment: 20 mg / kg ip on days 7 and 14 after MI. Endpoint: LV function 28 days after MI. n=6-7 / group (Figure 4).

[0095] 4.3 Results CDR132L treatment ameliorates left ventricular dysfunction after MI (Fig. 5A). Load-independent parameters of contractile function were also improved (Fig. 5B) (*p<0.05). CDR132L administration also improves longitudinal strain rate (LSR), thereby reversing post-MI contractile dysfunction in individual myocardial segments in remote parts of the heart (*p<0.05) (Figure 6). CDR123L treatment has been shown to be effective in cardiac tissue, e.g., in remote (non-infarct) regions and in the peri-infarct zone. , effectively silencing miR-132 expression ( Figure 7A ). At the histological level, CDR132L reduces cardiomyocyte size in remote regions of the heart after MI. decrease (Figure 7B). At the tissue level, CDR132L reduces the expression of the cardiac stress signal ANP in the post-MI heart (Figure 7C).

[0096] Example: Testing CDR132L in a porcine model of post-5-MI heart failure 5.1 Test Objective: Demonstration of the efficacy of CDR132L in vivo in a clinically relevant post-myocardial infarction model.

[0097] 5.2 Test Setup A porcine myocardial infarction model induced by 90 minutes of ischemia (LAD occlusion) followed by reperfusion. Group: placebo or CDR132L, n=6 per group. Treatment: 0.3 mg / kg intracoronary and 0.5 mg / kg intravenous administration twice, on days 3 and 28 after MI (Figure 8). Endpoint: 8 weeks post-MI. Primary outcome measures: EF and LV remodeling.

[0098] 5.3 Results CDR132L treatment was associated with improved cardiac function as measured by end-diastolic volume, end-systolic volume, ejection fraction, and left ventricular function. As determined, this avoids maladaptive remodeling and improves function (Figure 9A-D). CDR132L treatment improves segmental contractile force in segments corresponding to viable / remote myocardium Cardiac MRI at endpoint: n = 6 / group, red: p < 0.05 (Figure 9E). CDR132L treatment was evaluated by NOGA, an endpoint electroanatomical mapping As determined, it avoids maladaptive remodeling and improves LV survival in the distal region: n=6 / group, placebo vs. CDR132L: p<0.05 (FIG. 10). CDR132L normalizes the tissue expression of pathological heart failure markers, ANP and BNP, and reduces myocardial The shift in synheavy change isoforms, i.e., the MYH7 / 6 ratio, is provided (Figure 11). At the histological level, CDR132L treatment effectively reduces cardiomyocyte hypertrophy. Representative photographic images (WGA / DAPI staining 20x) (Figure 12A) and graphic depictions (Figure 12B) of the remote LV region, n=6 / group, placebo vs. CDR132L: p<0.05.

[0099] Example 6 - Pharmacodynamic profile / target engagement of CDR132L in pigs 6.1 Test Setup Treatment: 1x, day 0, intracoronary perfusion of 0.5 mg / kg or 5 mg / kg, n=3 pigs / group, placebo vs. CDR132L: p<0.05. · qPCR tissue miRNA assay at endpoint (24 hours post-treatment).

[0100] 6.2 Results A single dose of CDR132L silenced cardiac miR-132 levels in a dose-dependent manner (Figure 13).

[0101] Example 7 - Organ Toxicity Profiling 7.1 Test Setup Treatment: 0.3 mg / kg intracoronary and 0.5 mg / kg intravenous twice, on days 3 and 28 after MI. Serial blood sampling, endpoint: 72 hours after treatment, n=6 pigs / group, placebo vs. CDR132L: p<0.05.

[0102] 7.2 Results In vivo treatment with CDR132L did not induce organ toxicity in pigs (Figure 14).

[0103] Subsequent repeated dose toxicity studies were conducted in rats and minipigs.

[0104] In a 4-week toxicity study in rats administered CDR132L at 4, 20, and 100 mg / kg intravenous bolus doses on days 1 and 28, followed by a 4-week recovery period, the "no observed adverse effect level" (NOAEL) in rats was considered to be 20 mg / kg, which corresponds to a human equivalent dose of 3.23 mg / kg body weight. In a 4-week toxicity study in minipigs, where CDR132L was administered intravenously at doses of 4, 20, and 40 mg / kg on days 1 and 28, followed by a 4-week recovery period, the "no observed effect level" (NOEL) in minipigs was considered to be 40 mg / kg, which corresponds to a human equivalent dose of 36.36 mg / kg body weight.

[0105] Example 8 - Cardiac levels of FoxO3 and SERCA2 mRNA in a mouse model of heart failure Intraperitoneal injection of either a control scrambled oligonucleotide or CDR132L Cardiac FoxO3 and Serca2 mRNA in control and miR-132TG mice treated with Levels were measured four times weekly. All values ​​represent mean ± SEM. *P < 0.05 (Figures 15A and 15B).

[0106] Example 9 - Comparison of different oligonucleotides The purpose of this study was to evaluate the therapeutic effect of the novel miR-132-3p inhibitor CDR132L according to the present invention with two comparative oligonucleotides. The two comparative oligonucleotides have the same oligonucleotide sequence and phosphorothioate backbone as CDR132L, but differ in the distribution of LNA building blocks within the molecule. CDR2u1 has two LNA building blocks at the 5' and 3' ends, while CDR301 has one LNA building block per nucleotide.

[0107] To investigate the efficacy of these treatments, different oligonucleotides were administered to neonatal rat cardiomyocytes (NRCMs), and the effects of this treatment were monitored by quantitative real-time PCR (qRT-PCR) for changes in the expression of miR-132-3p and its known target gene, FoxO3 (Forkhead box O3).

[0108] The experimental design and results are shown in Figure 16: (A) Overview of the experimental setup. Neonatal rat myocardium Cells were seeded on day 0 and treated with oligonucleotides CDR132L, CDR2u1, or CDR301 (100 nM each) on day 1. At endpoint, cells were harvested for gene expression analysis. (B) Expression levels of miR-132-3p after treatment with CDR132L, CDR2u1, or CDR301. (C) Expression levels of the miR-132-3p target gene Forkhead box O3 (FoxO3) after treatment with CDR132L, CDR2u1, or CDR301. Data are means ± SD. P values ​​for oligonucleotides vs. placebo were determined by two-tailed Student's t-test.

[0109] Treatment of NRCM with CDR132L and CDR301 significantly reduced miR-132-3p levels by 96%. On the other hand, CDR2u1 reduced miRNA expression by 30% (Figure 16A-B). Furthermore, treatment with CDR132L resulted in significant suppression of the miR-132-3p target gene Foxo3, which was not achieved by CDR2u1 or CDR301 (Figure 16C).

[0110] In summary, our data demonstrated the superior inhibitory effect of CDR132L, indicated by significantly reduced expression levels of miR-132-3p and significant suppression of its target gene FoxO3, compared with CDR2u1 and CDR301.

[0111] Example 10 - Effect of CDR132L on cardiac fibrosis The aim of this study was to evaluate the antifibrotic therapeutic effect of CDR132L in an in vivo fibrosis model.

[0112] To demonstrate the antifibrotic activity in vivo, a myocardial infarction (MI) model was used in C57BL / 6N mice with permanent left anterior descending artery (LAD) ligation. CDR132L treatment consisted of placebo (a scrambled oligoanalog of CDR132L, 20 mg / kg) and CDR132L (20 mg / kg) administered on days 7 and 14. Control groups (sham-operated mice) were treated with either placebo or CDR132L. ) and LAD-ligated mice (MI, myocardial infarction): Sham + placebo, Sham + CDR132L, MI + placebo Placebo, MI+CDR132L. n=6-7 / group. The experimental design is described in Figure 17. In vivo evaluation of the anti-fibrotic effect of CDR132L in post-MI heart failure is shown in Figure 18. Fibrosis (% of collagen deposition detected by picrosirius red (PSR) staining) and β-acyltransferase activity were significantly reduced. The expression of collagen type III alpha 1 chain (Col3a1) gene relative to collagen type III was attenuated after MI by treatment with CDR132L. Groups included control-operated mice (sham-operated mice) and LAD-ligated mice (MI), and mice were treated with either placebo (black columns) or CDR132L (white columns). Mice were treated with one of the following: Sham + placebo, Sham + CDR132L, MI + placebo, or MI + CDR132L. Statistical tests (paired t-tests) were performed between MI mice receiving placebo or CDR132L. **p<0.01, n=6-7 / group.

[0113] Histological results showed that fibrosis was attenuated after CDR132L treatment (Fig. 18A), which was confirmed at the molecular level by reduced gene expression of fibrosis markers, such as collagen type III alpha 1 chain (Col3a1) (Fig. 18B).

[0114] Example 11 - Effect of CDR132L on pulmonary and liver fibrosis The antifibrotic effects of CDR132L were tested in in vitro models of pulmonary and liver fibrosis. To this end, we used liver (human primary liver fibroblasts, HPLF, PeloBiotech) and lung (normal human primary Human primary fibroblasts derived from human lung fibroblasts (NHLF, Lonza) were stimulated with profibrotic agents and treated with CDR132L. The therapeutic effects of CDRL132L were monitored by tracking key processes within the fibrotic pathway, including proliferation rate and changes in the expression of fibrosis marker genes at endpoints. Furthermore, miR-132-3p expression was assessed to demonstrate effective CDRL132L treatment.

[0115] To determine cell proliferation, a cell proliferation ELISA (enzyme-linked immunosorbent assay) kit (provided by Roche) was used. This colorimetric assay measures the amount of ATP incorporated into newly synthesized DNA in proliferating cells. Cell proliferation can be quantified based on the measurement of incorporated BrdU (bromodeoxyuridine). The amount of incorporated BrdU is detected and quantified. The absorbance value indicates the amount of DNA synthesis. The gene expression was measured by measuring the expression levels of miR-132-3p and fibrosis markers such as collagen 1A1 (COL1A1), collagen 1A2 (COL1A2), and matrix metallopeptidase 2 (MMP2). The activity of α-glucan was assessed using quantitative real-time PCR (qRT-PCR).

[0116] Figure 19. In vitro model of liver fibrosis: (A) Overview of the experimental setup. Human primary liver Fibroblasts (provided by PeloBiotech) were seeded on day 0 and stimulated with fibrosis (normal growth factor receptor 1 (GFAP)) on day 1. (B) Expression levels of miR-132-3p after treatment with CDR132L. (C) Proliferation assessed by monitoring BrdU incorporation during DNA synthesis. BrdU reagent was added to the medium 20 hours before the end point. (D) Expression levels of fibrotic marker genes (collagen 1A1 (COL1A1), collagen 1A2 (COL1A2), and matrix metallopeptidase 2 (MMP2)). The dashed lines in (B) and (D) indicate the time points after stimulation. Expression levels in untransfected control cells are shown. Data are means ± SD. P values ​​for CDR132L vs. control were determined by two-tailed Student's t-test.

[0117] Stimulation of HPLFs with transforming growth factor beta (TGF-β) (Fig. 19A) significantly increased the expression of miR-132-3p. This compound resulted in a slight induction of fibroblast proliferation (Figure 19C) and the expression of fibrotic genes such as COL1A1, COL1A2, and MMP2 (Figure 19D).

[0118] Figure 20 depicts an in vitro pulmonary fibrosis model: (A) Overview of the experimental setup. Normal human lung Fibroblasts (provided by Lonza) were seeded on day 0 and fibrotic stimulation (fibroblast proliferation) was performed on day 1. Cell proliferation and gene expression were assessed at the endpoint. (B) Expression levels of miR-132-3p after treatment with CDR132L. (C) Proliferation was assessed by monitoring BrdU incorporation during DNA synthesis. BrdU reagent was added to the medium 20 hours before the endpoint. The dashed line in (B) represents the expression of unstimulated control cells. Levels are shown. Data are means ± SD. P values ​​for CDR132L vs. control are calculated by two-tailed Student's t-test. was determined by.

[0119] In NHLF cells, after fibrosis stimulation with high FBS (5% compared to normal growth conditions of 2% FBS) (Figure 20A), no increase in miR-132-3p was observed (Figure 20B). Nevertheless, treatment with CDR132L resulted in a significant decrease in target microRNAs (Figure 20B) and fibroblast proliferation (Figure 20C).

[0120] In summary, our data demonstrate a significant antifibrotic effect of the oligonucleotide analog CDR132L in fibroblasts derived from liver or lung and in cardiac tissue. , presumably based on the antiproliferative potential of the drug and / or its effect on the expression of extracellular matrix proteins.

[0121] Example 12 - Human Clinical Trial Protocol and Results 12.1 Protocol: Rationale Current state-of-the-art pharmacotherapy for heart failure is primarily limited to less advanced stages (New York Heart Association stages I and II; NYHA I / II). However, medications cannot prevent progression to more advanced stages (NYHA III and IV), resulting in frequent hospitalizations and a 1-year mortality rate of over 70%. (13) As a last resort, implantable pumps (left ventricular assist devices, or LVADs) and ultimately heart transplantation may be the only life-saving options for a small minority of patients with end-stage heart failure.

[0122] Thus, novel, efficient, disease-fighting therapeutics that can reduce mortality and hospital stays are urgently needed to offer patients hope of a cure. Our approach offers new opportunities to revolutionize medical practice, improve patient care, and reduce the costs of heart failure care.

[0123] The mechanism of action of CDR132L has the following important elements, making it a promising next-generation therapeutic agent for heart failure. These form the basis of the role of: a) Normalization of miR-132 levels in abnormal hearts; b) normalization of calcium signaling, contractility, and cardiac function; c) improving cardiac autophagy and homeostasis, and d) Attenuation of maladaptive cardiac remodeling.

[0124] Preclinical studies have shown that the safety profile of CDR132L is suitable for clinical development. Something has been proven.

[0125] Therefore, this study is planned to evaluate the safety, pharmacokinetics, and some pharmacodynamic parameters in patients with stable heart failure of ischemic origin (NYHA I-III) based on the significant therapeutic effect shown in clinically relevant large animal studies. This study is planned to apply a dose escalation method.

[0126] First Objective Single-agent CDR132L in patients with stable heart failure of ischemic origin (NYHA stages I, II, and III) To evaluate the safety of single and multiple doses.

[0127] Second Objective To clarify the pharmacokinetic (PK) profile of CDR132L in patients with stable heart failure of ischemic origin. To make crab.

[0128] exploratory purpose To determine the effect of CDR132L on pharmacodynamic (PD) parameters.

[0129] First endpoint The primary endpoint is the safety of CDR132L, measured by: the incidence and severity of treatment-emergent adverse events (TEAEs); the proportion of subjects with clinically significant changes in experimental safety tests (hematology, chemistry, coagulation, and urinalysis); the proportion of subjects with morphological and / or rhythm abnormalities on electrocardiogram (ECG); -Subjects with clinically significant changes in ECG time intervals (PR, QRS, QT, and QTc intervals) ratio. The proportion of subjects who experienced clinically significant changes in vital signs (systolic blood pressure, diastolic blood pressure, and pulse rate), Heart (high-sensitivity cardiac troponin T), kidney (creatinine) and liver (asparagine) Impairment of acid transaminases and alanine transaminases, and organ damage markers of intravascular decongestion (N-terminal pro-b-type natriuretic peptide) Percentage of subjects who experienced a clinically significant change in

[0130] Secondary endpoint Maximum observed plasma concentration (C max ), time to maximum plasma concentration (t max ), the area under the plasma concentration-time curve from time zero to the last detected plasma concentration (AUC 0-t ), extrapolated from time zero to infinity The area under the plasma concentration-time curve (AUC 0-inf ), blood clearance (CL), terminal elimination rate constant (λz), terminal elimination half-life (t 1 / 2 PK parameters such as volume of distribution (Vdss) were analyzed using a non-compartmental method. Got it.

[0131] Exploratory Endpoints PD parameters included the following biomarkers: microRNA 132 (miR-132) for target engagement, N-terminal pro-b-type natriuretic peptide (NT-pro-BNP) for resolving congestion, and neutrophil gelatinase-binding lipocalin (N-GELA) as a marker of cardiac remodeling. GAL), additional parameters may be required. (1) predicting response to CDR132L therapy, (2) explaining PK / PD variability of drugs, and (3) drug-drug interactions (4) biomarkers that may predict susceptibility to side effects or the occurrence of safety issues The purpose of such exploratory studies is to identify potential pathways that may impair the pharmacokinetics of CDR132L in human subjects. The goal is to develop a deeper understanding of the intrinsic and extrinsic factors that may be causing harm. This does not include any genomic (DNA) sequence of the patient.

[0132] Study design This is a Phase 1, randomized, double-blind, placebo-controlled study evaluating the safety, pharmacokinetic, and pharmacodynamic parameters of CDR132L in patients with stable heart failure of ischemic origin (NYHA stages I-III). This is a controlled study.

[0133] A maximum of 28 patients will be enrolled. The largest cohort size planned is 4. The three cohorts are listed below. Each of these cohorts will consist of a maximum of seven patients. Patients will receive a 15-minute intravenous infusion of CDR132L or placebo in a maximum 5:2 ratio. Patients will be randomly assigned to receive the treatment. Treatment 1 (n=max 7) - 0.32 mg / kg CDR132L; placebo Treatment 2 (n=max 7) - 1.00 mg / kg CDR132L; placebo Treatment 3 (n=max 7) - 3.00 mg / kg CDR132L; placebo Treatment 4 (n=max 7) - 10.00 mg / kg CDR132L; placebo.

[0134] Patients will be screened on Day 1, within 41 days prior to study initiation. Subjects will receive written information and sign an informed consent form (ICF) before screening. Subjects will be admitted to the testing room on day 1, discharged on day 4, and examined on day 27. Patients will be readmitted to the hospital on day 1 and discharged on day 31. Volunteers will receive CDR132L or placebo on day 1, readmitted on day 27 for a second dose, and will receive the second matching dose on day 28. All patients will be receiving standard of care (SoC) for heart failure of ischemic origin in accordance with the latest European guidelines (14). CDR132L or placebo will be administered as an add-on treatment to SoC treatment.

[0135] All subjects will undergo scheduled outpatient visits on days 10-14, 56, 84, and 112. Patients will be seen at the study room for a consultation. All assessments performed during the study are detailed in the Study Assessment Schedule (Tables 2 and 3). The study design features Adaptive Features ( The study will use a sentinel dosing strategy. See Section 3.3.5 for details.

[0136] Number of subjects Twenty-eight patients with stable heart failure of ischemic origin were enrolled in treatment cohorts 1, 2, 3, and 4.

[0137] Main criteria for admission C18036_CDR132L-FIH01_Clinical Study Protocol_v1.0_17Apr2019 Clinical Study Protocol Template (Version 6) 14 MAR 2019 Page 16 of 85 Body mass index (BMI) 18.0~28.0kg / m 2 Patients aged 30 to 80 years and with confirmed stable heart failure of ischemic origin were included in the study. The main exclusion criteria were: heart failure of non-ischemic origin (hypertensive heart disease, myocarditis, alcoholic cardiomyopathy, cardiac dysfunction due to rapid atrial fibrillation), current or recurrent disease; CDR132L Do not include stable cardiac dysfunction (e.g., hematological, neurological, endocrine, immunological, renal, hepatic, or gastrointestinal or other conditions) that may affect the activity, absorption, or biodistribution of the drug or affect clinical or laboratory evaluations.

[0138] Study treatment and administration method Cohort 1: 0.32 mg / kg CDR132L intravenous infusion (15 min, 20 ml) on days 1 and 28 (n=5) Cohort 2: 1.00 mg / kg CDR132L intravenous infusion (15 min, 20 ml) on days 1 and 28 (n=5) Cohort 3: 3.00 mg / kg CDR132L intravenous infusion (15 min, 20 ml) on days 1 and 28 (n=5) Cohort 4: 10.00 mg / kg CDR132L intravenous infusion (15 min, 20 ml) on days 1 and 28 (n=5)

[0139] Reference treatment and administration method Placebo was administered intravenously (15 min; 20 ml) according to CDR132L (n = 8).

[0140] Evaluation criteria -Safety analysis Safety assessments will include standard laboratory safety tests (hematology, coagulation, biochemistry, and urinalysis), vital signs (systolic blood pressure [SBP], diastolic blood pressure [DBP], respiratory rate, pulse, tympanic temperature), physical examination, 12-lead ECG (RR, PR, QRS, QT, QTcF intervals, and heart rate [HR]), telemetry, biomarker assessment, and adverse event monitoring.

[0141] -Pharmacokinetic analysis The following pharmacokinetic parameters will be calculated from the measured plasma concentrations of CDR132L: Maximum observed plasma concentration (C max ), time to maximum plasma concentration (t max ), the area under the plasma concentration-time curve from time zero to the last detected plasma concentration (AUC 0-t ), the area under the plasma concentration-time curve extrapolated from time zero to infinity (AUC 0-inf ), blood clearance (CL), terminal elimination rate constant (λz), terminal elimination half-life (t1 / 2), volume of distribution (Vdss).

[0142] -Pharmacodynamic analysis Pharmacodynamic evaluation will be exploratory and will be assessed by blood sampling to determine the concentrations of the following biomarkers: microRNA 132 (miR-132) for target engagement, N-terminal pro-b-type natriuretic peptide (NT-pro-BNP) for vascular decongestion, and neutrophil gelatinase-binding lipocalin (NGAL) as a marker of cardiac remodeling. Other parameters may also be required.

[0143] statistical methods A statistical analysis plan (SAP) containing detailed statistical methods was prepared before the database was hard locked. This plan may be updated from time to time to reflect the adaptable characteristics of the study.

[0144] -Statistical analysis of safety parameters List adverse events (AEs), vital signs, ECG parameters and laboratory data , summarized using descriptive statistics.

[0145] The number (and %) of subjects who experienced an AE will be summarized for each dose. All AEs will be listed by the system organ class (SOC) and preferred term (PT) assigned to the event using the Medical Dictionary for Regulatory Activities (MedDRA). These events will be further summarized by maximum intensity. The number of subjects who experienced a drug-related AE will also be summarized. All serious adverse events (SAEs) and / or adverse events leading to treatment interruption will be listed.

[0146] -Statistical analysis of pharmacokinetic parameters Plasma concentrations will be described and summarized by time point. PK parameters will be listed for each subject and summarized for each treatment group using descriptive statistics. max and AUC To preliminarily evaluate the dose ratio, dose-normalized plasma C max , AUC 0-t and AUC 0-inf Calculate and descriptively summarize plasma C max , AUC 0-t and AUC 0-inf A power model is fitted to the data, using log(PK parameter) as the response variable and log(dose) as the predictor variable. A slope of 1 corresponds to dose proportionality. This slope is required for a two-sided 90% confidence interval. and estimates using 95% CI.

[0147] -Statistical analysis of pharmacodynamic parameters PD parameter data will be summarized using descriptive statistics, listing absolute values ​​along with changes from baseline.

[0148] For each dose group, the relationship between plasma concentrations and PD parameters is examined by graphically displaying each PD parameter against plasma concentration. This relationship is explored using the intersection of PK and relevant PD populations. If summary statistics and graphical displays indicate a relationship between plasma concentrations and PD parameters, an appropriate statistical model can be developed to further describe this relationship.

[0149] 12.2 Patient characteristics Twenty-eight patients with stable heart failure of ischemic origin (NYHA 1-3) were randomized, double-blind, placebo-controlled Further patient characteristics were type 2 diabetes, a history of myocardial infarction, and atrial fibrillation. , arterial hypertension, percutaneous intervention and / or coronary artery bypass grafting. Left ventricular ejection fraction ranged from 31% to 56%.

[0150] Patients were receiving background treatment for comorbidities at the physician's discretion and were receiving stable treatment for their individual HF status. Most patients were receiving double / triple therapy (beta-blockers). and either an ACE inhibitor or angiotensin receptor blocker, and mineral Two patients in the placebo group and two in the Verum group were receiving Three patients in the Verum group had a biventricular pacemaker and three had an implantable cardioverter defibrillator (ICD). there was.

[0151] 12.3 Preliminary results Pharmacokinetic (PK) profile The PK profile of CDR132L in humans has been shown to be safe with no signs of accumulation. The PK profile and translatability from pigs to humans were also confirmed. maxThe high dose linearity in α and AUC allowed prediction of PK parameters at other doses (e.g., 5 mg / kg). Based on the preliminary results of the Phase Ib study, the starting dose for Phase II clinical trials was suggested to be between 3 and 10 mg / kg, with subsequent maintenance doses between 3 and 5 mg / kg. .

[0152] Targeted Engagement Circulating miR-132 concentrations in Verum patients were significantly reduced in a dose-dependent manner and remained low over time (up to the study endpoint of day 112).

[0153] ECG results Most Verum patients had abnormal ECGs at screening, many of which normalized or showed significant dose-dependent improvement (e.g., normalization of T waves, narrowing of QRS, or Absence of left bundle branch block (LBBB) and / or right bundle branch block (RBBB), normalization of R progression No patients under CDR132L treatment experienced a worsening of ECG from baseline. Based on QT and QTc data, no indication of proarrhythmic potential was found.

[0154] Pharmacodynamic (PD) parameters A positive effect on ejection fraction (EF) was observed in most treated patients. NT-proBNP, a marker of BNP, was not adversely affected by CDR132L treatment. In patients receiving the highest dose (10 mg / kg), NT-proBNP levels were significantly lower at days 28 and 122 compared to baseline. Two treatments with CDR132L (doses of 1-10 mg / kg) resulted in improved EF and / or reduced NT-proBNP levels in more than 50% of all patients. Reductions in isovolumic relaxation time (IVRT), an important marker of left ventricular relaxation, were observed in patients with EF >45%, suggesting benefit in patients with diastolic dysfunction.

[0155] Biomarkers In CDR132L-treated HF patients, the cardiac fibrosis marker procollagen type I C-terminal propeptide The levels of PICP and galectin-3 (Gal-3) were reduced, indicating anti-fibrotic effects. Furthermore, the fibrosis biomarker matrix metalloproteinase 1 (MMP-1) was positively correlated with circulating levels of miR-132 and decreased in patients in the high-dose groups (cohorts 3 and 4). At the endpoint of this study, MMP-1 concentrations were below the detection limit in Cohort 4.

[0156] Safety and tolerability No serious adverse events (SAEs) were observed. No morbid effects or safety signals were identified in vital signs or ECG. It was.

[0157] 12.4 Conclusion CDR132L was well tolerated in human patients with heart failure and showed no signs of toxicity at doses up to 10 mg / kg.

[0158] Example 13 - CDR132L therapy monitoring by quantification of circulating miR-132-3p in plasma The aim of this study was to evaluate the level of miR-132-3p in plasma as a biomarker for CDR132L therapy monitoring.

[0159] Circulating miR-132-3p was measured in plasma samples from a placebo-controlled porcine model of myocardial infarction (MI)-induced heart failure (HF) (Figure 21). Animals underwent MI and received two applications (coronary artery puncture) at 3 days and 1 month. They were subjected to different treatment schemes of CDR132L, such as intravenous / intravenous (ICIV) vs intravenous / intravenous (IVIV) and three dose levels of CDR132L (low: 1 mg / kg, medium: 5 mg / kg, or high: 10 mg / kg). It was.

[0160] In this study, serial blood sampling was performed up to month 2. In plasma samples from the endpoint, circulating miR-132-3p levels were monitored by quantitative real-time PCR (qRT-PCR) using a TaqMan probe for miR-132-3p. Data were obtained using a synthetic microRNA added during the RNA extraction procedure. Normalized with (cel-miR-39) spike-in.

[0161] Treatment with CDR132L also resulted in a dose-dependent increase in drug substance in the target organ, the heart. This resulted in a significant decrease in functional miR-132-3p in plasma samples. Figure 22 shows the correlation analysis between CDR132L concentrations in cardiac tissue (regions remote from LV MI) of all included animals (IVIV and ICIV) and circulating levels of miR-132-3p normalized to cel-miR-39. Data are for individual animals as mean ± SEM. P values ​​are based on the nonparametric two-tailed Mann-Whitney U The correlation was assessed by Pearson product-moment correlation and Spearman rank correlation (right panel). Thus, circulating miR-132-3p was strongly correlated with the concentration of CDR132L in cardiac tissue of treated animals. These data suggest that circulating miR-132-3p is involved in cardiac tissue regulation. These results indicate that this gene can be used as an indicator marker for the presence of CDR132L in tissues.

[0162] Furthermore, we investigated whether other defined biomarkers indicate the presence of CDR132L in cardiac tissue. N-terminal pro-B-type natriuretic peptide (NT-proBNP) is a potent signaling pathway for cardiac stress. It is a well-known marker and correlates with the severity of heart failure, consistent with the level of miR-132-3p.

[0163] Figure 23 shows that in the cardiac tissue (regions remote from LV MI) of all animals included (IVIV and ICIV). Correlation analysis between CDR132L concentrations in mice and plasma levels of NT-proBNP (N-terminal pro-b-type natriuretic peptide) is shown. Data are individual animals as mean ± SEM. P values ​​were evaluated by the nonparametric two-tailed Mann-Whitney U test (left panel). Correlations were evaluated by Pearson Product-moment correlation and Spearman rank correlation were performed (right panel). In the 2000-mg dose-dependent study, a decrease in NT-proBNP was observed (Figure 23). The correlation with cardiac CDR132L was not very significant.

[0164] Apart from the relationship between circulating miR-132 in plasma and cardiac CDR132L, we evaluated whether this drug substance effectively inhibits its target microRNA, miR-132-3p. Functional miR-132-3p levels were significantly reduced in cardiac tissue (remote areas of LV MI), and this reduction was observed at high doses. It was so strong.

[0165] Figure 24 shows that in the cardiac tissue (regions remote from LV MI) of all animals included (IVIV and ICIV). Functional miR-132-3p levels in the circulating miRNA normalized to cel-miR-39 were compared. Correlation analysis with the IL-10001 is shown. Data are individual animals as mean ± SEM. P values ​​were assessed by the non-parametric two-tailed Mann-Whitney U test (left panel). Correlations were performed using the IL-100011 and IL-100011. The correlation between plasma and cardiac miR-132-3p was performed using Sonn product moment correlation and Spearman rank correlation (right panel). Correlation between plasma and cardiac miR-132-3p did not show a significant negative association between the two parameters, indicating that plasma miR-132-3p levels are an indicator of the activity of CDR132L against its target miR-132-3p.

[0166] CDR132L treatment has been shown to improve cardiac function after MI. We examined whether the change in left ventricular ejection fraction (EF) (delta EF) between day 3 and month 2 after myocardial infarction, which is an improvement, corresponded to circulating miR-132-3p. A significant negative correlation was observed between both parameters, indicating that circulating miR-132-3p levels are an indicator of improved cardiac function.

[0167] Figure 25 shows the correlation analysis between delta EF (EF improvement from day 3 to month 2) of all included animals (IVIV and ICIV) and the circulating levels of this microRNA normalized to cel-miR-39. Data are individual animals as mean ± SEM. P values ​​were evaluated by the nonparametric two-tailed Mann-Whitney U test (left panel). Correlations were evaluated by Pearson product-moment correlation and spinel correlation. This was performed by Aman rank correlation (right panel).

[0168] The negative correlation was as strong as the correlation between functional improvement and the cardiac stress marker NT-proBNP. .

[0169] Figure 26 shows the correlation analysis between plasma levels of NT-proBNP and delta EF (improvement in EF from day 3 to month 2) for all animals included (IVIV and ICIV). Data are individual animals as mean ± SEM. P values ​​were evaluated by the nonparametric two-tailed Mann-Whitney U test (left panel). Correlations were performed by Pearson product-moment correlation and Spearman rank correlation (right panel).

[0170] Furthermore, low levels of circulating NT-proBNP correspond linearly to low levels of circulating miR-132-3p. .

[0171] Figure 27 shows the correlation analysis between plasma levels of NT-proBNP and circulating levels of miR-132-3p normalized to cel-miR-39 in all animals included (IVIV and ICIV). Data are presented as mean ± SEM. The P values ​​were evaluated by the nonparametric two-tailed Mann-Whitney U test (left panel). Correlations were performed by Pearson product-moment correlation and Spearman rank correlation (right panel).

[0172] Example 14 - CDR132L in Subacute Heart Failure 14.1 Test Purpose: Testing the efficacy of CDR132L in a porcine model of post-MI subacute heart failure (HF)

[0173] 14.2 Test Overview: Post-myocardial infarction HF model (135 mice, 56 days follow-up) - Slow-gaining domestic pigs (Mangalica breed) Placebo control group and three treatment groups Two treatments of 1, 5, and 10 mg / kg bw were given on days 3 and 28 (Figure 28). Intracoronary vs. intravenous administration (IC / IV) and intravenous / intravenous (IV / IV) applications. · Animals considered for data analysis: 79.

[0174] 14.3 Results Change in ejection fraction delta EF (EF day 56 - EF day 3) Note: Inclusion criteria: EF < 40% on day 3 Significant changes in EF (delta EF) from day 3 to day 56 post-MI were observed in the medium and high dose IV / IV and IC / IV groups, indicating functional improvement (Figure 29).

[0175] Correlation between circulating NT-proBNP and delta EF (Day 56 - Day 3) The increase in NT-proBNP associated with post-MI HF was reversed at 56 days in the medium- and high-dose groups. Low levels of circulating NT-proBNP are associated with improved cardiac function, as indicated by increased delta EF. NT-proBNP is a potential biomarker for target engagement. (See Example 13).

[0176] Fibrosis (%) in the area remote from LV MI at endpoint (day 56) Note: Inclusion criteria: EF < 40% on day 3 In the mid- and high-dose groups, significant changes in the progression of fibrosis were observed, contributing to functional improvement (Figure 30).

[0177] Treatment response (day 56) NOTE: Inclusion criteria: EF < 40% on day 3 Responder analysis revealed a dose-dependent response in EF improvement: 85.7% of the IVIV high-dose group demonstrated a delta EF greater than 7%, whereas only 4.6% of all placebo animals demonstrated a recovery greater than 7% (Figure 31).

[0178] 14.4 Conclusion: CDR132L inhibits Goldstein-type HF in a clinically relevant and accepted large animal model of post-MI HF. Based on cardiac MRI measurements, it effectively improves cardiac function. A dose-related relationship was observed. In the high-dose IVIV group, EF increased by 10.38% on day 56 compared to day 3 (placebo-corrected). The demonstration of the efficacy of CDR132L is of high clinical significance ( By comparison, cardiac cell transplantation increases EF by at most 3–4%.)

[0179] Example 15 - CDR132L for the treatment of chronic heart failure 15.1 Test Purpose : Testing the efficacy of CDR132L in a porcine model of post-MI chronic heart failure (HF)

[0180] 15.2 Test Overview Validation of the efficacy of CDR132L in a porcine model of chronic heart failure (HF) after MI Chronic model of post-MI HF with 6-month follow-up (Figure 32) - Slow-gaining domestic pigs (Mangalica breed) Three treatment arms Placebo x 5 times per month CDR132L x 5 times per month CDR132L x 3 times per month Dosage: 5mg / kg Route of administration: IV Animals considered for data analysis: 29

[0181] 15.3. Results: Ejection fraction (EF) Note: Inclusion criteria: EF < 40% at 1 month There was a significant change in EF of >7% from 1 to 6 months post-MI compared with placebo. This was observed in all treatment groups (Figure 33).

[0182] Responder analysis Note: Inclusion criteria: EF < 40% at 1 month A strong correlation was observed between the treatment dose and the degree of EF improvement (Delta EF M6-M1) (Figure 33). 87.5% of the treatment group demonstrated a delta EF greater than 7%, whereas only 2 of 11 placebo animals demonstrated a recovery greater than 3%.

[0183] CDR132L and miR-132-3p levels after 6 months Note: Inclusion criteria: EF < 40% at 1 month Cardiac tissue distribution of CDR132L (regions remote from LV MI) was observed in a dose-dependent manner, with tissue concentrations of CDR132L corresponding to low functional levels of miR-132-3p (Figure 34).

[0184] Left ventricular end-systolic volume (LVESV) CDR132L treatment demonstrated beneficial effects on adverse left ventricular remodeling and was significantly superior to placebo. In comparison, both treatment groups significantly attenuated post-MI LVESV enlargement during the 6-month follow-up period.

[0185] Left atrium (LA) CDR132L treatment reduced chronic atrial remodeling after MI as assessed by imaging. Volume and LA index (LA volume normalized to body surface area) were significantly reduced in both treatment groups compared with placebo.

[0186] Contractile function and rate CDR132L treatment significantly improved contractile function and force in failing hearts after MI, as assessed by invasive hemodynamic measurements at a 6-month endpoint. Analysis of load-independent parameters demonstrated improved myocardial contractility (end-systolic pressure-volume relationship and Preload recruitable stroke work was revealed, and overall This was clearly associated with improved systolic function.

[0187] Extensions CDR132L treatment significantly improved diastolic function. Global diastolic parameters (cardiac The minimum rate of change of chamber pressure (MEP) and the load-independent parameter EDPVR (end-diastolic pressure-volume relationship), a sensitive marker of cardiac stiffness and capacitance, both improved with CDR321L treatment.

[0188] 15.4 Conclusion: CDR132L robustly improves cardiac function in a porcine model of chronic heart failure. Animals treated monthly with five doses of CDR132L demonstrated a 7.14% increase in EF at 6 months (placebo-corrected). 87.5% of animals responded to treatment with an improvement in EF of more than 7%. Treatment-related adverse events No changes in hematology or clinical chemistry were observed. This finding is of great clinical importance as a treatment option for chronic heart failure.

[0189] Furthermore, monthly treatment with CDR132L significantly improved the remodeling of peritoneal fibroblasts in a model of chronic heart failure after MI. Deling, effectively improving systolic function (e.g., myocardial contractile force) and diastolic function (e.g., myocardial relaxation force).

[0190] Example 16 - Pharmacokinetic Study To further evaluate the therapeutic potential of CDR132L, we investigated the effects of CDR132L on the target tissues of our compounds. We designed a large animal pharmacokinetic (PK) study in pigs to evaluate tissue exposure and distribution in 100% POIs. While intravenous (IV) administration is the clinically preferred route of administration, many novel therapeutics rely on alternative routes of administration, such as intracoronary (IC) perfusion, as is common in cardiac gene therapy studies. We demonstrated dose-dependent changes in cardiac tissue samples corresponding to both IV and IC administration of CDR132L. We found significant tissue exposure (Fig. 36a, b). The activity of CDR132L was confirmed by a reciprocal dose-dependent decrease in target miR-132 levels compared to untreated control animals. There was a strong inverse correlation between cardiac anti-miR-132 concentrations and functional miR-132 levels, independent of the route of administration (Fig. 36c). The half-life of the compound in myocardial tissue was calculated to be approximately 3 weeks (Fig. 36d), and plasma elimination was biphasic, with a rapid alpha phase and a prolonged beta phase (Fig. 36e). References 1. Barry, SP; Townsend, PA (2010). What causes a broken heart-Molecular insights into heart failure. Int Rev Cell Mol Biol 284, 113-179. 2. Datta, SR; Brunet, A.; Greenberg, ME (1999). Cellular survival: a play in three Akts. Genes Dev. 13, 2905-2927. 3. DeBosch, BJ; Muslin, AJ (2008). Insulin signaling pathways and cardiac growth. J Mol Cell Cardiol. 44, 855-864. 4. Frescas, D.; Valenti, L.; Accili, D. (2005). Nuclear trapping of the forkhead transcription factor FoxO1 via Sirt1-dependent deacetylation promotes expression of glucogenetic genes. J Biol Chem. 280, 20589-20595. 5. Glas, D.J. (2010). PI3 kinase regulation of skeletal muscle hypertrophy and atrophy. Curr Top Microbiol Immunol. 346, 267-278. 6. Gottlieb, R.A.; Gustafsson, A.B. (2011). Mitochondrial turnover in the heart. Biochim Biophys Acta. 1813, 1295-1301. 7. Kolk, M.V.; Meyberg, D.; DenseT.; Tang-Quam, K. R.; Robbins R.C.; Reichenspurner, H.; Schrepfer. S (2009), J. Vis Exp. 32, pii: 1438. doi: 103791 / 1438 8. McMullen, J.R.; Shioi, T.; Huang, W.Y.; Zhang, L.; Tarnavski, O.; Bisping, E.; Schinke, M.; Kong, S.; Sherwood, M.C.; Brown, J. et al. (2004). The insulin-like growth factor 1 receptor induces physiological heart growth via the phosphoinositide 3-kinase (p110alpha) pathway. J Biol Chem. 279, 4782-4793. 9. Ni, Y.G.; Berenji, K.; Wang, N.; Oh, M.; Sachan, N.; Dey, A.; Cheng, J.; Lu, G.; Morris, D.J.; Castrillon, D.H. et al. (2006). Foxo transcription factors blunt cardiac hypertrophy by inhibiting calcineurin signaling. Circulation. 114, 1159-1168. 10. Ronnebaum, S.M.; Patterson, C. (2010). The foxO family in cardiac function and dysfunction. Annu Rev Physiol. 72, 81-94. 11. Skurk, C.; Izumiya, Y.; Maatz, H.; Razeghi, P.; Shiojima, I.; Sandri, M.; Sa to, K.; Zeng, L.; Schiekofer, S.; Pimentel, D. et al. (2005). The FOXO3a transcription factor regulates cardiac myocyte size downstream of AKT signaling. J Biol Chem. 280, 20814-23. 12. Ucar, A. et al. (2012), Nat. Common. 3: 1078. doi: 10.1038 / ncomms2009. 13. Grech, ED & Ramsdale, DR (2003), BMJ 326: 1259-61. 14. Ponikoskwi, P. et al. (2016), Eur. Heart J. 37: 2129-2200.

Claims

1. 1. A method for preventing or treating cardiac disorders in a human subject, comprising administering to a subject a compound of formula III: 5’-dA * +T * dG * +G * dC * +T * dG * +T * dA * +G * dA * dC * dT * dG * +T * +T-3’ wherein dA is 2' deoxyadenosine, dG is 2' deoxyguanosine, dC is 2' deoxycytidine, dT is 2' deoxythymidine, T is thymidine and G is guanosine; +T is an LNA-T building block, and +G is an LNA-G building block; and * is a phosphorothioate bond) containing an oligonucleotide comprising the sequence The oligonucleotide is administered at a dose of 0.1 to 10 mg / kg body weight per application.

2. 10. The method of claim 1, wherein the human subject has heart failure and has an implanted pump.

3. 3. The method of claim 2, wherein the implanted pump is a left ventricular assist device (LVAD).

4. The agent of claim 1, wherein the oligonucleotide is administered directly or conjugated to a heterologous moiety.

5. 5. The method of claim 1, wherein the oligonucleotide is administered in combination with: (i) at least one diuretic; (ii) at least one angiotensin-converting enzyme inhibitor; and (iii) at least one beta-blocker.

6. 6. The agent of claim 5, wherein the oligonucleotide is administered in further combination with: (iv) an angiotensin II receptor blocker, (v) an If channel inhibitor, (vi) an angiotensin receptor neprilysin inhibitor, (vii) a glucose cotransporter 2 inhibitor, (viii) a stem cell therapeutic, (ix) an anti-miRNA targeting a different pathway, or (x) an SGLT-2 inhibitor.

7. The agent according to claim 1, wherein the cardiac disorder is selected from (i) acute or subacute heart failure, (ii) chronic and / or worsening chronic heart failure, (iii) stable heart failure, (iv) less advanced or advanced heart failure, (v) NYHA stage I and / or II, NYHA stage I, II and / or III, or NYHA stage III and / or IV heart failure, and (vi) left-sided or right-sided heart failure.

8. The method of claim 7, wherein the human subject suffers from chronic and / or worsening chronic heart failure.

9. The agent according to claim 7, wherein the stable heart failure is stable heart failure of non-ischemic or ischemic origin.

10. The method according to claim 7, wherein the left-sided heart failure is selected from systolic heart failure or diastolic heart failure, or a condition associated with systolic heart failure and / or diastolic heart failure.

11. 2. The method of claim 1, wherein the oligonucleotide is administered to a human subject at a dose of 3 to 10 mg / kg body weight per application.

12. The method of claim 1, wherein the oligonucleotide is administered parenterally.

13. The agent of claim 1, wherein the oligonucleotide is administered by intravenous or subcutaneous injection.

14. The method of claim 1, wherein the oligonucleotide is administered locally.

15. The oligonucleotide comprises: - Daily administration - Every 2 days - every 3 days, and - every 4 days, 10. The method of claim 1, wherein the oligonucleotide is administered parenterally by intravenous or subcutaneous injection to a human subject in a dosage regimen selected from the group consisting of:

16. 16. The method of claim 15, wherein the oligonucleotide is administered in a weight-dependent dose.

17. 16. The method of claim 15, wherein the oligonucleotide is administered at a dose of 0.1 mg / kg to 5 mg / kg of body weight per application.

18. 16. The method of claim 15, wherein the oligonucleotide is administered in a fixed dose.

19. Oligonucleotides are administered in fixed doses of 1 mg to 5000 mg per application, with doses of 2 mg to 1000 mg 19. The agent according to claim 18, wherein the agent is administered in a fixed dose of 5 mg to 500 mg or in a fixed dose of 5 mg to 500 mg.

20. The oligonucleotide comprises: - weekly administration - Every 2 weeks - Every 3 weeks - Every 4 weeks or every month - Every 6 weeks - Every 2 months - Every 3 months - every 6 months, and - once a year, The agent according to any one of claims 1 to 14, which is administered to a human subject parenterally by intravenous or subcutaneous injection in a dosage regimen selected from the group consisting of:

21. 21. The method of claim 20, wherein the oligonucleotide is administered in a weight-dependent dose.

22. 22. The method of claim 21, wherein the oligonucleotide is administered at a dose of 3 mg / kg to 10 mg / kg of body weight per application.

23. 21. The method of claim 20, wherein the oligonucleotide is administered in a fixed dose.

24. Oligonucleotides are available in fixed doses of 1 mg to 5000 mg per application, 5 mg to 2000 mg per application, 24. The agent of claim 23, administered in a fixed dose or in a fixed dose of 10 mg to 1000 mg.

25. The method of any one of claims 1 to 24, wherein the oligonucleotide is administered in an initial dose, followed by at least one maintenance dose different from the initial dose.

26. 26. The method of claim 25, wherein the oligonucleotide is administered in one or two starting doses.

27. The oligonucleotide comprises: (i) measuring the amount of miR132 in a body fluid sample of a subject to be treated with an oligonucleotide; (ii) administering the oligonucleotide at doses and / or time intervals between individual doses determined according to the amount of miR132 measured in step (i), and administering a new dose of the oligonucleotide if the amount of miR132 in the body fluid sample is found to exceed a predetermined value; The agent according to any one of claims 1 to 14, administered by an as-needed dosing regimen comprising:

28. The agent according to any one of claims 1 to 27, wherein the amount and / or activity of the marker is measured before, during and / or after administration of the agent.

29. 29. The method of claim 28, wherein the marker is selected from BNP, ANP, myosin heavy chain isoforms, FoxO3 SERCA2, collagen deposition and / or fibrosis markers such as collagen 1A1, collagen 1A2, collagen 3A1, procollagen type I C-terminal propeptide (PICP), galectin 3 (Gal-3), and matrix metalloproteinases.

30. 30. The method of claim 29, wherein the marker is selected from NT-proBNP, MYH7 / 6 ratio, procollagen type I C-terminal propeptide (PICP), galectin-3 (Gal-3), and matrix metalloproteinase 1 (MMP-1).

31. 31. The agent according to any one of claims 1 to 30, wherein ECG parameters are measured during the course of treatment before, during, and / or after administration of the agent, and the ECG parameters are selected from measurements of QRS, T waves, left bundle branch block (LBBB) and / or right bundle branch block (RBBB), and / or R-progression.

32. 1. A method for preventing or treating a fibrotic disorder in a human subject, comprising administering to a subject a compound having a length of 16 to 20 building blocks and having formula III: 5’-dA * +T * dG * +G * dC * +T * dG * +T * dA * +G * dA * dC * dT * dG * +T * +T-3’ wherein dA is 2' deoxyadenosine, dG is 2' deoxyguanosine, dC is 2' deoxycytidine, dT is 2' deoxythymidine, T is thymidine and G is guanosine; +T is an LNA-T building block, and +G is an LNA-G building block; * is a phosphorothioate bond) containing an oligonucleotide comprising the sequence the oligonucleotide is administered in one or two initial doses, followed by at least one maintenance dose that is different from the initial doses; The oligonucleotide is administered at a dose of 0.1 to 10 mg / kg body weight per application.

33. 33. The method of claim 32, wherein the fibrotic disorder is cardiac fibrosis, liver fibrosis, or pulmonary fibrosis.

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