Cholesteryl ester transfer protein inhibitors and pharmaceutical compositions containing same for treatment or prevention of cardiovascular diseases

Abstract

Cholesteryl ester transfer protein inhibitors and pharmaceutical compositions containing the same for use in the treatment or prevention of cardiovascular diseases. The present invention relates to a cholesteryl ester transfer protein (CETP) inhibitor for use in the treatment of a subject suffering from or at an increased risk of cardiovascular disease, in particular hyperlipidemia or mixed dyslipidemia. Another aspect of the invention relates to a pharmaceutical composition for treating a subject suffering from or having an increased risk of cardiovascular disease, wherein the composition comprises a therapeutically effective amount of the CETP inhibitor.

Description

[0001] The present application is a divisional application of the Chinese patent application with application number 201480074940.3 and invention name “Cholesterol ester transfer protein (CETP) inhibitors and pharmaceutical compositions containing said inhibitors for treating or preventing cardiovascular diseases”. The parent application is the PCT international patent application PCT / NL2014 / 050068 filed on February 5, 2014, which entered the Chinese national phase. Technical Field

[0002] The present invention relates to inhibitors of cholesteryl ester transfer protein (CETP) and pharmaceutical preparations comprising said CETP inhibitors for use in the treatment of individuals suffering from or at increased risk of cardiovascular disease, in particular hyperlipidemia or mixed dyslipidemia. Background Art

[0003] Prospective epidemiological studies have demonstrated a strong association between low-density lipoprotein cholesterol (LDL-C) levels and cardiovascular disease (CVF) risk (1). The subsequent use of statin therapy to lower atherogenic LDL-C levels has resulted in dramatic reductions in CVD-related morbidity and mortality: each 1 mmol / L reduction in LDL-C is associated with an approximately 22% reduction in CVD events and a 10% reduction in all-cause mortality (2). Despite these impressive benefits, the remaining disease burden remains substantial, with major implications for both individual patients and global health care costs (3). New therapies are needed to further reduce patients’ residual CVD risk.

[0004] A new approach to lowering LDL-C and raising HDL-C levels is to inhibit cholesteryl ester transfer protein (CETP). CETP is a plasma protein secreted primarily by the liver and adipose tissue. CETP regulates the transfer of cholesterol esters from HDL to apolipoprotein B (Apo B)-containing particles (primarily LDL and VLDL) by exchanging triglycerides, thereby resulting in a reduction in the cholesterol content of HDL in favor of the cholesterol content of (V)LDL. Therefore, inhibition of CETP is hypothesized to allow cholesterol esters to remain in HDL-C and reduce the cholesterol content of the Apo B fraction, which would lead to atherosclerosis.

[0005] Despite evidence supporting the potential of CETP inhibition in reducing cardiovascular morbidity, clinical development of CETP inhibitors has not proceeded. The first compound to enter Phase 3 clinical trials was torcetrapib at a dose of 60 mg. Torcetrapib was shown to increase HDL-C by 72% and reduce LDL-C by 25%, but its development was subsequently discontinued due to safety concerns, including an unanticipated increase in cardiovascular events and deaths when used in combination with atorvastatin compared to atorvastatin alone (11).

[0006] Although the mechanisms of these events are not fully understood, there is growing evidence that they may be due to off-target effects of torcetrap, such as increases in blood pressure, electrolyte changes (increases in sodium and bicarbonate and decreases in potassium), and increases in aldosterone, consistent with mineralocorticoid activity (11, 12, 13, 14, 15). There is also some evidence from animal studies that torcetrap increases endothelin-1 expression, which is postulated to contribute to the apparent (but not significant) increase in cancer deaths in the ILLUMINATE trial (16, 17). These observations may be related to the relatively high dose of torcetrap administered.

[0007] After this, another CETP inhibitor, dalcetrapib, entered Phase 2b clinical trials. Dalcetrapib was shown to be a weaker inhibitor that increased HDL-C by 30-40%, had minimal effect on LDL-C concentrations, but did not appear to show the off-target effects of torcetrapib (18, 19, 20). Recently, the development of dalcetrapib was also terminated due to its lack of effect in a Phase 3 study (drug dose of 600 mg). The lack of efficacy may be related to the only moderate inhibition of CETP (18).

[0008] There are also two CETP inhibitors, anacetrapib and evacetrapib, in Phase 3 clinical trials. Data from Phase 2 studies show that both are CETP inhibitors without mineralocorticoid activity. Anacetrapib 200 mg once daily has been shown to increase HDL C by 97% and reduce LDL-C by 36% in fasting healthy individuals (21), and anacetrapib 150 mg once daily increased HDL C by 139% and reduced LDL-C by 40% in patients (22). Evacetrapib (500 mg once daily monotherapy in patients) has been shown to increase HDL-C by 129% and reduce LDL-C by 36% (23).

[0009] In ongoing Phase 3 studies, anacetrapib is being evaluated at a 100 mg dose once daily, while evacetrapib is being evaluated at a 130 mg dose once daily. Such relatively high amounts of active ingredient may lead to several problems.

[0010] Since relatively high amounts of the aforementioned CETP inhibitors must be used, solid oral dosage forms (e.g. tablets or capsules) will be relatively large. This causes problems with swallowing such tablets and capsules. Alternatively, multiple smaller tablets or capsules may be used, but this has a negative impact on patient compliance and cost.

[0011] Another disadvantage of using current CETP inhibitors is that, since relatively high dosages must be used to achieve CETP inhibition, more and stronger side effects may be caused. This may have an adverse effect on the patient's health and patient compliance. In addition, due to the low bioavailability of known CETP inhibitors, pharmacokinetic changes between individuals may be caused. In addition, given that known CETP inhibitors (such as anacetrapib) require relatively high dosages to be effective, it takes several years to clear these CETP inhibitors from the body (see The American Journal of Cardiology: Evaluation of Lipids, Drug Concentration, and Safety Parameters Following Cessation of Treatment With the Cholesteryl Ester Transfer Protein Inhibitor Anacetrapib in Patients With or at High Risk for Coronary Heart Disease Antonio M. Gotto Jr. et al., which can be browsed online on October 4, 2013).

[0012] Therefore, there is still a need to provide CETP inhibitors and pharmaceutical compositions thereof that do not show the above-mentioned disadvantages, are potent and well tolerated. Summary of the invention

[0013] A first aspect of the present invention relates to a compound (hereinafter referred to as "Compound A") for use in treating a subject suffering from cardiovascular disease or having an increased risk of cardiovascular disease.

[0014]

[0015] or a pharmaceutically acceptable salt thereof, wherein the dosage of Compound A administered to the subject is in the range of about 1 to 25 mg per day.

[0016] A second aspect of the present invention relates to a pharmaceutical composition for treating an individual suffering from cardiovascular disease or having an increased risk of cardiovascular disease, wherein the composition comprises a therapeutically effective amount of Compound A or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient. The dose of Compound A administered to an individual with the pharmaceutical composition according to the present invention is preferably in the range of about 1 to 25 mg per day.

[0017] Clinical studies have shown that Compound A is a potent CETP inhibitor. Compared to other known CETP inhibitors, only relatively low doses of Compound A are required to achieve near-complete CETP inhibition. Typically, repeated administration of Compound A at doses as low as 2.5 mg once daily has been shown to be sufficient to achieve near-complete CETP inhibition. This is a relatively low dose compared to the amounts required for other CETP inhibitors.

[0018] In addition, clinical studies have shown that compound A is well tolerated and does not cause serious side effects. For example, no clinically significant effects were observed in blood pressure and heart rate, and compound A did not show an effect on serum electrolytes or aldosterone concentrations. Clinical studies also show that compound A is not plagued by food effects and does not show long-term residual effects after dosing is stopped at the claimed dose.

[0019] The third aspect of the present invention relates to the pharmaceutical composition itself, which comprises 1 to 25 mg of Compound A or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient.

[0020] A fourth aspect of the invention relates to a method of preparing such a composition.

[0021] definition

[0022] As used herein, the term "pharmaceutical composition" has its ordinary meaning and refers to a pharmaceutically acceptable composition.

[0023] The term "pharmaceutically acceptable" as used herein has its usual meaning and refers to compounds, materials, compositions and / or dosage forms that are suitable for contact with mammalian (especially human) tissues within the range judged medically sufficient, without excessive toxicity, irritation, allergic response and other problematic complications that exceed a reasonable benefit / risk ratio.

[0024] As used herein, the term "therapeutically effective amount" has its ordinary meaning and refers to an amount or concentration effective to produce a desired effect in a mammal, such as reducing, eliminating, treating, preventing or controlling the symptoms of a disease or condition affecting a mammal, especially a human.

[0025] The term "control" is intended to refer to all processes wherein the progression of diseases and conditions affecting mammals may be slowed, hindered, arrested or stopped. However, "control" does not necessarily mean complete elimination of all symptoms of diseases and conditions, and is intended to include preventative treatment.

[0026] The term "excipient" as used herein has its ordinary meaning and refers to a pharmaceutically acceptable ingredient commonly used in the pharmaceutical technology for preparing granular, solid or liquid oral dosage formulations.

[0027] The term "salt" used herein has its usual meaning and includes acid addition salts and base addition salts of Compound A.

[0028] The term "increased risk" has its usual meaning and refers to the condition in individuals, preferably humans, wherein the respective individual, male or female, has an LDL-cholesterol level of more than 2.6 mmol / l, thereby exposing them to an increased risk of cardiovascular events compared to individuals with lower levels.

[0029] As used herein, the term "treatment" has its ordinary meaning and refers to curative, palliative and prophylactic treatment.

[0030] The term "cardiovascular disease" has its usual meaning and includes arteriosclerosis, peripheral vascular disease, hyperlipidemia, mixed dyslipidemia, beta-lipoproteinemia, hypoalphalipoproteinemia, hypercholesterolemia, hypertriglyceridemia, familial-hypercholesteremia, angina pectoris, ischemia, myocardial ischemia, stroke, myocardial infarction, reperfusion injury, restenosis after angioplasty, hypertension, cerebral infarction and cerebral stroke.

[0031] The term "unit dosage form" has its usual meaning, referring to a dosage form that can be administered to an individual (preferably a human) to effect, and can be easily handled and packaged to maintain a physically and chemically stable unit dosage containing a therapeutic agent (ie, Compound A). DETAILED DESCRIPTION

[0032] The first aspect of the present invention relates to a compound (hereinafter referred to as "Compound A") for use in treating an individual (preferably a human) suffering from cardiovascular disease or having an increased risk of cardiovascular disease.

[0033]

[0034] or a pharmaceutically acceptable salt thereof, wherein the dosage of Compound A administered to the subject is in the range of about 1 to 25 mg per day.

[0035] This compound has been described in European patent application EP 1730152, in which it is described as a CETP inhibitor along with many other CETP inhibitors. It has now been surprisingly found that compound A has extremely excellent pharmacodynamic and pharmacokinetic properties compared to other CETP inhibitors mentioned in EP 1730152 or used clinically, and in particular, compound A has unexpectedly much better bioavailability than other known CETP inhibitors. It has also been found that compound A can be effectively used clinically at a relatively low dose of about 1 to 25 mg per day (preferably 1 to up to 10 mg (including 10 mg) per day). Such doses are preferably used in the form of a pharmaceutical composition comprising compound A and an excipient. The prior art does not disclose or suggest that CETP inhibitors can be effectively used at such a low dose. In this regard, see anacetrapib and evacetrapib, which both require a once-daily dose of more than 100 mg in a clinical setting.

[0036] Preferably, a daily dose of about 5 to up to 10 mg (inclusive) of Compound A is used, or a dose of about 5 mg of Compound A, a dose of about 10 mg of Compound A or a dose of about 25 mg of Compound A is used.

[0037] Clinical studies have shown that within the claimed daily dose range of about 1 to 25 mg, near complete CETP inhibition, significant increases in HDL-cholesterol concentrations, and remarkable reductions in LDL-cholesterol levels can be achieved in individuals administered Compound A. Clinical studies have also shown that these effects occur after a single dose of Compound A.

[0038] However, it is preferred that a dose of about 1 to 25 mg once daily, preferably about 5 to 10 mg once daily, is administered to a subject in need of Compound A for an extended period of time. Preferably, a daily dose of about 1 to 25 mg (preferably about 5 to 10 mg) is administered to a subject in need of Compound A for 1, 5, 10, 20, 40, 52, 100 or 200 weeks.

[0039] It is particularly preferred to administer to an individual in need thereof (ie a person suffering from cardiovascular disease or a person at increased risk of cardiovascular disease) a dose of 1 to 25 mg per day for at least one week, preferably at least three weeks.

[0040] Clinical studies have also shown that relatively low doses of about 1 to 25 mg per day (preferably about 5 to 10 mg per day) of Compound A do not exhibit severe adverse effects. For example, no clinically significant effects were observed in blood pressure and heart rate, and Compound A did not exhibit off-target effects in, for example, serum electrolytes or aldosterone concentrations. It has also been shown that the claimed daily doses for Compound A are not plagued by food effects, and at the claimed doses, they do not exhibit long-term residual effects due to incomplete drug clearance after dosing has ceased.

[0041] A daily dose of about 1 to 25 mg of compound A (preferably a dose of about 5 to 10 mg) is particularly suitable for treating individuals suffering from cardiovascular disease or having an increased risk of cardiovascular disease, such as arteriosclerosis, peripheral vascular disease, hyperlipidemia, mixed dyslipidemia, hyperβ-lipoproteinemia, hypoα-lipoproteinemia, hypercholesterolemia, hypertriglyceridemia, familial hypercholesterolemia, angina pectoris, ischemia, myocardial ischemia, stroke, myocardial infarction, reperfusion injury, restenosis after angioplasty, hypertension, cerebral infarction, and cerebral stroke.

[0042] Taking into account the drastic reduction in CETP activity, the drastic reduction in LDL-cholesterol plasma concentration and the significant increase in HDL-cholesterol plasma concentration, the lack of side effects and food effects, a daily dose of about 1 to 25 mg (preferably 1 to 10 mg) of compound A is particularly suitable for the treatment of patients suffering from mixed dyslipidemia, hyperlipidemia or especially primary hyperlipidemia or patients with an increased risk of mixed dyslipidemia, hyperlipidemia or especially primary hyperlipidemia.

[0043] In addition to compound A itself, its pharmaceutically acceptable salts can also be used. Pharmaceutically acceptable salts of compound A include acid addition salts and base addition salts thereof, such as preferably calcium salts, potassium salts or sodium salts. For a review of suitable salts, see Stahl and Wermuth's "Handbook of Pharmaceutical Salts: Properties, Selection, and Use" (Wiley-VCH, Weinheim, Germany, 2002).

[0044] Pharmaceutically acceptable salts of Compound A can be readily prepared by mixing a solution of Compound A with a desired acid or base in a suitable manner. The salt can be precipitated from the solution and collected by filtration, or the salt can be recovered by evaporating the solvent. The degree of ionization in the salt can vary from fully ionized to almost non-ionized.

[0045] The present invention also relates to pharmaceutically acceptable solvates of Compound A, and pharmaceutical compositions comprising such solvates, for use in treating individuals suffering from cardiovascular disease or having an increased risk of cardiovascular disease.

[0046] So-called "prodrugs" of compound A are also within the scope of the present invention. Thus, certain derivatives of compound A (which may have no or only very low pharmacological activity themselves) can be converted into compound A having the desired CETP inhibitory activity when administered to the body. Within the scope of the present invention, such derivatives are referred to as "prodrugs". Prodrugs according to the present invention can be prepared, for example, by replacing appropriate functional groups present in compound A with certain motifs known to those skilled in the art as "prodrug motifs" (such as those described in "Design of Prodrugs" by H. Bundgaard (Elsevier, 1985)).

[0047] The claimed dose of Compound A is preferably administered orally to an individual in need thereof. Preferably, Compound A is administered in the form of a pharmaceutical composition. Oral administration may involve swallowing, whereby the compound enters the gastrointestinal tract. Alternatively, buccal or sublingual administration may also be utilized, where Compound A enters the bloodstream directly from the oral cavity. Pharmaceutical formulations (as described below) may be developed to facilitate oral administration.

[0048] A second aspect of the present invention relates to a pharmaceutical composition for treating an individual suffering from cardiovascular disease or having an increased risk of cardiovascular disease, wherein the composition comprises a therapeutically effective amount of Compound A or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient. Compound A and its pharmaceutically acceptable salt or prodrug may be as described above.

[0049] The dosage of Compound A to be administered to an individual with the pharmaceutical composition according to the present invention is preferably in the range of about 1 to 25 mg per day (more preferably about 5 to 10 mg per day).

[0050] Alternatively, a dose of about 5 mg of Compound A, a dose of about 10 mg of Compound A, or a dose of about 25 mg of Compound A may be used.

[0051] As already described above, clinical studies have shown that a dramatic reduction in CETP activity, a dramatic reduction in LDL-cholesterol plasma concentrations, and a significant increase in HDL-cholesterol plasma concentrations are achieved with such relatively low doses of Compound A. Furthermore, it has been shown that no serious adverse effects occur with such doses, no food effect is observed, and Compound A shows no long-term residual effects after dosing has ceased.

[0052] The pharmaceutical composition used according to the present invention is preferably administered to an individual in need thereof for 1, 5, 10, 20, 40, 52, 100 or 200 weeks. It is particularly preferred that the pharmaceutical composition is administered to an individual in need thereof for at least one week, preferably at least three weeks.

[0053] In a preferred embodiment of the present invention, the pharmaceutical composition is formulated as a single unit dosage form. The single unit dosage form is preferably a solid oral dosage form, such as a tablet or capsule. Preferably, the single unit dosage form contains about 1 to 25 mg of Compound A, preferably about 5 to 10 mg of Compound A. It is particularly preferred to use a solid oral dosage form (e.g., tablet or capsule) containing about 1 to 25 mg (preferably 5 to 10 mg) of Compound A.

[0054] Solid oral dosage forms that can be used within the scope of the present invention include, in addition to tablets and capsules, caplets, lozenges, pills, mini-tablets, pellets, beads, and granules packaged in sachets. Liquid oral dosage forms that can be used for the pharmaceutical preparations of the present invention include, but are not limited to, drinks, solutions, beverages, and emulsions.

[0055] The pharmaceutical composition used in the present invention contains, in addition to Compound A, an excipient, ie, a pharmaceutically acceptable ingredient generally used in the pharmaceutical technology for preparing granular, solid or liquid oral dosage preparations.

[0056] Examples of classification of excipients include, but are not limited to, adhesives, disintegrants, lubricants, glidants, fillers, and diluents. One or more of the aforementioned excipients can be selected by routine experiments for the specific desired properties of particles and / or solid oral dosage forms without undue burden by those of ordinary skill in the art. The amount of each excipient used can vary within the range commonly used in the art. The following references incorporated herein by reference disclose techniques and excipients for preparing oral dosage forms. See "The Handbook of Pharmaceutical Excipients", 4th edition, Rowe et al., Eds., American Pharmaceuticals Association (2003) and "Remington: The Science and Practice of Pharmacy", 20th edition, Gennaro, Ed., Lippincott Williams & Wilkins (2000).

[0057] The third aspect of the present invention relates to a pharmaceutical composition comprising about 1 to 25 mg of Compound A or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier. Preferably, the pharmaceutical composition comprises 5 to 10 mg of Compound A or a pharmaceutically acceptable salt thereof.

[0058] Compound A and its pharmaceutically acceptable salts and possible prodrugs may be in the forms described above.

[0059] Preferably, the pharmaceutical composition is formulated as a single unit dosage form as described above. More preferably, the composition is formulated as a liquid oral dosage form or a solid oral dosage form, most preferably a tablet or a capsule.

[0060] In a preferred embodiment, the pharmaceutical composition comprises a tablet or capsule containing about 1 to 25 mg (preferably 5 to 10 mg) of Compound A or a pharmaceutically acceptable salt thereof.

[0061] The fourth aspect of the present invention relates to a method for preparing the pharmaceutical composition described above. The pharmaceutical composition of compound A can be prepared by means generally known to those skilled in the art.

[0062] The present invention will be further illustrated by the following non-limiting examples.

[0063] Example

[0064] In the following examples, Compound A was studied in vitro, ex vivo and clinically. Compound A was synthesized using the method described in International Patent Application WO2005095409.

[0065] Example 1: In vitro and ex vivo

[0066] In vitro testing methods

[0067] (a) Preparation of human plasma

[0068] Human blood was obtained from healthy male volunteers using 0.1% EDTA as an anticoagulant and centrifuged at 3,000 rpm for 15 minutes at 4°C. Human plasma was pooled and then used to prepare 3 H-labeled HDL was stored at -80°C until used in the CETP assay. Human plasma was used to prepare the enzymatic activity as described by Glenn and Melton (Methods in enzymology. 263; 339-351, 1996). 3H-labeled HDL. The plasma specific gravity was measured by a hydrometer, and its density was adjusted to 1.125 g / mL by adding solid KBr. Centrifuged at 100,000 rpm for 4 hours at 12°C (rotor: 100.4, Optima TLX, Beckman), and the fraction with d>1.125 g / mL was dialyzed against 4 L of Tris-saline-EDTA buffer (TSE; 50 mmol / L Tris, 150 mmol / L NaCl, 2 mmol / L EDTA, pH 7.4) at 4°C for 18 hours. [1,2- 3 H(N)]-cholesterol (37MBq / mL). The tube was tightly sealed under N2 flow and incubated at 37°C for 18 hours with gentle stirring to allow the radiolabeled cholesterol to be esterified by endogenous LCAT. The incubated plasma fraction was adjusted to d=1.21 g / mL using solid KBr and centrifuged at 12°C for 5 hours at 100,000 rpm. 3 The H-labeled HDL fraction was dialyzed against 2 L TSE for 18 h. 3 The radioactivity of H-labeled HDL was counted. 3 H-labeled HDL was stored at 4°C until use.

[0069] (b)CETP test

[0070] CETP activity as a 3 The transfer rate of H-labeled CE from donor HDL to acceptor VLDL / LDL was measured. Human plasma (94 μL) was preincubated with the compound dissolved in DMSO (1 μL) at 37°C for 24 h and then incubated with 5 μL of DMSO at 4°C or 37°C. 3 H-labeled HDL was incubated for 4 hours. 100 μL of tungsten phosphate / MgCl2 reagent (Wako pure chemical) was added to the precipitated apoB-containing lipoprotein. After centrifugation at 3,000 rpm for 10 minutes at room temperature, the radioactivity of the supernatant was counted in a liquid scintillation counter. CETP activity was determined as the difference in radioactivity between samples incubated at 37°C and 4°C as shown below: % inhibition = 100-{dpm (DMSO at 4°C - test compound at 37°C) / dpm (DMSO at 4°C - DMSO at 37°C)}×100. The concentration that achieves 50% inhibition of CETP activity (IC 50 ) for evaluation.

[0071] In vitro experimental methods

[0072] (a) Compound administration and blood collection

[0073] The experiment was conducted using Syrian Golden hamsters that had been acclimated for one week. After fasting overnight, the animals were orally administered a suspension of the compound in 0.5% sodium carboxymethylcellulose at a volume of 10 mL / kg. Three hours after administration, blood was collected from the abdominal aorta under deep ether anesthesia. To prepare serum, the collected blood was transferred to a plastic tube containing a clot activator, allowed to stand at room temperature for 15 minutes, and centrifuged. Serum CETP activity was measured immediately.

[0074] (b) Determination of serum CETP activity in vitro

[0075] 95 μL of serum was added to 5 μL of 0.1 mM sodium phosphate-buffered saline (pH 7.0) (containing 1.5 mM 5,5'-dithio-bis(2-nitrobenzoic acid)) in two 96-well V-bottom plates. One plate was incubated at 4°C and the other at 37°C. After incubation for 18 hours, each sample was mixed with 100 μL of a reagent for precipitating lipoproteins containing apolipoprotein B (tungsten phosphate / MgCl2 reagent, Wako pure chemical), allowed to stand at room temperature for 10 minutes, and centrifuged. Total cholesterol (TC) and free cholesterol (FC) in the supernatant were measured using commercial kits (Cholesterol E-test wako and Free Cholesterol E-test wako; Wako pure chemical). Cholesteryl esters (CE) were calculated by subtracting FC from TC. CETP activity was determined by the following formula:

[0076] CETP activity = [CETP transfer]* / [CE value in 4°C incubated sample]

[0077] *CETP transfer = [CE value in the sample incubated at 4°C] - [CE value in the sample incubated at 37°C]

[0078] (c) Results

[0079]

[0080] Example 2: Double-blind randomized study of subjects receiving multiple doses of Compound A or placebo

[0081] Study Design

[0082] The clinical study is a repeated dose study conducted in 5 groups of Caucasian male individuals aged 18 to 55 years. Each individual receives a single oral dose of Compound A / placebo on Day 1, followed by a once-daily dose on Days 8 to 35 (5 mg Compound A / placebo-Group 1) or Days 8 to 28 (1, 2.5, 10 and 25 mg Compound A / placebo-Groups 2 to 5). All doses are administered at the research center after a standard breakfast. For individuals in each dose group, 10 Compound A to 2 placebos are allocated for study treatment. Blood samples for pharmacokinetic and pharmacodynamic (CETP activity, CETP concentration, HDL-C, LDL-C, total cholesterol, triglycerides) assessments are collected at intervals before each dose and throughout the study until 336 hours after the last dose. Secondary pharmacodynamic endpoints (including apolipoprotein A1, A2, B and E, HDL2-C, HDL3-C, phospholipids, HDL-free cholesterol [HDL-FC], HDL-cholesterol esters [HDL-CE], HDL-phospholipids [HDL-PL], HDL-triglycerides [HDL-TG], and LDL particle size) were measured at intervals until the last dose day. Urine was collected for pharmacokinetic studies before dosing and at intervals up to 72 hours after the first and last doses. Safety assessments were performed throughout both studies, including adverse events, blood pressure and heart rate, ECG, laboratory safety tests (including aldosterone), and physical examinations.

[0083] Analytical methods

[0084] Plasma and urine concentrations of Compound A were determined by calibrated liquid chromatography with tandem mass spectrometry (LC / MS / MS). The lower limit of quantification (LLQ) for both tests was 0.500 ng / mL. Plasma CETP concentrations were determined using a calibrated enzyme-linked immunosorbent assay (ELISA) method with a lower limit of quantification (LLQ) of 0.500 μg / mL. CETP activity was measured as a 3 The transfer rate of H]-labeled CE from donor HDL to acceptor VLDL / LDL was measured. 3H]CE-labeled HDL was incubated at 37°C for 4 hours. Non-HDL lipoproteins were precipitated and separated from HDL, and the amount of radioactivity in the supernatant was quantified. CETP activity was determined as the difference in radioactivity between samples incubated at 37°C and 4°C. HDL-C and LDL-C were measured by homogenous enzymatic colorimetric assay using a Modular analyzer (Roche Diagnostics). Total cholesterol and triglycerides were measured by homogenous enzymatic colorimetric assay using a Modular analyzer using the cholesterol oxidase peroxidase-peroxidase aminophenazonephenol [CHOP-PAP] method and the glycerol phosphate oxidase [GPO-PAP] method, respectively. ApoA1, ApoA2, ApoB and ApoE were measured by immunoturbidimetry using reagents from RolfGreiner Biochemica (Germany) and N-apolipoprotein standard serum from Siemens (Germany). LDL particle size was determined by gradient gel electrophoresis. HDL fractions were separated by combined ultracentrifugation-precipitation method (β-quantification). HDL-2 and HDL-3 fractions were then separated by further ultracentrifugation. Total cholesterol in HDL, HDL-2 and HDL-3 fractions, free cholesterol in HDL fractions, triglycerides in HDL fractions, and phospholipids in plasma and HDL fractions were measured using enzymatic methods and reagents from Diasys Diagnostics (Germany). Measurements were performed on an Olympus AU600 automatic analyzer, and secondary standards from Roche Diagnostics (total cholesterol, triglycerides) and Diasys Diagnostics (free cholesterol, phospholipids) were used for calibration, respectively. Esterified cholesterol was calculated (as the difference between total and free cholesterol).

[0085] Statistical analysis

[0086] The sample size for the study was selected based on practical considerations rather than statistical power. The number of individuals in each group was considered sufficient to evaluate the primary purpose of each study. Individuals in each group were assigned to compound A or placebo by computer-generated random codes. Pharmacokinetic parameters were determined by non-compartmental methods using WinNonlin software version 4.1 (Pharsight Corporation, USA). Descriptive statistics were used to list and summarize all data by treatment group. In the study, the ANOVA model was used to compare the maximum percentage change from baseline for each compound A dose level with the combined placebo. All statistical analyses were performed using SAS version 6.12 or higher (SAS Institute Inc. USA).

[0087] Pharmacokinetic results

[0088] In this study, plasma concentrations increased in a roughly dose-proportional manner with single doses from 1 to 25 mg, although non-proportionality was observed at steady state: for a 25-fold dose increase, C min,ss , AUC 0-tau,ss and C max,ss The increases were 7, 9 and 12 times respectively. max Independent of dose, median values ​​were between 4 and 6 hours after dose administration. Variability between single and multiple doses was moderate, C max , C min The CV of the AUC parameter was ≤33%. The trough concentration was observed to suggest that Compound A reached steady state within 1 to 2 weeks of daily dosing. The average terminal half-life of Compound A after the last dose was 121 to 151 hours, which was independent of the dose. Respectively, similar half-lives were observed between single and multiple doses of Compound A of 5 to 25 mg. Compound A accumulated in a dose-dependent manner with a once-daily dose, increasing by approximately 6 times at 1 mg and 2 times at 25 mg.

[0089] Pharmacodynamic results

[0090] Baseline pharmacodynamic parameters were well balanced between the treatment groups in the study. Compound A strongly inhibited CETP activity in a dose-dependent manner after single and repeated doses. Nearly complete CETP inhibition (-92 to 99%) was observed with repeated doses of 2.5, 5, 10 to 25 mg of compound A once daily (Table 1). This level of inhibition was maintained throughout the repeated dose period, and the maximum effect of each dose was achieved within 1 week of once-daily doses. The duration of inhibition after the last dose was dose-dependent, with activity close to baseline levels 2 weeks after the lowest dose (1 mg), and still about 50% below baseline 2 weeks after 10 and 25 mg doses. Although CETP activity decreased with the dose of compound A, CETP concentrations increased in a dose-dependent manner after single and multiple doses. After 3 weeks of once-daily doses of 10 mg and 25 mg compound A, CETP concentrations increased 2.5 to 2.8 times from baseline. CETP concentrations decreased in parallel with plasma drug concentrations. After stopping dosing of Compound A, concentrations approached baseline within 2 weeks for 1 mg and 5 mg of Compound A, while concentrations at 2 weeks were still 1.4 times higher than baseline for 10 mg and 25 mg of Compound A. The maximum percentage changes in CETP activity and CETP concentrations at all Compound A dose levels (1 to 25 mg) were statistically significantly different from placebo (p<0.0001).

[0091] HDL-C concentrations increased in a dose-dependent manner in the case of multiple doses. Administration of compound A at a dose of 2.5 to 25 mg once daily resulted in a significant increase of about 96% to 140% from baseline HDL-C. When compound A was administered at 2.5 to 25 mg once daily, LDL-C concentrations decreased in a dose-dependent manner, with a maximum decrease of about 40% to 53% from baseline. For HDL-C, in the case of a dose of 5 to 25 mg of compound A once daily, the maximum percentage change from baseline was statistically significantly different from placebo (p<0.0001), and for LDL-C, in the case of a dose of 10 to 25 mg of compound A once daily, the maximum percentage change from baseline was statistically significantly different from placebo (p<0.0001). HDL-C and LDL-C concentrations returned to baseline after stopping the administration of compound A doses, which was consistent with the disappearance of CETP inhibition. There were trends showing dose-related increases in Apo A-1, Apo E, HDL2-C, and HDL3-C and dose-related decreases in Apo B concentrations. Variability was high for all of these variables; however, the data suggest that maximal effects were achieved with once daily dosing of Compound A at 5 to 10 mg. There were no dose-dependent trends for Apo A2 or phospholipids, but dose-related increases in HDL-FC, HDL-CE, and HDL-PL and dose-related decreases in HDL-TG were seen across the 1 to 10 mg dose range, with no further changes at 25 mg Compound A. There were no notable changes in LDL particle size. In addition, no evidence of any relevant effects of food, age, sex, or race on the pharmacodynamic variables was found.

[0092] Security

[0093] Repeated doses up to 25 mg once daily were well tolerated in all subjects. There were no serious adverse events and no subjects withdrew due to adverse events. There were no clinically significant effects on blood pressure or heart rate, ECG variables, physical examinations, or laboratory safety tests. In particular, Compound A had no effect on serum electrolytes or aldosterone concentrations.

[0094] Table 1 Maximum percent changes from baseline in primary pharmacodynamic variables of Compound A following repeated oral dosing in healthy Caucasian male subjects

[0095]

[0096] Values ​​are mean (SD)

[0097] Group 1 received 5 mg of Compound A / placebo on Day 1 and Days 8-42.

[0098] Groups 2-5 received 1, 2.5, 10 and 25 mg of Compound A / placebo on Day 1 and Days 8-35.

[0099] Chemical name and structural formula of compound A

[0100]

[0101] {4-[(2-{[3,5-bis(trifluoromethyl)phenyl][(2R,4S)-1-(ethoxycarbonyl)-2-ethyl-6-(trifluoromethyl)-1,2,3,4-tetrahydroquinolin-4-yl]amino}pyrimidin-5-yl)oxy]butanoic acid}

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Claims

1. A compound of the formula (hereinafter referred to as "Compound A") or a pharmaceutically acceptable salt thereof for use in treating an individual suffering from cardiovascular disease or having an increased risk of cardiovascular disease, wherein: The dosage of Compound A administered to the subject is in the range of about 1 to 25 mg per day, 2. Compound A according to claim 1, wherein The dosage of the compound administered to an individual in need thereof is in the range of about 5 to 10 mg per day.

3. Compound A according to any one of the preceding claims, wherein The compound is administered to a subject in need thereof at a dosage of about 5 mg per day or about 10 mg per day.

4. Compound A according to any one of the preceding claims, wherein The compound is administered to a subject in need thereof for 1, 5, 10, 20, 40, 52, 100, or 200 weeks.

5. Compound A according to any one of the preceding claims, wherein The compound is administered to a subject in need thereof for at least one week, preferably at least three weeks.

6. Compound A according to any one of the preceding claims, for use in treating a subject suffering from hyperlipidemia or mixed dyslipidemia, or having an increased risk of hyperlipidemia or mixed dyslipidemia.

7. A pharmaceutical composition for treating an individual suffering from cardiovascular disease or having an increased risk of cardiovascular disease, wherein: The composition comprises: a therapeutically effective amount of the following compound (hereinafter referred to as "Compound A") or a pharmaceutically acceptable salt thereof, and pharmaceutically acceptable excipients.

8. The pharmaceutical composition according to claim 7, wherein The composition is orally administered to a subject in need thereof, and wherein the dosage of Compound A administered to the subject is in the range of about 1 to 25 mg per day.

9. The pharmaceutical composition according to claim 8, wherein The dosage of Compound A administered to a subject in need thereof is in the range of about 5 to 10 mg per day.

10. The pharmaceutical composition according to claim 8 or 9, wherein The dosage of Compound A administered to a subject in need thereof is about 5 mg per day or about 10 mg per day.

11. A pharmaceutical composition according to any preceding claim, wherein The composition is administered to an individual in need thereof for 1, 5, 10, 20, 40, 52, 100, or 200 weeks.

12. A pharmaceutical composition according to any preceding claim, wherein The composition is administered to a subject in need thereof for at least one week, preferably at least three weeks.

13. A pharmaceutical composition according to any preceding claim, wherein The pharmaceutical composition is formulated as a single unit dosage form.

14. The pharmaceutical composition according to claim 13, wherein The single unit dosage form contains about 1 to 25 mg of Compound A, preferably about 5 to 10 mg of Compound A, optionally about 5 mg or 10 mg of Compound A.

15. A pharmaceutical composition according to any preceding claim, wherein The composition is formulated as a solid oral dosage form.

16. A pharmaceutical composition according to any preceding claim, wherein The composition is formulated as a tablet or a capsule.

17. A pharmaceutical composition according to any preceding claim for use in treating a subject suffering from, or at increased risk of developing, hyperlipidemia or mixed dyslipidemia.

18. A pharmaceutical composition comprising: about 1 to 25 mg of the following compound (hereinafter referred to as "Compound A") or a pharmaceutically acceptable salt thereof, and pharmaceutically acceptable excipients.

19. The pharmaceutical composition according to claim 18, wherein The composition comprises about 5 to 10 mg of Compound A.

20. The pharmaceutical composition according to claim 18 or 19, wherein The composition comprises about 5 mg of Compound A or about 10 mg of Compound A.

21. The pharmaceutical composition according to any one of claims 18 to 20, wherein The composition is formulated as a single unit dosage form.

22. The pharmaceutical composition according to any one of claims 18 to 21, wherein The composition is formulated as a liquid oral dosage form, or as a solid oral dosage form, such as a tablet or capsule.

23. A process for preparing a pharmaceutical composition according to any one of claims 18 to 22.

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