Cholesteryl ester transfer protein (CETP) inhibitors for use in the treatment or prevention of cardiovascular diseases and pharmaceutical compositions containing said inhibitors

Abstract

The present invention relates to a cholesteryl ester transfer protein (CETP) inhibitor for use in the treatment of an individual suffering from or at increased risk of cardiovascular disease, in particular hyperlipidemia or mixed dyslipidemia. Another aspect of the invention relates to a pharmaceutical composition for use in the treatment of an individual suffering from or at increased risk of cardiovascular disease, wherein the composition comprises a therapeutically effective amount of the CETP inhibitor.

Description

[0001] This application is a divisional application of the original application with application number 201480074940.3 and the invention title "Cholesterol ester transfer protein (CETP) inhibitor for the treatment or prevention of cardiovascular disease and a pharmaceutical composition containing said inhibitor". That original application was filed on February 5, 2014, under PCT international application PCT / NL2014 / 050068, and entered the Chinese national phase on August 4, 2016. Technical Field

[0002] This invention relates to cholesterol ester transfer protein (CETP) inhibitors for the treatment of individuals suffering from or at increased risk of cardiovascular disease, and pharmaceutical formulations comprising said CETP inhibitors, particularly hyperlipidemia or mixed dyslipidemia. Background Technology

[0003] Prospective epidemiological studies have demonstrated a strong association between low-density lipoprotein cholesterol (LDL-C) levels and cardiovascular disease (CVF) risk (1). Subsequent use of statins to lower LDL-C levels, which contribute to atherosclerosis, has resulted in significant reductions in CVD-related morbidity and mortality: each 1 mmol / L reduction in LDL-C leads to approximately a 22% decrease in CVD events and a 10% decrease in all-cause mortality (2). However, despite these impressive benefits, the remaining disease burden remains substantial, with significant implications for both individual patients and global healthcare costs (3). New therapies are needed to further reduce patients' remaining CVD risk.

[0004] A novel approach to lowering LDL-C and raising HDL-C levels is to inhibit cholesterol ester transfer protein (CETP). CETP is a plasma protein primarily secreted by the liver and adipose tissue. CETP regulates the transfer of cholesterol esters from HDL to apolipoprotein B (Apo B) particles (mainly LDL and VLDL) via triglyceride exchange, thereby reducing cholesterol levels in HDL and favoring cholesterol levels in (V)LDL. Therefore, inhibiting CETP is presumed to allow cholesterol esters to be retained as HDL-C and to reduce the levels of the Apo B fraction of cholesterol, which contributes to atherosclerosis.

[0005] Despite evidence supporting the potential of CETP inhibition in reducing cardiovascular morbidity, the clinical development of CETP inhibitors remains uncertain. The first compound to enter a phase 3 clinical trial was torcetrapib at a dose of 60 mg. Torcetrapib showed a 72% increase in HDL-C and a 25% decrease in LDL-C, but its development was subsequently halted due to safety concerns, including an unforeseen increase in cardiovascular events and mortality when used in combination with atorvastatin compared to atorvastatin alone (11).

[0006] While the mechanisms underlying these events are not fully understood, mounting evidence suggests they may be due to off-target effects of topchepus, such as increased blood pressure, electrolyte alterations (increased sodium and bicarbonate and decreased potassium), and increased aldosterone, consistent with mineralocorticoid activity (11, 12, 13, 14, 15). There is also some evidence from animal studies that topchepus increases endothelin-1 expression—which is presumed to contribute to a significant (non-significant) increase in cancer mortality in the ILLUMINATE trial (16, 17). These observations may be related to relatively high doses of topchepus.

[0007] Following this, another CETP inhibitor, dalcetrapib, entered a phase 2b clinical trial. Dacetrapib showed to be a weaker inhibitor, increasing HDL-C by 30-40% and having very little effect on LDL-C concentrations, but it did not appear to show the off-target effects of tochepib (18, 19, 20). Recently, development of dalcetrapib was also terminated due to its lack of efficacy in a phase 3 study (dose of 600 mg). The lack of efficacy may be related to its only moderate inhibition of CETP (18).

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

[0009] In ongoing Phase 3 studies, a 100 mg once-daily dose of acetrapil is being clinically evaluated, while a 130 mg once-daily dose of evacurapib is being evaluated. Such relatively high levels of active ingredient may present several challenges.

[0010] Because relatively high doses of the aforementioned CETP inhibitors must be used, solid oral dosage forms (such as tablets or capsules) will be relatively large. This leads to problems with swallowing such tablets and capsules. Alternatively, multiple smaller tablets or capsules could be used, but this negatively impacts patient compliance and cost.

[0011] Another disadvantage of using current CETP inhibitors is that the relatively high doses required to achieve CETP inhibition may result in more and more severe side effects. This can negatively impact patient health and adherence. Furthermore, the low bioavailability of known CETP inhibitors can lead to inter-individual pharmacokinetic variations. Additionally, given that known CETP inhibitors (such as acetrapib) require relatively high doses to be effective, it can take several years to clear these 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 EsterTransfer Protein Inhibitor Anacetrapib in Patients With or at High Risk for Coronary Heart Disease, available online October 4, 2013, by Antonio M. Gotto Jr.). et al .).

[0012] Therefore, there remains a need for CETP inhibitors and their pharmaceutical compositions that do not exhibit the aforementioned drawbacks, are highly effective, and are well-tolerated. Summary of the Invention

[0013] The first aspect of the present invention relates to a compound (hereinafter referred to as "Compound A") for treating individuals suffering from cardiovascular disease or with an increased risk of cardiovascular disease. Or a pharmaceutically acceptable salt thereof, wherein the dose of compound A administered to the individual is in the range of about 1 to 25 mg per day.

[0014] A second aspect of the invention relates to a pharmaceutical composition for treating an individual suffering from cardiovascular disease or with an increased risk of cardiovascular disease, said composition comprising 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 along with the pharmaceutical composition according to the invention is preferably in the range of about 1 to 25 mg daily.

[0015] 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 a dose as low as 2.5 mg once daily has been shown to achieve near-complete CETP inhibition. This is a considerably low dose compared to the amounts required for other CETP inhibitors.

[0016] Furthermore, 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 on blood pressure and heart rate, and compound A did not show any effect on serum electrolytes or aldosterone concentrations. Clinical studies also showed that compound A is not affected by food effects and does not show long-term residual effects after discontinuation of administration at the claimed dose.

[0017] A third aspect of the invention relates to a pharmaceutical composition comprising 1 to 25 mg of compound A or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient.

[0018] A fourth aspect of the invention relates to a method for preparing such compositions.

[0019] definition The term "pharmaceutical composition" as used in this article has its common meaning, referring to a pharmaceutically acceptable composition.

[0020] The term “pharmaceutically acceptable” as used herein has its common meaning and refers to compounds, materials, compositions and / or dosage forms that, within the limits of what is reasonably medically judged to be suitable for exposure to mammalian (especially human) tissues, do not have excessive toxicity, irritation, anaphylactic responses and other problematic complications beyond a reasonable benefit / risk ratio.

[0021] The term “therapeutic effective amount” as used in this article has its common meaning, referring to the amount or concentration that is effective in producing the desired effect in mammals (e.g., reducing, eliminating, treating, preventing or controlling symptoms of a disease or condition affecting mammals, especially humans).

[0022] The term "control" is intended to refer to all processes, including delaying, hindering, preventing, or stopping the progression of diseases and conditions affecting mammals. However, "control" does not necessarily mean the complete elimination of all symptoms of diseases and conditions, and is intended to include preventative treatment.

[0023] As used in this article, the term "excipient" has its common meaning and refers to a pharmaceutically acceptable ingredient typically used in pharmaceutical techniques for preparing oral dosage forms of granules, solids, or liquids.

[0024] The term “salt” as used in this article has its common meaning, including acid addition salts and base addition salts of compound A.

[0025] The term “increased risk” has its common meaning in referring to the following condition in an individual (preferably a person): an individual, male or female, has an LDL-cholesterol level greater than 2.6 mmol / L, thus exposing them to an increased risk of cardiovascular events compared to individuals with lower levels.

[0026] The term “treatment” as used in this article has its common meaning, referring to curative, remittent, and preventative treatment.

[0027] The term "cardiovascular disease" has its common meanings, including arteriosclerosis, peripheral vascular disease, hyperlipidemia, mixed dyslipidemia, beta-lipoproteinemia, hypoalpha-lipoproteinemia, hypercholesterolemia, hypertriglyceridemia, familial-hypercholesterolemia, angina pectoris, ischemia, myocardial ischemia, stroke, myocardial infarction, reperfusion injury, restenosis after angioplasty, hypertension, cerebral infarction, and stroke.

[0028] The term “unit dosage form” has its common meaning as a dosage form that can be administered to an individual (preferably a human) to exert its effect and can be easily manipulated and packaged to maintain a physically and chemically stable unit dose containing the therapeutic agent (i.e., compound A). Detailed Implementation

[0029] The first aspect of the present invention relates to a compound (hereinafter referred to as "Compound A") for treating individuals (preferably humans) suffering from cardiovascular disease or with an increased risk of cardiovascular disease. Or a pharmaceutically acceptable salt thereof, wherein the dose of compound A administered to the individual is in the range of about 1 to 25 mg per day.

[0030] This compound has been described in European patent application EP 1730152, where it is described as a CETP inhibitor along with many other CETP inhibitors. It has now been surprisingly discovered that compound A possesses exceptionally superior pharmacodynamic and pharmacokinetic properties compared to other CETP inhibitors mentioned in EP 1730152 or those used clinically. In particular, compound A exhibits unexpectedly much better bioavailability than other known CETP inhibitors. It has also been found that compound A can be effectively used clinically at relatively low doses of about 1 to 25 mg daily (preferably 1 to up to 10 mg daily, including 10 mg). 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 imply that CETP inhibitors can be effectively used at such low doses. For this, see acetrapib and evacetrapib, both of which require doses exceeding 100 mg once daily in clinical settings.

[0031] Preferably, a dose of about 5 to up to 10 mg (containing 10 mg) of compound A is used daily, or a dose of about 5 mg of compound A, about 10 mg of compound A, or about 25 mg of compound A is used.

[0032] Clinical studies have shown that, within the claimed daily dose range of approximately 1 to 25 mg, near-complete CETP inhibition, a significant increase in HDL-cholesterol concentration, and a remarkable decrease in LDL-cholesterol levels were achieved in individuals administered compound A. Clinical studies have also shown that these effects occurred after a single dose of compound A.

[0033] However, it is preferable to administer a dose of about 1 to 25 mg once daily to individuals requiring compound A over an extended period, more preferably a dose of about 5 to 10 mg once daily. Preferably, individuals requiring compound A are administered a daily dose of about 1 to 25 mg (preferably about 5 to 10 mg) for 1, 5, 10, 20, 40, 52, 100, or 200 weeks.

[0034] It is particularly preferred to administer it to individuals who need it (i.e., people with cardiovascular disease or those at increased risk of cardiovascular disease) at a dose of 1 to 25 mg daily for at least one week, preferably at least three weeks.

[0035] Clinical studies have also shown that at relatively low doses of compound A, such as approximately 1 to 25 mg daily (preferably approximately 5 to 10 mg daily), no serious adverse effects were observed. For example, no clinically significant effects were observed in blood pressure and heart rate, and compound A did not exhibit off-target effects, such as in serum electrolytes or aldosterone concentrations. It has also been shown that the claimed daily dose of compound A is not affected by food effects, and at the claimed dose, no long-term residual effects due to incomplete drug clearance were observed after discontinuation of administration.

[0036] A daily dose of approximately 1 to 25 mg of compound A (preferably approximately 5 to 10 mg) is particularly suitable for treating individuals with cardiovascular disease or 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 stroke.

[0037] Considering the dramatic reduction in CETP activity, the dramatic decrease in LDL-cholesterol plasma concentration and the significant increase in HDL-cholesterol plasma concentration, the lack of side effects and food effects, compound A at a daily dose of approximately 1 to 25 mg (preferably 1 to 10 mg) is particularly suitable for the treatment of patients with mixed dyslipidemia, hyperlipidemia, or especially primary hyperlipidemia, or patients with an increased risk of mixed dyslipidemia, hyperlipidemia, or especially primary hyperlipidemia.

[0038] In addition to compound A itself, its pharmaceutically acceptable salts may also be used. Pharmaceutically acceptable salts of compound A include its acid addition salts and base addition salts, such as preferably calcium, potassium, or sodium salts. For a review of suitable salts, see Stahl and Wermuth, “Handbook of Pharmaceutical Salts: Properties, Selection, and Use” (Wiley-VCH, Weinheim, Germany, 2002).

[0039] A pharmaceutically acceptable salt 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 it can be recovered by evaporating the solvent. The degree of ionization in the salt can vary between complete ionization and almost non-ionization.

[0040] The present invention also relates to pharmaceutically acceptable solvates of compound A, and pharmaceutical compositions comprising such solvates for treating individuals with cardiovascular disease or with an increased risk of cardiovascular disease.

[0041] The so-called "prodrug" of compound A is also within the scope of this invention. Thus, certain derivatives of compound A (which may have little or no pharmacological activity) can be converted into compound A with the desired CETP inhibitory activity upon administration to the body. Within the scope of this invention, such derivatives are referred to as "prodrugs." Prodrugs according to the invention can be manufactured, for example, by replacing suitable functional groups present in compound A with certain motifs known to those skilled in the art as "prodrug motifs" (e.g., described in H. Bundgaard's "Design of Prodrugs" (Elsevier, 1985)).

[0042] The claimed dosage of compound A is preferably administered orally to the individual in need. Preferably, compound A is administered in the form of a pharmaceutical composition. Oral administration may involve swallowing, thereby allowing the compound to enter the gastrointestinal tract. Alternatively, buccal or sublingual administration may be used, wherein compound A enters the bloodstream directly from the oral cavity. Pharmaceutical formulations (as described below) may be developed to facilitate oral administration.

[0043] A second aspect of the invention relates to a pharmaceutical composition for treating an individual suffering from or at 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.

[0044] The preferred dose of compound A administered to an individual along with the pharmaceutical composition according to the invention is about 1 to 25 mg per day (more preferably about 5 to 10 mg per day).

[0045] Alternatively, a dose of compound A of approximately 5 mg, approximately 10 mg, or approximately 25 mg may be used.

[0046] As described above, clinical studies have shown that even at relatively low doses of compound A, a dramatic decrease in CETP activity, a dramatic decrease in LDL-cholesterol plasma concentration, and a significant increase in HDL-cholesterol plasma concentration were achieved. Furthermore, no serious adverse effects were observed at such doses, no food effects were observed, and compound A showed no long-term residual effects after discontinuation of administration.

[0047] The pharmaceutical composition used according to the invention is preferably administered to individuals in need for 1, 5, 10, 20, 40, 52, 100, or 200 weeks. Particularly preferred is administration of the pharmaceutical composition to individuals in need for at least one week, preferably at least three weeks.

[0048] In a preferred embodiment of the 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, more preferably about 5 to 10 mg of compound A. Particularly preferred is the use of a solid oral dosage form (e.g., tablet or capsule) containing about 1 to 25 mg (preferably 5 to 10 mg) of compound A.

[0049] Solid oral dosage forms that can be used within the scope of this invention include, in addition to tablets and capsules, caplets, lozenges, pills, mini-tablets, pellets, beads, and granules packaged in pouches. Liquid oral dosage forms that can be used in the pharmaceutical formulations of this invention include, but are not limited to, drinks, solutions, beverages, and emulsions.

[0050] In addition to compound A, the pharmaceutical compositions used in this invention also contain excipients, which are pharmaceutically acceptable ingredients commonly used in pharmaceutical techniques for preparing orally administered granular, solid, or liquid formulations.

[0051] Examples of excipients include, but are not limited to, binders, disintegrants, lubricants, glidants, fillers, and diluents. Those skilled in the art can select one or more of the aforementioned excipients for the specific desired properties of granular and / or solid oral dosage forms without undue burden through routine experiments. The amount of each excipient used can vary within the range commonly used in the art. Techniques and excipients for formulating oral dosage forms are disclosed by way of reference in the following references incorporated herein by reference. 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 ed., Gennaro, Ed., Lippincott Williams&Wilkins (2000).

[0052] A third aspect of the invention relates to a pharmaceutical composition comprising approximately 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.

[0053] Compound A, its pharmaceutically acceptable salts, and possible prodrugs may be in the forms described above.

[0054] 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 capsule.

[0055] 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.

[0056] A fourth aspect of the invention relates to a method for preparing the pharmaceutical composition described above. The pharmaceutical composition of compound A can be prepared by means commonly known to those skilled in the art.

[0057] The invention will be further illustrated by the following non-limiting embodiments.

[0058] Example In the following examples, compound A was studied in vitro, in vitro, and clinical settings. Compound A was synthesized using the method described in International Patent Application WO2005095409.

[0059] Example 1: In vitro and isolated In vitro testing methods (a) Preparation of human plasma 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 then combined and used to prepare [the following treatment / treatment]: 3 H-labeled HDL, or stored at -80°C until used for CETP testing. Human plasma was used according to Glenn and Melton (…). Methods in enzymology The preparation method described in (263; 339-351, 1996) is as follows. 3H-labeled HDL. Plasma specific gravity was measured using a hydrometer and its density was adjusted to 1.125 g / mL by adding solid KBr. Fractions with d > 1.125 g / mL were centrifuged at 100,000 rpm for 4 hours at 12°C (rotor: 100.4, Optima TLX, Beckman), and dialyzed at 4°C for 18 hours against 4 L of Tris-saline-EDTA buffer (TSE; 50 mmol / L Tris, 150 mmol / L NaCl, 2 mmol / L EDTA, pH 7.4). [1,2- 3 [H(N)]-cholesterol (37 MBq / mL). The tubes were tightly sealed under a nitrogen gas flow and incubated at 37°C for 18 hours with gentle stirring to allow endogenous LCAT esterification of radiolabeled cholesterol. 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. The incubated plasma fraction was then centrifuged at 4°C. 3 H-labeled HDL fractions were dialyzed against 2L TSE for 18 hours. 3 The radioactivity of H-labeled HDL was counted. 3 H-labeled HDL is stored at 4°C before use.

[0060] (b) CETP test CETP activity as a result of 3 The rate of H-labeled CE transfer from donor HDL to recipient VLDL / LDL was determined. Human plasma (94 μL) was pre-incubated with the compound dissolved in DMSO (1 μL) at 37°C for 24 hours, followed by incubation at 4°C or 37°C with 5 μL of [unclear text - likely a typo, should be "incubated"]. 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 using a liquid scintillation counter. CETP activity was determined as the difference in radioactivity between samples incubated at 37°C and 4°C, as described 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 at which 50% inhibition of CETP activity was achieved (IC50) was also defined. 50 An evaluation will be conducted.

[0061] Experimental methods for in vitro testing (a) Compound administration and blood collection The experiment used Syrian golden hamsters that had undergone one week of environmental acclimatization. After fasting overnight, the animals were orally administered a suspension of the compound in 0.5% sodium carboxymethyl cellulose 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, incubated at room temperature for 15 minutes, and then centrifuged. Serum CETP activity was immediately measured.

[0062] (b) Determination of CETP activity in isolated serum 95 μL of serum was added to 5 μL of 0.1 mM sodium phosphate buffer (pH 7.0) (containing 1.5 mM 5,5'-dithiobis(2-nitrobenzoic acid)) in two 96-well V-bottom plates. One plate was incubated at 4°C and the other at 37°C. After 18 hours of incubation, each sample was mixed with 100 μL of reagent for precipitating lipoproteins containing apolipoprotein B (tungsten phosphate / MgCl2 reagent, Wako pure chemical), incubated at room temperature for 10 minutes, and then 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). Cholesterol esters (CE) were calculated by subtracting FC from TC. CETP activity was determined using the following formula: CETP activity = [CETP transfer] * / [CE value in sample incubated at 4°C] *CETP transfer = [CE value in sample incubated at 4°C] - [CE value in sample incubated at 37°C] (c) Results

[0063] Example 2: A double-blind randomized study of individuals receiving multiple doses of compound A or placebo. Research Design This clinical study was a repeated-dose study conducted in five groups of Caucasian men aged 18 to 55 years. Each individual received a single oral dose of compound A / placebo on day 1, followed by once-daily doses 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 were administered at a research center after a standard breakfast. Individuals in each dose group were allocated study treatment at a ratio of 10 compound A participants to 2 placebo participants. Blood samples were collected at intervals before each dose and throughout the study until 336 hours after the last dose for pharmacokinetic and pharmacodynamic assessments (CETP activity, CETP concentration, HDL-C, LDL-C, total cholesterol, triglycerides). Secondary pharmacodynamic endpoints (including apolipoproteins 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 up to 72 hours after the first and last doses for pharmacokinetic studies. Safety assessments were performed throughout both studies, including adverse events, blood pressure and heart rate, ECG, laboratory safety tests (including aldosterone), and physical examinations.

[0064] Analytical methods Plasma and urine concentrations of compound A were determined by calibrated liquid chromatography / MS / MS with tandem mass spectrometry. The limits of quantitation (LLQ) for both tests were 0.500 ng / mL. Plasma CETP concentrations were determined using a calibrated enzyme-linked immunosorbent assay (ELISA) with a limit of quantitation (LLQ) of 0.500 μg / mL. CETP activity was used as a measure of the concentration of compound A in plasma and urine. 3 The rate at which H-labeled CEs are transferred from donor HDL to recipient VLDL / LDL was determined. Adding H-labeled CEs to human plasma... 3H]CE-labeled HDL was incubated at 37°C for 4 hours. Non-HDL lipoproteins 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 using a Modular analyzer (Roche Diagnostics) via homogenous enzymatic colorimetric assay. Total cholesterol and triglycerides were measured using a Modular analyzer via homogenous enzymatic colorimetric assay, employing 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 Rolf Greiner Biochemica (Germany) and N-apolipoprotein standard serum from Siemens (Germany). LDL particle size was determined by gradient gel electrophoresis. HDL fractions were separated by a combined ultracentrifugation-precipitation method (β-quantification). HDL-2 and HDL-3 fractions were then separated by further ultracentrifugation. Total cholesterol, free cholesterol, triglycerides, 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 automated analyzer and calibrated using secondary standards from Roche Diagnostics (total cholesterol, triglycerides) and Diasys Diagnostics (free cholesterol, phospholipids), respectively. Calculate the esterified cholesterol (as the difference between total cholesterol and free cholesterol).

[0065] Statistical analysis The sample size for the studies was selected based on practical considerations rather than statistical power. The number of individuals in each group was considered sufficient to assess the primary objective of each study. Individuals in each group were assigned to either compound A or placebo using computer-generated random codes. Pharmacokinetic parameters were determined using non-compartmental methods with WinNonlin software version 4.1 (Pharsight Corporation, USA). Descriptive statistics were used to list and summarize all data by treatment group. In the studies, ANOVA models were used to compare the maximum percentage change from baseline in each compound A dose level with the pooled placebo. All statistical analyses were performed using SAS version 6.12 or later (SAS Institute Inc., USA).

[0066] Pharmacokinetic results In the study, plasma concentrations increased in a roughly dose-proportional manner with single doses ranging from 1 to 25 mg, although no proportionality was observed at steady state: C0.05 increased with a 25-fold increase in dose. min,ss AUC 0-tau,ss and C max,ss These represent increases of 7 times, 9 times, and 12 times, respectively. T max Dose-independent, the median was located 4 to 6 hours after administration. Variation between single-dose and multiple-dose administrations was moderate, C max C min The CV for the AUC parameter was ≤ 33%. The observed trough concentrations suggest that compound A reaches steady state within 1 to 2 weeks of daily administration. The mean terminal half-life of compound A after the last dose was 121 to 151 hours, independent of the dose. Similar half-lives were observed between single and multiple administrations of compound A, ranging from 5 to 25 mg. Compound A accumulates in a dose-dependent manner with once-daily administration, increasing approximately 6-fold at 1 mg and 2-fold at 25 mg.

[0067] Pharmacodynamic results Baseline pharmacodynamic parameters were well balanced across the treatment groups. Compound A strongly inhibited CETP activity in a dose-dependent manner following both single and repeated doses. Near-complete CETP inhibition (-92% to 99%) was observed with repeated doses of compound A at doses of 2.5, 5, 10, and 25 mg once daily (Table 1). This level of inhibition was maintained throughout the repeated doses, with the maximum effect of each dose reached within one week of the once-daily dose. The duration of inhibition after the last dose was dose-dependent, with activity approaching baseline levels two weeks after the lowest dose (1 mg), while remaining approximately 50% below baseline two weeks after doses of 10 and 25 mg. Although CETP activity decreased with increasing dose of compound A, CETP concentrations increased in a dose-dependent manner following both single and repeated doses. After three weeks of administration of compound A at doses of 10 mg and 25 mg once daily, CETP concentrations increased 2.5 to 2.8 times from baseline. CETP concentrations decreased in parallel with plasma drug concentrations. After discontinuation of compound A, concentrations remained close to baseline within 2 weeks for those given 1 mg and 5 mg of compound A, while concentrations at 2 weeks remained 1.4 times higher than baseline for those given 10 mg and 25 mg of compound A. At all compound A dose levels (1 to 25 mg), the maximum percentage changes in CETP activity and CETP concentration were statistically significant compared to placebo (p < 0.0001).

[0068] HDL-C concentrations increased in a dose-dependent manner with repeated doses. Administration of compound A at doses of 2.5 to 25 mg once daily resulted in a significant increase of approximately 96% to 140% from baseline HDL-C. With administration of compound A at doses of 2.5 to 25 mg once daily, LDL-C concentrations decreased in a dose-dependent manner, with a maximum decrease from baseline of approximately 40% to 53%. For HDL-C, the maximum percentage change from baseline at doses of compound A of 5 to 25 mg once daily was statistically significantly different from placebo (p < 0.0001), and for LDL-C, the maximum percentage change from baseline at doses of compound A of 10 to 25 mg once daily was statistically significantly different from placebo (p < 0.0001). Both HDL-C and LDL-C concentrations recovered towards baseline after discontinuation of compound A, consistent with the disappearance of CETP inhibition. A trend was observed showing increased dose-dependency for Apo A-1, Apo E, HDL2-C, and HDL3-C, and decreased dose-dependency for Apo B concentration. Variability was high for all these variables; however, the data suggested that compound A was most effective at once-daily doses of 5 to 10 mg. No dose-dependent trend was observed for Apo A2 or phospholipids, but increased dose-dependency for HDL-FC, HDL-CE, and HDL-PL and decreased dose-dependency for HDL-TG were observed across the 1 to 10 mg dose range, with no further changes at 25 mg of compound A. No notable changes were observed in LDL particle size. Furthermore, no evidence was found of any relevant effect of food, age, sex, or race on the pharmacodynamic variables.

[0069] Security Repeated doses up to 25 mg once daily were well tolerated in all individuals. No serious adverse events occurred, and no individuals 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.

[0070] Table 1. Maximum percentage change from baseline in primary pharmacodynamic variables of compound A with repeated oral doses in healthy Caucasian males.

[0071] Chemical name and structural formula of compound A

[0072] {4-[(2-{[3,5-bis(trifluoromethyl)phenyl][(2R,4S)-l-(ethoxycarbonyl)-2-ethyl-6-(trifluoromethyl)-l,2,3,4-tetrahydroquinolin-4-yl]amino}pyrimidin-5-yl)oxy]butyric acid} References 1.The Emerging Risk Factors Collaboration. Major lipids,apolipoproteins, and risk of vascular disease. JAMA 2009;302:1993-2000. 2. Cholesterol Treatment Trialists (CTT) Collaboration. Efficacy and safety of more intensive lowering of LDL cholesterol: a meta-analysis of data from 170,000 participants in 26 randomized trials. Lancet 2010;13:1670-1681. 3.Roger VL, Go AS, Lloyd-Jones DM et al . Heart disease and strokestatistics – 2012 Update: A report from the American HeartAssociation. Circulation 2012;125:e12-e230. 4.Johannsen TH, Frikke-Schmidt R, Schou J, Nordestgaard BG, Tybjærg-Hansen A. Genetic inhibition of CETP, ischemic vascular disease andmortality, and possible adverse effects. J Am Coll Cardio 2012;60:2041-2048. 5.Voight BF, Peloso GM, Orho-Melander M et al. Plasma HDL cholesteroland risk of myocardial infarction: a mendelian randomisation study. Lancet .2012;380:572-580. 6.Thompson A, Di Angelantonio E, Sarwar N, Erqou S, Saleheen D,Dullaart RPF, Keavney B, Ye Z, Danesh J. JAMA . 2008;299:2777-2788. 7.Ridker PM, Pare G, Parker AN, Zee RYL, Miletich JP, Chasman DI. Circ Cardiovasc Genet . 2009;2:26-33. 8.Okamoto H, Yonemori F, Wakitani K, Minowa T, Maeda K, Shinkai H. Acholesteryl ester transfer protein inhibitor attenuates atherosclerosis inrabbits. Nature . 2000;406:203-207. 9.Barter PJ, Rye KA. Cholesteryl ester transfer protein inhibition asa strategy to reduce cardiovascular risk. J Lipid Res . 2012;53:1755-1766. 10.Bochem AE, Kuivenhoven JA, Stroes ESG. The promise of cholesterylester transfer protein (CETP) inhibition in the treatment of cardiovasculardisease. Curr Pharm Des . 2013;19:3143-3149. 11.Barter PJ, Caulfield M, Eriksson M et al. Effects of torcetrapib inpatients at high risk for coronary events. N Engl J Med . 2007;357:21009-2122. 12.Kastelein JJP, van Leuven SI, Burgess L et al . Effect oftorcetrapib on carotid atherosclerosis in familial hypercholesterolemia. N Engl J Med . 2007;356:1620-1630. 13.Nicholls SJ, Tuzcu EM, Brennan DM, Tardif J-C, Nissen SE.Cholesteryl ester transfer protein inhibition, high-density lipoproteinraising, and progression of coronary atherosclerosis. Insights fromILLUSTRATE (Investigation of Lipid Level Management Using Coronary Ultrasoundto Assess Reduction of Atherosclerosis by CETP Inhibition and HDL Elevation). Circulation . 2008;118:2506-2514. 14.Vergeer M, Bots ML, van Leuven SI, Basart DC, Sijbrands EJ, EvansGW, Grobbee DE, Visseren FL, Stalenhoef AF, Stroes ES, Kastelein JJP.Cholesteryl ester transfer protein inhibitor torcetrapib and off-targettoxicity: pooled analysis of the rating atherosclerotic disease change byimaging with a new CETP inhibitor (RADIANCE) trials. Circulation. 2008;118:2515-2522. 15.Forrest MJ, Bloomfield D, Briscoe RJ et al . Torcetrapib-inducedblood pressure elevation is independent of CETP inhibition and is accompaniedby increasing circulating levels of aldosterone. Br J Pharmacol. 2008;154:1465-1473. 16.Simic B, Hermann M, Shaw SG et al . Torcetrapib impairs endothelialfunction in hypertension. Eur Heart J. 2012;33:1615-1624. 17.Barter PJ, Rye K-A, Beltangady MS et al . Relationship betweenatorvastatin dose and the harm caused by torcetrapib. J Lipid Res. 2012;53:2436-2442. 18.Schwartz GG, Olsson AG, Abt M et al. Effects of dalcetrapib inpatients with recent acute coronary syndrome. N Engl J Med . 2012;367:2089-2099. 19.Stein EA, Stroes ES, Steiner G, et al . Safety and tolerability ofdalcetrapib. Am J Cardiol . 2009;104:82-91. 20.Lüscher TF, Taddei S, Kaski JC, et al . Vascular effects and safetyof dalcetrapib in patients with or at risk of coronary heart disease: thedal-VESSEL randomized clinical trial. Eur Heart J. 2012;33:857-65. 21.Krishna R, Bergman AJ, Fallon et al . Multiple-dose pharmacodynamicsand pharmacokinetics of anacetrapib, a potent cholesteryl ester transferprotein (CETP) inhibitor, in healthy subjects. Clin Pharmacol Ther . 2008;84:679-683. 22.Bloomfield D, Carlson GL, Aditi Sapre BS et al . Efficacy and safetyof the cholesteryl ester transfer protein inhibitor anacetrapib asmonotherapy and coadministered with atorvastatin in dyslipidemic patients. Am Heart J . 2009;157:352-360. 23.Nicholls SJ, Brewer HB, Kastelein JJP et al . Effects of the CETPinhibitor evacetrapib administered as monotherapy or in combination withstatins on HDL and LDL cholesterol. JAMA . 2011;306:2099-2109. 24.Dansky HM, Bloomfield D, Gibbons P et al. Efficacy and safetyafter cessation of treatment with the cholesteryl ester transfer proteininhibitor anacetrapib (MK-0859) in patients with primary hypercholesterolemiaor mixed hyperlipidemia. Am Heart J. 2011;162:708-716. 25.Florvall G, Basu S, Larsson A. Apolipoprotein A1 is a strongerprognostic marker than HDL and LDL cholesterol for cardiovascular disease andmortality in elderly men. J Gerontol A Biol Sci Med Sci . 2006;61:1262-1266. 26.Walldiius G, Jungner I. Rationale for using apolipoprotein B andapolipoproteins A-1 as indicators of cardiac risk and as targets for lipid-lowering therapy. Eur Heart J. 2005;26:210-212. 27.Barter PJ, Ballantyne CM, Carmena R et al. Apo B versus cholesterolin estimating cardiovascular risk and in guiding therapy: report of thethriy-person / ten-country panel. J Intern Med . 2006;259:247-258. 28.Nordestgaard BG, Chapman MJ, Ray K et al. Lipoprotein(a) as acardiovascular risk factor: current status. Eur Heart J . 2010;31:2844-2853. 29.Kamstrup PR, Tybjaerg-Hansen A, Nordestgaard BG. Lipoprotein(a)and risk of myocardial infarction – genetic epidemiologic evidence ofcausality. Scand J Clin Lab Invest . 2011;71:87-93. 30.The Emerging Risk Factors Collaboration. Lipoprotein(a)concentration and the risk of coronary heart disease, stroke and nonvascularmortality. JAMA . 2009;302:412-423. 31.Kamstrup PR, Benn M, Tybjaerg-Hansen A, Nordestgaard BG. Extremelipoprotein(a) levels and risk of myocardial infarction in the generalpopulation: The Copenhagen city heart study. Circulation . 2008;117:176-184. 32.Thanassoulis G, Campbell CY, Owens DS et al. Genetic associationswith valvular calcification and aortic stenosis. N Engl J Med . 2013;368:503-512. 33.Jaeger BR, Richter Y, Nagel E et al. Longitudinal cohort study onthe effectiveness of lipid apheresis treatment to reduce high lipoprotein(a)levels and prevent major adverse coronary events. Nat Clin Pract Cardiovasc Med . 2009;6:229-239. 34.Krishna, Garg A, Panebianco D et al. 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Claims

1. Use of compound A of the following formula or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating individuals with cardiovascular disease or with an increased risk of cardiovascular disease. 。 2. The use according to claim 1, wherein, The treatment can reduce LDL-cholesterol plasma concentration.

3. The use according to claim 2, wherein, The treatment reduced the plasma concentration of LDL-cholesterol by 40% to 53% from baseline.

4. The use according to claim 1, wherein, The treatment can increase HDL-cholesterol plasma concentration.

5. The use according to claim 4, wherein, The treatment increased the HDL-cholesterol plasma concentration by 96% to 140% from baseline.

6. The use according to claim 1, wherein, The treatment can reduce CETP activity.

7. The use according to claim 6, wherein, The treatment reduced the CETP activity by 92% to 99% from baseline.

8. The use according to any one of claims 1-7, wherein, The drug is administered orally to the individual.

9. The use according to any one of claims 1-7, wherein, The drug is formulated as a single-unit dosage form.

10. The use according to claim 8, wherein, The drug is formulated into a solid oral dosage form.

11. The use according to claim 8, wherein, The drug is formulated into tablets or capsules.

12. The use according to any one of claims 1-7, wherein, The drug is administered orally, and wherein the dose of compound A is about 1 to 25 mg daily.

13. The use according to claim 12, wherein, The dosage of compound A is approximately 5 to 10 mg daily.

14. The use according to claim 12 or 13, wherein, The dosage of compound A is approximately 5 mg or 10 mg daily.

15. The use according to any one of claims 1-7, wherein, The drug is administered to individuals who require it for at least one week.

16. The use according to any one of claims 1-7, wherein, The drug is administered to individuals who require it for at least 3 weeks.

17. The use according to any one of claims 1-7, wherein, The drug is administered to individuals in need for 1 week, 5 weeks, 10 weeks, 20 weeks, 40 weeks, 52 weeks, 100 weeks, or 200 weeks.

18. Use of compound A of the following formula or a pharmaceutically acceptable salt thereof in the preparation of a medicament for the treatment or prevention of cardiovascular disease in individuals in need of it. 。

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