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

Abstract

Cholesteryl ester transfer protein inhibitors for use in the treatment or prevention of cardiovascular disease and pharmaceutical compositions containing the same. The present invention relates to cholesteryl ester transfer protein (CETP) inhibitors 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] The present application is a divisional application of the Chinese patent application No. 201480074940.3, entitled "Cholesteryl ester transfer protein (CETP) inhibitors for use in the treatment or prevention of cardiovascular diseases and pharmaceutical compositions containing said inhibitors", which parent application entered the Chinese national phase from the PCT international patent application PCT / NL2014 / 050068 filed on February 05, 2014. TECHNICAL FIELD

[0002] The present application relates to cholesteryl ester transfer protein (CETP) inhibitors for use in the treatment of an individual suffering from a cardiovascular disease, in particular hyperlipidemia or mixed dyslipidemia, or having an increased risk of developing a cardiovascular disease, and pharmaceutical formulations comprising said CETP-inhibitors. BACKGROUND

[0003] Prospective epidemiological studies have shown a strong association between low-density lipoprotein-cholesterol (LDL-C) levels and the risk of cardiovascular disease (CVD) (1). Subsequent application of statin therapy to lower the aforementioned atherogenic LDL-C levels has led to a significant reduction in CVD-related morbidity and mortality: a 1 mmol / L reduction in LDL-C leads to approximately a 22% reduction in CVD events and a 10% reduction in all-cause mortality (2). But despite these impressive benefits, the residual disease burden remains substantial, with major implications for both individual patients and global health care costs (3). New therapies are needed to further reduce the residual CVD risk in patients.

[0004] A new approach to lower LDL-C and raise HDL-C levels is the inhibition of cholesteryl ester transfer protein (CETP). CETP is a plasma protein that is mainly secreted by the liver and adipose tissue. CETP mediates the transfer of cholesteryl esters from HDL to apo B-containing particles (mainly LDL and VLDL) in exchange for triglycerides, thereby resulting in a decrease in the cholesterol content of HDL and, conversely, in the cholesterol content of (V)LDL. Inhibition of CETP is therefore hypothesized to allow cholesteryl esters to remain in HDL-C and to decrease the cholesterol content of the apo B fraction that leads to atherosclerosis.

[0005] Despite evidence supporting the potential of CETP inhibition to reduce cardiovascular morbidity, the clinical development of CETP inhibitors has been less than clear. The first compound to enter phase 3 clinical trials was Torcetrapib at a dose of 60 mg. Torcetrapib showed a 72% increase in HDL-C and a 25% reduction in LDL-C, but its development was subsequently halted due to safety concerns, including an unexpected increase in cardiovascular events and deaths with atorvastatin co-administration compared to atorvastatin alone (11).

[0006] While the mechanism of these events has not been fully understood, there is increasing evidence that they can be due to off-target effects of Torcetrapib, such as increased blood pressure, electrolyte changes (increased sodium and bicarbonate and decreased potassium) and increased aldosterone, which is consistent with mineralocorticoid activity (11, 12, 13, 14, 15). There is also some evidence from animal experiments that Torcetrapib increases endothelin-1 expression, which was postulated to contribute to the apparent (non-significant) increase in cancer deaths in the ILLUMINATE trial (16, 17). These observations can be related to the relatively high dose of Torcetrapib.

[0007] Following this, another CETP inhibitor, dalcetrapib, entered phase 2b clinical trials. Dalcetrapib appears to be a weaker inhibitor, showing a 30-40% increase in HDL-C with minimal effect on LDL-C concentration, but does not appear to show the off-target effects of Torcetrapib (18, 19, 20). Recently, the development of dalcetrapib has also been terminated due to lack of efficacy in a phase 3 study (drug dose of 600 mg). The lack of potency can be related to only moderate association with CETP inhibition (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 has been shown to increase HDL-C by 97% and decrease LDL-C by 36% in healthy subjects on a fasting basis with once-daily 200 mg (21), and in patients with once-daily 150 mg (22). Evacetrapib (monotherapy in patients with once-daily 500 mg) has been shown to increase HDL-C by 129% and decrease LDL-C by 36% (23).

[0009] In an ongoing phase 3 study, anacetrapib is being clinically evaluated at a once daily dose of 100 mg, while Evacetrapib is being evaluated at a once daily dose of 130 mg. Such relatively high amounts of active ingredient can cause several problems.

[0010] Due to the fact that relatively high amounts of the aforementioned CETP inhibitors have to be used, solid oral dosage forms, such as tablets or capsules, will be relatively large. This causes problems with the swallowing of such tablets and capsules. Alternatively, smaller tablets or capsules can be used, but this has a negative impact on patient compliance and costs.

[0011] Another disadvantage of using the current CETP inhibitors is that due to the fact that relatively high doses have to be used in order to achieve CETP inhibition, more and stronger side effects can occur. This can have a negative impact on the physical well-being of the patient and on patient compliance. Furthermore, due to the fact that the bioavailability of the known CETP inhibitors is low, inter-individual pharmacokinetic variations can occur. Moreover, in view of the fact that the known CETP inhibitors, such as anacetrapib, require relatively high doses to be effective, it takes 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., October 4, 2013, available online).

[0012] Therefore, there is still a need for CETP inhibitors and pharmaceutical compositions thereof which do not show the aforementioned disadvantages, which are potent and which are well tolerated. SUMMARY

[0013] The first aspect of the present application relates to a compound (hereinafter referred to as "Compound A") for use in the treatment of an individual suffering from a cardiovascular disease or having an increased risk of developing a cardiovascular disease

[0014]

[0015] 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.

[0016] A second aspect of the present application relates to a pharmaceutical composition for use in the treatment of an individual suffering from a cardiovascular disease or having an increased risk of developing a 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 to be administered to the individual with the pharmaceutical composition according to the present application 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. In comparison to other known CETP inhibitors, only a relatively low dose of Compound A is required to achieve near complete CETP inhibition. Typically, Compound A has been shown to be sufficient to achieve near complete CETP inhibition upon repeated administration in a dose of as low as 2.5 mg once daily. This is a considerably low dose in comparison to the amounts necessary for other CETP inhibitors.

[0018] Furthermore, clinical studies have shown that Compound A is well tolerated and does not lead to severe side effects. For example, no clinically significant effects on blood pressure and heart rate were observed, and Compound A did not appear to have an influence on serum electrolyte or aldosterone concentrations. Clinical studies have also shown that Compound A is not troubled by food effects, and does not show long-term residual effects upon discontinuation of administration at the claimed doses.

[0019] A third aspect of the present application relates to the pharmaceutical composition as such, comprising 1 to 25 mg of Compound A or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient.

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

[0021] Definitions

[0022] The term "pharmaceutical composition" as used herein has its usual meaning and denotes a pharmaceutically acceptable composition.

[0023] The term "pharmaceutically acceptable" as used herein has its usual meaning and denotes a compound, material, composition, and / or dosage form that is, within the scope of sound medical judgment, suitable for use in contact with the tissues of a mammal, especially a human, without excessive toxicity, irritation, allergic response, and other problematological complications commensurate with a reasonable benefit / risk ratio.

[0024] The term "therapeutically effective amount" as used herein has its usual meaning and denotes an amount or concentration effective for producing a desired effect in a mammal, for example, reducing, eliminating, treating, preventing or controlling the symptoms of a disease or condition affecting a mammal, preferably a human.

[0025] The term "controlling" is intended to mean all processes in which there can be a delay, hindering, stopping or halting of the progress of a disease or condition affecting a mammal. However, "controlling" does not necessarily mean complete elimination of all symptoms of the disease or condition and is intended to include prophylactic treatment.

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

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

[0028] The term "increased risk" has its usual meaning and denotes a condition in an individual, preferably a human, wherein each individual, male or female, has an LDL-cholesterol level of more than 2.6 mmol / l, so that they are exposed to an increased risk of a cardiovascular event compared to individuals having a lower level.

[0029] The term "treatment" as used herein has its usual meaning and denotes 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 and denotes a dosage form which can be administered to an individual, preferably a human, to exert an effect and which can be easily handled and packaged, maintaining a physically and chemically stable unit dose comprising a therapeutic agent, i.e. Compound A. DETAILED DESCRIPTION

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

[0033]

[0034] or pharmaceutically acceptable salts thereof, wherein the dose of Compound A administered to the individual is in the range of about 1 to 25 mg per day.

[0035] The compound has been described in European Patent Application EP 1730152, where it is described as a CETP inhibitor together with many other CETP inhibitors. It has now surprisingly been found that Compound A has extremely superior pharmacodynamic and pharmacokinetic properties compared to other CETP inhibitors mentioned in EP 1730152 or used clinically. In particular, Compound A has a much better bioavailability than other known CETP inhibitors. It has also been found that Compound A can be used clinically effectively 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 excipients. The prior art does not disclose or suggest that a CETP inhibitor can be used effectively at such low doses. In this respect, see anacetrapib and evacetrapib, which both require a once daily dose of more than 100 mg in a clinical setting.

[0036] Preferably, a dose of about 5 to up to 10 mg (including 10 mg) of Compound A per day 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 dose range of about 1 to 25 mg per day, near complete CETP inhibition, a significant increase in HDL-cholesterol concentration and a remarkable decrease in LDL-cholesterol levels can be achieved in individuals administered with Compound A. Clinical studies have also shown that these effects occur already after a single dose of Compound A.

[0038] However, it is preferred to administer a dose of about 1 to 25 mg per day, preferably a dose of about 5 to 10 mg per day, once daily, to an individual in need of Compound A for an extended period of time. Preferably, an individual in need of Compound A is 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.

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

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

[0041] The dose of about 1 to 25 mg, preferably about 5 to 10 mg per day of Compound A is particularly suitable for the treatment of individuals suffering from or at increased risk of cardiovascular diseases, such as arteriosclerosis, peripheral vascular disease, hyperlipidemia, combined dyslipidemia, hyperbetalipoproteinemia, hypoalphalipoproteinemia, hypercholesterolemia, hypertriglyceridemia, familial hypercholesterolemia, angina pectoris, ischemia, myocardial ischemia, stroke, myocardial infarction, reperfusion injury, restenosis after angioplasty, hypertension, cerebral infarction, cerebral stroke.

[0042] In view of the strong reduction of CETP activity, the strong reduction of LDL-cholesterol plasma concentration and the significant increase of HDL-cholesterol plasma concentration, the lack of side effects and food effects, Compound A in a daily dose of about 1 to 25 mg, preferably 1 to 10 mg is particularly suitable for the treatment of patients suffering from or at increased risk of combined dyslipidemia, hyperlipidemia or, in particular, primary hyperlipidemia.

[0043] In addition to Compound A itself, pharmaceutically acceptable salts thereof can also be used. Pharmaceutically acceptable salts of Compound A include acid addition salts and base addition salts thereof, for example, preferably calcium, potassium or sodium salts. For an overview of suitable salts see "Handbook of Pharmaceutical Salts: Properties, Selection, and Use" by Stahl and Wermuth (Wiley-VCH, Weinheim, Germany, 2002).

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

[0045] The present application also relates to pharmaceutically acceptable solvates of Compound A, as well as pharmaceutical compositions comprising such solvates, for use in the treatment of an individual suffering from, or at increased risk of, a cardiovascular disease.

[0046] So-called "prodrugs" of Compound A are also within the scope of the present application. Thus, certain derivatives of Compound A which can have little or no pharmacological activity themselves can, when administered into or onto the body, be converted into the active Compound A. Such derivatives are referred to as "prodrugs". The prodrugs of the present application can be formed according to methods known to those skilled in the art for the formulation of such derivatives, e.g. by replacing appropriate functionalities with groups known to be hydrolyzed under

[0047] The dosage of Compound A claimed is preferably administered orally to an individual in need thereof. Preferably, Compound A is administered in the form of a pharmaceutical composition. Oral administration can involve swallowing, in which case the compound would be ingested through the mouth and passed into the stomach. Alternatively, oral administration can involve sublingual or buccal administration, in which case the compound would be placed in the mouth, allowed to pass into the blood stream via the blood vessels under the tongue, and bypass the stomach altogether. Pharmaceutical formulations (described below) can be developed to facilitate oral administration.

[0048] A second aspect of the present application relates to a pharmaceutical composition for use in the treatment of an individual suffering from, or at increased risk of, a cardiovascular disease, wherein said 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 salts or prodrugs can be as described hereinbefore.

[0049] The dosage of Compound A to be administered to an individual in accordance with the present application 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 dosage of about 5 mg of Compound A, a dosage of about 10 mg of Compound A or a dosage of about 25 mg of Compound A can be used.

[0051] As already described hereinbefore, clinical studies have shown that with such relatively low dosages of Compound A, a strong reduction of CETP activity, a strong reduction of LDL-cholesterol plasma concentration and a significant increase of HDL-cholesterol plasma concentration are achieved. Furthermore, it has been shown that with such dosages no severe adverse effects occur, no food effect is observed and Compound A shows no long-term residual effects after the administration has been stopped.

[0052] The pharmaceutical composition used according to the present application 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 application, 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 a capsule. Preferably, the single unit dosage form comprises about 1 to 25 mg of Compound A, preferably about 5 to 10 mg of Compound A. It is particularly preferred that a solid oral dosage form (such as a tablet or a capsule) comprising about 1 to 25 mg (preferably 5 to 10 mg) of Compound A is used.

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

[0055] The pharmaceutical composition used in the present application comprises, in addition to Compound A, excipients, i.e. pharmaceutically acceptable ingredients that are commonly used in pharmaceutical technology for the preparation of granular, solid or liquid oral dosage formulations.

[0056] Examples of categories of excipients include, but are not limited to, binders, disintegrants, lubricants, glidants, fillers and diluents. One of ordinary skill in the art can select one or more of the foregoing excipients for a particular desired property of the granular and / or solid oral dosage form without undue burden of experimentation by routine experimentation. The amount of each excipient used can vary within ranges commonly used in the art. Techniques and excipients for formulating oral dosage forms are disclosed in the following references, which are incorporated herein by reference. See "The Handbook of Pharmaceutical Excipients", 4thEdition, Rowe et al., Eds., American Pharmaceuticals Association (2003) and "Remington: The Science and Practice of Pharmacy", 20thEdition, Gennaro, Ed., Lippincott Williams & Wilkins (2000).

[0057] The third aspect of the application relates to a pharmaceutical composition per se 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 can be in the form as described hereinbefore.

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

[0060] In a preferred embodiment, the pharmaceutical composition comprises a tablet or a capsule comprising 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 application relates to a method of preparing the pharmaceutical composition as described hereinbefore. The pharmaceutical composition of Compound A can be prepared by means generally known to the person skilled in the art.

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

[0063] Examples

[0064] In the following examples, Compound A was investigated in an in vitro test, ex vivo and in a clinical manner. Compound A was synthesized using the methods described in international patent application WO2005095409.

[0065] Example 1: In vitro and ex vivo

[0066] Experimental methods for in vitro testing

[0067] (a) Preparation of human plasma

[0068] Human blood was obtained from healthy male volunteers using 0.1% EDTA as anticoagulant, centrifuged at 3,000 rpm for 15 minutes at 4°C. Human plasma was pooled and then used to prepare in vitro incubations. 33H-labeled HDL, or stored at -80°C until used for CETP testing. 3H-labeled HDL was prepared using human plasma as described by Glenn and Melton (Methods in enzymology. 263; 339-351, 1996). The plasma specific gravity was measured using a hydrometer and 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 tube was tightly sealed under a nitrogen gas 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. The esterified cholesterol was then... 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.

[0069] (b) CETP testing

[0070] 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, and then incubated with 5 μL of the compound at 4°C or 37°C. 3H-labeled HDL was incubated for 4 hours. To the precipitated apoB-containing lipoprotein, 100 μL of tungstate / MgCl2 reagent (Wako pure chemical) was added. 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 follows: % inhibition = 100 - {dpm (DMSO at 4°C - test compound at 37°C) / dpm (DMSO at 4°C - DMSO at 37°C)} x 100. The concentration (IC50) at which 50% inhibition of CETP activity was achieved was evaluated. 50

[0071] Experimental methods for ex vivo testing

[0072] (a) Compound administration and blood collection

[0073] Experiments were performed using Syrian Golden hamsters that were acclimated for 1 week. After a night of fasting, animals were orally administered a suspension of the compound in 0.5% sodium carboxymethylcellulose at a volume of 10 mL / kg. At 3 hours after administration, blood was collected from the abdominal aorta under deep ether anesthesia. To prepare serum, the collected blood was transferred to plastic tubes containing a clot activator, left at room temperature for 15 minutes, and centrifuged. Serum CETP activity was determined immediately.

[0074] (b) Measurement of ex vivo serum CETP activity

[0075] Ninety-five μL of serum was added to two 96-well V-bottom plates of 5 μL of 0.1 mM sodium phosphate buffer saline (pH 7.0) containing 1.5 mM 5,5'-dithio-bis(2-nitrobenzoic acid). 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 apoB-containing lipoprotein (tungstate / MgCl2 reagent, Wako pure chemical), left 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). Cholesterol ester (CE) was calculated by subtracting FC from TC. CETP activity was determined by the following equation:

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

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

[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 was a repeat-dose study in 5 groups of 18 to 55 year old Caucasian male individuals. 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 the study center after a standard breakfast. Within each dose group, individuals were assigned in a 10 Compound A to 2 placebo ratio to be on study treatment. Blood samples for pharmacokinetic and pharmacodynamic (CETP activity, CETP concentration, HDL-C, LDL-C, total cholesterol, triglycerides) assessments were collected prior to each dose administration and at intervals throughout the study until 336 hours after the last dose administration. Secondary pharmacodynamic endpoints, including apolipoprotein Al, A2, B, and E, HDL2-C, HDL3-C, phospholipids, HDL-free cholesterol [HDL-FC], HDL-cholesterol ester [HDL-CE], HDL-phospholipid [HDL-PL], HDL-triglyceride [HDL-TG], and LDL particle size, were measured at intervals until the last dose administration day. Urine was collected for pharmacokinetic studies prior to dose administration and at intervals until 72 hours after the first and last dose administration. Safety assessments, including adverse events, blood pressure and heart rate, ECG, laboratory safety tests (including aldosterone), and physical examinations, were performed throughout the overall period of both studies.

[0083] Analytical methods

[0084] Plasma and urine concentrations of Compound A were determined using a calibrated liquid chromatography with tandem mass spectrometry (LC / MS / MS) method. The lower limit of quantitation (LLQ) for both assays was 0.500 ng / mL. CETP concentration in plasma was determined using a calibrated enzyme-linked immunosorbent assay (ELISA) method with a lower limit of quantitation (LLQ) of 0.500 μg / mL. CETP activity was determined as the ratio of CETP concentration to HDL-C concentration in plasma. 3The rate at which H-labeled CEs are transferred from donor HDL to recipient VLDL / LDL was determined. Adding H-labeled CEs to human plasma... 3 H]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-quantitative). 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).

[0085] Statistical analysis

[0086] 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).

[0087] Pharmacokinetic results

[0088] 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.

[0089] Pharmacodynamic results

[0090] Good balance among baseline pharmacodynamic parameters was observed across treatment groups. Compound A strongly inhibited CETP activity in a dose-dependent manner following single and repeat doses. Near complete inhibition of CETP (-92 to 99%) was observed with repeat doses of 2.5, 5, 10 to 25 mg of compound A once daily. This level of inhibition was maintained throughout the repeat dose period, with the maximum effect of each dose being achieved within 1 week of once daily dosing. The duration of inhibition following the last dose was dose dependent, with activity returning to near baseline levels 2 weeks after the lowest dose (1 mg), while it remained approximately 50% below baseline 2 weeks after the 10 and 25 mg doses. While CETP activity decreased with increasing doses of compound A, CETP concentration increased in a dose-dependent manner following both single and multiple doses. Following 3 weeks of once daily doses of 10 and 25 mg of compound A, CETP concentration increased 2.5- to 2.8-fold from baseline. CETP concentration decreased in parallel with plasma drug concentration. Following cessation of compound A doses, concentration returned to near baseline within 2 weeks for the 1 mg and 5 mg compound A doses, while it remained 1.4-fold above baseline 2 weeks after the 10 and 25 mg doses. The maximum percent change in CETP activity and CETP concentration was statistically significantly different from placebo (p<0.0001) at all compound A dose levels (1 to 25 mg).

[0091] HDL-C concentrations increased in a dose-dependent manner with repeated doses. Compound A administered at once-daily doses of 2.5 to 25 mg resulted in a mean increase of approximately 96% to 140% from baseline HDL-C. LDL-C concentrations decreased in a dose-dependent manner with Compound A administered at once-daily doses of 2.5 to 25 mg, with a maximum decrease from baseline of approximately 40% to 53%. The maximum percent change from baseline for HDL-C with Compound A doses of 5 to 25 mg once daily was statistically significantly different from placebo (p < 0.0001), and the maximum percent change from baseline for LDL-C with Compound A doses of 10 to 25 mg once daily was statistically significantly different from placebo (p < 0.0001). HDL-C and LDL-C concentrations returned toward baseline after cessation of Compound A doses, consistent with the disappearance of CETP inhibition. There was a trend toward dose-related increases in Apo A-1, Apo E, HDL2-C, and HDL3-C, and a dose-related decrease in Apo B concentrations. Variability was high for all of these variables; however, the data suggest that the greatest effects were achieved with Compound A doses of 5 to 10 mg once daily. There was no dose-dependent trend for Apo A2 or phospholipids, but there was a dose-related increase in HDL-FC, HDL-CE, and HDL-PL and a dose-related decrease in HDL-TG 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. Furthermore, there was no evidence of any relevant effects of food, age, gender, or race on pharmacodynamic variables.

[0092] Safety

[0093] Repeated doses of Compound A up to 25 mg once daily were well tolerated in all individuals. There were no serious adverse events, 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 electrolyte or aldosterone concentrations.

[0094] Table 1 Maximum percent change from baseline in primary pharmacodynamic variables for Compound A with repeated oral doses in Caucasian male healthy individuals

[0095]

[0096] Values are mean (SD)

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

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

[0099] Chemical name and structural formula of Compound A

[0100]

[0101] {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]butanoic acid}

[0102] References Definitions The following terms used in the specification and claims are intended to have the following meanings: "Compound A" means the compound having the chemical name and structural formula shown in the Examples section of this specification. "Compound B" means the compound having the chemical name and structural formula shown in the Examples section of this specification. "Compound C" means the compound having the chemical name and structural formula shown in the Examples section of this specification. "Compound D" means the compound having the chemical name and structural formula shown in the Examples section of this specification. "Compound E" means the compound having the chemical name and structural formula shown in the Examples section of this specification. "Compound F" means the compound having the chemical name and structural formula shown in the Examples section of this specification. "Compound G" means the compound having the chemical name and structural

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Claims

1. Use of a compound A of the formula ###0001### or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for the treatment of a subject suffering from hyperlipidemia, wherein, The medicament administered to the individual in need thereof comprises 5 to 10 mg of Compound A. 。 2. Use according to claim 1, wherein, The medicament administered to the individual in need thereof comprises 5 to 10 mg of Compound A.

3. Use according to claim 1 or 2, wherein, The medicament administered to the individual in need thereof comprises 5 mg or 10 mg of Compound A.

4. Use according to claim 1 or 2, wherein, The compound is administered to the individual in need thereof for at least one week.

5. Use according to claim 1 or 2, wherein, The compound is administered to the individual in need thereof for at least three weeks.

6. Use according to claim 1 or 2, wherein, The medicament is formulated as a single unit dosage form.

7. Use according to claim 1 or 2, wherein, The single unit dosage form comprises 1 to 25 mg of Compound A.

8. Use according to claim 7, wherein, The single unit dosage form comprises 5 to 10 mg of Compound A.

9. The use according to claim 7, wherein, The single unit dosage form comprises 5 mg or 10 mg of Compound A.

10. Use according to claim 7, wherein, The medicament is formulated as a solid oral dosage form.

11. Use according to claim 1 or 2, wherein, The medicament is formulated as a tablet or capsule.

12. Use according to claim 1 or 2, wherein, The medicament administered to the individual in need thereof comprises 1 to 25 mg of Compound A, 13. Use of a compound A of the formula ###00009### or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for the treatment of hyperlipidemia or mixed dyslipidemia in an individual in need thereof, wherein, ​ 。

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