ES2864350T3 - Dosage Guidelines for Use with PCSK9 Inhibitors - Google Patents

Abstract

An anti-proprotein convertase subtilisin / kexin type 9 antibody (anti-PCSK9) or antigen-binding fragment thereof for use in the treatment of a subject with primary hypercholesterolemia who is statin intolerant or not controlled by statins, wherein the antibody or antigen-binding fragment thereof is administered in a dose of 150 mg every 4 weeks for at least 3 doses in the absence of concomitant statin therapy, thereby reducing low-density lipoprotein cholesterol (LDL-C) in the subject and wherein the antibody or antigen-binding fragment thereof comprises six amino acid sequences of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3 complementary determining regions of heavy and light chain having SEQ ID NO: 2 , 3, 4, 7, 8 and 10, respectively.

Description

DESCRIPTION

Dosage Guidelines for Use with PCSK9 Inhibitors

FIELD OF THE INVENTION

The present invention relates to the field of therapeutic treatment for a PCSK9-mediated disease or a PCSK9-mediated condition. In particular, the invention relates to methods comprising the administration of an antibody against proprotein convertase subtilisin / kexin type 9 (PCSK9) or antigen-binding protein, in the absence of a statin, to a subject in need thereof. The invention also relates to methods comprising administering a low frequency, high dose regimen of an antibody to PCSK9 or antigen-binding protein to a subject not taking a concomitant statin.

SEQUENCE LIST

A sequence listing is attached herewith.

BACKGROUND

Hypercholesterolemia, particularly an increase in low-density lipoprotein cholesterol (LDL-C) levels, constitutes a significant risk for the development of atherosclerosis and coronary heart disease (CHD), the leading cause of death and disability in the Western world. Numerous studies have shown that reducing LDL-C levels, mainly with 3-hydroxy-3-methyl-glutaryl-CoA reductase (HMG-CoA) inhibitors (commonly called statins), reduces the risk of CHD, with a strong relationship direct between LDL-C levels and CHD events; For every 1 mmol / L (~ 40 mg / dL) reduction in LDL-C, cardiovascular disease (CVD) morbidity and mortality is reduced by 22%. Greater reductions in LDL-C produce a greater reduction in events, and comparative data from intense versus usual statin therapy suggest that the lower the LDL-C level, the greater the benefit in patients at high cardiovascular risk. .

Long-term elevations in LDL-C that lead to a progressive accumulation of coronary atherosclerosis require long-term treatment, which includes lifestyle measures as the main intervention. However, since style measures rarely reduce plasma LDL-C by> 15%, the use of drug therapies is needed to successfully treat dyslipidemic patients. Current medications that lower LDL-C include statins, ezetimibe (EZE), fibrates, niacin, and bile acid sequestrants, of which statins are the most commonly prescribed, as they have shown great ability to lower LDL-C and reduce ECC events. Since hypercholesterolemia is largely asymptomatic, the side effects of the pharmacological agents used to treat it can make patient compliance difficult. In multi-cohort studies, the reported 1-year statin adherence rate ranged from 26% to 85%, with a rapid decline in compliance rates normally seen in the first few months.

Despite the widespread availability of lipid-modifying treatments, such as statins, approximately 30% of all adult patients treated for hypercholesterolemia in the United States between 1999 and 2006 failed to meet their recommended LDL-C goals. Reasons for this include poor adherence to treatment, drug resistance / intolerance, and the positive relationship between adverse event rates and dose escalation. In addition, since the most effective lipid-modifying treatments can only reduce LDL-C levels by up to 55%, target success rates in patients requiring substantial reductions in LDL-C, such as those with familial hypercholesterolemia, are frequently significantly lower than what might be expected. Therefore, more effective lipid-modifying therapies and treatment regimens are required to improve target success rates in these patients.

Proprotein convertase subtilisin / kexin type 9 (PCSK9) is a proprotein convertase belonging to the proteinase K subfamily of the secretory subtylase family. The data indicate that PCSK9 increases plasma LDL cholesterol, promoting degradation of the LDL receptor, which mediates LDL endocytosis in the liver, the main route of clearance of LDL from the circulation.

The effect of a monoclonal antibody against PCSK9 in patients with heterozygous familial hypercholesterolaemia with stable statin administration with or without ezetimibe treatment is determined in a phase 2 trial (Stein EA et al. "Effect of a monoclonal antibody to PCSK9, REGN727 / SAR236553, to reduce LDL-C in patients with heterozygous familial hypercholesterolemia on stable statin dose with our without ezetimibe therapy: a phase 2 randomized controlled trial ", Lancet, July 7, 2012, vol. 380 no 9836, pages 29-36) . The randomized GAUSS trial examines the effect of an antibody against PCSK9 on statin-intolerant patients (Sullivan D. et al., "Effect of a monoclonal antibody to PCSK9 on Low-Density Lipoprotein Cholesterol Levels in Statin-Intolerant Patients"; JAMA, vol. 308, N ° 23, December 19, 2012, page 2497). The use of PCSK9 inhibitors (anti-PCSK9 antibodies) to reduce serum total cholesterol, LDL cholesterol, and serum triglycerides has been described in US Patent Nos. 8,062,640 and 8,357,371, and US Patent Application Publication No. 2013/0064834. However, there remains a need in the art for improved therapeutic methods.

BRIEF SUMMARY OF THE INVENTION

The present invention addresses the need in the art for therapeutic methods improved by establishing the beneficial effects of administering an antibody against PCSK9 or antigen-binding protein to a subject in the absence of a statin at a lower frequency. The present invention provides methods of treating hypercholesterolemia and / or lowering LDL cholesterol by administering a low frequency, high dose regimen of an anti-PCSK9 antibody, in the absence of a statin.

The present invention provides an anti-proprotein convertase subtilisin / kexin type 9 (anti-PCSK9) antibody or antigen-binding fragment thereof for use in treating a subject with primary hypercholesterolemia who is statin intolerant or not controlled by statins, where the antibody or antigen-binding protein is administered at a dose of 150 mg every 4 weeks for at least 3 doses in the absence of concomitant statin therapy, thereby reducing low-density lipoprotein cholesterol (C- LDL) in the subject and wherein the antibody or antigen-binding fragment thereof comprises the amino acid sequences of the complementarity determining regions (CDRs) of the heavy and light chains having SEQ ID NO: 2, 3, 4 , 7, 8 and 10.

One embodiment provides a method of lowering low-density lipoprotein (LDL-C) cholesterol in a subject in need thereof by administering to the subject, who is not taking a concomitant statin, a pharmaceutical composition comprising an anti-proprotein convertase antibody. subtilisin / kexin type 9 (anti-PCSK9) or antigen-binding protein in a dose of approximately 150 mg every 4 weeks for at least 3 doses, thus reducing LDL-C in the subject.

Another embodiment provides a method of treating hypercholesterolemia in a subject in need thereof by administering to the subject, who is not taking a concomitant statin, a pharmaceutical composition comprising an anti-proprotein convertase subtilisin / kexin type 9 antibody (anti-PCSK9) or antigen-binding protein in a dose of approximately 150 mg every 4 weeks for at least 3 doses, thus treating hypercholesterolemia in the subject.

One embodiment provides a method of maintaining a constant low-density lipoprotein cholesterol (LDL-C) reduction in an interval between administrations in a subject by administering to the subject, who is not taking a concomitant statin, a pharmaceutical composition comprising a antiproprotein convertase subtilisin / kexin type 9 antibody (anti-PCSK9) or antigen-binding protein at a dose of approximately 150 mg every 4 weeks for at least 3 doses. The invention also provides a method of increasing the duration of action of a proprotein convertase subtilisin / kexin type 9 (PCSK9) antagonist in a subject by administering to the subject a PCSK9 antagonist in the absence of a statin.

In some embodiments, the subject has heterozygous familial hypercholesterolemia (heHF). In other embodiments, the subject has a form of hypercholesterolemia that is unfamiliar hypercholesterolemia (HnF).

The present invention also includes a method of treating a form of hypercholesterolemia that is unfamiliar hypercholesterolemia in a subject in need thereof by administering to the subject a pharmaceutical composition comprising an anti-proprotein convertase subtilisin / kexin type 9 (anti-PCSK9) antibody. or antigen-binding protein in a dose of about 150 mg every 4 weeks for at least 3 doses, thus treating the form of hypercholesterolemia which is unfamiliar hypercholesterolemia in the subject.

In some embodiments, the subject is taking a non-statin lipid-lowering agent prior to and / or during administration of the antibody or antigen-binding protein. In some embodiments, the non-statin lipid-lowering agent is selected from the group consisting of: ezetimibe, a fibrate, fenofibrate, niacin, an omega-3 fatty acid, and a bile acid resin. In specific embodiments, the non-statin lipid-lowering agent is ezetimibe or fenofibrate. In other embodiments, the subject is not taking the non-statin lipid lowering agent prior to and / or during administration of the antibody or antigen-binding protein.

In some embodiments, the PCSK9 antibody or antigen-binding fragment thereof comprises the heavy and light chain complementarity determining regions (CDRs) of a heavy chain variable region / variable region amino acid sequence pair. light chain (HCVR / LCVR) selected from the group consisting of SEQ ID NO: 1/6. In some aspects, the antibody or antigen-binding fragment thereof comprises heavy and light chain CDR amino acid sequences having SEQ ID NO: 2, 3, 4, 7, 8 and 10. In some aspects, the antibody or antigen-binding fragment thereof comprises a HCVR having the amino acid sequence of SEQ ID NO: 1 and an LCVR having the amino acid sequence of SEQ ID NO: 6.

The methods of the invention include administering an anti-PCSK9 antibody or antigen-binding protein to a subject in a dose of about 150 mg every 4 weeks for at least three doses. In certain embodiments, the antibody is administered at a dose of about 150 mg every four weeks for three doses, and the dose is maintained at about 150 mg every four weeks if the subject's LDL-C value is less than a C-level. -LDL target. In some embodiments, the subject's LDL-C is measured twelve weeks after the subject has received the first dose of the antibody or antigen-binding protein. In some embodiments, the level of Target CLDL is less than 70 milligrams per deciliter (mg / dL). In alternate embodiments, the target LDL-C level is less than 70 milligrams per deciliter (mg / dL) and a 30% reduction in LDL-C. In other embodiments, the target LDL-C level is less than 100 milligrams per deciliter (mg / dL). In alternative embodiments, the target LDL-C level is less than 100 milligrams per deciliter (mg / dL) and a 30% reduction in LDL-C.

Methods of the invention include administering an anti-PCSK9 antibody or antigen-binding protein by injection, including by subcutaneous injection.

In some embodiments, the method reduces the levels of one or more apolipoprotein B (ApoB), non-high-density lipoprotein cholesterol (non-HDL-C), total cholesterol (TC), lipoprotein a (Lp (a)), high-density lipoprotein cholesterol (HDL-C), triglyceride (TG), or apolipoprotein A-1 (Apo A-1) in a subject.

In certain embodiments, the subject exhibits one or more symptoms or indications of hypercholesterolemia or has been diagnosed with hypercholesterolemia, or would benefit from a reduction in total serum cholesterol, LDL, triglycerides, VLDL, lipoprotein (a), or would benefit from a increase in HDL.

In certain embodiments, the PCSK9-mediated disease or PCSK9-mediated condition is selected from the group consisting of elevated levels of total cholesterol, elevated levels of low-density lipoprotein (LDL-C) cholesterol, hyperlipidemia, dyslipidemia, atherosclerosis, disease cardiovascular, hypercholesterolaemia, primary hypercholesterolaemia, familial hypercholesterolaemia, and hypercholesterolaemia that is not controlled by statin. In certain embodiments, the subject is classified into one or more of the following subject groups: (i) subjects who have a serum LDL cholesterol (LDL-C) level of at least 100 mg / dL, (ii) subjects who have a serum HDL-C level less than 40 mg / dL; (iii) subjects who have a serum cholesterol level of at least 200 mg / dL; and (iv) subjects having a serum triacylglycerol level of at least 150 mg / dL, wherein the triacylglycerol level is determined after fasting for at least 8 hours.

One embodiment provides a regimen of an anti-proprotein convertase subtilisin / kexin type 9 antibody (anti-PCSK9) or antigen-binding protein that maintains a constant low-density lipoprotein cholesterol (LDL-C) reduction in the range between administration in a human subject who, after administration of the anti-PCSK9 antibody or antigen-binding protein thereof in a dose of approximately 150 mg every 4 weeks for at least 3 doses, has one or more of the properties selected from the group consisting of: (a) an area under the plasma concentration vs. time curve calculated using the trapezium rule from zero time to real time ( ^ultimate AUC) from about 250 mg day / L to about 650 mg day / L; (b) a maximum plasma concentration (C ^max ) observed from about 15 mg / L to about 33 mg / L; (c) a first time to reach a maximum plasma concentration (t ^max ) of approximately 7 days; and (d) a time to reach the terminal half-life (t ^{1 / 2Z} ) from about 5.5 days to about 12 days.

Another embodiment provides a dosage regimen of an anti-proprotein convertase subtilisin / kexin type 9 antibody (anti-PCSK9) or antigen-binding protein that maintains a constant low-density lipoprotein (LDL-C) cholesterol reduction in the range between administrations in a human subject who, after administration of the anti-PCSK9 antibody or antigen-binding protein thereof in a dose of approximately 150 mg every 4 weeks for at least 3 doses, has one or more of the selected properties of group consisting of: (a) an area under the plasma concentration vs. time curve calculated using the trapezium rule from zero time to real time ( ^ultimate AUC) from about 150 mg / day / L to about 450 mg / day / L; (b) an observed maximum plasma concentration (C ^max ) from about 10.5 mg / L to about 24 mg / L; (c) a first time to reach a maximum plasma concentration (t ^max ) of approximately 7 days; and (d) a time to reach the terminal half-life (t ^{1 / 2Z} ) from about 5 days to about 9 days.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 is a graph of the state of the art showing the mean percentage change in baseline LDL-C versus weeks during the treatment and follow-up period for patients with heterozygous familial hypercholesterolemia administered with alirocumab who are taking a stable dose. of statin with or without ezetimibe treatment. In particular, the "sawtooth" profile of the plasma concentration of LDL-C is evident, since the treatments of the state of the art are not capable of maintaining the constant reduction of LDL-C in the interval between administrations.

Figure 2 is a graph showing the percent change in LDL-C from baseline from day -29 to day 120 for the three groups: alirocumab placebo, alirocumab ezetimibe, and alirocumab fenofibrate.

Figure 3 is a graph showing the percent change in LDL-C from baseline from day -1 to day 120 for the three groups: alirocumab placebo, alirocumab ezetimibe, and alirocumab fenofibrate.

Figure 4 is a graph showing the percentage change in LDL-C from the baseline plot of mean estimates of pairwise comparisons for: alirocumab 150 mg SC c / 4sem ezetimibe vs. alirocumab 150 mg SC every 4 weeks; and alirocumab 150 mg SC every 4 weeks fenofibrate versus alirocumab 150 mg SC every 4 weeks.

Figure 5A-D is a group of four graphs showing mean free PCSK9 levels, comparing the three treatment groups together (A) and compared to percentage changes in LDL-C from baseline on day -29 , by treatment group (BD) (N = 24 per group). Figure 5A shows the results of the three groups compared. Figure 5B shows the results for the placebo alirocumab group. Figure 5C shows the results for the alirocumab EZE group. Figure 5D shows the results for the alirocumab FENO group.

Figure 6A-C is a group of three graphs showing the percentage changes in LDL-C from the baseline level on day -29 (Figure 6A), and the levels of free PCSK9 (Figure 6B) and total alirocumab (Figure 6C ) from day 57 (time of 3rd injection of alirocumab) to day 85 (28 days after 3rd injection of alirocumab).

Figure 7 (top) are two graphs showing the mean serum-time alirocumab concentration profiles on day 1 after the first alirocumab administration on a linear (top left) and semi-log scale (top right) for the three groups: alirocumab placebo, alirocumab ezetimibe and alirocumab fenofibrate. Figure 7 (bottom) are two graphs showing the mean serum alirocumab concentration-time profiles on day 57 after the third administration of alirocumab on a linear (bottom left) and semi-log scale (bottom right) for the three groups. : alirocumab placebo, alirocumab ezetimibe and alirocumab fenofibrate.

DETAILED DESCRIPTION

Before describing the present invention, it should be understood that the present invention is not limited to the particular methods and experimental conditions described, as such methods and conditions may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

Definitions

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of ordinary skill in the art.

It is noted herein, that as used in this specification and the appended claims, the singular forms "a", "an", "the" and "the" also include reference to the plural, unless the context clearly dictates another way.

The term "approximately", when used in reference to a particular quoted numerical value, means that the value may vary from the quoted value by no more than 1%. For example, as used herein, the term "about 100" includes 99 and 101 and all values in between (eg, 99.1, 99.2, 99.3, 99.4, etc.).

The terms "administer" or "administration" refer to the act of physically injecting or otherwise administering a substance as it exists outside the body (eg, a formulation of the invention) into a patient, such as by mucosal, intradermal, administration. intravenous, subcutaneous, intramuscular and / or any other physical administration method described herein or known in the art. When treating a disease, or a symptom thereof, administration of the substance normally occurs after the onset of the disease or symptoms thereof. When a disease or symptoms thereof are prevented, the administration of the substance normally occurs before the onset of the disease or symptoms thereof.

The terms "composition" and "formulation" are intended to encompass a product that contains the specified components (eg, an anti-PCSK9 antibody) in, optionally, the specified amounts, as well as any product that results, directly or indirectly, from the combination. of the specified components in, optionally, the specified quantities.

The term "excipients" refers to inert substances that are commonly used as a diluent, carrier, preservative, binder, stabilizer, etc., for drugs and includes, but is not limited to, proteins (eg, serum albumin, etc.). ), amino acids (eg aspartic acid, glutamic acid, lysine, arginine, glycine, histidine, etc.), fatty acids and phospholipids (eg alkylsulfonates, caprylate, etc.), surfactants (eg SDS, polysorbate, nonionic surfactant, etc.), saccharides (eg, sucrose, maltose, trehalose, etc.), and polyols (eg, mannitol, sorbitol, etc.). See, therefore, Remington's Pharmaceutical Sciences (1990) Mack Publishing Co., Easton, Pa.

In the context of a peptide or polypeptide, the term "fragment" refers to a peptide or polypeptide that comprises less than the full length amino acid sequence. Said fragment can arise, for example, from a truncation at the amino terminus, a truncation at the carboxy terminus and / or an internal deletion of a residue (s) of the amino acid sequence. The fragments can result, for example, from alternative RNA splicing or from protease activity in vivo. In certain embodiments, PCSK9 fragments include polypeptides that comprise an amino acid sequence of at least 50, at least 100 amino acid residues, at least 125 contiguous amino acid residues, at least 150 contiguous amino acid residues, at least 175 amino acid residues. contiguous, at least 200 contiguous amino acid residues, or at least 250 contiguous amino acid residues from the amino acid sequence of a PCSK9 polypeptide. In a specific embodiment, a fragment of a PCSK9 polypeptide or an antibody that specifically binds to a PCSK9 antigen retains at least 1, at least 2, or at least 3 functions of the full-length polypeptide or antibody.

The term "pharmaceutically acceptable" means that it is licensed by a federal or state government regulatory agency, or listed in the United States Pharmacopoeia, European Pharmacopoeia, or other generally recognized pharmacopoeia for use in animals, and more particularly in humans.

The terms "prevent", "prevent" and "prevention" refer to the total or partial inhibition of the development, reappearance, appearance or spread of a PCSK9-mediated disease and / or a related symptom, resulting from the administration of a therapy or combination of therapies provided herein (eg, a combination of prophylactic or therapeutic agents).

The term "PCSK9 antigen" refers to that portion of a PCSK9 polypeptide to which an antibody specifically binds. A PCSK9 antigen also refers to an analog or derivative of a PCSK9 polypeptide or fragment thereof to which an antibody specifically binds. In some embodiments, a PCSK9 antigen is a PCSK9 monomeric antigen or a PCSK9 trimeric antigen. A region of a PCSK9 polypeptide that contributes to an epitope can be contiguous amino acids of the polypeptide, or the epitope can be derived from two or more non-contiguous regions of the polypeptide. The epitope may or may not be a characteristic three-dimensional surface of the antigen. A region located on the surface of a PCSK9 antigen that is capable of eliciting an immune response is a PCSK9 epitope. The epitope may or may not be a characteristic three-dimensional surface of the antigen.

The term "human PCSK9", "PCSK9h" or "PCSK9h polypeptide" and similar terms refer to polypeptides ("polypeptides", "peptides" and "proteins" are used interchangeably herein) that comprise the amino acid sequence of SEQ ID NO: 198 and related polypeptides, including SNP variants thereof. Related polypeptides include allelic variants (eg, SNP variants); splice variants; fragments; derivatives; substitution, deletion, and insertion variants; fusion polypeptides; and interspecies homologs, preferably, that retain PCSK9 activity and / or are sufficient to elicit an anti-PCSK9 immune response. Also encompassed are soluble forms of PCSK9 that are sufficient to elicit an anti-PCSK9 immune response. As those skilled in the art will appreciate, an anti-PCSK9 antibody can bind to a PCSK9 polypeptide, polypeptide fragment, antigen, and / or epitope, since an epitope is part of the large antigen, which is part of the large polypeptide fragment, which , in turn, is part of the large polypeptide. PCSK9h can exist in a trimeric (native) or monomeric (denatured) form.

The terms "PCSK9-mediated disease", "PCSK9-mediated condition" and "PCSK9-mediated disorder" are used interchangeably and refer to any disease that is fully or partially caused by PCSK9 or its result, eg, PCSK9h. In certain embodiments, PCSK9 is expressed aberrantly (eg, highly). In some embodiments, PCSK9 can be aberrantly up-regulated. In other embodiments, normal, aberrant, or excessive cell signaling is caused by the binding of PCSK9 to a PCSK9 ligand. In certain embodiments, the PCSK9 ligand is a PCSK9 receptor. In certain embodiments, the PCSK9-mediated disease or condition is selected from the group consisting of: elevated levels of total cholesterol; elevated levels of low-density lipoprotein cholesterol (LDL-C); hyperlipidemia; dyslipidemia; hypercholesterolemia, particularly hypercholesterolemia not controlled by statins, hypercholesterolemia, such as familial hypercholesterolemia or unfamiliar hypercholesterolemia, and hypercholesterolemia not controlled by statins; atherosclerosis; and cardiovascular diseases.

The terms "subject" and "patient" are used interchangeably. As used herein, a subject is preferably a mammal, such as a non-primate (eg, cows, pigs, horses, cats, dogs, rats, etc.) or a primate (eg, monkey and human). , most preferably a human. In one embodiment, the subject is a mammal, preferably a human, having a PCSK9-mediated disease. In another embodiment, the subject is a mammal, preferably a human, at risk of developing a PCSK9-mediated disease.

The term "therapeutic agent" refers to any agent that can be used in the treatment, management, or amelioration of a PCSK9-mediated disease and / or a symptom related thereto. In certain embodiments, the term "therapeutic agent" refers to an antibody against PCSK9 of the invention. In certain other embodiments, the term "therapeutic agent" refers to an agent other than an antibody to PCSK9 of the invention. Preferably, a therapeutic agent is an agent that is known to be useful, or has been or is currently being used for the treatment, management, or amelioration of a PCSK9-mediated disease or one or more symptoms related thereto.

The term "therapy" refers to any protocol, method, and / or agent that can be used in the prevention, management, treatment, and / or amelioration of a PCSK9-mediated disease (eg, atherosclerosis or hypercholesterolemia). In certain embodiments, the terms "therapies" and "therapy" refer to biological therapy, supportive therapy, and / or other therapies useful in the prevention, management, treatment, and / or amelioration of a known PCSK9-mediated disease. by one of skill in the art, such as medical personnel.

The terms "treat", "treatment" and "treating" refer to the reduction or amelioration of the course, intensity and / or duration of a PCSK9-mediated disease (eg, atherosclerosis) that results from the administration of one or more therapies (including, but not limited to, the administration of one or more prophylactic or therapeutic agents). In specific embodiments, such terms refer to the reduction or inhibition of the binding of PCSK9 to a PCSK9 ligand.

Although any methods and materials similar or equivalent to those described herein can be used in the practice of the present invention, the preferred methods and materials are now described.

Patient populations

The methods of the present invention comprise selecting subjects who have or are at risk of developing a PCSK9-mediated disease or condition, such as hypercholesterolemia or a related disorder (eg, atherosclerosis), and administering to these subjects, in the absence of a statin , a pharmaceutical composition comprising an antibody against PCSK9 or antigen-binding fragment thereof comprising heavy and light chain CDR amino acid sequences having SEQ ID NO: 2, 3, 4, 7, 8 and 10.

For example, the methods of the present invention comprise administering to a subject in need thereof a therapeutic composition comprising an anti-PCSK9 antibody, in the absence of a statin. The therapeutic composition can comprise any of the anti-PCSK9 antibodies, or fragments thereof, as disclosed herein.

As used herein, the term "a subject in need thereof" means a human or non-human animal exhibiting one or more symptoms or indications of hypercholesterolemia or who has been diagnosed with hypercholesterolemia, or who would otherwise benefit. and a reduction in total serum cholesterol, LDL, triglycerides, VLDL, lipoprotein (a) [Lp (a)], or that would benefit from an increase in HDL. Specific exemplary populations treatable by the therapeutic methods of the invention include patients indicated for LDL apheresis, subjects with activating PCSK9 mutations (GOF), patients with heterozygous or homozygous familial hypercholesterolemia (HFhe or HFho); subjects with primary hypercholesterolemia who are intolerant to statins or are not controlled by statins; and subjects at risk of developing hypercholesterolemia that can be treated preventively.

Although lifestyle modifications and conventional drug treatment are often successful in lowering cholesterol levels, not all patients are able to achieve recommended target cholesterol levels with such approaches. It appears that various conditions, such as familial hypercholesterolemia (FH), are resistant to the reduction of LDL-C levels despite the intensive use of conventional therapy. Homozygous and heterozygous familial hypercholesterolemia (HFho, HFhe) are conditions associated with premature atherosclerotic vascular disease. However, patients diagnosed with HoFH are highly insensitive to conventional drug therapy and have limited treatment options. In particular, treatment with statins, which lower LDL-C by inhibiting cholesterol synthesis and upregulating the hepatic LDL receptor, may have little effect in patients with non-existent or defective LDL receptors. A mean LDL-C reduction of only less than approximately 20% has recently been reported in genotype-confirmed HFH patients treated with the maximum dose of statins. The addition of ezetimibe 10 mg / day to this regimen produced a total reduction in LDL-C levels of 27%, which is still far from optimal. Also, many patients are insensitive to statins, are poorly controlled on statin therapy, or cannot tolerate statin therapy; in general, these patients are unable to achieve cholesterol control with alternative treatments. There is a great unmet medical need for new treatments that can address the shortcomings of current treatment options.

Thus, the invention includes therapeutic methods in which an antibody against PCSK9 or antigen-binding fragment thereof comprising heavy and light chain CDR amino acid sequences having SEQ ID NO: 2, 3, 4, 7, 8 and 10 is administered in the absence of a statin to a patient to treat or prevent hypercholesterolemia. As used herein, use of the PCSK9 inhibitor "in the absence of a statin" means that the subject is not taking a concomitant statin while being treated with the PCSK9 inhibitor of the invention, or was not recently taking a statin. prior to treatment with the PCSK9 inhibitor of the invention. The terms "in the absence of a statin" and "not taking a concomitant statin" mean that the subject should not have detectable levels of statin in the bloodstream, but, due to prior therapy, the subject may have a serum concentration of statin less than 0.1 mg / mL. As used herein, a "lipid-lowering" means any pharmaceutical agent other than an antibody to PCSK9 or antigen-binding fragment thereof that is administered for the purpose of modifying the lipid profile of a subject. Examples of lipid-lowering drugs include, but are not limited to: HMG-CoA reductase inhibitors, which They include statins (atorvastatin, cerivastatin, fluvastatin, lovastatin, mevastatin, pitavastatin, pravastatin, rosuvastatin, simvastatin, etc.), niacin, fibric acid, bile acid sequestrants (eg, cholestyramine), colesevelam, colestypal, and ezetimlestibaol. It follows that a "non-statin lipid-lowering agent" means any pharmaceutical agent other than an anti-PCSK9 antibody or antigen-binding fragment thereof and a statin. Examples of non-statin lipid-lowering drugs include, but are not limited to, niacin. , fibric acid, fenofibrate, bile acid sequestrants (eg, cholestyramine), colesevelam, colestipol, an omega-3 fatty acid, a bile acid resin, and ezetimibe.

In some cases, the patient who is treated with a therapeutic formulation disclosed herein is otherwise healthy, except for the presence of elevated levels of cholesterol, lipids, triglycerides, or lipoproteins. For example, the patient may not have any other risk factors for cardiovascular, thrombotic, or other diseases or disorders at the time of treatment. In other cases, however, the patient is selected on the basis that he or she has been diagnosed with, or is at risk of developing, a disease or disorder that is caused by, is correlated with, or is secondary to elevated serum cholesterol, lipids, triglycerides, or lipoproteins. For example, at the time of, or prior to administration of the pharmaceutical composition of the present invention, the patient may be diagnosed with or identified as being at risk of developing a cardiovascular disease or disorder, such as, for example, disease of the arteries. coronary arteries, acute myocardial infarction, asymptomatic carotid atherosclerosis, cerebrovascular accident, occlusive disease of the peripheral arteries, etc. The cardiovascular disease or disorder is, in some cases, hypercholesterolemia. For example, a patient may be selected for treatment with a pharmaceutical composition of the present invention if the patient is diagnosed with or identified as being at risk of developing a hypercholesterolemic condition, such as, for example, heterozygous familial hypercholesterolemia (HFhe ), homozygous familial hypercholesterolemia (HFho), as well as incidences of hypercholesterolemia that are different from familial hypercholesterolemia (HnF).

In other cases, at the time of, or prior to administration of the pharmaceutical composition of the present invention, the patient may be diagnosed with or identified as being at risk of developing a thrombotic occlusive disease or disorder, such as, for example, pulmonary embolism. , central retinal vein occlusion, etc. In certain embodiments, the patient is selected based on having been diagnosed with or is at risk of developing a combination of two or more of the aforementioned diseases or disorders. For example, at the time of or prior to administration of the pharmaceutical composition of the present invention, the patient can be diagnosed with or identified as being at risk of developing coronary artery disease and pulmonary embolism. Other diagnostic combinations (eg, atherosclerosis and central retinal vein occlusion, heHF and stroke, etc.) are also included in the definition of patient populations that are treatable with a pharmaceutical composition of the present invention.

The pharmaceutical compositions of the present disclosure are also useful for the treatment of hypercholesterolemia or dyslipidemia caused by or related to a pre-existing disease or disorder selected from the group consisting of metabolic syndrome, diabetes mellitus, hypothyroidism, nephrotic syndrome, renal failure, Cushing's syndrome. , biliary cirrhosis, glycogen storage diseases, hepatoma, cholestasis, growth hormone deficiency. The pharmaceutical compositions of the present disclosure are also useful for the treatment of hypercholesterolemia or dyslipidemia caused by or related to a previous therapeutic regimen, such as estrogen therapy, progestin therapy, beta-blockers, or diuretics.

In still other cases, the patient to be treated with a pharmaceutical composition of the present disclosure is selected according to one or more factors selected from the group consisting of age (eg, over 40, 45, 50, 55, 60, 65, 70, 75 or 80 years old), race, gender (male or female), exercise habits (for example, person who exercises regularly, person who does not exercise), other pre-existing medical conditions (for example, type diabetes II, high blood pressure, etc.) and current medication status (for example, currently taking statins [for example, cerivastatin, atorvastatin, simvastatin, pitavastatin, rosuvastatin, fluvastatin, lovastatin, pravastatin, etc.], beta-blockers, niacin, etc. .). Potential patients can be selected / screened based on one or more of these factors (eg, by questionnaire, diagnostic evaluation, etc.) prior to being treated with the methods of the present invention.

The present invention also includes methods of enhancing transintestinal cholesterol excretion (TTE) in a subject by administering an antibody against PCSK9 or antigen-binding fragment thereof to the subject. For example, the present invention provides methods of increasing ETIC in a subject by administering to the subject an anti-PCSK9 antibody with pH-dependent binding characteristics. According to certain embodiments, the present invention includes methods comprising identifying a subject for whom enhanced ETIC would be beneficial, or identifying a subject exhibiting altered ETIC, and administering an anti-PCSK9 antibody or antigen-binding fragment thereof to the subject.

Hypercholesterolemia is a precursor to atherosclerosis. Accordingly, the disclosure also includes therapeutic methods in which a PCSK9 inhibitor of the invention is administered in the absence of a statin to a patient to treat or prevent atherosclerosis. Risk factors for atherosclerosis are well known in the art and include, without limitation, elevated levels of low-density lipoprotein (LDL) cholesterol, low levels of high-density lipoprotein (HDL) cholesterol, hypertension, diabetes mellitus, family history, sex male, cigarette smoker and elevated serum cholesterol. Methods of evaluating these risk factors for a given subject are also well known in the art.

In certain embodiments, the selected subject is hyperlipidemic. A "hyperlipidemic" is a subject who is a hypercholesterolemic and / or hypertriglyceridemic subject. A "hypercholesterolemic" subject is one that meets the current criteria established for a hypercholesterolemic subject. A "hypertriglyceridemic" subject is one that meets current established criteria for a hypertriglyceridemic subject (see, eg, Harrison's Principles of Experimental Medicine, 13th edition, McGraw-Hill, Inc., Inc., N.Y). For example, a hypercholesterolemic subject typically has an LDL level of> 160 mg / dL, or> 130 mg / dL and at least two risk factors selected from the group consisting of male sex, family history of premature coronary heart disease, smoker of cigarettes (plus 10 a day), hypertension, low HDL (<35 mg / dL), diabetes mellitus, hyperinsulinemia, abdominal obesity, high lipoprotein (a) and a personal history of cerebrovascular disease or peripheral vascular occlusive disease. A hypertriglyceridemic subject normally has a triglyceride (TG) level of> 250 mg / dL. In certain embodiments, the selected subject is hyperlipidemic, but is not receiving treatment for hyperlipidemia.

PCSK9 inhibitors

The methods of the present invention comprise administering to a patient a therapeutic composition comprising an antibody against PCSK9 or antigen-binding fragment thereof. As used herein, a "PCSK9 inhibitor" is any agent that binds to or interacts with human PCSK9 and inhibits the normal biological function of PCSK9 in vitro or in vivo. Non-limiting examples of categories of PCSK9 inhibitors include small molecule PCSK9 antagonists, peptide-based PCSK9 antagonists (eg, "peptibody" molecules), and antibodies or antigen-binding fragments of antibodies that specifically bind to PCSK9. human.

The term "human proprotein convertase subtilisin / kexin type 9" or "human PCSK9" or "PCSK9h" refers to PCSK9 having the nucleic acid sequence shown in SEQ ID NO: 197 and the amino acid sequence of SEQ ID NO: 198 , or a biologically active fragment thereof.

The term "antigen-binding protein" means a protein that binds to an antigen. For example, an antigen-binding protein includes, but is not limited to, an antibody, an antigen-binding fragment of an antibody, a DVD-Ig, and a double variable domain immunoglobulin.

The term "antibody" is intended to refer to immunoglobulin molecules comprising four polypeptide chains, two heavy (H) chains, and two light (L) chains interconnected by disulfide bonds, as well as multimers thereof (eg, IgM). Each heavy chain comprises a heavy chain variable region (abbreviated herein as HCVR or V ^h ) and a heavy chain constant region. The heavy chain constant region comprises three domains, C ^h 1, C ^h 2, and C ^h 3. Each light chain comprises a light chain variable region (abbreviated herein as LCVR or V ^l ) and a constant region of the light chain. The constant region of the light chain comprises a domain ( ^C1 ). The V ^h and V ^l regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR). Each V ^h and V ^l is composed of three CDRs and four FRs, arranged from amino end to carboxy end in the following order: FR1, CDR1, Fr 2, c Dr 2, FR3, CDR3, FR4. In different embodiments of the invention, the FRs of the anti-PCSK9 antibody (or antigen-binding portion thereof) may be identical to human germline sequences, or may be naturally or artificially modified. A consensus amino acid sequence can be defined based on a comparative analysis of two or more CDRs.

The term "antibody" also includes antigen-binding fragments of whole antibody molecules. The terms "antigen-binding portion" of an antibody, "antigen-binding fragment" of an antibody, and the like, include any naturally occurring polypeptide or glycoprotein, whether enzymatically obtainable, synthetic, or genetically engineered, that is specifically binds to an antigen to form a complex. Antigen-binding fragments of an antibody can be derived, for example, from whole antibody molecules using any suitable conventional technique, such as proteolytic digestion or recombinant genetic engineering techniques that involve manipulation and expression of DNA encoding variable domains. and optionally antibody constants. Such DNA is known and / or readily available from, for example, commercial sources, DNA libraries (including, for example, phage-antibody libraries), or can be synthesized. DNA can be sequenced and manipulated chemically or using molecular biology techniques, for example, to arrange one or more variable and / or constant domains in a suitable configuration, or to introduce codons, create cysteine residues, modify, add or delete amino acids , etc.

Non-limiting examples of antigen-binding fragments include: (i) Fab fragments; (ii) F (ab ') 2 fragments; (iii) Fd fragments; (iv) Fv fragments; (v) single chain Fv molecules (scFv); (vi) dAb fragments; and (vii) minimal recognition units consisting of amino acid residues that mimic the hypervariable region of an antibody (eg, an isolated complementarity determining region (CDR), such as a CDR3 peptide), or a FR3-peptide. CDR3-FR4 limited. Other manipulated molecules, such as specific antibodies to domain, single domain antibodies, domain deleted antibodies, chimeric antibodies, CDR-grafted antibodies, diabodies, tribodies, tetrabodies, minibodies, nanobodies (e.g., monovalent nanobodies, bivalent nanobodies, etc.), small modular immunpharmaceuticals (SMIPs ) and shark variable IgNAR domains are also encompassed within the term "antigen-binding fragment".

An antigen-binding fragment of an antibody will typically comprise at least one variable domain. The variable domain can be of any size or amino acid composition and will generally comprise at least one CDR that is adjacent to or in frame with one or more framework region sequences. In antigen-binding fragments having a V ^h domain associated with a V ¹ domain, the V ^h and V ¹ domains can be located in any suitable arrangement relative to each other. For example, the variable region can be dimeric and contain V ^h -V ^h , V ^h -V ^l, or V ^l -V ^l dimers. Alternatively, the antigen-binding fragment of an antibody may contain a monomeric ^{V h} or V ^{l domain.}

In certain embodiments, an antigen-binding fragment of an antibody may contain at least one variable domain covalently linked to at least one constant domain. Exemplary non-limiting configurations of variable and constant domains that may be found within an antigen-binding fragment of an antibody of the present invention include: (i) V ^h -C ^h 1; (ii) V ^h -C ^h 2; (iii) V ^h -C ^h 3; (iv) V ^h -C ^h 1 -C ^h 2; (v) V ^h -C ^h 1 -C ^h 2 ^{-C h} 3;

(vi) V ^h -C ^h 2 ^{-C h} 3; (vii) V ^h -C ^l ; (viii) V ^l -C ^h 1; (ix) V ¹ -C ^h 2; (x) V ^l -C ^h 3; (xi) V ^l -C ^h 1 ^{-C h} 2; (xii) V ^l -C ^h 1 ^{-C h} 2 -C ^h 3; and (xiv) V ^l -C ^l . In any variable and constant domain configuration, including any of the exemplary configurations listed above, the variable and constant domains may be either directly linked to each other, or they may be linked by a complete or partial connector or hinge region. A hinge region can consist of at least 2 (eg, 5, 10, 15, 20, 40, 60 or more) amino acids that result in a flexible or semi-flexible bond between adjacent variable and / or constant domains in a single molecule of polypeptide. Furthermore, an antigen-binding fragment of an antibody usable in the present invention may comprise a homodimer or heterodimer (or other multimer) of any of the variable and constant domain configurations listed above in non-covalent association with each other and / or with one or more domains V ^h o

V ^l monomeric (eg, by disulfide bond (s)).

As with whole antibody molecules, antigen-binding fragments can be monospecific or multispecific (eg, bispecific). A multispecific antigen-binding fragment of an antibody will typically comprise at least two different variable domains, where each variable domain is capable of specifically binding a separate antigen or a different epitope on the same antigen.

Any multispecific antibody format, including the exemplary bispecific antibody formats disclosed herein, can be adapted for use in the context of an antigen-binding fragment of an antibody usable in the present invention using routine techniques. available in the art.

The constant region of an antibody is important in an antibody's ability to fix complement and mediate cell-dependent cytotoxicity. Thus, the isotype of an antibody can be selected based on whether it is desired that the antibody mediates cytotoxicity.

The term "human antibody" is intended to include antibodies that have variable and constant regions derived from human germline immunoglobulin sequences. The human antibodies of the invention may, however, include amino acid residues not encoded by human germline immunoglobulin sequences (eg, mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo), for example For example, in CDRs and in particular CDR3. However, the term "human antibody" is not intended to include antibodies into which CDR sequences derived from the germ line of other mammalian species, such as a mouse, have been grafted onto human framework region sequences.

The term "recombinant human antibody" is intended to include all human antibodies that are prepared, expressed, created, or isolated by recombinant means, such as antibodies expressed using a recombinant expression vector transfected into a host cell (described below), antibodies isolated from a combinatorial library of recombinant human antibodies (described below), antibodies isolated from an animal (eg, a mouse) that are transgenic for human immunoglobulin genes (see, eg, Taylor et al. (1992) Nucl. Acids Res. 20: 6287-6295) or antibodies prepared, expressed, created, or isolated by any other means involving splicing of human immunoglobulin gene sequences with other DNA sequences. Such recombinant human antibodies have variable and constant regions derived from human germline immunoglobulin sequences. In certain embodiments, however, said recombinant human antibodies are subjected to in vitro mutagenesis (or, when using a transgenic animal for human Ig sequences, in vivo somatic mutagenesis) and thus the amino acid sequences of the V ^H and V regions ^L of the recombinant antibodies are sequences that, while derived from and related to sequences V ^h and V ^L human germ line, may not naturally exist within the repertoire of human antibody germline in vivo.

Human antibodies can exist in two forms that are associated with hinge heterogeneity. In one form, an immunoglobulin molecule comprises a stable four-chain construct of approximately 150-160 kDa in which the dimers are held together by an interchain heavy chain disulfide bond. In a second form, the dimers are not linked by interchain disulfide bonds and a approximately 75-80 kDa molecule composed of a covalently coupled heavy and light chain (half antibody). These forms are extremely difficult to separate, even after affinity purification.

The frequency of occurrence of the second form in various isotypes of intact IgG is due to, but is not limited to, structural differences associated with the isotype of the hinge region of the antibody. A single amino acid substitution in the hinge region of human IgG4 can significantly reduce the occurrence of the second form (Angal et al. (1993) Molecular Immunology 30: 105) to levels normally seen using an IgG 1 hinge. human. The present invention encompasses antibodies that have one or more mutations in the hinge region, C ^h 2 or C ^h 3 that may be desirable, for example, in production, to improve the performance of the desired antibody form.

An "isolated antibody" means an antibody that has been identified and separated and / or recovered from at least one component of its natural environment. For example, an antibody that has been separated or removed from at least one component of an organism, or from a tissue or cell in which the antibody exists naturally or occurs naturally, is an "isolated antibody" for the purposes herein. invention. An isolated antibody also includes an antibody in situ within a recombinant cell. Isolated antibodies are antibodies that have undergone at least one purification or isolation step. According to certain embodiments, an isolated antibody can be substantially free of other cellular material and / or chemical.

The term "specifically binds to", or the like, means that an antibody or antigen-binding fragment thereof forms a complex with an antigen that is relatively stable under physiological conditions. Methods of determining whether an antibody specifically binds to an antigen are well known in the art and include, for example, equilibrium dialysis, surface plasmon resonance, and the like. For example, an antibody that "specifically binds to" PCSK9, as used in the context of the present invention, includes antibodies that bind to PCSK9 or a portion thereof with a K ^{d of} less than about 1000 nM, less than about 500 nM, less than about 300 nM, less than about 200 nM, less than about 100 nM, less than about 90 nM, less than about 80 nM, less than about 70 nM, less than about 60 nM, less than about 50 nM , less than about 40 n about 30 nM, less than about 20 nM, less than about 10 n about 5 nM, less than about 4 nM, less than about 3 nM, less than about 2 nM, less than about 1 nM or less at approximately 0.5 nM, as measured in a surface plasmon resonance assay. An isolated antibody that specifically binds to human PCSK9, however, is cross-reactive with other antigens, such as PCSK9 molecules from other (non-human) species.

Anti-PCSK9 antibodies useful for the methods of the present invention may comprise one or more amino acid substitutions, insertions, and / or deletions in the framework regions of the heavy and light chain variable domains compared to the corresponding germline sequences. of the antibodies from which they are derived. Such mutations can be readily determined by comparing the amino acid sequences disclosed herein with germline sequences available from, for example, public antibody sequence databases. The present invention includes methods involving the use of antibodies, and antigen-binding fragments thereof, which are derived from any of the amino acid sequences disclosed herein, wherein one or more amino acids within one or more framework regions are mutated to the corresponding residue (s) of the germline sequence from which the antibody was derived, or to the corresponding residue (s) of another human germline sequence, or to a conservative amino acid substitution of the corresponding germline residue (s) (such sequence changes are collectively referred to herein as "germline mutations"). One of ordinary skill in the art, from the heavy and light chain variable region sequences disclosed herein, can readily produce numerous antibodies and antigen-binding fragments comprising one or more individual germline mutations or combinations thereof. In certain embodiments, all framework region residues within the V ^h and / or V ^l domains are back-mutated to residues found in the original germline sequence from which the antibody was derived. In other embodiments, only certain residues are back-mutated to the original germline sequence, for example, only the mutated residues found within the first 8 amino acids of FR1 or within the last 8 amino acids of FR4. In other embodiments, one or more of the framework region residues are mutated to the corresponding residue (s) of a different germline sequence (i.e., a germline sequence that is different from the germline sequence). germ line from which the antibody was originally derived). Furthermore, the antibodies usable in the present invention may contain any combination of two or more germ line mutations within the framework region, for example, where certain individual residues mutate to the corresponding residue of a particular germ line sequence, while certain other residues that differ from the original germline sequence are maintained or mutated to the corresponding residue from a different germline sequence. Once obtained, antibodies and antigen-binding fragments containing one or more germline mutations can easily be tested for one or more desired properties, such as enhancement of binding specificity, increased binding affinity, enhancement or potentiation of antagonist or agonist biological properties (as the case requires), reduced immunogenicity, etc. The use of antibodies and antigen-binding fragments obtained in this general way is encompassed within the present invention.

The present disclosure also includes methods that involve the use of anti-PCSK9 antibodies comprising variants of any of the HCVR, LCVR and / or CDR amino acid sequences disclosed herein that have one or more conservative substitutions. For example, the present invention includes the use of anti-PCSK9 antibodies having HCVR, LCVR and / or CDR amino acid sequences with, for example, 10 or less, 8 or less, 6 or less, 4 or less, etc. Conservative, amino acid substitutions with respect to any of the HCVR, LCVR, and / or CDR amino acid sequences disclosed herein.

The term "surface plasmon resonance" refers to an optical phenomenon that allows the analysis of interactions in real time by detecting alterations in protein concentrations within an array of biosensors, for example, using the BIAcore ™ system (Biacore Life Sciences GE Healthcare Division, Piscataway, NJ).

The term "KD" is intended to refer to the equilibrium dissociation constant of a particular antibody-antigen interaction.

The term "epitope" refers to an antigenic determinant that interacts with a specific antigen-binding site in the variable region of an antibody molecule known as a paratope. A single antigen can have more than one epitope. Thus, different antibodies can bind to different areas on an antigen and can have different biological effects. Epitopes can be either conformational or linear. A conformational epitope is produced by spatially juxtaposed amino acids from different segments of the linear polypeptide chain. A linear epitope is one produced by adjacent amino acid residues in a polypeptide chain. In certain circumstances, an epitope can include saccharide moieties, phosphoryl groups, or sulfonyl groups on the antigen.

According to certain embodiments, the anti-PCSK9 antibody used in the methods of the present invention is an antibody with pH-dependent binding characteristics. As used herein, the term "pH-dependent binding" means that the antibody or antigen-binding fragment thereof exhibits "reduced binding to PCSK9 at acidic pH compared to neutral pH" (for the purposes herein disclosure, both expressions can be used interchangeably). For example, antibodies "with pH-dependent binding characteristics" include antibodies and antigen-binding fragments thereof that bind to PCSK9 with higher affinity at neutral pH than at acidic pH. In certain embodiments, the antibodies and antigen-binding fragments of the present invention bind to PCSK9 with an affinity of at least 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or more times higher at neutral pH than at acidic pH.

According to this aspect of the disclosure, anti-PCSK9 antibodies with pH-dependent binding characteristics may possess one or more amino acid variations relative to the parent anti-PCSK9 antibody. For example, an anti-PCSK9 antibody with pH-dependent binding characteristics may contain one or more histidine substitutions or insertions, for example, in one or more CDRs of a parent anti-PCSK9 antibody. Thus, according to the present disclosure, methods are provided which comprise administering an anti-PCSK9 antibody comprising CDR amino acid sequences (eg, heavy and light chain CDRs) that are identical to the CDR amino acid sequences of an antibody. parental anti-PCSK9, except for the substitution of one or more amino acids of one or more CDRs of the parental antibody with a histidine residue. Anti-PCSK9 antibodies with pH-dependent binding may possess, for example, 1,2,3,4,5,6,7,8,9 or more histidine substitutions, or within a single CDR of a parent antibody or distributed over multiple CDRs (eg, 2, 3, 4, 5, or 6) of a parental anti-PCSK9 antibody. For example, the present disclosure includes the use of pH-dependent binding anti-PCSK9 antibodies comprising one or more histidine substitutions in HCDR1, one or more histidine substitutions in HCDR2, one or more histidine substitutions in HCDR3, one or more more histidine substitutions in LCDR1, one or more histidine substitutions in LCDR2, and / or one or more histidine substitutions in LCDR3, of a parent anti-PCSK9 antibody.

As used herein, the term "acidic pH" means a pH of 6.0 or less (eg, less than about 6.0, less than about 5.5, less than about 5.0, and so on. ). The term "acidic pH" includes pH values of about 6.0, 5.95, 5.90, 5.85, 5.8, 5.75, 5.7, 5.65, 5.6, 5, 55, 5.5, 5.45, 5.4, 5.35, 5.3, 5.25, 5.2, 5.15, 5.1.5.05, 5.0, or less. As used herein, the term "neutral pH" means a pH of from about 7.0 to about 7.4. The term "neutral pH" includes pH values of about 7.0, 7.05, 7.1,7,15, 7.2, 7.25, 7.3, 7.35, and 7.4.

Preparation of human antibodies

Methods of generating human antibodies in transgenic mice are known in the art. Any such known method can be used in the context of the present invention to prepare human antibodies that specifically bind to human PCSK9.

Using VELOCIMMUNE ™ technology (see, for example, US Patent 6,596,541, Regeneron Pharmaceuticals) or any other known method for generating monoclonal antibodies, high affinity chimeric antibodies against PCSK9 having a human variable region are initially isolated and a constant region of mouse. The VELOCIMMUNE® technology involves the generation of a transgenic mouse having a genome comprising variable regions of human heavy and light chains operably linked to endogenous mouse constant region loci so that the mouse produces an antibody comprising a human variable region and a mouse constant region in response to antigenic stimulation. DNA encoding the antibody heavy and light chain variable regions is isolated and operably linked to DNA encoding the human heavy and light chain constant regions. The DNA is then expressed in a cell capable of expressing the fully human antibody.

In general, a VELOCIMMUNE® mouse is exposed to the antigen of interest, and lymphatic cells (such as B lymphocytes) are recovered from the mice expressing the antibodies. Lymphatic cells can be fused with a myeloma cell line to prepare immortal hybridoma cell lines, and such hybridoma cell lines are screened and selected to identify hybridoma cell lines that produce antibodies specific to the antigen of interest. DNA encoding the heavy chain and light chain variable regions can be isolated and linked to desirable isotypic heavy chain and light chain constant regions. Such an antibody protein can be produced in a cell, such as a CHO cell. Alternatively, DNA encoding antigen-specific chimeric antibodies or heavy and light chain variable domains can be isolated directly from antigen-specific lymphocytes.

Initially, high affinity chimeric antibodies having a human variable region and a mouse constant region are isolated. Antibodies are characterized and selected for desirable characteristics, including affinity, selectivity, epitope, etc., using standard procedures known to those of skill in the art. The mouse constant regions are substituted for a desired human constant region to generate the fully human antibody of the invention, eg, natural or modified IgG1 or IgG4. Although the constant region selected may vary depending on the specific use, high affinity antigen binding and target specificity characteristics reside in the variable region.

In general, the antibodies that can be used in the methods of the present invention possess high affinities, as described above, when measured by binding to antigen or immobilized in solid phase or in solution phase. The mouse constant regions are substituted for desired human constant regions to generate the fully human antibodies of the invention. Although the constant region selected may vary depending on the specific use, high affinity antigen binding and target specificity characteristics reside in the variable region.

Specific examples of human antibodies or antigen-binding fragments of the antibodies that specifically bind to PCSK9 that can be used in the context of the methods of the present disclosure include any antibody or antigen-binding fragment that comprises the three CDRs of heavy chain (HCDR1, HCDR2 and HCDR3) contained within a heavy chain variable region (HCVR) having an amino acid sequence selected from the group consisting of SEQ ID NO: 1 and 11, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity. The antibody or antigen-binding fragment may comprise all three light chain CDRs (LCVR1, LCVR2, LCVR3) contained within a light chain variable region (LCVR) having an amino acid sequence selected from the group consisting of SEQ ID NO: 6 and 15, or a substantially similar sequence thereof that has at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

In certain embodiments of the present invention, the antibody or antigen-binding fragment thereof comprises the six CDRs (HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3) of the heavy chain variable region amino acid sequence pairs. and light (HCVR / LCVR) selected from the group consisting of SEQ ID NO: 1/6.

In certain embodiments of the present invention, the anti-PCSK9 antibody, or antigen-binding fragment thereof, that can be used in the methods of the present invention has the amino acid sequences of HCDR1 / HCDR2 / HCDR3 / LCDR1 / LCDR2 / LCDR3 selected from SEQ ID NO: 2/3/4/7/8/10 (mAb316P).

In certain embodiments of the present invention, the antibody or antigen-binding fragment thereof comprises the HCVR / LCVR amino acid sequence pairs selected from the group consisting of SEQ ID NO: 1/6.

Pharmaceutical compositions and methods of administration

The present invention includes methods that comprise administering a PCSK9 inhibitor to a subject in the absence of a statin, wherein the PCSK9 inhibitor is contained within a pharmaceutical composition. The pharmaceutical compositions of the invention are formulated with suitable carriers, excipients, and other agents that provide adequate transfer, delivery, tolerance, and the like. A multitude of suitable formulations can be found in the formulary known to all pharmaceutical chemists: Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, PA. These formulations include, for example, powders, pastes, ointments, jellies, waxes, oils, lipids, lipid-containing vesicles (cationic or anionic) (such as LIPOFECTIN ™), DNA conjugates, anhydrous absorption pastes, oil emulsions in water and water in oil, Carbowax emulsions (polyethylene glycols of various molecular weights), semisolid gels and semisolid mixtures containing Carbowax. See also Powell et al. "Compendium of excipients for parenteral formulations" PDA (1998) J Pharm Sci Technol 52: 238-311.

Various delivery systems are known and can be used to deliver the pharmaceutical composition of the invention, for example, encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing the mutant viruses, receptor-mediated endocytosis (see, for example, Wu et al., 1987, J. Biol. Chem. 262: 4429-4432). Methods of administration include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The composition can be administered by any convenient route, eg, by infusion or bolus injection, by absorption through the epithelial or mucocutaneous linings (eg, oral mucosa, rectal and intestinal mucosa, etc.) and can be administered together with other biologically active agents.

A pharmaceutical composition of the present invention can be administered subcutaneously or intravenously with a standard needle and syringe. Furthermore, with respect to subcutaneous administration, a pen delivery device easily has applications in the administration of a pharmaceutical composition of the present invention. Such a pen delivery device can be reusable or disposable. A reusable pen delivery device generally uses a disposable cartridge that contains a pharmaceutical composition. Once all of the pharmaceutical composition has been delivered into the cartridge and the cartridge is empty, the empty cartridge can be easily disposed of and replaced with a new cartridge containing the pharmaceutical composition. The pen delivery device can then be reused. In a disposable pen delivery device, there is no replaceable cartridge. Rather, the disposable pen delivery device comes pre-filled with the pharmaceutical composition contained in a reservoir within the device. Once the pharmaceutical composition reservoir is emptied, the entire device is discarded.

Numerous reusable pen delivery devices and autoinjectors have applications in the subcutaneous administration of a pharmaceutical composition of the present invention. Examples include, but are not limited to, AUTOPEN ™ (Owen Mumford, Inc., Woodstock, Gb), DISETRONIC ™ pen (Disetronic Medical Systems, Burghdorf, Switzerland), HUMALOG MIX 75/25 ™ pen, HUMALOG ™ pen, HUMALIN 70/30 ™ (Eli Lilly and Co., Indianapolis, IN), NOVOPEN ™ I, II and III (Novo Nordisk, Copenhagen, Denmark), NOVOPEN JUNIOR ™ (Novo Nordisk, Copenhagen, Denmark), pen b D ™ ( Becton Dickinson, Franklin Lakes, NJ), OPTIPEN ™, OPTIPEN PRO ™, OPTIPEN STARLET ™, and OPTiCl IK ™ (sanofi-aventis, Frankfurt, Germany), to name a few. Examples of disposable pen delivery devices that have applications in the subcutaneous administration of a pharmaceutical composition of the present invention include, but are not limited to, SOLOSTAR ™ (sanofi-aventis), FLEXPEN ™ (Novo Nordisk), and KWIKPEN. ™ (Eli Lilly), SURECLICK ™ autoinjector (Amgen, Thousand Oaks, c A), PENLET ™ (Haselmeier, Stuttgart, Germany), EPlPEN (Dey, LP) and HUMIRA ™ pen (Abbott Labs, Abbott Park IL), to name a few.

It is disclosed that the pharmaceutical composition is administered in a controlled release system. It is disclosed that a pump can be used (see Langer, supra; Sefton, 1987, CRC Crit. Ref. Biomed. Eng. 14: 201). It is also disclosed that polymeric materials can be used; see Medical Applications of Controlled Release, Langer and Wise (eds.), 1974, CRC Pres., Boca Raton, Florida. It is also disclosed that a controlled release system can be brought into proximity to the target of the composition, thus requiring only a fraction of the systemic dose (see, for example, Goodson, 1984, in Medical Applications of Controlled Release, above, vol. 2, pp. 115-138). Other controlled release systems are discussed in the review by Langer, 1990, Science 249: 1527-1533.

Injectable preparations may include dosage forms for intravenous, subcutaneous, intracutaneous and intramuscular injections, drip infusions, etc. These injectable preparations can be prepared by known methods. For example, injectable preparations can be prepared, for example, by dissolving, suspending or emulsifying the antibody or its salt described above in a sterile aqueous medium or an oily medium conventionally used for injections. As an aqueous medium for injections, there is, for example, physiological saline solution, an isotonic solution containing glucose and other auxiliary agents, etc., which can be used in combination with an appropriate solubilizing agent, such as an alcohol (for example, ethanol), a polyalcohol (eg, propylene glycol, polyethylene glycol), a nonionic surfactant [eg, polysorbate 80, HCO-50 (polyoxyethylene adduct (50 moles) of hydrogenated castor oil], etc. As an oily medium, it is employ, for example, sesame oil, soybean oil, etc., which can be used in combination with a solubilizing agent, such as benzyl benzoate, benzyl alcohol, etc. In certain embodiments, the injection thus prepared is filled into a blister appropriate. In certain embodiments, the pharmaceutically acceptable carrier is a combination of histidine, pH 6.0, polysorbate 20, and sucrose.

Advantageously, the pharmaceutical compositions for oral or parenteral use described above are prepared in pharmaceutical forms in a unit dose suitable to correspond to a dose of the active principles. Such dosage unit dosage forms include, for example, tablets, pills, capsules, injections (ampoules), suppositories, etc.

Dosage and administration guidelines

The amount of PCSK9 inhibitor (eg, anti-PCSK9 antibody) administered to a subject according to the methods and compositions of the present invention is, in general, a therapeutically effective amount. The term "therapeutically effective amount" means a dose of PCSK9 inhibitor that results in a detectable reduction in one or more symptoms of hypercholesterolemia or a related disorder (eg, lipid levels and / or atherosclerotic lesions).

The amount of anti-PCSK9 antibody contained within individual doses can be expressed in terms of milligrams of antibody per kilogram of patient body weight (ie, mg / kg). For example, the anti-PCSK9 antibody can be administered to a patient at a dose of from about 0.0001 to about 10 mg / kg of patient body weight. Exemplary therapeutically effective amounts of antibody can be from about 75 mg, about 80 mg, about 90 mg, about 100 mg, about 110 mg, about 120 mg, about 130 mg, about 140 mg, about 150 mg, about 160mg, about 170mg, about 180mg, about 190mg, about 200mg, about 210mg, about 220mg, about 230mg, about 240mg, about 250mg, about 260mg, about 270mg, about 280mg , about 290 mg, about 300 mg, about 310 mg, about 320 mg, about 330 mg, about 340 mg, about 350 mg, about 360 mg, about 370 mg, about 380 mg, about 390 mg, about 400 mg, about 410 mg, about 420 mg, about 430 mg, about 440 mg, a about 450 mg, about 460 mg, about 470 mg, about 480 mg, about 490 mg, about 500 mg, about 510 mg, about 520 mg, about 530 mg, about 540 mg, about 550 mg, about 560 mg, about 570 mg, about 580 mg, about 590 mg, or about 600 mg, of the anti-PCSK9 antibody.

In certain embodiments, the anti-PCSK9 antibody is administered to a subject in a dose of about 150 mg every four weeks for at least three doses.

In some embodiments, the antibody is administered to a subject at a dose of approximately 150 mg every four weeks for 12 weeks, and the dose is maintained at 150 mg every four weeks for another 12 weeks if, at week 8, the value of Subject's LDL-C was less than 100 mg / dL and a 30% reduction in LDL-C.

In other embodiments, the antibody is administered to a subject at a dose of about 150 mg every four weeks for 12 weeks, and the dose is progressively increased to about 150 mg every two weeks for another 12 weeks if, at week 8, the The subject's LDL-C value was greater than or equal to 100 mg / dL.

In some embodiments, the antibody is administered to a subject at a dose of approximately 150 mg every four weeks for 12 weeks, and the dose is maintained at 150 mg every four weeks for another 12 weeks if, at week 8, the value of Subject's LDL-C was less than 70 mg / dL and a 30% reduction in LDL-C.

In another embodiment, the antibody is administered to a subject in a dose of approximately 300 mg every four weeks for 48 weeks.

In a further embodiment, the antibody is administered to a subject at a dose of approximately 300 mg every four weeks for a total of three doses, and the dose is changed to 150 mg every two weeks for another 36 weeks if, at week 8 , the subject did not reach a predetermined treatment goal or the subject did not have at least a 30% reduction in LDL-C from baseline.

Additional therapies

In some embodiments, the invention relates to a method of increasing the duration of action of an antibody against proprotein convertase subtilisin / kexin type 9 (PCSK9) or antigen-binding fragment thereof in a subject comprising administering to the subject an anti-PCSK9 antibody or antigen-binding fragment thereof in the absence of a statin. In some embodiments, the antagonist is an antibody or antigen-binding protein. For example, the examples show that administering an anti-PCSK9 antibody to a subject in the absence of a statin increases the duration of action of the anti-PCSK9 antibody.

In some embodiments, the invention relates to a method of maintaining a constant low-density lipoprotein cholesterol (LDL-C) reduction in an interval between administrations in a subject comprising administering to the subject, who is not taking a concomitant statin. , a pharmaceutical composition comprising an anti-proprotein convertase subtilisin / kexin type 9 antibody (anti-PCSK9) or antigen-binding protein in a dose of approximately 150 mg every 4 weeks for at least 3 doses. As shown in Figure 1, state-of-the-art therapies exhibit a "sawtooth profile" of LDL-C during treatment. In contrast, the c / 4sem dosage regimen maintains a constant reduction in LDL-C in the interval between administrations in patients who do not receive a statin.

In some embodiments, the invention relates to a method of lowering low-density lipoprotein cholesterol (LDL-C) in a subject in need thereof which comprises administering to the subject, who is not taking a concomitant statin, a pharmaceutical composition. comprising an anti-proprotein convertase subtilisin / kexin type 9 antibody (anti-PCSK9) or antigen-binding protein in a dose of approximately 150 mg every 4 weeks for at least 3 doses, thus reducing LDL-C in the subject.

In some embodiments, the invention relates to a method of treating hypercholesterolemia in a subject in need thereof which comprises administering to the subject, who is not taking a concomitant statin, a pharmaceutical composition comprising an anti-proprotein convertase subtilisin / kexin antibody. type 9 (anti-PCSK9) or antigen-binding protein in a dose of approximately 150 mg every 4 weeks for at least 3 doses, thus treating hypercholesterolemia in the subject.

In some embodiments, the invention relates to a method of treating a form of hypercholesterolemia that is unfamiliar hypercholesterolemia in a subject in need thereof comprising administering to the subject a pharmaceutical composition comprising a subtilisin / kexin-type anti-proprotein convertase antibody. 9 (anti-PCSK9) or antigen-binding protein in a dose of approximately 150 mg every 4 weeks for at least 3 doses, thus treating the form of hypercholesterolemia which is unfamiliar hypercholesterolemia in the subject.

Thus, the methods of the present invention, according to certain embodiments, comprise administering a pharmaceutical composition comprising an anti-PCSK9 antibody to a subject, in the absence of a statin.

The methods of the present invention, according to certain embodiments, also comprise administering a pharmaceutical composition comprising an anti-PCSK9 antibody or antigen-binding fragment thereof to a subject in combination with another non-statin lipid-lowering agent.

Lipid-lowering agents include, for example, agents that inhibit the uptake of cholesterol and or the reabsorption of bile acids (such as ezetimibe); agents that increase the catabolism of lipoproteins (such as niacin); and / or activators of the transcription factor LXR that play a role in the removal of cholesterol, such as 22-hydroxycholesterol.

It is disclosed that the subject was previously on a therapeutic regimen for the treatment of hypercholesterolemia prior to administration of the pharmaceutical composition of the invention. For example, a patient who had previously been diagnosed with hypercholesterolemia could have been prescribed and was taking a stable therapeutic regimen of another drug prior to administration of a pharmaceutical composition comprising an anti-PCSK9 antibody.

In some embodiments, the subject was pretreated with a statin or other lipid-lowering agent prior to treatment with a PCSK9 inhibitor described herein. In other embodiments, the subject has not previously been treated with a statin or other lipid-lowering agent.

Exemplary Realizations and Disclosure

In one aspect, the present disclosure provides a method of lowering low-density lipoprotein cholesterol (LDL-C) in a subject in need thereof, the method comprising administering to the subject, who is not taking a concomitant statin, a composition Pharmaceutical drug comprising an anti-proprotein convertase subtilisin / kexin type 9 antibody (anti-PCSK9) or antigen-binding protein in a dose of approximately 150 mg every 4 weeks for at least 3 doses, thus reducing LDL-C in the subject .

In certain embodiments, the antibody or antigen-binding fragment thereof comprises the complementarity determining regions (CDRs) of the heavy and light chains of a pair of amino acid sequences of the variable region of the heavy chain / variable region of the light chain (HCVR / LCVR) selected from the group consisting of SEQ ID NO: 1/6 and 11/15. The antibody or antigen-binding fragment thereof is disclosed to comprise heavy and light chain CDR amino acid sequences having SEQ ID NO: 12, 13, 14, 16, 17 and 18. The antibody or fragment is disclosed antigen-binding thereof comprises an HCVR having the amino acid sequence of SEQ ID NO: 11 and an LCVR having the amino acid sequence of SEQ ID NO: 15. In certain embodiments, the antibody or antigen-binding fragment of the itself comprises heavy and light chain CDR amino acid sequences having SEQ ID NO: 2, 3, 4, 7, 8 and 10. In certain embodiments, the antibody or antigen-binding fragment thereof comprises an HCVR which has the amino acid sequence of SEQ ID NO: 1 and an LCVR that has the amino acid sequence of SEQ ID NO: 6.

The antibody or antigen-binding fragment thereof is disclosed to bind to the same epitope on PCSK9 as an antibody comprising heavy and light chain CDR amino acid sequences having SEQ ID NO: 12, 13, 14, 16, 17 and 18; or SEQ ID NO: 2, 3, 4, 7, 8 and 10.

The antibody or antigen-binding fragment thereof is disclosed to compete for binding to PCSK9 with an antibody comprising heavy and light chain CDR amino acid sequences having SEQ ID NO: 12, 13, 14, 16, 17 and 18; or SEQ ID NO: 2, 3, 4, 7, 8 and 10.

In certain embodiments, the subject has a form of hypercholesterolemia that is unfamiliar hypercholesterolemia (HnF). In certain embodiments, the subject has heterozygous familial hypercholesterolemia (heHF). In certain embodiments, the diagnosis of heFH is made either by genotyping or by clinical criteria. In certain embodiments, the clinical criteria are either the Simon Broome Registry Diagnostic Criteria for Heterozygous Familial Hypercholesterolemia, or the WHO / Dutch Lipic Network criteria with a score> 8.

In certain embodiments, the subject is receiving a non-statin lipid lowering agent prior to and / or during administration of the antibody or antigen-binding protein. In certain embodiments, the non-statin lipid-lowering agent is selected from the group consisting of: ezetimibe, a fibrate, fenofibrate, niacin, an omega-3 fatty acid, and a bile acid resin. In certain embodiments, the non-statin lipid-lowering agent is ezetimibe or fenofibrate.

In certain embodiments, the subject is not receiving a non-statin lipid lowering agent prior to and / or during administration of the antibody or antigen-binding protein.

In certain embodiments, the antibody or antigen-binding protein is administered subcutaneously.

In certain embodiments, the dose of about 150 mg every 4 weeks is maintained if the subject's LDL-C measured after 4 or more doses is <70 mg / dL. In certain embodiments, the dose of approximately 150 mg every 4 weeks is discontinued if the subject's LDL-C measured after 4 or more doses is> 70 mg / dL, and the antibody or antigen-binding protein is subsequently administered to the subject. in doses of approximately 150 mg every 2 weeks.

In another aspect, the present disclosure provides a method of treating hypercholesterolemia in a subject in need thereof, the method comprising administering to the subject, who is not taking a concomitant statin, a pharmaceutical composition comprising a subtilisin / anti-proprotein convertase antibody. kexin type 9 (anti-PCSK9) or antigen-binding protein in a dose of approximately 150 mg every 4 weeks for at least 3 doses, thus treating hypercholesterolemia in the subject.

In certain embodiments, the antibody or antigen-binding fragment thereof comprises the complementarity determining regions (CDRs) of the heavy and light chains of a pair of amino acid sequences of the variable region of the heavy chain / variable region of the light chain (HCVR / LCVR) selected from the group consisting of SEQ ID NO: 1/6. In certain aspects of the disclosure, the antibody or antigen-binding fragment thereof comprises heavy and light chain CDR amino acid sequences having SEQ ID NO: 12, 13, 14, 16, 17 and 18. In certain aspects of the disclosure, the antibody or antigen-binding fragment thereof comprises an HCVR having the amino acid sequence of SEQ ID NO: 11 and an LCVR having the amino acid sequence of SEQ ID NO: 15. In certain embodiments, the The antibody or antigen-binding fragment thereof comprises heavy and light chain CDR amino acid sequences having SEQ ID NO: 2, 3, 4, 7, 8, and 10. In certain embodiments, the antibody or antibody-binding fragment antigen thereof comprises a HCVR having the amino acid sequence of SEQ ID NO: 1 and an LCVR having the amino acid sequence of SEQ ID NO: 6.

In certain aspects of the disclosure, the antibody or antigen-binding fragment thereof binds to the same epitope on PCSK9 as an antibody comprising heavy and light chain CDR amino acid sequences having SEQ ID NO: 12, 13, 14, 16, 17 and 18; or SEQ ID NO: 2, 3, 4, 7, 8 and 10.

In certain aspects of the disclosure, the antibody or antigen-binding fragment thereof competes for binding to PCSK9 with an antibody comprising heavy and light chain CDR amino acid sequences having SEQ ID NO: 12, 13, 14 , 16, 17 and 18; or SEQ ID NO: 2, 3, 4, 7, 8 and 10.

In certain embodiments, the subject has a form of hypercholesterolemia that is unfamiliar hypercholesterolemia (HnF). In certain embodiments, the subject has heterozygous familial hypercholesterolemia (heHF). It is revealed that the diagnosis of heHF is made either by genotyping or by clinical criteria. In certain embodiments, the clinical criteria are either the Simon Broome Registry Diagnostic Criteria for Heterozygous Familial Hypercholesterolemia or the WHO / Dutch Lipid Network criteria with a score> 8.

In certain embodiments, the subject receives a non-statin lipid lowering agent prior to and / or during administration of the antibody or antigen-binding protein. In certain embodiments, the non-statin lipid-lowering agent is selected from group consisting of: ezetimibe, a fibrate, fenofibrate, niacin, an omega-3 fatty acid and a bile acid resin. In certain embodiments, the non-statin lipid-lowering agent is ezetimibe or fenofibrate.

In certain embodiments, the subject does not receive a non-statin lipid lowering agent prior to and / or during administration of the antibody or antigen-binding protein.

In certain embodiments, the antibody or antigen-binding protein is administered subcutaneously.

In certain embodiments, the dose of about 150 mg every 4 weeks is maintained if the subject's LDL-C measured after 4 or more doses is <70 mg / dL. In certain embodiments, the dose of approximately 150 mg every 4 weeks is discontinued if the subject's LDL-C measured after 4 or more doses is> 70 mg / dL, and the antibody or antigen-binding protein is subsequently administered to the subject. in doses of approximately 150 mg every 2 weeks.

In another aspect, the present disclosure provides a method of treating a form of hypercholesterolemia that is unfamiliar hypercholesterolemia in a subject in need thereof, the method comprising administering to the subject a pharmaceutical composition comprising an anti-proprotein convertase subtilisin / kexin antibody. type 9 (anti-PCSK9) or antigen-binding protein in a dose of approximately 150 mg every 4 weeks for at least 3 doses, thus treating the form of hypercholesterolemia which is unfamiliar hypercholesterolemia in the subject.

The antibody or antigen-binding fragment thereof is disclosed to comprise the complementarity determining regions (CDRs) of the heavy and light chains of a pair of heavy chain variable region / chain variable region amino acid sequences. light (HCVR / LCVR) selected from the group consisting of SEQ ID NO: 1/6 and 11/15. The antibody or antigen-binding fragment thereof is disclosed to comprise heavy and light chain CDR amino acid sequences having SEQ ID NO: 12, 13, 14, 16, 17 and 18. The antibody or fragment is disclosed antigen-binding thereof comprises an HCVR having the amino acid sequence of SEQ ID NO: 11 and an LCVR having the amino acid sequence of SEQ ID NO: 15. In certain embodiments, the antibody or antigen-binding fragment of the itself comprises heavy and light chain CDR amino acid sequences having SEQ ID NO: 2, 3, 4, 7, 8, and 10. In certain embodiments, the antibody or antigen-binding fragment thereof comprises an HCVR having the amino acid sequence of SEQ ID NO: 1 and an LCVR having the amino acid sequence of s Eq ID NO: 6.

The antibody or antigen-binding fragment thereof is disclosed to bind to the same epitope on PCSK9 as an antibody comprising heavy and light chain CDR amino acid sequences having SEQ ID NO: 12, 13, 14, 16, 17 and 18; or SEQ ID NO: 2, 3, 4, 7, 8 and 10.

The antibody or antigen-binding fragment thereof is disclosed to compete for binding to PCSK9 with an antibody comprising heavy and light chain CDR amino acid sequences having SEQ ID NO: 12, 13, 14, 16, 17 and 18; or SEQ ID NO: 2, 3, 4, 7, 8 and 10.

In certain embodiments, the subject has a form of hypercholesterolemia that is unfamiliar hypercholesterolemia (HnF). In certain embodiments, the subject has heterozygous familial hypercholesterolemia (heHF). It is revealed that the diagnosis of heHF is made either by genotyping or by clinical criteria. In certain embodiments, the clinical criteria are either the Simon Broome Registry Diagnostic Criteria for Heterozygous Familial Hypercholesterolemia or the WHO / Dutch Lipid NetWork criteria with a score> 8.

In certain embodiments, the subject receives a non-statin lipid lowering agent prior to and / or during administration of the antibody or antigen-binding protein. In certain embodiments, the non-statin lipid-lowering agent is selected from the group consisting of: ezetimibe, a fibrate, fenofibrate, niacin, an omega-3 fatty acid, and a bile acid resin. In certain embodiments, the non-statin lipid-lowering agent is ezetimibe or fenofibrate.

In certain embodiments, the subject does not receive a non-statin lipid lowering agent prior to and / or during administration of the antibody or antigen-binding protein.

In certain embodiments, the antibody or antigen-binding protein is administered subcutaneously.

In certain embodiments, the dose of about 150 mg every 4 weeks is maintained if the subject's LDL-C measured after 4 or more doses is <70 mg / dL. In certain embodiments, the dose of approximately 150 mg every 4 weeks is discontinued if the subject's LDL-C measured after 4 or more doses is> 70 mg / dL, and the antibody or antigen-binding protein is subsequently administered to the subject. in doses of approximately 150 mg every 2 weeks.

In another aspect, the present disclosure provides a dosage regimen of an anti-proprotein convertase subtilisin / kexin type 9 antibody (anti-PCSK9) or antigen-binding protein that maintains a constant reduction in low-density lipoprotein cholesterol (C- LDL) in the interval between administrations in a human subject who, after administration of the anti-PCSK9 antibody or antigen-binding protein thereof in a dose of approximately 150 mg every 4 weeks for at least 3 doses, has one or more of the properties selected from the group consisting of: (a) an area under the plasma concentration vs. time curve calculated using the trapezoid rule from zero time to real time (ABC ^ultimate ) from about 250 mgday / L to about 650 mgday / L; (b) a maximum observed plasma concentration (C ^max ) from about 15 mg / L to about 33 mg / L; (c) a first time to reach a maximum plasma concentration (t ^max ) of approximately 7 days; and (d) a time to reach the terminal half-life (t ^{1 / 2Z} ) from about 5.5 days to about 12 days.

In another aspect, the present disclosure provides a dosage regimen of an anti-proprotein convertase subtilisin / kexin type 9 antibody (anti-PCSK9) or antigen-binding protein that maintains a constant reduction in low-density lipoprotein cholesterol (C- LDL) in the interval between administrations in a human subject who, after administration of the anti-PCSK9 antibody or antigen-binding protein thereof in a dose of approximately 150 mg every 4 weeks for at least 3 doses, has one or more of the properties selected from the group consisting of: (a) an area under the plasma concentration vs. time curve calculated using the trapezium rule from zero time to real time ( ^ultimate AUC) from about 150 mg / day / L to about 450 mgday / L; (b) a maximum observed plasma concentration (C ^max ) from about 10.5 mg / L to about 24 mg / L; (c) a first time to reach a maximum plasma concentration (t ^max ) of approximately 7 days; and (d) a time to reach the terminal half-life (t ^{1 / 2Z} ) from about 5 days to about 9 days.

In another aspect, the present disclosure provides a method of maintaining a constant reduction in low-density lipoprotein cholesterol (LDL-C) in an interval between administrations in a subject, the method comprising administering to the subject, who is not taking a statin. concomitantly, a pharmaceutical composition comprising an anti-proprotein convertase subtilisin / kexin type 9 antibody (anti-PCSK9) or antigen-binding protein in a dose of approximately 150 mg every 4 weeks for at least 3 doses.

In certain embodiments, the antibody or antigen-binding fragment thereof comprises the complementarity determining regions (CDRs) of the heavy and light chains of a pair of amino acid sequences of the variable region of the heavy chain / variable region of the light chain (HCVR / LCVR) selected from the group consisting of SEQ ID NO: 1/6 and 11/15. The antibody or antigen-binding fragment thereof is disclosed to comprise heavy and light chain CDR amino acid sequences having SEQ ID NO: 12, 13, 14, 16, 17, and 18. In certain embodiments, the antibody or antigen-binding fragment thereof comprises an HCVR having the amino acid sequence of SEQ ID NO: 11 and an LCVR having the amino acid sequence of SEQ ID NO: 15. In certain embodiments, the antibody or antigen-binding fragment thereof comprises heavy and light chain CDR amino acid sequences having SEQ ID NO: 2, 3, 4, 7, 8 and 10. In certain embodiments, the antibody or antigen-binding fragment thereof comprises an HCVR which has the amino acid sequence of SEQ ID NO: 1 and an LCVR that has the amino acid sequence of SEQ ID NO: 6.

The antibody or antigen-binding fragment thereof is disclosed to bind to the same epitope on PCSK9 as an antibody comprising heavy and light chain CDR amino acid sequences having SEQ ID NO: 12, 13, 14, 16, 17 and 18; or SEQ ID NO: 2, 3, 4, 7, 8 and 10.

The antibody or antigen-binding fragment thereof is disclosed to compete for binding to PCSK9 with an antibody comprising heavy and light chain CDR amino acid sequences having SEQ ID NO: 12, 13, 14, 16, 17 and 18; or SEQ ID NO: 2, 3, 4, 7, 8 and 10.

In certain embodiments, the subject has a form of hypercholesterolemia that is unfamiliar hypercholesterolemia (HnF). In certain embodiments, the subject has heterozygous familial hypercholesterolemia (heHF). It is revealed that the diagnosis of heHF is made either by genotyping or by clinical criteria. In certain embodiments, the clinical criteria are either the Simon Broome Registry Diagnostic Criteria for Heterozygous Familial Hypercholesterolemia or the WHO / Dutch Lipid NetWork criteria with a score> 8.

In certain embodiments, the subject receives a non-statin lipid lowering agent prior to and / or during administration of the antibody or antigen-binding protein. In certain embodiments, the non-statin lipid-lowering agent is selected from the group consisting of: ezetimibe, a fibrate, fenofibrate, niacin, an omega-3 fatty acid, and a bile acid resin. In certain embodiments, the non-statin lipid-lowering agent is ezetimibe or fenofibrate.

In certain embodiments, the subject does not receive a non-statin lipid lowering agent prior to and / or during administration of the antibody or antigen-binding protein.

In certain embodiments, the antibody or antigen-binding protein is administered subcutaneously.

In certain embodiments, the dose of about 150 mg every 4 weeks is maintained if the subject's LDL-C measured after 4 or more doses is <70 mg / dL. In certain embodiments, the dose of approximately 150 mg every 4 weeks is discontinued if the subject's LDL-C measured after 4 or more doses is> 70 mg / dL, and the antibody or antigen-binding protein is subsequently administered to the subject. in doses of approximately 150 mg every 2 weeks.

EXAMPLES

The following examples are presented so as to provide those of ordinary skill in the art with a full disclosure and description of how to prepare and use the methods and compositions of the invention, and are not intended to limit the scope of what the inventors consider their invention. .

Example 1: Generation of human antibodies against human PCSK9

Anti-human PCSK9 antibodies were generated as described in US Patent No. 8,062,640. The exemplary PCSK9 inhibitor used in the following examples is the anti-human PCSK9 antibody designated "alirocumab". Alirocumab has the following amino acid sequence characteristics: heavy chain variable region (HCVR) comprising SEQ ID NO: 90; light chain variable domain (LCVR) comprising SEQ ID NO: 92; heavy chain complementarity determining region 1 (HCDR1) comprising SEQ ID NO: 76; HCDR2 comprising SeQ ID NO: 78; HCDR3 comprising SEQ ID NO: 80; light chain complementarity determining region 1 (LCDR1) comprising SEQ ID NO: 84; LCDR2 comprising SEQ ID NO: 86; and LCDR3 comprising SEQ ID NO: 88.

Alirocumab is a fully human monoclonal antibody that binds to the proprotein convertase subtilisin kexin type 9 (PCSK9). The proprotein convertase subtilisin kexin type 9 belongs to the subtilisin family of serine proteases and is highly expressed in the liver. PCSK9 participates in the regulation of the levels of the low-density lipoprotein receptor (LDLR) protein. In animals and humans, alirocumab reduces circulating LDL-C levels. Alirocumab may be an effective treatment to lower LDL-C and to reduce the risk of cardiovascular disease.

Example 2: Randomized, partially concealed, 3-group parallel study of the pharmacodynamic profile of alirocumab administered as multiple subcutaneous 150 mg doses, both alone and in addition to ezetimibe or fenofibrate administered as multiple oral doses in healthy subjects

Introduction

A phase 1 clinical trial was conducted to evaluate the pharmacodynamics and safety of an anti-PCSK9 antibody, alirocumab, in healthy subjects. The main objective of the study was to evaluate the pharmacodynamic profile of alirocumab administered both alone and in addition to ezetimibe or fenofibrate, according to low-density lipoprotein cholesterol (LDL-C). The secondary objectives of the study were: 1) to evaluate the pharmacodynamic profile of alirocumab administered both alone and in addition to ezetimibe or fenofibrate, according to other lipid parameters, 2) to evaluate the pharmacokinetic profile of alirocumab administered both alone and in addition to ezetimibe or fenofibrate, 3 ) document exposure to ezetimibe and fenofibrate, 4) evaluate the effect of either ezetimibe or fenofibrate on PCSK9 levels, and 5) evaluate the safety of concomitant administration of alirocumab and / or ezetimibe or fenofibrate.

This phase 1 study was a randomized, partially concealed, controlled study conducted in 3 parallel groups, with a 4-week run-in period with either ezetimibe or fenofibrate or placebo ezetimibe, followed by a 17-week treatment period ( that is: group A: alirocumab ezetimibe placebo, group B: alirocumab ezetimibe, group C: alirocumab fenofibrate). The study investigated the impact of combining 150 mg every 4 weeks with ezetimibe (EZE) and fenofibrate (FENO) on the LDL-C lowering effect and on the circulating levels of free PCSK9 and total alirocumab.

Subjects

The study evaluated 72 healthy subjects. The inclusion criteria were: healthy men or women, aged between 18 and 65 years, with serum LDL-C levels> 130 mg / dL who did not receive lipid-lowering therapy at selection (day -29), and C levels Serum LDL> 100 mg / dL in baseline control on day -1 (before initiation of alirocumab). (Note: for practical reasons, the baseline LDL-C blood sample was taken at D-1, to verify that the subject still had an LDL-C level> 100 mg / dL before starting treatment with alirocumab).

Seventy-two subjects were randomized to three groups, with 24 subjects per group. The baseline characteristics of the subjects are shown in Table 1.

Table 1. Characteristics of the patients at baseline (day -29)

Alirocumab 150 mg Alirocumab Alirocumab 150 mg w / 4 weeks placebo 150 mg w / 4 weeks w / 4 weeks FENO Treatment group (N = 24) EZE (N = 24) (N = 24)

Age (years), mean (SD) 48.5 (12.8) 49.5 (10.7) 54.6 (7.6) Men (%) 46 46 42

Body mass index (kg / m2), mean (SD) 23.9 (2.0) 25.5 (2.7) 24.7 (2.5)

Calculated LDL-C (mg / dL), mean (SD) 183.3 (38.7) 181.7 (37.1) 180.6 (31.3) Total cholesterol (mg / dL), mean (SD) 264.5 (43.7) 250.6 (40.6) 263.7 (40.6) Apolipoprotein B (g / L), mean (SD) 1.3 (0.21) 1.3 (0, 22) 1.3 (0.17)

Non-HDL-C (mg / dL), mean (SD) 198.4 (40.6) 200.3 (39.8) 199.5 (31.7) HDL-C (mg / dL), mean (SD ) 65.7 (12.4) 60.3 (13.1) 64.2 (15.5) Triglycerides (mg / dL), median (range) 78.8 (44.3-177.1) 95, 7 (35.4-168.3) 94.8 (53.1-194.9) Lipoprotein (a) (g / L), median (range) 0.27 (0.0-1.6) 0, 33 (0.0-1.6) 0.17 (0.0-1.5) Free PCSK9 (ng / L), mean (SD) 146.5 (54.3) 150.7 (48.5) 152.1 (54.1) C-HDL, high-density lipoprotein cholesterol; SD, standard deviation

Study treatments

Alirocumab was formulated as a 150 mg / ml injectable solution. Alirocumab was administered subcutaneously in the abdomen. Alirocumab was administered at a dose of 150 mg, 1 injection every four weeks, for a total of three injections.

Ezetimibe was formulated as an additional 10 mg encapsulated tablet (and equivalent placebo ezetimibe capsule). Ezetimibe was administered orally. Ezetimibe was administered at a dose of 10 mg, administered once daily for a total duration of 21 weeks (4-week run-in followed by 17 weeks after initial alirocumab administration).

Fenofibrate was formulated into a 160 mg coated tablet. Fenofibrate was administered orally during a meal. Fenofibrate was administered at a dose of 160 mg, administered once daily for a total duration of 21 weeks (4-week run-in followed by 17 weeks after initial alirocumab administration).

The duration of treatment was as follows. Subjects received repeat doses (daily doses) of ezetimibe or fenofibrate or placebo ezetimibe for the duration of the run-in period (days -28 to -1), and throughout the treatment phase (days 1 to 120), and repeat doses of alirocumab (on days 1, 29 and 57), in addition to continuing to take ezetimibe or fenofibrate or placebo ezetimibe.

The duration of observation was up to a maximum of 25 weeks per subject (from screening visit to study completion visit [EF]) which included a 3-week screening period, a 4-week run-in period, a 17 week treatment period and one FE visit (a minimum of 4 days after the last administration with ezetimibe, placebo or fenofibrate).

Evaluation criteria

The pharmacodynamic criteria were as follows. The primary endpoint was the percent change in serum LDL-C calculated from baseline (d -29). Secondary endpoints were absolute change from baseline in calculated LDL-C, total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), non-HDL-C, triglycerides (TG), apolipoprotein B (ApoB), apolipoprotein A1 (ApoA1) and lipoprotein a (Lp [a]) analyzed in percentage and absolute change from baseline.

The following pharmacokinetic parameters (PK) were calculated for alirocumab, using non-compartmental methods: maximum observed serum concentration (C ^max ), serum concentrations just before administration of treatment during repeated administration (C ^trough ), serum concentration observed on the day 29 (C ^D29 ), area under the curve of serum concentration versus time calculated using the trapezium rule from time zero to real time ( ^last AUC), area under the curve of serum concentration vs. time extrapolated to infinity (AUC), time to reach C ^max (t ^max ), area under the curve of serum concentration vs. time calculated using the trapezoid rule from time zero to day 14 (AUC ^0-D14 ), area under the curve of serum concentration versus time calculated using the trapezoid rule from time zero to day 29 of study (AUC ^0-D28 ), terminal half-life associated with the terminal slope (t ^{1 / 2z} ), time corresponding to the last concentration above the limit of quantification ( ^last t), apparent total body clearance of a drug from serum (CL / F), volume of distribution in the steady state (V ^{ss / f} ), mean time that a molecule remains in the body (MRT) and volume of distribution in the terminal phase (V ^{z / f} ). Total serum alirocumab concentrations, total and free proprotein convertase subtilisin kexin type 9 (PCSK9) concentrations, and anti-alirocumab antibodies were measured.

Subjects were monitored for safety for adverse events (AA; including local tolerability) spontaneously reported by subjects or observed by the investigator, clinical laboratory evaluations (biochemistry, hematology, coagulation, and urinalysis), vital sign evaluations (frequency cardiac, systolic blood pressure and diastolic blood pressure), body weight, physical examination, automatic 12-lead electrocardiogram (ECG) on- site recordings, urine drug analysis; breath or plasma breathalyzer test; human chorionic p-gonadotropin levels in women; and immunogenicity evaluations (anti-alirocumab antibody titers).

Sampling times for pharmacodynamics

Blood tests for lipid parameters (i.e. LDL-C, CT, HDL-C, non-HDL-C, TG, ApoB, ApoA1, Lp [a]) were done in the morning, fasting (in fasting at least 10 hours and abstinence from smoking), before taking any drug.

Blood samples were collected at screening; day -1 of the run-in phase; and days 8, 15, 22, 29 ± 1, 57 ± 1, 64 ± 1.71 ± 1.78 ± 1.85 ± 1.99 ± 2 and 120 ± 3 of the treatment phase.

Sampling times and bioanalytical methods for pharmacokinetics

Blood samples were collected for pharmacokinetics for the alirocumab and total and free PCSK9 assay on day -28 and day -15 ± 2 of the run-in phase (total and free PCSK9 assay alone); on days 1, 8, 15, 22, 29, 57, 64, 71,78, 85, 99, 120 of the treatment phase; and the visit FE.

Pharmacokinetic blood samples were collected for the total and unconjugated ezetimibe assay, as well as fenofibric acid, on day -28, day -15, and day -1 of the run-in phase; and on days 1, 29, 57 and 64 of the treatment phase.

Samples were collected for determination of serum anti-drug antibody (ADA) levels on day -28 of the run-in phase; days 29, 57, 85 and 120 of the treatment phase; and the visit FE.

Serum alirocumab concentrations were determined using a validated enzyme-linked immunosorbent assay (ELISA) with a lower limit of quantification (LLOQ) of 0.078 pg / mL.

Plasma concentrations of total and unconjugated ezetimibe and fenofibric acid were determined using liquid chromatography validated with tandem mass spectrometry (LC-MS / MS) methods with lower limits of quantification of 0.2 ng / mL, 0 , 04 ng / mL and 49.9 ng / mL, respectively.

Anti-alirocumab antibody samples were analyzed using a validated electrochemiluminescence assay for the determination of anti-alirocumab antibody titers in acid-treated human serum. Based on the minimum dilution of the samples, the minimum titer for any anti-alirocumab antibody positive sample was 30. In pure serum samples, the validated lower limit of detection was approximately 1.5 ng / mL.

Summary

population characteristics

The study population included 72 subjects (24 in each treatment group); there were 32 men and 40 women between the ages of 21 and 65.

Pharmacodynamic results

LDL-C decreased similarly in the ezetimibe and fenofibrate groups during the run-in period (day -29 to day -1) reaching -19.8 (2.1)% and -25.9 (3.2)% , in the ezetimibe and fenofibrate groups, respectively, on day -1 and was stable in the placebo group (+1.6 (3.0)% on day -1) (Figure 2). After the run-in period, alirocumab treatment given from day 1 produced a further decrease, greater in the ezetimibe and fenofibrate groups. The relative change in LDL-C was parallel in the 3 treatment groups until day 15, then from day 15 a new and modest increase began in the fenofibrate group, while this was more stable until day 22 in the ezetimibe group, from which a slight further increase was also observed. This trend was not observed in the alirocumab group, only in which the decrease in CLDL. The 3rd administration of alirocumab produced an additional decrease in LDL-C, compared to the decrease observed after the 1st dose, and a maximum effect was achieved after the 3rd administration of alirocumab on day 71 (14 days after the administration ) in all treatment groups (p <0.0001), regardless of the baseline level considered (day -29 or day -1), and with a behavior similar to that observed 21 days after the 1st administration in the ezetimibe and fenofibrate (Figures 2 and 3; Table 2). The maximum decreases were -47.59%, -65.34%, and -66.75% in the alirocumab alone, ezetimibe, and fenofibrate groups, respectively (change from baseline on day -29). Using the change from baseline on day -1, the maximum decreases were -47.39%, -56.56%, and -54.34% in the alirocumab alone, ezetimibe, and fenofibrate groups, respectively. On day 28 after the 3rd administration of alirocumab (i.e .: day 85), the decreases were -47.03%, -49.57% and -43.17% for alirocumab administered either alone, or with ezetimibe or with fenofibrate, respectively (change from baseline on day -1). See Figures 2 and 3.

The overall effect of the treatment was significant (p <0.0001). Pairwise comparisons showed that the difference between alirocumab alone and alirocumab co-administered with ezetimibe remained significant at all time points (Figure 2). The difference between alirocumab alone and alirocumab co-administered with fenofibrate also remained significant at all times, except on day 99 (CI: -13.8174 to 0.1219%).

As shown in Figure 4, the analysis of the difference between the mean estimates of alirocumab co-administered with ezetimibe versus alirocumab alone and alirocumab co-administered with fenofibrate versus alirocumab alone does not show any difference with respect to the study time course between the fenofibrate and ezetimibe groups. The relative changes in TC paralleled the change in LDL-C. The treatment effect was significant for both TG and HDL-C. Compared to alirocumab alone, fenofibrate produced an increase in HDL-C and a decrease in TG, which were maintained and significant during most time points, while in comparison with alirocumab only the changes in the ezetimibe group they were, in general, not significant.

The effects on other lipid parameters are summarized in Table 2. Table 2 shows that: 1) mean percent reductions (SEM) from the main basal level on day -29 to day 71 were - 48.2 ( 2.3)% with alirocumab alone, and -65.3 (2.0)% and -66.8 (2.7)% with alirocumab EZE and FENO, respectively; and 2) mean percent LDL-C reductions (SEM) from baseline on day -1 to day 71 were -47.4 (3.2)% with alirocumab alone, and -56.6 (2, 5)% and -54.3 (3.5)% with alirocumab EZE and FENO, respectively.

Table 2. Mean percent change (SEM) in other lipid parameters from baseline on day -29 and baseline on day -1 to day 71 (14 days after the 3rd dose of alirocumab)

Main baseline (day -29) (from baseline Additional baseline (day -1) (from start of placebo run-in period, EZE injection of alirocumab; continued on of En O) placebo / EZE / treatment FENO)

Alirocumab group Alirocumab Alirocumab Alirocumab Alirocumab Alirocumab treatment 150 mg 150 mg 150 mg 150 mg 150 mg 150 mg q / 4 w / 4 w / 4 w / 4 w / 4 w / 4 w placebo EZE (N = 24) FENO placebo EZE ( N = 24) FENO (N = 24) (N = 24) (N = 24) (N = 24)

LDL-C -48.2 (2.3) -65.3 (2.0) -66.8 (2.7) -47.4 (3.2) -56.6 (2.5) -54 , 3 (3.5)

Total cholesterol -31.6 (1.4) -45.7 (1.5) -46.1 (1.9) -31.5 (2.6) -36.5 (1.4) -32, 4 (2.2)

C-no HDL -43.0 (1.7) -60.6 (1.9) -64.4 (2.5) -43.0 (2.7) -51.9 (2.1) - 50.5 (3.2)

Apolipoprotein B -39.1 (1.5) -53.5 (1.8) -58.3 (2.1) -38.4 (2.4) -44.9 (2.0) -44, 6 (2.5)

HDL-C 3.3 (3.4) 5.4 (3.2) 12.3 (3.1) 3.6 (2.9) 6.4 (3.1) 8.7 (3.0 )

Triglycerides * 5.7 (-48.5 to -13.8 (-53.4 to -36.0 (-57.9 to -3.9 (-41.3 to -16.5 (-37.2 a -3.5 (-58.0 a 266.7) 53.5) 11.3) 77.6) 24.2) 74.1)

Lipoprotein (a) * -20.3 (-63.2 -27.0 (-71.4 to -19.9 (-57.6 to -11.7 (-58.8 to -9.2 (- 67.0 to -20.4 (-56.8 to 33.3) 35.8) 38.3) 160.9) 66.7) to 17.7)

* Median (range)

Figure 5 is a group of four graphs showing mean free PCSK9 levels, comparing the three treatment groups together, and compared to percent changes in LDL-C from baseline on day -29, by group. treatment.

Figure 6 is a group of three graphs showing percentage changes in LDL-C from baseline on day -29, levels of free PCSK9, and total alirocumab from day 57 (time of 3rd injection of alirocumab) to day 85 (28 days after the 3rd injection of alirocumab).

During the placebo run-in period (ie, before alirocumab treatment), FENO produced elevated levels of free PCSK9 from 152 to 217 ng / mL. Corresponding changes in free PCSK9 were 147 to 119 ng / mL in the placebo group and 151 to 142 ng / mL in the EZE group (Figure 5A).

A complete suppression of free PCSK9 followed the 1st and 3rd injections of alirocumab (no action was taken after the 2nd injection) (Figure 5A).

After 7 to 15 days after alirocumab injection, free PCSK9 levels had increased again in the FENO group and, to a lesser extent, in the EZE group, compared to the alirocumab alone group (Figure 5A ). These increases corresponded with a modest decrease in efficacy observed in the EZE and FENO groups (Figure 5B-D; and close-up in Figure 6A + B).

Corresponding to changes in free PCSK9, alirocumab exposures in the presence of FENO were reduced (geometric mean ratio [95% confidence interval] versus alirocumab alone: 0.64 [0.53 to 0.77]), thus as in the presence of EZE (0.85 [0.70 to 1.03] versus alirocumab alone) (Figure 6C).

Safety results

There were no deaths or SAEs during the study. The incidence of AAAT was similar in the 3 treatment groups (50.0%, 58.3%, and 50.0% in the alirocumab alone, ezetimibe, and fenofibrate groups, respectively). Only 1 subject had an AAAT of intense intensity, recorded in the fenofibrate group (renal colic, which lasted approximately 11 hours, which occurred 64 days after the last administration of alirocumab, and was considered unrelated to treatment). The most common AAATs (that is, recorded in> 2 subjects in any treatment group) were headache (3/24, 5/24, and 2/24 subjects in the alirocumab alone, ezetimibe, and fenofibrate groups, respectively), nasopharyngitis ( 3/24, 4/24, and 4/24 subjects in the alirocumab alone, ezetimibe, and fenofibrate groups, respectively), influenza (2/24, 0/24, and 1/24 subjects in the alirocumab alone, ezetimibe, and fenofibrate groups , respectively), viral gastroenteritis (0/24, 0/24, and 2/24 subjects in the alirocumab alone, ezetimibe, and fenofibrate groups, respectively), influenza-like illness (1/24, 3/24, and 1/24 subjects in the alirocumab alone, ezetimibe, and fenofibrate groups, respectively) and abdominal pain (2/24, 1/24, and 1/24 subjects in the alirocumab alone, ezetimibe, and fenofibrate groups, respectively). All other AAATs were sporadic in all 3 groups.

There were few APCS in vital signs, with a low incidence in the 3 treatment groups. There were few APCS in the ECG parameters, however, no QTc> 450 ms (men) or QTc> 470 ms (women) was recorded, and no QTc prolongation (defined as increases from baseline> 60 ms) was detected. None of these abnormalities in vital signs or ECG parameters were considered clinically significant.

There were few APCS in the hematology and biochemical parameters with low incidence in the 3 treatment groups. None of these abnormalities were considered clinically significant, except for an increase in creatinine protein kinase (CPK)> 10 upper limit of normal (ULN) in 1 subject in the alirocumab alone group. This increase in CPK recorded at the FE visit was considered to be related to physical exercise, and was declared as AA. There was no APCS in liver function tests.

There were no serious adverse events (AA) or discontinuations due to AA. Adverse events occurring during treatment (AAAT) are summarized in Table 3.

One subject in the alirocumab FENO group had a severe AAAT (renal colic), which the investigator did not consider to be related to study treatment.

No clinically significant changes in vital signs, electrocardiogram, hematological or biochemical parameters were observed in this study. One subject in the placebo alirocumab group had an increase in creatine phosphokinase> 10 times the upper limit of normal, which the investigator did not consider to be related to physical exercise (Table 3).

Table 3. Safety Summary

Alirocumab 150 mg Alirocumab 150 mg Alirocumab 150 mg Treatment group w / 4sem placebo (N = 24) w / 4sem EZE (N = 24) w / 4sem FENO (N = 24) Patients with any AAAT, 12 (50.0) 14 (58.3) 12 (50.0)

n (%)

Most frequent AAAT

(recorded in> 2 subjects in

any group), n (%)

Headache 3 (12.5) 5 (20.8) 2 (8.3) Nasopharyngitis 3 (12.5) 4 (16.7) 4 (16.7)

Influenza 2 (8.3) 0 (0) 1 (4.2) Viral gastroenteritis 0 (0) 0 (0) 2 (8.3) Flu-like illnesses 1 (4.2) 3 (12.5) 1 (4 ,2)

Abdominal pain 2 (8.3) 1 (4.2) 1 (4.2)

Immunogenicity results

Four of the 24 subjects (16.7%) in the alirocumab alone group, 6/24 subjects (25%) in the ezetimibe group, and 7/24 (29.2%) subjects in the fenofibrate group tested positive for AAF with titers ranging from 30 (minimum detectable titer) to 240. A single titer of 240 was detected in a subject in the ezetimibe group on day 29, which decreased to a titer of 30 on day 85. On day 85 120, no AAFs were detected in this subject. All other titers from a to A f were low and between 30 and 120 for all other AAF positive subjects.

In all groups, 4 subjects showed positive AAF titers in the pre-administration samples. This suggests that these subjects exhibited pretreatment immunoreactivity in the trial, and not necessarily an AAF response arising during treatment to study drug administration. Only 1 subject positive for pretreatment (fenofibrate group) had elevated titers in the period after baseline, with a maximum titre of 120 on day 29 and day 120.

Pharmacokinetic ( PK) results

The descriptive statistics of the FC parameters of alirocumab after repeated SC doses c / 4sem of 150 mg of alirocumab are provided in the tables below (Tables 4-6) and in Figure 7.

Table 4

Table 5

Table 6

After the 1st injection, ^{alirocumab C max} values were similar when alirocumab ezetimibe was compared to alirocumab alone with a point estimate of 0.97 (90% CI = 0.82 to 1.14). Meanwhile, a trend for ^{reduced AUC 0-D28} was observed with a point estimate of 0.88 (90% CI = 0.76 to 1.03). Serum alirocumab exposure was reduced when alirocumab fenofibrate was compared to alirocumab alone with point estimates of 0.78 (90% CI = 0.66 to 0.92) and 0.74 (90% CI = 0.64 to 0.86) for C ^max and AUC ^0-D28 , respectively.

After the 3rd injection, ^{alirocumab C max} values were similar when alirocumab ezetimibe was compared to alirocumab alone with a point estimate of 0.92 (90% CI = 0.78 to 1.09). It was observed that AUC ^0-D28 was reduced with a point estimate of 0.85 (90% CI = 0.70 to 1.03). A trend for a shorter half-life (6.72 ± 1.56 versus 8.76 ± 3.12 days) was observed in the alirocumab ezetimibe versus alirocumab alone group with a point estimate of 0.80 (90% CI = 0.72 to 0.90).

Alirocumab exposure in serum remained low when alirocumab fenofibrate was compared to alirocumab alone with a point estimate of 0.71 (90% CI = 0.60 to 0.84) and 0.64 (90% CI = 0, 53 to 0.77) for C ^max and AUC ^{0 -D28} , respectively. The mean half-lives were reduced to a point estimate of 0.83 (90% CI = 0.74 to 0.93) when the alirocumab fenofibrate group was compared to alirocumab alone. Half-lives of 7.07 ± 1.68 days and 8.76 ± 3.12 days were calculated for the alirocumab fenofibrate and alirocumab alone groups, respectively.

Conclusions

The LDL-C reductions with EZE and FENO therapy alone observed during the run-in period were as expected.

Alirocumab 150 mg / 4 weeks monotherapy produced ~ 48% LDL-C reductions (regardless of baseline) that were maintained throughout the 28-day dosing interval. The combination of alirocumab with EZE or FENO produced greater reductions in LDL-C than with alirocumab alone: 1) Maximum reductions from the main baseline level, which included a 28-day run-in with placebo, EZE, or FENO, were ~ 65% with EZE or FENO; 2) Corresponding reductions using additional baseline (from first injection with alirocumab) were ~ 55% with EZE or FENO.

Treatment with FENO alone, and to a lesser extent EZE, produced modest increases in free PCSK9 compared to placebo.

The slight decrease in efficacy observed from 14 to 28 days after alirocumab administration in the FENO combination group may be the result of reduced alirocumab exposure due to the increase in free PCSK9 levels observed with FENO administration. . This also appeared to be the case, to a lesser degree, with the EZE combination.

Previous studies suggested that concomitant statin therapy reduces the duration of efficacy of alirocumab due to high target-mediated clearance, which requires administration every 2 weeks to overcome this. This study suggests that other THLs (EZE, FENO) may have less impact on the duration of efficacy of alirocumab, and thus could be used with lower / less frequent doses of alirocumab.

The effects on other lipid parameters were as expected based on previous experience and a similar incidence of AAAT was reported in all groups.

The results of this study suggest that EZE and FENO therapy results in modest increases in free PCSK9 levels that may explain slightly greater reductions in LDL-C with alirocumab administration, as well as a modest reduction in the duration of this study. maximum effect. However, these data indicate that, unlike in combination with statins, alirocumab 150 mg could be administered q / 4sem in the context of monotherapy and in combination with non-statin THL.

The maximum decreases were -47.59%, -65.34%, and -66.75% in the alirocumab alone, ezetimibe, and fenofibrate groups, respectively (change from baseline on day -29). The administration of alirocumab 150 mg every 4 weeks both alone and in addition to ezetimibe (10 mg / day) or fenofibrate (160 mg / day) in healthy subjects produced a decrease in LDL-C that reached -47.39%, -56 , 56% and -54.34% in the alirocumab alone, ezetimibe and fenofibrate groups, respectively, 14 days after the 3rd administration of alirocumab, compared to the baseline value prior to alirocumab. A reduction of -47.03%, -49.57% and -43.17% was still observed in the alirocumab alone, ezetimibe and fenofibrate groups, respectively, 28 days after this 3rd administration of alirocumab. This suggests a maintenance of effect when alirocumab was administered alone, while a small change from the maximum effect was observed between 2 and 4 weeks after administration in both combination groups. Coadministration of alirocumab with either ezetimibe or fenofibrate produced a similar decrease in LDL-C.

When alirocumab ezetimibe was compared to alirocumab alone, the C ^max values were similar, with a non-significant trend towards a ^{lower AUC 0-D28} in the alirocumab ezetimibe treatment group and, therefore, a faster elimination in this group. group.

The PK parameters of alirocumab were significantly reduced by the co-administration of fenofibrate. A daily dose of 160 mg of fenofibrate reduced the serum exposure of alirocumab as described above. An analysis of the median difference in t ^max in treatment showed no difference.

Incidences and AAF titers were similar for the 3 treatment groups. The serum concentrations of alirocumab in positive and negative subjects for AAF were comparable in the 3 treatment groups.

Continuous plasma ezetimibe or fenofibrate exposure was confirmed during the study period.

Alirocumab administered both alone and in addition to ezetimibe or fenofibrate at the dose of 150 mg q / 4 weeks for 3 administrations in healthy subjects was well tolerated.

Example 3: Randomized, double-blind, placebo-controlled, parallel group study evaluating the efficacy and safety of alirocumab in patients with primary hypercholesterolaemia not treated with a statin

Dosage selection

Based on the results of studies performed with statin as background treatment, the c / 2 wk dose regimen is appropriate to maintain a constant reduction in LDL-C in the interval between administrations in patients treated with statin, with maximum efficacy at 12 weeks. provided by the administration of 150 mg every 2 weeks. However, for many patients, the magnitude of the effect seen at the 150 mg twice weekly dose may not be needed to achieve the target LDL-C target, and a lower dose may be started.

The 150 mg twice daily dose regimen for alirocumab to be evaluated in this study is based on the longest duration of action observed in patients not receiving concomitant statins. Increased statin-stimulated PCSK9 production may affect the duration of action of alirocumab, since higher rates of PCSK9 production may result in greater target-mediated clearance of the antibody. Compared with statins, ezetimibe and fibrates appear to have little or no effect on PCSK9 levels, and a c / 4sem regimen is expected to maintain a sufficient LDL-C reduction in the interval between administrations in patients who do not they receive a statin, but they receive these lipid-lowering therapies.

Rationale for the design of the protocol

The objective of the present study is to evaluate the efficacy and safety of alirocumab 150 mg q / 4 weeks as a possible starting dose in patients not treated with a statin. The present study will provide information on efficacy, safety and tolerability for a dosage regimen every 4 weeks, and in patients receiving a background treatment of ezetimibe or fenofibrate or diet alone.

None of the patients selected in this study will receive a statin. A component of the population in the current study is statin intolerant patients.

In general, a target of 2/3 of patients should receive a background treatment with ezetimibe or fenofibrate.

In this study of patients maintaining their baseline treatment of ezetimibe or fenofibrate or diet alone, who do not / cannot receive a statin, the choice of placebo as a control during the double-blind treatment period seems appropriate for the purposes of this study. study, since it will provide the most robust efficacy and safety evaluation of 150 mg every 4 weeks. A calibrator group of alirocumab 75 mg q / 2 wk will provide a reference point for the initial dose.

To help tailor the alirocumab regimen to patient needs, in the phase 3 schedule, alirocumab is started in most studies with a dose that is expected to provide a 50% reduction in LDL-C in the steady state (75 mg every 2 weeks program). In all studies with 75 mg every 2 weeks as the starting dose, the dose is increased to 150 mg every 2 weeks in week 12, depending on the level of LDL-C reached in week 8. In the current study that evaluates 150 mg c / 4 weeks as a possible starting dose in statin-naïve patients, dose titration, if necessary, will also be done at week 12, as in all other studies in the program. In the full program, the main effectiveness is evaluated after the adjustment takes place, if necessary. In this study, the primary efficacy parameter will be assessed at week 24. It will be important to consider the 12-week efficacy assessment (ie, prior to dose titration) and will therefore be analyzed as an endpoint. secondary key.

A duration of 24 weeks for the double-concealment period is considered adequate to provide safety information on a dosage regimen that includes 150 mg every 4 weeks as the initial dose, since the exposure to alirocumab (C ^max and AUC) will be between that observed. with 75 mg every 2 weeks and 150 mg every 2 weeks, for which a large database will be available for a longer duration. In the post-adjustment period, patients who do not reach the LDL-C goal will receive 150 mg every 2 weeks, as in the rest of the program. For additional safety data with this dose every 4 weeks, patients can participate in an optional non-concealment treatment period from week 24 to June 2016.

This specific study is carried out to demonstrate the safety and reduction of low-density lipoprotein cholesterol (LDL-C) with a regimen that includes alirocumab 150 mg q / 4 weeks as an initial dose, as an additional treatment to the lipid-modifying base treatment. no statin (ezetimibe or fenofibrate) or diet alone compared to placebo in patients with primary hypercholesterolaemia not treated with a statin. The statin intolerant population that is not on target for LDL-C with optimized TML (ezetimibe or fenofibrate) or on diet only represents a group with an identified unmet medical need that can be treated by adding alirocumab to their C-lowering therapies. -LDL. At the end of the 24-week double-concealment treatment period, the background treatment can be adjusted as needed in patients who choose to enter the no-concealment treatment period.

Objectives of the study

The main objective of the study is to demonstrate the reduction of low-density lipoprotein cholesterol (LDL-C) by a regimen that includes an initial dose of alirocumab of 150 mg every 4 weeks as an add-on treatment to the basic non-lipid modifying treatment. statin or as monotherapy compared with placebo in patients with primary hypercholesterolaemia not treated with a statin.

The secondary objectives are: to evaluate the effect of alirocumab, with 150 mg every 4 weeks as a starting dose, compared with placebo on other lipid parameters (for example, apolipoprotein B (ApoB) levels, non-lipoprotein cholesterol of high density (non-HDL-C), total cholesterol (TC), lipoprotein (a) (Lp [a]), high-density lipoprotein cholesterol (HDL-C), triglycerides (TG), and apolipoprotein A-1 (Apo A -1); evaluate the safety and tolerability of alirocumab 150 mg every 4 weeks; evaluate the development of anti-alirocumab antibodies; and evaluate the pharmacokinetics (PK) of alirocumab 150 mg every 4 weeks.

Other objectives are to evaluate the efficacy and safety of a 75 mg alirocumab dose regimen every 2 weeks.

Study design

This is a phase 3, multicenter, randomized, double-blind, placebo-controlled, parallel-group study. Randomization was stratified by statin intolerance status and non-statin lipid modifying background therapy. Only patients not receiving a statin will be included. Statin intolerant patients at moderate, high, or very high CV risk, as defined in the population section below, will represent a target of approximately 50% of the study population. Statin intolerance is defined for the trial as the inability to tolerate at least two statins: a statin at the lowest daily starting dose (defined as rosuvastatin 5 mg, atorvastatin 10 mg, simvastatin 10 mg, lovastatin 20 mg, pravastatin 40 mg, fluvastatin 40 mg, or pitavastatin 2 mg or as the lowest authorized daily dose by country-specific labeling), and another statin at any dose, due to skeletal muscle-related symptoms, other than those due to exertion or trauma, such as such as aches, weakness, or cramps, which started or increased during statin treatment and stopped when statin therapy was stopped. Patients at moderate CV risk, who do not meet the definition of statin intolerance (IE), will comprise the rest of the study population.

For background treatment, a target of approximately 2/3 patients will receive a background treatment (fenofibrate or ezetimibe) and up to 1/3 patients will be treated with diet alone.

The study comprises four periods:

1) A selection period of up to 3 weeks,

2) A 24-week parallel group double-blind treatment period during which patients will receive double-blind study treatment as follows:

Alirocumab 150 mg subcutaneous every 4 weeks *

OR

Placebo for subcutaneous alirocumab every 2 weeks

OR

Alirocumab 75 mg subcutaneous every 2 weeks.

* the concealment will be maintained in alirocumab 150 mg every 4 weeks alternating with placebo SC every 4 weeks.

At the week 12 visit, based on their LDL-C at week 8 and baseline CV risk, patients, in a concealment mode, will either continue to receive alirocumab 150 mg every 4 weeks or 75 mg every 2 weeks or adjust your dose in ascending order, as follows:

i) Patients with very high CV risk, in a concealment mode, or:

will continue to receive alirocumab 150 mg every 4 weeks or 75 mg every 2 weeks from week 12 onwards until the last injection in week 22, if their LDL-C at week 8 is <70 mg / dL (1.81 mmol / L) and had at least a 30% reduction in LDL-C from baseline at week 8; or

will receive a dose that is titrated up to alirocumab 150 mg every 2 weeks from week 12 onwards until the last injection at week 22, if their week 8 LDL-C is> 70 mg / dL (1.81 mmol / L) or do not have at least a 30% reduction in LDL-C from baseline at week 8.

ii) Patients with high or moderate CV risk, in a concealment mode, or:

will continue to receive alirocumab 150 mg every 4 weeks or 75 mg every 2 weeks from week 12 onwards until the last injection in week 22, if their LDL-C at week 8 is <100 mg / dL (2.59 mmol / L) and had at least a 30% reduction in LDL-C from baseline at week 8; or

will receive a dose that is titrated up to alirocumab 150 mg every 2 weeks from week 12 onwards until the last injection at week 22, if their week 8 LDL-C is> 100 mg / dL (2.59 mmol / L) or do not have at least a 30% reduction in LDL-C from baseline at week 8.

3) A follow-up period of 8 weeks after the end of the double-blind treatment period.

Patients who do not qualify for the no-concealment treatment period will be followed for a period of 8 weeks after the end of the double-concealed treatment period if they do not elect to participate in the no-concealment treatment period or do not meet the requirements. . The 8-week follow-up period will not apply to patients who are eligible and choose to enroll in the no-concealment treatment period.

4) A period of treatment without optional concealment.

Patients who successfully complete the double-concealment treatment period will be eligible (provided they have had no treatment-related AE or had significant protocol deviations) to enter a no-concealment optional treatment period.

Patients will receive alirocumab 150 mg every 4 weeks at the beginning of the treatment period without concealment. The first injection during the no-concealment treatment period will be administered on site at the 24 week visit (the first no-concealment treatment period visit).

From the 36 week visit, based on the LDL-C value at week 32, the investigator will manage, at his discretion, the adjustment of the alirocumab dose. At week 36, patients will either continue to receive alirocumab 150 mg every 4 weeks or they will receive a dose that is titrated up to alirocumab 150 mg every 2 weeks. Subsequent downward adjustment will be allowed up to 150 mg every 4 weeks.

Although background therapies should be kept stable if possible, they could be adjusted at the discretion of the investigator, particularly in the case of tolerability issues.

For adjustments based on LDL-C values, the investigator can modify the background treatment as needed. However, simultaneous dose adjustments of alirocumab and any TML should be avoided.

Treatment of these patients will continue uninterrupted from the last study drug administration during the double-concealed treatment period (week 22) to week 24 (the first administration in the no-concealment treatment period) onwards, until June 2016. .

Duration of study participation

Study duration includes a screening period of up to 3 weeks, a 24-week double-blind treatment period for efficacy and safety evaluation, and an 8-week post-treatment follow-up period for non-compliant patients. the requirements for the period of treatment without concealment after the last FTDO visit. Thus, the study duration per patient is approximately 35 weeks a treatment period without optional concealment. The 8-week follow-up period will not apply to patients who meet the criteria and choose to enroll in the no-concealment treatment period. Patients who successfully complete the double-concealed treatment period will meet the criteria (provided they have had no treatment-related AE or significant protocol deviations) to enter a no-concealment optional treatment period.

Selection of patients

Patients who meet all of the following criteria will be considered for enrollment in the study. Patients with primary hypercholesterolemia (heFH or HnF) receiving fenofibrate or ezetimibe or diet alone. Only patients not receiving a statin will be included in the study, corresponding to patients: who are intolerant to statins * as defined below with moderate, high or very high CV risk; or they do not meet the definition of IE. Only moderate CV risk patients will be included in this stratum.

* Statin intolerance is defined as the inability to tolerate at least 2 statins: 1 statin at the lowest daily starting dose (defined as rosuvastatin 5 mg, atorvastatin 10 mg, simvastatin 10 mg, lovastatin 20 mg, pravastatin 40 mg, fluvastatin 40 mg or pitavastatin 2 mg or as the lowest authorized daily dose by country-specific labeling), AND another statin at any dose, due to skeletal muscle-related symptoms, other than those due to exertion or trauma, such as pain, weakness or cramps, which started or increased during statin treatment and stopped when statin treatment was stopped.

Moderate CV Risk is defined as a calculated 10-year fatal CVD Risk SCORE> 1 and <5% (ESC / EAS 2012).

High CV risk is defined as a calculated 10-year fatal CVD risk SCORE> 5% (ESC / EAS 2012), or moderate chronic kidney disease (CKD), or type 1 or type 2 diabetes mellitus without organ damage target, or HFhe (NCEP-ATP III, ESC / EAS 2012).

Very high CV risk is defined as a history of documented CHD, ischemic stroke, peripheral arterial disease (PAD), transient ischemic attack (TIA), abdominal aortic aneurysm, carotid artery occlusion> 50% without symptoms, carotid endarterectomy, or surgical procedure. carotid artery stent placement, renal artery stenosis, renal artery stent procedure, type 1 or type 2 diabetes mellitus with target organ damage (NCEP-ATP III, ESC / EAS 2012 ).

Documented history of CHD (includes 1 or more of the following): acute MI, asymptomatic MI, unstable angina, coronary revascularization procedure (eg, percutaneous coronary intervention [PCI] or coronary artery bypass graft [CRI] surgery) and CHD clinically significant diagnosed by invasive or non-invasive tests (such as coronary arteriography, stress test using treadmill, stress echocardiography, or nuclear medicine diagnostic test).

Patients who have met all the inclusion criteria will be selected for the exclusion criteria. Exclusion criteria for the double-blind period are: patients specified as statin intolerant and very high CV risk with LDL-C <70 mg / dL (1.81 mmol / L) at the screening visit (week- 3, V1); patients specified as statin intolerant and high or moderate CV risk with LDL-C <100 mg / dL (<2.59 mmol / L) at screening visit (week-3, V1); patients who do not meet the definition of statin intolerance and are at moderate CV risk with LDL-C <100 mg / dL (<2.59 mmol / L), at the screening visit (week-3, V1); patients with LDL-C> 160 mg / dL (> 4.1 mmol / L) at the screening visit (week -3, V1) if they are on diet alone, regardless of statin intolerance status or if they do not meet the definition of intolerance to statins at moderate CV risk and treated with ezetimibe or fenofibrate; with a 10-year fatal CVD risk SCORE <1% (ESC / EAS 2011) at the screening visit (week-3, V1); newly diagnosed diabetes (3 months before randomization visit [week 0]) or poorly controlled (HbA1c>9%); with use of statin, red yeast products, niacin, or bile acid sequestrant within 4 weeks of the screening visit (week -3) or between screening and randomization visits; do not take a stable dose of ezetimibe or fenofibrate for at least 4 weeks, before the screening visit (week-3, V1) or between screening and randomization visits; with use of fibrates, other than fenofibrate, within 4 weeks of the screening visit (week -3, V1) or between screening and randomization visits; with the use of nutraceuticals or over-the-counter treatments known to affect lipids, at a dose / amount that has not been stable for at least 4 weeks, prior to the screening visit (week-3, V1) or between screening and randomization visits; planning to undergo elective PCI, CRF, carotid or peripheral revascularization during the study; systolic BP> 160 mmHg or diastolic BP> 100 mmHg at screening visits (week-3, V1) and / or randomization (week 0); a history of New York Heart Association class III or IV heart failure in the past 12 months; history of MI, unstable angina leading to hospitalization, CRF, PCI, uncontrolled cardiac arrhythmia, carotid stent or surgery, stroke, transient ischemic attack, carotid revascularization, endovascular procedure, or surgery for peripheral vascular disease within 3 months before the screening visit (week-3, V1); history of hemorrhagic stroke; age <18 years or age of majority at screening visit (week-3, V1), whichever is older; patients previously not advised on a cholesterol-lowering diet before the screening visit (week-3, V1); presence of any clinically significant uncontrolled endocrine disease known to influence serum lipids or lipoproteins; history of bariatric surgery in the 12 months before the screening visit (week-3, V1); unstable weight specified by a variation> 5 kg in the 2 months before the screening visit (week-3, V1); history of homozygous HF; history of PCSK9 loss of function (ie, genetic mutation or sequence variation); use of systemic corticosteroids, unless used as replacement therapy for pituitary / adrenal disease with a stable regimen for at least 6 weeks prior to randomization; a history of cancer in the past 5 years, except sufficiently treated basal cell skin cancer, squamous cell skin cancer, or cervical cancer in situ; history of a positive HIV test; patient who has taken any active investigational drug in 1 month or 5 half-lives, whichever is longer; patient who has previously been treated with at least one dose of alirocumab or any other anti-PCSK9 monoclonal antibody in other clinical trials; use of continuous hormone replacement therapy, unless the regimen has been stable in the last 6 weeks prior to the screening visit (week -3) and there are no plans to change the regimen during the study; patient who withdraws consent during the selection period (patient who is unwilling to continue or stops coming back); conditions / situations or laboratory data such as: any clinically significant abnormalities identified at the time of selection that in the judgment of the investigator or any sub-investigator would preclude the safe completion of the study or limit the evaluation of the endpoints, such as major systemic diseases , patients with a short life expectancy, patients considered by the investigator or any sub-investigator as inappropriate for this study for any reason, for example: those deemed unable to meet the specific requirements of the protocol, such as planned visits, those deemed unable to administer o tolerate long-term injections according to the patient or investigator, investigator or any sub-investigator, pharmacist, study coordinator, other study personnel or relative of the study directly involved in the execution of the protocol, etc., presence of any other condition (p For example, geographic, social ...) current or anticipated, which the investigator considers would restrict or limit the patient's participation for the duration of the study; laboratory data during the selection period (not including randomization laboratories): positive hepatitis B surface antigen or hepatitis C antibody positive result, positive serum or urine pregnancy test result ( including week 0) in women who can become pregnant, triglycerides> 400 mg / dL (> 4.52 mmol / L) (1 repeat laboratory allowed), eGFR <30 ml / min / 1.73 m2 according to the equation MDRD study of 4 variables (calculated by central laboratory), ALT or AST> 3xULN (1 repeat laboratory allowed), CPK> 3xULN (1 repeat laboratory allowed), TSH <LLN or> ULN (for patients in treatment thyroid hormone replacement see exclusion criteria above; known hypersensitivity to monoclonal antibody or any component of the drug; and pregnant or breastfeeding women (women who can become pregnant without protection by contraceptive method (s) highly effective (effective) (as defined in the informed consent statement and / or in an annex to the local protocol) and / or who are unwilling or unable to take a pregnancy test)

The exclusion criteria for the no-concealment period are: significant deviation from the protocol in the double-concealed period at the discretion of the investigator, such as non-compliance by the patient; any patient who presents an adverse event that leads to permanent interruption of the period with double concealment; patients who have a new or worsening condition from an existing condition that in the opinion of the investigator would make the patient unsuitable for enrollment, or that could interfere with the patient's participation in or completion of the study; known hypersensitivity to monoclonal antibody or to any component of the drug; positive result of the pregnancy test in the last visit of the period with double concealment (week 24, visit 11); and women who may become pregnant who are unwilling to continue using highly effective (effective) contraceptive method (s) (as defined in the informed consent statement and / or in an addendum to the local protocol) and / or who are unwilling or unable to take a pregnancy test.

Study treatments

For the double-concealed treatment period, the study treatment is a single 1 mL SC injection for a 75 or 150 mg dose of alirocumab or placebo provided in an autoinjector, administered into the abdomen, thigh, or outer arm area. .

During the double-concealed treatment period (week 0 to 24), patients meeting criteria will be randomized to receive: alirocumab 150 mg subcutaneous every 4 weeks alternating with placebo of alirocumab subcutaneous every 4 weeks, or alirocumab 75 mg subcutaneous every 2 weeks, or subcutaneous placebo every 2 weeks.

Study drug will be administered by SC injection every 2 weeks, beginning at week 0 and continuing until the last injection (week 22), 2 weeks prior to the end of the double-concealed treatment period.

At the week 12 visit, depending on their LDL-C at week 8 and baseline CV risk, patients will either continue to receive alirocumab 150 mg every 4 weeks or 75 mg every 2 weeks or their dose will be adjusted upwards, as follows mode:

1) Patients with very high CV risk, in a concealment mode, or: they will continue to receive alirocumab 150 mg every 4 weeks or 75 mg every 2 weeks from week 12 onwards until the last injection in week 22, if their C- Week 8 LDL is <70 mg / dL (1.81 mmol / L) and had at least a 30% reduction in LDL-C from baseline at Week 8, or will receive a dose that is adjusted accordingly ascending to alirocumab 150 mg every 2 weeks from week 12 onwards until the last injection at week 22, if your LDL-C at week 8 is> 70 mg / dL (1.81 mmol / L) or you do not have at least a 30% reduction in LDL-C from baseline at week 8.

2) Patients with high or moderate CV risk, in a concealment mode, or: they will continue to receive alirocumab 150 mg every 4 weeks or 75 mg every 2 weeks from week 12 onwards until the last injection in week 22, if their C -LDL at week 8 is <100 mg / dL (2.59 mmol / L) and had at least a 30% reduction in CLDL from baseline in week 8, or they will receive a dose that is titrated up to alirocumab 150 mg every 2 weeks from week 12 onwards until the last injection in week 22, if their LDL-C for the week 8 is> 100 mg / dL (2.59 mmol / L) or does not have at least a 30% reduction in LDL-C from baseline at week 8.

During the non-concealed treatment period (from week 24 to June 2016), patients who meet the criteria will receive: alirocumab 150 mg every 4 weeks until week 36; and from week 36, according to the measurement of LDL-C at week 32 and the criteria of the investigator, patients will either: continue to receive alirocumab 150 mg every 4 weeks, or they will receive a dose that is adjusted up to alirocumab 150 mg every 2 weeks from week 36 onwards until June 2016. A subsequent reduction in the dose will be allowed to 150 mg every 4 weeks.

During the open-label treatment period, all patients will receive alirocumab 150 mg every 4 weeks from week 24 to week 36. At the week 36 visit, based on their LDL-C at week 32, patients will either continue to receive alirocumab 150 mg every 4 weeks or the dose will be increased to alirocumab 150 mg every 2 weeks at the discretion of the investigator. Subsequent reduction of the dose to 150 mg every 4 weeks will be allowed.

Although background therapies should be kept stable if possible, they could be adjusted at the discretion of the investigator, particularly in the case of tolerability issues. For adjustments based on LDL-C values, the investigator can modify the background treatment as needed. However, simultaneous dose adjustments of alirocumab and any TML should be avoided.

Evaluation of alirocumab

The primary efficacy endpoint will be the percent change in LDL-C calculated from baseline to Week 24, defined as: 100x (LDL-C value calculated at Week 24 - LDL-C value calculated at baseline) / LDL-C value calculated at baseline.

The LDL-C value calculated at baseline will be the last LDL-C level obtained before the first double-concealed injection of investigational drug (MI). For randomized and untreated patients, baseline is defined as the last available value obtained until randomization.

The calculated LDL-C at week 24 will be the level of LDL-C obtained within the analysis window of week 24.

The primary secondary efficacy endpoint (s) are: Percentage change in LDL-C calculated from baseline to week 12: similar definition and rules as for the primary efficacy assessment, except that the calculated LDL-C at week 12 will be the LDL-C level obtained within the analysis window of week 12; the percentage change in Apo B from baseline to week 24. Same definition and rules as for main endpoint; the percentage change in non-HDL-C from baseline to week 24. Same definition and rules as for primary endpoint; the percentage change in TC from baseline to week 24. Same definition and rules as for primary endpoint; percentage change in Apo B from baseline to week 12. Same definition and rules as for percent change in LDL-C calculated from baseline to week 12; percentage change in non-HDL-C from baseline to week 12. Same definition and rules as for percent change in LDL-C calculated from baseline to week 12; percentage change in TC from baseline to week 12. Same definition and rules as for percent change in LDL-C calculated from baseline to week 12; the proportion of patients achieving calculated LDL-C <70 mg / dL (1.81 mmol / L) at week 24 for very high CV risk patients or <100 mg / dL (2.59 mmol / L) for others patients using the definition and rules used for the primary endpoint; the percent change in Lp (a) from baseline to week 24; the percentage change in HDL-C from baseline to week 24; the percent change in HDL-C from baseline to week 12; the percentage change in Lp (a) from baseline to week 12; the percentage change in fasting TG from baseline to week 24; the percentage change in fasting TG from baseline to week 12; the percentage change in Apo A-1 from baseline to week 24; and the percentage change in Apo A-1 from baseline to week 12.

Other secondary efficacy endpoints are: proportion of patients achieving LDL-C <70 mg / dL (1.81 mmol / L) for very high CV risk patients or <100 mg / dL (2.59 mmol / L). L) for other patients at week 12; the proportion of patients with LDL-C <100 mg / dL (2.59 mmol / L) at weeks 12 and 24 regardless of the CV risk patients; the proportion of patients with LDL-C <70 mg / dL (1.81 mmol / L) at weeks 12 and 24 for high CV risk patients; the absolute change in LDL-C (mg / dL and mmol / L) from baseline to weeks 12 and 24; the change in the Apo B / Apo A-1 ratio from baseline to weeks 12 and 24; the proportion of patients with Apo B <80 mg / dL (0.8 g / L) at weeks 12 and 24; the proportion of patients with non-HDL-C <100 mg / dL (2.59mmol / L) at weeks 12 and 24; the proportion of very high CV risk patients with LDL-C <70 mg / dL (1.81 mmol / L) and / or> 50% reduction in LDL-C (if LDL-C> 70 mg / dL) in week 12 and 24; the proportion of patients who achieve at least a 50% reduction in LDL-C at weeks 12 and 24.

The lipid parameters will be evaluated as follows. Total C, HDL-C, TG, Apo B, Apo A-1 and Lp (a) will be measured directly. LDL-C will be calculated using Friedewald's formula at all visits (except for the week-1 and follow-up). If TG values exceed 400 mg / dL (4.52 mmol / L), then the laboratory will conditionally measure (by the beta quantification method) LDL-C instead of calculating it. LDL-C will also be measured (by beta quantification method) at week 0 and week 24 in all patients. Non-HDL-C will be calculated by subtracting HDL-C from total C. The Apo B / Apo A-1 ratio will be calculated.

Clinical laboratory data will consist of urinalysis and blood tests, hematology (number of red cells, red cell distribution width (RDW), number of reticulocytes, hemoglobin, hematocrit, platelets, number of leukocytes with leukocyte formula), routine biochemistry ( glucose, sodium, potassium, chloride, bicarbonate, calcium, phosphorus, urea nitrogen, creatinine, uric acid, total protein, LDH, albumin, Y-glutamyltransferase [yGT]), hepatitis C antibody, hepatogram (ALT, AST, ALP and total bilirubin) and CPK. Some additional safety laboratory parameters can be measured on a conditional basis, based on actual data.

The following vital signs will be measured: heart rate, systolic and diastolic BP while sitting.

ECG data will be measured.

Anti-alirocumab antibodies will be evaluated and will include antibody status (positive / negative) and antibody titers.

The percentage change in hs-CRP from baseline to week 12 and week 24 will be evaluated.

The absolute change in HbA1c (%) from baseline to week 12 and week 24 will be assessed.

EQ-5D is a standardized measure of health status developed by the EuroQol group to provide a simple and generic measure of health for clinical and economic assessment. EQ-5D, as a health-related quality of life measure, determines health in terms of 5 dimensions: mobility, personal care, daily activities, pain / discomfort, anxiety / depression. Each dimension can adopt one of three responses (3 ordinal levels of intensity): 'no problem' (1) "some problems" (2) "serious problems" (3). General health status is determined as a 5-digit number. The health states determined by the 5-dimensional ranking can be converted into corresponding index scores that quantify health status, where 0 represents 'death' and 1 represents 'perfect health'. EQ-5D variables include response to each EQ-5D point, index score, and change in index score from baseline.

Pharmacokinetic variables include total serum alirocumab concentration. Total and free PCSK9 concentrations will be measured from the same sample for FC.

An optional pharmacogenomic substudy will be performed to identify genetic associations with clinical or biomarker response to inhibition of PCSK9, hyperlipidemia, or CVD. If necessary, samples can also be used to identify markers associated with toxicity. The analyzes can include the determination of sequences or single nucleotide polymorphisms (SNPs) of the candidate genes. Candidate genes can include (but are not limited to) PCSK9, Apo B, and LDL-R. Hologenomic studies can also be performed, including (but not limited to) SNP analysis and / or genomic sequencing.

Study procedures

For all visits after day 1 / week 0 (randomization visit), a time period of a certain number of days will be allowed. The time frame for all visits up to week 24 is ± 3 days and for the follow-up period is ± 7 days. During the period without concealment, the window of visits is ± 5 days for the week of visits 28, 32, 36 and ± 7 days for the other visits.

Blood tests for determination of lipid parameters (i.e. total C, LDL-C, HDL-C, TG, non-HDL-C, Apo B, Apo A-1, Apo B / Apo A- ratio 1, Lp [a]) should be done in the morning, fasting (that is, overnight, fasting at least 10 to 12 hours and abstaining from smoking).

The following laboratory data are collected: hematology; biochemistry; lipidogram 1: CT, calculated LDL-C, HDL-C, TG, non-HDL-C; lipidogram 2: ApoB, ApoA-1, ApoB / ApoA-1 ratio and Lp (a); hepatogram: in case of total bilirubin values above the normal range, the differentiation into conjugated and unconjugated bilirubin will occur automatically; creatine phosphokinase (CPK); Hepatitis B surface antigen; Hepatitis C antibody: positive tests will be confirmed by conditional testing; serum pregnancy test.

Urinalysis - dipstick will be performed and evaluated for pH, specific gravity, and for the presence of blood, protein, glucose, ketones, nitrates, leukocyte esterase, urobilinogen, and bilirubin. If the test strip is abnormal, then regular microscopy will be done.

All other blood parameters will also be measured during the study. Glycemic parameters (HbA1c and serum glucose) will be measured. Blood samples for inflammatory parameters, hs-CRP, will be collected periodically during the study.

Serum samples for evaluation of alirocumab concentration will be obtained periodically during the study. Blood samples will be collected prior to IM injection during visits 3 (week 0), 4 (week 4), 5 (week 8), 6 (week 9), 7 (week 10), 8 (week 11), 9 (week 12), 10 (week 16) and 11 (week 24). Blood samples should be collected prior to injection of MI.

Library samples (plasma and serum) will be collected during the study. The first planned sample at the randomization visit will be obtained prior to IM injection (predose). Library samples may include the study of PCSK9 levels, PCSK9 function, effect (s) of PCSK9 inhibition with a monoclonal antibody, lipoprotein subfractionation, and mechanisms of hyperlipidemia and heart disease. If necessary, samples can also be used to identify markers associated with toxicity. Library samples will never be used for genomic analysis.

A general physical examination should be performed at various times during the study.

Sitting BP should be measured under standardized conditions, at approximately the same time of day, on the same arm, with the same device (after the patient has rested comfortably in a sitting position for at least 5 minutes). Heart rate will be measured at the time of BP measurement.

12-lead ECG should be performed after at least 10 minutes of rest and in the supine position.

Body weight should be obtained with the patient in underwear or with very light clothing and without shoes, and with an empty bladder. Height should also be measured.

Visits calendar

Only patients who meet the inclusion criteria should be selected. The screening period will take place up to 3 weeks or 21 days (and as short as possible, after receipt of laboratory eligibility criteria) prior to the randomization visit / day 1. The screening visit (visit 1 / week -3 / day -21 to -8) will include: evaluating the inclusion / exclusion criteria; obtain patient demographics - age, sex, race, and ethnicity; obtain medical history (including menopausal status), surgical history, alcoholic habit and smoking; obtain a family medical history (including risk factors for premature CHD (before age 55 in a male first degree relative, age 65 in a female relative), allergy, and type 2 diabetes); document history of previous medication in the previous 12 weeks, especially for lipid modifying therapy (including statin) and nutritional products that may affect lipids (e.g. omega-3 fatty acids, plant stanols, such as found in Benecol , flax seed oil, psyllium); note concomitant medication; measure body weight and height. Measure vital signs including HR and BP; perform a physical examination.

The screening injection training visit (visit 2 / week -1 / day -7 ± 3) will include the following: assess the inclusion / exclusion criteria; collect AAs; note concomitant medication; measure vital signs including HR and BP.

The randomization visit (visit 3 / week 0 / day 1 3) will include the following: assessing the inclusion / exclusion criteria; collect AAs; note concomitant medication; review the patient's diet. The patient must take a TLC NCEP-ATP III diet or equivalent; perform a physical examination; measure body weight; measure vital signs including HR and BP; urinalysis (dipstick and if abnormal then microscopy); urine pregnancy test (only women who can become pregnant); collect a fasting blood sample for: lipids: measurement and / or calculation of total C, LDL-C (calculated and measured LDL), HDL-C, TG, non-HDL-C, Apo B, Apo A-1, ratio Apo B / Apo A-1 and Lp (a); hs-CRP; library samples; hematology: number of erythrocytes including hematocrit, hemoglobin, red cell distribution width (RDW), number of reticulocytes, number of leukocytes with leukocyte formula and platelets; biochemistry: glucose, sodium, potassium, chloride, bicarbonate, calcium, phosphorus, urea nitrogen, creatinine, uric acid, LDH, total protein, albumin and yGT; hepatogram (ALT, AST, ALP and total bilirubin); CPK; anti-alirocumab antibodies; serum alirocumab concentration (FC); and collection of genomic specimens.

Visit 4 / week 4, (day 29 ± 3) will include the following: collect AA; note concomitant medication; measure vital signs including HR and BP; data collection on MI administration and monitoring of MI compliance by journal review; obtain fasting blood samples for: lipids: measurement or calculation of total C, LDL-C, HDL-C, TG, non-HDL-C; hepatogram (ALT, AST, ALP and total bilirubin); serum alirocumab concentration (FC); and anti-alirocumab antibodies.

Visit 5 / week 8 (day 57 ± 3) will include the following: collect AA; note concomitant medication; measure vital signs including HR and BP; data collection on MI administration and monitoring of MI compliance by journal review; obtain fasting blood samples for: hepatogram (ALT, AST, ALP and total bilirubin), lipids: measurement or calculation of total C, LDL-C, HDL-C, TG, non-HDL-C, Apo B, Apo A- 1, Apo B / Apo A-1 ratio, and Lp (a), serum alirocumab concentration (FC) and anti-alirocumab antibodies.

Visits 6, 7, 8 / week 9, 10, 11 (day 64, 71, 78 ± 3) will include the following: blood samples should be taken prior to IM injection; serum alirocumab concentration (FC); lipids: measurement or calculation of total C, LDL-C, HDL-C, TG, non-HDL-C; and concomitant medications.

Visit 9 / week 12 (day 85 ± 3) will include the following: collect AA; note concomitant medication; measure body weight; measure vital signs including HR and BP; review the patient's diet. The patient must be taking a TLC NCEP-ATP III diet or equivalent; perform 12-lead ECG; EQ-5D patient questionnaire; urinalysis (dipstick and if abnormal then microscopy); urine pregnancy test (only women who can become pregnant); obtain fasting blood samples for: lipids: measurement and / or calculation of total C, LDL-C, HD-C, TG, non-HDL-C, Apo B, Apo A-1, Apo B / Apo A- ratio 1, and Lp (a); library samples; hematology: number of erythrocytes including hematocrit, hemoglobin, red cell distribution width (RDW), number of reticulocytes, number of leukocytes with leukocyte formula and platelets; biochemistry: glucose, sodium, potassium, chloride, bicarbonate, calcium, phosphorus, urea nitrogen, creatinine, uric acid, LDH, total protein, albumin and yGT. HbA1c and hs-CRP; hepatogram (ALT, AST, ALP and total bilirubin); CPK; anti-alirocumab antibodies; serum alirocumab concentration (FC).

Visit 10 / week 16 (day 113 ± 3) will include the following: collect AA; note concomitant medication; measure body weight; measure vital signs including HR and BP; perform 12-lead ECG; data collection on MI administration and monitoring of MI compliance by journal review; EQ-5D patient questionnaire; urinalysis (dipstick and if abnormal then microscopy); urine pregnancy test (only women who can become pregnant); obtain fasting blood samples for: lipids: measurement and / or calculation of total C, LDL-C, HD-C, TG, non-HDL-C, Apo B, Apo A-1, Apo B / Apo A- ratio 1 and Lp (a); hematology: number of erythrocytes including hematocrit, hemoglobin, red cell distribution width (RDW), number of reticulocytes, number of leukocytes with leukocyte formula and platelets; biochemistry: glucose, sodium, potassium, chloride, bicarbonate, calcium, phosphorus, urea nitrogen, creatinine, uric acid, LDH, total protein, albumin and yGT.

HbA1c and hs-CRP; hepatogram (ALT, AST, ALP and total bilirubin); CPK; anti-alirocumab antibodies; and serum alirocumab concentration (FC).

Visit 11 / week 24 / end of period with double concealment (day 169 ± 3) will include the following: collect AA; note concomitant medication; measure body weight; measure vital signs including HR and BP; perform 12-lead ECG; perform a physical examination; EQ-5D patient questionnaire; review the patient's diet. The patient must be taking a TLC NCEP-ATP III diet or equivalent; urinalysis (dipstick and if abnormal then microscopy); urine pregnancy test (only women who can become pregnant); obtain fasting blood samples for: lipids: measurement and / or calculation of total C, LDL-C (calculated and measured LDL), HDL-C, TG, non-HDL-C, Apo B, Apo A-1, Apo ratio B / Apo A-1 and Lp (a); hs-CRP; library samples; hematology: number of erythrocytes including hematocrit, hemoglobin, red cell distribution width (RDW), number of reticulocytes, number of leukocytes with leukocyte formula and platelets; biochemistry: glucose, sodium, potassium, chloride, bicarbonate, calcium, phosphorus, urea nitrogen, creatinine, uric acid, total protein, albumin and yGT; Hepatitis B and C antibody test (with automatic confirmatory test if positive); HbA1c; hepatogram (ALT, AST, ALP and total bilirubin); CPK; anti-alirocumab antibodies; and serum concentration of alirocumab (FC).

The follow-up visit (visit 12 / week 32 / day 225 ± 7) will include the following: collect AA; note concomitant medication; measure vital signs including HR and BP; perform a physical examination (only in case of clinically relevant abnormality at the end of treatment visit); urinalysis (only in case of clinically relevant abnormal value at the end of treatment visit); urine pregnancy test (only women who can become pregnant); obtain fasting blood samples for: anti-alirocumab antibodies, only in case of clinically relevant abnormal values for these parameters at the end of treatment visit will the following be obtained at this visit: ■ hematology: number of erythrocytes including hematocrit, hemoglobin , Red blood cell distribution width (RDW), number of reticulocytes, number of leukocytes with leukocyte formula and platelets; biochemistry: glucose, sodium, potassium, chloride, bicarbonate, calcium, phosphorus, urea nitrogen, creatinine, uric acid, total protein, LDH, albumin and yGT; hepatogram (ALT, AST, ALP and total bilirubin); and CPK.

Treatment period without concealment ( optional)

Patients who successfully complete the double-concealed treatment period will meet the criteria (provided they have not had any AE not related to treatment-limiting skeletal muscle or had significant protocol deviations) to enter a treatment period without optional concealment. Treatment for these patients will not be interrupted from the last dose of study drug during the double-concealed treatment period (week 22) through week 24 (the first dose in the no-concealment period) onwards, until June 2016.

At visit 11 / week 24, patients will undergo the double-concealed end of treatment period assessments and the no-concealment treatment period baseline assessments simultaneously. Study site staff should review the no-concealment period treatment requirements with patients and remind patients that administration in the no-concealment period begins at that visit. The following information will be collected: evaluate the exclusion criteria for the treatment period without

Claims (11)

1. An anti-proprotein convertase subtilisin / kexin type 9 antibody (anti-PCSK9) or antigen-binding fragment thereof for use in treating a subject with primary hypercholesterolemia who is statin intolerant or not controlled by statins, wherein the antibody or antigen-binding fragment thereof is administered in a dose of 150 mg every 4 weeks for at least 3 doses in the absence of concomitant statin therapy, thus reducing low-density lipoprotein cholesterol (C- LDL) in the subject and wherein the antibody or antigen-binding fragment thereof comprises six amino acid sequences of heavy and light chain complementarity determining regions HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3 having SEQ iD NO : 2, 3, 4, 7, 8 and 10, respectively. 2. The antibody or antigen-binding fragment thereof for use according to claim 1, wherein the antibody or antigen-binding fragment thereof comprises an HCVR heavy chain variable region and an LCVR light chain variable region having the amino acid sequences of SEQ ID NO: 1 and 6. The antibody or antigen-binding fragment thereof for use according to claim 1, wherein the subject has (a) a form of hypercholesterolemia that is unfamiliar hypercholesterolemia (HnF); or (b) heterozygous familial hypercholesterolemia (heHF). The antibody or antigen-binding fragment thereof for use according to any one of claims 1-3, wherein the subject has diabetes mellitus. The antibody or antigen-binding fragment thereof for use according to claim 4, wherein the subject has type 2 diabetes mellitus. 6. The antibody or antigen-binding fragment thereof for use according to any one of claims 1-5, wherein the subject receives a non-statin lipid-lowering agent before and / or during administration of the antibody or antigen-binding fragment of the same. The antibody or antigen-binding fragment thereof for use according to claim 6, wherein the non-statin lipid-lowering agent is selected from the group consisting of: ezetimibe, a fibrate, fenofibrate, niacin, an omega-3 fatty acid and a bile acid resin. The antibody or antigen-binding fragment thereof for use according to claim 7, wherein the non-statin lipid-lowering agent is ezetimibe or fenofibrate. The antibody or antigen-binding fragment thereof for use according to any one of claims 1-5, wherein the subject does not receive a non-statin lipid-lowering agent prior to and / or during administration of the antibody or binding fragment. antigen thereof. The antibody or antigen-binding fragment thereof for use according to any one of claims 1-9, wherein the antibody or antigen-binding fragment thereof is administered subcutaneously. The antibody or antigen-binding fragment thereof for use according to any one of claims 1-10, wherein the dose of 150 mg every 4 weeks is maintained if the subject's LDL-C measured after 4 or more dose is less than 70 mg / dL, and wherein the 150 mg dose every 4 weeks is interrupted and the antibody or antigen-binding fragment thereof is subsequently administered to the subject at a dose of 150 mg every 2 weeks if the C The subject's LDL measured after 4 or more doses is greater than or equal to 70 mg / dL.