METHODS FOR REDUCING LIPOPROTEIN(a) LEVELS BY ADMINISTERING AN INHIBITOR OF PROPROTEIN CONVERTASE SUBTILISIN KEXIN-9 (PCSK9).

METHODS TO REDUCE LEVELS OF LIPOPROTEIN (a) ADMINISTRATING A PROVERTER INHIBITOR CONVERTASE SUBTILISINE KEXINA-9 (PCSK9)

FIELD OF THE INVENTION

The present invention relates to the field of therapeutic treatments of diseases and disorders that are associated with elevated levels of lipoproteins. More specifically, the invention relates to the administration of PCSK9 inhibitors to reduce serum Lp (a) levels in a patient.

BACKGROUND

Lipoprotein (a) [Lp (a) J is a low-density lipoprotein-like particle, formed by the association of apolipoprotein (a) [Apo (a)] with apolipoprotein B100 (ΑροΒΊΟΟ). In the assembled Lp (a) particle, the Apo (a) component is covalently bound to the ΑροΒΙΟΟ component through a disulfide bond. It has been shown in several studies that an elevated serum level of Lp (a) is correlated with a variety of atherosclerotic and thrombotic disorders [see, for example, Marcovina and Koschinsky (1998), Am. J. Cardiol. 82: 57U-66U; Ignatescu et al. (1998), Thromb. Haemost 80: 231-232; Lippi and Guidi (2000), QJ Med. 93: 75-84; Bennet et al. (2008), Arch. Intern. Med. 168: 598-608; The Emerging Risk Factors Collaboration (2009), J. Am. Med. Assoc. 302: 412-423; and Lamon-Fava et al. (2011), J. Lipid Res. 52: 1181-1187). Therefore, the therapeutic reduction of serum levels of Lp (a) has been suggested as a means to treat or reduce the risk of cardiovascular disorders. There are few therapeutic options available to reduce serum levels of Lp (a). Examples of treatments that have been tested and / or proposed to reduce serum levels of Lp (a) include the administration of acetylsalicylic acid, L-carnitine, niacin or anacetrapib, and LDL apheresis [see, for example, Parhofer ( 2011), Curr. Pharm Des. 17: 871-876). However, no currently available treatment provides adequate and practical therapy for elevated levels of Lp (a).

Proprotein convertase subtilisin / kexin type 9 (PCSK9) is a proprotein convertase that belongs to the protein kin subfamily of the family of secretory subtylases. In U.S. Patent Application Publication No. 2010/0166768 The use of PCSK9 inhibitors (anti-PCSK9 antibodies) to reduce serum levels of total cholesterol, LDL cholesterol and triglycerides is described. However, it has not been shown to date that PCSK9 inhibitors reduce Lp (a) levels in patients. Therefore, there is still a need in the art for therapeutic methods to reduce serum levels of Lp (a).

BRIEF SUMMARY OF THE INVENTION

The present invention addresses the aforementioned need for the technique by providing methods to reduce serum levels of Lp (a) in an individual. The methods of the invention comprise selecting a patient having elevated serum Lp (a) levels and administering to the patient a pharmaceutical composition comprising a PCSK9 inhibitor. The patient is selected based on having a high serum level of Lp (a) that is indicative of an enhanced risk of occlusive cardiovascular and / or thrombotic diseases and disorders. The patient can also be selected based on presenting additional risk factors for such diseases and disorders, in which a reduction in the levels of Lp (a) would be beneficial or reduce the risk. For example, patients with hypercholesterolemia (for example, heFH, noFH, etc.) may be good candidates for treatment with the therapeutic methods of the present invention.

PCSK9 inhibitors that can be administered according to the methods of the present invention include, for example, anti-PCSK9 antibodies or antigen binding fragments thereof. Specific exemplary anti-PCSK9 antibodies that can be used in the practice of the methods of the present invention include any antibodies or antigen binding fragments such as those set forth in US Patent Application Publication No. 2010/0166768, and / or those described herein.

The PCSK9 inhibitor can be administered subcutaneously or intravenously to a subject. In addition, the PCSK9 inhibitor can be administered to a patient who is under a therapeutic statin regimen at the time of therapeutic intervention.

Other embodiments of the present invention will become apparent from a review of the following detailed description.

DETAILED DESCRIPTION

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

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as that commonly understood by those who have normal experience in the technique to which this invention belongs. As used herein, the term approximately, when used in reference to a particular cited numerical value, means that the value may vary with respect to the quoted value by no more than 1%. For example, as used herein, the expression about 100 includes 99 and 101 and all intermediate values ​​(for example, 99,1,99,2, 99,3, 99,4, etc.).

Although any methods and materials similar or equivalent to those described herein can be used in the practice of the present invention, preferred methods and materials are now described. All publications mentioned in this report are incorporated by reference to describe them in their entirety.

Elevated serum levels of Lp (a)

The present invention provides methods for reducing serum levels of Lp (a) in a patient. The methods of the invention comprise selecting a patient having elevated serum levels of Lp (a) and administering to the patient a pharmaceutical composition comprising a PCSK9 inhibitor. As used herein, the terms "Lp (a)" and lipoprotein (a) refer to a particle of low density lipoprotein type, formed by the association of apolipoprotein (a) [apo (a) j with apolipoprotein B100 ( apo B100).

In the context of the present invention, elevated serum level of Lp (a) means a serum level of Lp (a) greater than about 14 mg / dl. In certain embodiments, a patient is considered to have an elevated serum level of Lp (a) if the level of serum Lp (a) measured in the patient is greater than about 15 mg / dl, 20 mg / dl, 25 mg / dl , 30 mg / dl, 35 mg / dl, 40 mg / dl, 45 mg / dl, 50 mg / dl, 60 mg / dl, 70 mg / dl, 80 mg / dl, 90 mg / dl, 100 mg / dl , 120 mg / dl, 140 mg / dl, 160 mg / dl, 180 mg / dl or 200 mg / dl. The serum level of Lp (a) can be measured in a patient postprandially. In certain embodiments, the level of Lp (a) is measured after a period of fasting time (for example, after fasting for 6 hours, 8 hours, 10 hours, 12 hours or more). An exemplary method for measuring serum Lp (a) in a patient is by immunonephelometry, although any clinically acceptable diagnostic method can be used in the context of the present invention.

Patient population

The methods of the present invention are useful for reducing serum levels of Lp (a) in human subjects presenting an elevated level of serum Lp (a). In certain cases, the patient is otherwise healthy except for presenting a high serum level of Lp (a). For example, the patient may not present any other risk factor for cardiovascular, thrombotic or other diseases or disorders at the time of treatment. However, in other cases, the patient is selected on the basis that he has been diagnosed, or is at risk of developing, a disease or disorder that is caused by, or correlated with, an elevated serum level of Lp (a). For example, at the time or before administration of the pharmaceutical composition of the present invention, the patient may have been diagnosed, or may have been identified as presenting a risk of developing, a cardiovascular disease or disorder, such as, for for example, coronary artery disease, acute myocardial infarction, asymptomatic carotid atherosclerosis, stroke, occlusive disease of peripheral arteries, etc. In certain cases, the cardiovascular disease or disorder is hypercholesterolemia. For example, a patient may be selected for treatment with the methods of the present invention if he has been diagnosed, or has been identified as having a risk of developing, a state of hypercholesterolemia such as, for example, heterozygous familial hypercholesterolemia (heFH; from English, heterozyqous Familial Hypercholesterolemia), homozygous familial hypercholesterolemia (hoFH; from English, homozyqous Familial JHypercholesterolemia), as well as incidents of hypercholesterolemia that are different from familial hypercholesterolemia (nonFH).

In other cases, at the time or before administration of the pharmaceutical composition of the present invention, the patient may have been diagnosed, or may have been identified as presenting a risk of developing, a thrombotic disease or occlusive disorder, such as , for example, pulmonary embolism, occlusion of the central retinal vein, etc. In certain embodiments, the patient is selected on the basis that he has been diagnosed, or is at risk of developing, a combination of two or more of the above-mentioned diseases or disorders. For example, at the time or before administration of the pharmaceutical composition of the present invention, the patient may have been diagnosed, or may have been identified as presenting a risk of developing, coronary artery disease and pulmonary embolism. Other diagnostic combinations (for example, atherosclerosis and occlusion of the central retinal vein, heFH and stroke, etc.) are also included in the definition of patient populations that are treatable by the methods of the present invention.

In still other cases, the patient to be treated with the methods of the present invention is selected based on one or more factors selected from the group consisting of age (for example, over 40, 45, 50, 55, 60 , 65, 70, 75 or 80 years), race, sex (male or female), exercise habits (for example, regular exercise practitioner, non-exercise practitioner), other pre-existing medical conditions (for example, diabetes Type II, high blood pressure, etc.) and the current state of medication [for example, currently taking statins (for example, cerivastatin, atorvastatin, simvastatin, pitavastatin, rosuvastatin, fluvastatin, lovastatin, pravastatin, etc.), beta blockers, niacin, etc.]. The present invention also includes methods for reducing serum Lp (a) levels in patients who are intolerant or non-sensitive, or respond inappropriately, to conventional statin therapy. Potential patients may be selected / screened based on one or more of these factors (for example, through a questionnaire, a diagnostic evaluation, etc.) before being treated with the methods of the present invention.

The present invention also includes methods for increasing transintestinal cholesterol excretion (TICE) in a subject by administering a PCSK9 inhibitor to the subject. For example, the present invention provides methods for increasing the TICE in a subject by administering to the subject an anti-PCSK9 antibody, for example, the antibody referred to herein as mAb316P. In accordance with certain embodiments, the present invention includes methods comprising identifying a subject for which an enhanced TICE would be beneficial, or identifying a subject having a deteriorated TICE, and administering to the subject a PCSK9 inhibitor (eg, mAb316P).

PCSK9 inhibitors

The methods of the present invention comprise administering to a patient a therapeutic composition comprising a PCSK9 inhibitor. 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-restrictive examples of categories of PCSK9 inhibitors include small molecule PCSK9 antagonists, peptide-based PCSK9 antagonists (eg, peptibody molecules), and antibodies or antibody antigen binding fragments that specifically bind human PCSK9.

The term "proprotein convertase subtilisin / human type 9 kexin" or human PCSK9 or hPCSK9, as used herein, refers to PCSK9 having the nucleic acid sequence shown in the ID. SEC. No. 754 and the amino acid sequence of ID. SEC. No. 755, or a biologically active fragment thereof.

With the term antibody, as used herein, reference is made to immunoglobulin molecules comprising four polypeptide chains, two heavy chains (H; from English, heavy) and two light chains (L; from English, light) interconnected by disulfide bonds, as well as to multimers thereof (eg, IgM). Each heavy chain comprises a heavy chain variable region [abbreviated herein as HCVR (English, heavy chain variable region) or V_H] and a constant heavy chain region. The heavy chain constant region comprises three domains, C_H1 C_Htwo and C_H3. Each light chain comprises a light chain variable region [abbreviated herein as LCVR (English, light chain variable region) or VJ and a constant light chain region. The light chain constant region comprises a domain (C_Lone). V regions_H and V_L they can be further subdivided into regions of hypervariability, called complementarity determining regions (CDRs), intermingled with regions that are more conserved, called framework regions (FR), framework regions. Every V_H and V_L It is composed of three CDRs and four FRs, arranged from amino to carboxylic end in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 In different embodiments of the invention, the FRs of the anti-PCSK9 antibody (or of the antigen binding portion thereof) may be identical to the human germline sequences or they may be naturally or artificially modified. A consensus amino acid sequence can be defined based on an analysis of two or more CDRs against each other.

The term antibody, as used herein, 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, as used herein, include any naturally occurring, enzymatically obtainable, synthetically or genetically engineered polypeptide or glycoprotein that specifically binds to an antigen to form a complex. Antigen binding fragments of an antibody can, for example, come from complete antibody molecules using any suitable standard techniques such as proteolytic digestion or recombinant genetic engineering techniques that lead to manipulation and expression of a DNA encoding variable domains and optionally constants of an antibody. Said DNA is known and / or easily obtainable from, for example, commercial sources or DNA banks (including, for example, phage-antibody banks), or it can be synthesized. The DNA can be sequenced and chemically manipulated or using molecular biology techniques to, for example, arrange one or more variable and / or constant domains in a suitable configuration or to introduce codons, create cysteine ​​residues, modify, add or suppress amino acids, etc.

Non-restrictive examples of antigen binding fragments include: (i) Fab fragments; (i¡) F (ab ') 2i fragments (ü¡) Fd fragments; (iv) Fv fragments; (v) single chain Fv molecules (scFv; from English, single-chain Fv); (vi) dAb fragments; and (vii) minimum 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 FR3CDR3-FR4 peptide forced. Other designed molecules, such as domain-specific antibodies, single domain antibodies, antibodies with suppressed domains, chimeric antibodies, antibodies with grafted CDRs, diabody, triabody, tetrabody, minibody, nanobody (e.g., monovalent nanobody, bivalent nanobody, etc. .), small modular immunopharmaceutical products (SMIPs; English, small modular immunopharmaceuticals) and shark variable IgNAR domains, are also encompassed by the expression antigen binding fragment, as used herein.

An antigen binding fragment of an antibody will typically comprise at least one variable domain. The variable domain may have any size or composition of amino acids and will generally comprise at least one CDR that is adjacent to, or is in frame with, one or more framework sequences. In antigen binding fragments that have a V domain_H associated with a domain V_L, the domains V_H and V_L they can be located with each other in any suitable arrangement. For example, the variable region can be dimer and contain Vh-V dimers_h, V_h-V_l or Vl-V_l. Alternatively, the antigen binding fragment of an antibody may contain a V domain._H or V_L monomer

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-restrictive configurations of variable and constant domains that can be found in an antigen binding fragment of an antibody of the present invention include: (i) V_H-C_Hone; (¡I) V_H-C_Htwo; (iii) V_H-C_H3; (iv) V_HC_h1 C_htwo; (v) Vh-C_h1-Ch2-Ch3; (vi) V_H-C_H2 C_H3; (vii) V_H-C_L; (viii) V_L-C_Hone; (ix) V_L-C_Htwo; (x) V_LC_h3; (x¡) V_l-C_h1 C_htwo; (xii) V_l-Ch1-C_h2 C_h3; (xiii) V_L-C_H2 C_H3; y (xiv) V_L-C_L. In any configuration of variable and constant domains, including any of the exemplary configurations listed above, the variable and constant domains may be directly linked to each other or may be linked by a complete or partial hinge or connector region. A hinge region may consist of at least 2 (for example, 5, 10, 15, 20, 40, 60 or more) amino acids that give rise to a flexible or semi-flexible bond between adjacent variable and / or constant domains in a single polypeptide molecule . In addition, an antigen binding fragment of an antibody of the present invention may comprise a homodimer or heterodimer (or other multimer) of any of the configurations of variable and constant domains listed above, in non-covalent association with one another and / or with one or more domains V_H or V_L monomers [for example, by means of a disulfide bond (s)].

As with the complete antibody molecules, the 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, each variable domain being able to specifically bind a different antigen or a different epitope of the same antigen. Any multispecific antibody format, including the exemplary bispecific antibody formats described herein, can be adapted for use in the context of an antigen binding fragment of an antibody of the present invention using routine techniques available in this technical field.

The constant region of an antibody is important in terms of the ability of an antibody to fix complement and mediate cell-dependent cytotoxicity. In this way, the sotype of an antibody can be selected based on whether it is desirable for the antibody to mediate cytotoxicity.

With the term "human antibody," as used herein, it is intended to include antibodies that have variable and constant regions from human germline immunoglobulin sequences. However, the human antibodies of the invention may include amino acid residues not encoded by human germline immunoglobulin sequences (for example, mutations introduced by random or site-specific mutagenesis in vitro or by an in vivo somatic mutation), for example , in the CDRs and, in particular, in CDR3. However, with the term "human antibody", as used herein, it is not intended to include antibodies in which CDR sequences from the germ line of another mammalian species, such as a mouse, have been grafted into scaffold sequences human.

The term "recombinant human antibody", as used herein, 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 with which a host cell (additionally described below), antibodies isolated from a combinatorial bank of recombinant human antibodies (further described below), antibodies isolated from an animal (eg, a mouse) that is transgenic for human immunoglobulin genes [see, for example, Taylor et al. (1992) Nucí. Acids Res. 20: 6287-6295], or antibodies prepared, expressed, created or isolated by any other means involving the support of human immunoglobulin gene sequences to other DNA sequences. Such recombinant human antibodies have variable and constant regions from immunoglobulin sequences of the human germ line. However, in certain embodiments, such recombinant human antibodies are subjected to in vitro mutagenesis (or, when a transgenic animal is used for human Ig sequences, to in vivo somatic mutagenesis) and, therefore, amino acid sequences may of regions V_H and V_L of the recombinant antibodies are sequences that, although they come from, and are related to, V sequences_H and V_L of the human germ line, they do not naturally exist in the in vivo repertoire of the human antibody germ line.

Human antibodies can exist in two forms that are associated with the heterogeneity of hinges. In one form, an immunoglobulin molecule comprises a stable four-chain construction, of approximately 150-160 kDa, in which the dimers are held together by a disulfide bond between the heavy chains. In a second form, the dimers are not linked through intercatenary disulfide bonds and an approximately 75-80 kDa molecule composed of a covalently connected heavy and light chain (hemianticibody) is formed. It has been very difficult to separate these forms, even after affinity purification.

The frequency of occurrence of the second form in various intact IgG sotypes is due to, but not limited to, structural differences associated with the hinge region of the antibody. A single amino acid substitution in the hinge region of human lgG4 can significantly reduce the occurrence of the second form [Angal et al. (1993), Molecular Immunology 30: 105] to levels typically observed when using a hinge of human IgG1. The present invention encompasses antibodies that have one or more mutations in the hinge region, C_Htwo or C_H3, which may be desirable production, for example, to improve the performance of the desired antibody form.

An isolated antibody, as used herein, 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 of the present 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 operation. According to certain embodiments, an isolated antibody may be substantially free of other cellular material and / or other chemicals.

The expression specifically binds to, and the like, means that an antibody or an antigen binding fragment in the same way with an antigen a complex that is relatively stable under physiological conditions. Methods for 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, as used in the context of the present invention, an antibody that specifically binds to PCSK9 includes antibodies that bind to PCSK9 or a portion thereof with a K_D 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 nM, less than about 30 nM, less than about 20 nM, less than about 10 nM, less than 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 than about 0.5 nM, as measured in a surface plasmon resonance test. However, an isolated antibody that specifically binds to human PCSK9 exhibits cross reactivity 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 and / or CDR regions of the variable domains of the heavy and light chains compared to the corresponding germline sequences from which the antibodies came from. Such mutations can be easily determined by comparing the amino acid sequences described herein with the germline sequences available from, for example, public databases of antibody sequences. The present invention includes methods that involve the use of antibodies, and antigen binding fragments thereof, which are derived from any of the amino acid sequences described herein, wherein one or more amino acids from one or more framework regions and / or CDR are mutated with respect to the corresponding rest (s) of the germline sequence from which the antibody came, or with respect to the corresponding (s) rest (s) of another sequence of the human germ line, or with respect to a conservative amino acid substitution of the corresponding (s) rest (s) of the line germinal (these changes in the sequence are collectively referred to herein as germline mutations). A person with normal experience in the art, starting with the sequences of variable regions of heavy and light chains described herein, can easily produce numerous antibodies and antigen binding fragments that comprise one or more individual germline mutations or combinations of the same. In certain embodiments, all framework and / or CDR residues of the V domains_H and / or V_L they are mutated back to the remains found in the original germline sequence from which the antibody came. In other embodiments, only certain residues are mutated back to the original germline sequence; for example, only the mutated residues found in the first 8 amino acids of FR1 or in the last 8 amino acids of FR4, or only the mutated residues found in CDR1, CDR2 or CDR3. In other embodiments, one or more of the framework and / or CDR residues are mutated with respect to the corresponding residue (s) of a different germline sequence (i.e., a germline sequence which is different from the germline sequence from which the antibody originally originated). In addition, the antibodies of the present invention may contain any combination of two or more germline mutations within the framework and / or CDR regions, where, for example, certain individual residues are mutated with respect to the corresponding remainder of a particular germline sequence while other certain residues that differ from the original germline sequence are maintained or mutated with respect to the corresponding remainder of a different sequence of the line germinal. Once obtained, antibodies and antigen binding fragments that contain one or more germline mutations can be easily examined for one or more desired properties, such as, improved binding specificity, increased binding affinity, biological properties antagonists or agonists better bound or enhanced (as the case may be), reduced immunogenicity, etc. The use of antibodies and antigen binding fragments obtained in this general manner is encompassed by the present invention.

The present invention also includes methods that involve the use of anti-PCSK9 antibodies comprising variants of any of the amino acid sequences of HCVR, LCVR and / or CDR described herein that have one or more conservative substitutions. For example, the present invention includes the use of anti-PCSK9 antibodies having amino acid sequences of HCVR, LCVR and / or CDR 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 amino acid sequences of HCVR, LCVR and / or CDR described herein.

The term "surface plasmon resonance", as used herein, refers to an optical phenomenon that allows the analysis of real-time interactions by detecting alterations in protein concentrations in a biosensor matrix, using, for example, the BIAcore ™ system (Biacore Life Sciences, division of GE Healthcare, Piscataway, New Jersey, USA).

With the term K_D, as used herein, reference is made to the equilibrium dissociation constant of a particular antigen antibody interaction.

The term epitope refers to an antigenic determinant that interacts with an antigen binding site specific to the variable region of an antibody molecule, a site known as a paratope. A single antigen can have more than one epitope. In this way, different antibodies can bind to different areas of an antigen and can produce different biological effects. The epitopes can be conformational or linear. A conformational epitope is one produced by spatially juxtaposed amino acids of different segments of the linear polypeptide chain. A linear epitope is one produced by adjacent amino acid residues of a polypeptide chain. Under certain circumstances, an epitope may include components of saccharides, phosphoryl groups or sulfonyl groups in the antigen.

Preparation of human antibodies

Methods for generating human antibodies in transgenic mice are known in the art. Any of said known methods may be used in the context of the present invention to prepare human antibodies that specifically bind human PCSK9.

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

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

Initially, high affinity chimeric antibodies that have 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 skilled in the art. The mouse constant regions are replaced by a desired human constant region to generate the fully human antibody of the invention, such as, for example, modified or wild-type IgG1 or IgG4. Although the constant region selected may vary according to a specific use, the binding characteristics of high affinity to the antigen and specificity for the target lie in the variable region.

In general, antibodies that can be employed in the methods of the present invention have high affinities, as described above, as measured by binding to an antigen in the dissolution phase or immobilized in a solid phase. The mouse constant regions are replaced by desired human constant regions to generate the fully human antibodies of the invention. Although the constant region selected may vary according to a specific use, the binding characteristics of high affinity to the antigen and specificity for the target lie in the variable region.

Specific examples of human antibodies or antibody antigen binding fragments that specifically bind PCSK9, which may be used in the context of the methods of the present invention, include any antibody or antigen binding fragment comprising the three chain CDRs. heavy (HCDR1, HCDR2 and HCDR3) contained in a heavy chain variable region (HCVR) having an amino acid sequence selected from the group consisting of the IDs. SEC. Numbers: 2, 18, 22, 26, 42, 46, 50, 66, 70, 74, 90, 94, 98, 114, 118, 122, 138, 142, 146, 162, 166, 170, 186, 190, 194, 210, 214, 218, 234, 238, 242, 258, 262, 266, 282, 286, 290,

306, 310, 314, 330, 334, 338, 354, 358, 362, 378, 382, 386, 402, 406, 410, 426, 430, 434,

450, 454, 458, 474, 478, 482, 498, 502, 506, 522, 526, 530, 546, 550, 554, 570, 574, 578,

594, 598, 602, 618, 622, 626, 642, 646, 650, 666, 670, 674, 690, 694, 698, 714, 718, 722,

738 and 742, or a substantially similar sequence thereof that has a sequential identity of at least 90%, at least 95%, at least 98% or at least 99%. The antibody or antigen binding fragment may comprise the three light chain CDRs (LCVR1, LCVR2 and LCVR3) contained in a light chain variable region (LCVR) having an amino acid sequence selected from the group consisting of the IDs. SEC. Numbers: 10, 20, 24, 34, 44, 48, 58, 68, 72, 82, 92, 96, 106, 116, 120, 130, 140, 144, 154, 164, 168, 178, 188, 192, 202, 212, 216, 226, 236, 240, 250, 260,

264, 274, 284, 288, 298, 308, 312, 322, 332, 336, 346, 356, 360, 370, 380, 384, 394, 404,

408, 418, 428, 432, 442, 452, 456, 466, 476, 480, 490, 500, 504, 514, 524, 528, 538, 548,

552, 562, 572, 576, 586, 596, 600, 610, 620, 624, 634, 644, 648, 658, 668, 672, 682, 692,

696, 706, 716, 720, 730, 740 and 744, or a substantially similar sequence thereof having a sequential identity of at least 90%, at least 95%, at least 98% or at least 99%.

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 pairs of amino acid sequence pairs of heavy and light chain variable regions (HCVR / LCVR) selected from the group consisting of the IDs. SEC. Numbers: 2/10, 18/20, 22/24, 26/34, 42/44, 46/48, 50/58, 66/68, 70/72, 74/82, 90/92, 94/96, 98/106, 114/116, 118/120, 122/130, 138/140, 142/144, 146/154, 162/164, 166/168, 170/178, 186/188, 190/192, 194 / 202, 210/212, 214/216, 218/226, 234/236, 238/240,

242/250, 258/260, 262/264, 266/274, 282/284, 286/288, 290/298, 306/308, 310/312,

314/322, 330/332, 334/336, 338/346, 354/356, 358/360, 362/370, 378/380, 382/384,

386/394, 402/404, 406/408, 410/418, 426/428, 430/432, 434/442, 450/452, 454/456,

458/466, 474/476, 478/480, 482/490, 498/500, 502/504, 506/514, 522/524, 526/528,

530/538, 546/548, 550/552, 554/562, 570/572, 574/576, 578/586, 594/596, 598/600,

602/610, 618/620, 622/624, 626/634, 642/644, 646/648, 650/658, 666/668, 670/672,

674/682, 690/692, 694/696, 698/706, 714/716, 718/720, 722/730, 738/740 and 742/744.

In certain embodiments of the present invention, the anti-PCSK9 antibody, or the antigen binding fragment thereof, that can be used in the methods of the present invention has amino acid sequences of HCDR1 / HCDR2 / HCDR3 / LCDR1 / LCDR2 / LCDR3 selected from the IDs. SEC. Numbers: 76/78/80/84/86/88 (mAb316P) and 220/222/224/228/230/232 (mAb300N) (see Publication No. 2010/0166768 of US Patent Application) .

In certain embodiments of the present invention, the antibody or antigen binding fragment thereof comprises pairs of HCVR / LCVR amino acid sequences selected from the group consisting of the IDs. SEC. Numbers: 2/10, 18/20, 22/24, 26/34, 42/44, 46/48, 50/58, 66/68, 70/72, 74/82, 90/92, 94/96, 98/106, 114/116, 118/120, 122/130, 138/140, 142/144, 146/154, 162/164, 166/168, 170/178, 186/188, 190/192, 194 / 202, 210/212, 214/216, 218/226, 234/236, 238/240, 242/250, 258/260, 262/264,

266/274, 282/284, 286/288,

338/346, 354/356, 358/360,

410/418, 426/428, 430/432,

290/298, 306/308, 310/312,

362/370, 378/380, 382/384,

434/442, 450/452, 454/456,

314/322, 330/332, 334/336,

386/394, 402/404, 406/408,

458/466, 474/476, 478/480,

482/490, 498/500, 502/504,

554/562, 570/572, 574/576,

626/634, 642/644, 646/648,

506/514, 522/524, 526/528,

578/586, 594/596, 598/600,

650/658, 666/668, 670/672,

530/538, 546/548, 550/552,

602/610, 618/620, 622/624,

674/682, 690/692, 694/696,

698/706, 714/716, 718/720, 722/730, 738/740 and 742/744.

Pharmaceutical compositions and administration methods

The present invention includes methods comprising administering a PCSK9 inhibitor to a patient, wherein the PCSK9 inhibitor is contained in a pharmaceutical composition. The pharmaceutical compositions of the invention are formulated with suitable carriers, excipients and other agents that provide adequate transfer, delivery and tolerance and the like. In the form known to all pharmaceutical chemists: Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pennsylvania, USA, a multitude of appropriate formulations can be found. These formulations include, for example, powders, pastes, ointments, ointments, waxes, oils, lipids, vesicles containing lipids (cationic or anionic) (such as LIPOFECTIN ™), DNA conjugate products, anhydrous pastes for absorption, emulsions of oil in water and water in oil, Carbowax emulsions (polyethylene glycols of various molecular weights), semi-solid gels, and semi-solid 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 that can be used to administer the pharmaceutical composition of the invention, such as, for example, encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing mutant viruses, and 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, for example, by infusion, by bowling injection or by absorption through epithelial or mucocutaneous covers (e.g., 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 supplied subcutaneously or intravenously with a standard needle and syringe. Furthermore, with respect to subcutaneous delivery, a pen-shaped delivery device then has applications in the delivery of a pharmaceutical composition of the present invention, said pen-shaped delivery device can be reusable or disposable. A replaceable cartridge containing a pharmaceutical composition is generally used in a reusable pen-shaped delivery device. Once the entire pharmaceutical composition of the cartridge has been administered and the cartridge is empty, the empty cartridge can be easily discarded and replaced with a new cartridge containing the pharmaceutical composition. The pen-shaped delivery device can then be reused. In a disposable pen-shaped delivery device there is no replaceable cartridge. Instead, the disposable pen delivery device is pre-filled with the pharmaceutical composition contained in a reservoir within the device. Once the pharmaceutical composition of the reservoir has been exhausted, the entire device is discarded.

Numerous reusable pen and autoinjector delivery devices have applications in the subcutaneous delivery of a pharmaceutical composition of the present invention. Examples include, but are not limited to, AUTOPEN ™ (Owen Mumford, Inc., Woodstock, UK), the DISETRONIC ™ pen (Disetronic Medical Systems, Bergdorf, Switzerland), the HUMALOG MIX 75/25 ™ pen, the pen HUMALOG ™ and the HUMALIN 70/30 ™ pen (Eli Lilly and Co., Indianapolis, Indiana, USA), NOVOPEN ™ I, II and lll (Novo Nordisk, Copenhagen, Denmark), NOVOPEN JUNIOR ™ (Novo Nordisk, Copenhagen, Denmark), the BD ™ pen (Becton Dickinson, Franklin Lakes, New Jersey, USA), and OPTIPEN ™, OPTIPEN PRO ™, OPTIPEN STARLET ™ and OPTICLIK ™ (Sanofi-Aventis, Frankfurt, Germany), to name just a few. Examples of disposable pen delivery devices that have applications in the subcutaneous delivery of a pharmaceutical composition of the present invention include, but are not limited to, the SOLOSTAR ™ pen (SanofiAventis), the FLEXPEN ™ (Novo Nordisk) and KWIKPEN ™ (Eli Lilly), SURECLICK ™ autoinjector (Amgen, Thousand Oaks, California, USA), PENLET ™ (Haselmeier, Stuttgart, Germany), EPIPEN (Dey, LP) and HUMIRA ™ pen ( Abbott Labs, Abbott

Park, Illinois, USA), to name just a few.

In certain situations, the pharmaceutical composition may be supplied in a controlled release system. In one embodiment, a pump can be used (see Langer, supra; and Sefton, 1987, CRC Crit. Ref. Biomed. Eng. 14: 201). In another embodiment, polymeric materials can be used; see Medical Applications of Controlled Release, written by Langer and Wise, 1974, CRC Pres., Boca Raton, Florida, USA. In yet another embodiment, a controlled release system may be placed in the immediate vicinity of the composition target, thus requiring only a fraction of the systemic dose (see, for example, Goodson, 1984, in Medical Applications of Controlled Release, supra , volume 2, pages 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 oily medium conventionally used for injections. As for the aqueous medium for injections, there are, for example, the 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 (for example, propylene glycol or polyethylene glycol), a non-ionic surfactant [for example, polysorbate 80 or HCO-50 (hydrogenated castor oil adduct and polyoxyethylene (50 moles)], etc. As for the oily medium, sesame oil, soybean oil, etc., can be used, for example, which can be used in combination with a solubilizing agent such as benzyl benzoate, benzyl alcohol, etc. The injection thus prepared is preferably loaded into an appropriate vial.

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

Dosage

The amount of PCSK9 inhibitor (eg, anti-PCSK9 antibody) administered to a subject according to the methods of the present invention is, in general, a therapeutically effective amount. As used herein, the phrase "therapeutically effective amount" means a dose of PCSK9 inhibitor that results in a detectable reduction in serum levels of Lp (a). For example, a therapeutically effective amount of a PCSK9 inhibitor includes, for example, an amount of PCSK9 inhibitor that causes a reduction of at least 2%, 5%, 10%, 15%, 20%, 25%, 30 %, 40%, 50% or more at the levels of Lp (a) when administered to a human patient, as illustrated, for example, in Example 2 of this specification. Alternatively, animal models can be used to establish whether a particular amount of a candidate for PCSK9 inhibitor is a therapeutically effective amount.

In the case of an anti-PCSK9 antibody, a therapeutically effective amount may

<td colspan="9">be from about 0.05 mg to about 600 mg, for example,</td>

<td>approximately</td><td> 0,05</td><td>mg,</td><td>approximately</td><td> 0,1</td><td>mg,</td><td>approximately</td><td> 1.0</td><td>mg,</td>

<td>approximately</td><td> 1,5</td><td>mg,</td><td>approximately</td><td> 2,0</td><td>mg,</td><td>approximately</td><td> 10</td><td>mg,</td>

<td>approximately</td><td> 20</td><td>mg,</td><td>approximately</td><td> 30</td><td>mg,</td><td>approximately</td><td> 40</td><td>mg,</td>

<td>approximately</td><td> 50</td><td>mg,</td><td>approximately</td><td> 60</td><td>mg,</td><td>approximately</td><td> 70</td><td>mg,</td>

<td>approximately</td><td> 80</td><td>mg,</td><td>approximately</td><td> 90</td><td>mg,</td><td>approximately</td><td> 100</td><td>mg,</td>

<td>approximately</td><td> 110</td><td>mg,</td><td>approximately</td><td> 120</td><td>mg,</td><td>approximately</td><td> 130</td><td>mg,</td>

<td>approximately</td><td> 140</td><td>mg,</td><td>approximately</td><td> 150</td><td>mg,</td><td>approximately</td><td> 160</td><td>mg,</td>

<td>approximately</td><td> 170</td><td>mg,</td><td>approximately</td><td> 180</td><td>mg,</td><td>approximately</td><td> 190</td><td>mg,</td>

<td>approximately</td><td> 200</td><td>mg,</td><td>approximately</td><td> 210</td><td>mg,</td><td>approximately</td><td> 220</td><td>mg,</td>

<td>approximately</td><td> 230</td><td>mg,</td><td>approximately</td><td> 240</td><td>mg,</td><td>approximately</td><td> 250</td><td>mg,</td>

<td>approximately</td><td> 260</td><td>mg,</td><td>approximately</td><td> 270</td><td>mg,</td><td>approximately</td><td> 280</td><td>mg,</td>

<td>approximately</td><td> 290</td><td>mg,</td><td>approximately</td><td> 300</td><td>mg,</td><td>approximately</td><td> 310</td><td>mg,</td>

<td>approximately</td><td> 320</td><td>mg,</td><td>approximately</td><td> 330</td><td>mg,</td><td>approximately</td><td> 340</td><td>mg,</td>

<td>approximately</td><td> 350</td><td>mg,</td><td>approximately</td><td> 360</td><td>mg,</td><td>approximately</td><td> 370</td><td>mg,</td>

<td>approximately</td><td> 380</td><td>mg,</td><td>approximately</td><td> 390</td><td>mg,</td><td>approximately</td><td> 400</td><td>mg,</td>

<td>approximately</td><td> 410</td><td>mg,</td><td>approximately</td><td> 420</td><td>mg,</td><td>approximately</td><td> 430</td><td>mg,</td>

<td>approximately</td><td> 440</td><td>mg,</td><td>approximately</td><td> 450</td><td>mg,</td><td>approximately</td><td> 460</td><td>mg,</td>

<td>approximately</td><td> 470</td><td>mg,</td><td>approximately</td><td> 480</td><td>mg,</td><td>approximately</td><td> 490</td><td>mg,</td>

approximately 500 mg, approximately 510 mg, approximately 520 mg, approximately 530 mg, approximately 540 mg, approximately 550 mg, approximately 560 mg, approximately 570 mg, approximately 580 mg, approximately 590 mg or approximately 600 mg, of the anti-PCSK9 antibody.

The amount of anti-PCSK9 antibody contained in the individual doses can be expressed in terms of milligrams of antibody per kilogram of the patient's body weight (i.e. mg / kg). For example, the anti-PCSK9 antibody can be administered to a patient at a dose of about 0.0001 to about 10 mg / kg of the patient's body weight.

Combination therapies

In accordance with certain embodiments, the methods of the present invention may comprise administering a pharmaceutical composition comprising an antiPCSK9 antibody to a patient who is under a therapeutic regimen for the treatment of hypercholesterolemia at the time or just before administration of the composition. Pharmaceutical of the invention. For example, a patient who has been previously diagnosed with hypercholesterolemia may have been prescribed, and may be following, a stable therapeutic regimen of another drug before, and / or concurrently with, the administration of a pharmaceutical composition comprising a antiPCSK9 antibody. The prior or concurrent therapeutic regimen may comprise, for example, (1) an agent that causes a cellular reduction in cholesterol synthesis by inhibiting 3-hydroxy-3-methylglutaryl (HMG) -coenzyme A (CoA) reductase, such as a statin (for example, cerivastatin, atorvastatin, simvastatin, pitavastatin, rosuvastatin, fluvastatin, lovastatin, pravastatin, etc.); (2) an agent that inhibits the incorporation of cholesterol and / or the reabsorption of bile acids; (3) an agent that increases the lipoprotein catabolism (such as niacin); and / or (4) activators of the transcription factor LXR that plays a role in the elimination of cholesterol, such as 22-hydroxycholesterol. In certain embodiments, the patient, before, or concurrently with, the administration of an anti-PCSK9 antibody, is under a fixed combination of therapeutic agents, such as ezetimibe plus simvastatin; a statin with a biliary resin (for example, cholestyramine, colestipol or colesevelam); niacin plus a statin (for example, niacin with lovastatin); or with other agents reducing lipid levels, such as ethyl esters of omega-3 fatty acids (eg, omacor).

Administration Regimes

In accordance with certain embodiments of the present invention, multiple doses of a PCSK9 inhibitor can be administered to a subject over a defined time course. The methods according to this aspect of the invention comprise sequentially administering multiple doses of a PCSK9 inhibitor to a subject. As used herein, administering sequentially means that each dose of PCSK9 inhibitor is administered to the subject at a different time point, for example, on different days separated by a predetermined interval (eg, hours, days, weeks or months). . The present invention includes methods comprising sequentially administering to the patient a single initial dose of a PCSK9 inhibitor, followed by one or more secondary doses of the PCSK9 inhibitor and, optionally, followed by one or more tertiary doses of the PCSK9 inhibitor.

The terms initial dose, secondary doses and tertiary doses refer to the temporal sequence of administration of the PCSK9 inhibitor. Thus, the initial dose is the dose that is administered at the beginning of the treatment regimen (which is also referred to as a baseline dose); the secondary doses are the doses that are administered after the initial dose; and tertiary doses are the doses that are administered after the secondary doses. The initial, secondary and tertiary doses may all contain the same amount of PCSK9 inhibitor, but will generally differ from one another in terms of frequency of administration. However, in certain embodiments, the amounts of PCSK9 inhibitor contained in the initial, secondary and / or tertiary doses will vary from one another (for example, adjusted up or down as appropriate) during the course of treatment.

In an exemplary embodiment of the present invention, each secondary and / or tertiary dose is administered from 1 to 30 (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more) days after the immediately preceding dose. The phrase the immediately preceding dose, as used herein, means, in a sequence of multiple administrations, the dose of PCSK9 inhibitor that is administered to a patient before administration of the dose just following the sequence without any dose. intermediate.

The methods according to this aspect of the invention may comprise administering to a patient any number of secondary and / or tertiary doses of a

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ΠΑ PHO-PhEDAD 42 »: heeetp: al! \ 3 inhibitor of PCSK9. For example, in certain embodiments, only a single secondary dose is administered to the patient. In other embodiments, two or more (eg, 2, 3, 4, 5, 6, 7, 8 or more) are administered secondary doses to the patient. Also, in certain embodiments, only a single tertiary dose is administered to the patient. In other embodiments, two or more (eg, 2, 3, 4, 5, 6, 7, 8 or more) are administered tertiary doses to the patient.

In embodiments involving multiple secondary doses, each secondary dose may be administered with the same frequency as the other secondary doses. For example, each secondary dose can be administered to the patient 1 to 29 days after the immediately preceding dose. Similarly, in embodiments involving multiple tertiary doses, each tertiary dose may be administered with the same frequency as the other tertiary doses. For example, each tertiary dose can be administered to the patient 1 to 60 days after the immediately preceding dose. Alternatively, the frequency with which secondary and / or tertiary doses are administered to a patient may vary throughout the course of the treatment regimen. The frequency of administration can also be adjusted during the course of treatment by a doctor depending on the needs of the individual patient after a clinical examination.

EXAMPLES

The following examples are set forth in order to provide those with ordinary experience in the art 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 your invention. Efforts have been made to ensure accuracy with respect to the numbers used (eg, quantities, temperature, etc.), but certain errors and experimental deviations should be available. Unless stated otherwise, the parts are parts by weight, the molecular weight is the average molecular weight, the temperature is in degrees Celsius, and the pressure is atmospheric or close to it.

Example 1. Generation of human antibodies to human PCSK9

Human anti-PCSK9 antibodies were generated in the manner described in Publication η 2010/0166768 of US Patent Application. The exemplary inhibitor of PCSK9 used in the following Example is the human anti-PCSK9 antibody called mAb316P. The mAb316P has the following amino acid sequence characteristics: heavy chain variable region (HCVR) comprising the ID. SEC. No. 90; Light chain variable domain (LCVR) comprising the ID. SEC. n ° 92; region 1 determining the heavy chain complementarity (HCDR1) comprising the ID. SEC. No. 76; HCDR2 comprising the ID. SEC. No. 78; HCDR3 comprising the ID. SEC. No. 80; light chain complementarity determining region 1 (LCDR1) comprising the ID. SEC. No. 84; LCDR2 comprising the ID. SEC. No. 86; and LCDR3 comprising the ID. SEC. No. 88.

Example 2: Clinical trials of an anti-PCSK9 monoclonal antibody, such as monotherapy or complementary therapy in heterozygous and unfamiliar familial hypercholesterolemias

Introduction

This example describes the results of two clinical trials in which single doses of the anti-PCSK9 mAb316P monoclonal antibody were administered intravenously (IV) or subcutaneously (SC) to healthy volunteers, and a multiple dose clinical trial of mAb316P in subjects with hypercholesterolemia familial (FH) or nonFH, who received stable doses of atorvastatin or only dietary intervention.

Methods

A. Study design

Three clinical tests were carried out using mAb316P administered to human patients. Two of the tests were single-dose, placebo-controlled studies of mAb316P administered IV or SC, respectively. The third test was an assessment with multiple increasing doses, placebo-controlled, randomized, double-blind, phase 1b, of mAb316P administered SC to subjects with FH or nonFH. The third test was carried out in two parts: Part A and Part B. In Part A of the third test, each subject received a total of 3 administrations of mAb316P (50 mg, 100 mg or 150 mg) or equivalent placebo. In Part B of the third test, each subject received 2 administrations of mAb316P (200 mg) or equivalent placebo. In both parts of the test with multiple SC doses, the first dose was administered in a unit of hospitalized patients where the subjects were confined and observed for 48 hours after the dose. Subsequent doses were administered in an outpatient setting with at least 2 hours of observation after the dose.

B. Dose and dose increase

The single dose IV study included 5 sequential cohorts (0.3, 1.0, 3.0, 6.0 or

12.0 mg / kg of mAb316P), and the single dose SC test included 4 sequential dose cohorts (50, 100, 150 and 250 mg of mAb316P). Based on the initial results of these tests, in the third test SC doses of mAb316P of 50 mg, 100 mg, 150 mg or placebo were administered on days 1, 29 and 43 to 7 groups of subjects with FH 10 or no FH (Part A), and 200 mg or placebo administered on days 1 and 29 to an eighth group of subjects with FH or nonFH (Part B). Table 1 summarizes the dosage regimens and patient compositions of the three tests.

Table 1

<td>Dose of mAb 316</td><td>Patient group</td><td>Total number of patients (mAb316: Pbo)</td><td>Kind of patient</td><td>LDL-C scan</td><td>Dose of atorvastatin</td>

<td colspan="6">Single IV dose</td>

<td>0.3 mg / kg</td><td> 1</td><td rowspan="5"> 8 (6:2)</td><td rowspan="5">Healthy volunteers</td><td rowspan="5">&gt; 100 mg / dl</td><td rowspan="5">None</td>

<td>1.0 mg / kg</td><td> 2</td>

<td>3.0 mg / kg</td><td> 3</td>

<td>6.0 mg / kg</td><td> 4</td>

<td>12.0 mg / kg</td><td> 5</td>

<td colspan="6">Single dose SC</td>

<td>50 mg</td><td> 1</td><td rowspan="3"> 8 (6:2)</td><td rowspan="3">Healthy volunteers</td><td rowspan="3">&gt; 100 mg / dl</td><td rowspan="3">None</td>

<td>100 mg</td><td> 2</td>

<td>150 mg</td><td> 3</td>

i

<td>250 mg</td><td> 4</td><td></td><td></td><td></td><td></td>

<td colspan="6">Multiple doses SC</td>

<td colspan="6">Part A</td>

<td rowspan="2">50 mg</td><td> 1</td><td> 7(5:2)</td><td>Fh</td><td rowspan="6">&gt; 100 mg / dl</td><td rowspan="6">10-40 mg OD</td>

<td> 2</td><td> 10(8:2)</td><td>noFH</td>

<td rowspan="2">100 mg</td><td> 3</td><td> 7(5:2)</td><td>Fh</td>

<td> 4</td><td> 10(8:2)</td><td>noFH</td>

<td rowspan="3">150 mg</td><td> 5</td><td> 7(5:2)</td><td>Fh</td>

<td> 6</td><td> 10(8:2)</td><td>noFH</td>

<td> 7</td><td> 10(8:2)</td><td>noFH</td><td>&gt; 130 mg / dl</td><td>None</td>

<td colspan="6">Part B</td>

<td>200 mg</td><td> 8</td><td> 10(8:2)</td><td>FH and noFH</td><td>&gt; 100 mg / dl</td><td>10-40 g OD</td>

In the multiple dose SC test, each dose level began with a sentinel group of 3 subjects (FH, noFH, or both), receiving mAb316P at least 1 subject but no more than 2 subjects. The incorporation of additional subjects at a given dose level 5 only took place after the initial 3 subjects had safely completed their evaluations on day 3 after treatment. The incorporation to the next higher dose level was opened once 10 subjects had completed the safety assessments on day 15. The incorporation, once started at each dose level, continued regardless of the decision to increase the dose and the review. of 10 security on day 15.

C. Subjects

In studies with a single dose, the subjects were healthy men and women [1865 years old, 50-95 kg body weight, body mass index (BMI; body mass index) = 18-30 kg / m²] with serum LDL-C levels> 100 mg / dL (2.59 15 mmol / L). Throughout these studies the use of all agents to alter lipids that were not in the study was prohibited.

In the multi-dose study, subjects had heterozygous FH or non-FH (18-65 years of age, BMI = 18.0-35.0 kg / m²) and were under a stable therapy of atorvastatin (10 to 40 mg daily) with LDL cholesterol levels> 100 mg / dl (2.59 mmol / l), or noFH and were only on a diet with LDL cholesterol levels> 130 mg / dl (3.36 mmol / l) (Table 1). All subjects were examined and signed an informed consent previously approved by an institutional examination committee before any procedure related to the study. The subjects of the FH group met Simón Broome's criteria for a true or possible FH (Simón Broome Register Group (1991), BMJ 303: 893-896; Neil et al., (2000), BMJ 321: 148), while that subjects with noFH did not. Those taking atorvastatin were under a stable dose, 10 to 40 mg per day, for at least 28 days before baseline, and remained with the same dose throughout the study. Patients with noFH and only on a diet could not have received lipid-reducing therapy for at least 28 days before baseline, and remained without such treatment throughout the study. Potentially eligible patients who did not take atorvastatin were able to enter a 4-week preparation period during which they were transferred to a dose of atorvastatin (10 to 40 mg daily) with which it was possible to maintain an LDL cholesterol level close to its level prior to the study and> 100 mg / dl (2.59 millimoles / l). All subjects had triglyceride levels at 300 mg / dl and a controlled blood pressure with less than 3 antihypertensive medications. Patients with a history of peripheral cerebrovascular, cardiovascular or vascular atherosclerotic disease, diabetes, or disorders known to produce secondary elevations in the level of LDL cholesterol were excluded, as were those with hepatic transaminase levels (ALT or AST)> 1,2 times the upper limit of normal (x ULN; English, x upper limit of normal), proteinuria> 2+ or creatine kinase (CK; from English, creatine kinase)> 3 x ULN, unless clearly related to the exercise, in which case it was necessary to repeat the test with CK <3 x ULN.

D. Laboratory methods and measurements

In the multi-dose study, laboratory tests of serum lipid levels (total-cholesterol, -HDL, -LDL, -no HDL and -VLDL), apolipoproteins (Apo) B and A1, Lp (a) were carried out. and hs-CRP and security after 12 hours of overnight fasting (water only) on exploration days (days -21 to -3), day -2 (for those under atorvastatin), day -1, days 1 , 2, 3, 8, 15, 29, 43, 57, 71, 85, 99 and 120, and at the end of the study (day 148). All laboratory measurements were carried out by a central laboratory that maintained the Part III certification of the CDC Lipid Standardization Program and the accreditation of the College of American Pathologists. Triglycerides and cholesterol were measured by enzymatic colorimetric assays (Olympus AU2700 or AU5400 analyzer, Olympus, Center Valley, Pennsylvania, USA), with a directly traceable calibration to CDC reference procedures. Lipoproteins containing Apo B were precipitated with dextran sulfate and HDL cholesterol was measured in the supernatant [Warnick et al., (1978), J. Lipid Res. 19: 65-76). Cholesterols -LDL and -VLDL were measured after preparative ultracentrifugation (beta quantification). Apo A1 and B, Lp (a) and hs-CRP were measured by kinetic immunonephelometry (Dade Behring BNII nephelometer, Siemens Healthcare Diagnostics, Deerfield, Illinois, USA). All lipid, apolipoprotein and hs-CRP values ​​were hidden from researchers, study staff and patients from after day -2 for the groups treated with atorvastatin or after day -1 for the diet-only group.

E. Statistical plan

In studies with a single dose, the effects of mAb316P on lipid parameters were evaluated using covariance analysis models (ANCOVA). Within the ANCOVA framework, the means by least squares of the differences between the treatment groups and the placebo groups gathered, the 95% confidence intervals, and the p values ​​for the comparison between the treatment groups and of placebo per visit. Significance was adjusted to 0.05 for all trials.

In the multi-dose study, all subjects who received placebo and atorvastatin were gathered in two placebo groups of subjects with FH or with noFH. The 6 subjects of the placebo groups of both FH and nonFH (treated with atorvastatin), and the 5 and 8 subjects of each of the respective dose groups with FH and nonFH treated with mAb316P, provided at least 80% of the ability to detect a treatment difference of 30% [standard deviation (SD; English standard deviation) = 15%] versus placebo in the average percentage change from baseline LDL cholesterol at each study visit, when each dose was compared with placebo with a two-way trial for 5% significance level. No adjustments were made for multiple comparisons. The results of Group 7 were summarized separately by treatment and placebo and the two groups were compared individually. To analyze the continuous variables, ANCOVA was used with the treatment arm as a fixed effect and with the relevant baseline value as a covariate. The lost values ​​were imputed by the method of the Last Observation Performed (LOCF; from English, Lost Observed Carried Forward). All p values, except for triglycerides and Lp (a), which were obtained from the Covariables analysis based on Ranges, were taken from the ANCOVA model.

To summarize the effects on lipids and lipoproteins, the results of day 57 of the study were selected, in which the effects of two subsequent dosing periods of 2 weeks could be observed. The study was not driven by a direct statistical comparison of the response of subjects with FH versus that of subjects with noFH.

Results

A. Study population

A total of 40 subjects were randomly distributed in the study with a single IV dose and 32 were randomly distributed in the study with a single SC dose. For Part A of the multi-dose SC study, a total of 97 subjects were screened and 62 (21 FH and 41 noFH) were randomized. A total of 10 subjects (4 FH and 6 noFH) were included in Part B of the study with multiple SC doses. Table 2A shows the baseline characteristics of the subjects in studies with a single dose and in Part A of the study with multiple doses. Table 2B shows the baseline characteristics of the subjects in Part B of the multi-dose study.

Table 2A

<td rowspan="2"></td><td rowspan="2">Single dose IV</td><td rowspan="2">Single dose SC</td><td colspan="3">Multiple doses SC - Part A</td>

<td>Fh</td><td>noFH</td><td>noFH, only diet (without atorvastatin)</td>

<td>No. of patients</td><td> 40</td><td> 32</td><td> 21</td><td> 30</td><td> 10</td>

<td>Middle ages</td><td> 36</td><td> 34</td><td> 40</td><td> 52</td><td> 52</td>

<td colspan="6">Sex</td>

<td>Male</td><td> 65%</td><td> 74%</td><td> 81%</td><td> 59%</td><td> 56%</td>

<td>Female</td><td> 35%</td><td> 26%</td><td> 19%</td><td> 41%</td><td> 44%</td>

<td>BMI medium (kg / m²)</td><td> 26</td><td> 25</td><td> 27</td><td> 27</td><td> 27</td>

<td colspan="6">Race (%)</td>

<td>White</td><td> 55%</td><td> 44%</td><td> 86%</td><td> 93%</td><td> 100%</td>

<td>Black / African American</td><td> 37,5%</td><td> 50%</td><td> 14%</td><td> 7%</td><td> 0%</td>

<td>American Indian / Alaskan Native</td><td> 7,5%</td><td> 6%</td><td> 0%</td><td> 0%</td><td> 0%</td>

<td colspan="6">Atorvastatin dose</td>

<td>10 mg</td><td>None</td><td>None</td><td> 14%</td><td> 63%</td><td>None</td>

<td>20 mg</td><td>None</td><td>None</td><td> 33%</td><td> 33%</td><td>None</td>

<td>40 mg</td><td>None</td><td>None</td><td> 52%</td><td> 4%</td><td>None</td>

<td colspan="6">Baseline Value</td>

<td>LDL-C (mg / dl)</td><td> 133</td><td> 127</td><td> 134</td><td> 111</td><td> 174</td>

<td>ApoB (mg / dl)</td><td> 1,1</td><td> 1,0</td><td> 1,1</td><td> 1,0</td><td> 1,3</td>

<td>C-HDL (mg / dl)</td><td> 54</td><td> 55</td><td> 44</td><td> 51</td><td> 51</td>

<td>TG (mg / dl)</td><td> 108</td><td> 103</td><td> 113</td><td> 136</td><td> 133</td>

Table 2B

<td></td><td colspan="6">Multiple doses SC - Part B (200 mg dose)</td>

<td></td><td colspan="2">FH and noFH combined</td><td colspan="2">Fh</td><td colspan="2">NoFH</td>

<td></td><td>Pbo</td><td>mAb316P</td><td>Pbo</td><td>mAb316P</td><td>Pbo</td><td>mAb316P</td>

<td>No. of patients</td><td> 2</td><td> 8</td><td> 1</td><td> 3</td><td> 1</td><td> 5</td>

<td>Middle ages</td><td> 35</td><td> 45,5</td><td> 27</td><td> 50</td><td> 43</td><td> 41</td>

<td colspan="7">Sex</td>

<td>Male</td><td> 50%</td><td> 37,5%</td><td> 100%</td><td> 66,7%</td><td> 0%</td><td> 20%</td>

<td>Female</td><td> 50%</td><td> 62,5%</td><td> 0%</td><td> 33,3%</td><td> 100%</td><td> 80%</td>

<td>BMI median (kg / m²)</td><td> 29,39</td><td> 26,02</td><td> 28,26</td><td> 25,43</td><td> 30,52</td><td> 26,61</td>

<td colspan="7">Race (%)</td>

<td>White</td><td> 100%</td><td> 100%</td><td> 100%</td><td> 100%</td><td> 100%</td><td> 100%</td>

<td colspan="7">Atorvastatin dose</td>

<td>10 mg</td><td> 50%</td><td> 75%</td><td> 0%</td><td> 33,3%</td><td> 100%</td><td> 100%</td>

<td>20 mg</td><td> 0%</td><td> 12,5%</td><td> 0%</td><td> 33,3%</td><td> 0%</td><td> 0%</td>

<td>40 mg</td><td> 50%</td><td> 12,5%</td><td> 100%</td><td> 33,3%</td><td> 0%</td><td> 0%</td>

<td colspan="7">Baseline Value</td>

<td>C-LDL (millimoles / l)</td><td> 3,05</td><td> 3,32</td><td> 3,76</td><td> 3,78</td><td> 2,33</td><td> 3,08</td>

<td>ApoB (g / l)</td><td> 0,85</td><td> 1,06</td><td> 0,92</td><td> 1,05</td><td> 0,77</td><td> 1,07</td>

<td>C-HDL (millimoles / l)</td><td> 1,03</td><td> 1,23</td><td> 0,88</td><td> 1,19</td><td> 1,17</td><td> 1,30</td>

<td>TG (millimoles / l)</td><td> 1,17</td><td> 1,23</td><td> 1,01</td><td> 0,98</td><td> 1,33</td><td> 1,30</td>

B. Lipid and lipoprotein response

The administration of a single IV or SC dose of mAb316P to healthy volunteers produced similar maximum average percentages of LDL cholesterol reductions of 55-60%. The degree and duration of LDL cholesterol reduction were dose dependent, and LDL cholesterol reductions were maintained at least up to 29 and 22 days after administration of higher dose levels of mAb316P IV or SC, respectively. Similarly, in the multi-dose study, the average percentage of LDL cholesterol reduction from baseline was dose dependent and exceeded 50% two weeks after 150 mg administration in the FH and nonFH populations under atorvastatin. and of noFH only with diet. All subjects except 1 in each population who received 150 mg in the multi-dose study experienced a reduction of at least 40% from baseline.

Tables 3A (subjects treated with atorvastatin) and 3B (diet only - without treatment with atorvastatin) show baseline lipid and apolipoprotein levels on day 57 for all treatment groups in part A of the study with multiple doses

Table 3A

<td colspan="2" rowspan="3"></td><td colspan="4">Subjects treated with atorvastatin</td>

<td rowspan="2">Placebo (Pbo)</td><td colspan="3">Dose of mAb316P</td>

<td>50 mg</td><td>100 mg</td><td>150 mg</td>

<td colspan="2">Parameter</td><td>N = 12</td><td>N = 13</td><td>N = 13</td><td>N = 13</td>

<td rowspan="4">Half of LDL (SD) cholesterol mg / dl</td><td>Baseline</td><td> 125(19)</td><td> 114(15)</td><td> 121(31)</td><td> 123(27)</td>

<td>Day 57</td><td> 195(17)</td><td> 148 (29)</td><td> 130(16)</td><td> 127 (26)</td>

<td>% change vs. Pbo</td><td></td><td> -39,2</td><td> -53,7</td><td> -61,0</td>

<td>P value vs. Pbo</td><td></td><td> &lt; 0,0001</td><td> &lt; 0,0001</td><td> &lt; 0,0001</td>

<td rowspan="4">Half of total cholesterol (SD) mg / dl</td><td>Baseline</td><td> 193 (20)</td><td> 191 (24)</td><td> 192 (27)</td><td> 197(32)</td>

<td>Day 57</td><td> 195(17)</td><td> 148 (29)</td><td> 130(16)</td><td> 127 (26)</td>

<td>% change vs. Pbo</td><td></td><td> -24,6</td><td> -33,2</td><td> -36,4</td>

<td>P value vs. Pbo</td><td></td><td> &lt; 0,0001</td><td> &lt; 0,0001</td><td> &lt; 0,0001</td>

<td rowspan="4">Half of HDL (SD) cholesterol mg / dl</td><td>Baseline</td><td> 43(10)</td><td> 52 (16)</td><td> 48(10)</td><td> 48(11)</td>

<td>Day 57</td><td> 42(10)</td><td> 55(14)</td><td> 51 (10)</td><td> 54(12)</td>

<td>% change vs. Pbo</td><td></td><td> 13,2</td><td> 11,3</td><td> 18,2</td>

<td>P value vs. Pbo</td><td></td><td> 0,0153</td><td> 0,0336</td><td> 0,0009</td>

<td>Half of</td><td>Baseline</td><td> 125 (20)</td><td> 115(16)</td><td> 119(30)</td><td> 123 (29)</td>

<td rowspan="3">LDL cholesterol (SD) mg / dl</td><td>Day 57</td><td> 129(15)</td><td> 74(15)</td><td> 58 (14)</td><td> 52 (21)</td>

<td>% change vs. Pbo</td><td></td><td> -41,7</td><td> -55,8</td><td> -62,4</td>

<td>P value vs. Pbo</td><td></td><td> &lt; 0,0001</td><td> &lt; 0,0001</td><td> &lt; 0,0001</td>

<td>Medium of</td><td>Baseline</td><td> 113(60:244)</td><td> 118(43:171)</td><td> 135 (46:210)</td><td> 117 (65:278)</td>

<td>triglycerides (min: max)</td><td>Day 57</td><td> 111 (55:186)</td><td> 97 (40:189)</td><td> 111 (31:173)</td><td> 92 (60:181)</td>

<td>mg / dl</td><td>% change vs. Pbo</td><td></td><td> -16,3</td><td> -7,5</td><td> -11,7</td>

<td></td><td>P value vs. Pbo</td><td></td><td> 0,0142</td><td> 0,2515</td><td> 0,0609</td>

<td>Half of</td><td>Baseline</td><td> 107(12)</td><td> 104(15)</td><td> 100 (21)</td><td> 106(22)</td>

<td>apolipopro-</td><td>Day 57</td><td> 108(14)</td><td> 74(14)</td><td> 60(12)</td><td> 58(14)</td>

<td rowspan="2">Theine B (SD) mg / dl</td><td>% change vs. Pbo</td><td></td><td> -31,5</td><td> -42,0</td><td> -46,4</td>

<td>P value vs. Pbo</td><td></td><td> &lt; 0,0001</td><td> &lt;0,0001</td><td> &lt; 0,0001</td>

<td>Half of</td><td>Baseline</td><td> 132(19)</td><td> 155 (36)</td><td> 144(23)</td><td> 145 (23)</td>

<td>apolipopro-</td><td>Day 57</td><td> 132 (20)</td><td> 162(31)</td><td> 150(20)</td><td> 161 (24)</td>

<td rowspan="2">Theine A1 (SD) mg / dl</td><td>% change vs. Pbo</td><td></td><td> 9,9</td><td> 6,9</td><td> 13,5</td>

<td>P value vs. Pbo</td><td></td><td> 0,019</td><td> 0,086</td><td> 0,013</td>

<td>Medium of</td><td>Baseline</td><td> 22 (2:121)</td><td> 46(5:151)</td><td> 50 (7:142)</td><td> 61 (5:154)</td>

<td>lipoprotein (a) (min: max)</td><td>Day 57</td><td> 13 (2:119)</td><td> 42 (4:145)</td><td> 26 (3:95)</td><td> 47 (5:119)</td>

<td>mg / dl</td><td>% change vs. Pbo</td><td></td><td> -15,5</td><td> -24,1</td><td> -18,3</td>

<td></td><td>P value vs. Pbo</td><td></td><td> 0,1109</td><td> 0,0015</td><td> 0,1226</td>

Table 3B

<td colspan="2" rowspan="2"></td><td colspan="2">Diet only - No treatment with atorvastatin</td>

<td>Placebo (Pbo)</td><td>150 mg mAb316P</td>

<td colspan="2">Parameter</td><td>N = 2</td><td>N = 8</td>

<td rowspan="4">Half of LDL (SD) cholesterol mg / dl</td><td>Baseline</td><td> 152(16)</td><td> 179 (49)</td>

<td>Day 57</td><td> 242 (45)</td><td> 154 (29)</td>

<td>% change vs. Pbo</td><td></td><td> -57,0</td>

<td>P value vs. Pbo</td><td> &lt; 0,0001</td><td> 0,0023</td>

<td rowspan="4">Half of total cholesterol (SD) mg / dl</td><td>Baseline</td><td> 228(6)</td><td> 257(58)</td>

<td>Day 57</td><td> 242(45)</td><td> 154 (29)</td>

<td>% change vs. Pbo</td><td></td><td> -43,3</td>

<td>P value vs. Pbo</td><td></td><td> 0,0025</td>

<td rowspan="4">Half of HDL (SD) cholesterol mg / dl</td><td>Baseline</td><td> 54 (14</td><td> 50 (8)</td>

<td>Day 57</td><td> 64 (8)</td><td> 51 (10)</td>

<td>% change vs. Pbo</td><td></td><td> -18,9</td>

<td>P value vs. Pbo</td><td></td><td> 0,17</td>

<td rowspan="4">Half of LDL cholesterol (SD) mg / dl</td><td>Baseline</td><td> 152(12)</td><td> 177 (49)</td>

<td>Day 57</td><td> 159 (30)</td><td> 79 (24)</td>

<td>% change vs. Pbo</td><td></td><td> -58,4</td>

<td>P value vs. Pbo</td><td></td><td> &lt; 0,0025</td>

<td rowspan="4">Medium of triglycerides (min: max) mg / dl</td><td>Baseline</td><td> 116(86:146)</td><td> 127 (82:268)</td>

<td>Day 57</td><td> 93 (68:118)</td><td> 116(70:186)</td>

<td>% change vs. Pbo</td><td></td><td> -16,86</td>

<td>P value vs. Pbo</td><td></td><td> 0,4820</td>

<td>Half of</td><td>Baseline</td><td> 118(23)</td><td> 138(37)</td>

<td rowspan="3">apolipoprotein B (SD) mg / dl</td><td>Day 57</td><td> 118(6)</td><td> 76(19)</td>

<td>% change vs. Pbo</td><td></td><td> -45,0</td>

<td>P value vs. Pbo</td><td></td><td> 0,003</td>

<td rowspan="4">Half of apolipoprotein A1 (SD) mg / dl</td><td>Baseline</td><td> 138 (6)</td><td> 151 (15)</td>

<td>Day 57</td><td> 170 (32)</td><td> 154(16)</td>

<td>% change vs. Pbo</td><td></td><td> -19,2</td>

<td>P value vs. Pbo</td><td></td><td> 0,073</td>

<td rowspan="4">Medium of lipoprotein (a) (minimax) mg / dl</td><td>Baseline</td><td> 47 (30:63)</td><td> 34 (5:111)</td>

<td>Day 57</td><td> 58 (41:75)</td><td> 39 (4:119)</td>

<td>% change vs. Pbo</td><td></td><td> -43,8</td>

<td>P value vs. Pbo</td><td></td><td> 0,0415</td>

The LDL cholesterol response on day 57 was similar in all subjects regardless of whether they had FH or nonFH, were treated with atorvastatin or were only on a diet. The corresponding changes were also observed in total and non-HDL cholesterol 5 and in Apo B (Tables 3A and 3B). It is important to indicate that reductions in Lp (a) were also observed. In addition, favorable changes in both HDL cholesterol and ApoA1 were seen in patients taking atorvastatin.

Similar improvements were observed in Part B of the multi-dose study in patients treated with mAb316P. That is, the subcutaneous administration of 200 mg of 10 mAb316P caused rapid, substantial and sustained reductions in the levels of CLDL, total cholesterol, C-non HDL and ApoB and in the ApoB / ApoA ratio. It also appeared that patients who had received SC 200 mg of mAb316P had a tendency towards higher levels of C-HDL and ApoA1.

Discussion

This Example describes human trials of the anti-PCSK9 antibody, referred to herein as mAb316P, beginning with two studies with increasing single dose in healthy volunteers and continuing with a large proof-of-concept trial with multiple doses in patients. both with FH and with noFH, treated with a statin or only under diet. These trials confirm the potential to cause rapid and significant reductions in LDL cholesterol levels with PCSK9 inhibition in both familial and unfamiliar hypercholesterolemia, whether with a statin or only on a diet. The populations examined cover the vast majority of patients with hypercholesterolemia.

The robust and reproducible effect of mAb316P on LDL cholesterol from a single IV administration, through a single SC administration, to multiple SC doses is seen in an unexpectedly short time after administration, reaching a maximum at approximately 2 weeks. This speed exceeds that of all other therapeutic modalities for hypercholesterolemia other than the elimination of LDL cholesterol by apheresis, and is greater than that of statins. The fact that large reductions in LDL cholesterol levels with mAb316P can be achieved in patients already at relatively high doses of atorvastatin, known to produce reductions in LDL cholesterol levels from 40% to 50%, clearly distinguishes the potential of mAb316P from others. investigational therapies that have been added to statins, such as ezetimibe, bile acid sequestrants and squalene synthase inhibitors. The efficacy of mAb316P in reducing LDL cholesterol levels, combined with apparent tolerability and safety, highlights the surprising therapeutic advantages of mAb316P over other different agents reducing the levels of Apo B / C-LDL levels in development, such as Apo B antisense, MTP inhibitors and thyroid hormone analogues. In fact, the achievement, in many of the subjects of these studies, of low LDL cholesterol levels of 50 mg per deciliter (1.29 mmol / l) or less without any increase in liver transaminases is in stark contrast to the results obtained with agents that inhibit the hepatic formation of VLDL and LDL. Epidemiological studies that show that it does not appear that genetic under-expression, or even the absence, of PCSK9 and low levels of LDL cholesterol throughout life are associated with unexpected morbidity or mortality, provide a level of comfort with respect to the therapeutic potential of Pharmacological inhibition of PCSK9.

The anti-PCSK9 antibodies of the present invention, such as mAb316P, also provide patients unable to tolerate statins (which are, as is now estimated, from about 5% to 10% of all patients treated with statins) a significant opportunity to achieve large reductions (of at least 50%) in the level of LDL cholesterol. For many of the estimated 10 million patients with FH in the world, the anti-PCSK9 antibodies of the invention, including rr »Ab316P, offer the real possibility that their use together with that of statins can finally achieve optimal control of LDL cholesterol and possibly further reduce your substantial risk of early and recurrent cardiovascular disease. The therapeutic methodologies currently described can also offer many patients the potential to suspend LDL apheresis, an invasive, expensive procedure that takes a long time and takes place every 2 weeks, which only provides a reduction in the level of LDL cholesterol in the short term. .

Additional beneficial effects on other lipids were seen in HDL cholesterol, Apo A1 and, most surprisingly, Lp (a), which tended to, or reached, statistical significance even in patients treated with atorvastatin. It is important to indicate that, before now, it had not been clinically shown that therapeutic biological agents (for example, anti-PCSK9 antibodies) reduced the levels of Lp (a), from which it has been thought that it is not suppressed through the receptor of LDL. Thus, current experiments are the first demonstration of the ability of a PCSK9 inhibitor to reduce serum Lp (a) levels in patients.

In terms of safety, reactions at the injection site of mAb316P were minimal. MAb316P was also generally safe and well tolerated, without any tendency to negative processes related to the drug and without any evidence of hepatotoxicity or toxicity. The few increases in CK that were seen were related to the exercise.

In summary, this Example shows that the inhibition of PCSK9 by mAb316P, in addition to effectively reducing the level of LDL cholesterol in human patients, also surprisingly reduced the levels of Lp (a).

Example 3: A 12-week randomized, double-blind, placebo-controlled study of the safety and efficacy of an anti-PCSK9 monoclonal antibody in patients with heterozygous familial hypercholesterolemia.

A clinical trial was conducted to evaluate the efficacy of subcutaneous doses (SC) and variable dosing regimens of mAb316P on serum levels of low-density lipoprotein (C-LDL) cholesterol and other lipids / apolipoproteins [eg cholesterol total, high density lipoprotein cholesterol, triglycerides, apo B, Apo A1 and

Lp (a) J in patients with heterozygous familial hypercholesterolemia (heFH).

Patient population

The patient population for this study included men and women, 18-75 years of age, who had been diagnosed with heFH, had LDL-C levels of 100 mg / dl or more, and were under a stable daily dose of statin (with or without ezetimíba) at the beginning of the study. Patients were excluded if they were taking any additional compounds that reduce lipid levels, such as fibrates, niacin, omega-3 fatty acids, bile acid resins, vegetable stains (e.g., Benecol, flaxseed oil, zaragatone) and rice fermented red. A total of 77 patients completed the study.

Pharmacological Formulation

The pharmacological formulation comprised 150 mg / ml of mAb316P, 10 mM histidine, 0.2% polysorbate 20 and 10% sucrose, and had a pH of 6.0.

Administration method

The pharmacological formulation (or placebo) was administered to patients by subcutaneous injection into the abdomen. Each treatment included two subcutaneous injections, each 1 ml.

Administration Regimes

The patients were divided into five groups. Group 1 included 15 patients who received placebo once every two weeks; Group 2 included 16 patients who received 150 mg of mAb316P once every two weeks; Group 3 included 15 patients who received 150 mg of mAb316P once every four weeks; Group 4 included 16 patients who received 200 mg of mAb316P once every four weeks; and Group 5 included 15 patients who received 300 mg of mAb316P once every four weeks. The duration of the study was 12 weeks.

Effectiveness evaluation

Blood samples were collected from patients throughout the study and for a continuation period of 8 weeks. Samples were collected after a fast of at least 12 hours (in the morning, before ingestion of any drug). In the 5 blood samples measurements of the following parameters were made: (1) total cholesterol; (2) C-LDL; (3) C-HDL; (4) triglycerides (TG); (5) Apo B; (6) Apo A1; and (7) Lp (a). Table 4 summarizes the medians of the percentage changes of these parameters compared to placebo at the end of the study (week 12).

Table 4

<td>Group</td><td> 1</td><td> 2</td><td> 3</td><td> 4</td><td> 5</td>

<td>No. of patients</td><td> 15</td><td> 16</td><td> 15</td><td> 16</td><td> 15</td>

<td>Treatment</td><td>Placebo</td><td>mAb316P</td><td>mAb316P</td><td>mAb316P</td><td>mAb316P</td>

<td>Dose</td><td>N / A</td><td>150 mg</td><td>150 mg</td><td>200 mg</td><td>300 mg</td>

<td>Frequency</td><td>Every 2</td><td>Every 2</td><td>Every 4</td><td>Every 4</td><td>Every 4</td>

<td>administration</td><td>weeks</td><td>weeks</td><td>weeks</td><td>weeks</td><td>weeks</td>

<td>Total cholesterol</td><td> -6,52</td><td> -38,27</td><td> -18,89</td><td> -15,31</td><td> -28,84</td>

<td>LDL-C</td><td> -4,90</td><td> -66,71</td><td> -24,30</td><td> -24,07</td><td> -49,35</td>

<td>C-HDL</td><td> +2,48</td><td> +14,63</td><td> +6,33</td><td> +6,53</td><td> +4,48</td>

<td>Triglycerides</td><td> -10,55</td><td> -16,23</td><td> -16,73</td><td> -9,87</td><td> -4,92</td>

<td>Apo B</td><td> -7,32</td><td> -48,97</td><td> -19,09</td><td> -14,69</td><td> -36,27</td>

<td>Apo A1</td><td> -7,24</td><td> +9,98</td><td> +3,13</td><td> +0,33</td><td> +5,39</td>

<td>Lp (a)</td><td> -11,07</td><td> -22,50</td><td> -11,30</td><td> -8,00</td><td> -14,29</td>

Example 4: A randomized 12-week, placebo-controlled, parallel and double-blind study on the safety and efficacy of an anti-PCSK9 monoclonal antibody in patients with primary hypercholesteroiemia under stable atorvastatin therapy.

A clinical trial was conducted to evaluate the efficacy of five doses and two administration regimens of mAb316P for 12 weeks in patients with i

Primary hypercholesterolemia and LDL-C levels above 100 mg / dL under stable therapy with ongoing atorvastatin. Efficacy was assessed based on changes in serum levels of low density colesteroi lipoprotein (C-LDL) and other lipids / apolipoproteins [eg, total cholesteroi, high density cholesteroi lipoprotein, triglycerides, Apo B, Apo A1 and Lp (a)] throughout the course of the study.

Patient population

The patient population for this study included men and women, 18-75 years of age, who had been diagnosed with primary hypercholesterolemia, had LDL-C levels of 100 mg / dL or more, and were under stable atorvastatin therapy. 10, 20 or 40 mg at least six weeks before the start of the study. Patients were excluded if they were taking any additional compounds that reduce lipid levels, such as fibrates, niacin, omega-3 fatty acids, bile acid resins, vegetable stains (e.g., Benecol, flaxseed oil, zaragatone) and rice fermented red. A total of 118 patients completed the study.

Pharmacological Formulation

The pharmacological formulation comprised 150 mg / ml of mAb316P, 10 mM histidine, 0.2% polysorbate 20 and 10% sucrose, and had a pH of 6.0.

Administration method

The pharmacological formulation (or placebo) was administered to patients by subcutaneous injection into the abdomen. Each treatment included two subcutaneous injections, each 1 ml.

Administration Regimes

The patients were divided into six groups. Group 1 included 20 patients who received placebo once every two weeks; Group 2 included 19 patients who received 50 mg of mAb316P once every two weeks; Group 3 included 20 patients who received 100 mg of mAb316P once every two weeks; Group 4 included 18 patients who received 150 mg of mAb316P once every two weeks; Group 5 included 20 patients who received 200 mg of mAb316P once every four weeks; and Group 6 included 21 patients who received 300 mg of mAb316P once every four weeks. The duration of the study was 12 weeks.

Effectiveness evaluation

Blood samples were collected from patients throughout the study and for a continuation period of 8 weeks. Samples were collected after a fast 5 of at least 12 hours (in the morning, before ingestion of any drug). In blood samples, measurements of the following parameters were made: (1) total cholesterol; (2) C-LDL; (3) C-HDL; (4) triglycerides (TG); (5) Apo B; (6) Apo A1; and (7) Lp (a). Table 5 summarizes the medians of the percentage changes of these parameters compared to placebo at the end of the study (week 12).

Table 5

<td>Group</td><td> 1</td><td> 2</td><td> 3</td><td> 4</td><td> 5</td><td> 6</td>

<td>No. of patients</td><td> 20</td><td> 19</td><td> 20</td><td> 18</td><td> 20</td><td> 21</td>

<td>Treatment</td><td>Placebo</td><td>mAb316P</td><td>mAb316P</td><td>mAb316P</td><td>mAb316P</td><td>mAb316P</td>

<td>Dose</td><td>N / A</td><td>50 mg</td><td>100 mg</td><td>150 mg</td><td>200 mg</td><td>300 mg</td>

<td>Frequency</td><td>Every 2</td><td>Every 2</td><td>Every 2</td><td>Every 2</td><td>Every 4</td><td>Every 4</td>

<td>administration</td><td>weeks</td><td>weeks</td><td>weeks</td><td>weeks</td><td>weeks</td><td>weeks</td>

<td>Total cholesterol</td><td> -3,73</td><td> -23,34</td><td> -40,21</td><td> -45,03</td><td> -29,51</td><td> -33,48</td>

<td>LDL-C</td><td> -6,92</td><td> -37,04</td><td> -64,28</td><td> -74,83</td><td> -49,46</td><td> -51,98</td>

<td>C-HDL</td><td> +1,81</td><td> +5,08</td><td> +7,93</td><td> +8,96</td><td> +5,59</td><td> +12,09</td>

<td>Triglycerides</td><td> +22,42</td><td> -7,85</td><td> -11,41</td><td> -22,93</td><td> +1,27</td><td> -9,17</td>

<td>Apo B</td><td> +0,76</td><td> -28,67</td><td> -48,67</td><td> -54,16</td><td> -30,67</td><td> -32,95</td>

<td>Apo A1</td><td> +0,58</td><td> + 1,40</td><td> -0,88</td><td> +1,11</td><td> +0,66</td><td> +3,42</td>

<td>Lp (a)</td><td> 0,00</td><td> -13,33</td><td> -27,27</td><td> -28,57</td><td> -18,92</td><td> -11,11</td>

Example 5: A randomized, fixed-dose, placebo-controlled, parallel-blind, double-blind study on the safety and efficacy of an anti-PCSK9 monoclonal antibody administered together with 80 mg of atorvastatin in patients with primary hypercholesterolemia.

A clinical trial was conducted to evaluate the efficacy of mAb316P when administered together with 80 mg of atorvastatin for 8 weeks in patients with primary hypercholesterolemia and LDL-C levels above 100 mg / dl. Efficacy was evaluated based on changes in serum levels of low density lipoprotein cholesterol (C-LDL) and other lipids / apolipoproteins [eg, total cholesterol, high density lipoprotein cholesterol, triglycerides, Apo B, Apo A1 and Lp (a)] throughout the course of the study.

Patient population

The patient population for this study included men and women, 18-75 years of age, who had been diagnosed with primary hypercholesterolemia, had LDL-C levels of 100 mg / dl or more, and were under stable atorvastatin therapy. 10 mg from at least six weeks before the start of the study. Patients were excluded if they were taking any additional compounds that reduce lipid levels, such as fibrates, niacin, omega-3 fatty acids, bile acid resins, vegetable stains (e.g., Benecol, flaxseed oil, zaragatone) and rice fermented red. A total of 88 patients completed the study.

Pharmacological Formulation

The pharmacological formulation comprised 150 mg / ml of mAb316P, 10 mM histidine, 0.2% polysorbate 20 and 10% sucrose, and had a pH of 6.0.

Administration method

The pharmacological formulation (or placebo) was administered to patients by subcutaneous injection into the abdomen. Each treatment included a 1 ml subcutaneous injection.

Administration Regimes

The patients were divided into three groups. Group 1 included 29 patients who received placebo once every two weeks plus 80 mg of atorvastatin per day; Group 2 included 30 patients who received 150 mg of mAb316P once every two weeks plus 80 mg of atorvastatin per day; and Group 3 included 29 patients who received 150 mg of mAb316P once every two weeks plus 10 mg of atorvastatin daily. The duration of the study was 8 weeks.

Effectiveness evaluation

Blood samples were collected from patients throughout the study and for a continuation period of 8 weeks. Samples were collected after a fast of at least 12 hours (in the morning, before ingestion of any drug). In the 5 blood samples measurements of the following parameters were made: (1) total cholesterol; (2) C-LDL; (3) C-HDL; (4) triglycerides (TG); (5) Apo B; (6) Apo A1; (7) Apo B / Apo A1 ratio; and (8) Lp (a). In Table 6 the percentage changes in these parameters are summarized compared with placebo at the end of the study.

Table 6

<td>Group</td><td> 1</td><td> 2</td><td> 3</td>

<td>No. of patients</td><td> 29</td><td> 30</td><td> 29</td>

<td>Treatment</td><td>Placebo</td><td>mAb316P</td><td>mAb316P</td>

<td>Dose</td><td>N / A</td><td>150 mg</td><td>150 mg</td>

<td>Frequency</td><td>Every 2</td><td>Every 2</td><td>Every 2</td>

<td>administration</td><td>weeks</td><td>weeks</td><td>weeks</td>

<td>Daily atorvastatin</td><td>80 mg</td><td>80 mg</td><td>10 mg</td>

<td>Total cholesterol</td><td> -16,59</td><td> -47,21</td><td> -40,45</td>

<td>LDL-C</td><td> -26,87</td><td> -70,62</td><td> -70,37</td>

<td>C-HDL</td><td> -5,71</td><td> +5,17</td><td> +1,39</td>

<td>Triglycerides</td><td> -11,89</td><td> -24,67</td><td> -3,98</td>

<td>Apo B</td><td> -12,00</td><td> -58,00</td><td> -54,39</td>

<td>Apo A1</td><td> -5,56</td><td> -4,55</td><td> -0,69</td>

<td>Lp (a)</td><td> -2,70</td><td> -31,01</td><td> -34,65</td>

Taken together, the results of these studies (Examples 3-5) confirm the ability of mAb316P to effectively reduce LDL cholesterol levels and beneficially affect other lipid / apolipoprotein parameters in patients when administered in different doses and under different doses. administration regimes. These Clinical Examples also confirm the unexpected finding that mAb316P is able to significantly reduce Lp (a) levels in patients under various circumstances.

The present invention will not be limited in scope by the specific embodiments described herein. In fact, various modifications of the invention in addition to those described herein will be apparent to those skilled in the art from the foregoing description and the attached figures. Such modifications are intended to fall within the scope of the appended claims.