Inhibitors of pcsk9 for treatment of lipoprotein metabolism disorders

Abstract

The present disclosure relates to inhibitors of proprotein convertase subtilisin-like/kexin type 9 (PCSK9) for the treatment of lipoprotein metabolism disorders.

1 . A compound capable of inhibiting binding of heparan sulfate proteoglycan receptors (HSPGs) to PCSK9. 
     
     
         2 . The compound according to  claim 1 , wherein said compound specifically recognises and binds a region within the HSPG binding site of PCSK9 (SEQ ID NO: 1) that comprises at least one of amino acid residues 78 to 167 of PCSK9 (SEQ ID NO: 1), such as at least one of amino acid residues 78 to 92, such as at least one of amino acid residues 93 to 97, such as at least one of amino acid residues 98 to 103, such as at least one of amino acid residues 104 to 105, such as at least one of amino acid residues 106 to 135, such as at least one of amino acid residues 136 to 139, such as at least one of amino acid residues 140 to 164, such as at least one of amino acid residues 165 to 167 of PCSK9 (SEQ ID NO: 1). 
     
     
         3 . The compound according to any one of the preceding claims, wherein said compound binds to one or more amino acids selected from the group consisting of R93, R96, R97, R104, R105, K136, H139, R165 and R167 of PCSK9 (SEQ ID NO: 1). 
     
     
         4 . The compound according to any one of the preceding claims, wherein said compound binds to at least one, such as to at least two, such as to at least three, such as to at least four, such as to at least five, such as to all six of R93, R96, R97, R104, R105 and H139 of PCSK9 (SEQ ID NO: 1). 
     
     
         5 . The compound according to any one of the preceding claims, wherein said compound is an antibody or functional equivalent thereof. 
     
     
         6 . The compound according to any one of the preceding claims wherein said compound is a monoclonal antibody. 
     
     
         7 . The compound according to any one of the preceding claims, wherein the antibody or functional equivalent thereof is a murine monoclonal antibody. 
     
     
         8 . The compound according to any one of the preceding claims, wherein the antibody or functional equivalent thereof has been humanised. 
     
     
         9 . The compound according to any one of the preceding claims, wherein the functional equivalent of said antibody comprises an antigen-binding fragment of said antibody which is capable of binding to the HSPG binding site of PCSK9 (SEQ ID NO: 1). 
     
     
         10 . The compound according to any one of the preceding claims, wherein the fragment is selected from the group consisting of Fab, Fab′, F(ab′) 2  and Fv fragments. 
     
     
         11 . The compound according to any one of the preceding claims, wherein the antibody is a single chain antibody. 
     
     
         12 . The compound according to any one of the preceding claims, wherein the antibody or functional equivalent thereof specifically recognises and binds an epitope within the HSPG binding site of PCSK9 (SEQ ID NO: 1) that comprises at least one of amino acid residues 93, 96, 97, 104, 105, 136, 139, 165 or 167 of PCSK9 (SEQ ID NO: 1), such as an epitope comprising at least one, such as at least two, such as at least three, such as at least four, such as at least five, such as all six of amino acid residues 93, 96, 97, 104, 105 and 139 of PCSK9 (SEQ ID NO: 1). 
     
     
         13 . The compound according to any one of the preceding claims, wherein the antibody or antigen-binding fragment thereof comprises:
 (i) a heavy chain variable region or a humanized version thereof, comprising at least one of the three CDRs as defined by SEQ ID NO: 10, SEQ ID NO: 11 or SEQ ID NO: 12, and   (ii) a light chain variable region comprising or a humanized version thereof, comprising at least one of the three CDRs as defined by SEQ ID NO: 13, SEQ ID NO: 14 or SEQ ID NO: 15.   
     
     
         14 . The compound according to any one of the preceding claims, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable region or a humanized version thereof as defined by SEQ ID NO: 7 and/or a light chain variable region or a humanized version thereof as defined by SEQ ID NO: 5, 
     
     
         15 . The compound according to any one of the preceding claims, wherein the antibody or antigen-binding fragment thereof comprises:
 (i) a heavy chain variable region or a humanized version thereof, comprising at least one of the three CDRs as defined by SEQ ID NO: 16, SEQ ID NO: 17 or SEQ ID NO: 18, and   (ii) a light chain variable region comprising or a humanized version thereof, comprising at least one of the three CDRs as defined by SEQ ID NO: 19, SEQ ID NO: 20 or SEQ ID NO: 21.   
     
     
         16 . The compound according to any one of the preceding claims, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable region or a humanized version thereof as defined by SEQ ID NO: 9 and/or a light chain variable region or a humanized version thereof as defined by SEQ ID NO: 3. 
     
     
         17 . The compound according to any one of the preceding claims, wherein the antibody is constructed by domain shuffling. 
     
     
         18 . The compound according to any one of the preceding claims, wherein the antibody is an antibody variant. 
     
     
         19 . The compound according to any one of the preceding claims, wherein the antibody variant has been modified to increase half-life, stability, solubility, and/or bioavailability. 
     
     
         20 . The compound according to any one of the preceding claims, wherein the antibody variant comprises engineered intradomain disulphide bonds. 
     
     
         21 . The compound according to any one of the preceding claims, wherein one or more Fv fragment comprises a peptide linker between the V H  and the V L  domains. 
     
     
         22 . The compound according to any one of the preceding claims, wherein binding of the antibody to PCSK9 results in reduced binding of PCSK9 to LDLR compared to the binding in the absence of said antibody. 
     
     
         23 . The compound according to any one of the preceding claims, wherein the antibody is further capable of recognising and binding a region within the LDLR binding site of PCSK9 (SEQ ID NO: 1), wherein said region comprises at least one of residues 194 or 367-381 of PCSK9. 
     
     
         24 . The compound according to any one of the preceding claims, wherein binding of the antibody to PCSK9 results in increased levels of LDLR of LDLR-expressing cells such as hepatocytes compared to the levels in the absence of said antibody. 
     
     
         25 . The compound according to any one of the preceding claims, wherein binding of the antibody to PCSK9 results in decreased lysosomal degradation of LDLR compared to the degradation in the absence of said antibody. 
     
     
         26 . The compound according to any one of the preceding claims, wherein binding of the antibody to PCSK9 results in decreased plasma levels of LDL-C compared to the levels in the absence of said antibody. 
     
     
         27 . The compound according to any one of the preceding claims, wherein the plasma levels of LDL-C are determined in vitro. 
     
     
         28 . The compound according to any one of the preceding claims, wherein the plasma levels of LDL-C are determined by Western Blot, immuno-staining, ELISA, ultracentrifugation, FPLC, or electrophoresis. 
     
     
         29 . The compound according to any one of the preceding claims, wherein the antibody is stable in serum. 
     
     
         30 . The compound according to any one of the preceding claims, wherein the antibody is non-toxic after administration. 
     
     
         31 . The compound according to any one of the preceding claims, wherein the heparin analogue is a competitive antagonist, an uncompetitive antagonist, a non-competitive antagonist, a silent antagonist, a partial agonist or an inverse agonist of HSPGs. 
     
     
         32 . The compound according to any one of the preceding claims, wherein said heparin analogue is heparin. 
     
     
         33 . The compound according to according to any one of the preceding claims, wherein the heparin analogue is a peptide. 
     
     
         34 . The compound according to according to any one of the preceding claims, wherein the heparin analogue is capable of inhibiting binding of heparin to PCSK9. 
     
     
         35 . The compound according to any one of the preceding claims, wherein the compound is a heparin mimetic or a heparin analogue. 
     
     
         36 . The compound according to any one of the preceding claims, wherein the compound has a general structure of formula (I): 
       
         
           
           
               
               
           
         
         wherein:
 R 1  is selected from the group consisting of COOH and —O 3 SO, 
 R 2 , R 3 , R 5  and R 7  are selected from the group consisting of O and OH 
 R 4  is selected from the group consisting of a sulfate group, 
 R 6  is selected from the group consisting of a sulphamate group, OH and O, 
 R 8  is selected from the group consisting of a sulfate group, 
 n is an integer equal or greater than 1, 
 
         and wherein R 2  and/or R 7  optionally can act as linkers linking monomer units. 
       
     
     
         37 . The compound according to any one of the preceding claims, wherein the compound of formula (I) is
 a) Fondaparinux and has the formula:   
       
         
           
           
               
               
           
         
         b) a low molecular weight heparin such as dalteparin sodium or tinzaparin sodium; and 
         c) a compound of formula (II): 
       
       
         
           
           
               
               
           
         
         
           wherein R 9  and R 10  are independently selected from the group consisting of H, COOH, —O 3 SO, O, OH, sulfate and sulfamate, wherein n is an integer equal to or greater than 1. 
         
       
     
     
         38 . The compound of  claim 37 , wherein the compound of formula (II) is pentosan and has the formula: 
       
         
           
           
               
               
           
         
         where n is an integer equal to or greater than 1. 
       
     
     
         39 . The compound according to any one of the preceding claims, wherein the compound has a general structure of formula (III): 
       
         
           
           
               
               
           
         
         wherein R 11 , R 12 , R 13 , R 14 , R 15 , R 16 , R 17 , R 18 , and R 19  are independently selected from the group consisting of COOH, —O 3 SO, O, OH, sulfate and sulfamate, and n is an integer equal to or greater than 1, and wherein R14 optionally can act as linkers linking monomer units. 
       
     
     
         40 . The compound according to  claim 39 , wherein the compound is dextran sulphate and has the formula: 
       
         
           
           
               
               
           
         
         wherein n is an integer equal to or greater than 1. 
       
     
     
         41 . The compound according to any one of the preceding claims, wherein the compound is suramin and has the formula: 
       
         
           
           
               
               
           
         
       
     
     
         42 . The compound according to any one of the preceding claims, wherein the compound is a phosphorothioate oligonucleotide S-dNn, where n is the number of phosphorothioate linkages in said phosphorothioate oligonucleotide. 
     
     
         43 . The compound according to  claim 42 , wherein n is between 12 and 60. 
     
     
         44 . The compound according to  claim 43 , wherein n is between 15 and 55, such as between 20 and 50, such as between 25 and 45, such as between 30 and 40, such as 36. 
     
     
         45 . The compound according to any one of the preceding claims, wherein said compound is not capable of binding to the LDLR-binding domain of PCSK9 (SEQ ID NO: 1). 
     
     
         46 . Use of the compound as defined in any one of the preceding claims for the preparation of a medicament for the treatment of a disorder of lipoprotein metabolism. 
     
     
         47 . A compound according to any one of  claims 1  to  45  for use as a medicament. 
     
     
         48 . A compound according to any one of  claims 1  to  45  for use in a method of treatment of a disorder of lipoprotein metabolism in a subject in need thereof. 
     
     
         49 . The compound for the use according to  claim 48 , wherein the disorder of lipoprotein metabolism is linked to abnormal levels of LDL-C, such as elevated levels of LDL-C. 
     
     
         50 . The compound for the use according to any one of  claims 48  to  49 , wherein the disorder of lipoprotein metabolism is linked to abnormal PCSK9 plasma levels and/or abnormal LDLR levels of LDLR-expressing cells such as hepatocytes. 
     
     
         51 . The compound for the use according to any one of  claims 48  to  50 , wherein the disorder of lipoprotein metabolism is selected from the group consisting of dyslipidemia, hypercholesterolemia and coronary heart diseases. 
     
     
         52 . The compound for the use according to any one of  claims 48  to  51 , wherein the treatment is prophylactic. 
     
     
         53 . The compound for the use according to any one of  claims 48  to  52 , wherein the subject is a mammal. 
     
     
         54 . The compound for the use according to any one of  claims 48  to  53 , wherein the mammal is a human. 
     
     
         55 . A method of treatment of a disorder of lipoprotein metabolism in a subject in need thereof, said method comprising administering to said subject a compound as defined in any one of  claims 1  to  45 . 
     
     
         56 . The method according to  claim 49 , wherein the compound is administered at a daily dosage of between 0.1 mg and 1000 mg per kg bodyweight. 
     
     
         57 . A method of treatment of a disorder of lipoprotein metabolism such as dyslipidemia, hypercholesterolemia and heart coronary diseases in an individual in need thereof, the method comprising the steps of:
 i. providing a tissue sample or a plasma sample isolated from said individual,   ii. determining the levels of LDLR of said tissue and/or the levels of PCSK9 and/or the levels of LDL-C in said plasma sample,   iii. correlating the expression level of step ii) with the levels of a control tissue sample or a control plasma sample,   iv. assessing a treatment regime,   v. administering to the individual a composition comprising a therapeutically effective amount of a compound as defined in any one of  claims 1  to  45 .   
     
     
         58 . A method of inhibiting degradation of LDLR, said method comprising administering a compound as defined in any one of  claims 1  to  45  or a pharmaceutical composition as defined in any one of  claims 59  to  69 . 
     
     
         59 . A pharmaceutical composition comprising at least one compound as defined in any one of  claims 1  to  45 . 
     
     
         60 . The pharmaceutical composition according to  claim 59 , wherein said composition further comprises at least one of a statin or an anti-PCK9 antibody, such as an antibody binding to the LDLR-binding site of PCSK9. 
     
     
         61 . The pharmaceutical composition according to any one of  claims 59  to  60 , wherein said composition further comprises at least one further compound, such as a compound selected from the group consisting of lodelcizumab, ralpancizumab, alirocumab, evolocumab and bococizumab 
     
     
         62 . The pharmaceutical composition according to any one of  claims 59  to  61 , wherein the composition further comprises at least one of:
 an antibody inhibiting binding of PCSK9 to LDLR; 
 a statin; 
 cholestyramine; 
 ezetimibe. 
 
     
     
         63 . The pharmaceutical composition according to any one of  claims 59  to  62 , further comprising one or more pharmaceutically acceptable excipients. 
     
     
         64 . The pharmaceutical composition according to any one of  claims 59  to  63 , wherein the pH of the composition is between pH 4 and pH 10. 
     
     
         65 . The pharmaceutical composition according to any one of  claims 59  to  64 , wherein the composition is formulated for oral administration. 
     
     
         66 . The pharmaceutical composition according to any one of  claims 59  to  65 , wherein the composition is formulated for parenteral administration. 
     
     
         67 . The pharmaceutical composition according to any one of  claims 59  to  64  or  66 , wherein the parenteral administration is by injection. 
     
     
         68 . The pharmaceutical composition according to any one of  claims 59  to  64  or  66  to  67 , wherein the parenteral administration is intravenous, intramuscular, intraspinal, intraperitoneal, subcutaneous, a bolus or a continuous administration. 
     
     
         69 . The pharmaceutical composition according to any one of  claims 59  to  68 , wherein the compound is administered at a daily dosage of between 0.1 mg and 1000 mg per kg bodyweight. 
     
     
         70 . A method of selecting an antibody specifically recognising and binding an epitope within the HSPG binding site of PCSK9 (SEQ ID NO: 1), said method comprising the steps of:
 i. administering to a mammal a polypeptide fragment of PCSK9 or a polynucleotide encoding a polypeptide fragment of PCSK9 comprising at least one amino acid residue of the domain consisting of amino acid residues 78 to 167 of SEQ ID NO: 1, such as at least 5, such as at least 10, such as at least 15, such as at least 20, such as at least 25, such as at least 30, such as at least 35, such as at least 40, such as at least 45, such as at least 50, such as at least 55, such as at least 60, such as at least 65, such as at least 70, such as at least 75, such as at least 80, such as at least all amino acids of the domain consisting of amino acid residues 78 to 167 of SEQ ID NO: 1;   ii. identifying and selecting antibodies recognising said polypeptide, and   iii. determining whether said selected antibodies are capable of displacing one or more reference antibodies in a competitive ELISA assay.   
     
     
         71 . The method according to  claim 70 , wherein the method further comprises a step of selecting antibodies capable of displacing at least one of said reference antibodies. 
     
     
         72 . The method according to any one of  claims 70  to  71 , wherein the step of determining whether said selected antibodies are capable of displacing one or more reference antibodies in a competitive ELISA comprises the steps of:
 i. providing PCSK9 or a fragment thereof comprising the epitope recognised by said reference antibody; and 
 ii. adding a test antibody and said reference antibody to said PCSK9 or fragment thereof, wherein either the test antibody or the reference antibody is labelled with a detectable label or both antibodies are labelled with different detectable labels; and 
 iii. detecting the presence of the detectable label at PCSK9; 
 
       thereby detecting whether the test antibody is capable of displacing the reference antibody. 
     
     
         73 . The method according to any one of  claims 70  to  72 , wherein the method further comprises testing the antibodies to determine whether said antibody is capable of inhibiting LDLR degradation and selecting antibodies capable of inhibiting LDLR degradation. 
     
     
         74 . A method of selecting a peptide or a heparin analogue capable of inhibiting binding of HSPGs such as heparin to PCSK9, said method comprising the steps of:
 i. providing a plurality of peptides or heparin analogues;   ii. incubating said plurality of peptides or heparin analogues with cells derived from an LDLR-expressing cell line such as a hepatocyte-derived cell line in a medium;   iii. determining the levels of PCSK9 in said medium and/or the levels of LDLR of said cells;   iv. selecting the peptides or heparin analogues resulting in the highest levels of PCSK9 and/or LDLR as determined in step iii.   
     
     
         75 . A compound capable of selectively binding the compound as defined in any one of  claims 1  to  45 . 
     
     
         76 . The compound according to  claim 75 , wherein said compound is an antibody. 
     
     
         77 . A method for producing the antibody as defined in any one of  claims 1  to  45 , said method comprising the steps of:
 i. administering to a mammal a protein or a polynucleotide encoding a protein comprising the HSPG binding domain of PCSK9 or a fragment thereof or a functional equivalent thereof; 
 ii. selecting said antibody if it is able to bind to PCSK9 (SEQ ID NO: 1); 
 iii. selecting said antibody if it is unable to bind to mutated PCSK9, wherein said mutated PCSK9 is unable to bind to HSPG. 
 
     
     
         78 . The method according to  claim 77 , wherein the mutated PCSK9 is mutated at position 93, 96, 97, 104, 105, 136, 139, 165 or 167. 
     
     
         79 . The method according to any one of  claims 77  to  78 , wherein the mutated PCSK9 is mutated at positions 93, 96, 97, 104, 105 and 139. 
     
     
         80 . The method according to any one of  claims 77  to  79 , wherein the mutated PCSK9 is selected from the group consisting of mutants R93A, R96A, R97A, R104A, R105A, K136A, H139A, R165A or R167A. 
     
     
         81 . The method according to any one of  claims 77  to  80 , wherein the mutated PCSK9 is a R93A, R96A, R97A, R104A, R105A, R39A mutant. 
     
     
         82 . The method according to any one of  claims 77  to  81 , wherein the mammal is a rodent. 
     
     
         83 . The method according to any one of  claims 77  to  82 , wherein the method further comprises isolating antibody producing cells from said mammal, preparing hybridoma cells from said antibody producing cells, cultivating said hybridomas and isolating antibodies produced by said hybridomas. 
     
     
         84 . The method according to any one of  claims 77  to  83 , wherein said compound is an antibody according to any one of the preceding claims, and wherein said method comprises the steps of transfecting a host cell with a nucleic acid construct encoding said antibody. 
     
     
         85 . The method according to any one of  claims 77  to  84 , wherein the antibody is produced by a recombinant cell. 
     
     
         86 . The method according to any one of  claims 77  to  85 , wherein the recombinant cell is a microorganism selected from the group comprising bacteria and eukaryotic microorganisms. 
     
     
         87 . The method according to any one of  claims 77  to  86 , wherein the microorganism is a bacterium selected from the group comprising  Escherichia coli, Lactobacillus zeae, Bacillus subtilis, Streptomyces lividans, Staphylococcus carnosus, Bacillus megaterium  and  Corynebacterium glutamicum.    
     
     
         88 . The method according to any one of  claims 77  to  87 , wherein the microorganism is a eukaryotic microorganism selected from the group comprising  Saccharomyces cerevisiae, Aspergillus niger, Pichia pastoris, Schizosaccharomyces pombe, Yarrowia lipolytica  and  Kluyveromyces lactis.    
     
     
         89 . The method according to any one of  claims 77  to  88 , wherein the host cell is selected from the group comprising plant cells and animal cells. 
     
     
         90 . The method according to  claim 89 , wherein the plant cell is selected from the group comprising  Arabidopsis  sp., pea, rice, maize, tobacco, barley, or seeds thereof. 
     
     
         91 . The method according to  claim 89 , wherein the animal cell is derived from a mammal selected from the group comprising Chinese Hamster Ovary, mouse and human. 
     
     
         92 . The method according to  claim 89 , wherein the animal cell is derived from an insect or an avian cell line. 
     
     
         93 . The method according to any one of  claims 77  to  92 , further comprising the steps of identifying and selecting the antibody. 
     
     
         94 . A method for reducing plasma LDL-C levels in a subject in need thereof, said method comprising the step of administering to said subject a compound as defined in any one of  claims 1  to  45  or a pharmaceutical composition as defined in any one of  claims 59  to  69 . 
     
     
         95 . The method of  claim 94 , wherein said subject does not respond to statin treatment.