Combination therapy for cardiovascular diseases

Abstract

To provide methods of treating or reducing the risk of a cardiovascular disease using a lipid lowering agent (e.g., statin and / or PCSK9 inhibitor) and an anti-inflammatory agent (e.g., a pro-inflammatory cytokine inhibitor), and further provide methods of predicting the recurrence rate of a subject who has received or is undergoing therapy for a cardiovascular disease with a lipid lowering agent on the basis of the C-reactive protein (CRP) level in the subject.SOLUTION: In some embodiments, the recurrence rate can be reduced using an anti-inflammatory agent.SELECTED DRAWING: None

Description

Technical Field

[0001] Related Applications This application claims the benefit under 35 U.S.C. § 119(e) of U.S. Provisional Patent Application No. 62 / 640,918, entitled "COMBINATION THERAPY FOR CARDIOVASCULAR DISEASES," filed on Mar. 9, 2018, and claims the benefit under 35 U.S.C. § 119(e) of U.S. Provisional Patent Application No. 62 / 733,960, entitled "COMBINATION THERAPY FOR CARDIOVASCULAR DISEASES," filed on Sep. 20, 2018, the entire contents of each of which are incorporated herein by reference.

Background Art

[0002] Background Lipid lowering is a mainstay in the treatment of atherosclerotic cardiovascular disease. Some patients continue to have cardiovascular disease despite receiving lipid-lowering treatment. There is a need to develop new treatments.

Summary of the Invention

[0003] Summary In some aspects, the present disclosure is based on the surprising finding that in patients who have received aggressive lipid-lowering treatment, there is a residual inflammatory risk and that high-sensitivity C-reactive protein (hsCRP) levels (markers of inflammation) in these patients correlate with the likelihood of recurrence of cardiovascular disease and / or mortality. Provided herein is a method of treating cardiovascular disease using a lipid-lowering agent and an anti-inflammatory agent.

[0004] Some aspects of the present disclosure provide a method of treating cardiovascular disease, the method comprising administering to a subject in need thereof a therapeutically effective amount of a lipid-lowering agent and an anti-inflammatory agent.

[0005] In some embodiments, the anti-inflammatory agent is a pro-inflammatory cytokine inhibitor. In some embodiments, the anti-inflammatory agent includes an IL-1 inhibitor, an IL-1 receptor (IL-1R) inhibitor, an IL-6 inhibitor, an IL-6 receptor (IL-6R) inhibitor, an NLRP3 inhibitor, a TNF inhibitor, an IL-8 inhibitor, an IL-18 inhibitor, a natural killer cell inhibitor, or a combination thereof. In some embodiments, the anti-inflammatory agent is a nucleic acid, an aptamer, an antibody or antibody fragment, an inhibitory peptide, or a small molecule.

[0006] In some embodiments, the anti-inflammatory agent includes an IL-1 inhibitor. In some embodiments, the IL-1 inhibitor is an IL-1α inhibitor. In some embodiments, the IL-1α inhibitor is an antisense oligonucleotide against IL-1α, MABp1, or sIL-1RI. In some embodiments, the IL-1 inhibitor is an IL-1β inhibitor. In some embodiments, the IL-1β inhibitor is an antisense oligonucleotide against IL-1β, canakinumab, diacerein, gevokizumab, LY2189102, CYT013, sIL-1RII, VX-740, or VX-765. In some embodiments, the IL-1 inhibitor is sodium slamine, methotrexate-methyl-d3, methotrexate-methyl-d3 dimethyl ester, or diacerein.

[0007] In some embodiments, the anti-inflammatory agent includes an IL-1R inhibitor. In some embodiments, the IL-1R inhibitor is an IL-1R antagonist. In some embodiments, the IL-1R inhibitor is an antisense oligonucleotide against IL-1R, anakinra, rilonacept, MEDI-8968, sIL-1RI, EBI-005, interleukin-1 receptor antagonist (IL-1RA), or AMG108.

[0008] In some embodiments, the anti-inflammatory agent includes an IL-6 inhibitor. In some embodiments, the IL-6 inhibitor is an antisense oligonucleotide against IL-6, siltuximab, sirukumab, clazakizumab, olokizumab, elsilimomab, IG61, BE-8, CNTO328 PGE1 and its derivatives, PGI2 and its derivatives, or cyclophosphamide.

[0009] In some embodiments, the anti-inflammatory agent includes an IL-6R inhibitor. In some embodiments, the IL-6R inhibitor is an IL-6R antagonist. In some embodiments, the IL-6R inhibitor is an antisense oligonucleotide against IL-6R, tocilizumab, sarilumab, PM1, AUK12-20, AUK64-7, AUK146-15, MRA, or AB-227-NA.

[0010] In some embodiments, the anti-inflammatory agent includes an NLRP3 inhibitor. In some embodiments, the NLRP3 inhibitor is an antisense oligonucleotide against NLRP3, colchicine, MCC950, CY-09, the ketone metabolite β-hydroxybutyric acid (BHB), type I interferon, resveratrol, arglabin, CB2R, glibenclamide, isoliquiritigenin, Z-VAD-FMK, or microRNA-223.

[0011] In some embodiments, the anti-inflammatory agent includes a TNF inhibitor. In some embodiments, the TNF inhibitor is an antisense oligonucleotide against TNF, infliximab, adalimumab, certolizumab pegol, golimumab, etanercept (Enbrel), thalidomide, lenalidomide, pomalidomide, xanthine derivatives, bupropion, 5-HT2A agonists or hallucinogens.

[0012] In some embodiments, the anti-inflammatory agent comprises an IL-8 inhibitor. In some embodiments, the IL-8 inhibitor is an antisense oligonucleotide against IL8, HuMab-10F8, reparixin, curcumin, Antileukinate, a macrolide, or a trifluoroacetate salt.

[0013] In some embodiments, the anti-inflammatory agent comprises an IL-18 inhibitor. In some embodiments, the IL-18 inhibitor is selected from the group consisting of: an antisense oligonucleotide against IL-18, an IL-18 binding protein, an IL-18 antibody, NSC201631, NSC61610, and NSC80734.

[0014] In some embodiments, the anti-inflammatory agent comprises an inhibitor of natural killer cells. In some embodiments, the inhibitor of natural killer cells is an antibody that targets natural killer cells.

[0015] In some embodiments, the anti-inflammatory agent comprises methotrexate. In some embodiments, the anti-inflammatory agent comprises alhalofenate.

[0016] In some embodiments, the lipid-lowering agent includes a proprotein convertase subtilisin / kexin type 9 (PCSK9) inhibitor. In some embodiments, the PCSK9 inhibitor is a natural PCSK9 inhibitor, an anti-PCSK9 antibody, an antisense nucleic acid, a peptide inhibitor, a PCSK9 vaccine, or a small molecule inhibitor. In some embodiments, the natural PCSK9 inhibitor is berberine, annexin A2, or adnectin. In some embodiments, the small molecule inhibitor is PF-06446846, anacetrapib, or K-312. In some embodiments, the PCSK9 antibody is alirocumab, evolocumab, 1D05-IgG2, RG-7652, LY3015014, or bococizumab. In some embodiments, the antisense nucleic acid is an RNAi molecule. In some embodiments, the RNAi molecule is inclisiran or ALN-PCS. In some embodiments, the peptide inhibitor is a peptide that mimics the EGFa domain of the low density lipoprotein receptor (LDL-R). In some embodiments, the PCSK9 vaccine includes an antigenic PCSK9 peptide.

[0017] In some embodiments, the lipid-lowering agent includes an HMG-CoA reductase inhibitor. In some embodiments, the HMG-CoA reductase inhibitor is a statin. In some embodiments, the statin is simvastatin, lovastatin, pravastatin, fluvastatin, atorvastatin, cerivastatin, rosuvastatin, or pitivastatin.

[0018] In some embodiments, the lipid-lowering agent is a fibrate, a bile acid sequestrant or resin, a nicotinic acid agent, a cholesterol absorption inhibitor, an acyl coenzyme A:cholesterol acyltransferase (ACAT) inhibitor, a cholesteryl ester transfer protein (CETP) inhibitor, an LDL receptor antagonist, a farnesoid X receptor (FXR) antagonist, a sterol regulatory binding protein cleavage activating protein (SCAP) activator, a microsomal triglyceride transfer protein (MTP) inhibitor, a squalene synthase inhibitor, or a peroxisome proliferator-activated receptor (PPAR) agonist.

[0019] In some embodiments, the lipid-lowering agent and the anti-inflammatory agent are administered together. In some embodiments, the lipid-lowering agent and the anti-inflammatory agent are administered separately. In some embodiments, the lipid-lowering agent and / or the anti-inflammatory agent are administered intravenously, intramuscularly, subcutaneously, or orally.

[0020] In some embodiments, the level or activity of pro-inflammatory cytokines in the subject is decreased. In some embodiments, the level or activity of C-reactive protein (CRP) in the subject is decreased. In some embodiments, the level or activity of non-high density lipoprotein (HDL)-cholesterol in the subject is decreased. In some embodiments, the level or activity of LDL-cholesterol in the subject is decreased. In some embodiments, the level or activity of total cholesterol in the subject is decreased. In some embodiments, the level or activity of apolipoprotein B (ApoB) in the subject is decreased. In some embodiments, the level or activity of triglycerides in the subject is decreased. In some embodiments, the ratio of total cholesterol to HDL-cholesterol in the subject is decreased. In some embodiments, the occurrence of non-fatal myocardial infarction is decreased. In some embodiments, the occurrence of non-fatal stroke is decreased. In some embodiments, the rate of cardiovascular mortality is decreased.

[0021] In some embodiments, the cardiovascular disease is myocardial infarction, stroke, acute coronary syndrome, myocardial ischemia, chronic stable angina, unstable angina, cardiovascular death, coronary restenosis, coronary stent restenosis, coronary stent thrombosis, revascularization, angioplasty, transient ischemic attack, pulmonary embolism, vascular occlusion, or venous thrombosis.

[0022] Another aspect of the present disclosure provides a method of reducing the recurrence rate of cardiovascular disease in a subject who has been or is being treated with a lipid-lowering agent, the method comprising administering to the subject an effective amount of an anti-inflammatory agent.

[0023] Other aspects of the present disclosure provide a method for predicting the recurrence rate of cardiovascular disease in a subject who has received or is receiving treatment with a lipid-lowering agent, the method comprising measuring the level of C-reactive protein (CRP) in the subject and determining that the subject may have a recurrence of cardiovascular disease if the CRP level is higher than a predetermined value. In some embodiments, the predetermined value is 3 mg / L. In some embodiments, the predetermined value is 2 mg / L. In some embodiments, the predetermined value is 1 mg / L.

[0024] Further provided herein is a method for treating cardiovascular disease, the method comprising administering to a subject in need thereof a therapeutically effective amount of a bispecific antibody comprising a first antigen-binding domain that binds to a pro-inflammatory cytokine and a second antigen-binding domain that binds to proprotein convertase subtilisin / kexin type 9 (PCSK9). In some embodiments, the pro-inflammatory cytokine is selected from IL-1, IL-1 receptor (IL-1R), IL-6, IL-6 receptor (IL-6R), NLRP3, TNF, IL-8, or IL-18.

[0025] In some embodiments, the first antigen-binding domain binds to IL-1. In some embodiments, the first antigen-binding domain binds to IL-1α. In some embodiments, the first antigen-binding domain is derived from MABp1. In some embodiments, the first antigen-binding domain binds to IL-1β. In some embodiments, the first antigen-binding domain is derived from canakinumab, diacerein, gevokizumab, or LY2189102. In some embodiments, the first antigen-binding domain binds to IL-1R. In some embodiments, the first antigen-binding domain is derived from MEDI-8968 or AMG108. In some embodiments, the first antigen-binding domain binds to IL-6. In some embodiments, the first antigen-binding domain is derived from siltuximab, sirukumab, clazakizumab, olokizumab, or elsilimomab. In some embodiments, the first antigen-binding domain binds to IL-6R. In some embodiments, the first antigen-binding domain is derived from tocilizumab, sarilumab, PM1, AUK12-20, AUK64-7, AUK146-15, or AB-227-NA. In some embodiments, the first antigen-binding domain binds to NLRP3. In some embodiments, the first antigen-binding domain is derived from an NLRP3 antibody. In some embodiments, the first antigen-binding domain binds to TNF. In some embodiments, the first antigen-binding domain is derived from infliximab, adalimumab, certolizumab pegol, golimumab, or etanercept (Enbrel). In some embodiments, the first antigen-binding domain binds to IL-8. In some embodiments, the first antigen-binding domain is derived from HuMab-10F8. In some embodiments, the first antigen-binding domain binds to IL-18. In some embodiments, the first antigen-binding domain is derived from an IL-18 antibody.

[0026] In some embodiments, the second antigen-binding domain is derived from alirocumab, evolocumab, 1D05-IgG2, RG-7652, LY3015014, or bococizumab.

[0027] In some embodiments, the bispecific antibody comprises a common Fc region. In some embodiments, the bispecific antibody is a monoclonal bispecific antibody.

[0028] In some embodiments, the method further comprises administering to the subject a therapeutically effective amount of an HMG-CoA reductase inhibitor. In some embodiments, the HMG-CoA reductase inhibitor is a statin. In some embodiments, the statin is simvastatin, lovastatin, pravastatin, fluvastatin, atorvastatin, cerivastatin, rosuvastatin, or pitavastatin.

[0029] In some embodiments, the bispecific antibody is administered intravenously, intramuscularly, subcutaneously, or orally. In some embodiments, the level or activity of a pro-inflammatory cytokine in the subject is decreased. In some embodiments, the level or activity of C-reactive protein (CRP) in the subject is decreased. In some embodiments, the level or activity of non-high density lipoprotein (HDL)-cholesterol in the subject is decreased. In some embodiments, the level or activity of LDL-cholesterol in the subject is decreased. In some embodiments, the level or activity of total cholesterol in the subject is decreased. In some embodiments, the level or activity of apolipoprotein B (ApoB) in the subject is decreased. In some embodiments, the level or activity of triglycerides in the subject is decreased. In some embodiments, the ratio of total cholesterol to HDL-cholesterol in the subject is decreased. In some embodiments, the occurrence of non-fatal myocardial infarction is decreased. In some embodiments, the occurrence of non-fatal stroke is decreased. In some embodiments, the rate of cardiovascular mortality is decreased.

[0030] Further provided herein is a method of treating a cardiovascular disease, the method comprising administering to a subject in need thereof a therapeutically effective amount of a bispecific antibody comprising a first antigen-binding domain that binds to IL-1 and a second antigen-binding domain that binds to proprotein convertase subtilisin / kexin type 9 (PCSK9).

[0031] In some embodiments, the first antigen-binding domain binds to IL-1α. In some embodiments, the first antigen-binding domain is derived from MABp1. In some embodiments, the first antigen-binding domain binds to IL-1β. In some embodiments, the first antigen-binding domain is derived from canakinumab, diacerein, gevokizumab, or LY2189102. In some embodiments, the second antigen-binding domain is derived from alirocumab, evolocumab, 1D05-IgG2, RG-7652, LY3015014, or bococizumab.

[0032] Each limitation of the present disclosure can encompass various aspects of the present disclosure. Thus, it is understood that each limitation of the present disclosure that includes any one element or combination of elements can be included in each aspect of the present disclosure. The present disclosure is not limited in its application to the details of the construction and arrangement of components described in the following description or illustrated in the drawings. The present disclosure is capable of other aspects and of being practiced or carried out in various ways. Also, the phraseology and terminology used herein are for the purpose of description and should not be regarded as limiting. The use of "including", "comprising", "having", "containing", "involving", and variations thereof herein is intended to cover the recited items and their equivalents and further items. BRIEF DESCRIPTION OF THE DRAWINGS

[0033]

Fig. 1A-1B

Fig. 1C-1D

[0034]

Fig. 2

[0035]

Fig. 3

DETAILED DESCRIPTION OF THE INVENTION

[0036] DETAILED DESCRIPTION OF CERTAIN ASPECTS Despite aggressive lipid-lowering therapy, patients continue to have cardiovascular disease. The inventors have found clear evidence of residual inflammatory risk based on on-treatment hsCRP levels, not only in primary prevention but also during secondary prevention where patients are already undergoing aggressive LDL-C lowering therapy with both statins and PCSK9 inhibitors. Prior to the present disclosure, it was still unclear whether the residual inflammatory risk persists after extremely aggressive LDL-C lowering.

[0037] Some aspects of the present disclosure are based, at least in part, on the surprising finding that in a population of 9,738 high-risk patients who were actively treated with lipid-lowering agents (e.g., treated simultaneously with statins and PCSK9 inhibition), a large percentage of the patients were still at a persistent gradient of risk for future cardiovascular disease despite exceptionally aggressive lipid lowering. Such patients exhibit higher on-treatment hsCRP levels than normal. Compared to those without evidence of subclinical inflammation, those with on-treatment hsCRP > 3 mg / L had a 62% increase in the risk of future vascular events. Elevated hsCRP is significantly associated with an increased rate of myocardial infarction, cardiovascular death, and / or death from any cause. The inventors believe that the inflammatory risk persists despite maximal aggressive LDL-C lowering, and that reduction of inflammation provides additional benefit for reduction of cardiovascular disease.

[0038] Accordingly, some aspects of the present disclosure provide a method of treating cardiovascular disease, the method comprising administering to a subject in need thereof a therapeutically effective amount of a lipid-lowering agent and an anti-inflammatory agent.

[0039] "Anti-inflammatory agent" refers to an agent that reduces inflammation or an inflammatory response. In some embodiments, the anti-inflammatory agent is an inhibitor of pro-inflammatory cytokines. "Inhibitor of pro-inflammatory cytokines" refers to an agent that inhibits the inflammatory signaling pathway induced by pro-inflammatory cytokines. An inhibitor of pro-inflammatory cytokines can inhibit the level or activity of any protein or nucleic acid involved in the inflammatory signaling pathway. For example, in some embodiments, the inhibitor of pro-inflammatory cytokines inhibits the level of pro-inflammatory cytokines (e.g., IL-1 such as IL-1α and Il-1β, IL-6, IL-8, and IL-18). In some embodiments, the inhibitor of pro-inflammatory cytokines inhibits the activity of pro-inflammatory cytokines by inhibiting, for example, the level or activity of cytokine receptors (e.g., IL-1R and IL-6R).

[0040] In some embodiments, the pro-inflammatory cytokine inhibitor inhibits the inflammasome. The inflammasome is a multi-protein oligomer expressed in myeloid cells and is a component of the innate immune system. The exact composition of the inflammasome depends on the activator that initiates the assembly of the inflammasome. For example, dsRNA induces the composition of one inflammasome, while asbestos assembles different variants. The inflammasome promotes the maturation of the inflammatory cytokines interleukin 1β (IL-1β) and interleukin 18 (IL-18). In some embodiments, the inflammasome consists of caspase 1, PYCARD or ASC, NALP and sometimes caspase 5 (also known as caspase 11 or ICH-3). In some embodiments, the inflammasome includes the NOD-like receptor protein 3 (NLRP3).

[0041] In some embodiments, the anti-inflammatory agent is a nucleic acid, an aptamer, an antibody or antibody fragment, an inhibitory peptide, or a small molecule. In some embodiments, the anti-inflammatory agent is an inhibitory nucleic acid such as an antisense nucleic acid designed to target a pro-inflammatory cytokine gene.

[0042] As used herein, the term "antisense nucleic acid" describes a nucleic acid that hybridizes to DNA containing a particular gene or to the mRNA transcript of that gene under physiological conditions, thereby inhibiting the transcription of that gene and / or the translation of its mRNA. Antisense molecules are designed to interfere with the transcription or translation of a target gene by hybridization to the target gene or transcript. One of ordinary skill in the art will understand that the exact length of the antisense oligonucleotide and the degree of complementarity to its target will depend on the particular target selected (including the sequence of the target and the particular bases included in that sequence). Antisense nucleic acids bind to target RNA by Watson-Crick base pairing and block gene expression by preventing translation of the bound sequence by ribosomes, by steric hindrance, or by activating RNase H enzymes. Antisense molecules can also alter protein synthesis by interfering with RNA processing or transport from the nucleus to the cytoplasm (Mukhopadhyay & Roth, 1996, Crit. Rev. in Oncogenesis 7, 151-190).

[0043] In some embodiments, the antisense nucleic acid is an RNAi molecule. An RNAi molecule is an antisense molecule that inhibits the expression of pro-inflammatory cytokine signaling components. The nucleic acid sequences of pro-inflammatory cytokines are known in the art. Inhibitory nucleic acids can be designed using conventional methods in the art.

[0044] Inhibitory nucleic acids (e.g., antisense oligonucleotides) against pro-inflammatory cytokine genes typically cause specific gene knockdown while avoiding off-target effects. To inhibit gene expression, various strategies for gene knockdown known in the art can be used. For example, gene knockdown strategies utilizing the RNA interference (RNAi) and / or microRNA (miRNA) pathways, including small interfering RNA (siRNA), small hairpin RNA (shRNA), double-stranded RNA (dsRNA), miRNA, and other molecules based on small interfering nucleic acids known in the art, may be employed. In some embodiments, a vector-based RNAi modality (e.g., an shRNA or shRNA-mir expression construct) is used to reduce the expression of a gene (e.g., a target nucleic acid such as a pro-inflammatory cytokine nucleic acid) in a cell. In some embodiments, the inhibitory nucleic acid comprises an isolated plasmid vector (e.g., any isolated plasmid vector known in the art or disclosed herein) that expresses a small interfering nucleic acid such as shRNA. The isolated plasmid may optionally include a specific promoter operably linked to a gene encoding the small interfering nucleic acid. In some embodiments, the isolated plasmid vector is packaged within a virus that can infect the individual. Exemplary viruses include adenovirus, retrovirus, lentivirus, adeno-associated virus, and others known in the art and disclosed herein.

[0045] A wide range of RNAi-based molecules, such as oligonucleotides based on siRNA and / or oligonucleotides based on modified siRNA, can be used to inhibit the expression of genes (e.g., pro-inflammatory cytokine genes) in cells. Oligonucleotides based on modified siRNA are modified, for example, to make them resistant or partially resistant to intracellular degradation in order to modify their potency, target affinity, safety profile and / or stability. Modifications such as phosphorothioate can be made to the oligonucleotides to increase resistance to nuclease degradation, binding affinity and / or uptake. In addition, hydrophobization and bioconjugation reactions enhance the delivery and targeting of siRNA (De Paula et al., RNA. 13(4):431-56, 2007), and siRNAs with ribo-difluorotoluyl nucleotides maintain gene silencing activity (Xia et al., ASC Chem. Biol. 1(3):176-83, (2006)). siRNAs with amide-linked oligoribonucleosides have been made that are more resistant to S1 nuclease degradation than unmodified siRNAs (Iwase R et al. 2006 Nucleic Acids Symp Ser 50: 175-176). In addition, modification of siRNA at the 2'-sugar position and in the phosphodiester linkage confers improved serum stability without loss of potency (Choung et al., Biochem. Biophys. Res. Commun. 342(3):919-26, 2006). Other molecules that can be used to inhibit the expression of genes (e.g., CSC-related genes) include sense and antisense nucleic acids (single-stranded or double-stranded), ribozymes, peptides, DNAzymes, peptide nucleic acids (PNA), triplex-forming oligonucleotides, antibodies, and aptamers, and modified forms thereof (single or plural) directed to sequences in genes (single or plural), RNA transcripts, or proteins.

[0046] Antisense and ribozyme strategies have led to reversal of the tumor phenotype by reducing the expression of gene products or by cleaving mutant transcripts at the site of the mutation (Carter and Lemoine Br. J. Cancer. 67(5):869-76, 1993; Lange et al., Leukemia. 6(11):1786-94, 1993; Valera et al., J. Biol. Chem. 269(46):28543-6, 1994; Dosaka-Akita et al., Am. J. Clin. Pathol. 102(5):660-4, 1994; Feng et al., Cancer Res. 55(10):2024-8, 1995; Quattrone et al., Cancer Res. 55(1):90-5, 1995; Lewin et al., Nat Med. 4(8):967-71, 1998). Ribozyme has also been proposed as a means of inhibiting gene expression of mutant genes and as a means of correcting mutants by targeted trans-splicing (Sullenger and Cech Nature 371(6498):619-22, 1994; Jones et al., Nat. Med. 2(6):643-8, 1996). Ribozyme activity can be enhanced, for example, by the use of non-specific nucleic acid binding proteins or facilitator oligonucleotides (Herschlag et al., Embo J. 13(12):2913-24, 1994; Jankowsky and Schwenzer Nucleic Acids Res. 24(3):423-9,1996)). Multi-target ribozymes (linked or shotgun type) have been suggested as a means of improving the efficiency of ribozymes for gene suppression (Ohkawa et al., Nucleic Acids Symp Ser. (29):121-2, 1993).

[0047] In some embodiments, inhibitory nucleic acids include modified or unmodified RNA, DNA, or mixed polymer nucleic acids, and function primarily by specifically binding to a matching sequence to effect regulation of peptide synthesis (Wu-Pong, November 1994, BioPharm, 20-33).

[0048] In some embodiments, the inhibitory nucleic acids of the present disclosure are 100% identical to the nucleic acid target. In other embodiments, it is at least 99%, 95%, 90%, 85%, 80%, 75%, 70% or 50% identical to the nucleic acid target. The term "% identity" refers to sequence identity between two nucleotide sequences. Percent identity can be determined by comparing positions in each sequence that can be aligned for purposes of comparison. The expression as a percentage of identity refers to a function of the number of identical amino acids or nucleic acids at positions shared by the sequences being compared. A variety of alignment algorithms and / or programs may be used, including FASTA, BLAST or ENTREZ-FASTA, and BLAST is available as part of the GCG sequence analysis package (University of Wisconsin, Madison, Wis.) and may be used, for example, with default settings. ENTREZ is available through the National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health (Bethesda, Md.). In one embodiment, the percent identity of two sequences may be determined by the GCG program with a gap weight of 1 (e.g., each amino acid gap is weighted as a single amino acid or nucleotide mismatch between the two sequences).

[0049] Other techniques for alignment are described in Methods in Enzymology, Volume 266: Computer Methods for Macromolecular Sequence Analysis (1996) (edited by Doolittle, Academic Press, Inc. (a division of Harcourt Brace & Co., San Diego, Calif., USA)). Preferably, an alignment program that allows gaps in the sequence is utilized to align the sequences. Smith-Waterman is one type of algorithm that allows gaps in sequence alignment. See Meth. Mol. Biol. 70: 173-187 (1997). Also, the GAP program using the alignment method of Needleman and Wunsch can be utilized to align the sequences. An alternative search strategy uses the MPSRCH software that runs on a MASPAR computer. MPSRCH scores sequences on a massively parallel computer using the Smith-Waterman algorithm. This approach improves the ability to pick up distantly related matches and is particularly tolerant of small gaps and nucleotide sequence errors. The amino acid sequences encoded by nucleic acids can be used to search both protein and DNA databases.

[0050] Inhibitory nucleic acids useful in the present disclosure will generally be designed to have partial or complete complementarity with one or more target genes (i.e., complementarity with one or more transcripts of a pro-inflammatory cytokine gene). The target gene may be a gene derived from a cell, an endogenous gene, a transgene, or a gene of a pathogen present in the cell after its infection. Depending on the particular target gene, the nature of the inhibitory nucleic acid, and the level of expression of the inhibitory nucleic acid (e.g., depending on copy number, promoter strength), the procedure can result in a partial or complete loss of function for the target gene. Quantification of gene expression in a cell can indicate a similar amount of inhibition at the level of accumulation of the target mRNA or translation of the target protein.

[0051] "Inhibition of gene expression" refers to the absence or observable decrease in the level of protein and / or mRNA products from a target gene. The results of inhibition can be confirmed by testing the outward characteristics of cells or organisms, or by biochemical techniques such as RNA solution hybridization, nuclease protection, Northern hybridization, reverse transcription, gene expression monitoring by microarray, antibody binding, enzyme-linked immunosorbent assay (ELISA), Western blotting, radioimmunoassay (RIA), other immunoassays, and fluorescence activated cell analysis (FACS). For RNA-mediated inhibition in a cell line or whole organism, gene expression can be conveniently assayed by use of a reporter or drug resistance gene whose protein product is readily assayed. Such reporter genes include acetohydroxyacid synthase (AHAS), alkaline phosphatase (AP), beta-galactosidase (LacZ), beta-glucuronidase (GUS), chloramphenicol acetyltransferase (CAT), green fluorescent protein (GFP), horseradish peroxidase (HRP), luciferase (Luc), nopaline synthase (NOS), octopine synthase (OCS), and derivatives thereof. A plurality of selectable markers that confer resistance to ampicillin, bleomycin, chloramphenicol, gentamicin, hygromycin, kanamycin, lincomycin, methotrexate, phosphinothricin, puromycin, and tetracycline are available.

[0052] Depending on the assay, the degree of inhibition can be determined by quantifying the amount of gene expression, which may be higher than 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99% compared to cells not treated according to the present disclosure. As an example, the efficiency of inhibition can be determined by assessing the amount of gene product in the cell: mRNA can be detected by a hybridization probe having a nucleotide sequence outside the region used for the inhibitory nucleic acid, or the translated polypeptide can be detected by an antibody produced against the polypeptide sequence of that region.

[0053] "Antibody" and "antibody fragment" include the whole antibody and any antigen-binding fragment (i.e., "antigen-binding portion") or single chain thereof. "Antibody" refers to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains linked interchain by disulfide bonds, or an antigen-binding portion thereof. Each heavy chain consists of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region consists of three domains, CH1, CH2 and CH3. Each light chain consists of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region consists of one domain, CL. The VH and VL regions can be further subdivided into regions of hypervariability called complementarity determining regions (CDRs) interspersed with regions called more conserved framework regions (FRs). Each VH and VL consists of three CDRs and four FRs, which are arranged from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain the binding domains that interact with the antigen. The constant region of the antibody can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component of the classical complement system (C1q). The antibody may be a polyclonal antibody or a monoclonal antibody. The antibody may be a chimeric antibody or a humanized antibody.

[0054] An "antibody fragment" for use in accordance with the present disclosure includes the antigen-binding portion of an antibody. The antigen-binding portion of an antibody refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen. It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments included within the term "antigen-binding portion" of an antibody include: (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL, and CH1 domains; (ii) an F(ab’)2 fragment, a bivalent fragment comprising two Fab fragments linked by disulfide bridges in the hinge region; (iii) an Fd fragment consisting of the VH and CH1 domains; (iv) an Fv fragment consisting of the VL and VH domains of one arm of an antibody; (v) a dAb fragment consisting of a VH domain (e.g., as described in Ward et al., (1989) Nature 341:544-546, which is incorporated herein by reference); and (vi) an isolated complementarity-determining region (CDR). Furthermore, although the two domains of an Fv fragment, VL and VH, are encoded by separate genes, they can be linked using recombinant methods with a synthetic linker, which can join them into a single protein chain in which the VL and VH regions pair to form a monovalent molecule (known as a single-chain Fv (scFv); see, e.g., Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883, which are incorporated herein by reference). Such single-chain antibodies are also intended to be included within the term "antigen-binding portion" of an antibody. These antibody fragments can be obtained using conventional techniques known to those of skill in the art, and the fragments are screened for their utility in the same manner as full antibodies.

[0055] "Inhibitory peptide" refers to a peptide that specifically binds to a target molecule. In some embodiments, the binding of the inhibitory peptide to the target molecule inhibits the biological activity of the target molecule. For example, if the target molecule functions in a signal transduction pathway, the binding of the inhibitory peptide may inhibit the signal transduction pathway. Those skilled in the art are proficient in methods for developing inhibitory peptides or inhibitory peptides against selected target molecules thereof. For example, a peptide derived from the receptor-binding portion of a pro-inflammatory cytokine can competitively bind to the receptor, interfere with the binding of the cytokine, and inhibit downstream signal transduction. The inhibitory peptide may also be synthetic (i.e., a synthetic peptide). Those skilled in the art are proficient in methods for designing and synthesizing inhibitory peptides.

[0056] "Aptamer" refers to an oligonucleotide or peptide molecule that binds to a specific target molecule. Aptamers are usually prepared by selecting them from a large random sequence pool.

[0057] As used herein, a "small molecule" is a molecule of an organic or inorganic compound having a low molecular weight (e.g., < 900 Daltons), whether naturally occurring or artificially produced (e.g., via chemical synthesis), that has a relatively low molecular weight and is capable of functioning in the control of biological processes. Typically, organic compounds contain carbon. Organic compounds may contain multiple carbon-carbon bonds, stereocenters, and other functional groups (e.g., amines, hydroxyls, carbonyls, or heterocyclic rings). In some embodiments, a small molecule is a monomeric organic compound having a molecular weight of less than about 1500 g / mol. In one embodiment, the molecular weight of the small molecule is less than about 1000 g / mol or less than about 500 g / mol. In one embodiment, the small molecule is a drug, e.g., a drug that has already been considered safe and effective for use in humans or animals by the appropriate government authority or regulatory agency. In one embodiment, the organic molecule is known to bind to and / or cleave nucleic acids. In some embodiments, the organic compound is an endiyne. Non-limiting examples of small molecules include lipids, monosaccharides, second messengers, other natural products and metabolites, as well as drugs and other xenobiotics.

[0058] "Lipid" refers to a group of naturally occurring molecules including fats, waxes, sterols, fat-soluble vitamins (such as vitamins A, D, E, and K), monoglycerides, diglycerides, triglycerides, phospholipids, and others. "Monosaccharide" refers to a class of sugars (such as glucose) that cannot be hydrolyzed to yield simpler sugars. Non-limiting examples of monosaccharides include glucose (dextrose), fructose (levulose), and galactose. "Second messenger" refers to a molecule that relays a signal received at a receptor on the cell surface (such as from a protein hormone, growth factor, etc.) to target molecules in the cytosol and / or nucleus. Non-limiting examples of second messenger molecules include cyclic AMP, cyclic GMP, inositol trisphosphate, diacylglycerol, and calcium. "Metabolite" refers to a molecule that forms an intermediate product of metabolism. Non-limiting examples of metabolites include ethanol, glutamic acid, aspartic acid, 5'-guanylic acid, isoascorbic acid, acetic acid, lactic acid, glycerol, and vitamin B2. "Xenobiotic" refers to an exogenous chemical substance that is not naturally produced or expected to be present inside an organism and is found within the organism. Non-limiting examples of xenobiotics include drugs, antibiotics, carcinogens, environmental pollutants, food additives, carbohydrates, and pesticides.

[0059] In some embodiments, the anti-inflammatory agent is selected from the group consisting of: an IL-1 inhibitor, an IL-1 receptor (IL-1R) inhibitor, an IL-6 inhibitor, an IL-6 receptor (IL-6R) inhibitor, an NLRP3 inhibitor, a TNF inhibitor, an IL-8 inhibitor, an IL-18 inhibitor, or an inhibitor of natural killer cells. Combinations of different anti-inflammatory agents described herein are contemplated. In some embodiments, the anti-inflammatory agent comprises an inhibitor against one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) pro-inflammatory cytokines. Each inhibitor against one pro-inflammatory cytokine may be a different type of inhibitor, such as a combination of an inhibitory nucleic acid, an inhibitory peptide, an antibody, or a small molecule.

[0060] In some embodiments, the anti-inflammatory agent may be any of the following: a combination of an IL-1 inhibitor and an IL-1R inhibitor; a combination of an IL-1 inhibitor and an IL-6 inhibitor; a combination of an IL-1 inhibitor and an IL-6R inhibitor; a combination of an IL-1 inhibitor and an NLRP3 inhibitor; a combination of an IL-1 inhibitor and a TNF inhibitor; a combination of an IL-1 inhibitor and an IL-8 inhibitor; a combination of an IL-1 inhibitor and an IL-18 inhibitor; a combination of an IL-1R inhibitor and an IL-6 inhibitor; a combination of an IL-1R inhibitor and an IL-6R inhibitor; a combination of an IL-1R inhibitor and an NLRP3 inhibitor; a combination of an IL-1R inhibitor and a TNF inhibitor; a combination of an IL-1R inhibitor and an IL-8 inhibitor; a combination of an IL-1R inhibitor and an inhibitor of natural killer cells; a combination of an IL-1R inhibitor and an IL-18 inhibitor; a combination of an IL-6 inhibitor and an IL-6R inhibitor; a combination of an IL-6 inhibitor and an NLRP3 inhibitor; a combination of an IL-6 inhibitor and a TNF inhibitor; a combination of an IL-6 inhibitor and an IL-8 inhibitor; a combination of an IL-6 inhibitor and an IL-18 inhibitor; a combination of an IL-6 inhibitor and an inhibitor of natural killer cells; a combination of an IL-6R inhibitor and an NLRP3 inhibitor; a combination of an IL-6R inhibitor and a TNF inhibitor; a combination of an IL-6R inhibitor and an IL-8 inhibitor; a combination of an IL-6R inhibitor and an IL-18 inhibitor; a combination of an IL-6R inhibitor and an inhibitor of natural killer cells; a combination of an NLRP3 inhibitor and a TNF inhibitor; a combination of an NLRP3 inhibitor and an IL-8 inhibitor; a combination of an NLRP3 inhibitor and an IL-18 inhibitor; a combination of an NLRP3 inhibitor and an inhibitor of natural killer cells; a combination of an IL-8 inhibitor and an IL-18 inhibitor; a combination of an IL-8 inhibitor and an inhibitor of natural killer cells; a combination of an IL-1 inhibitor, an IL-1R inhibitor, and an IL-6 inhibitor; a combination of an IL-1 inhibitor, an IL-1R inhibitor, and an IL-6R inhibitor; a combination of an IL-1 inhibitor, an IL-1R inhibitor, and an NLRP3 inhibitor; a combination of an IL-1 inhibitor, an IL-1R inhibitor, and a TNF inhibitor; a combination of an IL-1 inhibitor, an IL-1R inhibitor, and an IL-8 inhibitor;Combinations of an IL-1 inhibitor, an IL-1R inhibitor, and an IL-18 inhibitor; combinations of an IL-1 inhibitor, an IL-1R inhibitor, and an inhibitor of natural killer cells; combinations of an IL-1 inhibitor, an IL-6 inhibitor, and an IL-6R inhibitor; combinations of an IL-1 inhibitor, an IL-6 inhibitor, and an NLRP3 inhibitor; combinations of an IL-1 inhibitor, an IL-6 inhibitor, and a TNF inhibitor; combinations of an IL-1 inhibitor, an IL-6 inhibitor, and an IL-8 inhibitor; combinations of an IL-1 inhibitor, an IL-6 inhibitor, and an IL-18 inhibitor; combinations of an IL-1 inhibitor, an IL-6R inhibitor, and an NLRP3 inhibitor; combinations of an IL-1 inhibitor, an IL-6R inhibitor, and a TNF inhibitor; combinations of an IL-1 inhibitor, an IL-6R inhibitor, and an IL-8 inhibitor; combinations of an IL-1 inhibitor, an IL-6R inhibitor, and an IL-18 inhibitor; combinations of an IL-1 inhibitor, an IL-6R inhibitor, and an inhibitor of natural killer cells; combinations of an IL-1 inhibitor, an NLRP3 inhibitor, and a TNF inhibitor; combinations of an IL-1 inhibitor, an NLRP3 inhibitor, and an IL-8 inhibitor; combinations of an IL-1 inhibitor, an NLRP3 inhibitor, and an inhibitor of natural killer cells; combinations of an IL-1 inhibitor, an NLRP3 inhibitor, and an IL-18 inhibitor; combinations of an IL-1 inhibitor, a TNF inhibitor, and an IL-8 inhibitor; combinations of an IL-1 inhibitor, a TNF inhibitor, and an IL-18 inhibitor; combinations of an IL-1 inhibitor, a TNF inhibitor, and an inhibitor of natural killer cells; combinations of an IL-1 inhibitor, an IL-8 inhibitor, and an IL18 inhibitor; combinations of an IL-1 inhibitor, an IL-8 inhibitor, and an inhibitor of natural killer cells; combinations of an IL-1 inhibitor, an NLRP3 inhibitor, and a TNF inhibitor; combinations of an IL-1R inhibitor, an IL-6 inhibitor, and an IL-6R inhibitor; combinations of an IL-1R inhibitor, an IL-6 inhibitor, and an NLRP3 inhibitor; combinations of an IL-1R inhibitor, an IL-6 inhibitor, and a TNF inhibitor; combinations of an IL-1R inhibitor, an IL-6 inhibitor, and an IL-8 inhibitor; combinations of an IL-1R inhibitor, an IL-6 inhibitor, and an IL-18 inhibitor; combinations of an IL-1R inhibitor, an IL-6 inhibitor, and an inhibitor of natural killer cells;Combinations of an IL-1R inhibitor, an IL-6R inhibitor, and an NLRP3 inhibitor; combinations of an IL-1R inhibitor, an IL-6R inhibitor, and a TNF inhibitor; combinations of an IL-1R inhibitor, an IL-6R inhibitor, and an IL-8 inhibitor; combinations of an IL-1R inhibitor, an IL-6R inhibitor, and an IL-18 inhibitor; combinations of an IL-1R inhibitor, an IL-6R inhibitor, and an inhibitor of natural killer cells; combinations of an IL-1R inhibitor, an NLRP3 inhibitor, and a TNF inhibitor; combinations of an IL-1R inhibitor, an NLRP3 inhibitor, and an IL-8 inhibitor; combinations of an IL-1R inhibitor, an NLRP3 inhibitor, and an inhibitor of natural killer cells; combinations of an IL-1R inhibitor, an NLRP3 inhibitor, and an IL-18 inhibitor; combinations of an IL-1R inhibitor, a TNF inhibitor, and an IL-8 inhibitor; combinations of an IL-1R inhibitor, a TNF inhibitor, and an Il-18 inhibitor; combinations of an IL-1R inhibitor, a TNF inhibitor, and an inhibitor of natural killer cells; combinations of an IL-1R inhibitor, an IL-8 inhibitor, and an IL18 inhibitor; combinations of an IL-1R inhibitor, an IL-8 inhibitor, and an inhibitor of natural killer cells; combinations of an IL-6 inhibitor, an NLRP3 inhibitor, and a TNF inhibitor; combinations of an IL-6 inhibitor, an IL-6R inhibitor, and an NLRP3 inhibitor; combinations of an IL-6 inhibitor, an IL-6R inhibitor, and a TNF inhibitor; combinations of an IL-6 inhibitor, an IL-6R inhibitor, and an IL-8 inhibitor; combinations of an IL-6 inhibitor, an IL-6R inhibitor, and an IL-18 inhibitor; combinations of an IL-6 inhibitor, an IL-6R inhibitor, and an inhibitor of natural killer cells; combinations of an IL-6 inhibitor, an NLRP3 inhibitor, and a TNF inhibitor; combinations of an IL-6 inhibitor, an NLRP3 inhibitor, and an IL-8 inhibitor; combinations of an IL-6 inhibitor, an NLRP3 inhibitor, and an IL-18 inhibitor; combinations of an IL-6 inhibitor, an NLRP3 inhibitor, and an inhibitor of natural killer cells; combinations of an IL-6 inhibitor, a TNF inhibitor, and an IL-8 inhibitor; combinations of an IL-6 inhibitor, a TNF inhibitor, and an IL-18 inhibitor; combinations of an IL-6 inhibitor, a TNF inhibitor, and an inhibitor of natural killer cells;Combinations of an IL-6 inhibitor, an IL-8 inhibitor, and an IL18 inhibitor; combinations of an IL-6 inhibitor, an IL-8 inhibitor, and an inhibitor of natural killer cells; combinations of an IL-6 inhibitor, an IL-18 inhibitor, and an inhibitor of natural killer cells; combinations of an IL-6 inhibitor, an NLRP3 inhibitor, and a TNF inhibitor; combinations of an IL-6R inhibitor, an NLRP3 inhibitor, and a TNF inhibitor; combinations of an IL-6R inhibitor, an NLRP3 inhibitor, and an IL-8 inhibitor; combinations of an IL-6R inhibitor, an NLRP3 inhibitor, and an IL-18 inhibitor; combinations of an IL-6R inhibitor, an NLRP3 inhibitor, and an inhibitor of natural killer cells; combinations of an IL-6R inhibitor, a TNF inhibitor, and an IL-8 inhibitor; combinations of an IL-6R inhibitor, a TNF inhibitor, and an IL-18 inhibitor; combinations of an IL-6R inhibitor, an IL-8 inhibitor, and an IL18 inhibitor; combinations of an IL-6R inhibitor, an IL-8 inhibitor, and an inhibitor of natural killer cells; combinations of an NLRP3 inhibitor, a TNF inhibitor, and an IL-18 inhibitor; combinations of an NLRP3 inhibitor, a TNF inhibitor, and an inhibitor of natural killer cells; combinations of an NLRP3 inhibitor, an IL-8 inhibitor, and an IL18 inhibitor; combinations of an NLRP3 inhibitor, an IL-8 inhibitor, and an inhibitor of natural killer cells; combinations of a TNF inhibitor, an IL-8 inhibitor, and an IL18 inhibitor; combinations of a TNF inhibitor, an IL-8 inhibitor, and an inhibitor of natural killer cells; combinations of an IL-8 inhibitor, an IL18 inhibitor, and an inhibitor of natural killer cells; or any suitable combination of the previous combinations. Any combination may be used. One of ordinary skill in the art can identify suitable combinations using conventional methods.;

[0061] In some embodiments, the anti-inflammatory agent includes an IL-1 inhibitor. In some embodiments, the IL-1 inhibitor can be any protein or molecule that can specifically interfere with the activation of the IL-1 cell receptor, which can result from any number of mechanisms. Exemplary mechanisms include, but are not limited to, downregulating IL-1 production, binding to free IL-1, interfering with the binding of IL-1 to its receptor, interfering with the formation of the IL-1 receptor complex (i.e., the association of the IL-1 receptor with the IL-1 receptor accessory protein), and interfering with the regulation of signal transduction after the binding of IL-1 to its receptor.

[0062] Specific interleukin-1 inhibitors include, but are not limited to, the following: IL-1 binding proteins (including, but not limited to, soluble IL-1 receptors (see, for example, U.S. Patent Nos. 5,492,888, 5,488,032, 5,464,937, 5,319,071, and 5,180,812, which are incorporated herein by reference)); anti-IL-1 monoclonal antibodies (see, for example, WO9501997, WO9402627, WO9006371, U.S. Patent No. 4,935,343, EP364778, EP267611, and EP220063, which are incorporated herein by reference); IL-1 receptor accessory proteins and antibodies thereto (see, for example, WO96 / 23067 and WO99 / 37773, which are incorporated herein by reference); inhibitors of interleukin-1 beta converting enzyme (ICE) or caspase I used to inhibit the production and secretion of IL-1 beta (see, for example, WO99 / 46248, WO99 / 47545, and WO99 / 47154, which are incorporated herein by reference); interleukin-1 beta protease inhibitors; and other compounds and proteins that block the synthesis or extracellular release of IL-1 in vivo.

[0063] Exemplary IL-1 inhibitors are disclosed, for example, in U.S. Patent Nos. 5,747,444; 5,359,032; 5,608,035; 5,843,905; 5,359,032; 5,866,576; 5,869,660; 5,869,315; 5,872,095; 5,955,480; 5,965,564; International (WO) Patent Applications 98 / 21957, 96 / 09323, 91 / 17184, 96 / 40907, 98 / 32733, 98 / 42325, 98 / 44940, 98 / 47892, 98 / 56377, 99 / 03837, 99 / 06426, 99 / 06042, 91 / 17249, 98 / 32733, 98 / 17661, 97 / 08174, 95 / 34326, 99 / 36426, 99 / 36415; European (EP) Patent Applications 534978 and 894795; and French Patent Application FR2762514. The entire disclosure of the foregoing references is incorporated herein by reference for all purposes.

[0064] In some embodiments, the IL-1 inhibitor is an IL-1α inhibitor. In some embodiments, the IL-1α inhibitor is an antisense oligonucleotide against IL-1α, for example, an RNAi molecule such as miRNA, siRNA, or shRNA. In some embodiments, the IL-1 inhibitor is an IL-1β inhibitor. In some embodiments, the IL-1β inhibitor is an antisense oligonucleotide against IL-1α, for example, an RNAi molecule such as miRNA, siRNA, or shRNA. The nucleic acid sequences of the IL-1A and IL-1B genes are known. One of ordinary skill in the art can design such antisense oligonucleotides using conventional methods.

[0065] In some embodiments, the IL-1α inhibitor is an antibody against IL-1α, such as MABp1 (e.g., as described in Hong et al., Lancet Oncol. 2014 May;15(6):656-66, which is incorporated herein by reference). In some embodiments, the IL-1α inhibitor is a protein that binds to IL-1α. In some embodiments, the protein that binds to IL-1α is soluble interleukin-1 receptor type I (sIL-1RI, as described in Okamoto et al., J Clin Lab Anal. 2009;23(3):175-8, which is incorporated herein by reference).

[0066] In some embodiments, the IL-1β inhibitor is an antibody against IL-1β, such as canakinumab (e.g., as described in Ridker et al., N Engl J Med 2017; 377:1119-1131, incorporated herein by reference), gevokizumab (e.g., as described in Knickelbein et al., Am J Ophthalmol. 2016 Dec;172:104-110, incorporated herein by reference), LY2189102 (e.g., as described in Sloan-Lancaster et al., Diabates Care 2013 Mar; DC_121835, incorporated herein by reference), CYT013 (e.g., as described in Dinarello et al., Nature Reviews Drug Discovery 11, 633-652, 2012, incorporated herein by reference). In some embodiments, the IL-1β inhibitor is a protein that binds to IL-1β. In some embodiments, the protein that binds to IL-1β is serum-soluble interleukin-1 receptor type II (sIL-1RII, e.g., as described in Jouvenne et al., Arthritis Rheum. 1998 Jun;41(6):1083-9, incorporated herein by reference). In some embodiments, the IL-1β inhibitor inhibits caspase I required in the production of IL-1β. In some embodiments, the caspase I inhibitor is VX-70 or VX-765 or belnacasan (e.g., as described in Boxer et al., ChemMedChem. 2010 May 3; 5(5): 730-738, incorporated herein by reference).

[0067] In some embodiments, the IL-1 inhibitor is a small molecule inhibitor selected from the group consisting of sodium slamine, methotrexate-methyl-d3, methotrexate-methyl-d3 dimethyl ester, and diacerein. All of these are commercially available, for example, from Santa Cruz Biotechnology, Inc., Texas, USA.

[0068] In some embodiments, the anti-inflammatory agent includes an IL-1R inhibitor, such as an IL-1R antagonist. An "antagonist" is a type of receptor ligand or drug that, upon binding to a receptor, does not cause a response like an agonist but rather blocks or attenuates a biological response. They are sometimes also referred to as blockers: examples include alpha blockers, beta blockers, and calcium channel blockers. In pharmacology, antagonists have an affinity for their cognate receptors but no efficacy, and the binding interferes with the interaction of an agonist or inverse agonist at the receptor and inhibits its function. Antagonists mediate their effects by binding to an active orthosteric (= right place) site on the receptor or an allosteric (= other place) site, or they can interact at unique binding sites that are not normally involved in the biological control of receptor activity. Antagonist activity can be reversible or irreversible depending on the lifetime of the antagonist-receptor complex, which in turn depends on the nature of the antagonist-receptor binding. The majority of drug antagonists achieve their efficacy by competing with the endogenous ligand or substrate at a structurally defined binding site on the receptor.

[0069] Of course, IL-1R antagonists include IL-1RA, IL-1RA variants, and IL-1RA derivatives, which are collectively referred to as "IL-1ra proteins". Interleukin-1 receptor antagonist (IL-1ra) is a human protein that acts as a natural inhibitor of interleukin-1 and is a member of the IL-1 family, including IL-1α and IL-1β. Certain receptor antagonists, including IL-1ra and its variants and derivatives, and methods of making and using them are incorporated herein by reference to U.S. Patent No. 5,075,222; WO91 / 08285; WO91 / 17184; AU9173636; WO92 / 16221; WO93 / 21946; WO94 / 06457; WO94 / 21275; FR2706772; WO94 / 21235; DE4219626, WO94 / 20517; WO96 / 22793; WO97 / 28828; and WO99 / 36541. In certain embodiments, the IL-1 receptor antagonist may be glycosylated. In certain embodiments, the IL-1 receptor antagonist may be non-glycosylated.

[0070] Three forms of IL-1ra and its variants are described in U.S. Patent No. 5,075,222, which is incorporated herein by reference. Methods of isolating genes encoding inhibitors, cloning those genes into suitable vectors, transforming and transducing those genes into specific cell types, and expressing those genes to produce the inhibitors are known to those of skill in the art.

[0071] In some embodiments, the IL-1R inhibitor is an antisense oligonucleotide to IL-1R, such as an RNAi molecule such as miRNA, siRNA, or shRNA. The nucleic acid sequence of the IL-1R gene is known. One of skill in the art can design such antisense oligonucleotides using conventional methods.

[0072] In some embodiments, the IL-1R inhibitor is an antibody against IL-1R (e.g., a monoclonal antibody). Exemplary IL-1 antibodies that can be used in accordance with the present disclosure include, without limitation, anakinra (e.g., as described in Mertens et al., Cochrane Database Syst Rev. 2009 Jan 21;(1):CD005121, which is incorporated herein by reference), MEDI-8968 (e.g., as described in Dinarello et al, Nature Reviews Drug Discovery 11, 633-652, 2012, which is incorporated herein by reference), and AMG108 (e.g., as described in Cohen et al., Arthritis Res Ther. 2011 Jul 29;13(4):R125, which is incorporated herein by reference).

[0073] In some embodiments, the IL-1R inhibitor is an inhibitory protein or peptide. Such inhibitory proteins or peptides include, without limitation, rilonacept, sIL-1RI (e.g., as described in Okamoto et al., J Clin Lab Anal. 2009;23(3):175-8; and in European Patent EP623674, which are incorporated herein by reference), and EBI-005 (e.g., as described in Kovalchin et al., Eye Contact Lens. 2017 Jul 18. doi: 10.1097 / ICL.0000000000000414, which is incorporated herein by reference).

[0074] In some embodiments, the anti-inflammatory agent includes an IL-6 inhibitor. In some embodiments, the IL-6 inhibitor is an antisense oligonucleotide against IL-6, e.g., an RNAi molecule such as miRNA, siRNA, or shRNA. The nucleic acid sequence of the IL-6 gene is known. One of ordinary skill in the art can design such antisense oligonucleotides using conventional methods.

[0075] In some embodiments, the IL-6 inhibitor is an antibody against IL-6. Antibodies against IL-6 are known in the art and include BE-8 and CNTO328 (see, for example, Trikha et al., Clin Cancer Res 2003, 9: 4653 or US20090022726). As IL-6 neutralizing antibodies, both polyclonal antibodies and monoclonal antibodies can be used, and monoclonal antibodies are preferred. An example of an anti-IL-6 antibody having the ability to neutralize IL-6 is IG61 described in Japanese Laid-open Patent Application (Kokai) No. 3-139292 and in European Patent Publication 0 399 429 A1, but the IL-6 neutralizing antibody is not limited to this antibody. IG61 was deposited on April 27, 1990 at the National Institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology, 1-3, Higashi 1-chome, Tsukuba, Ibaraki, Japan, under accession number FERM BP-2878. Other non-limiting examples of IL-6 antibodies include siltuximab, sirukumab, clazakizumab, olokizumab, and elsilimomab. Those skilled in the art are familiar with these IL-6 antibodies.

[0076] In some embodiments, the IL-6 inhibitor is a small molecule. Non-limiting exemplary small molecule IL-6 inhibitors include PGE1 and its derivatives, PGI2 and its derivatives, and cyclophosphamide.

[0077] In some embodiments, the anti-inflammatory agent includes an IL-6R inhibitor (e.g., an IL-6R antagonist). In some embodiments, the IL-6R inhibitor is an antisense oligonucleotide against IL-6R, such as an RNAi molecule like miRNA, siRNA, or shRNA. The nucleic acid sequence of the IL-6R gene is known. A person skilled in the art can design such antisense oligonucleotides using conventional methods.

[0078] In some embodiments, the IL-6R inhibitor includes an IL-6R antibody. Antibodies against IL-6R are known in the art and include PM1 (Hirata et al., J Immunol 143, 2900, 1986, incorporated herein by reference), AUK12-20, AUK64-7, AUK146-15 (WO92 / 19759), MRA (US5,888,510), AB-227-NA, and tocilizumab (see, e.g., Hashizume, Rheumat Int 2009, Jul 29, epub, incorporated herein by reference). These antibodies can neutralize IL-6 signaling by binding to either IL-6 or its receptor. Such antibodies can also be prepared via conventional techniques. In some embodiments, the IL-6R antibody is sarilumab (such as those described in Raimondo et al., Drug Des Devel Ther. 2017 May 24;11:1593-1603, incorporated herein by reference).

[0079] In some embodiments, the anti-inflammatory agent includes an NLRP3 inhibitor. In some embodiments, the NLRP3 inhibitor is an antisense oligonucleotide against NLRP3, such as an RNAi molecule like miRNA, siRNA, or shRNA. The nucleic acid sequence of the NLRP3 gene is known. A person skilled in the art can design such antisense oligonucleotides using conventional methods.

[0080] Other NLRP3 inhibitors are described in the art, for example, in Shao et al., Front Pharmacol. 2015; 6: 262, which is incorporated herein by reference. Non-limiting examples of NLRP3 inhibitors include colchicine, CY-09, the ketone metabolite β-hydroxybutyric acid (BHB), type I interferon, resveratrol, arglabin, CB2R, glibencamide, isoliquiritigenin, Z-VAD-FMK, and microRNA-223. Some of the NLRP3 inhibitors described herein, for example, glibencamide, isoliquiritigenin, and Z-VAD-FMK, are commercially available, for example, from Invivogen Inc. (California, USA).

[0081] In some embodiments, the anti-inflammatory agent comprises a TNF inhibitor, such as TNFα. In some embodiments, the TNFα inhibitor is an antisense oligonucleotide against TNFα, such as an RNAi molecule such as miRNA, siRNA, or shRNA. The nucleic acid sequence of the TNFα gene is known. One of ordinary skill in the art can design such antisense oligonucleotides using conventional methods.

[0082] In some embodiments, the TNF inhibitor can act by at least one of downregulating or inhibiting the production of TNF, binding to free TNF, interfering with the binding of TNF to its receptor, and interfering with the regulation of signaling subsequent to the binding of TNF to its receptor. Examples of TNF inhibitors include, without limitation: soluble tumor necrosis factor receptor type I (sTNF-RI; also known as the p55 receptor), soluble tumor necrosis factor receptor type II (also known as the p75 receptor), and solubilized TNF receptors including, but not limited to, Enbrel™; antibodies to TNF including, but not limited to, Remicade™ and D2E7 (e.g., U.S. Pat. Nos. 6,090,382 and 6,258,562); antibodies to TNF receptors; sTNF-RI (see, e.g., WO 98 / 24463), etanercept (Enbrel™), Avakine™; inhibitors of TNF-α converting enzyme (TACE); and other molecules that affect the activity of TNF.

[0083] Exemplary TNF-α inhibitors are described in the art, for example, as follows: European Patent Applications EP 308 378; EP 422 339; EP 393 438; EP 398 327; EP 412 486; EP 418 014, EP 417 563, EP 433 900; EP 464 533; EP 512 528; EP 526 905; EP 568 928; EP 607 776, which describes the use of leflunomide for the inhibition of TNF-α; EP 663 210; EP 542 795; EP 818 439; EP 664 128; EP 542 795; EP 741 707; EP 874 819; EP 882 714; EP 880 970; EP 648 783; EP 731 791; EP 895 988; EP 550 376; EP 882 714; EP 853 083; EP 550 376; EP 943 616; EP 939 121; EP 614 984; EP 853 083; U.S. Patents 5,136,021; 5,929,117; 5,948,638; 5,807,862; 5,695,953; 5,834,435; 5,817,822; 5,830,742; 5,834,435; 5,851,556; 5,853,977; 5,359,037, 5,512,544; 5,695,953; 5,811,261; 5,633,145; 5,863,926; 5,866,616; 5,641,673; 5,869,677; 5,869,511; 5,872,146; 5,854,003; 5,856,161; 5,877,222; 5,877,200; 5,877,151; 5,886,010; 5,869,660; 5,859,207; 5,891,883; 5,877,180; 5,955,480; 5,955,476; 5,955,435; 5,994,351; 5,990,119; 5,952,320; 5,962,481;International Patent Applications WO90 / 13575, WO91 / 03553, WO92 / 01002, WO92 / 13095, WO92 / 16221, WO93 / 07863, WO93 / 21946, WO93M19777, WO95 / 34326, WO96 / 28546, WO98 / 27298, WO98 / 30541, WO96 / 38150, WO96 / 38150, WO97 / 18207, WO97 / 15561, WO97 / 12902, WO96 / 25861, WO96 / 12735, WO96 / 11209, WO98 / 39326, WO98 / 39316, WO98 / 38859, WO98 / 39315, WO98 / 42659, WO98 / 39329, WO98 / 43959, WO98 / 45268, WO98 / 47863, WO96 / 33172, WO96 / 20926, WO97 / 37974, WO97 / 37973, WO97 / 47599, WO96 / 35711, WO98 / 51665, WO98 / 43946, WO95 / 04045, WO98 / 56377, WO97 / 12244, WO99 / 00364, WO99 / 00363, WO98 / 57936, WO99 / 01449, WO99 / 01139, WO98 / 56788, WO98 / 56756, WO98 / 53842, WO98 / 52948, WO98 / 52937, WO99 / 02510, WO97 / 43250, WO99 / 06410, WO99 / 06042, WO99 / 09022, WO99 / 08688, WO99 / 07679, WO99 / 09965, WO99 / 07704, WO99 / 06041, WO99 / 37818, WO99 / 37625, WO97 / 11668, WO99 / 50238, WO99 / 47672, WO99 / 48491; Japanese Patent Applications 10147531, 10231285, 10259140 and 10130149, 10316570, 11001481 and 127,800 / 1991; German Application No. 19731521; and UK Application Nos. 2 218 101, 2 326 881, 2 246 569. The entire disclosure of the foregoing references is hereby incorporated by reference for all purposes.;

[0084] In some embodiments, the TNF inhibitor is a TNF antibody, for example, without limitation, infliximab, adalimumab, certolizumab pegol, and golimumab. In some embodiments, the TNF inhibitor is etanercept (Enbrel). Non-limiting examples of small molecule TNF inhibitors include thalidomide, lenalidomide, pomalidomide, xanthine derivatives, bupropion, 5-HT2A agonist hallucinogens (e.g., (R)-DOI, TCB-2, LSD, and LA-SS-Az).

[0085] In some embodiments, the anti-inflammatory agent includes an IL-8 inhibitor. In some embodiments, the IL-8 inhibitor is an antisense oligonucleotide against IL-8, such as an RNAi molecule such as miRNA, siRNA, or shRNA. The nucleic acid sequence of the IL-8 gene is known. One of ordinary skill in the art can design such antisense oligonucleotides using conventional methods.

[0086] In some embodiments, the IL-8 inhibitor is an antibody against IL-8, for example, without limitation, HuMab-10F8 described in Skov et al., J Immunol. 2008 Jul 1;181(1):669-79, which is incorporated herein by reference. In some embodiments, the IL-8 inhibitor is reparixin, for example, described in Leitner et al., Int J Immunopathol Pharmacol. 2007 Jan-Mar;20(1):25-36, which is incorporated herein by reference. Non-limiting examples of small molecule IL-8 inhibitors include curcumin, antiroquinat, macrolides (e.g., those described in Kohyama et al., Antimicrob. Agents Chemother. April 1999 vol. 43 no. 4 907-911, which is incorporated herein by reference), and trifluoroacetate salts.

[0087] In some embodiments, the anti-inflammatory agent includes an IL-18 inhibitor. In some embodiments, the IL-18 inhibitor is an antisense oligonucleotide against IL-18, such as an RNAi molecule such as miRNA, siRNA or shRNA. The nucleic acid sequence of the IL-18 gene is known. Those skilled in the art can design such antisense oligonucleotides using conventional methods.

[0088] Exemplary IL-18 inhibitors include, but are not limited to: antibodies that bind to IL-18; antibodies that bind to IL-18R; antibodies that bind to IL-18RAcP; IL-18bp; IL-18R fragments (e.g., the solubilized extracellular domain of the IL-18 receptor); peptides that bind to IL-18 and reduce or interfere with its interaction with the IL-18R; peptides that bind to IL-18R and reduce or interfere with its interaction with IL-18 or IL-18RAcP; peptides that bind to IL-18RAcP and reduce or interfere with its interaction with the IL-18R; and small molecules that reduce or interfere with the production of IL-18 or the interaction between any of IL-18, IL-18R and IL-18RAcP.

[0089] Specific IL-18 inhibitors are described, for example, in the following: U.S. Patent No. 5,912,324 issued on July 14, 1994; EP 0 962 531 published on December 8, 1999; EP 712 931 published on November 15, 1994; U.S. Patent No. 5,914,253 issued on July 14, 1994; WO97 / 24441 published on July 10, 1997; U.S. Patent No. 6,060,283 issued on May 9, 2000; EP 850 952 published on December 26, 1996; EP 864 585 published on September 16, 1998; WO98 / 41232 published on September 24, 1998; U.S. Patent No. 6,054,487 issued on April 25, 2000; WO99 / 09063 published on August 14, 1997; WO99 / 22760 published on November 3, 1997; WO99 / 37772 published on January 23, 1998; WO99 / 37773 published on March 20, 1998; EP 0 974 600 published on January 26, 2000; WO00 / 12555 published on March 9, 2000; Japanese Patent Application JP111,399 / 94 published on October 31, 1997; Israeli Patent Application IL121554 A0 published on February 8, 1998, which are incorporated herein by reference.

[0090] In some embodiments, the IL-18 inhibitor is an IL-18 binding protein, such as that described in Dinarello et al., Front Immunol. 2013; 4: 289, which is incorporated herein by reference. In some embodiments, the IL-18 inhibitor is a small molecule such as NSC201631, NSC61610, and NSC80734 described in Krumm et al., Scientific Reports 7, Article number: 483, 2017, which is incorporated herein by reference.

[0091] In some embodiments, the anti-inflammatory agent comprises an inhibitor of natural killer cells. In some embodiments, the inhibitor of natural killer cells is an antibody (e.g., the MKp46 antibody described in Yossef et al., The Journal of Immunology, Vol. 192, No. 1, Supplement 1 (May 2014), which is incorporated herein by reference). In some embodiments, the inhibitor of natural killer cells is a viral major histocompatibility complex (MHC) class I homolog (e.g., those described in Farrell et al., Nature volume 386, pages 510-514, 1997, which is incorporated herein by reference). In some embodiments, the inhibitor of natural killer cells is a dietary lipid (e.g., those described in Yaqoob et al., Immunology Letters, Vol. 41, Nos. 2-3 (July 1994) pages 241-247, which is incorporated herein by reference). One of ordinary skill in the art can select an appropriate inhibitor of natural killer cells.

[0092] In some embodiments, the anti-inflammatory agent comprises any other cytokine inhibitor described in the art, for example, in PCT application publications WO2007075896, WO2008021388, WO2007056016 and WO2007056016, and in U.S. patent application publication US20040033535, which are incorporated herein by reference. In some embodiments, the anti-inflammatory agent comprises methotrexate. In some embodiments, the anti-inflammatory agent comprises an alhalofenate as described, for example, in Poiley et al., Arthritis & Rheumatology, Vol. 68, No. 8 (August 2016) pp2027-2034, which is incorporated herein by reference.

[0093] The methods described herein are methods of combination therapy. An anti-inflammatory agent and a lipid-lowering agent are administered to a subject. A "lipid-lowering agent" refers to an agent that reduces the level of one or more lipids (e.g., by at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or more) in a subject (e.g., a subject having or at risk of developing cardiovascular disease). Examples of lipids whose levels can be reduced by the lipid-lowering agents described herein include, without limitation: cholesterol (e.g., total cholesterol), LDL-C, very low density lipoprotein cholesterol (VLDL-C), non-high density lipoprotein cholesterol (non-HDL-C), and triglycerides. In an important aspect, the lipid is LDL-C. In some aspects, the lipid-lowering agent increases the level of high density lipoprotein cholesterol (HDL-C) in the subject (e.g., by at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, 2-fold, 5-fold, 10-fold, or more).

[0094] Non-limiting examples of lipid-lowering agents include, without limitation, HMG-CoA reductase inhibitors (e.g., statins), proprotein convertase subtilisin / kexin type 9 (PCSK9) inhibitors, other lipid-lowering agents and / or combinations thereof. In some aspects, the lipid-lowering agent of the disclosure comprises an HMG-CoA reductase inhibitor. By reducing the amount of cholesterol synthesized by cells through inhibition of the HMG-CoA reductase gene, a cycle of events is initiated that maximizes the uptake of LDL-C by hepatocytes. As the uptake of LDL-C increases, the levels of total cholesterol and LDL-C in the blood decrease.

[0095] In some embodiments, the HMG-CoA reductase inhibitor is a statin. Non-limiting examples of statins include: simvastatin (Zocor), lovastatin (Mevacor), pravastatin (Pravachol), fluvastatin (Lescol), atorvastatin (Lipitor), cerivastatin (Baycol), rosuvastatin (Crestor), pitavastatin, and many others described below: U.S. Patent No. 4,444,784, U.S. Patent No. 4,231,938, U.S. Patent No. 4,346,227, U.S. Patent No. 4,739,073, U.S. Patent No. 5,273,995, U.S. Patent No. 5,622,985, U.S. Patent No. 5,135,935, U.S. Patent No. 5,356,896, U.S. Patent No. 4,920,109, U.S. Patent No. 5,286,895, U.S. Patent No. 5,262,435, U.S. Patent No. 5,260,332, U.S. Patent No. 5,317,031, U.S. Patent No. 5,283,256, U.S. Patent No. 5,256,689, U.S. Patent No. 5,182,298, U.S. Patent No. 5,369,125, U.S. Patent No. 5,302,604, U.S. Patent No. 5,166,171, U.S. Patent No. 5,202,327, U.S. Patent No. 5,276,021, U.S. Patent No. 5,196,440, U.S. Patent No. 5,091,386, U.S. Patent No. 5,091,378, U.S. Patent No. 4,904,646, U.S. Patent No. 5,385,932, U.S. Patent No. 5,250,435, U.S. Patent No. 5,132,312, U.S. Patent No. 5,130,306, U.S. Patent No. 5,116,870, U.S. Patent No. 5,112,857, U.S. Patent No. 5,102,911, U.S. Patent No. 5,098,931, U.S. Patent No. 5,081,136, U.S. Patent No. 5,025,000, U.S. Patent No. 5,021,453, U.S. Patent No. 5,017,716, U.S. Patent No. 5,001,144, U.S. Patent No. 5,001,128, U.S. Patent No. 4,997,837, U.S. Patent No. 4,996,234, U.S. Patent No. 4,994,494, U.S. Patent No. 4,992,429, U.S. Patent No. 4,970,231, U.S. Patent No. 4,968,693, U.S. Patent No. 4,963,538, U.S. Patent No. 4,957,940, U.S. Patent No. 4,950,675, U.S. Patent No. 4,946,864, U.S. Patent No. 4,U.S. Patent Nos. 946,860; 4,940,800; 4,940,727; 4,939,143; 4,929,620; 4,923,861; 4,906,657; 4,906,624; and 4,897,402.

[0096] Non-limiting examples of statins already approved for use in humans include atorvastatin, cerivastatin, fluvastatin, pravastatin, simvastatin, and rosuvastatin. HMG-CoA reductase inhibitors are also described in Drugs and Therapy Perspectives (May 12, 1997), 9: 1-6; Chong (1997) Pharmacotherapy 17:1 157- 1177; Kellick (1997) Formulary 32: 352; Kathawala (1991) Medicinal Research Reviews, 11 : 121-146; Jahng (1995) Drugs of the Future 20: 387-404, and Current Opinion in Lipidology, (1997), 8, 362-368, each of which is incorporated herein by reference. Another notable statin drug is compound 3a(S-4522) described in Watanabe (1997) Bioorganic and Medicinal Chemistry 5: 437-444, which is incorporated herein by reference.

[0097] In some embodiments, the lipid-lowering agent includes a proprotein convertase subtilisin / kexin type 9 (PCSK9) inhibitor. "Proprotein convertase subtilisin / kexin type 9 (PCSK9)" is an enzyme encoded by the PCSK9 gene in humans. PCSK9 binds to the receptor for low-density lipoprotein (LDL) particles. In the liver, the LDL receptor removes LDL particles from the blood through the endocytosis pathway. When PCSK9 binds to the LDL receptor, the receptor channels open towards the lysosomal pathway and is degraded by proteolytic enzymes, limiting the number of times a given LDL receptor can take up LDL particles from the blood. By inhibiting the level or activity of PCSK9, more LDL receptors are recycled and present on the surface of hepatocytes, removing more LDL cholesterol from the blood, which in turn lowers blood cholesterol levels.

[0098] A variety of therapeutic approaches to the inhibition of PSCK9 have been proposed, including: gene silencing agents, such as inhibition of PSCK9 synthesis by RNAi; inhibition of the binding of PCSK9 to LDL-R by monoclonal antibodies, small molecule peptides or adnectins; and inhibition of PCSK9 autocatalytic processing by small molecule inhibitors. These strategies are described in Hedrick et al., Curr Opin Investig Drugs 2009;10:938-46; Hooper et al., Expert Opin Biol Ther, 2013;13:429-35; Rhainds et al., Clin Lipid, 2012;7:621-40; Seidah et al;, Expert Opin Ther Targets 2009;13:19-28; and Seidah et al., Nat Rev Drug Discov 2012;11:367-83, each of which is incorporated herein by reference.

[0099] The term "PCSK9 inhibitor" refers to an agent that reduces the level or activity of PCSK9 (e.g., in a subject). In some embodiments, the PCSK9 inhibitor reduces the expression of PCSK9 (e.g., by at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or 100%). In some embodiments, the PCSK9 inhibitor reduces the activity of PCSK9 (e.g., by at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or 100%). In some embodiments, the PCSK9 inhibitor is selected from the group consisting of: natural PCSK9 inhibitors, PCSK9 antibodies, antisense nucleic acids, peptide inhibitors, PCSK9 vaccines, and small molecule inhibitors.

[0100] In some embodiments, the PCSK9 inhibitor is a natural PCSK9 inhibitor. A "natural PCSK9 inhibitor" refers to a molecule that exists in nature (e.g., in plants or in mammals) and has inhibitory activity against PCSK9. For example, berberine, a plant alkaloid, inhibits the transcription of the PCSK9 gene in immortalized human hepatocytes in vitro (e.g., as described in Li et al., The Journal of Biological Chemistry. 284 (42): 28885-95, 2009, which is incorporated herein by reference) and reduces serum PCSK9 in vivo in mice and hamsters (e.g., as described in Dong et al., The Journal of Biological Chemistry. 290 (7): 4047-58, 2015, which is incorporated herein by reference). In another example, annexin A2, an endogenous protein, inhibits the activity of PCSK9 (e.g., as described in Seidah et al., PLoS ONE. 7 (7): e41865, 2012, which is incorporated herein by reference). In some embodiments, the PCSK9 inhibitor is adnectin (BMS-962476, as described in Mitchell et al., J Pharmacol Exp Ther. 2014 Aug;350(2):412-24, which is incorporated herein by reference).

[0101] In some embodiments, the PCSK9 inhibitor is a PCSK9 antibody. Non-limiting examples of PCSK9 antibodies include: alirocumab (Praluent (registered trademark) as described in Robinson et al., N Engl J Med 2015; 372:1489-1499, 2015, incorporated herein by reference), evolocumab (Repatha (registered trademark) as described in Sabatine et al., N Engl J Med 2017; 376:1713-1722, 2017, incorporated herein by reference), 1D05-IgG2 (e.g., as described in Ni et al., J Lipid Res. 2011 Jan;52(1):78-86m, incorporated herein by reference), RG-7652 (e.g., as described in Baruch et al., Am J Cardiol. 2017 May 15;119(10):1576-1583, incorporated herein by reference), LY3015014 (e.g., as described in Eur Heart J. 2016 May 1;37(17):1360-9, incorporated herein by reference), and bococizumab (e.g., as described in Ridker et al., N Engl J Med 2017; 376:1527-1539, incorporated herein by reference). The examples described herein are not intended to be limiting. Any PCSK9 antibody that inhibits its activity can be used in accordance with the present disclosure.

[0102] In some embodiments, the PCSK9 inhibitor is an antisense nucleic acid. In some embodiments, the antisense nucleic acid is an RNAi molecule (a molecule based on microRNA, siRNA, shRNA, dsRNA, and other small interfering nucleic acids known in the art). The nucleic acid sequence of PCSK9 is known in the art (e.g., human PCSK9, NCBI Gene ID: 255738). Those skilled in the art are proficient in methods for making and using antisense nucleic acids that target the PCSK9 gene. In some embodiments, the RNAi molecule that inhibits the expression of PCSK9 is inclisiran (e.g., as described in Ray et al., N Engl J Med 2017; 376:1430-1440, which is incorporated herein by reference) or ALN-PCS (e.g., as described in Fitzgerald et al., N Engl J Med. 2017 Jan 5;376(1):41-51, which is incorporated herein by reference).

[0103] In some embodiments, the PCSK9 inhibitor is a peptide inhibitor. In some embodiments, the peptide inhibitor is a peptide that mimics the EGFa domain of the low-density lipoprotein receptor (LDL-R) (e.g., as described in Kwon et al., PNAS, February 2008, 105 (6) 1820-1825; and Schroeder et al., Chemistry & Biology, Volume 21, Number 2, February 20, 2014, pages 284-294, which are incorporated herein by reference). In some embodiments, the peptide inhibitor is Pep2-8 as described in Zhang et al., The Journal of Biological Chemistry 289, 942-955, which is incorporated herein by reference).

[0104] In some embodiments, the PCSK9 inhibitor is a small molecule. In some embodiments, the small molecule PCSK9 inhibitor is PF-06446846 (e.g., as described in Lintner et al., PLoS Biol 15(3): e2001882, which is incorporated herein by reference). In some embodiments, the small molecule PCSK9 inhibitor is an inhibitor of cholesteryl ester transfer protein (CETP), such as anacetrapib (e.g., as described in Barter et al., J Lipid Res. 2015 Nov; 56(11): 2045-2047, which is incorporated herein by reference) or K-312 (e.g., as described in Miyosawa et al., Am J Physiol Endocrinol Metab. 2015 Jul 15;309(2):E177-90, which is incorporated herein by reference). Other examples of small molecule PCSK9 inhibitors are described in Petersen et al., Cell Chemical Biology, Volume 23, Issue 11, p1362-1371 (2016) and Halford et al., Chemical & Engineering News, Volume 94, Issue 44|p.12 (2016), which are incorporated herein by reference).

[0105] In some embodiments, the PCSK9 inhibitor is a PCSK9 vaccine. In some embodiments, the PCSK9 vaccine comprises an antigenic peptide from PCSK9. For example, the PCSK9 vaccine may be the AT04A vaccine described in Landlinger et al. (European Heart Journal, Volume 38, Issue 32, (August 21, 2017), pages 2499-2507, which is incorporated herein by reference). In some embodiments, the PCSK9 vaccine may be a virus-like particle peptide vaccine (e.g., the PCSK9Qβ-003 vaccine described in Pan et al., Scientific Reports volume 7, Article number: 12534 (2017), which is incorporated herein by reference).

[0106] Any other known PCSK9 inhibition strategy can be used in accordance with the present disclosure. For example, the PCSK9 gene can be modified to yield a non-functional PCSK9 variant in a subject, thereby inhibiting its activity. A number of PCSK9 variants are described, for example, in PCT Publication Nos. WO2001031007, WO2001057081, WO2002014358, WO2001098468, WO2002102993, WO2002102994, WO2002046383, WO2002090526, WO2001077137, and WO2001034768; U.S. Publication Nos. US2004 / 0009553 and US2003 / 0119038, as well as European Publication Nos. EP 1 440 981, EP 1 067 182, and EP 1 471 15, each of which is incorporated herein by reference.

[0107] Several mutant forms of PCSK9, including S127R, N157K, F216L, R218S, and D374Y, are well characterized, and S127R, F216L, and D374Y have been associated with autosomal dominant hypercholesterolemia (ADH). See Benjannet et al. (J. Biol. Chem., 279(47):48865-48875 (2004)); Rashid et al., PNAS, 102(15):5374-5379 (2005); Abifadel et al., 2003 Nature Genetics 34:154-156; Timms et al., 2004 Hum. Genet. 114:349-353; Leren, 2004 Clin. Genet. 65:419-422; Cohen et al., 2006 N. Engl. J. Med. 354:1264-1272; Lalanne et al. (J. Lipid Research, 46:1312-1319 (2005)); each of which is incorporated herein by reference.

[0108] In some embodiments, the lipid-lowering agent comprises one or more (e.g., 1, 2, 3, or more) HMG-CoA reductase inhibitors (e.g., statins) and one or more (e.g., 1, 2, 3, or more) PCSK9 inhibitors known in the art or described herein. For example, the lipid-lowering agent may comprise one or more (e.g., 1, 2, 3, or more) of simvastatin, lovastatin, pravastatin, fluvastatin, atorvastatin, cerivastatin, rosuvastatin, and pitavastatin, and one or more (e.g., 1, 2, 3, or more) of berberine, annexin A2, adiponectin, PF-06446846, anacetrapib, K-312, alirocumab, evolocumab, 1D05-IgG2, RG-7652, LY3015014, bococizumab, inclisiran, ALN-PCS, and PCSK9 vaccine. All possible combinations are contemplated herein.

[0109] In some embodiments, the lipid-lowering agents described herein further comprise one or more of other agents having a lipid-lowering effect, such as, without limitation: fibrate derivatives (fibric acid derivatives), bile acid sequestrants or resins, nicotinic acid agents, cholesterol absorption inhibitors, acyl coenzyme A: cholesterol acyltransferase (ACAT) inhibitors, cholesteryl ester transfer protein (CETP) inhibitors, LDL receptor antagonists, farnesoid X receptor (FXR) antagonists, SREBP cleavage-activating protein (SCAP) activators, microsomal triglyceride transfer protein (MTP) inhibitors, squalene synthase inhibitors, and peroxisome proliferator-activated receptor (PPAR) agonists.

[0110] Non-limiting examples of fibrate derivatives include the following: gemfibrozil (Lopid), fenofibrate (Tricor), clofibrate (Atromid), and bezafibrate. Non-limiting examples of bile acid sequestrants or resins include the following: colesevelam (WelChol), cholestyramine (Questran or Prevalite), and colestipol (Colestid), DMD-504, GT-102279, HBS-107, and S-8921. Non-limiting examples of nicotinic acid agents include niacin and probucol. Examples of cholesterol absorption inhibitors include, but are not limited to, ezetimibe (Zetia). Non-limiting examples of ACAT inhibitors include avasimibe, CI-976 (Parke Davis), CP-113818 (Pfizer), PD-138142-15 (Parke Davis), F1394, and numerous others described in U.S. Patent Nos. 6,204,278, 6,165,984, 6,127,403, 6,063,806, 6,040,339, 5,880,147, 5,621,010, 5,597,835, 5,576,335, 5,321,031, 5,238,935, 5,180,717, 5,149,709, and 5,124,337.Non-limiting examples of CETP inhibitors include torcetrapib, CP-529414, CETi-I, JTT-705, as well as those described in U.S. Patent Nos. 6,727,277, 6,723,753, 6,723,752, 6,710,089, 6,699,898, 6,696,472, 6,696,435, 6,683,099, 6,677,382, 6,677,380, 6,677,379, 6,677,375, 6,677,353, 6,677,341, 6,605,624, 6,586,448, 6,521,607, 6,482,862, 6,479,552, 6,476,075, 6,476,057, 6,462,092, 6,458,852, 6,458,851, 6,458,850, 6,458,849, 6,458,803, 6,455,519, 6,451,830, 6,451,823, 6,448,295, 5,512,548, and many others. One non-limiting example of an FXR antagonist is guggulsterone. One non-limiting example of an SCAP activator is GW532 (GlaxoSmithKline). Non-limiting examples of MTP inhibitors include Implitapide and R-103757. One non-limiting example of a squalene synthase inhibitor is zaragozic acid. Non-limiting examples of PPAR agonists include GW-409544, GW-501516, and LY-510929.

[0111] In some embodiments, a method of treating cardiovascular disease is further combined with other treatments for reducing the risk of future cardiovascular events, such as, without limitation, diet, and / or exercise, and / or treatment with antihyperlipidemic agents, anti-inflammatory agents, antithrombotic agents, thrombolytic agents, antiplatelet agents, direct thrombin inhibitors, glycoprotein Ilb / IIIa receptor inhibitors, agents that bind to cell adhesion molecules and inhibit the ability of leukocytes to adhere to molecules involved (e.g., anti-cell adhesion molecule antibodies), alpha-adrenergic receptor blockers, beta-adrenergic receptor blockers, cyclooxygenase-2 inhibitors, angiotensin system inhibitors, antiarrhythmic agents, calcium channel blockers, diuretics, inotropic agents, vasodilators, pressor agents, thiazolidinediones, cannabinoid-1 receptor blockers, and / or any combination thereof.

[0112] In some aspects, the present disclosure provides a strategy for treating cardiovascular disease by reducing inflammation and simultaneously lowering lipid levels using a bispecific antibody that targets both pro-inflammatory cytokines and PCSK9. In some embodiments, the method comprises administering to a subject in need thereof a therapeutically effective amount of a bispecific antibody comprising a first antigen-binding domain that binds to a pro-inflammatory cytokine and a second antigen-binding domain that binds to PCSK9.

[0113] A "bispecific antibody" is an antibody that has dual antigen-binding specificities. A bispecific antibody can be formed by linking two antigen-binding domains with different binding specificities. Thus, a bispecific antibody comprises a first antigen-binding domain that binds to a first antigen and a second antigen-binding domain that binds to a second antigen different from the first antigen.

[0114] An "antigen-binding domain" is also referred to herein as an "antigen-binding fragment" or "antigen-binding portion" and refers to a polypeptide having a binding affinity specific for an epitope of an antigen. In some embodiments, such a polypeptide is encoded by an immunoglobulin gene. Non-limiting examples of immunoglobulin genes include kappa, lambda, alpha, gamma, delta, epsilon, and mu constant region genes, as well as numerous immunoglobulin variable region genes. Immunoglobulins can exist in a variety of forms including, in addition to antibodies; for example, Fv, Fab, and F(ab)2, as well as single-chain (e.g., those described in Huston, et al., Proc. Nat. Acad. Sci. U.S.A., 85:5879-5883 (1988) and Bird, et al., Science, 242:423-426 (1988), which are incorporated herein by reference). Other examples of antigen-binding domains include the CD4 protein that binds to epitopes on T cell antigen receptors and MHC proteins. In addition to the naturally occurring forms of immunoglobulin chains, antigen-binding domains can be designed and produced using a variety of recombinant DNA techniques well known to those of skill in the art.

[0115] Bispecific antibodies may exist in a variety of forms. In some embodiments, the bispecific antibody is an Ig-G-like molecule. That is, the bispecific antibody comprises a first antigen-binding domain, a second antigen-binding domain, and a common fragment crystallizable region (Fc region). In some embodiments, the bispecific antibody is a monoclonal bispecific antibody. A monoclonal bispecific antibody retains the classical monoclonal antibody (mAb) structure of two antigen-binding domains and one Fc region, with the exception that the two antigen-binding domains bind to different antigens. The most common type of monoclonal bispecific antibody has three unique binding sites on the antibody: two Fab regions and the Fc region, and is thus referred to as a trifunctional antibody. Each antigen-binding domain (e.g., a pair of heavy and light chains) of the monoclonal bispecific antibody is derived from a unique monoclonal antibody. The Fc region, made from two heavy chains, forms a third binding site that binds to cell surface Fc receptors. These bispecific monoclonal antibodies are often produced using the quadroma, or hybrid hybridoma method.

[0116] In some embodiments, the bispecific antibody is non-IgG-like. There are other bispecific antibodies that completely lack the Fc region. Non-IgG-like bispecific antibodies include chemically linked Fabs consisting only of Fab regions, various types of bivalent and trivalent single-chain variable fragments (scFvs), and fusion proteins that mimic the variable domains of two antibodies. An example of a non-IgG-like bispecific antibody is a bispecific T-cell engager (BiTE), for example, as described in Yang et al, International Journal of Molecular Sciences. 18 (1): 48, 2016; Baeuerle et al., Cancer Res. 69 (12): 4941-4944, 2009; and Wozniak-Knopp et al., Protein Engineering Design and Selection. 23 (4): 289-297, 2010, which are incorporated herein by reference.

[0117] Bispecific antibodies can be generated by a variety of methods known to those of skill in the art. The two antigen-binding domains of the bispecific antibody may be derived from an antibody against a pro-inflammatory cytokine or from an antibody against PCSK9. "Derive from" means using the antigen-binding domain of an antibody against a pro-inflammatory cytokine described herein as the first antigen-binding domain of the bispecific antibody and using the antigen-binding domain of a PCSK9 antibody as the second antigen-binding domain of the bispecific antibody. The two antigen-binding domains can be attached to each other by chemical cross-linking through a pair of epitopes that interact with each other (e.g., leucine zipper), by hybrid-hybridoma (Milstein and Cuello, (1984) Immunol. Today 5:299) or transfectoma, or by disulfide exchange in the hinge region. Those of skill in the art are familiar with methods for generating bispecific antibodies.

[0118] In some embodiments, the pro-inflammatory cytokine targeted by the first antigen-binding domain is a pro-inflammatory cytokine described herein, such as, without limitation, any of IL-1, IL-1 receptor (IL-1R), IL-6, IL-6 receptor (IL-6R), NLRP3, TNF, IL-8, or IL-18.

[0119] In some embodiments, the bispecific antibody comprises a first antigen-binding domain that binds to IL-1 (e.g., IL-1α or IL-1β) and a second antigen-binding domain that binds to PCSK9. In some embodiments, the first antigen-binding domain binds to IL-1α. In some embodiments, the first antigen-binding domain is derived from an IL-1α antibody (e.g., without limitation, MABp1). In some embodiments, the first antigen-binding domain binds to IL-1β. In some embodiments, the first antigen-binding domain is derived from an IL-1β antibody (e.g., without limitation, canakinumab, gevokizumab, diacerein, or LY2189102).

[0120] In some embodiments, the first antigen-binding domain binds to IL-1R. In some embodiments, the first antigen-binding domain is derived from an IL-1R antibody (e.g., without limitation, MEDI-8968 or AMG108).

[0121] In some embodiments, the first antigen-binding domain binds to IL-6. In some embodiments, the first antigen-binding domain is derived from an IL-6 antibody (e.g., without limitation, siltuximab, sirukumab, clazakizumab, olokizumab, or elsilimomab).

[0122] In some embodiments, the first antigen-binding domain binds to IL-6R. In some embodiments, the first antigen-binding domain is derived from an IL-6R antibody (e.g., without limitation, tocilizumab, sarilumab, PM1, AUK12-20, AUK64-7, AUK146-15, or AB-227-NA).

[0123] In some embodiments, the first antigen-binding domain binds to NLRP3. In some embodiments, the first antigen-binding domain is derived from an NLRP3 antibody.

[0124] In some embodiments, the first antigen-binding domain binds to TNF. In some embodiments, the first antigen-binding domain is derived from a TNF antibody (e.g., without limitation, infliximab, adalimumab, certolizumab pegol, golimumab, or etanercept (Enbrel)).

[0125] In some embodiments, the first antigen-binding domain binds to IL-8. In some embodiments, the first antigen-binding domain is derived from an IL-8 antibody (e.g., without limitation, HuMab-10F8).

[0126] In some embodiments, the first antigen-binding domain binds to IL-18. In some embodiments, the first antigen-binding domain is derived from an IL-18 antibody.

[0127] In some embodiments, the second antigen-binding domain is derived from a PCSK9 antibody (e.g., without limitation, alirocumab, evolocumab, 1D05-IgG2, RG-7652, LY3015014, or bococizumab).

[0128] In some embodiments, the subject may further be administered a therapeutically effective amount of an HMG-CoA reductase inhibitor in addition to the bispecific antibodies described herein. In some embodiments, the HMG-CoA reductase inhibitor is a statin (e.g., but not limited to, simvastatin, lovastatin, pravastatin, fluvastatin, atorvastatin, cerivastatin, rosuvastatin, or pitavastatin). In some embodiments, in a subject treated with the lipid-lowering agent and anti-inflammatory agent described herein, the level or activity of a pro-inflammatory cytokine is decreased as compared to before the treatment. "Decreasing the level or activity of a pro-inflammatory cytokine" means that when the composition is administered to a subject, the level or activity of a cytokine (e.g., IL-1, IL-6, TNF, IL-8, or IL-18) is decreased by at least 20%, more lower, as compared to without the composition. For example, the level or activity of a cytokine (e.g., IL-1, IL-6, TNF, IL-8, or IL-18) may be decreased by at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or 100%, more lower, in a subject treated with the lipid-lowering agent and anti-inflammatory agent described herein as compared to before the treatment. In some embodiments, the level or activity of a cytokine (e.g., IL-1, IL-6, TNF, IL-8, or IL-18) is decreased by 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100% in a subject treated with the lipid-lowering agent and anti-inflammatory agent described herein as compared to before the treatment. The activity of a pro-inflammatory cytokine can be reflected in the degree of a signaling pathway. One of ordinary skill in the art can evaluate the activity of a pro-inflammatory cytokine using conventional methods.

[0129] In some embodiments, the level or activity of C-reactive protein is reduced in a subject treated with a lipid-lowering agent and an anti-inflammatory agent as described herein, compared to before the treatment. "C-reactive protein (CRP)" is a substance produced by the liver that increases in the presence of inflammation in the body. Elevated C-reactive protein levels are identified by a blood test and are considered a non-specific "marker" of disease.

[0130] In some embodiments, a subject having or at risk of developing cardiovascular disease has a CRP level that is at least 20% higher than that of a control subject. For example, a subject having or at risk of developing cardiovascular disease may have a CRP level that is at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 2-fold, at least 5-fold, at least 10-fold, at least 100-fold, or at least 1000-fold higher than that of a control subject. In some embodiments, a subject having or at risk of developing cardiovascular disease has a CRP level that is 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 2-fold, 5-fold, 10-fold, 100-fold, or 1000-fold higher than that of a control subject. In some embodiments, the control subject is a healthy subject.

[0131] "Reducing the level or activity of CRP" means that the level or activity of CRP is reduced by at least 20% in a subject treated with the lipid-lowering agent and anti-inflammatory agent described herein, compared to before the treatment. For example, the level or activity of CRP can be reduced by at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or 100% in a subject treated with the lipid-lowering agent and anti-inflammatory agent described herein, compared to before the treatment. In some embodiments, the level or activity of CRP is reduced by 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100% in a subject treated with the lipid-lowering agent and anti-inflammatory agent described herein, compared to before the treatment.

[0132] In some embodiments, the level or activity of one or more lipids (e.g., one or more of non-HDL-C, LDL-C, VLDL-C, total cholesterol, and triglycerides) is reduced in a subject treated with the lipid-lowering and anti-inflammatory agents described herein, compared to before the treatment. "Reducing the level or activity of one or more lipids" means that the level or activity of one or more lipids (e.g., one or more of non-HDL-C, LDL-C, VLDL-C, total cholesterol, and triglycerides) is reduced by at least 20% lower when the composition is administered to the subject, compared to without the composition. For example, the level or activity of one or more lipids (e.g., one or more of non-HDL-C, LDL-C, VLDL-C, total cholesterol, and triglycerides) can be reduced by at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or 100% lower in a subject treated with the lipid-lowering and anti-inflammatory agents described herein, compared to before the treatment. In some embodiments, the level or activity of one or more lipids (e.g., one or more of non-HDL-C, LDL-C, VLDL-C, total cholesterol, and triglycerides) is reduced by 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100% in a subject treated with the lipid-lowering and anti-inflammatory agents described herein, compared to before the treatment. One of ordinary skill in the art can evaluate the activity of lipids using conventional methods.

[0133] In some embodiments, the level or activity of apolipoprotein B (ApoB) is reduced in a subject treated with the lipid-lowering and anti-inflammatory agents described herein, as compared to before the treatment. "Reducing the level or activity of apolipoprotein B (ApoB)" means that the level or activity of ApoB is reduced by at least 20%, lower, when the composition is administered to the subject, as compared to without the composition. For example, the level or activity of ApoB can be reduced by at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or 100%, lower in a subject treated with the lipid-lowering and anti-inflammatory agents described herein, as compared to before the treatment. In some embodiments, the level or activity of ApoB is reduced by 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100%, in a subject treated with the lipid-lowering and anti-inflammatory agents described herein, as compared to before the treatment. One of ordinary skill in the art can evaluate the activity of ApoB using conventional methods, such as immunostaining or Western blotting.

[0134] In some embodiments, the ratio of total cholesterol to HDL-C decreases in a subject treated with the lipid-lowering and anti-inflammatory agents described herein as compared to before the treatment. "Decreasing the ratio of total cholesterol to HDL-C" means that the ratio of total cholesterol to HDL-C decreases by at least 20%, lower, when the composition is administered to the subject as compared to without the composition. For example, the ratio of total cholesterol to HDL-C can decrease by at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or 100%, lower in a subject treated with the lipid-lowering and anti-inflammatory agents described herein as compared to before the treatment. In some embodiments, the ratio of total cholesterol to HDL-C decreases by 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100% in a subject treated with the lipid-lowering and anti-inflammatory agents described herein as compared to before the treatment.

[0135] In some embodiments, the occurrence of non - lethal myocardial infarction and / or cardiovascular mortality rate is reduced in subjects treated with the lipid - lowering agents and anti - inflammatory agents described herein, compared to before the treatment. "Reducing the occurrence of non - lethal myocardial infarction and / or cardiovascular mortality rate" means that the occurrence of non - lethal myocardial infarction and / or cardiovascular mortality rate is reduced by at least 20% lower, compared to without the composition, when the composition is administered to a subject. For example, the occurrence of non - lethal myocardial infarction and / or cardiovascular mortality rate can be reduced by at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or 100% lower in subjects treated with the lipid - lowering agents and anti - inflammatory agents described herein, compared to before the treatment. In some embodiments, the occurrence of non - lethal myocardial infarction and / or cardiovascular mortality rate is reduced by 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100% in subjects treated with the lipid - lowering agents and anti - inflammatory agents described herein, compared to before the treatment. In some embodiments, the incidence of non-lethal stroke is reduced in a subject treated with the lipid-lowering agent and anti-inflammatory agent described herein as compared to before the treatment. "Reducing the incidence of non-lethal stroke" means that the incidence of non-lethal stroke is reduced by at least 20%, lower, when the composition is administered to a subject as compared to without the composition. For example, the incidence of non-lethal stroke can be reduced by at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or 100%, lower in a subject treated with the lipid-lowering agent and anti-inflammatory agent described herein as compared to before the treatment. In some embodiments, the incidence of non-lethal stroke is reduced by 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100% in a subject treated with the lipid-lowering agent and anti-inflammatory agent described herein as compared to before the treatment.

[0136] In some embodiments, the lipid-lowering agent and anti-inflammatory agent are administered together (e.g., in the same composition). In some embodiments, the lipid-lowering agent and anti-inflammatory agent are administered separately (e.g., sequentially). For example, in some embodiments, the lipid-lowering agent is administered first and the anti-inflammatory agent is administered second. In some embodiments, the anti-inflammatory agent is administered first and the lipid-lowering agent is administered second.

[0137] In some embodiments, the lipid-lowering agent and / or anti-inflammatory agent is formulated in one or more compositions for administration to a subject. In some embodiments, the composition is a pharmaceutical composition. In some embodiments, the composition further comprises a pharmaceutically acceptable carrier. The pharmaceutical composition may further comprise additional agents (e.g., for specific delivery, for extending the half-life, or other therapeutic agents). As used herein, the term “pharmaceutically acceptable carrier” means a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, manufacturing aid (e.g., lubricant, talc, magnesium stearate, calcium stearate or zinc stearate, or stearic acid), or a solvent encapsulating material involved in carrying or transporting the composition containing the anti-inflammatory agent from one part of the body (e.g., the delivery site) to another part (e.g., an organ, tissue, or part of the body). A pharmaceutically acceptable carrier is “acceptable” in the sense of being compatible with the other ingredients of the formulation and not injurious to the tissues of the subject (e.g., physiologically compatible, sterile, physiological pH, etc.).Some examples of materials that can serve as pharmaceutically acceptable carriers are as follows: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose and its derivatives, such as sodium carboxymethyl cellulose, methyl cellulose, ethyl cellulose, microcrystalline cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) lubricants, such as magnesium stearate, sodium lauryl sulfate and talc; (8) excipients, such as cocoa butter and suppository waxes; (9) fats and oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol, and polyethylene glycol (PEG); (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) pH buffering solutions; (21) polyesters, polycarbonates, and / or polyanhydrides; (22) bulking agents, such as polypeptides and amino acids; (23) serum components, such as serum albumin, HDL and LDL; (22) C2-C12 alcohols, such as ethanol; and (23) other non-toxic compatible substances used in pharmaceutical formulations. Wetting agents, coloring agents, release agents, coating agents, sweetening agents, flavoring agents, fragrances, preservatives and antioxidants may also be present in the formulation. Terms such as "excipient", "carrier", "pharmaceutically acceptable carrier" and the like are used interchangeably herein.

[0138] In some embodiments, a composition comprising an anti-inflammatory agent of the present disclosure is administered by injection, by catheter, by suppository, or by implant, and the implant is of a membrane such as a silicone membrane, or of a porous, non-porous, gelatinous material containing fibers. Typically, when administering the composition, a material in which the composition comprising the anti-inflammatory agent of the present disclosure is not absorbed is used.

[0139] In other embodiments, a composition comprising a lipid-lowering agent and / or an anti-inflammatory agent is delivered in a controlled release system. In one embodiment, a pump may be used (see, e.g., Langer, 1990, Science 249:1527-1533; Sefton, 1989, CRC Crit. Ref. Biomed. Eng. 14:201; Buchwald et al., 1980, Surgery 88:507; Saudek et al., 1989, N. Engl. J. Med. 321:574). In another embodiment, a polymeric material may be used. (See, e.g., Medical Applications of Controlled Release (ed. Langer and Wise, CRC Press, Boca Raton, Fla., 1974); Controlled Drug Bioavailability, Drug Product Design and Performance (ed. Smolen and Ball, Wiley, New York, 1984); Ranger and Peppas, 1983, Macromol. Sci. Rev. Macromol. Chem. 23:61; also see Levy et al., 1985, Science 228:190; During et al., 1989, Ann. Neurol. 25:351; Howard et al., 1989, J. Neurosurg. 71:105). Other controlled release systems are discussed, for example, in Langer supra.

[0140] In some embodiments, the pharmaceutical composition is formulated according to conventional procedures as a pharmaceutical composition adapted for intravenous or subcutaneous administration to a subject, e.g., a human. Typically, the composition for administration by injection is a solution in a sterile isotonic aqueous buffer. If necessary, the pharmaceutical may also contain solubilizing agents and local anesthetics such as lidocaine to relieve pain at the site of injection. Generally, the components are supplied separately or mixed together as a dry lyophilized powder or an anhydrous concentrate in a unit dosage form, e.g., in an airtight sealed container such as an ampoule or sachet indicating the amount of the active agent. When the pharmaceutical is administered by infusion, it can be dispensed by an infusion bottle containing sterile pharmaceutical grade water or saline. When the pharmaceutical is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the components can be mixed before administration.

[0141] The pharmaceutical composition for systemic administration may be a liquid, e.g., sterile saline, Ringer's lactate or Hank's solution. Additionally, the pharmaceutical composition may be in solid form and may be redissolved or suspended immediately prior to use. Lyophilized forms are also contemplated.

[0142] The pharmaceutical composition may be contained in lipid particles or vehicles such as liposomes or microcrystals, which are also suitable for parenteral administration. The particles may be of any suitable structure, such as monolayer or multilayer, as long as the composition is contained therein. Compositions containing lipid-lowering agents and / or anti-inflammatory agents may be encapsulated in "stabilized plasmid-lipid particles" (SPLP) containing dioleoylphosphatidylethanolamine (DOPE), a membrane-fusogenic lipid, and a low level (5-10 mol%) of cationic lipid, and may be stabilized by polyethylene glycol (PEG) coating (Zhang Y. P. et al., Gene Ther. 1999, 6:1438-47). Positively charged lipids such as N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethyl-ammonium methyl sulfate, or "DOTAP", are particularly preferred for such particles and vehicles. The preparation of such lipid particles is well known. See, for example, U.S. Patent Nos. 4,880,635; 4,906,477; 4,911,928; 4,917,951; 4,920,016; and 4,921,757.

[0143] The pharmaceutical compositions of the present disclosure may be administered or packaged, for example, as unit dosages. The term "unit dosage", as used in reference to the pharmaceutical compositions of the present disclosure, refers to physically discrete units suitable as unitary dosages for a subject, each unit containing a predetermined quantity of the active material calculated to produce the desired therapeutic effect in association with the required diluent; i.e., carrier or vehicle.

[0144] In some embodiments, the pharmaceutical composition can be provided as a pharmaceutical kit that includes (a) a container containing the composition comprising the anti-inflammatory agent of the present disclosure in lyophilized form, and (b) a second container containing a pharmaceutically acceptable diluent (e.g., sterile water) for injection. The pharmaceutically acceptable diluent can be used for reconstitution or dilution of the lyophilized composition of the present disclosure. Optionally, the container(s) may be combined with a notice in a form prescribed by a government agency that regulates the manufacture, use, or sale of pharmaceuticals or biologic products, reflecting the approval by the agency for manufacture, use, or sale for administration to humans.

[0145] In some embodiments, a product is included that contains materials useful for treating the above-mentioned diseases. In some embodiments, the product includes a container and a label. Suitable containers include, for example, bottles, vials, syringes, and test tubes. The container may be formed from a variety of materials such as glass or plastic. In some embodiments, the container holds a composition effective for treating the diseases described herein and may have a sterile access port. For example, the container may be a bag or vial of intravenous solution having a stopper penetrable by a hypodermic needle. The active agent in the composition is a lipid-lowering agent and / or an anti-inflammatory agent. In some embodiments, the label on or associated with the container indicates that the composition is used for treating a selected disease. The product may further include a second container containing a pharmaceutically acceptable buffer such as phosphate-buffered saline, Ringer's solution, or dextrose solution. It may further include other materials desirable from a commercial or user perspective, which include other buffers, diluents, fillers, needles, syringes, and package inserts with instructions for use.

[0146] Other aspects of the present disclosure provide a method for predicting the recurrence rate of cardiovascular disease in a subject who has received or is receiving treatment with a lipid-lowering agent, the method comprising measuring the level of C-reactive protein (CRP) in the subject and determining that the subject is likely to have a recurrence of cardiovascular disease if the CRP level is higher than a predetermined value.

[0147] In some embodiments, a subject (e.g., a human subject) has previously had a primary (first) cardiovascular event such as a myocardial infarction or has had angioplasty. A subject (e.g., a human subject) who has had a primary cardiovascular event is at risk of an increased secondary (second) cardiovascular event. In some embodiments, a subject (e.g., a human subject) has not had a primary cardiovascular event, but the subject (e.g., a human subject) has one or more risk factors for a cardiovascular event and thus is at risk of having a cardiovascular event. Examples of risk factors for a primary cardiovascular event include hyperlipidemia, obesity, type 2 diabetes, hypertension, prehypertension, elevated levels of a marker(s) of systemic inflammation, age, family history of a cardiovascular event, and smoking. The degree of risk of a cardiovascular event depends on the number and severity or degree of risk factors that the subject (e.g., a human subject) has. Based on the presence and severity of risk factors, risk charts and prediction algorithms for assessing the risk of a cardiovascular event in a subject (e.g., a human subject) are available. One such example is the Framingham Heart Study risk prediction score. A subject (e.g., a human subject) has an increased risk of having a cardiovascular event if the subject's calculated 10-year Framingham Heart Study risk score is higher than 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20%. In some embodiments, a subject who has or is at risk of developing cardiovascular disease has elevated CRP levels compared to a healthy subject. Other methods for assessing the risk of a cardiovascular event in a subject (e.g., a human subject) include coronary calcium scan, cardiac magnetic resonance imaging, and / or magnetic resonance angiography.

[0148] "Recurrence rate of cardiovascular disease" refers to the likelihood that a subject will experience future cardiovascular events after treatment with a lipid-lowering agent (e.g., a statin and / or a PCSK9 inhibitor). In some embodiments, the subject has been diagnosed with cardiovascular disease and has received treatment or is currently receiving treatment with a lipid-lowering agent. In some embodiments, the subject has been diagnosed as being at risk of developing cardiovascular disease and has received treatment or is currently receiving treatment with a lipid-lowering agent. In some embodiments, the subject is also receiving another therapeutic agent to treat or reduce the risk of a cardiovascular event (e.g., any of the treatment methods described herein). In some embodiments, the treatment may also be a non-pharmacological treatment such as diet and / or exercise.

[0149] In some embodiments, the subject has received treatment with a lipid-lowering agent for at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months or longer.

[0150] "Predetermined value" can take various forms. It may be a single cutoff value such as a median or an average value. It can be established based on a comparison group (e.g., when the risk in one defined group is twice the risk in another defined group). It may be a range, where, for example, the population being tested is evenly (or unevenly) divided into groups such as a low-risk group, a medium-risk group, and a high-risk group, or into quartiles (the lowest quartile having individuals with the lowest risk and the highest quartile having individuals with the highest risk), or into tertiles (the lowest tertile having individuals with the lowest risk and the highest tertile having individuals with the highest risk).

[0151] The pre-determined value may depend on a particular population of subjects to be selected (e.g., human subjects). For example, an ostensibly healthy population will have a “normal” range of systemic inflammation markers that is different from what a population of subjects (e.g., human subjects) with prior cardiovascular events will have. Thus, the selected pre-determined value may take into account the category to which the subject (e.g., human subject) belongs. One of ordinary skill in the art can select the appropriate range and category using only routine experimentation.

[0152] In some embodiments, the method further includes measuring a lipid level, such as a cholesterol level or a level of a cholesterol fraction such as LDLC, to characterize the risk that a subject (e.g., human subject) will develop a future cardiovascular event. Obtaining a level of a systemic inflammation marker in the subject (e.g., human subject). Comparing the level of the marker to a pre-determined value to establish a first risk value. Also obtaining a lipid level in the subject (e.g., human subject). Comparing the lipid level in the subject (e.g., human subject) to a second pre-determined value to establish a second risk value. Then characterizing the risk profile that the subject (e.g., human subject) will develop a cardiovascular event based on a combination of the first risk value and the second risk value, where the combination of the first risk value and the second risk value establishes a third risk value that is different from the first and second risk values. In some embodiments, the third risk value is greater than either of the first and second risk values. The cardiovascular event can be any cardiovascular event such as those described above.

[0153] As is known in the art, cholesterol is an important normal body component and is used in the structure of cell membranes, the synthesis of bile acids, and the synthesis of steroid hormones. Since cholesterol is water-insoluble, most serum cholesterol is transported by lipoproteins (chylomicrons, VLDL-C, LDL-C, and HDL-C). Excess cholesterol in the blood is associated with cardiovascular events. LDL is sometimes referred to as "bad" cholesterol because an increase in LDL levels most directly correlates with cardiovascular events such as coronary heart disease. HDL is sometimes referred to as "good" cholesterol because high levels of HDL correlate with a reduced risk for cardiovascular events such as coronary heart disease. The term cholesterol means "total" cholesterol, i.e., VLDL-C + LDL-C + HDL-C cholesterol.

[0154] In some embodiments, after a patient has been treated with a lipid-lowering agent, cholesterol levels are measured. Cholesterol measurements are typically reported in milligrams per deciliter (mg / dL). Typically, the higher the total cholesterol, the higher the risk for a subject (e.g., a human subject) for cardiovascular events. A total cholesterol value of less than 200 mg / dL is a "desirable" level and places the subject (e.g., a human subject) in a group with a lower risk for cardiovascular event(s). For example, a level above 240 mg / dL can be considered to double the risk for cardiovascular events such as coronary heart disease for a subject (e.g., a human subject) compared to an individual having a level of less than 200 mg / dL.

[0155] In some embodiments, the level of LDL-C is one of the predictors of the risk of cardiovascular events. Typically, the higher the LDL-C, the higher the risk for the subject (e.g., a human subject) of a cardiovascular event. A level of LDL-C above 160 mg / dL can be considered to place the subject (e.g., a human subject) at a higher risk of cardiovascular event(s) as compared to an individual having a level below 160 mg / dL. A level of LDL-C above 130 mg / dL in a subject (e.g., a human subject) having one or more risk factors for future cardiovascular events can be considered to place the subject (e.g., a human subject) at a higher risk of cardiovascular event(s) as compared to an individual having a level below 130 mg / dL. A level of LDL-C below 100 mg / dL is desirable in a subject (e.g., a human subject) who has had a prior cardiovascular event and is receiving treatment to reduce the risk of future cardiovascular events, positioning the subject (e.g., a human subject) in a group with a lower risk for cardiovascular events. In such a subject (e.g., a human subject), a level of LDL-C below 70 mg / dL is even more desirable to reduce the risk of future cardiovascular events.

[0156] In some embodiments, a subject who has been or is receiving treatment with a lipid-lowering agent has a healthy lipid (e.g., LDL-C or total cholesterol) level. As described herein, a subject who has been or is receiving treatment with a lipid-lowering agent and has a healthy lipid level may still be at risk of re-experiencing a cardiovascular event (i.e., having a high recurrence rate of cardiovascular disease) if the subject has a CRP level higher than a predetermined value. A subject can be determined to have a low recurrence rate of cardiovascular disease if both the lipid level and the CRP level are lower than predetermined healthy levels.

[0157] The CRP level in a subject can be determined by a CRP blood test(s). Tests and methods for measuring the CRP level in blood, particularly serum samples, and tests and methods for interpreting the results of such tests are widely used today in clinical practice. Since CRP is an acute-phase protein synthesized in the liver during inflammation and released into the bloodstream, its level can be low in subjects without severe inflammation (e.g., inflammation caused by infection). Thus, in some embodiments, the CRP level is measured by a high-sensitivity method (hsCRP) that can detect low levels of CRP (e.g., those in healthy subjects) to assess the risk for cardiovascular disease.

[0158] In some embodiments, the pre-determined value of the CRP level is about 3 mg per liter of blood (i.e., a blood sample from a subject, e.g., a human subject). In some embodiments, the pre-determined value of the CRP level is about 2 mg per liter of blood. In some embodiments, the pre-determined value of the CRP level is about 1.75 mg per liter of blood. In some embodiments, the pre-determined value of the CRP level is about 1.50 mg per liter of blood. In some embodiments, the pre-determined value of the CRP level is about 1.25 mg per liter of blood. In some embodiments, the pre-determined value of the CRP level is about 1 mg per liter of blood. When ranges are used, in some embodiments, the pre-determined value of the CRP level is lower than about 1-3 mg / L (e.g., 1-3, 2-3, 1-3 mg / L) per liter of blood, and another of the ranges is higher than about 3 mg per liter of blood.

[0159] A subject who has been or is receiving treatment with a lipid-lowering agent is determined to have a high recurrence rate of cardiovascular events if the subject has a CRP level higher than a pre-determined level. Another aspect of the present disclosure provides a method of reducing the recurrence rate of cardiovascular disease in a subject who has been or is receiving treatment with a lipid-lowering agent, the method comprising administering to the subject an effective amount of an anti-inflammatory agent.

[0160] The term "treatment" or "treating" refers to both therapeutic and prophylactic treatments. When a subject is in need of treatment for a cardiovascular disease, "treating the condition" refers to alleviating, reducing, or eliminating one or more symptoms associated with the cardiovascular disease or the severity of the cardiovascular disease, or preventing any further progression of the cardiovascular disease. When a subject in need of treatment is at risk of having a cardiovascular disease, treating the subject refers to reducing the risk that the subject has a cardiovascular disease or preventing the subject from developing a cardiovascular disease.

[0161] "Subject" should mean a human, a rodent such as a rat or mouse, a dog, a cat, a horse, a cow, a pig, a sheep, a goat, a chicken, a domestic fowl, and a primate such as a monkey, including but not limited to these, or a vertebrate, or a mammal. The methods of the present disclosure are useful for treating a subject in need thereof. The subject in need thereof may be a subject having or at risk of developing a cardiovascular disease.

[0162] The agents described herein (e.g., anti-inflammatory agents, lipid-lowering agents, and / or bispecific antibodies) can be formulated in pharmaceutical compositions for administration to a subject. The pharmaceutical compositions that can be used according to the present disclosure may be administered directly to a subject or to a subject in need thereof in a therapeutically effective amount. The term "therapeutically effective amount" refers to an amount necessary or sufficient to achieve the desired biological effect. For example, a therapeutically effective amount of a composition comprising a lipid-lowering agent and / or an anti-inflammatory agent according to the present disclosure may be an amount sufficient to relieve one or more symptoms of a disease or disorder. In combination with the teachings provided herein, by selecting from a variety of active compounds and weighting factors (such as potency, relative bioavailability, patient weight, severity of adverse side effects, and preferred mode of administration), an effective prophylactic or therapeutic treatment regimen can be planned that is effective overall for treating a particular subject without causing substantial toxicity. The effective amount for any particular application can vary depending on factors such as the disease or condition being treated, the particular pharmaceutical composition being administered, the size of the subject, or the severity of the disease or condition. One of ordinary skill in the art can empirically determine the effective amount of a particular therapeutic compound in relation to the present disclosure without undue experimentation.

[0163] The dosage for a subject for delivery of a composition comprising a lipid-lowering agent and / or an anti-inflammatory agent described herein typically ranges from about 0.1 μg to 10 mg per administration, which depends on whether the application is administered daily, weekly, or monthly, and at any other time in between. In some embodiments, a single dose is administered during a clinical intensification or re-intensification period. The dosage for these purposes can range from about 10 μg to 5 mg, most typically from about 100 μg to 1 mg per administration, and can be administered 2 to 4 times at intervals separated by several days or weeks, or more. In some embodiments, however, parenteral dosages for these purposes may be used in a range 5 to 10,000 times higher than the typical dosages described above.

[0164] In some embodiments, the compositions containing a lipid-lowering agent and / or an anti-inflammatory agent, or a bispecific antibody described herein are administered at a dosage of about 1 to 10 mg per kg of body weight of a mammal. In other embodiments, the compositions containing a lipid-lowering agent and / or an anti-inflammatory agent, or a bispecific antibody described herein are administered at a dosage of about 0.001 to 1 mg per kg of body weight of a mammal. In still other embodiments, the compositions containing a lipid-lowering agent and / or an anti-inflammatory agent, or a bispecific antibody described herein are administered at a dosage of about 10 to 100 ng, 100 to 500 ng, 500 ng to 1 mg, or 1 to 5 mg per kg of body weight of a mammal, or any individual dosage therein.

[0165] The formulations of the present disclosure are administered in a pharmaceutically acceptable solution, which may conventionally contain pharmaceutically acceptable concentrations of salts, buffering agents, preservatives, compatible carriers, and optionally other therapeutic components.

[0166] For use in therapy, an effective amount of the compositions containing a lipid-lowering agent and / or an anti-inflammatory agent, or a bispecific antibody described herein can be administered to a subject by any mode that delivers the composition to the desired site, such as, for example, mucosally, by injection, systemically, etc. Administration of the pharmaceutical compositions of the present disclosure can be accomplished by any means known to those of skill in the art. In some embodiments, the compositions containing an anti-inflammatory agent and / or an anti-inflammatory agent, or a bispecific antibody described herein are administered subcutaneously, intradermally, intravenously, intramuscularly, intra-articularly, intra-arterially, intrasynovially, intrasternally, intrathecally, intralesionally, or intracranially.

[0167] For oral administration, the composition can be readily formulated by combining the active compound(s) with a pharmaceutically acceptable carrier well-known in the art. Such carriers enable the compounds of the present disclosure to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, etc. for oral ingestion by the subject to be treated. Pharmaceutical preparations for oral use can be obtained as solid excipients, the resulting mixture is optionally comminuted and, if desired, after adding suitable auxiliaries, the mixture of granules is processed to obtain cores of tablets or dragees. Suitable excipients are, in particular, fillers such as sugars like lactose, sucrose, mannitol or sorbitol; cellulose preparations such as corn starch, wheat starch, rice starch, potato starch, gelatin, tragacanth gum, methylcellulose, hydroxypropylmethylcellulose, sodium carboxymethylcellulose, and / or polyvinylpyrrolidone (PVP), etc. Disintegrants such as cross-linked polyvinylpyrrolidone, agar, or alginic acid, or salts thereof such as sodium alginate, etc. may be added if desired. Optionally, the oral formulation may be formulated in saline or a buffer, i.e., EDTA for neutralizing internal acidic conditions, or administered without any carrier.

[0168] Also particularly contemplated is an oral dosage form of the above-described component(s). The component(s) may be chemically modified such that oral delivery of the derivative is effective. Generally, the chemical modification contemplated is the attachment of at least one moiety to the molecule of the component itself, where the moiety (a) inhibits proteolysis; and (b) enables uptake from the stomach or intestine into the bloodstream. Also desired is an increase in the overall stability of the component(s) and an extension of the circulation time in the body. Examples of such moieties include polyethylene glycol, copolymers of ethylene glycol and propylene glycol, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, and polyproline (Abuchowski and Davis, 1981, "Soluble Polymer-Enzyme Adducts", (in Hocenberg and Roberts, eds., Enzymes as Drugs, Wiley-Interscience, New York, NY, pp. 367-383; Newmark, et al., 1982, J. Appl. Biochem. 4:185-189). Other polymers that can be used are poly-1,3-dioxolane and poly-1,3,6-tioxocane. Preferred for pharmaceutical use as shown above is the polyethylene glycol moiety.

[0169] The site of release may be the stomach, small intestine (duodenum, jejunum or ileum), or large intestine. One of ordinary skill in the art has available formulations that will not dissolve in the stomach but will release the material in the duodenum or another location in the intestine. Preferably, the release will avoid the deleterious effects of the gastric environment by protection of the therapeutic agent or by release of the biologically active material, e.g., in the intestine, after crossing the gastric environment.

[0170] To ensure complete gastric resistance, a coating that is at least impermeable to pH 5.0 is preferred. Examples of more common inert ingredients used as enteric coatings are cellulose acetate trimellitate (CAT), hydroxypropylmethylcellulose phthalate (HPMCP), HPMCP 50, HPMCP 55, polyvinyl acetate phthalate (PVAP), Eudragit L30D, Aquateric, cellulose acetate phthalate (CAP), Eudragit L, Eudragit S, and Shellac. These coatings may be used as a blended film.

[0171] The coating or mixture of coatings may also be used for tablets that are not intended to provide protection against the stomach. This includes sugar coatings or coatings that facilitate swallowing of the tablet. Capsules may consist of a hard shell (such as gelatin) for delivery of a dry therapeutic agent, i.e., powder; for a liquid form, a soft gelatin shell may be used. The shell material of cachets may be thick starch or other edible paper. For pills, lozenges, wet granules or tablet triturates, wet massing techniques may be used.

[0172] In some embodiments, the composition may be included in the formulation in the form of fine multi-particles, granules or pellets with a particle size of about 1 mm. The formulation of materials for capsule administration may also be in the form of a powder, a lightly compressed plug, or even a tablet. The therapeutic agent can be prepared by compression.

[0173] Colorants and flavorants may all be included. For example, lipid-lowering agents and / or anti-inflammatory agents may be formulated (such as by encapsulation into liposomes or microspheres) and then further included in an edible product such as a refrigerated beverage that also contains colorants and flavorants.

[0174] The therapeutic agent can be diluted or its volume increased with an inert material. These diluents include carbohydrates, especially mannitol, lactose, lactose anhydrous, cellulose, sucrose, modified dextran and starch. Certain inorganic salts can also be used as fillers, including tricalcium phosphate, magnesium carbonate and sodium chloride. Some commercially available diluents are Fast-Flo, Emdex, STA-Rx 1500, Emcompress and Avicell.

[0175] Disintegrants may be included in formulations that render the therapeutic agent in a solid dosage form. Materials used as disintegrants include, but are not limited to, starch, including the commercially available starch-based disintegrant Explotab. Sodium starch glycolate, Amberlite, sodium carboxymethyl cellulose, ultramylopectin, sodium alginate, gelatin, orange peel, acid carboxymethyl cellulose, natural sponge and bentonite can all be used. Another form of disintegrant is an insoluble cation exchange resin. Powdered gums can be used as disintegrants and as binders, including powdered gums such as agar, karaya or tragacanth. Alginate and its sodium salts are also useful as disintegrants.

[0176] Binders can be used to bring the therapeutic agent together to form hard tablets and include materials from natural products such as gum arabic, tragacanth, starch and gelatin. Others include methylcellulose (MC), ethylcellulose (EC) and carboxymethylcellulose (CMC). Polyvinylpyrrolidone (PVP) and hydroxypropylmethylcellulose (HPMC) can both be used to granulate the therapeutic agent in an alcoholic solution.

[0177] The anti-friction agent may be included in the formulation of the therapeutic agent to prevent sticking during the formulation process. The lubricant can be used as a layer between the therapeutic agent and the pigment wall, and these include, but are not limited to, stearic acid (including its magnesium and calcium salts), polytetrafluoroethylene (PTFE), liquid paraffin, vegetable oil, and wax. Soluble lubricants such as sodium lauryl sulfate, magnesium lauryl sulfate, polyethylene glycols of various molecular weights, Carbowax 4000, and 6000 can also be used.

[0178] A glidant that can improve the flow characteristics of the drug during formulation and assist in rearrangement during compression may also be added. Examples of glidants can include starch, talc, calcined silica, and hydrated silicoaluminate.

[0179] To assist in the dissolution of lipid-lowering agents and / or anti-inflammatory agents into the aqueous environment, a surfactant may be added as a wetting agent. Examples of surfactants can include anionic detergents such as sodium lauryl sulfate, dioctyl sodium sulfosuccinate, and dioctyl sodium sulfonate. Cationic detergents may also be used, which include benzalkonium chloride or benzethonium chloride. The list of possible non-ionic detergents that can be included as surfactants in the formulation is lauromacrogol 400, polyoxyl 40 stearate, polyoxyethylene hydrogenated castor oil (10, 50, and 60), glyceryl monostearate, polysorbate 40, 60, 65, and 80, sucrose fatty acid ester, methylcellulose, and carboxymethylcellulose. These surfactants may exist alone or as mixtures in various ratios in the formulation of the therapeutic agent.

[0180] Pharmaceutical preparations that can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer (such as glycerol or sorbitol). The push-fit capsules may contain the active ingredient in a mixture with a filler such as lactose, a binder such as starch, and / or a lubricant such as talc or magnesium stearate, and optionally a stabilizer. In the soft capsules, the active compound may be dissolved or suspended in a suitable liquid such as an oil, liquid paraffin, or liquid polyethylene glycol. In addition, a stabilizer may be added. Microspheres formulated for oral administration can also be used. Such microspheres are well defined in the art. All formulations for oral administration are in suitable dosages for such administration.

[0181] For buccal administration, the composition may take the form of tablets or lozenges formulated in a conventional manner.

[0182] For administration by inhalation, the compounds for use according to the present disclosure can be conveniently delivered in the form of an aerosol spray from a pressurized pack or a nebulizer using a suitable propellant, such as dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide, or other suitable gas. In the case of a pressurized aerosol, the dosage unit can be determined by providing a valve for delivering a metered amount. Capsules and cartridges (e.g., of gelatin) for use in an inhaler or insufflator containing a powder mixture of the compound and a suitable powder base such as lactose or starch can be formulated.

[0183] The pharmaceutical compositions of the present disclosure may be formulated for parenteral administration, for example by injection, such as by bolus injection or continuous infusion, when systemic delivery thereof is desired. Formulations for injection may be presented in unit dosage form, for example in ampoules or in multi-dose containers, and may contain a preservative. The compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and / or dispersing agents.

[0184] Pharmaceutical formulations for parenteral administration include aqueous solutions of the active compound in water-soluble form. In addition, suspensions of the active compound can be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fats and oils such as sesame oil, synthetic fatty acid esters such as ethyl oleate or triglycerides, or liposomes. Aqueous injection suspensions may contain substances that increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol or dextran. Optionally, the suspension may also contain suitable stabilizers or agents that increase the solubility of the compound and enable the preparation of highly concentrated solutions.

[0185] In addition to the formulations described above, the compositions may also be formulated as depot preparations. Such long-acting formulations may be formulated with suitable polymeric or hydrophobic materials (such as emulsions in acceptable oils), or ion exchange resins, or as slightly insoluble derivatives, for example as slightly insoluble salts.

[0186] The pharmaceutical compositions may also contain suitable solid or gel phase carriers or excipients. Examples of such carriers or excipients include, but are not limited to, polymers such as calcium carbonate, calcium phosphate, various sugars, starch, cellulose derivatives, gelatin, and polyethylene glycol.

[0187] Suitable liquid or solid pharmaceutical preparation forms are, for example, aqueous solutions or saline solutions for inhalation, microencapsulated, encochleated, coated on microscopic gold particles, contained in liposomes, sprayed, aerosols, pellets for implantation into the skin, or dried on sharp objects for scratching into the skin. The pharmaceutical composition also includes granules, powders, tablets, coated tablets, (micro)capsules, suppositories, syrups, emulsions, suspensions, creams, droplets, or preparations having a delayed release of the active compound, in which auxiliaries such as excipients and additives and / or disintegrants, binders, coating agents, swelling agents, lubricants, flavoring agents, sweetening agents or solubilizing agents are customarily used as described above. The pharmaceutical composition is suitable for use in various drug delivery systems. For a brief overview of methods for drug delivery, see Langer, Science 249:1527-1533, 1990, which is incorporated herein by reference.

[0188] The pharmaceutical compositions of the present disclosure and any other therapeutic agent may be administered per se (neat) or in the form of a pharmaceutically acceptable salt. When used in a pharmaceutical, the salt should be pharmaceutically acceptable, but pharmaceutically unacceptable salts can be conveniently used to prepare their pharmaceutically acceptable salts. Such salts include, but are not limited to, those prepared from the following acids: hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, maleic acid, acetic acid, salicylic acid, p-toluenesulfonic acid, tartaric acid, citric acid, methanesulfonic acid, formic acid, malonic acid, succinic acid, naphthalene-2-sulfonic acid and benzenesulfonic acid. Also, such salts can be prepared as salts of alkali metals or alkaline earth metals such as sodium, potassium or calcium salts of carboxylic acid groups.

[0189] Suitable buffering agents include the following: acetic acid and salts (1 - 2% w / v); citric acid and salts (1 - 3% w / v); boric acid and salts (0.5 - 2.5% w / v); and phosphoric acid and salts (0.8 - 2% w / v). Suitable preservatives include benzalkonium chloride (0.003 - 0.03% w / v); chlorobutanol (0.3 - 0.9% w / v); parabens (0.01 - 0.25% w / v) and thimerosal (0.004 - 0.02% w / v).

[0190] The subject of the present disclosure has or is at risk of developing cardiovascular disease. "Cardiovascular disease (CVD)" is a class of diseases related to the heart or blood vessels. Non - limiting examples of cardiovascular diseases include: coronary artery disease (CAD) such as angina and myocardial infarction (commonly known as a heart attack), stroke, heart failure, hypertensive heart disease, rheumatic heart disease, cardiomyopathy, cardiac arrhythmia, congenital heart disease, valvular heart disease, myocarditis, aortic aneurysm, peripheral arterial disease, thromboembolic disease, venous thrombosis, acute coronary syndrome, myocardial ischemia, chronic stable angina, unstable angina, coronary restenosis, coronary stent restenosis, coronary stent thrombosis, revascularization, angioplasty, transient ischemic attack, pulmonary embolism, vascular occlusion, and cardiovascular death.

[0191] Coronary artery disease (CAD), also known as ischemic heart disease (IHD), is a group of diseases including stable angina, unstable angina, myocardial infarction, and sudden cardiac death. Risk factors for CAD include hypertension, smoking, diabetes, lack of exercise, obesity, high blood cholesterol, a diet high in fat, and excessive alcohol, and / or depression. The underlying mechanism involves a reduction in blood flow and oxygen due to atherosclerotic plaque in the arteries of the heart.

[0192] Myocardial infarction (MI), commonly known as a heart attack, occurs when blood flow to a part of the heart is reduced or stopped, causing damage to the myocardium. Risk factors for MI include hypertension, smoking, diabetes, lack of exercise, obesity, high blood cholesterol, a diet high in fat, and excessive alcohol intake.

[0193] Myocardial ischemia occurs when blood flow to the heart is reduced, preventing it from receiving sufficient oxygen. The reduced blood flow is usually the result of partial or complete blockage of the arteries (coronary arteries) of the heart.

[0194] Angina is the medical term for chest pain or discomfort caused by coronary artery disease. It occurs when the heart muscle does not receive enough blood as needed. This usually happens when one or more of the arteries of the heart are narrowed or blocked, also known as ischemia. Unstable angina (UA) is a type of angina that is irregular.

[0195] A stroke is a medical condition in which insufficient blood flow to the brain results in cell death. There are two main types of stroke: ischemic, caused by a lack of blood flow, and hemorrhagic, caused by bleeding. Risk factors for stroke include high blood pressure, smoking, obesity, high blood cholesterol, type 2 diabetes, previous TIA, and atrial fibrillation. Acute coronary syndrome is a term used to describe a range of conditions related to a sudden decrease in blood flow to the heart. A transient ischemic attack (TIA) is similar to a stroke, causing similar symptoms, but usually lasts only a few minutes and does not cause permanent damage.

[0196] Heart failure (HF), often referred to as congestive heart failure, occurs when the heart is unable to pump enough blood to meet the body's needs. Common causes of heart failure include coronary artery disease, including previous myocardial infarction (heart attack), high blood pressure, atrial fibrillation, heart valve disease, excessive alcohol use, infection, and cardiomyopathy of unknown cause.

[0197] Rheumatic heart disease is a complication of rheumatic fever in which the heart valves are damaged. Rheumatic fever (RF) is an inflammatory disease that can affect the heart, joints, skin, and brain.

[0198] Cardiomyopathy is a group of diseases that affect the myocardium. Types of cardiomyopathy include hypertrophic cardiomyopathy, dilated cardiomyopathy, restrictive cardiomyopathy, arrhythmogenic right ventricular dysplasia, and takotsubo cardiomyopathy (broken heart syndrome). Dilated cardiomyopathy can also be caused by alcohol, heavy metals, coronary heart disease, cocaine use, and viral infections. Restrictive cardiomyopathy can be caused by amyloidosis, hemochromatosis, and the treatment of some cancers.

[0199] Peripheral arterial disease (PAD) is the narrowing of the arteries that supply areas other than the heart or brain. Risk factors for PAD include tobacco smoking, diabetes, hypertension, and high blood cholesterol. The underlying mechanism is usually atherosclerosis.

[0200] Congenital heart disease (CHD), also known as congenital heart anomaly or congenital heart disorder, is a problem in the structure of the heart that is present at birth. Valvular heart disease is any disease process involving one or more of the four heart valves (the aortic and mitral valves on the left side, and the pulmonary and tricuspid valves on the right side). Myocarditis is inflammation of the heart or its surrounding area. An aortic aneurysm is an enlargement (dilation) of the aorta to a size more than 1.5 times its normal size.

[0201] Thrombosis is the formation of blood clots within blood vessels, which impedes the flow of blood through the circulatory system. A venous thrombus is a blood clot (thrombus) that forms within a vein. Pulmonary embolism is the sudden blockage of a major blood vessel (artery) in the lungs, usually by a blood clot. Vascular occlusion is the blockage of a blood vessel, usually by a blood clot. It differs from thrombosis in that it is used to describe any form of blockage, not just those formed by blood clots. When it occurs in a major vein, it can sometimes cause deep vein thrombosis.

[0202] Coronary restenosis is the recurrence of stenosis, which is the narrowing of blood vessels, resulting in blood flow restriction. Coronary stent restenosis occurs when a stent is implanted and restenosis develops inside the stent. Coronary stent thrombosis occurs when a stent is implanted and thrombosis develops inside the stent.

[0203] Revascularization is the restoration of perfusion to a body part or organ affected by ischemia. It is typically achieved by surgical means. Coronary artery bypass and angioplasty are two main means of revascularization.

[0204] The present disclosure will be described by reference to the following examples, but the present disclosure is not limited thereto.

[0205] Example Patients with residual inflammation risk have a high rate of recurrent cardiovascular events due to persistently elevated levels of high-sensitivity C-reactive protein (hsCRP), despite the aggressive use of statin therapy. 1~7 . Such patients are generally defined as those who are receiving statin therapy and have hsCRP ≥ 2 mg / L and LDL cholesterol < 70 mg / dL 8 , and currently account for nearly 30 percent of patients and are twice as common as patients with residual cholesterol risk (defined by LDL level ≥ 70 mg / dL and hsCRP < 2 mg / L). 9 . Recently, the Canakinumab Anti-inflammatory Thrombosis Outcomes Study (CANTOS) showed that inhibition of IL-1β by canakinumab significantly reduces both hsCRP and cardiovascular events 10, the data provides a first specific treatment to patients with residual cholesterol risk. In fact, the degree of risk reduction in CANTOS was substantially the same as that achieved in the protein convertase subtilisin-kexin type 9 (PCSK9) trials of FOURIER and SPIRE, despite no change in LDL cholesterol. 11、12 . Importantly, the absolute event rates of 5.3% and 9.1% at 1 and 2 years of follow-up in FOURIER inform the inventors that many patients achieving very low LDL-C levels will continue to experience vascular events. Whether residual inflammatory risk remains an important clinical problem among statin-treated patients who additionally receive PCSK9 inhibition is unknown. This issue was addressed in the recently completed SPIRE-1 and SPIRE 2 trials described herein.

[0206] Method Study Population and Procedures The SPIRE bococizumab development program consisted of two parts: six SPIRE lipid-lowering studies and the SPIRE-1 and SPIRE-2 event-driven cardiovascular trials. The design and initial findings of SPIRE-1 and SPIRE-2 have been previously published. 12、13Two trials of substantially the same design allowed them to be combined according to an integrated statistical analysis plan. Briefly, patients were eligible for enrollment if they had a history of prior cardiovascular events (secondary prevention cohort) or diabetes, chronic kidney disease, or peripheral vascular disease with additional cardiovascular risk, or a history of familial hypercholesterolemia (high-risk primary prevention cohort). All patients were required to have received at least 4 weeks of stable statin therapy (atorvastatin ≥40 mg / day, rosuvastatin ≥20 mg / day, or simvastatin ≥40 mg / d) unless they were unable to take these doses without side effects and thus were on lower-intensity statin therapy or had complete statin intolerance (eligible only for SPIRE-2). Patients were required to have directly measured LDL-C levels of at least 70 mg / dL in SPIRE-1 and >100 mg / dL in SPIRE-2. Patients also became eligible according to their non-HDL cholesterol levels at the time of entry (≥100 mg / dL for SPIRE-1 and ≥130 mg / dL for SPIRE-2). In a double-blind fashion, patients were randomized 1:1 to treatment with subcutaneous bococizumab 140 mg every 2 weeks or matching placebo. The SPIRE program was funded by Pfizer.

[0207] The study population for this analysis included a subgroup of patients who were on statin therapy, assigned to active bococizumab, and had hsCRP available at baseline and at 14 weeks available for analysis (n = 9,738). All patients provided written informed consent. The ethics committee at each center approved the protocol.

[0208] Endpoint The two pre-specified primary endpoints for the two trials were declared and confirmed non-fatal myocardial infarction, non-fatal stroke, hospital admission for unstable angina requiring urgent revascularization, or a composite of cardiovascular death. The events of all the constituents of these endpoints were declared by a committee whose members were unaware of the treatment assignment.

[0209] Statistical analysis Of the 13,675 patients randomized to the active treatment arm, 12,711 (93.0%) had received statin therapy and 9,738 (71.2%) also had hsCRP OT levels available at 14 weeks. The corresponding proportions of patients randomized to placebo, receiving statin therapy, and having follow-up biomarker levels were 9,785 (71.6%).

[0210] The study population was then restricted to individuals assigned to bococizumab and divided into three groups according to hsCRP OT levels <1, 1–3, and >3 mg / dL, which included 30.4%, 34.8%, and 34.9% of the patients, respectively. When using cut points of <2 and ≥2 mg / dL, these percentages were 52.8% and 47.2%. The baseline characteristics by the three initial hsCRP OT groups were summarized using category values and percentages of the median (interquartile range) for continuous variables. The trends in these characteristics across the organized hsCRP OT categories were evaluated using the Cochran–Armitage trend test for differences in proportions and the Jonckheere–Terpstra test for differences in medians.

[0211] To evaluate the treatment effect of bococizumab on lipid levels and on hsCRP, the median on-treatment level was determined at baseline and at 14 weeks of treatment. A linear mixed model repeated measures analysis, conditional on the baseline value, was constructed using a log transformation, which was considered appropriate for non-normal distributions, with the biomarker of interest as the independent variable. The percent change in mean and the treatment effect of bococizumab were estimated by fitting the conditions corresponding to the assignment of the study drug.

[0212] For each hsCRP among patients assigned to bococizumab OT the percent change in lipid levels in the group was then conditioned on the baseline value using a mixed model as before and fitting the conditions corresponding to the hsCRP OT group.

[0213] Using the Cox proportional hazards analysis model, hazard ratios (HRs) were estimated according to the hsCRP OT group. Three adjusted models are presented, adjusted for the following: 1) age and sex, 2) age, sex, classical cardiovascular risk factors (including current smoking, diabetes, hypertension, and body mass index) plus statin intensity at enrollment (moderate intensity or high intensity), and 3) the variables of model 2 and plus on-treatment LDL-C (LDL OT ). For each model, after assigning the median to each group, a test for trend across the entire category of hsCRP OT was conducted. All analyses were stratified by study (SPIRE-1 or SPIRE-2), region, and screening LDL-C threshold (<70 or <100 mg / dL). Additional tests were performed to assess the heterogeneity of the treatment effect of bococizumab relative to placebo by hsCRP OT group using an interaction term (bococizumab × hsCRP OT group).

[0214] To enable comparison of the associations for on-treatment LDL-C measured at 14 weeks, the study population was further divided into LDL OT groups (approximate tertiles) using categories of <30, 30-50 and >50 mg / dL, and adjusted HRs in each of these groups were estimated using comparable Cox models. Cut points of < or ≥2 mg / L for hsCRP and < or ≥40 mg / dL for LDL-C were also used. Finally, to examine the risk associations across the range of hsCRP OT levels, a smooth function was used on the mean of the estimated event rates at each hsCRP OT level, based on the adjusted Cox model, to plot the relationship between hsCRP OT and the cardiovascular event rate.

[0215] Results Study population by on-treatment hsCRP level The study population included 2958 (30.4%) with hsCRP OT <1 mg / L, 3385 (34.8%) with hsCRP OT of 1-3 mg / L, and 3395 (34.9%) with hsCRP OT >3 mg / L. Baseline characteristics by hsCRP OT are shown in Table 1. Patients with higher hsCRP OT groups were more likely to be female, obese, have diabetes or diagnosed hypertension, and be current smokers, but less likely to have pre-existing cardiovascular disease. Some baseline lipid parameters, including higher levels of LDL-C, total cholesterol (TC), non-HDL cholesterol (non-HDL-C), triglycerides, total:HDL-C ratio, and apolipoprotein B (apoB), and lower levels of HDL-C, also differed significantly across groups with increasing hsCRP.

Table 1

[0216] Effect of bococizumab treatment on lipid levels, hsCRP, and cardiovascular events Compared with placebo, bococizumab was associated with statistically significant decreases in LDL-C (-60.5%), TC (-37.6%), non-HDL-C (-54.9%), TC:HDL-C ratio (-41.1%), apoB (-56.0%), and triglycerides (-19.9%), as well as an increase in HDL-C (+6.4%) (Table 2; all p<0.001). In contrast, there was no significant effect on hsCRP: at 14 weeks, percent change in mean +6.6% (95% CI: -1.0 to 14.1; p=0.09; median change 0.0%), and at 52 weeks +6.7% (-9.3 to 16.9%; p=0.57; median change 0.0%) (n=3267). The percent change in lipid fractions was somewhat lower in those with higher hsCRP OT groups (Figure 1). However, even among those with hsCRP>3mg / L, median LDL-C OT at 14 weeks was 41.7 (IQR 25.9, 67.0) mg / L. The effect of bococizumab treatment by hsCRP OT was similar in degree and there was no evidence of heterogeneity among hsCRP OT groups (p-interaction=0.87).

Table 2-1

Table 2-2

[0217] Event rates by on-treatment hsCRP and on-treatment LDL Overall, a monotonic increase in event probability adjusted for primary CVD endpoints was observed as on-treatment hsCRP levels increased (Figure 2). hsCRP OTThe event rates in the cohort were 1.96, 2.50, and 3.59 per 100 person-years for hsCRP <1, 1–2, and >3 mg / L, respectively (Table 3). In the multivariate model adjusted for age and sex, the corresponding HRs for CVD were 1.0 (ref), 1.23 (95% CI: 0.86–1.75), and 1.79 (95% CI: 1.28–2.50); p-trend <0.001. In the model further adjusted for classical cardiovascular risk factors and baseline intensity of statin treatment, the HR comparing the highest hsCRP OT category with the lowest one (>3 vs <1 mg / dL) was 1.67 (95% CI: 1.18–2.37; p = 0.02). Further adjustment for LDL OT minimally attenuated this risk (Model 3, Table 1 and Figure 3A). In the model further adjusted for on-treatment TC:HDL-C ratio, the adjusted HRs were 1.0 (ref), 1.13, and 1.58 (p-trend = 0.002). Examining the individual components of the composite endpoint, the hsCRP OT category was significant for nonfatal myocardial infarction (adjusted HR: 1.0, 0.91, 1.46, p-trend = 0.017), cardiovascular mortality (adjusted HR: 1.0, 1.60, 3.76, p-trend = 0.002), and total mortality (adjusted HR: 1.0, 1.58, 3.45, p-trend <0.001). Similar but nonsignificant trends were noted for stroke and unstable angina requiring urgent coronary revascularization.

[0218] Patients were classified according to LDL-C OT (<30, 30–50, >50 mg / dl) in a parallel analysis. The HRs for the primary CVD endpoint were 1.0 (ref), 0.87 (95% CI: 0.62–1.22), and 1.21 (0.87–1.68) in the analysis adjusted for the covariates of Model 3 and hsCRP OT instead of LDL-C OT (Figure 3B and Table 4), with p-trend = 0.16. hsCRP OTFor those with ≥ 2 mg / L of LDL-C OT Similar findings were observed when using alternative cut-off points of ≥ 40 mg / dL (Tables 5 and 6).

Table 3

[0219]

Table 4

[0220]

Table 5

[0221]

Table 6

[0222] Discussion In this population of 9,738 high-risk patients simultaneously treated with statins and LDL-PCSK9 inhibition, 47.2% had a residual inflammatory risk defined by on-treatment hsCRP levels ≥ 2 mg / L, and 34.9% had values > 3 mg / L. Individuals with persistent CRP elevation tended to have multiple risk factors, including mixed dyslipidemia, a condition known (if not caused) to be correlated with diabetes, obesity, hypertension, and pro-inflammatory states. Inhibition of PCSK9 by bococizumab had no effect on hsCRP over time. Despite an exceptional reduction in LDL-C, there was a continuous gradient of risk for future vascular events based on on-treatment hsCRP. Those with on-treatment hsCRP > 3 mg / L had a 62% increased risk of future vascular events compared to those without evidence of subclinical inflammation. Elevated hsCRP was significantly associated with an increased rate of myocardial infarction, cardiovascular death, and death from all causes.

[0223] There is a broad consensus that atherosclerosis is both a disorder of lipid accumulation and inflammation. From a clinical perspective, large previous studies have found that hsCRP is an independent predictor of cardiovascular events in both primary prevention and high-risk secondary prevention. Furthermore, in patients with residual inflammatory risk, randomized clinical trials have demonstrated the efficacy of statin therapy in primary prevention 14 and anti-inflammatory therapy in secondary prevention. 10 However, it was unclear whether the residual inflammatory risk persists with the extremely aggressive LDL-C reduction achievable with the combination of statin therapy and PCSK9 inhibition. Importantly, in an era when increasingly specialized therapies in cardiovascular medicine will continue to emerge, the need for biomarkers to inform physicians about risk stratification, drug selection and dosing, treatment response, and ultimately individualized interventions will only be amplified.

[0224] In this context, these data have several important implications. First, these data reveal that inhibition of PCSK9 has no effect on plasma measurement of hsCRP, despite its profound effect on atherogenic lipids. Second, these data indicate that the combination of high-intensity statin therapy and inhibition of PCSK9 does not fully resolve the inflammatory mechanisms of atherothrombosis, despite the interrelationship between LDL oxidation and inflammation. Separately, the post hoc findings are associative and can still be explained by underlying states that promote subclinical inflammation. Therefore, the inventors believe that combination therapy of PCSK9 inhibition and anti-inflammatory therapy will provide an optimal approach to resolving residual cardiovascular risk. Canakinumab is the only anti-inflammatory agent currently proven to reduce cardiovascular events, while clinical trials using colchicine and low-dose methotrexate are currently ongoing 16、17The inventors also consider that agents that inhibit the activation of the upstream NLRP3 inflammasome and downstream IL-6 may also be useful in resolving the remaining cardiovascular risk, and have this in mind.

[0225] The SPIRE cardiovascular outcome trial was stopped early due to the high rate of development of neutralizing anti-drug antibodies. 18 Bococizumab immunogenicity was associated with a less persistent reduction in LDL, while treatment with bocozizumab in the longer-term SPIRE-2 outcome trial was nonetheless associated with an overall 21% (95% CI: 3–35%; p = 0.02) relative risk reduction in major cardiovascular events and a 14% (95% CI: 2–~25%) relative risk reduction per mmol / l reduction in LDL-C. These data were fully consistent with the benefits observed in the FOURIER trial. 12、19 Accordingly, the findings presented above in this specification are unlikely to be explained by the attenuated LDL-C lowering efficacy of bocozizumab and are more broadly likely to apply to this class of drugs. As with any post-hoc analysis, the findings presented above in this specification are likely to be subject to the influence of residual confounding. In particular, subjects with a persistent inflammatory risk were more likely to have cardiovascular risk factors and a higher median on-treatment LDL-C. However, multivariate analysis adjusted for the achieved LDL-C level showed a reduction in risk (if any). Furthermore, as shown in CANTOS enrolled on the basis of elevated hsCRP, this risk group may benefit from anti-inflammatory treatment.

[0226] In summary, these recent randomized trial data indicate that elevated on-treatment hsCRP levels continue to be a significant predictor of future vascular risk among patients with atherosclerotic disease treated simultaneously with statins and PCSK9 inhibition. This evidence of residual inflammatory risk despite maximal LDL-C lowering suggests that a combination of anti-inflammatory agents in addition to lipid-lowering agents may offer additional opportunities for reducing cardiovascular risk at all cholesterol levels.

[0227] References

Table 7-1

Table 7-2

Table 7-3

Claims

**Claim 1** A method of treating cardiovascular disease, comprising administering a therapeutically effective amount of a lipid-lowering agent and an anti-inflammatory agent to a subject in need thereof. **Claim 2** The method according to claim 1, wherein the anti-inflammatory agent is an inflammatory cytokine inhibitor. **Claim 3** The method according to claim 1 or 2, wherein the anti-inflammatory agent comprises an IL-1 inhibitor, an IL-1 receptor (IL-1R) inhibitor, an IL-6 inhibitor, an IL-6 receptor (IL-6R) inhibitor, an NLRP3 inhibitor, a TNF inhibitor, an IL-8 inhibitor, an IL-18 inhibitor, an inhibitor of natural killer cells, or a combination thereof. **Claim 4** The method according to any one of claims 1 to 3, wherein the anti-inflammatory agent is a nucleic acid, an aptamer, an antibody or antibody fragment, an inhibitory peptide, or a small molecule. **Claim 5** The method according to claim 3 or 4, wherein the anti-inflammatory agent comprises an IL-1 inhibitor. **Claim 6** The method according to claim 5, wherein the IL-1 inhibitor is an IL-1α inhibitor. **Claim 7** The method according to claim 6, wherein the IL-1α inhibitor is an antisense oligonucleotide against IL-1α, MABp1, or sIL-1RI. **Claim 8** The method according to claim 5, wherein the IL-1 inhibitor is an IL-1β inhibitor. **Claim 9** The method according to claim 8, wherein the IL-1β inhibitor is an antisense oligonucleotide against IL-1β, canakinumab, gevokizumab, diacerein, LY2189102, CYTO013, sIL-1RII, VX-740 or VX-765. **Claim 10** The method according to claim 5, wherein the IL-1 inhibitor is sodium slamine, methotrexate-methyl-d3, methotrexate-methyl-d3 dimethyl ester, or diacerein. **Claim 11** The method according to any one of claims 3 to 10, wherein the anti-inflammatory agent comprises an IL-1R inhibitor. **Claim 12** The method according to claim 11, wherein the IL-1R inhibitor is an IL-1R antagonist. **Claim 13** The method according to claim 11 or 12, wherein the IL-1R inhibitor is an antisense oligonucleotide against IL-1R, anakinra, rilonacept, MEDI-8968, sIL-1RI, EBI-005, interleukin-1 receptor antagonist (IL-1RA), or AMG108. **Claim 14** The method according to any one of claims 3 to 13, wherein the anti-inflammatory agent comprises an IL-6 inhibitor. **Claim 15** The method according to claim 14, wherein the IL-6 inhibitor is an antisense oligonucleotide against IL-6, siltuximab, sirukumab, clazakizumab, olokizumab, elsilimomab, IG61, BE-8, CNTO328, PGE1 and its derivatives, PGI2 and its derivatives, or cyclophosphamide.

16. The method according to any one of claims 3 to 15, wherein the anti-inflammatory agent comprises an IL-6R inhibitor.

17. The method according to claim 16, wherein the IL-6R inhibitor is an IL-6R antagonist.

18. The method according to claim 16 or claim 17, wherein the IL-6R inhibitor is an antisense oligonucleotide against IL-6R, tocilizumab, sarilumab, PM1, AUK12-20, AUK64-7, AUK146-15, MRA, or AB-227-NA.

19. The method according to any one of claims 1 to 18, wherein the anti-inflammatory agent comprises an NLRP3 inhibitor.

20. The method according to claim 19, wherein the NLRP3 inhibitor is an antisense oligonucleotide against NLRP3, colchicine, MCC950, CY-09, ketone metabolite β-hydroxybutyric acid (BHB), type I interferon, resveratrol, arglabin, CB2R, glibenclamide, isoliquiritigenin, Z-VAD-FMK, or microRNA-223.

21. The method according to any one of claims 3 to 20, wherein the anti-inflammatory agent comprises a TNF inhibitor.

22. The method according to claim 21, wherein the TNF inhibitor is an antisense oligonucleotide against TNF, infliximab, adalimumab, certolizumab pegol, golimumab, etanercept (Enbrel), thalidomide, lenalidomide, pomalidomide, xanthine derivative, bupropion, 5-HT2A agonist, or hallucinogen.

23. The method according to any one of claims 3 to 22, wherein the anti-inflammatory agent comprises an IL-8 inhibitor.

24. The method according to claim 23, wherein the IL-8 inhibitor is an antisense oligonucleotide against IL8, HuMab-10F8, reparixin, curcumin, antiloikinate, macrolide, or trifluoroacetate.

25. The method according to any one of claims 3 to 24, wherein the anti-inflammatory agent comprises an IL-18 inhibitor.

26. The method according to claim 25, wherein the IL-18 inhibitor is an antisense oligonucleotide against IL-18, an IL-18 binding protein, an IL-18 antibody, NSC201631, NSC61610, or NSC80734.

27. The method according to any one of claims 3 to 26, wherein the anti-inflammatory agent comprises an inhibitor of natural killer cells.

28. The method according to claim 27, wherein the inhibitor of natural killer cells is an antibody targeting natural killer cells.

29. The method according to any one of claims 1 to 28, wherein the anti-inflammatory agent comprises methotrexate.

30. The method according to any one of claims 1 to 29, wherein the anti-inflammatory agent comprises alclofenate.

31. The method according to any one of claims 1 to 30, wherein the lipid-lowering agent comprises a proprotein convertase subtilisin / kexin type 9 (PCSK9) inhibitor.

32. The method according to any one of claims 1 to 30, wherein the PCSK9 inhibitor is a natural PCSK9 inhibitor, a PCSK9 antibody, an antisense nucleic acid, a peptide inhibitor, a PCSK9 vaccine, or a small molecule inhibitor.

33. The method according to claim 32, wherein the natural PCSK9 inhibitor is berberine, annexin A2, or adnectin.

34. The method according to claim 32, wherein the small molecule inhibitor is PF-06446846, anacetrapib, or K-312.

35. The method according to claim 32, wherein the PCSK9 antibody is alirocumab, evolocumab, 1D05-IgG2, RG-7652, LY3015014, or bococizumab.

36. The method according to claim 32, wherein the antisense nucleic acid is an RNAi molecule.

37. The method according to claim 36, wherein the RNAi molecule is inclisiran or ALN-PCS.

38. The method according to claim 32, wherein the peptide inhibitor is a peptide mimicking the EGFa domain of the low density lipoprotein receptor (LDL-R).

39. The method according to claim 32, wherein the PCSK9 vaccine comprises an antigenic PCSK9 peptide.

40. The method according to any one of claims 1 to 39, wherein the lipid-lowering agent comprises an HMG-CoA reductase inhibitor.

41. The method according to claim 40, wherein the HMG-CoA reductase inhibitor is a statin.

42. The method according to claim 41, wherein the statin is simvastatin, lovastatin, pravastatin, fluvastatin, atorvastatin, cerivastatin, rosuvastatin, or pitavastatin.

43. The method according to any one of claims 1 to 42, wherein the lipid-lowering agent is a fibrate, a bile acid sequestrant, a resin, a nicotinic acid agent, a cholesterol absorption inhibitor, an acyl coenzyme A:cholesterol acyltransferase (ACAT) inhibitor, a cholesteryl ester transfer protein (CETP) inhibitor, an LDL receptor antagonist, a farnesoid X receptor (FXR) antagonist, a sterol regulatory element-binding protein cleavage-activating protein (SCAP) activator, a microsomal triglyceride transfer protein (MTP) inhibitor, a squalene synthase inhibitor, or a peroxisome proliferator-activated receptor (PPAR) agonist.

44. The method according to any one of claims 1 to 43, wherein the lipid-lowering agent and the anti-inflammatory agent are administered together.

45. The method according to any one of claims 1 to 43, wherein the lipid-lowering agent and the anti-inflammatory agent are administered separately.

46. The method according to any one of claims 1 to 45, wherein the lipid-lowering agent and / or the anti-inflammatory agent is administered intranasally, intravenously, intramuscularly, subcutaneously, or orally.

47. The method according to any one of claims 1 to 46, wherein the level or activity of a pro-inflammatory cytokine in the subject is decreased.

48. The method according to any one of claims 1 to 47, wherein the level or activity of C-reactive protein (CRP) in the subject is decreased.

49. The method according to any one of claims 1 to 48, wherein the level or activity of non-high density lipoprotein (HDL)-cholesterol in the subject is decreased.

50. The method according to any one of claims 1 to 49, wherein the level or activity of LDL-cholesterol in the subject is decreased.

51. The method according to any one of claims 1 to 50, wherein the level or activity of total cholesterol in the subject is decreased.

52. The method according to any one of claims 1 to 51, wherein the level or activity of apolipoprotein B (ApoB) in the subject is decreased.

53. The method according to any one of claims 1 to 52, wherein the level or activity of triglyceride in the subject is decreased.

54. The method according to any one of claims 1 to 53, wherein the ratio of total cholesterol to HDL-cholesterol in the subject decreases.

55. The method according to any one of claims 1 to 54, wherein the occurrence of non-fatal myocardial infarction decreases.

56. The method according to any one of claims 1 to 54, wherein the occurrence of non-fatal stroke decreases.

57. The method according to any one of claims 1 to 54, wherein the rate of cardiovascular mortality decreases.

58. The method according to any one of claims 1 to 57, wherein the cardiovascular disease is myocardial infarction, stroke, acute coronary syndrome, myocardial ischemia, chronic stable angina, unstable angina, cardiovascular death, coronary restenosis, coronary stent restenosis, coronary stent rethrombosis, recurrent cardiovascular event, revascularization, angioplasty, transient ischemic attack, pulmonary embolism, vascular occlusion, or venous thrombosis.

59. A method for reducing the recurrence rate of cardiovascular disease in a subject who has been or is being treated with a lipid-lowering agent, the method comprising administering to the subject an effective amount of an anti-inflammatory agent.

60. A method for predicting the recurrence rate of cardiovascular disease in a subject who has been or is being treated with a lipid-lowering agent, the method comprising measuring the level of C-reactive protein (CRP) in the subject, and determining that the subject has a potential for recurrence of cardiovascular disease when the CRP level is higher than a predetermined value.

61. The method according to claim 60, wherein the predetermined value is 3 mg / L.

62. The method according to claim 60, wherein the predetermined value is 2 mg / L.

63. The method according to claim 60, wherein the predetermined value is 1 mg / L.

64. A method for treating cardiovascular disease, the method comprising administering to a subject in need thereof a therapeutically effective amount of a bispecific antibody comprising a first antigen-binding domain that binds to a proinflammatory cytokine and a second antigen-binding domain that binds to proprotein convertase subtilisin / kexin type 9 (PCSK9).

65. The method according to claim 64, wherein the proinflammatory cytokine is IL-1, IL-1 receptor (IL-1R), IL-6, IL-6 receptor (IL-6R), NLRP3, TNF, IL-8, or IL-18.

66. The method according to claim 65, wherein the first antigen-binding domain binds to IL-1. **Claim 67** The method according to claim 66, wherein the first antigen-binding domain binds to IL-1α. **Claim 68** The method according to claim 66, wherein the first antigen-binding domain is derived from MABp1. **Claim 69** The method according to claim 66, wherein the first antigen-binding domain binds to IL-1β. **Claim 70** The method according to claim 69, wherein the first antigen-binding domain is derived from canakinumab, diacerein, gevokizumab, or LY2189102. **Claim 71** The method according to claim 65, wherein the first antigen-binding domain binds to IL-1R. **Claim 72** The method according to claim 71, wherein the first antigen-binding domain is derived from MEDI-8968 or AMG108. **Claim 73** The method according to claim 65, wherein the first antigen-binding domain binds to IL-6. **Claim 74** The method according to claim 73, wherein the first antigen-binding domain is derived from siltuximab, sirukumab, clazakizumab, orolizumab, or elsilimomab. **Claim 75** The method according to claim 65, wherein the first antigen-binding domain binds to IL-6R. **Claim 76** The method according to claim 75, wherein the first antigen-binding domain is derived from tocilizumab, sarilumab, PM1, AUK12-20, AUK64-7, AUK146-15, or AB-227-NA. **Claim 77** The method according to claim 65, wherein the first antigen-binding domain binds to NLRP3. **Claim 78** The method according to claim 77, wherein the first antigen-binding domain is derived from an anti-NLRP3 antibody. **Claim 79** The method according to claim 65, wherein the first antigen-binding domain binds to TNF. **Claim 80** The method according to claim 79, wherein the first antigen-binding domain is derived from infliximab, adalimumab, certolizumab pegol, golimumab, or etanercept (Enbrel). **Claim 81** The method according to claim 65, wherein the first antigen-binding domain binds to IL-8. **Claim 82** The method according to claim 81, wherein the first antigen-binding domain is derived from HuMab-10F8 **Claim 83** The method according to claim 65, wherein the first antigen-binding domain binds to IL-18. **Claim 84** The method according to claim 83, wherein the first antigen-binding domain is derived from an IL-18 antibody. **Claim 85** The method according to any one of claims 64 to 84, wherein the second antigen-binding domain is derived from alemtuzumab, evolocumab, 1D05-IgG2, RG-7652, LY3015014, or bococizumab.

86. The method according to any one of claims 64 to 85, wherein the bispecific antibody comprises a common Fc region.

87. The method according to any one of claims 64 to 86, wherein the bispecific antibody is a monoclonal bispecific antibody.

88. The method according to any one of claims 64 to 87, further comprising administering to the subject a therapeutically effective amount of an HMG-CoA reductase inhibitor.

89. The method according to claim 88, wherein the HMG-CoA reductase inhibitor is a statin.

90. The method according to claim 89, wherein the statin is simvastatin, lovastatin, pravastatin, fluvastatin, atorvastatin, cerivastatin, rosuvastatin, or pitavastatin.

91. The method according to any one of claims 64 to 89, wherein the bispecific antibody is administered intravenously, intramuscularly, subcutaneously, or orally.

92. The method according to any one of claims 64 to 91, wherein the level or activity of pro-inflammatory cytokines in the subject is decreased.

93. The method according to any one of claims 64 to 92, wherein the level or activity of C-reactive protein (CRP) in the subject is decreased.

94. The method according to any one of claims 64 to 93, wherein the level or activity of non-high density lipoprotein (HDL)-cholesterol in the subject is decreased.

95. The method according to any one of claims 64 to 94, wherein the level or activity of LDL-cholesterol in the subject is decreased.

96. The method according to any one of claims 64 to 95, wherein the level or activity of total cholesterol in the subject is decreased.

97. The method according to any one of claims 64 to 96, wherein the level or activity of apolipoprotein B (ApoB) in the subject is decreased.

98. The method according to any one of claims 64 to 97, wherein the level or activity of triglycerides in the subject is decreased.

99. The method according to any one of claims 64 to 98, wherein the ratio of total cholesterol to HDL-cholesterol in the subject is decreased.

100. The method according to any one of claims 64 to 99, wherein the occurrence of non-fatal myocardial infarction is reduced.

101. The method according to any one of claims 64 to 99, wherein the occurrence of non-fatal stroke is reduced.

102. The method according to any one of claims 64 to 101, wherein the rate of cardiovascular mortality is reduced.

103. A method of treating a cardiovascular disease, comprising administering to a subject in need thereof a therapeutically effective amount of a bispecific antibody comprising a first antigen-binding domain that binds to IL-1 and a second antigen-binding domain that binds to proprotein convertase subtilisin / kexin type 9 (PCSK9).

104. The method according to claim 103, wherein the first antigen-binding domain binds to IL-1α.

105. The method according to claim 104, wherein the first antigen-binding domain is derived from MABp1.

106. The method according to claim 103, wherein the first antigen-binding domain binds to IL-1β.

107. The method according to claim 106, wherein the first antigen-binding domain is derived from canakinumab, diacerein, gevokizumab, or LY2189102.

108. The method according to any one of claims 103 to 107, wherein the second antigen-binding domain is derived from alirocumab, evolocumab, 1D05-IgG2, RG-7652, LY3015014, or bococizumab.

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