WO2015003246A1

WO2015003246A1 - Combination of a statin with an inflammasome inhibitor

Info

Publication number: WO2015003246A1
Application number: PCT/CA2014/000559
Authority: WO
Inventors: Jonathan SCHERTZER; Gregory STEINBERG; Mark TARNOPOLSKY
Original assignee: Mcmaster University
Priority date: 2013-07-09
Filing date: 2014-07-09
Publication date: 2015-01-15

Abstract

A composition comprising a statin in combination with an NLRP3 inflammasome inhibitor is provided, as well as a treatment method which includes administration of a statin and NLRP3 inflammasome inhibitor to a mammal. The composition and method are useful to overcome undesirable side effects associated with the use of statin.

Description

COMBINATION OF A STATIN WITH AN INFLAMMASOME INHIBITOR Field of the Invention

[0001] The present invention generally relates to statin treatments and compositions, and more particularly, to compositions of statin combined with an inflammasome inhibitor.

Background of the Invention

[0002] Statins inhibit 3-hydroxy-3-methyl-glutaryl-CoA (HMG-CoA) reductase, a rate-limiting enzyme in cholesterol biosynthesis and reduce low-density lipoprotein (LDL) cholesterol levels. Statins have actions beyond lipid lowering effects, including modulation of immune function. Statins decrease intermediates in the mevalonate pathway that lie upstream of cholesterol formation; reducing protein prenylation, a post-translational lipid modification that occurs on many proteins, including those involved in immune responses. Immune proteins such as pattern recognition receptors (PRRs) have emerged as integrators of nutrient and pathogen sensing systems, and the inflammation during obesity (i.e. metaflammation) has been characterized in terms of excess nutrients and energy. Drug-mediated changes in inflammation via engagement of PRRs should also be considered, particularly for therapeutics such as statins that are used to treat aspects of metabolic disease. Statin-mediated decreases in protein prenylation are generally associated with anti-inflammatory responses and can reduce TNF and IL-6 in lipopolysaccharide (LPS)-treated peripheral blood.

[0003] In contrast, statins have been associated with increased secretion of the proinflammatory cytokine IL-Ι β; an effect that requires caspase-1 activity and priming with another immunogenic agent such as LPS. Statin therapy has also been associated with increased incidence of type 2 diabetes; as high as 48% in certain populations.

[0004] Accordingly, it would be desirable to overcome one or more of the limitations associated with statin therapy.

Summary of the Invention

[0005] It has now been determined that drawbacks associated with statin treatment, e.g. risk of type 2 diabetes and/or myopathy, are attenuated when statin is used in conjunction with an NLRP3 inflammasome inhibitor. [0006] Thus, in one aspect of the present invention, a composition comprising a statin in combination with an NLRP3 inflammasome inhibitor is provided.

[0007] In another aspect of the present invention, a treatment method is provided comprising the step of administering statin to a mammal in conjunction with an NLRP3 inflammasome inhibitor.

[0008] These and other aspects will become apparent from the following detailed description by reference to the figures.

Brief Description of the Figures

[0009] Figure 1 graphically illustrates quantity of IL-l (A) in media when macrophages treated with various statins (10 μΜ, 18 h) and/or LPS (200 ng/mL, final 4 h); quantity of IL-Ι β (B) and IL-6 (C) in media when macrophages treated with various doses of fluvastatin and/or LPS; quantity of IL-i (D) in media when macrophages treated with LPS and 1 μΜ of fluvastatin combined with vehicle or 10 μΜ GGPP; quantity of IL-Ι β (E) in media when macrophages treated with LPS and 1 μΜ of fluvastatin combined with vehicle or 10 μΜ z-WHED-FMK or 200 μΜ glyburide; transcript levels of cytokines (F) and PRRs (G) in macrophages after 1 μΜ fluvastatin (18h) and 200 ng/mL LPS (4 h) treatments; and quantity of IL-Ι β (H) and IL-6 (I) in the media when macrophages from NLRP3^"7" mice were treated various doses of fluvastatin and/or 200 ng/mL LPS. *-significantly different from LPS alone or as indicated by connecting bars, φ-significantly different from conditions without LPS. #- significantly different from fluvastatin plus LPS. ^A-significantly different from control or statin alone. Statin = fluvastatin. Values are means + SEM, n > 3 for all conditions.

[0010] Figure 2 illustrates in vivo insulin-stimulated 2-deoxy-D-glucose uptake (A) in brown adipose tissue (BAT) and white adipose tissue (WAT) from ob/ob mice orally treated with vehicle (Control) or fluvastatin (Statin) for 6 weeks (n > 3 mice per group); caspase-1 (B) and caspase-3 (C) activity in adipose tissue explants from WT mice and NLRP3^"/_ mice (n>7), where explants were treated with vehicle (control), LPS ^g/mL) plus 10 μΜ fluvastatin (L+S) or L+S plus 10 μΜ glyburide (L+S+Glyb); quantification of IL-Ι β (D) in adipose tissue lysates from WT and NLRP3^"7" mice after treatment of explants with vehicle (control), LPS, fluvastatin or LPS plus fluvastatin; representative immunoblots (E) and quantification (F) of basal (Bas; i.e. no insulin) and insulin- mediated phosphorylation of Akt (serine 473) after treatment with fluvastatin and/or LPS in adipose tissue explants from WT and NLRP3^"A mice (n>6); and representative immunoblots (G) and quantification (H) basal and insulin-mediated phosphorylation of Akt after treatment with fluvastatin and LPS with various doses of glyburide in adipose tissue explants from WT mice, φ-significantly different from control or basal; ε-significantly different from L+S. ^A-significantly different from LPS alone in WT. "-significantly different from vehicle control (with insulin). Statin = fluvastatin. Values are means + SEM, n > 8 explants per group.

[001 1] Figure 3 illustrates time-course (A) and quantification (B) of relative caspase-1 activity in 3T3-L1 adipocytes after treatment with vehicle (Control) or LPS plus fluvastatin (L+S) (n > 7 / group); quantification of IL-Ι β (C) and IL-6 (D) secreted in the media after control or L+S (n > 8 / group); representative immunoblots (E) and quantification (F) of 0.3 nM and 100 nM insulin- stimulated phosphorylation of Akt (serine 473) in 3T3-L1 adipocytes after treatment with control or L+S (E; n = 4 /group). * Significantly different from control (no insulin). "Significantly different from control at the same dose of insulin. Statin = fluvastatin. nd = not detected. Values are means + SEM.

[0012] Figure 4 graphically demonstrates transcript levels of various genes in muscle biopsy tissue from control versus statin-induced myopathy patients (4A-E); caspase-1 activity in muscle biopsy tissue control versus statin-induced myopathy patients (4G); and the effect of a statin with or without inflammasome inhibition (with glyburide) on the levels of an atrogene involved in myopathy in muscle cells (4H).

[0013] Figure 5 graphically displays the expression of fatty acid synthase (FASN) in liver cells from WT and NLRP3-/- mice with and without statin treatment.

[0014] Figure 6 illustrates the nucleic acid (A) and protein (B) sequence of human NLRP3.

[0015] Figure 7 illustrates the nucleic acid (A) and protein (B) sequence of human caspase-1.

Detailed Description of the Invention

[0016] A composition comprising a statin in combination with an NLRP3 inflammasome inhibitor is provided.

[0017] The term "statin" refers to compounds that inhibit HMG-CoA reductase. Examples of suitable statins for use in the present composition include, but are not limited to, fluvastatin, atorvastatin, simvastatin, cerivastatin, pitavastatin, rosuvastatin, lovastatin, pravastatin, compactin and dalvastatin.

[0018] The term "NLRP3" refers to NOD-like receptor family, pyrin domain containing 3) inflammasome or NACHT, LRR and PYD domains-containing protein 3 (NALP3), also known as cryopyrin, cold induced autoinflammatory syndrome 1 (CIAS1), caterpiller-like receptor 1.1 (CLR1.1) or Pyrin Domain-Containing Apafl-Like Protein 1 (PYPAF1). NLRP3 is a component of a multiprotein oligomer consisting of the NLRP3 protein, ASC (apoptosis-associated speck-like protein containing a CARD) and pro-caspase 1. It is involved in inflammation and the immune response. NLRP3 is encoded by the NLRP3 gene, the sequence of which is shown in Fig. 6. As one of skill in the art will appreciate, variants of the NLRP3 gene may exist which encode functionally equivalent NLRP3 which maintain function, at least in part, to activate caspase-1 and/or to promote the maturation of inflammatory cytokines such as Interleukin 1 β and Interleukin 18. Such functionally equivalent NLRP3 may, thus, incorporate amino acid substitutions, deletions or additions that do not abolish activity.

[0019] NLRP3 inflammasome inhibitor refers to compounds which inhibit or at least reduce the activity of the inflammasome, including glyburide and functionally equivalent precursors or derivatives thereof, caspase-1 inhibitors, adenosine monophosphate-activated protein kinase (AMPK) activators and P2X7 inhibitors. Inhibition of NLRP3 inflammasome may be achieved by a single compound or a combination of compounds that inhibit the inflammasome or caspase-1, but which do not result in changes to cytochrome P450 (cyp) enzyme activity, including cyp isoforms, 3A4, 2C9 and 2C19, that would adversely affect the metabolism of statins and thereby reduce the bioavailabilty of statins.

[0020] In one embodiment, the inhibitor is a sufonylurea drug such as glyburide, including functionally equivalent derivatives thereof, for example, glyburide precursors or derivatives that lack the cyclohexylurea moiety, or functionally equivalent precursors or derivatives that contain the sulfonyl and benamido groups. Examples include 5-chloro-2-methoxy-N-[2-(4-sulfamoylphenyl)-ethyl]- benzamide and 1 - [(4-methylbenzene)sulfonyl] - 1 H- 1 ,3 -benzodiazol-2-amine. Functionally equivalent precursors or derivatives of glyburide include precursors or derivatives that retain the activity of glyburide, at least in part, to inhibit or reduce the activity of NLRP3 inflammasome, e.g. retain at least about 25% of the activity of glyburide, preferably about 50% of glyburide activity, for example, at least about 70%, 80%, or 90% if glyburide activity. [0021] In another embodiment, the inhibitor is a caspase-1 inhibitor. The caspase-1 inhibitor may be a direct inhibitor of caspase-1 enzymatic activity, or may be an indirect inhibitor that inhibits initiation of inflammasome assembly or infiammasome signal propagation. Caspase-1 inhibitors for use in the present invention may be antioxidants, including reactive oxygen species (ROS) inhibitors. Examples of such caspase-1 inhibitors include, but are not limited to, flavonoids including flavones such as apigenin, luteolin, and diosmin; flavonols such as myricetin, fisetin and quercetin; flavanols and polymers thereof such as catechin, gallocatechin, epicatechin, epigallocatechin, epigallocatechin-3- gallate and theaflavin; isoflavone phytoestrogens; and stilbenoids such as resveratrol. Also included are phenolic acids and their esters such as gallic acid and salicyclic acid; terpenoids or isoprenoids such as andrographolide and parthenolide; vitamins such as vitamins A, C and E; vitamin cofactors such as co-enzyme Q10, manganese and iodide, other organic antioxidants such as citric acid, oxalic acid, phytic acid and alpha-lipoic acid, and Rhus verniciflua stokes extract. The caspase-1 inhibitor may be a combination of these compounds, for example, a combination of a-lipoic acid, co-enzyme Q10 and vitamin E, or a combination of a caspase 1 inhibitor(s) with another inflammasome inhibitor such as glyburide or a functionally equivalent precursor or derivative thereof.

[0022] The caspase-1 inhibitor may be a small molecule inhibitor, as one of skill in the art will appreciate. Non-limiting examples include cyanopropanate-containing molecules such as (S)-3-((S)-l- ((S)-2-(4-amino-3-chlorobenzamido)-3,3-dimethylbutanoyl)pyrrolidine-2-carboxamido)-3-cyano- propanoic acid, as well as other small molecule caspase-1 inhibitors such as (S)-l-((S)-2-{[l-(4-amino- 3 -chloro-phenyl)-methanoyl] -amino } -3 ,3 -dimethyl-butanoyl)-pyrrolidine-2-carboxylic acid ((2R,3 S)- 2-ethoxy-5-oxo-tetrahydro-furan-3-yl)-amide. Such inhibitors may be chemically synthesized.

[0023] NLRP3 inflammasome or caspase-1 may also be inhibited using immunological inhibitors such as monoclonal antibodies prepared using the well-established hybridoma technology developed by Kohler and Milstein (Nature 256, 495-497(1975)). Hybridoma cells can be screened immunochemically for production of antibodies specifically reactive with a selected NLRP3 or caspase-1 protein region, the full amino acid sequences of which is known in the art and provided herein (see Figs. 6 and 7) and the monoclonal antibodies can be isolated. The term "antibody" as used herein is intended to include fragments thereof which also specifically react with a NLRP3 or caspase-1 protein according to the invention, as well as chimeric antibody derivatives, i.e., antibody molecules resulting from the combination of a variable non-human animal peptide region and a constant human peptide region. [0024] The inflammasome inhibitor may also be an oligonucleotide inhibitor using, for example, anti-sense or RNA interference inhibitors such as siRNA. NLRP3- or caspase-1- encoding nucleic acid molecules, such as that provided herein, may be used to prepare oligonucleotide inhibitors effective to bind to NLRP3 or caspase-1 nucleic acid and inhibit the expression thereof. The term "antisense oligonucleotide" as used herein means a nucleotide sequence that is complementary to at least a portion of a target NLRP3 or caspase-1 nucleic acid sequence. The term "oligonucleotide" refers to an oligomer or polymer of nucleotide or nucleoside monomers consisting of naturally occurring bases, sugars, and intersugar (backbone) linkages. The antisense oligonucleotides of the present invention may be ribonucleic or deoxyribonucleic acids and may contain naturally occurring bases including adenine, guanine, cytosine, thymidine and uracil, or modified bases such as xanthine, hypoxanthine, 2-aminoadenine, 6-methyl, 2-propyl and other alkyl adenines, 5-halo uracil, 5-halo cytosine, 6-aza thymine, pseudo uracil, 4-thiouracil, 8-halo adenine, 8-aminoadenine, 8-thiol adenine, 8-thiolalkyl adenines, 8-hydroxyl adenine and other 8-substituted adenines, 8-halo guanines, 8-amino guanine, 8-thiol guanine, 8-thiolalkyl guanines, 8-hydrodyl guanine and other 8-substituted guanines, other aza and deaza uracils, thymidines, cytosines, adenines, or guanines, 5-tri-fluoromethyl uracil and 5-trifluoro cytosine. Antisense oligonucleotides of the invention may also contain modified phosphorous, oxygen heteroatoms in the phosphate backbone, short chain alkyl or cycloalkyl intersugar linkages or short chain heteroatomic or heterocyclic intersugar linkages. For example, the antisense oligonucleotides may contain phosphorothioates, phosphotriesters, methyl phosphonates and phosphorodithioates. In addition, the antisense oligonucleotides may contain a combination of linkages, for example, phosphorothioate bonds may link only the four to six 3 '-terminal bases, may link all the nucleotides or may link only 1 pair of bases.

[0025] The antisense oligonucleotides of the invention may comprise nucleotide analogs that may be better suited for therapeutic use. An example of an oligonucleotide analogue is a peptide nucleic acid (PNA) in which the deoxribose (or ribose) phosphate backbone in the DNA (or RNA), is replaced with a polymide backbone which is similar to that found in peptides. Other oligonucleotide analogues may contain nucleotides having polymer backbones, cyclic backbones, or acyclic backbones. For example, the nucleotides may have morpholino backbone structures (U.S. Pat. No. 5,034,506). Oligonucleotide analogues may also contain groups such as reporter groups, protective groups and groups for improving the pharmacokinetic properties of the oligonucleotide. Antisense oligonucleotides may also incorporate sugar mimetics as will be appreciated by one of skill in the art. [0026] Antisense nucleic acid molecules may be constructed using chemical synthesis and enzymatic ligation reactions using procedures known in the art based on a given NLRP3 or caspase-1 nucleic acid sequence such as that provided herein. The antisense nucleic acid molecules of the invention, or fragments thereof, may be chemically synthesized using naturally occurring nucleotides or variously modified nucleotides. The antisense sequences may also be produced biologically. In this case, an antisense encoding nucleic acid is incorporated within an expression vector that is then introduced into cells in the form of a recombinant plasmid, phagemid or attenuated virus in which antisense sequences are produced under the control of a high efficiency regulatory region, the activity of which may be determined by the cell type into which the vector is introduced.

[0027] In another embodiment, siRNA technology can be applied to inhibit expression of

NLRP3 or caspase-1. Application of nucleic acid fragments such as siRNA fragments that correspond with regions in an NLRP3 or caspase-1 gene and which selectively target an NLRP3 or caspase-1 gene may be used to block NLRP3 or caspase-1 expression, and thereby inhibit NLRP3 activity. Such blocking occurs when the siRNA fragments bind to the NLRP3 or caspase-1 gene, thereby preventing translation of the gene to yield functional NLRP3 or caspase-1. SiRNA fragments are generally 15-40 ribonucleotides in length, and preferably 20-30 ribonucleotides.

[0028] SiRNA, small interfering RNA molecules, corresponding to NLRP3 are made using well-established methods of nucleic acid syntheses as outlined above with respect to antisense oligonucleotides. Since the structure of target NLRP3 and caspase-1 genes is known, fragments of RNA that correspond therewith can readily be made. The effectiveness of selected siRNA to block NLRP3 or caspase-1 activity can be confirmed using a NLRP3- or caspase-1- expressing cell line, respectively. Briefly, selected siRNA may be incubated with a NLRP3- or caspase-1- expressing cell line under appropriate growth conditions. Following a sufficient reaction time, i.e. for the siRNA to bind with the target mRNA to result in decreased levels of translatable mRNA, the reaction mixture is tested to determine if such a decrease has occurred. Suitable siRNA will prevent processing of the NLRP3 or caspase-1 gene to yield functional NLRP3 or caspase-1 protein. This can be detected by assaying for NLRP3 or caspase-1 activity in a cell-based assay, as described herein.

[0029] It will be appreciated by one of skill in the art that siRNA fragments useful in the present method may be derived from specific regions of NLRP3- or caspase-1- encoding nucleic acid which may provide more effective inhibition of gene expression, for example, the 5' end of the gene. In addition, as one of skill in the art will appreciate, useful siRNA fragments may not correspond exactly with a NLRP3 or caspase-1 target gene, but may incorporate sequence modifications, for example, addition, deletion or substitution of one or more of the nucleotide bases therein, provided that the modified siRNA retains it ability to bind to the target NLRP3 or caspase-1 gene. Selected siRNA fragments may additionally be modified in order to yield fragments that are more desirable for use. For example, siRNA fragments may be modified to attain increased stability in a manner similar to that described for antisense oligonucleotides.

[0030] The present composition may additionally include a pharmaceutically acceptable carrier or adjuvant. The expression "pharmaceutically acceptable" means acceptable for use in the pharmaceutical arts, i.e. not being unacceptably toxic, or otherwise unsuitable for administration to a mammal. Examples of pharmaceutically acceptable adjuvants include, but are not limited to, diluents, excipients and the like. Reference may be made to "Remington's: The Science and Practice of Pharmacy", 21st Ed., Lippincott Williams & Wilkins, 2005, for guidance on drug formulations generally. The selection of adjuvant depends on the intended mode of administration of the composition. In one embodiment of the invention, the compounds are formulated for administration by infusion, or by injection either subcutaneously or intravenously, and are accordingly utilized as aqueous solutions in sterile and pyrogen-free form and optionally buffered or made isotonic. Thus, the compounds may be administered in distilled water or, more desirably, in saline, phosphate-buffered saline or 5% dextrose solution. Compositions for oral administration via tablet, capsule, lozenge, solution or suspension in an aqueous or non-aqueous liquid, an oil-in-water or water-in-oil liquid emulsion, an elixir or syrup are prepared using adjuvants including sugars, such as lactose, glucose and sucrose; starches such as corn starch and potato starch; cellulose and derivatives thereof, including sodium carboxymethylcellulose, ethylcellulose and cellulose acetates; powdered tragancanth; malt; gelatin; talc; stearic acids; magnesium stearate; calcium sulfate; vegetable oils, such as peanut oils, cotton seed oil, sesame oil, olive oil and corn oil; polyols such as propylene glycol, glycerine, sorbital, mannitol and polyethylene glycol; agar; alginic acids; water; isotonic saline and phosphate buffer solutions. Wetting agents, lubricants such as sodium lauryl sulfate, stabilizers, tableting agents, disintegrating agents, antioxidants, preservatives, colouring agents and flavouring agents may also be present. In another embodiment, the composition may be formulated for application topically as a cream, lotion or ointment. For such topical application, the composition may include an appropriate base such as a triglyceride base. Such creams, lotions and ointments may also contain a surface-active agent and other cosmetic additives such as skin softeners and the like as well as fragrance. Aerosol formulations, for example, for nasal delivery, may also be prepared in which suitable propellant adjuvants are used. Compositions of the present invention may also be administered as a bolus, electuary, or paste. Compositions for mucosal administration are also encompassed, including oral, nasal, rectal or vaginal administration for the treatment of infections, which affect these areas. Such compositions generally include one or more suitable non-irritating excipients or carriers comprising, for example, cocoa butter, polyethylene glycol, a suppository wax, a salicylate or other suitable carriers. Other adjuvants may also be added to the composition regardless of how it is to be administered, which, for example, may aid to extend the shelf-life thereof.

[0031] The present composition may be formulated for immediate, sustained-release or a combination of immediate-release and sustained-release dosing, using technology well-established in the art.

[0032] The present composition includes a therapeutically effective amount of statin, i.e. an amount of statin effective, for example, to inhibit HMG-CoA, reduce protein prenylation, reduce plasma levels of low-density lipoprotein cholesterol, or increase plasma levels of high density lipoprotein/cholesterol. Generally, the composition will include an amount of statin in the range of about 1-100 mg. The amount of statin in the composition will vary with the particular statin in the composition and the treatment to be effected. For example, effective doses for pitavastatin are in the range of about 1-5 mg, and for rosuvastatin, effective dosages range from about 5-40 mg.

[0033] The composition also includes a therapeutically effective amount of inflammasome inhibitor, namely, an amount sufficient to reduce or inhibit statin-induced NLRP3 inflammasome activation so as to reduce caspase-1 activity and/or reduce secretion of pro-inflammatory cytokines such as IL-Ι β or IL-18, or an amount sufficient to minimize, prevent or reverse statin-induced decreases in insulin secretion or decreases in insulin action such as cellular glucose uptake or protein kinase B phosphorylation. In this regard, it will be appreciated that the dosage of inflammasome inhibitor will vary with the inhibitor utilized as would be appreciated by those of skill in the art. Examples of dosages of some inflammasome inhibitors are as follows: apigenin (about 0.1-10 mg/kg), Luteolin (about 1-100 mg), Diosmin (about 100-900 mg), Myricetin (about 10-300 mg), Quercetin (about 10-1000 mg), Fisetin (1-200 mg/kg), Rhus verniciflua stokes extract (1 -100 mg/kg), Catechin (about 50-500 mg), Gallocatechin (about 100-1000 mg), Epicatechin (about 0.1-10 mg/kg), Epigallocatechin (about 100-1000 mg), epigallocatechin-3-gallate (about 100-1000 mg), theaflavin (about 75-750mg), isoflavone phytoestrogens (about 25-250 mg), resveratrol (about 100-lOOOmg), andrographolide (about 100-500mg), parthenolide (about 0.1-50 mg), vitamin A (about 5000-20000 IU), vitamin C (about 100 -2000 mg), co-enzyme Q10 (about 30-500mg), vitamin E (about 10-1000 IU), a-lipoic acid (about 10-lOOOmg), co-enzyme Q10 (30-500 mg), manganese (about 1 -100 mg), a- lipoic acid, co-enzyme Q10 and vitamin E (about 10-lOOOmg, 30-500mg, 10-1000 IU, respectively), glyburide (about 1 -20 mg), and glyburide derivative lacking cyclohexylurea moiety (about 1 -200 mg).

[0034] The present composition may also include one or more additional therapeutic agents which enhance the cholesterol-lowering effect of the statin, an antidiabetic effect, or which provide a related treatment. Examples of such additional therapeutic agents include, but are not limited to, a drug which inibits dietary cholesterol absorption such as ezetimibe, a blood pressure-lowering drug such as amlodipine, drugs which increase HDL (high density lipoproteins) such as niacin, dipeptidyl peptidase-4 (DPP-4) inhibitors such as sitagliptin, GLP-1 (glucagon-like peptide- 1) agonists such as exenatide and liraglutide, antihyperglycemic agents such as metformin or a thiazolidinedione. Suitable dosages of such compounds would be known in the art, but will generally be an amount sufficient to enhance the therapeutic effect of the selected statin, while not affecting the effect of the inflammasome inhibitor.

[0035] In another aspect, a method of treatment or use of the present composition is provided.

The method comprises administering to a mammal in need of statin treatment, a therapeutically effective amount of a statin in conjunction with an NLRP3 inflammasome inhibitor. The term "in conjunction" refers to administration of the statin and inflammasome inhibitor together in combination, or administration thereof independently, either simultaneously or at different times. The term "mammal" refers to human and non-human mammals.

[0036] The statin and NLRP3 inflammasome inhibitor may be administered using any suitable administrable form, and are formulated accordingly, including one or more appropriate carriers and/or adjuvants. Suitable administrable forms include, for example, oral, subcutaneous, intravenous, intraperitoneal, intranasal, enteral, topical, sublingual, intramuscular, intra-arterial, intramedullary, intrathecal, inhalation, ocular, transdermal, vaginal or rectal forms. Thus, the treatment may include administration of each of the statin and NLRP3 inflammasome inhibitor together via the same route of administration, separately via the same route of administration or separately via different routes of administration. In this regard, the statin and inflammasome inhibitor may be administered together in a single oral composition or intravenous solution, together as separate oral dosages forms that may be provided is separate containers or in a blister pack, together in a transdermal patch, rectal suppository or inhalational device.

[0037] The present treatment method advantageously provides a treatment which exhibits one or more advantages over statin treatment alone, including prevention of statin-induced diabetes, prevention or reduction of statin-induced myopathy, enhancement of statin-induced lipid lowering effects, and/or reduction of effective statin dosage.

[0038] Embodiments of the invention are described by way of the following specific examples which are not to be construed as limiting.

Example

[0039] Mice and materials. McMaster University Animal Ethics Review Board approved all procedures. Male WT C57BL/6 (#000664) and leptin-deficient ob/ob (# 000632) mice were from JAX. NLRP3^_/~ mice (>10 generations backcrossed to C57BL/6) were from Professor Nicolas Fasel (Universite de Lausanne, Switzerland) and were kindly provided by Dr. Dana Philpott (University of Toronto, Canada). To determine the effect of chronic statin treatment on insulin-stimulated tissue glucose uptake, ob/ob mice were orally administered 40-50 mg kg fluvastatin or vehicle 5 days a week for 6 weeks, a dose of fluvastatin used in other mouse models. 24 h after the last dose, mice were injected with 2 μθ of H-2-deoxy-D-glucose (2DG) via tail vein, immediately followed by 4U/kg of insulin ( . >.)· Blood samples were taken at baseline, 5, 10, 15, 20 min and analyzed for 2DG. Mice were sacrificed by cervical dislocation and tissues were snap frozen in liquid nitrogen. Brown adipose tissue (BAT) and gonadal white adipose tissue (WAT) were analyzed for 2DG radioactivity with and without deproteinization (0.3M BaOH and 0.3M ZnS04) to calculate rates of tissue specific glucose uptake. Statins were from Cayman Chemical (Ann Arbor, MI). Invivogen (San Diego, CA) supplied ultra-pure LPS {Escherichia coli 0111 :B4). Z-WHED-FMK and caspase-1/3 kits were from R&D Systems (Denver, CO). The MTT kit and all other chemicals were from Sigma (St. Louis, MO).

[0040] Macrophages. Bone-marrow derived macrophages (BMDMs) were cultured 7-10 d in

DMEM containing 10% FBS and 15% L929 conditioned-media. BMDMs were washed in serum- free media and exposed to statin (ΙμΜ fluvastatin, unless otherwise stated) for 18 h in serum-free DMEM and LPS (200ng/mL) was added during the final 4 h. GGPP (Geranylgeranyl-pyrophosphate, 10μΜ),

Z-WHED-FMK (caspase-1 inhibitor, 10μΜ), and glyburide (200μΜ), were used during the statin treatment period. IL-Ι β and IL-6 were quantified by ELISA. Transcript levels were analyzed by quantitative PCR, as described in Schertzer et al. (Diabetes. 2011;60:2206-2215), the relevant contents of which are incorporated herein by reference.

[0041] Adipose explants and adipocytes. Mice were killed by cervical dislocation and PBS- rinsed gonadal adipose tissue was minced into ~5mg pieces in DMEM containing 10% FBS. After 2 h equilibration, explants were placed in serum-free DMEM and exposed to 10μΜ fluvastatin (18 h) and 2μg/mL of LPS (4 h) and were stimulated with 0.3 nM insulin for 10 minutes. Adipose tissue lysates were used for caspase-1/3 activity (over 4 h), immunoblotting or ELISA determination of cytokines, as described in Schertzer et al. 3T3-L1 pre-adipocytes (ATCC, Rockville, MD, USA) were differentiated, and fluvaststin/LPS treated similar to explants. 3T3-L1 media was used for ELISAs and lysates were used to measure caspase-1 enzymatic activity fluorometrically or for immunoblotting after insulin stimulation at 0.3nM or 100 nM for 10 min.

[0042] Statistical Analysis. Significance was determined by unpaired, two-tailed T-tests or

ANOVA, as appropriate. Bonferroni or Tukey's post hoc test was used when appropriate (Prism 4-6, GraphPad Software, USA).

RESULTS

[0043] Statins were found to activate the NLRP3 inflammasome. All statins (10μΜ, 18 h) increased secretion of IL-Ιβ from WT BMDMs compared to LPS alone (Fig 1A). Fluvastatin increased IL-Ι β secretion in a dose-dependent manner only with LPS priming (Fig IB), but LPS alone increased IL-6 secretion in BMDMs (Fig 1C). Fluvastatin up to 100 μΜ did not lower BMDM viability detected using the MTT assay (data not shown). The isoprenyl intermediate GGPP prevented fluvastatin- induced IL-Ι β secretion in LPS-primed BMDMs (Fig ID), indicating that decreased prenylation drives statin-mediated inflammasome activation. Inhibition with z-WHED or glyburide prevented statin- induced IL-Ι β secretion in LPS-primed BMDMs (Fig IE). LPS, but not fluvastatin treatment alone, increased transcript levels of NLRP3, IL-Ι β and IL-6 (Fig IF, G). Therefore, statins alone did not alter inflammasome priming events such as increased NLRP3 transcript levels. The combination of fluvastatin and LPS synergistically increased both IL-Ιβ and IL-6 transcript levels (Fig IF). Fluvastatin did not increase IL-Ι β in LPS primed BMDMs from NLRP3^_/" mice (Fig 1H). LPS exposure increased IL-6 secretion in BMDMs from NLRP3^_/" mice (Fig II). This shows that IL-Ιβ, a cytokine known to be regulated by the NLRP3 inflammasome, is regulated via statins through NLRP3, and that IL-6, a cytokine known to be regulated independently of the NLRP3 inflammation, is not dependent on NLRP3 during statin exposure.

[0044] Fluvastatin impairs adipose tissue insulin signaling via the NLRP3 inflammasome. It was first established that chronic oral administration of fluvastatin impaired insulin-simulated glucose disposal into adipose tissue using an in vivo mouse model of obesity. 2DG uptake was >50% lower in WAT, but not BAT of fluvastatin-treated ob/ob mice (Fig 2A). WAT explants were then used to determine the mechanisms of statin-induced insulin resistance. Fluvastatin increased caspase-1 activity in LPS-primed adipose tissue from WT, but not from NLRP3^"/_ mice (Fig 2B). Glyburide prevented this increased caspase-1 activity (Fig. 2B). Fluvastatin increased caspase-3 activity in LPS-primed adipose tissue from WT and NLRP3^"7" mice independently of glyburide treatment (Fig. 2C). Therefore, fluvastatin activated a NLRP3 -dependent, glyburide sensitive caspase-1 inflammasome in adipose tissue.

[0045] Surprisingly, LPS alone increased IL-Ι in adipose explants from both WT and NLRP3

^~'^~ mice (Fig 2D). Fluvastatin plus LPS further increased IL-Ι β levels compared to LPS in adipose explants from WT, but not NLRP3^"7" mice (Fig 2D). LPS alone did not change the ability of insulin to phosphorylate protein kinase B (pAkt) at serine 473 in adipose tissue explants (Fig 2E). Fluvastatin alone impaired insulin-mediated pAkt in adipose tissue from WT, but not NLRP3^"7" mice (Fig 2E). The combination of LPS and fluvastatin prevented insulin's ability to increase pAkt in adipose tissue explants from WT, but not NLRP3^7" mice (Fig 2E/F). Glyburide reversed fluvastatin-induced suppression of insulin-mediated pAkt in LPS-primed adipose explants, but glyburide did not increase pAkt on its own (Fig. 2G/H).

[0046] Interestingly, changes in caspase-1 activity, but not II- 1 β secretion mirrored statin- induced insulin action in adipose explants. There are many non-adipocyte cell types and potential sources of IL- 1 β processing in adipose tissue, so the adipocyte cell autonomous response was next tested. Fluvastatin plus LPS increased caspase-1 activity in 3T3-L1 adipocytes, but did not increase IL-Ι β or IL-6 secretion (Fig 3A-D). However, fluvastatin plus LPS significantly lowered insulin- stimulated pAkt in 3T3-L1 adipocytes (Fig 3E/F).

[0047] The major side effect of statins, skeletal muscle myopathy, was also linked to the

NLRP3 inflammasome. Vastus lateralis muscle biopsies from statin myopathy patients showed increased levels of NLRP3 transcript levels (P < 0.05), an effect that was not seen for other pattern recognition receptors (PRRs) such as nucleotide-binding oligomerization domain-containing protein 1 (NODI), NOD2 or Toll-like receptor 4 (TLR4) (Fig 4A-D). This increase in NLRP3 transcript is indicative of priming for this inflammasome. Interestingly, IL-18 (P < 0.05), but not IL-Ιβ, transcripts were increased in the muscles of statin myopathy patients (Fig 4E, F). Further, muscle biopsies from statin myopathy patients showed greater than 100% higher caspase-1 activity (P < 0.05, Fig 4E). This indicates that both priming and activation of this inflammasome are increased in statin myopathy. It was further shown that statins increase MuRFl transcripts in C2C12 cells, an effect that is attenuated with glyburide co-treatment (P < 0.05, Fig 4F). This shows that muscle cell-autonomous activation of an atrogene involved in myopathy can be attenuated by the inflammasome inhibitor, glyburide.

[0048] The NLRP3 inflammasome was also shown to be required for statin-induced increases in specific SREBP target genes in hepatocytes. Fluvastatin increased expression of fatty acid synthase (FASN) by over 2-fold (P < 0.05) in primary hepatocytes from WT mice, but not in hepatocytes from NLRP3^"A mice (Figure 5). Certain SREBP-target genes are known to limit the ability of statins to promote favorable lipid profiles. Therefore, combining NLRP3 inflammasome inhibition with statins will augment lipid lowering properties and/or lower the effective dose of statin-therapy.

DISCUSSION

[0049] While statins lower blood lipids and reduce cardiovascular disease-related events, they have also been associated with increased incidence of diabetes. It has been determined that statins activate the NLRP3 inflammasome in various immune and metabolic cells of adipose tissue, and that statin-induced impairments in insulin signaling were dependent upon the NLRP3 inflammasome. Statin combined with an inflammasome-inhibiting compound inhibited statin-induced inflammasome activation and prevented defects in adipose tissue insulin action.

[0050] Statin myopathy was associated with increased NLRP3 levels and increased caspase-1 activity. Statin combined with an inflammasome-inhibiting compound attenuated the statin-induced increase in a muscle cell atrogene involved in statin-induced myopathy.

Claims

1. A composition comprising a statin in combination with an NLRP3 inflammasome inhibitor.

2. The composition of claim 1 , wherein the statin is selected from the group consisting of fluvastatin, atorvastatin, simvastatin, cerivastatin, pitavastatin, rosuvastatin, lovastatin, pravastatin, compactin and dalvastatin.

3. The composition of claim 1, wherein the inflammasome inhibitor is selected from the group consisting of glyburide or functionally equivalent precursors or derivatives thereof, a caspase 1 inhibitor, adenosine monophosphate-activated protein kinase (AMPK) activators, P2X7 inhibitors, and combinations thereof.

4. The composition of claim 3, wherein the inflammasome inhibitor is selected from glyburide, glyburide precursors or derivatives that lack the cyclohexylurea moiety, and glyburide precursors or derivatives that contain sulfonyl and benamido groups.

5. The composition of claim 3, wherein the caspase- 1 inhibitor is selected from the group consisting of flavones, flavonols, flavanols and polymers thereof, isoflavone phytoestrogens, stillbenoids, phenolic acids and their esters, terpenoids, vitamins, vitamin cofactors, citric acid, oxalic acid, phytic acid, alpha-lipoic acid and combinations thereof.

6. The composition of claim 5, wherein the caspase- 1 inhibitor comprises a-lipoic acid, coenzyme Q10 and vitamin E.

7. The composition of claim 1 , wherein the caspase- 1 inhibitor is selected from the group consisting of (S)-3-((S)-l-((S)-2-(4-amino-3-chlorobenzamido)-3,3-dimethylbutanoyl)pyrrolidine-2- carboxamido)-3-cyanopropanoic acid; (S)-l-((S)-2-{[l-(4-amino-3-chloro-phenyl)-methanoyl]- amino} -3, 3 -dimethyl -butanoyl)-pyrrolidine-2-carboxylic acid ((2R,3S)-2-efhoxy-5-oxo-tetrahydro- furan-3-yl)-amide.

8. The composition of claim 1, wherein the statin is fluvastatin, and the inflammasome inhibitor is selected from the group consisting of glyburide, glyburide precursors or derivatives that lack the cyclohexylurea moiety, and glyburide precursors or derivatives that contain sulfonyl and benamido groups.

9. The composition of claim 1, comprising about 1-lOOmg of a statin and an amount of an inflammasome inhibitor sufficient to reduce or inhibit statin-induced NLRP3 inflammasome activation.

10. The composition of claim 1, additionally comprising at least one therapeutic agent selected from an agent which inibits dietary cholesterol absorption, a blood pressure-lowering drug, an agent which increases high density lipoproteins, a dipeptidyl peptidase-4 (DPP-4) inhibitor, a glucagon-like peptide- 1 agonist, an antihyperglycemic agent.

11. The composition of claim 1 , formulated for oral, subcutaneous, intravenous, intraperitoneal, intranasal, enteral, topical, sublingual, intramuscular, intra-arterial, intramedullary, intrathecal, nasal, ocular, transdermal, vaginal or rectal adminstration.

12. A treatment method comprising the step of administering to a mammal a statin in conjunction with an NLRP3 inflammasome inhibitor.

13. The method of claim 12, wherein the statin is selected from the group consisting of fluvastatin, atorvastatin, simvastatin, cerivastatin, pitavastatin, rosuvastatin, lovastatin, pravastatin, compactin and dalvastatin.

14. The method of claim 12, wherein the inflammasome inhibitor is selected from the group consisting of glyburide or functionally equivalent precursors or derivatives thereof, a caspase 1 inhibitor, adenosine monophosphate-activated protein kinase (AMPK) activators, P2X7 inhibitors, and combinations thereof.

15. The method of claim 12, wherein the inflammasome inhibitor is selected from glyburide, glyburide precursors or derivatives that lack the cyclohexylurea moiety, and glyburide precursors or derivatives that contain sulfonyl and benamido groups.

16. The method of claim 12, wherein the caspase- 1 inhibitor is selected from the group consisting of flavones such as apigenin, luteolin, and diosmin; flavonols such as myricetin, fisetin and quercetin; flavanols and polymers thereof such as catechin, gallocatechin, epicatechin, epigallocatechin, epigallocatechin-3-gallate and theaflavin; isoflavone phytoestrogens; stilbenoids such as resveratrol; phenolic acids and their esters such as gallic acid and salicyclic acid; terpenoids such as

andrographolide and parthenolide; vitamins such as vitamins A, C and E; vitamin cofactors such as coenzyme Q10, manganese and iodide; citric acid, oxalic acid, phytic acid, alpha-lipoic acid, Rhus verniciflua stokes extract, and combinations thereof.

17. The method of claim 15, wherein the caspase-1 inhibitor comprises a-lipoic acid, co-enzyme Q10 and vitamin E.

18. The method of claim 12, additionally comprising administering to the mammal at least one therapeutic agent selected from an agent which inibits dietary cholesterol absorption, a blood pressure- lowering drug, an agent which increases high density lipoproteins, a dipeptidyl peptidase-4 (DPP-4) inhibitor, a glucagon-like peptide- 1 agonist, an antihyperglycemic agent.

19. The method of claim 12, wherein the statin is administered in an amount of about 1-lOOmg, and the inflammosome inhibitor is administered in an amount sufficient to reduce or inhibit statin-induced NLRP3 inflammasome activation.

20. The method of claim 12, wherein the statin and inflammasome inhibitor are administered to the mammal either separately or together.