METHODS OF DECREASING VASCULAR CALCIFICATION USING IL l INHIBITORS

This application claims the benefit of U.S. Provisional Application No. 60/729,305, filed October 21, 2005, which is hereby incorporated by reference.

FIELD OF THE INVENTION

This invention relates generally to the field of medicine and, more specifically, to methods of decreasing, treating or preventing vascular calcification.

BACKGROUND OF THE INVENTION

The IL-I system

One of the most potent inflammatory cytokines yet discovered is interleukin-1 (IL-I). IL-I is thought to be a key mediator in many diseases and medical conditions. It is manufactured (though not exclusively) by cells of the macrophage/monocyte lineage and may be produced in two forms: IL-I alpha (IL-lα) and IL-I beta (IL-I β). A third cytokine in the system acts as an antagonist and is referred to as IL-I receptor antagonist (IL- Ira). There are three known IL-I receptor subunits. The active receptor complex consists of the type I receptor (IL-I RI) and IL-I receptor accessoiy protein (IL- IRAcP). IL-I RI is responsible for binding the three naturally occurring ligands (IL-I alpha, IL-I beta and IL- Ira) and is able to do so in the absence of the IL- IRAcP. However, signal transduction requires interaction of IL-I alpha or IL-I beta with IL-IRAcP. IL-lra does not interact with the IL-IRAcP and hence cannot signal. A third receptor subunit, the type II receptor (IL-I RII), binds IL-I alpha or IL-I beta but cannot signal because it lacks an intracellular domain. Instead, it inhibits IL-I bioactivity by acting as a decoy receptor in both a membrane-bound form and a cleaved, secreted form. See Dinarello (1996) Blood 87:2095-2147. IL-lra inhibits IL-I alpha and IL-I beta by binding to IL-I RI but not transducing an intracellular signal or a biological response. IL-lra inhibits the

biological activities of IL-I both in vitro and in vivo, and has been shown to be effective in animal models of septic shock, rheumatoid arthritis, graft versus host disease, stroke, and cardiac ischemia. A recombinant form of IL- Ira produced in E. coli is currently approved for pharmaceutical use in the United States and Europe. This drug has the generic name anakinra and is marketed under the trade name Kineret ^® .

Vascular calcification

Vascular calcification, a well-recognized and common complication of chronic kidney disease (CKD), increases the risk of cardiovascular morbidity and mortality (Giachelli, C. J. Am. Soc. Nephrol. 15: 2959-64, 2004; Raggi, P. et al. J. Am. Coll. Cardiol. 39: 695-701, 2002). While the causes of vascular calcification in CKD remain to be elucidated, associated risk factors include age, gender, hypertension, time on dialysis, diabetes and glucose intolerance, obesity, and cigarette smoking (Zoccali C. Nephrol. Dial. Transplant 15: 454-7, 2000). These conventional risk factors, however, do not adequately explain the high mortality rates from cardiovascular causes in the patient population. Recent observations suggest that certain abnormalities in calcium and phosphorus metabolism, resulting in a raised serum calcium-phosphorus product (Ca x P) contribute to the development of arterial calcification, and possibly to cardiovascular disease, in patients with end-stage renal disease (Goodman, W. et al. N. Engl. J. Med. 342: 1478-83, 2000; Guerin, A. etjl. Nephrol. Dial- Transplant 15:1014-21, 2000; Vattikuti, R. & Towler, D. Am. J. Physiol. Endocrinol. Metab.. 286: E686-96, 2004).

Another hallmark of advanced CKD is secondary hyperparathyroidism (HPT), characterized by elevated parathyroid hormone (PTH) levels and disordered mineral metabolism. The elevations in calcium, phosphorus, and Ca x P observed in patients with secondary HPT have been associated with an increased risk of vascular calcification (Chertow, G. et al. Kidney Int. 62: 245-52, 2002; Goodman, W. eyd. N. Engl. J. Med. 342: 1478-83, 2000; Raggi, P. eUl. J. Am. Coll. Cardiol. 39: 695-701, 2002). Commonly used therapeutic interventions for secondary HPT, such as calcium-based phosphate binders and doses of active

vitamin D sterols can result in hypercalcemia and hyperphosphatemia (Chertow, G. et al- Kidney Int. 62: 245-52, 2002; Tan, A. eUL Kidney Int 51 : 317-23, 1997; Gallieni, M. et al. Kidney Int 42: 1191-8, 1992), which are associated with the development or exacerbation of vascular calcification. . Vascular calcification is an important and potentially serious complication of chronic renal failure. Two distinct patterns of vascular calcification have been identified (Proudfoot, D & Shanahan, C. Herz 26: 245-51, 2001), and it is common for both types to be present in uremic patients (Chen, N. & Moe, S. Semin Nephrol 24: 61-8, 2004). The first, medial calcification, occurs in the media of the vessel in conjunction with a phenotypic transformation of smooth muscle cells into osteoblast-like cells, while the other, atherogenesis, is associated with lipid-laden macrophages and intimal hyperplasia.

Medial wall calcification can develop in relatively young persons with chronic renal failure, and it is common in patients with diabetes mellitus even in the absence of renal disease. The presence of calcium in the medial wall of arteries distinguishes this type of vascular calcification from that associated with atherosclerosis (Schinke T. & Karsenty G. Nephrol Dial Transplant 15: 1272-4, 2000). Atherosclerotic vascular calcification occurs in atheromatous plaques along the intimal layer of arteries (Farzaneh-Far A. JAMA 284: 1515-6, 2000). Calcification is usually greatest in large, well-developed lesions, and it increases with age (Wexler L. et al. Circulation 94: 1175-92, 1996; Rumberger J. et al. Mayo Clin Proc 1999; 74: 243-52.). The extent of arterial calcification in patients with atherosclerosis generally corresponds to severity of disease. Unlike medial wall calcification, atherosclerotic vascular lesions, whether or not they contain calcium, impinge upon the arterial lumen and compromise blood flow. The localized deposition of calcium within atherosclerotic plaques may happen because of inflammation due to oxidized lipids and other oxidative stresses and infiltration by monocytes and macrophages (Berliner J. et al. Circulation 91 : 2488- 96, 1995). Some patients with end-stage renal disease develop a severe form of occlusive arterial disease called calciphylaxis or calcific uremic arteriolopathy.

This syndrome is characterized by extensive calcium deposition in small arteries (Gipstein R. etjL Arch Intern Med 136: 1273-80, 1976; Richens G. et al. J Am Acad. Dermatol. 6: 537-9, 1982). In patients with this disease, arterial calcification and vascular occlusion lead to tissue ischemia and necrosis. Involvement of peripheral vessels can cause ulceration of the skin of the lower legs or gangrene of the digits of the feet or hands. Ischemia and necrosis of the skin and subcutaneous adipose tissue of the abdominal wall, thighs and/or buttocks are features of a proximal form of calcific uremic arteriolopathy (Budisavljevic M. et al. J Am Soc Nephrol. 7: 978-82, 1996; Ruggian J. et al- Am. J. Kidney Pis. 28: 409-14, 1996). This syndrome occurs more frequently in obese individuals, and women are affected more often than men for reasons that remain unclear (Goodman W. J. Nephrol. 15(6): S82-S85, 2002).

Current therapies to normalize serum mineral levels or to decrease, inhibit, or prevent calcification of vascular tissues or implants are of limited efficacy and cause unacceptable side effects. Therefore, there exists a need for an effective method of inhibiting and preventing vascular calcification.

SUMMARY OF THE INVENTION

The present invention provides methods of inhibiting, decreasing, or preventing vascular calcification in a subject comprising administering a therapeutically effective amount of an IL-I inhibitor to the subject. In one aspect, the vascular calcification can be atherosclerotic calcification. In another aspect, the vascular calcification can be medial calcification.

In one aspect, the subject can be suffering from chronic renal insufficiency or end-stage renal disease. In another aspect, the subject can be pre-dialysis. In a further aspect, the subject can be suffering from uremia. In another aspect, the subject can be suffering from diabetes mellitus I or II. In another subject, the subject can be suffering from a cardiovascular disorder. In one aspect, the subject can be human. In one aspect, the IL-I inhibitor can be the molecule having the generic name anakinra. In another aspect, the IL-I inhibitor can be a molecule having the

sequence shown in Figure 4 hereinafter (SEQ ID NO: 1), hereinafter referred to as "Fc-IL- Ira." In yet another aspect, the IL-I inhibitor can be an antibody to the IL- 1 receptor. Preferred antibodies to the IL-I receptor are described in U.S. Pat. App. 2004/0097712, published May 20, 2004 (U.S. Ser. No. 10/656,769). In a still further aspect, the IL-I inhibitor can be an IL-I trap molecule.

In one aspect, the IL-I inhibitor used in the methods of the invention can be N- ((6-(methyloxy)-4'-(trifluoromethyl)-l,r-biphenyl-3-yl) methyl)- 1- phenylethanamine, or a pharmaceutically acceptable salt thereof.

In one aspect, the invention provides methods of inhibiting, decreasing, or preventing vascular calcification, wherein a vitamin D sterol had been previously administered to the subject. In one aspect, the vitamin D sterol can be calcitriol, alfacalcidol, doxercalciferol, maxacalcitol or paricalcitol. In one aspect, the IL-I inhibitor can be administered prior to or following administration of a vitamin D sterol. In another aspect, the IL-I inhibitor can be administered in combination with a vitamin D sterol.

In one aspect, the IL-I inhibitor can be administered in combination with RENAGEL®.

The invention further provides methods of decreasing serum creatinine levels in a subject, comprising administering a therapeutically effective of an IL-I inhibitor to the subject. In one aspect, the subject can be suffering from increased serum creatinine levels induced by the administration of a vitamin D sterol to the subject.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 shows an experimental paradigm for adenine model of chronic kidney disease (CKD) and secondary hyperparathyroidism (SHPT).

Figure 2 shows prevention of aortic vascular calcification with FC-IL- Ira in an animal model of CKD. The asterisk in Figure 2 indicates that under the conditions tested (p=0.005; unpaired t-test; control (A5S) vehicle (red; n=4); Fc- IL- Ira (blue hatched, n=7)), there was no calcification (0 g/cm2 bone mineral density) in seven of seven Fc-IL- Ira-treated animals.

Figure 3 shows reduction of parathyroid gland size (A) and serum PTH (B) by Fc-IL- Ira in an animal model of CKD/SHPT. In Figure 3 A, the asterisk (*) denotes the parathyroid wild type/body wild type for A5S vehicle control vs. Fc- IL-lra; p=0.009; unpaired t-test; control (vehicle, A5S), red; n=8); Fc-IL-lra, , (blue, n=13). In Figure 3B, the pound sign (#) denotes serum PTH for A5S vehicle control vs. Fc-IL-lra; p=0.028; unpaired t-test; control (vehicle, A5S), red; n=4); Fc-IL-lra, (blue, n=7).

Figure 4 shows the amino acid sequence (SEQ ID NO: 1) of the molecule referred to herein as Fc-IL-lra. Figure 5 shows the experimental paradigm for the 5/6-nephrectomy model of CKD and secondary HPT.

Figure 6 shows attenuation of calcitriol-induced aortic vascular calcification in uremic rats treated with Fc-IL-lra.

DETAILED DESCRIPTION OF THE INVENTION Recent scientific literature includes suggestions to study the effects of cytokines on vascular calcification. Yao et al. (2004), Scandinavian J. Urol. Nephrol. 38: 405-16, found what they considered a "strong association between inflammation and increased oxidative stress and endothelial dysfunction" in end- stage renal disease (ESRD) patients. Malberti and Ravini (2005), Giornale Italiano di Nefrologia (22 Suppl.) 31 : S47-52, noted that vascular calcifications are more frequent in dialysis patients than in the general population or in patients with cardiovascular disease with normal renal function, which led these authors to suggest study of the effects of anti-inflammatory treatments on the nutritional and cardiovascular status of ESRD patients. Moe and Chen (2005), Blood Purif. 23: 64-71, noted that cytokines and other mediators of inflammation may have a direct stimulatory effect on vascular calcification, leading them to suggest that inhibition of cytokiiie-mediated inflammation represents "a plausible therapeutic approach to limit vascular calcification." The literature does not identify, however, which inflammatory cytokines may mediate vascular calcification. Elsewhere in the literature, Nicklin et al. (2000), J. Exper. Med. 191 : 303-

11, found that IL- Ira deficient mice develop lethal arterial inflammation in

flexpoints and branch points of the aorta. Arai et al. (1998), J. Toxicological Sciences 23: 121-8, found that 1,25 dihydroxyvitamin D3 has been shown to increase IL-I synthesis. The literature does not clarify, however, a definitive role for IL-I receptor antagonism in prevention of vascular calcification. The present invention is directed to methods of reducing, inhibiting, or preventing vascular calcification using IL-I inhibitors.

IL-I inhibitors in general

"IL-I" refers to IL-I α and IL-I β.

"IL-I inhibitors" as used throughout this specification refers to molecules that decrease the bioactivity of IL-I α, IL-I β, or IL-I receptor type I (IL-I RI), whether by direct or indirect interaction with IL-I α, IL-I β, IL-I RI, IL-I receptor accessory protein (IL-lRacP), interleukin-1 converting enzyme (ICE), with proteins that mediate signaling through a receptor for IL-I α or β, with proteins controlling the expression or release of IL-I α, IL-I β, IL-I RI or IL-I RII. Inhibition of IL-I may result from a number of mechanisms, including down- regulation of IL-I transcription, expression, or release from cells that produce IL- 1 ; binding of free IL-I ; interference with binding of IL-I to its receptor; interference with formation of the IL-I receptor complex (i.e., association of the IL-I receptor type I with IL-I RacP); and interference with modulation of IL-I signaling after binding to its receptor. Thus, the term "IL-I inhibitor" includes, but is not limited to, IL-I beta inhibitors and IL-I receptor antagonists (IL- Ira), such as anakinra and antibodies to IL-I RI.

Classes of IL-I inhibitors include the following, which are described in detail further hereinbelow: Interleukin-1 receptor antagonists such as IL- Ira and anti-IL-1 receptor monoclonal antibodies, as described below;

IL-I binding proteins such as soluble IL-I receptors, anti-IL-1 monoclonal antibodies;

Inhibitors of interleukin-1 beta converting enzyme (ICE) or caspase I (e.g., WO 99/46248, WO 99/47545, and WO 99/47154, the disclosures of which are

hereby incorporated by reference), which can be used to inhibit IL-I beta production and secretion;

Interleukin-lbeta protease inhibitors; and compounds and proteins that block in vivo synthesis or extracellular release of IL-I .

The term "IL-I binding proteins" refers to molecules that bind to IL-I and thus prevent IL-I beta from exerting bioactivity when bound to IL-I RI. Thus, IL- 1 beta inhibitors include, but are not limited to, IL-I beta antibodies, peptides that bind to IL-I beta, peptibodies that bind to IL-I beta, soluble IL-I receptor molecules, and IL-I trap molecules.

The term "IL-I receptor antagonists" refers to molecules that bind to IL-I RI or IL-lRacP or otherwise prevent the interaction of IL-I RI and IL-lRacP. Thus, the term "IL-I receptor antagonists" includes, but is not limited to anakinra, Fc-IL- Ira, IL-I RI antibodies, IL-lRacP antibodies, peptides that bind to IL-I RI or to IL- 1 RAcP, and peptibodies that bind to IL- 1 RI or IL- 1 RAcP.

Exemplary IL-I inhibitors are disclosed in the following references:

US Pat. 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 FR 2762514. IL-I receptor antagonists

For purposes of the present invention, IL- Ira and variants and derivatives thereof as discussed hereinafter are collectively termed "IL-lra protein(s)". The molecules described in the above references and the variants and derivatives thereof discussed hereinafter are collectively termed "IL-I inhibitors." IL-lra is a human protein that acts as a natural inhibitor of interleukin-1 and which is a member of the IL-I family member that includes IL- lα and IL-I β.

Preferred receptor antagonists (including IL- Ira and variants and derivatives thereof), as well as methods of making and using thereof, are described in WO 91/08285; WO 91/17184; AU 9173636; WO 92/16221; WO93/21946; WO 94/06457; WO 94/21275; FR 2706772; WO 94/21235; DE 4219626, WO 94/20517; WO 96/22793 ;WO 97/28828; WO 99/36541, and U.S. Patent Nos. 5,075,222 and 6,599,873 (incorporated herein by reference). The proteins include glycosylated as well as non-glycosylated IL-I receptor antagonists.

Specifically, three useful forms of IL- Ira and variants thereof are disclosed and described in the 5,075,222 patent. The first of these, called "IL-Ii" in the '222 patent, is characterized as a 22-23 kD molecule on SDS-PAGE with an approximate isoelectric point of 4.8, eluting from a Mono Q FPLC column at around 52 mM NaCl in Tris buffer, pH 7.6. The second, IL-lraβ, is characterized as a 22-23 kD protein, eluting from a Mono Q column at 48 mM NaCl. Both IL- lraα and IL-lraβ are glycosylated. The third, IL-lrax, is characterized as a 20 kD protein, eluting from a Mono Q column at 48 mM NaCl, and is non-glycosylated. 5,075,222 patent also discloses methods for isolating the genes responsible for coding the inhibitors, cloning the gene in suitable vectors and cell types, and expressing the gene to produce the inhibitors.

Those skilled in the art understand that many combinations of deletions, insertions and substitutions (individually or collectively "variant(s)") can be made within the amino acid sequences of IL- Ira, provided that the resulting molecule is biologically active (e.g., possesses the ability to inhibit IL-I). Particular variants are described in U.S. Pat. No. 5,075,222 and U.S. Ser. No. 11/097,453, which are hereby incorporated by reference. The term "IL-I receptor antagonist" further includes modified IL- Ira and fusion proteins comprising IL- Ira. Exemplary fusion proteins include Fc-IL- Ira (Figure 4, SEQ ID NO: 1), and molecules as described in U.S. Pat. No. 6,294,170. Antibodies "IL-I beta antibodies" and "antibodies to IL-I beta" refer to antibodies that specifically bind to IL-I beta. One example of an IL-I beta antibody is known as MAb 201 and is commercially available. Additional IL-I beta antibodies may be

produced as described hereinafter. Further examples of IL-I antibodies are described in WO 9501997, WO 9402627, WO 9006371, EP 364778, EP 267611, EP 220063, and U.S. Pat. No. 4,935,343 (incorporated by reference).

Antibodies having specific binding affinity for IL- lβ can be produced through standard methods. Alternatively, antibodies may be commercially available, for example, from R&D Systems, Inc., Minneapolis, Minn. The terms "antibody" and "antibodies" include polyclonal antibodies, monoclonal antibodies, humanized or chimeric antibodies, single chain Fv antibody fragments, Fab fragments, and F(ab) ₂ fragments. Polyclonal antibodies are heterogeneous populations of antibody molecules that are specific for a particular antigen, which are contained in the sera of the immunized animals. Polyclonal antibodies are produced using well-known methods.

Likewise, "IL-I RI antibodies" and "antibodies to IL-I RI" refer to antibodies that specifically bind to IL-I RI. Examples of IL-I RI antibodies are described in EP 623 674 and U.S. Pat. App. 2004/0097712, published May 20, 2004 (U.S. Ser. No. 10/656,769), the disclosure of which is hereby incorporated by reference. Additional IL-I RI antibodies may be produced as described hereinafter.

The terms "antibody" and "antibodies" as used herein refer to intact antibody, or a binding fragment thereof that competes with the intact antibody for specific binding and includes chimeric, humanized, fully human, and bispecific antibodies. In certain embodiments, binding fragments are produced by recombinant DNA techniques. In additional embodiments, binding fragments are produced by enzymatic or chemical cleavage of intact antibodies. Binding fragments include, but are not limited to, Fab, Fab', F(ab') ₂ , Fv, and single-chain antibodies.

The term "heavy chain" includes a full-length heavy chain and fragments thereof having sufficient variable region sequence to confer specificity for IL-IRl . A full-length heavy chain includes a variable region domain, V _H , and three constant region domains, C _H I , C _H 2, and C _H 3. The V _H domain is at the amino- terminus of the polypeptide, and the C _H 3 domain is at the carboxyl-terminus.

The term "light chain" includes a full-length light chain and fragments thereof having sufficient variable region sequence to confer specificity for IL-IRl.

A full-length light chain includes a variable region domain, V _L , and a constant region domain, C _L . Like the heavy chain, the variable region domain of the light chain is at the amino-terminus of the polypeptide.

Monoclonal antibodies, which are homogeneous populations of antibodies to a particular epitope contained within an antigen, can be prepared using standard hybridoma technology. In particular, monoclonal antibodies can be obtained by any technique that provides for the production of antibody molecules by continuous cell lines in culture such as described by Kohler, G. et al.. Nature, 1975, 256:495, the human B-cell hybridoma technique (Kosbor et al.. Immunology Today, 1983, 4:72; Cole et aL, Proc. Natl. Acad. ScL USA, 1983, 80:2026), and the EBV-hybridoma technique (Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., 1983, pp. 77-96). Such antibodies can be of any immunoglobulin class including IgG, IgM, IgE, IgA, IgD, and any subclass thereof. The hybridoma producing the monoclonal antibodies of the invention can be cultivated in vitro or in vivo.

A chimeric antibody is a molecule in which different portions are derived from different animal species, such as those having a variable region derived from a murine monoclonal antibody and a human immunoglobulin constant region. Chimeric antibodies can be produced through standard techniques.

Antibody fragments that have specific binding affinity for IL-I β can be generated by known techniques. For example, such fragments include, but are not limited to, F(ab') ₂ fragments that can be produced by pepsin digestion of the antibody molecule, and Fab fragments that can be generated by reducing the disulfide bridges of F(ab') ₂ fragments. Alternatively, Fab expression libraries can be constructed. See, for example, Huse et al., 1989, Science, 246: 1275. Single chain Fv antibody fragments are formed by linking the heavy and light chain fragments of the Fv region via an amino acid bridge (e.g., 15 to 18 amino acids),

resulting in a single chain polypeptide. Single chain Fv antibody fragments can be produced through standard techniques. See, for example, U.S. Pat. No. 4,946,778.

A "Fab fragment" is comprised of one light chain and the C _H I and variable regions of one heavy chain. The heavy chain of a Fab molecule cannot form a disulfide bond with another heavy chain molecule.

A "Fab' fragment" contains one light chain and one heavy chain that contains more of the constant region, between the C _H I and C _R 2 domains, such that an interchain disulfide bond can be formed between two heavy chains to form a F(ab') ₂ molecule. A "F(ab') ₂ fragment" contains two light chains and two heavy chains containing a portion of the constant region between the C _H I and C _H 2 domains, such that an interchain disulfide bond is formed between two heavy chains.

The "Fv region" comprises the variable regions from both the heavy and light chains, but lacks the constant regions. "Single-chain antibodies" are Fv molecules in which the heavy and light chain variable regions have been connected by a flexible linker to form a single polypeptide chain, which forms an antigen-binding region. Single chain antibodies are discussed in detail in International Patent Application Publication No. WO 88/01649 and U.S. Patent Nos. 4,946,778 and 5,260,203 (hereby incorporated by reference).

A "bivalent antibody" other than a "multispecific" or "multifunctional" antibody, in certain embodiments, is understood to comprise binding sites having identical antigenic specificity.

A "bispecific" or "bifunctional" antibody is a hybrid antibody having two different heavy/light chain pairs and two different binding sites. Bispecific antibodies may be produced by a variety of methods including, but not limited to, fusion of hybridomas or linking of Fab' fragments. See, e _^ g., Songsivilai & Lachmann (1990), Clin. Exp. Immunol. 79:315-321; Kostelny et al. (1992). J ₁ Immunol. 148:1547-1553.

Preferred antibodies are described in U.S. Pat. App. 2004/0097712, published May 20, 2004 (hereinafter referred to as the '712 application). Specifically preferred are antibodies having a heavy chain variable region selected from the following: Met GIu Phe GIy Leu Ser Trp VaI Phe Leu 10

VaI Ala Leu Leu Arg GIy VaI GIn Cys Gin 20

VaI GIn Leu VaI GIu Ser GIy GIy GIy VaI 30

VaI GIn Pro GIy Arg Ser Leu Arg Leu Ser 40

Cys Ala Ala Ser GIy Phe Thr Phe Ser Asn 50 Tyr GIy Met His Trp VaI Arg GIn Ala Pro 60

GIy Lys GIy Leu GIu Trp VaI Ala GIy lie 70

Trp Asn Asp GIy lie Asn Lys Tyr His Ala 80

His Ser VaI Arg GIy Arg Phe Thr lie Ser 90

Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu 100 GIn Met Asn Ser Pro Arg Ala GIu Asp Thr 110

Ala VaI Tyr Tyr Cys Ala Arg Ala Arg Ser 120

Phe Asp Trp Leu Leu Phe GIu Phe Trp GIy 130

GIn GIy Thr Leu VaI Thr VaI Ser Ser 139

(SEQ ID NO: 408) Met GIu Phe GIy Leu Ser Trp VaI Phe Leu 10

VaI Ala Leu Leu Arg GIy VaI Gin Cys Gin 20

VaI Gin Leu VaI GIu Ser GIy GIy GIy VaI 30

VaI GIn Pro GIy Arg Ser Leu Arg Leu Ser 40

Cys Ala VaI Ser GIy Phe Thr Phe Ser Asn 50 Tyr GIy Met His Trp VaI Arg Gin Ala Pro 60

GIy Lys GIy Leu GIu Trp VaI Ala Ala He 70

Trp Asn Asp GIy GIu Asn Lys His His Ala 80

GIy Ser VaI Arg GIy Arg Phe Thr He Ser 90

Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu 100 GIn Met Asn Ser Leu Arg Ala GIu Asp Thr 110

Ala VaI Tyr Tyr Cys Ala Arg GIy Arg Tyr 120 Phe Asp Trp Leu Leu Phe GIu Tyr Trp GIy 130 GIn GIy Thr Leu VaI Thr VaI Ser Ser 139

(SEQ ID NO: 409) Met GIy Ser Thr Ala lie Leu Ala Leu Leu 10

Leu Ala VaI Leu GIn GIy VaI Cys Ala GIu 20

VaI GIn Leu Met GIn Ser GIy Ala GIu VaI 30

Lys Lys Pro GIy GIu Ser Leu Lys lie Ser 40

Cys Lys GIy Ser GIy Tyr Ser Phe Ser Phe 50 His Trp lie Ala Trp VaI Arg GIn Met Pro 60

GIy Lys GIy Leu GIu Trp Met GIy lie lie 70

His Pro GIy Ala Ser Asp Thr Arg Tyr Ser 80

Pro Ser Phe Gin GIy GIn VaI Thr lie Ser 90

Ala Asp Asn Ser Asn Ser Ala Thr Tyr Leu 100 GIn Trp Ser Ser Leu Lys Ala Ser Asp Thr 110

Ala Met Tyr Phe Cys Ala Arg GIn Arg GIu 120

Leu Asp Tyr Phe Asp Tyr Trp GIy GIn GIy 130

Thr Leu VaI Thr VaI Ser Ser 137

(SEQ ID NO: 410) The foregoing are SEQ ID NOS: 10, 14, and 16 of the '712 application.

Antibodies incorporating these sequences may be prepared as described therein. Most preferred are antibodies having all or an immunologically functional fragment of a heavy chain having a sequence selected from SEQ ID NOS: 20, 22, 24, 26, 28, 30, 32, 34, and 36 of the '712 application, all of which are specifically incorporated by reference.

Also specifically preferred are antibodies having a light chain variable region selected from the following:

Met GIu Ala Pro Ala Gin Leu Leu Phe Leu 10

Leu Leu Leu Trp Leu Pro Asp Thr Thr GIy 20 GIu lie VaI Leu Thr GIn Ser Pro Ala Thr 30

Leu Ser Leu Ser Pro GIy GIu Arg Ala Thr 40

Leu Ser Cys Arg Ala Ser GIn Ser VaI Ser 50

Ser Tyr Leu Ala Trp Tyr GIn Gin Lys Pro 60

GIy GIn Ala Pro Arg Leu Leu lie Tyr Asp 70 Ala Ser Asn Arg Ala Thr GIy lie Pro Ala 80

Arg Phe Ser GIy Ser GIy Ser GIy Thr Asp 90

Phe Thr Leu Thr lie Ser Ser Leu GIu Pro 100

GIu Asp Phe Ala VaI Tyr Tyr Cys Gin GIn 110

Arg Ser Asn Trp Pro Pro Leu Thr Phe GIy 120 GIy GIy Thr Lys VaI GIu lie Lys 128

(SEQ ID NO: 411)

Met Ser Pro Ser Gin Leu lie GIy Phe Leu 10

Leu Leu Trp VaI Pro Ala Ser Arg GIy GIu 20 lie VaI Leu Thr GIn Ser Pro Asp Phe GIn 30 Ser VaI Thr Pro Lys GIu Lys VaI Thr lie 40

Thr Cys Arg Ala Ser GIn Ser lie GIy Ser 50

Ser Leu His Trp Tyr GIn GIn Lys Pro Asp 60

GIn Ser Pro Lys Leu Leu lie Lys Tyr Ala 70

Ser GIn Ser Phe Ser GIy VaI Pro Ser Arg 80 Phe Ser GIy Ser GIy Ser GIy Thr Asp Phe 90

Thr Leu Thr lie Asn Ser Leu GIu Ala GIu 100

Asp Ala Ala Ala Tyr Tyr Cys His Gin Ser 110

Ser Ser Leu Pro Leu Thr Phe GIy GIy GIy 120

Thr Lys VaI GIu lie Lys 126 (SEQ ID NO: 412)

The foregoing are SEQ ID NOS: 12 and 18 of the '712 application.

Antibodies incorporating these sequences may be prepared as described therein.

Most preferred are antibodies having all or an immunologically functional fragment of a light chain having a sequence selected from SEQ ID NOS: 38 and 40 of the '712 application, all of which are specifically incorporated by reference.

Although SEQ ID NOS: 408 through 410 are described as heavy chain variable regions, persons skilled in the art may employ those sequences for IL-IR binding at a different position in an antibody (e.g., as a light chain variable region) or in another molecular (e.g., an Fc fusion molecule). Likewise, although SEQ ID NOS: 411 and 412 are described as light chain variable regions, persons skilled in the art may employ those sequences for IL-IR binding at a different position in an antibody (e.g., as a heavy chain variable region) or in another molecular (e.g., an Fc fusion molecule). The same techniques can be used to prepare additional IL-I beta antibodies or molecules derived from IL-I beta antibodies. Thus, the MAb201 heavy and light chain variable regions can be used at different positions in an antibody framework and in different molecular forms. All such molecules described in this paragraph are within the scope of this invention.

Peptides and Peptibodies

Phage display peptide libraries have emerged as a powerful method in identifying peptide agonists and antagonists of proteins of interest. See, for example, Scott et al. (1990), Science 249: 386; Devlin et al. (1990), Science 249: 404; WO 96/40987, published Dec. 19, 1996; WO 98/15833, published Apr. 16, 1998; and U.S. Pat. Nos. 5,223,409; 5,733,731; 5,498,530; 5,432,018; 5,338,665; and 5,922,545 (each of which is incorporated by reference). In such libraries, random peptide sequences are displayed by fusion with coat proteins of filamentous phage. Typically, the displayed peptides are affinity-eluted against an antibody-immobilized extracellular domain of a receptor. The retained phages may be enriched by successive rounds of affinity purification and repropagation. The best binding peptides may be sequenced to identify key residues within one or more structurally related families of peptides. See, e.g., Cwirla et al. (1997),

Science 276: 1696-9, in which two distinct families were identified. The peptide sequences may also suggest that residues may be safely replaced by alanine scanning or by mutagenesis at the DNA level. Mutagenesis libraries may be created and screened to further optimize the sequence of the best binders. Lowman (1997), Ann. Rev. Biophys. Biomol. Struct. 26: 401-24.

Phage display and other techniques may be used to generate peptide IL-I inhibitors. Such peptides have been generated as described in U.S. Pat. Nos. 5,608,035, 5,786,331, 5,880,096, and 6,660,843, each of which is hereby incorporated by reference. Such peptides may be linked to Fc domains, polyethylene glycol, or other half-life extending moieties (see U.S. Pat. No. 6,660,843). Such peptides linked to Fc domains are referred to as "peptibodies." Peptibodies directed to targets other than IL-I and IL-I receptor have shown efficacy in human clinical trials. A number of peptides suitable for use in peptibodies are described in Table 1 below.

Table 1 — IL-I antagonist peptide sequences

W

A-1009-PCT

O£

OO

The peptides above correspond to the peptides of Table 4 and SEQ ID NOS: 212, 907-910, 917, 979, 213 to 271, 671 to 906, and 911 to 978, and 980 to 1023 (incorporated herein by reference) of the aforementioned U.S. Pat. No. 6,660,843 and may be prepared by methods known in the art. Such peptides are within the scope of IL-I inhibitors in this invention.

Peptides such as those described in Table 1 may be used to make molecules of the formulae

I II

X ¹ -F ¹ III

F ¹ -X ²

IV

F ¹ -(L ¹ ) _C -P ¹ v F ¹ -(L ¹ ) _c -P ¹ -(L ² ) _d -P ² and multimers thereof wherein:

F ¹ is a half-life extending vehicle, such as polyethylene glycol (PEG), dextran, or preferably an Fc domain;

X ¹ and X ² are each independently selected from -(L ¹ ^-P ¹ , -(L '^-P ¹ -^ ² ^ - P ² , -(LVP'-CLVP'-CLVP ³ , and -P ³ -(L ⁴ )HP ⁴

P ¹ , P ² , P ³ , and P ⁴ are each independently sequences of pharmacologically active IL-I antagonist peptides;

L ¹ , L ² , L ³ , and L ⁴ are each independently linkers; and a, b, c, d, e, and f are each independently 0 or 1, provided that at least one of a and b is 1.

Molecules of the foregoing formulae in which F ¹ is an Fc domain have been named "peptibodies." Peptibodies may be prepared as described in the aforementioned U.S. Pat. No. 6,660,843. All vehicle-linked peptide molecules, including peptibodies, are IL-I inhibitors within the meaning of this specification. Soluble IL-I receptors

"Soluble IL-I receptor molecules" refers to soluble IL-I RI (sIL-1 RI), soluble IL-I RII (sIL-1 RII), and soluble IL-lRacP (sIL-lRacP); fragments of sIL- 1 RI, sIL-1 RII, and sIL-lRacP; and fusion proteins of sIL-1 RI, sIL-1 RII, sIL- lRacP and fragments of any thereof, including "IL-I trap" molecules and fusion proteins with human serum albumin, transthyretin or an Fc domain; and

1 λ

derivatives of any of the foregoing (e.g., soluble receptor linked to polyethylene glycol). Soluble IL-I receptor molecules are described in U. S. Pat. Nos. 5,492,888; 5,488,032; 5,464,937; 5,319,071; and 5,180,812, the disclosures of which are hereby incorporated by reference. Fragments of the IL-I receptor include, but are not limited to, synthetic polypeptides corresponding to residues 86-93 of the human type I IL-I receptor, which bind IL- lα and β and inhibit IL-I activity in vitro and in vivo. See Tanihara et al. (1992) Biochem. Biophvs. Res. Commun. 188: 912. IL-I trap The IL-I trap is as essentially described in U.S. Pat. No. 5,844,099, which is hereby incorporated by reference. Briefly, the IL-I trap is a fusion protein comprising the human cytokine receptor extracellular domains and the Fc portion of human IgGl. The IL-I trap incorporates into a single molecule the extracellular domains of both receptor components required for IL-I signaling; the IL-I Type I receptor (IL-IRI) and the IL-I receptor accessory protein (AcP). Since it contains both receptor components, the IL-I trap binds IL- lα and IL- lβ with picomolar affinity, while the IL-IRI alone in the absence of AcP binds with about 1 nM affinity. The IL-I trap was created by fusing the sequences encoding the extracellular domains of the AcP, IL-IRI, and Fc in line without any intervening linker sequences. An expression construct encoding the fusion protein is transfected into Chinese hamster ovary (CHO) cells, and high producing lines are isolated that secrete the IL-I trap into the medium. The IL-I TRAP is a dimeric glycoprotein with a protein molecular weight of 201 kD and including glycosylation has a total molecular weight of .about.252 kD. Disulfide bonds in the Fc region covalently link the dimer. Vascular calcification

"Vascular calcification," as used herein, means formation, growth or deposition of extracellular matrix hydroxyapatite (calcium phosphate) crystal deposits in blood vessels. Vascular calcification encompasses coronary, valvular, aortic, and other blood vessel calcification. The term includes atherosclerotic and medial wall calcification.

"Atherosclerotic calcification" means vascular calcification occurring in atheromatous plaques along the intimal layer of arteries.

"Medial calcification," "medial wall calcification," or "Mδnckeberg's sclerosis," as used herein, means calcification characterized by the presence of calcium in the medial wall of arteries.

"Inhibiting," in connection with inhibiting vascular calcification, is intended to mean preventing, retarding, or reversing formation, growth or deposition of extracellular matrix hydroxyapatite crystal deposits.

The term "treatment" or "treating" includes the administration, to a person in need, of an amount of an IL-I inhibitor, which will inhibit or reverse development of a pathological vascular calcification condition.

The term "prevention" or "preventing" includes either preventing the onset or preventing / slowing the progression of clinically evident vascular calcification disorders altogether or preventing the onset of a preclinically evident stage of vascular calcification disorder in individuals. This includes prophylactic treatment of those at risk of developing a vascular calcification disorder.

The phrase "therapeutically effective amount" is the amount of the IL-I inhibitor that will achieve the goal of improvement in disorder severity and the frequency of incidence. The improvement in disorder severity includes the reversal of vascular calcification, as well as slowing down the progression of vascular calcification. In one aspect, "therapeutically effective amount" means the amount of the IL-I inhibitor that decreases serum creatinine levels or prevents an increase in serum creatinine levels.

As used herein, the term "subject" is intended to mean a human or other mammal, exhibiting, or at risk of developing, calcification. Such an individual can have, or be at risk of developing, for example, vascular calcification associated with conditions such as atherosclerosis, stenosis, restenosis, renal failure, diabetes, prosthesis implantation, tissue injury or age-related vascular disease. The prognostic and clinical indications of these conditions are known in the art. An individual treated by a method of the invention can have a systemic

mineral imbalance associated with, for example, diabetes, chronic kidney disease, renal failure, kidney transplantation or kidney dialysis.

Animal models that are reliable indicators of human atherosclerosis, renal failure, hyperphosphatemia, diabetes, age-related vascular calcification and other conditions associated with vascular calcification are known in the art. For example, Yamaguchi et aL, Exp. Path., describe an experimental model of calcification of the vessel wall. 25: 185-190, 1984.

Assessment of vascular calcification

Methods of detecting and measuring vascular calcification are well known in the art. In one aspect, methods of measuring calcification include direct methods of detecting and measuring extent of calcium-phosphorus depositions in blood vessels.

In one aspect, direct methods of measuring vascular calcification comprise in vivo imaging methods such as plain film roentgenography, coronary arteriography; fluoroscopy, including digital subtraction fluoroscopy; cinefluorography; conventional, helical, and electron beam computed tomography; intravascular ultrasound (IVUS); magnetic resonance imaging; and transthoracic and transesophageal echocardiography. Persons skilled in the art most commonly use fluoroscopy and EBCT to detect calcification noninvasively. Coronary interventionalists use cinefluorography and IVUS to evaluate calcification in specific lesions before angioplasty.

In one aspect, vascular calcification can be detected by plain film roentgenography. The advantage of this method is availability of the film and the low cost of the method, however, the disadvantage is its low sensitivity. Kelley M. & Newell J. Cardiol Clin. 1: 575-595, 1983.

In another aspect, fluoroscopy can be used to detect calcification in coronary arteries. Although fluoroscopy can detect moderate to large calcifications, its ability to identify small calcific deposits is low. Loecker et al. L Am. Coll. Cardiol. 19: 1167-1172, 1992. Fluoroscopy is widely available in both inpatient and outpatient settings and is relatively inexpensive, but it has several disadvantages. In addition to only a low to moderate sensitivity, fluoroscopic

detection of calcium is dependent on the skill and experience of the operator as well as the number of views studied. Other important factors include variability of fluoroscopic equipment, the patient's body habitus, overlying anatomic structures, and overlying calcifications in structures such as vertebrae and valve annuli. With fluoroscopy, quantification of calcium is not possible, and film documentation is not commonly obtained.

In yet another aspect, vascular detection can be detected by conventional computed tomography (CT). Because calcium attenuates the x-ray beam, computed tomography (CT) is extremely sensitive in detecting vascular calcification. While conventional CT appears to have better capability than fluoroscopy to detect coronary artery calcification, its limitations are slow scan times resulting in motion artifacts, volume averaging, breathing misregistration, and inability to quantify amount of plaque. Wexler et al. Circulation 94: 1175- 1192, 1996. In a further aspect, calcification can be detected by helical or spiral computer tomography, which has considerably faster scan times than conventional CT. Overlapping sections also improve calcium detection. Shemesh et al. reported coronary calcium imaging by helical CT as having a sensitivity of 91% and a specificity of 52% when compared with angiographically significant coronary obstructive disease. Shemesh et al. Radiology 197: 779-783, 1995. However, other preliminary data have shown that even at these accelerated scan times, and especially with single helical CT, calcific deposits are blurred due to cardiac motion, and small calcifications may not be seen. Baskin et al. Circulation 92(suppl I): 1-651, 1995. Thus, helical CT remains superior to fluoroscopy and conventional CT in detecting calcification. Double-helix CT scanners appear to be more sensitive than single-helix scanners in detection of coronary calcification because of their higher resolution and thinner slice capabilities. Wexler et al, supra.

In another aspect, Electron Beam Computed Tomography (EBCT) can be used for detection of vascular calcification. EBCT uses an electron gun and a stationary tungsten "target" rather than a standard x-ray tube to generate x-rays,

permitting very rapid scanning times. Originally referred to as cine or ultrafast CT, the term EBCT is now used to distinguish it from standard CT scans because modern spiral scanners are also achieving subsecond scanning times. For purposes of detecting coronary calcium, EBCT images are obtained in 100 ms with a scan slice thickness of 3 mm. Thirty to 40 adjacent axial scans are obtained by table incrementation. The scans, which are usually acquired during one or two separate breath-holding sequences, are triggered by the electrocardiographic signal at 80% of the RR interval, near the end of diastole and before atrial contraction, to minimize the effect of cardiac motion. The rapid image acquisition time virtually eliminates motion artifact related to cardiac contraction. The unopacifϊed coronary arteries are easily identified by EBCT because the lower CT density of periarterial fat produces marked contrast to blood in the coronary arteries, while the mural calcium is evident because of its high CT density relative to blood. Additionally, the scanner software allows quantification of calcium area and density. An arbitrary scoring system has been devised based on the x-ray attenuation coefficient, or CT number measured in Hounsfield units, and the area of calcified deposits. Agatston et al. J. Am. Coll. Cardiol. 15:827-832. 1990. A screening study for coronary calcium can be completed within 10 or 15 minutes, requiring only a few seconds of scanning time. Electron beam CT scanners are more expensive than conventional or spiral CT scanners and are available in relatively fewer sites.

In one aspect, intravascular ultrasound (IVUS) can be used for detecting vascular calcification, in particular, coronary atherosclerosis. Waller et al. Circulation 85: 2305-2310, 1992. By using transducers with rotating reflectors mounted on the tips of catheters, it is possible to obtain cross-sectional images of the coronary arteries during cardiac catheterization. The sonograms provide information not only about the lumen of the artery but also about the thickness and tissue characteristics of the arterial wall. Calcification is seen as a hyperechoic area with shadowing: fϊbrotic noncalcified plaques are seen as hyperechoic areas without shadowing. Honve et al. Trends Cardiovasc Med. 1: 305-311. 1991. The disadvantages in use of IVUS, as opposed to other imaging modalities, are that it

is invasive and currently performed only in conjunction with selective coronary angiography, and it visualizes only a limited portion of the coronary tree. Although invasive, the technique is clinically important because it can show atherosclerotic involvement in patients with normal findings on coronary arteriograms and helps define the morphological characteristics of stenotic lesions before balloon angioplasty and selection of atherectomy devices. Tuzcu et al J. Am. Coll. Cardiol. 27: 832-838, 1996.

In another aspect, vascular calcification can be measured by magnetic resonance imaging (MRI). However, the ability of MRI to detect coronary calcification is somewhat limited. Because microcalcifications do not substantially alter the signal intensity of voxels that contain a large amount of soft tissue, the net contrast in such calcium collections is low. Therefore, MRI detection of small quantities of calcification is difficult, and there are no reports or expected roles for MRI in detection of coronary artery calcification. Wexler et al., supra.

In another aspect, vascular calcification can be measured by transthoracic (surface) echocardiography, which is particularly sensitive to detection of mitral and aortic valvular calcification; however, visualization of the coronary arteries has been documented only on rare occasions because of the limited available external acoustic windows. Transesophageal echocardiography is a widely available methodology that often can visualize the proximal coronary arteries. Koh et al. Int. J. Cardiol. 43: 202-206, 1994. Fernandes et al. Circulation 88: 2532-2540, 1993.

In another aspect, vascular calcification can be assessed ex vivo by Van Kossa method. This method relies upon the principle that silver ions can be displaced from solution by carbonate or phosphate ions due to their respective positions in the electrochemical series. The argentaffin reaction is photochemical in nature and the activation energy is supplied from strong visible or ultra-violet light. Since the demonstrable forms of tissue carbonate or phosphate ions are invariably associated with calcium ions the method may be considered as demonstrating sites of tissue calcium deposition.

Other methods of direct measuring calcification may include, but not limited to, immunofluorescent staining and densitometry. In another aspect, methods of assessing vascular calcification include methods of measuring determinants and/or risk factors of vascular calcification. Such factors include, but are not limited to, serum levels of phosphorus, calcium, and calcium x phosphorus product, parathyroid hormone (PTH), low-density lipoprotein cholesterol (LDL), high-density lipoprotein cholesterol (HDL), triglycerides, and creatinine. Methods of measuring these factors are well known in the art. Other methods of assessing vascular calcification include assessing factors of bone formation. Such factors include bone formation markers such as bone-specific alkaline phosphatase

(BSAP), osteocalcin (OC), carboxyterminal propeptide of type I collagen (PICP), and aminoterminal propeptide of type I collagen (PINP); serum bone resorption markers such as cross-linked C-telopeptide of type I collagen (ICTP), tartrate- resistant acid phosphatase, TRACP and TRAP5B, N-telopeptide of collagen cross- links (NTx), and C-telopeptide of collagen cross-links (CTx); and urine bone resorption markers, such as hydroxyproline, free and total pyridinolines (Pyd), free and total deoxypyridinolines (Dpd), N-telopeptide of collagen cross-links (NTx), and C-telopeptide of collagen cross-links (CTx). Methods of treatment In one aspect, the invention provides a method of inhibiting, decreasing or preventing vascular calcification in an individual. The method comprises administering to the individual a therapeutically effective amount of the IL-I inhibitor of the invention. In one aspect, administration of the compound of the invention retards or reverses the formation, growth or deposition of extracellular matrix hydroxyapatite crystal deposits. In another aspect of the invention, administration of the compound of the invention prevents the formation, growth or deposition of extracellular matrix hydroxyapatite crystal deposits.

Methods of the invention may be used to prevent or treat atherosclerotic calcification and medial calcification and other conditions characterized by vascular calcification. In one aspect, vascular calcification may be associated with chronic renal insufficiency or end-stage renal disease. In another aspect, vascular

calcification may be associated with pre- or post-dialysis or uremia. In a further aspect, vascular calcification may be associated with diabetes mellitus I or II, In yet another aspect, vascular calcification may be associated with a cardiovascular disorder. In one aspect, administration of an effective amount of an IL-I inhibitor can reduce serum PTH without causing aortic calcification. In another aspect, administration of an IL-I inhibitor can reduce serum creatinine level or can prevent increase of serum creatinine level. In another aspect, administration of an IL-I inhibitor can attenuates parathyroid (PT) hyperplasia. In one aspect of combination therapy, the IL-I inhibitors of the invention may be used with calcimimetics, vitamins and their analogs, such as vitamin D and analogs thereof (including vitamin D sterols such as calcitriol, alfacalcidol, doxercalciferol, maxacalcitol and paricalcitol), antibiotics, lanthanum carbonate, lipid-lowering agents, such as LIPITOR , anti-hypertensives, anti-inflammatory agents (steroidal and non-steroidal), inhibitors of pro-inflammatory cytokine (ENBREL ^® , KINERET ^® ), and cardiovascular agents, vitamin D sterols and/or RENAGEL ^® . In one aspect, the compositions of the invention may be administered before, concurrently, or after administration of calcimimetics, vitamin D sterols and/or RENAGEL ^® . The dosage regimen for treating a disease condition with the combination therapy of this invention is selected in accordance with a variety of factors, including the type, age, weight, sex and medical condition of the patient, the severity of the disease, the route of administration, and the particular compound employed, and thus may vary widely.

In accordance with this invention, IL-I inhibitors may be administered alone or in combination with other drags for treating vascular calcification, such as vitamin D sterols and/or RENAGEL®. Vitamin D sterols can include calcitriol, alfacalcidol, doxercalciferol, maxacalcitol or paricalcitol. In one aspect, IL-I inhibitors can be administered before or after administration of vitamin D sterols. In another aspect, IL-I inhibitors can be co-administered with vitamin D sterols. The methods of the invention can be practiced to attenuate the mineralizing effect of calcitriol on vascular tissue. In one aspect, the methods of

the invention can be used to reverse the effect of calcitriol of increasing the serum levels of calcium, phosphorus and Ca x P product thereby preventing or inhibiting vascular calcification. In another aspect, the methods of the invention can be used to stabilize or decrease serum creatinine levels. In one aspect, in addition to creatinine level increase due to a disease, a further increase in creatinine level can be due to treatment with vitamin D sterols such as calcitriol.

In addition, IL-I inhibitors may be administered in conjunction with surgical and non-surgical treatments. In one aspect, the methods of the invention can be practiced in injunction with dialysis. The following examples are offered to more fully illustrate the invention, but are not to be construed as limiting the scope thereof.

Example 1

Adenine-induced secondary hyperparathyroidism (SHPT) and calcification in rats and prevention of aortic vascular calcification by Fc-IL-lra

This experiment used the protocol shown in Figure 1. Adenine, included as a dietary supplement (0.75%), was fed to adult, male Sprague-Dawley rats. Blood for chemistry analyses (total serum calcium, phosphorous, blood urea nitrogen [BUN], creatinine, PTH) was collected before and again on drug treatment days 0 (pretreatment) and 21 from the retro-orbital sinus of anesthetized rats. Blood (0.5 ml) was collected for PTH levels into SST (clot activator) brand blood tubes and allowed to clot. Serum was removed and stored at -70°C until assayed. PTH levels were quantified according to the vendor's instructions using rat PTH _(1-34) immunoradiometric assay kit (Immutopics, San Clemente, CA). Calcium and phosphorous were measured using a blood chemistry analyzer (AU 400; Olympus, Melville, NY).

Vascular calcification was assessed by quantifying the bone mineral density from fixed (formalin, PBS), isolated aortas using a Dxa scanner (Piximus densitomer, GE Healthcare). In this model, CKD/SHPT induced by dietary adenine leads to significant renal impairment (increased BUN, creatinine), and aortic vascular calcification.

Fc-IL- Ira prevented the development of aortic vascular calcification (decreased bone mineral density content of the aorta) in this model (Figure 2), but did not affect BUN, creatinine levels. In addition, Fc-IL- Ira reduced the size of the parathyroid gland in this model (Figure 3A) and decreased serum PTH levels (Figure 3B).

Example 2

Effect of IL-lra on vitamin D-induced vascular calcification The protocol used in this experiment may be summarized as follows. PILOT: IL-lra effects on vascular calcification. Vitamin D 100 ng x 3 weeks + IL-IRa (subcutaneous) → aortas for VC

Female, Lewis rats 25O g (pump implantation) Vitamin D ₃ , supplied as lα, 25-dihydroxycholecalciferol from Sigma- Aldrich, Corp (St. Louis, MO), was dissolved in 90% ethanol to create a ImM stock solution that was stored at -20 ⁰ C until final dilution in phosphate buffered saline (PBS). Vitamin D ₃ (0.1 μg, in a dose volume of 0.2 ml PBS) was administered by subcutaneous (s.c.) injection. IL-lra

Dose 5mg/kg/h SC infusion Endpoint: Von Kossa Groups (n=6/group)

1. Vitamin D lOOng

2. Vitamin D 100ng + IL 1 -Ra (5mg/kg/h SC)

3. Vitamin D lOOng + vehicle (pump)

4. vehicle (n=2) 21 days treatment

Sacrifice: remove aortas for von Kossa staining

Example 3 Effect of Fc-IL-lra on vitamin D-induced vascular calcification The protocol used in this experiment may be summarized as follows.

PILOT: Fc IL-lra effects on vascular calcification

Vitamin D lOOng x 3 weeks + Fc IL- Ira (subcutaneous) → aortas for VC

Female, Lewis rats 250 g and/or male, SD rats (25Og)

Vitamin D ₃ , supplied as lα, 25-dihydroxycholecalciferol from Sigma- Aldrich, Corp (D-1530 -. lmg, St. Louis, MO), was dissolved in 90% ethanol to create a ImM stock solution that was stored at -20°C until final dilution in phosphate buffered saline (PBS). Vitamin D ₃ (0.1 μg, in a dose volume of 0.2 ml PBS) was administered by subcutaneous (s.c.) injection.

Fc IL-lra Dose 100 mg/kg SC/day

Endpoint: Von Kossa

Groups (n=6/group)

1. Vitamin D lOOng

2. Vitamin D 100ng + Fc ILl-Ra (100mg/kg/d SC) 3. Vitamin D lOOng + vehicle (pump)

4. vehicle (n=2)

21 days treatment

Sacrifice: remove aortas for von Kossa staining

Example 4

Attenuation of calcitriol (vitamin D3) -induced aortic vascular calcification with IL-lra-Fc (inhibitor of IL-I)

We administered the IL-I receptor antagonist Fc-IL- Ira, calcitriol, the combination of Fc-IL- Ira + calcitriol) or their corresponding vehicles to a rodent animal model of chronic kidney disease (CKD) and secondary hyperparathyroidism (SHPT) induced by subtotal nephrectomy (5/6Nx). The experimental paradigm is shown in Figure 5, with the treatment groups set out in Table 2 below.

Table 2 Treatment groups (n=10/group): 5/6 Nx, male, SD rats (n=45)

• FC-IL-lra 100 mg/kg/day s.c. in A5S buffer n=10 at week 4

• Vehicle (A5S buffer; 1 ml/kg/day s.c) n=10 at week 4

• FC-IL-lra 100 mg/kg/day s.c. + Calcitriol (30ng) n=10 at week 4

• Calcitriol (30ng) n=10 at week 4

• Calcitriol Vehicle n=5

5

Aortas were removed at treatment week 4 (trt wk4), fixed and stained for mineralization (Von Kossa) and the severity determined and scored by a pathologist blinded to the treatments (Calcification Scores: 0=no calcification; l=minimal; 2=mild; 3=moderate; 4=marked; 5=severe).

10 Fc-IL- Ira administered systemically to a rodent animal model of established chronic kidney disease accompanied with secondary hyperparathyroidism (induced by subtotal (5/6) nephrectomy) did not cause vascular calcification, whereas calcitriol caused marked to severe aortic calcification. Fc-IL- Ira attenuated calcitriol (Vitamin D3)-induced aortic vascular

15 calcification, in this model whereby uremia was established for 8 weeks before treatments were started (Figure 6). In addition, the incidence of severe calcification in calcitriol-treated uremic subjects (4/8 or 50%) was reduced in animals receiving calcitriol in combination with Fc-IL- Ira (1/8 or 12.5%).

of) a|s s|c s|e s|e s|s s|s H= H= H= H=

The foregoing specification includes numerous definitions. These definitions apply to the terms as used throughout this specification, unless otherwise limited in specific instances. Definitions apply equally to the plural and singular forms of each term.

25 All publications, patents and patent applications cited in this specification are herein incorporated by reference as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference.

Although the foregoing invention has been described in some detail by way

30 of illustration and example for purposes of clarity of understanding, it will be readily apparent to those of ordinary skill in the art in light of the teachings of this

invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.