MXPA99005227A - Combination therapy using an il-1 inhibitor for treating il-1 mediated diseases - Google Patents

Abstract

The invention relates to methods for treating or preventing arthritis. The method comprises administering to patients in need thereof therapeutically effective amounts of an IL-1 inhibitor and methotrexate. In a preferred embodiment, the IL-1 inhibitor is human recombinant IL-1ra and the methotrexate is N-[4-[(2,4-diamino-6-pteridinyl)methylamino]benzoyl]-L-glutamic acid. The invention also relates to pharmaceutical compositions containing an IL-1 inhibitor and methotrexate useful in such methods.

Description

PHARMACEUTICAL COMPOSITION COMPRISING AN IL-1 INHIBITOR TO TREAT MEDIUM TRANSTORNES BY IL-1 Field of Invention The present invention relates to the field of disorders mediated by IL-1. More specifically, the present invention relates to a combination therapy for the purpose of preventing or treating disorders mediated by IL-1.

Background of the Invention Inflammation is the defense reaction for injuries such as those caused by mechanical damage, infection or antigenic stimulation. An inflammatory reaction can be expressed pathologically when inflammation is induced by an inappropriate stimulus such as an autoantigen, is expressed in an exaggerated manner or persists even after the agents of injury have been removed. Such an inflammatory reaction may include the production of certain cytokines.

Ref: 030496 While the etiology of inflammation is poorly understood, considerable information has recently been appreciated regarding the molecular aspects of inflammation. This research has led to the identification of certain cytokines that are considered to figure prominently in the mediation of inflammation. Cytokines are extracellular proteins that modify the behavior of cells, particularly those cells that are in the immediate area of cytokine synthesis and are released. One of the most potent inflammatory cytokines has already discovered and a cytokine that is thought to be a mediating key in many disorders and medical conditions is interleukin-1 (IL-1). IL-1, which is made (although not exclusively) by cells of the macrophage / monocyte lineage, can be produced in two forms: IL-1 alpha (IL-la) and IL-1 beta (IL-lß).

A disorder or medical condition is considered to be an "interleukin-1-mediated disease" if the spontaneous or experimental disease is associated with elevated levels of IL-1 in blood fluids or tissues, or if the cells or tissues taken of the body produce high levels of IL-1 in culture. In many cases, such disorders mediated by interleukin-1 are also recognized by the following two additional conditions: (1) the pathological findings associated with the disease or medical condition can be mimicked in animals by the administration of IL-1; and (2) the pathology induced in models of disorders in experimental animals can be inhibited or abolished by treatment with agents that inhibit the action of IL-1. In many disorders mediated by interleukin-1 at least two of these three conditions are covered, and many disorders mediated by interleukin-1 can cover all three of the complete conditions.

IL-1 mediated disorders such as rheumatoid arthritis and psoriatic arthritis are chronic disorders of the joints that afflict and incapacitate, in a variety of degrees, millions of people worldwide. Rheumatoid arthritis is a disease of joint joints in which cartilage and bone are slowly worn out incessantly by an invading and proliferative connective tissue called pannus, which is derived from the synovial membrane. The disease may involve peri-articular structures such as bags, tendon and tendon sheaths or extra-articular tissues such as the sub-complex, cardiovascular system, lungs, spleen, lymph nodes, skeletal muscles, nervous system (central and peripheral) ) and eyes (Silberberg (1985), Anderson's Pathology, Kissane (ed.), II: 1828).

It is believed that rheumatoid arthritis results from the presentation of a relevant antigen to an immunogenetically susceptible host. Antigens that could potentially initiate an immune response resulting in rheumatoid arthritis may be endogenous or exogenous. Possible endogenous antigens include collagen, mucopolysaccharides and rheumatoid factors. Exogenous antigens include mycoplasmas, mycobacteria, spirochetes and viruses. The byproducts of the immune reaction inflame the synovium (eg, prostaglandins and oxygen radicals) and activate destructive changes of the junctions (eg, collagenase).

There is a broad spectrum of severity of the disease, but several patients go through the course of intermittent relapses and remissions with a global pattern of destruction and slowly progressive deformity of the joints. Clinical manifestations may include symmetric polyarthritis of the peripheral junctions with pain, tenderness, swelling and loss of function of the affected joints; morning stiffness; and loss of cartilage, erosion of bone matter and subluxation of joints after persistent inflammation. Extra-articular manifestations include rheumatoid nodules, rheumatoid vasculitis, pleuropulmonary inflammations, scleritis, sicca syndrome, Felty's syndrome (splenomegaly and neutropenia), osteoporosis, and weight loss (Katz (1985), Am. J. Med., 79: 24 and Krane and Simon (1986), Advances in Rheumatology, Synderman (ed.), 70 (2): 263-284). The manifestations result in a high degree of morbidity that results in a disturbed daily life of the patient.

One generally accepted theory that is used to explain the linkage between IL-1 and arthritis is that IL-1 stimulates several cell types, such as fibroblasts and chondrocytes, to produce and secrete pro-inflammatory and degradative compounds such as prostaglandin E2. and metalloproteinases. The involvement of interleukin-1 in arthritis has been implicated by two distinct lines of evidence.

First, the increased levels of interleukin-1, and mRNA encoding, are found in the synovial tissue and fluid of the arthritic junctions. See, for example, Buchan et al., "Third Annual General Meeting of the British Society for Rheumatology", London, England, November 19-21, 1988, J. Rhe uma t ol. , 25 (2); Fontana et al., (1982), Rh e uma t ol o gy In t. ,, 2: 49-53; and Duff et al., (1988), Mon oki n es and Oth er Non-Lymph ocyti c Cyt okines, M. Powanda et al., (eds), pages 387-392 (Alan R. Liss, Inc).

Second, the administration of interleukin-1 to healthy binding tissues is shown on numerous occasions to result in the erosion of cartilage and bone. In one experiment, intra-articular injections of IL-1 in rabbits were shown to cause cartilage destruction in vivo (Pettipher et al., (1986), Proc. Nat'l Acad. Sci. USA, d: 8749- 8753). In other studies, IL-1 was shown to cause the degradation of both cartilage and bone in explanted tissues (Saklatavala et al., (1987), Development of Diseases of Cartilage and Bone Matrix, Sen and Thornhill (eds.) , pages 291-298 (Alan R. Liss, Inc.) and Stashenko et al. (1987), The American Association of Immunologists, 183: 1464-1468). On the other hand, recent preliminary human experiments in rheumatoid arthritis with an inhibitor of IL-1 have shown prominent results (Bresnihan, et al., (1996), Arthritis and Rheumatism, 39 (9): S73; and Watt et al., (1996), Arthritis and Rheumatism, 39 (9): S123).

It is an object of the present invention to provide methods and therapeutic compositions for the treatment of inflammatory conditions of a joint. This and other objects of the present invention will be apparent from the description that follows.

Brief description of the invention.

The present invention relates to a combination therapy for preventing and treating disorders mediated by IL-1 in a patient. The present invention relates specifically to combination therapy using an IL-1 inhibitor to prevent and treat disorders mediated by IL-1, including rheumatic diseases, and the systemic inflammation and body weight loss associated therewith. The type of treatment referred to herein is intended for mammals, including humans.

Brief Description of the Figures.

Various aspects and advantages of the present invention will be apparent when reviewing the figures wherein: Figure 1 describes a nucleic acid sequence (SEQ ID NO: 1) encoding Arg ^ Glu153, the recombinant human mature IL-lra. Also detailed is the amino acid sequence (NO.

SEC ID: 2) of rhuIL-lra, with the initial amino acid being Mn where n is equal to o or 1.

Figure 2 describes the effects of rhuIL-lra alone, methotrexate alone and the combination of rhuIL-lra and methotrexate in bound diameters in adjuvant arthritic rats in Example 1.

Figure 3 details the effects of rhuIL-lra alone, methotrexate alone and the combination of rhuIL-lra and methotrexate on final weights of the legs (index of arthritis), espleniomegalia (index of systemic inflammation) and change in body weight the adjuvant arthritic rats in Example 1.

Figure 4 details the final analysis (inhibition at termination) of the effects of rhuIL-lra alone, methotrexate alone and the combination of rhuIL-lra with metotraxato on diámtero binding in arthritic rats adjuvants in Example 1 .

Figure 5 details the effects of rhuIL-lra alone, methotrexate alone and the combinacióin of rhuIL-lra with methotrexate on final weights of the legs (index of arthritis), espleniomegalia (index of systemic inflammation) and weight change in the adjuvant arthritic rats in Example 2.

Figure 6 details the effects on the clinical severity of rhuIL-lra or r-met I FN-conl in an EAE model in Example 4.

Figure 7 details the effects on clinical severity of the rhuIL-lra or r-met IFN-conl therapy combination in an EAE model in Example 4.

Figure 8 details the effects on gained weight of the combination therapy of rhuIL-lra or r-metlFN-conl in an EAE model in Example 4.

Detailed description of the invention.

The methods and compositions of the invention include administration to a afflicted animal with a disorder mediated by IL-1, an effective amount of an IL-1 inhibitor in combination with any of one or more medications and / or anti-inflammatory therapies. The preferred animal is a human.

Inhibitors of interleukin-1 can be formed from any protein capable of specifically preventing the activation of cellular receptors for IL-1. Classes of interlecun-1 inhibitors include: interleukin-1 receptor antagonists such as IL-lra, as described below; monoclonal antibodies to the anti-IL-1 receptor (e.g., EP 623674), the disclosure of which is incorporated herein by reference; binding proteins such as IL-1 receptors soluble IL-1 (e.g., U.S.P. 5,492,888, U.S.P. 5,488,032, U.S.P. 5,464,937 and, U.S.P. 5,319,071, U.S.P. 5,180,812 and the disclosure of which is incorporated herein by reference); anti-IL-1 monoclonal antibodies (e.g., WO 9501997, WO 9402627, WO 9006371, U.S.P. 4935343, EP 364778, EP 267611 and EP 220063, the disclosure of which is incorporated herein by reference); acessory proteins of the IL-1 receptor (eg, WO 96/23067, the disclosure of which is incorporated herein for reference), and other compounds and proteins with synthesis bloruq in vi o extracellular release of IL-1.

The interleukin-1 receptor antagonist (IL-lra) is a human protein that acts as a natural inhibitor of interleukin-1 and which is a member of the IL-1 family that includes IL-la and IL-1. Preferred receptor antagonists, as well as methods for making and using them, are described in U.S. Patent No. 5,075.22; WO 91/08285; WO 91/17184; AU 9173636; WO 92/16221; WO 93/21946; WO 94/06457; WO 94/21275; FR 2706772; WO 94/21235; DE 4219626; WO 94/20517; WO 96/22793 and WO 97/28828, the disclosure of which is incorporated herein by reference. The proteins include glycosylated as well as non-glycosylated IL-1 receptor antagonists. Specifically, three useful forms of IL-lra and variants thereof are disclosed and described in the '575,222 patent. The first of these, IL-lraa, is characterized as a 22-23 kD molecule in PAGE-SDS with an approximate isoelectric point of 4.8, eluting from a Mono Q FPLC column at around 52 mM NaCl in a Tris buffer, pH 7.6. The second, IL-lraß, is characterized as a 23 kD protein, eluting from a Mono Q column to 48 mM NaCl. As much as IL-lraa and IL-lraß 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. The 5,075,222 patent also discloses methods for isolating the genes responsible for coding the inhibitors, cloning the gene into appropriate vectors and cell types, and expressing the gene to produce the inhibitors.

For the purposes of this invention, IL-lra and modified forms of IL-lra in which IL-lra amino acids have been (1) removed from ("elimination variants"), (2) inserted into ( "addition variants") or (3) substituted by ("substitution variants") are collectively referred to as "IL-lra protein (s)". (Unless indicated otherwise, the amino acid numbering for the molecules described here will correspond to those presented for the mature form of the molecule (ie, minus the sequence signal), as detailed by the amino acids Argl-Glul52 of NO SEQ ID: 2, with any initial MET in each sequence being the residue number "0").

It will be appreciated by those skilled in the art that various combinations of deletions, insertions and substitutions (individually or collectively "variant (s)") can be made within the amino acid sequences of IL-lra, with the proviso that the molecule resulting is biologically active (for example, see the ability to inhibit IL-1).

One variant (s) of IL-ra can be quickly monitored to evaluate its physical properties. It will be appreciated that such variant (s) will demonstrate similar IL-1 inhibitory properties, but not necessarily all the same properties and not necessarily to the same extent as IL-lra.

These are two main variables in the construction of the variant (s) of the amino acid sequence: the location of the mutation site and the nature of the mutation. When designing the variant (s), the location of each mutation site and the nature of each mutation will depend on the biochemical characteristics to be modified. Each mutation site can be modified individually or in series, for example, by (1) elimination of the target amino acid residue, (2) inserting one or more amino acid residues adjacent to the localized site, or (3) substitution first with the Conservative choices of amino acids and, depending on the results achieved, then with more selections of radicals.

Removals of the amino acid sequence generally is in the range of about 1 to 30 amino acid residues, preferably from about 1 to 20 amino acid residues and more preferably from about 1 to 10 residues and more preferably from about from 1 to 5 contiguous residues. The eliminations of internal intrasequences, carboxy terminal, amino terminal are contemplated. Deletions can be made within the amino acid sequences of the IL-lra, for example, in regions of low homology with the sequences of other members of the IL-1 family. Deletions within the amino acid sequences of IL-lra in areas of substantial homology to the sequences of other members of the IL-1 family will be more likely to significantly modify biological activity.

An amino acid sequence addition may include insertions of an amino- and / or carboxy-terminal fusion in a length range from one residue to one hundred or more residues, as well as internal int sequence insertions of single or multiple amino acid residues . The internal additions may generally be in the range of from about 1 to 20 amino acid residues, preferably from about 1 to 10 amino acid residues, more preferably from about 1 to 5 amino acid residues, and more preferably from about from 1 to 3 amino acid residues. Additions within the amino acid sequences of IL-lra can be made in the regions of low homology with the sequences of other members of the IL-1 family. Additions within the amino acid sequence of IL-lra in areas of substantial homology to the sequences of the other members of the IL-1 family will be more likely to significantly modify biological activity. Additions preferably include amino acid sequences derived from the sequences of members of the IL-1 family.

An addition of amino terminus is contemplated to include the addition of a methionine (eg, as an artifact of direct expression in cultures of recombinant bacterial cells). A further example of an amino-terminal addition includes the fusion of a signal sequence to the amino terminus of the IL-lra in order to facilitate the secretion of the protein from the recombinant host cells. Said signal sequences will generally be obtained from and will thus be homologous to the intended host cell species. For prokaryotic host cells that do not recognize and process the signal sequence of origin of IL-1ra, the sequence can be replaced by a prokaryotic signal sequence selected from, for example, the alkaline phosphatase group, penicillinase or enterotoxin leader sequences. II stable to heat. For expression in yeast cells, each polypeptide can have the signal sequence, for example, from the yeast invertase group, the alpha factor or the acid phosphatase leader sequences. In the expression of mammalian cells, the signal sequences of IL-lra origin (US 5,075,222) are satisfactory, although other mammalian signal sequences may be appropriate, for example, sequences derived from other members of the IL family. -1.

An example of an amino- or carboxy-terminus addition includes chimeric proteins comprising the amino terminal or carboxy terminal fusion of an IL-lra protein (s) with all or part of the constant domain of the heavy chain or of human immunoglobulin (individually or collectively, ("IL-lra Fe (s)"), Such chimeric polypeptides are preferred wherein the immunoglobulin portion of each comprises all domains except the first domain of the constant region of the chain of a human immunoglobulin such as IgG (eg, IgGl or IgG3), IgA, IgM or IgE A skilled technician will appreciate that any amino acid from the immunoglobulin portion can be removed or replaced with one or more amino acids, or one or more amino acids can be added as long as the portion of IL-10 protein (s) is still inhibited by IL-1 and the immunoglobulin portion shows one or more of its characteristic properties.

Another variant group (s) is the amino acid substitution variant (s) of the amino acid sequence of IL-lra. These are variants in which at least one amino acid residue in an IL-lra is removed and a different residue is inserted in its place. The substitution variant includes allelic variants that are characterized by changes in the nucleotide sequence that occur naturally in the population of species that may or may not result in an amino acid change. One skilled in the art can use any known information about the link or the active site of the polypeptide in the selection of possible mutation sites. Exemplary substitution variants are taught in WO 91/17184, WO 92/16221 and WO 96/09323.

A method for identifying amino acid residues or regions for the mutagenesis of a protein is termed "alanine scanning mutagenesis", as described by Cunningham and Wells (1989), Sci en ce, 244: 1081-1085, the description of which is incorporated herein by reference. In this method, an amino acid residue or group of target residues is identified (eg, charged residues such as Arg, Asp, His, Lys and Glu) and replaced by a neutral or negatively charged amino acid (more preferably alanine or polyalanine), to affect the interaction of the amino acids with the surrounding aqueous medium inside or outside the cell. Those domains / residues that demonstrate functional sensitivity to substitutions are then refined by introducing alternate or additional residues at the substitution sites. Thus, the site is predetermined to introduce an amino acid sequence modification. To optimize the functionality of a mutation at a given site, random mutagenesis or alanine scanning can be carried out and the variant (s) can be monitored for the optimal combination of the desired activity and degree of activity. The binding sites of the receptor in IL-lra is marked by an extensive site of direct mutagenesis (Evans et al., Th e Jo u lna of Bi ol ogi ca l Ch em i s try, 270 (19): 1147-11483.

The sites of greatest interest for substitutional mutagenesis include sites in which particular amino acid residues within an IL-ra are substantially different from other species or other members of the IL-1 family in terms of chain-side mass, load and / or hydrophobicity. Other sites of interest include those in which particular IL-1ra residues are identical among other species or other IL-1 family members, since such positions are generally important for the biological activity of a protein. A qualified technician will appreciate that initially the sites should be modified by substitution in a relatively conservative manner.

Such conservative substitutions are shown in Table 1 under the heading of "Preferred Substitutions." If such substitutions result in a change in biological activity, then more substantial changes (Exemplary Substitutions) and / or other additions / deletions can be made and the resulting polypeptides can be monitored.

TABLE 1: Substitutions of Amino Acids Residue Substitutions Substitutions Original Preferred Ex emplares Ala (A) Val Val; Leu; I have Arg (R) Lys Lys; Gln; Asn Asn (N) Gln Gln; His; Lys; Arg Asp (D) Glu Glu Cys (C) Ser Ser Gln (Q) Asn Asn Glu (E) Asp Asp Gly (G) Pro Pro His (H) Arg Asn; Gln; Lys; Arg He (I) Leu Leu; Val; Met; To; Phe; Norleucine Leu (L) He Ñorleucina; lie; Val; Met; To; Phe Lys (K) Arg Arg; Gln; Asn Met (M) Leu Leu; Phe; He Phe (F) Leu Leu; Val; lie; Wing Pro (P) Gly Gly Ser (S) Thr Thr Thr (T) Ser Ser Trp (W) Tyr Tyr Tyr (Y) Phe Trp; Phe; Thr Ser Val (V) Leu He; Leu; Met; Phe; To; Norleucine In making such changes, the hydropathic index of amino acids can be considered. The importance of the hydropathic amino acid index in providing interactive biological function on a protein is generally understood in the art (Kyte and Doolittle) (1982), J. Mol. Bil., 157: 105-131, the description of which is incorporated herein by reference). It is known that certain amino acids "can be substituted by other amino acids that have a similar hydropathic index or record and still retain a similar biological activity.

It is also understood in the art that the substitution of similar amino acids can be effectively done on the basis of hydrophilic affinity, particularly where the functionally equivalent protein or peptide created thereby is intended for use in immunological modalities, as in the present case . The U.S. patent 4,554,101, the description of which is incorporated herein by reference, states that the highest local average hydrophilic affinity of a protein, as governed by the hydrophilic affinity of its adjacent amino acids, correlates with its immunogenicity and antigenicity, ie, with a biological property of the protein.

U.S. Pat. 4,554,101, also teaches the identification and preparation of epitopes from primary sequences of amino acids on the basis of their hydrophilic affinity. Through the methods described in U.S. Pat. 4,554,101, a skilled technician could identify the epitopes for example, within the amino acid sequence of an IL-lra. These regions are also referred to as "epitopic core regions." Several scientific publications have been devoted to the prediction of the secondary structure, and to the identification of the epitopes, from analysis of amino acid sequences (Chou and Fasman (1974), Biochemistry, 13 (2): 222 -245; Chou and Fas an (1974), Biochemistry, 13 (2): 221-222, Chou and Fasman (1978), Adv. Enzymol, Realt, Areas Mol. Biol., 47: 45-148, Chou and Fasman (1978), Ann. Rev. Biochem., 47: 251-276 and Chou and Fasman (1979), Biphys. J., 26: 367-384, the descriptions of which are incorporated herein by reference). In addition, computer programs are currently available to support the prediction of antigenic portions and epitopic core regions of proteins. Examples include those programs based on the Jameson-Wolf analysis (Jameson and Wolf (1988), Comput.Appl.Biosci., 4 (1): 181-186 and Wolf et al. (1988), Comput. Appl. Biosci. , 4 (1): 187-191, the descriptions of which are incorporated herein by reference); the PepPlot® program (Brutlag et al. (1990), CABS, 6: 237-245 and Weinberger et al (1985), Science, 228: 740-742, the descriptions of which are incorporated herein by reference); and other programs for the prediction of the tertiary structure of proteins (Fetrow and Bryant (1993), BIOTECHNOLOGY, 11: 479-483, the description of which is incorporated herein by reference).

In contrast, substantial modifications in the functional and / or chemical characteristics of IL-lra can be carried out by selecting substitutions that differ significantly in their effect of maintaining (a) the structure of the polypeptide backbone in the area of substitution, for example, as a sheet or helix conformation, (b) the relative charge or hydrophobic affinity of the protein at the target site or (c) the mass of the side chain. The naturally occurring residues are divided into groups based on the common properties of the side chain: 1) hydrophobic: norleucine, Met, Ala, Val, Leu, He; 2) neutral hydrophilic: Cys, Ser, Thr; 3) Acids: Asp, Glu; 4) Basic: Asn, Gln, His, Lys, Arg; 5) aromatics: Trp, Tyr, Phe; and 6) residues that influence the orientation of the chain: Gly, Pro.

Non-conservative substitutions may involve the exchange of a member of one of these groups by another. Said substituted resins can be introduced into regions of the IL-lra which, for example, are homologous with other members of the IL-1 family or within the non-homologous regions of the protein.

A variety of amino acid substitutions or deletions can be made to modify or add O-linked or N-linked glycosylation sites, resulting in a protein with altered glycosylation. The sequence can be altered to add glycosylation sites or to eliminate glycosylation sites bound in O or linked in N from IL-lra. A glycosylation recognition site ligated with aspargine comprises a tripeptide sequence that is specifically recognized by its appropriate cellular glycosylation enzymes. These tripeptide sequences are either Asn-Xaa-Thr or Asn-Xaa-Ser, wherein Xaa can be any amino acid other than Pro.

Specific mutations of the IL-lra sequences may involve the substitution of a non-original amino acid at the amino terminus, carboxy terminus or at any site of the protein that is modified by the addition of an N-linked carbohydrate or ligated with 0. Such modifications may be of particular utility in the addition of an amino acid (eg, cysteine), which is advantageous for the binding of a water soluble polymer to form a derivative. For example, WO 92/16221 describes the preparation of IL-lra muteins, for example, wherein an original residue is changed to cysteine.

In a specific embodiment, a variant polypeptide may preferably be substantially homologous to the amino acid of IL-lra (SEQ ID NO: 2). The term "substantially homologous" as used herein means a degree of homology that is in excess of 80%, preferably in excess of 90%, and more preferably in excess of 95% or more preferably still 99%. The percentage of homology as described here is calculated as the percentage of amino acid residues found in the smallest of the two sequences which are aligned with identical amino acid residues in the sequence that is compared when four spaces in one length of 100 amino acids can be introduced to assist in that alignment, as established by Dayhoff in Atlas of Protein Sequence and S tru ctu re, 5_: 124, (1972), National Biochemical Research Foundation, Washington, DC, the description of the which is incorporated here as a reference. Also included within the term "substantially homologous" are the variant (s) of IL-lra which can be isolated by virtue of their cross-reactivity with antibodies to the amino acid sequences of SEQ ID NO: 2 or whose genes are they can isolate through hybridization with the DNA of SEQ ID NO: it with the segments thereof.

Polypeptide derivatives The chemically modified derivatives of the IL-lra protein (s) in which the protein is linked to a polymer in order to modify the properties of the protein (referred to herein as "derivatives"), are included within the scope of the present invention. Such derivatives can be prepared by someone skilled in the art given the included descriptions. The conjugates can be prepared using appropriate chemical moieties and glycosylated, non-glycosylated or de-glycosylated IL-lra protein (s). Typically non-glycosylated proteins and water soluble polymers will be used.

Water-soluble polymers are desirable because the protein to which they are placed does not precipitate in an aqueous medium, such as a physiological medium. Preferably the polymer will be pharmaceutically acceptable for the preparation of a therapeutic product or composition. One skilled in the art will be able to select the desired polymer based on such considerations as if the polymer / protein conjugate will be used therapeutically and if so, the therapeutic profile of the protein (e.g., duration of prolonged release, resistance to proteolysis). effects if any, on dosage, biological activity, ease of handling, degree or lack of antigenicity and other known effects of a water soluble polymer on therapeutic proteins).

Suitably, clinically acceptable water soluble polymers include, but are not limited to, polyethylene glycol (PEG), polyethylene glycol propionaldehyde, ethylene glycol / propylene glycol copolymers, monomethoxy polyethylene glycol, carboxymethyl cellulose, dextran, polyvinyl alcohol (PVA), polyvinyl pyrrolidone, poly-1,3-dioxolane, poly i-1,3,6-trioxane, copolymer of ethylene / maleic anhydride, poly (ß-amino acids) (either homopolymers or random copolymers), poly (n-vinyl) pyrrolidone) polyethylene glycol, homopolymers of polypropylene glycol (PPG) and other polyalkylene oxides, copolymers of ethylene oxide / polypropylene oxide, polyoxyethylated polyols (POG) (for example, glycerol) and other polyoxyethylated polyols, polyoxyethylated sorbitol or polyoxyethylated glucose, colonic acids or other polymers of carbohydrates, Ficoll or dextran and mixtures thereof. As used herein, polyethylene glycol means that it groups any of the forms that have been used to derive other proteins, such as mono- (Cl-Cl 0) alkoxy-aryloxy-polyethylene glycol. Polyethylene glycol propionaldehyde may have advantages in manufacturing due to its stability in water.

The water-soluble polymers can each be of any molecular weight and can be branched or unbranched. Generally, the higher the molecular weight or the higher the branches, the higher the polymer: protein ratio. Water-soluble polymers each typically have an average molecular weight of between about 2kDa to about 100kDa (the term "about" indicates that in the preparations of a water-soluble polymer, some molecules will weigh more, some less, than the molecular weight established). The average molecular weight of each water soluble polymer is preferably between 5kDa and about 40kDa, more preferably between about 10kDa and about 35kDa and most preferably between about 15kDa and about 30kDa.

A variety of placement methods are available to those skilled in the art, including acylation reactions or alkylation reactions (preferably to generate a chemically modified amino terminal protein) with a water-soluble reactive molecule. See, for example, EP 0 401 384; Malik et al. (1992), Exp. Hema t ol. , 20: 1028-1035; Francis (1992), Fo c u s on Growth Fa c tors, 3 (2): 4-10, published by Mediscript, Mountain Court, Friern Barnet Lane, London N20 OLD, United Kingdom; EP 0 154 316; EP 0 401 384; WO 92/16221; WO 95/34326; WO 95/13312; WO 96/11953; WO 96/19459 and WO 96/19459 and the other publications cited herein which relate to pegylation, the descriptions of which are hereby incorporated by reference.

A specific embodiment of the present invention is an unbranched aldehyde molecule of monomet oxy-polyethylene glycol having an average molecular weight of either about 20kDa or about 33kDa (for example, between 30kDa and 35kDa) , or a tertiary butyl aldehyde and polyethylene glycol having an average molecular weight of about 33 kDa (eg, between 30 kDa and 35 kDa) conjugated by means of reductive alkylation to the IL-lra protein (s) .

PEGylation can also be carried out specifically using water-soluble polymers having at least one reactive hydroxy group (eg, polyethylene glycol). The water-soluble polymer can react with an activating group, thereby forming an "activated linker" useful in the modification of various proteins. Functional linkers can be monofunctional, bi-functional or multifunctional.

Activating groups that can be used to link the water soluble polymer to two or more proteins include the following: sulfone, maleimide, sulfhydryl, thiol, triflate, tresylate, azidyrine, oxirane and 5-pyridyl. Useful reagents having a reactive sulfone group that can be used in the methods include, without limitation, chlorosulfone, vinylsulfone and divinyl sulfone. These PEG derivatives are stable against hydrolysis for prolonged periods in aqueous media at pHs of about 11 or less, and can form bonds with molecules to form conjugates that are also hydrolytically stable. Two particularly useful homofunctional derivatives are PEG-jbis-chlorosulfone and PEG-jbis-vinyl sulfone (WO 95/13312).

Polyvalent forms Polyvalent forms, that is, molecules comprising more than one active portion, can be constructed. In one embodiment, the molecule can possess multiple sites of the interleukin-1 receptor antagonist. Additionally, the molecule may possess at least one site of the interleukin-1 receptor antagonist and, depending on the desired characteristic of the polyvalent form, at least one site of another molecule (eg, one IL-protein (s)). lra), and a TNFbp product as described below).

In one embodiment, the polyvalent form can be constructed for example, by chemical coupling of at least one IL-lra protein (s) and another portion with some clinically accepted linker (e.g., a water soluble polymer). In principle, the linker must not impart new immunogenicity either by virtue of the new amino acid residues, alter the hydrophobic affinity and the charge balance of the structure, which affects its biodistribution and spacing.

The water-soluble polymers can be, based on the monomers listed here, homopolymers, block or random copolymers, straight or branched chain terpolymers, substituted or unsubstituted. The polymer can be of any length or molecular weight, but these characteristics can affect the biological properties. The average polymer molecular weights particularly useful for decreasing spacing rates in pharmaceutical applications are in the range of 2,000 to 35,000 daltons. In addition, the length of the polymer can be varied to optimize or confer the desired biological activity.

The active portions can be linked using conventional coupling techniques (see WO 92/16221, WO 95/13312 and WO 95/34326, the disclosures of which are incorporated herein by reference). For example, patents WO 92/16221 and WO 95/34326 describe the preparation of various dimerized molecules of IL-lra.

Alternatively, a divalent molecule may consist of two tandem repeats of IL-lra protein (s) separated by a polypeptide linker region. The design of the polypeptide linkages is similar in design to the insertion of short circuit sequences between domains in the non-vo protein design (Mutter (1988), TIBS, 13: 260-265 and Regan and DeGrado (1988), S ci en ce, 241: 976-978, the descriptions of which are incorporated herein by reference).

Several different binder constructs have been assembled and shown to be useful for forming single chain antibodies; the most functional bonds vary in size from 12 to 25 amino acids (amino acids have non-reactive side groups, eg, alanine, serine and glycine), which together constitute a hydrophilic sequence, have a few oppositely charged residues to increase the solubility and are flexible (Whitlow and .Filpula (1991), Me thods: A Compani on to Me thods in En zym ol ogy, 2_: 97-105; and Brigido etal. (1993), J. Imm un ol., 150 : 469-479, the descriptions of which are incorporated herein by reference).

Additionally, an IL-1ra protein (s) can be chemically coupled to the biotin, and the resulting conjugate can then be allowed to bind avidin, resulting in avidin / biotin / IL-11 tetravalent protein (s) molecules. An IL-1ra protein (s) may also be covalently coupled to dinitrophenol (DNP) or trini-troenol (TNP) and the resulting conjugates precipitated with anti-DNP or anti-TNP-IgM to form decamer conjugates.

In yet another embodiment, recombinant fusion proteins can be produced wherein each recombinant chimeric molecule has an amino terminal or terminally carboxylated IL-lra protein sequence (s) fused to all or part of the constant domains, but at least a constant domain, of the light chain of human immunoglobulin. For example, a chimeric fusion protein (s) IL-Ira / IgGl (or IgGl / protein (s) IL-lra) can be produced from a chimeric gene containing a light chain: a kappa light chain chimera IL-lra / human protein (s) (IL-Ira / Ck protein (s)) or a human kappa light chain / chimera IL-lra protein (s) (Ck / IL-lra protein (s)); or a chimeric gene containing a heavy chain: a heavy chain chimera gamma-1 protein (s) IL-lra / humama (IL-lra / Cg-1 protein (s)) or a gamma-1 human heavy chain / chimera IL-lra protein (s) (Cg-1 / IL-lra protein (s)). Following the transcription and translation of a chimeric heavy chain gene, or a gene containing a light chain and a heavy chain chimeric gene, the gene products can be assembled into a simple chimeric molecule having one (s) protein ( s) IL-lra displayed in bivalent form. Additional details related to the construction of such dimeric molecules are described in U.S. Patent 5,116,964, WO 89/09622, WO 91/16437 and EP 315062, the disclosures of which are incorporated herein by reference.

In yet another embodiment, recombinant fusion proteins can also be produced in which each recombinant chimeric molecule has at least one IL-lra protein (s) as described herein, and at least a portion of the region 186-401 of the teoprotogerin, as described in European Patent Application No. 96309363.8, the descriptions of which are incorporated herein by reference. Either the IL-lra protein (s) or the portion of teoprotogerin may be at the amino terminus or the carboxy terminus of the chimeric molecule Synthesis of the IL-lra protein (s).

The production of the IL-lra protein (s) is described in more detail below. Such proteins can be prepared, for example, by recombinant techniques or by chemical synthesis in vi t ro.

Polynucleotides Based on the present disclosure and using the universal codon table, one of ordinary skill in the art can easily determine all the nucleic acid sequences encoding the amino acid sequence of the IL-lra protein (s).

Recombinant expression techniques carried out in accordance with the descriptions set forth below, can be followed to produce each such polynucleotide and to express the encoded proteins. For example, by inserting a nucleic acid sequence encoding an IL-lra protein (s) into an appropriate vector, someone with skill in art can. easily produce large quantities of the desired nucleotide sequence. The sequences can then be used to generate detection probes or amplification primers. Alternatively, a polynucleotide encoding an IL-lra protein (s) can be inserted into an expression vector. By introducing the expression vector into an appropriate host, the desired protein can be produced in large quantities.

As further described herein, various vector / host systems are available for the propagation of the desired nucleic acid sequences and / or the production of the desired proteins. These include but are not limited to, plasmids, insertional or viral vectors, and prokaryotic and eukaryotic hosts. One of skill in the art can adapt a host / vector system that is capable of propagating or expressing heterologous DNA to produce or express the sequences of the present invention.

Furthermore, it will be appreciated by those skilled in the art that, in view of the present disclosure, the nucleic acid sequences within the scope of the present invention include the nucleic acid of Figure 1, as well as degenerate nucleic acid sequences thereof. , nucleic acid sequences encoding the variant (s) of IL-lra, and those nucleic acid sequences that hybridize (under hybridization conditions, or equivalent conditions or stringent conditions) to the acid sequence complements nucleic of Figure 1.

Also provided with the present invention are recombinant DNA constructs that involve the DNA vector together with the DNA sequences encoding the desired proteins. In each such DNA construct, the nucleic acid sequence encoding a desired protein (with or without signal peptides) is in operative association with an appropriate expression control or regulatory sequence capable of directing replication and / or expression of the desired protein in a selected host.

Recombinant Expression Preparation of Polynucleotides A nucleic acid sequence encoding an IL-lra protein (s) can be easily obtained in a variety of ways including without limitation, chemical synthesis, genomic or cDNA collection screening, screening of the expression collection and / or PCR amplification of the cDNA. These methods and others that are useful for the isolation of such nucleic acid sequences are set forth in Sambrook et al. (1989), Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Habror, NY; by Ausubel et al. (1994), Current Protocols in Molecular Biology, Current Protocols Press; and by Berger and Kimmel (1987), Methods in Enzymology: Guide to Molecular Cloning Techniques, Vol. 152, Academic Press, Inc., San Diego, CA, The descriptions of which are incorporated herein by reference.

The chemical synthesis of a nucleic acid sequence encoding a desired protein can be carried out using methods well known in the art, such as those established by Engels et al., (1989), Angew. Chem. Intl. Ed., 2_8: 716-734 and Wells et al. (1985), Gene 3_4_: 315, the descriptions of which are incorporated herein by reference. These methods include, inter alia, methods of phosphotris ter, fos foramitate and H-phosphonate of the synthesis of the nucleic acid sequence. Large nucleic acid sequences, for example, those larger than about 100 nucleotides in length, can be synthesized as various fragments. The fragments can be ligated together to form an appropriate nucleic acid sequence. A preferred method is the supported polymer synthesis using the normal phosphoramidite chemistry.

Alternatively, an appropriate nucleic acid sequence can be obtained by screening an appropriate collection of cDNA (i.e., a library prepared from one or more tissue sources thought to express the protein) or a genomic library ( a collection prepared from total genomic DNA). The source of the cDNA collection is typically a source of tissue or cell of some species that is believed to express a desired protein in reasonable amounts. The source of the genomic collection can be any tissue or tissues of a mammal or other species that is believed to harbor a gene that encodes the desired protein.

Each hybridization medium can be screened for the presence of a DNA encoding a desired protein using one or more nucleic acid probes (oligonucleotides, cDNA fragments or genomic DNA that possess an acceptable level of homology with the cDNA or the be cloned) that will hybridize selectively with the cDNA (s) or gene (s) present in the collection. The probes typically used for such screening encode a small region of equal or similar species as the species from which the collection is prepared. Alternatively, the probes may degenerate as discussed here.

"Hybridization is typically carried out by softening the oligonucleotide probe or cDNA to the clones, under conditions of astringency that prevent non-specific binding but allow the binding of those clones that have a significant level of homology with the probe. or primary. Typical conditions of hybridization and wash stringency depend in part on the size (ie, the number of nucleotides in length) of the cDNA or oligonucleotide probe and on whether the probe is degenerate.

The probability of identifying a clone is also considered when designing the hybridization medium (for example, if a genomic collection or cDNA is being screened).

Where a DNA fragment (such as the cDNA) is used as a probe, typical hybridization conditions include those set forth in Ausubel et al. (1994), upra. After hybridization, the hybridization medium is washed to an appropriate astringency, depending on various factors such as the size of the probe, the expected homology of the probe to the clone, the hybridization medium being screened, the number of clones that are being sifted and similar.

The exemplary conditions of astringent hybridization are hybridization in 6 x SSC at 62-67CC, followed by washing in 2.5 x SSC at 62-67 ° C for about one hour. Alternatively, exemplary astringent hybridization conditions are hybridization to 45-55% formamide, 6 x SSC at 40-45 ° C, followed by washing in 0.1 x SSC at 62-67 ° C for about one hour. Also included are the DNA sequences which hybridize to the nucleic acid sequences set forth in Figures 1 and 3 under relaxed hybridization conditions and which encode an IL-lra protein (s). Examples of such relaxed astringency hybridization conditions are 6 x SSC at 45-55 ° C or hybridization with 30-40% of formamide at 40-45 ° C, followed by washing in 1-2 x SSC at 55 ° C for approximately 30 minutes. See Maniatis et al. (1982), Mol ecul a r Cl on ing (A laboratory manual), Cold Spring Harbor Laboratory, pages 387 to 389, the description of which is incorporated herein by reference.

There are also exemplary protocols for stringent washing conditions wherein the oligonucleotide probes are used to screen the hybridization media. For example, a first protocol uses 6 x SSC with 0.05 percent sodium pyrophosphate at a temperature between about 35 ° C and 63 ° C, depending on the length of the probe. For example, the base 14 probes are washed at 35-40 ° C, the base probes 17 at 45-50 ° C, the base probes 20 at 52-57 ° C and the base probes 23 to 57-63 ° C. The temperature can be increased 2-3 ° C where the non-specific back-up link appears high. A second protocol uses tetramethyl ammonium chloride (TMAC) for washing. One such astringent wash solution is TMAC 3 M, 50 mM tris-HC1, pH 8.0 and 0.2% SDS.

Another method for obtaining an appropriate nucleic acid sequence encoding an IL-lra protein (s) is the polymerase chain reaction (PCR). In this method, the cDNA is prepared from poly (A) + RNA or total RNA using the reverse enzyme transcriptase. The two primaries, typically complementary to the two separate regions of cDNA (oligonucleotides) encoding the desired protein, are then added to the cDNA together with a polymerase such as the Ta q polymerase and the polymerase amplifies the cDNA region between the two primaries.

The sequences of oligonucleotides selected as probes or primaries must be of adequate length and sufficiently unambiguous in order to minimize the amount of non-specific binding that may occur during screening or PCR amplification. The current sequence of the probes or primaries is usually based on highly homologous or conserved sequences or regions. Optionally, the oppressive probes may be completely or partially degenerate, that is, they may contain a mixture of probes / primaries, all encoding the same amino acid sequence but using different codons to do so. An alternative for the preparation of degenerated probes is to place an inosine in some or all codon positions that vary by species. Oligonucleotide or primary probes can be prepared by chemical synthesis methods for DNA as described herein.

Vector The DNA encoding the desired proteins can be inserted into vectors for further cloning (amplification of the DNA) or for expression. Suitable vectors are commercially available or can be specifically constructed. The selection or construction of an appropriate vector will depend on (1) whether it is to be used for DNA amplification or for DNA expression, (2) the size of the DNA to be inserted into the vector and (3) the intended host cell. .a transform with the vector.

The vectors each typically involve a nucleic acid sequence encoding a desired protein operably linked to one or more of the following regulatory or expression control sequences, capable of directing, controlling or otherwise effecting the expression of a desired protein by a selected host cell. Each vector contains various components depending on its function (DNA amplification or DNA expression) and its compatibility with the intended host cell.

The components of the vector generally include, but are not limited to, one or more of the following: a sequence signal, an origin of replication, one or more marker or selection genes, a promoter, an enrichment element, a transcription sequence of termination and the like. These components can be obtained from natural sources or synthesized by known methods.

Examples of suitable cloning prokaryotic vectors include bacteriophages such as lambda derivatives, or plasmids of E. Col i (e.g., pBR322, col El, pUC, F-factor derivatives and Bluescript® plasmid (Stratagene, La Jolla, CA) Other appropriate expression vectors, of which numerous types are known in the art for host cells described below, can also be used for this purpose.

Signal Sequence The nucleic acid encoding a sequence signal can be inserted 5 'of the sequence encoding a desired protein, for example, it can be a component of a vector or it can be part of a nucleic acid encoding the desired protein. Nucleic acids encoding the sequences of the original IL-lra signal are known (U.S. Patent No. 5, 075, 222).

Origin of the Replication.

The expression and cloning vectors generally each include a nucleic acid sequence that allows the vector to replicate in one or more selected host cells. In a cloning vector, this sequence is typically one that allows the vector to replicate independently of the host chromosome DNA and includes an origin of replication or an autonomous replicating sequence. Such sequences are well known. The origin of replication from plasmid pBR322 is appropriate for most Gram-negative bacteria, and various origins (eg, Simian Virus 40 (SV40), polyoma, adenovirus, VSV or BPV) are useful for vectors in the cells of mammals. Generally, the origin of replication is not needed for mammalian expression vectors (for example, the SV40 origin is often used only because it contains the early promoter).

Gene of Selection.

The expression and cloning vectors each typically contain a selection gene. This gene encodes a "marker" protein necessary for the survival or growth of the trans-fused host cells when they grow in selective culture media. The cells that are not transformed with the vector will not contain the selection gene and therefore will not survive in the culture media. Typical selection genes encode proteins that (a) confer resistance to antibiotics or other toxins, for example, ampicillin, neomycin, methotrexate or tetracycline; (b) auxotropic complement deficiencies; or (c) provide critical nutrients not available from the culture media.

Other selection genes can be used to amplify the genes to be expressed. Amplification is the process in which the genes that have a greater demand for the production of a protein critical for growth, are repeated in tandem within the chromosomes of successive generations of recombinant cells. Examples of suitable selectable markers for mammalian cells include dihydrofolate reductase (DHFR) and thymidine kinase. The cell transformants are placed under selection pressure to which only the transformants are particularly adapted to survive by virtue of the label that is present in the vector. The selection pressure is imposed by culturing the transformed cells under conditions in which the concentration of the selection agent in the media is successively changed, thereby leading to the amplification of both the selection gene and the DNA encoding the protein desired. As a result, the increasing amounts of the desired protein are synthesized from the amplified DNA.

For example, cells transformed with the DHFR selection gene are first identified by culturing all transformants in culture media containing methotrexate, a competitive antagonist of DHFR. An appropriate host cell when wild-type DHFR is used is the Chinese hamster ovary cell line deficient in DHFR activity (Urlaub and Chasin (1980), Pro c. Na ti. Acad. Sci., USA, 77 (7): 4216-4220, the description of which is incorporated herein by reference). The transformed cells are then exposed to increasing levels of methotrexate. This leads to the synthesis of multiple copies of the DHFR gene and concomitantly, multiple copies of other DNAs present in the expression vector, such as the DNA encoding a desired protein.

Promoter The expression and cloning vectors each will typically contain a promoter that is recognized by the host organism and is likely linked to a nucleic acid sequence encoding the desired protein. A promoter is a non-translated sequence located upstream (5 ') of the starting codon of a structural gene (generally within about 100 to 1000 bp) that controls the transcription and translation of a particular nucleic acid sequence. A promoter can be conventionally grouped into one of two classes, inducible promoters and constitutive promoters. An inducible promoter initiates increasing levels of transcription of the DNA under its control in response to some change in culture conditions, such as the presence or absence of a nutrient or a change in temperature. A large number of promoters, recognized by a variety of potential host cells are well known. A promoter can be operably linked to the DNA encoding a desired protein by removing the promoter from the source DNA by digestion of restriction enzymes and inserting the desired sequence of the promoter. The original sequences of the IL-lra promoter can be used to direct the amplification and / or expression of the DNA encoding a desired protein. A heterologous promoter is preferred however, if it allows for greater transcription and higher yields of the expressed protein, compared to the original promoter and if it is compatible with the host cell system that has been selected for use. For example, any of the original promoter sequences of other members of the IL-lra family can be used for direct amplification and / or expression of the DNA encoding a desired protein.

Suitable promoters for use with prokaryotic hosts include beta-lactamase and lactose-promoting systems; alkaline phosphotase; a tryptophan (trp) promoter system, a bacterial luminescence (luxR) gene system and hybrid promoters such as the tac promoter. Other known bacterial promoters are also suitable. Their nucleotide sequences have been published, thereby allowing someone with skill in the art to link each selected sequence to the desired DNA sequence using links or adapters as needed to supply some required sites of restriction.

Proper promoter sequences for use with yeast hosts are also well known in the art. Promoters suitable for use with mammalian host cells are well known and include those obtained from the genomes of viruses such as polyoma virus, poultry pox virus, adenovirus (such as Adenovirus 2), papilloma virus bovine, bird sarcoma virus, cytomegalovirus and retrovirus, hepatitis B virus, most preferably SV 40. Other suitable mammalian promoters include promoters from heterologous mammals, eg, heat shock promoters and the actin promoter.

Enriching Element.

The cloning and expression vectors will each typically contain an enrichment sequence to enhance transcription by higher eukaryotes of a DNA sequence encoding a desired protein. Enrichers are cis-acting elements of DNA, usually from about 10-300 bp in length, which act on the promoter to increase its transcription. The enrichments are relatively independent of orientation and position. 5 'and 3' have been found in the transcription unit. Yeast enrichments are advantageously used with yeast promoters. Various enrichment sequences available from mammalian genes (eg, globin, elastase, albumin, alpha-fetus-protein and insulin) are known. Additionally, viral enrichments such as the SV40 enrichment, the cytomegalovirus early promoter enrichment, the polyoma enrichment and the adenovirus enhancers are exemplary enriching elements for the activation of eukaryotic promoters. While an enrichment can be spliced with a vector at the 5 'or 3' position to a DNA encoding the desired protein, it is typically located at the 5 'site of the promoter.

Termination of the Transcript.

The expression vectors used in the eukaryotic host cells, each will typically contain a sequence necessary for the termination of transcription and to stabilize the mRNA. Such sequences are commonly available from the 5 'and occasionally 3' untranslated regions of eukaryotic DNAs and cDNAs. These regions contain nucleotide segments transcribed as polyadenylated fragments in the non-translated portion of the mRNA that encodes a desired portein.

Construction of the Vector.

The construction of an appropriate vector containing one or more of the compounds listed here (together with the desired coding sequence) can be carried out by normal binding techniques. The isolated plasmids or DNA fragments are cleaved, adjusted and re-ligated in the desired order to generate the required vector. To confirm that the correct sequence has been constructed, the binding mixture can be used to transform E. Col i. , and successful transformants can be selected by known techniques as described herein. The vector amounts of the transformants are then prepared, analyzed by digestion of restriction endonucleases and / or sequenced to confirm the presence of the desired construct.

A vector that provides transient expression of the DNA encoding a desired protein in mammalian cells, it can also be used. In general, transient expression involves the use of an expression vector that is capable of efficiently replicating in a host cell, such that the host cell accumulates various copies of the expression vector and in turn, synthesizes high levels of the desired protein encoded by the expression vector. Each transient expression system, comprising an appropriate expression vector and a host cell, allows convenient positive identification of proteins encoded by cloned DNAs, as well as for rapid screening of such proteins for desired biological and physiological properties.

Host Cells Any of a variety of recombinant host cells, each of which contains a nucleic acid sequence for use in the expression of the desired protein, is also provided by the present invention. Exemplary eukaryotic and prokaryotic host cells include bacterial, mammalian, fungal, insect, yeast or plant cells.

Prokaryotic host cells include, but are not limited to, eubacteria such as Gram-negative or Gram-positive organisms (e.g., E. coli (HB101, DH5a, DH10 and MC1061), Bacilli spp., Such as B. subtilis; Pseudomonas spp. P. aeruginosa, Streptomyces spp., Salmonella spp., Such as S. Typhimurium, or Serratia spp., Such as S. Marcescans In a specific embodiment, a desired protein can be expressed in E. coli.

In addition to the prokaryotic host cells, the IL-lra protein (s) can be expressed in glycosylated form by any of a number of appropriate host cells derived from multicellular organisms. Such host cells are capable of activities of complex processing and glycosylation. In principle, any culture of eukaryotic higher cells can be used, whether said culture involves vertebrate or invertebrate cells, including insect and plant cells. Eukaryotic microbes such as fungal strains or yeasts may be appropriate hosts for the expression of a desired protein. Saccharomyces cerevisiae, or common baker's yeast, is the most commonly used among lower eukaryotic host microorganisms, but a variety of other genera, species, and strains are well known and commonly available.

The vertebrate cells can be used since the propagation of vertebrate cells in cultures (tissue culture) is a well-known procedure. Examples of useful mammalian host cell lines include, but are not limited to, simian kidney line transformed by the SV40 human embryonic kidney line (COS-7) (293 cells or 293 cells subcloned for growth in suspension cultures). , newborn hamster kidney cells and Chinese hamster ovary cells. Other suitable mammalian cell lines include, but are not limited to, HeLa, mouse L-929 cells, 3T3 lines derived from Swiss mice, Balb-c or NIH, and hamster cell lines BHK or HaK. In a specific embodiment, a desired protein can be expressed in COS cells or in baculovirus cells.

A host cell can be transfected and preferably transformed with a desired nucleic acid under appropriate conditions that allow the expression of the nucleic acid. The selection of appropriate host cells and methods for transformation, culture, amplification, screening and product production and purification are well known in the art (Gething and Sambrook (1981), Na t ure, 293: 620-625 or alternatively, Kaufman et al. (1985), Mol.Cel. L Bi. Bi., 5 (7): 1750-1759, or US Pat. No. 4,419,446, the descriptions of which are incorporated herein by reference). For example, for mammalian cells without cell walls, the calcium phosphate precipitation method can be used. Electroporation, micro-injection and other known techniques can also be used.

It is also possible that a desired protein can be produced by homologous recombination or by recombinant production methods using control elements introduced into cells that already contain the DNA encoding a desired protein. Homologous recombination is a technique originally developed to search for target genes for inducing or correcting mutations in transcriptionally active genes (Kucherlapati (1989), Prog. In Nu. Aci d Res. An d Mol. Bi ol., 36: 301, the description of which is incorporated herein by reference). The basic technique was developed as a method to introduce specific mutations within specific regions of the mammalian genome (Thomas et al. (1986), Cel l, 4_4_: 419-428; Thomas and Capecchi (1987), Cel l, 5 ^: 503-512 and Doetschman et al. (1988), Pro c. Na t i. Aca d. Sci. , 85: 8583-8587, the descriptions of which are incorporated herein by reference) or to correct for specific mutations within the defective genes (Doetschman et al. (1987), Narure, 330: 576-578, the description of the which is incorporated here as a reference). Exemplary techniques are described in U.S. Pat. No. 5,272,071; WO 92/01069; WO 93/03183; WO 94/12650 and ~ WO 94/31560, the descriptions of which are incorporated herein by reference.

Cultivation of host cells.

The method for cultivating each or more recombinant host cells for production will vary depending on many factors and considerations; The optimal production procedure for a given situation will be apparent to those skilled in the art through minimal experimentation. Said recombinant host cells are cultured in appropriate media and the expressed protein is then recovered, isolated and optionally purified from the culture media (or from the cell if expressed intracellularly) by appropriate means known to those skilled in the art.

Specifically, each of the recombinant cells used to produce a desired protein can be cultured in appropriate culture media to induce promoters, by selecting appropriate recombinant host cells or by amplifying the gene encoding the desired protein. The culture media can be supplemented as necessary with hormones and / or other growth factors (such as insulin, transferrin or epidermal growth factor), salts (such as sodium chloride, calcium, magnesium and phosphate), buffer solutions ( such as HEPES), nucleosides (such as adenosine and thymidine), antibiotics (such as gentamicin), trace elements (defined as inorganic compounds usually present in final concentrations in the micromolar range) and glucose or other energy source. Other supplements may be included at appropriate concentrations, as will be appreciated by those skilled in the art. Suitable culture conditions such as temperature, pH and the like are also well known in the art for use with the selected host cells.

The product resulting from the expression can then be purified to near homogeneity by using procedures known in the art. Exemplary purification techniques are taught in U.S. Patents. No. 5,075,222, and WO 91/08285, the descriptions of which are incorporated herein by reference. Preferably, the expression product is produced in a substantially pure form. By "substantially pure" it means an IL-lra, in an unmodified form, as a highly comparative specific activity, preferably in the range of about 150,000-500,000 receptor units / mg as defined in Hannum et al. (1990), Na t ure, 343: 336-340 and Eisenberg et al. (1990), Na t ure, 343: 341-346, both of which are incorporated herein by reference.

Pharmaceutical compositions.

The present invention groups together pharmaceutical compositions, each containing therapeutically or prophylactically effective amounts of an IL-lra protein (s) or a chemically modified derivative thereof (collectively, "protinin product (s) IL-lra") in combination with a vehicle. The carrier preferably includes one or more pharmaceutically or physiologically acceptable formulation materials in combination with the IL-lra protein product (s).

The primary solvent in a vehicle can be aqueous or non-aqueous in nature. In addition, the carrier may contain pharmaceutically acceptable excipients to modify or maintain the pH preferably between 6.0 and 7.0, more preferably 6.5 (for example, buffer solutions such as citrates or phosphates, and amino acids such as glycine); viscosity; clarity; color; sterility; stability (for example, sucrose or sorbitol); odor; dissolution rate (for example, solubilizers or solubilizing agents such as alcohols, polyethylene glycols and sodium chloride); speed of release; as well as volume agents for lyophilized formulation (e.g., mannitol and glycine); surfactants (for example, polysorbate 20, polysorbate 80, triton and pluronics); antioxidants (for example, sodium sulfite and sodium acid sulfite); preservatives (for example, benzoic acid and salicylic acid); flavoring agents and diluents; emulsifying agents; suspension agents; solvents; fillings; delivery vehicles and other adjuvants and / or pharmaceutical excipients. Other forms of effective administration such as parenteral slow release formulations, inhalant nebulizations, orally active formulations or suppositories are also envisioned. The composition may also involve particular preparations of polymeric compounds such as bulk erosion polymers (eg, poly (lactic-co-glycolic acid) copolymers (PLGA), mixtures of PLGA polymers, PEG block copolymers, and lactic acid and glycolic, poly (cyanoacrylates)); surface erosion polymers (e.g., poly (anhydrides) and poly (ortho esters)); hydrogel esters (for example, pluronic polyols, polyvinyl alcohol, polyvinylpyrrolidone, alkyl vinyl ether-maleic anhydride copolymers, cellulose, derivatives of hyaluronic acid, alginate, collagen, gelatin, albumin, and starches and dextrans) and systems of compositions thereof; or liposome or microsphere preparations. Such compositions can influence the physical state, stability, rapidity of the in vivo release, and rapidity of the spacing of the current proteins and derivatives. The optimal pharmaceutical formulation for a desired protein will be determined by one skilled in the art depending on the route of administration and the desired dose. Exemplary pharmaceutical compositions are described in Remington's Pharmaceutical Sciences, Ed. 18a (1990), Mack Publishing Co., Easton, PA 18042, pages 1435-1712; Gombotz and Pettit (1995), Bioconj ugate Chem., 6: 332-351; Leone-Bay et al. (1995), Journal of Medicine Chemistry, 38: 4263-4269; Haas, et al. (1995), Clinical Immunology and Immunopathology, 76 (1): 93; WO 94/21275; FR 2706772; WO 94/21235, and WO 97/28828, the descriptions of which are incorporated herein by reference.

Specific sustained release compositions are available from the following providers: Depotech (Depofoam ™, a multivesicular liposome) and Alkermes (ProLease ™, a PLGA microsphere). Exemplary forms of hyaluronan are described er. Peyron and Balazs (1974), Path. Biol., 22 (8): 731-736; Isdale et al. (1991), J. Drug. Dev., 4 (2): 93-99; Larsen et al. (1993), Journal of Biomedical Materials Research, 27_: 1129-1134; Namiki, et al. ("1982), International Journal of Clinical Pharmacology, Therapy and Toxicology, 20 (11): 501-507, Meyer et al. (1995), Journal of Controlled Relay, 3_5_: 67-72, Kikuchi et al. (1996). ), Osteoarthritis and Cartilage, 4: 99-110, Sakakibara et al. (1994), Clinical Orthopedics and Related Research, 299: 282-292, Meyers and Brandt (1995), 22 (9): 1732-1739, Laurent et al. al. (1995), Acta Orthop Scand, 66 (266): 116-120, Cascone et al. (1995), Biomaterials, 16 (7): 569-574, Yerashalmi et al. (1994), Archives of Biochemistry and Biophysics, 313 (2): 267-273; Bernatchez et al. (1993), Journal of Biomedical Materials research, 27 (5): 677-681; Tan et al. (1990), Australian Journal of Biotechnology, 4 (1): 38 -43; Gomobotz and Petit (1995), Bioconjugate Chem., 6 ^: 332-351; U.S. 4,582,865, 4,605,691, 4,636,524, 4,713,448, 4,716,154, 4,716,224, 4,772,419, 4,851,521, 4,957,774, 4,863,907, 5,128,326, 5,202,431, 5,336,767, 5,356,883; European Patent Application Nos. 0 507 604 A2 and 0 718 312 A2; and WO 96/05845, the descriptions of which are incorporated herein by reference. Specific hyaluronan compositions are available from the following suppliers: BioMatrix, Inc. Ridgefield, NJ (Synvisc ™, a 90:10 mixture of a hilane fluid and a hilane gel); Fidia S.p.A., Abano Terme, Italy (Hyalgan ™, the sodium salt of a hyaluronic acid derived from the crest of the cock (~ 500,000 up to ~ 700,000 PM)); Kaken Pharmacuetical Co., Ltd., Tokyo, Japan (Artz ™, a 1% solution of a hyaluronic acid derived from the crest of the cock ~ 700,000 PM); Pharmacia AB, Stockholm, Sweden (Healon ™, a "ialuronic acid derived from the crest of the cock, 4 x 106 MP); Genzyme Corporation, Cambridge, MA (Surgicoat ™, a recombinant hyaluronic acid); Pronova Biopolymer, Inc. Portsmouth, NH (Hyaluronic Acid FCH, a high molecular weight hyaluronic acid (for example, ~ 1.5-2.2 x 106 PM) hyaluronic acid prepared from cultures of S trep t ococcus z oo epi dem i c u s; MV sodium hyaluronate, ~ 1.0-1-6 x 106 PM and LV sodium hyaluronate, ~ 1.5-2.2 x 106 PM); Calbiochem-Novabiochem AB, Lautelfingen, Switzerland (Hyaluronic Acid), sodium salt (catalog number 1997 of company 385908) prepared from S trep to coccus sp.); Intergen Company, Purchase, NY (a hyaluronic acid derived from crest of the rooster, > 1 x 106 MP); Diosynth Inc., Chicago IL; Amerchol Corp., Edison, NJ and Kyowa Hakko Kogyo Co., Ltd., Tokyo, Japan.

Another specific pharmaceutical formulation is 10 millimolar of sodium citrate, 140 millimolar of sodium chloride, 0.5 millimolar of EDTA, 0.1% of polysorbate (by weight) in water, pH 6.5 ("formulation of citrate buffer"). Another specific formulation is 10 millimolar of sodium phosphate, 140 millimolar of sodium chloride, between 0.1% (by weight) and 0.01% of polysorbate (by weight) in water, and, optionally, 0.5 millimolar of EDTA, pH 6.5 (" forulation of phosphate buffer "). Yet another specific formulation is Hilan H-10 ™ fluid, a cross-linked hyaluronic acid (Biomatrix, Inc. Ridgefield, Inc.) to carry out 100 mg / ml IL-lra and 2% hyaluronic acid (Mr3 4xl06).

Once the pharmaceutical composition has been formulated, it can be stored in sterile vials as a solution, suspension, gel, emulsion, solid or a lyophilized or dehydrated powder. Such compositions each may be stored either in an easy-to-use form or in a form (eg, lyophilized) that requires reconstitution prior to administration.

In a specific embodiment, the present invention is directed to kits for producing a single dose administration unit. The kits may contain a first container having a dry protein and a second container having an aqueous formulation. The kits included within the scope of this invention are simple syringes and multi-chambered pre-filled syringes; Exemplary pre-filled syringes (eg, liquid syringes, and lyo-syringes such as Lyo-Ject®, a pre-filled dual-chamber lyo-syringe) are available from Vetter GmbH, Ravensburg, Germany.

Applications The IL-lra protein product (s) may be useful as research reagents and as diagnostic and therapeutic agents. In this way, the IL-lra protein product (s) can be used in in vitro and / or in vi ve diagnostic tests to quantify the amount of IL-lra in a tissue or organ sample. or to determine and / or isolate cells expressing IL-1. In tissue or organ testing, there will be less radioactivity from a 125I-IL-1 product (s) bound to IL-1, compared to a standardized binding curve of a 125-protein product (s). lra, due to unlabeled original IL-lra linked to IL-1. Similarly, the use of a 125I-IL-lra protein product (s) can be used to detect the presence of IL-1 in various cell types.

This invention also contemplates the use of the IL-lra protein product (s) in the generation of antibodies and the resulting antibodies (specifically including those that also bind to the original IL-lra). The antibodies can develop which bind to the IL-lra protein product (s). One of ordinary skill in the art can use well-known published procedures to obtain monoclonal and polyclonal antibodies or recombinant antibodies which specifically recognize and bind to various proteins encoded by the amino acid sequences of the present invention. Such antibodies can then be used to purify and characterize the original IL-Ira.

The present invention also relates to methods for the treatment of certain diseases and medical conditions (many of which may be characterized as inflammatory disorders) that are measured by IL-1, as well as the related sequelae and symptoms associated therewith. . A non-exclusive list of acute and chronic disorders mediated by interleukin 1 (IL-1) include but are not limited to the following: ALS; Alzheimer disease; asthma; arterosclerosis; cachexia / anorexia; Chronic Fatigue Syndrome; depression; diabetes (for example, diabetes mellitus and juvenile Type 1 attack); fever; fibromyalgia or anal gesia; glomerulonepri tis; rejection of grafts against host; hemorrhagic shock; hyperalgesia; inflammatory bowel disease, ischemic injury, including cerebral ischemia (for example, brain damage resulting from trauma, epilepsy, hemorrhage or infarction, each of which leads to neurodegeneration); lung disorders (eg, ARDS adult respiratory distress syndrome and pulmonary fibrosis); multiple myeloma; multiple sclerosis; myelogenous (for example, AML and CML) and other leukemias; myopathies (eg, muscle protein metabolism, esp., in sepsis); eye disorders; osteoporosis; Parkinson's disease; pain; pancreatitis; pulmonary fibrosis; labor; psoriasis; reperfusion damage; rheumatic disorders (eg, rheumatoid arthritis, osteoarthritis, juvenile arthritis (rheumatoid), seronegative polyarthritis, ankylosing spondylitis, Reiter's syndrome and reactive arthritis, psoriatic arthritis, enteropathic arthritis, polymyositis, dermatomyositis, scleroderma, systemic sclerosis, vasculitis, cerebral vasculitis, Lyme disease, staphylococcal-induced arthritis ("septic"), Sjögren's syndrome, rheumatic fever, polychondritis, and polymyalgia rheumatica and giant cell arteritis); septic shock; side effects of radiation therapy; disease of the temporal mandibular union, tumor metastasis or an inflammatory condition resulting from damage to the cartilage, jerks or twists, trauma, orthopedic surgery, infection or other disease processes.

IL-1 inhibitors (e.g., preferably an IL-lra protein product (s) and more preferably IL-lra) can be administered to a patient in therapeutically effective amounts for the prevention or treatment of IL-1 mediated diseases. including rheumatic diseases. The term "patient" is intended to group animals (eg cats, dogs and horses) as well as humans.

In addition IL-1 inhibitors (e.g., preferably an IL-lra protein product (s) and more preferably IL-lra) can be administered topically, enterally or parenterally including, without limitation, infusion, intraarterial, intraarticular, intracapsular , intracardiac, intradermal, intramuscular, intorbital, intrathecal, intravenous, intraperitoneal, intraspinal, intrasternal, intraventricular, subcutaneous, subcuticular, subcapsular, subarachnoid and trans tracheal injection. IL-1 inhibitors (e.g., preferably an IL-lra protein product (s) and more preferably IL-lra) may also be administered via oral administration or administered through mucosal membranes, i.e., buccally, intranasally, rectally or sublingually by systemic delivery.

It is preferred that IL-1 inhibitors (e.g., preferably an IL-lra protein product (s) and more preferably IL-lra) be administered intra-articularly, intramuscularly, intravenously or subcutaneously. Additionally, IL-1 inhibitors (e.g., preferably an IL-lra protein product (s) and more preferably IL-lra) can be administered by continuous infusion (eg, constant or intermittently implanted or flow modulating devices). for external infusion) so as to continuously provide the desired level of IL-1 inhibitors (eg, preferably an IL-lra protein product (s) and more preferably IL-lra) in the blood for the duration of administration. This can be carried out by means of a mini-pump such as an osmotic mini-pump. In these forms, one can ensure that the amount of the drug remains at the desired level and one can take blood samples and monitor the amount of medication in the bloodstream. Various pumps are commercially available, for example, from suppliers such as MiniMed Inc., Sylmar, CA (e.g., MT507) and Alza Corp., Palo Alto, CA (eg, Alzet osmotic pump, 2MLI model).

It is also contemplated that other forms of continuous or almost continuous dosing can be practiced. For example, chemical derivatization can result in sustained release forms of protein that have the effect of a continuous presence in the bloodstream, in predictable amounts based on a predetermined dosage regimen.

The modes of use of IL-1 inhibitors (e.g., preferably an IL-lra protein product (s) and more preferably IL-lra) for the treatment of IL-1 mediated diseases, including rheumatic disorders (e.g. osteoarthritis, psoriatic arthritis and rheumatoid arthritis), are set forth in Australian Patent Application AU 9173636, the teachings of which are incorporated herein by reference. By way of example but not limitation, in a specific embodiment, IL-1 inhibitors (e.g., preferably an IL-lra protein product (s) and more preferably IL-lra) may be administered intra-articularly for treatment of rheumatoid arthritis and osteoarthritis. By way of example but not limitation in another specific embodiment, the IL-1 inhibitors (eg, preferably an IL-1ra protein product (s) and, more preferably IL-1RA) can be administered subcutaneously or intramuscularly for the treatment of rheumatoid arthritis, inflammatory bowel disease, multiple sclerosis, multiple myeloma, or myelogenous (for example, AML and CML) and other leukemias. By way of example but not limitation, in yet a specific additional embodiment, the IL-1 inhibitors (eg, preferably an IL-lra protein product (s) and more preferably IL-lra) can be administered intravenously for tarting of brain damage as a result of trauma, epilepsy, haemorrhage or infarction, or for the treatment of disorders of host versus graft disorder or administered int raventricularly for the treatment of brain damage as a result of trauma.

In another embodiment, cell therapy is also contemplated, for example, implantation of cells that produce an IL-1 inhibitor (e.g., preferably an IL-lra protein product (s) and more preferably IL-lra). This embodiment of the present invention may include implanting in cells patients that are capable of synthesizing and secreting an IL-1 inhibitor (e.g., preferably an IL-lra protein product (s) and more preferably IL-lra). Such cells that produce an IL-1 inhibitor (e.g., preferably an IL-lra protein product (s) and more preferably IL-lra) may be cells that do not normally produce an IL-1 inhibitor but have been modified to produce an IL-1 inhibitor (e.g., preferably an IL-lra protein product (s) and more preferably IL-lra). The cells can also be cells whose ability to produce an IL-1 inhibitor (e.g., preferably an IL-lra protein product (s) and more preferably IL-lra) has been enhanced by transformation with an appropriate polynucleotide for expression. and secretion of an IL-1 inhibitor (e.g., preferably an IL-lra protein product (s) and more preferably IL-lra). In order to minimize a potential immune reaction in patients by the administration of an IL-1 inhibitor (e.g., preferably an IL-lra protein product (s) and more preferably IL-lra) of a foreign species, it is preferred that the cells are of the same species as the patient (e.g., humans) or that the cells are encapsulated with material that provides a barrier against immune recognition, or that the cells are placed in an immunologically privileged anatomic location, such as in the testicles, eyes or the central nervous system.

Human or non-human animal cells can be implanted in patients in biocompatible, semipermeable polymeric membranes or membranes to allow the release of the IL-1 inhibitor (e.g., preferably an IL-lra protein product (s) and more preferably IL-lra) , but to avoid the destruction of the cells by the patient's immune system or by other detrimental factors from the surrounding tissue. Alternatively, the patient's own cells, transformed ex vi ve to produce an IL-1 inhibitor (e.g., preferably an IL-lra protein product (s) and more preferably IL-lra), can be implanted directly into the patient. patient without such encapsulation. The methodology for membrane encapsulation of living cells is familiar to those of ordinary skill in the art, and the preparation of the encapsulated cells and their implantation in patients can be carried out.

In yet another embodiment, in vivo gene therapy is also envisioned, wherein a nucleic acid sequence encoding an IL-1 inhibitor (e.g., preferably an IL-lra protein product (s) and more preferably IL-lra ), is introduced directly into the patient. For example, a nucleic acid sequence encoding an IL-1 inhibitor (e.g., preferably an IL-lra protein product (s) and more preferably IL-lra) is introduced into the target cells by local injection of an acid construct. nucleic acid, with or without an appropriate delivery vector, such as an adeno-associated virus vector. Alternate viral vectors include but are not limited to retroviruses, adenoviruses, herpes simplex viruses and papilloma virus vectors. The physical transfer can be achieved in vivo by local injection of the desired nucleic acid construct or other appropriate delivery vector containing the desired nucleic acid sequence, liposome-mediated transfer, direct injection (naked DNA), receptor-mediated transfer ( DNA-ligand complex) or microparticle bombardment (gene gun).

Exemplary gene and cell therapy techniques are described in U.S. Pat. No. 4,892,538; U.S. Patent No. 5,011,472; U.S. Patent No. 5,106,627; DE 4219626, WO 94/20517 and 96/22793, the descriptions of which are incorporated herein by reference.

Regardless of the manner of administration, treatment of IL-1 mediated disease requires a dose or a total dose regimen of an IL-1 inhibitor (e.g., preferably an IL-lra protein product (s) and more preferably IL-lra) effective in reducing or improving the symptoms of the disease. The factors that determine the appropriate dose or total dose regimen may include the disease or condition to be treated or prevented, the severity of the disease, the manner of administration and the age, sex and medical condition of the patient.

Further refinement of the calculations necessary to determine the appropriate dose for the treatment is routinely done by those skilled in the art, especially in light of the dose information and the assays included herein. The dosing frequency also depends on the pharmacokinetic parameters of the IL-1 inhibitor (eg, preferably an IL-lra protein product (s) and more preferably IL-lra) in the formulation used. The IL-1 inhibitor (e.g., preferably an IL-lra protein product (s) and more preferably IL-lra) may be administered once or in cases of severe and prolonged disease, administered daily at less frequent doses or administered with an initial dose of bolus followed by a continuous dose or sustained supply. It is also contemplated that other forms of continuous or almost continuous dosing can be practiced. For example, chemical derivatization can result in prolonged release forms that have the effect of a continuous presence in the bloodstream, in predictable amounts based on a given dosage or total dose regimen. The dosage or total dose regimen can also be determined through the use of known assays to determine the doses used in conjunction with the appropriate data of the dose response.

When administered parenterally, each unit dose for example, can be up to 200 mg, generally up to 150 mg and more generally up to 100 mg. When administered in an articular cavity, the pharmaceutical composition is preferably administered as a single injection of for example, a 3 to 10 ml syringe containing a dose for example of more than 150 mg / ml and more generally more than 100. mg / ml of IL-1 product dissolved in an isotonic phosphate buffered saline solution. The preparation can be administered into an articular cavity at a frequency of, for example, once every 7 to 10 days. In such a way that the administration is carried out continuously for example, 4 to 5 times while the dose is varied if necessary.

As contemplated by the present invention, an IL-1 inhibitor product (eg, preferably an IL-lra protein product (s) and more preferably IL-lra) can be administered as an adjunct to another therapy and also with other pharmaceutical formulations appropriate for the indication in question. An IL-1 inhibitor product (e.g., preferably an IL-lra protein product (s) and more preferably IL-lra) and one or more additional therapies or pharmaceutical formulations may be administered separately or in combination.

In a specific embodiment, the present invention is directed to the use of an IL-1 inhibitor (e.g., preferably an IL-lra protein product (s) and more preferably IL-lra) in combination (pre-treatment, post-treatment or concurrent treatment) with one or more additional TNF inhibitors for the treatment of IL-1 mediated diseases, including acute and chronic inflammation, cachexia / anorexia; Chronic Fatigue Syndrome; depression; diabetes (for example, diabetes mellitus and juvenile Type 1 attack); fever; fibromyalgia or analgesia; glomerulonepritis; rejection of grafts against host; hemorrhagic shock; hyperalgesia; inflammatory bowel disease, ischemic injury, including cerebral ischemia (for example, brain damage as a result of trauma, epilepsy, hemorrhage or infarction, each of which leads to neurodegeneration); lung disorders (eg, ARDS adult respiratory distress syndrome and pulmonary fibrosis); multiple myeloma; multiple sclerosis; myelogenous (for example, AML and CML) and other leukemias; myopathies (eg, muscle protein metabolism, esp., in sepsis); eye disorders; osteoporosis; Parkinson's disease; pain; pancreatitis; pulmonary fibrosis; labor; psoriasis; reperfusion damage; rheumatic disorders (eg, rheumatoid arthritis, osteoarthritis, juvenile arthritis (rheumatoid), seronegative polyarthritis, ankylosing spondylitis, Reiter's syndrome and reactive arthritis, psoriatic arthritis, enteropathic arthritis, polymyositis, dermatomyositis, scleroderma, systemic sclerosis, vasculitis, vasculitis cerebral, Lyme disease, staphylococcal-induced arthritis ("septic"), Sjögren's syndrome, rheumatic fever, polychondritis, and polymyalgia rheumatica and giant cell arteritis); septic shock; side effects of radiation therapy; disease of the temporal mandibular union, tumor metastasis or an inflammatory condition resulting from damage to the cartilage, jerks or twists, trauma, orthopedic surgery, infection or other disease processes. TNF inhibitors include compounds and proteins that block in vivo synthesis or extracellular release of TNF, in a specific embodiment, the present invention is directed to the use of an IL-1 inhibitor (e.g., preferably a product (s) of IL-lra protein and more preferably IL-lra) in combination (pretreatment, post-treatment or current treatment) with one or more of the following TNF inhibitors: TNF binding proteins (type I soluble TNF receptor and type II soluble TNF receptor ( "sTNFRs"), as defined herein), anti-TNF antibodies, granulocyte colonial stimulation factor; thalidomide; BN 50730; tenidap; E 5531; tiapafant PCA 4248; nimesulide; panavir; rolipram; RP 73401; T peptide; MDL 201,449A; hydrochloride (IR, 3S) -cis-1 - [9- (2,6-diaminopurinyl)] - 3-hydroxy-4-cyclopentene; (IR, 3R) -trans-1- [9- (2,6-diamino) purine] -3-acetoxycyclopentane; hydrochloride (IR, 3R) -trans-1- [9-adenyl) -3-azidocyclopentane and (IR, 3R) -trans-1- [6-hydroxy-purin-9-yl) -3-azidocyclopentane.

TNF binding proteins are described in the art (EP 308 378, EP 422 339, GB 2 218 101, EP 393 438, WO 90/13575, EP 398 327, EP 412 486, WO 91/03553, EP 418 014, JP 127,800 / 1991, EP 433 900, US Patent No. 5,136,021, GB 2 246 569, EP 464 533, WO 92/01002, WO 92/13095, WO 92/16221, EP 512 528, EP 526 905, WO 93 / 07863, EP 568 928, WO 93/21946, WO 93/19777, EP 417 563, International Patent Application No. PCT / US97 / 12244, the descriptions of which are incorporated by reference).

For example, EP 393 438 and EP 422 339 teaches the amino acid and nucleic acid sequence of an sTNFR-I (referred to in the application as "30 kDa TNF inhibitor") and an sTNFR-II (referred to in the application as "inhibitor"). 40kDa ") or as modified forms thereof (eg, fragments, functional derivatives and variants). EP 393 438 and EP 422 339 also describe methods for isolating the genes responsible for coding the inhibitors, cloning the gene into appropriate vectors and cell types and expressing the gene to produce the inhibitors. Additionally, polyvalent forms (ie, molecules comprising more than one active portion) of sTNFR-I and sTNFR-II are also described. In one embodiment, the polyvalent form can be constructed, for example, by chemical coupling of at least one TNF inhibitor and another portion with any clinically acceptable linkage, for example polyethylene glycol (WO 92/16221 and WO 95/34326), by a link peptide (Nevé et al. (1996), Ci t okin e, 8 (5): 365-370, the description of which are incorporated by reference), by chemical binding for biotin and then binding to avidin (WO 91/03553 , the description of which is incorporated by reference) and, finally, by construction of chimeric antibody molecules (US Patent 5,116,964, WO 89/09622, WO 91/16437 and EP 315062, the descriptions of which are incorporated by reference) .

Anti-TNF antibodies include MAK 195F Fab antibody (Holler et al. (1993), First International Symposium of Cytokines in Bone Marrow Transplantation, 147); anti-TNF monoclonal antibody CDP 571 (Rankin et al (1995), British Journal of Rheumatology, 34: 334-342); monoclonal antibody to murine antitumor necrosis factor BAY X 1351 (Kieft et al. (1995) 1 ° European Congress of Clinical Microbiology and Infectious Diseases, 9); monoclonal anti-TNF antibody CenTNF cA2 (Elliott et al. (1994), Lancet, 344: 1105-1110).

In a specific embodiment, the present invention is directed to the use of an IL-1 inhibitor (e.g., preferably an IL-lra protein product (s) and more preferably IL-lra) in combination (pre-treatment, post-treatment or concurrent treatment) with human or secreted soluble fas antigen or recombinant versions thereof (WO 96/20206 and Mountz et al., J. Imm un olgy, 155: 4829-4837; and EP 510 691, the descriptions of which are they are incorporated here by reference). WO 96/20206 discloses secreted human fas antigen (original and recombinant, including an Ig fusion protein), methods for ailing the genes responsible for encoding the soluble recombinant human fas antigen, methods for cloning the gene into appropriate vectors and cell types, and methods to express the gene to produce the inhibitors. EP 510 691 teaches coding of DNAs for the human fas antigen, including soluble fas antigen, vectors expressing the DNAs and transformants transfected by the vector. When administered parenterally, the doses of a soluble or secreted Fas antigen fusion protein are each generally from about 1 microgram / kg to about 100 microgram / kg.

The present treatment of disorders mediated by IL-1, include acute and chronic inflammation such as rheumatic disorders which include the use of a first line of drugs for the control of pain and inflammation classified as non-steroidal, anti-inflammatory drugs ( NSAIDs). Secondary treatments include corticosteroids, slow-acting anti-rheumatic medications (SAARDs) or disorder-modifying medications (DM). The information appreciated in the following compounds can be found in the Merck Diagnostic and Therapy Manual, 16th edition, Merck, Sharp & Dohme Research Laboratories, Merck & Co. Rahway, NJ (1992) and Pharmaproj ects, PJB Publications Ltd.

In a specific embodiment, the present invention is directed to the use of an IL-1 inhibitor (e.g., preferably an IL-lra protein product (s) and more preferably IL-lra) and any of one or more NSAIDs for the treatment of disorders measured by IL-1, including acute and chronic inflammation such as rheumatic disorders and graft-versus-host disorders. The NSAIDs possess their anti-inflammatory action at least in part, for the inhibition of prostaglandin synthesis (Goodman and Gilman in "The Pharmacological Bases of Therapeutics" MacMillan Edition 7a. (1985).) The NSAIDs can be characterized into nine groups: (1) salicylic acid derivatives, (2) propionic acid derivatives, (3) acetic acid derivatives, (4) phenamic acid derivatives, (5) carboxylic acid derivatives, (6) butyric acid derivatives; ) oxicams, (8) pyrazoles and (9) pyrazolones.

In a more specific embodiment, the present invention is directed to the use of an IL-1 inhibitor (for example, preferably an IL-lra protein product (s) and more preferably IL-lra) in combination (pretreatment, post-treatment or concurrent treatment) with any of one or more salicylic acid derivatives, prodrug esters or pharmaceutically salts acceptable from them. Such salicylic acid derivatives, prodrug esters and pharmaceutically acceptable salts thereof include: acetaminosalol, alloxiprine, aspirin, benorilate, bromosaligenin, calcium acetylsalicylate, magnesium trisalicylate diflusinal choline, ethersalate, fendosal, gentisic acid, glycol salicylate, salicylate of imidazole, lysine acetylsalicylate, mesalamine, morpholine salicylate, 1-naphthyl salicylate, olsalazine, parsalmide, phenyl acetylsalicylate, phenyl salicylate, salacetamide, O-acetic acid salicylamide, salsalate and sulfasalazine. Derivatives are structurally related salicylic acid that have similar analgesic and anti-inflammatory properties are also intended to be grouped in this group.

In a more specific embodiment, the present invention is directed to the use of an IL-1 inhibitor (e.g., preferably an IL-lra protein product (s) and more preferably IL-lra) in combination (pretreatment, post-treatment or concurrent treatment) with any of one or more propionic acid derivatives, prodrug esters or pharmaceutically acceptable salts thereof. The propionic acid derivatives, prodrug esters and pharmaceutically acceptable salts thereof include: alminoprofen, benoxaprofen, bucilloxic acid, carprofen, dexindoprofen, fenoprofen, flunoxaprofen, fluprofen, flurbiprofen, furcloprofen, ibuprofen, ibuprofen aluminum, ibuproxam, indoprofen, isoprofen , ketoprofen, loxoprofen, miroprofen, naproxen, oxaprozin, pice toprofen, pimeprofen, pirprofen, pranoprofen, protizinic acid, pyridoxiprofen, suprofen, thiaprofenic acid and thioxaprofen. The structurally related derivatives of propionic acid that have similar analgesic and anti-inflammatory properties are also intended to be covered by this group.

In a more specific embodiment, the present invention is directed to the use of an IL-1 inhibitor (e.g., preferably an IL-lra protein product (s) and more preferably IL-lra) in combination (pre-treatment, post-treatment or treatment). concurrent) with any of one or more acetic acid derivatives, prodrug esters or pharmaceutically acceptable salts thereof. The acetic acid derivatives, prodrug esters and pharmaceutically acceptable salts thereof include: acetaminophen, alclofenac, amfenac, bufexamac, cinmetacin, clopyra, delmatacin, sodium diclofenac, etodolac, felbinaco, fenclofenac, phencloraco, fenclozic acid, fentiazaco, furofenac , glucametacin, ibufenac, indomethacin, isophenolac, isoxepac, lonazolac, metyazinic acid, oxametacin, oxpinac, pimetacin, proglumetacin, sulindac, talmetacin, thiaramide, tiopinac, tolmetin, zidometacin and zomepirac. Structurally related derivatives of acetic acid that have similar analgesic and anti-inflammatory properties are also intended to be covered by this group.

In a more specific embodiment, the present invention is directed to the use of an IL-1 inhibitor (e.g., preferably an IL-lra protein product (s) and more preferably IL-lra) in combination (pre-treatment, post-treatment or treatment). concurrent) with any of one or more phenamic acid derivatives, prodrug esters or pharmaceutically acceptable salts thereof. The phenamic acid derivatives, prodrug esters and pharmaceutically acceptable salts thereof comprise: enfenamic acid, etofenamate, flufenamic acid, isonixin, meclofenamic acid, sodium meclofenamate, medofenamic acid, mefanamic acid, niflumic acid, talniflumate, terofenamate, tolfenamic acid and ufenamate. The structurally related derivatives of fenamic acid that have similar analgesic and anti-inflammatory properties are also intended to be covered by this group.

In a more specific embodiment, the present invention is directed to the use of an IL-1 inhibitor (e.g., preferably an IL-lra protein product (s) and more preferably IL-lra) in combination (pretreatment, post-treatment or concurrent treatment) with any of one or more carboxylic acid derivatives, prodrug esters or pharmaceutically acceptable salts thereof. The carboxylic acid derivatives, prodrug esters and pharmaceutically acceptable salts thereof which may be used comprise: clidanaco, diflunisal, flufenisal, inoridine, ketorolac and tinoridine.

, Structurally related derivatives of the carboxylic acid that have similar analgesic and anti-inflammatory properties are also intended to be covered by this group.

In a more specific embodiment, the present invention is directed to the use of an IL-1 inhibitor (for example, preferably an IL-lra protein product (s) and more preferably IL-lra) in combination (pretreatment, post-treatment or concurrent treatment) with any of one or more butyric acid derivatives, prodrug esters or salts pharmaceutically acceptable thereof. The butyric acid derivatives, prodrug esters and pharmaceutically acceptable salts thereof include: bumadizon, butibufen, fenbufen and xenbucin. Structurally related derivatives of butyric acid that have similar analgesic and anti-inflammatory properties are also intended to be covered by this group.

In a more specific embodiment, the present invention is directed to the use of an IL-1 inhibitor (e.g., preferably an IL-lra protein product (s) and more preferably IL-lra) in combination (pretreatment, post-treatment or concurrent treatment) with any of one or more oxicams, esters of prodrugs or pharmaceutically acceptable salts thereof. The oxicams, prodrug esters and pharmaceutically acceptable salts thereof include: droxicam, enolicam, isoxicam, piroxicam, sudoxicam, tenoxicam and 4-hydroxyl-1,2-benzothiazine 1,1-dioxide 4- (N-phenyl) -carboxamide . The structurally related derivatives of the oxicamos that have similar analgesic and anti-inflammatory properties are also intended to be covered by this group.

In a more specific embodiment, the present invention is directed to the use of an IL-1 inhibitor (e.g., preferably an IL-lra protein product (s) and more preferably IL-lra) in combination (pretreatment, post-treatment or concurrent treatment) with any of one or more pyrazoles, esters of prodrugs or pharmaceutically acceptable salts thereof. The pyrazoles, prodrug esters and pharmaceutically acceptable salts thereof comprise: diphenamizole and epirizol. The structurally related derivatives of pyrazoles that have similar analgesic and anti-inflammatory properties are also intended to be covered by this group.

In a more specific embodiment, the present invention is directed to the use of an IL-1 inhibitor (for example, preferably an IL-lra protein product (s) and more preferably IL-lra) in combination (pretreatment, post-treatment or concurrent treatment) with any of one or more pyrazolones, prodrug esters or pharmaceutically acceptable salts of the same. The pyrazolones, prodrug esters and pharmaceutically acceptable salts thereof include: apazone, azapropazone, bencipiperilone, feprazone, mofebutazone, morazone, oxifenbutazone, phenylbutazone, pipebuzone, propylphenazone, ramifenazone, suxibuzone and thiazolinobutazone. The structurally related derivatives of pyrazalones that have similar analgesic and anti-inflammatory properties are also intended to be covered by this group.

In a more specific embodiment, the present invention is directed to the use of an IL-1 inhibitor (e.g., preferably an IL-lra protein product (s) and more preferably IL-lra) in combination (pretreatment, post-treatment or concurrent treatment) with any one or more of the following NSAIDs: e-acetamidocaproic acid, S-adesilmethionine, 3-amino-4-hydroxybutyral acid, amixetrine, anitrazafen, anthrafenin, bendazaco, bendazaco lisinate, benzidamine, beprozin, broperamol, bucoloma, bufezolac, ciprocuazone, cloximate, dazidamine, deboxamet, detomidine, diphenpyramide, diphenpyramide, difisalamine, ditazole, emorfazone, fanetizol mesylate, fenflumizol, floctafenin, flumizol, flunixin, fluprocuazone, fopirtoline, phosfosal, guaimesal, guaiazole, isonixirin, lefetamine hydrochloride, leflunomide, lofemizol, lotifazol, lysine clonixinate, meseclazone, nabumetone, nictindola, nimese, orgotein, orpanoxin, oxaceprolm, oxapadol, paraniline, perisoxal, perisoxal citrate, pifoxime, piproxene, pyrazolac, pirfenidone, procuazone, proxazole, tielavin B, tiflamizol, timegadine, tolectin, tolpadol, triptamide and those designated by the company code number such as 480156S, AA861, AD1590, AFP802, AFP860, AI77B, AP504, AU8001, BPPC, BW540C, CHINOIN 127, CN100, EB382, EL508, F1044, FK-506, GV3658, ITF182, KCNTEI6090, KME4, LA2851, MR714, MR897, MY309, ON03144, PR823, PV102, PV108, R830, RS2131 , SCR152, SH440, SIR133, SPAS510, SQ27239,. ST281, SY6001, TA60, TAI-901 (4-benzoyl-l-indanecarboxylic acid), TVX2706, U60257, UR2301 and WY41770. The structurally related derivatives of NSAIDs that have similar analgesic, anti-inflammatory, 4, * properties are also intended to be covered by this group.

In a more specific embodiment, the present invention is directed to the use of an IL-1 inhibitor (e.g., preferably an IL-lra protein product (s) and more preferably IL-lra) in combination (pretreatment, post-treatment or concurrent treatment) with any of one or more corticosteroids, prodrug esters or pharmaceutically acceptable salts thereof for the treatment of TNF-mediated diseases, including acute and chronic inflammation such as rheumatic disorders, graft-versus-host disease and multiple sclerosis. Corticosteroids, prodrug esters and pharmaceutically acceptable salts thereof comprise hydrocortisone and hydrocortisone derivatives such as 21-acetoxipregnenolone, alclomerasone, algestone, amcinonide, beclomethasone, betamethasone, betamethasone valerate, budesonide, chloroprednisone, clobetasol, propionate. of clobetasol, clobetasone, clobetasone butyrate, clocortolone, cloprednol, corticosterone, cortisone, cortivazole, deflazacon, desonide, deoximerasone, dexamethasone, diflorasone, diflucortolone, difluprednate, enoxolone, fluazacort, flucloronide, flumetasone, flumethasone pivalate, flunisolide, flucinolone acetonide , fluocinonide, fluoroquinolone acetonide, butyl fluocorrin, fluocortolone, fluorocortolone hexanoate, diflucortolone valerate, fluorometholone, fluperolone acetate, fluprednidene acetate, fluprednione, flurandenolide, formocorthal, halcinonide, halometasone, halopredone acetate, hydrocortamate, hydrocortisone, hydrocortisone acetate, hydrocortisone butyrate, hydrocortisone phosphate, hydrocortisone sodium-succinate, hydrocortisone tebutate, mazipredone, medrisone, meprednisone, methylprednicolone, mometasone furoate, parametasone, prednicarbate, prednisolone, prednisolone-21-diureriaminoacetate , prednisolone sodium phosphate, prednisolone sodium succinate, prednisolone sodium 21-m-sulfobenzoate, 21-prednisolone sodium thearoglycolate, prednisolone tebutate, prednisolone-21-trimethylacetate, prednisone, prednival, prednilidene, 21- prednilidene diethylaminoacetate, tixocortol, triamcinolone, triamcinolone acetonide, triamcinolone benetonide and triamcinolone hexacetonide. The structurally related derivatives of corticosteroids that have similar analgesic and anti-inflammatory properties are also intended to be covered by this group.

In a more specific embodiment, the present invention is directed to the use of an IL-1 inhibitor (e.g., preferably an IL-lra protein product (s) and more preferably IL-lra) in combination (pretreatment, post-treatment or concurrent treatment) with any of one or more of low action antirheumatic drugs (SAARDs) or disease modifying antirheumatic drugs (DMARDS), prodrug esters or pharmaceutically acceptable salts thereof, for the treatment of TNF mediated disorders, including acute and chronic inflammation such as rheumatic disorders, multiple sclerosis and graft versus host. The SAARDs or DMARDS, prodrug esters and pharmaceutically acceptable salts thereof include: sodium alocupreido, auranofino, aurothioglucose, aurothioglycanide, azathioprine, brequinar sodium, bucillamine, calcium 3-aurothio-2-propanol-l-sulfonate, chloranbucil, chloroquine, clobuzarit, cuproxoline, cyclophosphamide, cyclosporine, dapsone, 15-deoxyspergualin, diacerein, glucosamine, gold salts (eg, cyclokine gold salt, gold sodium thiomalate, gold sodium thiosulfate), hydroxychloroquine, hydrofroxyurea, cebuzone, levamisole, lobenzarit, melitin, 6-mercaptopurine, methotrexate, mizoribine, mycophenolate mofetil, mioral, nitrogen mustard, D-penicillamine, pyridinol imidazoles such as SKNF86002 and SB203580, rapamycin, thiols, thimopoietin and vincristine. SAARDs or structurally related DMARDs that have similar analgesic and anti-inflammatory properties are also intended to be covered by this group.

In a more specific embodiment, the present invention is directed to the use of an IL-1 inhibitor (for example, preferably an IL-lra protein prod(s) and more preferably IL-lra) in combination (pretreatment, post-treatment or concurrent treatment) with any one or more C0X2 inhibitors, prodrug esters or pharmaceutically acceptable salts. of them for the treatment of TNF-mediated disorders, including acute and chronic inflammation. Examples of the C0X2 inhibitors, prodrug esters or pharmaceutically acceptable salts thereof include, for example, celecoxib. The strrally related C0X2 inhibitors that have analgesic and anti-inflammatory properties are also intended to be covered in this group.

In a more specific embodiment, the present invention is directed to the use of an IL-1 inhibitor (e.g., preferably an IL-lra protein prod(s) and more preferably IL-lra) in combination (pretreatment, post-treatment or concurrent treatment) with any of one or more antimicrobials, prodrug esters or pharmaceutically acceptable salts thereof for the treatment of TNF-mediated disorders, including acute and chronic inflammation. Examples of the antimicrobials include, for example, ampicillin, amoxicillin, aureomycin, bacitracin, ceftazimide, ceftriaxone, cefotaxime, cefaclor, cephalexin, cephradine, ciprofloxacin, clavulanic acid, cloxacillin, dicloxacillin, erythromycin, flucloxacilane, gentamicin, gramicidin, methicilane, neomycin, oxacilane , penicillin and vancomycin. Strrally related antimicrobials that have analgesic and anti-inflammatory properties are also intended to be covered in this group.

It is especially advantageous to formulate compositions of the additional anti-inflammatory compounds in unit dose form for ease of administration and uniformity of dosage. The "unit dose form" as used herein, refers to physically discrete units adjusted as unit doses for the patients to be treated, each unit containing a predetermined amount of additional anti-inflammatory compounds calculated to produce the desired therapeutic effect in association with the pharmaceutical protractor required. As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and fungicidal agents, isotonic and absorption delaying agents and the like which are compatible with the active ingredient and mode of administration. administration and other ingredients of the formulation and not harmful to the container.

For therapeutic oral administration, the additional anti-inflammatory compound can be incorporated with excipients and used in the form of ingestible tablets, buccal tablets, lozenges, capsules, elixirs, suspensions, syrups, wafers and the like, or can be directly incorporated with the food in the diet . The tablets, pills, pills, capsules and the like may also contain the following: a binder such as gum tragacanth, acacia, corn starch or gelatin; excipients such as dicalcium phosphate; a disintegrating agent such as a corn starch, alginic acid and the like, a lubricant such as magnesium stearate; a sweetening agent such as sucrose, lactose or saccharin; or a flavoring agent such as peppermint, oil of wintergreen or cherry or orange flavorings. When the unit dose form is a capsule, it may contain in addition to the material of the type described herein, a liquid carrier. Various other materials may be present as a coating or otherwise modify the physical form of the unit dose. For example, tablets, pills or capsules can be replenished with shellac, sugar or both. Of course, any material used in the preparation of any unit dosage form must be pharmaceutically pure and substantially non-toxic in the amounts employed. In addition, the additional anti-inflammatory compound can be incorporated into a sustained release preparation and formulation. The amount of additional anti-inflammatory compound in such a therapeutically useful composition is such that an appropriate dose will be obtained.

For parenteral therapeutic administration, each additional anti-inflammatory compound can be incorporated with a sterile injectable solution. The sterile injectable solution can be prepared by incorporating the additional anti-inflammatory compound in the required amount in a pharmaceutically acceptable carrier, with various other ingredients, followed by filtered sterilization. In the case of dispersions, each can be prepared by incorporating the additional anti-inflammatory compound in a sterile vehicle containing the basic dispersion medium and the other ingredients required from those listed herein. In the case of sterile injectable solutions, each can be prepared by incorporating a powder of the additional anti-inflammatory compound and optionally, any additional desired ingredients from a previously filtered sterile solution, wherein the powder is prepared by any appropriate technique (for example, vacuum drying and vacuum freezing The use of such media and agents is well known in the art (see, for example, Rem in gt on 's Pha rma ce uti ca l Sci en ces, Ed. 18. (1990), Mack Publishing Co. Easton, PA 18042 , pages 1435-1712, the description of which is incorporated herein by reference). The complementary active ingredients can also be incorporated into the compositions.

The specific dose of the additional anti-inflammatory compound is calculated in accordance with the approximate body weight or surface area of the patient. Other factors that determine the appropriate dose may include acute and chronic inflammatory disease or condition to be treated or avoided, the severity of the disease, the route of administration and the age, sex and medical condition of the patient. Further refinement of the calculations necessary to determine the appropriate dose for the treatment involving each of the formulations mentioned herein is routinely done by those skilled in the art. Doses can also be determined through the use of known assays to determine the doses used in conjunction with the appropriate dose response data.

Thus, for example, it is within the scope of the invention that the doses of additional anti-inflammatory compounds selected for the treatment of a particular chronic or acute inflammatory disease such as rheumatic disorders may vary to achieve the desired therapeutic effect. Where one of the additional anti-inflammatory compounds has side effects, it can be given to patients during alternate periods of combination therapy treatment. For example, chronic treatment with methotrexate is associated with gastrointestinal, hepatic, bone marrow and pulmonary toxicity (Sandoval et al. (1995), Bri ti sh Jo urna l of Rh e uma t ol ogy, 34: 49-56 , the description of which is incorporated herein by reference).

Tests to monitor the improvement of a disease may include specific tests directed, for example, to the determination of the systemic response to inflammation, which includes the rapidity of erythrocyte sedimentation (ESR) and acute phase reagents (APR). Observations are made for the swelling, etc., of the affected parts of the body. Improvement in stiffness and control (where applicable), and reduction in patient pain is also observed. If the patient's condition is stable, the patient is treated again with the same dose weekly and evaluated weekly. As long as the patient's condition is stable, treatment can be continued. After six months of treatment, the anatomical changes of the skeleton are determined by radiological images, for example by X-ray radiography.

At the end of each period, the patient is evaluated again. The comparison of the evaluation of the pre-treatment and the radiological post-treatment, ESR and APR indicates the effectiveness of the treatments. In accordance with the effectiveness of the treatments and the patient's condition, the dose can be increased or kept constant for the duration of the treatment.

Preferably the present invention is directed to a method with optionally, one of the following combinations for treating or preventing diseases mediated by IL-1, including acute and chronic inflammation such as rheumatic disorders and the symptoms associated therewith. A combination is an IL-1 inhibitor (e.g., preferably an IL-lra protein product (s) and more preferably IL-lra Fe, optionally formulated with a controlled release 4A polymer (e.g., a dextran or hyaluronan) , the formulation of citrate buffer solution or the phosphate buffer solution formulation, with one or more of methotrexate, leflunomide, an immunosuppressant (eg, cyclosporin), ciprofloxacin, soluble or secreted fas antigen and a TNF inhibitor (eg, sTNFRa.) Preferred combinations include the IL-lra product (s) and methotrexate or the IL-lra product (s) and leflunomide Another combination is an IL-lra protein product (s) with one or more of methotrexate, leflunomide, sulfasazine and hydroxychlorine.

In a preferred specific embodiment, the method comprises administration (eg, intra-articular, subcutaneous or intramuscular) of an IL-lra inhibitor (e.g., preferably an IL-lra protein product (s) and more preferably IL-lra or IL -lra Fe, optionally formulated with a controlled release polymer (eg, a dextran or hyaluronan), the formulation of citrate buffer solution or the phosphate buffer solution) in combination (pretreatment, post-treatment or concurrent treatment) ) with sTNFRa for the treatment of disorders mediated by IL-1, including including acute and chronic inflammation, cachexia / anorexia; Chronic Fatigue Syndrome; depression; diabetes (for example, diabetes mellitus and juvenile Type 1 attack); fever; fibromyalgia or analgesia; glomerulonepritis; rejection of grafts against host; hemorrhagic shock; hyperalgesia; inflammatory bowel disease, ischemic injury, including cerebral ischemia (for example, brain damage as a result of trauma, epilepsy, hemorrhage or infarction, each of which leads to neurodegeneration); lung disorders (eg, ARDS adult respiratory distress syndrome and pulmonary fibrosis); multiple myeloma; multiple sclerosis; myelogenous (for example, AML and CML) and other leukemias; myopathies (eg, muscle protein metabolism, esp., in sepsis); eye disorders; osteoporosis; Parkinson's disease; pain; pancreatitis; pulmonary fibrosis; labor; psoriasis; reperfusion damage; rheumatic disorders (eg, rheumatoid arthritis, osteoarthritis, juvenile arthritis (rheumatoid), seronegative polyarthritis, ankylosing spondylitis, Reiter's syndrome and reactive arthritis, psoriatic arthritis, enteropathic arthritis, polymyositis, dermatomyositis, scleroderma, systemic sclerosis, vasculitis, cerebral vasculitis Lyme disease, staphylococcal ("septic") arthritis, Sjögren's syndrome, rheumatic fever, polychondritis, and polymyalgia rheumatica and giant cell arteritis); septic shock; side effects of radiation therapy; disease of the temporal mandibular union, tumor metastasis or an inflammatory condition resulting from damage to the cartilage, jerks or twists, trauma, orthopedic surgery, infection or other disease processes.

In a specific preferred embodiment, the method comprises administering (e.g., intraventricular, subcutaneous or intramuscular) that of an IL-lra inhibitor (e.g., preferably an IL-lra protein product (s) and more preferably IL-lra or IL-lra Fe, optionally formulated with a controlled release polymer (eg, a dextran or hyaluronan) citrate buffer solution or phosphate buffer solution) in combination (pretreatment, post-treatment or concurrent treatment) with methotrexate and / or leflunomide and / or sTNFRs to treat arthritis (e.g., osteoarthritis, psoriatic arthritis and / or rheumatoid arthritis), and the symptoms associated therewith.

In a preferred specific embodiment, the method comprises administering (e.g., subcutaneously or intramuscularly) an IL-lra inhibitor (e.g., preferably an IL-lra protein product (s) and more preferably IL-lra or IL-lra. Fe, optionally formulated with a controlled release polymer (eg, a dextran or hyaluronan) the citrate buffer solution formulation or the phosphate buffer solution) in combination (pretreatment, post-treatment or concurrent treatment) with an activator tissue plasminogen and / or sTNFRs to treat brain injuries as a result of trauma, epilepsy, hemorrhage or infarction, each of which cause neurodegeneration.

In a preferred specific embodiment, the method comprises administration (eg, intravenous) of an IL-lra inhibitor (e.g., preferably an IL-lra protein product (s) and more preferably IL-lra or IL-lra Fe, optionally formulated with a controlled release polymer (eg, a dextran or hyaluronan) the formulation of citrate buffer solution or the phosphate buffer solution) in combination (pretreatment, post-treatment or concurrent treatment) with one or more of methotrexate, leflunomide, a corticosteroid, FK506, ciclosporin, a soluble fas protein and / or sTNFRs to treat graft-versus-host rejection.

In a specific preferred embodiment, the method comprises the administration (e.g., subcutaneous or intramuscular) of an IL-lra inhibitor. (e.g., preferably an IL-lra protein product (s) and more preferably IL-lra or IL-lra Fe, optionally formulated with a controlled release polymer (e.g., a dextran or hyaluronan) buffer formulation. citrate or phosphate buffer solution) in combination (pretreatment, post-treatment or concurrent treatment) with G-CSF and / or sTNFRs to treat inflammatory bowel disease.

In a specific preferred embodiment, the method comprises the administration (e.g., subcutaneous or intramuscular) of an IL-lra inhibitor. (e.g., preferably an IL-lra protein product (s) and more preferably IL-lra or IL-lra Fe, optionally formulated with a controlled release polymer (e.g., a dextran or hyaluronan) buffer formulation. citrate or phosphate buffer solution) in combination (pretreatment, post-treatment or concurrent treatment) with interferon (for example, alpha interferon, beta interferon, gamma interferon and consensus interferon), to treat multiple or myelogenous myeloma (e.g. , AML and CML) and other leukemias.

In a preferred specific embodiment, the method comprises administration (e.g., subcutaneous, intraventricular or intrathecal) of an IL-lra inhibitor (e.g., preferably an IL-lra protein product (s) and more preferably IL-lra or IL -lra Fe, optionally formulated with a controlled release polymer (for example, a dextran or hyaluronan) the formulation of citrate buffer solution or the phosphate buffer solution) in combination (pretreatment, posttreatment or concurrent treatment) with NSAIDs (eg, indomethacin) and / or sTNFRs to treat the Alzheimer disease.

In a preferred specific embodiment, the method comprises administration (eg, local, subcutaneous or intramuscular injection) of an IL-lra inhibitor (e.g., preferably an IL-lra protein product (s) and more preferably IL-lra or IL-lra Fe, optionally formulated with a controlled release polymer (for example, a dextran or hyaluronan) the citrate buffer solution formulation or the phosphate buffer solution) optionally with standard treatments, to treat the disease of the union of the temporal jaw.

In a preferred specific embodiment, the method comprises administration (eg, local, subcutaneous or intramuscular injection) of an IL-lra inhibitor (e.g., preferably an IL-lra protein product (s) and more preferably IL-lra or IL-lra Fe, optionally formulated with a controlled release polymer (eg, a dextran or hyaluronan) citrate buffer solution or phosphate buffer solution) in combination (pretreatment, post-treatment or concurrent treatment) with osteoprotogerin (European Patent Application No. 96309363.8) in the treatment of osteoporosis or Paget's disorder.

In a preferred specific embodiment, the method comprises administration (eg, local, subcutaneous or intramuscular injection) of an IL-lra inhibitor (e.g., preferably an IL-lra protein product (s) and more preferably IL-lra or IL-lra Fe, optionally formulated with a controlled release polymer (for example, a dextran or hyaluronan) the formulation of citrate buffer solution or the phosphate buffer solution) in combination (pretreatment, posttreatment or concurrent treatment) in combination with gene therapy (for example, using the human adenovirus) ) to modulate the inflammatory response to vector antigens (Zhang et al (1997), Arthritis &Rheumatism, 40 (9): S220 (1138)).

The surprising and unexpected result described here is the ability of IL-lra and methotrexate to act synergistically in the treatment of various symptoms associated with IL-1 mediated disorders, including acute and chronic inflammation such as rheumatic diseases. "Mg synergistic" is used herein to refer to a situation wherein the benefit provided by the coadministration of an IL-1 inhibitor (eg, preferably an IL-lra protein product (s) and one or more additional anti-inflammatory compounds) , is greater than the algebraic sum of the effects resulting from the separate administration of the components of the combination. As shown in the experiments below, in the adjuvant arthritis model, the combination treatment of rhuIL-lra and methotrexate is synergistic with respect to the systemic inflammation of the treatment (ie, splenomegaly) and weight loss associated with rheumatoid arthritis. Thus, the combined treatment with rhuIL-lra and methotrexate has the advantage of achieving the same result with a lower dose or a less frequent administration of methotrexate, thereby reducing any toxic effect and potentially the advantage of persisting even after that the treatment is over.

Methotrexate is an anti-metabolite and immunosuppressant medication. Methotrexate is an effective anti-inflammatory agent with utility in the treatment of severe and disabling psoriasis and rheumatoid arthritis (Hoffmeister (1983), Th e Ameri can Jo lna of Medi cin e, 30: 69-73 and Jaffe (1988 ), Arthri tis and Rheuma ti sm, 31: 299). Methotrexate is N- [4 - [(2,4-diamino-6-pteridinyl) methylamino] benzoyl] -L-glutamic acid and has the structural formula: CJ ^ CHjCO H H The following references describe the preparation of methotrexate (Seeger et al (1949), J. Am. Chem. Soc., 71: 1753; metabolism of methotrexate (Freeman (1958), J. Pharmacol. Exp. Ther. 122: 154 and Henderson et al. (1965), Cancer Res., 25: 1008), the toxicity of methotrexate Condit et al., (1960), Cancer, 13: 222-249; the faramcokinetic models of methotrexate (Bischoff et al. (1970), J. Pharm, Sci., 59: 149); Metabolism and pharmacokinetics of methotrexate (Evans (1980), Appl. Pharmacokinet., Williams et al. (Eds.), Pp- 518-548 (Appl. Ther. Inc.), the clinical pharmacology of methotrexate (bertin) (1981), Cancer Chemother., 3: 359-375 and Jolivet et al. (1983), N. Eng. J. Med., 309: 1094-1104J; and the clinical experience of methotrexate in rheumatoid arthritis (Weiunblatt et al. (1985), N. Eng. J.

Med., 312: 818-822; Furst (1985), J. Rheumatol., 12 (12): 1-14; Williams et al. (1985), Arthritis Rheum., 28: 721-730 and Seitz et al. (1995), British Journal of Rheumatology, 34: 602-609). Additionally, various patents have been issued describing the active agent of methotrexate and methods for synthesizing methotrexate or potential intermediates in the synthesis of methotrexate: U.S. Nos. 2,512,572, 3,892,801, 3,989,703, 4,057,548, 4,067,867, 4,079,056, 4,080,325, 4,136,101, 4,224,446, 4,306,064, 4,374,987, 4,421,913 and 4,767,859.

The mechanism of action of methotrexate is poorly understood, however, various activities of this drug have been shown to probably contribute to its efficacy (Segal et al (1990), Seminars in Arthritis and Rheumatism, 20: 190-198). The following mechanisms of action of methotrexate have been postulated: inhibition of folate-dependent pathways and protein metabolism (Morgan et al. (1987), Arthritis and Rheumatism, 30: 1348-1356); inhibition of neutrophil migration in arthritic junctions (van der Kerkhof et al. (1985), British Journal of Dermatology, 113: 251-255; Ternowitz et al. (1987), Journal of Inves tigative Dermatology, 89: 192-196 and Sperling (1992), Arthritis and Rheumatism, 35: 376-384); inhibitory activity IL-6 (Segal (1991), Arthritis and Rheumatism, 34 (2): 1 6-152) and the specific local anti-proliferative effect on the cells involved in arthritis (Rodenhuis et al. (1987), Arthritis and Rheumatism, 30: 369-374). Methotrexate has been shown to block the pathway of the interleukin-1 beta / interleukin-1 receptor (Brody et al. (1993), European Journal of Clinical Chemistry and Clinical Biochemistry, 31 (10): 667-674); however, although methotrexate can inhibit the proliferative effects of IL-1 and decrease the production of IL-1 monocytes in the short term in certain patients, this effect is not sustained and is unlikely to explain the long-term efficacy of methotrexate ( Barrera et al. (1996), Seminars in Arthritis and Rheumatism, 25 (4): 234-253).

Methotrexate can be administered orally, intraperitoneally, subcutaneously or intravenously. Oral administration is preferred. The following is an example of the procedure for the combined administration of an IL-lra protein product (s) (e.g., IL-lra) and methotrexate to treat a human patient. The patient takes a tablet or capsule of methotrexate three times a week, at a total weekly dose of 5 to 50 mg / patient / week. The patient is also injected intravenously with IL-lra protein product (s) (eg, IL-lra), at a daily dose of 50 to 150 mg. It will be appreciated by those skilled in the art that the doses presented herein are the preferred doses. The starting dose of the particular compound used is reduced for a patient exhibiting an adverse reaction, or the drug used in combination with the compound (s) can be changed or reduced, for example, depending on the different formulations, routes, programs of doses and / or other variables known to those skilled in the art, such as the patient's individual tolerance to the drug, its efficacy and toxicity.

Preferably, the patient is treated with a weekly starting dose of methotrexate at between 5 mg and 7.5 mg (orally or intramuscularly) and a daily dose of IL-lra protein product (s) (eg, IL-lra) a between 50 mg and 150 mg (intravenously). The dose of methotrexate is increased by 5 mg every 2 to 3 weeks. The maximum dose level is determined at a point at which the patient shows improvement, which is generally preferable less than about 25 mg of methotrexate per week, more preferably between 5 and 25 mg of methotrexate per week. The patient is then evaluated by a physical examination and detailed laboratory tests. The tests include toxicity evaluation. Additional laboratory monitoring in the case of methotrexate includes a complete blood cell count every 2 weeks for the first three months and monthly thereafter. Additional precautions preferably include monthly assessments of the levels of serum albumin, amino alanine transferase, bilirubin, creatinine and nitrogen and blood urea. A monthly analysis of urine is also preferred.

The foregoing is by way of example and does not exclude other treatments to be used concurrently with these anti-inflammatory compounds that are known to those skilled in the art or that can be reached by those skilled in the art using the guidelines set forth in this specification.

Eg emplos Normal methods for many of the methods described in the following examples, or appropriate alternate procedures, are provided in widely recognized molecular biology manuals such as, for example, Sambrook et al., Molecular Cloning, Second Edition, Cold Spring Harbor Laboratory Press ( 1987) and Ausabel et al., Current Protocols in Molecular Biology, Greene Publishing Associates / Wiley Interscience, New York (1990). All the chemicals were analytical grade or USP grade.

Example 1.

An animal model of rheumatoid arthritis induced by an adjuvant was used to investigate the combination therapy of an inhibitor IL-1 and methotrexate. Male Lewis rats (5 per group) weighing at least 200 g were cannulated with jugular catheters and allowed to recover for several days. These were then placed in infusion cages and acclimated for a week before starting the adjuvant injections.

On day zero, all rats were injected with 100 μl of Freunds Complete Adjuvant (Sigma Chemical Co., St Louis, MO) to which was added a synthetic adjuvant N, N-dioctyldecyldecyl-N ', N-bis ( 2-hydroxyethyl) propandiamine, 75 mg / ml. On days 0-14, methotrexate in 1% carboxymethylcellulose (Sigma) was orally administered daily (0.06 mg / kg) to the two groups of rats. On day 8, treatment with the human recombinant IL-1 receptor antagonist derived from E. coli (generally prepared in accordance with the teachings of US Patent No. 5,075,222, rhuIL-lra) formulated in the pharmaceutical composition (10 millimoles of Sodium citrate, 140 millimoles of sodium chloride, 0.5 millimoles of EDTA, 0.1% polysorbate (by weight) in water, pH 6.5), was administered by continuous IV infusion (5 4 .A mg / kg / hr) to a group of rats that were treated with the Complete Freunds Adjuvant and methotrexate and another group of rats was treated with the Freunds Complete Adjuvant only.

Body weights were taken on day 0 every other day from day 8 until completion on day 15. Calibrator measurements and clinical record were taken on day 8 and every other day until completion. At this time, the body weight, legs and spleen of the animal were determined.

As seen in Figures 2 and 3, the rats were treated with rhuIL-lra only, exhibited around 57% inhibition of arthritis (swelling of the legs), without a significant benefit in splenomegaly and 25% inhibition in the change of body weight. The rats treated with methotrexate had 55% inhibition in the swelling of the legs, no inhibition in the weight of the spleen and 23% inhibition in the change of body weight. Combination therapy provided 100% inhibition of foot swelling, 49% inhibition of splenomegaly and 106% inhibition of body weight change.

E j us 2.

Male Lewis rats (Charles River, Portage, MI) (5-7 / group) weighing at least 200 g were cannulated with jugular catheters and allowed to recover for a few days. These were then placed in infusion cages and acclimated for a week before starting the adjuvant injections.

On day zero, all rats were injected with 100 μl of Freunds Complete Adjuvant (Sigma Chemical Co., St Louis, MO) to which was added a synthetic adjuvant N, N-dioctyldecyldecyl-N ', N-bis ( 2-hydroxyethyl) propandiamine, 75 mg / ml. On days 0-14 methotrexate in 1% carboxymethylcellulose (Sigma) treatment was initiated and administered orally (0.06 mg / kg). On day 8, treatment with rhuIL-lra formulated in a pharmaceutical composition (10 millimolar sodium citrate, 140 millimoles of sodium chloride, 0.5 millimoles of EDTA, 0.1% of polysorbate (by weight) in water, pH 6.5), "was administered by continuous IV infusion (5 mg / kg / hr). on day 0 and every other day from day 8 until completion on day 15. Calibrator measurements and clinical record were made from day 8 and every other day until completion, at this time, body weight , legs and spleen of the animal were determined.

As seen in Figures 4 and 5, rats treated with rhuIL-la alone exhibited about 22% inhibition of leg swelling, 24% inhibition of splenomegaly and did not inhibit the change in the body weight. The rats treated with methotrexate had 57% inhibition in swelling of the legs, 22% inhibition in splenomegaly and 59% inhibition in the change of body weight. The combination of rhuIL-lra and methotrexate resulted in 90% inhibition of leg swelling, 77% inhibition of splenomegaly and 65% inhibition of body weight change associated with arthritis / systemic inflammation.

Example 3 Male Lewis rats (Charles River, Portage, MI) (5-7 / group) weighing at least 250-300 g were cannulated in the subcitis of the spine and allowed to recover for several days. These were then placed in infusion cages and acclimated for at least 4 days before initiating the adjuvant injections.

On day zero, all rats were injected with 100 μl of Freunds Complete Adjuvant (Sigma Chemical Co., St. Louis, MO) to which was added a synthetic adjuvant N, N-dioctyldecyldecyl-N ', N-bis ( 2-hydroxyethyl) propandiamine, 75 mg / ml. On days 0-14 methotrexate in 1% carboxymethylcellulose (Sigma) treatment was initiated and administered orally (0.06 or 0.075 mg / kg). On day 8, the treatment with rhuIL-lra formulated in a pharmaceutical composition (10 millimolar of sodium citrate, 140 millimoles of sodium chloride, 0.5 millimoles of EDTA, 0.1% of polysorbate (by weight) in water, pH 6.5 ), was administered by continuous subcutaneous infusion (5 mg / kg / hr, 0.1 ml / hr). Body weights were taken on day 0 and every other day from day 8 until completion on day 15. Calibrator measurements and clinical record were taken from day 8 and every other day until completion. At this time, the body weight, legs and spleen of the animal were determined.

Continuous subcutaneous infusion of rhuIL-lra in the range of 5mg / ml / hr, days 8-15 in adjuvant arthritic rats, resulted in a 6% inhibition of final leg weight. The treatment of adjuvant arthritic rats with daily dose of methotrexate (0.075, 0.060 or 0.048 mg / kg) on days 0-14 resulted in 47, 27 or 0% (respectively) of inhibition in final leg weight . The concurrent treatment with rhuIL-lra and methotrexate at these same doses increased the inhibition of the passage of the final paw to 84, 44 or 21%. Therefore, statistically significant clinical benefits are noted when methotrexate is dosed at 0.075 or 0.06 mg / kg. In the case of the therapy combination, the inhibition of foot swelling was significantly greater than when it occurred with either rhuIL-lra or methotrexate only when the dose of methotrexate was 0.075 mg / kg. The paw swelling analysis was measured sequentially with the ankle joint gauge revealing an inhibition of arthritis when the data was analyzed as an area under the curve. The rats treated with rhuIL-lra only resulted in a 6% inhibition of the swelling while those given with 0.075 mg / kg of methotrexate resulted in a 45% decrease in the diameter of the ankle joint over time. In contrast, those given with the combination therapy resulted in a 78% inhibition of arthritis. The benefits of statistical significant combination in the area under the curve of inhibition of ankle diameter seen in rats gave 0.048 but not 0.06 mg / kg methotrexate.

Histological evaluation of the ankle joints of rats treated with rhuIL-lra showed 8% inhibition of inflammation and 53% inhibition of bone resorption. Treatment with methotrexate resulted in a significant inhibition in responsive doses of these parameters at 0.075 (44% inhibition of inflammation and 58% inhibition of bone resorption) or 0.06 (26% inhibition of inflammation and 55% inhibition). inhibition of bone resorption) but not 0.048 mg / kg (8% inhibition of inflammation and 11% inhibition of bone resorption). The benefits of the combination were more remarkable in rats giving them the 0.075 mg / kg dose of methotrexate in combination with rhuIL-lra. In these rats, inflammation was inhibited by 85% and bone resorption decreased by 97%. These differences are significantly increased over those seen in either the treatment only for both parameters. Similar benefits of the combination were also seen at doses of 0.06 mg / kg methotrexate in combination with rhuIL-lra (44% inhibition of inflammation and 84% inhibition of bone resorption) and at doses of 0.048 mg / kg of methotrexate in combination with rhuIL-lra (2% inhibition of inflammation and 68% inhibition of bone resorption).

Example 4 Male Lewis rats (Charles River, Portage, MI) (5-7 / group) weighing at least 250-300 g were cannulated in the subcitis of the spine and allowed to recover for several days. These were then placed in infusion cages and acclimated for at least 4 days before the adjuvant injections were started.

On day zero, all rats were injected with 100 μl of Freunds Complete Adjuvant (Sigma Chemical Co., St. Louis, MO) to which was added a synthetic adjuvant N, N-dioctyldecyldecyl-N ', N-bis ( 2-hydroxyethyl) propandiamine, 75 mg / ml. On days 0-14 methotrexate in 1% carboxymethylcellulose (Sigma) treatment was initiated and administered orally (0.048, 0.06 or 0.075 mg / kg). On day 8, the treatment with rhuIL-lra formulated in a pharmaceutical composition (10 millimolar of sodium citrate, 140 millimoles of sodium chloride, 0.5 millimoles of EDTA, 0.1% of polysorbate (by weight) in water, pH 6.5 ), was administered by continuous IV subcutaneous infusion (5 mg / kg / hr, 0.1 ml / hr). Body weights were taken on day 0 and every other day from day 8 until completion on day 15. Calibrator measurements and clinical record were taken from day 8 and every other day until completion. At this time, the body weight, legs and spleen of the animal were determined.

Continuous subcutaneous infusion of rhuIL-lra in the range of 5mg / ml / hr, days 8-15 in adjuvant arthritic rats, resulted in a 6% inhibition of final leg weight. The treatment of adjuvant arthritic rats with daily dose of methotrexate (0.075, 0.060 or 0.048 mg / kg) on days 0-14 resulted in 47, 27 or 0% (respectively) of inhibition in final leg weight . The concurrent treatment with rhuIL-lra and methotrexate at these same doses increased the inhibition of the passage of the final paw to 84, 44 or 21%. Therefore, statistically significant clinical benefits are noted when methotrexate is dosed at 0.075 or 0.06 mg / kg. In the case of the therapy combination, the inhibition of foot swelling was significantly greater than when it occurred with either rhuIL-lra or methotrexate only when the dose of methotrexate was 0.075 mg / kg. The paw swelling analysis was measured sequentially with the ankle joint gauge revealing an inhibition of arthritis when the data was analyzed as an area under the curve. The rats treated with rhuIL-lra only resulted in a 6% inhibition of the swelling while those given with 0.075 mg / kg of methotrexate resulted in a 45% decrease in the diameter of the ankle joint over time. In contrast, those given with the combination therapy resulted in a 78% inhibition of arthritis. The benefits of statistical significant combination in the area under the curve of inhibition of ankle diameter seen in rats gave 0.048 but not 0.06 mg / kg methotrexate.

Histological evaluation of the ankle joints of rats treated with rhuIL-lra showed 8% inhibition of inflammation and 53% inhibition of bone resorption. Treatment with methotrexate resulted in a significant inhibition in responsive doses of these parameters at 0.075 (44% inhibition of inflammation and 58% inhibition of bone resorption) or 0.06 (26% inhibition of inflammation and 55% inhibition). inhibition of bone resorption) but not 0.048 mg / kg (8% inhibition of inflammation and 11% inhibition of bone resorption). The benefits of the combination were more remarkable in rats giving them the 0.075 mg / kg dose of methotrexate in combination with rhuIL-lra. In these rats, inflammation was inhibited by 85% and bone resorption decreased by 97%. These differences are significantly increased over those seen in either the treatment only for both parameters. Similar benefits of the combination were also seen at doses of 0.06 mg / kg methotrexate in combination with rhuIL-lra (44% inhibition of inflammation and 84% inhibition of bone resorption) and at doses of 0.048 mg / kg of methotrexate in combination with rhuIL-lra (2% inhibition of inflammation and 68% inhibition of bone resorption).

Example 4 Materials: Interferon consensus (r-metlFN-conl) A synthetic interferon was generated substantially in accordance with the techniques of US Pat. No. 4,695,623.

Methods: Female guinea pigs of strain 13 (175-200 g) were immunized with 0.5 ml of an emulsion containing 12 g of brain and spine doral (guinea pig) in 24 ml of saline phosphate buffer, 24 ml of adjuvant of Freund complete containing 2.5 mg / ml M. Tuberculosis H37Ra. The emulsion was injected intradermally (4-5 places) in the neck region of the guinea pigs. All injections of r-met IFN-conl, rhuIL-Ira vehicle were given subcutaneously The evaluation of the clinical disorder was based on a standard 0-5 record system and was conducted every day for a period of 14 days. The spectrum of the range was 0, normal; 1, weaknesses in the hind limbs; 2, paralysis in 2 hind limbs or paralysis in 1 hind limb; 3, paralysis both hind limbs; 4, dying and 5, dead. The guinea pigs that survived were sacrificed on day 14, and their spine and brain were removed for histological examination. 4 ? The integrated clinical record for each guinea pig during the entire course of the disorder was calculated as the area under the curve of daily clinical records against time (arbitration units). The values of the treated groups were compared statistically against those of the control vehicle group using the Mann-Whitney test.

Results: rhuIL-lra at 100 mg / kg or 10 mg / kg s.c., 3 x a day starting on day 0 attenuated clinical symptoms by 53% and 49% respectively (Figure 6). In the same study, the e-met I FN-conl given each day beginning on the day gave clinical symptoms by 30% (Figure 6). The combination of rhuIL-lra (100 mg / kg sc) + r-metlFN-conl (0.03 mg / kg sc) or rhuIL-lra (10 mg / kg sc) + r-met I FN-conln (0.03 mg / kg sc) attenuated clinical signs by 73% and 84% respectively (Figure 7). In addition, the combination significantly improves the weight gained by these animals compared to the treated vehicle animals (Figure 8).

The foregoing description of the invention is exemplary for purposes of illustration and explanation. It will be apparent to those skilled in the art, that changes and modifications are possible without departing from the spirit and scope of the invention. It is intended that the following claims be construed as covering said changes and modifications.

LIST OF SEQUENCE (1) GENERAL INFORMATION (i) APPLICANT: Amgen Inc. (ii) TITLE OF THE INVENTION: COMBINATION THERAPY USING A PROTEIN FOR THE TREATMENT OF MEDIATED DISORDERS BY TNF. (iii) NUMBER OF SEQUENCES: 4 (iv) ADDRESS OF CORRESPONDENCE: (A) RECIPIENT: Amgen Inc. (B) STREET: 1840 DeHavilland Drive (C) CITY: Thousand Oaks (D) STATE: CA (E) COUNTRY: E.U. (F) POSTAL CODE: 91320-1789 (v) COMPUTER READING FORM (A) TYPE OF MEDIA: Flexible disk (B) COMPUTER: IBM compatible PC (C) OPERATING SYSTEM: PC-DOS / MS- DOS (D) PACKAGE: Patentln Release No. 1, Version o. 1.30 (vi) CURRENT DATA OF THE APPLICATION (A) NUMBER OF APPLICATION: DO NOT KNOW STILL (B) DATE OF SUBMISSION: 8-DEC-1997 (C) CLASSIFICATION: (vii) PREVIOUS APPLICATION DATA (A) APPLICATION NUMBER: US 60 / 032,790 (B) DATE OF SUBMISSION: 6-DEC-1996 (vii) PREVIOUS APPLICATION DATA (A) APPLICATION NUMBER: US 60 / 036,353 (C) DATE OF SUBMISSION: 23-JAN-1997 (vii) PRIOR APPLICATION DATA (A) APPLICATION NUMBER: US 60 / 039,311 (B) DATE OF SUBMISSION: 7-FEB-1997 (vii) PREVIOUS APPLICATION DATA (A) APPLICATION NUMBER: US 60 / 052,025 (B) DATE OF SUBMISSION: 9-JUL-1997 (viii) INFORMATION OF AGENT / LAWYER (A) NAME: Zindrick, Thomas K. (C) REFERENCE NUMBER / FILE: A-430D (2) INFORMATION FOR SEQ ID NO: l (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 462 base pairs (B) TYPE: nucleic acid (C) STRIP AFFILITY: unknown (D) TOPOLOGY: unknownF MOLECULE: cDNA (ix) CHARACTERISTICS (A) NAME / KEY: CDS (B) LOCATION: 1.4662 (x) DESCRIPTION OF THE SEQUENCE: SEQ ID NO. 1 ATG CGA CCC TCT GGG AGA AAA TCC AGC AAG ATG CAA GCC TTC AGA ATC 48 Met Arg Pro Ser Gly Arg Lys Ser Ser Lys Met Gn Wing Phe Arg He 1 5 10 15 TGG GAT GTT AAC CAG AAG ACC TTC TAT CTG AGG AAC AAC CAA CTA GTT 96 Trp Asp Val Asn Gln Lys Thr Phe Tyr Leu Arg Asn Asn Gln Leu Val 20 25 30 GCT GGA TAC TTG CAA GGA CCA AAT GTC AAT TTA GAA GAA AAG ATA GAT 144 Wing Gly Tyr Leu Gln Gly Pro Asn Val Asn Leu Glu Glu Lys He Asp 35 40 45 GTG GTA CCC ATT GAG CCT CAT GCT CTG TTC TTG GGA ATC CAT GGA GGG 192 Val Val Pro He Glu Pro His Ala Leu Phe Leu Gly He His Gly Gly 50 55 60. AAG ATG TGC CTG TCC TGT GTC AAG TCT GGT GAT GAG ACC AGA CTC CAG 240 Lys Met Cys Leu Ser Cys Val Lys Ser Gly Asp Glu Thr Arg Leu Gln 65 70 75 80 CTG GAG GCA GTT AAC ATC ACT GAC CTG AGC GAG AAC AGA AAG CAG GAC 288 Leu Glu Wing Val Asn He Thr Asp Leu Ser Glu Asn Arg Lys Gln Asp 85 90 95 'AAG CGC TTC GCC TTC ATC CGC TCA GAC AGT GGC CCC ACC ACC AGT TTT 336 Lys Arg Phe Wing Phe He Arg Ser Asp Be Gly Pro Thr Thr Ser Phe 100 105 110 GAG TCT GCC GCC TGC CCC GGT TGG TTC CTC TGC ACA GCG ATG GAA GCT 384 Glu Be Ala Wing Cys Pro Gly Trp Phe Leu Cys Thr Wing Met Glu Wing 115 120 125 GAC CAG CCC GTC AGC CTC ACC AAT ATG CCT GAC GAA GGC GTC ATG GTC 432 Asp Gln Pro Val Ser Leu Thr Asn Met Pro Asp Glu Gly Val Met Val 130 135 140 ACC AAA TTC TAC TTC CAG GAG GAC GAG TAG_462_Thr Lys Phe Tyr Phe Gln Glu Asp Glu * 145 150 (2) INFORMATION FOR SEQ ID NO: 2 (i) CHARACTERISTICS OF THE SEQUENCE (A) LENGTH: 154 amino acids (B) TYPE: amino acid (C) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO. 2 Met Arg Pro Be Gly Arg Lys Be Ser Lys Met Gln Wing Phe Arg He 1 5 10 15 Trp Asp Val Asn Gln Lys Thr Phe Tyr Leu Arg Asn Asn Gln Leu Val 20 25 30 Wing Gly Tyr Leu Gln Gly Pro Asn Val Asn Leu Glu Glu Lys He Asp 35 40 45 Val Val Pro He Glu Pro His Wing Leu Phe Leu Gly He His Gly Gly 50 55 60 Lys Met Cys Leu Ser Cys Val Lys Ser Gly Asp Glu Thr Arg Leu Gln 65 70 75 80 Leu Glu Ala Val Asn He Thr Asp Leu Ser Glu? Sn Arg Lys Gln Asp 85 90 95 Lys Arg Phe Wing Phe He Arg Ser Asp S x Gly Pro Thr Thr Ser Phß 100 105 110 Glu Ser Ala Ala Cys Pro Gly Trp Phe Leu Cys Thr Wing Met Glu Wing 115 120 125 Asp Gln Pro Val Ser Leu Thr Asn Met Pro Asp Glu Gly Val Met Val 130 135 140 Thr Lys Phe Tyr Phe Gln Glu Asp Glu * 145 150 It is noted that in relation to this date the best method to carry out the invention is that which is clear from the present description of the invention.

Claims (26)

Claims

1. A method for the treatment of an acute or chronic inflammatory disorder, characterized in that it comprises administering to a patient in need thereof, therapeutically effective amounts of an inhibitor IL-1 and at least one additional anti-inflammatory drug, wherein the inhibitor IL-1 and at least one additional anti-inflammatory drug are administered separately or in combination.

2. The method according to claim 1, characterized in that the anti-inflammatory compound is methotrexate (N- [4- [(2,4-diamino-6-pteridinyl) methylamino] benzoyl] -L-glutamic acid).

3. The method according to claim 1, characterized in that the inhibitor IL-1 is an IL-1 receptor antagonist.

4. The method according to the rei indication 3, characterized in that the IL-1 receptor antagonist comprises at least one compound selected from the group consisting of: IL-lraa, IL-lraa and IL-rax.

5. The method according to claim 4, characterized in that the IL-lra is a human recombinant IL-lra ..

6. The method according to any of claims 1 to 5, characterized in that the inflammatory disorder is an inflammatory disorder of a joint.

7. The method according to the rei indication 6, characterized in that the inflammatory disorder of a joint is rheumatoid arthritis.

8. The method according to claim 2, characterized in that the inhibitor IL-1 and methotrexan are administered in a pharmaceutically acceptable carrier.

9. A pharmaceutical composition characterized in that it comprises an inhibitor IL-1 and at least one additional anti-inflammatory compound.

10. The pharmaceutical composition according to claim 9, characterized in that the anti-inflammatory compound is methotrexate (N- [4- [(2,4-diamino-6-pteridinyl) methylamino] benzoyl] -L-glutamic acid).

11. The pharmaceutical composition according to claim 9, characterized in that the inhibitor IL-1 is an IL-1 receptor antagonist.

12. The pharmaceutical composition according to claim 11, characterized in that the IL-1 receptor antagonist comprises at least one compound from the group consisting of: IL-lraa, IL-lraß and IL-lrax.

13. The pharmaceutical composition according to the rei indication 12, characterized in that the IL-1 receptor antagonist is a human recombinant IL-1ra.

14. The pharmaceutical composition according to claim 13, characterized in that the human recombinant IL-lra is present in an amount of more than about 200 mg.

15. The pharmaceutical composition according to claim 13, characterized in that the methotrexan is present in an amount of more than about 25 mg.

16. The use of an anti-inflammatory compound, other than an IL-1 inhibitor, in the preparation of a medicament for the treatment of an acute or chronic inflammatory disorder, in a mammal in combination with the administration of an IL-1 inhibitor.

17. The use of rei indication 16, wherein the anti-inflammatory compound is methotrexate.

18. The use according to claim 17, wherein the methotrexate in the medicament is up to about 25 mg.

19. The use according to claims 16 to 18, characterized in that methotrexate is administered orally.

20. The use of an IL-1 inhibitor in the preparation of a medicament for the treatment of an acute or chronic inflammatory disorder in a mammal in combination with the administration of an additional anti-inflammatory compound.

21. The use according to claim 20, wherein the anti-inflammatory compound is methotrexate.

22. The use according to claim 20, wherein the inhibitor IL-1 is an IL-1 receptor antagonist.

23. The use according to claim 22, wherein the IL-1 receptor antagonist comprises at least one compound selected from the group consisting of: IL-lraa, IL-lraß and IL-lrax.

24. The use according to claim 23, wherein the IL-1 receptor antagonist is a human recombinant IL-1ra.

25. The use according to claims 20 to 24, wherein the inhibitor IL-1 in the medicament is present in an amount of up to about 200 mg.

26. The use according to claims 21 to 25, wherein the methotrexate is administered orally.