SI20110A - Methods and compositions for modulating responsiveness to corticosteroids - Google Patents

Abstract

Method for modulating responsiveness to corticosteroids in a subjet are provided. In the method of the invention, an agent which antagonizes a target that regulates production of IFN-'gamma' in the subject is administered to the subject in combination with a corticosteroid such that responsiveness of the subject to the corticosteroid is modulated as compared to when a corticosteroid alone is administered to the subject. In one embodiment, the agent is an IL-18 antagonist. In another embodiment, the agent is an interleukin-12 (IL-12) antagonist. In yet another embodiment, the agent is an NK cell antagonist. In a preferred embodiment, the agent is an inhibitor of a caspase family protease, preferably an ICE inhibitor. In another preferred embodiment, the agent is an anti-IL-12 monoclonal antibody. In yet another preferred embodiment, the agent is an anti-asialo-GM1 antibody or an NK1.1 antibody. Other preferred agents include phosphodiesterase IV inhibitors and beta-2 agonists. The methods of the invention can be used in the treatment of a variety of inflammatory and immunological diseases and disorders. Pharmaceutical compositions comprising an agent which antagonizes a target that regulates production of IFN-'gamma' in a subject, a corticosteroid and a pharmaceutically acceptable carrier are also provided. A preferred composition comprises an ICE inhibitor, a corticosteroid and a pharmaceutically acceptable carrier.

Description

BASF Aktiengesellschaft

Methods and compositions for modulating corticosteroid responsiveness

Standard therapy for many immune and inflammatory disorders involves the administration of corticosteroids that have the ability to suppress immune and inflammatory responses. Corticosteroids are used in the treatment of disorders such as. asthma, autoimmune diseases (eg rheumatoid arthritis, systemic lupus erythematosus) and transplant rejection (corticosteroid screening can be seen, for example, in: Truhan AP et a / (1989) Annals of Allergy 62375-391; Baxter JD (1992) Hospital Practice 27: 111-134; Kimberly RP (1992) Curr. Opin Rheumatol. 4: 325-331; Weisman MH (1995) Curr. Opin. Rheumatol. 7: 183-190). Corticosteroids are also used topically in the treatment of various dermatological disorders, such as. contact dermatitis, psoriasis, lichen planus, keloids, and urticaria pigmentosis (reviewed in: Sterry W. (1992) Arch. Dermatol. Res. 284 (suppl.): S27-S29).

Although the use of corticosteroids is therapeutically beneficial, it is associated with a number of side effects ranging from medium to potential life-threatening effects. Complications associated with prolonged use and / or use of high doses of steroids include musculoskeletal effects (eg osteoporosis, myopathy, aseptic bone necrosis), ophthalmic effects (eg posterior subcapsular cataracts), gastrointestinal effects (eg ulcers, pancreatitis, pancreatitis, pancreatitis) vomiting), cardiovascular effects (eg hypertension, atherosclerosis), effects of the central nervous system (eg pseudotumor cerebri, psychiatric reactions), dermatological effects (eg hirsutism, redistribution of subcutaneous fat, impaired skin healing, thinning of the skin, thinning of the skin, thinning pituitame-adrenal axes (e.g., Truhan AP et al (1989) Annals of Allergy 62: 375-391). Many of the side effects of corticosteroid use appear to be dose dependent (Kimberly R.P. (1992) Curr. Opin. Rheumatol. 4: 325-331).

Therefore, methods and compositions that would allow the use of lower effective doses of corticosteroids (referred to as a steroid sparing effect) to avoid undesirable side effects would be highly desirable.

Another problem limiting the usefulness of corticosteroids is the phenomenon of steroid resistance. Some inflammatory or immunological diseases show an unresponsiveness to steroid treatment. Attempts to use corticosteroid therapy e.g. for the treatment of septic shock in humans have led to unsatisfactory results, so corticosteroids are generally not recommended as adjunctive therapy for severe sepsis or septic shock (as seen, for example: Putterman C. (1989) Israel J. Med. Sci. 25 : 332338; Bone, RC and Brown RC (1990) in Vincent JL (etc.) Update in Intensive Charms and Emergency Medicine 10 ”Heidelberg: Springer Verlag, p. 121). Other disorders that often show resistance to corticosteroid treatment include inflammatory bowel disease (e.g. Hibi T. el «/.(1995) J. Gastroenterol. 30: 121-123) and graft-versus-host disease (Antin JH et. al. (1994) Blood 84: 1342-1348; Racadot E. et al. (1995) Bone Marrow Transplantation 15: 669-677). Thus, methods and compositions that could be used to overcome or reverse corticosteroid resistance in inflammatory and immunological disorders are still needed.

A further disadvantage of corticosteroid therapy is that the vindicutomy effect of steroids occurs when corticosteroid administration is interrupted. The syndicomic effect of steroids is indicated by the worsening of the inflammatory state (s) being treated (cured) after discontinuation of steroid therapy. Methods and compositions that could be used to improve the syndromic effect of steroids are still needed.

The present invention provides methods and compositions for modulating corticosteroid responsiveness in a subject. The methods and compositions of the invention may e.g. used to reverse steroid resistance in the subject and thereby allow Ie to be treated with corticosteroids. The methods and compositions of the invention can also be used to increase steroid sensitivity in a subject, thereby achieving the therapeutic efficacy of corticosteroid treatment at lower doses (e.g., to avoid the harmful side effects of high doses of corticosteroids or to allow the treatment of steroid-dependent diseases , with lower doses).

The methods and compositions of the invention can further be used to improve the vindicative effect of steroids when corticosteroids are deprived of a subject exposed to corticosteroid treatment.

In the modulation methods of the invention, the subject is administered an agent that antagonizes the target that regulates IFN-γ production in the subject, in combination with a corticosteroid, such that the subject's responsiveness to the corticosteroid is modulated as compared to the subject's corticosteroid alone. The target to be antagonized may be e.g. a cytokine or enzyme that regulates IFN-γ production, or a cell that regulates IFN-γ production. The agent is administered at a dose and in a manner sufficient to inhibit IFN-γ production in the subject.

In various embodiments, the agent and corticosteroid are administered simultaneously, the agent is administered first and only then the corticosteroid, or the corticosteroid is administered first and then the agent. The methods can be used for prophylactic and therapeutic regimens of corticosteroid treatment.

In one embodiment, the method involves administering an agent that is an IL-18 antagonist. The IL-18 antagonist is administered at a dose and in a manner sufficient to inhibit the activity of IL-18 in the subject. The IL-18 antagonist may e.g. acts in the stage of IL-18 synthesis, IL-18 cytokine activity or IL-18 interaction with the IL-18 receptor. In a preferred embodiment, the IL-18 antagonist is a caspase family protease inhibitor, preferably an interleukin--β conversion enzyme (ICE) inhibitor. In another embodiment, the IL-18 antagonist is an antibody, antibody fragment, or engineered binding protein that binds to the IL-18 or IL-18 receptor.

In another embodiment, the method involves administering an agent that is an interleukin-12 (IL-12) antagonist. The IL-12 antagonist is administered at a dose and in a manner sufficient to inhibit IL12 activity in the subject. The IL-12 antagonist may e.g. acts in the stage of IL-12 synthesis, IL12 cytokine activity or IL-12 interaction with the IL-12 receptor. In a preferred embodiment, the IL-12 antagonist is an antibody, antibody fragment, or engineered binding protein that binds to the IL-12 or IL-12 receptor. In another preferred embodiment, the IL-12 antagonist is an agent that stimulates the production of cyclic AMP (cAMP) in IL-12-producing cells. Examples of agents that can be used to stimulate cAMP include phosphodiesterase IV inhibitors and beta-2 agonists. In another embodiment, the IL-12 antagonist is a STAT4 inhibitor.

In a further embodiment, the method involves administering an agent that depletes or eliminates NK cells and NK-like cells (referred to herein as an NK cell antagonist) from a subject. The NK cell antagonist is administered at a dose and in a manner sufficient to inhibit IFN-γ production in the subject. Preferred NK cell antagonists are antibodies specific for NK- / NK-like cells that deplete these cells in vivo. Examples of preferred antibodies for use as NK cell antagonists are anti-asialoGM1 antibodies and NK1.1 antibodies.

A further aspect of the present invention relates to a method for modulating corticosteroid responsiveness in a subject where a caspase family protease inhibitor is administered to the subject, preferably ICE, together with a corticosteroid, such that the subject's response to a corticosteroid is modulated compared to giving the subject a corticosteroid alone .

A further aspect of the present invention relates to a method for modulating corticosteroid responsiveness in a subject, wherein the IL-12 antagonist is administered to the subject together with a corticosteroid, such that the subject's responsiveness to the corticosteroid is modulated compared to giving the subject a corticosteroid alone. Still further, the invention relates to a method for modulating corticosteroid responsiveness in a subject, wherein the subject is administered an NK cell antagonist (e.g., anti-NK- / NK-like cellular antibody) together with a corticosteroid, such that the subject's response to the corticosteroid is modulated, in when compared to giving a subject a corticosteroid alone.

From a further point of view, the invention relates to a method for modulating corticosteroid responsiveness in a subject, wherein a subject in need of modulating corticosteroid responsiveness is selected and provided with a target antagonizing agent that regulates IFN-γ production in the subject such that the response is subject to a corticosteroid modulated, compared to giving the subject a corticosteroid alone. The agent is administered to the subject at a dose and in a manner sufficient to inhibit IFN-γ production in the subject. The entity we select is e.g. may be one who is steroid resistant to treatment, an entity that responds to steroids and in which steroid sensitivity is to be increased, or an entity that is required to withdraw steroids and ameliorate the viral effects of steroids.

The present invention also provides pharmaceutical compositions for modulating corticosteroid responsiveness in a subject. In one embodiment, the composition of the invention comprises a target antagonizing agent that regulates the production of IFN-γ in a subject, a corticosteroid and a pharmaceutically acceptable carrier. In another embodiment, the composition of the invention comprises an IL18 antagonist (such as a caspase family protease inhibitor, preferably an ICE inhibitor or anti-IL-18 or anti-IL-18 receptor monoclonal antibody), a corticosteroid, and a pharmaceutically acceptable carrier. In a further embodiment, the composition of the invention comprises an IL-12 antagonist (e.g., anti-IL-12 or anti-IL-12 receptor monoclonal antibody, phosphodiesterase IV inhibitor, beta-2 agonist, STAT4 inhibitor), a corticosteroid, and a pharmaceutically acceptable carrier. In a further embodiment, the composition of the invention comprises an NK cell antagonist (e.g., anti-NK- / NK-like cellular antibody) a corticosteroid and a pharmaceutically acceptable carrier. The pharmaceutical compositions of the invention can be formulated for administration in a preferred mode of administration that achieves the desired therapeutic effect. In one preferred embodiment, a pharmaceutical composition for topical administration is formulated. In another preferred embodiment, the pharmaceutical composition is formulated for administration by inhalation. Other preferred routes of administration include oral and intravenous administration.

The methods and compositions of the invention may be used in the treatment of any disease or disorder in which it is desired to modulate steroid responsiveness. In preferred embodiments, the methods and compositions of the invention are used to treat a subject suffering from septic shock. In another embodiment, the methods and compositions of the invention are used to treat a subject suffering from Crohn's disease. In another embodiment, methods and compositions are used to treat a subject suffering from asthma. In another embodiment, methods and compositions are used to treat a subject suffering from an autoimmune disease or disorder. In another embodiment, methods and compositions are used to treat a subject suffering from a graft-versus-host disease or transplant rejection. In yet another embodiment, methods and compositions are used to treat a subject suffering from an acute inflammatory disorder. In another embodiment, methods and compositions are used to treat a subject suffering from chronic inflammatory disorder.

Short description of the pictures

FIG. 1 is a bar chart showing serum TNFα levels (in ng / ml) in wild-type and ICE-deficient (ICE KO) mice treated with vehicle or dexamethasone alone (4 mg / kg) 30 minutes after LPS in LPS / P. acw & s-septic shock model, which shows that ICE-deficient mice, but not wild-type mice, exhibit suppression of TNFα production and are thus steroidally responsive.

FIG. 2 is a bar chart showing serum TNFα levels (in ng / ml) in wild-type mice (dotted columns) and ICE-deficient mice (dashed lines) pretreated with solvent alone or decreasing amounts of dexamethasone (0.05, 0.005 or 0.0005 mg / kg) in LPS / P. ac «es model of septic shock, which shows that ICE-deficient mice retain steroid responsiveness to falling steroid doses compared with wild-type mice.

FIG. 3 is a bar chart showing LPS-induced serum IL-12 (in pg / ml) in B6 mice pretreated with solvent or phosphodiesterase IV inhibitor rolipram alone, showing that phosphodiesterase IV inhibitor treatment inhibits IL- 12.

FIG. 4 is a bar chart showing serum TNFα levels (in ng / ml) in B6 mice treated with solvent alone (saline) or ICE inhibitor (AcYVAD-CHO) in combination with dexamethasone treatment in LPS / P. acnev-septic shock model.

FIG. 5 is a bar chart showing serum IL-6 levels (in ng / ml) in B6 mice treated with solvent alone (saline) or ICE inhibitor (AcYVAD-CHO) in combination with dexamethasone treatment in LPS / P. ac «cj-model of septic shock.

FIG. 6 is a bar chart showing serum IL-1 β levels (in ng / ml) in vehicle-treated B6 (saline) or ICE inhibitor (AcYVAD-CHO) combined with dexamethasone treatment in LPS / P . acnes model of septic shock.

The present invention is based, at least in part, on the discovery that ICE-deficient mice are corticosteroid responsive to LPS-challenged mice compared to wild-type control mice (Example 1). In addition, ICE-deficient mice exhibit increased sensitivity to low doses of corticosteroids compared with wild-type control mice when corticosteroid treatment prior to the LPS challenge is used in a septic shock model (Example 2). The present invention is further based, at least in part, on the discovery that depletion of NK- / NK-like cells in wild-type LPS-induced mice results in substantially reduced IFN-γ production (compared to control untreated mice) and substantially increases survival ( Example 10).

It has been previously described that administration of interferon-γ (IFN-γ) can overcome corticosteroid suppression of TNFα biosynthesis by murine macrophages (Leudke, C.E. and Cerami, A. (1990) J. Ciin. Invest 86: 1234-1240). In addition, ICE and other proteases of the caspase family can cleave the precursor form of IL-18 to its mature active form (Example 4). Although not intended to be limiting by the mechanism of the present invention, it is believed that the ability to confer corticosteroid responsiveness by inhibiting ICE activity in a subject is a result of inhibiting IL-18 processed by ICE, thus producing the production of mature IL-18 inhibited, thereby leading to reduced of IFN-γ production in the subject. In addition, IL-18, in conjunction with IL-12, stimulates NK- / NK-like cells to produce more IFN-γ. Thus, NK / NK-like cells are considered to form a positive feedback loop in the production of IFN-γ, which is degradatively modulated by depletion or elimination of NK- / NK-like cells.

In the light of the foregoing, the present invention generally provides methods and compositions for modulating corticosteroid responsiveness, wherein the target that regulates IFN-γ production is antagonized in the subject. This target, which regulates IFN-γ production and which is antagonized, may be IL-18 (which can be antagonized, for example, indirectly by inhibiting ICE activity or directly by using anti-IL-18 antibody). Alternatively, another factor can be antagonized that regulates IFN-γ production, such as e.g. IL-12 to thus modulate corticosteroid responsiveness in the subject. Further, an agent that depletes or eliminates NK- / NK-like cells can be used to inhibit IFN-γ production to modulate corticosteroid responsiveness in the subject.

In order to better understand the present invention, we first explain a number of terms.

As used herein, the term corticosteroid refers to a class of therapeutic agents useful in treating inflammatory conditions, including those resulting from infections, transplant rejection, and autoimmune disorders. Corticosteroids include those that are natural, synthetic or semi-synthetic in origin and are indicated by the presence of a steroid core from four fused rings, e.g. as found in the structures of cholesterol, dihydroxycholesterol, stigmasterol and lanosterol. Corticosteroid medications include: cortisone, cortisol, hydrocortisone (11317-dihydroxy-21- (phosphonooxy) -pregn-4-ene-3,20-dione disodium), dihydroxycortisone, dexamethasone (21 - (acetyloxy) -9-fluoro-11 β , 17-dihydroxy-16amethylpregna-1,4-diene-3,20-dione) and highly derivatized steroid drugs such as e.g. beconase (beclomethasone dipropionate, which is 9-chloro-1 1β, 17,21-trihydroxy-16pmethylpregna-1,4-diene-3,20-dione-17,21-dipropionate). Other examples of corticosteroids include flunizide, prednisone, prednisolone, methylprednisolone, triamcinolone, deflazortort and betamethasone.

The term target that regulates IFN-γ production is intended to include chemical factors (e.g., cytokines, enzymes, etc.) and cells that directly or indirectly control IFN-γ synthesis in a subject. Examples of factors that regulate IFN-γ production include IL-18 (e.g., Okamura, H. et al. (1995) Nature 378: 88-91; Ushio, S. et al. (1996) J. Immunol. 156 : 4274-4279) and interleukin-12 (IL-12) (see, e.g., Schoenhaut, D. et al. (1992) J. Immunol. 148: 3433; PCT Publication WO 90/05147; European Patent Application EP 433 827 A2). Examples of cells that regulate IFN-γ production include NK- and NK-like cells.

As used herein, factor antagonizing agents are intended to include agents that inhibit factor activity and agents that degrade (i.e. inhibit) the synthesis or production of the factor.

The term IL-18 refers to a cytokine having an amino acid sequence as shown by: Okamura H. et al. (1995) Nature 378: 88-91 (mouse) or Ushio S. et al. (1996) J. Immunol. 156: 4274-4279 (human), and other mammalian homologs thereof. The cytokine IL18 is also referred to as interferon γ inducer factor (IGIF) and IL-Ιγ in the art.

The term IL-18 antagonist is intended to include agents that inhibit the synthesis or production of IL-18, agents that inhibit IL-18 activity after being synthesized, agents that inhibit the interaction of IL-18 with the IL-18 receptor, and agents that inhibit IL-18 receptor activity. Examples of IL-18 antagonists include caspase family protease inhibitors (e.g., ICE inhibitors) and antibodies, antibody fragments, and engineered binding proteins that bind to either the IL-18 or IL-18 receptor.

The term interleukin-12 (IL-12) refers to a cytokine having an amino acid sequence as shown by: Schoenhaut, D. et al. (1992) J. Immunol. 148: 3433, PCT Publication

WO 90/05147, and European Patent Application EP 433 827 A2, and other mammalian homologs thereof.

The term IL-12 antagonist is intended to include: agents that inhibit the synthesis or production of IL-12, agents that inhibit IL-12 activity after being synthesized, agents that inhibit the interaction of IL-12 with the IL-12 receptor, and agents that inhibit IL-12 receptor activity. Examples of IL-12 antagonists include antibodies, antibody fragments, and engineered binding proteins that bind to either the IL-12 or IL-12 receptor, agents that stimulate intracellular cAMP production in IL-12-producing cells (such as phosphodiesterase IV inhibitors or beta-2 agonists) and STAT4 inhibiting agents.

The term protease of the caspase family is intended to include members of caspase proteases as described by: Alnemri, E. et al. (1996) Whole 87: 171, including caspase-1 (ICE), caspase-2 (ICH-1), caspase-3 (CPP32, Yama, apopain), caspase-4 (TX, ICH-2, ICE _re i- II), caspase-5 (ICE _re i-III, TY), caspase-6 (Mch2), caspase-7 (Mch3, ICE-LAP3, CMH-1), caspase-8 (MACH, FLICE, Mch5), caspase -9 (ICE-LAP6, Mch6) m caspase-10 (Mch4). Furthermore, the term protease of the caspase family is intended to include any protein that shares an amino acid sequence identity greater than 20% with ICE in the protease active domains (i.e., the active domains of p 10 and p20 subunits of ICE), containing the peptide sequence of glutamine-alanine- cysteine-X-glycine (QACXG), wherein the cysteine (C) is a catalytic active cysteine residue and X represents any amino acid, and contains a sequence of serine-histidine-glycine (SHG) positioned N-terminal with respect to the QACXG motif in which the histidine (H) catalytically essential histidine residue. The caspase family proteases typically show a strong preference for hydrolysis of peptide bonds directly by the acidic amino acid (i.e. aspartic or glutamic acid).

The proteases of the caspase family are known in humans and other organisms, including mice and Caenorhabditis elegans. Examples of caspase family proteases include, for example: Ich-1 (Wang, L. et al. (1994) Whole 78: 739-750); ICH-2 (Kamens, J. et al. (1995) J. Biol. Chem. 270: 15250-15256); Mch2 (Femandes-Alnemri, T. et al. (1995) Cancer Res. 55: 2737-2742); CPP32 (Femandes-Alnemri, T. et al., 1994) J. Biol. Chem.

269: 30761-30764); Yama / CPP32p (Tewari, M. et al. (1995) Whole 8L801-809); product of the mouse Nedd2 gene (Kumar, S. et al. (1992) Biochem. Biophys. Res. Commun. 185: 1155-1161; Kumar, S. et al. (1994) Genes Dev. 8: 1613-1626); the product of the C. elegans gene, ced-3 (Yuan, J. et al. (1993) Celi 75: 641-652); human TX protein (Faucheu, C., et al., (1995) EMBO J. 14: 1914-1922); ICE _rel II and ICE _re iIII (Munday, NA et al. (1995) 7 Biol. Chem. 270: 15870-15876).

The term interleukin-1β conversion enzyme (ICE) is intended to refer to a protease having an amino acid sequence as described by: Cerretti D.P. et al. (1992) Science 256: 97-100 (Human) or Nett, M.A. et al. (1992) J. Immunol. 149: 3254-3259 (mouse) and its other mammalian homologs.

The term ICE inhibitor is intended to include chemical agents that inhibit the proteolytic activity of ICE. Examples of ICE inhibitors are known in the art, including e.g. agents disclosed in: U.S. Pat. 5,585,357 (pyrazolyl derivatives); U.S. Pat. No. 5,677,283 (pyrazolyl derivatives); U.S. Pat. No. 5,656,627 (inhibitors comprising hydrogen bonding group, hydrophobic group and electronegative group); U.S. Pat. No. 5,411,985 (Gamma-pyrone-3-acetic acid compounds); U.S. Pat. 5,430,128 (tripeptidyl derivatives); U.S. Pat. 5,434,248 (tripeptidyl compounds); U.S. Pat. No. 5,565,430 (Ν, Ν'-diacylhydrazinoacetic acid compounds); U.S. Pat. 5,416,013 (peptidyl derivatives); PCT Publication WO 94/21673 (Alpha-Ketoamide Derivatives); PC Publication WO 97/22619 (N-acylamino compounds); PCT Publication WO 97/22618 (Amino Acid or Di- or Tripeptide Amide Derivatives); PCT Publication WO 95/35308 (inhibitors comprising hydrogen bonding group, hydrophobic compound and electronegative group); PCT Publication WO 93/14777 (peptidyl derivatives); PCT Publication WO 93/16710 (peptidyl derivatives); PCT Publication WO 95/05152 (substituted ketone derivatives); PCT Publication WO 94/03480 (4-Amino-2,2-difluoro-3-oxo-1,6-hexanedioic acid derivatives); PCPublication WO 94/00154 (peptidyl derivatives); PCT Publication WO 93/05071 (peptidyl derivatives); European application EP 519 748 (peptidyl derivatives); European application EP 590 650 (cyclopropene derivatives); European application EP 628 550 (pyridazines); European application EP 644 198 (alpha-heteroaryloxymethyl ketones); European application EP 644 197 (peptide phosphinyloxymethyl ketones); European patent application EP 547 699 (peptidyl derivatives); British application GB 2,278,276 (gamma-pyrone-3-acetic acid compounds) and Canadian application 2,109,646 (para-nitroanilide peptides). The present invention encompasses the use of ICE inhibitors shown in any of the foregoing publications in the methods described herein.

Additional preferred ICE inhibitors for use in the methods of the invention include ICE inhibitors aspartic acid substituted with sulfonamides of formula I:

R * o _{/ z} 0

X '' ^S ^ N

R ²

RR

OR where it is

R ^{1 is} hydrogen, C 1 -C 6 alkyl or benzyl;

R ² is -CHO-, -COR ^a or -CN;

each R ^a is independently hydrogen or C 1-6 alkyl;

X is a bond, CH ₂ , CHR ⁵ , NH, NR ⁵ or O;

R ³ is aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocycle or substituted heterocycle;

Y is absent, NR ⁵ , CO, S, O, SO 2, -O (CHR ⁵ ) '-, CHR ⁵ , NR ⁵ CO, NCR ⁵ , CONR ⁵ , OCHR ⁵ , CHR ⁵ O, SCHR ⁵ , CHR ⁵ S , SO2NR ^5, C ^ -alkyl, NR ⁵ SO 2, CH2CHR ^5, CHR ⁵ CH 2, COCH ₂ or CH ₂ CO;

R ⁴ is absent, aryl, substituted aryl, C 1 -C 8 alkyl, heteroaryl, substituted heteroaryl, cycloalkyl, C 1 -C 6 alkyl, substituted cycloalkyl, heterocycloalkyl or substituted heterocycloalkyl;

each R 'is independently hydrogen, C 1 -C 6 alkyl, aryl, - (CH ₂ ) _n -aryl or - (CH ₂ ) _n -cycloalkyl;

each n is independently 0 to 5, m is 1 or 2, and pharmaceutically acceptable salts, esters, amides and prodrugs thereof.

In one embodiment of the invention, R ^{2 is} CHO.

In another embodiment of the invention, R ^{1 is} hydrogen.

In another embodiment of the invention, R ^{a is} hydrogen.

In another embodiment of the invention, X is a bond.

In another embodiment of the invention, R is phenyl or substituted phenyl.

In another embodiment of the invention, Y is a bond.

In another embodiment of the invention is Y O.

In another embodiment of the invention, Y is CH ₂ .

In another embodiment of the invention, R ^{4 is} phenyl or substituted phenyl.

In another embodiment of the invention, R ^{2 is} CHO, R ^a is H, R ¹ is hydrogen, X is a bond, R ³ and R ⁴ are phenyl or substituted phenyl and Y is a bond, CH ₂ or O.

In another embodiment of the invention, m is 1 and R ⁵ is hydrogen.

Other preferred ICE inhibitors substituted with sulfonamides have the formula II:

where it is

R ^{1 is} hydrogen, C 1 -C 6 alkyl or benzyl;

R ² is -CHO, -COR ^a or -CN;

each R ^a is independently hydrogen or C 1 -C 6 alkyl;

X is a bond, CH ₂ , CHR ⁵ , NH, NR ⁵ or O;

Y is a bond, NR ⁵ , CO, S, O, SO ₂ , CHR ⁵ , NR ⁵ CO, CONR ⁵ , OCHR ⁵ , CHR ⁵ O,

-O (CHR ⁵⁾ n, Schram ^5, CHR ⁵ with, SO2NR ^5, NR ⁵ SO 2, CH2CHR ^5, CHR ⁵ CH 2, COCH ₂ or CH ₂ CO;

each R ⁵ is independently hydrogen, C 1 -C 8 alkyl, aryl or - (CH ₂ ) _n- aryl;

each n is independently from 0 to 5;

m is 1 or 2;

each Z is independently hydrogen or aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclic or substituted heterocyclic group attached to a phenyl group containing Z as a substituent;

R ^b , R ^c , R ^d and R ^e are each independently hydrogen, C 1 -C 6 alkyl, C 1 -C 6 alkoxy, -OH, C 1 -C 6 thioalkoxy, halogen, trifluoromethyl, dialkylamino, -NO2, -CN, -CF3, -CO2-alkyl, -SO3H, -CHO, -CO-alkyl, -CONH-alkyl, -CONHR ^q , -CON (alkyl) 2, - (CH ₂ ) _n -NH ₂ , - (CH ₂ ) _n -NHalkyl -NHR ^q , -NHCOR ^q , - (CH ₂ ) _n OH, - (CH ₂ ) _n -CONH ₂ or - (CH ₂ ) _n CO ₂ H; and R ^q is hydrogen or C 1 -C 8 alkyl, and their pharmaceutically acceptable salts, esters, amides and prodrugs.

In another embodiment, with respect to the compounds of formula II, R ^{1 is} hydrogen.

In another embodiment, with respect to the compounds of formula II, R ^{2 is} CHO.

In another embodiment, with respect to the compounds of formula II, R ^{a is} hydrogen.

In another embodiment, with respect to the compounds of formula II, X is a bond.

In another embodiment, with respect to the compounds of formula II, Y is a bond, O or CH ₂ .

In another embodiment, with respect to the compounds of formula II, R ^b and R c are hydrogen.

In another embodiment, with respect to the compounds of formula II, wherein R ^b , R ^c and R ^{d are} hydrogen, R ^{e is} C 1 -C 6 alkyl.

In another preferred embodiment with respect to the compounds of formula II, R ^b or R ^{c is} positioned in the para position of the phenyl ring with respect to X and R ^b or R ^{c is} -OCH ₃ .

In another embodiment, with respect to the compounds of formula II, m is 1 and R ⁵ is hydrogen.

Preferred compounds include:

3- (Biphenyl-2-sulfoamino) -4-oxo-butyric acid

3- (2-Benzyl-benzenesulfonylamino) -4-oxo-butyric acid

4- Oxo-3- (2-phenoxy-benzenesulfonylamino) -butyric acid

4-Oxo-3- (2-p-tolyloxy-benzenesulfonylamino) -butyric acid

3- [2- (4-Isopropyl-phenoxy) -benzenesulfonylamino] -4-oxo-butyric acid

4- Oxo-3- (2-m-tolyloxy-benzenesulfonylamino) -butyric acid

3- [2- (3-Isopropyl-phenoxy) -benzenesulfonylamino] -4-oxo-butyric acid and 3- (4'-methyl-biphenyl-2-sulfonylamino) -4-oxo-butyric acid.

Other ICE inhibitors include compounds of formula III:

where it is

R ^{1 is} hydrogen, C 1 -C 8 alkyl or benzyl;

R ² is -CHO, -COR ^a or -CN;

each R ^a is independently hydrogen or C 1 -C 8 alkyl;

X is a bond, CH ₂ , CHR ⁵ , NH, NR ⁵ ab O;

R ³ is hydrogen, C 1 -C ₆ -alkyl, aryl or - (CH ₂ ) _n -aryl;

each independently from 0 to 5;

m is 1 or 2;

Z is absent or is an aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclic or substituted heterocyclic group attached to a phenyl group containing Z as a substituent;

R ^{1 and} R ^s are each independently hydrogen, C 1 -C 6 alkyl, hydroxy, halogen, trifluoromethyl, dialkylamino, -NO 2, -CN, -CO 2 H, -CO 2 -alkyl, -SO 3 H, -CHO, -CO-alkyl, - CONH2, -CONH (CH2) n-aryl, -CONH (CH2) '- substituted aryl, -CONH-alkyl, -CONHR ^q , -CON (alkyl) 2, - (CH ₂ ) _n -NH ₂ , - (CH ₂ ) _n -NH-alkyl, -NHR ^q , -NHCOR ^q , -OR ^q , -SR ^q or - (CH ₂ ) _n -aryl, and

R ^q is hydrogen or C 1 -C ₈ alkyl, and their pharmaceutically acceptable salts, esters, amides and prodrugs.

In a preferred embodiment of the compounds of formula III, R ^{f is} in the ortho position with respect to X on the phenyl ring and R ^{8 is} hydrogen.

In a preferred embodiment of the compounds of formula III, Z is hydrogen, m is 1, R ⁵ is hydrogen and R ^a is hydrogen.

In a preferred embodiment of the compounds of Formula III, the compound is tribenzenesulfonylamino-4-oxobutyric acid.

The ICE inhibitors of the sulfonamide substituted aspartic acid described above can generally be done as follows:

(1: 4: next)

Nu

Oh

Nu = nldfiofil

Other sulfonamide ICE inhibitors that can be used according to the invention are compounds of formula IV:

where R is ¹

R ³ is hydrogen,

C1-C8-alkyl,

- (CH ₂ ) '- aryl or - (CH ₂ ) _n -heteroaryl;

R ⁴ is C _r C ₆ -alkyl,

- (CH ₂ ) _n -aryl or - (CH ₂ ) _n -heteroaryl;

R ⁵ and R ⁶ are each independently hydrogen, C 1 -C 6 alkyl,

- (CH ₂ ) _n -aryl or - (CH ₂ ) _n -heteroaryl;

R ⁷ is C 1 -C 6 alkyl,

- (CH ₂ ) _n -aryl or

- (CH ₂ ) _n -heteroaryl; each n O to 6; each m is O, 1, 2 or 3;

A is alanine, leucine, isoleucine, proline, phenylalanine, glycine, tyrosine, serine, threonine, tryptophan, cysteine, methionine, valine, asparagine, glutamine, aspartic acid, lysine, glutamic acid, arginine or histidine;

R ² is - (CH ₂ ) _n -Zin

Z is aryl, heteroaryl, cycloalkyl, C1-C6-alkyl, -

(CH ₂ ) _n H < ^CH 2> 7 < ^{R <} \ '

ⁿ fluorenyl, substituted fluorenyl, substituted aryl, substituted heteroaryl or substituted cycloalkyl, and pharmaceutically acceptable salts, esters, amides and prodrugs thereof.

In a preferred embodiment of the compounds of formula IV, R ^{1 is} O

R ⁷ 0 '( ^A ) m

In another preferred embodiment of the compounds of formula IV, R ^{1 is} O

m is 0 and R ⁷ is - (CH ₂ ) _n -aryl.

In another preferred embodiment of the compounds of formula IV, R ^{1 is} O

m is 0 and R ⁷ is -CH ₂ -aryl.

In another preferred embodiment of the compounds of formula IV, R ^{2 is} - (CH ₂ ) _n -aryl.

In another preferred embodiment of the compounds of formula IV, aryl is phenyl or naphthyl.

In another preferred embodiment of the compounds of formula IV, R ^{2 is} - (CH ₂ ) _n -cycloalkyl.

In another preferred embodiment of the compounds of formula IV, R ^{1 is}

O / (CITpH - phenyl or -SO ₂ -phenyl.

In another preferred embodiment of the compounds of formula IV, R ^{2 is}

In another preferred embodiment of the compounds of formula IV, R ^{2 is}

Other sulfonamide ICE inhibitors include compounds of formula V:

wherein R 'is -CH ₂ -CH ₂ -aryl, -CH ₂ -cycloalkyl, -CH ₂ CH ₂ -cycloalkyl, or -CH ₁ CH ₂ -heteroaryl;

R is

R ^a

Oh

Oh

II bdi '

R ^e O o

R ^L ; n

Oh

R ^L o ch ₃

or C.

O o

Oh

R ^a is - (CH ₂ ) _n -aryl or - (CH ₂ ) _n -heteroaryl; R ^b is aryl or heteroaryl;

R ^c is -CH ₂ -aryl or aryl;

R ^d is hydrogen or C 1 -C 6 alkyl;

R ^e is -CH ₂ -aryl or -CH ₂ -heteroaryl, and their pharmaceutically acceptable salts, amide esters and prodrugs.

In a preferred embodiment of the compounds of formula V, R ^{1 is}

R ^e O

In another preferred embodiment of the compounds of formula V is R ¹

Oh

II .s. ·

Rb ^ ll ^ ^R o

In another preferred embodiment of the compounds of formula V, R ^is - (CH ₂ ) _n -aryl.

In another preferred embodiment of the compounds of formula V, aryl is phenyl or naphthyl.

In another preferred embodiment of the compounds of formula V, R ^{b is} aryl.

Preferred compounds include:

3-Benzyloxycarbonylamino-4-oxo-5- (2-phenoxy-ethanesulfonylamino) -pentanoic acid;

3-Benzyloxycarbonylamino-4-oxo-5- (3-phenyl-propane-1-sulfonylamino) -pentanoic acid;

3-Benzenesulfonylamino-4-oxo-5- (2-phenylethan-1-sulfonylainino) -pentanoic acid;

5-Benzenesulfonylamino-3-benzyloxycarbonylamino-4-oxo-pentanoic acid;

3-Benzyloxycarbonylamino-5-methanesulfonylamino-4-oxo-pentanoic acid;

-benzyloxycarbonylamino-5- (naphthalene-1-sulfonylamino) -4-oxo-pentanoic acid;

3-Benzyloxycarbonylamino-5- (2-cyclohexyl-ethanesulfonylamino) -4-oxo-pentanoic acid;

-benzyloxycarbonylamino-5- (2-naphthalen-1-yl-ethanesulfonylamino) -4-oxo-pentanoic acid;

3-Benzyloxycarbonylamino-5- (7,7-dimethyl-2-oxo-bicyclo [2.2.1] hept-1- (R) ylmethanesulfonylamino) -4-oxo-pentanoic acid;

3-Benzyloxycarbonylamino-5- (indan-1-ylmethanesulfonylamino) -4-oxo-pentanoic acid;

3-Benzyloxycarbonylamino-5- (9-fluoro-9H-fluoren-9-ylmethanesulfonylamino) -4-oxopentanoic acid;

3-Benzyloxycarbonylamino-5- (7,7-diinethyl-2-oxo-bicyclo [2.2.1] hept-1- (S) ylmethanesulfonylamino) -4-oxo-pentanoic acid;

- [2- (2-Benzyloxycarbonylamino-3-methyl-butyrylamino) -propionylamino] 4-oxo-5- (2-phenyl-ethanesulfonylamino) -pentanoic acid;

3- [2- (2-Benzyloxycarbonylamino-4-carboxy-butyrylamino) -3-methylbutyrylamino] -4-oxo-5- (2-phenyl-ethanesulfonylamino) -pentanoic acid;

- {2- [4-Carboxy-2- (3-phenyl-propionylamino) -butyrylamino] -3-methylbutyrylamino} -4-oxo-5- (2-phenyl-ethanesulfonylamino) -pentanoic acid;

3- (2- {2- [2-acetylamino-3- (4-hydroxy-phenyl) -propionylamino] -4-carboxybutyrylamino} 3-methyl-butyrylamino) -4-oxo-5- (2-phenyl-ethanesulfonylamino) -pentanoic acid;

3- (2-acetylamino-3-methyl-butyrylamino) -5- (7,7-dimethyl-2-oxo-bicyclo [2.2.1] hept-1- (S) ylmethanesulfonylamino) -4-oxo-pentanoic acid;

3- (2-acetylamino-propylamino) -5- (7,7-dimethyl-2-oxo-bicyclo [2.2.1] hept-1- (S) -ylmethane sulfonylamino) -4-oxo-pentanoic acid;

3- [2- (2-Benzyloxycarbonylamino-3-methyl-butyrylamino) -propionylamino] -5- (7,7-dimethyl-2-oxo-bicyclo [2.2.1] hept-1-ylmethanesulfonylamino) -4-oxo-pentanoic acid acid;

3- {2- [4-carboxy-2- (3-phenyl-propionylamino) -butyrylamino] -3-methyl-butyrylamino} -5 (7,7-dimethyl-2-oxo-bicyclo [2.2.1] hept- 1-ylmethanesulfonylamino) -4-oxo-pentanoic acid;

- (2- {2- [2-acetylamino-3- (4-hydroxy-phenyl) -propionylamino] -4-carboxy-butyrylamino} 3-methyl-butyrylamino) -5- (7,7-dimethyl-2-oxo -bicyclo [2.2.hept-1ylmethanesulfonylamino) -4-oxo-pentanoic acid;

3- [2- (2-Benzyloxycarbonylamino-4-carboxy-butyrylamino) -3-methyl-butyrylamino] -5 (7,7-dimethyl-2-oxo-bicyclo [2.2.1] hept-1-ylmethanesulfonylamino) -4 -oxo-pentanoic acid;

3- (1,2,3,4-tetrahydro-1-oxo-isoquinolin-2-yl) -acetanino-5-benzenesulfonylamino-4-hydroxy-pentanoic acid;

(S) -5- (bicyclo [2.2.1hept-1-ylmethanesulfonylamino) -4-oxo-3- [2- (1-oxo-3 _J 4-dihydro-1H-isoquinolin-2-yl) -acetylamino] -pentanoic acid sko acid; (S) -4-Oxo-3- [2- (1-oxo-3,4-dihydro-1H-isoquinolin-2-yl) -acetylamino] -5- (2-phenylethanesulfonylamino) -pentanoic acid; and

4- Oxo-3- [2- (1-oxo-3,4-dihydro-1H-isoquinolin-2-yl) -acetylamino] -5phenibnethanesulfonylamino-pentanoic acid.

Other sulfonamide ICE inhibitors include compounds of formula VI:

.COOH

o

CH · o

where R '

VI

Ο

II

ο

C ο ch ₃

ο or

R ^e O

C

Oh

R ^a is - (CH ₂ ) _n -aryl or - (CH ₂ ) _n -heteroaryl

R ^b is aryl or heteroaryl

R ^c is -CH ₂ -aryl or aryl

R ^d is hydrogen or C 1 -C ₆ -alkyl

R ^e is -CH ₂ -aryl or -CH ₂ -heteroaryl, and their pharmaceutically acceptable salts, esters, amides and prodrugs.

In a preferred embodiment of the compounds of formula VI, R ^{1 is} O

In a preferred embodiment of the compounds of formula VI, R ^{1 is} O

II

In a preferred embodiment of the compounds of formula VIe R ^is - (CH ₂ ) _n -aryl.

In a preferred embodiment, the compounds of the formula are aryl phenyl or naphthyl.

In a preferred embodiment of the compounds of formula VI, R ^{b is} aryl.

The compounds of formulas IV, V or VI described above can be prepared generally by converting the corresponding starting sulfonamide i into Boc sulfonamide 2 using a reagent such as e.g. di-tert.butyl dicarbonate. Boc sulfonamide 2 can then be reacted with the appropriately substituted aspartic acid bromomethylketone β-tert.butyl ester 3 in the presence of a base and then treated with acid to give the desired product 4.

Scheme 1

Oh

H ₂ N - S - R ² --► Boc - N nuq di tert.-butyl dicarbonate ⁿ 1

O u

-sII

Oh

*>

J

O ¹¹ o

1. Boc-N-S-R ²

H and 'base O

2. HCiaIiCF ₃ COOH

Alternatively, compounds of formulas IV, V or VI can be prepared generally by the reaction of a suitably substituted aspartic acid aldehyde j. with nitromethane in the presence of a base such as potassium tert.butoxide to give nitroalcohol 2. By reducing 2 to amine 3 and then reacting with the corresponding sulfonyl chloride to give product 4, which can be oxidized to ketone 5 with a reagent such as Dess Martin periodic or with Swem oxidation. Acid deprotection of t-butyl ester with HCl or trifluoroacetic acid gives the desired product 6.

Scheme 2, 1 H, 1 H

R — N ^ COOMe ^R —N ^ COOH: £ ^Ξ —COOt-Bu COOt-Bu ih

R ¹ - K / CHO

R ¹ - N ^ C ^ OH ⁼ —COOt-Bu

OH, H? ^H „OH ^RN > A \ / ^N0 2 ^{rI — N} x / O _ / - NH ₂ —COOt-Bu 1 —COOt-Bu 2

OH —COOt-Bu 3

R ^ -SO ^ Cl R ¹ ~ ^N \ _r xk ^ / N ~ SO ₂ —R ²

-COOt-Bu ^{rI S0} 2 R ² R ¹ ——SO? —R ²

-—COOt-Bu 5 • a ^ H

Other ICE inhibitor compounds that may be used in the invention include hydroxamate compounds, including compounds of formula VII:

t ^a (CH ₂ ) _n —Q

VII where R ¹

ο

II

R ^{4 / X} (A) _m r ⁷ o (A)

O ^{11 R} \ / S. / or 11 ^X (A) Jf _r 6 ^ o ^m each R is independently hydrogen or C 1 -C 6 alkyl;

R ³ is hydrogen, C 1 -C ₆ -alkyl, - (CH ₂ ) _n -aryl, - (CH ₂ ) _n -heteroaryl, - (CH ₂ ) _p -X-aryl, or (CH ₂ ) _p -X-heteroaryl ;

R ⁴ is C 1 -C 6 alkyl, - (CH ₂ ) _n -aryl, - (CH ₂ ) _n -heteroaryl, - (CH ₂ ) _r X-aryl, or - (CH ₂ ) _r Xheteroaryl;

R ⁵ and R ⁶ are each independently hydrogen, C 1 -C 8 alkyl, - (CH ₂ ) _n -aryl, - (CH ₂ ) _n -heteroaryl, (CH ₂ ) jX-aryl, or - (CH ₂ ) jX-heteroaryl ;

R ⁷ is C 1 -C ₆ -alkyl, - (CH ₂ ) _p- aryl, - (CH ₂ ) _p -heteroaryl, - (CH ₂ ) jX-aryl, or - (CH ₂ ) j-Xheteroaryl;

each n is independently 0 to 6;

each p is independently 1 to 6;

each j is independently 2 to 6;

each m is 0 to 2;

A is alanine, valine, serine, threonine, glutamic acid, lysine, arginine, histidine, glutamine or alpha amino butyric acid;

R ^a is hydrogen, C 1 -C 6 alkyl or - (CH ₂ ) '- phenyl;

X is O or S and

Q is C1-C6 alkyl, - (CH ₂ ) _n -aryl, - (CH ₂ ) _n -heteroaryl, and their pharmaceutically acceptable salts, esters, amides and prodrugs.

In one embodiment of the compounds of formula VII, each R is hydrogen.

In another embodiment, compounds of formula Vlia R ¹

Ο

and m is 0.

In another embodiment of the compounds of formula VII, R ^{1 is}

m is 0 and R ⁷ is - (CH ₂ ) _n -aryl.

In another embodiment of the compounds of formula Vlia Q is - (CH ₂ ) _n -phenyl or (CH ₂ ) _n -naphthyl.

In another embodiment of the compounds of formula VII, R ^{a is} hydrogen or methyl.

In another embodiment of the compounds of formula Vlia Q is -CH ₂ -phenyl, -CH ₂ -naphthyl, -CH ₂ CH ₂ -phenyl, or CH ₂ CH ₂ -naphthyl.

Other hydroxamate ICE inhibitor compounds include compounds of formula VIII

where Z is

Cntn each g is independently hydrogen, C _r C ₆ -alkyl, C _r C ₆ -alkoxy, - (CH ₂ ) _n CO ₂ R, - (CH ₂ ) _n -aryl, aryl, - (CH ₂ ) _n -heteroaryl or -heteroaryl;

U is O or CH ₂ ;

'I ^R5 \ / S or _R 6 of ^m ^ each R is independently hydrogen or a Ci-C ₆ -alkyl;

R ³ is hydrogen, C 1 -C ₆ -alkyl, - (CH ₂ ) _n -aryl, - (CH ₂ ) _n -heteroaryl, - (CH ₂ ) _p -X-aryl, or - (CH ₂ ) _p -X- heteroaryl;

R ⁴ is C 1 -C ₆ alkyl, - (CH ₂ ) '- aryl, - (CH ₂ ) _n -heteroaryl, - (CH ₂ ) jX-aryl, or - (CH ₂ ) j-Xheteroaryl;

R ⁵ and R ⁶ are each independently hydrogen, C 1 -C ₆ -alkyl, - (CH ₂ ) _n -aryl, - (CH ₂ ) _n -heteroaryl, (CH ₂ ) jX-aryl, or - (CH ₂ ) jX- heteroaryl;

R ⁷ is C 1 -C ₆ -alkyl, - (CH ₂ ) _p- aryl, - (CH ₂ ) _p -heteroaryl, - (CH ₂ ) jX-aryl, or - (CH ₂ ) j-Xheteroaryl;

each n is independently 0 to 6;

each p is independently 1 to 6;

each j is independently 2 to 6;

each m is 0 to 2;

A is alanine, valine, serine, threonine, glutamic acid, lysine, arginine, histidine, glutamine or alpha amino butyric acid, and

X is O or S and their pharmaceutically acceptable salts, esters, amides and prodrugs.

In one embodiment of the compounds of formula VIII is Z O

g g and each g is hydrogen.

In another embodiment of the compounds of formula VIII is R or ⁷ o

(A) τη and m is 0.

In one embodiment of the compounds of formula VIII, R ^{1 is} r ⁷ om is 0 and R ⁷ is - (CH ₂ ) _n -aryl.

In one embodiment of the compounds of the formula William R ⁷ - (CH ₂ ) _n -phenyl.

In a preferred embodiment of the compounds of formula VIII is Z

and each g is hydrogen.

Preferred hydroxamate ICE inhibitor compounds include:

3-Benzyloxycarbonyl-amino-4-oxo-5-phenylacetylaminooxy-pentanoic acid; 3-Benzyloxycarbonylamino-4-oxo-5- (2-oxo-pyrrolidin-1-yloxy) -pentanoic acid; 3-Benzyloxycarbonylamino-5- (3,5-dioxo-10-oxa-4-aza-tricyclo [5.2.1.0 ^2,6 ] dec-8-en-4 yloxy) -4-oxo-pentanoic acid;

3-Benzyloxycarbonylamino-5- (2-oxo-2,3-dihydro-indol-1-yloxy) -4-oxo-pentanoic acid;

3-Benzyloxycarbonylamino-5- (7-methoxycarbonylmethyl-2-oxo-octahydro-indole-lyloxy) -4-oxo-pentanoic acid;

3-Benzyloxycarbonylamino-4-oxo-5- (2-oxo-octahydro-indol-1-yloxy) -pentanoic acid;

3- [2- (2-Benzyloxycarbonylamino-3-methyl-butyrylamino) -propionylamino] -5- (7methoxycarbonylmethyl-2-oxo-octahydro-indol-1-yloxy) -4-oxo-pentanoic acid; 3- [2- (2-Benzyloxycarbonylamino-3-methyl-butyrylamino) -propionylamino] -4-oxo-5- (2-oxo-2,3-dihydro-indol-1-yloxy) -pentanoic acid;

3-Benzyloxycarbonylamino-5- (2,5-dioxo-pyrrolidin-1-yloxy) -4-oxo-pentanoic acid;

3-Benzyloxycarbonylamino-4-oxo-5- (2,2,3-trinethyl-5-oxo-pyrrolidin-1-yloxy) pentanoic acid;

3-Benzyloxycarbonylamino-5- (1,3-dioxo-octahydro-isoindol-2-yloxy) -4-oxopentanoic acid;

3-Benzyloxycarbonylamino-5- (1,3-dioxo-1,3-dihydro-isoindol-2-yloxy) -4-oxopentanoic acid;

3-Benzyloxycarbonylamino-5- [3- (4-bromo-phenyl) -2,5-dioxo-2,5-dihydro-pyrrol-lyloxy] -4-oxo-pentanoic acid;

3-Benzyloxycarbonylamino-5- (3,5-dioxo-4-aza-tricyclo [5.2.1.0 ^2,6 ] dec-8-en-4-yloxy) 4-oxo-pentanoic acid;

3-Benzyloxycarbonylamino-5- (2,4-dioxo-3-aza-spiro [5.5] undec-3-yloxy) -4-oxopentanoic acid;

5- (2-Biphenyl-4-yl-5-oxo-pyrrolidin-1-yloxy) -4-oxo-3- (2-propenyl-penta-2,4-dienyloxycarbonyl amino) -pentanoic acid;

5-Benzoylaminooxy-3-benzyloxycarbonylamino-4-oxo-pentanoic acid;

3-Benzyloxycarbonylamino-4-oxo-5- (3-phenyl-propionyl-aninoxy) -pentanoic acid;

3-Benzyloxycarbonylamino-5- (2-naphthalen-1-yl-acetyl-aminooxy) -4-oxo-pentanoic acid;

3-Benzyloxycarbonylamino-5- (3-naphthalen-1-yl-propionylaminooxy) -4-oxo-pentanoic acid;

3-Benzyloxycarbonylamino-5- [methyl- (3-phenyl-propionyl) -aminoxy] -4-oxopentanoic acid;

5- (Benzoyl-methyl-aminooxy) -3-benzyloxycarbonylamino-4-oxo-pentanoic acid; 3-Benzyloxycarbonylamino-5- [methyl- (3-naphthalen-1-yl-propionyl) -aminoxy] -4-oxopentanoic acid;

3-Benzyloxycarbonylamino-5- [methyl- (naphthalen-1-yl-acetyl) -aminoxy] -4-oxopentanoic acid;

3-Benzyloxycarbonylamino-5- [benzyl- (3-phenyl-propionyl) -aminoxy] -4-oxopentanoic acid;

5- [Benzyl- (3-naphthalen-1-yl-propionyl) -aminoxy] -3-benzyloxycarbonylamino-4-21 pentanoic acid;

5- (3-Benzyl-2-oxo-pyrrolidin-1-yloxy) -4-oxo-3- (2-propenyl-penta-2,4-dienyloxycarbonylamino-pentanoic acid;

5- (3-Methyl-2-oxo-pyrrolidin-1-yloxy) -4-oxo-3- (2-propenyl-penta-2,4-dienyloxycarbonylamino) -pentanoic acid;

3-Benzyloxycarbonylamino-4-oxo-5- [methyl- (phenylacetyl) -aminoxy] -pentanoic acid or

3-Benzyloxycarbonylamino-4-oxo-5- (1-oxo-1,3-dihydro-isoindol-2-yloxy) pentanoic acid.

Hydroxamate ICE inhibitors as described above may e.g. prepared as described in Example 12.

Still further types of ICE inhibitors that can be used according to the invention include aspartate ester inhibitors, including compounds of formula IX:

IX where R ¹ R ^{3 is} OC (O) -,

R ³ CO-, r ³ are ₂ -,

R ⁵ N (R ^a ) CHR ⁶ CO-,

Ο

ο · νη ₂ ο

ιι

C ο

ο —c

ιι

C ο

ιι

R each R ^a is independently hydrogen, C 1 -C 6 alkyl or - (CH ₂ ) _n -aryl; R ² is - (CRR) '- aryl,

- (CRR) '- X-aryl,

- (CRR) _n- heteroaryl,

- (CRR) _n -X-heteroaryl,

- (CRR) _n - (substituted heteroaryl), - (CRR) _n - (substituted aryl), - (CRR) _n -X- (substituted aryl),

- (CRR) _n -aryl-aryl,

- (CRR) _n- aryl-heteroaryl,

- (CRR) _n- aryl- (CH ₂ ) _n -aryl,

- (CRR) '- CH (aryl) ₂ ,

- (CRR) '- cycloalkyl,

- (CRR) _n -X-cycloalkyl,

- (CRR) _n -heterocycle,

- (CRR) _n -X-heterocycle,

- (CRR) n-substituted heterocycle,

- (CRR) -CH / CH;) -aryl ^(CH 2) n ^-aryl .

- substituted aryl

- (CRR) - CH (CH ^ -aryl

O (CRR) -N

, (ch ₂ ) /

- (CRR) -CH - heteroaryl ( ^CH 2) -,

Oh

Oh

N-S- [aryl or substituted aryl), 'II O

H

N-CO (CH ₂ ) - [aryl or substituted aryl) O ν ^Λ

N- aru _f

a „γΚ ° (εΗ ₂ ) _η Ητΐι - (CRR) —N

- (CRR) _n CH

or

each R is independently hydrogen, Ci-C ₆ -alkyl, halogen or hydroxy; X is O or S;

R ³ is C 1 -C 6 alkyl, aryl, heteroaryl,

- (CHR) _n -aryl,

- (CHR) _n -heteroaryl,

- (CHR) _n -substituted heteroaryl,

- (CHR) _n -substituted aryl,

- (CRR) _n C (O) ORa,

- (CRR) _n S (CH ₂ ) _n -aryl, cycloalkyl, substituted cycloalkyl, heterocycle, substituted heterocycle,

- (CRR) _n C (O) NR ^a R ^a ,

- (CRR) _n -SO ₂ - (CH ₂ ) '- aryl,

- (CRR) _n -SO ₂ -C _r C ₆ -alkyl,

J-CH ₂ C (R ^a ) H-,

-CHR ⁶ C (O) -heteroaryl,

- (CRR) _n S (CH ₂ ) „C (O) OR ^a ,

- (CRR) _n -SO ₂ - (CH ₂ ) _n C (O) OR ^a ,

- (CRR) _n S (CH ₂ ) _n -aryl,

- (CRR) _n -SO ₂ - (CH ₂ ) _n -aryl,

- (CRR) _n SC (O) C _r C ₆ -alkyl,

- (CRR) _n S (O) (CH ₂ ) _n -aryl,

- (CRR) _n S (O) (CH ₂ ) _n CO ₂ R ^a ,

- (CH ₂ ) _n NHC (O) C _r C ₆ -alkyl

- (CH ₂ ) _n C (O) NR ^b R ^b .

O R

" ^V (CH ₂ )"

ο ιι aryl —S

Oh

II

I '^ jr ^{C_ <CRR,} ' or ^ ril

O = S = O I 0 N. ¹¹ each R is independently C 1 -C 6 alkyl, C 1 -C 8 alkylaryl, aryl or hydrogen;

each J is independently -CO ₂ R ^b ,

-CONR ^b R ^b ,

-SO ₂ NR ^b R ^b or -SO ₂ R ^b ;

each R ^b is independently hydrogen, C 1 -C ₆ -alkyl, aryl, substituted aryl; arylalkyl, heteroarylalkyl, substituted arylalkyl or substituted heteroarylalkyl;

R ⁴ is hydrogen,

C1-C6-alkyl,

CH ₃ OC (O) -,

-phenyl or

C _r C ₆ -alkyl C (O) -;

R ⁵ is C 1 -C 6 -alkyl-CO-,

- (CH ₂ ) _n -aryl,

C _r C ₆ -alkyl-OC (O) -,

Ci-C ₆ -alkyl-X- (CH _{2) n} CO,

C ₁ -C ₆ -alkyl-X- (CH ₂ ) _n OC (O) -,

-C (O) (CRR) '- aryl,

-C (O) NR ^a R ^a ,

-SO ₂ -C 1 -C 6 alkyl,

-C (O) (CH ₂ ) _n C (O) NR ^a R ^a ,

-C (O) O (CH ₂ ) _n -aryl,

-C (O) O (CH ₂ ) _n -substituted aryl,

-C (O) (CRR) _n NHC (O) O (CH ₂ ) _n -aryl,

- (CH ₂ ) _n X (CH ₂ ) _n -aryl, -C 1 -C 6 alkyl-X-C 1 -C 6 alkyl aryl, or

0 0

II II U

-C-CH-NHC-CH-NHCCJ-C6 alkyl

II (CH 2) _n CH 2 - ^heteroaryl I

CO ₂ R ^a

R ^5a is

C (O) C _r C ₆ -alkyl,

-C (O) OC, -C ₆ -alkyl,

Ο Ο β η

-C-CH-NHCCI-C ₆ alkyl (CH ₂ ) _n aryl or substituted aryl,

C (O) O (CH ₂ ) '- aryl,

C (O) (CH ₂ ) _n -aryl or O 0

And! II

CCHNHCCl-C5 alkyl I

CH ₂ heteroaryl

R ⁶ is hydrogen,

C 1 -C 6 alkyl, - (CH ₂ ) '- aryl, - (CH ₂ ) _n CO ₂ R ^a , hydroxyl substituted C 1 -C ₆ alkyl or imidazole substituted C 1 -C ₆ alkyl;

each n is independently 0 to 3, and their pharmaceutically acceptable salts, esters, amides and prodrugs.

In one embodiment of the compounds of formula IX, R ^{1 is} phenyl-CH ₂ -OC (O) -.

In another embodiment of the compounds of formula IX, R ^{1 is} phenyl-SO ₂ -.

In another embodiment of the compounds of formula IX, R ^{1 is} CH ₃ -OC (O) -.

In another embodiment of the compounds of formula IX, R ^{1 is} phenyl-CH ₂ CH ₂ -CO-.

In another embodiment of the compounds of formula IX, R ^{1 is}

In another embodiment of the compounds of formula IX, R ^{1 is}

In another embodiment of the compounds of formula IX, R ^{1 is} phenyl-CH ₂ -CO-.

In another embodiment of the compounds of formula IX, R ^{1 is}

In another embodiment of the compounds of formula IX, each R ^{a is} hydrogen.

In another embodiment of the compounds of formula IX, R ^{2 is} - (CH ₂ ) _n -phenyl.

In one embodiment of the compounds of formula IX, R ^{2 is} - (CH ₂ ) _n- naphthyl.

In another embodiment of the compounds of formula IX, R ^{2 is} - (CH ₂ ) _n -O-phenyl.

In another embodiment, the compound of formula IX is R ² - (CH ₂ ) _n -O-naphthyl.

In another embodiment of the compounds of formula IX, R is - (CH ₂ ) _n -S-phenyl. ·

In another embodiment of the compounds of formula IX, R ^{2 is} - (CH ₂ ) _n -CH- (phenyl) ₂ .

In another embodiment of the compounds of formula IX, each R ^{a is} hydrogen; R ¹ is benzyloxycarbonyl; R ² is aryl-X (CRR) _n -, aryl- (CRR) _n -, heteroaryl- (CRR) _n - or cycloalkyl- (CRR) _n -; n is 1, 2 or 3; X is O or S; and R is hydrogen, methyl or benzyl.

In another embodiment of the compounds of formula IX, each R ^{a is} hydrogen;

R ¹ is benzyloxycarbonyl and R ² is- (CH ₂ ) _n- naphthyl,

- (CH ₂ ) '- phenyl,

- (CH ₂ ) _n -cycloalkyl,

- (CH ₂ ) _n O (CH ₂ ) _n- naphthyl,

- (CH ₂ ) 'O (CH ₂ ) _n -phenyl or - (CH ₂ ) _n S (CH ₂ ) _n -phenyl.

In another embodiment of the compounds of formula IX, each R ^{a is} hydrogen; R ¹ is benzyloxycarbonyl and R ² is -CFk-naphthyl.

In another embodiment of the compounds of formula IX, each R ^{a is} hydrogen; R ² is benzyloxycarbonyl,

Oh

Oh

II-c-CH-NHC-CHNHC-CHNHCC, -C <alkyl,

I \.

CH ^ CH ₃ CH ₂ CH ₂ CO ₂ H ^CH 2 ^heteroaryl- C-CH-NHC-CHNHC CHNHCC, -C 2 -alkyl,

I \, I pu arn Ί

CH ₃ ch ₃ CH ₂ CH ₂ CO ₂ H 2

-C-CH-CH2-S-aryl or I ch ₃ oo

II II

-C-CHCH ₂ -S-aryl.

I II ch ₃ o

Other aspartate ester ICE inhibitor compounds that can be used according to the invention include compounds of formula X:

/ CO ₀ H r ² O

Oh

X where R is ¹

-C (O) OCH ₂ -phenyl,

-SO ₂ -phenyl,

-C (O) OCH ₃ ,

C (O) CH ₂ CH ₂ -phenyl,

O O i «

Č-CHNHCCH3, ch ₃

-C (O) CH ₂ -thienyl,

-C (O) C _r C ₆ -alkyl,

- (CH ₂ ) ₃ -phenyl,

Oh

C-C-CH-NHCOCH ₂ phenyl h ₃ c ^x ch ₃ 0 0

-CCH-NHS-CH3,

I II ch ₃ o

000 II II II

-C-CH-NH-C-CH-NHCCH3, ch ₃ (ch ₂ ) ₂ phenyl

0

-C-CH-NHCCH3, ch ₂ ch ₃

0 0

D II II

-CCHNHC (CH ₂ ) 3CNH ₂ ch ₃

Ο Ο

II II

C-CHCH ₂ S-phenyl,

I β

CH ₃ 0

Ο

-C-CH-NHCNH ₂ , ch ₃ ο ιι

C — CHi ι -ο.

-CCH-C- thienyl,

I ch ₃ ο ο

II β

-c-chch ₂ -s-ch ₃

I i ch ₃ ο ο ο

II β

-C (CH ₂ ) 2CNH ₂ , ο ο

II II

-C (CH ₂ ) ₃ CNH ₂ ,

Ο Ο

II I

-cchch ₂ cnh ₂ , ch ₃

-S (CH ₂ ) _n SCH ₃ ,

In II o o o o

-cchch ₂ sch _3} ch ₃ o

-CCHNHC-CHNHCOCHnphenyl,

I II ² ch ₃ ooo-C-CH-NHCOCH, phenyl

Λ

H ₃ C with ^x CH ₃ phenyl

O = S = O ^{0 0} Λ

II II N —ch ₂ ch ₂ c— / \

0 0

II II II

C-CH-NHC-CHNHCCH ₃ ,

CH ₃ (CH ₂ ) 2

I

C0 ₂ H χ

N-CH-N N-phenyl

I V_7 CH,

II

II

-CCH-SCH ₂ phenyl,

I ch ₃ o

II • c0

X

CH-N N-phenyl

U,

CH

II

-cO

II

Oh

II

CH-NHC-CH-NHCOCH / enyl h ₃ c

CH, (CH ₂ ) ₂ co ₂ h

Oh

II o

II

Oh

II — C — CH — NHC — CH — NHCCHNHCCH, h ₃ c ch ₃

II

OH

Oh

II —c

cnh46

Ο

-C-CH-NHC (CH ₂ ) ₂ phenyl, I

CH ₃

Oh

II

-c • CH — NHC — CH ^{1 x} ch ₂ ch ₃

CH,

Oh

II

-cO

II

Oh

II

CH — NHC— CH —NHCCHNHCCH, h ₃ c

CH,

O O

II II

-C-CH-NHCCH, (CH ₂ ) 2 CH ₂ CO ₂ H imidazoyl h ₃ c

OH

O O

II II

-CCHCH ₂ CNH (CH ₂ ) ₂ phenyl, I ch ₃ o

II o

II

Oh

II

C — CH — NHC — CH — NHCCHNHCCHh ₃ c

CH,

OH

Β

CCH-CH ₂ -S-phenyl,

I ch ₃ oo

B

CCHCH ₂ -S-phenyl,

IB ch ₃ o

II

C-CHCH ₂ -S- (CH ₂ ) ₂ phenyl,

I ch ₃ oo

II II

-C-CH-CH ₂ -S (CH ₂ ) ₂ phenyl,

I II ch ₃ oo

II

-C-CHCH ₂ SCH ₂ phenyl,

I ch ₃

O O

II U

-C-CH-CH ₂ -S (CH ₂ ) ₂ phenyl,

I II ch ₃ ooo »II

-C-CH-SCH ₂ phenyl,

I II ch ₃ ooo-C-CH-NHCCH ₉ CH ₉ phenyl

Λ

Η ₃ σΥΗ ₃

-S-CHCH ₂ SCH ₃ ,

II I II o ch ₃ o

O o

II II

-cchch ₂ scch ₃ , ch ₃ o

II

-cchch ₂ co ₂ h,

I ch ₃

II

-CCHCH ₂ S- (CH ₂ ) ₃ - phenyl,

I ch ₃ oo

II II: C-CH-CH ₂ S- (CH ₂ ) ₃ phenyl, 'I II ch ₃ o

C-CH-CH ₂ S (CH 2) 2 CO ₂ H, ch ₃

O O

II II

C-CHCH2S (CH2) 2CO ₂ H or

I II ch ₃ oo

-CCHCH ₂ S- (CH ₂ ) 2CO ₂ H in

I II ch ₃ o

R ² is

-CH ₂ CH ₂ -phenyl,

-CH ₂ -naphthyl,

-CH ₂ CH ₂ -cyclohexyl,

-CH ₂ O-naphthyl,

-CH ₂ O-phenyl,

-CH ₂ S-phenyl,

-CH ₂ -substituted naphthyl,

-CH ₂ CH (phenyl) ₂ ,

-CH ₂ -imidazole,

- (CH ₂ ) ₃ -phenyl,

-C (CH ₃ ) H-naphthyl,

-CH [CH ₂ -phenyl] ₂ ,

-C (OH) H-naphthyl,

-CH ₂ -NH-phenyl, /

- CH ₉ - CH ⁴ \

CH ₂ substituted phenyl naphthyl

-CH ₂ -naphthyl-phenyl,

-CH ₂ -fluorenyl,

-CH ₂ -naphthyl-thienyl,

Ο II, N-S - substituted phenyl, 0

Oh

II, N-COCH2 phenyl,

-CH ₂ -benzofuranyl, -CH ₂ -benzothienyl, -CH ₂ -naphthyl-CH ₂ -phenyl, -CH ₂ -substituted phenyl,

-CH2- substituted indolyl,

Oh

C- phenyl

Oh

COCHO 1 2 phenyl

Z - CH ₉ - CH ^z \ phenyl

CH ₂ phenyl phenyl

Z

each n is independently O to 3, and pharmaceutically acceptable salts, esters, amides and prodrugs thereof.

Preferred aspartate ester ICE inhibitor compounds include:

3-Benzyloxycarbonylamino-5- (naphthalen-1-yl-acetoxy) -4-oxo-pentanoic acid; 3-Benzyloxycarbonylamino-4-oxo-5- (3-phenyl-propionyloxy) -pentanoic acid; 3-Benzyloxycarbonylamino-5- (3-cyclohexyl-propionyloxy) -4-oxo-pentanoic acid;

3-Benzyloxycarbonylamino-5 - [(naphthalen-1-yl-oxy) -acetoxy] -4-oxo-pentanoic acid;

-benzyloxycarbonylamino-4-oxo-5-phenoxyacetoxy-pentanoic acid; 3-bezyloxycarbonylamino-4-oxo-5-phenylsulfanylacetoxy-pentanoic acid; 3-Benzyloxycarbonylamino-5 [(6-methoxy-naphthalen-1-yl) -acetoxy] -4-oxo-pentanoic acid;

3-Benzyloxycarbonylamino-5- (naphthalen-2-yl-acetoxy) -4-oxo-pentanoic acid;

3-Benzyloxycarbonylamino-5- (3-naphthalen-2-yl-propionyloxy) -4-oxo-pentanoic acid;

-benzyloxycarbonylamino-5- (3,3-diphenyl-propionyloxy) -4-oxo-pentanoic acid; 3-Benzyloxycarbonylamino-5 - [(1H-indol-3-yl) -acetoxy] -4-oxo-pentanoic acid;

-benzyloxycarbonylamino-5- (indol-1-yl-acetoxy) -4-oxo-pentanoic acid;

3-Benzyloxycarbonylamino-5- (2-naphthalen-1-yl-propiomyloxy) -4-oxo-pentanoic acid;

3-Benzyloxycarbonylamino-4-oxo-5 - [(2-oxo-pyrrolidin-1-yl) -acetoxy] -pentanoic acid;

5 - [(acetyl-phenyl-amino) -acetoxy] -3-benzyloxycarbonyl-amino-4-oxo-pentanoic acid;

3-Benzyloxycarbonylamino-5- (2-benzyl-3-phenyl-propionyloxy) -4-oxo-pentanoic acid;

3-Benzyloxycarbonylamino-5- (hydroxy-naphthalen-1-yl-acetoxy) -4-oxo-pentanoic acid;

3-Benzyloxycarbonylamino-4-oxo-5 - [(phenyl-amino) -acetoxy] -pentanoic acid;

3-Benzyloxycarbonylamino-5 - [(6-hydroxy-naphthalen-1-yl) -acetoxy] -4-oxo-pentanoic acid;

3-Benzyloxycarbonylamino-5- [3- (4-hydroxy-phenyl) -2-naphthalen-1-yl-propionyloxy) -4-oxo-pentanoic acid;

(S) -3-Benzyloxycarbonylamino-4-oxo-5-phenylacetoxy-pentanoic acid; (S) -3-Benzyloxycarbonylamino-4-oxo-5- (4-phenyl-butyryloxy) -pentanoic acid; 3-Benzyloxycarbonylamino-4-oxo-5 - [(4-phenyl-naphthalen-1-yl) -acetoxy] -pentanoic acid;

3-Benzyloxycarbonylamino-5 - [(4-methyl-naphthalen-1-yl) -acetoxy] -4-oxo-pentanoic acid;

3-Benzyloxycarbonylamino-4-oxo-5 - [(4-thiophen-2-yl-naphthalen-1-yl) -acetoxy] pentanoic acid;

3-Benzyloxycarbonylamino-5 - [(4-fluoro-naphthalen-1-yl) -acetoxy] -4-oxo-pentanoic acid;

3-Benzyloxycarbonylamino-5 - [(2-methyl-naphthalen-1-yl) -acetoxy] -4-oxo-pentanoic acid;

3-Benzyloxycarbonylamino-5 - [(2-fluoro-N-naphthalen-1-yl) -acetoxy] -4-oxo-pentanoic acid;

5- (Benzofuran-4-yl-acetoxy) -3-benzyloxycarbonylamino-4-oxo-pentanoic acid;

5- (Benzo [b] thiophen-7-yl-acetoxy) -3-benzyloxycarbonylamino-4-oxo-pentanoic acid;

5- (Benzo [b] thiophen-4-yl-acetoxy) -3-benzyloxycarbonylamino-4-oxo-pentanoic acid;

5 - [(4-Benzyl-naphthalen-1-yl) -acetoxy] -3-benzyloxycarbonylarnino-4-oxo-pentanoic acid;

3-Benzyloxycarbonylamino-5 - [(3,4-dihydro-naphthalen-1-yl) -acetoxy] -4-oxo-pentanoic acid;

3-Benzyloxycarbonylamino-5 - [(5-bromo-1H-indol-3-yl) -acetoxy] -4-oxo-pentanoic acid;

3-Benzyloxycarbonylamino-5- (3,4-diphenyl-butyryloxy) -4-oxo-pentanoic acid;

-benzyloxycarbonylamino-4-oxo-5- (3-phenyl-3-phenylamino-propionyloxy) -pentanoic acid;

3-Benzyloxycarbonylamino-4-oxo-5 - [(1,2,3,4-tetrahydro-naphthalen-2-yl) -acetoxy] pentanoic acid;

3-Benzyloxycarbonylamino-5 - [(1-methanesulfonyl-piperidin-4-yl) -acetoxy] -4-oxopentanoic acid;

3-benzyloxycarbonylamino-4-oxo-5 - [(2 ₇ 3,5,6-tetramethyl-phenyl) acetoxy] -pentanoic acid;

5- (Benzothiazol-4-yl-acetoxy) -3-benzyloxycarbonylamino-4-oxo-pentanoic acid;

5- (Benzofuran-3-yl-acetoxy) -3-benzyloxycarbonylamino-4-oxo-pentanoic acid;

5- (Benzo [b] thiophen-3-yl-acetoxy) -3-benzyloxycarbonylamino-4-oxo-pentanoic acid;

3-Benzyloxycarbonylamino-4-oxo-5- (3-phenyl-3-pyridin-2-yl-propionyloxy) -pentanoic acid;

3-Benzyloxycarbonylamino-5 - [(2,3-dichloro-phenyl) -acetoxy] -4-oxo-pentanoic acid;

3-Benzyloxycarbonylamino-5 - [(5-methyl-naphthalen-1-yl) -acetoxy] -4-oxo-pentanoic acid;

3-Benzyloxycarbonylamino-5 - [(2-iodo-phenyl) -acetoxy] -4-oxo-pentanoic acid;

3-Benzyloxycarbonylamino-4-oxo-5- (3-pyridin-3-yl-propionyloxy) -pentanoic acid;

3-Benzyloxycarbonylamino-5 - [(5-methoxy-naphthalen-1-yl) -acetoxy] -4-oxo-pentanoic acid;

3-Benzyloxycarbonylamino-5 - [(8-methyl-naphthalen-1-yl) -acetoxy] -4-oxo-pentanoic acid;

3-Benzyloxycarbonylamino-5 - [(9H-fluoren-9-yl) -acetoxy] -4-oxo-pentanoic acid; 3-benzyloxycarbonylamino-5 - [(10 _{J-dihydro-5H-dibenzo} [a, d] cyclohepten-5-yl) acetoxy] -4-oxo-pentanoic acid;

5-Oxo-1- (toluene-4-sulfonyl) -pbolidine-2-carboxylic acid 3-benzyloxycarbonylamino-4-carboxy-2-oxo-butyl ester;

5-Oxo-pyrrolidine-1,2-dicarboxylic acid 1-benzyl ester 2- (3-benzyloxycarbonylamino-4-carboxy-2-oxo-butyl) -ester;

-benzoyl-pyrrolidine-2-carboxylic acid 3-benzyloxycarbonylamino-4-carboxy-2-oxo-butyl ester;

pyrrolidine-1,2-dicarboxylic acid 1-benzyl ester 2- (3-benzyloxycarbonylamino-4carboxy-2-oxo-butyl) ester;

3-Benzyloxycarbonylamino-5- (2-benzyl-3-phenyl-propionyloxy) -4-oxo-pentanoic acid;

3-Benzyloxycarbonylamino-5 - [(5-cyano-naphthalen-1-yl) -acetoxy] -4-oxo-pentanoic acid;

3-Benzyloxycarbonylamino-4-oxo-5- (3-phenyl-3-pyridin-3-yl-propionyloxy) -pentanoic acid;

3-Benzyloxycarbonylamino-4-oxo-5- (3-phenyl-3-pyridin-4-yl-propionyloxy) -pentanoic acid; and

3-Benzyloxycarbonylamino-4-oxo-5 - [(1-oxo-3,4-dihydro-1H-isoquinolin-2-yl) acetoxy-pentanoic acid;

3-Benzenesulfonylainino-5- (naphthalen-1-yl-acetoxy) -4-oxo-pentanoic acid;

-methoxycarbonylamino-5- (naphthalen-1-yl-acetoxy) -4-oxo-pentanoic acid; 5- (Naphthalen-1-yl-acetoxy) -4-oxo-3- (3-phenyl-propionylamino) -pentanoic acid; 3-methoxycarbonylamino-4-oxo-5-phenoxyacetoxy-pentanoic acid; and 3- (2-methanesulfonyl-1-methyl-ethylsulfanylamino) -5- (naphthalen-1-yl-acetoxy) -4-oxopentanoic acid;

[S- (R *, R *)] - 3- (2-acetylamino-propionylamino) -5- (naphthalen-1-yl-acetoxy) -4-oxopentanoic acid;

5- (Naphthalen-1-yl-acetoxy) -4-oxo-3 - [(thiophene-3-carbonyl) -amino] -pentoic acid;

- [(furan-3-carbonyl) -amino] -5- (naphthalen-1-yl-acetoxy) -4-oxo-pentanoic acid;

5- (Naphthalen-1-yl-acetoxy) -4-oxo-3- [2- (4-phenyl-butyrylamino) -propylamino] -pentanoic acid;

3- (2-Methanesulfonylamino-propionylamino) -5- (naphthalen-1-yl-acetoxy) -4-oxopentanoic acid;

3- [2- (2-Acetylamino-4-phenyl-butyrylamino) -propionylamino] -5- (naphthalen-1-yl-acetoxy) -4-oxo-pentanoic acid;

3- (2-acetylamino-butyrylamino) -5- (naphthalen-1-yl-acetoxy) -4-oxo-pentanoic acid; 3- [2- (4-Carbamoyl-butyrylamino) -propionylamino] -5- (naphthalen-1-yl-acetoxy) -4-oxopentanoic acid;

3- (2-Benzyloxycarbonylamino-propionylamino) -5- (naphthalen-1-yl-acetoxy) -4-oxopentanoic acid;

5- (Naphthalen-1-yl-acetoxy) -4-oxo-3- (2-ureido-propionylamino) -pentanoic acid;

3- (2-acetylamino-propionylamino) -5- (naphthalen-1-yl-acetoxy) -4-oxo-pentanoic acid;

3 - [(1-acetyl-pyrrolidine-2-carbonyl) -amino] -5- (naphthalen-1-yl-acetoxy) -4-oxopentanoic acid;

3- (2-methyl-3-oxo-3-thiophen-2-yl-propionylamino) -5- (naphthalen-1-yl-acetoxy) -4-oxopentanoic acid;

3- (2-acetylamino-acetylamino) -5- (naphthalen-1-yl-acetoxy) -4-oxo-pentanoic acid;

- (2-acetylamino-propionylamino) -5- (3,3-diphenyl-propionyloxy) -4-oxo-pentanoic acid;

3- [2- (2-Acetylanoic-4-carboxy-butyrylamino) propionylamino] -5- (naphthalen-1-ylacetoxy) -4-oxo-pentanoic acid;

5- (Naphthalen-1-yl-acetoxy) -4-oxo-3- [2- (3-phenyl-propionylamino) -propionylamino] pentanoic acid;

3- [2- (3-Methyl-butyrylamino) -propionylamino] -5- (naphthalen-1-yl-acetoxy) -4-oxopentanoic acid;

3 - [(1-acetyl-4-benzyloxy-pyrrolidine-2-carbonyl) -amino] -5- (naphthalen-1-yl-acetoxy) -4-oxo-pentanoic acid;

3- (4-Carbamoyl-butyrylamino) -5- (naphthalen-1-yl-acetoxy) -4-oxo-pentanoic acid; and 3- [2- (1-methyl-1H-imidazol-4-yl) -acetylamino] -5- (naphthalen-1-yl-acetoxy) -4-oxopentanoic acid;

(S) -5- (Naphthalen-1-yl-acetoxy) -4-oxo-3-phenylacetylamino-pentanoic acid; (S) -5- (Naphthalen-1-yl-acetoxy) -4-oxo-3- (2-thiophen-2-yl-acetylamino) -pentanoic acid; 3 - [(2-Carbamoyl-cyclopentanecarbonyl) -anino] -5- (naphthalen-1-yl-acetoxy) -4-oxopentanoic acid;

3 - [(3-Carbamoyl-bicyclo [2.2.1] heptane-2-carbonyl) -amino] -5- (naphthalen-1-yl-acetoxy) -4-oxo-pentanoic acid;

3- (3-Methanesulfonyl-2-methyl-propionylamino) -5- (naphthalen-1-yl-acetoxy) -4-oxopentanoic acid;

3- (3-Benzenesulfonyl-2-methyl-propionylamino) -5- (naphthalen-1-yl-acetoxy) -4-oxopentanoic acid;

-butyrylamino-5- (naphthalen-2-yl-acetoxy) -4-oxo-pentanoic acid;

3-acetylamino-5- (naphthalen-1-yl-acetoxy) -4-oxo-pentanoic acid;

3- (3-Methanesulfonyl-2-methyl-propionylamino) -5- (naphthalen-1-yl-acetoxy) -4-oxopentanoic acid;

3- (3-methyl-butyrylamino) -5- (naphthalen-1-yl-acetoxy) -4-oxo-pentanoic acid; 3- (3-Carbamoyl-propionylamino) -5- (naphthalen-1-yl-acetoxy) -4-oxo-pentanoic acid; [S- (R, R)] -3- (3-acetylsulfanyl-2-methyl-propionylamino) -5- (naphthalen-1-yl-acetoxy) -4-oxo-pentanoic acid; and N &apos; 5-3 - [(3-Carbamoyl-cyclopentanecarbonyl) -amino] -5- (naphthalen-1-yl-acetoxy) -4-oxopentanoic acid

3- (1,2,3,4-tetrahydro-1-oxo-isoquinolin-2-yl) -acetamino-5- (naphthalen-1-yl-acetoxy) -4-oxo-pentanoic acid;

3- (2-methyl-3-phenethylcarbamoyl-propionylamino) -5- (naphthalen-1-yl-acetoxy) -4-oxopentanoic acid;

5- (Naphthalen-2-yl-acetoxy) -4-oxo-3- [2- (2-oxo-6-phenyl-piperidin-1-yl) -acetylamino] pentanoic acid;

5- (Naphthalen-1-yl-acetoxy) -4-oxo-3- [2- (2-oxo-6-phenyl-piperidin-1-yl) -acetylamino] pentanoic acid;

3- [3-methyl-2- (3-phenyl-propionylamino) -butyrylamino] -4-oxo-5 - [(1-oxo-1,2,3,4tetrahydro-naphthalen-2-yl) -acetoxy] - pentanoic acid; 5- (Naphthalen-2-yl-acetoxy) -4-oxo-3- [2- (1-oxo-3,4-dihydro-1H-isoquinolin-2-yl) acetylamino-pentanoic acid;

5- (2-Benzyl-3-phenyl-propionyloxy) -4-oxo-3- [2- (1-oxo-3,4-dihydro-1H-isoquinolin-2yl) -acetylamino] -pentanoic acid;

5- (2-Benzyl-3-phenyl-propionyloxy) -4-oxo-3- [2- (2-oxo-6-phenyl-piperidin-1-yl) acetylamino] -pentanoic acid;

5- (Naphthalen-1-yl-acetoxy) -4-oxo-3- [2- (1-oxo-1,2,3,4-tetrahydro-naphthalen-2-yl) acetylamino] -pentanoic acid;

5- (Naphthalen-1-yl-acetoxy) -4-oxo-3- [2- (1-oxo-3,4-dihydro-1H-isoquinolin-2-yl) propiolaminic-pentanoic acid;

5- (Naphthalen-2-yl-acetoxy) -4-oxo-3- [2- (1-oxo-3,4-dihydro-1H-isoquinolin-2-yl) propionylamino-petanoic acid;

3- [4- (1-Benzenesulfonyl-1H-pyrrol-2-yl) -4-oxo-butyrylamino] -5- (naphthalen-1-yl-acetoxy) 4-oxo-pentanoic acid;

5- (2-Benzyl-3-phenyl-propionyloxy) -4-oxo-3- [2- (1-oxo-1,2,3,4-tetrahydronaphthalen-2-yl) acetylamino-pentanoic acid;

5- (2-Benzyl-3-phenyl-propionyloxy) -4-oxo-3- [2- (1-oxo-3,4-dihydro-1H-isoquinolin-2yl) -propionylamino] -pentanoic acid;

4-oxo-3- [2- (1-oxo-3,4-dihydro-1H-isoquinolin-2-yl) -propionylamino] -5 - [(1-oxole, 2,3,4-tetrahydro-naphthalene) -2-yl) -acetoxy] -pentanoic acid;

- [4- (1-Benzenesulfonyl-1H-pyrrol-2-yl) -4-oxo-butyrylamino] -5- (2-benzyl-3-phenylpropionyloxy) -4-oxo-pentanoic acid;

4- oxo-5 - [(1-oxo-1,2,3,4-tetrahydro-naphthalen-2-yl) -acetoxy] -3- [2- (1-oxo-1,2,3,4tetrahydro- Naphthalen-2-yl) -acetylamino] -pentanoic acid;

5- (Naphthalen-1-yl-acetoxy) -4-oxo-3- [2- (2-oxo-3-phenyl-imidazolidin-1-yl) propionylamino] -pentanoic acid;

5- (Naphthalen-1-yl-acetoxy) -4-oxo-3- [2- (2-oxo-3-phenyl-tetrahydropyrimidin-1-yl) propioylamino] -pentanoic acid;

5- (Naphthalen-1-yl-acetoxy) -4-oxo-3- [2- (2-oxo-3-phenyl-tetrahydropyrimidin-1-yl) acetylamino] -pentanoic acid;

- (2-acetylamino-3-methylbutyrylamino) -5- (naphthalen-1-yl-acetoxy) -4-oxo-pentanoic acid;

3- (2-acetylamino-3-methyl-butyrylamino) -5- (2-benzyl-3-phenylpropionyloxy) -4-oxopentanoic acid;

3- (2-acetylamino-3-methylbutyrylamino) -5- (3-benzyl-4-phenylbutyryloxy) -4-oxopentanoic acid;

3- (2-acetylamino-3-methyl-butyrylamino) -5- (4-benzyl-5-phenyl-pentanoyloxy) -4-oxopentanoic acid;

3- (2-acetylamino-3-methyl-butyrylamino) -4-oxo-5 - [(1-oxo-1,2,3,4-tetrahydro-naphthalene)

2-yl) -acetoxy] -pentanoic acid;

5- (3-Benzyl-4-phenyl-butyryloxy) -3- [3-methyl-2- (3-phenyl-propionylamino) -butyrylamino] -4-oxo-pentanoic acid;

3- [2- (3-acetylamino-2-oxo-2H-pyridin-1-yl) -acetylamino] -5- (3,3-diphenylpropionyloxy) -4-oxo-pentanoic acid; and

3- [2- (3-acetylamino-2-oxo-2H-pyridin-1-yl) -acetylamino] -5- (2-benzyl-3-phenylpropionyloxy) -4-oxo-pentanoic acid;

3- [2- (2-Benzyloxycarbonylamino-4-carboxy-butyrylamino) -3-methyl-butyrylanilino] -5 (naphthalen-1-yl-acetoxy) -4-oxo-pentanoic acid;

3- [2- (2-Benzyloxycarbonylamino-3-methyl-butyrylanilino) -propionylamino] -5- (naphthalen-1-yl-acetoxy) -4-oxo-pentanoic acid;

3- (2-acetylamino-3-methyl-butyrylamino) -5- (naphthalen-1-yl-acetoxy) -4-oxo-pentanoic acid;

3- [2- (2-Benzyloxycarbonylamino-3-methyl-butyrylamino) -propionylamino] -5- (3,3-diphenyl propionyloxy) -4-oxo-pentanoic acid;

3- [2- (2-Benzyloxycarbonylamino-3-methyl-butyrylamino) -pyropionylamino] -5- (2-benzyl-3 · phenyl-propionyloxy) -4-oxo-pentanoic acid;

3- [2- (2-Benzyloxycarbonylamino-3-methyl-butyrylamino) -propionylamino] -5- (naphthalen-1-yl-acetoxy) -4-oxo-pentanoic acid;

5- (2-Benzyl-3-phenyl-propionyloxy) -3- {2- [4-carboxy-2- (3-phenyl-propionylamino) butyrylanilino] -3-methyl-butyrylamino} -4-oxo-pentanoic acid ; 3- (2-Benzyloxycarbonylamino-3-methyl-butyrylamino) -5- (3,3-diphenyl-propiomyloxy) -4-oxo-pentanoic acid;

- (2-acetylamino-3-hydroxy-butyrylamino) -5- (naphthalen-1-yl-acetoxy) -4-oxopentanoic acid;

3- (2-acetylamino-3-hydroxy-butyrylamino) -5- (3,3-diphenyl-propionyloxy) -4-oxopentanoic acid;

3- (2- {2- [2-acetylamino-3- (1H-indol-3-yl) -proponylamino] -4-carboxy-butyrylane ]ino} -3methyl-butyrylanilino) -5- (2-benzyl-3 -phenyl-pyrroloxy) -4-oxo-pentanoic acid; and 5- (3,3-diphenyl-propionyloxy) -4-oxo-3- [2- (4-phenyl-butyrylamino) -proponylamino] pentanoic acid;

3- (2- (2- [2-acetylamino-3- (1H-indol-3-yl) -proponylamino] -4-carboxy-butyrylamino} -3-methyl-butyrylamino) -5- (naphthalen-1-yl- acetoxy) -4-oxo-pentanoic acid; and 3- (2- (2- [2- [acetylamino-3- (4-hydroxy-phenyl) -proponylamino] -4-carboxy-butyrylamino} 3-methyl-butyrylamino) - 5- (Naphthalen-1-yl-acetoxy) -4-oxo-pentanoic acid; 3 - [(2-carboxy-cyclohexanecarbonyl) -amino] -5- (naphthalen-1-yl-acetoxy) -4-oxopentanoic acid;

3 - [(2-methoxycarbonyl-cyclohexanecarbonyl) -amino] -5- (naphthalen-1-yl-acetoxy) -4-oxopentanoic acid; and

3 - [(2-Carbamoyl-cyclohexanecarbonyl) -amino] -5- (naphthalen-1-yl-acetoxy) -4-oxopentanoic acid;

3- (3-Benzylsulfanyl-2-methyl-propionylamino) -5- (naphthalen-1-yl-acetoxy) -4-oxopentanoic acid;

3- (2-methyl-3-phenylmethanesulfonyl-propionylanino) -5- (naphthalen-1-yl-acetoxy) -4-oxopentanoic acid;

3- [3- (2-Carboxy-ethanesulfanyl) -2-methyl-propionylamino] -5- (naphthalen-1-yl-acetoxy) -4-oxo-pentanoic acid;

5- (2-Benzyl-3-phenyl-propionyloxy) -3- [3- (2-carboxy-ethanesulfonyl) -2-methylpropionylamino] -4-oxo-pentanoic acid;

5- (2-Benzyl-3-phenyl-propionyloxy) -3- [3- (3-carboxy-propane-1-sulfinyl) -2-methylpropionylamino] -4-oxo-pentanoic acid;

5- (Naphthalen-1-yl-acetoxy) -4-oxo-3- (2-phenylmethanesulfanyl-propionylamino) -pentanoic acid;

3- (2-methyl-3-phenylsulfanyl-propionylamino) -5- (naphthalen-1-yl-acetoxy) -4-oxopentanoic acid;

5- (2-Benzyl-3-phenyl-propionyloxy) -3- (2-methyl-3-phenylsulfanyl-propionylamino) -4-oxopentanoic acid;

3- (2-methyl-3-phenethylsulfanyl-propionylamino) -5- (naphthalen-1-yl-acetoxy) -4-oxopentanoic acid;

5- (2-Benzyl-3-phenyl-propionyloxy) -3- (2-methyl-3-phenethylsulfanyl-propionylamino) -4-oxopentanoic acid;

5- (2-Benzyl-3-phenyl-propionyloxy) -3- (3-benzylsulfanyl-2-methyl-propionylamino) -4-oxopentanoic acid;

5- (2-Benzyl-3-phenyl-propionyloxy) -3- (2-benzylsulfanyl-propionylamino) -4-oxopentanoic acid;

3- [2-methyl-3- (3-phenyl-propylsulfanyl) -propionylamino] -5- (naphthalen-1-yl-acetoxy) -4-oxo-pentanoic acid;

3- (3-Benzenesulfonyl-2-methyl-propionylamino) -5- (naphthalen-1-yl-acetoxy) -4-oxopentanoic acid;

3- (3-Benzenesulfonyl-2-methyl-propionylamino) -5- (2-benzyl-3-phenyl-propionyloxy) -4-oxo-pentanoic acid;

5- (2-Benzyl-3-phenyl-propionyloxy) -3- [2-methyl-3- (2-phenyl-ethanesulfonyl) -propionylamino] 4- oxo-pentanoic acid;

3- [2-methyl-3- (2-phenyl-ethanesulfonyl) -propionylamino] -5- (naphthalen-1-yl-acetoxy) -4-oxopentanoic acid;

5- (Naphthalen-1-yl-acetoxy) -4-oxo-3- (2-phenylmethanesulfonyl-propionylamino) -pentanoic acid;

5- (2-Benzyl-3-phenyl-propionyloxy) -3- (2-methyl-3-phenylmethanesulfonyl-propionylamino) -4-oxo-pentanoic acid;

5- (2-Benzyl-3-phenyl-propionyloxy) -4-oxo-3- (2-phenylmethanesulfonyl-propionylamino) pentanoic acid;

3- [2-methyl-3- (3-phenyl-propane-1-sulfonyl) -propionylamino] -5- (naphthalen-1-yl-acetoxy) -4-oxo-pentanoic acid;

5- (2-Benzyl-3-phenyl-propionyloxy) -3- [2-methyl-3- (3-phenyl-propane-1-sulfonyl) propionylamino] -4-oxo-pentanoic acid;

5- (2-Benzyl-3-phenyl-propionyloxy) -3- [3- (2-carboxy-ethylsulfanyl) -2-methylpropionylamino] -4-oxo-pentanoic acid;

3- [3- (3-Carboxy-propylsulfanyl) -2-methyl-propionylamino] -5- (naphthalen-1-yl-acetoxy) -4-oxo-pentanoic acid;

5- (2-Benzyl-3-phenyl-propionyloxy) -3- [3- (3-carboxy-propylsulfanyl) -2-methylpropionylamino] -4-oxo-pentanoic acid;

3- (3-carboxymethylsulfanyl-2-methyl-propionylamino) -5- (naphthalen-1-yl-acetoxy) -4-oxopentanoic acid;

5- (2-Benzyl-3-phenyl-propionyloxy) -3- (3-carboxymethylsulfanyl-2-methyl-propionylamino) 4- oxo-pentanoic acid;

3- [3- (2-Carboxy-ethanesulfonyl) -2-methyl-propionylamino] -5- (naphthalen-1-yl-acetoxy) -4-oxo-pentanoic acid;

3- [3- (3-Carboxy-propane-1-sulfonyl) -2-methyl-propionylamino] -5- (naphthalen-1-ylacetoxy) -4-oxo-pentanoic acid;

3- (3-carboxyethanesulfonyl-2-methyl-propionylainino) -5- (naphthalen-1-yl-acetoxy) -4-oxopentanoic acid;

5- (2-Benzyl-3-phenyl-propionyloxy) -3- [3- (3-carboxy-propane-1-sulfonyl) -2-methylpropionylamino] -4-oxo-pentanoic acid;

5- (2-Benzyl-3-phenyl-propionyloxy) -3- (3-carboxymethanesulfonyl-2-methylpropionylamino) -4-oxo-pentanoic acid;

3- [3- (3-Carboxy-propane-1-sulfinyl) -2-methyl-propionylamino] -5- (naphthalen-1-yl-acetoxy)

4- oxo-pentanoic acid;

3- [2-methyl-3- (3-phenyl-propane-1-sulfinyl) -propionylamino] -5- (naphthalen-1-yl-acetoxy) -4-oxo-pentanoic acid; and

5- (2-Benzyl-3-phenyl-propionyloxy) -3- [2-methyl-3- (3-phenyl-propane-1-sulfonyl) propionylamino] -4-oxo-pentanoic acid;

3- [3-methyl-2- (phenethylcarbamoyl-methyl) -butyrylamino] -5- (naphthalen-1-yl-acetoxy) -4-oxo pentanoic acid; and

3- (3-carboxy-2-methyl-propionylamino) -5- (naphthalen-1-yl-acetoxy) -4-oxo-pentanoic acid;

3- (2-methyl-3-sulfamoyl-propionylamino) -5- (naphthalen-1-yl-acetoxy) -4-oxo-pentanoic acid;

3- (3-Carbamoyl-2-methyl-propionylamino) -5- (naphthalen-1-yl-acetoxy) -4-oxo-pentanoic acid;

- (2-Benzyloxycarbonylamino-3-methyl-naphthalen-1-yl-acetoxy) -4-oxo-pentanoic acid;

3 - [(2-Carbamoyl-cyclopentanecarbonyl) -amino] -5- (naphthalen-1-yl-acetoxy) -4-oxopentanoic acid;

3 - [(1-Carbamoyl-pyrrolidine-2-carbonyl) -aminol-5- (naphthalen-1-yl-acetoxy) -4-oxopentanoic acid;

3- (2- {2- [2-acetylamino-3- (4-hydroxy-phenyl) -propionylamino] -4-carboxybutylamino} 3-methyl-butyrylamino) -5- (2-benzyl-3-phenyl-propionyloxy) -4-Oxo-pentanoic acid; 3- (3-Carbamoyl-2-methyl-propionylamino) -5- (naphthalen-1-yl-acetoxy) -4-oxo-pentanoic acid;

3- (2-Carbamoylmethyl-3-methyl-butyrylamino) -5- (naphthalen-1-yl-acetoxy) -4-oxopentanoic acid;

3- (3-Benzyloxy-2-ureido-propionylamino) -5- (naphthalen-1-yl-acetoxy) -4-oxopentanoic acid;

3- [2- (2-Benzyloxycarbonylamino-4-carboxy-butyrylamino) -3-methyl-butynlamino] -5- (2-benzyl-3-phenyl-propionyloxy) -4-oxo-pentanoic acid;

- {2- [4-carboxy-2- (3-phenyl-propionylamino) -butyrylamino] -3-methyl-butyrylamino} -5 (naphthalen-1-yl-acetoxy) -4-oxo-pentanoic acid; and

3- [2- (2-Acetylamino-4-carboxy-butyrylamino) -3-methyl-butyrylamino] -5- (naphthalen-1-ylacetoxy) -4-oxo-pentanoic acid.

The aspartate ester ICE inhibitors described above can be made according to Schemes 1 below:

Level A rco ₂ h, base (KF or K ₂ CO ₃ ) DMF

3-Benzyloxycarbonylamino-5-bromo-4-oxo-pentanoic acid tert-butyl ester, also known as Z-Asp (OtBu) -bromomethyl ketone, can be commercially available or prepared by the process of: Dolle et al., J. Med. Chem., 1994: 37: 563-564. The methyl bromo ketone is treated with a suitably substituted carboxylic acid and a base such as e.g. with potassium fluoride. Alternatively, other bases could be used, such as. potassium carbonate, cesium carbonate or potassium t-butoxide. The reagents should be mixed in dimethyl formamide (DMF), dimethylacetamide (DMA), dimethyl sulfoxide (DMSO), acetonitrile or other suitable solvent and then stirred at room temperature for 8 to 24 hours. The t-butyl ester protecting group may be removed in an acidic medium, preferably trifluoroacetic acid, to produce the carbobenzoxy aspartyl esters shown in Scheme 1.

bb

Scheme 2

or

ROCOCI

A mixture of the appropriately substituted acyloxymethyl ketone carbobenzoxy aspartyl t-butyl ester is hydrogenated with an equivalent of hydrochloric or other acid in the presence of a catalyst, such as e.g. palladium on charcoal to give amine salt. The salt may be acylated with suitably substituted isocyanate, sulfonyl chloride, chloroformate or phenyl propionyl chloride to give N-substituted derivatives. These isocyanates, sulfonyl chlorides or chloro formates can be commercially available or synthesized by methods described in the chemical literature, and the t-butyl ester protecting group can be ultimately eliminated using an acidic medium, preferably trifluoroacetic acid, to produce the acyloxymethyl ketone derivatives shown in the shemi derivatives 2.

Scheme 3

Level A

HCI · H ₂ N

CO ₂ ¹ Bu coupling reagent HOBT, base

Level B cf ₃ co ₂ h

R

The amine salt of acyloxymethyl ketone Z-Asp (Ot-Bu) OH is synthesized and treated with the appropriately substituted carboxylic acid and the coupling reagent. Coupling reagents may, but are not limited to, for example: 1,3-dicyclohexylcarbodiimide (DCC), 1 (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDCI), Ι, Γ-carbonyldiimidazole (CDI), 1,1'-carbonylbis ( 3-methylimidazolium) triflate (CBMIT), isobutylchloroformate, benzotriazol-1-yloxytris (dimethylamino) -phosphonium hexafluorophosphate (BOP), 2- (3,4-dihydro-4-oxo-1,2,3-benzotriazin-3-yl) - 1,1,3,3-tetramethyluronium tetrafluoroborate (TDBTU) and 2- (1H-benzotriazol-1-yl) -1,3,3,3-tetramethyluronium hexafluorophosphate (HBTU). 1-Hydroxybenzotriazole hydrate should be added to the reaction mixture to improve yield and limit the isomerization, base, preferably amine, e.g. such as trimethylamine or methyl morpholine, however, should be added as an acid remover. The resulting amide product is treated with an acidic medium, preferably trifluoroacetic acid, to remove the t-butyl ester and produce the final products as shown in Scheme 3.

Level A

ΖσΟΧ (X = C1, F) - -----— ►

Level B cf ₃ co ₉ h --— ►

The amine salt of acyloxymethyl ketone Cbz-Asp (OtBu) OH is synthesized and treated with appropriately substituted acid chloride or acid fluoride to produce the amide product. Acid chlorides are commercially available or prepared by treating carboxylic acids with agents such as e.g. thionyl chloride, phosphorus tribromide or oxalyl chloride / DMF. Acid fluorides are prepared by treating the carboxylic acid with cyanuric acid fluoride. The penultimate amide product is treated with an acidic medium, preferably trifluoroacetic acid, to remove the t-butyl ester to give the final products as shown in Scheme 4.

Scheme 5

Level A

1. idcc ^ H, coupling reagent

------ 2. NaOH

R

Level B Level C

The hydrochloride salt of H-Asp (OtBu) OMe is treated with the appropriately substituted carboxylic acid and the coupling reagent. 1-Hydroxybenzotriazole hydrate should be added to the reaction mixture to improve yield and limit the isomerization, base, preferably amine, such as e.g. trimethylamine or methylmorpholine, however, should be added as an acid scavenger. The resulting amide product is treated with an alkaline reagent such as e.g. sodium hydroxide to hydrolyze the methyl ester into the carboxylic acid. The resulting acid is treated with chloroformate, such as e.g. isobutyl chloroformate followed by diazomethane and then hydrobromic acid to give methyl bromo ketone. By treating methyl bromo ketone with a suitably substituted carboxylic acid and base such as e.g. potassium fluoride produces the desired acyloxymethyl ketones which are deprotected with trifluoroacetic acid to give the final compounds as shown in Scheme 5.

Scheme 6

Levels A-D Level E

i. annexation of Cbz-AA li. removing Qby.

iii. annexation of Cbz-AA iv. etc.

formation of bromo methyl ketone '- --►

Level F

RCO ₂ H, KF. DMF - *

P

Level G removal of remaining ester_ protecting groups = protecting group

The hydrochloride salt of H-Asp (OtBu) OMe is treated with an appropriately protected amino acid and a coupling reagent. 1-Hydroxybenzotriazole hydrate should be added to the reaction mixture to improve yield and limit the isomerization, base, preferably amine, such as e.g. trimethylamine or methyl morpholine, however, should be added as an acid scavenger. The resulting amide product is treated with an alkaline reagent such as e.g. with sodium hydroxide to hydrolyze the methyl ester into the carboxylic acid. The Cbz-amine protecting group is removed using standard conditions for catalytic hydrogenation, and coupling of another protected aminic acid can be carried out as described above. This procedure is repeated until the peptide has the desired length. The resulting peptide product is treated with an alkaline reagent such as e.g. with sodium hydroxide to hydrolyze the methyl ester into the carboxylic acid. The resulting acid is then treated with chloroformate, such as e.g. isobutyl chloroformate, followed by diazomethane and then hydrobromic acid to give methyl bromo ketone. By treating methyl bromo ketone with a suitably substituted carboxylic acid and base such as e.g. with potassium fluoride, the desired acyloxymethyl ketones are produced, which is deprotected with trifluoroacetic acid to give the final compounds as described in Scheme 6.

Scheme 7

Level A

H ₂ , 20% Pd / C -h ₂ n-aa

the coupling reagent

Level B rco ₂ h,

Level C cf ₃ co ₂ h

The appropriately substituted acyloxymethyl ketone of the protected amino acid is synthesized. The cbzamine protecting group is removed using standard conditions for catalytic hydrogenation, and the amine product is treated with the appropriately substituted carboxylic acid and the coupling reagent. 1-Hydroxybenzotriazole hydrate should be added to the reaction mixture to improve yield and limit the isomerization, base, preferably amine, such as e.g. trimethylamine or methyl morpholine, however, should be added as an acid scavenger. The penultimate amide product is treated with an acidic medium, preferably trifluoroacetic acid, to remove the t-butyl ester to give the final products as shown in Scheme 7.

Scheme 8

HCI ·

Trans-1,2-cyclohexanedicarboxylic acid anhydride is treated with the amine salt of the appropriately substituted acyloxymethyl ketone aspartyl t-butyl ester in the presence of pyridine and 4-dimethylaminopyridine (DMAP) to give the amide product. The carboxylic acid can be fimctionalized with appropriately substituted amines or alcohols and standard coupling reagents to give amide and ester products. The penultimate product is treated with an acid medium, preferably trifluoroacetic acid, to remove the t-butyl ester to give the final products as shown in Scheme 8.

Scheme 9

Me

2. CF ₃ CO ₇ H

I. oxidation

Methyl methacrylate is treated with the anion of the appropriately substituted sulfide to give a Michael adduct which is hydrolyzed in a basic medium, such as e.g. in sodium hydroxide to produce carboxylic acid. This acid is combined with the amine salt of acyloxymethyl ketone aspartyl t-butyl ester and the coupling reagent to give the amide product. If the desired product is sulfide (where n = 0) then no oxidation step is used and the t-butyl ester amide is deprotected in an acidic medium, preferably trifluoroacetic acid, to give the final product. Alternatively, when the end product is sulfoxide (n = 1) or sulfone (n = 2), the amide intermediate is treated with an oxidizing agent, which can be, but is not limited to, m-chloroperbenzoic acid, potassium monoperoxysulfate or sodium perborate to obtain the desired oxidized the product, the t-butyl ester of the penultimate intermediate, is deprotected in an acidic medium, preferably trifluoroacetic acid, to give the final compounds as shown in Scheme 9.

R.

Scheme 10 ₀ A _nh i. n-BuLi.-78 ° C ^ Jt ^ R ,.

_R ii. R, CH ₂ COCl

H ° </ ,, · Ύ LiOH.H ₂ O ₂ . _t

H ' _R ^X ' CO ₂ ^t Bu ^THF ' ^water

HCI-H ₂ N \ ^ X ^ξ ύ o

'BuO.

NaN (SiMe ₃ ) ₂ , -78 ° C

-—--- », ii. BrCH ₂ CO ₂ ^l Bu

R,

i. ^l BuO ₂ C

co ₂ h coupling reagent O Ji oov Ό

i.cf ₃ co ₂ h

1. PhSiH ₃ , Pd (PPh ₃ ) ₄

2. Bromo methyl ketone formation

3. R ₉ CO _o H, KF, DMF O fl

Ck ^ Ph

2. R ₃ R ₂ NH or

PhCH ₂ ONH _second

the coupling reagent

1. PhSiH ₃ , Pd (PPh ₃ ) ₄

2. Bromo methyl ketone formation

3. R ₂ CO ₂ H, KF, DMF r ₃ n

O »1 O

O o ^l BuO

CL ^ Ph

H, cf ₃ co ₂ h

2. ester formation

3. H _o , 20% Pd / C

O fl O oo co ₂ h

A 4-substituted-2-oxosolidinone chiral auxiliary as described by Evans et al., J.Org. Chem ,, 1985; 50: 1830 is mixed with a base such as, but not limited to, n-butyl lithium, and then treated with the appropriately substituted acid chloride or other activated carboxylic acid to give the N-acylated product. The product is then treated with a base such as, but not limited to, sodium bis (trimethylsilyl) amide and t-butyl bromoacetate to produce an alkylated chiral product. The chiral auxiliary is removed using lithium hydroxide and hydrogen peroxide to give chiral acid. Treatment of the acid with the H-Asp (OBz) O-allyl amine salt and the coupling reagent yields the succinyl amide product.

At this stage of the process, we can process the product in one of two ways. After the first t-butyl ester is removed in an acidic medium, preferably trifluoroacetic acid, and the resulting acid is combined with a suitably substituted amine in the presence of a coupling reagent to form a new amide product. The allyl ester is removed with phenylsilane and tetrakis (triphenylphosphine) palladium or other catalyst Pd (0) to give carboxylic acid and the acid is converted to methyl bromo ketone and then acyloxymethyl ketone. The penultimate intermediate is subjected to catalytic hydrogenation to remove the benzyl ester to give the final amide products, as shown in Scheme 10.

Alternatively, in the second end product method, the allyl ester is removed using phenylsilane and tetrakistetrakis (triphenylphosphine) palladium or another Pd (0) catalyst to give the carboxylic acid. This acid is converted to methyl bromo ketone and then acyloxymethyl ketone. Removal of the acyloxymethyl ketone t-butyl ester with trifluoroacetic acid and then conversion of the resulting carboxylic acid to ester gives a new ester product. The esterification can be carried out using various literatum techniques, which include, but are not limited to, treatment of the carboxylic acid with the appropriately substituted alcohol in the presence of a coupling reagent. The penultimate intermediate is subjected to catalytic hydrogenation to remove the benzyl ester to give the final ester products as shown in Scheme 10.

Scheme 11> cr

O * _, θ

Ri

CO ₂ CH ₂ Ph

Rj l.PhCH ₂ Br,

DBU

Y ^^ co ₂ hO 2. Cl ₂ (gas)

EtOH: CCI ₄ (l: 9)

1. SOC1 ₂ or cyanic acid fluoride, 2. H-Asp ^ Buj-OMe-HCl NMM, CH ₂ C1 ₂

3. NaOH, H ₂ , EtOH

R.

H

1 .. formation of bromo methyl ketone

2. RCO ₂ H, KF, DMF

N ^ xC0 ₂ H CO ₉ ¹ Bu

The appropriately substituted S-acetyl mercapto carboxylic acid is treated with benzyl bromide and 1,8-diazobicyclo [5.4.0] undec-7-ene (DBU) to produce the benzyl ester which is then subjected to the reaction with chlorine gas to give sulfonyl chloride . The sulfonyl chloride is treated with N, N-bis (p-methoxybenzyl) amine to give sulfonamide, which is subjected to catalytic hydrogenation to give the intermediate carboxylic acid. The acid is activated by the use of cyuran fluoride, which is then mixed with the amine salt H-Asp (Ot-Bu) OM to produce the amide prodrug. The methyl ester is hydrolyzed with sodium hydroxide and the carboxylic acid is converted to acyloxymethyl ketone. The p-methoxybenzyl sulfonamide protecting group is removed under oxidation conditions, preferably but not limiting, with cerium ammonium nitrate, and the t-butyl ester protecting group is removed in acidic medium, preferably with trifluoroacetic acid, to give the desired sulfonamide products, as shown in Scheme 11 .

As used herein, the term alkyl is a straight or branched chain hydrocarbon. Representative examples of alkyl groups are: methyl, ethyl, propyl, isopropyl, isobutyl, butyl, tert-butyl, sec-butyl, pentyl and hexyl.

The term alkoxy means an alkyl group attached to an oxygen atom. Representative examples of alkoxy groups include: methoxy, ethoxy, tert-butoxy, propoxy and isobutoxy.

The term halogen includes chlorine, fluorine, bromine and iodine.

The term aryl means an aromatic hydrocarbon. Representative examples of aryl groups include phenyl and naphthyl.

The term heteroatom includes oxygen, nitrogen, sulfur and phosphorus.

The term heteroaryl means an aryl group in which one or more aromatic hydrocarbon carbon atoms are replaced by heteroatoms. Examples of heteroaryl groups include: furan, thiophene, pyrrole, thiazole, pyridine, pyrimidine, pyrazine, benzofuran, indole, coumarin, quinoline, isoquinoline and naphthyridine.

The term cycloalkyl means a cyclic alkyl group. Examples of cycloalkyl groups include: cyclopropane, cyclobutane, cyclopentane and cyclohexane.

The term heterocycle means a cycloalkyl group in which one or more carbon atoms are replaced by a heteroatom. Examples of heterocycles include piperazine, morpholine, and piperidine.

The aryl, heteroaryl or cycloalkyl group may be substituted by one or more substituents which may be the same or different. Examples of suitable substituents include: alkyl, alkoxy, thioalkoxy, hydroxy, halogen, trifluoromethyl, amino, alkylamino, dialkylamino, -NO ₂ -, -CN, -CO ₂ H, -CO ₂ alkyl, -SO ₃ H, -CHO. -COalkyl, CONH ₂ , -CONH-alkyl, -CONHR ^q , -CON (alkyl) 2, - (CH 2) n-NH 2, -OH, -CF 3, -OC 1 -C 6 alkyl, - (CH 2) n-NH-alkyl, - NHR ^q , -NHCOR ^q , phenyl, - (CH2) _n OH, - (CH ₂ ) _n C (O) NH ₂ , or (CH ₂ ) _n CO ₂ H, where n is 1 to 5 and R ^q is hydrogen or alkyl.

Symbolic embroidery.

Examples of other caspase family inhibitors approved for use according to the invention include Ich-1 inhibitors, such as those described in PCT Publication 97/27220.

The term phosphodiesterase IV inhibitor is intended to refer to agents that inhibit the activity of the phosphodiesterase IV enzyme. Examples of phosphodiesterase IV inhibitors are known in the art and include: 4-arylpyrrolidinones such as e.g. rolipram (see, e.g., Sekut L. et al. (1995) Ciin. Exp. Immunol. 100: 126-132), nitroquazone (see, e.g., Van Wauwe J. et al. (1995) Inflamm. Res. 44: 400- 405), denbufilin, tibenelast (see, e.g., Banner KH et al. (1996) No. J. Pharmacol. 119: 1255-1261), CP-80633 (see, e.g., Cohan VL et al. (1996) J. Pharmacol. Exp. Therap. 278: 1356-1361) and quinazolindione, e.g. CP-77059 (see, e.g., Sekut L. et al. (1995) Ciin. Exp. Immunol. 100: 126-132),

The term beta-2-agonist is intended to refer to agents that stimulate the beta-2 adrenergic receptor. Examples of beta-2-agonists are known in the art and include salmeterol (see, e.g., Sekut L. et al. (1995) Ciin. Exp. Immunol 99: 461-466). phenoterol and isoproterenol (see, e.g., Severin A. et al. (1992) 7. Immunol. 148: 3441-3445).

STAT4 expression is intended to refer to the transcription factor involved in IL-12 responses (see, e.g., Thierfelder WE et al. (1996) Nature 382: 171-174: Kaplan MH et al. (1996) Nature 382: 174- 177). The term STAT4 inhibitor refers to an agent that inhibits the activity of the STAT4 transcription factor by inhibiting responses to IL12.

The term antibody as used herein refers to immunoglobulin molecules and immuno-active determinants of immunoglobulin molecules, i. molecules that contain an antigen binding site that specifically binds (reacts with it) to the antigen. The term antibody is further intended to include bispecific and chimeric molecules having at least one antigen binding determinant derived from an antibody molecule. Although the H and L chains of the Fv-fagment are encoded by separate genes, a synthetic lyricer can be made that allows them to be made as a single protein chain (known as a single-chain antibody, sAb; Bird et al. 1988 Science 242 : 423-426 and Huston et al 1988 PNAS 85: 5879-5883) by recombinant methods. Such single chain antibodies are also covered by the term antibody and can be used as binding determinants in the model and in constructing a multispecific binding molecule.

The term antibody fragment, as used herein, refers to an active antibody fragment that retains the ability to bind (react with it) antigen. Examples of antibody fragments include: the Fab fragment consisting of the V _L -, V _H -, Cl- and Cni domains, the Fd fragment consisting of the V _H - and Cm domains, the Fv fragment which consists of the V _L - and Vn domains of the single arm of the antibody, the dAb fragment (Ward et al., 1989 Nature 341: 544-546), which consists of the Vn domain, an isolated complementarity determining region (CDR), and F (ab ') ₂ fragment, a bivalent fragment comprising a Fab'80 fragment bound by a disulfide bridge at a moving region. These antibody fragments are obtained using conventional techniques well known to those skilled in the art, and fragments are selected for use in the same manner as intact antibodies.

The term engineered binding protein as used herein is intended to include molecules derived from an antibody or other binding molecule (e.g., receptor or ligand) that retains the desired binding specificity but is constructed by recombinant DNA techniques and / or expressed using recombinant DNA techniques. Examples of engineered binding proteins include soluble and truncated forms of receptors, receptor dimers (e.g., p40 IL-12 receptor dimers), and modified or mutated forms of antibodies, ligands, or receptors selected using combinatorial libraries (eg, phage display techniques).

The term NK cell antagonist as used herein is intended to include antibodies, antibody fragments and engineered binding proteins capable of depleting NK- / NK-like cells when administered to a subject. Examples of NK cell antagonists include antiasialo-GMl antibodies and NK1.1 antibodies.

The terms steroid-resistant disease and the steroid-resistant subject as used herein are intended to refer to diseases and subjects that do not significantly respond to corticosteroid therapy prior to treatment according to the methods of the invention. Steroid resistance also refers to steroid resistance.

The term immunoinflammatory disease or disorder is intended to include inflammatory diseases and disorders in which immune cells and / or cytokines are involved in the pathophysiology of the disease or disorder. The term acute inflammatory disorder is intended to include disorders and episodes of disorders characterized by the rapid onset of symptoms associated with an inflammatory response and the relatively short duration of symptoms, while the term chronic inflammatory disorder is intended to include disorders characterized by the continued presence of associated symptoms with an inflammatory response and continued duration of symptoms.

I. Methods of the Invention

In one embodiment, the invention provides a method for modulating corticosteroid responsiveness in a subject comprising administering to the subject:

- a target antagonizing agent that regulates interferon-γ (IFN-γ) production in the subject, the agent being administered at a dose and in a manner sufficient to inhibit IFN-γ production in the subject, and

- a corticosteroid such that the response of the subject to the corticosteroid is modulated as compared to when the subject is administered a corticosteroid alone.

In one embodiment, the method comprises administering an agent that is an IL-18 antagonist. The IL-18 antagonist is administered to the subject at a dose and in a manner sufficient to inhibit IL-18 activity in the subject. The IL-18 antagonist may function e.g. by inhibiting IL-18 synthesis in the subject, inhibiting IL-18 cytokine activity in the subject, inhibiting the interaction of IL-18 with the IL-18 receptor, or inhibiting the activity of the IL-18 receptor.

In a preferred embodiment, the IL-18 antagonist is a caspase family protease inhibitor. Proteases of the caspase family, especially ICE, convert the precursor form of IL-18 to a mature (i.e., active) form (e.g., Example 4). A caspase family protease inhibitor is considered, although not intended to be restricted by a mechanism, to antagonize IL-18 activity by inhibiting the processing of IL-18 from its precursor form into its mature (i.e., active) form. A preferred caspase family protease inhibitor for use in the methods of the invention is an ICE inhibitor. Other caspase family proteases capable of grafting IL-18 precursor into mature IL-18 (such as Ich-2 (caspase-4) and ICE _re iIII (caspase-5)) may be inhibited additionally or alternatively. Chemical agents that can inhibit the activity of ICE and other proteases of the caspase family are known in the art, including peptidyl derivatives, azaaspartic acid analogues, and gamapirone-3-acetic acid (see, e.g., U.S. Patent No. 5,411,985, U.S. Patent No. 5,430,128 , U.S. Patent No. 5,434,248, U.S. Patent No. 5,565,430, U.S. Patent No. 5,416,013, PCT Publication WO

94/00154, PCT Publication WO 93/16710, PCT Publication WO 93/14777, PCT Publication WO 93/05071, PCT Publication WO 95/35308, European Patent Application EP 547 699 and European Patent Application EP 519 748) . Additional suitable ICE inhibitors and other caspase family inhibitors are shown in US application serial no. No. 08 / 700,716 and US Provisional Applications Serial no. 60 / 028,322, 60 / 028,324, 60 / 028,313 and 60 / 028,323. The exact doses and regimen for administration of an ICE or ICE protease inhibitor family necessarily depend on the needs of the subject to be treated, the type of treatment, the efficacy of the compound, and the severity of the disease in the subject. However, the non-limiting example of a dosage range for an ICE inhibitor and other proteases of the caspase family is from about 0.05 to about 150 mg / kg body weight / day.

In other embodiments, the IL-18 antagonist is an antibody, antibody fragment or engineered binding protein that binds the IL-18 or IL-18 receptor. Such binding agents can be prepared by standard methods known in the art for the production of poly- and monoclonal antibodies and recombinant binding proteins, and are further described, for example, in: European Patent Application 692 536, European Patent Application 712 931, PCPublication WO 97/24441, and PCT Publication WO 97/44468.

In another embodiment, the method of the invention comprises administering an agent that is an IL-12 antagonist. The IL-12 antagonist is administered to the subject at a dose and in a manner sufficient to inhibit IL-12 activity in the subject. The IL-12 antagonist may function e.g. by inhibiting IL-12 synthesis in a subject, inhibiting IL-12 cytokine activity in a subject, inhibiting the interaction of IL-12 with the IL-12 receptor, or inhibiting IL-12 receptor activity.

In one embodiment, the IL-12 antagonist is an antibody, antibody fragment, or engineered binding protein that binds the IL-12 or IL-12 receptor. The preferred IL-12 antagonist is anriIL-12 monoclonal antibody. Such antibodies are described in the art (e.g., Chizzonite R. et al. (1991) J. Immunol. 147: 1548-1556). The ability of anti-IL-2 monoclonal antibodies to inhibit disease responses is also described in the art (e.g., Leonard JP et al. (1995) J. Exp. Med. 181: 381-386; Neurath MF et al. ( 1995) J. Exp Med 182: 1281-1290). Another hp IL-12 antagonist is the p40 homodimer (e.g.:

Gillessen S. et al. (1995) Eur. J. Immunol. 25: 200-206; Gately M.K. et al. (1996) Ann. NY Acad. Sci. 795: 1-12; Ling P. et al. (1995) J. Immunol. 154: 116-127). A further type of IL-12 antagonist is the low-affinity form of the IL-12 receptor, as described in European Patent Application EP 638 644 and U.S. Pat. No. 5,536,657.

Non-limiting examples of IL-12 antagonists for use in the methods of the invention include mono- and polyclonal antibodies and fragments thereof, chimeric antibodies and fragments thereof, soluble IL-12 receptors and fragments thereof, reactive peptides or fragments thereof, chemically or genetically modified peptides of IL -12, IL12 subunits and fragments thereof, homopolymers of IL-12 subunits and fragments thereof, and small organic molecules designed to inhibit the bioactivity of IL-12 or IL-12 receptors. The preparation of IL-12 antagonists, including: (i) a species that binds IL-12 or their biologically active fragments and (ii) a species that interferes with the binding of IL-12 to receptors or other binding proteins is described in the art (e.g. PCT Publication WO 95/24918, Leonard et al., The contents of which are expressly incorporated herein by reference; and also Presky DH et al. (1995) Res. Immunol. 146: 439-445).

In another embodiment, the IL-12 antagonist used in the method of the invention is an agent that stimulates the production of cyclic AMP (cAMP) in IL-12-producing cells. IL-12 production has been shown to be inhibited by increased intracellular cAMP production (e.g., van der Pouw Kraan et al. (1995) J. Exp. Med. 181: 775-779). Examples of agents that can be used to stimulate the production of intracellular cAMP include phosphodiesterase IV inhibitors and beta-2 agonists. As shown in Example 3, administration of a phosphodiesterase IV inhibitor in a septic shock model inhibits LPS-induced IL-12 production. Examples of suitable phosphodiesterase IV inhibitors for use in the methods of the invention include rolipram, denbufilin, tibenelast, nitroquasone, and CP-80633. Examples of beta-2 agonists for use in the methods of the invention include salmeterol, phenoterol and isoproterenol. The exact dosage and regimen for administration of a phosphodiesterase IV inhibitor or beta-2 agonist necessarily depend on the need of the subject to be treated, the type of treatment, the efficacy of the compound, and the severity of the disease in the subject. However, a non-limiting example of a dosage range for phosphodiesterase IV inhibitors or beta-2 agonists of ca.

0.05 to about 150 mg / kg body weight / day. In a preferred embodiment, an agent that stimulates the production of cyclic AMP (cAMP) (e.g., phosphodiesterase IV inhibitor or beta-2 agonist) is administered systemically (e.g., orally or intravenously) to inhibit IL-12 production systemically by monocytes and macrophages.

In another embodiment, the IL-12 antagonist used in the method of the invention is an STAT4 inhibitor. STAT4 is a transcription factor that has been shown to be involved in IL-12 responses (e.g., Thierfelder W.E. et al. (1996) Nature 382: 171-174). Kaplan M.H. et al. (1996) Nature 382: 174-177). Thus, IL-12 responses in a subject can be inhibited by administration of a STAT4 inhibitor.

Other IL-12 activity inhibitors known in the art can also be used in the methods of the invention. PCT Publication WO 96/40093 discloses biphenyl derivatives for antagonizing IL-12 induced immune responses. Such biphenyl derivatives can be used as IL-12 antagonists in the methods of the invention.

In another embodiment, the method of the invention comprises administering an agent that is an NK cell antagonist. The NK cell antagonist is administered to the subject at a dose and in a manner sufficient to inhibit IFN-γ activity in the subject. Preferably, the NK cell antagonist is an antibody, antibody fragment, or engineered binding protein that specifically binds to NK- / NK-like cells so that the cells are depleted or eliminated in the subject. Thus, preferred NK cell antagonists bind to specific surface markers present on NK- / NK-like cells. Particularly preferred NK cell antagonists are antiasialo-GMl antibodies and NK 1.1 antibodies, which have been shown to be effective in depleting NK- / NK-like activity in a subject (Example 10; Axelsson LG et al. (1996) Inflamm. Res. 45: 181-191; Heremans H. et al. (1994) Eur. J. Immunol. 24: 1155-1160).

Other antibodies that target surface markers that identify NK- / NK-like cells include those that are reactive with the Fc-IgG receptors B73.1 and Leu 11 (CD16) (Lancer LL et al. (1983) J. Immunol 131: 1789-1796; Perussia B. et al.

(1983) J. Immunol. 130: 2133-2141), Leu 7 (anti-HNKl identifying 40-60% NK cells, Abo T. and Balch CM (1981) J. Immunol. 127: 1024-1029) and OKT11 (CD2 identifying 50 % or more NK cells; Lancer, LL et al. supra; Perussia, B. et al., supra). Other NK-cell specific surface antigens and antibodies described include DX1-antigen (PCT release WO 95/02611), PEN5-alpha and PEN5-beta glycoprotein pair (PCT release WO 95/06247) and NKBl-antigen (PCT Publication WO 95/20604).

The exact dosage and regimen for administration of the NK cell antagonist necessarily depend on the needs of the subject to be treated, the type of treatment, the efficacy of the compound, and the severity of the disease in the subject. However, the non-limiting example of the dosage range for anti-NK- / NK-like cell antibodies is from about 0.01 to about 150 mg / kg body weight / day. A single dose of antibody may be sufficient to deplete or eliminate NK- / NK-like cellular activity, or alternatively, multiple doses may be administered as needed to deplete or eliminate NK- / NK-like cellular activity. Preferably, the NK antagonist is administered intravenously or intraperitoneally.

In the methods of the invention, a target antagonizing agent that regulates interferon-γ (IFN-γ) production is administered to a subject in combination with one or more corticosteroids. The term in combination with a corticosteroid is intended to include: simultaneous administration of an agent and a corticosteroid, first administration of an agent and then a corticosteroid, and first administration of a corticosteroid and then an agent. Any of the therapeutically useful corticosteroids known in the art can be used in the methods of the invention. Corticosteroids are typically classified by the duration of their tissue effects: short acting compounds (eg beclomethasone, flunisolid, hydrocortisone, cortisone), medium acting compounds (eg prednisone, prednisolone, methylprednisolone, triamcinolone, deflazortone) and long acting compounds (e.g. beta metazone). The subject may be administered one or more corticosteroids in a manner and at a dose that is effective to achieve the desired therapeutic results. Examples of suitable routes of administration include intravenous administration, oral, topical, administration by inhalation (eg bronchial administration), and topical injections (eg, into the joint). The exact dosage and regimen of corticosteroid administration to a subject necessarily depends on the needs of the subject to be treated, the type of treatment, the efficacy of the compound, and the severity of the disease in the subject. However, the non-limiting example of a dosage range for corticosteroids is from about 0.05 mg / day to about 1 g / day, depending on the particular corticosteroid used. Alternative daily dosing (eg, high-dose intravenous pulse therapy) is used in certain preferred dosage regimens.

Suitable corticosteroid formulations are well known in the art and are commercially available. Dexamethasone acetate, 16 mg / ml aqueous suspension, is e.g. suitable for intramuscular injection in the treatment of rheumatoid, dermatological, ophthalmic, gastrointestinal, hematologic, neoplastic, allergic conditions and collagen disorders. Non-limiting examples of doses include 0.8 mg, 1.6 mg, 4 mg and 16 mg dexamethasone per injection.

Hydroxycortisone is available as a sterile aqueous solution for intravenous, intramuscular and subcutaneous injection and is a potent anti-inflammatory agent for conditions such as osteoarthritis, rheumatoid arthritis, juvenile rheumatoid arthritis, acute and chronic bursitis. Preferred starting doses can range from 15 mg to 250 mg per person per day. Preferred doses are oral or parenteral and may be given in half daily doses twice daily or as other multiple doses. Hydrocortisone injection can be added to sodium chloride injection or dextrose injection and given intravenously. Hydrocortisone valerate 0.2% by weight is formulated as a topical cream called Westkort. Preferred dosages comprise application to damaged surfaces several times a day as a thin layer.

Baconaz (beclomethasone) is available for inflammation of the nasal passages and sinuses, as

8.4 mg for 200 dosed spray doses, in 0.042% aqueous suspension, for administration at 100 mg doses containing 42 pg per dose, such that daily nasal administration consists preferably of 42 pg per nostril, 84 pg per nostril, 168 pg per nostril, 336 pg per nostril, 672 pg per nostril, or 1344 pg per nostril. Preferably, we give it e.g. in aqueous medium in suspension with microcrystalline cellulose, sodium carboxymethylcellulose, dextrose, benzalkonium chloride, polysorbate 80 and 0.25 vol./wt. % phenylethyl alcohol. Some formulations also include additional propellants and media.

In some embodiments, wherein the agents of the invention are administered together with a corticosteroid, the agent is administered systemically to regulate IFN-γ production systemically, while the corticosteroid is administered either locally or systemically. In some embodiments, e.g. when a phosphodiesterase IV inhibitor or beta-2 agonist is administered together with a corticosteroid, a phosphodiesterase IV inhibitor or beta-2 agonist is administered systemically, such as e.g. intravenously or orally, and the corticosteroid is administered either systemically or topically. In addition, the use of a phosphodiesterase IV inhibitor or beta-2 agonist in combination with a corticosteroid for the treatment of asthma is specifically excluded from the scope of the invention in certain embodiments of the methods of the invention.

The methods of the invention can be used in the treatment of various inflammatory and immunological disorders. In a preferred embodiment, e.g. the subject to be treated is suffering from septic shock (i.e., the methods of the invention allow corticosteroids to be used in the treatment of septic shock). In another preferred embodiment, the subject to be treated suffers from Crohn's disease. In yet another preferred embodiment, the subject to be treated is suffering from asthma. In yet another preferred embodiment, the subject to be treated is suffering from a graft-versus-host disease or transplant rejection.

In another preferred embodiment, the subject to be treated is suffering from an autoimmune disease.

In another embodiment, the subject to be treated is suffering from an immunoinflammatory disease or disorder. Non-limiting examples of immunoinflammatory diseases and disorders that can be treated according to the invention include: asthma, adult respiratory distress syndrome, systemic lupus erythematosus, inflammatory bowel disease (including Crohn's disease and ulcerative colitis), multiple sclerosis, diabetes mellitus, diabetes mellitus, , autoimmune arthritis, (including rheumatoid arthritis, juvenile rheumatoid arthritis, psoriatic arthritis), inflammatory pulmonary syndrome, pemphigus vulgaris, idiopathic thrombocytopenic purpura, autoimmune meningitis, myasthenia gravis, dermatitis, dermatitis, dermatitis, dermatitis, dermatitis, dermatitis syndrome, (including keratoconjunctivitis sicca secondary to Sjogrens syndrome), alopecia areata, allergic reactions due to reactions to arthropod spots, aphthous ulcer, iritis, conjunctivitis, keratoconjunctivitis, cutaneous lupus erythematosus, vag einfarction, Steven erythematosus, scleroderma, Stevenle. - Johnson syndrome), leprosy reverse reactions, erythema nodosum leprosum, autoimmune uveitis, allergic encephalomyelitis, aplastic anemia, pure red cell anemia, idiopathic thrombocytopenia, polychondritis, Wegener granulomatosis, chronic active posterior hepatitis, chronic active posterior hepatitis, chronic active posterior cirrhosis, the lungs.

In another embodiment, the subject to be treated suffers from an acute inflammatory disorder. Examples of acute inflammatory disorders include graft-versus-host disease, graft rejection, septic shock, endotoxemia, Lyme arthritis, infectious meningitis (eg, viral, bacterial, Lyme disease-related), acute asthma, and acute episodes of autoimmune disease.

In another embodiment, the subject to be treated suffers from a chronic inflammatory disorder. Non-limiting examples of chronic inflammatory disorders that can be treated include asthma, rubella arthritis, and chronic autoimmune diseases, such as. systemic lupus erythematosus, psoriasis, inflammatory bowel disease, including Crohn's disease and ulcerative colitis, multiple sclerosis and rheumatoid arthritis.

In some cases, agents that antagonize a specific target that regulates IFN-γ in a subject may be preferred for the treatment of a specific disorder. Thus, e.g. disorders, although not intended to be restricted by a mechanism in which IFN-γ is preferentially or predominantly produced by NK cells, are preferentially treated using an agent that antagonizes IL-18 (such as an ICE inhibitor) or directly antagonizes NK- cells, (i.e., an NK cell antagonist such as an anti-NK- / NK-like cellular antibody) in combination with a corticosteroid. Alternatively, disorders in which IFN-γ is preferentially or predominantly produced by T cells are preferentially treated using an agent that antagonizes IL-12 (e.g., anti-IL-12 antibody or an agent that stimulates intracellular cAMP production) in combined with a corticosteroid. In other circumstances, it may be advantageous to use both an IL-18 antagonist and an IL-12 antagonist (e.g., in the treatment of disorders that contribute to the production of IFN-γ by both T cells and NK cells).

The agent and corticosteroid are administered to the subject in need of treatment according to standard routes of administration, well known in the art, and the particular mode and dose for agent and corticosteroid are selected depending on the needs of the subject to be treated, the type of treatment, the effectiveness of the compound and the severity of the disease in the subject. The agent and corticosteroid are administered at an effective therapeutic dose, which means that the amount of therapeutic composition that, when administered to a subject, produces improved disorders compared to those of the subjects not receiving the drug. One skilled in the art can determine and prescribe the effective amount of therapeutic agents and corticosteroids required. The agents would be administered to the subject in the invention according to the invention in biologically compatible forms suitable for pharmaceutical administration in vivo to produce the desired therapeutic response. A biologically compatible formulation suitable for administration in vivo is intended to be a formulation of a medicament to be administered which therapeutic effects overcome any toxic and side effects of the composition. In addition, an agent of the invention is administered that antagonizes a target that regulates IFN-γ production in a dose-administered subject and in a manner sufficient to inhibit IFN-γ production in the subject. Similarly, an IL-12 antagonist or IL-18 antagonist of the invention is administered at a dose and in a manner sufficient to inhibit IL-12 activity or IL-18 activity in a subject. Animal models of inflammatory and immunological disorders acceptable in the art as models for human diseases can be used to evaluate the various therapeutic regimens of the invention. Thus, e.g. we use the P.ncnes / LPS septic shock model described in the Examples to evaluate the effectiveness of therapeutic regimens for the treatment of septic shock. Many animal models of autoimmune diseases are known in the art and can be used in the methods of the present invention to evaluate the effectiveness of therapeutic regimens, and non-limiting examples of these include experimental colitis (e.g., Neurath MF et al. (1995) J. Exp. Med. 182: 1281-1290), experimental allergic encephalomyelitis (e.g., Leonard JP et al. (1995) J. Exp. Med. 181: 381-386), halogen-induced arthritis (Banerjee S. et al. (1989) ) J. Immunol. 142: 2237-2243) and the human TNF-α transgenic model of polyarthritis (e.g., Keffer J. et al. EMBO J (1991) 10: 4025-4031). For therapeutic regimens involving the inhibition of ICE activity, ICE-deficient mice can be used as a model for complete inhibition of ICE activity. Such ICE - / - mice are described in the art (e.g., Li P. et al. (1995) Whole 80: 401-411 and PCT Publication No. WO 96/12025).

The methods of the invention are useful for modulating corticosteroid responsiveness in various clinical determinations. In one embodiment, e.g. the methods of the invention to reverse steroid resistance in a subject compared to when administered to a subject only a corticosteroid. In another embodiment, the method of the invention is used to increase steroid sensitivity in a subject compared to giving a subject a corticosteroid alone. In a further embodiment, the corticosteroid is administered to a subject in accordance with a program to reduce the corticosteroid dose over time, and the method improves the vindicative effect associated with the administration of reduced corticosteroid doses. The ability of the methods of the invention to increase steroid sensitivity (i.e., to have a steroid-sparing effect) may thus allow the use of corticosteroid therapy in clinical situations in which such therapy has been previously contraindicated. The use of the methods of the invention may e.g. allow corticosteroid therapy in patients who have not been previously treated for the adverse side effects of corticosteroid therapy, such as. young children (eg, juvenile rheumatoid arthritis), patients with uncontrolled diabetes, and patients with hypertension.

Another aspect of the present invention relates to a method for modulating corticosteroid responsiveness in a subject comprising:

- selection of a subject in need of corticosteroid response modulation and

- administering to the subject an agent that antagonizes the target that regulates interferon-γ (IFN-γ) production in the subject, administering the agent at a dose and in a manner sufficient to inhibit IFN-γ production in the subject, thereby giving the subject responsiveness per corticosteroid modulated, compared to when administered to a subject with only a corticosteroid.

The subject selected for treatment according to the method may e.g. a corticosteroid-resistant subject prior to administration of the agent. Alternatively, the subject selected for treatment may be one that responds to the corticosteroid prior to administration of the agent but exhibits increased sensitivity to the corticosteroid after administration of the agent. One example of such a subject is patients suffering from a disorder of steroid dependence, the disorder being treated with lower doses of corticosteroids if treated according to the methods of the invention. Another example of such an entity is a patient for whom steroid therapy is contraindicated due to side effects when administered corticosteroid alone, but which may tolerate a lower dose of corticosteroid when administered in

a corticosteroid according to the methods of the invention. Furthermore, the subject selected for treatment according to the method may be one exposed to corticosteroid therapy, but corticosteroid therapy should be discontinued so that administration of the agent improves the subject's effect on steroids in the subject. Target antagonizing agents that regulate IFN-γ production in a subject have been described previously.

II. Pharmaceutical compositions

Another aspect of the invention relates to pharmaceutical compositions for modulating corticosteroid responsiveness. In one embodiment, the pharmaceutical composition of the invention comprises a target antagonizing agent that regulates interferon-γ (IFN-γ) production in a subject, a corticosteroid, and a pharmaceutically acceptable carrier. As shown above, the target to be antagonized may e.g. IL-18, IL-12, or NK cells (i.e., the pharmaceutical composition may comprise an IL-18 antagonist, an IL-12 antagonist, or an NK cell antagonist, as previously described, a corticosteroid and a pharmaceutically acceptable carrier).

In a preferred embodiment, the pharmaceutical composition of the invention comprises a caspase family protease inhibitor, a corticosteroid, and a pharmaceutically acceptable carrier. Examples of caspase family protease inhibitors and non-limiting dose examples have been described previously. In a preferred embodiment, the protease inhibitor of the caspase family is an ICE inhibitor.

In another embodiment, the pharmaceutical composition of the invention comprises an IL-12 antagonist, a corticosteroid, and a pharmaceutically acceptable carrier. Examples of such IL-12 antagonists are described previously. In a preferred embodiment, the IL-12 antagonist is an anti-IL-12 monoclonal antibody. In another preferred embodiment, the IL-12 antagonist is a phosphodiesterase IV inhibitor.

In another preferred embodiment, the IL-12 antagonist is a beta-2 agonist.

In another embodiment, the pharmaceutical composition of the invention comprises an NK cell antagonist, a corticosteroid, and a pharmaceutically acceptable carrier. Examples of such NK cell antagonists are described previously. In a preferred embodiment, the anti-NK cell antagonist is an anti-NK- / NK-like cellular antibody, preferably an anti-asialoGM1 antibody, or an NK 1.1 antibody.

As used herein, the term pharmaceutically acceptable carrier is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption-inhibiting agents, etc. compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is well known in the art. If the conventional medium or agent is incompatible with the active compound, then its use in the compositions is not confirmed. Additional active compounds may also be incorporated into the compositions.

The pharmaceutical composition of the invention is formulated to be compatible with the intended route of administration. E.g. solutions or suspensions used for parenteral, intradermal or subcutaneous administration may include the following components: a sterile diluent such as e.g. water for injection, saline, fixed oils, polyethylene glycols, glycerol, propylene glycol or other synthetic solvents; antibacterial agents such as e.g. benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulphite; kelima agents, such as e.g. ethylenediamine tetraacetic acid; buffers, such as acetates, citrates or phosphates, and tonicity adjusters such as e.g. sodium chloride or dextrose. The pH can be adjusted with acids or bases, such as e.g. with hydrochloric acid or with sodium hydroxide. The parenteral preparation may be contained in an ampoule, disposable needles or multiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (in the case of water solubility) or dispersions and sterile powders for the improvised preparation of sterile injectable solutions or dispersions. For intravenous administration include suitable saline carriers, bacteriostatic water, Cremophor EL ™ (BASF, Parsippany, NJ) or phosphate saline (PBS) buffers. In all cases, the composition should be sterile and should be liquid to such an extent that there is an easy injection option. It must be as stable under the conditions of manufacture and storage as it is protected against contamination by micro-organisms, such as. bacteria and fungi. The carrier may be a solvent or dispersion medium containing e.g. water, ethanol, polyol (e.g., glycerol, propylene glycol and liquid polyethylene glycol, etc.) and suitable mixtures thereof. Proper fluidity can be maintained e.g. using coatings such as e.g. lecithin, maintaining the required particle size in the case of dispersion and using surfactants. The prevention of microorganisms can be achieved by various antibacterial and antifungal agents, such as e.g. parabens, chlorobutanol, phenol, ascorbic acid, thimerosal and the like. In many cases, it is preferable to include isotonic agents in the composition, e.g. sugars, polyalcohols such as mannitol, sorbitol, sodium chloride. Prolonged absorption of injectable compositions can be achieved by including absorption retardants, e.g. aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in a suitable solvent with one ingredient or combination of the ingredients listed above as needed and then filtered sterilization. Generally, dispersions are prepared by incorporating the active ingredient into a sterile carrier containing a basic dispersion medium and the necessary other ingredients, of those mentioned above. In the case of sterile powders for the preparation of sterile injectable solutions, vacuum drying and lyophilisation methods are preferred, where a powder of the active ingredient and any additional desired constituents are obtained from its pre-sterile filtered solution.

Oral compositions generally include an inert diluent or an edible carrier. They may be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active ingredient may be incorporated with excipients and used in the form of tablets, lozenges or capsules. Oral compositions can also be prepared using a liquid carrier for use as mouthwash, whereby the compound in the liquid carrier is administered orally, rinsed with the mouth and spit or swallowed. Pharmaceutically compatible binders and / or adjuvants may be included as part of the composition. Tablets, pills, capsules, lozenges and the like. may contain any of the following ingredients or compounds having similar properties: a binder, such as e.g. microcrystalline cellulose, tragacanth gum or gelatin; excipient, such as e.g. starch or lactose; disintegrating agent such as e.g. alginic acid, Primogel or corn starch; lubricant such as e.g. magnesium stearate or Sterotes; a sliding means such as e.g. colloidal silica; sweetener, such as sucrose or saccharin; aroma, such as peppermint, methylsalicylate or orange aroma.

In one embodiment, the active compounds are prepared with carriers that protect the compound from rapid elimination from the body, such as e.g. a controlled release formulation that includes implants and microencapsulated delivery systems. Biodegradable biocompatible polymers such as e.g. ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, poliortoesters and polylactic acid. Methods for preparing such formulations are clear to those skilled in the art. The materials can also be obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes targeting infected cells with monoclonal antibodies for viral antigens) can also be used as pharmaceutically acceptable carriers. They can be prepared according to procedures known to those skilled in the art, such as e.g. described in U.S. Pat. 4,522,811.

The pharmaceutical compositions of the invention may be formulated for administration in a particular manner, such as e.g. oral, intravenous, ophthalmic administration, and the like.

In a preferred embodiment, the pharmaceutical composition of the invention is formulated for topical administration. A target antagonizing agent that regulates interferon-γ (IFN-γ) production in a subject, a corticosteroid and a pharmaceutically acceptable carrier can be formulated as a cream, pomade, ointment, etc., suitable for application to the skin.

In another preferred embodiment, the pharmaceutical composition of the invention is formulated for administration by inhalation. A target antagonizing agent that regulates interferon-γ (IFN-γ) production in a subject, a corticosteroid and a pharmaceutically acceptable carrier can be formulated into a nasal spray or inhaler to allow the administration of therapeutic agents into the nasal or sinus passages or lungs (e.g., bronchial passages). ) by inhalation.

The present invention is further illustrated by the following Examples, which are not intended to be construed as limiting. The contents of all references, published patents and patent applications cited throughout this application are incorporated herein by reference.

EXAMPLE 1:

Inhibition of ICE activity in a septic shock model results in steroid responsiveness.

In this Example, we investigate the effect of inhibiting ICE activity on steroid responsiveness in septic shock. The septic shock model is induced in ICE-deficient (ICE - / -) and wild-type (ICE + / +) mice, which are then treated with a corticosteroid. (ICE - / -) - Mice are used as a model for complete inhibition of ICE activity (see Li P. et al. (1995) Whole 80: 401-411 for further description of ICE-deficient mice. Animal response to corticosteroid treatment is determined by recording levels TNFα inflammatory cytokine in mouse sera.

ICE-deficient and wild-type mice were first sensitized with Propionibacterium acnes cell wall material (1 mg per mouse) to induce low grade inflammation, and challenged 6 days later with lipopolysaccharide (LPS) (1 μg per mouse 0.1 ml saline) salts iv). 30 min after LPS administration, mice were treated with corticosteroid dexamethasone (4 mg / kg per mouse in 0.5 ml of 95% saline / 0.5% ethanol, i.p.). Control mice were treated with vehicle only. All mice were sampled 90 minutes after LPS administration and serum samples were analyzed for the presence of TNFα by standard ELISA.

The results are shown in FIG. 1. The vehicle-treated wild-type and ICE-deficient mice have similar levels of serum TNFα. Treatment of wild-type mice with dexamethasone did not significantly affect serum TNFα levels, indicating their resistance to steroid treatment in this septic shock model. In comparison, treatment of ICE-deficient mice with dexamethasone suppressed serum TNFα levels by 74% (p <0.002). These data suggest that inhibition of ICE activity obscures resistance to steroid therapy in a septic shock model.

EXAMPLE 2:

Inhibition of ICE activity in a septic shock model increases steroid sensitivity.

In this Example, we investigate the effect of inhibiting ICE activity on steroid sensitivity in septic shock. We use the same LPS / P. from the <? "es-septic shock model as described in Example 1, except that ICE-deficient and wild-type mice were pretreated with vehicle or corticosteroid 15 minutes before challenge with LPS. The animal's responsiveness to corticosteroid therapy is again determined by recording the levels of inflammatory cytokine TNFα in mouse sera.

ICE-deficient and wild-type mice were first sensitized with Propiombacterium acnes cell wall material (1 mg per mouse) to induce low grade inflammation and were challenged 6 days later with lipopolysaccharide (LPS) (1 pg per mouse in 0.1 ml of physiological saline solutions, iv). Animals were treated with decreasing amounts of corticosteroid dexamethasone (0.5, 0.005, or 0.0005 mg / kg per mouse in 0.5 ml of 95% saline / 0.5% ethanol, i.p.) 15 minutes before the challenge with LPS. Control mice were treated with vehicle only. Blood was drawn on all mice 90 minutes after LPS administration and serum samples were analyzed for the presence of TNFα using standard ELISA.

The results are shown in FIG. 2. Both wild-type and ICE-deficient mice exhibit a pretreatment response of 0.05 mg / kg dexamethasone. In contrast, ICE deficient mice pretreated with only 0.005 or 0.0005 mg / kg dexamethasone showed 76% and 78% (p <0.005) lower levels of serum TNFα, respectively, compared to lack of TNFα suppression in similarly treated animals. wild type. These data suggest that inhibition of ICE activity results in increased steroid sensitivity in the septic shock model because 10- to 100-fold lower doses of dexamethasone are therapeutically effective in ICE-deficient animals compared with live wild-type animals.

EXAMPLE 3:

Phosphodiesterase IV inhibitor impairs IL-12 production.

In this case, we investigate the effect of the phosphodiesterase IV rolipram inhibitor on LPS-induced IL-12 production. B6 mice were pretreated with solvent or rolipram (30 mg / kg in 0.5 ml of 0.1% methyl cellulose, i.p.) 15 minutes before challenge with LPS (10 pg / mouse, i.v.). 90 minutes after LPS administration, blood was collected from mice and serum levels of IL-12 were determined by standard ELISA.

The results are shown in FIG. 3. Mice pretreated with rolipram have 70% lower levels of serum IL-12 than mice pretreated with vehicle alone. These data indicate that phosphodiesterase IV inhibitors are effective for inhibiting LPS-induced IL-12 production.

EXAMPLE 4:

Cleavage of IL-18 by caspase family proteases.

The ability of various recombinant proteases of the caspase family (i.e. expressed in E. coli) to cleave precursor IL-18 (proIL-18) in mature IL-18 was tested by in vitro proteolysis assay. The cleavage of poly (ADP-ribose) polymerase (PARP) was used as a positive control. The results are summarized below in Table 1.

Table 1. ProILolysis of proIL-18 with recombinant caspases% caspase cleavage * concentration (ug / ml) of proIL-18 PARP

ICE (1) 1,25 100 99 ICH-2 (4) 5,00 82 93 ICE _rel III (5) 20,00 55 90 CPP32 (3) 5,00 0 ^a 100 Mch2 (6) 10,00 2 96 Mch3 (7) 5,00 32 ^b 97 ICH-1 (2) 75.00 5 98

Caspases are numbered in parentheses as suggested by: Alnemri et al. (1996) Cel 87: 171.

^a with CPP32 (5 μg / Inl) grafted proIL-18 producing a 12 kDa and 10 kDa fragment instead of the expected 18 kDa-fagment.

L unlike other caspases, the Mch3 precursor expressed in E. coli is not exposed to autocatalysis to produce active protease. ICE supplementation is required for the initiation of Mch3 autocatalysis and the production of active Mch3 protease. Partial cleavage of proIL-18 with Mch3 is mediated by the presence of ICE in Mch3 preparation.

EXAMPLE 5:

Treatment of septic shock.

Patients exhibiting septic shock in clinical settings (eg in connection with infected abrasions, projectile damage or systemic bacteraemia from other sources) are administered an agent selected from an ICE inhibitor, a phosphodiesterase IV inhibitor (eg rolipram, 30 mg / kg) and anti -IL-12-monoclonal antibody, together with corticosteroid (eg high dose methylprednisolone, 1 g / day, iv). Corticosteroid and

100 may be administered concurrently or alternatively, the agent may be administered before or after corticosteroid administration. Patients are also treated with appropriate antibiotic therapy.

EXAMPLE 6:

Treatment of transplant rejection.

Patients who will receive a kidney transplant are given an agent selected from an ICE inhibitor, a phosphodiesterase IV inhibitor (e.g., rolipram, 30 mg / kg), and an anti-IL-12 monoclonal antibody, together with a corticosteroid (e.g., oral prednisone, 25-75 mg / day). Preferably, start treatment before receiving kidney from the donor (eg, drug administration can be started 24 hours before receiving kidney from the donor) with doses to be repeated if necessary (eg every 12 hours). The corticosteroid and agent may be administered concomitantly or alternatively, the agent may be administered before or after corticosteroid administration. Patients are also treated with additional immunosuppressive therapy (such as ciclosporin A treatment or OKT3 antibody treatment) so that the immune rejection and inflammatory response are simultaneously suppressed.

EXAMPLE 7:

Improving the effect of steroid rebound.

Patients with asthma, allergic rhinitis or rheumatoid arthritis who will be exposed to treatment with a corticosteroid inhaler or systemic corticosteroids and who initiate programmed discontinuation of steroid therapy are given an agent selected from an ICE inhibitor, a phosphodiesterase IV inhibitor (eg 30 mg / kg rolipram ) and anti-IL-12 monoclonal antibody. Patients are preferentially treated prior to discontinuation or discontinuation of steroid therapy to improve the vindicative effect of steroids, which may be due to discontinuation of steroid therapy. If necessary, patients can be treated with additional NSAIDs.

101

EXAMPLE 8:

Treatment of an acute episode of autoimmune disease.

Patients suffering from acute inflammation of an autoimmune disease, such as inflammatory bowel diseases (eg ulcerative colitis or Crohn's disease), administered an agent selected from an ICE inhibitor, a phosphodiesterase IV inhibitor (eg rolipram, 30 mg / kg) and an anti-IL-12 monoclonal antibody together with a corticosteroid (eg oral prednisone , 25-75 mg / day). The corticosteroid and agent may be administered concurrently or alternatively, the agent may be administered before or after corticosteroid administration. Patients can also be treated with additional immunosuppressive therapy to control acute inflammation of an autoimmune disease.

EXAMPLE 9;

Treatment of chronic autoimmune disease.

Patients suffering from chronic autoimmune disease, such as Crohn's disease, administered an agent selected from an ICE inhibitor, a phosphodiesterase IV inhibitor (e.g., rolipram, 30 mg / kg), and an anti-IL-12 monoclonal antibody together with a corticosteroid (eg, oral prednisone, 25-75 mg / day). The corticosteroid and agent may be administered concomitantly or alternatively, the agent may be administered before or after corticosteroid administration. Patients can also be treated with additional immunosuppressive therapy to control autoimmune disease.

EXAMPLE 10;

Inhibition of IFN-γ production by elimination of NK cells.

In this case, shock was induced in mice by treatment with a high dose of LPS (40 mg / kg LPS administered intravenously). The effect of NK cell depletion on the production of various cytokines in mice and on mortality is investigated by administration of antiasialo-GMl (anti-ASGMl) antibody intravenously 10 minutes before LPS administration. Control animals receive rabbit IgG. The effect of ASGM1 treatment on IL-Ιβ production,

TNFα and IFN-γ, as well as mortality, are shown below in Table 2.

Table 2

102

Processing # mice cytokine production (pg / ml) mortality (30 h) % of survivors IL-Ιβ TNFα IFN-γ LPS + rabbits IgG (control) 6 19461483 585711071 16631811 0 (n = 10) * LPS + ASGM1 antibodies 6 16471482 545311103 3631108 90 (n = 10)

* All animals die within 15 hours.

These results suggest that the elimination of NK cells by anti-asialo-GMl antibody treatment reduces IFN-γ production and prolongs survival after administration of LPS in high-dose LPS shock.

EXAMPLE:

Effect of ICE inhibitor and corticosteroid in a septic shock model.

In this example, LPS / P is used. the acnes septic shock model described in Examples 1 and 2 to investigate the effect of an ICE inhibitor in combination with a corticosteroid. The B6 mice were implanted for 24 hours with an osmotic pump containing an ICE inhibitor acetyl-tyrosinvalin-alanine-aspartic acid-CHO (Ac-YVAD-CHO) (100 mg / kg), or a control solvent, subcutaneously 18 hours before LPS injection. LPS was injected intravenously (0.01 μg / mouse or 10 μg / mouse) at time zero. All mice were injected with 5 mg / kg dexamethasone intraperitoneally 30 minutes after LPS injection. The response of animals to corticosteroid treatment is determined by recording the level of the inflammatory cytokine TNFα as

103 also interleukin-6 (IL-6) and interleukin-ΐβ (IL-Ιβ) in mouse sera. Blood was collected from all mice 90 minutes after LPS administration and serum samples were analyzed for the presence of TNFα, IL-6 and IL-1 β by standard methods.

The results are shown in FIG. 4, 5 and 6 for TNFα, IL-6, or. IL-Ιβ. The data showed that mice treated with both ICE inhibitor Ac-YVAD-CHO and dexamethasone had significantly lower serum levels of TNFα, IL-6 and IL-Ιβ when using either 0.01 pg or. 10 pg LPS to induce septic shock. As previously shown in Example 1, treatment of mice with dexamethasone alone did not significantly affect serum TNFα levels, indicating the resistance of mice to steroid-only treatment in this model of septic shock. In contrast, treatment of mice with both dexamethasone and ICE inhibitors suppressed serum TNFα levels by 96% (p <0.005) in 0.01 pg LPS treated mice and 86% (p <0.005) in treated mice with 10 pg LPS. In addition, serum IL-6 levels are reduced by 95% (p <0.00005) or. 91% (p <0.00005) and serum IL-Ιβ levels decreased 94% (p <0.001) and 92% (p <0.0002). These data indicate that inhibition of ICE activity using an ICE inhibitor obviates resistance to steroid treatment in a septic shock model.

EXAMPLE 12:

Synthesis of hydroxamate ICE inhibitors.

3-Benzyloxycarbonylamino-4-oxo-5-phenylacetylaminooxy-pentanoic acid

Level A

N- (phenylmethoxy) -benzenacetamide [(0.760 g, 3.15 mmol) prepared by the method of Heam MTW and Ward AD (Aust. J. Chem., 1969: 22: 1731)] was taken up in 10 ml of CH ₃ CN and treated with dimethylamino-pyridine (DMAP) (50 mg) and diterbutyl dicarbonate (0.824 g, 3.78 mmol). The reaction mixture was allowed to stir under argon for 12 hours, then diluted with ethyl acetate (EtOAc) and washed with 3M K2S2O5 (1 x 10 ml), NaHCO ₃ (1 x 10 ml). The organic layer is dried over Na ₂ SO4,

104 filtered and concentrated. Purification by chromatography (SiO ₂ , 9: 1 hexane-EtOAc) gave 0.910 g (84%) of 1,1-dimethylethyl (phenylacetyl) phenyl-methoxy) carbamate as a clear viscous oil.

1 H NMR (400 MHz, DMSO-do):? 7.41 [m, 5Η], 7.32 [m, 2Η], 7.24 [m, 3Η], 4.90 [s, 2Η], 4 , 09 [s, 2Η], 1.48 [s, 9Η], IR (thin film), 3063, 3032, 2979, 2935, 2886, 1777, 1736, 1497, 1455, 1370, 1302 cm ^-1 . Mass spectrum (MS) (chemical ionization [Cl] NH ₃ ) 342 (M ⁺ + H).

Elemental analysis:

Calc. for C ₂₀ H ₂₃ NO ₄ .0.051 CH ₂ C1 ₂ : C 69.66; H, 6.74; N, 4.05.

Found: C, 69.66; H, 6.83; N, 3.99.

Level B

1,1-Dimethylethyl (phenylacetyl) (phenyl-methoxy) carbamate (810 mg, 2.37 mmol) was dissolved in 75 ml of dry THF and 90 mg of 5% Pd / BaSO _{4 was added} . The reaction mixture was treated with H ₂ (1, 3.10 ⁵ Pa) for 20 hours. The reaction mixture was filtered through Celite and concentrated to give 588 mg (99%) of 1,1-dimethyl-ethylhydroxy (phenylacetyl) carbamate as an oil. No further purification was performed.

1 H NMR (400 MHz, CDCl ₃ ):? 8.22 [s, 1Η], 7.31 [m, 5Η], 4.24 [s, 2Η], 1.55 [s, 9Η].

Step C (S) -5-Bromo-4-oxo-3 - [[(phenylmethoxy) -carbonyl] amino] -pentanoic acid 1,1-dimethylethyl ester [(297 mg, 0.742 mmol) prepared by Dolle RE et al. , (J. Med, Chem., 1994; 37: 563-4)], 1,1-dimethylethylhydroxy (phenylacetyl) carbamate (187 mg, 0.742 mmol) and KF (104 mg, 1.85 mmol) were combined in 5 ml. of dimethylformamide (DMF) and allowed to stir under argon for 12 hours. The reaction mixture was diluted with EtOAc (15 ml) and washed with water (3 x 15 ml) and brine (1 x 15 ml). The organic layer was dried over Na ₂ SO ₃ and concentrated. Purification by chromatography (SiO ₂ , 4: 1 hexane-EtOAc) gave 168 mg (40%) of [[[(1,1-dimethylethoxy) -carbonyl] (phenylacetyl) amino] oxy] -4-oxo-3 - [[( (phenylmethoxy) carbonyl] amino] -pentanoic acid 1,1-dimethylethyl ester as a clear oil.

1 H NMR (300 MHz, CDCl ₃ ):? 7.35 [m, 5Η], 5.84 [d, J = 9.0 Hz, 1Η], 4.79, [A of AB, J = 15. 3 Hz, 1Η], 4.70 [m, 1Η], 4.57 [B of AB, J = 15.3 Hz, 1Η], 4.10 [s, 2Η],

105

3.09 [dd, J = 16.8, 4.6 Hz, 1Η], 2.79 [dd, J = 16.8, 4.9 Hz, 1Η], 1.52 [s, 9Η], 1 , 39 [s, 9Η], IR (thin film) 3374, 2980, 2935, 1726 (br), 1499, 1370, 1298, 1150 cm ^-1 . MS (APCI, methanol (MeOH)) 571.5 (M ⁺ + H).

Elemental analysis:

calcd. for C ₃ oH ₃₈ N ₂ 0 ₉ : C 63.15; H, 6.71; N, 4.91.

Found: C, 62.76; H, 6.70; N, 4.69.

Level D

3-Benzyloxycarbonylamino-4-oxo-5-phenylacetylaminooxy-pentanoic acid, 1,1-dimethylethyl ester (208 mg, 0.365 mmol) was taken up in 3 ml of 1: 1 trifluoroacetic acid (TFA) / CH ₂ Cl ₂ and allowed to stir for 2 hours . The reaction mixture was diluted with acetonitrile (MeCN) (10 ml) and concentrated. The residue was separated from MeCN five times. Purification by chromatography (SiO ₂ , 90: 9: 1 CH ₂ Cl ₂ -acetone-formic acid) gave 3-benzyloxycarbonylamino-4-oxo-5-phenylacetylaminooxy-pentanoic acid (51 mg, 34%) as a white foam.

1 H NMR (300 MHz, CDCl ₃ ):? 8.63 [s, 1Η], 7.34 [broad (br), s, 10Η], 5.48 [br, d, J = 4 Hz, 1Η] , 5.08 [br dd, J = 16, 12 Hz, 2Η], 4.23 [m, 1Η], 3.97 [m, 2Η], 3.58 [br, s, 2Η], 2.80 [m, 1Η], 2.64 [m, 1H].

IR (KBr) 3305 (br), 2928, 1791, 1772, 1717, 1699, 1685, 1674, 1654, 1521,

1455 cm ¹ .

MS (APCI, MeOH) 415 (M ⁺ + H).

Elemental analysis:

calc .: C ₂₁ H ₂₂ N ₂ O ₇ .0,106 CF ₃ CO ₂ H: C, 59.73; H, 5.22; N, 6.57.

Found: C, 59.73; H, 5.46; N, 6.28.

The compounds listed below are prepared from (S) -5-bromo-4-oxo-3 - [[(phenibnetoxy) carbonyl] amino] -pentanoic acid 1,1-dimethylethyl ester as described above in step C and step D.

106

3-Benzyloxycarbonylamino-4-oxo-5- (2-oxo-pyrrolidin-1-yloxy) -pentanoic acid

Level A

Prepared from l-hydroxy-2-pyrrolidinone [Biswas A. and Miller M.J. (Heterocycles, 1987; 26: 2849)] in the manner described above in step C to give 3-benzyloxycarbonylamino-4-oxo-5- (2-oxo-pyrrolidin-1-yloxy) -pentanoic acid 1,1-dimethylethyl ester (74% ).

1 H NMR (400 MHz, CDCl ₃ ):? 7.37 [m, 5H]; 5.88 [br, d, J = 8.9 Hz] 5.16 [A of AB, J = 12.2 Hz, 1H]; 5.11 [B from AB, J = 12.2 Hz, 1H]; 4.95 [A from AB, J = 17.1 Hz, 1Η], 4.81 [B from AB, J = 17.1 Hz, 1Η], 4.60 [m, 1Η], 3.62 [m , 2Η], 3.01 [dd, J = 17.1, 4.6 Hz, 1Η], 2.75 [dd, J = 17.1, 4.8 Hz, 1Η], 2.30 [t, J = 7.95 Hz, 2Η], 1.99 [quint, J =

7.5 Hz, 2Η], 1.41 [s, 9Η], IR (KBr) 3328 (br), 2976, 2932, 1717, 1701, 1522, 1256 cm ^-1 .

MS (APCI, MeOH) 421 (M ⁺ + H).

Elemental analysis:

calcd. for C ₂ H ₂₈ N ₂ O 7.0.096 DMF: C 59.81; H, 6.76; N, 6.87.

Found: C, 59.56; H, 7.00; N, 6.52.

Level B:

Prepared from 3-benzyloxycarbonylamino-4-oxo-5- (2-oxo-pyrrolidin-1-yloxy) pentanoic acid 1,1-dimethylethyl ester in the manner described above in step D to give 3-benzyloxycarbonylamino-4-oxo- 5- (2-Oxo-pyrrolidin-1-yloxy) -pentanoic acid (72%).

1 H NMR (400 MHz, CDCl ₃ ):? 8.55 [br s, 1Η], 7.36 [m, 5Η], 5.46 [br d, J = 9.4 Hz, 1Η], 5. 14 [A of AB, J = 5.2 Hz, 1Η], 5.11 [B of AB, J = 5.2 Hz, 1Η], 4.23 [m, 1Η],

4.19 [A of AB, J = 13.3 Hz, 1Η], 3.96 [B of AB, J = 13.3 Hz, 1Η], 3.67 [m, 1Η], 3.52 [dd , J = 15.1, 7.9 Hz, 1Η], 2.84 [dd, J = 16.9, 8.2 Hz, 1Η], 2.61 [dd, J = 16.9, 10.9 Hz, 1Η], 2.42 [m, 2Η], 2.11 [m, 2H]. IR (KBr) 3408 (br), 2926, 1791, 1717, 1700, 1540, 1268, 1054 cm ^-1 . MS (APCI, MeOH) 365 (M ⁺ + H).

Analysis: Calc. for Ci ₇ H ₂ oN ₂ 0 ₇ .0.32 C ₃ H ₇ OC ₃ H ₇ : C 57.27; H, 6.24; N, 7.04.

107

Found: C, 57.27; Η 6.24; N, 6.74.

-Benzyloxycarbonylamino-5- (3, 5-dioxo-10-oxa-4-aza-tricyclo [5.2.1.0 ^2,6 ldec-8-ene-4yloxy) -4-oxo-pentanoic acid

Level A

Prepared from 3a, 4,7,7a-tetrahydro-2-hydroxy-4,7-epoxy-1H-isoindole-1,3 (2H) -dione [Narita M., Teramoto T, Okawara M (Buli. Chem. Soc. Jap., 1971; 44: 1084)] in the manner described above in step C to give 3-benzyloxycarbonylamino-5- (3,5-dioxo10-oxa-4-aza-tricyclo [5.2.1.0 ^{2 , 6} ] dec-8-en-4-yloxy) -4-oxo-pentanoic acid 1,1-dimethylethyl ester (64%).

^! H NMR (400 MHz, DMSO-d ₆ ): a 7.84 [d, J = 8.2 Hz, 1Η], 7.34 [m, 5Η], 6.54 [s, 2Η], 5.16 [s, 2H]; 5.07 [A from AB, J = 12.5 Hz, 1Η], 5.03 [B from AB, J = 12.5 Hz, 1Η],

4.93 [A from AB, J = 16.2 Hz, 1Η], 4.87 [B from AB, J = 16.2 Hz, 1Η], 4.52 [m, 1Η], 2.87 [s , 2Η], 2.73 [dd, J = 16.2, 5.8 Hz, 1Η], 2.50 [dimmed by dimethyl sulfoxide (DMSO) resonance], 1.37 [s, 9Η], IR ( KBr) 3421, 2979, 2930, 1790, 1726, 1520, 1368 cm ^-1 . MS (APCI, MeOH) 445 (M ⁺ -C ₄ H ₈ ).

Analysis: Calc. for C25H28N2O9; C, 59.65; H, 5.70; N, 5.35.

Found: C, 59.99; H, 5.64; N, 5.60.

Level B

Prepared from 3-benzyloxycarbonylamino-5- (3,5-dioxo-10-oxa-4-azatricyclo [5.2.1.0 ^2,6 ] dec-8-en-4-yloxy) -4-oxo-pentanoic acid 1,1-dimethylethyl ester method described above in step C to give 3-benzyloxycarbonylamino-4-oxo-5-phenylacetylaminooxy-pentanoic acid (78%). IR (thin film) 3360, 1789, 1723, 1530, 1220 cm ^-1 . MS (APCI, MeOH) 445 (M ⁺ + H). Elemental analysis:

Calc. for C ₂ H ₂₀ N ₂ O 9.0, 194 CF ₃ CO ₂ H: C 55.06; H, 4.36; N, 5.96.

Found: C 55.06; H, 4.58; N, 5.99.

3-Benzyloxycarbonylamino-5- (2-oxo-2,3-dihydro-indol-1-yloxy) -4-oxo-pentanoic acid

108

Prepared from 1-hydroxyoxyindole [Kende A.S. and Thurston J. (Svnthetic Communications, 1990; 20: 2133-8)] to give 3-benzyloxycarbonylamino-4-oxo-5 (2-oxo-2,3-dihydro-indol-1-yloxy) -pentanoic acid ( 24%), mp 58-70 ° C (decomposition).

Elemental analysis:

Calc. for C2iH ₂ oN ₂ 0 _7: C, 61.16; H, 4.89; N, 6.79.

Found: C, 60.84; H, 4.72; N, 6.46.

3-Benzyloxy · carbonylamino-5- (7-methoxycarbonylmethyl-2-oxo-octabutro-indole-lyloxy) -4-oxo-pentanoic acid

Level A

Hydroxylamine hydrochloride (200 mmol, 13.8 g) was dissolved in pyridine (200 mmol, 16 ml) and methanol (10 ml) and this solution was added to a mixture of cis-2-oxo-1,3-cyclohexanedioacetic acid, dimethyl ester [(35 mmol, 8.5 g) prepared according to the procedure described by: Grieco PA, Noguez JA, Masaki Y., Hiroi K., Nishizawa M., Rosowsky A., Oppenheim S ,, Lazarus HJ Med. Chem, 1977; 20:71] in 200 ml MeOH. NaCNBH ₄ (30 mmol, 1.9 g) was added to this solution in approximately 1 hour and the resulting solution was stirred at room temperature for 4 days. The reaction mixture was then concentrated to dryness, redissolved in 500 ml of ethyl acetate and washed with 3 x 50 ml of saturated NaCl, dried with Na ₂ SO ₄ , filtered and concentrated to give the crude solid, which is mainly the desired product and pyridine. The crude octahydro-1-hydroxy-2-oxo-1H-indole7-acetic acid methyl ester was recrystallized from EtOAc to give 4.05 g (51%) of a white solid.

1 H-NMR: 9.26 [1H, s], 3.64 [1H, dd], 3.59 [3H, s], 2.65 [1H, dd], 2.49 [1H, dd], 2.34 [IH, dd], 2.18 [IH, m], 2.04 [IH, m], 1.79 [IH, d], 1.62 [IH, m], 1.60 [IH , s-br], 1.42 [1H, m], 1.25 [2H, m], 1.06 [1H, m]. MS (CI, NH ₃ ) 228 (M ⁺ + H).

109

Level B

Prepared from octahydro-1-hydroxy-2-oxo-1H-indole-7-acetic acid methyl ester in the manner described above in step C to give 3-benzyloxycarbonylamino-5- (7methoxycarbonylmethyl-2-oxo-octahydro-indole- 1-yloxy) -4-oxo-pentanoic acid 1,1-trimethylethyl ester as a glassy oil (45%).

1 H NMR (400 MHz, DMSO-d ₆ , 1: 1 mixture of diastereomers): a 7.85 [d, J = 5.8 Hz, 0.5 Η], 7.83 [d, J = 5.8 Hz, 0.5 Η], 7.35 [m, 5Η], 5.06 [s, 2Η], 4.94 [A of AB, J = 16.9 Hz, 0.25 Η], 4.87 [A from AB, J = 17.6 Hz, 0.25 Η], 4.82 [B from AB, J = 17.6 Hz, 0.25 Η], 4.74 [B from AB, J = 16 , 9 Hz, 0.25 Η], 4.23 [m, 1Η], 3.82 [m, 0.5 Η], 3.79 [m, 0.5 Η], 3.57 [s, 1 , 5 H]; 3.57 [s, 1.5 Η], 2.72 [m, 0.5 Η], 2.70 [m, 0.5 Η], 2.52 [m, shaded by DMSO], 2.39 [m, 2Η], 2.22 [br m, 1Η], 2.10 [br m, 1Η], 1.88 [br s, 0.5 Η], 1.84 [br s, 0.5 Η ], 1.61 [m, 2Η], 1.42 [m, 1Η], 1.36 [s, 9 Η], 1.25 [m, 2 Η], 1.06 [m, 1H]. IR (thin film) 3418, 3344, 3017, 2979, 2934, 2860, 1725, 1506 cm ^-1 . MS (APCI, MeOH) 547.6 (M ⁺ + H).

Level C

Prepared from 3-benzyloxycarbonylamino-5- (7-methoxycarbonylmethyl-2-oxooctahydro-indol-1-yloxy) -4-oxo-pentanoic acid 1,1-trimethylethyl ester in the manner described above in step D to give 3-benzyloxy -carbonylamino-5- (7methoxycarbonylmethyl-2-oxo-octahydro-indol-1-yloxy) -4-oxo-pentanoic acid (45%), mp 55-58 ° C.

^! H NMR (400 MHz, DMSO-d ₆ , 1: 1 mixture of diastereomers): a 12.4 [s, 1Η], 7.84 [m, 1Η], 7.35 [m, 5Η], 5.05 [ s, 2Η], 4.86 [m, 2Η], 4.45 [m, 1Η], 3.83 [m, 0.5H], 2.79 [m, 0.5H], 3.59 [s , 1.5H], 3.58 [s, 0.5H], 2.57 [m, shadowed by DMSO], 2.41 [complex m, 4Η], 2.20 [m, 2Η], 1.88 [m, 1Η], 1.62 [m, 2Η], 1.43 [m, 2Η], 1.23 [m, 2Η], 1.05 [m, 1H]. IR (KBr) 3337, 2931, 1790, 1726, 15384 cm. * ¹ MS (ES, NH4OH) 489.5 (M ⁺ -H). Elemental analysis:

Calc. for C24H30N2O9: C 58.77; H, 6.16; N, 5.71.

Found. Stk #: C 59.19; H, 6.40; N, 5.34.

110

-Benzyloxycarbonylamino-4-oxo-5- (2-oxo-octahydro-indol-1-yloxy-pentanoic acid

Level A

Ethyl 2-cyclohexanonacetate (4.28 g, 23.3 mmol) and O-benzyl hydroxylamine hydrochloride were combined in 100 ml of ethanol (EtOH) and 2.59 g (25.6 mmol, 3.55 ml) of triethyl amine (Et ₃ N). The reaction mixture was stirred at room temperature for 12 hours and concentrated in vacuo after that time. The residue was taken up in EtOAc and washed with 1N HCl (2 x 20 ml), saturated NaHCO ₃ (1 x 20 ml), dried over Na 2 SO 4, filtered and concentrated. Purification by chromatography (SiO ₂ , 90: 1 hexanes EtOAc) afforded (2-benzyloxyimino-cyclohexyl) -acetic acid ethyl ester (4.76 g, 72%) as a mixture of oxime isomers.

1 H NMR (400 MHz, CDCl _{3 3} 7: 1 mixture of oxime isomers): a 7.32 [complex m, 5Η], 5.05 [s, 0.25 Η], 5.02 [s, 1Η], 4 , 05 [q, J = 7.2 Hz, 2Η], 3.20 [m, 1Η], 2.73 [complex m, 2Η], 2.46 [d, J = 8.0 Hz, 0.125 Η] , 2.21 [dd, J = 15.4, 6.3 Hz, 0.875H], 1.92 [m, 1Η], 1.79 [complex m, 3Η], 1.43 [m, 1Η], 1.38 [complex m, 2Η], 1.22 [t, J = 7.2 Hz, 3H]. IR (thin film) 2931, 1735, 1638, 1451 cm ^-1 . MS (CI, NH3) 290 (M ⁺ + H). Calc. for Ci7H ₂₃ NiO _3: C, 70,56; H, 8.01; N, 4.84.

Found: C, 70.47; H, 7.92; N, 4.78.

Step B (2-Benzyloxyimino-cyclohexyl) -acetic acid ethyl ester (4.66 g, 16.1 mmol) was taken up in 15 ml of acetic acid (AcOH) and NaBH ₃ CN and stirred for 72 hours. The reaction mixture was poured into NaHCO ₃ and extracted into EtOAc (3x30 ml). The combined organic layers were washed once with brine, dried over Na ₂ SO4, filtered and concentrated. The clear oil was dissolved in 50 ml of MeOH and K ₂ CO ₃ (5.55 g, 40.2 mmol) was added and the reaction mixture was stirred for 12 hours. The reaction mixture was concentrated, the residue was taken up in CHCl ₃ , filtered and concentrated. Purification by chromatography (SiO ₂ , 4: 1 hexanes / EtOAc) gave 1.72 g (43%) of cis- (2-benzyloxyamino-cyclohexyl) acetic acid ethyl ester and 0.441 g (11%) of trans- (2-benzyloxyamino) -cyclohexyl) -acetic acid ethyl ester.

Cis isomer data:

111 ^J H NMR (400 MHz, CDCl 3):? 7.44 [complex m, 2Η], 7.37 [complex m, 3Η], 5.05 [A from AB, J = 10.4 Hz, 1Η]. 4.94 [B from AB, J = 10.4 Hz, 1H), 3.47 [dd, J = 10.6, 5.3 Hz, 1Η], 2.33 [dd, J = 16.4 Hz , 1Η], 2.20 [m, 1Η], 2.08 [dd, J = 16.4, 4.6 Hz, 1Η], 1.74 [complex m, 2Η], 1.60 [m, 1Η ], 1.32 [complex m, 5Η], IR (solution, CHCl3) 3031, 2932, 2856, 1717, 1453 cm ^-1 . MS (CI, NH 3) 246 (M ⁺ + H).

Trans isomer data: mp: 79-82 ° C.

Elemental analysis:

Calc. for Ci ₅ H ₉ NiO ₂ : C 73.44; H, 7.81; N, 5.71.

Found: C, 73.38; H, 7.89; N, 5.63.

Level C

Prepared from cis- (2-benzyloxyamino-cyclohexyl) -acetic acid ethyl ester in the manner described above in step B to give cis-1-hydroxy-octahydro-indol-2-one (85%), m.p .: 85- 86 ° C.

'H NMR (400 MHz, CDC1 _3); a 9.86 [br s, 1H), 3.75 [dd, J = 10.4, 4.8 Hz, 1Η], 2.41 [dd, J = 16.1, 7.7 Hz, 1Η] , 2.33 [m, 1Η], 1.97 [m, 1Η], 1.71 [complex m, 2Η], 1.54 [m, 1Η], 1.44 [complex m, 2Η], 1. 31 [complex m, 2Η], IR (KBr) 3037, 2936, 2856, 2710, 1690, 1659, 1548 cm ^-1 . MS (CI, NH ₃ ) 156 (M ⁺ + H).

Elemental analysis:

Calc. for C ₈ Hi ₃ NiO ₂ : C 61.91; H, 8.44; N, 9.03.

Found: C, 61.94; H, 8.49; N, 8.96.

Level D

Prepared from cis-1-hydroxy-octahydro-indol-2-one in the manner described above in step C to give 3-benzyloxycarbonylamino-4-oxo-5- (2-oxo-octahydro-indole-lyloxy) pentanoic acid 1 , 1-dimethylethyl ester (41%). IR (thin film) 2933, 1723, 1516, 1367 cm ^-1 .

Elemental analysis:

Calc. for C ₂₅ Hi34N ₂ O ₇ : C 63.28; H, 7.22; N, 5.90.

Found: C 63.03; H, 7.36; N, 5.65.

112

Level E

Prepared from 3-benzyloxycarbonylamino-4-oxo-5- (2-oxo-octahydro-indol-1-yloxy) pentanoic acid 1,1-dimethylethyl ester in the manner described above in step D to give 3-benzyloxycarbonylamino-4- oxo-5- (2-oxo-octahydro-indol-1-yloxy) -pentanoic acid (72%). IR (KBr) 3352 (br), 2935, 2869, 1789, 1704, 1535 cm ^-1 . MS (APCI, MeOH) 419.5 (M ⁺ + H).

Elemental analysis:

Calc. for C ₂ iH26 N ₂ O7.0,12 H ₂ O.0,322 CH ₂ C1 ₂ :

C, 57.17; H, 6.05; N, 6.26.

Found: C 57.17; H, 6.05; N, 5.89.

The compounds below are prepared from 5-bromo-3- [2- (2-benzyloxycarbonylamino-3methyl-butyrylamino) -propionylamino] -) -4-oxo-pentanoic acid 1,1-dimethyl ester [Dolle R.E. et al. (J. Med. Chem. 1994; 37: 563-4)] in the manner described above in step C and step D.

3 "[2- (2-Benzyloxycarbonylamino-3-methyl-butyrylamino) -propionylaminol-5- (7-methoxy- (1) -1126 ^ 12220 ^ 8020 ^^ 11 ^ 0411401214) ^ 51) 24: ^ 80 ^ 621311018 ^ 3 ^ 1811113

Prepared from octahydro-1-hydroxy-2-oxo-1H-indole-7-acetic acid methyl ester (65%), mp: 162-167 ° C, decomposition. Elemental analysis:

Calc. for C ₂₉ H ₃₄ N ₄ O ₉ .0.75 H ₂ O (596.127): C 58.43; H, 6.00; N, 9.40.

Found: C 58.40; H, 5.68; N, 9.19.

3- [2- (2-Benzyloxycarbonylamino-3-methyl-butyrylamino) -propionylaminol-4-oxo-5-fluoro-2,3-dihydro-indol-1-yloxy-pentanoic acid

Prepared from 1-hydroxyoxyindole [Kende A.S. and Thurston J. (Svnthetic Communications, 1990; 20: 2133-8] to give 3- [2- (2-benzyloxycarbonylamino-3methyl-butyrylamino) -propionylamino] -4-oxo-5- (2-oxo-2, 3-Dihydro-indol-1-yloxy) pentanoic acid (67%), mp: 162-167 ° C, dec.

113

Elemental analysis:

Calc'd: for C29H34N4O9.O75 H ₂ 0 (596.127):

C, 58.43; H, 6.00; N, 9.40.

Found: C 58.40; H, 5.68; N, 9.19.

Other compounds are prepared using automated parallel synthesis as follows:

To a 7 ml screw-coated glass vial containing 17 mg (0.3 mmol, 3 eq) of potassium fluoride was added 500 μΐ (0.1 mmol, 1 eq) of a 0.2 M solution of the corresponding hydroxamate in DMF. The reaction vial was stirred for a few minutes without completely dissolving the potassium fluoride. At this point, 500 μΐ (0.1 mmol, 1 eq) of 0.2 M solution of (S) -5-bromo-4-oxo-3 - [[(phenylmethoxy) carbonyl] amino] -pentanoic acid 1,1-dimethylethyl of esters in DMF. Cover the vials and place a rack of 30 to 40 vials on top of a circular mixer for 12 hours.

The reaction mixtures were diluted with 2 ml of ethyl acetate and then with 2 ml of deionized water. Two milliliters of fluid is withdrawn from the center of the vial and quickly injected twice back. Leave the vials for 30 minutes and remove the organic layer from the upper half of the vial. Add 2 ml of ethyl acetate twice more, mix and separate. The combined organic layers were evaporated under a constant stream of nitrogen overnight.

The crude residue from the reaction mixtures was dissolved in 3 to 4 ml of 40% TFA in methylene chloride. Mix the vials to ensure complete dissolution, in a digestion without lids. After 2 hours, the vials were again placed under a constant stream of nitrogen overnight.

The crude reaction mixture was taken up in 1 ml of chloroform (sometimes MeOH was added for complete dissolution). The solutions were applied to 500-μm preparative silica gel TLC plates and then eluted with acetone (20%) in methylene chloride with 1% to 2% acetic acid. The product bands are visualized by UV absorption, scraped off the plates, and the silica gel washed with methanol in a tared vial. Put the vials under nitrogen stream overnight. The weighed purified products were then diluted to 10 mM in 25% methanol in chloroform

114 and aliquots are loaded onto plates, for both analytical analysis and biological evaluation. The solutions were allowed to evaporate in the digestion room for 72 hours.

Equivalents

It will be apparent to those skilled in the art that they will be able to determine, by routine experimentation, many equivalents for specific embodiments of the invention described herein. These equivalents are also intended to be covered by the attached claims.

Claims (25)

PATENT APPLICATIONS A combination preparation for simultaneous, separate or sequential use in modulating a corticosteroid response in a subject, characterized in that it comprises: a) a target antagonizing agent that regulates interferon-γ (IFN-γ) production in a subject, m b) a corticosteroid. A combination preparation for simultaneous, separate or sequential use in modulating corticosteroid responsiveness in a subject according to claim 1, characterized in that the agent is an IL-18 antagonist. A combination preparation for simultaneous, separate or sequential use in modulating corticosteroid responsiveness in a subject according to claim 2, characterized in that the agent is a caspase family protease inhibitor, e.g. ICE inhibitor. A combination preparation for simultaneous, separate or sequential use in modulating corticosteroid responsiveness in a subject according to claim 2 or 3, characterized in that the agent is an antibody, antibody fragment or engineered binding protein that binds the IL-18 or IL-18 receptor . A combination preparation for simultaneous, separate or sequential use in modulating corticosteroid responsiveness in a subject according to claim 1, characterized in that the agent is an interleukin-12 (IL-12) antagonist. A combination preparation for simultaneous, separate or sequential use in modulating corticosteroid responsiveness in a subject according to claim 5, characterized in that the agent is an antibody, antibody fragment ah engineered binding protein that binds the IL-12 or IL-12 receptor. 116 A combination preparation for simultaneous, separate or sequential use in modulating corticosteroid responsiveness in a subject according to claim 5 or 6, characterized in that the agent stimulates the production of cyclic AMP in IL-12-producing cells. A combination preparation for simultaneous, separate or sequential use in modulating corticosteroid responsiveness in a subject according to claim 7, characterized in that the agent is a phosphodiesterase IV inhibitor, e.g. selected from the group consisting of 4-arylpyrrolidinones, rolipram, denbufilin, tibenelast, mtracasone, CP-80633, kmazolindions and CP-77059. A combination preparation for simultaneous, separate or sequential use in modulating corticosteroid responsiveness in a subject according to claim 7, characterized in that the agent is a beta-2 agonist, e.g. selected from the group consisting of salmeterol, phenoterol and isoproterenol. A combination preparation for simultaneous, separate or sequential use in modulating corticosteroid responsiveness in a subject according to claim 5, characterized in that the agent is a STAT4 inhibitor. A combination preparation for simultaneous, separate or sequential use in modulating corticosteroid responsiveness in a subject according to claim 1, characterized in that the agent is an NK cell antagonist. A combination preparation for simultaneous, separate or sequential use in modulating corticosteroid responsiveness in a subject according to claim 11, characterized in that the agent is an anti-NK- / NK-like cellular antibody, e.g. anti-asialo-GMl antibody or NK1.1 antibody. A combination preparation for simultaneous, separate or sequential use in modulating corticosteroid responsiveness in a subject according to any preceding claim, characterized in that the corticosteroid is selected from the group consisting of: cortisone, hydrocortisone, beclomethasone, flunizide, prednisone, prednisolone, methylprednisolone, triamcinolone, deflazortort, betamethasone and dexamethasone. 117 A combination preparation for simultaneous, separate or sequential use in modulating corticosteroid responsiveness in a subject according to any preceding claim, characterized in that the subject suffers from septic shock, Crohn's disease, asthma, graft-versus-host disease, or transplant rejection or autoimmune diseases or disorders. A combination preparation for simultaneous, separate or sequential use in modulating corticosteroid responsiveness in a subject according to any one of claims 1-13, characterized in that the subject suffers from an immunoinflammatory disease or disorder selected from the group comprising: asthma, respiratory syndrome adult distress, systemic lupus erythematosus, inflammatory bowel disease, Crohn's disease, ulcerative colitis, multiple sclerosis, diabetes mellitus, insulin dependent, autoimmune arthritis, rheumatoid arthritis, juvenile rheumatoid arthritis, psonatic pulmonary arthritis, inflammatory syndrome, inflammatory syndrome , autoimmune meningitis, myasthenia gravis, autoimmune thyroiditis, dermatitis, atopic dermatitis, eczematous dermatitis, psoriasis, Sjogrens syndrome, keratoconjunctivitis sicca, secondary to Sjogrens syndrome, alopecia arectivitis, allergic reaction, allergic aphthous, conjunctivitis, conjunctivitis tis, cutaneous lupus erythematosus, scleroderma, vagimtis, proctitis, drug eruptions, Stevens-Johnson syndrome, leprosy reverse reactions, erythema nodosum leprosum, autoimmune uveitis, allergic encephalomyelitis, aplastic anemia, pure anemia cytophthia, cellulitis, chronic active hepatitis, Graves' ophthalmopathy, primary biliary cirrhosis, posterior uveitis, and interstitial lung fibrosis. A combination preparation for simultaneous, separate or sequential use in modulating a corticosteroid response in a subject according to any one of claims 1-13, characterized in that the subject suffers from an acute or chronic inflammatory disorder. A combination preparation for simultaneous, separate or sequential use in the modulation of corticosteroid responsiveness in a subject according to any one of claims 1 to 13, characterized 118, in that the steroid resistance in the subject is reversed or increased compared to when the corticosteroid is administered to the subject alone. A combination preparation for simultaneous, separate or sequential use in modulating corticosteroid responsiveness in a subject according to any one of claims 1 to 13, characterized in that the corticosteroid is administered to the subject according to a program that lowers the corticosteroid dose over time, and the method ameliorates steroid syndromes The effect associated with administering reduced doses of corticosteroid. 19. A corticosteroid response modulating agent in a subject, characterized in that the agent antagonizes a target that regulates interferon-γ (IFN-γ) production in the subject. 20. A corticosteroid responsive modulator in a subject according to claim 19, characterized in that a) a corticosteroid resistant subject before administration of the agent b) that the subject responds to a corticosteroid prior to administration of the agent but exhibits increased sensitivity to the corticosteroid after administration of the agent; c) treatment of the subject with a corticosteroid should be stopped and administration of the agent ameliorates the steroid syndrome of effect in the subject. 21. A corticosteroid responsive agent in a subject according to claim 19, wherein the agent is an IL-18 antagonist or an interleukin-12 (IL-12) antagonist. A pharmaceutical composition comprising an agent as described in any one of claims 1 to 12 or 19 to 21, a corticosteroid and a pharmaceutically acceptable carrier. 119 Pharmaceutical composition according to claim 22, characterized in that it is formulated for topical administration or for administration by inhalation. Use of an agent as described in any one of claims 1 to 12 or 19 to 21 (optionally when combined with a corticosteroid) in the manufacture of a medicament for modulating corticosteroid responsiveness in a subject. Use of an agent as described in any one of claims 1 to 12 or 19 to 21 (optionally when combined with a corticosteroid) in the manufacture of a medicament for the treatment of one or more of the disorders described in any of claims 14 to 16.