MXPA00002938A

MXPA00002938A - PROTEASOME INHIBITORS, UBIQUITIN PATHWAY INHIBITORS OR AGENTS THAT INTERFERE WITH THE ACTIVATION OF NF-&kgr;B VIA THE UBIQUITIN PROTEASOME PATHWAY TO TREAT INFLAMMATORY AND AUTOIMMUNE DISEASES

Info

Publication number: MXPA00002938A
Application number: MXPA/A/2000/002938A
Authority: MX
Inventors: Peter Elliot; Julian Adams; Louis Plamondon
Original assignee: Julian Adams; Peter Elliot; Louis Plamondon; Proscript Inc
Priority date: 1997-09-25
Filing date: 2000-03-24
Publication date: 2001-07-09

Abstract

This invention is directed to the treatment of inflammatory and autoimmune diseases by administering proteasome inhibitors, ubiquitin pathway inhibitors, agents that interfere with the activation of NF-kB via the ubiquitin proteasome pathway, or mixtures thereof. The invention is further directed to the treatment of inflammatory and autoimmune diseases by administering an effective combination of a glucocorticoid and a proteasome inhibitor, ubiquitin pathway inhibitor, agent that interferes with the activation of NF-kB via the ubiquitin proteasome pathway, or mixture thereof. Pharmaceutical compositions comprising a combination of a glucocorticoid and a proteasome inhibitor, ubiquitin pathway inhibitor, agent that interferes with the activation of NF-kB via the ubiquitin proteasome pathway, or mixture thereof are also contemplated within the scope of the invention.

Description

PROTEASOMA INHIBITORS, INHIBITORS OF THE VIA DE THE UBIQUITINE OR AGENTS THAT INTERFERE WITH THE ACTIVATION OF NF-KB THROUGH THE ROUTE OF THE ÜBIQUITINE-PROTEASOMA TO TREAT INFLAMMATORY AND AUTOIMMUNE DISEASES BACKGROUND OF THE INVENTION Field of the Invention This invention is directed to compositions and methods for the treatment of inflammatory and autoimmune diseases.

Brief Description of the Related Art Eukaryotic cells contain multiple proteolytic systems, including lysosomal proteases, calpains, the ATP-ubiquitin-proteasome dependent pathway and a non-lysosomal process independent of ATP. The highest neutral proteolytic activity in the cytosol and in the nucleus is the proteasome, a 20S particle (700 kDa) with multiple peptidase activities. The 20S complex is the proteolytic nucleus of a 26S complex (1500 REF .: 119012 kDa) that degrades or processes proteins conjugated to ubiquitin. Ubiquitination marks a protein for hydrolysis by the 26S proteasome complex. Many abnormal or normal short-lived polypeptides are degraded by the proteasome-dependent pathway of ubiquitin. Abnormal peptides include proteins damaged by oxidants (for example, those that have oxidized disulfide bonds), products of premature translational termination (for example, those that have exposed hydrophobic groups that are recognized by the proteasome, and denatured or damaged proteins). induced by stress or stress (where stress is induced for example by changes in pH or temperature, or exposure to metals.) The proteasome also participates in the rapid elimination and post-translational processing of the proteins involved in cellular regulation (eg, cell cycle, gene transcription, and metabolic pathways), intercellular communication, and immune response (eg, antigen presentation) Transcriptional factor NF-? B is a member of the Reí family of proteins. The Reí family of transcriptional activator proteins can be divided into two groups. The first group requires a proteolytic processing, and includes pl05 and plOO, which are processed at p50 and p52, respectively. The second group does not require proteolytic processing and includes p65 (Reí A), Reí (c-Rel), and Reí B. NF-? B comprises two subunits, p50 and an additional member of the Reí gene family, for example, p65. pl05 unprocessed can also be associated with p65 and other members of the Reí family. In most cells, the p50-p65 heterodimer is present in an inactive form in the cytoplasm, linked to I? B-a. The ternary complex can be activated by the dissociation and destruction of I? B-a, while the p65 / pl05 heterodimer can be activated by pl05 processing. The ubiquitin-proteasome pathway plays an essential role in the regulation of NF-? B activity, being responsible for the processing of pl05 to p50 and for the degradation of the inhibitor protein I? B-a. In order to be targeted for degradation by the proteasome, I? Ba must first undergo selective phosphorylation at serine residues 32 and 33, followed by ubiquitination (Chen et al., Genes &Devel opmen t (1995) 9 : 1586; Chen et al., Cel l (1996) 84: 853; Brockman et al., Mol. Cel l. Biol. (1995) 15: 2809; Brown et al. Sci ence (1995) 267: 1485). Once activated, NF-? B translocates to the nucleus, where it plays a central role in the regulation of a remarkably diverse group of genes involved in immune and inflammatory responses (Grilli et al., In t erna ti onal Revi ew of Cyt olgy (1993) 143: 1-62). For example, NF-? B is required for the expression of a number of genes involved in the inflammatory response, such as the TNF-a gene and the genes encoding the cell adhesion molecules E-selectin, P-selectin, ICAM and VCAM (Collins, T., Lab. In ves t. (1993) 68: 499. NF-? B is also required for the expression of a large number of cytokine genes such as IL-2. , IL-6, the granulocyte colony stimulation factor, and IFN-ß, inducible nitric oxide synthetase, is also under the regulatory control of NF- [alpha] B. Proteasome inhibitors block the degradation of I? Ba and the activation of NF-? B (Palombella et al., W095 / 25533 published on 9/28/95; Traenckner et al., EMBO J. (1994) 13: 5433). Proteasome inhibitors also block TNF-α-induced expression of adhesion molecules to leukocytes, E-selectin, VCAM-1 and ICAM-1 (Read et al., Immuni ty (1995) 2: 493). Cyclins are proteins involved in the control of the cell cycle. The proteasome participates in the degradation of cyclins. The degradation of cyclin makes it possible for a cell to leave one stage of the cell cycle (for example mitosis) and enter another (for example, division). There is evidence that cyclin is converted to a form vulnerable to a ubiquitin ligase or that a specific cyclin ligase is activated during mitosis (Ciechanover Ceil (1994) 79: 13). Inhibition of the proteasome inhibits the degradation of cyclin, and therefore inhibits cell proliferation (Kumatori et al, Proc.Na.i.Ac.d.Sci.USA (1990) 87: 7071). The continuous conversion of cellular proteins via the ubiquitin-proteasome is also used by the immune system to select the presence of abnormal intracellular proteins (Goldberg and Rock Na t ure (1993) 357: 375). In this process, the lymphocytes continually verify the small fragments of the cellular protein that are presented on the molecules of the major histocopathy complex (MHC) of class I. Proteasomes initially degrade proteins to small peptides, most of which they are rapidly hydrolyzed to amino acids by cytosolic exopeptidases. But some of these peptides are transported to the endoplasmic reticulum where they bind to MHC molecules and are then transported to the cell surface in a process known as antigen presentation. If the peptides are abnormal (for example, if they are derived from viral proteins), they cause cell destruction by cytotoxic T cells. Inhibitors that prevent proteasome function have been shown to block the generation of most of the peptides presented on MHC class I molecules (Rock et al., Cel l (1994) 78: 761). Multiple sclerosis (MS) is an incurable neurological disease that frequently causes chronic disability. MS is the most common demyelinating disease of the human central nervous system and typically affects young people and women more than men. Clinically, the disease is characterized in a stage of relapsing-remission or chronic progressive, although more precisely there are stages defined for research purposes. This tends to follow a highly unpredictable course, leading to chronic and sometimes devastating disability. It is widely believed that MS is the result of an autoimmune disorder in a genetically susceptible individual, mediated by autoreactive T cells that migrate to the central nervous system and initiate the inflammatory demyelination lesion. The hyper-reactivity of the respiratory tract to a variety of spasmogens and pulmonary inflammation, characterized by eosinophilia, are the pathologies that are characteristic of asthma (Beasely et al., Am. Rev. Resp. Di s. (1989) 139: 806) . Asthma is a chronic condition of the airways that involves many types of inflammatory cells and the release of many mediators and neurotransmitters that have multiple effects on the various target cells in the respiratory tract. The degree and extent of inflammation in the wall of the respiratory tract are largely related to the clinical severity of asthma. The inflammatory response of asthma consists of the activation of resident airway mastocytes / increased number of lymphocytes (which are mainly CD4 + T lymphocytes) and an infiltration with eosinophils, which seem to degranulate. There is a need in the art for effective therapies for the treatment of multiple sclerosis or asthma.

BRIEF DESCRIPTION OF THE INVENTION The present invention is directed to methods for the treatment of a patient afflicted with multiple sclerosis or asthma, comprising administering to said patient an effective amount of an agent selected from the group consisting of proteasome inhibitors, inhibitors of the ubiquitin, agents that interfere with the activation of NF-? B by means of the ubiquitin-proteasome pathway and mixtures thereof. In certain embodiments of the invention, the agent is a proteasome inhibitor. Preferably, the proteasome inhibitor is selected from the group consisting of peptidyl aldehydes, boronic acids, boronic esters, lactacystin and lactacystin analogues. In a preferred embodiment, the proteasome inhibitor is lactacystin or a lactacystin analogue, more preferably lactacystin, clasto-lactacystin β-lactone, 7-ethyl-clasto-lactacystin β-lactone, β-lactone 7-n-propyl-clasto-lactacystin, or β-lactone of 7-n-butyl-cyasto-lactacystin. More preferably, the proteasome inhibitor is the β-lactone of 7-n-propyl-clasto-lactacystin. In other embodiments of the invention, the agent is an inhibitor of the ubiquitin pathway. In other additional embodiments of the invention, the agent is one that interferes with the activation of NF-? B by the ubiquitin-proteasome pathway. Preferably, the agent that interferes with the activation of NF-? B is an agent that inhibits the phosphorylation of I? B-a. The invention is further directed to methods for the treatment of a patient afflicted with asthma, comprising administering to said patient an effective combination of a glucocorticoid and an agent selected from the group consisting of proteasome inhibitors, inhibitors of the ubiquitin, agents that interfere with the activation of NF-? B by means of the ubiquitin-proteasome pathway, and mixtures thereof. In a preferred embodiment, the combination comprises an amount of the agent, sufficient to reduce the frequency of dose or treatment, required for the glucocorticoid. In certain preferred embodiments, the combination comprises an amount of the glucocorticoid that is less than its standard recommended dose. In another preferred aspect, the combination comprises an amount of the glucocorticoid, sufficient to reduce the frequency of dose or treatment required for the agent. In certain preferred embodiments, the glucocorticoid is selected from the group consisting of flunisolide, triamcinolone-acetonide, beclomethasone dipropionate, dexamethasone sodium phosphate, fluticasone propionate, budesonide, hydrocortisone, prednisone, prednisolone, mometasone, tipredane, and butixicort. In other preferred embodiments, the combination used to treat a patient afflicted with asthma comprises a glucocorticoid and a proteasome inhibitor. More preferably, the proteasome inhibitor is lactacystin or a lactacistin analogue. More preferably, the combination comprises the β-lactone of 7-n-propyl-clasto-lactacystin and budesonide. The invention is further directed to pharmaceutical compositions comprising a combination of a glucocorticoid and an agent selected from the group consisting of proteasome inhibitors, inhibitors of the ubiquitin pathway, agents that interfere with the activation of NF-? B by means of the ubiquitin-proteasome pathway, or mixtures thereof. In certain embodiments, the pharmaceutical composition is provided in a unit dosage form. Preferably, the unit dose form comprises the glucocorticoid in an amount that is less than its standard recommended dose.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a graphical representation of the mean clinical score as a function of time in an experimental autoimmune encephalomyelitis model. These data demonstrate that treatment with 3b (β-lactone 7-n-propyl-clasto-lactacystin) causes a reduction in the rate of relapse and the average clinical score compared to animals treated with vehicle. Figure 2 is a graphic representation of the rate or rate of relapse, as a function of time in a model of experimental autoimmune encephalomyelitis. These data demonstrate that treatment with .3b causes a reduction in the proportion and severity of relapse. Figure 3 is a graphic representation of the leukocyte count in the bronchoalveolar lavage fluid from clean Brown (N) or actively sensitized (AS) rats, 72 hours after exposure to aerosolized ovalbumin (10 mg / ml) . Treatment with 3b causes a dose-dependent reduction in the influx of leukocytes. Figure 4 is a graphical representation of the eosinophil count in the bronchoalveolar lavage fluid from clean Brown (N) or actively sensitized (AS) rats, 72 hours after exposure to aerosolized ovalbumin (10 mg / ml) ). Treatment with 3b causes a dose-dependent inhibition of eosinophilia in this model. Figure 5 is a graphical representation of the leukocyte count in the bronchoalveolar lavage fluid from clean, untreated Brown Norway rats (N); actively sensitized, treated with vehicle (V); or actively sensitized, treated with drug (A-H), 72 hours after exposure to aerosolized ovalbumin (10 mg / ml). Treatment with budesonide alone (0.1 mg / kg) or 3b alone (0.03 or 0.1 mg / kg) was not effective. However, the combination of budesonide (0.1 mg / kg) with 3b (0.03 or 0.1 mg / kg) causes a reduction in leukocyte influx in this model. High dose budesonide (0.5 mg / kg) is effective with or without added 3b. Figure 6 is a graphic representation of the eosinophil count in the bronchoalveolar lavage fluid from clean, untreated Brown Norway (N) rats; actively sensitized, treated with vehicle (V); or actively sensitized, treated with drug (A-H), 72 hours after exposure to aerosolized ovalbumin (10 mg / ml). Treatment with budesonide alone (0.1 mg / kg) or 3b alone (0.03 or 0.1 mg / kg) was not effective. However, the combination of budesonide (0.1 mg / kg) with 3b (0.03 or 0.1 mg / kg) causes a reduction in eosinophilia in this model. High dose budesonide (0.5 mg / kg) is effective with or without added 3b.

DETAILED DESCRIPTION OF THE PREFERRED MODALITIES This invention is directed to the compositions and methods for the treatment of inflammatory and autoimmune diseases. All patent applications, patents and literature references cited herein are incorporated by reference in their entirety. In the case of inconsistencies, the present description will prevail. The invention provides methods for the treatment of an affected patient with multiple sclerosis or asthma, comprising administering to said patient an effective amount of an agent selected from the group consisting of inhibitors of the proteasome, inhibitors of the ubiquitin pathway, agents that interfere with the activation of NF-? B by means of the ubiquitin-proteasome pathway, and mixtures thereof. It has now been unexpectedly discovered that the ubiquitin-proteasome pathway is an objective for the treatment of multiple sclerosis, asthma and rheumatoid arthritis. In the present description, the following definitions will be used. "Treatment" will mean any improvement of any symptom according to the administration of any inhibitor of the proteasome, inhibitor of the ubiquitin pathway, or agent that interferes with the activation of NF-? B by means of the ubiquitin pathway. proteasome "Ubiquitin pathway inhibitor" will mean any substance that specifically inhibits ubiquitination or the transfer of ubiquitin to proteins. "Proteasome inhibitor" shall mean any substance that specifically inhibits the proteasome or the activity thereof. "Agents that interfere with the activation of NF-? B by the ubiquitin-proteasome pathway" will mean any substance that 1) specifically inhibits the proteasome or the activity of the same; 2) specifically inhibits the ubiquitination of I? B-a or pl05; or 3) specifically inhibits the phosphorylation of I? B-a or pl05.

"Specifically inhibit" will mean that it interferes with the ability of a protein to mediate its biological function at a concentration of inhibitor that is less than the concentration of inhibitor required to produce another, unrelated biological effect. Preferably, the concentration of the inhibitor, required for such interference is at least 2 times lower, more preferably at least 5 times lower, still more preferably at least 10 times lower, and more preferably at least 20 times lower than the concentration required to produce a biological effect not related. Such inhibitors can act by any of a variety of mechanisms, including without limitation, interference with the active site or conformation of the protein, interference with the ability of the protein to interact with another protein, the substrate or a co-factor , either by an effect on the protein itself or on another protein, substrate or co-factor, and altering the microenvironment in which the biological function of the protein normally occurs. In a first aspect, the invention provides methods for treating multiple sclerosis. Multiple sclerosis (MS) is an incurable neurological disease that frequently causes chronic diseapacity. It is widely believed that MS is the result of an autoimmune disorder in a genetically susceptible individual, mediated by the autoreactive T cells that migrate to the central nervous system and initiate the inflammatory deslininization lesion. The observation that MS is an autoimmune disease is derived in part from the systemic abnormalities of immune function observed in patients with the disease, and partly through the similarity with experimental autoimmune encephalomyelitis (EAE), which in turn It serves as a model for human disease (Kennedy et al., J. Ne uroimmun ol. (1987) 16: 345; Arnason et al., Ne urol. Cl in. (1983) 1: 765; van der Veen et al., J Neuroimmunol. (1989) 48: 213; Gonatas et al., Immunol. Today (1986) 7 121; Wckerle Ac ta Neurol. (1991) 13: 197). EAE is a disease of autoimmune, inflammatory demyelination, mediated by the T cell, of the CNS. The disease can be induced in a number of experimental laboratory animals, including primates, by injection of the whole brain homogenate, a purified preparation of myelin basic protein (MBP) or proteolipoprotein (PLP) in adjuvant. EAE is a disease mediated by T cells, and the passive transfer of T cells reactive with MBP or PLP is sufficient to induce the disease. Experimental relapsing-remitting autoimmune encephalomyelitis (R-EAE) is induced in SJL / J mice by immunization with the immunodominant epitope on the proteolipid protein (PLP139-151) or by adoptive transfer of CD4 + T cells specific for PLP139-151 ( McRac et al., J. Neuroimmun ol. (1992) 38: 229). Clinical disease is characterized by an acute paralytic phase followed by recovery and subsequent relapses. This pattern of relapse and spontaneous recovery in the experimental animal model, which occurs over a period of weeks to months, is very similar to the clinical signs of the disease observed in patients with multiple sclerosis (MS), in many years. The method according to this aspect of the invention comprises the administration to a patient affected with MS of an effective amount of an agent selected from the group consisting of proteasome inhibitors, inhibitors of the ubiquitin pathway, agents that interfere with the activation of NF-? B by means of the ubiquitin-proteasome pathway, and mixtures thereof. In a preferred embodiment, the agent is administered in an amount sufficient to reduce the frequency or severity of the relapse of the disease. When administered during the remission phase at doses of 0.3 to 1.0 mg / kg i.p., the proteasome inhibitor 3b reduces the proportion and severity of relapse in the R-EAE model (Figures 1-2). In a second aspect, the invention provides a method for treating asthma. Asthma is an obstructive disease of the lungs characterized by hyper-responsiveness of the respiratory tract, which is an exaggerated narrowing of the airways in response to many different stimuli, such as histamine, exercise, cold air and allergens . Due to the constriction in episodes of the bronchial tubes, the treatment has been partially based on bronchodilation by ß-adrenergic agonist drugs. More recently, however, it has come to be appreciated that asthma is a chronic condition of the airways that involve many types of inflammatory cells and. the release of many mediators and neurotransmitters that have multiple effects on the various target cells in the respiratory tract. The degree and extent of inflammation in the wall of the respiratory tract are largely related to the clinical severity of asthma. The inflammatory response of asthma consists of the activation of mast cells residing in the respiratory tract, increased numbers of lymphocytes (which are mainly CD4 + T lymphocytes) and an infiltration with eosinophils, which seem to degranulate. The increased total eosinophil count in the peripheral blood is almost invariably present unless it is suppressed by corticosteroids or sympathomimetic drugs. The sputum examination also reveals eosinophils. Several animal models have been developed to study pulmonary inflammation with characteristic manifestations of eosinophilia of the respiratory tract. One of the animal models frequently used is coballo sensit with ovalbumin (Dunn et al., Am. Rev. Respir Dis. (1990) 142: 680; Sanjar et al., Br. J. Pharmacol. (1990) 99: 679; Gulbenkian et al., Am. Rev. Respir Dis. (1990) 142: 680). Selective accumulation of neutrophils and eosinophils has also been described in acutely sensit Brown Norway rats (Kips et al., Am. Rev. Respir Dis. (1992) 145: 1306; Richards et al., Agents Actions. Suppl. 34 (1991) 34: 359; Chapman et al., Am. J. Resp. Crit. Care Med (1996) 153; A219). The allergen-induced pulmonary eosinophilia in actively sensit Brown Norway rats is inhibited by the steroid dexamethasone. Glucocorticoid therapy remains one of the most effective anti-inflammatory treatments available, and these drugs have been shown to reduce pulmonary eosinophilia in asthmatic patients (Holgate et al., Int.Arch.Allergy Appl. Immunol. (1991) 94: 210). The method according to this aspect of the invention comprises the administration to an affected patient with asthma, an effective amount of an agent selected from the group consisting of proteasome inhibitors, inhibitors of the ubiquitin pathway, agents that interfere with activation of NF-? B by means of the ubiquitin-proteasome pathway, and mixtures thereof. In a preferred embodiment, the agent is administered in an amount sufficient to reduce the frequency or severity of asthmatic attacks. When administered intratracheally one hour before and 24 hours and 48 hours after challenge with the allergen, 3b (0.1 or 0.3 mg / kg) inhibited eosinophilia in actively sensit Brown Norway rats (Figures 3 and 4). Further contemplated within the scope of the invention, is administration combined with another drug or drugs used to treat asthma. Currently accepted therapies for asthma include cromoglycate, nedrocromil, theophylline, short-acting and long-acting β2-adrenergic receptor agonists, and inhaled or oral glucocorticoids. The most recently developed therapeutics include inhibitors of leukotriene biosynthesis, leukotriene receptor antagonists, and thromboxane antagonists. Anti-IL-5 and anti-IgE antibodies are being developed (Sci in ce (1997) 276: 1643), and antisense procedures are also being investigated (Nyce and Metzger, Na t ure (1997) 383: 721). In a preferred embodiment, the agent of the invention is used in an amount sufficient to reduce the dose or frequency of treatment required for the other drug or drugs. In another preferred embodiment, the other drug or drugs are used in an amount sufficient to reduce the dose or frequency of treatment required for the agent of the invention. The agent can be administered at the same time as the other drug or drugs, or it can be administered at a different time. Steroid therapy is particularly effective for the treatment of asthma, and is an essential therapy line for severe asthmatics. Unfortunately, however, a number of unwanted side effects result from long-term steroid use, including suppression of bone growth, adrenal insufficiency, Cushing's syndrome, cataracts, immunosuppression, and excessive contusions. Many of these effects can be minim by topical administration of the drug to the lung, by inhalation. However, high doses, such as those required in severe cases, result in significant systemic exposure and an increase in associated collateral increases. The drugs that allow the reduction in the steroid dose ("steroid shortage") thus offers very real clinical benefit. Unexpectedly, it has been found that intratracheal administration of 3b (0.03 or 0.1 mg / kg) in combination with the glucocorticoid budesonide (0.1 mg / kg) 1 hour before and 24 hours and 48 hours after challenge with the allergen inhibits eosinophilia in Brown Norway rats actively sensitized (Figures 5-6). 'Surprisingly, no drug was effective when administered alone at these doses, suggesting the synergistic action of the two drugs. In a third aspect, the invention provides methods for treating an affected patient with asthma, which comprises administering to the patient a combination of a glucocorticoid and an agent selected from the group consisting of proteasome inhibitor, inhibitors of the ubiquitin, agents that interfere with the activation of NF-? B by means of the ubiquitin-proteasome pathway, and mixtures thereof. The glucocorticoid and the agent can be administered at the same or at different times, on the same or different days, and with the same or different frequency. Preferably, the doses of each drug are spaced apart to achieve a combined physiological effect. Preferably, the glucocorticoid is administered between 0 minutes and about one month before or after the inhibiting agent, more preferably between 0 minutes and about one week before or after the agent of the invention, more preferably between 0 minutes and 24 hours before or after the agent of the invention. Glucocorticoids for use in the invention include, but are not limited to, flunisolide, triamcinolone acetonide, beclomethasone dipropionate, dexamethasone sodium phosphate, fluticasone propionate, budesonide, hydrocortisone, prednisone, prednisolone, mometasone, tipredane, and butixicort. Preferably, the glucocorticoid is budesonide. Suitable formulations, dosages and routes of administration for glucocorticoids are known in the art (Physi ci an 's Desk Reference, 51 Edition, 1997, Medical Economics: Montvale, NJ).

In certain preferred embodiments, the agent of the invention is administered in an amount sufficient to reduce the dose Q the frequency of treatment required for the glucocorticoid. Preferably, the amount of glucocorticoid administered does not exceed the standard recommended dose, and more preferably the amount of glucocorticoid administered is less than the standard dose recommended for the drug, when administered alone. In certain preferred embodiments, the amount of glucocorticoid administered is sufficient to reduce the dose or frequency of treatment required for the agent selected from the group consisting of proteasome inhibitors, inhibitors of the ubiquitin pathway, agents that interfere with the activation of NF-? B by means of the ubiquitin-proteasome pathway, and mixtures thereof. More preferably, the treatment of a patient afflicted with asthma with a combination of a glucocorticoid and an agent selected from the group consisting of proteasome inhibitors, inhibitors of the ubiquitin pathway, agents that interfere with the activation of NF-? B by means of the ubiquitin-proteasome pathway, and mixtures thereof produce efficacy with few or fewer severe side effects or less toxicity than treatment with the drug alone. In a fourth aspect, the invention provides the pharmaceutical compositions comprising a combination of a glucocorticoid and an agent selected from the group consisting of proteasome inhibitors, inhibitors of the ubiquitin pathway, agents that interfere with the activation of NF-? B by means of the ubiquitin-proteasome pathway, and mixtures thereof, are further contemplated within the scope of the invention. The pharmaceutical compositions of the invention can be provided in unit dosage form. In a preferred embodiment, the unit dosage form contains an amount of glucocorticoid that is less than its recommended standard dose, when administered alone. The following description of non-limiting examples of suitable proteasome inhibitors, inhibitors of the ubiquitin pathway, and agents that interfere with the activation of NF-? B by means of the ubiquitin-proteasome pathway, apply to pharmaceutical formulations as well. as to the methods according to the invention.

Non-limiting examples of proteasome inhibitors for use in the present invention include peptidyl aldehydes (Orlowski et al., US Patent No. 5,580,854, Stein et al., W095 / 24914, Siman et al., O91 / 13904, Iqbal et al. J. Med. Chem. 38: 2276-2277 (1995)), peptidylboronic acids (Ada et al., 096/13266; Siman et al., O91 / 13904), other peptidyl derivatives with proteasome inhibitory activity (Iqbal et al. U.S. Patent No. 5,614,649, Iqbal et al, U.S. Patent No. 5,550,262, Spaltenstein et al, Tetrahedron Letters 1996, 37, 1343), and lactacystin and lactacystin analogs (Fenteany et al., Proc. Na ti. Acad. Sci. USA (1994) 91: 3358; Fenteany et al., O96 / 32105; Soucy et al., U.S. Patent Application Serial No. 08 / 912,111, filed on 8/15/97)). The agents described herein can be administered by any route, including intradermally, intraperitoneally, intranasally, itratracheally, subcutaneously, orally or intravenously. For asthma indications, administration is preferably by the inhalation route. The peptide-aldehyde proteasome inhibitors for use in the present invention are preferably those described in Stein et al., 095/24914 published on September 21, 1995, or Siman et al., O91 / 13904 published on September 19. of 1991, both incorporated by reference herein in their entirety. Boronic acid or ester compounds for use in the present invention preferably include those described in Adams et al., W096 / 13266, Siman et al., WO91 / 13904, or Iqbal et al., US Patent No. 5,614,649, each of which is hereby incorporated by reference in its entirety. In certain preferred embodiments, the boronic acid compound for use in the present invention is selected from the group consisting of: N-acetyl-L-leucine-β- (1-naphthyl) -L-alanine-L-leucine boronic, ß- (1-naphthyl) -L-alanine-L-leucine-boronic acid, N- (4-mproline) carbonyl-β- (1-naphthyl) -L-alanine-L-leucine-boronic acid, acid N- (8-quinoline) sulfonyl-β- (1-naphthyl) -L-alanine-L-leucine-boronic acid and N- (4-morpholine) carbonyl- [O- (2-pyridylmethyl)] -L-tyrosine -L-leucine-boronic. The lactacystin and lactacystin analog compounds for use in the present invention preferably include those described in Fenteany et al., WO96 / 32105 or Soucy et al., U.S. Patent Application No. (08 / 912,111; filed on 08/15/97), each of which is incorporated by reference herein. its entirety In certain preferred embodiments, the lactacystin analog compound is selected from the group consisting of lactacystin, β-lactone from lactate to lactase, β-lactone from 7-ethyl-clasto-lactacetine, β-lactone from 7-n- propyl-clasto-lactacystin, and ß-lactone of 7-n-butyl-clasto-lactacy tub. These compounds can be prepared as illustrated in Schemes 1 and 2. More preferably, the lactacystin analog compound is the β-lactone of 7-n-propyl-clasto-lactacystin (3b (Reaction Scheme 2)). In a preferred embodiment, the agent used to treat an affected patient with multiple sclerosis or asthma is a proteasome inhibitor. Preferably, the proteasome inhibitor is lactacystin or a lactacystin analogue, more preferably 7-n-propyl-clasto-lactacystin β-lactone. Preferably, the combination used to treat a patient affected with asthma comprises a glucocorticoid and a proteasome inhibitor. More preferably, the proteasome inhibitor is lactacystin or a lactacystin analog. More preferably, the combination comprises β-lactone of 7-n-propyl-clasto-lactacystin and budesonide.

Reaction Scheme 2 1. 0.1 VNaOH, EtOH 2. TBTU, MeCN 3a -3e Reaction Scheme 1 EtjNH, TBT?, DEA MtCWCH.Ct-. ri Non-limiting examples of inhibitors of the ubiquitin pathway include those described in Berleth et al., Bi och em. 35 (5): 1664-1671 (1996). Inhibitors of I? B-a phosphorylation are also known (Chen, Cel l 84: 853 (1996); Chen, US Patent Application No. 08 / 825,559). The concentration of a described compound in a pharmaceutically acceptable mixture will vary depending on various factors, including the dose of the compound to be administered, the pharmacokinetic characteristics of the compound (s) employed, and the route of administration.

The effective amounts of the agents for treating multiple sclerosis, asthma or rheumatoid arthritis would be broadly in the range between about 10 μg and about 50 μg per kg of body weight of a mammalian patient. The agent can be administered in a single dose or in repeated doses. The treatments may be administered daily or more frequently depending on a number of factors, including the age and general health of a patient, and the formulation of the route of administration of the selected compound (s). Other factors that will be considered in the determination of the dose include the type of concurrent treatment, if any; the frequency of the treatment and the nature of the desired effect; the degree of tissue damage; the sex the duration of the symptoms; the contraindications, if any; and other variables that are going to be evaluated by the individual doctor. In certain preferred embodiments, the concentration of the proteasome inhibitor is determined by measuring the activity of the proteasome ex vi ve after administering the proteasome inhibitor to the mammal. Such measurement comprises obtaining one or more biological test samples from a mammal at one or more specified times after administration of the proteasome inhibitor; the measurement of proteasome activity in the biological sample or in the samples; determining the amount of proteasome activity in the sample or biological samples tested; and comparing the amount of proteasome activity in the biological sample tested to that in a biological reference sample obtained from a mammal to which the proteasome inhibitor has not been administered. Biological samples that are obtained from the mammal may include, without limitation, blood, urine, organ and tissue samples. In certain preferred embodiments, the biological sample is a blood sample, more preferably a blood sample from which the white blood cells are isolated before measuring proteasome activity. Methods for fractionating blood cells are known in the art and are further described in the examples. Hemoglobin strongly interferes with fluorescence measurements and is thus preferably excluded from test samples when fluorometric assays are used for proteasome activity. Once fractionated, the white blood cells are used by standard procedures. The variability in lysis efficiency can be corrected by determining the total protein content in the sample, using standard procedures and normalizing the proteasome activity measured in the sample in relation to the protein content in the sample. Preferably, a substrate having a detectable level is provided to the reaction mixture and the proteolytic cleavage of the substrate is checked periodically following the disappearance of the substrate or the appearance of a cleavage product. The detection of the marker can be achieved, for example, by fluorometric, colorimetric and radiometric assay. More preferably, the substrate is a peptide substrate and the reaction mixture further comprises a 20S proteasome activator. preferably, the activator is one shown in Coux et al.

(Ann.Rev. Biochem. 65: 801-847 (1995)), more preferably PA28 or sodium dodecylsulfate (SDS). Preferably, the peptide substrate contains a cleavable fluorescent label and the release of the label is periodically verified by fluorometric assay. More preferably, the substrate is N-succinyl-leucyl-valyl-tyrosyl-7-amino-4-methylcoumarin (Suc-Leu-Leu-Val-Tyr-AMC). The activity measured in the biological test samples is compared to that measured in a biological reference sample obtained from a mammal to which the inhibitor has not been administered. In some preferred embodiments, the biological test sample and the reference biological sample each separately comprise a plurality of combined samples from a group of mammals, preferably mice, undergoing treatment. In other preferred embodiments, the biological test sample and the reference biological sample each comprise a simple sample obtained from an individual mammal. The testing of individual samples is currently preferred except when it is impractical due to the small size of the mammal. In some preferred embodiments, a statistical sample is obtained by combining the data from the individual biological test samples or the individual reference biological samples. The day-to-day variability in the test can result from factors such as differences in buffer solutions, operator variability, variability in instrument performance, and temperature variability. Such variability can be minimized by standardizing the proteasome activity in the biological sample and in the reference sample relative to a standard proteasome sample. In certain preferred embodiments, the standard sample comprises the purified 20S proteasome, more preferably the purified 20S proteasome from rabbit reticulocytes. In some preferred embodiments, the reference sample is obtained from the mammal treated before the start of treatment. This modality is currently preferred for higher mammals, in order to minimize the impact of inter-mammals variability. The clinical verification of the action of the drug preferably involves, currently, this modality of the invention, with each patient serving as its own baseline control.

A decrease in the inhibitory activity in the biological sample, in comparison to the reference sample, is indicative of an inhibitory effect of the inhibitor at the time in which the biological sample was obtained. In some preferred embodiments, biological samples are obtained at multiple time points after administration of the inhibitor. In these embodiments, the measurement of proteasome activity in the biological samples provides an indication of the extent and duration of the inhibitory effect of the inhibitor. In certain other preferred embodiments, multiple biological samples are obtained from a single mammal at one or more time points. In these embodiments, the measurement of the proteasome activity in the biological samples provides an indication of the distribution of the inhibitor in the mammal. In certain preferred embodiments, the amount of dose and the dose frequency of the proteasome inhibitor are selected to avoid excessive inhibition of the proteasome. In some embodiments, excessive inhibition of proteasome results in a toxic effect, the toxic effect includes, but is not limited to, vomiting, diarrhea, hypovolemia, hypotension and lethality. Preferably, the amount of dose and the dose frequency of the proteasome inhibitor are selected such that the inhibition of proteasome in any future biological sample does not exceed about 95%. In certain other preferred embodiments, the amount of dose and the dose frequency of the proteasome inhibitor are selected such that proteasome inhibition is achieved., therapeutically useful. Preferably, therapeutically useful proteasome inhibition results in an antitumor, antiinflammatory, antiviral and antiparasitic, therapeutically beneficial effect. Preferably, the dose amount and the dose frequency of the proteasome inhibitor are selected such that the proteasome inhibition is at least about 15%, preferably about 20%, more preferably about 30%, even more preferably about 40%, and still more preferably about 50%, and most preferably from about 50% to about 80%, is achieved in a future biological sample, although in some cases inhibition of proteasome as high as 95% may be preferred. Agents for the use of this invention can be prepared for administration by any of the methods well known in the pharmaceutical art, for example, as described in Remington Pharmaceuticals Scells (Marek Pub. Co., Easton, PA, 1980). The agents can be prepared for use in parenteral administration in the form of liquid solutions or suspensions; for oral administration in the form of tablets or capsules; for intranasal or intratracheal administration in the form of powders, gels, oily solutions, nasal drops, aerosols, or mists. Formulations for parenteral administration may contain common excipients such as sterile water or sterile saline, polyalkylene glycols such as polyethylene glycol, oils of vegetable origin, hydrogenated naphthalenes, and the like. The controlled release of an agent can be obtained, in part, by the use of biocompatible, biodegradable, lactide polymers, and lactide / glycolide or polyoxyethylene / polyoxypropylene copolymers. Additional parenteral administration systems include ethylene-vinyl acetate copolymer particles, osmotic pumps, implantable infusion systems, and liposomes. Formulations for administration by inhalation may contain lactose, polyoxyethylene-9-lauryl ether, glycocholate or deoxycholate. For the treatment of asthma, the route of administration by inhalation is preferred in order to minimize potential side effects or toxicity resulting from systemic exposure to the agent. According to the present invention, an "effective amount" of an agent is an amount sufficient to produce any improvement of any symptom or sign of the disease (Sites et al., Ba si c &Cl ini cal Immunol ogy Lange Medical Publications, Los Altos, California, 1982). The use of any of the agents described herein in combination with another agent or agents used in the treatment of multiple sclerosis or asthma is further contemplated within the scope of the present invention. The invention is further exemplified by the following non-limiting examples: EXAMPLES Example 1: Experimental Relapsed-Remission Autoimmune Encephalomyelitis Materials and methods Mice 6-week-old SJL / J female mice were purchased from Harlan Laboratories (Indianapolis, IN), housed in the Northwestern animal care facility, and maintained in a standard laboratory with food and water ad libitum. The paralyzed mice were given easier access to food and water. Peptides PLP139-151 '(HSLGK LGHPDKF) was purchased from Peptides International (Louisville, KY). The amino acid composition was verified by mass spectrometry and the purity (> 98%) was evaluated by HPLC. Induction of R-EAE. Mice were immunized by subcutaneous injection of PLP139-151 in complete Freund's adjuvant (CFA) as previously described (McRae et al., J. Neuroimmunol. (1992) 38: 229). Each mouse received 50 μg of PLP139-151 distributed over 2 sites on each hind flank. Tra tami on to Drug. Beginning on day 22, the animals (10 per group) were dosed once daily i.p. (5 ml / kg) with vehicle or with 3b (0.3 or 1.0 mg / kg). Treatment continued until day 40. Clinical evaluation. Mice were observed daily for clinical signs of the disease. The mice were classified according to their clinical severity as follows: grade 0, without abnormality; grade 1, flaccid tail; grade 2, flaccid tail and weakness of hind legs (wavering gait); grade 3, partial paralysis of hind legs; grade 4, complete paralysis of the hind legs; and grade 5, dying.

Results The data are graphically plotted as the daily, mean clinical score for all the animals in a particular treatment group (Figure 1). A relapse was defined as an increase of at least one full degree in the clinical score after the animal had previously improved at least one complete clinical score and had stabilized. Animals treated with 3b (both groups of doses) showed reduced clinical scores compared to animals treated with vehicle. The incidence of relapse was 5/10 for the group of 0.3 mg / kg and 2/10 for the group of 1.0 mg / kg, compared to 6/10 for the group treated with vehicle. The data are also treated graphically as the average incidence of daily relapse for all animals in a particular treatment group (Figure 2). The average maximum clinical score per group is also provided as an indication of the severity of the disease. Animals treated with 3b (both groups of doses) showed reduced relapse rate and reduced severity of the disease compared to animals treated with vehicle.

Example 2: Effect of Treatment with 3b on the Accumulation of Pulmonary Leukocytes Induced by Allergen in Brown Norway Rats Actively Sensitized Materials and methods .Ratings. Brown Norway male rats were supplied by Harlan Olac Limited (Bicester, Oxon, UK) and distributed within the weight range of 180-200 g. After acclimation for at least five days, the animals were actively sensitized in a period of 3 weeks and were within the weight range of 250 to 300 grams at the time of exposure to the allergen. Food and water were provided ad libitum. Sensitization . Ovalbumin (OA, 10 μg) mixed with aluminum hydroxide gel (10 mg) will be injected (0.5 ml, i.p.) in Brown Norway rats and repeated 7 and 14 days later. Drug treatment. On day 21, the sensitized rats were anesthetized (5% halothane in 02) and 3b, dexamethasone, or vehicle (lactose) was instilled through a cannula placed directly into the trachea 1 hour before exposure to OA. This procedure was repeated at 24 hours and 48 hours after exposure to OA. Challenge After recovery, the sensitized animals were restrained in plastic tubes and exposed (60 minutes) to an OA aerosol (10 mg / ml) using a nose-only exposure system. The animals were sacrificed 72 hours later with pentobarbital (250 mg / kg i.p.). Analysis . Lungs were washed using 3 aliquots of 4 ml of Hank's solution (HBSS X 10, 100 ml; EDTA 100 M, 100 ml; HEPES 1M, ml graduated to 11 ml with water); the recovered cells were combined and the total volume of the recovered fluid was adjusted to 12 ml by the addition of Hank's solution. Total cells were counted (Sysmex Counter Microcell F-500, TOPA Medical Electronics Ltd., Japan). Smears were made by diluting the recovered fluid (up to approximately 10 6 cells / ml) and centrifuging a 100 μl aliquot in a centrifuge (Cytospin, Shandon UK). The smears were air-dried, fixed using a solution of bright green in methanol (2 mg / liter) for 5 seconds and stained with eosin G (5 seconds) and thiazine (5 seconds) (Diff-Quik, Baxter Dade Ltd, Switzerland) in order to differentiate eosinophils, neutrophils, macrophages and lymphocytes. A total of 500 cells were counted per smear by light microscopy under immersion oil (x 1000).

Results The challenge with ovalbumin resulted in a significant increase in the eosinophils, neutrophils and total leukocytes in the BAS fluid from actively sensitized Brown Norway rats (AS) compared to the clean rats (N) Dexamethasone treatment (0.1 mg / kg, it) prevented this increase, at doses of 0.1 and 0.3 mg / kg, 3b reduced the influx of eosinophils and total leukocytes, and a significant decrease in the lymphocyte count was observed at all doses (Figures 3 and 3). 4) . conclusion Compound 3b is effective in preventing the influx of leukocytes after challenge with the allergen in an animal model of asthma.

Example 3: Effect of Treatment with a Combination of 3b and Budesonide on the Accumulation of Pulmonary Leukocytes Induced by Allergen in Brown Norway Rats Actively Sensitized Materials and methods JRatas. Male Brown Norway rats were supplied by Harlan Olac Limited (Bicester, Oxon, United Kingdom) and distributed within the weight range of 180 to 200 g. After acclimation for at least five days, the animals were actively sensitized in a period of 3 weeks and were within the weight range of 250 to 300 g at the time of exposure to the allergen. Food and water were provided ad libitum. Sensitization . Ovalbumin (OA, 10 μg) mixed with aluminum hydroxide gel (10 mg) will be injected (0.5 ml, i.p.) in Brown Norway rats and repeated 7 and 14 days later. Tra tami on to Drug. On day 21 ,. the sensitized rats were anesthetized (halothane at 5% in 02) and treated intratracheally (it) 1 hour before exposure to OA with vehicle (group V; lactose, 1 mg), budesonide (group C, 0.1 mg / kg; group F, 0.5 mg / kg), 3b (group A, 0.03 mg / kg, group B, 0.1 mg / kg), or mixtures of budesonide and 3b (group D, 0.1 / 0.3 mg / kg, group E, 0.1 / 0.1 mg / kg, group G, 0.5 / 0.03 mg / kg, group H, 0.5 / 0.1 mg / kg). The drug was instilled by means of a cannula placed directly into the trachea. This procedure was repeated at 24 hours and 48 hours after exposure to OA. Re t o. After recovery, sensitized animals were restrained in plastic tubes and exposed for 60 minutes to an OA aerosol (10 mg / ml) using a nose-only exposure system. The animals were sacrificed 72 hours later with pentobarbital (250 mg / kg, i.p.). Analysis . The lungs were washed using 3 aliquots of 4 ml in Hank's solution (HBSS X 10, 100 ml, 100 mM EDTA, 100 ml, 1M HEPES, 10 ml graduated to 11 ml with water); the recovered cells were recovered and the total volume of the recovered fluid was adjusted to 12 ml by the addition of Hank's solution. The total cells were counted (Sysmex Counter Microcell F-500, TOPA Medical Electronics Ltd., Japan). Smears were made by diluting the recovered fluid (up to approximately 10 6 cells / ml) and centrifuging a 100 μl aliquot in a centrifuge (Cytospin, Shandon, UK). The smears were air-dried, fixed using a solution of bright green in methanol (2 mg / liter) for 5 seconds and stained with eosin G (5 seconds) and thiazine (5 seconds) (Diff-Quik, Baxter Dade Ltd ., Switzerland) in order to differentiate eosinophils, neutrophils, macrophages and lymphocytes. A total of 500 cells were counted per smear by light microscopy under immersion oil (x 1000).

Results The challenge with ovalbumin resulted in a significant increase in eosinophils, neutrophils, and total leukocytes in the BAS fluid from Brown Norway rats treated with vehicle (V) actively sensitized, as compared to the untreated, clean rats (N ). At doses of 0.03 mg / kg (A) and 0.1 mg / kg (B), 3b failed to prevent this increase. At a dose of 0.1 mg / kg (C), budesonide also had no effect when administered alone. However, the combination of 0.1 mg / kg of budesonide with 3b at 0.03 mg / kg of (D) or 0.1 mg / kg of (E) produced a significant reduction in the eosinophil count. The statistically significant reduction in the neutrophil count was achieved only in group (D) at 0.1 / 0.03 mg / kg. At a higher dose (0.5 mg / kg, group F), treatment with budesonide alone was effective in preventing the increase in eosinophils, neutrophils, and total leukocytes, and the combination of budesonide (0.5 mg / kg) with 0.03 mg was also effective. / kg of (G) or 0.1 mg / kg of (H) of 3b (Figures 5-6). conclusion The combination of compound 3b with the glucocorticoid budesonide is effective in preventing the leukocyte influx after challenge with the allergen in an animal model of asthma at doses where no drug only had any effect.

Example 4: Preparation of form ami s 14 (Reacting Scheme 1) Acyloxazolidinone 9b (R = n-Pr) A solution cooled to -78 ° C of (S) - (-) - 4-benzyl-2-oxazolidinone (4.0 g, 22.6 mmol) in 75 ml of anhydrous tetrahydrofuran was treated with a 2.5 M solution of n-BuLi in hexane (9.1 ml, 22.6 mmol) in 15 minutes. After 5 minutes, pure valeryl chloride (2.95 ml, 24.9 mmol) was added dropwise, and the mixture was stirred for another 45 minutes at -78 ° C. The mixture was then allowed to reach room temperature, stirred for another 90 minutes, and then treated with 50 ml of saturated ammonium chloride solution. Then 50 ml of dichloromethane was added and the organic layer was washed with 2 30 ml portions of brine, dried over magnesium sulfate, and concentrated in vacuo. This afforded 5.94 g (100%) of the desired acyloxazolidinone 9b as a colorless oil. NMR A (300 MHz, CDCl) d 7.36-7.20 (m, 5H), 4.71-4.64 (m, 1H), 4.23-4.14 (m, 1H), 3.40 (dd, J = 13.3, 3.2 Hz, 1H), 3.04-2.84 (m, 2H), 2.77 (dd, J = 13.3, 9.6 Hz, 1H), 1.74-1.63 (m, 2H), 1.46-1.38 (m, 2H), 0.96 (t, J = 7.3 Hz, 3H).

Acyloxazolidinone 9a (R = Et) By an analogous procedure to that described for the preparation of acyloxazolidinone 9b, the lithium anion of (S) - (-) -4-benzyl-2-oxazolidinone was treated with butyryl chloride to provide acyloxazolidinone 9a with 94% of performance. NMR A (300 MHz, CDC13) d 7.37-7.20 (m, 5H), 4.68 (ddd, J = 13.1, 7.0, 3.4 Hz, 1H), 4.23-4.13 (m, 2H), 3.30 (dd, J = 13.3 , 9.6 Hz, 1H), 3.02-2.82 (m, 2H), 2.77 (dd, J = 13.3, 9.6 Hz, 1H), 1.73 (q, J = 7.3 Hz, 2H), 1.01 (t, J = 7.3 Hz , 3H).

Acyloxazolidinone 9c (R = n-Bu) By an analogous procedure to that described for the preparation of acyloxazolidinone 9b, the lithium anion of (S) - (-) -4-benzyl-2-oxazolidinone was treated with hexanoyl chloride to provide acyloxazolidinone 9a with 96% performance. NMR A (300 MHz, CDC13) d 7.36-7.20 (m, 5H), 4.68 (m, 1H), 4.23-4.14 (m, 2H), 3.30 (dd, J = 13.3, 3.3 Hz, 1H), 3.02- 2.83 (m, 2H), 2.76 (dd, J = 13.3, 9.6 Hz, 1H), 1.70 (m, 2H), 1.43-1.34 (m, 4H), 0.92 (t, J = 3.3 Hz, 3H). 4-Methylvaleryl Chloride 4-Methylvaleryl chloride was prepared from commercially available 4-methylvaleric acid in the following manner: to a cold solution of 0 ° C of 4-methylvaleric acid (1.85 ml, 15.0 mmol) in 50 ml of anhydrous methylene chloride which containing 10 ml of dimethylformamide was treated with 1.95 μl of oxalyl chloride (22.5 mmol). The mixture was then stirred for 3 hours at room temperature, concentrated in vacuo and filtered to provide 1.6 g (100%) of the desired acid chloride as a colorless liquid.

Acyloxazolidinone 9d (R = i-Bu) By an analogous procedure to that described for the preparation of acyloxazolidinone 9b, the lithium anion of (S) - (-) -4-benzyl-2-oxazolidinone was treated with 4-methylvaleryl chloride to provide acyloxazolidinone 9d with 85 Performance% NMR A (300 MHz, CDC13) d 7.37-7.20 (, 5H), 4.70-4.63 (m, 1H), 4.23-4.15 (m, 2H), 3.30 (dd, J = 13.2, 3.2 Hz, 1H), 2.98 -2.90 (m, 2H), 2.76 (dd, J = 13.3, 9.6 Hz, 1H), 1.68-1.54 (m, 3H), 0.94 (d, J = 6.2 Hz, 3H).

Acyloxazolidinone 9e. { R = CH2Ph) By an analogous procedure to that described for the preparation of acyloxazolidinone 9b, the lithium anion of (S) - (-) -4-benzyl-2-oxazolidinone was treated with hydrocinmoyl chloride to provide acyloxazolidinone 9e with 82% performance. RMN A (300 MHz, CDC13) d 7.35-7.16 (m, 10H), 4.70-4.63 (, 1H), 4.21-4.14 (m, 2H), 3.38-3.19 (, 3H), 3.08-2.98 (m, 2H), 2.75 (dd, J = 13.4, 9.5 Hz, 1H).

Acyloxazolidinone 10b (R = n-Pr) A cold solution at 0 ° C of acyloxazolidinone 9b (5.74 g, 22.0 mmol) in 110 ml of anhydrous methylene chloride was treated with 2.52 ml of TiCl 4 (23.1 mmol) resulting in the formation of an abundant precipitate. After 5 minutes, diisopropylethylamine (4.22 ml, 24.2 mmol) was slowly added and the resulting dark brown solution was stirred at room temperature for 35 minutes. The chloromethyl benzyl ether (6.0 ml, 44.0 mmol) was rapidly added and the mixture was stirred for 5 hours at room temperature. Then 50 ml of methylene chloride and 75 ml of 10% aqueous ammonium chloride were added, resulting in the formation of a yellow, rubber-like material. After stirring the suspension vigorously for 10 minutes, the supernatant was transferred to a separatory funnel and the gummy-like residue was collected in 100 ml of 10% aqueous ammonium chloride / methylene chloride 1: 1. The combined organic layers were then washed successively with saturated aqueous HCl, saturated sodium bicarbonate and brine, dried over magnesium sulfate, and concentrated in vacuo. The crude solid material was recrystallized from ethyl acetate / hexane to provide 6.80 g of acyloxazolidinone 10b as a white solid in 81% yield. NMR A (300 MHz, CDC13) d 7.34-7.18 (m, 10H), 4.77-4.69 (m, 1H), 4.55 (s, 2H), 4.32-4.23 (m, 1H), 4.21-4.10 (m, 2H) ), 3.80 (t, J = 9.0 Hz, 1H), 3.65 (dd, J = 9.0, 5.0 Hz, 1H), 3. 23 (dd, J = 13.5, 3.3 Hz, 1H), 2.69 (dd, J = 13.5, 9. 3 Hz, 1H), 1.74-1.64 (m, 1H), 1.54-1.44 (m, 1H), 1.40-1.28 (m, 2H), 0.91 (t, J = 7.3 Hz, 3H). LRMS (FAB) m / e 382 (M + H ") Acyloxazolidinone 10a (R = Et) By an analogous procedure to that described for the preparation of acyloxazolidinone 10b, acyloxazolidinone 10a was obtained in 80% yield. NMR A (300 MHz, CDC13) d 7.36-7.18 (m, 10H), 4.55 (s, 2H), 4.21-4.11 (m, 3H), 3.81 (t, J = 9.0 Hz, 1H), 3.66 (dd, J = 9.0, 5.0 Hz, 1H), 3.23 (dd, J = 13.5, 3.2 Hz, 1H), 2.70 (dd, J = 13.5, 9.3 Hz, 1H), 1.78-1.57 (, 2H), 0.94 (t, J = 7.5 Hz, 3H).

Acyloxazolidinone 10c (R = n-Bu) By an analogous procedure to that described for the preparation of acyloxazolidinone 10b, acyloxazolidinone 10c was obtained in 91% yield. NMR A (300 MHz, CDC13) d 7.38-7.17 (, 10H), 4.72 (, 1H), 4.54 (s, 2H), 4.27-4.10 (m, 2H), 3.79 (t, J = 8.7 Hz, 1H) , 3.65 (dd, J = 9.1, 5.0 Hz, 1H), 3.23 (dd, J = 13.5, 3.3 Hz, 1H), 2.68 (dd, J = 13.5, 9.3 Hz, 1H), 1.75-1.68 (m, 1H ), 1.31-1.26 (m, 4H), 0.87 (t, J = 6.8 Hz, 3H).

Acyloxazolidinone lOd (R = i-Bu) By an analogous procedure to that described for the preparation of acyloxazolidinone 10b, acyloxazolidinone IOd was obtained in 98% yield1. NMR A (300 MHz, CDC13) d 7.38-7.17 (m, 10H), 4.75-4.67 (m, 1H), 4.57 (d, J = 12.0 Hz, 1H), 4.51 (d, J = 12.0 Hz, 1H) , 4.41-4.36 (m, 1H), 4.20-4.09 (m, 2H), 3.74 (t, J = 9.0 Hz, 1H), 3.65 (dd, J = 9.0, 5.1 Hz, 1H), 3.23 (dd, J = 13.5, 3.2 Hz, 1H), 2.63 (dd, J = 13.5, 9.5 Hz, 1H), 1.74-1.52 (m, 2H), 1.35 (dd, J = 13.1, 6.1 Hz, 1H), 0.92 (d, J = 2.9 Hz, 3H), 0.90 (d, J = 2.9 Hz, 3H).

Acyloxazolidinone IOe (R = CH2Ph) By an analogous procedure to that described for the preparation of acyloxazolidinone 1.0b, acyloxazolidinone IOe was obtained in 84% yield. NMR A (300 MHz, CDC13) d 7.38-7.15 (m, 15H), 4.62-4.50 (m, 4H), 4.03 (dd, J = 9.0, 2.7 Hz, 1H), 3.93-3.82 (, 2H), 3.66 (dd, J = 9.2, 4.8 Hz, 1H), 3.19 (dd, J = 13.5, 3.2 Hz, 1H), 2.98 (dd, J = 13.4, 8.2 Hz, 1H), 2.88 (dd, J = 13.4, 7.3 Hz, 1H), 2.68 (dd, J = 13.5, 9.3 Hz, 1H).

Carboxylic acid 11b (R = n-Pr) A cold solution at 0 ° C of 6.60 g (17.3 mmol) of acyloxazolidinone 10b in 320 ml of THF / water was treated successively with 6.95 ml of 35% aqueous H202 and a solution of lithium hydroxide monohydrate (1.46 g, 34.6 mmol). ) in 20 ml of water. The mixture was stirred for 16 hours at 0 ° C and then carefully treated with a solution of Na 2 SO 3 (10.5 g) in 55 ml of water and then with a solution of sodium bicarbonate (4.35 g) in 100 ml of water. The mixture was stirred for 30 minutes at room temperature and concentrated in vacuo to remove THF. The resulting aqueous mixture was then washed with 4 portions of 75 ml of methylene chloride, cooled to 0 ° C, acidified with 6N aqueous HCl and extracted with methylene chloride (1 x 200 ml and 3 x 100 ml). The combined organic layers were then dried over magnesium sulfate and concentrated in vacuo to provide 3.47 g (90%) of the desired acid 11b with a colorless oil. NMR A (30 MHz, CDC13) d 7.38-7.26 (m, 5H), 4.55 (s, 2H), 3.67 (, 1H), 3.57 (dd, J = 9.2, 5.2 Hz, 1H), 2.75 (m, 1H) ), 1.72-1.31 (m, 4H), 0.93 (t, = 7.2 Hz, 3H). LRMS (FAB) m / c 223 (M + H +) Carboxylic acid lia (R = Et) By an analogous procedure to that described for the preparation of acyloxazolidinone IIb, acyloxazolidinone was obtained in a yield of 48%. NMR A (300 MHz, CDC13) d 7.36-7.27 (m, 5H), 4.55 (s, 2H), 3.68 (dd, J = 9.2, 7.9 Hz, 1H), 3.59 (dd, J = 9.2, 5.4 Hz, 1H), 2.68-2.65 (m, 1H), 1.71-1.62 (m, 2H), 0.97 (t, J = 7.5 Hz, 3H).

Carboxylic acid 11c (R = n-Bu) By an analogous procedure to that described for the preparation of acyloxazolidinone IIb, acyloxazolidinone 11c was obtained in 96% yield. NMR A (300 MHz, CDC13) d 7.37-7.28 (m, 5H), 4.55 (s, 2H), 3.67 (dd, J = 9.1, 8.1 Hz, 1H), 3.57 (dd, J = 9.2, 5.3 Hz, 1H), 2.72 (m, 1H), 1.67-1.51 (m, 2H), 1.36-1.27 (m.4H), 0.89. (t, J = 6.9 Hz, 3H).

Carboxylic acid lid (R = i-Bu) By an analogous procedure to that described for the preparation of acyloxazolidinone IIb, acyloxazolidinone lid was obtained with a yield of 80%. NMR A (300 MHz, CDC13) d 7.37-7.28 (m, 5H), 4.55 (s, 2H), 3.64 (t, J = 9.1 Hz, 1H), 3.54 (dd, J = 9.1, 5.1 Hz, 1H) , 2.81 (, 1H), 1.68-1.54 (m, 2H), 1.36-1.27 (, 1H), 0.92 (d, J = 4.9 Hz, 3H), 0.90 (d, J = 4.9 Hz, 3H).

Carboxylic acid lie (R = CH2Ph) By an analogous procedure to that described for the preparation of acyloxazolidinone IIb, acyloxazolidinone was obtained in a yield of 92%. NMR A (300 MHz, CDC13) d 7.38-7.16 (m, 10H), 4.53 (d, J = 12.1 Hz, 1H), 4.50 (d, J = 12.1 Hz, 1H), 3.68-3.57 (, 2H), 3.09-2.85 (m, 3H).

Diethylamide 12b (R = n-Pr) A cold solution at 0 ° C of the carboxylic acid llb (3.40 g, 15.3 mmol) in 150 ml of 1: 1 MeCN / CH 2 Cl 2, containing diethylamine (2.36 ml, 23.0 mmol) and 2- (1 H-benzotriazole tetrafluoroborate) -yl) -1, 1, 3, 3-tetramethyluronium (TBTU, 5.89 g, 18.4 mmol), was treated with diisopropylethylamine (6.7 ml, 38.2 mmol) in 1.5 hours (syringe pump). The mixture was then concentrated in vacuo and partitioned between 200 ml of ether and 100 ml of water. The aqueous layer was extracted with more ether (2 x 100 ml) and the combined organic layers were washed with aqueous IN HCl (3 x 50 ml), with saturated aqueous sodium bicarbonate, and with brine, dried over magnesium sulfate and they concentrated in vacuum. Chromatographic purification (230-400 Si02 mesh, elution with ethyl acetate / hexane 1: 3) gave 4.24 g (97%) of diethylamide 12b as a clear, colorless oil. NMR A (300 MHz, CDC13) d 7.35-7.23 (, 5H), 4.52 (d, J = 12.0 Hz, 1H), 4.44 (d, J = 12.0 Hz, 1H), 3.67 (t, J = 8.6 Hz, 1H), 3.51 (dd, J = 8.7, 5.5 Hz, 1H), 3.46-3.27 (m, 4H), 2.96 (m, 1H), 1.67-1.57 (m, 1H), 1.48-1.22 (m, 4H) , 1.20-1.10 (, 6H), 0.90 (t, J = 7.2 Hz, 3H). LRMS (FAB) m / e 278 (M + H +) Diethylamine 12a (R = Et) By means of a procedure analogous to that described for the preparation of diethylamide 12b, diethylamine 12a was obtained with a yield of 73%. NMR A (300 MHz, CDC13) d 7.33-7.26 (m, 5H), 4.52 (d, J = 12.0 Hz, 1H), 4.44 (d, J = 12.20 Hz, 1H), 3.68 (t, J = 8.6 Hz , 1H), 3.53-3.33 (m, 5H), 2.90 (m, 1H), 1.75-1.50 (m, 2H), 1.18 (t, J = 7.1 Hz, 3H), 1.13 (t, J = 7.1 Hz, 3H), 0.89 (t, J = 7.4 Hz, 3H).

Diethylamine 12s (R = n-Bu) By means of a procedure analogous to that described for the preparation of diethylamide 12b, diethylamine 12c was obtained with a yield of 94%. NMR A (300 MHz, CDC13) d 7.35-7.25 (m, 5H), 4.51 (d, J = 12.0 Hz, 1H), 4.44 (d, J = 12.0 Hz, 1H), 3.67 (t, J = 8.6 Hz , 1H), 3.51 (dd, J = 8.8, 5.5 Hz, 1H), 3.46-3.29 (m, 1H), 2.94 (m, 1H), 1.66-1.62. (m, 2H), 1.33-1.10 (m, 9H), 0.85 (t, J = 7.0 Hz, 3H).

Diethylamine 12d (R = i-Bu) By means of a procedure analogous to that described for the preparation of diethylamide 12b, diethylamine 12d was obtained with a yield of 95%. NMR aH (300 MHz, CDC13) d 7.35-7.23 (m, 5H), 4.51 (d, J = 12.0 Hz, 1H), 4.44 (d, J = 12.0 Hz, 1H), 3.65 (t, J = 8.7 Hz , 1H), 3.54-3.28 (m, 5H), 3.03 (, 1H), 1.63-1.49 (m, 2H), 1.33-1.24 (m, 1H), 1.18 (t, J = 7.1 Hz, 3H), 1.12 (t, J = 7.1 Hz, 3H), 0.90 (t, J = 6.4 Hz, 3H).

Diethylamine 12e (R = CH2Ph) By means of a procedure analogous to that described for the preparation of diethylamide 12b, diethylamine 12d was obtained with a yield of 89%. NMR A (300 MHz, CDC13) d 7.35-7.16 (, 10H), 4.53 (d, J = 12.1 Hz, 1H), 4.47 (d, J = 12.1 Hz, 1H), 3.77 (t, J = 8.5 Hz, 1H), 3.59 (dd, J = 8.8, 5.7 Hz, 5H), 3.40 (m, 1H), 3.22-2.89 (m, 5H), 2.79 (dd, J = 13.0, 5.1 Hz, 3H), 1.01 (t, J 7.1 Hz, 3H), 0.85 (t, J = 7.2 Hz, 3H).

Alcohol 13b (R = n-Pr) To a solution of the diethylamide 12b (4.08 g, 14.7 mmol) in 140 ml of methanol was added 20% Pd (0H) 2 / C (400 ml) and the suspension was hydrogenated at atmospheric pressure and at room temperature for 15 h. Filtration of the catalyst and concentration of the filtrate in vacuo afforded 2.84 g (100%) of the desired primary alcohol 13b: NMR A (300 MHz, CDCli) d 3.74 (broad d, J = 4.2 Hz, 1H), 3.61-3.15 ( m 5H), 2.71 (m, 1H), 1.69-1.24 (m, 4H), 1.20 (t, J = 7.1 Hz, 3H), 1.12 (t, J = 7'.1 Hz, 3H), 0.92 (t , J = 7.2 Hz, 3H). LRMS (FAB) m / e 188 (M + H *) Alcohol 13a (R = Et) By means of a procedure analogous to that described for the preparation of alcohol 13b, alcohol 13a was obtained with a yield of 100% NMR A (300 MHz, CDC13) d 3.76 (m, 2H), 3.58-3.19 (m 4H), 2.64 (m, 1H), 1.71 - 1.65 (m, 2H, 1.21 (t, J = 7.1 Hz, 3H), 0.96 (t, J = 7.4 Hz, 3H).

Alcohol 13c (R = n-Bu) By an analogous procedure to that described for the preparation of alcohol 13b, alcohol 13c was obtained in 100% yield. NMR A (300 MHz, CDC13) d 3.76 (d, J = 4.5 Hz, 2H), 3.58-3.19 ( m 4H), 2.72 - 2.65 (m, 2H), 1.68 - 1.65 (m, 2H), 1.40 - 1.24 (m, 4H), 1.20 (t, J = 7.1 Hz, 3H), 1.12 (t, J = 7.1 Hz, 3H), 0.90 (t, J = 6.9 Hz, 3H).

Alcohol 13d (R = i-Bu) By means of a procedure analogous to that described for the preparation of alcohol 13b, alcohol 13d was obtained with a yield of 100%. NMR A (300 MHz, CDC13) d 3.78 - 3.68 (m, 2H), 3.57 - 3.15 (m 4H), 2.81 - 2.73 (m, 1H), 1.70 - 1.60 (m, 2H), 1.40 - 1.28 (m, 1H), 1.21 (t, J = 7.1 Hz, 3H), 1.12 (t, J = 7.1 Hz, 3H), 0.92 (, 6H).

Alcohol 13e (R = CH2Ph) By means of a procedure analogous to that described for the preparation of alcohol 13b, alcohol 13e was obtained with a yield of 100%. NMR A (300 MHz, CDC13) d 7.29 - 7.16 (m, 5H), 3.81 - 3.71 (m, 2H), 3.61 - 3.50 (m, 1H), 3.15 - 2.87 (m, 6H), 1.05 (t, J = 7.1 Hz, 3H), 0.98 (t, J = 7.1 Hz, 3H).

Aldehyde 14b (R = n-Pr) To a solution of the alcohol 13b (2.34 g, 12.7 mmol) in wet methylene chloride (125 ml, prepared by stirring methylene chloride with water and separating the organic layer was added the Dess-Martin periodinan (8.06 g, 19.0 The mixture was stirred at room temperature for 40 minutes and then poured into a mixture of 250 ml of 5% aqueous Na2S203 containing 5.2 g of NaHCO3 and 200 ml of ether.The biphasic mixture was stirred vigorously for 5 minutes and the aqueous layer was extracted with 15% methylene chloride / diethyl ether (2 x 100 ml) The combined organic layers were then washed with three 75 ml portions of water and brine., dried over magnesium sulfate, filtered and concentrated in vacuo to provide 2.06 g of (88%) of the desired aldehyde 14b, a clear, colorless oil. NMR XH (300 MHz, CDC13) d 9.60 (d J = Hz, 1H), 3.49 - 3.30 (5H), 1.96 - 1.85 (m, 2H), 1.39 - 1.31 (m, 2H), 1.19 (t, J = 7.1 Hz, 3H), 1.13 (t, J = 7.1 Hz, 3H), 0.95 (t, J = 7.3 Hz, 3H).

Aldehyde 14a (R = Et) By means of a procedure analogous to that described for the preparation of alcohol 14b, aldehyde 14a was obtained with a yield of 80% NMR A (300 MHz, CDCl 3) d 9.61 (d, J = 3.6 Hz, 1H), 3.48-3.29 ( m, 5H), 2.02-1.90 (m, 2H), 1.19 (t, J = 7.1 Hz, 3H), 1.14 (t, J = 7.1 Hz, 3H), 0.96 (t, J = 7.4 Hz, 3H).

Aldehyde 14c (R = n-Bu) By means of a procedure analogous to that described for the preparation of alcohol 14b, aldehyde 14c was obtained in a yield of 98% NMR A (300 MHz, CDC13) d 9.59 (d, J = 3.6 Hz, 1H), 3.48-3.29 ( m, 5H), 1.97 - 1.87 (, 2H), 1.39 -1.22 (, 4H), 1.18 (t, J = 7.2 Hz, 3H), 1.13 (t, J = 7.2 Hz, 3H), 0.90 (t, J = 7.0 Hz, 3H).

Aldehyde 14d (R = i-Bu) By an analogous procedure to that described for the preparation of alcohol 14b, aldehyde 14d was obtained in 96% yield NMR A (300 MHz, CDC13) d 9.57 (d, J = 3.7 Hz, 1H), 3.51 - 3.27 ( m, 5H), 1.83 (t, J = 7.1 Hz, 3H), 1.66 - 1.55 (m, 1H), 1.20 (t, J = 7.1 Hz, 3H), 1.13 (t, J = 7.1 Hz, 3H) 0.93 (d, J = 6.6 Hz, 6H).

Aldehyde 14e (R = CH2Ph) By an analogous procedure to that described for the preparation of alcohol 14b, aldehyde 14e was obtained in a yield of 97% NMR A (300 MHz, CDC13) d 9.69 (d, J = 2.9 Hz, 1H), 7.29 - 7.16 ( m, 5H), 3.65 (m, 1H), 3.53 - 3.42 (m, 1H), 3.30 (dd, J = 13.5, 9.3 Hz, 1H), 3.23 - 3.13 (M, 2H), 3.06 - 2.91 (m, 2H), 1.04 (t, J = 7.1 Hz, 3H), 0.93 (t, J = 7.1 Hz, 3H).

Example 5: Preparation of β-lactones 3 (Reaction scheme 2) Aldol 5b (R = n-Pr) To a cold solution (-78 ° C) of the trans-oxazoline 4 in 35 ml of ether was added lithium bis (trimethylsilyl) amide (2.17 of a 1 M solution in hexane, 2.17 mmol). After 30 minutes, the orange solution was treated dropwise with a 1M solution of dimethylaluminum chloride in hexane (4.55 ml, 4.55 mmol) and the mixture was stirred for another 60 minutes before it was cooled to 85 ° C. added liquid nitrogen to the dry ice / acetone bath). A solution of aldehyde 14b (4.20 mg 2.27 mmol) in 4 ml of ether was added 10 minutes, along the side of the flask. The mixture was then allowed to warm to minus 40 ° C in 2.5 hours, and then quenched by the addition of 35 ml of saturated aqueous ammonium chloride and 25 ml of ethyl acetate. Sufficient 2 N HCl was then added until 2 clear phases were obtained (approximately 15 ml were added). The aqueous layer was extracted with 2 portions of 20 ml of ethyl acetate and the combined organic layers were washed successively with 20 ml of 0.5 N aqueous hydrochloric acid, 20 ml of water, 0.5 M aqueous NaHS03 (2 x 15 ml), saturated aqueous sodium bicarbonate, and finally with brine, then dried over sodium sulfate, and concentrated in vacuo to give 879 mg (> 100%) of crude aldol product 5b which was pure enough to yield uti-1-lifted directly in the subsequent step. 1 H NMR (300 MHz, CDC13) d 8.02-1. 91"and 7.53 - 7.39 (m, 5H, 6.58 (d, J = 9.9 Hz, 1H, 4.82 (d, J = 2.4 Hz, 1H), 3.73 (s, 3H), 3.69 - 3.61 (m, 2H), 3.49 - 3.39 (m, 2H), 3.24 - 3.16 (m, 1H), 3.05 (, 1H), 2.89 (m, 1H); 2.28 - 2.23 (m, 1H), 1.37-1.20 (m, 6H), 1.19-1.06 (m, 6H), 0.87. (t, J = 7.1 Hz, 3H), 0.70 (d, J = 6.7 Hz, 3H).

The aldol 5b product was also obtained in 100% yield by an analogous procedure to that described above, but using cis-oxazoline 21 (see below) instead of trans-oxazoline 4.

Aldol 5a (R = Et) By a procedure analogous to that described for the preparation of aldol 5b, the lithium anion of the trans-oxazoline 4 was treated successively with dimethylaluminum chloride and the aldehyde 14a to provide the aldehyde 5a in 95% yield. NMR A (300 MHz, CDCl 3) d 8.00 - 7.97 and 7.51 - 7.39 (m, 5H), 6.50 (d, J = 9.9 Hz, 1H), 4.80 (d, J = 2.4 Hz, 1H), 3.81 - 3.64 ( m, 2H), 3.74 (s, 3H), 3.45 (m, 2H), 3.19 (, 2H), 2.93 - 2.84 (m, 2H), 2.24 (m, 1H) 1.89 (, 1H), 1.73 - 1.64 ( m, 4H), 1.29 (t, J = 7.2 Hz, 3H), 1.12 (d, J = 6.9 Hz, 3H), 1.07 (d, J = 7.2 Hz, 3H), 0.70 (d, J = Hz, 3H ).

Aldol 5c (R = n-Bu) By a procedure analogous to that described for the preparation of aldol 5b, the lithium anion of the trans-oxazoline 4 was treated successively with dimethylaluminum chloride and the aldehyde 14c to provide the aldehyde 5c in a yield of 100%. NMR A (300 MHz, CDC13) d 8.02-7.98 and 7.53-7.33 (m, 5H), 6.57 (d, J = 10.0 Hz, 1H), 4.81 (d, J = 2.3 Hz, 1H), 3.73 (s, 3H), 3.68 - 3.60 (m, 2H), 3.49 - 3.17 (m, 2H), 3.00 (m, 1H), 2.90 (m, 1H), 1.98 - 1.87 (m, 2H), 1.38 - 0.83 (m, 16H), 0.70 (d, J = 6.7 Hz, 3H).

Aldol 5d < R = i -Bu) By a procedure analogous to that described for the preparation of aldol 5b, the lithium anion of the trans-oxazoline 4 was treated successively with dimethylaluminum chloride and the aldehyde 14d to provide the aldehyde 5d in 100% yield. NMR A (300 MHz, CDC13) d 8.01 - 7.80 and 7.55 - 7.20 (, 5H), 4.87 (d, J = 2.3 Hz, 1H), 3.73 (s, 3H), 3.69 - 3.58 (m, 2H), 3.51 - 3.32 (m, 2H), 2.98 - 2.87 (m, 1H), 2.33 - 2.24 (, 1H), 2.12 -2.02 (m, 1H), 1.83 (t, J = 7.1 Hz, 1H), 1.35 (t, J = 7.1 Hz, 3H), 1.25 - 1.05 (, 5H), 0.93 (d, J = 6.6 Hz, 3H), 0.89 (d, J = 6.5 Hz, 3H), 0.80 (d, J = 6.5 Hz , 3H), 0.69 (d, J = 6.7 Hz, 3H).

Aldol 5e (R = CH2Ph) By an analogous procedure to that described for the preparation of aldol 5b, the lithium anion of the trans-oxazoline 4 was treated successively with dimethylaluminum chloride and the aldehyde 14c to provide the aldehyde 5e in 100% yield. NMR A (300 MHz, CDC13) d 8.01 - 7.93 and 7.54 - 7.10 (, 10H), 4.71 (d, J = 2.5 Hz, 1H), 3.73 (s, 2H), 3.48 - 2.79 (m, 6H), 2.17 (m, 1H), 1.12 -0.91 (, 9H), 0.68 (d, J = 6.7 Hz, 3H). ? -lactam 7b (R = n-Pr) A solution of aldol 5b (4.72 g 10.9 mmol) in 100 ml of AcOH / MeOH 1: 9, to which 4.8 g of 20% Pd / (0H) 2 / C was added, was stirred vigorously under 3.87 kg / cm2 ( 55 psi) of H2 for 60 hours The mixture was brought to atmospheric temperature before being filtered and concentrated in vacuo. The solid obtained was purified by flash chromatography (SiO elution with 1% AcOH in ethyl acetate / hexane 1: 1) to provide 2.23 g (75%) of the desired β-lactam, 7b as a white solid. NMR A (300 MHz, CDC13) d 7.89 (s broad 1H), 4.77 (d, broad J = 11.5 Hz, 1H), 4.47 (dd, J = 11.5 5.6 Hz, 1H), 4.08 (dd, J = 9.4, 5.0 Hz, 1H), 3.83 (s, 3H), 2.93 (m, 1H), 1.78 - 1.39 (, 6H), 1.02 - 0.88 (m, 9H). ? -lactama 7a (R = Et) By a procedure analogous to that described for the preparation of β-lactam 7b, aldol 5a was hydrogenated at 3.86 kg / cm 2 (55 psi) for 48 hours to provide β-lactam 7a in 72% yield. NMR A (300 MHz, CDC13) d 7.79 (s broad 1H), 4.62 (d, broad J = 11.2 Hz, 1H), 4.51 (dd, J = 11.2, 5.4 Hz, 1H), 3.83 (s, 3H), 2.85 (m, 1H), 1.77 - 1.64 (, 3H), 1.01 (t, J = 7.4 Hz, 3H), 0.98 (d, J = 6.9 Hz, 3H), 0.95 (d, J = 6.9 Hz, 3H) . ? -stam 7c (R = n-Bu) A solution of aldol 5c (361 mg 0.80 mmol) in 6 ml of AcOH / MeOH 1: 9, to which was added 250 mg of 20% Pd (OH) 2 / C, was vigorously stirred under 3.51 kg / cm 2 (50 psi) of H2 for 24 hours. Then more catalyst (100 mg) was added and the mixture was stirred again at 3.53 kg / cm2 (50 psi) for another 24 hours, after which it was brought to atmospheric temperature before being filtered. The filtrate was heated to reflux for 30 minutes, cooled to room temperature and concentrated in vacuo. The solid obtained was co-evaporated once with toluene and purified by flash chromatography (Si02 elution with 4% methanol / chloroform) to provide 140 mg (61%) of the desired β-lactam 7c as a white solid. NMR A (300 MHz, CDC13) d 8.02 (s broad 1H), 4.93 (d, broad J = 11.3 Hz, 1H), 4.46 (dd, J = 11.3, 5.5 Hz, 1H), 4.15 - -4.08 (m, 1H), 3.83 (s, 3H), 2.94 - 2.87 (m, 1H), 1.80 - 1.34 (m, 6H), 0.94 (d, J = 6.9 Hz, 3H), 0.89 (t, J = 7.2 Hz, 3H ). ? -lactama 7d (R = i-Bu) By a procedure analogous to that described for the preparation of β-lactam 7c, aldol 5d was hydrogenated at 3.53 kg / cm 2 (50 psi) for 40 hours and heated to reflux for 30 minutes to give the β-lactam 7d with a Performance 61% NMR A (300 MHz, CDC13) d 7.92 (s broad 1H), 4.81 (d, broad J = 11.5 Hz, 1H) ', 4.46 (m, 1H), 4.09 (m, 1H), 3.83 (s, 3H), 3.04 (m, 1H), 1.78 - 1.73 (, 2H), 1.66 - 1.47 (m, 3H), 1.00-0.90 (m, 12H). ? -lactam 7e (R = CH3Ph) By an analogous procedure to that described for the preparation of β-lactam 7c, aldol 5e was hydrogenated at 3.53 kg / cm 2 (50 psi) for 24 hours and heated to reflux for 30 minutes to provide the β-lactam 7e with a 71% yield. NMR A (300 MHz, CDC13) d 8.01 (s broad 1H), 7.35 - 7.15 (m, 5H), 5.02 (broad d, J = 11.7 Hz, 1H), 4.40 - 4.34 (m, 1H), 4.06 - 4.01 (m, 1H), 3.84 (s, 3H), 3.34 - 3.27 (m, 1H), 3.10 - 3.04 (, 2H), 1.84 - 1.72 (m, 1H), 0.98 (d, J = 6.7 Hz, 3H) , 0.93 (d J = 6.9 Hz, 3H). β-lactone 3b (R = n-Pr; β-lactone 7-n-propyl-clasto-lactacystin).

To a cold solution at (0 ° C) of the β-lactam 7b (2.20 g, 8.06 mmol) in 100 mL of ethanol was added 0.1 N aqueous sodium hydroxide (100 mL, 10.0 mmol). The mixture was stirred at room temperature for 15 hours after which 50 ml of water and 100 ml of ethyl acetate were added, the aqueous layer was then washed with 2 50 ml portions of ethyl acetate, acidified with 6N hydrochloric acid. and concentrated in vacuo to a volume of approximately 60 ml. This solution was then frozen and lyophilized. The solid obtained was suspended in tetrahydrofuran, filtered to remove sodium chloride and concentrated in vacuo to afford 2.05 g (98%) of the desired dihydroxy acid as a white solid. NMR A (300 MHz, CDC13) d 4.42 (d, J = 5.58 Hz, 1H), 3.90 (d, J = 6.5 Hz, 1H), 2.84 (m, 1H), 1.70 - 1.24 (m, 6H), 0.95 -0.84 (m, 9H).

To a solution of the dihydroxy acid (1.90 g, 7.33 mmol) in 36 ml of anhydrous THF was added a solution of 2- (1H-benzotriazol-1-yl) -1,3,3-tetramethyluronium tetrafluoroborate (TBTU, 2.59, 8.06 mmol) in 36 ml of anhydrous MeCN, followed by triethylamine (0.72 ml, 22.0 mmol). After stirring for 70 minutes at room temperature, some toluene was added and the mixture was concentrated in vacuo and co-evaporated 2 times more with toluene. Purification by flash chromatography (Si02 elution with ethyl acetate / hexane) 1: 3 afforded 1.44 g (81%) of the desired β-lactone 3b as a white solid. NMR XH (300 MHz, CDC13) d 6.07 (s broad 1H), 5.26 (d, broad J = 6.1 Hz, 1H), 3.97 (dd, J = 6.4, 4.4 Hz, 1H), 2.70-2.63 (m, 1H) ), 2.03 (d, J = 6.4 Hz, 3H), 1.93 - 1.44 (m, 5H), 1.07 (d, J = 7.0 Hz, 3H), 0.99 (d, J = 7.3 Hz, 3H), 0.91 (d , J = 6.7 Hz, 3H). LRMS (FAB) m / e 242 (M + H *) β-lactone 3a (R = Et; β-lactone of 7-ethyl-cla = rto-lastacystin) Hydrolysis of 7a, as described for 7b above, provided the corresponding dihydroxy acid in 100% yield. NMR XH (300 MHz, CDC13) d 4.45 (d, J = 5.8 Hz, 1H), 3.90 (d, J = 6.4 Hz, 1H), 2.74 (, 1H), 1.71 - 1.53 (m, 3H), 0.94 ( t, J = 7.4 Hz, 3H), 0.92 (d, J = 6.8 Hz, 3H), 0.88 (d, J = 6.8 Hz, 3H).

By means of a procedure analogous to that described for the preparation of β-lactone 3b, β-lactone 3a was obtained with a yield of 79%. NMR A (300 MHz, CDC13) d 6.17 (broad s, 1H), 5.30 (d, J = 6.0 Hz, 1H), 3.98 (dd, J = 6.4, 4.4 Hz, 1H), 2.60 (m, 1H), 2.08 (d, J = 6.4 Hz, 3H), 1.97 (m, 2H), 1.75 (m, 1H), 1.12 (t, J = 7.5 Hz, 3H), 1.07 (d, J = 6.8 Hz, 3H), 0.92 (d, J = 6.8 Hz, 3H). β-lactone 3c (R = n-Bu; β-lactone of 7-n-butyl-clasto-lactacystin) Hydrolysis of 7c, as described for 7b above, provided the corresponding dihydroxy acid in 100% yield. NMR XH (300 MHz, CDC13) d 4.42 (d, J = 5.8 Hz, 1H), 3.90 (d, J = 6.4 Hz, 1H), 2.86-2.79 (m, 1H), 1.70-1.24 (m, 8H) , 0.97 - 0.86 (m, 9H).

By means of a procedure analogous to that described for the preparation of β-lactone 3b, ß-lactone 3c was obtained with a yield of 40%. NMR XH (300 MHz, CDC13) d 6.14 (broad s, 1H), 5.27 (d, J = 6.1 Hz, 1H), 3.97 (d, J = 4.4 Hz, 1H), 2.68-2.61 (m, 1H), 1.94 - 1.86 (m, 2H), 1.72 - 1.36 (m, 7H), 1.07 (d, J = 7.0 Hz, 3H), 0.93 (t, J = 7.1 Hz, 3H), 0.91 (d, J = 6.8 Hz , 3H). LRMS (FAB) m / e 256 (M + H *) β-lactone 3d (R = i-Bu; ß-lactone of 7-i-c2asto-lactacis ina) Hydrolysis of 7d, as described for 7b above, provided the corresponding dihydroxy acid in 100% yield. 1 H NMR (300 MHz, CDC13) d 4.50 (d, J = 5.8 Hz, 1 H), 4.00 (d, J = 6.5 Hz, 1 H), 3.09 - 3.02 (m, 1 H), 1.90 -1.61 (m, 3 H) , 1.49 - 1.40 (m, 2H), 1.02 (d, J = 6.7 Hz, 3H) 0.98 (d, J = 6.5 Hz, 3H), 0.97 (d, J = 6.7 Hz, 3H).

By means of a procedure analogous to that described for the preparation of β-lactone 3b, the β-lactone 3d was obtained with a 62% yield. NMR XH (300 MHz, CDC13) d 6.16 (broad s, 1H), 5.25 (d, J = 6.1 Hz, 1H), 3.97 (d, J = 4.4 Hz, 1H), 2.71 (dd, J = 15.1, 6.2 Hz, 1H), 1.95 - 1.66 (m, 5H), 1.08 (d, J = 6.9 Hz, 3H), 0.99 (d, J = 6.3 Hz, 3H), 0.98 (d, J = 6.3 Hz, 3H), 0.92 (d, J = 6.7 Hz, 3H). LRMS (FAB) m / e 256 (M + H *) β-lastone 3e (R = CH2Ph; 7-benzyl-clasto-lactacystin β-lactone) Hydrolysis of 7e, as described for 7b above, provided the corresponding dihydroxy acid with 88% yield. NMR A (300 MHz, CDC13) d 7.25 - 7.04 (m, 5H), 4.29 (d, J = 5.7 Hz, 1H), 3.83 (d, J = 6.4 Hz, 1H), 3.01 - 2.82 (m, 3H) , 1.65 (m, 1H), 0.90 (d, J = 6.6 Hz, 3H), 0.86 (d, J = 6.8 Hz, 3H).

By means of a procedure analogous to that described for the preparation of β-lactone 3b, β-lactone 3e was obtained with a yield of 77%. NMR A (300 MHz, CDC13) d 7.36-7.20 (m, 5H), 6.57 (broad s, 1H), 5.08 (d, J = 5.4 Hz, 1H), 3.94 (d, J = 4.5 Hz, 1H), 3.01 - 2.89 (m, 2H), 1.92 - 1.81 (m, 1H), 1.05 (d, J = 6.9 Hz, 3H), 0.86 (d, J = 6.7 Hz, 3H). LRMS (FAB) m / e 290 (M + H *) Example 6: Pharmacokinetics of the acid N- (rubric) carbonyl-L-phenol to the anin-L-l eucin-Boron (1) in Rats and Primates A simple dose intravenous pharmacokinetic study with N- (pyrazine) carbonyl-L-phenylalanine-L-leucine-boronic acid (1) was conducted in Sprague-Dawley rats (140 to 280 g weight). The animals were assigned to 3 groups (6 / sex in groups 1 and 2; 9 / group sex 3). Animals in groups 1, 2 and 3 received 0.03, 0.1 or 0.3 mg / kg of 1, respectively, in the same dose volume. Blood samples (approximately 1.0 ml) were collected from the jugular vein of the predose animals and approximately 10 and 30 minutes and 1, 3 and 24 hours after the dose on day 1. The samples were evaluated for 1 using a method of chromatography / mass spectroscopy (LC / MS / MS). The lower limit of the quantification for analysis was set at 2.5 ng / ml for 1 in rat plasma and whole blood. After single intravenous doses, plasma whole blood levels of 1 were only measured at a dose level of 0.3 mg / kg. The observed Cmax occurred at the first time point; therefore, the time to peak concentration (Tmax) was estimated as 10 minutes in the male and female rats. The males in general had slightly higher peak concentration (Cma?) And slightly higher values of the area under the concentration-time curve (AUC0-t) than the females. The Cmax values in plasma and whole blood in males were 51.8 and 22.7 ng / ml, respectively, and in females were 36.9 and 19.1 ng / ml, respectively. The values of AuC0-t in whole blood plasma in males were 14.0 and 18.6 ng * h / ml, respectively, and in females were 12.9 and 17.7 ng * h / ml, respectively. The estimation of the elimination half-life (t? / 2) was not possible due to the fluctuation of the levels of compound 1 during the terminal phase. Observations suggest that 1 is rapidly cleared of blood.

Example 7; Preparation of blood samples for the measurement of the proteasome actiVity 20S This method of preparation applies to blood samples collected from mammals, particularly human subjects, Cynomologus monkeys, rats and mice. Peripheral white blood cells are separated from blood samples after collection for storage at approximately -70 ° C until tested. To prevent interference with the fluorescence assay, it is important that the sample preparation remove all of the hemoglobin.

PROCESS The required amount of blood is collected in a tube containing anticoagulant.

For human and primate subjects, approximately 5 ml of blood is required; for rats, approximately 4 ml of blood is needed; for mice, approximately 1 ml of blood is needed from each of the five mice, and all five blood samples are combined to provide approximately 5 ml. The blood sample is diluted 1: 1 (v / v) with sterile saline, the blood-saline mixture is layered on about 1 ml of NycoprepMR separation medium (GIBCO BRL Products) in a polystyrene test tube 14 of 75 mm. The sample is centrifuged at 500 g for about 30 minutes at room temperature. The upper layer is removed, leaving approximately 2 to 3 mm of the cellular band between the upper and lower layers. The remaining cell band is transferred by pipette to a clean centrifuge tube. The cell band is washed with 3 ml of cold saline buffered with phosphate and centrifuged at 400 x g for 5 minutes at 4 ° C. The supernatant is emptied and the button is resuspended in approximately 1 ml of cold phosphate-buffered saline. The suspension is transferred to a 1.5 ml Eppendorf microcentrifuge tube and microcentrifuged at 6600 x g for approximately 10 minutes at 4 ° C. The supernatant is aspirated and the cell button is stored at -70 ° C.

Example 8: Assay for Measuring 20S Proteasome Acivity in Peripheral White Blood Cells The assay is based on activity similar to chymotrypsin inducible by SDS of free 20S particles. It uses fluorometry to measure the rate at which the 20S proteasome hydrolyzes an amide bond in a small peptide substrate. The measurement of this speed in the absence and presence of an inhibitor allows a determination of how the enzyme is linked by the inhibitor. This assay is used to measure 20S proteasome activity in peripheral white blood cells in mammals, particularly humans, Cynomolgus monkeys, rats and mice.

ABBREVIATIONS AMC 7-amino-4-methylcoumarin DMF dimethyl formamide BSA bovine serum albumin DMSO dimethyl sulfoxide DTT Dithiothreitol EDTA Disodium ethylenediaminetetraacetate HEPES N- (2-hydroxyethyl) piperazine-N- (2-ethanesulfonic acid); pH adjustments with NaOH Hgb Hemoglobin SDS Sodium Dodecyl Sulfate SDS-grade 99% Sodium Dodecyl Sulfate Lauryl grade Dodecyl Sulfate approximately 70% with the rest as tetradecyl and hexadecyl sulfates TMB 3, 3, 5, 5-tetramethylbenzidine CSB White blood cells Substrate Ys N-succinyl-leucyl-leucyl-valyl-tyrosyl-7-amino-4-methyl-ramin (Suc-Leu-Leu-Va1-Tyr-AMC) PROCESS White blood cells, prepared as described in Example 1, are used by the addition of 200 μl of 5 mM EDTA to each sample. Samples are left to stand on ice for at least 1 hour. The substrate Ys is dissolved in DMSO. The standard purified 20S proteasome from mouse reticulocytes is diluted 1: 9 (v / v) in 20 mM HEPES / 0.5-mM EDTA (pH 7.8). The Ys substrate buffer contains 20 mM HEPES, 0.5 mM EDTA, 0.35% SDS, and 60 μM Ys substrate. The Coomassie protein assay (measurement of total protein content) and the hemoglobin assay are performed on the test sample following standard procedures using commercially available equipment. The amount of white blood cell protein present is calculated according to the following formula: CSB protein (μg mL) = total protein (μg / mL) - [hemoglobin (μg mL) = 0.56] The 20S proteasome standard is diluted 1:10 in 20 mM HEPES / 0.5 mM EDTA (pH 7.8) to form a stock solution of 12 μg / mL and placed on ice. 10 μL of the standard 20S proteasome solution is added to a cuvette and the reaction is run for 10 minutes. The maximum linear slope is measured on a florometer and provides a measurement of the standard proteasome activity. 5 μl of a test sample are added to a cuvette containing 2 mL of Ys substrate buffer at 37 ° C, and the reaction is allowed to run for 10 minutes. Complete activation of the 20S proteasome is achieved in 10 minutes. Consistent results are obtained for the readings taken after 3 minutes and up to 10 minutes. The maximum linear slope for at least 1 minute of data is measured in a fluorometer. If the velocity is less than 1 pmol of AMC / sec, the measurement is repeated using 10 μl of the test sample. The amount of 20S proteasome activity in the test sample is calculated according to the following formula: Activity of 20S (pmol / s) = Speed (FU / min) x calibration YS (pmol / FU) 0.0001 * protein CSB (mg) * 60 s / min Example 9: Negativeness of Proteasome Activity in Blood Cells The Human Peripheral Blood Cells Blood samples (approximately 2 mL each) were obtained on five occasions from five human volunteers in a period of ten weeks. After harvesting, the white blood cells were isolated from individual blood samples using a Nycoprop ™. The resulting pellet or button was stored in a tight freezer to maintain -60 ° C to -80 ° C until the day of the test. The samples collected on each occasion were tested together, and each sample was tested in duplicate. The 20S proteasome activity was determined by measuring the rate of proteolytic hydrolysis of a peptide substrate by labeling with fluorescent (AMC), by sample and normalizing activity to the amount of cell-specific protein present in the lysate, as described in Example 2 above. The 20S proteosome activity in the sample was determined from the equation: S proteasome activity (pmol (FU / sec) / (5 x 10"6 mL) (μg protein / L) AMC / sec / mg protein) where C = conversion factor that equals the amount of fluorescence at the concentration of free AMC (FU / pmol AMC).

RESULTS AND DISCUSSION The individual test data and the average +/- standard error mean (SEM) for each sample are presented in Appendix A. The values of the average 20S proteasome activity found for each human volunteer were in the range of 10.73 to 14.79 AMC / sec / mg of protein (Table 1 and Figure 1). The average 20S proteasome activity found in the population was 12.12 +/- 0.81 pmol ADC / sec / mg protein with the individual values observed in the range of 53% to 165% of the average value. A test was performed in duplicate in each sample. The variation in the test duplicates (% SEM) after five days of testing was 9.9%, 11.9%, 10.3%, 8.2% and 9.5%. For duplicates of individual tests this was in the range of 0.8% to 22.9%. The average variation between test duplicates up to four days of testing was 10.0%. In addition, the development of the test to reduce variation is in progress.

Table 1: Levels of Proteasome Activity in Human Volunteers Table 2: Variation in the Test Duplicates of the Proteasome Activity in Human Volunteers Example 10: Activity of Temporary 20S Proteasome in Isolated White Blood Cells, - and Tissues After N- (Pyrazine) Carbonyl-L- F-in-L-Leu Cin-Boronic Acid Administration (I) GENERAL PROCEDURES The dose formulations of 1 were prepared daily during the course of the study. The dilutions were prepared from a stock solution. The stock solution of 1 was constituted in 98% saline solution (0.9%), 2% ethanol with 0.1% ascorbic acid. The dilutions of the reserve were made in the same vehicle. Female CD2-F1 mice (18 to 20 g), female BALB / c mice (18 to 20 g), female Wistar rats (150 to 200 g) and male Sprague-Dawley rats (250 to 450 g) were obtained from Taconic Farms (Germantown, NY). The animals were observed for at least a week and examined for general health before the start of the study. The animals used in these studies were asymptomatic. The mice were housed 5 per cage and the rats 3 per cage in polycarbonate cages. A Corn Cob bed (AND-1005; Farmers Exchange, Framingham, MA) was used during the observation and study periods. Fluorescent lighting was controlled to automatically provide alternate cycles of light and dark of approximately 12 hours each. The temperature and humidity were centrally controlled and recorded daily, and the readings were in the range between 21 +/- 2 ° C and 45 +/- 5 ° C, respectively. The standard rodent food pellets (# 5001, Purina, St. Louis, MO) were available ad libitum throughout the observation and study periods. Water from the key of the City of Cambridge was provided by water bottles ad libi t um. There are no known food and water contaminants that could be expected to interfere with the study. The drugs were administered intravenously (IV) using a dose volume of 100 μl per mouse or 1.9 ml / kg in rats. The control groups were administered with the vehicle (98% saline [0.9%], 2% ethanol, 0.1% ascorbic acid). The animals were dosed with 1 as a single bolus given either once or on multiple occasions. The animals that show dying activity were sacrificed with inhalation with C02. After IV dosing with 1, blood was withdrawn at various time points and peripheral white blood cells were isolated. The tissues collected were brain, colon, liver, muscle (gastrocnemius), prostate and testes.

Proteasome Activity 20s g Vi vo Determined In Peripheral White Blood Cells of Mice, After the simple intravenous administration of 1 In two combined studies, female CD2-F1 mice (18 to 20 g) and BALB / c female mice (18 to 20 g) were given a single intravenous dose of 1 (0.1 to 30 mg / kg in one volume). of 100 μL dose).

The vehicle was 98% saline [0.9%], 2% ethanol, 0.1% ascorbic acid. Blood samples were collected at 1.0 and 24 h after administration. Due to the volume of blood required in the 20S proteasome activity assay, groups of five mice were sacrificed at the same time and their blood was combined to generate simple data points.

There was a significant dose-related decrease (p <0.05) in 20S proteasome activity for all dose groups at 1.0 h after intravenous administration of 1 (Figure 1) which begins to recover at 24 h (Figure 1). 2) . These studies demonstrated a reversible and dose-dependent inhibition of 20S proteasome activity in mouse peripheral white blood cells, after administration of a single intravenous injection of 1.

Activity of Proteasome 20s Ex Vi Vo determined in Cell, as Peripheral White Sanguineas of Rats, After the simple intravenous administration of 1 In four combined studies, female Wistar rats (150 to 200 g) were administered with a single intravenous dose of 1 (0.03 to 0.3 mg (kg in a dose volume of 1.0 mL / kg) .The vehicle was 0.1% of ascorbic acid / 2% ethanol / 98% saline solution (0.9%) Blood samples were collected at 1.0, 24 and 48 h after the administration of 1. There was a significant dose-related decrease (p <0.05) in the 20S proteasome activity at 1.0 h after intravenous administration of 1 (Figure 3) Twenty-four hours after administration, the dose-related decreases in 20S proteasome activity were smaller, but remained significant (p <0.05) in the highest-dose groups (0.2 mg / kg (Figure 4).) At 48 h after administration, the 20S proteasome activity was no longer significantly decreased (Figure 5). they showed an inhibition depending of the dose and reversible 20S proteasome activity in the peripheral white blood cells of rats, after the administration of a simple intravenous injection of 1. A smaller rate of return to the baseline for the levels of proteasome activity 20S was observed in rats, possibly indicating the fastest metabolism of 1 in mice. 20s Proteasome Activity Ex Vivo Determined In Peripheral White Blood Cells of Rats After Of the Iintravenous Administration Repeated 1 When compound 1 was administered intravenously daily for 7 days, a dose-related decrease in activity of the 20S proteasome was observed 24 h after the last dose was administered. Significant inhibition was observed for the 0.05 mg / kg doses. The degree of inhibition of 20S proteasome observed 24 h after the administration of 7 daily intravenous doses, was higher than that observed 24 h after the administration of a single intravenous dose and probably reflects a cumulative effect of daily administration of 1 on its objective biological, the proteasome. A significant dose-related decrease in 20S proteasome activity was observed 24 h after the last dose administration for alternate daily intravenous administration of compound 1 for 14 days. The dose-related decreases in 20S proteasome activity were significant (p <0.05) in the dose groups 0.2 mg / kg. A significant dose-related decrease was also observed (p >0.05) in the 20S proteasome activity, 24 h after the administration of the last dose for intravenous administration once a week of compound 1 for 8 weeks. The dose-related decreases in protease activity at 20S were significant (p <0.05) in the dose groups > 0.1 mg / kg. In a fourth repeated dose study, male Sprague-Dawley rats (250 to 450 g, n = 6 per group) were treated with intravenous doses, twice a week, of compound 1 (0.01 to 0.35 mg / kg / day). in a dose volume of 1.0 mL / kg) for two weeks. The vehicle was 0.1% ascorbic acid / 2% ethanol / 98% saline (0.9%). Blood samples were collected 1 h after the last dose for the evaluation of 20S proteasome activity. When administered 1 twice a week for 2 weeks (a total of 4 doses), a dose-related decrease in 20S proteasome activity was observed one hour after the last dose (Figure 6). The dose-related decreases in 20S proteasome activity were significant (p <0.05) for all dose groups 0.03 mg / kg. The results indicate that administration of repeated dose of 1 causes a dose-related decrease in 20S proteasome activity in white blood cells of rats. The degree of inhibition of 20S proteasome activity is greater than that observed after a single dose when administered 1 daily or every third day. When the interval between doses of 1 is increased to allow recovery (eg, once-a-week regimens), the degree of inhibition equivalent to the simple administration of 1. The pharmacokinetic profile supports dosing twice a week with 1, where transient inhibition is observed.

Proteasome Activity 20s Ex Vi, Determined in Rat Tissues After Repeated Intravenous Administration of 1 In two studies, female Wistar rats (150 to 200 g) were given a single intravenous dose of 1 (0.03, 0.1 and 0.3 mg / kg / in a dose volume of 1.0 mL / kg). The vehicle was 0.1% ascorbic acid / 2% ethanol / 98% saline (0.9%). Liver and brain tissue samples were collected at 1.0, 24 and 48 h after administration for the evaluation of 20S proteasome activity. There was a significant dose-related decrease (p <0.05) in 20S proteasome activity in rat liver at 1.0 h after intravenous administration of 1. Twenty-four hours after administration, dose-related decreases in the activity of proteasome 2 OS were smaller, but remained significant (p <0.05) in the high dose group, 0.3 mg / kg. At 48 h after the administration, the 20S proteasome activity in the rat liver had returned to baseline. The degree of inhibition of the 20S proteasome in the liver returned to baseline levels faster than that observed for peripheral white blood cells. No inhibition of 20S proteasome was observed in the brain tissue, reflecting the lack of penetration of 1 into this tissue. In a third study, rats were administered Male Sprague-Dawley (250 to 450 g) with a single intravenous dose of 1 (0.1 and 0.3 mg / kg in a dose volume of 1.0 mL / kg). The vehicle was 0.1% ascorbic acid / 2% ethanol / 98% saline (0.9%). Blood and tissue samples were collected 1.0 h after administration for the evaluation of 20S proteasome activity. The tissues collected were brain, colon, liver, muscle (gastrocnemius), prostate and testes.

Significant dose-related decreases (p <0.05) were observed in 20S proteasome activity in peripheral white blood cells in colon, liver, muscle (gastrocnemius), and prostate at 1.0 h after intravenous administration of 1. No Inhibition of 20S proteasome was observed in brain and testes, reflecting the lack of penetration of 1 into these tissues. Inhibition of 20S proteasome in tissues 1.0 h after administration of the intravenous dose, except for brain and testis, was similar to that observed for peripheral white blood cells.

Activity of Proteasome 2 Ex Vivo Determined in Primates After Simple Intravenous Administration of 1 Male and female Cynomolgus monkeys (from 2.2 to 3.5 kg) were assigned to four groups (5 / sex / group). Each group received 0 (control with vehicle), 0.045, 0.067 or 0.100 mg / kg / dose of 1 as a single intravenous injection in a dose volume of 0.3 mL / kg twice a week for 4 weeks (days 1, 5 , 8, 12, 15, 19, 22 and 26). The vehicle was 0.1% ascorbic acid / 2% ethanol / 98% saline (0.9%). Three males from the control groups, low and medium dose, two high dose males, and three females / group were sacrificed at the end of treatment on day 27. Two animals / sex / group were designated as animals in recovery and received treatment for 4 weeks followed by 2 weeks of recovery; these were sacrificed on Day 41. Blood was collected for the determination of 20S proteasome activity before treatment, at 1.0 h after dosing on Days 1, 8, 15 and 22, and 1.0 h before the treatment. dosage on Days 5, 12, 19 and 26; and on Days 31, 34, 38 and 41 (recovery animals slaughtered). Blood was also collected for determination of 20S proteasome activity from the high dose male before it was sacrificed in dying condition on day 26 after receiving 8 doses. The determination of 20S proteasome activity in white saline cells, 1.0 h after dosing, revealed a significant and dose-related decrease in enzyme activity that was recovered before subsequent dosing (Figures 7 and 8). The dying animal was found to have low residual 20S proteasome activity in their white blood cells at slaughter at day 26. These data support a twice-weekly treatment regimen for 1, as 20S proteasome levels recover between the doses. While the above invention has been described in certain details for purposes of clarity and understanding, it may be appreciated by a person skilled in the art from reading the description, that various changes in form and detail may be made. without departing from the true spirit of the invention and the appended claims.

It is noted that in relation to this date, the best method known to the applicant to carry out the aforementioned invention, is that which is clear from the present description of the invention.

Claims

CLAIMS Having described the invention as above, the content of the following claims is claimed as property:

1. A method for treating an affected patient with multiple sclerosis, characterized in that it comprises administering to the patient an effective amount of an agent selected from the group consisting of proteasome inhibitors, inhibitors of the ubiquitin pathway, agents that interfere with the activation of NF -? B by means of the ubiquitin-proteasome pathway, and mixtures thereof.

2. The method according to claim 1, characterized in that the agent is administered in an amount sufficient to reduce the frequency or severity of the relapse.

3. The method according to claim 1, characterized in that the agent is a proteasome inhibitor.

4. The method according to claim 3, characterized in that the proteasome inhibitor is lactacystin or an analogous compound of lactacystin.

5. The method according to claim 4, characterized in that the lactacistin analog compound is selected from the group consisting of lactacystin, clasto-lactacystin β-lactone, 7-ethyl-clasto-lactacystin β-lactone, β-lactone 7-n-propyl-clasto-lactacystin, and 7-n-butyl-clasto-lactacystin β-lactone.

6. The method according to claim 5, characterized in that the lactacistin analog compound is 7-n-propyl-clasto-lactacystin β-lactone.

7. A method for treating a patient affected with asthma, characterized in that it comprises administering to the patient an effective amount of an agent selected from the group consisting of inhibitors of .proteasome, inhibitors of the ubiquitin pathway, agents that interfere with the activation of NF-? B by means of the ubiquitin-proteasome pathway, and mixtures thereof.

8. The method according to claim 7, characterized in that the agent is administered in an amount sufficient to reduce the frequency or severity of the asthmatic attack.

9. The method according to claim 7, characterized in that the agent is a proteasome inhibitor.

10. The method according to claim 9, characterized in that the proteasome inhibitor is lactacystin or an analogous compound of lactacystin.

11. The method according to claim 10, characterized in that the lactacistin analog compound is selected from the group consisting of lactacystin, clasto-lactacystin ß-lactone, 7-ethyl-clasto-lactacystin ß-lactone, ß-lactone ß-lactone. -n-propyl-clasto-lactacystin, and 7-n-butyl-clasto-lactacystin ß-lactone.

12. The method according to claim 11, characterized in that the lactacistin analog compound is 7-n-propyl-clasto-lactacystin β-lactone.

13. A method for the treatment of a patient afflicted with asthma, characterized in that it comprises administering to the patient an effective combination of a glucocorticoid and an agent selected from the group consisting of proteasome inhibitors, inhibitors of the ubiquitin pathway, agents that interfere with the activation of NF-? B by means of the ubiquitin-proteasome pathway, and mixtures thereof.

14. The method according to claim 13, characterized in that the combination is administered in an amount sufficient to reduce the frequency of the severity of the asthmatic attack.

15. The method according to claim 13, characterized in that the glucocorticoid and the agent are administered at the same time.

16. The method according to claim 13, characterized in that the glucocorticoid and the agent are administered at different times.

17. The method according to claim 13, characterized in that the combination comprises an amount of glucocorticoid that is less than its standard recommended dose.

18. The method according to claim 13, characterized in that the combination comprises an amount of the agent, sufficient to reduce the dose or the frequency of the treatment required for the glucocorticoid.

19. The method according to claim 13, characterized in that the combination comprises an amount of glucocorticoid, sufficient to reduce the dose or frequency of treatment, required for the agent.

20. The method according to claim 13, characterized in that the agent is a proteasome inhibitor.

21. The method according to claim 20, characterized in that the proteasome inhibitor is lactacystin or an analogous compound of lactacystin.

22. The method according to claim 21, characterized in that the lactacistin analog compound is selected from the group consisting of lactacystin, clasto-lactacystin ß-lactone, 7-ethyl-clasto-lactacystin ß-lactone, ß-lactone 7-n-propyl-clasto-lactacystin, and 7-n-butyl-clasto-lactacystin ß-lactone.

23. The method according to claim 22, characterized in that the lactacistin analog compound is 7-n-propyl-clasto-lactacystin β-lactone.

24. The method according to claim 13, characterized in that the glucocorticoid is selected from the group consisting of flunisolide, triamcinolone acetonide, beclomethasone dipropionate, dexamethasone sodium phosphate, fluticasone propionate, budesonide, hydrocortisone, prednisone, prednisolone, mometasone and tipredane. butixicort

25. The method according to claim 24, characterized in that the glucocorticoid is budesonide.

26. The method according to claim 13, characterized in that the agent is the β-lactone of 7-n-propyl-clasto-lactacystin and the glucocorticoid is budesonide.

27. A pharmaceutical composition, characterized in that it comprises an effective combination of a glucocorticoid and an agent selected from the group consisting of proteasome inhibitors, inhibitors of the ubiquitin pathway, agents that interfere with the activation of NF-? B via the pathway of ubiquitin-proteasome, and mixtures thereof.

28. The composition according to claim 27, characterized in that the composition is provided in a unit dosage form.

29. The composition according to claim 28, characterized in that the unit dose form comprises an amount of the glucocorticoid that is less than its standard recommended dose.

30. The composition according to claim 27, characterized in that the composition comprises the agent in an amount sufficient to reduce the dose or frequency of treatment required for the glucocorticoid.

31. The composition according to claim 27, characterized in that the agent is a proteasome inhibitor.

32. The composition according to claim 31, characterized in that the proteasome inhibitor is lactacystin or a lactacistin analog.

33. The method according to claim 32, characterized in that the lactacistin analog compound is selected from the group consisting of lactacystin, clasto-lactacystin β-lactone, 7-ethyl-clas to-lactacystin β-lactone, β-lactone of 7-n-propyl-clasto-lactacystin, and β-lactone of "7-n-butyl-clasto-lactacystin.

34. The method according to claim 33, characterized in that the lactacistin analog compound is 7-n-propyl-clasto-lactacystin β-lactone.

35. The method according to claim 27, characterized in that the glucocorticoid is selected from the group consisting of flunisolide, triamcinolone acetonide, beclomethasone dipropionate, dexamethasone sodium phosphate, fluticasone propionate, budesonide-, hydrocortisone, prednisone, prednisolone, mometasone tipredane and butixicort.

36. The composition according to claim 35, characterized in that the glucocorticoid is budesonide.

37. The composition according to claim 27, characterized in that the agent is the β-lactone of 7-n-propyl-to-lactacystin and the glucocorticoid is budesonide.