Composition for proteasome inhibition

[0001] The Proteasome has been approved as a therapeutic target, as evidenced by the recent FDA approval for the introduction of bortezomib, a proteasome inhibitor, a boronic acid derivative, for the treatment of multiple myeloma. Recently, however, other inhibitors with higher proteasome specificity have been reported that may have less toxic side effects. These compounds include peptide epoxy-ketones, such as epoxomycin and peptide (b), described in US Patent 6,831,099 and peptide (a) described in US Patent Provisional Application No. 60 / 569,096, issued May 7, 2004, published as WO 2005105827. However the poor water solubility of some of these compounds makes it difficult to make compositions with optimal bioavailability. Additional methods for making epoxy-ketone peptide preparations are therefore needed.

Summary of the invention [0002] It has now been found that the solubility of proteasome inhibitors such as peptide epoxy ketones as described in claim 1. 1, increases significantly when its cyclodextrin preparation is made.

[0003] In one embodiment, the invention provides a pharmaceutical composition comprising a practically insoluble proteasome inhibitor, cyclodextrin and optionally a buffer. Such pharmaceutical compositions typically contain a pharmaceutically effective amount of a proteasome inhibitor, e.g. one that, when administered to a patient, alleviates the effects of, inter alia, neurodegenerative disease (such as Alzheimer's disease), immune disease conditions, muscle-destroying diseases, cancer, chronic infectious diseases, fever, muscle diseases, denervation, nerve damage and / or malnutrition.

[0004] In another aspect, the invention provides anti-inflammatory compositions that comprise a therapeutically effective amount of a proteasome inhibitor), cyclodextrin, and optionally a buffer.

[0005] In another aspect, the invention provides the use of these formulations for the manufacture of a medicament for use in methods that include, but are not limited to, inhibiting or reducing an HIV infection in a subject; effect on the level of expression of a viral gene in a person; altering many different antigenic peptides produced by the proteasome in the body; determining whether the cellular, developmental or physiological process or efficiency of this process in the body is regulated by the proteolytic activity of a particular Ntn hydrolase; treating Alzheimer's in a subject; reducing the rate of muscle protein degradation in a cell; reducing the rate of intracellular protein degradation in a cell; reducing the rate of p53 protein degradation in a cell; inhibiting the growth of p53-dependent tumors in a subject; inhibiting antigen presentation in a cell; suppression of a person's immune system (e.g. conditions such as septic shock, psoriasis, transplant rejection and rheumatoid arthritis); inhibiting the breakdown of IkBα in the body; reducing the NF-κΒ content in a cell, muscle, organ or person; influencing cyclin-dependent cell cycles in eukaryotic cells; treating a proliferative disease in a subject; effect on proteasome-dependent regulation of oncogenic proteins in the cell; treating tumor growth in a person; treatment in a p53-dependent person of apoptosis; and screening for proteins in the cell processed by the nucleophilic N-terminus hydrolases.

[0006] Other features and advantages of the invention will become apparent from the following detailed description and claims.

Brief description of the drawings [0007]

Fig. 1 shows the solubility of peptide (a) at various pH values ​​in 10% (w / v) solutions: beta-cyclodextrin sulfobutyl ether (SBECD) / 10 mM sodium citrate.

Fig. 2 shows the percentage of peptide (a) remaining in aqueous 10% (w / v) SBECD solutions / 10 mM sodium citrate versus time at different pH values.

Detailed description of the invention [0008] The pharmaceutical compositions of the invention contain a practically insoluble proteasome inhibitor, cyclodextrin and optionally a buffer.

[0009] The amount of proteasome inhibitor that can be solubilized depends on several parameters. One such parameter is the pH value. As shown in Figure 1, a higher pH value causes a lower solubility of the basic compound and it would be expected that a lower pH value would reduce the solubility of the acid compound, as is well known in the art. However, pH should be chosen to ensure adequate stability of the proteasome inhibitor. For example, a lower pH value reduces the chemical stability of one such compound, as shown in Figure 2. Effect

ΡΗ the stability and solubility of a compound can easily be determined by methods widely known in the art and disclosed herein. For formulations intended for use in a mammal, the pH is preferably from pH 2.5 to pH 9. [0010] In many compositions of the invention, the main source of pH adjustment is buffer. Typically, the buffer is present as an acid or base and the base or acid, respectively, coupled thereto. In one embodiment, the buffering salt concentration is in the range of 1 to 100 mM, preferably 5 to 50 mM, most preferably it is about 10 mM (in solid preparations the amount of buffer is selected to achieve such a concentration after reconstitution / dilution). The buffer concentration and pH of the solution are preferably selected so as to obtain an optimal balance of solubility and stability.

[0011] Examples of suitable buffers include mixtures of weak acids and alkali metal salts (e.g., sodium, potassium), coupling a base with a weak acid, such as sodium tartrate and sodium citrate. The preferred buffer is sodium citrate / citric acid. [0012] The cyclodextrins of the present invention include hydroxylpropylbetacyclodextrin (HPBCD) and beta-cyclodextrin sulfobutyl ether (SBECD), preferably SBECD.

[0013] One specific embodiment of the invention is a pharmaceutical formulation containing from 1 to 5 mg / ml of a proteasome inhibitor, 5% to 25% by weight / volume of cyclodextrin such as HPBCD and SBECD, and from 5 mM to 20 mM buffer giving a pH from about pH 3 to about pH 6, e.g. 2 mg / ml solution of the proteasome inhibitor (peptide (a)), 10% by weight / volume SBECD, 10 mM sodium citrate, pH 3.5. The use of cyclodextrins to dissolve a water-insoluble drug preparation is described in Liu (Waterinsoluble drug formulation) Interpharm Press, Denver, pages 111-130 (2009).

Proteasome inhibitors [0014] Some proteasome inhibitors similar to those described in claim 1 are described in Chemistry and Biology, 6 (11), pages 411-420 (1995).

[0015] Proteasome inhibitors have a structure described by formula (XIII), or a pharmaceutically acceptable salt thereof,

<img file="PL2260835T3_D0001.tif" />

(ΧΤΠ) in which;

Ar is each a 5-6 membered heteroaromatic group optionally substituted with 1-4 substituents;

L is selected from the group consisting of C = O and SO2;

X is O;

Y is absent;

Z is absent;

R¹, R², and R³ each independently selected from the group consisting of C1-6 alkyl, C1-₆hydroxyalkyl, C 1-4 alkoxyakyl, aryl, C 1-4 aralkyl, heteroaryl, C 1-7 heteroalkyl, heterocyclyl, and C 1-6 heterocycloalkyl;

R⁴ is N (R⁵) LZR⁶;

R⁵ is hydrogen;

R⁶ is heterocyclyl;

R and R are hydrogen;

[0016] In preferred embodiments, the inhibitor is represented by formula (XV), or a pharmaceutically acceptable salt thereof;

<img file="PL2260835T3_D0002.tif" />

wherein ;

R and R are each independently selected from the group consisting of C 1-4 alkyl, C 1-8 hydroxyalkyl, C 1-4 alkoxyalkyl, aryl, C 1-4 arylalkyl.

Definitions [0017] The term "C_x._salkyl "refers to a substituted or unsubstituted saturated hydrocarbon group containing straight chain alkyl groups and branched chain alkyl groups that contain from x to y carbon atoms in the chain, including haloalkyl groups such as trifluoromethyl and 2,2,2-trifluoroethyl, etc.

[0018] The term "alkoxy" refers to an alkyl group with oxygen attached. Suitable alkoxy groups include methoxy, ethoxy, Pronoxy, tert-butoxy, and the like.

[0019] The term "C 1-6 alkoxyalkyl" refers to a C 1-6 alkyl group substituted with an alkoxy group, thereby forming an ether.

[0020] The term "Ci.₆ arylalkyl "refers to a group Ci.₆aryl substituted alkyl.

(0021] The term "aryl" as used herein includes aromatic groups with a 5-, 6-, 7- membered substituted or unsubstituted single ring in which each ring atom is carbon. The term "aryl" also includes ring polycyclic systems containing two or more cyclic rings in which two or more carbons are common to two conjugated rings, where at least one of the rings is aromatic, e.g. other cyclic rings may be cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl, and / or heterocyclyl. Aryl groups include benzene, naphthalene, phenanthrene, phenol, aniline, and the like.

[0022] The term "buffer" means a substance that, by its presence in solution, increases the amount of acid or base that must be added to cause a unit change in pH. Therefore, the buffer is a substance that accompanies the regulation of the pH of the composition. In general, the buffer is selected based on the required pH level and is compatible with the other ingredients of the composition. Generally, the buffer has a pKa that is no more than 1 unit lower or higher than the desired pH of the composition (or when the composition will be formed after dissolution).

The term "C 1-6 heteroarylalkyl" refers to a C 1-6 alkyl group substituted with a heteroaryl group.

[0024] The term "heteroaryl" includes an unsubstituted aromatic 5-7 membered ring structure, more preferably 5-6 membered rings whose ring structure contains from 1 to 4 heteroatoms. The term "heteroaryl" also includes polycyclic ring systems containing two or more cyclic rings in which two or more carbon atoms are common to two adjacent rings, wherein at least one of the rings is heteroaromatic, e.g. other cyclic rings may be cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, hetero aryl, and / or heterocyclyl. Heteroaryl groups include, for example, pyrrole, furan, thiophene, imidazolol, oxazole, thiazole, triazole, pyrazole, pyridine, pyrazine, pyridazine and pyrimidine, and the like.

[0025] The term "heteroatom" as used herein means an atom other than carbon and hydrogen. Preferred heteroatoms are nitrogen, oxygen, phosphorus, and sulfur.

[0026] The term "heterocyclyl" or "heterocyclic group" means a substituted or unsubstituted non-aromatic 3-10 membered ring structure, more preferably 3-7 membered rings whose ring structure contains from 1 to 4 heteroatoms. The term "heterocyclyl" or "heterocyclic group" also includes polycyclic ring systems containing two or more cyclic rings in which two or more carbon atoms are common to two adjacent rings, wherein at least one of the rings is heterocyclic, e.g., other cyclic rings may be cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, hetero aryl, and / or heterocyclyl. Heterocyclic groups include, for example, piperidine, piperazine, pyridazine and pyrrolidine, morpholine, lactones, lactams and the like.

[0027] The term "C 1-6 hydroxylalkyl" refers to a C 1-6 alkyl group substituted with a hydroxyl group.

[0028] The term "inhibitor" as used herein, means a compound that blocks or reduces the activity of an enzyme (e.g., inhibits the proteolytic cleavage of standard fluorogenic peptide substrates such as suc-LLVY-AMC, BoxLLR-AMC and Z-LLE-AMC, inhibits various catalytic activities of the 20S proteasome). An inhibitor may act on the basis of competitive, anti-competitive and non-competitive inhibition. An inhibitor can bind reversibly or irreversibly, and thus, the term includes compounds that are suicide substrates for the enzyme. An inhibitor may modify one or more sites at or near the active site of an enzyme, or may cause a conformational change in another part of this enzyme.

[0029] The term "peptide" as used herein includes not only standard amide bonding with standard α-substituents but also commonly used peptidomimetics, other modified bonds, non-naturally occurring side chains, and side chain modifications, which are described in detail in this description.

[0030] The term "polycyclyl" or "polycyclic" refers to two or more rings (e.g., cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl and / or heterocyclic compounds) in which two or more carbons are shared by two adjacent rings, ie, these rings are "condensed rings." Each of the rings in this polycyclic compound may be substituted or unsubstituted.

[0031] The term "practically insoluble" refers to proteasome inhibitors whose water solubility is generally less than 0.1 mg / ml. The invention also encompasses proteasome inhibitors with a water solubility below 0.05 mg / ml and even below 0.01 mg / ml.

[0032] The term: "prophylactic or therapeutic" treatment is known in the art and includes administering to the host one or more of the compositions of the invention. If the composition is applied prior to the appearance of symptoms of an undesirable condition (e.g. disease or other undesirable condition in an animal host), then the treatment is prophylactic (i.e. it protects the host against the development of an undesirable condition), whereas if it is given after the appearance of the symptoms of an undesirable condition then the treatment is therapeutic (i.e. its purpose is to reduce, alleviate or stabilize existing undesirable condition or its side effects).

[0033] The term "proteasome" as used herein includes immunological and constitutive proteasomes.

[0034] The term "substituted" refers to residues containing substituents replacing a hydrogen atom on one or more non-hydrogen atoms in the molecule. It should be understood that the term "substituted" or "substituted with" includes the condition that such substitution is in accordance with the acceptable valence of the substituted atom and substituent, and that such substitution is formed in a stable compound, e.g. which does not undergo Pontanic transformation such as rearrangement, cyclization, elimination, etc. The term "substituted" as used herein is considered to include all acceptable substituents of organic compounds. In a broad aspect, acceptable substituents include acyclic and cyclic, branched and unbranched. carbocyclic and heterocyclic, aromatic and non-aromatic substituents of organic compounds. Acceptable substituents can be one or more, they can be the same or different for the corresponding organic compounds. For the purposes of this invention, heteroatoms such as nitrogen may have hydrogen substituents and / or acceptable substituents of the organic compounds described herein that meet the valences of the heteroatoms. Substituents may include, for example, halogen, hydroxyl, carbonyl (e.g. carboxy, alkoxycarbonyl, forms 1 or acyl), thiocarbonyl (such as thioeser, thiooclan or thioformate), alkoxy, phosphoryl, phosphate, phosphonate, phosphinate, amino, amido, amidine, imine. cyano, nitro, azide, sulfhydryl. alkylthio, sulfate, sulfonate, sulfamoyl, sulfonamido, sulfonyl, heterocyclyl, aralkyl, or an aromatic or heteroaromatic moiety. It will be understood by those skilled in the art that the moieties substituted in the hydrocarbon chain may themselves be substituted, if desired.

[0035] A "therapeutically effective amount" of a compound with respect to the above method of treatment, refers to the amount of compound (s) in a formulation that, when administered as part of a desired dosage regimen (to a mammal, preferably a human) alleviates the symptom, improves the condition, or slows down the onset of conditions sickness according to accepted medical standards for the disorder or condition to be treated, or which is used for cosmetic purposes, e.g. with a reasonable benefit / risk ratio applicable to all medical treatments.

[0036] As used herein, the term "treatment" or "treatment" includes reversing, reducing or stopping the symptoms, clinical signs and causal pathology of the condition in a way that improves or stabilizes the condition of the person.

Uses of the composition [0037] The biological consequences of proteasome inhibition are numerous. Inhibition of the proteasome is proposed as a method of preventing and / or treating a wide variety of diseases, including but not limited to proliferative diseases, neuronal intoxication and degeneration diseases, Alzheimer's disease, ischemic conditions, inflammation, autoimmune diseases, HIV, cancer, organ transplant rejection, septic shock, inhibition of antigen presentation, reduction of viral gene expression, control of parasitic infections, treatment of acidosis-related conditions, treatment of macular degeneration, treatment of pulmonary conditions, muscle-destroying diseases, fibrosis, bone and hair growth diseases. Pharmaceutical preparations of very strong proteasome specific compounds, such as epoxy-ketone class molecules, provide tools for the patient to use the drug and to treat these conditions.

[0038] At the cellular level, the accumulation of polyubiquitinated proteins, cell morphological changes and apoptosis were reported as a result of treating cells with various proteasome inhibitors. Proteasome inhibition has also been suggested as a possible therapeutic strategy for cancer. The fact that epoxymycin was initially identified in screening for compounds with anti-tumor activity gives importance to the proteosome as a target for cancer chemotherapy. Hence, these compositions are suitable for the treatment of cancer. Inhibition of the proteasome is also associated with inhibition of NF-κΒ activation and stabilization of p53 levels. Thus, the compositions of the invention can also be used to inhibit NF-κΒ activation and stabilize p53 levels in cell culture. As NF-κΒ is a key regulator of inflammation, it is an attractive target for therapeutic anti-inflammatory intervention. The compositions of the invention may therefore be useful in the treatment of inflammation-related conditions, including but not limited to obstructive pulmonary disease (COPD), psoriasis, bronchitis, emphysema and cystic fibrosis.

[0039] The disclosed compositions can be used to treat conditions directly caused by the proteolytic activity of the proteasome, such as muscle loss or induced indirectly, through proteins processed by the proteasome, such as NF-kB. Proteasome is involved in the rapid elimination and post-translational processing of proteins (e.g. enzymes) involved in cellular regulation (e.g. in cell cycle, gene transcription and metabolic pathways), in intercellular communication and in immune response (e.g. in antigen presentation). The specific examples described below include β-amyloid protein and regulatory proteins such as cyclins and NF-kB transcription factor.

[0040] Another object of the invention is to use compositions disclosed herein for the treatment of neurodegenerative diseases and conditions, including, but not limited to, stroke, ischemic nervous system injury, nervous system injury (e.g., percussion brain injury, spinal cord injury, and traumatic nervous system injury), multiple sclerosis and other immune-mediated neuropathies (e.g. Guillain-Barre syndrome and its variants, acute motor-axonal neuropathy, acute inflammatory demyelinating polyneuropathy and Fisher's syndrome), dementia in HIV / AIDS, axomonia, diabetic neuropathy, Parkinson's disease, Huntington's chorea, multiple sclerosis, meningitis bacterial, parasitic, fungal and viral background, encephalitis, vascular dementia, dementia after multiple heart attacks, dementia with Lewy bodies, fronto-temporal dementia, such as Pieka's disease, subcortical dementia (such as Huntington's chorea or progressive supranuclear palsy), focal cortical atrophy syndromes (such as primary aphasia), metabolic toxic dementia (such as chronic hypothyroidism or vitamin BI2 deficiency) and dementia infections (such as syphilis or chronic meningitis).

[0041] Alzheimer's disease is characterized by extracellular deposits of βamyloid (β-ΑΡ) protein in senile plaques and cerebral vessels. β-ΑΡ is a peptide fragment consisting of 39 to 42 amino acids, derived from the precursor of amyloid protein (APP). At least three APP isoforms (consisting of 695, 751 and 770 amino acids) are known. These isoforms result from alternative mRNA splicing;

normal processing involves part of the β-sekwencji sequence, thereby preventing β-ΑΡ formation. It is believed that the abnormal protein processing by the proteasome contributes to an abundance of β-ΑΡ in the brain of an Alzheimer patient. The APP processing enzyme in rats contains about ten different subunits (22 kDa to 32 kDa). The 25 kDa subunit has the N-terminal sequence X-Gln-Asn-Pro-Met-X-ThrGly-Thr-Ser, which is identical to the β-subunit of human macropain (Kojima S. et al., Fed. Eur. Biochem. Soc. (1992), 304: 57-60). The APP processing enzyme cleaves on Gln binding¹⁵Lys¹⁶; in the presence of calcium ion, this enzyme also cleaves at 11 12 Met -Asp and Asp -Ala bonds, releasing the extracellular domain of β-ΑΡ.

[0042] One embodiment of the invention is a method of treating Alzheimer's disease comprising administering to the person an effective amount of a composition disclosed herein. This treatment includes a reduction in β-przetwarzania processing speed, a decrease in β-szybkości plaque formation rate, a reduction in β-ΑΡ generation rate, and a reduction in clinical signs of Alzheimer's disease.

[0043] Other embodiments of the invention relate to cachexia and muscle-destroying diseases. Proteasomes break down many proteins in maturing reticulocytes and growing fibroblasts. In cells deprived of insulin or serum, the rate of proteolysis almost doubles. Inhibition of the proteasome inhibits proteolysis, thereby reducing the loss of muscle protein and the nitrogen load on the kidneys or liver. The inhibitors of the invention are useful in the treatment of conditions such as cancer, chronic infectious diseases, fever, inactivity (atrophy) and muscle denervation, nerve damage, malnutrition, acidosis-related renal failure and liver failure. See, e.g., Goldberg, US Patent No. 5,340,736. An embodiment of the invention thus includes methods for: reducing the rate of muscle protein degradation in a cell; reducing the rate of intracellular protein degradation; reducing the rate of p53 protein degradation in a cell; and inhibiting the growth of p53-dependent tumors. Each of these methods involves contacting the cell (in vivo or in vitro, e.g., muscle of a subject) with an effective amount of the pharmaceutical composition disclosed herein.

[0044] Fibrosis is the excessive and persistent formation of scar tissue following hyperproliferative growth of fibroblasts and is associated with activation of the TGF-β signaling pathway. In fibrosis, excessive intercellular deposition occurs. It can actually appear in any tissue or spread across several different tissues. Normally, the level of intracellular signaling protein (Smad) that activates TGF-β-stimulated transcription of target genes is regulated by proteasome activity (Xu et al., 2000). However, accelerated degradation of TGF-β signaling components has been observed in cancer and other hyperproliferative conditions. Therefore, some embodiments of the invention relate to a method of treating hyperproliferative conditions such as diabetic retinopathy, macular degeneration, diabetic nephropathy, glomerulosclerosis, IgA nephropathy, cirrhosis, biliary atresia, congestive heart failure, scleroderma, fibrosis, and radiation fibrosis idiopathic pulmonary fibrosis, vascular collagenosis, sarcoidosis, interstitial lung diseases and vascular lung disorders). Treatment of burn victims is often hampered by fibrosis, and thus, an additional embodiment of the invention is the local or general use of inhibitors in the treatment of burns. Wound closure after surgery is often associated with unsightly scars that can be prevented by inhibiting fibrosis. Thus, some embodiments of the invention relate to a method of preventing or reducing scarring.

[0045] Another protein processed by the proteasome is NF-κΒ, a member of the Rei protein family. The Rei family of transcription activator proteins can be divided into two groups. The first group requires proteolytic processing and includes p50 protein (NF-kB1, 105 kDa) and p52 protein (NF-k2, 100 kDa). The second group does not require proteolytic processing and includes p65 (RelA, Rei (c-Rel) and RelB). Members of the Rei family can form both homo and heterodimers; for example, NF-κΒ is a p-50-p65 heterodimer. After phosphorylation and ubiquitination of IkB and p05, these two proteins degrade and process, respectively, to give active NF-κΒ, which travels from the cytoplasm to the nucleus. The ubiquitinated p05 protein is also processed by purified proteasomes (Palombełla et al., Cell (1994), 78: 773-785). Active NF-κΒ forms with other transcription activators and e.g. with IIMG I (Y) a stereospecific enhancer complex, inducing the selective expression of a particular gene.

[0046] NF-κΒ regulates genes involved in the immune and inflammatory response as well as mitotic events. For example, NF-κΒ is needed for the expression of the immunoglobulin κ light chain gene, IL-2 receptor chain gene, the major histocompatibility complex class I (MHC-I) gene and many genes for cytokines encoding for example IL-2, IL-6, granulocyte colony stimulating factor and IFN-β (Palombella et al., Cell (1994), 78: 773-785). The compositions of the invention are useful in methods of affecting the level of expression of IL-2, MHC-I, IL-6, TNF-α, IFN-β and other proteins mentioned above, each of which involves administering to the individual an effective amount of the composition disclosed in this description. Complexes containing p50 are rapid mediators of acute inflammatory and immune responses (Thanos D. and Maniatis T., Cell (1995) 80: 529-532).

[0047] NF-κΒ also participates in the expression of cell adhesion genes encoding Eselectin, P-selectin, intercellular adhesion molecule (ICAM) and vascular cell adhesion molecule (VCAM-1) (Collins T., Lab. Invest. (1993) 68 : 499-508). One embodiment of the invention is a method of inhibiting cellular adhesion (e.g. cell adhesion mediated by E-selectin, P-selectin, ICAM or VCAM-I) by contacting the cell with an effective amount of the composition disclosed herein (or administering the composition to a person).

[0048] Ischemic and reperfusion injury causes hypoxia (hypoxia), a condition in which there is a deficiency of oxygen reaching tissues in the body. This condition causes increased rozkładκ-Βα degradation, which in turn causes NF-κΒ activation (Koong et al. 1994). It has been shown that the severity of damage resulting from hypoxia can be reduced by administering a proteasome inhibitor (Gao et al. 2000; Bao et al., 2001; Pye et al., 2003). Thus, some embodiments of the invention relate to a method of treating an ischemic or reperfusion injury condition, comprising administering to a person in need of such treatment an effective amount of a compound disclosed herein. Examples of such conditions or injuries include, but are not limited to, acute coronary syndrome (plaques sensitive to damage), arterial occlusive disease (cardiac, cerebral, peripheral and vascular occlusion), atherosclerosis (coronary atherosclerosis), coronary artery disease), heart attacks, heart failure. pancreatitis, myocardial hypertrophy, narrowing and recurrence of stenosis.

[0049] NF-κΒ also binds specifically to the HIV enhancer / promoter. Compared with Nef from mac239, the HIV Nef regulatory protein from pbj 14 differs by two amino acids in the region that controls protein kinase binding. This protein kinase is thought to signal IkB phosphorylation, releasing IkB degradation via the ubiquitin-proteasome pathway. After degradation, NF-κΒ is released into the nucleus, thereby enhancing HIV transcription (Cohen J., Science (1995) 267: 960). Two suitable uses of the compounds of the invention are a method of inhibiting or reducing HIV infection in a person and a method of reducing the level of expression of a viral gene. Each of these methods involves administering to the individual an effective amount of the composition disclosed herein.

[0050] Overproduction of lipopolysaccharide (LPS) -induced cytokines such as TNF-α is believed to be a major phenomenon in processes associated with septic shock. In addition, it is widely recognized that the first step in cell activation by LPS is the binding of LPS to specific membrane receptors. The α and β subunits of the 20S proteasome complex have been identified as LPS binding proteins, suggesting that LPS-induced signal transduction may be an important therapeutic target in the treatment or prevention of sepsis (Qureshi N. et al., J. Immun. (2003) 171: 1515-1525). Therefore, in some embodiments, the compositions of the invention may therefore be useful in inhibiting TNFα to prevent and / or treat septic shock.

[0051] As a result of intracellular proteolysis, small peptides are generated for presentation to T lymphocytes to elicit MHC class I immune responses. The immune system is screening for the presence of autologous cells that are infected with a virus or have undergone oncogenic transformation. One suitable embodiment of the invention is a method of inhibiting the presentation of an antigen in a cell, comprising exposing said cell to the composition disclosed herein. A further suitable embodiment of the invention is a method of suppressing the immune system in a person (e.g., inhibiting transplant rejection, allergies, asthma), comprising administering to that person an effective amount of a composition disclosed herein. The compositions of the invention may also be used to treat autoimmune diseases such as lupus, rheumatoid arthritis, multiple sclerosis and inflammatory bowel diseases such as ulcerative colitis and Crohn's disease.

[0052] Another further suitable embodiment of the invention is a method for changing the repertoire of antigenic peptides produced by the proteasome or other Ntn hydrolase with multicatalytic activity. For example, if PGPH activity of the 20S proteasome is selectively inhibited, then this proteasome will produce a set of antigenic peptides then presented in the MHC molecule system on the cell surface, different from the set that would be produced and presented either without inhibition of any enzyme or with e.g. selective inhibiting the chymotrypsin-like activity of this proteasome.

[0053] Some proteasome inhibitors block both the degradation and processing of ubiquitinated NF-κΒ in vitro and in vivo. Proteasome inhibitors also block rozkładκΒ-α degradation and NF-κΒ activation (Palombella et al., Cell (1994), 78: 773-785; and Traenckner et al., EMBO J. (1994) 13: 5433-5441). One suitable application of the compositions of the invention is a method of inhibiting ΙκΒ-α degradation, comprising contacting the cell with the composition described herein. A further suitable embodiment of the invention is a method of reducing the cellular content of NF-κΒ in a cell, muscle, organ or person, comprising contacting that cell, muscle, organ or person with the composition described herein.

[0054] Other eukaryotic transcription factors that require proteolytic processing include: general TFIIA transcription factor, herpes simplex virus VP 16 helper protein (host cell factor), protein induced IFN regulatory factor 2 protein and membrane binding protein sterol regulatory element. [0055] Another embodiment of the invention is a method of affecting cyclin-dependent cell cycles in eukaryotic cells, comprising exposing the cell (in vitro and in vivo) to the composition disclosed herein. Cyclins are proteins involved in cell cycle control. Proteasome is involved in cyclin degradation. Examples of cyclins include mitotic cyclins, cyclins G1 and cyclin B. Degradation of cyclins allows the cell to exit from one stage of the cell cycle (e.g. mitosis) and entering another (e.g. division). All cyclins are thought to be associated with p34 protein kinase^cdc2 or with related kinases. The signal for proteolysis is located in amino acids 42-RAALGNISEN-50 (destructive sequence). There is evidence that cyclin is converted into a ubiquitin ligase sensitive form or that cyclin-specific ligase is activated during mitosis (Ciechanover A., ​​Cell (1994) 79: 13-21). Inhibition of the proteasome inhibits cyclin degradation and thus, inhibits cell proliferation, for example, in cyclin-dependent tumors (Kumatori et al., Proc. Natl. Acad. Sci. USA (1990) 87: 7071-7075). One suitable embodiment of the invention is a method of treating a proliferative disease in a subject (e.g., cancer, psoriasis or recurrence of vasoconstriction), comprising administering to the individual an effective amount of a composition disclosed herein. The invention also encompasses a method of treating cyclin-dependent inflammation, comprising administering to said person a therapeutically effective amount of a composition described herein.

[0056] Additional suitable embodiments of the invention are methods of affecting proteasome-dependent oncoprotein regulation and methods of treating or inhibiting tumor growth; each of these methods involves exposure of the cell (in vivo, e.g. in a person or in vitro} to the composition disclosed herein. HPV-16 and HPV-18 E6 proteins stimulate ATP and ubiquitin-dependent p53 coupling and degradation in crude lysates reticulocyte. It turned out that the recessive p53 oncogene accumulates at an illegal temperature in a cell line with mutated thermolabile El. Elevated levels of p53 may lead to apoptosis. Examples of proto-oncoproteins degraded by the ubiquitin system include c-Mos, c-Fos and c-Jun. One embodiment of the invention is a method of treating p53-dependent apoptosis, comprising administering to the individual an effective amount of the composition disclosed herein.

[0057] In one embodiment of the invention, the disclosed compositions are useful in the treatment of a parasitic infection, such as an infection caused by protozoal parasites. It is assumed that the proteasome of these parasites is mainly involved in cell differentiation and replication-related activities (Paugam et al., Trends Parasitol. 2003, 19 (2): 55-59). In addition, it was found that amoebas lose the ability to encapsulate when exposed to proteasome inhibitors (Gonzales et al., Arch. Med. Res. 1997, 28, Spec No 139-140). In such embodiments, the disclosed compositions are useful in the treatment of human parasitic infections caused by protozoal parasites selected from the genus Plasmodium (including P. falciparum, P. vivax, P. malariae and P. ovale, causing malaria), the genus Trypanosoma (including T. cruzi, which causes Chagas and T. brucei, which causes African coma) of the genus Leishmania (including L. amazonesis, L. donovani, L. infantum, L. Mexiana and the like), Pneumocystis carinii (a protozoan that is known to cause pneumonia in AIDS and other immunosuppressive patients), Toxoplasma gondii, Entamoeba histolytica, Entamoeba invadens and Giardia lamblia. In some embodiments, the disclosed compositions are useful in the treatment of parasitic infections in animals and in livestock caused by protozoal parasites selected from Plasmodium hermani, genus Cryptosporidium, Echinococcus granulosus, Eimeria tenella, Sarcocystis neurona and Neurospora crassa. Other compounds useful as proteasome inhibitors in the treatment of parasitic infections are described in WO 98/10779, which is incorporated herein by reference.

[0058] In some embodiments, the disclosed compositions inhibit the proteasome activity irreversibly. Such irreversible inhibition has been shown to induce the closure of enzymatic activity irretrievably in red blood cells and in white blood cells. In some such embodiments, the long half-life of blood cells may provide extended protection with respect to therapy in the event of repeated exposure to parasites. In some such embodiments, the long half-life of blood cells may provide extended protection with respect to the prophylactic use of drugs against future infection.

[0059] Inhibitors that bind to the 20S proteasome have been shown to stimulate bone formation in bone tissue cultures. Also, when such inhibitors were administered systemically to mice, then some proteasome inhibitors increased bone volume and bone formation rate by 70% (Garrett IR et al., J. Clin. Invest. (2003) 111: 1771-1782), which indicated that the ubiquitin-proteasome machinery regulates osteoblast differentiation and bone formation. The disclosed compositions may therefore be useful in the treatment and / or prevention of diseases associated with bone loss such as osteoporosis.

(0060] Bone tissue is an excellent source of factors that have the ability to stimulate bone cells. Thus, bovine bone tissue extracts contain not only the structural proteins responsible for maintaining bone structural integrity but also biologically active bone growth factors that have the ability to stimulate bone cells Of the latter factors, recently a family of proteins called bone morphogenetic proteins (BMPs) has been described. All these growth factors affect other cell types, as well as bone cells, including Hardy Μ. H. et al., Trans. Genet. (1992) 8: 55-61 described the evidence that bone morphogenetic proteins (BMPs) are produced differently in hair follicles during development. Harris SE et al. (J. Bona Miner. Res. (1994) 9: 855-863) described the effect of TGF-β on the expression of BMP-2 and other substances in bone cells. BMP-2 expression in mature follicles also occurs during maturation and after a period of cell proliferation (Hardy et al. (1992, supra). Thus, the compounds of the invention may also be useful to stimulate hair follicle growth.

[0061] Finally, the disclosed compositions are also useful as diagnostic agents (e.g., in diagnostic kits or for use in clinical laboratories) for screening for proteins (e.g., enzymes, transcription factors) processed by Ntn-hydrolases, including proteasome. The disclosed compositions are also useful as reagents for testing for specific binding of the Χ / ΜΒ1 subunit or the α chain and for inhibition of associated proteolytic activities. For example, the activity of other proteasome subunits (and their specific inhibitors) can be determined.

[0062] Most cellular proteins undergo proteolytic processing during maturation or activation. The enzyme inhibitors disclosed herein can be used to determine whether a cellular, developmental or physiological process or its efficiency is regulated by the proteolytic activity of a particular Ntn hydrolase. One such method involves obtaining an organism, intact cell preparation or cell extract; exposing the organism, cell preparation or cell extract to the composition disclosed in the description; exposure of the exposed body compound, intact cell preparation or cell extract to a signal and monitoring of this process or performance. The high selectivity of the compounds disclosed in the description allows for rapid and accurate exclusion or detection of Ntn (e.g. 20S proteasome) in a given cellular, developmental or physiological process.

Administration [0063] The compositions prepared as described herein can be ordained in a variety of forms depending on the condition to be treated, the age, health and weight of the patient, which is a matter well known in the art. For example, if the compositions are to be used orally, they may be prepared in the form of tablets, capsules, granules, powders or syrups; if they are to be administered parenterally, they may be prepared in the form of injections (intravenous, intramuscular or subcutaneous), drip infusions or suppositories. For use through the mucous membrane of the eye, they can be prepared as eye drops and eye ointments. These preparations can be prepared by conventional methods in conjunction with the methods described herein and, if desired, in addition to cyclodextrin and buffer, the active ingredient may be mixed with a conventional additive or excipient such as a binder, disintegrant, glidant, flavor enhancer, a solubilizing, suspending, emulsifying or coating agent. Although the dosage will vary, depending on the patient's symptoms, age and weight, type and severity of the condition to be treated or prevented, route of administration and form of the drug, daily doses of 0.01 to 0.01 are generally recommended. 2000 mg of the compound for an adult human can be given as a single dose or as divided doses. The amount of active ingredient that can be combined with a carrier material to produce a single-dose drug form will generally be the amount of compound that produces the therapeutic effect. In general, compositions intended for parenteral use (e.g., as intravenous or subcutaneous injections) contain substituted cyclodextrin. Compositions used by other routes, especially oral, include substituted or unsubstituted cyclodextrin.

[0064] The specific duration of use and / or amount of the composition that will give the best results in terms of the effectiveness of treatment in a given patient will depend on the activity, pharmacokinetics and bioavailability of the particular compound, the patient's physiological condition (including age, sex, type and stage of the disease, general physical condition, response to the given dosage and type of treatment), route of administration and other factors. However, the above guidelines can serve as a basis for fine-tuning treatment, and thus determining the optimal time and / or amount of drug administered, which will not require more than routine experimentation of monitoring the patient and adjusting the dosage and / or synchronizing the time of use.

[0065] The expression "pharmaceutically acceptable" is used herein to refer to ligands, materials, compositions and / or dosage forms which, according to reasonable medical judgment, are suitable for use in contact with human and animal tissues without causing excessive toxicity, irritation. , allergic response or other problem or complication commensurate with a reasonable benefit-risk balance.

[0066] The term "pharmaceutically acceptable carrier" as used herein means a pharmaceutically acceptable material, composition or carrier such as a liquid or solid filler, diluent, excipient, solvent or material used for encapsulation. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and must not harm the patient. Some examples of materials that can serve as pharmaceutically acceptable carriers include: (1) sugars such as lactose, glucose and sucrose; (2) starches, such as corn starch, potato starch and substituted or unsubstituted β-cyclodextrin; (3) cellulose and its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) tragacanth powder; (5) malt; (6) gelatin; (7) talc; (8) excipients such as cocoa butter and wax suppository bases; (9) oils, such as peanut oil, cottonseed oil, sunflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols such as propylene glycol; (11) polyols such as glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's fluid; (19) ethyl alcohol; (20) phosphate buffer solutions; and (21) other non-toxic, non-incompatible substances used in pharmaceutical preparations. In some embodiments, the pharmaceutical compositions of the invention are non-pyrogenic, i.e., they do not cause a significant increase in temperature when administered to a patient.

[0067] The term "pharmaceutically acceptable salt" refers to relatively non-toxic inorganic and organic acid addition salts of the inhibitor (s). Such salts can be prepared in situ during the final isolation and purification of the inhibitor (s) or by separately subjecting the purified inhibitor (s) in the form of the free base, reacting with the appropriate organic or inorganic acid, and isolating the salt thus formed. Representative salts include the salts of: hydrobromide, hydrochloride, sulfate, disulfate, phosphate, nitrate, acetate, valerate, oleate, palmitate, stearate, laurate, benzoate, lactate, phosphate, toluenesulfonate, citrate, maleate, fumarate, succinate, tartrate, succinate, tartrate methylsulfonate, glucoheptonate, lactobionate, lauryl sulfonate and salts with amino acids and the like (See, for example, Berge et al. (1977) "Pharmaceutical Salts", J. Pharm. Sci. 66: 1-19).

[0068] In other cases, inhibitors useful in the methods of the present invention may contain one or more acidic functional groups and are therefore capable of forming pharmaceutically acceptable salts with pharmaceutically acceptable bases. The term "pharmaceutically acceptable salts" in these cases refers to relatively non-toxic, inorganic and organic base addition salts of the inhibitor (s). These salts can also be prepared in situ during the final isolation and purification of the inhibitor (s), or by separately treating the purified inhibitor (s) in the form of the free acid with a suitable base, such as hydroxide, carbonate or bicarbonate of a pharmaceutically acceptable metal cation, with ammonia or from a pharmaceutically acceptable organic primary, secondary, or tertiary amine. Representatives of alkaline or alkaline earth salts are lithium, sodium, potassium, calcium, magnesium and aluminum salts, and the like. Representatives of an organic amine useful for forming the base addition salt include ethylamine. diethylamine. ethylenediamine, ethanolamine, diethanolamine, piperazine, and the like (see, for example, Berge et al. as above). [0069] These compositions may also contain wetting, emulsifying and lubricating agents, such as sodium lauryl sulfate and magnesium stearate, and coloring, release enhancing agents, coating agents, sweeteners, flavors and flavors, preservatives and antioxidants.

[0070] Examples of pharmaceutically acceptable antioxidants include: (1) water soluble antioxidants such as ascorbic acid, cysteine ​​hydrochloride, sodium disulfate, sodium metabisulfate, sodium sulfite and the like; (2) oil-soluble antioxidants such as ascorbyl palmitate, utilized hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol and the like; and (3) metal chelating agents such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid and the like.

[0071] Formulations suitable for oral use may be in the form of capsules, sachets, pills, tablets, lozenges (using a base with a pleasant taste, usually sucrose and acacia or tragacanth), powders, granules or a solution or suspension in an aqueous liquid or non-aqueous or as an oil-in-water liquid emulsion or water-in-oil, or an elixir or syrup or lozenges (using an inert matrix, such as gelatin and glycerin or sucrose and acacia) and / or mouthwash and similar forms, each containing a specific amount of inhibitor (s) as the active ingredient. The composition can also be used as one large dose (bolus), butter or paste.

[0072] In solid dosage forms for oral use (capsules, tablets, pills, dragees, powders, granules and the like), the active ingredient is mixed with one or more pharmaceutically acceptable carriers such as sodium citrate or dicalcium phosphate and / or with any of the following excipients: (1) bulking and bulking agents such as starches, cyclodextrins, lactose, sucrose, glucose, mannitol and / or silicic acid; (2) binders such as, for example, carboxymethyl cellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose and / or acacia; (3) humectants such as glycerol; (4) disintegrants such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates and sodium carbonate; (5) solution retarding agents such as paraffin; (6) absorption accelerators, such as quaternary ammonium compounds; (7) wetting agents such as, for example, acetyl alcohol and glycerol monostearate; (8) adsorbents, such as kaolin and bentonite clay; (9) lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate and mixtures thereof; and (10) coloring agents. In the case of capsules, tablets and pills, these pharmaceutical compositions may also contain buffering agents. Solid compositions of a similar type may also be employed for filling soft and hard-filled gelatin capsules, using lactose or milk sugars as well as high molecular weight polyethylene glycols and the like as excipients.

[0073] A tablet may be made by compression or molding, optionally with the addition of one or more accessory ingredients. Compressed tablets can be made using a binder (e.g., gelatin or hydroxylpropyl methylcellulose), a lubricant, an inert diluent, a preservative, a disintegrant (e.g., sodium starch glycolate or cross-linked sodium carboxymethyl cellulose), a surfactant or dispersing agent. Extruded tablets can be made by molding in a suitable machine a mixture of the powdered inhibitor (s) moistened with an inert liquid diluent.

[0074] Tablets and other solid dosage forms such as dragees, capsules, pills and granules may optionally be grooved or provided with coatings or coatings, such as an enteric coating and other coatings known in the art of making pharmaceutical preparations. These preparations may also be prepared so as to provide slow or controlled release of the active ingredient therefrom, using, for example, hydroxypropyl methylcellulose in various proportions suitable for providing the desired release profile, other polymer matrices, liposomes and / or microspheres. They can be sterilized, for example, by filtration through a bacterial-retaining filter or by incorporating sterilizing agents in the form of sterile solid compositions that can be dissolved in sterile water or some other sterile injectable medium immediately before use. These compositions may also optionally contain opacifying agents and may be compositions releasing the active ingredient (s) exclusively or preferentially in a certain part of the gastrointestinal tract, optionally in a slow manner. Examples of coating compositions that can be used include polymeric substances and waxes. The active ingredient may also be in micro-encapsulated form, with the addition of one or more of the excipients described above, as desired.

[0075] Liquid dosage forms for oral use include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. In addition to the active ingredient, these liquid dosage forms may contain inert diluents commonly known in the art, such as, for example, water or other solvents, solubilizers and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzoate benzyl, propylene glycol, 1,3-butylene glycol, oils (especially oils: cotton, peanut, corn, germ, oil, rapeseed and sesame oil), glycerol, tetrahydrofuryl alcohol, polyethylene glycols and sorbitan fatty acid esters and mixtures thereof.

[0076] In addition to inert diluents, these oral compositions may also contain adjuvants such as wetting, emulsifying, suspending, sweetening, flavoring, coloring, flavoring and preserving agents.

[0077] Suspensions, in addition to the active inhibitor (s) may also contain suspending agents such as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agaragar and tragacanth, and mixtures thereof.

[0078] Preparations for rectal or vaginal use may be in the form of a suppository, which may be prepared by mixing one or more inhibitors with one or more suitable non-irritating excipients or carriers, which are, for example, cocoa butter, polyethylene glycol, a wax suppository base or Salicylate, which are solid at room temperature and become liquid at body temperature and therefore they will melt in the rectal or vaginal space and release the active agent.

[0079] Formulations that are adapted for vaginal use also include pessaries, tampons, creams, gels, pastes, foams or sprays containing such carriers as are known in the art to be suitable.

[0080] Dosage forms for topical or transdermal use (s) include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches and inhalants. The active ingredient may be mixed under sterile conditions with a pharmaceutically acceptable carrier and with such preservatives, buffers or propellants as may be required.

[0081] Ointments, pastes, creams and gels may contain, in addition to the inhibitor (s), auxiliary substances such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc, zinc oxide or mixtures thereof.

[0082] Powders and sprays may contain, in addition to the inhibitor (s), excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicates and polyamide powder, and mixtures of these substances. The sprays may additionally contain commercial propellants such as chlorofluoro hydrocarbons and volatile, unsubstituted hydrocarbons such as butane and propane.

[0083] The inhibitor (s) may alternatively be used as an aerosol. This can be done by preparing an aqueous aerosol, liposome preparation or composition containing solid particles. A non-aqueous suspension may be used (e.g. with a fluorocarbon propellant). Ultrasonic nebulizers are recommended as they minimize exposure of the agent to shear forces that can cause compound degradation.

[0084] Typically, an aqueous aerosol is prepared by preparing an aqueous solution or suspension of this agent with conventional, pharmaceutically acceptable carriers and stabilizers. Carriers and stabilizers differ in requirements for a particular composition, but generally include nonionic surfactants (products under the names Tween, Pluronic, sorbitan esters, lecithin, Cremophor), pharmaceutically acceptable co-solvents such as polyethylene glycol, harmless proteins such as serum albumin, sorbitan esters, oleic acid, lecithin, amino acids such as glycine, buffers, salts, sugars or sugar alcohols. Aerosols are generally prepared from isotonic solutions.

[0085] Transdermal patches have the additional advantage of allowing controlled delivery of the inhibitor (s) to the body. Such dosage forms can be made by dissolving or dispersing the agent in a suitable medium. Absorption enhancers may also be used to increase the penetration of the inhibitor (s) through the skin. The permeation rate can be controlled either by introducing a rate controlling membrane or by dispersing the inhibitor (s) in the polymer matrix or gel.

[0086] Pharmaceutical compositions of the invention suitable for parenteral use contain peptide (a) in combination with one or more pharmaceutically acceptable, sterile, aqueous or non-aqueous solutions, dispersions, suspensions or emulsions, or sterile powders, which can be reconstituted to form sterile injectable solutions or dispersions immediately before use, which compositions may contain antioxidants, buffers, bacteriostatic agents, solutes that render the formulation isotonic with the recipient's blood, or suspending or thickening agents.

[0087] Examples of suitable aqueous and non-aqueous carriers that can be used in the pharmaceutical compositions of the invention include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol and the like) and suitable mixtures thereof, vegetable oils such as oil from olive oil and injectable organic esters such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.

[0088] These compositions may also contain auxiliary agents such as preserving, wetting, emulsifying and dispersing agents. Protection against the action of microorganisms can be provided by the addition of various antibacterial and antifungal agents, for example paraben, chlorobutanol, phenol sorbic acid and the like. It is also desirable to include isotonic agents, such as sugars, sodium chloride, and the like into the composition. In addition, prolonged absorption of the injectable pharmaceutical preparation can be brought about by the inclusion of agents that delay absorption such as aluminum monostearate and gelatin.

[0089] In some cases, in order to prolong the effect of a drug, it is desirable to slow the absorption of this drug from subcutaneous or intramuscular injection. For example, slowed absorption of the parenterally administered drug form can be achieved by dissolving or suspending the drug in an oil carrier.

[0090] Injectable depot forms are made by forming microencapsule matrices of the inhibitor (s) in biodegradable polymers such as polylactide-polyglycolide. The rate of drug release can be controlled by the ratio of drug to polymer and the type of polymer specifically used. Examples of other biodegradable polymers include poly (orthoesters) and poly (anhydrides). Injectable depot preparations are also prepared by entrapping the drug in liposomes or in microemulsions that are compatible with body tissue.

[0091] Formulations of agents may be administered orally, parenterally, topically, or rectally. Of course, they are given in forms adapted to the given route of administration. For example, they are administered in the form of tablets or capsules, by injection, inhalation, as eye lotion, ointment, suppository, infusion; topically in the form of a lotion or ointment; and rectally in the form of suppositories. Oral administration is preferred.

[0092] The terms "parenteral" and "parenterally administered" as used herein mean non-gastrointestinal and topical methods, usually by injection, and include, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intrathecal, intrathecal, intrathecal injection orbital, intracardiac, intradermal, intraperitoneal, endotracheal, subcutaneous, subcutaneous, intraocular, subcapsular, subarachnoid, intrathecal and intrasternal and by infusion.

[0093] The terms "general use", "generally used", "peripheral use" and "peripherally used" mean in the present description the use of a ligand, drug or other material other than directly into the central nervous system in such a way that it enters to the patient's system and then becomes subject to metabolism and other such processes, for example, subcutaneous use.

[0094] The inhibitor (s) can be used in humans and other animals for therapeutic purposes by any convenient route of administration, including orally, intranasally, in the form of, for example, spray, rectal, vaginal, post-elution, topical and topical, in the form of powders, ointments or drops, including buccal and sublingual.

[0095] Regardless of the chosen route of use, pharmaceutically acceptable dosage forms are prepared from the inhibitor that can be used in an appropriately hydrated form and / or from the pharmaceutical compositions of the invention by conventional methods known to those skilled in the art.

[0096] Actual dosage levels of the active ingredients in the pharmaceutical compositions of the invention may be different, so as to obtain an amount of active ingredient that is effective to achieve the desired therapeutic response for a given patient, composition and method of application without causing toxicity to that patient.

[0097] The concentration of the disclosed compound in the pharmaceutically acceptable mixture will vary and will depend on a number of factors, including the dose of the compound to be administered, the pharmacokinetic properties of the compound used (compounds used) and the route of administration. In principle, the compositions of the invention may be provided in an aqueous solution for parenteral use containing, among other substances, from about 0.1 to 10% by weight / volume of the compound disclosed herein. A typical dosage range is from about 0.01 to about 50 mg / kg body weight per day, given in 14 divided doses. Each divided dose may contain the same or different compounds of the invention. This dose will be an effective amount, depending on a number of factors, including the patient's overall health, type of preparation, and route of administration of the selected compound (s).

[0098] In a preferred embodiment, the proteasome inhibitor is a dry powder before it is added to the cyclodextrin solution. The process of dissolving the drug in the cyclodextrin solution can be enhanced by mixing, mixing or shaking. In the most preferred embodiment, the solvent in the cyclodextrin solution is "water for injection" (WFI), which means that it is purified, sterile, low-endotoxin water. Such a preparation is suitable for both parenteral and oral use. [0099] In another preferred embodiment, the pharmaceutical composition is an oral solution or parenteral solution. Another solution is a freeze dried preparation that can be reconstituted prior to administration. As a solid form, this formulation may also include tablets, capsules and powders.

[0100] Another aspect of the invention is combination therapy in which one or more other therapeutic agents are administered with a proteasome inhibitor composition. Such combined treatment can be obtained by simultaneous, sequential or separate dosing of the individual components of this treatment.

[0101] In some embodiments, the composition of the invention is used in combination with one or more other proteasome inhibitors.

[0102] In some embodiments, the composition is used in combination with a chemotherapeutic agent. Suitable chemotherapeutics may include natural products such as Vinca alkaloids (e.g. vinblastine, vincristine and vinorelbine), paclitaxel, epidipodophyllotoxins (e.g. etoposide, teniposide), antibiotics (dactinomycin (actinomycin D), daunorubicin, doxorubicin and idarubicin), anthracyclines, mitoxantrone, bleomycins, fileamycin (mitramycin) and mitomycin, enzymes, which L-asparagine they are not able to synthesize their own asparagine); anti-platelet agents; alkylating antiproliferative / antimitotic agents such as mustard derivatives (mechlorethamine, cyclophosphamide and its analogues, melphalan, chlorambucil), ethyleneimines and methylmelamines (hexamethylmelamine and thiotepa), alkyl sulfonates (busulfan), nitrosinyl urea (BC) trazenes - dacarbazine (DTIC); antiproliferative / antimitotic antimetabolites such as folic acid analogues (methotrexate), pyrimidine analogues (fluorouracil, floxuridine and cytarabine), purine analogues and related inhibitors (mercaptopurine, thioguanine, pentostatin and 2-chlorodeoxyadenosine); aromatase inhibitors (anastrozole, exemestane and letrozole); platinum coordination complexes (cisplatin, carboplatin), procarbazine, hydroxyurea, mitotane, aminoglutethimide; histone-deacetylase (HDAC) inhibitors (trichostatin, sodium butyrate, epicidan, subverylanilide hydroxamic acid); hormones (e.g. estrogen) and hormone antagonists such as luteinizing hormone releasing hormone (LHRH) antagonists (goserelin, leuprolide and triptorelin). Other chemotherapeutic agents may include mechloretamine, camptothecin, ifosfamide, tamoxifen, raloxifene, gemcitabine, navebin or any analogue or derivative of the above.

[0103] In some embodiments, the composition of the invention can be used in combination with a cytokine. Cytokines include, but are not limited to, interferon-γ, -a and -β, interleukins 18, 10 and 12, granulocyte and macrophage colony stimulating factor (GM-CSF), TNF-α and -β, and TGF-β.

[0104] In some embodiments, the composition of the invention can be used in combination with a steroid. Suitable steroids may include 21acetoxypregnenolone, alclometasone, algestone, amcinonide, beclometasone, betamethasone, budesonide, chloroprednisone, clobetasol, clocortolone, cloprednol, corticosterone, cortisone, cortivazole, deflazonone, desclasonone, desclasonazone, fluazacort, flucloronide, flumethazone, flunisolide, fluocinolone acetonide, fluocinonide, butyl-fluokortin, fluocortolone, fluometolone, fluderolone acetate, fluprednidene acetate, fluprednisolone, flurandrenolide, fluticasone propionate, formocortal, halcinonide, halobetazole propionate, halometasone, hydrocortisone, loteprednol etabonate, mazipredone, medrison, meprednisone, methylprednisolone, predanazone, 2-carcinone sodium salt of prednisolone phosphate, prednisone, prednival, predniliden, rimexolone, thixocortol, triamcinolone, triamcinolone acetonide, triamcinolone benetonide, triamcinolone hexacetonide and their salts and / or derivatives.

[0105] In some embodiments, the composition of the invention can be used in combination with an immunotherapeutic agent. Suitable immunotherapeutic agents may include, but are not limited to, MDR modulators (verapamil, valspordar, biricodar, tarididar, lanididar), cyclosporin, thalidomide and monoclonal antibodies. Monoclonal antibodies can be either naked or conjugated, such as rituximab, tositumomab, alemtuzumab, epratuzumab, ibritumomab, thiuxetan, gemtuzumab, ozogamicin, bevacizumab, cetuximab, erlotinib and trastuzumab.

Production Methods [0106] In another aspect, the invention provides a method of making a proteasome inhibitor pharmaceutical composition. This method involves determining the desired volume, preparing a cyclodextrin solution containing cyclodextrin and acid buffer in 20% to 90% (e.g. 75%) of the desired final volume of H2O, suspending the appropriate amount of proteasome inhibitor in this cyclodextrin solution and stirring until dissolved, adjusting the pH (eg. base solution, preferably sodium hydroxide solution) and the next addition of an aqueous diluent in such an amount as to obtain the desired final volume. [0107] In a further aspect, the invention relates to a process for preparing a proteasome inhibitor pharmaceutical composition. This method involves determining the desired volume, preparing a cyclodextrin solution containing cyclodextrin and a basic buffer in 20% to 90% (e.g. 75%) of the desired final volume H₂O, suspending the appropriate amount of proteasome inhibitor in this cyclodextrin solution and stirring until dissolved, adjusting the pH (e.g. with acid solution, and then adding an aqueous diluent in such an amount to achieve the desired final volume.

[0108] An alternative method of preparing the pharmaceutical composition of the invention is by dissolving the proteasome inhibitor in a suitable solvent (e.g., in an alcohol such as ethanol), dissolving the cyclodextrin in a miscible, preferably in the same solvent, and mixing the two solutions. The solvent is then removed, for example by rotary evaporation, by spray drying or by freeze-drying to form a precipitate. This precipitate is then dissolved in a suitable aqueous diluent and the pH adjusted to the appropriate value if necessary.

[0109] Some time may elapse between receiving the precipitate and its redissolving in aqueous buffer. In one example, the pellet is sterilized (to allow storage and / or shipping, essentially in a contamination-free and sealed package) and dissolved immediately before use.

[0110] The compositions obtained above are generally sterilized before use, unless the method of preparation included a sterilization step and no contamination has occurred prior to use. [0111] The proteasome inhibitor dissolved in the aqueous buffer, preferably after sterilization, can optionally be lyophilized (in an impurity-free and sealed container) and reconstituted in a suitable aqueous diluent immediately before use. The preferred diluent is water for injection (WFI).

Examples

Example 1

Preparation Peptide (b) / HPBCD [0112, 100 pg / ml peptide (b) was placed in an aqueous solution containing 130 mg / ml hydroxypropyl beta cyclodextrin (HPBCD) and about 0.9% (w / v) NaCl. A supersaturated (metastable) solution was obtained at room temperature and could not be prepared simply by dissolving peptide (b) in an aqueous solution of 13% HPBCD / 0.9% (w / v) NaCl.

| 0113 | The required amount of peptide (b) was weighed and dissolved in absolute 1 mg / ml ethanol. For each 1 mg of peptide (b) to be produced, 1.3 g of HPBCD was dissolved in absolute ethanol at a concentration of 10 mg / ml. This ethanol / HPBCD solution was then combined with the ethanol / peptide solution (b), stirred for 5 minutes at room temperature, and then evaporated under reduced pressure (on a rotary evaporator) to give a white solid. This solid was placed in a high vacuum (approx. 1 mTorr) for 24 hours, then pulverized and subjected to high vacuum (approx. 1 mTorr) for another 4 hours to give a dry solid. The final formulation was prepared by dissolving this solid medicinal product to a final concentration of 100 pg Peptide (b) / mL solid medicinal product in ice cold aqueous 0.9% (w / v) NaCl grade USP, followed by sterile filtration. Stability at room temperature of the prepared drug product lasted about 12 hours.

Example 2

Preparation Peptide (a) / HPBCD [0114 | Peptide (a) solution at a concentration of 2 mg / ml was prepared in an aqueous solution containing 10% w / v SB SB and 10 mM citric acid, adjusted to pH 3.5 with 0.1 M aqueous sodium hydroxide solution.

[0115, Appropriate weight amounts of SBECD and citric acid were added to a volume of WFI corresponding to about 75% of the volume of the final formulation. The mixture was then stirred at room temperature until SB SB and citric acid were completely dissolved. Then the appropriate amount of peptide (a) was added and the resulting mixture was stirred at room temperature until the added peptide (a) was dissolved. The pH electrode was then immersed in the solution and the pH was adjusted to 3.5 by vigorous stirring by slowly adding a 0.1 M solution of sodium hydroxide in WFI; slow addition of the sodium hydroxide solution with adequate stirring was necessary to prevent precipitation of peptide (a). With vigorous stirring, the resulting solution was diluted with WFI to a final peptide (a) concentration of 2.0 mg / ml. This solution was sterilized by filtration to give the final preparation.

[0116] For the peptide (a) / SBECD preparations, several other final pH values ​​were used. FIG.

shows the solubility of peptide (a) at different pH values ​​in aqueous 10% (weight / volume%) solutions: beta-cyclodextrin sulfobutyl ether (SBECD) / 10 mM sodium citrate.

[0117] In these preparations, the stability of peptide (a) was also determined. Fig. 2 shows the percentage of peptide (a) remaining in aqueous 10% (w / v) solutions: SBECD / 10 mM sodium citrate versus time at various pH values.

Example 3

Preparation (3X) peptide (a) / SBECD, lyophilization of the preparation and reconstitution [0118] A solution of peptide (a) at a concentration of 6 mg / ml was prepared in an aqueous solution containing 30% w / v SB SB and 30 mM citric acid, adjusted to pH 3 , With 0.5 M aqueous sodium hydroxide solution.

[0119] The appropriate weight amounts of SBECD and citric acid were added to a volume of WFI corresponding to 70% of the volume of the final formulation. The mixture was then stirred at room temperature until SB SB and citric acid were completely dissolved. Then the appropriate amount of peptide (a) was added and the resulting mixture was stirred at room temperature until the added peptide (a) was dissolved. The pH electrode was then immersed in the solution and the pH was adjusted to 3.5 by vigorous stirring by slowly adding a 0.5 M solution of sodium hydroxide in WFI; slow addition of the sodium hydroxide solution with adequate stirring was necessary to prevent precipitation of peptide (a). With vigorous stirring, the resulting solution was diluted with WFI to a final peptide (a) concentration of 6.0 mg / ml. This solution was sterilized by filtration to give the final preparation.

[0120] The vials containing the product were placed on shelves and the temperature was set to 5 ° C for 2 hours. The shelves were then chilled at a rate of 30 ° C per hour to a target temperature of -45 ° C, which was then held for 4 hours to complete freezing. The cold trap was set at a temperature below -50 ° C and the chamber was evacuated to a target pressure of 60 pm Hg. The chamber pressure was controlled by blowing nitrogen filtered through 0.2 μ filters and the corresponding American Pharmacopoeia (NF) into the chamber. The shelves were then warmed to a target temperature of -18 ° C at an average controlled rate of 30 ° C per hour and held at this Control setting until the end of the pre-drying. The shelves were then heated to a target temperature of 30 ° C at an average controlled rate of 12 ° C per hour and held on this control setting for 12 hours until the end of drying.

[0121] The chamber was refilled with NF nitrogen filtered through 0.2 µm filters and then the vials were capped at 1 atm.

[0122] Reconstitution was carried out by adding 9.75 ml of WFI water corresponding to the American Pharmacopoeia, so as to obtain the desired filling volume of the 10.5 ml vial. The vial was inverted several times. The time to dissolution was less than 2 minutes. The vial was then allowed to stand for a few minutes to get rid of bubbles. A clear, colorless solution containing no visible solid particles was produced. The pH of this solution was measured to be 3.5 ± 0.1.

Example 4

Dry powder preparation: peptide (a) / SBECD

A peptide (a) preparation was prepared as a cyclodextrin complexed dry powder for dissolution in 10 mM citric acid prior to intravenous administration.

[0123] The appropriate weight amounts of peptide and SBECD were added to the appropriate volume of anhydrous ethanol, so as to obtain an ethanolic solution containing 4 mg / ml of peptide (a) and 200 mg / ml of SBECD. This solution was sterilized by filtration (0.2 µm diameter filters) and evaporated (under sterile conditions) to a dry residue at room temperature under reduced pressure, resulting in a white to off-white precipitate. This pellet was then crushed (under sterile conditions) to obtain a powder with good flow properties. A suitable amount of this powder was filled under sterile conditions a sterile vial of appropriate capacity. The vial was then sealed with a sterile, pierceable elastomeric stopper and sealed with an aluminum cap in a flip-off closure system. Then, by puncturing the pierced elastomeric cork with a sterile syringe needle, an appropriate volume of a solution of 10 mM citric acid in WFI water (adjusted to pH 3.2 with 0.1 M sodium hydroxide in WFI) was added and shaken at room temperature until complete dissolution. sediment so as to obtain a final solution containing 2 mg / ml peptide (a), 100 mg / ml SBECD and 10 mM citric acid / sodium citrate buffer concentration, pH 3.2-4.0. \