MX2008001590A - Treatment of proliferative disorders - Google Patents

Abstract

Inhibitors of cIAP-1 and methods and compositions for treating proliferative disorders.

Description

TREATMENT OF PROLIFERATIVE DISORDERS RECIPROCAL REFERENCE This request claims priority with respect to the Request Provisional of the U.S. No. 60 / 706,649 entitled "PEPTIDOMIMETIC OF SMAC AS clAP INHIBITORS" presents August 9, 2005. Apoptosis (programmed cell death) plays a central role in the development and homeostasis of all multi-cellular organisms. Apoptosis can be initiated within a cell from an external factor such as a chemokine (an extrinsic pathway) or via an intracellular event such as DNA damage (an intrinsic pathway). Alterations in apoptotic pathways are implicated in many types of human pathologies, including developmental disorders, cancer, autoimmune diseases, as well as neurodegenerative disorders. One mode of action of chemotherapeutic drugs is cell death via apoptosis. Apoptosis is conserved across species and is mainly carried out by activated caspases, a family of cysteine proteases with aspartate specificity in their substrates. These aspartate-specific proteases containing cysteine ("caspases") are produced in cells as catalytically inactive zymogens and are processed proteolytically to become activated proteases during apoptosis. Once activated, the effector caspases are responsible for the excision proteolytic of a broad spectrum of cellular targets that eventually lead to cell death. In normal surviving cells that have not received an apoptotic stimulus, most caspases remain inactive. If the caspases are aberrantly activated, their proteolytic activity can be inhibited by a family of evolutionarily conserved proteins called lAPs (proteins inhibiting apoptosis). The IAP family of proteins suppresses apoptosis by preventing the activation of procaspases and by inhibiting the enzymatic activity of mature caspases. Several different mammalian LAPs have been identified including XIAP, c-IAPI, C-IAP2, ML-IAP, NAIP (neuronal apoptosis inhibitory protein), Bruce, and survivin, and all exhibit anti-apoptotic activity in cell culture. The lAPs were originally discovered in baculovirus because of their functional capacity to replace the P35 protein, an anti-apoptotic gene. LAPs have been discovered in organisms ranging from Drosophila to human, and are known to be overexpressed in many human cancers. Generally speaking, lAPs comprise one to three repeating IAP Baculovirus (BIR) domains, and most of these also possess a terminal carboxyl terminal RING motif. The BIR domain itself is a zinc binding domain of approximately 70 residues comprising 4 alpha helices and 3 beta chains, with cysteine and histidine residues that coordinate the zinc ion. It is the BIR domain that is believed to cause the anti-apoptotic effect by inhibiting caspases and thus inhibiting apoptosis. XIAP is expressed ubiquitously in most tissues adults and fetal Overexpression of XIAP in tumor cells has been shown to confer protection against a variety of pro-a poptotic stimuli and promotes resistance to chemotherapy. Consistent with this, a strong correlation between XIAP protein levels and survival for patients with acute myelogenous leukemia has been demonstrated. Sub-regulation of XIAP expression by antisense oligonucleotides has been shown to sensitize tumor cells to death induced by a wide range of pro-apoptotic agents, both in vitro and in vivo. It has also been shown that peptides derived from Smac / DIABLO to sensitize numerous different tumor cell lines towards apoptosis induced by a variety of pro-apoptotic drugs. However, in normal cells signalized to carry out apoptosis, the inhibitory effect mediated by IAP must be removed, a procedure that is carried out at least in part by a mitochondrial protein called Smac (second mitochondrial activator of caspases) . Smac (or, DIABLO), is synthesized as a 239 amino acid precursor molecule; and the 55 N-terminal residues serve as the mitochondrial targeting sequence that is removed after the amount. The mature form of Smac contains 184 amino acids and behaves like an oligomer in solution. Smac and various fragments thereof have been proposed for use as targets for the identification of therapeutic agents. Smac is synthesized in the cytoplasm with an N-terminal mitochondrial targeting sequence that is proteolytically removed during maturation towards the mature polypeptide and then it is directed towards the inter-membrane space of the mitochondria. At the time of the induction of apoptosis, Smac is released from the mitochondria into the cytosol, together with cytochrome c, where it binds to the lAPs, and allows the activation of caspase, thus eliminating the inhibitory effect of the lAPs on apoptosis. While cytochrome c induces the multimerization of Apaf-1 to activate procaspase-9 and -3, Smac eliminates the inhibitory effect of multiple lAPs. Smac interacts with essentially all the lAPs that have been examined to date including XIAP, c-IAPI, C-IAP2, ML-IAP, and survivin. Therefore, Smac appears to be a master regulator of apoptosis in mammals. It has been shown that Smac promotes not only the proteolytic activation of procaspasses, but also the enzymatic activity of mature caspase, both of which depend on their ability to physically interact with lAPs. X-ray crystallography has shown that the first four amino acids (AVPI) of the mature Smac bind to a portion of the lAPs. This N-terminal sequence is essential for the binding of lAPs and for blocking their anti-apoptotic effects. Current trends in the design of drugs for cancer focus on selective targeting to activate apoptotic signaling pathways within tumors while not being sent to normal cells. The tumor-specific properties of specific chemotherapeutic agents, such as TRAIL, have been reported. The ligand inducer of apoptosis related to tumor necrosis factor (TRAIL) is one of several members of the tumor necrosis factor (TNF) superfamily that induces apoptosis through the commitment of death recipients. TRAIL interacts with an unusually complex receptor system, which in humans comprises two death receptors and three decoy receptors. TRAIL has been used as an anti-cancer agent alone and in combination with other agents including ionizing radiation. TRAIL can initiate apoptosis in cells that overexpress the Bcl-2 and Bcl-XL survival factors, and may represent a treatment strategy for tumors that have an acquired resistance to chemotherapeutic drugs. TRAIL binds to its cognate receptors and activates the caspase cascade using adapter molecules such as TRADD. TRAIL signaling can inhibit overexpression of clAP-1 or 2, indicating an important role for these proteins in the signaling pathway. Currently, five TRAIL recipients have been identified. Two receptors of TRAEL-R1 (DR4) and TRABL-R2 (DR5) mediate apoptotic signaling, and three non-functional receptors, DcR1, DcR2, and osteoprotegerin (OPG) can act as decoy receptors. Agents that increase the expression of DR4 and DR5 may exhibit synergistic antitumor activity when combined with TRAIL. The basic biology of how IAP antagonists work suggests that they may complement or synergize with other chemotherapeutic / antineoplastic agents and / or radiation. It could be expected that chemotherapeutic / anti-neoplastic agents and radiation could induce apoptosis as a result of DNA damage and / or alteration of cellular metabolism. Inhibiting the ability of a cancer cell to replicate and / or repair damage to DNA will promote the fragmentation of nuclear DNA and thus promote the cell entering the apoptotic pathway. Topoisomerases, a class of enzymes that reduce supercoiling in DNA by fragmenting and pooling one or both strands of the DNA molecule, are vital for cellular processing, such as DNA replication and repair. Inhibition of this class of enzymes impairs the ability of cells to replicate as well as to repair damaged DNA and activates the intrinsic apoptotic pathway. The major pathways leading from topoisomerase-mediated DNA damage to cell death include the activation of caspases in the cytoplasm by proapoptotic molecules released from the mitochondria., such as Smac. The compromise of these apoptotic effector pathways is tightly controlled by upstream regulatory pathways that respond to DNA lesions induced by topoisomerase inhibitors in cells that carry out apoptosis. The initiation of cellular responses to DNA lesions induced by topoisomerase inhibitors is ensured by the protein kinases that bind to the DNA fragments. These kinases (the limiting examples of which include Akt, JNK and P38) commonly referred to as "DNA sensors" mediate DNA repair, cell cycle arrest and / or apoptosis through phosphorylation of a large number of substrates, including several downstream kinases. The drugs for platinum chemotherapy belong to a general group of DNA modifying agents. The DNA modifying agents can be any highly reactive chemical compound that binds to various nucleophilic groups in nucleic acids and proteins and causes mutagenic, carcinogenic, or cytotoxic effects. DNA modifying agents work through different mechanisms, the alteration of DNA function and cell death; DNA damage / the formation of crosslinked bonds or bonds between atoms in DNA; and induction of uncoupling of the nucleotides leading to mutations, to achieve the same end result: Three non-limiting examples of platinum-containing DNA modifying agents are cisplatin, carboplatin and oxaliplatin. It is believed that cisplatin kills cancer cells by binding to DNA and interfering with its repair mechanism, eventually leading to cell death. Carboplatin and oxaliplatin are cisplatin derivatives that share the same mechanism of action. Highly reactive platinum complexes are formed intracellularly and inhibit DNA synthesis by covalently attaching DNA molecules to form intrachain and interchain DNA crosslinks. It has been shown that non-steroidal anti-inflammatory drugs (NSAIDs) induce apoptosis in colorectal cells. It seems that NSAIDS induce apoptosis via the release of Smac from the mitochondria (PNAS, November 30, 2004, vol.101: 16897-16902). Therefore, one might expect that the use of NSAIDs in combination with certain LAP antagonists will increase the activity of each drug in comparison to the activity of each drug independently. The drug discovery process typically comprises selecting compounds to identify those compounds that have a desirable biological activity, for example, binding to a certain receptor or other protein, and then, based on said activity, identifying the compound as a leader for fur development. Such additional development may be, for example, by chemical modification of the compound to improve its properties (sometimes referred to as leader optimization) or by placing the compound through other tests and analyzes to profile the compound and therefore to fur evaluate its potential as a candidate for the development of the drug. At a certain point, if the procedure is successful, then a compound is selected for human clinical trials, which are ultimately designed to demonstrate safety and efficiency to a degree of acceptability by a drug regulatory agency. A regulatory agency for the drug is a governmental, or quasi-governmental agency, empowered to receive and review applications for marketing approval for a drug. Examples include the U.S. Food and Drug Administration in the United States ("FDA"), the European Agency for the Evaluation of Medicines in the European Union ("EMEA"), and the Ministry of Health in Japan ("MOH"). The applicant for the approval of the marketing of a drug presents information and data related to the safety and efficiency of the compound for which approval is sought. Such data may include data indicating the mechanism by which the compound causes a particular pharmacological result. Thus, for example, the applicant may submit data showing that the compound binds to a given ligand.

BRIEF DESCRIPTION OF THE INVENTION The present invention provides methods for the discovery of compounds for development as useful agents in the treatment of proliferative disorders and related methods for obtaining the regulatory approval thereof and the treatment of patients with these, as well as pharmaceutical compositions useful in said methods DETAILED DESCRIPTION OF THE ILLUSTRATIVE MODALITIES OF THE INVENTION This invention relates to the discovery of those compounds that bind and thus degrade clAP-1 referred to below as antagonists of clAP-1, which are particularly useful for the treatment of proliferative disorders. In one aspect of the invention, said compounds are useful in the treatment of cancers, such as, but not limited to, bladder cancer, breast cancer, prostate cancer, lung cancer, pancreatic cancer, gastric cancer, colon cancer , ovarian cancer, kidney cancer, hepatoma, melanoma, lymphoma, sarcoma, and combinations thereof. In another aspect, said compounds act as chemo-enhancing agents. The term "chemo-enhancing agent" refers to an agent that acts to increase the sensitivity of an organism, tissue, or cells to a chemical compound or treatment, ie, "chemotherapeutic agents" or "chemo drugs" or radiation treatment. In addition to the defects in apoptosis found in tumors, defects in the ability to eliminate auto-reactive cells of the immune system due to resistance to apoptosis are considered to play a key role in the pathogenesis of autoimmune diseases. Autoimmune diseases are characterized because the cells of the immune system produce antibodies against their own organs and molecules or directly attack tissues resulting in the destruction of the latter. An insufficiency of these self-reactive cells to carry out apoptosis leads to the manifestation of the disease. Defects in the regulation of apoptosis have been identified in autoimmune diseases such as systemic lupus erythematosus or rheumatoid arthritis.

The pathogenic cells can be those of any disease or proliferative autoimmune diseases, said cells are resistant to apoptosis due to the expression of clAPs. Examples of said autoimmune diseases are collagen diseases such as rheumatoid arthritis, systemic lupus erythematosus, Sharp syndrome, CREST syndrome (calcinosis, Raynaud's syndrome, esophageal dysmotility, telangiectasia), dermatomyositis, vasculitis (Morbus de Wegener) and Sjogren's syndrome, kidney diseases such as Goodpasture's syndrome, rapid progressing glomerulonephritis and membranoproliferative type II glomerulonephritis, endocrine diseases such as type I diabetes, autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED), autoimmune parathyroidism, pernicious anemia, gonadal insufficiency, idiopathic Addison's Morbus, hyperthyroidism, Hashimoto's thyroiditis and primary myxedema, skin diseases such as pemphigus vulgaris, bullous pemphigoid, gestational herpes, epidermolysis bullosa and erythema multiforme major, liver diseases such as primary biliary cirrhosis, autoimmune cholangitis, hep autoimmune atitis type-1, autoimmune hepatitis type-2, primary sclerosing cholangitis, neuronal diseases such as multiple sclerosis, myasthenia gravis, myasthenic syndrome of Lambert-Eaton, acquired neuromyotonia, Guillain-Barre syndrome (Muller-Fischer syndrome), syndrome of stifff-man, cerebellar degeneration, ataxia, opsoclonus, sensory neuropathy and achalasia, blood diseases such as autoimmune hemolytic anemia, idiopathic thrombocytopenic purpura (Morbus Werlhof), Infectious diseases with associated autoimmune reactions such as ADDS, Malaria and Chagas disease. In certain proliferative disorders, for example, in certain cancers, the aberrant regulation of apoptosis associated with the disorders may be due to a higher degree of clAP-1 activity compared to the activity of XIAP, despite the fact that The inhibition of apoptosis by XIAP may also be a factor in the disorder. In this case, said patients are preferably selected for treatment with compounds that preferably bind and degrade clAP-1 in relation to XIAP, because the treatment with said compound will be more effective than the treatment with a compound that binds preferably to XIAP. Compositions useful in the practice of the invention include pharmaceutical compositions comprising an effective amount (ie, an amount that when administered during a complete course of therapies is effective to inhibit the progress of the disease and / or result in regression of the symptoms of the disease) of a clAP-1 antagonist, i.e., an IAP antagonist, which binds clAP-1, in a dosage form and a pharmaceutically acceptable carrier. Another embodiment of the present invention are compositions comprising an effective amount of said clAP-1 antagonist in a dosage form and a pharmaceutically acceptable carrier, in combination with a chemotherapeutic and / or radiotherapy, wherein the clAP-1 antagonist inhibits the activity of an inhibitor of apoptosis protein (IAP), thus promoting apoptosis and improving the effectiveness of chemotherapy and / or radiotherapy. Smac mimetics, ie, small molecules that mimic the binding activity of the four N-terminal amino acids of mature Smac, are described, for example, in WO04005248, WO04007529, WO05069894, WO05069888, WO05097791, WO06010118, WO06069063, US20050261203, US20050234042, US 20060014700, US2006017295, US20060025347, US20050197403, and the US Application Serial number 11 / 363,387 filed on 2/27/2006, all of which are incorporated herein by reference even though they have been fully established above. The compounds of the structures described therein may be selected for binding affinity to clAP-1 or degradation, or both, and are selected or rejected for further development based thereon. Preferably, said compounds have a higher affinity for clAP-1 than for other lAPs, for example, they have a higher affinity for clAP-1 than for XIAP. Preferably, the difference in relative affinities as measured by the binding constants is at least 3 times greater for clAP-1 than for XIAP. More preferably, the binding affinity is at least about one order of magnitude greater, ie, at least about 10 times greater, and more preferably is at least about two orders of magnitude greater, ie, at least about 100 times greater. "Mimetics" or "peptidomimetics" are synthetic compounds having a three-dimensional structure (i.e. a "peptide core motif") based on the three-dimensional structure of a selected peptide. A variety of techniques are available to construct peptide mimetics with the same desired biological activity or a similar biological activity as the corresponding native mimetic peptide but with a more favorable activity than the peptide with respect to solubility, stability, and / or susceptibility to hydrolysis or proteolysis (see, for example, Morgan &Gainor, Ann. Rep. Med. Chem. 24, 243-252, 1989). Certain peptidomimetic compounds are based on the amino acid sequence of the peptides of the invention. Frequently, peptidomimetic compounds are synthetic compounds having a three-dimensional structure (ie, a "peptide motif") based on the three-dimensional structure of a selected peptide. The peptide motif provides the peptidomimetic compound with the desired biological activity, i.e., binding to IAP, wherein the binding activity of the mimetic compound is not substantially reduced, and is often the same as or greater than the activity of the native peptide in the which mimetic is modeled. The peptidomimetic compounds may have additional characteristics that improve their therapeutic application, such as the increase in cell permeability, greater affinity and / or avidity and prolonged biological half-life. The mimetic design strategies, specifically, the peptidomimetic design strategies are readily available in the They can be easily adapted for use in the present invention (see, for example, Ripka &Rich, Curr. Op. Chem. Biol. 2, 441-452, 1998; Hruby et al., Curr. Op. Chem. Biol. 1, 114-119, 1997; Hruby &Balse, Curr. Med. Chem. 9, 945-970, 2000). A mimetic class mimics a base structure that is partially or completely non-peptidic, but mimics the atom-by-atom peptide base structure and comprises the side groups that similarly mimic the functionality of the side groups of the amino acid residues native people. Various types of chemical bonds are known in the art, for example ester, thioester, thioamide, retroamide, reduced carbonyl, dimethylene and ketomethylene linkages as generally useful substitutes for peptide bonds in the construction of protease-resistant peptidomimetics. Another class of peptidomimetics comprises a small non-peptidic molecule that binds to another peptide or protein, but that is not necessarily a structural mimetic of the native peptide. Even another class of peptidomimetics has been generated from combinatorial chemistry and the generation of massive chemical libraries. These generally comprise novel templates which, although not structurally related to the native peptide, possess necessary functional groups located in a non-peptide base structure to serve as "topographic" mimetics of the original peptide (Ripka & amp;; Rich, 1998, previously mentioned). For example, the IAP-binding peptides of the invention can be modified to produce peptide mimetics by replacement of a or more naturally occurring side chains of the 20 genetically encoded amino acids, or D amino acids with other side chains, for example with groups such as alkyl, lower alkyl, cyclic alkyl of 4-, 5-, 6-, to 7 -members, amide, lower alkyl amide, di (lower alkyl) amide, lower alkoxy, hydroxy, carboxy and the lower ester derivatives thereof, and with 4-, 5-, 6-, to 7-membered heterocyclics. For example, proline analogs can be made in which the ring size of the proline residue is modified from 5 members to 4, 6, or 7 members. The cyclic groups may be saturated or unsaturated, and if they are saturated, they may be aromatic or non-aromatic. The heterocyclic groups may contain one or more heteroatoms of nitrogen, oxygen, and / or sulfur. Examples of such groups include furazanyl, imidazolidinyl, imidazolyl, imidazolinyl, isothiazolyl, isoxazolyl, morpholinyl (for example morpholino), oxazolyl, piperazinyl (for example 1-piperazinyl), piperidyl (for example 1 -piperidyl, piperidino), pyranyl, pyrazinyl, pyrazolidinyl, pyrazolinyl, pyrazolyl, pyridazinyl, pyridyl, pyrimidinyl, pyrrolidinyl (for example 1-pyrrolidinyl), pyrrolinyl, pyrrolyl, thiadiazolyl, thiazolyl, thienyl, thiomorpholinyl (for example thiomorpholino), and triazolyl. These heterocyclic groups can be substituted or unsubstituted. When a group is substituted, the substituent may be alkyl, alkoxy, halogen, oxygen, or substituted or unsubstituted phenyl. The peptidomimetics may also have amino acid residues that have been chemically modified by phosphorylation, sulfonation, biotinylation, or the addition or removal of other portions.

The present invention provides compounds that bind to clAP-1. The stereoisomers of the mimetic compounds described in the present invention are also encompassed by the present invention. The invention also provides methods for the use of these mimetics to modulate apoptosis and additionally for therapeutic purposes.

Binding affinities and MTT To illustrate this invention, compounds A through R were synthesized and evaluated in a biochemical binding assay using purified BIR-3 domains of XIAP and c-IAP-1.

TABLE 1 TABLE 2 TABLE 3 TABLE 4 TABLE 5 TABLE 6 TABLE 7 Binding constants were measured using fluorescence polarization as described previously (Zaneta Nikolovska-Coleska et al., (2004) Analytical Biochemistry, 332, 261-273). Briefly, peptides tested at various concentrations for binding measurements were mixed with 5 nM of fluorescently labeled peptide (AbuRPF-K (5-Fam) -NH2; FP peptide) and 40 nM of XIAP-Bir3, and clAPI-BIR3 by 15 minutes at room temperature (approximately 22 ° C) in 100 μL of 0.1 M pH potassium phosphate buffer, pH 7.5 containing 100 μg / ml bovine β-globulin. After incubation, the polarization values (mP) were measured in a Victoi ^ V using an excitation filter at 485 nm and an emission filter at 535 nm. The IC50 values (Table 1) were determined from the graph using non-linear least squares analysis using GraphPad Prism. The inventors also evaluated the ability of these compounds to inhibit the growth of an ovarian cancer cell line, SK-OV-3 (Table 1). The MTT assay is an example of an assay that has been used to measure cell growth as previously described (Hansen, MB, Nielsen, SE, and Berg, K. (1989) J. Immunol. Methods 119, 203-210) and was incorporated in the present invention as a reference in its entirety. Briefly, SK-OV-3 cells were seeded in 96-well plates in McCoy's medium containing 10% fetal bovine serum albumin (10,000 per well) and incubated overnight at 37 ° C. The next day, the test compounds were added at various concentrations (0.003-10 μM) and the plates were incubated at 37 ° C for an additional 72 hours. This incubation time was optimal to measure the inhibitory effects of different analogues. Fifty microliters of 5 mg / mL of the MTT reagent was added to each well and the plates were incubated at 37 ° C for 3 hours. At the end of the incubation period, 50 microliters of DMSO was added to each well to dissolve the cells and the optical density (OD) of the wells was measured with a microplate reader (Victor2 1420, Wallac, Finland) at 535 nm. Cell survival (CS) was calculated by the following equation: CS = (DO of the treated well / average DO of the control wells) X 100%.

The EC50 (Table 1), defined as the concentration of the drug resulting in 50% CS, was derived by calculating the point where the dose-response curve crosses the 50% point of CS using GraphPad Prism.

TABLE 8 IAP antagonists bind (IC50) to the BIR-3 domain of clAP-1 with higher affinity than with XIAP ++++ = < o.Q1 μM; +++ = = O.OI - 0.1 μM; ++ = > 0.1 μM; - = > 1 μM; ND = not determined The homology between the XIAP and clAP-1 BIR3 domains is high. Therefore, it is not surprising that IAP antagonists that are specifically synthesized bind to XIAP also bind to clAP-1. However, the binding data show that certain IAP antagonists bind to clAP-1 from three to more than 100 times more closely than to XIAP.

Degradation of IAP SKOV3 cells were passed through six dishes for tissue culture of 60 x 15 mm 2 days before the experiment. The cells appeared to be ~ 80% confluent at the time of harvest. A freshly prepared 100 nM solution of compound (B or Q) in 10% FBS / 90% McCoys 5a (medium A) was used for each time point. This solution was prepared by diluting 1 μl of a 10 mM storage solution of compound (B or Q) DMSO in 10 mL of medium A to generate a 1 μM solution. A 10-fold dilution of this solution in medium A produced the working solution 100 nM. The cells were treated at 0.5, 2, 4, 6 and 8 hours before lysis for western blot analysis by removing the existing medium and adding 3 mL of the freshly prepared 100 nM solution of the compound (B or Q) in medium A. Western blot analysis was carried out using standard techniques. Briefly, the cells were used using the MPER mammalian cell lysis solution (Bio-Rad # 78503) to which 10 μl / mL of a 100x solution of the HALT protease inhibitor cocktail (Bio- Rad # 78410). To each of the dishes of the cells, 200 μl of the lysis solution plus protease inhibitors were added. The cells in each dish were scraped using a cell scraper and allowed to incubate with the reagent for 10 minutes. The used ones were transferred to pre-cooled microfuge tubes and centrifuged for 20 minutes at 15,000 x g at 4 ° C. the supernatant was transferred to a clean, cooled microfuge tube. Next, the total protein content of those used was determined using the BCA protein assay according to the manufacturer's protocol and using interpolation from a standard curve generated with BSA. The samples were normalized for protein content during the preparation for gel electrophoresis. Samples were prepared using pH regulator for Laemmli 2x sample to which 200 mM DTT was added. Samples were loaded on 4-15% polyacrylamide gels HCI (10 lines, 50 μl wells) and electrophoresis was carried out at 200 V for 35 minutes in 25 mM Tris, 192 mM Glycine and 0.1% p / v of SDS pH 8.3. For each protein tested a separate gel / blot was used for it and its charge control only. No elimination and re-testing was performed for lAPs. The gels were removed from the cartridge and incubated in pH buffer for transfer for at least 15 minutes. The pH regulator for transfer was prepared by mixing 100 mL of pH regulator for 10x transfer (24.2 g of Tris base, 112.6 g of glycine in 1 L of water), 200 mL of methanol and 700 mL of water. A piece of PVDF of the size of the gel was cut and briefly pre-moistened in methanol before soaking in pH buffer for transfer. The filter paper was also cut to the exact size of the membrane and the gel and moistened in pH buffer for transfer. The fiber pads were also moistened. One sandwich consisted of the fiber pad assembly, filter paper, gel, membrane, filter paper, fiber pad. After placing the last piece of the filter paper, a glass tube was rolled over the sandwich to remove any air bubbles. The strut containing the sandwich was closed, secured and placed inside the transfer unit with the side of the membrane facing the positive side of the chamber. A stir bar and a Bio-lce unit were placed inside the chamber. The unit was filled with pH regulator for transfer which had been pre-cooled to 4 ° C and a stir bar was added. The pH regulator was stirred while transferring at 100 V, 200 mA (maximum) for 75 minutes. The back sides of the blots were marked with a pen or pencil and the blots were blocked in 5% w / v of skim milk powder in TBS-T for 3 hours at room temperature. The blots were placed in primary antibody solution overnight at 4 ° C (anti-XIAP R & amp; amp;; D Systems Cat # MAB822, lot DYJOI; anti-clAP-1 R &D Systems Cat # AF8181, lot KHSOI). The blots were washed with at least 5 x 100 mL of TBS-T and thenv. were incubated for 1 hour at room temperature with the appropriate secondary antibody (anti-mouse-HRP for the XIAP bot and anti-goat-HRP for clAP-1 and clAP-2; ImmunoPure Goat Anti-Mouse IgG (H + L) -conjugated peroxidase Pierce Biotechnology (Cat # 31430) lot GI964019; Anti-goat IgG-HRP antibody R &D Systems Cat # HAF109, lot FKA09). The blots were washed with 5 x 100 mL of TBS-T, changing the containers frequently. For detection, the Amersham ECL and ECL Hyperfilm equipment was used in accordance with the manufacturer's specifications. The analysis of the time course of the disappearance of clAP-1 and XIAP showed that clAP-1 was completely degraded in the first hour of treatment with the IAP antagonist while XIAP does not begin to degrade until 6 to 8 hours. Therefore, the preferred clAP-1 antagonists of the invention will, upon administration to the patient, result in the degradation of clAP that occurs more rapidly than the degradation of XIAP, for example, at a rate that is 2 times 3. times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, or more, faster than the degree of XIAP degradation.

Effect of Proteosome Inhibitor SK-O V-3 cells in McCoy's medium containing 10% fetal bovine serum albumin were treated with clAP-1 antagonists (compounds B and Q) for 20 hours in the presence and absence of bortezomib, a proteosome inhibitor. Cells were harvested after trypsinization by centrifugation at 2000 rpm for 10 minutes. The cell concentrate was washed with PBS and smoothed with RIPA to ferment the cell membrane. The lysate after centrifugation was loaded on a 5-20% polyacrylamide gel to separate the proteins. The Western blot was carried out using standard techniques and tested for the XIAP and clAP-1 proteins as described above. Cells treated with compounds B and Q in the absence of bortezomib, a proteosome inhibitor, showed complete disappearance of both clAP-1 and XIAP. The degradation of clAP-1 can be canceled with bortezomib. This indicates that the degradation is mediated by ubiquitination possibly due to the crosslinking of the RING domains of XIAP and clAP-1.

TRAIL synergy Two distinct clAP-1 antagonists were chosen for this experiment in which compound I binds to clAP-1 117 times more closely than to XIAP while compound S binds to XIAP and clAP-1 with a comparable affinity (table 2). The MTT assays were established by testing a matrix of concentrations of both drugs.

TABLE 9 These two compounds were tested for synergistic toxicity in MDA-MB231 cells with TRAIL. The inventories observed that the amount of synergistic toxicity as measured by the volume of synergy using the MACSYNERGY II program was identical. Compounds S and I were also evaluated for synergistic toxicity in the OVCAR-3 cell line with an inhibitor of topoisomerase I, SN-38, an active portion of irinotecan was used. The synergistic volume was comparable again suggesting that clAP-1 plays more significant role than XIAP showing synergistic toxicity.

TABLE 10 IAP antagonists that bind more tightly to the domains BIR-3 of clAP-1 compared to XIAP show cell deletion equivalent to SKO V-3 cells and equivalent synergistic toxicity with TRAIL and SN-38 Another unexpected observation made by the inventors was with respect to TRAIL sensitivity. SK-OV-3 cells resistant to the IAP antagonist (SK-OV-3 <R>) were generated by exposure of the parental SK-OV-3 cells (SK-OV-3s) to an antagonist compound of IAP at a concentration that eliminates 95% of the cells. Three days later, the viable cells were transferred to new bottles and grown to confluence. Two weeks later, the cells that were evaluated for sensitivity to the IAP antagonist in an MTT assay as described above and as expected, were found to be resistant to cytotoxicity by the IAP antagonist. SK-OV-3R cells were subsequently evaluated for TRAIL sensitivity in an MTT assay and found to be sensitive to TRAIL while SK-OV-3s cells are resistant to TRAIL (Figure 1). Similar results were also observed in a breast cancer cell line: MDA-MB-231.

Western blot analysis of the cell listings obtained from both SK-OV-3s and SK-OV-3R cell lines was carried out as described above. The cell lysate from the SK-OV-3 cell line showed the presence of clAP-1 protein while no clAP-1 band was observed in the cell lysate obtained from the SK-OV-3R cell line. These results suggest that clAP-1 plays an important role in TRAIL resistance, that is, the presence of clAP-1 protein in SK-OV-3s cells leads to TRAIL resistance that can be overcome by the addition of a antagonist of the clAP-1 compound that binds clAP-1 in combination with TRAIL while the degradation of clAP-1 in SK-OV-3R cells makes them more sensitive to TRAIL. In this way, a clAP-1 antagonist that binds clAP-1 acts synergistically with TRAIL. For simplicity and for illustrative purposes, the principles of the invention are described by reference to the illustrative embodiments thereof. In addition, in the foregoing description and below, numerous specific details are set forth in order to provide an understanding throughout the invention. However, it will be apparent to one skilled in the art that the invention can be practiced without limitation to these specific details. In other cases, well-known methods and structures have not been described in detail so as not to create unnecessary confusion of the invention. It should also be noted that as used in the present invention and in the appended claims, the singular forms "a", "an", and "he" includes plural references unless the context clearly dictates otherwise. Unless defined otherwise, all the technical and scientific thermals used in the present invention have the same meanings commonly understood by one skilled in the art. Although any method similar or equivalent to those described in the present invention can be used in the practice or evaluation of the embodiments of the present invention, preferred methods are described below. All publications and references mentioned in the present invention are incorporated as references. Nothing in the present invention is considered as an admission that the invention has been previously entitled in said description by virtue of a prior invention. The present invention is generally directed to the use of Smac mimetics that have affinity for clAP-1, said affinity is preferably higher than for XIAP. In one embodiment of the invention, the clAP binding affinity data is submitted to a regulatory agency as part of a dossier to seek approval to conduct clinical trials in humans with a clAP-1 antagonist. In the United States, such approval is referred to as an IND or an IND release, because it is a release, for a novel drug in research, from laws that prohibit the administration of drugs not approved to humans. Such binding data may also include absolute or relative binding affinity for other lAPs, for example, XIAP. In certain modalities, said data show that the binding of a given agent whose approval is sought is greater for clAP-1 compared to XIAP, as discussed elsewhere in this specification. Alternatively, or in addition to such data, an entity seeking such approval (or release) may provide data showing the degradation of clAP-1. Such data could also include data showing the relative or absolute degradation of other lAPs, such as XIAP. Alternatively, or in addition, such binding data, degradation data, or both may be submitted to a regulatory agency to support an application for approval for the commercialization of a clAP-1 antagonist. For example, such data may be submitted as part of a New Drug Approval Application (NDA) with the United States Food and Drug Administration (FDA). Alternatively, or in addition, said binding data, degradation data, or both can be used as decision points of advance or not advance in the discovery and development of drugs. For example, a compound may be selected for further development based on whether or not it exhibits binding to clAP-1 and / or degradation of a clAP-1. As discussed elsewhere in this specification, said binding affinity may be greater than for other lAPs and the rate of degradation may be greater than for the other lAPs. Alternatively, or in addition, such data can be used to characterizing a given agent that has been selected for further development based on other data, such as cellular toxicity data. In any case, binding to clAP-1 or to other lAPs can be determined using the standard binding affinity assays, as illustrated above. The crystallization of a full-length Smac protein with XIAP-BIR3 and the NMR spectroscopy of an N-terminal 9-element Smac peptide with the BIR3 domain of XIAP has revealed that the N-terminal Smac AVPI residues are critical for binding to XIAP. The homologous residues in processed caspase 9 and other proteins define these four residues as the "lAP binding motif". It has been shown that peptides containing this configuration bind to XIAP at the same site as the N-terminal ATPF of the p12 subunit of active caspase-9, thereby relieving the XIAP inhibition of caspase-9 and allowing it to proceed. apoptosis The inventors have used the specificity of this lAP binding motif in a fluorescence polarization assay to measure binding activities for clAP-1 antagonists. The fluorescence polarization assay consists of the FP peptide. { Sri: What is "FP Peptide" ?} (What is the FP peptide?), And the recombinant BIR3 domain of the XIAP protein. The FP peptide and the N-terminal clAP-1 mimics compete for binding to the BIR3 protein. However, if the compound does not compete with the FP peptide, the labeled peptide remains bound to BIR3 and there is a high mP value (micipolarization). If a peptide, peptidomimetic, or other small molecule evaluated is a competitor, then it is successful in displacing the FP peptide, resulting in a low mP value. The molecules that compete with the FP peptide can be titrated and the IC50 values (non-linear regression curve fitting program with GraphPad Prism) are determined by plotting the mp value as the direct measurement of the fraction against the log of the concentration of the compound. Similarly, lAP degradation assays can be carried out by well-known techniques, as illustrated above. Comparable with the phosphorylation of the protein, ubiquitination is a reversible procedure, regulated by the activities of the ubiquitin E3 protein ligases that function to covalently bind the ubiquitin molecules to direct the proteins. clAP-1 contains a c-terminal ring domain that allows clAP-1 to catalyze this same and selected target proteins. The ubiquitinated protein is then escorted to the 26S proteosome where it carries out its final degradation and the ubiquitin is released and recycled. Once clAP-1 antagonists bind to clAP-1, this results in disruption of cell survival complexes or dissociation of natural ligands, signaling of lAPs either to self-ubiquitate or to become targets for ubiquitination followed by proteosomal degradation. As previously mentioned, western blot analysis of the cell lysate after treatment with the clAP-1 antagonist results in the disappearance of the clAP-1 and XIAP bands when the treatment is compared without the drug. To further elucidate the machinery involved in this phenomenon, the inventors focused on the regulating the stability of lAP and wondered whether or not the proteosome participates in the degradation of clAP-1 and XIAP. The inventors found that the addition of botezomib to the cells during treatment with the clAP-1 antagonist completely prevented the degradation of clAP-1 and XIAP as detected by western blot. This experiment suggests that clAP-1 and XIAP are ubiquitinated and directed towards degradation by the proteasome. Preferably, after internal administration to a human (or other animal) suffering from a pro-iterative disorder, said clAP-1 antagonist causes the degradation of clAP-1. Preferably, the clAP-1 antagonist is selected to be one that causes such degradation more rapidly than the degradation of XIAP, as discussed above. In one embodiment, clAP-1 antagonists act as chemo-potentiating agents. The term "chemoenhancing agent" refers to an agent that acts to increase the sensitivity of an organism, tissue, or cell to a chemical compound, or treatment ie "chemotherapeutic agents" or "chemo drugs" or radiation treatment. A further embodiment of the invention is a pharmaceutical composition of a clAP-1 antagonist, which can act as a chemo-potentiating agent, and a chemotherapeutic agent or chemoradiation. Another embodiment of the invention is a method for inhibiting tumor growth in vivo by administering said clAP-1 antagonist. Another embodiment of the invention is a method for inhibiting tumor growth in vivo at administer a chemo-potentiator antagonist of clAP-1 and a chemotherapeutic agent or chemoradiation. Another embodiment of the invention is a method for the treatment of a patient with a cancer by administering clAP-1 antagonists of the present invention alone or in combination with a chemotherapeutic agent or chemoradiation. In one embodiment of the invention, a chemotherapeutic composition, i.e., a pharmaceutical composition, for promoting apoptosis can be a therapeutically effective amount of a clAP-1 antagonist which binds at least one lAP different from a clAP. In another modality the lAP can be XIAP. Any of the aforementioned therapeutic compositions may additionally include a pharmaceutical carrier. The embodiments of the invention also include a method for treating a patient with a condition in need thereof wherein a therapeutically effective amount of a clAP-1 antagonist is administered to the patient, and the clAP-1 antagonist binds to clAP- 1. The embodiments of the invention also include a method of treating a cancer patient by promoting apoptosis by administering an effective amount of a clAP-1 antagonist, and the clAP-1 antagonist binds to clAP-1. The embodiments of the invention also include a method for treating a patient with an autoimmune disease by administering an effective amount of a clAP-1 antagonist.

In each of the aforementioned illustrative embodiments, the composition or method may additionally include a chemotherapeutic agent. The chemotherapeutic agent can be, but is not limited to, alkylating agents, antimetabolites, anti-tumor antibiotics, taxanes, hormonal agents, monoclonal antibodies, glucocorticoids, mitotic inhibitors, topoisomerase I inhibitors, topoisomerase II inhibitors, immunomodulatory agents , cell growth factors, cytokines, and nonsteroidal non-estrogenic analogues. The invention described in the present invention provides methods and compositions for improving apoptosis in pathogenic cells. The general method comprises contacting the cells with an effective amount of a clAP-1 antagonist. In some embodiments, the cells are in situ in an individual and the contact step is affected by administration to the individual of a pharmaceutical composition comprising an effective amount of the clAP-1 antagonist wherein the individual can be subjected to radiation or chemotherapy. concurrent or antecedent for the treatment of a neoproliferative pathology. Pathogenic cells are from a tumor such as, but not limited to, breast cancer, prostate cancer, lung cancer, pancreatic cancer, gastric cancer, colon cancer, ovarian cancer, kidney cancer, hepatoma, melanoma, lymphoma, and sarcoma. In addition to apoptosis, the defects found in tumors, defects in the ability to eliminate autoreactive cells from Immune system due to resistance to apoptosis is considered to play a key role in the pathogenesis of autoimmune diseases. Autoimmune diseases are characterized because the cells of the immune system produce antibodies against their own organs and molecules or directly attack the tissues which results in the destruction of the latter. A failure of those self-reactive cells to carry out apoptosis leads to the manifestation of the disease. Defects in the regulation of apoptosis have been identified in autoimmune diseases such as systemic lupus erythematosus or rheumatoid arthritis. The subject compositions include pharmaceutical compositions comprising a therapeutically effective amount of a clAP-1 antagonist in a dosage form with a pharmaceutically acceptable carrier, wherein the clAP-1 antagonist inhibits the activity of an apoptosis protein inhibitor., thus promoting apoptosis. Another embodiment of the present invention are compositions comprising a therapeutically effective amount of a clAP-1 antagonist in dosage form and a pharmaceutically acceptable carrier, in combination with a chemotherapeutic and / or radiotherapy, wherein the clAP-1 antagonist inhibits the activity of an inhibitor of the protein of apoptosis (lAP), thus promoting apoptosis and improving the effectiveness of chemotherapy and / or radiotherapy.

Administration of clAP-1 antagonists. ClAP-1 antagonists are administered in effective amounts. An effective amount is that amount of a preparation that alone, or together with additional doses, produces the desired response. This may include only slowing down the progression of the disease temporarily, but preferably, including stopping the progression of the disease permanently or delaying the onset of or preventing the occurrence of the disease or condition. This can be monitored by routine methods. Generally, the doses of the active compounds could be from about 0.01 mg / kg per day to 1000 mg / kg per day. It is expected that doses having a range of 50-500 mg / kg will be suitable, preferably intravenously, intramuscularly, or intradermally, and in one or more administrations per day. Administration of the clAP-1 antagonist may occur concurrently with, subsequent to, or prior to chemotherapy or radiation until the chemotherapeutic agent or radiation sensitizes the system to the clAP-1 antagonist. In general, routine experimentation in clinical trials will determine the specific intervals for the optimal therapeutic effect for each therapeutic agent and each administrative protocol, and administration to specific patients will be adjusted within effective and safe intervals depending on the patient's condition and the response to the initial administrations. However, the final administration protocol will be regulated in accordance with the judgment of the attending physician considering factors such as the age, condition and size of the patient, the potency of the clAP-1 antagonist, the duration of treatment and the severity of the disease to be treated. For example, a dose regimen of the clAP-1 antagonist can be oral administration of 1 mg to 2000 mg / day, preferably 1 to 1000 mg / day, more preferably 50 to 600 mg / day, in two to four ( preferably two) divided doses, to reduce tumor growth. Intermittent therapy (for example, a week of three weeks or three of four weeks) can also be used. In the event that a response in a subject is insufficient at the initial doses applied, higher doses (or effectively higher doses by a different, more localized route of administration) can be employed to the extent that it allows patient tolerance. Multiple doses per day are contemplated to achieve the appropriate systemic levels of the compounds. Generally, a maximum dose is used, which is the highest safe dose in accordance with medical judgment. However, those skilled in the art will understand that a patient may persist after a lower dose or tolerable dose for medical reasons, psychological reasons or virtually for any other reason.

Administration routes. A variety of administration routes are available. The particular mode selected will, of course, depend on the particular chemotherapeutic drug selected, the severity of the condition to be treated and the dose required for therapeutic efficiency. The Methods of the invention, generally speaking, can be practiced using any mode of administration that is medically acceptable, meaning any mode that produces effective levels of the active compounds without causing clinically unacceptable adverse effects. Such modes of administration include, but are not limited to, oral, rectal, topical, nasal, intradermal, inhalation, intraperitoneal, or parenteral routes. The term "parenteral" includes subcutaneous, intravenous, intramuscular, or infusion routes. Intravenous or intramuscular routes are particularly suitable for purposes of the present invention. In one aspect of the invention, a clAP-1 antagonist as described in the present invention, with or without chemotherapeutic agents or additional radiotherapy, does not adversely affect normal tissues, while sensitizing the tumor cells to additional chemotherapeutic / radiation protocols. Although one does not wish to stick to the theory, it would appear that because this specific tumor induced apoptosis, marked lateral and adverse defects such as inappropriate vasodilation or shock are minimized. Preferably, the composition or method is designed to allow sensitization of the cell or tumor to chemotherapeutic or radiation therapy by the administration of at least a portion of the clAP-1 antagonist prior to chemotherapeutic or radiation therapy. Radiation therapy, and / or the inclusion of chemotherapeutic agents, may be included as part of the therapeutic regimen to further enhance the clearance of the cells tumors by the clAP-1 antagonist.

Pharmaceutical compositions In one embodiment of the invention, an additional chemotherapeutic agent (below) or radiation may be added before, together with, or after the clAP-1 antagonist. The term "pharmaceutically acceptable carrier" as used in the present invention means one or more compatible solid or liquid fillers, diluents or encapsulating substances that are suitable for administration to a human. The term "vehicle" denotes an organic or inorganic ingredient, natural or synthetic, with which the active ingredient is combined to facilitate the application. The components of the pharmaceutical compositions are also capable of being co-mixed with the molecules of the present invention, and with each other, so that there is no interaction that could substantially alter the desired pharmaceutical efficiency. The delivery systems of the invention are designed to include delivery, time-release, delayed-release or sustained release delivery systems such that administration of the clAP-1 antagonist occurs earlier, and with sufficient time, to cause sensitization. of the site to be treated. A clAP-1 antagonist can be used in conjunction with radiation and / or with additional anti-cancer chemical agents. Such systems can avoid repeated administrations of the clAP-1 antagonist, increasing convenience for the subject and the patient. medical, and may be particularly suitable for certain compositions of the present invention. Many types of delivery delivery systems are available and are known to those skilled in the art. These include polymer based systems such as poly (lactide-glycolide), copolyoxalates, polycaprolactones, polyesteramides, polyorthoesters, polyhydroxybutyric acid, and polyanhydrides. Microcapsules of the foregoing drug-containing polymers are described in, for example, U.S. Pat. No. 5,075,109. Administration systems also include non-polymeric systems which are: liquids including sterols such as cholesterol, cholesterol esters and fatty acids or neutral fats such as mono-, di- and triglycerides; hydrogel release systems; silastic systems; peptide-based systems; wax coatings; compressed tablets using conventional binders and excipients; partially fused implants; and the similar ones. Specific examples include, but are not limited to: (a) erodible systems in which the active compound is contained in a form within a matrix such as those described in U.S. Pat. Nos. 4,452,775, 4,667,014, 4,748,034 and 5,239,660 and (b) diffusion systems in which an active component permeates at a controlled rate from a polymer as described in US Pat. Nos. 3,832,253, and 3,854,480. In addition, pump-based hardware management systems, some of which are adapted for implantation, can be used.

The use of a long-term sustained release implant may be desirable. Long-term release, as used in the present invention, means that the implant is constructed and arranged to administer therapeutic levels of the active ingredient for at least 30 days, and preferably 60 days. Long-term sustained release implants are well known to those skilled in the art and include some of the delivery systems described above. The pharmaceutical compositions may contain suitable pH regulating agents, including: acetic acid in a salt; citric acid in a salt; boric acid in a salt; and phosphoric acid in a salt. The pharmaceutical compositions may also contain, optionally, suitable preservatives, such as: benzalkonium chloride, chlorobutanol, parabens and thimerosal. The pharmaceutical compositions can be conveniently presented in unit dosage form and can be prepared by any of the methods well known in the pharmacy art. All methods include the step of placing the active agent in association with a vehicle that constitutes one or more accessory ingredients. In general, the compositions are prepared by uniformly and intimately placing the active compound in association with a liquid carrier, a finely divided solid carrier, or both, and then, if necessary, shaping the product. The compositions suitable for parenteral administration conveniently comprise a sterile aqueous preparation of a chemo-potentiating agent (eg clAP-1 antagonist), which is preferably isotonic with the blood of the container. This aqueous preparation can be formulated in accordance with known methods using suitable agents for dispersion or humectants and suspending agents. The sterile injectable preparation can also be a sterile injectable solution or suspension in a non-toxic pharmaceutically acceptable diluent or solvent, for example, as a solution in 1,3-butane diol. Among the acceptable vehicles and solvents that can be used are water, Ringer's solution, and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or medium for suspension. For this purpose any mixture of fixed oil can be employed including synthetic mono- or di-glycerides. In addition, fatty acids such as oleic acid can be used in the preparation of the injectables. The formulation of the vehicle suitable for oral, subcutaneous, intravenous, intramuscular, etc. administrations it can be found in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, PA which is incorporated herein by reference in its entirety.

Additional guimiotherapeutic agents. Chemotherapeutic agents are suitable, include but are not limited to the chemotherapeutic agents described in "Modem Pharmacology with Clinical Applications ", sixth edition, Craig &Stitzel, chapter 56, page 639-656 (2004), incorporated herein by reference This reference describes chemotherapeutic drugs that include alkylating agents, antimetabolites, anti-tumor antibiotics, anti-tumor products, plants such as taxanes, enzymes, hormonal agents such as glucocorticoids, miscellaneous agents such as cisplatin, monoclonal antibodies, immunomodulatory agents such as interferons, and cell growth factors Other suitable classifications for chemotherapeutic agents include mitotic inhibitors and anti-estrogenic analogs Non-steroidal agents Other suitable chemotherapeutic agents include inhibitors of toposiomerase I and II: CPT (8-Cyclopentyl-1,3-dimethylxanthine, inhibitor of topoisomerase I) and VP 16 (etoposide, inhibitor of topoisomerase II). of suitable chemotherapeutic agents include, but are not limited to, cisplatin, carmustine (BCNU), 5-flourouracil (5-FU), cytarabine (Ara-C), gemcitabine, methotrexate, daunorubicin, doxorubicin, dexamethasone, topotecan, etoposide, paclitaxel, vincristine, tamoxifen, TNF- alpha, TRAIL, interferon (both in its alpha and beta forms), thalidomide, and melphalan. Other specific examples of suitable chemotherapeutic agents include nitrogen mustards such as cyclophosphamide, alkyl sulfonates, nitrosoureas, ethylene imines, triazenes, folate antagonists, purine analogs, pyrimidine analogs, anthracyclines, bleomycins, mitomycins, dactinomycins, plicamycin, vinca. alkaloids, epipodophyllotoxins, taxanes, glucocorticoids, L-asparaginase, estrogens, androgens, progestins, luteinizing hormones, octreotide acetate, hydroxyurea, procarbazine, mitotane, hexamethylmelamine, carboplatin, mitoxantrone, monoclonal antibodies, levamisole, interferons, interleukins, filgrastim and sargramostima. The chemotherapeutic compositions also comprise other members, ie, others other than TRAIL, of the TNF superfamily of compounds.

Radiotherapy protocols. Additionally, in various embodiments of the method of the present invention, clAP-1 antagonist therapy can be used in connection with chemoradiation or other cancer treatment protocols used to inhibit the growth of the tumor cell. For example, but not limited to, Radiation therapy (or radiotherapy) is the medical use of ionizing radiation as part of the treatment for cancer to control malignant cells that is suitable for use in the embodiments of the present invention. Although radiotherapy is often used as part of curative therapy, it is occasionally used as a palliative treatment, when cure is not possible and the goal is relief of symptoms. Radiation therapy is commonly used to treat tumors. This can be used as the primary therapy. It is also common to combine radiotherapy with surgery and / or chemotherapy. The most common tumors treated with radiotherapy are the breast cancer, prostate cancer, rectal cancer, head and neck cancers, gynecological tumors, bladder cancer and lymphoma. Radiation therapy is commonly applied right to the localized area involved with the tumor. Frequently the radiation fields also include the draining of the lymphatic modules. It is possible but uncommon to provide radiation therapy to the entire body, or to the entire surface of the skin. Radiation therapy is usually provided daily for up to 35-38 fractions (a daily dose is a fraction). These frequent small doses allow the healthy cells to have time to grow again, repair the damage inflicted by the radiation. The three main divisions of radiotherapy are external beam radiotherapy or teletherapy, brachytherapy or sealed source radiotherapy, and unsealed source radiotherapy, which are suitable examples of the treatment protocol in the present invention. Administration of the clAP-1 antagonist may occur before, after, or concurrently with the treatment protocol. The foregoing describes the illustrative embodiments of the invention. However, the invention is not limited to the precise aspects described above, but rather includes modifications to them and alternatives to them that fall within the scope of the following claims.

Claims (21)

NOVELTY OF THE INVENTION CLAIMS

1. A method for identifying a compound for developing it as a candidate drug for the treatment of a proliferative disorder comprising evaluating the compound for its binding affinity to clAP-1, and selecting the compounds that bind to clAP-1.

2. The method according to claim 1, further characterized in that the compound is preferably linked to clAP-1 in relation to XIAP.

3. The method according to claim 1, further characterized in that the binding affinity for clAP-1 is at least three times greater than the binding affinity for XIAP.

4. The method according to claim 1, further characterized in that the binding affinity for clAP-1 is at least 100 times greater than the binding affinity for XIAP.

5. A method to identify a compound to develop it as a candidate drug for the treatment of a proliferative disorder comprising evaluating the compound for its ability to cause the degradation of clAP-1 and select the compounds that cause the degradation of clAP-1 .

6. The method according to claim 5, characterized further because the degradation rate of clAP is faster than that of XIAP.

7. A method for obtaining regulatory approval of the drug for a compound for the treatment of a proliferative disorder comprising presenting to a drug regulatory agency data demonstrating that the compound binds to clAP-1.

8. The method according to claim 7, further characterized in that the compound is preferably linked to clAP-1 in relation to XIAP.

9. The method according to claim 7, further characterized in that the binding affinity for clAP-1 is at least three times greater than the binding affinity for XIAP.

10. The method according to claim 7, further characterized in that the binding affinity for clAP-1 is at least 100 times greater than the binding affinity for XIAP.

11. The method according to any of the preceding claims, further characterized in that the compound is a mimic of SMAC.

12. The method according to claim 11, further characterized in that the compound is a peptidomimetic of the four N-terminal amino acids of the mature SMAC. 13.- The use of a compound that binds to clAP-1, for the elaboration of a drug useful to treat a proliferative disorder in a subject. 14. The use as claimed in claim 13, wherein the compound is preferably linked to clAP-1, in relation to XIAP. 15. The use as claimed in claim 13, wherein the compound is a Smac peptidomimetic. 16. A pharmaceutical composition comprising a clAP-1 antagonist that preferably binds to clAP-1 in connection with XIAP, and a pharmaceutically acceptable carrier. 17. The pharmaceutical composition according to claim 16, further characterized in that it comprises an effective amount of the clAP-1 antagonist that is less than the effective amount of a XIAP antagonist. 18. The use of a Smac peptidomimetic for the preparation of a drug useful for treating a patient with a condition that requires it, wherein said Smac peptidomimetic binds to clAP-1. 19. The use as claimed in claim 18, wherein the condition is a proliferative disorder caused to a greater degree by the expression of clAP than by the expression of XIAP. 20. The use of a compound that preferably binds to clAP-1 in relation to XIAP, for the preparation of a medicament useful for treating a proliferative disorder in a human or animal subject, whose proliferative disorder is mediated primarily by the activity of clAP-1. 21.- The use of a clAP-1 antagonist, for the elaboration of a medicament useful for treating a subject suffering from a proliferative disorder that is sensitive to the inhibition of a clAP, wherein said drug is formulated to be internally administrable.