EP1129108A1 - Caspases et apoptose - Google Patents

Caspases et apoptose

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Publication number
EP1129108A1
EP1129108A1 EP99953073A EP99953073A EP1129108A1 EP 1129108 A1 EP1129108 A1 EP 1129108A1 EP 99953073 A EP99953073 A EP 99953073A EP 99953073 A EP99953073 A EP 99953073A EP 1129108 A1 EP1129108 A1 EP 1129108A1
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EP
European Patent Office
Prior art keywords
acid
acetyl
apoptosis
aspartylglutamylvalylaspartic
excessive
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Pending
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EP99953073A
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German (de)
English (en)
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EP1129108A4 (fr
Inventor
Dennis Lee
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SmithKline Beecham Corp
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SmithKline Beecham Corp
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Publication of EP1129108A1 publication Critical patent/EP1129108A1/fr
Publication of EP1129108A4 publication Critical patent/EP1129108A4/fr
Pending legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/02—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing at least one abnormal peptide link
    • C07K5/0202—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing at least one abnormal peptide link containing the structure -NH-X-X-C(=0)-, X being an optionally substituted carbon atom or a heteroatom, e.g. beta-amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
    • A61P19/10—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease for osteoporosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention is to the discovery of a new method to block excessive or inappropriate apoptosis in a mammal.
  • necrosis is usually the result of severe trauma and is a process that involves loss of membrane integrity and uncontrolled release of cellular contents, often giving rise to inflammatory responses.
  • apoptosis is a more physiological process that occurs in a controlled manner and is generally noninflammatory in nature. For this reason apoptosis is often referred to as programmed cell death.
  • the name itself (apoptosis: Greek for "dropping off", for example leaves from trees) implies a cell death that is part of a normal physiological process (Kerr et al., Br. J. Cancer, 26: 239-257 (1972)).
  • Apoptosis appears to be a carefully controlled series of cellular events which ultimately leads to death of the cell. This process for elimination of unwanted cells is active and requires expenditure of cellular energy.
  • the morphological characteristics of apoptosis include cell shrinkage and loss of cell-cell contact, condensation of nuclear chromatin followed by fragmentation, the appearance of membrane ruffling, membrane blebbing and apoptotic bodies. At the end of the process, neighboring cells and macrophages phagocytose the fragments from the apoptotic cell. The process can be very fast, occurring in as little as a few hours (Bright et al., Biosci. Rep., 14: 67-82 (1994)).
  • the best defined biochemical event of apoptosis involves the orderly destruction of nuclear DNA.
  • Signals for apoptosis promote the activation of specific calcium- and magnesium-dependent endonucleoases that cleave the double stranded DNA at linker regions between nucleosomes. This results in production of DNA fragments that are multiples of 180-200 base pair fragments (Bergamaschi et al., Haematologica, 79: 86-93 (1994): Stewart, JNCI, 86: 1286-1296 (1994)). When examined by agarose gel electrophoresis, these multiple fragments form a ladder pattern that is characteristic for most cells undergoing apoptosis.
  • TNFa tumor necrosis factor
  • growth factor deprivation some viral proteins
  • radiation and anticancer drugs Some of these stimuli can induce their signals through a variety of cell surface receptors, such as the TNF / nerve growth factor family of receptors, which include CD40 and Fas/Apo-1 (Bright et al., supra).
  • TNF / nerve growth factor family of receptors which include CD40 and Fas/Apo-1 (Bright et al., supra).
  • genes that appear to be required for induction of apoptosis are Ced-3 and Ced-4. These genes must function in the dying cells and, if either gene is inactivated by mutation, cell death fails to occur (Yuan et al., Devel. Biol., 138: 33-41 (1990)).
  • genes that have been linked with induction of apoptosis include the proto-oncogene c-myc and the tumor suppresser gene p53 (Bright et al., supra; Symonds et al., Cell, 78: 703-71 1 (1994)).
  • Ced-9 An example in C. elegans is Ced-9. When it is abnormally activated, cells survive that would normally die and, conversely, when Ced-9 is inactivated cells die that would normally live (Stewart, B.W., supra).
  • bcl-2 A mammalian counterpart is bcl-2, which had been identified as a cancer-causing oncogene. This gene inhibits apoptosis when its product is overexpressed in a variety of mammalian cells, rendering them less sensitive to radiation, cytotoxic drugs and apoptotic signals such as c-myc (Bright et al., supra).
  • Some virus protein have taken advantage of this ability of specific proteins to block apoptosis by producing homologous viral proteins with analogous functions.
  • An example of such a situation is a protein produced by the Epstein Barr virus that is similar to bcl-2, which prevents cell death and thus enhances viral production (Wells et al., J. Reprod. Fertil, 101: 385-391 (1994)).
  • some proteins may bind to and inhibit the function of bcl-2 protein, an example being the protein bax (Stewart, B.W., supra).
  • the overall picture that has developed is that entry into apoptosis is regulated by a careful balancing act between specific gene products that promote or inhibit apoptosis (Barinaga, Science, 263: 754-756 (1994).
  • Apoptosis is an important part of normal physiology. The two most often sited examples of this are fetal development and immune cell development. In development of the fetal nervous system, over half of the neurons that exist in the early fetus are lost by apoptosis during development to form the mature brain (Bergamaschi et al., Haematologica, 79: 86-93 (1994)). In the production of immune competent T cells (and to a lesser extent evidence exists for B cells), a selection process occurs that eliminates cells that recognize and react against self This selection process is thought to occur in an apoptotic manner within areas of immune cell maturation (Williams, G T., J. Pathol., 173 1-4 (1994): Krammer et al.. Curr. Opin. Immunol . 6 279-289 (1994)).
  • Dysregulation of apoptosis can play an important role in disease states, and diseases can be caused by both excessive or too little apoptosis occurring
  • diseases associated with too little apoptosis would be certain cancers
  • There is a follicular B-cell lymphoma associated with an aberrant expression of functional bcl-2 and an inhibition of apoptosis in that cell (Bergamaschi et al , supra)
  • p53 associated with the inhibition of apoptosis and the production of cancerous cells
  • one example of excessive or inappropriate apoptosis is the loss of neuronal cells that occurs in Alzheimer disease, possible induced by b-amyloid peptides (Barr et al., BioTechnology, 12' 487-493 (1994)).
  • apoptosis is the loss of neurons and ohgondendrogha that occur in traumatic spinal cord injury ((Springer er al) Nature Medicine 5 943-946 1999)).
  • Other examples include excessive apoptosis of CD4 + T cells that occurs in HIV infection, of cardiac myocytes during infarction / reperfusion and of neuronal cells during ischemia (Bergamaschi et al , supra); Barr et al , supra).
  • Some pharmacological agents attempt to counteract the lack of apoptosis that is observed in cancers.
  • examples include topoisomerase II inhibitors, such as the epipodophyllotoxins, and antimetabohtes, such as ara-c, which have been reported to enhance apoptosis in cancer cells (Ashwell et al , supra) In many cases with these anti- cancer drugs, the exact mechanism for the induction of apoptosis remains to be elucidated.
  • This gene of the cowpox virus encodes a serpin protein, a family of proteins that are inhibitors of proteases (Ray et al dislike Cell, 69: 597-604 (1992)).
  • the protein of crmA has been shown to inhibit processing of pro-interleukin -lb by ICE. (Gagliardini et al. Science, 263: 826-828 (1994)) showed that microinjection of the crmA gene into dorsal root ganglion neurons prevented cell death induced by nerve growth factor deprivation. This result shows that ICE is involved in neuronal cell apoptosis.
  • protease and ICE appear to be different and to act on different substrate proteins. Blockade of protease activity in the system, using non-selective cysteine protease inhibitors, resulted in inhibition of apoptosis.
  • Brain interleukin-1 has been reported to be elevated in Alzheimer disease and Down syndrome (Griffin et al., Proc. Natl. Acad. Sci. U. S. A., 86: 7611-7615 (1989)).
  • interleukin-1 can increase the mRNA and production of b-amyloid protein, a major component of senile plaques in Alzheimer disease as well as in brains of people with Down syndrome and with aging (Forloni et al., Mol. Brain Res., J6: 128-134 (1992); Buxbaum et al., Proc. Natl.
  • the present invention is to the novel compounds of Formula (I), their pharmaceutical compositions, and to the novel inhibition of caspases for use in the treatment of apoptosis, and disease states caused by excessive or inappropriate cell death.
  • the compounds of Formula I are most effective in inhibiting caspases three, seven and eight.
  • Another aspect of the present invention is to a pharmaceutical composition comprising a compound of Formula (I), or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier or diluent.
  • Another aspect of the present invention is to a method for the treatment of diseases or disorders associated with excessive IL-lb convertase activity, in a mammal in need thereof, which method comprises administering to said mammal an effective amount of a compound of Formula (I), or a pharmaceutically acceptable salt thereof.
  • Another aspect of the present invention is to a method of preventing or reducing apoptosis in a mammal, preferably a human, in need of such treatment which method comprises administering to said mammal or human an effective amount of a compound of Formula (I), or a pharmaceutically acceptable salt thereof.
  • Another aspect of the present invention is to a method of blocking or decreasing the production of IL-lb and/or TNF, in a mammal, preferably a human, in need of such treatment which method comprises administering to said mammal or human an effective amount of a compound of Formula (I), or a pharmaceutically acceptable salt thereof.
  • Z is C , S0 2 , "N Rj is alkyl, alkylaryl or aryl;
  • R 2 , R3, and R4 are selected from naturally occuring amino acids; X is sulfur, oxygen or nitrogen and when X is sulfur, R5 is C 1. alkyl, alkylaryl or aryl
  • R5 is Y-R and Y is SO2, or C .
  • R j is alkyl, alkylaryl or aryl
  • R 2 is valine, isoleucine or threonine
  • R3 is glutamic acid or valine.
  • R4 is aspartic acid;
  • R 5 is Y ⁇ Rf5 and Y is S0 2 , or C ; and X is Nitrogen.
  • Acetyl-aspartylglutamylvalylaspartic acid (3-phenylpropionyl)aminomethylketone
  • Acetyl-aspartylglutamylvalylaspartic acid (5-acetamido-3-methylthiophene)-2- sulfonamidomethylketone
  • Acetyl-aspartylglutamylvalylaspartic acid (5-benzoylaminomethylthiophene)-2- sulfonamidomethylketone
  • Acetyl-aspartylglutarnylvalylaspartic acid (3-phenylpropyl)thiomethylketone
  • excessive IL-lb convertase activity is used herein to mean an excessive expression of the protein, or activation of the enzyme.
  • C ⁇ _ alkyl or “alkyl” is used herein to mean both straight and branched chain radicals of 1 to 6 carbon atoms, unless the chain length is otherwise specified, including, but not limited to, methyl, ethyl, n-propyl, .so-propyl, ..-butyl, sec-butyl, iso- butyl, .erf-butyl, and the like.
  • heteroaryl (on its own or in any combination, such as “heteroaryloxy”, or “heteroaryl alkyl”) is used herein to mean a 5-10 membered aromatic ring system in which one or more rings contain one or more heteroatoms selected from the group consisting of N, O or S, such as, but not limited, to pyrrole, pyrazole, furan, thiophene, quinoline, isoquinoline, quinazolinyl, pyridine, pyrimidine, oxazole, oxadiazole, tetrazole, thiazole, thiadiazole, triazole, imidazole, benzimidazole, benzothiaphene, benzopyrrole, or benzofuran.
  • aryl (on its own or in any combination, such as “aryloxy”, or “arylalkyl”) is used herein to mean a phenyl and naphthyl ring.
  • cycloalkyl is used herein to mean cyclic radicals, preferably of 3 to 7 carbons, including but not limited to cyclopropyl, cyclopentyl, cyclohexyl, and the like.
  • halo or halogens, is used herein to include, unless otherwise specified, chloro, fluoro, bromo and iodo.
  • the present invention contains the inhibition of caspases by compounds of Formula (I).
  • caspases are fragment, homologs, analogs and derivatives of the polypeptides Interleukin-1 b converting enzyme (or convertase). These analogs are structurally related to the caspase family. They generally encode a protein (s) which exhibits high homology to the human ICE over the entire sequence. Preferably, the pentapeptide QACRG is conserved.
  • the caspases which may include many natural allelic variants (such as substitutions, deletion or addition of nucleotides) does not substantially alter the function of the encoded polypeptide.
  • Suitable caspases encompasses within this invention are those described in PCT US94/07127 filed 23 June 1994, Attorney Docket No.: 325800-184; and in USSN 08/334,251, filed 1 November 1994, Attorney Docket No.: 325800-249 whose disclosures are incorporated herein by reference in their entirety.
  • blocking or inhibiting, or decreasing the production of IL-lb and/or TNF refers to: a) a decrease of excessive levels, or a down regulation, of the cytokine in a human to normal or sub-normal levels by inhibition of the in vivo release of the cytokine; or b) a down regulation, at the genomic level, of excessive in vivo levels of the cytokine (IL-1 or TNF) in a human to normal or sub-normal levels; or c) a down regulation, by inhibition of the direct synthesis of the cytokine (IL- 1 , or TNF) as a postranslational event; or d) a down regulation, at the translational level, of excessive in vivo levels of the cytokine (IL-1, or TNF) in a human to normal or sub-normal levels.
  • the blocking or inhibiting, or decreasing the production of IL-lb and/or TNF is a discovery that the compounds of Formula (I) are inhibitors of the cytokines, IL-1 and TNF is based upon the effects of the compounds of Formulas (I) on the production of the IL- 1 and TNF in in vitro and in vivo assays which are well known and recognized in the art, some of which are described herein.
  • This amine is coupled to the tripeptide AcD(OtBu)E(OtBu)V-OH (Prepared using standard Fmoc-amino acid coupling chemistry from the following monomers: FmocD(OtBu)-OH, FmocE(OtBu)-OH, V-OBn.
  • N -terminal functionalization as an amide, sulfonamide or urea is effected under standard conditions on the appropriately protected tripeptide) using standard amino acid coupling conditions to give the tetrapeptide azidomethyl alcohol 6.
  • the azide is then reduced to the primary amine using reducing condition such as hydrogenation using Pd/C in methanol or PPh3/H 2 0 in THF to give the corresponding amino alcohol 7.
  • the amine may then be reacted with an electrophile such as an acylchloride or sulfonychloride in an organic solvent such as THF or CH 2 C1 2 using a tertiary amine such as Et3N or Hunig's base to give amides or sulfonamides 8.
  • This intermediate is then oxidized to the ketones 9 with an oxidizing agent such as the tetrapropylammonium perruthenate/4-methylmorpholine N- oxide, pyridine sulfur trioxide or Dess Martin reagent.
  • the tetrapeptide derivative may then be deprotected with TFA to give the desired amidomethylketone or sulfonamidomethylketone tetrapeptide derivatives 10.
  • Acetyl-aspartyl(t-Bu)glutamyl(t-Bu)valylaspartic(t-Bu) acid benzoylaminomethylalcohol To a solution of acetyl-aspartyl(t-Bu)glutamyl(t-Bu)valylaspartic(t-Bu) acid aminomethylalcohol (0.22 g, 0.31 mmol) in 10 mL of CH 2 C1 2 and 0.5 mL of DMF was added pyridine (0.076 mL, 0.94 mmol) followed by benzoyl chloride (0.044 mL, 0.38 mmol). The solution was stirred for 3 h, and 20 mL of CH 2 C1 2 was added.
  • Acetyl-aspartylglutamylvalylaspaitic acid (5-benzoylaminomethylthiophene)-2- sulfonamidomethylketone
  • E.coli cells were lysed in 10 ml/g of cells of lysis buffer (50 mM Na phosphate pH 7.2, 0.1 M NaCl, 0.1 % Tween 20, and 10 mM b-mercaptoethanol) using Microfluidics Ml 10Y homogenizer at 10,000 psi. After centrifugation, Caspase 3 activity was detected in lysate supernatant.
  • lysis buffer 50 mM Na phosphate pH 7.2, 0.1 M NaCl, 0.1 % Tween 20, and 10 mM b-mercaptoethanol
  • Caspase 3 was assayed at 30 degrees C in 96-well plates using the fluorogenic tetrapeptide substrate N-acetyl-L-aspartyl-L-glutamyl-L-valyl-L-aspartyl-7-amido-4- methylcoumarin (Ac-DEVD-AMC).
  • the assays were conducted at pH 7.5 in a buffered system containing 25 mM Hepes, 10% sucrose, 0.1 % CHAPS, and 1-50 uM DTT. The concentration of substrate was fixed at 10 uM. Fluorescence of the liberated 7-amino-4- methylcoumarin was continuously monitored at 460 nm following excitation at 360 nm.
  • DMSO dimethylsulf oxide
  • Jurkat cells were obtained from American Type Culture Collection and grown in RPMI-1640 media supplemented with 10% fetal bovine serum at 37°, 5% C0 2 . Cells were seeded in T-flasks at 0.03 to 0.08 x 10" cells / ml and used for experiments at 0.5 to 1.0 x 10" cells / ml. Other proliferative cells can be used with apoptosis induced by anti-fas, campto hecine, cerimide or TNF.
  • a method for measuring apoptosis is to quantitate the amount of broken DNA fragments using a fluorescent end-labeling method, a system used in the ApopTag kit from Oncor (Gaithersburg, MD).
  • the enzyme terminal deoxynucleotidyl transferase extends the DNA fragments with digoxigenin-containing nucleotides, which are then dected with an antidigoxigenin antibody earring fluorescein to allow dection by fluorescence (494 nm excitation and 523 nm emission).
  • Propidium iodide is used as counter stain to measure total DNA content.
  • Flow cytometric analysis was done on Becton-Dickinson (Rutherfor, NJ) FACScan instrument using CellQuest software.
  • the compounds of the present invention will generally be administered in a standard pharmaceutical composition obtained by admixture with a pharmaceutical carrier or diluent selected with regard to the intended route of administration and standard pharmaceutical practice.
  • a pharmaceutical carrier or diluent selected with regard to the intended route of administration and standard pharmaceutical practice.
  • they may be administered orally in the form of tablets containing such excipients as starch or lactose, or in capsule, ovules or lozenges either alone or in admixture with excipients, or in the form of elixirs or suspensions containing flavouring or colouring agents.
  • They may be injected parenterally, for example, intravenously, intramuscularly or subcutaneously.
  • parenteral administration they are best used in the form of a sterile aqueous solution which may contain other substances, for example, enough salts or glucose to make the solution isotonic with blood.
  • a sterile aqueous solution which may contain other substances, for example, enough salts or glucose to make the solution isotonic with blood.
  • the choice of form for administration as well as effective dosages will vary depending, inter alia, on the condition being treated. The choice of mode of administration and dosage is within the skill of the art.
  • the compounds of the present invention can be formulated as liquids, for example syrups, suspensions or emulsions, tablets, capsules and lozenges.
  • a liquid formulation will generally consist of a suspension or solution of the compound or pharmaceutically acceptable salt in a suitable liquid carrier(s) for example, ethanol, glycerin, non-aqueous solvent, for example polyethylene glycol, oils, or water with a suspending agent, preservative, flavouring or colouring agent.
  • a composition in the form of a tablet can be prepared using any suitable pharmaceutical carrier(s) routinely used for preparing solid formulations.
  • suitable pharmaceutical carrier(s) routinely used for preparing solid formulations.
  • examples of such carriers include magnesium stearate, starch, lactose, sucrose and cellulose.
  • a composition in the form of a capsule can be prepared using routine encapsulation procedures.
  • pellets containing the active ingredient can be prepared using standard carriers and then filled into a hard gelatin capsule; alternatively, a dispersion or suspension can be prepared using any suitable pharmaceutical carrier(s), for example aqueous gums, celluloses, silicates or oils and the dispersion or suspension then filled into a soft gelatin capsule.
  • the composition is in unit dose form such as a tablet or capsule.
  • Typical parenteral compositions consist of a solution or suspension of the compound or pharmaceutically acceptable salt in a sterile aqueous carrier or parenterally acceptable oil, for example polyethylene glycol, polyvinyl pyrrolidone, lecithin, arachis oil or sesame oil.
  • a sterile aqueous carrier or parenterally acceptable oil for example polyethylene glycol, polyvinyl pyrrolidone, lecithin, arachis oil or sesame oil.
  • the solution can be lyophilized and then reconstituted with a suitable solvent just prior to administration.
  • a typical suppository formulation comprises a compound or a pharmaceutically acceptable salt thereof which is active when administered in this way, with a binding and/or lubricating agent such as polymeric glycols, gelatins or cocoa butter or other low melting vegetable or synthetic waxes or fats.
  • a binding and/or lubricating agent such as polymeric glycols, gelatins or cocoa butter or other low melting vegetable or synthetic waxes or fats.
  • the pharmaceutically acceptable compounds of the invention will normally be administered to a subject in a daily dosage regimen.
  • a daily dosage regimen for a patient this may be, for example, from about .001 to about lOOmg/kg, preferably from about 0.001 to about lOmg/kg animal body weight.
  • a daily dose, for a larger mammal is preferably from about 1 mg to about 1000 mg, preferably between 1 mg and 500 mg or a pharmaceutically acceptable salt thereof, calculated as the free base, the compound being administered 1 to 4 times per day.
  • Unit dosage forms may contain from aobut 25 ⁇ g to about 500mg of the compound.
  • Bone remodeling involves the initial resorption by osteoclasts, followed by bone formation by osteoblasts. Recently, there have been a number of reports of apoptotic events occurring during this process. Apoptotic events have been observed in both the bone forming and bone resorbing cells in vitro and indeed at the sites of these remodeling units in vivo.
  • Apoptosis has been suggested as one of the possible mechanisms of osteoclast disappearance from reversal sites between resorption and formation.
  • TGF- ⁇ l induces apoptosis (approx. 30%) in osteoclasts of murine bone marrow cultures grown for 6 days in vitro .
  • the anti-resorptive bisphosphonates (clodronate, pamidronate or residronate) promote apoptosis in mouse osteoclasts in vitro and in vivo . (Hughes, et al., supra at S347).
  • M-CSF which has previously been found to be essential for osteoclast formation can suppress apoptosis, suggesting not only that maintenance of osteoclast populations, but also that formation of these multinucleated cells may be determined by apoptosis events.
  • Fuller, et al., J. Bone Min. Res. 8 , S384 (1993); Perkins, et al., J. Bone Min. Res. 8 , S390 (1993) Local injections of IL- 1 over the calvaria of mice once daily for 3 days induces intense and aggressive remodeling. (Wright, et al., J. Bone Min. Res. 9 , S 174 (1994)).
  • one aspect of the present invention is the promotion of apoptosis in osteoclasts as a novel therapy for inhibiting resorption in diseases of excessive bone loss, such as osteoporosis, using compounds of Formula (I) as defined herein.
  • apoptosis may play a role in loss of osteoblasts and chondrocytes and inhibition of apoptosis could provide a mechanism to enhance bone formation. Therefore, another aspect of the present invention is the inhibition of apoptosis as a novel therapy to enhance bone formation using compounds of Formula (I) as defined herein.
  • Osteoarthritits is a degenerative disease characterized by progressive erosion of articular cartilage.
  • Chondrocytes are the single cell-type found in articular cartilage and perturbations in metabolism of these cells may be involved in the pathogenesis of OA.
  • Damage to cartilage initiates a specific reparative response which involves an increase in the production of proteoglycan and collagen in an attempt to reestablish normal matrix homeostasis.
  • the 3-dimensional collagen network is disrupted and cell death of chondrocytes occurs in OA lesions (Malemud, et al.: Regulation of chondrocytes in osteoarthritis. In: Adolphe, M. ed. Biological Regulation of Chondrocytes.
  • Another aspect of the present invention is the inhibition of apoptosis as a novel therapy to treat osteoarthritis, using compounds of Formula (I) as defined herein.
  • apoptosis as a novel therapy to treat osteoarthritis, using compounds of Formula (I) as defined herein.
  • Recent evidence shows that chronic, degenerative conditions of the liver are linked to hepatocellular apoptosis. These conditions include chemical-, infectious- and immune/inflammatory-induced hepatocellular degeneration.
  • Apoptosis of liver cells has been observed in liver degenerative states induced by a variety of chemical agents, including acetaminophen (Ray, et a l.,(1993) FASEB. J.
  • Another aspect of the present invention is the inhibition of apoptosis as a novel therapy to treat degenerative liver diseases., using compounds of Formula (I) as defined herein.
  • Caspase-3 apoptotic pathway are activated after traumatic spinal cord injury in rats, and occur early in neurons in the injury site and hours to days later in the oligodendroglia adjacent to and distant from the injury site.
  • Another aspect of the present invention is the inhibition of apoptosis as a novel therapy to treat spinal cord injury using compounds of Formula (I) as defined herein.
  • Apoptosis is recognized as a fundamental process within the immune system where cell death shapes the immune system and effects immune functions. Apoptosis also is implicated in viral diseases (e.g AIDS). Recent reports indicate that HIV infection may produce an excess of apoptosis, contributing to the loss of CD4 T cells. Of additional interest is the observation that APO-1/Fas shares sequence homology with HIV-1 gpl20.
  • Another aspect of the present invention is the inhibition of apoptosis as a novel therapy to treat viral diseases, using compounds of Formula (I) as defined herein.
  • IL- 1 and TNF inhibiting effects of compounds of the present invention are determined by the following in vitro assays: Interleukin - 1 (IL-1)
  • Human peripheral blood monocytes are isolated and purified from either fresh blood preparations from volunteer donors, or from blood bank buffy coats, according to the procedure of Colotta et al, J Immunol, 132, 936 (1984). These monocytes (lxlO 6 ) are plated in 24-well plates at a concentration of 1-2 million/ml per well. The cells are allowed to adhere for 2 hours, after which time non-adherent cells are removed by gentle washing. Test compounds are then added to the cells for about lhour before the addition of lipopolysaccharide (50 ng/ml), and the cultures are incubated at 37°C for an additional 24 hours. At the end of this period, culture super-natants are removed and clarified of cells and all debris.
  • Culture supernatants are then immediately assayed for IL-1 biological activity, either by the method of Simon et al., J. Immunol. Methods, 84, 85, (1985) (based on ability of IL-1 to stimulate a Interleukin 2 producing cell line (EL-4) to secrete IL-2, in concert with A23187 ionophore) or the method of Lee et al, J. ImmunoTherapy, 6 (1), 1-12 (1990) (ELISA assay).
  • TNF Tumour Necrosis Factor
  • Human peripheral blood monocytes are isolated and purified from either blood bank buffy coats or platelet pheresis residues, according to the procedure of Colotta, R. et al., J Immunol, 132(2), 936 (1984).
  • the monocytes are plated at a density of 1x10 ⁇ cells/ml medium/well in 24-well multi-dishes. The cells are allowed to adhere for 1 hour after which time the supernatant is aspirated and fresh medium (1ml, RPMI-1640, Whitaker Biomedical Products, Whitaker, CA) containing 1% fetal calf serum plus penicillin and streptomycin (10 units/ml) added.
  • the cells are incubated for 45 minutes in the presence or absence of a test compound at InM-lOmM dose ranges (compounds are solubilized in dimethyl sulfoxide/ethanol, such that the final solvent concentration in the culture medium is 0.5% dimethyl sulfoxide/0.5% ethanol).
  • Bacterial lipopoly-saccharide E. coli 055:B5 [LPS] from Sigma Chemicals Co.
  • E. coli 055:B5 [LPS] from Sigma Chemicals Co.
  • culture supematants are removed from the cells, centrifuged at 3000 rpm to remove cell debris. The supernatant is then assayed for TNF activity using either a radio-immuno or an ELISA assay, as described in WO 92/10190 and by Becker et al, J Immunol, 1991, 147, 4307.

Abstract

La présente invention concerne de nouveaux composés représentés par la formule (I), leurs compositions pharmaceutiques, ainsi qu'une nouvelle forme d'inhibition des caspases utile dans le traitement de l'apoptose et des états pathologiques provoqués par une mort cellulaire excessive ou inappropriée.
EP99953073A 1998-10-06 1999-10-06 Caspases et apoptose Pending EP1129108A4 (fr)

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US10342898P 1998-10-06 1998-10-06
US103428P 1998-10-06
PCT/US1999/023271 WO2000020440A1 (fr) 1998-10-06 1999-10-06 Caspases et apoptose

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EP1129108A4 EP1129108A4 (fr) 2002-01-09

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WO2006017295A2 (fr) * 2004-07-12 2006-02-16 Idun Pharmaceuticals, Inc. Analogues de tetrapeptide
US7709370B2 (en) 2007-09-20 2010-05-04 International Business Machines Corporation Spin-on antireflective coating for integration of patternable dielectric materials and interconnect structures
US8084862B2 (en) 2007-09-20 2011-12-27 International Business Machines Corporation Interconnect structures with patternable low-k dielectrics and method of fabricating same
US8618663B2 (en) 2007-09-20 2013-12-31 International Business Machines Corporation Patternable dielectric film structure with improved lithography and method of fabricating same
WO2012019003A1 (fr) 2010-08-06 2012-02-09 Bristol-Myers Squibb Company Dérivés d'oxoacétyl pipérazinamide à substitution indole et azaindole
CN110759966B (zh) * 2019-11-22 2021-01-26 中国药科大学 caspase-3抑制剂及用途

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WO1995035308A1 (fr) * 1994-06-17 1995-12-28 Vertex Pharmaceuticals Incorporated INHIBITEURS DE L'ENZYME DE CONVERSION D'INTERLEUKINE-1$g(b)
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WO1995035308A1 (fr) * 1994-06-17 1995-12-28 Vertex Pharmaceuticals Incorporated INHIBITEURS DE L'ENZYME DE CONVERSION D'INTERLEUKINE-1$g(b)
GB2292149A (en) * 1994-08-09 1996-02-14 Ferring Res Ltd Peptide inhibitors of pro-interleukin-1beta converting enzyme
WO1998016505A1 (fr) * 1996-10-11 1998-04-23 Warner-Lambert Company INHIBITEURS DE L'ENZYME DE CONVERSION DE L'INTERLEUKINE 1β A BASE DE SULFONAMIDE
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