Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D501/00—Heterocyclic compounds containing 5-thia-1-azabicyclo [4.2.0] octane ring systems, i.e. compounds containing a ring system of the formula:, e.g. cephalosporins; Such ring systems being further condensed, e.g. 2,3-condensed with an oxygen-, nitrogen- or sulfur-containing hetero ring
• • 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
• • 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
• • 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
• • 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
• • 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

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 non ⁇ inflammatory 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: 703-711 (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 viral proteins 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)).
• Apoptosis is an important part of normal physiology.
• the two most often cited examples of this are fetal development and immune cell development.
• 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)).
• 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..
• Dysregulation of apoptosis can play an important role in disease states, and diseases can be caused by both excessive or too little apoptosis occurring.
• An example of 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).
• 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).
• topoisomerase II inhibitors such as the epipodophyllotoxins
• antimetabohtes such as ara-c
• ICE play a key role in apoptosis.
• This area of research has been spurred by the observation of homology between the protein coded by Ced-3, a gene known to be critical for C. Elegans apoptosis, and ICE. These two proteins share 29% amino acid identity, and complete identity in the 5 amino acid portion thought to be responsible for protease activity (QACRG) (Yuan et al., CeU, 75: 641-652 (1993)). Additional homologies are observed between ICE and the product of the nedd-2 gene in mice, a gene suspected of involvement in apoptosis in the developing brain (Kumar et al., Genes Dev..
• This gene of the cowpox virus encodes a serpin protein, a family of proteins that are inhibitors of proteases (Ray et al., 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.
• ICE-like genes In addition to ICE, researchers have examined the ability of ICE-like genes to promote apoptosis. (Kumar et al. supra) demonstrated that over expression of nedd-2 in fibroblasts and neuroblastoma cells resulted in cell death by apoptosis and that this apoptosis could also be suppressed by expression of the bcl-2 gene. Most recently,
• 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)). There are also reports that 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
• EPO 0 533 226 a novel peptide structure is diclosed which is said to be useful for determining the activity of ICE, and therefore useful in the diagnoses and monitoring of IL-1 mediated diseases.
• the present invention is to the novel compounds of Formula (I), their pharmaceutical compositions, and to the novel inhibition of ICE and ICE-like proteins for use in the treatment of apoptosis, and disease states caused by excessive or inappropriate cell death.
• Another aspect of the present invention is to a pharmaceutical composition
• 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 (i.e.blocking excess or inappropriate apoptosis) in a mammal, preferably a human, in need of such treatment which method comprises adininistering 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 decresing 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.
• the compounds of the present invention may contain one or more asymmetric carbon atoms, in particular positions 6 and 7, and may exist in racemic and optically active forms. All of these compounds are included within the scope of the present invention.
• the compound has a 6R, 7S configuration.
• Rl is hydrogen, an optionally substituted alkoxy, or halogen
• R 2 is OR a
• R a is Ci-4alkyl, or optionally substituted aryl C ⁇ galkyl
• R3 is hydrogen, -OC(O)R5, S(O) n R6 > or bromine; provided that when R3 is hydrogen, R4 is other than hydrogen;
• R4 is hydrogen;
• R5 is Ci-6 alkyl, C3-7 cycloalkyl, optionally substituted aryl, or optionally substituted arylalkyl;
• R6 is optionally substituted aryl, or optionally substituted heteroaryl;
• m is an integer having a value of 1 or 2;
• n is 0, or an integer having a value of 1 or 2; or a pharmaceutically acceptable salt thereof.
• Ri is hydrogen, an optionally substituted Ci-4 alkoxy or halogen.
• the carbon chain may be optionally substituted, one or more times, suitably one to three times, independently by hydroxy, halogen, alkoxy, C(O)H, C(O)2Rc, or C(O)CH3 moieties; wherein Rc is hydrogen, Cl-6 alkyl, aryl, or arylC ⁇ _4alkyl.
• Ri is methoxy.
• R2 is OR a ; wherein R a is Ci-4alkyl, or an optionally substituted arylCi-4alkyl, preferably benzyl. It is recognized that the alkyl group in the arylalkyl moiety may be branched or straightsuch as a methylene or substituted methylene group , i.e., -CH(CH3) - aryl. When R a is an optionally substituted arylC ⁇ _4alkyl, the aryl ring may be substituted one or more times independently by hydroxy, halogen, alkyl or alkoxy. When R a is an alkyl, it is preferably methyl or t-butyl.
• n is 1 or 2.
• m is 2.
• R3 is hydrogen, -OC(O)R5, S(O)n-R6, or bromo; provided that when R3 is hydrogen, then R4 is other than hydrogen.
• R3 is -OC(O)R5, the R5 group is suitably Cl-6 alkyl, C3-7 cycloalkyl, optionally substituted aryl, or optionally substituted arylalkyl; preferably R5 is Cl-6 alkyl, more preferably methyl.
• R6 is suitably an optionally substituted aryl, or an optionally substituted aryl heteroaryl; and n is 0, or an integer having a value of 1 or 2.
• R6 is heteroaryl, as defined below, it is preferably a triazole, oxadiazole, or tetrazole moiety.
• R6 is aryl, as also defined below, it is preferably a phenyl; the n value is preferably 1 or 2.
• R6 is a heteroaryl, n is preferably 0.
• the heteroaryl or aryl ring may be optionally substituted one or more times independently by hydroxy, halogen, alkyl or alkoxy, preferably alkyl, more preferably methyl.
• Compounds of Formula (I) for use in the methods of the present invention include those noted above and: tert-Butyl (6R,7S)-3-acetoxymethyl-7-methoxy-3-cephem-4-carboxylate- 1 , 1 -dioxide tert-Butyl (6R,7R)-3-acetoxymethyl-7-methoxy-3-cephem-4-carboxylate- 1 , 1 -dioxide
• excessive IL-lb convertase activity is used herein to mean an excessive expression of the protein, or activation of the enzyme.
• C ⁇ _6 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, iso-propyl, n-butyl, sec- butyl, iso-butyl, tert-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, or benzimidazole.
• 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 is to the inhibition of ICE and ICE-like proteases by compounds of Formula (I).
• ICE-like proteases are fragment, homologs, analogs and derivatives of the polypeptides Interleukin- 1 b converting enzyme (or convertase). These analogs are structurally related to the ICE 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 ICE like proteases 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.
• ICE protease retain essentially the same biological function or activity as the ICE protease, although it is recognized that the biological function may be enhanced or reduced activity.
• the suitable activity is not IL-lb convertase activity, but the ability to induce apoptosis or involved in programmed cell death in some manner.
• Suitable ICE like proteases 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.
• the t-Butyl ester, 2-Scheme I is synthesized by treating commercially available 7-aminocephalosporonic acid (1 -Scheme I) with isobutylene and sulfuric acid in dioxane. Following the procedure of Doherty et al. (J. Med. Chem. 1990, 33, 2513- 2521, which is inco ⁇ orated herein by reference), 7-alkoxy substituted 3a-Scheme I and 3b-Scheme I are produced as a separable mixture. Deprotection of 3-Scheme I with trifluoroacetic acid/anisole at 0 C gives the free acid 5-Scheme I or the sodium salt 4- Scheme I upon titration with aqueous sodium bicarbonate.
• Alkoxy derivative 9-Scheme 2 is obtained in one step from 8-Scheme 2 by treatment with NaNO2 and the alcohol in perchloric acid (Alpegiani et al. US
• Ester 10-Scheme I is formed by esterification of 9-Scheme 2 by procedures described for 6-Scheme 1 ; m- chloroperoxybenzoic acid or oxone oxidation of 10-Scheme I yields 11 -Scheme 2.
• the following derivatives can be synthesized according to procedures outlined by Alpegiani et al. J. Med. Chem.
• Example 2(a) To the ester (83 mg, 186 umol) of Example 2(a) in methylene chloride (3 mL) was added 85% m-chloroperoxybenzoic acid (114 mg, 558 umol) and the solution was stirred for 4 h. To the solution was added 20% sodium metabisulfite, followed by saturated sodium bicarbonate and the mixture was extracted with methylene chloride.
• Example 5 Methyl (6R.7S)-3-acetoxymethyl-7-methoxy-3-cephem-4-carboxylate- 1.1-dioxide a) (6R,7S)-3-Acetoxymethyl-7-methoxy-3-cephem-4-carboxylic acid
• Example 9 3.4-Dichlorobenzyl (lRS.6R.7SV7-methoxy-3-acetoxymethyl-3-cephem-4-carboxylate 1 -oxide Following the procedure of Example 3,. except one equivalent of m- chloroperoxybenzoic acid is used. MS(ES") m/e 460 [M-H]".
• Example 15 3.4-Dichlorobenzyl -(6R.7SV7-ln-butoxy1-3-acetoxymethyl-3-cephem-4-carboxylate- 1.1 -dioxide Following the procedure of Example 16, except substituting n-butanol for ethylene glycol, the title compound was prepared. MS(ES “ ) m/e 518 [M-H] " .
• Example 17 3.4-Dichlorobenzyl-(6R.7SV3-bromomethyl-7-methoxy-3-cephem-4-carboxylate- 1.1- dioxide a) 3,4-Dichlorobenzyl-(6R,7S)-7-methoxy-3-methyl-3-cephem-4-carboxylate To a solution of (6R,7S)-7-methoxy-3-methyl-3-ce ⁇ hem-4-carboxylic acid ( 1 g) in ethyl acetate (30 mL) is added water (30 mL).
• Example 17b To the sulfone of Example 17b (91 mg) in carbon tetrchloride (4 mL) is added AIBN (5 mg) and N-bromosuccinimide (44 mg), and solution was reluxed under argon for 3 h. the reaction mixture was cooled, saturated sodium bicarbonate was added, and the mixture was extracted with methylene chloride. The organic extract was dried (MgSO4) and concentrated in vacuo. The residue was purified by flash chromatography (silica gel, 15-35% ethyl acetate/hexanes) to yield the title compound (45 mg). MS(ES-) m/e 496 [M-H]".
• Example 18 3.4-Dichlorobenzyl-(6R.7S)-3-phenylsulfonylmethyl -7-methoxy-3-cephem-4- carboxylate- 1.1 -dioxide a) 3,4-Dichlorobenzyl-(6R,7S)-3-phenylthiomethyl -7-methoxy-3-cephem-4- carboxylate- 1,1 -dioxide
• Example 17 To the bromide of Example 17 (110 mg) in dimethylformamide (15 mL) at 0°C was added thiophenol (25 uL) and N,N-diisopropyl-N-ethylamine (42 uL). The solution was stirred until the disappearance of starting material. Water was added, and the solution was extracted with ether. The organic extract concentrated in vacuo, and the residue was purified by flash chromatography (silica gel, ethyl acetate/hexanes) to yield the title compound. MS(ES+) m/e 528 [M+H] + .
• the present invention utilizes a model that measures apoptosis, by measuring the production of DNA ladders visualized on agarose gels.
• the observation of DNA ladders has been a hallmark of the apoptosis response for many years.
• the model used in our studies is the production of apoptosis in human monocytic HL-60 cells by the anti-cancer ether lipid l-O-octadecyl-2-O-methyl-5n-3-phosphocholine (ET-I8-OCH3) and tumor necrosis factor a (TNF).
• ET-I8-OCH3 anti-cancer ether lipid l-O-octadecyl-2-O-methyl-5n-3-phosphocholine
• TNF tumor necrosis factor a
• the general method is to treat HL-60 cells with 6 ⁇ M ET-I8-OCH3 or 10 units of TNF for 24 hours, followed by extraction of small molecular weight DNA and removal of protein and RNA. The DNA is separated on a agarose gel and visualized with ethidium bromide staining. An internal standard is added to the cells just prior to extraction and preparation of DNA. Drugs are provided to cells 10 minutes prior to the apoptotic insult. This method provides a qualitative assessment of the ability of compounds to inhibit apoptosis.
• HL-60 cells American Type Cell Culture
• the desired number of cells for example, 5 x 10 ⁇ cells/treatment group
• RPMI RPMI complete
• a final cell concentration of approximately 0.5 x 10 ⁇ cells/ml For each treatment group, 10 mis of cell suspension were placed in a culture flask. Cells were incubated for 2 hours at 37°C. Exposures:
• Human ICE was cloned and expressed in E. coli as its inactive precursor (p45) bearing a hexa-His flag on its ⁇ /nmo-terminal end. Following harvesting, the cells were lysed, centrifuged, and the pellet containing the p45 solubilized with phosphate buffered 7 M urea at pH 7.5. The flagged p45 was applied to a Ni-nitrilo-acetic acid column, washed, and eluted with 300 mM imidazole. This yielded a highly enriched proenzyme preparation ( ⁇ 0% pure p45).
• Catalytic autoproteolytic activation to pl0/p20 dimer was achieved by concentrating the p45 on a Centricon ultrafiltration membrane (Amicon) at 10 _C for several hours.
• the formation of the catalytic subunits (plO and p20) in activated samples was demonstrated by correlating time-dependent generation of ICE activity with pl0/p20 signals in Westem blots and by reversed-phase HPLC. Formation of authentic plO and p20 was also confirmed by N-terminal sequence and MALD-mass spectral analyses of samples purified by reversed-phase HPLC.
• the activated enzyme was stored frozen at -80 C.
• ICE was assayed at 25 _C using the fluorogenic tetrapeptide substrate N-acetyl- L-tyrosyl-L-valyl-L-alanyl-L-aspartyl-7-amido-4-mefhylcoumarin (Ac-YVAD-AMC).
• the assays were conducted at pH 7.5 in a buffered system containing 25 mM Hepes, 10% sucrose, 0.1% CHAPS, and 2 mM DTT. The concentration of substrate was fixed at 25 uM. Fluorescence of the liberated 7-amino-4-methylcoumarin was continuously monitored at 460 nm following excitation at 335 nm.
• Assay III Inhibition of ICE ICE was assayed at 25 _C in 96-well plates using the fluorogenic tetrapeptide substrate N-acetyl-L-tyrosyl-L-valyl-L-alanyl-L-aspartyl-7-amido-4-methylcoumarin (Ac-YVAD-AMC).
• the assays were conducted at pH 7.5 in a buffered system containing 25 mM Hepes, 10% sucrose, 0.1% CHAPS, and 20-50 uM DTT. The concentration of substrate was fixed at 20 uM. Fluorescence of the liberated 7-amino-4- methy Icoumarin was continuously monitored at 460 nm following excitation at 360 nm.
• 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. 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;
• 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. Altematively, the solution can be lyophilized and then reconstituted with a suitable solvent just prior to administration.
• 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.
• the pharmaceutically acceptable compounds of the invention will normally be administered to a subject in 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 reso ⁇ tion 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 reso ⁇ tion and formation. TGF- ⁇ 1 induces apoptosis (approx.
• one aspect of the present invention is the promotion of apoptosis in osteoclasts as a novel therapy for inhibiting reso ⁇ tion in diseases of excessive bone loss, such as osteoporosis, using compounds of Formula (I) as defined herein.
• Fetal rat calvaria derived osteoblasts grown in vitro undergo apoptosis and this is localized to areas of nodule formation as indicated by in situ end-labeling of fragmented DNA. (Lynch, et al., (1994) J. Sone Min. Res.9, S352).
• 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.
• liver 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. 7, 453-463), cocaine (Cascales, et al., (1994) Hepatology 20, 992-1001) and ethanol (Baroni, etal., (1994) J. Hepatol. 20, 508-513).
• Infectious agents and their chemical components that have been shown to induce apoptosis include hepatitis ((Hiramatsu, et al., (1994) Hepatology 19, 1354- 1359; Mita, et al., (1994) Biochem. Biophys. Res. Commun. 204, 468-474)), tumor necrosis factor and endotoxin . (Leist, et al., (1995) J. Immunol. 154, 1307-1316; and Decker, K. (1993) Gastroenterology 28(S4), 20-25).
• hepatocellular apoptosis is central to degenerative liver diseases. Therefore, 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.
• 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-l 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 (1x10 ⁇ ) 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 supematants 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 supematant is aspirated and fresh medium (lml, 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 lnM-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

Landscapes

• Health & Medical Sciences (AREA)
• Chemical & Material Sciences (AREA)
• Organic Chemistry (AREA)
• Life Sciences & Earth Sciences (AREA)
• Veterinary Medicine (AREA)
• Engineering & Computer Science (AREA)
• Chemical Kinetics & Catalysis (AREA)
• General Chemical & Material Sciences (AREA)
• Medicinal Chemistry (AREA)
• Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
• Pharmacology & Pharmacy (AREA)
• Bioinformatics & Cheminformatics (AREA)
• Animal Behavior & Ethology (AREA)
• General Health & Medical Sciences (AREA)
• Public Health (AREA)
• Physical Education & Sports Medicine (AREA)
• Biomedical Technology (AREA)
• Communicable Diseases (AREA)
• Rheumatology (AREA)
• Heart & Thoracic Surgery (AREA)
• Cardiology (AREA)
• Neurology (AREA)
• Neurosurgery (AREA)
• Orthopedic Medicine & Surgery (AREA)
• Oncology (AREA)
• Urology & Nephrology (AREA)
• Virology (AREA)
• Psychiatry (AREA)
• Hospice & Palliative Care (AREA)
• Vascular Medicine (AREA)
• Immunology (AREA)
• Gastroenterology & Hepatology (AREA)
• Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Applications Claiming Priority (3)

Publications (2)

Family

ID=21704039

Family Applications (1)

Country Status (3)

Families Citing this family (15)

Citations (10)

Family Cites Families (1)

• 1996
  • 1996-08-29 EP EP96932941A patent/EP0859779A4/de not_active Withdrawn
  • 1996-08-29 WO PCT/US1996/013967 patent/WO1997008174A1/en not_active Application Discontinuation
  • 1996-08-29 JP JP9510590A patent/JPH11511463A/ja active Pending

Patent Citations (10)

Non-Patent Citations (16)

Also Published As

Similar Documents

Legal Events