Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/33—Heterocyclic compounds
  • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
  • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
  • A61K31/403—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil condensed with carbocyclic rings, e.g. carbazole
• • 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
  • 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
  • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00

Definitions

• the present invention is to the discovery of a new method to block excessive or inappropriate apoptosis of neurons and oligodendrophia that occur in traumatic spinal cord injury cases 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., _____ 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 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. Qpin. 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.
• 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).
• There are numerous reports that associate deletion or mutation of p53 with the inhibition of apoptosis and the production of cancerous cells (Kerr et al., Cancer. 73: 2013-2026 (1994); Ashwell et al., Immunol. Today. 15: 147-151, (1994)).
• apoptosis In contrast, 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)). Another example of excessive or inappropriate apoptosis is the loss of neurons and oligondendroglia that occur in traumatic spinal cord injury ((Springer et 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 antimetabolites, such as ara-c, which have been reported to enhance apoptosis in cancer cells (Ashwell et al., supra).
• ara-c antimetabolites
• the exact mechanism for the induction of apoptosis remains to be elucidated.
• evidence has built that ICE and proteins homologous to ICE (Caspases) play a key role in apoptosis.
• the protein of crmA has been shown to inhibit processing of pro-interleukin -lb by ICE.
• ICE pro-interleukin -lb
• FIG. 1 A more direct demonstration of ICE involvement comes from experiments in which ICE transfection is coupled with the co-expression of crmA, demonstrating a crmA-induced suppression of the ICE-induced apoptosis response (Miura et al., supra; Wang et al., supra).
• 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., 16: 128-134 (1992); Buxbaum et al.. Proc. Natl. Acad.
• the present invention is to a method of blocking excess or inappropriate apoptosis of neurons and oligondendroglia in a mammal which method comprises administering to said mammal an effective amount of a compound of Formula (I), and to the novel inhibition of caspases for use in the treatment of apoptosis of neurons and oligondendroglia.
• the compounds of Formula I are most effective in inhibiting caspases 3 and 7.
• Another aspect of the present invention is to a method of blocking excess or on inappropriate apoptosis of neurons and oligondendroglia in a mammal which method comprises administering to said mammal an effective amount of 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 of preventing or reducing apoptosis of neurons and oligondendroglia that occurs in traumatic spinal cord injury 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), a pharmaceutical corporation 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 of blocking or decreasing the production of IL-lb and/or TNF, in a mammal, preferably a human suffering from a traumatic spinal cord injury 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 Formula I are represented by the structure
• R j is hydrogen, or C 1.4 alkyl
• R2 is C ] _ ⁇ alkyl, optionally substituted arylCj ⁇ alkyl, optionally substituted heteroaryl C ⁇ _4 alkyl, optionally substituted C3.7 cycloalkyl, or Ri and R2 together with the nitrogen to which they are attached from a 3 to 10 membered ring which optionally contains an additional heteroatom selected from oxygen, nitrogen or sulfur;
• R3 and R4 are Ci.galkyl, hydrogen, nitro or halogen and
• R5 is C j .galkyl, hydrogen, arylalkyl or heteroarylalkyl.
• Rj and R2 are joined for form a five membered nitrogen containing ring.
• the alkyl group in the arylalkyl or heteroalkyl moiety may be branched or straight, such as a methylene or a substituted methylene group , i.e., - CH(CH3) - aryl.
• the optionally substituted aryl moiety of the arylalkyl group may be substituted one to three times independently by hydroxy, halogen, alkyl or alkoxy.
• R5 is preferably benzyl.
• excessive IL-lb convertase activity is used herein to mean an excessive expression of the protein, or activation of the enzyme.
• C j .g 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, z ' jo-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. That is they 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.
• Suitable activity is not IL-lb convertase activity, but the ability to induce apoptosis or involved in programmed cell death in some manner.
• 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 inco ⁇ orated 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.
• N-Alkylisatin derivatives are prepared either by direct alkylation of isatin or via syntheses of the isatin ring using one of the procedures described by Popp, F. D., Advances in Heterocyclic Chemistry, 1975, 18, 1-58.
• 5-Isatinsulfonic acid, sodium salt or their N-alkyl derivatives can be treated with phosphorus oxychloride in organic solvents such as sulfalane at temperatures ranging from 50-80 °C in order to obtain the corresponding 5-chlorosulfonylisatins (Martinez, F; Naarmann; H, Synth. Met., 1990, 39, 195), the direct precursors to the novel compounds of this invention.
• a secondary amine in organic solvents such as tetrahydrofuran, methylene chloride or dimethylformamide
• a tertiary amine base such as triethylamine
• treatment of the alkylaminosulfonyl-3,3-dichloro-2-oxindole derivative with an aqueous acid such as HCl with or without the presence of a cosolvent such as tetrahydrofuran, dimethylformamide, or methanol also yields the 5-alkylaminosulfonylisatin.
• the 5-alkylaminosulfonylisatin derivative may be alkylated (Tacconi, Von G; Righetti, P. P.; Desimoni, G.; J. Prakt. Chem., 1973, 315, 339) by deprotonation by a base such as sodium hydride in organic solvents such as dimethylformamide and treatment of the resulting salt with an alkyl halide at temperatures ranging from 25-80 °C to give l-alkyl-5-alkylaminosulfonylisatin.
• a base such as sodium hydride
• organic solvents such as dimethylformamide
• Example 20 (+/-)-5-f l-(3-(N-Methyl-N-phenethylcarbonylamino)pyrrolidinyl)sulfonvnisatin 20a) (+/-)-5-ri-(3-(N-Methylamino)pyrrolidinyl)sulfonvnisatin trifluoro acetic acid salt
• Example 29 (S)-(+)-l-Benzyl-5-ri-(2-thiophenoxymethylpyrrolidinyl)sulfonyl1isatin Prepared according to the procedure of example 15) without heating except using (S)-(+)-5-[l-(2-thiophenoxymethylpyrrolidinyl)sulfonyl]isatin and benzylbromide. Purification by silica gel chromatography with 2% CH 3 0H CH 2 C1 2 afforded the title compound as an orange oil in 86% yield.
• Example 30 (+/-)-5-ri-(3-(N-methylbenzamide)pyrrolidinyl)sulfonyllisatin Prepared according to the procedure of example 20b) except using benzoyl chloride afforded the title compound as a yellow solid in 47% yield.
• ES (+) MS m/e 414 (M+H).
• Example 31 5-ri-(2-(Phenylsulfinylmethyl)pyrrolidinyl)sulfonvnisatin
• Example 56 (S)-(+)-l -(Acetic acid)-5-ri-(2-phenoxymethylpyrrolidinyl)sulfonvnisatin
• To a solution of (S)-(+)-l-(t-butoxycarbonylmethyl)-5-[l-(2- phenoxymethylpyrrolidinyl)sulfonyl]isatin (0.052 g, 0.104 mmol) in 5 mL of CH 2 C1 2 at 0 °C was added 5 mL of TFA. The solution was warmed to room temperature and stirred for 1.5 hours. The organic layer was concentrated under reduced pressure and redissolved in CH 2 C1 2 and toluene was added.
• Example 70 5-ri-(Thiazolidine)sulfonyllisatin Prepared according to the procedure of example lb) except using thiazolidine and purification by silica gel chromatography with 50% EtOAc/Hexanes to afford the title compound as a yellow solid in 14% yield.
• Example 80 (S)- 1 -r4-(t-Butoxycarbonyl)phenylmethvn-5-r 1 -(2- phenoxymethylpyrrolidinvDsulfonvnisatin 80a) t-Butyl 4-(bromomethyl)benzoate Prepared according to the procedure of example 72) except using 4- chloromethylbenzoic acid and purification by silica gel chromatography with 25% EtOAc/Hexanes to afford the title compound as a white solid in 97% yield.
• ES (+) MS m/e 227 (M+H).
• 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.
• Jurkat cells were obtained from American Type Culture Collection and grown in RPMI-1640 media supplemented with 10% fetal bovine serum at 37°, 5% CO2. 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
• proliferative cells 10" cells / ml.
• Other proliferative cells can be used with apoptosis induced by anti-fas, camptothecine, 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 transf erase 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 particularly those noted herein or their pharmaceutically acceptable salts which are active when given orally, 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 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, poly vinyl pyrrolidone, lecithin, arachis oil or sesame oil.
• a sterile aqueous carrier or parenterally acceptable oil for example polyethylene glycol, poly vinyl 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.
• the present invention provides for the inhibition of apoptosis as a novel therapy to treat spinal cord injury using compounds of Formula (I) as defined herein.
• 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 supernatants are removed from the cells, centrifuged at 3000 ⁇ m 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.

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