EP1011654A1 - A method of modulating cellular activity - Google Patents
A method of modulating cellular activityInfo
- Publication number
- EP1011654A1 EP1011654A1 EP98941157A EP98941157A EP1011654A1 EP 1011654 A1 EP1011654 A1 EP 1011654A1 EP 98941157 A EP98941157 A EP 98941157A EP 98941157 A EP98941157 A EP 98941157A EP 1011654 A1 EP1011654 A1 EP 1011654A1
- Authority
- EP
- European Patent Office
- Prior art keywords
- sphingosine
- sphingosine kinase
- molecule expression
- adhesion molecule
- components
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Withdrawn
Links
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/5308—Immunoassay; Biospecific binding assay; Materials therefor for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/43—Enzymes; Proenzymes; Derivatives thereof
- A61K38/45—Transferases (2)
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
-
- 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
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
Definitions
- the present invention relates generally to a method of modulating cellular activity and agents useful for same. More particularly, the present invention contemplates a method of modulating endothelial cell activity and even more particularly endothelial cell adhesion molecule expression. Most particularly, the present invention provides a method of treating coronary heart disease by preventing or reducing endothelial cell adhesion molecule expression.
- Atherosclerotic coronary heart disease is one of the major causes of death in the western world (World Health Statistics Annual).
- An earlier event in atherogenesis is the adhesion of monocytes to the endothelium via adhesion molecules such as VCAM-1, ICAM-1 and E-selectin, all of which are rapidly synthesised in response to cytokines.
- VCAM-1 is primarily involved in the adhesion of mononuclear leukocytes to the endothelium. It is rapidly induced by the inflammatory cytokines IL-1 and TNF- , and its induction is sustained for 48 to 72 hours.
- ICAM-1 is expressed on many cells types and is involved in both monocyte and lymphocyte adhesion to activated endothelium.
- E-selectin is an endothelial specific adhesion molecule important in capturing leukocytes from the axial stream to roll along the endothelium (Abbassi et a , 1993).
- the inventors have identified a sphingosine kinase signalling pathway via which cellular activities such as adhesion molecule expression are achieved By regulating the expression and activity of individual components of this pathway, these cellular activities can be modulated
- the inventors have also developed a rapid, high volume assay for detecting agents exhibiting sphingosine kinase activity and agents which can act as agonists and antagonists of sphingosine kinase activity
- One aspect of the present invention relates to a method of modulating cellular activity in a mammal said method comp ⁇ sing administering to said mammal an effective amount of an agent for a time and under conditions sufficient to modulate the activity of one or more components of a sphingosine kinase signalling pathway
- Another aspect of the present invention provides a method of modulating adhesion molecule expression in a mammal said method comprising administering to said mammal an effective amount of an agent for a time and under conditions sufficient to modulate the activity of one or more components of a sphingosine kinase signalling pathway
- Yet another aspect of the present invention provides a method of modulating adhesion molecule expression in a mammal said method comprising administering to said mammal an effective amount of an agent for a time and under conditions sufficient to modulate the activity of one or both of sphingosine kinase and/or Sph- l-P
- Still yet another aspect of the present invention provides a method of downregulating endothelial cell adhesion molecule expression in a mammal said method comprising administering to said mammal an effective amount of an agent for a time and under conditions sufficient to downregulate the activity of one or both of sphingosine kinase and/or Sph-l-P
- Another further aspect of the present invention relates to a method of treatment or prophylaxis of a disease condition involving inflammatory mechanisms said method comprising administering to said mammal an effective amount of an agent for a time and under conditions sufficient to modulate the activity of one or more components of a sphingosine kinase signalling pathway wherein said modulation results in modulation of adhesion molecule expression
- Still another further aspect of the present invention provides a method of treating a mammal exhibiting coronary heart disease said method comprising administering to said mammal an effective amount of an agent for a time and under conditions sufficient to downregulate the activity of one or more components of a sphingosine kinase signalling pathway wherein said downregulation results in downregulation of endothelial cell adhesion molecule expression.
- Still yet another further aspect of the present invention provides a method of treatment or prophylaxis of a disease condition involving inflammatory mechanisms said method comprising administering an effective amount of one or more components of a sphingosine kinase signalling pathway or functional equivalents thereof to said mammal.
- the present invention relates to the use of an agent capable of modulating the activity of one or more components of a sphingosine kinase signalling pathway in the manufacture of a medicament for the modulation of adhesion molecule expression in a mammal.
- Another aspect of the present invention relates to the use of one or more components of a sphingosine kinase signalling pathway or functional equivalents thereof in the manufacture of a medicament for the modulation of adhesion molecule expression in a mammal.
- Yet another aspect of the present invention relates to agents for use in modulating one or more components of a sphingosine kinase signalling pathway wherein modulating said components modulates adhesion molecule expression.
- Yet another aspect of the present invention relates to one or more components of a sphingosine kinase signalling pathway or functional equivalents thereof for use in modulating adhesion molecule expression in a mammal.
- Still another aspect of the present invention relates to a pharmaceutical composition
- a pharmaceutical composition comprising an agent capable of modulating one or more components of a sphingosine kinase signalling pathway wherein said modulation results in modulation of adhesion molecule expression, together with one or more pharmaceutically acceptable carriers and/or diluents.
- Still yet another further aspect of the present invention relates to a pharmaceutical composition
- a pharmaceutical composition comprising one or more components of a sphingosine kinase signalling pathway or functional equivalents thereof together with one or more pharmaceutically acceptable carriers and/or diluents.
- a further aspect of the present invention provides a method of detecting an analyte structurally or functionally reactive with a lipid, said method comprising the steps of contacting either:
- Another further aspect of the present invention relates to a method of detecting an analyte structurally or functionally reactive with sphingosine, said method comprising the steps of contacting either:
- Still another further aspect of the present invention provides a method for detecting an analyte exhibiting sphingosine kinase activity, said method comprising the steps of contacting said analyte and P-ATP with sphingonsine in the presence of a scintillant, said analyte being contacted with said sphingosine simultaneously with or separately to said 3 P-ATP, for a time and under conditions sufficient for 3 P-sphingosine complex to form and to excite said scintillant, and detecting said excited scintillant.
- Figure 1 is a graphical representation of the effect of high density lipoprotein (HDL) on tumour necrosis factor (TNF) mediated induction of adhesion proteins VCAM-1 and E-selectin.
- Mean fluorscence intensity is a flow cytometric measurement signifying the intensity of expression of adhesion molecules on the cell surface.
- Figure 2 is a graphical representation of the effect of sphingosine kinase inhibitor on adhesion protein expresssion. Intensity of cell surface expression was measured as a percent of the mean fluorescence intensity stimulated by 100U of TNF.
- Figure 2 A filled symbols show the effect of the cell-permeable ceramide (C2-ceramide) on adhesion proteins. The open symbols show the effect of ceramide in the presence of lOOU/ml TNF.
- Sph-l-P the effect of sphingosine- 1- phosphate
- Figure 2C shows the effect dimethylsphingosine (DMS) on adhesion protein expression (MFI) stimulated by TNF or Sph-l-P.
- DMS dimethylsphingosine
- MFI adhesion protein expression
- Figure 3 is a graphical representation of the time course of changes after TNF treatment of
- HUVECs on key components of sphingomyelin pathway are shown.
- Open symbols in Figure 3 A show hydrolysis of sphingomyelin, in Figure 3B generation of ceramide, in Figure 3C sphingosine kinase activity and in Figure 3D formation of sphingosine- 1 -phosphate, all as percent of amount at time 0. Filled circles represent the effect of pre-treatment of HUVECs with HDL.
- Figure 4 is a photographic representation of the effect of Sph-l-P, TNF and HDL on generation of ERK (Figure 4A) and NF- ⁇ B (Figure 4B).
- the bar graph provides a summary of three independent experiments on ERK activation ( ⁇ 1 SEM). * show p value of difference from TNF by ⁇ 0.01 and f by ⁇ 0.05, J shows p ⁇ 0.01 versus nil counted.
- Figure 4B top shows gel retardations with NF- ⁇ B.
- Figure 4B bottom shows supershifts with antibodies specific for p50 and p65 components of NF- ⁇ B and demonstrates the similar composition of NF- ⁇ B stimulated by TNF and Sph-l-P.
- Figure 5 is a schematic representation of the sphingosine kinase pathway by which HDL inhibits TNF ⁇ -induced adhesion protein expression.
- FIG. 6 is a schematic representation of the multi-well assay for sphingosine-kinase activity.
- the basis of the assay is the phosphorylation of sphingosine bound by absorption to the walls of a multi-well plate in which the walls have been impregnated with scintillant (Flashplates, New England Nuclear).
- scintillant Flashplates, New England Nuclear.
- ⁇ - 33 P labelled ATP is incubated in the presence of the source of enzyme. If active enzyme is present the 33 P is transferred to the sphingosine.
- the wells are then washed to remove unbound JJ P.
- the 3 P bound to the sphingosine excites the scintillant and a signal is produced that is measured in a scintillation counter.
- Figure 7 is a graphical representation showing that sphingosine kinase activity is linear for up to 400 minutes when measured in the multi-well assay.
- 50 ⁇ of an endothelial cell extract prepared as described above in example 8, containing sphingosine kinase activity
- 50 ⁇ of an ATP solution containing 400 ⁇ M ATP/20 Ci/ml 33 P-ATP was incubated with flashplates coated with phospholipid mixes either with (filled squares) or without (open squares) sphingosine and incubated at 37 °C for the indicated lengths of time before washing.
- Figure 8 is a graphical representation of a survey of tissues for sphingosine-kinase activity. High speed “supernatant” (open bar) and “pellet” (filled bar) fractions from 50 ⁇ of extract were then assayed for sphingosine kinase activity. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
- one aspect of the present invention relates to a method of modulating cellular activity in a mammal said method comprising administering to said mammal an effective amount of an agent for a time and under conditions sufficient to modulate the activity of one or more components of a sphingosine kinase signalling pathway.
- mammal includes humans, primates, livestock animals (e.g. horses, cattle, sheep, pigs, donkeys), laboratory test animals (e.g. mice, rats, rabbits, guinea pigs), companion animals (e.g. dogs and cats) and captive wild animals (e.g. kangaroos, deer, foxes).
- livestock animals e.g. horses, cattle, sheep, pigs, donkeys
- laboratory test animals e.g. mice, rats, rabbits, guinea pigs
- companion animals e.g. dogs and cats
- captive wild animals e.g. kangaroos, deer, foxes.
- the mammal is a human or a laboratory test animal. Even more preferably the mammal is a human.
- Reference to "modulating cellular activity” is a reference to up-regulating, down-regulating or otherwise altering any one or more of the activities which a cell is capable of performing such as, but not limited to, one or more of chemokine production, cytokine production, nitric oxide synthesase production, adhesion molecule expression and production of other inflammatory modulators.
- Preferably said cellular activity is adhesion molecule expression.
- Reference hereinafter to adhesion molecule expression should be read as including reference to other cellular activities.
- a method of modulating adhesion molecule expression in a mammal comprising administering to said mammal an effective amount of an agent for a time and under conditions sufficient to modulate the activity of one or more components of a sphingosine kinase signalling pathway.
- expression refers to the transcription and translation of a nucleic acid molecule resulting in the synthesis of a polypeptide.
- adhesion molecule should be understood as a reference to a molecule which mediates the binding of a cell to another cell or to a protein such as an extracellular matrix protein.
- adhesion molecules include, but are not limited to, integrins, selectins (e.g. E-selectin, P-selectin), members of the immunoglobulin-gene superfamily (e.g. VCAM-1, ICAM- l) and CD44.
- Reference to modulating the "activity" of sphingosine kinase signalling pathway components is a reference to modulating the functions which said components are capable of performing such as but not limited to increasing or decreasing the role or extent to which a given component performs its function or modifying the nature of the function which a given component performs. Modulating said "activity” should also be understood to encompass increasing or decreasing the concentration levels of a given component. Modulation of said "activity” may be achieved by:
- lipid modification non-covalent coupling to a required co-activator such as a protein, lipid or ion subcellular localisation of the enzyme.
- a "sphingosine kinase signalling pathway” is defined as a signalling pathway which utilises one or both of sphingosine kinase and/or sphingosine- 1 -phosphate. Without limiting the present invention to any one theory or mode of action, it is thought a sphingosine kinase signalling pathway cascade which results in adhesion molecule expression may take the form of:
- components should be understood to refer to any molecule which is or may be involved in a sphingosine kinase signalling pathway cascade and includes, but is not limited to cellular proteins, metabolites (e g sphingomyelin, ceramide, sphingosine and Sph-1- P), kinases (e g SMase, sphingosine kinase, protein kinase C and ERK) and transcription factors (e g NF- ⁇ B)
- said components are sphingosine kinase and/or Sph-l-P
- a method of modulating adhesion molecule expression in a mammal comprising administering to said mammal an effective amount of an agent for a time and under conditions sufficient to modulate the activity of one or both of sphingosine kinase and/or Sph-l-P
- a key event in many disease conditions is the upregulation of cellular activities such as those which lead to inflammation
- upregulation of the production of inflammatory mediators such as cytokines chemokines, eNOS and upregulation of adhesion molecule expression
- Said upregulation may be induced by a number of stimuli including, for example, inflammatory cytokines such as tumour necrosis factor- ⁇ (TNF- ) and interleukin- 1 (IL-1), endotoxin, oxidised or modified hpids, radiation or tissue injury
- TNF- tumour necrosis factor- ⁇
- IL-1 interleukin- 1
- endotoxin oxidised or modified hpids
- radiation or tissue injury In a preferred aspect said cellular activity is endothelial cell activity
- the present invention relates to a method of modulating endothelial cell adhesion molecule expression in a mammal said method comprising administering to said mammal an effective amount of an agent for a time and under conditions sufficient to modulate the activity of one or more components of a sphingosine kinase signalling pathway Most preferably said components are sphingosine kinase and/or Sph-l-P.
- Sph-l-P is generated from sphingosine by sphingosine kinase. Stimulation of endothelial cells causes a rapid and transient increase in cytosolic sphingosine kinase activity which leads to upregulation of adhesion molecule expression. The production of Sph-l-P is induced in parallel with sphingosine kinase activity.
- inhibitory components of a sphingosine kinase signalling pathway result in inhibition of adhesion molecule expression.
- the term "modulate" in relation to the activity of any one or more components of the sphingosine kinase signalling pathway refers to upregulating or downregulating or otherwise altering said activity.
- the preferred method is to downregulate sphingosine kinase pathway activity either by inhibiting or reducing the activity of one or more stimulatory components of a sphingosine kinase signalling pathway or by upregulating the activity of one or more inhibitory components of said pathway.
- modulation of the activity of said components wherein adhesion molecule expression is upregulated may be desired under certain circumstances.
- the biological activity of said stimulatory components is down-regulated.
- a method of downregulating endothelial cell adhesion molecule expression in a mammal comprising administering to said mammal an effective amount of an agent for a time and under conditions sufficient to downregulate the activity of one or both of sphingosine kinase and/or Sph-l-P.
- Modulation of said activity by the administration of an agent to a mammal can be achieved by one of several techniques, including but in no way limited to introducing into said mammal a proteinaceous or non-proteinaceous molecule which:
- Said proteinaceous molecule may be derived from natural, recombinant or synthetic sources including fusion proteins or following, for example, natural product screening.
- Said non- proteinaceous molecule may be derived from natural sources, such as for example natural product screening or may be chemically synthesised.
- the present invention contemplates chemical analogs of said components capable of acting as agonists or antagonists of said components.
- Chemical agonists may not necessarily be derived from said components but may share certain conformational similarities. Alternatively, chemical agonists may be specifically designed to mimic certain physiochemical properties of said components.
- Antagonists may be any compound capable of blocking, inhibiting or otherwise preventing said components from carrying out their normal biological functions.
- Antagonists include monoclonal antibodies specific for said components, or parts of said components, and antisense nucleic acids which prevent transcription or translation of genes or mRNA in mammalian cells.
- HDL high density lipoprotein
- Treatment of endothelial cells with HDL inhibits both the amplitude and duration of TNF- ⁇ induced sphingosine kinase activation.
- HDL treatment substantially blunts the amplitude and duration of Sph-l-P formation by inhibiting sphingosine kinase activity.
- the inhibition of Sph- 1 -P formation results in blunting of downstream pathway events including blunting of MEK ERK activation and NF- ⁇ B nuclear translocation thereby reducing adhesion protein expression.
- N,N-dimethyl sphingosine decreases TNF- ⁇ induced adhesion protein expression and mRNA levels by competitively inhibiting sphingosine kinase activity. Interruption of the sphingosine kinase signalling pathway at this point prevents the downstream formation of intracellular Sph-l-P thereby preventing the further downstream events of MEK/ERK and NF- ⁇ B stimulation which lead to adhesion protein expression.
- the present invention relates to a method of downregulating endothelial cell adhesion molecule expression in a mammal said method comprising administering to said mammal an effective amount of HDL for a time and under conditions sufficient to downregulate sphingosine kinase activity.
- An "effective amount” means an amount necessary to at least partly obtain the desired response or to delay the onset or inhibit progression or halt altogether the onset or progression of adhesion molecule expression. This amount varies depending upon the health and physical condition of the individual to be treated, the taxonomic group of the individual to be treated, the assessment of the medical situation and other relevant factors. It is expected that the amount will fall in a relatively broad range that can be determined through routine trials.
- Modulation of cellular adhesion molecule expression can also be achieved by the administration of one or more sphingosine kinase signalling pathway components or functional equivalents thereof.
- another aspect of the present invention relates to a method of modulating adhesion molecule expression in a mammal said method comprising administering to said mammal an effective amount of one or more components of a sphingosine kinase signalling pathway or functional equivalents thereof.
- Derivatives include fragments, parts, portions, chemical equivalents, mutants, homologs, mimetics from natural, synthetic or recombinant sources including fusion proteins. Derivatives may be derived from insertion, deletion or substitution of amino acids. Amino acid insertional derivatives include amino and/or carboxylic terminal fusions as well as intrasequence insertions of single or multiple amino acids. Insertional amino acid sequence variants are those in which one or more amino acid residues are introduced into a predetermined site in the protein although random insertion is also possible with suitable screening of the resulting product. Deletional variants are characterized by the removal of one or more amino acids from the sequence.
- Substitutional amino acid variants are those in which at least one residue in the sequence has been removed and a different residue inserted in its place. Additions to amino acid sequences including fusions with other peptides, polypep tides or proteins.
- the derivatives of said components include fragments having particular epitopes or parts of the entire component fused to peptides, polypeptides or other proteinaceous or non- proteinaceous molecules. For example, said components or derivative thereof may be fused to a molecule to facilitate its entry into a cell.
- Analogs of said components contemplated herein include, but are not limited to, modification to side chains, incorporating of unnatural amino acids and/or their derivatives during peptide, polypeptide or protein synthesis and the use of crosslinkers and other methods which impose conformational constraints on the proteinaceous molecules or their analogs.
- Derivatives of nucleic acid sequences may similarly be derived from single or multiple nucleotide substitutions, deletions and/or additions including fusion with other nucleic acid molecules.
- the derivatives of the nucleic acid molecules of the present invention include oligonucleotides, PCR primers, antisense molecules, molecules suitable for use in cosuppression and fusion of nucleic acid molecules.
- side chain modifications contemplated by the present invention include modifications of amino groups such as by reductive alkylation by reaction with an aldehyde followed by reduction with NaBH ⁇ .; amidination with methylacetimidate; acylation with acetic anhydride; carbamoylation of amino groups with cyanate; trinitrobenzylation of amino groups with 2, 4, 6-trinitrobenzene sulphonic acid (TNBS); acylation of amino groups with succinic anhydride and tetrahydrophthalic anhydride; and pyridoxylation of lysine with pyridoxal-5-phosphate followed by reduction with NaBH
- modifications of amino groups such as by reductive alkylation by reaction with an aldehyde followed by reduction with NaBH ⁇ .; amidination with methylacetimidate; acylation with acetic anhydride; carbamoylation of amino groups with cyanate; trinitrobenzylation of amino groups with 2, 4, 6-trinitrobenzene sulphonic acid (TNBS); acylation
- the guanidine group of arginine residues may be modified by the formation of heterocyclic condensation products with reagents such as 2,3-butanedione, phenylglyoxal and glyoxal.
- the carboxyl group may be modified by carbodiimide activation via O-acylisourea formation followed by subsequent derivitisation, for example, to a corresponding amide.
- Sulphydryl groups may be modified by methods such as carboxymethylation with iodoacetic acid or iodoacetamide; performic acid oxidation to cysteic acid; formation of a mixed disulphides with other thiol compounds; reaction with maleimide, maleic anhydride or other substituted maleimide, formation ot mercurial derivatives using 4-chloromercu ⁇ benzoate, 4-chloromercu ⁇ phenylsulphon ⁇ c acid, phenylmercury chloride, 2-chloromercu ⁇ -4-mtrophenol and other mercurials, carbamoylation with cyanate at alkaline pH
- Tryptophan residues may be modified by, for example, oxidation with N-bromosuccinimide or alkylation ot the indole ring with 2-hydroxy-5-mtrobenzyl bromide or sulphenyl ha des Tyrosine residues on the other hand, may be altered by nitration with tetramtromethane to form a 3-n ⁇ trotyros ⁇ ne derivative
- Modification of the imidazole ring ot a histidine residue may be accomplished by alkylation with lodoacetic acid derivatives or N-carboethoxylation with diethylpyrocarbonate
- Examples ot incorporating unnatural amino acids and derivatives during protein synthesis include, but are not limited to, use ot norleucine, 4-ammo butyric acid, 4-ammo-3-hydroxy-5- phenylpentanoic acid, 6-am ⁇ nohexano ⁇ c acid, t-butylglycine. norvaline, phenylglycine, ornithine. sarcosme, 4-am ⁇ no-3-hydroxy-6-methylheptano ⁇ c acid, 2-th ⁇ enyl alanine and/or D- lsomers of amino acids
- Table 1 A list ot unnatural amino acid contemplated herein is shown in Table 1
- Non-conventional Code Non-conventional Code amino acid amino acid
- peptides can be contormationally constrained by, tor example, inco ⁇ oration ot C, and N , -methylamino acids, introduction ot double bonds between C, t and C,, atoms of amino acids and the formation ot cyclic peptides or analogues by introducing covalent bonds such as torming an amide bond between the N and C termini, between two side
- the present invention is useful in relation to human disease conditions
- the present invention is particularly useful but in no way limited to, use as a prophylactic or as a therapy in relation to disease conditions which involve inflammatory mechanisms, such as coronary heart disease
- another aspect of the present invention relates to a method of treatment or prophylaxis of a disease condition involving inflammatory mechanisms in a mammal said method comprising administering to said mammal an effective amount of an agent for a time and under conditions sufficient to modulate the activity of one or more components of a sphingosine kinase signalling pathway wherein said modulation results in modulation of adhesion molecule expression
- modulation of said adhesion molecule expression is modulation of endothelial cell adhesion molecule expression
- said components are sphingosine kinase and/or Sph-l-P.
- said components are downregulated said downregulation resulting in downregulation of adhesion molecule expression.
- the method of treating a mammal exhibiting coronary heart disease comprising administering to said mammal an effective amount of an agent for a time and under conditions sufficient to downregulate the activity of one or more components of a sphingosine kinase signalling pathway wherein said downregulation results in downregulation of endothelial cell adhesion molecule expression.
- said components are sphingosine kinase and/or Sph-l-P.
- Another aspect of the present invention provides a method of treatment or prophylaxis of a disease condition involving inflammatory mechanisms in a mammal said method comprising administering to said mammal an effective amount of one or more components of a sphingosine kinase signalling pathway or functional equivalents thereof wherein said modulation results in modulation of adhesion molecule expression.
- Administration of the agent or component or functional equivalent thereof, in the form of a pharmaceutical composition may be performed by any convenient means.
- the agent or component or functional equivalent thereof of the pharmaceutical composition is contemplated to exhibit therapeutic activity when administered in an amount which depends on the particular case. The variation depends, for example, on the human or animal and the agent chosen.
- a broad range of doses may be applicable. Considering a patient, for example, from about 0.1 mg to about 1 mg of the agent or component or functional equivalent thereof may be administered per kilogram of body weight per day. Dosage regimes may be adjusted to provide the optimum therapeutic response. For example, several divided doses may be administered daily, weekly, monthly or at other suitable time intervals or the dose may be proportionally reduced as indicated by the exigencies of the situation.
- agent or component or functional equivalent thereof may be administered in a convenient manner such as by the oral, intravenous (where water soluble), intraperitoneal, intramuscular, subcutaneous, intradermal or suppository routes or implanting (e.g. using slow release molecules).
- agent or component or functional equivalent thereof may be administered in the form of pharmaceutically acceptable nontoxic salts, such as acid addition salts or metal complexes, e.g. with zinc, iron or the like (which are considered as salts for pu ⁇ oses of this application).
- the tablet may contain a binder such as tragacanth, com starch or gelatin; a disintegrating agent, such as alginic acid; and a lubricant, such as magnesium stearate.
- the agent used in the method is linked to an antibody specific for coronary endothelial cells to enable specific delivery of said agent to these cells.
- the present invention relates to the use of an agent capable of modulating the activity of one or more components of a sphingosine kinase signalling pathway in the manufacture of a medicament for the modulation of adhesion molecule expression in a mammal.
- modulation of said adhesion molecule expression is modulation of endothelial cell adhesion molecule expression.
- said components are sphingosine kinase and/or Sph-l-P. Most preferably said components are downregulated said downregulation resulting in downregulation of adhesion molecule expression.
- Another aspect of the present invention relates to the use of one or more components of a sphingosine kinase signalling pathway or functional equivalents thereof in the manufacture of a medicament for the modulation of adhesion molecule expression in a mammal.
- Yet another aspect of the present invention relates to agents for use in modulating one or more components of a sphingosine kinase signalling pathway wherein modulating said components modulates adhesion molecule expression.
- modulation of said adhesion molecule expression is modulation of endothelial cell adhesion molecule expression.
- said components are sphingosine kinase and/or Sph-l-P. Most preferably said components are downregulated said downregulation resulting in downregulation of adhesion molecule expression.
- a further aspect of the present invention relates to one or more components of a sphingosine kinase signalling pathway or functional equivalents thereof for use in modulating adhesion molecule expression in a mammal.
- Yet another further aspect of the present invention relates to a pharmaceutical composition
- a pharmaceutical composition comprising an agent capable of modulating one or more components of a sphingosine kinase signalling pathway wherein said modulation results in modulation of adhesion molecule expression, together with one or more pharmaceutically acceptable carriers and/or diluents.
- the present invention relates to a pharmaceutical composition
- a pharmaceutical composition comprising one or more components of a sphingosine kinase signalling pathway or functional equivalents thereof together with one or more pharmaceutically acceptable carriers and/or diluents.
- the pharmaceutical forms suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion or may be in the form of a cream or other form suitable for topical application. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi.
- the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils.
- the proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of superfactants.
- the preventions of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride.
- Prolonged abso ⁇ tion of the injectable compositions can be brought about by the use in the compositions of agents delaying abso ⁇ tion, for example, aluminum monostearate and gelatin.
- Sterile injectable solutions are prepared by inco ⁇ orating the active compounds in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilisation.
- dispersions are prepared by inco ⁇ orating the various sterilised active ingredient into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above.
- the preferred methods of preparation are vacuum drying and the freeze-drying technique which yield a powder of the active ingredient plus any additional desired ingredient from previously sterile-filtered solution thereof.
- the active ingredients When the active ingredients are suitably protected they may be orally administered, for example, with an inert diluent or with an assimilable edible carrier, or it may be enclosed in hard or soft shell gelatin capsule, or it may be compressed into tablets, or it may be inco ⁇ orated directly with the food of the diet.
- the active compound For oral therapeutic administration, the active compound may be inco ⁇ orated with excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. Such compositions and preparations should contain at least 1 % by weight of active compound.
- compositions and preparations may, of course, be varied and may conveniently be between about 5 to about 80% of the weight of the unit.
- the amount of active compound in such therapeutically useful compositions in such that a suitable dosage will be obtained.
- Preferred compositions or preparations according to the present invention are prepared so that an oral dosage unit form contains between about 0.1 ⁇ g and 2000 mg of active compound.
- the tablets, troches, pills, capsules and the like may also contain the components as listed hereafter: a binder such as gum, acacia, corn starch or gelatin; excipients such as dicalcium phosphate; a disintegrating agent such as corn starch, potato starch, alginic acid and the like; a lubricant such as magnesium stearate; and a sweetening agent such as sucrose, lactose or saccharin may be added or a flavouring agent such as peppermint, oil of wintergreen, or cherry flavouring.
- a binder such as gum, acacia, corn starch or gelatin
- excipients such as dicalcium phosphate
- a disintegrating agent such as corn starch, potato starch, alginic acid and the like
- a lubricant such as magnesium stearate
- a sweetening agent such as sucrose, lactose or saccharin
- a flavouring agent such as peppermint, oil of wintergreen, or
- tablets, pills, or capsules may be coated with shellac, sugar or both.
- a syrup or elixir may contain the active compound, sucrose as a sweetening agent, methyl and propylparabens as preservatives, a dye and flavouring such as cherry or orange flavour.
- any material used in preparing any dosage unit form should be pharmaceutically pure and substantially non-toxic in the amounts employed.
- the active compound(s) may be inco ⁇ orated into sustained-release preparations and formulations.
- the pharmaceutical composition may also comprise genetic molecules such as a vector capable of transfecting target cells where the vector carries a nucleic acid molecule capable of modulating the expression of a component of the sphingosine kinase signalling pathway.
- the vector may, for example, be a viral vector.
- this assay provides a means of easily and rapidly identifying agents which either mimic the activity of a component of sphingosine kinase signalling pathway or act as an agonist or antagonist to a component of sphingosine kinase signalling pathway.
- another aspect of the present invention provides a method of detecting an analyte structurally or functionally reactive with a lipid, said method comprising the steps of contacting either:
- analyte or analyte and reporter molecule are contacted with lipid and scintillant coupled to a solid support.
- lipid should be understood as a reference to any molecule comprising a lipid component.
- lipid is a lipid component of the sphingosine kinase signalling pathway and even more preferably sphingosine.
- This aspect of the present invention is exemplified herein with respect to sphingosine, however this should not be understood as imposing any limitation on the application of this method.
- the present invention more particularly provides a method of detecting an analyte structurally or functionally reactive with sphingosine, said method comprising the steps of contacting either: (i) said analyte which analyte is radio labelled; or
- said analyte or analyte and reporter molecule are contacted with sphingosine and scintillant which sphingosine may be coupled to a solid support having a scintillant already coupled thereto using a method as described in Example 9.
- references to an analyte which is "structurally" reactive with a lipid should be understood as a reference to a molecule which either binds, links or otherwise associates with said lipid. Said association may be due to the formation of a peptide bond, ionic bond, hydrogen bond or other interactive bonding mechanisms.
- Reference to an analyte which is "functionally” reactive with a lipid should be understood as a molecule which, when it interacts with said lipid, directly or indirectly causes a functional process to take place. A functionally reactive analyte may or may not also structurally interact with said lipid.
- an example of a functionally reactive analyte is an enzyme (for example, a sphingosine kinase equivalent) the substrate of which is a lipid (for example, sphingosine).
- an enzyme exhibiting sphingosine kinase activity is functionally reactive in that it will phosphorylate sphingosine in the presence of ATP.
- the occurrence of the strucmral or functional activity is detected by the method of this aspect of the present invention.
- the detection of functional or structural reactivity is based on the formation, by the lipid, of a radio labelled complex. Due to the proximity of the complexed radio label with the scintillant, the scintillant becomes excited.
- the analyte which is being assayed may be directly radio labelled. This is useful where, for example, said analyte directly associates with said lipids.
- a reporter molecule may be radio labelled.
- Reference to "reporter molecule” should be understood as a reference to any molecule, other than the analyte, which detects reactivity between the analyte and lipid.
- the reporter molecule may be an antibody which recognises one or more epitopes present on the surface of the analyte of interest.
- This method of detection is an indirect method of detection, commonly referred to as a "sandwich assay".
- the reporter molecule may be a molecule required to both facilitate reactivity between the analyte and the lipid, for example the reporter molecule may be a substrate, in addition to facilitating detection.
- the reporter molecule is 33 P-ATP. An analyte exhibiting sphingosine kinase activity will phosphorylate sphingosine in the presence of 33 P-ATP resulting in the coupling of 3 P to the sphingosine.
- the analyte In the absence of a phosphate reporter molecule, the analyte would be unable to phosphorylate the lipid. Further, since the analyte does not structurally interact with the lipid sufficiently to facilitate detection, the method of detection of the functional reactivity is based on the phosphorylation of the lipid with radio labelled phosphate.
- analyte is radio labelled and said analyte binds to the lipid. It may nevertheless be necessary that a molecule other than the analyte is present in the reaction mixture to facilitate reactivity of the analyte with the lipid.
- Solid supports suitable for use in the present invention include, but are not limited to, micro- titre plates, 96 well plates, columns and micro-beads.
- the present invention provides a method for detecting an analyte exhibiting sphingosine kinase activity, said method comprising the steps of contacting said analyte and 33 P-ATP with sphingosine in the presence of a scintillant.
- said analyte being contacted with said sphingosine simultaneously with or separately to said 33 P-ATP, for a time and under conditions sufficient for 33 P-sphingosine complex to form and to excite said scintillant, and detecting said excited scintillant.
- said analyte and 33 P-ATP are contacted with sphingosine and scintillant coupled to a solid support.
- Detection of the excited scintillant may be by any suitable means including, but not limited to, a scintillation counter.
- the analyte which is assayed in the method of this aspect of the present invention may be in any suitable form.
- a fluid form is particularly useful as are tissue extracts such as lysates or homogenates.
- Said analyte may be a proteinaceous or a non-proteinaceous molecule derived from natural, recombinant or synthetic sources.
- the method of the present invention should be understood to extend to the detection of agents which act as agonists or antagonists of analytes which either structurally or functionally react with a lipid.
- the present invention is particularly useful for detecting agonists or antagonists of sphingosine kinase activity. This may be achieved, in accordance with this embodiment of the present invention, by contacting the sphingosine with sphingosine kinase in the presence of the potential agonistic or antagonistic agent. Comparison of the degree of sphingosine-radio label complex formation relative to a control assay in which addition of said agent was omitted is indicative of agonistic or antagonistic agent activity.
- an increase in sphingosine 33 P complex formation relative to the control is indicative of agonistic activity while a decrease in sphingosine 33 P complex activity is indicative of antagonistic activity.
- This aspect of the present invention should not in any way be taken as limited to the embodiment described above. Rather this aspect of the present invention can be applied to detect antagonists or agonists of any analyte which either structurally or functionally react with a lipid.
- Agents suitable for testing in the method of the present invention include, but are not limited to compounds isolated from chemical libraries or broths produced from fermentation of an organism.
- sources of sphingosine kinase suitable for use in the method of this aspect of the present invention include, but are not limited to: (i) partially purified sphingosine kinase derived from mammalian tissue, (ii) crude homogenates of mammalian tissue exhibiting enzyme activity, (iii) recombinant enzyme or (iv) HUVEC crude lysate.
- HDL were isolated from normal healthy adult donors by sequential ultracentrifugation in the density range of 1.07-1.21 g/ml. Resulting preparation of HDL contained two main populations: one with particles of Stokes' diameter 10.45 nm (HDL 2 ) and one with particles of diameter 8.6 nm (HDL 3 ). Since the inhibition of adhesion molecules expression of HDL 3 was stronger than that induced by other native or reconstituted HDL particles from purified
- HDL 3 Apo-A-1 and egg phosphatidylcholin or sphingomyelin, HDL 3 was thus used in this study.
- Figure 1 HDL, inhibited the
- VCAM-1 and E-selectin TNF ⁇ -induced expression of VCAM-1 and E-selectin by human umbilical vein endothelial cells (HUVECs) by > 80% .
- Confluent monolayers of HUVECs were preincubated with or without HDL that was isolated as the fraction of density 1.13-1.21 g/ml from human plasma at a concentration of 1 mg/ml of apoA-1. After 16-h preincubation, the cells were treated with TNF ⁇ (100 U/ml). Following 4-h treatment of TNF ⁇ , the cell-surface expression of VCAM-1 and E-selectin was measured by using flow cytometry.
- HUVECs were treated with an increasing concentration of C 2 -ceramide with or without TNF ⁇ (100 U/ml), or an increasing concentration of Sph-l-P for 4 h.
- the cell-surface expression of VCAM-1 or E-selectin was measured by flow cytometry Effect of sphingosine kinase inhibitor (DMS) on the expression of VCAM-1 and E-selectin
- DMS sphingosine kinase inhibitor
- the cells were treated with a vehicle (Nil), DMS (5 ⁇ M), Sph-l-P (5 / xM), C 2 -ceram ⁇ de (10/ ⁇ M) or TNF ⁇ (100 U/ml) tor 4 h, then measure VCAM-1 or E-selectin expression Effect ot agents on E-selectm mRNA levels and the comparison between the effect ot HDL and DMS After the indicated treatment tor 4 h, E-selectin mRNA levels were measured by Northern blotting as
- HUVECs were labelled with [ J HJsph ⁇ ngomyelm was measured at the desired time point ot TNF ⁇ treatment
- the unlabelled cells were treated with HDL and/or TNF ⁇ as indicated, cells were lysed to measure ceramide levels and sphingosine kinase activity, respectively
- the cells were permeabihzed to measure the production ot Sph-l-P in vivo
- ERK activities were assayed with myeline basic protein (MBP) as substrate after immunoprecipitation with antibodies against p42/p44 MAPK (Berra et al . 1995, Li et al , 1996, Lee et al , 1997)
- MBP myeline basic protein
- MAPK MAPK
- the kinase reaction products were separated on 10% SDS-polyacrylamide gels
- Bar graph depicting ERK activities were quantified by Phosphoimager NF- ⁇ B binding activity was measured by electrophoretic mobility shift assay after 30 mm treatment as indicated
- the specific NF- ⁇ B binding complexes were identified by the super-shift gel assay with ant ⁇ -p50 and ant ⁇ -p65 antibodies and by competition analyses with the addition of a 50-told molar excess ot unlabelled NF- ⁇ B ohgonucleotides EXAMPLE 3 Sphingosine kinase signalling pathway
- C 2 -ceramide a cell-permeable form of ceramide
- sphingomyelinase that generates endogenous ceramide
- Sph-l-P a cell-permeable form of ceramide
- C 2 -ceramide ( Figure 2A) or sphingomyelinase were poor stimulators of E-selectin and VCAM-1 expression, reaching levels that were less than 10% of that stimulated by TNF- ⁇ .
- Sph-l-P was a potent and dose-dependent inducer of E-selectin and VCAM-1 , reaching levels at 5 ⁇ M that were approximately equivalent to lOOU/ml TNF- ⁇ ( Figure 2B).
- FIG. 3A shows that TNF ⁇ stimulation of HUVECs rapidly reduced cellular sphingomyelin content to 40% of control within 30 min with return to near basal levels by 2 h.
- the cellular ceramide levels were rapidly increased (approximately 2 fold) peaking at 30 min after TNF ⁇ treatment (Figure 3B).
- Treatment of endothelial cells with HDL had a potent effect: it delayed the reversion of post TNF ⁇ sphingomyelin levels to base line and sustained the increased ceramide levels after TNF ⁇ stimulation.
- TNF ⁇ stimulation of HUVECs caused a rapid and transient increase in cytosolic sphingosine kinase activity, reaching a maximum of 165 ⁇ 13% (p ⁇ 0.01) of basal within 5 min.
- HDL pretreatment profoundly inhibited both the amplitude and duration of TNF ⁇ -induced sphingosine kinase activation (Figure 3C).
- the production of Sph-l-P was induced in parallel with the sphingosine kinase activity.
- HDL treatment again substantially blunted the amplitude and duration of Sph-l-P formation (Figure 3D).
- FIG 4A shows that both TNF ⁇ and Sph-l-P were approximately equipotent in stimulating ERK activities, whereas C 2 -ceramide did not.
- Treatment with DMS inhibited TNF ⁇ - activated ERK by 50% (p ⁇ 0.02), showing a role for sphingosine kinase in the TNF ⁇ - activated ERK signal cascade.
- Preincubation of HUVECs with HDL also reduced TNF ⁇ - stimulated ERK activation, consistent with its effect on reducing cellular levels of Sph-l-P ( Figure 4 A, bar 4).
- NF- ⁇ B binding activity was measured.
- Nuclear extracts were prepared from HUVECs treated for 30 min with vehicle or the indicated agents.
- the double-stranded oligonucleotides used as a probe in these experiements included 5'-GGATGCCATTGGGGATTTCCTCTTTACTGGATGT-3' (SEQ ID NO: 1) which contains a consensus NF- ⁇ B binding site in E-selection promoter that is underlined.
- Gel mobility shift of a consensus NF- ⁇ B oligonucleotide was performed by incubating a 32 P-labelled NF- ⁇ B probe with 4mg of nuclear proteins.
- Electrophoretic mobility shift assay shows that treatment of HUVECs with Sph-l-P induced a significant nuclear NF- ⁇ B accumulation (Figure 4B).
- the composition of Sph-l-P induced NF- ⁇ B specific protein-DNA complexes were identical to that induced by TNF ⁇ which was revealed to be p50/p65 heterodimer by antibody supershift assay and by competition analyses ( Figure 4B, the bottom).
- Treatment of cells with HDL or DMS markedly inhibited the TNF ⁇ -induced activation of NF- ⁇ B by 45-60% , but did not inhibit that induced by Sph-l-P.
- Total RNA was prepared from HUVECs that was treated with the indicated agents for 4h. Equal aliquots of total RNA (12 g) were electrophoresed in a 1 % formaldehyde gel and transferred to nylon membrane. The blots were hybridized with ⁇ 32 P-labeled E-selection cDNA probe. The mRNA levels of E-selectin were quantified by Phosphoimager and normalised to radiolabeled GAPDH probe.
- the HUVECs were labeled with [ 3 H]serine (5 Ci/ml) for 48h and preincubated with or without HDL for another 6h. The cells were then washed three times and incubated for additional 2h in the presence or absence of HDL. After the treatment with TNF ⁇ for the indicated times, cellular lipids were extracted and resolved by thin-layer chromatography (TLC) with chloroform: methanol: acetic acid:water (50:30:8:5, v/v). Sphingomyelin spots were visualized by fluorography, quantitated by scintillation
- Sphingosine kinase activity was measured by incubating the supernatant with 20mM sphingosine-BSA complex and [ ⁇ 2 P]ATP (ImM, 5 Ci/ml) for 15min at 37°C. Labeled lipids were extracted and separated by TLC along with Sph-l-P standard. Radioactivity of the spot corresponding to Sph-l -P was quantified using Phosphoimager system. The measurement of Sph-l-P formation in vivo was performed as previously described (Olivier,
- the cells were permeubilized for 15min at 37 °C in hypotonic buffer (25mM HEPES. 5mM MgCl 2 , 20 M ZnCl 2 , 20 M Na 3 VO 4 , lO g/ml leupeptin and aprotinin, ImM PMSF and 0.5mM 4-deoxypyridoxine, pH 7.2) with sphingosine (lO ⁇ M) and [ ⁇ 32 P]ATP (1/zM, 10 ⁇ Ci/ml). Lipids were extracted and resolved by TLC, and [ ⁇ 2 P]Sph-l-P was quantified as described above.
- hypotonic buffer 25mM HEPES. 5mM MgCl 2 , 20 M ZnCl 2 , 20 M Na 3 VO 4 , lO g/ml leupeptin and aprotinin, ImM PMSF and 0.5mM 4-deoxypyridoxine, pH 7.2
- EXAMPLE 9 A multi-well assay for sphingosine-kinase activity.
- Flashplates were coated with lipid substrate by adding 200 ⁇ of a mix of 30 phosphatidylcholine (19.5 / g/ml), phosphatidylserine (5.5 ⁇ g/ml), and sphingosine (25 / g/ml) in methanol and evaporating at 30°C under N 2 for 3 hours.
- a source of sphingosine kinase is added in a buffer consisting of 50mM HEPES pH 7.2/20% glycerol/lOmM MgCyimM DTT/20 /M ZnCl 2 /lmM Na 3 VO 4 /15mM NaF/0.5mM 4-deoxypyridoxine along with [ 33 P]- ⁇ -ATP (Bresatech, l//Ci/well/200 /M) and incubated for up to 2 hours at 37 °C. 5 The plates are then washed with 400 ⁇ of 50mM sodium pyrophosphate, 4 times and radioactivity was measured in a scintillation counter (Top count, Packard).
- Tissues were collected from freshly sacrificed animals and rapidly frozen in liquid nitrogen, 2-5 grams of wet tissue were homogenized with a Polytron tissue homogenizer in 2 volumes of 50mM Tris pH 7.4/20% giycerol/lmM DDT/lmM EDTA, containing a protease inhibitor cocktail (Boehringer-Mannheim). The material was subjected to centrifugation at
- EXAMPLE 11 20 The multi-well assay is linear over time and specific.
- Figure 7 illustrates the results of assaying for sphingosine kinase activity from a lysate made from human umbilical endothelial cells. It can be seen that the signal increases linearly over a period of approximately 400 minutes. This signal is specific to sphingosine kinase since
- Plates are prepared as described in Example 9. Sphingosine kinase (prepared in buffer together with 33 P-ATP as described in Example 9) is added in the presence of an agent to be tested for antagonistic or agonistic activity. The agent is added to a level of enzyme activity yielding a linear response with respect to time from 0 - 3 hours of incubation. Plates are incubated for two hours at 37 °C, washed as in Example 9 and phosphorylation of sphingosine is determined by scintillation counting.
- Inhibition is detected as diminshed inco ⁇ oration of 33 P and stimulation (agonistic activity) is detected as increased inco ⁇ oration of 33 P relative to a control incubation containing an equivalent amount of the vehicle solvent used to deliver the agents tested.
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Immunology (AREA)
- General Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Medicinal Chemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- Pharmacology & Pharmacy (AREA)
- Animal Behavior & Ethology (AREA)
- Zoology (AREA)
- Urology & Nephrology (AREA)
- Biotechnology (AREA)
- Biochemistry (AREA)
- Hematology (AREA)
- Biomedical Technology (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Epidemiology (AREA)
- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Microbiology (AREA)
- Gastroenterology & Hepatology (AREA)
- Wood Science & Technology (AREA)
- Heart & Thoracic Surgery (AREA)
- General Physics & Mathematics (AREA)
- General Engineering & Computer Science (AREA)
- Genetics & Genomics (AREA)
- Cardiology (AREA)
- Biophysics (AREA)
- Tropical Medicine & Parasitology (AREA)
- Rheumatology (AREA)
- Cell Biology (AREA)
- Pain & Pain Management (AREA)
Abstract
Description
Claims
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
AUPO900297 | 1997-09-08 | ||
AUPO9002A AUPO900297A0 (en) | 1997-09-08 | 1997-09-08 | A method of modulating cellular activity |
PCT/AU1998/000730 WO1999012533A1 (en) | 1997-09-08 | 1998-09-08 | A method of modulating cellular activity |
Publications (2)
Publication Number | Publication Date |
---|---|
EP1011654A1 true EP1011654A1 (en) | 2000-06-28 |
EP1011654A4 EP1011654A4 (en) | 2004-09-15 |
Family
ID=3803288
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
EP98941157A Withdrawn EP1011654A4 (en) | 1997-09-08 | 1998-09-08 | A method of modulating cellular activity |
Country Status (5)
Country | Link |
---|---|
EP (1) | EP1011654A4 (en) |
JP (1) | JP4570246B2 (en) |
AU (2) | AUPO900297A0 (en) |
CA (1) | CA2302838C (en) |
WO (1) | WO1999012533A1 (en) |
Families Citing this family (13)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CA2372541A1 (en) * | 1999-05-13 | 2000-11-23 | Stuart Maxwell Pitson | Sphingosine kinase enzyme |
AUPQ744700A0 (en) * | 2000-05-11 | 2000-06-01 | Medvet Science Pty. Ltd. | A method of treatment and agents useful for same |
EP1195604A1 (en) * | 2000-09-29 | 2002-04-10 | Warner-Lambert Company | Methods and compositions for screening modulators of lipid kinases |
US6610534B2 (en) * | 2000-10-05 | 2003-08-26 | Novartis Ag | Induction of blood vessel formation through administration of polynucleotides encoding sphingosine kinases |
NZ529983A (en) * | 2001-06-07 | 2006-09-29 | Medvet Science Pty Ltd | Sphingosine kinase interacts with TRAF2 and modulates tumor necrosis factor-induced cellular activity |
EP1499343A4 (en) * | 2002-03-28 | 2005-12-28 | Medvet Science Pty Ltd | A method of modulating cellular activity |
BR0315330A (en) * | 2002-10-14 | 2005-08-16 | Medvet Science Pty Ltd | Methods for modulating one or more functional characteristics of an endothelial cell and for treatment and / or prophylaxis of a condition distinguished by aberrant or otherwise unwanted endithelial cell functioning in a mammal, uses of an agent capable of modulating the functional level of sphingosine kinase and sphingosine kinase or a nucleic acid encoding sphingosine kinase and pharmaceutical composition |
EP1481680A1 (en) | 2003-05-30 | 2004-12-01 | Aventis Pharma Deutschland GmbH | Use of S1P |
AU2005253643B2 (en) * | 2004-06-15 | 2011-12-01 | Medvet Science Pty. Ltd. | Methods of modulating cellular activity involving sphingosine kinase and agents for same, and sphingosine kinase variants |
EP1765384A4 (en) * | 2004-06-15 | 2009-07-08 | Medvet Science Pty Ltd | Method of modulating cellular activity involving sphingosine kinase and agents for same, and sphingosine kinase variants |
WO2006076767A1 (en) * | 2005-01-21 | 2006-07-27 | Medvet Science Pty. Ltd. | A method of treating cellular damage |
JP2013533286A (en) | 2010-07-30 | 2013-08-22 | セントルイス ユニバーシティ | How to treat pain |
US10877045B2 (en) | 2015-07-21 | 2020-12-29 | Saint Louis University | Compositions and methods for diagnosing and treating endometriosis-related infertility |
Family Cites Families (9)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US4766111A (en) * | 1985-10-31 | 1988-08-23 | Trustees Of Boston University | Lipids with plasmin inhibitory properties |
WO1988009345A1 (en) * | 1987-05-20 | 1988-12-01 | The Rogosin Institute | Reconstituted hdl particles and uses thereof |
US5190876A (en) * | 1988-12-27 | 1993-03-02 | Emory University | Method of modifying cellular differentiation and function and compositions therefor |
CA2007507C (en) * | 1989-02-03 | 1998-05-19 | Yasuyuki Igarashi | Sphingosine and n-methyl-sphingosine as inhibitor of cell growth |
US5466716A (en) * | 1990-12-31 | 1995-11-14 | The Biomembrane Institute | Liposomal trimethylsphingosine |
WO1995003039A1 (en) * | 1993-07-23 | 1995-02-02 | The Biomembrane Institute | N,n,n-trimethylsphingosine derivatives |
WO1996020710A1 (en) * | 1994-12-29 | 1996-07-11 | The Regents Of The University Of California | Compounds for inhibition of ceramide-mediated signal transduction |
US5677189A (en) * | 1995-06-29 | 1997-10-14 | Oncomembrane, Inc. | Method for quantifying sphingosine and for diagnosing platelet activation |
JPH09176083A (en) * | 1995-12-28 | 1997-07-08 | Sankyo Co Ltd | New compound f-12509a |
-
1997
- 1997-09-08 AU AUPO9002A patent/AUPO900297A0/en not_active Abandoned
-
1998
- 1998-09-08 AU AU89658/98A patent/AU757358B2/en not_active Expired
- 1998-09-08 JP JP2000510431A patent/JP4570246B2/en not_active Expired - Lifetime
- 1998-09-08 EP EP98941157A patent/EP1011654A4/en not_active Withdrawn
- 1998-09-08 WO PCT/AU1998/000730 patent/WO1999012533A1/en active IP Right Grant
- 1998-09-08 CA CA2302838A patent/CA2302838C/en not_active Expired - Lifetime
Non-Patent Citations (3)
Title |
---|
AFTAB D T ET AL: "A semiautomated 96-well plate assay for protein kinase C", ANALYTICAL BIOCHEMISTRY, ACADEMIC PRESS INC, NEW YORK, vol. 187, no. 1, 15 May 1990 (1990-05-15), pages 84-88, XP024819783, ISSN: 0003-2697, DOI: 10.1016/0003-2697(90)90420-E [retrieved on 1990-05-15] * |
No further relevant documents disclosed * |
See also references of WO9912533A1 * |
Also Published As
Publication number | Publication date |
---|---|
JP4570246B2 (en) | 2010-10-27 |
CA2302838C (en) | 2010-03-16 |
AU8965898A (en) | 1999-03-29 |
AU757358B2 (en) | 2003-02-20 |
JP2001515857A (en) | 2001-09-25 |
EP1011654A4 (en) | 2004-09-15 |
CA2302838A1 (en) | 1999-03-18 |
WO1999012533A1 (en) | 1999-03-18 |
AUPO900297A0 (en) | 1997-10-02 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US7172879B2 (en) | Detection of sphingosine kinase activity and sphingosine kinase agonist or antagonist activity | |
US20120277166A1 (en) | Conotoxin peptides useful as inhibitors of neuronal amine transporters | |
WO1999012533A1 (en) | A method of modulating cellular activity | |
EP1290182B1 (en) | Sphingosine kinase and uses thereof | |
US20080044428A1 (en) | Glycosylphosphatidylinositol Glycan Signalling Via Integrins Functioning as Glycan Specific Receptors | |
AU2001256001A1 (en) | Sphingosine kinase and uses thereof | |
US20060234914A1 (en) | Fragments of proinsulin c-peptide | |
WO2000024417A1 (en) | A method of regulation | |
US20050100547A1 (en) | Sphingosine kinase interacts with traf2 and modulates tumor necrosis factor-induced cellular activity | |
AU2006200646B2 (en) | A method of modulating cellular activity | |
WO2006135968A1 (en) | Modulation of sphingosine kinase signalling | |
US20080039364A1 (en) | Modulating serum amyloid a interaction with tanis and agents useful for same | |
US20080279897A1 (en) | Method of Treating Cellular Damage | |
US20070265196A1 (en) | Method of Modulating Pro-Inflammatory and Inflammatory Activity Mediated by C-Reactive Protein | |
WO2006135969A1 (en) | Modulation of sphingosine kinase signalling | |
AU2003204254B2 (en) | A Method of Modulating Cellular Activity | |
US6849601B1 (en) | Peptides | |
US20080131406A1 (en) | Angiotensin I Derivatives | |
US6884617B1 (en) | Isolated nucleic acid encoding murine musculin | |
AU2002344670A1 (en) | Modulating serum amyloid A interaction with tanis and agents useful for same | |
Xia | 2. An isolated polynucleotide according to claim 1, the polynucleotide comprising a sequence at least 95% identical to SEQ ID NO: 1. 3. An isolated polynucleotide sequence that hybridizes to the nucleotide sequence of claim 2 stringency conditions of about 65 C C. and about 50% v/v formamide and about 0.15 M salt. 4. An isolated polynucleotide according to claim 1, the polynucleotide encoding a polypeptide having the sequence of SEQ ID NO: 2. 5. An isolated polynucleotide according to claim 1, the polynucleotide comprising the sequence of SEQ ID NO: 1. 6. An expression system comprising a polynucleotide according to claim 1. | |
EP1545589A1 (en) | A method of modulating endothelial cell activity |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
PUAI | Public reference made under article 153(3) epc to a published international application that has entered the european phase |
Free format text: ORIGINAL CODE: 0009012 |
|
17P | Request for examination filed |
Effective date: 20000404 |
|
AK | Designated contracting states |
Kind code of ref document: A1 Designated state(s): AT BE CH CY DE DK ES FI FR GB GR IE IT LI LU MC NL PT SE |
|
RAP1 | Party data changed (applicant data changed or rights of an application transferred) |
Owner name: JOHNSON & JOHNSON RESEARCH PTY LIMITED |
|
RAP1 | Party data changed (applicant data changed or rights of an application transferred) |
Owner name: JOHNSON & JOHNSON PHARMACEUTICAL RESEARCH AND |
|
A4 | Supplementary search report drawn up and despatched |
Effective date: 20040730 |
|
17Q | First examination report despatched |
Effective date: 20070725 |
|
STAA | Information on the status of an ep patent application or granted ep patent |
Free format text: STATUS: THE APPLICATION IS DEEMED TO BE WITHDRAWN |
|
18D | Application deemed to be withdrawn |
Effective date: 20150106 |