WO2004100986A2 - Utilisation pharmaceutique d'une substance inhibant l'enzyme adam 10 - Google Patents

Utilisation pharmaceutique d'une substance inhibant l'enzyme adam 10 Download PDF

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WO2004100986A2
WO2004100986A2 PCT/GB2004/002098 GB2004002098W WO2004100986A2 WO 2004100986 A2 WO2004100986 A2 WO 2004100986A2 GB 2004002098 W GB2004002098 W GB 2004002098W WO 2004100986 A2 WO2004100986 A2 WO 2004100986A2
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adam
substrate
use according
substance
protein
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WO2004100986A3 (fr
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John Bird
Sheila Bridget Mcloughlin
Matthew Joseph Barnes
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Celltech R&D Ltd
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Celltech R&D Ltd
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    • 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
    • C12Q1/37—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving peptidase or proteinase
    • 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]
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value

Definitions

  • the invention relates to the use of a substance capable of inhibiting ADAM 10 for the manufacture of a medicament for the treatment of allergic disease. Such substances are capable of inhibiting ADAM 10-mediated cleavage of CD23.
  • the invention also relates to a method of identifying substances capable of binding ADAM 10.
  • CD23 is the low affinity receptor for IgE (Fc ⁇ RII) with homology to the c-type lectin family of proteins and consists of a transmembrane domain, a stalk region and a lectin domain which is responsible for the binding of IgE.
  • Fc ⁇ RII the low affinity receptor for IgE
  • two isoforms of CD23 have been identified which differ only marginally at the intracellular N-terminus of the protein although the expression of the isoforms is notably differential (Yokota et al., Proc. Natl. Acad. Sci. USA, 1 ;89 (11 ):50, pp30-4, 1992).
  • CD23 is not only an IgE receptor, but also a membrane-bound precursor of soluble molecules that still bind IgE (sCD23 or IgE-binding factors) (Sarfati. M. et al., Immunol. Res., 1992, 11 , pp. 260-272; Letellier et al., Mol Immunol, 26(12), 1105-12, 1989). These soluble fragments of CD23 are thought to be involved in the regulation of IgE by inducing the differentiation of B-cells into IgE secreting plasma cells via a receptor-ligand interaction with CD21 (Aubry et al., Nature, 358(6386), 505-7, 1992).
  • Soluble fragments are generated via multiple cleavage events, for example sCD23 of molecular weights 37, 33, 29, 25 and 17kDa arise by an autocatalytic cleavage process postulated to involve a metalloprotease cleavage of membrane-bound CD23 (Marolewski et al., Biochem J, 333 (Pt 3), 573-9, 1998) in a process known as ectodomain shedding, although the identity of the precise metalloproteinase enzyme involved is currently unknown.
  • sCD23 fragments may exert several effects, either alone or in conjunction with other cytokines, on a large variety of haematopoietic cells. These effects include the regulation of IgE synthesis, promotion of B- and T-cell proliferation, inhibition of monocyte migration and in synergy with interleukin 1 (IL1 ) it may be implicated in the differentiation of early thymoctes, myeloid cell precursors and some germinal centre B cells.
  • IL1 interleukin 1
  • sCD23 fragments 37, 33 and 29 kDa
  • IgE production the three higher molecular weight sCD23 fragments (37, 33 and 29 kDa) have multifunctional cytokine properties which appear to play a major role in IgE production.
  • the excessive formation of sCD23 has been implicated in the overproduction of IgE, which is the hallmark of allergic diseases such as extrinsic asthma, rhinitis, allergic conjunctivitis, eczema, atopic dermatitis and anaphylaxis (Sutton and Gould, Nature, 1993, 366, pp421-428).
  • Elevated levels of sCD23 have also been observed in the synovial fluids of patients with rheumatoid arthritis (Chomarat P et al., Arthritis and Rheumatism, 1993, 36, pp. 234-242).
  • Metalloproteinases represent a class of the proteinase enzyme family with zinc-dependent catalytic activity. They can be broadly divided into matrix metalloproteinases, which catalyse the degradation of protein constituents of basement membrane and the extracellular matrix, and non-matrix metalloproteinases.
  • the ADAM (a disintegrin and metalloproteinase) family represent a sub-class of non-matrix metalloproteinase enzymes of which 33 have been identified to date that are involved in various biological processes including ectodomain shedding, cell-cell interactions and development (Primakoff and Myles, Trends Genet, 16(2), 83-7, 2000).
  • ADAM zinc-dependent catalytic activity typical of metalloenzymes and have been shown to exist in a membrane- associated form both of which are consistent with their inherent structure containing a pro-domain, catalytic domain, disintegrin domain, cysteine-rich, epidermal growth factor (EGF)-like, transmembrane and cytoplasmic domains (Wolfsberg et al, Dev Biol, 169(1 ), 378-83, 1995).
  • EGF epidermal growth factor
  • ADAM 10 has been identified as a catalytically active ADAM through the cleavage of a 12-amino acid peptide based around the pro-tumour necrosis factor (TNF) cleavage site (Rosendahl et al., J Biol Chem, 272(39), 24588-93, 1997) and also been implicated in the ectodomain shedding of protein (Qi et al., Science, 283, 91-94, 1999).
  • TNF pro-tumour necrosis factor
  • ADAM 9 has also been shown as catalytically active through the cleavage of peptides, myelin basic protein (Amour et al., FEBS Letters, 524, 154-158, 2002) and has also been proposed to have a role in the ectodomain shedding of CD23 (European Patent Application EP1142910).
  • ADAM 10 a member of the ADAM family
  • ADAM 10 activity results in the inhibition of CD23 shedding and thus the subsequent generation of IgE, thus providing a possible target for drug therapy in allergic diseases.
  • the invention provides the means to identify compounds for use in the treatment of allergic disease.
  • one aspect of the invention encompasses the use of a substance capable of inhibiting ADAM 10 for the manufacture of a medicament for the treatment of allergic disease.
  • a further aspect of the invention encompasses the use of a substance capable of inhibiting ADAM 10 for the manufacture of a medicament for the treatment of allergic disease, characterised in that said substance is obtained by an assay comprising the steps of: i) incubating a test substance with ADAM 10 and a substrate ii) determining whether the test substance inhibits the activity of ADAM 10 mediated cleavage of said substrate.
  • Such substances may be of use in the treatment or prevention of conditions in which IgE up-regulation is implicated, in particular allergic diseases.
  • Figure 1 shows that ADAM 10 protein is expressed in the RPMI8866 cell line and primary human tonsillar B-cells
  • Figure 2 shows the cleavage site for ADAM 10-mediated cleavage of a peptide based on the N-terminus of the 33 kDa cleavage site CD23 (Substrate
  • Figure 3 shows ADAM 10 mediated cleavage of a recombinant CD23 protein
  • ADAM 10 refers to a metal dependent proteinase (Rosendahl et al., J Biol Chem, 272(39), 24588-93, 1997), a fragment, a mutant or homologue thereof, which still maintains its catalytic function.
  • the invention is intended to encompass ADAM 10 from any source, including the native protein from any species, and those prepared by recombinant methods, in vitro methods, or standard peptide synthesis.
  • the proteinase is human or a mutant thereof.
  • a recombinant ADAM 10 especially human (e.g. R+D Systems 936-AD-020), produced in, for example, an insect cell line or a mammalian cell line may be used.
  • substrate refers to any peptide or protein, comprising one or more amino acids, that possesses a cleavage site that is capable of being cleaved by ADAM 10.
  • the substrate is "CD23-based", such that it includes one or more cleavage sites of membrane- bound CD23 (see for example Letellier M et al., Mol. Immunology, Vol 26, pp 1105-1112, 1989).
  • the substrate is based on the 37, 33, 29, 25 or 17 kDa cleavage sites, especially the 37 or 33 kDa cleavage sites, most especially the 33 kDa cleavage site, of membrane-bound CD23.
  • the said CD23-based substrate is a peptide of greater than 1 amino acid.
  • a peptide of this type is the 12-amino acid Ac-RAEQQRLKSQDL-NH2 (Substrate 1 ).
  • the CD23-based substrate is a protein, containing the amino acid sequence of CD23 or a fragment, mutant or homologue thereof, such that it includes one or more cleavage sites of membrane-bound CD23.
  • the invention is intended to encompass CD23 protein from any source, including the native protein from any species, and those prepared by recombinant methods, in vitro methods, or standard peptide synthesis.
  • the protein is human or a mutant thereof.
  • a protein of this type is the ectodomain of CD23, which comprises the amino acid residues 48-321.
  • the cleavage of said substrates may be followed by conventional methods, either directly with such methods as high-performance liquid chromatography (HPLC) or mass spectrometry (MS), or indirectly with such methods as immunoblotting.
  • the substrate may, in a further aspect of the invention, contain a suitable label in order to provide a more facile means of detecting the cleavage of said substrate.
  • label refers to any atom or molecule which can be used to provide a detectable (preferably quantifiable) signal, and which can, for example, be attached to the substrate to help detect cleavage of said substrate.
  • Suitable labels include, but are not limited to, fluorophores, chromophores, radioactive isotopes, or chemical groups, peptides or proteins which act as affinity ligands through which cleavage products can be identified.
  • useful labels include fluorophores such as 7- methoxycoumarin-4-acetic acid (Mca), chromophores such as p-nitroanilide, 14 C labelled amino acids, affinity ligands such as biotin, poly-histidine, immunoreactive peptide sequences e.g. c-myc (EQKLISEEDLGGC), glutathione-S transferase (GST), or the FC domain of IgG.
  • fluorophores such as 7- methoxycoumarin-4-acetic acid (Mca)
  • chromophores such as p-nitroanilide
  • 14 C labelled amino acids such as biotin, poly-histidine
  • immunoreactive peptide sequences e.g. c-myc (EQKLISEEDLGGC), glutathione-S transferase (GST), or the FC domain of IgG.
  • EQKLISEEDLGGC immunoreactive peptide sequences e.g. c-myc
  • the course of the assay may be followed by measuring the change in fluorescence intensity of the assay medium directly due to the accumulation of the label, e.g. Mca, comprising product as a result of cleavage, at an appropriate wavelength, e.g. 328/396 nm, or by using any convenient filter pair, such as 340/405 nm.
  • the assay can be used as a discontinuous assay in the visible spectrum it has the advantage that cheap plastic microtitre plates can be used to detect the effects of test substances on ADAM 10 activity.
  • a fluorometric method based on Fluorescence Resonance Energy Transfer (FRET) utilizing a synthetic peptide substrate for measuring protease activity may be used to generate a spectroscopic response to protease cleavage.
  • FRET Fluorescence Resonance Energy Transfer
  • both an acceptor molecule and a fluorescent molecule are attached to the peptide or protein.
  • the acceptor molecule is carefully chosen so that its absorbance overlaps with the fluorophore's excited-state fluorescence, thus ensuring that the fluorescence is quenched through resonance energy transfer.
  • Enzyme hydrolysis of the substrate results in spatial separation of the fluorophore and the acceptor molecule, thereby restoring the fluorophore's fluorescence.
  • Substrate 2 is a peptide that includes a CD23 protease cleavage site for ADAM 10, along with two covalently modified amino acid residues: Mca (7-methoxycoumarin-4- acetic acid), which acts as the fluorophore; and Dpa (3-(2,4-dinitrophenyl)-L- 2,3-diaminopropionic acid), which acts as the quenching group.
  • ADAM 10 cleavage of Substrate 2 releases a fragment containing only the Mca fluorophore, thus liberating it from the quenching effect of the nearby Dpa.
  • ADAM 10 protease activity can be measured by exciting the sample at 328 nm and measuring the fluorescence emission at 396 nm. This fluoromethc peptide substrate has been demonstrated to be cleaved quantitatively by ADAM 10.
  • a suitable fluorometric substrate of this type is the commercially available generic metalloprotease peptide Mca- PLAQAVDap(Dnp)RSSSR (Substrate 3, (Bachem (UK) Ltd) M2255) [(7- methoxycoumarin-4-yl) acetyl-Pro-Leu-Ala-Gln-Ala-Val-(3-[2,4-dinitrophenyl]- L-2,3-diaminopropionyl)-Arg-Ser-Ser-Ser-Arg-NH 2 (R&D Systems' Catalog # ES003)].
  • Mca- PLAQAVDap(Dnp)RSSSR Substrate 3, (Bachem (UK) Ltd) M2255) [(7- methoxycoumarin-4-yl) acetyl-Pro-Leu-Ala-Gln-Ala-Val-(3-[2,4-dinitrophenyl]- L-2,3-diaminopro
  • the substrate in particular a protein substrate, may contain labels that can be detected using, amongst other methods, immunoblotting techniques.
  • Appropriate labels may include, but are not limited to, short sequences of amino acids or proteins, which have been co-engineered with the substrate at either the N-terminal or the C-terminal.
  • suitable labels include a Flag, c-myc, poly-histidine, V5, glutathione-S-transferase, green fluorescent protein, maltose binding protein (MBP), influenza A virus haemaglutinin (HA), ⁇ -galactosidase, and GAL4, which are capable of interacting with highly specific antibodies to the engineered label.
  • Suitable examples may include tagged recombinant ectodomain CD23 protein, which can also contain one or more tags.
  • a suitable substrate of this type is a dual tagged protein, H 2 N-c-myc-[human CD23 residues 48-321]-V5-[hexa-histidine]-COOH (Substrate 4).
  • Substrate 4 is a recombinant protein that includes the extracellular portion of the CD23 protein containing all the known cleavage sites of CD23, along with one peptide tag at the N-terminus and two separate tags at the C-terminus.
  • any suitable format known to those skilled in the art, may be used for the assay for identifying inhibitors of ADAM 10.
  • a typical enzyme reaction may be commenced by the addition of a substrate for ADAM 10 and if assaying for ADAM 10 inhibitors, addition of the substrate may be preceded by addition of a medium, typically a buffered medium, containing a test substance and ADAM 10.
  • the assay may, for example, be followed by depletion of the substrate or by increase of the cleaved product, using the detection methods described herein, depending on substrate.
  • the assay may be carried out at any temperature or pH at which ADAM 10, in the absence of any inhibitor, is active, and is typically carried out in the range from 25-37°C, pH7-8, in any suitable buffer compatible for use in the assay.
  • a detergent may also be employed in the assay.
  • the buffer may, for example, be a Hepes buffer, preferably 50 mM Hepes, pH7.2 and 0.0015% Brij 35.
  • Final assay conditions may therefore consist of an assay mixture containing 50 mM Hepes, pH7.2, 0.0015% Brij 35, 0.15 ⁇ g/well recombinant human ADAM 10 and 10 ⁇ M substrate which is incubated at 37°C for 2 hrs. Cleavage may be measured by comparing with controls.
  • Appropriate controls may include wells monitoring the progress of the assay in the absence of the test substance; and wells including substrate and buffer in the absence of ADAM 10.
  • the assay is suitable for adaptation to 96 well and 384 well plate technologies and can be automated using liquid handling robots, allowing modern high throughput screening techniques to be applied.
  • the invention therefore permits high throughput, flexible and inexpensive screening for substances which inhibit ADAM 10. This will increase the likelihood of potent bioavailable drugs being identified.
  • a substance which inhibits the activity of ADAM 10 may be a reversible, irreversible, competitive, non-competitive or mixed inhibitor, or may inhibit by binding to the substrate thus preventing enzyme-substrate binding.
  • Suitable test substances may include defined chemical entities, peptides or peptide mimetics, oligonucleotides, and antibodies.
  • Particular antibodies include anti-ADAM 10 antibodies or fragments thereof, for example monoclonal or polyclonal antibodies, any antigen binding fragment such as scFv, Fv, Fab, Fab' or F(ab) 2 , or recombinant antibodies such as chimehc, CDR-grafted or other engineered antibodies.
  • the assay of the invention may be used to screen libraries, such as combinatorial libraries, defined chemical entity libraries, peptide or peptide mimetic libraries, oligonucleotide libraries or natural product libraries.
  • Test substances including libraries, may be obtained using conventional chemical or biological means, for example by simple chemical synthesis, peptide synthesis or recombinant DNA technology, or from naturally occurring sources, for example a microorganism.
  • a substance capable of inhibiting ADAM 10 reduces the ability of ADAM 10 to catalyse the cleavage of membrane-bound CD23 or a suitable substrate thereof.
  • Such substances inhibit ADAM 10 with a high affinity, having an IC 50 of less than 20 x 10 "6 M, preferably less than 1 x 10 "6 M, most especially less than 1 x 10 "7 M, or even lower.
  • the present invention enables a substance to be identified that is capable of inhibiting the activity of ADAM 10 and consequently the shedding of soluble CD23 fragments.
  • a substance may be used in a method of treating or modulating allergic or IgE-mediated diseases.
  • Typical diseases include allergic diseases such as asthma, atopic dermatitis and other atopic diseases, allergic rhinitis, gastrointestinal allergies such as food allergies, eosinophilia, conjunctivitis, glomerular nephritis, graft-v-host disease, systemic anaphylaxis or hypersensitivity responses, urticaria, shock, drug allergies, insect sting allergies and parasite infections.
  • Particular conditions include asthma, atopic dermatitis and allergic rhinitis .
  • compositions identified according to the invention will depend upon the nature of the identified substance.
  • pharmaceutical composition would comprise the identified substance together with one or more pharmaceutically acceptable carriers, excipients or diluents.
  • compositions may comprise the identified substance or a salt thereof; an additional agent selected from an immunosuppressant or an anti- inflammatory agent; and any pharmaceutically acceptable carrier, adjuvant or vehicle.
  • compositions may take a form suitable for oral, buccal, parenteral, nasal, topical, vaginal or rectal administration, or a form suitable for administration by inhalation or insufflation.
  • the pharmaceutical compositions may take the form of, for example, tablets, lozenges or capsules prepared by conventional means with pharmaceutically acceptable excipients such as binding agents (e.g. pregelatinised maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose); fillers (e.g. lactose, microcrystalline cellulose or calcium hydrogen phosphate); lubricants (e.g. magnesium stearate, talc or silica); disintegrants (e.g. potato starch or sodium glycolate); or wetting agents (e.g. sodium lauryl sulphate).
  • binding agents e.g. pregelatinised maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose
  • fillers e.g. lactose, microcrystalline cellulose or calcium hydrogen phosphate
  • lubricants e.g. magnesium stearate, talc or silica
  • disintegrants e.g. potato starch or sodium glycolate
  • Liquid preparations for oral administration may take the form of, for example, solutions, syrups or suspensions, or they may be presented as a dry product for constitution with water or other suitable vehicle before use.
  • Such liquid preparations may be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents, emulsifying agents, non-aqueous vehicles and preservatives.
  • the preparations may also contain buffer salts, flavouring, colouring and sweetening agents as appropriate.
  • Preparations for oral administration may be suitably formulated to give controlled release of the active compound.
  • compositions may take the form of tablets or lozenges formulated in conventional manner.
  • the identified substance may be formulated for parenteral administration by injection, e.g. by bolus injection or infusion.
  • Formulations for injection may be presented in unit dosage form, e.g. in glass ampoules, or multi-dose containers, e.g. glass vials.
  • the compositions for injection may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilising, preserving and/or dispersing agents.
  • the active ingredient may be in powder form for constitution with a suitable vehicle, e.g. sterile pyrogen-free water, before use.
  • a suitable vehicle e.g. sterile pyrogen-free water
  • the substance may be coated on particles such as microscopic gold particles.
  • the identified substance may also be formulated as a depot preparation.
  • Such long-acting formulations may be administered by implantation or by intramuscular injection.
  • the compounds for use according to the present invention are conveniently delivered in the form of an aerosol spray presentation for pressurised packs or a nebuliser, with the use of suitable propellant, e.g. dichlorodifluoromethane, trichloro- fluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas or mixture of gases.
  • suitable propellant e.g. dichlorodifluoromethane, trichloro- fluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas or mixture of gases.
  • the identified substance may be formulated as a suppository.
  • These formulations may be prepared by mixing the active ingredient with a suitable non-irritating excipient which is a solid at room temperature but liquid at the body temperature.
  • suitable non-irritating excipient include, for example, cocoa butter and polyethylene glycols.
  • the compositions may, if desired, be presented in a pack or dispenser device which may contain one or more unit dosage forms containing the active ingredient.
  • the pack or dispensing device may be accompanied by instructions for administration.
  • daily dosages may range from around 100 ng/kg to 100 mg/kg, e.g. around 0.01 mg/kg to 40 mg/kg body weight for oral or buccal administration, from around 10 ng/kg to 50 mg/kg body weight for parenteral administration, and around 0.05 mg to around 1000 mg, e.g. around 0.5 mg to around 1000 mg, for nasal administration or administration by inhalation or insufflation.
  • ADAM 10 is co-expressed with CD23 in physiologically relevant human cellular systems and that ADAM 10 can mediate the cleavage of a recombinant ectodomain CD23 protein in vitro.
  • ADAM 10 was also incubated with a peptide (Substrate 1 ) based on a region flanking the previously identified N- terminus of the 33 kDa soluble CD23 fragment and found to cleave at the same site exhibited during the shedding of soluble CD23, suggesting a relationship between ADAM 10 and the unknown CD23 sheddase.
  • Example 1 ADAM 10 is expressed in physiologically relevant cellular systems
  • ADAM 10 was expressed in physiologically relevant cellular systems (RPMI8866 cell line and isolated primary human B-cells) immunoblotting experiments were carried out using a commercially available purified rabbit polyclonal antibody (Chemicon-AB19026), which had been raised against a peptide corresponding to amino acids 732-748 of human ADAM 10.
  • RPMI8866 cell line Cells of the human B lymphoid RPMI8866 cell line were maintained in RPMI1640 medium (Sigma, Catalogue Number R0883) supplemented with 2 mM L-glutamine, 100 units/ml penicillin, 100 ⁇ g/ml streptomycin and 10% foetal bovine serum (FBS) at 37°C with 5% CO 2 until they reached a density of 8 x 10 5 cells/ml.
  • FBS foetal bovine serum
  • the cells were resuspended in unsupplemented RPMI1640 medium at a density of 1 x 10 7 cells/ml and maintained for 1 hour at 37°C with 5% CO 2 .
  • DPBS Dulbecco's phosphate buffered saline
  • a pair of human tonsils were roughly chopped, mixed with 30 ml DPBS and forced through a 40 micron sieve.
  • the resulting cell suspension was then layered onto 20 ml Ficoll-Plaque Plus (Amersham Biosciences) and centrifuged at 1180 x g for 30 minutes.
  • the tonsillar mononuclear cells were then recovered from the DPBS/Ficoll-Plaque Plus boundary, washed with 50 ml DPBS and resuspended in FBS/EDTA (0.99% (v/v) FBS prepared using 0.02% EDTA in DPBS without Ca 2+ and Mg 2+ ; Sigma, Catalogue Number E8008) (1.6 ml/2 x 10 8 cells).
  • the resulting cell suspension was then mixed with MACS CD19 Microbeads (Miltenyi Biotec, Catalogue Number 503-01) (0.4 ml/2 x 10 8 cells) and incubated for 15 minutes at 4°C.
  • MACS CD19 Microbeads (Miltenyi Biotec, Catalogue Number 503-01) (0.4 ml/2 x 10 8 cells) and incubated for 15 minutes at 4°C.
  • the cell suspension was diluted to 50 ml with FBS/EDTA, the cells recovered by centrifugation and resuspended in FBS/EDTA (1 ml/2 x 10 8 cells).
  • An autoMACS machine (Miltenyi Biotec) was then used to separate the tonsillar B-cells, which express CD19, from other mononuclear cells, which lack CD19.
  • the isolated tonsillar B-cells were diluted to 50 ml with DPBS, recovered by centrifugation and resuspended at a density of 1 x 10 7 cells/ml in RPM11640/2% FBS (RPMI1640 medium (Sigma, Catalogue Number R0883) supplemented with 2 mM L-glutamine, 100 units/ml penicillin, 100 ⁇ g/ml streptomycin and 2% (v/v) FBS).
  • RPM11640/2% FBS RPMI1640 medium (Sigma, Catalogue Number R0883) supplemented with 2 mM L-glutamine, 100 units/ml penicillin, 100 ⁇ g/ml streptomycin and 2% (v/v) FBS.
  • the resulting cell suspensions which contained 5 x 10 6 cells/ml in RPMI1640 medium with 2 mM L-glutamine, 100 units/ml penicillin, 100 ⁇ g/ml streptomycin, 1% (v/v) FBS ⁇ 100 ng/ml IL-4, were then maintained for 24 hours at 37°C with 5% C0 2 .
  • the cells were then washed with DPBS (1 ml/2 x 107 cells), snap frozen and stored at -80°C until use.
  • the nitrocellulose Western blot was blocked overnight at 4°C in 50 ml Milk Mix (PBS (10 mM phosphate buffer, 2.7 mM KCI, 137 mM NaCI, pH 7.4 at 25°C; Sigma, Catalogue Number P4417) containing 0.05% (v/v) Tween 20 (Sigma) and 5% (w/v) 'Marvel' non-fat dried milk) and then incubated for 3.5 hours at 20°C with 3 ml Milk Mix containing 1 ⁇ g/ml of anti-ADAM 10 antibody (Chemicon International).
  • the blot was maintained at 20°C, washed 5 times (each 6 minutes) with 20 ml Milk Mix, then incubated for 45 minutes with 3 ml of Milk Mix containing a 1 :1000 dilution of protein A-horseradish peroxidase (Amersham Biosciences). Finally, blots were washed 5 times (each 6 minutes) with 20 ml PBS/0.05% (v/v) Tween 20, then washed 5 times (each 6 minutes) with 20 ml PBS and antigen-antibody complexes located using enhanced chemiluminescence (ECL). This was performed using ECL Western Blotting Detection Reagents and Hyperfilm ECL (both from Amersham Biosciences). The results shown in Figure 1 were obtained during a 10 minute period of ECL.
  • ADAM 10 was found to be expressed (60 kDa and 85 kDa) in both the RPMI8866 cell line and primary human B-cells isolated from tonsillar tissue both in the presence and absence of IL-4 stimulation ( Figure 1 ). Expression in the Jurkat cell line was included as a positive control for expression of ADAM 10 (Wu et al., Biochem Biophys Research Comm, 235(2), 437-442, 1997).
  • ADAM 10 mediated cleavage of a peptide based on 33KDa cleavage site of CD 23
  • Substrate 1 was supplied lyophilized and reconstituted to 1 mM using sterile ultrapure H 2 O.
  • Recombinant human ADAM 10 amino acids 214-672 of human ADAM 10 with a C-terminal 10X His tag was obtained from R&D Systems; this was supplied lyophilized and reconstituted to 100 ⁇ g/ml using sterile ultrapure H 2 O.
  • Assays were carried out in a final volume of 200 ⁇ l, containing 50 mM Hepes, pH 7.2, 0.0015% Brij 35, recombinant human ADAM 10 (200 ng), Substrate 1 (20 ⁇ l of a 1 mM stock solution) and either DMSO (20 ⁇ l of 20%) or one of the test compounds, SB257702, Mahmastat or Trocade (20 ⁇ l of 30 ⁇ M stock solution in 20% DMSO). The resulting reaction mixtures were then incubated for 18 h at 37°C, and then stored at -80°C prior to analysis.
  • LC-MS Gradient LC was performed using a Vydac 300A Protein C18 column (250 x 4.6 mm) with a mobile phase of 0.1 % TFA (aqueous) and 0.1 % TFA (MeCN). Mass spectra were determined under electrospray ionisaton conditions (cone voltage 25V), using a Micromass LC-Z mass spectrometer.
  • Substrate 2 (Mca-RAEQQRLKSQDLE-Dpa, a fluorogenic version of Substrate 1 ) was prepared and used subsequently to develop a method for identifying inhibitors of ADAM 10-mediated cleavage of CD23.
  • ADAM 10 was incubated with Substrate 2 and inhibition (IC 50 values) determined in the presence of a panel of metalloproteinase inhibitors.
  • ADAM 10 activity was also demonstrated following incubation with a commercially available fluorescent metalloproteinase peptide substrate, Mca-PLAQAVDap(Dnp)RSSSR (Bachem (UK) Ltd) (Substrate 3), which has previously been shown to be cleaved by human recombinant ADAM 10 (Rosendahl et al., J Biol Chem, 272(39), 24588-93, 1997).
  • ADAM 10 was incubated with Substrate 3 and inhibition (IC 50 values) determined in the presence of a panel of metalloproteinase inhibitors.
  • Assays were carried out in a 96-well plate format, in a final volume of 100 ⁇ l, containing 50 mM Hepes, pH 7.2, 0.0015% Brij 35, recombinant human ADAM 10 (150 ng), Substrate 2 or Substrate 3 (10 ⁇ l of 100 ⁇ M) and either DMSO (10 ⁇ l of 20%) or one of the test compounds, SB257702, SB435466, Marimastat or Trocade (10 ⁇ l of 30 ⁇ M stock solution, or dilution thereof, in 20% DMSO), or one of the Tissue Inhibitors of Metalloproteinases (TIMP1 , 2 & 3; obtained from R&D Systems) (10 ⁇ l of 2.5 ⁇ M stock solution, or a dilution thereof, in assay buffer). After incubation at 37°C for 1-2 h, protease activities were determined by measuring the change in fluorescence intensity of the assay medium in a fluorescence plate reader (Tecan Ultra) using
  • IC 50 values generated in either the Substrate 2- or Substrate 3-based assay were compared to those obtained from a CD23 shedding membrane preparation assay which was based on a previously published method (Bailey et al., Bioorg Med Chem Lett, 8(1 ), 23-8, 1998; Marolewski et al., Biochem J, 333 (Pt 3), 573-9, 1998).
  • IC 50 values were defined as the concentration of drug required to inhibit the enzyme or shedding activity by 50%. The enzyme or shedding activity for a given concentration of drug was divided by the mean value obtained from the control (without drug) and multiplied by 100 to give values equivalent to the percentage of the control count. Non-linear least squares regression was then used to fit the Hill equation to the data. The results are shown in the Table of Biological Results below.
  • ADAM 10 mediated cleavage of recombinant CD23 protein
  • ADAM 10 could cleave a peptide based on 33 kDa cleavage site of CD23 at an identical position to the CD23 sheddase and that there was a relationship between the activity of metalloproteinase inhibitors in CD23 sheddase and ADAM 10 assays, subsequent experiments attempted to confirm these observations on the protein level (Figure 3).
  • Reaction mixtures were subjected to SDS-PAGE and then transferred onto nitrocellulose using the NuPAGE Bis-Tris Electrophoresis System (Invitrogen) according the manufacturer's instructions.
  • Samples of reaction mixture containing 7 ⁇ l of medium containing Substrate 4 were mixed with enough NuPAGE LDS Sample Buffer and NuPAGE Sample Reducing Agent to give a 4-fold dilution of the former and a 10-fold dilution of the latter.
  • samples were loaded onto a NuPAGE Novex 10% Bis-Tris Gel (1.0 mm thick with 10 wells) and subjected to SDS-PAGE under reducing conditions using NuPAGE MOPS SDS Running Buffer. After electrophoresis, proteins were then transferred onto Hybond ECL nitrocellulose (Amersham Biosciences) by Western blotting.
  • the nitrocellulose Western blot was blocked by incubating for 1 hour at 20°C in 50 ml Milk Mix (PBS (10 mM phosphate buffer, 2.7 mM KCI, 137 mM NaCI, pH 7.4 at 25°C; Sigma, Catalogue Number P4417) containing 0.05% (v/v) Tween 20 (Sigma) and 5% (w/v) 'Marvel' non-fat dried milk) and then incubated for 1 hour at 20°C with 10 ml PBS/0.05% (v/v) Tween 20 containing a 1 :5000 dilution of anti-V5-horseradish peroxidase (Invitrogen, Catalogue Number R961-25).
  • ECL enhanced chemiluminescence
  • Dual tagged ectodomain CD23 (Substrate 4) was found to be cleaved into lower molecular weight fragments following co-incubation with recombinant ADAM 10 (Lane 2) but found to be stable when incubated alone (Lane 1 ). This apparent cleavage into lower molecular weight fragments was inhibited by 1 ⁇ M SB257702 (a specific CD23 sheddase inhibitor) and 1 ⁇ M Marimastat (a broad spectrum metalloproteinase inhibitor) but not by 1 ⁇ M Trocade (a specific matrix metalloproteinase inhibitor), a profile consistent with inhibition of CD23 shedding (see Table of Biological Results below).
  • IC 50 values of CD23 shedding and ADAM 10 following exposure to various metalloproteinase inhibitors IC 50 values of CD23 shedding and ADAM 10 following exposure to various metalloproteinase inhibitors.

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Abstract

L'invention concerne l'utilisation d'une substance pouvant inhiber ADAM 10, une enzyme impliquée dans l'élimination de l'ectodomaine de CD23, pour produire un médicament servant à traiter des maladies allergiques parmi lesquelles figurent l'asthme, la dermatite atopique ainsi que la rhinite allergique.
PCT/GB2004/002098 2003-05-16 2004-05-13 Utilisation pharmaceutique d'une substance inhibant l'enzyme adam 10 Ceased WO2004100986A2 (fr)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007025999A1 (fr) * 2005-08-31 2007-03-08 Serentis Limited Utilisation d'un inhibiteur de l'aureolysine pour le traitement de maladies inflammatoires de la peau caracterisees par une colonisation par staphylococcus aureus
GB2453589A (en) * 2007-10-12 2009-04-15 King S College London Protease inhibition
EP2637679A4 (fr) * 2010-11-09 2015-09-23 Univ Chicago Rôle d'adam10 et sa pertinence au plan pathologique et thérapeutique

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* Cited by examiner, † Cited by third party
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US5922546A (en) * 1997-08-25 1999-07-13 Smithkline Beecham Corporation Human disintegrin metalloprotease KUZ gene
DE19910108C2 (de) * 1999-03-08 2001-02-22 Falk Fahrenholz Zellen, die ein Amyloidvorläuferprotein und eine alpha-Sekretase coexprimieren, ein Testverfahren zur Identifikation alpha-Sekretase-aktiver Substanzen sowie eines zur Identifikation weiterer Sekretasen, ein Testverfahren zur Bestimmung der Anfälligkeit gegenüber Morbus Alzheimer und Verwendung von Nukleinsäuren, die für eine alpha-Sekretase codieren, für die Gentherapie
ES2331177T3 (es) * 2001-12-14 2009-12-23 Exelixis, Inc. Inhibidores de adam-10 humana.

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007025999A1 (fr) * 2005-08-31 2007-03-08 Serentis Limited Utilisation d'un inhibiteur de l'aureolysine pour le traitement de maladies inflammatoires de la peau caracterisees par une colonisation par staphylococcus aureus
GB2453589A (en) * 2007-10-12 2009-04-15 King S College London Protease inhibition
US9546198B2 (en) 2007-10-12 2017-01-17 Cancer Research Technology Limited Cyclic peptides as ADAM protease inhibitors
US10472393B2 (en) 2007-10-12 2019-11-12 Cancer Research Technology Limited Method for inhibiting ADAM proteases with cyclic peptides
EP2637679A4 (fr) * 2010-11-09 2015-09-23 Univ Chicago Rôle d'adam10 et sa pertinence au plan pathologique et thérapeutique
US9150865B2 (en) 2010-11-09 2015-10-06 The University Of Chicago Role of ADAM10 and its relevance to disease and therapeutics

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