WO1994023038A1 - Synthase d'oxyde nitrique inductible et gene s'appliquant a celle-ci - Google Patents

Synthase d'oxyde nitrique inductible et gene s'appliquant a celle-ci Download PDF

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Publication number
WO1994023038A1
WO1994023038A1 PCT/GB1994/000621 GB9400621W WO9423038A1 WO 1994023038 A1 WO1994023038 A1 WO 1994023038A1 GB 9400621 W GB9400621 W GB 9400621W WO 9423038 A1 WO9423038 A1 WO 9423038A1
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synthase
leu
sequence
val
ser
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PCT/GB1994/000621
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English (en)
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Salvador Enrique Moncada
Ian George Charles
Richard Michael John Palmer
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The Wellcome Foundation Limited
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Priority to AU62878/94A priority Critical patent/AU6287894A/en
Publication of WO1994023038A1 publication Critical patent/WO1994023038A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0071—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14)
    • C12N9/0073—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14) with NADH or NADPH as one donor, and incorporation of one atom of oxygen 1.14.13
    • C12N9/0075—Nitric-oxide synthase (1.14.13.39)
    • 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
    • C12Q1/527—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving lyase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y114/00—Oxidoreductases acting on paired donors, with incorporation or reduction of molecular oxygen (1.14)
    • C12Y114/13—Oxidoreductases acting on paired donors, with incorporation or reduction of molecular oxygen (1.14) with NADH or NADPH as one donor, and incorporation of one atom of oxygen (1.14.13)
    • C12Y114/13039—Nitric-oxide synthase (NADPH dependent) (1.14.13.39)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the invention relates to a novel nitric oxide (NO) synthase, DNA coding therefor, methods for detection of the NO synthase and the DNA, a method for screening for compounds capable of inhibiting or stimulating the NO synthase, and the compounds thereby identified.
  • NO nitric oxide
  • NO is synthesised from the amino acid L-arginine by the enzyme NO synthase.
  • NO is synthesised by the vascular endothelium to regulate smooth muscle tone and thus blood pressure.
  • Nitric oxide synthase is also present in the central nervous system, where NO is a neurotransmitter/neuromodulator mediating the action of glutamate on NMDA receptors and mediating/modulating transmission in nerves previously recognised as nonadrenergic and noncholinergic.
  • NO can also act as an autocrine regulator on some cells, including platelets, where it modulates their activation.
  • NO generated by activated macrophages is also an important effector molecule in host defence. In this role, NO has been shown to possess anti-tumour and anti-microbial activity against various parasites in vitro and in vivo.
  • NO synthases can be classified into two types, namely inducible and constitutive NO synthases.
  • NO synthase activity is constitutively expressed in a variety of cells including endothelial cells, neurons, platelets, adrenal gland cells, and endocardium cells.
  • NO synthase is inducible in macrophages, hepatocytes, Kupffer cells, vascular smooth muscle and vascular endothelium following activation with endotoxin and/or cyto ines. More recently, NO synthase has been shown to be induced in rabbit articular chondrocytes (Stadler e_£. al- (1991) J. Immunol. 147. 3915-3920 and Palmer e£ al. (1992) Biochem.
  • the NO synthase enzymes comprise a family that can be distinguished on the basis of comparative DNA sequence analysis. Sequences have been reported for the constitutive neuronal NO synthase cDNAs from rat and man (Bredt e_L al.
  • a novel human inducible NO synthase has been characterized.
  • the full length cDNA encoding the NO synthase has been cloned and expressed using human articular chondrocytes activated with IL-l ⁇ .
  • the NO synthase enzyme has utility in a number of settings as described hereinafter, but is of particular value in a research environment in which it can be used in a screen or assay to identify compounds that are capable of inhibiting or stimulating the activity of the enzyme. Such compounds have utility in the clinic for the treatment of indications as described hereinafter.
  • the present invention provides an NO synthase having the sequence of SEQ ID NO: 2, or an NO synthase having a sequence at least 85% identical to the sequence of SEQ ID NO: 2.
  • the NO synthase may, for example, have a sequence at least 90%, at least 95%, at least 98% or at least 99% identical to SEQ ID NO: 2.
  • the NO synthase is generally of human origin, preferably human chondrocyte origin.
  • the expression 'NO synthase 1 includes the full length cDNA encoding the NO synthase and any fragment thereof.
  • the fragment of the NO synthase may be a fragment at least 6 amino acids in length.
  • the length of the fragment may be, for example, at least 8, at least 12, at least 24, at least 48 or at least 96 amino acids, and is intended to include the fragment corresponding to the active domain of the enzyme, i.e. the domain which catalyses the synthesis of NO from L-arginine.
  • the NO synthase is generally in substantially pure form.
  • the NO synthase in substantially pure form comprises a preparation in which at least 90%, at least 95%, at least 98% or at least 99% of the weight of protein in the preparation is the NO synthase of the invention.
  • the NO synthase will usually be obtained by recombinant DNA techniques. However, the NO synthase may be obtained using biochemical purification of the protein from its natural origin.
  • the invention provides a DNA molecule encoding the NO synthase.
  • the DNA molecule may contain the sequence of nucleotides 226 to 3687 of SEQ ID NO: 1.
  • the DNA molecule may contain a sequence at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to the sequence of nucleotides 226 to 3687 of SEQ ID NO: 1.
  • DNA molecules according to the invention could be produced by various means, such as, for example, DNA synthesis, or more preferably, by recombinant DNA techniques. Techniques for synthesising DNA molecules are described by, for example, Wu e_£ al (Prog. Nucl. Acid. Res. Molec. Biol. 21, 101-141 (1978)) . Techniques for constructing recombinant molecules are described by Sambrook __________ al (1989) Molecular Cloning: A Laboratory
  • the present NO synthase may be obtained by making a library of replicable expression vectors.
  • the library may be created by cloning genomic DNA or, more preferably, cDNA into a parent vector.
  • the cDNA may be obtained using the poly A + mRNA of a cell (e.g. a chondrocyte) in which NO synthase production has been induced, e.g. by a cytokine such as IL-l ⁇ .
  • the library is screened for members containing the desired nucleic acid sequence, e.g. by means of a DNA probe or antibody.
  • a probe having a sequence identical to a portion of the sequence of nucleotides 226 to 3687 of SEQ ID NO: 1 or exactly complementary to a portion of the sequence nucleotides 226 to 3687 of SEQ ID NO: 1 may be used to identify an NO synthase coding sequence not identical to the sequence of nucleotides 226 to 3687 of SEQ ID NO: 1 (e.g. from 85% to 99% identical to nucleotides 226 to 3687 of SEQ ID NO: 1) by low stringency hybridization.
  • a replicable expression vector is a vector which contains the appropriate origin of replication sequence for replication of the vector and the appropriate sequences for expression of the foreign nucleotide sequence in the vector.
  • the sequences for expression of a foreign sequence will generally include a transcription promoter operably linked to the foreign sequence.
  • operably linked refers to a linkage in which the promoter and foreign sequence are connected in such a way to permit expression of the foreign sequence.
  • the transcription promoter sequence may be part of the parent vector sequence into which the foreign sequence is inserted. Alternatively, the promoter sequence may be a native promoter sequence of a gene encoding an NO synthase of the invention, so that the NO synthase is inducible by IL-l ⁇ .
  • a vector may be, for example, a plasmid, virus or phage vector.
  • a vector may contain one or more selectable markers, for example an ampicillin resistance gene in the case of a bacterial vector or a neomycin resistance gene in the case of a mammalian vector.
  • a foreign gene sequence inserted into a vector may be transcribed in vitro or the vector may be used to transform or transfect a host cell.
  • a host cell transformed or transfected with a vector there is provided a host cell transformed or transfected with a vector.
  • a vector and host cell will be chosen so as to be compatible with each other, and may be prokaryotic or eukaryotic.
  • a prokaryotic host may, for example, be E. coli. in which case the vector may, for example, be a bacterial plasmid or a phage vector.
  • a eukaryotic host may, for example, be a yeast (e.g. S. cerevisiae) a Chinese hamster ovary (CHO) cell or an insect cell (e.g. Spodpptera frugiperda) .
  • yeast e.g. S. cerevisiae
  • CHO Chinese hamster ovary
  • insect cell e.g. Spodpptera frugiperda
  • the vector is generally a baculovirus vector (reviewed by Luckow and Summers (1988) in BIO/TECHNOLOGY,
  • An NO synthase according to the invention may be produced by a method comprising
  • expression of NO synthase may be induced by a cytokine such as IL-l ⁇ .
  • the NO synthase may be recovered from either the host cell or, where the NO synthase is secreted by the host cell, from the culture supernatant.
  • the invention includes a an oligonucleotide fragment having a sequence of a portion of a DNA molecule encoding an NO synthase of the invention, and an oligonucleotide fragment having a sequence at least 85%, at least 90%, at least 95%, at least 98%, or at least 99%, identical to a sequence encoding an NO synthase of the invention or fragment thereof.
  • the fragment is at least 12 nucleotides in length, e.g. at least 15, at least 18, at least 30, at least 60, at least 180, or at least 720 nucleotides in length.
  • the fragment may be single or double stranded.
  • the fragment When the fragment is single stranded, it may have a sequence from either a sense or antisense strand of a DNA molecule encoding an NO synthase of the invention.
  • An antisense fragment may be useful in the therapeutic treatment of a disease involving over-expression of NO synthase, whilst a full-length expression fragment may be of use in treatment of conditions requiring stimulation of the NO synthase, for example treatment of some viral diseases or solid tumours.
  • the fragment will generally be DNA, although other types of nucleic acid may be used, for example RNA or modified DNA.
  • a number of different types of nucleic acid modification are known in the art. These include methylphosphonate and phosphorothioate backbones, and addition of acridine or polylysine chains at the 3' and/or 5' ends of the molecule.
  • the oligonucleotide fragment may be an oligonucleotide probe or an oligonucleotide polymerase chain reaction (PCR) primer, which will hybridise to a nucleic acid molecule (e.g. a DNA or RNA molecule) encoding an NO synthase of the invention.
  • PCR oligonucleotide polymerase chain reaction
  • the probe or a pair of primers may be used to detect or quantitatively determine the nucleic acid sequence. This has diagnostic utility in detecting and quantitatively determining NO synthase mRNA associated with a disease state, for example arthritic disease, high blood pressure, disorders of the central nervous system, and cancers such as breast cancer.
  • a fragment which is a probe or primer may carry a revealing label, such as 32 P, digoxigenin or biotin.
  • the probe or primer will specifically hybridise only to its target sequence, e.g. a portion of SEQ ID NO: 1, and not to other sequences. However, it will be appreciated that this will not always be the case, and the probe or primer may only be selective for its target sequence.
  • a probe or primer which hybridises only to its target sequence will generally be exactly complementary to the target sequence (e.g.
  • a probe or primer which is only selective may have a sequence which is, for example, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% complementary to the target sequence.
  • a probe which is not exactly complementary to its target sequence has utility in the identification of new NO synthase nucleotide sequences having a sequence similar to the sequence of nucleotides 226 to 3687 of SEQ ID NO: 1.
  • a fragment which is a probe or primer may have from 12 to 60 nucleotides, e.g. from 12 to 40 nucleotides, or from 15 to 30 nucleotides.
  • Primers for PCR are generally provided as a pair.
  • a first primer hybridises to a sense sequence 3 ' to the sequence to be amplified and a second primer hybridises to an antisense sequence 5' to the sequence to be amplified. This allows synthesis of double stranded DNA representing the region between the two primers.
  • the invention includes a method of amplifying a target nucleic acid sequence present in a nucleic acid encoding an NO synthase of the invention, which method comprises carrying out PCR employing a primer of the invention.
  • Such a method generally comprises carrying out cycles of
  • the number of cycles is suitably from 10 to 50, preferably 20 to 40, more preferably 25 to 35.
  • the method may be carried out starting from a double stranded nucleic acid (e.g. dsDNA) or a single stranded nucleic acid (e.g. mRNA) .
  • the target sequence may be a complete NO synthase encoding sequence or a partial NO synthase encoding sequence.
  • PCR polymerase chain reaction
  • RT-PCR reverse transcriptase-PCR
  • An oligonucleotide probe according to the invention has utility in detecting or quantitatively determining a nucleic acid (e.g. a DNA or RNA) encoding an NO synthase according to the invention.
  • a nucleic acid e.g. a DNA or RNA
  • Conventional methods for detecting or quantitatively determining a nucleic acid may be used, for example in situ hybridization, Southern blotting or Northern blotting. Accordingly, there is provided a method of detecting or quantitatively determining in a sample a target nucleic acid sequence present in a nucleic acid encoding an NO synthase according to the invention, which method comprises
  • the sample containing the target nucleotide sequence may, for example, be a tissue specimen, a tissue extract or cell extract from a patient suffering from a disease associated with abnormal NO synthase activity such as arthritis, high blood pressure, a disorder of the central nervous system, or a cancer such as breast cancer.
  • the sample may, for example, be a sample produced as a result of a recombinant DNA procedure, in which case the sample may be a vector or an extract of host cells.
  • the target nucleic acid sequence may be a complete NO synthase encoding sequence or a partial NO synthase encoding sequence.
  • a preferred method of detecting or quantitatively determining a target nucleic acid sequence in a sample comprises
  • a probe can be used in an in situ hybridization procedure to locate a nucleic acid sequence encoding an NO synthase of the invention. This can be done to determine the spatial distribution of NO synthase encoding DNA or RNA sequences in a cell or tissue. In the case of mRNA detection, the tissue is gently fixed so that its RNA is retained in an exposed form and the tissue is then incubated with a labelled complementary probe.
  • a polypeptide fragment of the present invention has utility in, for example, producing antibodies against an NO synthase.
  • the invention includes an antibody specific for an NO synthase according to the invention.
  • the antibody has utility in detecting and quantitatively determining NO synthases, and hence is useful in diagnosis of diseases associated with NO synthase, such as the diseases listed herein.
  • An antibody of the present invention may also be of use in identifying which NO synthase enzyme is responsible for a particular condition.
  • the antibody also has utility in production of NO synthases by recombinant DNA procedures, for example in detection of positive clones containing a target sequence.
  • the antibody may be of use as a therapeutic agent.
  • the antibody is preferably monoclonal, but may also be polyclonal.
  • the antibody may be labelled.
  • suitable antibody labels include radiolabels, biotin (which may be detected by avidin or streptavidin conjugated to peroxidase), alkaline phosphatase and fluorescent labels (e.g. fluorescein and rhodamine) .
  • the term "antibody” is used herein to include both complete antibody molecules and fragments thereof. Preferred fragments contain at least one antigen binding site, such as Fab and F(ab') 2 fragments. Humanised and chimaeric antibodies and fragments thereof are also included within the term "antibody".
  • the antibody is produced by raising antibody in a host animal against an NO synthase according to the invention or an antigenic epitope thereof (hereinafter "the immunogen") .
  • the immunogen an antigenic epitope thereof.
  • a method for producing a polyclonal antibody comprises immunising a suitable host animal, for example an experimental animal, with the immunogen and isolating immunoglobulins from the serum. The animal may therefore be inoculated with the immunogen, blood subsequently removed from the animal and the IgG fraction purified.
  • a method for producing a monoclonal antibody comprises immortalising cells which produce the desired antibody. Hybridoma cells may be produced by fusing spleen cells from an inoculated experimental animal with tumour cells (Kohler and Milstein, Nature 256. 495- 497, 1975) .
  • An immortalized cell producing the desired antibody may be selected by a conventional procedure.
  • the hybridomas may be grown in culture or injected intraperitoneally for formation of ascites fluid or into the blood stream of an allogenic host or immunocompromised host.
  • Human antibody may be prepared by in vitro immunisation of human lymphocytes, followed by transformation of the lymphocytes with Epstein-Barr virus.
  • the experimental animal is suitably a goat, rabbit, rat or mouse.
  • the immunogen may be administered as a conjugate in which the immunogen is coupled, for example via a side chain of one of the amino acid residues, to a suitable carrier.
  • the carrier molecule is typically a physiologically acceptable carrier.
  • the antibody obtained may be isolated and, if desired, purified.
  • the invention includes a method of detecting or quantitatively determining in a sample an NO synthase of the invention, which method comprises
  • a preferred method for detecting or quantitatively determining an NO synthase is Western blotting. Such a method can comprise the steps of
  • ELISA enzyme-linked immunoassay
  • a non- competitive ELISA method Preferred methods of quantitative determination are ELISA (enzyme-linked immunoassay) methods such as a non- competitive ELISA methods.
  • an ELISA method comprises the steps of
  • An antibody of the invention may be employed histologically for in situ detection of an NO synthase, e.g. by immunofluorescence or immunoelectron micropsy.
  • In situ detection may be accomplished by removing a histological specimen from a patient, and allowing a labelled antibody to bind to the specimen. Through use of such a procedure, it is possible to determine not only the presence of an NO synthase but also its distribution.
  • An antibody of the invention may be used to purify a target NO synthase.
  • Conventional methods of purifying an antigen using an antibody may be used. Such methods include immunoprecipitation and immunoaffinity column methods.
  • an antibody in accordance with the invention is coupled to the inert matrix of the column and a sample containing the target NO synthase is passed down the column, such that the target NO synthase is retained. The NO synthase is then eluted.
  • the sample containing the target NO synthase used in the detection, determination and purification methods may be a tissue specimen, a tissue extract or a cell extract from a patient suffering from a disease associated with NO synthase, such as a disease listed above.
  • the sample may be one produced as a result of recombinant DNA procedures, e.g. a vector or an extract of host cells.
  • An NO synthase of the invention is useful for screening for substrates which inhibit or stimulate the enzyme.
  • the invention includes a method for identifying a substrate which inhibits or stimulates the NO synthase, which method comprises
  • the activity of a substrate as an inhibitor or stimulator of the NO synthase of the present invention can be determined by an assay in which activated chondrocytes are incubated with the substrate, and NO synthase activity recorded using a dual wavelength spectrophotometer, reading at 401 and 421.
  • the invention also extends to substrates identified by the use of a screen hereinbefore described.
  • the substrate is a chemical molecule of relatively low molecular weight, for example, less than about 1000.
  • suitable classes of molecule include arginine analogues and isothiourea derivatives.
  • the substrate can be a macromolecule, for example an antibody.
  • the invention also includes an enzyme-substrate complex which comprises an NO synthase as described herein and a substrate capable of inhibiting or stimulating the activity of the NO synthase.
  • the enzyme-substrate complex optionally exists in an ⁇ x. vivo situation.
  • a substrate which inhibits or stimulates the NO synthase enzyme is of utility in medical therapy.
  • Inhibition of the inducible NO synthase may have many clinical utilities, for example in the treatment of septic shock and in particular in the treatment of hypotension assosiated therewith, in therapy with cytokines such as TNF, IL-1 and IL-2 or therapy with cytokine-inducing agents, for example 5, 6-dimethylxanthenone acetic acid, as an adjuvant to short term immunosuppression in transplant therapy, in patients suffering from inflammatory conditions in which an excess of NO contributes to the pathophysiology of the condition, for example adult respiratory distress syndrome and myocarditis, and in autoimmune and/or inflammatory conditions such as arthritis and rheumatoid arthritis.
  • Inhibition of the NO synthase enzyme may also be of use in the treatment of cerebral ischemia, CNS trauma, epilepsy, AIDS dementia, chronic neurodegenerative disease and chronic pain, and conditions in which non-adrenergic non- cholinergic nerve may be implicated such as priapism, obesity and hyperphagia.
  • stimulation of the inducible NO synthase would lead to increased NO levels in the body and may be of use in treating parasitic and/or viral diseases, and killing tumour cells.
  • a pharmaceutical formulation which comprises one or more of a substrate identified using the present invention, an NO synthase as described herein, or an antibody of the present invention in combination with a pharmaceutically acceptable carrier or diluent therefor, and optionally one or more further therapeutic agents.
  • Formulations comprising a substrate include those suitable for oral, parenteral (including subcutaneous, intradermal, intramuscular, intravenous and intraarticular) , rectal and topical (including dermal, buccal, sublingual and intraocular) administration although the most suitable route may depend upon for example the condition and disorder of the recipient.
  • Formulations comprising an NO synthase as described herein, or an antibody are those suitable for parenteral administration. All formulations may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. All methods include the step of bringing into association a substrate, an NO synthase as described herein, or an antibody ("active ingredient") with the carrier which constitutes one or more accessory ingredients.
  • formulations are prepared by uniformly and intimately bringing into association the active ingredient with liquid carriers or finely divided solid carriers or both and then, if necessary, shaping the product into the desired formulation.
  • Formulations of the present invention suitable for oral administration may be presented as discrete units such as capsules, cachets or tablets each containing a predetermined amount of the active ingredient; as a powder or granules; as a solution or a suspension in an aqueous liquid or a non-aqueous liquid; or as an oil-in-water liquid emulsion or a water-in-oil liquid emulsion.
  • the active ingredient may also be presented as a bolus, electuary or paste.
  • Formulations for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents.
  • the formulations may be presented in unit-dose or multi-dose containers, for example sealed ampoules and vials, and may be stored in a freeze-dried (lyophilised) condition requiring only the addition of the sterile liquid carrier, for example, saline, water-for-injection, immediately prior to use.
  • Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the kind previously described.
  • Formulations for rectal administration may be presented as a suppository with the usual carriers such as cocoa butter or polyethylene glycol.
  • Formulations for topical administration in the mouth include lozenges comprising the active ingredient in a flavoured basis such as sucrose and acacia or tragacanth, and pastilles comprising the active ingredient in a basis such as gelatin and glycerin or sucrose and acacia.
  • Preferred unit dosage formulations are those containing an effective dose, as hereinbelow recited, or an appropriate fraction thereof, of the active ingredient.
  • formulations of this invention may include other agents conventional in the art having regard to the type of formulation in question, for example those suitable for oral administration may include flavouring agents.
  • the substrates are preferably administered orally or via injection at a dose of from 0.1 to 500mg/kg per day.
  • the dose range for adult humans is generally from 5mg to 35g/day and preferably 5mg to 2g/day.
  • Tablets or other forms of presentation provided in discrete units may conveniently contain an amount of compound of the invention which is effective at such dosage or as a multiple of the same, for instance, units containing 5mg to 500mg, usually around lOmg to 200mg.
  • the NO synthase as described herein or antibody are administered parenterally at a dose of from 1 to about 100 mg for an adult patient, preferably 1 - 10 mg, usually administered daily for a period between 1 and 30 days.
  • a two part dosing regime may be preferable wherein 1 - 5 mg are administered for 5 - 10 days followed by 6 - 15mg for a further 5 - 10 days.
  • the precise amount of active ingredient administered to a patient will be the responsibility of the attendant physician. However the dose employed will depend on a number of factors, including the age and sex of the patient, the precise disorder being treated, and its severity. Also the route of administration may vary depending on the condition and its severity.
  • Figure 1 shows the results of Northern blot analysis of inducible NO synthase specific mRNA from human chondrocytes.
  • PolyA + mRNA (0.25 ⁇ g) , extracted from induced and uninduced cells, was electrophoresed through a formaldehyde-agarose gel and transferred to a nylon membrane. The blot was hybridized with a full-length cDNA probe labelled with digoxigenin and washed under high stringency conditions. A positively hybridizing band at 4.4 kb) is apparent only in track 2 (induced) .
  • Track 1 was loaded with the same amount of polyA + mRNA from uninduced cells and shows no positively hybridizing band. The positions of molecular mass markers are indicated.
  • Figure 2 shows the results of expression of recombinant human chondrocyte inducible NO synthase in CHO cells.
  • CHO cells were transfected with pSVL-NO containing the full-length cDNA for human iNOS, and NO synthase activity assayed as NO in the culture supernatant after (A) 24 h or (B) 96 h.
  • Controls were parent cells alone (i.e. untransfected; closed box) and an unrelated CHO-recombinant (pSVLS; hatched box) .
  • Human chondrocytes were isolated and cultured. In order to induce NO synthase activity, cells were incubated with IL-l ⁇ (1 ng/ml) for 24 h. The methods of culture and induction used were analogous to those described in relation to rabbit chondrocytes by Stadler fit al. (1991) J. Immunol 147, 3915-3920 and Palmer ⁇ t al. (1992) Biochem. Biophys. Res. Commun. 188, 209-215. Cells were harvested with trypsin, washed with growth medium, pelleted and frozen at -70°C.
  • PolyA + mRNA was extracted with a Micro Fast-Track kit (Trade Name, Invitrogen) from chondrocytes (l-2xlO ⁇ cells) incubated for 24 h with or without IL-l ⁇ (1 ng/ml) . Typically 1-2 ⁇ g polyA + mRNA was purified from lxlO 6 cells.
  • the murine macrophage cell line J774 was cultured and induced to express NO synthase with interferon ⁇ and lipopolysaccharide from Escherichia £ ⁇ JLi strain 026.B6 as described previously (Cunha fit al (1993) J. Immunol 150, 1-6. PolyA + mRNA was extracted as described above.
  • DHFR ' Dihydrofolate reductase " (DHFR ' )CHO cells were maintained in 75 cm flasks in Dulbecco's MEM (Trade Name, Gibco) , 10% foetal calf serum (FCS) , 1 mM L-glutamine, non essential amino acids, antibiotics, 100 ⁇ M hypoxanthine and 16 ⁇ m thymidine. pSVL transfected cells were cultured in the absence of hypoxanthine and thymidine, but in the presence of dialysed FCS and 100 ⁇ M methotrexate.
  • FCS foetal calf serum
  • L-N- iminoethyl-ornithine (L-NIO) or N-guanidino-monomethyl-L- arginine (L-NMMA) was added to some cultures to a final concentration of 100 ⁇ M. All cultures were then incubated at 37°C in a humidified 5% C0 2 atmosphere. Samples (100 ⁇ l) were removed from triplicate cultures at 24 h intervals and stored at 4°C before determination of NO by chemiluminescence (Palmer fit al. (1987), Nature 327, 524-526)
  • RT-PCR reverse transcriptase- polymeraae chain reaction
  • the primers BB3 5 ' -CGGGATCCGGNACNGGNATHGCNCCNTT-3 '
  • RT-PCR was carried out on polyA + mRNA extracted from induced J774 cells using oligonucleotide primers derived from, the RAW 264.7 sequence (Lyons fit al. (1992) J. Biol. Chem. 267 6370-6374; Xie fit al- (1992) Science 256, 225-228; and Lowenstein fit al. (1992) Proc. Natl. Acad. Sci. USA 89, 6711- 6715) .
  • PCR products were digested with Hindlll and BamHI purified and cloned into Hindlll-BairiHI digested Bluescript pBS SKII+ (Sambrook fit al- • supra) .
  • Recombinant DNA was sequenced using double standard DNA as template (Stephen fit al (1991) Nucleic Acids Res. 24, 7463-7464) . An overlapping series of deletions was made in template DNA (Henikoff (1984) Gene 28, 351-359 using the exonuclease III kit (Pharmacia) . Sequencing was carried out using universal primer, [ ⁇ 35 S] dATP and wedge gels (Sanger fit al (1983) Proc. Natl. Acad. Sci. USA 74, 5463-5467) . Clones were sequenced with modified T7 DNA polymerase (Tabor and Richardson (1987) Proc. Natl. Acad. Sci. USA 84, 4767-4771) .
  • CHO cells were co-transfected with 10 ⁇ g of the full-length cDNA for the human inducible NO synthase, pSVL-NO, cloned as an Xbal fragment into the vector pSVL (Pharmacia, UK) and with 1 ⁇ g of the DHFR encoding plasmid pRDN2 (Dr. N. Sharp, Wellcome Research Laboratories, Beckenham, Kent) . Cells were seeded at 10 6 per 100 cm petri dish and individual recombinants cloned by dilution cloning. Individual clones were assayed for the ability to increase the N0 2 - concentration in growth medium. One cell line, CHO-INOS-20, expressing the highest levels of inducible NO synthase was selected for further study.
  • RT-PCR was carried out using the primer set BB3 and BB4.
  • PolyA + mRNA (50 ng) from induced human cells was used as a template and cDNA was generated by random priming.
  • PCR resulted in a 350 bp band which was purified, cloned and sequenced. Analysis of the sequence demonstrated that the human chondrocyte iNOS cDNA had high (>80%) identity with the murine inducible NOS cDNA over this region.
  • a cDNA library was constructed in lambda ZAPII using oligo dT and random primed polyA + mRNA isolated from induced cells.
  • a [ ⁇ 32 P] -labelled probe was prepared form a 650 bp 5 '-fragment of the murine inducible NOS cDNA cloned from mRNA isolated from the cell line J774. This cell line has a cytokine-inducible NOS cDNA sequence that is identical to that described for the RAW 264.7 cell line (our unpublished observations) .
  • the murine enzyme comprises 1144 amino acids with a calculated molecular mass of 130,556 daltons. Overall, the two proteins have 81% identity and 88% similarity as determined by the GAP align program (Trade Name, Wisconsin GCG) . Both molecules have consensus recognition sites for the co-factors FAD, FMN and NADPH and in addition have a calmodulin recognition motif, although both enzymes are Ca 2+" independent.
  • Figure 3 SDS-PAGE of baculovirus/insect cell expressed human inducible NO synthase.
  • Tracks 1 and l ⁇ Amersham rainbow molecular weight markers; Track 2. total cell lysate (soluble supernatant fraction;) Tracks 2.-2. are fractions from an ADP column eluted with lOmM NADPH. The arrow indicates the position of the 135kDa band corresponding to the iNOS. Samples were run on a 10% SDS-PAGE gel.
  • a full-length human iNOS cDNA fragment was cloned as an Xbal fragment into the baculovirus transfer vector pVL1393 to generate pVLHINOS.
  • This vector directs the expression of recombinant proteins under the control of the strong polyhedrin promoter.
  • the human iNOS construct pVLMINOS was used to generate recombinant Autographa californica baculovirus using the Baculogold transfection kit (Pharmingen) .
  • S. frugiperda (Sf-21) cells were maintained as stirred cultures at 27°C in TC100 medium. Roller cultures of Sf-21 cells (5 x 10 8 cells/800cm 2 roller) were infected with human iNOS baculovirus (>10 8 pfu/ml) at a ratio of 5pfu/cell for 24 hours at 27°C.
  • Cytosol preparations containing iNOS were prepared from 2 x 10 9 cells as described below. Briefly, the S. frugiperda cell pellet was harvested and washed in a buffer containing 0.1M Hepes pH7.4, 1.OmM dithiothreitol. Cells were resuspended (10 8 /ml) in the same buffer and lysed by 3 freeze- thaw cycles. The resulting lysate was centrifuged at 100,000g for 30 min, and the supernatant mixed at 4°C for 45 mins with one ml of 2 '-5' ADP sepharose-4B (Pharmacia), (Charles fit al .. Biochem. Biophys. Res.
  • NO sythase activity was measured spectrophotometrically as described previously (Palmer et al.. Biochem. Biophys. Res. Commun., 188, 209-215 and Feelisch fit al. , Eur. J Pharmac, 139, 19-30) .
  • the effects of altering the arginine concentrations were investigated as was the inhibition of NO synthase with L-NMMA (N G -monomethyl-L-arginine) .
  • a cDNA clone encoding human inducible NO synthase has been isolated from a ⁇ ZAPII cDNA library and cloned into the baculovirus expression vector pVL1393 as an Xbal fragment.
  • Transfection into S. frugiperda (Sf-21) cells results in the expression of NO synthase activity that can be isolated by a freeze-thaw procedure.
  • Fig 3 shows 10% SDS-PAGE gel showing the NADPH elution profile of NO synthase from an ADP sepharose column.
  • Track 2 shows the S. frugiperda iNOS cell lysate following high speed centrifugation.
  • Table 1 Summary of the characteristics of recombinant human iNOS compared with its native counterpart.
  • the native iNOS was fneasured from IL- 1 induced human chondrocytes and megakaryocytes (Meg- 01 ) and a human adenocarcinoma cell -line SW480 .
  • Vmax chondrocytes (pmol/min per mg) 430 ⁇ 150 180 (SW480 cells)
  • ORGANISM Homo sapiens
  • G CELL TYPE: chondrocyte

Abstract

L'invention se rapporte à une enzyme de la synthase NO d'une forme pratiquement pure ayant une séquence qui est au moins identique à 85 % à la séquence SEQ ID No 2, de préférence exprimée à partir de chondrocytes humains; à l'ADN codant pour celle-ci; à des procédés de détection ou de détermination quantitative de la synthase N0 ou de l'ADN; à des procédés de dépistage s'appliquant à des substrats pouvant inhiber ou stimuler la synthase NO; et à des substrats identifiés par celle-ci.
PCT/GB1994/000621 1993-03-26 1994-03-25 Synthase d'oxyde nitrique inductible et gene s'appliquant a celle-ci WO1994023038A1 (fr)

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WO1996040785A1 (fr) * 1995-06-07 1996-12-19 Merck & Co., Inc. Anticorps monospecifiques et leurs procedes d'utilisation
WO1996039858A1 (fr) * 1995-06-07 1996-12-19 Merck & Co., Inc. Anticorps monospecifiques et leurs procedes d'utilisation
WO1996041885A1 (fr) * 1995-06-09 1996-12-27 Schering Corporation Expression de la synthetase d'oxyde nitrique humaine inductible
EP0769903A1 (fr) * 1994-06-24 1997-05-02 University Of Pittsburgh Of The Commonwealth System Of Higher Education Gene pour synthase d'oxyde nitrique inductible pour le traitement de maladies
EP0864648A1 (fr) * 1996-09-20 1998-09-16 Suntory Limited Procede de tri de composes regulateurs de l'expression de la synthase induisant chez l'homme la production d'acide nitrique
US5830461A (en) * 1992-11-25 1998-11-03 University Of Pittsburgh Of The Commonwealth System Of Higher Education Methods for promoting wound healing and treating transplant-associated vasculopathy
US5908842A (en) * 1995-12-08 1999-06-01 Merck & Co., Inc. Substituted 2-acylamino-pyridines as inhibitors of nitric oxide synthase
US6090839A (en) * 1996-12-23 2000-07-18 Merck & Co., Inc. Antidiabetic agents
WO2000066725A1 (fr) * 1999-05-04 2000-11-09 Aventis Pharma S.A. Utilisation d'oligonucleotides antisens de no-synthase inductible dans la prevention et le traitement de l'ischemie cerebrale
FR2793142A1 (fr) * 1999-05-04 2000-11-10 Aventis Pharma Sa Utilisation d'oligonucleotides antisens de no-synthase inductible dans la prevention et le traitement de l'ischemie cerebrale
US6146887A (en) * 1994-03-31 2000-11-14 Jurgen Schrader DNA expression vectors for use in the gene therapeutic treatment of vascular disorders
US6160000A (en) * 1996-12-23 2000-12-12 Merck & Co., Inc. Antidiabetic agents based on aryl and heteroarylacetic acids
WO2001066791A1 (fr) * 2000-03-10 2001-09-13 Fujisawa Pharmaceutical Co., Ltd. Criblage de l'inhibiteur d'activation de la synthetase d'oxyde nitrique inductible
EP1250157A1 (fr) * 2000-01-24 2002-10-23 Isis Pharmaceuticals, Inc. Modulation antisens de l'expression de l'oxyde nitrique synthase inductible
US6531578B1 (en) * 1996-04-12 2003-03-11 Robert Webber Immunoassay method employing monoclonal antibody reactive to human iNOS
US7198904B1 (en) * 1996-04-12 2007-04-03 Robert Webber Immunoassay method employing monoclonal antibody reactive to human iNOS

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Cited By (22)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5830461A (en) * 1992-11-25 1998-11-03 University Of Pittsburgh Of The Commonwealth System Of Higher Education Methods for promoting wound healing and treating transplant-associated vasculopathy
US6103230A (en) * 1992-11-25 2000-08-15 University Of Pittsburgh Of The Commonwealth System Of Higher Education Methods for promoting wound healing and treating transplant-associated vasculopathy
US6149936A (en) * 1994-03-31 2000-11-21 Joseph Schrader DNA expression vectors for the use in the gene therapeutic treatment of vascular disorders
US6146887A (en) * 1994-03-31 2000-11-14 Jurgen Schrader DNA expression vectors for use in the gene therapeutic treatment of vascular disorders
EP0769903A1 (fr) * 1994-06-24 1997-05-02 University Of Pittsburgh Of The Commonwealth System Of Higher Education Gene pour synthase d'oxyde nitrique inductible pour le traitement de maladies
EP0769903A4 (fr) * 1994-06-24 1999-10-27 Univ Pittsburgh Gene pour synthase d'oxyde nitrique inductible pour le traitement de maladies
WO1996040785A1 (fr) * 1995-06-07 1996-12-19 Merck & Co., Inc. Anticorps monospecifiques et leurs procedes d'utilisation
WO1996039858A1 (fr) * 1995-06-07 1996-12-19 Merck & Co., Inc. Anticorps monospecifiques et leurs procedes d'utilisation
US5744340A (en) * 1995-06-09 1998-04-28 Schering Corporation Expression of human inducible nitric oxide synthase
WO1996041885A1 (fr) * 1995-06-09 1996-12-27 Schering Corporation Expression de la synthetase d'oxyde nitrique humaine inductible
US5908842A (en) * 1995-12-08 1999-06-01 Merck & Co., Inc. Substituted 2-acylamino-pyridines as inhibitors of nitric oxide synthase
US7198904B1 (en) * 1996-04-12 2007-04-03 Robert Webber Immunoassay method employing monoclonal antibody reactive to human iNOS
US6531578B1 (en) * 1996-04-12 2003-03-11 Robert Webber Immunoassay method employing monoclonal antibody reactive to human iNOS
EP0864648A1 (fr) * 1996-09-20 1998-09-16 Suntory Limited Procede de tri de composes regulateurs de l'expression de la synthase induisant chez l'homme la production d'acide nitrique
EP0864648A4 (fr) * 1996-09-20 2002-10-30 Suntory Ltd Procede de tri de composes regulateurs de l'expression de la synthase induisant chez l'homme la production d'acide nitrique
US6160000A (en) * 1996-12-23 2000-12-12 Merck & Co., Inc. Antidiabetic agents based on aryl and heteroarylacetic acids
US6090839A (en) * 1996-12-23 2000-07-18 Merck & Co., Inc. Antidiabetic agents
FR2793142A1 (fr) * 1999-05-04 2000-11-10 Aventis Pharma Sa Utilisation d'oligonucleotides antisens de no-synthase inductible dans la prevention et le traitement de l'ischemie cerebrale
WO2000066725A1 (fr) * 1999-05-04 2000-11-09 Aventis Pharma S.A. Utilisation d'oligonucleotides antisens de no-synthase inductible dans la prevention et le traitement de l'ischemie cerebrale
EP1250157A1 (fr) * 2000-01-24 2002-10-23 Isis Pharmaceuticals, Inc. Modulation antisens de l'expression de l'oxyde nitrique synthase inductible
EP1250157A4 (fr) * 2000-01-24 2004-12-29 Isis Pharmaceuticals Inc Modulation antisens de l'expression de l'oxyde nitrique synthase inductible
WO2001066791A1 (fr) * 2000-03-10 2001-09-13 Fujisawa Pharmaceutical Co., Ltd. Criblage de l'inhibiteur d'activation de la synthetase d'oxyde nitrique inductible

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