WO1998035055A1 - Method for identifying selective inhibitors of nitric oxide synthase - Google Patents
Method for identifying selective inhibitors of nitric oxide synthase Download PDFInfo
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- WO1998035055A1 WO1998035055A1 PCT/GB1998/000353 GB9800353W WO9835055A1 WO 1998035055 A1 WO1998035055 A1 WO 1998035055A1 GB 9800353 W GB9800353 W GB 9800353W WO 9835055 A1 WO9835055 A1 WO 9835055A1
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- C—CHEMISTRY; METALLURGY
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- 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
- C12Q1/26—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase
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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
Definitions
- the present invention relates to methods for identifying compounds which selectively inhibit nitric oxide synthase (NOS). It also relates to the use of such compounds in methods of treatment.
- NOS nitric oxide synthase
- NO nitric oxide
- iNOS inducible NOS
- eNOS endothelial NOS
- nNOS neuronal NOS
- iNOS In rodent cells, expression of iNOS occurs readily in vascular smooth muscle cells in response to bacterial endotoxin or certain inflammatory cytokines and largely accounts for the hypotension seen in experimental septic shock. Despite the evidence for involvement of iNOS in rodent models, reproducible and consistent induction of functionally active iNOS in human vascular cells and tissues in vitro has not been achieved.
- the present invention is based on the surprising finding that cytokine induced vasodilatation in humans caused by an increase in NO generation is due to activation of eNOS and not induction of iNOS. This phenomenon appears to be as a result of transcriptional regulation of GTP cyclohydrolase 1 leading to an increase in levels of the pterin tetrahydrobiopterin (BH 4 ), a co-factor for all three known isoforms of NOS. The increase in levels of BH 4 in turn leads to increased eNOS activity.
- the present invention provides a method for identifying a compound which selectively inhibits a nitric oxide synthase (NOS) which method comprises: (a) determining the inhibition of the activity of the NOS in the presence of a candidate compound and a first concentration of BH 4 ; (b) determining the inhibition of the activity of the NOS in the presence of the candidate compound and a second concentration BH 4 ; (c) determining whether the candidate compound inhibits the activity of the NOS to a different extent in (a) than in (b).
- NOS nitric oxide synthase
- step (a) comprises: (i) determining the activity of said NOS in the absence of the candidate compound and in the presence of a first concentration of BH 4 ; (ii) determining the activity of said NOS in the presence of the candidate compound and the first concentration of BH 4 ; (iii) comparing the activity of said NOS in steps (i) and (ii).
- step (b) comprises: (i) determining the activity of said NOS in the absence of the candidate compound and in the presence of a second concentration of BH 4 ; (ii) determining the activity of said NOS in the presence of the candidate compound and the second concentration of BH 4 ; (iii) comparing the activity of said NOS in steps (i) and (ii).
- the second concentration of BF£ 4 used in step (b) is preferably lower than the first concentration of BF£ 4 used in step (a).
- the NOS is preferably a human NOS, more preferably human eNOS.
- nitric oxide synthase activity may be used to treat a variety of conditions in which overproduction of nitric oxide has been implicated, for example septic shock, asthma, arthritis, inflammatory bowel disease, heart failure and acute systemic inflammation.
- this approach might be of value in the treatment of neurological disease states in which nNOS has been implicated (for example, strokes, dementia and Parkinsons disease).
- the present invention also provides the use of a compound identified by the method of the invention in the manufacture of a medicament for use in selectively inhibiting a human nitric oxide synthase.
- the present invention further provides a pharmaceutical composition
- a pharmaceutical composition comprising a compound identified by the method of the invention together with a pharmaceutically acceptable carrier or diluent.
- Cytokine-induced stimulation of NO production by NOS appears to be mediated via an increase in the activity of GTP cyclohydrolase 1 which is the rate-limiting enzyme in the synthesis of BH 4 . Since BH 4 is required for NOS activity, and therefore increases in
- BH 4 levels in response to cytokines result in increased NOS activity
- inhibition of GTP cyclohydrolase activity may prevent or reduce NO production by any isoform of NOS.
- the present invention further provides the use of a compound in the manufacture of a medicament for use in inhibiting a human nitric oxide synthase wherein said compound is capable of inhibiting or reducing synthesis of BH 4 by GTP cyclohydrolase 1.
- the compound is identified by an assay method which method comprises:
- NOS nitric oxide synthase
- the phrase "nitric oxide synthase” is intended to include all naturally occuring forms of eNOS, iNOS and nNOS as well as variants which retain NOS activity, for example functional forms of iNOS encoded by additional copies of the iNOS gene or variants produced by mutagenesis techniques.
- the NOS is preferably of mammalian origin, for example rodent (including rat and mouse) or primate (such as human).
- the NOS is of human origin.
- NOS used in the assays may be obtained from mammal cellular extracts or produced recombinantly from, for example, bacteria, yeast or higher eukaryotic cells including mammalian cell lines and insect cell lines.
- NOS used in the assays is recombinant (see, for example, Charles et al, 1996).
- Suitable candidate compounds may include analogues of arginine, aminoguanidine or analogues thereof, guanidine compounds, isothiourea, derivatives of L-N 6 - (l-iminoethyl)lysine (L-NMMA), 2-nitroaryl and 2-cyanoaryl compounds or ⁇ -(S-methylisothioureido)-L-norvaline.
- the inventors have found that the sensitivity of NOS to different inhibitors may vary depending on the physiological pterin concentration. This may be due to an allosteric effect on the conformation of NOS resulting in a BH 4 -modif ⁇ ed isoform.
- the inventors have found that the rise in physiological BH 4 levels caused by cytokine/endotoxin stimulation increases the sensitivity of eNOS to aminoguanidine. Therefore to provide maximum specificity and clinical efficacy it is very important to screen for potential inhibitors, for use in therapeutic methods, at a physiologically or pathophysiologically relevant pterin concentration.
- the principle behind the assay methodologies is to screen for compounds which inhibit a particular NOS isoform at a particular physiologically or pathophysiologically relevant concentration or concentration range of BH 4 but which have a reduced inhibitory effect at other concentrations.
- the human eNOS isoform which is present at normal BH 4 concentrations is essential for maintaining homeostatic pressure. Any compound which dramatically inhibits this isoform will have a potentially deleterious effect on the patient.
- eNOS is converted into a form, possibly due to an allosteric conformational change, which is responsible for generating the NO which causes refractory hypotension. It is this latter form which needs to be targeted.
- a suitable inhibitor would inhibit eNOS at the pathophysiological concentration of BH 4 present as a result of the action of, for example, inflammatory cytokines but would not inhibit eNOS at normal BH 4 concentrations.
- Step (a) of the method of the invention comprises determining the inhibition of NOS activity by the candidate compound in the presence of a first concentration of BH 4 .
- the amount of NOS used in the assay has a specific activity of from 10 to 600 pmol/min.
- the concentration of BH 4 used in the assay is from 1 ⁇ M to 1000 ⁇ M, preferably from 10 ⁇ M to 1000 ⁇ M, more preferably from 100 ⁇ M to 1000 ⁇ M or 500 ⁇ M to 1000 ⁇ M, most preferably from 500 ⁇ M to 1000 ⁇ M.
- the concentration of candidate compound used in the assay is from 1 ⁇ M to 1000 ⁇ M, preferably from 1 ⁇ M to 100 ⁇ M, more preferably from 1 ⁇ M to 10 ⁇ M.
- inhibition, if any, of NOS activity is determined by firstly measuring NOS activity in the absence of the candidate compound but in the presence of the first concentration of BH 4 . This provides a control value for NOS activity under the assay conditions. Secondly, NOS activity is measured in the presence of both the candidate compound and the first concentration of BH 4 . The inhibition of NOS activity due to the candidate compound can be determined by comparing the control value with the value obtained in the presence of the candidate compound. These assay steps are conducted by contacting NOS with the candidate compound and/or BH 4 where appropriate. The extent and/or the rate of reaction can be measured quantitatively, semi-quantitatively or qualitatively. If, for example, a fast throughput screening assay format is required, the control reaction in the absence of the candidate compound need not be performed for every candidate compound tested.
- NOS activity can be measured in a number of ways (see Packer, 1996). For example, NOS activity can be measured directly by conversion of radiolabelled arginine to citrulline or indirectly by assaying for NO production. Assays for NO include chemical assays or bioassays. A typical chemical assay for NO utilises the change in absorbance at 405 nm and 420 nm when NO oxidises oxyhaemoglobin to methemoglobin.
- the reaction mixture comprises: 3 ⁇ M oxyhaemoglobin, 200 ⁇ M CaCl 2 (for eNOS and nNOS), 1 mM MgCl 2 , 1 ⁇ M FAD, 1 ⁇ M FMN, 100 ⁇ M NADPH, 0.1 ⁇ M calmodulin, 30 ⁇ M L-arginine (substrate for NOS), from 1 ⁇ M to 1000 ⁇ M BH 4 and 100 ⁇ M DTT; in 100 mM HEPES buffer, pH 7.4 and in a final reaction volume of 250 ⁇ l (Charles et al, 1996).
- This assay is suitable for use in a microtitre plate format.
- Step (b) of the method of the invention comprises determining the extent to which the activity of the NOS is inhibited by the candidate compound in the presence of a second concentration of BH 4 .
- This step is similar to step (a) except that the concentration of BH 4 used is different to that used in step (a), preferably lower.
- the amount of NOS used in the assay has a specific activity of from 10 to 600 pmol/min.
- the second concentration of BH 4 used in the assay is from 1 ⁇ M to 1000 ⁇ M, preferably from 1 ⁇ M to 100 ⁇ M, more preferably from 1 ⁇ M to 10 ⁇ M.
- the concentration of candidate compound used in the assay is from 1 ⁇ M to 1000 ⁇ M, preferably from 1 ⁇ M to 100 ⁇ M, more preferably from 1 ⁇ M to 10 ⁇ M.
- the steady-state inhibition is determined after between 15 to 30 minutes incubation.
- Step (c) of the method of the invention comprises determining whether the candidate compound inhibits the NOS activity to a different extent in (a) than in (b).
- a candidate compound may inhibit eNOS by 70% in the presence of a high concentration of BH 4 , but only by 5% in the presence of low concentrations of BH 4 .
- Such a compound would be a useful therapeutic agent because it does not inhibit the normal physiological form of eNOS involved in maintaining homeostatic pressure, but does inhibit the isoform of eNOS which is generated in the presence of high levels of BH 4 caused by inflammatory cytokines/endotoxins.
- NOS activity in the presence of an inhibitor is dependent on
- the inhibitor is preferably selective with respect to different isoforms of NOS, for example eNOS, iNOS and nNOS, whether from the same organism or different organisms.
- an inhibitor may inhibit human eNOS but not human iNOS.
- inhibition of eNOS activity at a higher level of BH 4 by an inhibitor compound is typically at least 20%, preferably at least 30%, more preferably at least 40, 50, 75, 90 or 95% greater than the inhibition of eNOS activity by the inhibitor compound at a lower level of BH 4 .
- concentrations typically envisaged for the higher and lower levels of BH 4 are discussed above.
- assays may include in vivo assays such as those described in WO95/34534.
- the inhibition of eNOS and iNOS in situ in rat aortic rings can assessed by measuring the increases in ring tension caused by NO synthase inhibition.
- endothelium-denuded rings are typically exposed to LPS (0.1 ⁇ g/ml from S.
- eNOS and iNOS in vivo can also be assessed by the effects of inhibitors on blood pressure in either normal or endotoxin shocked conscious mice. For example, one suitable method is as follows. Mice are anaesthetised briefly with isoflourane (2%).
- Cannula lines are implanted in a femoral vein, tunnelled subcutaneously to exit at the top of the back and connected to a swivel tether system for continuous monitoring of blood pressure and for inhibitor administration respectively.
- animals with mean blood pressures in the normal range 90-110 mm Hg are used to obtain cumulative concentration curves for inhibitors on blood pressure either without further treatment ("normal mice") or 7 h after administration of lipopolysaccharide (12.5 mg/kg of LPS from E. coli 026:B6 intravenously over 30 s) to induce shock (“shocked mice”).
- lipopolysaccharide 12.5 mg/kg of LPS from E. coli 026:B6 intravenously over 30 s
- cytokine-induced stimulation of NO production by NOS appears to be mediated via an increase in the activity of GTP cyclohydrolase 1 which is the rate-limiting enzyme in the synthesis of BH 4 . Since BH 4 is required for NOS activity, and therefore increases in BH 4 levels in response to cytokines result in increased NOS activity, inhibition of GTP cyclohydrolase activity may prevent or reduce NO production by NOS. Thus compounds which inhibit GTP cyclohydrolase 1 activity may also inhibit NOS activity as a result of the inhibition or reduction in BH 4 production.
- inhibition, if any, of GTP cyclohydrolase 1 is determined by firstly measuring GTP cyclohydrolase 1 activity in the absence of the candidate compound. This provides a control value for GTP cyclohydrolase 1 activity under the assay conditions. Secondly, GTP cyclohydrolase 1 activity is measured in the presence of the candidate compound. The inhibition of GTP cyclohydrolase 1 activity due to the candidate compound can be determined by comparing the control value with the value obtained in the presence of the candidate compound. The assays are conducted by contacting GTP cyclohydrolase 1 with the candidate compound. The extent and/or the rate of reaction can be measured quantitatively, semi-quantitatively or qualitatively.
- the control reaction in the absence of the candidate compound need not be performed for every candidate compound tested.
- the amount of GTP cyclohydrolase 1 used in the assay is from 1 to 10 ng of pure protein.
- the concentration of candidate compound used in the assay is from 1 ⁇ M to 1000 ⁇ M, preferably from 1 ⁇ M to 500 ⁇ M or 1 ⁇ M to 100 ⁇ M, more preferably from 1 ⁇ M to 10 ⁇ M.
- GTP cyclohydrolase 1 activity can be measured, for example, by assessing the production of BH 4 or GTP consumption.
- BH 4 production can be determined, for example, by oxidation to biopterin using iodine and measurement of biopterin using HPLC (mobile phase is 5% methanol run at 1.5 ml/min on a 10 micron ODS reverse phase column (4.6 mm x 25 cm) with excitation at 350 nm and emission at 410 nm).
- nitric oxide synthases can lead to potentially fatal vasodilatation and hypotension.
- Selective inhibition of nitric oxide synthases by a compound identified by the method of the invention may be useful in alleviating this potentially fatal response.
- Compounds identified by the method of the invention may be used therapeutically to treat conditions in which over-production of NO is implicated, for example septic shock, asthma, rheumatoid arthritis, inflammatory bowel disease, heart failure or acute systemic inflammation. NOS is also implicated in various neurological disease states including strokes, dementia and Parkinsons disease.
- compounds which inhibit or reduce synthesis of BH 4 by GTP cyclohydrolase 1 may also be used therapeutically to treat the conditions described above in which NOS is implicated.
- the compound is used to treat diseases in which eNOS is implicated.
- the formulation will depend upon the nature of the compound identified but typically the compounds may be formulated for clinical administration by mixing them with a pharmaceutically acceptable carrier or diluent.
- a pharmaceutically acceptable carrier or diluent for example they can be formulated for topical, parenteral, intravenous, intramuscular, subcutaneous, intraocular or transdermal administration.
- the compound is used in an injectable form. It may therefore be mixed with any vehicle which is pharmaceutically acceptable for an injectable formulation, preferably for a direct injection at the site to be treated.
- the pharmaceutically carrier or diluent may be, for example, sterile or isotonic solutions. It is also preferred to formulate that compound in an orally active form.
- the dose of compound used may be adjusted according to various parameters, especially according to the compound used, the age, weight and condition of the patient to be treated, the mode of administration used and the required clinical regimen.
- the amount of compound administered by injection is suitably from 0.01 mg/kg to 30 mg/kg, preferably from 0.1 mg/kg to 10 mg/kg.
- FIG. 2 Panel A - Two adjacent vessels were studied simultaneously and at a distension pressure of 40 mmHg.
- the sympathetic nervous system was activated by asking subjects to take a deep breath. This manoeuvre produced transient constriction in both vessels. Incubation of one vessel (lower trace) with IL-l ⁇ virtually abolished sympathetically mediated constriction 6 h later. Sympathetic constriction was restored by infusion of L-NMMA. Congesting cuff deflation and re-inflation is marked by solid circle.
- Panel B Traces showing effects of L-NMMA on basal vessel tone in control vessel (upper trace) and vein treated with IL- l ⁇ (lower trace). The effects of L-NMMA were reversed by L-arginine.
- Panel B - Original trace showing effects of BH 4 infusion (upper trace) and reversal by aminoguanidine (lower trace). Congesting cuff deflation and re-inflation is marked by a solid circle.
- Figure 4 - PCR reactions were carried out in 20 ⁇ l volumes, and 5 ⁇ l samples analysed by electrophoresis through 2% agarose gels.
- Panel A shows the result with the iNOS primer pair BB52 and BB71.
- Track 1 size markers tracks 2 & 3 niRNA from uninduced hand vein; track 4 and 5 mRNA from cytokine-induced hand vein; track 6 positive control (cytokine-induced CAPAN-1 mRNA).
- a PCR product of 499 bp is only visible in track 6.
- Panel B shows the result of PCR amplification with the eNOS specific primer pair
- Panel C shows the ⁇ -actin specific PCR products from the primer set BB27 and BB28.
- Traces 1-4 are the hand vein mRNA samples amplified with an initial reverse transcriptase step.
- Tracks 5-8 are negative controls, amplified in the absence of a reverse transcriptase step.
- Track 9 is a positive control using, placental mRNA.
- Track 10 is size markers. Bands corresponding to ⁇ -actin can be detected in tracks 1-4 confirming that mRNA had been isolated from hand vein biopsies.
- the volume of blood in the isolated vein is in the order of 1-2 ml and the calculated concentration of cytokine was in the order of 300-1000 pg/ml (TNF ⁇ and IL-l ⁇ ) and 30-100 pg/ml (IL-6).
- RT-PCR Reverse transcriptase polymerase chain reaction
- Poly A mRNA extracted from hand veins using Fast Track reagents (InvitrogenTM).
- Positive control poly A + mRNA for eNOS was carried out on human placental preparations (ClontechTM).
- Positive controls for iNOS and GTPCH-1 were carried out on poly A + mRNA isolated from the human pancreatic adenocarcinoma line CAPAN-1 stimulated with cytokines. The following primer sets were used:
- GTPCH-1 igcl 5'-ttggttatcttcctaacaag-3' igc2 5'-gtgctggtcacagttttgct-3'
- PCR was carried out in thin-walled tubes using a Gene AmpTM RT PCR kit (Perkin-ElmerTM) in a Gene Amp PCR system 9600 thermocycler (Perkin-Elmer). The following conditions were used: 95°C for lmin 45s, followed by 30 cycles of 95°C, 15s, 60°C, 30s. An elongation step of 72°C was carried out for 7 min at the end of the cycle.
- tumour necrosis factor ⁇ TNF ⁇
- IL-6 100 pg, in 1 ml of saline
- co-instillation of these cytokines with IL-l ⁇ (1 ng/ml) for 1 h caused a prolonged (>24 h) attenuation of the response to noradrenaline ( Figure lb).
- eNOS is sensitive to inhibition by an iNOS inhibitor, aminoguanidine. in the presence of
- aminoguanidine was inhibited by aminoguanidine.
- the ability of aminoguanidine to inhibit eNOS activity is dependent on the prevailing concentration of BH 4 .
- IL-l ⁇ induces expression of GTP cyclohydrolase 1 leading to increased generation of BH,.
- the rate limiting enzyme for BH 4 synthesis is GTP cyclohydrolase 1.
- This enzyme is transcriptionally regulated in response to cytokines or endotoxin, and its induction can be prevented by glucocorticoids. It is clear, therefore that the pharmacological changes we observed in the hand vein in vivo might be explained equally well by activation of eNOS by BH 4 synthesised following induction of GTP cyclohydrolase 1, as by induction of iNOS.
- To explore further the mechanism of the functional induction of NOS activity we excised a portion of hand vein. As expected, mRNA encoding eNOS was detected in all of the samples ( Figure 4).
- eNOS is an endothelial enzyme that has not been detected in smooth muscle cells.
- An implication of our findings is that the endothelium can generate sufficient NO to cause profound vasodilatation and that expression of NOS throughout the much larger smooth muscle cell layer is not necessary to cause septic hypotension.
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Priority Applications (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
AU59962/98A AU5996298A (en) | 1997-02-05 | 1998-02-05 | Method for identifying selective inhibitors of nitric oxide synthase |
JP53399198A JP2001511009A (en) | 1997-02-05 | 1998-02-05 | Methods for identifying selective inhibitors of nitric oxide synthase |
EP98903134A EP0961835A1 (en) | 1997-02-05 | 1998-02-05 | Method for identifying selective inhibitors of nitric oxide synthase |
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GB9702312.1 | 1997-02-05 | ||
GBGB9702312.1A GB9702312D0 (en) | 1997-02-05 | 1997-02-05 | Method for identifying selective inhibitors of nitric oxide synthase |
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WO1998035055A1 true WO1998035055A1 (en) | 1998-08-13 |
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PCT/GB1998/000353 WO1998035055A1 (en) | 1997-02-05 | 1998-02-05 | Method for identifying selective inhibitors of nitric oxide synthase |
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EP (1) | EP0961835A1 (en) |
JP (1) | JP2001511009A (en) |
AU (1) | AU5996298A (en) |
GB (1) | GB9702312D0 (en) |
WO (1) | WO1998035055A1 (en) |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1999059566A1 (en) * | 1998-05-15 | 1999-11-25 | Glaxo Group Limited | Use of nitric oxide synthase inhibitors in the manufacture of a medicament for the prophylaxis or treatment of bacterial infection |
US7906520B2 (en) | 2003-11-13 | 2011-03-15 | The General Hospital Corporation | Methods for treating pain |
Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1995013803A1 (en) * | 1993-11-15 | 1995-05-26 | Cornell Research Foundation, Inc. | Blocking induction of tetrahydrobiopterin to block induction of nitric oxide synthesis |
-
1997
- 1997-02-05 GB GBGB9702312.1A patent/GB9702312D0/en active Pending
-
1998
- 1998-02-05 AU AU59962/98A patent/AU5996298A/en not_active Abandoned
- 1998-02-05 JP JP53399198A patent/JP2001511009A/en active Pending
- 1998-02-05 WO PCT/GB1998/000353 patent/WO1998035055A1/en not_active Application Discontinuation
- 1998-02-05 EP EP98903134A patent/EP0961835A1/en not_active Withdrawn
Patent Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1995013803A1 (en) * | 1993-11-15 | 1995-05-26 | Cornell Research Foundation, Inc. | Blocking induction of tetrahydrobiopterin to block induction of nitric oxide synthesis |
Non-Patent Citations (5)
Title |
---|
A.C.F. GORREN, B.M. LIST, A. SCHRAMMEL, E. PITTERS, B. HEMMENS, E.R. WERNER, K. SCHMIDT, B. MAYER: "Tetrahydrobiopterin-Free Neuronal Nitric Oxide Synthase: Evidence for Two Indentical Highly Anticooperative Pteridine Binding Sites", BIOCHEMISTRY, vol. 35, no. 51, 1996, pages 16735 - 16745, XP002066036 * |
B. M. LIST, P. KLATT, E. R. WERNER, K. SCHMIDT, B. MAYER: "Overexpression of neuronal nitric oxide synthase in insect cells reveals requirement of haem for tetrahydrobiopterin binding", BIOCHEMICAL JOURNAL, vol. 315, no. 1, 1 April 1996 (1996-04-01), pages 57 - 63, XP002066037 * |
G. WERNER-FELMAYER, E.R. WERNER, D. FUCHS, A. HAUSEN, G. REIBNEGGER, K. SCHMIDT, G. WEISS, H. WACHTER: "Pteridine Biosynthesis in Human Endothelial Cells", JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 268, no. 3, 25 January 1993 (1993-01-25), pages 1842 - 1846, XP002066039 * |
P. KLATT, K. SCHMIDT, G. URAY, B. MAYER: "Multiple Catalytic Functions of Brain Nitric Oxide Synthase", JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 268, no. 20, 20 July 1993 (1993-07-20), pages 14781 - 14787, XP002066038 * |
P. KLATT, M. SCHMID, E. LEOPOLD, K. SCHMIDT, E. R. WERNER, B. MAYER: "The Pteridine Binding Site of Brain Nitric Oxide Synthase", JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 269, no. 19, 13 May 1994 (1994-05-13), pages 13861 - 13866, XP002066035 * |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1999059566A1 (en) * | 1998-05-15 | 1999-11-25 | Glaxo Group Limited | Use of nitric oxide synthase inhibitors in the manufacture of a medicament for the prophylaxis or treatment of bacterial infection |
US7906520B2 (en) | 2003-11-13 | 2011-03-15 | The General Hospital Corporation | Methods for treating pain |
Also Published As
Publication number | Publication date |
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GB9702312D0 (en) | 1997-03-26 |
JP2001511009A (en) | 2001-08-07 |
AU5996298A (en) | 1998-08-26 |
EP0961835A1 (en) | 1999-12-08 |
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