JP2001511009A - Methods for identifying selective inhibitors of nitric oxide synthase - Google Patents

Abstract

(57) a method for identifying compounds that selectively inhibit the [Summary] Human nitric oxide synthase (NOS) is measuring the inhibition of (a) a candidate compound and NOS activity in the presence of a first concentration of tetrahydrobiopterin BH ₄ to it; (b) measuring the inhibition of NOS activity in the presence of a second concentration of the candidate compound and BH _4; (c) in the candidate compound (a) NOS activity to a different extent than in (b) Deciding whether or not to inhibit

Description

DETAILED DESCRIPTION OF THE INVENTION   Methods for identifying selective inhibitors of nitric oxide synthaseField of the invention   The present invention provides a method for identifying compounds that selectively inhibit nitric oxide synthase (NOS). About the law. It also relates to the use of such compounds in therapy.Background of the Invention   Septic shock is refractory hypotension, with over 100,000 live births in the United States each year. Take your life. Vasodilation is a ubiquitous and conservative counterpart of the cardiovascular system against acute systemic inflammation. And is the leading cause of hypotension associated with septic shock. Nitric oxide (N Overproduction of O) is associated with the pathogenesis of septic shock in both rodents and humans. Related to the business. NO synthesis is catalyzed by the enzyme nitric oxide synthase (NOS). Medium. There are three known types of mammalian NOS: inducible NOS (iNOS), endothelial NOS (eNOS) and neuronal NOS (nNOS).   In rodent cells, iNOS expression is controlled by bacterial endotoxin or certain inflammatory cytokines. Occurs easily in vascular smooth muscle cells in response to and is seen in experimental septic shock Is a major reason for hypotension. INOS is involved in rodent models Despite evidence, the renewal of functionally active iNOS in human vascular cells and tissues No persistent persistence induction has been achieved in vitro.   NO overproduction is associated with the pathophysiology of septic shock in humans, and iNOS is But iNOS is directly expressed in the vasculature of patients with sepsis. There is no proof. In addition, plasma concentrations of NOx in patients with sepsis were found in rodent models. This is about a 0.5-fold increase compared to a 10-fold increase. These observations were made on different species Is supported by molecular evidence for important functional differences between iNOS.Summary of the Invention   The present invention induces cytokines in humans caused by increased NO production Vasodilation is due to activation of eNOS and not to induction of iNOS Based on the surprising discovery that no. This phenomenon is related to the three known isoforms of NOS. Pterin, tetrahydrobiopterin (BH_Four)Of level Appears to be a consequence of transcriptional regulation of GTP cyclohydrolase 1 leading to an increase You. BH_FourIncreased levels, in turn, result in increased eNOS activity.   Physiological pathway of NO production by NOSs depends on changes in pterin concentration The finding that it can be adjusted is defeated Specific for designing drugs to treat bloody hypotension and inflammatory vasodilation Has a great suggestion. It also helps to reduce excess NO during systemic inflammation in humans. Extensive inflammatory or tumors with multiple cell types as well as altered pterin synthesis It may be more commonly associated with pathways generated in ulcerative conditions.   Accordingly, the present invention provides compounds that selectively inhibit nitric oxide synthase (NOS). Providing a method of determining (a) Candidate compound and BH_FourMeasuring inhibition of NOS activity in the presence of a first concentration of (b) Candidate compound and BH_FourMeasuring inhibition of NOS activity in the presence of a second concentration of (c) the candidate compound inhibits NOS activity to a different extent in (a) than in (b) To decide whether to do   Is included.   Preferably, step (a) comprises: (i) In the absence of candidate compound and BH_FourThe NOS activity in the presence of a first concentration of That; (ii) Candidate compound and BH_FourMeasuring the NOS activity in the presence of a first concentration of (iii) comparing the NOS activity in steps (i) and (ii),   Is included.   Preferably, step (b) comprises: (i) In the absence of candidate compound and BH_FourThe NOS activity in the presence of a second concentration of That; (ii) Candidate compound and BH_FourMeasuring the NOS activity in the presence of a second concentration of: (iii) comparing the NOS activity in steps (i) and (ii),   Is included.   BH used in step (b)_FourOf the BH used in step (a)_Four Is lower than the first concentration. NOS is preferably human NOS, more preferably human eNOS. is there.   Compounds identified by the method of the invention that selectively inhibit nitric oxide synthase activity Is associated with various conditions involving overproduction of nitric oxide, such as septic shock, asthma Used to treat arthritis, inflammatory bowel disease, heart failure and acute systemic inflammation obtain. In addition, this approach can be used to address nNOS-related neurological disease states (e.g., , Seizures, dementia and Parkinson's disease) may be of value. Therefore The present invention relates to a medicament for use in selectively inhibiting human nitric oxide synthase. Also provided is the use of a compound identified by the method of the invention in the construction.   The present invention further provides a compound identified by the method of the present invention as a pharmaceutically acceptable carrier. There is provided a pharmaceutical composition comprising the body or a diluent.   The stimulation induced by cytokines of NO production by NOS is BH_FourThe rate-limiting enzyme in synthesis It appears to be mediated through increased GTP cyclohydrolase 1 activity. BH_Four Is required for NOS activity and therefore BH in response to cytokines_FourLevel increase Inhibition of GTP cyclohydrolase activity is responsible for NOS, as it results in increased NOS activity. It may prevent or reduce NO production by any of the isoforms.   Accordingly, the present invention is further used to inhibit human nitric oxide synthase. There is provided the use of a compound in the manufacture of a medicament, wherein the compound comprises a GTP cyclohydride BH by Lolase 1_FourSynthesis can be prevented or reduced. Preferably, the compound is (i) contacting GTP cyclohydrolase 1 with a candidate compound; and (ii) BH mediated by GTP cyclohydrolase by the compound_FourDetermining synthesis inhibition That   Identified by an assay method, including:Detailed description of the invention   The term “nitric oxide synthase” (NOS) refers to eNOS, iNOS and And all naturally occurring forms of nNOS, as well as variants that retain NOS activity, such as The functional form or mutation of iNOS encoded by a further copy of the iNOS gene It is intended to include variants produced by the mutagenesis technique. NOS preferred Or of mammalian origin, such as rodents (including rats and mice) or Primates (such as humans). Preferably, the NOS is of human origin. The NOS used in the assay may be obtained from mammalian cell extracts or may be recombinant. Specifically, for example, bacteria, yeast or higher, including mammalian and insect cell lines. It can be produced from eukaryotic cells. Preferably, the NOS used in the assay is recombinant (See, eg, Charles et al., 1996).   Suitable candidate compounds are arginine analogs, aminoguanidines or Analogue, guanidine compound, isothiourea, L-N⁶-(1-Iminoethyl) lithi (L-NMMA), 2-nitroaryl and 2-cyanoaryl compounds or δ- (S-methyl (Lisothioureido) -L-norvaline.Assay methodology   The inventors have shown that the sensitivity of NOS to different inhibitors Discover that they can change depending on Was. This is BH_FourAllostery for the conformation of NOS resulting in modified isoforms It may be due to the lock effect. In particular, as in the embodiment, the inventor Physiological BH caused by stimulation of endotoxin_FourIncreased levels are eNOS It has been found to increase sensitivity to noguanidine. Therefore, the largest singular Potential for use in treatment methods to provide clinical and clinical benefits. Inhibitors at physiologically or pathophysiologically relevant concentrations of pterin. Is very important.   The principles behind the assay methodology are relevant for certain physiological or pathophysiological BH_FourInhibits specific NOS isoforms at concentrations or concentration ranges, but decreases at other concentrations Screening for compounds having the above-mentioned inhibitory effect. According to the embodiment , Normal BH_FourHuman eNOS isoforms present at concentrations are essential for maintaining constant pressure It is. Any compound that dramatically inhibits this isoform is potentially harmful to the patient It has a great effect. However, for example, induced by inflammatory cytokines Increased BH_FourAt the concentration, eNOS is probably an allosteric conformational change Is converted to a form by the change, which is responsible for NO production that results in intractable hypotension. You. What needs to be targeted is This is the latter form. Thus, suitable inhibitors include, for example, inflammatory cytokines BH exists as a consequence of the action of_FourWill inhibit eNOS at pathophysiological concentrations But normal BH_FourConcentrations will not inhibit eNOS.   Step (a) of the method of the present invention comprises the step of BH_FourNOS activity by candidate compounds in the presence of a first concentration of Measuring the inhibition of sex. Typically, the amount of NOS used in the assay Has a specific activity of 10-600 pmol / min. Typically, the BH used in the assay_Four The concentration is 1 μM to 1000 μM, preferably 10 μM to 1000 μM, more preferably 100 μM to 1 μM. 000 μM or 500 μM to 1000 μM, most preferably 500 μM to 1000 μM. Typical The concentration of the candidate compound used in the assay is between 1 μM and 1000 μM, preferably 1 μM. It is from μM to 100 μM, more preferably from 1 μM to 100 μM.   Generally, inhibition of NOS activity, if any, will occur only in the absence of the candidate compound. BH_FourIs determined by first measuring NOS activity in the presence of a first concentration of This provides a control value for NOS activity under the assay conditions. next , NOS activity, candidate compounds and BH_FourIs measured in the presence of both first concentrations. Weather Inhibition of NOS activity by the complement compound provided a control value in the presence of the candidate compound. It can be measured by comparing with a given value. This These assay steps involve the conversion of NOS into candidate compounds and / or BH_FourWhen This is done by contact. The extent and / or rate of the reaction may be quantitative, It can be measured semi-quantitatively or qualitatively. If, for example, rapid throughput If a cleaning assay format is required, control in the absence of candidate compounds Roll reactions need not be performed for every candidate compound to be tested.   NOS activity can be measured in a number of ways (see Packer, 1996). For example, NOS activity Sex is directly or directly converted from radiolabeled arginine to citrulline. Alternatively, it can be measured indirectly by assaying NO production. NO assay Includes chemical or bioassays. A typical chemical assay for NO is When NO oxidizes oxyhemoglobin to methemoglobin, 405 nm and 420 nm The change in absorbance at is used. Typically, the reaction mixture contains 3 μM oxyhemoglobin. Bottle, 200 μM CaCl_Two(for eNOS and nNOS), 1 mM MgCl_Two, 1μM FAD, 1μM FM N, 100 μM NADPH, 0.1 μM calmodulin, 30 μM L-arginine (NOS substrate), 1 μM to 1000 μM BH_FourAnd 100 μM DTT; in 100 mM HEPES buffer, pH 7.4, and 250 In a final reaction volume of μl (Charles et al., 1996). This assay uses micro Is used in the plate format Suitable for.   All three isoforms have activity for L-arginine, NADPH, FMN, FAD, BH_Four And calmodulin. eNOS and nNOS also require calcium (Charles et al., 1996).   In step (b) of the method of the present invention, the NOS activity is BH_FourIn the presence of a second concentration of Measuring the degree of more inhibition. This process is used for BH_Fourof The concentration should be different and preferably lower than that used in step (a). Is the same as step (a), with the exception of Typically, the NOS used in the assay The amount has a specific activity of 10-600 pmol / min. Typically, the BH used in the assay_Four The second concentration of 1 μM to 1000 μM, preferably 1 μM to 100 μM, more preferably 1 μM 1010μ. Typically, the concentration of the candidate compound used in the assay is between 1 μM and It is 1000 μM, preferably 1 μM to 100 μM, more preferably 1 μM to 10 μM. General Alternatively, steady-state inhibition is measured after 15-30 minutes of incubation.   In step (c) of the method of the present invention, the candidate compound exhibits NOS activity, Also determine whether they inhibit to different degrees. This is the control Inhibition in step (a) as shown by comparing the Is the degree of inhibition in step (b), which is also indicated by comparing the control value with the second value. Can be achieved by simply comparing Thus, for example, a candidate compound is eNOS with high concentration of BH_FourCan inhibit 70% in the presence of_FourIn the presence of Inhibits only 5%. Such a compound may not be able to maintain a constant pressure. Does not inhibit the normal physiological form of the associated eNOS, but is an inflammatory cytokine / endotoxin High levels of BH caused by elementary_FourENOS isoforms produced in the presence of Would be a useful therapeutic agent.   In other words, NOS activity in the presence of an inhibitor_FourDepends on the concentration of ). The inhibitors are preferably from different NOS isoforms, e.g., from the same organism Or eNOS, iNOS and nNOS from different organisms. For example, Inhibitors can inhibit human eNOS but do not inhibit human iNOS.   Higher levels of BH due to examples of numerical form_FourActivity by inhibitory compounds in the environment Inhibition of lower levels of BH_FourThan inhibition of eNOS activity by inhibitory compounds at Typically, at least 20%, preferably at least 30%, more preferably less Both are 40, 50, 75, 90 or 95% higher. Typically higher and lower levels BH_FourIntended for Practical concentrations are discussed above.   Screen candidate compounds for NOS inhibition in the treatment of cardiovascular disease When using eNOS, it is preferred to use eNOS in the assay. Conversely, cure neurological disease NNOS when screening candidate compounds for NOS inhibition in therapy. It is preferable to use it in the assay.   Other types of assays are in vivo assays, such as those described in WO95 / 34534. Essays. For example, in situ eNOS in the rat aortic ring Inhibition of iNOS and iNOS, the ring tension caused by NO synthase inhibition It can be assessed by measuring the increase. Study basal tone To do so, thoracic aortic rings with intact endothelium were typically described in Rees et al. (1989). And the cumulative concentration curve was adjusted to the threshold concentration of phenylephrine (ED_Ten= 10nM ) In the presence of an inhibitor. To study the induced smooth muscle tone, Typically, about ED₉₀In the presence of phenylephrine as described in Rees et al. (1990). And exposed to LPS (0.1 μg / ml from S. thphosa) for 6 hours. During this time, the tension progressively Loss usually occurs due to iNOS induction. In general, the cumulative concentration curve Obtained against harmful agents.   In vivo inhibition of eNOS and iNOS is normal or internal. Effects of inhibitors on blood pressure in any conscious mice under toxic shock Can also be evaluated. For example, one suitable method is as follows. Mau The animals are briefly anesthetized with isoflourane (2%). Cannula Rye Implant into the femoral vein and create a subcutaneous tunnel to emerge at the top of the back. For continuous monitoring of blood pressure and for each dose of inhibitor. Connect to swivel tether system. Average blood after recovery from surgery Use animals with normal pressures (90-110 mmHg) for further treatment (`` `` Normal mice '') or lipopolysaccharide administration (E. coli  026: 7 hours after (12.5 mg / kg LPS from B6 intravenously over 30 seconds) (see Mice)) to obtain a cumulative concentration curve of the inhibitor on blood pressure . Inhibitors of NOS activity can be used in vivo (or in practice, as described in the Examples). Other suitable assays can be used to measure (in vitro), and And the above assays are examples.   The present inventors have found that the stimulation of cytokine-induced NO production by NOS_FourFor synthesis Mediated by increasing the activity of the rate-limiting enzyme GTP cyclohydrolase 1 I also found something that looked like. BH_FourIs required for NOS activity and BH in response to cytokines_FourSince increasing levels result in increased NOS activity, Inhibition of GTP cyclohydrolase activity can block or reduce NO production by NOS You. Thus, compounds that inhibit GTP cyclohydrolase 1 activity are BH_FourInhibition of production Or, as a consequence of the decrease, NOS activity may also be inhibited.   In general, inhibition of GTP cyclohydrolase 1, if any, Measured by first measuring GTP cyclohydrolase 1 activity in the presence . This provides control over GTP cyclohydrolase 1 activity under assay conditions. Provide the default value. Next, GTP cyclohydrolase 1 activity was measured in the presence of the candidate compound. Measure. Inhibition of GTP cyclohydrolase 1 activity by candidate compounds is controlled Values in the presence of the candidate compound can be determined by comparing . This assay involves contacting GTP cyclohydrolase 1 with a candidate compound. This is done. The extent and / or rate of the reaction may be quantitative, semi-quantitative or constant. It can be measured sexually. If, for example, a rapid throughput screening If an assay format is required, a control reaction in the absence of the candidate compound It need not be done for every candidate compound to be tested.   Typically, the amount of GTP cyclohydrolase 1 used in the assay is 1-10 ng Is a pure protein. Typically, the concentration of the candidate compound used in the assay Is 1 μM to 1000 μM, preferably 1 μM to 500 μM or 1 μM to 100 μM, more preferably Or 1 μM to 10 μM.   GTP cyclohydrolase 1 activity is, for example, BH_FourAssess production or GTP consumption And can be measured by: BH_FourProduction is, for example, in response to biopterin using iodine. Oxidation or HPLC (mobile phase 1.5 ml / min, 10 micron ODS reverse phase column (4.6 mm x 2 (5 cm) run with 5% methanol, with excitation at 350 nm and emission at 410 nm) Can be measured by biopterin measurement usingTherapeutic use   For example, overproduction of nitric oxide as a consequence of the effects of bacterial endotoxin is potentially lethal. It can lead to fatal vasodilation and hypotension. Chemicals identified by the method of the invention Selective inhibition of nitric oxide synthase by compounds slows this potentially fatal reaction. It may be useful to sum. The compound identified by the method of the present invention has an excess of NO Conditions associated with production, such as septic shock, asthma, rheumatoid arthritis, inflammation Can be used to treat symptomatic bowel disease, heart failure or acute systemic inflammation therapeutically . NOS, seizures, dementia And various neurological disease states, including Parkinson's disease.   Similarly, BTP by GTP cyclohydrolase 1_FourInhibiting or reducing synthesis The compounds can also be used to therapeutically treat the above conditions in which NOS is associated. Good Preferably, the compounds are used to treat diseases associated with eNOS.   The dosage form will depend on the nature of the compound being identified, but typically the compound will By mixing them with pharmaceutically acceptable carriers or diluents, Can be formulated. For example, they can be topical, parenteral, intravenous, intramuscular , May be formulated for subcutaneous, intraocular or transdermal administration. Preferably, the compound is Used in injectable form. It is therefore preferred for injectable dosage forms Is mixed with any pharmaceutically acceptable vehicle for direct injection at the site to be treated. Can be combined. The pharmaceutically acceptable carrier or diluent can be, for example, sterile or It can be a tonic solution. It is also preferred to formulate the compound in an orally active form .   The dose of the compound used depends on various parameters, in particular the The compound, the age, weight and condition of the patient being treated, the mode of administration used and requirements Required It can be adjusted according to clinical living regulations. As a guideline, administration by injection The amount of the compound is suitably 0.01 mg / kg to 30 mg / kg, preferably 0.1 mg / kg to 10 mg / kg. It is.   Routes and dosages described will be determined by the skilled practitioner for any particular patient. Guidelines and guidelines can be used to easily determine the optimal route and dosage for It is only intended.   The present invention is described with reference to the following examples, which are intended to be illustrative only and not limiting. Will be revealed. In the accompanying drawings: Figure 1-(a) IL-1β (n = 5) (b) IL-1β and TNFα (n = 5) or (c) IL-1β, TNFα and Dose response curves to noradrenaline before and after IL-6 (n = 5) (a) Dose response curves were obtained before (o) and before IL-1β (1 ng) instillation. Created against (b) Dose response curves were obtained prior to IL-1β and TNFα (1 ng) and IL-6 (100 pg) instillation, And made to noradrenaline after 1, 6, and 24 hours. (c) Dose response curves were IL-1β (1 ng) and TNFα (1 ng) and IL-6 (100 pg) instillation. Before, and 1, 6, and 24 Was. (d) Dose response curves were obtained before (o) and before IL-1β (1 ng) infusion. Was done. Repeated dose response 6 hours after IL-1β instillation And then flushed with saline for 15 minutes to produce noradrenaline. Dose-response curve for It was made by co-injection. Six hours after IL-1β instillation, a repeated dose response curve was developed using aminoguanidine (1 μmo l / min-x) with noradrenaline, followed by a saline wash For 15 minutes, repeated dose-response curve for noradrenaline It was made by co-injection. Figure 2-Panel A-Two adjacent vessels were studied simultaneously at an inflation pressure of 40 mm Hg. Exchange The sensory nervous system was activated by asking the subject to take a deep breath. This operation is A temporary contraction of the other blood vessel occurred. One blood vessel (lower trace) with IL-1β After incubation, sympathetic mediated contraction was substantially achieved after 6 hours. Was abolished. Exchange Sensory contractions were restored by infusion of L-NMMA. Congestion cuff deflation Inflation and reinflation are indicated by solid circles.     Panel B-traces show control vessels (upper trace) and IL-1β treatment. Shows the effect of L-NMMA on basal vascular tone in treated vessels (lower trace) You. The effects of L-NMMA were reversed by L-arginine. Figure 3-Panel A-BH_FourInfusion into control vessels for 20 minutes results in sustained vasodilation (Fine parallel lines; n = 6). Aminoguanidine co-injection Reverse the tension (solid line; n = 6)     Panel B-BH_FourEffect of injection (top trace) and reverse with aminoguanidine First trace showing rollover (lower trace). Congestion cuff deflation and re Inflation is indicated by solid circles. Figure 4-Perform PCR reaction in 20 μl volume and swim 5 μl sample on 2% agarose gel And analyzed.   Panel A shows the results using the iNOS primer pair BB52 and BB71. Track 1   Size markers; tracks 2 and 3 mRNA from uninduced hand vein; Tracks 4 and 5 mRNA from hand vein induced by cytokines; Track 6 Positive control (induced by cytokines) CAPAN-1 mRNA). The 499 bp PCR product is found only on track 6.   Panel B shows the results of PCR amplification using eNOS-specific primer pairs BB158 and BB159. Is shown. Tracks 1 and 2 Uninduced mRNA from hand vein; Tracks 3 and 2 From hand vein induced by cytokines 4 and 4; Track 5 positive control (Human placenta); Track 6 size marker. Bands that support eNOS It can be detected in racks 1-5.   Panel C shows β-actin-specific PCR from primer sets BB27 and BB28. Indicates the product. Traces 1-4 show hand vein mRNA amplified in the first reverse transcriptase step. Here is a sample. Tracks 5-8 are the negative controls, the reverse transcriptase step. Amplified in the absence. Track 9 is a positive control using placental mRNA. You. Track 10 is a size marker. The band corresponding to β-actin Can be detected on tracks 1-4 and confirms that mRNA was isolated from hand vein biopsies. Admit.Example Materials and methods In vivo pharmacology   The study was approved by the local ethics committee and was conducted on male and female subjects aged 19-40 . Subjects included that they were healthy and had not received any drug treatment He also gave written informed consent. subject Lay down in a supine position in a temperature controlled (28-30 ° C.) laboratory. Congestion cuff in upper arm Wound and inflated to 40 mmHg. Place a drug or saline solution in the back vein of the hand Injected with a 23 gauge needle placed. When the pressure of the congestion cuff drops from 40 to 0 mmHg On the skin overlaying the top of the blood vessel, 5-1Omm downstream from the tip of the injection needle Vein diameter was measured by recording the linear displacement of the lightweight probe . In all studies, diets should be maintained until a stable baseline vein diameter is recorded. Brine was injected for at least 15 minutes. Under study to instill cytokines A length of vein, 2-3 cm above the skin overlaying the vessel as described above Separated from circulation by two wedges spaced apart, one at a time or individually Intravenous infusion was performed. At the end of the infusion time, remove wedges and assess reactivity The vein was reconnected to the circulation to complete. This method of instillation is topical to the study vein. However, adjacent blood vessels remain unaffected. Static isolated Pulse blood volume is on the order of 1-2 ml And the calculated concentration of cytokine is 300-1000 pg / ml (TNFα and IL-1 β) and 30-100 pg / ml (IL-6). biopsy Surgical removal of the vein is performed under local anesthesia (1% lignocaine^TM). All sumps Were minimally frozen and immediately frozen at -80 ° C. PCR   Reverse transcriptase polymerase chain reaction (RT-PCR) was performed using Fast Track reagent (Invitrogen^TM) Poly A extracted from hand vein using⁺Performed on mRNA. eNOS Positive Con Troll Poly A⁺mRNA is prepared from a human placenta preparation (Clontech^TM). iNOS and Positive controls for GTPCH-1 and GTPCH-1 were compared with human pancreatic glands stimulated with cytokines. Poly A isolated from the cancer strain CAPAN-1⁺Performed on mRNA. The following primer set used: β actin: CTPCH-1 iNOS eNOS   PCR was performed in a Gene Amp PCR System 9600 thermocycler (Perkin-Elmer). e Amp^TM RT PCR kit (Perkin-Elmer^TM) In thin-walled test tubes. next Conditions were used: 1 minute 45 seconds at 95 ° C, then 30 cycles of 95 ° C, 15 seconds, 60 ° C, 30 seconds. 7 A 2 ° C. extension step was performed at the end of the cycle for 7 minutes. resultEffects of inflammatory cytokines on the vasoconstrictor response to noradrenaline   We investigated the mechanism of septic vasodilation in humans by A bo model has been developed (Bhagat et al., 1996). In this model, drug or media One eater at a very low dose sufficient to produce a study vessel change Put in the superficial vein. This may initiate a systemic cytokine cascade. Without the blood vessels Allows in situ exposure to individual cytokines.   First, we consider an inflammatory cytokine for the vasoconstrictor response to noradrenaline. Tried to determine the effect of Involved in systemic cardiovascular response to sepsis 3 One cytokine was studied. Infusion of IL-1β into one superficial blood vessel (1 ml physiology) (1 ng in saline for 1 hour) shows the dose response curve for noradrenaline To reduce the maximum shrinkage obtained (FIG. 1a). This effect progresses over several hours And reached a maximum 6 hours after exposing the vessels to IL-1β. By 24 hours, Norad Renalin's efficacy was fully restored. Tumor necrosis factor α (TNFα); 1 ml saline Intravenous infusion of either 1 ng) or IL-6 (100 pg, 1 ml in saline) Did not produce any significant changes in response to noradrenaline ( These cytokines were simultaneously administered for 1 hour with IL-1β (1 ng / ml). Instillation resulted in prolonged (> 24 hours) decay of response to noradrenaline (FIG. 1b). Poorly responsive to noradrenaline, which progresses slowly, Lutisone (100 mg, given 2 hours before cytokine instillation; n = 6, data (Not shown) was prevented by pre-treating the subject. These studies Is the major cytochrome that induces vascular changes in humans Identified IL-1β as a glucocorticoid, and therapeutically relevant doses of glucocorticoids Shows to prevent the progression of hypotonia.Effects of NOS inhibitors on IL-1β-induced hyporesponsiveness to noradrenaline   Determine if increased production of NO contributes to low reactivity to noradrenaline To make us N^GMonomethyl-L-arginine (L-NMMA; standard NOS inhibitor), And aminoguanidine (a compound widely used as a selective inhibitor of iNOS) Tested the effect. L-NMMA (1 μmol / min) and aminoguanidine (1 pmol / min) Both reverse the IL-1β-induced decay of the dose-response curve to noradrenaline (Figure 1). When NOS substrate (L-arginine; 1 μmol / min) is injected, L-NMMA or The effect of aminoguanidine was reversed (FIG. 1). Control vein (treated with IL-1β L-NMMA (1 μmol / min) and aminoguanidine (1 μmol / min) Lacks constriction (Figure 2B) and does not alter the response to noradrenaline (data shown Confirms previous observations that these vessels do not essentially produce NO. You. As expected, in control veins not exposed to cytokines , L-NMMA (1 μmol / min) is an expansion caused by the endothelium-dependent dilator Bradykinin Was inhibited by 88%, but aminoguanidine (1 μmol / min) did not. these Combined studies demonstrate that low reactivity induced by IL-1β is due to NO production You. Time course and pharmacological profile of the reaction (predicted by glucocorticoids) Prevention, reversal by L-NMMA and aminoguanidine) was consistent with iNOS expression, Was suggested as suggestive of them in previous studies.   The potential physiological significance of IL-1β-induced increase in NO synthesis is shown in Figure 2A. Is done. Cytokine instillation is a temporary activation of the sympathetic nervous system (deep Substantially abolishes intrinsic venous contractions (obtained by inhaling and asking to maintain) The activity of the sympathetic nervous system is a major determinant of human venous tone. Lockers produce sufficient venous dilation and postural hypotension. Our discovery is sepsis Or deep vein dilation with systemic inflammatory response alters venous tone Being mediated by triggering basal production of NO within the vessel wall by dulling the system Is shown. Local injection of L-NMMA alters control response to sympathetic activation Of the sympathetic nervous system that did not induce venous contraction in blood vessels exposed to IL-1β The ability was restored (FIG. 2A). This is because systemic L-NMMA administration has been It does not change pulse pressure, but Explain the observation that it increases that of patients with shock.eNOS Sensitive der to inhibition by aminoguanidine is iNOS inhibitor BH ₄ presence is To In another series of studies, we introduced pre-contracted healthy control veins. BH_FourInjection (cofactor for all three isoforms of NOS) results in rapid vasodilation (Figure 3). In contrast to bradykinin-induced vasodilation, BH_FourWhat This expansion reaction was well reversed by aminoguanidine injection (1 μmol / min; FIG. 3). Turned over. Control vessels are expected to express eNOS but not iNOS Ah, BH_FourVasodilation caused by rapid and consistent activation of constitutive enzymes So this finding clearly presents a challenge. However, aminoguanidine is , BH_FourIn the absence, it has little effect on the activity of purified human eNOS, but BH_FourIs added to the enzyme When performed, aminoguanidine became an effective inhibitor (data not shown). Absent). In addition, BH to human endothelial cells in culture_FourAddition increases NO production, which Was inhibited by aminoguanidine. Therefore, amino acids that inhibit eNOS activity Anidine's ability is BH_FourDepends on the effective concentration ofIL-1 β is GTP Shikurohidoro lead to increased generation of BH ₄ Induces expression of Rase 1   BH_FourThe rate-limiting enzyme for synthesis is GTP cyclohydrolase 1. This enzyme is It is transcriptionally regulated in response to Cain or endotoxin and its induction is controlled by glucocortico. Blocked by id. Therefore, the pharmacological we have observed in hand veins in vivo The alteration is induced following GTP cyclohydrolase 1 induction, such as by induction of iNOS. BH made_FourCan be explained equally well by activation of eNOS. NOS activity To further investigate the mechanism of functional induction, we resected a portion of the hand vein did. As expected, mRNA encoding eNOS was detected in all samples (Figure 4). Messenger RNA encoding GTP cyclohydrolase 1 Detected in veins exposed to heat (Figure 4). In contrast, under the same conditions, iNOS No encoding mRNA was detected (FIG. 4). Taken together, pharmacological and The simplest explanation for molecular studies is that IL-1β induces GTP cyclohydrolase 1 expression. GTP cyclohydrolase 1 is BH_FourProduction and its consequences of eNOS activity Is to bring about sex. This interpretation implies IL-1β in human cultured endothelial cells. Treatment resulted in the appearance of GTPCH-1 mRNA but not iNOS, and nitrides in the culture medium. Findings that lead to the induction of NO production as assessed by measurements Therefore, it is further strengthened. FACS analysis shows that cells express eNOS protein, iNOS protein was not detected. GTP sick we detected in hand vein Lohydrolase 1 mRNA was present throughout vascular endothelium, smooth muscle or the entire vascular wall It is not clear whether BH produced in one cell_FourNOS enters neighboring cells Has been shown to be able to activate Neopterin levels are Human macrophages, which were greatly increased in the plasma of Make opterin and biopterin.   Our study clearly shows that IL-1β can be used in humans by increasing NO production. Identified as the major cytokines that cause physiologically significant vasodilation in E. coli. We attribute this phenomenon to the transcriptional regulation of GTP cyclohydrolase 1 and its consequences. BH_FourSuggest that this is explained by activation of eNOS. eNOS is smooth Endothelial enzyme not detected in muscle cells. Implications of our discovery are: That the endothelium can produce enough NO to produce deep vasodilation, and even greater NOS expression across the smooth muscle cell layer is not required to produce septic hypotension That is to say.

────────────────────────────────────────────────── ─── Continuation of front page    (81) Designated countries EP (AT, BE, CH, DE, DK, ES, FI, FR, GB, GR, IE, IT, L U, MC, NL, PT, SE), OA (BF, BJ, CF) , CG, CI, CM, GA, GN, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, M W, SD, SZ, UG, ZW), EA (AM, AZ, BY) , KG, KZ, MD, RU, TJ, TM), AL, AM , AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN, CU, CZ, DE, DK, EE, E S, FI, GB, GE, GH, GM, GW, HU, ID , IL, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MD, M G, MK, MN, MW, MX, NO, NZ, PL, PT , RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, UA, UG, US, UZ, V N, YU, ZW (72) Inventor Bagat Kiran             United Kingdom 1P 9LN               London Tottenham Court Row             De 140 University College             London Wolfson Institute             To Four Biochemical Research (72) Inventor Balance Patrick John Thompso             N             United Kingdom 1P 9LN               London Tottenham Court Row             De 140 University College             London Wolfson Institute             To Four Biochemical Research (72) Hingolani Arun Dinesh             United Kingdom 1P 9LN               London Tottenham Court Row             De 140 University College             London Wolfson Institute             To Four Biochemical Research

Claims (1)

[Claims] 1. A method for identifying a compound that selectively inhibits nitric oxide synthase (NOS), comprising: (a) measuring the inhibition of NOS activity in the presence of a candidate compound and a first concentration of tetrahydrobiopterin (BH ₄ ); (b) measuring the inhibition of NOS activity in the presence of a second concentration of the candidate compound and BH _4; in (c) candidate compound (a) inhibits NOS activity to a different extent than in (b) Determining whether the method comprises. 2. Step (a), (i) in the presence of a first concentration in the absence and BH ₄ of a candidate compound, measuring the NOS activity; (ii) the presence of a first concentration of the candidate compound and BH ₄ Measuring the NOS activity; (iii) comparing the NOS activity in steps (i) and (ii). 3. Step (b), (i) in the presence of a second concentration of the absence and BH ₄ of a candidate compound, measuring the NOS activity; (ii) the presence of a second concentration of the candidate compound and BH ₄ The method of claim 1 or 2, further comprising: measuring the NOS activity; (iii) comparing the NOS activity in steps (i) and (ii). 4. The method of claim 1, wherein the second concentration is lower than the first concentration. 5. The method according to any one of the preceding claims, wherein the NOS is human eNOS. 6. The method according to any one of claims 1 to 5, wherein the NOS is human iNOS. 7. The method according to any one of claims 1 to 5, wherein the NOS is human nNOS. 8. Use of a compound identified by a method according to any one of the preceding claims in the manufacture of a medicament for use in selectively inhibiting human nitric oxide synthase. 9. 9. Use according to claim 8, wherein said NOS is eNOS. 10. 9. Use according to claim 8, wherein said NOS is iNOS. 11. 9. Use according to claim 8, wherein said NOS is nNOS. 12. Use of a compound identified by the method of any one of claims 1 to 7 in the manufacture of a medicament for use in treating toxic shock syndrome. 13. A method of identifying a compound which is an inhibitor of BH ₄ synthesis mediated GTP cyclohydrolase 1, (i) contacting a GTP cyclohydrolase 1 candidate compound; and (ii) mediated GTP cyclohydrolase 1 determining the inhibition by the compound BH ₄ synthesis as a method including. 14． The use of a compound in a pharmaceutical preparation that is used to inhibit human nitric oxide synthase, use the compound capable of inhibiting or reducing the BH ₄ synthesis by GTP cyclohydrolase 1. 15. The compound, (i) GTP contacting with cyclohydrolase 1 candidate compound; by the assay method comprising, determining the inhibition by and (ii) the compound BH ₄ synthesis mediated GTP cyclohydrolase 1 15. Use according to claim 14, which is identified. 16. The use according to claim 14 or 15, wherein the NOS is eNOS. 17． A compound identified by the method of any one of claims 1 to 7 or 13. 18. 18. A compound according to claim 17 for use in therapy. 19. 19. A compound according to claim 18 for use in treating toxic shock syndrome. 20. 18. A compound according to claim 17 for use in the manufacture of a medicament for preventing or treating a clinical condition caused by excessive NO production. 21. A pharmaceutical composition comprising a compound according to any one of claims 17 to 20 together with a pharmaceutically acceptable carrier or diluent. 22. 22. A method for preventing or treating a patient's clinical condition caused by excessive NO production, comprising an effective amount of a compound according to any one of claims 17 to 20 or a pharmaceutical composition according to claim 21. Administering to the patient.