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WO2006130090A1 - Utilisation d'une substance inhibant la regulation a la hausse des recepteurs de l'endotheline de type b et de la 5-hydroxytryptamine de type 1b dans le traitement de troubles ischemiques - Google Patents

Utilisation d'une substance inhibant la regulation a la hausse des recepteurs de l'endotheline de type b et de la 5-hydroxytryptamine de type 1b dans le traitement de troubles ischemiques Download PDF

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Publication number
WO2006130090A1
WO2006130090A1 PCT/SE2006/000661 SE2006000661W WO2006130090A1 WO 2006130090 A1 WO2006130090 A1 WO 2006130090A1 SE 2006000661 W SE2006000661 W SE 2006000661W WO 2006130090 A1 WO2006130090 A1 WO 2006130090A1
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inhibitor
receptor
type
endothelin
use according
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Lars Edvinsson
Saema Beg
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Pronas Pharma AB
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Pronas Pharma AB
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5082—Supracellular entities, e.g. tissue, organisms
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/275—Nitriles; Isonitriles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
    • A61K31/403—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil condensed with carbocyclic rings, e.g. carbazole
    • A61K31/404—Indoles, e.g. pindolol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/4353—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems
    • A61K31/437—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems the heterocyclic ring system containing a five-membered ring having nitrogen as a ring hetero atom, e.g. indolizine, beta-carboline
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6887—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids from muscle, cartilage or connective tissue

Definitions

  • the invention relates to the use of at least one inhibitor, which prevents or reverts endothelin type B and 5-hydroxytryptamine type IB receptor up regulation, for the manufacturing of a medicament for the treatment of an ischemic disorder or disease, as well as a screening method to identify such an inhibitor.
  • Stroke is the third leading cause of death in many industrial countries. Around 20 % of the patients do not survive the first month and >30% who are alive 6 months later will be dependent on other people. Stroke is often ischemic and the majority of ischemic strokes are the result of an occlusion of a major cerebral artery by a thrombus or an embolism, which give rise to loss of blood flow in one or more specific regions.
  • rt-PA tissue-plasminogen activator
  • agents to be used for the treatment of ischemic diseases or disorders, such as stroke as well as screening methods to enable the finding of new bioactive agents that can be used to efficiently treat ischemic brain injury, to save the penumbral area and help the patient to get a better quality of life.
  • agents are not available, agents, which are safe, non- addictive and effective, and also to which the body in the long-term is not refractory.
  • the invention relates to the finding that by inhibiting of endothelin type B and 5-hydroxytryptamine type IB it is possible to prevent the cerebral blood flow (CBF) reduction associated with subarachnoid haemorrhage (SAH) in a model system of a rat.
  • CBF cerebral blood flow
  • SAH subarachnoid haemorrhage
  • the invention relates to the use of at least one inhibitor, which prevents or reverts endothelin type B and 5-hydroxytryptamine type IB receptor up regulation for the manufacturing of a medicament for the treatment of an ischemic disorder or disease, such as stroke.
  • the invention relates to a screening method to identify an inhibitor of endothelin type B and 5-hydroxytryptamine type IB receptor up regulation comprising the steps of; providing dissected arteries from an animal, inducing at least the endothelin receptors, immersing said dissected arteries in a solution and applying a tension on said arteries, adding a bioactive agent to said immersed dissected arteries, determining the contractile response of said dissected arteries and obtaining a bioactive agent, which inhibits endothelin type B and 5-hydroxytryptamine type IB receptor up regulation.
  • the invention relates to the use of at least one biomarker to screen for an inhibitor of endothelin type B and 5- hydroxytryptamine type IB receptor up regulation.
  • the invention relates to a method to identify a bioactive agent obtainable by the disclosed method.
  • the bioactive agent may be used alone or in combination with the therapeutic agents commonly used today and mentioned above for the treatment of ischemic brain injury.
  • an endothelin type B and 5-hydroxytryptamine type IB inhibitor is intended to mean any natural, synthetic or semisynthetic compound that may act as an inhibitor of the altered expression of endothelin type B and 5- hydroxytryptamine type IB receptors and prevent reduction of CBF.
  • the inhibitor may for example be a protein kinase inhibitor or part of a protein kinase inhibitor or a fusion of a protein kinas inhibitor and another component or part as long as it has the capability to inhibit endothelin type B and 5-hydroxytryptamine type IB receptor upregulation and hence the function.
  • ischemic brain injury is intended to mean any kind of disorder or disease involving any ischemic event.
  • bioactive agent is intended to mean an agent being capable of acting as an endothelin type B and 5-hydroxytryptamine type IB inhibitor, such as being a protein kinase inhibitor or a MAPK inhibitor.
  • the inhibitor may for example inhibit ERK1/2, rafox protein kinase alpha or delta and being capable of normalising vascular tone by decreasing vasoconstriction or increasing vasodilatation of blood vessels such as arteries after ischemic injury, such as ischemic brain injury, angina pectoris or ischemic heart desease.
  • physiological solution is intended to mean a solution, in which receptors of dissected arteries may be upregulating.
  • receptors are endothelin, such A 1 and A 2 , angiotensin, such as AT 1 and AT 2 , serotonin, such as 5-HTi B and bradykinin, such as Bi and B 2 .
  • physiological solutions will be obvious for a person skilled in the art. Examples are modified KREB 's, DMEM, fysiological NaCl, phosphate buffer, etc.
  • treatment means that by inhibiting the signal resulting in transcription and translation, the receptor upregulation will be prevented and, hence, the sequele leading to penumbral zone damage and neuronal loss will be revoked.
  • screening method means that the applicability of said method to inhibit upregulation of receptors can be shown, e.g., by using certain commercially available PK inhibitors and related compounds (for reference see, e.g., Hansen-Schwartz et al., Br J Pharmacol, 2002;137(l):l 18-126; Uddman et al, Eur J Pharmacol, 2002; 452(2):215-22).
  • Endothelin type B and 5-hydroxytryptamine type IB upregulation inhibitor The present invention relates to the area of treatment of vasoconstriction and/or vasodilation of arteries, such as human or animal arteries, including cerebral arteries.
  • the idea being based on the hypothesis that down regulation of the endothelin type B (ET 3 ) as well as the 5-hydroxytryptamine type IB (5-HT 1B ) receptors prevent the cerebral blood flow (CBF) reduction associated with subarachnoid haemorrhage (SAH) or stroke after occlusion of a cerebral artery.
  • CBF cerebral blood flow
  • SAH subarachnoid haemorrhage
  • stroke after occlusion of a cerebral artery.
  • the cerebral ischemia induce by either SAH or artery occlusion results in a change in the receptor expression in cerebral arteries involved.
  • the receptor upregulation cause further contraction, which limits the oxygen delivery to the ischemic artery and thus worsens the area at risk; in SAH a second ischemia and in occlusion the development of the penumbra.
  • the receptor upregulation By inhibiting the process by e.g., raf, ERKl/2 or a PKC inhibitor we can not only reverse or counteract the vascular receptor upregulation but also reduce and prevent the neuronal death and cerebral blood flow reduction. Thus by using this mechanism we can limit the damage in man after a stroke.
  • the invention relates to the use of an inhibitor, which prevents or reverts endothelin type B and 5-hydroxytryptamine type IB receptor up regulation for the manufacturing of a medicament for the treatment of an ischemic disorder or disease.
  • disorders or diseases are ischemic brain injury, such as stroke, angina pectoris, claudication and ischemic heart disease or disorder.
  • the inhibitors may be a protein kinase C, MAPKKK, MAPKK or
  • the inhibitors may for example inhibit raf, MEK1/2 or extracellular signal-regulated kinase (ERK1/2). Examples of inhibitors are SB
  • the inhibitor is a protein kinase C inhibitor, such as a PKC alpha or delta inhibitor.
  • PKC alpha or delta inhibitor examples of inhibitors are RO-31-8220, RO-31-7549 or
  • a mixture of one or more inhibitors selected from the group consisting of protein kinase C, MAPKKK, MAPKK and MAPK may be used for the treatment of a ischemic disorder or disease.
  • the inhibitor is used for the inhibition of at least one transcription factor activity, wherein said transcription factor is selected from the group consisting of EIk-I, ATF-2 and c-Jun. These transcription factors become activated by p38, ERK 1/2 and SAPK/JNK and increase the expression of inflammatory and extracellular-matrix-related genes.
  • the invented medicament may further comprise a pharmaceutically acceptable diluent, excipient, buffer of carrier.
  • a pharmaceutically acceptable diluent, excipient, buffer of carrier means a non-toxic material that does not decrease the effectiveness of the biological activity of the active ingredients.
  • Such pharmaceutically acceptable buffers, carriers or excipients are well-known in the art
  • the invented medicament may be placed in a container, such as an ampoule, syringe or the like.
  • a second protein kinase inhibitor was also evaluated, i.e., Ro-32-0432.
  • a MAPK inhibitor evaluated, an inhibitor, which inhibits of ERK1/2.
  • the MAPKKK inhibitor SB386023(-b) used. It was injected intracisternally in conjunction with and after the induced SAH. Two days after the SAH cerebral arteries were harvested for quantitative real-time PCR, immunohistochemistry and the contractile responses to endothelin-1 (ET-I; ET A and ET B receptor agonist) and 5-carboxamidotryptamine (5-CT; 5-HT 1 receptor agonist) were carried out in a sensitive myograph.
  • the inhibitor may be any inhibitor which can act as a MAPK inhibitor or a protein kinase inhibitor, such as being synthetic or semisynthetic or part thereof.
  • the inhibitor may also be small peptides or a small chemical compound as long as it can act as an inhibitor as mentioned above.
  • the invention relates to a screening method to identify an endothelin type B and 5-hydroxytryptamine type IB inhibitor, such as by the use of one or more biomarker as shown under the examples.
  • a screening method to identify an endothelin type B and 5-hydroxytryptamine type IB inhibitor, such as by the use of one or more biomarker as shown under the examples.
  • bioactive agents there is a need for one or more model system in which vasoconstriction and/or vasodilatation may be induced in parallel or in sequence with upregulation of one or more receptors, such as endothelin, angiotensin, serotonin and/or bradykinin.
  • the inventors have surprisingly found that blood vessels of tissue subjected to ischemia, such as the basilar artery and the middle cerebral artery, undergoes phenotypic changes so that the vasoconstrictor receptor population is upregulated, which result in excessive arterial constriction, and that for example the endothelin and the 5- carboxamidotryptamine receptors are among those becoming upregulated.
  • Vasoconstriction and/or vasodilatation can be induced in a mammalian animal such as rat, mouse, guinea-pig, dog, cat, horse or a synthetic version thereof.
  • intraluminal occlusion technique originally described by Longa et al., Stroke, 1989; 20(l):84-91.
  • Another example is the use of isolated dissected arteries from a mammalian animal such as rat, mouse, guinea-pig, dog, cat, horse or a synthetic version thereof (see Example 3 and 4).
  • the isolated arteries are exposed to a physiological solution, in which the endothelial receptors can be upregulated.
  • a physiological solution is the nutrition solution Dulbecco's modified Eagle's medium (DMEM).
  • DMEM Dulbecco's modified Eagle's medium
  • the arteries may be dissected into smaller segments or discs either prior to or after upregulation of one or more receptors, such as the endothelin receptors.
  • the tissue, organ, cells or arteries have the same characteristics as for example a patient who has suffered an ischemic brain injury.
  • the arteries with upregulated receptors will then be immersed in a solution to maintain their biological activities, also exposing the preparations to a tension, in order to enable the possibility to study vasoconstriction and/or vasodilatation.
  • One way to study isometric tension is to use a Mulvany-Halpern Myograph (Danish Myo Technology AJS, Denmark).
  • the arteries are then exposed to a bioactive agent and the contractile response studied. If the response is reduced the bioactive agent is determined as being a candidate able of acting as a kinas inhibitor, which may be used for the treatment of disorders involving vasoconstric
  • MCA Middle cerebral artery occlusion
  • Focal cerebral ischemia was induced by the intraluminal occlusion technique originally described by Longa et al., 1989. Male Wistar Hannover rats (M ⁇ llegaard Breeding Center, Copenhagen, Denmark), weighing 350-420 g were used for the study. The experimental procedures used have been approved by the Ethics Committee for use of Laboratory Animal Experiments at the University of Lund (M217-00). The animals were fasted overnight with free access to water. Anaesthesia was induced with 4.5% halothane in N 2 O: O 2 (70: 30). They were kept anaesthetized on a mask, inhaling 1-1.5% halothane in N 2 O: O 2 (70: 30) during the surgical procedure.
  • the right MCA was occluded by inserting a thin filament into the internal carotid artery and advance this to the origin of the MCA (further details are described by Stenman et al., Stroke, 2002; 33(9):2311-16).
  • anaesthesia was discontinued and the animals were allowed to wake up.
  • the animals were briefly re-anaesthetized to allow withdrawal of the filament to achieve reperfusion.
  • Sham- operated rats went through the same operative procedure as the MCA-occluded rats, except that the filament was immediately withdrawn after insertion. These animals were re-anaesthetized for 15 min after two hours.
  • Subarachnoid haemorrhage was induced by a model originally devised by Svendgaard (Prunell et al., 2002) and carefully described by Prunell (Prunell et al, 2003).
  • mice Male Sprague-Dawley rats (350-400 g) were anaesthetized using 5% halothane (Halocarbon Laboratories, River Edge, New Jersey) in N 2 O/O 2 (30:70). The rat was intubated and artificially ventilated with inhalation of 0.5-1.5% halothane in N 2 O/O 2 (70:30) during the surgical procedure. The depth of anaesthesia was carefully monitored and the respiration checked by regularly withdrawing arterial blood samples for blood gas analysis (Radiometer, Copenhagen, Denmark). An arterial catheter to measure blood pressure was placed in the tail artery and a catheter to monitor intracranial pressure (ICP) was placed in the subocciptal membrane.
  • ICP intracranial pressure
  • RO-31-7549 (Calbiochem, Sweden) was injected intracisternally at 30 minutes prior to the induced SAH and after the SAH (20 ⁇ L; 10 "6 M) RO-31-7549 was given at the time points 3, 6, 24 and 32 hour after the first RO-31-7549 injection.
  • Rat subarachnoid haemorrhage model with MAPK ERKl/2 inhibition This group of animals went through the same procedure as the above- mentioned SAH animals. In addition they were treated with SB386023 -b (a kind gift from Dr A A Parsons, GSK, UK) in conjunction with the operation and after the induced SAH. SB386023-b (50 ⁇ l; 1O -6 M) was injected intracisternally at 30 minutes prior to the induced SAH and after the SAH (20 ⁇ l; 1O -6 M) SB386023 -b was given at time points 3, 6, 24 and 32 hours after the first SB386023-b injection.
  • a distal branch of the rat superior mesenteric artery (outer diameter ⁇ 1 mm) or basilar arteries(outer diameter 0.4-0.5 mm) was chosen as experimental target since previous studies have shown that endothelin ET B receptors are up- regulated in this vessel following 24 h of incubation with Dulbecco's modified Eagle's medium (DMEM).
  • DMEM Dulbecco's modified Eagle's medium
  • Male Wistar-Kyoto rats 200-35Og, M & B, Denmark) were anaesthetized with CO2 and killed by decapitation. The artery was removed, dissected free from adherent tissue and cut into 1 mm long circular segments.
  • Segments were placed in a well containing 1 ml of DMEM 5 supplemented with penicillin (100 U/ml) and streptomycin (100 mg/ml), and incubated at 37° C in humidified 5% CO2 in air (pH 7.4).
  • Each segment was mounted on two L-shaped prongs, one of which was attached to a Grass FT-03 transducer (Grass Instr., Quincy, USA) connected to a MacLab unit (ADInstruments, Hastings, UK) for continuous recording of isometric tension.
  • a tension of 2 mN was applied to each segment and the segments were allowed to stabilise at this tension for one hour before being exposed to a K + -rich (63,5 mM) buffer solution with the same composition as the standard solution except that NaCl was replaced by an equimolar concentration of KCl.
  • Standard buffer solution NaCl 119, NaHCO 3 15, KCl 4.6, MgC12 1.2, NaH 2 PO 4 1.2, CaCl 2 1.5, glucose 5.5.
  • Analytical grade chemicals and double-distilled water were used for preparing all solutions.
  • S6c and ET-I (Auspeptide, Aus) were dissolved in water with bovine serum albumin (Kabi, Sweden) (0.1% w/v). All MAPK inhibitors were dissolved in DMSO and diluted in water.
  • PD98059 was purchased from Sigma, St. Louis, USA. SB239063, SB408039, SB386023b and SB203580 were a generous gift from Dr A.A. Parsons, GSK, UK. Calculations and statistics
  • the vessels were cut into cylindrical segments and the endothelium was removed mechanically by inserting a thin thread and rubbing the endothelium.
  • the vessel segments were threaded on two 40 ⁇ m diameter stainless steel wires and mounted in a Mulvany-Halpern myograph (Danish Myo Technology A/S, Denmark) for recording of isometric tension.
  • One wire was connected to a force displacement-transducer attached to an analogue-digital converter unit (ADInstraments, Hastings UK).
  • the other wire was attached to a movable displacement device allowing fine adjustments of vascular tension by varying the distance between the wires.
  • the experiments were recorded on a computer by use of the software program ChartTM (ADInstruments, UK).
  • the segments were immersed in temperature-controlled (37 0 C) tissue baths containing a bicarbonate buffer solution of the following composition (mmol/L): NaCl 119; NaHCO 3 15; KCl 4.6; MgCl 2 1.2; NaH 2 PO 4 1.2; CaCl 2 1.5 and glucose 5.5.
  • the solution was continuously gassed with 5% CO 2 in O 2 , resulting in a pH of 7.4.
  • the vessels were given an initial tension of 1.2 mN, and were adjusted to this level of tension for 1 hour.
  • the contractile capacity was determined by exposure to a potassium-rich (63.5 mmol/L) buffer solution with the same composition as the bicarbonate buffer solution except that NaCl was exchanged for KCl.
  • Segments to be used for real-time PCR were cultured, or removed from stroke or SAH rats, snap frozen in liquid nitrogen and subsequently stored at -8O 0 C until use.
  • the total cellular RNA was extracted using the FastRNA, Pro Green kit (Qbiogene) for 60 seconds in the FastPrep FP 120 instrument (Qbiogene) following the suppliers' instructions.
  • the resulting pellet was finally washed with 70% ethanol, air-dried and redissolved in 50 ⁇ L diethyl-pyrocarbonate (DEPC)-treated water.
  • Reverse transcription of total RNA to cDNA was carried out using the GeneAmp RNA PCR kit (PE Applied Biosystems, Foster City, CA, USA) in a Perkin-Elmer DNA Thermal cycler.
  • First strand cDNA was synthesized from 1 ⁇ g total RNA in a 20 ⁇ L reaction volume using random hexamers as primers. The reaction mixture was incubated at 25° C for 10 min, 48° C for 15 min, heated to 95° C for 5 min and chilled to 5° C for 5 min.
  • Real-time PCR was performed in a GeneAmp 5700 Sequence Detection System using the GeneAmp SYBR ® Green kit (Perkin-Elmer, Applied Biosystems Foster City, CA, USA) with the cDNA synthesised above as template in a 50 ⁇ l reaction volume. A no template control was included in all experiments.
  • the GeneAmp 5700 Sequence Detection System monitors the growth of DNA in real-time using an optics- and imaging system, via the binding of a fluorescent dye to double-stranded DNA. Specific primers were designed as follows:
  • Elongation factor- 1 (EF-I) mRNA was used as a reference, since it is the product of a housekeeping gene, continuously expressed to a constant amount in cells.
  • the EF-I primers were designed as follows:
  • the real-time PCR was carried out with the following profile: 50° C for 2 min, 95° C for 10 min, followed by forty cycles with 95° C for 15 sec and 60° C for 1 min.
  • standard curves were made where the C ⁇ - values were plotted against cDNA concentration based on the equation:
  • E is the amplification efficiency with the optimal value of 1.
  • the amplification efficiency can be calculated from the slope of the standard curve with an ideal slope close to 3.3 (Fig. 1).
  • the amount of endothelin ET B receptor mRNA was calculated as relative to the amount of EF-I mRNA in the same sample by the formula:
  • X 0 original amount of ET receptor mRNA
  • R 0 original amount of EF-I mRNA
  • CtR C ⁇ -value for EF-I
  • CtX C ⁇ -value for the ET receptor.
  • DEPC diethyl-pyrocarbonate
  • the reaction mixture was incubated at 25°C for 10 min, heated to 42 0 C for 15 min, further heated to 99 0 C for 5 min and chilled to 5°C for 5 min.
  • Real-time PCR was performed in a GeneAmp 5700 Sequence Detection System using the GeneAmp SYBR ® Green kit (Perkin- Elmer, Applied Biosystems Foster City, CA, USA) with the cDNA synthesized above as template in a 50 ⁇ L reaction volume. A no template control was included in all experiments.
  • the GeneAmp 5700 Sequence Detection System monitored the growth of DNA in real-time using an optic and imaging system, via the binding of a fluorescent dye to double-stranded DNA.
  • Specific primers for the AT 1 and AT 2 receptors, ACE and NF- ⁇ B were designed as follows:
  • GAGAGCCAGTAGCACGCATG-3 reverse: 5 '-CCTGGGTTCGTGGAATGAGT- y
  • Elongation factor- 1 (EF-I) and glyceraldehyde 3 -phosphate dehydrogenase (GAPDH) mRNA were used as endogenous standards, and the primers were designed as follows:
  • GAPDH forward 5 '-GGCCTTCCGTGTTCCTACC-S ' reverse: 5 '-CGGCATGTCAGATCCACAAC- 3 '
  • the real-time PCR was carried out with the following profile: 50 0 C for
  • C ⁇ -values we refer to the number of PCR cycles performed per gene product in one sample at a specific point of time. Data are expressed as mean values ⁇ SEM. Statistical analyses were performed with unpaired Students' t-test and P ⁇ 0.05 was considered significant.
  • Total protein concentration was determined using a BioRad DC kit (Hercules, CA, USA) and measuring absorbance at 750 nm on a Genesys 10 spectrophotometer (Thermo, Waltham, MA, U.S.A.). 25 ⁇ g total protein was loaded per lane on a 10% SDS-PAGE gel and blotted onto a Hybond PVDF membrane for 30 min at 1.5 mA/cm 2 . Subsequently the membrane was blocked in 5% non-fat milk for 1 hour at room temperature and incubated with primary antibody over-night at 4°C and with secondary antibody (Pierce, Rockford, IL, U.S.A.) for 1 hour at room temperature, with 3x5 min wash after antibody incubation.
  • the membranes were developed using the Supersignal Dura kit (Pierce, Rockford, IL, U.S.A.) and visualized using a Fujifilm LAS-1000 Luminiscent Image Analyzer (Stamford, CT, U.S.A.). Antibodies against pPKC ⁇ TH R638 5 pPKC ⁇ S ER645, pPKC ⁇ SER729; (Biosource, Camarillo, CA, U.S.A.), and ET A and ET B (Chemicon, Temecula, CA, U.S.A.) were used at 1:1000.
  • the animals were rapidly decapitated, the brain removed.
  • the whole brain was gently homogenized in PBS with a glass homogenizer several times.
  • Cerebral vessels were then isolated by 15% dextran density centrifugation at 3500 x g for 45 min.
  • the cerebral vessels were collected from the bottom of the centrifugation tubes and incubated with collagenase/dispase (1 mg/ml) at 370 C for 1 h.
  • the collagenase/dispase was removed by centrifugation 2000 x g for 10 min.
  • the segments of cerebral vessel were then explanted into Dulbecco's modified Eagle's medium (DMEM) (GibcoBRL).
  • DMEM Dulbecco's modified Eagle's medium
  • the DMEM was supplemented with 10% fetal bovine serum (FBS) (GibcoBRL, heated inactivated), bFGF 2 ng/ml, EGF 5 ng/ml, penicillin 100 U/ml and streptomycin 100 mg/ml. Subcultures were obtained by use of 0.25% trypsin-lmM EDTA (GibcoBRL).
  • Rat aortic and cerebral SMC were grown from explanted segments of the vessels.
  • the endothelium was removed by scraping the intimal surface with a surgical blade.
  • the medial layer of the vessel wall was removed and cut into 1x1 mm segments.
  • the segments were grown in the same medium as mentioned above.
  • SMC were identified by more than 95% positive reaction of immunofluorescence staining with monoclonal antibody (mouse IgG) against alpha- smooth muscle actin and a secondary anti-mouse antibody labelled with FITC (Boehringer Mannheim, Germany) and in addition of a typical "hill and valley" growth pattern. Cell viability was checked through all experiments by trypan blue (GibcoBRL) exclusion (>95%).
  • SAH and SAH treated with SB386023-b were anaesthetized using 5% halothane in N 2 O/O 2 (30:70).
  • the animal was intubated and artificially ventilated with inhalation of 0.5 -1.5% halothane in N 2 O/O 2 (70:30) during the surgical procedure.
  • the anaesthesia and the respiration were monitored by regularly withdrawing arterial blood samples for blood gas analysis.
  • a catheter to measure MABP was placed in the right femoral artery and a catheter for blood sampling was placed in the left femoral artery. This catheter was connected to a constant velocity withdrawal pump (Harvard apparatus 22, USA) for mechanical integration of tracer concentration.
  • a catheter was inserted in one femoral vein for injection of heparin and for infusion of the radioactive tracer.
  • the MABP was continuously monitored and a temperature probe was inserted into the rectum to record the temperature, which was regularly maintained at 37°C by a heating table.
  • the haematocrit was measured by a haematocrit centrifuge (Beckman Microfuge 11, USA).
  • Arterial blood (122 ⁇ l) was withdrawn over 20 seconds. Immediately after this the animal was decapitated, the brain removed and immersed in isopentane (J.T. Baker, Deventer, Netherlands) chilled to - 5O 0 C.
  • the arterial blood sample was transferred to liquid scintillation counting vials containing 1 ml mixture of Soluene-350 (Perkin-Elmer, Boston, USA) and Isopropanol (J.T. Baker, Deventer, Netherlands) (1:1). After 2 hours at 6O 0 C 0.2 ml of 30% hydrogen peroxide was added to the vials, and the samples were maintained at room temperature for 15-30 minutes. Thereafter the samples were kept at 6O 0 C for 30 minutes and 10-15 ml Hionic-Fluor (Perkin-Elmer, Foster, CA, USA) was added. The ⁇ -radioactivity scintillation counting was performed on the samples with a program that included quench correction ( Packard 2000 CA, Denmark).
  • the 14 C activity in the tissue was determined after sectioning the brain in 20 ⁇ m sections at -2O 0 C in a cryostat (Wild Leitz A/S, Glostrup, Denmark). The sections were exposed to x-ray films (Kodak, Denmark) together with C methylmethacrylate standards (Amersham Life Science, England) which exposed the films for 20 days. Densities of the autoradiograms were measured with a Macintosh computer equipped with an analog CF 4/1 camera (Kaiser, Germany) and a transparency flat viewer (Color-Control 5000, Weilheim, Germany). The 14 C content was determined in several brain regions (see Table 1). The CBF was calculated from the brain tissue 14 C activity determined by autoradiography using Gjedde et al.'s equation (Gjedde et al. 1980):
  • the aim was to examine the effect of inhibition of the MAP kinase/ERK kinase (MEK) 1/2 on endothelin receptor alteration, brain damage and neurology in experimental cerebral ischaemia.
  • MEK MAP kinase/ERK kinase
  • Transient middle cerebral artery occlusion was induced in male Wistar rats by the intraluminal filament technique as described above.
  • the animals received 100 mg/kg i.p of the MEKl/2 inhibitor U0126 or vehicle in conjunction with the occlusion. After 24 hours the rats were decapitated and the brains removed.
  • the middle cerebral arteries were dissected out and examined with myographs as described above or immunohistochemistry. The ischaemic areas of the brains were compared.
  • the MCAs were placed onto Tissue TEK (Gibco, Sweden), frozen and subsequently sectioned into 10 ⁇ m slices.
  • the primary antibodies used were rabbit anti- ⁇ hos ⁇ ho-p44/42 MAPK ( ⁇ ERKl/2; Cellsignalling, Santa Cruz, USA) and rabbit anti-phospho-Elk-1 (Cellsignalling) diluted 1:50.
  • the secondary antibody used was biotin conjugated goat anti-rabbit (JacksonlmmunoResearch, Sohan, UK) diluted 1:100. All dilutions were done in PBS with 0.3% Triton-XIOO and 5% foetal calf serum.
  • the Vectastain ABC kit (Vector Laboratories, Burlinggame, USA) together with DAB substrate (Rockland, Gilbertsville, USA) were used for detection according to the manufacturers' instructions. Pictures were taken at 4Ox magnification. As control, only secondary antibodies were used. After the middle cerebral artery occlusion, the contractile responses of the endothelin A and B receptors were augmented in the ipsilateral middle cerebral artery. UO 126 decreased this alteration in endothelin receptor response. Furthermore, treatment with UO 126 significantly decreased the brain damage and improved neurological scores.
  • This example aimed to examine the effect of PKC inhibition on endothelin receptor upregulation, brain damage and neurology in experimental cerebral ischemia.
  • Transient middle cerebral artery occlusion was induced in male Wistar rats by the intraluminal filament technique as described above. The animals received
  • the rat MCA was dissected out, treated as described above and placed in Tissue TEK (Gibco), frozen and subsequently sectioned into 10 Dm thick slices in a calibrated Microm HM500M cryostat (Microm, Germany).
  • the primary antibodies used were rabbit antiphospho p38 (Cellsignalling #4631), rabbit antiphopho ERK 1/2 MAPK (Cellsignalling, #4376), rabbit antiphospho SAPK/JNK (Cellsignalling, #9251) rabbit antiphospho ATF-2 (Cellsignalling #9221), rabbit antiphospho EIk-I (Cellsignalling, #9181) and rabbit antiphospho c-Jun(Cellsignalling, #9261) each diluted 1:50.
  • the secondary antibodies used were biotin-conjugated goat anti-rabbit antibodies (Jackssonlmmuno, 111-065-003) diluted 1:100. All dilutions were with PBS containing 0.3% Triton-XIOO and 5% fetal calf serum.
  • the Vectastain ABC kit (Vector Laboratories) together with DAB substrate (Rockland) were used for detection in accordance with the manufacturers' instructions. Pictures were taken at 40X magnification. Secondary antibodies alone served as a control. The results show that ATF-2, EIk-I as well as c-Jun is activated.

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Abstract

L'invention concerne l'utilisation d'au moins un inhibiteur permettant de prévenir ou d'inverser la régulation à la hausse des récepteurs de l'endothéline de type B et de la 5-hydroxytryptamine de type 1B pour la fabrication d'un médicament destiné à traiter un trouble ou une maladie ischémique, ainsi qu'une méthode de criblage destinée à identifier cet inhibiteur.
PCT/SE2006/000661 2005-06-03 2006-06-02 Utilisation d'une substance inhibant la regulation a la hausse des recepteurs de l'endotheline de type b et de la 5-hydroxytryptamine de type 1b dans le traitement de troubles ischemiques Ceased WO2006130090A1 (fr)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008121044A1 (fr) 2007-03-30 2008-10-09 Pronas Pharma Ab Trouble ischémique ou inhibiteurs de maladie

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0365863A2 (fr) * 1988-10-24 1990-05-02 Merrell Dow Pharmaceuticals Inc. Quinolyloxazole 2-ones utiles comme inhibiteurs de protéinkinase-C
WO2000036150A1 (fr) * 1998-12-17 2000-06-22 Isis Pharmaceuticals, Inc. Modulation antisens de l'expression d'elk-1
WO2003068754A1 (fr) * 2002-02-13 2003-08-21 Astrazeneca Ab Derives d'indazole therapeutiques a substitution
WO2004064733A2 (fr) * 2003-01-15 2004-08-05 Board Of Regents, University Of Texas System Utilisation d'inhibiteurs de c-raf dans le traitement de maladies neurodegeneratives

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0365863A2 (fr) * 1988-10-24 1990-05-02 Merrell Dow Pharmaceuticals Inc. Quinolyloxazole 2-ones utiles comme inhibiteurs de protéinkinase-C
WO2000036150A1 (fr) * 1998-12-17 2000-06-22 Isis Pharmaceuticals, Inc. Modulation antisens de l'expression d'elk-1
WO2003068754A1 (fr) * 2002-02-13 2003-08-21 Astrazeneca Ab Derives d'indazole therapeutiques a substitution
WO2004064733A2 (fr) * 2003-01-15 2004-08-05 Board Of Regents, University Of Texas System Utilisation d'inhibiteurs de c-raf dans le traitement de maladies neurodegeneratives

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
HANSEN-SCHWARTZ J. ET AL.: "Protein kinase mediated upregulation of endothelin A, endothelin B and 5-hydroxytryptamine 1B/1D receptors during organ culture in rat basilar artery", BRITISH JOURNAL OF PHARMACOLOGY, vol. 137, 2002, pages 118 - 126, XP003000332 *
HANSEN-SCHWARTZ J.: "Receptor changes in cerebral arteries after subarachnoid haemorrhage", ACTA NEUROLOGICA SCANDINAVICA, vol. 109, no. 1, 2004, pages 33 - 44, XP003000331 *
HENRIKSSON M. ET AL.: "Importance of ERK1/2 in upregulation of endothelin type B receptors in cerebral arteries", BRITISH JOURNAL OF PHARMACOLOGY, vol. 142, 2004, pages 1155 - 1161, XP003000333 *
NAMURA S. ET AL.: "Intravenous administration of MEK inhibitor U0126 affords brain protection against forebrain ischemia and focal cerebral ischemia", PNAS, vol. 98, no. 20, 25 September 2001 (2001-09-25), pages 11569 - 11574, XP003000335 *
SATOH M. ET AL.: "Inhibitory Effect With Antisense Mitogen-Activated Protein Kinase Oligodeoxynucleotide Against Cerebral Vasospasm in Rats", STROKE JOURNAL OF THE AMERICAN HEARTH ASSOCIATION, vol. 33, 2002, pages 775 - 781, XP003000334 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008121044A1 (fr) 2007-03-30 2008-10-09 Pronas Pharma Ab Trouble ischémique ou inhibiteurs de maladie
US8273771B2 (en) 2007-03-30 2012-09-25 Pronas Pharma Ab Ischemic disorder or disease inhibitors

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