EP1144050A2 - Binding partners for 5-ht5-receptors for the treatment of migraine - Google Patents

Binding partners for 5-ht5-receptors for the treatment of migraine

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Publication number
EP1144050A2
EP1144050A2 EP00901526A EP00901526A EP1144050A2 EP 1144050 A2 EP1144050 A2 EP 1144050A2 EP 00901526 A EP00901526 A EP 00901526A EP 00901526 A EP00901526 A EP 00901526A EP 1144050 A2 EP1144050 A2 EP 1144050A2
Authority
EP
European Patent Office
Prior art keywords
binding
receptors
binding partner
binding partners
treatment
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
EP00901526A
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German (de)
French (fr)
Inventor
Francisco Javier Garcia-Ladona
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Abbott GmbH and Co KG
Original Assignee
Knoll GmbH
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Filing date
Publication date
Application filed by Knoll GmbH filed Critical Knoll GmbH
Publication of EP1144050A2 publication Critical patent/EP1144050A2/en
Ceased legal-status Critical Current

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Classifications

    • 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
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • G01N33/6896—Neurological disorders, e.g. Alzheimer's disease
    • 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
    • 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
    • A61K31/13—Amines
    • A61K31/135—Amines having aromatic rings, e.g. ketamine, nortriptyline
    • 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
    • A61K31/4045—Indole-alkylamines; Amides thereof, e.g. serotonin, melatonin
    • 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
    • A61K31/47—Quinolines; Isoquinolines
    • A61K31/48—Ergoline derivatives, e.g. lysergic acid, ergotamine
    • 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
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/496—Non-condensed piperazines containing further heterocyclic rings, e.g. rifampin, thiothixene
    • 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
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/506—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim not condensed and containing further heterocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/06—Antimigraine agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • 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

Definitions

  • the present invention relates to binding partners for 5-HT5 receptors, methods for identifying and characterizing such binding partners, and pharmaceutical compositions containing them and their use for the treatment of cerebrovascular diseases such as migraines.
  • At least seven different receptor classes mediate the diverse physiological activities that are ascribed to the involvement of the neurotransmitter serotonin (5-hydroxytryptamine, 5-HT for short). According to an internationally recognized classification, they are designated 5-HT1, 5-HT2, 5-HT3, 5-HT4, 5-HT5, 5-HT6 and 5-HT7. Most of these classes also include other distinguishable receptor types.
  • the 5-HTI class includes receptors, which can be divided into at least five subclasses, which are designated 5-HT1A, 5-HT1B, 5-HT1C, 5-HT1D and 5-HT1E (Boess FG and Martin IL, Neuropharmacology 33: 275-317 (1994)).
  • the 5-HT5 class was first described by Plassat et al., The EMBO Journal Vol. 11 No. 13, pp. 4779-4786 (1992). A distinction is made between 5-HT5A and 5-HT5B receptors (Erlander et al., Proc. Natl. Acad. Sci. USA 90: 3452-3456 (1993)). There are only minor sequence homologies between 5-HT5 and other 5-HT receptors. The pharmacological profile of these receptors differs significantly. Molecular biological techniques successfully localized 5-HT5 receptors in the olfactory bulb, in the hippocampus, in the cortex, in the cerebral ventricles, in the corpus callosum and in the cerebellum.
  • 5-HT5 receptors are primarily expressed on astrocytes (Carson et al., GLIA 17: 317-326 (1996)).
  • Astrocytes lie directly on the basement membrane of brain capillaries of the blood-brain barrier.
  • An abnormal astrocyte endothelium structure is associated with a loss of the blood-brain barrier.
  • the exact meaning of the astrocytes is unclear. They seem to carry out transport tasks and connective functions.
  • Reactive astrocytes have been observed in association with reactive gliosis in a number of pathological brain changes and neuropsychiatric disorders. As a result of brain injuries, they change their morphology. The protein expression pattern changes and growth factors are produced.
  • Migraine is a CNS disease that affects large parts of the population. It manifests itself in most cases by a recurring headache, of which an estimated 8 million people, i.e. 3-5% of all children, 7% of all men and 14% of all women are affected. Although a genetic predisposition is propagated, the causes seem to be complex (Diener H.C. et al., Arzneiffentherapie 15: 387-394 (1997)).
  • the vascular theory which has been known for a long time, suggests a dilation process of the inner and outer cerebral vascular system as the cause.
  • the neurogenic theory is based on a release of vasoactive neurotransmitters, primarily neuropeptides such as substance p and neurokinin, from axons of the vasculature as a result of stimulation of certain brain tissue innervating ganglia, which is said to lead to inflammatory reactions and thus to pain.
  • Prophylactic migraine-specific active ingredients such as Sanmigran R , Nocerton R , Desernil R and Vidora R , but also active ingredients commonly used for other indications, such as beta-blockers, antiemetic active ingredients such as Sibelium R , antidepressants such as Laroxyl R , or anti-epileptic active ingredients such as Depakin R , administered.
  • analgesics such as Aspirin R , Paracetamol or Optalidon R , non-steroidal anti-inflammatory drugs such as Cebutid R , Voltaren R , B-40 R , Ponstyl R , Profenid R , Apranx R and Na-prosin R are used to relieve pain and inflammation , Ergot alkaloids, such as ergotamine, dihydroergotamine, which can trigger a vasoconstriction, or substances of the triptan family, such as sumatriptan, Naramig R , and AscoTop R with high affinity for 5-HTID receptors. The latter substances act as an agonist and block vasodilation.
  • Non-opioid analgesics often have side effects.
  • the complex mechanism of action of the ergot alkaloids leads to side effects such as hypertension and gangrene due to the strong peripheral vasoconstriction.
  • Compounds belonging to the triptan family are also not entirely satisfactory (Pfaffenrath V. Münch. Med. Wschr. 625-626 (1998)).
  • Sumatriptan Imigran R
  • This active ingredient is ineffective in 28% of the patients; the oral doses are very high at 50-100 mg. Coronary vasopasms, hypertension, kidney and liver disorders have become known as contraindications.
  • the present invention therefore relates to selective binding partners for 5-HT5 receptors, which are characterized in that their binding affinity for 5-HT5 receptors is greater than for one or more 5-HT5 receptors different from 5-HT5.
  • binding partner for 5-HT5 receptors describes substances which bind to 5-HT5 receptors and can therefore also be referred to as 5-HT5 receptor ligands.
  • irreversible interactions between binding partner and receptor can also be considered, e.g. covalent bonds.
  • Selectivity means the property of a binding partner, preferably to bind to 5-HT5 receptors.
  • binding partners have binding affinities for 5-HT5 receptors that are greater than for one or more 5-HT5 receptors other than 5-HT5, in particular the 5-HT receptor classes 5-HT1, 5-HT2, 5 mentioned above -HT3, 5-HT4, 5-HT6 and 5-HT7 assignable receptors. If the binding affinity for 5-HT5 receptors of a binding partner is greater than that of a 5-HT receptor different from 5-HT5, one speaks of a selective binding of the 5-HT receptor different from 5-HT5 Binding partner to 5-HT5 receptors.
  • Special binding partners are those whose binding affinity for 5-HT5 receptors is greater than for at least one and in particular all 5-HTI receptors, in particular for 5-HT1D and / or 5-HTIB receptors. Binding partners whose binding affinity for 5-HT5 receptors is greater than for all 5-HT5 receptors different from 5-HT5 represent another special class of binding partners.
  • the binding affinities for 5-HT5 receptors on the one hand and for one or more 5-HT5 receptors different from 5-HT5 on the other hand differ sufficiently. Affinity differences are preferred, according to which binding affinity ratios of at least 2, more advantageously of at least 5, particularly advantageously of at least 10, preferably of at least 20, particularly preferably of at least 50 and in particular of at least 100 are present.
  • binding partners according to the invention competitively inhibit the binding of comparative binding partners, such as 5-HT (5-hydroxytryptamine) or 5-CT (5-carboxamidotryptamine), to 5-HT5 receptors.
  • comparative binding partners such as 5-HT (5-hydroxytryptamine) or 5-CT (5-carboxamidotryptamine
  • ком ⁇ онентs according to the invention with a comparative binding partner, in the present case e.g. 5-HT or 5-CT to compete for binding to the receptor, i.e. the bond of one hinders the bond of the other.
  • a comparative binding partner in the present case e.g. 5-HT or 5-CT to compete for binding to the receptor, i.e. the bond of one hinders the bond of the other.
  • binding partners according to the invention inhibit the binding of comparison binding partners, such as 5-HT (5-hydroxytryptamine) or 5-CT (5-carboxamidotryptamine), to 5-HT5 receptors in a non-competitive manner.
  • comparison binding partners such as 5-HT (5-hydroxytryptamine) or 5-CT (5-carboxamidotryptamine
  • Non-competitive inhibition means that binding partners according to the invention bind a comparative binding partner via their binding to the receptor, in the present case, for example 5-HT or 5-CT, modulate, especially reduce its binding affinity.
  • binding partners that can be used according to the invention therefore have a high binding affinity for 5-HT5 receptors.
  • Such a binding affinity allows, on the one hand, an effective displacement of naturally occurring binding partners for 5-HT5 receptors, such as, for example, serotonin (5-hydroxytryptamine, 5-HT) itself, the required concentration of binding partner which can be used according to the invention being used to bind a certain amount of this binding partner 5-HT5 receptors decrease with increasing binding affinity.
  • Binding partners are therefore preferably administered in daily doses of approximately 0.01 to 100 mg / kg body weight and in particular of approximately 0.1 to 15 mg / kg body weight when administered parenterally and 1 to 30 mg / kg body weight when administered orally.
  • the competitive inhibition of the binding of 5-CT to 5-HT5 receptors can also be evaluated to the extent that binding partners preferred according to the invention have half-maximum inhibition constants IC 50 of less than 10 ⁇ 7 M, preferably less than 10 -8 M and in particular have less than 10- 9 M.
  • binding affinity of binding partners according to the invention can also be expressed by means of the inhibition constant Ki, which is generally also determined using competition experiments in vitro.
  • Ki inhibition constant
  • binding partners according to the invention preferably have Ki values of less than 10 -8 M, advantageously less than 10 -9 M and particularly preferably less than 10 " 10 M. Ki values of compounds according to the invention are for example in the range of l-lO -7 M to 7-10 -7 M or in the range of 1-10- 8 M to 1-10 "7 M.
  • binding partners according to the invention bind selectively to 5-HT5 receptors with the advantageous binding affinities described above with respect to one or more 5-HT5 receptors other than 5-HT5.
  • binding partners according to the invention bind selectively to 5-HT5 receptors with the advantageous binding affinities described above with respect to all 5-HT receptors other than 5-HT5.
  • Binding partners which bind to the 5-HT5 receptors with the affinities and selectivities described above and which are expressed by glial cells and in particular by astrocytes are particularly advantageous.
  • the human receptor variant is the preferred target for the binding partners used.
  • binding partners according to the invention to 5-HT5 receptors is coupled to an effector function.
  • Binding partners can act agonistically or antagonistically as well as partially agonistically and / or partially antagonistically.
  • agonists are compounds which completely or partially mimic the activity of 5-HT at 5-HT5 receptors.
  • antagonists are compounds which can block the agonistic activity of 5-HT at 5-HT5 receptors.
  • 5-HT5 agonists are provided as binding partners.
  • the expression "5-HT5 agonist” denotes binding partners which trigger a partially to fully agonistic effect.
  • a binding partner that triggers a partially agonistic effect on the 5-HT5 receptor has sufficient agonistic activity to be able to be administered in an acceptable amount in the context of an effective medical treatment.
  • binding partners with at least one are preferred.
  • binding partners with at least 80% agonistic activity and particularly those with an essentially fully agonistic activity (E max ) are particularly preferred.
  • binding partners are made available whose binding at least to 5-HT5 receptors of h5-HT5-transfected CHO cells stimulates GTP binding to membrane-bound G proteins, an agonist-induced change in intracellular calcium Level, an induction of phospholipase C activity and / or an agonist-induced change in cAMP production.
  • the use of binding partners which bring about an increase in intracellular calcium levels represents a particular embodiment of the invention.
  • This embodiment also includes binding partners which are effective in known animal models for cerebrovascular diseases, in particular for migraines, and / or which induce certain in vivo effects in brain areas, in particular which cause genomic responses in the brain, for example the expression of Transcription factors such as c-fos, c-jun, zif268 or Homergen isoforms (Brakeman PR et al., Nature 386: 284-288 (1997)).
  • binding partners which are effective in known animal models for cerebrovascular diseases, in particular for migraines, and / or which induce certain in vivo effects in brain areas, in particular which cause genomic responses in the brain, for example the expression of Transcription factors such as c-fos, c-jun, zif268 or Homergen isoforms (Brakeman PR et al., Nature 386: 284-288 (1997)).
  • Binding partners are preferred which are also selective for 5-HT5 receptors with regard to their effector function in the sense described above.
  • 5-HT5 binding partners according to the invention are low molecular weight, mostly synthetic compounds.
  • 5-HT5 binding partners according to the invention are 5-HT5-specific antibodies. It can be polyclonal antisera, monoclonal antibodies, antibody fragments such as F (ab), Fc, etc. , chimeric and recombinant antibodies. Such antibodies can be produced in a manner known per se. 5-HT5 receptor as such or antigenic fragments thereof, which are generally coupled to customary carrier proteins, can be used as the immunogen.
  • 5-HT5 binding partners according to the invention are aptamers, ie nucleic acids, usually oligonucleotides, with sufficient affinity for 5-HT5 receptors.
  • Assays for determining the binding affinities of binding partners according to the invention for 5-HT5 receptors are known in principle. This can be done, for example, by evaluating the competitive inhibition of the binding of a comparative binding partner to 5-HT5 receptors by the substance to be examined. As a sales Similar binding partners are known ligands for 5-HT receptors, such as 5-HT or 5-CT or LSD. These are appropriately marked so that their binding to 5-HT receptors can be followed analytically using standard methods. Radioactive and optical markings are preferred. At attachment studies
  • 5-HT5 receptors are used according to the invention 5-CT or LSD, in particular in the form of [ 3 H] -LSD.
  • the binding affinities can be expressed as the half-maximum inhibition constant IC 50 or as the inhibition constant Ki. This method is used primarily for primary screening. SPA or FlashPlate technology is preferred.
  • the binding partner to be examined can also be determined directly on 5-HT receptors.
  • Inhibitory constants Ki expressing the binding affinity can, for example, be calorimetric, i.e. by measuring the released binding energy.
  • the binding affinity of the binding partners to be investigated for other 5-HT receptors is determined in the same way, if appropriate using the ligands specific for the respective receptor, and the values obtained are compared.
  • Effector functions can also be evaluated qualitatively or quantitatively using known functional assays both in vitro and in vivo.
  • the assessment of agonistic activity can be based on all those effects which are caused by the binding of 5-HT to 5-HT5 receptors. It is preferred according to the invention to evaluate the effects on the binding of GTP to G proteins, on intercellular calcium levels, on the phospholipase C activity and / or on the cAMP production. These methods are preferably used for secondary screening. Here too, SPA or FlashPlate technology is used to advantage.
  • GTP binding to G proteins can be examined using a non-hydrolyzable analog of GTP, for example [ 35 S] GTP ⁇ S, the binding of which can be examined radiologically. This investigation is preferably carried out on membranes having 5-HT5 receptor.
  • Suitable calcium probes usually calcium chelators, for example fluorescent compounds such as fura-2-acetyl methyl ester or fluo-3-AM, can be used to measure intracellular calcium levels. This investigation is preferred 5-HT5 receptor-containing cell cultures, in particular carried out on single cells.
  • the phospholipase C activity can be determined on the basis of the reactions it catalyzes, for example the incorporation of myo-inositol, which is preferably radioactively labeled as [ 3 H] -myo-inosol for detection purposes, or the conversion of PPIP 2 to IP 3 , with PPIP 2 also preferably being radioactively labeled as [ 32 P] PIP 2 .
  • myo-inositol which is preferably radioactively labeled as [ 3 H] -myo-inosol for detection purposes
  • PPIP 2 also preferably being radioactively labeled as [ 32 P] PIP 2 .
  • cAMP The production of cAMP can be determined using the cAMP binding protein. This investigation is preferably carried out on single cells having 5-HT5 receptor.
  • the effector function is also determined, i.e. the activity of binding partners according to the invention for other 5-HT receptors. This is expediently done taking into account the binding affinities determined for 5-HT5 and other 5-HT receptors, that is to say in particular taking into account the selectivity.
  • the present invention therefore also relates to methods for identifying and characterizing binding partners according to the invention.
  • These and other similarly suitable methods can form the basis for in vitro screening methods which can be used to select from a large number of different compounds those which appear to be the most promising with regard to future use.
  • combinatorial chemistry extensive substance banks can be created that include myriads of potential active substances.
  • the screening of combinatorial substance libraries for substances with the desired activity can be automated. Screening robots are used for the efficient evaluation of the individual assays, which are preferably arranged on microtiter plates.
  • the present invention also relates to screening methods, i.e. Both primary ice and secondary screening methods, in which at least one of the methods described below is preferably used. If several methods are used, this can be done at different times or simultaneously on one and the same sample or on different samples of a substance to be examined.
  • a particularly effective technology for carrying out such methods is the scintillation proximity assay known in the field of active substance screening, or SPA for short. Kits and components for performing this assay can be obtained commercially at Amersham Pharmacia Biotech.
  • solubilized or membrane-bound receptors are immobilized on small fluoromicrospheres containing scintillating substance. If, for example, a radioligand binds to the immobilized receptors, the scintillation substance is stimulated to emit light, since the spatial proximity between the scintillation substance and the radioligand is given.
  • FlashPlate R Another particularly effective technology for carrying out such processes is the FlashPlate R technology known in the field of active substance screening. Kits and components for performing this assay can be obtained commercially, for example from NEN R Life Science Products. This principle is also based on microtiter plates (96 or 384), which are coated with scintillation substance.
  • a first method according to the invention serves to determine the affinity and / or selectivity of binding partners for 5-HT5 receptors.
  • the binding partner is brought into contact with 5-HT5 receptors and the binding affinity is determined.
  • Another method according to the invention relates to the determination of the activity of binding partners for 5-HT5 receptors, i.e. the determination of agonistic, partially agonistic, antagonistic and / or partially antagonistic effects.
  • the binding partner is brought into contact with 5-HT5 receptors and the effects caused by the binding are evaluated.
  • binding partners are subjected to primary screening by determining their binding affinity for 5-HT5 receptors with the above-described [ 3 H] -5-CT or [ 3 H] -LSD competition experiment. Those binding partners which have an inhibition constant IC 50 in the range of 10 ⁇ 6 M or less are then subjected to a secondary screening by their effector function, in particular with regard to the GTP binding and / or the intracellular calcium levels in the evaluated way described above. Finally, the binding partners selected in this way are subjected to a counter-screening for the determination of selectivity by determining their binding affinity for further 5-HT receptors essentially in the manner described above - if appropriate using the ligands specific for the respective receptor.
  • [ 3 H] -8-hydroxy-di-propylamino- Use tetralin [ 3 H] -8-DPAT) for binding studies on 5-HTlA receptors, while 5-HT1B and 5-HTID receptors can be tested with [ 3 H] -5-CT competition experiments.
  • 5 5-HT5 receptors are preferably provided in the form of cellular systems, i.e. in the form of membranes, cells, cell groups, tissues or organs that carry 5-HT5 receptors.
  • cellular systems can naturally express 5-HT5 receptors, but they can also be expressed by suitable genetic methods.
  • 15 glioma cell lines are preferred as natural, 5-HT5 receptor cellular systems.
  • h5-HT5 transfected heterologous cell lines preference is given to those which stably express the h5-HT5 gene. Examples include h5-HT5-transfected CHO cells, h5-HT5-transfected human cells
  • h5-HT5 transfected HEK293 cells or h5-HT5 transfected C-6 glioma cells.
  • Brain tissue sections and native membranes from brain parts can also be used to determine the selectivity, affinity and activity of binding partners according to the invention. If radioactive markers are used, tissue sections are preferably evaluated autoradiographically.
  • binding partners according to the invention for the treatment of migraines is preferably determined using animal models which are based on mechanisms which can be the basis for the development of migraines.
  • This assay can be carried out in a manner known per se, for example by fluorescent staining (Johnson KW and Phebus LA (1998) J. Neurosci. Methods 81: 19-24) or isotopically labeled albumin (Petty MA et al., (1997) Eur. J. Pharmacol. 336: 40 127-136).
  • the animals are treated successively with the test compound and with [ 125 I] albumin or fluorescent dye.
  • the trigeminal ganglion is then electrically stimulated.
  • the radioactivity or fluorescence accumulated in the dura mater is determined.
  • the anti-migraine activity of binding partners according to the invention is shown by the protein extravasation induced by stimulation of the triminal ganglion.
  • Another model is based on the distribution of carotid blood flow. This test is also known per se (Saxena PR et al., (1998) Eur. J. Pharmacol. 351: 329-339).
  • radioactive microspheres are injected into the carotid. Following a stabilization phase, the animals are treated with a test compound and the radioactivity accumulated in the brain is determined in a micro-ß counter.
  • nitroglycerin-induced c-fos gene expression and translocation is also suitable.
  • animals are treated with nitroglycerol and the compound to be tested.
  • the c-fos gene expression and protein translocation into the nucleus is determined with in situ hybridization or immunohistochemically (Garcia-Ladona FJ et al., (1994) Mol. Brain Res. 21: 75-84; Garcia-Ladona FJ et al., (1997) J. Neurosci. Res 50: 50-61).
  • 5-HT5 binding partners includes a method in the context of the treatment.
  • An effective amount of one or more 5-HT5 binding partners is administered to the individual to be treated, preferably a mammal, in particular a human, useful or domestic animal. Whether such treatment is indicated and in what form it must be carried out depends on the individual case and is subject to a medical assessment (diagnosis) to develop the existing signs, symptoms and / or malfunctions, risks, certain signs, symptoms and / or malfunctions , and other factors.
  • the treatment is usually carried out by single or repeated daily administration, optionally together or alternating with other active substances or preparations containing active substances, so that an individual to be treated receives a daily dose of about 0.001 g to 10 g, preferably of about 0.001 g to about 1 g - to be led.
  • the invention also relates to the production of pharmaceutical compositions for the treatment of an individual, preferably a mammal, in particular a human, useful or domestic animal.
  • the binding partners according to the invention are usually administered in the form of pharmaceutical compositions which contain a pharmaceutically acceptable excipient with at least one include binding partner and optionally other active ingredients.
  • These compositions can be administered, for example, by the oral, rectal, transdermal, subcutaneous, intravenous, intramuscular or intranasal routes.
  • suitable pharmaceutical formulations are solid pharmaceutical forms, such as powders, powders, granules, tablets, troches, sachets, cachets, dragees, capsules such as hard and soft gelatin capsules, suppositories or vaginal pharmaceutical forms, semi-solid pharmaceutical forms such as ointments, creams, hydrogels, pastes or plasters, and liquid pharmaceutical forms, such as solutions, emulsions, in particular oil-in-water emulsions, suspensions, for example lotions, injection and infusion preparations, eye and ear drops.
  • Implanted delivery devices can also be used for the administration of binding partners according to the invention. Liposomes, microspheres or polymer matrices can also be used.
  • binding partners according to the invention are usually mixed or diluted with an excipient.
  • Excipients can be solid, semi-solid or liquid materials that serve as vehicles, carriers or media for the active ingredient.
  • Suitable excipients include, for example, lactose, dextrose, sucrose, sorbitol, mannitol, starches, acacia, calcium phosphate, alginates, tragacanth, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup and methyl cellulose.
  • the formulations can be pharmaceutically acceptable carriers or customary auxiliaries, such as lubricants, for example tallow, magnesium stearate and mineral oil; Wetting agents; emulsifying and suspending agents; preservatives such as methyl and propyl hydroxybenzoates; Antioxidants; Anti-irritants; Chelating agents; Coating aids; Emulsion stabilizers; Film formers; Gelling agent; Odor masking agents; Taste corrections; Resins; Hydrocolloids; Solvents; Solubilizer; Neutralizing agents; Permeation accelerator; Pigments; quaternary ammonium compounds; Refatting and overfatting agents; Ointment, cream or oil base materials; Silicone derivatives; Spreading aids; Stabilizers; Sterilizers; Fundamentals of the suppository; Tablet excipients such as binders, fillers, lubricants, disintegrants or coatings; Blowing agent; Desiccants; Opacifiers; Thickeners; Waxes;
  • the present invention also relates to the use of a binding partner for 5HT-5 receptors and, in particular, the above-mentioned particular and preferred embodiments of binding partners according to the invention for the treatment of migraine-like cerebrovascular disorders, especially for the treatment of migraines and other vascular headaches, in particular seizure-like headaches of the migraine type.
  • migraine-like cerebrovascular disorders especially for the treatment of migraines and other vascular headaches, in particular seizure-like headaches of the migraine type.
  • migraine-like cerebrovascular disorders especially for the treatment of migraines and other vascular headaches, in particular seizure-like headaches of the migraine type.
  • migraine-like cerebrovascular disorders especially for the treatment of migraines and other vascular headaches, in particular seizure-like headaches of the migraine type.
  • migraine-like cerebrovascular disorders especially for the treatment of migraines and other vascular headaches, in particular seizure-like headaches of the migraine type.
  • migraine-like cerebrovascular disorders especially for the treatment of migraines and other vascular headaches
  • Treatment is understood to mean both prophylactic therapy, in particular the prevention of recurring attacks, for example as an interval treatment, and treatment for acute symptoms, in particular during the headache phases.
  • Successful treatment leads to a reduction in the intensity of symptoms, in particular headache, neurological dysfunction, e.g. visual, sensory, motor and speech disorders, nausea and nausea, and / or the frequency of attacks.
  • Acute treatment of migraines with attachment partners is preferred.
  • the invention in particular also relates to the use of the binding partners mentioned for the treatment of such forms of the diseases listed above, in whose formation and / or course 5-HT5 receptors are involved, i.e. Diseases that are modulated by 5-HT5 receptor activity.
  • disorder in the sense according to the invention denotes anomalies which are generally regarded as pathological conditions and can be identified in the form of certain signs, symptoms and / or malfunctions.
  • the treatment according to the invention can be directed to individual disorders, i.e. anomalies or pathological conditions, but it is also possible for a number of anomalies which may be causally linked to one another, i.e. Syndromes can be summarized, which can be treated according to the invention.
  • Treatment in the sense of the invention includes not only the treatment of acute or chronic signs, symptoms and / or malfunctions but also preventive treatment (prophylaxis), in particular as relapse or phase prophylaxis.
  • Thieves- action can be symptomatic, for example as symptom suppression. It can be short-term, medium-term, or it can also be long-term treatment, for example in the context of maintenance therapy.
  • Binding partners which can be used for migraine treatment can bind to 5-HT5 with a lower, substantially the same or a higher affinity than to a specific receptor different from 5-HT5.
  • binding partners for 5-HT5 receptors include, in particular, those whose binding affinity for 5-HT5 receptors compared to the affinity for 5-HTI receptors, in particular 5-HTIB and / or 5-HTID is so high that they are advantageously suitable for the use according to the invention. This does not necessarily require a comparatively more selective binding to 5-HT5 receptors, although selective binding partners for 5-HT5 receptors are a special embodiment of the present invention.
  • binding partners can be used which are highly affine to both 5-HT5 and 5-HTI receptors, in particular to 5-HT1B and / or 5-HTID.
  • high affinity means Ki values generally in the range from 1'10 -9 M to 1'10 -6 M.
  • useful binding partners in the high-affinity range to 5-HT receptors have a binding profile that is characterized by a binding affinity for 5-HT5 which is essentially the same or only slightly less than other binding affinities in this area. Factors of 10 or less can be beneficial.
  • the selective 5-HT5 binding partners described above are used.
  • binding partners are used whose binding at least to 5-HT5 receptors of h5-HT5-transfected CHO cells is a change in the agonist-induced stimulation of GTP binding to membrane-bound G proteins, a change in intracellular Calcium levels, a change in the agonist-induced induction of phospholipase C activity and / or a change in cAMP production.
  • the change in intracellular calcium levels the use of binding partners which bring about an increase in intracellular calcium levels represents a particular embodiment of the invention. The present invention is explained in more detail with reference to the following examples, without being restricted thereto.
  • the gene coding for the human 5-HT5 receptor was isolated from human tissues in a manner known per se using 3'-5'-RT-PCR (RACE system, Boehringer Mannheim). The gene sequence was then inserted into a plasmid carrying the neomycin resistance gene (pcDNA3; Invitrogen, Germany) and amplified in E. coli according to the manufacturer's instructions. A preparation of the resulting plasmid was mixed with Lipofectamin R (Gibco Life Sciences, Germany) and HEK293 cells were incubated in Petri dishes (2.5 cm) with a thin layer of this transfection mixture. The transfection mixture was then replaced by culture medium containing neomycin.
  • RACE system 3'-5'-RT-PCR
  • Surviving cells were further cultured in DMEM-F12 medium supplemented with 10% fetal calf serum, 2 mM glutamine and antibiotics (90 mg streptomycin, 90 mg penicillin). The cells were grown to confluence at 5% CO 2 , 95% humidity and 37 ° C.
  • CHO cells expressing h5-HT5 receptor are obtained in an analogous manner.
  • the method used here is essentially based on known methods for the preparation of cell membranes from cells (Findlay JBC and Evans WH Biological Membranes, Practical Approach (1987)).
  • the cells cultured according to reference example 1 were carefully scraped off the surface of the culture vessel and centrifuged for 10 min at 180 ⁇ g in DMEM-F12 medium.
  • the cell pellets obtained were resuspended in 5 mM Tris-HCl buffer (pH: 7.6; buffer A) containing 5 mM EDTA, 5 mM EGTA, 0.1 mM PMSF and 3 mM benzamidine and at 15 ° C. for 15 min incubated.
  • the cell suspension was homogenized in an Ultraturrax R (15000 rpm) (6x3s) and centrifuged for 1 min at 100Xg and 4 ° C.
  • the pellet was resuspended in buffer A and homogenized and centrifuged as described above.
  • the supernatants from both steps were collected and centrifuged for 20 min at 40000xg and 4 ° C.
  • the pellet was resuspended in buffer A and homogenized (1x15s).
  • the membrane suspension was centrifuged at 40000xg and 4 ° C for 20 min. yaws.
  • the resulting pellet was resuspended in buffer A containing 10% glycerin and 1% bovine serum albumin. Aliquots were frozen and stored at -80 ° C until use.
  • the nonspecific binding was defined as the binding of [ 3 H] -5-CT that was observed in the presence of 10 ⁇ M methiothepin. Similar systems can also be used which, through the use of microtiter plates, permit high sample throughput and secondary screening.
  • the saturation parameters of the [ 3 H] -5-CT binding were determined both by non-linear regression analysis and from linear plots using the Sig aPlot software (Jandel Scientific, Germany). Competition curves were set up in which the radioactive binding is expressed as a percentage of the total binding.
  • Half-maximum inhibition constants IC 50 and Hill coefficients ( ⁇ x ü ) were determined by means of non-linear regression analysis.
  • 96-well FlashPlates coated with h5-HT5 membranes are available from Bio Signal Inc. (Canada).
  • [3H] -LSD was diluted to an appropriate concentration in Tris-HCl buffer containing 10 mM MgCl 2 , 0.5 mM EDTA and 0.5% BSA. The radio ligand solution was added to the wells (25 ml), which either contained test compound or not. The plates were incubated for 180 minutes at room temperature and the radioactive signal was measured with a micro- ⁇ counter (Wallac). The non-specific binding was determined with methiothepin.
  • [3H] -LSD has an affinity of 12 nM. The radioactive signal of [3H] -LSD decreased with increasing binding affinity of the test compound.
  • [ 35 S] GTP ⁇ S binding assays are known. The present assay was carried out based on the previously described method by Hilf, G. and Jakobs, KH (Eur. J. Mol. Pharmacol. 225: 245-252 (1992)). Drug-induced changes in the [ 35 S] GTP ⁇ S binding to membranes from HEK293 cells stably transfected with the h5HT5 receptor gene were measured (see reference examples 1 and 2).
  • the cell membranes (12 ⁇ g) were incubated with 6.75 mM MgCl 2 , 150 mM NaCl, 1 mM DTT, 1 mM EDTA, 10 ⁇ M GDP and 50 mM triethanolamine-HCl buffer (pH: 7.5) containing [ 35 S] GTP ⁇ S . Following a 60-minute incubation at 30 ° C. with or without the addition of the active substances to be tested, the test mixture (100 ⁇ l) was quickly filtered through GF-B filters using a Skatron R filtration device.
  • the filters were quickly washed with 50 mM Tris-HCl buffer (9 ml; pH: 7.5; 4 ° C) containing 100 mM NaCl and 5 mM MgCl 2 .
  • the radioactivity retained on the filters was determined by scintillation spectrometry, using Ultima Gold scintillation fluid. Similar systems can also be used. allow high throughput and secondary screening.
  • the drug activities were expressed as a percentage of the basic bond measured in the absence of the drug.
  • HEK293 cells expressing h5HT5 receptor were grown in culture vessels. The cells were carefully scraped off before they were confluent. The cells were labeled with Fura-2 by incubating with Fura-2-acetylmethyl ester (Sigma) at room temperature. The cells were centrifuged at 180 ⁇ g for 10 min and resuspended in DMEM-F12 medium without serum and incubated at 37 ° C., 5% CO 2 and 95% atmospheric humidity for 45 min.
  • Intracellular calcium levels were determined using a fluorescence microscope equipped with a suitable filter exchange system (Olympus / Hamamatsu). The fluorescence ratio (340 n / 380 nm) was determined using the Argus R software. The intracellular calcium levels were observed for a short time in individual cells without the addition of active substances and then 30 minutes after the addition of the active substance to be tested. Similar systems can also be used, which allow high throughput and secondary screening through the use of microtiter plates.
  • h5-HT5 receptor-expressing CHO cells were cultivated overnight in 96-well plates (30,000-80,000 cells / well). The cells were labeled with HEPES buffer containing 1mM Fluo-3-AM, 10% pluronic acid and 2.5mM sample eep for one hour and washed. A test compound was added to each well. To determine the calcium level, the fluorescence intensity was measured using a fluorometric Read fluorometric imaging plate reader (FLIPR).
  • FLIPR fluorometric Read fluorometric imaging plate reader
  • the method is essentially known (Garcia-Ladona FJ et al., Neuroreport 4: 691-694 (1993)).
  • the cells were incubated with 0.125 ⁇ M [ 3 H] myo-inositol for 24 h. Unincorporated [ 3 H] myo-insitol was removed from the medium and replaced with Krebs-Henseleit buffer containing 10 mM LiCl. After 10 minutes of incubation, the active ingredient to be tested was added. After 45 minutes, the reaction was stopped by replacing the stimulation medium with distilled water.
  • a similar procedure is used when using tissue samples (Garcia-Ladona et al., 1993). The cells were frozen and stored at -80 ° C.
  • test animals were decapitated 90 min after administration of the active ingredient (oral, intraperitoneal, intravenous or intracerebroventricular).
  • the entire brain was quickly removed from the skull, frozen on dry ice and stored at -80 ° C.
  • Rat brain sections (15 ⁇ m) were obtained at -20 ° C in a cryostat, applied to gelatin-coated slides and stored at -30 ° C until use.
  • FIG. 1 shows a plot of bound [ 3 H] -5-CT as a function of the [ 3 H] -5-CT concentration.
  • a dissociation constant of K ⁇ 0.570 nM was determined.
  • the receptor binding density (B) varied in a range of 900-28000 fmol / mg protein.
  • Ki IC 50 / (1 + C / K d )
  • the drug-induced binding of GTP to G proteins was investigated.
  • the coupling of 5-HT5 receptors to G proteins in HEK293 cells was evident.
  • the typical serotoninergic agonists 5-HT and 5-CT induced an increase in [ 35 S] GTP ⁇ S binding to the cell membranes of more than 40% above the basic value (see FIG. 2).
  • the 5-HT5 receptor requires GDP for the agonist-mediated coupling to G proteins (see FIG. 3A).
  • the 5-HT effect was dose-dependent (see FIG. 4) with an EC 50 of 2.6 ⁇ M.

Abstract

The invention relates to selective binding partners for 5-HT5-receptors, methods, in particular screening methods, for identifying and characterizing such binding partners, as well as pharmaceutical compositions containing said binding partners and their use in the treatment of cerebrovascular diseases such as migraine.

Description

Bindungspartner für 5-HT5-Rezeptoren zur MigränebehandlungBinding partner for 5-HT5 receptors for migraine treatment
Die vorliegende Erfindung betrifft Bindungspartner für 5-HT5-Re- zeptoren, Verfahren zur Identifizierung und Charakterisierung solcher Bindungspartner, sowie diese enthaltende pharmazeutische Zusammensetzungen und deren Verwendung zur Behandlung cerebrovas- kulärer Erkrankungen wie Migräne.The present invention relates to binding partners for 5-HT5 receptors, methods for identifying and characterizing such binding partners, and pharmaceutical compositions containing them and their use for the treatment of cerebrovascular diseases such as migraines.
Wenigstens sieben verschiedene Rezeptorklassen vermitteln die mannigfaltigen physiologischen Aktivitäten, die einer Beteiligung des Neurotransmitters Serotonin (5-Hydroxytryptamin, kurz 5-HT) zugeschrieben werden. Sie werden entsprechend einer international anerkannten Klassifikation mit 5-HT1, 5-HT2, 5-HT3, 5-HT4, 5-HT5, 5-HT6 und 5-HT7 bezeichnet. Die meisten dieser Klassen umfassen darüber hinaus weitere unterscheidbare Rezeptortypen. So gehören zur 5-HTl-Klasse Rezeptoren, welche sich in wenigstens fünf Unterklassen einteilen lassen, die mit 5-HT1A, 5-HT1B, 5-HT1C, 5-HT1D und 5-HT1E bezeichnet werden (Boess F.G. und Martin I.L., Neuropharmacology 33:275-317 (1994)).At least seven different receptor classes mediate the diverse physiological activities that are ascribed to the involvement of the neurotransmitter serotonin (5-hydroxytryptamine, 5-HT for short). According to an internationally recognized classification, they are designated 5-HT1, 5-HT2, 5-HT3, 5-HT4, 5-HT5, 5-HT6 and 5-HT7. Most of these classes also include other distinguishable receptor types. The 5-HTI class includes receptors, which can be divided into at least five subclasses, which are designated 5-HT1A, 5-HT1B, 5-HT1C, 5-HT1D and 5-HT1E (Boess FG and Martin IL, Neuropharmacology 33: 275-317 (1994)).
Die 5-HT5-Klasse wurde erstmals beschrieben von Plassat et al., The EMBO Journal Bd. 11 Nr. 13, S. 4779-4786 (1992). Man unterscheidet 5-HT5A- und 5-HT5B-Rezeptoren (Erlander et al., Proc. Natl. Acad. Sei. USA 90:3452-3456 (1993)). Es bestehen nur geringe Sequenzhomologien zwischen 5-HT5 und anderen 5-HT-Rezepto- ren. Das pharmakologische Profil dieser Rezeptoren unterscheidet sich deutlich. Mit molekularbiologischen Techniken gelang die Lokalisierung von 5-HT5-Rezeptoren im Riechkolben, im Hippocampus, im Cortex, in den Zerebralventrikeln, im Corpus Callosum und im Kleinhirn. Mittels immunhistochemischer Methoden konnte gezeigt werden, daß 5-HT5-Rezeptoren vornehmlich auf Astrocyten expri- miert werden (Carson et al., GLIA 17:317-326 (1996)). Astrocyten liegen direkt an der Basalmembran von Gehirnkapillaren der Blu- thirnschranke an. Eine anormale Astrocyten-Endothelium-Struktur geht mit einem Verlust der Bluthirnschranke einher. Die genaue Bedeutung der Astrocyten ist unklar. Sie scheinen Transportaufgaben und konnektive Funktionen wahrzunehmen. Reaktive Astrocyten wurden in Zusammenhang mit reaktiver Gliosis bei einer Reihe von pathologischen Gehirnveränderungen und neuropsychiatrischen Erkrankungen beobachtet. Infolge von Gehirnverletzungen verändern sie ihre Morphologie. Das Proteinexpressionsmuster ändert sich und Wachstumsfaktoren werden produziert. In vitro-Untersuchungen an kultivierten Astrocyten haben 5-HT5-Rezeptor-vermittelte Ant- worten erkennen lassen. So wird einerseits vermutet, daß sie an Erholungsprozessen des Gehirns nach. Störungen beteiligt sind, an- dererseits ist aber auch nicht auszuschließen, daß sie zur Schadensentstehung oder sogar zu einer Schadensvermehrung beitragen.The 5-HT5 class was first described by Plassat et al., The EMBO Journal Vol. 11 No. 13, pp. 4779-4786 (1992). A distinction is made between 5-HT5A and 5-HT5B receptors (Erlander et al., Proc. Natl. Acad. Sci. USA 90: 3452-3456 (1993)). There are only minor sequence homologies between 5-HT5 and other 5-HT receptors. The pharmacological profile of these receptors differs significantly. Molecular biological techniques successfully localized 5-HT5 receptors in the olfactory bulb, in the hippocampus, in the cortex, in the cerebral ventricles, in the corpus callosum and in the cerebellum. Using immunohistochemical methods it could be shown that 5-HT5 receptors are primarily expressed on astrocytes (Carson et al., GLIA 17: 317-326 (1996)). Astrocytes lie directly on the basement membrane of brain capillaries of the blood-brain barrier. An abnormal astrocyte endothelium structure is associated with a loss of the blood-brain barrier. The exact meaning of the astrocytes is unclear. They seem to carry out transport tasks and connective functions. Reactive astrocytes have been observed in association with reactive gliosis in a number of pathological brain changes and neuropsychiatric disorders. As a result of brain injuries, they change their morphology. The protein expression pattern changes and growth factors are produced. In vitro studies on cultured astrocytes have shown 5-HT5 receptor-mediated responses. On the one hand, it is suspected that they follow brain recovery processes. Disturbances are involved on the other hand, it cannot be ruled out that they contribute to the occurrence of damage or even to an increase in damage.
Migräne ist eine ZNS-Erkrankung, die große Bevölkerungsteile be- trifft. Sie äußert sich in den meisten Fällen durch immer wiederkehrende Kopfschmerzen, von denen schätzungsweise 8 Mio Personen, d.h. 3-5 % aller Kinder, 7% aller Männer und 14% aller Frauen, betroffen sind. Wenngleich eine genetische Prädisposition propagiert wird, scheinen die Ursachen doch vielschichtig zu sein (Diener H.C. et al., Arzneimitteltherapie 15:387-394 (1997)).Migraine is a CNS disease that affects large parts of the population. It manifests itself in most cases by a recurring headache, of which an estimated 8 million people, i.e. 3-5% of all children, 7% of all men and 14% of all women are affected. Although a genetic predisposition is propagated, the causes seem to be complex (Diener H.C. et al., Arzneimitteltherapie 15: 387-394 (1997)).
Es dominieren zwei Hypothesen. Die schon seit langem bekannte Gefäß-Theorie schlägt als Ursache einen Dilatationsvorgang des inneren und äußeren zerebralen Gefäßsystems vor. Die neurogene Theorie stützt sich auf eine Ausschüttung vasoaktiver Neurotrans- mitter, vornehmlich Neuropeptide, wie Substanz p and Neurokinin, aus Axonen der Vaskulatur infolge einer Stimulierung bestimmter Gehirngewebe innervierender Ganglien, was zu entzündlichen Reaktionen und somit zu Schmerz führen soll.Two hypotheses dominate. The vascular theory, which has been known for a long time, suggests a dilation process of the inner and outer cerebral vascular system as the cause. The neurogenic theory is based on a release of vasoactive neurotransmitters, primarily neuropeptides such as substance p and neurokinin, from axons of the vasculature as a result of stimulation of certain brain tissue innervating ganglia, which is said to lead to inflammatory reactions and thus to pain.
Eine Kausaltherapie zur Behandlung von Migräne gibt es derzeit noch nicht. Zwei verschiedene Behandlungsmethoden kommen momentan zur Anwendung. Eine erste, prophylaktische, Therapie zur Vorbeugung gegen wiederkehrende Migräneattacken und eine zweite, symp- tomatische, Therapie zur Unterdrückung akuter Symptome bei Attak- ken. Prophylaktisch werden migränespezifische Wirkstoffe, wie SanmigranR, NocertonR, DesernilR und VidoraR, aber auch gewöhnlich für andere Indikation verwendete Wirkstoffe, wie Beta-Blocker, antiemetische Wirkstoffe wie SibeliumR, Antidepressiva wie Laro- xylR, oder antiepileptsche Wirkstoffe wie DepakinR, verabreicht. Im Rahmen der Akuttherapie gibt man Analgetika, wie AspirinR, Pa- racetamol oder OptalidonR, nichtsteroidale Antiinflammatika, wie CebutidR, VoltarenR, BrufenR, PonstylR, ProfenidR, ApranxR und Na- prosinR gegen den Schmerz und Entzündungen, Ergotalkaloide, wie Ergotamin, Dihydroergotamin, die eine Vasokonstriktion auslösen können, oder Substanzen der Triptan-Familie, wie Sumatriptan, Na- ramigR, und AscoTopR mit hoher Affinität für 5-HTlD-Rezeptoren. Letztere Substanzen wirken als Agonist und blockieren die Vasodi- latation.There is currently no causal therapy for the treatment of migraines. Two different treatment methods are currently used. A first, prophylactic, therapy to prevent recurring migraine attacks and a second, symptomatic, therapy to suppress acute symptoms of attacks. Prophylactic migraine-specific active ingredients, such as Sanmigran R , Nocerton R , Desernil R and Vidora R , but also active ingredients commonly used for other indications, such as beta-blockers, antiemetic active ingredients such as Sibelium R , antidepressants such as Laroxyl R , or anti-epileptic active ingredients such as Depakin R , administered. In the context of acute therapy, analgesics such as Aspirin R , Paracetamol or Optalidon R , non-steroidal anti-inflammatory drugs such as Cebutid R , Voltaren R , Brufen R , Ponstyl R , Profenid R , Apranx R and Na-prosin R are used to relieve pain and inflammation , Ergot alkaloids, such as ergotamine, dihydroergotamine, which can trigger a vasoconstriction, or substances of the triptan family, such as sumatriptan, Naramig R , and AscoTop R with high affinity for 5-HTID receptors. The latter substances act as an agonist and block vasodilation.
Die genannten Wirkstoffe sind allerdings nicht optimal für die Behandlung von Migräne geeignet. Nichtopioide Analgetika haben häufig Nebenwirkungen. Der komplexe Wirkungsmechanismus der Ergotalkaloide führt infolge der starken peripheren Vasokonstriktion zu Nebenwirkungen wie Hypertonie and Gangrän. Zu der Triptan-Familie gehörende Verbindungen sind ebenfalls nicht völlig zufriedenstellend (Pfaffenrath V. Münch. med. Wschr. 625-626 (1998)). Sumatriptan (ImigranR), einer der effektivsten und am häufigsten eingesetzten Wirkstoffe gegen akute Migräneattacken, passiert aufgrund ausgeprägter Hydrophilie die Bluthirnschranke nicht. In 28 % der Patienten ist dieser Wirkstoff wirkungslos, die oralen Dosen liegen mit 50-100 mg recht hoch. Als Gegenanzeigen sind ko- ronare Vasopasmen, Hypertonie, Nieren- und LeberStörungen bekannt geworden.However, the active ingredients mentioned are not ideal for the treatment of migraines. Non-opioid analgesics often have side effects. The complex mechanism of action of the ergot alkaloids leads to side effects such as hypertension and gangrene due to the strong peripheral vasoconstriction. Compounds belonging to the triptan family are also not entirely satisfactory (Pfaffenrath V. Münch. Med. Wschr. 625-626 (1998)). Sumatriptan (Imigran R ), one of the most effective and most frequently used agents against acute migraine attacks, does not cross the blood-brain barrier due to its pronounced hydrophilicity. This active ingredient is ineffective in 28% of the patients; the oral doses are very high at 50-100 mg. Coronary vasopasms, hypertension, kidney and liver disorders have become known as contraindications.
Bislang hat man Verbindungen mit selektiver Affintät für 5-HTl-Rezeptoren zur Behandlung von Migräne in Betracht gezogen (Goadsby P.J. CNS Drugs 10:271-286 (1998); Saxena P.R. Exp. Opin. Invest. Drugs 581-593 (1996)). Beispielsweise gilt Sumatriptan als ausgesprochen selektiver Ligand für 5-HTlD-Rezeptoren, weshalb die Fachwelt bemüht war, diese Bindungseigenschaften und die dadurch vermittelten vasokonstriktiven Aktivitäten zu optimieren. Ähnliches gilt für 5-HT1B- und 5-HTlF-Rezeptorliganden. So wurden Serien von Wirkstoffen entwickelt, die den geschilderten Problemen allerdings auch nicht abhelfen konnten.So far, compounds with selective affinity for 5-HTI receptors for the treatment of migraines have been considered (Goadsby PJ CNS Drugs 10: 271-286 (1998); Saxena PR Exp. Opin. Invest. Drugs 581-593 (1996)) . For example, sumatriptan is considered to be a very selective ligand for 5-HTID receptors, which is why experts have tried to optimize these binding properties and the vasoconstrictive activities mediated by them. The same applies to 5-HT1B and 5-HTIF receptor ligands. This is how series of active ingredients were developed, which, however, could not remedy the problems described.
Aufgabe der vorliegenden Erfindung ist es daher, die, vorzugsweise akute, Behandlung migräneartiger cerebrovaskulärer Erkrankungen mit ausreichender Wirksamkeit und geringen Nebenwirkungen zu ermöglichen.It is therefore an object of the present invention to enable the, preferably acute, treatment of migraine-like cerebrovascular diseases with sufficient effectiveness and few side effects.
Überraschenderweise wurde nun gefunden, daß Substanzen mit vergleichsweise hoher Bindungsaffinität für 5-HT5-Rezeptoren die gewünschten Wirkungen vermitteln können.Surprisingly, it has now been found that substances with a comparatively high binding affinity for 5-HT5 receptors can mediate the desired effects.
Ein Gegenstand der vorliegenden Erfindung sind daher selektive Bindungspartner für 5-HT5-Rezeptoren, die dadurch gekennzeichnet sind, daß deren Bindungsaffinität für 5-HT5-Rezeptoren größer ist als für einen oder mehrere von 5-HT5 verschiedene 5-HT-Rezepto- ren.The present invention therefore relates to selective binding partners for 5-HT5 receptors, which are characterized in that their binding affinity for 5-HT5 receptors is greater than for one or more 5-HT5 receptors different from 5-HT5.
Der Begriff "Bindungspartner für 5-HT5-Rezeptoren" beschreibt Substanzen, welche an 5-HT5-Rezeptoren binden und daher auch als 5-HT5-Rezeptorliganden bezeichnet werden können.The term "binding partner for 5-HT5 receptors" describes substances which bind to 5-HT5 receptors and can therefore also be referred to as 5-HT5 receptor ligands.
oder metallkomplexartige koordinative Bindungen gehören. Zusätz- lieh zu den vorstehend genannten, reversiblen molekularen Wechselwirkungen können auch irreversible Wechselwirkungen zwischen Bindungspartner und Rezeptor in Betracht kommen, wie z.B. kova- lente Bindungen.or metal complex-like coordinative bonds. In addition to the reversible molecular interactions mentioned above, irreversible interactions between binding partner and receptor can also be considered, e.g. covalent bonds.
Unter Selektivität versteht man die Eigenschaft eines Bindungspartners, vorzugsweise an 5-HT5-Rezeptoren zu binden. So besitzen Bindungspartner Bindungsaffinitäten für 5-HT5-Rezep- toren, die größer sind als für einen oder mehrere von 5-HT5 verschiedene 5-HT-Rezeptoren, also insbesondere den obengenannten 5-HT-Rezeptorklassen 5-HT1, 5-HT2, 5-HT3, 5-HT4, 5-HT6 und 5-HT7 zuzuordnenden Rezeptoren. Ist die Bindungsaffinität für 5-HT5-Re- zeptoren eines Bindungspartners größer als die eines von 5-HT5 verschiedenen 5-HT-Rezeptors, so spricht man von einer in Bezug auf den von 5-HT5 verschiedenen 5-HT-Rezeptor selektiven Bindung dieser Bindungspartner an 5-HT5-Rezeptoren. Besondere Bindungs- partner sind diejenigen, deren Bindungsaffinität für 5-HT5-Rezep- toren größer ist als für wenigstens einen und insbesondere sämtliche 5-HTl-Rezeptoren, insbesondere für 5-HT1D- und/oder 5-HTlB- Rezeptoren. Bindungspartner, deren Bindungsaffinität für 5-HT5-Rezeptoren größer ist als für sämtliche von 5-HT5 verschie- dene 5-HT-Rezeptoren, stellen eine weitere besondere Klasse von Bindungspartnern dar.Selectivity means the property of a binding partner, preferably to bind to 5-HT5 receptors. For example, binding partners have binding affinities for 5-HT5 receptors that are greater than for one or more 5-HT5 receptors other than 5-HT5, in particular the 5-HT receptor classes 5-HT1, 5-HT2, 5 mentioned above -HT3, 5-HT4, 5-HT6 and 5-HT7 assignable receptors. If the binding affinity for 5-HT5 receptors of a binding partner is greater than that of a 5-HT receptor different from 5-HT5, one speaks of a selective binding of the 5-HT receptor different from 5-HT5 Binding partner to 5-HT5 receptors. Special binding partners are those whose binding affinity for 5-HT5 receptors is greater than for at least one and in particular all 5-HTI receptors, in particular for 5-HT1D and / or 5-HTIB receptors. Binding partners whose binding affinity for 5-HT5 receptors is greater than for all 5-HT5 receptors different from 5-HT5 represent another special class of binding partners.
Für die vorstehend geschilderte Selektivität ist maßgebend, daß sich die Bindungsaffinitäten für 5-HT5-Rezeptoren einerseits und für einen oder mehrere von 5-HT5 verschiedene 5-HT-Rezeptoren andererseits hinreichend unterscheiden. Bevorzugt sind Affinitätsunterschiede, wonach Bindungsaffinitäts-Verhältnisse von wenigstens 2 , vorteilhafter von wenigstens 5 , besonders vorteilhaft von wenigstens 10, vorzugsweise von wenigstens 20, besonders be- vorzugt von wenigstens 50 und insbesondere von wenigstens 100 vorliegen.For the selectivity described above it is decisive that the binding affinities for 5-HT5 receptors on the one hand and for one or more 5-HT5 receptors different from 5-HT5 on the other hand differ sufficiently. Affinity differences are preferred, according to which binding affinity ratios of at least 2, more advantageously of at least 5, particularly advantageously of at least 10, preferably of at least 20, particularly preferably of at least 50 and in particular of at least 100 are present.
Gemäß einer Ausführungsform hemmen erfindungsgemäße Bindungspartner die Bindung von Vergleichsbindungspartnern, wie 5-HT (5-Hy- droxytryptamin) oder 5-CT (5-Carboxamidotryptamin) , an 5-HT5-Re- zeptoren kompetitiv.According to one embodiment, binding partners according to the invention competitively inhibit the binding of comparative binding partners, such as 5-HT (5-hydroxytryptamine) or 5-CT (5-carboxamidotryptamine), to 5-HT5 receptors.
Unter kompetitiver Hemmung versteht man, daß erfindungsgemäße Bindungspartner mit einem Vergleichsbindungspartner, im vorlie- genden Fall z.B. 5-HT oder 5-CT, um die Bindung an den Rezeptor konkurrieren, d.h. die Bindung des einen behindert die Bindung des anderen.Competitive inhibition means that binding partners according to the invention with a comparative binding partner, in the present case e.g. 5-HT or 5-CT to compete for binding to the receptor, i.e. the bond of one hinders the bond of the other.
Gemäß einer weiteren Ausführungsform hemmen erfindungsgemäße Bin- dungspartner die Bindung von Vergleichsbindungspartnern, wie 5-HT (5-Hydroxytryptamin) oder 5-CT (5-Carboxamidotryptamin) , an 5-HT5-Rezeptoren nicht-kompetitiv.According to a further embodiment, binding partners according to the invention inhibit the binding of comparison binding partners, such as 5-HT (5-hydroxytryptamine) or 5-CT (5-carboxamidotryptamine), to 5-HT5 receptors in a non-competitive manner.
Unter nicht-kompetitiver Hemmung versteht man, daß erfindungsge- mäße Bindungspartner über ihre Bindung an den Rezeptor die Bindung eines Vergleichsbindungspartner, im vorliegenden Fall z.B. 5-HT oder 5-CT, modulieren, insbesondere dessen Bindungsaffinität verringern.Non-competitive inhibition means that binding partners according to the invention bind a comparative binding partner via their binding to the receptor, in the present case, for example 5-HT or 5-CT, modulate, especially reduce its binding affinity.
Zumindest für den Fall der kompetitiven Hemmung, also der rever- siblen Bindung gilt der Grundsatz, daß die Verdrängung eines Bindungspartners durch einen anderen mit abnehmender Bindungsaffinität des einen bzw. zunehmender Bindungsaffinität des anderen im Hinblick auf den Rezeptor zunimmt. Zweckmäßigerweise besitzen daher erfindungsgemäß verwendbare Bindungspartner eine hohe Bin- dungsaffinitat für 5-HT5-Rezeptoren. Eine derartige Bindungsaffinität gestattet einerseits eine wirksame Verdrängung natürlich vorkommender Bindungspartner für 5-HT5-Rezeptoren, wie beispielsweise Serotonin (5-Hydroxytryptamin, 5-HT) selbst, wobei die erforderliche Konzentration an erfindungsgemäß verwendbarem Bin- dungspartner zur Bindung einer bestimmten Menge dieses Bindungspartners an 5-HT5-Rezeptoren mit zunehmender Bindungsaffinität abnimmt. Im Hinblick auf die medizinische Anwendung werden daher Bindungspartner bevorzugt, deren Bindungsaffinität so groß ist, daß diese als Wirkstoff im Rahmen einer wirksamen medizinischen Behandlung in vertretbaren Mengen verabreicht werden können. Erfindungsgemäße Bindungspartner werden daher vorzugsweise in Tagesdosen von etwa 0,01 bis 100 mg/kg Körpergewicht und insbesondere von etwa 0,1 bis 15 mg/kg Körpergewicht bei parenteraler Gabe und 1 bis 30 mg/kg Körpergewicht bei oraler Gabe vera- breicht.At least in the case of competitive inhibition, that is to say reversible binding, the principle applies that the displacement of one binding partner by another increases with decreasing binding affinity of one or increasing binding affinity of the other with regard to the receptor. Appropriately, binding partners that can be used according to the invention therefore have a high binding affinity for 5-HT5 receptors. Such a binding affinity allows, on the one hand, an effective displacement of naturally occurring binding partners for 5-HT5 receptors, such as, for example, serotonin (5-hydroxytryptamine, 5-HT) itself, the required concentration of binding partner which can be used according to the invention being used to bind a certain amount of this binding partner 5-HT5 receptors decrease with increasing binding affinity. With regard to medical use, preference is therefore given to binding partners whose binding affinity is so great that they can be administered as an active ingredient in an effective medical treatment in acceptable amounts. Binding partners according to the invention are therefore preferably administered in daily doses of approximately 0.01 to 100 mg / kg body weight and in particular of approximately 0.1 to 15 mg / kg body weight when administered parenterally and 1 to 30 mg / kg body weight when administered orally.
Eine Möglichkeit, die Bindungsaffinität auszudrücken, bieten die oben angesprochenen Kompetitionsexperimente, mit denen man in vitro diejenige Konzentration an erfindungsgemäßem Bindungspartner ermittelt, die einen anderen Vergleichsbindungspartner zu 50% von der Rezeptorbindungsstelle verdrängt (IC50-Werte) . So läßt sich auch die kompetitive Hemmung der Bindung von 5-CT an 5-HT5-Rezep- toren dahingehend auswerten, daß erfindungsgemäß bevorzugte Bindungspartner halbmaximale Hemmkonstanten IC50 von weniger als 10~7 M, vorzugweise von weniger als 10-8 M und insbesondere von weniger als 10-9 M aufweisen.One possibility of expressing the binding affinity is provided by the competition experiments mentioned above, with which the concentration of binding partner according to the invention is determined in vitro which displaces another comparative binding partner by 50% from the receptor binding site (IC 50 values). Thus, the competitive inhibition of the binding of 5-CT to 5-HT5 receptors can also be evaluated to the extent that binding partners preferred according to the invention have half-maximum inhibition constants IC 50 of less than 10 ~ 7 M, preferably less than 10 -8 M and in particular have less than 10- 9 M.
Die Bindungsaffinität erfindungsgemäßer Bindungspartner kann auch über die Hemmkonstante Ki ausgedrückt werden, die man im allgemei- nen ebenfalls mit Kompetitionsexperimenten in vitro bestimmt. Für die Bindung an 5-HT5-Rezeptoren weisen erfindungsgemäße Bindungspartner vorzugsweise Ki-Werte von weniger als 10-8 M, vorteilhafterweise von weniger als 10-9 M und insbesondere bevorzugt von weniger als 10"10 M auf. Ki-Werte erfindungsgemäßer Verbindungen liegen z.B. im Bereich von l-lO-7 M bis 7-10-7 M oder im Bereich von 1-10-8 M bis 1-10"7 M. Gemäß einer weiteren Ausführungsform binden erfindungsgemäße Bindungspartner in Bezug auf einen oder mehrere von 5-HT5 verschiedene 5-HT-Rezeptoren selektiv an 5-HT5-Rezeptoren mit den oben beschriebenen vorteilhaften Bindungsaffinitäten.The binding affinity of binding partners according to the invention can also be expressed by means of the inhibition constant Ki, which is generally also determined using competition experiments in vitro. For binding to 5-HT5 receptors, binding partners according to the invention preferably have Ki values of less than 10 -8 M, advantageously less than 10 -9 M and particularly preferably less than 10 " 10 M. Ki values of compounds according to the invention are for example in the range of l-lO -7 M to 7-10 -7 M or in the range of 1-10- 8 M to 1-10 "7 M. According to a further embodiment, binding partners according to the invention bind selectively to 5-HT5 receptors with the advantageous binding affinities described above with respect to one or more 5-HT5 receptors other than 5-HT5.
Gemäß einer weiteren Ausführungsform binden erfindungsgemäße Bindungspartner in Bezug auf alle von 5-HT5 verschiedenen 5-HT-Re- zeptoren selektiv an 5-HT5-Rezeptoren mit den oben beschriebenen vorteilhaften Bindungsaffinitäten.According to a further embodiment, binding partners according to the invention bind selectively to 5-HT5 receptors with the advantageous binding affinities described above with respect to all 5-HT receptors other than 5-HT5.
Besonders vorteilhaft sind Bindungspartner, die mit den vorstehend beschriebenen Affinitäten und Selektivitäten an 5-HT5-Rezep- toren binden, die von Gliazellen und insbesondere von Astrocyten exprimiert werden.Binding partners which bind to the 5-HT5 receptors with the affinities and selectivities described above and which are expressed by glial cells and in particular by astrocytes are particularly advantageous.
Erfindungsgemäß ist die humane Rezeptorvariante bevorzugtes Target für die eingesetzten Bindungspartner.According to the invention, the human receptor variant is the preferred target for the binding partners used.
Die Bindung erfindungsgemäßer Bindungspartner an 5-HT5-Rezeptoren ist an eine Effektorfunktion gekoppelt. Bindungspartner können agonistisch oder antagonistisch sowie teilagonistisch und/oder teilantagonistisch wirken.The binding of binding partners according to the invention to 5-HT5 receptors is coupled to an effector function. Binding partners can act agonistically or antagonistically as well as partially agonistically and / or partially antagonistically.
Als Agonisten werden erfindungsgemäß Verbindungen bezeichnet, die ganz oder teilweise die Aktivität von 5-HT an 5-HT5-Rezeptoren nachahmen.According to the invention, agonists are compounds which completely or partially mimic the activity of 5-HT at 5-HT5 receptors.
Als Antagonisten werden erfindungsgemäß Verbindungen bezeichnet, welche die agonistische Aktivität von 5-HT an 5-HT5-Rezeptoren blockieren können.According to the invention, antagonists are compounds which can block the agonistic activity of 5-HT at 5-HT5 receptors.
Gemäß einer besonderen Ausführungsform der vorliegenden Erfindung werden als Bindungspartner 5-HT5-Agonisten bereitgestellt. Der Ausdruck "5-HT5-Agonist" bezeichnet Bindungspartner, die eine teil- bis vollagonistische Wirkung auslösen. Ein Bindungspartner, der eine teilagonistische Wirkung am 5-HT5-Rezeptor auslöst, besitzt erfindungsgemäß ausreichend agonistische Aktivität, um im Rahmen einer wirksamen medizinischen Behandlung in vertretbaren Mengen verabreicht werden zu können. Im Rahmen dieser Ausfüh- rungsform bevorzugt werden Bindungspartner mit wenigstensAccording to a particular embodiment of the present invention, 5-HT5 agonists are provided as binding partners. The expression "5-HT5 agonist" denotes binding partners which trigger a partially to fully agonistic effect. According to the invention, a binding partner that triggers a partially agonistic effect on the 5-HT5 receptor has sufficient agonistic activity to be able to be administered in an acceptable amount in the context of an effective medical treatment. In this embodiment, binding partners with at least one are preferred
50%-iger agonistischer Wirkung. Besonders bevorzugt sind diejenigen Bindungspartner mit wenigstens 80%-iger agonistischer Wirkung und insbesondere diejenigen mit im wesentlichen vollagonistiseher Wirkung (Emax) . Gemäß einer besonderen Ausführungsform der vorliegenden Erfindung werden Bindungspartner zur Verfügung gestellt, deren Bindung zumindest an 5-HT5-Rezeptoren h5-HT5-transfizierter CHO-Zellen eine Stimulierung der GTP-Bindung an membrangebundene G-Proteine, eine Agonist-induzierte Veränderung intrazellulärer Calcium-Spiegel, eine Induktion der Phospholipase C-Aktivität und/oder eine Agonist-induzierte Veränderung der cAMP-Produktion bewirkt. Was die Veränderung intrazellulärer Calcium-Spiegel angeht, so stellt die Verwendung von Bindungspartnern, die eine Erhöhung intrazellulä- rer Calcium-Spiegel bewirken, eine besondere Ausführungsform der Erfindung dar.50% agonistic effect. Those binding partners with at least 80% agonistic activity and particularly those with an essentially fully agonistic activity (E max ) are particularly preferred. According to a particular embodiment of the present invention, binding partners are made available whose binding at least to 5-HT5 receptors of h5-HT5-transfected CHO cells stimulates GTP binding to membrane-bound G proteins, an agonist-induced change in intracellular calcium Level, an induction of phospholipase C activity and / or an agonist-induced change in cAMP production. With regard to the change in intracellular calcium levels, the use of binding partners which bring about an increase in intracellular calcium levels represents a particular embodiment of the invention.
Zu dieser Ausführungsform gehören auch Bindungspartner, die in bekannten Tiermodellen für cerebrovaskuläre Erkrankungen, insbe- sondere für Migräne, wirksam sind, und/oder die bestimmte in vi- vo-Wirkungen in Gehirnbereichen induzieren, insbesondere genomische Antworten im Gehirn hervorrufen, beispielsweise die Expression von Transkriptionsfaktoren wie c-fos, c-jun, zif268 oder Homergen-Isoformen (Brakeman P.R. et al., Nature 386:284-288 (1997)).This embodiment also includes binding partners which are effective in known animal models for cerebrovascular diseases, in particular for migraines, and / or which induce certain in vivo effects in brain areas, in particular which cause genomic responses in the brain, for example the expression of Transcription factors such as c-fos, c-jun, zif268 or Homergen isoforms (Brakeman PR et al., Nature 386: 284-288 (1997)).
Bevorzugt sind Bindungspartner, die auch in Bezug auf ihre Effektorfunktion im oben beschriebenen Sinn selektiv für 5-HT5-Rezep- toren sind.Binding partners are preferred which are also selective for 5-HT5 receptors with regard to their effector function in the sense described above.
Gemäß einer Ausführungsform sind erfindungsgemäße 5-HT5-Bindungs- partner niedermolekulare, meist synthetische Verbindungen.According to one embodiment, 5-HT5 binding partners according to the invention are low molecular weight, mostly synthetic compounds.
Gemäß einer weiteren Ausführungsform sind erfindungsgemäße 5-HT5-Bindungspartner 5-HT5-spezifische Antikörper. Es kann sich um polyklonale Antiseren, monoklonale Antikörper, Antikörperfrag- mente, wie F( ab) , Fc, etc . , chimäre und rekombinante Antikörper handeln. Die Herstellung solcher Antikörper kann in an sich bekannter Weise erfolgen. Als Immunogen kann man 5-HT5-Rezeptor als solchen oder antigene, in der Regel an übliche Trägerproteine gekoppelte Fragmente davon verwenden.According to a further embodiment, 5-HT5 binding partners according to the invention are 5-HT5-specific antibodies. It can be polyclonal antisera, monoclonal antibodies, antibody fragments such as F (ab), Fc, etc. , chimeric and recombinant antibodies. Such antibodies can be produced in a manner known per se. 5-HT5 receptor as such or antigenic fragments thereof, which are generally coupled to customary carrier proteins, can be used as the immunogen.
Gemäß einer weiteren Ausführungsform sind erfindungsgemäße 5-HT5-Bindungspartner Aptamere, das sind Nukleinsäuren, in der Regel Oligonukleotide, mit ausreichender Affinität zu 5-HT5-Re- zeptoren.According to a further embodiment, 5-HT5 binding partners according to the invention are aptamers, ie nucleic acids, usually oligonucleotides, with sufficient affinity for 5-HT5 receptors.
Assays zur Bestimmung von Bindungsaffinitäten erfindungsgemäßer Bindungspartner zu 5-HT5-Rezeptoren sind im Prinzip bekannt. Dies kann beispielsweise dadurch geschehen, daß man die kompetitive Hemmung der Bindung eines Vergleichsbindungspartner an 5-HT5-Re- zeptoren durch die zu untersuchende Substanz bewertet. Als Ver- gleichsbindungspartner eignen sich bekannte Liganden für 5-HT-Re- zeptoren, wie 5-HT oder 5-CT oder LSD. Diese werden zweckmäßigerweise so markiert, daß sich deren Bindung an 5-HT-Rezeptoren analytisch mit Standardmethoden verfolgen läßt. Bevorzugt sind ra- dioaktive und optische Markierungen. Bei BindungsStudien anAssays for determining the binding affinities of binding partners according to the invention for 5-HT5 receptors are known in principle. This can be done, for example, by evaluating the competitive inhibition of the binding of a comparative binding partner to 5-HT5 receptors by the substance to be examined. As a sales Similar binding partners are known ligands for 5-HT receptors, such as 5-HT or 5-CT or LSD. These are appropriately marked so that their binding to 5-HT receptors can be followed analytically using standard methods. Radioactive and optical markings are preferred. At attachment studies
5-HT5-Rezeptoren wird erfindungsgemäß 5-CT oder LSD insbesondere in Form von [3H]-LSD verwendet. Die Bindungsaffinitäten können als halbmaximale Hemmungkonstante IC50 oder als Hemmkonstante Ki ausgedrückt werden. Dieses Verfahren dient vorzugsweise zum primären Screening. Die SPA- oder die FlashPlate-Technologie kommt bevorzugt zur Anwendung.5-HT5 receptors are used according to the invention 5-CT or LSD, in particular in the form of [ 3 H] -LSD. The binding affinities can be expressed as the half-maximum inhibition constant IC 50 or as the inhibition constant Ki. This method is used primarily for primary screening. SPA or FlashPlate technology is preferred.
Die Bindung zu untersuchender Bindungspartner kann man auch an 5-HT-Rezeptoren direkt bestimmen. Die Bindungsaffinität ausdrük- kende Hemmkonstanten Ki können beispielsweise kalorimetrisch, d.h. durch Messung der freigesetzten Bindungsenergie, bestimmt werden.The binding partner to be examined can also be determined directly on 5-HT receptors. Inhibitory constants Ki expressing the binding affinity can, for example, be calorimetric, i.e. by measuring the released binding energy.
Zur Bestimmung von Selektivitäten bestimmt man in gleicher Weise - gegebenenfalls unter Verwendung der für den jeweiligen Rezeptor spezifischen Liganden - die Bindungsaffinität der zu untersuchenden Bindungspartner für andere 5-HT-Rezeptoren und vergleicht die erhaltenen Werte.To determine selectivities, the binding affinity of the binding partners to be investigated for other 5-HT receptors is determined in the same way, if appropriate using the ligands specific for the respective receptor, and the values obtained are compared.
Auch Effektorfunktionen können mit Hilfe bekannter funktioneller Assays sowohl in vitro als auch in vivo qualitativ oder quantitativ bewertet werden.Effector functions can also be evaluated qualitatively or quantitatively using known functional assays both in vitro and in vivo.
Der Beurteilung einer agonistischen Aktivität können all diejenigen Effekte zugrundegelegt werden, die durch die Bindung von 5-HT an 5-HT5-Rezeptoren hervorgerufen werden. Erfindungsgemäß bevorzugt ist es, die Auswirkungen auf die Bindung von GTP an G-Pro- teine, auf interzelluläre Calcium-Spiegel, auf die Phospholipase C-Aktivität und/oder auf die cAMP-Produktion zu bewerten. Diese Verfahren dienen vorzugsweise zum sekundären Screening. Auch hier kommt die SPA- oder die FlashPlate-Technologie vorteilhaft zur Anwendung.The assessment of agonistic activity can be based on all those effects which are caused by the binding of 5-HT to 5-HT5 receptors. It is preferred according to the invention to evaluate the effects on the binding of GTP to G proteins, on intercellular calcium levels, on the phospholipase C activity and / or on the cAMP production. These methods are preferably used for secondary screening. Here too, SPA or FlashPlate technology is used to advantage.
Die GTP-Bindung an G-Proteine kann untersucht werden, indem man ein nicht hydrolysierbares Analogon von GTP verwendet, beispiels- weise [35S]GTPγS, dessen Bindung radiologisch untersucht werden kann. Diese Untersuchung wird vorzugsweise an 5-HT5-Rezeptor aufweisenden Membranen durchgeführt.GTP binding to G proteins can be examined using a non-hydrolyzable analog of GTP, for example [ 35 S] GTPγS, the binding of which can be examined radiologically. This investigation is preferably carried out on membranes having 5-HT5 receptor.
Zur Messung intrazellulärer Calcium-Spiegel kann man geeignete Calcium-Sonden, in der Regel Calcium-Chelatoren, beispielsweise fluoreszierende Verbindungen, wie Fura-2-acetylmethylester oder Fluo-3-AM, einsetzen. Diese Untersuchung wird vorzugsweise an 5-HT5-Rezeptor aufweisenden Zellkulturen, insbesondere an Einzelzellen durchgeführt.Suitable calcium probes, usually calcium chelators, for example fluorescent compounds such as fura-2-acetyl methyl ester or fluo-3-AM, can be used to measure intracellular calcium levels. This investigation is preferred 5-HT5 receptor-containing cell cultures, in particular carried out on single cells.
Die Phospholipase C-Aktvität läßt sich anhand der von ihr kataly- sierten Reaktionen bestimmen, beispielsweise dem Einbau von Myo- Inositol, das zu Nachweiszwecken vorzugsweise als [3H]-Myo-Inosi- tol radioaktiv markiert ist, oder die Umsetzung von PPIP2 zu IP3, wobei auch das PPIP2 vorzugsweise als [32P]PIP2 radioaktiv markiert ist. Diese Untersuchungen werden vorzugsweise an 5-HT5-Re- zeptor aufweisenden Einzelzellen durchgeführt.The phospholipase C activity can be determined on the basis of the reactions it catalyzes, for example the incorporation of myo-inositol, which is preferably radioactively labeled as [ 3 H] -myo-inosol for detection purposes, or the conversion of PPIP 2 to IP 3 , with PPIP 2 also preferably being radioactively labeled as [ 32 P] PIP 2 . These investigations are preferably carried out on single cells having 5-HT5 receptors.
Die cAMP-Produktion kann mit Hilfe des cAMP-Bindungsproteins bestimmt werden. Diese Untersuchung wird vorzugsweise an 5-HT5-Re- zeptor aufweisenden Einzelzellen durchgeführt.The production of cAMP can be determined using the cAMP binding protein. This investigation is preferably carried out on single cells having 5-HT5 receptor.
Gegebenfalls bestimmt man auch die Effektorfunktion, d.h. die Aktivität von erfindungsgemäßen Bindungspartnern für andere 5-HT- Rezeptoren. Dies geschieht zweckmäßigerweise unter Berücksichtigung der für 5-HT5 und andere 5-HT-Rezeptoren ermittelten Bin- dungsaffinitäten, also insbesondere unter Berücksichtigung der Selektivität.If necessary, the effector function is also determined, i.e. the activity of binding partners according to the invention for other 5-HT receptors. This is expediently done taking into account the binding affinities determined for 5-HT5 and other 5-HT receptors, that is to say in particular taking into account the selectivity.
Gegenstand der vorliegenden Erfindung sind daher auch Verfahren zur Identifizierung und Charakterisierung erfindungsgemäßer Bin- dungspartner. Diese und weitere in ähnlicher Weise geeignete Verfahren können die Grundlage bilden für in vitro-Screening-Verfah- ren, mit denen man aus einer Vielzahl verschiedener Verbindungen diejenigen auslesen kann, die im Hinblick auf eine künftige Anwendung am aussichtsreichsten zu sein scheinen. Beispielsweise können mittels kombinatorischer Chemie umfangreiche Stoffbanken angelegt werden, die Myriaden potentieller Wirkstoffe umfassen. Das Durchmustern kombinatorischer Substanzbibliotheken nach Stoffen mit gewünschter Aktivität ist automatisierbar. Screening-Ro- boter dienen der effizienten Auswertung der vorzugsweise auf Mi- krotiterplatten angeordneten Einzelassays. So betrifft die vorliegende Erfindung auch Screening-Verfahren, d.h. sowohl Primärais auch Sekundärscreening-Verfahren, bei denen vorzugsweise wenigstens eines der nachfolgend beschriebenen Verfahren zur Anwendung kommt. Kommen mehrere Verfahren zur Anwendung, so kann das zeitlich versetzt oder gleichzeitig an ein und derselben Probe oder an verschiedenen Proben einer zu untersuchenden Substanz geschehen.The present invention therefore also relates to methods for identifying and characterizing binding partners according to the invention. These and other similarly suitable methods can form the basis for in vitro screening methods which can be used to select from a large number of different compounds those which appear to be the most promising with regard to future use. For example, using combinatorial chemistry, extensive substance banks can be created that include myriads of potential active substances. The screening of combinatorial substance libraries for substances with the desired activity can be automated. Screening robots are used for the efficient evaluation of the individual assays, which are preferably arranged on microtiter plates. Thus, the present invention also relates to screening methods, i.e. Both primary ice and secondary screening methods, in which at least one of the methods described below is preferably used. If several methods are used, this can be done at different times or simultaneously on one and the same sample or on different samples of a substance to be examined.
Eine besonders effektive Technologie zur Durchführung derartiger Verfahren ist der im Bereich des Wirkstoffscreenings bekannte Scintillation Proximity Assay, kurz SPA genannt. Kits und Komponenten zur Durchführung dieses Assays können kommerziell bezogen werden, beispielweise bei Amersham Pharmacia Biotech. Im Prinzip werden solubilisierte oder membrangebundene Rezeptoren auf Scin- tillationssubstanz enthaltenden, kleinen Fluoromikrosphären immo- bilisert. Bindet beispielsweise ein Radioligand an die immobili- sierten Rezeptoren, so wird die Scintillationssubstanz zur Lichtemission angeregt, da die räumliche Nähe zwischen Scintillationssubstanz und Radioligand gegeben ist.A particularly effective technology for carrying out such methods is the scintillation proximity assay known in the field of active substance screening, or SPA for short. Kits and components for performing this assay can be obtained commercially at Amersham Pharmacia Biotech. In principle, solubilized or membrane-bound receptors are immobilized on small fluoromicrospheres containing scintillating substance. If, for example, a radioligand binds to the immobilized receptors, the scintillation substance is stimulated to emit light, since the spatial proximity between the scintillation substance and the radioligand is given.
Eine weitere besonders effektive Technologie zur Durchführung derartiger Verfahren ist die im Bereich des Wirkstoffscreenings bekannte FlashPlateR-Technologie. Kits und Komponenten zur Durchführung dieses Assays können kommerziell bezogen werden, beispielweise bei NENR Life Science Products. Dieses Prinzip basiert ebenfalls auf Mikrotiterplatten (96er oder 384er), die mit Scin- tillationssubstanz beschichtet sind.Another particularly effective technology for carrying out such processes is the FlashPlate R technology known in the field of active substance screening. Kits and components for performing this assay can be obtained commercially, for example from NEN R Life Science Products. This principle is also based on microtiter plates (96 or 384), which are coated with scintillation substance.
Die vorstehend genannten Assays sind dem Fachmann im Prinzip bekannt.The assays mentioned above are known in principle to the person skilled in the art.
Ein erstes erfindungsgemäßes Verfahren dient zur Bestimmung der Affinität und/oder Selektivität von Bindungspartnern für 5-HT5-Rezeptoren. Zu diesem Zweck bringt man den Bindungspartner mit 5-HT5-Rezeptoren in Kontakt und bestimmt die Bindungsaffinität.A first method according to the invention serves to determine the affinity and / or selectivity of binding partners for 5-HT5 receptors. For this purpose, the binding partner is brought into contact with 5-HT5 receptors and the binding affinity is determined.
Ein weiteres erfindungsgemäßes Verfahren betrifft die Bestimmung der Aktivität von Bindungspartnern für 5-HT5-Rezeptoren, d.h. die Bestimmung agonistischer, teilagonistischer, antagonistischer und/oder teilantagonistischer Wirkung. Zu diesem Zweck bringt man den Bindungspartner mit 5-HT5-Rezeptoren in Kontakt und bewertet die durch die Bindung hervorgerufenen Effekte.Another method according to the invention relates to the determination of the activity of binding partners for 5-HT5 receptors, i.e. the determination of agonistic, partially agonistic, antagonistic and / or partially antagonistic effects. For this purpose, the binding partner is brought into contact with 5-HT5 receptors and the effects caused by the binding are evaluated.
Gemäß einer bevorzugten Ausführungsform werden Bindungspartner einem primären Screening unterzogen, indem man ihre Bindungsaffi- nität zu 5-HT5-Rezeptoren mit dem oben beschriebenen [3H]-5-CT- oder [3H]-LSD-Kompetitionsexperiment bestimmt. Diejenigen Bindungspartner, die eine Hemmkonstante IC50 im Bereich von 10~6 M oder weniger aufweisen, werden dann einem sekundären Screening unterzogen, indem man ihre Effektorfunktion insbesondere im Hin- blick auf die GTP-Bindung und/oder die intrazellulären Calcium- Spiegel in der oben beschriebenen Weise bewertet. Schließlich werden die so ausgewählten Bindungspartner zur Selektivitätbestimmung einem Gegen-Screening unterzogen, indem man ihre Bindungsaffinität zu weiteren 5-HT-Rezeptoren im wesentlichen in der vorstehend beschriebenen Art und Weise - gegebenenfalls unter Verwendung der für den jeweiligen Rezeptor spezifischen Liganden - bestimmt. Beispielsweise kann man [3H]-8-Hydroxy-di-propylamino- tetralin ( [3H]-8-DPAT) für Bindungsstudien an 5-HTlA-Rezeptoren verwenden, während 5-HT1B- und 5-HTlD-Rezeptoren mit [3H]-5-CT- Kompetitionsexperimenten untersucht werden können.According to a preferred embodiment, binding partners are subjected to primary screening by determining their binding affinity for 5-HT5 receptors with the above-described [ 3 H] -5-CT or [ 3 H] -LSD competition experiment. Those binding partners which have an inhibition constant IC 50 in the range of 10 ~ 6 M or less are then subjected to a secondary screening by their effector function, in particular with regard to the GTP binding and / or the intracellular calcium levels in the evaluated way described above. Finally, the binding partners selected in this way are subjected to a counter-screening for the determination of selectivity by determining their binding affinity for further 5-HT receptors essentially in the manner described above - if appropriate using the ligands specific for the respective receptor. For example, [ 3 H] -8-hydroxy-di-propylamino- Use tetralin ([ 3 H] -8-DPAT) for binding studies on 5-HTlA receptors, while 5-HT1B and 5-HTID receptors can be tested with [ 3 H] -5-CT competition experiments.
5 5-HT5-Rezeptoren werden vorzugsweise in Form zellulärer Systeme zur Verfügung gestellt, d.h. in Form von Membranen, Zellen, Zellverbänden, Geweben oder Organen, die 5-HT5-Rezeptoren tragen. Derartige zelluläre Systeme können 5-HT5-Rezeptoren von Natur aus exprimieren, sie können aber auch durch geeignete genetische Ma-5 5-HT5 receptors are preferably provided in the form of cellular systems, i.e. in the form of membranes, cells, cell groups, tissues or organs that carry 5-HT5 receptors. Such cellular systems can naturally express 5-HT5 receptors, but they can also be expressed by suitable genetic methods.
10 nipulation, z.B. durch Transfektion, zur 5-HT5-Expression veranlaßt sein. Im Rahmen der h5-HT5 betreffenden bevorzugten Ausführungsform der vorliegenden Erfindung kann man hierzu insbesondere die in Rees S. et al, FEBS Letters 335:242-246 (1994) beschriebenen kodierende Sequenz verwenden (Zugangsnummer X81411). Humane10 nipulation, e.g. by transfection to be induced to 5-HT5 expression. In the context of the preferred embodiment of the present invention relating to h5-HT5, the coding sequence described in Rees S. et al, FEBS Letters 335: 242-246 (1994) can be used (accession number X81411). Humane
15 Gliom-Zellinien werden als natürliche, 5-HT5-Rezeptoren aufweisende zelluläre Systeme bevorzugt. Von den h5-HT5-transfizierten heterologen Zellinien werden diejenigen bevorzugt, die das h5-HT5-Gen stabil exprimieren. Zu nennen sind beispielsweise h5-HT5-transfizierte CHO-Zellen, h5-HT5-transfizierte humane Nie-15 glioma cell lines are preferred as natural, 5-HT5 receptor cellular systems. Of the h5-HT5 transfected heterologous cell lines, preference is given to those which stably express the h5-HT5 gene. Examples include h5-HT5-transfected CHO cells, h5-HT5-transfected human cells
20 renzellen, insbesondere h5-HT5-transfizierte HEK293-Zellen, oder h5-HT5-transfizierte C-6-Gliomzellen.20 cells, in particular h5-HT5 transfected HEK293 cells, or h5-HT5 transfected C-6 glioma cells.
Zur Bestimmung von Selektivität, Affinität und Aktivität erfindungsgemäßer Bindungspartner können auch Gehirngewebeschnitte und 25 native Membranen aus Gehirnteilen verwendet werden. Werden radioaktive Marker eingesetzt, so erfolgt die Auswertung von Gewebeschnitten vorzugsweise autoradiographisch.Brain tissue sections and native membranes from brain parts can also be used to determine the selectivity, affinity and activity of binding partners according to the invention. If radioactive markers are used, tissue sections are preferably evaluated autoradiographically.
Die Wirksamkeit erfindungsgemäßer Bindungspartner zur Behandlung 30 von Migräne wird vorzugsweise an Tiermodellen bestimmt, die auf Mechanismen aufbauen, die der Entstehung von Migräne zugrunde liegen können.The effectiveness of binding partners according to the invention for the treatment of migraines is preferably determined using animal models which are based on mechanisms which can be the basis for the development of migraines.
Beispielsweise kann man die durch erfindungsgemäße Bindungspart- 35 ner induzierte Proteinextravasation bestimmen. Dieser Assay kann in an sich bekannter Weise durchgeführt werden, beispielsweise indem man eine Fluoreszenzfärbung (Johnson K.W. and Phebus L.A. (1998) J. Neurosci. Methods 81: 19-24) oder isotopisch markiertes Albumin (Petty M.A. et al., (1997) Eur. J. Pharmacol. 336: 40 127-136) verwendet. Hierzu werden die Tiere nacheinander mit der Testverbindung und mit [125I]-Albumin oder Fluoreszenzfarbstoff behandelt. Danach wird das trigeminale Ganglion elektrisch stimuliert. Die in der Dura Mater akkumulierte Radioaktivität oder Fluoreszenz wird bestimmt. Die Antimigräne-Aktivität erfindungs- 45 gemäßer Bindungspartner zeigt sich an der durch Stimulation tri- geminaler Ganglion induzierten Proteinextravasation. Ein weiteres Modell basiert auf der Verteilung des carotiden Blutflusses. Auch dieser Test ist an sich bekannt (Saxena P.R. et al., (1998) Eur. J. Pharmacol. 351: 329-339). Hierzu werden radioaktive Mikrosphären in die Carotis injiziert. Im Anschluß an eine Stabilisierungsphase werden die Tiere mit einer Testverbindung behandelt, und die im Gehirn akkumulierte Radioaktivität wird in einem Mikro-ß-Zähler bestimmt.For example, one can determine the protein extravasation induced by binding partners according to the invention. This assay can be carried out in a manner known per se, for example by fluorescent staining (Johnson KW and Phebus LA (1998) J. Neurosci. Methods 81: 19-24) or isotopically labeled albumin (Petty MA et al., (1997) Eur. J. Pharmacol. 336: 40 127-136). For this purpose, the animals are treated successively with the test compound and with [ 125 I] albumin or fluorescent dye. The trigeminal ganglion is then electrically stimulated. The radioactivity or fluorescence accumulated in the dura mater is determined. The anti-migraine activity of binding partners according to the invention is shown by the protein extravasation induced by stimulation of the triminal ganglion. Another model is based on the distribution of carotid blood flow. This test is also known per se (Saxena PR et al., (1998) Eur. J. Pharmacol. 351: 329-339). For this purpose, radioactive microspheres are injected into the carotid. Following a stabilization phase, the animals are treated with a test compound and the radioactivity accumulated in the brain is determined in a micro-ß counter.
Geeignet ist auch die Messung der Nitroglycerin-induzierten c-fos-Genexpression und -Translokation. Dazu werden Tiere mit Ni- troglycerin und der zu testenden Verbindung behandelt. Die c-fos- Genexpression und -Proteintranslokation in den Kern wird mit in- situ Hybridisierung oder immunhistochemisch bestimmt (Garcia-La- dona F.J. et al., (1994) Mol. Brain Res. 21: 75-84; Garcia-Ladona F.J. et al., (1997) J. Neurosci. Res 50: 50-61).The measurement of the nitroglycerin-induced c-fos gene expression and translocation is also suitable. For this, animals are treated with nitroglycerol and the compound to be tested. The c-fos gene expression and protein translocation into the nucleus is determined with in situ hybridization or immunohistochemically (Garcia-Ladona FJ et al., (1994) Mol. Brain Res. 21: 75-84; Garcia-Ladona FJ et al., (1997) J. Neurosci. Res 50: 50-61).
Weitere Modelle sind aus Fernandes de Lima V.M. et al. (1993) Brain Res. 614: 445-51 (Retinal Spreading depression) and aus Ka- wahara N. et al. (1999) Exp. Neurol. 108: 27-36 (Cortical Sprea- ding Depression) bekannt.Other models are from Fernandes de Lima V.M. et al. (1993) Brain Res. 614: 445-51 (retinal spreading depression) and from Kawahara N. et al. (1999) Exp. Neurol. 108: 27-36 (cortical spreading depression).
Die erfindungsgemäße Verwendung von 5-HT5-Bindungspartnern beinhaltet im Rahmen der Behandlung ein Verfahren. Dabei wird dem zu behandelnden Individuum, vorzugsweise einem Säuger, insbesondere einem Menschen, Nutz- oder Haustier, eine wirksame Menge eines oder mehrerer 5-HT5-Bindungspartner, in der Regel der pharmazeutischen und tierarzneilichen Praxis entsprechend formuliert, verabreicht. Ob eine solche Behandlung angezeigt ist und in welcher Form sie zu erfolgen hat, hängt vom Einzelfall ab und unterliegt einer medizinischen Beurteilung (Diagnose), die vorhandene Anzeichen, Symptome und/oder Fehlfunktionen, Risiken, bestimmte Anzeichen, Symptome und/oder Fehlfunktionen zu entwickeln, und weitere Faktoren miteinbezieht.The use of 5-HT5 binding partners according to the invention includes a method in the context of the treatment. An effective amount of one or more 5-HT5 binding partners, generally formulated in accordance with pharmaceutical and veterinary practice, is administered to the individual to be treated, preferably a mammal, in particular a human, useful or domestic animal. Whether such treatment is indicated and in what form it must be carried out depends on the individual case and is subject to a medical assessment (diagnosis) to develop the existing signs, symptoms and / or malfunctions, risks, certain signs, symptoms and / or malfunctions , and other factors.
Die Behandlung erfolgt in der Regel durch einmalige oder mehrmalige tägliche Verabfolgung gegebenenfalls zusammen oder im Wechsel mit anderen Wirkstoffen oder Wirkstoffhaltigen Präparaten, so daß einem zu behandelnden Individuum eine Tagesdosis von etwa 0,001 g bis 10 g, vorzugsweise von etwa 0,001 g bis etwa 1 g zu- geführt wird.The treatment is usually carried out by single or repeated daily administration, optionally together or alternating with other active substances or preparations containing active substances, so that an individual to be treated receives a daily dose of about 0.001 g to 10 g, preferably of about 0.001 g to about 1 g - to be led.
Die Erfindung betrifft auch die Herstellung pharmazeutischer Mittel zur Behandlung eines Individuums, vorzugsweise eines Säugers, insbesondere eines Menschen, Nutz- oder Haustieres. Die erfin- dungsgemäßen Bindungspartner werden gewöhnlich in Form von pharmazeutischen Zusammensetzungen verabreicht, die einen pharmazeutisch verträglichen Exzipienten mit wenigstens einem erfindungs- gemäßen Bindungspartner und gegebenenfalls weiteren Wirkstoffen umfassen. Diese Zusammensetzungen können beispielsweise auf oralem, rektalem, transdermalem, subkutanem, intravenösem, intramuskulärem oder intranasalem Weg verabreicht werden.The invention also relates to the production of pharmaceutical compositions for the treatment of an individual, preferably a mammal, in particular a human, useful or domestic animal. The binding partners according to the invention are usually administered in the form of pharmaceutical compositions which contain a pharmaceutically acceptable excipient with at least one include binding partner and optionally other active ingredients. These compositions can be administered, for example, by the oral, rectal, transdermal, subcutaneous, intravenous, intramuscular or intranasal routes.
Beispiele geeigneter pharmazeutischer Formulierungen sind feste Arzneiformen, wie Pulver, Puder, Granulate, Tabletten, Pastillen, Sachets, Cachets, Dragees, Kapseln wie Hart- und Weichgelatinekapseln, Suppositorien oder vaginale Arzneiformen, halbfeste Arz- neiformen, wie Salben, Cremes, Hydrogele, Pasten oder Pflaster, sowie flüssige Arzneiformen, wie Lösungen, Emulsionen, insbesondere Öl-in-Wasser-Emulsionen, Suspensionen, beispielsweise Lotionen, Injektions- und Infusionszubereitungen, Augen- und Ohrentropfen. Auch implantierte Abgabevorrichtungen können zur Verab- reichung erfindungsgemäßer Bindungspartner verwendet werden. Ferner können auch Liposomen, Mikrosphären oder Polymermatrizes zur Anwendung kommen.Examples of suitable pharmaceutical formulations are solid pharmaceutical forms, such as powders, powders, granules, tablets, troches, sachets, cachets, dragees, capsules such as hard and soft gelatin capsules, suppositories or vaginal pharmaceutical forms, semi-solid pharmaceutical forms such as ointments, creams, hydrogels, pastes or plasters, and liquid pharmaceutical forms, such as solutions, emulsions, in particular oil-in-water emulsions, suspensions, for example lotions, injection and infusion preparations, eye and ear drops. Implanted delivery devices can also be used for the administration of binding partners according to the invention. Liposomes, microspheres or polymer matrices can also be used.
Bei der Herstellung der Zusammensetzungen werden erfindungsgemäße Bindungspartner gewöhnlich mit einem Exzipienten vermischt oder verdünnt. Exzipienten können feste, halbfeste oder flüssige Materialien sein, die als Vehikel, Träger oder Medium für den Wirkstoff dienen.In the preparation of the compositions, binding partners according to the invention are usually mixed or diluted with an excipient. Excipients can be solid, semi-solid or liquid materials that serve as vehicles, carriers or media for the active ingredient.
Zu geeigneten Exzipienten gehören beispielsweise Lactose, Dextrose, Sucrose, Sorbitol, Mannitol, Stärken, Akaziengummi, Calci- umphosphat, Alginate, Traganth, Gelatine, Calciumsilikat, mikrokristalline Cellulose, Polyvinylpyrrolidon, Cellulose, Wasser, Sirup und Methylcellulose. Ferner können die Formulierungen phar- mazeutisch akzeptable Träger oder übliche Hilfsstoffe, wie Gleitmittel, beispielsweise Talg, Magnesiumstearat und Mineralöl; Netzmittel; emulgierende und suspendierende Mittel; konservierende Mittel, wie Methyl- und Propylhydroxybenzoate; Antioxidan- tien; Antireizstoffe; Chelatbildner; Dragierhilfsmittel; Emul- sionsstabilisatoren; Filmbildner; Gelbildner; Geruchsmaskierungs- mittel; Geschmackskorrigentien; Harze; Hydrokolloide; Lösemittel; Lösungsvermittler; Neutralisierungsmittel; Permeationsbeschleuni- ger; Pigmente; quaternäre Ammoniumverbindungen; Rückfettungs- und Überfettungsmittel; Salben-, Creme- oder Öl-Grundstoffe; Silikon- Derivate; Spreithilfsmittel; Stabilisatoren; Sterilanzien; Suppo- sitoriengrundlagen; Tabletten-Hilfsstoffe, wie Bindemittel, Füllstoffe, Gleitmittel, Sprengmittel oder Überzüge; Treibmittel; Trocknungsmittel; Trübungsmittel; Verdickungsmittel; Wachse; Weichmacher; Weißöle umfassen. Eine diesbezügliche Ausgestaltung beruht auf fachmännischem Wissen, wie beispielsweise in Fiedler, H.P., Lexikon der Hilfsstoffe für Pharmazie, Kosmetik und angren- zende Gebiete, 4. Auflage, Aulendorf: ECV-Editio-Kantor-Verlag, 1996, dargestellt ist.Suitable excipients include, for example, lactose, dextrose, sucrose, sorbitol, mannitol, starches, acacia, calcium phosphate, alginates, tragacanth, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup and methyl cellulose. Furthermore, the formulations can be pharmaceutically acceptable carriers or customary auxiliaries, such as lubricants, for example tallow, magnesium stearate and mineral oil; Wetting agents; emulsifying and suspending agents; preservatives such as methyl and propyl hydroxybenzoates; Antioxidants; Anti-irritants; Chelating agents; Coating aids; Emulsion stabilizers; Film formers; Gelling agent; Odor masking agents; Taste corrections; Resins; Hydrocolloids; Solvents; Solubilizer; Neutralizing agents; Permeation accelerator; Pigments; quaternary ammonium compounds; Refatting and overfatting agents; Ointment, cream or oil base materials; Silicone derivatives; Spreading aids; Stabilizers; Sterilizers; Fundamentals of the suppository; Tablet excipients such as binders, fillers, lubricants, disintegrants or coatings; Blowing agent; Desiccants; Opacifiers; Thickeners; Waxes; Plasticizers; Include white oils. A relevant design is based on professional knowledge, such as in Fiedler, HP, encyclopedia of excipients for pharmacy, cosmetics and zende territories, 4th edition, Aulendorf: ECV-Editio-Kantor-Verlag, 1996.
Gegenstand der vorliegenden Erfindung ist auch die Verwendung ei- nes Bindungspartner für 5HT-5-Rezeptoren und insbesondere der vorstehnd genannten besonderen und bevorzugten Ausführungsformen erfindungsgemäßer Bindungspartner zur Behandlung migräneartiger cerebrovasculärer Störungen , vor allem zur Behandlung von Migräne und anderen vaskulär bedingten Kopfschmerzen, insbesondere anfallartigen Kopfschmerzen vom Migränetyp. Hierzu gehören die als einfache und als klassische Migräne bezeichneten Erkrankungen ohne bzw. mit begleitenden neurologischen Funktionsstörungen, echte und atypische Migräne, und auch speziellere Erkrankungen dieses Typs, beispielsweise Migraine accompagnee, sogenannte Mi- gräneäquivalente, Digestivmigräne, Migraine ophtalmique, ophtal- moplegische Migräne, Migraine rouge, auch als Cluster-Kopfschmerz (Horton-Syndrom) bezeichnet, Zervikalmigräne. Unter Behandlung wird sowohl eine prophylaktische Therapie, insbesondere die Vorbeugung gegen wiederkehrende Attacken, beispielsweise als Inter- vallbehandlung, als auch die Behandlung bei akuten Symptomen, insbesondere während der Kopfschmerzphasen, verstanden. Eine erfolgreiche Behandlung führt zu einer Minderung der Intensität der Symptome, insbesondere des Kopfschmerzes, neurologischer Funktionsstörungen, z.B. visueller, sensorischer, motorischer Störungen und Sprachstörungen, Übelkeit und Brechreiz, und/oder der Anfallhäufigkeit. Eine akute Behandlung von Migräne mit Bindungspartnern ist bevorzugt. Gegenstand der Erfindung ist insbesondere auch die Verwendung der genannten Bindungspartner für die Behandlung solcher Formen oben aufgeführter Erkrankungen, an deren Ent- stehung und/oder Verlauf 5-HT5-Rezeptoren beteiligt sind, d.h. Erkrankungen, die durch eine 5-HT5-Rezeptoraktivität moduliert werden.The present invention also relates to the use of a binding partner for 5HT-5 receptors and, in particular, the above-mentioned particular and preferred embodiments of binding partners according to the invention for the treatment of migraine-like cerebrovascular disorders, especially for the treatment of migraines and other vascular headaches, in particular seizure-like headaches of the migraine type. These include the diseases described as simple and classic migraines without or with accompanying neurological functional disorders, real and atypical migraines, and also more specific diseases of this type, for example migraine accompagnee, so-called migraine equivalents, digestive migraines, migraine ophthalmics, ophthalmic moplegic migraines, Migraine rouge, also known as cluster headache (Horton syndrome), cervical migraine. Treatment is understood to mean both prophylactic therapy, in particular the prevention of recurring attacks, for example as an interval treatment, and treatment for acute symptoms, in particular during the headache phases. Successful treatment leads to a reduction in the intensity of symptoms, in particular headache, neurological dysfunction, e.g. visual, sensory, motor and speech disorders, nausea and nausea, and / or the frequency of attacks. Acute treatment of migraines with attachment partners is preferred. The invention in particular also relates to the use of the binding partners mentioned for the treatment of such forms of the diseases listed above, in whose formation and / or course 5-HT5 receptors are involved, i.e. Diseases that are modulated by 5-HT5 receptor activity.
Der Begriff "Störung" im erfindungsgemäßen Sinne bezeichnet Ano- malien, die in der Regel als krankhafte Zustände angesehen werden und sich in Form bestimmter Anzeichen, Symptome und/oder Fehlfunktionen zu erkennen geben können. Die erfindungsgemäße Behandlung kann auf einzelne Störungen sprich Anomalien bzw. krankhafte Zustände gerichtet sein, es können aber auch mehrere gegebenen- falls ursächlich miteinander verbundene Anomalien zu Mustern, d.h. Syndromen, zusammengefaßt sein, die erfindungsgemäß behandelt werden können.The term “disorder” in the sense according to the invention denotes anomalies which are generally regarded as pathological conditions and can be identified in the form of certain signs, symptoms and / or malfunctions. The treatment according to the invention can be directed to individual disorders, i.e. anomalies or pathological conditions, but it is also possible for a number of anomalies which may be causally linked to one another, i.e. Syndromes can be summarized, which can be treated according to the invention.
Eine Behandlung im erfindungsgemäßen Sinne umfasst nicht nur die Behandlung akuter oder chronischer Anzeichen, Symptome und/oder Fehlfunktionen sondern auch eine vorbeugende Behandlung (Prophylaxe) insbesondere als Rezidiv- oder Phasen-Prophylaxe. Die Be- handlung kann symptomatisch, beispielsweise als Symptomsuppres- sion ausgerichtet sein. Sie kann kurzzeitig erfolgen, mittelfristig ausgerichtet sein, oder es kann sich auch um eine Langzeitbehandlung, beispielsweise im Rahmen einer Erhaltungstherapie, handeln.Treatment in the sense of the invention includes not only the treatment of acute or chronic signs, symptoms and / or malfunctions but also preventive treatment (prophylaxis), in particular as relapse or phase prophylaxis. Thieves- action can be symptomatic, for example as symptom suppression. It can be short-term, medium-term, or it can also be long-term treatment, for example in the context of maintenance therapy.
Zur Migränebehandlung brauchbare Bindungspartner können mit einer geringeren, einer im wesentlichen gleichen, oder einer höheren Affinität an 5-HT5 binden als an einen bestimmten von 5-HT5 ver- schiedenen Rezeptor.Binding partners which can be used for migraine treatment can bind to 5-HT5 with a lower, substantially the same or a higher affinity than to a specific receptor different from 5-HT5.
So gehören zu Bindungspartnern für 5-HT5-Rezeptoren im Hinblick auf die erfindungsgemäße Verwendung insbesondere diejenigen, deren Bindungsaffinität zu 5-HT5-Rezeptoren verglichen mit der Af- finität zu 5-HTl-Rezeptoren, insbesondere 5-HTlB und/oder 5-HTlD, so hoch ist, daß sie für die erfindungsgemäße Verwendung in vorteilhafter Weise geeignet sind. Dies setzt nicht notwendigerweise eine vergleichsweise selektivere Bindung an 5-HT5-Rezptoren voraus, wenngleich selektive Bindungspartner für 5-HT5-Rezptoren eine besondere Ausführungsform der vorliegenden Erfindung sind.With regard to the use according to the invention, binding partners for 5-HT5 receptors include, in particular, those whose binding affinity for 5-HT5 receptors compared to the affinity for 5-HTI receptors, in particular 5-HTIB and / or 5-HTID is so high that they are advantageously suitable for the use according to the invention. This does not necessarily require a comparatively more selective binding to 5-HT5 receptors, although selective binding partners for 5-HT5 receptors are a special embodiment of the present invention.
Beispielsweise kann man Bindungspartner verwenden, die hochaffin sind sowohl zu 5-HT5 als auch zu 5-HTl-Rezeptoren, insbesondere zu 5-HT1B und/oder 5-HTlD. Hochaffin bedeutet in diesem Zusammen- hang Ki-Werte in der Regel im Bereich von 1'10-9 M bis 1'10-6 M. Gemäß einer besonderen Ausführungsform besitzen brauchbare Bindungspartner im hochaffinen Bereich zu 5-HT-Rezeptoren ein Bindungsprofil, daß durch eine Bindungsaffinität zu 5-HT5 gekennzeichnet ist, die im Vergleich zu anderen Bindungsaffinitäten dieses Bereichs im wesentlichen gleich oder nur wenig geringer ist. Faktoren von 10 oder weniger können von Vorteil sein.For example, binding partners can be used which are highly affine to both 5-HT5 and 5-HTI receptors, in particular to 5-HT1B and / or 5-HTID. In this context, high affinity means Ki values generally in the range from 1'10 -9 M to 1'10 -6 M. According to a particular embodiment, useful binding partners in the high-affinity range to 5-HT receptors have a binding profile that is characterized by a binding affinity for 5-HT5 which is essentially the same or only slightly less than other binding affinities in this area. Factors of 10 or less can be beneficial.
Gemäß einer weiteren Ausführungsform der Erfindung werden die zuvor beschriebenen selektiven 5-HT5-Bindungspartner eingesetzt.According to a further embodiment of the invention, the selective 5-HT5 binding partners described above are used.
Gemäß einer besonderen Ausführungsform der vorliegenden Erfindung werden Bindungspartner eingesetzt, deren Bindung zumindest an 5-HT5-Rezeptoren h5-HT5-transfizierter CHO-Zellen eine Veränderung der Agonist-induzierten Stimulierung der GTP-Bindung an mem- brangebundene G-Proteine, eine Veränderung intrazellulärer Calcium-Spiegel, eine Veränderung der Agonist-induzierten Induktion der Phospholipase C-Aktivität und/oder eine Veränderung der cAMP- Produktion bewirkt. Was die Veränderung intrazellulärer Calcium- Spiegel angeht, so stellt die Verwendung von Bindungspartnern, die eine Erhöhung intrazellulärer Calcium-Spiegel bewirken, eine besondere Ausführungsform der Erfindung dar. Die vorliegende Erfindung wird anhand der nachfolgenden Beispiele näher erläutert, ohne darauf beschränkt zu sein.According to a particular embodiment of the present invention, binding partners are used whose binding at least to 5-HT5 receptors of h5-HT5-transfected CHO cells is a change in the agonist-induced stimulation of GTP binding to membrane-bound G proteins, a change in intracellular Calcium levels, a change in the agonist-induced induction of phospholipase C activity and / or a change in cAMP production. As regards the change in intracellular calcium levels, the use of binding partners which bring about an increase in intracellular calcium levels represents a particular embodiment of the invention. The present invention is explained in more detail with reference to the following examples, without being restricted thereto.
Referenzbeispiel 1Reference example 1
h5-HT5-Rezeptor exprimierende HEK293-Zellen und CHO-ZellenHEK293 cells and CHO cells expressing h5-HT5 receptor
Das für den humanen 5-HT5-Rezeptor kodierende Gen wurde in an sich bekannter Weise über 3'-5'-RT-PCR (RACE-System, Boehringer Mannheim) aus menschlichen Geweben isoliert. Die Gensequenz wurde dann in ein das Neomycinresistenz-Gen tragendes Plasmid inser- tiert (pcDNA3; Invitrogen, Deutschland) und in E. coli den Herstellerhinweisen entsprechend amplifiziert. Eine Präparation des resultierenden Plasmids wurde mit LipofectaminR (Gibco Life-Scien- ces, Deutschland) vermischt, und HEK293-Zellen wurden in Petri- schalen (2,5 cm) mit einer dünnen Schicht dieses Transfektionsge- misches inkubiert. Danach wurde das Transfektionsgemisch durch Neomycin-haltiges Kulturmedium ersetzt. Überlebende Zellen wurden weiter in DMEM-F12-Medium kultiviert, das mit 10% fötalem Kälberserum, 2 mM Glutamin und Antibiotika (90 mg Streptomycin, 90 mg Penicillin) ergänzt war. Die Zellen wurden bei 5% C02, 95% Luftfeuchtigkeit und 37°C bis zur Konfluenz aufgezogen.The gene coding for the human 5-HT5 receptor was isolated from human tissues in a manner known per se using 3'-5'-RT-PCR (RACE system, Boehringer Mannheim). The gene sequence was then inserted into a plasmid carrying the neomycin resistance gene (pcDNA3; Invitrogen, Germany) and amplified in E. coli according to the manufacturer's instructions. A preparation of the resulting plasmid was mixed with Lipofectamin R (Gibco Life Sciences, Germany) and HEK293 cells were incubated in Petri dishes (2.5 cm) with a thin layer of this transfection mixture. The transfection mixture was then replaced by culture medium containing neomycin. Surviving cells were further cultured in DMEM-F12 medium supplemented with 10% fetal calf serum, 2 mM glutamine and antibiotics (90 mg streptomycin, 90 mg penicillin). The cells were grown to confluence at 5% CO 2 , 95% humidity and 37 ° C.
In analoger Weise erhält man h5-HT5-Rezeptor exprimierende CHO- Zellen.CHO cells expressing h5-HT5 receptor are obtained in an analogous manner.
Referenzbeispiel 2Reference example 2
Zellmembran-PräparationCell membrane preparation
Die hier verwendete Methode lehnt sich im wesentlichen an bekannte Methoden zur Präparation von Zellmembranen aus Zellen an (Findlay J.B.C. und Evans W.H. Biological Membranes, Practical Approach (1987)). Die gemäß Referenzbeispiel 1 kultivierte Zellen wurden vorsichtig von der Oberfläche des Kulturgefäßes abgeschabt und 10 min bei 180xg in DMEM-F12-Medium zentrifugiert. Die gewonnenen Zellpellets wurden in 5 mM EDTA, 5 mM EGTA, 0,1 mM PMSF und 3 mM Benzamidin enthaltendem 5 mM Tris-HCl-Puffer (pH: 7,6; Puf- fer A) resuspendiert und 15 min bei 4°C inkubiert. Die Zellsuspension wurde in einem UltraturraxR (15000 UPM) homogenisiert (6x3s) und 1 min bei lOOOxg und 4°C zentrifugiert. Das Pellet wurde in Puffer A resuspendiert und, wie vorstehend beschrieben, homogenisiert und zentrifugiert. Die Überstände aus beiden Schritten wur- den gesammelt und 20 min bei 40000xg und 4°C zentrifugiert. Das Pellet wurde in Puffer A resuspendiert und homogenisiert (1x15s). Die Membransuspension wurde 20 min bei 40000xg und 4°C zentrifu- giert. Das resultierende Pellet wurde in 10% Glycerin und 1% Rinderserumalbumin enthaltendem Puffer A resuspendiert. Es wurden Aliquots eingefroren und bei -80°C bis zum Gebrauch aufbewahrt.The method used here is essentially based on known methods for the preparation of cell membranes from cells (Findlay JBC and Evans WH Biological Membranes, Practical Approach (1987)). The cells cultured according to reference example 1 were carefully scraped off the surface of the culture vessel and centrifuged for 10 min at 180 × g in DMEM-F12 medium. The cell pellets obtained were resuspended in 5 mM Tris-HCl buffer (pH: 7.6; buffer A) containing 5 mM EDTA, 5 mM EGTA, 0.1 mM PMSF and 3 mM benzamidine and at 15 ° C. for 15 min incubated. The cell suspension was homogenized in an Ultraturrax R (15000 rpm) (6x3s) and centrifuged for 1 min at 100Xg and 4 ° C. The pellet was resuspended in buffer A and homogenized and centrifuged as described above. The supernatants from both steps were collected and centrifuged for 20 min at 40000xg and 4 ° C. The pellet was resuspended in buffer A and homogenized (1x15s). The membrane suspension was centrifuged at 40000xg and 4 ° C for 20 min. yaws. The resulting pellet was resuspended in buffer A containing 10% glycerin and 1% bovine serum albumin. Aliquots were frozen and stored at -80 ° C until use.
Referenzbeispiel 3Reference example 3
Kinetik der Sättigungsbindung von [3H]-5-CTKinetics of saturation binding of [ 3 H] -5-CT
Die Methodik ist im wesentlichen bekannt (Ress S. et al., FEBS Letters 335:242-246 (1994)). Gemäß Referenzbeispiel 2 gewonnene Membranen (200 μl) wurden bei einem Gesamtvolumen von 600 μl in 1 mM EDTA enthaltendem 100 mM Tris-HCl (pH: 7,7; Puffer B) mit ansteigenden Konzentrationen an [3H]-5-CT (96 Ci/mmol) inkubiert, wobei zur Bestimmung der spezifischen Bindung 10 μM Methiothepin zugesetzt wurde, während zur Bestimmung der Gesamtbindung Methiothepin nicht zugesetzt wurde. Es wurde 90 min bei 30°C inkubiert. Danach wurden die Proben filtriert, wobei man ein SkatronR-Filtra- tionssystem und in 0,3% Polyethylenimid eingebettete GF/B-Filter verwendete. Die Filter wurden mit 9 ml Puffer B bei 4°C gewaschen. Die auf den Filtern zurückgehaltene Radioaktivität wurde mittels Flüssigscintillationszählung gemessen, wobei man 5 ml Ultima-Gold (Packard) verwendete.The methodology is essentially known (Ress S. et al., FEBS Letters 335: 242-246 (1994)). Membranes obtained according to reference example 2 (200 μl) were mixed with a total volume of 600 μl in 100 mM Tris-HCl (pH: 7.7; buffer B) containing 1 mM EDTA with increasing concentrations of [ 3 H] -5-CT (96 Ci / mmol), whereby 10 μM methiothepin was added to determine the specific binding, while methiothepin was not added to determine the total binding. It was incubated at 30 ° C. for 90 min. The samples were then filtered using a Skatron R filtration system and GF / B filters embedded in 0.3% polyethyleneimide. The filters were washed with 9 ml buffer B at 4 ° C. Radioactivity retained on the filters was measured by liquid scintillation counting using 5 ml Ultima Gold (Packard).
Referenzbeispiel 4Reference example 4
a) [3H]-5-CT-Bindungskompetitiona) [ 3 H] -5-CT binding competition
Die Experimente zur Bindungskompetition erfolgten im wesentlichen in Anlehnung an bekannte Untersuchungen (Rees et al., 1994). Ge- maß Beispiel 2 gewonnene Membranen (200 μl) wurden in einem Gesamtvolumen von 600 μl in Puffer B mit ansteigenden Konzentrationen ausgewählter Verbindungen in Gegenwart von 2 nM [3H]-5-CT inkubiert. Nach einer Inkubationszeit von 75 min bei 30°C wurden die Proben mit Puffer B bei 4°C über in 0,3% Polyethylenimid eingebet- teten GF/B-Filtern filtriert. Die Filter wurden mit 9 ml Puffer B gewaschen. Die auf den Filtern zurückgehaltene Radioaktivität wurde wie in Referenzbeispiel 3 bestimmt. Die Gesamtbindung wurde definiert als diejenige Bindung des Radioliganden, die ohne Zusatz weiterer Verbindungen beobachtet wurde. Die nicht-spezifi- sehe Bindung wurde definiert als diejenige Bindung von [3H]-5-CT, die in Gegenwart von 10 μM Methiothepin beobachtet wurde. Es können auch ähnliche Systeme verwendet werden, die durch Verwendung von Mikrotiterplatten einen hohen Probendurchsatz und ein Sekun- därscreening gestatten. Die Sättigungsparameter der [3H]-5-CT-Bindung wurden sowohl durch nicht-lineare Regressionsanalyse als auch aus linearen Auftragungen bei Verwendung der Sig aPlot-Software (Jandel Scientific, Germany) bestimmt. Es wurden Kompetitionskurven aufgestellt, in denen die radioaktive Bindung als prozentualer Anteil der Gesamtbindung ausgedrückt ist. Halbmaximale Hemmkonstanten IC50 und Hill-Koeffizienten ( ιxü) wurden mittels nicht-linearer Regressionsanalyse ermittelt.The experiments on binding competition were essentially based on known studies (Rees et al., 1994). Membranes obtained in accordance with Example 2 (200 μl) were incubated in a total volume of 600 μl in buffer B with increasing concentrations of selected compounds in the presence of 2 nM [ 3 H] -5-CT. After an incubation time of 75 min at 30 ° C, the samples were filtered with buffer B at 4 ° C over GF / B filters embedded in 0.3% polyethyleneimide. The filters were washed with 9 ml of Buffer B. The radioactivity retained on the filters was determined as in Reference Example 3. The total binding was defined as the binding of the radioligand that was observed without the addition of further compounds. The nonspecific binding was defined as the binding of [ 3 H] -5-CT that was observed in the presence of 10 μM methiothepin. Similar systems can also be used which, through the use of microtiter plates, permit high sample throughput and secondary screening. The saturation parameters of the [ 3 H] -5-CT binding were determined both by non-linear regression analysis and from linear plots using the Sig aPlot software (Jandel Scientific, Germany). Competition curves were set up in which the radioactive binding is expressed as a percentage of the total binding. Half-maximum inhibition constants IC 50 and Hill coefficients (ιx ü ) were determined by means of non-linear regression analysis.
b) Identifizierung von h5-HT5-Rezeptorliganden durch HTS unter Verwendung der FlashPlate-Technologie.b) Identification of h5-HT5 receptor ligands by HTS using FlashPlate technology.
96-Well-FlashPlates, die mit h5-HT5-Membranen beschichtet sind, können von Bio Signal Inc. (Kanada) bezogen werden. [3H]-LSD wurde in Tris-HCl-Puffer, der 10 mM MgCl2, 0,5 mM EDTA und 0,5% BSA enthält, auf eine geeignete Konzentration verdünnt. Die Ra- dioligand-Lösung wurde in die Wells (25 ml) gegeben, die entweder TestVerbindung enthielten oder nicht. Die Platten wurden 180 Minuten bei Raumtemperatur inkubiert und das radioaktive Signal wurde mit einem Mikro-ß-Zähler (Wallac) gemessen. Die nicht spezifische Bindung wurde mit Methiothepin bestimmt. [3H]-LSD besitzt eine Affinität von 12 nM. Mit zunehmender Bindungsaffinität der Testverbindung nahm das radioaktive Signal von [3H]-LSD ab.96-well FlashPlates coated with h5-HT5 membranes are available from Bio Signal Inc. (Canada). [3H] -LSD was diluted to an appropriate concentration in Tris-HCl buffer containing 10 mM MgCl 2 , 0.5 mM EDTA and 0.5% BSA. The radio ligand solution was added to the wells (25 ml), which either contained test compound or not. The plates were incubated for 180 minutes at room temperature and the radioactive signal was measured with a micro-β counter (Wallac). The non-specific binding was determined with methiothepin. [3H] -LSD has an affinity of 12 nM. The radioactive signal of [3H] -LSD decreased with increasing binding affinity of the test compound.
Referenzbeispiel 5Reference example 5
Bestimmung der Agonist-induzierten Stimulierung der [35S]GTPγS- BindungDetermination of agonist-induced stimulation of [ 35 S] GTPγS binding
[35S]GTPγS-Bindungsassays sind bekannt. Der vorliegende Assay wurde in Anlehnung an die zuvor beschriebene Methode von Hilf, G. und Jakobs, K.H. (Eur. J. Mol. Pharmacol. 225:245-252 (1992)) durchgeführt. Es wurden Wirkstoff-induzierte Veränderungen der [35S]GTPγS-Bindung an Membranen aus stabil mit dem h5HT5-Rezeptor- gen transfizierten HEK293-Zellen gemessen (siehe Referenzbeispiele 1 und 2). Die Zellmembranen (12 μg) wurden mit 6,75 mM MgCl2, 150 mM NaCl, 1 mM DTT, 1 mM EDTA, 10 μM GDP und [35S]GTPγS enthaltendem 50 mM Triethanolamin-HCl-Puffer (pH: 7.5) inkubiert. Im Anschluß an eine 60-minütige Inkubation bei 30°C mit oder ohne Zusatz der zu testenden Wirkstoffe wurde das Testgemisch (100 μl) rasch über GF-B-Filtern bei Verwendung einer SkatronR-Filtrations- vorrichtung filtriert. Die Filter wurden rasch mit 100 mM NaCl und 5 mM MgCl2 enthaltendem 50 mM Tris-HCl-Puffer (9 ml; pH: 7,5; 4°C) gewaschen. Die auf den Filtern zurückgehaltene Radioaktivität wurde mittels Scintillationsspektrometrie bestimmt, wobei man Ul- tima Gold-Scintillationsflüssigkeit verwendete. Ebenfalls verwendet werden können ähnliche Systeme, die durch Verwendung von Mi- krotiterplatten einen hohen Durchsatz und ein Sekundärscreening gestatten.[ 35 S] GTPγS binding assays are known. The present assay was carried out based on the previously described method by Hilf, G. and Jakobs, KH (Eur. J. Mol. Pharmacol. 225: 245-252 (1992)). Drug-induced changes in the [ 35 S] GTPγS binding to membranes from HEK293 cells stably transfected with the h5HT5 receptor gene were measured (see reference examples 1 and 2). The cell membranes (12 μg) were incubated with 6.75 mM MgCl 2 , 150 mM NaCl, 1 mM DTT, 1 mM EDTA, 10 μM GDP and 50 mM triethanolamine-HCl buffer (pH: 7.5) containing [ 35 S] GTPγS . Following a 60-minute incubation at 30 ° C. with or without the addition of the active substances to be tested, the test mixture (100 μl) was quickly filtered through GF-B filters using a Skatron R filtration device. The filters were quickly washed with 50 mM Tris-HCl buffer (9 ml; pH: 7.5; 4 ° C) containing 100 mM NaCl and 5 mM MgCl 2 . The radioactivity retained on the filters was determined by scintillation spectrometry, using Ultima Gold scintillation fluid. Similar systems can also be used. allow high throughput and secondary screening.
Die Wirkstoffaktivitäten wurden als prozentualer Anteil der in Abwesenheit des Wirkstoffes gemessenen Grundbindung ausgedrückt. Die Anpassung der Kurven erfolgte mit einer Software zur nichtlinearen Regressionsanalyse (SigmaPlot, Jandel Scientific, Deutschland) gemäß der allgemeinen Gleichung E=(L*Emax) / (L+EC50) , worin E die Wirkung, L die Ligandkonzentration, Emax die Maximal- Wirkung und EC50 diejenige Konzentration, die 50% der Maximalwirkung induziert, bedeutet.The drug activities were expressed as a percentage of the basic bond measured in the absence of the drug. The curves were adjusted using software for non-linear regression analysis (SigmaPlot, Jandel Scientific, Germany) according to the general equation E = (L * E max ) / (L + EC 50 ), in which E is the effect, L the ligand concentration, E max the maximum effect and EC 50 means the concentration which induces 50% of the maximum effect.
Referenzbeispiel 6Reference example 6
Bestimmung der Agonist-induzierten Veränderung intrazellulärer CalciumspiegelDetermination of the agonist-induced change in intracellular calcium levels
Die Methode ist bekannt (Kao J.P.Y. Methods in Cell Biology 40:155-181 (1994)). Wie in Referenzbeispiel 1 beschrieben, wurden h5HT5-Rezeptor exprimierende HEK293-Zellen in Kulturgefäßen aufgezogen. Die Zellen wurden vorsichtig abgeschabt, bevor sie kon- fluent waren. Die Zellen wurden mit Fura-2 markiert, indem man mit Fura-2-acetylmethylester (Sigma) bei Raumtemperatur inkubierte. Die Zellen wurden bei 180xg 10 min zentrifugiert und in DMEM-F12-Medium ohne Serum resuspendiert und bei 37°C, 5% C02 und 95% Luftfeuchtigkeit 45 min inkubiert.The method is known (Kao JPY Methods in Cell Biology 40: 155-181 (1994)). As described in reference example 1, HEK293 cells expressing h5HT5 receptor were grown in culture vessels. The cells were carefully scraped off before they were confluent. The cells were labeled with Fura-2 by incubating with Fura-2-acetylmethyl ester (Sigma) at room temperature. The cells were centrifuged at 180 × g for 10 min and resuspended in DMEM-F12 medium without serum and incubated at 37 ° C., 5% CO 2 and 95% atmospheric humidity for 45 min.
Intrazelluläre Calciumspiegel wurden mit einem Fluoreszenzmikroskop bestimmt, das mit einem geeigneten Filteraustauschsystem aus- gestattet war (Olympus/Hamamatsu) . Es wurde das Fluoreszenzverhältnis (340 n / 380 nm) mit der ArgusR-Software bestimmt. Die intrazellulären Calciumspiegel wurden über kurze Zeit in einzelnen Zellen ohne den Zusatz von Wirkstoffen und dann 30 min nach Zugabe des zu testenden Wirkstoffs beobachtet. Ebenfalls verwen- det werden können ähnliche Systeme, die durch die Verwendung von Mikrotiterplatten einen hohen Durchsatz und ein Sekundärscreening gestatten.Intracellular calcium levels were determined using a fluorescence microscope equipped with a suitable filter exchange system (Olympus / Hamamatsu). The fluorescence ratio (340 n / 380 nm) was determined using the Argus R software. The intracellular calcium levels were observed for a short time in individual cells without the addition of active substances and then 30 minutes after the addition of the active substance to be tested. Similar systems can also be used, which allow high throughput and secondary screening through the use of microtiter plates.
In analoger Weise kann die Modulierung intrazellulärer Ca2+-Spie- gel im HTS bewertet werden. Dazu wurden h5-HT5-Rezeptor exprimierende CHO-Zellen über Nacht in 96-Well-Platten kultiviert (30.0000 - 80.000 Zellen/Well). Die Zellen wurden eine Stunde mit 1 mM Fluo-3-AM, 10% Pluronsäure und 2,5 mM Probeneid enthaltendem HEPES-Puffer markiert und gewaschen. Zu jedem Well wurde eine Testverbindung gegeben. Zur Bestimmung der Calciumspiegel wurde die Fluoreszenz-Intensität mit einem fluorometrisch arbeitendem Plattenlesegerät (Fluorometric Imaging Plate Reader; FLIPR) abgelesen.The modulation of intracellular Ca 2+ levels in the HTS can be evaluated in an analogous manner. For this purpose, h5-HT5 receptor-expressing CHO cells were cultivated overnight in 96-well plates (30,000-80,000 cells / well). The cells were labeled with HEPES buffer containing 1mM Fluo-3-AM, 10% pluronic acid and 2.5mM sample eep for one hour and washed. A test compound was added to each well. To determine the calcium level, the fluorescence intensity was measured using a fluorometric Read fluorometric imaging plate reader (FLIPR).
Referenzbeispiel 7Reference example 7
Bestimmung der Agonist-induzierten Phospholipase C-AktivitätDetermination of agonist-induced phospholipase C activity
Die Methode ist im wesentlichen bekannt (Garcia-Ladona F.J. et al., Neuroreport 4:691-694 (1993)). Die Zellen wurden 24 h mit 0,125 μM [3H]Myo-Inositol inkubiert. Nicht eingebautes [3H]Myo-In- ositol wurde aus dem Medium entfernt und durch 10 mM LiCl enthaltendem Krebs-Henseleit-Puffer ersetzt. Nach 10-minütiger Inkubation wurde der zu testende Wirkstoff zugesetzt. Nach 45 min wurde die Reaktion gestoppt, indem man das Stimulationsmedium durch de- stilliertes Wasser ersetzte. Verwendet man Gewebeproben, geht man in ähnlicher Weise vor (Garcia-Ladona et al., 1993). Die Zellen wurden eingefroren und bei -80°C gelagert. Es wurde die Produktion von [3H]Inositolmonophosphat mittels bekannter chromatographischer Methoden bestimmt. Eine ähnliche Methode kann mit Gewebe-Mini- prismen zur Anwendung kommen. Die Bestimmung der Phospholipase C- Stimulierung wurde ebenfalls in ähnlicher Weise durchgeführt, indem man Membranfraktionen, wie in Referenzbeispiel 2 beschrieben, präparierte und mit [32P]PIP2 und Wirkstoffen inkubiert. In diesem Fall wurde die Produktion von IP3 bestimmt. Es wurden auch be- kannte Verfahren optimiert, um auf Mikrotiterplatten basierende Systeme zu verwenden. Kommerziell erhältliche Materialien gestatten die Ausdehnung auf Analysen mit hohem Durchsatz und die Durchführung eines Sekundärscreenings .The method is essentially known (Garcia-Ladona FJ et al., Neuroreport 4: 691-694 (1993)). The cells were incubated with 0.125 μM [ 3 H] myo-inositol for 24 h. Unincorporated [ 3 H] myo-insitol was removed from the medium and replaced with Krebs-Henseleit buffer containing 10 mM LiCl. After 10 minutes of incubation, the active ingredient to be tested was added. After 45 minutes, the reaction was stopped by replacing the stimulation medium with distilled water. A similar procedure is used when using tissue samples (Garcia-Ladona et al., 1993). The cells were frozen and stored at -80 ° C. The production of [ 3 H] inositol monophosphate was determined using known chromatographic methods. A similar method can be used with tissue mini-prisms. The determination of the phospholipase C stimulation was also carried out in a similar manner by preparing membrane fractions as described in reference example 2 and incubating them with [ 32 P] PIP 2 and active ingredients. In this case, the production of IP3 was determined. Known methods have also been optimized in order to use systems based on microtiter plates. Commercially available materials allow expansion to high throughput analysis and secondary screening.
Referenzbeispiel 8Reference example 8
Bestimmung der Agonist-induzierten Veränderung der cAMP-Produk- tionDetermination of the agonist-induced change in cAMP production
Die verwendete Methode ist im wesentlichen bekannt (Strada S.S. et al., Methods in Neurotransmission receptor analysis: 89-110 (1990)). Zellen wurden 10 min in Kulturmedium ohne Serum und Antibiotika inkubiert. Es wurde 15 min auf 95°C erwärmt, um die Reaktion zu stoppen. Die Zellproben wurden eingefroren und bei -80°C gelagert. cAMP-Spiegel wurden mit kommerziell erhältlichen Kits bestimmt, die das cAMP-Bindungsprotein verwenden. Es wurden auch bekannte Verfahren optimiert, um auf Mikrotiterplatten basierende Systeme zu verwenden. Kommerziell erhältliche Materialien gestatten die Ausdehnung auf Analysen mit hohem Durchsatz und die Durchführung eines Sekundärscreenings. Referenzbeispiel 9The method used is essentially known (Strada SS et al., Methods in Neurotransmission receptor analysis: 89-110 (1990)). Cells were incubated for 10 min in culture medium without serum and antibiotics. The mixture was warmed to 95 ° C for 15 minutes to stop the reaction. The cell samples were frozen and stored at -80 ° C. cAMP levels were determined using commercially available kits that use the cAMP binding protein. Known methods have also been optimized to use microtiter plate based systems. Commercially available materials allow expansion to high throughput analysis and secondary screening. Reference example 9
GewebepräparationTissue preparation
90 min nach Verabreichung des Wirkstoffs (oral, intraperitoneal, intravenös oder intracerebroventriculär) wurden die Versuchstiere enthauptet. Das gesamte Gehirn wurde rasch aus dem Schädel entfernt, auf Trockeneis gefroren und bei -80°C gelagert. Rattenhirnschnitte (15 um) wurden bei -20°C in einem Cryostat erhalten, auf Gelatine-beschichtete Objektträger aufgebracht und bei -30°C bis zum Gebrauch gelagert.The test animals were decapitated 90 min after administration of the active ingredient (oral, intraperitoneal, intravenous or intracerebroventricular). The entire brain was quickly removed from the skull, frozen on dry ice and stored at -80 ° C. Rat brain sections (15 µm) were obtained at -20 ° C in a cryostat, applied to gelatin-coated slides and stored at -30 ° C until use.
Beispiel 1example 1
Gemäß Referenzbeispiel 3 wurde die Bindungsaffinität von [3H]-5-CT an 5-HT5-Rezeptoren bestimmt. Figur 1 zeigt eine Auftragung von gebundenem [3H]-5-CT in Abhängigkeit der [3H]-5-CT-Konzentration. Es wurde eine Dissoziationskonstante von K^ = 0.570 nM bestimmt. Die Rezeptorbindungsdichte (B) variierte je nach klonaler Zelli- nie in einem Bereich von 900-28000 fmol/mg Protein.The binding affinity of [ 3 H] -5-CT to 5-HT5 receptors was determined according to reference example 3. FIG. 1 shows a plot of bound [ 3 H] -5-CT as a function of the [ 3 H] -5-CT concentration. A dissociation constant of K ^ = 0.570 nM was determined. Depending on the clonal cell line, the receptor binding density (B) varied in a range of 900-28000 fmol / mg protein.
Beispiel 2Example 2
Gemäß Referenzbeispiel 4 wurden die Bindungsaffinitäten serotoni- nerger Verbindungen anhand der [3H]-5-CT-Bindungskompetition bestimmt. Folgende iCso-Werte wurden erhalten:According to Reference Example 4, the binding affinities of serotoninergic compounds were determined using the [ 3 H] -5-CT binding competition. The following iCso values were obtained:
In einer weiteren Testreihe wurden auch die Hemmkonstanten Ki folgender Verbindungen bestimmt (Ki = IC50/ ( 1+C/Kd) ) , wobei C die Konzentration an [3H]-5-CT ist und Kd gemäß Beispiel 1 bestimmt wurde ) : In a further series of tests, the inhibition constants Ki of the following compounds were also determined (Ki = IC 50 / (1 + C / K d )), where C is the concentration of [ 3 H] -5-CT and K d was determined in accordance with Example 1 ):
Beispiel 3Example 3
Gemäß Referenzbeispiel 5 wurde die Wirkstoff-induzierte Bindung von GTP an G-Proteine untersucht. Die Kopplung von 5-HT5-Rezepto- ren an G-Proteine in HEK293-Zellen war evident. Die typischen se- rotoninergen Agonisten 5-HT und 5-CT induzierten einen Anstieg der [35S]GTPγS-Bindung an die Zellmembranen von über 40 % über dem Grundwert (siehe Figur 2). Der 5-HT5-Rezeptor benötigt GDP für die durch Agonisten vermittelte Kopplung an G-Proteine (siehe Figur 3A) . Der 5-HT-Effekt war dosisabhängig (siehe Figur 4) mit einer EC50 von 2,6 μM.According to reference example 5, the drug-induced binding of GTP to G proteins was investigated. The coupling of 5-HT5 receptors to G proteins in HEK293 cells was evident. The typical serotoninergic agonists 5-HT and 5-CT induced an increase in [ 35 S] GTPγS binding to the cell membranes of more than 40% above the basic value (see FIG. 2). The 5-HT5 receptor requires GDP for the agonist-mediated coupling to G proteins (see FIG. 3A). The 5-HT effect was dose-dependent (see FIG. 4) with an EC 50 of 2.6 μM.
Beispiel 4Example 4
Gemäß Referenzbeispiel 6 wurde die Wirkstoff-induzierte Auswir- kung auf intrazelluläre Calcium-Spiegel untersucht. Die Stimulierung der 5-HT5-Rezeptoren mit R(+)-Lisurid (1 μM) in HEK293-Zellen induzierte einen Anstieg von intrazellulärem Ca2+ (siehe Figur 5). According to reference example 6, the drug-induced effect on intracellular calcium levels was investigated. Stimulation of the 5-HT5 receptors with R (+) - lisuride (1 μM) in HEK293 cells induced an increase in intracellular Ca 2+ (see FIG. 5).

Claims

Patentansprüche claims
1. Selektiver Bindungspartner für 5-HT5-Rezeptoren, dadurch ge- kennzeichnet, daß dessen Bindungsaffinität für 5-HT5-Rezepto- ren größer ist als für einen oder mehrere von 5-HT5 verschiedene 5-HT-Rezeptoren.1. Selective binding partner for 5-HT5 receptors, characterized in that its binding affinity for 5-HT5 receptors is greater than for one or more 5-HT5 receptors different from 5-HT5.
2. Bindungspartner nach Anspruch 1, dadurch gekennzeichnet, daß dessen Bindungsaffinität für 5-HT5-Rezeptoren größer ist als für 5-HT1D- und/oder 5-HTlB-Rezeptoren.2. Binding partner according to claim 1, characterized in that its binding affinity for 5-HT5 receptors is greater than for 5-HT1D and / or 5-HTIB receptors.
3. Bindungspartner nach Anspruch 1 oder 2 , dadurch gekennzeichnet, daß er die Bindung von 5-CT an 5-HT5-Rezeptoren kompeti- tiv hemmt.3. Binding partner according to claim 1 or 2, characterized in that it competitively inhibits the binding of 5-CT to 5-HT5 receptors.
4. Bindungspartner nach einem der Ansprüche 1 bis 3 , dadurch gekennzeichnet, daß die Ki-Werte für dessen Bindung an 5-HT5-Rezeptoren weniger als 10-8 M, vorzugweise weniger als 10-9 M und insbesondere weniger als 10-10 M betragen.4. Binding partner according to one of claims 1 to 3, characterized in that the Ki values for its binding to 5-HT5 receptors less than 10 -8 M, preferably less than 10 -9 M and in particular less than 10 -10 M. be.
5. Bindungspartner nach einem der Ansprüche 1 bis 4 , dadurch gekennzeichnet, daß dessen Bindung an 5-HT5-Rezeptoren die GTP- Bindung an G-Proteine stimuliert.5. Binding partner according to one of claims 1 to 4, characterized in that its binding to 5-HT5 receptors stimulates GTP binding to G proteins.
6. Bindungspartner nach einem der Ansprüche 1 bis 5, dadurch gekennzeichnet, daß dessen Bindung an 5-HT5-Rezeptoren eine Erhöhung des intrazellulären Calcium-Spiegels bewirkt.6. Binding partner according to one of claims 1 to 5, characterized in that its binding to 5-HT5 receptors causes an increase in the intracellular calcium level.
7. Bindungspartner nach einem der Ansprüche 1 bis 6, dadurch gekennzeichnet, daß dessen Bindung an 5-HT5-Rezeptoren eine Induktion der Phospholipase C-Aktivität bewirkt.7. Binding partner according to one of claims 1 to 6, characterized in that its binding to 5-HT5 receptors causes an induction of the phospholipase C activity.
8. Bindungspartner nach einem der Ansprüche 1 bis 7 , dadurch ge- kennzeichnet, daß dessen Bindung an 5-HT5-Rezeptoren eine Induktion der cAMP-Produktion bewirkt.8. Binding partner according to one of claims 1 to 7, characterized in that its binding to 5-HT5 receptors causes an induction of cAMP production.
9. Pharmazeutische Zusammensetzung, umfassend wenigstens einen Bindungspartner nach einem der Ansprüche 1 bis 8 und einen pharmazeutisch verträglichen Exzipienten sowie gegebenenfalls weitere Wirkstoffe. 9. A pharmaceutical composition comprising at least one binding partner according to one of claims 1 to 8 and a pharmaceutically acceptable excipient and optionally further active ingredients.
10. Verwendung eines Bindungspartners für 5-HT5-Rezeptoren zur Herstellung eines Mittels zur Behandlung cerebrovasculärer Störungen.10. Use of a binding partner for 5-HT5 receptors for the preparation of an agent for the treatment of cerebrovascular disorders.
5 11. Verwendung nach Anspruch 10 zur Behandlung von Migräne, insbesondere zur akuten Behandlung von Migräne.5 11. Use according to claim 10 for the treatment of migraine, in particular for the acute treatment of migraine.
12. Verwendung nach Anspruch 10 oder 11 eines Bindungspartners nach einem der Ansprüche 1 bis 8.12. Use according to claim 10 or 11 of a binding partner according to one of claims 1 to 8.
1010
13. Verfahren zur Bestimmung der Affinität von Bindungspartnern für 5-HT5-Rezeptoren, wobei man den Bindungspartner mit 5-HT5-Rezeptoren aufweisenden zellulären Systemen in Kontakt bringt und die Bindungsaffinität bestimmt.13. A method for determining the affinity of binding partners for 5-HT5 receptors, wherein the binding partner is brought into contact with cellular systems having 5-HT5 receptors and the binding affinity is determined.
1515
14. Verfahren zur Bestimmung der Aktivität von Bindungspartnern für 5-HT5-Rezeptoren, wobei man den Bindungspartner mit 5-HT5-Rezeptoren aufweisenden zellulären Systemen in Kontakt bringt und wenigstens eine Bindungspartner-induzierte agoni-14. A method for determining the activity of binding partners for 5-HT5 receptors, wherein the binding partner is brought into contact with cellular systems having 5-HT5 receptors and at least one agoni-
20 stische Wirkung bestimmt.20 determined static effect.
15. Verfahren nach Anspruch 14, wobei die Bindung von GTP an G- Proteine, intrazelluläre Calcium-Spiegel, die Phospholipase C-Aktivität und/oder die cAMP-Produktion bestimmt werden.15. The method according to claim 14, wherein the binding of GTP to G proteins, intracellular calcium levels, the phospholipase C activity and / or the cAMP production are determined.
2525
16. Verfahren nach einem der Ansprüche 14 oder 15, dadurch gekennzeichnet, daß man humane Gliom-Zellinien oder h5-HT5-transfizierte heterologe Zellinien verwendet.16. The method according to any one of claims 14 or 15, characterized in that one uses human glioma cell lines or h5-HT5-transfected heterologous cell lines.
30 17. Verfahren nach Anspruch 16, dadurch gekennzeichnet, daß man h5-HT5-transfizierte CHO-Zellen, h5-HT5-transfizierte humane Nierenzellen, oder h5-HT5-transfizierte C-6-Gliomzellen verwendet.17. The method according to claim 16, characterized in that h5-HT5-transfected CHO cells, h5-HT5-transfected human kidney cells, or h5-HT5-transfected C-6 glioma cells are used.
35 18. In vitro-Screening-Verfahren zur Identifizierung eines35 18. In vitro screening method to identify a
5-HT5-Rezeptor-Bindungspartners, wobei wenigstens ein Verfahren nach den Ansprüchen 13 bis 17 zur Anwendung kommt.5-HT5 receptor binding partner, at least one method according to claims 13 to 17 being used.
4040
45 45
EP00901526A 1999-01-11 2000-01-11 Binding partners for 5-ht5-receptors for the treatment of migraine Ceased EP1144050A2 (en)

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PCT/EP2000/000142 WO2000041472A2 (en) 1999-01-11 2000-01-11 Binding partners for 5-ht5-receptors for the treatment of migraine

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DE102004008141A1 (en) 2004-02-19 2005-09-01 Abbott Gmbh & Co. Kg Guanidine compounds and their use as binding partners for 5-HT5 receptors
US8921406B2 (en) 2005-08-21 2014-12-30 AbbVie Deutschland GmbH & Co. KG 5-ring heteroaromatic compounds and their use as binding partners for 5-HT5 receptors
WO2007022964A2 (en) 2005-08-24 2007-03-01 Abbott Gmbh & Co. Kg Hetaryl-substituted guanidine compounds and use thereof as binding partners for 5-ht5-receptors

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AU8770282A (en) * 1981-08-27 1983-03-03 Shaun R. Coughlin Composition for the treatment of atherosclerosis
US5360735A (en) * 1992-01-08 1994-11-01 Synaptic Pharmaceutical Corporation DNA encoding a human 5-HT1F receptor, vectors, and host cells
FR2693200B1 (en) * 1992-07-01 1994-08-19 Inst Nat Sante Rech Med New polypeptides having serotonergic receptor activity, nucleic acids encoding these polypeptides and uses.
FR2701265B1 (en) * 1993-02-09 1995-04-07 Inst Nat Sante Rech Med New polypeptides having serotoninergic receptor activity, nucleic acids encoding these polypeptides and uses.

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Title
FORTH W.; HENSCHLER D.; RUMMEL W.: "Allgemeine und spezielle Pharmakologie und Toxikologie", 1990, BI WISSENSCHAFTSVERLAG, MANNHEIM, WIEN, ZÜRICH *
See also references of WO0041472A3 *

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