EP1144050A2 - Bindungspartner für 5-ht5-rezeptoren zur migränebehandlung - Google Patents

Bindungspartner für 5-ht5-rezeptoren zur migränebehandlung

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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)
English (en)
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/de
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 or sparfloxacin
    • 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.

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