DE19900674A1 - Binding partner for 5-HT5 receptors for migraine treatment - Google Patents

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

The present invention relates to binding partners for 5-HT5-Re receptors, identification and characterization methods such binding partner, as well as pharmaceutical containing them Compositions and their use in the treatment of cerebrovas culinary disorders such as migraines.

At least seven different receptor classes mediate the diverse physiological activities that involve participation of the neurotransmitter serotonin (5-hydroxytryptamine, short 5-HT) be attributed. You will become one internationally recognized classification with 5-HT1, 5-HT2, 5-HT3, 5-HT4, 5-HT5, 5-HT6 and 5-HT7 designated. Most of these classes include in addition, other distinguishable receptor types. So belong to the 5-HT1 class receptors, which can be found in at least five Un categorize with 5-HT1A, 5-HT1B, 5-HT1C, 5-HT1D and 5-HT1E (Boess F. G. and Martin I. L., 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). One under secretes 5-HT5A and 5-HT5B receptors (Erlander et al., Proc. Natl. Acad. Sci. USA 90: 3452-3456 (1993)). There are only ge ring sequence homologies between 5-HT5 and other 5-HT receptors ren. The pharmacological profile of these receptors differs themselves clearly. The Lo calibration of 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 was possible to show be that 5-HT5 receptors primarily on astrocytes expri be lubricated (Carson et al., GLIA 17: 317-326 (1996)). Astrocytes lie directly on the basement membrane of brain capillaries of the Blu brain barrier. An abnormal astrocyte endothelium structure is associated with a loss of the blood brain barrier. The exact Meaning of astocytes is unclear. They seem to be transport tasks and perform connective functions. Reactive astrocytes have been associated with reactive gliosis in a number of pathological brain changes and neuropsychiatric Er diseases observed. Change as a result of brain injuries their morphology. The protein expression pattern changes and growth factors are produced. In vitro studies on cultured astrocytes, 5-HT5 receptor-mediated Ant let words recognize. On the one hand, it is suspected that it is Brain recovery processes after disorders are involved  on the other hand, it can not be ruled out that they are to the Scha emergence or even contribute to an increase in damage.

Migraine is a CNS disease that affects large sections of the population meets. In most cases, it manifests itself again and again sweeping headache, of which an estimated 8 million people d. H. 3-5% of all children, 7% of all men and 14% of all women, are affected. Although a genetic predisposition propa The causes seem to be complex (Diener H.C. et al., Drug Therapy 15: 387-394 (1997)).

Two hypotheses dominate. The long-known Ge barrel theory suggests a dilation process of the in inner and outer cerebral vasculature. The neurogenic Theory is based on the release of vasoactive neurotrans middle, mainly neuropeptides, like substance P and neurokinin, from axons of the vasculature as a result of stimulation of certain Brain tissue innervating ganglia, leading to inflammatory reak tion and thus lead to pain.

There is currently a causal therapy for the treatment of migraines not yet. Two different treatment methods are currently coming to use. A first, prophylactic, preventive therapy against recurring migraine attacks and a second, sym ptomatic, therapy to suppress acute symptoms in At tack. Prophylactic migraine-specific active ingredients, such as Sanmigran®, Nocerton®, Desernil® and Vidora®, but also usually active ingredients used for other indications, such as beta-blockers, antiemetic agents like Sibelium®, antidepressants like Laro xyl®, or anti-epileptic agents such as Depakin®. In the context of acute therapy, analgesics such as Aspirin®, Pa racetamol or Optalidon®, non-steroidal anti-inflammatory drugs, such as Cebutid®, Voltaren®, Brufen®, Ponstyl®, Profenid®, Apranx® and Na prosin® for pain and inflammation, ergot alkaloids, such as Ergotamine, dihydroergotamine, which cause vasoconstriction can, or substances of the triptan family, such as sumatriptan, Na ramig®, and AscoTop® with high affinity for 5-HT1D receptors. The latter substances act as an agonist and block the vasodi latation.

However, the active ingredients mentioned are not optimal for the Suitable for treating migraines. Have non-opioid analgesics often side effects. The complex mechanism of action of the Ergo talcaloids result from the strong peripheral vasoconstriction to side effects such as hypertension and gangrene. To the Triptan Fa connections belonging to milie are also not entirely satisfied providing (Pfaffenrath V. Münch. med. Wschr. 625-626 (1998)).  

Sumatriptan (Imigran®), one of the most effective and most common active ingredients used against acute migraine attacks happens due to pronounced hydrophilicity, the blood-brain barrier does not. In 28% of patients find this drug ineffective, the oral one Doses are quite high at 50-100 mg. As contraindications are ko known ronar vasopasms, hypertension, kidney and liver disorders become.

So far, one has connections with selective affinity for 5-HT1 receptors considered for the treatment of migraines (Goadsby P.J. CNS Drugs 10: 271-286 (1998); Saxena P.R. Exp. Opin. Invest. Drugs 581-593 (1996)). For example, sumatriptan applies as a very selective ligand for 5-HT1D receptors, wes half of the experts were concerned with these binding properties and the thereby optimizing mediated vasoconstrictive activities. The same applies to 5-HT1B and 5-HT1F receptor ligands. So were Series of active ingredients developed that the described problem but could not help either.

The object of the present invention is therefore, the preferred wise acute, treatment of migraine-like cerebrovascular disease with sufficient effectiveness and few side effects to enable.

Surprisingly, it has now been found that substances with ver equally high binding affinity for 5-HT5 receptors can convey desired effects.

An object of the present invention are therefore selective Binding partner for 5-HT5 receptors, which are characterized are that their binding affinity for 5-HT5 receptors is greater than for one or more 5-HT5 receptors other than 5-HT5 ren.

Binding partners according to the invention therefore bind preferentially 5-HT5 receptors. Binding means any molecular Interaction between the binding partner and the receptor, especially under physiological conditions. These are in the Usually classic interactions, to which electrostatic an drawing, hydrogen bonding, hydrophobic bonds, van der Waals forces or metal complex-like coordinative bonds belong. In addition to the reversible mo lecular interactions can also cause irreversible interactions considerations between binding partner and receptor, like covalent bonds.  

Selectivity is the property of a bond partners, preferably to bind to 5-HT5 receptors.

For example, binding partners have binding affinities for 5-HT5 recipes gates that are larger than one or more of 5-HT5 ver different 5-HT receptors, in particular those mentioned above 5-HT receptor classes 5-HT1, 5-HT2, 5-HT3, 5-HT4, 5-HT6 and 5-HT7 assignable receptors. Is the binding affinity for 5-HT5-Re binding partner receptors larger than that of 5-HT5 different 5-HT receptor, one speaks of one in relation on the 5-HT receptor that is more selective than 5-HT5 extension of these binding partners to 5-HT5 receptors. Special bin End partners are those whose binding affinity for 5-HT5 receptors is larger than for 5-HT1 receptors, in particular for 5-HT1D and / or 5-HT1B receptors. Attachment partner, de binding affinity for 5-HT5 receptors is greater than for all 5-HT5 receptors other than 5-HT5 represent one represents another special class of attachment partners.

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 other than 5-HT5 on the other hand make a sufficient distinction. Affinity is preferred distinguished, according to which binding affinity ratios of little least 2, more advantageous of at least 5, particularly advantageous of at least 10, preferably at least 20, especially be preferably at least 50 and in particular at least 100 available.

According to a preferred embodiment, inhibit according to the invention Binding partner the binding of comparative binding partners, such as 5-HT (5-hydroxytryptamine) or 5-CT (5-carboxamidotryptamine) 5-HT5 receptors competitive.

Competitive inhibition means that the invention Binding partner with a comparison binding partner, in the present preferably 5-CT to bind to the receptor compete, d. H. the bond of one hinders the bond of the other.

The reason applies at least in the case of reversible binding sentence that the displacement of a binding partner by one which increase or decrease with decreasing binding affinity of the one the binding affinity of the other with respect to the receptor increases. Appropriately therefore have bin according to the invention partner a high binding affinity for 5-HT5 receptors. On the one hand, such a binding affinity allows an effective  same displacement of naturally occurring binding partners for 5-HT5 receptors, such as, for example, serotonin (5-hydroxytrypta min. 5-HT) itself, taking the required concentration of it binding partner according to the invention for binding a specific Amount of this binding partner to 5-HT5 receptors with increasing Attachment affinity decreases. With regard to medical an Therefore binding partners are preferred whose binding aff is so great that it acts as an active ingredient in a effective medical treatment in acceptable amounts can be enough. Binding partners according to the invention are there forth preferably in daily doses of about 0.01 to 100 mg / kg body perweight and in particular from about 0.1 to 15 mg / kg body weight important for parenteral administration and about 1 to 30 mg / kg body weight administered by oral administration.

One way 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 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 be evaluated in such a way that binding partners preferred according to the invention have half-maximal inhibitory constants IC ₅₀ of less than 10 ^-7 M, preferably less than 10 ^-8 M and have in particular less than 10 ^-9 M.

The binding affinity of binding partners according to the invention can also be expressed via the inhibition constant K _i , which is also generally determined using competition experiments. For the binding to 5-HT5 receptors, binding partners according to the invention preferably have Kl values of less than 10 ^-8 M, advantageously less than 10 ^-9 M and particularly preferably less than 10 ^-10 M.

According to a further embodiment, bin according to the invention bind partner in relation to one or more of 5-HT5 den 5-HT receptors more selective to 5-HT5 receptors with the above described advantageous binding affinities.

According to a further embodiment, bin according to the invention bind partner for all 5-HT-Re different from 5-HT5 receptors more selective at 5-HT5 receptors with the above described beneficial binding affinities.  

Binding partners with the first one are particularly advantageous affinities and selectivities described for 5-HT5 recipe bind gates from glial cells and especially from astrocytes be expressed.

According to the invention, the human receptor variant is the preferred tar get for the binding partner used.

The binding of binding partners according to the invention to 5-HT5 receptors is linked to an effector function. Binding partners can agonistic or antagonistic as well as partially agonistic and / or partially antagonistic.

According to the invention, agonists are compounds which all or part of the activity of 5-HT at 5-HT5 receptors imitate.

According to the invention, compounds are referred to as antagonists which shows the agonistic activity of 5-HT on 5-HT5 receptors can block.

According 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 which triggers a partially agonistic effect on the 5-HT5 receptor has sufficient agonistic activity in order to be able to be administered in an acceptable amount as part of an effective medical treatment. Binding partners with at least 50% agonistic activity are preferred. Particularly preferred are those binding partners with at least 80% agonistic activity and especially those with essentially fully agonistic activity (E _max ).

Within this particular embodiment, in particular Provided binding partners, their binding to 5-HT5 receptors stimulate GTP binding to membranes bound G proteins, an agonist-induced change intracelally lular calcium level, an induction of phospholipase C-Akti vity and / or an agonist-induced change in cAMP-Pro production causes.

This embodiment also includes binding partners that are in known animal models for cerebrovascular diseases, esp especially for migraines, are effective, and / or the specific in vi induce vo effects in brain areas, especially genomi Cause responses in the brain, such as the expresses  sion of transcription factors such as c-fos, c-jun, zif268 or Ho mergen isoforms (Brakeman P.R. et al., Nature 386: 284-288 (1997)).

Binding partners are preferred which also relate to their effect gate function in the sense described above is more selective for 5-HT5-Re are receptors.

Such effector functions can be performed using known functions qualitative assays both in vitro and in vivo or be assessed quantitatively.

The present invention therefore also relates to methods for identifying and characterizing bin according to the invention partner. These procedures can form the basis for in vitro screening method, with which one ver from a variety different connections can read out those that are in view Seems to be the most promising for a future application nen. For example, using combinatorial chemistry catchy fabric banks are created, the myriads of potential Active ingredients include. Screening combinatorial substance libraries by substance with the desired activity is automatic sizable. Screening robots are used for the efficient evaluation of preferably individual assays arranged on microtiter plates. So the present invention also relates to screening methods, i. H. both primary and secondary screening procedures where preferably at least one of the ver described below driving comes into play. Several procedures are used dung, it can be staggered or at the same time to one and the same sample or on different samples to be examined happening substance.

A particularly effective technology for performing such The method is the one known in the field of active substance screening Scintillation Proximity Assay, SPA for short. Kits and compo Components for performing this assay can be obtained commercially at Amersham Pharmacia Biotech. Basically become solubilized or membrane bound receptors on Scin small fluoromicrospheres containing tillation substance immo bilizes. For example, binds a radioligand to the immobili based receptors, the scintillation substance becomes Lich temission stimulated because of the spatial proximity between Scintillati substance and radio ligand is given.

Another particularly effective technology for carrying out such processes is the FlashPlate® 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. For this purpose, the binding partner is brought into contact with 5-HT5 receptors and the binding affinity is determined. This can be done, for example, by evaluating the com petitive inhibition of the binding of a comparative binding partner to 5-HT5 receptors by the substance to be examined. Known ligands for 5-HT receptors, such as 5-HT or 5-CT, are suitable as comparison binding partners. These are appropriately marked so that their binding to 5-HT receptors can be analyzed analytically using standard methods. Radioactive and optical markings are preferred. In binding studies on 5-HT5 receptors, 5-CT is used according to the invention, in particular in the form of [ ³ H] -5-CT. The binding affinities can be expressed as a half maximum inhibition constant IC ₅₀ or as an inhibition constant K _i . This method is used primarily for primary screening. SPA technology is preferred.

The binding partner to be examined can also be determined directly on 5-HT receptors. Inhibitory constants K _i expressing the binding affinity can be determined, for example, calorimetrically, ie by measuring the released binding energy.

Selectivity is determined in the same way - If necessary, using that for the respective receptor specific ligands - to examine the binding affinity of the the binding partner for other 5-HT receptors and compares them received values.

Another method according to the invention relates to the determination the activity of binding partners for 5-HT5 receptors, d. H. the Determination of agonistic, partially agonistic, antagonistic and / or partially antagonistic effect. For this purpose you bring the binding partner in contact with 5-HT5 receptors and evaluated the effects caused by the bond. The assessment An agonistic activity can have all those effects be determined by the binding of 5-HT to 5-HT5-Re receptors are caused. According to the invention, it is preferred the effects on the binding of GTP to G proteins on in tercellular calcium levels, on phospholipase C activity and / or to evaluate cAMP production. This procedure  are used primarily for secondary screening. Here too comes the SPA technology beneficial to use.

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 5-HT5 receptor on pointing membranes.

Suitable for measuring intracellular calcium levels Calcium probes, usually calcium chelators, for example fluorescent compounds such as fura-2-acetylmethyl ester put. This test is preferably done on 5-HT5 receptor having single cells performed.

The phospholipase C activity can be determined on the basis of the reactions catalyzed by it, 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 , the 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.

The cAMP production can be with the help of the cAMP binding protein be true. This test is preferably carried out on 5-HT5-Re single cells having zeptor performed.

If necessary, one also determines the effector function, i. H. the Ak activity of binding partners according to the invention for other 5-HT Receptors. This is expediently taken into account of the bin determined for 5-HT5 and other 5-HT receptors affinity, especially taking into account the Selectivity.

The above-mentioned methods are in principle a person skilled in the art known.

In a preferred embodiment, binding partners are subjected to primary screening by determining their binding affinity for 5-HT5 receptors with the [ ³ H] -5-CT competition experiment described above. Those binding partners that have an inhibitory constant IC ₅₀ in the range of 10 ^-6 M or less are then subjected to secondary screening by examining their effector function, particularly with regard to GTP binding and intracellular calcium levels written way. Finally, the binding partners selected in this way are subjected to 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 particular receptor . For example, [ ³ H] -8-hydroxy-di-propylaminotetralin [ ³ H] -8-DPAT can be used for binding studies on 5-HT1A receptors, while 5-HT1B and 5-HT1D receptors with [ ³ H] -5-CT competition experiments can be examined.

5-HT5 receptors are preferably in the form of cellular systems provided, d. H. in the form of membranes, cells, cells associations, tissues or organs that carry 5-HT5 receptors. Such cellular systems can inherently 5-HT5 receptors express, but they can also by appropriate genetic Ma nipulation, e.g. B. by transfection to 5-HT5 expression let it be. Human glioma cell lines are considered natural, Cellular systems having 5-HT5 receptors are preferred. Of the h5-HT5 transfected heterologous cell lines will be those preferred that stably express the h5-HT5 gene. To be mentioned for example h5-HT5 transfected CHO cells, h5-HT5 transfi graced human kidney cells, especially h5-HT5 transfected HEK293 cells, or h5HT5 transfected C-6 glioma cells.

To determine selectivity, affinity and activity invented According to the binding partner can also brain tissue sections and native membranes from brain parts are used. Be radio If active markers are used, tissue is evaluated cut preferably autoradiographically.

The binding partners of the invention are usually in the form of pharmaceutical compositions administered a pharmaceutically acceptable excipients with at least one he binding partner according to the invention and optionally further active ingredient include fabrics. These compositions can, for example on oral, rectal, transdermal, subcutaneous, intravenous, administered intramuscularly or intranasally.

Examples of suitable pharmaceutical formulations are solid Pharmaceutical forms, such as powder, powder, granules, tablets, lozenges, Sachets, cachets, coated tablets, capsules such as hard and soft gelatin capsules, suppositories or vaginal dosage forms, semi-solid medic neiforms, such as ointments, creams, hydrogels, pastes or plasters, as well as liquid pharmaceutical forms, such as solutions, emulsions, in particular other oil-in-water emulsions, suspensions, for example Lotio NEN, injection and infusion preparations, eyes and ears drops. Implanted delivery devices can also be used for administration range of binding partners according to the invention can be used. Fer  ner can also liposomes, micro-spheres or polymer matrices Application come.

In the preparation of the compositions according to the invention Binding partners are usually mixed with an excipient or diluted. Excipients can be solid, semi-solid or liquid mate rialien, as a vehicle, carrier or medium for the active serve fabric.

Suitable excipients include, for example, lactose, dex trose, succrose, sorbitol, manitol, starches, acacia, calci umphosphate, alginates, tragacanth, gelatin, calcium silicate, micro crystalline cellulose, polyvinylpyrrolidone, cellulose, water, Syrup and methyl cellulose. Furthermore, the formulations can be phar pharmaceutically acceptable carriers or conventional excipients such as glide medium, for example tallow, magnesium stearate and mineral oil; Wetting agents; emulsifying and suspending agents; conserve agents such as methyl and propyl hydroxybenzoates; Antioxidant tien; Anti-irritants; Chelating agents; Coating aids; Emul ion stabilizers film formers; Gelling agent; Odor masking medium; Taste corrections; Resins; Hydrocolloids; Solvents; Solubilizer; Neutralizing agents; Permeation acceleration ger; Pigments; quaternary ammonium compounds; Regreasing and Superfatting agents; Ointment, cream or oil base materials; Silicone- Derivatives; Spreading aids; Stabilizers; Sterilizers; Suppo territorial foundations; Tablet excipients, such as binders, fillers fabrics, lubricants, disintegrants or coatings; Blowing agent; Desiccants; Opacifiers; Thickeners; Waxes; Plasticizers; Include white oils. A design in this regard is based on professional knowledge, such as in Fiedler, H. P., Lexicon of excipients for pharmacy, cosmetics and angren zende areas, 4th edition, Aulendorf: ECV-Editio-Kantor-Verlag, 1996.

The present invention also relates to the use of egg Nes binding partner for 5HT-5 receptors and especially the above-mentioned special and preferred embodiments binding partner according to the invention for the treatment of migraine-like cerebrovascular diseases, especially for the treatment of Mi tears and other vascular headaches, in particular seizure-like headache of the migraine type. These include the diseases known as simple and classic migraines without or with accompanying neurological dysfunction, real and atypical migraines, and also more specific diseases of this type, for example Migraine accompagnée, so-called Mi equine equivalent, digestive migraine, migraine ophtalmique, ophthalmic mophlegic migraine, migraine rouge, also as a cluster head  pain (Horton syndrome), cervical migraine. Under Be act becomes both a prophylactic therapy, in particular the prevention of recurring attacks, for example as Interval treatment, as well as treatment for acute symptoms men, especially during the headache phases. A successful treatment leads to a reduction in intensity symptoms, especially headache, neurological Malfunctions, e.g. B. visual, sensory, motor Disorders and speech disorders, nausea and nausea, and / or the frequency of attacks. An acute treatment for migraines with bin partner is preferred. The invention relates in particular special also the use of the binding partners mentioned for the Treatment of such forms of the diseases listed above, de their formation and / or course 5-HT5 receptors are involved, d. H. Diseases caused by 5-HT5 receptor activity be lated.

Binding partners for 5-HT5 receptors include with regard to the use according to the invention in particular those de their binding affinity for 5-HT5 receptors compared to the Af 5-HT1 receptors, especially 5-HT1B and / or 5-HT1D, is so high that it is suitable for use in accordance with the invention are suitably suitable. This does not necessarily mean comparatively more selective binding to 5-HT5 receptors from, albeit selective binding partners for 5-HT5 receptors are a particular embodiment of the present invention.

The present invention is illustrated by the following examples explained in more detail without being limited thereto.

Reference example 1 HEK293 cells expressing h5-HT5 receptor

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 accordance with the manufacturer's instructions in E. coli. A preparation of the resulting plasmid was mixed with Lipofectamin® (Gibco Life Science, 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% atmospheric humidity and 37 ° C.

Reference example 2 Cell membrane preparation

The method used here is essentially based on Known methods for the preparation of cell membranes from cells (Findlay J.B.C. and Evans W.H. 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 x g in DMEM-F12 medium. The won The cell pellets were in 5 mM EDTA, 5 mM EGTA, 0.1 mM PMSF and 3 mM Tris-HCl buffer containing 3 mM benzamidine (pH: 7.6; Puf fer A) resuspended and incubated for 15 min at 4 ° C. The cell suspensions sion was homogenized in an Ultraturrax® (15,000 rpm) (6 × 3 s) and centrifuged for 1 min at 1000 x g and 4 ° C. The pellet was in Buffer A resuspended and, as described above, homogeneous centrifuged and centrifuged. The supernatants from both steps were the collected and centrifuged for 20 min at 40,000 × g and 4 ° C. The The pellet was resuspended in buffer A and homogenized (1 × 15 s). The membrane suspension was centrifuged at 40,000 × g and 4 ° C. for 20 min yaws. The resulting pellet was in 10% glycerin and 1% Rin buffer A containing serum albumin resuspended. There were Frozen aliquots and stored at -80 ° C until use.

Reference example 3 Kinetics of saturation binding of [ ³ H] -5-CT

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), with 10 µM methiothepin being added to determine the specific binding, while methio thepin 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® 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).

Reference example 4 [ ³ H] -5 CT binding competition

The experiments on binding competition were essentially based on known studies (Rees et al., 1994). Ge membranes obtained according to 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 period 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 non-specific binding was defined as that 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 SigmaPlot software (Jandel Scientific, Germany). Competition curves were drawn up in which the radioactive binding is expressed as a percentage of the total binding. Half-maximum inhibition constants IC ₅₀ and Hill coefficients (n _H ) were determined by means of non-linear regression analysis.

Reference example 5 Determination of agonist-induced stimulation of [ ³⁵ S] GTPγS binding

[ 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 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 [ ³⁵ S] GTPγS containing 50 mM triethanolamine-HCl buffer (pH: 7.5) . 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® 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 Ulima Gold scintillation fluid. Similar systems can also be used, which allow high throughput and secondary screening through the use of microtiter plates.

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 = (LE _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 that induces 50% of the maximum effect.

Reference example 6 Determination of agonist-induced change in intracellular Calcium level

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-acetyl methyl 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 measured using a fluorescence microscope Kop determines that with a suitable filter exchange system was allowed (Olympus / Hamamatsu). It was the fluorescence ver Ratio (340 nm / 380 nm) determined with the Argus® software. The intracellular calcium levels were measured briefly in single NEN cells without the addition of active ingredients and then after 30 min Addition of the drug to be tested observed. Also use Similar systems can be detected by using Microtiter plates have a high throughput and secondary screening allow.  

Reference example 7 Determination of agonist-induced phospholipase C activity

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-in ositol was removed from the medium and replaced by 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 substances. 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.

Reference example 8 Determination of the agonist-induced change in cAMP product tion

The method used is essentially known (Strada S. S. et al., Methods in Neurotransmission receptor analysis: 89-110 (1990)). Cells were grown in culture medium without serum and AN for 10 min incubated tibiotics. The mixture was heated to 95 ° C. for 15 min in order to remove the Re stop action. The cell samples were frozen and at -80 ° C stored. cAMP levels were measured using commercially available kits who use the cAMP binding protein. It was too known methods optimized to be based on microtiter plates Systems to use. Commercially available materials allowed extension to high throughput analysis and Implementation of a secondary screening.  

Reference example 9 Tissue preparation

90 min after administration of the active ingredient (oral, intraperitoneal, intravenous or intracerebroventricular) were the experimental animals beheaded. The entire brain was quickly removed from the skull distant, frozen on dry ice and stored at -80 ° C. Rat brain Sections (15 µm) were obtained in a cryostat at -20 ° C Gelatin-coated slides applied and at -30 ° C to stored for use.

example 1

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 ^[3 H] -5-CT concentration. A dissociation constant of K _d = 0.570 nM was determined. Depending on the clonal cell, the receptor binding density (B) never varied in a range of 900-28,000 fmol / mg protein.

Example 2

According to reference example 4, the binding affinities of serotoninergic compounds were determined using the [ ³ H] -5-CT binding competition. The following IC ₅₀ values were obtained:

In a further series of tests, the inhibition constants K _{i following} compounds were determined (K _i = IC ₅₀ / (1 + C / K _d )), where C is the concentration of [ ³ H] -5-CT and K _d according to the example 1 was determined):

Example 3

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 se rotoninergic 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 agonist-mediated coupling to G proteins (see Figure 3A). The 5-HT effect was dose-dependent (see FIG. 4) with an EC ₅₀ of 2.6 μM.

Example 4

According to reference example 6, the drug-induced effect on intracellular calcium levels was examined. The 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 (18)

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 of 5-HT5 different 5-HT receptors. 2. binding partner according to claim 1, characterized in that whose binding affinity for 5-HT5 receptors is greater than for 5-HT1D and / or 5-HT1B receptors. 3. binding partner according to claim 1 or 2, characterized net that he competi the binding of 5-CT to 5-HT5 receptors tiv inhibits. 4. Binding partner according to one of claims 1 to 3, characterized in that the K _i 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 ^{- 1} m. 5. binding partner according to any one of claims 1 to 4, characterized ge indicates that its binding to 5-HT5 receptors Binding to G proteins stimulated. 6. binding partner according to any one of claims 1 to 5, characterized ge indicates that its binding to 5-HT5 receptors is an Er causes an increase in the intracellular calcium level. 7. binding partner according to any one of claims 1 to 6, characterized ge indicates that its binding to 5-HT5 receptors is an In reduction in phospholipase C activity. 8. binding partner according to one of claims 1 to 7, characterized ge indicates that its binding to 5-HT5 receptors is an In production of cAMP. 9. A pharmaceutical composition comprising at least one Binding partner according to one of claims 1 to 8 and one pharmaceutically acceptable excipients and optionally other active ingredients. 10. Use of a binding partner for 5-HT5 receptors Treatment of cerebrovascular diseases.   11. Use according to claim 10 for the treatment of migraine, ins especially for the acute treatment of migraines. 12. Use according to claim 10 or 11 of a binding partner according to one of claims 1 to 8. 13. Method for determining the affinity of binding partners for 5-HT5 receptors, whereby the binding partner with Cellular systems having 5-HT5 receptors in contact brings and determines the binding affinity. 14. Method for determining the activity of binding partners for 5-HT5 receptors, whereby the binding partner with Cellular systems having 5-HT5 receptors in contact brings and at least one agoni static effect determined. 15. The method of claim 14, wherein the binding of GTP to G- Proteins, intracellular calcium levels, the phospholipase C activity and / or cAMP production can be determined. 16. The method according to any one of claims 14 or 15, characterized ge indicates that human glioma cell lines or h5-HT5 transfected heterologous cell lines were used. 17. The method according to claim 16, characterized in that one h5-HT5 transfected CHO cells, h5-HT5 transfected human Kidney cells, or h5HT5 transfected C-6 glioma cells ver turns. 18. In vitro screening method to identify a 5-HT5 receptor binding partner, at least one method ren is used according to claims 13 to 17.