MXPA01006986A

MXPA01006986A - Binding partners for 5-ht5-receptors for the treatment of migraine.

Info

Publication number: MXPA01006986A
Application number: MXPA01006986A
Authority: MX
Inventors: Francisco Javier Garcia-Ladona
Original assignee: Knoll Ag
Priority date: 1999-01-11
Filing date: 2000-01-11
Publication date: 2002-09-18
Also published as: WO2000041472A2; CN1342070A; AU2289100A; DE19900674A1; CA2359360A1; WO2000041472A3; CN1321635C; CA2359360C; JP2002540063A; EP1144050A2

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

UNION COUNTERPARTS FOR RECEIVERS 5-HT5 FOR MIGRAINE TREATMENT The present invention relates to ing counterparts for 5-HT5 receptors, processes for the identification and characterization of ing counterparts of this type, and also to pharmaceutical compositions containing them and their use for the treatment of disorders cerebrovascular diseases such as migraine. At least seven different classes of receptors mediate the multiple physiological activities ascribed to an involvement of the neurotransmitter serotonin (5-hydroxytryptamine, abbreviated 5-HT). According to a classification with international recognition, these are designated by 5-HT1, 5-HT2, 5-HT3, 5-HT4, 5-HT5, 5-HT6 and 5-HT7. Most of these classes also include types of receptors that can be more differentiated. Thus, class 5-HT1 includes receptors that can be divided into at least five subclasses, which are designated by 5-HT1A, 5-HT1B, 5-HT1C, 5-HT1D and 5-HT1E (Boess FG and Martin IL, Neuropharmacology 33: 275-317 (1994)). Class 5-HT5 was first described by Plasta et al., The EMBO Journal vol. 11, No. 13, pp. 4779-4786 (1992). The 5-TH5A and 5-HT5B receptors are differentiated (Erlander et al., Proc. Nati, Acad. Sci. USA 90: 3542-3456 (1993)). There are only small sequence homologies between the 5-HT5 receptors and the other 5-HT receptors. The pharmacological profile of these receptors differs markedly. With the use of molecular biology techniques, the location of 5-HT5 receptors was possible in the olfactory bulb, the hippocampus, the cortex, the cerebral ventricles, the corpus callosum, and the cerebellum. By means of immunohistochemical methods it was possible to show that 5-HT5 receptors are mainly expressed on astrocytes (Carson et al., GLIA 17: 317-326 (1996)). The astrocytes are directly adjacent to the basement membrane of the cerebral capillaries of the blood-brain barrier. An abnormal structure of the endothelium of an astrocyte accompanies a loss of the blood-brain barrier. The exact importance of astrocytes is not clear. They seem to have transport tasks and connectivity functions. Reactive astrocytes were observed in connection with reactive gliosis in different pathological changes of the brain and neuropsychiatric disorders. As a result of brain injuries, they change their morphologies; there are changes in the pattern of protein expression and growth factors. In vitro research on cultured astrocytes has allowed the detection of responses mediated by the 5-HT5 receptor. Thus, on the one hand it is suspected that these are involved in the recovery processes of the brain after disorders, but on the other hand, it should not be excluded that they contribute to the creation of damage or even to an increase in damage. Migraine is a disorder of the CNS that affects a large number of the population. In most cases, it is manifested by headaches that recur over and over again, affecting, in an approximate amount, 8 million people, that is, 3-5% of children, 7% men and 14% women. Although it is considered that there is a genetic predisposition, the cause seems to be complex (Diener H. C. et al., Arzneimitteltherapie 15: 387-394 (1997)). Two hypotheses are dominant. The vascular theory that has been known for a long time proposes as cause and process of dilatation of the internal and external cerebral vascular system. The neurogenic theory is based on the secretion of vasoactive transmitters, mainly neuropeptides, such as substance P and neurokinin, from the axons of the vasculature as a result of the stimulation of certain ganglia that innervate brain tissue, and which causes inflammatory reactions and in that way pain. At present, there is still no causal therapy for the treatment of migraine. Today two different methods of treatment are used, the first is a prophylactic therapy for the prevention of recurrent migraine attacks, and the second is symptomatic therapy for the suppression of acute symptoms during attacks. Migraine-specific active compounds, such as Sanmigran®, Nocerton®, Desernil® and Vidora®, but also active compounds commonly used for other indications, such as beta blockers, antiemetic active compounds, such as Sibeliurp®, antidepressants such as Laroxyl® or active antiepileptic compounds such as Depakin® are administered as a prophylactic method. Acute treatment includes analgesics such as aspirin, paracetamol or Optalidon®, non-steroidal anti-inflammatory drugs such as Cebutid®, Voltaren®, Brufen®, Ponstyl®, Profenid®, Apranx® and Naprosin® for pain and inflammation , ergot alkaloids such as ergotamine, dihydroergotamine, which can induce vasoconstriction, or substances of the triptan family, such as sumatriptan, Naramig® and AscoTop® having a high affinity for 5-HT1D receptors. These latter substances act as agonists and block vasodilation. The mentioned active compounds, however, are not very suitable for the treatment of migraine. Non-opioid analgesics usually have side effects. As a result of the strong peripheral vasoconstriction, the complex action mechanism of ergot alkaloids gives rise to side effects such as hypertension and gangrene. Compounds belonging to the triptan family are also not very satisfactory (Pfaffenrath V., Münch, Med. Wschr 625-626 (1998)). The compound Sumatriptan (Imigran®), one of the most effective active compounds and most commonly used against acute migraine attacks, does not cross the blood-brain barrier because of its remarkable hydrophilicity. In 28% of patients, this active compound is ineffective and oral doses of 50-100 mg are very high. The contraindications that have been known are coronary vasospasm, hypertension, kidney and liver disorders. Heretofore, compounds that have selective affinity for 5-HT1 receptors have been considered for the treatment of migraine (Goadsby, P.J., CNS Drugs 10: 271-286 (1998)).; Saxena P. R., Exp. Opin. Invest. Drugs 581-593 (1996)). For example, sumatriptan represents a very selective ligand for 5-HT1D receptors, which is why the professional world wants to optimize these binding properties and the vasoconstrictive activities mediated by them. The same applies to the ligands of the 5-HT1B and 5-HT1F receptors. Thus, a series of active compounds was developed which, however, were not able to remedy the problems described. Therefore, an objective of the present invention is to enable the acute treatment of migraine type cerebrovascular disorders with adequate efficacy and slight side effects. Surprisingly, it has now been found that substances that have comparatively high binding affinity for 5-HT5 receptors can provide the desired effects. The present invention, therefore, relates to selective binding counterparts for 5-HT5 receptors, whose binding affinity for 5-HT5 receptors is greater than for one or more of the 5-HT receptors other than 5-HT5. . The term "binding partner for 5-HT5 receptors" describes substances that bind to 5-HT5 receptors and, therefore, can also be referred to as ligands of 5-HT5 receptors. Or they include coordinate coordinated metallic type [sic]. In addition to the aforementioned, reversible molecular interactions, irreversible interactions between binding partners and the receptor are also possible, such as, for example, covalent bonds. By selectivity is meant the property of a binding partner to preferentially bind to 5-HT5 receptors. Thus, the binding counterparts have binding affinities for 5-HT5 receptors that are greater than for one or more of the 5-HT receptors other than 5-HT5, i.e., in particular, the receptors that will be assigned to the classes of receptors for 5-HT mentioned above 5-HT1, 5-HT2, 5-HT3, 5-HT4, 5-HT6 and 5-HT7. If the binding affinity for the 5-HT5 receptors of a binding partner is greater than that of a 5-HT receptor other than 5-HT5, in relation to the 5-HT receptor which is different from 5-HT5 it is refers to a more selective binding of these binding partners to 5-HT5 receptors. Particular binding partners are those whose binding affinity for 5-HT5 receptors is greater than for at least one and, in particular, all 5-HT1 receptors, in particular for 5-HT1D and / or 5-HT1B receptors . The binding counterparts whose binding affinity for 5-HT5 receptors is higher than for all 5-HT receptors other than 5-HT5 are a more particular class of binding counterparts according to the invention. For the aforementioned selectivity it is decisive that the binding affinities for the 5-HT5 receptors, on the one hand, and for one or more of the 5-HT receptors other than 5-HT5, on the other hand, differ by a sufficient amount . Affinity differences are preferred by means of which binding affinity ratios of at least 2 are present, of greatest convenience at least 5, particularly for convenience of at least 10, preferably at least 20, particularly preferably when minus 50 and in particular at least 100. According to one embodiment, the binding counterparts according to the invention competitively inhibit the binding of the comparative binding counterparts, such as 5-HT5 (5-hydroxytryptamine) or 5-CT (5-carboxamidotriptamina), for 5-HT5 receptors. By competitive inhibition it is understood that the binding counterparts according to the invention compete with a comparative binding partner, in the present case, for example, 5-HT or 5-CT, to bind to the receptor, ie, the binding of one prevents the union of the other.

According to another embodiment, the binding counterparts according to the invention inhibit the binding of the comparative binding counterparts, such as 5-HT. (5-hydroxytryptamine) or 5-CT (5-carboxamidotriptamine) to the 5-HT5 receptors in a non-competitive manner. By non-competitive inhibition it is understood that the binding counterparts according to the invention modulate the binding of a comparative binding partner, in the present case, for example, 5-HT or 5-CT, in particular reducing its binding affinity by its union with the receiver. At least in the case of competitive inhibition, that is, reversible binding, the principle is applied that the displacement of one binding partner by another increases with the decrease of binding affinity of one or the increase of affinity of union of the other with respect to the receiver. Therefore, it is desirable that the binding counterparts that can be used according to the invention have a high binding affinity for the 5-HT5 receptors. A binding affinity of this type allows, on the one hand, an effective displacement of the binding counterparts that occur in nature for 5-HT5 receptors such as, for example, serotonin itself (5-hydroxytryptamine, 5-HT ), where the necessary concentration of the binding counterpart that can be used according to the invention for the binding of a certain amount of this binding counterpart to the 5-HT5 receptors decreases with increasing binding affinity. With respect to medical use, the binding counterparts are therefore preferred whose binding affinity is so great that they can be administered in justifiable amounts in the course of effective medical treatment as an active compound. The binding counterparts according to the invention are therefore preferably administered in daily doses of from about 0.01 to 100 mg / kg of body weight, in particular from about 0.1 to 15 mg / kg of body weight in parenteral administration and of 1 to 30 mg / kg of body weight in oral administration. The aforementioned competition experiments, with which this concentration of the binding counterpart according to the invention is determined in vitro, which displaces 50% of the other counterpart of comparative binding of the receptor binding site (IC50 values), they offer a possibility to express union affinity. Thus, the competitive inhibition of 5-CT binding to 5-HT5 receptors can also be evaluated for the effect that the binding counterparts that can be used preferably according to the invention have half-maximal inhibition constants IC50 less than 10"7 M, preferably less than 10 ~ 8 M and, in particular, less than 10" 9 M. The binding affinity of the binding counterparts according to the invention can also be expressed by means of the inhibition constant Kl. r which, in general, is determined in the same way in vitro using competition experiments. For the binding of 5-HT 5 receptors, the binding counterparts according to the invention preferably have K-values less than 10 ~ 8 M, for convenience less than 10 ~ 9 M and particularly preferably less than 10 -10 M. The K values of the compounds according to the invention are, for example, in the range from 1 x 10 ~ 7 M to 7 x 10"7 M or in the range from 1 x 10 ~ 8 M to 1 x 10" 7 M. According to another embodiment, the binding counterparts according to the invention bind more selectively to the 5-HT 5 receptors having the advantageous binding affinities described above in relation to one or more 5-HT receptors other than 5-HT 5. -HT5 According to another embodiment, the binding counterparts according to the invention bind more selectively to the 5-HT 5 receptors having the advantageous binding affinities described above in relation to all the 5-HT receptors other than 5-HT 5. Particularly suitable are binding counterparts that bind to 5-HT5 receptors that are expressed by glial cells and, in particular, by astrocytes, with the affinities and selectivities described above. According to the invention, the human receptor variant is a preferred target for the binding counterparts that are employed according to the invention. The binding of the binding counterparts according to the invention to the 5-HT 5 receptors is coupled for an effector function. The binding counterparts can act in an agonist or antagonist form and partially agonist and / or partially antagonist. Agonists are defined as compounds according to the invention that completely or partially mimic the activity of 5-HT at 5-HT5 receptors. Agonists are defined as compounds according to the invention that can block the 5-HT agonist activity on 5-HT5 receptors. According to a particular embodiment of the invention, 5-HT5 agonists are provided as binding counterparts. The term "5-HT5 agonist" designates the binding counterparts that induce a partial or compound agonist action. A binding counterpart that induces a partial agonist action at the 5-HT5 receptor has sufficient agonist activity according to the invention so that it can be administered in justified amounts in the course of an effective medical treatment. Preferred binding counterparts in the context of this embodiment are those that have at least 50% agonist action. Particularly preferred binding counterparts are those that have at least 80% agonist action and in particular those that have virtually complete agonist action (Emax). According to a particular embodiment of the present invention, binding counterparts are made available whose binding, at least for the 5-HT5 receptors of CHO cells transfected with h5-HT5, produces a stimulation of the binding of GTP to proteins. G bound to the membrane, a change induced by the agonist in intracellular calcium concentrations, an induction of phospholipase C activity and / or a change induced by the agonist in the production of cAMP. Regarding the change in intracellular calcium concentrations, the use of the binding counterparts leading to an increase in intracellular calcium concentrations is a particular embodiment of the invention. This embodiment also includes counterparts that are active in known animal models of cerebrovascular disorders, in particular for migraine and / or that induce certain in vivo actions in areas of the brain, in particular genomic responses in the brain, for example factor expression. of transcription as the isoforms of the c-fos gene, c-jun, zif268 or Homer (Brakeman PR et al., Nature 386: 284-288 (1997)). Preferred binding partners are those which, also with respect to their effector function, are selective in the sense described above for 5-HT5 receptors. According to one embodiment, the binding partners of 5-HT5 are usually synthetic compounds of low molecular weight. According to another embodiment, the 5-HT5 binding partners according to the invention are 5-HT5 specific antibodies. These can be polyclonal antisera, monoclonal antibodies, fragments of antibodies, such as F (ab), Fe, etc., chimeric and recombinant antibodies. Such antibodies can be prepared in a manner known per se. The immunogen that is used can be a 5- HT5 receptor as such or antigenic fragments, as a rule, fragments coupled to the customary carrier proteins. According to another embodiment, the binding partners of 5-HT5 according to the invention are aptamers, ie nucleic acids, as a general rule, oligonucleotides with sufficient affinity for 5-HT5 receptors. In principle, tests for determining the binding affinities of the binding counterparts according to the invention to 5-HT5 receptors are known. These can be done, for example, by testing the competitive inhibition of the binding of a comparative binding counterpart with the 5-HT5 receptors for the substance to be investigated. Suitable comparative binding partners are known ligands for 5-HT receptors, such as 5-HT or 5-CT or LSD. These are conveniently labeled so that their binding to 5-HT receptors can be analytically tested using traditional methods. Radioactive markers and optical markers are preferred. In binding studies on 5-HT5 receptors, according to the invention, 5-CT or LSD is used, particularly in the form of [3 H] -LSD. The binding affinities can be expressed as the constants of half-maximal inhibition IC ^ o or as constants of Ki inhibition. This process of preference is used for primary detection. The use of SPA technology or FlashPlate technology is preferred. The binding to the binding counterparts to be investigated can also be determined directly at the 5-HT receptors. The inhibition constants Ki that express the binding affinity can be determined, for example, by calorimetry, that is, by measuring the energy released at the junction. For the determination of the selectivities, the binding affinity of the binding counterparts 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 compared the values obtained. It is also possible to assess the effector functions qualitatively or quantitatively in vitro and in vivo with the help of known functional assays. The evaluation of an agonist activity can be based on all those effects that are produced by the binding of 5-HT to the 5-HT5 receptors. According to the invention, it is preferred to assess the effects on the binding of GTP to G proteins, on intracellular calcium concentrations, on the activity of phospholipase C and / or on the production of cAMP. These processes are preferred for secondary detection. In this case, the SPA or 5 FlashPlate technology is also conveniently used. The binding of GTP to the G proteins can be investigated by the use of a non-hydrolysable analog of GTP, for example [35S] GTP? S, whose binding can be investigated by radiology. This investigation of Preference is carried out on membranes having 5-HT5 receptors. For the measurement of intracellular calcium concentrations, suitable calcium probes, as a general rule calcium chelators, for example compounds Fluorescents, such as Fura 2-acetylmethyl ester or fluo-3-AM, can be used. This research is preferably carried out in cell cultures that have a 5-HT 5 receptor, in particular an individual cell. It is possible to determine the activity of phospholipase 20 C by means of its catalytic reactions, for example, the incorporation of myoinositol, which for purposes of detection preferably is added a radioactive label such as [3H] -mioinositol, or the conversion of PPIP2 to IP3, where the PPIP2 also preferably is 25 radiolabelled as [32P] PIP2. These investigations of preference are carried out in individual cells having 5-HT5 receptors. It is possible to determine the production of cAMP with the help of the cAMP binding protein. This preferential investigation is carried out in individual cells having 5-HT5 receptors. If appropriate, the effector function is also determined, ie the activity of the binding counterparts according to the invention for other 5-HT receptors. This takes place conveniently taking into consideration the binding affinities determined for 5-HT5 receptors and other receptors for 5-HT, that is, in particular taking into consideration the selectivity. The present invention, therefore, also relates to the processes for the identification and characterization of the binding counterparts according to the invention. These and other equally convenient processes can form the basis for the in vitro detection processes with which these are possible from a large number of different compounds to select those which, with respect to future use, appear to be more promising. For example, by means of combinatorial chemistry, it is possible to prepare extensive banks of substances comprising multiple potential active compounds. The inspection of libraries of combinatorial substances for substances that have the desired activity can be automated. Detector robots are used for efficient evaluation of individual assays, which are preferably arranged in microtitre plates. Thus, the present invention also relates to the detection processes, that is, to the primary and secondary detection processes, in which preferably at least one of the processes described above is used. If different processes are used, they can be displaced in terms of time or performed simultaneously in one and the same sample or in different samples of a substance to be investigated. A particularly effective technology for carrying out processes of this type is the scintillation proximity test, called SPA for brevity, known in the field of detection of active compounds. It is possible to obtain the kits and components in commerce to carry out this test, for example, from Amersham Pharmacia Biotech. In principle, solubilized or membrane-bound receptors are immobilized in small fluoromicrospheres containing the substance for scintillation. For example, if a radioligand binds to the immobilized receptors, the scintillation substance is stimulated to emit light, as the spatial proximity between the scintillation substance and the radioligand [sic] occurs. Another particularly effective technology for carrying out processes of this type is the FlashPlate® technology known in the field of detection of active compounds. The kits and components for carrying out this test can be obtained commercially, for example from NEN® Life Science Products. This principle is similarly based on microtiter plates (96-well or 384-well), which are coated with the scintillating substance. The aforementioned assays are known in principle to those skilled in the art. A first process according to the invention is used for the determination of the affinity and / or selectivity of the binding counterparts for the 5-HT5 receptors. For this purpose, the binding counterparts are contacted with the 5-HT5 receptors and the binding affinity is determined. Another process according to the invention relates to the determination of the activity of the binding counterparts for 5-HT5 receptors, that is, the determination of the agonist, partially agonist, antagonist and / or partially antagonist action. For this purpose, the binding counterparts are contacted with 5-HT5 receptors and the effects caused by the binding are evaluated. According to a preferred embodiment, the binding counterparts are subjected to a primary detection by determining their binding affinity for 5-5 HT5 receptors using the competition experiment with [3H] -5-CT or [3H] -LSD described above . These binding counterparts having an IC5o inhibition constant in the range of 10"6 M or less are then subjected to secondary detection by evaluating their effector function in the The above-described form, in particular with respect to GTP binding and / or intracellular calcium concentrations. Finally, the binding counterparts selected in this way can be subjected to a reverse detection for determination of the selectivity 15 determining its binding affinity to other receptors for 5-HT mainly in the manner described above - but optionally using the ligands specific for the respective receptors. For example, it is possible to use [3H] -8-hydroxydipropylaminotetralin ([3H] -8-20 DPAT) for studies of binding to 5-HT1A receptors, whereas 5-HT1B and 5-HT1D receptors can be investigated using [ 3H] -5-CT. Preferably, 5-HT5 receptors are available in the form of cellular systems, ie, in 25 the shape of membranes, cells, cell colonies, • "j ^ f ^ *" -'- "tissues or organs that carry 5-HT5 receptors Cell systems of this type can express 5-HT5 receptors by nature, but can also be induced to express 5-HT5 by suitable genetic manipulation, for example by transfection In the context of the preferred embodiment of the present invention related to h5-HT5, it is possible to use for this purpose, in particular, the coding sequence described in Rees S. et al., FEBS Letters 335: 242-246 (1994) (accession number X81411). Glio to human cell lines are preferred as natural cellular systems having 5-HT5 receptors.From heterologous cell lines transfected with h5-HT5, those expressing are preferred. the h5-HT5 gene Mention can be made, for example, of CHO cells transfected with h5-HT5, human kidney cells transfected with h5-HT5, in particular HEK293 cells transfected with h5-HT5 or transfected C-6 glioma cells with h 5-HT5 To determine the selectivity, affinity and activity of the binding counterparts according to the invention, it is also possible to use sections of brain tissue and natural membranes of parts of the brain. If radioactive labels are used, the evaluation of the tissue sections preferably is carried out by autoradiography.

The effectiveness of the binding counterparts according to the invention for the treatment of migraine preferably is determined in animal models that are based on mechanisms that can support the formation of migraine. For example, it is possible to determine the extravasation of the protein induced by binding counterparts according to the invention. This assay can be performed in a manner known per se, for example using fluorescence staining (Johnson K. "and Phebus LA (1998) J. Neurosci. Methods 81: 19-24) or isotope-labeled albumin (Petty MA et al. (1997) Eur. J. Pharmacol 336: 127-136) For this, the animals are treated in succession with the test compound and with [125 I] -albumin or fluorescent dye, then the trigeminal ganglion is electrically stimulated. The radioactivity or accumulated fluorescence in the dura is determined The antimigraine activity of the binding counterparts according to the invention is observed in the extravasation of the protein induced by the stimulation of the trigeminal ganglion Another model is based on the distribution of the flow This test is also known per se (Saxena PR et al., (1998) Eur. J. Pharmacol., 351: 329-339) For this, radioactive micro spheres are injected into the carotid. stabilize The animals are treated with a test compound, and the radioactivity accumulated in the brain is determined in a micro-counter ß. It is also convenient to measure the expression and translocation of the c-fos gene induced by nitroglycerin. For this, the animals are treated with nitroglycerin and the compound that will be tested. The expression of the c-fos gene and the translocation of the protein in the nucleus is determined using in situ or immunohistochemical hybridization (Garcia-Ladona FJ et al., (1994) Mol. Brain Res. 21: 75-84; Garcia-Ladona FJ et al. (1997) J. Neurosci Res 50: 50-61). Other models are known from Fernandes de Lima V. M. et al., (1993) Brain Res. 614: 445-51 (Retinal Spreading depression) and Kawahara N. et al., (1999) Exp. Neurol. 108: 27-36 (Cortical Spreading Depression).

The use according to the invention of the 5-HT 5 binding partners consists, in the context of the treatment, in a process. In this process, an effective amount of one or more binding partners of 5-HT5, as a rule, formulated in accordance with pharmaceutical and veterinary medical practice, is administered to the individual to be treated, preferably a mammal, in particular a mammal. human, productive animal or pet. If the treatment is indicated and how it will take place, it will depend on individual cases and is subject to medical evaluation (diagnosis), the signs, symptoms and / or dysfunctions present, the risks of developing certain signs, symptoms and / or dysfunctions, and includes other additional factors [sic]. As a general rule, the treatment is carried out by single or repeated daily administration, if suitable together or alternating with other active compounds or preparations containing active compound, so that the individual to be treated is administered with a daily dose from about 0.001 g to 10 g, preferably from about 0.001 g to about 1 g. The invention also relates to the production of a pharmaceutical agent for the treatment of an individual, preferably a mammal, in particular a human, a productive animal or a pet. The binding counterparts according to the invention are usually administered in the form of pharmaceutical compositions containing a pharmaceutically tolerable excipient with at least one inhibitor according to the invention and, if appropriate, other active compounds. These compositions can be administered, for example, orally, rectally, transdermally, subcutaneously, intravenously, intramuscularly or intranasally. Examples of suitable pharmaceutical formulations are solid dosage forms such as powders, granules, tablets, lozenges, sachets, wafers, coated tablets, capsules such as hard and soft gelatin capsules, suppositories or vaginal dosage forms, semisolid pharmaceutical forms as ointments, creams , hydrogels, pastes or plasters, and also liquid pharmaceutical forms such as solutions, emulsions, in particular oil-in-water emulsions, suspensions, for example lotions, injection and infusion preparations, ophthalmic and otic drops. It is also possible to use devices implanted for the delivery, for the administration of the binding counterparts according to the invention. In addition, it is also possible to use liposomes, microspheres or polymer matrices. In the production of the compositions, the binding counterparts according to the invention are usually mixed or diluted with an excipient. The excipients may be solid, semi-solid or liquid materials that are used as vehicle or medium for the active compound. Suitable excipients include, for example, lactose, dextrose, sucrose, sorbitol, mannitol, starches, acacia gum, calcium phosphate, alginates, tragacanth, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, and methylcellulose. . In addition, the formulations may contain the acceptable vehicles for pharmaceutical use or customary excipients such as lubricants, for example tallow, magnesium stearate and mineral oil; wetting agents; emulsifying and suspending agents; 10 preservatives such as methyl and propyl hydroxybenzoates; antioxidants; anti-irritants; chelating agents; auxiliaries for drum coating; emulsion stabilizers, film-forming agents; gel forming agents; taste masking agents; 15 flavor correctors; resins; hydrocolloids; solvents; solubilizers; neutralizing agents; permeation accelerators; pigments; quaternary ammonium compounds; re-greasing and superfatting agents; ointment, cream or oil bases; silicone derivatives, 20 auxiliaries for dispersion; stabilizers, stabilizing agents; suppository bases; excipients for tablets such as binders, fillers, lubricants, disintegrants or coatings; propellants; drying agents; opacifying agents; thickeners; 25 waxes; plasticizers; white oils. One modality iA * ^ A «i > Relevant is based on expert knowledge, such as that presented for example in Fiedler, HP, Lexikon der Hilfsstoffe für Pharmazie, Kosmetik und angrenzende Gebiete [Encyclopedia of pharmacy excipients, cosmetics and related areas], 4th edition, Aulendorf: ECV- Editio-Kantor-Verlag, 1996. The present invention also relates to the use of binding counterparts for 5-HT5 receptors and, in particular, of the aforementioned particular and preferred embodiments of the binding counterparts according to the invention for the treatment of migrainous cerebrovascular disorders, especially for the treatment of migraine and other headaches related to vascular problems, in particular paroxysmal headaches of the migraine type. These include the disorders described as simple and traditional migraine without or with concomitant neurological functional disorders, true and atypical migraine, and also more specific disorders of this type, for example, associated migraine, called migraine equivalents, digestive migraine, migraine ophthalmic, migraine ophthalmoplegic, migraine redness, also described as acuminate headache (Broton's syndrome) and cervical migraine. By treatment is meant prophylactic therapy, in particular prophylaxis against recurrent attacks, for example as treatment at intervals and treatment in the case of acute symptoms, in particular during the phases of headache. Successful treatment leads to a reduction in the intensity of symptoms, in particular of headache, of neurological functional disorders, for example of visual, sensory, motor and speech disorders, nausea and dry vomiting and / or attack frequency. Acute migraine treatment with the binding counterparts is preferred. The invention also relates in particular to the use of the aforementioned binding partners for the treatment of those forms of the aforementioned disorders in whose formation and / or course the 5-HT5 receptors are involved, ie disorders that are modulated by the activity of a 5-HT5 receptor. The term "disorder" in the sense according to the invention describes anomalies which, as a general rule, are considered as pathological conditions and may be manifested in the form of certain signs, symptoms and / or abnormal functions. The treatment according to the invention can be directed to individual disorders, that is, abnormalities or pathological conditions, but also to different anomalies that are connected to each other by their etiology and that can be summarized to give patterns, that is, syndromes, which they can be treated according to the invention. A treatment within the meaning according to the invention consists not only in the treatment of signs, symptoms and / or acute or chronic abnormal functions but also in a preventive treatment (prophylaxis) in particular a relapse or phase prophylaxis. The treatment can be carried out in a symptomatic way, for example as suppression of the symptoms. It can be carried out in the short term, it can be done in the medium term, or it can also be a long-term treatment, for example in the context of maintenance treatment. The binding counterparts that can be used for migraine treatment can bind 5-HT5 with a lower affinity, virtually identical or greater than a specific receptor that is different from 5-HT5. The binding counterparts for 5-HT5 receptors with respect to use according to the invention, thus, in particular, include those whose binding affinity for 5-HT5 receptors compared with affinity for 5-HT1 receptors, in particular 5-HT1B and / or 5-HT1D, is so high that they are conveniently suitable for use according to the invention. This does not necessarily presuppose a comparatively more selective binding to 5-HT5 receptors, even those selective binding counterparts for the 5-HT5 receptor are a particular embodiment of the present invention. For example, it is possible to use binding partners having a high affinity for the 5-HT 5 and 5-HT 1 receptors, in particular for 5-HT 1 B and / or 5-HT 1 D. In this sense, high affinity means Kl values as a general rule, in the range from 1-10"9 M to 1-10" 6M. According to a particular embodiment, in the high affinity range the useful binding counterparts have, relative to the 5-HT receptors, a binding profile which is marked by a binding affinity for 5-HT5 which, in comparison with other union affinities this interval is practically identical or 15 only slightly smaller. Factors of 10 or less can be advantageous. According to another embodiment of the invention, the selective 5-HT5 binding partners described above are employed. According to a particular embodiment of the present invention, binding counterparts are used whose binding, at least for the 5-HT 5 receptors of CHO cells transfected with h5-HT5, cause a change in the stimulation induced by the binding agonist. GTP 25 to G proteins bound to the membrane, a change in the intracellular calcium concentrations, a change in the induction induced by the agonist of phospholipase C activity and / or a change in the production of cAMP. As regards the change in intracellular calcium concentrations, the use of binding counterparts that cause an increase in intracellular calcium concentrations is a particular embodiment of the invention. The present invention is illustrated in more detail by means of the following examples, without being limited thereto.

Reference example 1 HEK293 cells and CHO cells expressing the h5-HT5 receptor The gene encoding the human 5-HT5 receptor was isolated from human tissues in a known manner by means of the 3'-5 '-RT-PCR system (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® (Gibco Life-Sciences, Germany), and the HEK293 cells were incubated with a thin layer of this transfection mixture in petri dishes (2.5 cm).

. The transfection mixture was then replaced by culture medium containing neomycin. The surviving cells were also cultured in DMEM-F12 medium that was supplemented with 10% of fetal bovine serum, 2 mM glutamine and antibiotics (90 mg of streptomycin, 90 mg of penicillin). The cells were grown to confluence in 5% C02, 95% atmospheric humidity and 37 ° C.

In the same way CHO cells were obtained expressing the h5-HT5 receptor.

Reference example 2 Preparation of cell membranes The method that was used practically follows the known methods for the preparation of cell membranes from cells (Findlay J. B. C. and Evans, H. Biological Membranes, Practical Approach (1987)). The cells cultured according to Reference Example 1 were carefully scraped from the surface of the culture vessel and centrifuged in DMEM-F12 medium at 180 x g for 10 minutes. The cell packets obtained were resuspended in 5 mM Tris-HCl buffer containing 5 mM EDTA, 5 mM EGTA, 0.1 mM PMSF and 3 mM benzamidine (pH: 7.6, buffer A) and incubated at 4 ° C for 15 minutes . The cell suspension was homogenized (6 x 3s) in an Ultraturrax® (15,000 rpm) and centrifuged at 1000 x g and 4 ° C for one minute. The package was resuspended in buffer A and, as already described, homogenized and centrifuged. Supernatants from both steps were collected and centrifuged at 40,000 x g and 4 ° C for 20 minutes. The package was resuspended and homogenized in buffer solution A (1 x 15s). the membrane suspension was centrifuged at 40,000 x g and 4 ° C for 20 minutes. The resulting package was resuspended in buffer A containing 10% glycerol and 1% bovine serum albumin. The aliquots were frozen and stored at -80 ° C until use.

Reference example 3 Kinetics of binding to saturation of [3 H] -5-CT The method is practically known (Ress, S. et al., FEBS Letters 335: 242-246 (1994)). The membranes that were obtained according to Reference Example 2 (200 μl) were incubated in a total volume of 600 μl in 100 mM tris-HCl with a content of 1 mM EDTA (pH 7.7, buffer B) with increasing concentrations of [3 H] -5-CT (96 Ci / mmol), adding 10 μM methiotepine for the determination of the specific binding, while no metiotepine was added for the determination of the total union. The mixture was incubated at 30 ° C for 90 minutes. The samples were then filtered using a Skatron® filtration system and GF / B filters embedded in 0.3% polyethylene imide. The filters were washed at 4 ° C with 9 ml of the buffer B. The radioactivity retained in the filters was measured by means of liquid scintillation counting using 5 ml of Ultima-Gold (Packard).

Reference example 4 a) [3H] -5-CT binding competition The binding competition experiments were performed practically following the known investigations (Rees et al., 1994). The membranes that were obtained according to Example 2 (200 μl) were incubated in the presence of [2 H] -5-CT 2 nM in a total volume of 600 μl in buffer B with increasing concentrations of the selected compounds. After an incubation time of 75 minutes at 30 ° C, the samples were filtered at 4 ° C through GF / B filters embedded in 0.3% polyethyleneimide using buffer B. The filters were washed with 9 ml of buffer B. The radioactivity retained in the filters was determined as in Reference Example 3. The total binding was defined as that radioligand binding that was observed without the addition of other compounds. This non-specific binding was defined as that binding of [3 H] -5-CT that was observed in the presence of 10 μM methiotepine. It is also possible to use similar systems that, as a result of the use of microtiter plates, allow high sample throughput and secondary detection. The saturation parameters of the binding of [3H] -5-CT were determined by non-linear regression analysis and from linear graphs using the SigmaPlot software (Jandel Scientific, Germany). The competition curves were established in which the radioactive union is expressed as a proportion in percent of the total union. The constants of half-maximal inhibition IC50 and Hill coefficients (nH) were determined by means of non-linear regression analysis. b) Identification of the h5-HT5 receptor ligands by HTS using the FlashPlate technology. 96 well HPP-coated h5-HT5 membranes can be obtained from Bio Signal Inc. (Canada). [3H] -LSD was diluted to a convenient concentration in Tris-HCl buffer solution containing 10 mM MgCi 2, 0.5 mM EDTA and 0.5% BSA. The radioligand solution was added to the wells (25 ml) [sic], which contained or did not contain the test compound. The plates were incubated at room temperature for 180 minutes and the radioactive signal was measured using a micro-β counter (Wallac) Non-specific binding was determined using methiothepin. [3H] -LSD has an affinity of 12 nM. With the increasing binding affinity of the test compound decreases the radioactive signal of [3 H] -LSD.

Reference example 5 Determination of Agonist-induced Stimulation of [35 S] GTP? S Binding assays [35 S] GTP? S binding assays are known. The present assay was carried out following the method described above of Hilf, G and Jakobs. K. H. (Eur.J. Mol.Pharmacol. 225: 245-252 (1992)). The changes induced by the active compound in the binding of [35S] GTP? S to the membranes of HEK293 cells transfected stably with the h5-HT5 receptor gene were measured (see reference examples 1 and 2). The cell membranes (12 μg) were incubated with 50 mM triethanolamine HCl buffer solution (pH 7.5) containing 6.75 mM MgCl 2, 150 mM NaCl, 1 mM DTT, 1 mM EDTA, 10 μM GDP and [35 S] GTPγS. After incubation for 60 minutes at 30 ° C with or without addition of the active compounds 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 solution (9 ml, pH 7.5, 4 ° C) containing 100 mM NaCl and 5 mM MgCl2. The radioactivity retained in the filters was determined by means of scintillation spectrometry, using the Ultima Gold scintillation liquid. In the same way it is possible to use similar systems that allow high performance and secondary detection as a result of the use of microtitre plates. The activities of the active compound were expressed as a proportion in percent of the basic linkage measured in the absence of the test compound. The correspondence of the curves was performed using a software for non-linear regression analysis (SigmaPlot, Jandel Scientific, Germany) according to the general equation E = (LxEmax) / (L + EC50), in which E is the action, L is the concentration of the ligand, Emax is the maximum action and EC50 is that concentration that induces 50% of the maximum action.

Reference example 6 Determination of changes induced by the agonist of intracellular calcium concentrations The method is known (Kao, J. P. Y. Methods in Cell Biology 40: 155-181 (1994)). As described in Reference Example 1, HEK293 cells expressing the h5-HT5 receptor were grown in culture vessels. The cells were carefully scraped before they were confluent. Cells were labeled with Fura 2 by incubating at room temperature with Fura 2-acetylmethyl ester (Sigma). The cells were centrifuged at 180 x g for 10 minutes and resuspended in DMEM-F12 medium without serum and incubated at 37 ° C, with 5% C02 and 95% atmospheric humidity for 45 minutes. Intracellular calcium concentrations were determined with a fluorescence microscope that was equipped with a convenient filter remover system (Olympus / Hamamatsu). The fluorescence ratio (240 nm / 380 nm) was determined using the Argus® software. The intracellular calcium concentrations were observed for a short time in individual cells without the addition of the active compounds and then 30 minutes after the addition of the active compound to be tested. In the same way it is possible to use similar systems that allow a high performance and secondary detection as a result of the use of microtitre plates. It is also possible to evaluate the modulation of intracellular Ca2 + concentrations in HTS. For this, the CHO cells expressing the h5-HT5 receptor were cultured overnight in 96-well plates (30,000-80,000 cells / well). The cells were labeled for one hour using HEPES buffer solution containing 1 mM fluor-3-AM, 10% pluronic acid and 2.5 mM probenecid, and washed. A test compound was added to each well, for the determination of the calcium concentrations, the intensity of the fluorescence was read using a Fluorometric Imaging Plate reader (FLIPR).

Reference example 7 Determination of phospholipase C activity induced by the agonist The method is practically known (Garcia-Ladona F. J. et al., Neuroreport 4: 691-694 (1993)). The cells were incubated with 0.125 [mu] M [3 H] myo-inositol for 24 hours. Unincorporated [3H] myoinositol was removed from the medium and replaced with Krebs-5 Henseleit buffer containing 10 mM LiCl. After the incubation for 10 minutes, the active compound that was to be tested was added. After 45 minutes the reaction was stopped by replacing the stimulation medium with distilled water. If samples are used 10 of tissue, a similar procedure is employed (Garcia-Ladona et al., 1993). The cells were frozen and stored at -80 ° C. The production of [3H] -inositol monophosphate was determined by means of known chromatographic methods. It is possible to use a method 15 similar with tissue miniprismas. The determination of the stimulation of phospholipase C in the same way was carried out in the same way by preparing membrane fractions, as described in Reference Example 2, and incubating with [32 P] PIP2 and the active compounds. In In this case, the production of IP3 was determined. Known processes are also optimized for using microtiter plate based systems. The materials that can be obtained in the trade allow the extension to analysis with a high yield and the 25 embodiment of secondary detection. ÍÜ --ÍÉÉÉÉÍff lili lll'lil íl i, Reference example 8 Determination of the change induced by the agonist in the production of cAMP The method used is practically known (Strada S. S. et al., Methods in Neurotransmission receptor analysis: 89-110 (1990)). The cells were incubated in culture medium without serum and antibiotics for 10 minutes. The medium was heated at 95 ° C for 15 minutes to interrupt the reaction. The cell samples were frozen and stored at -80 ° C. The cAMP levels were determined using commercial kits that use the cAMP binding protein. The known processes were also optimized to use systems based on microtitre plates. The materials that can be obtained commercially allow the extension for analysis with high performance and perform secondary detection.

Reference example 9 Preparation of the tissue 90 minutes after administration of the active compound (orally, intraperitoneally, intravenously or intracerebroventricular), the experimental animals were decapitated. The entire brain was quickly removed from the skull, frozen in dry ice and stored at -80 ° C. sections of rat brain (15 μ) were obtained in a cryostat at -20 ° C, these were applied to gelatin-coated slides and stored at -30 ° C until use.

Example 1 According to Reference Example 3, the binding affinity of [3 H] -5-CT to the 5-HT 5 receptors was determined. Figure 1 shows a graph of [3 H] -5-CT bound as a function of the concentration of [3 H] -5-CT. A dissociation constant of Kd = 0.570 nM was determined. Depending on the clonal cell line, the receptor binding density (B) varied in a range of 900-28,000 fmol / mg of the protein.

Example 2 According to Reference Example 4, the binding affinities of the serotonergic compounds were determined by means of the binding competition of [3 H] -5-CT. The following IC5Q values were obtained: In another test series, the Kx inhibition constants of the following compounds were also determined (Ki = IC50 / (l + C / kd)), where C is the concentration of [3H] - 5-CT and Kd was determined from according to Example 1 Example 3 According to Reference Example 5, the binding induced by the active compound of GTP to the G proteins was investigated. The coupling of 5-HT5 receptors to G proteins in HEK293 cells was evident. The common serotonergic agonists 5-HT5 and 5-CT induced an increase in the binding of [35S] GTP? S to cell membranes about 40% above the baseline value (see Figure 2). The 5-HT5 receptor needs GDP for coupling to G proteins, which is mediated by agonists (see Figure 3a). The effect of 5-HT5 was dose-dependent (see Figure 4) with an EC50 of 2.6 μM.

Example 4 The effect induced by the active compound on intracellular calcium concentrations was investigated according to Reference Example 6. Stimulation of 5-HT5 receptors with R (+) -lisuride (1 μM) in HEK293 cells induced a increase in intracellular Ca2 + (see Figure 5). . ^ rfB- AJ

Claims

1. A process of in vitro detection for the identification of compounds for the treatment of 5 cerebrovascular disorders, which consists of determining the affinity of the compounds for the 5-HT5 receptors and the reading of these 5-HT5 binding counterparts whose binding affinity for the 5-HT5 receptors is greater than their affinity for the 5-HT5 receptors. 5-HT1D receptors.

2. The process as claimed in claim 1, wherein those compounds whose binding affinity for 5-HT5 receptors is higher are read when 15 minus the factor of 2 compared to its binding affinity for 5-HT1D receptors.

3. The process as claimed in claim 1, wherein those compounds whose affinity of The binding by the 5-HT5 receptors is higher at at least the factor of 5 compared to its binding affinity for the 5-HT1D receptors.

4. The process as claimed in one of the previous 25 claims, where those are read riMM "a" '' '• "- compounds whose Kx value by binding to 5-HT5 receptors is also less than 10" 8 M.

The process as claimed in one of the preceding claims, wherein at least one action induced by the binding partner of 5-HT5 is also determined.

The process as claimed in claim 5, wherein those compounds whose action is agonist are read.

The process as claimed in claim 5 or claim 6, wherein the binding of GTP to G proteins, intracellular calcium concentrations, phospholipase C activity and / or the production of cAMP is determined.

The process as claimed in one of the preceding claims, wherein, in order to determine the binding affinity and / or activity, the compounds are contacted with cellular systems having 5-HT5 receptors.

9. The process as claimed in claim 8, wherein human glioma cell lines or heterologous cell lines transfected with h5-HT5 are used.

10. The process as claimed in claim 9, wherein CHO cells transfected with h5-HT5, human kidney cells transfected with h5-HT5 or C-6 glioma cells transfected with h5-HT5 are used.

11. The use of at least one binding partner for 5-HT5 receptors whose binding affinity for 5-HT5 receptors is greater than their binding affinity for 5-HT1D receptors, for the production of an agent for the treatment of disorders cerebrovascular

12. The use as claimed in claim 11, wherein the binding affinity of the binding counterparts for the 5-HT5 receptors is greater by at least the factor of 2 compared to their binding affinity for the 5-HT1D receptors. -utal--

13. The use as claimed in claim 11, wherein the binding affinity of the binding counterparts for the 5-HT5 receptors is greater by at least the factor of 2 compared to their binding affinity for the 5-HT1D receptors.

14. The use as claimed in one of claims 11 to 13, wherein the Ki value for the binding of the binding counterparts to 5-HT5 receptors is less than 10"6 M.

15. The use as claimed in one of claims 11 to 14, wherein the binding counterpart is a 5-HT5 agonist.

16. The use as claimed in one of claims 11 to 15, for the treatment of migraine.

17. The use as claimed in claim 16 for the acute treatment of migraine.