Methods for determining efficacy of ligands of sodium/proton antiporters

FIELD: medicine.

SUBSTANCE: invention refers to methods for determining the efficacy of an ion channel ligand. The ex vivo methods for determining the efficacy of the ion channel ligand in vivo depending on plasma, involves the stages as follows: a) contacting a cell expressing the ion channel with i) animal's plasma and ii) the ion channel ligand and b) determining the effect of the ion channel ligand on the cell or a) contacting the cell expressing the ion channel with i) animal's plasma and ii) a compound that is defined as the ion channel ligand, and b) determining the effect of the compound on the cell, or a) contacting the cell expressing the ion channel with animal's plasma wherein the ion channel ligand has been administered, and b) determining the effect of the ion channel ligand on the cell. The method according to the invention may be used for screening of a therapeutic preparation for preventing and/or treating a disease involving the ion channel dysfunction, especially for preventing and/or treating a cardiovascular disease or cancer.

EFFECT: invention provides higher effectiveness of the method and lower costs of screening of therapeutic preparations.

14 cl, 4 tbl, 2 ex, 3 dwg

The present invention relates to a method of determining the effectiveness of the ligand ion channel.

The family of Na⁺/H⁺-antiporters or sodium-proton antiporter (NHE) of membrane transporters uses Na⁺-gradient directed from the outside to the inside of the cell, to control the leakage of H⁺from the cells. Essential functions carried out by these groups transporters (or ion channels), are to regulate the volume of cells and pH, resolution, role in cell proliferation and transepithelial transport of Na⁺. NHE mammals is an integral membrane protein, whose function is to exchange one intracellular proton for one extracellular sodium ion. Through his involvement in ion fluxes NHE serves to regulate intracellular pH and volume of the cells and to initiate changes in growth or functional status of the cells. In addition to its physiological role NHE plays an important role in human pathology. Due NHE transport plays a major role in the damage done to the myocardium of a person during and after myocardial infarction, and it is believed, represents a key step in carcinogenic transformation of malignant cells. Consequently, for many years NHE was a purpose in cardiovascular and cancer research, and thousands of soybeans is ineni were tested for inhibition and selectivity NHE.

The standard way of determining the effectiveness of antagonists NHE is the analysis of thein vitrodescribed Schwark with employees (Schwarket al., 1998, Pflügers Arch., 336(5):797-800) and Wiemann with employees (Wiemannet al., 1999, Pflügers Arch., 436(3):255-262). After identifying the IC₅₀ligand selected NHE selectivity against other NHE can be determined using the same test. As a rule, effective ligands with good selectivity was selected for further studies by determining the pharmacokinetics of the test ligandin vivoetc. However, some very potent compounds showed only weak effects in studies ofin vivo. The reason could be the strong binding of the protein, low bioaccumulation or other effects that reduce the effective concentration of the compounds in plasma. Preferably very early to exclude these compounds during drug development to save time and resources.

Due to a mismatch between the IC₅₀in vitroand pharmacological efficacy (efficiency) of some compounds there was a need to develop a method of determining or modeling the effectiveness of ligands NHE or ligands of other ion channels in the body.

Accordingly, the first object of the present invention is the provision of a method definition wide-angle the efficiency of the ligand ion channel. Preferably, this test should be efficient in terms of time and monetary costs. In the context of this application, the term "ion channel", as implied, includes passive ion channels, as well as the active ion channels (ion transporters).

It was surprisingly found that the efficiency of the ligands of ion channels, especially antagonists NHE, can be identified by contacting cells expressing the ion channel, animal plasma and ligand ion channel. In this test it was possible to determine the effect of ligand ion channel on the cell where this effect reflects the efficiency of ligandin vivo.

Accordingly, the first aspect of the present invention provides a method of determining the effectiveness of the ligand ion channel, which includes stages:

(a) contacting cells expressing the ion channel, with

i) animal plasma and

ii) a ligand ion channel;

and

b) determining the effect of the ligand ion channel on the cell.

Briefly, the method can be made as follows: the Appropriate cells expressing the considered ion channel support (e.g., cultured) in suitable conditions, and subjected to the contacting (incubated) with the plasma of the animal and the ligand ion channel. Both components can be added simultaneously or together. Che is ez for some time, sufficient to ensure that the effect on the cell, determine the effect on the cell. After or simultaneously with contacting him see the effect (signal), where the detection effect is an indication of the validity of the ligand ion channel on the cell.

The term "efficiency of the ligand ion channel" in the context of the present invention relates to the pharmacological efficacy of the ligand ion channel. Unlike testin vitroin the absence of plasma, the method according to the invention, performed in the presence of plasma, takes into account the amount of bound protein, the extent and rate of absorption, distribution, metabolism and excretion of the suitability of the ligand and/or other effects that reduce the effective concentration of the compounds in plasma. When the ligand was injected animal took a plasma, the method also takes into account the extent and rate of absorption, distribution, metabolism and excretion of the ligand. These effects may depend, as non-limiting examples, the physical properties of the drug (hydrophobicity, pKa, solubility), the composition of the drug injection during a meal or on an empty stomach, daily features and/or interaction with other drugs, food or endogenous substances (e.g. enzymes). Accordingly, the term "efficiency of the ligand ion channel" refers not only to binding the human ligand ion channel and the subsequent induction of signal transduction but also take into account the above-mentioned effects of the ligand in plasma and/or living animal. This allows for more convenient identification of suitable drug candidates, for example, for the treatment ofin vivoand/or exclusion of compounds with low efficiencyin vivo.

The effect of the ligand on the cell can be any effect on the cell, including, as non-limiting examples, the modified morphology, viability or composition of intracellular compounds. The effect can take place at any level of signal transduction ion channel, including the binding of ligand to the ion channel, the flow of ions, the binding of the corresponding ion with a target, such as the intracellular target, the determination of the number of cellular connections, such as a second messenger (e.g. camp, cGMP, CA²⁺IP₃, diacylglycerol, and so on), the status of the activation of intracellular protein, such as protein kinase A, protein kinase C or MAR-kinase, determining the amount of mRNA or protein, any modified cell function (such as induction of apoptosis or cell cycle arrest), etc.

This method can be used to determine or model the efficiency of the ligand ion channelin vivo. Examples of the range of applications of the method include the following: first, can be measured effective is the concentration of ligand ion channel in the plasma sample, obtained from animals that were injected ligands ion channel (see, e.g., Example 2). If the ligand is injected animal, low concentrations of ligand in plasma compared with the entered amount of the ligand can be interpreted as low bioaccumulation (for example, because of the small time half-life in plasma).

Secondly, it is possible to determine the effect of ligand ion channel in the plasma, such as human plasma (see, e.g., Example 2). From comparison with IC₅₀for samples not containing plasma (as is well known in this area), we can conclude that the binding in plasma and suitability of the ligandin vivo. Compounds with a very high difference in the IC₅₀and efficiency in the plasma can be excluded in advance, which is effective for reducing the cost of the development of medicines. Currently, these compounds may be excluded only after experimentsin vivo.

Ion channel is a protein, usually forming pores, which helps to establish and control the flow of ions across plasma membrane. It is an integral protein or, more typically, an ensemble of several proteins. Such ensembles of several subunits typically include a circular configuration identical or homologous proteins, tightly Packed around the water-filled pore through the plasma of memb the Anu. Despite the fact that some channels allow the passage of ions exclusively in charge, archetypal channel pore is wide at its narrowest point, only one or two atoms. She spends a certain type ions, such as sodium or potassium, and transports them across the membrane. In some ion channels passing through the pore is governed by the "gate"that can be opened or closed by chemical or electrical signals, temperature or mechanical effort, depending on the types of ion channel.

Typically, ion channels are characterized depending on the opening of membrane channel, types of ions passing through this gate, and the number of pores.

If classified by the opening of the membrane channel, the channels are divided into two classes of ion channels with the potential-dependent gate (activation/inactivation depending on the potential gradient across the membrane and ion channels ligand-dependent gate (activation/inactivation depending on the binding of ligand to the channel). Examples of channels with the potential-dependent gates include sodium channels with the potential-dependent gate calcium channels with the potential-dependent gate potassium channel with potential-dependent gate, some transient receptor potential channels, aktiviseerumine the hyperpolarization channels with dependent cyclic nucleotide gate and proton channels with potential-dependent gate. Examples of channels with the ligand-dependent gates include potassium channels, such as internal rectified potassium channels activated by calcium potassium channels and potassium channels with two pore regions, the channels with the light-induced gate, such as channel rhodopsin and canals with gates that are dependent on cyclic nucleotides. In the case of classification by ions passing through the channels, ion channels, normally classified as follows:

chloride channels, potassium channels, such as potassium channel with potential-dependent gate, calcium-activated potassium channels, internal rectified potassium channels and potassium channels with two pore regions, sodium channels, calcium channels, proton channels, such as proton channels with potential-dependent gate and common ion channels, which are relatively non-specific ions, including most unstable receptor potential channels.

Some ion channels affect intracellular pH, like sodium-bicarbonate of cotransporter and sodium-proton antiporters (NHE). On the activity of ion channels can be influenced by natural or missing in the nature of the ligands that are associated with the considered ion channels. Known examples thereof include blocked tet is ototoxins, the saxitoxin, lidocaine and novocaine channels of sodium ions and blocked dendrologia, iberiotoxin and heteroepitaxial potassium channels.

In accordance with this ligand ion channel is any chemical substance that specifically binds to an ion channel. "Specifically bind to the ion channel of the present invention includes, as non-limiting examples, communicating with the dissociation constant K_Dnot exceeding 10^-4mol/l, preferably not exceeding 10^-5mol/L. the dissociation Constant K_Dyou can define, for example, in experiments on competitive binding, as is known to the person skilled in the art, in accordance with the following equation:

B[L] = [L]/{[L] + K_DL(1+[L^*]/K_DL*},

where [L] and [L^*] represent the concentration of the considered ligand ion channel and the concentration detected (e.g., labeled) of the ligand ion channel, such as radioligand for ion channel, respectively. K_DLand K_DL*represent the dissociation constants of the considered ligand ion channel and program ligand, respectively, and B[L] (from 0% to 100%) represents the binding at a certain concentration of the ligand ion channel.

In a preferred variant is NTE embodiment of the invention identifiable ligand is an agonist or antagonist. Agonists are associated with ion channel and activate it (for example, causing a conformational change). Antagonists or blockers are also associated with ion channel and inactivate it. Activation and inactivation can lead to a detectable signal if the status (active or inactive) ion channel is changed by the ligand. Preferably, the ligand is an antagonist, inactivating the considered ion channel. The ligand is characterized by the method according to the invention, can be used as a potential drug suitable for the treatment and prevention-related ion channel disorders or diseases.

In a preferred variant of the invention, the ion channel is a sodium-proton antiporters (NHE) or sodium bicarbonate cotransporter. Since the structure of the protein such as an enzyme which functions in cells largely depends on the pH, there is an optimum pH for protein function. For this reason, the maintenance and regulation of intracellular pH is extremely important for the cells for maintenance of homeostasis of cellular functions. NHE, as well as sodium bicarbonate cotransporter involved in the regulation of the pH of the cell. Sodium bicarbonate cotransporter controlled by the concentration gradient of Na⁺inside and outside of the cell membrane is wound and takes one Na ⁺in the cell together with one or more ions HCO₃^-. Because sodium bicarbonate cotransporter is located in the cell membrane and HCO₃^-trapped in a cage due to the sodium bicarbonate of cotransporter, neutralizes H⁺in the cytoplasm, it plays an important role in the regulation of intracellular pH.

NHE also play an important role in the regulation of intracellular pH. Up to this time had been identified nine isoforms (NHE1 to NHE9) within the family of mammalian NHE. The isoforms have approximately 25-75%sequence identity, with a calculated relative molecular masses ranging from approximately 74000 up to 93000. Structural analysis of antiporters suggests that they have similar membrane topologies, with aminocentesis membrane domain, consisting of twelve transmembrane segments, and a more divergent carboxykinase cytoplasmic domain.

For a long time it was known that NHE important for the growth of the tumor because the tumor cells are defective in Na⁺/H⁺- exchanging activity, were either unable to tumor growth, or showed severe growth retardation during implantation in mice with immune deficiency. Now it is obvious that NHE1 causes reversal of the pH gradient in the many types of transformed and/or malignant cells thus what intracellular environment becomes alkaline and the extracellular environment becomes acidic. This "malignant disorders", as I believe, represents a key stage of the carcinogenic transformation and necessary for the development and maintenance of the altered phenotype.

In addition, NHE, especially NHE1 was involved in the physiology of certain diseases, most studies concentrated on the role of NHE1 in heart disease and cancer. In the myocardium under normal conditions NHE1 removes excess intracellular acid in exchange for extracellular sodium. Increased intracellular sodium is removed regulatory membrane proteins, including Na⁺/K⁺-ATPase and Na⁺/Ca²⁺-antiporters. In the myocardium problems arise when increased production of protons, which occurs in the myocardium of human rights during and after myocardial infarction.

Isoform NHE1 is the isoforms of "household" antiporters and is expressed everywhere in the plasma membrane of virtually all tissues. This initial NHE isoforms detected in the plasma membrane of the myocardium. Isoforms from NHE2 to NHE5 also localized in the plasma membrane, however, have more limited distributions in tissues. NHE2 and NHE3 mainly located in the apical membrane of the epithelium and much expressroute the kidney and the intestine. In contrast, NHE4 most strongly represented in the stomach, but is also expressed in the intestine, kidney, brain, uterus and skeletal muscle, whereas NHE5 is expressed predominantly in the brain (but may also be present at low levels in other non-epithelial tissues, including spleen, testes and skeletal muscle). Isoforms from NHE6 to NHE9 expressed ubiquitously and are present in intracellular compartments. These are present in the membranes of organelles NHE is assumed to regulate luminally pH and concentration of cations intracellular compartments. Expression of NHE6 is the highest in the heart, brain and skeletal muscle, and it is localized in early recirculating endosomes. Isoform NHE7 predominantly localized in the TRANS-Golgi-apparatus and is different from other NHE-isoforms of the fact that it mediates the influx of either Na⁺or K⁺in exchange for H⁺. The highest NHE8 expression was detected in skeletal muscle and kidney, and this isoform is localized mainly in the area from the middle to the TRANS Golgi compartments. It was found that the newly identified isoform NHE9 localized in late recirculating endosomes.

NHE are targets for inhibition diuretic compound amiloride and its analogues and derivatives benzoylpyridine. Comparison R is slichnih NHE isoforms show they have different affiniscape to these inhibitors, with the following order of sensitivity under similar experimental conditions: NHE1≥NHE2>NHE5>NHE3>NHE4. Since NHE1 is the isoform that is most sensitive to inhibition and seems to be the most important isoform present in the plasma membrane of the myocardium, the selective properties of these inhibitors can be used in therapeutic purposes.

In a preferred embodiment of the present invention, the ion channel is a NHE, such as NHE1, NHE2, NHE3, NHE4, NHE5, NHE6, NHE7, NHE8 or NHE9, preferably NHE1, NHE2, NHE3 or NHE5, especially NHE1 or NHE3, especially NHE1.

Isoform NHE1 is the most well-described isoforms of NHE family. NHE1 is 815 amino acids in length, with residues 1 to 500, representing the membrane domain, and residues with 501 on 815, representing the cytoplasmic tail. Membrane domain of NHE1 is both necessary and sufficient for ion transport, whereas the cytosolic region involved in the regulation of the activity of antiporters. Ion flow through the antiporters is controlled by the transmembrane gradient of Na⁺and, apparently, does not require any direct metabolic energy arrival.

As described in detail above, the ligand ion channel p is edocfile is an agonist or antagonist. However, due to the clinical significance NHE, the ligand is preferably a agonist NHE, more preferably antagonist NHE. If this ion channel is inactive in the absence of ligand, it may be necessary to control the effect of the antagonist in the presence of agonist. In this case, the effect of the antagonist represents the inactivation of activated ion channel agonist.

In accordance with the present invention, the cell is incubated with the ligand and plasma. The cell can be any suitable cell, expressing the considered ion channel. Cells expressing the considered ion channel may be cells that naturally Express the ion channel. Alternatively, cells can be genetically modified to Express the ion channel.

The cell can be selected (randomly genetically modified), to maintain and cultivate, as known to the person skilled in the art. In addition to temperature and gas mixture, most often a variable factor in cell culture systems is the environment for growth. Recipe for growth can vary in pH, glucose concentration, growth factors and the presence of other nutrients, among others. The growth factors used as additives to the environment, cha is then obtained from the blood of animals, such as calf serum. Genetically modified cells can be obtained by incorporating the full coding sequence of the ion channel, as known to the expert. The person skilled in the art knows how to obtain a sequence of nucleic acids encoding a protein ion channel, and how to select or produce the following sequence of nucleic acids using standard methods of molecular biology. This can be accomplished, for example, by using and combining existing sequences using restriction enzymes. Nucleic acid can be combined with other elements, for example, the promoter and the start of transcription and the stop signal and the beginning of a broadcast and a stop signal, to ensure the expression of the sequence of the ion channel. The resulting sequence of nucleic acid can be introduced into cells, for example, by using a virus as a carrier or transfection, including, for example, electroporation, heat shock, magneticly, nucleofection and the use of transfection agents.

Optionally, the cell may be a part of the fabric; however, the method according to the invention is a method ofex vivo. In a preferred embodiment of the invention the cell is a is tapped from the cell line (many of which are well characterized and provide constant conditions and appropriate treatment), in particular, the line of cells of the mammal, more specifically, a line of human cells or cell line of the mouse, especially the line LTK-cells mouse LAP1 (Franchi et al., 1986, Proc Natl Acad Sci USA 83(24): 9388 - 9392). Examples of suitable cell lines include, as non-limiting examples, HEK 293, 745-A, A-431, BxPC3, C5N, Caco-2, Capan-1, CC531, CFPAC, CHO, CHO K1, COS-1, COS-7, CV-1, EAHY, EAHY 926, F98, GH3, H-295 R, H-4-II-E, HACAT, HACAT A131, HEK, HEL, HeLa, Hep G2, High Five, Hs 766T, HT29, HUV-EC R24, HUV-EC-C, IEC 17, 18 IEC, Jurkat, K 562, KARPAS-299, L 929, LIN 175, MAt-LYLU, MCF-7, MNEL, MRC-5, MT4, N64, NCTC 2544, NDCK II, Neuro 2A, NIH 3T3, NT2/D1, P19, SF9, SK-UT-1, ST, SW 480, SWU-2 OS, U-373, U-937 and Y-1. Other suitable cells are cells known specialist in this field.

Preferred cell lines are cells HEK 293 (primary embryonic human kidney)cells, 3T3 (embryonic rat fibroblasts, CHO cells (Chinese hamster ovary)cells, COS-7 (cell line of African green monkey)cells, HeLa (epithelioma human cervical carcinoma), JURKAT cells (T-cell leukemia human)cells, BHK 21 (normal hamster kidney, a fibroblast) cells and MCF-7 (breast cancer man), especially LTK-cell line mouse LAP1.

Within the method according to the invention the cell (as defined above) is incubated with plasma of the animal. The animal may be a vertebrate animal, in particular a mammal, especially a rat, mouse, rabbit, Guinea pig or cat that p is educativos safe and convenient handling in the laboratory.

To get the most significant results for human medicine plasma can also be obtained from a person. This venous blood is obtained by venipuncture from a human donor, where, as a rule, just a small sample of blood, for example, the sample from 5 ml to 25 ml, for the method according to the present invention (see Examples). The blood is usually obtained from the median cubital vein on the anterior forearm (side within the bend of the elbow). This vein runs close to the surface of the skin where there is no strong innervation. In industrialized countries, the blood sample is mainly carried out through a system of drainage pipes, consisting of a plastic center, syringe for subcutaneous injection and vacuum tubes.

In accordance with the method according to the invention determine the effectiveness of the ligand ion channel. Efficiency is determined by detecting the effect of the ligand ion channel on the cell in the presence of plasma. As described in detail above, the effect of the ligand on the cell can be evaluated at each level of signal transduction, including the binding of the ligand ion channel, linking the corresponding ion with the target, the determination of the number of cellular connections, such as a second messenger, the amount of mRNA or protein, any modified cell function (such as induction of apoptosis or the rest of the cell cycle), etc. Methods for determining binding of the ligand to the target are well known in the art and include the methods defined here. Methods for determining the amount of mRNA or protein is also known to specialists. Methods observations of altered cellular functions largely depend on the type of cellular functions and is also known to the skilled practice.

Methods for determining the effect of the ligand ion channel on the cell may include a heterogeneous or homogeneous assay. As used here, heterogeneous represents an analysis that includes one or more stages of washing, whereas in a homogeneous analysis of such stages of cleaning are not necessary. Only mix the reagents and compounds and provide definition. The method according to the invention can also include antibodies specific for the signal, acting downstream than an ion channel. Analysis can be an ELISA (ELISA analysis using fixed enzymes), kits are used (latenightalumni analysis with enhanced dissociation), SPA (scintillation proximity assay), the analysis of the "flash in the die, the analysis of FRET (resonance energy transfer fluorescence)analysis of TR-FRET (resonance energy transfer fluorescence with a time resolution), analysis of FP (fluorescence polarization), ALPHA (amplified luminescent makontak the hydrated homogeneous analysis), analysis of EFC (fragment complementation enzyme), two-hybrid analysis or coimmunoprecipitation analysis.

In another embodiment of the invention, the method of determination of efficiency may include measuring one or more second messengers such as camp or phospholipid(s), preferably phosphatidylinositols. The measurement may include determining the concentration of one or more second messengers. Means for determining the concentration of one or more second messengers known in the art and include such involvement, for example, labeled precursors, preferably labeled precursors of second messengers (e.g.,³²P-ATP or³H-Inositol), including cleaning, for example, by chromatography on columns or mass spectrometry. Change phospholipids, as a rule, especially justified for calcium channels.

If this ion channel, for example, NHE or sodium bicarbonate cotransporter affects the pH, the effect is determined by the method according to the present invention, is a change in the pH value, preferably, the change in intracellular pH values.

In a preferred embodiment of the invention the effect of the ligand ion channel on the ion channel, n is the sample, NHE or sodium bicarbonate cotransporter, determined by fluorescence.

Fluorescence is an optical phenomenon in which the molecular absorption of a photon triggers the emission of another photon with a longer wavelength. The energy difference between the absorbed and emitted photons is emitted in the form of molecular vibrations or heat. Typically, the absorbed photon is in the ultraviolet range, and the emitted light is in the visible range, but this depends on the adsorption curve and the Stokes frequency shift of a particular fluorophore. The number of applications of fluorescence increases in biomedical, biological and related Sciences. Also, an increasing number of methods of analysis in these areas, although with more and more bad item in the form of acronyms, such as FLIM, FLI, FLIP, CALI, FLIE, FRET, FRAP, FCS, PFRAP, smFRET, FIONA, FRIPS, SHREK, SHRIMP, TIRF. Most of these methods are based on the use of fluorescent microscopes. In such microscopes use light sources with high intensity, usually mercury or xenon lamps, LEDs or lasers for excitation of fluorescence in the sample under observation. Optical filters then separate the excitation light from the emitted fluorescence to detect by eye or camera on charge-coupled devices or other speedmachine (optoelektronnye multipliers, the spectrographs etc).

As a rule, to detect the effect of the considered signal (here the effect of the ligand ion channel) is used fluorescent dye or marker, or label. Fluorescent dye comprises a fluorophore. The fluorophore, by analogy with the chromophore, is a component of a molecule which causes a molecule to be fluorescent. He represents a functional group in the molecule, which absorbs radiation of a specific wavelength and re-emits radiation with a different (but strict) wavelength. The number and the wavelength of the emitted radiation depend on the fluorophore and the chemical environment of the fluorophore. Fluoresceinisothiocyanate, the reactive derivative of fluorescein, was one of the most common fluorophores, chemically attached to other afluorescent molecules for the formation of new and fluorescent molecules for a variety of applications. Other historically common fluorophores are derivatives of rhodamine, coumarin and cyanine. Examples of fluorescent dyes include, as non-limiting examples, 7-aminooctanoic D, acridine orange, acridine yellow, Alexa Fluor, AnaSpec, auramine O, rhodamine-Euromoney dye, benzanthrone, 9,10-bis(phenylethynyl)anthracene, 5,12-bis(phenylethynyl)on TACAN, CFDA-SE, CFSE, calcein, carboxyfluorescein, 1-chloro-9,10-bis(phenylethynyl)anthracene, 2-chloro-9,10-bis(phenylethynyl)anthracene, coumarin, cyanin, DAPI, Dioc6, DyLight Fluor, ethidiumbromid, fluorescein, fu-2, fu-2-acetoxymethyl ether, green fluorescent protein, Hilyte Fluor dye Hoechst, Indian yellow, Indo-1, luciferin, fixed, phycobilin, phycoerythrin, phycoerythrin, propyliodide, Pyramin, rhodamine, RiboGreen, Rubin, ruthenium(II)Tris(bathophenanthrolinedisulfonic), SYBR Green, stilbene, sulforhodamine 101, TSQ, Texas Red, umbelliferon, yellow fluorescent protein or BCECF.

In accordance with the preferred embodiment uses BCECF. BCECF (2',7'-bis-(carboxyethyl)-5(6)-carboxyfluorescein, C27H20011), M 520,45 g/mol CAS-Nr.: 85138-49-4, storage: 2-8°C protected from light), BCECF is an analogue of carboxyfluorescein with improved characteristics to determine the pH in lymphocytes due to its increased retention within cells. For efficient accumulation in intact cells BCECF can be used in the form acetoxymethyl ether BCECF/AM, which is broken down within cells with the formation of more non-penetrating BCECF. The maximum excitability to 70 nm-aqueous solution of BCECF corresponds to 500 nm. The intensity of BCECF fluorescence may be under the influence of several substances, such as glycerol, sucrose, polietilen the Kohl or polyvinylpyrrolidone. A suitable concentration of BCECF is 5 μm (final concentration).

In the context of the present invention a fluorescent dye is sensitive to the action of the ligand ion channel ion channel. The effect may be a direct impact directly on the channel (e.g., conformational change, changing the characteristics of the binding and so on) or a change in signaling pathways downstream of the considered ion channel, especially in the case of intracellular ion concentration is transported considered ion channel. Ion can represent, for example, H⁺, HCO₃^-, K⁺, Na⁺, Cl^-Ca²⁺. Suitable pH-dependent fluorescent markers (for H⁺and HCO₃^-include, as non-limiting examples H⁺-selective fluorescent chromoionophores ETH 5294, S NAFL, SNARF, HPTS, fluoresceine, carbofuran, BCECF (2',7'-bis(2-carboxyethyl) 5 (and 6) carboxyfluorescein) and BCPCF (2',7'-bis(2-carboxypropyl) 5 (and 6) carboxyfluorescein). Calcium ions can be detected using aquaria (first selected photoblog), calmoduline, bulleted Alexa-488 or photina (Axxam SpA, Milan, Italy), Fluo4 (Glycine, N-[4-[6-[(atomic charges)methoxy]-2,7-debtor-3-oxo-3H-xanthene-9-yl]-2-[2-[2-[bis[2-[(atomic charges)methoxy]-2-ethoxyl]amino]-5-methylphenoxy]ethoxy]phenyl-N-[2-[(atomic charges)methoxy]-2-ethoxyl]-(atomic charges) methyl ether, C₅₁H₅₀F₂N₂O₂₃), M=1096,95 g/mol CAS-Nr.: 273221-67-3, storage: 2-8°C protected from light), or Fura2 (5-Oxazolidinecarboxylate acid,2-(6-(bis(2-((atomic charges)methoxy)-2-oxoethyl)amino)-5-(2-(2-(bis(2-((atomic charges)methoxy)-2-oxoethyl)amino)-5-methylphenoxy)ethoxy)-2-benzofuranyl)-(atomic charges) methyl ester, C₄₄H₄₇N₃O₂₄), M=1001,86 g/mol CAS-Nr.: 108964-32-5, storage: -20°C, protected from light). For detection of sodium and potassium ions can be used ionophor X and ionophor BME-44.

Such methods can be performed as follows, where the following is provided for illustrative purposes only. The specialist will understand that one or more stages can be performed in other ways (for example, as presented in detail in the present description of the invention):

For the culture ofex vivocells can be obtained by extraction from tissues. For example, pieces of cloth can be placed in the growth medium, and cells that grow available for cultivation. This method is known as explanata culture. An alternative can be used cell line (e.g., traditional or immortal cell line). There are numerous examples of traditional cell lines specific cell types (see also above).

Cells can be grown in a suitable medium (for example, commercial and accessible environment, containing serum and antibiotics). Cells are typically cultured in a suitable atmosphere (e.g., 5%CO₂), relative humidity (e.g., 90%) and temperature (e.g., 37°C). For large-scale screening and/or proper treatment of the cells can be grown in advance tablets, such as 96-well plates, 24-hole tablets etc.

As described above, the change in intracellular pH can be determined using fluorescent markers. Suitable markers include SNARF-1, BCECF and CMFDA. Excitation and emission are Exc 485, Em 590 nm for carboxyS ARF-1; Exc 500, Em 538 nm for BCECF; and Exc 485, Em 538 nm for CMFDA. The width of the bands of excitation and emission can be 20 nm and 25 nm, respectively. A suitable device for determining intracellular pH is, for example, fluorometric reader advance tablets FLUOstar 97 (BMG LabTechnologies, Inc, Durham, North Carolina) or device described in the Examples.

One option implementation uses a fluorescent dye BCECF (2',7'-bis-(carboxyethyl)-5-(6)-carboxyfluorescein, C₂₇H₂₀O₁₁), M=520,45 g/mol CAS-Nr.: 85138-49-4, storage: 2-8°C protected from light), BCECF - analog carboxyfluorescein with improved characteristics to determine the pH in lymphocytes due to its increased retention within cells. For effective proniknovenie is in undamaged cells BCECF can be used in the form acetoxymethyl ether BCECF/AM, which is broken down in cells with more impervious BCECF. The maximum excitability to 70 nm solution of BCECF is necessary to 500 nm. The intensity of BCECF fluorescence can be influenced by several agents, such as glycerol, sucrose, polyethylene glycol or polyvinylpyrrolidone. A suitable concentration of BCECF is 5 μm (final concentration).

Next is one typical procedure for the preparation of cells for intracellular determination of pH: the Cells can be grown, for example, up to 90%confluently, to collect, for example, using trypsin and immediately block the culture medium containing, for example, 10%fetal bovine serum, precipitated by centrifugation and then washed. Precipitated cells can be re-resuspendable in the new environment, to give a chance to recover (for example, 5%CO₂at 37°C for 1 hour, rinse, for example, twice, for example, not containing bicarbonate buffer (130 mm NaCl, of 4.7 mm KCl, 1.2 mm MgSO₄, 1.2 mm KH₂PO₄, 11.7 mm D-glucose, 1.3 mm CaCl₂, 10 mm HEPES, pH 7,4) and then loading the above-mentioned marker (dye). In another variant implementation, the cells not treated with trypsin before loading dye.

Loading dye can be, for example, as follows: cells incubated with 5 μm 5-(and 6)-carbox the SNARF-1/AM (Molecular Probes, Eugene, Oregon) for 30 minutes at room temperature in not containing bicarbonate-buffered Krebs-Hepes (pH 7,4)containing 1% (weight/vol) Pluronic F-127 (Sigma Chemicals, St. Louis, MO). Cells can be loaded, for example, 1 μm 2-,7-bis-(2-carboxy)-5-(and 6)-carboxyfluorescein (BCECF/AM) (Molecular Probes) for 45 minutes at room temperature in the buffer. Cells can be loaded with 5 μm CellTracker Green CMFDA (5-chloromethylfluorescein) (Molecular Probes) for 45 min at 37°C in the buffer. Load one or more other identified above dyes can be performed in a similar manner in accordance with the relevant and well-known protocols.

After loading, the cells may be washed, for example, one or more times (e.g. twice) buffer and re-suspended in fresh medium, and to enable recovery in 5%CO₂at 37°C for 1 hour or longer (e.g. during the night). After loading dye and the time of recovery of cells may be washed one or more (e.g., 3) times the appropriate buffer, for example, not containing bicarbonate-buffered Krebs-Hepes (pH of 7.4 or 6.0), re-suspended to a final concentration of 2.5×10⁶cells/ml and left at 4°C.

The cells can be diluted and spread evenly (approximately 35,000 cells/well) in opaque white 96-well tablet (or any other suitable PLA is shite, for example, black with clear bottom). Plasma and buffer alone or buffer containing the ligand, can be sequentially injected into individual wells and record the intensity of fluorescence at appropriate intervals (for example, 20 seconds). You can make several (e.g. five) of the control readings at appropriate intervals (for example, a 20-second intervals) before each injection.

At the end of each experiment, you can apply the calibration procedurein situwith the known ligand ion channel (e.g., nigericin for H⁺/K⁺-antiporters)to link the intensity of fluorescence with pH. This can be accomplished, for example, so that K⁺/H⁺-antiporters ionophor sets [K⁺]o=[K⁺]i and pHo=pHi by exposure of cells in buffers with different pH in depolarization buffer with a high content of K⁺(140 mm KCl, 1.2 mm MgSO₄, 1.2 mm KH₂PO₄, 11.7 mm D-glucose, 1.3 mm CaCl₂, 10 mm HEPES, pH from 6.0 to 8.0, in the presence of 20 μm of nigericin). For correcting small changes in the density of cells and the instability of the light intensity between calibration and experiments the concentration of CMFDA was measured by perselisihan cells at the end of each experiment, 0.1% Triton X-100 and brought the pH to 11 by using KOH.

In addition, leakage of dye can is to follow the separation of the contributions of the issued and intracellular dye. For this, cells can turn with different time intervals, for example, within 30 seconds. Then you can determine the intensity of fluorescence of each of the solutions supernatant and lysate re resuspending cells. The intensity of the fluorescence of the supernatant solutions reflects the leakage of dye from the cells, and the total intensity of the fluorescence from the supernatant and the cell lysate can be used to determine the total amount of dye. The fluorescence intensity of the control (containing no dye) can be subtracted from each fraction, and the leak can be described as the ratio of fluorescence intensity (supernatant/total) depending on time.

Additional typical method is described in the Examples.

In addition, sensitive to ions microelectrodes (including ionoobmennymi ionophor, such as sodium ionophor bis(12-crown-4), kalibrasi ionophor bis(benzo-15-crown-5), calciummagnesium ionophor HDOPP-Sa) or ion-sensitive enzymes (such as potassium-dependent uric Hamidreza, piruwatkinaza (U.S. Patent Nos. 5501958 and 5334507) or glyceraldehydes (U.S. Patent No. 5719036)), can be used for detection of ion flow across cell membranes. Additional methods with sensitivity to pH include the distribution of weak acids or bases,³¹RAMR spectroscopy and pH-sensitive green fluorescent protein [GFP].

Preferably, the method was adapted to large-scale screening. In this way sceneroot a large number of compounds in relation to the considered ion channel in tests with whole cells. Typically, these screenings performed in 96-well tablets, using technology based on automated, robotic devices or formats matrices (chip) with high density.

In one embodiment of the method according to the invention the ligand ion channel, such as a ligand NHE add to the cell together with the plasma. This can be achieved by introducing a ligand animal (non-human). The ligand can be entered by any suitable means, including parenteral (such as intravenous, intraarterial, intramuscular, intracardiac, subcutaneous, intradermal, intrathecal, intraperitoneal), enteral, such as oral or rectal), local (such as episodically, inhalation, nasal, or vaginal), etc. Preferably the introduction along with the food.

After a period of time sufficient to allow the ligand to be present in the plasma, the animal take the plasma. For carrying out the method according to the invention containing ligand plasma is added to the cell and determine the effect of the ligand on the cell. Determination of effect may include the step determining the concentration of ligand in plasma (see, also Example 2). If the number of ligand introduced animal, comparable to the amount of ligand present in the plasma, it is possible to obtain a conclusion from the comparison, for example, the extent and rate of absorption, distribution, metabolism and excretion of the ligand.

In accordance with the present invention the method for determining the effect of the ligand ion channel can be used to determine plasma concentrations of ligand. This can be done by comparing the effect of plasma with an unknown ligand concentration standard, for example, the standard curve (see, e.g., Example 2).

The method according to the invention can be used for screening of a drug for preventing and/or treating diseases involving dysfunction of ion channels, especially for preventing and/or treating cardiovascular diseases or cancer. In the case of screening checked the ligand can be either a known ligand ion channel, or a substance whose function is still unknown, or not yet established communication with the ion channel. Accordingly, the method can be used to identify the new ligand ion channel. Alternatively, a known ion channel ligands can be tested for their efficiency using the method according to the invention. Effectivetime to be associated with the action of ligands in the system, not containing plasma (such as the binding affinity of)to evaluate the effect of plasma on the activity of the ligand and to evaluate the activity of the ligandin vivo.

The ligand may be provided in the form of a library of chemical compounds. Libraries of chemical compounds include a variety of chemical compounds and collected from any number of sources, including chemically synthesized molecules and natural products, or formed combinatorial chemical methods. They are particularly suitable for large-scale screening. They can consist of chemical compounds with a specific structure or compounds of a particular organism such as a plant. In the context of the present invention a library of chemical compounds represents preferably a library consisting of small molecules. Further, the present invention is illustrated by the figures and examples which are not intended to limit the scope of the present invention.

Figure 1 shows a calibration curve calculated efficiency cariporide in the plasma of rats (EC_5O=115 nm).

Figure 2 shows the calibration curve of the estimated effectiveness of the new compound X in the plasma of rats (EC₅₀=662 nm).

Figure 3 shows the estimated plasma concentration cariporide and connections X using thein vitrothe test is on NHE1.

EXAMPLES

Example 1: analysis to determine the effective concentrations of the antagonists NHE in plasma samples of animals

Plasma samples were obtained from male new Zealand rabbits (2.5 to 3.5 kg), supported on a standard forage for rabbits with 0.3% cariporide (NHE inhibitor). A blood sample was taken after 1 week of auditory artery for determination of plasma concentrations cariporide. Determination of plasma concentrations cariporide conducted as described below:

Gene NHE (SLC9A1, A. Franchi et al., Proc Natl Acad Sci USA, 1986 Dec; 83(24):9388-92.) was cloned into the pMamneo vector and introduced into cells LAP1 (LTK-cell line mouse). Stable cell lines were obtained from transfected cells.

Cells LAP1 (LTK-cell line mouse), stably expressing the human NHE1 (hNHE1), were seeded with a density of 25,000 cells/well/100 μl medium (Iscove medium, 10%FCS, 2 mm L-glutamine, 100 units/ml penicillin/streptomycin, 50 μg/ml gentamicin, 400 μg/ml G418) in 96-well microplates with black clear bottom (Costar®, Corning Inc., Corning, NY). Cells were incubated over night at 37°C, 5% CO₂and 90%humidity. Before measurement, the medium was removed and added to 100 μl/well of buffer with dye (20 mm HEPES, brought to a pH of 7.4 using KOH, 20 mm NH₄Cl 115 mm choline chloride, 1 mm MgCl₂, 1 mm CaCl₂, 5 mm KCl, 5 mm glucose, 5 μm BCECF). After incubation for 20 min at 37°C the cells 3 times washed with buffer, not containing Na⁺(5 mm HEPES, brought to a pH of 7.4 using KOH, 133,8 mm choline chloride, a 4.7 mm KCl, 1.25 mm CaCl₂, 1.25 mm MgCl₂, 0,97 mm K₂HPO₄, 0.23 mm KH₂PO₄, 5 mm glucose), leaving 90 ál wash buffer per well. Restoring the pH was measured using FLIPR (Fluoromethc Imaging Plate Reader, Molecular Devices, Sunnyvale, Calif.; a laser power of 0.3 W, aperture 4, the measurement interval 2, the measurement time 120 sec). During the measurement, the hole was added to 90 μl of solution plasma (90%plasma of animals from animals not treated antagonists with food, 10%Na⁺buffer: 10 mm HEPES, brought to a pH of 7.4 using KOH, 133,8 mm NaCl, of 4.7 mm KCl, 1.25 mm CaCl₂, 1.25 mm MgCl₂, 0,97 mm K₂HPO₄, 0.23 mm KH₂PO₄, 5 mm glucose). Because of the presence of Na⁺in the buffer NHE1 begins to transport the H⁺from the cells, leading to increased intracellular pH and fluorescence. Upper control solution plasma without antagonist. Lower control solution plasma with 20 μm cariporide (final concentration 10 μm). Cariporide was added to the plasma after training. The addition of 10 μm cariporide leads to a complete blocking of the activity of NHE1. For calibration to the plasma after preparation were added different amounts of cariporide and check antagonists to determine the efficiency in the plasma of animals.

To calculate the activity of NHE1 must count the Ali increase in fluorescence between the 12th and 32nd seconds. Upper control was taken as 100%activity, whereas the lower control determines 0% NHE1 activity. % activity of the samples was calculated by comparison with the controls.

Results: As shown in Tables 1 and 2, the example showed good results. The test work was very good (z'=0,74, table 1). All studies showed complete blockage of NHE1, indicating that the concentration cariporide ≥10 ám (table 2, figure 1).

Example 2: Test for determination of plasma levels of antagonists NHE

Animals used to study the plasma was subjected to pre-processing, as follows:

The plasma was obtained from groups I through VII through 85 minutes after injection (groups I-IV, VII) or 200 minutes after treatment (group V-VI) and from untreated rats. To analyze the plasma was stored at -20°C.

To determine the effectiveness of the ligand NHE1 cell-based LAP1 (LTK-cell line mouse) cells, stably expressing hNHE1 (see, Example 1), were seeded to a density of 25,000 cells/well/100 μl medium (Iscove medium, 10%FCS, 2 mm L-glutamine, 100 units/ml of interest is Illin/streptomycin, 50 µg/ml gentamicin, 400 μg/ml G418) in 96-well microplates with black clear bottom (Costar®, Corning Inc., Corning, NY). Cells were incubated over night at 37°C, 5% CO₂and 90%humidity. Before measurement, the medium was removed and added to 100 μl/well of buffer with dye (20 mm HEPES, brought to a pH of 7.4 using KOH, 20 mm NH₄Cl 115 mm choline chloride, 1 mm MgCl₂, 1 mm CaCl₂, 5 mm KCl, 5 mm glucose, 5 μm BCECF). After incubation for 20 minutes at 37°C the cells 3 times washed with buffer not containing Na⁺(5 mm HEPES, brought to a pH of 7.4 using KOH, 133,8 mm choline chloride, a 4.7 mm KCl, 1.25 mm CaCl₂, 1.25 mm MgCl₂, 0,97 mm K₂HPO₄, 0.23 mm KH₂PO₄, 5 mm glucose), leaving 90 ál wash buffer per well. Plasma samples were prepared as follows: 10% (volume/volume) Na⁺buffer (10 mm HEPES, brought to a pH of 7.4 using KOH, 133,8 mm NaCl, of 4.7 mm KCl, 1.25 mm MgCl₂, 1.25 mm CaCl₂, 0,97 mm K₂HPO₄, 0.23 mm KH₂PO₄, 5 mm glucose) was added to 90% (volume/volume) plasma of the rat.

Were prepared upper controls (solution plasma without antagonist) and lower controls (solution plasma with 20 μm cariporide, with a final concentration of 10 μm). A series of dilutions cariporide and CONNECTIONS X 10% (vol/vol) Na⁺buffer/90% (volume/volume) of rat plasma was used for the calibration curve to calculate the concentration is connected to the I in the plasma samples of rats (Figure 1 and 2).

Restoring the pH was measured using FLIPR (Fluoromethc Imaging Plate Reader, Molecular Devices, Sunnyvale, Calif.; a laser power of 0.3 W, aperture 4, the measurement interval 2, the measurement time 120 sec). During the measurement, the hole was added to 90 μl of a solution of plasma.

To calculate the activity of NHE1 was calculated increase in fluorescence from the 12th to 32 seconds. Upper control was taken as 100%activity and lower control - 0% NHE1 activity. Activity (%) of the samples was calculated by comparison with the controls. The levels of compounds in plasma were calculated with respect to the calibration curve.

Results: the results are summarized in Table 3. The factor Z' was greater than 0.75 for all measurements.

Typical calibration curves for cariporide and compound X is shown in Figures 1 and 2.

Additionally, the calibration curves were used to calculate the levels of compounds in plasma in sets of samples. Calibration curves were calculated using the following formula:

The variables were set as shown in Table 4:

The estimated levels of compounds in plasma are shown in Figure 3.

Accordingly, it is possible to determine the effectiveness of NHE1 inhibitors in the plasma of rats, using the analysis of NHE1 of the present invention. It is also possible to calculate the active plasma concentration of these compounds in the calibration curve. The NHE1 inhibitors in the plasma of rats were active after training. The data show a good dose dependence of inhibition of NHE1.

1. Ex vivo method of determining the effectiveness of the ligand ion channel in vivo could be the cost from the presence of the plasma, incorporating the following stages:
a) bringing the cells expressing the ion channel in contact with
i) animal plasma and
ii) a ligand ion channel and
b) determining the effect of the ligand ion channel on the cell
or
a) bringing the cells expressing the ion channel in contact with
i) animal plasma and
ii) connection, which is defined as the ligand ion channel,and
b) determining the effect of compounds on the cell
or
a) bringing the cells expressing the ion channel in contact with the plasma of the animal, which was introduced ligand ion channel, and
b) determining the effect of the ligand ion channel on the cell.

2. The method according to claim 1, where the ion channel is a sodium-proton antiporters (NHE), or sodium bicarbonate cotransporter.

3. The method according to claim 1, where NHE is a NHE1, NHE2, NHE3 or NHE5, in particular NHE1 or NHE3, especially NHE1.

4. The method according to claim 1, where the ligand ion channel, in particular a ligand NHE, is an agonist or antagonist, particularly an antagonist NHE.

5. The method according to claim 1, where the cell is a cell line, in particular the cell line of mammalian, in particular the cell line of human or mouse, especially LTK-cell line mouse LAP1.

6. The method according to claim 1, where the animal is a vertebrate animal, in particular a mammal, especially a rat, mouse, rabbit, Guinea pig, or to the specifications.

7. The method according to claim 1, where the animal is a human.

8. The method according to claim 1, where the effect is a change in the pH value.

9. The method according to claim 1, where the effect is a change in the values of intracellular pH.

10. The method according to claim 1, where the effect is determined by fluorescence.

11. The method according to claim 1, where the ligand NHE is administered to an animal, where the animal is an animal other than human.

12. The method according to claim 1, where the method is used to determine the concentration of ligand in plasma.

13. The method according to claim 1, where the method is used for screening of a drug for prevention and/or treatment of a condition that causes impaired function of the ion channel.

14. The method according to claim 1, where the method is used for screening of a drug for preventing and/or treating cardiovascular diseases or cancer.