EP2193377A2 - Method of screening for compounds that can be used for the treatment of respiratory conditions - Google Patents

Abstract

The present invention relates to the use of a screening method for identifying candidate molecules that can be used for the treatment of respiratory conditions in a mammal, wherein said screening method comprises a step which comprises determining whether the functional activity of a TASK-2 polypeptide in the presence of a test molecule is decreased or eliminated compared with the functional activity of said TASK-2 polypeptide in the absence of said test molecule, the test molecule being considered to be a candidate molecule when it decreases or eliminates said functional activity.

Description

 METHOD OF SCREENING COMPOUNDS USEFUL FOR THE TREATMENT OF RESPIRATORY DISORDERS

Technical field of the invention

[1] The present invention relates to the use of a method of screening compounds for the treatment of respiratory disorders in a mammal. It finds particular application in the identification of new candidate molecules for the treatment of respiratory disorders.

[2] In the description below, the references in parentheses (Ref) refer to the list of references presented after the examples.

PRIOR ART [3] The central adaptation of breathing to physiological needs is a chemosensitive phenomenon that involves changes in the electrical activity of specialized neurons located mainly in the brainstem. These respiratory neurons divide into small groups forming columns in the brain stem that extend from the ventrolateral part of the caudal rachidian bulb to the dorsolateral portion of the annular protuberance (Richter DW, Spyer KM (2001) "Studying rhythmogenesis of breathing: In Neurosci, 24, 464-472 (Ref 1) and Feldman JL, DeI Negro (2006) "Looking for inspiration: new perspectives on respiratory rhythm." Nat Rev Neurosci, 7, 232 -242 (Ref 2)). It is therefore not surprising that ion channel deficiencies are involved in respiratory physiopathologies, for example linked to prolonged stay at altitude, or to various diseases such as sleep apnea syndrome and sudden death syndrome. infant.

[4] In mammals, respiration is regulated by three chemical parameters of the arterial blood: i) the increase in carbon dioxide, or hypercapnia. ii) decreased blood pH, or acidosis. iii) the decrease of oxygen concentration in the blood, or hypoxia.

[5] The activity of the neuron network controlling respiration is therefore adapted according to the various parameters mentioned above (Feldman JL et al., 2003) "Breathing: rhythmicity, plasticity, chemosensivity": Annu Rev Neurosci, 26, 239-266 (Ref 3)). [6] The variations of these parameters are measured by chemoreceptors. These chemoreceptors detect changes in arterial CO ₂ pH and partial pressure at the peripheral level through carotid body chemoreceptors, and at the brainstem with central chemoreceptors located in the raphe nuclei and the retrotrapezoid nucleus (Severson et al. (2003) "Midbrainserotonergic neurons are central pH chemoreceptors." Nat Neurosci, 6, 1139-1140 (Ref 4)). The electrical signals of the chemoreceptors contribute to the adaptation of the respiratory activity to the physiological needs of the organism.

[7] Among the pathologies related to defects in the regulation of respiration, sleep apnea syndrome is a real public health problem. This syndrome is often associated with obesity. For example, in the United States, about 3 million men and 1.5 million women suffer from sleep apnea syndrome. This syndrome could have negative consequences on the state of health of the people suffering from it, for example by worsening the cardiovascular diseases as indicated by the article of Namen and collaborators ((2002) «Increase Physician-reported sleep apnea: the National Ambulatory Medical Care Survey. "Chest 121 (6): 1741-1747 (Ref 5)). [8] The different forms of sleep apnea have the common occurrence during sleep of pathological breaks in breathing (more than 10 seconds in adults, more than 8 seconds in children), which causes hypoxia with reduced oxygen supply to the brain and peripheral tissues. Sleep apnea syndromes have a heterogeneous etiology and can be classified according to the likely underlying pathologies. For example, the subgroup of obstructive sleep apnea is characterized by upper airway obstruction that prevents proper and efficient ventilation. The subgroup of central sleep apnea is characterized by defects in the regulation of respiration at the level of the brainstem, the nerve control of the respiratory muscles no longer functioning transiently. The mixed apnea subgroup corresponds to central apnea followed by obstructive sleep apnea.

[9] Interestingly, one of the central forms of apnea ("non-hypercapnic central apnea") is characterized by periods of hyperventilation that result in a reduction in the concentration of arterial carbon dioxide called hypocapnia. The reduction of carbon dioxide, in turn, results in a lack of stimulation of the brainstem breathing centers which results in longer breaks in breathing, and therefore, causes a reduction in arterial oxygen concentration.

[10] Apparently, patients suffering from these forms of sleep apnea have a delay in stimulating hypoxia-induced breathing.

[11] Several factors leading to reduced stimulation of breathing may aggravate sleep apnea syndromes: eg, substances having an effect on respiration, such as alcohol and tranquillizers, or a stay at high altitude. altitude. [12] Currently, sleep apnea syndrome therapy includes surgical procedures and devices for establishing positive pressure in the upper respiratory tract.

[13] Continuous nasal positive airway pressure (nCPAP) therapy during sleep is the most effective method to date to improve clinical symptoms. However, this treatment requires that the patient be permanently connected to the system via a mask. Thus, the fact that the technique is impractical limits its therapeutic success.

[14] In addition to surgical interventions and nCPAP treatment, some pharmacological approaches have been made to improve breathing in patients with sleep apnea, for example using progesterone, theophylline, acetazolamide and protriptyline.

[15] Although these substances are capable of stimulating respiration in at least some of the patients, none of them has proved to be really effective for the treatment of sleep apnea syndromes. In addition, some of these substances have significant side effects. As a result, many pharmaceutical industries are looking for new molecules to treat sleep apnea syndrome.

[16] In the past, researchers have focused on the development of TASK-1 potassium channel blockers that appear to stimulate respiration. The TASK-1 channels are part of a family comprising the TASK 1, TASK 2 and TASK 3 channels. These are K + channels with 4 transmembrane domains and two channel domains (K2P channels) (Goldstein SA et al (2005), "International Union Pharmacology, LV. Nomemclature and molecular relationship of two-pot potassium channels ". Pharmacol Rev., 57, 527-540. (Ref 6)). These channels are active in the form of monomers, heterodimers and homodimers (Czirjak G, Enyedi P (2001), "Formation of functionnal heterodimers between the TASK-1 and TASK-3 two-pore domain potassium channel subunits". Biol Chem, 277, 5426-5432 (Ref 7), see Kang D et al (2004), "Functionnal expression of TASK-1 / TASK-3 heteromers in cerebral granule cells." J.Physiol, 554, 64- 77 (Ref 8) and Berg AP et al. (2004), "TWIK-related acid sensitive K + (TASK) heteroneric express motorkines channels TASK-1 (KNCK3) and TASK-3 (KNCK9) subunits." J. Neurosci, 24, 6693-6702 (Ref 9)). These channels produce a K + current that is inhibited by external acidification and subsequent activation of G protein-coupled receptors (Mathie A (2007) "Neuronal two pore domain potassium channels and their regulation by G protein-coupled receptors". J. Physiol, 578, 377-385 (Ref 10)). They are activated by volatile anesthetics (eg halothane, isoflurane) (see Patel AJ, Honore E (2001) "Properties and Modulation of Mammalian 2P Domain K + Channels." Trends Neurosci, 24, 339-346 (Ref. 11). [17] TASK-1 channels are abundant in the brain, adrenal glands, blood vessels, and heart, and based on observations of knock-out mice for TASK-1, it has been shown that noted that the genetic inactivation of TASK-1 leads to aldosterone secretion disorders, arterial hypertension and changes in the electrocardiogram, and pulmonary hypertension can also be observed. it has been found that the use of TASK-1 inhibitors for therapeutic treatment is not an acceptable solution. [18] It has recently been shown that central chemosensitivity in response to hypercapnia persists in TA double mutant mice. SK1 / TASK3 whereas the chemosensitivity of raphe neurons is suppressed (Mulkey et al. (2007) "TASK channel determines pHsensitivity in select respiratory neurons but do not contribute to central respiratory chemosensitivity. J Neurosci, 27, 14049-14058. (Ref 12)).

[19] The term "Sudden Infant Death Syndrome" is used for unexplained cases of infant death, which is the leading cause of death among children aged one month to one year.

In most cases, the cause of death is not clearly identified, but breathing disorders seem to play an important role. For children at risk, a respiratory monitoring system is available, but there is no pharmaceutical treatment today.

[20] At high altitudes, hypoxia leads to an increase in ventilation which leads to a reduction in arterial carbon dioxide (hypocapnia) and results in an increase in arterial pH (respiratory alkalosis). Respiratory alkalosis affects the regulation of breathing and leads to irregular breathing patterns, especially during sleep phases. It has been shown that substances capable of inhibiting respiratory alkalosis, for example acetazolamide, can improve the respiratory patterns and symptoms associated with high altitude by increasing the elimination of bicarbonate by the kidneys, which has as a consequence the decrease of the arterial pH. However, the use of acetazolamide may cause side effects, for example by disrupting electrolyte homeostasis.

[21] In summary, few substances are currently known to treat these respiratory disorders, and those known are either insufficiently effective or have negative side effects that limit their use for treatment over time. In addition, researchers do not have sufficient means to identify new candidate substances for the treatment of respiratory disorders. [22] There is therefore a real need for new ways to identify new candidate molecules for the treatment of respiratory disorders.

Presentation of the invention

[23] The present invention specifically addresses the aforementioned need by providing a method of screening ^• for the identification of candidate molecules for the treatment of respiratory disorders in a mammal, said screening method comprising a step of determining whether the functional activity of a TASK-2 ion channel polypeptide (KCNK5) in the presence of a test molecule is decreased or suppressed with respect to the functional activity of said TASK-2 ion channel in the absence of said test molecule.

[24] If it is determined that the activity of TASK-2 is decreased or suppressed in carrying out the method of the invention, then the test molecule is considered a candidate molecule for the treatment of respiratory disorders in a mammal.

[25] The inventors of the present invention are indeed the first to have demonstrated that the TASK-2 channels, also called KCNK5, are present in certain regions of the respiratory centers of the brain stem and play a surprising role in the breathing. In vitro experiments on a brainstem preparation from neonatal mice show a marked decrease in respiratory activity during anoxia. This decrease is no longer observed on brainstem preparations from mice disabled for the TASK-2 channel. Inhibition or absence of TASK-2 therefore protects against anoxia. These data indicate that the expression of these channels at the level of certain neurons is directly or indirectly associated with chemoreception. [26] The inventors of the present invention have further demonstrated the expression of these TASK-2 channels at certain neurons known to be directly or indirectly associated with chemoreception.

[27] The inventors have furthermore shown that mutant mice invalidated for the TASK-2 inactive channel ("TASK-2 knockout") exhibit a remarkable maintenance of respiration during hypoxia, unlike mouse-type wild that have a strong respiratory depression. These results demonstrate that in the mouse under in vivo condition inactivation of TASK-2 channels profoundly changes the ventilatory response to hypoxia and results in stimulation of respiration.

[28] Interestingly, it has been demonstrated from an in vitro cell culture system that the expression of TASK-2 is reduced after several hours of hypoxia due to hypoxia sensitivity of the promoter. TASK-2 (Brazier et al., (2005) "Cloning of the human TASK-2 (KCNK5) promoter and its regulation by chronic hypoxia." Biochem Biophys Res Commun 336: 1251-1258 (Ref 13)).

[29] Thus, adaptation to hypoxia (for example during a high altitude stay), may include a body defense mechanism by reducing the expression of TASK-2 channels. However, this physiological adaptation probably requires several hours and does not work for an immediate response to short-term hypoxia.

[30] In these cases of short-term hypoxia, the pharmacological inactivation of TASK-2 may make it possible to anticipate the down-regulation defense mechanisms of TASK-2 expression. [31] In addition, unlike the TASK-1 and TASK-3 channels, which are probably also involved in the regulation of respiration due to their expression in multiple groups of chemosensitive respiratory neurons (Sirois et al., 2000). "The TASK-1 two-pore domain K + channel is a molecular substrate for neuronal effects of anesthetic inhalation." J. Neurosci, 20, 6347-6354 (Ref 14)), the expression of TASK-2 in the central nervous system is extremely weak and limited to restricted groups of neurons in the breathing circuits (Reyes R et al (1998) "Cloning and expression of a new pH-sensitive two-pore domain K + channel from human kidney." J. Biol Chem, 273, 30863-30869 (Ref. 15)). Moreover, TASK-2 is not or very weakly expressed in the heart where TASK-1 is very strongly expressed. Thus, the very limited expression of TASK-2 in the central nervous system and its virtual absence in the heart constitutes a huge advantage for the specific inhibition of TASK-2.

[32] The peptide sequence of TASK-2, its organization at the cell membrane level and the coding gene are described in WO 00/27871 (PCTYI B99 / 01886 - belonging to the CNRS, filed on November 9, 1999 and published on May 18, 2000 ). This document also describes the cellular distribution of TASK-2 in humans and in mice, TASK-2 mRNA distribution in the adult kidney, the TASK-2 chromosomal map, the expression of TASK-2 in COS cells transfected and in xenopus oocytes, the sensitivity of TASK-2 currents to pH, as well as the regulation of TASK-2 as a function of pH, as well as the biophysical and pharmacological properties of TASK-2. These elements are usable herein for understanding and practicing the present invention.

[33] "Respiratory disorders" means respiratory deficiencies due to dysfunction of the central nervous system, particularly any respiratory insufficiency related to a malfunction of brain respiratory centers located at the level of the brainstem.

[34] Respiratory disorders include, but are not limited to: sleep apnea syndrome, respiratory forms of sudden infant death syndrome, altitude sickness breathing models, respiratory syndrome Ondine or congenital alveolar hypoventilation syndrome, disorders due to accidental or intentional drug intoxication (eg absorption of barbiturates or opioids), respiratory depression related to general anesthesia, acute respiratory failure and severe hypoxemia.

[35] The term "test molecule" means a molecule tested by the method of the invention to determine whether it decreases or suppresses the activity of the TASK-2 channel or its expression.

[36] The term "candidate molecule" means a molecule identified by the practice of the present invention as decreasing or suppressing the activity of the TASK-2 channel or its expression.

[37] The present invention makes it possible to identify candidate molecules for the treatment of respiratory disorders in a mammal, whether human or animal.

[38] The test molecules for the practice of the present invention may be selected for example randomly in molecular libraries or, for example, from biologically acceptable molecules capable of interacting with an ion channel.

[39] As test molecules capable of decreasing or suppressing the activity of TASK-2, mention may be made, for example, of quinine, quinidine, clofilium, lidocaine, bupivacaine, doxapram and volatile anesthetics such as halothane.

[40] As a candidate molecule, for example, an antibody against TASK-2 may also be mentioned. This antibody can be manufactured by techniques known to those skilled in the art. This antibody may be, for example, a polyclonal, monoclonal, chimeric, or Fab fragment.

[41] By "functional activity" is meant the ability of the potassium channel to drive and control ionic movements across the cell membrane.

[42] According to the invention, the step of determining the functional activity of TASK-2 can be carried out according to one of the methods known to those skilled in the art for determining the activity of an ion channel. It can be as an example of a method such as that described for the KCNK2 channel in WO 05/054866 (PCT / EP / 2004/012823 - Bayer Healthcare

AG, filed November 12, 2004 and published June 16, 2005), a method such as that described for channels TASK-2, TWIK-1, TREK-1 and TASK-1 in WO 00/27871 cited above, or a method such as that described for the TREK-2 channel described in WO

02/00715 (PCT / IB01 / 01436 - CNRS, filed on June 27, 2001 and published on

January 3, 2002).

[43] According to the invention, the step of determining whether the functional activity of the TASK-2 polypeptide is decreased or suppressed may include:

i) contacting a cell expressing a polypeptide

TASK-2 having functional activity, with said test molecule, and ii) a measurement of the functional activity of the TASK-2 polypeptide and / or its expression in the presence of said test molecule.

[44] Examples of implementation of this step are described in particular in the three aforementioned documents.

[45] According to the invention, the cell expressing the TASK-2 polypeptide may express it endogenously or in recombinant form. Methods for expressing this polypeptide by cells for the practice of the present invention are described for example in WO 00/27871 (COS cells or xenopus oocytes) and in the other aforementioned documents.

[46] In general, according to the invention, the functional activity can be measured by one or more parameter (s) chosen from the group comprising: the ion current flowing through the TASK-2 polypeptide, the change of potential membrane of the cell expressing the TASK-2 polypeptide, the change in the intracellular ionic concentration of the cell expressing the TASK-2 polypeptide. Examples of functional activity measurement of a channel usable in the present invention are given for example in each of the three aforementioned documents.

[47] According to the invention, the functional activity can be determined, for example, by measuring the ionic current flowing through the TASK-2 polypeptide, the ion current flowing through the TASK-2 polypeptide possibly being, for example, an efflux of rubidium ions. Preferably, the measurement of the ion current passing through the TASK-2 polypeptide is carried out by measuring the efflux of rubidium ions, this measurement being able to be carried out for example by atomic absorption spectroscopy, for example as indicated in FIG.

GiII et al. (2007) (GiII S et al., 2007 "A cell-based rb (+) - flow assay of the potassium channel kv1.3. Assay Drug Dev Technol 5, 373-80 (Ref 16)).

[48] According to the invention, a test molecule which decreases the activity of TASK-2 preferably by at least 10%, preferably by at least 50%, and even more preferably by 75, 90 or 100% is identified. as a candidate molecule to decrease the activity of TASK-2.

[49] The invention notably allows the identification of candidate molecules for the treatment of disorders. Respiratory selected from the group consisting of sleep apnea syndrome, respiratory forms of sudden infant death syndrome, models of pathological breathing due to altitude.

[50] A step subsequent to the identification of a candidate molecule may be a step of studying the effect of said molecule on respiratory disorders, for example after administration of the candidate molecule to an animal model presenting breathing or put in conditions causing respiratory disorders.

[51] Another aspect of the invention thus relates to the use of a molecule modulating the functional activity of the TASK-2 polypeptide and / or inhibiting its expression, for the preparation of a composition for the treatment of respiratory disorders.

[52] According to a variant of the present invention, it is also possible to screen for molecules which have the capacity to increase, decrease or eliminate the gene expression of TASK-2. to determine, for each molecule tested, the level of mRNA encoding TASK-2 or TASK-2 generated. This determination can be carried out by any method known to those skilled in the art. It can be a qualitative or quantitative method. Methods of Determinations useful in the present invention can be found for example in WO 05/054866, WO 00/27871 and WO 02/00715. The presence of an mRNA encoding TASK-2 or TASK-2 can be determined for example by one of the immunochemical methods known to those skilled in the art, for example by immunoassay, a Western blot technique or immunohistochemistry.

[53] The screening of the present invention can be carried out in a cell-free or cell-free system. Any cell expressing TASK-2 can be used. The TASK-2 polypeptide may be naturally expressed in the cell or may be introduced therein by a genetic recombination technique known to those skilled in the art. Examples of genetic recombination protocols that can be used for the implementation of the present invention are described in the three aforementioned documents.

[54] Thus, according to this variant, a candidate molecule may for example be a sequence complementary to the polynucleotide sequence encoding TASK-2 (or KCNK5) which is capable of blocking the channel transcription.

[55] Expression vectors derived from retroviruses, adenovirus, vaccinia or herpes virus or other bacterial viruses can be used to deliver nucleotide sequences complementary to target organs, tissues or cell populations. Methods known to those skilled in the art can be used for the construction of vectors that express the nucleic acid sequence complementary to the polynucleotides of the TASK-2 coding genes (Scott JK, Smith GP (1990). epitope library. "Science, 249: 386-390 (Ref 17)). Methods known to those skilled in the art can be used to construct expression vectors containing TASK-2 coding sequences as well as transcriptional and translational control elements. These methods include DNA techniques recombinant in vitro, synthetic techniques and in vivo genetic recombination. The three aforementioned documents describe methods that can be used for constructing vectors for implementing the present invention.

[56] Other advantages will become apparent upon reading the following examples given by way of illustration with reference to the appended figures.

Brief Description of the Figures [57] Figure 1 is a photograph of TASK-2 channels present on the ventral surface of the rostral spinal bulb.

[58] Figure 2 is a histogram showing the experimental results of Example 2 below in vitro demonstration of TASK-2 channel intervention in central chemosensitivity mechanisms. In this figure, the ordinate axis represents the frequency of the respiratory rhythmic activity (expressed in number of cycles per minute), and the abscissa axis, the various tests implemented in this example. The white bars correspond to the results obtained on the wild mice and the black bars on the mutant TASK-2 mice.

[59] Figure 3 is a histogram showing the experimental results of Example 3 below in vivo demonstration of intervention of TASK-2 channels in the mechanisms of respiration. In this figure, the ordinate axis represents the ventilatory flow rate per minute ("minute volume" or MV) expressed in ml / min / g of body weight, and the abscissa axis, the various tests implemented in this example. .

[60] Figure 4 shows a recording "patch-clamp" the TASK-2 current (intensity I in picoamperes (pA)) on transfected HEK cells with ¹ I DNA encoding human TASK-2 channel. [61] Figure 5 depicts the location of TASK-2 channels present in the brainstem of an adult mouse. A: whole brain, ventral surface of the brain stem around the facial motor nucleus (VII) and enlargement showing the retrotrapezoid nucleus and the parafacial respiratory group (RTN / pfRG). From B to E: localization of cells expressing TASK-2 on coronal sections. B: mesencephalon, dorsal raphe (DR). C: rostral bridge, lateral nucleus of the superior olive (LSO). D: caudal bridge, ventral surface (RTN / pfRG) and parvocellular reticular nucleus (PCRtA). E: rostral spinal bulb, caudal end of VII. F; Summary diagram showing the distribution of TASK-2 expressing cells on a sagittal section of the brainstem. Other abbreviations: 3N, oculomotor nucleus; 4V, 4 ^th ventricle; 7N, facial nucleus; 1ON, vagal motor dorsal nucleus; 12N, hypoglossal nucleus; Amb, ambiguous core; AP, area postrema; CIC, caudal nucleus of lower colliculus; ILL, intermediate nucleus of lateral lemniscus; IO, lower olive; me5, mesencephalic trigeminal tract; Self, nucleus of the solitary tract.

[62] Figure 6 illustrates the respiratory adaptation measured by the plethysmography technique in response to hypercapnia and short-lived hypoxia in awake mice. This adaptation is similar in wild mice or mice disabled for TASK-2. In this figure, the ordinate axis represents the ventilatory flow rate per minute ("minute volume" or MV) expressed in ml / min / g of body weight, the respiratory rate (RF) and the tidal volume (VT), the x-axis, the different tests implemented in this example as a function of time.

[63] Figures 7A and B illustrate the respiratory adaptation measured by the plethysmography technique in response to long-term hypoxia. The strong respiratory depression observed in wild mice is totally absent in TASK-2-disabled mice. In A, the y-axis represents the tidal volume (VT), the frequency respiratory (RF) and ventilatory flow rate per minute (MV, expressed in ml / min / g); the abscissa axis represents the different tests implemented in this example as a function of time. In B, the same parameters are represented on a longer time scale; TE, expiration time. In C are shown examples of original traces for individual animals at different times (1, 2, 3 and 4 as indicated in B, last trace of the bottom). Immediately after switching to a hypoxic atmosphere, wild-type and mutant mice show increased respiration (point 2). This response is followed by depression (point 3) observable only in wild mice. After 12 hours of hypoxia (point 4) all mice show similar ventilation.

[64] Figure 8 is a histogram showing the expression of messenger RNAs of TASK-1, TASK-2 and erythropoietin (EPO) in kidney and brainstem in mice maintained for 12 hours in an environment hypoxic. These are quantitative PCR results (8 mice per group).

[65] Figure 9A illustrates the expression of TASK-2 in the area around the facial motor nucleus (retrotrapezoid nucleus and parafacial respiratory group) on a whole mouse brain aged 1 day. This area is preserved in the block recording technique. B: schematic representation of the block preparation where the structures of the medulla oblongata and the most caudal part of the bridge are preserved. An electrode placed on the cervical root C4 makes it possible to record the activity of the phrenic nerve representing the respiratory activity. C: example of recording trace (C4) and integration (JC4) inspiratory puffs from which the amplitude, the surface and the duration of the inspiratory (Tl) and expiratory (TE) phases are measured. D: examples of respiratory activity (JC4) under normoxic (control) and anoxic conditions obtained on block preparations of wild mice (task2 + / +) or mutants (task2 - / -). E: Histogram of respiratory frequencies (RF) under control conditions, anoxia, respiratory or metabolic acidosis and alkalosis for wild (WT), heterozygous (task2 +/-) or homozygous (task2 - / -) mice.

EXAMPLES

Example 1 Demonstration of the Expression of TASK-2 in the Neuronal Cells of the Brainstem

[66] The experiments in this example demonstrate the presence of TASK-2 channels in a major nerve structure belonging to the respiratory centers of the brainstem. These experiments were performed on adult heterozygous mutant mice (TASK-2 ^{+ / -} ), and the TASK-2 gene, which is invalidated, is replaced by a coding sequence for an enzyme called beta-galactosidase. Cells that normally express the TASK-2 channels can be easily identified by the standard histochemical revelation technique using I ¹ X-GaI as a substrate that turns blue when hydrolyzed.

[67] After a para-formaldehyde fixation protocol, the nerve tissue is removed and the X-GAL revelation technique is performed on 30 μm thick brainstem cross sections obtained using a cryostat. . At the end of this reaction, the cells expressing the TASK-2 channels are stained blue (arrow in FIG. 1).

[68] The results obtained show the presence of expressing cells

TASK-2 in discrete areas of the brainstem. By way of example, FIG. 1 illustrates the TASK-2 channels present on the ventral surface of the rostral spinal bulb. This is a cross-section illustrating the presence of X-gal labeling in neurons of the ventral surface of the rostral spinal bulb (retotrapezoidal nucleus, RTN).

[69] As shown by numerous studies, this zone plays a fundamental role in central chemosensitivity, that is to say, in the processes of adaptation of ventilation in response to a chemical variation of the internal environment (blood, cerebrospinal fluid or CSF). Thanks to the unique location of these channels in areas as soon as involved in the control of respiration, it is therefore possible to specifically modulate respiration by a method acting on TASK-2.

Example 2: In Vitro Demonstration of the Intervention of TASK-2 Channels in the Central Mechanisms of Respiratory Adaptation

[70] In this example, the inventors used "wild-type" mice (active TASK-2 channels) and mutant TASK-2 mice that do not express active TASK-2 channels ("TASK-2 ^"; ). These mice were initially produced by the W. SKARNES laboratory in San Francisco in the public domain. They were backcrossed in the C57BI / 6J genetic background and then reared and produced within the inventors' laboratories.

[71] The experiments in this example demonstrate changes in respiratory activity during anoxia. These experiments were performed on isolated brainstem preparation in vitro from 1 to 3 day old mice ("en bloc" preparation).

[72] The brainstem of wild-type or mutant mice (TASK-2 ^"Λ ) is rapidly dissected in ice, isolated from adjacent tissues, and placed in artificial (ARF) equilibrated CFL (95% O ₂ , 5). % CO ₂ ). The ventral root of the C4 spinal segment is at the origin of the phrenic nerve which innervates the diaphragm. This nerve root is sucked inside a glass electrode to collect the spontaneous rhythmic activity generated spontaneously by the preparation. The electrical activity of the root C4 is filtered, amplified, visualized and stored on computer to analyze a posteriori the respiratory parameters (frequency, amplitude, duration). The tests consisted in modifying the quantity of dissolved gases in the LCRa or its chemical composition and comparing the respiratory activities.

[73] Figure 2 is a histogram showing the experimental results of this example ("bulk" preparation: respiratory rate and electrical activity of the phrenic nerve C4 root recorded in wild-type mice and task2 - / -). Control: Artificial CSF balanced 95% O ₂ , 5% CO ₂ . Anoxia: 95% N ₂ , 5% CO ₂ . The ordinate axis represents the frequency of the respiratory rhythmic activity (expressed in number of cycles per minute), and the abscissa axis, the various tests implemented in this example. The white bars correspond to the results obtained on the wild mice and the black bars on the mutant TASK-2 mice.

[74] The results obtained show that in control conditions (95% O ₂ , 5% CO ₂ ), the respiratory rate does not differ between wild animals and TASK-2 mutants. In anoxia (LCRa equilibrated in a gas devoid of oxygen: 95% N ₂ , 5% CO ₂ ), the respiratory rate of wild animals falls by about 40%. This classic response, called hypoxic respiratory depression, is no longer observed in TASK-2 ^'Λ mutant mice (statistical comparison, ** ^* : p <0.001).

[75] These experiments demonstrate that the in vitro respiratory activity of anoxic mice is significantly more common after invalidation of the gene coding for TASK-2 channels. Thus, under conditions of respiratory depression, it is conceivable to restore the central respiratory activity by a method acting on TASK-2.

Example 3 In Vivo Demonstration of the Intervention of TASK-2 Channels in the Mechanisms of Respiration

[76] In this example, the inventors also used "wild-type" mice and mutant TASK-2 mice. [77] The respiration of the mice was measured with a plethysmographic apparatus (EMKA technologies, France).

[78] Figure 3 is a histogram showing the experimental results of this example illustrating the ventilatory responses of wild-type and TASK-2 - / - mice on prolonged exposure to hypoxia (8% O2). In this figure, the ordinate axis represents the ventilatory flow rate per minute ("minute volume" or MV, expressed in ml / min / g), and the abscissa axis, the various tests implemented in this example. The white bars correspond to the results obtained on the wild mice and the black bars on the mutant TASK-2 mice.

[79] Different conditions of exposure to oxygen were tested. The ventilation rate per minute (ml / min / g) was measured during the control (con) (21% oxygen) and during hypoxia at 8% oxygen corresponding to an altitude of 6500m above sea level. Hypoxia was tested for 1-6 hours.

[80] Hypoxia caused a transient increase in respiration in all animals (hyperventilation secondary to peripheral chemoreceptor stimulation) (not shown). Secondarily, prolonged exposure to hypoxia resulted in a reduction in respiratory movements or depression that is only seen in wild-type mice. The reduction of arterial carbon dioxide subsequent to initial hyperventilation was an important factor in wild type mice (in white on the histogram of Figure 1) to reduce their respiration.

[81] The invalidated animals for the TASK2 channel have on the contrary a sustained ventilation. throughout the duration of hypoxia.

[82] It is clear that following the inactivation of TASK-2 channels, hypoxia is a powerful stimulus for respiration, including under conditions of hypocapnia (respiratory alkalosis). It is likely that respiratory alkalosis is no longer able to activate TASK-2 canals of the central nervous system.

[83] The results of these experiments clearly demonstrate that a "TASK-2 knockout" mouse exhibits increased respiration during hypoxia compared to a wild-type mouse. [84] Thus, inhibition of TASK-2 appears as a means of treating breathing disorders.

[85] In conclusion, it appears that the inactivation of potassium channels TASK-2 as a means to stimulate breathing under hypoxic conditions is a new strategy that differs from the therapeutic approaches used in the prior art.

Example 4: Localization of the expression of TASK-2 in the brainstem

[86] The mice and conditions used in this experiment were identical to those of Example 1.

[87] The vector used for the generation of TASK-2 mice ^";" contains a gene encoding beta-galactosidase as described in Mitchell KJ et al. (2001) "Functional analysis of secreted and transmembrane proteins critical to mouse development". Nat Genet, 28, 241-249 (Ref 18). The expression of TASK-2 cells was visualized using a TASK-2 promoter activity in directing the TASK-2 mice ^{"/ +.} Surprisingly, specific labeling with X-gal Ie is restricted few regions of the brainstem. No cell expressing TASK-2 has found in other areas of the brain.

[88] The marking present at the level of the medulla oblongata is limited to the ventrolateral part of the ventral surface.

[89] The cells expressing TASK-2 form a bilateral column extending over 1.5 mm, from 500-700 μm in front of the obex to the rostral pole of the facial motor nucleus (VII). These cells form clusters located in the marginal zone on the surface of the brainstem and in the tissue parenchyma between 100 to 300 μm deep (see Figure 5).

This region corresponds to the peri-facial region comprising the retrotrapezoid nucleus and the parafacial respiratory group (RTN / pfRG).

[90] In the annular protuberance (or bridge), cells expressing TASK-2 are observed in the lateral part of the superior olive (Figure 5C), the parvocellular reticular nucleus (Figure 5D), and the raphe nuclei dorsal and intermediate lateral lemniscus. Partial staining is detected in the lower caudal colliculus.

[91] No markings were detected in the cervical spinal cord corresponding to the phrenic motor nucleus.

[92] As demonstrated in Example 1 and in this experiment, the TASK-2 channels are present on the ventral surface of the rostral spinal bulb, more particularly in the region corresponding to the retrotrapezoid nucleus (RTN) and the parafacial respiratory group (pfRG). ).

[93] The results obtained show the presence of cells expressing TASK-2 in discrete areas of the brainstem. [94] Due to the unique localization of these channels in key areas involved in the control of respiration, it is therefore possible to specifically modulate respiration by a method acting on TASK-2.

Example 5 Effect of hypoxia and hypercapnia on the respiration of mice under in vivo condition

[95] The mice used in this experiment are identical to that of Example 2 above.

[96] For this experiment, 10 TASK-2 ^{+ / +} mice and 7 TASK-2 ^~ ' ^~ mice were used.

[97] Central and peripheral chemoreceptors contribute to the adaptation of respiration by detecting changes in pH and blood gases (Vizek M et al (1987) "Biphasic ventilatory response of adult cats to sustained hypoxia has central origin" J Appl Physiol, 63, 1658-

1664. (Ref 19) The peripheral chemoreceptor cells located in the carotid bodies specifically control the rapid increase of ventilation in response to hypoxia. [98] Respiratory adaptation in response to short-term hypoxia or hypercapnia was studied in wild-type and mutant mice (TASK-2 ^";" ).

[99] The respiration of the mice was measured with a plethysmographic apparatus (EMKA technologies, France).

[100] Under normal conditions (21% oxygen), respiratory parameters were identical in TASK-2 ^'A mice and wild-type mice.

[101] Both types of mice show a similar increase in respiration in response to a 5% CO2 environment

(hypercapnia) (Figure 6A). A decrease in oxygen concentration 21% to 9% also induced an identical increase in respiration in both types of mice (Figure 6B). [102] Hypoxia was transient and the maximum increase in breathing occurred after a few minutes.

[103] This experiment shows normal respiratory adaptation in response to short-term hypoxia, which is related to the oxygen sensitivity of peripheral chemotopic cells in carotid bodies. The response to hypercapnia is also unchanged in the mutant mice.

Example 6 Effect of Prolonged Hypoxia on the Transcription of TASK-2

[104] The response to prolonged hypoxia has also been studied. This response was measured at 8% oxygen over a period of several hours.

[105] The mice used in this experiment are identical to those of the previous example. [106] For this experiment, 8 TASK-2 ^{+ / +} mice and 7 TASK-2 ^{'7 "} mice were used.

[107] During a 2-hour hypoxia, a decrease in respiration was observed in control mice (Figure 7A). A hypoxic depression of the ventilatory flow rate (minute volume or MV, expressed in ml / min / g) was caused in particular by a decrease in the respiratory rate (RF). This induced respiratory depression in response to chronic hypoxia was absent in mice TASK-2 ^{'' ".}

[108] This example shows that the initial response to hypoxia is normal in TASK-2 ^"7" mice. This response corresponds to the stimulation of peripheral chemoreceptors (Takakura AC et al., 2006) "Peripheral Chemoreceptor Inputs to Retrotrapezoid Nucleus (RTN) CO2-sensitive neurons in rats ". J. Appl Physiol, 63, 1658-1664 (Ref. 20). Instead, the central respiratory depression induced by chronic hypoxia is totally absent in the TASK-2 mice ^{~ ~.}

[109] This experience clearly indicates that the TASK-2 channel is a critical element for medullary hypoxia sensitivity.

[11O] As demonstrated in this experiment and in previous Example 2, inactivation of TASK-2 potassium channels is therefore a means for inhibiting respiratory depression and stimulating respiration under hypoxic conditions. This inactivation is therefore a new strategy that differs from the therapeutic approaches used in the prior art.

[111] Respiratory adaptation to chronic hypoxia as described in Powell et al (Powell FL et al., 1998) "Time domain of the hypoxia ventilatory response." Respir Physiol, 112, 123-134 (1998). Ref 21)) was studied under the following conditions: 10% oxygen corresponding to an altitude of 5300m for 20 hours. [112] During the first three to four hours of hypoxia, the control mice showed a strong depression of the respiratory rate with an extension of the expiration time and a reduction of the respiratory rate. This phase was followed by an adaptation phase characterized by a decrease in the expiration time which returned to normal values after 10 to 12 hours (Figure 8 B and C). [113] During all the time of hypoxia, the respiratory parameters remained unchanged for the TASK-2 ^' mice. ^" These mice thus have a respiratory phenotype similar to that of the control mice after adaptation.

[114] In addition, a measure of the expression of TASK-2 was performed. [115] The mice were submitted in an environment with 10% oxygen for 24 hours, the measurement of TASK-2 messenger RNA expression (as well as TASK-1 and erythropoietin in controls) was then performed. [116] RNAs were isolated from the kidney and brainstem of mice using the Mini RNeasy Kit (Qiagen). For reverse transcription, a reverse transcriptase (Promega) was used according to the protocol provided by the manufacturer. A real time polymerase chain reaction was performed with the LightCycler system (Roche) using SYBR Green PCR Kit (Qiagen). The transcription of the TASK-2, TASK-1 and erythropoietin genes was measured and normalized with respect to the expression of beta-actin.

[117] The primers used and the conditions for performing the real time polymerization reaction were as follows. Primers for beta-actin: sense primer, 5 'CCACCGATCCACACAGAGTACTT 3' (SEQ ID NO: 1); Antisense primer, 5 'GACAGGATGCAGAAGGAGATTACTG 3 (SEQ ID NO: 2)'. Primers for erythropoietin: sense primer, 5 'AGAATGGAGGTGGAAGAACAG 3' (SEQ ID NO: 3); Antisense primer, 5 'TGTCTATATGAAGCTGAAGGGT 3' (SEQ ID NO: 4). Primers for TASK-2: sense primer, 5 'GCTTTGGGGACTTTGTGG 3' (SEQ ID NO: 5); Antisense primer, 5 'AAAGAGGGACAGCCAAGC 3' (SEQ ID NO: 6).

[118] The hybridization temperature of the primers was 55 ^° C.

[119] The results showed that the amount of mRNA encoding TASK-2 remained unchanged in the kidney and brainstem during hypoxia. (Figure 8 A and B).

[120] Transcription of the TASK-2 gene therefore remains unchanged during prolonged hypoxia. Thus, the potassium channel TASK-2 is therefore a new target for inhibiting respiratory depression and stimulating breathing under hypoxic conditions. As demonstrated previously, the inactivation of TASK-2 is therefore a new strategy which differs from the therapeutic approaches used in the prior art. Example 7: In Vitro Demonstration of the Intervention of TASK-2 Channels in the Central Adaptation Mechanisms of Respiration

[121] This experiment was performed under conditions and on mice identical to those of Example 2.

[122] This experiment demonstrates changes in respiratory activity during anoxia, acidosis and alkalosis. [123] A bulk preparation of neonatal mouse brain trunks that include central chemoreceptors was performed (Figure 9 AC). [124] The bulk preparation was carried out on neonates from 1 to 3 days. Each mouse was anesthetized with halothane. The brainstem and cervical spinal cord were isolated and transferred to a recording chamber, the ventral part upwards, and perfused with an artificial cerebrospinal fluid at a flow rate of 4 ml. min ^"1 .

[125] A measurement of respiratory rate, duration of puffs, amplitude and surface area of puffs was performed.

[126] For this experiment, 10 TASK-2 ^{+ / +} mice, 8 mice TASK-2 ^{+ / "and} 7 TASK-2 ^{mice" "were} used.

[127] The basic respiratory rate was identical in all individuals under normal conditions. In control mice, metabolic or respiratory acidosis induced an increase in respiratory rate (approximately + 40%), while alkalosis and anoxia caused a significant decrease in respiratory rate (approximately - 40%).

[128] TASK-2 - / - mice showed decreases in respiratory rate similar to the change in pH. However, this response to anoxia was completely suppressed in TASK-2 - / - mice (Figure 9 D and E). [129] This experiment shows that there exists in TASK-2 - / - mice a response to hypoxia which results in an increase in the duration of the inspirations without a change in the respiratory rate. [130] Thus, the potassium channel TASK-2 is therefore a new target for inhibiting respiratory depression and stimulating breathing under hypoxic conditions. 2

Example 8: Implementation of a Screen According to the Present Invention

[131] The distribution of TASK-2 in an adult human being, its chromosomal mapping, its biophysical and pharmacological properties, its regulation by external pH have been studied according to WO 00/27871.

[132] The following screen has been implemented. Human Embryonic Kidney cells (HEK) were transfected with a plasmid containing the DNA complementary to the human TASK-2 channel gene (pRES-CD8-hTASK2) by the lipofectamine method. After 24 hours of culture, the TASK-2 currents are recorded by the voltage-clamp method (patch-clamp in whole cell configuration). The currents are activated by voltage jumps from a rest potential at -95V up to variable values between -80 and + 45mV.

[133] The currents are recorded in whole cell configuration using an EPC-10 amplifier (HEKA). The "patch" pipette contains a 95 K-gluconate solution, 30 mM KCl, 4.8 mM Na ₂ HPO ₄ , 1.2 mM

NaH ₂ PO ₄ , 5 mM glucose, 2.38 mM MgCl ₂ , 0.726 mM CaCl ₂ , 1 mM EGTA,

3 mM ATP, pH 7.2. The external solution is a RINGER solution: 145 mM

NaCl, 0.4 mM KH ₂ PO ₄ , 1.6 mM K ₂ HPO ₄ , 5 mM glucose, 1 mM MgCl ₂ , 1, 3 mM CaCl ₂ , 5 mM HEPES, pH 7.4. [134] Different drugs can be applied at different concentrations by infusion of the external bath and their effects are evaluated by the change of currents with respect to the control conditions.

[135] In the example of Figure 4, we see that the application of Doxapram 0.9 mM in the bath caused a 25% decrease in the current evoked by potential jumps.

List of references

(Ref 1) Richter DW, Spyer KM (2001) "Studying rhythmogenesis of breathing: comparison of in vivo and in vitro models". Trends

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(Ref 2) Feldman JL, DeI Negro (2006) "Looking for inspiration: new perspectives on respiratory rhythm". Nat Rev Neurosci, 7, 232-242. (Ref 3) Feldman JL et al. (2003) "Breathing: rhythmicity, plasticity, chemosensivity". Annu Rev Neurosci, 26, 239-266.

(Ref 4) Severson et al. (2003) "Midbrainserotonergic neurons are central pH chemoreceptors". Nat Neurosci, 6, 1139-1140.

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(Ref 6) Goldstein SA et al. (2005), "International Union Pharmacology. LV. Nomemclature and molecular relationship of two-pot potassium channels ". Pharmacol Rev., 57, 527-540. (Ref 7) Czirjak G, Enyedi P (2001), "Formation of functionnal heterodimers between the TASK-1 and TASK-3 two-pore-domain potassium channel subunits". J Biol Chem, 277, 5426-5432.

(Ref 8) Kang D et al. (2004), "Functionnal expression of TASK-1 / TASK-3 heteromers in cerebral granule cells". J.Physiol, 554,

64-77.

(Ref 9) Berg AP et al. (2004), "TWIK-related acid sensitive K + (TASK) heteromeric express motorkines channels TASK-1 (KNCK3) and TASK-3 (KNCK9) subunits." J. Neurosci, 24, 6693-6702. (Ref 10) Mathie A (2007) "Neuronal two pore domain potassium channels and their regulation by G protein coupled receptors". J. Physiol, 578, 377-385.

(Ref 11) Patel AJ, Honore E (2001) "Properties and modulation of mammalian 2P domain K + channels". Trends Neurosci, 24,

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(Ref 12) Mulkey et al. (2007) "TASK channel determines pHsensitivity in selective neurons but does not contribute to central respiratory chemosensitivity". J Neurosci, 27, 14049-14058. (Ref 13) ^" Brazier et al (2005)" Cloning of the human TASK-2 (KCNK5) promoter and its regulation by chronic hypoxia. "Biochem Biophys Res Commun 336: 1251-1258.

(Ref 14) Sirois et al. (2000) "The TASK-1 two-pore domain K + channel is a molecular substrate for neuronal effects of anesthetic inhalation". J. Neurosci, 20, 6347-6354.

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Claims

1. Use of a screening method for identifying candidate molecules for the treatment of respiratory disorders due to central nervous system dysfunction in a mammal, said screening method comprising a step of determining whether the functional activity of the a TASK-2 ion channel polypeptide in the presence of a test molecule is decreased or suppressed with respect to the functional activity of said TASK-2 ion channel in the absence of said test molecule, the test molecule being considered as a molecule candidate when it decreases or removes the said functional activity. Use according to claim 1, wherein the step of determining whether the functional activity of the TASK-2 polypeptide is decreased or suppressed comprises: i) bringing into contact a cell expressing a TASK-2 polypeptide having a functional activity, with said test molecule, and ii) a measurement of the functional activity of the TASK-2 polypeptide and / or its expression in the presence of said test molecule. The use of claim 2, wherein said cell expresses the TASK-2 polypeptide endogenously. The use of claim 3, wherein said cell expresses the TASK-2 polypeptide in recombinant form. The use according to any one of claims 1 to 4, wherein said functional activity is determined by a measurement selected from the group consisting of: a measurement of the ion current flowing through the TASK-2 polypeptide, a measure of the change in potential membrane of the cell expressing the TASK-2 polypeptide, a measure of the change in intracellular ionic concentration of the cell expressing the TASK-2 polypeptide. The use of claim 5, wherein said functional activity is determined by measuring the ion current flowing through the TASK-2 polypeptide, and wherein the ion current flowing through the TASK-2 polypeptide is an efflux of rubidium ions. 7. Use according to any one of claims 1 to 6, for the identification of molecules usable for the treatment of respiratory disorders due to central nervous system dysfunction selected from the group consisting of sleep apnea syndrome, respiratory forms of sudden infant death syndrome, breathing patterns pathological due to altitude.