Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
  • G01N33/6872—Intracellular protein regulatory factors and their receptors, e.g. including ion channels

Definitions

• the present invention relates to the treatment and prevention of pain. More particularly, the present invention demonstrates the involvement of potassium channels K2P in the analgesic effect of morphine. The present invention thus provides a screening method for the identification of analgesics.
• Pain can be defined as an unpleasant sensory and emotional experience related to existing or potential tissue injury or described in terms of such injury.
• the management of the pain perceived by the patient results from the interaction between the phenomenon that generates the pain, the capacity of the individual to integrate it, and the response of the professionals in situation to recognize it and to treat it.
• Pain is also the most common symptom related to cancer. It concerns 30 to 50% of cancer patients in all stages, and 65 to 90% of patients with advanced stages. The vast majority of patients interviewed suffering from chronic pain report receiving medical analgesic treatment. The most frequently prescribed prescription drugs are anti-inflammatories and weak opioids, while strong opioids are at a frequency of about 5%. For over-the-counter medicines, drugs mentioned are anti-inflammatories and paracetamol, and to a lesser extent weak opioids in countries where they can be obtained without prescription (Breivik et al., 2006 Eur J Pain 10: 287-333).
• the opioid analgesics include morphine and all hemi-synthetic or synthetic derivatives of this alkaloid. Morphine remains the reference product in the treatment of intense pain of nociception excess, including post-operative pain and cancer pain. Opioids have analgesic effects but also psychodysleptics, respiratory depressants, emetic, cardiovascular, on the smooth musculature and on the immune system.
• the adverse effects associated with the use of opioids are characterized by almost systematic constipation, nausea and vomiting in more than half of the patients, frequent sedation, the risk of respiratory depression requiring monitoring, and hallucinations or confusion at home. the elderly who, although rare, require a reduction in doses. In addition, constipating effects are a major handicap, especially for the elderly and oncology.
• Morphine produces its effects via the activation of opioid receptors, of which three subtypes are known ( ⁇ , ⁇ and ⁇ ).
• Therapeutic (analgesic) and undesirable effects preferentially involve activation of the ⁇ receptor (Roy et al., 1998 Brain Res Mol Brain Res 56: 281-3). The desire to dissociate these effects therefore requires working downstream of this receiver.
• opioid analgesics with no action on ⁇ receptors or peripheral ⁇ -receptor antagonists which, however, oppose only constipating effects, and not effects respiratory depressants.
• the former may have less analgesic efficacy than ⁇ receptor agonists, and the latter must be co-prescribed with opioids.
• analgesic as effective as morphine, but devoid of constipating effects and respiratory depressant, two major adverse effects in terms of frequency and severity, respectively.
• the present invention demonstrates, for the first time, the involvement of potassium channels K2P, and more particularly TREK-1 and TRAAK, in the analgesic effect of morphine.
• K2P potassium channels
• TREK-1 and TRAAK the deletion of the gene encoding TREK-1 or TRAAK leads to the suppression of the analgesic effect of the opiate.
• the deletion of the gene encoding TREK-1 or TRAAK does not reduce the constipating effect and / or respiratory depressant of morphine.
• the TREK-1 and TRAAK channels are therefore necessary for the analgesic action but do not participate in these undesirable effects.
• the K2P potassium channels of the TREK / TRAAK family can therefore be used as targets to screen new analgesics as effective as morphine with a better benefit / risk ratio.
• the present invention thus provides a screening method for isolating new effective and well tolerated analgesics, and therefore allows the synthesis, validation, development and formulation of the analgesics thus selected.
• K2P potassium channels as targets
• K2P potassium channels of the TREK / TRAAK family are useful as targets in screening to identify antalgic compounds.
• the analgesic compounds thus identified are characterized in that they have no constipating effect and / or respiratory depressant.
• the invention therefore relates to a screening method for identifying an analgesic compound comprising the following steps:
• the method according to the invention may comprise the additional step of selecting an analgesic compound devoid of constipating effect and / or respiratory depressant.
• analgesic compound is meant here a compound capable of reducing or abolishing pain.
• potassium channel K2P is meant here the UniProt family No. TC 1 .A.1 .8 (also called “two pore domain potassium channel family”).
• the potassium channel K2P according to the invention is chosen from the members of the TREK / TRAAK family, more particularly from TREK-1, TREK-2 and TRAAK.
• TREK-1 channel is meant here a potassium channel K2P comprising at least one subunit whose sequence is shown in Swiss-Prot entry No. O95069 (SEQ ID NO: 1) or whose sequence is d from this sequence.
• the TREK-1 channel is a channel consisting of two subunits of sequences chosen from the sequence SEQ ID NO: 1 and its derived sequences.
• the channel TREK-1 may for example be a homodimeric channel consisting of two subunits of sequence SEQ ID NO: 1.
• TREK-2 channel is meant here a potassium channel K2P comprising at least one subunit whose sequence is shown in Swiss-Prot entry No. P57789 (SEQ ID NO: 2) or whose sequence is d from this sequence.
• the TREK-2 channel is a channel consisting of two subunits of sequences selected from the sequence SEQ ID NO: 2 and its derived sequences.
• the channel TREK-2 may for example be a homodimeric channel consisting of two subunits of sequence SEQ ID NO: 2.
• TRAAK channel is meant here a potassium channel K2P comprising at least one subunit whose sequence is shown in the Swiss-Prot entry No. Q9NYG8 (SEQ ID NO: 3) or whose sequence is derived from this sequence.
• the channel TRAAK is a channel consisting of two subunits of sequences chosen from the sequence SEQ ID NO: 3 and its derived sequences.
• the TRAAK channel may for example be a homodimeric channel consisting of two subunits of sequence SEQ ID NO: 3.
• “Derived sequences” include splice variants, allelic variants, and homologous sequences in non-human mammalian species.
• the derived sequences include especially sequences at least 50, 60, 70, 80, 85, 90, 95 or 99% identical to one of the sequences SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3.
• amino acid sequence at least 95% (for example) identical to a reference sequence is meant a sequence identical to the reference sequence except that this sequence may comprise up to five mutations (substitutions , deletions and / or insertions) for each 100 amino acid portion of the reference sequence.
• substitutions substitutions , deletions and / or insertions
• sequences 95% identical to the reference sequence are two examples of sequences 95% identical to the reference sequence.
• the percentage of identity is usually determined using sequence analysis software.
• the amino acid sequences to be compared are aligned to obtain the maximum degree of identity. To this end, it may be necessary to artificially introduce gaps in the sequence.
• the degree of identity is established by recording all the positions for which the amino acids of the two compared sequences are identical, relative to the total number of positions.
• the "needle” program which uses the global alignment algorithm “Needleman-Wunsch” to find the optimal alignment (with gaps) of two sequences over their entire length, can for example be used. This program is available on the ebi.ac.uk website.
• the derived sequences may differ from the reference sequence by substitution, deletion and / or insertion of one or more amino acids, and this at positions such that these modifications do not significantly affect the biological activity of the peptides.
• the substitutions may in particular correspond to conservative substitutions.
• the sequence of the derivatives differs from the sequence SEQ ID NO: 1, 2 or 3 solely by the presence of conservative substitutions.
• Conservative substitutions are amino acid substitutions of the same class, such as amino acid substitutions to uncharged side chains (such as asparagine, glutamine, serine, cysteine, and tyrosine), amino acids with basic side chains (such as lysine, arginine, and histidine), amino acids with acid side chains (such as aspartic acid and glutamic acid), chain amino acids apolar side effects (such as alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine and tryptophan).
• K2P potassium channel activator is meant herein a compound capable of increasing the biological activity of a potassium channel K2P.
• the activators according to the invention do not have the effect of activating the opioid receptors. As a result, they are devoid of constipating effect and / or respiratory depressant.
• the activators act directly on the potassium channel K2P, that is to say that they interact directly with said channel.
• Activators can also activate the K2P potassium channel via a signaling cascade.
• the activators necessarily act downstream of the opioid receptors, that is to say they modulate the activity of a component of the cascade which is situated upstream of the potassium channel K2P, but downstream of the opioid receptors.
• K2P potassium channel activator acts downstream of the opioid receptors
• K2P potassium channel activity in the presence and absence of an opioid receptor inhibitor such as, for example, naloxone.
• K2P potassic activity is the same in the presence and absence of the opioid receptor inhibitor indicates that the activator acts downstream of the opioid receptors.
• a cell expressing a TREK-1 channel for example a DRG neuron
• an increase in the current in the presence of the candidate compound indicates that the candidate compound is an activator of the TREK-1 channel.
• Said stream may for example be induced by a compound such as arachidonic acid or by intracellular acidification.
• the activators of the TREK-1 channel are capable of producing an increase in the intensity of dorsal spinal ganglion neuron current from wild mice when the intensity of the current is evaluated by the so-called patch technique. clamp in whole cell condition "under the conditions described in Alloui et al. (EMBO J. 2006 25: 2368-76).
• analgesic compounds thus identified are characterized in that they have no constipating effect and / or respiratory depressant. In addition, they may also be devoid of other undesirable effects.
• compound having an "analgesic effect” is meant here a compound capable of reducing or abolishing pain.
• the analgesic effect of a compound can be measured by any method well known to those skilled in the art, for example by the von Frey mechanical susceptibility test or by the immersion test of the tail in the body. hot water (see Examples 1 and 2).
• compound having a “constipating effect” is meant here a compound capable of decreasing defecation.
• the constipating effect of a compound can be measured by any method well known to those skilled in the art, for example using a comprehensive method of collecting faeces (see Examples 1 and 4).
• compound having a "respiratory depressant effect” is meant herein a compound capable of decreasing the respiratory rate.
• the respiratory depressant effect of a compound can be measured by any method well known to those skilled in the art, for example by measuring the respiratory rate by means of a barometric plethysmograph (see Examples 1 and 5).
• devoid of constipating effect and / or respiratory depressant means a compound in the presence of which the defecation and / or respiratory rate is greater than or equal to 70%, 80%, 90% or 95% of the value observed in the absence of said compound. Preferably, defecation and / or respiratory rate is not statistically significantly decreased in the presence of said compound.
• Constipating effects and / or respiratory depressant being dose-dependent, the absence of constipating effect and / or respiratory depressant of an analgesic compound is preferably evaluated at an equianalgesic dose to that at which morphine produces an analgesic effect.
• the candidate compounds may for example correspond to natural ligands of a potassium channel. K2P, chemical molecules, aptamers, peptides and antibodies.
• the candidate compounds are small chemical molecules ("small molecules").
• the invention provides a screening method for the identification of an analgesic compound comprising the following steps:
• This method may further comprise the step of measuring the analgesic effect of said candidate compound which activates said potassium channel K2P, and / or the step of measuring the constipating effect and / or respiratory depressant of said compound activating said potassium channel K2P.
• the method according to the invention may comprise the additional step of selecting an analgesic compound devoid of constipating effect and / or respiratory depressant.
• K2P potassium channel as a target for identifying an analgesic compound during screening of candidate compounds is another object of the invention. More specifically, such use is directed to the identification of potassium channel activators K2P, which have an analgesic effect.
• the screening methods according to the invention can be carried out in vitro or in vivo. When these methods involve experiments on model animals such as rats or mice, these animals are sacrificed at the end of these methods. Thus, these methods may include an additional step of sacrificing model animals that may have been used.
• the activators of the TREK-1 and TRAAK channels are analgesics without these effects adverse effects of morphine.
• the invention thus relates to an activator of a K2P potassium channel of the TREK / TRAAK family for use in the treatment or prevention of pain. Such activators are particularly advantageous for treating or preventing pain since they are devoid of constipating effect and / or respiratory depressant.
• the pain can be of various natures. It can be acute or chronic. For example, it may be postoperative pain, pain associated with a hyperalgesic attack, pain associated with cancer, osteoarticular pain, visceral pain or pain. neuropathic, said pain occurring at any stage of life. In a preferred embodiment, it is a chronic pain, and more particularly a chronic pain syndrome such as fibromyalgia, irritable bowel or sine materia pain. In another preferred embodiment, it is intestinal, stomach or lung pain, whether or not linked to cancer, such as pain associated with colorectal cancer, colon cancer, cancer stomach, ulcerative colitis, inflammatory bowel disease (abbreviated as "IBD” or "IBD”), irritable bowel, lung cancer, or asthma.
• IBD inflammatory bowel disease
• the pain can be treated at any stage.
• treatment is meant curative treatment (aimed at at least relieving, curbing or stopping pain).
• prevention means prophylactic treatment (aimed at reducing the risk of developing pain).
• Activators may, for example, correspond to natural K2P potassium channel ligands, chemical molecules, aptamers, peptides, or antibodies.
• the activator according to the invention may, for example, correspond to one of the potassium channel activators K2P already known in the art.
• the channels of the TREK / TRAAK family are reversibly open by lysophospholipids which have large polar heads (lysophosphatidylcholine, lysophosphatidylinositol) and polyunsaturated fatty acids (linolenic acid, arachidonic acid) (Fink et al., 1998 EMBO J. 17: 3297-308).
• esters of caffeic acid especially cinnamyl 1 -3,4-dihydroxy- ⁇ -cyanocinnamate (CDC) and caffeic acid phenethyl ester (CAPE), increase the TREK-1 current on adreno-fascicular bovine cells (Danthi et al., 2004 Mol Pharmacol 65: 599-610).
• fenamates such as flufenamic acid, niflumic acid and mefenamic acid activate the TREK-1, TREK-2 and TRAAK channels in heterologous expression systems (Takahira et al., 2005 Pflugers Arch. : 474-8).
• the compounds specifically mentioned above are examples of activator according to the invention.
• the activator according to the invention may for example be chosen from a lysophospholipid, a polyunsaturated fatty acid, a caffeic acid ester or a fenamate.
• K2P potassium channel activators can be isolated by the screening methods described above in the section titled "Using K2P Potassium channels as targets”.
• the invention also relates to a method of manufacturing a medicament containing an activator of a potassium channel K2P comprising the following steps:
• Such a manufacturing method which makes it possible to test the quality of different batches of production, is useful during the industrial production of drugs. More particularly, this method makes it possible to verify that a given batch of medicinal product containing an activator of a potassium channel K2P has an analgesic effect, and has no constipating effect and / or respiratory depressant.
• the drug can be produced by any method known to those skilled in the art. The production of such a drug typically comprises the synthesis of K2P potassium channel activator and then its formulation into a pharmaceutical product.
• the drug according to the invention may be intended to be administered by any suitable route, for example oral, sub-lingual, nasal, oral, transdermal, intravenous, subcutaneous, intramuscular and / or rectal.
• a medicament the active ingredient (i.e., the activator according to the invention) is combined with a pharmaceutically acceptable carrier (i.e. say any solvent, dispersion medium, absorption retarding agent, etc., which does not produce a side reaction, for example allergic, in humans or animals).
• a pharmaceutically acceptable carrier i.e. say any solvent, dispersion medium, absorption retarding agent, etc., which does not produce a side reaction, for example allergic, in humans or animals.
• Pharmaceutically acceptable carriers are well known to those skilled in the art, and include those described in "Remington's Pharmaceutical Sciences” (Mack Publishing Company, Easton, USA, 1985).
• the invention also relates to a method of treating or preventing pain, comprising administering a therapeutically effective amount of a K2P potassium channel enhancer to an individual in need thereof.
• the individual is preferably a mammal, more particularly a human.
• the effective therapeutic dose can easily be determined by those skilled in the art.
• the invention further relates to the use of a potassium channel activator K2P for the preparation of a medicament for treating or preventing pain.
• Figure 4 Antinociceptive effect of morphine (5mg / kg, sc) in the von Frey mechanical test (A) and the immersion test of the tail in water at 46 ⁇ (B) in wild animals and TRAAK " ' " .
• the results are expressed in variations of nociceptive thresholds with respect to the values of the mice of the same genotype treated by the vehicle.
• SEQ ID NO: 1 is the sequence of a subunit of a TREK-1 channel.
• SEQ ID NO: 2 is the sequence of a subunit of a TREK-2 channel.
• SEQ ID NO: 3 is the sequence of a sub-unit of a TRAAK channel.
• mice Male mice C57BI / 6J mice (Charles River Lab, France), TREK-1 " '" Trek- 2 "” and TRAAK “'” (IPMC, Nice Sophia Antipolis), weighing 20 to 30 grams, were placed caged with food and water ad libitum in thermo-regulated environment at 22 ° C with a day / night cycle of 12h / 12h. The details concerning the generation of KO animals have been described previously (Heurteaux et al., 2004 EMBO J. 23: 2684-95 and Guyon et al., 2009 J. Neurosc 29: 2528-33). The experiments were carried out blind in a quiet room by the same experimenter, taking care to comply with the regulatory requirements on animal testing.
• morphine hydrochloride (Cooper, Melun, France), naloxone hydrochloride (Sigma Chemical Co., St. Louis, MO).
• the solutions were prepared extemporaneously in NaCl (0.9%).
• Naloxone (1 mg / kg, s.c.) was administered 15 minutes prior to morphine or vehicle injection.
• Thermal sensitivity is measured by the immersion test of the tail in water to nociceptive temperature of 46 ° C (Janssen et al 1963 Arzneiffenforschung 13:.. 502-7).
• the animal is kept manually and the tail is immersed in a water bath until the withdrawal of the animal by the animal or until "off", set at 30 s.
• the basal threshold (pre-treatment) is defined by the average of the first two latency periods not differing by more than one second. The animals were used to restraint for a week before the start of the experiment.
• the evaluation of the intestinal transit was carried out using a global method of collecting feces in kinetics for 2 hours following the injection of morphine or vehicle (NaCl 0.9%).
• the animals are placed in boxes (85 x 35 mm, opaque separation between the mice, mesh bottom for the collection of feces) 20 minutes before the test, for habituation.
• the faeces are collected and weighed immediately after collection every hour for 2 hours after the injection of morphine or vehicle.
• Respiratory rate was measured non-invasively (Drorbaugh and Fenn, 1955 Pediatrics, 16: 81-7, Matthes et al., 1998 J Neurosci 18: 7285-95) using a barometric plethysmograph (Emka Technologies, VA, USA) with 8 chambers allowing measurement in parallel on several animals.
• the IOX software (Emka Technologies, VA, USA) was used to calculate the respiratory rate.
• Each dose of morphine or vehicle was administered, and evaluated their effects on respiratory rate in four wild mice and four TREK-1 mice "" in parallel. The animals were used to the rooms for 15 minutes before the injection. Analysis of the respiratory rate during this 15-minute period revealed a stable respiratory rate. The mean respiratory rate during this period was therefore used to normalize the effect of morphine or vehicle on the respiratory rate during the test period (90 minutes).
• the experimental data was analyzed using Sigma STAT software, version 3.0 for Windows (STAT32 Software Inc., San Diego, California).
• Example 2 Role of TREK-1, TREK-2 and TRAAK channels in the physiology of nociception
• TREK-1 animals “" having pain threshold before injection of morphine or vehicle, significantly lower than those of wild animals (fig.l and 1B), the differences between the pre and post values injection were calculated for each animal and then compared.
• Opioid receptors are key players in the inhibition of morphine-induced gastrointestinal transit (Reisine and Pasternak, 1996, “The Pharmacological Basis of Therapeutics", Publishers: Hardman JG, Gilman AG, and Limbird, pp. 521-555 ).
• Morphine-induced respiratory depression also occurs via activation of opioid receptors (Santiago and Edelman, 1985 J Appl Physiol 59: 1675-85, Reisine and Pasternak, 1996, "The Pharmacological Basis of Therapeutics", Publishers: Hardman JG, Gilman AG, and Limbird, pages 521-555), in addition mice whose gene encoding the ⁇ receptor has been invalidated are protected against this adverse effect (Matthes et al., 1998 J Neurosci.
• the basal respiratory rate is not different in the two genotypes. Moreover, regardless of the genotype, neither the vehicle nor the morphine at the dose of 5 mg / kg influence the respiratory rate. On the other hand, whatever genotype, administration of morphine at doses of 10, 20 and 50 mg / kg results in a decrease in the dose-dependent respiratory rate with no difference between genotypes (Fig. 3B).
• the deletion of the gene that codes for TREK-1 does not reduce the respiratory depressant effect of morphine.
• the TREK-1 channel is therefore not involved in this adverse effect.
• Constipation induced by morphine in animals TRAAK " '" has also been studied.
• the effects of morphine on gastrointestinal functions were assessed by measuring the production of faeces harvested hourly for 2 hours after morphine or vehicle injection. To ensure that the two genotypes do not differ in their food and water consumption, this consumption was measured and standardized by animal (cages containing 3 to 5 animals) and data averaged for 3 cages per genotype.

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