WO2015189342A1 - Pharmaceutical compositions comprising an at2r agonist for treating pain - Google Patents

Pharmaceutical compositions comprising an at2r agonist for treating pain Download PDF

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Publication number
WO2015189342A1
WO2015189342A1 PCT/EP2015/063085 EP2015063085W WO2015189342A1 WO 2015189342 A1 WO2015189342 A1 WO 2015189342A1 EP 2015063085 W EP2015063085 W EP 2015063085W WO 2015189342 A1 WO2015189342 A1 WO 2015189342A1
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WIPO (PCT)
Prior art keywords
pain
at2r
mycolactone
traak
screening
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PCT/EP2015/063085
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English (en)
French (fr)
Inventor
Priscille Brodin
Estelle MARION
Laurent MARSOLLIER
Guillaume Sandoz
Song OK-RYUL
Yannick COMOGLIO
Original Assignee
INSERM (Institut National de la Santé et de la Recherche Médicale)
Institut Pasteur De Lille
Université De Lille 1 Sciences Et Technologies
Université De Droit Et De La Santé De Lille 2
Centre National De La Recherche Scientifique (Cnrs)
Universite De Nice Sophia Antipolis
Université de Nantes
Université d'Angers
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Application filed by INSERM (Institut National de la Santé et de la Recherche Médicale), Institut Pasteur De Lille, Université De Lille 1 Sciences Et Technologies, Université De Droit Et De La Santé De Lille 2, Centre National De La Recherche Scientifique (Cnrs), Universite De Nice Sophia Antipolis, Université de Nantes, Université d'Angers filed Critical INSERM (Institut National de la Santé et de la Recherche Médicale)
Priority to EP15728519.8A priority Critical patent/EP3154540A1/en
Publication of WO2015189342A1 publication Critical patent/WO2015189342A1/en

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    • 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
    • G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/10—Screening for compounds of potential therapeutic value involving cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/28—Neurological disorders
    • G01N2800/2842—Pain, e.g. neuropathic pain, psychogenic pain

Definitions

  • the present invention relates to methods and pharmaceutical compositions for treating pain in subject in need thereof.
  • Pain is an unpleasant feeling often caused by intense or damaging stimuli.
  • the International Association for the Study of Pain's widely used definition states: "Pain is an unpleasant sensory and emotional experience associated with actual or potential tissue damage, or described in terms of such damage.”
  • Chronic pain is a common problem that presents a major challenge to healthcare providers because of its complex natural history, unclear etiology, and poor response to therapy.
  • Chronic pain is a poorly defined condition. Most authors consider ongoing pain lasting longer than 6 months as diagnostic, and others have used 3 months as the minimum criterion. In chronic pain, the duration parameter is used arbitrarily. Some authors suggest that any pain that persists longer than the reasonable expected healing time for the involved tissues should be considered chronic pain.
  • the pathophysiology of chronic pain is multifactorial and complex and still is poorly understood.
  • Chronic pain can result from musculoskeletal disorders such as osteoarthritis/degenerative joint disease/spondylosis, rheumatoid arthritis, lyme disease, reiter syndrome, disk herniation facet osteoarthropathy, fractures/compression fracture of lumbar vertebrae, faulty or poor posture, fibromyalgia, polymyalgia rheumatica, mechanical low back pain, chronic coccygeal pain, muscular strains and sprains, pelvic floor myalgia (levator ani spasm), piriformis syndrome, rectus tendon strain, hernias (e.g.
  • obturator sciatic, inguinal, femoral, spigelian, perineal, umbilical), abdominal wall myofascial pain (trigger points), chronic overuse syndromes (e.g., tendinitis, bursitis); neurological disorders such as, brachial plexus traction injury, cervical radiculopathy, thoracic outlet syndrome, spinal stenosis, arachnoiditis syndrome, metabolic deficiency myalgias, polymyositis, neoplasia of spinal cord or sacral nerve, cutaneous nerve entrapment in surgical scar, postherpetic neuralgia (shingles), neuralgia (e.g., iliohypogastric, ilioinguinal, or genitofemoral nerves), polyneuropathies, polyradiculoneuropathies, mononeuritis multiplex, chronic daily headaches, muscle tension headaches, migraine headaches, temporomandibular joint dysfunction, temporalis tendonitis,
  • Pain is generally controlled by the administration of short acting analgesic agents, steroids and non-steroidal anti- inflammatory drugs.
  • Analgesic agents include opiates, agonistic-antagonistic agents, and antiinflammatory agents.
  • all opiates have a wide variety of side effects that can decrease their clinical utility in certain situations.
  • opiates include respiratory depression, reduced cough reflex, bronchial spasms, nausea, vomiting, release of histamine, peripheral vasodilation, orthostatic hypotension, alteration of vagal nerve activity of the heart, hyperexcitability of smooth muscles (sphincters), reduction of peristaltic motility in the gastrointestinal tract and urinary retention.
  • Opiates also stimulate the release of adrenaline, anti-diuretic hormone, cause changes in the regulation of body temperature and sleep pattern, and are liable to promote the development of tolerance and addiction.
  • the present invention relates to methods and pharmaceutical compositions for treating pain in subject in need thereof
  • Mycobacterium ulcerans the etiological agent of Buruli ulcer, causes extensive skin lesions, which despite their severity are not accompanied by pain. It was previously thought that this remarkable analgesia is ensured by direct nerve cell destruction.
  • the inventors demonstrate here that M. ulcerans '-induced hypoesthesia is instead achieved through a specific neurological pathway triggered by the secreted mycobacterial polyketide mycolactone. They decipher this pathway at the molecular level, showing that mycolactone elicits signalling through type 2 angiotensin II receptors (AT2RS), leading to potassium-dependent hyperpolarization of neurons.
  • AT2RS type 2 angiotensin II receptors
  • the inventors further validate the physiological relevance of this mechanism with in vivo studies of pain sensitivity in mice infected with M.
  • a first object of the present invention relates to a method of treating pain in a subject thereof comprising administering the subject with a therapeutically effective amount of at least one agonist of the type 2 angiotensin II receptors (AT2Rs)- TWIK-related arachidonic acid stimulated K+ channel (TRAAK) pathway.
  • Treatment may be for any purpose, including the therapeutic treatment of subjects suffering from pain, as well as the prophylactic treatment of subjects who do not suffer from pain (e.g., subjects identified as being at high risk pain).
  • treatment refers to reversing, alleviating, inhibiting the progress of a disease or disorder as described herein (i.e. pain), or delaying, eliminating or reducing the incidence or onset of a disorder or disease as described herein, as compared to that which would occur in the absence of the measure taken.
  • prophylaxis or “prophylactic use” and “prophylactic treatment” as used herein, refer to any medical or public health procedure whose purpose is to prevent the disease herein disclosed (i.e. pain).
  • prevent refers the reduction in the risk of acquiring or developing a given condition (i.e.
  • the method of the present invention is suitable in the treatment of a wide range of pain disorders, particularly acute pain, chronic pain, neuropathic pain, inflammatory pain, iatrogenic pain including cancer pain, infectious pain including herpetic pain visceral pain, central pain, dysfunctioning pain including fibromyalgia, nociceptive pain including post-surgical pain, and mixed pain types involving the viscera, gastrointestinal tract, cranial structures, musculoskeletal system, spine, urogenital system, cardiovascular system and CNS, including cancer pain, back and orofacial pain.
  • Pain is generally be classified as acute or chronic. Acute pain begins suddenly and is short-lived (usually twelve weeks or less). It is usually associated with a specific cause such as a specific injury and is often sharp and severe. It is the kind of pain that can occur after specific injuries resulting from surgery, dental work, a strain or a sprain. Acute pain does not generally result in any persistent psychological response. In contrast, chronic pain is long-term pain, typically persisting for more than three months and leading to significant psychological and emotional problems. Common examples of chronic pain are neuropathic pain (e.g. painful diabetic neuropathy, postherpetic neuralgia), carpal tunnel syndrome, back pain, headache, cancer pain, arthritic pain and chronic post-surgical pain.
  • neuropathic pain e.g. painful diabetic neuropathy, postherpetic neuralgia
  • carpal tunnel syndrome e.g. painful diabetic neuropathy, postherpetic neuralgia
  • back pain e.g. painful diabetic neuropathy, postherpetic neuralgia
  • Clinical pain is present when discomfort and abnormal sensitivity feature among the patient's symptoms. Patients tend to be quite heterogeneous and may present with various pain symptoms. Such symptoms include: 1) spontaneous pain which may be dull, burning, or stabbing; 2) exaggerated pain responses to noxious stimuli (hyperalgesia); and 3) pain produced by normally innocuous stimuli. Although patients suffering from various forms of acute and chronic pain may have similar symptoms, the underlying mechanisms may be different and may, therefore, require different treatment strategies. Pain can also therefore be divided into a number of different subtypes according to differing pathophysiology, including nociceptive, inflammatory and neuropathic pain.
  • Nociceptive pain is induced by tissue injury or by intense stimuli with the potential to cause injury. Pain afferents are activated by transduction of stimuli by nociceptors at the site of injury and activate neurons in the spinal cord at the level of their termination. This is then relayed up the spinal tracts to the brain where pain is perceived (Meyer et al., 1994, Textbook of Pain, 13-44). The activation of nociceptors activates two types of afferent nerve fi res. Myelinated A-delta fihres transmit rapidly and are responsible for sharp and stabbing pain sensations, whilst unmyelinated C fibres transmit at a slower rate and convey a dull or aching pain.
  • Moderate to severe acute nociceptive pain is a prominent feature of pain from central nervous system trauma, strains/sprains, burns, myocardial infarction and acute pancreatitis, postoperative pain (pain following any type of surgical procedure), posttraumatic pain, renal colic, cancer pain and back pain.
  • Cancer pain may be chronic pain such as tumour related pain (e.g. bone pain, headache, facial pain or visceral pain) or pain associated with cancer therapy (e.g. postchemotherapy syndrome, chronic postsurgical pain syndrome or post radiation syndrome). Cancer pain may also occur in response to chemotherapy, immunotherapy, hormonal therapy or radiotherapy.
  • Back pain may be due to herniated or ruptured intervertebral discs or abnormalities of the lumber facet joints, sacroiliac joints, paraspinal muscles or the posterior longitudinal ligament. Back pain may resolve naturally but in some patients, where it lasts over 12 weeks, it becomes a chronic condition which can be particularly debilitating.
  • Neuropathic pain is currently defined as pain initiated or caused by a primary lesion or dysfunction in the nervous system. Nerve damage can be caused by trauma and disease and thus the term 'neuropathic pain' encompasses many disorders with diverse aetiologies. These include, but are not limited to, peripheral neuropathy, diabetic neuropathy, post herpetic neuralgia, trigeminal neuralgia, back pain, cancer neuropathy, HIV neuropathy, phantom limb pain, carpal tunnel syndrome, central post- stroke pain and pain associated with chronic alcoholism, hypothyroidism, uremia, multiple sclerosis, spinal cord injury, Parkinson's disease, epilepsy and vitamin deficiency. Neuropathic pain is pathological as it has no protective role.
  • neuropathic pain are difficult to treat, as they are often heterogeneous even between patients with the same disease (Woolf & Decosterd, 1999, Pain Supp., 6, S141-S147; Woolf and Mannion, 1999, Lancet, 353, 1959-1964). They include spontaneous pain, which can be continuous, and paroxysmal or abnormal evoked pain, such as hyperalgesia (increased sensitivity to a noxious stimulus) and allodynia (sensitivity to a normally innocuous stimulus).
  • the inflammatory process is a complex series of biochemical and cellular events, activated in response to tissue injury or the presence of foreign substances, which results in swelling and pain (Levine and Taiwo, 1994, Textbook of Pain, 45-56).
  • Arthritic pain is the most common inflammatory pain.
  • Rheumatoid disease is one of the commonest chronic inflammatory conditions in developed countries and rheumatoid arthritis is a common cause of disability. The exact aetiology of rheumatoid arthritis is unknown, but current hypotheses suggest that both genetic and microbiological factors may be important (Grennan & Jayson, 1994, Textbook of Pain, 397-407).
  • Visceral pain is pain associated with the viscera, which encompass the organs of the abdominal cavity. These organs include the sex organs, spleen and part of the digestive system. Pain associated with the viscera can be divided into digestive visceral pain and non-digestive visceral pain.
  • Gl gastrointestinal
  • FBD functional bowel disorder
  • IBD inflammatory bowel disease
  • Gl disorders include a wide range of disease states that are currently only moderately controlled, including, in respect of FBD, gastro-esophageal reflux, dyspepsia, irritable bowel syndrome (IBS) and functional abdominal pain syndrome (FAPS), and, in respect of IBD, Crohn's disease, ileitis and ulcerative colitis, all of which regularly produce visceral pain.
  • Other types of visceral pain include the pain associated with dysmenorrhea, cystitis and pancreatitis and pelvic pain.
  • the method of the present invention is suitable for the treatment of pain resulting from musculo-skeletal disorders, including myalgia, fibromyalgia, spondylitis, sero-negative (non-rheumatoid) arthropathies, non-articular rheumatism, dystrophinopathy, glycogenosis, polymyositis and pyomyositis; - heart and vascular pain, including pain caused by angina, myocardical infarction, mitral stenosis, pericarditis, Raynaud's phenomenon, scleredoma and skeletal muscle ischemia.
  • musculo-skeletal disorders including myalgia, fibromyalgia, spondylitis, sero-negative (non-rheumatoid) arthropathies, non-articular rheumatism, dystrophinopathy, glycogenosis, polymyositis and pyomyositis;
  • the method of the present invention is suitable for the treatment of head pain, such as migraine (including migraine with aura and migraine without aura), cluster headache, tension-type headache mixed headache and headache associated with vascular disorders.
  • head pain such as migraine (including migraine with aura and migraine without aura), cluster headache, tension-type headache mixed headache and headache associated with vascular disorders.
  • the method of the present invention is suitable for the treatment of orofacial pain, including dental pain, otic pain, burning mouth syndrome and temporomandibular myofascial pain.
  • the method of the present invention is particularly suitable for the treatment of chronic pain which results from musculoskeletal disorders such as osteoarthritis/degenerative joint disease/spondylosis, rheumatoid arthritis, lyme disease, reiter syndrome, disk herniation facet osteoarthropathy, fractures/compression fracture of lumbar vertebrae, faulty or poor posture, fibromyalgia, polymyalgia rheumatica, mechanical low back pain, chronic coccygeal pain, muscular strains and sprains, pelvic floor myalgia (levator ani spasm), piriformis syndrome, rectus tendon strain, hernias (e.g.
  • musculoskeletal disorders such as osteoarthritis/degenerative joint disease/spondylosis, rheumatoid arthritis, lyme disease, reiter syndrome, disk herniation facet osteoarthropathy, fractures/compression fracture of lumbar vertebrae,
  • obturator sciatic, inguinal, femoral, spigelian, perineal, umbilical), abdominal wall myofascial pain (trigger points), chronic overuse syndromes (e.g., tendinitis, bursitis); neurological disorders such as, brachial plexus traction injury, cervical radiculopathy, thoracic outlet syndrome, spinal stenosis, arachnoiditis syndrome, metabolic deficiency myalgias, polymyositis, neoplasia of spinal cord or sacral nerve, cutaneous nerve entrapment in surgical scar, postherpetic neuralgia (shingles), neuralgia (e.g., iliohypogastric, ilioinguinal, or genitofemoral nerves), polyneuropathies, polyradiculoneuropathies, mononeuritis multiplex, chronic daily headaches, muscle tension headaches, migraine headaches, temporomandibular joint dysfunction, temporalis tendonitis,
  • the method of the present invention is particularly suitable for the treatment of pain which results from autoimmune diseases including multiple sclerosis, neurodegenerative disorders, neurological disorders including epilepsy and senso-motor pathologies, irritable bowel syndrome, osteoarthritis, rheumatoid arthritis, neuropathological disorders, functional bowel disorders, inflammatory bowel diseases, pain associated with dysmenorrhea, pelvic pain, cystitis, pancreatitis, migraine, cluster and tension headaches, diabetic neuropathy, peripheral neuropathic pain, sciatica, causalgia, and conditions of lower urinary tract dysfunction.
  • autoimmune diseases including multiple sclerosis, neurodegenerative disorders, neurological disorders including epilepsy and senso-motor pathologies, irritable bowel syndrome, osteoarthritis, rheumatoid arthritis, neuropathological disorders, functional bowel disorders, inflammatory bowel diseases, pain associated with dysmenorrhea, pelvic pain, cystitis, pancreatitis, migraine, cluster and tension headaches, diabetic neuropathy, peripheral
  • the prophylactic methods of the invention are particularly suitable for subjects who are identified as at high risk for pain.
  • subject that are risk for pain include patient that will have a surgical operation.
  • TRAAK has its general meaning in the art and refers to TWIK-related arachidonic acid stimulated K+ channel.
  • TRAAK is also known as Homo sapiens potassium channel, subfamily K, member 4 (KCNK4). This protein is a member of the superfamily of potassium channel proteins containing two pore-forming P domains. The encoded protein dimerizes and functions as an outwardly rectifying channel. It is expressed primarily in neural tissues and is stimulated by membrane stretch and polyunsaturated fatty acids. Accordingly, the term "TRAAK agonist” used herein relates to any compound binding specifically to, and thus stimulating, TRAAK.
  • AT2R has its general leaning in the art and refers to type 2 angiotensin II receptor. Accordingly, the term “angiotensin II type 2 receptor agonist” used herein relates to any compound binding specifically to, and thus stimulating, angiotensin II type 2 receptors.
  • type 2 angiotensin II receptors (AT2Rs)- TWIK-related arachidonic acid stimulated K+ channel (TRAAK) pathway refers to the pathway deciphered in the EXAMPLE. This novel pathway consists of the activation of TRAAK by the stimulation of AT2R receptors in neurons. This pathway leads to the hyperpolarization of the neurons.
  • agonist of the type 2 angiotensin II receptors (AT2Rs)- TWIK-related arachidonic acid stimulated K+ channel (TRAAK) pathway refers to any compounds which activate this pathway.
  • the agonist of the type 2 angiotensin II receptors (AT2Rs)- TWIK-related arachidonic acid stimulated K+ channel (TRAAK) pathway is an AT2R agonist or a TRAKK agonist.
  • the agonist used according to the invention may be any substance, derived from natural sources or from synthesis by chemical and/or genetic engineering methods.
  • Agonists typically include but are not limited to small organic molecule, peptides, polypeptides (including antibodies), nucleic acids (e.g. aptamers).
  • AT2R agonists are well known in the art (Steckelings UM, Unger T. Angiotensin II type 2 receptor agonists—where should they be applied? Expert Opin Investig Drugs. 2012 Jun;21(6):763-6. doi: 10.1517/13543784.2012.681046. Epub 2012 Apr 21.; Steckelings UM, Paulis L, Unger T, Bader M. Emerging drugs which target the renin-angiotensin-aldosterone system. Expert Opin Emerg Drugs. 2011 Dec; 16(4):619-30. doi: 10.1517/14728214.2011.618495. Epub 2011 Sep 12.
  • AT2R agonists include compounds of formula I which are disclosed in US 20120035232, I
  • R 1 represents H
  • R 2 and R 3 independently represent H, Ci- ⁇ alkyl, Ci- ⁇ alkoxy, Ci-6 alkoxy-Ci-6-alkyl or halo;
  • Yi , Y2, Y3 and Y4 independently represent— CH— or— CF— ;
  • Z2 represents— CH— or— N— ;
  • R 4 represents— S(0) 2 N(H)C(0)R 6 ,— S(0) 2 N(H)S(0) 2 R 6 ,— C(0)N(H)S(0) 2 R 6 ;
  • R 5 represents Ci-6 alkyl, C1-5 alkoxy, Ci-e alkoxy-Ci-6-alkyl or di-Ci-3-alkylamino-Ci-4- alkyl;
  • R 6 represents C 1-5 alkyl, C1-5 alkoxy, Ci-6 alkoxy-Ci-6-alkyl, C1-3 alkoxy-Ci-6-alkoxy, Ci- 6 alkylamino or di-Ci-6 alkylamino;
  • the AT2R agonist of the present invention is selected from the group consisting of:
  • AT2R agonists include those described in WO1999043339, and WO1996039164, WO2012070936.
  • the AT2R agonist is a peptide or a peptide mimetic with high selectivity for the AT2R.
  • Examples of peptides functioning as AT2R agonist and thus suitable for use according to the present invention are p-aminophenylalanine6- angiotensin II or N- - nicotinoyl-Tyr-(N-a-CBZ-Arg)-Lys-His-Pro-Ile-OH.
  • a peptide mimetic may contain elements that enforce steric constraints of a peptide and a peptide mimetic may retain some peptidic character.
  • a peptide mimetic may alternatively be lacking peptidic fragments and consist of an organic molecule.
  • a peptide mimetic can be an organic molecule comprising biaryl, arylheteroaryl, or biheteroaryl fragments that can be attached to a nitrogen containing monocyclic or bicyclic heterocycle by a one, two or three atom linker.
  • a selective AT2R agonist may be an analogue of the non- selective angiotensin II type 2 receptor ligand 5,7- dimethyl-2-ethyl-3- [ [4- [2 (n-butyloxycarbonylsulfonamido) - 5-isobutyl-3-thienyl] phenyl] methyl] -imidazo [4 , 5-b] - pyridine.
  • the compound of the present invention is administered to the patient with a therapeutically effective amount.
  • a therapeutically effective amount is meant a sufficient amount of the compound of the present invention to treat the disease (i.e. pain) at a reasonable benefit/risk ratio applicable to any medical treatment.
  • the total daily usage of the compounds and compositions of the present invention will be decided by the attending physician within the scope of sound medical judgment.
  • the specific therapeutically effective dose level for any particular patient will depend upon a variety of factors including the age, body weight, general health, sex and diet of the patient; the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidental with the specific polypeptide employed; and like factors well known in the medical arts. For example, it is well known within the skill of the art to start doses of the compound at levels lower than those required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved.
  • the daily dosage of the products may be varied over a wide range from 0.01 to 1,000 mg per adult per day.
  • the compositions contain 0.01, 0.05, 0.1 , 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 25.0, 50.0, 100, 250 and 500 mg of the active ingredient for the symptomatic adjustment of the dosage to the patient to be treated.
  • a medicament typically contains from about 0.01 mg to about 500 mg of the active ingredient, typically from 1 mg to about 100 mg of the active ingredient.
  • An effective amount of the drug is ordinarily supplied at a dosage level from 0.0002 mg/kg to about 20 mg/kg of body weight per day, especially from about 0.001 mg/kg to 7 mg/kg of body weight per day.
  • the compound of the present invention is typically combined with pharmaceutically acceptable excipients, and optionally sustained-release matrices, such as biodegradable polymers, to form pharmaceutical compositions.
  • pharmaceutically acceptable excipients such as biodegradable polymers
  • sustained-release matrices such as biodegradable polymers
  • “Pharmaceutically” or “pharmaceutically acceptable” refer to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to a mammal, especially a human, as appropriate.
  • a pharmaceutically acceptable carrier or excipient refers to a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type.
  • the active principle in the pharmaceutical compositions of the present invention for oral, sublingual, subcutaneous, intramuscular, intravenous, transdermal, local or rectal administration, can be administered in a unit administration form, as a mixture with conventional pharmaceutical supports, to animals and human beings.
  • Suitable unit administration forms comprise oral-route forms such as tablets, gel capsules, powders, granules and oral suspensions or solutions, sublingual and buccal administration forms, aerosols, implants, subcutaneous, transdermal, topical, intraperitoneal, intramuscular, intravenous, subdermal, transdermal, intrathecal and intranasal administration forms and rectal administration forms.
  • the pharmaceutical compositions contain vehicles which are pharmaceutically acceptable for a formulation capable of being injected.
  • vehicles which are pharmaceutically acceptable for a formulation capable of being injected.
  • These may be in particular isotonic, sterile, saline solutions (monosodium or disodium phosphate, sodium, potassium, calcium or magnesium chloride and the like or mixtures of such salts), or dry, especially freeze-dried compositions which upon addition, depending on the case, of sterilized water or physiological saline, permit the constitution of injectable solutions.
  • the pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions; formulations including sesame oil, peanut oil or aqueous propylene glycol; and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions.
  • the form In all cases, the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi.
  • Solutions comprising compounds of the invention as free base or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.
  • the polypeptide (or nucleic acid encoding thereof) can be formulated into a composition in a neutral or salt form.
  • Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like.
  • the carrier can also be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetables oils.
  • the proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • the prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like.
  • isotonic agents for example, sugars or sodium chloride.
  • Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminium monostearate and gelatin.
  • Sterile injectable solutions are prepared by incorporating the active polypeptides in the required amount in the appropriate solvent with several of the other ingredients enumerated above, as required, followed by filtered sterilization.
  • dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above.
  • sterile powders for the preparation of sterile injectable solutions
  • the preferred methods of preparation are vacuum- drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
  • solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective.
  • the formulations are easily administered in a variety of dosage forms, such as the type of injectable solutions described above, but drug release capsules and the like can also be employed.
  • parenteral administration in an aqueous solution for example, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose.
  • aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration.
  • sterile aqueous media which can be employed will be known to those of skill in the art in light of the present disclosure.
  • one dosage could be dissolved in 1 ml of isotonic NaCl solution and either added to 1000 ml of hypodermoclysis fluid or injected at the proposed site of infusion. Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject.
  • a further object of the present invention relates to a method for screening a plurality of test compounds useful for treatment of pain comprising the steps consisting of (a) testing each of the test compounds for its ability to activate the angiotensin II receptors (AT2Rs)- TWIK- related arachidonic acid stimulated K+ channel (TRAAK) pathway and (b) positively selecting the test compounds capable of said activation.
  • the screening method of the invention comprises a first step of determining whether the test compound is able to bind to TRAAK or AT2R. Methods for determining whether a test compound binds to a protein are well known in the art.
  • Binding of the test compound to the amyloid channel protein(s) can be detected by any of a number of methods known to those of skill in the art.
  • the test compounds are labeled with a detectable label (e.g., a fluorescent label, a colorimetric label, a radioactive label, a spin (spin resonance) label, a radioopaque label, etc.).
  • a detectable label e.g., a fluorescent label, a colorimetric label, a radioactive label, a spin (spin resonance) label, a radioopaque label, etc.
  • the membrane comprising the protein of interest i.e. AT2R or TRAAK
  • a secondary binding moiety e.g.
  • the label on the test agent and the label on the secondary agent can be labels selected that undergo fluorescent resonance energy transfer (FRET) so that excitation of one label results in emission from the second label thereby providing an efficient means of detecting association of the labels.
  • FRET fluorescent resonance energy transfer
  • a competitive binding assay as described in the EXAMPLE is used. In such assays, a "competitive" agent known to bind to the protein of interest is also utilized.
  • the competitive agent can be labeled and the amount of such agent displaced when the bilayer containing the protein of interest is contacted with a test agent provides a measure of the biding of the test agent.
  • Methods of detecting specific binding are well known and commonly used, e.g. in various immunoassays. Any of a number of well recognized immunological binding assays (see, e.g., U.S. Patents 4,366,241 ; 4,376,110; 4,517,288; and 4,837, 168) are well suited to detection of test agent binding to proteins in a lipid bilayer.
  • U.S. Patents 4,366,241 ; 4,376,110; 4,517,288; and 4,837, 168 are well suited to detection of test agent binding to proteins in a lipid bilayer.
  • the screening method of the present invention is performed in screening cells.
  • said screening cells are neurons, such as neurons derived from PC 12 cells or are macrophages, such as Raw267.4 as described in the EXAMPLE.
  • the screening cells express a first DNA that encodes a TRAAK channel and optionally a second DNA encoding for AT2R.
  • the screening cells express a first DNA that encodes or express a TRAAK channel and a second DNA encoding for AT2R. Method for introducing a nucleic acid sequence encoding for a protein of interest are well known in the art.
  • the screening method consists in determining whether the test compound is able to induce hyperpolarisation of the screening cells.
  • the step of contacting the screening cells with a test compound is accomplished for example by adding the test compound to the saline solution used for the patch clamp assay.
  • the step of contacting a screening cell with a test compound also includes contacting the cell with a plurality of test compounds simultaneously, such as two or more test compounds, or three or more test compounds, etc.
  • an ex vivo system comprising two chambers separated by a lipid bilayer, that contains channel of the present invention (i.e. TRAAK).
  • TRAAK channel of the present invention
  • the conductance across the lipid bilayer is monitor continuously.
  • the TRAKK channel is activated so that the resting membrane potential becomes deeper in the negative direction, or in other words, so that the negative potential increases.
  • the resting membrane potential is preferably deepened in the negative direction to a degree that does not affect cell viability.
  • the induced hyperpolarization is -5; -6; -7; -8; -9; -10; -11; -12; -13; -14; -15; -16; -17; -18; -19; -20; -21; - 22; -23; -24; -25; -26; -27; -28; -29; -30; -31; -32; -33; -34; -35; -36; -37; -38; -39; -40; -41; - 42; -43; -44; -45; -46; -47; -48; -49; or -50 mV.
  • the induced hyperpolarization is compared to a predetermined reference value and when the hyperpolarization induced by the test compound is higher than the predetermined reference value then the test compound is selected.
  • the predetermined reference value represents the hyperpolarization determined in the absence of the test compound or the hyperpolarization induced by a reference compound (i.e. a compound which is known to activate the pathway of the present invention).
  • Said reference compound is for example the myco lactone toxin as described in the EXAMPLE.
  • a phenotypic assay could be used for analysing hyperpolarization of a screening cell.
  • Such a phenotypic assay may consist in the cell-based fluorescence assay coupled with image acquisition by automated confocal microscopy as described in the EXAMPLE. Briefly this assay consists of measuring changes in the fluorescent intensity of a potential-sensitive fluorochrome when the screening cells are contacted with the test compound.
  • the potential-sensitive fluorochrome as used herein may be any of the types that are generally available in the art concerned and a suitable one may be selected from among the following: styryl-based potential-sensitive fluorochromes comprising ANEPPSs, ANRPEQs and RHs; cyanine- or oxonol-based potential- sensitive fluorochromes comprising DiSC's, DiOC's, DilC's, DiBAC's, and DiSBAC's; and rhodamine-derived potential-sensitive fluorochrome such as Rh 123, TMRM, and TMRE.
  • styryl-based potential-sensitive fluorochromes comprising ANEPPSs, ANRPEQs and RHs, which are specifically exemplified by di-8-ANEPPS, di-4-ANEPPS, RH-237, RH-1691 , di-5-ASP, RH-160, RH-421, RH-795, di-4-ANEPPDHQ, ⁇ -5, and ⁇ -6, and a preferred potential-sensitive fluorochrome may be selected from among these.
  • any type of fluorescent microscope that can be used in the art concerned may be applied in the present invention and a typical example is 1X71 (OLYMPUS Corporation).
  • 1X71 (OLYMPUS Corporation) was used as a fluorescent microscope and combined with a suitable light source unit such as a mercury lamp (OLYMPUS Corporation) or an LED assembly (OLYMPUS Corporation).
  • a suitable light source unit such as a mercury lamp (OLYMPUS Corporation) or an LED assembly (OLYMPUS Corporation).
  • any models of analysis software for imaging and numerical calculations that can be used in the art concerned may be applied in the present invention.
  • confocal images are recorded on an automated fluorescent confocal microscope OperaTM (Evotec) as described in the EXAMPLE. Each image are then processed using a dedicated image analysis software.
  • the screening cells e.g. Raw267.4 cells
  • the screening method of the present invention comprises the steps of i) labeling the screening cells with the potential- sensitive fluorochrome into contact with screening cells of the present invention previously, ii) determining the fluorescence intensity [Tlint] of the screening cells, iii) bringing the screening cells into contact with the test compound, iv) determining the fluorescence intensity [T2int] of the screening cells, v) calculating the intensity ratio [T2int] / [Tlint] and vi) selecting the test compound when the intensity ratio is inferior to 1 ( ⁇ 1).
  • the intensity ratio is compared with the intensity ratio determined for a compound of reference (e.g.
  • step i) is performed in presence of a blocker of AT2R or TRAA well known in the art (e.g. a siRNA specific for AT2R or TRAAK) and when the intensity ratio is equal or superior to 1 then it is concluded that the test compound (for which a decrease in the intensity ratio was previously determined) is a specific agonist of angiotensin II receptors (AT2Rs)- TWIK-related arachidonic acid stimulated K+ channel (TRAAK) pathway.
  • AT2Rs angiotensin II receptors
  • TRAAK arachidonic acid stimulated K+ channel
  • the test compound of may be selected from the group consisting of peptides, peptidomimetics, small organic molecules, antibodies, aptamers or nucleic acids.
  • the test compound according to the invention may be selected from a library of compounds previously synthesized, or a library of compounds for which the structure is determined in a database, or from a library of compounds that have been synthesized de novo.
  • the test compound may be selected form small organic molecules.
  • small organic molecule refers to a molecule of size comparable to those organic molecules generally sued in pharmaceuticals.
  • the screening methods of the invention are very simple. It can be performed with a large number oftest compounds, serially or in parallel. The method can be readily adapted to robotics.
  • the above assays may be performed using high throughput screening techniques for identifying test compounds for developing drugs that may be useful to the treatment or prevention of pain.
  • High throughput screening techniques may be carried out using multi-well plates (e.g., 96-, 384-, or 1536-well plates), in order to carry out multiple assays using an automated robotic system.
  • multi-well plates e.g., 96-, 384-, or 1536-well plates
  • stably-transfected cells growing in wells of micro-titer plates can be adapted to high through-put screening of libraries of compounds.
  • Compounds in the library will be applied one at a time in an automated fashion to the wells of the microtitre dishes containing the transgenic cells described above.
  • the test compounds that have been positively selected may be subjected to further selection steps in view of further assaying its properties in vitro assays or in an animal model organism, such as a rodent animal model system, for the desired therapeutic activity prior to use in humans.
  • an animal model organism such as a rodent animal model system
  • Any well-known animal model may be used for exploring the in vivo therapeutic effects of the screened test compounds.
  • the therapeutic activity of the screened test compounds can be determined by using various experimental animal models of pain known in the art such as those described in the EXAMPLE (e.g. Pain Receptive Assay).
  • the screened test compound may be compared to a reference compound such as a well-known analgesic or the myco lactone toxin. If the screened test compound provide the same effects or even better effects than the reference compound, said test compound could be typically selected for clinical investigation.
  • FIGURES
  • FIG. 1 Absence of Pain in Burn I i Ulcer Lesions Results from a Direct Anaesthetic Effect of Mycolactone.
  • A The nociceptive reflex was quantified using a tail- flick Unit. Mice infected with ulcerans (Mu) showed redness at day 35 and oedema at day 45.
  • B Histological analysis of cutaneous tissue from the footpad inoculated with M. ulcerans- GFP (green). Blue, DAPI; red, ⁇ 3 tubulin staining with Texas red-conjugated anti- 3 tubulin antibody.
  • Ax axon; Mi: mitochondria; Sch N: Schwann cell nucleus; Mt: microtubule; Mf: microfilament; My: myelin; CF: C-Fibre; SLI: Schmidt-Lanterman incisures.
  • the scale bars in C are 40 ⁇ , and those in D are 5 ⁇ .
  • E The nociceptive reflex was measured in mice after injection of 5 ⁇ g of purified myco lactone (black bars), synthetic myco lactone (hatched bars), lidocaine and prilocaine anaesthetics (grey bars) or absolute ethanol alone (vehicle control) (white bars).
  • FIG. 1 Mycolactone Provokes Hyperpolarization via K+ Channels in Murine Primary Neurons.
  • A Representative chart recordings of the membrane potential of PC 12 cells challenged either by ethanol (vehicle), purified mycolactone or synthetic mycolactone.
  • B Pooled data illustrating a 10 mV-hyperpolarization triggered by continuous application of purified mycolactone or synthetic mycolactone for 10-20 min.
  • C Photomicrographs of hippocampal neurons loaded with bis-oxonol (DiSBAC2(3)) and inoculated with mycolactone at 350 ng mL or with ethanol (vehicle) at various times. Scale bars: 100 ⁇ .
  • AT2R is Targeted by Mycolactone.
  • A Fluorescence ratio of Raw267.4 cells treated either with siRNA targeting AT 2 R or with scrambled siRNA, and further loaded with bis-oxonol (DiSBAC 2 (3)) before incubation with mycolactone. The ratio is normalised to that of the scrambled siRNA control.
  • B Fluorescence ratio of neurons from AT2R deficient (AT2R KO) or wild-type (WT) mice; the neurons were loaded with bis-oxonol (DiSBAC2(3)) before incubation with mycolactone.
  • C Competitive binding of mycolactone to human AT2R.
  • D Fluorescence ratio of neurons incubated with PD123,319, a selective inhibitor of AT2R and loaded with bis-oxonol (DiSBAC2(3)), before addition of mycolactone.
  • E Fluorescence ratio of neurons incubated with Ga inhibitor pertussis toxin (PTX) and loaded with bis-oxonol (DiSBAC2(3)) before addition of mycolactone.
  • F Fluorescence ratios of Raw267.4 cells treated with various siRNAs or scrambled siRNA and loaded with bis-oxonol DiSBAC2(3) before incubation with mycolactone. The ratios are normalised to that of the scrambled siRNA control.
  • Pla2g2f, Pla2glbr and Pla2gl0 are members of three different groups of PLA2- encoding genes.
  • Ptgsl COX-1 ;
  • Ptges2 prostaglandin E synthase 2
  • Kcnk4 potassium channel subfamily K member 4;
  • cnkl3 potassium channel subfamily K member 13.
  • G Fluorescence ratio of neurons incubated with FR122047, a selective inhibitor of Ptgsl, before addition of myco lactone.
  • H Current increase in inside-out patches containing TRAA after application of 10 ⁇ ⁇ 2.
  • FIG. 4 M. ulcerans (Mu) and Mycolactone-mediated Hypoesthesia Impaired in vivo Upon Blockage of the AT2R Signalling Cascade.
  • A Detection of AT2R by western blot in neurons of wild-type (WT) or AT2R-deficient mice (AT2R KO). PC12 cells, strongly expressing AT2R were used as a positive control.
  • B The nociceptive reflex was measured in AT2R-KO (grey bars) or wild-type (white bars) mice, which had been inoculated with 5 ⁇ g of mycolactone.
  • C The nociceptive reflex was measured in AT2R- O (grey bars) or wild-type (white bars) mice, which had been inoculated with M.
  • FIG. 1 Model of the Signalling Cascade triggered by Mycolactone. Upon binding to the AT2R receptor, coupled to God, mycolactone triggers a signalling cascade leading to PLA2 activation and inducing the synthesis of arachidonic acid. Arachidonic acid is then metabolized by COX-1 into PGE2, leading to the opening of TRAAK channels.
  • AA arachidonic acid
  • PTGS1 COX-1
  • PGE2 prostaglandin E2
  • TRAAK potassium channel subfamily K member 4.
  • TRAAK current densities before and after C21 application (Fig 7A). Representative TRAAK current before and after C21 application (5 ⁇ ) (Fig 7B). Current was elicited by voltage-ramps (from -100 to 50 mV, Is in duration). Student's t test (*P ⁇ 0.05). The numbers of cells tested are indicated in parentheses.
  • Figure 8. TREK1 is not sensitive to AT2R activation by Mycolactone. TREK1 current density before and after mycolactone application. Representative TREK1 current before and after mycolactone application (5 ⁇ ) Current was elicited by voltage-ramps (from -100 to 50 mV, Is in duration). The numbers of cells tested are indicated in parentheses.
  • M. ulcerans (strain no. 1615) was originally isolated from a skin biopsy of a hu man patient from Malaysia (George et al., 1999)
  • the recombinant M. ulcerans-GFF bacterium (strain no. JKD8083) was derived from an Australian strain in which green fluorescent protein (GFP) expression is controlled by the mis SigA-like promoter (Tobias et al., 2009).
  • GFP green fluorescent protein
  • Mycolactones were purified from M. ulcerans (strain 1615) extracts as previously described (George et al., 1999). Mycolactone purity was estimated to be better than 98%, based on HPLC profiles. Mycolactone was diluted to 4 mg/mL in absolute ethanol and stored in the dark in amber glass tubes. Synthesis of mycolactone was described elsewhere (Gersbach et al, 201 1; Scherr et al, 2013). Synthetic mycolactone was diluted to 500 ng/niL.
  • AT2Rknockout mice were generated and provided by L. Hein (Hein et al, 1995). Six- week-old female FV BN wild-type (Charles River France, http://www.criver.com/ico) and AT2Rknockout (Hein et al., 1 95) mice were maintained under conventional conditions in the animal house facility of the Centre Hospitalier Universitaire, Angers, France (Agreement A 49 007 002), adhering to the institution's guidelines for animal husbandry.
  • the pain receptive assay was performed by adapting the tail- flick procedure, using the tail- flick Unit (UGO BASILE). The sensitivity of the tissue to a noxious thermal stimulus was measured. The radiant heat stimulus was focused on the left footpad. The assay was performed twice each week on mice that had been previously anesthetized. This assay was validated by local application of Emla® analgesic cream containing 5% lidocaine/prilocaine (Astra Zeneca).
  • a perfusion with phosphate buffer was performed after section of the femoral artery. Fixation of the tissue was then performed with a second perfusion with 4% paraformaldehyde and 0.1 % glutaraldehyde in phosphate buffer fixation. The sciatic nerve was subsequently isolated by dissection. The samples were fixed for 30 min in 0.1 M cacodylate buffer (pH 7.2) containing 2.5% glutaraldehyde for 1 h at 4°C and left to stand for 12 h at 20°C in cacodylate buffer. Specimens were progressively dehydrated and embedded in Araldite (Fluka). After dehydration, thin sections were stained with uranyl acetate and Reynold's lead citrate and then examined on a JEOL 120 EX electron microscope.
  • Macrophage cells (Raw264.7, ATCC TIB-71) were cultured in RPMI 1640 (Invitrogen) supplemented with 10% heat-inactivated foetal calf serum (Invitrogen).
  • Pheochromocytoma cell 12 (PC 12) cells were plated on collagen IV-coated culture dishes in DMEM supplemented with 10% horse serum and 5% foetal bovine serum. After 6 days, differentiation to a neuronal phenotype was induced by incubation in DMEM supplemented with 2% horse serum, 1% foetal bovine serum, and 100 ng/mL nerve growth factor (Invitrogen). The culture medium was changed every two days until differentiation was complete (6-7 days).
  • Primary cultures of hippocampal neurons were prepared from neonatal (P0 to P2) BALB/c and FVB N mice according to an established protocol (Banker and Cowan, 1977) with minor modifications.
  • PC-12 cells were grown for 7 days on glass coverslips before being harvested. After an overnight incubation, the glass coverslips were transferred into a recording chamber and continuously superfused with Ringer's saline buffer containing 125 mM NaCl, 2.5 mM KC1, 2 mM CaCl 2 , 1 mM MgCb, 1.25 mM NaH 2 P0 4 , 26 mM NaHCOa, 12 mM glucose, buffered to pH 7.4, at 30-32°C and bubbled with 95% 0 2 and 5% CO2.
  • Ringer's saline buffer containing 125 mM NaCl, 2.5 mM KC1, 2 mM CaCl 2 , 1 mM MgCb, 1.25 mM NaH 2 P0 4 , 26 mM NaHCOa, 12 mM glucose, buffered to pH 7.4, at 30-32°C and bubbled with 95% 0 2 and 5% CO2.
  • Membrane potential was continuously monitored for 10- 20 min before and 10-20 min after my co lactone application at 1.4 g/mL using a pneumatic drug ejection system (PDES, npi electronic GmbH, Germany). Control cells were stimulated with 2% ethanol as vehicle.
  • PDES pneumatic drug ejection system
  • TRAAK defolliculated Xenopus oocytes were injected with 50 nL of cRNA at 0.02-0.4 ⁇ g/ ⁇ L encoding either mTRAAK and recorded 2-4 days later.
  • oocytes were placed in a 0.3-mL perfusion chamber and impaled with two standard microelectrodes (1-2.5 ⁇ resistance) filled with 3 M KC1 and voltage clamped with a Dagan CA-1 amplifier, in ND96 solution (96 mM NaCl, 2 mM KC1, 1.8 mM CaCl 2 , 2 mM MgCl 2 , 5 mM Hepes, pH 7.4 with NaOH). Stimulation of the preparation, data acquisition, and analysis were performed using pClamp software (Axon Instruments). Cell-based Fluorescence Assay, Image Acquisition by Automated Confocal Microscopy and Data Analysis
  • aw264.7 macrophages were harvested with Versene (GibcoTM) and seeded at a density of 10 4 cells per well in 384-well plates (Evotec, Hamburg, Germany) in 50 ⁇ RPMI 1640 supplemented with 10% heat-inactivated foetal calf serum (FCS) (all from Invitrogen). After overnight incubation, the medium was removed, and the cells were labelled for 2 hours at 37°C with bis-(l ,3-diethylthiobarbituric acid) trimethine oxonol (DiSBAC2(3), Invitrogen, B413) at 62.5 g mL.
  • FCS heat-inactivated foetal calf serum
  • the cells were then washed three times with imaging buffer containing 150 mM NaCl, 5 mM C1, 10 mM HEPES, 2 mM CaCk, 2 mM MgCl 2 , 5.5 mM glucose, 2.9 mM sucrose.
  • a first series of image acquisition was conducted to ascertain the proper labelling of the cells (Tlint); myco lactone was then added to the cells. After 20 minutes incubation at room temperature under mild shaking (500 rpm), a second scan was performed to measure the intensity of the fluorescence of the cells (T2int).
  • Tetraethylammonium an inhibitor of various potassium channels and Piroxicam, a highly active inhibitor of prostaglandin were from Sigma.
  • EMLA cream containing 5% lidocaine and prilocaine anaesthetics was from Astra Zeneca. FR122047, a selective inhibitor of COX-1 signalling and PD123,319, a selective inhibitor of AT2R signalling were from Tocris Bioscience.
  • 200 (daily renewed) solution of piroxicam at 0.5 mg/mL in 1 % gelosed water was administered by gavage every day starting 30 days after M. ulcer cms inoculation.
  • Osmotic mini-pumps (AlzetVR model 2002, Palo Alto, CA) containing PD123,319 were implanted subcutaneously under anaesthesia in the back of the neck of mice 30 days after M. ulcer ans inoculation, with a delivery rate of 20 mg/kg/day in water.
  • mice We developed a mouse model to assess the relationship between hypoesthesia and nerve degeneration under low dose conditions of ulcerans infection.
  • M. ulcerans was injected into the footpads of mice, and hypoesthesia was monitored with the pain receptive tail- flick assay at different time points. Under such conditions, mice exhibited redness at day 35 post-infection and oedema at day 4 . At both time points, hypoesthesia was observed in M. ulcerans- fected animals, with a significant tail- flick latency of 4 seconds, as compared to non-infected controls (Figure 1A). Histological analysis of mice inoculated with M.
  • GFP green fluorescent protein
  • Hyperpolarization is typically associated with K+ channels, and we checked that such channels were involved in the mycolactone-induced hyperpolarization. To this end we treated cells with the two + channel blockers teftaemylammonium (TEA) and barium chloride (BaCh). As shown in Figures 2E and 2F, TEA and BaCb abolished the mycolactone-mediated hyperpolarization demonstrating that it is mediated by K+ efflux. At this level, the analgesic action of mycolactone appears to share common features with the non-opioid analgesic flupirtine, which also hyperpolarises neurons by activation of + channels (Szelenyi, 2013).
  • the + channels involved in the mycolactone-mediated hyperpolarization appear to be sensitive to TEA and BaCb. only at relatively high concentrations (above 10 mM and 1 mM, respectively), suggesting that they belong to the K2P channel family (Noel et al., 2011; Sandoz et al, 2012).
  • angiotensin II type 2 receptor (AGTR2 or AT2R) represented a favourable candidate to account for the observed analgesia effects.
  • AGTR2 or AT2R represents a favourable candidate to account for the observed analgesia effects.
  • This receptor has been reported recently, in a completely different context, to be specifically involved in nociception in the course of experiments testing the effects of the AT2R chemical antagonist PD 123,319 (Anand et al, 2012). Accordingly we focused our subsequent investigations on AT2R receptors.
  • AT2R-selective antagonist PD 123,319 inhibits mycolactone- induced hyperpolarization in neuronal cells (Figure 3D).
  • AT2R is a G-protein-coupled receptor, which couples selectively to pertussis toxin (PTX)-sensitive Gia2 and Gia3 proteins (Kang et al, 1994; Sumners and Gelband, 1998).
  • PTX pertussis toxin
  • Figure 3E we showed that mycolactone-mediated hyperpolarization was abolished, thus demonstrating that G-ai proteins are involved in mycolactone-induced AT 2 R signalling.
  • the second-pass reanalysis of our siRNA primary screen further allowed refining the characterization of the K+ channels in terms of subfamily assignment. More precisely such reanalysis allowed to pinpoint two possible candidate subfamilies to which the potassium channels involved in the mycolactone induced hyperpolarization belong, namely CNK4 (TRAAK) and KCNK13 (THIK1) from the K2P channels family (Figure 3F). Channels from the K2P family function as regulatory hubs for the generation of negative resting membrane potentials and therefore they constitute appropriate final targets of the signaling pathway involved in the mycolactone-induced hyperpolarization.
  • CNK4 TRAAK
  • KCNK13 TKIK1
  • Mycolactone triggers TRAAK activation in heterologous system
  • Mycolactone is able to induce a 3 fold increase of the TRAAK current obtained from HEK cells expressing AT2R and TRAAK channel. It is worth to note that Mycolactone as no effect on TRAAK current in the absence of AT2R. In addition, the TRAAK current density shows no modification induced by AT2R expression showing that the AT2R has no basal activity for the AT2R- TRAAK pathway.
  • Mycolactone triggers a very specific signaling to activate TRAAK
  • TREKl is not sensitive to AT2R activation by Mycolactone.
  • TREKl which belongs to the same channel subfamily as TRAAK, sharing a high degree of homology with TRAAK, could be activated by Mycolactone.
  • TREKl is not sensitive to AT2R activation by Mycolactone ( Figure 8, P>0.8), thus the role of TREKl in our system will not be investigated any further.
  • TREKl is known to be activated through a canonical GiPCR pathway, the absence of regulation in our system provides an additional argument to rule out a major contribution of a classical G protein pathway.
  • the focus of the work here is the molecular dissection of a previously unanticipated infection strategy employed by a bacterium that uses the mycolactone toxin as an effective analgesic to annul the pain of the lesions it causes. It was previously believed that absence of pain in Buruli ulcer disease could be attributed to nerve damage, caused by the action of the toxin mycolactone secreted by M. ulcerans. Accordingly we first revisited this hypothesis to test it in detail. We demonstrated, with purified as well as synthetic mycolactone, that the analgesic effect of the toxin was not accompanied by nerve degeneration. In addition, it is clear that M. ulcerans does not associate with nerve fibres and it is most likely that the toxin reaches neurons through diffusion.
  • the findings here can be cast in more general perspectives relevant to the theme of counteracting pain on health-oriented grounds. It is particularly significant to notice that the pathway dissected here involves a natural effector that does not belong to the different classes of analgesics of common use today, such as paracetamol (or acetaminophen), opiate molecules such as morphine or non-steroidal antiinflammatory drugs (NSAID) such as salicylates. Notably because of series of, more or less severe, secondary effects it is rather unanimously recognized that new potent analgesics, showing less adverse effects, are highly desirable.
  • analgesics of common use today such as paracetamol (or acetaminophen), opiate molecules such as morphine or non-steroidal antiinflammatory drugs (NSAID) such as salicylates.
  • NSAID non-steroidal antiinflammatory drugs
  • AT2R antagonist PD 123,319 also called EMA200
  • AT2R antagonist PD 123,319 also called EMA200
  • EMA300 and EMA401 alleviate neuropathic pain through AT 2 R (Rice et al., 2014; Smith et al., 2013).
  • Mycolactone is responsible for the painlessness of Mycobacterium ulcerans infection (buruli ulcer) in a murine study. Infect Immun 76, 2002-2007.
  • Mycolactone a polyketide toxin from Mycobacterium ulcerans required for virulence. Science 283, 854-857. Gersbach, P., Jantsch, A., Feyen, F., Scherr, N., Dangy, J.P., Pluschke, G., and Altmann, .H. (2011). A ring-closing metathesis (RCM)-based approach to mycolactones A/B. Chemistry 17, 13017-13031.
  • Angiotensin II type 2 receptor stimulation of neuronal K+ currents involves an inhibitory GTP binding protein. Am J Physiol 267, C 1389- 1397.
  • EMA401 an orally administered highly selective angiotensin II type 2 receptor antagonist, as a novel treatment for postherpetic neuralgia: a randomised, double-blind, placebo-controlled phase 2 clinical trial. Lancet.

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WO2019183513A1 (en) * 2018-03-22 2019-09-26 University Of Iowa Research Foundation Compositions and methods for the treatment and prevention of muscular dystrophy
US10550156B2 (en) 2014-07-21 2020-02-04 Arizona Board Of Regents On Behalf Of The University Of Arizona Ang (1-7) derivative oligopeptides and methods for using and producing the same
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US10550156B2 (en) 2014-07-21 2020-02-04 Arizona Board Of Regents On Behalf Of The University Of Arizona Ang (1-7) derivative oligopeptides and methods for using and producing the same
US10881708B2 (en) 2014-07-21 2021-01-05 Arizona Board Of Regents On Behalf Of The University Of Arizona Ang (1-7) derivative oligopeptides for the treatment of pain
US11104706B2 (en) 2014-07-21 2021-08-31 Arizona Board Of Regents On Behalf Of The University Of Arizona Ang (1-7) derivative oligopeptides and methods for using and producing the same
WO2018129506A3 (en) * 2017-01-09 2018-08-16 Arizona Board Of Regents On Behalf Of The University Of Arizona Ang (1-7) derviative oligopeptides for the treatment of pain
WO2019183513A1 (en) * 2018-03-22 2019-09-26 University Of Iowa Research Foundation Compositions and methods for the treatment and prevention of muscular dystrophy
US11931341B2 (en) 2018-03-22 2024-03-19 University Of Lowa Research Foundation Compositions and methods for the treatment and prevention of muscular dystrophy
WO2024236051A1 (fr) 2023-05-16 2024-11-21 Centre National De La Recherche Scientifique Formulation antidouleur à effet prolongé
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